**Meet the editor**

Dr. Mohammad Mahdi Najafpour received his PhD in Inorganic Chemistry from Sharif University of Technology, Tehran, Iran, in 2009. He has been a guest scientist in Dr Philipp Kurz's group in the Institut für Anorganische Chemie, Christian-Albrechts-Universität zu Kiel in Germany for six months. Mahdi is a recipient of several awards and fellowships, notably the gold medal of the

National Chemistry Olympiad in 2004; he ranked 1st in the Khwarizmi Youth Festival in 2010. Currently, he is a faculty member in the Chemistry Department and also the Center of Climate Change and Global Warming in the Institute for Advanced Studies in Basic Sciences (IASBS) (Zanjan, Iran). Mahdi and his research group explore transition-metal compounds as water oxidizing catalysts for artificial photosynthesis. He is the author of over 60 publications in international journals in these and other areas.

Contents

**Preface IX** 

**How and Why? 3** 

**Part 2 Light and Photosynthesis 13** 

Chapter 2 **The Guiding Force of Photons 15** 

Chapter 4 **Carotenoids and Photosynthesis -** 

Chapter 5 **Mechanisms of Photoacclimation** 

Claudia Stange and Carlos Flores

Sabina Jodłowska and Adam Latała

Chapter 6 **Photosynthesis in Microalgae as Measured** 

Ana Serrano and Milagros Medina

Chapter 7 **Fast Kinetic Methods with Photodiode** 

**and Ferredoxin NADP+**

Kevin M. Folta

Chapter 3 **Energy Conversion** 

Mohammad Mahdi Najafpour and Babak Pashaei

**in Purple Bacteria Photosynthesis 43**  Felipe Caycedo-Soler, Ferney J. Rodríguez, Luis Quiroga, Guannan Zhao and Neil F. Johnson

**on Photosynthesis Level in Cyanobacteria 97** 

**with Delayed Fluorescence Technique 109**  Maja Berden-Zrimec, Marina Monti and Alexis Zrimec

**Array Detection in the Study of the Interaction and Electron Transfer Between Flavodoxin** 

**Regulation of Carotenoid Biosyntesis by Photoreceptors 77** 

**-Reductase 129** 

**Part 1 Introduction 1** 

Chapter 1 **Photosynthesis:** 

### Contents

#### **Preface XIII**

**Part 1 Introduction 1**  Chapter 1 **Photosynthesis: How and Why? 3**  Mohammad Mahdi Najafpour and Babak Pashaei **Part 2 Light and Photosynthesis 13**  Chapter 2 **The Guiding Force of Photons 15**  Kevin M. Folta Chapter 3 **Energy Conversion in Purple Bacteria Photosynthesis 43**  Felipe Caycedo-Soler, Ferney J. Rodríguez, Luis Quiroga, Guannan Zhao and Neil F. Johnson Chapter 4 **Carotenoids and Photosynthesis - Regulation of Carotenoid Biosyntesis by Photoreceptors 77**  Claudia Stange and Carlos Flores Chapter 5 **Mechanisms of Photoacclimation on Photosynthesis Level in Cyanobacteria 97**  Sabina Jodłowska and Adam Latała Chapter 6 **Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 109**  Maja Berden-Zrimec, Marina Monti and Alexis Zrimec Chapter 7 **Fast Kinetic Methods with Photodiode** 

**Array Detection in the Study of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP+ -Reductase 129**  Ana Serrano and Milagros Medina

X Contents


Contents VII

**Part 4 Special Topics in Photosynthesis 401** 

**Maquis During Winter 403**  Carmen Arena and Luca Vitale

Chapter 20 **The Core- and Pan-Genomes**

Shinji Masuda

Chapter 19 **Photosynthetic Adaptive Strategies in Evergreen** 

**of Photosynthetic Prokaryotes 423** Jeffrey W. Touchman and Yih-Kuang Lu

Chapter 21 **A Review: Polyamines and Photosynthesis 439** Sheng Shu, Shi-Rong Guo and Ling-Yun Yuan

Chapter 22 **An Overview of Plant Photosynthesis Modulation** 

Kumarakurubaran Selvaraj and Bourlaye Fofana

**in Juvenile Tropical Trees Under Contrasting** 

**by Pathogen Attacks 465** 

Chapter 23 **The Stringent Response in Phototrophs 487** 

Chapter 24 **Morphological and Physiological Adjustments** 

Chapter 25 **Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids 519**

> Nikolaos E. Ioannidis, Josep Maria Torné, Kiriakos Kotzabasis and Mireya Santos

Chapter 26 **The Plant–Type Ferredoxin-NADP<sup>+</sup> Reductases 539**  Matías A. Musumeci, Eduardo A. Ceccarelli

and Daniela L. Catalano-Dupuy

Chapter 27 **Primary Production in the Ocean 563**  Daniel Conrad Ogilvie Thornton

**Sunlight Irradiance 501**  Geraldo Rogério Faustini Cuzzuol and Camilla Rozindo Dias Milanez

**and Semi-Deciduous Species of Mediterranean** 


Daniel Conrad Ogilvie Thornton

VI Contents

Chapter 8 **Photosynthesis in Lichen:** 

**Light Reactions and Protective Mechanisms 149**

Alfonso Ros-Barceló, Eva Barreno, José Miguel Zapata and Alfredo Guéra

Lea Vojta and Hrvoje Fulgosi

Chapter 11 **Chloroplast Photorelocation Movement:** 

Chapter 10 **The Photomorphogenic Signal:** 

Mansour Matloobi

Chapter 9 **Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents 175**

Francisco M. Iglesias and Pablo D. Cerdán

**A Sophisticated Strategy for Chloroplasts to Perform Efficient Photosynthesis 215**  Noriyuki Suetsugu and Masamitsu Wada

Chapter 12 **Light Harvesting and Photosynthesis by the Canopy 235** 

Chapter 13 **The Path of Carbon in Photosynthesis – XXVIII – Response of Plants to Polyalkylglucopyranose and** 

Arthur M. Nonomura, Barry A. Cullen and Andrew A. Benson

**Part 3 The Path of Carbon in Photosynthesis 257** 

**Polyacylglucopyranose 259** 

Chapter 14 **The Role of C to N Balance in the Regulation of Photosynthetic Function 273** 

Vladimir I. Chikov and Svetlana N. Batasheva

Chapter 15 **High-CO2 Response Mechanisms in Microalgae 299**  Masato Baba and Yoshihiro Shiraiwa

**Seedlings to Elevated Carbon Dioxide 321** 

Chapter 17 **Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 341** 

Chapter 16 **Photosynthesis and Quantum Yield of Oil Palm** 

H.Z.E. Jaafar and Mohd Hafiz Ibrahim

Alexander A. Ivlev

Roghieh Hajiboland

Chapter 18 **Photosynthetic Carbon Metabolism: Plasticity and Evolution 367** 

Francisco Gasulla, Joaquín Herrero, Alberto Esteban-Carrasco,

**An Essential Component of Photoautotrophic Life 191**  Sabrina Iñigo, Mariana R. Barber, Maximiliano Sánchez-Lamas,

Preface

chapter.

Basic Sciences (Zanjan, Iran) for support.

throughout the time that the book was being prepared.

Photosynthesis is one of the most important reactions on Earth. It is estimated that photosynthesis produces more than 100 billion tons of dry biomass annually. These fossil fuels are also derived from millions of years of photosynthetic activity. Now, the advances in characterization techniques and their application to the field have improved our understanding of photosynthesis. This book is aimed at providing the fundamental aspects of photosynthesis, and the results collected from different research groups. We have three sections in this book: light and photosynthesis, the path of carbon in photosynthesis, and special topics in photosynthesis. In each section important topics in the subject are discussed and (or) reviewed by experts in each book

I would like to take this opportunity to thank all the contributors for their chapters.

I wish to express my gratitude to the staff at In Tech, and in particular Mr. Vedran Greblo, for his kind assistance. I am grateful to the Institute for Advanced Studies in

Finally I want to thank my wife, Mary, for her encouragement and infinite patience

**Mohammad Mahdi Najafpour**  Department of Chemistry,

Gava Zang, Zanjan,

Iran

Institute for Advanced Studies in Basic Sciences,

### Preface

Photosynthesis is one of the most important reactions on Earth. It is estimated that photosynthesis produces more than 100 billion tons of dry biomass annually. These fossil fuels are also derived from millions of years of photosynthetic activity. Now, the advances in characterization techniques and their application to the field have improved our understanding of photosynthesis. This book is aimed at providing the fundamental aspects of photosynthesis, and the results collected from different research groups. We have three sections in this book: light and photosynthesis, the path of carbon in photosynthesis, and special topics in photosynthesis. In each section important topics in the subject are discussed and (or) reviewed by experts in each book chapter.

I would like to take this opportunity to thank all the contributors for their chapters.

I wish to express my gratitude to the staff at In Tech, and in particular Mr. Vedran Greblo, for his kind assistance. I am grateful to the Institute for Advanced Studies in Basic Sciences (Zanjan, Iran) for support.

Finally I want to thank my wife, Mary, for her encouragement and infinite patience throughout the time that the book was being prepared.

> **Mohammad Mahdi Najafpour**  Department of Chemistry, Institute for Advanced Studies in Basic Sciences, Gava Zang, Zanjan, Iran

**Part 1** 

**Introduction** 

## **Part 1**

### **Introduction**

**1** 

 *Iran* 

**Photosynthesis: How and Why?** 

Mohammad Mahdi Najafpour\* and Babak Pashaei

*Chemistry Department, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan,* 

The total solar energy absorbed by Earth is approximately 3,850,000 exajoules per year. This was more energy in one hour than the world used in one year! *Nature* uses very wonderful and interesting strategies to capture the energy in an interesting process: *Photosynthesis*. To know more about photosynthesis, the first we should know about phototrophy. Phototrophy is the process by which organisms trap photons and store energy as chemical energy in the form of adenosine triphosphate (ATP). ATP transports chemical energy within cells for metabolism. There are three major types of phototrophy: Oxygenic and Anoxygenic photosynthesis, and Rhodopsin-based phototrophy. Photosynthesis is a chemical process that converts carbon dioxide into different organic compounds using solar energy. Oxygenic and anoxygenic photosynthesis undergo different reactions in the presence and absence of light (called light and dark reactions, respectively). In anoxygenic photosynthesis, light energy is captured and stored as ATP, without the production of oxygen. This means water is not used as primary electron donor. Phototrophic green bacteria, phototrophic purple bacteria, and heliobacteria are three groups of bacteria that use anoxygenic photosynthesis. Anoxygenic phototrophs have photosynthetic pigments called bacteriochlorophylls. Bacteriochlorophyll a and b have maxima wavelength absorption at 775 nm and 790 nm, respectively in ether. Unlike oxygenic phototrophs, anoxygenic photosynthesis only functions using a single photosystem. This restricts them to cyclic electron flow only, and they are therefore unable to produce O2 from the oxidization of H2O. In plants, algae and cyanobacteria, the photosynthetic processes results not only in the fixation of carbon dioxide (CO2) from the atmosphere but also release of molecular oxygen to the atmosphere. This

Photosynthesis captures approximately 3,000 EJ per year in biomass and produces more than 100 billion tons of dry biomass annually (Barber, 2009). Photosynthesis is also

It is believed that the first photosynthetic organisms evolved about 3,500 million years ago. In that condition, the atmosphere had much more carbon dioxide and organisms used hydrogen or hydrogen sulfide as sources of electron (Olson, 2006). Around 3,000 million years ago, Cyanobacteria appeared later and changed the Earth when they began to oxygenate the atmosphere, beginning about 2,400 million years ago. This new atmosphere was a revolution for complex life. The chloroplasts in modern plants are the descendants of

necessary for maintaining the normal level of oxygen in the atmosphere.

**1. Introduction** 

\* *Corresponding Author*

process is known as oxygenic photosynthesis.

### **Photosynthesis: How and Why?**

Mohammad Mahdi Najafpour\* and Babak Pashaei

*Chemistry Department, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, Iran* 

#### **1. Introduction**

The total solar energy absorbed by Earth is approximately 3,850,000 exajoules per year. This was more energy in one hour than the world used in one year! *Nature* uses very wonderful and interesting strategies to capture the energy in an interesting process: *Photosynthesis*.

To know more about photosynthesis, the first we should know about phototrophy. Phototrophy is the process by which organisms trap photons and store energy as chemical energy in the form of adenosine triphosphate (ATP). ATP transports chemical energy within cells for metabolism. There are three major types of phototrophy: Oxygenic and Anoxygenic photosynthesis, and Rhodopsin-based phototrophy. Photosynthesis is a chemical process that converts carbon dioxide into different organic compounds using solar energy. Oxygenic and anoxygenic photosynthesis undergo different reactions in the presence and absence of light (called light and dark reactions, respectively). In anoxygenic photosynthesis, light energy is captured and stored as ATP, without the production of oxygen. This means water is not used as primary electron donor. Phototrophic green bacteria, phototrophic purple bacteria, and heliobacteria are three groups of bacteria that use anoxygenic photosynthesis. Anoxygenic phototrophs have photosynthetic pigments called bacteriochlorophylls. Bacteriochlorophyll a and b have maxima wavelength absorption at 775 nm and 790 nm, respectively in ether. Unlike oxygenic phototrophs, anoxygenic photosynthesis only functions using a single photosystem. This restricts them to cyclic electron flow only, and they are therefore unable to produce O2 from the oxidization of H2O. In plants, algae and cyanobacteria, the photosynthetic processes results not only in the fixation of carbon dioxide (CO2) from the atmosphere but also release of molecular oxygen to the atmosphere. This process is known as oxygenic photosynthesis.

Photosynthesis captures approximately 3,000 EJ per year in biomass and produces more than 100 billion tons of dry biomass annually (Barber, 2009). Photosynthesis is also necessary for maintaining the normal level of oxygen in the atmosphere.

It is believed that the first photosynthetic organisms evolved about 3,500 million years ago. In that condition, the atmosphere had much more carbon dioxide and organisms used hydrogen or hydrogen sulfide as sources of electron (Olson, 2006). Around 3,000 million years ago, Cyanobacteria appeared later and changed the Earth when they began to oxygenate the atmosphere, beginning about 2,400 million years ago. This new atmosphere was a revolution for complex life. The chloroplasts in modern plants are the descendants of

<sup>\*</sup> *Corresponding Author*

Photosynthesis: How and Why? 5

of electrons down an electron transport chain that leads to the ultimate reduction of

Fig. 2. Plants absorb light primarily using some pigments.

water molecule and oxidizes it to dioxygen (O2):

The proton gradient across the chloroplast membrane is used by ATP synthase for the concomitant synthesis of ATP. The chlorophyll molecule regains the lost electron from a

NADP to NADPH.

these ancient symbiotic cyanobacteri (Gould et al., 2008). In plants and algae, photosynthesis takes place in chloroplasts. Each plant cell contains about 10 to 100 chloroplasts (Fig. 1).

Fig. 1. In photosynthesis, organic synthesis and oxygen evolution reactions performs in two distinct enzymatic systems.

The chloroplast is composed of two membranes (phospholipid inner and outer membrane) and an intermembrane space between them. Within the membrane is an aqueous fluid called the stroma contains stacks (grana) of thylakoids, which are the site of photosynthesis. The thylakoids are flattened disks, bounded by a membrane with a lumen or thylakoid space within it. The site of photosynthesis is the thylakoid membrane, which contains integral and peripheral membrane protein complexes, including the pigments that absorb light energy, which form the photosystems. The first step in photosynthesis is the absorption of light by a pigment molecule of photosynthetic antenna resulting in conversion of the photon energy to an excited electronic state of pigment molecule. Plants absorb light primarily using the pigment chlorophyll. Besides chlorophyll, organisms also use pigments such as, phycocyanin, carotenes, xanthophylls, phycoerythrin and fucoxanthin (Fig. 2).

The most useful decay pathway is "energy transfer" to a photochemical reaction centers, and it is important to photosynthetic reactions. Excitons trapped by a reaction center provide the energy for the primary photochemical reactions. Subsequent electron transfer reactions occur in the dark which results in accumulation of chemical bound energy. In the other words, photosynthesis occurs in two stages. In the first stage, *light-dependent reactions* or *light reactions* capture the energy of light and use it to make the energy-storage molecules (ATP). During the second stage, the *light-independent reactions* use these products to capture and reduce carbon dioxide (Govindjee et al., 2010). The dark reaction doesn't directly need light, but it does need the products of the light reaction.

In the light reactions, a chlorophyll molecule of reaction center absorbs one photon and loses one electron. This electron is passed to a modified form of chlorophyll called pheophytin, which passes the electron to a quinone molecule, allowing the start of a flow

these ancient symbiotic cyanobacteri (Gould et al., 2008). In plants and algae, photosynthesis takes place in chloroplasts. Each plant cell contains about 10 to 100 chloroplasts (Fig. 1).

Fig. 1. In photosynthesis, organic synthesis and oxygen evolution reactions performs in two

The chloroplast is composed of two membranes (phospholipid inner and outer membrane) and an intermembrane space between them. Within the membrane is an aqueous fluid called the stroma contains stacks (grana) of thylakoids, which are the site of photosynthesis. The thylakoids are flattened disks, bounded by a membrane with a lumen or thylakoid space within it. The site of photosynthesis is the thylakoid membrane, which contains integral and peripheral membrane protein complexes, including the pigments that absorb light energy, which form the photosystems. The first step in photosynthesis is the absorption of light by a pigment molecule of photosynthetic antenna resulting in conversion of the photon energy to an excited electronic state of pigment molecule. Plants absorb light primarily using the pigment chlorophyll. Besides chlorophyll, organisms also use pigments such as, phycocyanin, carotenes, xanthophylls, phycoerythrin and fucoxanthin (Fig. 2). The most useful decay pathway is "energy transfer" to a photochemical reaction centers, and it is important to photosynthetic reactions. Excitons trapped by a reaction center provide the energy for the primary photochemical reactions. Subsequent electron transfer reactions occur in the dark which results in accumulation of chemical bound energy. In the other words, photosynthesis occurs in two stages. In the first stage, *light-dependent reactions* or *light reactions* capture the energy of light and use it to make the energy-storage molecules (ATP). During the second stage, the *light-independent reactions* use these products to capture and reduce carbon dioxide (Govindjee et al., 2010). The dark reaction doesn't directly need

In the light reactions, a chlorophyll molecule of reaction center absorbs one photon and loses one electron. This electron is passed to a modified form of chlorophyll called pheophytin, which passes the electron to a quinone molecule, allowing the start of a flow

distinct enzymatic systems.

light, but it does need the products of the light reaction.

of electrons down an electron transport chain that leads to the ultimate reduction of NADP to NADPH.

Fig. 2. Plants absorb light primarily using some pigments.

The proton gradient across the chloroplast membrane is used by ATP synthase for the concomitant synthesis of ATP. The chlorophyll molecule regains the lost electron from a water molecule and oxidizes it to dioxygen (O2):

Photosynthesis: How and Why? 7

Recently, Umena et al. (Umena et al., 2011) reported crystal structure of this calciummanganese cluster of photosystem II at an atomic resolution. In this structure one calcium and four manganese ions are bridged by five oxygen atoms. Four water molecules were found also in this structure that two of them are suggested as the substrates for water

Fig. 4. The structure of water oxidizing complex (WOC) (Umena et al., 2011).

Light-dependent reactions occur in the thylakoid membranes of the chloroplasts in plants and use light energy to synthesize ATP and NADPH. Cyclic and non-cyclic are two forms of the light-dependent reaction. In the non-cyclic reaction, the photons are captured in the light-harvesting antenna complexes of photosystem II by different pigments (Fig. 5 and

oxidation (Fig. 4).

Fig. 6).

#### 2H2O + 2NADP+ + 3ADP + 3Pi + light → 2NADPH + 2H+ + 3ATP + O2

A good method to study of oxygen evolution in this process is to activate a photosynthetic system with short and intense light flashes and study of oxygen evolution reaction. Joliot's experiments in 1969 showed that flashes produced an oscillating pattern in the oxygen evolution and a maximum of water oxidation occurred on every fourth flash (Satoh et al., 2005). These patterns were very interesting because splitting of two water molecules to produce one oxygen molecule requires the removal of also four electrons. In 1970, Kok proposed an explanation for the observed oscillation of the oxygen evolution pattern (Kok et al., 1970). Kok's hypothesis (Kok et al., 1970) is that in a cycle of water oxidation succession of oxidizing equivalents is stored on each separate and independent water oxidizing complex, and when four oxidizing equivalents have been accumulated one by one an oxygen is spontaneously evolved (Kok et al., 1970). Each oxidation state of the water oxidizing complex is known as an "S-state" and S0 being the most reduced state and S4 the most oxidized state in the catalytic cycle (Fig. 3) (Kok et al., 1970). The S1 state is dark-stable. The S4 → S0 transition is light independent and in this state oxygen is evolved. Other S-state transitions are induced by the photochemical oxidation of oxidized chlorophyll (P680+) (Satoh et al., 2005).

Fig. 3. Catalytic cycle proposed by Joliot and Kok for water oxidation, protons and electrons at photosystem II. The figure was reproduced from Sproviero et al., 2008.

2H2O + 2NADP+ + 3ADP + 3Pi + light → 2NADPH + 2H+ + 3ATP + O2 A good method to study of oxygen evolution in this process is to activate a photosynthetic system with short and intense light flashes and study of oxygen evolution reaction. Joliot's experiments in 1969 showed that flashes produced an oscillating pattern in the oxygen evolution and a maximum of water oxidation occurred on every fourth flash (Satoh et al., 2005). These patterns were very interesting because splitting of two water molecules to produce one oxygen molecule requires the removal of also four electrons. In 1970, Kok proposed an explanation for the observed oscillation of the oxygen evolution pattern (Kok et al., 1970). Kok's hypothesis (Kok et al., 1970) is that in a cycle of water oxidation succession of oxidizing equivalents is stored on each separate and independent water oxidizing complex, and when four oxidizing equivalents have been accumulated one by one an oxygen is spontaneously evolved (Kok et al., 1970). Each oxidation state of the water oxidizing complex is known as an "S-state" and S0 being the most reduced state and S4 the most oxidized state in the catalytic cycle (Fig. 3) (Kok et al., 1970). The S1 state is dark-stable. The S4 → S0 transition is light independent and in this state oxygen is evolved. Other S-state transitions are induced by the photochemical oxidation of oxidized chlorophyll (P680+)

Fig. 3. Catalytic cycle proposed by Joliot and Kok for water oxidation, protons and electrons

at photosystem II. The figure was reproduced from Sproviero et al., 2008.

(Satoh et al., 2005).

Recently, Umena et al. (Umena et al., 2011) reported crystal structure of this calciummanganese cluster of photosystem II at an atomic resolution. In this structure one calcium and four manganese ions are bridged by five oxygen atoms. Four water molecules were found also in this structure that two of them are suggested as the substrates for water oxidation (Fig. 4).

Fig. 4. The structure of water oxidizing complex (WOC) (Umena et al., 2011).

Light-dependent reactions occur in the thylakoid membranes of the chloroplasts in plants and use light energy to synthesize ATP and NADPH. Cyclic and non-cyclic are two forms of the light-dependent reaction. In the non-cyclic reaction, the photons are captured in the light-harvesting antenna complexes of photosystem II by different pigments (Fig. 5 and Fig. 6).

Photosynthesis: How and Why? 9

Z-scheme diagram of oxygenic photosynthesis demonstrates the relative redox potentials of

Fig. 6. Schematic representation of photosystem II and its components embedded in the

thylakoid membrane. The figure was reproduced from Sproviero et al., 2008.

the co-factors in the linear electron transfer from water to NADP+.

Fig. 5. Map of the main cofactors of PSII. The arrows show the electron transfer steps and the numbers indicate the order in which they occur. The figure was reproduced from Herrero et al., 2010.

When a chlorophyll molecule in reaction center of the photosystem II obtains sufficient excitation energy from the adjacent antenna pigments, an electron is transferred to the primary electron-acceptor molecule, pheophytin, through a process called photoinduced charge separation. These electrons are shuttled through an electron transport chain, the socalled *Z-scheme* shown in Fig. 7, that initially functions to generate a chemiosmotic potential across the membrane.

Fig. 5. Map of the main cofactors of PSII. The arrows show the electron transfer steps and the numbers indicate the order in which they occur. The figure was reproduced from

When a chlorophyll molecule in reaction center of the photosystem II obtains sufficient excitation energy from the adjacent antenna pigments, an electron is transferred to the primary electron-acceptor molecule, pheophytin, through a process called photoinduced charge separation. These electrons are shuttled through an electron transport chain, the socalled *Z-scheme* shown in Fig. 7, that initially functions to generate a chemiosmotic potential

Herrero et al., 2010.

across the membrane.

Z-scheme diagram of oxygenic photosynthesis demonstrates the relative redox potentials of the co-factors in the linear electron transfer from water to NADP+.

Fig. 6. Schematic representation of photosystem II and its components embedded in the thylakoid membrane. The figure was reproduced from Sproviero et al., 2008.

Photosynthesis: How and Why? 11

In these reactions, the enzyme RuBisCO captures CO2 from the atmosphere and in a process that requires the newly formed NADPH, releases three-carbon sugars, which are later combined to form sucrose and starch. The overall equation for the light-independent

3 CO2 + 9 ATP + 6 NADPH + 6 H+ → C3H6O3-phosphate + 9 ADP + 8 Pi + 6 NADP+ + 3 H2O

It is believed that photosynthesis is the most important biological process on earth. Our food, energy, environment and culture, directly or indirectly, depend on the important process. Really, the relationship between living organisms and the balance of atmosphere and life on earth needs knowledge of the molecular mechanisms of photosynthesis. The process also provides paradigms for sustainable global energy production and efficient energy transformation. Research into the nature of photosynthesis is necessary because by understanding photosynthesis, we can control it, and use its strategies for the improvement

Barber, J. (2009). Photosynthetic energy conversion: natural and artificial. Chem. Soc. Rev,

Fig. 8. Schematic representation of photosynthesis.

**2. Why is photosynthesis important?** 

Vol. 38, pp. 185-196.

reactions in green plants is:

of human's life.

**3. References** 

Fig. 7. Z-Scheme of Electron Transport in Photosynthesis (the picture provided by Govindjee and Wilbert Veit in http://www.life.illinois.edu/govindjee/photoweb/subjects.html#antennas).

An ATP synthase enzyme uses the chemiosmotic potential to make ATP during photophosphorylation, whereas NADPH is a product of the terminal redox reaction in the *Z-scheme*. Photosystem I operates at the final stage of light-induced electron transfer. It reduces NADP+ via a series of intermediary acceptors that are reduced upon excitation of the primary donor P700 and oxidize plastocyanin. The cyclic reaction is similar to that of the non-cyclic, but differs in the form that it generates only ATP, and no reduced NADP+ (NADPH) is created. The stored energy in the NADPH and ATP is subsequently used by the photosynthetic organisms to drive the synthesis in the Calvin - Benson cycle in the lightindependent or dark reactions (Fig. 8).

Fig. 7. Z-Scheme of Electron Transport in Photosynthesis (the picture provided by

An ATP synthase enzyme uses the chemiosmotic potential to make ATP during photophosphorylation, whereas NADPH is a product of the terminal redox reaction in the *Z-scheme*. Photosystem I operates at the final stage of light-induced electron transfer. It reduces NADP+ via a series of intermediary acceptors that are reduced upon excitation of the primary donor P700 and oxidize plastocyanin. The cyclic reaction is similar to that of the non-cyclic, but differs in the form that it generates only ATP, and no reduced NADP+ (NADPH) is created. The stored energy in the NADPH and ATP is subsequently used by the photosynthetic organisms to drive the synthesis in the Calvin - Benson cycle in the light-

http://www.life.illinois.edu/govindjee/photoweb/subjects.html#antennas).

Govindjee and Wilbert Veit in

independent or dark reactions (Fig. 8).

Fig. 8. Schematic representation of photosynthesis.

In these reactions, the enzyme RuBisCO captures CO2 from the atmosphere and in a process that requires the newly formed NADPH, releases three-carbon sugars, which are later combined to form sucrose and starch. The overall equation for the light-independent reactions in green plants is:

3 CO2 + 9 ATP + 6 NADPH + 6 H+ → C3H6O3-phosphate + 9 ADP + 8 Pi + 6 NADP+ + 3 H2O

#### **2. Why is photosynthesis important?**

It is believed that photosynthesis is the most important biological process on earth. Our food, energy, environment and culture, directly or indirectly, depend on the important process. Really, the relationship between living organisms and the balance of atmosphere and life on earth needs knowledge of the molecular mechanisms of photosynthesis. The process also provides paradigms for sustainable global energy production and efficient energy transformation. Research into the nature of photosynthesis is necessary because by understanding photosynthesis, we can control it, and use its strategies for the improvement of human's life.

#### **3. References**

Barber, J. (2009). Photosynthetic energy conversion: natural and artificial. Chem. Soc. Rev, Vol. 38, pp. 185-196.

**Part 2** 

**Light and Photosynthesis** 


## **Part 2**

**Light and Photosynthesis** 

12 Advances in Photosynthesis – Fundamental Aspects

Gould, S.; Waller, R. & Mcfadden, G. (2008). Plastid evolution. Annu. Rev. Plant. Biol, Vol.

Govindjee; Kern, J.F.; Messinger, J.& Whitmarsh, J. (2010) Photosystem II. In: Encyclopedia

Herrero, Ch.; Lassalle-Kaiser, B.; Leibl, W.; Rutherford, A.W.; Ally Coord, A. (2008).

Kok, B.; Forbush, B. & McGloin, M. (1970). Cooperation of charges in photosynthetic O2

Satoh, K.; Wydrzynski, T.J. & Govindjee, (2005). Introduction to photosystem II. In:

Sproviero, E.M.; Gascón, J. A.; McEvoy, J.P.; Brudvig, G.W. & Batista V.S. (2008) Coord.

Umena, Y.; Kawakami, K.; Shen, J.R. & Kamiya, N. (2011). Crystal structure of oxygenevolving photosystem II at a resolution of 1.9Ǻ. Nature, v. 473, p. 55-60.

http://www.life.illinois.edu/govindjee/photoweb/subjects.html#antennas).

Olson, J. (2006). Photosynthesis in the archean. Photosyn. Res, Vol. 88(2), pp. 109-117.

Artificial systems related to light driven electron transfer processes in PSII. Chem.

evolution: I. A linear four-step mechanism. Photochem. Photobiol, v. 11, p. 457 –

Wydrzynski, T.J. & Satoh, K. (eds) Photosystem II: The light-driven water: plastoquinone oxidoreductase. Advances in Photosynthesis and Respiration, v. 22.

of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester.

59, pp. 491-517.

475.

Rev, Vol. 252, pp. 456-468.

Springer, Dordrecht, p. 111-227.

Chem. Rev., v. 252, p. 395–415.

**2** 

*USA*

Kevin M. Folta

**The Guiding Force of Photons** 

*and Cellular Biology, University of Florida, Gainesville, FL* 

*Horticultural Sciences Department, Graduate Program in Plant Molecular* 

In a book titled *Photosynthesis* it is easy to forget that light is not simply the energy driving plant metabolism. Light also is the central environmental factor that affects plant size, shape and development. In fact, light activation of photomorphogenic signaling pathways sets the stage for photosynthesis and ensures the maintenance of the apparatus. The effects of specific wavebands of light exert their influence on plant biology from the molecular level all the way up to the higher morphological level, and even contribute to the canopy form as a whole. The wide influence is based on the fact that the light wavelengths that optimally activate photosynthesis also strongly modulate mechanisms that control plant morphology, such as the length of internodes, expansion of leaves or even leaf position. The same light qualities also guide the development, activity and maintenance of the chloroplast, as the demands of light-driven autotrophy require specialized communication and coordination between the plastid and nucleus to ensure full function of the organelle. Contrastingly, the light qualities that lend relatively little power to photosynthesis provide important information about the ambient environment as well that lead to adaptive adjustments in

Clearly, integrating information from the light environment is an important prerequisite of photosynthesis, as it sets the stage for photosynthetic activity and later maintains the core apparatus. Precise regulatory mechanisms guide the non-photosynthetic plastid, the etioplast, toward photosynthetic competence. Sensing of the first photons of light sparks a rapid cascade of events that shift the role of the plastid from a warehouse of essential materials to a dynamic center of metabolism. The control of gene expression associated with the conversion of etioplast to chloroplast has been well described, and is a central theme of

Nuclear genes are required for photosynthetic competence. Studies of Ribulose bisphosphosphate carboxylase-oxygenase small subunit (*Rbcs)* and Chlorophyll a/b binding protein (*cab*; synonymous with Light-harvesting, chlorophyll-binding protein or *Lhcb*) transcript accumulation have been models of photomorphogenic gene expression going back almost three decades. The current literature describes the transition in the parlance of genome-wide changes, and a complementary proteomics literature adds additional understanding of how the dark-state of the plastid matures rapidly into a light-harvesting

**1. Introduction** 

physiology.

this chapter.

sub-cellular machine.

### **The Guiding Force of Photons**

Kevin M. Folta

*Horticultural Sciences Department, Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL USA*

#### **1. Introduction**

In a book titled *Photosynthesis* it is easy to forget that light is not simply the energy driving plant metabolism. Light also is the central environmental factor that affects plant size, shape and development. In fact, light activation of photomorphogenic signaling pathways sets the stage for photosynthesis and ensures the maintenance of the apparatus. The effects of specific wavebands of light exert their influence on plant biology from the molecular level all the way up to the higher morphological level, and even contribute to the canopy form as a whole. The wide influence is based on the fact that the light wavelengths that optimally activate photosynthesis also strongly modulate mechanisms that control plant morphology, such as the length of internodes, expansion of leaves or even leaf position. The same light qualities also guide the development, activity and maintenance of the chloroplast, as the demands of light-driven autotrophy require specialized communication and coordination between the plastid and nucleus to ensure full function of the organelle. Contrastingly, the light qualities that lend relatively little power to photosynthesis provide important information about the ambient environment as well that lead to adaptive adjustments in physiology.

Clearly, integrating information from the light environment is an important prerequisite of photosynthesis, as it sets the stage for photosynthetic activity and later maintains the core apparatus. Precise regulatory mechanisms guide the non-photosynthetic plastid, the etioplast, toward photosynthetic competence. Sensing of the first photons of light sparks a rapid cascade of events that shift the role of the plastid from a warehouse of essential materials to a dynamic center of metabolism. The control of gene expression associated with the conversion of etioplast to chloroplast has been well described, and is a central theme of this chapter.

Nuclear genes are required for photosynthetic competence. Studies of Ribulose bisphosphosphate carboxylase-oxygenase small subunit (*Rbcs)* and Chlorophyll a/b binding protein (*cab*; synonymous with Light-harvesting, chlorophyll-binding protein or *Lhcb*) transcript accumulation have been models of photomorphogenic gene expression going back almost three decades. The current literature describes the transition in the parlance of genome-wide changes, and a complementary proteomics literature adds additional understanding of how the dark-state of the plastid matures rapidly into a light-harvesting sub-cellular machine.

The Guiding Force of Photons 17

Plants interact with the ambient light environment through a series of light sensors. These specialized molecules capture photons of discrete wavelengths and initiate downstream signaling events that ultimately lead to changes in gene expression, development, and/or morphology. It is important to remember that plants rely on these environmental cues to drive, or in some cases constrain, their development. In the context of photosynthesis, light signaling is important to consider in two general contexts. The transition from etiolate to autotrophic growth is driven by light. As the developing seedling meanders through the soil, sensitive light receptors are in place and prepared to ignite a downstream flow of events upon capture of a photon. These signaling events in many cases prepare the plastid, shifting its function from that of an etioplast to the metabolic center of the chloroplast. The sensory networks that transduce information starting with the capture of a photon into a suite of downstream responses are well known. Historically these pigments were postulated to control various aspects of plant growth and development, particularly germination, phototropic movements and the transition between vegetative and reproductive growth. Long before the genes and proteins were identified and characterized, a tremendous body of work produced evidence of their activities and effects on physiology. Light quality effects on germination were examined by Lewis H. Flint where he demonstrated the promotive effects of red light (Flint, 1936). These studies were expanded through collaboration with E.D. McAlister, a physicist that utilized a spectrograph to split the spectrum and illuminate seeds with discrete wavelengths. Together Flint and McAlister generated elegant action spectra that illustrated how red light promoted germination, while blue, green and far-red light were inhibitory (Flint and McAlister, 1937). Work E.S. Johnson followed earlier studies by Blaauw that implicated that shorter wavelengths of blue light were more effective in generating phototropic responses in oat coleoptiles (Johnson, 1937). Monochromatic light studies of this period are well documented in the book, *Pigment of the Imagination*, by Linda Sage (1992). The text documents the quest for higher fluence rates of pure monochromatic light, noting barriers like countless blown circuit breakers and generation of deadly gases, along with the use of all kinds of light sources from arc lamps and 200W incandescent filaments. One interesting passage describes the development of a large spectrograph, cobbled together from parts obtained from streetcars, movie theatres and other sundry sources, assembled in a windowless wine-racking room at the USDA laboratories in Beltsville, MD. This spectrograph projected a 14 m rainbow of light onto an adjacent wall powered by a 10,000 W cabon-arc lamp. This large spectral projection allowed great advances in understanding how specific light qualities affected discrete plant processes. Clearly different parts of the spectrum had unique abilities to spur developmental or morphological changes—and even plants placed outside of the visible spectrum exhibited treatment effects, indicating that plants responded to a wider span of

In discussion of photoreceptor families it is important to define the nomenclature, as first presented in a Plant Cell Letter to the Editor (Quail et al., 1994). In this report the notation is as follows: wild-type gene: *PHY, PHYA, PHYD* mutant gene: *phy, phyA, phyD* apoprotein:

In the following pages the discussion on photosensory systems is broken down into sections

PHY, PHYA, PHYD chromoprotein (apoprotein + chromophore) phy, phyA, phyD.

**2.2 The light sensor collection** 

wavelengths than the human eye.

on discovery, mechanism, and associated physiology.

The first major heading of this chapter will cover the qualities of light and the receptors that sense them. The approach will be more historical and provide an understanding of how each receptor system delivers a signal and some of the processes that are controlled. Development and competence of the plastid is the second area of emphasis, describing events that transform the etioplast to the chloroplast. The final portion of the chapter will discuss the communication between the chloroplast and the nucleus. These separate compartments must be in constant and precise communication to ensure coordinated gene expression that fulfills the requirements of the plastid for new proteins. While many of the proteins required for photosynthesis are encoded in the plastid itself, a subset of important genes reside in the nucleus, and their precise expression is required for normal chloroplast operation. Many of these are subunits of chloroplast protein complexes that are non-functional in the absence of nuclear encoded subunits. Careful communication between these compartments has been the subject of interest for decades, and recent findings have illuminated how antero- and retrograde mechanisms might mediate this critical network.

#### **2. Connecting light to gene expression and development**

#### **2.1 Not all wavebands are created equal**

Back in seventh grade I was introduced to the Spectronic 20, or "Spec 20" for short. For those readers that are unfamiliar, it is essentially a tan breadbox with two dials, an analog meter, a dial to determine the wavelength transmitted, and a chamber for introducing a sample in a test tube. The device was originally made by Bausch & Lomb back in 1954, and even modern iterations reflect the basic, industrial, sturdy simplicity of the original model.

Back in 1980 we were given the charge to determine which wavelengths chlorophyll absorbed best. I don't remember how we purified our sample, I just know that I zeroed the machine using a blank filled with water using the dial on the left, lowered the sample into the chamber, closed the lid, and then recorded the values for light absorption as we marched across the dial—from the UV to past the red wavelengths. From these readings we'd build a graph that would reflect an absorption spectrum for chlorophyll, absorbing light in the blue and red most efficiently, while offering little to no absorption in the green, yellow, orange and far-red regions of the spectrum.

Some basic hypotheses could have been constructed from these findings. Certainly the qualities of light that excite chlorophyll must provide information to the plant as well. In my third year of college I learned that this was so. I learned that red and blue light would trigger photomophogenic development. We discussed effects in a variety of plants, from peas to mung beans, to tobacco, to *Lemna* as well as studies of chloroplast orientation in the green alga *Mougeotia*. There were even studies in a strange plant called *Arabidopsis thaliana*. All showed developmental effects of light, but mostly blue and red wavelengths. The correlation between the wavelengths that stimulated development and drove lightregulated metabolism was no surprise. Some wavelengths impart valuable information to the plant that promotes growth, development and photosynthetic capacity. Other wavelengths, like far-red and green, are not so important for metabolism but they are not benign- they shape plant processes in other ways that optimize light capture and adaptation.

The first major heading of this chapter will cover the qualities of light and the receptors that sense them. The approach will be more historical and provide an understanding of how each receptor system delivers a signal and some of the processes that are controlled. Development and competence of the plastid is the second area of emphasis, describing events that transform the etioplast to the chloroplast. The final portion of the chapter will discuss the communication between the chloroplast and the nucleus. These separate compartments must be in constant and precise communication to ensure coordinated gene expression that fulfills the requirements of the plastid for new proteins. While many of the proteins required for photosynthesis are encoded in the plastid itself, a subset of important genes reside in the nucleus, and their precise expression is required for normal chloroplast operation. Many of these are subunits of chloroplast protein complexes that are non-functional in the absence of nuclear encoded subunits. Careful communication between these compartments has been the subject of interest for decades, and recent findings have illuminated how antero- and retrograde mechanisms might mediate this

Back in seventh grade I was introduced to the Spectronic 20, or "Spec 20" for short. For those readers that are unfamiliar, it is essentially a tan breadbox with two dials, an analog meter, a dial to determine the wavelength transmitted, and a chamber for introducing a sample in a test tube. The device was originally made by Bausch & Lomb back in 1954, and even modern iterations reflect the basic, industrial, sturdy simplicity of the original model. Back in 1980 we were given the charge to determine which wavelengths chlorophyll absorbed best. I don't remember how we purified our sample, I just know that I zeroed the machine using a blank filled with water using the dial on the left, lowered the sample into the chamber, closed the lid, and then recorded the values for light absorption as we marched across the dial—from the UV to past the red wavelengths. From these readings we'd build a graph that would reflect an absorption spectrum for chlorophyll, absorbing light in the blue and red most efficiently, while offering little to no absorption in the green, yellow, orange

Some basic hypotheses could have been constructed from these findings. Certainly the qualities of light that excite chlorophyll must provide information to the plant as well. In my third year of college I learned that this was so. I learned that red and blue light would trigger photomophogenic development. We discussed effects in a variety of plants, from peas to mung beans, to tobacco, to *Lemna* as well as studies of chloroplast orientation in the green alga *Mougeotia*. There were even studies in a strange plant called *Arabidopsis thaliana*. All showed developmental effects of light, but mostly blue and red wavelengths. The correlation between the wavelengths that stimulated development and drove lightregulated metabolism was no surprise. Some wavelengths impart valuable information to the plant that promotes growth, development and photosynthetic capacity. Other wavelengths, like far-red and green, are not so important for metabolism but they are not benign- they shape plant processes in other ways that optimize light capture and

**2. Connecting light to gene expression and development** 

**2.1 Not all wavebands are created equal** 

and far-red regions of the spectrum.

adaptation.

critical network.

#### **2.2 The light sensor collection**

Plants interact with the ambient light environment through a series of light sensors. These specialized molecules capture photons of discrete wavelengths and initiate downstream signaling events that ultimately lead to changes in gene expression, development, and/or morphology. It is important to remember that plants rely on these environmental cues to drive, or in some cases constrain, their development. In the context of photosynthesis, light signaling is important to consider in two general contexts. The transition from etiolate to autotrophic growth is driven by light. As the developing seedling meanders through the soil, sensitive light receptors are in place and prepared to ignite a downstream flow of events upon capture of a photon. These signaling events in many cases prepare the plastid, shifting its function from that of an etioplast to the metabolic center of the chloroplast.

The sensory networks that transduce information starting with the capture of a photon into a suite of downstream responses are well known. Historically these pigments were postulated to control various aspects of plant growth and development, particularly germination, phototropic movements and the transition between vegetative and reproductive growth. Long before the genes and proteins were identified and characterized, a tremendous body of work produced evidence of their activities and effects on physiology. Light quality effects on germination were examined by Lewis H. Flint where he demonstrated the promotive effects of red light (Flint, 1936). These studies were expanded through collaboration with E.D. McAlister, a physicist that utilized a spectrograph to split the spectrum and illuminate seeds with discrete wavelengths. Together Flint and McAlister generated elegant action spectra that illustrated how red light promoted germination, while blue, green and far-red light were inhibitory (Flint and McAlister, 1937). Work E.S. Johnson followed earlier studies by Blaauw that implicated that shorter wavelengths of blue light were more effective in generating phototropic responses in oat coleoptiles (Johnson, 1937). Monochromatic light studies of this period are well documented in the book, *Pigment of the Imagination*, by Linda Sage (1992). The text documents the quest for higher fluence rates of pure monochromatic light, noting barriers like countless blown circuit breakers and generation of deadly gases, along with the use of all kinds of light sources from arc lamps and 200W incandescent filaments. One interesting passage describes the development of a large spectrograph, cobbled together from parts obtained from streetcars, movie theatres and other sundry sources, assembled in a windowless wine-racking room at the USDA laboratories in Beltsville, MD. This spectrograph projected a 14 m rainbow of light onto an adjacent wall powered by a 10,000 W cabon-arc lamp. This large spectral projection allowed great advances in understanding how specific light qualities affected discrete plant processes. Clearly different parts of the spectrum had unique abilities to spur developmental or morphological changes—and even plants placed outside of the visible spectrum exhibited treatment effects, indicating that plants responded to a wider span of wavelengths than the human eye.

In discussion of photoreceptor families it is important to define the nomenclature, as first presented in a Plant Cell Letter to the Editor (Quail et al., 1994). In this report the notation is as follows: wild-type gene: *PHY, PHYA, PHYD* mutant gene: *phy, phyA, phyD* apoprotein: PHY, PHYA, PHYD chromoprotein (apoprotein + chromophore) phy, phyA, phyD.

In the following pages the discussion on photosensory systems is broken down into sections on discovery, mechanism, and associated physiology.

The Guiding Force of Photons 19

Interactors were termed PHYTOCHROME INTERACTING FACTORS, or PIFs (for review Castillon et al., 2007; Leivar and Quail, 2011). Analysis of PIF function would prove complex. For instance, PIF3 (a bHLH protein) binds phyA or phyB upon illumination, yet through separate domains and with distinct affinities (Leivar and Quail, 2011). PIF3, PIF1 and PIF5 have been shown to be rapidly phosphorylated and degraded via a ubiquitindependent process, with half lives between 5-20 min. One interpretation is that PIFs repress photomorphognesis in darkness and they are degraded rapidly upon light exposure to

Phytochromes are relevant to just about all aspects of light-mediated development because they absorb well in red, blue, far-red and UV portions of the spectrum. The sum of molecular and physiological processes controlled is too extreme to list here. The most relevant roles to applied phy biology are in regulation of plant stature. Phytochromes repress stem elongation, promote leaf expansion and alter plant body form in response to crowding. The phytochromes also contribute to flowering. In Arabidopsis phyA has a role in promoting flowering in response to far-red signals whereas phyB works against it after

The role of phytochrome in establishing a platform for photosynthesis is clearly observed during photomorphogenic development. During this time there is a substantial contribution of phy to chloroplast developmental processes. Phytochromes regulate the accumulation of transcripts encoding CAB (LHCB) proteins required for anchoring the photosynthetic apparatus (Kaufman et al., 1985; Karlin-Neumann et al., 1988), as well as the small subunit of RUBISCO (Kaufman et al., 1984). Global analysis of gene expression shows that many transcripts encoding proteins destined for the plastid are induced within minutes to hours of phy activation (Tepperman et al., 2001; Tepperman et al., 2004). The major role of phy in plastid development is in de-repression of the PIF-mediated constraint of transcription and will be discussed later in this chapter. The effect is strong as developing seedlings treated with far-red light can actually be permanently disabled from greening and chloroplast

While the participation of phytochromes defined a mechanism for red light effects in many plant processes, there were clear effects of blue light that could not be easily ascribed to phytochromes. In fact, phototropic curvature, had been described as blue-favored by Charles Darwin (Darwin, 1897), complementing a battery of blue light responses characterized in the early part of the century (Briggs, 2006). Analysis of plant actions in response to red and/or blue light provided clear evidence that more than one photosensory pigment was involved in light responses. Throughout the 20th century there was considerable discussion about the nature of the pigment, fueled by experimentation in plants and fungi. Analysis of countless action spectra drove speculation that the receptor was based on a carotenoid, a flavin, or a pterin, since there were general peaks at 450, 475 and 420 nm that supported these possibilities, yet fine structure of action spectra left the

initiate developmental responses (Leivar et al., 2008).

**2.2.1.3 Associated physiology** 

absorbing red (Valverde et al., 2004).

development (Barnes et al., 1996).

absolute identification of the chromophore(s) ambiguous.

**2.2.2 The cryptochromes** 

**2.2.2.1 Discovery** 

#### **2.2.1 The phytochromes**

#### **2.2.1.1 Discovery**

The USDA spectrograph and tools like it led to the discovery that red and far-red light presented opposing effects on biological processes. Within a short time the red/far-red reversibility of Grand Rapids lettuce seed germination was described (Borthwick et al., 1952), and the floodgates of phytochrome research were open. Within several decades a series of light-sensing mutants were obtained from mutagenized *Arabidopsis thaliana* collections (Koornneef et al., 1980). Ultimately several of these would be shown to encode light-signaling components. The *hy3* mutation was shown to be a lesion in the phytochrome B receptor (Somers et al., 1991). Soon after, a separately-isolated mutant called *hy8* was shown to encode phytochrome A (Parks and Quail, 1993). These genetic studies now attached genes and their cognate proteins to processes controlled by red and far-red light. Additional phytochromes were isolated, a total of five in Arabidopsis, phyA-phyE (Clack et

al., 1994). Phytochromes may be grouped by their stability in light. They Type I phytochromes are light-labile while the Type II's are light stable. In Arabidopsis phyA is the only Type I phytochrome, and it is also thought of as the dark phytochrome because of its abundance (Jordan et al., 1997). The other phytochromes are stable in light, where phyB makes up the majority of phytochrome in the cell (Chen et al., 2004; Franklin and Quail, 2010). The individual phytochromes form functional hetero- and homodimers (Sharrock and Clack, 2004; Clack et al., 2009).

#### **2.2.1.2 Signaling mechanism**

The hallmark photoreversiblity is achieved from switching phytochromes between two conformational states. In darkness, phytochromes exist in a form known as Pr. This form is biologically inactive and has an absorption peak of approximately 660 nm. When illuminated the Pr form converts to the Pfr form, which initiates biological activity (for review see Chen et al., 2004). The Pfr form may be photoconverted back to the Pr form immediately by illumination with far-red light, or over a longer period of time in darkness. Both conformations maintain some overlap in their spectral absorption profiles while maintaining their distinct sensitivities. When illuminated with light and an equilibrium is established between Pr and Pfr.

After conversion to Pfr phytochromes travel from the cytosol to the nucleus. The phyA receptor moves quickly to the nucleus. The phyA::GFP fusion proteins are detected only minutes after illumination, while the phyB receptor moves with different kinetics, showing up hours after light treatment (Kircher et al., 1999; Hisada et al., 2000). The timing of movement to the nucleus matches well with the earliest detected responses to phytochrome activation. Using high-resolution imaging and phytochrome mutants, Parks and Spalding (Parks et al., 1998) demonstrated that phyA and phyB activity control inhibition of stem elongation by red light with similar kinetics. The phyA receptor exerts a transient influence within minutes while phyB involvement is evident later, but persists longer. Here localization kinetics overlap impeccably with physiological events, suggesting that rate of nuclear localization is directly influencing plant growth and development. Later, regulated nuclear import of phyB using a steroid-inducible system demonstrated that both light and nuclear localization were required for phyB to induce its effects (Huq et al., 2003).

Once in the nucleus phytochromes interact with a suite of other proteins. Some of these were first identified in interaction screens using the C-terminal PHYB as bait (Ni et al., 1998).

The USDA spectrograph and tools like it led to the discovery that red and far-red light presented opposing effects on biological processes. Within a short time the red/far-red reversibility of Grand Rapids lettuce seed germination was described (Borthwick et al., 1952), and the floodgates of phytochrome research were open. Within several decades a series of light-sensing mutants were obtained from mutagenized *Arabidopsis thaliana* collections (Koornneef et al., 1980). Ultimately several of these would be shown to encode light-signaling components. The *hy3* mutation was shown to be a lesion in the phytochrome B receptor (Somers et al., 1991). Soon after, a separately-isolated mutant called *hy8* was shown to encode phytochrome A (Parks and Quail, 1993). These genetic studies now attached genes and their cognate proteins to processes controlled by red and far-red light. Additional phytochromes were isolated, a total of five in Arabidopsis, phyA-phyE (Clack et al., 1994). Phytochromes may be grouped by their stability in light. They Type I phytochromes are light-labile while the Type II's are light stable. In Arabidopsis phyA is the only Type I phytochrome, and it is also thought of as the dark phytochrome because of its abundance (Jordan et al., 1997). The other phytochromes are stable in light, where phyB makes up the majority of phytochrome in the cell (Chen et al., 2004; Franklin and Quail, 2010). The individual phytochromes form functional hetero- and homodimers (Sharrock and

The hallmark photoreversiblity is achieved from switching phytochromes between two conformational states. In darkness, phytochromes exist in a form known as Pr. This form is biologically inactive and has an absorption peak of approximately 660 nm. When illuminated the Pr form converts to the Pfr form, which initiates biological activity (for review see Chen et al., 2004). The Pfr form may be photoconverted back to the Pr form immediately by illumination with far-red light, or over a longer period of time in darkness. Both conformations maintain some overlap in their spectral absorption profiles while maintaining their distinct sensitivities. When illuminated with light and an equilibrium is

After conversion to Pfr phytochromes travel from the cytosol to the nucleus. The phyA receptor moves quickly to the nucleus. The phyA::GFP fusion proteins are detected only minutes after illumination, while the phyB receptor moves with different kinetics, showing up hours after light treatment (Kircher et al., 1999; Hisada et al., 2000). The timing of movement to the nucleus matches well with the earliest detected responses to phytochrome activation. Using high-resolution imaging and phytochrome mutants, Parks and Spalding (Parks et al., 1998) demonstrated that phyA and phyB activity control inhibition of stem elongation by red light with similar kinetics. The phyA receptor exerts a transient influence within minutes while phyB involvement is evident later, but persists longer. Here localization kinetics overlap impeccably with physiological events, suggesting that rate of nuclear localization is directly influencing plant growth and development. Later, regulated nuclear import of phyB using a steroid-inducible system demonstrated that both light and

nuclear localization were required for phyB to induce its effects (Huq et al., 2003).

Once in the nucleus phytochromes interact with a suite of other proteins. Some of these were first identified in interaction screens using the C-terminal PHYB as bait (Ni et al., 1998).

**2.2.1 The phytochromes** 

Clack, 2004; Clack et al., 2009). **2.2.1.2 Signaling mechanism** 

established between Pr and Pfr.

**2.2.1.1 Discovery** 

Interactors were termed PHYTOCHROME INTERACTING FACTORS, or PIFs (for review Castillon et al., 2007; Leivar and Quail, 2011). Analysis of PIF function would prove complex. For instance, PIF3 (a bHLH protein) binds phyA or phyB upon illumination, yet through separate domains and with distinct affinities (Leivar and Quail, 2011). PIF3, PIF1 and PIF5 have been shown to be rapidly phosphorylated and degraded via a ubiquitindependent process, with half lives between 5-20 min. One interpretation is that PIFs repress photomorphognesis in darkness and they are degraded rapidly upon light exposure to initiate developmental responses (Leivar et al., 2008).

#### **2.2.1.3 Associated physiology**

Phytochromes are relevant to just about all aspects of light-mediated development because they absorb well in red, blue, far-red and UV portions of the spectrum. The sum of molecular and physiological processes controlled is too extreme to list here. The most relevant roles to applied phy biology are in regulation of plant stature. Phytochromes repress stem elongation, promote leaf expansion and alter plant body form in response to crowding. The phytochromes also contribute to flowering. In Arabidopsis phyA has a role in promoting flowering in response to far-red signals whereas phyB works against it after absorbing red (Valverde et al., 2004).

The role of phytochrome in establishing a platform for photosynthesis is clearly observed during photomorphogenic development. During this time there is a substantial contribution of phy to chloroplast developmental processes. Phytochromes regulate the accumulation of transcripts encoding CAB (LHCB) proteins required for anchoring the photosynthetic apparatus (Kaufman et al., 1985; Karlin-Neumann et al., 1988), as well as the small subunit of RUBISCO (Kaufman et al., 1984). Global analysis of gene expression shows that many transcripts encoding proteins destined for the plastid are induced within minutes to hours of phy activation (Tepperman et al., 2001; Tepperman et al., 2004). The major role of phy in plastid development is in de-repression of the PIF-mediated constraint of transcription and will be discussed later in this chapter. The effect is strong as developing seedlings treated with far-red light can actually be permanently disabled from greening and chloroplast development (Barnes et al., 1996).

#### **2.2.2 The cryptochromes**

#### **2.2.2.1 Discovery**

While the participation of phytochromes defined a mechanism for red light effects in many plant processes, there were clear effects of blue light that could not be easily ascribed to phytochromes. In fact, phototropic curvature, had been described as blue-favored by Charles Darwin (Darwin, 1897), complementing a battery of blue light responses characterized in the early part of the century (Briggs, 2006). Analysis of plant actions in response to red and/or blue light provided clear evidence that more than one photosensory pigment was involved in light responses. Throughout the 20th century there was considerable discussion about the nature of the pigment, fueled by experimentation in plants and fungi. Analysis of countless action spectra drove speculation that the receptor was based on a carotenoid, a flavin, or a pterin, since there were general peaks at 450, 475 and 420 nm that supported these possibilities, yet fine structure of action spectra left the absolute identification of the chromophore(s) ambiguous.

The Guiding Force of Photons 21

flowering time is probably the cryptochrome's most agriculturally relevant attribute. The transition is controlled by blue light activation of cry2, followed by its nuclear localization and enhanced stability (Valverde et al., 2004). The cry2 receptor contributes to seedling height under certain fluence rates (Lin et al., 1998), yet has potent effects on stem elongation during early development (Folta and Spalding, 2001). Perhaps the most well-studied output of cryptochrome activation is the modulation of gene expression. A number of reports have examined the role of cryptochromes using genomic-level analyses. Studies during blue light induced de-etiolation show that crys alter gene expression associated with the plant hormone gibberellic acid (Folta et al., 2003; Zhao et al., 2007), providing a means to connect

What is the mechanism of cryptochrome action? Great steps were made to pinpoint the transduction mechanism when the c-terminus of the CRY1 protein was overexpressed in transgenic plants (Yang et al., 2000). Such transgenic seedlings, known as CCT for CRY C-Terminus, exhibited *cop*-like phenotypes, meaning that that were presenting light-grown phenotypes even in darkness. This finding was exciting because it potentially linked the COP1 protein, a regulator known to repress the light response in darkness, to cry function. Two hybrid interactions and co-immunoprecipitation analysis in vitro would confirm the

The mutant *hy5* locus was isolated as part of the original screen of Arabidopsis photomorphogenic mutants (Koornneef et al., 1980). The mutant exhibited light-insensitivity symptoms especially under blue light. Later it was observed that *HY5* exhibited epistatic interactions with *COP1* (Ang and Deng, 1994), a gene encoding a ring-finger E3 ubiquitin ligase that shows a constitutive photomorphogenic phenotype. *HY5* transcripts and proteins accumulated rapidly after illumination, presenting the hypothesis that they were causal to development. It was demonstrated that *HY5* was transcribed and translated in darkness, yet the protein did not accumulate (Osterlund et al., 2000). The lack of accumulation could be reversed with the application of proteosome inhibitors, indicating that HY5 was likely being degraded via a ubiquitin-mediated mechanism. Moreover, the protein also accumulated in the *cop1* mutant. The stage was set- a positive regulator of photomorphogenesis, HY5, was destabilized when it was not needed and mutation or pharmacological block of the degradation system caused hyperaccumulation. It was possible to infer a mechanism. Now

Studies soon after tested the possibility that the cry receptor itself interacted with the COP1 degradation system. Examination of COP1-cry interaction showed that the receptor did interact with COP1 through the CCT domain, and interaction between the receptor and COP1 would interrupt ubiquitin-mediated degradation of the positive regulator HY5 (Wang

While this mechanism is supported by many lines of evidence it is important to remember that crys also have effects outside of the nucleus. Constructs that exclude cry from the nucleus show physiological function (Wu and Spalding, 2007) and events at the depolarization events at the cell membrane seconds after illumination (Folta and Spalding,

The flavin chromophore of the cryptochromes, when activated, opens new absorption properties and signaling states for cryptochrome receptors. When treated with blue light the chromophore takes on a different oxidation state that absorbs in the green, yellow and into

et al., 2001). The effect appears to take place predominantly in the nucleus.

2001) suggest that crys are indeed functional in other contexts.

interaction between COP1 and the cryptochrome light sensors (Wang et al., 2001).

light and cryptochromes to growth responses.

how to connect it with the light sensor?

**2.2.2.4 Signaling mechanism** 

In 1979 Jonathan Gressel gave a name to the illusive photoreceptor controlling plant and cryptogam form and function in blue light, appropriately, *cryptochrome* (Gressel, 1979). In his review he notes that the name was "despised by many". Yet his moniker was quite accurate, as blue light responses would later be shown to be transduced by a series of receptors (including phytochromes) some requiring phytochrome co-activation. These ambiguities would hide the genetic nature of the cryptochrome gene for another fourteen years. Gressel also contended that the cryptochrome receptor would be the single blue-light receptor.

#### **2.2.2.2 Molecular structure**

The actual structure of the cryptochrome receptor was eventually elucidated in 1993, yet the path to its characterization was laid with a series of plants that grew long and tall under blue light in a 1980 report. A screen for light sensing mutants in a mutagenized *Arabidopsis thaliana* population revealed a seedling that failed to suppress elongation in light. This particular seedling, noted as *hy4* (the fourth of the hypocotyl elongation mutants), showed an especially strong presentation of the long-hypocotyl phenotype under blue light, moreso than red, green, far-red, or white light (Koornneef et al., 1980). These findings suggested a lesion in the blue-light sensing pathway. Later, several T-DNA mutants with long hypocotyls under blue light revealed the first sequence identity of the HY4 protein- a sequence that matched convincingly with the long-wavelength microbial DNA photolyases. DNA photolyases are chromophore-bound proteins that catalyze repair of pyrimidine dimers in DNA (REF). Later studies would show that the HY4 protein (later renamed to CRY1 for CRYPTOCHROME1) was the receptor controlling these blue light responses. The receptor maintains two chromophores—flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF), with the photon exciting MTHF and shuttling the excitation energy to FAD to initiate the signaling process (Cashmore et al., 1999).

#### **2.2.2.3 Various types of cryptochromes**

The cry proteins are distinguished by two domains that underlie its diverse functions (Lin and Shalitin, 2003). The first is an N-terminal photolyase related (PHR) domain. The other domain is a C-terminal extension. This latter domain is variable between the different cryptochromes and defines the function. While exhibiting variation in this extension there are short islands of conserved sequence. These motifs (from N to C) are DQXVP, an acidic region high in D and E, and a STAES sequence followed by GGXVP. Because their order and sequences are so highly conserved they are noted in the literature together as a DAS domain. The DAS organization has been conserved from the most rudimentary mosses to angiosperms, so cryptochromes date back approximately 400 million years. The DAS domain also dictates cry localization and interaction that defines how individual crys contribute to physiology. A comprehensive report on cry structure and function is presented by Lin and Shalitin (2003).

In Arabidopsis there are three cryptochromes. The CRY1 and CRY2 proteins are translated and then localized to the nucleus upon activation by light. The third cryptochrome is called cry3 or cry-DASH. This member is localized to the chloroplast and performs DNA repair, much like prototypical photolyases (Kleine et al., 2003). No other signaling role has been proposed.

A late flowering mutant (known at the time as *fha1*) would connect the cry2 receptor to control of the flowering transition (Guo et al., 1998; Mockler et al., 2003). The contribution to

In 1979 Jonathan Gressel gave a name to the illusive photoreceptor controlling plant and cryptogam form and function in blue light, appropriately, *cryptochrome* (Gressel, 1979). In his review he notes that the name was "despised by many". Yet his moniker was quite accurate, as blue light responses would later be shown to be transduced by a series of receptors (including phytochromes) some requiring phytochrome co-activation. These ambiguities would hide the genetic nature of the cryptochrome gene for another fourteen years. Gressel also contended that the cryptochrome receptor would be the single blue-light receptor.

The actual structure of the cryptochrome receptor was eventually elucidated in 1993, yet the path to its characterization was laid with a series of plants that grew long and tall under blue light in a 1980 report. A screen for light sensing mutants in a mutagenized *Arabidopsis thaliana* population revealed a seedling that failed to suppress elongation in light. This particular seedling, noted as *hy4* (the fourth of the hypocotyl elongation mutants), showed an especially strong presentation of the long-hypocotyl phenotype under blue light, moreso than red, green, far-red, or white light (Koornneef et al., 1980). These findings suggested a lesion in the blue-light sensing pathway. Later, several T-DNA mutants with long hypocotyls under blue light revealed the first sequence identity of the HY4 protein- a sequence that matched convincingly with the long-wavelength microbial DNA photolyases. DNA photolyases are chromophore-bound proteins that catalyze repair of pyrimidine dimers in DNA (REF). Later studies would show that the HY4 protein (later renamed to CRY1 for CRYPTOCHROME1) was the receptor controlling these blue light responses. The receptor maintains two chromophores—flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF), with the photon exciting MTHF and shuttling the excitation energy to FAD to initiate the signaling process

The cry proteins are distinguished by two domains that underlie its diverse functions (Lin and Shalitin, 2003). The first is an N-terminal photolyase related (PHR) domain. The other domain is a C-terminal extension. This latter domain is variable between the different cryptochromes and defines the function. While exhibiting variation in this extension there are short islands of conserved sequence. These motifs (from N to C) are DQXVP, an acidic region high in D and E, and a STAES sequence followed by GGXVP. Because their order and sequences are so highly conserved they are noted in the literature together as a DAS domain. The DAS organization has been conserved from the most rudimentary mosses to angiosperms, so cryptochromes date back approximately 400 million years. The DAS domain also dictates cry localization and interaction that defines how individual crys contribute to physiology. A comprehensive report on cry structure and function is presented

In Arabidopsis there are three cryptochromes. The CRY1 and CRY2 proteins are translated and then localized to the nucleus upon activation by light. The third cryptochrome is called cry3 or cry-DASH. This member is localized to the chloroplast and performs DNA repair, much like prototypical photolyases (Kleine et al., 2003). No other signaling role has been

A late flowering mutant (known at the time as *fha1*) would connect the cry2 receptor to control of the flowering transition (Guo et al., 1998; Mockler et al., 2003). The contribution to

**2.2.2.2 Molecular structure** 

(Cashmore et al., 1999).

by Lin and Shalitin (2003).

proposed.

**2.2.2.3 Various types of cryptochromes** 

flowering time is probably the cryptochrome's most agriculturally relevant attribute. The transition is controlled by blue light activation of cry2, followed by its nuclear localization and enhanced stability (Valverde et al., 2004). The cry2 receptor contributes to seedling height under certain fluence rates (Lin et al., 1998), yet has potent effects on stem elongation during early development (Folta and Spalding, 2001). Perhaps the most well-studied output of cryptochrome activation is the modulation of gene expression. A number of reports have examined the role of cryptochromes using genomic-level analyses. Studies during blue light induced de-etiolation show that crys alter gene expression associated with the plant hormone gibberellic acid (Folta et al., 2003; Zhao et al., 2007), providing a means to connect light and cryptochromes to growth responses.

#### **2.2.2.4 Signaling mechanism**

What is the mechanism of cryptochrome action? Great steps were made to pinpoint the transduction mechanism when the c-terminus of the CRY1 protein was overexpressed in transgenic plants (Yang et al., 2000). Such transgenic seedlings, known as CCT for CRY C-Terminus, exhibited *cop*-like phenotypes, meaning that that were presenting light-grown phenotypes even in darkness. This finding was exciting because it potentially linked the COP1 protein, a regulator known to repress the light response in darkness, to cry function. Two hybrid interactions and co-immunoprecipitation analysis in vitro would confirm the interaction between COP1 and the cryptochrome light sensors (Wang et al., 2001).

The mutant *hy5* locus was isolated as part of the original screen of Arabidopsis photomorphogenic mutants (Koornneef et al., 1980). The mutant exhibited light-insensitivity symptoms especially under blue light. Later it was observed that *HY5* exhibited epistatic interactions with *COP1* (Ang and Deng, 1994), a gene encoding a ring-finger E3 ubiquitin ligase that shows a constitutive photomorphogenic phenotype. *HY5* transcripts and proteins accumulated rapidly after illumination, presenting the hypothesis that they were causal to development. It was demonstrated that *HY5* was transcribed and translated in darkness, yet the protein did not accumulate (Osterlund et al., 2000). The lack of accumulation could be reversed with the application of proteosome inhibitors, indicating that HY5 was likely being degraded via a ubiquitin-mediated mechanism. Moreover, the protein also accumulated in the *cop1* mutant. The stage was set- a positive regulator of photomorphogenesis, HY5, was destabilized when it was not needed and mutation or pharmacological block of the degradation system caused hyperaccumulation. It was possible to infer a mechanism. Now how to connect it with the light sensor?

Studies soon after tested the possibility that the cry receptor itself interacted with the COP1 degradation system. Examination of COP1-cry interaction showed that the receptor did interact with COP1 through the CCT domain, and interaction between the receptor and COP1 would interrupt ubiquitin-mediated degradation of the positive regulator HY5 (Wang et al., 2001). The effect appears to take place predominantly in the nucleus.

While this mechanism is supported by many lines of evidence it is important to remember that crys also have effects outside of the nucleus. Constructs that exclude cry from the nucleus show physiological function (Wu and Spalding, 2007) and events at the depolarization events at the cell membrane seconds after illumination (Folta and Spalding, 2001) suggest that crys are indeed functional in other contexts.

The flavin chromophore of the cryptochromes, when activated, opens new absorption properties and signaling states for cryptochrome receptors. When treated with blue light the chromophore takes on a different oxidation state that absorbs in the green, yellow and into

The Guiding Force of Photons 23

phosphorylation reaction is complete just prior to the development of phototropic curvature. Such correlations were observed in other species as well (Palmer et al., 1993a;

The Arabidopsis mutants and the phosphorylation activity would become linked. Reymond et al. (1992) tested the diminutive Arabidopsis plants for the phosphorylation activity detected in the epicotyls of peas, zucchini, tomato and sunflower hypocotyls, and the coleoptiles of maize, barley and oat coleoptiles. The non-phototropic JK224 mutant exhibited low levels of phosphorylation upon illumination, suggesting that JK224 was in fact the receptor. On the other hand, JK218's levels were not significantly altered. This reported tied

With a mutant genotype possessing defects in biochemistry and phenotype it would seem simple to move to the process of gene discovery. In the early 90's the Arabidopsis system was emerging as a tractable genetic system (Konieczny and Ausubel, 1993), it would be possible to map the gene if the phenotypes were robust. This was the problem. Poff's mutant phenotypes were solid, yet subtle, as first-positive phototropism would bend a seedling only 6-10 degrees. Furthermore, the defective seedlings did eventually bend over time. Because of the subtle differences, and the fact that these were not null mutants, it would have been extremely difficult to screen reliably in large populations suitable for

Liscum and Briggs also performed a screen in the Arabidopsis system, yet they resorted to fast-neutron treated seeds in an attempt to find strong non-phototropic alleles (Liscum and Briggs, 1995, 1996). Four loci were identified. The *nph1* mutant was allelic to JK224 and the *nph3* mutant proved to correspond to JK218. The *nph1* mutant had no detectable phosphorylation of the 120 kDa protein, leading to the hypothesis that it was locus encoding

The NPH1 protein contains two highly similar domains reminiscent of LOV (Light, Oxygen and Voltage) PAS domains. These domains bind flavins (FAD) and perform a variety of functions relative to environmental sensing from bacterial aerotaxis to modulating K+ currents in *Drosophila*. NPH1 also possessed a serine-threonine kinase domain in the Cterminus. The NPH1 protein was shown to preferably bind FMN as a chromophore, and the absorption spectrum for the purified receptor mirrored that of phototropism (Christie et al., 1998). These findings prompted a functionalized name change from the locus *NPH1* to the

Based on sequence homology the *NPH-LIKE* (*NPL1*) gene was soon identified (Jarillo et al., 1998). This sequence has a similar topology to that of *NPH1*, with the same conserved kinase region and LOV domains. The individual LOV domains (LOV1 and LOV2) have distinct roles in phototropin action. Mutation of Cys39 of the LOV domain in the LOV1 domain has no effect on phototropism, whereas this mutation in the LOV2 domain abolishes curvature. The LOV2 domain also is the critical domain for promoting leaf expansion (Cho et al., 2007). There is evidence that the role of the LOV1 domain is to attenuate LOV2 effects by acting as

Separating the LOV domains from the kinase domain is an alpha-helical hinge that holds LOV domains in proximity to the kinase domain. Upon activation with light, the protein opens around this hinge region, allowing the kinase to be phosphorylated (Tokutomi et al.,

2008). This is the basis for the phototropin signaling mechanism.

the receptor for phototropism. The gene was eventually cloned (Huala et al., 1997).

Palmer et al., 1993b).

genetic mapping.

**2.2.3.2 Phototropin structure** 

gene encoding the receptor, *PHOT1*.

a site for dimerization.

the autophosphorylation to phototropic curvature.

the red. Several lines of evidence show that the treatment of plants with green light can reverse cryptochrome mediated responses (Banerjee et al., 2007; Bouly et al., 2007), including anthocyanin accumulation, hypocotyl elongation and flowering. In this way the cry responses to blue light may be attenuated much like the red/far-red responses of phytochromes.

The cryptochromes are a stellar example of why examination of plant processes can have large-scale impacts. Cryptochromes were first identified in plants, yet since have been shown to have central positions in the animal circadian oscillator, and in magnetoperception that guides bird migration. Fungal cryptochromes have been identified, yet their precise functions remain elusive for the most part, and understood members bind DNA reminiscent of the cry3 (CRY-DASH) proteins of Arabidopsis. The cryptochrome receptors clearly control a great swath of responses relevant to all eukaryotes.

#### **2.2.3 The phototropins**

#### **2.2.3.1 Discovery**

Characterization of the cryptochromes gave plant science discrete receptors for red, far-red and blue light responses. A number of lines of evidence indicated that the effects of cryptochrome activation were distinct from those that regulated phototropism (Liscum et al., 1992; Liscum and Briggs, 1996; Lasceve et al., 1999), suggesting the existence of an additional blue light receptor class. A report in *Nature* showed that the *cry1cry2* double mutant was deficient in first-positive phototropism (Ahmad et al., 1998). Yet the results of this work did not bear out with further tests.

The pursuit for the blue-light photosensor controlling phototropism was heating up in concert with the characterization of the first cryptochrome receptor in the years leading up to 1993. Several independent research tracks were racing toward receptor identification that would ultimately converge in an Arabidopsis mutant with defects in the receptor. One approach was an attempt to identify the receptor genetically using the Arabidopsis system. The photophysiological characteristics of curvature were well understood in this species (Steinitz and Poff, 1986), and formed a sound basis for a mutant screen. Two noncomplementing mutants with defects in phototropic curvature (JK224 and JK218) were isolated (Khurana and Poff, 1989). JK224 was defective in first-positive curvature, requiring substantially higher fluences to induce measurable change. The JK218 mutant also showed resistance to phototropic curvature, only bending after long treatments with unilateral blue light. Both mutants were perfectly gravitropic, suggesting that they were sensory mutants and not simply unable to respond to stimuli that induce differential growth (Khurana and Poff, 1989).

With a separate approach a team of scientists working under the direction of Winslow R. Briggs used biochemical methods to characterize the blue light sensor for phototropism. A 120 k-Da phosphorylated protein was identified in association with plasma membranes of pea epicotyls (Gallagher and Ellis, 1982; Short and Briggs, 1990; Short et al., 1993; Short et al., 1994). When biochemistry and physiology were compared through time and space, some important correlations were uncovered. The threshold and saturation in the phototropic fluence response (Baskin, 1986) mirrored the parameters of light induced phosphorylation (Short and Briggs, 1990; Short et al., 1992). The regions of the seedling that exhibit the strongest phototropic response show the highest degree of phosphorylation (Short and Briggs, 1990). Both responses obeyed the Bunsen-Roscoe Law of Reciprocity, and the

the red. Several lines of evidence show that the treatment of plants with green light can reverse cryptochrome mediated responses (Banerjee et al., 2007; Bouly et al., 2007), including anthocyanin accumulation, hypocotyl elongation and flowering. In this way the cry responses to blue light may be attenuated much like the red/far-red responses of

The cryptochromes are a stellar example of why examination of plant processes can have large-scale impacts. Cryptochromes were first identified in plants, yet since have been shown to have central positions in the animal circadian oscillator, and in magnetoperception that guides bird migration. Fungal cryptochromes have been identified, yet their precise functions remain elusive for the most part, and understood members bind DNA reminiscent of the cry3 (CRY-DASH) proteins of Arabidopsis. The cryptochrome receptors clearly

Characterization of the cryptochromes gave plant science discrete receptors for red, far-red and blue light responses. A number of lines of evidence indicated that the effects of cryptochrome activation were distinct from those that regulated phototropism (Liscum et al., 1992; Liscum and Briggs, 1996; Lasceve et al., 1999), suggesting the existence of an additional blue light receptor class. A report in *Nature* showed that the *cry1cry2* double mutant was deficient in first-positive phototropism (Ahmad et al., 1998). Yet the results of

The pursuit for the blue-light photosensor controlling phototropism was heating up in concert with the characterization of the first cryptochrome receptor in the years leading up to 1993. Several independent research tracks were racing toward receptor identification that would ultimately converge in an Arabidopsis mutant with defects in the receptor. One approach was an attempt to identify the receptor genetically using the Arabidopsis system. The photophysiological characteristics of curvature were well understood in this species (Steinitz and Poff, 1986), and formed a sound basis for a mutant screen. Two noncomplementing mutants with defects in phototropic curvature (JK224 and JK218) were isolated (Khurana and Poff, 1989). JK224 was defective in first-positive curvature, requiring substantially higher fluences to induce measurable change. The JK218 mutant also showed resistance to phototropic curvature, only bending after long treatments with unilateral blue light. Both mutants were perfectly gravitropic, suggesting that they were sensory mutants and not simply unable to respond to stimuli that induce differential growth (Khurana and

With a separate approach a team of scientists working under the direction of Winslow R. Briggs used biochemical methods to characterize the blue light sensor for phototropism. A 120 k-Da phosphorylated protein was identified in association with plasma membranes of pea epicotyls (Gallagher and Ellis, 1982; Short and Briggs, 1990; Short et al., 1993; Short et al., 1994). When biochemistry and physiology were compared through time and space, some important correlations were uncovered. The threshold and saturation in the phototropic fluence response (Baskin, 1986) mirrored the parameters of light induced phosphorylation (Short and Briggs, 1990; Short et al., 1992). The regions of the seedling that exhibit the strongest phototropic response show the highest degree of phosphorylation (Short and Briggs, 1990). Both responses obeyed the Bunsen-Roscoe Law of Reciprocity, and the

control a great swath of responses relevant to all eukaryotes.

this work did not bear out with further tests.

phytochromes.

**2.2.3 The phototropins** 

**2.2.3.1 Discovery** 

Poff, 1989).

phosphorylation reaction is complete just prior to the development of phototropic curvature. Such correlations were observed in other species as well (Palmer et al., 1993a; Palmer et al., 1993b).

The Arabidopsis mutants and the phosphorylation activity would become linked. Reymond et al. (1992) tested the diminutive Arabidopsis plants for the phosphorylation activity detected in the epicotyls of peas, zucchini, tomato and sunflower hypocotyls, and the coleoptiles of maize, barley and oat coleoptiles. The non-phototropic JK224 mutant exhibited low levels of phosphorylation upon illumination, suggesting that JK224 was in fact the receptor. On the other hand, JK218's levels were not significantly altered. This reported tied the autophosphorylation to phototropic curvature.

With a mutant genotype possessing defects in biochemistry and phenotype it would seem simple to move to the process of gene discovery. In the early 90's the Arabidopsis system was emerging as a tractable genetic system (Konieczny and Ausubel, 1993), it would be possible to map the gene if the phenotypes were robust. This was the problem. Poff's mutant phenotypes were solid, yet subtle, as first-positive phototropism would bend a seedling only 6-10 degrees. Furthermore, the defective seedlings did eventually bend over time. Because of the subtle differences, and the fact that these were not null mutants, it would have been extremely difficult to screen reliably in large populations suitable for genetic mapping.

Liscum and Briggs also performed a screen in the Arabidopsis system, yet they resorted to fast-neutron treated seeds in an attempt to find strong non-phototropic alleles (Liscum and Briggs, 1995, 1996). Four loci were identified. The *nph1* mutant was allelic to JK224 and the *nph3* mutant proved to correspond to JK218. The *nph1* mutant had no detectable phosphorylation of the 120 kDa protein, leading to the hypothesis that it was locus encoding the receptor for phototropism. The gene was eventually cloned (Huala et al., 1997).

#### **2.2.3.2 Phototropin structure**

The NPH1 protein contains two highly similar domains reminiscent of LOV (Light, Oxygen and Voltage) PAS domains. These domains bind flavins (FAD) and perform a variety of functions relative to environmental sensing from bacterial aerotaxis to modulating K+ currents in *Drosophila*. NPH1 also possessed a serine-threonine kinase domain in the Cterminus. The NPH1 protein was shown to preferably bind FMN as a chromophore, and the absorption spectrum for the purified receptor mirrored that of phototropism (Christie et al., 1998). These findings prompted a functionalized name change from the locus *NPH1* to the gene encoding the receptor, *PHOT1*.

Based on sequence homology the *NPH-LIKE* (*NPL1*) gene was soon identified (Jarillo et al., 1998). This sequence has a similar topology to that of *NPH1*, with the same conserved kinase region and LOV domains. The individual LOV domains (LOV1 and LOV2) have distinct roles in phototropin action. Mutation of Cys39 of the LOV domain in the LOV1 domain has no effect on phototropism, whereas this mutation in the LOV2 domain abolishes curvature. The LOV2 domain also is the critical domain for promoting leaf expansion (Cho et al., 2007). There is evidence that the role of the LOV1 domain is to attenuate LOV2 effects by acting as a site for dimerization.

Separating the LOV domains from the kinase domain is an alpha-helical hinge that holds LOV domains in proximity to the kinase domain. Upon activation with light, the protein opens around this hinge region, allowing the kinase to be phosphorylated (Tokutomi et al., 2008). This is the basis for the phototropin signaling mechanism.

The Guiding Force of Photons 25

They regulate guard cells that gate gas exchange may be restricted or opened to admit carbon dioxide. At the molecular level the transcripts of specific genes necessary for photosynthetic activity accumulate and decay in light-dependent ways. All of these diverse processes share phototropins as primary photoreceptors. All of these responses have the potential to affect on optimizing photosynthesis, which appears to be the major codifying

The physiology regulated by phot1 and phot2 may be broken down into responses that have contrasting fluence thresholds, time courses and areas of action. The phot receptors have been implicated in phototropism (Christie et al., 1998), chloroplast relocation (Jarillo et al., 2001a; Kagawa et al., 2001), stomatal opening (Kinoshita et al., 2001), leaf expansion (Sakamoto and Briggs, 2002), control of stem elongation (Folta and Spalding, 2001), inflorescence, stem and petiole positioning (Kagawa et al., 2009), leaf positioning (Inoue et al., 2008), growth responses in low-light environments (Takemiya et al., 2005) and posttranslational stability of transcripts encoding chloroplast-targeted proteins (Folta and

Its isolation as a genetic mutant proved the importance of phot1 to phototropism in Arabidopsis. The null *phot1* mutants show severe defects in phototropic curvature. There is some evidence of redundant function between the two phot receptors. For instance, while *phot1-5* is a null mutant, it eventually will bend toward unilateral light based on

While the phot2 receptor has a clear role in phototropism in response to higher fluence blue light at lengthy time course, the receptor controls the predominance of other functions. The control of stomatal opening is also mediated by redundant function of the two phot receptors (Kinoshita et al., 2001). Both receptors are capable of modulating the response with similar light sensitivity and time course. The phot2 receptor also controls the accumulation of chloroplasts into a plane perpendicular to low fluence rate light. The chloroplasts move in the cell to orient themselves to optimize position for photosynthesis. This is known as the accumulation response. In times of low light the chloroplasts will align themselves to intercept incoming light. When light is extreme, the chloroplasts retreat to positions perpendicular to incoming light, shielding themselves essentially by hiding behind other chloroplasts. This is known as the avoidance response. It has been demonstrated that both phot1 and phot2 contribute to the accumulation response to low light, but the avoidance

The phot1 receptor solely mediates the first phase of hypocotyl growth inhibition in response to blue light. Upon first illumination hypocotyl growth slows significantly within minutes. This primary, sensitive and early response is due to phot1 (Folta and Spalding, 2001) Sustained effects are cry dependent. The phot1 receptor also controls the stability of the *Lhcb* transcript in response to a short, single pulse of blue light. Whereas the accumulation from low fluence blue light requires the plant g-protein and the GCR1 receptor (Warpeha et al., 2007), the transcript is destabilized in a manner that requires phot1 (Folta and Kaufman, 2003). Phots have also been shown to control leaf expansion (Sakamoto and Briggs, 2002), leaf and petiole position (Kagawa et al., 2009), and will probably be shown to control solar tracking (Briggs and Christie, 2002). All of these responses, from molecular to macroscopic, utilize the phot system to optimize the position and content of the

responses are controlled by phot2 (Jarillo et al., 2001a; Kagawa et al., 2001).

theme for the phototropins.

hardware for photosynthesis.

compensatory activity of phot2 (Sakai et al., 2001).

Kaufman, 2003).

#### **2.2.3.3 Signaling mechanism**

A tremendous wealth of information has arisen concerning the photocycle of the LOV domains and how it is translated into receptor function. The field of LOV domain receptors exploded from two labs in 2000 to at least 42 by 2004 (Letter to Plant Physiol. October 2010, Vol. 154, p. 1,). There are literally hundreds today.

The most progress has been made on understanding the light induced activation of the photosensor itself. The phot proteins associate with the plasma membrane. Here a photon of blue light is captured by the FMN chromophore bound to the LOV2 domain of the receptor. This excitation establishes a covalent bond between the FMN and the aforementioned Cys39 of the LOV domain. A conformational change occurs and an adjacent alpha helix (termed the J-domain) opens access to the kinase domain. The protein then autophosphorylates on multiple serine residues. These events are a simple sketch of how the receptor begins to excite the downstream events mediated by phototropins.

The mechanism that controls phototropism toward unilateral blue light relies on a simple starting point—the plant must transform a gradient of blue light into a chemical gradient capable of inducing differential growth. A framework for inter-molecular signaling in phototropic curvature was deduced from the members of the original genetic screen. The *nph1* (*phot1*), *nph3* and *nph4* mutants were all defective in phototropic curvature (Liscum and Briggs, 1996). As mentioned earlier, *NPH1* encodes the phototropin receptor. NPH3 is a phot1 interacting protein thought to be an adapter or scaffold protein (Motchoulski and Liscum, 1999). The action of NPH3 has remained unclear for the last decade, but recent studies show that members of a family that are likely involved in ubiquitination of substrates. The NPH3 protein is phosphorylated in darkness and upon light activation of phot1 it is dephosphorylated (Pedmale and Liscum, 2007).

Recent studies have shown that two of the PHYTOCHROME KINASE SUBSTRATE proteins, PKS1 and PKS2, co-immunoprecipitate with phot1 and phot2 (de Carbonnel et al., 2010). PKS1 binds to both phototropin1 and NPH3, and is required for phototropic curvature (Lariguet et al., 2006). These proteins have roles in the phot transduction to leaf position and flatness, but do not affect chloroplast relocation (de Carbonnel et al., 2010). A specific isoform of the 14-3-3 protein class also binds to phot1, but does not interact with phot2 (Sullivan et al., 2009). A growing list of proteins have been confirmed as interactors (reviewed in Inoue et al., 2010).

One confirmed interactor ties phototropin to redistribution of auxin. The auxin efflux carrier ABCB19 was shown to interact with phot1, and is a substrate for its kinase activity (Christie et al., 2011). The phosphorylated carrier fails to translocate auxin it accumulates in the cells, leading to lateral efflux by PIN3, developing the onset of phototropic curvature.

A variety of other downstream signaling events may be required for phot resposnes. Various reports have implicated calcium release from endomembranes or insolitol phosphates as potential links in phot signal transduction. These studies involved the use of pharmacological agents or reporters, so while coincident with phot signaling events, it is unclear how they precisely contribute to the processes.

#### **2.2.3.4 Associated physiology in plants**

Photosynthesis is constantly tuned on the biochemical and molecular level, yet many other adjustments happen at a level that one may witness simply with the naked eye and time. Phototropins dictate the position of plant organs and organelles to optimize light intercept.

A tremendous wealth of information has arisen concerning the photocycle of the LOV domains and how it is translated into receptor function. The field of LOV domain receptors exploded from two labs in 2000 to at least 42 by 2004 (Letter to Plant Physiol. October 2010,

The most progress has been made on understanding the light induced activation of the photosensor itself. The phot proteins associate with the plasma membrane. Here a photon of blue light is captured by the FMN chromophore bound to the LOV2 domain of the receptor. This excitation establishes a covalent bond between the FMN and the aforementioned Cys39 of the LOV domain. A conformational change occurs and an adjacent alpha helix (termed the J-domain) opens access to the kinase domain. The protein then autophosphorylates on multiple serine residues. These events are a simple sketch of how the receptor begins to

The mechanism that controls phototropism toward unilateral blue light relies on a simple starting point—the plant must transform a gradient of blue light into a chemical gradient capable of inducing differential growth. A framework for inter-molecular signaling in phototropic curvature was deduced from the members of the original genetic screen. The *nph1* (*phot1*), *nph3* and *nph4* mutants were all defective in phototropic curvature (Liscum and Briggs, 1996). As mentioned earlier, *NPH1* encodes the phototropin receptor. NPH3 is a phot1 interacting protein thought to be an adapter or scaffold protein (Motchoulski and Liscum, 1999). The action of NPH3 has remained unclear for the last decade, but recent studies show that members of a family that are likely involved in ubiquitination of substrates. The NPH3 protein is phosphorylated in darkness and upon light activation of

Recent studies have shown that two of the PHYTOCHROME KINASE SUBSTRATE proteins, PKS1 and PKS2, co-immunoprecipitate with phot1 and phot2 (de Carbonnel et al., 2010). PKS1 binds to both phototropin1 and NPH3, and is required for phototropic curvature (Lariguet et al., 2006). These proteins have roles in the phot transduction to leaf position and flatness, but do not affect chloroplast relocation (de Carbonnel et al., 2010). A specific isoform of the 14-3-3 protein class also binds to phot1, but does not interact with phot2 (Sullivan et al., 2009). A growing list of proteins have been confirmed as interactors

One confirmed interactor ties phototropin to redistribution of auxin. The auxin efflux carrier ABCB19 was shown to interact with phot1, and is a substrate for its kinase activity (Christie et al., 2011). The phosphorylated carrier fails to translocate auxin it accumulates in the cells,

A variety of other downstream signaling events may be required for phot resposnes. Various reports have implicated calcium release from endomembranes or insolitol phosphates as potential links in phot signal transduction. These studies involved the use of pharmacological agents or reporters, so while coincident with phot signaling events, it is

Photosynthesis is constantly tuned on the biochemical and molecular level, yet many other adjustments happen at a level that one may witness simply with the naked eye and time. Phototropins dictate the position of plant organs and organelles to optimize light intercept.

leading to lateral efflux by PIN3, developing the onset of phototropic curvature.

**2.2.3.3 Signaling mechanism** 

Vol. 154, p. 1,). There are literally hundreds today.

excite the downstream events mediated by phototropins.

phot1 it is dephosphorylated (Pedmale and Liscum, 2007).

unclear how they precisely contribute to the processes.

**2.2.3.4 Associated physiology in plants** 

(reviewed in Inoue et al., 2010).

They regulate guard cells that gate gas exchange may be restricted or opened to admit carbon dioxide. At the molecular level the transcripts of specific genes necessary for photosynthetic activity accumulate and decay in light-dependent ways. All of these diverse processes share phototropins as primary photoreceptors. All of these responses have the potential to affect on optimizing photosynthesis, which appears to be the major codifying theme for the phototropins.

The physiology regulated by phot1 and phot2 may be broken down into responses that have contrasting fluence thresholds, time courses and areas of action. The phot receptors have been implicated in phototropism (Christie et al., 1998), chloroplast relocation (Jarillo et al., 2001a; Kagawa et al., 2001), stomatal opening (Kinoshita et al., 2001), leaf expansion (Sakamoto and Briggs, 2002), control of stem elongation (Folta and Spalding, 2001), inflorescence, stem and petiole positioning (Kagawa et al., 2009), leaf positioning (Inoue et al., 2008), growth responses in low-light environments (Takemiya et al., 2005) and posttranslational stability of transcripts encoding chloroplast-targeted proteins (Folta and Kaufman, 2003).

Its isolation as a genetic mutant proved the importance of phot1 to phototropism in Arabidopsis. The null *phot1* mutants show severe defects in phototropic curvature. There is some evidence of redundant function between the two phot receptors. For instance, while *phot1-5* is a null mutant, it eventually will bend toward unilateral light based on compensatory activity of phot2 (Sakai et al., 2001).

While the phot2 receptor has a clear role in phototropism in response to higher fluence blue light at lengthy time course, the receptor controls the predominance of other functions. The control of stomatal opening is also mediated by redundant function of the two phot receptors (Kinoshita et al., 2001). Both receptors are capable of modulating the response with similar light sensitivity and time course. The phot2 receptor also controls the accumulation of chloroplasts into a plane perpendicular to low fluence rate light. The chloroplasts move in the cell to orient themselves to optimize position for photosynthesis. This is known as the accumulation response. In times of low light the chloroplasts will align themselves to intercept incoming light. When light is extreme, the chloroplasts retreat to positions perpendicular to incoming light, shielding themselves essentially by hiding behind other chloroplasts. This is known as the avoidance response. It has been demonstrated that both phot1 and phot2 contribute to the accumulation response to low light, but the avoidance responses are controlled by phot2 (Jarillo et al., 2001a; Kagawa et al., 2001).

The phot1 receptor solely mediates the first phase of hypocotyl growth inhibition in response to blue light. Upon first illumination hypocotyl growth slows significantly within minutes. This primary, sensitive and early response is due to phot1 (Folta and Spalding, 2001) Sustained effects are cry dependent. The phot1 receptor also controls the stability of the *Lhcb* transcript in response to a short, single pulse of blue light. Whereas the accumulation from low fluence blue light requires the plant g-protein and the GCR1 receptor (Warpeha et al., 2007), the transcript is destabilized in a manner that requires phot1 (Folta and Kaufman, 2003). Phots have also been shown to control leaf expansion (Sakamoto and Briggs, 2002), leaf and petiole position (Kagawa et al., 2009), and will probably be shown to control solar tracking (Briggs and Christie, 2002). All of these responses, from molecular to macroscopic, utilize the phot system to optimize the position and content of the hardware for photosynthesis.

The Guiding Force of Photons 27

observed for the protein UV RESISTANCE LOCUS 8 (UVR8; Kaiserli and Jenkins, 2007), and the results were UV-B specific (Favory et al., 2009). Mutations in UVR8 that abolished UV-B induced photomorphogenesis also impaired interaction with COP1, and interaction in yeast was UV-B dependent (Rizzini et al., 2011) presenting support for the hypothesis that UVR8 was the UV-B receptor. The mechanism of action was shown to be dependent on UVR8 dimers splitting to monomers when specific aromatic amino acids were activated by UV-B radiation. The UVR8 protein is constitutively expressed throughout the plant (Kaiserli and Jenkins, 2007; Favory et al., 2009), allowing all cells to maintain a system to respond to

A quick glance at the current receptor collection shows that the visible light spectrum is well blanketed with the absorption spectra of photosensors to receive it. As noted, the sensor collection extends plant signal perception clearly into the UV and far-red. Are there truly responses that cannot be account for by the current set of receptors? Are there likely to be more ways that a plant can sense the light environment? A series of green light responses that persist in the absence of known sensors suggest that there are additional players in the

Green light can excite phytochrome, cryptochrome and phototropin responses, depending of course on fluence rate and time of illumination. Green wavebands can induce phytochrome-mediated germination (Shinomura et al., 1996), several effects via cryptochromes as discussed earlier (Banerjee et al., 2007; Bouly et al., 2007; Sellaro et al., 2011), and even phototropic curvature (Steinitz et al., 1985) that in retrospect must be phototropin dependent. Green light has also been shown to be transmitted efficiently within the plant body and efficiently drive photosynthesis in deeper layers of the leaf (Terashima et

However, examination of the literature presents a suite of green-light-dependent phenomena that cannot easily be described as the action of cryptochromes, phytochromes or LOV domain receptors. These actions are induced specifically by green wavebands (~500- 540 nm) and tend to oppose those of red and blue light (for review, Folta and Maruhnich, 2007). Some of the first evidence was noted when plants were grown under white light, or the same light source with various parts of the spectrum filtered to skew the quality of illumination. In early studies Frits Went observed that tomato seedlings grown under white light (red, blue and green) had a lower dry mass than tomato plants grown under red and blue light alone (Went, 1957). The effect was observed across fluence rates, so it was not simply an effect of limiting photosynthetic capacity. It was as if the presence of green

Later, similar "reversal" effects in plant growth and the performance of tissue cultures were observed (Klein et al., 1965; Klein and Edsall, 1967). A curious blue-green reversal of stomatal open was described in Arabidopsis (Frechilla et al., 2000), sunflower (Wang et al., 2011a), and other species (Talbott, 2002). During experiments testing Arabidopsis stem growth kinetics in response to blue and red light, effects of green illumination were observed that were quite unusual. Unlike the inhibition caused by other wavebands, green light caused an increase in stem elongation rate. This finding was surprising because the etiolated elongation rate was always presumed to be the most rapid. The response was analyzed for its photophysiological and genetic parameters (Folta, 2004) and the results

potentially damaging wavelengths.

plant sensorium.

al., 2009).

**2.2.6 Hypothetical green light receptors** 

wavebands contradicted the effects of red and blue.

#### **2.2.4 The other LOV domain photosensors**

The central flavin-binding, photocycling domain of the phototropin receptor, the LOV domain, has emerged as a recurrent theme in many proteins spanning many species. Their direct connection to processes germane to photosynthesis is limited at this point, but their existence merits discussion. LOV domain proteins have been identified in non-vascular plants, several types of algae, in fungi and bacteria. They are found within transcription factors, kinases, phosphatases, and proteins with undefined function. Their function outside of plants is diverse, with LOV domain proteins regulating processes as ranging from plant light signaling to virulence in *Brucella* (Swartz et al., 2007), to transcriptional changes in fungi (Ballario et al., 1998).

In Arabidopsis three non-phot, LOV domain proteins reside in the genome. These same genes were identified in genetic screens for defects in the circadian clock and flowering time. These are ZEITLUPE/ADAGIO (Somers et al., 2000; Jarillo et al., 2001b), FKF1 (Nelson et al., 2000), and LKP2 (Schultz et al., 2001). All three undergo a photocycle that mirrors that of the phototropin LOV domains (Salomon et al., 2000). The three proteins share a common role in using light to coordinate the stability and accumulation of regulatory proteins.

The other main class of LOV domain proteins comes from studies in *Adiantum*. In these organisms phototropic curvature and chloroplast relocation, canonical blue light responses in plants, are induced by red light (Kawai et al., 2003). Genetic analysis of phototropic deficient mutants showed that the fern receptor is a hybrid between the red/far-red sensor phytochrome and the LOV-domain sensors. The receptor is a fusion between two receptor types that has adapted to exploitation of the understory.

#### **2.2.5 A UV-B receptor**

#### **2.2.5.1 Discovery, structure and physiology**

The light from the sun presents the plant with a double-edged problem. While necessary for photosynthetic growth, the mixture of light energies contain parcels of poison that could impart damage to DNA to the detriment of the organism. Plants being anchored to the earth by a root must therefore have means to detect ultraviolet (UV) light energies and tailor appropriate physiological and molecular countermeasures to combat the problems associated with UV exposure. Growing evidence to support this hypothesis has mounted for decades and recently resolved in the elucidation of a UV-B (280-320 nm) photosensor.

Observation of many plant physiological and molecular responses pointed to the existence of this receptor (for review, Ulm and Nagy, 2005; Jenkins, 2009). A suite of plant responses to UV-B were reported, including increases in intercellular calcium (Frohnmeyer et al., 1999), strong effects on hypocotyl growth inhibition (Shinkle et al., 2004; Shinkle et al., 2005), induction of genes associated with disease (Green and Fluhr, 1995), as well as patterns of global gene expression that differ from those observed from activation of cryptochromes or phytochromes (Ulm et al., 2004). Effects on stomatal opening have also been observed (Eisinger et al., 2003), and synergistic interactions with phytochromes have been long documented (Yatsuhashi and Hashimoto, 1985).

The quest for identification of the UV-B receptor followed a trail established from studies of other light sensors. As mentioned earlier, interaction between receptors and the ubiquitin E3 ligase COP1 is a regulatory node of light signaling. Additionally, photoreceptors have been shown to move to, or reside in, the nucleus upon illumination. The same patterns were

The central flavin-binding, photocycling domain of the phototropin receptor, the LOV domain, has emerged as a recurrent theme in many proteins spanning many species. Their direct connection to processes germane to photosynthesis is limited at this point, but their existence merits discussion. LOV domain proteins have been identified in non-vascular plants, several types of algae, in fungi and bacteria. They are found within transcription factors, kinases, phosphatases, and proteins with undefined function. Their function outside of plants is diverse, with LOV domain proteins regulating processes as ranging from plant light signaling to virulence in *Brucella* (Swartz et al., 2007), to transcriptional changes in

In Arabidopsis three non-phot, LOV domain proteins reside in the genome. These same genes were identified in genetic screens for defects in the circadian clock and flowering time. These are ZEITLUPE/ADAGIO (Somers et al., 2000; Jarillo et al., 2001b), FKF1 (Nelson et al., 2000), and LKP2 (Schultz et al., 2001). All three undergo a photocycle that mirrors that of the phototropin LOV domains (Salomon et al., 2000). The three proteins share a common role in using light to coordinate the stability and accumulation of

The other main class of LOV domain proteins comes from studies in *Adiantum*. In these organisms phototropic curvature and chloroplast relocation, canonical blue light responses in plants, are induced by red light (Kawai et al., 2003). Genetic analysis of phototropic deficient mutants showed that the fern receptor is a hybrid between the red/far-red sensor phytochrome and the LOV-domain sensors. The receptor is a fusion between two receptor

The light from the sun presents the plant with a double-edged problem. While necessary for photosynthetic growth, the mixture of light energies contain parcels of poison that could impart damage to DNA to the detriment of the organism. Plants being anchored to the earth by a root must therefore have means to detect ultraviolet (UV) light energies and tailor appropriate physiological and molecular countermeasures to combat the problems associated with UV exposure. Growing evidence to support this hypothesis has mounted for decades and recently resolved in the elucidation of a UV-B (280-320 nm) photosensor. Observation of many plant physiological and molecular responses pointed to the existence of this receptor (for review, Ulm and Nagy, 2005; Jenkins, 2009). A suite of plant responses to UV-B were reported, including increases in intercellular calcium (Frohnmeyer et al., 1999), strong effects on hypocotyl growth inhibition (Shinkle et al., 2004; Shinkle et al., 2005), induction of genes associated with disease (Green and Fluhr, 1995), as well as patterns of global gene expression that differ from those observed from activation of cryptochromes or phytochromes (Ulm et al., 2004). Effects on stomatal opening have also been observed (Eisinger et al., 2003), and synergistic interactions with phytochromes have been long

The quest for identification of the UV-B receptor followed a trail established from studies of other light sensors. As mentioned earlier, interaction between receptors and the ubiquitin E3 ligase COP1 is a regulatory node of light signaling. Additionally, photoreceptors have been shown to move to, or reside in, the nucleus upon illumination. The same patterns were

**2.2.4 The other LOV domain photosensors** 

types that has adapted to exploitation of the understory.

**2.2.5.1 Discovery, structure and physiology** 

documented (Yatsuhashi and Hashimoto, 1985).

fungi (Ballario et al., 1998).

regulatory proteins.

**2.2.5 A UV-B receptor** 

observed for the protein UV RESISTANCE LOCUS 8 (UVR8; Kaiserli and Jenkins, 2007), and the results were UV-B specific (Favory et al., 2009). Mutations in UVR8 that abolished UV-B induced photomorphogenesis also impaired interaction with COP1, and interaction in yeast was UV-B dependent (Rizzini et al., 2011) presenting support for the hypothesis that UVR8 was the UV-B receptor. The mechanism of action was shown to be dependent on UVR8 dimers splitting to monomers when specific aromatic amino acids were activated by UV-B radiation. The UVR8 protein is constitutively expressed throughout the plant (Kaiserli and Jenkins, 2007; Favory et al., 2009), allowing all cells to maintain a system to respond to potentially damaging wavelengths.

#### **2.2.6 Hypothetical green light receptors**

A quick glance at the current receptor collection shows that the visible light spectrum is well blanketed with the absorption spectra of photosensors to receive it. As noted, the sensor collection extends plant signal perception clearly into the UV and far-red. Are there truly responses that cannot be account for by the current set of receptors? Are there likely to be more ways that a plant can sense the light environment? A series of green light responses that persist in the absence of known sensors suggest that there are additional players in the plant sensorium.

Green light can excite phytochrome, cryptochrome and phototropin responses, depending of course on fluence rate and time of illumination. Green wavebands can induce phytochrome-mediated germination (Shinomura et al., 1996), several effects via cryptochromes as discussed earlier (Banerjee et al., 2007; Bouly et al., 2007; Sellaro et al., 2011), and even phototropic curvature (Steinitz et al., 1985) that in retrospect must be phototropin dependent. Green light has also been shown to be transmitted efficiently within the plant body and efficiently drive photosynthesis in deeper layers of the leaf (Terashima et al., 2009).

However, examination of the literature presents a suite of green-light-dependent phenomena that cannot easily be described as the action of cryptochromes, phytochromes or LOV domain receptors. These actions are induced specifically by green wavebands (~500- 540 nm) and tend to oppose those of red and blue light (for review, Folta and Maruhnich, 2007). Some of the first evidence was noted when plants were grown under white light, or the same light source with various parts of the spectrum filtered to skew the quality of illumination. In early studies Frits Went observed that tomato seedlings grown under white light (red, blue and green) had a lower dry mass than tomato plants grown under red and blue light alone (Went, 1957). The effect was observed across fluence rates, so it was not simply an effect of limiting photosynthetic capacity. It was as if the presence of green wavebands contradicted the effects of red and blue.

Later, similar "reversal" effects in plant growth and the performance of tissue cultures were observed (Klein et al., 1965; Klein and Edsall, 1967). A curious blue-green reversal of stomatal open was described in Arabidopsis (Frechilla et al., 2000), sunflower (Wang et al., 2011a), and other species (Talbott, 2002). During experiments testing Arabidopsis stem growth kinetics in response to blue and red light, effects of green illumination were observed that were quite unusual. Unlike the inhibition caused by other wavebands, green light caused an increase in stem elongation rate. This finding was surprising because the etiolated elongation rate was always presumed to be the most rapid. The response was analyzed for its photophysiological and genetic parameters (Folta, 2004) and the results

The Guiding Force of Photons 29

photosynthesis, it is certainly not the sole entity that is required for the process. A cast of additional factors must be recruited to the rapidly developing plastid to facilitate photosynthetic functions. Their coordinated manufacture and assembly underlie maturation

Two of the phytochrome interacting factors (PIF1 and PIF3) have been generally shown to limit chloroplast development, primarily by interacting with the promoters of target genes. Their repression is lifted by activation of phytochrome as the PIFs are degraded by

Its counterpart, PIF1, is generally regarded as a negative regulator of phytochrome activity. It also has been shown to be an active repressor of blue light response It also has been shown to repress chlorophyll biosynthesis (Huq et al., 2004) by binding to the G-box in genes associated with chlorophyll synthesis (Moon et al., 2008), as well as limit carotenoid biosynthesis by

The phytochrome interacting bHLH protein PIF3 has been described to promote chloroplast development (Monte et al., 2004). Other reports examined early chloroplast development in *pif* mutants, unveiling clear roles as repressors of chloroplast development (Stephenson et al., 2009). The *pif1pif3* mutant exhibited a constitutively photomorphogenic phenotype in the dark. The plants accumulates protochlorophyllide, and showed more evidence of thylakoid stacking. Genes associated with chlorophyll and heme synthesis were also mis-regulated in the mutant, permitting accumulation of protochlorophyllide in darkness. The transition from darkness to light is phytochrome mediated, but the precise mechanisms that control the cross talk between compartments are now being elucidated.

The hardware of photosynthesis is composed of many components that are encoded in the nucleus. Some of these components are labile, requiring a constant reloading of the plastid from parts transcribed in the nucleus, translated in the cytosol and located to the chloroplast. How do these two separate intracellular entities coordinate activities to ensure

Earlier in this chapter there was a discussion of the etioplast and its light-driven transition to the chloroplast. This transition is critical, the timing is important, and the requirements of the plastid tax the cell as a whole. These organellar demands increase with the maintenance of autotrophy, as many proteins required for chloroplast function are encoded by genes in the nucleus. These two retrograde mechanisms have been referred to as developmental control and operational control, respectively (Pogson et al., 2008). To satisfy these demands, lines of careful biochemical coordination network the chloroplast and nucleus. This feedback between cellular genomes come as no surprise, as at some point (or probably many points) there was genetic exchange between the genes of the endosymbiont and the new resident cell. Evidence of this is rich, with islands of plastid genes present in the nucleus, likely benefiting from a finer control of gene expression, splicing and useful economic

binding directly to the *PHYTOENE SYNTHASE* promoter (Toledo-Ortiz et al., 2010).

of the chloroplast during the transition to the light environment.

**3.2 Molecular control of plastid development** 

This is the subject of the next section of this chapter.

**4. Biochemical communication between compartments** 

ubiquitin-mediated proteolysis

efficient interaction?

properties of the nuclear environment.

indicated that the green light induced stimulation of hypocotyl growth rate was not likely mediated by known photosensors.

Based on the results of this study a microarray experiment assessed the state of the transcriptome in green light treated, etiolated seedlings. Surprisingly, a dim pulse of green light, far below "safelight" energies, excited large-scale changes in the transcriptome. The most conspicuous difference observed as the lower abundance of transcripts associated with the chloroplast, especially those playing a role in photosynthesis. Green light incuded reduction of steady-state transcripts encoding (among many others) the large subunit of RUBISCO (RbcL), psaA, and psbD was observed (Dhingra et al., 2006). This response was shown to be excited by low fluence pulses of light, occur within minutes, happen only in response to green light, and persist in the suite of photoreceptor mutants tested. These findings also indicated that a response to dim green light could drive a series of counterintuitive adaptive responses.

Additional observations now show that the addition of green wavebands to a background of red and blue light can attenuate light responses. Green light can induce shade avoidance phenotypes (Mullen et al., 2006; Zhang et al., 2011) and directly antagonize the effects of red light on stem growth inhibition, but not blue light (Y. Wang and K. Folta, unpublished). These effects point to the presence of a yet-to-be-characterized green light sensor that works in concert with other light sensing systems to optimize plant physiology in low-light environments.

#### **3. The transition to photosynthetic competence**

#### **3.1 The plastic plastid**

As mentioned previously, the plastid housed within the cells of the etiolate seedling is simply a structure poised to rapidly mature into a center of light-driven metabolic activity. In darkness the etiolated plastid, or etioplast, maintains a process known as skotomorphogenesis, or the developmental state occurring in the absence of light. The etioplast should not be considered simply a default, ground state. When we consider that the plastid has evolved from an endosymbiont that was photosynthetic (Margulis, 1970), the etioplast must be a derived state, a structure that provides a selective advantage for the emerging plant. This interpretation is supported by the observation that etioplasts are specialized. They feature a unique arrangement of thylakoid membrane precursors into a highly-ordered pro-lamellar body (Selstam and Widell-Wigge, 1993). This structure contains a storehouse of lipids and proteins (Selstam and Widell-Wigge, 1993; Kleffmann et al., 2007) required for the greening process. The prolamellar body's paracrystalline matrix also contains carotenoids, chlorophyll precursors and NADPH:protochlorophyllide oxidoreductase (Rosinski and Rosen, 1972 ; Selstam and Sandelius, 1984; Masuda and Takamiya, 2004). In angiosperms, POR is the central enzyme required for the production of chlorophyll via a light dependent reaction (Lebedev and Timko, 1998). In the etioplast POR complexes with protochlorophyllide which is immediately (within 2 ms) converted to chlorophyllide upon activation with light (Heyes and Hunter, 2005). POR is responsible for the majority of chlorophyll synthesis as the prolamellar body with unstacked prothylakoids transitions to the mature thylakoids of photosynthetically active chloroplasts (Solymosi et al., 2007). Not only is POR activity light dependent, but the expression of POR-encoding genes has also been shown to be driven by light. Here a handful of photons steers the competence of the developing chloroplast by generating chlorophyll. While necessary for

indicated that the green light induced stimulation of hypocotyl growth rate was not likely

Based on the results of this study a microarray experiment assessed the state of the transcriptome in green light treated, etiolated seedlings. Surprisingly, a dim pulse of green light, far below "safelight" energies, excited large-scale changes in the transcriptome. The most conspicuous difference observed as the lower abundance of transcripts associated with the chloroplast, especially those playing a role in photosynthesis. Green light incuded reduction of steady-state transcripts encoding (among many others) the large subunit of RUBISCO (RbcL), psaA, and psbD was observed (Dhingra et al., 2006). This response was shown to be excited by low fluence pulses of light, occur within minutes, happen only in response to green light, and persist in the suite of photoreceptor mutants tested. These findings also indicated that a response to dim green light could drive a series of

Additional observations now show that the addition of green wavebands to a background of red and blue light can attenuate light responses. Green light can induce shade avoidance phenotypes (Mullen et al., 2006; Zhang et al., 2011) and directly antagonize the effects of red light on stem growth inhibition, but not blue light (Y. Wang and K. Folta, unpublished). These effects point to the presence of a yet-to-be-characterized green light sensor that works in concert with other light sensing systems to optimize plant physiology in low-light

As mentioned previously, the plastid housed within the cells of the etiolate seedling is simply a structure poised to rapidly mature into a center of light-driven metabolic activity. In darkness the etiolated plastid, or etioplast, maintains a process known as skotomorphogenesis, or the developmental state occurring in the absence of light. The etioplast should not be considered simply a default, ground state. When we consider that the plastid has evolved from an endosymbiont that was photosynthetic (Margulis, 1970), the etioplast must be a derived state, a structure that provides a selective advantage for the emerging plant. This interpretation is supported by the observation that etioplasts are specialized. They feature a unique arrangement of thylakoid membrane precursors into a highly-ordered pro-lamellar body (Selstam and Widell-Wigge, 1993). This structure contains a storehouse of lipids and proteins (Selstam and Widell-Wigge, 1993; Kleffmann et al., 2007) required for the greening process. The prolamellar body's paracrystalline matrix also contains carotenoids, chlorophyll precursors and NADPH:protochlorophyllide oxidoreductase (Rosinski and Rosen, 1972 ; Selstam and Sandelius, 1984; Masuda and Takamiya, 2004). In angiosperms, POR is the central enzyme required for the production of chlorophyll via a light dependent reaction (Lebedev and Timko, 1998). In the etioplast POR complexes with protochlorophyllide which is immediately (within 2 ms) converted to chlorophyllide upon activation with light (Heyes and Hunter, 2005). POR is responsible for the majority of chlorophyll synthesis as the prolamellar body with unstacked prothylakoids transitions to the mature thylakoids of photosynthetically active chloroplasts (Solymosi et al., 2007). Not only is POR activity light dependent, but the expression of POR-encoding genes has also been shown to be driven by light. Here a handful of photons steers the competence of the developing chloroplast by generating chlorophyll. While necessary for

mediated by known photosensors.

counterintuitive adaptive responses.

**3. The transition to photosynthetic competence** 

environments.

**3.1 The plastic plastid** 

photosynthesis, it is certainly not the sole entity that is required for the process. A cast of additional factors must be recruited to the rapidly developing plastid to facilitate photosynthetic functions. Their coordinated manufacture and assembly underlie maturation of the chloroplast during the transition to the light environment.

#### **3.2 Molecular control of plastid development**

Two of the phytochrome interacting factors (PIF1 and PIF3) have been generally shown to limit chloroplast development, primarily by interacting with the promoters of target genes. Their repression is lifted by activation of phytochrome as the PIFs are degraded by ubiquitin-mediated proteolysis

Its counterpart, PIF1, is generally regarded as a negative regulator of phytochrome activity. It also has been shown to be an active repressor of blue light response It also has been shown to repress chlorophyll biosynthesis (Huq et al., 2004) by binding to the G-box in genes associated with chlorophyll synthesis (Moon et al., 2008), as well as limit carotenoid biosynthesis by binding directly to the *PHYTOENE SYNTHASE* promoter (Toledo-Ortiz et al., 2010).

The phytochrome interacting bHLH protein PIF3 has been described to promote chloroplast development (Monte et al., 2004). Other reports examined early chloroplast development in *pif* mutants, unveiling clear roles as repressors of chloroplast development (Stephenson et al., 2009). The *pif1pif3* mutant exhibited a constitutively photomorphogenic phenotype in the dark. The plants accumulates protochlorophyllide, and showed more evidence of thylakoid stacking. Genes associated with chlorophyll and heme synthesis were also mis-regulated in the mutant, permitting accumulation of protochlorophyllide in darkness. The transition from darkness to light is phytochrome mediated, but the precise mechanisms that control the cross talk between compartments are now being elucidated. This is the subject of the next section of this chapter.

#### **4. Biochemical communication between compartments**

The hardware of photosynthesis is composed of many components that are encoded in the nucleus. Some of these components are labile, requiring a constant reloading of the plastid from parts transcribed in the nucleus, translated in the cytosol and located to the chloroplast. How do these two separate intracellular entities coordinate activities to ensure efficient interaction?

Earlier in this chapter there was a discussion of the etioplast and its light-driven transition to the chloroplast. This transition is critical, the timing is important, and the requirements of the plastid tax the cell as a whole. These organellar demands increase with the maintenance of autotrophy, as many proteins required for chloroplast function are encoded by genes in the nucleus. These two retrograde mechanisms have been referred to as developmental control and operational control, respectively (Pogson et al., 2008). To satisfy these demands, lines of careful biochemical coordination network the chloroplast and nucleus. This feedback between cellular genomes come as no surprise, as at some point (or probably many points) there was genetic exchange between the genes of the endosymbiont and the new resident cell. Evidence of this is rich, with islands of plastid genes present in the nucleus, likely benefiting from a finer control of gene expression, splicing and useful economic properties of the nuclear environment.

The Guiding Force of Photons 31

were isolated that affected retrograde signaling. It was well demonstrated that application of norflurazon treatment actively repressed *Lhcb* accumulation by disrupting chlorophyll synthesis. Therefore, mutagenized plants with lesions in the repressing pathway should be allow expression of a reporter from an *Lhcb* promoter in the presence of the norflurazon. Susek et al. (1993) utilized this approach, using a truncation of the Arabidopsis *CAB3* promoter to drive hygromycin resistance and the *uidA* (GUS) gene. Seedlings growing on hygromycin and norflurazon would be candidates for genotypes possibly deficient in the plastid to nuclear signal. Results could be confirmed using the colormetric detection. The results of this screen identified non-complementing alleles called *gun* (for genomes uncoupled) mutants—*gun1*, *gun2* and *gun3*. These mutants were deficient in *Lhcb* and *Rbcs* repression, indicating that nuclear gene expression could be uncoupled from the chloroplast, allowing light-mediated changes in gene expression while the chloroplast remained undeveloped (Susek et al., 1993). Three other loci, *gun4, gun5* (Mochizuki et al.,

Analysis of *GUN1* shows it to encode a pentatricopeptide repeat protein localized to the chloroplast (Koussevitzky et al., 2007). Analysis of promoters affected by *gun1* (and also *gun5*) mutation presented a suite of genes that shared an abscisic acid response element in the promoter, suggesting a role for ABA in retrograde signaling. The *gun2* and *gun3* mutants were shown to possess lesions in heme oxygenase and biliverdin reducase, respectively. Later it was shown that *GUN4* encodes a protein required for normal Mg chelatase activity, while *GUN5* encodes a required subunit of the Mg chelatase enzyme (Mochizuki et al., 2001). The *gun2-gun5* loss-of-function mutants disrupt genes essential for tetrapyrrole metabolism. Genetic evidence shows that they participate in the same signaling pathway, supporting the hypothesis that accumulation of a precursor could be the retrograde signal. *GUN6*-1D is a gain-of-function mutant that overexpresses a plastidic ferrochelatase (Woodson et al., 2011). Its overexpression leads to the hyper-accumulation of heme that could serve as a retrograde signal. In addition to the *GUN* genes, the *GOLDEN2-LIKE* (*GLK*) genes also have been shown to control similar sets of genes relevant to chlorophyll synthesis and antenna binding (Waters et al., 2009), playing central roles in communication between

When considered together the mutants and pharmacological treatments demonstrate that blocks in tetrapyrrole and/or heme synthesis may cause accumulation of precursor compounds that would leave the plastid (or trigger another mobile signal) leading to repression of plastid-associated, nuclear-encoded genes. While attractive, several lines of evidence reject this hypothesis. Mainly, there is no observed difference in Mg-ProtoIX or Mg-ProtoIXme is detected in plants with disrupted signaling responses (Mochizuki et al., 2008). Using sensitive LC/MS methods to identify chlorophyll precursors in norflurazon treated plants, it was shown that there was no effect on MgProtoIX when chlorophyll

There is an undeniable chemical communication link between the chloroplast and nucleus. Genetic and biochemical tools suggest that chlorophyll precursors and/or heme play a part in the process, yet clearly it is not as simple as over-accumulation of a compound like MgProtoIX. While many careers and high-profile publications frame this question, there are answers to be resolved before a complete picture of retrograde

2001) and *gun6* (Woodson et al., 2011), were later isolated.

plastid and nucleus (Fitter et al., 2002).

synthesis was disrupted (Moulin et al., 2008).

signaling is understood.

The cell requires the chloroplast to be in harmony with the nucleus- the two compartments working in concert with great precision. The chloroplast contains genome fewer than onehundred open reading frames, yet function of the chloroplast requires over three thousand proteins. Regulatory steps that communicate demands to the nucleus to start construction of these proteins need to be precise. There are challenges to fluid exchange of signals between chloroplasts and the nucleus, in particular the presence of membranes that block passage of the vast majority of ions, peptides or other small molecules. The co-evolution between the endosymbiont and the plant cell had to involve a way to bypass these barriers.

There are many lines of evidence that show evidence of communication between chloroplast and nucleus. Pharmacological disruption of transcription in the plastid transcription or chlorophyll synthesis results in aberrant nuclear gene expression. Using tagetitoxin (a potent phytotoxin that inhibits select RNA polymerases including the plastidic one) to repress transcription in the chloroplast, Rapp and Mullet (1991) illustrated that the nuclear *cab* (now *Lhcb*) and *RbcS* transcripts failed to normally accumulate when the chloroplast was impaired. Other nuclear-encoded transcripts like actin responded normally, and the plants grew in a typical fashion. Disruption of plastid translation with lincomycin or disruption of carotenoid biosynthesis with norflurazon, an inhibitor of phytoene desaturase, also inhibits the normal accumulation of *Lhcb* and *RbcS* transcripts (Gray et al., 2003). The same treatments do not affect mitochondrial gene expression patterns. The chloroplast specificity was demonstrated through the use of erythromycin in peas, a compound that inhibits plastid translation but does not affect translation in the mitochondrion (Sullivan and Gray, 1999). The use of thujaplicin arrests the production of protochlorophyllide, causing a backaccumulation of Mg-ProtoIX and Mg-ProtoIXme. The treatment also hinders *Lhcb* accumulation (Oster et al., 1996), a result that is important to underscore, as chlorophyll biosynthetic mutants will later show similar effects when this step is interrupted.

While these pharmacological studies had utility, the introduction of nucleus-chloroplast signaling mutants brought new illumination to the processes of intra-compartmental feedback. The barley genotypes *albostrians* and *Saskatoon* fail to accumulate chlorophyll in various sectors. Analysis of nuclear gene expression indicated that *RbcS* and *Lhcb* gene expression levels were repressed in these regions (Hess et al., 1991; Hess et al., 1994). Early studies in other plants, including maize (Mayfield and Taylor, 1984) and mustard (Oelmüller et al., 1986), showed that plants deficient in carotenoid synthesis similar breakdowns in nuclear gene expression. There is also evidence that the redox state of photosynthetic electron transport affects the expression of these transcripts (Pfannschmidt et al., 2001; Masuda et al., 2003; Brautigam et al., 2009). Other evidence suggests that an accumulation of nuclear encoded proteins that fail to localize to the chloroplast is a retrograde signal (Kakizaki et al., 2009).

Together the observation that *Lhcb, RbcS,* and other nuclear genes are repressed when the chloroplast is not functioning correctly is significant because these transcripts accumulate rapidly in response to phytochrome activation. Active repression of these transcripts indicated that some factor reflecting the state of the chloroplast was overriding the normal response. The repression was selectively affecting specific nuclear genes, impairing activity of those required for chloroplast function. In times of internal dystrophy the plastid is instructing the nucleus that there is no need for various gene products.

The understanding of chloroplast-nuclear communication was accelerated with studies in *Arabidopsis thaliana*. By exploiting the powerful genetics of this system a series of mutants

The cell requires the chloroplast to be in harmony with the nucleus- the two compartments working in concert with great precision. The chloroplast contains genome fewer than onehundred open reading frames, yet function of the chloroplast requires over three thousand proteins. Regulatory steps that communicate demands to the nucleus to start construction of these proteins need to be precise. There are challenges to fluid exchange of signals between chloroplasts and the nucleus, in particular the presence of membranes that block passage of the vast majority of ions, peptides or other small molecules. The co-evolution between the

There are many lines of evidence that show evidence of communication between chloroplast and nucleus. Pharmacological disruption of transcription in the plastid transcription or chlorophyll synthesis results in aberrant nuclear gene expression. Using tagetitoxin (a potent phytotoxin that inhibits select RNA polymerases including the plastidic one) to repress transcription in the chloroplast, Rapp and Mullet (1991) illustrated that the nuclear *cab* (now *Lhcb*) and *RbcS* transcripts failed to normally accumulate when the chloroplast was impaired. Other nuclear-encoded transcripts like actin responded normally, and the plants grew in a typical fashion. Disruption of plastid translation with lincomycin or disruption of carotenoid biosynthesis with norflurazon, an inhibitor of phytoene desaturase, also inhibits the normal accumulation of *Lhcb* and *RbcS* transcripts (Gray et al., 2003). The same treatments do not affect mitochondrial gene expression patterns. The chloroplast specificity was demonstrated through the use of erythromycin in peas, a compound that inhibits plastid translation but does not affect translation in the mitochondrion (Sullivan and Gray, 1999). The use of thujaplicin arrests the production of protochlorophyllide, causing a backaccumulation of Mg-ProtoIX and Mg-ProtoIXme. The treatment also hinders *Lhcb* accumulation (Oster et al., 1996), a result that is important to underscore, as chlorophyll

endosymbiont and the plant cell had to involve a way to bypass these barriers.

biosynthetic mutants will later show similar effects when this step is interrupted.

retrograde signal (Kakizaki et al., 2009).

While these pharmacological studies had utility, the introduction of nucleus-chloroplast signaling mutants brought new illumination to the processes of intra-compartmental feedback. The barley genotypes *albostrians* and *Saskatoon* fail to accumulate chlorophyll in various sectors. Analysis of nuclear gene expression indicated that *RbcS* and *Lhcb* gene expression levels were repressed in these regions (Hess et al., 1991; Hess et al., 1994). Early studies in other plants, including maize (Mayfield and Taylor, 1984) and mustard (Oelmüller et al., 1986), showed that plants deficient in carotenoid synthesis similar breakdowns in nuclear gene expression. There is also evidence that the redox state of photosynthetic electron transport affects the expression of these transcripts (Pfannschmidt et al., 2001; Masuda et al., 2003; Brautigam et al., 2009). Other evidence suggests that an accumulation of nuclear encoded proteins that fail to localize to the chloroplast is a

Together the observation that *Lhcb, RbcS,* and other nuclear genes are repressed when the chloroplast is not functioning correctly is significant because these transcripts accumulate rapidly in response to phytochrome activation. Active repression of these transcripts indicated that some factor reflecting the state of the chloroplast was overriding the normal response. The repression was selectively affecting specific nuclear genes, impairing activity of those required for chloroplast function. In times of internal dystrophy the plastid is

The understanding of chloroplast-nuclear communication was accelerated with studies in *Arabidopsis thaliana*. By exploiting the powerful genetics of this system a series of mutants

instructing the nucleus that there is no need for various gene products.

were isolated that affected retrograde signaling. It was well demonstrated that application of norflurazon treatment actively repressed *Lhcb* accumulation by disrupting chlorophyll synthesis. Therefore, mutagenized plants with lesions in the repressing pathway should be allow expression of a reporter from an *Lhcb* promoter in the presence of the norflurazon. Susek et al. (1993) utilized this approach, using a truncation of the Arabidopsis *CAB3* promoter to drive hygromycin resistance and the *uidA* (GUS) gene. Seedlings growing on hygromycin and norflurazon would be candidates for genotypes possibly deficient in the plastid to nuclear signal. Results could be confirmed using the colormetric detection. The results of this screen identified non-complementing alleles called *gun* (for genomes uncoupled) mutants—*gun1*, *gun2* and *gun3*. These mutants were deficient in *Lhcb* and *Rbcs* repression, indicating that nuclear gene expression could be uncoupled from the chloroplast, allowing light-mediated changes in gene expression while the chloroplast remained undeveloped (Susek et al., 1993). Three other loci, *gun4, gun5* (Mochizuki et al., 2001) and *gun6* (Woodson et al., 2011), were later isolated.

Analysis of *GUN1* shows it to encode a pentatricopeptide repeat protein localized to the chloroplast (Koussevitzky et al., 2007). Analysis of promoters affected by *gun1* (and also *gun5*) mutation presented a suite of genes that shared an abscisic acid response element in the promoter, suggesting a role for ABA in retrograde signaling. The *gun2* and *gun3* mutants were shown to possess lesions in heme oxygenase and biliverdin reducase, respectively. Later it was shown that *GUN4* encodes a protein required for normal Mg chelatase activity, while *GUN5* encodes a required subunit of the Mg chelatase enzyme (Mochizuki et al., 2001). The *gun2-gun5* loss-of-function mutants disrupt genes essential for tetrapyrrole metabolism. Genetic evidence shows that they participate in the same signaling pathway, supporting the hypothesis that accumulation of a precursor could be the retrograde signal. *GUN6*-1D is a gain-of-function mutant that overexpresses a plastidic ferrochelatase (Woodson et al., 2011). Its overexpression leads to the hyper-accumulation of heme that could serve as a retrograde signal. In addition to the *GUN* genes, the *GOLDEN2-LIKE* (*GLK*) genes also have been shown to control similar sets of genes relevant to chlorophyll synthesis and antenna binding (Waters et al., 2009), playing central roles in communication between plastid and nucleus (Fitter et al., 2002).

When considered together the mutants and pharmacological treatments demonstrate that blocks in tetrapyrrole and/or heme synthesis may cause accumulation of precursor compounds that would leave the plastid (or trigger another mobile signal) leading to repression of plastid-associated, nuclear-encoded genes. While attractive, several lines of evidence reject this hypothesis. Mainly, there is no observed difference in Mg-ProtoIX or Mg-ProtoIXme is detected in plants with disrupted signaling responses (Mochizuki et al., 2008). Using sensitive LC/MS methods to identify chlorophyll precursors in norflurazon treated plants, it was shown that there was no effect on MgProtoIX when chlorophyll synthesis was disrupted (Moulin et al., 2008).

There is an undeniable chemical communication link between the chloroplast and nucleus. Genetic and biochemical tools suggest that chlorophyll precursors and/or heme play a part in the process, yet clearly it is not as simple as over-accumulation of a compound like MgProtoIX. While many careers and high-profile publications frame this question, there are answers to be resolved before a complete picture of retrograde signaling is understood.

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#### **5. Conclusions**

The last two decades have brought tremendous resolution about how the guiding force of photons shapes plant biology, especially processes germane to photosynthesis. The 1990's produced a wellspring of genetic tools that would define several major classes of photosensors and their contiguous protein transduction partners. The last decade brought the utility of genomics tools that would help define the mechanisms and targets of light signal transduction events. New methods in imaging and improved reporter genes have allowed researchers to monitor small changes in plant growth and development, as well as localization and interaction between proteins in vivo. The challenge of the next decade will be to apply these basic discoveries in meaningful ways that escape the models. Here the rules that integrate light signals, change gene expression, alter development, and shape plant form may be manipulated to improve the production of food with less environmental impact.

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**0**

**3**

<sup>1</sup>*Germany* <sup>2</sup>*Colombia* <sup>3</sup>*USA*

**Photosynthesis**

Guannan Zhao<sup>3</sup> and Neil F. Johnson3

<sup>1</sup>*Ulm University, Institute of Theoretical Physics, Ulm* <sup>2</sup>*Departamento de Física, Universidad de los Andes, Bogotá*

**Energy Conversion in Purple Bacteria**

Felipe Caycedo-Soler1,2, Ferney J. Rodríguez2, Luis Quiroga2,

<sup>3</sup>*Department of Physics, University of Miami, Coral Gables, Miami, Florida*

The study of how photosynthetic organisms convert light offers insight not only into nature's evolutionary process, but may also give clues as to how best to design and manipulate artificial photosynthetic systems – and also how far we can drive natural photosynthetic systems beyond normal operating conditions, so that they can harvest energy for us under otherwise extreme conditions. In addition to its interest from a basic scientific perspective, therefore, the goal to develop a deep quantitative understanding of photosynthesis offers the potential payoff of enhancing our current arsenal of alternative energy sources for the future. In the following Chapter, we consider the excitation dynamics of photosynthetic membranes in *Rps. Photometricum* purple bacteria. The first studies on purple bacteria photosynthetic membranes were concerned with the complex underlying detailed structure (Jamieson & al, 2002; McDermott et al., 1995; Roszack & et al, 2003; Waltz et al., 1998). The interested reader might find helpful the first section of this chapter where we present a summary of structures and processes that allow photosynthesis in purple bacteria. As improved resolution became available for light-harvesting structures, so too the community's interest increased in understanding the details of the rapid femto- to picosecond timescales for the excitation transfer process within a given harvesting complex – even to the regime where quantum effects are expected. Indeed such quantum effects have recently been confirmed in, for instance, the Fenna-Matthews-Olson complex of green sulfur bacteria (Engel et al., 2007). However, the processes occurring at this level of detail in terms of both structure and properties of the excitation harvesting, have not yet been shown as being crucial to the performance of the full harvesting membrane, nor with the primary goal of any given photosynthetic organism: to fuel its metabolism. We focus on the transfer among different complexes, in particular the inter-complex excitation transfer. We consider first a system of few complexes in order to understand the consequences on the dynamics of the complexes' connectedness. We also consider the relative amounts of harvesting complexes (i.e. stoichiometry) on small sized networks in order to help establish our understanding of

**1. Introduction**

the behavior of complete chromatophore vesicles.


### **Energy Conversion in Purple Bacteria Photosynthesis**

Felipe Caycedo-Soler1,2, Ferney J. Rodríguez2, Luis Quiroga2, Guannan Zhao<sup>3</sup> and Neil F. Johnson3 <sup>1</sup>*Ulm University, Institute of Theoretical Physics, Ulm* <sup>2</sup>*Departamento de Física, Universidad de los Andes, Bogotá* <sup>3</sup>*Department of Physics, University of Miami, Coral Gables, Miami, Florida* <sup>1</sup>*Germany* <sup>2</sup>*Colombia* <sup>3</sup>*USA*

#### **1. Introduction**

42 Advances in Photosynthesis – Fundamental Aspects

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symptoms. Plant Physiology. In Press

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The study of how photosynthetic organisms convert light offers insight not only into nature's evolutionary process, but may also give clues as to how best to design and manipulate artificial photosynthetic systems – and also how far we can drive natural photosynthetic systems beyond normal operating conditions, so that they can harvest energy for us under otherwise extreme conditions. In addition to its interest from a basic scientific perspective, therefore, the goal to develop a deep quantitative understanding of photosynthesis offers the potential payoff of enhancing our current arsenal of alternative energy sources for the future. In the following Chapter, we consider the excitation dynamics of photosynthetic membranes in *Rps. Photometricum* purple bacteria. The first studies on purple bacteria photosynthetic membranes were concerned with the complex underlying detailed structure (Jamieson & al, 2002; McDermott et al., 1995; Roszack & et al, 2003; Waltz et al., 1998). The interested reader might find helpful the first section of this chapter where we present a summary of structures and processes that allow photosynthesis in purple bacteria. As improved resolution became available for light-harvesting structures, so too the community's interest increased in understanding the details of the rapid femto- to picosecond timescales for the excitation transfer process within a given harvesting complex – even to the regime where quantum effects are expected. Indeed such quantum effects have recently been confirmed in, for instance, the Fenna-Matthews-Olson complex of green sulfur bacteria (Engel et al., 2007). However, the processes occurring at this level of detail in terms of both structure and properties of the excitation harvesting, have not yet been shown as being crucial to the performance of the full harvesting membrane, nor with the primary goal of any given photosynthetic organism: to fuel its metabolism. We focus on the transfer among different complexes, in particular the inter-complex excitation transfer. We consider first a system of few complexes in order to understand the consequences on the dynamics of the complexes' connectedness. We also consider the relative amounts of harvesting complexes (i.e. stoichiometry) on small sized networks in order to help establish our understanding of the behavior of complete chromatophore vesicles.

Fig. 1. Representation of a lake containing both aerobic and anaerobic phototrophic organisms. Note that purple bacterial photosynthesis is restricted to the lower anaerobic layer and so they only receive solar energy that has been filtered, mainly by chlorophylls

due to the electronic excitation transferred from the surrounding LH1.

Light absorption occurs through organic molecules, known as chromophores, inserted in protein complexes. Bacteriochlorophyls (BChl) and carotenoids (Car) chromophores are the main absorbers in purple bacteria photosynthesis, principally in the far red and green, respectively. The light absorption process occurs through chromophore's *Qy* electronic transition excitation. Several chromophores are embedded in protein helices, named *α* and *β* apoproteins, inside complexes, classified by their absorption spectral maximum. Light Harvesting complex 2 (LH2) reveals two concentric subunits that according to their absorption maxima are called B800 or B850, composed of nine pairs of apoproteins ( McDermott et al., 1995) comprising an inner *α*-helix and an outer *β*-helix both crossing the harvesting membrane from periplasm to cytoplasm, in an *αβ* unit that serves to anchor a highly interacting B850 dimer and one B800 chromophore. Hence, the B800 is composed of nine chromophores, while B850 include eighteen BChl*a* chromophores, having dipole moments parallel and nearly perpendicular to the membrane plane, respectively. Raman spectra using different excitation wavelength (Gall et al., 2006) and stoichiometry analysis (Arellano et al., 1998), indicated that one carotenoid (Car) is present per each *αβ* unit. Light Harvesting complex 1 (LH1) absorbs maximally at 883 nm, and contain 32 BChls, arranged in 16 bi-chromophore *αβ* units, surrounding an RC ( Karrasch et al., 1995) in the same geometrical arrangement as B850 chromophores. An RC presents a highly interacting dimer, the special pair (P), that is ionized

Energy Conversion in Purple Bacteria Photosynthesis 45

The complexes' photon absorption cross section has been calculated for LH1 and LH2 complexes, where all absorbing molecules and extinction coefficients (Francke & Amesz, 1995) have been taken into account. A photon of wavelentht *λ*, is part of the power spectrum of a

belonging to algae, cyanobacteria and plants.

**2.1 Harvesting function**

As a whole, chromatophore vesicles comprise sections of the purple bacteria cytoplasmic membrane where a large number of harvesting complexes accommodate. The current capabilities to dissect these vesicles on the nanoscale through Atomic Force Microscopy (AFM) has provided evidence of changes in the conformation of chromatophores as a result of different environmental conditions. According to Ref.(Scheuring & Sturgis, 2005) , membranes grown under Low Light Intensity (LLI, 10 Watt/m2) present a relative amount of harvesting complexes which is different to the stoichiometry observed for bacteria grown under High Light Intensity (HLI, 100 Watt/m2), thereby pinpointing the importance of global changes in the complete vesicles as an important means of fulfilling the bacteria's metabolic requirements. In order to study these global conformational changes – given the fact that the inter-complex transfer time-scale involves several picoseconds and the excitation delocalization length is not expected to be beyond a single harvesting complex – we choose a model of excitation dynamics which is based on a classical random walk. This random walk is coupled to the main processes leading to electron/chemical energy transformation, and hence the bacteria's metabolic demands.

This Chapter is organized as follows. Section 2 provides a review of the basic structures involved in excitation transfer, along with a summary of the processes required for electron/chemical energy conversion. A discussion of the dynamics of excitations in a few model architectures is explored in section 3, in order to understand the results from complete LLI and HLI adapted chromatophores in section 4 and to guide our development of an analytical model in section 5 for determining both the efficiency and power output of any given chromatophore vesicle under arbitrary light intensity regimes. Lastly, in section 6 we explore the effect of incident light with extreme photon arrival statistics, on the resulting electronic/chemical energy conversion, in order to heuristically provide a survivability margin beyond which terrestrial bacteria could not survive. This is motivated by the fact that one day, it may be necessary to send simple bacteria into deep space and/or cope with extreme photon conditions here on Earth as a result of a catastrophic solar change.

#### **2. Important processes for solar/chemical energy conversion**

Purple bacteria sustain their metabolism using photosynthesis in anaerobic conditions and under the dim light excitation proper of several meters deeps at ponds, lagoons and streams (Pfenning, 1978). As depicted in Fig.1, aerobic organisms are present near the surface of water reservoirs, collect blue and red spectral components of sun's light, leaving only the green and far red (> 750nm) components, from where purple bacteria must fulfill their energy requirements.

The light energy absorption is accomplished through intracytoplasmic membranes where different pigment-protein complexes accommodate. Light Harvesting complexes (LHs) have the function of absorbing light and transfer it to Reaction Centers (RCs), where a charge separation process is initiated (Codgell et al., 2006). The unpaired charge reduces a quinone, which using a periplasmic hydrogen, converts to quinol (Q*B*H2). RCs neutrality is restablished thanks to cytochrome *cyt* charge carrier, which after undocking from the RC, must find the *bc*<sup>1</sup> complex to receive an electron and start its cycle all over again. The electron in *bc*<sup>1</sup> is given due to cytoplasmic Quinol delivery in *bc*1. The proton gradient induced by the charge carriers cycling becomes the precursor of adenosyn triphosphate (ATP) synthesis: ADP+P→ ATP+Energy, where ADP and P refer respectively, to adenosine diphosphate and phosphorous. The cycle is depicted in Fig.2.

2 Will-be-set-by-IN-TECH

As a whole, chromatophore vesicles comprise sections of the purple bacteria cytoplasmic membrane where a large number of harvesting complexes accommodate. The current capabilities to dissect these vesicles on the nanoscale through Atomic Force Microscopy (AFM) has provided evidence of changes in the conformation of chromatophores as a result of different environmental conditions. According to Ref.(Scheuring & Sturgis, 2005) , membranes grown under Low Light Intensity (LLI, 10 Watt/m2) present a relative amount of harvesting complexes which is different to the stoichiometry observed for bacteria grown under High Light Intensity (HLI, 100 Watt/m2), thereby pinpointing the importance of global changes in the complete vesicles as an important means of fulfilling the bacteria's metabolic requirements. In order to study these global conformational changes – given the fact that the inter-complex transfer time-scale involves several picoseconds and the excitation delocalization length is not expected to be beyond a single harvesting complex – we choose a model of excitation dynamics which is based on a classical random walk. This random walk is coupled to the main processes leading to electron/chemical energy transformation, and hence

This Chapter is organized as follows. Section 2 provides a review of the basic structures involved in excitation transfer, along with a summary of the processes required for electron/chemical energy conversion. A discussion of the dynamics of excitations in a few model architectures is explored in section 3, in order to understand the results from complete LLI and HLI adapted chromatophores in section 4 and to guide our development of an analytical model in section 5 for determining both the efficiency and power output of any given chromatophore vesicle under arbitrary light intensity regimes. Lastly, in section 6 we explore the effect of incident light with extreme photon arrival statistics, on the resulting electronic/chemical energy conversion, in order to heuristically provide a survivability margin beyond which terrestrial bacteria could not survive. This is motivated by the fact that one day, it may be necessary to send simple bacteria into deep space and/or cope with

extreme photon conditions here on Earth as a result of a catastrophic solar change.

Purple bacteria sustain their metabolism using photosynthesis in anaerobic conditions and under the dim light excitation proper of several meters deeps at ponds, lagoons and streams (Pfenning, 1978). As depicted in Fig.1, aerobic organisms are present near the surface of water reservoirs, collect blue and red spectral components of sun's light, leaving only the green and far red (> 750nm) components, from where purple bacteria must fulfill their energy

The light energy absorption is accomplished through intracytoplasmic membranes where different pigment-protein complexes accommodate. Light Harvesting complexes (LHs) have the function of absorbing light and transfer it to Reaction Centers (RCs), where a charge separation process is initiated (Codgell et al., 2006). The unpaired charge reduces a quinone, which using a periplasmic hydrogen, converts to quinol (Q*B*H2). RCs neutrality is restablished thanks to cytochrome *cyt* charge carrier, which after undocking from the RC, must find the *bc*<sup>1</sup> complex to receive an electron and start its cycle all over again. The electron in *bc*<sup>1</sup> is given due to cytoplasmic Quinol delivery in *bc*1. The proton gradient induced by the charge carriers cycling becomes the precursor of adenosyn triphosphate (ATP) synthesis: ADP+P→ ATP+Energy, where ADP and P refer respectively, to adenosine diphosphate and

**2. Important processes for solar/chemical energy conversion**

the bacteria's metabolic demands.

requirements.

phosphorous. The cycle is depicted in Fig.2.

Fig. 1. Representation of a lake containing both aerobic and anaerobic phototrophic organisms. Note that purple bacterial photosynthesis is restricted to the lower anaerobic layer and so they only receive solar energy that has been filtered, mainly by chlorophylls belonging to algae, cyanobacteria and plants.

#### **2.1 Harvesting function**

Light absorption occurs through organic molecules, known as chromophores, inserted in protein complexes. Bacteriochlorophyls (BChl) and carotenoids (Car) chromophores are the main absorbers in purple bacteria photosynthesis, principally in the far red and green, respectively. The light absorption process occurs through chromophore's *Qy* electronic transition excitation. Several chromophores are embedded in protein helices, named *α* and *β* apoproteins, inside complexes, classified by their absorption spectral maximum. Light Harvesting complex 2 (LH2) reveals two concentric subunits that according to their absorption maxima are called B800 or B850, composed of nine pairs of apoproteins ( McDermott et al., 1995) comprising an inner *α*-helix and an outer *β*-helix both crossing the harvesting membrane from periplasm to cytoplasm, in an *αβ* unit that serves to anchor a highly interacting B850 dimer and one B800 chromophore. Hence, the B800 is composed of nine chromophores, while B850 include eighteen BChl*a* chromophores, having dipole moments parallel and nearly perpendicular to the membrane plane, respectively. Raman spectra using different excitation wavelength (Gall et al., 2006) and stoichiometry analysis (Arellano et al., 1998), indicated that one carotenoid (Car) is present per each *αβ* unit. Light Harvesting complex 1 (LH1) absorbs maximally at 883 nm, and contain 32 BChls, arranged in 16 bi-chromophore *αβ* units, surrounding an RC ( Karrasch et al., 1995) in the same geometrical arrangement as B850 chromophores. An RC presents a highly interacting dimer, the special pair (P), that is ionized due to the electronic excitation transferred from the surrounding LH1.

The complexes' photon absorption cross section has been calculated for LH1 and LH2 complexes, where all absorbing molecules and extinction coefficients (Francke & Amesz, 1995) have been taken into account. A photon of wavelentht *λ*, is part of the power spectrum of a

**2.2 Excitation transfer**

where *c*<sup>+</sup>

Coulomb integral:

*σ*�

(van Amerongen et al., 2000):

�*φmφp*|*V*|*φnφq*� =

*Vij* <sup>=</sup> <sup>1</sup>

Hamiltonian *H* in the chromophore site basis |*i*�,

<sup>2</sup> ∑ *m*,*n*,*p*,*q*

*<sup>d</sup><sup>r</sup>*<sup>1</sup> *<sup>d</sup><sup>r</sup>*<sup>2</sup> *<sup>φ</sup>*<sup>∗</sup>

∑ *σ*,*σ*�

*H* = *�*∑ *i*

�*i*|*H*|*j*� = *Vij* = *C*

Excitation transfer happens through Coulomb interaction of electrons, excited to the Q*y* electronic transition in chromophores. The interaction energy can be formally written

Energy Conversion in Purple Bacteria Photosynthesis 47

�*φmφn*|*V*|*φpφq*�*c*<sup>+</sup>

*<sup>m</sup>σ*, *cn<sup>σ</sup>*� are fermion creation and annihilation operators of electrons with spin *σ* and

*<sup>m</sup>*(*<sup>r</sup>*<sup>1</sup> <sup>−</sup>*ri*)*φp*(*<sup>r</sup>*<sup>1</sup> <sup>−</sup>*ri*) *<sup>e</sup>*<sup>2</sup>

, in the mutually orthogonal atomic orbitals *φ<sup>m</sup>* and *φn*. The overlap �*φmφn*|*V*|*φpφq*� is the

that accounts on inter-molecular exchange contribution when donor and acceptor are at a distance comparable to the extent of the molecules, and the direct Coulomb contribution for an electron that makes a transition between *φm* and *φp*, having both a finite value near the position of the donnor *r*<sup>1</sup> ≈ *rD*, while another electron is excited between *φ<sup>n</sup>* and *φ<sup>q</sup>* at the acceptor coordinate *r*<sup>2</sup> ≈ *rA*. In this latter situation, a commonly used framework concerns a tight-binding Hamiltonian, where details in the specific molecular orbitals |*φn*� involving mainly the *Qy* orbitals is left aside, and emphasis relies on occupation with a single index labeling an electronic state |*i*� concerning occupation in a given chromophore. The

> |*i*��*i*| + ∑ *i*,*j*

has diagonal elements �*i*|*H*|*i*� = *�*, concerning the energy of the excitation, usually measured from the ground electronic state. Neighboring chromophores are too close to neglect their charge distribution and its interaction is determined such that the effective Hamiltonian spectrum matches the spectrum of an extensive quantum chemistry calculation (Hu et al., 1997). In the LH2 complex, the B850 ring, with nearest neighbors coupling �*i*|*H*|*i* + 1� = 806 cm−<sup>1</sup> or 377 cm <sup>−</sup><sup>1</sup> for chromophores respectively, within or in different neighboring *αβ* units. For next-to-neighboring chromophores the dipole-dipole approximation is usually used,

> *μ<sup>i</sup>* · *μ<sup>j</sup> r*3 *ij*

where *μ<sup>i</sup>* is the dipole moment and *r* is the distance between the interacting dipoles with constants: *�* = 13059 cm−<sup>1</sup> and *C* = 519 044 <sup>3</sup> cm−1. The LH1 complex has a spectrum maximum near 875 nm, with inter-complex distances and nearest neighbor interactions equal to the ones provided in the B850 LH2 ring. The molecular nature of chromophores involve vibrational degrees of freedom that provide a manifold within each electronic states which should be accounted. However, given these parameters within a harvesting complex, intra-complex energy transfer involves sub-picosecond time-scales, that imply relevance of electronic quantum features over the influence of thermalization in the vibrational manifold. If decoherence sources are not important within the time-scale of excitation dynamics on donor

*mσc*<sup>+</sup>


<sup>−</sup> <sup>3</sup>(*rij* · *<sup>μ</sup>i*)(*rij* · *<sup>μ</sup>j*) *r*5

with *i* �= *j*, *i* = *j* �= ±1 (6)

*ij*

*φ*∗

*Vij*|*i*��*j*| (5)

*<sup>p</sup>σ*� *cqσcnσ*, (3)

*<sup>n</sup>*(*r*<sup>2</sup> −*rj*)*φq*(*r*<sup>2</sup> −*rj*) (4)

Fig. 2. Schematic representation of the photosynthetic apparatus in the intracytoplasmic membrane of purple bacteria. The reaction center (RC, red) is surrounded by the light-harvesting complex 1 (LH1, green) to form the LH1+RC complex, which is surrounded by multiple light-harvesting complexes (LH2s) (blue), forming altogether the photosynthetic unit (PSU). Photons are absorbed by LHs and excitation is transferred to the RC initiating a charge (electron-hole) separation. Electrons are shuttled back by the cytochrome *c*<sup>2</sup> charge carrier (blue) from the ubiquinone-cytochrome *bc*<sup>1</sup> complex (yellow) to the RC. The electron transfer across the membrane produces a proton gradient that drives the synthesis of ATP from ADP by the ATPase (orange). Electron *e*− is represented in blue, and quinones *QB*, likely confined to the intramembrane space, in black.

source with occupation numbers *n*(*λ*). Normalized to 18 W/m<sup>2</sup> intensity, the rate of photon absorption for circular LH1 complexes in *Rb. sphaeroides* (Geyer & Heims, 2006):

$$
\gamma\_1^A = \int \mathfrak{n}(\lambda) \sigma\_{\rm LH1}(\lambda) d\lambda = 18 \text{s}^{-1}.\tag{1}
$$

The same procedure applied to LH2 complexes, yields a photon capture rate of *γ<sup>A</sup>* <sup>2</sup> <sup>=</sup> 10s−1. Since these rates are normalized to 18 W/m2, the extension to arbitrary light intensity *I* is straightforward. The rate of photon absorption normalized to 1 W/m<sup>2</sup> intensity, will be *<sup>γ</sup>*1(2) = <sup>1</sup>(0.55)s−1. From now on, subindexes 1 and 2 relate to quantities of LH1 and LH2 complexes, respectively. Vesicles containing several hundreds of complexes will have an absorption rate:

$$
\gamma\_A = I(\gamma\_1 N\_1 + \gamma\_2 N\_2) \tag{2}
$$

where *N*1(2) is the number of LH1 (LH2) complexes in the vesicle.

#### **2.2 Excitation transfer**

4 Will-be-set-by-IN-TECH

Fig. 2. Schematic representation of the photosynthetic apparatus in the intracytoplasmic membrane of purple bacteria. The reaction center (RC, red) is surrounded by the

likely confined to the intramembrane space, in black.

absorption rate:

*γA* <sup>1</sup> = 

where *N*1(2) is the number of LH1 (LH2) complexes in the vesicle.

light-harvesting complex 1 (LH1, green) to form the LH1+RC complex, which is surrounded by multiple light-harvesting complexes (LH2s) (blue), forming altogether the photosynthetic unit (PSU). Photons are absorbed by LHs and excitation is transferred to the RC initiating a charge (electron-hole) separation. Electrons are shuttled back by the cytochrome *c*<sup>2</sup> charge carrier (blue) from the ubiquinone-cytochrome *bc*<sup>1</sup> complex (yellow) to the RC. The electron transfer across the membrane produces a proton gradient that drives the synthesis of ATP from ADP by the ATPase (orange). Electron *e*− is represented in blue, and quinones *QB*,

source with occupation numbers *n*(*λ*). Normalized to 18 W/m<sup>2</sup> intensity, the rate of photon

Since these rates are normalized to 18 W/m2, the extension to arbitrary light intensity *I* is straightforward. The rate of photon absorption normalized to 1 W/m<sup>2</sup> intensity, will be *<sup>γ</sup>*1(2) = <sup>1</sup>(0.55)s−1. From now on, subindexes 1 and 2 relate to quantities of LH1 and LH2 complexes, respectively. Vesicles containing several hundreds of complexes will have an

*<sup>n</sup>*(*λ*)*σ*LH1(*λ*)*d<sup>λ</sup>* <sup>=</sup> 18s<sup>−</sup>1. (1)

*γ<sup>A</sup>* = *I*(*γ*1*N*<sup>1</sup> + *γ*2*N*2) (2)

<sup>2</sup> <sup>=</sup> 10s−1.

absorption for circular LH1 complexes in *Rb. sphaeroides* (Geyer & Heims, 2006):

The same procedure applied to LH2 complexes, yields a photon capture rate of *γ<sup>A</sup>*

Excitation transfer happens through Coulomb interaction of electrons, excited to the Q*y* electronic transition in chromophores. The interaction energy can be formally written (van Amerongen et al., 2000):

$$V\_{ij} = \frac{1}{2} \sum\_{m,n,p,q} \sum\_{\sigma,\sigma'} \langle \phi\_m \phi\_n | V | \phi\_p \phi\_q \rangle \langle c\_{m\sigma}^+ c\_{p\sigma'}^+ c\_{q\sigma} c\_{n\sigma'} \tag{3}$$

where *c*<sup>+</sup> *<sup>m</sup>σ*, *cn<sup>σ</sup>*� are fermion creation and annihilation operators of electrons with spin *σ* and *σ*� , in the mutually orthogonal atomic orbitals *φ<sup>m</sup>* and *φn*. The overlap �*φmφn*|*V*|*φpφq*� is the Coulomb integral:

$$\langle \phi\_{\rm m} \phi\_{p} | V | \phi\_{\rm n} \phi\_{q} \rangle = \int \int d\vec{r}\_{1} \, d\vec{r}\_{2} \, \phi\_{\rm m}^{\*}(\vec{r}\_{1} - \vec{r}\_{i}) \phi\_{p}(\vec{r}\_{1} - \vec{r}\_{i}) \frac{e^{2}}{|\vec{r}\_{1} - \vec{r}\_{2}|} \phi\_{n}^{\*}(\vec{r}\_{2} - \vec{r}\_{j}) \phi\_{q}(\vec{r}\_{2} - \vec{r}\_{j}) \tag{4}$$

that accounts on inter-molecular exchange contribution when donor and acceptor are at a distance comparable to the extent of the molecules, and the direct Coulomb contribution for an electron that makes a transition between *φm* and *φp*, having both a finite value near the position of the donnor *r*<sup>1</sup> ≈ *rD*, while another electron is excited between *φ<sup>n</sup>* and *φ<sup>q</sup>* at the acceptor coordinate *r*<sup>2</sup> ≈ *rA*. In this latter situation, a commonly used framework concerns a tight-binding Hamiltonian, where details in the specific molecular orbitals |*φn*� involving mainly the *Qy* orbitals is left aside, and emphasis relies on occupation with a single index labeling an electronic state |*i*� concerning occupation in a given chromophore. The Hamiltonian *H* in the chromophore site basis |*i*�,

$$H = \epsilon \sum\_{i} |i\rangle\langle i| + \sum\_{i,j} V\_{ij} |i\rangle\langle j| \tag{5}$$

has diagonal elements �*i*|*H*|*i*� = *�*, concerning the energy of the excitation, usually measured from the ground electronic state. Neighboring chromophores are too close to neglect their charge distribution and its interaction is determined such that the effective Hamiltonian spectrum matches the spectrum of an extensive quantum chemistry calculation (Hu et al., 1997). In the LH2 complex, the B850 ring, with nearest neighbors coupling �*i*|*H*|*i* + 1� = 806 cm−<sup>1</sup> or 377 cm <sup>−</sup><sup>1</sup> for chromophores respectively, within or in different neighboring *αβ* units. For next-to-neighboring chromophores the dipole-dipole approximation is usually used,

$$
\langle i|H|j\rangle = \mathcal{V}\_{ij} = \mathcal{C}\left(\frac{\vec{\mu}\_i \cdot \vec{\mu}\_j}{r\_{ij}^3} - \frac{\Im(\vec{r}\_{ij} \cdot \vec{\mu}\_i)(\vec{r}\_{ij} \cdot \vec{\mu}\_j)}{r\_{ij}^5}\right)
$$

$$
\text{with } i \neq j, i = j \neq \pm 1\tag{6}
$$

where *μ<sup>i</sup>* is the dipole moment and *r* is the distance between the interacting dipoles with constants: *�* = 13059 cm−<sup>1</sup> and *C* = 519 044 <sup>3</sup> cm−1. The LH1 complex has a spectrum maximum near 875 nm, with inter-complex distances and nearest neighbor interactions equal to the ones provided in the B850 LH2 ring. The molecular nature of chromophores involve vibrational degrees of freedom that provide a manifold within each electronic states which should be accounted. However, given these parameters within a harvesting complex, intra-complex energy transfer involves sub-picosecond time-scales, that imply relevance of electronic quantum features over the influence of thermalization in the vibrational manifold. If decoherence sources are not important within the time-scale of excitation dynamics on donor

assumed as products of electronic |*φ*� and vibrational |*χ*� molecular states:

*D*)|*χ*(*wD*)��*χ*(*wA*)|*χ*(*w*<sup>∗</sup>

*<sup>h</sup>*¯ <sup>|</sup>*UDA*<sup>|</sup>

where *UDA* = �*φD*∗*ψA*|*VDA*|*ψDψA*<sup>∗</sup> �. Using the above approximation, the expression (8) now including the overlap between vibrational levels can be cast in a more illustrative form:

Energy Conversion in Purple Bacteria Photosynthesis 49

2 

Here, *GD*(*E*) and *GA*(*E*) are often called the Franck-Condon weighted and thermally averaged

*<sup>D</sup>*) exp(−*w*<sup>∗</sup>

with equal expression for acceptor molecule by replacement *D* → *A*. Förster showed (Förster, 1965) that these distributions are related to extinction coefficient *�*(*E*) and fluorescence

where *<sup>N</sup>*<sup>0</sup> <sup>=</sup> 6.022 <sup>×</sup> 1020 is the number of molecules per mol per cm3, *<sup>n</sup>* is the refractive index of the molecule sample, *c* the speed of light, and *τ*<sup>0</sup> the mean fluorescence time of the donnor

*dE �A*(*E*)/*E*, <sup>ˆ</sup>*fD*(*E*) = *fD*(*E*)

*<sup>D</sup>* − *E* and *w*<sup>∗</sup>

*dE �*ˆ*A*(*E*) <sup>ˆ</sup>

*fD*(*E*)

exp(−*Ek*/*kBT*)|*φk*��*φk*| (14)

exp(−*Ek*/*kBT*)�*φ<sup>k</sup>* |*VDA*|*φp*�

*Vij* (15)

<sup>2</sup>*E GA*(*E*), *fD*(*E*) = 3¯*h*4*c*3*τ*<sup>0</sup>

*D*)|*χ*(*wD*)��*χ*(*wA*)|*χ*(*w*<sup>∗</sup>

*D*/*kBT*)|�*χ*(*w*<sup>∗</sup>

<sup>4</sup>*<sup>n</sup>* <sup>|</sup>*μD*<sup>|</sup>

*Z*∗ *D* *<sup>A</sup>*)�

*<sup>A</sup>*)� (9)

*dE GD*(*E*)*GA*(*E*) (10)

*D*)|*χ*(*wD*)�|<sup>2</sup>

<sup>2</sup>*E*<sup>3</sup>*GD*(*E*) (11)

*dE fD*(*E*)/*E*<sup>3</sup> (12)

*<sup>A</sup>* = −*EA*<sup>0</sup> + *wA* + *E* schematically

*<sup>E</sup>*<sup>4</sup> . (13)

*<sup>U</sup>*<sup>ˆ</sup> *DA* ≈ �*φD*∗*φA*|*VDA*|*φDφA*<sup>∗</sup> ��*χ*(*w*<sup>∗</sup>

*kDA* <sup>=</sup> <sup>2</sup>*<sup>π</sup>*


*<sup>A</sup>*(*E*) = *�A*(*E*)

*kDA* <sup>=</sup> <sup>2</sup>*<sup>π</sup>*

calculation in eq.(9) involves an statistical thermal mixture, explicitly:

<sup>=</sup> <sup>1</sup>

Tr {·} <sup>∑</sup> *k*

*<sup>ρ</sup>* <sup>=</sup> <sup>1</sup>

*UDA* <sup>=</sup> Tr{*ρVDA*} <sup>=</sup> <sup>1</sup>

*<sup>h</sup>*¯ <sup>|</sup>*UDA*<sup>|</sup>

2 

Therefore, whenever fluorescence and absorption spectra are available, an estimate for the

Thermalization occurs firstly in the vibrational manifold of the electronic states involved. Due to the greater energy gap of electronic transitions compared with the one of vibrational nature, on a longer time-scale thermalization also occurs in excitonic states. Accordingly, the

where Tr {·} is trace of the numerator operator, used to normalize the state. Hence, in a

*k* ∑ *p*

*k*,*p*,*i*,*j*

*ak*,*ia*<sup>∗</sup> *p*,*j*

<sup>∑</sup>*<sup>k</sup>* exp(−*Ek*/*kBT*) <sup>∑</sup>

<sup>∑</sup>*<sup>k</sup>* exp(−*Ek*/*kBT*) <sup>∑</sup>

≈ *UDA*�*χ*(*w*<sup>∗</sup>

*ED*<sup>∗</sup> 0 *dw*∗ *D g*∗ *D*(*w*<sup>∗</sup>

spectrum *fD*(*E*) of direct experimental verification:

3 ln 10¯*h*<sup>2</sup> *nc*

combined density of states. Explicitly:

*GD*(*E*) =

*�*(*E*) = <sup>2</sup>*πN*<sup>0</sup>

excited state. For normalized spectra

*�*ˆ

and from the relations *wD* = *ED*<sup>0</sup><sup>∗</sup> + *w*<sup>∗</sup>

presented in Fig.3, the Förster rate is cast

excitation transfer rate can be calculated.

straightforward fashion

Fig. 3. Energy level scheme of donor and acceptor molecules. Although the zero phonon line might be different between both, energy conservation on the transfer applies due to vibrational levels.

or acceptor aggregates, they can be known from the eigenvector problem *H*|*φk*� = *Ek*|*φk*� whose solution provides excitonic fully delocalized states

$$|\phi\_k\rangle = \sum\_{n} a\_{k,i} |i\rangle$$

$$\langle \mathcal{T} |$$

considered to best describe the B850 ring. A slightly less interaction strength allows thermalization through the vibrational manifold in comparable time-scales, degrading delocalization in the B800 ring over 2-3 pigments, however, able to improve robustness of B800→B850 energy transfer (Jang et al., 2004).

The fact that states involved in excitation transfer in molecules include the vibrational manifold, makes enormous the Hilbert space over which the sums of eq.(3) should be formally performed. When the transfer occurs with chromophores that belong to different complexes, say inter-complex energy transfer, rapid thermalization out stands direct Coulomb mechanism, and no defined phase relationship between donor and acceptor electronic states is expected. The hermitian nature of excitonic exchange is replaced due to decoherence, by a rate to describe electronic excitation transfer within thermalization in the vibrational manifold. According to Fermi's golden rule, adapted to the vibrational continuum, the rate of transfer is given by (Hu et al., 2002):

$$k\_{\rm DA} = \frac{2\pi}{\hbar} \int dE \int\_{E\_{A\_0}} dw\_A \int\_{E\_{D\_0^\*}} dw\_D^\* \frac{g\_D^\*(w\_D^\*) \exp(-w\_D^\*/k\_B T)}{Z\_D^\*} \frac{g\_A(w\_A) \exp(-w\_A/k\_B T)}{Z\_A} |\tilde{M}\_{\rm DA}|^2,\tag{8}$$

interpreted as a the sum of Coulomb contributions from electrons at donnor (D) and acceptor (A) aggregates in ground or excited states (these latter dennoted by <sup>∗</sup>), *U*˜ *DA* = �Ψ*D*∗Ψ*A*|*VDA*|Ψ*D*Ψ*A*<sup>∗</sup> �, weighted by Boltzmann factor (*kB* is Botlzmann constant) and vibrational manifold multiplicity (*gD*(*A*)(*wD*(*A*)) for donnor (acceptor)) at the electronic energies *wD*(*A*) measured from the donnor (acceptor) zero phonon lines *ED*(*A*)<sup>0</sup> . With the Born-Oppenheimer approximation (Hu et al., 2002; van Amerongen et al., 2000) |Ψ� is 6 Will-be-set-by-IN-TECH

Fig. 3. Energy level scheme of donor and acceptor molecules. Although the zero phonon line

or acceptor aggregates, they can be known from the eigenvector problem *H*|*φk*� = *Ek*|*φk*�

considered to best describe the B850 ring. A slightly less interaction strength allows thermalization through the vibrational manifold in comparable time-scales, degrading delocalization in the B800 ring over 2-3 pigments, however, able to improve robustness of

The fact that states involved in excitation transfer in molecules include the vibrational manifold, makes enormous the Hilbert space over which the sums of eq.(3) should be formally performed. When the transfer occurs with chromophores that belong to different complexes, say inter-complex energy transfer, rapid thermalization out stands direct Coulomb mechanism, and no defined phase relationship between donor and acceptor electronic states is expected. The hermitian nature of excitonic exchange is replaced due to decoherence, by a rate to describe electronic excitation transfer within thermalization in the vibrational manifold. According to Fermi's golden rule, adapted to the vibrational continuum, the rate of transfer is

*<sup>D</sup>*) exp(−*w*<sup>∗</sup>

*Z*∗ *D*

interpreted as a the sum of Coulomb contributions from electrons at donnor (D) and acceptor (A) aggregates in ground or excited states (these latter dennoted by <sup>∗</sup>), *U*˜ *DA* = �Ψ*D*∗Ψ*A*|*VDA*|Ψ*D*Ψ*A*<sup>∗</sup> �, weighted by Boltzmann factor (*kB* is Botlzmann constant) and vibrational manifold multiplicity (*gD*(*A*)(*wD*(*A*)) for donnor (acceptor)) at the electronic

the Born-Oppenheimer approximation (Hu et al., 2002; van Amerongen et al., 2000) |Ψ� is

energies *wD*(*A*) measured from the donnor (acceptor) zero phonon lines *ED*(*A*)<sup>0</sup>

*<sup>D</sup>*/*kBT*)

*ak*,*i*|*i*� (7)

*gA*(*wA*) exp(−*wA*/*kBT*) *ZA*

<sup>|</sup>*U*˜ *DA*<sup>|</sup> 2

. With

(8)

<sup>|</sup>*φk*� <sup>=</sup> <sup>∑</sup>*<sup>n</sup>*

might be different between both, energy conservation on the transfer applies due to

whose solution provides excitonic fully delocalized states

B800→B850 energy transfer (Jang et al., 2004).

vibrational levels.

given by (Hu et al., 2002):

*kDA* <sup>=</sup> <sup>2</sup>*<sup>π</sup> h*¯ *dE EA*<sup>0</sup> *dwA ED*<sup>∗</sup> 0 *dw*∗ *D g*∗ *D*(*w*<sup>∗</sup> assumed as products of electronic |*φ*� and vibrational |*χ*� molecular states:

$$\begin{split} \mathcal{U}\_{DA} &\approx \langle \phi\_{\mathcal{D}^\*} \phi\_A | V\_{DA} | \phi\_{\mathcal{D}} \phi\_{A^\*} \rangle \times \langle \chi(w\_D^\*) | \chi(w\_D) \rangle \langle \chi(w\_A) | \chi(w\_A^\*) \rangle \\ &\approx \mathcal{U}\_{DA} \langle \chi(w\_D^\*) | \chi(w\_D) \rangle \langle \chi(w\_A) | \chi(w\_A^\*) \rangle \end{split} \tag{9}$$

where *UDA* = �*φD*∗*ψA*|*VDA*|*ψDψA*<sup>∗</sup> �. Using the above approximation, the expression (8) now including the overlap between vibrational levels can be cast in a more illustrative form:

$$k\_{DA} = \frac{2\pi}{\hbar} |\mathcal{U}\_{DA}|^2 \int dE \, \mathcal{G}\_D(E) \mathcal{G}\_A(E) \tag{10}$$

Here, *GD*(*E*) and *GA*(*E*) are often called the Franck-Condon weighted and thermally averaged combined density of states. Explicitly:

$$G\_D(E) = \int\_{E\_{D\_0^\*}} dw\_D^\* \frac{g\_D^\*(w\_D^\*) \exp(-w\_D^\*/k\_B T) |\langle \chi(w\_D^\*)| \chi(w\_D) \rangle|^2}{Z\_D^\*}$$

with equal expression for acceptor molecule by replacement *D* → *A*. Förster showed (Förster, 1965) that these distributions are related to extinction coefficient *�*(*E*) and fluorescence spectrum *fD*(*E*) of direct experimental verification:

$$\epsilon(E) = \frac{2\pi N\_0}{3\ln 10\hbar^2 nc} |\mu\_A|^2 E \,\mathrm{G}\_A(E), \quad f\_D(E) = \frac{3\hbar^4 c^3 \tau\_0}{4n} |\mu\_D|^2 E^3 \mathrm{G}\_D(E) \tag{11}$$

where *<sup>N</sup>*<sup>0</sup> <sup>=</sup> 6.022 <sup>×</sup> 1020 is the number of molecules per mol per cm3, *<sup>n</sup>* is the refractive index of the molecule sample, *c* the speed of light, and *τ*<sup>0</sup> the mean fluorescence time of the donnor excited state. For normalized spectra

$$
\varepsilon\_A(E) = \frac{\varepsilon\_A(E)}{\int dE \, \varepsilon\_A(E)/E}, \quad f\_D(E) = \frac{f\_D(E)}{\int dE \, f\_D(E)/E^3} \tag{12}
$$

and from the relations *wD* = *ED*<sup>0</sup><sup>∗</sup> + *w*<sup>∗</sup> *<sup>D</sup>* − *E* and *w*<sup>∗</sup> *<sup>A</sup>* = −*EA*<sup>0</sup> + *wA* + *E* schematically presented in Fig.3, the Förster rate is cast

$$k\_{DA} = \frac{2\pi}{\hbar} |\mathcal{U}\_{DA}|^2 \int d\mathbf{E} \frac{\hat{\varepsilon}\_A(\mathbf{E}) \hat{f}\_D(\mathbf{E})}{E^4}. \tag{13}$$

Therefore, whenever fluorescence and absorption spectra are available, an estimate for the excitation transfer rate can be calculated.

Thermalization occurs firstly in the vibrational manifold of the electronic states involved. Due to the greater energy gap of electronic transitions compared with the one of vibrational nature, on a longer time-scale thermalization also occurs in excitonic states. Accordingly, the calculation in eq.(9) involves an statistical thermal mixture, explicitly:

$$\rho = \frac{1}{\text{Tr}\left\{ \cdot \right\}} \sum\_{k} \exp(-E\_k/k\_B T) |\phi\_k\rangle\langle\phi\_k| \tag{14}$$

where Tr {·} is trace of the numerator operator, used to normalize the state. Hence, in a straightforward fashion

$$\langle LQA = \text{Tr}\{\rho V\_{DA}\} = \frac{1}{\sum\_{k} \exp(-E\_k/k\_B T)} \sum\_{k} \sum\_{p} \exp(-E\_k/k\_B T) \langle \phi\_k | V\_{DA} | \phi\_p \rangle$$

$$= \frac{1}{\sum\_{k} \exp(-E\_k/k\_B T)} \sum\_{k, p, i, j} a\_{k, i} a\_{p, j}^\* V\_{ij} \tag{15}$$

from\ to LH1 LH2 RC LH1 20.0/N.A. 15.5/N.A. 15.8/30-50 LH2 7.7/3.3 10.0/N.A. N.A RC 8.1/8 N.A N.A

Energy Conversion in Purple Bacteria Photosynthesis 51

Table 1. Theoretical estimation/experimental evidence of inter-complex transfer times in

Fig. 4. Dissipation mechanisms. In (a), the electron de-excites due to its interaction with the quantized electromagnetic vacuum field through a fluorescent photon. In (b) internal conversion mechanism, where the vibrational levels overlap induces a transition between electronic excited and ground states. Dissipation overcomes when thermal equilibrium is

Excitation in chromophores might be dissipated by two main mechanisms. The first is fluorescence, where the electronic excited state has a finite lifetime on the nanosecond time-scale, due to its interaction with the electromagnetic vacuum (Scully & Zubairy, 1997). The second is internal conversion, where the electronic energy is transferred to vibrational

Within the Born-Oppenheimer approximation, the molecular state Ψ, can be decomposed into purely electronic *φ* and (nuclear) vibrational *χ* states. The transition probability between initial state Ψ*<sup>i</sup>* and final state Ψ*<sup>f</sup>* , is proportional to �Ψ*i*|*H*|Ψ*f*� ∝ �*χi*|*χf*�. Note that *χ<sup>i</sup>* (*χf*) are vibrational levels in the ground (excited) electronic state manifold (see Fig.4). If the energy difference is small, and the overlap between vibrational levels of different electronic states is appreciable, the excitation can be transferred from the excited electronic state, to an excited vibrational level in the ground electronic state. This overlap increases with decreasing energy difference between electronic states. As higher electronic levels have smaller energy difference among their zero phonon lines, internal conversion process is more probable the higher energy electronic states have. Fluorescence and internal conversion between first excited singlet and ground electronic states, induce dissipation in a range of hundreds of picoseconds and a few nanoseconds. Numerical simulations are performed with a dissipation time including both

fluorescence and internal conversion of 1/*γ<sup>D</sup>* = 1 ns, also used in (Ritz et al., 2001).

From the LH1 complex excitation reaches the RC, specifically the special pair (SP) dimer. The excitation can be transferred back to its surrounding LH1, or initiate a chain of ionizations

reached in the vibrational manifold of electronic ground state.

picoseconds. N.A are not available data.

**2.3 Dissipation**

degrees of freedom.

**2.4 Special pair (SP) ionization**

where the element �*φk*|*VDA*|*φp*� are the elements of interaction among exciton states in molecules on different complexes and use is made of the individual contributions of excitonic states in chromophore's site basis eq.(7).

Summarizing, excitation transfer occurs through induced dipole transfer, among BChls transitions. The common inter-complex BChl distances 20-100 (Bahatyrova et al., 2004; Scheuring & Sturgis, 2005) cause excitation transfer to arise through the Coulomb interaction on the picosecond time-scale (Hu et al., 2002), while vibrational dephasing destroys coherences within a few hundred femtoseconds (Lee et al., 2007; Panitchayangkoona et al., 2010). As noted, the Coulomb interaction as dephasing occurs, makes the donor and acceptor phase become uncorrelated pointing into a classical rate behavior. Transfer rate measures from pump-probe experiments agree with the just outlined generalized Förster calculated rates (Hu et al., 2002), assuming intra-complex delocalization along thermodynamical equilibrium. LH2→LH2 transfer has not been measured experimentally, although an estimate of *t*<sup>22</sup> = 10 ps has been calculated (Hu et al., 2002). LH2→ LH1 transfer has been measured for *R. Sphaeroides* as *t*<sup>21</sup> = 3.3ps (Hess et al., 1995). Due to formation of excitonic states (Jang et al., 2007), back-transfer LH1→ LH2 is enhanced as compared to the canonical equilibrium rate for a two-level system, up to a value of *t*<sup>12</sup> = 15.5ps. The LH1→LH1 mean transfer time *t*<sup>11</sup> has not been measured, but the just mentioned generalized Förster calculation (Ritz et al., 2001) has reported an estimated mean time *t*<sup>11</sup> of 20 ps. LH1→ RC transfer occurs due to ring symmetry breaking through optically forbidden (within ring symmetry) second and third lowest exciton lying states (Hu et al., 1997), as suggested by agreement with the experimental transfer time of 35-37 ps at 77 K (Visscher et al., 1989; 1991). Increased spectral overlap at room temperature improves the transfer time to *t*1,*RC* = 25 ps (van Grondelle et al., 1994). A photo-protective design makes the back-transfer from an RC's fully populated lowest exciton state to higher-lying LH1 states occur in a calculated time of *tRC*,1 =8.1 ps (Hu et al., 1997), close to the experimentally measured 7-9 ps estimated from decay kinetics after RC excitation (Timpmann et al., 1993).

Table 1 shows the results of mean transfer times presented in Ref.(Ritz et al., 2001) through the above mentioned calculation, compared with the experimental evidence restricted to different complex kind from the spectral resolution requirement of pump-probe spectroscopy. Since LH1↔ LH1 and LH2↔ LH2 transfer steps involve equal energy transitions, no experimental evidence is available regarding the rate at which these transitions occur. The experimentally determined B800→ B850 rate was 1/700fs (Shreve et al., 1991). The inter-complex transfer rate between LH2→ LH1 have been determined experimentally to be 1/3.3ps (Hess et al., 1995). Experimentally, LH1↔ RC forward transfer rate ranges between 1/50ps and 1/35ps, while back-transfer rate ranges between 1/12ps and 1/8ps (Timpmann et al., 1995; 1993; Visscher et al., 1989). It is interesting to note that exists a two fold difference in the experimental and theoretical determined LH2→LH1, ascribed to BChl*a* Q*<sup>y</sup>* dipole moment underestimation. It is assumed for theoretical calculation a value of 6.3 Debye, while a greater BChl*a* Q*y* dipole moment in Prostecochloris aestuarii (not a purple bacterium) of 7.7 Debye has been determined (Ritz et al., 2001). On the other hand, LH1→RC theoretical calculation gives a greater value for tranfer rate, thought to arise due to an overestimate of LH1 exciton delocalization (Ritz et al., 2001). This rate decreases when delocalization is assumed over fewer BChl's, therefore, further research is needed to understand the effect of decoherence sources (static inhomogeneities and dynamical disorder due to thermal fluctuations) on the delocalization length.


Table 1. Theoretical estimation/experimental evidence of inter-complex transfer times in picoseconds. N.A are not available data.

Fig. 4. Dissipation mechanisms. In (a), the electron de-excites due to its interaction with the quantized electromagnetic vacuum field through a fluorescent photon. In (b) internal conversion mechanism, where the vibrational levels overlap induces a transition between electronic excited and ground states. Dissipation overcomes when thermal equilibrium is reached in the vibrational manifold of electronic ground state.

#### **2.3 Dissipation**

8 Will-be-set-by-IN-TECH

where the element �*φk*|*VDA*|*φp*� are the elements of interaction among exciton states in molecules on different complexes and use is made of the individual contributions of excitonic

Summarizing, excitation transfer occurs through induced dipole transfer, among BChls transitions. The common inter-complex BChl distances 20-100 (Bahatyrova et al., 2004; Scheuring & Sturgis, 2005) cause excitation transfer to arise through the Coulomb interaction on the picosecond time-scale (Hu et al., 2002), while vibrational dephasing destroys coherences within a few hundred femtoseconds (Lee et al., 2007; Panitchayangkoona et al., 2010). As noted, the Coulomb interaction as dephasing occurs, makes the donor and acceptor phase become uncorrelated pointing into a classical rate behavior. Transfer rate measures from pump-probe experiments agree with the just outlined generalized Förster calculated rates (Hu et al., 2002), assuming intra-complex delocalization along thermodynamical equilibrium. LH2→LH2 transfer has not been measured experimentally, although an estimate of *t*<sup>22</sup> = 10 ps has been calculated (Hu et al., 2002). LH2→ LH1 transfer has been measured for *R. Sphaeroides* as *t*<sup>21</sup> = 3.3ps (Hess et al., 1995). Due to formation of excitonic states (Jang et al., 2007), back-transfer LH1→ LH2 is enhanced as compared to the canonical equilibrium rate for a two-level system, up to a value of *t*<sup>12</sup> = 15.5ps. The LH1→LH1 mean transfer time *t*<sup>11</sup> has not been measured, but the just mentioned generalized Förster calculation (Ritz et al., 2001) has reported an estimated mean time *t*<sup>11</sup> of 20 ps. LH1→ RC transfer occurs due to ring symmetry breaking through optically forbidden (within ring symmetry) second and third lowest exciton lying states (Hu et al., 1997), as suggested by agreement with the experimental transfer time of 35-37 ps at 77 K (Visscher et al., 1989; 1991). Increased spectral overlap at room temperature improves the transfer time to *t*1,*RC* = 25 ps (van Grondelle et al., 1994). A photo-protective design makes the back-transfer from an RC's fully populated lowest exciton state to higher-lying LH1 states occur in a calculated time of *tRC*,1 =8.1 ps (Hu et al., 1997), close to the experimentally measured 7-9 ps estimated from decay kinetics after RC excitation

Table 1 shows the results of mean transfer times presented in Ref.(Ritz et al., 2001) through the above mentioned calculation, compared with the experimental evidence restricted to different complex kind from the spectral resolution requirement of pump-probe spectroscopy. Since LH1↔ LH1 and LH2↔ LH2 transfer steps involve equal energy transitions, no experimental evidence is available regarding the rate at which these transitions occur. The experimentally determined B800→ B850 rate was 1/700fs (Shreve et al., 1991). The inter-complex transfer rate between LH2→ LH1 have been determined experimentally to be 1/3.3ps (Hess et al., 1995). Experimentally, LH1↔ RC forward transfer rate ranges between 1/50ps and 1/35ps, while back-transfer rate ranges between 1/12ps and 1/8ps (Timpmann et al., 1995; 1993; Visscher et al., 1989). It is interesting to note that exists a two fold difference in the experimental and theoretical determined LH2→LH1, ascribed to BChl*a* Q*<sup>y</sup>* dipole moment underestimation. It is assumed for theoretical calculation a value of 6.3 Debye, while a greater BChl*a* Q*y* dipole moment in Prostecochloris aestuarii (not a purple bacterium) of 7.7 Debye has been determined (Ritz et al., 2001). On the other hand, LH1→RC theoretical calculation gives a greater value for tranfer rate, thought to arise due to an overestimate of LH1 exciton delocalization (Ritz et al., 2001). This rate decreases when delocalization is assumed over fewer BChl's, therefore, further research is needed to understand the effect of decoherence sources (static inhomogeneities and dynamical disorder due to thermal fluctuations) on the

states in chromophore's site basis eq.(7).

(Timpmann et al., 1993).

delocalization length.

Excitation in chromophores might be dissipated by two main mechanisms. The first is fluorescence, where the electronic excited state has a finite lifetime on the nanosecond time-scale, due to its interaction with the electromagnetic vacuum (Scully & Zubairy, 1997). The second is internal conversion, where the electronic energy is transferred to vibrational degrees of freedom.

Within the Born-Oppenheimer approximation, the molecular state Ψ, can be decomposed into purely electronic *φ* and (nuclear) vibrational *χ* states. The transition probability between initial state Ψ*<sup>i</sup>* and final state Ψ*<sup>f</sup>* , is proportional to �Ψ*i*|*H*|Ψ*f*� ∝ �*χi*|*χf*�. Note that *χ<sup>i</sup>* (*χf*) are vibrational levels in the ground (excited) electronic state manifold (see Fig.4). If the energy difference is small, and the overlap between vibrational levels of different electronic states is appreciable, the excitation can be transferred from the excited electronic state, to an excited vibrational level in the ground electronic state. This overlap increases with decreasing energy difference between electronic states. As higher electronic levels have smaller energy difference among their zero phonon lines, internal conversion process is more probable the higher energy electronic states have. Fluorescence and internal conversion between first excited singlet and ground electronic states, induce dissipation in a range of hundreds of picoseconds and a few nanoseconds. Numerical simulations are performed with a dissipation time including both fluorescence and internal conversion of 1/*γ<sup>D</sup>* = 1 ns, also used in (Ritz et al., 2001).

#### **2.4 Special pair (SP) ionization**

From the LH1 complex excitation reaches the RC, specifically the special pair (SP) dimer. The excitation can be transferred back to its surrounding LH1, or initiate a chain of ionizations

Fig. 5. Schematic of the biomolecular photosynthetic machinery in purple bacteria, together with relevant inter-complex mean transfer times *tij*, dissipation rate *γD*, and normalized light

Energy Conversion in Purple Bacteria Photosynthesis 53

The theoretical framework used to describe the excitation transfer must be built around the experimental (if available) and theoretical parameters just outlined. Remind that the thermalization process occurs faster than inter-complex energy transfer, and provides the support to rely in a classical hopping process, since phase information is lost well within the time frame implied by direct Coulomb coupling. Accordingly, we base our analysis on a classical random walk for excitation dynamics along the full vesicle, by considering a collective state with *N* = *N*<sup>2</sup> + 2*N*<sup>1</sup> sites – resulting from *N*<sup>2</sup> LH2s, *N*<sup>1</sup> LH1s and hence *N*<sup>1</sup> RC complexes in the vesicle. The state vector *ρ* = (*ρ*1, *ρ*2, ..., *ρM*) has in each element the probability of occupation of a collective state comprising several excitations. If a single excitation is allowed in each complex, both excited and ground states of any complex should

*N*

excitation that wanders in the whole network of complexes is allowed, a site basis can be used where each element of the state vector gives the probability of residence in the respective complex, and reduces the state vector size to *M* = *N*. In either case the state vector time

> *M* ∑ *j*=1

where *Gi*,*<sup>j</sup>* is the transition rate from a collective state or site *i*– whether many or a single excitation are accounted, respectively – to another collective state or site *j*. Since the transfer rates do not depend on time, this yields a formal solution *ρ*(*t*) = ˜*eGtρ*(0). However, the required framework depends on exciton abundance within the whole chromatophore at the

*∂tρi*(*t*) =

= 2*N*. On the other hand, if only one

*Gi*,*jρj*(*t*). (16)

be accounted and the state space size is *<sup>M</sup>* <sup>=</sup> <sup>2</sup> <sup>×</sup> <sup>2</sup> <sup>×</sup> 2...

evolution obeys a master equation

regime of interest.

intensity rate *γ*1(2)

**3.1 Model**

along the A branch, probably, due to a tyrosine residue strategically positioned instead of a phenylalanine present in the B branch (Lia et al., 1993). Once the special pair is excited, it has been determined experimentally (Fleming et al., 1988) that takes 3-4 ps for the special pair to ionize and produce a reduced bacteriopheophytin, *H*− *<sup>A</sup>* , in a reaction *SP*<sup>∗</sup> <sup>→</sup> *SP*+*H*<sup>−</sup> *A* . This reaction initiates an electron hop, to a quinone *QA* in about 200 ps, and to a second quinone, *QB* if available. Initially, the ionized quinol *Q*<sup>+</sup> *<sup>B</sup>* captures an introcytoplasmic proton and produces hydroxiquinol *QBH*, which after a second ionization that produces *QBH*<sup>+</sup> to form quinol *QBH*2. After any SP ionization a neutrality restablishment is required, provided by the cytochrome *cyt* charge carrier. After SP ionization, the cytochrome diffuses from the bc1 complex to a RC in order to replenish its neutrality SP<sup>+</sup> <sup>→</sup>SP, within several microseconds (Milano et al., 2003). The first electron transfer step *<sup>P</sup>*<sup>∗</sup> <sup>→</sup> *<sup>P</sup>*<sup>+</sup> occurs in the RC within *<sup>t</sup>*<sup>+</sup> <sup>=</sup><sup>3</sup> ps, used for quinol (*QBH*2) production (Hu et al., 2002).

#### **2.5 Quinone-quinol cycling**

The RC cycling dynamics also involves undocking of *QBH*<sup>2</sup> from the RC due to lower affinity among RC and this new product. Quinol starts a migration to the *bc*1 complex where enables the ionization of the cytochrome *cyt* charge carrier, while a new quinone *QB* molecule docks into the RC. The time before quinol unbinds, and a new *QB* is available, has been reported within milliseconds (Osváth & Maróti, 1997) to highlight quinol removal o as the rate limiting step (Osváth & Maróti, 1997) if compared to special pair restablishment.

Even though it has been reported that excitation dynamics change as a function of the RCs state (Borisov et al., 1985; Comayras et al., 2005), at a first glance the several orders of magnitude difference among the picosecond transfer, the nanosecond dissipation and the millisecond RC cycling, seems to disregard important effects due to these mechanisms' interplay. However, the quinol-quinone dynamics leaves the RC unable to promote further quinol production and eventually enhances the influence of dissipation of a wandering excitation, evident when none RC is available and the unique fate of any excitation is to be dissipated.

Interestingly, the quinone-quinol mechanism has been well established and thought to be of priority on adaptations of bacteria, that seem to respond to its dynamics. For instance, an observed trend for membranes to form clusters of same complex type (Scheuring, Rigaud & Sturgis, 2004) seems to affect diffusion of quinones, enhanced when, due to higher mobility of LH1s, left void spaces help travel quinones to the periplasm. Negligible mobility of LH2s in their domains, would restrict metabolically active quinones to LH1 domains (Scheuring & Sturgis, 2006). Easier diffusion of quinones, quinol and cytochromes promotes higher availability of charge carriers in RC domains under LLI conditions, increasing the rate at which RCs can cycle. The RC cycling dynamics and its connection to the membranes performance has been accounted in (Caycedo-Soler et al., 2010a;b) in a quantitative calculation to understand the effect of core clustering and stoichiometry variation in the RC supply or in the efficiency of the membranes from experimentally obtained Atomic Force Microscopy images, to be presented in this chapter.

#### **3. Exciton kinetics**

Figure 5 summarizes the relevant biomolecular complexes in purple bacteria *Rsp. Photometricum* (Scheuring, Rigaud & Sturgis, 2004), together with experimental– theoretical if the former are not available– timescales governing the excitation kinetics: absorption and transfer; and reaction center dynamics: quinol removal.

Fig. 5. Schematic of the biomolecular photosynthetic machinery in purple bacteria, together with relevant inter-complex mean transfer times *tij*, dissipation rate *γD*, and normalized light intensity rate *γ*1(2)

#### **3.1 Model**

10 Will-be-set-by-IN-TECH

along the A branch, probably, due to a tyrosine residue strategically positioned instead of a phenylalanine present in the B branch (Lia et al., 1993). Once the special pair is excited, it has been determined experimentally (Fleming et al., 1988) that takes 3-4 ps for the special

This reaction initiates an electron hop, to a quinone *QA* in about 200 ps, and to a second

and produces hydroxiquinol *QBH*, which after a second ionization that produces *QBH*<sup>+</sup> to form quinol *QBH*2. After any SP ionization a neutrality restablishment is required, provided by the cytochrome *cyt* charge carrier. After SP ionization, the cytochrome diffuses from the bc1 complex to a RC in order to replenish its neutrality SP<sup>+</sup> <sup>→</sup>SP, within several microseconds (Milano et al., 2003). The first electron transfer step *<sup>P</sup>*<sup>∗</sup> <sup>→</sup> *<sup>P</sup>*<sup>+</sup> occurs in the RC within *<sup>t</sup>*<sup>+</sup> <sup>=</sup><sup>3</sup>

The RC cycling dynamics also involves undocking of *QBH*<sup>2</sup> from the RC due to lower affinity among RC and this new product. Quinol starts a migration to the *bc*1 complex where enables the ionization of the cytochrome *cyt* charge carrier, while a new quinone *QB* molecule docks into the RC. The time before quinol unbinds, and a new *QB* is available, has been reported within milliseconds (Osváth & Maróti, 1997) to highlight quinol removal o as the rate limiting

Even though it has been reported that excitation dynamics change as a function of the RCs state (Borisov et al., 1985; Comayras et al., 2005), at a first glance the several orders of magnitude difference among the picosecond transfer, the nanosecond dissipation and the millisecond RC cycling, seems to disregard important effects due to these mechanisms' interplay. However, the quinol-quinone dynamics leaves the RC unable to promote further quinol production and eventually enhances the influence of dissipation of a wandering excitation, evident when none RC is available and the unique fate of any excitation is to be

Interestingly, the quinone-quinol mechanism has been well established and thought to be of priority on adaptations of bacteria, that seem to respond to its dynamics. For instance, an observed trend for membranes to form clusters of same complex type (Scheuring, Rigaud & Sturgis, 2004) seems to affect diffusion of quinones, enhanced when, due to higher mobility of LH1s, left void spaces help travel quinones to the periplasm. Negligible mobility of LH2s in their domains, would restrict metabolically active quinones to LH1 domains (Scheuring & Sturgis, 2006). Easier diffusion of quinones, quinol and cytochromes promotes higher availability of charge carriers in RC domains under LLI conditions, increasing the rate at which RCs can cycle. The RC cycling dynamics and its connection to the membranes performance has been accounted in (Caycedo-Soler et al., 2010a;b) in a quantitative calculation to understand the effect of core clustering and stoichiometry variation in the RC supply or in the efficiency of the membranes from experimentally obtained Atomic Force Microscopy images, to be presented in this chapter.

Figure 5 summarizes the relevant biomolecular complexes in purple bacteria *Rsp. Photometricum* (Scheuring, Rigaud & Sturgis, 2004), together with experimental– theoretical if the former are not available– timescales governing the excitation kinetics: absorption and

*<sup>A</sup>* , in a reaction *SP*<sup>∗</sup> <sup>→</sup> *SP*+*H*<sup>−</sup>

*<sup>B</sup>* captures an introcytoplasmic proton

*A* .

pair to ionize and produce a reduced bacteriopheophytin, *H*−

quinone, *QB* if available. Initially, the ionized quinol *Q*<sup>+</sup>

ps, used for quinol (*QBH*2) production (Hu et al., 2002).

step (Osváth & Maróti, 1997) if compared to special pair restablishment.

**2.5 Quinone-quinol cycling**

dissipated.

**3. Exciton kinetics**

transfer; and reaction center dynamics: quinol removal.

The theoretical framework used to describe the excitation transfer must be built around the experimental (if available) and theoretical parameters just outlined. Remind that the thermalization process occurs faster than inter-complex energy transfer, and provides the support to rely in a classical hopping process, since phase information is lost well within the time frame implied by direct Coulomb coupling. Accordingly, we base our analysis on a classical random walk for excitation dynamics along the full vesicle, by considering a collective state with *N* = *N*<sup>2</sup> + 2*N*<sup>1</sup> sites – resulting from *N*<sup>2</sup> LH2s, *N*<sup>1</sup> LH1s and hence *N*<sup>1</sup> RC complexes in the vesicle. The state vector *ρ* = (*ρ*1, *ρ*2, ..., *ρM*) has in each element the probability of occupation of a collective state comprising several excitations. If a single excitation is allowed in each complex, both excited and ground states of any complex should be accounted and the state space size is *<sup>M</sup>* <sup>=</sup> <sup>2</sup> <sup>×</sup> <sup>2</sup> <sup>×</sup> 2... *N* = 2*N*. On the other hand, if only one

excitation that wanders in the whole network of complexes is allowed, a site basis can be used where each element of the state vector gives the probability of residence in the respective complex, and reduces the state vector size to *M* = *N*. In either case the state vector time evolution obeys a master equation

$$
\partial\_t \rho\_{\dot{l}}(t) = \sum\_{j=1}^{M} G\_{\dot{l},j} \rho\_{\dot{j}}(t). \tag{16}
$$

where *Gi*,*<sup>j</sup>* is the transition rate from a collective state or site *i*– whether many or a single excitation are accounted, respectively – to another collective state or site *j*. Since the transfer rates do not depend on time, this yields a formal solution *ρ*(*t*) = ˜*eGtρ*(0). However, the required framework depends on exciton abundance within the whole chromatophore at the regime of interest.

<sup>a</sup> <sup>b</sup>

line; (b) is a dotted line; (c) is a dashed line.

**3.2.2 Relative amount of complexes: Stoichiometry**

they will not be taken into account. Populations can be written as:

*<sup>p</sup>*ˆ1(*<sup>t</sup>* <sup>→</sup> <sup>∞</sup>) = *<sup>f</sup>*1(*s*) *<sup>N</sup>*<sup>1</sup>

*<sup>p</sup>*ˆ2(*<sup>t</sup>* <sup>→</sup> <sup>∞</sup>) = *<sup>f</sup>*2(*s*) *<sup>N</sup>*<sup>2</sup>

*N*<sup>1</sup> + *N*<sup>2</sup>

*N*<sup>1</sup> + *N*<sup>2</sup>

<sup>=</sup> *<sup>f</sup>*1(*s*)

<sup>=</sup> *s f*2(*s*)

<sup>1</sup> <sup>+</sup> *<sup>s</sup>* (18)

<sup>1</sup> <sup>+</sup> *<sup>s</sup>* (19)

and architecture.

c

(e)

0 2 4 6 8 10 12 14 t [ps]

0.25 0.3 0.35 0.4

pLH1

ˆ

Fig. 6. Top panel: Three example small network architectures. The bottom panel shows the normalized probabilities for finding an excitation at an RC (see (d)), an LH1 (see (e)), or an LH2 (see (f)). In panels (d)-(f), we represent these architectures as follows: (a) is a continuous

Energy Conversion in Purple Bacteria Photosynthesis 55

somehow pinpoints other mechanisms as the rulers of harvesting membranes conformation

We can also address with use of small architectures the effect of variation in the relative amount of LH1/LH2 complexes, able to change the population of the available states. Fig.7 shows small networks of LH-RC nodes, where the relative amount of LH2 and LH1 complexes quantified by stoichiometry *s* = *N*2/*N*<sup>1</sup> is varied, in order to study the exciton dynamics. In Fig.8(a) the population ratio at stationary state of LHs demonstrate that as stoichiometry *s* becomes greater, the population of LH1s, becomes smaller, since their amount is reduced. It is apparent that RC population is quite small, and although their abundance increases the exciton trend to be found in any RC (Fig.8(b)), generally, excitations will be found in harvesting complexes. The population of LHs should be dependent on the ratio of complexes type. As verified in Fig.8(b), RCs have almost no population, and for the discussion below,

For instance, purple bacteria ecosystem concerns several meters depths, and should be reminded as a low light intensity environment. Within a typical range of 10-100 W/m<sup>2</sup> and a commonly sized chromatophore having ≈ 400 LH complexes, eq.(2) leads to an absorption rate *<sup>γ</sup><sup>A</sup>* <sup>≈</sup>100-1000 s−1, which compared with the dissipation mechanisms (rates of <sup>≈</sup> 109 s−1) imply that an absorption event occurs and then the excitation will be trapped by a RC or become dissipated within a nanosecond, and other excitation will visit the membrane not before some milliseconds have elapsed. However, it is important to remind the nature of thermal light where the possibility of having bunched small or long inter-photon times is greater than evenly spread, with greater deviations from poissonian statistics the grater its mean intensity is. Therefore, regardless of such deviations, under the biological light intensity conditions, the event of two excitations present simultaneously along the membrane will rarely occur and a single excitation model is accurate.

#### **3.2 Small architectures**

Small absorption rates lead to single excitation dynamics in the whole membrane, reducing the size of *ρ*(*t*) to the total number of sites *N*. The probability to have one excitation at a given complex initially, is proportional to its absorption cross section, and can be written as *ρ*(0) = <sup>1</sup> *<sup>γ</sup><sup>A</sup>* (*γ*1, ... *N*<sup>1</sup> , *γ*2, ... *N*<sup>2</sup> , 0, .. *N*<sup>1</sup> ), where subsets correspond to the *N*<sup>1</sup> LH1s, the *N*<sup>2</sup> LH2s and the

*N*<sup>1</sup> RCs respectively.

#### **3.2.1 Complexes arrangement: architecture**

To gain physical insight on the global behavior of the harvesting membrane, our interest lies in the probability to have an excitation at a given complex kind *k* ∈ LH1,LH2 or RC, namely *p*ˆ*k*, given that at least one excitation resides in the network:

$$
\hat{p}\_k(t) = \frac{\rho\_k(t)}{\sum\_{i=1}^N \rho\_i(t)}\ . \tag{17}
$$

The effects that network architecture might have on the model's dynamics, are studied with different arrangements of complexes in small model networks, focusing on architectures which have the same amount of LH1, LH2 and RCs as shown in the top panel of Fig.6(a), (b) and (c). The bottom panel Fig.6 (d)-(e)-(f) shows that *p*ˆ*<sup>k</sup>* values for RC, LH1 and LH2 complexes, respectively. First, it is important to notice that excitations trend is to stay within LH1 complexes, and not in the RC. Fig.6(d) shows that the highest RC population is obtained in configuration (c), followed by configuration (a) and (b) whose ordering relies in the connectedness of LH1s to antenna complexes. Clustering of LH1s will limit the number of links to LH2 complexes, and reduce the probability of RC ionization. For completeness, the probability of occupation in LH1 and LH2 complexes (Figs.6(e) and (f), respectively), shows that increased RC occupation benefits from population imbalance between LH1 enhancement and LH2 reduction. As connections among antenna complexes become more favored, the probability of finding an excitation on antenna complexes will become smaller, while the probability of finding excitations in RCs is enhanced. This preliminary result, illustrates that if the apparent requirement to funnel excitations down to RCs in bacterium were of primary importance, the greatest connectedness of LH1-LH2 complexes should occur in nature as a consequence of millions of years evolution. However, as will be presented, the real trend to form LH1 clusters, reduces its connectedness to antenna LH2 complexes and 12 Will-be-set-by-IN-TECH

For instance, purple bacteria ecosystem concerns several meters depths, and should be reminded as a low light intensity environment. Within a typical range of 10-100 W/m<sup>2</sup> and a commonly sized chromatophore having ≈ 400 LH complexes, eq.(2) leads to an absorption rate *<sup>γ</sup><sup>A</sup>* <sup>≈</sup>100-1000 s−1, which compared with the dissipation mechanisms (rates of <sup>≈</sup> 109 s−1) imply that an absorption event occurs and then the excitation will be trapped by a RC or become dissipated within a nanosecond, and other excitation will visit the membrane not before some milliseconds have elapsed. However, it is important to remind the nature of thermal light where the possibility of having bunched small or long inter-photon times is greater than evenly spread, with greater deviations from poissonian statistics the grater its mean intensity is. Therefore, regardless of such deviations, under the biological light intensity conditions, the event of two excitations present simultaneously along the membrane will

Small absorption rates lead to single excitation dynamics in the whole membrane, reducing the size of *ρ*(*t*) to the total number of sites *N*. The probability to have one excitation at a given complex initially, is proportional to its absorption cross section, and can be written as

To gain physical insight on the global behavior of the harvesting membrane, our interest lies in the probability to have an excitation at a given complex kind *k* ∈ LH1,LH2 or RC, namely

> *<sup>p</sup>*ˆ*k*(*t*) = *<sup>ρ</sup>k*(*t*) ∑*<sup>N</sup>*

The effects that network architecture might have on the model's dynamics, are studied with different arrangements of complexes in small model networks, focusing on architectures which have the same amount of LH1, LH2 and RCs as shown in the top panel of Fig.6(a), (b) and (c). The bottom panel Fig.6 (d)-(e)-(f) shows that *p*ˆ*<sup>k</sup>* values for RC, LH1 and LH2 complexes, respectively. First, it is important to notice that excitations trend is to stay within LH1 complexes, and not in the RC. Fig.6(d) shows that the highest RC population is obtained in configuration (c), followed by configuration (a) and (b) whose ordering relies in the connectedness of LH1s to antenna complexes. Clustering of LH1s will limit the number of links to LH2 complexes, and reduce the probability of RC ionization. For completeness, the probability of occupation in LH1 and LH2 complexes (Figs.6(e) and (f), respectively), shows that increased RC occupation benefits from population imbalance between LH1 enhancement and LH2 reduction. As connections among antenna complexes become more favored, the probability of finding an excitation on antenna complexes will become smaller, while the probability of finding excitations in RCs is enhanced. This preliminary result, illustrates that if the apparent requirement to funnel excitations down to RCs in bacterium were of primary importance, the greatest connectedness of LH1-LH2 complexes should occur in nature as a consequence of millions of years evolution. However, as will be presented, the real trend to form LH1 clusters, reduces its connectedness to antenna LH2 complexes and

), where subsets correspond to the *N*<sup>1</sup> LH1s, the *N*<sup>2</sup> LH2s and the

*<sup>i</sup>*=<sup>1</sup> *<sup>ρ</sup>i*(*t*) . (17)

rarely occur and a single excitation model is accurate.

**3.2 Small architectures**

*<sup>γ</sup><sup>A</sup>* (*γ*1, ... *N*<sup>1</sup>

*N*<sup>1</sup> RCs respectively.

, *γ*2, ... *N*<sup>2</sup>

**3.2.1 Complexes arrangement: architecture**

, 0, .. *N*<sup>1</sup>

*p*ˆ*k*, given that at least one excitation resides in the network:

*ρ*(0) = <sup>1</sup>

Fig. 6. Top panel: Three example small network architectures. The bottom panel shows the normalized probabilities for finding an excitation at an RC (see (d)), an LH1 (see (e)), or an LH2 (see (f)). In panels (d)-(f), we represent these architectures as follows: (a) is a continuous line; (b) is a dotted line; (c) is a dashed line.

somehow pinpoints other mechanisms as the rulers of harvesting membranes conformation and architecture.

#### **3.2.2 Relative amount of complexes: Stoichiometry**

We can also address with use of small architectures the effect of variation in the relative amount of LH1/LH2 complexes, able to change the population of the available states. Fig.7 shows small networks of LH-RC nodes, where the relative amount of LH2 and LH1 complexes quantified by stoichiometry *s* = *N*2/*N*<sup>1</sup> is varied, in order to study the exciton dynamics. In Fig.8(a) the population ratio at stationary state of LHs demonstrate that as stoichiometry *s* becomes greater, the population of LH1s, becomes smaller, since their amount is reduced. It is apparent that RC population is quite small, and although their abundance increases the exciton trend to be found in any RC (Fig.8(b)), generally, excitations will be found in harvesting complexes. The population of LHs should be dependent on the ratio of complexes type. As verified in Fig.8(b), RCs have almost no population, and for the discussion below, they will not be taken into account. Populations can be written as:

$$\not{p}\_1(t \to \infty) = f\_1(s) \frac{N\_1}{N\_1 + N\_2} = \frac{f\_1(s)}{1+s} \tag{18}$$

$$\not{p}\_2(t \to \infty) = f\_2(s) \frac{N\_2}{N\_1 + N\_2} = \frac{sf\_2(s)}{1+s} \tag{19}$$

<sup>10</sup> <sup>20</sup> <sup>30</sup> <sup>40</sup> <sup>s</sup>

c

(circles) are presented.

individual LH1 complexes.

**3.2.3 Special pair ionization**

LH1s.

a

Energy Conversion in Purple Bacteria Photosynthesis 57

<sup>10</sup> <sup>20</sup> <sup>30</sup> <sup>40</sup> <sup>s</sup>

Fig. 8. In (a) stationary state populations for LH2s (circles), LH1s (diamonds) and RCs (crosses), as a function of the stoichiometry of membranes presented in Fig.7. In (b) a zoom of RC populations is made, and in (c) the enhancement factors *f*1(*s*) (diamonds) and *f*2(*s*)

has a broader range, and increases with *s*. This result reflects the fact that population of individual LH1s will become greater as more LH2 complexes surround a given LH1. An unconventional architecture (third column, second row in Fig.7) has an outermost line of LH1 complexes, whose connectedness to LH2s is compromised. In all the results in Fig. 8 (sixth point), this architecture does not follow the trends just pointed out, as LH1 and RC population, and enhancement factors, are clearly reduced. The population of LH1 complexes depends on their neighborhood and connectedness. Whenever connectedness of LH1 complexes is lowered, their population will also be reduced. Hence, deviations from populations trend with variation of stoichiometry, are a consequence of different degrees of connectedness of

Up to this point, the master equation approach has helped us understand generally the effect

2. The relative amount of LH2/LH1 complexes, namely, stoichiometry *s* = *N*2/*N*1, when augmented, induces smaller population on LH1-RC complexes. On the other hand, smaller *s* tends to increase the connectedness of LH1s to LH2s and hence, the population of

Another basic process involved in the solar energy conversion is the ionization of the special pair in the RC, and eventual quinol *QBH*<sup>2</sup> formation. Remind that once quinol is formed,

of stoichiometry and architecture in small networks. Two conclusions can be made:

1. Connectedness of LH2 complexes to LH1s, facilitates transfer to RCs

0.2 0.4 0.6 0.8 1

pk


Fig. 7. Networks with different stoichiometries, from left to right, top to bottom, *s*={1.04, 2.06, 3.08, 4.44, 5.125, 6, 7.16, 8.8, 11.25, 15.33, 23.5, 48}, and equal number of harvesting complexes.

where the dependence on the amount of complexes is made explicit with the ratio *Nk <sup>N</sup>*1+*N*<sup>2</sup> , and where *f*1(*s*) and *f*2(*s*) are enhancement factors. This factor provides information on how the population on individual complexes changes, beyond the features arising from their relative abundance. With use of eqs.(18-19), *f*1(*s*) and *f*2(*s*) can be numerically calculated provided that *p*ˆ*k*(*t* → ∞) can be known from the master equation, while *s* is a parameter given for each network. The results for enhancement factors are presented in Fig.8(c). The enhancement factor *f*2(*s*) for LH2 seems to saturate at values below one, as a consequence of the trend of excitations to remain in LH1s. This means that increasing further the number of LH2s will not enhance further the individual LH2 populations. On the other hand *f*1(*s*) 14 Will-be-set-by-IN-TECH

Fig. 7. Networks with different stoichiometries, from left to right, top to bottom, *s*={1.04, 2.06, 3.08, 4.44, 5.125, 6, 7.16, 8.8, 11.25, 15.33, 23.5, 48}, and equal number of harvesting

where the dependence on the amount of complexes is made explicit with the ratio *Nk*

and where *f*1(*s*) and *f*2(*s*) are enhancement factors. This factor provides information on how the population on individual complexes changes, beyond the features arising from their relative abundance. With use of eqs.(18-19), *f*1(*s*) and *f*2(*s*) can be numerically calculated provided that *p*ˆ*k*(*t* → ∞) can be known from the master equation, while *s* is a parameter given for each network. The results for enhancement factors are presented in Fig.8(c). The enhancement factor *f*2(*s*) for LH2 seems to saturate at values below one, as a consequence of the trend of excitations to remain in LH1s. This means that increasing further the number of LH2s will not enhance further the individual LH2 populations. On the other hand *f*1(*s*)

*<sup>N</sup>*1+*N*<sup>2</sup> ,

complexes.

Fig. 8. In (a) stationary state populations for LH2s (circles), LH1s (diamonds) and RCs (crosses), as a function of the stoichiometry of membranes presented in Fig.7. In (b) a zoom of RC populations is made, and in (c) the enhancement factors *f*1(*s*) (diamonds) and *f*2(*s*) (circles) are presented.

has a broader range, and increases with *s*. This result reflects the fact that population of individual LH1s will become greater as more LH2 complexes surround a given LH1. An unconventional architecture (third column, second row in Fig.7) has an outermost line of LH1 complexes, whose connectedness to LH2s is compromised. In all the results in Fig. 8 (sixth point), this architecture does not follow the trends just pointed out, as LH1 and RC population, and enhancement factors, are clearly reduced. The population of LH1 complexes depends on their neighborhood and connectedness. Whenever connectedness of LH1 complexes is lowered, their population will also be reduced. Hence, deviations from populations trend with variation of stoichiometry, are a consequence of different degrees of connectedness of LH1s.

Up to this point, the master equation approach has helped us understand generally the effect of stoichiometry and architecture in small networks. Two conclusions can be made:


#### **3.2.3 Special pair ionization**

Another basic process involved in the solar energy conversion is the ionization of the special pair in the RC, and eventual quinol *QBH*<sup>2</sup> formation. Remind that once quinol is formed,

**3.3 Full vesicles**

*t*

Once *t*

**3.3.1 Simulation algorithm**

a mean inter-photon time of *γ*−<sup>1</sup>

A real vesicle involves several hundreds of harvesting complexes. Given the large state-space needed to describe such amount of complexes and our interest to inquire on a variety of incoming light statistics in the sections ahead, our subsequent model analysis will be based on a discrete-time random walk for excitation hopping between neighboring complexes.

Energy Conversion in Purple Bacteria Photosynthesis 59

In particular, we use a Monte Carlo method to simulate the events of excitation transfer, the photon absorption, the dissipation, and the RC electron transfer. We have checked that our Monte Carlo simulations accurately reproduce the results of the population-based calculations described above, as can be seen from Figs.9(a) and (b). The Monte Carlo simulations proceed as follows. In general, any distribution of light might be used with the restriction of having

membrane and the time for the next absorption is set by inverting the cumulative distribution function from a [0,1] uniformly distributed (Unit Uniformly Distributed, UUD) random number. This inversing procedure is used for any transfer, dissipation or quinol removal event as well. The chosen absorbing complex is randomly selected first among LH1 or LH2 by a second UUD number compared to the probability of absorption in such complex kind, say *N*1(2)*γ*1(2)/*γ<sup>A</sup>* for LH1 (LH2), and a third UUD random number to specifically select any of the given complexes, with probability 1/*N*1(2). Once the excitation is within a given complex, the conditional master equation given that full knowledge of the excitation residing in site *i*, only involves transfers outside such site, say *∂tρ<sup>i</sup>* = −(∑*<sup>j</sup>* 1/*ti*,*<sup>j</sup>* + *γD*)*ρi*, whose solution is straightforward to provide the survival probability and its inverse, of use to choose the time

<sup>∗</sup> for the next event according to eq.(16) from a UUD number *r*: − log *r*/(∑*<sup>j</sup>* 1/*ti*,*<sup>j</sup>* + *γD*) = *t*∗.

∗ is found, a particular event is chosen: transfer to a given neighboring complex *j* with probability (1/*ti*,*j*)/(∑*<sup>j</sup>* 1/*ti*,*<sup>j</sup>* + *γD*) or dissipation with probability *γD*/(∑*<sup>j</sup>* 1/*ti*,*<sup>j</sup>* + *γD*), which are assigned a proportional segment within [0,1] and compared with another UUD number to pinpoint the particular event. If the chosen event is a transfer step, then the excitation jumps to the chosen complex and the transfer-dissipation algorithm starts again. If dissipation occurs, the absorption algorithm is called to initiate a new excitation history. In a RC, the channel of quinol ionization is present with a rate 1/*t*+ in an event that if chosen, produces the same effect as dissipation. Nonetheless, the number of excitations that become SP ionizations are counted on each RC, such that when two excitations ionize a given RC and produce quinol, it becomes closed by temporally setting 1/*t*+ = 0 at such RC. Quinol unbinding will set "open" the RC, not before the RC-cycling time with mean *τ*, has elapsed, chosen according to a poissonian distribution. The algorithm can be summarized as follows: 1. Create the network: Obtain coordinates and type of LHs, and label complexes, for instance, by solely numerating them along its type, say complex 132 is of type 2 (we use 1 for LH1, 2 for LH2 and 3 for RC). Choose the *j* neighbors of complex *i* according to a maximum center to center distance less than *r*<sup>1</sup> + *r*<sup>2</sup> + *δ*, *r*<sup>2</sup> + *r*<sup>2</sup> + *δ* and *r*<sup>1</sup> + *r*<sup>1</sup> + *δ* for respective complexes. We use *δ* = 20, chosen such that only nearest neighbors are accounted and further increase of *δ* makes no difference on the amount of nearest neighbor connections, although further increase may include non-physical next to near-neighbors. In practice, the network creation was done by three arrays, one, say *neigh*(*i*, *j*) with size *M* × *S*, with *M* complexes as described above, and *S* as the maximum number of neighboring complexes among all the sites, hence requiring several attempts to be determined. Minimally *j* ≤ 1 for an LH2, concerning the dissipation channel, *j* ≤ 2 for LH1 including both dissipation and

*<sup>A</sup>* from eq.(2). Accordingly, a first photon is captured by the

Fig. 9. Normalized probabilities *p*ˆ*<sup>k</sup>* for finding the excitation at an LH2 (dashed), LH1 (dotted) or at an RC (continuous), for (a) *t*<sup>+</sup> = 3ps, and (b) *t*<sup>+</sup> → ∞. Crosses are the results from the Monte Carlo simulation.

the special pair is unable to use further incoming excitations before quinol undocks and a new quinone replaces it. Even though the RC neutrality-diffusion process is propelled by complicated dynamics and involved mechanisms, in an easy approach, let us assume that the RC dynamics will proceed through a dichotomic process of "open" and "closed" RC states. In the open state, special pair oxidation is possible, while when closed, special pair oxidation to form quinol never happens, hence *t*<sup>+</sup> → ∞

The effect of open and closed RC states changes the exciton kinetics. We start with a minimal configuration corresponding to a basic photosynthetic unit: one LH2, one LH1 and its RC. Figure 9(a) shows that if the RC is open, excitations will mostly be found in the LH1 complex, followed by occurrences at the LH2 and lastly at the RC. On the other hand, Figure 9(b)) shows clearly the different excitation kinetics which arise when the RC is initially unable to start the electron transfer *<sup>P</sup>*<sup>∗</sup> <sup>→</sup> *<sup>P</sup>*+, and then after <sup>≈</sup> 15ps the RC population becomes greater with respect to the LH2's. This confirms that a faithful description of the actual photosynhesis mechanism, even at the level of the minimal unit, must resort into RC cycling, given that its effects are by no means negligible. Moreover, comparison among Figs.6(d) and 9 also presents a feature that is usually undermined when small architectures are used to straightforward interpret its results as truth for greater, real biological vesicles. Energy funneling becomes smaller with the number of antenna LH2 complexes, thereby, in architectures with many harvesting antenna complexes, excitation will find it more difficult to arrive to any of the relatively spread RCs. Besides, although LH2→LH1 transfer rate is five-fold the back-transfer rate, the amount of smaller sized LH2s neighboring a given LH1 will increase the net back-transfer rate due to site availability. Hence, the funneling concept might be valid for small networks (Hu et al., 2002; Ritz et al., 2001), however, in natural scenarios involving entire chromatophores with many complexes, energy funneling might not be priority due to increased number of available states, provided from all LH2s surrounding a core complex, and globally, from the relative low RC abundance within a real vesicle.

It is important to mention that results for master equation calculations require several minutes in a standard computer to yield the results shown in Fig.8, and that these networks have an amount of nodes an order of magnitude smaller than the actual chromatophore vesicles. Dynamics concerning the RC cycling have not been described yet, fact that would increase further the dimension of possible membrane's states. To circumvent this problem, further analysis will proceed from *stochastic simulations*, and observables will be obtained from ensemble averages.

#### **3.3 Full vesicles**

16 Will-be-set-by-IN-TECH

t ps

Fig. 9. Normalized probabilities *p*ˆ*<sup>k</sup>* for finding the excitation at an LH2 (dashed), LH1 (dotted) or at an RC (continuous), for (a) *t*<sup>+</sup> = 3ps, and (b) *t*<sup>+</sup> → ∞. Crosses are the results

the special pair is unable to use further incoming excitations before quinol undocks and a new quinone replaces it. Even though the RC neutrality-diffusion process is propelled by complicated dynamics and involved mechanisms, in an easy approach, let us assume that the RC dynamics will proceed through a dichotomic process of "open" and "closed" RC states. In the open state, special pair oxidation is possible, while when closed, special pair oxidation to

The effect of open and closed RC states changes the exciton kinetics. We start with a minimal configuration corresponding to a basic photosynthetic unit: one LH2, one LH1 and its RC. Figure 9(a) shows that if the RC is open, excitations will mostly be found in the LH1 complex, followed by occurrences at the LH2 and lastly at the RC. On the other hand, Figure 9(b)) shows clearly the different excitation kinetics which arise when the RC is initially unable to start the electron transfer *<sup>P</sup>*<sup>∗</sup> <sup>→</sup> *<sup>P</sup>*+, and then after <sup>≈</sup> 15ps the RC population becomes greater with respect to the LH2's. This confirms that a faithful description of the actual photosynhesis mechanism, even at the level of the minimal unit, must resort into RC cycling, given that its effects are by no means negligible. Moreover, comparison among Figs.6(d) and 9 also presents a feature that is usually undermined when small architectures are used to straightforward interpret its results as truth for greater, real biological vesicles. Energy funneling becomes smaller with the number of antenna LH2 complexes, thereby, in architectures with many harvesting antenna complexes, excitation will find it more difficult to arrive to any of the relatively spread RCs. Besides, although LH2→LH1 transfer rate is five-fold the back-transfer rate, the amount of smaller sized LH2s neighboring a given LH1 will increase the net back-transfer rate due to site availability. Hence, the funneling concept might be valid for small networks (Hu et al., 2002; Ritz et al., 2001), however, in natural scenarios involving entire chromatophores with many complexes, energy funneling might not be priority due to increased number of available states, provided from all LH2s surrounding a core complex,

It is important to mention that results for master equation calculations require several minutes in a standard computer to yield the results shown in Fig.8, and that these networks have an amount of nodes an order of magnitude smaller than the actual chromatophore vesicles. Dynamics concerning the RC cycling have not been described yet, fact that would increase further the dimension of possible membrane's states. To circumvent this problem, further analysis will proceed from *stochastic simulations*, and observables will be obtained from

and globally, from the relative low RC abundance within a real vesicle.

0.2 0.4 0.6 0.8 p t

5 10 15 20 25 30

t ps

b

a

5 10 15 20 25 30

0.2 0.4 0.6 0.8 p t

ensemble averages.

from the Monte Carlo simulation.

form quinol never happens, hence *t*<sup>+</sup> → ∞

A real vesicle involves several hundreds of harvesting complexes. Given the large state-space needed to describe such amount of complexes and our interest to inquire on a variety of incoming light statistics in the sections ahead, our subsequent model analysis will be based on a discrete-time random walk for excitation hopping between neighboring complexes.

#### **3.3.1 Simulation algorithm**

In particular, we use a Monte Carlo method to simulate the events of excitation transfer, the photon absorption, the dissipation, and the RC electron transfer. We have checked that our Monte Carlo simulations accurately reproduce the results of the population-based calculations described above, as can be seen from Figs.9(a) and (b). The Monte Carlo simulations proceed as follows. In general, any distribution of light might be used with the restriction of having a mean inter-photon time of *γ*−<sup>1</sup> *<sup>A</sup>* from eq.(2). Accordingly, a first photon is captured by the membrane and the time for the next absorption is set by inverting the cumulative distribution function from a [0,1] uniformly distributed (Unit Uniformly Distributed, UUD) random number. This inversing procedure is used for any transfer, dissipation or quinol removal event as well. The chosen absorbing complex is randomly selected first among LH1 or LH2 by a second UUD number compared to the probability of absorption in such complex kind, say *N*1(2)*γ*1(2)/*γ<sup>A</sup>* for LH1 (LH2), and a third UUD random number to specifically select any of the given complexes, with probability 1/*N*1(2). Once the excitation is within a given complex, the conditional master equation given that full knowledge of the excitation residing in site *i*, only involves transfers outside such site, say *∂tρ<sup>i</sup>* = −(∑*<sup>j</sup>* 1/*ti*,*<sup>j</sup>* + *γD*)*ρi*, whose solution is straightforward to provide the survival probability and its inverse, of use to choose the time *t* <sup>∗</sup> for the next event according to eq.(16) from a UUD number *r*: − log *r*/(∑*<sup>j</sup>* 1/*ti*,*<sup>j</sup>* + *γD*) = *t*∗. Once *t* ∗ is found, a particular event is chosen: transfer to a given neighboring complex *j* with probability (1/*ti*,*j*)/(∑*<sup>j</sup>* 1/*ti*,*<sup>j</sup>* + *γD*) or dissipation with probability *γD*/(∑*<sup>j</sup>* 1/*ti*,*<sup>j</sup>* + *γD*), which are assigned a proportional segment within [0,1] and compared with another UUD number to pinpoint the particular event. If the chosen event is a transfer step, then the excitation jumps to the chosen complex and the transfer-dissipation algorithm starts again. If dissipation occurs, the absorption algorithm is called to initiate a new excitation history. In a RC, the channel of quinol ionization is present with a rate 1/*t*+ in an event that if chosen, produces the same effect as dissipation. Nonetheless, the number of excitations that become SP ionizations are counted on each RC, such that when two excitations ionize a given RC and produce quinol, it becomes closed by temporally setting 1/*t*+ = 0 at such RC. Quinol unbinding will set "open" the RC, not before the RC-cycling time with mean *τ*, has elapsed, chosen according to a poissonian distribution. The algorithm can be summarized as follows:

1. Create the network: Obtain coordinates and type of LHs, and label complexes, for instance, by solely numerating them along its type, say complex 132 is of type 2 (we use 1 for LH1, 2 for LH2 and 3 for RC). Choose the *j* neighbors of complex *i* according to a maximum center to center distance less than *r*<sup>1</sup> + *r*<sup>2</sup> + *δ*, *r*<sup>2</sup> + *r*<sup>2</sup> + *δ* and *r*<sup>1</sup> + *r*<sup>1</sup> + *δ* for respective complexes. We use *δ* = 20, chosen such that only nearest neighbors are accounted and further increase of *δ* makes no difference on the amount of nearest neighbor connections, although further increase may include non-physical next to near-neighbors. In practice, the network creation was done by three arrays, one, say *neigh*(*i*, *j*) with size *M* × *S*, with *M* complexes as described above, and *S* as the maximum number of neighboring complexes among all the sites, hence requiring several attempts to be determined. Minimally *j* ≤ 1 for an LH2, concerning the dissipation channel, *j* ≤ 2 for LH1 including both dissipation and

In this preliminary study it is of interest to understand the excitation kinetics in complete chromatophores. In particular, it is useful to understand if any important feature arises according to nature's found tendency of forming clusters of the same complex type. In AFM images (Scheuring & Sturgis, 2005) it has been found that there is an apparent trend to form clusters of LH1 complexes with simultaneous formation of LH2 para-chrystalline domains. The reason that has been argued for this trend (Scheuring & Sturgis, 2005) involves the RC

Energy Conversion in Purple Bacteria Photosynthesis 61

The charge carrier quinone-quinol require diffusing through the intracytoplasmic membrane within the void spaces left among harvesting complexes, in order to reach the bc1 complex and complete the electric charge cycle with cytochorme that however, diffuses through the periplasm as schematically shown in Fig.2. The closeness among LH2 complexes in these para-chrystalline domains restricts the void spaces required for diffusion of quinone-quinol to the LH1 domains, where charge separation is taking place. Then, such aggregation indeed helps to improve the time it takes to quinone perform the whole RC-bc1-RC cycle, by restricting its presence to RC domains. However, an advantage concerning excitation dynamics has been heuristically proposed, where the path an excitation has to travel to reach an open RC once it encounters a closed RC is reduced due to LH1 clustering. In this section we investigate this latter possibility from a more quantitative point of view and address the

In order to understand exciton kinetics, for instance, let us fix our attention on the probability of a RC ionization when excitations start in a given LH2 on the configurations shown in Fig.10. To that end, let us introduce first the number *nRCij* of excitations that are absorbed at site *i* and become ionization of the special pair at a given RC, labeled *j*. Also let *r*ˆ*ij* be the unitary vector pointing in the direction from LH2 site *i* to RC site *j*. As an analogous to a force field, let vector field **v** correspond to a weighted sum of the directions on which RC ionizations occur, starting

> **<sup>v</sup>***<sup>i</sup>* <sup>=</sup> <sup>∑</sup>*<sup>j</sup> nRCijr*ˆ*ij* ∑*<sup>j</sup> n*<sup>2</sup> *RCij*

of help as a purely numerical calculation of graphical interpretation. The normalization gives a basis to compare only the directions for RC ionizations independent whether excitations are dissipated or not. Note that *r*ˆ*ij* has both positive and negative signs, hence the vector **v***<sup>i</sup>* will have greater magnitude as RC ionizations occur more frequently at a given direction, or less

Several features can be understood from this vector field. Figure 10 shows the vector fields attracted to the LH1 most abundant regions. The arrows illustrate the favored statistical direction in which excitations will become RC ionization, and they do not imply that an excitation will deterministically move in that direction if it were absorbed in such place. Firstly, in all cases, a tendency to point on clustered LH1s stands from distant LH2 complexes, a fact that emphasizes the very rapid transfer rates that lead to excitation's rapid global membrane sampling. Secondly, it is evident that better excitation attractors are made with increasing the core centers cluster size, easily observed in the top panel. Hence, according to this result one may naively state that a funneling effect is apparent and strongly dependent on cluster size. However, on the middle panel, it can be seen that this funneling effect is vanishingly small at sites where some degree of symmetry is present concerning the distribution of neighboring RCs, even though if these latter display some clustering degree. Third, a trend is displayed where the spreading of the flux becomes smaller with a lesser

magnitude as ionizations occur in more spread out directions.

. (20)

cycling dynamics and can be explained as follows.

former in the next section.

for a given LH2 complex

transfer to its RC, and *j* ≤ 3 for a RC accounting on dissipation, RC ionization an transfer to its surrounding LH1. The other arrays are built, say *size*(*i*), with *M* positions, that keep on each the number of neighbors of the respective *i* labelled complex, and *rates*(*i*, *j*) where at each position the inter-complex rate *i* → *j* is saved. For instance, *rates*(*i*, 1) of any RC will be the ionization rate 1/*t*+.


The language used to program this algorithm was FORTRAN77, just to point out that these calculations do not require any high-level language.

#### **3.3.2 Excitation dynamics trends in many node-complexes networks**

In order to understand at a qualitative degree the excitation dynamics trends involved in full network chromatophores, a few toy architectures have been studied, shown in Fig.10. 18 Will-be-set-by-IN-TECH

2. Send photons to the network: On a time *t*<sup>∗</sup> = − log(*r*)/*γ<sup>A</sup>* according to eq.(2), with *r* being an UUD number. Choose an LH2 or an LH1, according to the probability of absorption from the cross section of complex type *N*1(2)*γ*1(2)/*γA*. Add one excitation to the network, say *n* = *n* + 1, and assign the initial position *pos*(*n*) = *i* of the excitation according to another UUD that selects an specific labelled *i* complex. Remind that *n* is bounded by the maximum amount of excitations allowed to be at the same time within the membrane,

3. If the *i*th complex is excited, the construction of the above mentioned arrays make the cycle of excitation dynamics straightforward since the network is created only once, and dynamics only require to save the complex *i* where the excitation is, and then go through cycles of size *size*(*n*, *i*) to acknowledge the stochastically generated next time for a given event. Excitation can be transferred to the available neighbors, become dissipated or a RC ionization event. Order all times for next events in order to know which will be the next in the array, say *listimes*(*p*) with *p* ≤ *n*, where *t*min = *listemp*(1). In parallel, update an organized array that saves the next process with the number of the neighbor to which hopping occurs, or say a negative number for RC ioinization and another negative number

4. Jump to next event: By cycling over the *n* present excitations, increase time up to the next event *t*min. If RC cycling is accounted, check which time among *tmin* and the next opening RC time *tRC* (its algorithm is to be discussed in the following) is the closest, and jump to it.

5. Change state of excitations or that of RCs: Update the current site of the excitation *n*, or whether it becomes a dissipation or a RC ionization. If the latter process occurs, keep in an array, say *rcstate*(*k*) whose size equals the total amount of RCs, the number of excitations that have become ionizations from the last time the *k*th RC was opened.If *rcstate*(*k*) = 2 then the *k*th RC is closed by redefinition of *rate*(*i*, 1) = 0 and a poissonian stochastically generated opening time with mean *τ* is generated. This time interval is kept in an array *rctimes*(*k*). Now, introduce this time interval into an ascending ordered list among all

closed RCs opening times such that the minimum *tRC* is obtained. If *tRC* < *t*

<sup>∗</sup> < (*tRC*, *t*min) or (*tRC*, *t*min) < *t*

membrane, finish all processes and write external files.

**3.3.2 Excitation dynamics trends in many node-complexes networks**

calculations do not require any high-level language.

jump to that time and open the *k*th RC by letting *rcstate*(*k*) = 0 and *rates*(*i*, 1) = 1/*t*+. 6. Look which is minimum among *t*∗, *tRC* and *t*min and jump to steps 2 or 4 according to

7. If the maximum amount of excitations chosen from the initialization, have been sent to the

The language used to program this algorithm was FORTRAN77, just to point out that these

In order to understand at a qualitative degree the excitation dynamics trends involved in full network chromatophores, a few toy architectures have been studied, shown in Fig.10.

∗, respectively.

min then

will be the ionization rate 1/*t*+.

usually being one.

for dissipation.

whether *t*

transfer to its RC, and *j* ≤ 3 for a RC accounting on dissipation, RC ionization an transfer to its surrounding LH1. The other arrays are built, say *size*(*i*), with *M* positions, that keep on each the number of neighbors of the respective *i* labelled complex, and *rates*(*i*, *j*) where at each position the inter-complex rate *i* → *j* is saved. For instance, *rates*(*i*, 1) of any RC

In this preliminary study it is of interest to understand the excitation kinetics in complete chromatophores. In particular, it is useful to understand if any important feature arises according to nature's found tendency of forming clusters of the same complex type. In AFM images (Scheuring & Sturgis, 2005) it has been found that there is an apparent trend to form clusters of LH1 complexes with simultaneous formation of LH2 para-chrystalline domains. The reason that has been argued for this trend (Scheuring & Sturgis, 2005) involves the RC cycling dynamics and can be explained as follows.

The charge carrier quinone-quinol require diffusing through the intracytoplasmic membrane within the void spaces left among harvesting complexes, in order to reach the bc1 complex and complete the electric charge cycle with cytochorme that however, diffuses through the periplasm as schematically shown in Fig.2. The closeness among LH2 complexes in these para-chrystalline domains restricts the void spaces required for diffusion of quinone-quinol to the LH1 domains, where charge separation is taking place. Then, such aggregation indeed helps to improve the time it takes to quinone perform the whole RC-bc1-RC cycle, by restricting its presence to RC domains. However, an advantage concerning excitation dynamics has been heuristically proposed, where the path an excitation has to travel to reach an open RC once it encounters a closed RC is reduced due to LH1 clustering. In this section we investigate this latter possibility from a more quantitative point of view and address the former in the next section.

In order to understand exciton kinetics, for instance, let us fix our attention on the probability of a RC ionization when excitations start in a given LH2 on the configurations shown in Fig.10. To that end, let us introduce first the number *nRCij* of excitations that are absorbed at site *i* and become ionization of the special pair at a given RC, labeled *j*. Also let *r*ˆ*ij* be the unitary vector pointing in the direction from LH2 site *i* to RC site *j*. As an analogous to a force field, let vector field **v** correspond to a weighted sum of the directions on which RC ionizations occur, starting for a given LH2 complex

**<sup>v</sup>***<sup>i</sup>* <sup>=</sup> <sup>∑</sup>*<sup>j</sup> nRCijr*ˆ*ij* ∑*<sup>j</sup> n*<sup>2</sup> *RCij* . (20)

of help as a purely numerical calculation of graphical interpretation. The normalization gives a basis to compare only the directions for RC ionizations independent whether excitations are dissipated or not. Note that *r*ˆ*ij* has both positive and negative signs, hence the vector **v***<sup>i</sup>* will have greater magnitude as RC ionizations occur more frequently at a given direction, or less magnitude as ionizations occur in more spread out directions.

Several features can be understood from this vector field. Figure 10 shows the vector fields attracted to the LH1 most abundant regions. The arrows illustrate the favored statistical direction in which excitations will become RC ionization, and they do not imply that an excitation will deterministically move in that direction if it were absorbed in such place. Firstly, in all cases, a tendency to point on clustered LH1s stands from distant LH2 complexes, a fact that emphasizes the very rapid transfer rates that lead to excitation's rapid global membrane sampling. Secondly, it is evident that better excitation attractors are made with increasing the core centers cluster size, easily observed in the top panel. Hence, according to this result one may naively state that a funneling effect is apparent and strongly dependent on cluster size. However, on the middle panel, it can be seen that this funneling effect is vanishingly small at sites where some degree of symmetry is present concerning the distribution of neighboring RCs, even though if these latter display some clustering degree. Third, a trend is displayed where the spreading of the flux becomes smaller with a lesser

<sup>a</sup> <sup>b</sup>

Fig. 11. Vector field **v** for HLI (a) and LLI (b) membranes (100000 runs each site).

flux when many complexes are accounted.

quantitatively investigated.

LH1 complexes, and disregard a funneling LH2→LH1 as the unique direction of excitation

Energy Conversion in Purple Bacteria Photosynthesis 63

In detail, from the reasons just provided, in Fig.11(a) it is apparent that in HLI membranes, individual LH1 complexes are not efficient attractors of excitations. Here, not even the LH2s closest to a core center complex are notably attracted to such cores. On the other hand, a more uniform flux seems to be shared on clusters of LH2s in the LLI membrane (Fig.11(b)), that points to locations where more core centers are displayed. Hence, it is more evident the directionality of the flux to the most LH1 abundant locations that gives a statistical preference to ionize the closest RCs due their low global abundance in LLI membranes. The greater amount of LH1 complexes in the HLI membrane induces no significant preference for excitations to hop to the closest lying RCs and provides evidence that the excitation process proceeds without a clear funneling effect. If a single excitation can become an ionization in a single RC, the flux is clear, but if there are many and spread, the flux becomes random, as would be expected from any random walk. In summary, these results allow a mental picture of the excitation transfer process: Excitations become absorbed and start wandering along the membrane in a random walk regardless of a funneling effect, up to the moment where they reach a RC and become ionized, in a process where statistical preference is present in LLI membranes due to the reduced amount of RC, and presenting more clearly the random wandering on HLI due to higher spread out possibilities of ionization. This fact can also be

Suppose that a given LH1 is surrounded by a few LH2 complexes. If an excitation is absorbed into one of these neighboring LH2s, it will have a survival probability decaying with rate 1/*t*<sup>21</sup> + *n*/*t*<sup>22</sup> where *n* wold be 3-4 corresponding to the number of LH2s neighboring such absorbing LH2. Given the presented transfer times (remind *t*<sup>21</sup> = 3.3ps and *t*<sup>22</sup> = 10ps) both terms in the survival decay rate become almost equal for *n* = 3, and hence no preference will occur on the excitation to be transferred to the closest LH1. If LH2→LH1 transfer would actually happen, the same exercise can be done for the survival probability within the LH1 complex, decaying with a rate *m*/*t*<sup>12</sup> + 1/*t*1,*RC* (remind *t*<sup>12</sup> = 15.5ps and *t*1,*RC* ≈ 30ps) which assuming a single surrounding LH2 complex (*m* = 1) would give a preference for excitations to return to antenna complexes. Hence, if the availability of neighbors is accounted,

Fig. 10. Vector field **v** for different configurations, where the effect of core centers clustering is investigated (100000 runs starting in each site).

amount of RCs. However it is important to be aware that in more detail and in a general fashion, one also sees that even the LH2s closest to a given RC, "feel" the presence of further RCs. This implies that a significant amount of excitations that reach LH2 complexes neighboring a given LH1, will better prefer hopping to neighboring LH2 complexes to eventually reach a distant RC: no "funneling", as usually understood.

In a more realistic situation, the clustering of LH1 complexes is just a trend, and in average any cluster is formed by a few 3-4 core complexes in HLI situations and somehow less for LLI membranes as shown in Fig.11, where empirical architectures are presented in complete accordance with experimental data, taken from Ref.(Scheuring, Rigaud & Sturgis, 2004). Along these figures, the vector field calculated for the HLI and LLI empirical membranes is made, to highlight no clear trend of excitations to be transferred immediately to neighboring 20 Will-be-set-by-IN-TECH

200 400 600 800 1000

100 200 300 400 500 600 700

100 200 300 400 500 600 700

Fig. 10. Vector field **v** for different configurations, where the effect of core centers clustering

amount of RCs. However it is important to be aware that in more detail and in a general fashion, one also sees that even the LH2s closest to a given RC, "feel" the presence of further RCs. This implies that a significant amount of excitations that reach LH2 complexes neighboring a given LH1, will better prefer hopping to neighboring LH2 complexes to

In a more realistic situation, the clustering of LH1 complexes is just a trend, and in average any cluster is formed by a few 3-4 core complexes in HLI situations and somehow less for LLI membranes as shown in Fig.11, where empirical architectures are presented in complete accordance with experimental data, taken from Ref.(Scheuring, Rigaud & Sturgis, 2004). Along these figures, the vector field calculated for the HLI and LLI empirical membranes is made, to highlight no clear trend of excitations to be transferred immediately to neighboring

200

400

600

800

1000

200 400 600 800 1000

100 200 300 400 500 600

100 200 300 400 500 600 700

200 400 600 800 1000

100 200 300 400 500 600 700

100 200 300 400 500 600 700

is investigated (100000 runs starting in each site).

200

eventually reach a distant RC: no "funneling", as usually understood.

400

600

800

1000

200

400

600

800

1000

Fig. 11. Vector field **v** for HLI (a) and LLI (b) membranes (100000 runs each site).

LH1 complexes, and disregard a funneling LH2→LH1 as the unique direction of excitation flux when many complexes are accounted.

In detail, from the reasons just provided, in Fig.11(a) it is apparent that in HLI membranes, individual LH1 complexes are not efficient attractors of excitations. Here, not even the LH2s closest to a core center complex are notably attracted to such cores. On the other hand, a more uniform flux seems to be shared on clusters of LH2s in the LLI membrane (Fig.11(b)), that points to locations where more core centers are displayed. Hence, it is more evident the directionality of the flux to the most LH1 abundant locations that gives a statistical preference to ionize the closest RCs due their low global abundance in LLI membranes. The greater amount of LH1 complexes in the HLI membrane induces no significant preference for excitations to hop to the closest lying RCs and provides evidence that the excitation process proceeds without a clear funneling effect. If a single excitation can become an ionization in a single RC, the flux is clear, but if there are many and spread, the flux becomes random, as would be expected from any random walk. In summary, these results allow a mental picture of the excitation transfer process: Excitations become absorbed and start wandering along the membrane in a random walk regardless of a funneling effect, up to the moment where they reach a RC and become ionized, in a process where statistical preference is present in LLI membranes due to the reduced amount of RC, and presenting more clearly the random wandering on HLI due to higher spread out possibilities of ionization. This fact can also be quantitatively investigated.

Suppose that a given LH1 is surrounded by a few LH2 complexes. If an excitation is absorbed into one of these neighboring LH2s, it will have a survival probability decaying with rate 1/*t*<sup>21</sup> + *n*/*t*<sup>22</sup> where *n* wold be 3-4 corresponding to the number of LH2s neighboring such absorbing LH2. Given the presented transfer times (remind *t*<sup>21</sup> = 3.3ps and *t*<sup>22</sup> = 10ps) both terms in the survival decay rate become almost equal for *n* = 3, and hence no preference will occur on the excitation to be transferred to the closest LH1. If LH2→LH1 transfer would actually happen, the same exercise can be done for the survival probability within the LH1 complex, decaying with a rate *m*/*t*<sup>12</sup> + 1/*t*1,*RC* (remind *t*<sup>12</sup> = 15.5ps and *t*1,*RC* ≈ 30ps) which assuming a single surrounding LH2 complex (*m* = 1) would give a preference for excitations to return to antenna complexes. Hence, if the availability of neighbors is accounted,

complex. If more dissipation is found in some regions, it can there be supposed that excitations remain more time at such domains. Hence the tendency for excitations to dissipate implies they reside longer in LH2 complex domains and justify the view of LH2 clusters as excitation reservoirs. Although RC cycling has not been accounted yet, a given history might include an event where the excitation reaches closed clustered RCs and then jumps back to these LH2 domains waiting before the RCs become available again. On the other hand, HLI membranes display an evenly distributed dissipation. For instance, at HLI not all excitations might be required and dissipation can be used as a photo-protective mechanism. In this case, if dissipation were highly concentrated, vibrational recombination would overheat such highly

Energy Conversion in Purple Bacteria Photosynthesis 65

Beyond these local details, an analysis regarding average values of dissipation *Dk* and residence *pRk* probabilities on a complex type *k* (*k* = 1, 2 are respectively LH1, LH2 complexes) better supports the view of a completely random excitation hopping process. Table 2 shows the numerical results of *pRk* , that concerns the probability to find an excitation in a given complex type and is calculated from the sum of the residence times of excitations at a given complex type. It is straightforward to see that *pRk* is closely related to the probability of dissipation at such complex type, therefore, dissipation can correctly measure where excitations are to be found in general. The randomness of excitation dynamics is illustrated by realizing that dissipation in a given complex type depends primarily on its relative abundance,

behave equivalently, and no dynamical feature arises to set a difference among LH1s and LH2s able to argue on the stoichiometry adaptation experimentally found. Lastly, as expected from the dynamical equivalence of sites, along the result obtained with toy architectures of varying stoichiometry where RC population heavily rose due their abundance but relatively invariant to arrangement, the probability to reach a RC solely depends on its abundance, and

therefore the global efficiency *η* is greater in HLI membranes.

Membrane *pR*<sup>2</sup> *pR*<sup>1</sup> *D*<sup>2</sup> *D*<sup>1</sup>

, and justifies that apart from LH2 clusters local variations, in the mean, all sites

*D*<sup>2</sup> *<sup>D</sup>*<sup>1</sup> *<sup>s</sup>* <sup>=</sup> *<sup>N</sup>*<sup>2</sup>

LLI 0.72 0.25 0.74 0.26 9.13 9.13 0.86 HLI 0.50 0.46 0.52 0.48 3.88 3.92 0.91

Table 2. Dissipation *Dk*, residence probability *pRk* , on *k* = {1, 2} corresponding to *N*<sup>1</sup> LH1 and *N*<sup>2</sup> LH2 complexes respectively. Stoichiometry *s* and efficiency *η* are also shown.

equally on LLI and HLI membranes, therefore, keeping the best performance to HLI.

Hence, for the present discussion, the most important finding from these numerical simulations is that the adaptation of purple bacteria does *not* lie in the single excitation kinetics. In particular, LLI membranes are seen to reduce their efficiency globally at the point where photons are becoming scarcer – hence the answer to adaptation must lie in some more fundamental trade-off (as we will later show explicitly). Due to the dissimilar timescales between millisecond absorption from eq.(2) and nanosecond dissipation, multiple excitation dynamics are also unlikely to occur within a membrane. However, multiple excitation dynamics cannot be regarded *a priori* not to be a reason for purple bacteria adaptation. A numerical study in Ref.(Caycedo-Soler et al., 2010a) involving blockade in which two excitations can not occupy the same site and annihilation, where two excitations annihilate due to vibrational recombination occurring due to significant Frank-Condon overlap on higher exciton states, shows that these mechanisms decrease the efficiency of membranes

*<sup>N</sup>*<sup>1</sup> *<sup>η</sup>* <sup>=</sup> <sup>1</sup> <sup>−</sup> *nD*

*nA*

dissipating domains.

since *Dk*

*Dj* <sup>≈</sup> *Nk Nj*

Fig. 12. Contour plots for dissipation in LLI (a) and HLI (b) membranes. Greater contrast means higher values for dissipation. The simulation is shown after 10<sup>6</sup> excitations were absorbed by the membrane with rate *γA*.

the funneling LH2→LH1 is lost, which along the enhanced back-transfer rate LH1→LH2 as compared to LH1→RC, provides the basis for understanding complete chromatophores as networks where actual performance depends upon the RC availability, since the event of ionization only depends on the probability to get to RC sites having no preference to be visited within the network. Excitations sample great portions of the whole membrane in its hopping, hence become able to reach RCs far away from the absorption sites before dissipation overcomes.

Dissipation itself also provides interesting features. The dissipation *di* measures the probability for excitations to dissipate at site *i* from the the total amount of excitations being absorbed in the membrane *nA*:

$$d\_{\bar{l}} = \frac{n\_{D\_l}}{n\_A} \tag{21}$$

that has an straightforward relation with the global efficiency *η* of the membrane, that accounts on the probability of any excitation to be used as an SP ionization. Given that excitations can only become RC ionizations or be dissipated, the sum over all complexes of dissipation probability will give the probability of any excitation to be dissipated, hence

$$\eta = 1 - \sum\_{i} d\_{i} \tag{22}$$

Figure 12 shows the dissipation on the membranes shinned with the respective illumination rate of their growth (LLI 10 W/m2 and HLI 100 W/m2). Figure 12(a) shows that the LLI membrane has highly dissipative clusters of LH2s, in contrast to the uniform dissipation in the HLI membrane (see Fig.12(b)).

This result addresses distinct features probably connected with the own requirements for bacteria when different light intensity is used during growth. Under LLI, bacterium might require use of all the available solar energy to promote its metabolism. In the numerical simulations (as expected from nature), the dissipation rate is set equal on any site, and therefore dissipation is only dependent on the probability for an excitation to be in a given 22 Will-be-set-by-IN-TECH

Fig. 12. Contour plots for dissipation in LLI (a) and HLI (b) membranes. Greater contrast means higher values for dissipation. The simulation is shown after 10<sup>6</sup> excitations were

the funneling LH2→LH1 is lost, which along the enhanced back-transfer rate LH1→LH2 as compared to LH1→RC, provides the basis for understanding complete chromatophores as networks where actual performance depends upon the RC availability, since the event of ionization only depends on the probability to get to RC sites having no preference to be visited within the network. Excitations sample great portions of the whole membrane in its hopping, hence become able to reach RCs far away from the absorption sites before dissipation

Dissipation itself also provides interesting features. The dissipation *di* measures the probability for excitations to dissipate at site *i* from the the total amount of excitations being

> *di* <sup>=</sup> *nDi nA*

that has an straightforward relation with the global efficiency *η* of the membrane, that accounts on the probability of any excitation to be used as an SP ionization. Given that excitations can only become RC ionizations or be dissipated, the sum over all complexes of dissipation probability will give the probability of any excitation to be dissipated, hence

*<sup>η</sup>* = <sup>1</sup> − ∑

Figure 12 shows the dissipation on the membranes shinned with the respective illumination rate of their growth (LLI 10 W/m2 and HLI 100 W/m2). Figure 12(a) shows that the LLI membrane has highly dissipative clusters of LH2s, in contrast to the uniform dissipation in

This result addresses distinct features probably connected with the own requirements for bacteria when different light intensity is used during growth. Under LLI, bacterium might require use of all the available solar energy to promote its metabolism. In the numerical simulations (as expected from nature), the dissipation rate is set equal on any site, and therefore dissipation is only dependent on the probability for an excitation to be in a given

*i*

(21)

*di* (22)

<sup>a</sup> <sup>b</sup>

absorbed by the membrane with rate *γA*.

absorbed in the membrane *nA*:

the HLI membrane (see Fig.12(b)).

overcomes.

complex. If more dissipation is found in some regions, it can there be supposed that excitations remain more time at such domains. Hence the tendency for excitations to dissipate implies they reside longer in LH2 complex domains and justify the view of LH2 clusters as excitation reservoirs. Although RC cycling has not been accounted yet, a given history might include an event where the excitation reaches closed clustered RCs and then jumps back to these LH2 domains waiting before the RCs become available again. On the other hand, HLI membranes display an evenly distributed dissipation. For instance, at HLI not all excitations might be required and dissipation can be used as a photo-protective mechanism. In this case, if dissipation were highly concentrated, vibrational recombination would overheat such highly dissipating domains.

Beyond these local details, an analysis regarding average values of dissipation *Dk* and residence *pRk* probabilities on a complex type *k* (*k* = 1, 2 are respectively LH1, LH2 complexes) better supports the view of a completely random excitation hopping process. Table 2 shows the numerical results of *pRk* , that concerns the probability to find an excitation in a given complex type and is calculated from the sum of the residence times of excitations at a given complex type. It is straightforward to see that *pRk* is closely related to the probability of dissipation at such complex type, therefore, dissipation can correctly measure where excitations are to be found in general. The randomness of excitation dynamics is illustrated by realizing that dissipation in a given complex type depends primarily on its relative abundance, since *Dk Dj* <sup>≈</sup> *Nk Nj* , and justifies that apart from LH2 clusters local variations, in the mean, all sites behave equivalently, and no dynamical feature arises to set a difference among LH1s and LH2s able to argue on the stoichiometry adaptation experimentally found. Lastly, as expected from the dynamical equivalence of sites, along the result obtained with toy architectures of varying stoichiometry where RC population heavily rose due their abundance but relatively invariant to arrangement, the probability to reach a RC solely depends on its abundance, and therefore the global efficiency *η* is greater in HLI membranes.


Table 2. Dissipation *Dk*, residence probability *pRk* , on *k* = {1, 2} corresponding to *N*<sup>1</sup> LH1 and *N*<sup>2</sup> LH2 complexes respectively. Stoichiometry *s* and efficiency *η* are also shown.

Hence, for the present discussion, the most important finding from these numerical simulations is that the adaptation of purple bacteria does *not* lie in the single excitation kinetics. In particular, LLI membranes are seen to reduce their efficiency globally at the point where photons are becoming scarcer – hence the answer to adaptation must lie in some more fundamental trade-off (as we will later show explicitly). Due to the dissimilar timescales between millisecond absorption from eq.(2) and nanosecond dissipation, multiple excitation dynamics are also unlikely to occur within a membrane. However, multiple excitation dynamics cannot be regarded *a priori* not to be a reason for purple bacteria adaptation. A numerical study in Ref.(Caycedo-Soler et al., 2010a) involving blockade in which two excitations can not occupy the same site and annihilation, where two excitations annihilate due to vibrational recombination occurring due to significant Frank-Condon overlap on higher exciton states, shows that these mechanisms decrease the efficiency of membranes equally on LLI and HLI membranes, therefore, keeping the best performance to HLI.

will tend to wander along the membrane and eventually be dissipated - hence reducing *η*. For the configurations resembling the empirical architectures (Fig.12), this effect is shown as a function of *τ* in Fig. 13(a) yielding a wide range of RC-cycling times at which LLI membrane is more efficient than HLI. Interestingly, this range corresponds to the measured time-scale for *τ* of milliseconds (Milano et al., 2003), and supports the suggestion that bacteria improve their performance in LLI conditions by enhancing quinone-quinol charge carrier dynamics as opposed to manipulating exciton transfer. As mentioned, a recent proposal (Scheuring & Sturgis, 2006) has shown numerically that the formation of LH2 para-crystalline domains produces a clustering trend of LH1 complexes with enhanced quinone availability –

Energy Conversion in Purple Bacteria Photosynthesis 67

However, the crossover of efficiency at *τ* ≈ 3 ms implies that even if no enhanced RC-cycling occurs, the HLI will be less efficient than the LLI membranes on the observed *τ* time-scale. The explanation is quantitatively related to the number *No* of open RCs. Figs. 13(b), (c) and (d) present the distribution *p*(*No*) of open RCs, for both HLI and LLI membranes and for the times shown with arrows in Fig.13(a). When the RC-cycling is of no importance (Fig. 13(b)) almost all RCs remain open, thereby making the HLI membrane more efficient than LLI since having more (open) RCs induces a higher probability for special pair oxidation. Near the crossover in Fig. 13, both membranes have distributions *p*(*No*) centered around the same value (Fig. 13(c)), indicating that although more RCs are present in HLI vesicles, they are more frequently closed due to the ten fold light intensity difference, as compared to LLI conditions. Higher values of *τ* (Fig. 13(d)) present distributions where the LLI has more open RCs, in order to yield a better performance when photons are scarcer. Note that distributions become wider when RC cycling is increased, reflecting the mean-variance correspondence of Poissonian statistics used for simulation of *τ*. Therefore the trade-off between RC-cycling, the actual number of RCs and the light intensity, determines the number of open RCs and hence the performance of a given

Hence, even though these adaptations show such distinct features in the experimentally relevant regimes for the RC-cycling time and illumination intensity magnitude (Milano et al., 2003; Osváth & Maróti, 1997; Scheuring, Sturgis, Prima, Bernadac & Rigaud, 2004), Figs.13(c) and (d) show that the distributions of open RCs actually overlap implying that despite such differences in growing environments, due to the adaptations arising, the resulting dynamics of the membranes become quite similar. Growth conditions generate adaptations that allow on LLI membrane to have a larger number of open RCs than the HLI adaptation and therefore LLI membrane will perform better than HLI with respect to RC ionization irrespective of any funneling dynamics. The inclusion of RC dynamics implies that the absorbed excitation will not find all RCs available, and somehow funneling would limit the chance of a necessary membrane sampling to explore further open RCs. Globally, a given amount of closed RCs will eventually alter the excitation's fate since probable states of oxidization are readily reduced. In a given lifetime, an excitation will find (depending on *τ* and current light intensity *I*) a number of available RCs – the *effective stoichiometry* – which is different from the actual number reported by Atomic Force Microscopy (Bahatyrova et al.,

The effect of incident light intensity variations relative to the light intensity during growth with both membranes, presents a similar behavior. In Ref. (Caycedo-Soler et al., 2010a;b) such study is performed and it is concluded that: the LLI membrane performance starts to diminish well beyond the growth light intensity, while the HLI adaptation starts diminishing just above its growth intensity due to rapid RC closing that induce increased dissipation. Hence, in LLI

a fact that would reduce the RC cycling time.

photosynthetic vesicle architecture (i.e. HLI versus LLI).

2004; Scheuring, Sturgis, Prima, Bernadac & Rigaud, 2004).

Fig. 13. (a) Monte Carlo calculation of efficiency *η* of HLI (diamonds) and LLI (crosses) grown membranes, as a function of the RC-cycling time *τ*. Continuous lines give the result of the analytical model. (b), (c) and (d) show the distributions *p*(*No*) of the number of open RCs for the times shown with arrows in the main plot for HLI (filled bars) and LLI (white bars).

Nevertheless a more comprehensive study to be presented in the next section taking into account the coupled exciton-RC cycling is able to justify nature's choice of proceeding with the observed bacterial adaptations.

#### **4. Complete chromatophores: Exciton and RC cycling coupled dynamics**

It has been here repeatedly commented that RCs perform a cycle that provides the required exciton-chemical energy conversion. We now explain that the answer as to how adaptation can prefer the empirically observed HLI and LLI structures under different illumination conditions, lies in the *interplay* between the excitation kinetics and RC cycling dynamics. By virtue of quinones-quinol and cytochrome charge carriers, the RC dynamics features a 'dead' (or equivalently 'busy') time interval during which quinol is produced, removed and then a new quinone becomes available (Milano et al., 2003; Osváth & Maróti, 1997). Once quinol is produced, it leaves the RC and a new quinone becomes attached. These dynamics are introduced into the simulation algorithm as presented in section 3.3.1, by closing an RC for a (random) poissonian distributed time with mean *τ* after two excitations form quinol. The cycle can be sketched as follows: open RC→ 2 ionizing excitations form quinol → closed RC in a time with mean *τ*→ open RC. This RC cycling time *τ* implies that at any given time, not all RCs are available for turning the electronic excitation into a useful charge separation. Therefore, the number of useful RCs decreases with increasing *τ*. Too many excitations will rapidly close RCs, implying that any subsequently available nearby excitation 24 Will-be-set-by-IN-TECH

1 10 100

<sup>35</sup> No

c

0.2

0.4 <sup>p</sup>No a

<sup>10</sup> 35No

d

<sup>Τ</sup>ms

<sup>b</sup> <sup>c</sup> <sup>d</sup>

Fig. 13. (a) Monte Carlo calculation of efficiency *η* of HLI (diamonds) and LLI (crosses) grown membranes, as a function of the RC-cycling time *τ*. Continuous lines give the result of the analytical model. (b), (c) and (d) show the distributions *p*(*No*) of the number of open RCs for the times shown with arrows in the main plot for HLI (filled bars) and LLI (white bars).

**4. Complete chromatophores: Exciton and RC cycling coupled dynamics**

Nevertheless a more comprehensive study to be presented in the next section taking into account the coupled exciton-RC cycling is able to justify nature's choice of proceeding with

It has been here repeatedly commented that RCs perform a cycle that provides the required exciton-chemical energy conversion. We now explain that the answer as to how adaptation can prefer the empirically observed HLI and LLI structures under different illumination conditions, lies in the *interplay* between the excitation kinetics and RC cycling dynamics. By virtue of quinones-quinol and cytochrome charge carriers, the RC dynamics features a 'dead' (or equivalently 'busy') time interval during which quinol is produced, removed and then a new quinone becomes available (Milano et al., 2003; Osváth & Maróti, 1997). Once quinol is produced, it leaves the RC and a new quinone becomes attached. These dynamics are introduced into the simulation algorithm as presented in section 3.3.1, by closing an RC for a (random) poissonian distributed time with mean *τ* after two excitations form quinol. The cycle can be sketched as follows: open RC→ 2 ionizing excitations form quinol → closed RC in a time with mean *τ*→ open RC. This RC cycling time *τ* implies that at any given time, not all RCs are available for turning the electronic excitation into a useful charge separation. Therefore, the number of useful RCs decreases with increasing *τ*. Too many excitations will rapidly close RCs, implying that any subsequently available nearby excitation

0.2

<sup>35</sup> <sup>60</sup> No

the observed bacterial adaptations.

b

0.2

0.4 <sup>p</sup>No

0.4

0.6

Η

0.2

0.4 <sup>p</sup>No 0.8

1.0

will tend to wander along the membrane and eventually be dissipated - hence reducing *η*. For the configurations resembling the empirical architectures (Fig.12), this effect is shown as a function of *τ* in Fig. 13(a) yielding a wide range of RC-cycling times at which LLI membrane is more efficient than HLI. Interestingly, this range corresponds to the measured time-scale for *τ* of milliseconds (Milano et al., 2003), and supports the suggestion that bacteria improve their performance in LLI conditions by enhancing quinone-quinol charge carrier dynamics as opposed to manipulating exciton transfer. As mentioned, a recent proposal (Scheuring & Sturgis, 2006) has shown numerically that the formation of LH2 para-crystalline domains produces a clustering trend of LH1 complexes with enhanced quinone availability – a fact that would reduce the RC cycling time.

However, the crossover of efficiency at *τ* ≈ 3 ms implies that even if no enhanced RC-cycling occurs, the HLI will be less efficient than the LLI membranes on the observed *τ* time-scale. The explanation is quantitatively related to the number *No* of open RCs. Figs. 13(b), (c) and (d) present the distribution *p*(*No*) of open RCs, for both HLI and LLI membranes and for the times shown with arrows in Fig.13(a). When the RC-cycling is of no importance (Fig. 13(b)) almost all RCs remain open, thereby making the HLI membrane more efficient than LLI since having more (open) RCs induces a higher probability for special pair oxidation. Near the crossover in Fig. 13, both membranes have distributions *p*(*No*) centered around the same value (Fig. 13(c)), indicating that although more RCs are present in HLI vesicles, they are more frequently closed due to the ten fold light intensity difference, as compared to LLI conditions. Higher values of *τ* (Fig. 13(d)) present distributions where the LLI has more open RCs, in order to yield a better performance when photons are scarcer. Note that distributions become wider when RC cycling is increased, reflecting the mean-variance correspondence of Poissonian statistics used for simulation of *τ*. Therefore the trade-off between RC-cycling, the actual number of RCs and the light intensity, determines the number of open RCs and hence the performance of a given photosynthetic vesicle architecture (i.e. HLI versus LLI).

Hence, even though these adaptations show such distinct features in the experimentally relevant regimes for the RC-cycling time and illumination intensity magnitude (Milano et al., 2003; Osváth & Maróti, 1997; Scheuring, Sturgis, Prima, Bernadac & Rigaud, 2004), Figs.13(c) and (d) show that the distributions of open RCs actually overlap implying that despite such differences in growing environments, due to the adaptations arising, the resulting dynamics of the membranes become quite similar. Growth conditions generate adaptations that allow on LLI membrane to have a larger number of open RCs than the HLI adaptation and therefore LLI membrane will perform better than HLI with respect to RC ionization irrespective of any funneling dynamics. The inclusion of RC dynamics implies that the absorbed excitation will not find all RCs available, and somehow funneling would limit the chance of a necessary membrane sampling to explore further open RCs. Globally, a given amount of closed RCs will eventually alter the excitation's fate since probable states of oxidization are readily reduced. In a given lifetime, an excitation will find (depending on *τ* and current light intensity *I*) a number of available RCs – the *effective stoichiometry* – which is different from the actual number reported by Atomic Force Microscopy (Bahatyrova et al., 2004; Scheuring, Sturgis, Prima, Bernadac & Rigaud, 2004).

The effect of incident light intensity variations relative to the light intensity during growth with both membranes, presents a similar behavior. In Ref. (Caycedo-Soler et al., 2010a;b) such study is performed and it is concluded that: the LLI membrane performance starts to diminish well beyond the growth light intensity, while the HLI adaptation starts diminishing just above its growth intensity due to rapid RC closing that induce increased dissipation. Hence, in LLI

their own dynamycs, closing at a rate *λC*(*No*) × *NE* and individually opening at a rate 1/*τ*. The dependance *λC*(*No*) is numerically found aidded by the stochastic model and shown in

Energy Conversion in Purple Bacteria Photosynthesis 69

*<sup>τ</sup>* (*N*<sup>1</sup> <sup>−</sup> *No*) <sup>−</sup> *<sup>λ</sup>C*(*No*)

*<sup>C</sup>*(*γ<sup>D</sup>* <sup>−</sup> *<sup>λ</sup>*<sup>0</sup>

*<sup>C</sup>* is the rate of RC ionization when no RC-cycling is accounted, dependent only on the amount of RCs present in the vesicle (Caycedo-Soler et al., 2010a). This analytical expression is shown in Fig.14(b) and illustrates that *η* ≥ 0.9 if the transfer-P reduction time is less than a tenth of the dissipation time, not including RC cycling. As can be seen in Figs. 13(a), the analytical solution is in good quantitative agreement with the numerical stochastic simulation, and provides support for the assumptions made. Moreover, this model shows directly that the efficiency is driven by the interplay between the RC cycling time and light intensity. Figure 14(b) shows up an entire region of parameter space where LLI membranes are better than HLI in terms of causing P ionization, even though the actual number of RCs that they have is smaller. In view of these results, it is interesting to note how clever Nature has been in tinkering with the efficiency of LLI vesicles and the dissipative behavior of HLI adaptation, in order to meet the needs of bacteria subject to the illumination conditions of the growing

Photosynthetic membranes must provide enough energy to fulfill the metabolic requirements

requirement of two ionizations to form a single quinol molecule. Although these membranes were grown under continuous illumination, the adaptations themselves are a product of millions of years of evolution. Using RC cycling times that preserve quinol rate in both adaptations, different behaviors emerge when the illumination intensity is varied (see Fig. 15(a). The increased illumination is readily used by the LLI adaptation, in order to profit from excess excitations in an otherwise low productivity regime. On the other hand, the HLI

*<sup>W</sup>* <sup>=</sup> <sup>1</sup> 2 *dnRC*

which depends directly on the excitations that ionize RCs *nRC*. The factor <sup>1</sup>

to be solved, and of use for the calculation of the steady-state efficiency *η* = *nRC*/*nA*:

*γAλ*<sup>0</sup> *Cτ* 2*N*1(*λ*<sup>0</sup>

*<sup>η</sup>* <sup>=</sup> *<sup>λ</sup>C*(*No*)*NE γA*

*dt* <sup>=</sup> <sup>−</sup>(*λC*(*No*) + *<sup>γ</sup>D*)*NE* <sup>+</sup> *<sup>γ</sup><sup>A</sup>* (23)

*<sup>C</sup>* <sup>+</sup> *<sup>γ</sup>D*) + *<sup>γ</sup>Aλ*<sup>0</sup>

*<sup>C</sup>*)*<sup>τ</sup>* + (*γAλ*<sup>0</sup>

<sup>2</sup> *NE*. (24)

. (25)

*Cτ*)<sup>2</sup> 

*dt* (28)

<sup>2</sup> accounts for the

*<sup>C</sup>τ*− (26)

(27)

Fig.14(a). Rate equations can therefore be written:

*dNE*

*dNo dt* <sup>=</sup> <sup>1</sup>

A linear fit for *λC*(*No*) allows an analytical expression for *η*:

 4*N*<sup>2</sup> <sup>1</sup> (*λ*<sup>0</sup>

where *λ*<sup>0</sup>

environment.

**5.2 Bacterial metabolic demands**

*<sup>η</sup>*(*τ*, *<sup>γ</sup>A*(*I*)) = <sup>1</sup>

of the living bacteria quantified by the quinol output or quinol rate

*<sup>C</sup>* <sup>+</sup> *<sup>γ</sup>D*)<sup>2</sup> <sup>+</sup> <sup>4</sup>*N*1*γAλ*<sup>0</sup>

Fig. 14. (a) Numerical results showing the rate of ionization *λC*(*No*) of an RC for HLI (diamonds) and LLI (crosses) membranes, together with a quadratic (dashed line) and linear (continuous) dependence on the number of closed RCs (*N*<sup>1</sup> − *No*). The fitting parameters for *<sup>a</sup>* <sup>+</sup> *bNo* are *<sup>a</sup>* <sup>=</sup> {15.16, 7.72}ns−1, *<sup>b</sup>* <sup>=</sup> {−0.21, <sup>−</sup>0.21} ns−1; and for *<sup>a</sup>* <sup>+</sup> *bNo* <sup>+</sup> *cN*<sup>2</sup> *o* , *<sup>a</sup>* <sup>=</sup> {16.61, 8.21}ns−1, *<sup>b</sup>* <sup>=</sup> {−0.35, <sup>−</sup>0.33}, and *<sup>c</sup>* <sup>=</sup> {3.6, 1.5}*μ*s−1, for HLI and LLI membranes respectively. (b) *η* as function of *τ* and *α* = *I*/*I*0, obtained from the complete analytical solution for LLI (white) and HLI (grey) membranes

membranes excess photons are readily used for bacterial metabolism, and HLI membranes exploit dissipation in order to limit the number of processed excitations. In the same work, it is found that the effect of the arrangement itself is lost due to RC dynamics, since the effective stoichiometry with spread out open RCs becomes alike among different membranes sharing the same amount of RCs, equal cycling time *τ* and incident light intensity.

To summarize so far, the arrangement of complexes changes slightly the efficiency of the membranes when no RC dynamics is included – but with RC dynamics, the most important feature is the number of open RCs. Although the longer RC closing times make membranes more prone to dissipation and decreased efficiency, it also makes the architecture less relevant for the overall dynamics. The relevant network architecture instead becomes the dynamical one including sparse open RCs, not the static geometrical one involving the actual clustered RCs.

#### **5. Analytical model**

Within a typical fluorescence lifetime of 1 ns, a single excitation has travelled hundreds of sites and explored the available RCs globally. The actual arrangement or architecture of the complexes seems not to influence the excitation's fate, since the light intensity and RC cycling determine the number of open RCs and the availability for P oxidation.

#### **5.1 Excitation transfer-RC cycling rate model**

Here, we present an alternative rate model which is inspired by the findings of the numerical simulations, but which (1) globally describes the excitation dynamics and RC cycling, (2) leads to analytical expressions for the efficiency of the membrane and the rate of quinol production, and (3) sheds light on the trade-off between RC-cycling and exciton dynamics (Caycedo-Soler et al., 2010a;b).

Shortly, *NE* excitations area absorbed by the membrane at a rate *γA*, and will find its way to become RC ionizations with a rate per particle *λC*(*No*) whose dependence on the number of open RCs is made explicit, or be dissipated at a rate *γD*. On the other hand, RCs have

26 Will-be-set-by-IN-TECH

0.1

0.3

0.7

Η

1

II0

1

10

<sup>1</sup> 0.1

*o* ,

10 Τms

3<sup>b</sup>

a

Fig. 14. (a) Numerical results showing the rate of ionization *λC*(*No*) of an RC for HLI (diamonds) and LLI (crosses) membranes, together with a quadratic (dashed line) and linear (continuous) dependence on the number of closed RCs (*N*<sup>1</sup> − *No*). The fitting parameters for

*<sup>a</sup>* <sup>+</sup> *bNo* are *<sup>a</sup>* <sup>=</sup> {15.16, 7.72}ns−1, *<sup>b</sup>* <sup>=</sup> {−0.21, <sup>−</sup>0.21} ns−1; and for *<sup>a</sup>* <sup>+</sup> *bNo* <sup>+</sup> *cN*<sup>2</sup>

*<sup>a</sup>* <sup>=</sup> {16.61, 8.21}ns−1, *<sup>b</sup>* <sup>=</sup> {−0.35, <sup>−</sup>0.33}, and *<sup>c</sup>* <sup>=</sup> {3.6, 1.5}*μ*s−1, for HLI and LLI membranes respectively. (b) *η* as function of *τ* and *α* = *I*/*I*0, obtained from the complete

the same amount of RCs, equal cycling time *τ* and incident light intensity.

determine the number of open RCs and the availability for P oxidation.

**5.1 Excitation transfer-RC cycling rate model**

(Caycedo-Soler et al., 2010a;b).

membranes excess photons are readily used for bacterial metabolism, and HLI membranes exploit dissipation in order to limit the number of processed excitations. In the same work, it is found that the effect of the arrangement itself is lost due to RC dynamics, since the effective stoichiometry with spread out open RCs becomes alike among different membranes sharing

To summarize so far, the arrangement of complexes changes slightly the efficiency of the membranes when no RC dynamics is included – but with RC dynamics, the most important feature is the number of open RCs. Although the longer RC closing times make membranes more prone to dissipation and decreased efficiency, it also makes the architecture less relevant for the overall dynamics. The relevant network architecture instead becomes the dynamical one including sparse open RCs, not the static geometrical one involving the actual clustered

Within a typical fluorescence lifetime of 1 ns, a single excitation has travelled hundreds of sites and explored the available RCs globally. The actual arrangement or architecture of the complexes seems not to influence the excitation's fate, since the light intensity and RC cycling

Here, we present an alternative rate model which is inspired by the findings of the numerical simulations, but which (1) globally describes the excitation dynamics and RC cycling, (2) leads to analytical expressions for the efficiency of the membrane and the rate of quinol production, and (3) sheds light on the trade-off between RC-cycling and exciton dynamics

Shortly, *NE* excitations area absorbed by the membrane at a rate *γA*, and will find its way to become RC ionizations with a rate per particle *λC*(*No*) whose dependence on the number of open RCs is made explicit, or be dissipated at a rate *γD*. On the other hand, RCs have

20 40 60 N1�No

analytical solution for LLI (white) and HLI (grey) membranes

16

12

8

ΛCns

RCs.

**5. Analytical model**

�1

4

their own dynamycs, closing at a rate *λC*(*No*) × *NE* and individually opening at a rate 1/*τ*. The dependance *λC*(*No*) is numerically found aidded by the stochastic model and shown in Fig.14(a). Rate equations can therefore be written:

$$\frac{dN\_E}{dt} = -(\lambda\_\mathbb{C}(N\_o) + \gamma\_D)N\_E + \gamma\_A \tag{23}$$

$$\frac{dN\_o}{dt} = \frac{1}{\tau}(N\_1 - N\_0) - \frac{\lambda\_C(N\_0)}{2}N\_E.\tag{24}$$

to be solved, and of use for the calculation of the steady-state efficiency *η* = *nRC*/*nA*:

$$\eta = \frac{\lambda\_{\mathbb{C}}(N\_{\bullet})N\_{\mathrm{E}}}{\gamma\_{\mathrm{A}}}.\tag{25}$$

A linear fit for *λC*(*No*) allows an analytical expression for *η*:

$$\eta(\tau, \gamma\_A(I)) = \frac{1}{\gamma\_A \lambda\_\mathcal{C}^0 \tau} \left\{ 2N\_1(\lambda\_\mathcal{C}^0 + \gamma\_D) + \gamma\_A \lambda\_\mathcal{C}^0 \tau - \tag{26}$$

$$\sqrt{4N\_1^2(\lambda\_\mathbb{C}^0 + \gamma\_D)^2 + 4N\_1\gamma\_A\lambda\_\mathbb{C}^0(\gamma\_D - \lambda\_\mathbb{C}^0)\tau + (\gamma\_A\lambda\_\mathbb{C}^0\tau)^2} \tag{27}$$

where *λ*<sup>0</sup> *<sup>C</sup>* is the rate of RC ionization when no RC-cycling is accounted, dependent only on the amount of RCs present in the vesicle (Caycedo-Soler et al., 2010a). This analytical expression is shown in Fig.14(b) and illustrates that *η* ≥ 0.9 if the transfer-P reduction time is less than a tenth of the dissipation time, not including RC cycling. As can be seen in Figs. 13(a), the analytical solution is in good quantitative agreement with the numerical stochastic simulation, and provides support for the assumptions made. Moreover, this model shows directly that the efficiency is driven by the interplay between the RC cycling time and light intensity. Figure 14(b) shows up an entire region of parameter space where LLI membranes are better than HLI in terms of causing P ionization, even though the actual number of RCs that they have is smaller. In view of these results, it is interesting to note how clever Nature has been in tinkering with the efficiency of LLI vesicles and the dissipative behavior of HLI adaptation, in order to meet the needs of bacteria subject to the illumination conditions of the growing environment.

#### **5.2 Bacterial metabolic demands**

Photosynthetic membranes must provide enough energy to fulfill the metabolic requirements of the living bacteria quantified by the quinol output or quinol rate

$$W = \frac{1}{2} \frac{dn\_{RC}}{dt} \tag{28}$$

which depends directly on the excitations that ionize RCs *nRC*. The factor <sup>1</sup> <sup>2</sup> accounts for the requirement of two ionizations to form a single quinol molecule. Although these membranes were grown under continuous illumination, the adaptations themselves are a product of millions of years of evolution. Using RC cycling times that preserve quinol rate in both adaptations, different behaviors emerge when the illumination intensity is varied (see Fig. 15(a). The increased illumination is readily used by the LLI adaptation, in order to profit from excess excitations in an otherwise low productivity regime. On the other hand, the HLI

0.1 1 10 II0

dynamics within the empirical membrane.

following Barabasi, we define as:

0 20 40 60 80 100 120 140 <sup>I</sup>Wattm2

b

2

4

6

s

Energy Conversion in Purple Bacteria Photosynthesis 71

Fig. 15. (a) Quinol rate *W* in HLI (diamonds, *I*<sup>0</sup> = 100W/m2) and LLI (crosses,

*I*<sup>0</sup> = 10W/m2) grown membranes, as a function of incident intensity *I*. (b) Quinol rate contours of *<sup>W</sup>* <sup>=</sup> {1900, 2000, 2100, 2200} <sup>s</sup>−<sup>1</sup> in black, blue, red and pink, respectively.

anti-Stokes Raman scattering in silicon, or (iv) the external membrane skin of the bacterium is modified in such a way that the absorption of photons takes on extreme statistical properties. Our discussion is qualitatively different from previous work looking at life in extreme conditions, since those discussions have tended to focus on *environmental* extremes affecting the biochemical metabolism or extremes in the incident light intensity. By contrast, our current discussion focuses on extremes in the incident photon statistics. Although the setting is largely hypothetical, our theoretical predictions are based on a realistic semi-empirical model which incorporates (i) high precision empirical AFM information about spatial locations of LH1 and LH2 biomolecular complexes in the membrane architecture of *Rsp. Photometricum* purple bacteria, and (ii) full-scale stochastic simulations of the excitation kinetics and reaction center

As with any process involving events occurring in a stochastic way over time, the statistical properties of arriving photons may show deviations from a pure coin-toss process in two broad ways: burstiness and memory (Goh & Barabasi, 2008). First consider the simplest process in which the rate of arrival of a photon has a constant probability per unit time. It is well known that this so-called Poisson process produces a distribution of the waiting time for the next photon arrival which is exponential in form, given by *P*P(*τ*) ∼ exp(−*τ*/*τ*0). The extent to which the observed arrival time distribution *P*(*τ*) deviates from exponential, indicates how non-Poissonian the photon arrival is. Following Barabasi (Goh & Barabasi, 2008), we refer to this as 'burstiness' *B* and define it by its deviation from a purely Poisson

(*στ*/*m<sup>τ</sup>* <sup>+</sup> <sup>1</sup>) <sup>=</sup> (*στ* <sup>−</sup> *<sup>m</sup>τ*)

where *στ* and *m<sup>τ</sup>* are the standard deviation and mean respectively of the empirical distribution *P*(*τ*). For a pure Poisson process, the mean and standard deviation of the arrival time distribution are equal and hence *B* = 0. The other property which can be noticeable for a non-Poisson process is the memory *M* between consecutive inter-arrival times which,

(*στ* + *mτ*)

(*τ<sup>i</sup>* − *m*1)(*τi*+<sup>1</sup> − *m*2) *σ*1*σ*<sup>2</sup>

*<sup>B</sup>* <sup>≡</sup> (*στ*/*m<sup>τ</sup>* <sup>−</sup> <sup>1</sup>)

*nτ*−1 ∑ *i*=1

*<sup>M</sup>* <sup>≡</sup> <sup>1</sup> *n<sup>τ</sup>* − 1 8

10

20

a

2 W103s

process:

membrane maintains the quinone rate constant, thereby avoiding the risk of pH imbalance in the event that the light intensity suddenly increased. We stress that the number of RCs synthesized does not directly reflect the number of available states of ionization in the membrane. LLI synthesizes a small amount of RCs in order to enhance quinone diffusion, such that excess light intensity is utilized by the majority of special pairs. In HLI, the synthesis of more LH1-RC complexes slows down RC-cycling, which ensures that many of these RCs are unavailable and hence be advantageous of evenly distributed dissipation to steadily supply quinol independent of any excitation increase. The very good agreement between our analytic results and the stochastic simulations, yields additional physical insight concerning the stoichiometries found experimentally in *Rsp. Photometricum*.

A closed form expression regarding all dynamical parameters involved can be obtained (Caycedo-Soler et al., 2010a):

$$2\mathcal{W}(s,I) = \frac{\gamma\_A(s,I)}{2} + \frac{1}{B(s)}\left(1 + \frac{\gamma\_D}{\lambda\_t^0}\right) + \sqrt{[\frac{\gamma\_A(s,I)}{2} + \frac{1}{B(s)}\left(1 + \frac{\gamma\_D}{\lambda\_t^0}\right)]^2 + \frac{\gamma\_A(s,I)}{2B(s)}}\tag{29}$$

where the dependence on stoichiometry is made explicit due to absoprtion cross section in *γ<sup>A</sup>* and on *B*(*s*) = *<sup>τ</sup>*(*s*)(*A*1+*sA*2) *<sup>f</sup>*(*s*)*A*<sup>0</sup> , which is a parameter that depends on area *<sup>A</sup>*1(2) of individual LH1 (LH2) complexes and filling fraction *f*(*s*). The filling chromatophore fraction dependence on *s* is available from experimental data of Ref.(Scheuring & Sturgis, 2006) and *τ*(*s*) is constructed from an interpolation scheme (Caycedo-Soler et al., 2010a).

As emphasized in Ref. (Scheuring & Sturgis, 2005), membranes with *s*=6 or *s*=2 were not observed, which is to be compared with the contour plots regarding constant quinol output, of *s* as a function of growing light intensity *I*0, shown in Fig.15(b). These results support a dichotomic observation where *s* ≈ 4 predominantly on a great range for growing light intensity. However, a prediction can be made for 30-40 W/m2 where a great sensitivity of stoichiometry ratios rapidly build up the number of antenna LH2 complexes. Very recently (Liu et al., 2009), membranes were grown with 30W/m<sup>2</sup> and an experimental stoichiometry of 4.8 was found. The contour of 2200 s−<sup>1</sup> predicts a value for stoichiometry of 4.72 at such growing light intensities. This agreement is quite remarkable, since a simple linear interpolation among the values *<sup>s</sup>* <sup>≈</sup> 4, *<sup>I</sup>*<sup>0</sup> <sup>=</sup> 100 W/m<sup>2</sup> and *<sup>s</sup>* <sup>≈</sup> 8, *<sup>I</sup>*<sup>0</sup> <sup>=</sup> 10 W/m<sup>2</sup> would wrongly predict *s* = 7.1 at 30 W/m2. We encourage experimentalists to confirm the full range of predicted behaviors as a function of light-intensity and stoichiometry. Such exercise would without doubt confirm/expand the understanding on RC-exciton dynamics trade off, pinpointing a direction to pursue solar energy conversion research, provided by Nature itself.

#### **6. Performance of photosynthetic membranes under extreme photon statistics**

Photosynthetic (e.g purple) bacteria provide the crucial coupling between the Sun's energy and the production of food on Earth, and have adapted successfully to a variety of terrestrial conditions since the beginnings of life on Earth several billion years ago. In this section we explore whether terrestrial bacteria, which are the product of millions of years of evolutionary pressure on Earth, could survive if suddenly exposed to incident light with extreme statistics. We are therefore mimicking a scenario in which purple bacteria are either (i) suddenly transported to some unknown extreme solar environment elsewhere in the universe, or (ii) where our own Sun suddenly picks up extremal behavior in terms of the temporal statistics of its emitted photons, or (iii) the bacteria are subjected to extreme artificial light sources such as that in Ref. (Borlaug et al., 2009) involving stimulated Raman scattering, as well as in coherent 28 Will-be-set-by-IN-TECH

membrane maintains the quinone rate constant, thereby avoiding the risk of pH imbalance in the event that the light intensity suddenly increased. We stress that the number of RCs synthesized does not directly reflect the number of available states of ionization in the membrane. LLI synthesizes a small amount of RCs in order to enhance quinone diffusion, such that excess light intensity is utilized by the majority of special pairs. In HLI, the synthesis of more LH1-RC complexes slows down RC-cycling, which ensures that many of these RCs are unavailable and hence be advantageous of evenly distributed dissipation to steadily supply quinol independent of any excitation increase. The very good agreement between our analytic results and the stochastic simulations, yields additional physical insight concerning

A closed form expression regarding all dynamical parameters involved can be obtained

where the dependence on stoichiometry is made explicit due to absoprtion cross section in *γ<sup>A</sup>*

(LH2) complexes and filling fraction *f*(*s*). The filling chromatophore fraction dependence on *s* is available from experimental data of Ref.(Scheuring & Sturgis, 2006) and *τ*(*s*) is constructed

As emphasized in Ref. (Scheuring & Sturgis, 2005), membranes with *s*=6 or *s*=2 were not observed, which is to be compared with the contour plots regarding constant quinol output, of *s* as a function of growing light intensity *I*0, shown in Fig.15(b). These results support a dichotomic observation where *s* ≈ 4 predominantly on a great range for growing light intensity. However, a prediction can be made for 30-40 W/m2 where a great sensitivity of stoichiometry ratios rapidly build up the number of antenna LH2 complexes. Very recently (Liu et al., 2009), membranes were grown with 30W/m<sup>2</sup> and an experimental stoichiometry of 4.8 was found. The contour of 2200 s−<sup>1</sup> predicts a value for stoichiometry of 4.72 at such growing light intensities. This agreement is quite remarkable, since a simple linear interpolation among the values *<sup>s</sup>* <sup>≈</sup> 4, *<sup>I</sup>*<sup>0</sup> <sup>=</sup> 100 W/m<sup>2</sup> and *<sup>s</sup>* <sup>≈</sup> 8, *<sup>I</sup>*<sup>0</sup> <sup>=</sup> 10 W/m<sup>2</sup> would wrongly predict *s* = 7.1 at 30 W/m2. We encourage experimentalists to confirm the full range of predicted behaviors as a function of light-intensity and stoichiometry. Such exercise would without doubt confirm/expand the understanding on RC-exciton dynamics trade off, pinpointing a direction to pursue solar energy conversion research, provided by Nature itself.

**6. Performance of photosynthetic membranes under extreme photon statistics**

Photosynthetic (e.g purple) bacteria provide the crucial coupling between the Sun's energy and the production of food on Earth, and have adapted successfully to a variety of terrestrial conditions since the beginnings of life on Earth several billion years ago. In this section we explore whether terrestrial bacteria, which are the product of millions of years of evolutionary pressure on Earth, could survive if suddenly exposed to incident light with extreme statistics. We are therefore mimicking a scenario in which purple bacteria are either (i) suddenly transported to some unknown extreme solar environment elsewhere in the universe, or (ii) where our own Sun suddenly picks up extremal behavior in terms of the temporal statistics of its emitted photons, or (iii) the bacteria are subjected to extreme artificial light sources such as that in Ref. (Borlaug et al., 2009) involving stimulated Raman scattering, as well as in coherent

1 *B*(*s*) <sup>1</sup> <sup>+</sup> *<sup>γ</sup><sup>D</sup> λ*0 *c* 

*<sup>f</sup>*(*s*)*A*<sup>0</sup> , which is a parameter that depends on area *<sup>A</sup>*1(2) of individual LH1

]<sup>2</sup> <sup>+</sup> *<sup>γ</sup>A*(*s*, *<sup>I</sup>*)

<sup>2</sup>*B*(*s*) (29)

the stoichiometries found experimentally in *Rsp. Photometricum*.

1 *B*(*s*) <sup>1</sup> <sup>+</sup> *<sup>γ</sup><sup>D</sup> λ*0 *c* + [ *γA*(*s*, *I*) <sup>2</sup> <sup>+</sup>

from an interpolation scheme (Caycedo-Soler et al., 2010a).

(Caycedo-Soler et al., 2010a):

and on *B*(*s*) = *<sup>τ</sup>*(*s*)(*A*1+*sA*2)

<sup>2</sup>*W*(*s*, *<sup>I</sup>*) = *<sup>γ</sup>A*(*s*, *<sup>I</sup>*)

<sup>2</sup> <sup>+</sup>

Fig. 15. (a) Quinol rate *W* in HLI (diamonds, *I*<sup>0</sup> = 100W/m2) and LLI (crosses, *I*<sup>0</sup> = 10W/m2) grown membranes, as a function of incident intensity *I*. (b) Quinol rate contours of *<sup>W</sup>* <sup>=</sup> {1900, 2000, 2100, 2200} <sup>s</sup>−<sup>1</sup> in black, blue, red and pink, respectively.

anti-Stokes Raman scattering in silicon, or (iv) the external membrane skin of the bacterium is modified in such a way that the absorption of photons takes on extreme statistical properties. Our discussion is qualitatively different from previous work looking at life in extreme conditions, since those discussions have tended to focus on *environmental* extremes affecting the biochemical metabolism or extremes in the incident light intensity. By contrast, our current discussion focuses on extremes in the incident photon statistics. Although the setting is largely hypothetical, our theoretical predictions are based on a realistic semi-empirical model which incorporates (i) high precision empirical AFM information about spatial locations of LH1 and LH2 biomolecular complexes in the membrane architecture of *Rsp. Photometricum* purple bacteria, and (ii) full-scale stochastic simulations of the excitation kinetics and reaction center dynamics within the empirical membrane.

As with any process involving events occurring in a stochastic way over time, the statistical properties of arriving photons may show deviations from a pure coin-toss process in two broad ways: burstiness and memory (Goh & Barabasi, 2008). First consider the simplest process in which the rate of arrival of a photon has a constant probability per unit time. It is well known that this so-called Poisson process produces a distribution of the waiting time for the next photon arrival which is exponential in form, given by *P*P(*τ*) ∼ exp(−*τ*/*τ*0). The extent to which the observed arrival time distribution *P*(*τ*) deviates from exponential, indicates how non-Poissonian the photon arrival is. Following Barabasi (Goh & Barabasi, 2008), we refer to this as 'burstiness' *B* and define it by its deviation from a purely Poisson process:

$$B \equiv \frac{(\sigma\_{\tau}/m\_{\tau} - 1)}{(\sigma\_{\tau}/m\_{\tau} + 1)} = \frac{(\sigma\_{\tau} - m\_{\tau})}{(\sigma\_{\tau} + m\_{\tau})}$$

where *στ* and *m<sup>τ</sup>* are the standard deviation and mean respectively of the empirical distribution *P*(*τ*). For a pure Poisson process, the mean and standard deviation of the arrival time distribution are equal and hence *B* = 0. The other property which can be noticeable for a non-Poisson process is the memory *M* between consecutive inter-arrival times which, following Barabasi, we define as:

$$M \equiv \frac{1}{n\_{\tau} - 1} \sum\_{i=1}^{n\_{\tau}-1} \frac{(\tau\_{i} - m\_{1})(\tau\_{i+1} - m\_{2})}{\sigma\_{1}\sigma\_{2}}.$$

of the kinetic interplay between exciton migration and trapping, as discussed earlier in this chapter. For example, panel (c) shows that a bacterium which is only tuned to survive on Earth can have a maximally negative burstiness *B* = −1 and a maximal memory *M* = 1, meaning that the incident photons arrive like the regular ticks of a clock. The same applies to all the incident photon conditions demonstrated by the blank spaces. If we make the survival criterion more generous (i.e. *�* > 0.01) the blank spaces corresponding to survivability of the terrestrial bacterium increase in size until no dotted space remains (i.e. when *�* = 1). All the dotted areas in Fig. 16 become blank spaces and hence represent survivable incident photon

Energy Conversion in Purple Bacteria Photosynthesis 73

Fig. 16. The (*M*, *B*) phase diagram governing survivability of a terrestrial bacteria under a wide range of incident photon time-series with a given burstiness *B* and memory *M*. The sum of the blank and dotted areas in each diagram is the region accessible by each of the three photon processes shown. For each (*M*, *B*) value, a time-series of photons is created with these properties and used to generate excitations within the membrane. The resulting quinol output time-series is then calculated. Assuming that this output needs to be similar to that on Earth, the dotted region corresponds to photon (*M*, *B*) input values for which the bacteria would die, while the blank region corresponds to photon input for which the bacteria would survive. The trajectories represent the range of quinol outputs from the reaction centers (RC) as we span the range of physically reasonable RC closure times, given a particular input (shown as a star or cross of the same color). As the time during which the RC is closed increases, the *B* and *M* output values tend to move toward the origin (i.e. toward *B* = 0 and *M* = 0). Bottom: Photon inputs correspond to (a) step input, (b) bunched input and (c) power-law step input. Lower row in each case shows photon arrival process (left, black barcode) and waiting time *τ* between photon arrivals (right, red histogram).

conditions for the bacteria.

where *n<sup>τ</sup>* is the number of inter-arrival times measured from the signal and *m*1(*m*2) and *σ*1(*σ*2) are sample mean and sample standard deviation of *τi*(*τi*+1)'s respectively (*i* = 1, . . . , *n<sup>τ</sup>* − 1). For a pure Poisson process, *M* = 0. Both *B* and *M* range from −1 to +1, with the value *B* = 0 and *M* = 0 for a strict Poisson process. We will assume for simplicity that the arriving photons are all absorbed and hence one exciton created within the membrane by each photon. This can easily be generalized but at the expense of adding another layer of statistical analysis to connect the statistics of arriving photons to the statistics of the excitons being created within the LH2/LH1 membrane – indeed, taking a constant absorption probability less than unity would not change our main conclusions. We will also neglect the possibility that several photons arrive at exactly the same time. In principle, incident photons can be generated numerically with values of *B* and *M* which are arbitrarily close to any specific (*M*, *B*) value – however this is extremely time-consuming numerically. Instead, we focus here on specific processes where the *B* and *M* can be calculated analytically. Although this means that the entire (*M*, *B*) parameter space is not accessed, most of it can indeed be – and with the added advantage that analytic values for *B* and *M* are generated.

Figure 16 summarizes our findings in terms of the incident photons (i.e. excitation input) and metabolic output from the LH2/LH1 membrane, over the entire (*M*, *B*) parameter space. The subspace shown by the combination of the white and dotted regions (i.e. the region in the (*M*, *B*) space which is *not* diagonally shaded) comprises points with (*M*, *B*) values that can be generated by one of three relatively simple types of photon input: (a) step input, (b) bunched input and (c) power-law step input, as shown in the three panels respectively. Each point in the blank or dotted region denotes a time-series of initial excitations in the membrane with those particular burstiness and memory values (*B* and *M*). This train of initial excitations then migrates within the membrane of LH2/LH1 complexes, subject to the dynamical interplay of migration and trapping as discussed earlier in this paper, and gives rise to a given output time-series of 'food' (quinol) to the bacteria. Figure 16 shows explicitly a variety of initial (*M*, *B*) values (crosses and stars) and the trajectory represents the locus of resulting quinol outputs from the reaction centers (RC) for this particular (*M*, *B*) input. The trajectory is generated by varying the RC closing time within the range of physically reasonable values. The trajectory is finite since the range of physically reasonable RC closing times is also finite (20 to 1000Hz). As the time during which the RC is closed increases, the output becomes more Poisson-like, i.e. the *B* and *M* output values from the membrane after absorption at the RC, tend towards 0. Hence the trajectories head toward the center as shown.

The (*M*, *B*) values for natural sunlight on Earth lie near *M* = 0 and *B* = 0 since the incident photon arrival from the Sun is approximately a Poisson process. This produces a quinol output which is also a Poisson process (*M* = 0 and *B* = 0). Now suppose a terrestrial bacteria is suddenly subjected to an extreme incident light source with (*M*, *B*) values which lie at a general point in (*M*, *B*) parameter space. Can it survive? It is reasonable to expect that the bacteria will not survive if the resulting quinol output is very different from that on Earth (i.e. very different from *B* = 0 and *M* = 0) since the bacteria is well-tuned for Life on Earth only. Hence we say that the bacteria can only survive if the quinol output has (*M*, *B*) values within 0.01 of the terrestrial values of *B* = 0 and *M* = 0. The blank spaces denote (*M*, *B*) values for which the bacteria would survive, while the dotted spaces are where the bacteria would die. Remarkably, there are therefore substantial regions of extreme incident photon statistics (i.e.

*B* �= 0 and *M* �= 0) where the bacteria would survive, producing a quinol output for the rest of the organism which resembles that on Earth (i.e. |*B*| < *�* and |*M*| < *�* where *�* = 0.01). The fact that these blank spaces have an irregular form is due to the highly nonlinear nature 30 Will-be-set-by-IN-TECH

where *n<sup>τ</sup>* is the number of inter-arrival times measured from the signal and *m*1(*m*2) and *σ*1(*σ*2) are sample mean and sample standard deviation of *τi*(*τi*+1)'s respectively (*i* = 1, . . . , *n<sup>τ</sup>* − 1). For a pure Poisson process, *M* = 0. Both *B* and *M* range from −1 to +1, with the value *B* = 0 and *M* = 0 for a strict Poisson process. We will assume for simplicity that the arriving photons are all absorbed and hence one exciton created within the membrane by each photon. This can easily be generalized but at the expense of adding another layer of statistical analysis to connect the statistics of arriving photons to the statistics of the excitons being created within the LH2/LH1 membrane – indeed, taking a constant absorption probability less than unity would not change our main conclusions. We will also neglect the possibility that several photons arrive at exactly the same time. In principle, incident photons can be generated numerically with values of *B* and *M* which are arbitrarily close to any specific (*M*, *B*) value – however this is extremely time-consuming numerically. Instead, we focus here on specific processes where the *B* and *M* can be calculated analytically. Although this means that the entire (*M*, *B*) parameter space is not accessed, most of it can indeed be – and with the added

Figure 16 summarizes our findings in terms of the incident photons (i.e. excitation input) and metabolic output from the LH2/LH1 membrane, over the entire (*M*, *B*) parameter space. The subspace shown by the combination of the white and dotted regions (i.e. the region in the (*M*, *B*) space which is *not* diagonally shaded) comprises points with (*M*, *B*) values that can be generated by one of three relatively simple types of photon input: (a) step input, (b) bunched input and (c) power-law step input, as shown in the three panels respectively. Each point in the blank or dotted region denotes a time-series of initial excitations in the membrane with those particular burstiness and memory values (*B* and *M*). This train of initial excitations then migrates within the membrane of LH2/LH1 complexes, subject to the dynamical interplay of migration and trapping as discussed earlier in this paper, and gives rise to a given output time-series of 'food' (quinol) to the bacteria. Figure 16 shows explicitly a variety of initial (*M*, *B*) values (crosses and stars) and the trajectory represents the locus of resulting quinol outputs from the reaction centers (RC) for this particular (*M*, *B*) input. The trajectory is generated by varying the RC closing time within the range of physically reasonable values. The trajectory is finite since the range of physically reasonable RC closing times is also finite (20 to 1000Hz). As the time during which the RC is closed increases, the output becomes more Poisson-like, i.e. the *B* and *M* output values from the membrane after absorption at the RC,

The (*M*, *B*) values for natural sunlight on Earth lie near *M* = 0 and *B* = 0 since the incident photon arrival from the Sun is approximately a Poisson process. This produces a quinol output which is also a Poisson process (*M* = 0 and *B* = 0). Now suppose a terrestrial bacteria is suddenly subjected to an extreme incident light source with (*M*, *B*) values which lie at a general point in (*M*, *B*) parameter space. Can it survive? It is reasonable to expect that the bacteria will not survive if the resulting quinol output is very different from that on Earth (i.e. very different from *B* = 0 and *M* = 0) since the bacteria is well-tuned for Life on Earth only. Hence we say that the bacteria can only survive if the quinol output has (*M*, *B*) values within 0.01 of the terrestrial values of *B* = 0 and *M* = 0. The blank spaces denote (*M*, *B*) values for which the bacteria would survive, while the dotted spaces are where the bacteria would die. Remarkably, there are therefore substantial regions of extreme incident photon statistics (i.e. *B* �= 0 and *M* �= 0) where the bacteria would survive, producing a quinol output for the rest of the organism which resembles that on Earth (i.e. |*B*| < *�* and |*M*| < *�* where *�* = 0.01). The fact that these blank spaces have an irregular form is due to the highly nonlinear nature

advantage that analytic values for *B* and *M* are generated.

tend towards 0. Hence the trajectories head toward the center as shown.

of the kinetic interplay between exciton migration and trapping, as discussed earlier in this chapter. For example, panel (c) shows that a bacterium which is only tuned to survive on Earth can have a maximally negative burstiness *B* = −1 and a maximal memory *M* = 1, meaning that the incident photons arrive like the regular ticks of a clock. The same applies to all the incident photon conditions demonstrated by the blank spaces. If we make the survival criterion more generous (i.e. *�* > 0.01) the blank spaces corresponding to survivability of the terrestrial bacterium increase in size until no dotted space remains (i.e. when *�* = 1). All the dotted areas in Fig. 16 become blank spaces and hence represent survivable incident photon conditions for the bacteria.

Fig. 16. The (*M*, *B*) phase diagram governing survivability of a terrestrial bacteria under a wide range of incident photon time-series with a given burstiness *B* and memory *M*. The sum of the blank and dotted areas in each diagram is the region accessible by each of the three photon processes shown. For each (*M*, *B*) value, a time-series of photons is created with these properties and used to generate excitations within the membrane. The resulting quinol output time-series is then calculated. Assuming that this output needs to be similar to that on Earth, the dotted region corresponds to photon (*M*, *B*) input values for which the bacteria would die, while the blank region corresponds to photon input for which the bacteria would survive. The trajectories represent the range of quinol outputs from the reaction centers (RC) as we span the range of physically reasonable RC closure times, given a particular input (shown as a star or cross of the same color). As the time during which the RC is closed increases, the *B* and *M* output values tend to move toward the origin (i.e. toward *B* = 0 and *M* = 0). Bottom: Photon inputs correspond to (a) step input, (b) bunched input and (c) power-law step input. Lower row in each case shows photon arrival process (left, black barcode) and waiting time *τ* between photon arrivals (right, red histogram).

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#### **7. Perspectives: Photosynthetic membranes of purple bacteria as a basis to develop stable energy conversion**

This chapter has covered a comprehensive review of purple bacteria adaptation to light intensity conditions and has provided a basis to understand the chromatic adaptation of photosynthetic harvesting membranes as a consequence of the requirement imposed by nature on living organisms to develop a machinery capable of fuel production in a stable manner. It is worth to emulate, and certainly interesting to develop in artificial devices to surpass the usual bleaching on semiconductor panels, an analogous mechanism to the excitation kinetics and RC dynamics interplay, in order to shift the emphasis of requirements of fuel production and allow highly efficient energy transfer to available charge carriers (quinones) in low light intensity conditions, while consistently, a leveled fuel (quinol) production statistics is accomplished when photon arrival varies both in intensity and photon waiting time statistics.

#### **8. Acknowledgments**

The authors acknowledges financial support from Research Projects Facultad de Ciencias, Universidad de los Andes, Banco de la Republica and Fundacion Mazda.

#### **9. References**


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*Physics* 12: 095008.

*Biophys.* 39: 227–324.

*Chem.* 280: 11203–11213.

**7. Perspectives: Photosynthetic membranes of purple bacteria as a basis to**

**develop stable energy conversion**

waiting time statistics.

**8. Acknowledgments**

**9. References**


**4** 

*Chile* 

**Carotenoids and Photosynthesis - Regulation of** 

Carotenoids are isoprenoid molecules of 40 carbons which are synthesized in a wide variety of photosynthetic (plants, algae) and non photosynthetic (some fungi and bacteria) organisms. So far, over 750 carotenoid structures are known, and these are divided into nonoxygenated molecules designated as carotenes and into oxygenated carotenoids referred

In photosynthetic organisms, carotenoids are synthesized in the plastids, such as chloroplasts. They are localized and accumulated in the thylakoid membranes of chloroplasts (Cunningham & Gantt, 1998), near the reaction center of photosystem II in the light harvesting complexes (LHC), along with other pigments such as chlorophyll *a* and *b*. Carotenoids act as accessory pigments in the LHC, where they absorb light in a broader range of the blue spectrum (400-500 nm) than chlorophyll. Carotenoids transfer the absorbed energy to chlorophyll *a* during photosynthesis (Britton, 1995). Carotenoids also protect plant cells from photo-oxidative damage as a result of their antioxidant characteristic giving by the conjugate bonds of the polyene chain (Britton, 1995; Britton et al., 1998). In this context carotenoids absorb the excess of energy from reactive oxygen species (ROS) and quench singlet oxygen produced from the chlorophyll triplet in the reaction center of photosystem II (Telfer, 2005). Carotenoids also protect the plant from photo-oxidative damage through thermal dissipation by means of the xanthophyll cycle (Baroli & Nigoyi, 2000). This process occurs when excessive light increases the thylakoid ΔpH, which activatates the enzyme violaxanthin de-epoxidase (VDE), converting violaxanthin to zeaxanthin. Zeaxanthin molecules and protons may change the conformation in the LHC,

Carotenoids are also synthesized and accumulated in chromoplasts, plastids that accumulate pigments in flowers, fruits and storage roots. Carotenoids are stored in lipid bodies or in crystalline structures inside the chromoplasts where they are more stable because they are protected from light (Vishnevetsky et al., 1999). In addition, carotenoids are precursors for apocarotenoids such as the phytohormones abscisic acid (ABA) and stringolactones. ABA is involved in dormancy, development and differentiation of plant embryos, stomata open-closure and in tolerance to abiotic stress (Crozier et al*.*, 2000). The stringolactones act as shoot branching inhibitor hormones. Also they are involved in plant signaling to both harmful (parasitic weeds) and beneficial (arbuscular mycorrhizal fungi)

**1. Introduction** 

to as xanthophylls.

favoring the thermal dissipation.

rhizosphere residents (Walter et al, 2010).

**Carotenoid Biosyntesis by Photoreceptors** 

Claudia Stange and Carlos Flores

*Universidad de Chile* 


### **Carotenoids and Photosynthesis - Regulation of Carotenoid Biosyntesis by Photoreceptors**

Claudia Stange and Carlos Flores *Universidad de Chile Chile* 

#### **1. Introduction**

34 Will-be-set-by-IN-TECH

76 Advances in Photosynthesis – Fundamental Aspects

Osváth, S. & Maróti, P. (1997). Coupling of cytochrome and quinone turnovers in the

Panitchayangkoona, G., Hayes D., Fransteda, K. A., Caram J. R., Harel, E., Wen, J.,

Scheuring, S., Rigaud, J. L. & Sturgis, J. N. (2004). Variable lh2 stoichiometry and core clustering in native membranes of rhodospirillum photometricum, *EMBO* 23: 4127.

Scheuring, S., Sturgis, J. N., Prima, V., Bernadac, Levi, D. & Rigaud, J. L. (2004). Watching

Shreve, A. P., Trautman, J. K., Franck, H. A., Owens, T. G. & Albrecht, A. C. (1991).

rhodobacter sphaeroides 2.4.1, *Biochimica et Biophysica Acta* 1058: 280–288. Timpmann, K., Freiberg, A. & Sundström, V. (1995). Energy trapping and detrapping in

Timpmann, K., Zhang, F., Freiberg, A. & Sundström, V. (1993). Detrapping of excitation

van Amerongen, H., Valkunas, L. & R, G. (2000). *Photosynthetic Excitons*, World Scientific

van Grondelle, R. Dekker, J., Gillbro, T. & Sundstrom, V. (1994). Energy transfer and trapping

Visscher, K., Bergström, H., Sündstrom, V., Hunter, C. & van Grondelle, R. (1989).

Visscher, K. J., Chang, M. C. van Mourik, F., Parkes-Loach, P. S., Heller, B. A., Loach,

Waltz, T., Jamieson, S. J., Bowers, C. M., Bullough, P. A. & Hunter, C. N. (1998). *Projection*

the photosytnhetic apparatus in native membranes, *Proceeding of National Academy of*

Femtosecond energy-transfer processes in the b800-b850 light-harvesting complex of

the photosynthetic bacterium rhosopseudomonas viridis: transfer to trap limited

energy transfer from the reaction center in the photosynthetic purple bacterium

Temperature dependence of energy transfer from the long wavelength antenna bchl-896 to the reaction center in rhodospirillum rubrum, rhodobacter sphaeroides (w.t. and m21 mutant) from 77 to 177k, studied by picosecond absorption

P. A. & van Grondelle, R. (1991). Fluorescence polarization and low-temperature absorption spectroscopy of a subuntit form of light harvesting complex I from purple

*structure of three photosynthetic complexes from Rhodobacter sphaeroides: LH2 at 6 , LH1*

Scheuring, S. & Sturgis, J. (2005). Chromatic adaptation of purple bacteria, *Science* 309: 484. Scheuring, S. & Sturgis, J. N. (2006). Dynamics and diffusion in photosynthetic membranes

sphaeroides, *Biophys. Journ* 73: 972–982.

Rhodopseudomonas palustris, *Science* 302: 1969.

*Sciences USA* 101: 1193.

dynamics, *Chemical Physics* 194: 275.

Publishing Co., Singapore.

*and RC-LH1 at 25* 282: 883.

from rhodospirillum photometricum., *Biophys. J.* 91: 3707.

Scully, M. O. & Zubairy, M. S. (1997). *Quantum Optics*, Cambrige University Press.

rhodospirillum rubrum, *Biochemical et Biophysica Acta* 1183: 185.

in photosynthesis, *Biochim. et Biophys. Acta* 11087: 1–65.

spectroscopy, *Photosynthesys Research* 22: 211.

photosynthetic bacteria, *Biochemistry* 30: 5734.

photocycle of reaction centers from the photosynthetic bacterium rhodobacter

Blankenship, R. & Engel, G. S. (2010). Long-lived quantum coherence in photosynthetic complexes at physiological temperature, *PNAS* 107: 12766–12770. Pfenning, N. (1978). *The Photosynthetic Bacteria*, Plenum Publishing Corporation, New York. Ritz, T., Park, S. & Schulten, K. (2001). Kinetics of exciton migration and trapping in the photosynthetic unit of purple bacteria, *Journal of Physical Chemistry B* 105: 8259. Roszack, A. W. & et al (2003). Crystal Structure the RC-LH1 core complex from

> Carotenoids are isoprenoid molecules of 40 carbons which are synthesized in a wide variety of photosynthetic (plants, algae) and non photosynthetic (some fungi and bacteria) organisms. So far, over 750 carotenoid structures are known, and these are divided into nonoxygenated molecules designated as carotenes and into oxygenated carotenoids referred to as xanthophylls.

> In photosynthetic organisms, carotenoids are synthesized in the plastids, such as chloroplasts. They are localized and accumulated in the thylakoid membranes of chloroplasts (Cunningham & Gantt, 1998), near the reaction center of photosystem II in the light harvesting complexes (LHC), along with other pigments such as chlorophyll *a* and *b*. Carotenoids act as accessory pigments in the LHC, where they absorb light in a broader range of the blue spectrum (400-500 nm) than chlorophyll. Carotenoids transfer the absorbed energy to chlorophyll *a* during photosynthesis (Britton, 1995). Carotenoids also protect plant cells from photo-oxidative damage as a result of their antioxidant characteristic giving by the conjugate bonds of the polyene chain (Britton, 1995; Britton et al., 1998). In this context carotenoids absorb the excess of energy from reactive oxygen species (ROS) and quench singlet oxygen produced from the chlorophyll triplet in the reaction center of photosystem II (Telfer, 2005). Carotenoids also protect the plant from photo-oxidative damage through thermal dissipation by means of the xanthophyll cycle (Baroli & Nigoyi, 2000). This process occurs when excessive light increases the thylakoid ΔpH, which activatates the enzyme violaxanthin de-epoxidase (VDE), converting violaxanthin to zeaxanthin. Zeaxanthin molecules and protons may change the conformation in the LHC, favoring the thermal dissipation.

> Carotenoids are also synthesized and accumulated in chromoplasts, plastids that accumulate pigments in flowers, fruits and storage roots. Carotenoids are stored in lipid bodies or in crystalline structures inside the chromoplasts where they are more stable because they are protected from light (Vishnevetsky et al., 1999). In addition, carotenoids are precursors for apocarotenoids such as the phytohormones abscisic acid (ABA) and stringolactones. ABA is involved in dormancy, development and differentiation of plant embryos, stomata open-closure and in tolerance to abiotic stress (Crozier et al*.*, 2000). The stringolactones act as shoot branching inhibitor hormones. Also they are involved in plant signaling to both harmful (parasitic weeds) and beneficial (arbuscular mycorrhizal fungi) rhizosphere residents (Walter et al, 2010).

Carotenoids and Photosynthesis - Regulation of Carotenoid Biosyntesis by Photoreceptors 79

Fig. 1. **Scheme of the Isoprenoid Biosynthetic Pathways in Plants**. The non-mevalonate pathway (MEP) takes place in plastids and the mevalonate route (MEV) ocurrs in the cytoplasm of the cell. Isopentenylpyrophosphate (IPP) and geranylgeranyl pyrophosphate

(GGPP) are key metabolites in the biosynthesis of chlorophylls and carotenoids. Abbreviations: 2-C-methyl-D-erythritol-4-phosphate (MEP), 1-deoxy-D-xylulose-5 phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), 2Cmethyl-D-erythritol 4-phosphate cytidyltransferase (CMS), 4-(cytidine 5#-diphospho)-2-Cmethyl-D-erythritol kinase (CMK), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2 methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), isopentenyl pyrophosphate isomerase

(IPI), dimethylallylpyrophosphate (DMAPP), geranylgeranyl pyrophosphate syntase (GGPPS), phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene isomerase (Z-ISO), ζ-carotene desaturase (ZDS), carotenoid isomerase (CrtISO), lycopene β cyclase (LCYB), lycopene ε cyclase (LCYE), β-carotene hydroxylase (CHYB), -carotene hydroxylase (CHYE), zeaxanthin epoxidase (ZEP), neoxanthin synthase (NSY), abscisic acid (ABA), geranylgeranyl reductase (GGDR), Chlorophyll synthetase (CHLG), light .

In flowers and fruits, the presence of carotenoids serve also to attract pollinators and seed dispersal agents by the intense yellow, orange and red colors that they provide to these organs (Grotewold, 2006).

Animals are not able to synthesize carotenoids, so they have to be included in their diet. In animals, carotenoids are precursors of vitamin A (retinal) and retinoic acid, which play essential roles in nutrition, vision and cellular differentiation, respectively (Krinsky et al., 1994). These molecules have also antioxidant properties (Bartley & Scolnik, 1995) and therefore, oxidative damage, associated with several pathologies, including aging (Esterbauer et al., 1992), carcinogenesis (Breimer, 1990) and degenerative processes in humans, among others, can be resisted by the ingestion of carotenoids (Rao and Rao, 2007; von Lintig 2010).

#### **2. Biosynthesis of carotenoid in plants**

Carotenogenic genes are encoded in the nuclear genome and the synthesized proteins are targeted as preproteins to the plastids, where they are post-translationally processed.

Chlorophyll, carotenoids, and prenylquinones are key molecules that share early steps in the biosynthesis and directly derive from the plastidic isoprenoid biosynthetic pathway. This pathway starts within the 2-C-methyl-D-erythritol-4-phosphate (MEP) which provides isopentenylpyrophosphate (IPP) for the synthesis of the primal intermediate geranylgeranyl diphosphate (GGDP). The MEP pathway is involved in the IPP biosynthesis for plastidial isoprenodid, and the mevalonate (MEV) pathway is required for the synthesis of IPP for cytoplasmic sterols (brassinoesteroids, cytoquinins, ubiquinones, Figure 1). Despite these biosynthetic routes appear as independent and compartmentalized, a regulated metabolic cross-talk has been reported between them (Flügge & Gao, 2005).

The first step of the MEP pathway condenses glyceraldehyde- 3-phosphate and pyruvate—a reaction catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS)- to produce deoxy-Dxylulose 5-phosphate (DOXP). Then, a reductive isomerization by a DOXP reductoisomerase (DXR) yields MEP; the introduction of a cytidyl moiety by 2-C-methyl-D-erythritol 4 phosphate cytidylyl transferase (CMS) produces 4-(cytidine 5'-diphospho)-2-C-methyl-Derythritol that is further phosphorylated by 4-(cytidine 5'-diphospho)- 2-C-methyl-Derythritol kinase (CMK) and then cyclised by 2-C-methyl-Derythritol 2,4-cyclodiphosphate synthase (MCS) to form 2C-methyl-D-erythritol 2,4-cyclo-diP. The final two reactions leading to IPP and DMAPP are carried out by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS) and reductase (HDR), respectively. All the enzymes of the MEP pathway reside in the stroma. Functional data suggest that the enzymes responsible for the biosynthesis of IPP and DMAPP via the MEP pathway in plants are soluble and localized to plastids (Lange & Ghassemian, 2003). IPP molecules synthesized in the plastids are isomerized to the allylic isomer, dimethylallyl pyrophosphate (DMAPP) through IPP isomerase (IPI). Three molecules of IPP condense with DMAPP to generate geranylgeranyl pyrophosphate (GGPP), in a process involving GGPP synthase (GGPPS, Figure 1). GGPPS is a central intermediate in the synthesis of plastidic isoprenoids: chlorophylls (phytyl sidechain), carotenoids and prenylquinones (isoprenoid side-chains, Figure 1).

For chlorophyll biosynthesis, the enzyme geranylgeranyl reductase (GGDR) catalyzes the formation of phytyl pyrophosphate (Phytul-PP) from GGPP and chlorophyll synthase (CHLG) catalyses the synthesis of chlorophyll *a* from Phytyl-PP and chlorophyllide (Figure 1). Chlorophyll a and b are precursors for tocopherols (Joyard et al. 2009).

In flowers and fruits, the presence of carotenoids serve also to attract pollinators and seed dispersal agents by the intense yellow, orange and red colors that they provide to these

Animals are not able to synthesize carotenoids, so they have to be included in their diet. In animals, carotenoids are precursors of vitamin A (retinal) and retinoic acid, which play essential roles in nutrition, vision and cellular differentiation, respectively (Krinsky et al., 1994). These molecules have also antioxidant properties (Bartley & Scolnik, 1995) and therefore, oxidative damage, associated with several pathologies, including aging (Esterbauer et al., 1992), carcinogenesis (Breimer, 1990) and degenerative processes in humans, among others, can be resisted by the ingestion of carotenoids (Rao and Rao, 2007;

Carotenogenic genes are encoded in the nuclear genome and the synthesized proteins are

Chlorophyll, carotenoids, and prenylquinones are key molecules that share early steps in the biosynthesis and directly derive from the plastidic isoprenoid biosynthetic pathway. This pathway starts within the 2-C-methyl-D-erythritol-4-phosphate (MEP) which provides isopentenylpyrophosphate (IPP) for the synthesis of the primal intermediate geranylgeranyl diphosphate (GGDP). The MEP pathway is involved in the IPP biosynthesis for plastidial isoprenodid, and the mevalonate (MEV) pathway is required for the synthesis of IPP for cytoplasmic sterols (brassinoesteroids, cytoquinins, ubiquinones, Figure 1). Despite these biosynthetic routes appear as independent and compartmentalized, a regulated metabolic

The first step of the MEP pathway condenses glyceraldehyde- 3-phosphate and pyruvate—a reaction catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS)- to produce deoxy-Dxylulose 5-phosphate (DOXP). Then, a reductive isomerization by a DOXP reductoisomerase (DXR) yields MEP; the introduction of a cytidyl moiety by 2-C-methyl-D-erythritol 4 phosphate cytidylyl transferase (CMS) produces 4-(cytidine 5'-diphospho)-2-C-methyl-Derythritol that is further phosphorylated by 4-(cytidine 5'-diphospho)- 2-C-methyl-Derythritol kinase (CMK) and then cyclised by 2-C-methyl-Derythritol 2,4-cyclodiphosphate synthase (MCS) to form 2C-methyl-D-erythritol 2,4-cyclo-diP. The final two reactions leading to IPP and DMAPP are carried out by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS) and reductase (HDR), respectively. All the enzymes of the MEP pathway reside in the stroma. Functional data suggest that the enzymes responsible for the biosynthesis of IPP and DMAPP via the MEP pathway in plants are soluble and localized to plastids (Lange & Ghassemian, 2003). IPP molecules synthesized in the plastids are isomerized to the allylic isomer, dimethylallyl pyrophosphate (DMAPP) through IPP isomerase (IPI). Three molecules of IPP condense with DMAPP to generate geranylgeranyl pyrophosphate (GGPP), in a process involving GGPP synthase (GGPPS, Figure 1). GGPPS is a central intermediate in the synthesis of plastidic isoprenoids: chlorophylls (phytyl side-

targeted as preproteins to the plastids, where they are post-translationally processed.

cross-talk has been reported between them (Flügge & Gao, 2005).

chain), carotenoids and prenylquinones (isoprenoid side-chains, Figure 1).

1). Chlorophyll a and b are precursors for tocopherols (Joyard et al. 2009).

For chlorophyll biosynthesis, the enzyme geranylgeranyl reductase (GGDR) catalyzes the formation of phytyl pyrophosphate (Phytul-PP) from GGPP and chlorophyll synthase (CHLG) catalyses the synthesis of chlorophyll *a* from Phytyl-PP and chlorophyllide (Figure

organs (Grotewold, 2006).

von Lintig 2010).

**2. Biosynthesis of carotenoid in plants** 

Fig. 1. **Scheme of the Isoprenoid Biosynthetic Pathways in Plants**. The non-mevalonate pathway (MEP) takes place in plastids and the mevalonate route (MEV) ocurrs in the cytoplasm of the cell. Isopentenylpyrophosphate (IPP) and geranylgeranyl pyrophosphate (GGPP) are key metabolites in the biosynthesis of chlorophylls and carotenoids. Abbreviations: 2-C-methyl-D-erythritol-4-phosphate (MEP), 1-deoxy-D-xylulose-5 phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), 2Cmethyl-D-erythritol 4-phosphate cytidyltransferase (CMS), 4-(cytidine 5#-diphospho)-2-Cmethyl-D-erythritol kinase (CMK), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2 methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), isopentenyl pyrophosphate isomerase (IPI), dimethylallylpyrophosphate (DMAPP), geranylgeranyl pyrophosphate syntase (GGPPS), phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene isomerase (Z-ISO), ζ-carotene desaturase (ZDS), carotenoid isomerase (CrtISO), lycopene β cyclase (LCYB), lycopene ε cyclase (LCYE), β-carotene hydroxylase (CHYB), -carotene hydroxylase (CHYE), zeaxanthin epoxidase (ZEP), neoxanthin synthase (NSY), abscisic acid (ABA), geranylgeranyl reductase (GGDR), Chlorophyll synthetase (CHLG), light .

Carotenoids and Photosynthesis - Regulation of Carotenoid Biosyntesis by Photoreceptors 81

(PHYA-PHYE), cryptochromes (CRY) and phototropins. The reaction catalysed by psy has been shown to be the rate limiting step of carotenoid biosynthesis in plants and most studies on psy have been focused on the induction of its transcription by PHY and CRY during plant de-etiolation in *A. thaliana, maize*, tomato and tobacco. The expression of other carotenogenic genes such as lcyb, bhx, zep y vde is also induced in the presence of white light or during plant de-etiolation (Simkin et al., 2003; Woitsch & Römer, 2003; Briggs &

Plant photoreceptors, include the family of phytochromes (PHYA-PHYE) that absorb in the red and far red range and cryptochromes (CRY) and phototropins that absorb in the blue

Phytochrome (PHY) is the most characterized type of photoreceptor and their photosensitivity is due to their reversible conversion between two isoforms: the Pr isoform that absorbs light at 660 nm (red light) resulting in its transformation to the Pfr isoform that absorbs light radiation at 730 nm (far red). Once Pr is activated, it is translocated to the nucleus as a Pfr homodimer or heterodimer (Franklin et al., 2005; Sharrock & Clack, 2004; Huq et al., 2003;) where it accumulates in subnuclear bodies, called speckles (Nagatani, 2004). PHY acts as irradiance sensor through its active Pfr form, contributing to the regulation of growth and development in plants (Franklin et al., 2007). A balance between these two isoforms regulates the light-mediated activation of signal transduction in plants

The signal transduction machinery activated by PHYA and PHYB promotes the binding of transcription factors such as HY5, HFR1 and LAF1 and the release of PIFs factors from light responsive elements (LREs) located in the promoter of genes that are up regulated during the de-etiolation process, such as the psy gene. The most common type of LREs that are present in genes activated by light are the ATCTA element, the G box1 (CACGAG) and G box (CTCGAG). PHYA, PHYB and CRY1, can also activate the Z-box (ATCTATTCGTATACGTGTCAC), another LRE present in light inducible promoters (Yadav et al., 2002). In *A.thaliana*, it has been shown that PHYA, but not PHYB, plays a role in the transcriptional induction of psy by promoting the binding of HY5 to white, blue, red and far red light responsive elements (LREs) located in its promoter (von Lintig et al., 1997). The involvement of the b-zip transcription factor HY5 in tomato carotenogenesis was proven with LeHY5 transgenic tomatoes that carry an antisense sequence or RNAi of the HY5 transcription factor gene. The transgenic Lehy5 antisense plants contained 24–31% less leaf chlorophyll compared with non-transgenic plants (Liu et al., 2004), while, immature fruit from Lehy5 RNAi plants exhibited an even greater reduction in chlorophyll and

Photosynthetic development and the production of chlorophylls and carotenoids are coordinately regulated by phytochrome –interacting factor (PIF) family of basic helix-loophelix transcription factors (bHLH, Shin et al., 2009; Leivar et al. 2009) PIFs are negative regulators of photomorphogenesis in the dark. In darkness, PIF1 directly binds to the promoter of the psy gene, resulting in repression of its expression. Once etiolated seedlings are exposed to R light, the activated conformation of PHY, the Pfr, interacts and phosphorylates PIF, leading to its proteasome-mediated degradation (Figure 2). Lighttriggered degradation of PIFs results in a rapid de repression of psy gene expression and a

Olney, 2001; Franklin et al., 2005; Briggs et al., 2007, Toledo-Ortiz et al., 2010).

and UV-A range (Briggs and Olney, 2001; Franklin et al., 2005; Briggs et al., 2007).

**3.1 Carotenoid gene activation mediated by photoreceptors in plants** 

(Bae and Choi, 2008), Figure 2.

carotenoid accumulation.

With regard to the carotenoid pathway, two molecules of GGPP give rise to the colorless phytoene by means of phytoene synthase (PSY, Figure 1). The biosynthesis continues with the desaturation of phytoene to produce the pink-colored trans-lycopene. These reactions are catalyzed by two desaturases and two isomerases. The first desaturase, phytoene desaturase (PDS), catalyzes the biosynthesis of 9,15,9'-tri-cis-ζ-carotene, substrate of the 15 cis- ζ-carotene isomerase (Z-ISO) to produce 9,9'-di-cis- ζ-carotene. After the 15-cis- ζcarotene isomerization, the second desaturase termed ζ –carotene desaturasa (ZDS) leads to the formation of 7,9,9'-*cis*-neurosporene and 7',9'-*cis*-lycopene. Finally, the carotene isomerasa (CRTISO) catalyzes the isomerization of this compound resulting in all-*trans* lycopene (Isaacson et al., 2004; Chen et al., 2010). Although isomerization can be mediated by light, carotenoid biosynthesis in "dark grown" tissues such as roots and etiolates leaves required Z-ISO and CRTISO enzymes.

Subsequently, lycopene is transformed into different bicyclic molecules by means of lycopene cyclases. Lycopene-β-cyclase (LCYB) converts lycopene into γ-carotene and afterward to β-carotene. Lycopene is also cyclized by lycopene--cyclase (LCYE) and by LCYB toproduce α-carotene. The β-carotene is hydroxylated by the enzyme β-carotene hydroxylase (CβHx*,* CRTZ) to give rise zeaxanthin, while the hydroxylation of α-carotene by the -carotene hydroxylase (CHx) and CβHx results in the formation of lutein. Abscisic acid is synthesized in the cytoplasm at the end of the pathway by the cleavage of violaxanthin and neoxanthin by carotenoid cleavage dioxygenases (CDE and NCED, Cunningham, 2002).

Some carotenoid enzymes act in multienzyme complexes in the stroma (isopentenyl pyrophosphate isomerase (IPI), geranylgeranyl pyrophosphate synthase (GGPPS) and phytoene synthase (PSY) and others are associated with the thylakoid membrane (phytoene desaturase (PDS), z-carotene desaturase (ZDS), lycopene -cyclase (LCYB) and lycopene εcyclase (LCYE) (Cunningham & Gantt, 1998).

#### **3. Regulation of the carotenogenic pathway**

Due to the importance of carotenoids for plant and animal health, carotenoid biosynthesis regulation has been studied for the last 40 years both at the pure and applied levels. Nearly all carotenogenic genes in diverse plant species, algae, fungi and bacteria have been identified and characterized (Cunningham & Gantt, 1998; Cunningham, 2002; Howitt & Pogson, 2006; Cazzonelli & Pogson, 2010). The knowledge generated has been used to improve the nutritional value of several organisms, preferentially to metabolically engineer -carotene and ketocarotenoid formation in plants (Ye et al., 2000; Davuluri et al., 2005; Aluru et al., 2008; Apel & Bock, 2009).

The regulation of carotenoid biosynthesis has been studied in photosynthetic organs (leaves) and in non-photosynthetic organs (fruits, flowers) of traditional plant models such as *Arabidopsis thaliana*, *Nicotiana tabacum* (tobacco) and *Solanum lycopersicon* (tomato) (Römer and Fraser, 2005; Howitt & Pogson, 2006).

Almost all of these studies show that carotenogenic genes are expressed in photosynthetic organs exposed to different light qualities, during the transition of etioplasts to chloroplasts (de-etiolation) which correlates with a high and concomitant increase in the carotenoid and chlorophyll levels (Römer & Fraser, 2005; Toledo-Ortiz et al., 2010).

During these processes, carotenogenic gene expression is mostly regulated at the transcriptional level mediated by photoreceptors such as the family of phytochromes

With regard to the carotenoid pathway, two molecules of GGPP give rise to the colorless phytoene by means of phytoene synthase (PSY, Figure 1). The biosynthesis continues with the desaturation of phytoene to produce the pink-colored trans-lycopene. These reactions are catalyzed by two desaturases and two isomerases. The first desaturase, phytoene desaturase (PDS), catalyzes the biosynthesis of 9,15,9'-tri-cis-ζ-carotene, substrate of the 15 cis- ζ-carotene isomerase (Z-ISO) to produce 9,9'-di-cis- ζ-carotene. After the 15-cis- ζcarotene isomerization, the second desaturase termed ζ –carotene desaturasa (ZDS) leads to the formation of 7,9,9'-*cis*-neurosporene and 7',9'-*cis*-lycopene. Finally, the carotene isomerasa (CRTISO) catalyzes the isomerization of this compound resulting in all-*trans* lycopene (Isaacson et al., 2004; Chen et al., 2010). Although isomerization can be mediated by light, carotenoid biosynthesis in "dark grown" tissues such as roots and etiolates leaves

Subsequently, lycopene is transformed into different bicyclic molecules by means of lycopene cyclases. Lycopene-β-cyclase (LCYB) converts lycopene into γ-carotene and afterward to β-carotene. Lycopene is also cyclized by lycopene--cyclase (LCYE) and by LCYB toproduce α-carotene. The β-carotene is hydroxylated by the enzyme β-carotene hydroxylase (CβHx*,* CRTZ) to give rise zeaxanthin, while the hydroxylation of α-carotene by the -carotene hydroxylase (CHx) and CβHx results in the formation of lutein. Abscisic acid is synthesized in the cytoplasm at the end of the pathway by the cleavage of violaxanthin and neoxanthin by carotenoid cleavage dioxygenases (CDE and NCED,

Some carotenoid enzymes act in multienzyme complexes in the stroma (isopentenyl pyrophosphate isomerase (IPI), geranylgeranyl pyrophosphate synthase (GGPPS) and phytoene synthase (PSY) and others are associated with the thylakoid membrane (phytoene desaturase (PDS), z-carotene desaturase (ZDS), lycopene -cyclase (LCYB) and lycopene ε-

Due to the importance of carotenoids for plant and animal health, carotenoid biosynthesis regulation has been studied for the last 40 years both at the pure and applied levels. Nearly all carotenogenic genes in diverse plant species, algae, fungi and bacteria have been identified and characterized (Cunningham & Gantt, 1998; Cunningham, 2002; Howitt & Pogson, 2006; Cazzonelli & Pogson, 2010). The knowledge generated has been used to improve the nutritional value of several organisms, preferentially to metabolically engineer -carotene and ketocarotenoid formation in plants (Ye et al., 2000; Davuluri et al., 2005;

The regulation of carotenoid biosynthesis has been studied in photosynthetic organs (leaves) and in non-photosynthetic organs (fruits, flowers) of traditional plant models such as *Arabidopsis thaliana*, *Nicotiana tabacum* (tobacco) and *Solanum lycopersicon* (tomato) (Römer

Almost all of these studies show that carotenogenic genes are expressed in photosynthetic organs exposed to different light qualities, during the transition of etioplasts to chloroplasts (de-etiolation) which correlates with a high and concomitant increase in the carotenoid and

During these processes, carotenogenic gene expression is mostly regulated at the transcriptional level mediated by photoreceptors such as the family of phytochromes

chlorophyll levels (Römer & Fraser, 2005; Toledo-Ortiz et al., 2010).

required Z-ISO and CRTISO enzymes.

cyclase (LCYE) (Cunningham & Gantt, 1998).

Aluru et al., 2008; Apel & Bock, 2009).

and Fraser, 2005; Howitt & Pogson, 2006).

**3. Regulation of the carotenogenic pathway** 

Cunningham, 2002).

(PHYA-PHYE), cryptochromes (CRY) and phototropins. The reaction catalysed by psy has been shown to be the rate limiting step of carotenoid biosynthesis in plants and most studies on psy have been focused on the induction of its transcription by PHY and CRY during plant de-etiolation in *A. thaliana, maize*, tomato and tobacco. The expression of other carotenogenic genes such as lcyb, bhx, zep y vde is also induced in the presence of white light or during plant de-etiolation (Simkin et al., 2003; Woitsch & Römer, 2003; Briggs & Olney, 2001; Franklin et al., 2005; Briggs et al., 2007, Toledo-Ortiz et al., 2010).

#### **3.1 Carotenoid gene activation mediated by photoreceptors in plants**

Plant photoreceptors, include the family of phytochromes (PHYA-PHYE) that absorb in the red and far red range and cryptochromes (CRY) and phototropins that absorb in the blue and UV-A range (Briggs and Olney, 2001; Franklin et al., 2005; Briggs et al., 2007).

Phytochrome (PHY) is the most characterized type of photoreceptor and their photosensitivity is due to their reversible conversion between two isoforms: the Pr isoform that absorbs light at 660 nm (red light) resulting in its transformation to the Pfr isoform that absorbs light radiation at 730 nm (far red). Once Pr is activated, it is translocated to the nucleus as a Pfr homodimer or heterodimer (Franklin et al., 2005; Sharrock & Clack, 2004; Huq et al., 2003;) where it accumulates in subnuclear bodies, called speckles (Nagatani, 2004). PHY acts as irradiance sensor through its active Pfr form, contributing to the regulation of growth and development in plants (Franklin et al., 2007). A balance between these two isoforms regulates the light-mediated activation of signal transduction in plants (Bae and Choi, 2008), Figure 2.

The signal transduction machinery activated by PHYA and PHYB promotes the binding of transcription factors such as HY5, HFR1 and LAF1 and the release of PIFs factors from light responsive elements (LREs) located in the promoter of genes that are up regulated during the de-etiolation process, such as the psy gene. The most common type of LREs that are present in genes activated by light are the ATCTA element, the G box1 (CACGAG) and G box (CTCGAG). PHYA, PHYB and CRY1, can also activate the Z-box (ATCTATTCGTATACGTGTCAC), another LRE present in light inducible promoters (Yadav et al., 2002). In *A.thaliana*, it has been shown that PHYA, but not PHYB, plays a role in the transcriptional induction of psy by promoting the binding of HY5 to white, blue, red and far red light responsive elements (LREs) located in its promoter (von Lintig et al., 1997). The involvement of the b-zip transcription factor HY5 in tomato carotenogenesis was proven with LeHY5 transgenic tomatoes that carry an antisense sequence or RNAi of the HY5 transcription factor gene. The transgenic Lehy5 antisense plants contained 24–31% less leaf chlorophyll compared with non-transgenic plants (Liu et al., 2004), while, immature fruit from Lehy5 RNAi plants exhibited an even greater reduction in chlorophyll and carotenoid accumulation.

Photosynthetic development and the production of chlorophylls and carotenoids are coordinately regulated by phytochrome –interacting factor (PIF) family of basic helix-loophelix transcription factors (bHLH, Shin et al., 2009; Leivar et al. 2009) PIFs are negative regulators of photomorphogenesis in the dark. In darkness, PIF1 directly binds to the promoter of the psy gene, resulting in repression of its expression. Once etiolated seedlings are exposed to R light, the activated conformation of PHY, the Pfr, interacts and phosphorylates PIF, leading to its proteasome-mediated degradation (Figure 2). Lighttriggered degradation of PIFs results in a rapid de repression of psy gene expression and a

Carotenoids and Photosynthesis - Regulation of Carotenoid Biosyntesis by Photoreceptors 83

expression of the key GA-biosynthetic genes GA3ox1 and GA3ox2 and promoting the expression of the GA catabolic genes. PIF1 also promotes the expression of the abscisic acid (ABA)-biosynthetic genes, and represses the expression of the ABA catabolic gene, resulting in high ABA levels. PIF4 and PI5 also promote ii) Shade Avoidance Syndrome (SAS); the abundance of these proteins increases rapidly upon transfer of white-light grown seedlings to simulated shade. Pif4, pif5 and pif4 pif5 mutants have reduced hypocotyl-elongation and marker-gene responsiveness to this signal compared with wild type (Leivar & Quail, 2011). The cryptochrome CRY, another type of photoreceptor, is also involved in carotenoid light mediated gene activation. Phytochrome and cryptochrome signal transduction events are coordinated (Casal, 2000); PHYA phosphorylates cryptochrome *in vitro* (Ahmad et al., 1998) and blue and UV-A light trigger the phosphorylation of CRY1 and CRY2 (Shalitin et al., 2002; Shalitin et al., 2003). CRY1 localizes in the cytoplasm during darkness and when plants are exposed to light, CRY1 is exported to the nucleus (Guo et al., 1999; Yang et al., 2000; Schepens et al., 2004). CRY2 which belongs to the same family as CRY1, is localized in the nucleus of plant cells during both light and dark periods (Guo et al., 1999). Overexpression of cry2 in tomato causes repression of lycopene cyclase genes, resulting in an overproduction of flavonoids and lycopene in fruits (Giliberto et al. 2005). It has been reported that zeaxanthin acts as a chromophore of CRY1 and CRY2, leading to stomatal opening when guard cells are exposed to light (Briggs, 1999). The blue/green light absorbed by these photoreceptors induces a conformational change in the zeaxanthin molecule, resulting in the formation of a physiologically active isomer leading to the opening and

CRY and PHY bind and inactivate COP1 through direct protein-protein contact (Wang et al., 2001; Seo et al., 2004). COP1 is a ring finger ubiquitin ligase protein associated with the signalosome complex involved in protein degradation processes via the 26S proteasome (Osterland et al., 2000; Seo et al., 2003). During darkness, COP1 triggers degradation of transcription factors committed in light regulation, such as HY5 and HFR1 (Yang et al., 2001; Holm et al., 2002; Yanawaga et al., 2004) whose colocalize with COP1 in nuclear bodies and are marked for post-translational degradation during repression of photomorphogenesis (Ang et al., 1998; Jung et al., 2005). Light promotes conformational changes of COP1, inducing the release of photomorphogenic transcription factors. Once these factors are released, they accumulate and bind to LREs located in the promoters of genes activated by light (Wang et al., 2001;Lin & Shalitin, 2003, Figure 2). Transgenic tomatoes over expressing a Lecop1 RNAi have a reduced level of cop1 transcripts and significantly higher leaf and fruit chlorophyll and carotenoid content than the corresponding non-transformed controls

The UV-damaged DNA binding protein 1 (DDB1) and the de-etiolated-1 (DET1) factors are also negative regulators of light-mediated gene expression, they interact with COP1 and other proteins from the signalosome complex, and lead to ubiquitination of transcription factors (Osterlund et al., 2000; Yanawaga et al., 2004). Post transcriptional gene silencing of det1 leads to an accumulation of carotenoids in tomato fruits (Davuluri et al., 2005). Highly pigmented tomato mutants, *hp1* and *hp2* display shortened hypocotyls and internodes, anthocyanin accumulation, strongly carotenoid colored fruits and an excessive response to light (Mustilli et al., 1999). HP1 and HP2 encode the tomato orthologs of DDB1 and DET1 in *A. thaliana*, respectively (Liu et al., 2004). Carotenoid biosynthesis in *hp2* mutants increased during light treatments, due to the inactivation of the signalosome, decreasing the

closing of stomata (Talbott et al., 2002).

(Liu et al. 2004),.

burst in the production of carotenoids in coordination with chlorophyll biosynthesis and chloroplast development, leading to an optimal transition to the photosynthetic metabolism (Toledo-Ortíz et al., 2010).

Fig. 2. **Ligh-mediated activation of the signal transduction involved in** 

**photomorphogenesis in plants.** The transition from dark conditions (A) to light conditions (B) allows the photosynthetic metabolism. Abbreviations: activated phytochromoe (PHY-Pr), cryptochrome 1 (CRY1), transcription factor LONG HYPOCOTYL 5 (HY5), constitutive photomorphogenic 1 (COP1), phytochrome interacting factor (PIF1), light response element (LRE).

Microarray transcriptome analysis during seedling deetiolation indicated that the majority of the gene expression changes elicited by the absence of the PIFs in dark grown pifq seedlings (pif1 pif3 pif4 pif5 quadruple mutants) are normally induced by prolonged light in wild-type seedlings, such as the induction of numerous photosynthetic genes related to the biogenesis of active chloroplasts, auxin, gibberellins (GA), cytokinin and ethylene hormone pathway-related genes, potentially mediating growth responses and metabolic genes involved in the transition from heterotrophic to autotrophic growth.

Besides, other functions associated with PIFs have been described as: i) regulating seed germination; dormant Arabidopsis seeds require both light activation of the phytochrome system and cold treatment (stratification) to induce efficient germination. PIF1 repress germination in the dark and exerts this function, at least in part, by repressing the

burst in the production of carotenoids in coordination with chlorophyll biosynthesis and chloroplast development, leading to an optimal transition to the photosynthetic metabolism

Fig. 2. **Ligh-mediated activation of the signal transduction involved in** 

involved in the transition from heterotrophic to autotrophic growth.

**photomorphogenesis in plants.** The transition from dark conditions (A) to light conditions (B) allows the photosynthetic metabolism. Abbreviations: activated phytochromoe (PHY-Pr), cryptochrome 1 (CRY1), transcription factor LONG HYPOCOTYL 5 (HY5), constitutive photomorphogenic 1 (COP1), phytochrome interacting factor (PIF1), light response element

Microarray transcriptome analysis during seedling deetiolation indicated that the majority of the gene expression changes elicited by the absence of the PIFs in dark grown pifq seedlings (pif1 pif3 pif4 pif5 quadruple mutants) are normally induced by prolonged light in wild-type seedlings, such as the induction of numerous photosynthetic genes related to the biogenesis of active chloroplasts, auxin, gibberellins (GA), cytokinin and ethylene hormone pathway-related genes, potentially mediating growth responses and metabolic genes

Besides, other functions associated with PIFs have been described as: i) regulating seed germination; dormant Arabidopsis seeds require both light activation of the phytochrome system and cold treatment (stratification) to induce efficient germination. PIF1 repress germination in the dark and exerts this function, at least in part, by repressing the

(Toledo-Ortíz et al., 2010).

(LRE).

expression of the key GA-biosynthetic genes GA3ox1 and GA3ox2 and promoting the expression of the GA catabolic genes. PIF1 also promotes the expression of the abscisic acid (ABA)-biosynthetic genes, and represses the expression of the ABA catabolic gene, resulting in high ABA levels. PIF4 and PI5 also promote ii) Shade Avoidance Syndrome (SAS); the abundance of these proteins increases rapidly upon transfer of white-light grown seedlings to simulated shade. Pif4, pif5 and pif4 pif5 mutants have reduced hypocotyl-elongation and marker-gene responsiveness to this signal compared with wild type (Leivar & Quail, 2011).

The cryptochrome CRY, another type of photoreceptor, is also involved in carotenoid light mediated gene activation. Phytochrome and cryptochrome signal transduction events are coordinated (Casal, 2000); PHYA phosphorylates cryptochrome *in vitro* (Ahmad et al., 1998) and blue and UV-A light trigger the phosphorylation of CRY1 and CRY2 (Shalitin et al., 2002; Shalitin et al., 2003). CRY1 localizes in the cytoplasm during darkness and when plants are exposed to light, CRY1 is exported to the nucleus (Guo et al., 1999; Yang et al., 2000; Schepens et al., 2004). CRY2 which belongs to the same family as CRY1, is localized in the nucleus of plant cells during both light and dark periods (Guo et al., 1999). Overexpression of cry2 in tomato causes repression of lycopene cyclase genes, resulting in an overproduction of flavonoids and lycopene in fruits (Giliberto et al. 2005). It has been reported that zeaxanthin acts as a chromophore of CRY1 and CRY2, leading to stomatal opening when guard cells are exposed to light (Briggs, 1999). The blue/green light absorbed by these photoreceptors induces a conformational change in the zeaxanthin molecule, resulting in the formation of a physiologically active isomer leading to the opening and closing of stomata (Talbott et al., 2002).

CRY and PHY bind and inactivate COP1 through direct protein-protein contact (Wang et al., 2001; Seo et al., 2004). COP1 is a ring finger ubiquitin ligase protein associated with the signalosome complex involved in protein degradation processes via the 26S proteasome (Osterland et al., 2000; Seo et al., 2003). During darkness, COP1 triggers degradation of transcription factors committed in light regulation, such as HY5 and HFR1 (Yang et al., 2001; Holm et al., 2002; Yanawaga et al., 2004) whose colocalize with COP1 in nuclear bodies and are marked for post-translational degradation during repression of photomorphogenesis (Ang et al., 1998; Jung et al., 2005). Light promotes conformational changes of COP1, inducing the release of photomorphogenic transcription factors. Once these factors are released, they accumulate and bind to LREs located in the promoters of genes activated by light (Wang et al., 2001;Lin & Shalitin, 2003, Figure 2). Transgenic tomatoes over expressing a Lecop1 RNAi have a reduced level of cop1 transcripts and significantly higher leaf and fruit chlorophyll and carotenoid content than the corresponding non-transformed controls (Liu et al. 2004),.

The UV-damaged DNA binding protein 1 (DDB1) and the de-etiolated-1 (DET1) factors are also negative regulators of light-mediated gene expression, they interact with COP1 and other proteins from the signalosome complex, and lead to ubiquitination of transcription factors (Osterlund et al., 2000; Yanawaga et al., 2004). Post transcriptional gene silencing of det1 leads to an accumulation of carotenoids in tomato fruits (Davuluri et al., 2005). Highly pigmented tomato mutants, *hp1* and *hp2* display shortened hypocotyls and internodes, anthocyanin accumulation, strongly carotenoid colored fruits and an excessive response to light (Mustilli et al., 1999). HP1 and HP2 encode the tomato orthologs of DDB1 and DET1 in *A. thaliana*, respectively (Liu et al., 2004). Carotenoid biosynthesis in *hp2* mutants increased during light treatments, due to the inactivation of the signalosome, decreasing the

Carotenoids and Photosynthesis - Regulation of Carotenoid Biosyntesis by Photoreceptors 85

Chlorophyll and carotenoid biosynthesis are also regulated indirectly by light through the redox potential generated during photosynthesis. In this process, plastoquinone acts as a redox potential sensor responsible for the induction of carotenogenic genes, indicating that the biosynthesis of carotenoids is under photosynthetic redox control (Jöet et al., 2002;

Different experimental approaches were used to determine the regulatory mechanism in which carotenoid and photosynthetic components are involved to determine the chloroplast biogenesis. Arabidopsis *pds3* knockout mutant, or plants treated by norflurazon (NF) exert white tissues (photooxidized plastids) due to inactivation of PDS. The *immutans (im)*  variegation mutant, that has a defect in plastoquinol terminal oxidase IMMUTANS (IM) termed PTOX that transfers electrons from the plastoquinone (PQ) pool to molecular oxygen, presents variegated leaves. Considering the PQ pool as a potent initiator of retrograde signaling, a plausible hypothesis is that PDS activity exerts considerable control on excitation pressure, especially during chloroplast biogenesis when the photosynthetic electron transport chain is not yet fully functional and electrons from the desaturation reactions of carotenogenesis cannot be transferred efficiently to acceptors downstream of the

Several different types of electronic interactions between carotenoids and chlorophylls have

In Arabidopsis, the carotenoids–chlorophyll interactions parameter correlates with the nonphotochemical quenching (NPQ), and the fluorescence quenching of isolated major light-harvesting complex of photosystem II (LHCII). During the regulation of photosynthesis, the carotenoids excitation occurs after selective chlorophylls excitation. Furthermore, the new possibility to quantify the carotenoids–chlorophyll interactions in real time in intact plants will allow the identification of the exact site of these regulating interactions, using plant mutants in which specific chlorophyll and carotenoide binding sites

Light is a stimulus that activates a broad range of plant genes that participate in photosynthesis and photomorphogenesis. Carotenoids are required during photosynthesis in plants and algae and therefore, genes that direct the biosynthesis of carotenoids in these organisms are also regulated by light (von Lintig et al., 1997; Welsch et al., 2000; Simkin et

The process of de-etiolation of leaves has been used to compare the levels of carotenoids and gene expression in dark-grown plants versus plants that were transferred to light after being in darkness. During de-etiolation of *A. thaliana*, the expression of *ggpps* and *pds* genes are relatively constant, whereas expression of the single copy gene, *psy* and *hdr* are significantly enhanced (von Lintig et al*.*, 1997; Welsch et al*.*, 2000, Botella-Pavía et al*.,* 2004). Evidence indicates that the transcriptional activation of *psy, dxs and dxr* is essential for the induction of carotenoid biosynthesis in green organs (Welsch et al*.*, 2003; Toledo-Ortiz et al., 2010). During de-etiolation of tobacco (*Nicotiana tabacum*) and pepper, xanthophyll biosynthesis genes are transcriptionally activated after 3 or 5 h of continuous white-light illumination

(Simkin et al., 2003; Woitsch & Römer, 2003). In *A. thaliana* and tomato, *lcy*

expression increases 5 times when seedlings are transferred from a low light to a high light environment (Hirschberg, 2001). With the onset of red, blue or white light illumination,

mRNA

been proposed to play a key role as dissipation valves for excess excitation energy.

**3.3 Regulation of carotenoid expression in photosynthetic organs** 

al., 2003; Woitsch & Römer, 2003, Ohmiya et al., 2006; Briggs et al., 2007).

Steinbrenner & Linden, 2003; Woitsch & Römer, 2003).

PQ pool (Foudree et al., 2010).

are disrupted (Bode et al., 2009).

ubiquitination of transcription factors involved in phytochrome/cryptochrome transduction mechanisms.

The involvement of other photoreceptors such as phototropins, phytochrome C and E or CRY2 in the activation of carotenogenic genes has been evaluated through mutants. PhyC mutants, revealed that PHYC is involved in photomorphogenesis throughout the life cycle of *A. thaliana* playing a role in the perception of day length and acting with PHYB in the regulation of seedling de-etiolation in response to constant red light (Monte et al., 2003). As outlined above, regulation of light-mediated gene expression at the transcriptional level is the key mechanism controlling carotenogenesis in the plastids. Nonetheless, Schofield & Paliyath (2005) demonstrated post-translational control of PSY mediated by phytochrome. In red light exposed seedlings, PHY is activated which lead to an increase in PSY activity (Schofield & Paliyath, 2005). Therefore, light by means of photoreceptors, regulates carotenoid biosynthesis through transcriptional and post-transcriptional mechanisms.

#### **3.2 Carotenoid and chlorophyll biosynthesis are simultaneously regulated**

As mentioned previously, carotenoids carry out an essential function during photosynthesis in the antennae complexes of chloroplasts from green organs. Therefore, the regulation of the biosynthesis of chlorophyll and carotenoid biosynthesis are associated in photosynthetic organs (Woitsch & Römer, 2003; Joyard et al., 2009).

The photosynthetic machinery is composed of large multisubunit protein complexes composed of both plastidial and nuclear gene products, therefore a proper coordination and regulation of photosynthesis-associated nuclear genes (PhANG) and photosynthesisassociated plastidic genes is thought to be critical for proper chloroplast biogenesis. Light and plastidial signals trigger PhANG expression using common or adjacent promoter elements. A plastidial signal may convert multiple light signaling pathways, that perceive distinct qualities of light, from positive to negative regulators of some but not all PhANGs. Part of this remodeling of light signaling networks involves converting HY5, a positive regulator of PhANGs, into a negative regulator of PhANGs. In addition, mutants with defects in both plastid-to-nucleus and CRY1 signaling exhibited severe chlorophyll deficiencies.

Thus, the remodeling of light signaling networks induced by plastid signals is a mechanism that permits chloroplast biogenesis through the regulation of PhANG expression (Rucke et al., 2007)

White light induces a moderate stimulation of the expression of ppox, that encodes for protophorphirine oxidase (PPOX), an enzyme involved in chlorophyll biosynthesis, and simultaneously induces the expression of several carotenogenic genes (lcyβ, cβhx, violaxanthin de-epoxidase (vde) and zeaxanthin epoxidase (zep) genes). In addition, the psy gene, the fundamental gene that controls the biosynthesis of carotenoids, is co-expressed with photosynthetic genes that codify for plastoquinone, NAD(P)H deshydrogenase, tiorredoxin, plastocianin and ferredoxin (Meier et al, 2011). Moreover, according to the induction of carotenogenic genes during de-etiolation, chloroplyll genes are also induced (Woitsh & Römer, 2003) and the inhibition of lycopene cyclase with 2-(4 chlorophenylthiotriethyl-amine (CPTA) leads to accumulation of non-photoactive protochlorophyllide *a* (La Rocca et al., 2007). Also, PIF1 has been shown to bind to the promoter of PORC gene encoding Pchilide oxidoreductase whose activity is to convert Pchlide into chlorophylls (Moon et al., 2008).

ubiquitination of transcription factors involved in phytochrome/cryptochrome transduction

The involvement of other photoreceptors such as phototropins, phytochrome C and E or CRY2 in the activation of carotenogenic genes has been evaluated through mutants. PhyC mutants, revealed that PHYC is involved in photomorphogenesis throughout the life cycle of *A. thaliana* playing a role in the perception of day length and acting with PHYB in the regulation of seedling de-etiolation in response to constant red light (Monte et al., 2003). As outlined above, regulation of light-mediated gene expression at the transcriptional level is the key mechanism controlling carotenogenesis in the plastids. Nonetheless, Schofield & Paliyath (2005) demonstrated post-translational control of PSY mediated by phytochrome. In red light exposed seedlings, PHY is activated which lead to an increase in PSY activity (Schofield & Paliyath, 2005). Therefore, light by means of photoreceptors, regulates carotenoid biosynthesis through transcriptional and post-transcriptional mechanisms.

**3.2 Carotenoid and chlorophyll biosynthesis are simultaneously regulated** 

organs (Woitsch & Römer, 2003; Joyard et al., 2009).

As mentioned previously, carotenoids carry out an essential function during photosynthesis in the antennae complexes of chloroplasts from green organs. Therefore, the regulation of the biosynthesis of chlorophyll and carotenoid biosynthesis are associated in photosynthetic

The photosynthetic machinery is composed of large multisubunit protein complexes composed of both plastidial and nuclear gene products, therefore a proper coordination and regulation of photosynthesis-associated nuclear genes (PhANG) and photosynthesisassociated plastidic genes is thought to be critical for proper chloroplast biogenesis. Light and plastidial signals trigger PhANG expression using common or adjacent promoter elements. A plastidial signal may convert multiple light signaling pathways, that perceive distinct qualities of light, from positive to negative regulators of some but not all PhANGs. Part of this remodeling of light signaling networks involves converting HY5, a positive regulator of PhANGs, into a negative regulator of PhANGs. In addition, mutants with defects in both plastid-to-nucleus and CRY1 signaling exhibited severe chlorophyll

Thus, the remodeling of light signaling networks induced by plastid signals is a mechanism that permits chloroplast biogenesis through the regulation of PhANG expression (Rucke et

White light induces a moderate stimulation of the expression of ppox, that encodes for protophorphirine oxidase (PPOX), an enzyme involved in chlorophyll biosynthesis, and simultaneously induces the expression of several carotenogenic genes (lcyβ, cβhx, violaxanthin de-epoxidase (vde) and zeaxanthin epoxidase (zep) genes). In addition, the psy gene, the fundamental gene that controls the biosynthesis of carotenoids, is co-expressed with photosynthetic genes that codify for plastoquinone, NAD(P)H deshydrogenase, tiorredoxin, plastocianin and ferredoxin (Meier et al, 2011). Moreover, according to the induction of carotenogenic genes during de-etiolation, chloroplyll genes are also induced (Woitsh & Römer, 2003) and the inhibition of lycopene cyclase with 2-(4 chlorophenylthiotriethyl-amine (CPTA) leads to accumulation of non-photoactive protochlorophyllide *a* (La Rocca et al., 2007). Also, PIF1 has been shown to bind to the promoter of PORC gene encoding Pchilide oxidoreductase whose activity is to convert Pchlide into chlorophylls

mechanisms.

deficiencies.

al., 2007)

(Moon et al., 2008).

Chlorophyll and carotenoid biosynthesis are also regulated indirectly by light through the redox potential generated during photosynthesis. In this process, plastoquinone acts as a redox potential sensor responsible for the induction of carotenogenic genes, indicating that the biosynthesis of carotenoids is under photosynthetic redox control (Jöet et al., 2002; Steinbrenner & Linden, 2003; Woitsch & Römer, 2003).

Different experimental approaches were used to determine the regulatory mechanism in which carotenoid and photosynthetic components are involved to determine the chloroplast biogenesis. Arabidopsis *pds3* knockout mutant, or plants treated by norflurazon (NF) exert white tissues (photooxidized plastids) due to inactivation of PDS. The *immutans (im)*  variegation mutant, that has a defect in plastoquinol terminal oxidase IMMUTANS (IM) termed PTOX that transfers electrons from the plastoquinone (PQ) pool to molecular oxygen, presents variegated leaves. Considering the PQ pool as a potent initiator of retrograde signaling, a plausible hypothesis is that PDS activity exerts considerable control on excitation pressure, especially during chloroplast biogenesis when the photosynthetic electron transport chain is not yet fully functional and electrons from the desaturation reactions of carotenogenesis cannot be transferred efficiently to acceptors downstream of the PQ pool (Foudree et al., 2010).

Several different types of electronic interactions between carotenoids and chlorophylls have been proposed to play a key role as dissipation valves for excess excitation energy.

In Arabidopsis, the carotenoids–chlorophyll interactions parameter correlates with the nonphotochemical quenching (NPQ), and the fluorescence quenching of isolated major light-harvesting complex of photosystem II (LHCII). During the regulation of photosynthesis, the carotenoids excitation occurs after selective chlorophylls excitation.

Furthermore, the new possibility to quantify the carotenoids–chlorophyll interactions in real time in intact plants will allow the identification of the exact site of these regulating interactions, using plant mutants in which specific chlorophyll and carotenoide binding sites are disrupted (Bode et al., 2009).

#### **3.3 Regulation of carotenoid expression in photosynthetic organs**

Light is a stimulus that activates a broad range of plant genes that participate in photosynthesis and photomorphogenesis. Carotenoids are required during photosynthesis in plants and algae and therefore, genes that direct the biosynthesis of carotenoids in these organisms are also regulated by light (von Lintig et al., 1997; Welsch et al., 2000; Simkin et al., 2003; Woitsch & Römer, 2003, Ohmiya et al., 2006; Briggs et al., 2007).

The process of de-etiolation of leaves has been used to compare the levels of carotenoids and gene expression in dark-grown plants versus plants that were transferred to light after being in darkness. During de-etiolation of *A. thaliana*, the expression of *ggpps* and *pds* genes are relatively constant, whereas expression of the single copy gene, *psy* and *hdr* are significantly enhanced (von Lintig et al*.*, 1997; Welsch et al*.*, 2000, Botella-Pavía et al*.,* 2004). Evidence indicates that the transcriptional activation of *psy, dxs and dxr* is essential for the induction of carotenoid biosynthesis in green organs (Welsch et al*.*, 2003; Toledo-Ortiz et al., 2010).

During de-etiolation of tobacco (*Nicotiana tabacum*) and pepper, xanthophyll biosynthesis genes are transcriptionally activated after 3 or 5 h of continuous white-light illumination (Simkin et al., 2003; Woitsch & Römer, 2003). In *A. thaliana* and tomato, *lcy* mRNA expression increases 5 times when seedlings are transferred from a low light to a high light environment (Hirschberg, 2001). With the onset of red, blue or white light illumination,

Carotenoids and Photosynthesis - Regulation of Carotenoid Biosyntesis by Photoreceptors 87

genes, *psy1* and *psy2* code for phytoene synthase, and the former was found to be transcriptionally activated only in petals and ripening tomato fruits after continuous blue and white-light illumination (Welsch et al., 2000; Schofield & Paliyath, 2005; Giorio et al*.*, 2008). Transgenic tomato plants expressing an antisense fragment of *psy1* showed a 97% reduction in carotenoid levels in the fruit, while leaf carotenoids remained unaltered due to the expression of *psy2* (Fraser et al., 1999). *psy2* is expressed in all plant organs, preferentially in tomato leaves and petals (Giorio et al., 2008), but in green or ripe fruits it is only expressed at low levels (Bartley & Scolnik, 1993; Fraser et al., 1999; Giorio et al., 2008). *psy1* is also induced in the presence of ethylene, the major senescence hormone implicated in fruit ripening, indicating that PSY is a branch point in the regulation of carotenoid synthesis (Lois

Evidence emphasizing the importance of light effectors during fruit ripening and carotenoid accumulation was obtained through post-transcriptionally silencing of negative regulators of light signal transduction such as HP1 and HP2, as described above (Mustilli et al., 1999, Liu et al., 2004, Giovannoni, 2004). These high–pigment tomato mutants (*hp1* and *hp2*) have increased total ripe fruit carotenoids and are hypersensitive to light, having little effect on other ripening characteristics, similar to transgenic tomato plants that overexpress CRY

The up regulation of carotenoid gene expression during ripening has also been reported in

accumulate in parallel with the synthesis of carotenoids (Kita et al., 2007). In juice sacs of Satsuma mandarin (*Citrus reticulata*), Valencia orange (*C. sinensis*) and Lisbon lemon (*C. limon*) the expression of carotenoid biosynthetic genes such as CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP increases during fruit maturation, co-ordinately with the synthesis of carotenes and xanthophylls (Kato et al., 2004). In citrus of the "Star Ruby" cultivar, the high level of lycopene was correlated with a decrease in *CβHx* and *lcyb2* expression, genes associated to the synthesis of carotenoids in chromoplast (Alquezar et al., 2009). In *G. lutea* analysis of the expression of carotenogenic genes during flower development and in different plant organs indicated that *psy* was expressed in flowers

concomitant with carotenoid synthesis but not in stems and leaves (Zhu et al., 2002).

Carotenoids are also present in amyloplasts of potato and cereal seeds such as maize and wheat (*Triticum aestivum*; Panfili et al., 2004, Howitt & Pogson 2006; Nesterenko & Sink, 2003). Both potatoes and cereals accumulate low levels of carotenoid in the dark (Nesterenko and Sink, 2003) in contrast to the highly pigmented modified root of carrots. *Daucus carota* L. (carrot, 2n=18) is a biennal plant whose orange storage or modified root is consumed worldwide. Orange carrot contains high levels of -carotene and -carotene (8 mg/g dry weight, Fraser, 2004) that together constitute up to 95% of total carotenoids in the storage root Baranska et al., 2006). The kinetics of the transcript accumulation of some of the carotenogenic genes correlates with total carotenoid composition during the development of

We are focused in the study of carotenoid regulation in this novel plant model, taken in account that carotenoids in carrot are synthesized in leaves exposed to light, and also in the storage root that develops in darkness. All carotenogenic genes in carrot are expressed in both, leaves and roots during plant development, but the expression level is higher in leaves maybe due the faster exchange rate of carotenoids during photosynthesis (Beisel y et al., 2010). *Lcyb1* gene presents the higher increase in transcript level during leaves development and the paralogous genes, *psy1* and *psy2* are differentially expressed during development.

, *cβhx* and *zep* transcripts

et al., 2000).

(Davuluri et al., 2004; Giliberto et al. 2005).

other species. In Japanese apricot (*Prunus mume*) *psy*, *lcy*

storage roots grown in the dark (Clotault et al., 2008).

significant induction of the expression of carotenogenic genes was documented in etiolated seedlings of tobacco*,* regardless of the light quality used (Woitsch & Römer, 2003). The expression level was dependent of phytochrome and cryptochrome activities. However, considerable differences in expression levels were observed with respect to the type of light used to irradiate the seedlings. For example, *psy* gene expression was significantly induced after continuous red and white light illumination, pointing to an involvement of different photoreceptors in the regulation of their expression (Woitsch & Römer, 2003). PHY is involved in mediating the up-regulation of *psy2* gene expression during maize (*Zea mays*) seedling photoinduction (Li et al, 2008). Also *Lcy*, *cβhx* and *vde* are induced upon red light illumination. However, *zep* shows similar transcriptional activation in the presence of red or blue light (Woitsch & Römer, 2003).

Compared to normal carotenogenic gene induction mediated by light, the contribution of photo-oxidation to the amount of carotenoids produced in leaves is also important. Carotenoids are synthesized during light exposure but when light intensity increases from 150 to 280 mol/m2/s, the rate of photo oxidation is higher than the rate of synthesis and carotenoids are destroyed, reaching a certain basal level (Simkin et al., 2003). The level of expression of some carotenogenic genes is also reduced following prolonged illumination at moderate light intensities (Woitsch & Römer, 2003). During darkness, when photo oxidation of carotenoids does not occur, biosynthesis of carotenoids in leaves is stopped due principally to the very low level of expression of carotenogenic genes. In *C. annum*, *psy*, *pds*, *zds* and *lcy* genes are down regulated in darkness (Simkin et al., 2003) while in *A. thaliana* the *psy* and *hdr* are active in darkness only at basal levels (Welsch et al., 2003, Botella-Pavía et al., *2004*).

#### **3.4 Effect of light in non-photosyntetic organs**

Light has not only been analysed in photosynthetic tissue as a regulatory agent. In actual fact, light effect on carotenogenic pathway has been report in a number of species during physiological processes like fruit ripening and flower development (Zhu et al., 2003; Giovanonni, 2004; Adams-Phillips et al., 2004; Ohmiya et al., 2006).

In tomato, normal pigmentation of the fruits requires phytochrome-mediated light signal transduction, a process that does not affect other ripening characteristics, such as flavor (Alba et al., 2000). During tomato fruit ripening, carotenoid concentration increases 10 to 14 times, due mainly to accumulation of lycopene (Fraser et al., 1994). An increase in the synthesis of carotenoids is required during the transition from mature green to orange in tomato fruits. During this process, a coordinated upregulation of *dxs*, *hdr*, *pds* and *psy1* is observed, whilst at the same time the expression of *lcy, cyc* and *lcy* decreased (Fraser et al., 1994; Pecker et al., 1996; Ronen et al., 1999; Lois et al., 2000; Botella-Pavía et al., 2004). Two *lcy* genes have been identified in tomato, *cyc* and *lcy*. The first is responsible for carotenoid biosynthesis in chromoplasts whereas *lcy* performs this role preferentially in chloroplasts (Ronen et al., 1999). The down regulation of *lcy* and *cyc* in tomato during ripening leads to an accumulation of lycopene in chromoplasts of ripe fruits (Pecker et al., 1996; Ronen et al., 1999). In *C. annuum*, *lcy* is constitutively expressed during fruit ripening leading to an accumulation of -carotene and the red-pigmented capsanthin (Hugueney et al., 1995). The *psy* gene also plays a considerable role in controlling carotenoid synthesis during fruit development and ripening (Fraser et al., 1999, Giuliano et al., 1993) and during flower development (Zhu et al., 2002, Zhu et al., 2003). In tomato, two distantly-related

significant induction of the expression of carotenogenic genes was documented in etiolated seedlings of tobacco*,* regardless of the light quality used (Woitsch & Römer, 2003). The expression level was dependent of phytochrome and cryptochrome activities. However, considerable differences in expression levels were observed with respect to the type of light used to irradiate the seedlings. For example, *psy* gene expression was significantly induced after continuous red and white light illumination, pointing to an involvement of different photoreceptors in the regulation of their expression (Woitsch & Römer, 2003). PHY is involved in mediating the up-regulation of *psy2* gene expression during maize (*Zea mays*)

genes are down regulated in darkness (Simkin et al., 2003) while in *A. thaliana*

*, cyc* and *lcy*

and *cyc*

is constitutively expressed during fruit ripening

and *lcy*

decreased (Fraser et

in tomato during

. The first is responsible for

performs this role preferentially in

illumination. However, *zep* shows similar transcriptional activation in the presence of red or

Compared to normal carotenogenic gene induction mediated by light, the contribution of photo-oxidation to the amount of carotenoids produced in leaves is also important. Carotenoids are synthesized during light exposure but when light intensity increases from 150 to 280 mol/m2/s, the rate of photo oxidation is higher than the rate of synthesis and carotenoids are destroyed, reaching a certain basal level (Simkin et al., 2003). The level of expression of some carotenogenic genes is also reduced following prolonged illumination at moderate light intensities (Woitsch & Römer, 2003). During darkness, when photo oxidation of carotenoids does not occur, biosynthesis of carotenoids in leaves is stopped due principally to the very low level of expression of carotenogenic genes. In *C. annum*, *psy*, *pds*,

the *psy* and *hdr* are active in darkness only at basal levels (Welsch et al., 2003, Botella-Pavía

Light has not only been analysed in photosynthetic tissue as a regulatory agent. In actual fact, light effect on carotenogenic pathway has been report in a number of species during physiological processes like fruit ripening and flower development (Zhu et al., 2003;

In tomato, normal pigmentation of the fruits requires phytochrome-mediated light signal transduction, a process that does not affect other ripening characteristics, such as flavor (Alba et al., 2000). During tomato fruit ripening, carotenoid concentration increases 10 to 14 times, due mainly to accumulation of lycopene (Fraser et al., 1994). An increase in the synthesis of carotenoids is required during the transition from mature green to orange in tomato fruits. During this process, a coordinated upregulation of *dxs*, *hdr*, *pds* and *psy1* is

al., 1994; Pecker et al., 1996; Ronen et al., 1999; Lois et al., 2000; Botella-Pavía et al., 2004).

ripening leads to an accumulation of lycopene in chromoplasts of ripe fruits (Pecker et al.,

leading to an accumulation of -carotene and the red-pigmented capsanthin (Hugueney et al., 1995). The *psy* gene also plays a considerable role in controlling carotenoid synthesis during fruit development and ripening (Fraser et al., 1999, Giuliano et al., 1993) and during flower development (Zhu et al., 2002, Zhu et al., 2003). In tomato, two distantly-related

, *cβhx* and *vde* are induced upon red light

seedling photoinduction (Li et al, 2008). Also *Lcy*

**3.4 Effect of light in non-photosyntetic organs** 

observed, whilst at the same time the expression of *lcy*

carotenoid biosynthesis in chromoplasts whereas *lcy*

1996; Ronen et al., 1999). In *C. annuum*, *lcy*

genes have been identified in tomato, *cyc*

chloroplasts (Ronen et al., 1999). The down regulation of *lcy*

Giovanonni, 2004; Adams-Phillips et al., 2004; Ohmiya et al., 2006).

blue light (Woitsch & Römer, 2003).

*zds* and *lcy*

et al., *2004*).

Two *lcy*

genes, *psy1* and *psy2* code for phytoene synthase, and the former was found to be transcriptionally activated only in petals and ripening tomato fruits after continuous blue and white-light illumination (Welsch et al., 2000; Schofield & Paliyath, 2005; Giorio et al*.*, 2008). Transgenic tomato plants expressing an antisense fragment of *psy1* showed a 97% reduction in carotenoid levels in the fruit, while leaf carotenoids remained unaltered due to the expression of *psy2* (Fraser et al., 1999). *psy2* is expressed in all plant organs, preferentially in tomato leaves and petals (Giorio et al., 2008), but in green or ripe fruits it is only expressed at low levels (Bartley & Scolnik, 1993; Fraser et al., 1999; Giorio et al., 2008). *psy1* is also induced in the presence of ethylene, the major senescence hormone implicated in fruit ripening, indicating that PSY is a branch point in the regulation of carotenoid synthesis (Lois et al., 2000).

Evidence emphasizing the importance of light effectors during fruit ripening and carotenoid accumulation was obtained through post-transcriptionally silencing of negative regulators of light signal transduction such as HP1 and HP2, as described above (Mustilli et al., 1999, Liu et al., 2004, Giovannoni, 2004). These high–pigment tomato mutants (*hp1* and *hp2*) have increased total ripe fruit carotenoids and are hypersensitive to light, having little effect on other ripening characteristics, similar to transgenic tomato plants that overexpress CRY (Davuluri et al., 2004; Giliberto et al. 2005).

The up regulation of carotenoid gene expression during ripening has also been reported in other species. In Japanese apricot (*Prunus mume*) *psy*, *lcy*, *cβhx* and *zep* transcripts accumulate in parallel with the synthesis of carotenoids (Kita et al., 2007). In juice sacs of Satsuma mandarin (*Citrus reticulata*), Valencia orange (*C. sinensis*) and Lisbon lemon (*C. limon*) the expression of carotenoid biosynthetic genes such as CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP increases during fruit maturation, co-ordinately with the synthesis of carotenes and xanthophylls (Kato et al., 2004). In citrus of the "Star Ruby" cultivar, the high level of lycopene was correlated with a decrease in *CβHx* and *lcyb2* expression, genes associated to the synthesis of carotenoids in chromoplast (Alquezar et al., 2009). In *G. lutea* analysis of the expression of carotenogenic genes during flower development and in different plant organs indicated that *psy* was expressed in flowers concomitant with carotenoid synthesis but not in stems and leaves (Zhu et al., 2002).

Carotenoids are also present in amyloplasts of potato and cereal seeds such as maize and wheat (*Triticum aestivum*; Panfili et al., 2004, Howitt & Pogson 2006; Nesterenko & Sink, 2003). Both potatoes and cereals accumulate low levels of carotenoid in the dark (Nesterenko and Sink, 2003) in contrast to the highly pigmented modified root of carrots.

*Daucus carota* L. (carrot, 2n=18) is a biennal plant whose orange storage or modified root is consumed worldwide. Orange carrot contains high levels of -carotene and -carotene (8 mg/g dry weight, Fraser, 2004) that together constitute up to 95% of total carotenoids in the storage root Baranska et al., 2006). The kinetics of the transcript accumulation of some of the carotenogenic genes correlates with total carotenoid composition during the development of storage roots grown in the dark (Clotault et al., 2008).

We are focused in the study of carotenoid regulation in this novel plant model, taken in account that carotenoids in carrot are synthesized in leaves exposed to light, and also in the storage root that develops in darkness. All carotenogenic genes in carrot are expressed in both, leaves and roots during plant development, but the expression level is higher in leaves maybe due the faster exchange rate of carotenoids during photosynthesis (Beisel y et al., 2010). *Lcyb1* gene presents the higher increase in transcript level during leaves development and the paralogous genes, *psy1* and *psy2* are differentially expressed during development.

Carotenoids and Photosynthesis - Regulation of Carotenoid Biosyntesis by Photoreceptors 89

development of carrot modified roots in a reversible manner (Stange et al., 2008). Light inhibited storage root development, possibly because some transcriptional or growth factors are repressed, although more extensive studies are needed to investigate this

Light induces photomorphogenesis, chlorophyll and carotenoid biosynthesis through the signal transduction mediated by photoreceptors such as PHYA, PHYB and CRY in photosynthetic organs. At present, the principal components involved in the carotenogenic pathway have been described in many plant models, but fundamental knowledge regarding to the regulation is still necessary. In fact, *psy* gene may be the rate limiting step on carotenoid biosynthesis in leaves and also in chromoplasts accumulating organs. In addition, the highly regulated machinery on carotenoid biosynthesis can also be displayed through the organ specificity associated with carotenogenic gene function and their

New strategies aimed to elucidate the regulation of carotenoid pathway could be associated with transcriptome analysis which could provide insights into regulatory branch points of the pathway. Conventional studies focused on the identification and characterization of carotenogenic gene promoters could also help to understand the regulation of the expression of the genes in photosynthetic and in non-photosynthetic organs. In fact, light responsive elements (LRE) in such promoters could be associated with transcription factors involved in carotenogenic and chlorophyll gene expression. On the other hand, research focused in the adjustment of the light- mediated signal transduction machinery would also be an effective metabolic approach for modulating chlorophyll and fruit carotenoid

Adams-Philips, L.; Barry, C. & Giovannoni, J. (2004). Signal transduction system regulating

Ahmad, M.; Jarillo, JA.; Smirnova O. & Cashmore, AR. (1998). The CRY1 blue light

Alba, R.; Cordonnier-Pratt MM. & Pratt LH. (2000). Fruit localized phytochromes regulate

Alquézar, B.; Zacarías, L. & Rodrigo, MJ. (2009). Molecular and functional characterization

photoreceptor of Arabidopsis interacts with phytochrome A in vitro. *Mol Cell*,

lycopene accumulation independently of ethylene production in tomato. *Plant* 

of a novel chromoplast-specific lycopene β-cyclase from *Citrus* and its relation to lycopene accumulation. *Journal of Experimental Botany*, Vol.60, No.6, (March 2009),

fruit ripening. *Trends Plant Sci*, Vol.9, No.7, (July 2004), pp. 331-338.

phenomenon.

**4. Conclusion** 

correlation with chlorophyll biosynthesis.

composition in economically valuable plants.

Acknowledgements to the Chilean Grant Fondecyt 11080066

Vol.1, No.7, (June 1998), pp. 939–948.

*Physiol*, Vol.123, No.1, (May 2000), pp. 363-370.

**5. Acknowledgements** 

pp.1783-97.

**6. References** 

In roots, the expression of almost all carotenogenic genes are induced during storage root development and it correlates with carotenoid accumulation. In this organ carotenoids are stored in plastoglobuli in the chromoplasts, where they are more photo-stable than in chloroplasts (Merzlyak & Solovchenko, 2002). Therefore, photo-oxidation does not affect carotenoid content in these organs, even when they are exposed to light.

When roots were exposed to light, they did not develop normally and the expression of almost all genes differs from the pattern obtained in dark-grown roots during development (Figure 3A). In addition, the roots developed in the presence of light have the same carotenoid composition and amount as in leaves (Stange et al., 2008; Fuentes et al., 2011 in preparation). The thin non-orange carrot root also accumulates chloroplasts instead of chromoplasts, as leaves, and the carotenoid gene expression profile is almost the same as those expressed in the photosynthetic organ.

+++: high gene expression level, ++: middle gene expression level, +: low gene expression level. : expression increases during development, : expression decreases during development

Fig. 3. **Light affects morphology and carotenogenic gene expression in carrot roots A**; a comparison of carotenogenic gene expression in roots under light (R/L) and dark (R/D) conditions during the developmental process from 4 weeks to 12 weeks. Abbreviations: phytoene synthase 1 (psy 1), phytoene synthase 2 (psy 2), phytoene desaturase (pds), ζcarotene desaturase 1 (zds1), ζ-carotene desaturase 2 (zds2), lycopene β cyclase 1 (lcyb1), Develoment (Develop). **B**; changes in the phenotype of a 8 weeks old carrot root grown in light (R/L) and then transferred to dark conditions (R/D) until 12 weeks and 24 weeks. The root normal development is inhibited by light in a reversible manner (Modified from Stange et al., 2008)**.** 

Also, when the carrot root of an 8 weeks old plant was transferred from light to darkness, the root started to develop (Figure 3B). Therefore, light alters the morphology and

### **4. Conclusion**

phenomenon.

88 Advances in Photosynthesis – Fundamental Aspects

In roots, the expression of almost all carotenogenic genes are induced during storage root development and it correlates with carotenoid accumulation. In this organ carotenoids are stored in plastoglobuli in the chromoplasts, where they are more photo-stable than in chloroplasts (Merzlyak & Solovchenko, 2002). Therefore, photo-oxidation does not affect

When roots were exposed to light, they did not develop normally and the expression of almost all genes differs from the pattern obtained in dark-grown roots during development (Figure 3A). In addition, the roots developed in the presence of light have the same carotenoid composition and amount as in leaves (Stange et al., 2008; Fuentes et al., 2011 in preparation). The thin non-orange carrot root also accumulates chloroplasts instead of chromoplasts, as leaves, and the carotenoid gene expression profile is almost the same as

+++: high gene expression level, ++: middle gene expression level, +: low gene expression level. :

Fig. 3. **Light affects morphology and carotenogenic gene expression in carrot roots A**; a comparison of carotenogenic gene expression in roots under light (R/L) and dark (R/D) conditions during the developmental process from 4 weeks to 12 weeks. Abbreviations: phytoene synthase 1 (psy 1), phytoene synthase 2 (psy 2), phytoene desaturase (pds), ζcarotene desaturase 1 (zds1), ζ-carotene desaturase 2 (zds2), lycopene β cyclase 1 (lcyb1), Develoment (Develop). **B**; changes in the phenotype of a 8 weeks old carrot root grown in light (R/L) and then transferred to dark conditions (R/D) until 12 weeks and 24 weeks. The root normal development is inhibited by light in a reversible manner (Modified from Stange

Also, when the carrot root of an 8 weeks old plant was transferred from light to darkness, the root started to develop (Figure 3B). Therefore, light alters the morphology and

expression increases during development, : expression decreases during development

carotenoid content in these organs, even when they are exposed to light.

those expressed in the photosynthetic organ.

et al., 2008)**.** 

Light induces photomorphogenesis, chlorophyll and carotenoid biosynthesis through the signal transduction mediated by photoreceptors such as PHYA, PHYB and CRY in photosynthetic organs. At present, the principal components involved in the carotenogenic pathway have been described in many plant models, but fundamental knowledge regarding to the regulation is still necessary. In fact, *psy* gene may be the rate limiting step on carotenoid biosynthesis in leaves and also in chromoplasts accumulating organs. In addition, the highly regulated machinery on carotenoid biosynthesis can also be displayed through the organ specificity associated with carotenogenic gene function and their correlation with chlorophyll biosynthesis.

New strategies aimed to elucidate the regulation of carotenoid pathway could be associated with transcriptome analysis which could provide insights into regulatory branch points of the pathway. Conventional studies focused on the identification and characterization of carotenogenic gene promoters could also help to understand the regulation of the expression of the genes in photosynthetic and in non-photosynthetic organs. In fact, light responsive elements (LRE) in such promoters could be associated with transcription factors involved in carotenogenic and chlorophyll gene expression. On the other hand, research focused in the adjustment of the light- mediated signal transduction machinery would also be an effective metabolic approach for modulating chlorophyll and fruit carotenoid composition in economically valuable plants.

#### **5. Acknowledgements**

Acknowledgements to the Chilean Grant Fondecyt 11080066

#### **6. References**


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**5** 

*Poland* 

**Mechanisms of Photoacclimation on** 

Sabina Jodłowska and Adam Latała

*University of Gdańsk, Gdynia* 

**Photosynthesis Level in Cyanobacteria** 

*Department of Marine Ecosystems Functioning, Institute of Oceanography,* 

Cyanobacteria are oxygenic photoautotrophic prokaryotes, which develop in many aquatic environments, both freshwater and marine. They successfully grow in response to increasing eutrophication of water, but also because of shifts in the equilibrium of ecosystems (Stal et al., 2003). Cyanobacteria possess many unique adaptations allowing optimal growth and persistence, and the ability to out-compete algae during favorable conditions. For instance, many species are buoyant due to the possession of gas vesicles, some of them are capable of fixing N2, and unlike algae, which require carbon dioxide gas for photosynthesis, most cyanobacteria can utilize other sources of carbon, like bicarbonate, which are more plentiful in alkaline or high pH environments. The cyanobacteria live in a dynamic environment and are exposed to diurnal fluctuations of light. Planktonic species experience differences in irradiance when mixed in the water column (Staal et al., 2002), whereas mat-forming cyanobacteria are exposed to changes in light intensity caused by sediment covering or sediment dispersion. Such rapidly changing environmental factors forced photoautotrophic organisms to develop many acclimation mechanisms to minimize stress due to low and high light intensities. High irradiance may damage photosynthetic apparatus by photooxidation of chlorophyll *a* molecules. Some carotenoid pigments may provide effective protection against such disadvantageous influence of light (Hirschberg & Chamovitz, 1994; Steiger et al., 1999; Lakatos et al., 2001; MacIntyre et al., 2002). Photosynthetic organisms respond to decreased light intensity by increasing the size or/and the number of photosynthetic units (PSU) whose changes, in turn, can be reflected in characteristic patterns of P-E curves (Platt et al., 1980; Prézelin, 1981; Ramus, 1981; Richardson et al., 1983; Henley, 1993; Dring, 1998; Mouget et al., 1999; MacIntyre et al., 2002; Jodłowska & Latała, 2010). Variation in α and Pm (expressed per biomass or per chlorophyll *a* unit) plays a key part in interpreting physiological responses to changes in environmental

The aim of this review was to present exceptional properties of two different cyanobacteria, planktonic and benthic, their abilities to changing environmental condition, especially to irradiance. This information would be helpful in understanding the phenomenon of mass formation of cyanobacterial blooms worldwide, and would be very useful to interpret the

domination of cyanobacteria in water ecosystem in summer months.

**1. Introduction** 

conditions.


### **Mechanisms of Photoacclimation on Photosynthesis Level in Cyanobacteria**

Sabina Jodłowska and Adam Latała

*Department of Marine Ecosystems Functioning, Institute of Oceanography, University of Gdańsk, Gdynia Poland* 

#### **1. Introduction**

96 Advances in Photosynthesis – Fundamental Aspects

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synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development. *Biochem et Biophys Acta*, Vol.1625, No.3, (February Cyanobacteria are oxygenic photoautotrophic prokaryotes, which develop in many aquatic environments, both freshwater and marine. They successfully grow in response to increasing eutrophication of water, but also because of shifts in the equilibrium of ecosystems (Stal et al., 2003). Cyanobacteria possess many unique adaptations allowing optimal growth and persistence, and the ability to out-compete algae during favorable conditions. For instance, many species are buoyant due to the possession of gas vesicles, some of them are capable of fixing N2, and unlike algae, which require carbon dioxide gas for photosynthesis, most cyanobacteria can utilize other sources of carbon, like bicarbonate, which are more plentiful in alkaline or high pH environments. The cyanobacteria live in a dynamic environment and are exposed to diurnal fluctuations of light. Planktonic species experience differences in irradiance when mixed in the water column (Staal et al., 2002), whereas mat-forming cyanobacteria are exposed to changes in light intensity caused by sediment covering or sediment dispersion. Such rapidly changing environmental factors forced photoautotrophic organisms to develop many acclimation mechanisms to minimize stress due to low and high light intensities. High irradiance may damage photosynthetic apparatus by photooxidation of chlorophyll *a* molecules. Some carotenoid pigments may provide effective protection against such disadvantageous influence of light (Hirschberg & Chamovitz, 1994; Steiger et al., 1999; Lakatos et al., 2001; MacIntyre et al., 2002). Photosynthetic organisms respond to decreased light intensity by increasing the size or/and the number of photosynthetic units (PSU) whose changes, in turn, can be reflected in characteristic patterns of P-E curves (Platt et al., 1980; Prézelin, 1981; Ramus, 1981; Richardson et al., 1983; Henley, 1993; Dring, 1998; Mouget et al., 1999; MacIntyre et al., 2002; Jodłowska & Latała, 2010). Variation in α and Pm (expressed per biomass or per chlorophyll *a* unit) plays a key part in interpreting physiological responses to changes in environmental conditions.

The aim of this review was to present exceptional properties of two different cyanobacteria, planktonic and benthic, their abilities to changing environmental condition, especially to irradiance. This information would be helpful in understanding the phenomenon of mass formation of cyanobacterial blooms worldwide, and would be very useful to interpret the domination of cyanobacteria in water ecosystem in summer months.

Mechanisms of Photoacclimation on Photosynthesis Level in Cyanobacteria 99

of marine algae grown at different light intensities and different light spectrum suggest that phenotypic variations in pigment composition with depth are controlled by the irradiance level and not by the quality of the light (Ramus, 1981; Dring, 1992). However, the effect of light wavelength on pigment content of the cells appears to be restricted to some cyanobacteria. Changes in cell pigmentation in response to spectral quality of light result from modifications of the relative amounts of phycoerythrin (PE) and phycocyanin (PC). These phycobiliproteins are the major light-harvesting pigments used to drive photosynthesis. Only cyanobacteria which are able to synthesize PE can undergo

Light quality may be important factor controlling and regulating metabolic processes in algae and cyanobacteria, however in this study we consider to discuss the effect of changes

The photosynthesis rate achieved by a photoautotroph cell depends on the rate of capture of quantum of light. Certainly, this is determined by the light absorption properties of the photosynthetic biomass, composition of photosynthetic pigments in the cell and by the

The relationship between photosynthesis rate and light intensity is well described by photosynthetic light response curves (P-E). P-Es are very useful tools to predict primary productivity, and their analysis provides a lot of valuable information about photoacclimation mechanisms of cells (Platt et al., 1980; Ramus, 1981; Richardson, 1983; Henley, 1993; Dring, 1998; MacIntyre et al., 2002). The changes in concentration of chlorophyll *a* and other photosynthetic pigments in cell influence the course of P-E curves, which illustrate maximum rate of photosynthesis (Pm), the initial slope of photosynthetic curves (), the compensation (Pc) and saturation irradiances (Ek; the intercept between the initial slope of P-E and Pm) and dark respiration (R). The initial slope of photosynthetic curves at limiting light intensity (α) is a function of both light-harvesting efficiency and photosynthetic energy conversion efficiency (Henley, 1993). An increase of chlorophyll *a* concentration in the cell of photoautotrophic organism improves the effective absorption of light, what causes an increase of the P-E slope, in case the rate of photosynthesis is expressed in biomass unit. However, if photosynthesis is expressed in chlorophyll unit, it will not be observed the variability of α parameter. It is because of the photosynthetic rate at the limiting light intensity is proportional to the concentration of chlorophyll *a* (Dring, 1998). The chlorophyll *a* is arranged in the thylakoid membranes in photosynthetic units (PSU), each of which consists of 300-400 chlorophyll molecules associated with a single reaction centre. An increase in pigment concentration could be achieved either by building extra molecules into the existing PSUs without changing the number of reaction centers (called as a change of PSU size) or by building up complete new PSUs without changing their size (called as a change of PSU number) (Dring, 1998). These two mechanisms of photoacclimation have quite different effects on values of Pm. Since Pm is related to the number of reaction centers available, an increase in the size of PSUs have no effect on the maximum photosynthesis in biomass unit, that is why the photoautotrophs with different concentration of chlorophyll *a* have different P-E slope, but the same value of Pm. If, however, the photosynthesis rate is expressed in chlorophyll *a*, the value of Pm will drop with an increase of chlorophyll *a* concentration, but the P-E slope remains constant.

complementary chromatic adaptation (Tandeau de Marsac & Houmard, 1988).

of light intensity on photoacclimation on photosynthesis level.

**3. Photosynthesis and light response curves** 

intensity and spectral quality of the field (Kirk, 1996).

#### **2. Light as a major factor controlling distribution, growth and functionality of photoautotrophic organisms**

Light is one of the main trophic and morphogenetic factors in the life of photosynthetic organisms. Intensity, quality and the time of light impact affect photosynthesis, which is responsible for producing organic matter, cell division and the growth rate of organism. The positive effect of increasing light intensity states only to a point, after which stabilization or even a drop in cell division takes place (Ostroff et al., 1980; Latała & Misiewicz, 2000).

Effect of light on organisms can be investigated in two ways, firstly in ecological perspective i.e. paying special attention to the influence of this factor on distribution, and secondly in physiological point of view studying mechanisms of acclimation facilitating to survive in changing environmental conditions. Light is one of the main factors controlling the distribution of photoautotrophs in water column demonstrating their light preferences. Similarly to vascular plants, algae and cyanobacteria may also indicate their shadowtolerant features or heliophylous character (Falkowski & La Roche, 1991). This phenomenon is observed both in case of benthic forms attached to the bottom and planktonic form floated in water column. Shadow-tolerant autotrophs concentrate in deeper water, where intensity of light is significantly lower, whereas heliophylous ones, preferring higher light intensity to growth and functionality, live in shallows. However, at surface layer of water phototrophic organisms are exposed to harmful effect of very high intensity of light and also effect of ultraviolet radiation, what may cause photoinhibition. Moreover, at deep water body we can define the level, below which photoautotrophic life gradually disappeared. The depth, at which anabolic and catabolic processes are balanced, is named as compensation level. Below this level light condition are insufficient, so that photosynthetic organisms are not able to normally develop and reproduce (Falkowski & La Roche, 1991).

In natural conditions, phytoplankton which is vertically transferred within the euphotic zone experience not only changes of intensity but as well spectral quality (Rivkin, 1989). Spectral composition of downwelling light change progressively with increasing depth. Changes in spectral quality depend on the absorption spectra and scattering of the suspended particles within the water column and on absorption of water itself and the angle at which light impinges the water surface (Staal et al., 2002). In regions poor in nutrients the longer wavelengths are selectively absorbed within the upper ca 10 m and only blue-green light remains. However, in coastal regions rich in biogenic substances and yellow substances the shorter wavelengths are rapidly absorbed (Kirk, 1983; Rivkin, 1989).

When autotrophic organisms experience changes in light regime, both intensity and spectral quality, acclimation of photosynthetic apparatus to variable light condition is observed. Photoacclimation of these organisms is linked to alterations in total cellular concentration of light-harvesting and reaction centre pigments, and also in the ratio of different pigments. Generally, one observes an inverse correlation between light intensity and pigment content: the less light energy available, the more photosynthetic pigments are synthesized by the cells (Tandeau de Marsac & Houmard, 1988). According to the idea proposed by Engelmann in 1883 different groups of marine algae dominate the benthic vegetation at different depths because their pigment composition adapts them for absorption, and hence photosynthesis, in light quality that prevails at that depth. This phenomenon is known as chromatic adaptation. It has often been pointed out that this concept is based on extremely superficial generalizations about the vertical distribution of benthic algae, since the representatives of most of the major groups can be observed at most depths (Dring, 1992). Laboratory studies

**2. Light as a major factor controlling distribution, growth and functionality of** 

Light is one of the main trophic and morphogenetic factors in the life of photosynthetic organisms. Intensity, quality and the time of light impact affect photosynthesis, which is responsible for producing organic matter, cell division and the growth rate of organism. The positive effect of increasing light intensity states only to a point, after which stabilization or even a drop in cell division takes place (Ostroff et al., 1980; Latała & Misiewicz, 2000). Effect of light on organisms can be investigated in two ways, firstly in ecological perspective i.e. paying special attention to the influence of this factor on distribution, and secondly in physiological point of view studying mechanisms of acclimation facilitating to survive in changing environmental conditions. Light is one of the main factors controlling the distribution of photoautotrophs in water column demonstrating their light preferences. Similarly to vascular plants, algae and cyanobacteria may also indicate their shadowtolerant features or heliophylous character (Falkowski & La Roche, 1991). This phenomenon is observed both in case of benthic forms attached to the bottom and planktonic form floated in water column. Shadow-tolerant autotrophs concentrate in deeper water, where intensity of light is significantly lower, whereas heliophylous ones, preferring higher light intensity to growth and functionality, live in shallows. However, at surface layer of water phototrophic organisms are exposed to harmful effect of very high intensity of light and also effect of ultraviolet radiation, what may cause photoinhibition. Moreover, at deep water body we can define the level, below which photoautotrophic life gradually disappeared. The depth, at which anabolic and catabolic processes are balanced, is named as compensation level. Below this level light condition are insufficient, so that photosynthetic organisms are not

able to normally develop and reproduce (Falkowski & La Roche, 1991).

the shorter wavelengths are rapidly absorbed (Kirk, 1983; Rivkin, 1989).

In natural conditions, phytoplankton which is vertically transferred within the euphotic zone experience not only changes of intensity but as well spectral quality (Rivkin, 1989). Spectral composition of downwelling light change progressively with increasing depth. Changes in spectral quality depend on the absorption spectra and scattering of the suspended particles within the water column and on absorption of water itself and the angle at which light impinges the water surface (Staal et al., 2002). In regions poor in nutrients the longer wavelengths are selectively absorbed within the upper ca 10 m and only blue-green light remains. However, in coastal regions rich in biogenic substances and yellow substances

When autotrophic organisms experience changes in light regime, both intensity and spectral quality, acclimation of photosynthetic apparatus to variable light condition is observed. Photoacclimation of these organisms is linked to alterations in total cellular concentration of light-harvesting and reaction centre pigments, and also in the ratio of different pigments. Generally, one observes an inverse correlation between light intensity and pigment content: the less light energy available, the more photosynthetic pigments are synthesized by the cells (Tandeau de Marsac & Houmard, 1988). According to the idea proposed by Engelmann in 1883 different groups of marine algae dominate the benthic vegetation at different depths because their pigment composition adapts them for absorption, and hence photosynthesis, in light quality that prevails at that depth. This phenomenon is known as chromatic adaptation. It has often been pointed out that this concept is based on extremely superficial generalizations about the vertical distribution of benthic algae, since the representatives of most of the major groups can be observed at most depths (Dring, 1992). Laboratory studies

**photoautotrophic organisms** 

of marine algae grown at different light intensities and different light spectrum suggest that phenotypic variations in pigment composition with depth are controlled by the irradiance level and not by the quality of the light (Ramus, 1981; Dring, 1992). However, the effect of light wavelength on pigment content of the cells appears to be restricted to some cyanobacteria. Changes in cell pigmentation in response to spectral quality of light result from modifications of the relative amounts of phycoerythrin (PE) and phycocyanin (PC). These phycobiliproteins are the major light-harvesting pigments used to drive photosynthesis. Only cyanobacteria which are able to synthesize PE can undergo complementary chromatic adaptation (Tandeau de Marsac & Houmard, 1988).

Light quality may be important factor controlling and regulating metabolic processes in algae and cyanobacteria, however in this study we consider to discuss the effect of changes of light intensity on photoacclimation on photosynthesis level.

#### **3. Photosynthesis and light response curves**

The photosynthesis rate achieved by a photoautotroph cell depends on the rate of capture of quantum of light. Certainly, this is determined by the light absorption properties of the photosynthetic biomass, composition of photosynthetic pigments in the cell and by the intensity and spectral quality of the field (Kirk, 1996).

The relationship between photosynthesis rate and light intensity is well described by photosynthetic light response curves (P-E). P-Es are very useful tools to predict primary productivity, and their analysis provides a lot of valuable information about photoacclimation mechanisms of cells (Platt et al., 1980; Ramus, 1981; Richardson, 1983; Henley, 1993; Dring, 1998; MacIntyre et al., 2002). The changes in concentration of chlorophyll *a* and other photosynthetic pigments in cell influence the course of P-E curves, which illustrate maximum rate of photosynthesis (Pm), the initial slope of photosynthetic curves (), the compensation (Pc) and saturation irradiances (Ek; the intercept between the initial slope of P-E and Pm) and dark respiration (R). The initial slope of photosynthetic curves at limiting light intensity (α) is a function of both light-harvesting efficiency and photosynthetic energy conversion efficiency (Henley, 1993). An increase of chlorophyll *a* concentration in the cell of photoautotrophic organism improves the effective absorption of light, what causes an increase of the P-E slope, in case the rate of photosynthesis is expressed in biomass unit. However, if photosynthesis is expressed in chlorophyll unit, it will not be observed the variability of α parameter. It is because of the photosynthetic rate at the limiting light intensity is proportional to the concentration of chlorophyll *a* (Dring, 1998). The chlorophyll *a* is arranged in the thylakoid membranes in photosynthetic units (PSU), each of which consists of 300-400 chlorophyll molecules associated with a single reaction centre. An increase in pigment concentration could be achieved either by building extra molecules into the existing PSUs without changing the number of reaction centers (called as a change of PSU size) or by building up complete new PSUs without changing their size (called as a change of PSU number) (Dring, 1998). These two mechanisms of photoacclimation have quite different effects on values of Pm. Since Pm is related to the number of reaction centers available, an increase in the size of PSUs have no effect on the maximum photosynthesis in biomass unit, that is why the photoautotrophs with different concentration of chlorophyll *a* have different P-E slope, but the same value of Pm. If, however, the photosynthesis rate is expressed in chlorophyll *a*, the value of Pm will drop with an increase of chlorophyll *a* concentration, but the P-E slope remains constant.

Mechanisms of Photoacclimation on Photosynthesis Level in Cyanobacteria 101

increased temperatures. Optimal growth conditions were noted at about 180-290 mol photonsm-2s-1 and 15-17°C. The maximum number of filament units (about 5·105 filament units·ml-1) was about 10x greater than the minimum one. Similarly, the factorial experiments on *G. amphibium* showed that both irradiance and temperature have promoting effect on cyanobacterial culture concentration, but this positive effect was observed up to 120 µmol photons·m-2·s-1 at 35°C as well as up to 170 µmol photons·m-2·s-1 at 30°C (Fig. 1B). However, the interaction between temperature of 35°C and light intensity of 170 µmol photons·m-2·s-1 resulted in growth inhibition (Latała & Misiewicz, 2000). An excess of light energy absorbed by photosynthetic pigments together with high-temperature stress may accelerate photoinhibition by inhibiting the repair of photodamaged PSII (Allakhverdiev et al., 2008; Takahashi & Murata, 2008; Takahashi & Badger, 2011). Richardson & her co-workers (1983)

Fig. 1. Response-surface estimation at different temperatures (°C) and irradiances (mol photonsm-2s-1) of: A) *N. spumigena* filament concentration (1 filament unit = 100 µm), B) *G. amphibium* optical density at 750 nm. Experimental cultures were grown in three replicates and the measurements of culture were done on the last day of incubation in the exponential

growth phase.

However, when the number of PSUs increases proportionally to the increase in chlorophyll *a* concentration, the maximum photosynthesis in biomass unit will also increase in proportion to the chlorophyll *a* concentration, but this means that Pm in chlorophyll unit will be constant (Dring, 1998). According to the Ramus (1981) model, if the number of PSUs increases, more quantum of light is necessary to saturate the photosynthesis (higher Ek and Pm), but if only the size of PSUs increases without any change of their number, not much light will be need to saturate the photosynthesis (lower Ek and Pm). In the second case, PSU will be working more effectively (Lobban and Harrison, 1997).

#### **4. Photoacclimation strategies in two strains of cyanobacteria: Planktonic** *Nodularia spumigena* **and benthic** *Geitlerinema amphibium*

Cyanobacteria are very interesting material to study relationships between biological activity of organism and various environmental factors, because they often exist in extreme circumstances and can acclimate efficiently to changing environmental conditions (Latała & Misiewicz, 2000).

The experiments were conducted on two different strains of Baltic cyanobacteria: planktonic *Nodularia spumigena* and benthic *Geitlerinema amphibium*. The investigated strains were isolated from southern Baltic Sea, exactly from the coastal zone of the Gulf of Gdańsk, and now they were maintained as an unialgal cultures in the Culture Collection of Baltic Algae (CCBA) (http://ccba.ug.edu.pl) at the Institute of Oceanography, University of Gdańsk, Poland (Latała et al., 2006). Buoyant cyanobacteria, like *N. spumigena*, previously mixed throughout the water column, float to the surface of water, where their cells are exposed to full sunlight, and this abrupt change in irradiance may induce photoinhibition (Ibelings, 1996). This species of cyanobacterium always occurs at water temperatures over 18ºC and during weather conditions conductive to water column thermal stratification (Hobson et al., 1999). It is one of the filamentous cyanobacteria, which regularly occurs in the Baltic water in summer and often forms toxic blooms. *N. spumigena* grows especially well in the illuminated upper layer of the euphotic zone. In the Baltic Sea, it is most abundant to depths of 5 m (Hajdu et al., 2007), but it is also observed as deep as 18 m (Stal & Walsby 2000; Jodłowska & Latała, 2010). Similarly, mat-forming cyanobacterium, like *G. amphibium*, experience changes in light regime. These organisms may periodically be covered by a layer of sediment, which limits availability of light. On the other hands, sediment dispersion can lead to a rise in light intensity. *G. amphibium*, as a permanent element of summer microbial mats in the Gulf of Gdańsk (Southern Baltic) (Witkowski, 1986), may be an example of how organisms make use of their outstanding acclimation ability in the best possible way (Jodłowska & Latała, 2010).

According to literature data cyanobacteria are generally recognized to prefer low light intensity for growth and photosynthesis (Fogg & Thake, 1987; Ibelings, 1996). However, the investigated strain of *N. spumigena* was found to be well acclimated to relatively high light intensity (290 mol photonsm-2s-1), which was especially evident within the range of temperature 15-20ºC (Fig. 1A). The factorial experiments with *N. spumigena* showed that irradiance had a promoting effect on cyanobacterial concentration, but the interaction between increasing irradiance of over about 100 mol photonsm-2s-1 and increasing temperature over about 23ºC inhibited the filament concentration. Prolonged exposure to high light intensity may cause photoinhibition, and induce harmful effects resulting from

However, when the number of PSUs increases proportionally to the increase in chlorophyll *a* concentration, the maximum photosynthesis in biomass unit will also increase in proportion to the chlorophyll *a* concentration, but this means that Pm in chlorophyll unit will be constant (Dring, 1998). According to the Ramus (1981) model, if the number of PSUs increases, more quantum of light is necessary to saturate the photosynthesis (higher Ek and Pm), but if only the size of PSUs increases without any change of their number, not much light will be need to saturate the photosynthesis (lower Ek and Pm). In the second case, PSU

will be working more effectively (Lobban and Harrison, 1997).

Misiewicz, 2000).

(Jodłowska & Latała, 2010).

**4. Photoacclimation strategies in two strains of cyanobacteria:** 

**Planktonic** *Nodularia spumigena* **and benthic** *Geitlerinema amphibium*

Cyanobacteria are very interesting material to study relationships between biological activity of organism and various environmental factors, because they often exist in extreme circumstances and can acclimate efficiently to changing environmental conditions (Latała &

The experiments were conducted on two different strains of Baltic cyanobacteria: planktonic *Nodularia spumigena* and benthic *Geitlerinema amphibium*. The investigated strains were isolated from southern Baltic Sea, exactly from the coastal zone of the Gulf of Gdańsk, and now they were maintained as an unialgal cultures in the Culture Collection of Baltic Algae (CCBA) (http://ccba.ug.edu.pl) at the Institute of Oceanography, University of Gdańsk, Poland (Latała et al., 2006). Buoyant cyanobacteria, like *N. spumigena*, previously mixed throughout the water column, float to the surface of water, where their cells are exposed to full sunlight, and this abrupt change in irradiance may induce photoinhibition (Ibelings, 1996). This species of cyanobacterium always occurs at water temperatures over 18ºC and during weather conditions conductive to water column thermal stratification (Hobson et al., 1999). It is one of the filamentous cyanobacteria, which regularly occurs in the Baltic water in summer and often forms toxic blooms. *N. spumigena* grows especially well in the illuminated upper layer of the euphotic zone. In the Baltic Sea, it is most abundant to depths of 5 m (Hajdu et al., 2007), but it is also observed as deep as 18 m (Stal & Walsby 2000; Jodłowska & Latała, 2010). Similarly, mat-forming cyanobacterium, like *G. amphibium*, experience changes in light regime. These organisms may periodically be covered by a layer of sediment, which limits availability of light. On the other hands, sediment dispersion can lead to a rise in light intensity. *G. amphibium*, as a permanent element of summer microbial mats in the Gulf of Gdańsk (Southern Baltic) (Witkowski, 1986), may be an example of how organisms make use of their outstanding acclimation ability in the best possible way

According to literature data cyanobacteria are generally recognized to prefer low light intensity for growth and photosynthesis (Fogg & Thake, 1987; Ibelings, 1996). However, the investigated strain of *N. spumigena* was found to be well acclimated to relatively high light intensity (290 mol photonsm-2s-1), which was especially evident within the range of temperature 15-20ºC (Fig. 1A). The factorial experiments with *N. spumigena* showed that irradiance had a promoting effect on cyanobacterial concentration, but the interaction between increasing irradiance of over about 100 mol photonsm-2s-1 and increasing temperature over about 23ºC inhibited the filament concentration. Prolonged exposure to high light intensity may cause photoinhibition, and induce harmful effects resulting from increased temperatures. Optimal growth conditions were noted at about 180-290 mol photonsm-2s-1 and 15-17°C. The maximum number of filament units (about 5·105 filament units·ml-1) was about 10x greater than the minimum one. Similarly, the factorial experiments on *G. amphibium* showed that both irradiance and temperature have promoting effect on cyanobacterial culture concentration, but this positive effect was observed up to 120 µmol photons·m-2·s-1 at 35°C as well as up to 170 µmol photons·m-2·s-1 at 30°C (Fig. 1B). However, the interaction between temperature of 35°C and light intensity of 170 µmol photons·m-2·s-1 resulted in growth inhibition (Latała & Misiewicz, 2000). An excess of light energy absorbed by photosynthetic pigments together with high-temperature stress may accelerate photoinhibition by inhibiting the repair of photodamaged PSII (Allakhverdiev et al., 2008; Takahashi & Murata, 2008; Takahashi & Badger, 2011). Richardson & her co-workers (1983)

Fig. 1. Response-surface estimation at different temperatures (°C) and irradiances (mol photonsm-2s-1) of: A) *N. spumigena* filament concentration (1 filament unit = 100 µm), B) *G. amphibium* optical density at 750 nm. Experimental cultures were grown in three replicates and the measurements of culture were done on the last day of incubation in the exponential growth phase.

Mechanisms of Photoacclimation on Photosynthesis Level in Cyanobacteria 103

The lower cellular content of chlorophyll *a* noted in the population acclimated to high light is associated with a decrease in the size and/or the number of photosynthetic units (PSUs) that can be reflected by P-E curves (Platt et al., 1980; Prézelin, 1981; Ramus, 1981; Richardson et al., 1983; Henley, 1993; Dring, 1998; Mouget et al., 1999; MacIntyre et al., 2002; Jodłowska & Latała, 2010). P-E curves obtained for *N. spumigena* and *G. amphibium* grown under different light and temperature conditions illustrate that these two strains conforms to more than one of the photoadaptive models used to categorize species (Fig. 3) (Jodłowska & Latała, 2010). Photosynthetic rates, normalized to biomass and chlorophyll unit, were plotted against irradiance. The P-E curves were fitted to the data with Statistica using the mathematical function by Platt & Jassby (1976), as follows: P=Pmtanh(αE/Pm)+Rd. To illustrate the course of P-E curves, the results recorded at 15°C were chosen (Fig. 3 and 4). In *N. spumigena* culturing at 15ºC, the biomass-specific Pm was about 70% higher in the low light treatment than in the high light treatment (Fig. 3A), whereas in *G. amphibium* the same difference was about 60% (Fig. 3B). Since the biomass-specific Pm noted in the strains acclimated to low light was higher than that grown in high light, it indicates a change in the number of the PSUs. However, higher chl*a*-specific Pm in the strains acclimated to high light in comparison to that in the low light strain indicates there was a change in the size of the PSUs. In *N. spumigena* culturing at 15ºC, the chlorophyll-specific Pm was about 45% higher in the high light treatment than in the low light treatment (Fig. 3B), whereas in *G. amphibium*

These two mechanisms of photoacclimation, concerning the changes in the size and the number of PSUs, explain significant changes in photosynthesis rate and its parameters upon the influence of different light intensities and temperatures (Fig. 5). It is noteworthy that *N. spumigena* and *G. amphibium* exhibit substantial changes in Ek and Pc within the range of irradiance tested. However, it is typical for Ek values to increase in phototrophic populations as irradiance increases (Richardson et al., 1983), whereas the link between the evolution of compensation light intensity (Pc) and increased irradiance is somewhat more characteristic of Chlorophyta (Falkowski & Owens, 1980). The results of variance analysis showed that both irradiance and temperature were important factors contributing to the variation of Ek and Pc parameters at the investigated strains, but the influence of irradiance was higher. In *N. spumigena*, the minimum values of Ek (about 145 mol photonsm-2s-1) were about 47% lower than the maximum ones (about 275 mol photonsm-2s-1) (Fig. 5A), but in *G. amphibium* the differences were higher, and were about 94% (Fig. 5B). In contrast, in cyanobacterium *N. spumigena*, the minimum values of Pc (about 10 mol photonsm-2s-1) were lower about 95% than the maximum ones (about 180 mol photonsm-2s-1) (Fig. 5C), whereas in *G. amphibium* the minimum Pc (about 5 mol photonsm-2s-1) were lower about 93% than the maximum ones (about 70 mol photonsm-2s-1) (Fig. 5D). The obtained minimum values of Ek and Pc are close to those reported for shadow-tolerant plants, while the maximum ones are close to those noted in heliophylous plants (Rabinowitch, 1951; Wallentinus, 1978). Achieved results for both parameters showed good acclimation capacity of the investigated species to irradiance changes. It is also noteworthy that P-E curves for investigated cyanobacteria did not indicate photosynthetic photoinhibition until approximately 700-1000 mol photons m-2 s-1, even if the cyanobacteria were acclimated to very low light intensity (5 -10 mol photons m-2 s-1). Moisander et al. (2002) did not also note

this phenomenon in *N. spumigena* at irradiances exceeding 1000 mol photons m-2 s-1.

the same difference was about 70% (Fig. 4B).

suggested that photoinhibition of the growth and photosynthesis occurred in the natural populations at above 200 mol photonsm-2s-1, although a lot of species can be subjected to this phenomenon at lower irradiance. *Aphanizomenon ovalisporum* indicated inhibition of the cell concentration even at 50 mol photonsm-2s-1 and 20-35ºC (Hadas et al., 2002).

The experiments on *N. spumigena* and *G. amphibium* showed that both irradiance and temperature were important factors contributing to the variation of chlorophyll *a* content at the investigated strains, but the influence of irradiance was higher (Latała & Misiewicz, 2000; Jodłowska & Latała, 2010) (Fig. 2). In two investigated strains*,* pigment content was negatively affected by irradiance and positively by temperature. The highest values of pigment content at low light treatment and high temperature treatment were almost 95% and 85% for *N. spumigena* and *G. amphibium,* respectively, higher than the lowest ones at high light treatment and low temperature treatment.

Fig. 2. Response-surface estimation of chlorophyll *a* content (pg·filament unit-1): A) *N. spumigena* and B) *G. amphibium* at different temperatures (°C) and irradiances (mol photonsm-2s-1).

suggested that photoinhibition of the growth and photosynthesis occurred in the natural populations at above 200 mol photonsm-2s-1, although a lot of species can be subjected to this phenomenon at lower irradiance. *Aphanizomenon ovalisporum* indicated inhibition of the

The experiments on *N. spumigena* and *G. amphibium* showed that both irradiance and temperature were important factors contributing to the variation of chlorophyll *a* content at the investigated strains, but the influence of irradiance was higher (Latała & Misiewicz, 2000; Jodłowska & Latała, 2010) (Fig. 2). In two investigated strains*,* pigment content was negatively affected by irradiance and positively by temperature. The highest values of pigment content at low light treatment and high temperature treatment were almost 95% and 85% for *N. spumigena* and *G. amphibium,* respectively, higher than the lowest ones at

cell concentration even at 50 mol photonsm-2s-1 and 20-35ºC (Hadas et al., 2002).

Fig. 2. Response-surface estimation of chlorophyll *a* content (pg·filament unit-1): A) *N. spumigena* and B) *G. amphibium* at different temperatures (°C) and irradiances (mol

photonsm-2s-1).

high light treatment and low temperature treatment.

The lower cellular content of chlorophyll *a* noted in the population acclimated to high light is associated with a decrease in the size and/or the number of photosynthetic units (PSUs) that can be reflected by P-E curves (Platt et al., 1980; Prézelin, 1981; Ramus, 1981; Richardson et al., 1983; Henley, 1993; Dring, 1998; Mouget et al., 1999; MacIntyre et al., 2002; Jodłowska & Latała, 2010). P-E curves obtained for *N. spumigena* and *G. amphibium* grown under different light and temperature conditions illustrate that these two strains conforms to more than one of the photoadaptive models used to categorize species (Fig. 3) (Jodłowska & Latała, 2010). Photosynthetic rates, normalized to biomass and chlorophyll unit, were plotted against irradiance. The P-E curves were fitted to the data with Statistica using the mathematical function by Platt & Jassby (1976), as follows: P=Pmtanh(αE/Pm)+Rd. To illustrate the course of P-E curves, the results recorded at 15°C were chosen (Fig. 3 and 4). In *N. spumigena* culturing at 15ºC, the biomass-specific Pm was about 70% higher in the low light treatment than in the high light treatment (Fig. 3A), whereas in *G. amphibium* the same difference was about 60% (Fig. 3B). Since the biomass-specific Pm noted in the strains acclimated to low light was higher than that grown in high light, it indicates a change in the number of the PSUs. However, higher chl*a*-specific Pm in the strains acclimated to high light in comparison to that in the low light strain indicates there was a change in the size of the PSUs. In *N. spumigena* culturing at 15ºC, the chlorophyll-specific Pm was about 45% higher in the high light treatment than in the low light treatment (Fig. 3B), whereas in *G. amphibium* the same difference was about 70% (Fig. 4B).

These two mechanisms of photoacclimation, concerning the changes in the size and the number of PSUs, explain significant changes in photosynthesis rate and its parameters upon the influence of different light intensities and temperatures (Fig. 5). It is noteworthy that *N. spumigena* and *G. amphibium* exhibit substantial changes in Ek and Pc within the range of irradiance tested. However, it is typical for Ek values to increase in phototrophic populations as irradiance increases (Richardson et al., 1983), whereas the link between the evolution of compensation light intensity (Pc) and increased irradiance is somewhat more characteristic of Chlorophyta (Falkowski & Owens, 1980). The results of variance analysis showed that both irradiance and temperature were important factors contributing to the variation of Ek and Pc parameters at the investigated strains, but the influence of irradiance was higher. In *N. spumigena*, the minimum values of Ek (about 145 mol photonsm-2s-1) were about 47% lower than the maximum ones (about 275 mol photonsm-2s-1) (Fig. 5A), but in *G. amphibium* the differences were higher, and were about 94% (Fig. 5B). In contrast, in cyanobacterium *N. spumigena*, the minimum values of Pc (about 10 mol photonsm-2s-1) were lower about 95% than the maximum ones (about 180 mol photonsm-2s-1) (Fig. 5C), whereas in *G. amphibium* the minimum Pc (about 5 mol photonsm-2s-1) were lower about 93% than the maximum ones (about 70 mol photonsm-2s-1) (Fig. 5D). The obtained minimum values of Ek and Pc are close to those reported for shadow-tolerant plants, while the maximum ones are close to those noted in heliophylous plants (Rabinowitch, 1951; Wallentinus, 1978). Achieved results for both parameters showed good acclimation capacity of the investigated species to irradiance changes. It is also noteworthy that P-E curves for investigated cyanobacteria did not indicate photosynthetic photoinhibition until approximately 700-1000 mol photons m-2 s-1, even if the cyanobacteria were acclimated to very low light intensity (5 -10 mol photons m-2 s-1). Moisander et al. (2002) did not also note this phenomenon in *N. spumigena* at irradiances exceeding 1000 mol photons m-2 s-1.

Mechanisms of Photoacclimation on Photosynthesis Level in Cyanobacteria 105

Fig. 4. P-E curves for *G. amphibium* growing at three light intensity (mol photonsm-2s-1)

and at 15ºC: A) in biomass unit, B) in chlorophyll unit.

Fig. 3. P-E curves for *N. spumigena* growing at three light intensity (mol photonsm-2s-1) and at 15ºC: A) in filament unit, B) in chlorophyll unit (Jodłowska & Latała, 2010).

Fig. 3. P-E curves for *N. spumigena* growing at three light intensity (mol photonsm-2s-1) and at 15ºC: A) in filament unit, B) in chlorophyll unit (Jodłowska & Latała, 2010).

Fig. 4. P-E curves for *G. amphibium* growing at three light intensity (mol photonsm-2s-1) and at 15ºC: A) in biomass unit, B) in chlorophyll unit.

Mechanisms of Photoacclimation on Photosynthesis Level in Cyanobacteria 107

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Latała, A.; Jodłowska, S. & Pniewski, F. (2006). Culture Collection of Baltic Algae (CCBA)

Lobban, C. S. & Harrison, P. J. (1997). *Seaweed Ecology and Physiology*. Cambridge University

MacIntyre, H.L.; Kana, T.M.; Anning, T. & Geider, R.J. (2002). Photoacclimation of

Moisander, P.H.; McClinton III, E. & Paerl, H.W. (2002). Salinity effects on growth,

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Sukenik, A. & Kaplan, A. (2002). Physiological variables determined under laboratory conditions may explain the bloom of *Aphanizomenon ovalisporum* in Lake

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irradiance on growth and toxin production by *Nodularia spumigena*. *Journal of* 

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Fig. 5. Response-surface estimation at different temperatures (°C) and irradiances (mol photonsm-2s-1) of Ek (mol photons m-2 s-1) for: A) *N. spumigena*, B) *G. amphibium* and Pc (mol photons m-2 s-1) for: C) *N. spumigena*, D) *G. amphibium*.

#### **5. Conclusions**

Every phototrophic organisms, including cyanobacteria, differ in their light intensity range within which they grow and photosynthesize. This is determined by their optimal ecological requirements for light and controlled by metabolic properties of each species (Collier et al., 1978; Richardson et al., 1983). The P-E response curves, derived from short-term measurement, provides an information about condition of photosynthetic apparatus and yields insight into the regulation of energy and material balance of the cell (MacIntyre et al., 2002).

In this review, we give importance of interpreting P-Es in the context photoacclimation mechanisms in cyanobacteria. The current experiments on two different strains of cyanobacteria demonstrated their capacity to acclimate to irradiance, which is reflected in the wide range of changes in the growth, pigment composition as well as photosynthetic activity of the examined cyanobacteria. The studied parameters demonstrated their exceptional adaptability of changes, which explain why these strains grow successfully in well-illuminated habitats and also can grow well in position with limited access of light. The identification of factors that regulate the growth and photosynthetic activity of

cyanobacteria can be helpful for understanding the ecological triggers of cyanobacterial blooms.

#### **6. Reference**

Allakhverdiev, S.I.; Kreslavski, V.D.; Klimov, V.V.; Los, D.A.; Carpentier, R. & Mohanty, P. (2008). Heat stress: an overview of molecular responses in photosynthesis. *Photosynthesis Research* 98: 541-550.

Fig. 5. Response-surface estimation at different temperatures (°C) and irradiances (mol photonsm-2s-1) of Ek (mol photons m-2 s-1) for: A) *N. spumigena*, B) *G. amphibium* and Pc

Every phototrophic organisms, including cyanobacteria, differ in their light intensity range within which they grow and photosynthesize. This is determined by their optimal ecological requirements for light and controlled by metabolic properties of each species (Collier et al., 1978; Richardson et al., 1983). The P-E response curves, derived from short-term measurement, provides an information about condition of photosynthetic apparatus and yields insight into

In this review, we give importance of interpreting P-Es in the context photoacclimation mechanisms in cyanobacteria. The current experiments on two different strains of cyanobacteria demonstrated their capacity to acclimate to irradiance, which is reflected in the wide range of changes in the growth, pigment composition as well as photosynthetic activity of the examined cyanobacteria. The studied parameters demonstrated their exceptional adaptability of changes, which explain why these strains grow successfully in well-illuminated habitats and also can grow well in position with limited access of light. The identification of factors that regulate the growth and photosynthetic activity of cyanobacteria can be helpful for understanding the ecological triggers of cyanobacterial blooms.

Allakhverdiev, S.I.; Kreslavski, V.D.; Klimov, V.V.; Los, D.A.; Carpentier, R. & Mohanty, P.

(2008). Heat stress: an overview of molecular responses in photosynthesis.

the regulation of energy and material balance of the cell (MacIntyre et al., 2002).

(mol photons m-2 s-1) for: C) *N. spumigena*, D) *G. amphibium*.

*Photosynthesis Research* 98: 541-550.

**5. Conclusions** 

**6. Reference** 

Dring, M.J. (1998). *The biology of marine plants*. Cambridge University Press, Cambridge. 199 pp.


**6** 

*1Slovenia 2Italy* 

*1Institute of Physical Biology,* 

**Photosynthesis in Microalgae as Measured** 

Two to three percent of the absorbed sun energy is re-emitted from the pigment systems as fluorescence. Delayed fluorescence (DF) represents only 0.03 % of that emission (Jursinic, 1986). But although DF reflects an insignificant loss of the total energy stored by photosynthesis, it is a sensitive indicator of the many steps in photosynthesis processes (Jursinic, 1986). This sensitivity makes DF an extremely complex phenomenon, however with awareness and control of the variables, DF becomes an important intrinsic probe

Delayed fluorescence, also termed delayed luminescence or delayed light emission, is a long-lived light emission by plants, algae and cyanobacteria after being illuminated with light and placed in darkness (Strehler & Arnold, 1951). It can last from milliseconds to several minutes, which is quite a long time in a nanosecond world of classical fluorescence. The main source of DF is the photosystem II (PSII) (Jursinic, 1986), whereas the photosystem I (PSI) contributes much less to the emission. Delayed fluorescence emission spectrum resembles the fluorescence emission spectrum of chlorophyll a (Arnold & Davidson, 1954; Jursinic, 1986; Van Wijk et al., 1999). The main difference from prompt fluorescence is in the origin of the excited single state of the emitting pigment molecule (Jursinic, 1986). Delayed fluorescence originates from the repopulation of excited states of chlorophyll from the stored energy after charge separation (Jursinic, 1986), whereas prompt fluorescence reflects the radiative de-excitation of excited chlorophyll molecules before charge separation. This is why delayed and prompt fluorescence contain information about different fundamental

An important feature of DF is that it is emitted only by a functionally active chlorophyll – in other words, when photosynthesis is active (Bertsch, 1962). The emission depends on the number of PSII centers and the rate of back reactions in the photosynthetic chain, which are influenced by the membrane potential and pH gradient (Avron & Schreiber, 1979; Joliot &

Delayed fluorescence is affected by many chemical and physical variables, such as ATP (Avron & Schreiber, 1979), proton gradient in the thylakoids (Wraight & Crofts, 1971), chill stress (Melcarek & Brown, 1977), different xenobiotics (Berden-Zrimec et al., 2007; Drinovec

processes of the photosynthetic apparatus (Goltsev et al., 2003).

**1. Introduction** 

(Jursinic, 1986).

Joliot, 1980).

**with Delayed Fluorescence Technique** 

Maja Berden-Zrimec1, Marina Monti2 and Alexis Zrimec1

*2Istituto Nazionale di Oceanografia e Geofisica Sperimentale,* 

on growth rates, pigment content and photosynthesis. *European Journal of Phycology* 34: 109-115.


### **Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique**

Maja Berden-Zrimec1, Marina Monti2 and Alexis Zrimec1 *1Institute of Physical Biology, 2Istituto Nazionale di Oceanografia e Geofisica Sperimentale, 1Slovenia 2Italy* 

#### **1. Introduction**

108 Advances in Photosynthesis – Fundamental Aspects

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Platt, T. & Jassby, A.D. (1976). The relationship between photosynthesis and light for natural assemblages of coastal marine phytoplankton. *Journal of Phycology* 12: 421-430. Platt, T.; Gallegos, C.L. & Harrison, W.G. (1980). Photoinhibition of photosynthesis in

Prézelin, B.B. (1981). Light reactions in photosynthesis. In: Platt T (ed) Physiological bases of

Rabinowitch, E.I. (1951). *Photosynthesis and Related Processes*. Vol. II, part 1. Interscience

Ramus, J. (1981). The capture and transduction of light energy. In: *The biology of seaweeds* (Ed. by C.S. Lobban & M.J. Wynne), Blackwell Scientific, Oxford. 458-492 pp. Richardson, K.; Beardall, J. & Raven, J.A. (1983). Adaptation of unicellular algae to

Rivkin, R.B. (1989). Influence of irradiance and spectral quality on the carbon metabolism of

Staal, M.; te Lintel Hekkert, S.; Herman, P. & Stal, L.J. (2002). Comparison of model

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salinity. *Journal of Phycology* 16: 421-423.

on growth rates, pigment content and photosynthesis. *European Journal of Phycology*

*pyriforme* (Chrysophyceae) in response to 75 combination of light, temperature and

natural assemblages of marine phytoplankton. *Journal of Marine Research* 38: 687-

phytoplankton ecology, *Canadian Bulletin of Fisheries and Aquatic Sciences,* no. 210,

phytoplankton. I. Photosynthesis, chemical composition and growth. *Marine* 

describing light dependence of N2 fixation in heterocystous cyanobacteria. *Applied* 

Walsby, A.E. (2003). BASIC: Baltic Sea cyanobacteria. An investigation of the structure and dynamic of water blooms of cyanobacteria in the Baltic Sea – response to a changing environment. *Continental Shelf Research* 23: 1695-1714. Steiger, S.; Schäfer, L. & Sandmann, G. (1999). High-light-dependent upregulation of

carotenoids and their antioxidative properties in the cyanobacterium *Synechocystis*

II damage. *Trends in Plant Science* 16(1): 53-60. *Plant Science* 13(4): 178-182. Tandeau de Marsac, N. & Houmard, J. (1988). Complementary chromatic adaptation: physiological conditions and action spectra. In: *Methods in Enzymology* (Ed. by L.

water environment, the Puck Bay, Poland, a possible analog of an "advanced anaerobic ecosystem"?. *Origins of Life and Evolution of Biospheres* 16(3-4): 337-338.

Two to three percent of the absorbed sun energy is re-emitted from the pigment systems as fluorescence. Delayed fluorescence (DF) represents only 0.03 % of that emission (Jursinic, 1986). But although DF reflects an insignificant loss of the total energy stored by photosynthesis, it is a sensitive indicator of the many steps in photosynthesis processes (Jursinic, 1986). This sensitivity makes DF an extremely complex phenomenon, however with awareness and control of the variables, DF becomes an important intrinsic probe (Jursinic, 1986).

Delayed fluorescence, also termed delayed luminescence or delayed light emission, is a long-lived light emission by plants, algae and cyanobacteria after being illuminated with light and placed in darkness (Strehler & Arnold, 1951). It can last from milliseconds to several minutes, which is quite a long time in a nanosecond world of classical fluorescence.

The main source of DF is the photosystem II (PSII) (Jursinic, 1986), whereas the photosystem I (PSI) contributes much less to the emission. Delayed fluorescence emission spectrum resembles the fluorescence emission spectrum of chlorophyll a (Arnold & Davidson, 1954; Jursinic, 1986; Van Wijk et al., 1999). The main difference from prompt fluorescence is in the origin of the excited single state of the emitting pigment molecule (Jursinic, 1986). Delayed fluorescence originates from the repopulation of excited states of chlorophyll from the stored energy after charge separation (Jursinic, 1986), whereas prompt fluorescence reflects the radiative de-excitation of excited chlorophyll molecules before charge separation. This is why delayed and prompt fluorescence contain information about different fundamental processes of the photosynthetic apparatus (Goltsev et al., 2003).

An important feature of DF is that it is emitted only by a functionally active chlorophyll – in other words, when photosynthesis is active (Bertsch, 1962). The emission depends on the number of PSII centers and the rate of back reactions in the photosynthetic chain, which are influenced by the membrane potential and pH gradient (Avron & Schreiber, 1979; Joliot & Joliot, 1980).

Delayed fluorescence is affected by many chemical and physical variables, such as ATP (Avron & Schreiber, 1979), proton gradient in the thylakoids (Wraight & Crofts, 1971), chill stress (Melcarek & Brown, 1977), different xenobiotics (Berden-Zrimec et al., 2007; Drinovec

Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 111

reduction state of PQ pool is influenced by many reactions including electron transport in

The reaction rate is additionally influenced by changes in pH gradient and electric field in thylakoid membrane of chloroplasts. In the case of monotonous DF decay kinetics (without the transition peak), both electric field and pH gradient decay slowly after the initial

Fig. 2. Slow fluorescence induction kinetics of two marine algae (*Prorocentrum minimum* and

The slow components probably provide information on temporary energy storage during the photosynthetic electron transport (Desai et al., 1983), because they depend on electron distribution in the plastoquinone pool (PQ) and photosystem I (PSI) (Katsumata et al., 2006). By a measurement of the fluorescent yield it is possible to monitor the reduction state of quinone QA in PSII. We measured slow florescence induction kinetics by switching on an actinic source of the same intensity as was used for illumination in the DF measurement (Drinovec et al., 2004b). The fluorescence induction curves of marine dinoflagellate *Prorocentrum minimum* had a distinct transient peak and in marine diatom *Skeletonema costatum* a monotonous decay kinetics (Figure 2). In *S. costatum*, a slow decay of fluorescent yield after the initial sharp increase was observed. This is a consequence of progressive oxidation of quinones as Calvin cycle is initiated. *P. minimum* produces a maximum fluorescent yield about 15 seconds after the start of actinic illumination. At this point, the reduction state of the quinones is at the maximum and starts decreasing slowly. This experiment showed the occurrence of transient peaks represents an important physiological

The occurrence of the transient peak in DF decay kinetics probably depends on the rates of back reactions and possibly the organization of the thylakoid membrane (Desai et al., 1983). The exact physiological interpretation of transient peaks is quite difficult due to complex electron pathways and their interactions, however they appear to be formed when the

*Skeletonema costatum*) after switching on actinic light (Drinovec et al., 2004b).

parameter for investigation of photosynthetic processes.

PSI and dark reactions of photosynthesis.

increase caused by single light pulse excitation.

et al., 2004a; Katsumata et al., 2006), excitation light spectral and intensity characteristics (Wang et al., 2004; Zrimec et al., 2005), cell culture growth stage (Berden-Zrimec et al., 2008b; Monti et al., 2005), and nutrient status (Berden-Zrimec et al., 2008a). The changes in chemical and physical parameters affect the reduction state of the plastoquinone pool or its coupling with PSII by modulating the reversed electron flow (Avron & Schreiber, 1979; Mellvig & Tillberg, 1986).

In the field studies, the intensity of delayed fluorescence is used as a measure of photosynthetic activity and living algal biomass (Berden-Zrimec et al., 2009; Krause & Gerhardt, 1984; Kurzbaum et al., 2007; Schneckenburger & Schmidt, 1996). Additionally, DF excitation spectra can be utilized for the analysis of taxonomical changes in the algal communities (Greisberger & Teubner, 2007; Hakanson et al., 2003; Istvanovics et al., 2005; Yacobi et al., 1998) (Figure 1).

#### **2. Basic characteristics of delayed fluorescence decay kinetics**

Delayed fluorescence shows monotonic decay kinetics in the first seconds, sometimes followed by a more or less pronounced transient peak (Bertsch & Azzi, 1965). The emission is composed of several components, characterized by different decay rates (Bjorn, 1971; Desai et al., 1983). The faster decaying components (first few seconds) provide information about the fate of energy absorbed by PSII (Desai et al., 1983). The slow components (from few seconds to minutes or hours) originate in back reactions in the photosynthetic chain as well as between the S2 and S3 states of the oxygen evolving complex (OEC) and quinones QA and QB (Joliot et al., 1971). OEC reacts with quinone molecules and their reduction state is in the equilibrium with PQ. The increase of PQH2 concentration induces reverse electron flow, producing QA- and QB- states (Joliot & Joliot, 1980), which contribute to DF. The

Fig. 1. Delayed fluorescence excitation spectra of marine phytoplankton species. cps – counts per second, DFI 0.4-1s – delayed fluorescence intensity in the interval 0.4 – 1 s after sample illumination (Berden-Zrimec et al., 2010).

et al., 2004a; Katsumata et al., 2006), excitation light spectral and intensity characteristics (Wang et al., 2004; Zrimec et al., 2005), cell culture growth stage (Berden-Zrimec et al., 2008b; Monti et al., 2005), and nutrient status (Berden-Zrimec et al., 2008a). The changes in chemical and physical parameters affect the reduction state of the plastoquinone pool or its coupling with PSII by modulating the reversed electron flow (Avron & Schreiber, 1979;

In the field studies, the intensity of delayed fluorescence is used as a measure of photosynthetic activity and living algal biomass (Berden-Zrimec et al., 2009; Krause & Gerhardt, 1984; Kurzbaum et al., 2007; Schneckenburger & Schmidt, 1996). Additionally, DF excitation spectra can be utilized for the analysis of taxonomical changes in the algal communities (Greisberger & Teubner, 2007; Hakanson et al., 2003; Istvanovics et al., 2005;

Delayed fluorescence shows monotonic decay kinetics in the first seconds, sometimes followed by a more or less pronounced transient peak (Bertsch & Azzi, 1965). The emission is composed of several components, characterized by different decay rates (Bjorn, 1971; Desai et al., 1983). The faster decaying components (first few seconds) provide information about the fate of energy absorbed by PSII (Desai et al., 1983). The slow components (from few seconds to minutes or hours) originate in back reactions in the photosynthetic chain as well as between the S2 and S3 states of the oxygen evolving complex (OEC) and quinones QA and QB (Joliot et al., 1971). OEC reacts with quinone molecules and their reduction state is in the equilibrium with PQ. The increase of PQH2 concentration induces reverse electron flow, producing QA- and QB- states (Joliot & Joliot, 1980), which contribute to DF. The

Fig. 1. Delayed fluorescence excitation spectra of marine phytoplankton species. cps – counts per second, DFI 0.4-1s – delayed fluorescence intensity in the interval 0.4 – 1 s after

sample illumination (Berden-Zrimec et al., 2010).

**2. Basic characteristics of delayed fluorescence decay kinetics** 

Mellvig & Tillberg, 1986).

Yacobi et al., 1998) (Figure 1).

reduction state of PQ pool is influenced by many reactions including electron transport in PSI and dark reactions of photosynthesis.

The reaction rate is additionally influenced by changes in pH gradient and electric field in thylakoid membrane of chloroplasts. In the case of monotonous DF decay kinetics (without the transition peak), both electric field and pH gradient decay slowly after the initial increase caused by single light pulse excitation.

Fig. 2. Slow fluorescence induction kinetics of two marine algae (*Prorocentrum minimum* and *Skeletonema costatum*) after switching on actinic light (Drinovec et al., 2004b).

The slow components probably provide information on temporary energy storage during the photosynthetic electron transport (Desai et al., 1983), because they depend on electron distribution in the plastoquinone pool (PQ) and photosystem I (PSI) (Katsumata et al., 2006). By a measurement of the fluorescent yield it is possible to monitor the reduction state of quinone QA in PSII. We measured slow florescence induction kinetics by switching on an actinic source of the same intensity as was used for illumination in the DF measurement (Drinovec et al., 2004b). The fluorescence induction curves of marine dinoflagellate *Prorocentrum minimum* had a distinct transient peak and in marine diatom *Skeletonema costatum* a monotonous decay kinetics (Figure 2). In *S. costatum*, a slow decay of fluorescent yield after the initial sharp increase was observed. This is a consequence of progressive oxidation of quinones as Calvin cycle is initiated. *P. minimum* produces a maximum fluorescent yield about 15 seconds after the start of actinic illumination. At this point, the reduction state of the quinones is at the maximum and starts decreasing slowly. This experiment showed the occurrence of transient peaks represents an important physiological parameter for investigation of photosynthetic processes.

The occurrence of the transient peak in DF decay kinetics probably depends on the rates of back reactions and possibly the organization of the thylakoid membrane (Desai et al., 1983). The exact physiological interpretation of transient peaks is quite difficult due to complex electron pathways and their interactions, however they appear to be formed when the

Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 113

(Berden-Zrimec et al., 2008a; Zrimec et al., 2005) (Figure 3). In our experiments, *Dunaliella tertiolecta* Butcher (Chlorophyta) exhibited a peak at the illuminations below 600 nm and above 650 nm (Zrimec et al., 2005) (Figure 3b), whereas *Desmodesmus* (*Scenedesmus*) *subspicatus* Chodat 1926 (Chlorophyta) did not exhibit the peak at all (Berden-Zrimec et al., 2007). *Prorocentrum minimum* (Pavillard) Schiller (Dinophyta) exhibited a peak only when illuminated with wavelengths above 650 nm (Figure 3a). Bertsch (1962) observed the peak in *Chlorella sp.* (Chlorophyta) at the illumination wavelength of 700 nm, but not

The presence of a peak in DF decay curves after a pulse of light of longer wavelengths indicates PSI involvement in DF generation (Bertsch, 1962; Desai et al., 1983; Hideg et al., 1991; Mellvig & Tillberg, 1986), because far-red light is predominantly absorbed by PSI. If cyclic electron flow produces excess ATP over NADPH, back electron flow from PSI can be generated, resulting in the transient peak from a few to tens of seconds after their being

Fig. 4. Normalized delayed fluorescence decay curves of different algal species after a 3 s white-light illumination pulse. (1) *Prorocentrum minimum* (Dinophyta), (2) *Scrippsiella trochoidea* (Dinophyta), (3) *Gyrodinium* sp. (Dinophyta), (4) *Skeletonema costatum*

(Bacillariophyceae), (5) *Pyrenomonas* sp. (Chryptophyta), (6) *Isochrysis galbana* (Chrysophyta).

The peak position varies greatly between species (Figure 4). In algae, the peak is usually positioned in first minute after the illumination (Figure 4) (Mellvig & Tillberg, 1986), whereas in higher plants it lies in the range of minutes after excitation (Desai et al., 1983). DF decay kinetics can differ even among strains (Berden-Zrimec et al., 2008b; Monti et al., 2005), due to the kinetic rate constants of the electron back reactions depending on the physiological and organizational state of the entire photosynthetic apparatus. Several peaks appear when algae are put in low CO2 conditions or as a consequence of phosphorous starvation (Mellvig & Tillberg, 1986). In both cases, dark reactions of photosynthesis are

at 650 nm.

illuminated (Mellvig & Tillberg, 1986).

(Berden-Zrimec et al., 2010).

affected.

metabolic conditions affect the redox status of QA and QB directly or indirectly. It has been reported that ATP and NADPH can reduce quinones (Joliot & Joliot, 1980). Degradation of starch to PGA begins immediately after switching off the light. ATP and NADPH thus formed may enhance the reduction of quinones and induce the formation of charge pairs with higher S states as long as they exist (Mellvig & Tillberg, 1986). ATP concentration oscillates after switching cells from light to darkness which is a consequence of feedback mechanisms in the reactions of photosynthesis. A strong coupling of biochemical reactions in thylakoid membrane is also an essential prerequisite for hyperbolic decay.

Fig. 3. Delayed fluorescence decay kinetics after illumination with two different wavelengths. a) *Prorocentrum minimum*, b) *Dunaliella tertiolecta*. a.u. – arbitrary units.

The transient peak is preferentially stimulated by far-red excitation (Desai et al., 1983; Hideg et al., 1991), but in some species it can also be induced by shorter wavelengths

metabolic conditions affect the redox status of QA and QB directly or indirectly. It has been reported that ATP and NADPH can reduce quinones (Joliot & Joliot, 1980). Degradation of starch to PGA begins immediately after switching off the light. ATP and NADPH thus formed may enhance the reduction of quinones and induce the formation of charge pairs with higher S states as long as they exist (Mellvig & Tillberg, 1986). ATP concentration oscillates after switching cells from light to darkness which is a consequence of feedback mechanisms in the reactions of photosynthesis. A strong coupling of biochemical reactions

in thylakoid membrane is also an essential prerequisite for hyperbolic decay.

Fig. 3. Delayed fluorescence decay kinetics after illumination with two different wavelengths. a) *Prorocentrum minimum*, b) *Dunaliella tertiolecta*. a.u. – arbitrary units.

The transient peak is preferentially stimulated by far-red excitation (Desai et al., 1983; Hideg et al., 1991), but in some species it can also be induced by shorter wavelengths (Berden-Zrimec et al., 2008a; Zrimec et al., 2005) (Figure 3). In our experiments, *Dunaliella tertiolecta* Butcher (Chlorophyta) exhibited a peak at the illuminations below 600 nm and above 650 nm (Zrimec et al., 2005) (Figure 3b), whereas *Desmodesmus* (*Scenedesmus*) *subspicatus* Chodat 1926 (Chlorophyta) did not exhibit the peak at all (Berden-Zrimec et al., 2007). *Prorocentrum minimum* (Pavillard) Schiller (Dinophyta) exhibited a peak only when illuminated with wavelengths above 650 nm (Figure 3a). Bertsch (1962) observed the peak in *Chlorella sp.* (Chlorophyta) at the illumination wavelength of 700 nm, but not at 650 nm.

The presence of a peak in DF decay curves after a pulse of light of longer wavelengths indicates PSI involvement in DF generation (Bertsch, 1962; Desai et al., 1983; Hideg et al., 1991; Mellvig & Tillberg, 1986), because far-red light is predominantly absorbed by PSI. If cyclic electron flow produces excess ATP over NADPH, back electron flow from PSI can be generated, resulting in the transient peak from a few to tens of seconds after their being illuminated (Mellvig & Tillberg, 1986).

Fig. 4. Normalized delayed fluorescence decay curves of different algal species after a 3 s white-light illumination pulse. (1) *Prorocentrum minimum* (Dinophyta), (2) *Scrippsiella trochoidea* (Dinophyta), (3) *Gyrodinium* sp. (Dinophyta), (4) *Skeletonema costatum* (Bacillariophyceae), (5) *Pyrenomonas* sp. (Chryptophyta), (6) *Isochrysis galbana* (Chrysophyta). (Berden-Zrimec et al., 2010).

The peak position varies greatly between species (Figure 4). In algae, the peak is usually positioned in first minute after the illumination (Figure 4) (Mellvig & Tillberg, 1986), whereas in higher plants it lies in the range of minutes after excitation (Desai et al., 1983). DF decay kinetics can differ even among strains (Berden-Zrimec et al., 2008b; Monti et al., 2005), due to the kinetic rate constants of the electron back reactions depending on the physiological and organizational state of the entire photosynthetic apparatus. Several peaks appear when algae are put in low CO2 conditions or as a consequence of phosphorous starvation (Mellvig & Tillberg, 1986). In both cases, dark reactions of photosynthesis are affected.

Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 115

 Data: duckweed Model: I(*t*)=*I* 0 /(*t*-*t* 0 ) *m*

Chi^2 = 505820.11094 R^2 = 0.99968

<sup>0</sup> 1461220 ±23148

<sup>0</sup> 1.25 ±0.016 *m* 1.71 ±0.0062

 *I*

*t*

Fig. 6. Delayed fluorescence kinetics of duckweed in log- log scale. A hyperbolic curve was

*t* (s)

To model the different positions and amplitudes of the transient peak, we introduced a variable parameter, *a*, that continuously variates the different equation parameters. The variation in the peak amplitude (Figure 7) is best ascribed to the different pumping of the system, therefore *a* variates the initial distribution of excited electrons among the three pools – where *a=1* when the quinone pool is maximized. The location of the peak (Figure 8) depends on the reactions rate, namely the kinetic constants – where *a=1* when the *ki* are maximized. The combined effect of both, the pumping rate and the relaxation rate, best models the variations in temperature dependence of DF (Figure 9). In this case, the initial distribution of electrons among the three pools as well as the kinetic constants variate in

fitted to the measured data. cps – counts per second (Drinovec et al., 2004b).

1 10

1000

10000

*Delayed luminescence* (cps)

100000

parallel. Higher temperatures are modeled with higher values of the parameter *a*.

Fig. 7. Dependence of the transient peak amplitude on the initial pumping of the electron pools. DF is in relative units, *t* is in seconds, and *a* is the variable parameter, where *a=1*

when the quinone pool is maximized.

#### **3. Modelling of delayed fluorescence kinetics**

There are several proposed phenomenological models of long-term DF decay kinetics: the "multiexponential models" (Schmidt & Schneckenburger, 1992), the "hyperbolic models" (Lavorel & Dennery, 1982; Scordino et al., 1993), the "coherence models" (Yan et al., 2005). Recently, Qiang Li and co-workers presented a very interesting mathematical–physical analysis where they modeled the electron reflux for photosynthetic electron transport chain (Li et al., 2007). Unfortunately, all the presently published models fail to include all the experimental data available in the literature. Especially problematic is the modeling of DF decay kinetics with the transient peaks.

The monotonous decay kinetics (Figure 5) is simply described as a hyperbolic decay using a function *I*=*I*0/(*t*+*t*0)*m* (Scordino et al., 1996) (Figure 6). In usual decay processes the relaxation kinetics is exponential. A hyperbolic decay kinetics was reported to be a sign of a coupled system (Lavorel & Dennery, 1982).

We modeled the non-monotonous relaxation kinetics of DF – the decay curves with the transient peak – as a multiexponential relaxation among three pools of electrons in metastable states. The first pool are the excited electrons, stabilized on quinones, *q(t)*, that relaxes as delayed fluorescence emission or by transfer to the plastoquinone pool, *p(t)*, which in turn preferably relaxes further to the slower reactions, *d(t)*, or back to the quinones (Eqs. 1, 2 and 3):

$$q'(t) = -k\_1 q(t) + k\_2 p(t) \, , \tag{1}$$

$$p'(t) = -k\_2 p(t) + k\_3 d(t) \, \text{ } \tag{2}$$

$$d'(t) = -k\_3 d(t) \, , \tag{3}$$

where *t* is time in seconds, and *ki* are the kinetic constants, and we assume only the pool of electrons that eventually relaxes back to the ground state by emitting delayed fluorescence.

Fig. 5. A monotonous delayed fluorescence of duckweed (*Lemna minor*). cps – counts per second.

There are several proposed phenomenological models of long-term DF decay kinetics: the "multiexponential models" (Schmidt & Schneckenburger, 1992), the "hyperbolic models" (Lavorel & Dennery, 1982; Scordino et al., 1993), the "coherence models" (Yan et al., 2005). Recently, Qiang Li and co-workers presented a very interesting mathematical–physical analysis where they modeled the electron reflux for photosynthetic electron transport chain (Li et al., 2007). Unfortunately, all the presently published models fail to include all the experimental data available in the literature. Especially problematic is the modeling of DF

The monotonous decay kinetics (Figure 5) is simply described as a hyperbolic decay using a function *I*=*I*0/(*t*+*t*0)*m* (Scordino et al., 1996) (Figure 6). In usual decay processes the relaxation kinetics is exponential. A hyperbolic decay kinetics was reported to be a sign of a coupled

We modeled the non-monotonous relaxation kinetics of DF – the decay curves with the transient peak – as a multiexponential relaxation among three pools of electrons in metastable states. The first pool are the excited electrons, stabilized on quinones, *q(t)*, that relaxes as delayed fluorescence emission or by transfer to the plastoquinone pool, *p(t)*, which in turn preferably relaxes further to the slower reactions, *d(t)*, or back to the quinones

where *t* is time in seconds, and *ki* are the kinetic constants, and we assume only the pool of electrons that eventually relaxes back to the ground state by emitting delayed fluorescence.

Fig. 5. A monotonous delayed fluorescence of duckweed (*Lemna minor*). cps – counts per

0 10 20 30 40 50 60

*t* (s)

1 2 *q t kqt kpt* () () () , (1)

2 3 *p*() () () *t kpt kdt* , (2)

<sup>3</sup> *d t kdt* () () , (3)

Duckweed

**3. Modelling of delayed fluorescence kinetics** 

decay kinetics with the transient peaks.

system (Lavorel & Dennery, 1982).

(Eqs. 1, 2 and 3):

second.

0

50000

100000

150000

200000

*Delayed luminescence* (cps)

250000

300000

350000

Fig. 6. Delayed fluorescence kinetics of duckweed in log- log scale. A hyperbolic curve was fitted to the measured data. cps – counts per second (Drinovec et al., 2004b).

To model the different positions and amplitudes of the transient peak, we introduced a variable parameter, *a*, that continuously variates the different equation parameters. The variation in the peak amplitude (Figure 7) is best ascribed to the different pumping of the system, therefore *a* variates the initial distribution of excited electrons among the three pools – where *a=1* when the quinone pool is maximized. The location of the peak (Figure 8) depends on the reactions rate, namely the kinetic constants – where *a=1* when the *ki* are maximized. The combined effect of both, the pumping rate and the relaxation rate, best models the variations in temperature dependence of DF (Figure 9). In this case, the initial distribution of electrons among the three pools as well as the kinetic constants variate in parallel. Higher temperatures are modeled with higher values of the parameter *a*.

Fig. 7. Dependence of the transient peak amplitude on the initial pumping of the electron pools. DF is in relative units, *t* is in seconds, and *a* is the variable parameter, where *a=1* when the quinone pool is maximized.

Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 117

rate of back reactions, which are influenced by the membrane potential and pH gradient (Avron & Schreiber, 1979; Joliot et al., 1971; Joliot & Joliot, 1980; Wraight & Crofts, 1971). In DF kinetics, the changes are most obvious when observing the position and intensity of the transient peak. The peak is culture-state dependent – the peak position and intensity

The results presented here were acquired by a 3 seconds long illumination and a sensor with a red-light-sensitive photomultiplier tube (Hamamatsu R1104) with a Hamamatsu C3866 Photon Counting Unit for signal conditioning and amplification (Monti et al., 2005; Zrimec

Fig. 10. Temperature dependence of delayed fluorescence. a) Delayed fluorescence decay kinetics at different temperatures in *Prorocentrum minimum* (Dinophyta); inset: log-log scale of DF decay kinetics, a.u. – arbitrary units; b) temperature dependence of the peak position:

change during culture growth (Berden-Zrimec et al., 2008a; Monti et al., 2005).

et al., 2005).

the line represents a linear fit.

Fig. 8. Dependence of the transient peak position on the reaction rates. DF is in relative units, *t* is in seconds, and *a* is the variable parameter, where *a=1* when the kinetic constants are maximized.

Fig. 9. Dependence of the transient peak on temperature. Both, peak position and amplitude change with temperature, with higher *a* standing for higher temperatures. DF is in relative units, *t* is in seconds.

#### **4. Physiology**

In delayed fluorescence, specific changes of physiological state are reflected in its intensity and kinetics. Delayed fluorescence intensity (DFI) is represented by an integral under the DF decay curve. In many cases, DFI can be utilized as a measure of living cell concentration (Berden-Zrimec et al., 2009). It also reflects the number of PSII centers, the fluorescence yield, and the

Fig. 8. Dependence of the transient peak position on the reaction rates. DF is in relative units, *t* is in seconds, and *a* is the variable parameter, where *a=1* when the kinetic constants

Fig. 9. Dependence of the transient peak on temperature. Both, peak position and amplitude change with temperature, with higher *a* standing for higher temperatures. DF is in relative

In delayed fluorescence, specific changes of physiological state are reflected in its intensity and kinetics. Delayed fluorescence intensity (DFI) is represented by an integral under the DF decay curve. In many cases, DFI can be utilized as a measure of living cell concentration (Berden-Zrimec et al., 2009). It also reflects the number of PSII centers, the fluorescence yield, and the

are maximized.

units, *t* is in seconds.

**4. Physiology** 

rate of back reactions, which are influenced by the membrane potential and pH gradient (Avron & Schreiber, 1979; Joliot et al., 1971; Joliot & Joliot, 1980; Wraight & Crofts, 1971).

In DF kinetics, the changes are most obvious when observing the position and intensity of the transient peak. The peak is culture-state dependent – the peak position and intensity change during culture growth (Berden-Zrimec et al., 2008a; Monti et al., 2005).

The results presented here were acquired by a 3 seconds long illumination and a sensor with a red-light-sensitive photomultiplier tube (Hamamatsu R1104) with a Hamamatsu C3866 Photon Counting Unit for signal conditioning and amplification (Monti et al., 2005; Zrimec et al., 2005).

Fig. 10. Temperature dependence of delayed fluorescence. a) Delayed fluorescence decay kinetics at different temperatures in *Prorocentrum minimum* (Dinophyta); inset: log-log scale of DF decay kinetics, a.u. – arbitrary units; b) temperature dependence of the peak position: the line represents a linear fit.

Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 119

Fig. 11. Delayed fluorescence decay kinetics of *Dunaliella tertiolecta* after 3 s illumination

a)

with white light of different intensities*.* 

#### **4.1 Temperature and illumination intensity dependence**

The temperature and illumination intensity strongly influence the transient peak position and the intensity. With increasing temperature, peak position is moving towards the beginning of the relaxation curve (Figure 10a) and average delayed fluorescence intensity increases until a maximum around 28 - 30°C (Wang et al., 2004; Yan et al., 2005; Zrimec et al., 2005). Due to changes in the kinetics, the temperature dependence of DFI is more complicated because it also depends on the time interval on which it is averaged.

The peak position has a typical temperature dependency for metabolic biochemical reactions (Zrimec et al., 2005). In the Arrhenius plot the natural logarithm of the peak position is linearly dependent on temperature (Figure 10b). Q10 of 2.6 and the activation energy of 71.5 kJ/mol calculated from the plot are in the expected range of plastoquinone-PSII reactions (Zrimec et al., 2005).

The illumination intensity profoundly influences only the peak intensity and less the peak position (Figure 11). Delayed emission can already be observed at relatively low illumination intensities. In the experiment with *Dunaliella tertiolecta* Butcher (Chlorophyta), DF was saturated already by a 3 s excitation pulse of 15 µmol m-2 s-1 PAR (Zrimec et al., 2005), which is even lower than obtained by Wang et al. (2004) for isolated spinach chloroplasts. At the excitation light intensity of 3.75 µmol m-2 s-1 PAR, DF showed slight differences in decay kinetics in the region of the transient peak, probably due to the changed oxidation state of the plastoquinone pool (Zrimec et al., 2005). DFI at 3.75 µmol m-2 s-1 PAR was only approximately 3% lower than at 15 and 60 µmol m-2 s-1 PAR (Zrimec et al., 2005). The maximal peak intensity was observed at the light intensity which was used for growing of batch cultures.

Dependence on light intensity can be explained by its influence on the ratio of the light captured by PSI and PSII: at low light intensity more light is absorbed by PSI, because its absorption spectrum has a maximum at longer wavelengths compared to the PSII. Thus cyclic electron transport producing only ATP is stronger than linear transport and an excess of ATP over NADPH is produced. The electron flow through PSI also causes oxidation of the plastoquinone pool. At higher light intensities the PSI get saturated and the ratio of the light absorbed by PSI compared to PSII is decreased.

#### **4.2 Salinity**

Changes in salinity influence photosynthesis in several ways. Increased salinity studies in the red alga *Porphyra perforata* showed there are at least three sites in the photosynthetic apparatus that are affected (Satoh et al., 1983). The first site, photoactivation and darkinactivation of electron flow on the reducing side of PSI, was completely inhibited at high salinity. The second site, electron flow on the oxidizing side (water side) of PSII, was inhibited as was the re-oxidation of Q in the presence of 3-(3,4-dichlorophenyl)-1,1 dimethylurea. The third site affected by high salinity was the transfer of light energy probably from pigment system II to I. High salinity also reduced the amount of light energy that reached the reaction centers of PSII.

Photosynthetic activity was reduced by lowered salinity in two brown algae *Ascophyllum nodosum* and *Fucus versiculosus* (Connan & Stengel, 2011). Chlorophyll and phycobiliprotein concentrations were lower in changed salinity conditions in red alga *Gelidium coulteri* (Macler, 1988).

The temperature and illumination intensity strongly influence the transient peak position and the intensity. With increasing temperature, peak position is moving towards the beginning of the relaxation curve (Figure 10a) and average delayed fluorescence intensity increases until a maximum around 28 - 30°C (Wang et al., 2004; Yan et al., 2005; Zrimec et al., 2005). Due to changes in the kinetics, the temperature dependence of DFI is more

The peak position has a typical temperature dependency for metabolic biochemical reactions (Zrimec et al., 2005). In the Arrhenius plot the natural logarithm of the peak position is linearly dependent on temperature (Figure 10b). Q10 of 2.6 and the activation energy of 71.5 kJ/mol calculated from the plot are in the expected range of plastoquinone-PSII reactions

The illumination intensity profoundly influences only the peak intensity and less the peak position (Figure 11). Delayed emission can already be observed at relatively low illumination intensities. In the experiment with *Dunaliella tertiolecta* Butcher (Chlorophyta), DF was saturated already by a 3 s excitation pulse of 15 µmol m-2 s-1 PAR (Zrimec et al., 2005), which is even lower than obtained by Wang et al. (2004) for isolated spinach chloroplasts. At the excitation light intensity of 3.75 µmol m-2 s-1 PAR, DF showed slight differences in decay kinetics in the region of the transient peak, probably due to the changed oxidation state of the plastoquinone pool (Zrimec et al., 2005). DFI at 3.75 µmol m-2 s-1 PAR was only approximately 3% lower than at 15 and 60 µmol m-2 s-1 PAR (Zrimec et al., 2005). The maximal peak intensity was observed at the light intensity which was used for growing

Dependence on light intensity can be explained by its influence on the ratio of the light captured by PSI and PSII: at low light intensity more light is absorbed by PSI, because its absorption spectrum has a maximum at longer wavelengths compared to the PSII. Thus cyclic electron transport producing only ATP is stronger than linear transport and an excess of ATP over NADPH is produced. The electron flow through PSI also causes oxidation of the plastoquinone pool. At higher light intensities the PSI get saturated and the ratio of the

Changes in salinity influence photosynthesis in several ways. Increased salinity studies in the red alga *Porphyra perforata* showed there are at least three sites in the photosynthetic apparatus that are affected (Satoh et al., 1983). The first site, photoactivation and darkinactivation of electron flow on the reducing side of PSI, was completely inhibited at high salinity. The second site, electron flow on the oxidizing side (water side) of PSII, was inhibited as was the re-oxidation of Q in the presence of 3-(3,4-dichlorophenyl)-1,1 dimethylurea. The third site affected by high salinity was the transfer of light energy probably from pigment system II to I. High salinity also reduced the amount of light energy

Photosynthetic activity was reduced by lowered salinity in two brown algae *Ascophyllum nodosum* and *Fucus versiculosus* (Connan & Stengel, 2011). Chlorophyll and phycobiliprotein concentrations were lower in changed salinity conditions in red alga *Gelidium coulteri*

complicated because it also depends on the time interval on which it is averaged.

**4.1 Temperature and illumination intensity dependence** 

light absorbed by PSI compared to PSII is decreased.

that reached the reaction centers of PSII.

(Zrimec et al., 2005).

of batch cultures.

**4.2 Salinity** 

(Macler, 1988).

Fig. 11. Delayed fluorescence decay kinetics of *Dunaliella tertiolecta* after 3 s illumination with white light of different intensities*.* 

a)

Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 121

a)

b)

Fig. 12. Delayed fluorescence decay kinetics at different salinities in three strains of *Prorocentrum minimum.* a) strain from the Adriatic Sea, original salinity 32 PSU; b) strain from the Chesapeake Bay, USA, original salinity 16 PSU; c) strain from the Baltic Sea, original salinity 8 PSU. a.u. – arbitrary units.

0

b)

c)

Fig. 12. Delayed fluorescence decay kinetics at different salinities in three strains of *Prorocentrum minimum.* a) strain from the Adriatic Sea, original salinity 32 PSU; b) strain from the Chesapeake Bay, USA, original salinity 16 PSU; c) strain from the Baltic Sea,

0

original salinity 8 PSU. a.u. – arbitrary units.

Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 123

plastoquinone and plastoquinol for the QB binding site, preventing the electron flow between PSII and the plastoquinone pool, or 3,5-dichlorophenol (3,5-DCP), which is used as an unspecific reference toxicant in toxicity tests (Berden-Zrimec et al., 2007). DNP locks the ATP-ase in open state, thus allowing H+ ions to pass freely. The backreactions in PSII are enhanced by pH gradient in thylakoid membrane and there is a report that a permanent pH gradient in thylakoid membrane is present even in the dark (Joliot and Joliot 1980). The reason for the reduction of DF intensity in the region before peak formation is most likely the collapse of pH gradient caused by DNP. The disappearance of transient peak after 10 minutes of DNP action confirms the idea that pH gradient is directly or indirectly

DF is a very good parameter in rapid toxicity tests (Berden-Zrimec et al., 2007; Katsumata et al., 2006). DFI was equally or more sensitive to the tested toxicants compared with the cell concentration and absorbance, which are standard parameters in algal growth inhibition tests (Berden-Zrimec et al., 2007). The advantage of the delayed fluorescence is that only living cells are measured, the sensitivity in toxicity tests thus being increased. Additionally, minimal disturbance to the cells, small sample volumes enabling homogenous illumination of all samples, and short test duration minimize a negative influence of changing physicochemical properties of the medium on the results, being the most important features for the

The nutrient status of algal cells markedly influences delayed fluorescence decay kinetics (Berden-Zrimec et al., 2008a; Burger & Schmidt, 1988; Mellvig & Tillberg, 1986). Phosphorus starvation can induce one or several transient peaks (Mellvig & Tillberg, 1986) or change the peak position (Berden-Zrimec et al., 2008a) (Figure 15). Nitrogen limitation also influences the peak position as well as causes the peak cessation (Figure 15) (Burger & Schmidt, 1988). DFI per cell significantly increases due to both, phosphorus and nitrogen limitation (Figure 16).

Fig. 14. The response of delayed fluorescence decay kinetics to geometrical series of diuron

responsible for peak formation.

quality toxicity tests.

(DCMU) concentrations.

**4.4 Nutrients** 

Fig. 13. Influence of photosynthetic inhibitors on *P. minimum* DF decay kinetics: a) 15 µM dicyclohexylcarbodiimide (DCCD) – inhibition of ATP synthesis inhibition; b) 2 µM dinitrophenol (DNP) - destruction of proton gradient and inhibition of electron flow in thylacoid membrane; c) 30 mM NaCN - inhibition of respiration and photosynthesis.

In DF kinetics, higher salinity delayed the transient peak in three strains of *Prorocentrum minimum* (Figure 12). The strains were originally growing at different salinities, namely the Baltic strain (BAL) at 8 PSU, the Chesapeake Bay strain (D5) at 16 PSU, and the Adriatic strain (PmK) at 32 PSU (Monti et al., 2005). In our study, all strains were incubated in all three salinities for their whole growth period. BAL had in general earliest peak onset and PmK the latest in all salinities. The transient peak ceased at the end of the growth period, disappearing sooner at 32 PSU than in the lower salinities.

#### **4.3 Influence of toxins**

Toxic effects of photosynthesis inhibitors can be measured by DF already after a few minutes of incubation (Figure 13) (Berden-Zrimec et al., 2007). The transient peak can disappear soon after the addition of photosynthetic inhibitors like ATP synthesis inhibitior dicyclohexylcarbodiimide (DCCD) – (Figure 13a), dinitrophenol (DNP), which destroys the proton gradient and inhibits electron flow in thylakoid membranes (Figure 13b), or NaCN, an inhibitior of respiration and photosynthesis (Figure 13c) (Berden-Zrimec et al., 2010). These toxins influence photosynthesis in different ways, but at the end they affect the reduction state of the plastoquinone pool or its coupling with PSII by inhibiting the reversed electron flow and thus DF (Wang et al., 2004).

Delayed fluorescence response to toxins is dose-dependent (Figure 14), like in the case of herbicide diuron (DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea), which competes with plastoquinone and plastoquinol for the QB binding site, preventing the electron flow between PSII and the plastoquinone pool, or 3,5-dichlorophenol (3,5-DCP), which is used as an unspecific reference toxicant in toxicity tests (Berden-Zrimec et al., 2007). DNP locks the ATP-ase in open state, thus allowing H+ ions to pass freely. The backreactions in PSII are enhanced by pH gradient in thylakoid membrane and there is a report that a permanent pH gradient in thylakoid membrane is present even in the dark (Joliot and Joliot 1980). The reason for the reduction of DF intensity in the region before peak formation is most likely the collapse of pH gradient caused by DNP. The disappearance of transient peak after 10 minutes of DNP action confirms the idea that pH gradient is directly or indirectly responsible for peak formation.

DF is a very good parameter in rapid toxicity tests (Berden-Zrimec et al., 2007; Katsumata et al., 2006). DFI was equally or more sensitive to the tested toxicants compared with the cell concentration and absorbance, which are standard parameters in algal growth inhibition tests (Berden-Zrimec et al., 2007). The advantage of the delayed fluorescence is that only living cells are measured, the sensitivity in toxicity tests thus being increased. Additionally, minimal disturbance to the cells, small sample volumes enabling homogenous illumination of all samples, and short test duration minimize a negative influence of changing physicochemical properties of the medium on the results, being the most important features for the quality toxicity tests.

#### **4.4 Nutrients**

122 Advances in Photosynthesis – Fundamental Aspects

c)

In DF kinetics, higher salinity delayed the transient peak in three strains of *Prorocentrum minimum* (Figure 12). The strains were originally growing at different salinities, namely the Baltic strain (BAL) at 8 PSU, the Chesapeake Bay strain (D5) at 16 PSU, and the Adriatic strain (PmK) at 32 PSU (Monti et al., 2005). In our study, all strains were incubated in all three salinities for their whole growth period. BAL had in general earliest peak onset and PmK the latest in all salinities. The transient peak ceased at the end of the growth period,

Toxic effects of photosynthesis inhibitors can be measured by DF already after a few minutes of incubation (Figure 13) (Berden-Zrimec et al., 2007). The transient peak can disappear soon after the addition of photosynthetic inhibitors like ATP synthesis inhibitior dicyclohexylcarbodiimide (DCCD) – (Figure 13a), dinitrophenol (DNP), which destroys the proton gradient and inhibits electron flow in thylakoid membranes (Figure 13b), or NaCN, an inhibitior of respiration and photosynthesis (Figure 13c) (Berden-Zrimec et al., 2010). These toxins influence photosynthesis in different ways, but at the end they affect the reduction state of the plastoquinone pool or its coupling with PSII by inhibiting the reversed

Delayed fluorescence response to toxins is dose-dependent (Figure 14), like in the case of herbicide diuron (DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea), which competes with

disappearing sooner at 32 PSU than in the lower salinities.

electron flow and thus DF (Wang et al., 2004).

**4.3 Influence of toxins** 

Fig. 13. Influence of photosynthetic inhibitors on *P. minimum* DF decay kinetics: a) 15 µM dicyclohexylcarbodiimide (DCCD) – inhibition of ATP synthesis inhibition; b) 2 µM dinitrophenol (DNP) - destruction of proton gradient and inhibition of electron flow in thylacoid membrane; c) 30 mM NaCN - inhibition of respiration and photosynthesis.

The nutrient status of algal cells markedly influences delayed fluorescence decay kinetics (Berden-Zrimec et al., 2008a; Burger & Schmidt, 1988; Mellvig & Tillberg, 1986). Phosphorus starvation can induce one or several transient peaks (Mellvig & Tillberg, 1986) or change the peak position (Berden-Zrimec et al., 2008a) (Figure 15). Nitrogen limitation also influences the peak position as well as causes the peak cessation (Figure 15) (Burger & Schmidt, 1988). DFI per cell significantly increases due to both, phosphorus and nitrogen limitation (Figure 16).

Fig. 14. The response of delayed fluorescence decay kinetics to geometrical series of diuron (DCMU) concentrations.

Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 125

chlorophyll a and increases non-photochemically active carotenoid pigments (Berges et al., 1996; Geider et al., 1998). Phosphate availability is connected to regulation of Calvin cycle activity or by interdependence of light and dark reactions via ATP/ADP, with consequent

DF decay kinetics is influenced differently by nitrogen and phosphorus starvation, making it a potential method of discriminating various nutrient conditions. Such a discriminating technique is still missing in the monitoring of oceanic phytoplankton population changes.

Delayed fluorescence has been used for researching photosynthesis since 1951. Nevertheless, not many publications can be found in the literature, mostly due to lack of commercially available measuring devices. Delayed fluorescence provides different information about photosynthesis as prompt fluorescence. It is emitted only from living cells therefore the problems with fluorescent background in field samples are omitted. The measurements can be successfully utilized in toxicity tests, biomass monitoring, primary productivity measurements and following changes in phytoplankton composition. Some more research, however, must be done on better understanding of the complex processes

Our colleague Luka Drinovec has assembled the delayed fluorescence measuring apparatus and prepared the software. Alfred Beran has provided us with the algal cultures. Lidija Berden revised the language. The work was financed by the Slovenian Research Agency

Arnold W., Davidson J.B. (1954). The identity of the fluorescent and delayed light emission

Avron M., Schreiber U. (1979). Properties of ATP induced chlorophyll luminescence in

Berden-Zrimec M., Drinovec L., Molinari I., Zrimec A., Fonda S., & Monti M. (2008a).

Delayed fluorescence as a measure of nutrient limitation in *Dunaliella tertiolecta*.

*fluorescence in aquatic sciences: methods and applications,* D.J. Sugget, O. Prasil& M.

delayed fluorescence and pigment composition of four *Prorocentrum minimum*

New approach in studies of microalgal cell lysis. *Central European Journal of Biology.*

spectra in *Chlorella*. *Journal of General Physiology.* Vol.37, pp. 677-684

*Journal of Photochemistry and Photobiology B-Biology.* Vol.92, pp. 13-18 Berden-Zrimec M., Drinovec L., & Zrimec A. (2010). Delayed fluorescence, In: *Chlorophyll a* 

Borowitzka, (Eds.), 293-309, Springer,ISBN 978-90-481-9267-0, Dordrecht Berden-Zrimec M., Drinovec L., Zrimec A., & Tisler T. (2007). Delayed fluorescence in algal growth inhibition tests. *Central European Journal of Biology.* Vol.2, pp. 169-181 Berden-Zrimec M., Flander V., Drinovec L., Zrimec A., & Monti M. (2008b). Growth,

strains growing at two salinities. *Biological Research.* Vol.41, pp. 11-23 Berden-Zrimec M., Kozar-Logar J., Zrimec A., Drinovec L., Franko M., & Malej A. (2009).

chloroplasts. *Biochimica et Biophysica Acta.* Vol.546, pp. 448-454

reductions in the efficiency of photosynthetic electron transfer (Geider et al., 1998).

**5. Conclusions** 

influencing the delayed fluorescence kinetics.

(grants #P1-0237, L4-6222, and V4-0106).

Vol.4, pp. 313-320

**6. Acknowledgements** 

**7. References** 

Fig. 15. Peak position dependence on nutrient status in *Dunaliella tertiolecta* cells. P – phosphorus, N – nitrogen, a.u. – arbitrary units.

Fig. 16. DFI per cell dependence on nutrient status in *Dunaliella tertiolecta* cells. P – phosphorus, N – nitrogen, a.u. – arbitrary units.

Nitrogen and phosphorus starvation influence DF decay kinetics by changing the back reactions rates in the electron backflow. Nitrogen starvation causes reduction in the number of active PSII reactive centers and linear electron flow, but does not influence active PSI leading to relatively higher rates of cyclic photophosphorylation (Berges et al., 1996). Nitrogen starvation also influences thylakoid organization, facilitating trapped energy transfer from PSII to PSI (Berges et al., 1996). In marine phytoplankton, nitrogen limitation affects photosynthesis by reducing the efficiency of energy collection due to loss of chlorophyll a and increases non-photochemically active carotenoid pigments (Berges et al., 1996; Geider et al., 1998). Phosphate availability is connected to regulation of Calvin cycle activity or by interdependence of light and dark reactions via ATP/ADP, with consequent reductions in the efficiency of photosynthetic electron transfer (Geider et al., 1998).

DF decay kinetics is influenced differently by nitrogen and phosphorus starvation, making it a potential method of discriminating various nutrient conditions. Such a discriminating technique is still missing in the monitoring of oceanic phytoplankton population changes.

#### **5. Conclusions**

124 Advances in Photosynthesis – Fundamental Aspects

Fig. 15. Peak position dependence on nutrient status in *Dunaliella tertiolecta* cells. P –

Fig. 16. DFI per cell dependence on nutrient status in *Dunaliella tertiolecta* cells. P –

Nitrogen and phosphorus starvation influence DF decay kinetics by changing the back reactions rates in the electron backflow. Nitrogen starvation causes reduction in the number of active PSII reactive centers and linear electron flow, but does not influence active PSI leading to relatively higher rates of cyclic photophosphorylation (Berges et al., 1996). Nitrogen starvation also influences thylakoid organization, facilitating trapped energy transfer from PSII to PSI (Berges et al., 1996). In marine phytoplankton, nitrogen limitation affects photosynthesis by reducing the efficiency of energy collection due to loss of

phosphorus, N – nitrogen, a.u. – arbitrary units.

phosphorus, N – nitrogen, a.u. – arbitrary units.

Delayed fluorescence has been used for researching photosynthesis since 1951. Nevertheless, not many publications can be found in the literature, mostly due to lack of commercially available measuring devices. Delayed fluorescence provides different information about photosynthesis as prompt fluorescence. It is emitted only from living cells therefore the problems with fluorescent background in field samples are omitted. The measurements can be successfully utilized in toxicity tests, biomass monitoring, primary productivity measurements and following changes in phytoplankton composition. Some more research, however, must be done on better understanding of the complex processes influencing the delayed fluorescence kinetics.

#### **6. Acknowledgements**

Our colleague Luka Drinovec has assembled the delayed fluorescence measuring apparatus and prepared the software. Alfred Beran has provided us with the algal cultures. Lidija Berden revised the language. The work was financed by the Slovenian Research Agency (grants #P1-0237, L4-6222, and V4-0106).

#### **7. References**


Photosynthesis in Microalgae as Measured with Delayed Fluorescence Technique 127

Istvanovics V., Honti M., Osztoics A., Shafik H.M., Padisak J., Yacobi Y., & Eckert W. (2005).

Joliot P., Joliot A. (1980). Dependence of delayed luminescence upon adenosine triphosphatase activity in *Chlorella*. *Plant Physiology.* Vol.65, pp. 691-696 Joliot P., Joliot A., Bouges B., & Barbieri G. (1971). Studies of system-II photocenters by

Jursinic P.A. (1986). Delayed fluorescence: Current concepts and status, In: *Light emission by* 

Katsumata M., Koike T., Nishikawa M., Kazumura K., & Tsuchiya H. (2006). Rapid

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light from chloroplasts is emitted from photosystem II - Evidence from emission


**7** 

*Spain* 

**-Reductase** 

Ana Serrano and Milagros Medina

**Fast Kinetic Methods with Photodiode Array Detection in the Study of the Interaction and** 

**and Ferredoxin NADP+**

The primary function of Photosystem I (PSI)1 during photosynthesis is to provide reducing equivalents, in the form of NADPH, that will then be used in CO2 assimilation (Golbeck, 2006). In plants, this occurs via reduction of the soluble [2Fe-2S] Ferredoxin (Fd) by PSI. Subsequent reduction of NADP+ by Fdrd is catalysed by the FAD-dependent Ferredoxin-NADP+ reductase (FNR) (*E*ox/hq = -374 mV at pH 8.0 and 25ºC) through the formation of a ternary complex (Arakaki *et al.*, 1997; Nogués *et al.*, 2004; Sancho *et al.*, 1990). In most cyanobacteria and some algae under low iron conditions the FMN-dependent Flavodoxin (Fld), particularly its Fldsq/Fldhq couple (*E*ox/sq = -256 mV, *E*sq/hq = -445 mV at pH 8.0 and 25ºC), substitutes for the Fdox/Fdrd pair in this reaction (Bottin & Lagoutte, 1992; Goñi *et al.*, 2009; Medina & Gómez-Moreno, 2004). Thus, two Fldhq molecules transfer two electrons to one FNRox that gets fully reduced after formation of the FNRsq intermediate. FNRhq transfers

Additionally to their role in photosynthesis, Fld and FNR are ubiquitous flavoenzymes that deliver low midpoint potential electrons to redox-based metabolisms in plastids, mitochondria and bacteria in all kingdoms (Müller, 1991). They are also basic prototypes for a large family of di-flavin electron transferases that display common functional and structural properties, where the electron transfer (ET) flow supported by the Fld/FNR modules occurs in reverse direction to the photosynthesis (Brenner *et al.*, 2008; Wolthers & Scrutton, 2004). The better understanding of the ET flavin-flavin mechanism in this system should witness a greater comprehension of the many physiological roles that Fld and FNR, either free or as modules in multidomain proteins, play. In these chains there are still many

<sup>1</sup> PSI, Photosystem I; FNR, ferredoxin-NADP+ reductase; FNRox, FNR in the fully oxidised state; FNRsq, FNR in the semiquinone state; FNRhq, FNR in the hydroquinone (fully reduced) state; Fld, Fladoxin; Fldox, Fld in the fully oxidised state; Fldsq, Fld in the semiquinone state; Fldhq, Fld in the hydroquinone (fully reduced) state; Fd, ferredoxin; Fdox, Fd in the oxidised state; Fdrd, Fd in the reduced state; *E*ox/sq, midpoint reduction potential for the ox/sq couple; *E*sq/hq, midpoint reduction potential for the sq/hq couple; ET, electron transfer; WT, wild-type; *k*<sup>A</sup>B, *k*<sup>B</sup>C, *k*<sup>C</sup>D, apparent rate constants obtained by global analysis of spectral kinetic data; I, ionic strength; UV/Vis, ultraviolet/visible; PDA, photodiode array

then both electrons simultaneously to NADP+ (Medina, 2009) (Figure 1).

**1. Introduction** 

detector; SVD, single value decomposition.

*Department of Biochemistry and Molecular and Cellular Biology and Institute of Biocomputation and Physics of Complex Systems, University of Zaragoza* 

**Electron Transfer Between Flavodoxin** 


### **Fast Kinetic Methods with Photodiode Array Detection in the Study of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP+ -Reductase**

Ana Serrano and Milagros Medina

*Department of Biochemistry and Molecular and Cellular Biology and Institute of Biocomputation and Physics of Complex Systems, University of Zaragoza Spain* 

#### **1. Introduction**

128 Advances in Photosynthesis – Fundamental Aspects

Scordino A., Grasso F., Musumeci F., & Triglia A. (1993). Physical aspects of delayed luminescence in *Acetabularia acetabulum*. *Experientia.* Vol.49, pp. 702-705 Scordino A., Triglia A., Musumeci F., Grasso F., & Rajfur Z. (1996). Influence of the presence

Van Wijk R., Scordino A., Triglia A., & Musumeci F. (1999). 'Simultaneous' measurements of

Yacobi Y.Z., Gerhardt V., Gonen-Zurgil Y., & Sukenik A. (1998). Delayed fluorescence

Yan Y., Popp F.A., Sigrist S., Schlesinger D., Dolf A., Yan Z.C., Cohen S., & Chotia A. (2005).

Zrimec A., Drinovec L., & Berden-Zrimec M. (2005). Influence of chemical and physical

*Journal of Photochemistry and Photobiology B-Biology.* Vol.49, pp. 142-149 Wang C., Xing D., & Chen Q. (2004). A novel method for measuring photosynthesis using delayed fluorescence of chloroplast. *Biosens Bioelectron.* Vol.20, pp. 454-459 Wraight C.A., Crofts A.R. (1971). Delayed fluorescence and the high-energy state of

*Journal of Photochemistry and Photobiology B: Biology.* Vol.32, pp. 11-17 Strehler B.L., Arnold W. (1951). Light production by green plants. *Journal of General* 

*Physiology.* Vol.34, pp. 809-820

2577-2582

chloroplasts. *Eur J Biochem.* Vol.19, pp. 386-397

*Photobiology B-Biology.* Vol.78, pp. 235-244

*Biology and Medicine.* Vol.24, pp. 309-318

of atrazine in water on the in-vivo delayed luminescence of *Acetabularia acetabulum*.

delayed luminescence and chloroplast organization in *Acetabularia acetabulum*.

excitation spectroscopy: A rapid method for qualitative and quantitative assessment of natural population of phytoplankton. *Water Research.* Vol.32, pp.

Further analysis of delayed luminescence of plants. *Journal of Photochemistry and* 

factors on long-term delayed fluorescence in *Dunaliella tertiolecta*. *Electromagnetic* 

The primary function of Photosystem I (PSI)1 during photosynthesis is to provide reducing equivalents, in the form of NADPH, that will then be used in CO2 assimilation (Golbeck, 2006). In plants, this occurs via reduction of the soluble [2Fe-2S] Ferredoxin (Fd) by PSI. Subsequent reduction of NADP+ by Fdrd is catalysed by the FAD-dependent Ferredoxin-NADP+ reductase (FNR) (*E*ox/hq = -374 mV at pH 8.0 and 25ºC) through the formation of a ternary complex (Arakaki *et al.*, 1997; Nogués *et al.*, 2004; Sancho *et al.*, 1990). In most cyanobacteria and some algae under low iron conditions the FMN-dependent Flavodoxin (Fld), particularly its Fldsq/Fldhq couple (*E*ox/sq = -256 mV, *E*sq/hq = -445 mV at pH 8.0 and 25ºC), substitutes for the Fdox/Fdrd pair in this reaction (Bottin & Lagoutte, 1992; Goñi *et al.*, 2009; Medina & Gómez-Moreno, 2004). Thus, two Fldhq molecules transfer two electrons to one FNRox that gets fully reduced after formation of the FNRsq intermediate. FNRhq transfers then both electrons simultaneously to NADP+ (Medina, 2009) (Figure 1).

Additionally to their role in photosynthesis, Fld and FNR are ubiquitous flavoenzymes that deliver low midpoint potential electrons to redox-based metabolisms in plastids, mitochondria and bacteria in all kingdoms (Müller, 1991). They are also basic prototypes for a large family of di-flavin electron transferases that display common functional and structural properties, where the electron transfer (ET) flow supported by the Fld/FNR modules occurs in reverse direction to the photosynthesis (Brenner *et al.*, 2008; Wolthers & Scrutton, 2004). The better understanding of the ET flavin-flavin mechanism in this system should witness a greater comprehension of the many physiological roles that Fld and FNR, either free or as modules in multidomain proteins, play. In these chains there are still many

<sup>1</sup> PSI, Photosystem I; FNR, ferredoxin-NADP+ reductase; FNRox, FNR in the fully oxidised state; FNRsq, FNR in the semiquinone state; FNRhq, FNR in the hydroquinone (fully reduced) state; Fld, Fladoxin; Fldox, Fld in the fully oxidised state; Fldsq, Fld in the semiquinone state; Fldhq, Fld in the hydroquinone (fully reduced) state; Fd, ferredoxin; Fdox, Fd in the oxidised state; Fdrd, Fd in the reduced state; *E*ox/sq, midpoint reduction potential for the ox/sq couple; *E*sq/hq, midpoint reduction potential for the sq/hq couple; ET, electron transfer; WT, wild-type; *k*<sup>A</sup>B, *k*<sup>B</sup>C, *k*<sup>C</sup>D, apparent rate constants obtained by global analysis of spectral kinetic data; I, ionic strength; UV/Vis, ultraviolet/visible; PDA, photodiode array detector; SVD, single value decomposition.

Fast Kinetic Methods with Photodiode Array Detection in the Study

**electron transfer processes** 

stopped-flow with photodiode detection.

Martínez-Júlvez *et al.*, 2001; Medina *et al.*, 1998).

**2.2 Stopped-flow pre-steady-state kinetic measurements** 

of ~300 µM NADP+ at the indicated ionic strength conditions.

**2.3 Kinetic analysis of multiple-wavelength absorption data** 

of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP<sup>+</sup>

**2.1 Biological material and steady-state spectroscopic measurements** 

**2. Experimental methods improving the kinetic analysis of pre-steady-state** 

ET has been analysed at different ionic strengths (I) and in both directions; the physiological photosynthetic ET, from Fldhq to FNRox, and the reverse ET, from FNRhq to Fldox, used to provide reducing equivalents to different metabolic pathways. Different *Anabaena* FNR and Fld variants have been used in the kinetic characterization of these ET processes using

The different FNR and Fld variants were purified from *Luria-Bertani* IPTG-induced *E. coli* cultures containing, respectively, the pTrc99a-Fld and pET28a-FNR plasmids encoding *Anabaena* WT or E301A FNRs and WT, E16K/E61K or E16K/E61K/D126K/D150K Flds as previously described (Casaus *et al.*, 2002; Goñi *et al.*, 2009; Martínez-Júlvez *et al.*, 2001; Medina *et al.*, 1998; Tejero *et al.*, 2003). UV/Vis spectra were recorded in a Cary 100 spectrophotometer at 25 ºC. Unless otherwise stated, all measurements were recorded in 50 mM Tris/HCl, pH 8.0. Binding ability between WT FNRox and WT Fldox was determined using difference absorption spectroscopy as previously described (Goñi *et al.*, 2009;

Fast ET processes between Fld and FNR were followed by stopped-flow under anaerobic conditions in 50 mM Tris/HCl, pH 8.0 at 12ºC. Reactions were analysed by collecting multiple wavelength absorption data (360-700 nm) using an Applied Photophysics SX17.MV stopped-flow and a photodiode array detector (PDA) with the X-Scan software (*App. Photo. Ltd.*). Typically, 400 spectra *per* second were collected for each reaction. Anaerobic conditions were obtained by several cycles of evacuation and bubbling with O2-free argon. FNRhq and Fldhq samples were obtained by photoreduction in the presence of 1 mM EDTA and 5 µM 5-deazariboflavin (Medina *et al.*, 1998). The use of PDA allowed detecting that under our experimental conditions the produced Fldhq samples usually contained a small amount of Fldsq (detected at 580 nm), taken into account in subsequent analysis. Unless otherwise stated, the mixing molar ratio was 1:1 with a final concentration of 10 µM for each protein. Molar ratios of 1:1, 1:2, 1:4 and 1:8 were also assayed for the reaction of FNRhq and Fldox, with a final FNRhq concentration of 10 µM. Reactions were also analysed at different ionic strengths, obtained using variable NaCl concentrations ranging from 0 to 300 mM in 50 mM Tris/HCl, pH 8.0. The reaction between FNRox and Fldhq was also studied in presence

Spectral intermediates formed during reactions were resolved by singular value decomposition (SVD) using Pro-Kineticist (*App. Photo. Ltd.*). Data collected were fit either to a single step, AB, to a two steps ABC, or to a three steps, ABC, model allowing estimation of the conversion rate constants (*k*<sup>A</sup>B, *k*<sup>B</sup><sup>C</sup>*, k*<sup>C</sup>D) (Tejero *et al.*, 2007). Estimated errors in the determined rate constant values were 15%. It should be stressed that SVD analysis of PDA spectra over a selected time domain resolves the spectra in the minima number of spectral intermediates species that are formed during the reaction, reflecting a


open questions in understanding not only the ET mechanisms, but also the role that the flavin itself might play.

Fig. 1. Proteins involved in the photosynthetic electron transfer from PSI to NADP+ via Flavodoxin (Fld, orange) and Ferredoxin-NADP+ reductase (FNR, blue).

Recent studies on the FNR:Fld interaction and ET indicate that the orientation driven by the alignment of the Fld molecular dipole moment with that of FNR contributes to the formation of a bunch of alternative binding modes competent for the efficient ET reaction (Frago *et al.*, 2010; Goñi *et al.*, 2009; Medina *et al.*, 2008). FNR, Fld and NADP+ are able to form a ternary complex, indicating that NADP+ is able to occupy a site on FNR without displacing Fld (Martínez-Júlvez *et al.*, 2009; Velázquez-Campoy *et al.*, 2006). The two binding sites are not completely independent, and the overall reaction is expected to work in an ordered two-substrate process, with the pyridine nucleotide binding first, as reported for the Fd system (Batie & Kamin, 1984a, 1984b). Complex formation of Fdrd with FNR:NADP+ was found to increase the ET rate by facilitating the rate-limiting step of the process, the dissociation of the product (Fdox) (Carrillo & Ceccarelli, 2003). Binding equilibrium and steady-state studies in WT and mutant proteins envisaged similar mechanisms for Fld, but the less specific FNR:Fld interaction might alter this behaviour (Medina, 2009).

Fast kinetic stopped-flow methods have been used for the analysis of the mechanisms involving binding and ET between FNR and Fld. So far only single wavelength stoppedflow spectrophotometry studies (mainly at 600 nm) have been reported (Casaus *et al.*, 2002; Goñi *et al.*, 2008; Goñi *et al.*, 2009; Nogués *et al.*, 2003; Nogués *et al.*, 2005), not involving the effects of NADP+, the ionic strength of the media, or the evaluation of the intermediate and final compounds of the equilibrium mixture. In this work, we use stopped-flow with photodiode array detection (PDA) to better evaluate the intermediate and final species in the equilibrium mixture during the ET process in the binary Fld:FNR and ternary Fld:FNR:NADP+ systems.

#### **2. Experimental methods improving the kinetic analysis of pre-steady-state electron transfer processes**

ET has been analysed at different ionic strengths (I) and in both directions; the physiological photosynthetic ET, from Fldhq to FNRox, and the reverse ET, from FNRhq to Fldox, used to provide reducing equivalents to different metabolic pathways. Different *Anabaena* FNR and Fld variants have been used in the kinetic characterization of these ET processes using stopped-flow with photodiode detection.

#### **2.1 Biological material and steady-state spectroscopic measurements**

The different FNR and Fld variants were purified from *Luria-Bertani* IPTG-induced *E. coli* cultures containing, respectively, the pTrc99a-Fld and pET28a-FNR plasmids encoding *Anabaena* WT or E301A FNRs and WT, E16K/E61K or E16K/E61K/D126K/D150K Flds as previously described (Casaus *et al.*, 2002; Goñi *et al.*, 2009; Martínez-Júlvez *et al.*, 2001; Medina *et al.*, 1998; Tejero *et al.*, 2003). UV/Vis spectra were recorded in a Cary 100 spectrophotometer at 25 ºC. Unless otherwise stated, all measurements were recorded in 50 mM Tris/HCl, pH 8.0. Binding ability between WT FNRox and WT Fldox was determined using difference absorption spectroscopy as previously described (Goñi *et al.*, 2009; Martínez-Júlvez *et al.*, 2001; Medina *et al.*, 1998).

#### **2.2 Stopped-flow pre-steady-state kinetic measurements**

130 Advances in Photosynthesis – Fundamental Aspects

open questions in understanding not only the ET mechanisms, but also the role that the

**e-**

**h**

Fig. 1. Proteins involved in the photosynthetic electron transfer from PSI to NADP+ via

Recent studies on the FNR:Fld interaction and ET indicate that the orientation driven by the alignment of the Fld molecular dipole moment with that of FNR contributes to the formation of a bunch of alternative binding modes competent for the efficient ET reaction (Frago *et al.*, 2010; Goñi *et al.*, 2009; Medina *et al.*, 2008). FNR, Fld and NADP+ are able to form a ternary complex, indicating that NADP+ is able to occupy a site on FNR without displacing Fld (Martínez-Júlvez *et al.*, 2009; Velázquez-Campoy *et al.*, 2006). The two binding sites are not completely independent, and the overall reaction is expected to work in an ordered two-substrate process, with the pyridine nucleotide binding first, as reported for the Fd system (Batie & Kamin, 1984a, 1984b). Complex formation of Fdrd with FNR:NADP+ was found to increase the ET rate by facilitating the rate-limiting step of the process, the dissociation of the product (Fdox) (Carrillo & Ceccarelli, 2003). Binding equilibrium and steady-state studies in WT and mutant proteins envisaged similar mechanisms for Fld, but

Fast kinetic stopped-flow methods have been used for the analysis of the mechanisms involving binding and ET between FNR and Fld. So far only single wavelength stoppedflow spectrophotometry studies (mainly at 600 nm) have been reported (Casaus *et al.*, 2002; Goñi *et al.*, 2008; Goñi *et al.*, 2009; Nogués *et al.*, 2003; Nogués *et al.*, 2005), not involving the effects of NADP+, the ionic strength of the media, or the evaluation of the intermediate and final compounds of the equilibrium mixture. In this work, we use stopped-flow with photodiode array detection (PDA) to better evaluate the intermediate and final species in the equilibrium mixture during the ET process in the binary Fld:FNR and ternary

Flavodoxin (Fld, orange) and Ferredoxin-NADP+ reductase (FNR, blue).

the less specific FNR:Fld interaction might alter this behaviour (Medina, 2009).

**FNR**

**NADPH**

**Fld**

**e-**

**PSI**

flavin itself might play.

Fld:FNR:NADP+ systems.

Fast ET processes between Fld and FNR were followed by stopped-flow under anaerobic conditions in 50 mM Tris/HCl, pH 8.0 at 12ºC. Reactions were analysed by collecting multiple wavelength absorption data (360-700 nm) using an Applied Photophysics SX17.MV stopped-flow and a photodiode array detector (PDA) with the X-Scan software (*App. Photo. Ltd.*). Typically, 400 spectra *per* second were collected for each reaction. Anaerobic conditions were obtained by several cycles of evacuation and bubbling with O2-free argon. FNRhq and Fldhq samples were obtained by photoreduction in the presence of 1 mM EDTA and 5 µM 5-deazariboflavin (Medina *et al.*, 1998). The use of PDA allowed detecting that under our experimental conditions the produced Fldhq samples usually contained a small amount of Fldsq (detected at 580 nm), taken into account in subsequent analysis. Unless otherwise stated, the mixing molar ratio was 1:1 with a final concentration of 10 µM for each protein. Molar ratios of 1:1, 1:2, 1:4 and 1:8 were also assayed for the reaction of FNRhq and Fldox, with a final FNRhq concentration of 10 µM. Reactions were also analysed at different ionic strengths, obtained using variable NaCl concentrations ranging from 0 to 300 mM in 50 mM Tris/HCl, pH 8.0. The reaction between FNRox and Fldhq was also studied in presence of ~300 µM NADP+ at the indicated ionic strength conditions.

#### **2.3 Kinetic analysis of multiple-wavelength absorption data**

Spectral intermediates formed during reactions were resolved by singular value decomposition (SVD) using Pro-Kineticist (*App. Photo. Ltd.*). Data collected were fit either to a single step, AB, to a two steps ABC, or to a three steps, ABC, model allowing estimation of the conversion rate constants (*k*<sup>A</sup>B, *k*<sup>B</sup><sup>C</sup>*, k*<sup>C</sup>D) (Tejero *et al.*, 2007). Estimated errors in the determined rate constant values were 15%. It should be stressed that SVD analysis of PDA spectra over a selected time domain resolves the spectra in the minima number of spectral intermediates species that are formed during the reaction, reflecting a

Fast Kinetic Methods with Photodiode Array Detection in the Study

**400 450 500 550 600 650 700**

**-0.005 -0.004 -0.003 -0.002 -0.001 0.000 0.001**

**0.00 0.05 0.10 0.15 0.20 0.25**

**458 - 465 nm**

**507 - 517 nm**

**Time (s)**

**Wavelength (nm)**

shows the evolution of these species along the time.

**(mM-1cm-1**

**)**

**C**

**0.00**

**A**

**0.02**

**0.04**

**0.06**

**Absorbance (AU)**

**0.08**

**0.10**

of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP<sup>+</sup>

**3. Spectral evolution of the electron transfer from Fldhq to FNRox** 

Mixing Fldhq with FNRox at I ≤ 125 mM produced a slight increase in the global amount of neutral semiquinone species within the instrumental dead-time. Then, absorbance decreased in the region of the flavin band-I (458 and 464 nm), while additionally increased in the 507- 650 nm range (Fig. 3A and 3B). The overall reaction best fits a single ET step (described by an apparent *k*<sup>B</sup>C rate constant), without detection of a protein-protein interaction step (*k*<sup>A</sup>B). Resolution of the spectroscopic properties of B was consistent with FNRox and Fldhq as the main components, while those of the final species C indicated Fld accumulated mainly as Fldsq, while FNR consisted mainly of FNRox (80%) in equilibrium with smaller amounts of FNRsq and FNRhq (10% each). Deviation of the lineal combination of the

**-0.008**

**B**

**400 450 500 550 600 650 700**

**Concentration (M)**

**0.00 0.05 0.10 0.15 0.20 0.25**

**Time (s)**

**Wavelength (nm)**

Fig. 3. Evolution of spectral changes during the reaction of Fldhq with FNRox in 50 mM Tris/HCl, pH 8.0, 100 mM NaCl at 12ºC. (A). Time course for the reaction. Spectra recorded at 0.00128, 0.0064, 0.01664, 0.03712, 0.05504, 0.0832 and, 0.2547 s after mixing. The spectrum of Fldhq before mixing is shown as a dashed line, and the first spectrum after mixing is in black. The inset shows differences between kinetic traces at the indicated wavelengths. (B). Kinetics of the absorbance changes at 458, 464, 507, 517 and 577 nm. (C). Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis. Intermediate B and C species are shown in green and red lines, respectively. The inset

**0.000**

**0.008**

**0.016**


**577 nm 517 nm 507 nm**

**465 nm 458 nm**

**0.00 0.05 0.10 0.15 0.20 0.25**

**Time (s)**

distribution of protein intermediates (reactants, complexes, products...) at a certain point along the reaction time course, and not discrete enzyme intermediates. Moreover, none of them represent individual species and, their spectra cannot be included as fixed values in the global-fitting. Consequently, a spectral intermediate, in particular one that is formed in the middle of a reaction sequence, is an equilibrium distribution of protein species that are formed in a resolvable kinetic phase. Model validity was assessed by lack of systematic deviation from the residual plot at different wavelengths, inspection of calculated spectra and consistence among the number of significant singular values with the fit model. Simulations using Pro-Kineticist were also performed in order to validate the determined ET kinetics constants for the direct and reverse processes.

#### **2.4 Determination of the absorbance spectra of FNRsq and Fldsq, and of protein discrete species contained in the spectral intermediates**

Due to the high maximum level for Fldsq stabilisation, the spectrum from this species was easily determined from photoreduction experiments (Fig. 2A) (Frago *et al.*, 2007). The spectrum of FNRsq was determined by following the one-electron oxidation of FNRhq with ferricyanide in the spectral range between 360 and 700 nm (Batie & Kamin, 1984a). Molar ratios FNRhq:ferricyanide of 1:10, 1:15 and 1:20 were used, with a final FNRhq concentration of 10 µM (Fig. 2B). Analysis of the spectroscopic data along the time was performed by Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS).

Intermediate A, B, C and D species were deconvoluted initially considering they are produced by the lineal combination of FNRox, FNRsq, FNRhq, Fldox, Fldsq and Fldhq spectra (Fig. 2), and having into account the mass balance for total FNR and Fld. Deviations from the linear combination of the different redox states of FNR and Fld were observed in all cases. Visible spectra of FNR-Fld reaction mixtures slightly differ from the lineal combination of the individual components due to modulation of the flavin spectroscopic properties when changing its environment polarity upon complex formation. This indicates contribution of FNR:Fld complexes to the spectra of intermediate and final species (Casaus *et al.*, 2002; Martínez-Júlvez *et al.*, 2001; Nogués *et al.*, 2003). Nevertheless, simulations proved that the method was adequate to estimate the percentage of the different redox states of each protein.

Fig. 2. Extinction coefficient of (A) Fld and (B) FNR in their different oxidation states. Oxidised, semiquinone and, reduced species are shown in black, green and red, respectively.

#### **3. Spectral evolution of the electron transfer from Fldhq to FNRox**

132 Advances in Photosynthesis – Fundamental Aspects

distribution of protein intermediates (reactants, complexes, products...) at a certain point along the reaction time course, and not discrete enzyme intermediates. Moreover, none of them represent individual species and, their spectra cannot be included as fixed values in the global-fitting. Consequently, a spectral intermediate, in particular one that is formed in the middle of a reaction sequence, is an equilibrium distribution of protein species that are formed in a resolvable kinetic phase. Model validity was assessed by lack of systematic deviation from the residual plot at different wavelengths, inspection of calculated spectra and consistence among the number of significant singular values with the fit model. Simulations using Pro-Kineticist were also performed in order to validate the determined ET

**2.4 Determination of the absorbance spectra of FNRsq and Fldsq, and of protein** 

Due to the high maximum level for Fldsq stabilisation, the spectrum from this species was easily determined from photoreduction experiments (Fig. 2A) (Frago *et al.*, 2007). The spectrum of FNRsq was determined by following the one-electron oxidation of FNRhq with ferricyanide in the spectral range between 360 and 700 nm (Batie & Kamin, 1984a). Molar ratios FNRhq:ferricyanide of 1:10, 1:15 and 1:20 were used, with a final FNRhq concentration of 10 µM (Fig. 2B). Analysis of the spectroscopic data along the time was performed by

Intermediate A, B, C and D species were deconvoluted initially considering they are produced by the lineal combination of FNRox, FNRsq, FNRhq, Fldox, Fldsq and Fldhq spectra (Fig. 2), and having into account the mass balance for total FNR and Fld. Deviations from the linear combination of the different redox states of FNR and Fld were observed in all cases. Visible spectra of FNR-Fld reaction mixtures slightly differ from the lineal combination of the individual components due to modulation of the flavin spectroscopic properties when changing its environment polarity upon complex formation. This indicates contribution of FNR:Fld complexes to the spectra of intermediate and final species (Casaus *et al.*, 2002; Martínez-Júlvez *et al.*, 2001; Nogués *et al.*, 2003). Nevertheless, simulations proved that the method was adequate to estimate the percentage of the different redox

**0**

Fig. 2. Extinction coefficient of (A) Fld and (B) FNR in their different oxidation states. Oxidised, semiquinone and, reduced species are shown in black, green and red,

**2**

**4**

**6**

**8**

**10**

**400 450 500 550 600 650 700**

**A B**

**Wavelength (nm)**

kinetics constants for the direct and reverse processes.

**400 450 500 550 600 650 700**

**Wavelength (nm)**

states of each protein.

**0**

respectively.

**2**

**4**

**(mM-1cm-1**

**)**

**6**

**8**

**10**

**discrete species contained in the spectral intermediates** 

Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS).

Mixing Fldhq with FNRox at I ≤ 125 mM produced a slight increase in the global amount of neutral semiquinone species within the instrumental dead-time. Then, absorbance decreased in the region of the flavin band-I (458 and 464 nm), while additionally increased in the 507- 650 nm range (Fig. 3A and 3B). The overall reaction best fits a single ET step (described by an apparent *k*<sup>B</sup>C rate constant), without detection of a protein-protein interaction step (*k*<sup>A</sup>B). Resolution of the spectroscopic properties of B was consistent with FNRox and Fldhq as the main components, while those of the final species C indicated Fld accumulated mainly as Fldsq, while FNR consisted mainly of FNRox (80%) in equilibrium with smaller amounts of FNRsq and FNRhq (10% each). Deviation of the lineal combination of the

Fig. 3. Evolution of spectral changes during the reaction of Fldhq with FNRox in 50 mM Tris/HCl, pH 8.0, 100 mM NaCl at 12ºC. (A). Time course for the reaction. Spectra recorded at 0.00128, 0.0064, 0.01664, 0.03712, 0.05504, 0.0832 and, 0.2547 s after mixing. The spectrum of Fldhq before mixing is shown as a dashed line, and the first spectrum after mixing is in black. The inset shows differences between kinetic traces at the indicated wavelengths. (B). Kinetics of the absorbance changes at 458, 464, 507, 517 and 577 nm. (C). Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis. Intermediate B and C species are shown in green and red lines, respectively. The inset shows the evolution of these species along the time.

Fast Kinetic Methods with Photodiode Array Detection in the Study

**400 450 500 550 600 650 700**

**-0.004 0.000 0.004 0.008 0.012**

**0.0 0.5 1.0 1.5 2.0**

**Time (s)**

**Wavelength (nm)**

**Absorbance (AU)**

**0.00 0.02 0.04 0.06 0.08 0.10 0.12**

**C**

shown as a dashed line, and the first spectra after mixing are in black.

**0.00**

**A**

**0.02**

**0.04**

**0.06**

**Absorbance (AU)**

**0.08**

**0.10**

of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP<sup>+</sup>

and the band-I of the flavin with regard to B. The different spectroscopic properties of the intermediate species in the absence and presence of the nucleotide suggests that the nicotinamide portion of NADP+ must contribute to the catalytically competent active site, probably by modulating the orientation and/or distance between the reacting flavins. This is in agreement with the negative cooperative effect observed for simultaneous binding of Fld and NADP+ to FNR (Velázquez-Campoy *et al.*, 2006). Additionally, C slowly evolved with a slight absorbance increase at the band-I of the flavin with minor changes in the semiquinone. These observations still indicate initial production of Fldsq and reduction of FNRox, but suggest an additional step consistent with further FNR reoxidation. Analysis of the kinetic traces at 340 nm, where absorbance increase upon reduction of NADP+ to

**B**

**0.0 0.5 1.0 1.5 2.0**

**Time (s)**

**400 450 500 550 600 650 700**

**0.000 0.005 0.010 0.015 0.020**

**Wavelength (nm)**

Fig. 5. Ionic strenght dependence of spectral evolution. (A). Time course for the reaction Fldhq with FNRox in 50 mM Tris/HCl, pH 8.0, 200 mM NaCl at 12ºC. Spectra recorded at 0.00384, 0.007552, 0.1626, 0.265, 0.5514 and, 2.047 s after mixing. The inset shows kinetic traces at the

distinguishable species obtained by global analysis of the reaction in A. The inset shows the species evolution along the time. B, C, and D species are shown in green, red and purple lines, respectively. (C). Time course for the reaction at 300 mM NaCl. Spectra recorded at 0.00384, 0.08576, 0.2035, 0.3315, 0.5158 and, 2.047 s after mixing. The inset shows kinetic traces at the indicated wavelengths. In A and C, the spectra corresponding to the Fldhq before mixing are

indicated wavelengths. (B). Absorbance spectra for the pre-steady-state kinetically

**(mM-1cm-1**

**)**

**577 nm**

**465 nm**


**400 450 500 550 600 650 700**

**Concentration (M)**

**0.0 0.5 1.0 1.5 2.0**

**Time (s)**

**Wavelength (nm)**

**577 nm**

**465 nm**

different FNR and Fld redox spectra was observed for B and C (Fig. 2), indicating mutual modulation of the spectroscopic properties of each one of the flavins, and, therefore, of its environment, by the presence of the second flavoprotein. This indicates a number of molecules must be forming FNR:Fld interactions, which might even be in different oxidoreduction states.

Noticeably, increasing ionic strength up to 125 mM had a drastic deleterious impact in the ET apparent *k*<sup>B</sup>C rate constant (Fig. 4), consistent with the lack of interaction observed by difference spectroscopy when WT FNRox was titrated with WT Fldox at I = 125 mM (not shown). These results indicated a considerable decrease in the FNR:Fld affinity upon increasing the ionic strength of the media.

Fig. 4. Ionic strength dependence of the apparent rate constants for the reduction of FNRox by Fldhq in absence (black) and presence of NADP+ (red). *k*<sup>B</sup>C rate constants in absence (black closed circles) and in presence (red closed circles) of NADP+. *k*<sup>C</sup>D rate constant in presence of NADP+ (red open circles). The ionic strength profile reported for the reduction of FNRox by Fdrd is plotted on the right axis of the figure (open green circles) (Medina *et al.*, 1998).

Further increases in the ionic strength (I > 225 mM) produced the appearance of an additional final slow absorbance increase in the band-I, and, therefore, two ET steps described the process (Fig. 5). Thus, a slight absorbance bleaching of the band-I was observed from species B into C (as before described at lower I), but C further evolved with absorbance increases of this band and minor changes in the semiquinone one (Fig. 5A and 5C). Resolution of the spectroscopic properties of B was also consistent with FNRox and Fldhq as the main components, those of C indicated Fldsq and FNRox in equilibrium with some amounts of FNRsq (10%), and FNRhq (20%), while D was mainly composed by Fldsq and FNRox with 15% of Fldox and FNRhq. The ionic strength additionally contributed to considerably diminish the apparent *k*<sup>B</sup>C ET rates (Fig. 4).

ET from Fldhq to the FNRox:NADP+ preformed complex was consistent with two ET steps at all the assayed ionic strengths, and the presence of the pyridine nucleotide apparently modulated the electronic properties of the flavins (compare Fig. 6 with 3A and 3C). These observations correlated with the changes produced in the FNR spectrum upon NADP+ complexation (Tejero *et al.*, 2005). Despite the presence of NADP+ barely affected apparent *k*<sup>B</sup>C between Fld and FNR (Table 1, Fig. 4), C showed increments in both the semiquinone

different FNR and Fld redox spectra was observed for B and C (Fig. 2), indicating mutual modulation of the spectroscopic properties of each one of the flavins, and, therefore, of its environment, by the presence of the second flavoprotein. This indicates a number of molecules must be forming FNR:Fld interactions, which might even be in different oxido-

Noticeably, increasing ionic strength up to 125 mM had a drastic deleterious impact in the ET apparent *k*<sup>B</sup>C rate constant (Fig. 4), consistent with the lack of interaction observed by difference spectroscopy when WT FNRox was titrated with WT Fldox at I = 125 mM (not shown). These results indicated a considerable decrease in the FNR:Fld affinity upon

**4 8 12 16 20 24 28**

Further increases in the ionic strength (I > 225 mM) produced the appearance of an additional final slow absorbance increase in the band-I, and, therefore, two ET steps described the process (Fig. 5). Thus, a slight absorbance bleaching of the band-I was observed from species B into C (as before described at lower I), but C further evolved with absorbance increases of this band and minor changes in the semiquinone one (Fig. 5A and 5C). Resolution of the spectroscopic properties of B was also consistent with FNRox and Fldhq as the main components, those of C indicated Fldsq and FNRox in equilibrium with some amounts of FNRsq (10%), and FNRhq (20%), while D was mainly composed by Fldsq and FNRox with 15% of Fldox and FNRhq. The ionic strength additionally contributed to

ET from Fldhq to the FNRox:NADP+ preformed complex was consistent with two ET steps at all the assayed ionic strengths, and the presence of the pyridine nucleotide apparently modulated the electronic properties of the flavins (compare Fig. 6 with 3A and 3C). These observations correlated with the changes produced in the FNR spectrum upon NADP+ complexation (Tejero *et al.*, 2005). Despite the presence of NADP+ barely affected apparent *k*<sup>B</sup>C between Fld and FNR (Table 1, Fig. 4), C showed increments in both the semiquinone

**I 1/2 (mM1/2)** Fig. 4. Ionic strength dependence of the apparent rate constants for the reduction of FNRox by Fldhq in absence (black) and presence of NADP+ (red). *k*<sup>B</sup>C rate constants in absence (black closed circles) and in presence (red closed circles) of NADP+. *k*<sup>C</sup>D rate constant in presence of NADP+ (red open circles). The ionic strength profile reported for the reduction of FNRox by Fdrd is plotted on the right axis of the figure (open green circles) (Medina *et al.*, 1998).

**0**

**1**

**2**

*k***obs·10-3 (s-1**

**)**

**3**

**4**

reduction states.

increasing the ionic strength of the media.

**0**

considerably diminish the apparent *k*<sup>B</sup>C ET rates (Fig. 4).

**40**

**80**

**120**

*k***obs (s-1**

**)**

**160**

**200**

and the band-I of the flavin with regard to B. The different spectroscopic properties of the intermediate species in the absence and presence of the nucleotide suggests that the nicotinamide portion of NADP+ must contribute to the catalytically competent active site, probably by modulating the orientation and/or distance between the reacting flavins. This is in agreement with the negative cooperative effect observed for simultaneous binding of Fld and NADP+ to FNR (Velázquez-Campoy *et al.*, 2006). Additionally, C slowly evolved with a slight absorbance increase at the band-I of the flavin with minor changes in the semiquinone. These observations still indicate initial production of Fldsq and reduction of FNRox, but suggest an additional step consistent with further FNR reoxidation. Analysis of the kinetic traces at 340 nm, where absorbance increase upon reduction of NADP+ to

Fig. 5. Ionic strenght dependence of spectral evolution. (A). Time course for the reaction Fldhq with FNRox in 50 mM Tris/HCl, pH 8.0, 200 mM NaCl at 12ºC. Spectra recorded at 0.00384, 0.007552, 0.1626, 0.265, 0.5514 and, 2.047 s after mixing. The inset shows kinetic traces at the indicated wavelengths. (B). Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis of the reaction in A. The inset shows the species evolution along the time. B, C, and D species are shown in green, red and purple lines, respectively. (C). Time course for the reaction at 300 mM NaCl. Spectra recorded at 0.00384, 0.08576, 0.2035, 0.3315, 0.5158 and, 2.047 s after mixing. The inset shows kinetic traces at the indicated wavelengths. In A and C, the spectra corresponding to the Fldhq before mixing are shown as a dashed line, and the first spectra after mixing are in black.

Fast Kinetic Methods with Photodiode Array Detection in the Study

of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP<sup>+</sup>

strength again had deleterious kinetic effects in the overall ET (Table 1).

WT:NADP+ WT 25 160 14 WT:NADP+ WT 125 23 10

**400 450 500 550 600 650 700**

**Concentration (M)**

**A B**

**0.0 0.2 0.4 0.6 0.8 1.0**

**Time (s)**

species in the reaction of FNRox with (A) E16K/E61K Fldhq and (B)

**Wavelength (nm)**

Processes studied in 50 mM Tris/HCl, pH 8.0 at 12ºC.

FNR form Fld form

instrumental dead time.

**0**

**2**

**4**

**(mM-1cm-1**

**)**

**6**

**8**

**10**

**12**

Despite process for E16K/E61K Fldhq fits a single ET step, with B showing similar characteristics to those in the WT reaction, a 17-fold decrease in *k*<sup>B</sup>C was observed (Table 1, Fig. 7A). Moreover, the final species C showed the appearance of Fldox (25%) in equilibrium with Fldsq (75%), while the amount of FNRox (40%) decreased with regard to the WT reaction with the consequent increase in the proportions of FNRsq (20%) and FNRhq (40%). The process for E16K/E61K/D126K/D150K Fldhq fits a two ET model, with *k*<sup>B</sup>C being hindered up to 190 fold with regard to WT (Table 1, Fig. 7B). Surprisingly, C shows important amounts of the reduced species of both proteins and its additional transformation is consistent with Fldsq and FNRhq as the main products of the reaction. For both Fld mutants the increase in the ionic

> (mM) *<sup>k</sup>*<sup>B</sup><sup>C</sup> (s-1)

WT WT 25 190a 2.0 0.5 WT WT 125 22 0.7 0.3 E301A WT 25 >300a 0.4 0.1 E301A WT 125 >300a 0.4 0.03

WT E16K/E61K 25 11.3 0.9 0.3 WT E16K/E61K 125 <0.005 1.4 0.3 WT E16K/E61K/D126K/D150K 25 1.0 0.4 1.2 0.1

Table 1. Kinetic parameters for the electron transfer between *Anabaena* Fld and FNR variants determined by stopped-flow and PDA detection. aMost of the reaction took place in the

**0**

Fig. 7. Absorbance spectra for the pre-steady-state kinetically distinguishable intermediate

E16K/E61K/D126K/D150K Fldhq. The insets show the time evolution of these species. Species A, B, C and D species are shown in black, green, red and purple lines, respectively.

**2**

**4**

**6**

**8**

**10**

WT E16K/E61K/D126K/D150K 125 <0.005 <0.001

I FNRox + Fldhq FNRhq + Fldox

*k*<sup>C</sup><sup>D</sup> (s-1)

*k*<sup>B</sup><sup>C</sup> (s-1)

**400 450 500 550 600 650 700**

**Concentration (M)**

**02468**

**Time (s)**

**Wavelength (nm)**


*k*<sup>C</sup><sup>D</sup> (s-1)

NADPH is expected, shows that amplitudes are considerably larger for samples containing the coenzyme (not shown). This indicates the last detected step must be related with the hydride transfer from FNRhq to NADP+ to produce its reduced form. Additionally, while the ionic strength of the media decreases the amplitudes at 340 nm for the reactions in the absence of NADP+, those in its presence are nearly unaffected (not shown).

This process was also analysed for some FNR or Fld mutants previously produced and showing altered interaction or ET properties. This includes the FNR variant produced when Ala substituted for E301, a residue involved in the FNR catalytic mechanism as proton donor as well as for the stabilisation of reaction intermediates (Dumit *et al.*, 2010; Medina *et al.*, 1998). Analysis of the reduction of this variant by WT Fldhq using the PDA detector showed that most of the ET took place within the instrumental dead time (not shown), confirming this is a very fast process as previously suggested (Medina *et al.*, 1998). Moreover, in contrast to the WT process, no changes in rate constants or species were observed upon increasing the ionic strength of the media (Table 1). Thus, only information of the final spectra was obtained, being this similar to those for the WT reaction with main accumulation of Fldsq and FNRox. In the presence of NADP+ production of NADPH was observed at 340 nm with similar rates to those reported for the WT reaction.

Fig. 6. Evolution of spectral changes during the reaction of Fldhq with FNRox in 50 mM Tris/HCl, pH 8.0, 100 mM NaCl at 12ºC and NADP+ ~300 M. (A). Time course for the reaction. Spectra recorded at 0.00128, 0.0064, 0.01664, 0.03712, 0.05504 and, 0.5107 s after mixing. The inset shows kinetic traces at the indicated wavelengths. (B). Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis. The inset shows the evolution of these species along the time. Species B, C and D are shown in green, red and purple lines, respectively.

The process was similarly analysed for two Fld mutants for which the interaction step with FNR was reported to be modified by the introduction of multiple mutations (Goñi *et al.*, 2009). Spectral evolution for the ET process between E16K/E61K Fldhq and FNRox at low ionic strength (I = 25 mM) presented similar features to those above described for WT Fld (not shown). Despite a similar starting behaviour was observed for the reaction of E16K/E61K/D126K/D150K Fldhq, final absorbance increase and decrease in the flavin I and semiquinone bands, respectively, were observed (not shown). In both cases, but particularly with E16K/E61K/D126K/D150K Fldhq, evolution of the system was considerably hindered.

NADPH is expected, shows that amplitudes are considerably larger for samples containing the coenzyme (not shown). This indicates the last detected step must be related with the hydride transfer from FNRhq to NADP+ to produce its reduced form. Additionally, while the ionic strength of the media decreases the amplitudes at 340 nm for the reactions in the

This process was also analysed for some FNR or Fld mutants previously produced and showing altered interaction or ET properties. This includes the FNR variant produced when Ala substituted for E301, a residue involved in the FNR catalytic mechanism as proton donor as well as for the stabilisation of reaction intermediates (Dumit *et al.*, 2010; Medina *et al.*, 1998). Analysis of the reduction of this variant by WT Fldhq using the PDA detector showed that most of the ET took place within the instrumental dead time (not shown), confirming this is a very fast process as previously suggested (Medina *et al.*, 1998). Moreover, in contrast to the WT process, no changes in rate constants or species were observed upon increasing the ionic strength of the media (Table 1). Thus, only information of the final spectra was obtained, being this similar to those for the WT reaction with main accumulation of Fldsq and FNRox. In the presence of NADP+ production of NADPH was

**(mM-1cm-1**

**)**

**400 450 500 550 600 650 700**

**Concentration (M)**

**0.0 0.1 0.2 0.3 0.4 0.5**

**Time (s)**

**Wavelength (nm)**

absence of NADP+, those in its presence are nearly unaffected (not shown).

observed at 340 nm with similar rates to those reported for the WT reaction.

**577 nm**

**465 nm**

Fig. 6. Evolution of spectral changes during the reaction of Fldhq with FNRox in 50 mM Tris/HCl, pH 8.0, 100 mM NaCl at 12ºC and NADP+ ~300 M. (A). Time course for the reaction. Spectra recorded at 0.00128, 0.0064, 0.01664, 0.03712, 0.05504 and, 0.5107 s after mixing. The inset shows kinetic traces at the indicated wavelengths. (B). Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis. The inset shows the evolution of these species along the time. Species B, C and D are shown in

The process was similarly analysed for two Fld mutants for which the interaction step with FNR was reported to be modified by the introduction of multiple mutations (Goñi *et al.*, 2009). Spectral evolution for the ET process between E16K/E61K Fldhq and FNRox at low ionic strength (I = 25 mM) presented similar features to those above described for WT Fld (not shown). Despite a similar starting behaviour was observed for the reaction of E16K/E61K/D126K/D150K Fldhq, final absorbance increase and decrease in the flavin I and semiquinone bands, respectively, were observed (not shown). In both cases, but particularly with E16K/E61K/D126K/D150K Fldhq, evolution of the system was considerably hindered.

**0.0 0.1 0.2 0.3 0.4 0.5**

**Time (s)**

**A B**

**400 450 500 550 600 650 700**

**0.000 0.004 0.008 0.012 0.016**

**Wavelength (nm)**

green, red and purple lines, respectively.

**0.00**

**0.02**

**0.04**

**0.06**

**Absorbance (AU)**

**0.08**

**0.10**

Despite process for E16K/E61K Fldhq fits a single ET step, with B showing similar characteristics to those in the WT reaction, a 17-fold decrease in *k*<sup>B</sup>C was observed (Table 1, Fig. 7A). Moreover, the final species C showed the appearance of Fldox (25%) in equilibrium with Fldsq (75%), while the amount of FNRox (40%) decreased with regard to the WT reaction with the consequent increase in the proportions of FNRsq (20%) and FNRhq (40%). The process for E16K/E61K/D126K/D150K Fldhq fits a two ET model, with *k*<sup>B</sup>C being hindered up to 190 fold with regard to WT (Table 1, Fig. 7B). Surprisingly, C shows important amounts of the reduced species of both proteins and its additional transformation is consistent with Fldsq and FNRhq as the main products of the reaction. For both Fld mutants the increase in the ionic strength again had deleterious kinetic effects in the overall ET (Table 1).


Table 1. Kinetic parameters for the electron transfer between *Anabaena* Fld and FNR variants determined by stopped-flow and PDA detection. aMost of the reaction took place in the instrumental dead time.

Fig. 7. Absorbance spectra for the pre-steady-state kinetically distinguishable intermediate species in the reaction of FNRox with (A) E16K/E61K Fldhq and (B) E16K/E61K/D126K/D150K Fldhq. The insets show the time evolution of these species. Species A, B, C and D species are shown in black, green, red and purple lines, respectively. Processes studied in 50 mM Tris/HCl, pH 8.0 at 12ºC.

Fast Kinetic Methods with Photodiode Array Detection in the Study

of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP<sup>+</sup>

behaviour and a linear dependence of *k*<sup>B</sup>C and *k*<sup>C</sup>D rates (Fig. 9B).

FNRhq at I = 25 mM.

Fig. 9. (A). Ionic strength dependence of the apparent rate constants for the reduction of Fldox by FNRhq. The reaction was fit to a two-step process, *k*<sup>B</sup>C (black) and *k*<sup>C</sup>D (green). (B). Fldox concentration dependence of the apparent rate constants for the reduction of Fldox by

Reactions of FNRhq with E16K/E61K or E16K/E61K/D126K/D150K Fldox variants showed similar features to the WT Fld (Fig. 10). Transformation of A into B appeared slightly slower for E16K/E61K (*k*<sup>A</sup><sup>B</sup>11 s-1), while those species could not be resolved for E16K/E61K/D126K/D150K. These observations are in agreement with the 23-fold decrease and the absence of interaction reported for the complexes of E16K/E61K or E16K/E61K/D126K/D150K Fldox, respectively, with FNRox (Goñi *et al.*, 2009). B evolved to C in an ET process only 2-fold slower than for WT Fldox (Table 1) and similarly consistent with FNRsq and Fldsq as the main components of C. On conversion of C into D, absorbance increments are observed in the band-I and the semiquinone band of the flavin, particularly

When globally analysed this reaction best fits to a three steps process (A B C D) (Fig. 8C). Conversion of species A into B was relatively fast (*k*<sup>A</sup>B >50 s-1) with very little absorbance changes. Both species had FNRhq and Fldox as the main components, but deviation of mathematical combination of individual redox spectra (Fig. 2) indicated their spectroscopic properties were modulated by the presence of each other. This suggests a number of FNRhq and Fldox molecules must be forming a FNRhq:Fldox complex. This step appears, therefore, rather indicative of a protein-protein interaction or reorganisation event than of an ET one. B evolved to C in a relatively slow process (Table 1), in which the intensity of the band-I of the flavin gets decrease by 20% and displaced to shorter wavelengths, whereas accumulation of semiquinone is produced (Fig. 8B). These observations are compatible with Fldox being consumed and FNRsq, Fldsq and FNRox as the main components of species C. On conversion of C into D, there is an increment in the band-I absorption intensity (with the maximum at 458 nm) and minor changes in the semiquinone band amplitude. This is consistent with FNRox and Fldsq as the main components of D. The faster ET rates are observed at the lower ionic strength, but the ionic strength effect is much more moderated than for the reverse reaction (Fig. 9A). Reaction of FNRhq with Fldox was also analysed at different protein-protein ratios showing similar


#### **4. Spectral evolution of the electron transfer from FNRhq to Fldox**

When FNRhq reacted with Fldox an initial bleaching and displacement of the maximum from 464 nm (typical of Fldox) to 458 nm (maximum for FNRox) was observed, together with the appearance of a neutral semiquinone band (Fig. 8A). Then, absorbance increased in both the band-I and the semiquinone one (Fig. 8A and 8B). During the overall process only minor absorbance changes were detected in the Fldox/sq isosbestic point (517 nm) at all the ionic strengths assayed, but an absorbance bleaching was initially observed at the FNRox/sq one (507 nm) (Fig. 8B).

Fig. 8. Evolution of spectral changes observed during the reaction of Fldox with FNRhq. (A). Time course with spectra recorded at 0.03968, 0.2035, 0.4083, 0.695, 1.187, 1.555, 2.456, 3.521, 16.38 and 12.61 s after mixing. The spectrum of FNRhq before mixing is shown as a dashed line, and the first spectrum after mixing is in black. The inset shows kinetic traces at the indicated wavelengths. (B). Kinetics of absorbance changes observed at 458, 464, 507, 516 and 577 nm. (C). Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis. The inset shows the evolution of these species along the time. A, B, C and, D species are shown in black, green, red and purple lines, respectively. Processes studied in 50 mM Tris/HCl, pH 8.0, 100 mM NaCl at 12ºC.

When FNRhq reacted with Fldox an initial bleaching and displacement of the maximum from 464 nm (typical of Fldox) to 458 nm (maximum for FNRox) was observed, together with the appearance of a neutral semiquinone band (Fig. 8A). Then, absorbance increased in both the band-I and the semiquinone one (Fig. 8A and 8B). During the overall process only minor absorbance changes were detected in the Fldox/sq isosbestic point (517 nm) at all the ionic strengths assayed, but an absorbance bleaching was initially observed at the FNRox/sq one

Fig. 8. Evolution of spectral changes observed during the reaction of Fldox with FNRhq. (A). Time course with spectra recorded at 0.03968, 0.2035, 0.4083, 0.695, 1.187, 1.555, 2.456, 3.521, 16.38 and 12.61 s after mixing. The spectrum of FNRhq before mixing is shown as a dashed line, and the first spectrum after mixing is in black. The inset shows kinetic traces at the indicated wavelengths. (B). Kinetics of absorbance changes observed at 458, 464, 507, 516 and 577 nm. (C). Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis. The inset shows the evolution of these species along the time. A, B, C and, D species are shown in black, green, red and purple lines, respectively.

Processes studied in 50 mM Tris/HCl, pH 8.0, 100 mM NaCl at 12ºC.

**4. Spectral evolution of the electron transfer from FNRhq to Fldox** 

(507 nm) (Fig. 8B).

When globally analysed this reaction best fits to a three steps process (A B C D) (Fig. 8C). Conversion of species A into B was relatively fast (*k*<sup>A</sup>B >50 s-1) with very little absorbance changes. Both species had FNRhq and Fldox as the main components, but deviation of mathematical combination of individual redox spectra (Fig. 2) indicated their spectroscopic properties were modulated by the presence of each other. This suggests a number of FNRhq and Fldox molecules must be forming a FNRhq:Fldox complex. This step appears, therefore, rather indicative of a protein-protein interaction or reorganisation event than of an ET one. B evolved to C in a relatively slow process (Table 1), in which the intensity of the band-I of the flavin gets decrease by 20% and displaced to shorter wavelengths, whereas accumulation of semiquinone is produced (Fig. 8B). These observations are compatible with Fldox being consumed and FNRsq, Fldsq and FNRox as the main components of species C. On conversion of C into D, there is an increment in the band-I absorption intensity (with the maximum at 458 nm) and minor changes in the semiquinone band amplitude. This is consistent with FNRox and Fldsq as the main components of D. The faster ET rates are observed at the lower ionic strength, but the ionic strength effect is much more moderated than for the reverse reaction (Fig. 9A). Reaction of FNRhq with Fldox was also analysed at different protein-protein ratios showing similar behaviour and a linear dependence of *k*<sup>B</sup>C and *k*<sup>C</sup>D rates (Fig. 9B).

Fig. 9. (A). Ionic strength dependence of the apparent rate constants for the reduction of Fldox by FNRhq. The reaction was fit to a two-step process, *k*<sup>B</sup>C (black) and *k*<sup>C</sup>D (green). (B). Fldox concentration dependence of the apparent rate constants for the reduction of Fldox by FNRhq at I = 25 mM.

Reactions of FNRhq with E16K/E61K or E16K/E61K/D126K/D150K Fldox variants showed similar features to the WT Fld (Fig. 10). Transformation of A into B appeared slightly slower for E16K/E61K (*k*<sup>A</sup><sup>B</sup>11 s-1), while those species could not be resolved for E16K/E61K/D126K/D150K. These observations are in agreement with the 23-fold decrease and the absence of interaction reported for the complexes of E16K/E61K or E16K/E61K/D126K/D150K Fldox, respectively, with FNRox (Goñi *et al.*, 2009). B evolved to C in an ET process only 2-fold slower than for WT Fldox (Table 1) and similarly consistent with FNRsq and Fldsq as the main components of C. On conversion of C into D, absorbance increments are observed in the band-I and the semiquinone band of the flavin, particularly

Fast Kinetic Methods with Photodiode Array Detection in the Study

FNRox + Fldhq FNRox:Fldhq FNRsq + Fldsq

*k*A B *k*B C

with Fldhq in the WT system (1) and with some of the Fldhq mutants (2).

*k*B C

Scheme 1. Reaction pathways describing the processes observed for the reaction of FNRox

When reduction of E301A FNRox by Fldhq was analysed a very similar behaviour to the WT one described the process, again suggesting quick FNRsq deproportionation. When using the same methodology to analyse the process for two Fld mutants, with interaction and ET parameters considerably hindered (Goñi *et al.*, 2009), intermediates and products of the reaction were in agreement with the mechanism previously proposed using singlewavelength detection and with the final production of FNRhq and Fldsq under the assayed conditions (Scheme 1 reaction (2)). The PDA analysis additionally allowed improving the determination of the ET rates (Table 1). In these cases the initially produced FNRsq appears unable to quickly react with traces of the Fldox mutants, preventing the quick relaxation after the initial ET. This effect can be explained since the *E*ox/sq for these Fld variants is more negative than in WT Fld, getting closer to the FNR *E*ox/sq and making ET from FNRsq to Fldox less favourable from the thermodynamic point of view than in the WT reaction (Table 2). Therefore, our observations suggest that the stopped-flow methodology, independently of the detector, does not allow to identify the initial acceptance by WT or E301A FNRox of a single electron from WT Fldhq, since under the experimental conditions (even upon increasing the ionic strength of the media) the subsequent relaxation of the putatively initially produced FNRsq is faster than the initial ET process. Moreover, the products of this relaxation consist of a mixture of species that might not have physiological relevance within the *Anabaena* cell, where FNRsq must be able to accept electrons from a second Fldhq

The reverse ET reaction, FNRhq with Fldox was reported as a slow process (when compared with the photosynthetic one) taking place in two sequential ET steps; production of both

might produce side energy transfer reactions.

molecule.

of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP<sup>+</sup>

appear to be further reduced to FNRhq, and apparently quickly relaxes to FNRox. Surprisingly, such behaviour was observed even when the overall ET reaction results slowed down by increasing the ionic strength. Thus, fast relaxation of the equilibrium distribution after the initial ET is produced with the consequent accumulation of FNRox and Fldsq. Previous fast kinetic studies using laser flash photolysis indicated that ET from WT FNRsq to WT Fldox is an extremely fast process (*k*obs ~ 7000 s-1), suggesting the produced FNRsq will quickly react with any traces of Fldox producing Fldsq and FNRox (Medina *et al.*, 1992). Moreover, the proper nature of the PDA experiment might also contribute to this effect, since the high intensity of the lamp simultaneously exciting a wavelength range


FNRox + Fldsq (1)

FNRhq + Fldsq (2)

*k*C D

in the E16K/E61K/D126K/D150K Fld variant. This might be consistent with FNRsq deproportionation into FNRox and FNRhq, with the FNRhq produced being able to reduce another Fldox molecule to the semiquinone state. Again processes of E301A FNRox with Fldhq resembled those for the native proteins, with rate constants just slightly decreased and in agreement with previous reported data (Medina *et al.*, 1998).

Fig. 10. Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis of the reaction of FNRhq with (A) E16K/E61K Fldox and (B) E16K/E61K/D126K/D150K Fldox. Insets show the evolution of these species along the time. Intermediate B, C and D species are shown in green, red and, purple lines, respectively. Processes were studied in 50 mM Tris/HCl, pH 8.0, at 12ºC.

#### **5. Insights into the electron transfer processes in the Fld:FNR system**

The physiological ET from WT Fldhq to WT FNRox was reported as a very fast process difficult to follow by single-wavelength stopped-flow methods. Nevertheless, this methodology resulted useful to study the processes for some FNR or Fld mutants with altered interaction or ET properties, but interpretation of the data was usually confuse due to the spectral similarities between the different oxido-reduction states of both proteins (Fig. 2) (Casaus *et al.*, 2002; Goñi *et al.*, 2008; Goñi *et al.*, 2009; Medina *et al.*, 1998; Nogués *et al.*, 2003; Nogués *et al.*, 2005). Analysis of mutants suggested that the reaction might take place in two steps interpreted as formation of the semiquinones of both proteins followed by further reduction of FNRsq to the fully reduced state, with further accumulation of Fldsq at the end of the reaction. Therefore, a similar behaviour was assumed for the WT system. In this work, we have taken advantage of stopped-flow with PDA detection to better evaluate the intermediate and final species in the equilibrium mixture during these ET processes for the reactions using the WT proteins and some of their mutants.

Analysis of the process for the reduction of WT FNRox by WT Fldhq, under similar conditions to those so far reported at single-wavelengths, indicates that even using PDA it is difficult to extract conclusions for this ET process. The data suggest a very fast interaction (or collisional) step unable to be observed (*k*<sup>A</sup>B), followed by an ET step than cannot be resolved from the subsequent equilibration to finally produce Fldsq and FNRox (process (1) in Scheme 1). However, despite FNRsq is hardly detected along the reaction, both semiquinones, Fldsq and FNRsq, must be initially produced. Moreover, FNRsq does not

in the E16K/E61K/D126K/D150K Fld variant. This might be consistent with FNRsq deproportionation into FNRox and FNRhq, with the FNRhq produced being able to reduce another Fldox molecule to the semiquinone state. Again processes of E301A FNRox with Fldhq resembled those for the native proteins, with rate constants just slightly decreased and in

Fig. 10. Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis of the reaction of FNRhq with (A) E16K/E61K Fldox and (B) E16K/E61K/D126K/D150K Fldox. Insets show the evolution of these species along the time. Intermediate B, C and D species are shown in green, red and, purple lines, respectively.

**5. Insights into the electron transfer processes in the Fld:FNR system** 

The physiological ET from WT Fldhq to WT FNRox was reported as a very fast process difficult to follow by single-wavelength stopped-flow methods. Nevertheless, this methodology resulted useful to study the processes for some FNR or Fld mutants with altered interaction or ET properties, but interpretation of the data was usually confuse due to the spectral similarities between the different oxido-reduction states of both proteins (Fig. 2) (Casaus *et al.*, 2002; Goñi *et al.*, 2008; Goñi *et al.*, 2009; Medina *et al.*, 1998; Nogués *et al.*, 2003; Nogués *et al.*, 2005). Analysis of mutants suggested that the reaction might take place in two steps interpreted as formation of the semiquinones of both proteins followed by further reduction of FNRsq to the fully reduced state, with further accumulation of Fldsq at the end of the reaction. Therefore, a similar behaviour was assumed for the WT system. In this work, we have taken advantage of stopped-flow with PDA detection to better evaluate the intermediate and final species in the equilibrium mixture during these ET processes for

Analysis of the process for the reduction of WT FNRox by WT Fldhq, under similar conditions to those so far reported at single-wavelengths, indicates that even using PDA it is difficult to extract conclusions for this ET process. The data suggest a very fast interaction (or collisional) step unable to be observed (*k*<sup>A</sup>B), followed by an ET step than cannot be resolved from the subsequent equilibration to finally produce Fldsq and FNRox (process (1) in Scheme 1). However, despite FNRsq is hardly detected along the reaction, both semiquinones, Fldsq and FNRsq, must be initially produced. Moreover, FNRsq does not

**400 450 500 550 600 650 700**

**Concentration (M)**

**0 4 8 12 16**

**Time (s)**

**Wavelength (nm)**

agreement with previous reported data (Medina *et al.*, 1998).

**02468**

**Time (s)**

**A B**

Processes were studied in 50 mM Tris/HCl, pH 8.0, at 12ºC.

the reactions using the WT proteins and some of their mutants.

**400 450 500 550 600 650 700**

**Concentration (M)**

**Wavelength (nm)**

**0**

**2**

**4**

**(mM-1cm-1**

**)**

**6**

**8**

**10**

**12**

appear to be further reduced to FNRhq, and apparently quickly relaxes to FNRox. Surprisingly, such behaviour was observed even when the overall ET reaction results slowed down by increasing the ionic strength. Thus, fast relaxation of the equilibrium distribution after the initial ET is produced with the consequent accumulation of FNRox and Fldsq. Previous fast kinetic studies using laser flash photolysis indicated that ET from WT FNRsq to WT Fldox is an extremely fast process (*k*obs ~ 7000 s-1), suggesting the produced FNRsq will quickly react with any traces of Fldox producing Fldsq and FNRox (Medina *et al.*, 1992). Moreover, the proper nature of the PDA experiment might also contribute to this effect, since the high intensity of the lamp simultaneously exciting a wavelength range might produce side energy transfer reactions.

Scheme 1. Reaction pathways describing the processes observed for the reaction of FNRox with Fldhq in the WT system (1) and with some of the Fldhq mutants (2).

When reduction of E301A FNRox by Fldhq was analysed a very similar behaviour to the WT one described the process, again suggesting quick FNRsq deproportionation. When using the same methodology to analyse the process for two Fld mutants, with interaction and ET parameters considerably hindered (Goñi *et al.*, 2009), intermediates and products of the reaction were in agreement with the mechanism previously proposed using singlewavelength detection and with the final production of FNRhq and Fldsq under the assayed conditions (Scheme 1 reaction (2)). The PDA analysis additionally allowed improving the determination of the ET rates (Table 1). In these cases the initially produced FNRsq appears unable to quickly react with traces of the Fldox mutants, preventing the quick relaxation after the initial ET. This effect can be explained since the *E*ox/sq for these Fld variants is more negative than in WT Fld, getting closer to the FNR *E*ox/sq and making ET from FNRsq to Fldox less favourable from the thermodynamic point of view than in the WT reaction (Table 2). Therefore, our observations suggest that the stopped-flow methodology, independently of the detector, does not allow to identify the initial acceptance by WT or E301A FNRox of a single electron from WT Fldhq, since under the experimental conditions (even upon increasing the ionic strength of the media) the subsequent relaxation of the putatively initially produced FNRsq is faster than the initial ET process. Moreover, the products of this relaxation consist of a mixture of species that might not have physiological relevance within the *Anabaena* cell, where FNRsq must be able to accept electrons from a second Fldhq molecule.

The reverse ET reaction, FNRhq with Fldox was reported as a slow process (when compared with the photosynthetic one) taking place in two sequential ET steps; production of both

Fast Kinetic Methods with Photodiode Array Detection in the Study

more efficient orientations between the flavin cofactors.

of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP<sup>+</sup>

an optimum ionic strength value and with the electrostatic stabilisation at low ionic strengths of non-optimal orientations within the intermediate ET complex. Thus, specific electrostatic and hydrophobic interactions play an important role in these association and dissociation processes, as well as in the rearrangement of the complex (Faro *et al.*, 2002a; Hurley *et al.*, 2006; Martínez-Júlvez *et al.*, 1998; Martínez-Júlvez *et al.*, 1999; Martínez-Júlvez *et al.*, 2001; Medina & Gómez-Moreno, 2004; Morales *et al.*, 2000; Nogués *et al.*, 2003). Despite some residues on the FNR surface are critical for the interaction with Fld and it is accepted that FNR interacts using the same region with Fld and Fd (Hurley *et al.*, 2002; Martínez-Júlvez *et al.*, 1999), the bell-shaped profile for the ionic strength dependence is not reproduced for ET reactions between FNR and Fld (Fig. 4). A strong deleterious influence of the ionic strength is observed on the overall ET process between Fld and FNR, particularly in the photosynthetic direction (Fig. 4 and 9A). This suggests re-arrangement of the initial FNR:Fld interaction either does not take place or does not increase the efficiency of the system, while at lower ionic strength the electrostatic interactions contribute to produce

Biochemical and docking studies suggested that the FNR:Fld interaction does not rely on a precise complementary surface of the reacting molecules. Thus, WT Fld might adopt different orientations on the FNR surface without significantly altering the relative disposition and contact between the FMN and FAD groups of Fld and FNR and, therefore, the distance between their methyl groups (Fig. 11A) (Goñi *et al.*, 2009; Medina *et al.*, 2008; Medina, 2009). Those studies suggested the molecular dipole moment alignment as one of the major determinants for the efficiency of this system (Fig. 11B). However, kinetic

Fig. 11. (A). Model for the interaction of Fld and FNR. The figure shows several positions determined by docking of Fld onto the FNR surface. (B). Magnitude and orientation of the

dipole moment of Fld and FNR in the model.


flavoprotein semiquinones (reaction (3) in Scheme 2), followed by the reduction of a second Fldox molecule by the FNRsq produced in the first step (reaction (4) in Scheme 2) (Casaus *et al.*, 2002; Medina *et al.*, 1998; Nogués *et al.*, 2005). The spectral evolutions acquired using the PDA detector confirm such mechanism, and allowed to improve the assignment of intermediates and the major contribution of apparent rate constants to particular steps of the process.


Table 2. Midpoint reduction potentials for the different *Anabaena* Fld and FNR forms. Data obtained in 50 mM Tris/HCl at pH 8.0 and 25 ºC for Flda and at 10ºC for FNRb. aData from (Goñi *et al.*, 2009). bData from (Faro *et al.*, 2002b). cData from (Medina *et al.*, 1998).

Scheme 2. Reaction pathways describing the processes observed for the reaction of the FNRhq variants with the Fldox variants.

Thus, for the process with the E16K/E61K Fld variant, the step corresponding to complex formation-reorganisation was erroneously related with an ET step in a previous study. At the lowest ionic strength assayed our data indicate that E16K/E61K Fldox and, particularly, E16K/E61K/D126/D150K Fldox are still able to accept electrons from FNRox with apparent rates that only decreased by 2-fold and with final production of FNRox occurring in higher degree that in the WT system (Table 1, Fig. 8 and 10). Their slightly more negative *E*ox/sq values (Table 2) makes them poorer electron acceptors from FNRhq than WT Fld and might explain the small differences in rates (Goñi *et al.*, 2009). Additionally, E301A FNRhq is also able to pass electrons to Fldox with a rate 5-fold slower than WT (Table 1). This behaviour might be related with the very low stability of the semiquinone form in this FNR mutant (Table 2), that makes the formation of this intermediate state non-favourable (Medina *et al.*, 1998).

A biphasic dependence of the observed rate constants on the protein concentration has been reported for the ET reaction from Fd to FNR (Fig. 4), and associated with the appearance of

flavoprotein semiquinones (reaction (3) in Scheme 2), followed by the reduction of a second Fldox molecule by the FNRsq produced in the first step (reaction (4) in Scheme 2) (Casaus *et al.*, 2002; Medina *et al.*, 1998; Nogués *et al.*, 2005). The spectral evolutions acquired using the PDA detector confirm such mechanism, and allowed to improve the assignment of intermediates and the major contribution of apparent rate constants to particular steps of the

*E*ox/sq -

*E*sq/hq -

*K*dFNRox:Fldox (µM)

(3)

(4)

*E*sq/hqWT (mV)

*E*ox/sqWT (mV)

process.

FNR form

Fld form *E*ox/sq

FNRhq variants with the Fldox variants.

(mV)

*E*sq/hq (mV)

WTa -256 -445 --- --- 2.6 E16K/E61Ka -301 -390 -45 55 46 E16K/E61K/D126K/D150Ka -297 -391 -41 54 ---

WTb -325 -338 --- --- 3c E301A -284b -358b 41 -20 4c

(Goñi *et al.*, 2009). bData from (Faro *et al.*, 2002b). cData from (Medina *et al.*, 1998).

FNRhq + Fldox FNRhq:Fldox FNRsq + Fldsq

*k*A B *k*B C

FNRsq + Fldox FNRsq:Fldox FNRox + Fldsq

Scheme 2. Reaction pathways describing the processes observed for the reaction of the

*k*B C +

the formation of this intermediate state non-favourable (Medina *et al.*, 1998).

*k*C D

Thus, for the process with the E16K/E61K Fld variant, the step corresponding to complex formation-reorganisation was erroneously related with an ET step in a previous study. At the lowest ionic strength assayed our data indicate that E16K/E61K Fldox and, particularly, E16K/E61K/D126/D150K Fldox are still able to accept electrons from FNRox with apparent rates that only decreased by 2-fold and with final production of FNRox occurring in higher degree that in the WT system (Table 1, Fig. 8 and 10). Their slightly more negative *E*ox/sq values (Table 2) makes them poorer electron acceptors from FNRhq than WT Fld and might explain the small differences in rates (Goñi *et al.*, 2009). Additionally, E301A FNRhq is also able to pass electrons to Fldox with a rate 5-fold slower than WT (Table 1). This behaviour might be related with the very low stability of the semiquinone form in this FNR mutant (Table 2), that makes

A biphasic dependence of the observed rate constants on the protein concentration has been reported for the ET reaction from Fd to FNR (Fig. 4), and associated with the appearance of

Table 2. Midpoint reduction potentials for the different *Anabaena* Fld and FNR forms. Data obtained in 50 mM Tris/HCl at pH 8.0 and 25 ºC for Flda and at 10ºC for FNRb. aData from an optimum ionic strength value and with the electrostatic stabilisation at low ionic strengths of non-optimal orientations within the intermediate ET complex. Thus, specific electrostatic and hydrophobic interactions play an important role in these association and dissociation processes, as well as in the rearrangement of the complex (Faro *et al.*, 2002a; Hurley *et al.*, 2006; Martínez-Júlvez *et al.*, 1998; Martínez-Júlvez *et al.*, 1999; Martínez-Júlvez *et al.*, 2001; Medina & Gómez-Moreno, 2004; Morales *et al.*, 2000; Nogués *et al.*, 2003). Despite some residues on the FNR surface are critical for the interaction with Fld and it is accepted that FNR interacts using the same region with Fld and Fd (Hurley *et al.*, 2002; Martínez-Júlvez *et al.*, 1999), the bell-shaped profile for the ionic strength dependence is not reproduced for ET reactions between FNR and Fld (Fig. 4). A strong deleterious influence of the ionic strength is observed on the overall ET process between Fld and FNR, particularly in the photosynthetic direction (Fig. 4 and 9A). This suggests re-arrangement of the initial FNR:Fld interaction either does not take place or does not increase the efficiency of the system, while at lower ionic strength the electrostatic interactions contribute to produce more efficient orientations between the flavin cofactors.

Biochemical and docking studies suggested that the FNR:Fld interaction does not rely on a precise complementary surface of the reacting molecules. Thus, WT Fld might adopt different orientations on the FNR surface without significantly altering the relative disposition and contact between the FMN and FAD groups of Fld and FNR and, therefore, the distance between their methyl groups (Fig. 11A) (Goñi *et al.*, 2009; Medina *et al.*, 2008; Medina, 2009). Those studies suggested the molecular dipole moment alignment as one of the major determinants for the efficiency of this system (Fig. 11B). However, kinetic

Fig. 11. (A). Model for the interaction of Fld and FNR. The figure shows several positions determined by docking of Fld onto the FNR surface. (B). Magnitude and orientation of the dipole moment of Fld and FNR in the model.

Fast Kinetic Methods with Photodiode Array Detection in the Study

**6. Conclusion** 

process.

**7. Acknowledgment** 

**8. References** 

2728.

of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP<sup>+</sup>

Single-wavelength fast kinetic stopped-flow methods have been widely used for the analysis of the mechanisms involving transient binding and ET between Fld and FNR. However, this methodology did not allow to un-ambiguously identifying the intermediate and final compounds of the reactions. PDA detection combined with fast kinetic stoppedflow methods results useful to better understand the mechanisms involving transient binding and ET between Fld and FNR. Despite the high similarity among the spectra for the same redox states within both proteins, the methodology here used allowed identifying the composition of the intermediate species and final species of the reactions, as long as the kinetics fits in the measurable instrumental time. The mechanism of these inter-flavin ET reactions is revisited, evaluating the evolution of the reaction along the time within a wavelength spectral range by using a PDA detector. Additionally, our analysis of the dependence of the inter-flavin ET mechanism on the ionic strength suggest that, under physiological conditions, the electrostatic alignment contributes to the overall orientation but it is not anymore the major determinant of the orientation of Fld on the protein partner surface. Additionally, the presence of the coenzyme reveals a complex modulation of the

This work has been supported by Ministerio de Ciencia e Innovación, Spain (Grant BIO2010- 14983 to M.M.). We thank to Dr. G. Goñi for the production of the Fld mutants and Dr. R.

Arakaki, A. K., Ceccarelli, E. A. & Carrillo, N. (1997). Plant-type ferredoxin-NADP+

Batie, C. J. & Kamin, H. (1984a). Electron transfer by ferredoxin:NADP+ reductase. Rapid-

Batie, C. J. & Kamin, H. (1984b). Ferredoxin:NADP+ oxidoreductase. Equilibria in binary and

Bottin, H. & Lagoutte, B. (1992). Ferredoxin and flavodoxin from the cyanobacterium

Brenner, S., Hay, S., Munro, A. W. & Scrutton, N. S. (2008). Inter-flavin electron transfer in

in mechanism. *Febs J*. Vol.275, No.18, pp. 4540-4557, ISSN 1742-464X. Carrillo, N. & Ceccarelli, E. A. (2003). Open questions in ferredoxin-NADP+ reductase catalytic mechanism. *Eur J Biochem*. Vol.270, No.9, pp. 1900-1915, ISSN 0014-2956. Casaus, J. L., Navarro, J. A., Hervás, M., Lostao, A., De la Rosa, M. A., Gómez-Moreno, C.,

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cytochrome P450 reductase - effects of solvent and pH identify hidden complexity

Sancho, J. & Medina, M. (2002). *Anabaena* sp. PCC 7119 flavodoxin as electron

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No.19, pp. 11976-11985, ISSN 0021-9258.

8832-8839, ISSN 0021-9258.


parameters reported to the date for these processes were only obtained at low ionic strength (0.03 M), conditions far away from those found in the thylakoid (0.15-0.3 M (Durán *et al.*, 2006)). Our data suggest that at physiological ionic strengths the ET efficiency, particularly in the photosynthetic direction, will be considerably hindered with regard to the data reported *in vitro* at low ionic strengths. The ionic strength will shield the dipole moment alignment contribution, making it just one additional contribution to proteins encounter. Among those contributions we might include electrostatic and hydrophobic interactions imposed by the thylakoid membrane, physical diffusional parameters and molecular crowding inside the cell. It is also worth to note that increasing the ionic strength makes reduction of FNR by Fldhq only 4-8 times faster than the reverse process (Compare Fig. 4A and 9A). Thus, shielding the effect of the dipole moment appears to have a larger impact in producing the competent ET orientation between the redox centres in the FNRox:Fldhq complex than in the FNRhq:Fldox one. In other words, it reduces the probability of obtaining the best FNRox:Fldhq orientations for ET.

The Fld mutants here studied, particularly E16K/E61K/D126/D150K, have lost the ability to efficiently reduce FNR (Table 1). More positive *E*sq/hq values (Table 2) might somehow contribute to this behaviour, but previous studies suggested the introduced mutations induced changes in the Fld electrostatic potential surfaces, as well as in the orientation and magnitude of the Fld molecular dipole moment (Goñi *et al.*, 2009). Despite the thermodynamic parameters favour the process, the observed reaction might only correlate with a collisional-type reaction. Therefore, it could exit the possibility that steering of the dipole moment contribution might produce a positive effect on the overall ET process. However, our analysis also shows that the increasing of the ionic strength again had a deleterious effect in the ET processes from these Fldhq mutants. Thus, electrostatic and hydrophobic interactions and the dipole moment still must contribute to the formation of productive interactions between both proteins at physiological ionic strengths. *In vivo* the presence of other proteins competing for Fldhq might also result in changes to electron channelling into distinct pathways. When going to physiological conditions Fld interaction with FNR is confirmed to be less specific than that of Fd. Subtle changes at the isoalloxazine environment influence the Fld binding abilities and modulate the ET processes by producing different orientations and distances between the redox centres. Therefore, ET reactions involving Fld might not have as much specific interaction requirements as other reactions involving protein-protein interactions. Thus, the bound state could be formed by dynamic ensembles instead of single conformations as has already been proposed for this system (Fig. 11) (Goñi *et al.*, 2009; Medina *et al.*, 2008) and also observed in other ET systems (Crowley & Carrondo, 2004; Worrall *et al.*, 2003). This further confirms previous studies suggesting that Fld interacts with different structural partners through non-specific interactions, which in turn decreased the potential efficiency in ET that could achieve if unique and more favourable orientations were produced with a reduced number of partners. Heterogeneity of ET kinetics is an intrinsic property of Fld oxido-reduction processes, and can be most probably ascribed to different conformations of FNR:Fld complexes (Medina *et al.*, 2008; Sétif, 2001). During Fld-dependent photosynthetic ET the Fld molecule must pivot between its docking sites in PSI and in FNR. Formation of transient complexes of Fld with FNR *in vivo* is useful during this process, though not critical, for promoting efficient reduction of Fld and FNR and for avoiding reduction of oxygen by the donor redox centres (Goñi *et al.*, 2008; Goñi *et al.*, 2009; Hurley *et al.*, 2006; Sétif, 2001, 2006).

#### **6. Conclusion**

144 Advances in Photosynthesis – Fundamental Aspects

parameters reported to the date for these processes were only obtained at low ionic strength (0.03 M), conditions far away from those found in the thylakoid (0.15-0.3 M (Durán *et al.*, 2006)). Our data suggest that at physiological ionic strengths the ET efficiency, particularly in the photosynthetic direction, will be considerably hindered with regard to the data reported *in vitro* at low ionic strengths. The ionic strength will shield the dipole moment alignment contribution, making it just one additional contribution to proteins encounter. Among those contributions we might include electrostatic and hydrophobic interactions imposed by the thylakoid membrane, physical diffusional parameters and molecular crowding inside the cell. It is also worth to note that increasing the ionic strength makes reduction of FNR by Fldhq only 4-8 times faster than the reverse process (Compare Fig. 4A and 9A). Thus, shielding the effect of the dipole moment appears to have a larger impact in producing the competent ET orientation between the redox centres in the FNRox:Fldhq complex than in the FNRhq:Fldox one. In other words, it reduces the probability of obtaining

The Fld mutants here studied, particularly E16K/E61K/D126/D150K, have lost the ability to efficiently reduce FNR (Table 1). More positive *E*sq/hq values (Table 2) might somehow contribute to this behaviour, but previous studies suggested the introduced mutations induced changes in the Fld electrostatic potential surfaces, as well as in the orientation and magnitude of the Fld molecular dipole moment (Goñi *et al.*, 2009). Despite the thermodynamic parameters favour the process, the observed reaction might only correlate with a collisional-type reaction. Therefore, it could exit the possibility that steering of the dipole moment contribution might produce a positive effect on the overall ET process. However, our analysis also shows that the increasing of the ionic strength again had a deleterious effect in the ET processes from these Fldhq mutants. Thus, electrostatic and hydrophobic interactions and the dipole moment still must contribute to the formation of productive interactions between both proteins at physiological ionic strengths. *In vivo* the presence of other proteins competing for Fldhq might also result in changes to electron channelling into distinct pathways. When going to physiological conditions Fld interaction with FNR is confirmed to be less specific than that of Fd. Subtle changes at the isoalloxazine environment influence the Fld binding abilities and modulate the ET processes by producing different orientations and distances between the redox centres. Therefore, ET reactions involving Fld might not have as much specific interaction requirements as other reactions involving protein-protein interactions. Thus, the bound state could be formed by dynamic ensembles instead of single conformations as has already been proposed for this system (Fig. 11) (Goñi *et al.*, 2009; Medina *et al.*, 2008) and also observed in other ET systems (Crowley & Carrondo, 2004; Worrall *et al.*, 2003). This further confirms previous studies suggesting that Fld interacts with different structural partners through non-specific interactions, which in turn decreased the potential efficiency in ET that could achieve if unique and more favourable orientations were produced with a reduced number of partners. Heterogeneity of ET kinetics is an intrinsic property of Fld oxido-reduction processes, and can be most probably ascribed to different conformations of FNR:Fld complexes (Medina *et al.*, 2008; Sétif, 2001). During Fld-dependent photosynthetic ET the Fld molecule must pivot between its docking sites in PSI and in FNR. Formation of transient complexes of Fld with FNR *in vivo* is useful during this process, though not critical, for promoting efficient reduction of Fld and FNR and for avoiding reduction of oxygen by the donor redox centres (Goñi *et al.*, 2008; Goñi *et al.*, 2009; Hurley *et al.*, 2006; Sétif, 2001, 2006).

the best FNRox:Fldhq orientations for ET.

Single-wavelength fast kinetic stopped-flow methods have been widely used for the analysis of the mechanisms involving transient binding and ET between Fld and FNR. However, this methodology did not allow to un-ambiguously identifying the intermediate and final compounds of the reactions. PDA detection combined with fast kinetic stoppedflow methods results useful to better understand the mechanisms involving transient binding and ET between Fld and FNR. Despite the high similarity among the spectra for the same redox states within both proteins, the methodology here used allowed identifying the composition of the intermediate species and final species of the reactions, as long as the kinetics fits in the measurable instrumental time. The mechanism of these inter-flavin ET reactions is revisited, evaluating the evolution of the reaction along the time within a wavelength spectral range by using a PDA detector. Additionally, our analysis of the dependence of the inter-flavin ET mechanism on the ionic strength suggest that, under physiological conditions, the electrostatic alignment contributes to the overall orientation but it is not anymore the major determinant of the orientation of Fld on the protein partner surface. Additionally, the presence of the coenzyme reveals a complex modulation of the process.

#### **7. Acknowledgment**

This work has been supported by Ministerio de Ciencia e Innovación, Spain (Grant BIO2010- 14983 to M.M.). We thank to Dr. G. Goñi for the production of the Fld mutants and Dr. R. Tauler for his help in initial spectral deconvolution of intermediate species.

#### **8. References**


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**8** 

**Photosynthesis in Lichen:** 

Alfonso Ros-Barceló3, Eva Barreno2, José Miguel Zapata1 and Alfredo Guéra1

> *1University of Alcalá 2University of Valencia 3University of Murcia*

> > *Spain*

**Light Reactions and Protective Mechanisms** 

Lichens are symbiotic associations (holobionts) established between fungi (mycobionts) and certain groups of cyanobacteria or unicellular green algae (photobionts). This symbiotic association has been essential in establishing the colonization of terrestrial and consequently dry habitats. About 44 genera of algae and cyanobacteria have been reported as lichen photobionts. Due to the uncertain taxonomy of many of these photobionts, these numbers were considered as approximations only. Ahmadjian (1993) estimates that only 25 genera were typical lichen photobionts. The most common cyanobionts are *Nostoc*, *Scytonema*, *Stigonema*, *Gloeocapsa*, and *Calothrix*, in order of frequency (Büdel, 1992). Green algal photobionts include *Asterochloris, Trebouxia*, *Trentepohlia*, *Coccomyxa*, and *Dictyochloropsis* (Gärtner, 1992). These authors assessed that more than 50% of all lichen species are associated with *Trebouxia* and *Asterochloris* species. However, this is just estimation since in only 2% of the described lichen species the photobiont genus is reported (Tschermak-Woess, 1989), mostly by the difficulties to isolate and then characterize the algae from the lichen thalli. Lichens are well known for their slow growth and longevity. Their radial growth is measured in millimetres per year (Hale, 1973), while individual lichens live for hundreds or even thousands of years. It is assumed that in lichens the photobiont population is under mycobiont control. Lichenologists have proposed some control mechanisms such as, cell division inhibitors (Honegger, 1987), phytohormones (Backor & Hudak, 1999) or nutrients

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competition (Crittenden et al., 1994; Schofield et al., 2003).

**1. Introduction** 

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photosystem I to ferredoxin-NADP+ reductase in *Anabaena* PCC 7119. *Biochemistry*. Vol.42, No.7, pp. 2036-2045, ISSN 0006-2960.


### **Photosynthesis in Lichen: Light Reactions and Protective Mechanisms**

Francisco Gasulla2, Joaquín Herrero1, Alberto Esteban-Carrasco1, Alfonso Ros-Barceló3, Eva Barreno2, José Miguel Zapata1 and Alfredo Guéra1 *1University of Alcalá 2University of Valencia 3University of Murcia Spain* 

#### **1. Introduction**

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Tejero, J., Peregrina, J. R., Martínez-Júlvez, M., Gutierrez, A., Gómez-Moreno, C., Scrutton,

Velázquez-Campoy, A., Goñi, G., Peregrina, J. R. & Medina, M. (2006). Exact analysis of

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of *Anabaena* ferredoxin-NADP+ reductase to NADP+ and to ferredoxin. *Eur J* 

flavodoxin: kinetic and structural properties of ferredoxin/flavodoxin reduction by photosystem I, In: *Photosystem I. The light-driven platocyanin:ferredoxin oxidoreductase*, J. H. Golbeck (Ed.), pp. 439-454, Springer, ISBN 1-4020-4255-8, Dordrecht, The

Hurley, J. K., Tollin, G., Gómez-Moreno, C. & Medina, M. (2003). Involvement of the pyrophosphate and the 2'-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity. *J Biol Chem*. Vol.278, No.49, pp. 49203-49214,

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reductase studied by stopped-flow spectrophotometry. *Biochemistry*. Vol.43, No.2,

studied by NMR spectroscopy: the case of cytochrome *c* and adrenodoxin.

Lichens are symbiotic associations (holobionts) established between fungi (mycobionts) and certain groups of cyanobacteria or unicellular green algae (photobionts). This symbiotic association has been essential in establishing the colonization of terrestrial and consequently dry habitats. About 44 genera of algae and cyanobacteria have been reported as lichen photobionts. Due to the uncertain taxonomy of many of these photobionts, these numbers were considered as approximations only. Ahmadjian (1993) estimates that only 25 genera were typical lichen photobionts. The most common cyanobionts are *Nostoc*, *Scytonema*, *Stigonema*, *Gloeocapsa*, and *Calothrix*, in order of frequency (Büdel, 1992). Green algal photobionts include *Asterochloris, Trebouxia*, *Trentepohlia*, *Coccomyxa*, and *Dictyochloropsis* (Gärtner, 1992). These authors assessed that more than 50% of all lichen species are associated with *Trebouxia* and *Asterochloris* species. However, this is just estimation since in only 2% of the described lichen species the photobiont genus is reported (Tschermak-Woess, 1989), mostly by the difficulties to isolate and then characterize the algae from the lichen thalli. Lichens are well known for their slow growth and longevity. Their radial growth is measured in millimetres per year (Hale, 1973), while individual lichens live for hundreds or even thousands of years. It is assumed that in lichens the photobiont population is under mycobiont control. Lichenologists have proposed some control mechanisms such as, cell division inhibitors (Honegger, 1987), phytohormones (Backor & Hudak, 1999) or nutrients competition (Crittenden et al., 1994; Schofield et al., 2003).

Similar to plants, all lichens photosynthesise. They need light to provide energy to make their own matter. More specifically, the algae in the lichen produce carbohydrates and the fungi take those carbohydrates to grow and reproduce. The amount of light intensity needed for optimal lichen growth varies widely among species. The optimum light intensity range of most algal photobionts in axenic cultures is very low, between 16-27 μmol m-2 s-1. If the response of cultured photobionts to light is similar to that of the natural forms (lichen), then there must be additional mechanisms protecting the algae in the lichen that are not developed under culture conditions. Pigments and crystal of secondary metabolites in the

Photosynthesis in Lichen: Light Reactions and Protective Mechanisms 151

On the other hand, non-axenic cultures can be used for studying algal metabolites or enzymatic activities of the lichenized photobionts. These methods consist in homogenization of the lichen thalli, followed by separation of the photobiont from the mycobiont and fragments of thalli using differential centrifugation (Richardson, 1971), gradient centrifugation on CsCl2/KI (Ascaso, 1980) or on Percoll® gradients (Calatayud et al., 2001),

Here we resume the fast and simple methods developed in our laboratories (Gasulla et al., 2010): a low-scale isolation method (micromethod) and a large scale one (macromethod). The micromethod for isolation of lichen photobions starts from 15–25 mg dry weight (DW) of lichen material that is washed first in tap water, and then slowly stirred in sterile distilled water in a bucket for 5 min. The fragments of thalli are homogenised in an sterile eppendorf tube with a pellet pestle and resuspended in sterile 1 ml of isotonic buffer (0.3 M sorbitol in 50 mM HEPES pH 7.5). After filtration through sterile muslin, the filtrate is centrifuged at 490×g in a bench-top microcentrifuge (Micro 20, Hettich, Germany) for 5 min. The pellet is resuspended in 200 μl of sterile isotonic buffer and then loaded on 1.5 ml, of sterile 80 % Percoll ® in isotonic buffer. After centrifugation at 10000×g for 10 min a clear green layer must be present near the top of the eppendorf tube and some grey particles and pellet at the bottom of the tube (Fig. 1). The green layer is recovered (ca. 400 μl), avoiding to take any drop of the upper interphase. Then, the green layer is diluted 2-fold with sterile distilled water and centrifuged at 1000×g for 10 min. The supernatant is discarded; the pellet is resuspended in 2 ml of sterile distilled water and a drop of Tween 20 is added. The resulting suspension is sonicated at 40 KHz (Elma Transsonic Digital 470 T, 140% ultrasound power) for 1 min and again centrifuged at 490×g for 5 min. This treatment is repeated five times. The final pellet containing the isolated algal cells is resuspended in 1 ml of sterile distilled water. This micromethod can be scaled up to a macromethod, which allows preparation of

Fig. 1. Separation of *Ramalina farinacea* fractions after centrifugation of the extract of thalli at 10,000×g for 10 min on 40 ml (macromethod), or 1.5 ml (micromethod) of 80 % Percoll ® in isotonic buffer. A–D, each optical micrograph refers to the corresponding Percoll layer; A and C phase contrast microscopy. Scale = 15 μm. \* Not real size. Photograph from Gasulla

and/or filtering (Weissman et al., 2005).

large amounts of photobiont cells.

et al., (2010).

upper cortex are supposed to decrease the intensity of light reaching the photobionts especially under desiccated conditions by absorbing certain wavelengths and by reflecting light (Heber et al,. 2007; Scott, 1969; Veerman et al., 2007). Apparently, the balance between energy conservation and energy dissipation is tilted towards dissipation in many poikilohydric autotrophs, whereas, in higher plants, energy conservation assumes dominance over energy dissipation. It thus appears that sensitivity to excess light is higher in the mosses and the lichens than in higher plants (Heber, 2008).

Lichens are found among poikilohydric organisms, those that cannot actively regulate their water content, but are capable of surviving long periods in a desiccated state (Kappen & Valladares, 2007). In the dry state many lichens exhibit an enhanced resistence to other stress. For instance, heat resistance up to 70-75 ºC in species from sheltered microhabitats and up to 90-100 ºC in species from exposed microhabitats (Lange, 1953). Desiccation tolerance was described in nematodes and in rotifers observed by van Leeuwenhoek in 1702, and has since been discovered in four other phyla of animals, algae, fungi, bacteria, in ca. 350 species of flowering plants and ferns and in most bryophytes and seeds of flowering plants (Alpert, 2006; Proctor & Tuba, 2002). Among them, algae, lichen and bryophytes can be considered fully desiccation-tolerant plants because can survive very rapid drying events (less than 1 h) and recover respiration and photosynthesis within a few minutes (Oliver & Wood, 1997; Proctor & Smirnoff, 2000). Most lichen-forming fungi and their photobionts are fully adapted to daily wetting and drying cycles, but die off under continuously moist conditions (Dietz & Hartung, 1999; Farrar, 1976a, 1976b). It is well known that photosynthesis in homoiohydric plants is very sensitive to water stress conditions (Heber et al., 2001), especially under high irradiance. Under these conditions, reactive oxygen species (ROS) generation associated to photosynthetic electron transport is enhanced. The question arises of how lichen algae can maintain the function of their photosynthetic machinery under continuous desiccation-rehydration processes. We will review in this chapter the possible mechanisms which should allow maintaining of photosynthesis performance under the life style of poikilohydric organisms.

#### **2. Methods for isolating lichen photobionts**

One of the main problems to study the mechanisms of photosynthesis in lichens under wellcontrolled conditions is to develop an appropriate method for isolating the lichen photobionts. Many chlorolichens contains more than one photobiont. For instance, *Ramalina farinacea* includes two different *Trebouxia* photobionts (TR1 and TR9) and isolation of these algae allowed to characterise physiological differences between both of them (Casano et al., 2010; del Hoyo et al., 2011). There are different methods in function of the objective of investigation. We can distinguish between those which allow and not allow obtaining axenic cultures.

Axenic cultures are useful to study the taxonomy, biochemical, molecular or physiological behaviour of microscopic algae outside the symbiosis. There are lots of methods, but the most popular isolation method was developed by Ahmadjian (1967a, 1967b) and consists of cutting the lichen photobiont layer into thin slices, then grinding it between two glass slides and finally spreading the homogenate on a solid agar medium. There are several variations to this method, but the main common problem to all of them is the long time required after isolation to obtain clones.

upper cortex are supposed to decrease the intensity of light reaching the photobionts especially under desiccated conditions by absorbing certain wavelengths and by reflecting light (Heber et al,. 2007; Scott, 1969; Veerman et al., 2007). Apparently, the balance between energy conservation and energy dissipation is tilted towards dissipation in many poikilohydric autotrophs, whereas, in higher plants, energy conservation assumes dominance over energy dissipation. It thus appears that sensitivity to excess light is higher

Lichens are found among poikilohydric organisms, those that cannot actively regulate their water content, but are capable of surviving long periods in a desiccated state (Kappen & Valladares, 2007). In the dry state many lichens exhibit an enhanced resistence to other stress. For instance, heat resistance up to 70-75 ºC in species from sheltered microhabitats and up to 90-100 ºC in species from exposed microhabitats (Lange, 1953). Desiccation tolerance was described in nematodes and in rotifers observed by van Leeuwenhoek in 1702, and has since been discovered in four other phyla of animals, algae, fungi, bacteria, in ca. 350 species of flowering plants and ferns and in most bryophytes and seeds of flowering plants (Alpert, 2006; Proctor & Tuba, 2002). Among them, algae, lichen and bryophytes can be considered fully desiccation-tolerant plants because can survive very rapid drying events (less than 1 h) and recover respiration and photosynthesis within a few minutes (Oliver & Wood, 1997; Proctor & Smirnoff, 2000). Most lichen-forming fungi and their photobionts are fully adapted to daily wetting and drying cycles, but die off under continuously moist conditions (Dietz & Hartung, 1999; Farrar, 1976a, 1976b). It is well known that photosynthesis in homoiohydric plants is very sensitive to water stress conditions (Heber et al., 2001), especially under high irradiance. Under these conditions, reactive oxygen species (ROS) generation associated to photosynthetic electron transport is enhanced. The question arises of how lichen algae can maintain the function of their photosynthetic machinery under continuous desiccation-rehydration processes. We will review in this chapter the possible mechanisms which should allow maintaining of photosynthesis performance under

One of the main problems to study the mechanisms of photosynthesis in lichens under wellcontrolled conditions is to develop an appropriate method for isolating the lichen photobionts. Many chlorolichens contains more than one photobiont. For instance, *Ramalina farinacea* includes two different *Trebouxia* photobionts (TR1 and TR9) and isolation of these algae allowed to characterise physiological differences between both of them (Casano et al., 2010; del Hoyo et al., 2011). There are different methods in function of the objective of investigation. We can distinguish between those which allow and not allow obtaining axenic

Axenic cultures are useful to study the taxonomy, biochemical, molecular or physiological behaviour of microscopic algae outside the symbiosis. There are lots of methods, but the most popular isolation method was developed by Ahmadjian (1967a, 1967b) and consists of cutting the lichen photobiont layer into thin slices, then grinding it between two glass slides and finally spreading the homogenate on a solid agar medium. There are several variations to this method, but the main common problem to all of them is the long time required after

in the mosses and the lichens than in higher plants (Heber, 2008).

the life style of poikilohydric organisms.

cultures.

isolation to obtain clones.

**2. Methods for isolating lichen photobionts** 

On the other hand, non-axenic cultures can be used for studying algal metabolites or enzymatic activities of the lichenized photobionts. These methods consist in homogenization of the lichen thalli, followed by separation of the photobiont from the mycobiont and fragments of thalli using differential centrifugation (Richardson, 1971), gradient centrifugation on CsCl2/KI (Ascaso, 1980) or on Percoll® gradients (Calatayud et al., 2001), and/or filtering (Weissman et al., 2005).

Here we resume the fast and simple methods developed in our laboratories (Gasulla et al., 2010): a low-scale isolation method (micromethod) and a large scale one (macromethod).

The micromethod for isolation of lichen photobions starts from 15–25 mg dry weight (DW) of lichen material that is washed first in tap water, and then slowly stirred in sterile distilled water in a bucket for 5 min. The fragments of thalli are homogenised in an sterile eppendorf tube with a pellet pestle and resuspended in sterile 1 ml of isotonic buffer (0.3 M sorbitol in 50 mM HEPES pH 7.5). After filtration through sterile muslin, the filtrate is centrifuged at 490×g in a bench-top microcentrifuge (Micro 20, Hettich, Germany) for 5 min. The pellet is resuspended in 200 μl of sterile isotonic buffer and then loaded on 1.5 ml, of sterile 80 % Percoll ® in isotonic buffer. After centrifugation at 10000×g for 10 min a clear green layer must be present near the top of the eppendorf tube and some grey particles and pellet at the bottom of the tube (Fig. 1). The green layer is recovered (ca. 400 μl), avoiding to take any drop of the upper interphase. Then, the green layer is diluted 2-fold with sterile distilled water and centrifuged at 1000×g for 10 min. The supernatant is discarded; the pellet is resuspended in 2 ml of sterile distilled water and a drop of Tween 20 is added. The resulting suspension is sonicated at 40 KHz (Elma Transsonic Digital 470 T, 140% ultrasound power) for 1 min and again centrifuged at 490×g for 5 min. This treatment is repeated five times. The final pellet containing the isolated algal cells is resuspended in 1 ml of sterile distilled water. This micromethod can be scaled up to a macromethod, which allows preparation of large amounts of photobiont cells.

Fig. 1. Separation of *Ramalina farinacea* fractions after centrifugation of the extract of thalli at 10,000×g for 10 min on 40 ml (macromethod), or 1.5 ml (micromethod) of 80 % Percoll ® in isotonic buffer. A–D, each optical micrograph refers to the corresponding Percoll layer; A and C phase contrast microscopy. Scale = 15 μm. \* Not real size. Photograph from Gasulla et al., (2010).

Photosynthesis in Lichen: Light Reactions and Protective Mechanisms 153

90 % of the maximum water content that peak photosynthesis occurs. Thereafter, with increasing temperatures and light intensities, both water content and net photosynthesis decline. Desiccation occurs reasonably slowly, over hours rather than minutes (Kappen, 1974). Lichen photobionts are able to maintain maximum photosynthetic activity until the water content reach the 20 % (Gasulla et al., 2009), thus, during this period lichens photosynthetize at rates that are sufficient to allow a net positive carbon gain over the year. Thalline growth rates depend on the frequency and length of this period per day and year (Lange & Matthes, 1981). On the other hand, although carbon fixation is inhibited during desiccation, electron flow through the photosystems continues, and excitation energy can be transferred from photoexcited chlorophyll pigments to 3O2, forming singlet oxygen (1O2), while superoxide and hydrogen superoxide can be produced at photosystem II and photosystem I by the Mehler reaction (Halliwell, 2006; Kranner & Lutzoni, 1999; Peltier et al., 2010). Likewise, rehydration of lichens produces a burst of ROS during the first minutes and then decrease (Minibayeva & Beckett, 2001; Weissman et al*.,* 2005). Thus, although lichens have adapted their carbon assimilation necessities to their living conditions, they will need specific mechanisms to avoid the development of oxidative damage during the desiccation and rehydration processes. We can follow two levels of protection mechanisms at the photobiont cellular level. First, processes directed to the dissipation of excess light energy as heat, which can be considered as oxidative stress avoidance mechanisms. Second, enzymatic or non-enzymatic antioxidant systems that

**4. Production of reactive oxygen species (ROS) and reactive nitrogen species** 

Aerobic organisms generate ROS as a side-product product of metabolism. In healthy cells occurs at a controlled rate, but many abiotic and biotic stress conditions lead to cellular redox imbalance and accumulation of ROS (Foyer & Noctor, 2003; Halliwell & Gutteridge, 1999; Mittler, 2002; Sharma & Dietz, 2009; Smirnoff, 1993) that causes molecular and cellular damage. Free radicals are atoms or molecules with an unpaired electron, which is easily donated, thus, most free radicals are very reactive (Elstner & Osswald, 1994; Halliwell & Gutteridge, 1999). Oxygen is a highly oxidizing molecule that forms free radicals and participates in other oxidative chemical reactions (Abele, 2002; Finkel & Holbrook, 2000). Oxygen radicals include singlet oxygen (1O2), superoxide (O2•−), the hydroxyl radical (OH•) (Elstner & Osswald, 1994; Finkel & Holbrook, 2000; Halliwell & Gutteridge, 1999). Together with hydrogen peroxide (H2O2) that is not a free radical but is also highly reactive. ROS accumulation is the most likely source of damage to nucleic acids, proteins and lipids that

Every free radical formed in a living organism can initiate a series of chain reactions that will continue until they are eliminated (Halliwell, 2006). Free radicals disappear from the organism only by reactions with other free radicals or, more important, due to the actions of the antioxidant system that will be treated in section 6 of this review. Any imbalance in the redox state, which altered equilibrium in the direction of pro-oxidant molecules production, may result in univalent reduction of molecular oxygen to the potentially dangerous radical anion superoxide (Foyer & Noctor, 2003). Its formation is an unavoidable consequence of aerobic respiration (Møller, 2001) and photosynthesis (Halliwell, 2006), but imbalances may also occur under changing environmental conditions such as desiccation-rehydration cycles experienced by lichens, causing oxidative damage in cells of the photobiont and mycobiont.

can constitute oxidative stress tolerance mechanisms.

can, as a final result, conduct to cell death (Zapata et al., 2005).

**(RNS) during desiccation/rehydration** 

In the macromethod, one to two g DW of lichen thallus are homogenised with a mortar and pestle in 20 ml of sterile isotonic buffer. The steps following are similar to the micromethod, but the volume for resuspension of the first pellet is 1 ml. The second centrifugation step is carried out on 40 ml of sterile 80% Percoll in isotonic buffer using a fixed-angle rotor (221.22 V01/V02, Hermle, Germany). After this centrifugation step in the macromethod, four layers are visible: a) a 2–3 ml dark green supernatant at the top of the tube on the Percoll layer; b) a large and diffuse light green layer in the upper part of the Percoll gradient; c) a thick layer at the bottom of the tube and d) a grey pellet (Fig. 1). Five millilitres of the "b" layer are recovered and the subsequent isolation steps for this layer are identical to those described for the micromethod.

The algal suspensions isolated with any one of both methods, are diluted 100 folds with sterile distilled water and 50 μl of this suspension are spread on sterile 1.5% agar 3xN (meaning three times more nitrogen content in the form of NaNO3) Bold's Basal Media (3NBBM) (Bischoff & Bold, 1963) in each of five Petri dishes using the streak method and sterile technique. The isolated algae are cultured under 15 μmol m−2s−1 (PPFD) with a 12 h photoperiod at 17°C. The number of algal colonies growing on each plate is counted after 45 days. Several colonies must be selected under the stereo-microscope and subcultured onto Petri dishes containing 1.5% agar 3NBBM medium supplemented with glucose (20 gl−1) and casein (10 gl−1) (Ahmadjian, 1967a) using a sterile toothpick.

#### **3. Effects of water content on the carbon budget of lichens**

The thallus water content is mainly determined by the water availability of the environment. When desiccated, their water status is frequently in the range of 10-20 % in respect to their fresh weight (Rundel, 1988). This state would be lethal for most of the vascular plants and organisms, however the vast majority of lichens are desiccation-tolerant and can survive in a suspended animation until water becomes available again, then they revive and resume normal metabolism (Kappen & Valladares, 2007). Upon rehydration they recover normal photosynthetic rates within a short time span, 15-60 min or less (Fos et al., 1999; Jensen et al., 1999; Tuba et al., 1996). Therefore, lichens may be the predominant life-form in extreme environments like cold and hot deserts. In lichens, photosynthetic activity of the photobiont partner is restricted to a short time when thalli are at least partly hydrated and solar radiation is available at temperatures within the range suitable for photosynthesis. Frequent drying and wetting cycles and the correlated in- and re-activation of photosynthesis is a pattern observed in most terrestrial habitats and produced by the nocturnal dewfall or fog (del Prado & Sancho, 2007; Kershaw, 1985; Lange, 1970; Lange et al., 2006). Typically, dewfall occurred in the night when temperatures had declined substantially from their daytime maximum value. Lichens readily absorb water from dewfall, and this water activates dark respiration (CO2 exchange below the zero line) through the remaining night time hours. Sunrise activates net photosynthesis (CO2 exchange above the zero line) but the peak was not reached after 1-2 h when the water content started to decrease. The net photosynthesis rate of lichens depends in large part on the water content of their thalli (Green et al., 1985; Lange & Matthes, 1981). In many lichens, when the thallus is fully saturated with water, diffusion of CO2 to the phycobiont is hindered and maximum rates of CO2 assimilation do not occur (Lange & Tenhunen, 1981). Furthermore, at maximum water saturation in continuous light, the photobiont eventually dies because all of its products are translocated to the fungus (Harris & Kershaw, 1971). It is only when the thallus dries to a 65-

In the macromethod, one to two g DW of lichen thallus are homogenised with a mortar and pestle in 20 ml of sterile isotonic buffer. The steps following are similar to the micromethod, but the volume for resuspension of the first pellet is 1 ml. The second centrifugation step is carried out on 40 ml of sterile 80% Percoll in isotonic buffer using a fixed-angle rotor (221.22 V01/V02, Hermle, Germany). After this centrifugation step in the macromethod, four layers are visible: a) a 2–3 ml dark green supernatant at the top of the tube on the Percoll layer; b) a large and diffuse light green layer in the upper part of the Percoll gradient; c) a thick layer at the bottom of the tube and d) a grey pellet (Fig. 1). Five millilitres of the "b" layer are recovered and the subsequent isolation steps for this layer are identical to those described

The algal suspensions isolated with any one of both methods, are diluted 100 folds with sterile distilled water and 50 μl of this suspension are spread on sterile 1.5% agar 3xN (meaning three times more nitrogen content in the form of NaNO3) Bold's Basal Media (3NBBM) (Bischoff & Bold, 1963) in each of five Petri dishes using the streak method and sterile technique. The isolated algae are cultured under 15 μmol m−2s−1 (PPFD) with a 12 h photoperiod at 17°C. The number of algal colonies growing on each plate is counted after 45 days. Several colonies must be selected under the stereo-microscope and subcultured onto Petri dishes containing 1.5% agar 3NBBM medium supplemented with glucose (20 gl−1) and

The thallus water content is mainly determined by the water availability of the environment. When desiccated, their water status is frequently in the range of 10-20 % in respect to their fresh weight (Rundel, 1988). This state would be lethal for most of the vascular plants and organisms, however the vast majority of lichens are desiccation-tolerant and can survive in a suspended animation until water becomes available again, then they revive and resume normal metabolism (Kappen & Valladares, 2007). Upon rehydration they recover normal photosynthetic rates within a short time span, 15-60 min or less (Fos et al., 1999; Jensen et al., 1999; Tuba et al., 1996). Therefore, lichens may be the predominant life-form in extreme environments like cold and hot deserts. In lichens, photosynthetic activity of the photobiont partner is restricted to a short time when thalli are at least partly hydrated and solar radiation is available at temperatures within the range suitable for photosynthesis. Frequent drying and wetting cycles and the correlated in- and re-activation of photosynthesis is a pattern observed in most terrestrial habitats and produced by the nocturnal dewfall or fog (del Prado & Sancho, 2007; Kershaw, 1985; Lange, 1970; Lange et al., 2006). Typically, dewfall occurred in the night when temperatures had declined substantially from their daytime maximum value. Lichens readily absorb water from dewfall, and this water activates dark respiration (CO2 exchange below the zero line) through the remaining night time hours. Sunrise activates net photosynthesis (CO2 exchange above the zero line) but the peak was not reached after 1-2 h when the water content started to decrease. The net photosynthesis rate of lichens depends in large part on the water content of their thalli (Green et al., 1985; Lange & Matthes, 1981). In many lichens, when the thallus is fully saturated with water, diffusion of CO2 to the phycobiont is hindered and maximum rates of CO2 assimilation do not occur (Lange & Tenhunen, 1981). Furthermore, at maximum water saturation in continuous light, the photobiont eventually dies because all of its products are translocated to the fungus (Harris & Kershaw, 1971). It is only when the thallus dries to a 65-

casein (10 gl−1) (Ahmadjian, 1967a) using a sterile toothpick.

**3. Effects of water content on the carbon budget of lichens** 

for the micromethod.

90 % of the maximum water content that peak photosynthesis occurs. Thereafter, with increasing temperatures and light intensities, both water content and net photosynthesis decline. Desiccation occurs reasonably slowly, over hours rather than minutes (Kappen, 1974). Lichen photobionts are able to maintain maximum photosynthetic activity until the water content reach the 20 % (Gasulla et al., 2009), thus, during this period lichens photosynthetize at rates that are sufficient to allow a net positive carbon gain over the year. Thalline growth rates depend on the frequency and length of this period per day and year (Lange & Matthes, 1981). On the other hand, although carbon fixation is inhibited during desiccation, electron flow through the photosystems continues, and excitation energy can be transferred from photoexcited chlorophyll pigments to 3O2, forming singlet oxygen (1O2), while superoxide and hydrogen superoxide can be produced at photosystem II and photosystem I by the Mehler reaction (Halliwell, 2006; Kranner & Lutzoni, 1999; Peltier et al., 2010). Likewise, rehydration of lichens produces a burst of ROS during the first minutes and then decrease (Minibayeva & Beckett, 2001; Weissman et al*.,* 2005). Thus, although lichens have adapted their carbon

assimilation necessities to their living conditions, they will need specific mechanisms to avoid the development of oxidative damage during the desiccation and rehydration processes. We can follow two levels of protection mechanisms at the photobiont cellular level. First, processes directed to the dissipation of excess light energy as heat, which can be considered as oxidative stress avoidance mechanisms. Second, enzymatic or non-enzymatic antioxidant systems that can constitute oxidative stress tolerance mechanisms.

#### **4. Production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during desiccation/rehydration**

Aerobic organisms generate ROS as a side-product product of metabolism. In healthy cells occurs at a controlled rate, but many abiotic and biotic stress conditions lead to cellular redox imbalance and accumulation of ROS (Foyer & Noctor, 2003; Halliwell & Gutteridge, 1999; Mittler, 2002; Sharma & Dietz, 2009; Smirnoff, 1993) that causes molecular and cellular damage. Free radicals are atoms or molecules with an unpaired electron, which is easily donated, thus, most free radicals are very reactive (Elstner & Osswald, 1994; Halliwell & Gutteridge, 1999). Oxygen is a highly oxidizing molecule that forms free radicals and participates in other oxidative chemical reactions (Abele, 2002; Finkel & Holbrook, 2000). Oxygen radicals include singlet oxygen (1O2), superoxide (O2•−), the hydroxyl radical (OH•) (Elstner & Osswald, 1994; Finkel & Holbrook, 2000; Halliwell & Gutteridge, 1999). Together with hydrogen peroxide (H2O2) that is not a free radical but is also highly reactive. ROS accumulation is the most likely source of damage to nucleic acids, proteins and lipids that can, as a final result, conduct to cell death (Zapata et al., 2005).

Every free radical formed in a living organism can initiate a series of chain reactions that will continue until they are eliminated (Halliwell, 2006). Free radicals disappear from the organism only by reactions with other free radicals or, more important, due to the actions of the antioxidant system that will be treated in section 6 of this review. Any imbalance in the redox state, which altered equilibrium in the direction of pro-oxidant molecules production, may result in univalent reduction of molecular oxygen to the potentially dangerous radical anion superoxide (Foyer & Noctor, 2003). Its formation is an unavoidable consequence of aerobic respiration (Møller, 2001) and photosynthesis (Halliwell, 2006), but imbalances may also occur under changing environmental conditions such as desiccation-rehydration cycles experienced by lichens, causing oxidative damage in cells of the photobiont and mycobiont.

Photosynthesis in Lichen: Light Reactions and Protective Mechanisms 155

On the other hand, less studied in algae and lichen but potentially important is Nitric Oxide (NO). NO is a relatively stable paramagnetic free radical molecule involved in many physiological processes in a very broad range of organisms. These functions include signal transduction, cell death, transport, basic metabolism, ROS production and degradation (Almagro et al., 2009; Curtois et al., 2008; Ferrer & Ros Barcelo, 1999; Palmieri et al., 2008). It is now clear that NO• and, in general, most of the Reactive Nitrogen Species (RNS) (NO•, NO+, NO−, NO•2, and ONOO−), are major signalling molecules in plants (Durner & Klessig, 1999) which can be synthesized during stress responses at the same time as H2O2 (Almagro et al., 2009). Feelisch & Martin (1995) suggested a role for NO in both the early evolution of aerobic cells and in symbiotic relationships involving NO efficacy in neutralizing ROS. In addition, NO is involved in the abiotic stress response of green algae such as *Chlorella pyrenoidosa* Pringsheim, by reducing the damage produced by photo-oxidative stress (Chen

The first work that focused on NO production in lichens was published, by Weissman et al. (2005), who carried out a microscopy study of *Ramalina lacera* (With.) J.R. Laundon. These authors described the occurrence of intracellular oxidative stress during rehydration together with the release of NO by the mycobiont, but not by the photobiont. We have recently reported evidence that NO is involved in oxidative stress in lichens exposed to the oxidative agent cumene hydroperoxide (Catalá et al., 2010) Our group has studied the role of NO during rehydration of the lichen *Ramalina farinacea* (L.) Ach., its isolated photobiont partner *Trebouxia sp*. and *Asterochloris erici* (formerly *Trebouxia erici*). The results showed that lichen NO plays an important role in the regulation of lipid peroxidation and photobiont photo-oxidative stress during rehydration. Its role is similar in plants and animals where NO is known to modulate the toxic potential of ROS and to limit lipid peroxidation, acting as a chain-breaking antioxidant to scavenge peroxyl radicals (Darley-Usmar et al., 2000; Kroncke et al., 1997; Miranda et al., 2000). Our data showed that rehydration is accompanied by ROS and NO generation and thus confirmed the results of Weissman et al. (2005). Moreover, the inhibition of NO action altered the photosynthetic activity of the photobionts suggesting that NO is involved in PSII stabilization and could be related with the limited role of classical antioxidant systems during desiccation-rehydration cycles in *Asterochloris* photobionts. These results point to the importance of NO in the early stages of lichen rehydration. However, there are needed further studies on NO function, or on the

Plants have developed mechanisms to prevent the formation of ROS by dissipating the excess of energy as heat. This phenomenon comprises several processes, which applying a nomenclature derived from Chl *a* fluorescence theory are globally known as nonphotochemical quenching (denoted as NPQ or qN). The light energy when reach the photosystems is transformed in heat, fluorescence or photochemistry. These processes compete among them and then make possible estimate the proportion of light energy employed in photosynthetic electron transport or dissipated as heat from direct measurement of the variation of fluorescence emission. The electron transfer from the reaction center chlorophyll of PSII (P680) to the primary quinone acceptor of PSII (QA) produces losses in fluorescence emission in a process known as photochemical quenching. Photochemical quenching (qP) reaches maximal values when all the quinone pool is oxidized and electron

et al., 2003).

occurrence of NO in other lichens.

**5. Mechanisms of light energy dissipation in lichen algae** 

Several reactions in chloroplast and mithochondria generate the free superoxide radical (O2 •− ) which can in turn react with hydrogen peroxide (H2O2) to produce singlet oxygen (1O2) and the hydroxyl radical (OH•) (Casano et al., 1999; Elstner, 1982). Moreover, in the presence of Fe or Cu (II), OH• radicals are formed as quickly that can attack and damage almost every molecule found in living cells as lipids, amino acids and even nucleic acids by direct attack or activation of endonucleases (Kranner & Birtic, 2005; Yruela et al., 1996). They can, for example, hydroxylate purine and pyrimidine bases in DNA (Aruoma et al., 1989), thus enhancing mutation rates. Oxidative damage to proteins changes their configuration, mostly by oxidizing the free thiol residues of cysteine to produce thiyl radicals. These can form disulphide bonds with other thiyl radicals, causing intra- or inter-molecular crosslinks. After oxidative modification, proteins become sensitive to proteolysis and/or may be inactivated, or may show reduced activity (Kranner & Birtic, 2005). Singlet oxygen and OH• can also initiate peroxidation chain reactions in lipids. Lipid peroxides decompose to give volatile hydrocarbons and aldehydes (Esterbauer et al., 1991; Valenzuela, 1991). Accumulation of malondialdehyde, an indicator of lipid peroxidation, has been observed in lichens during dehydration (Kranner & Lutzoni, 1999). The latter can act as secondary toxic messengers that disseminate initial free radical events (Esterbauer et al., 1991). On the other hand, hydrogen peroxide is a ubiquitous constituent of plant cells under a fine homeostatic control which prevents its accumulation (Foyer & Noctor, 2003; Ros Barceló, 1998).

In most organisms, desiccation is associated with production of ROS and associated deleterious effects (Weissman et al., 2005). ROS may modify the properties of the thylakoids, thereby changing the yield of Chl *a* fluorescence, leading to photoinhibition (Ort, 2001). Under normal growth conditions, the production of ROS in cells is low (240 μM s−1 O2− and a steady-state level of 0.5 μM H2O2 in chloroplasts, Polle, 2001). Many stresses such as drought stress and desiccation disrupt the cellular homeostasis of cells and enhance the production of ROS (240–720 μM s−1 O2− and a steady-state level of 5–15 μM H2O2, Polle, 2001). The production of ROS during desiccation results from pathways such as photorespiration, from the photosynthetic apparatus and from mitochondrial respiration (Mittler et al., 2002). Whether lichens have photorespiration process is not clear and likely it depends on the species (Ahmadjian, 1993). We hypothesize (from genomic data), that probably, in algae, the glycolate oxidase reaction in peroxisomes is prevented by the presence of the alternative enzyme glycolate dehydrogenase, which does not produce hydrogen peroxide.

As we have said, lichens can tolerate dehydration, but what happen in the chloroplast when water is unavailable as reductant? When desiccated in the light, chlorophyll molecules continue to be excited, but the energy not used in carbon fixation will cause formation of singlet oxygen (Kranner et al., 2005). In these cases, oxygen can be the electron acceptor forming superoxide and here can begin the generation of ROS (Heber et al., 2001). Desiccation stress in lichens shows features similar to reversible photoinhibition (Chakir & Jensen, 1999; Jensen & Feige, 1991) because ROS production is enhanced during dessication process (Weissman et al., 2005). In the same manner, excess of illumination causes either excessive reduction on the acceptor side or oxidation on the donor side (Anderson & Barber, 1996) producing ROS in the thylakoids. Therefore, under both high illumination and dehydration, reactive oxygen species are a major cause of damage in photosynthetic organisms (Demmig-Adams & Adams, 2000; Halliwell, 1984).

Several reactions in chloroplast and mithochondria generate the free superoxide radical

control which prevents its accumulation (Foyer & Noctor, 2003; Ros Barceló, 1998).

In most organisms, desiccation is associated with production of ROS and associated deleterious effects (Weissman et al., 2005). ROS may modify the properties of the thylakoids, thereby changing the yield of Chl *a* fluorescence, leading to photoinhibition (Ort, 2001). Under normal growth conditions, the production of ROS in cells is low (240 μM s−1 O2− and a steady-state level of 0.5 μM H2O2 in chloroplasts, Polle, 2001). Many stresses such as drought stress and desiccation disrupt the cellular homeostasis of cells and enhance the production of ROS (240–720 μM s−1 O2− and a steady-state level of 5–15 μM H2O2, Polle, 2001). The production of ROS during desiccation results from pathways such as photorespiration, from the photosynthetic apparatus and from mitochondrial respiration (Mittler et al., 2002). Whether lichens have photorespiration process is not clear and likely it depends on the species (Ahmadjian, 1993). We hypothesize (from genomic data), that probably, in algae, the glycolate oxidase reaction in peroxisomes is prevented by the presence of the alternative enzyme glycolate dehydrogenase, which does not produce

As we have said, lichens can tolerate dehydration, but what happen in the chloroplast when water is unavailable as reductant? When desiccated in the light, chlorophyll molecules continue to be excited, but the energy not used in carbon fixation will cause formation of singlet oxygen (Kranner et al., 2005). In these cases, oxygen can be the electron acceptor forming superoxide and here can begin the generation of ROS (Heber et al., 2001). Desiccation stress in lichens shows features similar to reversible photoinhibition (Chakir & Jensen, 1999; Jensen & Feige, 1991) because ROS production is enhanced during dessication process (Weissman et al., 2005). In the same manner, excess of illumination causes either excessive reduction on the acceptor side or oxidation on the donor side (Anderson & Barber, 1996) producing ROS in the thylakoids. Therefore, under both high illumination and dehydration, reactive oxygen species are a major cause of damage in photosynthetic

organisms (Demmig-Adams & Adams, 2000; Halliwell, 1984).

•− ) which can in turn react with hydrogen peroxide (H2O2) to produce singlet oxygen (1O2) and the hydroxyl radical (OH•) (Casano et al., 1999; Elstner, 1982). Moreover, in the presence of Fe or Cu (II), OH• radicals are formed as quickly that can attack and damage almost every molecule found in living cells as lipids, amino acids and even nucleic acids by direct attack or activation of endonucleases (Kranner & Birtic, 2005; Yruela et al., 1996). They can, for example, hydroxylate purine and pyrimidine bases in DNA (Aruoma et al., 1989), thus enhancing mutation rates. Oxidative damage to proteins changes their configuration, mostly by oxidizing the free thiol residues of cysteine to produce thiyl radicals. These can form disulphide bonds with other thiyl radicals, causing intra- or inter-molecular crosslinks. After oxidative modification, proteins become sensitive to proteolysis and/or may be inactivated, or may show reduced activity (Kranner & Birtic, 2005). Singlet oxygen and OH• can also initiate peroxidation chain reactions in lipids. Lipid peroxides decompose to give volatile hydrocarbons and aldehydes (Esterbauer et al., 1991; Valenzuela, 1991). Accumulation of malondialdehyde, an indicator of lipid peroxidation, has been observed in lichens during dehydration (Kranner & Lutzoni, 1999). The latter can act as secondary toxic messengers that disseminate initial free radical events (Esterbauer et al., 1991). On the other hand, hydrogen peroxide is a ubiquitous constituent of plant cells under a fine homeostatic

(O2

hydrogen peroxide.

On the other hand, less studied in algae and lichen but potentially important is Nitric Oxide (NO). NO is a relatively stable paramagnetic free radical molecule involved in many physiological processes in a very broad range of organisms. These functions include signal transduction, cell death, transport, basic metabolism, ROS production and degradation (Almagro et al., 2009; Curtois et al., 2008; Ferrer & Ros Barcelo, 1999; Palmieri et al., 2008). It is now clear that NO• and, in general, most of the Reactive Nitrogen Species (RNS) (NO•, NO+, NO−, NO•2, and ONOO−), are major signalling molecules in plants (Durner & Klessig, 1999) which can be synthesized during stress responses at the same time as H2O2 (Almagro et al., 2009). Feelisch & Martin (1995) suggested a role for NO in both the early evolution of aerobic cells and in symbiotic relationships involving NO efficacy in neutralizing ROS. In addition, NO is involved in the abiotic stress response of green algae such as *Chlorella pyrenoidosa* Pringsheim, by reducing the damage produced by photo-oxidative stress (Chen et al., 2003).

The first work that focused on NO production in lichens was published, by Weissman et al. (2005), who carried out a microscopy study of *Ramalina lacera* (With.) J.R. Laundon. These authors described the occurrence of intracellular oxidative stress during rehydration together with the release of NO by the mycobiont, but not by the photobiont. We have recently reported evidence that NO is involved in oxidative stress in lichens exposed to the oxidative agent cumene hydroperoxide (Catalá et al., 2010) Our group has studied the role of NO during rehydration of the lichen *Ramalina farinacea* (L.) Ach., its isolated photobiont partner *Trebouxia sp*. and *Asterochloris erici* (formerly *Trebouxia erici*). The results showed that lichen NO plays an important role in the regulation of lipid peroxidation and photobiont photo-oxidative stress during rehydration. Its role is similar in plants and animals where NO is known to modulate the toxic potential of ROS and to limit lipid peroxidation, acting as a chain-breaking antioxidant to scavenge peroxyl radicals (Darley-Usmar et al., 2000; Kroncke et al., 1997; Miranda et al., 2000). Our data showed that rehydration is accompanied by ROS and NO generation and thus confirmed the results of Weissman et al. (2005). Moreover, the inhibition of NO action altered the photosynthetic activity of the photobionts suggesting that NO is involved in PSII stabilization and could be related with the limited role of classical antioxidant systems during desiccation-rehydration cycles in *Asterochloris* photobionts. These results point to the importance of NO in the early stages of lichen rehydration. However, there are needed further studies on NO function, or on the occurrence of NO in other lichens.

#### **5. Mechanisms of light energy dissipation in lichen algae**

Plants have developed mechanisms to prevent the formation of ROS by dissipating the excess of energy as heat. This phenomenon comprises several processes, which applying a nomenclature derived from Chl *a* fluorescence theory are globally known as nonphotochemical quenching (denoted as NPQ or qN). The light energy when reach the photosystems is transformed in heat, fluorescence or photochemistry. These processes compete among them and then make possible estimate the proportion of light energy employed in photosynthetic electron transport or dissipated as heat from direct measurement of the variation of fluorescence emission. The electron transfer from the reaction center chlorophyll of PSII (P680) to the primary quinone acceptor of PSII (QA) produces losses in fluorescence emission in a process known as photochemical quenching. Photochemical quenching (qP) reaches maximal values when all the quinone pool is oxidized and electron

Photosynthesis in Lichen: Light Reactions and Protective Mechanisms 157

In vascular plants NPQ changes in response to diurnal variations in the light environment. Large changes in the composition of the xanthophyll cycle are observed over the course of the day. This consisted of increases and subsequent decreases in the zeaxanthin and antheraxanthin content of the leaves that paralleled the changes in incident PFD rather closely (Adams & Demmig-Adams, 1992). There are also acclimations to the plant light regime. Sun plants or sun acclimated leaves possess a higher capacity for the use of light in photosynthesis and also for rapid increases in xanthophyll cycle-dependent energy dissipation. Sun-grown leaves typically exhibit a larger total pool size of the xanthophyll cycle components as well as a greater ability to convert this pool to antheraxanthin and zeaxanthin rapidly under high light (Demmig-Adams & Adams, 1993, 1996). Finally, seasonal changes in NPQ have been described for several evergreen species (Adams et al.,

Most green algal photobiont cultures require low light intensities of 10-30 mol m-2 s-1 (Ahmadjian, 1967a; Friedl & Büdel, 2008). One reason which could justify the necessity of low intensities for lichen algae culture is that compared to surficial light measurements, the light reaching the photobiont is reduced by 54-79% when dry, and 24-54% when fully hydrated (Büdel & Lange, 1994; Dietz et al., 2000; Ertl, 1951; Green et al*.*, 2008). During desiccation a decrease in fluorescence emission is observed in lichens (Veerman et al., 2007). This decrease is likely associated with their phototolerance and could be caused by structural changes in the thallus that induce changes in light-scattering and shading properties or by changes in shape and aggregation of algae (de los Rios et al., 2007; Scheidegger et al., 1995; Veerman et al., 2007). The thallus also offers some protection against photodamage through the use of light-absorbing pigments (Gauslaa & Solhaug, 1999; Holder et al., 2000). All this kind of features decreases the exposure of the photosynthetic apparatus of the photobiont to light and can be described as sunshade mechanisms (Veerman et al., 2007). However, shading or the production of sun-protectant pigments reduce but cannot prevent photooxidation. Photosynthetic pigments absorb light, whether the organisms are hydrated or desiccated, but energy conservation by carbon assimilation is possible only in the presence of water. Photosynthetic reaction centers can remain intact during desiccation in lichens and then photoreactions threaten to cause severe

Piccotto & Tetriach (2010) found some similarities of lichens with the acclimation of vascular plants to the light regime, as the photobiont activity of lichen should be significantly modulated by the growth light regime of the thallus. According to these authors, lichen chlorobionts show the same general variation patterns described in ''sun'' and ''shade'' leaves, with optimisation of light absorption and harvesting capacity in chlorobionts of thalli grown in shaded habitat, and increased photosynthetic quantum conversion and lower Chl *a* fluorescence emission in chlorobionts of thalli grown under direct solar irradiation. However, this comparison should be complicated by the influence in lichens of extrinsic factors, as nitrogen availability, or intrinsic factors, as light transmittance through the peripheral mycobiont layer. These "sun" and "shade" patterns are maintained by the chlorobionts independently of the symbiosis. Our group isolated the two species of phycobionts that are always coexisting in the lichen *Ramalina farinacea*, and we observed that one species, so called TR9, was better adapted to high irradiances than the other one, so called TR1 (Casano et al., 2010). The proportion of the two phycobionts in the lichen changes depending on the local conditions, allowing the lichen behaves either as a "sun" or

2001; Zarter et al., 2006a, 2006b).

photooxidative damage (Heber & Lüttge, 2011).

transport is not impaired (open reaction centers) and minimal values if all the quinone pool is reduced (closed reaction centers). The value of qp usually maintains a non-linear relationship with the actual proportion of reduced quinone (Kramer et al., 2004). The rate of heat loss is correlated with the non-photochemical quenching of fluorescence; this is, with the loss of fluorescence emission that is independent of photochemical events and results in heat dissipation of light energy (Baker, 2008). In consequence, the maximum fluorescence emission (termed *Fm*) will be registered when all reaction centers are closed (what is obtained by a saturating flash of light) and mechanisms of thermal energy dissipation are inactivated (what is assumed to happen within a dark period of about 20 min for most vascular plants). After new exposure to light, the mechanisms of heat dissipation become active and after a new saturating flash the value of the fluorescence peak (Fm') will be lower. Hence, the expression NPQ= (*Fm/Fm'*)-1 (Bilger & Björkman, 1990) describes the extent of energy dissipation. When a leaf is kept in the dark, QA becomes maximally oxidized and the heat dissipation mechanisms are relaxed. Exposure of a dark-adapted leaf to a weak modulated measuring beam (photosynthetically active photon flux density -PPFD- of *ca.*0.1 μmol m−2 s−1) results in the minimal level of fluorescence*,* called Fo (Baker, 2008). If taken into account the maximal variable fluorescence after dark adaptation (*Fm-Fo*) and after a period in the light (*Fm'-Fo'*), an alternative expression (Schreiber et al., 1986) for non-photochemical quenching can be calculated like qN= 1-(*F'*m-*F'*o)/(*F*m-*F*o).

According to Krause & Weis (1991), non-photochemical quenching can be related to three different events: high trans-thylakoidal pH gradient (qE), to state I-state II transitions (qT) and to photosystem II photoinhibition (qI). The "energy-dependent" quenching (qE), originated by the formation of a proton gradient across the thylakoidal membranes, is the main mechanism implied in the development of NPQ (Krause & Weis, 1991; Müller et al*.,* 2001). This qE is characterized by its rapid relaxation kinetics, occurring within 3 min of darkness (Li et al., 2002; Munekage et al., 2002). Under excessive light, an elevated proton concentration in the thylakoid luminal space activates violaxanthin de-epoxidase, generating antheraxanthin and then zeaxanthin. The increase of violaxanthin deepoxidation (DPS) leads to an increase of thermal energy dissipation that is correlated with the NPQ parameter of chlorophyll fluorescence (Demmig-Adams et al., 1996). The generation of NPQ requires also the participation of a protein associated to PSII, the protein Psbs of the light harvesting complex antenna, which can change its conformation at lower pH and increase its affinity for zeaxanthin. De-epoxidated xanthophylls then binds to the subunit S where could accept the excitation energy transferred from chlorophyll and thereby act as a direct quencher in NPQ (Holt et al., 2004; Krause & Jahns 2004; Niyogi et al., 2004; Ruban et al., 2002; Spinall-O'Dea et al., 2002). An alternative explanation is that PsbS alone can cause the quenching and zeaxanthin acts as an allosteric activator, but not as the primary cause of the process (Crouchman et al., 2006; Horton et al. 2005).

The transthylakoidal proton gradient is generated by water splitting in the thylakoid lumen, but also by the Q cycle of quinones around cytochrome b6/f complex. The latter allows that the proton gradient and NPQ generation (like ATP production) can be maintained by cyclic electron flow and probably by a thylakoidal NADH dehydrogenase complex (Guéra et al., 2005). In some mosses and chlorolichens has been described the possibility of the activation of energy dissipation by CO2 dependent protonation. Carbon dioxide, which, at a pK of 6.31, acts as a very weak protonating agent, is capable of promoting NPQ in a light independent way, provided some zeaxanthin is present (Bukhov et al., 2001; Heber et al., 2000; Heber, 2008; Kopecky et al.,2005).

transport is not impaired (open reaction centers) and minimal values if all the quinone pool is reduced (closed reaction centers). The value of qp usually maintains a non-linear relationship with the actual proportion of reduced quinone (Kramer et al., 2004). The rate of heat loss is correlated with the non-photochemical quenching of fluorescence; this is, with the loss of fluorescence emission that is independent of photochemical events and results in heat dissipation of light energy (Baker, 2008). In consequence, the maximum fluorescence emission (termed *Fm*) will be registered when all reaction centers are closed (what is obtained by a saturating flash of light) and mechanisms of thermal energy dissipation are inactivated (what is assumed to happen within a dark period of about 20 min for most vascular plants). After new exposure to light, the mechanisms of heat dissipation become active and after a new saturating flash the value of the fluorescence peak (Fm') will be lower. Hence, the expression NPQ= (*Fm/Fm'*)-1 (Bilger & Björkman, 1990) describes the extent of energy dissipation. When a leaf is kept in the dark, QA becomes maximally oxidized and the heat dissipation mechanisms are relaxed. Exposure of a dark-adapted leaf to a weak modulated measuring beam (photosynthetically active photon flux density -PPFD- of *ca.*0.1 μmol m−2 s−1) results in the minimal level of fluorescence*,* called Fo (Baker, 2008). If taken into account the maximal variable fluorescence after dark adaptation (*Fm-Fo*) and after a period in the light (*Fm'-Fo'*), an alternative expression (Schreiber et al., 1986) for non-photochemical quenching can be

According to Krause & Weis (1991), non-photochemical quenching can be related to three different events: high trans-thylakoidal pH gradient (qE), to state I-state II transitions (qT) and to photosystem II photoinhibition (qI). The "energy-dependent" quenching (qE), originated by the formation of a proton gradient across the thylakoidal membranes, is the main mechanism implied in the development of NPQ (Krause & Weis, 1991; Müller et al*.,* 2001). This qE is characterized by its rapid relaxation kinetics, occurring within 3 min of darkness (Li et al., 2002; Munekage et al., 2002). Under excessive light, an elevated proton concentration in the thylakoid luminal space activates violaxanthin de-epoxidase, generating antheraxanthin and then zeaxanthin. The increase of violaxanthin deepoxidation (DPS) leads to an increase of thermal energy dissipation that is correlated with the NPQ parameter of chlorophyll fluorescence (Demmig-Adams et al., 1996). The generation of NPQ requires also the participation of a protein associated to PSII, the protein Psbs of the light harvesting complex antenna, which can change its conformation at lower pH and increase its affinity for zeaxanthin. De-epoxidated xanthophylls then binds to the subunit S where could accept the excitation energy transferred from chlorophyll and thereby act as a direct quencher in NPQ (Holt et al., 2004; Krause & Jahns 2004; Niyogi et al., 2004; Ruban et al., 2002; Spinall-O'Dea et al., 2002). An alternative explanation is that PsbS alone can cause the quenching and zeaxanthin acts as an allosteric activator, but not as the primary cause of the

The transthylakoidal proton gradient is generated by water splitting in the thylakoid lumen, but also by the Q cycle of quinones around cytochrome b6/f complex. The latter allows that the proton gradient and NPQ generation (like ATP production) can be maintained by cyclic electron flow and probably by a thylakoidal NADH dehydrogenase complex (Guéra et al., 2005). In some mosses and chlorolichens has been described the possibility of the activation of energy dissipation by CO2 dependent protonation. Carbon dioxide, which, at a pK of 6.31, acts as a very weak protonating agent, is capable of promoting NPQ in a light independent way, provided some zeaxanthin is present (Bukhov et al., 2001; Heber et al., 2000; Heber,

calculated like qN= 1-(*F'*m-*F'*o)/(*F*m-*F*o).

process (Crouchman et al., 2006; Horton et al. 2005).

2008; Kopecky et al.,2005).

In vascular plants NPQ changes in response to diurnal variations in the light environment. Large changes in the composition of the xanthophyll cycle are observed over the course of the day. This consisted of increases and subsequent decreases in the zeaxanthin and antheraxanthin content of the leaves that paralleled the changes in incident PFD rather closely (Adams & Demmig-Adams, 1992). There are also acclimations to the plant light regime. Sun plants or sun acclimated leaves possess a higher capacity for the use of light in photosynthesis and also for rapid increases in xanthophyll cycle-dependent energy dissipation. Sun-grown leaves typically exhibit a larger total pool size of the xanthophyll cycle components as well as a greater ability to convert this pool to antheraxanthin and zeaxanthin rapidly under high light (Demmig-Adams & Adams, 1993, 1996). Finally, seasonal changes in NPQ have been described for several evergreen species (Adams et al., 2001; Zarter et al., 2006a, 2006b).

Most green algal photobiont cultures require low light intensities of 10-30 mol m-2 s-1 (Ahmadjian, 1967a; Friedl & Büdel, 2008). One reason which could justify the necessity of low intensities for lichen algae culture is that compared to surficial light measurements, the light reaching the photobiont is reduced by 54-79% when dry, and 24-54% when fully hydrated (Büdel & Lange, 1994; Dietz et al., 2000; Ertl, 1951; Green et al*.*, 2008). During desiccation a decrease in fluorescence emission is observed in lichens (Veerman et al., 2007). This decrease is likely associated with their phototolerance and could be caused by structural changes in the thallus that induce changes in light-scattering and shading properties or by changes in shape and aggregation of algae (de los Rios et al., 2007; Scheidegger et al., 1995; Veerman et al., 2007). The thallus also offers some protection against photodamage through the use of light-absorbing pigments (Gauslaa & Solhaug, 1999; Holder et al., 2000). All this kind of features decreases the exposure of the photosynthetic apparatus of the photobiont to light and can be described as sunshade mechanisms (Veerman et al., 2007). However, shading or the production of sun-protectant pigments reduce but cannot prevent photooxidation. Photosynthetic pigments absorb light, whether the organisms are hydrated or desiccated, but energy conservation by carbon assimilation is possible only in the presence of water. Photosynthetic reaction centers can remain intact during desiccation in lichens and then photoreactions threaten to cause severe photooxidative damage (Heber & Lüttge, 2011).

Piccotto & Tetriach (2010) found some similarities of lichens with the acclimation of vascular plants to the light regime, as the photobiont activity of lichen should be significantly modulated by the growth light regime of the thallus. According to these authors, lichen chlorobionts show the same general variation patterns described in ''sun'' and ''shade'' leaves, with optimisation of light absorption and harvesting capacity in chlorobionts of thalli grown in shaded habitat, and increased photosynthetic quantum conversion and lower Chl *a* fluorescence emission in chlorobionts of thalli grown under direct solar irradiation. However, this comparison should be complicated by the influence in lichens of extrinsic factors, as nitrogen availability, or intrinsic factors, as light transmittance through the peripheral mycobiont layer. These "sun" and "shade" patterns are maintained by the chlorobionts independently of the symbiosis. Our group isolated the two species of phycobionts that are always coexisting in the lichen *Ramalina farinacea*, and we observed that one species, so called TR9, was better adapted to high irradiances than the other one, so called TR1 (Casano et al., 2010). The proportion of the two phycobionts in the lichen changes depending on the local conditions, allowing the lichen behaves either as a "sun" or

Photosynthesis in Lichen: Light Reactions and Protective Mechanisms 159

dissipation. In any case, all these results indicate that the activity of the algae is modulated by the symbiotic association with funghi, but also that other factors, as the speed of

Alternative mechanisms of energy dissipation have been described to take place in mosses and lichens, where new quenching centres are functional during desiccation (Heber et al., 2006a, 2006b, 2007; Heber, 2008). In a work based on changes on the emission spectra of chlorophylls, Bilger et al. (1989) proposed that with green algal symbionts desiccation induces a functional interruption of energy transfer between the light harvesting Chl *a/b*  pigment complex and PSII and that this can be largely restored by rehydration with humidified air. Heber et al. (2006a, 2006b) described that the recovery of fluorescence levels after drying was better in dark dried mosses than in sun-dried mosses, an effect that the authors can not ascribe to zeaxanthin and classical NPQ protection. Heber & Shuvalov (2005) found the existence in briophytes and lichens of an alternative quencher of chlorophyll fluorescence characterised by a long wavelenght (720 nm) emission. Helped by results obtained in dessicated spinach leaves, Heber (2008) proposed that electrons can be redirected in the dried state from pheophitin to a secondary chlorophyll placed very close to P680, implying the formation of the radical pair P680+Chl-. A further and slower recombination between Chl- and a carotene molecule positively charged should complete a photoprotective cycle in desiccated leaves. The authors argued that the similarity of fluorescence emission spectra of dessicated leaves with those obtained in a desiccated fern and a desiccated moss should justify the extrapolation of this model to poikilohydric plants. A stronger support for the hypothesis of alternative sinks for energy or electrons during desiccation was obtained by Veerman et al. (2007) with steady-state, low temperature, and time-resolved chlorophyll fluorescence spectroscopy. These authors presented a model in which a pigment molecule with a peak emission at 420 nm should act as a sink for energy accumulated on P680 in the lichen *Parmelia sulcata* under desiccation. Heber et al. (2007) afford more evidences for the existence of these alternative mechanisms, as they found in dehydrated mosses and lichens a quenching mechanism independent of light activation. This mechanism is probably dependent of conformational changes in a protein-pigment complex as is inhibited by glutaraldehyde or heat treatments. This mechanism is also dependent of the speed of desiccation, as fast drying is less effective in decreasing chlorophyll fluorescence than slow drying. Gasulla et al. (2009) found that the basal fluorescence (*Fo*) values in desiccated *Asterochloris erici* were significantly higher after rapid dehydration, than after slow dehydration, suggesting higher levels of light energy dissipation in slow-dried algae. Higher values of PSII electron transport were recovered after rehydration of slow-dried *A. erici* compared to rapid-dried algae. The authors suggest that there is probably a minimal period required to develop strategies which will facilitate transition to the desiccated state in chlorobionts. In this process, the xanthophyll cycle and classical antioxidant mechanisms play a very limited role. More recently, Komura et al. (2010) have found proof that the quencher of chlorophyll fluorescence under desiccation conditions is not a chlorophyll molecule and suggest a new kind of quenching in PSII antenna or aggregation in PSII. The mechanisms implicated in protection under desiccation should be dependent of the desiccation rate, independent of light and probably associated to conformational changes in a chlorophyllprotein complex (Heber et al., 2007; Heber, 2008; Heber et al., 2010; Heber & Lüttge, 2011). Finally, Heber et al. (2011) propose that photoprotection is achieved by the drainage of

desiccation can influence on the photosynthetic activity after rehydration.

light energy out of the reaction centers.

as "shade" species. The ecophysiological plasticity of this symbiosis allows the lichen proliferates in a wide variety of habitats.

In lichens, the exposure to high light in the hydrated state produces photoinhibition in chlorolichens and cyanolichens, an effect much more pronounced in cyanolichens, which do not reverse photosynthetic depression after a recovery period as chlorolichens do. Photoinhibition can be largely diminished if both kinds of lichens are in the desiccated state or become desiccated during the period of high light exposure (Demmig-Adams et al., 1990a). Demmig-Adams et al. (1990b) described significant increases of zeaxanthin content after two hours of exposure to high light of six chlorolichens previously adapted to a low light regime. This effect was not observed in cyanolichens. Following treatment of the thalli with an inhibitor of the violaxanthin de-epoxidase, dithiothreitol, the response of green algal lichens to light became very similar to that of the blue-green algal lichens. Thus, Demmig-Adams et al. (1990b) proposed that the higher light stress in blue-green algae lichens is related to the absence of an effective accumulation of zeaxanthin (lack of the xanthophyll cycle) in cyanobacteria. On the other hand, Heber & Shuvalov (2005) and Heber (2008) report differences in the recovery under different light tretments between mosses collected in autumn-winter or in spring-summer, indicating seasonal acclimation of poikilohydric organisms to the light regime, but they found that in this case it was independent of zeaxanthin accumulation. In lichens the xanthophyll cycle activation cannot either be assumed as a general response to light dissipation under stress conditions because some lichen species, like *Pseudevernia furfuracea* or the isolated lichen photobiont *Trebouxia excentrica,* do not increase the xanthophyll DPS upon dehydration or rehydration (Kranner et al., 2003, 2005).

Relationship between NPQ and desiccation tolerance in lichens has been studied during the last years. For instance, Calatayud et al. (1997) found that in the thallus of *Parmelia quercina*, NPQ increases during dehydration, which seems to be related with the conversion from violaxantin to zeaxanthin observed during the desiccation. These effects were also observed in *Ramalina maciformis* (Zorn et al., 2001). Fernández Marín et al. (2010) described in the lichen *Lobaria pulmonaria* that violaxanthin deepoxidase needs a time to be activated during desiccation and then de-epoxidation of violaxanthin to zeaxanthin occurs only when the tissue has lost most of its water and dehydration is slow. Kranner et al. (2005) described that activation of the xanthophyll cycle in *Cladonia vulcani* is dependent of the symbiosis because an effective accumulation of zeaxanthin can be observed in the lichen during desiccation and rehydration processes, but not in its isolated photobiont. In accordance, during a study carried out in the isolated photobiont *Asterochloris erici* (Gasulla et al., 2009) we did not found significant differences in the deepoxidation state of xanthophylls after rapid or slow desiccation treatments when compared with controls. On the other hand, a significant increase in the deepoxidation state was registered after exposure of the isolated *A. erici* to high light intensities. Kosugi et al. (2009) found that the responses to air drying and hypertonic treatments of *Ramalina yasudae* and its isolated *Trebouxia sp*. photobiont are different in three ways: 1) PSII activity is completely inhibited in the desiccated lichen but not in the isolated photobiont; 2) the dehydration induced quenching of PSII fluorescence was lower in the lichen than in the photobiont; and 3) the isolated *Trebouxi*a was more sensitive to photoinhibition than the *R. yasudae* thalli. The authors proposed that a lichen substance or mechanism lost during the photobiont isolation could be implied in light

as "shade" species. The ecophysiological plasticity of this symbiosis allows the lichen

In lichens, the exposure to high light in the hydrated state produces photoinhibition in chlorolichens and cyanolichens, an effect much more pronounced in cyanolichens, which do not reverse photosynthetic depression after a recovery period as chlorolichens do. Photoinhibition can be largely diminished if both kinds of lichens are in the desiccated state or become desiccated during the period of high light exposure (Demmig-Adams et al., 1990a). Demmig-Adams et al. (1990b) described significant increases of zeaxanthin content after two hours of exposure to high light of six chlorolichens previously adapted to a low light regime. This effect was not observed in cyanolichens. Following treatment of the thalli with an inhibitor of the violaxanthin de-epoxidase, dithiothreitol, the response of green algal lichens to light became very similar to that of the blue-green algal lichens. Thus, Demmig-Adams et al. (1990b) proposed that the higher light stress in blue-green algae lichens is related to the absence of an effective accumulation of zeaxanthin (lack of the xanthophyll cycle) in cyanobacteria. On the other hand, Heber & Shuvalov (2005) and Heber (2008) report differences in the recovery under different light tretments between mosses collected in autumn-winter or in spring-summer, indicating seasonal acclimation of poikilohydric organisms to the light regime, but they found that in this case it was independent of zeaxanthin accumulation. In lichens the xanthophyll cycle activation cannot either be assumed as a general response to light dissipation under stress conditions because some lichen species, like *Pseudevernia furfuracea* or the isolated lichen photobiont *Trebouxia excentrica,* do not increase the xanthophyll DPS upon dehydration or rehydration (Kranner

Relationship between NPQ and desiccation tolerance in lichens has been studied during the last years. For instance, Calatayud et al. (1997) found that in the thallus of *Parmelia quercina*, NPQ increases during dehydration, which seems to be related with the conversion from violaxantin to zeaxanthin observed during the desiccation. These effects were also observed in *Ramalina maciformis* (Zorn et al., 2001). Fernández Marín et al. (2010) described in the lichen *Lobaria pulmonaria* that violaxanthin deepoxidase needs a time to be activated during desiccation and then de-epoxidation of violaxanthin to zeaxanthin occurs only when the tissue has lost most of its water and dehydration is slow. Kranner et al. (2005) described that activation of the xanthophyll cycle in *Cladonia vulcani* is dependent of the symbiosis because an effective accumulation of zeaxanthin can be observed in the lichen during desiccation and rehydration processes, but not in its isolated photobiont. In accordance, during a study carried out in the isolated photobiont *Asterochloris erici* (Gasulla et al., 2009) we did not found significant differences in the deepoxidation state of xanthophylls after rapid or slow desiccation treatments when compared with controls. On the other hand, a significant increase in the deepoxidation state was registered after exposure of the isolated *A. erici* to high light intensities. Kosugi et al. (2009) found that the responses to air drying and hypertonic treatments of *Ramalina yasudae* and its isolated *Trebouxia sp*. photobiont are different in three ways: 1) PSII activity is completely inhibited in the desiccated lichen but not in the isolated photobiont; 2) the dehydration induced quenching of PSII fluorescence was lower in the lichen than in the photobiont; and 3) the isolated *Trebouxi*a was more sensitive to photoinhibition than the *R. yasudae* thalli. The authors proposed that a lichen substance or mechanism lost during the photobiont isolation could be implied in light

proliferates in a wide variety of habitats.

et al., 2003, 2005).

dissipation. In any case, all these results indicate that the activity of the algae is modulated by the symbiotic association with funghi, but also that other factors, as the speed of desiccation can influence on the photosynthetic activity after rehydration.

Alternative mechanisms of energy dissipation have been described to take place in mosses and lichens, where new quenching centres are functional during desiccation (Heber et al., 2006a, 2006b, 2007; Heber, 2008). In a work based on changes on the emission spectra of chlorophylls, Bilger et al. (1989) proposed that with green algal symbionts desiccation induces a functional interruption of energy transfer between the light harvesting Chl *a/b*  pigment complex and PSII and that this can be largely restored by rehydration with humidified air. Heber et al. (2006a, 2006b) described that the recovery of fluorescence levels after drying was better in dark dried mosses than in sun-dried mosses, an effect that the authors can not ascribe to zeaxanthin and classical NPQ protection. Heber & Shuvalov (2005) found the existence in briophytes and lichens of an alternative quencher of chlorophyll fluorescence characterised by a long wavelenght (720 nm) emission. Helped by results obtained in dessicated spinach leaves, Heber (2008) proposed that electrons can be redirected in the dried state from pheophitin to a secondary chlorophyll placed very close to P680, implying the formation of the radical pair P680+Chl-. A further and slower recombination between Chl- and a carotene molecule positively charged should complete a photoprotective cycle in desiccated leaves. The authors argued that the similarity of fluorescence emission spectra of dessicated leaves with those obtained in a desiccated fern and a desiccated moss should justify the extrapolation of this model to poikilohydric plants. A stronger support for the hypothesis of alternative sinks for energy or electrons during desiccation was obtained by Veerman et al. (2007) with steady-state, low temperature, and time-resolved chlorophyll fluorescence spectroscopy. These authors presented a model in which a pigment molecule with a peak emission at 420 nm should act as a sink for energy accumulated on P680 in the lichen *Parmelia sulcata* under desiccation. Heber et al. (2007) afford more evidences for the existence of these alternative mechanisms, as they found in dehydrated mosses and lichens a quenching mechanism independent of light activation. This mechanism is probably dependent of conformational changes in a protein-pigment complex as is inhibited by glutaraldehyde or heat treatments. This mechanism is also dependent of the speed of desiccation, as fast drying is less effective in decreasing chlorophyll fluorescence than slow drying. Gasulla et al. (2009) found that the basal fluorescence (*Fo*) values in desiccated *Asterochloris erici* were significantly higher after rapid dehydration, than after slow dehydration, suggesting higher levels of light energy dissipation in slow-dried algae. Higher values of PSII electron transport were recovered after rehydration of slow-dried *A. erici* compared to rapid-dried algae. The authors suggest that there is probably a minimal period required to develop strategies which will facilitate transition to the desiccated state in chlorobionts. In this process, the xanthophyll cycle and classical antioxidant mechanisms play a very limited role. More recently, Komura et al. (2010) have found proof that the quencher of chlorophyll fluorescence under desiccation conditions is not a chlorophyll molecule and suggest a new kind of quenching in PSII antenna or aggregation in PSII. The mechanisms implicated in protection under desiccation should be dependent of the desiccation rate, independent of light and probably associated to conformational changes in a chlorophyllprotein complex (Heber et al., 2007; Heber, 2008; Heber et al., 2010; Heber & Lüttge, 2011). Finally, Heber et al. (2011) propose that photoprotection is achieved by the drainage of light energy out of the reaction centers.

Photosynthesis in Lichen: Light Reactions and Protective Mechanisms 161

dismutase as well as the auxiliary enzyme glutathione reductase and the pentose-phospahte pathway key enzyme glucose-6-phosphate dehydrogenase were shown to increase, decrease, or remain unchanged in response to desiccation and rehydration, depending on the species and the experimental conditions (Weissman et al., 2005). For instance, Mayaba & Beckett (2001) observed that activities of SOD, CAT, and ascorbate peroxidase (AP) were similar during wetting and drying cycles in *Peltigera polydactyla*, *Ramalina celastri* and *Teloschistes capensis*, which grow in moist, xeric and extremely xeric habitats, respectively. Kranner (2002) neither observed a correlation between GR activity and the different degrees of desiccation-tolerance of three lichens, *Lobaria pulmonaria*, *Peltigera polydactyla* and *Pseudevernia furfuracea*. Weissman et al. (2005) even reported that after rehydration *Ramalina lacera* loss almost all CAT activity and SOD decreases by 50-70%. In our laboratories we have observed that during both desiccation period and recovery of *Asterochloris erici*, the levels of superoxide dismutase and peroxidase decreased under both slow and rapid dehydration. Thus, in *A. erici* a longer dehydration time does not lead to a higher accumulation or preservation of classical antioxidants during dehydration/desiccation (Gasulla et al., 2009). Therefore, as a general conclusion, enzymatic antioxidants are perhaps more likely to be involved in removing ROS produced during normal metabolism or by other stresses rather

than during rehydration following severe desiccation (Kranner et al., 2008).

ascorbate levels were undetectable in *A. erici* (Gasulla et al., 2009).

In many organisms, the major water-soluble low-molecular weight antioxidants are the tripeptide GSH (glutatione, γ-glutamyl-cysteinyl-glycine) and ascorbate (Noctor & Foyer, 1998). Ascorbate is known to be a non-enzymatic antioxidant of major importance to the assimilatory and photoprotective processes, its function being central to the defence system. Ascorbate acts as an antioxidant by removing hydrogen peroxide generated during photosynthetic processes in a group of reactions termed the "Mehler peroxidase reaction sequence" (Asada, 1994). Also, ascorbic acid may act as a direct electron donor in photosynthetic and mitochondrial electron transport (Miyake & Asada, 1992) in the ascorbate–glutathione cycle (Foyer & Halliwell, 1976). In addition, it is a cofactor for violaxanthin de-epoxidase in chloroplasts. In lichens, it has been reported that the ascorbate play an important antioxidant role against oxidative stress, such as excessive light or atmospheric pollution (Calatayud et al., 1999). In desiccation-tolerant higher plants, ascorbate forms the first line of defense against oxidative damage (Kranner et al., 2002). However, Kranner et al. (2005) did not find any relationship between ascorbate and the desiccation tolerance of the lichen *Cladonia vulcanii* nor its photobiont. Furthermore,

On the other hand, GSH not only can scavenge ROS reacting with OH• to form GS•; it can also react with another GS•, forming glutathione disulphide (GSSG) (Kranner, 2002). In addition, the redox couple of glutathione (GSH-GSSG) is involved in protecting protein thiol-groups by forming protein-bound glutathione (PSSG) (Kranner & Grill, 1996). In plants, accumulation of GSSG is often correlated with increased stress. Indeed, GSSG can be recycled by the NADPH dependent enzyme GR and in desiccation-tolerant organisms GSSG accumulates during desiccation and is re-reduced to GSH during rehydration (Kranner et al. 2006). Going back to the work of Kranner (2002), it is has been reported that desiccation caused oxidation of almost all GSH in the lichens *Lobaria pulmonaria*, *Peltigera polydactyla* and *Pseudevernia furfuracea*, and rehydration caused the inverse effect. However, after a long desiccation period, in *P. furfuracea* the recovery of the initial concentration of GSH was very rapid, while *P. polydactyla* did not re-establish the GSH pool initial level. It has been demonstrated that NADPH dependent enzyme GR activity is high during rehydration

#### **6. Antioxidant systems in lichen algae**

Photobionts, as aerobic organisms, have to prevent and control oxidative stress damages through a complex antioxidant system capable to maintain controlled the levels of ROS, what is known as redox homeostasis. This defence system has co-evolved with aerobic metabolism to counteract oxidative damage from ROS (Gülçin et al., 2002). It has been studied in detail in plants, and includes enzyme activities of the so-called ascorbateglutathione cycle (Asada, 1994; Foyer & Halliwell, 1976; Mittler, 2002), superoxide dismutase (SOD; Bowler et al., 1992), peroxidases (POX; Esteban-Carrasco et al., 2000, 2001; Ros Barceló et al., 2007; Zapata et al., 1998), catalase (CAT; Mittler, 2002) along with redox metabolites, like ascorbic acid, and glutathione (GSH; Noctor & Foyer, 1998). In the ascorbate-glutathione cycle, the two major antioxidant molecules, ascorbate and glutathione, play an important role as reductants, and they are involved in the scavenging of H2O2 produced by SOD (Kranner & Birtic, 2005; Lascano et al., 1999). Indeed, Superoxide dismutases catalyze the dismutation of O2•− to H2O2 and prevent the further and dangerous conversion into OH• (Casano et al., 1997). Peroxidases catalyse the oxidation of a wide range of substrates at the expense of H2O2 (Ros Barceló et al., 2007; Zapata et al., 2005). Finally, catalases break down H2O2 very rapidly producing H2O and O2, but are much less effective than peroxidases at removing H2O2 because of their lower affinity (high Km) to H2O2 (Kranner & Birtic, 2005).

Concerning to antioxidant metabolites, glutathione and ascorbate are the main antioxidants present in organisms. Glutathione are involved in scavenging of the highly reactive OH• by a cycle that includes the glutathione reductase enzyme (GR) to recover the biologically active glutathione molecule (Noctor & Foyer, 1998). On the other hand, ascorbate reacts rapidly with OH•, O2 •− and 1O2 (Halliwell & Gutteridge, 1999), forming monodehydroascorbate (MDA) and then dehydroascorbate (DHA). Regeneration of Asc may occur via a Mehler peroxidase reaction sequence or through the Asc-GSH cycle (Foyer & Halliwell, 1976).

In desiccation tolerant organisms, as lichens, the desiccated state is characterised by little intracellular water and almost no metabolic activity. Many deleterious effects are associated to this state, such as, irreversible damage to lipids, proteins and nucleic acids through Maillard reactions and ROS (Kranner et al., 2002). During their lifetime, lichens undergo continuous cycles of dehydration-rehydration and therefore they have to must be able to (i) limit the damage to a repairable level, (ii) maintain physiological integrity in the dried state, and (iii) mobilise mechanisms upon rehydration that repair damage suffered during desiccation and subsequent rehydration (Bewley, 1979; Oliver & Bewley, 1997; Oliver et al., 2000). In a first conclusion, desiccation tolerance and prolonged longevity in the desiccated state seems to depend on the ability to prevent light damage (as discussed in the previous section) and activate the biochemical mechanism to scavenge free radicals produced during dehydration-rehydration cycles, using the two described pathways: antioxidant molecules such as glutathione or ascorbate and antioxidant enzymes capable of scavenging free radicals (Kranner & Birtic, 2005). Particularly, ROS-antioxidant interactions has been described in lichens and it is well known that an enhancement of antioxidant status occurs in the symbiotic partnership being more resistant to environmental stress than either partner alone (Kranner et al., 2005). However, within the few studies carried out with lichens, there is not a clear relationship between desiccation tolerance and antioxidant levels. Cellular activities of the antioxidant enzymes ascorbate peroxidase, catalase, and superoxide

Photobionts, as aerobic organisms, have to prevent and control oxidative stress damages through a complex antioxidant system capable to maintain controlled the levels of ROS, what is known as redox homeostasis. This defence system has co-evolved with aerobic metabolism to counteract oxidative damage from ROS (Gülçin et al., 2002). It has been studied in detail in plants, and includes enzyme activities of the so-called ascorbateglutathione cycle (Asada, 1994; Foyer & Halliwell, 1976; Mittler, 2002), superoxide dismutase (SOD; Bowler et al., 1992), peroxidases (POX; Esteban-Carrasco et al., 2000, 2001; Ros Barceló et al., 2007; Zapata et al., 1998), catalase (CAT; Mittler, 2002) along with redox metabolites, like ascorbic acid, and glutathione (GSH; Noctor & Foyer, 1998). In the ascorbate-glutathione cycle, the two major antioxidant molecules, ascorbate and glutathione, play an important role as reductants, and they are involved in the scavenging of H2O2 produced by SOD (Kranner & Birtic, 2005; Lascano et al., 1999). Indeed, Superoxide dismutases catalyze the dismutation of O2•− to H2O2 and prevent the further and dangerous conversion into OH• (Casano et al., 1997). Peroxidases catalyse the oxidation of a wide range of substrates at the expense of H2O2 (Ros Barceló et al., 2007; Zapata et al., 2005). Finally, catalases break down H2O2 very rapidly producing H2O and O2, but are much less effective than peroxidases at removing H2O2 because of their lower affinity (high Km) to H2O2

Concerning to antioxidant metabolites, glutathione and ascorbate are the main antioxidants present in organisms. Glutathione are involved in scavenging of the highly reactive OH• by a cycle that includes the glutathione reductase enzyme (GR) to recover the biologically active glutathione molecule (Noctor & Foyer, 1998). On the other hand, ascorbate reacts

monodehydroascorbate (MDA) and then dehydroascorbate (DHA). Regeneration of Asc may occur via a Mehler peroxidase reaction sequence or through the Asc-GSH cycle (Foyer

In desiccation tolerant organisms, as lichens, the desiccated state is characterised by little intracellular water and almost no metabolic activity. Many deleterious effects are associated to this state, such as, irreversible damage to lipids, proteins and nucleic acids through Maillard reactions and ROS (Kranner et al., 2002). During their lifetime, lichens undergo continuous cycles of dehydration-rehydration and therefore they have to must be able to (i) limit the damage to a repairable level, (ii) maintain physiological integrity in the dried state, and (iii) mobilise mechanisms upon rehydration that repair damage suffered during desiccation and subsequent rehydration (Bewley, 1979; Oliver & Bewley, 1997; Oliver et al., 2000). In a first conclusion, desiccation tolerance and prolonged longevity in the desiccated state seems to depend on the ability to prevent light damage (as discussed in the previous section) and activate the biochemical mechanism to scavenge free radicals produced during dehydration-rehydration cycles, using the two described pathways: antioxidant molecules such as glutathione or ascorbate and antioxidant enzymes capable of scavenging free radicals (Kranner & Birtic, 2005). Particularly, ROS-antioxidant interactions has been described in lichens and it is well known that an enhancement of antioxidant status occurs in the symbiotic partnership being more resistant to environmental stress than either partner alone (Kranner et al., 2005). However, within the few studies carried out with lichens, there is not a clear relationship between desiccation tolerance and antioxidant levels. Cellular activities of the antioxidant enzymes ascorbate peroxidase, catalase, and superoxide

•− and 1O2 (Halliwell & Gutteridge, 1999), forming

**6. Antioxidant systems in lichen algae** 

(Kranner & Birtic, 2005).

rapidly with OH•, O2

& Halliwell, 1976).

dismutase as well as the auxiliary enzyme glutathione reductase and the pentose-phospahte pathway key enzyme glucose-6-phosphate dehydrogenase were shown to increase, decrease, or remain unchanged in response to desiccation and rehydration, depending on the species and the experimental conditions (Weissman et al., 2005). For instance, Mayaba & Beckett (2001) observed that activities of SOD, CAT, and ascorbate peroxidase (AP) were similar during wetting and drying cycles in *Peltigera polydactyla*, *Ramalina celastri* and *Teloschistes capensis*, which grow in moist, xeric and extremely xeric habitats, respectively. Kranner (2002) neither observed a correlation between GR activity and the different degrees of desiccation-tolerance of three lichens, *Lobaria pulmonaria*, *Peltigera polydactyla* and *Pseudevernia furfuracea*. Weissman et al. (2005) even reported that after rehydration *Ramalina lacera* loss almost all CAT activity and SOD decreases by 50-70%. In our laboratories we have observed that during both desiccation period and recovery of *Asterochloris erici*, the levels of superoxide dismutase and peroxidase decreased under both slow and rapid dehydration. Thus, in *A. erici* a longer dehydration time does not lead to a higher accumulation or preservation of classical antioxidants during dehydration/desiccation (Gasulla et al., 2009). Therefore, as a general conclusion, enzymatic antioxidants are perhaps more likely to be involved in removing ROS produced during normal metabolism or by other stresses rather than during rehydration following severe desiccation (Kranner et al., 2008).

In many organisms, the major water-soluble low-molecular weight antioxidants are the tripeptide GSH (glutatione, γ-glutamyl-cysteinyl-glycine) and ascorbate (Noctor & Foyer, 1998). Ascorbate is known to be a non-enzymatic antioxidant of major importance to the assimilatory and photoprotective processes, its function being central to the defence system. Ascorbate acts as an antioxidant by removing hydrogen peroxide generated during photosynthetic processes in a group of reactions termed the "Mehler peroxidase reaction sequence" (Asada, 1994). Also, ascorbic acid may act as a direct electron donor in photosynthetic and mitochondrial electron transport (Miyake & Asada, 1992) in the ascorbate–glutathione cycle (Foyer & Halliwell, 1976). In addition, it is a cofactor for violaxanthin de-epoxidase in chloroplasts. In lichens, it has been reported that the ascorbate play an important antioxidant role against oxidative stress, such as excessive light or atmospheric pollution (Calatayud et al., 1999). In desiccation-tolerant higher plants, ascorbate forms the first line of defense against oxidative damage (Kranner et al., 2002). However, Kranner et al. (2005) did not find any relationship between ascorbate and the desiccation tolerance of the lichen *Cladonia vulcanii* nor its photobiont. Furthermore, ascorbate levels were undetectable in *A. erici* (Gasulla et al., 2009).

On the other hand, GSH not only can scavenge ROS reacting with OH• to form GS•; it can also react with another GS•, forming glutathione disulphide (GSSG) (Kranner, 2002). In addition, the redox couple of glutathione (GSH-GSSG) is involved in protecting protein thiol-groups by forming protein-bound glutathione (PSSG) (Kranner & Grill, 1996). In plants, accumulation of GSSG is often correlated with increased stress. Indeed, GSSG can be recycled by the NADPH dependent enzyme GR and in desiccation-tolerant organisms GSSG accumulates during desiccation and is re-reduced to GSH during rehydration (Kranner et al. 2006). Going back to the work of Kranner (2002), it is has been reported that desiccation caused oxidation of almost all GSH in the lichens *Lobaria pulmonaria*, *Peltigera polydactyla* and *Pseudevernia furfuracea*, and rehydration caused the inverse effect. However, after a long desiccation period, in *P. furfuracea* the recovery of the initial concentration of GSH was very rapid, while *P. polydactyla* did not re-establish the GSH pool initial level. It has been demonstrated that NADPH dependent enzyme GR activity is high during rehydration

Photosynthesis in Lichen: Light Reactions and Protective Mechanisms 163

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### **7. Conclusion**

Two different ways to protect the photosynthetic machinary of lichen chlorobionts were considered in this review. The first one, based in the dissipation of light energy and the second based in the presence of antioxidant activities. Dissipation of light energy at the level of the chlorobiont´s PSII can be achieved by the classical mechanisms based in the xanthophyll cycle, but proof is increasing in favour of the presence of new sinks for conversion of light energy into heat. The activation of these sinks is independent of zeaxanthin accumulation and probably requires an additional pigment and conformational changes in some protein(s) associated to the reaction center or the antenna. Further research is needed to determine the chemical nature and action mechanism of this (or these) alternative energy sink(s).

The lichen is more resistant to oxidative stress than the photobiont or the mycobiont alone. The main antioxidant substance in lichens seems to be glutathione. However, there is not a clear relationship between desiccation tolerance and antioxidant levels. Glutathione, ascorbic acid and antioxidant activities such as SOD or POX can increase, decrease, or not change, depending on the desiccation tolerance of the organism as well as the mode and duration of the treatment. Probably, constitutive levels of these antioxidants are high enough to protect cells against abrupt changes in the environmental conditions, mainly light intensity and humidity.

#### **8. Acknowledgment**

This work was supported by grants from the MEC (BFU2006-11577/BFI and BFU2009- 08151)-FEDER and CARM (08610/PI/08), as well as grants funded by the Spanish Ministry of Education and Science (CGL2006-12917-C02-01/02), the Spanish Ministry of Science and Innovation (CGL2009-13429-C02-01/02), the AECID PCI\_A/024755/09) and the Generalitat Valenciana (PROMETEO 174/2008 GVA). Joaquín Herrero is a fellow of the spanish FPU program.

#### **9. References**


process and therefore it is not a limiting factor to explain the differences between different lichen species. It is more likely that the capacity to reduce GSSG was correlated with the reactivation or synthesis "de novo" of glucose-6-phosphate dehydrogenase, an enzyme of

Two different ways to protect the photosynthetic machinary of lichen chlorobionts were considered in this review. The first one, based in the dissipation of light energy and the second based in the presence of antioxidant activities. Dissipation of light energy at the level of the chlorobiont´s PSII can be achieved by the classical mechanisms based in the xanthophyll cycle, but proof is increasing in favour of the presence of new sinks for conversion of light energy into heat. The activation of these sinks is independent of zeaxanthin accumulation and probably requires an additional pigment and conformational changes in some protein(s) associated to the reaction center or the antenna. Further research is needed to determine the chemical nature and action mechanism of this (or these)

The lichen is more resistant to oxidative stress than the photobiont or the mycobiont alone. The main antioxidant substance in lichens seems to be glutathione. However, there is not a clear relationship between desiccation tolerance and antioxidant levels. Glutathione, ascorbic acid and antioxidant activities such as SOD or POX can increase, decrease, or not change, depending on the desiccation tolerance of the organism as well as the mode and duration of the treatment. Probably, constitutive levels of these antioxidants are high enough to protect cells against abrupt changes in the environmental conditions, mainly light

This work was supported by grants from the MEC (BFU2006-11577/BFI and BFU2009- 08151)-FEDER and CARM (08610/PI/08), as well as grants funded by the Spanish Ministry of Education and Science (CGL2006-12917-C02-01/02), the Spanish Ministry of Science and Innovation (CGL2009-13429-C02-01/02), the AECID PCI\_A/024755/09) and the Generalitat Valenciana (PROMETEO 174/2008 GVA). Joaquín Herrero is a fellow of the spanish FPU

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**7. Conclusion** 

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**8. Acknowledgment** 

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**9. References** 

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**9** 

*Croatia* 

**Energy Conductance from Thylakoid** 

Lea Vojta and Hrvoje Fulgosi

*Rudjer Boskovic Institute* 

**Complexes to Stromal Reducing Equivalents** 

Oxygenic photosynthesis is the basis of heterotrophic life on Earth. It produces carbohydrates and oxygen and may be dived into two sets of reactions: light reactions taking place in the thylakoid membranes, and carbon fixation reactions in soluble stroma. The light reactions involve highly reactive species, and if not controlled properly, they can produce deleterious reactive oxygen species. The structure and function of photosynthetic machinery must be extremely dynamic to enable flawless primary production under a wide spectrum of environmental conditions. The molecular mechanisms behind these dynamic changes remain largely uncharacterized, in particular because various auxiliary proteins

Cooperation of two photosystems in the chloroplast thylakoid membranes produces a linear electron flow (LEF) from H2O to NADP+. Efficient photosynthetic energy conversion requires a high degree of integration and regulation of various redox reactions in order to maximize the use of available light and to minimize damaging effects of excess light (Allen, 2002). The interplay between cyclic (CEF), linear, and pseudocyclic electron transport pathways is required for maintaining the poised state of the photosynthetic system (Allen, 2003). In the over-oxidized state there is no electron flow while in the over-reduced state photooxidation can cause damage to photosystems and eventually death. Common to all three pathways is the activity of PSI that transfers electrons from the plastocyanin located in the thylakoidal lumen to the stromal ferredoxin (Fd). This transfer is mediated by three subunits, C, D and E, of the so-called stromal ridge of PSI (Nelson and Yocum, 2006). In the reduced state Fd provides electrons for the ferredoxin:NADP+ oxidoreductase (FNR), which produces NADPH in a linear pathway (Carrillo & Ceccarelli, 2003), for the ferredoxin– thioredoxin reductase (FTR), which catalyses the reduction of chloroplast thioredoxins (Shaodong et al., 2007), for feeding of the CEF (Allen, 2003) or, alternatively, electrons can be transferred to superoxide, the terminal acceptor in pseudocyclic pathway (Allen, 2003). The generation of reducing power is crucial for all biosynthetic processes within chloroplasts. NAD(P)H and ATP may be considered cell's energetic equivalents and are the principal energetic links between membrane-associated redox reactions and metabolism in the cell soluble compartments. These two types of molecules are generated simultaneously in the chloroplast during light-dependent electron transport and photophosphorylation. They are utilized in the reductive assimilation of inorganic elements (carbon, nitrogen, sulphur) into cellular matter, from which ATP and reductant can be regenerated by oxidative

linking photosynthesis with physiological responses are still missing.

**1. Introduction** 


### **Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents**

Lea Vojta and Hrvoje Fulgosi *Rudjer Boskovic Institute Croatia* 

#### **1. Introduction**

174 Advances in Photosynthesis – Fundamental Aspects

Weissman, L.; Garty, J. & Hochman, A. (2005). Characterization of enzymatic antioxidants in

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the lichen *Ramalina lacera* and their response to rehydration. *Applied and Environmental Microbiology,* Vol. 71, No. 11, (November 2005), pp. 6508–6514. Yruela, I.; Gatzen, G.; Picorel, R. & Holzwarth, AR. (1996). Cu(II)-inhibitory efect on

Photosystem II from higher plants. A picosecond time-esolved fluorescence study.

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regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco. *Cell Death and Differentiation*, Vol. 12, (May 2005), pp. 1277–1284, ISSN 1350-9047. Zapata, JM.; Gasulla, F.; Esteban-Carrasco, A.; Barreno, E. & Guéra, A. (2007). Inactivation of

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(2006a). Winter acclimation of PsbS and related proteins in the evergreen *Arctostaphylos uva-ursi* as influenced by altitude and light environment. *Plant, Cell &* 

(2006b). Winter downregulation of intrinsic photosynthetic capacity coupled with upregulation of Elip-like proteins and persistent energy dissipation in a subalpine forest. *New Phytologist*, Vol. 172, No. 2, (October 2006), pp. 272-282, ISSN 0028-646X.

desiccation and rehydration in the desert lichen Ramalina maciformis. *Symbiosis* 31,

Oxygenic photosynthesis is the basis of heterotrophic life on Earth. It produces carbohydrates and oxygen and may be dived into two sets of reactions: light reactions taking place in the thylakoid membranes, and carbon fixation reactions in soluble stroma. The light reactions involve highly reactive species, and if not controlled properly, they can produce deleterious reactive oxygen species. The structure and function of photosynthetic machinery must be extremely dynamic to enable flawless primary production under a wide spectrum of environmental conditions. The molecular mechanisms behind these dynamic changes remain largely uncharacterized, in particular because various auxiliary proteins linking photosynthesis with physiological responses are still missing.

Cooperation of two photosystems in the chloroplast thylakoid membranes produces a linear electron flow (LEF) from H2O to NADP+. Efficient photosynthetic energy conversion requires a high degree of integration and regulation of various redox reactions in order to maximize the use of available light and to minimize damaging effects of excess light (Allen, 2002). The interplay between cyclic (CEF), linear, and pseudocyclic electron transport pathways is required for maintaining the poised state of the photosynthetic system (Allen, 2003). In the over-oxidized state there is no electron flow while in the over-reduced state photooxidation can cause damage to photosystems and eventually death. Common to all three pathways is the activity of PSI that transfers electrons from the plastocyanin located in the thylakoidal lumen to the stromal ferredoxin (Fd). This transfer is mediated by three subunits, C, D and E, of the so-called stromal ridge of PSI (Nelson and Yocum, 2006). In the reduced state Fd provides electrons for the ferredoxin:NADP+ oxidoreductase (FNR), which produces NADPH in a linear pathway (Carrillo & Ceccarelli, 2003), for the ferredoxin– thioredoxin reductase (FTR), which catalyses the reduction of chloroplast thioredoxins (Shaodong et al., 2007), for feeding of the CEF (Allen, 2003) or, alternatively, electrons can be transferred to superoxide, the terminal acceptor in pseudocyclic pathway (Allen, 2003). The generation of reducing power is crucial for all biosynthetic processes within chloroplasts. NAD(P)H and ATP may be considered cell's energetic equivalents and are the principal energetic links between membrane-associated redox reactions and metabolism in the cell soluble compartments. These two types of molecules are generated simultaneously in the chloroplast during light-dependent electron transport and photophosphorylation. They are utilized in the reductive assimilation of inorganic elements (carbon, nitrogen, sulphur) into cellular matter, from which ATP and reductant can be regenerated by oxidative

Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents 177

the large, shallow cleft between the two domains (Pascalis et al., 1993). The amino acids essential for the formation and activity of the Fd-FNR complex have been identified in detail and nuclear magnetic resonance and mutagenesis studies have further revealed that the flexible N-terminus of FNR is also involved in the interaction with Fd (Maeda et al., 2005). FNR is synthesized on cytosolic rybosomes as a precursor containing an amino-terminal transit peptide, which is responsible for targeting the protein to the chloroplasts (Newman & Gray, 1988). Upon import of FNR into chloroplasts cleavage of the transit peptide by a stromal processing peptidase occurs, followed by the interaction with stromal chaperones Hsp70 (heat shock protein of 70 kDa) and Cpn60 (chaperonin of 60 kDa), which assist in the proper folding of FNR (Tsugeki & Nishimura, 1993), and FAD incorporation, which is required for maintenance of the native structure. Binding of FAD is also a prerequisite for membrane binding of FNR (Onda & Hase, 2004). Regulation of the enzyme activity has been proposed to occur by binding of FNR to the thylakoid membrane (Nakatani & Shin, 1991). Although soluble and membrane-bound FNR form a complex with Fd with the same dissociation constant, the rate constant of NADP+ photoreduction has been shown to be much higher in the membrane bound than in the soluble complex *in vitro* (Forti & Bracale, 1984). But, since the *Arabidopsis fnr1* knock out mutant does not contain any membranebound FNR and still possesses normal photosynthetic performance, it may be concluded that *in planta* the soluble FNR is photosynthetically competent (Lintala et al., 2007), and thus

the solubility of FNR itself is not a crucial determinant of enzyme activity.

In higher plants, chloroplast-targeted FNR is encoded by a small nuclear gene family with one to three *FNR* genes sharing approximately 80% homology with each other. The chloroplast FNR proteins seem to be at least partly redundant, but they also possess unique properties, which are probably required for adjustment of chloroplast metabolism according to changes in the ambient environment (Mulo, 2011). FNR gene families have been well studied in the dicot C3 plant *Arabidopsis thaliana* (thale cress, Hanke et al., 2005), monocot C3 plant *Triticum aestivum* (wheat, Gummadova et al., 2007; Grzyb et al., 2008), and monocot C4

In *Arabidopsis* two genes, *At5g66190* and *At1g20020*, encode the two distinct ~ 32 kDa leaf isoforms FNR1 and FNR2 (Hanke et al., 2005; Lintala et al., 2007). Both genes are predominantly expressed in the rosette leaves, whereas only minor amount of mRNA could be detected in the stems, flowers and siliques, and no FNR proteins could be detected in the root tissue (Hanke et al., 2005). Chloroplast FNR1 has been shown to be more abundant in the membrane fraction (Hanke et al., 2005), especially at the stroma thylakoids (Benz et al., 2009), whereas FNR2 accumulates in higher amounts in the soluble stroma (Hanke et al., 2005). Indeed, FNR1 serves as a membrane anchor to FNR2, since upon inactivation of *FNR1* all the chloroplast FNR (FNR2) exists as a soluble protein (Lintala et al., 2007). It is very likely that FNR *in vivo* exists as a (hetero)dimer (Hanke et al., 2005). Recently, also formation of large (~ 330 kDa) FNR oligomers, devoid of other proteins, has been documented (Grzyb et al., 2008). Inactivation of one chloroplast *FNR* isoform did not result in upregulation of the expression of the other, neither at the level of transcription nor translation (Lintala et al., 2007; Lintala et al., 2009). Inactivation of either *FNR* gene resulted in general downregulation of the photosynthetic machinery, but neither of the isoforms showed any specific function in LEF or CEF of photosynthesis, or other alternative electron transfer

**2.2 FNR gene families** 

plant *Zea mays* (maize, Okutani et al., 2005).

phosphorylation in the mitochondria, which enables the reducing power of NAD(P)H to be converted into ATP. The synthesis of ATP and NADPH in linear electron flow is tightly coupled and if the substrates for the ATP synthase (ADP, inorganic phosphate) become limiting, then the proton motive force builds up, inhibiting electron transfer to NADP+. Likewise, if NADP+ is limiting, photosynthetic electron carriers become reduced, slowing electron transfer and associated proton translocation, thus limiting ATP synthesis (Kramer & Evans, 2006). Linear electron flow produces a fixed ATP/NADPH ratio, and each metabolic pathway directly powered by photosynthesis consumes different fixed ATP/NADPH ratios. Chloroplasts have very limited pools of ATP and NADPH and since mismatches in ATP/NADPH ratios rapidly (within seconds) inhibit photosynthesis (Kramer & Evans, 2006), chloroplasts must balance the production and consumption of both ATP and NADPH by augmenting production of the limiting intermediate (e.g. by CEF) or dissipating the intermediate in excess.

#### **2. Ferredoxin: NADP+ oxidoreductase**

Final electron transfer from ferredoxin to NADP+ is accomplished by the ferredoxin:NADP+ oxidoreductase (FNR), a key enzyme of photosynthetic electron transport required for generation of reduction equivalents. Reducing power derived this way may be further used for carbon assimilation (Calvin-Benson cycle), amino acid, lipid and chlorophyll biosynthesis or reduction of stromal redox-active components. FNR is a ubiquitous flavin adenine dinucleotide (FAD)-binding enzyme that has been identified in various organisms including heterotrophic and phototrophic bacteria, in mitochondria and plastids of higher plants and algae, as well as apicoplasts of some intracellular parasites (Ceccarelli et al., 2004). In higher plants FNR is encoded by a small nuclear gene family and has been found in various chloroplast compartments: at the thylakoid membrane, in the soluble stroma, and at the chloroplast inner envelope. Both the membrane-bound and the soluble FNR pools are photosynthetically active.

#### **2.1 Structure and localization of chloroplast FNRs**

FNR harbors one molecule of noncovalently bound FAD as a prosthetic group (Arakaki et al., 1997; Carillo & Ceccarelli, 2003) and it catalyzes reversible electron transfer between reduced Fd to NADP+ for production of NADPH according to the reaction 2Fdred + NADP+ + H+ ↔ 2Fdox + NADPH. The FAD cofactor of FNR functions as an one-totwo electron switch by reduction of FAD to a semiquinone form FADH. , followed by another round of reduction to FADH−, and hydride transfer from FADH− to NADP+.

Chloroplast FNR proteins are hydrophilic proteins with a molecular weight of approximately 35 kDa. Sequence similarity of FNRs from various plant species varies from 40% to 97% (Arakaki et al., 1997), and especially regions involved in FAD and NADP+ binding share high degree of identity. The topology of all chloroplast FNRs is highly conserved, consisting of two distinct domains connected by a loop (Dorowski et al., 2001), which shows the biggest variance between the species. The N-terminal domain (ca. 150 amino acids) is made up of a β-barrel structure built of six antiparallel β-strands and capped by an α-helix and a long loop and is involved in FAD binding, while the C-terminal domain (ca. 150 amino acids) consists of a central five-stranded parallel β-sheet surrounded by six αhelices and is mainly responsible for binding of NADP+ (Karplus et al., 1991). Fd is bound to the large, shallow cleft between the two domains (Pascalis et al., 1993). The amino acids essential for the formation and activity of the Fd-FNR complex have been identified in detail and nuclear magnetic resonance and mutagenesis studies have further revealed that the flexible N-terminus of FNR is also involved in the interaction with Fd (Maeda et al., 2005).

FNR is synthesized on cytosolic rybosomes as a precursor containing an amino-terminal transit peptide, which is responsible for targeting the protein to the chloroplasts (Newman & Gray, 1988). Upon import of FNR into chloroplasts cleavage of the transit peptide by a stromal processing peptidase occurs, followed by the interaction with stromal chaperones Hsp70 (heat shock protein of 70 kDa) and Cpn60 (chaperonin of 60 kDa), which assist in the proper folding of FNR (Tsugeki & Nishimura, 1993), and FAD incorporation, which is required for maintenance of the native structure. Binding of FAD is also a prerequisite for membrane binding of FNR (Onda & Hase, 2004). Regulation of the enzyme activity has been proposed to occur by binding of FNR to the thylakoid membrane (Nakatani & Shin, 1991). Although soluble and membrane-bound FNR form a complex with Fd with the same dissociation constant, the rate constant of NADP+ photoreduction has been shown to be much higher in the membrane bound than in the soluble complex *in vitro* (Forti & Bracale, 1984). But, since the *Arabidopsis fnr1* knock out mutant does not contain any membranebound FNR and still possesses normal photosynthetic performance, it may be concluded that *in planta* the soluble FNR is photosynthetically competent (Lintala et al., 2007), and thus the solubility of FNR itself is not a crucial determinant of enzyme activity.

#### **2.2 FNR gene families**

176 Advances in Photosynthesis – Fundamental Aspects

phosphorylation in the mitochondria, which enables the reducing power of NAD(P)H to be converted into ATP. The synthesis of ATP and NADPH in linear electron flow is tightly coupled and if the substrates for the ATP synthase (ADP, inorganic phosphate) become limiting, then the proton motive force builds up, inhibiting electron transfer to NADP+. Likewise, if NADP+ is limiting, photosynthetic electron carriers become reduced, slowing electron transfer and associated proton translocation, thus limiting ATP synthesis (Kramer & Evans, 2006). Linear electron flow produces a fixed ATP/NADPH ratio, and each metabolic pathway directly powered by photosynthesis consumes different fixed ATP/NADPH ratios. Chloroplasts have very limited pools of ATP and NADPH and since mismatches in ATP/NADPH ratios rapidly (within seconds) inhibit photosynthesis (Kramer & Evans, 2006), chloroplasts must balance the production and consumption of both ATP and NADPH by augmenting production of the limiting intermediate (e.g. by CEF) or dissipating

Final electron transfer from ferredoxin to NADP+ is accomplished by the ferredoxin:NADP+ oxidoreductase (FNR), a key enzyme of photosynthetic electron transport required for generation of reduction equivalents. Reducing power derived this way may be further used for carbon assimilation (Calvin-Benson cycle), amino acid, lipid and chlorophyll biosynthesis or reduction of stromal redox-active components. FNR is a ubiquitous flavin adenine dinucleotide (FAD)-binding enzyme that has been identified in various organisms including heterotrophic and phototrophic bacteria, in mitochondria and plastids of higher plants and algae, as well as apicoplasts of some intracellular parasites (Ceccarelli et al., 2004). In higher plants FNR is encoded by a small nuclear gene family and has been found in various chloroplast compartments: at the thylakoid membrane, in the soluble stroma, and at the chloroplast inner envelope. Both the membrane-bound and the soluble FNR pools are

FNR harbors one molecule of noncovalently bound FAD as a prosthetic group (Arakaki et al., 1997; Carillo & Ceccarelli, 2003) and it catalyzes reversible electron transfer between reduced Fd to NADP+ for production of NADPH according to the reaction 2Fdred + NADP+ + H+ ↔ 2Fdox + NADPH. The FAD cofactor of FNR functions as an one-to-

Chloroplast FNR proteins are hydrophilic proteins with a molecular weight of approximately 35 kDa. Sequence similarity of FNRs from various plant species varies from 40% to 97% (Arakaki et al., 1997), and especially regions involved in FAD and NADP+ binding share high degree of identity. The topology of all chloroplast FNRs is highly conserved, consisting of two distinct domains connected by a loop (Dorowski et al., 2001), which shows the biggest variance between the species. The N-terminal domain (ca. 150 amino acids) is made up of a β-barrel structure built of six antiparallel β-strands and capped by an α-helix and a long loop and is involved in FAD binding, while the C-terminal domain (ca. 150 amino acids) consists of a central five-stranded parallel β-sheet surrounded by six αhelices and is mainly responsible for binding of NADP+ (Karplus et al., 1991). Fd is bound to

, followed by

two electron switch by reduction of FAD to a semiquinone form FADH.

another round of reduction to FADH−, and hydride transfer from FADH− to NADP+.

 **oxidoreductase** 

the intermediate in excess.

**2. Ferredoxin: NADP+**

photosynthetically active.

**2.1 Structure and localization of chloroplast FNRs** 

In higher plants, chloroplast-targeted FNR is encoded by a small nuclear gene family with one to three *FNR* genes sharing approximately 80% homology with each other. The chloroplast FNR proteins seem to be at least partly redundant, but they also possess unique properties, which are probably required for adjustment of chloroplast metabolism according to changes in the ambient environment (Mulo, 2011). FNR gene families have been well studied in the dicot C3 plant *Arabidopsis thaliana* (thale cress, Hanke et al., 2005), monocot C3 plant *Triticum aestivum* (wheat, Gummadova et al., 2007; Grzyb et al., 2008), and monocot C4 plant *Zea mays* (maize, Okutani et al., 2005).

In *Arabidopsis* two genes, *At5g66190* and *At1g20020*, encode the two distinct ~ 32 kDa leaf isoforms FNR1 and FNR2 (Hanke et al., 2005; Lintala et al., 2007). Both genes are predominantly expressed in the rosette leaves, whereas only minor amount of mRNA could be detected in the stems, flowers and siliques, and no FNR proteins could be detected in the root tissue (Hanke et al., 2005). Chloroplast FNR1 has been shown to be more abundant in the membrane fraction (Hanke et al., 2005), especially at the stroma thylakoids (Benz et al., 2009), whereas FNR2 accumulates in higher amounts in the soluble stroma (Hanke et al., 2005). Indeed, FNR1 serves as a membrane anchor to FNR2, since upon inactivation of *FNR1* all the chloroplast FNR (FNR2) exists as a soluble protein (Lintala et al., 2007). It is very likely that FNR *in vivo* exists as a (hetero)dimer (Hanke et al., 2005). Recently, also formation of large (~ 330 kDa) FNR oligomers, devoid of other proteins, has been documented (Grzyb et al., 2008). Inactivation of one chloroplast *FNR* isoform did not result in upregulation of the expression of the other, neither at the level of transcription nor translation (Lintala et al., 2007; Lintala et al., 2009). Inactivation of either *FNR* gene resulted in general downregulation of the photosynthetic machinery, but neither of the isoforms showed any specific function in LEF or CEF of photosynthesis, or other alternative electron transfer

Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents 179

CEF supplies the ATP needed for driving the CO2 concentrating mechanism in the C4 plants. Recently, the significance of CEF has been shown in C3 plants as well, where under normal physiological conditions CEF might have a role in adjusting the stoichiometry of ATP/NADPH generated by photosynthesis (Munekage et al., 2004). FNR has been

Four possible routes of cyclic electron transfer have been proposed so far and may operate in parallel. **NAD(P)H dehydrogenase (NDH)-dependent route**, in which electrons are first transferred from NADPH to NAD(P)H dehydrogenase-1 complex, and secondly to the PQ pool (Kramer & Evans, 2006; Mulo, 2011). The redox reactions are coupled to proton translocation in two ways. First, protons are taken up on the negatively charged side of the membrane during quinone reduction and released on the positive side of the membrane during quinol oxidation. Four protons should be translocated for each electron transferred through the cycle, two via the reduction and oxidation of PQ and the Q cycle and two more via the NDH proton pump (Kramer & Evans, 2006). **Fd-dependent route**, in which electrons are funneled from Fd to Cyt *b6f* complex via a partly uncharacterized route involving PGR5 and PGRL1 proteins, which work together to catalyze cyclic electron flow, and possibly FNR and hypothetical FQR (ferredoxin-plastoquinone oxidoreductase) (Munekage et al., 2002; Kramer & Evans, 2006). **Nda2, a type 2 NAD(P)H:PQ oxidoreductase route** is active in some green algae and conifers that lack the chloroplast NDH complex. In *Chlamydomonas*, PQ reduction in CEF has been proposed to occur via a Nda2 (Desplats et al., 2009). It is related to those found in bacteria and mitochondria and does not pump protons. Since type 2 complexes are structurally much simpler than complex I (one subunit with a single flavin cofactor compared with at least 11 protein subunits, nine FeS clusters, and a flavin), Nda2 may be less efficient in energy balancing (Kramer & Evans, 2006). **The Cyt** *b6f* **complex and FNR route** uses the PQ reductase site of the Cyt *b6f* complex to reduce PQ (Zhang et al., 2004). Electron transfer to Qi probably involves the newly discovered heme *c*i, which allows electrons to flow from Fd or FNR to the bound PQ (Zhang et al., 2004). This pathway probably involves the formation of a special cyclic electron flow supercomplex (Iwai et al.,

PSII and PSI in the chloroplasts of higher plants are potential sources of harmful reactive oxygen species (ROS). In *E. coli* FNR is involved in quenching of ROS (Krapp et al., 2002), and in methyl viologen resistant *Chlamydomonas reinhardtii* strains the steady state level of chloroplast FNR transcripts has been shown to be increased as compared to wild type (Kitiyama et al., 1995). Moreover, expression of plant FNR has been proven to restore the oxidative tolerance of a mutant *E. coli* (Krapp et al., 1997). The research on the participation of FNR in oxidative stress responses of higher plants has been performed on wheat and has shown that, in contrast to bacterial cells, the content of FNR mRNA as well as protein in higher plants rather decreases than increases in response to induction of oxidative stress (Palatnik et al., 1997). However, production of ROS results in marked release of FNR from the thylakoid membrane followed by reduction of NADP+ photoreduction capacity, which might aim at maintaining the NADP+/NADPH homeostasis of the stressed plants (Palatnik et al., 1997). Recently, it has been shown that FNR releases from the thylakoids in the plants suffering from drought stress (Lehtimaki et al., 2010), and the FNR containing thylakoid protein complexes disassemble upon high

identified as a component of the Cyt *b6f* complex (Clark et al., 1984).

2010).

**2.4 FNR and oxidative stress tolerance** 

light illumination (Benz et al., 2009).

reactions (Lintala et al., 2007; Lintala et al., 2009). Growth of the *fnr* knock-out plants under unfavorable conditions revealed a unique role for FNR2 in redistribution of electrons to various redox reactions (Lintala et al., 2009). Beside two leaf-type FNR, two root-type FNR isoenzymes, encoded by genes *At1g30510* and *At4g05390* are present in *Arabidopsis* as well (Hanke et al., 2005). The growth of *Arabidopsis* on different nitrogen regimes induced differential expression of the two chloroplast *FNR* genes showing that multiple FNR isoenzymes have variable metabolic roles and differentially contribute to nitrogen assimilation (Hanke et al., 2005). Studies *in vivo* have revealed that suppression of FNR expression leads to increased susceptibility to photo-oxidative damage, impaired plant growth and lowered photosynthetic activity of transgenic plants (Hajirezaei et al., 2002; Palatnik et al., 2003). On the other hand, overexpression of FNR results in slightly increased rates of electron transport from water to NADP+ and increased tolerance to oxidative stress (Rodriguez et al., 2007).

Based on isoelectric focusing and SDS-PAGE the proteome of wheat has revealed four distinct leaf FNR isoforms that can be divided into two groups, FNRI and FNRII. Both groups contain two proteins, which differ from each other by truncation of the N-terminus (Gummadova et al., 2007; Grzyb et al., 2008). It has been demonstrated that the presence of mature wheat FNR proteins with alternative N-terminal start points, differing by a three amino acid truncation in pFNRI and a two amino acid truncation in pFNRII, have statistically significant differences in response to the physiological parameters of chloroplast maturity, nitrogen regime, and oxidative stress (Moolna et al., 2009). It has been suggested that these isoforms may be crucial to the regulation of reductant partition between carbon fixation and other metabolic pathways. Also, differences in the N-terminus of the wheat FNR isoforms seem to result in changes of FNR activity, subchloroplast localization as well as affinity of FNR to different Fd isoforms (Moolna et al., 2009). Results of Moolna et al. (2009) also suggest that four pFNR protein isoforms are each present in the chloroplast in phosphorylated and nonphosphorylated states, probably as a response to physiological parameters.

The genome of maize codes for three distinct leaf FNR genes that share 83–92% homology with each other, and are present in the leaves at approximately equivalent concentration. FNR1 is found at the thylakoid membrane, FNR3 is an exclusively soluble stromal protein, while FNR2 has a dual location (Okutani et al., 2005). The activity of FNR2 is similar to FNR3, and higher than that of the FNR1, and the mode of interaction between Fd(s) and the FNR isoforms is dependent on both pH and redox status of the chloroplast (Okutani et al., 2005). It has also been proposed that leaf FNR isoenzymes 1 and 2 are relatively more abundant under conditions of high demand for NADPH (Okutani et al., 2005).

#### **2.3 FNR and the cyclic electron flow**

FNR functions in the crossing of various electron transfer pathways. Besides its confirmed involvment in the last step of the LEF from water via PSII, plastoquinone (PQ) pool, Cyt *b6f* complex, PSI, Fd and FNR to NADP+, the role of FNR in cyclic electron transfer has not yet been defined. In CEF, electrons are transferred from PSI to Cyt *b6f* complex via Fd, with associated formation of proton gradient. PQ is reduced by Fd or NADPH via one or more enzymes collectively called PQ reductase, rather than by PSII, as in LEF. From hidroplastoquinone (PQH2), electrons return to PSI via the Cyt *b6f* complex. Thus, CEF around PSI produces ATP without accumulation of NADPH. It is generally accepted that

reactions (Lintala et al., 2007; Lintala et al., 2009). Growth of the *fnr* knock-out plants under unfavorable conditions revealed a unique role for FNR2 in redistribution of electrons to various redox reactions (Lintala et al., 2009). Beside two leaf-type FNR, two root-type FNR isoenzymes, encoded by genes *At1g30510* and *At4g05390* are present in *Arabidopsis* as well (Hanke et al., 2005). The growth of *Arabidopsis* on different nitrogen regimes induced differential expression of the two chloroplast *FNR* genes showing that multiple FNR isoenzymes have variable metabolic roles and differentially contribute to nitrogen assimilation (Hanke et al., 2005). Studies *in vivo* have revealed that suppression of FNR expression leads to increased susceptibility to photo-oxidative damage, impaired plant growth and lowered photosynthetic activity of transgenic plants (Hajirezaei et al., 2002; Palatnik et al., 2003). On the other hand, overexpression of FNR results in slightly increased rates of electron transport from water to NADP+ and increased tolerance to oxidative stress

Based on isoelectric focusing and SDS-PAGE the proteome of wheat has revealed four distinct leaf FNR isoforms that can be divided into two groups, FNRI and FNRII. Both groups contain two proteins, which differ from each other by truncation of the N-terminus (Gummadova et al., 2007; Grzyb et al., 2008). It has been demonstrated that the presence of mature wheat FNR proteins with alternative N-terminal start points, differing by a three amino acid truncation in pFNRI and a two amino acid truncation in pFNRII, have statistically significant differences in response to the physiological parameters of chloroplast maturity, nitrogen regime, and oxidative stress (Moolna et al., 2009). It has been suggested that these isoforms may be crucial to the regulation of reductant partition between carbon fixation and other metabolic pathways. Also, differences in the N-terminus of the wheat FNR isoforms seem to result in changes of FNR activity, subchloroplast localization as well as affinity of FNR to different Fd isoforms (Moolna et al., 2009). Results of Moolna et al. (2009) also suggest that four pFNR protein isoforms are each present in the chloroplast in phosphorylated and nonphosphorylated states, probably as a response to physiological

The genome of maize codes for three distinct leaf FNR genes that share 83–92% homology with each other, and are present in the leaves at approximately equivalent concentration. FNR1 is found at the thylakoid membrane, FNR3 is an exclusively soluble stromal protein, while FNR2 has a dual location (Okutani et al., 2005). The activity of FNR2 is similar to FNR3, and higher than that of the FNR1, and the mode of interaction between Fd(s) and the FNR isoforms is dependent on both pH and redox status of the chloroplast (Okutani et al., 2005). It has also been proposed that leaf FNR isoenzymes 1 and 2 are relatively more

FNR functions in the crossing of various electron transfer pathways. Besides its confirmed involvment in the last step of the LEF from water via PSII, plastoquinone (PQ) pool, Cyt *b6f* complex, PSI, Fd and FNR to NADP+, the role of FNR in cyclic electron transfer has not yet been defined. In CEF, electrons are transferred from PSI to Cyt *b6f* complex via Fd, with associated formation of proton gradient. PQ is reduced by Fd or NADPH via one or more enzymes collectively called PQ reductase, rather than by PSII, as in LEF. From hidroplastoquinone (PQH2), electrons return to PSI via the Cyt *b6f* complex. Thus, CEF around PSI produces ATP without accumulation of NADPH. It is generally accepted that

abundant under conditions of high demand for NADPH (Okutani et al., 2005).

(Rodriguez et al., 2007).

parameters.

**2.3 FNR and the cyclic electron flow** 

CEF supplies the ATP needed for driving the CO2 concentrating mechanism in the C4 plants. Recently, the significance of CEF has been shown in C3 plants as well, where under normal physiological conditions CEF might have a role in adjusting the stoichiometry of ATP/NADPH generated by photosynthesis (Munekage et al., 2004). FNR has been identified as a component of the Cyt *b6f* complex (Clark et al., 1984).

Four possible routes of cyclic electron transfer have been proposed so far and may operate in parallel. **NAD(P)H dehydrogenase (NDH)-dependent route**, in which electrons are first transferred from NADPH to NAD(P)H dehydrogenase-1 complex, and secondly to the PQ pool (Kramer & Evans, 2006; Mulo, 2011). The redox reactions are coupled to proton translocation in two ways. First, protons are taken up on the negatively charged side of the membrane during quinone reduction and released on the positive side of the membrane during quinol oxidation. Four protons should be translocated for each electron transferred through the cycle, two via the reduction and oxidation of PQ and the Q cycle and two more via the NDH proton pump (Kramer & Evans, 2006). **Fd-dependent route**, in which electrons are funneled from Fd to Cyt *b6f* complex via a partly uncharacterized route involving PGR5 and PGRL1 proteins, which work together to catalyze cyclic electron flow, and possibly FNR and hypothetical FQR (ferredoxin-plastoquinone oxidoreductase) (Munekage et al., 2002; Kramer & Evans, 2006). **Nda2, a type 2 NAD(P)H:PQ oxidoreductase route** is active in some green algae and conifers that lack the chloroplast NDH complex. In *Chlamydomonas*, PQ reduction in CEF has been proposed to occur via a Nda2 (Desplats et al., 2009). It is related to those found in bacteria and mitochondria and does not pump protons. Since type 2 complexes are structurally much simpler than complex I (one subunit with a single flavin cofactor compared with at least 11 protein subunits, nine FeS clusters, and a flavin), Nda2 may be less efficient in energy balancing (Kramer & Evans, 2006). **The Cyt** *b6f* **complex and FNR route** uses the PQ reductase site of the Cyt *b6f* complex to reduce PQ (Zhang et al., 2004). Electron transfer to Qi probably involves the newly discovered heme *c*i, which allows electrons to flow from Fd or FNR to the bound PQ (Zhang et al., 2004). This pathway probably involves the formation of a special cyclic electron flow supercomplex (Iwai et al., 2010).

#### **2.4 FNR and oxidative stress tolerance**

PSII and PSI in the chloroplasts of higher plants are potential sources of harmful reactive oxygen species (ROS). In *E. coli* FNR is involved in quenching of ROS (Krapp et al., 2002), and in methyl viologen resistant *Chlamydomonas reinhardtii* strains the steady state level of chloroplast FNR transcripts has been shown to be increased as compared to wild type (Kitiyama et al., 1995). Moreover, expression of plant FNR has been proven to restore the oxidative tolerance of a mutant *E. coli* (Krapp et al., 1997). The research on the participation of FNR in oxidative stress responses of higher plants has been performed on wheat and has shown that, in contrast to bacterial cells, the content of FNR mRNA as well as protein in higher plants rather decreases than increases in response to induction of oxidative stress (Palatnik et al., 1997). However, production of ROS results in marked release of FNR from the thylakoid membrane followed by reduction of NADP+ photoreduction capacity, which might aim at maintaining the NADP+/NADPH homeostasis of the stressed plants (Palatnik et al., 1997). Recently, it has been shown that FNR releases from the thylakoids in the plants suffering from drought stress (Lehtimaki et al., 2010), and the FNR containing thylakoid protein complexes disassemble upon high light illumination (Benz et al., 2009).

Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents 181

the membrane-bound Tic62–FNR protein complexes were most abundant in the dark, while increase in light intensity resulted in the disassembly of the complex. Similarly, *in vitro* alkalization of isolated thylakoids dissociated FNR and Tic62 (Benz et al., 2009). It is important to stess that the interaction of Tic62 with FNR stabilizes the activity of the FNR protein (Benz et al., 2009) and that FNR activity is lower in acidic than basic environment (Lee et al., 2007). These results indicate that Tic62 acts as a chaperone for FNR, and protects the flavoenzyme from inactivation and degradation during the photosynthetically inactive

By using antisense and gene inactivation strategies Jurić et al. (2009) identified a novel component of non-appressed thylakoid membranes which is responsible for anchoring of FNR. TROL (thylakoid rhodanese-like protein) is a 66 kDa nuclear encoded component of thylakoid membranes required for tethering of FNR and sustaining efficient LEF in vascular plants. TROL contains two transmembrane helices and a centrally positioned (inactive) rhodanese domain (Jurić et al., 2009). As an integral membrane protein, TROL is firmly attached to the thylakoid membrane and cannot be extracted from the membrane by high salt, urea or high pH treatments (Jurić et al., 2009). TROL possesses a unique fusion of two distinct modules: a centrally positioned rhodanese-like domain, RHO, which is found in all life forms, and a C-terminal single hydrophobic FNR-binding region, ITEP, which is ascribed to the vascular plants (Balsera et al., 2007). It is hypothesised that both N- and Cterminal parts of TROL face the stroma, while RHO faces the thylakoid lumen (Jurić et al., 2009). A closer investigation of the TROL protein sequence revealed an interesting region upstream of the ITEP domain. The Pro-Val-Pro repeat-rich region was designated PEPE. It consists of two identical repeats, followed by a possible PVP hinge. In membrane proteins, prolines are known to have a structural role in transmembrane helices, where they distort the alpha-helix due to the loss of at least one stabilising backbone hydrogen bond. Thus, PEPE region, which is presumed to be exposed into the stroma, is proposed to introduce flexibility in the helix that may result in kink and swivel motions of FNR-binding region. Localization in non-appressed regions places TROL in the vicinity of the site of Fd reduction. TROL has been found in several complexes, indicating the presence of several TROL subpools in the thylakoid membrane. Only a 190 kDa complex appears to contain TROL in association with the FNR. Complexes at about 110 and 120 kDa indicate the existence of a small ligand which may be associated with other TROL domains, namely the

large rhodanese-like domain which is predicted to be located in the thylakoid lumen.

The findings of Küchler et al. (2002) that Tic62 interacts with FNR prompted the analysis of TROL protein sequence in search for the similar binding module. Tic62 from *Pisum sativum*  (PsTic62) contains three Pro/Ser-rich repetitive motives at the C-terminus, S-P-Y-x(2)-Y-x-D/E-L-K-P(2)-S/T/A-S/T-P-S/T-P, involved in the binding of FNR (Küchler et al., 2002). PsTic62 homolog in *A. thaliana*, encoded by a single-copy gene (*At3g18890*), shows approximately 60% identity for the deduced mature sequence and has a calculated molecular weight of 62.1 kDa (Küchler et al., 2002). AtTic62 contains four repetitive motives at the C-terminus, but it has been shown previously that only one repeat is sufficient for the binding of FNR (Küchler et al., 2002; Balsera et al., 2007). As TROL possesses almost identical domain to the Tic62 FNR-binding repeats, a modified yeast-two hybrid assay was used to confirm ITEP-FNR interaction (Jurić et al., 2009). Predictably, ITEP strongly binds to

periods, e.g. in darkness (Benz et al., 2009).

**3.2 TROL** 

#### **3. Supramolecular FNR complexes**

It has been shown that FNR exists in soluble and membrane-bound forms (Palatnik et al., 1997; Lintala et al., 2007). Several potential FNR-binding partners have been discussed, which might be involved in membrane attachment of FNR. Various studies have shown interaction of FNR with the photosynthetic protein complexes Cyt *b6f* (Clark et al., 1984; Zhang et al., 2001), PSI (Andersen et al., 1992) or NDH complex (Quiles & Cuello, 1998), but also interaction with glyceraldehyde-3-phosphate dehydrogenase, or direct membrane attachment have been suggested. However, until recently no exact protein partner responsible for FNR tethering has been identified. Two chloroplast proteins, Tic62 and TROL, were recently identified and shown to form high molecular weight protein complexes with FNR at the thylakoid membrane, and thus seem to act as molecular anchors of FNR to the thylakoid membrane. Tic62 and TROL have been shown to bind FNR by specific interaction via a conserved Ser/Pro-rich motif. In darkness, FNR forms large protein complexes at the thylakoids together with Tic62 and TROL. Similarly, Tic62 and presumably TROL bind FNR at the envelope. FNR is released from the membranes upon illumination.

#### **3.1 Tic62**

During its import into chloroplasts from the site of synthesis on cytosolic rybosomes, FNR has been found to interact with Tic62 protein, a 62 kDa component of the Translocon at the inner envelope of chloroplast (Küchler et al., 2002; Balsera et al., 2007; Stengel et al., 2008; Benz et al. 2009). Proteomics studies have identified Tic62 in the chloroplast envelope, stroma and thylakoid fraction (Benz et al., 2009). Furthermore, Tic62 at the thylakoid membrane was found in several high molecular mass protein complexes (250–500 kDa), and it was shown to be tightly associated with both chloroplast FNR isoforms (Benz et al., 2009). The N-terminus of Tic62 binds pyridine nucleotides, while the stroma exposed C-terminus contains repetitive, highly conserved FNR-binding domains (Küchler et al., 2002). Database searches have verified the presence of the FNR-binding domains only in the Tic62 protein of vascular plants (Balsera et al., 2007) and it occurs in different numbers dependent on the respective plants species.

The function of FNR in the Tic complex has been suggested to link redox regulation to chloroplast protein import. Indeed, *in vitro* experiments with compounds interfering either with NAD binding or NAD(P)/NAD(P)H ratio modulate the import characteristics of the leaf FNR isoforms: FNR1 is translocated preferentially at high NAD(P)/NAD(P)H ratio, while the translocation of FNR2 is not influenced by the redox status (Küchler et al., 2002). In maize, import of pre-FdI to chloroplast stroma is independent on illumination, while pre-FdIII and preFNR were efficiently targeted into stroma only in darkness (Hirohashi et al., 2001). These results imply that the diurnal changes in the chloroplast redox poise may control import characteristics of the organelle. It was recently shown that Tic62 shuttles between the soluble stroma and the chloroplast membranes, and that oxidation of stroma results in stronger association of Tic62 to the membrane fraction (Stengel et al., 2008). FNR shows similar shuttling behavior, and therefore the Tic62–FNR interaction is dependent on chloroplast redox state (Stengel et al., 2008). The lack of Tic62 and consequently the lack of Tic62–FNR complexes did not have any effects on the plant phenotype or photosynthetic properties, neither on LEF nor CEF (Benz et al., 2009), implying that the Tic62–FNR complexes serve for some other purpose(s) than photosynthesis. It has been observed that the membrane-bound Tic62–FNR protein complexes were most abundant in the dark, while increase in light intensity resulted in the disassembly of the complex. Similarly, *in vitro* alkalization of isolated thylakoids dissociated FNR and Tic62 (Benz et al., 2009). It is important to stess that the interaction of Tic62 with FNR stabilizes the activity of the FNR protein (Benz et al., 2009) and that FNR activity is lower in acidic than basic environment (Lee et al., 2007). These results indicate that Tic62 acts as a chaperone for FNR, and protects the flavoenzyme from inactivation and degradation during the photosynthetically inactive periods, e.g. in darkness (Benz et al., 2009).

#### **3.2 TROL**

180 Advances in Photosynthesis – Fundamental Aspects

It has been shown that FNR exists in soluble and membrane-bound forms (Palatnik et al., 1997; Lintala et al., 2007). Several potential FNR-binding partners have been discussed, which might be involved in membrane attachment of FNR. Various studies have shown interaction of FNR with the photosynthetic protein complexes Cyt *b6f* (Clark et al., 1984; Zhang et al., 2001), PSI (Andersen et al., 1992) or NDH complex (Quiles & Cuello, 1998), but also interaction with glyceraldehyde-3-phosphate dehydrogenase, or direct membrane attachment have been suggested. However, until recently no exact protein partner responsible for FNR tethering has been identified. Two chloroplast proteins, Tic62 and TROL, were recently identified and shown to form high molecular weight protein complexes with FNR at the thylakoid membrane, and thus seem to act as molecular anchors of FNR to the thylakoid membrane. Tic62 and TROL have been shown to bind FNR by specific interaction via a conserved Ser/Pro-rich motif. In darkness, FNR forms large protein complexes at the thylakoids together with Tic62 and TROL. Similarly, Tic62 and presumably TROL bind FNR at the envelope. FNR is released from the membranes upon illumination.

During its import into chloroplasts from the site of synthesis on cytosolic rybosomes, FNR has been found to interact with Tic62 protein, a 62 kDa component of the Translocon at the inner envelope of chloroplast (Küchler et al., 2002; Balsera et al., 2007; Stengel et al., 2008; Benz et al. 2009). Proteomics studies have identified Tic62 in the chloroplast envelope, stroma and thylakoid fraction (Benz et al., 2009). Furthermore, Tic62 at the thylakoid membrane was found in several high molecular mass protein complexes (250–500 kDa), and it was shown to be tightly associated with both chloroplast FNR isoforms (Benz et al., 2009). The N-terminus of Tic62 binds pyridine nucleotides, while the stroma exposed C-terminus contains repetitive, highly conserved FNR-binding domains (Küchler et al., 2002). Database searches have verified the presence of the FNR-binding domains only in the Tic62 protein of vascular plants (Balsera et al., 2007) and it occurs in different numbers dependent on the

The function of FNR in the Tic complex has been suggested to link redox regulation to chloroplast protein import. Indeed, *in vitro* experiments with compounds interfering either with NAD binding or NAD(P)/NAD(P)H ratio modulate the import characteristics of the leaf FNR isoforms: FNR1 is translocated preferentially at high NAD(P)/NAD(P)H ratio, while the translocation of FNR2 is not influenced by the redox status (Küchler et al., 2002). In maize, import of pre-FdI to chloroplast stroma is independent on illumination, while pre-FdIII and preFNR were efficiently targeted into stroma only in darkness (Hirohashi et al., 2001). These results imply that the diurnal changes in the chloroplast redox poise may control import characteristics of the organelle. It was recently shown that Tic62 shuttles between the soluble stroma and the chloroplast membranes, and that oxidation of stroma results in stronger association of Tic62 to the membrane fraction (Stengel et al., 2008). FNR shows similar shuttling behavior, and therefore the Tic62–FNR interaction is dependent on chloroplast redox state (Stengel et al., 2008). The lack of Tic62 and consequently the lack of Tic62–FNR complexes did not have any effects on the plant phenotype or photosynthetic properties, neither on LEF nor CEF (Benz et al., 2009), implying that the Tic62–FNR complexes serve for some other purpose(s) than photosynthesis. It has been observed that

**3. Supramolecular FNR complexes** 

**3.1 Tic62** 

respective plants species.

By using antisense and gene inactivation strategies Jurić et al. (2009) identified a novel component of non-appressed thylakoid membranes which is responsible for anchoring of FNR. TROL (thylakoid rhodanese-like protein) is a 66 kDa nuclear encoded component of thylakoid membranes required for tethering of FNR and sustaining efficient LEF in vascular plants. TROL contains two transmembrane helices and a centrally positioned (inactive) rhodanese domain (Jurić et al., 2009). As an integral membrane protein, TROL is firmly attached to the thylakoid membrane and cannot be extracted from the membrane by high salt, urea or high pH treatments (Jurić et al., 2009). TROL possesses a unique fusion of two distinct modules: a centrally positioned rhodanese-like domain, RHO, which is found in all life forms, and a C-terminal single hydrophobic FNR-binding region, ITEP, which is ascribed to the vascular plants (Balsera et al., 2007). It is hypothesised that both N- and Cterminal parts of TROL face the stroma, while RHO faces the thylakoid lumen (Jurić et al., 2009). A closer investigation of the TROL protein sequence revealed an interesting region upstream of the ITEP domain. The Pro-Val-Pro repeat-rich region was designated PEPE. It consists of two identical repeats, followed by a possible PVP hinge. In membrane proteins, prolines are known to have a structural role in transmembrane helices, where they distort the alpha-helix due to the loss of at least one stabilising backbone hydrogen bond. Thus, PEPE region, which is presumed to be exposed into the stroma, is proposed to introduce flexibility in the helix that may result in kink and swivel motions of FNR-binding region. Localization in non-appressed regions places TROL in the vicinity of the site of Fd

reduction. TROL has been found in several complexes, indicating the presence of several TROL subpools in the thylakoid membrane. Only a 190 kDa complex appears to contain TROL in association with the FNR. Complexes at about 110 and 120 kDa indicate the existence of a small ligand which may be associated with other TROL domains, namely the large rhodanese-like domain which is predicted to be located in the thylakoid lumen.

The findings of Küchler et al. (2002) that Tic62 interacts with FNR prompted the analysis of TROL protein sequence in search for the similar binding module. Tic62 from *Pisum sativum*  (PsTic62) contains three Pro/Ser-rich repetitive motives at the C-terminus, S-P-Y-x(2)-Y-x-D/E-L-K-P(2)-S/T/A-S/T-P-S/T-P, involved in the binding of FNR (Küchler et al., 2002). PsTic62 homolog in *A. thaliana*, encoded by a single-copy gene (*At3g18890*), shows approximately 60% identity for the deduced mature sequence and has a calculated molecular weight of 62.1 kDa (Küchler et al., 2002). AtTic62 contains four repetitive motives at the C-terminus, but it has been shown previously that only one repeat is sufficient for the binding of FNR (Küchler et al., 2002; Balsera et al., 2007). As TROL possesses almost identical domain to the Tic62 FNR-binding repeats, a modified yeast-two hybrid assay was used to confirm ITEP-FNR interaction (Jurić et al., 2009). Predictably, ITEP strongly binds to

Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents 183

light conditions, TROL anchors FNR and stabilises it, similar to association between Tic62 and FNR (Benz et al., 2009). FNR could be gradually released by binding/releasing of certain signalling molecules to the luminal RHO domain, and electrons are transferred from ferredoxin to NADP+ at normal rates (Figure 1). Under the high-light/excess-light intensities TROL discharges FNR, possibly because of binding/releasing of certain signalling molecules to the luminal RHO domain, and FNR now catalyses the reverse reaction of transferring electrons from excess NADPH to potential electron-acceptor molecules (Figure 1) (Jurić et al., 2010). The PEPE swivel that preceeds the ITEP domain could maneuver the bound FNR protein due to the proline-introduced flexibility. For instance, the free-moving PEPE swivel could move FNR closer to the thylakoids to establish transient contacts with other transmembrane proteins, or it could move FNR away from the thylakoids. In addition, if the binding of FNR to the thylakoids is a precondition for efficient LEF (Forti & Bracale, 1984), then the TROL-bound FNR molecules could be easily displaced from TROL to the already discovered FNR-binding membrane proteins or the unknown ones. This is in accordance with the clearly visible TROL-FNR complex under dark

conditions and with its disappearance during light periods (Benz et al., 2009).

**4. FNR, TROL and Tic62** *Arabidopsis* **mutants** 

(Rodriguez et al., 2007).

The most interesting property of the FNR-Tic62/TROL interaction is the clear difference of the affinity at acidic (pH 6; KD ≈ 0.04 μM) compared to alkaline (pH 8; KD ≈ 3 μM) conditions (Alte et al., 2010). The pH variations reflect differences of the chloroplast stroma between light and dark cycles (Alte et al., 2010): During light phases, when photosynthetic activity is high, protons are transported into the thylakoid lumen, leading to an alkaline stromal pH. By contrast, when photosynthesis ceases during dark phases the stromal pH decreases again. Under these conditions, Tic62 and TROL are predominantly associated with the thylakoid membrane where they recruit FNR into stable high-molecular-weight complexes (Benz et al., 2009; Jurić et al., 2009). Light quantity can vary dramatically during the course of the day, therefore requiring constant adjustment of the light harvesting processes and the enzymatic reactions. Changes in light quantities alter stromal pH as well as the amount of FNR bound to the thylakoid membranes. Also, the membrane attachment of FNR is influenced by the stromal redox state (NADP+NADPH ratio), which mimics variations in environmental conditions (Stengel et al., 2008). Therefore, reversible attachment of FNR to the thylakoid membrane via Tic62/TROL provides an elegant way to store redundant molecules, not required when photosynthesis is less active or dormant.

When the amount of FNR was artificially reduced by antisense or silencing techniques (Hajirezaei et al., 2002; Lintala et al., 2009), or by interruption of a *FNR* gene by T-DNA (Lintala et al., 2007; Hanke & Hase, 2008), the plants suffered from chlorosis and reduced photosynthetic activity, which finally resulted in reduced growth. Although the level of total NADP(H) was not affected in the mutants, the NADPH/NADP+ ratio was strongly reduced (Hajirezaei et al., 2002; Hald et al., 2008). These mutants are prone to photooxidative damage, and suffer from oxidative stress (Palatnik et al., 2003; Lintala et al., 2009). The redox poise of the NADP(H) pool is also likely to regulate photosynthetic electron transfer activity in order to balance production and consumption of reducing equivalents, and thereby to limit production of ROS in the chloroplasts (Hald et al., 2008). Overexpression of FNR, however, did not markedly up-regulate the rate of NADP+ photoreduction or CO2 assimilation, but showed increased tolerance to photodamage

the FNR protein, even eight times stronger than Tic62. Established high-affinity interaction with FNR, together with the reports on TROL abundance at the thylakoid membranes (Peltier et al., 2004), implies that we are probably not dealing with highly dynamical and fast-responding interaction, but with more quantitative-based and rather inert interaction. The size of the complex is, however, smaller (190 kDa) than the Tic62–FNR complexes (250- 500 kDa), which is in line with only one FNR binding motif found in the amino acid sequence of *Arabidopsis* TROL protein, as compared to four of such motifs present in the sequence of *Arabidopsis* Tic62. Although *in vitro* experiments indicate that the interaction between TROL and FNR is stronger than the interaction between FNR and Tic62 (Jurić et al., 2009), the exposure of plants to high light intensity results in faster dissociation of FNR from the TROL–FNR complexes than from the Tic62–FNR complexes (Jurić et al., 2009).

Using a synthetic peptide, representing the conserved binding motif, called the FNRmembrane-recruiting-motif (FNR-MRM) found in Tic62 and TROL, Alte et al. (2010) determined the crystal structure of the FNR:peptide complex and concluded that the FNR-MRM induces self-assembly of two FNR molecules. Although FNR is commonly distributed among all three domains of life, FNR-MRM of both Tic62 and TROL exclusively exists in vascular plants, thus, membrane tethering of FNR by this motif seems to be a recent evolutionary invention (Alte et al., 2010). Whereas most TROL proteins comprise a single FNR-binding domain, its number varies to a higher extent in Tic62 proteins. However, the binding affinity to FNR did not change significantly when constructs comprising one or three FNR-interacting motifs were analyzed (Alte et al., 2010). This indicates that binding to each domain occurs independently of the other motifs and excludes cooperative binding effects.

#### **3.2.1 Redox regulation and tethering properties of TROL**

Besides being involved in sulfur metabolism, rhodanese-like domains are implicated in redox regulation of various intracellular processes (Horowitz & Falksen, 1986; Horowitz et al., 1992; Nandi et al., 2000). It has been speculated that the rhodanese-like domain of TROL is involved in redox regulation of FNR binding and release (Jurić et al., 2009). It has been proposed that the redox regulation of FNR binding and release could be important for balancing the redox status of stroma with the membrane electron transfer chain. Such regulation could be important for prevention of over-reduction of any of these two compartments and maintenance of the redox poise (Jurić et al., 2009).

TROL, as FNR, is mainly located at the stroma thylakoids, but it can be also found embedded in the chloroplast inner envelope membrane in the non-processed form (70 kDa). Localization of the TROL precursor at the chloroplast envelope in its unprocessed form indicates its possible role in electron transfer chain specific for this membrane. This dual localization might also be dependent on the NADP+/NADPH ratio in the chloroplasts, similar to the shuttling of the Tic62 protein (Stengel et al., 2008).

FNR is supposed to be the key protein in transferring electrons to the final destination in LEF and *trol* plants indeed exhibit decreased LEF. On the basis of the investigation of membrane-bound pool of FNR and the 190 kDa-complex containing TROL and FNR in the wild-type plants grown under growth-light and high-light intensities (Jurić, 2010), a role for TROL as the FNR anchor could be proposed. TROL anchors and thus stabilises FNR during the night, possibly to prevent FNR from extracting electrons from NADPH molecules and compromising the downstream metabolic reactions. During the light period, under growth-

the FNR protein, even eight times stronger than Tic62. Established high-affinity interaction with FNR, together with the reports on TROL abundance at the thylakoid membranes (Peltier et al., 2004), implies that we are probably not dealing with highly dynamical and fast-responding interaction, but with more quantitative-based and rather inert interaction. The size of the complex is, however, smaller (190 kDa) than the Tic62–FNR complexes (250- 500 kDa), which is in line with only one FNR binding motif found in the amino acid sequence of *Arabidopsis* TROL protein, as compared to four of such motifs present in the sequence of *Arabidopsis* Tic62. Although *in vitro* experiments indicate that the interaction between TROL and FNR is stronger than the interaction between FNR and Tic62 (Jurić et al., 2009), the exposure of plants to high light intensity results in faster dissociation of FNR from

the TROL–FNR complexes than from the Tic62–FNR complexes (Jurić et al., 2009).

**3.2.1 Redox regulation and tethering properties of TROL** 

compartments and maintenance of the redox poise (Jurić et al., 2009).

similar to the shuttling of the Tic62 protein (Stengel et al., 2008).

effects.

Using a synthetic peptide, representing the conserved binding motif, called the FNRmembrane-recruiting-motif (FNR-MRM) found in Tic62 and TROL, Alte et al. (2010) determined the crystal structure of the FNR:peptide complex and concluded that the FNR-MRM induces self-assembly of two FNR molecules. Although FNR is commonly distributed among all three domains of life, FNR-MRM of both Tic62 and TROL exclusively exists in vascular plants, thus, membrane tethering of FNR by this motif seems to be a recent evolutionary invention (Alte et al., 2010). Whereas most TROL proteins comprise a single FNR-binding domain, its number varies to a higher extent in Tic62 proteins. However, the binding affinity to FNR did not change significantly when constructs comprising one or three FNR-interacting motifs were analyzed (Alte et al., 2010). This indicates that binding to each domain occurs independently of the other motifs and excludes cooperative binding

Besides being involved in sulfur metabolism, rhodanese-like domains are implicated in redox regulation of various intracellular processes (Horowitz & Falksen, 1986; Horowitz et al., 1992; Nandi et al., 2000). It has been speculated that the rhodanese-like domain of TROL is involved in redox regulation of FNR binding and release (Jurić et al., 2009). It has been proposed that the redox regulation of FNR binding and release could be important for balancing the redox status of stroma with the membrane electron transfer chain. Such regulation could be important for prevention of over-reduction of any of these two

TROL, as FNR, is mainly located at the stroma thylakoids, but it can be also found embedded in the chloroplast inner envelope membrane in the non-processed form (70 kDa). Localization of the TROL precursor at the chloroplast envelope in its unprocessed form indicates its possible role in electron transfer chain specific for this membrane. This dual localization might also be dependent on the NADP+/NADPH ratio in the chloroplasts,

FNR is supposed to be the key protein in transferring electrons to the final destination in LEF and *trol* plants indeed exhibit decreased LEF. On the basis of the investigation of membrane-bound pool of FNR and the 190 kDa-complex containing TROL and FNR in the wild-type plants grown under growth-light and high-light intensities (Jurić, 2010), a role for TROL as the FNR anchor could be proposed. TROL anchors and thus stabilises FNR during the night, possibly to prevent FNR from extracting electrons from NADPH molecules and compromising the downstream metabolic reactions. During the light period, under growthlight conditions, TROL anchors FNR and stabilises it, similar to association between Tic62 and FNR (Benz et al., 2009). FNR could be gradually released by binding/releasing of certain signalling molecules to the luminal RHO domain, and electrons are transferred from ferredoxin to NADP+ at normal rates (Figure 1). Under the high-light/excess-light intensities TROL discharges FNR, possibly because of binding/releasing of certain signalling molecules to the luminal RHO domain, and FNR now catalyses the reverse reaction of transferring electrons from excess NADPH to potential electron-acceptor molecules (Figure 1) (Jurić et al., 2010). The PEPE swivel that preceeds the ITEP domain could maneuver the bound FNR protein due to the proline-introduced flexibility. For instance, the free-moving PEPE swivel could move FNR closer to the thylakoids to establish transient contacts with other transmembrane proteins, or it could move FNR away from the thylakoids. In addition, if the binding of FNR to the thylakoids is a precondition for efficient LEF (Forti & Bracale, 1984), then the TROL-bound FNR molecules could be easily displaced from TROL to the already discovered FNR-binding membrane proteins or the unknown ones. This is in accordance with the clearly visible TROL-FNR complex under dark conditions and with its disappearance during light periods (Benz et al., 2009).

The most interesting property of the FNR-Tic62/TROL interaction is the clear difference of the affinity at acidic (pH 6; KD ≈ 0.04 μM) compared to alkaline (pH 8; KD ≈ 3 μM) conditions (Alte et al., 2010). The pH variations reflect differences of the chloroplast stroma between light and dark cycles (Alte et al., 2010): During light phases, when photosynthetic activity is high, protons are transported into the thylakoid lumen, leading to an alkaline stromal pH. By contrast, when photosynthesis ceases during dark phases the stromal pH decreases again. Under these conditions, Tic62 and TROL are predominantly associated with the thylakoid membrane where they recruit FNR into stable high-molecular-weight complexes (Benz et al., 2009; Jurić et al., 2009). Light quantity can vary dramatically during the course of the day, therefore requiring constant adjustment of the light harvesting processes and the enzymatic reactions. Changes in light quantities alter stromal pH as well as the amount of FNR bound to the thylakoid membranes. Also, the membrane attachment of FNR is influenced by the stromal redox state (NADP+NADPH ratio), which mimics variations in environmental conditions (Stengel et al., 2008). Therefore, reversible attachment of FNR to the thylakoid membrane via Tic62/TROL provides an elegant way to store redundant molecules, not required when photosynthesis is less active or dormant.

#### **4. FNR, TROL and Tic62** *Arabidopsis* **mutants**

When the amount of FNR was artificially reduced by antisense or silencing techniques (Hajirezaei et al., 2002; Lintala et al., 2009), or by interruption of a *FNR* gene by T-DNA (Lintala et al., 2007; Hanke & Hase, 2008), the plants suffered from chlorosis and reduced photosynthetic activity, which finally resulted in reduced growth. Although the level of total NADP(H) was not affected in the mutants, the NADPH/NADP+ ratio was strongly reduced (Hajirezaei et al., 2002; Hald et al., 2008). These mutants are prone to photooxidative damage, and suffer from oxidative stress (Palatnik et al., 2003; Lintala et al., 2009). The redox poise of the NADP(H) pool is also likely to regulate photosynthetic electron transfer activity in order to balance production and consumption of reducing equivalents, and thereby to limit production of ROS in the chloroplasts (Hald et al., 2008). Overexpression of FNR, however, did not markedly up-regulate the rate of NADP+ photoreduction or CO2 assimilation, but showed increased tolerance to photodamage (Rodriguez et al., 2007).

Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents 185

molecule, possibly reduced PQ, competes for the RHO binding site and generates the signal for the FNR release, which, when soluble, acts as NADPH consumer. Protons are passed to

Overexpression of FNR in transgenic plants causes enhanced tolerance to photo-oxidative damage and herbicides that propagate reactive oxygen species (Rodriguez et al., 2007). On the other hand, antisense repression of FNR renders transgenic plants abnormally prone to

Analysis of *Arabidopsis* mutant lines indicates that, in the absence of TROL, relative electron transport rates at high-light intensities are severely lowered accompanied with significant increase in non-photochemical quenching (NPQ). If solubilization of FNR is necessary for the regulation of oxidative stress, then it is not surprising that, under high-light conditions, TROL-deficient plants exhibit increased rates of NPQ. This effect was also explained by recently proposed feedback redox regulation via the redox poise of the NADP(H) pool (Hald et al., 2008). Thus, TROL might represent a missing thylakoid membrane docking site for a complex between FNR, ferredoxin and NADP+. Such association might be necessary for maintaining photosynthetic redox poise and enhancement of the NPQ (Jurić et al., 2009). Inhibition of TROL accumulation by antisense expression results in quenching which is higher than that of the wild-type plants, but lower than that of the TROL knock-out plants (Jurić et al., 2009). This demonstrates dosage effect of TROL and indicates that FNR binding to the thylakoid membranes is dependent on the availability of tethering sites and that the

It has been proposed that the balance between NADP+ and NADPH regulates the photosynthetic electron transport at the level of cyt *b*6/*f* complex in a feedback manner (Hald et al., 2008). Interestingly, NADP-malic enzyme 2 that catalyses the oxidative decarboxylation of malate, producing pyruvate, carbon dioxide and NAD(P)H in cytosol (Wheeler et al., 2005) was significantly up-regulated in TROL-deficient plants grown under growth-light conditions, thus providing the possible pathway of maintaining NADP+/NADPH balance through the malate valve (Jurić et al., 2009). In this case, *trol* plants would act as efficient NADPH producers, and, in an effort not to hyperreduce the

thylakoids, they would export the reducing energy in a form of malate to the cytosol. Also, in *trol* plants, genes encoding proteins involved in stress management are strongly upregulated. As plant growth and development are driven by electron transfer reactions (Noctor, 2006), it is not surprising that leaf anatomy is altered in the knock-out. Furthermore, chloroplasts in the knock-out are small and have less developed thylakoids. These morphological changes reflect alterations in gene expression of a specific set of genes encoding chloroplast proteins. Many processes important for chloroplast morphogenesis could be influenced by NADPH production, or be dependent on metabolic retrograde

When Tic62 was knocked out, formation of high molecular weight FNR protein complexes was hindered, while some free FNR still was detected at the thylakoids of *tic62* plants. The amount of FNR in the soluble pool, however, remained more or less constant. *Vice versa*, if either one of the chloroplast targeted FNR isoforms was missing the membrane binding of the Tic62 protein was prevented. Since no changes in the *FNR* gene expression or in the FNR

NADPH production. In conditions of growth-light, FNR is bound to the thylakoids *via* TROL and acts as an efficient NADPH producer. In conditions of saturating light, a

an unknown scavenger A.

photo-oxidative stress (Palatnik et al., 2003).

amount of soluble FNR directly influences NPQ.

signaling (Jurić et al., 2009).

Fig. 1. The proposed mechanism of redox regulation of FNR binding and release. During the dark period, FNR is bound to the thylakoids *via* TROL. This stage could be sustained through the binding of small molecule, possibly oxidized PQ, to the RHO cavity. There is no

Fig. 1. The proposed mechanism of redox regulation of FNR binding and release. During the

dark period, FNR is bound to the thylakoids *via* TROL. This stage could be sustained through the binding of small molecule, possibly oxidized PQ, to the RHO cavity. There is no NADPH production. In conditions of growth-light, FNR is bound to the thylakoids *via* TROL and acts as an efficient NADPH producer. In conditions of saturating light, a molecule, possibly reduced PQ, competes for the RHO binding site and generates the signal for the FNR release, which, when soluble, acts as NADPH consumer. Protons are passed to an unknown scavenger A.

Overexpression of FNR in transgenic plants causes enhanced tolerance to photo-oxidative damage and herbicides that propagate reactive oxygen species (Rodriguez et al., 2007). On the other hand, antisense repression of FNR renders transgenic plants abnormally prone to photo-oxidative stress (Palatnik et al., 2003).

Analysis of *Arabidopsis* mutant lines indicates that, in the absence of TROL, relative electron transport rates at high-light intensities are severely lowered accompanied with significant increase in non-photochemical quenching (NPQ). If solubilization of FNR is necessary for the regulation of oxidative stress, then it is not surprising that, under high-light conditions, TROL-deficient plants exhibit increased rates of NPQ. This effect was also explained by recently proposed feedback redox regulation via the redox poise of the NADP(H) pool (Hald et al., 2008). Thus, TROL might represent a missing thylakoid membrane docking site for a complex between FNR, ferredoxin and NADP+. Such association might be necessary for maintaining photosynthetic redox poise and enhancement of the NPQ (Jurić et al., 2009).

Inhibition of TROL accumulation by antisense expression results in quenching which is higher than that of the wild-type plants, but lower than that of the TROL knock-out plants (Jurić et al., 2009). This demonstrates dosage effect of TROL and indicates that FNR binding to the thylakoid membranes is dependent on the availability of tethering sites and that the amount of soluble FNR directly influences NPQ.

It has been proposed that the balance between NADP+ and NADPH regulates the photosynthetic electron transport at the level of cyt *b*6/*f* complex in a feedback manner (Hald et al., 2008). Interestingly, NADP-malic enzyme 2 that catalyses the oxidative decarboxylation of malate, producing pyruvate, carbon dioxide and NAD(P)H in cytosol (Wheeler et al., 2005) was significantly up-regulated in TROL-deficient plants grown under growth-light conditions, thus providing the possible pathway of maintaining NADP+/NADPH balance through the malate valve (Jurić et al., 2009). In this case, *trol* plants would act as efficient NADPH producers, and, in an effort not to hyperreduce the thylakoids, they would export the reducing energy in a form of malate to the cytosol.

Also, in *trol* plants, genes encoding proteins involved in stress management are strongly upregulated. As plant growth and development are driven by electron transfer reactions (Noctor, 2006), it is not surprising that leaf anatomy is altered in the knock-out. Furthermore, chloroplasts in the knock-out are small and have less developed thylakoids. These morphological changes reflect alterations in gene expression of a specific set of genes encoding chloroplast proteins. Many processes important for chloroplast morphogenesis could be influenced by NADPH production, or be dependent on metabolic retrograde signaling (Jurić et al., 2009).

When Tic62 was knocked out, formation of high molecular weight FNR protein complexes was hindered, while some free FNR still was detected at the thylakoids of *tic62* plants. The amount of FNR in the soluble pool, however, remained more or less constant. *Vice versa*, if either one of the chloroplast targeted FNR isoforms was missing the membrane binding of the Tic62 protein was prevented. Since no changes in the *FNR* gene expression or in the FNR

Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents 187

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**6. Aknowledgement** 

**7. References** 

Science, Education and Sports of the Republic of Croatia.

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4148–4157.

pre-protein import could be detected in the *tic62* plants, reduction of FNR level most probably resulted from differences in the turnover of FNR isoforms inside the chloroplasts (Benz et al., 2009).

In contrast to *tic62* plants, *trol* knock-out mutants have a clear photosynthetic phenotype (Jurić et al., 2009). The appearance of the *trol* plants is slightly smaller than the WT, but with no distinct differences in pigment composition. However, the mutant chloroplasts are small, irregular in morphology and show deteriorated thylakoid structure. The abnormalities in chloroplast structure are reflected on the function with marked differences in electron transfer rate under high light intensity (500 to 800 μmol photons m−2 s−1). Since nonphotochemical quenching increases and variable chlorophyll fluorescence decreases in the *trol* leaves upon increasing illumination, it seems that the absence of TROL results in increased ability to dissipate excess absorbed energy.

The absence of TROL disables FNR from being tethered to the membrane, therefore a substantial amount of FNR remains soluble. Forti & Bracale (1984) demonstrated that the soluble form of FNR is very inefficient in NADP+ photoreduction by isolated thylakoids. It has been shown in TROL-deficient plants that linear photosynthetic flow can be sustained until light intensity exceeds 250 µmol photons m−2 s−1. As soluble FNR is no longer able to reduce NADP+ at high rates, this could lead to over-reduction of the entire electron transport chain. In this case, NPQ modulation could be particularly important to prevent photo-damage caused by build-up of reduced electron carriers which block LEF before the lumen could be significantly acidified (Kanazawa & Kramer, 2002). In conclusion, TROL is necessary for maintenance of efficient LEF, induction of NPQ, as well as for redoxregulation of key thylakoid signal-transduction pathways. Furthermore, discovery of TROL provides new information for linking leaf and chloroplast morphogenesis with photosynthetic cues.

#### **5. Conclusion**

The discovery of TROL protein and its important role in tethering of the flavoenzyme FNR not only answers the old dilemma about the position at which this crucial photosynthetic enzyme is located, but opens new approaches for the investigations of oxidative stress management in chloroplasts. FNR in bacteria acts as an important scavenger of free radicals and it will be interesting to see if it possesses similar function in plant cells. Linking FNR with cellular energetics and possibly with retrograde signaling will also be investigated. Is TROL the source element in signal-transduction cascade linking photosynthesis with plant growth and cellular responses? TROL possesses several elaborate elements of signal transduction, namely rhodanese-like domain located in lumen, proline-rich swivel involved in signal attenuation, and FNR membrane recruitment motif. Functions of each of these domains will be investigated *in planta*, by using genetic transformation techniques. Global gene expression analysis revealed that genes depending on NADPH synthesis and availability are up-regulated in TROL deficient plants. Among them are NADP-malic enzyme and protochlorophyllide oxidoreductase B. How is TROL linked with malate shuttle enzymes? What is the role of TROL and Tic62 in the inner envelope membrane? Finally, other proteins following the same expression pattern in correlation analyses will be investigated for their ability to interact with TROL and Tic62. Supramolecular complexes of TROL and membrane yeast-two-hybrid screens will likely reveal so far overlooked elements of thylakoid signal transduction. An exciting quest for auxiliary proteins involved in finetuning of photosynthetic energy conversion lies ahead.

#### **6. Aknowledgement**

The work of H.F. and L.V. has been funded by the Grant 098-0982913-2838 of the Ministry of Science, Education and Sports of the Republic of Croatia.

#### **7. References**

186 Advances in Photosynthesis – Fundamental Aspects

pre-protein import could be detected in the *tic62* plants, reduction of FNR level most probably resulted from differences in the turnover of FNR isoforms inside the chloroplasts

In contrast to *tic62* plants, *trol* knock-out mutants have a clear photosynthetic phenotype (Jurić et al., 2009). The appearance of the *trol* plants is slightly smaller than the WT, but with no distinct differences in pigment composition. However, the mutant chloroplasts are small, irregular in morphology and show deteriorated thylakoid structure. The abnormalities in chloroplast structure are reflected on the function with marked differences in electron transfer rate under high light intensity (500 to 800 μmol photons m−2 s−1). Since nonphotochemical quenching increases and variable chlorophyll fluorescence decreases in the *trol* leaves upon increasing illumination, it seems that the absence of TROL results in

The absence of TROL disables FNR from being tethered to the membrane, therefore a substantial amount of FNR remains soluble. Forti & Bracale (1984) demonstrated that the soluble form of FNR is very inefficient in NADP+ photoreduction by isolated thylakoids. It has been shown in TROL-deficient plants that linear photosynthetic flow can be sustained until light intensity exceeds 250 µmol photons m−2 s−1. As soluble FNR is no longer able to reduce NADP+ at high rates, this could lead to over-reduction of the entire electron transport chain. In this case, NPQ modulation could be particularly important to prevent photo-damage caused by build-up of reduced electron carriers which block LEF before the lumen could be significantly acidified (Kanazawa & Kramer, 2002). In conclusion, TROL is necessary for maintenance of efficient LEF, induction of NPQ, as well as for redoxregulation of key thylakoid signal-transduction pathways. Furthermore, discovery of TROL provides new information for linking leaf and chloroplast morphogenesis with

The discovery of TROL protein and its important role in tethering of the flavoenzyme FNR not only answers the old dilemma about the position at which this crucial photosynthetic enzyme is located, but opens new approaches for the investigations of oxidative stress management in chloroplasts. FNR in bacteria acts as an important scavenger of free radicals and it will be interesting to see if it possesses similar function in plant cells. Linking FNR with cellular energetics and possibly with retrograde signaling will also be investigated. Is TROL the source element in signal-transduction cascade linking photosynthesis with plant growth and cellular responses? TROL possesses several elaborate elements of signal transduction, namely rhodanese-like domain located in lumen, proline-rich swivel involved in signal attenuation, and FNR membrane recruitment motif. Functions of each of these domains will be investigated *in planta*, by using genetic transformation techniques. Global gene expression analysis revealed that genes depending on NADPH synthesis and availability are up-regulated in TROL deficient plants. Among them are NADP-malic enzyme and protochlorophyllide oxidoreductase B. How is TROL linked with malate shuttle enzymes? What is the role of TROL and Tic62 in the inner envelope membrane? Finally, other proteins following the same expression pattern in correlation analyses will be investigated for their ability to interact with TROL and Tic62. Supramolecular complexes of TROL and membrane yeast-two-hybrid screens will likely reveal so far overlooked elements of thylakoid signal transduction. An exciting quest for auxiliary proteins involved in fine-

(Benz et al., 2009).

photosynthetic cues.

**5. Conclusion** 

increased ability to dissipate excess absorbed energy.

tuning of photosynthetic energy conversion lies ahead.


Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents 189

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**10** 

*Argentina* 

**The Photomorphogenic Signal: An Essential** 

In a thermosolar plant, the engineers locate movable mirrors that concentrate solar radiation. These plants are designed to maximize energy capture. In green plants, their morphology changes to maximize energy capture as well, but also to avoid light in excess, which can damage plant tissues. Contrary to mirrors in thermosolar plants, located in desert land and organized in regular arrays, most green plants grow surrounded of vegetation and their own tissues are not regularly spaced, new leaves tend to shade older leaves. Hence, plants need to use light as a source of information in order to properly locate their "sunlight

collectors" and be able to efficiently use light as an energy source for photosynthesis.

shade avoidance, photoperiodic responses and senescence.

and the activity of the ROS scavenging system.

To monitor environmental light conditions, plants are equipped with several families of photoreceptors: the cryptochromes, the phytochromes and the LOV-domain bearing photoreceptors. While phytochromes perceive light most effectively in the red/far-red region of the spectrum, cryptochromes and LOV photoreceptors detect blue and UV-A light. Downstream these photoreceptors, plants have evolved sophisticated transcriptional networks that mediate metabolic and developmental changes in response to light. These light-regulated processes include seed germination, seedling photomophogenesis, greening,

Greening and chloroplast biogenesis are promoted after light exposure. Phytochromes and cryptochromes trigger to initiate this biogenesis, which includes the induction of photosynthesis-related genes at the transcriptional level, the import of nuclear-encoded proteins and the establishment of a thylakoid network fully assembled with photosynthetic electron transport complexes. Furthermore, these photoreceptors affect the synthesis of chlorophyll and other photosynthetic accessory pigments; modifying the photosynthetic apparatus properties as a result of light quality perception. On the other hand, phytochromes are also involved in the induction of Rubisco, a key enzyme of the Calvin Cycle. Light quality plays an important role in modulating the photosynthetic characteristics. It regulates chlorophyll degradation, modulates photosystem stoichiometry

Besides the role in the formation of the photosynthetic apparatus, the photoreceptors play significant roles in establishing how the photosynthetic pigments will be exposed to light to harvest its energy content. Under weak light, chloroplasts move towards light, in a blue

**1. Introduction** 

**Component of Photoautotrophic Life** 

*Fundación Instituto Leloir, IIBBA-CONICET and FCEN-UBA* 

Francisco M. Iglesias and Pablo D. Cerdán

Sabrina Iñigo, Mariana R. Barber, Maximiliano Sánchez-Lamas,

location, expression, and interaction with ferredoxin. *Plant Physiology*, Vol. 139, No. 3, pp. 1451–1459.


### **The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life**

Sabrina Iñigo, Mariana R. Barber, Maximiliano Sánchez-Lamas, Francisco M. Iglesias and Pablo D. Cerdán *Fundación Instituto Leloir, IIBBA-CONICET and FCEN-UBA Argentina* 

#### **1. Introduction**

190 Advances in Photosynthesis – Fundamental Aspects

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& Bosshard, H. R. (1993). Binding of ferredoxin to ferredoxin:NADP+ oxidoreductase: the role of carboxyl groups, electrostatic surface potential, and

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subunit of the chloroplast cytochrome b6f complex. *Journal of Biological Chemistry*,

Characterization of the high-spin heme x in the cytochrome b6f complex of

In a thermosolar plant, the engineers locate movable mirrors that concentrate solar radiation. These plants are designed to maximize energy capture. In green plants, their morphology changes to maximize energy capture as well, but also to avoid light in excess, which can damage plant tissues. Contrary to mirrors in thermosolar plants, located in desert land and organized in regular arrays, most green plants grow surrounded of vegetation and their own tissues are not regularly spaced, new leaves tend to shade older leaves. Hence, plants need to use light as a source of information in order to properly locate their "sunlight collectors" and be able to efficiently use light as an energy source for photosynthesis.

To monitor environmental light conditions, plants are equipped with several families of photoreceptors: the cryptochromes, the phytochromes and the LOV-domain bearing photoreceptors. While phytochromes perceive light most effectively in the red/far-red region of the spectrum, cryptochromes and LOV photoreceptors detect blue and UV-A light. Downstream these photoreceptors, plants have evolved sophisticated transcriptional networks that mediate metabolic and developmental changes in response to light. These light-regulated processes include seed germination, seedling photomophogenesis, greening, shade avoidance, photoperiodic responses and senescence.

Greening and chloroplast biogenesis are promoted after light exposure. Phytochromes and cryptochromes trigger to initiate this biogenesis, which includes the induction of photosynthesis-related genes at the transcriptional level, the import of nuclear-encoded proteins and the establishment of a thylakoid network fully assembled with photosynthetic electron transport complexes. Furthermore, these photoreceptors affect the synthesis of chlorophyll and other photosynthetic accessory pigments; modifying the photosynthetic apparatus properties as a result of light quality perception. On the other hand, phytochromes are also involved in the induction of Rubisco, a key enzyme of the Calvin Cycle. Light quality plays an important role in modulating the photosynthetic characteristics. It regulates chlorophyll degradation, modulates photosystem stoichiometry and the activity of the ROS scavenging system.

Besides the role in the formation of the photosynthetic apparatus, the photoreceptors play significant roles in establishing how the photosynthetic pigments will be exposed to light to harvest its energy content. Under weak light, chloroplasts move towards light, in a blue

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life 193

non-covalently to the apoprotein (Moglich *et al.*, 2010). As explained above, plants possess several classes of photoreceptors whose absorption properties match the spectrum of the incoming light: the red/far-red photoreversible phytochromes, the UV-A/blue-light absorbing cryptochromes, the phototropins, the members of the Zeitlupe family (Moglich *et al.*, 2010) and, more recently, a UV-B specific photoreceptor, UVR8, has been added to the

Phytochromes are the only red and far-red light photoreceptors in plants (Strasser *et al.*, 2010; Takano *et al.*, 2009) and, together with cryptochromes and phototropins, constitute one of the three mayor regulators of photomorphogenesis (Rockwell *et al.*, 2006). Phytochromes are synthesised in the cytosol as soluble dimers composed of two 125-kDa polypeptides. Each polypeptide folds into two main domains. The amino-terminal domain covalently binds phytochromobilin (PфB), a tetrapyrrole chromophore that confers the spectral properties characteristic of phytochromes. PфB is synthesised from haeme in plastids, haeme oxygenase encoded by *HY1* converts haeme into biliverdin IX, which is reduced to 3Z-PфB by the PфB synthase (*HY2*). Then 3Z-PфB isomerises to 3E-PфB and attaches

The carboxy-terminal part of the phytochrome molecule is involved in dimerisation and transfer of the signal to downstream components (Rockwell *et al.*, 2006). Phytochromes are synthesised in the dark in a biologically inactive red-light absorbing form (known as Pr). Biological activity is acquired upon red-light triggered photoconversion to the far-red light absorbing form (known as Pfr). Photoconversion of Pfr back to Pr is triggered by far-red light. Both reactions are fully reversible, and eventually results in a dynamic photoequilibrium of Pr and Pfr in natural light conditions that depends on the proportion of red to far-red light (Franklin & Quail, 2009). The conversion is due to a single photochemical isomerisation of the chromophore about a specific double bond between the rings C and D of the phytochromobilin (Rockwell *et al.*, 2006). Following conversion, Pfr translocates into

The phytochromes are encoded by a small gene family in angiosperms. The rice genome, for example, encodes three members, phyA, phyB and phyC, each representing one of the lineages found in plants (Sharrock, 2008). In Arabidopsis, the phytochrome family consists

Classical photobiological experiments established three phytochromes modes of acting, the Very Low Fluence Response mode (VLFR), where responses to phytochromes are already saturated at very low fluencies of light, the Low Fluence Response (LFR) showing the classical red and far-red light reversibility and the High Irradiance responses (HIR) that require prolonged exposures to light of relative high intensity (Casal *et al.*, 2003). Now that we know about each phytochrome species, the phytochrome action modes can be explained by the different phytochrome species and different signal transduction pathways. phyA is the most specialized of the phytochromes; it is responsible for the VLFR and the HIR. The extraordinary sensitivity of this photoreceptor to light allows phyA to control germination of buried seeds in the soil and to induce germination when seeds are exposed briefly to light. phyA importance is evident when plants germinate under a dense canopy (Yanovsky, 1995) or for example, when weeds are induced to germinate after soil tillage (Ballaré, 1992). The other phytochromes control the R/FR reversible LFR and the responses to continuous

of five members, designated phytochrome A (phyA) to phytochrome E (phyE).

list (Rizzini *et al.*, 2011).

**2.2.1 Generalities about phytochromes** 

covalently to phytochrome (Tanaka & Tanaka, 2007).

the nucleus (Fankhauser & Chen, 2008).

**2.2 Phytochromes** 

light dependent way, to optimize the light absorption and photosynthesis. However, under strong light, chloroplasts show an opposite response to avoid photodamage (Kodama *et al.*, 2011). Besides chloroplast movement, photoreceptors modulate plant architecture to maximize the photosynthetic surface exposed to light. When plants perceive the presence of plant neighbours, phytochromes trigger the elongation of the stem and petioles, a series of changes known as Shade Avoidance Syndrome (SAS). The manipulation of phytochrome levels has been used to improve the harvest index of tobacco plants (Robson *et al.*, 1996) by avoiding the diversion of resources to the SAS. However, the phytochromes are still very important to position the leaves in the canopy. Thus, manipulation of phytochrome activity must be precise, if used to improve crop performance (Maddonni *et al.*, 2002).

In this chapter, we focus on the role of the photomorphogenic signal to trigger the synthesis of photosynthetic genes and pigments during the greening process and later on, during photosynthetic plant development, with emphasis on the regulation of gene expression.

#### **2. Photomorphogenesis**

Plants are sessile organisms, and as such, have evolved a great deal of developmental plasticity to optimally respond to the immediate environment. Light is one of the most important cues for plant growth; plants respond to its intensity, wavelength, direction and periodicity (Franklin & Quail, 2009). The first physiological consequence of light perception is the reprogramming of the seedling development in a process termed deetiolation, or photomorphogenesis. In darkness, seedlings display a skotomorphogenesis development characterised by the following phenotypes: elongated hypocotyl, closed, pale and unexpanded cotyledons; the apical hook remains closed to protect the apical meristem before emerging from the soil and chlorophyll and anthocyanin biosynthesis do not take place. All these features allow the seedlings to grow through a layer of soil and eventually emerge into the light. Once the seedlings perceive sufficient light, they exhibit a photomorphogenic development. They undergo deetiolation that includes inhibition of hypocotyl elongation, unfolding and greening of cotyledons, opening of the apical hook, chlorophyll and anthocyanin biosynthesis and differentiation of chloroplasts; processes aimed to achieve full autotrophy. This transition from skotomorphogenic to photomorphogenic development is steered by a complex molecular network that includes upstream signalling components (photoreceptors) and intermediate factors transducing the signal to downstream regulators. These downstream components integrate the light signals from the various photoreceptors and bring about changes in metabolism and gene expression that eventually lead to photomorphogenesis (Casal *et al.*, 2003).

#### **2.1 Photomorphogenic photoreceptors**

Light is directly perceived by protein molecules known as photoreceptors. Photoreceptors are considered as such if upon photon absorption they are able to deliver a signal to downstream components. Because membranes are transparent to light, most photoreceptors are cytoplasmic and water soluble, contrary to other type of receptors whose ligands are not able to move through membranes.

The solar spectrum at Earth's surface extends from UV (about 280 nm) through the blue to red/far red (about 750 nm). Because the polypeptide backbone and amino acid side chains do not absorb in most of this range, most photoreceptors contain an organic, non-protein component, known as the chromophore. Chromophores can be attached either covalently or non-covalently to the apoprotein (Moglich *et al.*, 2010). As explained above, plants possess several classes of photoreceptors whose absorption properties match the spectrum of the incoming light: the red/far-red photoreversible phytochromes, the UV-A/blue-light absorbing cryptochromes, the phototropins, the members of the Zeitlupe family (Moglich *et al.*, 2010) and, more recently, a UV-B specific photoreceptor, UVR8, has been added to the list (Rizzini *et al.*, 2011).

#### **2.2 Phytochromes**

192 Advances in Photosynthesis – Fundamental Aspects

light dependent way, to optimize the light absorption and photosynthesis. However, under strong light, chloroplasts show an opposite response to avoid photodamage (Kodama *et al.*, 2011). Besides chloroplast movement, photoreceptors modulate plant architecture to maximize the photosynthetic surface exposed to light. When plants perceive the presence of plant neighbours, phytochromes trigger the elongation of the stem and petioles, a series of changes known as Shade Avoidance Syndrome (SAS). The manipulation of phytochrome levels has been used to improve the harvest index of tobacco plants (Robson *et al.*, 1996) by avoiding the diversion of resources to the SAS. However, the phytochromes are still very important to position the leaves in the canopy. Thus, manipulation of phytochrome activity

In this chapter, we focus on the role of the photomorphogenic signal to trigger the synthesis of photosynthetic genes and pigments during the greening process and later on, during photosynthetic plant development, with emphasis on the regulation of gene expression.

Plants are sessile organisms, and as such, have evolved a great deal of developmental plasticity to optimally respond to the immediate environment. Light is one of the most important cues for plant growth; plants respond to its intensity, wavelength, direction and periodicity (Franklin & Quail, 2009). The first physiological consequence of light perception is the reprogramming of the seedling development in a process termed deetiolation, or photomorphogenesis. In darkness, seedlings display a skotomorphogenesis development characterised by the following phenotypes: elongated hypocotyl, closed, pale and unexpanded cotyledons; the apical hook remains closed to protect the apical meristem before emerging from the soil and chlorophyll and anthocyanin biosynthesis do not take place. All these features allow the seedlings to grow through a layer of soil and eventually emerge into the light. Once the seedlings perceive sufficient light, they exhibit a photomorphogenic development. They undergo deetiolation that includes inhibition of hypocotyl elongation, unfolding and greening of cotyledons, opening of the apical hook, chlorophyll and anthocyanin biosynthesis and differentiation of chloroplasts; processes aimed to achieve full autotrophy. This transition from skotomorphogenic to photomorphogenic development is steered by a complex molecular network that includes upstream signalling components (photoreceptors) and intermediate factors transducing the signal to downstream regulators. These downstream components integrate the light signals from the various photoreceptors and bring about changes in metabolism and gene

must be precise, if used to improve crop performance (Maddonni *et al.*, 2002).

expression that eventually lead to photomorphogenesis (Casal *et al.*, 2003).

Light is directly perceived by protein molecules known as photoreceptors. Photoreceptors are considered as such if upon photon absorption they are able to deliver a signal to downstream components. Because membranes are transparent to light, most photoreceptors are cytoplasmic and water soluble, contrary to other type of receptors whose ligands are not

The solar spectrum at Earth's surface extends from UV (about 280 nm) through the blue to red/far red (about 750 nm). Because the polypeptide backbone and amino acid side chains do not absorb in most of this range, most photoreceptors contain an organic, non-protein component, known as the chromophore. Chromophores can be attached either covalently or

**2.1 Photomorphogenic photoreceptors** 

able to move through membranes.

**2. Photomorphogenesis** 

#### **2.2.1 Generalities about phytochromes**

Phytochromes are the only red and far-red light photoreceptors in plants (Strasser *et al.*, 2010; Takano *et al.*, 2009) and, together with cryptochromes and phototropins, constitute one of the three mayor regulators of photomorphogenesis (Rockwell *et al.*, 2006). Phytochromes are synthesised in the cytosol as soluble dimers composed of two 125-kDa polypeptides. Each polypeptide folds into two main domains. The amino-terminal domain covalently binds phytochromobilin (PфB), a tetrapyrrole chromophore that confers the spectral properties characteristic of phytochromes. PфB is synthesised from haeme in plastids, haeme oxygenase encoded by *HY1* converts haeme into biliverdin IX, which is reduced to 3Z-PфB by the PфB synthase (*HY2*). Then 3Z-PфB isomerises to 3E-PфB and attaches covalently to phytochrome (Tanaka & Tanaka, 2007).

The carboxy-terminal part of the phytochrome molecule is involved in dimerisation and transfer of the signal to downstream components (Rockwell *et al.*, 2006). Phytochromes are synthesised in the dark in a biologically inactive red-light absorbing form (known as Pr). Biological activity is acquired upon red-light triggered photoconversion to the far-red light absorbing form (known as Pfr). Photoconversion of Pfr back to Pr is triggered by far-red light. Both reactions are fully reversible, and eventually results in a dynamic photoequilibrium of Pr and Pfr in natural light conditions that depends on the proportion of red to far-red light (Franklin & Quail, 2009). The conversion is due to a single photochemical isomerisation of the chromophore about a specific double bond between the rings C and D of the phytochromobilin (Rockwell *et al.*, 2006). Following conversion, Pfr translocates into the nucleus (Fankhauser & Chen, 2008).

The phytochromes are encoded by a small gene family in angiosperms. The rice genome, for example, encodes three members, phyA, phyB and phyC, each representing one of the lineages found in plants (Sharrock, 2008). In Arabidopsis, the phytochrome family consists of five members, designated phytochrome A (phyA) to phytochrome E (phyE).

Classical photobiological experiments established three phytochromes modes of acting, the Very Low Fluence Response mode (VLFR), where responses to phytochromes are already saturated at very low fluencies of light, the Low Fluence Response (LFR) showing the classical red and far-red light reversibility and the High Irradiance responses (HIR) that require prolonged exposures to light of relative high intensity (Casal *et al.*, 2003). Now that we know about each phytochrome species, the phytochrome action modes can be explained by the different phytochrome species and different signal transduction pathways. phyA is the most specialized of the phytochromes; it is responsible for the VLFR and the HIR. The extraordinary sensitivity of this photoreceptor to light allows phyA to control germination of buried seeds in the soil and to induce germination when seeds are exposed briefly to light. phyA importance is evident when plants germinate under a dense canopy (Yanovsky, 1995) or for example, when weeds are induced to germinate after soil tillage (Ballaré, 1992). The other phytochromes control the R/FR reversible LFR and the responses to continuous

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life 195

(MTFG). The CCT domain appears to be important for cryptochrome function, it interacts with downstream effectors and promotes photomorphogenic development in the dark by itself (Li & Yang, 2007). cry1 and cry2 form homodimers; dimerisation is mediated by the PRH domain and appears to be essential for signalling (Moglich *et al.*, 2010). cry1 and cry2 are predominantly nuclear. However, cry1 is also found in the cytoplasm. They mediate the regulation of gene expression and together are responsible for blue-light dependent changes in gene expression of up to 10-20% of the Arabidopsis genome (Lin & Todo, 2005). cry1 and cry2 participate in many blue-light responses including inhibition of hypocotyl elongation, anthocyanin accumulation, regulation of flowering time, stem and internode elongation, blue-light regulated gene expression, and entrainment of circadian rhythms. The function of cry1 and cry2 partially overlap, but differences are evident at different light intensities or at different developmental stages. Under high intensities of blue light, cry2 is rapidly degraded, leaving cry1 as the predominant photoreceptor, so the role of cry2 during seedling deetiolation is more evident under low blue light intensities. In contrast, cry2 role is

ATE PAS GAF PHY PAS1 and PAS2 HKRD Phy FHR

Phot

predominant in the regulation of flowering time (Li & Yang, 2007).

PHR CCT Cry

LOV F Kelch Zeitlupe

terminal extension domain), FAD (flavin adenine dinucleotide), MTFG (5,10 methenyltetrahydrofolate), LOV (Light-oxygen-voltage domains), Ser/Thrk (serine threonine kinase domain), FMN (Flavin mononucleotide), F (F box), Kelch (Kelch repeat).

**2.4 LOV domain photoreceptors: The phototropins and the ztl family** 

The phototropic response of plants has been known at least since Darwin times. The photoreceptors involved were identified after finding mutants impaired in the phototropic response (Huala *et al.*, 1997), and were later named phot1 and phot2. The sequence revealed

Fig. 1. Schematic representation of domain structure and chromophores of the main photoreceptors: phytochromes (phy), cryptochromes (cry), phototropins (phot) and the zeitlupe family. Domain abbreviation are ATE (N-terminal extension); PAS: domain acronym derived from period clock (PER) protein, aromatic hydrocarbon receptor nuclear translocator (ARNT), and single minded (SIM); GAF: (domain acronym derived from vertebrate cGMP-specific phosphodiesterase, cyanobacterial adenylate cyclase and formate hydrogen lyase transcription activator FhlA); PHY (phytochrome); FHR (flexible hinge region); HKRD (Histidine kinase related domain), PHR (photolyase related region), CCT (C-

LOV1 LOV2 Ser/ThrK

phytocromobilin

MTFG FAD

FMN

FMN FMN

red light. phyB is involved in seed germination, deetiolation, stem elongation, the SAS, stomatal development and several other aspects of plant development. phyD and phyE act in SAS by controlling internode elongation and flowering time, and phyE is also involved in far-red HIR- mediated seed germination (Franklin & Quail, 2009).

Before even knowing of the existence of multiple phytochromes, they were classified in type I, the light-labile pool and type II, the light stable pool. Now we know that type I is represented by phyA and type II by the other phytochromes. As illustrated above, type I and II phytochromes play distinct roles. The rapid proteolytic degradation of phyA is believed to be responsible for the termination of signalling. The light stable phytochromes are not totally resistant to proteolytic degradation (Jang *et al.*, 2010), but dark reversion also emerges as a switch-off mechanism. Dark reversion is a thermal process in which the Pfr form is slowly converted to the Pr form in the dark. Although dark reversion is not yet well characterised, it makes an important contribution to the balance between Pr and Pfr and hence, to determine the output state for a given phytochrome (Rockwell *et al.*, 2006).

#### **2.2.2 Phytochrome structure and nuclear translocation**

The two phytochrome mayor domains mentioned above are separated by a flexible hinge region. The N-terminal photosensory region (70 kDa) contains an N-terminal extension (ATE) and three conserved subdomains: PAS, GAF and PHY. The ATE is poorly conserved, possibly accounting for some functional differences among phytochromes and it might be implicated in stabilization of the Pfr form of photoreceptors. The GAF domain is associated with the bilin chromophore and possesses bilin lyase activity. The PAS and PHY domains are important for tuning the spectroscopic properties of the bound bilin.

A flexible hinge region separates the N-terminal domains from the C-terminal regulatory region (55 kDa), which is composed of two PAS subdomains, called PAS 1 and PAS 2, and a histidine kinase related domain (HKRD) (Figure 1). The PAS and HKRD domains contribute to the high-affinity subunit-subunit interaction between the phytochrome monomers to form dimers, and both domains are required for the formation of nuclear speckles. Besides, the PAS domains contain the nuclear localization signal (NLS) responsible for the relocalisation to the nucleus after phytochrome photoconversion (Rockwell *et al.*, 2006). Finally, at least one domain must be responsible for the serine/threonine kinase activity that governs phytochrome autophosphorylation and phytochrome–directed phosphorylation of other proteins, such as PHYTOCHROME-INTERACTING FACTOR 3 (PIF3). The functional significance of this kinase activity remains unknown. HKRD domain was initially suggested to be a kinase because of its relatedness to bacterial histidin kinases (Figure 1). However, it was shown that the kinase activity resides in the N-terminal domain (Bae & Choi, 2008). Further, it was recently shown that a Casein Kinase II is involved in phosphorylating phytochrome interacting factor 1 (PIF1), one or the downstream effectors of phytochrome signalling (see below) (Bu *et al.*, 2011).

#### **2.3 Cryptochromes**

Cryptochromes are receptors for blue and ultraviolet light. Arabidopsis contains two cryptochromes, cry1 and cry2. They are composed of two domains, an N-terminal photolyase related region (PHR), without photolyase activity, and a C-terminal extension domain (CCT), more variable among family members (Figure 1). The PHR region binds two chromophores, flavin adenine dinucleotide (FAD) and 5,10-methenyltetrahydrofolate

red light. phyB is involved in seed germination, deetiolation, stem elongation, the SAS, stomatal development and several other aspects of plant development. phyD and phyE act in SAS by controlling internode elongation and flowering time, and phyE is also involved in

Before even knowing of the existence of multiple phytochromes, they were classified in type I, the light-labile pool and type II, the light stable pool. Now we know that type I is represented by phyA and type II by the other phytochromes. As illustrated above, type I and II phytochromes play distinct roles. The rapid proteolytic degradation of phyA is believed to be responsible for the termination of signalling. The light stable phytochromes are not totally resistant to proteolytic degradation (Jang *et al.*, 2010), but dark reversion also emerges as a switch-off mechanism. Dark reversion is a thermal process in which the Pfr form is slowly converted to the Pr form in the dark. Although dark reversion is not yet well characterised, it makes an important contribution to the balance between Pr and Pfr and

hence, to determine the output state for a given phytochrome (Rockwell *et al.*, 2006).

The two phytochrome mayor domains mentioned above are separated by a flexible hinge region. The N-terminal photosensory region (70 kDa) contains an N-terminal extension (ATE) and three conserved subdomains: PAS, GAF and PHY. The ATE is poorly conserved, possibly accounting for some functional differences among phytochromes and it might be implicated in stabilization of the Pfr form of photoreceptors. The GAF domain is associated with the bilin chromophore and possesses bilin lyase activity. The PAS and PHY domains

A flexible hinge region separates the N-terminal domains from the C-terminal regulatory region (55 kDa), which is composed of two PAS subdomains, called PAS 1 and PAS 2, and a histidine kinase related domain (HKRD) (Figure 1). The PAS and HKRD domains contribute to the high-affinity subunit-subunit interaction between the phytochrome monomers to form dimers, and both domains are required for the formation of nuclear speckles. Besides, the PAS domains contain the nuclear localization signal (NLS) responsible for the relocalisation to the nucleus after phytochrome photoconversion (Rockwell *et al.*, 2006). Finally, at least one domain must be responsible for the serine/threonine kinase activity that governs phytochrome autophosphorylation and phytochrome–directed phosphorylation of other proteins, such as PHYTOCHROME-INTERACTING FACTOR 3 (PIF3). The functional significance of this kinase activity remains unknown. HKRD domain was initially suggested to be a kinase because of its relatedness to bacterial histidin kinases (Figure 1). However, it was shown that the kinase activity resides in the N-terminal domain (Bae & Choi, 2008). Further, it was recently shown that a Casein Kinase II is involved in phosphorylating phytochrome interacting factor 1 (PIF1), one or the downstream effectors of phytochrome

Cryptochromes are receptors for blue and ultraviolet light. Arabidopsis contains two cryptochromes, cry1 and cry2. They are composed of two domains, an N-terminal photolyase related region (PHR), without photolyase activity, and a C-terminal extension domain (CCT), more variable among family members (Figure 1). The PHR region binds two chromophores, flavin adenine dinucleotide (FAD) and 5,10-methenyltetrahydrofolate

far-red HIR- mediated seed germination (Franklin & Quail, 2009).

**2.2.2 Phytochrome structure and nuclear translocation** 

signalling (see below) (Bu *et al.*, 2011).

**2.3 Cryptochromes** 

are important for tuning the spectroscopic properties of the bound bilin.

(MTFG). The CCT domain appears to be important for cryptochrome function, it interacts with downstream effectors and promotes photomorphogenic development in the dark by itself (Li & Yang, 2007). cry1 and cry2 form homodimers; dimerisation is mediated by the PRH domain and appears to be essential for signalling (Moglich *et al.*, 2010). cry1 and cry2 are predominantly nuclear. However, cry1 is also found in the cytoplasm. They mediate the regulation of gene expression and together are responsible for blue-light dependent changes in gene expression of up to 10-20% of the Arabidopsis genome (Lin & Todo, 2005). cry1 and cry2 participate in many blue-light responses including inhibition of hypocotyl elongation, anthocyanin accumulation, regulation of flowering time, stem and internode elongation, blue-light regulated gene expression, and entrainment of circadian rhythms. The function of cry1 and cry2 partially overlap, but differences are evident at different light intensities or at different developmental stages. Under high intensities of blue light, cry2 is rapidly degraded, leaving cry1 as the predominant photoreceptor, so the role of cry2 during seedling deetiolation is more evident under low blue light intensities. In contrast, cry2 role is predominant in the regulation of flowering time (Li & Yang, 2007).

Fig. 1. Schematic representation of domain structure and chromophores of the main photoreceptors: phytochromes (phy), cryptochromes (cry), phototropins (phot) and the zeitlupe family. Domain abbreviation are ATE (N-terminal extension); PAS: domain acronym derived from period clock (PER) protein, aromatic hydrocarbon receptor nuclear translocator (ARNT), and single minded (SIM); GAF: (domain acronym derived from vertebrate cGMP-specific phosphodiesterase, cyanobacterial adenylate cyclase and formate hydrogen lyase transcription activator FhlA); PHY (phytochrome); FHR (flexible hinge region); HKRD (Histidine kinase related domain), PHR (photolyase related region), CCT (Cterminal extension domain), FAD (flavin adenine dinucleotide), MTFG (5,10 methenyltetrahydrofolate), LOV (Light-oxygen-voltage domains), Ser/Thrk (serine threonine kinase domain), FMN (Flavin mononucleotide), F (F box), Kelch (Kelch repeat).

#### **2.4 LOV domain photoreceptors: The phototropins and the ztl family**

The phototropic response of plants has been known at least since Darwin times. The photoreceptors involved were identified after finding mutants impaired in the phototropic response (Huala *et al.*, 1997), and were later named phot1 and phot2. The sequence revealed

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life 197

substrates. Several of the COP1 substrates have been characterised and they are

Genetic and molecular approaches have identified several transcription factors acting positively on photomorphogenesis downstream of photoreceptors. As phyA is the main photoreceptor perceiving continuous far-red light (acting in the HIR mode), mutants with long hypocotyls under far-red light were isolated, leading to phyA signalling components. Two of the genes identified, *long after far-red light 1* (*laf1*) and *long hypocotyl in far-red* (*hfr*) encode an R2/R3 MYB and a bHLH transcription factor respectively. Other mutants helped to identify other phyA signalling components; among them, two small plant-specific proteins involved in light-regulated phyA import to the nuclei, FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL) (Fankhauser & Chen, 2008), and two transposase-derived transcription factors, FHY3 and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1), which are direct activators of *FHY1* and *FHL* transcription, promoting

Genetic screenings for enhancers of phyA signalling led to the identification of SUPPRESSOR OF PHYTOCHROME A-105 1 (SPA1), which belongs to a small family of four proteins (SPA1-4). The quadruple mutant defective for the four *SPA* genes shows a constitutive photomorphogenesis phenotype in the dark, similar to *cop1* mutants (Laubinger *et al.*, 2004). SPA proteins and COP1 form complexes and, as mentioned above, show E3 ligase activity (Zhu *et al.*, 2008). This SPA-COP1 complex targets HFR and LAF1 for degradation, explaining part of its negative role in photomorphogenesis (Henriques *et al.*,

The SPA1-COP1 E3 ligase complex targets other important transcription factors for degradation, like elongated hypocotyl 5 (hy5) and hy5 homolog (hyh), two bZIP transcription factors. These transcription factors promote photomorphogenesis under

*hy5* mutants display a long hypocotyl phenotype under diverse wavelengths of light, suggesting HY5 is a common promotor of photomorphogenesis downstream several photoreceptor signalling pathways. Interestingly, the association between HY5 and COP1 can be deduced from the overlapping set of differentially expressed genes in the respective mutants (Ma *et al.*, 2002). At the biochemical level, it was shown that both HY5 and HYH are targeted for degradation by COP1 (Holm *et al.*, 2002; Osterlund *et al.*, 2000). On the other hand, cryptochromes are known to interact with COP1 through its CCT domain and negatively regulate COP1 activity (Li & Yang, 2007). However, the precise light-mediated mechanism that controls COP1 activity remained unknown until recently. Three simultaneous publications addressed this issue (Lian *et al*., 2011; Liu *et al*., 2011; Zuo *et al*., 2011). They showed that CRY1 interacts with the SPA proteins in a blue-light dependent manner and inhibit the interaction between COP1 and SPA proteins. This mechanism disrupts the complex E3 ligase activity and avoids HY5 degradation, promoting photomorphogenesis. In the case of CRY2, a similar blue-light dependent interaction with SPA proteins inhibits the activity of the COP1-SPA complex. This inhibition leads to higher levels of CONSTANS, a transcription factor that promotes flowering in long-day conditions.

various wavelengths and will be explained in the following sections.

**3.2 COP1 targets positive regulators of photomorphogenesis for degradation** 

transcription factors that act positively on photomorphogenesis.

**3.2.1 COP1 in phyA signalling** 

phyA signalling (Lin *et al.*, 2007).

**3.2.2 COP1 in cryptochrome signalling** 

2009).

the presence of two domains showing homology to domains that are involved in sensing Light, Oxygen or Voltage, the LOV domains (Figure 1). These domains bind FMN, the chromophore for phototropism. Phototropins were important in the identification of other LOV-domain containing photoreceptors.

The second family of LOV photoreceptors is comprised by Zeitlupe/Adagio/LOV KELCH Protein 1 (ztl/ado/lkp1), fkf1 and lkp2. Contrary to phototropins, the ztl family contains a single LOV domain, an F-box and a C-terminal Kelch domain. F-box proteins play a role in recruiting specific substrates for ubiquitination and protein degradation, whereas the Kelch domain might aid in this function by mediating protein-protein interactions (Moglich *et al.*, 2010). These photoreceptors have important functions in flowering time and circadian clock function, as we will explain in the following sections, mainly by controlling the stability of important clock associated proteins (Harmer, 2009; Mas, 2005).

#### **2.5 The UV-B specific photoreceptor: The UVR8 protein**

UV-B radiation (280-315 nm) is an integral part of the sunlight reaching the surface of the Earth and induces a broad range of physiological responses that are mediated by a recently identified UV-B specific photoreceptor, UVR8 (Rizzini *et al.*, 2011). The most extensively studied examples of photomorphogenic responses are the suppression of hypocotyl extension by low fluences of UV-B and the induction of genes involved in flavonoid biosynthesis (Jenkins, 2009).

UVR8 is a β-propeller protein with similar sequence to the eukaryotic RCC1, a guanine nucleotide exchange factor (GEF) for the small GTP-binding protein Ran (Gruber *et al.*, 2010). Aromatic amino acids absorb UV-B radiation. Tryptophan, with an absorption maximum in solution at around 280 nm (which extends to 300 nm and is likely to be further shifted in a protein environment), is particularly suited as UV-B chromophore. Structure modelling according to structurally related RRC1, identified 14 tryptophans of UVR8 all located at the top of the predicted -propeller cluster in the centre of the protein structure. Evidence suggests UV-B perception is based on a tryptophan-based mechanism, an important difference with the other Photoreceptors that bear chromophores suited for visible light perception (Rizzini *et al.*, 2011).

#### **3. Transducers of light signalling**

#### **3.1 COP1 is a general repressor of photomorphogenesis**

Most of the photoreceptors mentioned above were identified after genetic screenings in Arabidopsis and led to the isolation of mutants defective in deetiolation. Other type of genetic screenings led to the isolation of mutants with constitutive photomorphogenic phenotypes in the dark (*cop*) or deetiolated (*det*). The phenotype of one of such mutants, *cop1*  (Deng *et al.,* 1992)*,* suggested that it was a negative regulator of photomorphogenesis. COP1 is an essential protein, null alleles are not viable. The overlap between the light-responsive transcriptome and the *cop1*-responsive transcriptome in dark grown seedlings clearly shows that COP1 is a general repressor of photomorphogenesis (Ma *et al.*, 2002). We now know that COP1 is a single unit E3 ubiquitin ligase, bearing both the substrate and E2 binding motifs. Ubiquitin ligation is the last step in the chain of events that leads to protein ubiquitination that marks proteins for degradation by the 26S proteasome. The COP1 protein bears three domains: a RING-finger motif, a coiled-coil domain and seven WD40 repeats. The RING domain is essential to recruit E2s and the other domains to recognize

the presence of two domains showing homology to domains that are involved in sensing Light, Oxygen or Voltage, the LOV domains (Figure 1). These domains bind FMN, the chromophore for phototropism. Phototropins were important in the identification of other

The second family of LOV photoreceptors is comprised by Zeitlupe/Adagio/LOV KELCH Protein 1 (ztl/ado/lkp1), fkf1 and lkp2. Contrary to phototropins, the ztl family contains a single LOV domain, an F-box and a C-terminal Kelch domain. F-box proteins play a role in recruiting specific substrates for ubiquitination and protein degradation, whereas the Kelch domain might aid in this function by mediating protein-protein interactions (Moglich *et al.*, 2010). These photoreceptors have important functions in flowering time and circadian clock function, as we will explain in the following sections, mainly by controlling the stability of

UV-B radiation (280-315 nm) is an integral part of the sunlight reaching the surface of the Earth and induces a broad range of physiological responses that are mediated by a recently identified UV-B specific photoreceptor, UVR8 (Rizzini *et al.*, 2011). The most extensively studied examples of photomorphogenic responses are the suppression of hypocotyl extension by low fluences of UV-B and the induction of genes involved in flavonoid

UVR8 is a β-propeller protein with similar sequence to the eukaryotic RCC1, a guanine nucleotide exchange factor (GEF) for the small GTP-binding protein Ran (Gruber *et al.*, 2010). Aromatic amino acids absorb UV-B radiation. Tryptophan, with an absorption maximum in solution at around 280 nm (which extends to 300 nm and is likely to be further shifted in a protein environment), is particularly suited as UV-B chromophore. Structure modelling according to structurally related RRC1, identified 14 tryptophans of UVR8 all located at the top of the predicted -propeller cluster in the centre of the protein structure. Evidence suggests UV-B perception is based on a tryptophan-based mechanism, an important difference with the other Photoreceptors that bear chromophores suited for

Most of the photoreceptors mentioned above were identified after genetic screenings in Arabidopsis and led to the isolation of mutants defective in deetiolation. Other type of genetic screenings led to the isolation of mutants with constitutive photomorphogenic phenotypes in the dark (*cop*) or deetiolated (*det*). The phenotype of one of such mutants, *cop1*  (Deng *et al.,* 1992)*,* suggested that it was a negative regulator of photomorphogenesis. COP1 is an essential protein, null alleles are not viable. The overlap between the light-responsive transcriptome and the *cop1*-responsive transcriptome in dark grown seedlings clearly shows that COP1 is a general repressor of photomorphogenesis (Ma *et al.*, 2002). We now know that COP1 is a single unit E3 ubiquitin ligase, bearing both the substrate and E2 binding motifs. Ubiquitin ligation is the last step in the chain of events that leads to protein ubiquitination that marks proteins for degradation by the 26S proteasome. The COP1 protein bears three domains: a RING-finger motif, a coiled-coil domain and seven WD40 repeats. The RING domain is essential to recruit E2s and the other domains to recognize

LOV-domain containing photoreceptors.

biosynthesis (Jenkins, 2009).

visible light perception (Rizzini *et al.*, 2011).

**3.1 COP1 is a general repressor of photomorphogenesis** 

**3. Transducers of light signalling** 

important clock associated proteins (Harmer, 2009; Mas, 2005).

**2.5 The UV-B specific photoreceptor: The UVR8 protein** 

substrates. Several of the COP1 substrates have been characterised and they are transcription factors that act positively on photomorphogenesis.

#### **3.2 COP1 targets positive regulators of photomorphogenesis for degradation 3.2.1 COP1 in phyA signalling**

Genetic and molecular approaches have identified several transcription factors acting positively on photomorphogenesis downstream of photoreceptors. As phyA is the main photoreceptor perceiving continuous far-red light (acting in the HIR mode), mutants with long hypocotyls under far-red light were isolated, leading to phyA signalling components. Two of the genes identified, *long after far-red light 1* (*laf1*) and *long hypocotyl in far-red* (*hfr*) encode an R2/R3 MYB and a bHLH transcription factor respectively. Other mutants helped to identify other phyA signalling components; among them, two small plant-specific proteins involved in light-regulated phyA import to the nuclei, FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL) (Fankhauser & Chen, 2008), and two transposase-derived transcription factors, FHY3 and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1), which are direct activators of *FHY1* and *FHL* transcription, promoting phyA signalling (Lin *et al.*, 2007).

Genetic screenings for enhancers of phyA signalling led to the identification of SUPPRESSOR OF PHYTOCHROME A-105 1 (SPA1), which belongs to a small family of four proteins (SPA1-4). The quadruple mutant defective for the four *SPA* genes shows a constitutive photomorphogenesis phenotype in the dark, similar to *cop1* mutants (Laubinger *et al.*, 2004). SPA proteins and COP1 form complexes and, as mentioned above, show E3 ligase activity (Zhu *et al.*, 2008). This SPA-COP1 complex targets HFR and LAF1 for degradation, explaining part of its negative role in photomorphogenesis (Henriques *et al.*, 2009).

The SPA1-COP1 E3 ligase complex targets other important transcription factors for degradation, like elongated hypocotyl 5 (hy5) and hy5 homolog (hyh), two bZIP transcription factors. These transcription factors promote photomorphogenesis under various wavelengths and will be explained in the following sections.

#### **3.2.2 COP1 in cryptochrome signalling**

*hy5* mutants display a long hypocotyl phenotype under diverse wavelengths of light, suggesting HY5 is a common promotor of photomorphogenesis downstream several photoreceptor signalling pathways. Interestingly, the association between HY5 and COP1 can be deduced from the overlapping set of differentially expressed genes in the respective mutants (Ma *et al.*, 2002). At the biochemical level, it was shown that both HY5 and HYH are targeted for degradation by COP1 (Holm *et al.*, 2002; Osterlund *et al.*, 2000). On the other hand, cryptochromes are known to interact with COP1 through its CCT domain and negatively regulate COP1 activity (Li & Yang, 2007). However, the precise light-mediated mechanism that controls COP1 activity remained unknown until recently. Three simultaneous publications addressed this issue (Lian *et al*., 2011; Liu *et al*., 2011; Zuo *et al*., 2011). They showed that CRY1 interacts with the SPA proteins in a blue-light dependent manner and inhibit the interaction between COP1 and SPA proteins. This mechanism disrupts the complex E3 ligase activity and avoids HY5 degradation, promoting photomorphogenesis. In the case of CRY2, a similar blue-light dependent interaction with SPA proteins inhibits the activity of the COP1-SPA complex. This inhibition leads to higher levels of CONSTANS, a transcription factor that promotes flowering in long-day conditions.

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life 199

dependent pathway, and thus relieving its negative regulation of photomorphogenesis. phyA is responsible for the rapid degradation of PIF3 in response to far-red light, whereas phyA, phyB and phyD are responsible for PIF3 degradation in response to red light (Bae

PIF1 (also known as PIL5), PIF4, PIF5 (also known as PIL6) and PIF6 (also known as PIL2) also have important roles in photomorphogenic development. Although they have highly similar sequences, their roles do not overlap completely. For example, PIF1 negatively regulates seed germination by inhibiting gibberelin (GA) biosynthesis and GA signalling, and simultaneously activating abscisic acid biosynthesis. In addition, PIF1 activates the expression of two DELLA genes, which are key negative GA signalling components. Phytochromes promote seed germination by inhibiting PIF1 activity. Conversely, PIF4 and PIF5 have important roles in the regulation of the SAS (Leivar & Quail, 2011). We will describe the roles of PIFs in chloroplast biogenesis and c chlorophyll synthesis in a

**4. The role of the circadian clock in photomorphogenic development** 

The circadian clocks are endogenous mechanisms that allow organisms to time their physiological changes to day/night cycles. These mechanisms are present in a wide range of organisms, from cyanobacteria to mammals. Circadian clocks generate rhythms with a ~24 hr period, which include changes in gene expression or protein activity. They regulate diverse aspects of plant growth and development, such as the movement of leaves and flowers, the production of volatiles, the stomatal opening, the hypocotyl expansion, the photosynthetic activity and the photoperiodic control of flowering, allowing plants to anticipate daily environmental changes and to synchronise their endogenous physiological processes to external environmental cues. Circadian rhythms persist with a period close to 24 hours after an organism is transferred from an environment that varies according to the time of the day (entraining condition) to an unchanging condition (free-running condition)

In a simple way, the circadian system can be divided into three main components: the *input pathways*, involved in the perception and transmission of environmental signals to synchronise the *central oscillator* that generates and maintains rhythmicity through multiple *output pathways*, connecting the oscillator to physiology and metabolism. However, this is an oversimplified model of the clock. The circadian system has to be considered as a complex network. The central clock is composed of multiple interlocked feedback loops, where clock *outputs* may be regulated directly by clock *input* signalling pathways and can also feedback to clock components and *input* signalling pathways. Clock genes have multiple functions, they can act within the *central oscillator* and in clock *input* and *output* signalling pathways (Mas, 2005). A key observation is that circadian clock mutants show defective developmental responses to red light (Harmer, 2009), but the endogenous clock oscillates in the absence of phyA phyB cry1 and cry2 (Yanovsky *et al.*, 2000) or in a mutant devoid of all phytochromes (Strasser *et al.*, 2010). These observations imply that the photoreceptors

In *Arabidopsis thaliana*, the current model for the circadian oscillator is composed of several interlocking positive and negative feedback loops. The first loop that was identified involves

modulate the clock but they are not themselves part of the *central oscillator*.

**4.1 Molecular basis of the circadian clock** 

& Choi, 2008).

following section.

(Harmer, 2009).

These facts also explain some of the differences between the roles of CRY1 and CRY2 in plant development that we mentioned before.

#### **3.2.3 COP1 in UV-B signalling**

UVR8 forms a dimer but rapidly dissociates as the result of direct perception of UV-B. This is followed by a rapid nuclear accumulation of UVR8 and UVR8 interaction with COP1 that depends on the C-terminal WD40-repeat domain. The UVR8-COP1 interaction mediates the activation of numerous genes, including HY5, inducing photomorphogenic responses (Favory *et al.*, 2009; Jenkins, 2009).

#### **3.3 The PIF family of bHLH transcription factor represses photomorphogenesis downstream of phytochromes**

The photoconversion of Pr to Pfr with red light leads to conformational changes that unmask the NLS to become accessible for the nuclear-transport machinery and also allow the interacting surfaces for partner proteins. Within the nucleus, phytochromes accumulate in subnuclear foci, the phytochrome Nuclear Bodies (NBs). The identification of HEMERA, a protein involved in the formation of NBs, supports the notion that NBs are the sites of phytochrome-induced protein degradation (Chen *et al.*, 2010). Phytochrome-induced protein degradation is important to control the activity of the Phytochrome interaction factor (PIF) family of bHLH transcription factors. The Pfr form is rapidly translocated into the nucleus, where it interacts with PIFs, more strongly with the Pfr form (Fankhauser & Chen, 2008). Upon binding Pfr, the PIFs are phosphorylated and degraded. This event initiates a gene expression cascade leading to photomorphogenesis (Bae & Choi, 2008; Leivar *et al.*, 2009; Shen *et al.*, 2008).

The PIFs belong to a transcription factor superfamily, which forms dimers to target specific DNA sites and are well characterised in nonplant eukaryotes as important regulatory components in diverse biological processes. In Arabidopsis, there are at least 133 bHLH protein–encoding genes. Phylogenetic analysis of the bHLH domain sequences allowed the classification of these genes into 21 subfamilies (Heim *et al.*, 2003; Toledo-Ortiz *et al.*, 2003). The PIFs subfamily, called PHYTOCHROME INTERACTING FACTORs (PIFs) is involved in the repression of seed germination, promotion of seedling skotomorphogenesis and SAS, by regulating the expression of over a thousand genes (Leivar & Quail, 2011). PIF3 was the first member identified in this subfamily, isolated by a two-hybrid assay as a PHYB interactor (Ni *et al*., 1998). Afterwards, other members of the family were identified by computational analysis (Leivar & Quail, 2011). Unlike other bHLHs, this subfamily have a characteristic active phytochrome binding motif (APB) in its N-terminal, that make them able to interact with the photoactivated phytochrome (Leivar & Quail, 2011).

PIFs can form homodimers and heterodimers that bind specifically to the G-box motif CACGTG (Toledo-Ortiz *et al.*, 2003) and, in some cases, HFR1 and other bHLH closely related to PIFs can form non-DNA binding bHLH heterodimers with some PIFs, preventing excessive responses (Hornitschek *et al.*, 2009). In addition, different PIFs are regulated preferentially by different phytochromes (Shen *et al.*, 2008).

As mentioned above, PIF3 is the founding member of this family. PIF3 acts as a negative regulator in both phyA and phyB-mediated seedling deetiolation processes such as hook opening and hypocotyl elongation. Both phyA and phyB bind to PIF3. This interaction leads to the phosphorylation of PIF3, triggering its degradation by the 26S proteasome-

These facts also explain some of the differences between the roles of CRY1 and CRY2 in

UVR8 forms a dimer but rapidly dissociates as the result of direct perception of UV-B. This is followed by a rapid nuclear accumulation of UVR8 and UVR8 interaction with COP1 that depends on the C-terminal WD40-repeat domain. The UVR8-COP1 interaction mediates the activation of numerous genes, including HY5, inducing photomorphogenic responses

The photoconversion of Pr to Pfr with red light leads to conformational changes that unmask the NLS to become accessible for the nuclear-transport machinery and also allow the interacting surfaces for partner proteins. Within the nucleus, phytochromes accumulate in subnuclear foci, the phytochrome Nuclear Bodies (NBs). The identification of HEMERA, a protein involved in the formation of NBs, supports the notion that NBs are the sites of phytochrome-induced protein degradation (Chen *et al.*, 2010). Phytochrome-induced protein degradation is important to control the activity of the Phytochrome interaction factor (PIF) family of bHLH transcription factors. The Pfr form is rapidly translocated into the nucleus, where it interacts with PIFs, more strongly with the Pfr form (Fankhauser & Chen, 2008). Upon binding Pfr, the PIFs are phosphorylated and degraded. This event initiates a gene expression cascade leading to photomorphogenesis (Bae & Choi, 2008; Leivar *et al.*, 2009;

The PIFs belong to a transcription factor superfamily, which forms dimers to target specific DNA sites and are well characterised in nonplant eukaryotes as important regulatory components in diverse biological processes. In Arabidopsis, there are at least 133 bHLH protein–encoding genes. Phylogenetic analysis of the bHLH domain sequences allowed the classification of these genes into 21 subfamilies (Heim *et al.*, 2003; Toledo-Ortiz *et al.*, 2003). The PIFs subfamily, called PHYTOCHROME INTERACTING FACTORs (PIFs) is involved in the repression of seed germination, promotion of seedling skotomorphogenesis and SAS, by regulating the expression of over a thousand genes (Leivar & Quail, 2011). PIF3 was the first member identified in this subfamily, isolated by a two-hybrid assay as a PHYB interactor (Ni *et al*., 1998). Afterwards, other members of the family were identified by computational analysis (Leivar & Quail, 2011). Unlike other bHLHs, this subfamily have a characteristic active phytochrome binding motif (APB) in its N-terminal, that make them

PIFs can form homodimers and heterodimers that bind specifically to the G-box motif CACGTG (Toledo-Ortiz *et al.*, 2003) and, in some cases, HFR1 and other bHLH closely related to PIFs can form non-DNA binding bHLH heterodimers with some PIFs, preventing excessive responses (Hornitschek *et al.*, 2009). In addition, different PIFs are regulated

As mentioned above, PIF3 is the founding member of this family. PIF3 acts as a negative regulator in both phyA and phyB-mediated seedling deetiolation processes such as hook opening and hypocotyl elongation. Both phyA and phyB bind to PIF3. This interaction leads to the phosphorylation of PIF3, triggering its degradation by the 26S proteasome-

able to interact with the photoactivated phytochrome (Leivar & Quail, 2011).

preferentially by different phytochromes (Shen *et al.*, 2008).

**3.3 The PIF family of bHLH transcription factor represses photomorphogenesis** 

plant development that we mentioned before.

**3.2.3 COP1 in UV-B signalling** 

(Favory *et al.*, 2009; Jenkins, 2009).

**downstream of phytochromes** 

Shen *et al.*, 2008).

dependent pathway, and thus relieving its negative regulation of photomorphogenesis. phyA is responsible for the rapid degradation of PIF3 in response to far-red light, whereas phyA, phyB and phyD are responsible for PIF3 degradation in response to red light (Bae & Choi, 2008).

PIF1 (also known as PIL5), PIF4, PIF5 (also known as PIL6) and PIF6 (also known as PIL2) also have important roles in photomorphogenic development. Although they have highly similar sequences, their roles do not overlap completely. For example, PIF1 negatively regulates seed germination by inhibiting gibberelin (GA) biosynthesis and GA signalling, and simultaneously activating abscisic acid biosynthesis. In addition, PIF1 activates the expression of two DELLA genes, which are key negative GA signalling components. Phytochromes promote seed germination by inhibiting PIF1 activity. Conversely, PIF4 and PIF5 have important roles in the regulation of the SAS (Leivar & Quail, 2011). We will describe the roles of PIFs in chloroplast biogenesis and c chlorophyll synthesis in a following section.

#### **4. The role of the circadian clock in photomorphogenic development**

The circadian clocks are endogenous mechanisms that allow organisms to time their physiological changes to day/night cycles. These mechanisms are present in a wide range of organisms, from cyanobacteria to mammals. Circadian clocks generate rhythms with a ~24 hr period, which include changes in gene expression or protein activity. They regulate diverse aspects of plant growth and development, such as the movement of leaves and flowers, the production of volatiles, the stomatal opening, the hypocotyl expansion, the photosynthetic activity and the photoperiodic control of flowering, allowing plants to anticipate daily environmental changes and to synchronise their endogenous physiological processes to external environmental cues. Circadian rhythms persist with a period close to 24 hours after an organism is transferred from an environment that varies according to the time of the day (entraining condition) to an unchanging condition (free-running condition) (Harmer, 2009).

In a simple way, the circadian system can be divided into three main components: the *input pathways*, involved in the perception and transmission of environmental signals to synchronise the *central oscillator* that generates and maintains rhythmicity through multiple *output pathways*, connecting the oscillator to physiology and metabolism. However, this is an oversimplified model of the clock. The circadian system has to be considered as a complex network. The central clock is composed of multiple interlocked feedback loops, where clock *outputs* may be regulated directly by clock *input* signalling pathways and can also feedback to clock components and *input* signalling pathways. Clock genes have multiple functions, they can act within the *central oscillator* and in clock *input* and *output* signalling pathways (Mas, 2005). A key observation is that circadian clock mutants show defective developmental responses to red light (Harmer, 2009), but the endogenous clock oscillates in the absence of phyA phyB cry1 and cry2 (Yanovsky *et al.*, 2000) or in a mutant devoid of all phytochromes (Strasser *et al.*, 2010). These observations imply that the photoreceptors modulate the clock but they are not themselves part of the *central oscillator*.

#### **4.1 Molecular basis of the circadian clock**

In *Arabidopsis thaliana*, the current model for the circadian oscillator is composed of several interlocking positive and negative feedback loops. The first loop that was identified involves

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life 201

(Kim *et al.*, 2007), contributing to a faster degradation of ztl, GI, TOC1 and PRR5 in darkness than in light (Loop 4, figure 2) (Kiba *et al.*, 2007; Kim *et al.*, 2007; Mas *et al.*, 2003). Within this loop 4, TOC1 binds to PRR3, interfering with TOC1 binding to ztl (Para *et al.*, 2007). Thus, PRR3 seems to stabilize TOC1 avoiding its recruitment to the SCF complex and its

Fig. 2. A model for the *Arabidopsis* clock. The circadian clock is composed of several

The other members of the ztl family, fkf1 and lkp2, were also studied. Mutant combinations showed that fkf1 and lkp2 play similar roles to ztl in the circadian clock when ztl is absent, and that both of them interact with TOC1 and PRR5. These results indicate that ztl, fkf1 and lkp2 regulate TOC1 and PRR5 degradation and are important to determine the period of

Cryptochromes also signal to the circadian clock. However, the mechanisms are still unclear. One possibility is through the regulation of COP1 activity; COP1 directly interacts with ELF3 and with GI to promote GI degradation by the proteasome. This could be a mechanism by which cryptochromes regulate the activity of GI, a protein closely associated with

As mentioned above, one interesting aspect of phytochrome and circadian clock is that mutants affected in the clock are also affected in phytochrome responses (Ito *et al.*, 2007). However, how phytochromes contribute to the entrainment of the clock is still unclear. It was previously suggested that PIF3 could directly induce *CCA1* and *LHY* mRNA expression (Martinez-Garcia *et al.*, 2000). Later, it was shown that TOC1 interacts with PIF3 and PIL1

degradation by the proteasome (Loop 4, figure 2).

interlocking positive and negative loops.

circadian oscillation (Baudry *et al.*, 2010).

circadian clock function (Yu *et al.*, 2008).

the Myb-related transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the pseudo-response regulator TIMING OF CAB EXPRESSION 1 (TOC1/PRR1) (Loop 1, figure 2). CCA1 and LHY proteins have partially redundant functions, bind directly to the TOC1 promoter and inhibit its expression during the day (Alabadi *et al.*, 2001). In turn, TOC1 promotes the expression of *CCA1* and *LHY* indirectly via a hypothetical component X in the morning. The mechanism by which TOC1 induces *CCA1* and *LHY1* is not completely understood, but it includes CCA1 HIKING EXPEDITION (CHE), a TCP type transcription factor, which associates with TOC1 to regulate *CCA1* (Pruneda-Paz *et al.*, 2009). Eventually, CHE also forms an additional loop with CCA1 (Imaizumi, 2010).

Mathematical modelling suggests that an evening-phased negative loop is coupled to the first loop, with an unknown component Y that positively regulates *TOC1* whereas *Y* is negatively regulated by TOC1, CCA1 and LHY (Locke *et al.*, 2005) (Loop 2, figure 2). It was suggested that a portion of Y activity is provided by the protein GIGANTEA (GI) (Locke *et al.*, 2005), but this is still unclear (Ito *et al.*, 2009; Martin-Tryon *et al.*, 2007).

The Arabidopsis genome contains four genes encoding proteins with similarity to TOC1: PSEUDORESPONSE REGULATOR (PRR), PRR3, 5, 7 and 9. All these *PRR* genes play a role in the circadian system, although the effect of single mutations is subtle. Multiple mutants generally have stronger phenotypes, for example the triple *prr5 prr7 prr9* mutants are essentially arrhythmic (Nakamichi *et al.*, 2005a; Nakamichi *et al.*, 2005b). Experimental and modelling studies suggest that morning expression of *CCA1* and *LHY* activates the transcription of *PRR7* and *PRR9* (Farre *et al.*, 2005; Nakamichi *et al.*, 2005b; Zeilinger *et al.*, 2006). This loop is called morning loop (Loop 3, figure 2) and is closed when PRR7 and PRR9 feedback to inhibit *CCA1* and *LHY* expression. Together the three interlinked feedback loops form an important part of the clock regulatory mechanism and enhance the robustness of the network against environmental perturbations (Harmer, 2009).

Other components that function within or close to the circadian oscillator have recently been identified: FIONA 1, TIME FOR COFFEE, LIGHT REGULATED WD-1 (LWD1) and LWD2 (Ding *et al.*, 2007; Kim *et al.*, 2008; Wu *et al.*, 2008). However, it is not known whether these clock proteins are part of pre-existing loops or constitute unidentified regulatory loops. It has been recently reported that LWD1/2 regulate the expression of multiple oscillator genes and attenuate light signals to adjust period length. Further, it was also proposed that LWD1 and PRR9 form a positive feedback loop within the central oscillator which is also involved in regulating the light input pathway (Wang *et al.*, 2011) (Figure 2). These results underscore the difficulties in dissecting which signalling events are part of the circadian oscillator and which ones are input pathways.

#### **4.2 Light signalling input to the circadian clock**

Several different photoreceptors mediate light input to the clock, including the phytochromes and the cryptochromes (Somers *et al.*, 1998; Devlin & Kay, 2000; Yanovsky *et al.*, 2001). However, the molecular mechanisms are only partially known. The ztl family of photoreceptors interacts with clock components and regulates their turnover; hence they are potentially part of input mechanisms. ztl interacts with TOC1 and PRR5, leading to their degradation via the proteasome pathway in the dark (Kiba *et al.*, 2007; Mas *et al.*, 2003). The TOC1-ztl interaction does not depend on light, but an interaction between ztl and GI is bluelight dependent, stabilizes both ztl and GI, and contributes to the robust rhythms of TOC1

the Myb-related transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the pseudo-response regulator TIMING OF CAB EXPRESSION 1 (TOC1/PRR1) (Loop 1, figure 2). CCA1 and LHY proteins have partially redundant functions, bind directly to the TOC1 promoter and inhibit its expression during the day (Alabadi *et al.*, 2001). In turn, TOC1 promotes the expression of *CCA1* and *LHY* indirectly via a hypothetical component X in the morning. The mechanism by which TOC1 induces *CCA1* and *LHY1* is not completely understood, but it includes CCA1 HIKING EXPEDITION (CHE), a TCP type transcription factor, which associates with TOC1 to regulate *CCA1* (Pruneda-Paz *et al.*, 2009). Eventually, CHE also forms an additional loop

Mathematical modelling suggests that an evening-phased negative loop is coupled to the first loop, with an unknown component Y that positively regulates *TOC1* whereas *Y* is negatively regulated by TOC1, CCA1 and LHY (Locke *et al.*, 2005) (Loop 2, figure 2). It was suggested that a portion of Y activity is provided by the protein GIGANTEA (GI) (Locke *et* 

The Arabidopsis genome contains four genes encoding proteins with similarity to TOC1: PSEUDORESPONSE REGULATOR (PRR), PRR3, 5, 7 and 9. All these *PRR* genes play a role in the circadian system, although the effect of single mutations is subtle. Multiple mutants generally have stronger phenotypes, for example the triple *prr5 prr7 prr9* mutants are essentially arrhythmic (Nakamichi *et al.*, 2005a; Nakamichi *et al.*, 2005b). Experimental and modelling studies suggest that morning expression of *CCA1* and *LHY* activates the transcription of *PRR7* and *PRR9* (Farre *et al.*, 2005; Nakamichi *et al.*, 2005b; Zeilinger *et al.*, 2006). This loop is called morning loop (Loop 3, figure 2) and is closed when PRR7 and PRR9 feedback to inhibit *CCA1* and *LHY* expression. Together the three interlinked feedback loops form an important part of the clock regulatory mechanism and enhance the robustness

Other components that function within or close to the circadian oscillator have recently been identified: FIONA 1, TIME FOR COFFEE, LIGHT REGULATED WD-1 (LWD1) and LWD2 (Ding *et al.*, 2007; Kim *et al.*, 2008; Wu *et al.*, 2008). However, it is not known whether these clock proteins are part of pre-existing loops or constitute unidentified regulatory loops. It has been recently reported that LWD1/2 regulate the expression of multiple oscillator genes and attenuate light signals to adjust period length. Further, it was also proposed that LWD1 and PRR9 form a positive feedback loop within the central oscillator which is also involved in regulating the light input pathway (Wang *et al.*, 2011) (Figure 2). These results underscore the difficulties in dissecting which signalling events are part of the circadian oscillator and

Several different photoreceptors mediate light input to the clock, including the phytochromes and the cryptochromes (Somers *et al.*, 1998; Devlin & Kay, 2000; Yanovsky *et al.*, 2001). However, the molecular mechanisms are only partially known. The ztl family of photoreceptors interacts with clock components and regulates their turnover; hence they are potentially part of input mechanisms. ztl interacts with TOC1 and PRR5, leading to their degradation via the proteasome pathway in the dark (Kiba *et al.*, 2007; Mas *et al.*, 2003). The TOC1-ztl interaction does not depend on light, but an interaction between ztl and GI is bluelight dependent, stabilizes both ztl and GI, and contributes to the robust rhythms of TOC1

*al.*, 2005), but this is still unclear (Ito *et al.*, 2009; Martin-Tryon *et al.*, 2007).

of the network against environmental perturbations (Harmer, 2009).

with CCA1 (Imaizumi, 2010).

which ones are input pathways.

**4.2 Light signalling input to the circadian clock** 

(Kim *et al.*, 2007), contributing to a faster degradation of ztl, GI, TOC1 and PRR5 in darkness than in light (Loop 4, figure 2) (Kiba *et al.*, 2007; Kim *et al.*, 2007; Mas *et al.*, 2003). Within this loop 4, TOC1 binds to PRR3, interfering with TOC1 binding to ztl (Para *et al.*, 2007). Thus, PRR3 seems to stabilize TOC1 avoiding its recruitment to the SCF complex and its degradation by the proteasome (Loop 4, figure 2).

Fig. 2. A model for the *Arabidopsis* clock. The circadian clock is composed of several interlocking positive and negative loops.

The other members of the ztl family, fkf1 and lkp2, were also studied. Mutant combinations showed that fkf1 and lkp2 play similar roles to ztl in the circadian clock when ztl is absent, and that both of them interact with TOC1 and PRR5. These results indicate that ztl, fkf1 and lkp2 regulate TOC1 and PRR5 degradation and are important to determine the period of circadian oscillation (Baudry *et al.*, 2010).

Cryptochromes also signal to the circadian clock. However, the mechanisms are still unclear. One possibility is through the regulation of COP1 activity; COP1 directly interacts with ELF3 and with GI to promote GI degradation by the proteasome. This could be a mechanism by which cryptochromes regulate the activity of GI, a protein closely associated with circadian clock function (Yu *et al.*, 2008).

As mentioned above, one interesting aspect of phytochrome and circadian clock is that mutants affected in the clock are also affected in phytochrome responses (Ito *et al.*, 2007). However, how phytochromes contribute to the entrainment of the clock is still unclear. It was previously suggested that PIF3 could directly induce *CCA1* and *LHY* mRNA expression (Martinez-Garcia *et al.*, 2000). Later, it was shown that TOC1 interacts with PIF3 and PIL1

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life 203

Genome-wide CHIP-chip analysis was used to identify HY5 binding regions and to compare this information to HY5-global expression data. This approach allowed the identification of more that 1100 direct targets where HY5 can either activate or repress transcription. However, not all the targets were light responsive genes, suggesting that HY5 must act in concert with other factors to confer light responsiveness (Zhang *et al.*,

**5.3 The dissection of single light responsive promoters reveals another layer of** 

paradigm of light-regulated gene expression (Akhilesh & Gaur, 2003).

different light inputs, as will be explained below.

Most of the photoreceptors, signalling components and transcription factors mentioned above were identified using genetic approaches, after Arabidopsis was established as the model plant. Another strategy to understand light signalling and photosynthetic gene expression has been underway since late mid 80s, after the first transgenic plants became available. This strategy was simple, the generation of transgenic plants bearing promoter:reporter gene fusions. With this approach, light responsive promoters were the subject of extensive research with the aim of finding the light responsive elements (LREs) and their cognate binding factors. The genes encoding the small subunits of the Rubisco (RbcS) and the light-harvesting chlorophyll a/b-binding proteins (Lhc; previously known as Cab), were considered a

Several LREs were described, as GT-1-Boxes (core sequence GGTTAA), I-Boxes (GATAA), G-Boxes (CACGTG), H-Boxes (CCTACC), AT-rich sequences (consensus AATATTTTTATT) (Akhilesh & Gaur, 2003). Using complementary approaches as Gel Shift analysis and DNA footprinting, some of the cognate binding factors were identified. However, three difficulties hampered this approaches. First, the LREs identified were not always enough to sustain light regulation. Hence, it was proposed that combinations of different motifs but not multimerisation of single motifs could function as LREs, confirming the complex nature of these regulatory elements (Chattopadhyay *et al.*, 1998; Puente *et al.*, 1996). Second, when the cognate transcription factors were studied in Arabidopsis with available mutants, a direct role in light signal was not evident. This can be illustrated by the GT-element binding factors, a small family of plant trihelix DNAbinding proteins comprising Arabidopsis GT2 (AT1G76890), DF1L (AT1G76880), PTL (At5g03680), GT-2-LIKE1 (GTL1, AT1G33240), GT2L (At5g28300), EDA31 (AT3G10000) and GTL1L (AT5G47660). Some of these transcription factors have roles in the fusion of the polar nuclei, in the development of the embryo sac or even perianth development (Brewer *et al.*, 2004; Pagnussat *et al.*, 2005), but were not involved in responses to light. The third difficulty was the apparent "redundancy" of LREs in single promoters. This redundancy could be just the consequence of a single promoter responding to several

In a few examples, thorough analysis of promoter sequences, combined with genetic approaches significantly advanced our understanding of light-regulated transcription, but also revealed the complex nature underneath this process. The Arabidopsis *Lhcb1\*1* (*Cab 2*) promoter fused to luciferase reporters has been extensively used as a marker for light and circadian expression. Genetic screenings using this construct led to the isolation of *toc1* mutants (Strayer *et al.*, 2000). Promoter analysis of *Lhcb1\*1* allowed the identification of a 78 bp fragment that was sufficient to confer phytochrome and circadian regulation to a minimal promoter (Anderson *et al.*, 1994). Further analysis of this promoter allowed the

2011).

**complexity** 

(Yamashino *et al.*, 2003). However, thorough analysis of PIF3 function has led to the conclusion that it does not play a significant role in controlling light input to the circadian clock (Viczian *et al.*, 2005).

Indeed, there is circumstantial evidence of phytochromes regulating CCA1 and LHY. Both genes are rapidly induced in a TOC1 dependent manner upon transfer of dark grown seedlings to red light. This induction requires *EARLY FLOWERING 4* (*ELF4*), which forms with CCA1 and LHY a negative feedback loop in an analogous manner to TOC1 (Kikis *et al.*, 2005) and ELF4 is itself a direct target of FHY3, FAR1 and HY5 (Li *et al.*, 2011). *ELF3*, is also necessary for light-induced expression of *CCA1* and *LHY* and this event seems to occur indirectly, through a direct repression of PRR9 by physically interacting with its promoter (Dixon *et al.*, 2011).

#### **5. Downstream targets of light and clock signalling**

#### **5.1 The impact of the circadian clock in the expression of photosynthesis related genes**

As presented above, the interconnections between the clock and light signalling are extremely complex. The regulation of outputs is not an exception. One unbiased measure of the impact of the circadian clock on plant development is the finding that at least one third of the Arabidopsis genome is circadian regulated (Covington *et al.*, 2008). The genes involved in photosynthesis are an important target group of the circadian clock, and tend to be expressed at the middle of the subjective day, together with genes involved in the phenylpropanoid pathway (Edwards *et al.*, 2006). In another global analysis it was shown that PRR5, PRR7 and PRR9 are negative regulators of the chlorophyll and carotenoid biosynthetic pathways (Fukushima *et al.*, 2009).

Despite what we know of the clock impact on photosynthetic gene expression, the mechanisms are still poorly understood. One such mechanism may involve CCA1. CCA1 was originally identified by its binding to an AA(CA)AATCT motif in the *lhcb1\*3* promoter, and also shown to be required for phytochrome responsivity (Wang *et al.*, 1997). Hence, CCA1 can represent one of the mechanisms by which the clock regulates photosynthetic gene expression. Nevertheless, the reality is more complex. CCA1 binding site is similar to the Evening Element (AAAATATCT) found in promoters of clock regulated genes that peak toward the end of the subjective day (Harmer *et al.*, 2000), including TOC1, which is repressed by CCA1 (Alabadi *et al.*, 2001). However, *lhcb1\*3* expression peaks earlier and is promoted by CCA1 (Wang *et al.*, 1997). These apparent contradictions can be reconciled by the finding that CCA1 effects depend on the context, showing also another level of complexity (Harmer & Kay, 2005).

#### **5.2 Global expression analysis identifies the targets of photomorphogenesis master regulators**

HY5, the bZIP targeted by COP1 for degradation, is necessary for responses to a broad spectrum of wavelengths of light and, as explained above, acts as a positive regulator in photomorphogenesis. Arabidopsis plants defective in HY5 show aberrant light mediated phenotypes, including an elongated hypocotyl, reduced chlorophyll/anthocyanin accumulation and reduced chloroplast development in greening hypocotyls (Lee *et al.*, 2007). HY5 regulates the transcription of multiple genes in response to light signals through binding to G-box elements in their promoters such as RBCS1A or CHS1 genes.

(Yamashino *et al.*, 2003). However, thorough analysis of PIF3 function has led to the conclusion that it does not play a significant role in controlling light input to the circadian

Indeed, there is circumstantial evidence of phytochromes regulating CCA1 and LHY. Both genes are rapidly induced in a TOC1 dependent manner upon transfer of dark grown seedlings to red light. This induction requires *EARLY FLOWERING 4* (*ELF4*), which forms with CCA1 and LHY a negative feedback loop in an analogous manner to TOC1 (Kikis *et al.*, 2005) and ELF4 is itself a direct target of FHY3, FAR1 and HY5 (Li *et al.*, 2011). *ELF3*, is also necessary for light-induced expression of *CCA1* and *LHY* and this event seems to occur indirectly, through a direct repression of PRR9 by physically interacting with its promoter

**5.1 The impact of the circadian clock in the expression of photosynthesis related** 

As presented above, the interconnections between the clock and light signalling are extremely complex. The regulation of outputs is not an exception. One unbiased measure of the impact of the circadian clock on plant development is the finding that at least one third of the Arabidopsis genome is circadian regulated (Covington *et al.*, 2008). The genes involved in photosynthesis are an important target group of the circadian clock, and tend to be expressed at the middle of the subjective day, together with genes involved in the phenylpropanoid pathway (Edwards *et al.*, 2006). In another global analysis it was shown that PRR5, PRR7 and PRR9 are negative regulators of the chlorophyll and carotenoid

Despite what we know of the clock impact on photosynthetic gene expression, the mechanisms are still poorly understood. One such mechanism may involve CCA1. CCA1 was originally identified by its binding to an AA(CA)AATCT motif in the *lhcb1\*3* promoter, and also shown to be required for phytochrome responsivity (Wang *et al.*, 1997). Hence, CCA1 can represent one of the mechanisms by which the clock regulates photosynthetic gene expression. Nevertheless, the reality is more complex. CCA1 binding site is similar to the Evening Element (AAAATATCT) found in promoters of clock regulated genes that peak toward the end of the subjective day (Harmer *et al.*, 2000), including TOC1, which is repressed by CCA1 (Alabadi *et al.*, 2001). However, *lhcb1\*3* expression peaks earlier and is promoted by CCA1 (Wang *et al.*, 1997). These apparent contradictions can be reconciled by the finding that CCA1 effects depend on the context, showing also another level of

**5.2 Global expression analysis identifies the targets of photomorphogenesis master** 

HY5, the bZIP targeted by COP1 for degradation, is necessary for responses to a broad spectrum of wavelengths of light and, as explained above, acts as a positive regulator in photomorphogenesis. Arabidopsis plants defective in HY5 show aberrant light mediated phenotypes, including an elongated hypocotyl, reduced chlorophyll/anthocyanin accumulation and reduced chloroplast development in greening hypocotyls (Lee *et al.*, 2007). HY5 regulates the transcription of multiple genes in response to light signals through binding to G-box elements in their promoters such as RBCS1A or CHS1 genes.

**5. Downstream targets of light and clock signalling** 

biosynthetic pathways (Fukushima *et al.*, 2009).

complexity (Harmer & Kay, 2005).

**regulators** 

clock (Viczian *et al.*, 2005).

(Dixon *et al.*, 2011).

**genes** 

Genome-wide CHIP-chip analysis was used to identify HY5 binding regions and to compare this information to HY5-global expression data. This approach allowed the identification of more that 1100 direct targets where HY5 can either activate or repress transcription. However, not all the targets were light responsive genes, suggesting that HY5 must act in concert with other factors to confer light responsiveness (Zhang *et al.*, 2011).

#### **5.3 The dissection of single light responsive promoters reveals another layer of complexity**

Most of the photoreceptors, signalling components and transcription factors mentioned above were identified using genetic approaches, after Arabidopsis was established as the model plant. Another strategy to understand light signalling and photosynthetic gene expression has been underway since late mid 80s, after the first transgenic plants became available. This strategy was simple, the generation of transgenic plants bearing promoter:reporter gene fusions. With this approach, light responsive promoters were the subject of extensive research with the aim of finding the light responsive elements (LREs) and their cognate binding factors. The genes encoding the small subunits of the Rubisco (RbcS) and the light-harvesting chlorophyll a/b-binding proteins (Lhc; previously known as Cab), were considered a paradigm of light-regulated gene expression (Akhilesh & Gaur, 2003).

Several LREs were described, as GT-1-Boxes (core sequence GGTTAA), I-Boxes (GATAA), G-Boxes (CACGTG), H-Boxes (CCTACC), AT-rich sequences (consensus AATATTTTTATT) (Akhilesh & Gaur, 2003). Using complementary approaches as Gel Shift analysis and DNA footprinting, some of the cognate binding factors were identified. However, three difficulties hampered this approaches. First, the LREs identified were not always enough to sustain light regulation. Hence, it was proposed that combinations of different motifs but not multimerisation of single motifs could function as LREs, confirming the complex nature of these regulatory elements (Chattopadhyay *et al.*, 1998; Puente *et al.*, 1996). Second, when the cognate transcription factors were studied in Arabidopsis with available mutants, a direct role in light signal was not evident. This can be illustrated by the GT-element binding factors, a small family of plant trihelix DNAbinding proteins comprising Arabidopsis GT2 (AT1G76890), DF1L (AT1G76880), PTL (At5g03680), GT-2-LIKE1 (GTL1, AT1G33240), GT2L (At5g28300), EDA31 (AT3G10000) and GTL1L (AT5G47660). Some of these transcription factors have roles in the fusion of the polar nuclei, in the development of the embryo sac or even perianth development (Brewer *et al.*, 2004; Pagnussat *et al.*, 2005), but were not involved in responses to light. The third difficulty was the apparent "redundancy" of LREs in single promoters. This redundancy could be just the consequence of a single promoter responding to several different light inputs, as will be explained below.

In a few examples, thorough analysis of promoter sequences, combined with genetic approaches significantly advanced our understanding of light-regulated transcription, but also revealed the complex nature underneath this process. The Arabidopsis *Lhcb1\*1* (*Cab 2*) promoter fused to luciferase reporters has been extensively used as a marker for light and circadian expression. Genetic screenings using this construct led to the isolation of *toc1* mutants (Strayer *et al.*, 2000). Promoter analysis of *Lhcb1\*1* allowed the identification of a 78 bp fragment that was sufficient to confer phytochrome and circadian regulation to a minimal promoter (Anderson *et al.*, 1994). Further analysis of this promoter allowed the

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life 205

(Tanaka & Tanaka, 2007). Phytochrome and cryptochrome mutants contain lower levels of chlorophyll (Strasser *et al.*, 2010) stressing out the importance of the photomorphogenic signal for proper assembly of the photosynthetic machinery. In the next paragraphs we

review how light signalling pathways regulate chlorophyll biosynthesis (Figure 4).

Fig. 3. Light interactions in plastid development. Phytochrome and PIFs roles during the

2004; McCormac *et al.*, 2001; McCormac & Terry, 2002a; McCormac & Terry, 2002b).

biogenesis with circadian and light signalling (Stephenson *et al.*, 2009).

Glutamyl-tRNA reductase activity is regulated by negative feedback loops; the accumulation of Haeme, Mg-Protoporphyrin IX or Divinyl protochlorofilide a antagonise Glutamyl-tRNA reductase activity (Srivastava *et al.*, 2005). At the transcriptional level, *HEMA1* expression is induced by red and far-red light, implicating at least phyA and phyB, and blue light perceived by cry1 (McCormac *et al.*, 2001; McCormac & Terry, 2002a). *pif1* and *pif3* mutants contain higher levels of *HEMA1* mRNA, higher levels of protochlorophyllide and partially developed chloroplasts in the dark, a phenotype observed in *cop* mutants. The effects of *pif1* and *pif3* mutations are essentially additive, suggesting a model where phytochromes promote chloroplast biogenesis by antagonizing the activity of at least PIF1 and PIF3. As PIF1 and PIF3 are regulated by the circadian clock, but do not seem to affect central clock components (TOC1, CCA1, LHY), these PIFs seem to integrate chloroplast

The expression of photosynthetic nuclear genes is repressed by plastid signals if chloroplast biogenesis is blocked (retrograde signalling). This finding led to the isolation of mutants that disrupt chloroplast to nucleus communication, the genomes uncoupled mutants (*gun*) (Nott *et al.*, 2006). These mutants show high levels of *lhcb1* mRNA in the presence of norfluorzazon and were named *gun1* to *gun5*. *gun2* and *gun3* are allelic to *hy1* and *hy2* and disrupt phytochromobilin synthesis, leading to haeme accumulation and feedback

Chlorophyll synthesis occurs in plastids; in the first step glutamate is activated to GlutamyltRNA by the Glutamyl-tRNA synthetase, a step shared with plastid protein synthesis. The following step, the reduction of the Glutamyl-tRNA to produce glutamate-1-semialdehyde is subjected to tight regulation (Figure 4). In Arabidopsis, the Glutamyl-tRNA reductases are encoded by a little family of nuclear genes called *HEMA*. Of this family, the expression of *HEMA1* correlates with the expression of *Lhcb1* genes, which encode light-harvesting proteins of the photosystem II; in some way the expression of *HEMA1* reflects the demand of chlorophyll synthesis. On the other hand, *HEMA2* is not light regulated (Matsumoto *et al.*,

transition from proplastid or amyloplast to chloroplast.

identification of HY5, CCA1 and a DET1 responsive elements (Maxwell *et al.*, 2003). Similarly, it has been shown that HY5 binds to the *Lhcb1\*3* promoter and physically interacts with CCA1 to synergistically regulate expression (Andronis *et al.*, 2008).

Another promoter analysed in more detail was the tobacco *Lhcb1\*2*. First, a 146 bp promoter fragment sufficed to confer VLFR (mediated by phyA), LFR (mediated by phyB) and HIR (mediated by phyA) to a minimal promoter (Cerdan *et al.*, 1997). Then, the motifs for VLFR and LFR were dissected from the HIR responsive motifs (Cerdan *et al.*, 2000) and finally, the TGGA motif was shown to bind Bell-like homeodomain 1 (BLH1) as part of the phyA mediated HIR (Staneloni *et al.*, 2009). This promoter is an example of how several different photoreceptors can regulate a single gene and integrate their signalling pathways at the promoter level; at least four different photoreceptors were shown to regulate this single promoter (Casal *et al.*, 1998; Cerdan *et al.*, 1999; Mazzella *et al.*, 2001) .

#### **6. Light promotes chloroplast development**

Proplastids are found in the embryo; they are undifferentiated plastids that are converted to other kind of plastids like chromoplasts, amyloplasts, chloroplasts and etioplasts. During skotomorphogenic development, proplastids turn into etioplasts, the chloroplast precursors. Etioplasts contain the prolamellar body, a structure rich in protochlorophyllide, the chlorophyll precursor, and the enzyme protochlorophyllide oxidoreductase (POR). During the development of etioplasts into chloroplasts, the POR is directly activated by light to convert protochlorophyllide into divinyl-chlorophyllide a, which is chlorophyll a and b precursor (Tanaka & Tanaka, 2007). This light-dependent step can be promoted by red-light in Arabidopsis, even in the absence of phytochromes (Strasser *et al.*, 2010). However, other events that occur during chloroplast biogenesis require the signals transduced by photoreceptors. These signals ensure proper coordination of synthesis and import of LHCB proteins, which are essential for the assembly of the photosynthetic complexes. These events are also coordinated with the synthesis of carotenoids, which are necessary for photoprotection (Cazzonelli & Pogson, 2010).

Phytochromes, through the action of PIFs, regulate the transition from amiloplasts to etioplasts and to chloroplasts. For example, the PIFs inhibit the conversion of endodermal amyloplasts to etioplasts, whereas the phytochromes antagonise this inhibition, promoting the formation of chloroplasts (Figure 3) (Kim *et al.*, 2011).

#### **6.1 Chlorophyll biosynthesis is regulated by light**

Chlorophyll biosynthesis and the synthesis of other components of the photosystems are tightly regulated by light and the circadian clock. This coordination is necessary because when the chlorophyll synthesis exceeds the accumulation of chlorophyll-binding apoproteins, reactive oxygen species are generated, ultimately leading to cell death. However, when the chlorophyll synthesis is not enough, the amount of fully functional chlorophyll-binding proteins is not sufficient to gain optimal photosynthetic activity. Another example highlighting the importance of proper coordination is that PIF deficient plants accumulate protochlorophyllide in the dark during skotomorphogenic development, but this accumulation leads to bleaching upon exposure to light (Stephenson *et al.*, 2009).

Plants have four classes of tetrapyrroles: chlorophyll, phytochromobilin, haeme and siroheme, all derived from the same biosynthetic pathway. The flow of the tetrapyrrole pathway is strictly regulated, keeping at low levels the potentially toxic intermediates

identification of HY5, CCA1 and a DET1 responsive elements (Maxwell *et al.*, 2003). Similarly, it has been shown that HY5 binds to the *Lhcb1\*3* promoter and physically interacts

Another promoter analysed in more detail was the tobacco *Lhcb1\*2*. First, a 146 bp promoter fragment sufficed to confer VLFR (mediated by phyA), LFR (mediated by phyB) and HIR (mediated by phyA) to a minimal promoter (Cerdan *et al.*, 1997). Then, the motifs for VLFR and LFR were dissected from the HIR responsive motifs (Cerdan *et al.*, 2000) and finally, the TGGA motif was shown to bind Bell-like homeodomain 1 (BLH1) as part of the phyA mediated HIR (Staneloni *et al.*, 2009). This promoter is an example of how several different photoreceptors can regulate a single gene and integrate their signalling pathways at the promoter level; at least four different photoreceptors were shown to regulate this single

Proplastids are found in the embryo; they are undifferentiated plastids that are converted to other kind of plastids like chromoplasts, amyloplasts, chloroplasts and etioplasts. During skotomorphogenic development, proplastids turn into etioplasts, the chloroplast precursors. Etioplasts contain the prolamellar body, a structure rich in protochlorophyllide, the chlorophyll precursor, and the enzyme protochlorophyllide oxidoreductase (POR). During the development of etioplasts into chloroplasts, the POR is directly activated by light to convert protochlorophyllide into divinyl-chlorophyllide a, which is chlorophyll a and b precursor (Tanaka & Tanaka, 2007). This light-dependent step can be promoted by red-light in Arabidopsis, even in the absence of phytochromes (Strasser *et al.*, 2010). However, other events that occur during chloroplast biogenesis require the signals transduced by photoreceptors. These signals ensure proper coordination of synthesis and import of LHCB proteins, which are essential for the assembly of the photosynthetic complexes. These events are also coordinated with the synthesis of carotenoids, which are necessary for

Phytochromes, through the action of PIFs, regulate the transition from amiloplasts to etioplasts and to chloroplasts. For example, the PIFs inhibit the conversion of endodermal amyloplasts to etioplasts, whereas the phytochromes antagonise this inhibition, promoting

Chlorophyll biosynthesis and the synthesis of other components of the photosystems are tightly regulated by light and the circadian clock. This coordination is necessary because when the chlorophyll synthesis exceeds the accumulation of chlorophyll-binding apoproteins, reactive oxygen species are generated, ultimately leading to cell death. However, when the chlorophyll synthesis is not enough, the amount of fully functional chlorophyll-binding proteins is not sufficient to gain optimal photosynthetic activity. Another example highlighting the importance of proper coordination is that PIF deficient plants accumulate protochlorophyllide in the dark during skotomorphogenic development, but this accumulation leads to bleaching upon exposure to light (Stephenson *et al.*, 2009). Plants have four classes of tetrapyrroles: chlorophyll, phytochromobilin, haeme and siroheme, all derived from the same biosynthetic pathway. The flow of the tetrapyrrole pathway is strictly regulated, keeping at low levels the potentially toxic intermediates

with CCA1 to synergistically regulate expression (Andronis *et al.*, 2008).

promoter (Casal *et al.*, 1998; Cerdan *et al.*, 1999; Mazzella *et al.*, 2001) .

**6. Light promotes chloroplast development** 

photoprotection (Cazzonelli & Pogson, 2010).

the formation of chloroplasts (Figure 3) (Kim *et al.*, 2011).

**6.1 Chlorophyll biosynthesis is regulated by light** 

(Tanaka & Tanaka, 2007). Phytochrome and cryptochrome mutants contain lower levels of chlorophyll (Strasser *et al.*, 2010) stressing out the importance of the photomorphogenic signal for proper assembly of the photosynthetic machinery. In the next paragraphs we review how light signalling pathways regulate chlorophyll biosynthesis (Figure 4).

Fig. 3. Light interactions in plastid development. Phytochrome and PIFs roles during the transition from proplastid or amyloplast to chloroplast.

Chlorophyll synthesis occurs in plastids; in the first step glutamate is activated to GlutamyltRNA by the Glutamyl-tRNA synthetase, a step shared with plastid protein synthesis. The following step, the reduction of the Glutamyl-tRNA to produce glutamate-1-semialdehyde is subjected to tight regulation (Figure 4). In Arabidopsis, the Glutamyl-tRNA reductases are encoded by a little family of nuclear genes called *HEMA*. Of this family, the expression of *HEMA1* correlates with the expression of *Lhcb1* genes, which encode light-harvesting proteins of the photosystem II; in some way the expression of *HEMA1* reflects the demand of chlorophyll synthesis. On the other hand, *HEMA2* is not light regulated (Matsumoto *et al.*, 2004; McCormac *et al.*, 2001; McCormac & Terry, 2002a; McCormac & Terry, 2002b).

Glutamyl-tRNA reductase activity is regulated by negative feedback loops; the accumulation of Haeme, Mg-Protoporphyrin IX or Divinyl protochlorofilide a antagonise Glutamyl-tRNA reductase activity (Srivastava *et al.*, 2005). At the transcriptional level, *HEMA1* expression is induced by red and far-red light, implicating at least phyA and phyB, and blue light perceived by cry1 (McCormac *et al.*, 2001; McCormac & Terry, 2002a). *pif1* and *pif3* mutants contain higher levels of *HEMA1* mRNA, higher levels of protochlorophyllide and partially developed chloroplasts in the dark, a phenotype observed in *cop* mutants. The effects of *pif1* and *pif3* mutations are essentially additive, suggesting a model where phytochromes promote chloroplast biogenesis by antagonizing the activity of at least PIF1 and PIF3. As PIF1 and PIF3 are regulated by the circadian clock, but do not seem to affect central clock components (TOC1, CCA1, LHY), these PIFs seem to integrate chloroplast biogenesis with circadian and light signalling (Stephenson *et al.*, 2009).

The expression of photosynthetic nuclear genes is repressed by plastid signals if chloroplast biogenesis is blocked (retrograde signalling). This finding led to the isolation of mutants that disrupt chloroplast to nucleus communication, the genomes uncoupled mutants (*gun*) (Nott *et al.*, 2006). These mutants show high levels of *lhcb1* mRNA in the presence of norfluorzazon and were named *gun1* to *gun5*. *gun2* and *gun3* are allelic to *hy1* and *hy2* and disrupt phytochromobilin synthesis, leading to haeme accumulation and feedback

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life 207

the cryptochromes, establishing GUN4 as a link between the phytochromes and the regulation of MgCH activity (Stephenson & Terry, 2008). *GUN5* encodes the H subunit of MgCH, known as CHLH (Nott *et al.*, 2006). The expression of *CHLH* is regulated at the mRNA level by light/dark cycles and by the circadian clock. Interestingly, this gene is co-regulated with *HEMA1*, *lhcb, Mg-protoporphyrin IX monomethyl estercyclase (MGCy)* and the gene encoding the chlorophyll(ide) a oxygenase (*CAO*) (Matsumoto *et al.*, 2004). On the other hand, *GUN1* encodes a pentatricopeptide repeat–containing protein that does not affect chlorophyll synthesis. GUN1 was proposed to generate a signal in chloroplast that represses nuclear photosynthetic gene expression; this repression on *lhcb* genes seems to be mediated by direct

Another connection between light signalling and the retrograde signalling was recently established. A sensitive genetic screening for the *gun* phenotype uncovered new cry1 alleles. These results establish that cry1 is necessary for maximal repression of *lhcb* genes, when

One of the latest steps in chlorophyll synthesis is the reduction of 3,8-divinyl protochlorophyllide to 3,8-divinyl chlorophyllide. This protochlorophyllide to chlorophyllide conversion is catalysed by the POR enzyme. In angiosperms, POR is light-dependent and it is likely the source of red-light promoted chlorophyll synthesis in the absence of phytochromes (Strasser *et al.*, 2010). Angiosperms carry three POR-encoding genes, *PorA, PorB* and *PorC*, which are differentially regulated by both light and developmental stage. *PORA* expression is high in etiolated seedlings and rapidly becomes undetectable after illumination with FR, a HIR response mediated by phyA, whereas *PORB* expression persists throughout greening and in adult plants (Runge *et al.*, 1996). PORC is expressed during the adult life and together with PORB is responsible for bulk chlorophyll synthesis in green plants (Paddock *et al.*, 2010). It has been recently shown that *PORC* expression is directly activated by PIF1 binding to a G-box in *PORC* promoter, whereas *PORA* and *PORB* are also induced by PIF1, presumably in an

During the last twenty years, plant biologists have witnessed major advances in our understanding of how plants use light as a source of information. These advances were possible thanks to the adoption of Arabidopsis as a model system. During these twenty years, 13 Arabidopsis photoreceptors were characterised In molecular terms and these findings extended to other species as well. A high number of signal transduction components were also characterised. With the advent of "omics" technologies, the networks that work downstream photoreceptors and their targets started to surface. However, with all these advances, we still do not know in detail how a single light responsive promoter works. How many transcription factors are sitting there? Which are their identities? How do they interact to fine tune expression under the diverse light conditions found in nature? If we multiply these questions by the number of light responsive promoters we can just have a

Akhilesh, K. T. &Gaur, T. (2003). Light regulation of nuclear photosynthetic genes in higher

plants. *Critical Reviews in Plant Sciences* 22(5): 417-452.

binding of *ABI4*, an AP2–type transcription factor (Koussevitzky *et al.*, 2007).

chloroplast biogenesis is blocked (Ruckle *et al.*, 2007).

indirect manner (Moon *et al.*, 2008).

hint of the enormous task ahead.

**7. Conclusion** 

**8. References** 

inhibition of Glutamyl-tRNA reductase (Nott *et al.*, 2006). The product of the *GUN4* gene, a 22 kD protein localized to Chloroplasts, promotes Magnesium chelatase (MgCH) activity which catalyses the insertion of Mg2+ into protoporphyrin IX (Tanaka & Tanaka, 2007). The *GUN4* gene is also under circadian clock regulation and is repressed by PIF1 and PIF3 suggesting a similar regulatory mechanism to HEMA1 (Stephenson *et al.*, 2009). The expression of GUN4 is primarily under the control of phyA and phyB with some input from

Fig. 4. Simplified chlorophyll biosynthesis pathway and light regulated steps. We emphasise how the light regulate directly the activity of NADPH:protochlorophyllide oxidoreductase (POR); or indirectly, through phytochrome and PIFs the expression of genes encoding the Glutamyl-tRNA reductases (HEMAs), Ferrum chelatase (FeCH), Magnesium chelatase (MgCH), NADPH:protochlorophyllide oxidoreductase (POR), Chlorophyllide a oxygenase (CAO), Mg-protoporphyrin IX methyltransferase (MgMT), and Mg-protoporphyrin IX monomethyl estercyclase (MgCy). The ATP/ADP ratio, the Mg2+ concentration and the thioredoxin levels also affect the MgCH activity, furthermore, these factors are light regulated (Tanaka & Tanaka, 2007). LHCs attach *chlorophyll a*, and CAO converts the *chlorophyll* a to *b* on the LHC apoprotein (Tanaka & Tanaka, 2007).

the cryptochromes, establishing GUN4 as a link between the phytochromes and the regulation of MgCH activity (Stephenson & Terry, 2008). *GUN5* encodes the H subunit of MgCH, known as CHLH (Nott *et al.*, 2006). The expression of *CHLH* is regulated at the mRNA level by light/dark cycles and by the circadian clock. Interestingly, this gene is co-regulated with *HEMA1*, *lhcb, Mg-protoporphyrin IX monomethyl estercyclase (MGCy)* and the gene encoding the chlorophyll(ide) a oxygenase (*CAO*) (Matsumoto *et al.*, 2004). On the other hand, *GUN1* encodes a pentatricopeptide repeat–containing protein that does not affect chlorophyll synthesis. GUN1 was proposed to generate a signal in chloroplast that represses nuclear photosynthetic gene expression; this repression on *lhcb* genes seems to be mediated by direct binding of *ABI4*, an AP2–type transcription factor (Koussevitzky *et al.*, 2007).

Another connection between light signalling and the retrograde signalling was recently established. A sensitive genetic screening for the *gun* phenotype uncovered new cry1 alleles. These results establish that cry1 is necessary for maximal repression of *lhcb* genes, when chloroplast biogenesis is blocked (Ruckle *et al.*, 2007).

One of the latest steps in chlorophyll synthesis is the reduction of 3,8-divinyl protochlorophyllide to 3,8-divinyl chlorophyllide. This protochlorophyllide to chlorophyllide conversion is catalysed by the POR enzyme. In angiosperms, POR is light-dependent and it is likely the source of red-light promoted chlorophyll synthesis in the absence of phytochromes (Strasser *et al.*, 2010). Angiosperms carry three POR-encoding genes, *PorA, PorB* and *PorC*, which are differentially regulated by both light and developmental stage. *PORA* expression is high in etiolated seedlings and rapidly becomes undetectable after illumination with FR, a HIR response mediated by phyA, whereas *PORB* expression persists throughout greening and in adult plants (Runge *et al.*, 1996). PORC is expressed during the adult life and together with PORB is responsible for bulk chlorophyll synthesis in green plants (Paddock *et al.*, 2010). It has been recently shown that *PORC* expression is directly activated by PIF1 binding to a G-box in *PORC* promoter, whereas *PORA* and *PORB* are also induced by PIF1, presumably in an indirect manner (Moon *et al.*, 2008).

#### **7. Conclusion**

206 Advances in Photosynthesis – Fundamental Aspects

inhibition of Glutamyl-tRNA reductase (Nott *et al.*, 2006). The product of the *GUN4* gene, a 22 kD protein localized to Chloroplasts, promotes Magnesium chelatase (MgCH) activity which catalyses the insertion of Mg2+ into protoporphyrin IX (Tanaka & Tanaka, 2007). The *GUN4* gene is also under circadian clock regulation and is repressed by PIF1 and PIF3 suggesting a similar regulatory mechanism to HEMA1 (Stephenson *et al.*, 2009). The expression of GUN4 is primarily under the control of phyA and phyB with some input from

Glutamate

**Light**

Phytochromes

PIFs

Protoheme

FeCH

Heme oxidases

Phytochromebilin

**Light**

Other Hemes

Glutamyl-tRNA

HemA

5-Aminoleculinic acid

Protoporphyrinogen IX

ATP Mg2+ Thiredoxin GUN4

PIFs Phytochromes

Chlorophyll b

Fig. 4. Simplified chlorophyll biosynthesis pathway and light regulated steps. We emphasise how the light regulate directly the activity of NADPH:protochlorophyllide oxidoreductase (POR); or indirectly, through phytochrome and PIFs the expression of genes encoding the Glutamyl-tRNA reductases (HEMAs), Ferrum chelatase (FeCH), Magnesium chelatase (MgCH), NADPH:protochlorophyllide oxidoreductase (POR), Chlorophyllide a oxygenase (CAO), Mg-protoporphyrin IX methyltransferase (MgMT), and Mg-protoporphyrin IX monomethyl estercyclase (MgCy). The ATP/ADP ratio, the Mg2+ concentration and the thioredoxin levels also affect the MgCH activity, furthermore, these factors are light regulated (Tanaka & Tanaka, 2007). LHCs attach *chlorophyll a*, and CAO converts the

Lchb1

Mg- protoporphyrin IX

FLU

Divinyl protochlorofilide a

**Light**

Divinyl chlorofilide a

Chlorophyll a

MgCH

MgCY MgMT

POR

CAO

*chlorophyll* a to *b* on the LHC apoprotein (Tanaka & Tanaka, 2007).

During the last twenty years, plant biologists have witnessed major advances in our understanding of how plants use light as a source of information. These advances were possible thanks to the adoption of Arabidopsis as a model system. During these twenty years, 13 Arabidopsis photoreceptors were characterised In molecular terms and these findings extended to other species as well. A high number of signal transduction components were also characterised. With the advent of "omics" technologies, the networks that work downstream photoreceptors and their targets started to surface. However, with all these advances, we still do not know in detail how a single light responsive promoter works. How many transcription factors are sitting there? Which are their identities? How do they interact to fine tune expression under the diverse light conditions found in nature? If we multiply these questions by the number of light responsive promoters we can just have a hint of the enormous task ahead.

#### **8. References**

Akhilesh, K. T. &Gaur, T. (2003). Light regulation of nuclear photosynthetic genes in higher plants. *Critical Reviews in Plant Sciences* 22(5): 417-452.

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life 209

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**11** 

*Japan* 

*Kyushu University* 

**Chloroplast Photorelocation Movement:** 

**to Perform Efficient Photosynthesis** 

Noriyuki Suetsugu and Masamitsu Wada

**A Sophisticated Strategy for Chloroplasts** 

Chloroplasts move to weak light so that they can perceive light efficiently (the accumulation response), whereas they escape from strong light to avoid photodamage (the avoidance

Fig. 1. Typical intracellular distribution pattern of chloroplasts by their photorelocation movement. In darkness, chloroplasts are located on the cell bottom in *Arabidopsis thaliana*. Note that the dark position varies among plant species. Weak light induces the chloroplast accumulation response along the peliclinal walls so that chloroplasts can perceive light efficiently. Strong light induces the chloroplast avoidance response toward the anticlinal

The phototropin photoreceptor family of proteins, which includes phototropin (phot) and neochrome (neo), mediate chloroplast photorelocation movement in green plants (reviewed

**1. Introduction** 

response) (Fig. 1).

walls to reduce photodamage.


### **Chloroplast Photorelocation Movement: A Sophisticated Strategy for Chloroplasts to Perform Efficient Photosynthesis**

Noriyuki Suetsugu and Masamitsu Wada *Kyushu University Japan* 

#### **1. Introduction**

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Fig. 1. Typical intracellular distribution pattern of chloroplasts by their photorelocation movement. In darkness, chloroplasts are located on the cell bottom in *Arabidopsis thaliana*. Note that the dark position varies among plant species. Weak light induces the chloroplast accumulation response along the peliclinal walls so that chloroplasts can perceive light efficiently. Strong light induces the chloroplast avoidance response toward the anticlinal walls to reduce photodamage.

The phototropin photoreceptor family of proteins, which includes phototropin (phot) and neochrome (neo), mediate chloroplast photorelocation movement in green plants (reviewed

Chloroplast Photorelocation Movement: A Sophisticated

mechanism and (iii) the contribution to photosynthesis.

discussed.

**detailed photobiological analyses** 

Strategy for Chloroplaststo Perform Efficient Photosynthesis 217

facilitate leaf photosynthesis by allowing greater light penetration to lower parts within the leaf (reviewed by Suetsugu & Wada, 2009) (Fig. 4b & 4c). However, these hypotheses are

In this chapter, we review three topics of chloroplast photorelocation movement: (i) the insights gained from physiological and photobiological analyses, (ii) the molecular

Light-induced chloroplast movement (chloroplast photorelocation movement) has fascinated plant biologists since its discovery in the mid-nineteenth century (Böhm, 1856). Comprehensive analyses by Gustav Senn (1875-1945) of chloroplast movement in various plant species revealed the general responses of chloroplasts to light intensity and direction; chloroplasts are distributed at a position that ensures more efficient light absorption under weak light conditions, and they are positioned away from strong light, as if they had escaped (Senn, 1908). In land plant species, which generally bear multiple small chloroplasts in a cell, low light induces chloroplast movement and distribution toward the periclinal walls (the accumulation response), whereas strong light induces chloroplast avoidance toward the anticlinal walls (the avoidance response) (Fig. 1). Blue light is most effective at inducing chloroplast movement in most plant species, but in some cryptogam plant species, red light as well as blue light is effective. This red light effect exhibits red/far-red light reversibility, suggestive of the involvement of a red/far-red light receptor phytochrome. Detailed photobiological analyses, especially by the research groups of Wolfgang Haupt (1921-2005) and Jan Zurzycki (1925-1984), have provided many important insights on putative photoreceptor molecules that regulate chloroplast movement (Haupt, 1999; Zurzycki, 1980). They found that membrane-bound blue light photoreceptors other than phytochrome mediate blue-light-induced chloroplast movement in most plant species and that membrane-bound phytochromes mediate red-light induced chloroplast movement in some cryptogam plants. These predictions were demonstrated by the recent identification of photoreceptor genes in various plant species. The blue light receptor phot mediates bluelight-induced chloroplast movement in various plant species (Jarillo et al., 2001; Kagawa et al., 2001, 2004; Kasahara et al., 2004; Sakai et al., 2001). Neo, the chimeric photoreceptor that is a fusion of phytochrome and phototropin, regulates red-light-induced chloroplast movement in ferns and some green alga (Kawai et al., 2003; Suetsugu et al., 2005b). The photoreceptors are not discussed here; for a comprehensive review, see Suetsugu & Wada, 2005, 2007a, 2007b, 2009. First, we show our attempts to elucidate the mechanism of chloroplast photorelocation movement by detailed photobiological analyses. Second, we review recent molecular biological analyses of chloroplast photorelocation movement. Finally, the contribution of chloroplast movement and positioning to photosynthesis is

**2.1 Elucidation of the mechanism of chloroplast photorelocation movement by** 

The underlying processes of chloroplast photorelocation movement can be categorized into three parts: photoperception, signal transduction and the motility system. Most of the photobiological analyses of chloroplast photorelocation movement were performed to

controversial and have not yet been clearly demonstrated experimentally.

**2. From physiological analyses to molecular genetic analyses** 

by Suetsugu & Wada, 2007b, 2009). Phot mediates blue-light-induced chloroplast movement in most green plant species, and neo mediates red-light-induced chloroplast movement in ferns and some green alga (reviewed by Suetsugu & Wada, 2005, 2007a). Like other plant organelle movement responses, chloroplast photorelocation movement depends on actin filaments (reviewed by Wada & Suetsugu, 2004). Detailed physiological and photobiological analyses revealed that chloroplasts could move in any direction without turning or rolling within a short lag time during both the accumulation and the avoidance responses (Tsuboi et al., 2009; Tsuboi & Wada, 2011a). This fact argued that chloroplasts move by utilizing preexisting actin filaments and myosins. However, recent detailed microscopic analyses in the flowering plant *A. thaliana* (Kadota et al., 2009), the fern *Adiantum capillus-veneris* (Tsuboi & Wada, 2011b) and the moss *Physcomitrella patens* (Yamashita et al., 2011) have revealed that short actin filaments around the periphery of chloroplasts (called as cp-actin filaments) but not cytoplasmic actin cables are involved in chloroplast photorelocation movement and in the attachment to the plasma membrane (Fig. 2). Furthermore, chloroplast photorelocation movement was normal in all of the examined multiple-myosin knockout plants and even in myosin mutant plants severely defective in movements of the mitochondria, Golgi bodies, peroxisomes, endoplasmic reticulum and cytoplasm (Suetsugu et al., 2010b). Molecular genetic analyses using *A. thaliana* are of a great benefit to the study of chloroplast movement. First, various molecular factors that regulate cp-actin filament generation and reorganization during chloroplast movement can be identified (Fig. 3). Two phototropins, phot1 and phot2, mediate chloroplast photorelocation movement (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001) by reorganizing cp-actin filaments (Kadota et al., 2009, Ichikawa et al., 2011). Two interacting coiled-coil proteins, WEB1 (weak chloroplast movement under blue light 1) and PMI2 (plastid movement impaired 2), regulate the velocity of chloroplast movement via light-induced cp-actin filament reorganization, possibly by suppressing JAC1 (J-domain protein required for chloroplast accumulation response 1) (Kodama et al., 2010, 2011; Luesse et al., 2006; Suetsugu et al., 2005a). A chloroplast outer envelope protein, CHUP1 (chloroplast unusual positioning 1), and two kinesin-like proteins, KAC1 (kinesin-like protein for actin-based chloroplast movement 1) and KAC2, are indispensable for cp-actin filament formation (Kadota et al., 2009; Oikawa et al., 2003; Suetsugu et al., 2010a). CHUP1 and KAC1 showed in vitro F-actin binding activity, and CHUP1 also interacted with G-actin and profilin in vitro (Oikawa et al., 2003; Schmidt von Braun & Schleiff, 2008; Suetsugu et al., 2010a), suggesting the direct involvement of these proteins in cp-actin filament generation and regulation. Most of these components are highly conserved in land plants from bryophytes to angiosperms (reviewed by Suetsugu et al., 2010b), suggesting that cp-actin filament-mediated chloroplast movement may facilitate the explosive evolution of land plants when in a fluctuating, ambient light environment. Second, the availability of mutants deficient in chloroplast movement encouraged us to verify a long-standing hypothesis that chloroplast movement is required for efficient photosynthesis in fluctuating light conditions. Experiments using mutants deficient in avoidance movement showed that the avoidance response is necessary for reducing photodamage under strong light conditions (Kasahara et al., 2002)(Fig. 4a). Some reports have suggested that the avoidance response (i.e. the distribution of chloroplasts on the anticlinal walls) affects CO2 diffusion by changing the chloroplast surface that is exposed to intercellular air spaces and that the avoidance response in the upper part of the leaf could

by Suetsugu & Wada, 2007b, 2009). Phot mediates blue-light-induced chloroplast movement in most green plant species, and neo mediates red-light-induced chloroplast movement in ferns and some green alga (reviewed by Suetsugu & Wada, 2005, 2007a). Like other plant organelle movement responses, chloroplast photorelocation movement depends on actin filaments (reviewed by Wada & Suetsugu, 2004). Detailed physiological and photobiological analyses revealed that chloroplasts could move in any direction without turning or rolling within a short lag time during both the accumulation and the avoidance responses (Tsuboi et al., 2009; Tsuboi & Wada, 2011a). This fact argued that chloroplasts move by utilizing preexisting actin filaments and myosins. However, recent detailed microscopic analyses in the flowering plant *A. thaliana* (Kadota et al., 2009), the fern *Adiantum capillus-veneris* (Tsuboi & Wada, 2011b) and the moss *Physcomitrella patens* (Yamashita et al., 2011) have revealed that short actin filaments around the periphery of chloroplasts (called as cp-actin filaments) but not cytoplasmic actin cables are involved in chloroplast photorelocation movement and in the attachment to the plasma membrane (Fig. 2). Furthermore, chloroplast photorelocation movement was normal in all of the examined multiple-myosin knockout plants and even in myosin mutant plants severely defective in movements of the mitochondria, Golgi bodies, peroxisomes, endoplasmic reticulum and cytoplasm (Suetsugu et al., 2010b). Molecular genetic analyses using *A. thaliana* are of a great benefit to the study of chloroplast movement. First, various molecular factors that regulate cp-actin filament generation and reorganization during chloroplast movement can be identified (Fig. 3). Two phototropins, phot1 and phot2, mediate chloroplast photorelocation movement (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001) by reorganizing cp-actin filaments (Kadota et al., 2009, Ichikawa et al., 2011). Two interacting coiled-coil proteins, WEB1 (weak chloroplast movement under blue light 1) and PMI2 (plastid movement impaired 2), regulate the velocity of chloroplast movement via light-induced cp-actin filament reorganization, possibly by suppressing JAC1 (J-domain protein required for chloroplast accumulation response 1) (Kodama et al., 2010, 2011; Luesse et al., 2006; Suetsugu et al., 2005a). A chloroplast outer envelope protein, CHUP1 (chloroplast unusual positioning 1), and two kinesin-like proteins, KAC1 (kinesin-like protein for actin-based chloroplast movement 1) and KAC2, are indispensable for cp-actin filament formation (Kadota et al., 2009; Oikawa et al., 2003; Suetsugu et al., 2010a). CHUP1 and KAC1 showed in vitro F-actin binding activity, and CHUP1 also interacted with G-actin and profilin in vitro (Oikawa et al., 2003; Schmidt von Braun & Schleiff, 2008; Suetsugu et al., 2010a), suggesting the direct involvement of these proteins in cp-actin filament generation and regulation. Most of these components are highly conserved in land plants from bryophytes to angiosperms (reviewed by Suetsugu et al., 2010b), suggesting that cp-actin filament-mediated chloroplast movement may facilitate the explosive evolution of land plants when in a fluctuating, ambient light environment. Second, the availability of mutants deficient in chloroplast movement encouraged us to verify a long-standing hypothesis that chloroplast movement is required for efficient photosynthesis in fluctuating light conditions. Experiments using mutants deficient in avoidance movement showed that the avoidance response is necessary for reducing photodamage under strong light conditions (Kasahara et al., 2002)(Fig. 4a). Some reports have suggested that the avoidance response (i.e. the distribution of chloroplasts on the anticlinal walls) affects CO2 diffusion by changing the chloroplast surface that is exposed to intercellular air spaces and that the avoidance response in the upper part of the leaf could facilitate leaf photosynthesis by allowing greater light penetration to lower parts within the leaf (reviewed by Suetsugu & Wada, 2009) (Fig. 4b & 4c). However, these hypotheses are controversial and have not yet been clearly demonstrated experimentally.

In this chapter, we review three topics of chloroplast photorelocation movement: (i) the insights gained from physiological and photobiological analyses, (ii) the molecular mechanism and (iii) the contribution to photosynthesis.

#### **2. From physiological analyses to molecular genetic analyses**

Light-induced chloroplast movement (chloroplast photorelocation movement) has fascinated plant biologists since its discovery in the mid-nineteenth century (Böhm, 1856). Comprehensive analyses by Gustav Senn (1875-1945) of chloroplast movement in various plant species revealed the general responses of chloroplasts to light intensity and direction; chloroplasts are distributed at a position that ensures more efficient light absorption under weak light conditions, and they are positioned away from strong light, as if they had escaped (Senn, 1908). In land plant species, which generally bear multiple small chloroplasts in a cell, low light induces chloroplast movement and distribution toward the periclinal walls (the accumulation response), whereas strong light induces chloroplast avoidance toward the anticlinal walls (the avoidance response) (Fig. 1). Blue light is most effective at inducing chloroplast movement in most plant species, but in some cryptogam plant species, red light as well as blue light is effective. This red light effect exhibits red/far-red light reversibility, suggestive of the involvement of a red/far-red light receptor phytochrome. Detailed photobiological analyses, especially by the research groups of Wolfgang Haupt (1921-2005) and Jan Zurzycki (1925-1984), have provided many important insights on putative photoreceptor molecules that regulate chloroplast movement (Haupt, 1999; Zurzycki, 1980). They found that membrane-bound blue light photoreceptors other than phytochrome mediate blue-light-induced chloroplast movement in most plant species and that membrane-bound phytochromes mediate red-light induced chloroplast movement in some cryptogam plants. These predictions were demonstrated by the recent identification of photoreceptor genes in various plant species. The blue light receptor phot mediates bluelight-induced chloroplast movement in various plant species (Jarillo et al., 2001; Kagawa et al., 2001, 2004; Kasahara et al., 2004; Sakai et al., 2001). Neo, the chimeric photoreceptor that is a fusion of phytochrome and phototropin, regulates red-light-induced chloroplast movement in ferns and some green alga (Kawai et al., 2003; Suetsugu et al., 2005b). The photoreceptors are not discussed here; for a comprehensive review, see Suetsugu & Wada, 2005, 2007a, 2007b, 2009. First, we show our attempts to elucidate the mechanism of chloroplast photorelocation movement by detailed photobiological analyses. Second, we review recent molecular biological analyses of chloroplast photorelocation movement. Finally, the contribution of chloroplast movement and positioning to photosynthesis is discussed.

#### **2.1 Elucidation of the mechanism of chloroplast photorelocation movement by detailed photobiological analyses**

The underlying processes of chloroplast photorelocation movement can be categorized into three parts: photoperception, signal transduction and the motility system. Most of the photobiological analyses of chloroplast photorelocation movement were performed to

Chloroplast Photorelocation Movement: A Sophisticated

actual signals.

Strategy for Chloroplaststo Perform Efficient Photosynthesis 219

accumulation response are long-lived. (iv) Signals for the avoidance response can be override those for the accumulation response, at least in the irradiated area. Alternatively, it is possible that the signals for the avoidance response can be generated only when chloroplasts are directly irradiated with strong light. In this case, it is likely that the photoreceptor is localized on chloroplasts or that a plasma membrane-localized receptor

However, it is clear that the signals for the accumulation response are generated by the activation of photoreceptors in the irradiated area and are subsequently transferred toward chloroplasts. If measuring the speed of signal transfer for the accumulation response were possible, we could guess as to what is the putative signal by comparing the speeds between the putative signal and the known signaling molecules. When the chloroplast accumulation response was induced by microbeam irradiation in *A. capillus-veneris* protonemal cells, the onset of the accumulation movement lagged in proportion to the increase in the distance between the irradiated area and the chloroplasts, suggesting that the speed of the signal can be calculated as the lag time before the onset of movement (Tsuboi & Wada, 2010a, b). Similar calculations were also performed in *A. capillus-veneris* prothallial and *A. thaliana* mesophyll cells (Tsuboi & Wada, 2010a, b). These analyses revealed three interesting features of the putative signals. First, in protonemal cells, the speed of the signals in the basal-to-apical directions (about 2.3-2.4 µm min-1) was about three times faster than that in the apical-to-basal direction (about 0.6-0.9 µm min-1). However, the speed of the signals was almost equal in each cell type (about 0.9-1.1 µm min-1 in *A. capillus-veneris* prothallial cells and about 0.7 µm min-1 in *A. thaliana* mesophyll cells) (Tsuboi & Wada, 2010a, b). This difference in speed could result from the difference in the cell growth pattern of the cells, i.e. polarized (protonemal cells) and diffusive (prothallial and mesophyll cells). Second, in fern gametophytic cells, the speed of the signal and the maximum distance over which the signals could be transferred were almost equal irrespective of the intensity of the red or blue light microbeam, although in this case, more chloroplasts that were located farther away responded under continuous irradiation, compared to those submitted to a 1 min pulse of irradiation (Tsuboi & Wada, 2010a, b). Interestingly, the velocity of chloroplast accumulation movement was constant, regardless of the intensity of the microbeam placed on the prothallial cells (Kagawa & Wada, 1996; Tsuboi & Wada, 2011a). These results suggested that the properties of the signal, such as the speed, the amount and the activity, do not change in proportion to the change of light intensity. However, chloroplasts in the protonemal cells accumulated in the area that had been irradiated by a beam with a higher fluence rate, compared to adjacent areas that had been irradiated with beam of lower fluence rates of blue or red light. This result suggests that the amount or activity of the signal was increased when exposed to a beam with a higher fluence rate (Yatsuhashi et al., 1987; Yatsuhashi, 1996). Third and most importantly, the speed of signals (about 0.6-2.4 µm min-1) was much slower than that caused by calcium ion spiking or waves known to occur in plant and animal systems (about several µm sec-1 to 100 µm sec-1) (Tsuboi & Wada, 2010a, b). Furthermore, the signal transfer must not be actomyosin-dependent because the transfer of the signal still occurred when actin filaments were disrupted by treatment with inhibitor (Sato et al., 2001). Collectively, although the signals for chloroplast movement remained to be determined, our detailed physiological analyses will provide the clue to identify the

generates the signals only when it exists in close proximity to the chloroplasts.

identify the photoreceptor molecules (reviewed by Haupt, 1999; Zurzycki, 1980; Wada et al., 1993). Many pharmacological (i.e. treatment with chemicals and inhibitors) and microscopic (i.e. staining of the cytoskeleton) analyses have provided valuable insights, such as the possible involvement of calcium ions in the signal transduction pathway and the actin filament-dependency of the motility system (reviewed by Suetsugu & Wada, 2007b, 2009). However, the data from pharmacological treatment and microscopic observation of fixed samples should be carefully considered because of possible artifactual results. Thus, we decided to analyze chloroplast relocation movement using detailed physiological and photobiological analyses of the gametophytic cells of a fern *A. capillus-veneris* as a model system (reviewed by Wada, 2007). By changing light conditions, we can easily obtain two types of gametophytes: a filamentous protonemal cell or a two-dimensional prothallus, which is a cell sheet made of a one-cell layer. This fern regulates chloroplast movement by utilizing phot family proteins and actin filaments, like *A. thaliana* (Kadota & Wada, 1992; Kagawa et al., 2004; Tsuboi & Wada, 2011b). Using a microbeam irradiation system, we analyzed chloroplast photorelocation movement in protonemal and phothallial cells and elucidated several aspects of chloroplast movement, especially putative signaling molecules and movement.

#### **2.1.1 Physiological properties of putative signals in chloroplast photorelocation movement**

Blue light mediates the influx of calcium ions (Ca2+) into the cytosol and this influx is dependent upon phots in *A. thaliana* and *P. patens* (reviewed by Harada & Shimazaki, 2007). Importantly, a Ca2+ chelator inhibited chloroplast movement and external Ca2+ ions and Ca2+ ionophores changed the distribution of chloroplasts when placed in darkness (reviewed by Suetsugu & Wada, 2009). However, plasma membrane Ca2+ channel blockers, which effectively inhibited phot-dependent blue light-mediated Ca2+ influx (reviewed by Harada & Shimazaki, 2007), were totally ineffective in suppressing chloroplast photorelocation movement in various plant species (reviewed by Suetsugu & Wada, 2009). Thus, the putative signals that control chloroplast movement remain to be determined. To characterize the properties of these putative signals, chloroplast photorelocation movement was induced by partial cell irradiation with a microbeam irradiator and analyzed in detail (Kagawa & Wada, 1999, 2000; Tsuboi & Wada, 2010a, b).

An open question was whether the signals were different between the accumulation and avoidance responses. When a dark-adapted cell of an *A. capillus-veneris* prothallus (Kagawa & Wada, 1999) and an *A. thaliana* leaf (Kagawa & Wada, 2000) (in this situation, a few chloroplasts were on the upper periclinal walls) were partially irradiated with strong blue light, chloroplasts moved to the irradiated area but could not enter the beam area. Immediately after the light was turned off, the chloroplasts moved into the formerly irradiated area. Similar responses were also found in filamentous protonemal cells in *A. capillus-veneris* (Yatsuhashi et al., 1985) and *P. patens* (Kadota et al., 2000; Sato et al., 2001). These results suggested several characteristics of putative signals (reviewed by Suetsugu & Wada, 2009): (i) Signals for both the accumulation and the avoidance responses are simultaneously generated by strong light. (ii) Signals for the avoidance response function only at the irradiated area whereas those for the accumulation response can be transferred toward chloroplasts when located far from the irradiated area. (iii) Signals for the avoidance response disappear immediately after the light is turned off, whereas those for the

identify the photoreceptor molecules (reviewed by Haupt, 1999; Zurzycki, 1980; Wada et al., 1993). Many pharmacological (i.e. treatment with chemicals and inhibitors) and microscopic (i.e. staining of the cytoskeleton) analyses have provided valuable insights, such as the possible involvement of calcium ions in the signal transduction pathway and the actin filament-dependency of the motility system (reviewed by Suetsugu & Wada, 2007b, 2009). However, the data from pharmacological treatment and microscopic observation of fixed samples should be carefully considered because of possible artifactual results. Thus, we decided to analyze chloroplast relocation movement using detailed physiological and photobiological analyses of the gametophytic cells of a fern *A. capillus-veneris* as a model system (reviewed by Wada, 2007). By changing light conditions, we can easily obtain two types of gametophytes: a filamentous protonemal cell or a two-dimensional prothallus, which is a cell sheet made of a one-cell layer. This fern regulates chloroplast movement by utilizing phot family proteins and actin filaments, like *A. thaliana* (Kadota & Wada, 1992; Kagawa et al., 2004; Tsuboi & Wada, 2011b). Using a microbeam irradiation system, we analyzed chloroplast photorelocation movement in protonemal and phothallial cells and elucidated several aspects of chloroplast movement, especially putative signaling molecules

**2.1.1 Physiological properties of putative signals in chloroplast photorelocation** 

(Kagawa & Wada, 1999, 2000; Tsuboi & Wada, 2010a, b).

Blue light mediates the influx of calcium ions (Ca2+) into the cytosol and this influx is dependent upon phots in *A. thaliana* and *P. patens* (reviewed by Harada & Shimazaki, 2007). Importantly, a Ca2+ chelator inhibited chloroplast movement and external Ca2+ ions and Ca2+ ionophores changed the distribution of chloroplasts when placed in darkness (reviewed by Suetsugu & Wada, 2009). However, plasma membrane Ca2+ channel blockers, which effectively inhibited phot-dependent blue light-mediated Ca2+ influx (reviewed by Harada & Shimazaki, 2007), were totally ineffective in suppressing chloroplast photorelocation movement in various plant species (reviewed by Suetsugu & Wada, 2009). Thus, the putative signals that control chloroplast movement remain to be determined. To characterize the properties of these putative signals, chloroplast photorelocation movement was induced by partial cell irradiation with a microbeam irradiator and analyzed in detail

An open question was whether the signals were different between the accumulation and avoidance responses. When a dark-adapted cell of an *A. capillus-veneris* prothallus (Kagawa & Wada, 1999) and an *A. thaliana* leaf (Kagawa & Wada, 2000) (in this situation, a few chloroplasts were on the upper periclinal walls) were partially irradiated with strong blue light, chloroplasts moved to the irradiated area but could not enter the beam area. Immediately after the light was turned off, the chloroplasts moved into the formerly irradiated area. Similar responses were also found in filamentous protonemal cells in *A. capillus-veneris* (Yatsuhashi et al., 1985) and *P. patens* (Kadota et al., 2000; Sato et al., 2001). These results suggested several characteristics of putative signals (reviewed by Suetsugu & Wada, 2009): (i) Signals for both the accumulation and the avoidance responses are simultaneously generated by strong light. (ii) Signals for the avoidance response function only at the irradiated area whereas those for the accumulation response can be transferred toward chloroplasts when located far from the irradiated area. (iii) Signals for the avoidance response disappear immediately after the light is turned off, whereas those for the

and movement.

**movement** 

accumulation response are long-lived. (iv) Signals for the avoidance response can be override those for the accumulation response, at least in the irradiated area. Alternatively, it is possible that the signals for the avoidance response can be generated only when chloroplasts are directly irradiated with strong light. In this case, it is likely that the photoreceptor is localized on chloroplasts or that a plasma membrane-localized receptor generates the signals only when it exists in close proximity to the chloroplasts.

However, it is clear that the signals for the accumulation response are generated by the activation of photoreceptors in the irradiated area and are subsequently transferred toward chloroplasts. If measuring the speed of signal transfer for the accumulation response were possible, we could guess as to what is the putative signal by comparing the speeds between the putative signal and the known signaling molecules. When the chloroplast accumulation response was induced by microbeam irradiation in *A. capillus-veneris* protonemal cells, the onset of the accumulation movement lagged in proportion to the increase in the distance between the irradiated area and the chloroplasts, suggesting that the speed of the signal can be calculated as the lag time before the onset of movement (Tsuboi & Wada, 2010a, b). Similar calculations were also performed in *A. capillus-veneris* prothallial and *A. thaliana* mesophyll cells (Tsuboi & Wada, 2010a, b). These analyses revealed three interesting features of the putative signals. First, in protonemal cells, the speed of the signals in the basal-to-apical directions (about 2.3-2.4 µm min-1) was about three times faster than that in the apical-to-basal direction (about 0.6-0.9 µm min-1). However, the speed of the signals was almost equal in each cell type (about 0.9-1.1 µm min-1 in *A. capillus-veneris* prothallial cells and about 0.7 µm min-1 in *A. thaliana* mesophyll cells) (Tsuboi & Wada, 2010a, b). This difference in speed could result from the difference in the cell growth pattern of the cells, i.e. polarized (protonemal cells) and diffusive (prothallial and mesophyll cells). Second, in fern gametophytic cells, the speed of the signal and the maximum distance over which the signals could be transferred were almost equal irrespective of the intensity of the red or blue light microbeam, although in this case, more chloroplasts that were located farther away responded under continuous irradiation, compared to those submitted to a 1 min pulse of irradiation (Tsuboi & Wada, 2010a, b). Interestingly, the velocity of chloroplast accumulation movement was constant, regardless of the intensity of the microbeam placed on the prothallial cells (Kagawa & Wada, 1996; Tsuboi & Wada, 2011a). These results suggested that the properties of the signal, such as the speed, the amount and the activity, do not change in proportion to the change of light intensity. However, chloroplasts in the protonemal cells accumulated in the area that had been irradiated by a beam with a higher fluence rate, compared to adjacent areas that had been irradiated with beam of lower fluence rates of blue or red light. This result suggests that the amount or activity of the signal was increased when exposed to a beam with a higher fluence rate (Yatsuhashi et al., 1987; Yatsuhashi, 1996). Third and most importantly, the speed of signals (about 0.6-2.4 µm min-1) was much slower than that caused by calcium ion spiking or waves known to occur in plant and animal systems (about several µm sec-1 to 100 µm sec-1) (Tsuboi & Wada, 2010a, b). Furthermore, the signal transfer must not be actomyosin-dependent because the transfer of the signal still occurred when actin filaments were disrupted by treatment with inhibitor (Sato et al., 2001). Collectively, although the signals for chloroplast movement remained to be determined, our detailed physiological analyses will provide the clue to identify the actual signals.

Chloroplast Photorelocation Movement: A Sophisticated

photorelocation movement.

Strategy for Chloroplaststo Perform Efficient Photosynthesis 221

chloroplast movement is mediated by neo in several ferns and probably in some green algae (reviewed by Suetsugu & Wada, 2005, 2007a, 2007b, 2009). Regardless of significant advances in photoreceptor identification, the molecular mechanism of signal transduction and the identity of the motility system for chloroplast movement have been obscure. However, molecular genetic analyses using *A. thaliana* have identified several components that regulate chloroplast movement. Furthermore, recent imaging analyses have revealed that a novel actin-based mechanism governs chloroplast photorelocation and positioning. By combining these results, we could imagine the molecular framework of chloroplast

**2.2.1 Unique actin-based mechanism for chloroplast movement in land plants** 

utilize an actin-based mechanism that is different from those of other organelles.

For many years, it was thought that chloroplasts moved along long cytosolic actin cables by myosin motor proteins, similar to the movements of other organelles. However, the aforementioned studies (Tsuboi & Wada, 2009, 2011a) suggested that chloroplasts could

To find the actin-based mechanism for chloroplast movement, we utilized Arabidopsis transgenic lines in which actin filaments could be visualized by various fusions of fluorescent proteins and actin binding proteins (such as GFP-talin and tdTomato-fimbrin) and analyzed the behavior of the actin filaments during chloroplast movement using a custom-made microscope and a confocal microscope (Kadota et al., 2009). Although cytoplasmic actin cables and filaments were associated with chloroplasts, they did not change much in response to light irradiation, and their behavior did not associate with directional chloroplast movement. Instead, we found that short actin filaments found around chloroplasts dynamically changed their structure in response to light irradiation and that their dynamics correlated with the direction and speed of chloroplast movement. We have named these actin filaments chloroplast-actin filaments (abbreviated as cp-actin filaments) (Kadota et al., 2009)(Fig. 2). When chloroplasts were stationary, cp-actin filaments were distributed around the chloroplast periphery. In response to strong blue light, cp-actin filaments transiently disappeared within about 30 seconds and then reappeared at the one side of the chloroplasts, which would eventually be the front region of the moving chloroplasts (Fig. 2). We called this pattern of localization of cp-actin filaments at the front region "biased" (Fig. 2). After biased cp-actin filaments were fully formed, chloroplasts moved toward the side where the cp-actin filaments accumulated (Kadota et al., 2009). The generation of biased cp-actin filaments was also found during the accumulation response that had been induced by weak blue light, but this was not accompanied by a transient disappearance of cp-actin filaments, unlike what occurred during the avoidance response (Kadota et al., 2009). Thus, the light-induced generation of biased cpactin filaments is a prerequisite for both the avoidance and the accumulation responses. Possibly, a transient disappearance of cp-actin filaments induced by strong blue light facilitated an acceleration of chloroplast avoidance movement. As more cp-actin filaments accumulated at the front halves of the chloroplasts, in relation to the rear halves, the velocity of chloroplast avoidance also increased. When irradiated with a higher fluence of blue light, even more cp-actin filaments accumulated at the front halves, and chloroplasts moved even faster (Kadota et al., 2009). Thus, strong light caused a greater difference in the amount of cp-actin filaments at certain locations on the chloroplasts because cp-actin filaments located at the rear halves of the chloroplasts did not increase after transient disappearance. Conversely, weak light could not induce transient disappearance of cp-actin filaments, so a greater difference in

#### **2.1.2 An actin-based motility system deduced by detailed observation of chloroplast movement**

For a long time, it was believed that the actomyosin system mediated chloroplast movement in various species. Many analyses using several kinds of techniques (such as inhibitor treatment, immunocytochemistry and observation of the in vivo dynamics of actin filaments) clarified the involvement of actin filaments in chloroplast movement in various plant species (reviewed by Suetsugu & Wada, 2009; Suetsugu et al., 2010b). However, the involvement of myosin motor proteins was still controversial (reviewed by Suetsugu et al., 2010b). If the actomyosin system is involved in chloroplast movement, it is expected that chloroplasts move along long actin cables that preexist or elongate in the direction of movement immediately after light exposure and, thus, that chloroplast movement should be polarized (i.e. parallel to actin cables). However, this was not the case with chloroplast photorelocation movement at least in *A. capillus-veneris* prothallial and *A. thaliana* mesophyll cells (Tsuboi & Wada, 2009, 2011a). Importantly, chloroplasts moved by sliding but not rolling during both the accumulation and the avoidance responses (Tsuboi & Wada, 2009, 2011a), suggesting that chloroplasts moved by attaching one side to the plasma membrane via actin filaments that spanned between the chloroplasts and the plasma membrane. When observed with a microscope, chloroplasts look elliptic (or dumbbell-shaped for dividing chloroplasts) but not completely round. Therefore, it is plausible that chloroplasts keep their long axis in parallel with the moving direction so that they can take the path of least resistance. If that is the case, then they should turn at an angle formed by an imaginary line spanning their long axis and a second imaginary line that connects the center of the chloroplast, at the original position, to the center of the irradiated area. However, chloroplasts were capable of moving in any direction even without turning. Even if chloroplasts turned immediately before or while they moved, the extent of their turning was so small (Tsuboi & Wada, 2009, 2011a). Exceptionally, chloroplasts of Arabidopsis mesophyll cells tended to adjust their short axis to be parallel with the moving direction during the avoidance movement, although they started to move without turning (Tsuboi & Wada, 2011a). Importantly, chloroplasts escaped from strong light by taking the shortest route, suggesting that they are capable of determining the location of the closest area that is out of the strong light (Tsuboi & Wada, 2011a). Moreover, when sequentially irradiated with weak or strong light, chloroplasts could change their moving direction according to the position of subsequent irradiated beam, with a short lag time (Tsuboi & Wada, 2009, 2011a). Collectively, these detailed microscopic analyses argued against the hypothesis that chloroplasts utilize of pre-existing actin filaments for photomovement and suggested that they move using actin filaments that dynamically reorganize in response to light irradiation.

#### **2.2 Conserved molecular mechanism of chloroplast photorelocation movement in land plants**

Generally, blue light is most effective in inducing chloroplast photorelocation movement, although red light can also induce the movement in some cryptogam plants (green algae, mosses and ferns). Phot is the blue light receptor for chloroplast movement and also mediates phototropism and stomatal opening (reviewed by Christie, 2007). Phototropins were identified in green plants, from green alga to seed plants, and were shown to regulate blue-light-induced chloroplast movement at least in *A. thaliana*, *A. capillus-veneris* and *P. patens* (reviewed by Suetsugu & Wada, 2005, 2007a, 2007b, 2009). Red-light-induced

**2.1.2 An actin-based motility system deduced by detailed observation of chloroplast** 

**2.2 Conserved molecular mechanism of chloroplast photorelocation movement in** 

Generally, blue light is most effective in inducing chloroplast photorelocation movement, although red light can also induce the movement in some cryptogam plants (green algae, mosses and ferns). Phot is the blue light receptor for chloroplast movement and also mediates phototropism and stomatal opening (reviewed by Christie, 2007). Phototropins were identified in green plants, from green alga to seed plants, and were shown to regulate blue-light-induced chloroplast movement at least in *A. thaliana*, *A. capillus-veneris* and *P. patens* (reviewed by Suetsugu & Wada, 2005, 2007a, 2007b, 2009). Red-light-induced

For a long time, it was believed that the actomyosin system mediated chloroplast movement in various species. Many analyses using several kinds of techniques (such as inhibitor treatment, immunocytochemistry and observation of the in vivo dynamics of actin filaments) clarified the involvement of actin filaments in chloroplast movement in various plant species (reviewed by Suetsugu & Wada, 2009; Suetsugu et al., 2010b). However, the involvement of myosin motor proteins was still controversial (reviewed by Suetsugu et al., 2010b). If the actomyosin system is involved in chloroplast movement, it is expected that chloroplasts move along long actin cables that preexist or elongate in the direction of movement immediately after light exposure and, thus, that chloroplast movement should be polarized (i.e. parallel to actin cables). However, this was not the case with chloroplast photorelocation movement at least in *A. capillus-veneris* prothallial and *A. thaliana* mesophyll cells (Tsuboi & Wada, 2009, 2011a). Importantly, chloroplasts moved by sliding but not rolling during both the accumulation and the avoidance responses (Tsuboi & Wada, 2009, 2011a), suggesting that chloroplasts moved by attaching one side to the plasma membrane via actin filaments that spanned between the chloroplasts and the plasma membrane. When observed with a microscope, chloroplasts look elliptic (or dumbbell-shaped for dividing chloroplasts) but not completely round. Therefore, it is plausible that chloroplasts keep their long axis in parallel with the moving direction so that they can take the path of least resistance. If that is the case, then they should turn at an angle formed by an imaginary line spanning their long axis and a second imaginary line that connects the center of the chloroplast, at the original position, to the center of the irradiated area. However, chloroplasts were capable of moving in any direction even without turning. Even if chloroplasts turned immediately before or while they moved, the extent of their turning was so small (Tsuboi & Wada, 2009, 2011a). Exceptionally, chloroplasts of Arabidopsis mesophyll cells tended to adjust their short axis to be parallel with the moving direction during the avoidance movement, although they started to move without turning (Tsuboi & Wada, 2011a). Importantly, chloroplasts escaped from strong light by taking the shortest route, suggesting that they are capable of determining the location of the closest area that is out of the strong light (Tsuboi & Wada, 2011a). Moreover, when sequentially irradiated with weak or strong light, chloroplasts could change their moving direction according to the position of subsequent irradiated beam, with a short lag time (Tsuboi & Wada, 2009, 2011a). Collectively, these detailed microscopic analyses argued against the hypothesis that chloroplasts utilize of pre-existing actin filaments for photomovement and suggested that they move using actin filaments that dynamically

**movement** 

reorganize in response to light irradiation.

**land plants** 

chloroplast movement is mediated by neo in several ferns and probably in some green algae (reviewed by Suetsugu & Wada, 2005, 2007a, 2007b, 2009). Regardless of significant advances in photoreceptor identification, the molecular mechanism of signal transduction and the identity of the motility system for chloroplast movement have been obscure. However, molecular genetic analyses using *A. thaliana* have identified several components that regulate chloroplast movement. Furthermore, recent imaging analyses have revealed that a novel actin-based mechanism governs chloroplast photorelocation and positioning. By combining these results, we could imagine the molecular framework of chloroplast photorelocation movement.

#### **2.2.1 Unique actin-based mechanism for chloroplast movement in land plants**

For many years, it was thought that chloroplasts moved along long cytosolic actin cables by myosin motor proteins, similar to the movements of other organelles. However, the aforementioned studies (Tsuboi & Wada, 2009, 2011a) suggested that chloroplasts could utilize an actin-based mechanism that is different from those of other organelles.

To find the actin-based mechanism for chloroplast movement, we utilized Arabidopsis transgenic lines in which actin filaments could be visualized by various fusions of fluorescent proteins and actin binding proteins (such as GFP-talin and tdTomato-fimbrin) and analyzed the behavior of the actin filaments during chloroplast movement using a custom-made microscope and a confocal microscope (Kadota et al., 2009). Although cytoplasmic actin cables and filaments were associated with chloroplasts, they did not change much in response to light irradiation, and their behavior did not associate with directional chloroplast movement. Instead, we found that short actin filaments found around chloroplasts dynamically changed their structure in response to light irradiation and that their dynamics correlated with the direction and speed of chloroplast movement. We have named these actin filaments chloroplast-actin filaments (abbreviated as cp-actin filaments) (Kadota et al., 2009)(Fig. 2). When chloroplasts were stationary, cp-actin filaments were distributed around the chloroplast periphery. In response to strong blue light, cp-actin filaments transiently disappeared within about 30 seconds and then reappeared at the one side of the chloroplasts, which would eventually be the front region of the moving chloroplasts (Fig. 2). We called this pattern of localization of cp-actin filaments at the front region "biased" (Fig. 2). After biased cp-actin filaments were fully formed, chloroplasts moved toward the side where the cp-actin filaments accumulated (Kadota et al., 2009). The generation of biased cp-actin filaments was also found during the accumulation response that had been induced by weak blue light, but this was not accompanied by a transient disappearance of cp-actin filaments, unlike what occurred during the avoidance response (Kadota et al., 2009). Thus, the light-induced generation of biased cpactin filaments is a prerequisite for both the avoidance and the accumulation responses. Possibly, a transient disappearance of cp-actin filaments induced by strong blue light facilitated an acceleration of chloroplast avoidance movement. As more cp-actin filaments accumulated at the front halves of the chloroplasts, in relation to the rear halves, the velocity of chloroplast avoidance also increased. When irradiated with a higher fluence of blue light, even more cp-actin filaments accumulated at the front halves, and chloroplasts moved even faster (Kadota et al., 2009). Thus, strong light caused a greater difference in the amount of cp-actin filaments at certain locations on the chloroplasts because cp-actin filaments located at the rear halves of the chloroplasts did not increase after transient disappearance. Conversely, weak light could not induce transient disappearance of cp-actin filaments, so a greater difference in

Chloroplast Photorelocation Movement: A Sophisticated

**cp-actin filaments** 

Strategy for Chloroplaststo Perform Efficient Photosynthesis 223

We identified various Arabidopsis mutants deficient in chloroplast photorelocation movement (Kagawa et al., 2001; Kodama et al., 2010; Oikawa et al., 2003; Suetsugu et al., 2005, 2010a), and thus, the analyses of cp-actin filament behavior in these mutants have shed light on the molecular mechanism of cp-actin filament-mediated chloroplast movement (Kadota et al., 2009; Kodama et al., 2010; Suetsugu et al., 2010a; Ichikawa et al., 2011)(Fig.3). Phototropin is a blue light receptor bearing two photosensory LOV (light, oxygen and voltage) domains at its N-terminus and a C-terminal serine/threonine kinase domain (reviewed by Christie, 2007). In *A. thaliana*, phot1 and phot2 redundantly mediated the chloroplast accumulation response (Sakai et al., 2001), and phot2 alone regulated the avoidance response (Kagawa et al., 2001; Jarillo et al., 2001). In *phot1phot2* double mutant plants, which are completely defective in chloroplast photorelocation movement (Sakai et al., 2001), blue-light-induced cp-actin filament reorganization did not occur, indicating that phototropins mediated chloroplast movement via the regulation of cp-actin filaments (Kadota et al., 2009; Ichikawa et al., 2011). The *phot1phot2* double mutant plants also did not change their amounts of cp-actin filaments in response to both weak and strong blue light and thus showed no light-induced motility changes (Kadota et al., 2009). This outcome indicated that phototropins mediated anchoring of the chloroplast to the plasma membrane via regulation of the amounts of cp-actin filaments. The strong-blue-light-induced transient disappearance of cp-actin filaments did not occurr at all in *phot2* mutant plants, which were impaired in the avoidance response. However, they showed normal biased cp-actin filament formation during the accumulation response (Kadota et al., 2009; Ichikawa et al., 2011), indicating that phot2 mediated the strong-blue-light-induced transient disappearance of cpactin filaments (Fig.3) and that this reorganization of cp-actin filaments could be a prerequisite for the avoidance response. In *phot1* mutant plants, chloroplast photorelocation movement was only slightly impaired in the accumulation response (Kagawa & Wada, 2000), and therefore light-induced reorganization of cp-actin filaments in these plants was mostly normal (Kadota et al., 2009). However, in response to strong blue light, the onset of biased cp-actin formation and the avoidance movement in *phot1* mutants occurred earlier than in wild-type plants (Ichikawa et al., 2011), suggesting a small inhibition of cp-actin

**2.2.2 Molecular components regulating the generation and/or reorganization of** 

filament reorganization by phot1 during the avoidance movement.

JAC1 has a J-domain at the C-terminus and is similar to a clathrin uncoating factor, auxilin (Suetsugu et al., 2005). The J-domain of JAC1 is necessary for JAC1 function and the crystal structure showed high similarity between that domain and that of the bovine auxilin J-domain (Takano et al., 2010; Suetsugu et al., 2010c). *jac1* mutant plants were completely defective in the accumulation response but retain the avoidance response (Suetsugu et al., 2005). In response to weak blue light, the reorganization of cp-actin filaments did not occur in most chloroplasts of *jac1* mutant plants, but a few chloroplasts that avoided weak light formed biased cp-actin filaments (Ichikawa et al., 2011), indicating that JAC1 is essential for the reorganization of cp-actin filaments during the accumulation response but not for biased cp-actin filament formation (Fig. 3). Interestingly, in *jac1* mutant plants, whole cell irradiation with strong blue light did not induce the disappearance and subsequent biased localization of cp-actin filaments and thus the avoidance movement did not occur. However, when part of a cell was irradiated, chloroplasts that were close to the beam edge showed the avoidance movement with biased cp-actin filament formation, although cp-actin filaments on chloroplasts inside the

the amount of cp-actin filaments at certain locations was not made. Actually, the velocity of chloroplast accumulation movement was constant irrespective of light intensity (Kagawa & Wada, 1996; Tsuboi & Wada, 2011a). Cp-actin filaments localized at the interface between the chloroplast and the plasma membrane, elongated from the edge of the chloroplast and shortened toward the chloroplast periphery, suggesting that the nucleation site of cp-actin filaments might exist on the chloroplast edge and that the force for chloroplast movement by cp-actin filaments might be generated there (Kadota et al., 2009). Cp-actin filaments mediated the anchoring of chloroplasts to the plasma membrane as well as their directional movement (Kadota et al., 2009). The strong-light-induced disappearance of cp-actin filaments was accompanied by increased chloroplast motility in random directions before avoidance movement, suggestive of the detachment of chloroplasts from the plasma membrane. Conversely, weak blue light induced the increase of cp-actin filaments around the chloroplast periphery and accompanied a decrease in chloroplast motility, likely facilitating chloroplast anchoring to the plasma membrane. In summary, there are three types of blue-light-induced rearrangements of cp-actin filaments that mediate both directional movement and the anchoring of chloroplasts to the plasma membrane: (i) the formation of biased cp-actin filaments during both the accumulation and the avoidance responses; (ii) a strong-blue-lightinduced transient disappearance of cp-actin filaments; (iii) a weak-blue-light-induced increase in cp-actin filaments. Importantly, cp-actin filament-mediated chloroplast movement is conserved in a fern, *A. capillus-veneris*, and in a moss, *P. patens* (Tsuboi & Wada, 2011b; Yamashita et al., 2011). Thus, the regulation of chloroplast movement by cp-actin filaments was likely to be utilized during the early stages of land plant evolution.

Fig. 2. Light-induced cp-actin filament reorganization during the chloroplast avoidance response. The chloroplast avoidance response was induced by scanning circular regions of interest (diameter 15 µm indicated by a shaded circle) with 2.8 mW of a 458 nm laser and using a confocal microscope (SP5, Leica). Time-lapse images of chloroplast movement and the associated cp-actin filament dynamics were captured at the indicated times (sec). Detailed dynamics of the cp-actin filaments of a chloroplast marked with a white circle are indicated below. After 40 sec of light irradiation, cp-actin filaments disappeared and then reappeared at the front region of the chloroplast, which had moved to the upper left side in this figure via the avoidance response.

the amount of cp-actin filaments at certain locations was not made. Actually, the velocity of chloroplast accumulation movement was constant irrespective of light intensity (Kagawa & Wada, 1996; Tsuboi & Wada, 2011a). Cp-actin filaments localized at the interface between the chloroplast and the plasma membrane, elongated from the edge of the chloroplast and shortened toward the chloroplast periphery, suggesting that the nucleation site of cp-actin filaments might exist on the chloroplast edge and that the force for chloroplast movement by cp-actin filaments might be generated there (Kadota et al., 2009). Cp-actin filaments mediated the anchoring of chloroplasts to the plasma membrane as well as their directional movement (Kadota et al., 2009). The strong-light-induced disappearance of cp-actin filaments was accompanied by increased chloroplast motility in random directions before avoidance movement, suggestive of the detachment of chloroplasts from the plasma membrane. Conversely, weak blue light induced the increase of cp-actin filaments around the chloroplast periphery and accompanied a decrease in chloroplast motility, likely facilitating chloroplast anchoring to the plasma membrane. In summary, there are three types of blue-light-induced rearrangements of cp-actin filaments that mediate both directional movement and the anchoring of chloroplasts to the plasma membrane: (i) the formation of biased cp-actin filaments during both the accumulation and the avoidance responses; (ii) a strong-blue-lightinduced transient disappearance of cp-actin filaments; (iii) a weak-blue-light-induced increase in cp-actin filaments. Importantly, cp-actin filament-mediated chloroplast movement is conserved in a fern, *A. capillus-veneris*, and in a moss, *P. patens* (Tsuboi & Wada, 2011b; Yamashita et al., 2011). Thus, the regulation of chloroplast movement by cp-actin filaments

was likely to be utilized during the early stages of land plant evolution.

Fig. 2. Light-induced cp-actin filament reorganization during the chloroplast avoidance response. The chloroplast avoidance response was induced by scanning circular regions of interest (diameter 15 µm indicated by a shaded circle) with 2.8 mW of a 458 nm laser and using a confocal microscope (SP5, Leica). Time-lapse images of chloroplast movement and the associated cp-actin filament dynamics were captured at the indicated times (sec). Detailed dynamics of the cp-actin filaments of a chloroplast marked with a white circle are indicated below. After 40 sec of light irradiation, cp-actin filaments disappeared and then reappeared at the front region of the chloroplast, which had moved to the upper left side in

this figure via the avoidance response.

#### **2.2.2 Molecular components regulating the generation and/or reorganization of cp-actin filaments**

We identified various Arabidopsis mutants deficient in chloroplast photorelocation movement (Kagawa et al., 2001; Kodama et al., 2010; Oikawa et al., 2003; Suetsugu et al., 2005, 2010a), and thus, the analyses of cp-actin filament behavior in these mutants have shed light on the molecular mechanism of cp-actin filament-mediated chloroplast movement (Kadota et al., 2009; Kodama et al., 2010; Suetsugu et al., 2010a; Ichikawa et al., 2011)(Fig.3). Phototropin is a blue light receptor bearing two photosensory LOV (light, oxygen and voltage) domains at its N-terminus and a C-terminal serine/threonine kinase domain (reviewed by Christie, 2007). In *A. thaliana*, phot1 and phot2 redundantly mediated the chloroplast accumulation response (Sakai et al., 2001), and phot2 alone regulated the avoidance response (Kagawa et al., 2001; Jarillo et al., 2001). In *phot1phot2* double mutant plants, which are completely defective in chloroplast photorelocation movement (Sakai et al., 2001), blue-light-induced cp-actin filament reorganization did not occur, indicating that phototropins mediated chloroplast movement via the regulation of cp-actin filaments (Kadota et al., 2009; Ichikawa et al., 2011). The *phot1phot2* double mutant plants also did not change their amounts of cp-actin filaments in response to both weak and strong blue light and thus showed no light-induced motility changes (Kadota et al., 2009). This outcome indicated that phototropins mediated anchoring of the chloroplast to the plasma membrane via regulation of the amounts of cp-actin filaments. The strong-blue-light-induced transient disappearance of cp-actin filaments did not occurr at all in *phot2* mutant plants, which were impaired in the avoidance response. However, they showed normal biased cp-actin filament formation during the accumulation response (Kadota et al., 2009; Ichikawa et al., 2011), indicating that phot2 mediated the strong-blue-light-induced transient disappearance of cpactin filaments (Fig.3) and that this reorganization of cp-actin filaments could be a prerequisite for the avoidance response. In *phot1* mutant plants, chloroplast photorelocation movement was only slightly impaired in the accumulation response (Kagawa & Wada, 2000), and therefore light-induced reorganization of cp-actin filaments in these plants was mostly normal (Kadota et al., 2009). However, in response to strong blue light, the onset of biased cp-actin formation and the avoidance movement in *phot1* mutants occurred earlier than in wild-type plants (Ichikawa et al., 2011), suggesting a small inhibition of cp-actin filament reorganization by phot1 during the avoidance movement.

JAC1 has a J-domain at the C-terminus and is similar to a clathrin uncoating factor, auxilin (Suetsugu et al., 2005). The J-domain of JAC1 is necessary for JAC1 function and the crystal structure showed high similarity between that domain and that of the bovine auxilin J-domain (Takano et al., 2010; Suetsugu et al., 2010c). *jac1* mutant plants were completely defective in the accumulation response but retain the avoidance response (Suetsugu et al., 2005). In response to weak blue light, the reorganization of cp-actin filaments did not occur in most chloroplasts of *jac1* mutant plants, but a few chloroplasts that avoided weak light formed biased cp-actin filaments (Ichikawa et al., 2011), indicating that JAC1 is essential for the reorganization of cp-actin filaments during the accumulation response but not for biased cp-actin filament formation (Fig. 3). Interestingly, in *jac1* mutant plants, whole cell irradiation with strong blue light did not induce the disappearance and subsequent biased localization of cp-actin filaments and thus the avoidance movement did not occur. However, when part of a cell was irradiated, chloroplasts that were close to the beam edge showed the avoidance movement with biased cp-actin filament formation, although cp-actin filaments on chloroplasts inside the

Chloroplast Photorelocation Movement: A Sophisticated

be determined.

Strategy for Chloroplaststo Perform Efficient Photosynthesis 225

were severely impaired in the strong-light-induced disappearance and subsequent biased localization of cp-actin filaments, it was concluded that the mutant phenotypes observed were a result of the impairment in cp-actin filament reorganization (Kodama et al., 2010)(Fig. 3). The defective avoidance response phenotype in these mutants was suppressed by a *jac1* mutation, suggesting a role for WEB1 and PMI2 in suppressing JAC1 activity, which regulates the accumulation response under high light conditions (Kodama et al., 2010). Given that the strong-light-induced reorganization of cp-actin filaments was severely impaired in *web1*, *pmi2* and *jac1* mutant plants (Kodama et al., 2010; Ichikawa et al., 2011), it is possible that WEB1/PMI2 and JAC1 cooperatively mediate the strong-light-induced reorganization of cp-actin filaments, although the detailed molecular mechanism remains to

Currently, two types of proteins, CHUP1 and KAC (KAC1 and KAC2), were identified as the factors necessary for the existence of cp-actin filaments, possibly serving as nucleators and/or stabilizers of cp-actin filaments (reviewed by Suetsugu et al., 2010b). CHUP1 is a multi-domain protein that bears an N-terminal hydrophobic region, a coiled-coil region, an F-actin-binding domain, a proline-rich region and a highly conserved C-terminal region (Oikawa et al., 2003). The hydrophobic region is essential for the localization of chloroplast outer envelope (Oikawa et al., 2003, 2008; Schmidt von Braun & Schleiff, 2008) and the coiled-coil region confer the ability of the protein to dimerize in vitro (Lehmann et al., 2011). The actin-binding domain was capable of interacting with F-actin in vitro (Oikawa et al., 2003), and the proline-rich region might serve as the profilin-interacting domain (Schmidt von Braun & Schleiff, 2008). KAC proteins belong to a microtubule motor kinesin-14 subfamily, but their motor and microtubule-binding activities have not yet been detected (Suetsugu et al., 2010a). A subset of KAC proteins was associated with the plasma membrane and chloroplast envelope although the bulk of the KAC proteins were found as soluble proteins (Suetsugu et al., 2010a). Both *chup1* and *kac1kac2* double mutant plants completely lacked cp-actin filaments but retained the normal cytosolic actin filament structure, indicating that the CHUP1 and KAC proteins are essential for cp-actin filament formation and/or maintenance (Kadota et al., 2009; Suetsugu et al., 2010a)(Fig. 3). Importantly, both mutants showed no chloroplast photorelocation movement and defects in the anchoring of chloroplasts to the plasma membrane. This result reinforced the notion that cp-actin filaments mediate photorelocation and the anchoring of chloroplasts to the plasma membrane. In *kac1* single mutant plants, significantly fewer amounts of cp-actin filaments were observed, the accumulation response was severely impaired and the velocity of the avoidance movement was much slower compared to wild-type plants (Suetsugu et al., 2010a). Thus, the amount of KAC proteins is an important factor that determines the

chloroplast velocity by regulating the amounts of cp-actin filaments.

Although PMI1 and THRUMIN1 were identified through the analyses of mutants deficient in chloroplast photorelocation movement (DeBlasio et al., 2005; Whippo et al., 2011), their involvement in cp-actin filament regulation is unknown. Because THRUMIN1 has an actin bundling activity (Whippo et al., 2011), analyses of mutants deficient in these factors will reveal a more detailed framework of cp-actin filament-dependent chloroplast movement. Three essential genes for cp-actin filament-mediated chloroplast movement, *PHOT*, *CHUP1* and *KAC*, are found in the genome of a liverwort, *Marchantia polymorpha*, and a moss, *P. patens*. Because cp-actin filament-mediated chloroplast movement is found in *P. patens*

beam did not disappear and their motility did not increase (Ichikawa et al., 2011). These results indicate that JAC1 is essential for an efficient chloroplast avoidance response by regulating the disappearance of cp-actin filaments (Fig. 3).

Fig. 3. A schematic model of cp-actin filament-mediated chloroplast movement. Weak light activates both phot1 and phot2, which are localized on the plasma membrane and subsequently generate an as yet unidentified signal that initiates the chloroplast accumulation response. JAC1 may be involved in signal generation, transport and/or perception. The signal activates a cp-actin filament nucleation complex, which is localized at the chloroplast edge, resulting in the polymerization of cp-actin filaments at the leading edge of the chloroplasts (black arrowheads indicate G-actins). CHUP1 could be the nucleation factor, and KAC proteins could be involved in cp-actin filament nucleation and/or maintenance. THRUMIN1 may interact with cp-actin filaments because THRUMIN1-YFP fusion protein decorated actin filaments in vivo. Strong-light-induced cpactin filament disappearance (indicated by broken-lined arrowheads) is mediated by phot2, JAC1 and WEB1/PMI2. After disappearance, cp-actin filaments reappeared at the leading edge, and the chloroplasts escaped from the strong light.

Recently, we identified two coiled-coil proteins, WEB1 and PMI2, as factors that regulate light-induced cp-actin filament reorganization (Kodama et al., 2010; Luesse et al., 2006). WEB1 and PMI2 belong to a coiled-coil protein family that contains a DUF827 (Domain of Unknown Function 827) domain (Kodama et al., 2010, 2011). WEB1 and PMI2 interacted with each other in yeast and plant cells, and WEB1 showed self-interaction activity, forming large complexes in plant cells, indicating that both WEB1 and PMI2 have protein-protein interaction activity (Kodama et al., 2010). Both *web1* and *pmi2* mutant plants showed severe defects in the avoidance response and slight defects in the accumulation response. Because the phenotypes of *web1pmi2* double mutant plants were very similar to those of *web1* and *pmi2* single-mutant plants, it was concluded that WEB1 and PMI2 probably function in the same pathway, possibly as a complex (Kodama et al., 2010, 2011). Because these mutants

beam did not disappear and their motility did not increase (Ichikawa et al., 2011). These results indicate that JAC1 is essential for an efficient chloroplast avoidance response by

Fig. 3. A schematic model of cp-actin filament-mediated chloroplast movement. Weak light

THRUMIN1-YFP fusion protein decorated actin filaments in vivo. Strong-light-induced cpactin filament disappearance (indicated by broken-lined arrowheads) is mediated by phot2, JAC1 and WEB1/PMI2. After disappearance, cp-actin filaments reappeared at the leading

Recently, we identified two coiled-coil proteins, WEB1 and PMI2, as factors that regulate light-induced cp-actin filament reorganization (Kodama et al., 2010; Luesse et al., 2006). WEB1 and PMI2 belong to a coiled-coil protein family that contains a DUF827 (Domain of Unknown Function 827) domain (Kodama et al., 2010, 2011). WEB1 and PMI2 interacted with each other in yeast and plant cells, and WEB1 showed self-interaction activity, forming large complexes in plant cells, indicating that both WEB1 and PMI2 have protein-protein interaction activity (Kodama et al., 2010). Both *web1* and *pmi2* mutant plants showed severe defects in the avoidance response and slight defects in the accumulation response. Because the phenotypes of *web1pmi2* double mutant plants were very similar to those of *web1* and *pmi2* single-mutant plants, it was concluded that WEB1 and PMI2 probably function in the same pathway, possibly as a complex (Kodama et al., 2010, 2011). Because these mutants

edge, and the chloroplasts escaped from the strong light.

activates both phot1 and phot2, which are localized on the plasma membrane and subsequently generate an as yet unidentified signal that initiates the chloroplast accumulation response. JAC1 may be involved in signal generation, transport and/or perception. The signal activates a cp-actin filament nucleation complex, which is localized at the chloroplast edge, resulting in the polymerization of cp-actin filaments at the leading edge of the chloroplasts (black arrowheads indicate G-actins). CHUP1 could be the nucleation factor, and KAC proteins could be involved in cp-actin filament nucleation and/or maintenance. THRUMIN1 may interact with cp-actin filaments because

regulating the disappearance of cp-actin filaments (Fig. 3).

were severely impaired in the strong-light-induced disappearance and subsequent biased localization of cp-actin filaments, it was concluded that the mutant phenotypes observed were a result of the impairment in cp-actin filament reorganization (Kodama et al., 2010)(Fig. 3). The defective avoidance response phenotype in these mutants was suppressed by a *jac1* mutation, suggesting a role for WEB1 and PMI2 in suppressing JAC1 activity, which regulates the accumulation response under high light conditions (Kodama et al., 2010). Given that the strong-light-induced reorganization of cp-actin filaments was severely impaired in *web1*, *pmi2* and *jac1* mutant plants (Kodama et al., 2010; Ichikawa et al., 2011), it is possible that WEB1/PMI2 and JAC1 cooperatively mediate the strong-light-induced reorganization of cp-actin filaments, although the detailed molecular mechanism remains to

be determined. Currently, two types of proteins, CHUP1 and KAC (KAC1 and KAC2), were identified as the factors necessary for the existence of cp-actin filaments, possibly serving as nucleators and/or stabilizers of cp-actin filaments (reviewed by Suetsugu et al., 2010b). CHUP1 is a multi-domain protein that bears an N-terminal hydrophobic region, a coiled-coil region, an F-actin-binding domain, a proline-rich region and a highly conserved C-terminal region (Oikawa et al., 2003). The hydrophobic region is essential for the localization of chloroplast outer envelope (Oikawa et al., 2003, 2008; Schmidt von Braun & Schleiff, 2008) and the coiled-coil region confer the ability of the protein to dimerize in vitro (Lehmann et al., 2011). The actin-binding domain was capable of interacting with F-actin in vitro (Oikawa et al., 2003), and the proline-rich region might serve as the profilin-interacting domain (Schmidt von Braun & Schleiff, 2008). KAC proteins belong to a microtubule motor kinesin-14 subfamily, but their motor and microtubule-binding activities have not yet been detected (Suetsugu et al., 2010a). A subset of KAC proteins was associated with the plasma membrane and chloroplast envelope although the bulk of the KAC proteins were found as soluble proteins (Suetsugu et al., 2010a). Both *chup1* and *kac1kac2* double mutant plants completely lacked cp-actin filaments but retained the normal cytosolic actin filament structure, indicating that the CHUP1 and KAC proteins are essential for cp-actin filament formation and/or maintenance (Kadota et al., 2009; Suetsugu et al., 2010a)(Fig. 3). Importantly, both mutants showed no chloroplast photorelocation movement and defects in the anchoring of chloroplasts to the plasma membrane. This result reinforced the notion that cp-actin filaments mediate photorelocation and the anchoring of chloroplasts to the plasma membrane. In *kac1* single mutant plants, significantly fewer amounts of cp-actin filaments were observed, the accumulation response was severely impaired and the velocity of the avoidance movement was much slower compared to wild-type plants (Suetsugu et al., 2010a). Thus, the amount of KAC proteins is an important factor that determines the chloroplast velocity by regulating the amounts of cp-actin filaments.

Although PMI1 and THRUMIN1 were identified through the analyses of mutants deficient in chloroplast photorelocation movement (DeBlasio et al., 2005; Whippo et al., 2011), their involvement in cp-actin filament regulation is unknown. Because THRUMIN1 has an actin bundling activity (Whippo et al., 2011), analyses of mutants deficient in these factors will reveal a more detailed framework of cp-actin filament-dependent chloroplast movement.

Three essential genes for cp-actin filament-mediated chloroplast movement, *PHOT*, *CHUP1* and *KAC*, are found in the genome of a liverwort, *Marchantia polymorpha*, and a moss, *P. patens*. Because cp-actin filament-mediated chloroplast movement is found in *P. patens*

Chloroplast Photorelocation Movement: A Sophisticated

Strategy for Chloroplaststo Perform Efficient Photosynthesis 227

Thus, chloroplasts should be located on the cell wall facing air spaces. Mesophyll cell chloroplasts tended to be located along intercellular air spaces in various plant species (Senn, 1908; Psaras et al., 1996) (Fig. 4c). Senn (1908) hypothesized that this positioning might result from the chemotaxis of chloroplasts to the CO2 in air spaces. Using different approaches and plant species, three groups examined whether chloroplast photorelocation movement, especially the avoidance response, influenced CO2 diffusion in leaves (Gorton et al., 2003; Loreto et al., 2009; Tholen et al., 2008). One research group hypothesized that chloroplast distribution on the anticlinal walls by the avoidance response facilitated CO2 utilization by shortening the CO2 diffusion path length from air spaces to the mesophyll chloroplasts (Gorton et al., 2003). This group measured oxygen diffusion times using pulsed photoacoustics (as a substitution for CO2 diffusion) between control and strong lightirradiated *Alocasia* leaf samples, but they could not find any differences in CO2 diffusion rates between the two samples (Gorton et al., 2003). Additionally, they could found no difference in the distance between the centers of the chloroplasts and the closest air spaces between two samples (Gorton et al., 2003). Another group found that blue light rapidly reduced CO2 diffusion from intercellular air spaces to the chloroplasts in both *Nicotiana* and *Platanus* leaves and that this reduction was completed before chloroplasts finished the avoidance movement to the aniticlinal walls (Loreto et al., 2009). Importantly, the blue-lightinduced reduction of CO2 diffusion was still normal in samples treated with an anti-actin inhibitor cytochalasin, which completely inhibits chloroplast movement (Loreto et al., 2009). Thus, results by two independent groups indicated that chloroplast movement did not significantly change the efficiency of CO2 diffusion from intercellular air spaces to the chloroplasts. However, the results by a third group suggested that the avoidance response reduced the CO2 diffusion rate rather than increased it (Tholen et al., 2008). In *Arabidopsis* wild-type plants, the surface area of chloroplasts facing air spaces was reduced after the induction of the chloroplast avoidance response and resulted in the reduction of CO2 diffusion. However, these reductions were not found in *phot2* and *chup1* mutant plants or in cytochalasin-treated plants (Tholen et al., 2008). Compared to wild type, the surface area of chloroplasts that faced air spaces and the rate of CO2 diffusion were constitutively lower in *chup1* mutant plants because of aberrant positioning of their chloroplasts (Tholen et al., 2008). Collectively, these results suggested that the chloroplast avoidance response was not involved in CO2 diffusion or that it possibly decreased, rather than increased, the diffusion rate. However, these three groups examined the contribution of chloroplast movement to CO2 diffusion using different plant species and techniques: pulsed photoacoustics (Gorton et al., 2003), a chlorophyll fluorescence-based method (Loreto et al., 2009) and a carbon isotope discrimination method (Tholen et al., 2008). The examination of one plant species using different techniques and/or that of various plant species by one technique are required to

uncover whether chloroplast photorelocation movement influences CO2 diffusion.

The two aforementioned hypotheses on the roles of the avoidance response are applicable only in multilayered leaf tissue and not in gametophytic cells that have single cell layers or in the filamentous structures of fern, moss, liverwort and green alga. During the early period of land plant evolution, plants were exposed directly to sunlight until seed plants eventually dominated terrestrial ecosystems and formed a dense canopy. Thus, it is plausible that the main role of the chloroplast avoidance response is to prevent chloroplasts

**2.3.3 Chloroplast avoidance response is essential for protection against** 

**photodamage by strong light** 

(Yamashita et al., 2011), the molecular mechanism of cp-actin filament-mediated chloroplast movement must have evolved early in land plant evolution.

#### **2.3 Contribution of chloroplast photorelocation movement to photosynthesis**

The intracellular distribution of chloroplasts is essential for the promotion of photosynthetic performance. For example, the chloroplast distribution in bundle sheath cells of C4 plants may be necessary for efficient C4 photosynthesis because it controlled CO2 diffusion and/or facilitated the metabolite exchange between mesophyll and bundle sheath cells (reviewed by von Caemmerer & Furbank, 2003). The chloroplast accumulation response could play an important role in efficient light capture under weak light conditions, although it has not been demonstrated experimentally (Zurzycki, 1955). The avoidance response is required for chloroplats to escape from photodamage under excess light conditions (Fig. 4a); however, two other hypotheses exist that could also explain the ecological advantages of chloroplast distribution on anticlinal walls by the avoidance response (Fig. 4b & 4c).

#### **2.3.1 Promotion of light penetration to deeper leaf cell layer by the avoidance response**

In the leaves of *Oxalis*, *Marah* and *Cyrtomium*, changes in leaf absorptance due to chloroplast movement positively correlated with changes in fluorescence emission; in particular, changes in fluorescence emissions increased during the avoidance response induced by strong blue light, whereas they decreased during subsequent relaxation in red light (Brugnoli & Björkman, 1992). Considering that leaves consist of multiple layers of photosynthetic cells and that the efficiency of net leaf photosynthesis depends on the efficient light utilization of chloroplasts in all cell layers, it is reasonable to conclude that chloroplast distribution to anticlinal cell walls, as a result of the avoidance response in the upper cell layer (or palisade layer), could facilitate the penetration of the incident light to a deeper cell layer (or sponge layer)(Fig. 4b). Light transmittance through the palisade layer was greater in high light-irradiated *Alocasia* leaves than in dark-adapted leaves. However, the difference in light transmittance through the spongy layer was not significant between high light- and dark-adapted leaves, indicating that chloroplast positioning on the anticlinal walls by the avoidance response in the palisade layer could facilitate light penetration to a deeper layer (Gorton et al., 1999). In *Tradescantia* leaves, which consist of three mesophyll cell layers (the first is a palisade layer, and the second and third are sponge layers), the chloroplasts in the second layer did not move to the anticlinal walls when irradiated with strong light of 100 µmol m-2 s-1 from either the adaxial or the abaxial side. However, by abaxial-side irradiation, chloroplasts in the third layer were positioned on the anticlinal walls by way of the avoidance response (Terashima & Hikosaka, 1995). These results suggest that the avoidance response in the surface mesophyll layer facilitates light capture in the deeper cell layers, resulting in a net increase of whole leaf photosynthesis. However, this hypothetical role of the avoidance movement in the enhancement of light penetration to the deeper cell layers has not yet been demonstrated conclusively.

#### **2.3.2 Influence of the chloroplast avoidance response on CO2 diffusion between air spaces and mesophyll cells**

The diffusion path length of CO2 from intercellular air spaces to the chloroplast stroma must be short so that mesophyll chloroplasts can efficiently utilize CO2 from those air spaces.

(Yamashita et al., 2011), the molecular mechanism of cp-actin filament-mediated chloroplast

The intracellular distribution of chloroplasts is essential for the promotion of photosynthetic performance. For example, the chloroplast distribution in bundle sheath cells of C4 plants may be necessary for efficient C4 photosynthesis because it controlled CO2 diffusion and/or facilitated the metabolite exchange between mesophyll and bundle sheath cells (reviewed by von Caemmerer & Furbank, 2003). The chloroplast accumulation response could play an important role in efficient light capture under weak light conditions, although it has not been demonstrated experimentally (Zurzycki, 1955). The avoidance response is required for chloroplats to escape from photodamage under excess light conditions (Fig. 4a); however, two other hypotheses exist that could also explain the ecological advantages of chloroplast

**2.3 Contribution of chloroplast photorelocation movement to photosynthesis** 

distribution on anticlinal walls by the avoidance response (Fig. 4b & 4c).

the deeper cell layers has not yet been demonstrated conclusively.

**spaces and mesophyll cells** 

**2.3.2 Influence of the chloroplast avoidance response on CO2 diffusion between air** 

The diffusion path length of CO2 from intercellular air spaces to the chloroplast stroma must be short so that mesophyll chloroplasts can efficiently utilize CO2 from those air spaces.

**response** 

**2.3.1 Promotion of light penetration to deeper leaf cell layer by the avoidance** 

In the leaves of *Oxalis*, *Marah* and *Cyrtomium*, changes in leaf absorptance due to chloroplast movement positively correlated with changes in fluorescence emission; in particular, changes in fluorescence emissions increased during the avoidance response induced by strong blue light, whereas they decreased during subsequent relaxation in red light (Brugnoli & Björkman, 1992). Considering that leaves consist of multiple layers of photosynthetic cells and that the efficiency of net leaf photosynthesis depends on the efficient light utilization of chloroplasts in all cell layers, it is reasonable to conclude that chloroplast distribution to anticlinal cell walls, as a result of the avoidance response in the upper cell layer (or palisade layer), could facilitate the penetration of the incident light to a deeper cell layer (or sponge layer)(Fig. 4b). Light transmittance through the palisade layer was greater in high light-irradiated *Alocasia* leaves than in dark-adapted leaves. However, the difference in light transmittance through the spongy layer was not significant between high light- and dark-adapted leaves, indicating that chloroplast positioning on the anticlinal walls by the avoidance response in the palisade layer could facilitate light penetration to a deeper layer (Gorton et al., 1999). In *Tradescantia* leaves, which consist of three mesophyll cell layers (the first is a palisade layer, and the second and third are sponge layers), the chloroplasts in the second layer did not move to the anticlinal walls when irradiated with strong light of 100 µmol m-2 s-1 from either the adaxial or the abaxial side. However, by abaxial-side irradiation, chloroplasts in the third layer were positioned on the anticlinal walls by way of the avoidance response (Terashima & Hikosaka, 1995). These results suggest that the avoidance response in the surface mesophyll layer facilitates light capture in the deeper cell layers, resulting in a net increase of whole leaf photosynthesis. However, this hypothetical role of the avoidance movement in the enhancement of light penetration to

movement must have evolved early in land plant evolution.

Thus, chloroplasts should be located on the cell wall facing air spaces. Mesophyll cell chloroplasts tended to be located along intercellular air spaces in various plant species (Senn, 1908; Psaras et al., 1996) (Fig. 4c). Senn (1908) hypothesized that this positioning might result from the chemotaxis of chloroplasts to the CO2 in air spaces. Using different approaches and plant species, three groups examined whether chloroplast photorelocation movement, especially the avoidance response, influenced CO2 diffusion in leaves (Gorton et al., 2003; Loreto et al., 2009; Tholen et al., 2008). One research group hypothesized that chloroplast distribution on the anticlinal walls by the avoidance response facilitated CO2 utilization by shortening the CO2 diffusion path length from air spaces to the mesophyll chloroplasts (Gorton et al., 2003). This group measured oxygen diffusion times using pulsed photoacoustics (as a substitution for CO2 diffusion) between control and strong lightirradiated *Alocasia* leaf samples, but they could not find any differences in CO2 diffusion rates between the two samples (Gorton et al., 2003). Additionally, they could found no difference in the distance between the centers of the chloroplasts and the closest air spaces between two samples (Gorton et al., 2003). Another group found that blue light rapidly reduced CO2 diffusion from intercellular air spaces to the chloroplasts in both *Nicotiana* and *Platanus* leaves and that this reduction was completed before chloroplasts finished the avoidance movement to the aniticlinal walls (Loreto et al., 2009). Importantly, the blue-lightinduced reduction of CO2 diffusion was still normal in samples treated with an anti-actin inhibitor cytochalasin, which completely inhibits chloroplast movement (Loreto et al., 2009). Thus, results by two independent groups indicated that chloroplast movement did not significantly change the efficiency of CO2 diffusion from intercellular air spaces to the chloroplasts. However, the results by a third group suggested that the avoidance response reduced the CO2 diffusion rate rather than increased it (Tholen et al., 2008). In *Arabidopsis* wild-type plants, the surface area of chloroplasts facing air spaces was reduced after the induction of the chloroplast avoidance response and resulted in the reduction of CO2 diffusion. However, these reductions were not found in *phot2* and *chup1* mutant plants or in cytochalasin-treated plants (Tholen et al., 2008). Compared to wild type, the surface area of chloroplasts that faced air spaces and the rate of CO2 diffusion were constitutively lower in *chup1* mutant plants because of aberrant positioning of their chloroplasts (Tholen et al., 2008). Collectively, these results suggested that the chloroplast avoidance response was not involved in CO2 diffusion or that it possibly decreased, rather than increased, the diffusion rate. However, these three groups examined the contribution of chloroplast movement to CO2 diffusion using different plant species and techniques: pulsed photoacoustics (Gorton et al., 2003), a chlorophyll fluorescence-based method (Loreto et al., 2009) and a carbon isotope discrimination method (Tholen et al., 2008). The examination of one plant species using different techniques and/or that of various plant species by one technique are required to uncover whether chloroplast photorelocation movement influences CO2 diffusion.

#### **2.3.3 Chloroplast avoidance response is essential for protection against photodamage by strong light**

The two aforementioned hypotheses on the roles of the avoidance response are applicable only in multilayered leaf tissue and not in gametophytic cells that have single cell layers or in the filamentous structures of fern, moss, liverwort and green alga. During the early period of land plant evolution, plants were exposed directly to sunlight until seed plants eventually dominated terrestrial ecosystems and formed a dense canopy. Thus, it is plausible that the main role of the chloroplast avoidance response is to prevent chloroplasts

Chloroplast Photorelocation Movement: A Sophisticated

conditions.

**3. Conclusion** 

Strategy for Chloroplaststo Perform Efficient Photosynthesis 229

responses were implicated in the protection from photodamage in plants that inhabit tropical and/or dry areas. Experiments using *Arabidopsis* wild-type and mutant plants definitely demonstrated that chloroplast avoidance movement is essential for the protection of plants from photodamage by strong light (Kasahara et al., 2002). Leaf transmittance in wild type and *phot1* mutant plants increased as light intensity was increased to about fivefold of the initial value (about 500 µmol m-2 s-1 of white light). However, little change in leaf transmittance occurred in *phot2* and *chup1* mutant leaves, even at 2000 µmol m-2 s-1 of white light, indicating that *phot2* and *chup1* mutant plants are defective in the avoidance response under a wide range of light intensity. When low-light-acclimated plants were shifted to a strong light condition, the leaves of *phot2* and *chup1* mutant plants were bleached after 10 h and were severely necrotic after 22 h. However, wild-type and *phot1* plants did not show leaf necrosis even after 31 h. When the change in the chlorophyll fluorescence parameter *F*v/*F*m (representing the maximal quantum yield of photosystem II photochemistry) was analyzed during strong light treatment, the *F*v/*F*m value in wild-type and *phot1* plants steeply declined to about 80% of the initial value within 1 h and then gradually decreased and finally reached about 70% of the initial value in 5 h. However, the *F*v/*F*m values in *phot2* and *chup1* mutant plants declined more rapidly than in wild type and consequently reached about 50% of the initial value in 5 h. In *phot2* mutants, the extent of the decrease of the *F*v/*F*<sup>m</sup> value after 1 h of light treatment was larger at all examined light intensities than that of wild-type plants. Furthermore, the *F*v/*F*m value in *phot2* mutant plants did not fully recover after 6 h, whereas the *F*v/*F*m values in wild-type plants almost fully recovered after 3 h under low light. Collectively, these results indicate that *phot2* and *chup1* mutant plants are highly susceptible to strong light stress and their photosystem IIs are much less tolerant of light stress resulting in leaf necrosis. Note that *phot2* and *chup1* mutant plants were normal in chlorophyll content, chlorophyll fluorescence parameters, antioxidant contents and the activities of reactive oxygen-scavenging enzymes. *phot2* mutant plants showed a slight defect in stomatal opening (Kinoshita et al., 2001), but this defect was less than that in *phot1* mutant plants and was negligible in the strong light conditions used by Kasahara et al. Recently, another research group confirmed that photosystem II of *phot2* mutant plants was more susceptible to strong light (Sztatelman et al., 2010). Overall, we conclude that chloroplast avoidance movement is indispensable for plant survival under strong light

Although chloroplast photorelocation movement has been extensively studied by many researchers, we still cannot accurately explain the molecular mechanism of chloroplast photorelocation movement. Some unanswered questions remain: what is the signal for the chloroplast accumulation response?; what protein(s) nucleate cp-actin filaments?; and how do cp-actin filaments generate the motive force for chloroplast movement? To answer these questions, chloroplast movement must be analyzed by combining various approaches: physiology, molecular biology, proteomics, crystallography and imaging techniques. Chloroplast movement under natural light conditions must also be examined because natural light is usually much more severe and always fluctuates, compared to laboratory conditions. Because various mutants deficient in chloroplast movement are available, the growth of these mutant plants under natural conditions must be analyzed, and the

ecological significance of chloroplast photorelocation movement must be verified.

from photodamage caused by strong light (Zurzycki, 1957) (Fig. 4a). Shade plants (such as *Oxalis oregana* and *Tradesdantia albiflora*) showed a greater avoidance response than that of non-shade plants (such as sunflower and pea) (Brugnoli & Björkman, 1992; Park et al., 1996), partly explaining why *T. albiflora* was more tolerant of strong light stress than pea plants (Park et al., 1996). Furthermore, cytochalasin-treated *Platanus* leaves, whose chloroplast movements were inhibited by the drug, but not untreated leaves showed a strong inhibition of photochemical efficiency (Loreto et al., 2009). Clumping of chloroplasts in succulent plants (Kondo et al., 2004) and the aggregative movement of C4 mesophyll chloroplasts (Yamada et al., 2009) were induced by drought stress in a light-dependent fashion, and these

Fig. 4. Three hypotheses for the ecological significance of the chloroplast avoidance response: (a) Protection from photodamage. Chloroplasts escape from strong light and distribute along the anticlinal walls so that they do not directly perceive excess light energy, which could cause photodamage. As a result, plants can tolerate strong light stress. Mutants deficient in the avoidance response, such as Arabidopsis *phot2* mutants, cannot survive under the strong light conditions because their chloroplasts are directly exposed to extremely strong light and are therefore severely damaged and die. (b) Promotion of light penetration in leaves. Chloroplasts distributed along the anticlinal walls in the upper cell layer facilitate light penetration to deeper cell layers. Consequently, light perception and thus photosynthesis in the deeper cell layers increases. (c) Modulation of CO2 diffusion from intercellular air spaces to the chloroplast. The chloroplast avoidance response can change the total chloroplast surface area facing airspace and thus the efficiency of CO2 diffusion from intercellular air spaces to the chloroplast may increase.

from photodamage caused by strong light (Zurzycki, 1957) (Fig. 4a). Shade plants (such as *Oxalis oregana* and *Tradesdantia albiflora*) showed a greater avoidance response than that of non-shade plants (such as sunflower and pea) (Brugnoli & Björkman, 1992; Park et al., 1996), partly explaining why *T. albiflora* was more tolerant of strong light stress than pea plants (Park et al., 1996). Furthermore, cytochalasin-treated *Platanus* leaves, whose chloroplast movements were inhibited by the drug, but not untreated leaves showed a strong inhibition of photochemical efficiency (Loreto et al., 2009). Clumping of chloroplasts in succulent plants (Kondo et al., 2004) and the aggregative movement of C4 mesophyll chloroplasts (Yamada et al., 2009) were induced by drought stress in a light-dependent fashion, and these

Fig. 4. Three hypotheses for the ecological significance of the chloroplast avoidance response: (a) Protection from photodamage. Chloroplasts escape from strong light and distribute along the anticlinal walls so that they do not directly perceive excess light energy, which could cause photodamage. As a result, plants can tolerate strong light stress. Mutants deficient in the avoidance response, such as Arabidopsis *phot2* mutants, cannot survive under the strong light conditions because their chloroplasts are directly exposed to extremely strong light and are therefore severely damaged and die. (b) Promotion of light penetration in leaves. Chloroplasts distributed along the anticlinal walls in the upper cell layer facilitate light penetration to deeper cell layers. Consequently, light perception and thus photosynthesis in the deeper cell layers increases. (c) Modulation of CO2 diffusion from intercellular air spaces to the chloroplast. The chloroplast avoidance response can change the total chloroplast surface area facing airspace and thus the efficiency of CO2 diffusion

from intercellular air spaces to the chloroplast may increase.

responses were implicated in the protection from photodamage in plants that inhabit tropical and/or dry areas. Experiments using *Arabidopsis* wild-type and mutant plants definitely demonstrated that chloroplast avoidance movement is essential for the protection of plants from photodamage by strong light (Kasahara et al., 2002). Leaf transmittance in wild type and *phot1* mutant plants increased as light intensity was increased to about fivefold of the initial value (about 500 µmol m-2 s-1 of white light). However, little change in leaf transmittance occurred in *phot2* and *chup1* mutant leaves, even at 2000 µmol m-2 s-1 of white light, indicating that *phot2* and *chup1* mutant plants are defective in the avoidance response under a wide range of light intensity. When low-light-acclimated plants were shifted to a strong light condition, the leaves of *phot2* and *chup1* mutant plants were bleached after 10 h and were severely necrotic after 22 h. However, wild-type and *phot1* plants did not show leaf necrosis even after 31 h. When the change in the chlorophyll fluorescence parameter *F*v/*F*m (representing the maximal quantum yield of photosystem II photochemistry) was analyzed during strong light treatment, the *F*v/*F*m value in wild-type and *phot1* plants steeply declined to about 80% of the initial value within 1 h and then gradually decreased and finally reached about 70% of the initial value in 5 h. However, the *F*v/*F*m values in *phot2* and *chup1* mutant plants declined more rapidly than in wild type and consequently reached about 50% of the initial value in 5 h. In *phot2* mutants, the extent of the decrease of the *F*v/*F*<sup>m</sup> value after 1 h of light treatment was larger at all examined light intensities than that of wild-type plants. Furthermore, the *F*v/*F*m value in *phot2* mutant plants did not fully recover after 6 h, whereas the *F*v/*F*m values in wild-type plants almost fully recovered after 3 h under low light. Collectively, these results indicate that *phot2* and *chup1* mutant plants are highly susceptible to strong light stress and their photosystem IIs are much less tolerant of light stress resulting in leaf necrosis. Note that *phot2* and *chup1* mutant plants were normal in chlorophyll content, chlorophyll fluorescence parameters, antioxidant contents and the activities of reactive oxygen-scavenging enzymes. *phot2* mutant plants showed a slight defect in stomatal opening (Kinoshita et al., 2001), but this defect was less than that in *phot1* mutant plants and was negligible in the strong light conditions used by Kasahara et al. Recently, another research group confirmed that photosystem II of *phot2* mutant plants was more susceptible to strong light (Sztatelman et al., 2010). Overall, we conclude that chloroplast avoidance movement is indispensable for plant survival under strong light conditions.

#### **3. Conclusion**

Although chloroplast photorelocation movement has been extensively studied by many researchers, we still cannot accurately explain the molecular mechanism of chloroplast photorelocation movement. Some unanswered questions remain: what is the signal for the chloroplast accumulation response?; what protein(s) nucleate cp-actin filaments?; and how do cp-actin filaments generate the motive force for chloroplast movement? To answer these questions, chloroplast movement must be analyzed by combining various approaches: physiology, molecular biology, proteomics, crystallography and imaging techniques. Chloroplast movement under natural light conditions must also be examined because natural light is usually much more severe and always fluctuates, compared to laboratory conditions. Because various mutants deficient in chloroplast movement are available, the growth of these mutant plants under natural conditions must be analyzed, and the ecological significance of chloroplast photorelocation movement must be verified.

Chloroplast Photorelocation Movement: A Sophisticated

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#### **4. Acknowledgment**

This work was supported in part by the Japanese Ministry of Education, Sports, Science, and Technology (MEXT 13139203 and 17084006 to M. W.) and the Japan Society of Promotion of Science (JSPS 13304061, 16107002, and 20227001 to M. W.; 20870030 to N. S.).

#### **5. References**


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**12** 

*Iran* 

**by the Canopy** 

Mansour Matloobi

**Light Harvesting and Photosynthesis** 

*Department of Horticulture, Faculty of Agriculture, University of Tbariz, Tabriz,* 

Photosynthesis is a life-sustaining process driven mostly by green plants to support not only life of plants, but also life on earth in general. The estimated dry matter produced by photosynthesis of land plants reaches as much as 125×109 per year (Field et al., 1998). About 40% of this material is composed of C, fixed in photosynthesis. Light has long been recognized as a source of energy to convert atmospheric CO2 into energetic chemical bands which finally appear as sucrose, starch and many other energy containing substances. This conversion will not happen until there is specialized light-harvesting system to capture and transfer light energy to low-energy compounds. Leaves are this specialized system with broad, laminar surface well suited to gather and absorb light. When a large number of leaves are arranged beside each other the canopy will be formed. Organization and spatial arrangement of leaves within the canopy directly affect the amount of light absorbed by this integrated system. Therefore, photosynthetic capacity at canopy level depends not only on factors affecting leaf level photosynthesis but also on factors which influence properties of canopy microclimate, particularly its light distribution profile. Estimating photosynthesis at canopy scale, however can be of great importance as it provides a tool to predict crop yield and help producer to make decision and planning of production. While photosynthesis mechanism in C4 plants differs virtually from that of C3 plants, there have been no significant differences in the methods implemented to investigate light harvesting and upscaling photosynthesis from leaf to canopy level, so in this chapter the issues related to

There are many factors that determine plant canopy architecture. Some of these factors are genetic and relate to the plant species while some are ecological and relate to the plantenvironment interactions. Under the influence of these factors plants develop their canopy so that they reach a compromise between affecting factors and internal physiological requirements. The resultant will be a volume composed of numerous leaves varying in size, thickness, inclination and many other physical and physiological properties distributed in space and time. Naturally, plants attempt to construct their canopy in a way that the highest ambient irradiance could be absorbed. This process is usually done by developing special branching system, efficient leaf arrangement; appropriate canopy dimension and

the photosynthesis of C3 plants will be emphasized and addressed.

**2. Canopy: An integrated foliage structure** 

**1. Introduction** 


### **Light Harvesting and Photosynthesis by the Canopy**

Mansour Matloobi

*Department of Horticulture, Faculty of Agriculture, University of Tbariz, Tabriz, Iran* 

#### **1. Introduction**

234 Advances in Photosynthesis – Fundamental Aspects

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von Caemmerer, S & Furbank, R.T. (2003). The C4 pathway: an efficient CO2 pump. *Photosynthesis Research,* Vol.77, No.2-3, (August 2003), pp. 191-207, ISSN 0166-8595 Wada, M. (2007). The fern as a model system to study photomorphogenesis. *Journal of Plant* 

Wada, M.; Grolig, F. & Haupt, W. (1993). New trends in photobiology: Light-oriented

Wada, M. & Suetsugu, N. (2004). Chloroplasts can move in any direction to avoid strong

Whippo, C.W.; Khurana, P.; Davis, P.A.; DeBlasio, S.L.; DeSloover, D.; Staiger, C.J. &

Yamada, M.; Kawasaki, M.; Sugiyama, T.; Miyake, H. & Taniguchi, M. (2009). Differential

Yamashita, H.; Sato, Y.; Kanegae, T.; Kagawa, T.; Wada, M. & Kadota, A. (2011). Chloroplast

Yatsuhashi, H. (1996). Photoregulation systems for light-oriented chloroplast movement. *Journal of Plant Research,* Vol.109, No.2, (June 1996), pp. 139-146, ISSN 0918-9440 Yatsuhashi, H.; Kadota, A. & Wada, M. (1985). Blue- and red-light action in photoorientation

Yatsuhashi, H.; Wada, M. & Hashimoto, T. (1987). Dichroic orientation of phytochrome and

movement. *Planta,* Vol.9, No.3, (September 1987), pp. 27-63, ISSN 0137-5881 Zurzycki, J. (1955). Chloroplast arrangement as a factor in photosynthesis. *Acta Societasis* 

Zurzycki, J. (1957). The destructive effect of intense light on the photosynthetic apparatus. *Acta Societasis Botanicorum Poloniae,* Vol.26, (1957), pp. 157-175, ISSN 0001-6977 Zurzycki, J. (1980). Blue light-induced intracellular movement, In: *Blue Light Syndrome,* H. Senger, (Ed.), 50-68, Springer-Verlag, ISBN 3-540-10075-X, Heidelberg, Germany

*Botanicorum Poloniae,* Vol.24, (1955), pp. 163-173, ISSN 0001-6977

*Research,* Vol.120, No.1, (January 2007), pp. 3-16, ISSN 0918-9440

doi:10.1007/s10265-011-0444-8, ISSN 0918-9440

9440

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0935

59-64, ISSN 0960-9822

50, ISSN 0032-0935

light. *Journal of Plant Research,* Vol.124, No.1, (January 2011), pp. 201-210, ISSN 0918-

movement in *Adiantum capillus-veneris*. *Journal of Plant Research,* (July 2011),

chloroplast positioning. Contribution to progress in photobiology. *Journal of Photochemistry and Photobiology B: Biology,* Vol.17, No.1, (January 1993), pp. 3-25,

light. *Current Opinion in Plant Biology,* Vol.7, No.6, (December 2004), pp. 626-631,

Hangarter, R.P. (2011). THRUMIN1 is a light-regulated actin-bundling protein involved in chloroplast motility. *Current Biology,* Vol.21, No.1, (January 2011), pp.

positioning of C4 mesophyll and bundle sheath chloroplasts: Aggregative movement of C4 mesophyll chloroplasts in response to environmental stresses. *Plant & Cell Physiology,* Vol.50, No.10, (October 2009), pp. 1736-1749, ISSN 0032-0781

actin filaments organize meshwork on the photorelocated chloroplasts in the moss *Physcomitrella patens*. *Planta,* Vol.233, No.2, (February 2011), pp. 357-368, ISSN 0032-

of chloroplasts in *Adiantum* protonemata. *Planta,* Vol.165, No.1, (July 1985), pp. 43-

blue-light photoreceptor in *Adiantum* protonemata as determined by chloroplast

Photosynthesis is a life-sustaining process driven mostly by green plants to support not only life of plants, but also life on earth in general. The estimated dry matter produced by photosynthesis of land plants reaches as much as 125×109 per year (Field et al., 1998). About 40% of this material is composed of C, fixed in photosynthesis. Light has long been recognized as a source of energy to convert atmospheric CO2 into energetic chemical bands which finally appear as sucrose, starch and many other energy containing substances. This conversion will not happen until there is specialized light-harvesting system to capture and transfer light energy to low-energy compounds. Leaves are this specialized system with broad, laminar surface well suited to gather and absorb light. When a large number of leaves are arranged beside each other the canopy will be formed. Organization and spatial arrangement of leaves within the canopy directly affect the amount of light absorbed by this integrated system. Therefore, photosynthetic capacity at canopy level depends not only on factors affecting leaf level photosynthesis but also on factors which influence properties of canopy microclimate, particularly its light distribution profile. Estimating photosynthesis at canopy scale, however can be of great importance as it provides a tool to predict crop yield and help producer to make decision and planning of production. While photosynthesis mechanism in C4 plants differs virtually from that of C3 plants, there have been no significant differences in the methods implemented to investigate light harvesting and upscaling photosynthesis from leaf to canopy level, so in this chapter the issues related to the photosynthesis of C3 plants will be emphasized and addressed.

#### **2. Canopy: An integrated foliage structure**

There are many factors that determine plant canopy architecture. Some of these factors are genetic and relate to the plant species while some are ecological and relate to the plantenvironment interactions. Under the influence of these factors plants develop their canopy so that they reach a compromise between affecting factors and internal physiological requirements. The resultant will be a volume composed of numerous leaves varying in size, thickness, inclination and many other physical and physiological properties distributed in space and time. Naturally, plants attempt to construct their canopy in a way that the highest ambient irradiance could be absorbed. This process is usually done by developing special branching system, efficient leaf arrangement; appropriate canopy dimension and

Light Harvesting and Photosynthesis by the Canopy 237

experiences some quantitative and qualitative alterations which favor life sustaining processes occurring on the planet. Upon reaching leaf surface light transferred and distributed through the leaf by a phenomenon called **lens effect** created by the planoconvex nature of epidermal cells covering leaf surface. The consequent of this effect is efficient redirecting of incoming radiation to the chloroplasts confined in mesophyll cells. The mesophyll tissue consisted of two distinct cells: palisade and spongy cells. Palisade cells are elongated and cylindrical with the long axis perpendicular to the surface of the leaf, while spongy cells situated below this layer and surrounded by the prominent air spaces (Hopkins & Huner, 2004). Although a large number of chloroplasts occupy the cell volume of palisades, there is still a significant proportion of cell volume that does not contain chloroplast. This chloroplast-free portion of the cell helps to distribute incoming light and maximize absorption by chlorophyll. Consequently, some of the incident light may pass through the first palisade layer without being absorbed, but more likely will be intercepted through successive layers by **the sieve effect.** Additionally, palisade cells help efficient distribution of incoming light by **light-guide effect**, a feature that assists light reaching the cell-air interfaces to be reflected and channeled through these layers to the spongy

A large portion of the light reaching the leaf surface then finally targets the chloroplasts, where photochemical reactions occur. Although the mesophyll layer is the main place hosting chloroplasts, these organelles may be also found in other organs such as; buds, the bark of stems and branches, flowers and fruits. Light interception in chloroplasts is carried out specifically by antenna complex or light harvesting complex (LHC), mainly consisted of chlorophylls (i.e. chlorophyll a and b) and several hundred accessory pigments clustered together in the thylakoid membrane. Carotenoides are one of the most important accessory pigments in green plants which absorb light at wavelength different from that of chlorophyll and so act together to maximize the light harvested. When a pigment molecule absorbed incoming photon energy and excited, it transfers the energy to two special chlorophyll molecules in the photosynthetic reaction center. The reaction center then passes on the energy as a high-energy electron to a chain of electron carriers in the thylakoid membrane. The high energy electrons are then exploited to produce high energy molecules

In response to changes of environmental conditions chloroplast may undergo some modifications in structure and biochemical composition in order to cope with new environment. Some of these environmental factors negatively affect chloroplast activity and therefore directly limit the photosynthetic rate. The consequence of most of these factors, such as high light intensity, UV radiation, air pollutants, herbicides, water and heavy metal stress will usually appear as oxidative stress and often leads to the symptoms of structural damage which emerges as swelling of thylakoids, plastoglobule and starch accumulation, photodestruction of pigments, and inhibition of photosynthesis (Mostowska, 1997). It was shown similarly that chloroplast property changes in accordance with the light gradient within a bifacial leaf (Terashima & Inoue, 1985). That is, near leaf surface facing to ambient light, the chloroplasts have higher rates of electron transport and Rubisco activities per unit of chlorophyll than chloroplasts farther away from the surface. Moreover, in plants acclimated to shade conditions, it was shown that chloroplasts migrate in response to

Plants try to increase their light absorption at leaf level by adjusting leaf weight to plant weight or leaf weight to leaf area. One of the parameters which can be very helpful in giving

mesophyll below (Hopkins & Huner, 2004).

which are eventually used to reduce RuBP by CO2.

inducing ambient light (lambers et al., 1998).

even sometimes by natural pruning and removing weak and underdeveloped organs. Consequently canopies appear to be a complex, dynamic and ever-changing volumes; being difficult to interpret and understand. The complexity of canopy becomes more apparent when we move from leaf level to pure stand to heterogeneous plant communities, since each level contains elements of the lower levels (Norman & Campbell 1994). In vast plant communities, when diverse plant species mixed together and form a very heterogeneous vegetation stand, description of canopy structure become much more difficult. Therefore canopies composed of single species or integrated of only a few species usually assumed to be homogeneous with uniform monotypic plant stand (Beyshlag & Ryel, 2007).

In order to interpret canopy in detail we may have to consider its components. Canopy structure can be defined in detail by including the size, shape, orientation and positional distribution of various plant organs such as leaves, stems, branches, flowers and fruits. Getting such information for each element in a canopy is not currently feasible, so quantitative description of the canopy by means of mathematical and statistical methods seems to be appropriate. Norman and Campbell (1994) summarized all the methods applied in describing canopy structure to two main groups: direct and indirect methods. They explained that direct methods involve usually much labor in the field and require very simple data reduction when compared to the indirect methods which use simple and rapid field measurements but complex algorithms for the reduction of data. In spite of recent considerable progresses achieved in 3D modeling by computers, this technique still requires a considerable effort to sample all the growing organs of a canopy. Because of this, only a few variables, such as the leaf area density, and the leaf inclination distribution function could be used to describe canopy structure (Weiss et al., 2004). Sound estimation of a crop whole canopy leaf area may be sufficient to predict crop productivity in large scale, but does not give an accurate estimation of vertical gradient of light or spatial distribution of materials applied to the plant canopy. Plant architectural models attempt to fill the gap caused by not considering the influence of plant functioning or environmental variables on the process of morphogenesis through including physiological processes of plant growth and development as well as the physical structure of plants. To do this, more precise and extensive data will be required than usually collected on the dynamics of production of individual organs of plants (Birch et al., 2003).

#### **3. Light harvesting**

Light harvesting by plants is influenced by many factors such as, diurnal variation in solar elevation and variation in leaf angle, leaf position in the canopy, sky cloudiness, degree of leaf clumping and amount of sunflecks penetrated through the canopy, and all the factors affecting gas flux properties of individual leaves. Photosynthesis occurs in leaves, the smallsized food factories constituting the majority of the canopy volume. Any disturbances in canopy microclimate such as variations occurred in ambient gas composition, light quantity and quality, temperature and humidity will clearly lead to corresponding changes in C uptake by the leaves. Therefore studying leaves as the primary light harvesting organ within the canopy could merit first priority.

#### **3.1 Light harvesting at the leaf scale**

Before being intercepted by leaves, light travels a long distance between the sun and the earth, passing through the atmosphere according to its composition and physical features, it

even sometimes by natural pruning and removing weak and underdeveloped organs. Consequently canopies appear to be a complex, dynamic and ever-changing volumes; being difficult to interpret and understand. The complexity of canopy becomes more apparent when we move from leaf level to pure stand to heterogeneous plant communities, since each level contains elements of the lower levels (Norman & Campbell 1994). In vast plant communities, when diverse plant species mixed together and form a very heterogeneous vegetation stand, description of canopy structure become much more difficult. Therefore canopies composed of single species or integrated of only a few species usually assumed to

In order to interpret canopy in detail we may have to consider its components. Canopy structure can be defined in detail by including the size, shape, orientation and positional distribution of various plant organs such as leaves, stems, branches, flowers and fruits. Getting such information for each element in a canopy is not currently feasible, so quantitative description of the canopy by means of mathematical and statistical methods seems to be appropriate. Norman and Campbell (1994) summarized all the methods applied in describing canopy structure to two main groups: direct and indirect methods. They explained that direct methods involve usually much labor in the field and require very simple data reduction when compared to the indirect methods which use simple and rapid field measurements but complex algorithms for the reduction of data. In spite of recent considerable progresses achieved in 3D modeling by computers, this technique still requires a considerable effort to sample all the growing organs of a canopy. Because of this, only a few variables, such as the leaf area density, and the leaf inclination distribution function could be used to describe canopy structure (Weiss et al., 2004). Sound estimation of a crop whole canopy leaf area may be sufficient to predict crop productivity in large scale, but does not give an accurate estimation of vertical gradient of light or spatial distribution of materials applied to the plant canopy. Plant architectural models attempt to fill the gap caused by not considering the influence of plant functioning or environmental variables on the process of morphogenesis through including physiological processes of plant growth and development as well as the physical structure of plants. To do this, more precise and extensive data will be required than usually collected on the dynamics of production of

Light harvesting by plants is influenced by many factors such as, diurnal variation in solar elevation and variation in leaf angle, leaf position in the canopy, sky cloudiness, degree of leaf clumping and amount of sunflecks penetrated through the canopy, and all the factors affecting gas flux properties of individual leaves. Photosynthesis occurs in leaves, the smallsized food factories constituting the majority of the canopy volume. Any disturbances in canopy microclimate such as variations occurred in ambient gas composition, light quantity and quality, temperature and humidity will clearly lead to corresponding changes in C uptake by the leaves. Therefore studying leaves as the primary light harvesting organ

Before being intercepted by leaves, light travels a long distance between the sun and the earth, passing through the atmosphere according to its composition and physical features, it

be homogeneous with uniform monotypic plant stand (Beyshlag & Ryel, 2007).

individual organs of plants (Birch et al., 2003).

within the canopy could merit first priority.

**3.1 Light harvesting at the leaf scale** 

**3. Light harvesting** 

experiences some quantitative and qualitative alterations which favor life sustaining processes occurring on the planet. Upon reaching leaf surface light transferred and distributed through the leaf by a phenomenon called **lens effect** created by the planoconvex nature of epidermal cells covering leaf surface. The consequent of this effect is efficient redirecting of incoming radiation to the chloroplasts confined in mesophyll cells. The mesophyll tissue consisted of two distinct cells: palisade and spongy cells. Palisade cells are elongated and cylindrical with the long axis perpendicular to the surface of the leaf, while spongy cells situated below this layer and surrounded by the prominent air spaces (Hopkins & Huner, 2004). Although a large number of chloroplasts occupy the cell volume of palisades, there is still a significant proportion of cell volume that does not contain chloroplast. This chloroplast-free portion of the cell helps to distribute incoming light and maximize absorption by chlorophyll. Consequently, some of the incident light may pass through the first palisade layer without being absorbed, but more likely will be intercepted through successive layers by **the sieve effect.** Additionally, palisade cells help efficient distribution of incoming light by **light-guide effect**, a feature that assists light reaching the cell-air interfaces to be reflected and channeled through these layers to the spongy mesophyll below (Hopkins & Huner, 2004).

A large portion of the light reaching the leaf surface then finally targets the chloroplasts, where photochemical reactions occur. Although the mesophyll layer is the main place hosting chloroplasts, these organelles may be also found in other organs such as; buds, the bark of stems and branches, flowers and fruits. Light interception in chloroplasts is carried out specifically by antenna complex or light harvesting complex (LHC), mainly consisted of chlorophylls (i.e. chlorophyll a and b) and several hundred accessory pigments clustered together in the thylakoid membrane. Carotenoides are one of the most important accessory pigments in green plants which absorb light at wavelength different from that of chlorophyll and so act together to maximize the light harvested. When a pigment molecule absorbed incoming photon energy and excited, it transfers the energy to two special chlorophyll molecules in the photosynthetic reaction center. The reaction center then passes on the energy as a high-energy electron to a chain of electron carriers in the thylakoid membrane. The high energy electrons are then exploited to produce high energy molecules which are eventually used to reduce RuBP by CO2.

In response to changes of environmental conditions chloroplast may undergo some modifications in structure and biochemical composition in order to cope with new environment. Some of these environmental factors negatively affect chloroplast activity and therefore directly limit the photosynthetic rate. The consequence of most of these factors, such as high light intensity, UV radiation, air pollutants, herbicides, water and heavy metal stress will usually appear as oxidative stress and often leads to the symptoms of structural damage which emerges as swelling of thylakoids, plastoglobule and starch accumulation, photodestruction of pigments, and inhibition of photosynthesis (Mostowska, 1997). It was shown similarly that chloroplast property changes in accordance with the light gradient within a bifacial leaf (Terashima & Inoue, 1985). That is, near leaf surface facing to ambient light, the chloroplasts have higher rates of electron transport and Rubisco activities per unit of chlorophyll than chloroplasts farther away from the surface. Moreover, in plants acclimated to shade conditions, it was shown that chloroplasts migrate in response to inducing ambient light (lambers et al., 1998).

Plants try to increase their light absorption at leaf level by adjusting leaf weight to plant weight or leaf weight to leaf area. One of the parameters which can be very helpful in giving

Light Harvesting and Photosynthesis by the Canopy 239

depending on the canopy architecture some may reach the most lower layers and form "sunflecks". These packages of high light intensity are not generally stable, but dynamically change their location due to movements of branches, and the changing angle of the sun. Their duration may range from less than a second to minutes. Small-sized sunflecks typically carry lower light energy than direct sunlight because of penumbral effects, but large ones can approach irradiances of direct sunlight (Lambers et al., 1998). Direct beam light predominantly absorbed by the leaves at the top of the canopy, some portion transmitted down with altered spectral quality, due to action of the various leaf pigments. Leaves typically transmit only a few percent of incident PAR in the green band at around 550 nm, and are otherwise efficiently opaque in the visible range. Transmittance of PAR is normally less than 10%, whereas transmittance of far-red light is substantial (Terashima & Hikosala, 1995). This spectral alteration affects the phytochrome photoequilibrum and allows plants to perceive shading by other plants to adjust their photomorphogenesis activities (Lambers et al., 1998). Leaves like to other biological surfaces not only transmit light but reflect a proportion. The amount of reflection depends on morphological and physical properties of leaves such as, leaf shape, thickness and shininess of the cuticle. However, it should be noted that reflected light then may be absorbed or transmitted by the

Rundel and Gibson (1996) found that leaf angle and orientation are the main factors which control daily integrated radiation, maximum irradiance and diurnal distribution of irradiance. Orientation of leaves at the top of the canopy is usually at oblique (acute or obtuse) to incident light. When leaves in the uppermost layer of the canopy arrange obliquely, they allow a given amount of light to distribute over a greater total leaf area of the plant than when they arrange at right angles to the direction of incoming light. While leaves on the canopy surface are most efficient at utilizing full sunlight when at an oblique angle to the sun's ray, the leaves located in lower parts do best in lower irradiance if the leaf area is at right angle to the light, intercepting the greatest sunlight per leaf surface. Ontogenetically change from sessile juvenile leaves to petiolate adult leaves is accompanied by a change in leaf orientation from horizontal to vertical (king, 1997). Research by Shelley and Bell (2000) on the heteroblastic species *Eucalyptus globules* Labill. ssp. Globules showed that there was no active diurnal orientation between juvenile and adult leaves twoard or away from incident radiation. They concluded that greater interception of light by juvenile leaves compared with vertical adult leaves, may be due to their high adaptation capacity to high

Beer's law has long been used by many authors to describe light penetration in plant canopy. With the assumption that the gaps are randomly distributed horizontally, the area of direct-beam irradiance penetrating to any depth in the canopy is an exponential function

 *I=IO exp(-KLAI)* (3) I and Io are respectively the irradiance beneath and above the canopy (umolm-2s-1), K is the extinction coefficient and LAI denotes leaf area index. The extinction coefficient is actually the ratio of horizontally projected shadow area per unit ground area per unit leaf area. Both leaf angle and solar elevation angle (β) affect the shadow projection of leaves. At any point

of the cumulative LAI from the top of the canopy (Boote & Loomis, 1991):

lower leaves similar to the radiation reaching the canopy surface.

incident light.

**3.2.1 Light profile within the canopy** 

good understanding of the plant manner of investment on light harvesting complexes is specific leaf area (SLA). It is defined as projected leaf area per unit leaf dry mass. This parameter relates with the other plant growth parameters as follows:

$$\text{LAR=LWR} \times \text{SLA} \tag{1}$$

LWR is the ratio of leaf weight to plant weight (gg-1), LAR is the ratio of leaf area to plant weight (m2g-1). The equation that links LAR to RGR is:

$$\text{RGR} = \text{NAR} \times \text{LAR} \tag{2}$$

Where RGR is relative growth rate (gg-1d-1) and NAR denotes net assimilation rate (gm-2d-1). This relationship implies that transferring a sun-acclimated plant to a shade environment will result in a reduction in RGR caused by a lowering in NAR, reflecting the effect of PAR on photosynthesis. In order to keep RGR unchanged, plant has to increase LAR with the assumption that there is no change in the light dependence of photosynthesis. LAR directly changes with any changes in LWR and/or SLA. It has been revealed that LWR may proportionally change in accordance with plant light regime alterations, having tendency to increase in shade-adapted plants, while showing decline in non-adapted plants in shade (Fitter & Hay, 2002). Studying with many plants indicated that SLA seems to change faster than LWR, playing an important role in acclimation process to varying environmental light regimes. Plants developed under high light usually have thick leaves with a low SLA (Bjorkman, 1981, as cited in Fitter & Hay, 2002). Light-saturated photosynthesis remained unchanged in plants acclimated to shade environment because of doubling SLA (Evans & Poorter, 2001). It can be deduced that SLA is more variable than LWR, or, leaf area is more plastic than leaf weight. Studying with *Cucumis sativa* , a light-demanding species, showed that leaf area changes proportionally with the total ambient light, with a maximum at about 4.2 Mjm-2d-1 (Newton, 1963, as cited in Fitter & Hay, 2002). Instantaneous light variations do not exert any immediate changes in SLA, while these changes generally occur in response to total radiation load; this is probably the case for *Impatients parviflora* which shows an almost threefold increase in SLA when grown in 7% of full daylight (Evans & Hughes, 1961, as cited in Fitter & Hay, 2002). Findings of Evans and Poorter (2001) indicated that increasing SLA is a very important means applied by plants to maximize carbon gain per unit leaf mass under different environmental light conditions.

#### **3.2 Light harvesting at the canopy scale**

Foliage density distribution and leaves orientation highly impact sunlight attenuation through the canopy. As described before, canopies normally are not solid sheets, but are loosely stacked formation of leaves which help plants to effectively absorb most of the incident light, with leaves near the top of the canopy absorbing near maximum solar radiation and the lower leaves perceiving sunlight of a reduced intensity and also an altered spectral composition. Therefore the amount of photosynthetically active radiation intercepted by a leaf usually depends on its position in the canopy and the angle it faces incoming solar radiation. Leaves within the canopy are generally subject to three types of radiation: light beam, reflected and transmitted radiation. Light beam penetrates through the gaps created in the canopy probably by instantaneous fluttering of leaves caused by wind, or sparse leaf arrangement which naturally forms gaps within the canopy. While passing through the canopy light beam is usually trapped by the lower leaf layers, however,

good understanding of the plant manner of investment on light harvesting complexes is specific leaf area (SLA). It is defined as projected leaf area per unit leaf dry mass. This

LWR is the ratio of leaf weight to plant weight (gg-1), LAR is the ratio of leaf area to plant

 RGR=NAR×LAR (2) Where RGR is relative growth rate (gg-1d-1) and NAR denotes net assimilation rate (gm-2d-1). This relationship implies that transferring a sun-acclimated plant to a shade environment will result in a reduction in RGR caused by a lowering in NAR, reflecting the effect of PAR on photosynthesis. In order to keep RGR unchanged, plant has to increase LAR with the assumption that there is no change in the light dependence of photosynthesis. LAR directly changes with any changes in LWR and/or SLA. It has been revealed that LWR may proportionally change in accordance with plant light regime alterations, having tendency to increase in shade-adapted plants, while showing decline in non-adapted plants in shade (Fitter & Hay, 2002). Studying with many plants indicated that SLA seems to change faster than LWR, playing an important role in acclimation process to varying environmental light regimes. Plants developed under high light usually have thick leaves with a low SLA (Bjorkman, 1981, as cited in Fitter & Hay, 2002). Light-saturated photosynthesis remained unchanged in plants acclimated to shade environment because of doubling SLA (Evans & Poorter, 2001). It can be deduced that SLA is more variable than LWR, or, leaf area is more plastic than leaf weight. Studying with *Cucumis sativa* , a light-demanding species, showed that leaf area changes proportionally with the total ambient light, with a maximum at about 4.2 Mjm-2d-1 (Newton, 1963, as cited in Fitter & Hay, 2002). Instantaneous light variations do not exert any immediate changes in SLA, while these changes generally occur in response to total radiation load; this is probably the case for *Impatients parviflora* which shows an almost threefold increase in SLA when grown in 7% of full daylight (Evans & Hughes, 1961, as cited in Fitter & Hay, 2002). Findings of Evans and Poorter (2001) indicated that increasing SLA is a very important means applied by plants to maximize carbon gain per unit leaf mass

Foliage density distribution and leaves orientation highly impact sunlight attenuation through the canopy. As described before, canopies normally are not solid sheets, but are loosely stacked formation of leaves which help plants to effectively absorb most of the incident light, with leaves near the top of the canopy absorbing near maximum solar radiation and the lower leaves perceiving sunlight of a reduced intensity and also an altered spectral composition. Therefore the amount of photosynthetically active radiation intercepted by a leaf usually depends on its position in the canopy and the angle it faces incoming solar radiation. Leaves within the canopy are generally subject to three types of radiation: light beam, reflected and transmitted radiation. Light beam penetrates through the gaps created in the canopy probably by instantaneous fluttering of leaves caused by wind, or sparse leaf arrangement which naturally forms gaps within the canopy. While passing through the canopy light beam is usually trapped by the lower leaf layers, however,

LAR=LWR×SLA (1)

parameter relates with the other plant growth parameters as follows:

weight (m2g-1). The equation that links LAR to RGR is:

under different environmental light conditions.

**3.2 Light harvesting at the canopy scale** 

depending on the canopy architecture some may reach the most lower layers and form "sunflecks". These packages of high light intensity are not generally stable, but dynamically change their location due to movements of branches, and the changing angle of the sun. Their duration may range from less than a second to minutes. Small-sized sunflecks typically carry lower light energy than direct sunlight because of penumbral effects, but large ones can approach irradiances of direct sunlight (Lambers et al., 1998). Direct beam light predominantly absorbed by the leaves at the top of the canopy, some portion transmitted down with altered spectral quality, due to action of the various leaf pigments. Leaves typically transmit only a few percent of incident PAR in the green band at around 550 nm, and are otherwise efficiently opaque in the visible range. Transmittance of PAR is normally less than 10%, whereas transmittance of far-red light is substantial (Terashima & Hikosala, 1995). This spectral alteration affects the phytochrome photoequilibrum and allows plants to perceive shading by other plants to adjust their photomorphogenesis activities (Lambers et al., 1998). Leaves like to other biological surfaces not only transmit light but reflect a proportion. The amount of reflection depends on morphological and physical properties of leaves such as, leaf shape, thickness and shininess of the cuticle. However, it should be noted that reflected light then may be absorbed or transmitted by the lower leaves similar to the radiation reaching the canopy surface.

Rundel and Gibson (1996) found that leaf angle and orientation are the main factors which control daily integrated radiation, maximum irradiance and diurnal distribution of irradiance. Orientation of leaves at the top of the canopy is usually at oblique (acute or obtuse) to incident light. When leaves in the uppermost layer of the canopy arrange obliquely, they allow a given amount of light to distribute over a greater total leaf area of the plant than when they arrange at right angles to the direction of incoming light. While leaves on the canopy surface are most efficient at utilizing full sunlight when at an oblique angle to the sun's ray, the leaves located in lower parts do best in lower irradiance if the leaf area is at right angle to the light, intercepting the greatest sunlight per leaf surface. Ontogenetically change from sessile juvenile leaves to petiolate adult leaves is accompanied by a change in leaf orientation from horizontal to vertical (king, 1997). Research by Shelley and Bell (2000) on the heteroblastic species *Eucalyptus globules* Labill. ssp. Globules showed that there was no active diurnal orientation between juvenile and adult leaves twoard or away from incident radiation. They concluded that greater interception of light by juvenile leaves compared with vertical adult leaves, may be due to their high adaptation capacity to high incident light.

#### **3.2.1 Light profile within the canopy**

Beer's law has long been used by many authors to describe light penetration in plant canopy. With the assumption that the gaps are randomly distributed horizontally, the area of direct-beam irradiance penetrating to any depth in the canopy is an exponential function of the cumulative LAI from the top of the canopy (Boote & Loomis, 1991):

$$\mathbf{I} \equiv \mathbf{I}\_O \exp(\mathbf{-KLAI}) \tag{3}$$

I and Io are respectively the irradiance beneath and above the canopy (umolm-2s-1), K is the extinction coefficient and LAI denotes leaf area index. The extinction coefficient is actually the ratio of horizontally projected shadow area per unit ground area per unit leaf area. Both leaf angle and solar elevation angle (β) affect the shadow projection of leaves. At any point

Light Harvesting and Photosynthesis by the Canopy 241

Photosynthesis process begins with absorbing light-energy photos and transferring them to the reaction centers of the photosystems where the second process starts. In the thylakoid membrane water splits to release electrons which are then transported along an electrontransport chain to produce NADPH and ATP. All the reactions occurred up to this point are called light reactions of photosynthesis as they depend on light energy to proceed. The produced NADPH and ATP during the light reactions then enter the carbon-reduction cycle (Calvin cycle), in which CO2 assimilated, leading to the synthesis of C3 compounds (triosephosphates). This reaction does not need light to proceed and therefore is referred to as the dark reaction of photosynthesis. In Calvin cycle, CO2 molecules are condensed with ribulose 1,5-bisphoshate ( a five-carbon molecule) to produce a transient six-carbon intermediate that immediately hydrolyzes to two molecules of 3-phosphoglycerate. This important process will not be complete without the mediation of the key enzyme ribulose bisphosphate carboxylase/oxygenase (often called Rubisco). Rubisco is known as a slow enzyme as it only fixes three molecules of its substrate every second and hence plants need a large amount of this enzyme to assimilate enough CO2 to sustain plant life. Approximately 50% of chloroplast protein content is Rubisco, probably the most abundant protein on the earth.

The rate of photosynthesis at leaf level varies widely and is influenced not only by leaf internal biochemical and physiological conditions, but also by many environmental variables such as, CO2 concentration, light intensity, temperature and humidity fluctuations. Temperature is an essential factor that control enzymatically catalyzed reactions and membrane processes and in this way it controls photosynthesis (Lambers et al., 1998). Photosynthetic response to temperature varies among species because of the different activation energy required by different reactions processed in various plants. Consequently, temperature-dependent photosynthesis of plants range widely from temperatures near freezing to over 40C, implying that the specific range depends on species and genotype, plant age, plant origin, and season (Pallardy, 2008). Optimal temperature for any plant is usually defined by the temperature that plant has experienced and adapted to during the entire growing period. High temperatures increase affinity of Rubisco to oxygen than carbon dioxide, consequently leading to enhancing photorespiration. In addition, the solubility of CO2 declined with increasing temperature more strongly than does that of O2. At temperatures below about 15C the rate of photosynthesis is often reduced in many (sub)tropical plants. For example, after exposing coffee trees to 4C at night, the rate of photosynthesis was reduced by more than half (Pallardy, 2008). This kind of damage is called chilling injury and differs from frost damage, a type of damage that only happens below 0C. Chilling injury generally results from a precipitate decrease in the activity of metabolic processes, notably respiration, which can be fatal within a few hours or days (Fitter & Hay, 2002). Part of the chilling injury is due to the depression of photosynthetic metabolism caused by: (i) decrease in membrane fluidity, (ii) changes in processes and activities of the enzymes related to the membrane, such as the photosynthetic electron transport, (iii) decline in activity of cold-sensitive enzymes (Lambers et al., 1998). Freezing injury commonly occurs to the woody plants of north temperate, subarctic, and alpine regions. It happens not because of low temperatures *per se*, but due to ice formation within tissues. If ambient temperature falls with intermediate rates (10C to 100C min-1), it will cause intracellular ice formation which disrupts the fine structure of the cells and invariably

**4.1 Factors affecting leaf level photosynthesis** 

results death.

within the canopy, radiation is composed of contributions from all directions. The angle between leaf surface and incident radiation depend on leaf orientation and the radiation direction. However, for horizontally positioned leaves, the fraction of radiation intercepted by any leaf will be proportional to the leaf area itself, independent of the radiation direction (Marcelis et al., 1998). Consequently, the extinction coefficient is high for horizontally inclined leaves, but low for vertical leaf arrangements. When all leaves distributed randomly in the horizontal plane and are perpendicular to the direct beam with solar elevation of 90, the value of K is 1. Solar position changes during the day influence the value of K by the factor 1/sin(β). Variations occurred in leaf angle also change K value dramatically, as vertically oriented leaves intercept less light than horizontal leaves.( Boote & Loomis, 1992). For greenhouse roses trained by arching system K ranged from 0.58 to 0.66 at different hours of day, with a daily average value of about 0.63 (Gonzalez –Real et al., 2007). Typical values for K are in the range of 0.5 to 0.8 (Marcelis et al., 1998). A canopy with low extinction coefficient allows more effective light reaches lower leaves. Some crops tend to arrange upper leaves at oblique angles to incident radiation to minimize the probability of photoinhibition and increase light penetration to lower leaves in high light environments, thereby maximizing whole-canopy photosynthesis. (Terashima & Hikosaka, 1995). It should be noted that direct and diffuse light have different extinction profiles in the canopy and due to light saturation of photosynthesis, direct beam should be singled out from the rest of the incoming radiation (Spitters, 1986). For this reason, experimentally determined values for total light extinction would not necessarily be the same as K. Leaf area index varies with the number and density of leaves within the canopy. In sparsely vegetated communities like deserts or tundra LAI value is less than 1, while for crops it is about 5 to 7 and for dense forests it estimated to range between 5 to 10 (Schulze et al. 1994). About 90% of PAR is absorbed by the canopy when LAI exceeds a value of 3. At leaf level absorption of PAR is approximately 80%-85% (Marcelis et al., 1998).

#### **4. Whole canopy photosynthesis**

Photosynthesis is a fundamental process occurring in green plants, algae and photosynthetic bacteria. During the process solar energy is trapped and utilized to drive the synthesis of carbohydrate from carbon dioxide and water. There are two distinct phases in the reactions of photosynthesis: the light reactions and the dark reactions. Light reactions use light energy to synthesize NADPH and ATP, which then transfer the energy to produce carbohydrate from CO2 and H2O during dark reactions. Chloroplast is an organelle in which photosynthesis takes place and has highly permeable outer membrane and an inner membrane that is impermeable to most molecules and ions. Light reactions occur in thylakoids, stacks of flattened chloroplast membranes extended into stroma, the place where the dark reactions are taken place. Two photosystems are involved in light reactions: photosystem I (PSI) and photosystm II (PS II). The difference is that PSI contains chlorophylls which have an absorption peak at 700 nm and so is called P700 but chlorophylls in the reaction center of PSII absorb light mostly at 680 nm and so is referred to as P680. The two photosystems are linked by a chain of electron carriers and when arranged in order of their redox potentials they form so-called Z scheme. Electrons released in PSII flow through Z scheme to reduce NADP+ in PSI. Pigments involved in the light harvesting complex (LHC) have already been discussed and here a brief explanation of overall photosynthesis reactions is presented:

within the canopy, radiation is composed of contributions from all directions. The angle between leaf surface and incident radiation depend on leaf orientation and the radiation direction. However, for horizontally positioned leaves, the fraction of radiation intercepted by any leaf will be proportional to the leaf area itself, independent of the radiation direction (Marcelis et al., 1998). Consequently, the extinction coefficient is high for horizontally inclined leaves, but low for vertical leaf arrangements. When all leaves distributed randomly in the horizontal plane and are perpendicular to the direct beam with solar elevation of 90, the value of K is 1. Solar position changes during the day influence the value of K by the factor 1/sin(β). Variations occurred in leaf angle also change K value dramatically, as vertically oriented leaves intercept less light than horizontal leaves.( Boote & Loomis, 1992). For greenhouse roses trained by arching system K ranged from 0.58 to 0.66 at different hours of day, with a daily average value of about 0.63 (Gonzalez –Real et al., 2007). Typical values for K are in the range of 0.5 to 0.8 (Marcelis et al., 1998). A canopy with low extinction coefficient allows more effective light reaches lower leaves. Some crops tend to arrange upper leaves at oblique angles to incident radiation to minimize the probability of photoinhibition and increase light penetration to lower leaves in high light environments, thereby maximizing whole-canopy photosynthesis. (Terashima & Hikosaka, 1995). It should be noted that direct and diffuse light have different extinction profiles in the canopy and due to light saturation of photosynthesis, direct beam should be singled out from the rest of the incoming radiation (Spitters, 1986). For this reason, experimentally determined values for total light extinction would not necessarily be the same as K. Leaf area index varies with the number and density of leaves within the canopy. In sparsely vegetated communities like deserts or tundra LAI value is less than 1, while for crops it is about 5 to 7 and for dense forests it estimated to range between 5 to 10 (Schulze et al. 1994). About 90% of PAR is absorbed by the canopy when LAI exceeds a value of 3. At leaf level absorption of PAR is

Photosynthesis is a fundamental process occurring in green plants, algae and photosynthetic bacteria. During the process solar energy is trapped and utilized to drive the synthesis of carbohydrate from carbon dioxide and water. There are two distinct phases in the reactions of photosynthesis: the light reactions and the dark reactions. Light reactions use light energy to synthesize NADPH and ATP, which then transfer the energy to produce carbohydrate from CO2 and H2O during dark reactions. Chloroplast is an organelle in which photosynthesis takes place and has highly permeable outer membrane and an inner membrane that is impermeable to most molecules and ions. Light reactions occur in thylakoids, stacks of flattened chloroplast membranes extended into stroma, the place where the dark reactions are taken place. Two photosystems are involved in light reactions: photosystem I (PSI) and photosystm II (PS II). The difference is that PSI contains chlorophylls which have an absorption peak at 700 nm and so is called P700 but chlorophylls in the reaction center of PSII absorb light mostly at 680 nm and so is referred to as P680. The two photosystems are linked by a chain of electron carriers and when arranged in order of their redox potentials they form so-called Z scheme. Electrons released in PSII flow through Z scheme to reduce NADP+ in PSI. Pigments involved in the light harvesting complex (LHC) have already been discussed and here a brief explanation of overall

approximately 80%-85% (Marcelis et al., 1998).

**4. Whole canopy photosynthesis** 

photosynthesis reactions is presented:

Photosynthesis process begins with absorbing light-energy photos and transferring them to the reaction centers of the photosystems where the second process starts. In the thylakoid membrane water splits to release electrons which are then transported along an electrontransport chain to produce NADPH and ATP. All the reactions occurred up to this point are called light reactions of photosynthesis as they depend on light energy to proceed. The produced NADPH and ATP during the light reactions then enter the carbon-reduction cycle (Calvin cycle), in which CO2 assimilated, leading to the synthesis of C3 compounds (triosephosphates). This reaction does not need light to proceed and therefore is referred to as the dark reaction of photosynthesis. In Calvin cycle, CO2 molecules are condensed with ribulose 1,5-bisphoshate ( a five-carbon molecule) to produce a transient six-carbon intermediate that immediately hydrolyzes to two molecules of 3-phosphoglycerate. This important process will not be complete without the mediation of the key enzyme ribulose bisphosphate carboxylase/oxygenase (often called Rubisco). Rubisco is known as a slow enzyme as it only fixes three molecules of its substrate every second and hence plants need a large amount of this enzyme to assimilate enough CO2 to sustain plant life. Approximately 50% of chloroplast protein content is Rubisco, probably the most abundant protein on the earth.

#### **4.1 Factors affecting leaf level photosynthesis**

The rate of photosynthesis at leaf level varies widely and is influenced not only by leaf internal biochemical and physiological conditions, but also by many environmental variables such as, CO2 concentration, light intensity, temperature and humidity fluctuations. Temperature is an essential factor that control enzymatically catalyzed reactions and membrane processes and in this way it controls photosynthesis (Lambers et al., 1998). Photosynthetic response to temperature varies among species because of the different activation energy required by different reactions processed in various plants. Consequently, temperature-dependent photosynthesis of plants range widely from temperatures near freezing to over 40C, implying that the specific range depends on species and genotype, plant age, plant origin, and season (Pallardy, 2008). Optimal temperature for any plant is usually defined by the temperature that plant has experienced and adapted to during the entire growing period. High temperatures increase affinity of Rubisco to oxygen than carbon dioxide, consequently leading to enhancing photorespiration. In addition, the solubility of CO2 declined with increasing temperature more strongly than does that of O2. At temperatures below about 15C the rate of photosynthesis is often reduced in many (sub)tropical plants. For example, after exposing coffee trees to 4C at night, the rate of photosynthesis was reduced by more than half (Pallardy, 2008). This kind of damage is called chilling injury and differs from frost damage, a type of damage that only happens below 0C. Chilling injury generally results from a precipitate decrease in the activity of metabolic processes, notably respiration, which can be fatal within a few hours or days (Fitter & Hay, 2002). Part of the chilling injury is due to the depression of photosynthetic metabolism caused by: (i) decrease in membrane fluidity, (ii) changes in processes and activities of the enzymes related to the membrane, such as the photosynthetic electron transport, (iii) decline in activity of cold-sensitive enzymes (Lambers et al., 1998). Freezing injury commonly occurs to the woody plants of north temperate, subarctic, and alpine regions. It happens not because of low temperatures *per se*, but due to ice formation within tissues. If ambient temperature falls with intermediate rates (10C to 100C min-1), it will cause intracellular ice formation which disrupts the fine structure of the cells and invariably results death.

Light Harvesting and Photosynthesis by the Canopy 243

reduction in substrate limitation of Rubsico catalysis (Farquahr, von Caemerer & Berry, 1980) , (ii) competitive reduction of RuBP oxygenation (Farquhar et al., 1980), and (iii) any adjustments in the photosynthetic apparatus (from light capture through starch and sucrose synthesis ) that change the RuBP limitation of Rubsico (Sage, Sharkey & Seemann,1989; Sage, 1990)". Modifications in leaf morphology and anatomy are the second way that influences the rate of leaf carboxylation. These alterations appear as changes in mesophyll cell number, carbohydrate concentration and leaf mass per unit area

Photosynthetic capacity at leaf level also depends highly on stomata density per unit leaf area and their gas exchange behavior controlled by environmental factors. Stomatal opening is bordered by a pair of unique guard cells which actively regulate the rate of aperture opening by means of swelling and shrinking mechanism, controlled by proton pump and potassium ion uptake processes. Outer surface of epidermis is coated with CO2 impermeable cuticle, therefore nearly all of the CO2 taken into the leaf for photosynthesis must enter only by diffusion through stomatal pores. The degree of stomata opening determines the rate of gas exchange between the leaf and environment which in turn results in direct influence on the rate of transpiration and CO2 assimilation. Of the environmental variables affecting stomatal movements, CO2 and light appear to make a substantial contribution to the rate of opening. Stomatal pores tend to be open when the leaves experience low CO2 concentration or light, and gradually begin to close when face high CO2 concentrations (Fig. 1). Although high environmental CO2 concentration may gradually stimulate closure of stomata, there are a number of studies which show elevated ambient CO2 enhances plant photosynthesis [Curtis, 1996; Gonzalez- Real & Baille, 2000; Tissues et al., 1997]. Gas concentration gradient between leaf intercellular air spaces and leaf boundary layer, together with the size of aperture determine the rate of gas movement across the stomata, referred to as stomatal conductivity. Water vapor and CO2 are the two main gases crossing stomata which directly influence the rate of transpiration and photosynthesis respectively. As the concentration gradient across the stomata differs considerably for H2O and CO2, and since they are not equal in the coefficient of diffusion, rate of gas exchange through the stomata will be different for them. As a result, gas exchange would affect photosynthesis and transpiration almost independently (Fig. 2). It has been recognized that elevating ambient CO2 increases plant water use efficiency. This term is defined as the ratio of CO2 molecules assimilated by photosynthesis to the number of water molecules lost *via* transpiration. Efficient water use by crops will result in increased agricultural products per liter of water consumed and therefore it can be highly beneficial to agriculture in arid and

Investigations have shown that all the stomata distributed over the entire leaf do not respond homogeneously to environmental factors at least in stressful condition (Pospisilova & Santrucek, 1997) ). This is called stomata patchiness which occurs when some stomata over the leaf close completely, whereas others are almost open. Meyer and Genty (1999) documented that inhibition of photosynthesis was mainly mediated through stomatal

Water stress is another important factor that controls the rate of leaf photosynthesis. It is carried out partially through regulating the size of stomatal aperture and thus limiting CO2 diffusion to the leaf air spaces, and partly by means of increasing diffusional resistance to CO2 movement from intercellular spaces to the chloroplast. Since water stress in drought conditions usually coincide with high solar radiation and higher temperatures, the

closure, when leaves undergo stress caused by dehydration or ABA treatment.

(Lambers et al., 1998).

semi-arid regions with elevated CO2.

Leaves absorb approximately about 85% to 90% of incident PAR (Nobel, 1999). The rate of absorption depends on leaf morphology and structure, especially on the number of palisade and spongy mesophyll layers (Vogelmann & Martin, 1993). In darkness there is no photosynthesis and leaves continue respiration, releasing CO2 to the atmosphere. In accordance with increasing light intensity, the rate of photosynthesis starts to increase until it reaches compensation point where the uptake of CO2 in photosynthesis equals releasing of CO2 in respiration. At this point there is no CO2 exchange between leaves and the atmosphere. When light intensity goes beyond the compensation point the photosynthetic rate starts to increase linearly. The initial slope of this line, located between compensation point and light saturation point, is referred to as quantum efficiency. Quantum efficiency describes the efficiency with which light is converted into fixed carbon. With further increase in light intensity, photosynthesis became saturated and is limited by the carboxylation rate. Increasing irradiance beyond the upper limits may even cause a decline in photosynthesis due to occurrence of photoinhibition, particularly in shade-adapted leaves. Photoinhibition may take place in both shade intolerant and shade tolerant plants. However, shade tolerant species and plants grown under shade are especially prone to photoinhibition (Pallardy, 2008). It was shown when willow leaves that previously developed in the shade were exposed to full sunlight, they showed more photoinhibition than leaves developed in the light (Ogren, 1988). Photoinhibition reduces plant quantum efficiency, therefore negatively influence photosynthetic productivity. Nevertheless, plants develop mechanisms to recover from photoinhibition, and it was indicated that the level of recovery is partly related to the duration of exposure to higher light environment.

Although under normal conditions the probability of photosynthesis reduction caused by decreased levels of enzyme Rubisco is very low, there are nevertheless circumstances under which Rubisco concentration exerts strong control over photosynthetic capacity, for example, in low plants transferred to high light (Lauerer et al., 1993). In addition, it was proven that sufficient amount of Rubsico may effectively regulate other components of photosynthetic apparatus. Anthisense plants with greatly reduced levels of Rubisco often suffer imbalances in electron transport and decreased water-use efficiency (Quick et al., 1991).

Availability of carbon dioxide at the carboxylation site within the chloroplast highly affects photosynthesis capacity. This availability is strongly limited by resistances in its diffusion path twoards the mesophyll cells. Resistance may be generated by boundary layer of air, cuticle, stomata, and mesophyll air space and liquid diffusion resistance. Regarding predicted atmospheric CO2 elevation up to 700-1000 μmol mol-1 by the end of the 21th century (Houghton et al., 2001), many researches have been conducted over the past decades on the effects of rising atmospheric CO2 on the physiological aspects of higher plants. These researches showed that leaf-level photosynthesis was often increased in plants developed under long-term exposure to increased levels of CO2 (Curtis, 1996; Gonzalez-Real & Baille, 2000; Tissues et al., 1997). Since Rubisco uses CO2 and RuBP as the principal substrate to catalyze the carboxylation reactions, it could be expected that any increase in the environmental CO2 concentration may cause increases in the rate of photosynthesis, assuming that there is no other limiting factor . The rate of carboxylation per unit leaf area can be governed by elevated CO2 through at least two fundamentally different ways: biochemical mechanisms and leaf morphological modifications (Peterson et al., 1999). The biochemical mechanism consists of three levels, as stated by Peterson et al. (1999):" (i) a

Leaves absorb approximately about 85% to 90% of incident PAR (Nobel, 1999). The rate of absorption depends on leaf morphology and structure, especially on the number of palisade and spongy mesophyll layers (Vogelmann & Martin, 1993). In darkness there is no photosynthesis and leaves continue respiration, releasing CO2 to the atmosphere. In accordance with increasing light intensity, the rate of photosynthesis starts to increase until it reaches compensation point where the uptake of CO2 in photosynthesis equals releasing of CO2 in respiration. At this point there is no CO2 exchange between leaves and the atmosphere. When light intensity goes beyond the compensation point the photosynthetic rate starts to increase linearly. The initial slope of this line, located between compensation point and light saturation point, is referred to as quantum efficiency. Quantum efficiency describes the efficiency with which light is converted into fixed carbon. With further increase in light intensity, photosynthesis became saturated and is limited by the carboxylation rate. Increasing irradiance beyond the upper limits may even cause a decline in photosynthesis due to occurrence of photoinhibition, particularly in shade-adapted leaves. Photoinhibition may take place in both shade intolerant and shade tolerant plants. However, shade tolerant species and plants grown under shade are especially prone to photoinhibition (Pallardy, 2008). It was shown when willow leaves that previously developed in the shade were exposed to full sunlight, they showed more photoinhibition than leaves developed in the light (Ogren, 1988). Photoinhibition reduces plant quantum efficiency, therefore negatively influence photosynthetic productivity. Nevertheless, plants develop mechanisms to recover from photoinhibition, and it was indicated that the level of

recovery is partly related to the duration of exposure to higher light environment.

1991).

Although under normal conditions the probability of photosynthesis reduction caused by decreased levels of enzyme Rubisco is very low, there are nevertheless circumstances under which Rubisco concentration exerts strong control over photosynthetic capacity, for example, in low plants transferred to high light (Lauerer et al., 1993). In addition, it was proven that sufficient amount of Rubsico may effectively regulate other components of photosynthetic apparatus. Anthisense plants with greatly reduced levels of Rubisco often suffer imbalances in electron transport and decreased water-use efficiency (Quick et al.,

Availability of carbon dioxide at the carboxylation site within the chloroplast highly affects photosynthesis capacity. This availability is strongly limited by resistances in its diffusion path twoards the mesophyll cells. Resistance may be generated by boundary layer of air, cuticle, stomata, and mesophyll air space and liquid diffusion resistance. Regarding predicted atmospheric CO2 elevation up to 700-1000 μmol mol-1 by the end of the 21th century (Houghton et al., 2001), many researches have been conducted over the past decades on the effects of rising atmospheric CO2 on the physiological aspects of higher plants. These researches showed that leaf-level photosynthesis was often increased in plants developed under long-term exposure to increased levels of CO2 (Curtis, 1996; Gonzalez-Real & Baille, 2000; Tissues et al., 1997). Since Rubisco uses CO2 and RuBP as the principal substrate to catalyze the carboxylation reactions, it could be expected that any increase in the environmental CO2 concentration may cause increases in the rate of photosynthesis, assuming that there is no other limiting factor . The rate of carboxylation per unit leaf area can be governed by elevated CO2 through at least two fundamentally different ways: biochemical mechanisms and leaf morphological modifications (Peterson et al., 1999). The biochemical mechanism consists of three levels, as stated by Peterson et al. (1999):" (i) a reduction in substrate limitation of Rubsico catalysis (Farquahr, von Caemerer & Berry, 1980) , (ii) competitive reduction of RuBP oxygenation (Farquhar et al., 1980), and (iii) any adjustments in the photosynthetic apparatus (from light capture through starch and sucrose synthesis ) that change the RuBP limitation of Rubsico (Sage, Sharkey & Seemann,1989; Sage, 1990)". Modifications in leaf morphology and anatomy are the second way that influences the rate of leaf carboxylation. These alterations appear as changes in mesophyll cell number, carbohydrate concentration and leaf mass per unit area (Lambers et al., 1998).

Photosynthetic capacity at leaf level also depends highly on stomata density per unit leaf area and their gas exchange behavior controlled by environmental factors. Stomatal opening is bordered by a pair of unique guard cells which actively regulate the rate of aperture opening by means of swelling and shrinking mechanism, controlled by proton pump and potassium ion uptake processes. Outer surface of epidermis is coated with CO2 impermeable cuticle, therefore nearly all of the CO2 taken into the leaf for photosynthesis must enter only by diffusion through stomatal pores. The degree of stomata opening determines the rate of gas exchange between the leaf and environment which in turn results in direct influence on the rate of transpiration and CO2 assimilation. Of the environmental variables affecting stomatal movements, CO2 and light appear to make a substantial contribution to the rate of opening. Stomatal pores tend to be open when the leaves experience low CO2 concentration or light, and gradually begin to close when face high CO2 concentrations (Fig. 1). Although high environmental CO2 concentration may gradually stimulate closure of stomata, there are a number of studies which show elevated ambient CO2 enhances plant photosynthesis [Curtis, 1996; Gonzalez- Real & Baille, 2000; Tissues et al., 1997]. Gas concentration gradient between leaf intercellular air spaces and leaf boundary layer, together with the size of aperture determine the rate of gas movement across the stomata, referred to as stomatal conductivity. Water vapor and CO2 are the two main gases crossing stomata which directly influence the rate of transpiration and photosynthesis respectively. As the concentration gradient across the stomata differs considerably for H2O and CO2, and since they are not equal in the coefficient of diffusion, rate of gas exchange through the stomata will be different for them. As a result, gas exchange would affect photosynthesis and transpiration almost independently (Fig. 2). It has been recognized that elevating ambient CO2 increases plant water use efficiency. This term is defined as the ratio of CO2 molecules assimilated by photosynthesis to the number of water molecules lost *via* transpiration. Efficient water use by crops will result in increased agricultural products per liter of water consumed and therefore it can be highly beneficial to agriculture in arid and semi-arid regions with elevated CO2.

Investigations have shown that all the stomata distributed over the entire leaf do not respond homogeneously to environmental factors at least in stressful condition (Pospisilova & Santrucek, 1997) ). This is called stomata patchiness which occurs when some stomata over the leaf close completely, whereas others are almost open. Meyer and Genty (1999) documented that inhibition of photosynthesis was mainly mediated through stomatal closure, when leaves undergo stress caused by dehydration or ABA treatment.

Water stress is another important factor that controls the rate of leaf photosynthesis. It is carried out partially through regulating the size of stomatal aperture and thus limiting CO2 diffusion to the leaf air spaces, and partly by means of increasing diffusional resistance to CO2 movement from intercellular spaces to the chloroplast. Since water stress in drought conditions usually coincide with high solar radiation and higher temperatures, the

Light Harvesting and Photosynthesis by the Canopy 245

light response curve was one of the cases that has been noted by many authors to be described by several mathematical functions. Three functions have been used to describe

This model was initially proposed by Rabinowitch in 1951, and later reviewed by Johnson and Thornley (1984) in order to describe photosynthesis light response. Non-rectangular hyperbola seems to be one of the best equations in prediction of leaf photosynthetic light

<sup>2</sup> ( )4

 

(4)

2 *max max max I P I P IP*

where *P* and Pmax are respectively the rate of leaf gross photosynthesis (μmolm-2s-1) and light-saturated photosynthesis (μmolm-2s-1), *α* is quantum efficiency (mol CO2 mol quanta-1), *I* is irradiance quantity (μmol qunta m-2s-1) and *θ* denotes curvature (convexity) of the light-

This model have three parameters to be estimated: (i) the quantum efficiency (α),the initial slope of the curve which relates the rate of CO2 uptake to absorbed or incident light at very low light intensity. Values of this parameter change with the species, leaf history of stress such as; low temperatures, drought and high irradiance and usually range between 0.040 to 0.075 mol CO2 mol quanta-1 at ambient CO2 concentrations (Cannell & Thornley, 1998), (ii) the light –saturated photosynthetic rate (Pmax), which varies extremely among species and is affected by the temperature and life history of leaves which influence leaf morphological and physiological properties like N content and leaf thickness, (iii) and the curvature factor (θ), which indicates how quickly the transition of the curve is made from Rubisco-limited rgion to RuBP-regeneration-limited region. There are many studies, indicating that nonrectangular hyperbola usually best fitted with θ ranging from 0.7 to 0.9 (Matloobi, 2007;

In non-rectangular hyperbola when θ closes zero, the equation appears to be a rectangular

*IP <sup>P</sup> I P* 

This equation recalls the famous Michaelis-Menten equation. With θ=1 the equation will be

Another equation that is used to model photosynthesis light response is asymptotic

In an experiment we measured leaf gas exchange parameters in a rose crop (*Rosa hybrid* "Habari") by a portable photosynthesis measurement system (Matloobi,2008). Obtained data then were used to estimate the models parameters by non-linear least squares regression method. A linear regression was fitted to the data obtained by direct measurement and those estimated by the models. Results showed that all the models had the potential to present good estimations of the leaf photosynthetic light response with roughly high R2 (coefficient of determination), but the non-rectangular hyperbola with the highest R2 (R2=0.968) was the best as it predicted values more closer to the observed ones

*max max*

(5)

(6)

Marshall & Biscoe, 1980; Cannel & Thornley, 1998; Kim et al., 2004).

Blackman response with two intersecting straight lines.

(/) (1 ) *max I P PP e max*

photosynthesis light response cure:

hyperbola (Fig. 4):

exponential equation (Fig. 5):

response. The function is as follows (Fig. 3):

photosynthesis relationship (dimensionless).

*P*

mechanism of this down-regulation of photosynthesis in response to water stress is not fully understood (Lambers et al., 1998).

Fig. 1. Effect of CO2 on stomatal conductance and water use efficiency of *Rosa hybrida* 'Habari'

Fig. 2. Effect of CO2 on the rate of photosynthesis and transpiration of *Rosa hybrida* 'Habari'

#### **4.1.1 Modeling photosynthesis**

Models in plant science can be divided into two main groups: mechanistic and empirical. Mechanistic models are descriptive and developed usually based on exhaustive and comprehensive studies which led to globally accepted findings. However, empirical models sometimes referred to as statistical, regression or black-box models are limited to time, location and species on which that model developed and cannot accurately be extrapolated to other conditions and species. Empirical models could be valuable in that they implicitly take into account all unknown effects (Marcelis et al., 1998). In modeling photosynthesis processes both mechanistic and empirical approaches have been considered. Photosynthetic 244 Advances in Photosynthesis – Fundamental Aspects

mechanism of this down-regulation of photosynthesis in response to water stress is not fully

Fig. 1. Effect of CO2 on stomatal conductance and water use efficiency of *Rosa hybrida* 'Habari'

Fig. 2. Effect of CO2 on the rate of photosynthesis and transpiration of *Rosa hybrida* 'Habari'

Models in plant science can be divided into two main groups: mechanistic and empirical. Mechanistic models are descriptive and developed usually based on exhaustive and comprehensive studies which led to globally accepted findings. However, empirical models sometimes referred to as statistical, regression or black-box models are limited to time, location and species on which that model developed and cannot accurately be extrapolated to other conditions and species. Empirical models could be valuable in that they implicitly take into account all unknown effects (Marcelis et al., 1998). In modeling photosynthesis processes both mechanistic and empirical approaches have been considered. Photosynthetic

understood (Lambers et al., 1998).

**4.1.1 Modeling photosynthesis** 

light response curve was one of the cases that has been noted by many authors to be described by several mathematical functions. Three functions have been used to describe photosynthesis light response cure:

This model was initially proposed by Rabinowitch in 1951, and later reviewed by Johnson and Thornley (1984) in order to describe photosynthesis light response. Non-rectangular hyperbola seems to be one of the best equations in prediction of leaf photosynthetic light response. The function is as follows (Fig. 3):

$$P = \frac{\alpha I + P\_{\text{max}} - \sqrt{(\alpha I + P\_{\text{max}})^2 - 4\alpha\theta I P\_{\text{max}}}}{2\theta} \tag{4}$$

where *P* and Pmax are respectively the rate of leaf gross photosynthesis (μmolm-2s-1) and light-saturated photosynthesis (μmolm-2s-1), *α* is quantum efficiency (mol CO2 mol quanta-1), *I* is irradiance quantity (μmol qunta m-2s-1) and *θ* denotes curvature (convexity) of the lightphotosynthesis relationship (dimensionless).

This model have three parameters to be estimated: (i) the quantum efficiency (α),the initial slope of the curve which relates the rate of CO2 uptake to absorbed or incident light at very low light intensity. Values of this parameter change with the species, leaf history of stress such as; low temperatures, drought and high irradiance and usually range between 0.040 to 0.075 mol CO2 mol quanta-1 at ambient CO2 concentrations (Cannell & Thornley, 1998), (ii) the light –saturated photosynthetic rate (Pmax), which varies extremely among species and is affected by the temperature and life history of leaves which influence leaf morphological and physiological properties like N content and leaf thickness, (iii) and the curvature factor (θ), which indicates how quickly the transition of the curve is made from Rubisco-limited rgion to RuBP-regeneration-limited region. There are many studies, indicating that nonrectangular hyperbola usually best fitted with θ ranging from 0.7 to 0.9 (Matloobi, 2007; Marshall & Biscoe, 1980; Cannel & Thornley, 1998; Kim et al., 2004).

In non-rectangular hyperbola when θ closes zero, the equation appears to be a rectangular hyperbola (Fig. 4):

$$P = \frac{\alpha I P\_{\text{max}}}{\alpha I + P\_{\text{max}}} \tag{5}$$

This equation recalls the famous Michaelis-Menten equation. With θ=1 the equation will be Blackman response with two intersecting straight lines.

Another equation that is used to model photosynthesis light response is asymptotic exponential equation (Fig. 5):

$$P = P\_{\max} \left( \mathbb{1} - e^{\left( -\alpha \mathbb{1} / P\_{\max} \right)} \right) \tag{6}$$

In an experiment we measured leaf gas exchange parameters in a rose crop (*Rosa hybrid* "Habari") by a portable photosynthesis measurement system (Matloobi,2008). Obtained data then were used to estimate the models parameters by non-linear least squares regression method. A linear regression was fitted to the data obtained by direct measurement and those estimated by the models. Results showed that all the models had the potential to present good estimations of the leaf photosynthetic light response with roughly high R2 (coefficient of determination), but the non-rectangular hyperbola with the highest R2 (R2=0.968) was the best as it predicted values more closer to the observed ones

Light Harvesting and Photosynthesis by the Canopy 247

*J C*

*j*

*A*

2

photosystem II, *I2* by:

the following equation:

transmittance of the leaf to PAR (~ 0.12).

estimates model efficiency (bottom)

 \* ( ) 4 2\* *i*

2 2 ?( ) 0 *J I J ax J I J ax m m* (10)

(9)

*i*

*C*

where *J* is the rate of electron transport, J is related to irradiance usefully absorbed by

where *Jmax* is the potential rate of electron transport, *I2* is related to the incident PAR, *Io* by

 *I2=Io(1-f)(1-r)/2* (11) where *f* is spectral correction factor (~ 0.15) and *r* is the reflectance plus any small

Fig. 4. Photosynthetic light response of *Rosa hybrida* 'Habari', rectangular hyperbola model was fitted to the observed data (top), regression between the model and observed data

The two key parameters in this model which vary among species are Vcmax and Jmax . The potential rate of electron transport, Jmax, is a property of the thylakoids that varies depending on growth conditions (Farquhar & Evans, 1991). Factors affecting the chlorophyll content per unit leaf area determine the rate of Jmax . There are many studies showing that the chlorophyll content of leaves dynamically change according to the environmental light availability [Kitajamia & Hogan, 2003; Matloobi et al., 2009; Walters, 2005). Plants acclimated to low irradiance are enriched in the light-harvesting chlorophyll a/b protein complex and

(Fig. 3). Non-rectangular hyperbola has been frequently used to describe observed leaf photosynthetic responses to environmental variables (e.g. Pasian, 1989; kim et al., 2004; Cannel & Thornley, 1998)

Fig. 3. Photosynthetic light response of *Rosa hybrida* 'Habari', non-rectangular hyperbola model was fitted to the observed data (top), regression between the model and observed data estimates model efficiency (bottom)

The model proposed by Farquahr et al. in 1980 for leaf photosynthesis of C3 plants is the only mechanistic model which is accepted and widely used for determination of the leaf CO2 assimilation capacity. This model developed based on the amount and kinetic properties of Rubisco and the ratio of RuBP to enzyme (Rubisco) active site (Harley & Tenhunen, 1991). In this model two limiting factors were assumed to control the leaf photosynthetic capacity:

$$A\_n = \min\left\{A\_{\upsilon}, A\_{\not\!\!/\!}\right\} - R\_d \tag{7}$$

where Av and Aj are the rate of gross photosynthesis limited by Rubisco activity and the rate of RuBP regeneration through electron transport, respectively, and Rd is the rate of mitochondrial respiration. Rubisco limited photosynthesis is given by:

$$A\_{\upsilon} = V\_{c\max} \frac{\mathbf{C}\_i - \boldsymbol{\Gamma}^\*}{\mathbf{C}\_i + \mathbf{K}\_c \text{ (}\left(1 + \mathbf{O} \,/\, K\_o\right)\text{)}}\tag{8}$$

where *Vcmax* is the maximum carboxylation rate, with Kc and Ko the Michaelis constants for carboxylation and oxygenation, respectively, and Ci and O are the partial pressure of CO2 and O2 in the intercellular air spaces, and Γ\* is the CO2 compensation point in the absence of mitochondrial respiration. The rate of photosynthesis limited by RuBP regeneration is given by:

(Fig. 3). Non-rectangular hyperbola has been frequently used to describe observed leaf photosynthetic responses to environmental variables (e.g. Pasian, 1989; kim et al., 2004;

Fig. 3. Photosynthetic light response of *Rosa hybrida* 'Habari', non-rectangular hyperbola model was fitted to the observed data (top), regression between the model and observed

The model proposed by Farquahr et al. in 1980 for leaf photosynthesis of C3 plants is the only mechanistic model which is accepted and widely used for determination of the leaf CO2 assimilation capacity. This model developed based on the amount and kinetic properties of Rubisco and the ratio of RuBP to enzyme (Rubisco) active site (Harley & Tenhunen, 1991). In this model two limiting factors were assumed to control the leaf

where Av and Aj are the rate of gross photosynthesis limited by Rubisco activity and the rate of RuBP regeneration through electron transport, respectively, and Rd is the rate of

> \* (1 /

*ic o*

*C K OK* 

mitochondrial respiration. Rubisco limited photosynthesis is given by:

*<sup>i</sup> v cmax*

where *Vcmax* is the maximum carboxylation rate, with Kc and Ko the Michaelis constants for carboxylation and oxygenation, respectively, and Ci and O are the partial pressure of CO2 and O2 in the intercellular air spaces, and Γ\* is the CO2 compensation point in the absence of mitochondrial respiration. The rate of photosynthesis limited by RuBP regeneration is given by:

*<sup>C</sup> A V*

*An vj d min A A R* , (7)

(8)

Cannel & Thornley, 1998)

data estimates model efficiency (bottom)

photosynthetic capacity:

$$A\_j = \left(\frac{J}{4}\right) \times \frac{\left(\mathbf{C}\_i - \boldsymbol{\Gamma}^\*\right)}{\left(\mathbf{C}\_i + 2\boldsymbol{\Gamma}^\*\right)}\tag{9}$$

where *J* is the rate of electron transport, J is related to irradiance usefully absorbed by photosystem II, *I2* by:

$$\text{(??)}\,^2 - (I\_2 + I\_m ax)f + I\_2 f\_m ax = 0 \tag{10}$$

where *Jmax* is the potential rate of electron transport, *I2* is related to the incident PAR, *Io* by the following equation:

$$\mathbf{I}\_2 = \mathbf{I}\_o(\mathbf{1} \cdot \mathbf{f})(\mathbf{1} \cdot \mathbf{r})/\mathbf{2} \tag{11}$$

where *f* is spectral correction factor (~ 0.15) and *r* is the reflectance plus any small transmittance of the leaf to PAR (~ 0.12).

Fig. 4. Photosynthetic light response of *Rosa hybrida* 'Habari', rectangular hyperbola model was fitted to the observed data (top), regression between the model and observed data estimates model efficiency (bottom)

The two key parameters in this model which vary among species are Vcmax and Jmax . The potential rate of electron transport, Jmax, is a property of the thylakoids that varies depending on growth conditions (Farquhar & Evans, 1991). Factors affecting the chlorophyll content per unit leaf area determine the rate of Jmax . There are many studies showing that the chlorophyll content of leaves dynamically change according to the environmental light availability [Kitajamia & Hogan, 2003; Matloobi et al., 2009; Walters, 2005). Plants acclimated to low irradiance are enriched in the light-harvesting chlorophyll a/b protein complex and

Light Harvesting and Photosynthesis by the Canopy 249

incorporate them to upscale photosynthesis from individual leaves to the canopy. To achieve this, the first challenge will be calculating the amount of radiation absorbed by

Beer's law (equation 3) has been used as a basis to develop more precise models in order to get a clear profile of light distribution within the canopy. Three approaches have been considered in modeling light absorption by the canopy: (i) big leaf model (ii) multi-layer

Big leaf model tries to simplify rather than increase canopy structural complexity (Beyschlag & Ryel, 2007). The concept comes from the findings of Farquhar (Farquhar, 1989, as cited in Evans & Farquhar, 1991) who demonstrated that the equation for whole-leaf photosynthesis would be the same form as for individual chloroplast provided that chloroplast photosynthetic capacity distributes in proportion to the profile of absorbed irradiance and that in all layers the shape of the response to irradiance become identical. This approach applies to predict canopy light absorption by reduction of properties of all leaves within the canopy to a single leaf. However, this prediction will not be accurate enough to ignore developing alternative models. While Beer's law describes time-averaged profile of absorbed irradiance and the spatially averaged instantaneous profiles in a canopy, it doesn't describe the actual instantaneous distribution of absorbed irradiance. In fact, some leaves located deep in the canopy receive much higher radiation than the amount that Beer's law would predict when they are subject to sunflecks. Generally Beer's law does not represent the instantaneous profiles of absorbed irradiance in canopies because of errors created by

Multi layer model of light penetration through the canopies was proposed by Goudriaan (Goudriaan,1977, as cited in De Pury & Farquhar, 1997). In this model the plant canopy is divided into multiple leaf layers (increments in *L* of 0.1) distributed horizontally and assumed to be homogeneous with respect to leaf angels. Two groups of leaves are identified in each layer: shade and sunlit leaves, and sunlit section is divided into nine leaf angle classes. Irradiance absorption by each leaf group (sunlit and shade leaves) and also by each angle class of sunlit leaves is then calculated separately and integrated to give the whole

Sun-shade model initially introduced by Sinclair et al. (Sinclair et al., 1976, as cited in De Pury & Farquhar, 1997) and then applied by Norman (Norman,1980, as cited in De Pury & Farquhar, 1997), recently improved by De Pury and Farquhar (1997). This model gives predictions of canopy photosynthesis that closely match estimations of multi layer model with far fewer calculations. Sun-shade model divides canopy into large foliage groups: sunlit foliage which receives direct beam, and shade foliage which is subject to diffuse and/or transmitted irradiation. Amount of irradiance absorbed by each of these parts is

Regardless of the way one calculates the rate of irradiance absorbed by the canopy, the next step in prediction of canopy photosynthesis will be estimation of the rate of photosynthesis undertaken by each group of leaves. In big leaf model an averaged value of light intercepted by the whole canopy enters photosynthesis model to calculate entire plant CO2 assimilation rate. The method may be quite complex with multi layer model and somehow with sunshade model as in these cases the calculations should be done in detail and more accurately for each leaf class. The performance of big leaf model in estimation of canopy photosynthesis depends in part on the accuracy by which the nitrogen distribution was

calculated as an integral of absorbed light and the corresponding leaf area fraction.

individual leaves and finally by the plant whole canopy.

both sunflecks and leaf angles (De Pury & Farquhar, 1997).

model (iii) sun and shade model.

canopy light absorption profile.

deplete in the photosystem II reaction-center complexes, therefore the electron-transport capacity per unit of chlorophyll is less in leaves acclimated to low irradiance (Farquhar & Evans, 1991).

It was found that there is a good correlation between the leaf N content and the photosynthetic maximum carboxylation rate, Vcmax. Gonzalez-Real and Baille (2000) documented that in rose crop there is a gradient in the leaf photosynthetic N concentration from the top of canopy down to the bottom layers according to the amount of light absorbed. The value of Vcmax decreased from 66 μmolm-2s-1 for leaves situated at the top of the canopy to 44 μmolm-2s-1 for leaves located at the bottom layers. The ratio of Jmax/Vcmax for all leaf layers within the canopy was almost constant and resulted 2.3. It should be noted that photosynthetic key parameters (Jmax and Vcmax ) change proportionally with seasonal variations in soil water content, air temperature and VPD (Xu & Baldocchi, 2003). Parameterization of the photosynthetic models for several plants have been previously done (Gonzalez-Real & Baille, 2000; Kim & Lieth, 2002; Kim & Lieth, 2003; Matloobi, 2007 ) and still it is noted and under research by many authors around the world.

Fig. 5. Photosynthetic light response of *Rosa hybrida* 'Habari', asymptotic exponential model was fitted to the observed data (top), regression between the model and observed data estimates model efficiency (bottom)

#### **4.2 Photosynthesis at canopy level**

Canopy photosynthesis at the first step highly depends on the rate of photosynthesis at leaf level which is controlled by interaction of many internal and some external factors, outlined earlier. Therefore in order to obtain an estimation of the plant whole-canopy photosynthesis one must first consider the factors affecting leaf level CO2 assimilation and then accurately

deplete in the photosystem II reaction-center complexes, therefore the electron-transport capacity per unit of chlorophyll is less in leaves acclimated to low irradiance (Farquhar &

It was found that there is a good correlation between the leaf N content and the photosynthetic maximum carboxylation rate, Vcmax. Gonzalez-Real and Baille (2000) documented that in rose crop there is a gradient in the leaf photosynthetic N concentration from the top of canopy down to the bottom layers according to the amount of light absorbed. The value of Vcmax decreased from 66 μmolm-2s-1 for leaves situated at the top of the canopy to 44 μmolm-2s-1 for leaves located at the bottom layers. The ratio of Jmax/Vcmax for all leaf layers within the canopy was almost constant and resulted 2.3. It should be noted that photosynthetic key parameters (Jmax and Vcmax ) change proportionally with seasonal variations in soil water content, air temperature and VPD (Xu & Baldocchi, 2003). Parameterization of the photosynthetic models for several plants have been previously done (Gonzalez-Real & Baille, 2000; Kim & Lieth, 2002; Kim & Lieth, 2003; Matloobi, 2007 ) and

Fig. 5. Photosynthetic light response of *Rosa hybrida* 'Habari', asymptotic exponential model was fitted to the observed data (top), regression between the model and observed data

Canopy photosynthesis at the first step highly depends on the rate of photosynthesis at leaf level which is controlled by interaction of many internal and some external factors, outlined earlier. Therefore in order to obtain an estimation of the plant whole-canopy photosynthesis one must first consider the factors affecting leaf level CO2 assimilation and then accurately

estimates model efficiency (bottom)

**4.2 Photosynthesis at canopy level** 

still it is noted and under research by many authors around the world.

Evans, 1991).

incorporate them to upscale photosynthesis from individual leaves to the canopy. To achieve this, the first challenge will be calculating the amount of radiation absorbed by individual leaves and finally by the plant whole canopy.

Beer's law (equation 3) has been used as a basis to develop more precise models in order to get a clear profile of light distribution within the canopy. Three approaches have been considered in modeling light absorption by the canopy: (i) big leaf model (ii) multi-layer model (iii) sun and shade model.

Big leaf model tries to simplify rather than increase canopy structural complexity (Beyschlag & Ryel, 2007). The concept comes from the findings of Farquhar (Farquhar, 1989, as cited in Evans & Farquhar, 1991) who demonstrated that the equation for whole-leaf photosynthesis would be the same form as for individual chloroplast provided that chloroplast photosynthetic capacity distributes in proportion to the profile of absorbed irradiance and that in all layers the shape of the response to irradiance become identical. This approach applies to predict canopy light absorption by reduction of properties of all leaves within the canopy to a single leaf. However, this prediction will not be accurate enough to ignore developing alternative models. While Beer's law describes time-averaged profile of absorbed irradiance and the spatially averaged instantaneous profiles in a canopy, it doesn't describe the actual instantaneous distribution of absorbed irradiance. In fact, some leaves located deep in the canopy receive much higher radiation than the amount that Beer's law would predict when they are subject to sunflecks. Generally Beer's law does not represent the instantaneous profiles of absorbed irradiance in canopies because of errors created by both sunflecks and leaf angles (De Pury & Farquhar, 1997).

Multi layer model of light penetration through the canopies was proposed by Goudriaan (Goudriaan,1977, as cited in De Pury & Farquhar, 1997). In this model the plant canopy is divided into multiple leaf layers (increments in *L* of 0.1) distributed horizontally and assumed to be homogeneous with respect to leaf angels. Two groups of leaves are identified in each layer: shade and sunlit leaves, and sunlit section is divided into nine leaf angle classes. Irradiance absorption by each leaf group (sunlit and shade leaves) and also by each angle class of sunlit leaves is then calculated separately and integrated to give the whole canopy light absorption profile.

Sun-shade model initially introduced by Sinclair et al. (Sinclair et al., 1976, as cited in De Pury & Farquhar, 1997) and then applied by Norman (Norman,1980, as cited in De Pury & Farquhar, 1997), recently improved by De Pury and Farquhar (1997). This model gives predictions of canopy photosynthesis that closely match estimations of multi layer model with far fewer calculations. Sun-shade model divides canopy into large foliage groups: sunlit foliage which receives direct beam, and shade foliage which is subject to diffuse and/or transmitted irradiation. Amount of irradiance absorbed by each of these parts is calculated as an integral of absorbed light and the corresponding leaf area fraction.

Regardless of the way one calculates the rate of irradiance absorbed by the canopy, the next step in prediction of canopy photosynthesis will be estimation of the rate of photosynthesis undertaken by each group of leaves. In big leaf model an averaged value of light intercepted by the whole canopy enters photosynthesis model to calculate entire plant CO2 assimilation rate. The method may be quite complex with multi layer model and somehow with sunshade model as in these cases the calculations should be done in detail and more accurately for each leaf class. The performance of big leaf model in estimation of canopy photosynthesis depends in part on the accuracy by which the nitrogen distribution was

Light Harvesting and Photosynthesis by the Canopy 251

and harvesting above third bud for the first-order flowering shoot and above first bud for the following flowering shoots, (iii) T.S. 3-3-2: after bending primary shoot above third bud, the first-order flower shoot was harvested above the third bud and the second-order one above the second bud, (iv) T.S. 5-1: primary shoot was bent above fifth bud and the bearing flower shoot was harvested above first bud, (v): T.S. 5-3: primary shoot was bent above fifth bud and the bearing shoot was harvested above third bud. Leaf photosynthetic measurements have been performed for three layers of upright shoots (top, middle and bottom layer), and bent layer. Results exhibited that training system did not affect whole canopy light absorption significantly, but affected photosynthetic rate at canopy level (Table 1). This implies that photosynthetic rate at canopy level was influenced particularly by the interaction between light distribution profile through the canopy and canopy leaf area distribution rather than the

> T.S 1-1 T.S 3-3 T.S 3-32 T.S 5-1 T.S 5-3 **Training system**

a a

a

a

T.S 1-1 T.S 3-3 T.S 3-32 T.S 5-1 T.S 5-3 **Training system**

a

ab

Fig. 7. Effect of different types of training system on the whole photosynthetic rate of *Rosa hybrida* 'Habari'. Different letters above columns indicate significantly difference according

Fig. 6. Effect of different types of training system on the leaf area of *Rosa hybrida* 'Habari'. Different letters above columns indicate significantly difference according to the Duncan's

ab

0

1000

to the Duncan's multiple range test (p<0.05).

**Photosynthesis (μmol m-2 s-1)**

b

2000

3000

b b

**Leaf area (cm2 plant-1)**

multiple range test (p<0.05).

4000

5000

6000

predicted in proportion to the daily irradiance. While sun-shade model gives predictions of canopy photosynthesis with best approximation to those predicted by multi layer model, the big leaf model usually shows deviations ranged from 10% to 45% (De Pury & Farquhar, 1997). Each model accompanies advantages and disadvantages, differing in the rate of accuracy and degree of complexity in calculations. Presently, computer software makes it so feasible to integrate many mathematical formulas into one distinctive program, facilitating calculations of even more complex equations.

#### **5. Training systems and canopy photosynthesis**

Pruning and training techniques are professional horticultural practices developed not only to control plant growth in some circumstances but also to modify plant canopy in such a way that increases the amount of light absorption. Fruit trees commonly are subject to training systems during their juvenile period when the plant canopy is being formed. Depend on the type of buds (vegetative or reproductive), abundance and method of distribution within tree crown, pruning and training practices are carefully adopted so that it ensures maximum light penetration through the canopy, and provides plants the highest growth and productivity. In an experiment with two cultivars of apple trees Mierowska et al. (2002) indicated that summer pruning enhances photosynthetic acclimation of spur leaves, previously developed under shade, by rapid increasing of the chlorophyll a/b ratio. Similarly, in *phalaenopsis* , it was shown that providing the lower shade-developed leaves with higher rates of light intensities caused increased rate of photosynthesis (Lin & Hsu, 2004). Pruning resulted in changes in light harvesting complexes of rose plants, showing that rose leaves are very plastic and acclimate rapidly to any changes in light intensity (Calatayud, et al., 2007).

Training systems alter canopies light harvesting hehaviour through changing the foliage density, spatial form; the ratio of sun/shade leaves, leaf angles and finally the canopy leaf distribution pattern. There are several training systems developed for fruit trees based on the tree reproductive biology such as, central leader and modified leader particularly appropriate for pome fruits (apple, pear and quince trees) and open center specifically developed for prunes (peach, plum, and cherry trees). Recently, most greenhouse cut rose producers apply a type of training system, called arching technique recognized as an effective method to improve marketable qualities of cut flowers (Lieth & Kim, 2001; Sarkka & Rita, 1999). In this system most weak and blind shoots (shoots without flower bud) are bent toward the aisle instead of being pruned, a common practice traditionally performed before introducing bending method. This training system divides the rose canopy into two different parts: upright shoots which comprise the crop harvesting stems and bent stems which consisted of unmarketable shoots devoted to extend plant leaf area facing high solar radiation and to act as a pool to store and reserve assimilates in order to be used in future production of high quality flower shoots. It was found that *K* value (equation 3) for bent layer of the rose canopy is higher than the value determined for upright canopy (Gonalez-Real, et al. 2007). Additionally, bent layer showed lower rate of photosynthesis than upright shoots (Kim et al., 2004; Gonzalez-Real et al., 2007).

In an experiment we examined effects of 5 training systems on the rate of canopy light absorption and photosynthesis of *Rosa hybrida* 'Habari'(Matloobi et al., 2009). Treatments were combinations of bending height on the mother stem and height of harvesting on the successive flower shoots: (i) T.S. 1-1: bending at the base of the primary shoot and harvesting all flowering shoots above the first bud, (ii) T.S. 3-3: bending above the third bud,

predicted in proportion to the daily irradiance. While sun-shade model gives predictions of canopy photosynthesis with best approximation to those predicted by multi layer model, the big leaf model usually shows deviations ranged from 10% to 45% (De Pury & Farquhar, 1997). Each model accompanies advantages and disadvantages, differing in the rate of accuracy and degree of complexity in calculations. Presently, computer software makes it so feasible to integrate many mathematical formulas into one distinctive program, facilitating

Pruning and training techniques are professional horticultural practices developed not only to control plant growth in some circumstances but also to modify plant canopy in such a way that increases the amount of light absorption. Fruit trees commonly are subject to training systems during their juvenile period when the plant canopy is being formed. Depend on the type of buds (vegetative or reproductive), abundance and method of distribution within tree crown, pruning and training practices are carefully adopted so that it ensures maximum light penetration through the canopy, and provides plants the highest growth and productivity. In an experiment with two cultivars of apple trees Mierowska et al. (2002) indicated that summer pruning enhances photosynthetic acclimation of spur leaves, previously developed under shade, by rapid increasing of the chlorophyll a/b ratio. Similarly, in *phalaenopsis* , it was shown that providing the lower shade-developed leaves with higher rates of light intensities caused increased rate of photosynthesis (Lin & Hsu, 2004). Pruning resulted in changes in light harvesting complexes of rose plants, showing that rose leaves are very plastic and acclimate

Training systems alter canopies light harvesting hehaviour through changing the foliage density, spatial form; the ratio of sun/shade leaves, leaf angles and finally the canopy leaf distribution pattern. There are several training systems developed for fruit trees based on the tree reproductive biology such as, central leader and modified leader particularly appropriate for pome fruits (apple, pear and quince trees) and open center specifically developed for prunes (peach, plum, and cherry trees). Recently, most greenhouse cut rose producers apply a type of training system, called arching technique recognized as an effective method to improve marketable qualities of cut flowers (Lieth & Kim, 2001; Sarkka & Rita, 1999). In this system most weak and blind shoots (shoots without flower bud) are bent toward the aisle instead of being pruned, a common practice traditionally performed before introducing bending method. This training system divides the rose canopy into two different parts: upright shoots which comprise the crop harvesting stems and bent stems which consisted of unmarketable shoots devoted to extend plant leaf area facing high solar radiation and to act as a pool to store and reserve assimilates in order to be used in future production of high quality flower shoots. It was found that *K* value (equation 3) for bent layer of the rose canopy is higher than the value determined for upright canopy (Gonalez-Real, et al. 2007). Additionally, bent layer showed lower rate of photosynthesis than upright

In an experiment we examined effects of 5 training systems on the rate of canopy light absorption and photosynthesis of *Rosa hybrida* 'Habari'(Matloobi et al., 2009). Treatments were combinations of bending height on the mother stem and height of harvesting on the successive flower shoots: (i) T.S. 1-1: bending at the base of the primary shoot and harvesting all flowering shoots above the first bud, (ii) T.S. 3-3: bending above the third bud,

calculations of even more complex equations.

**5. Training systems and canopy photosynthesis** 

rapidly to any changes in light intensity (Calatayud, et al., 2007).

shoots (Kim et al., 2004; Gonzalez-Real et al., 2007).

and harvesting above third bud for the first-order flowering shoot and above first bud for the following flowering shoots, (iii) T.S. 3-3-2: after bending primary shoot above third bud, the first-order flower shoot was harvested above the third bud and the second-order one above the second bud, (iv) T.S. 5-1: primary shoot was bent above fifth bud and the bearing flower shoot was harvested above first bud, (v): T.S. 5-3: primary shoot was bent above fifth bud and the bearing shoot was harvested above third bud. Leaf photosynthetic measurements have been performed for three layers of upright shoots (top, middle and bottom layer), and bent layer. Results exhibited that training system did not affect whole canopy light absorption significantly, but affected photosynthetic rate at canopy level (Table 1). This implies that photosynthetic rate at canopy level was influenced particularly by the interaction between light distribution profile through the canopy and canopy leaf area distribution rather than the

Fig. 6. Effect of different types of training system on the leaf area of *Rosa hybrida* 'Habari'. Different letters above columns indicate significantly difference according to the Duncan's multiple range test (p<0.05).

Fig. 7. Effect of different types of training system on the whole photosynthetic rate of *Rosa hybrida* 'Habari'. Different letters above columns indicate significantly difference according to the Duncan's multiple range test (p<0.05).

Light Harvesting and Photosynthesis by the Canopy 253

Beyschlag, W. and Ryel, R.J. (2007). Canopy Photosynthesis Modeling, In: *Functional Plant* 

Birch, C.J., Andrieu, B., Fournier, C., Vos, J. & Room, P. (2003). Modelling Kinetics of Plant

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Calatayud, A., Roca, D., Gorbe, E. & Martinez F.P. (2007). Light Acclimation in Rose (*Rosa* 

Cannell, M. G. R. & Thornley, J. H. M. (1998). Temperature and CO2 Response of Leaf and

Curtis, P. S., Vogel, C. S. Pregitzer, K. S., Zak, D. R. & Teeri, J. A. (1995). Interacting Effects of

De Pury D.G.G. & Farquhar G.D. (1997). Simple Scaling of Photosynthesis from Leaves to

Evans, J.R. & Poorter, H. (2001). Photosynthetic Acclimation of Plants to Growth Irradiance:

Farquhar, G. D. & Evans, J. R. (1991). Modeling Canopy Photosynthesis from the

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Fitter, A.H. & Hay, R. KM. (2002). Environmental Physiology of Plants. Academis Press,

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amount of light incident on the canopy. In other words, interaction between light intensity, light quality, leaf age, leaf area and many other intrinsic factors related to the leaf photosynthetic capacity have determined the canopy entire photosynthetic rate (Fig. 6 & Fig. 7). However, it was clearly deduced that canopy training system affected plant leaf area distribution between different leaf layers and accordingly impacted the rate of photosynthesis in each leaf layer. Plants developed by T.S. 1-1 method showed the lowest rate of canopy photosynthesis because they produced much less leaf area among the other training systems (Fig. 6). Although photosynthetic rate of the bent-shoots layer per unit leaf area was lower owing to the lower incident PAR, this layer accounted for about 40% of the whole plant photosynthetic capacity as a result of increased leaf area. Increasing leaf area does not enhance canopy assimilation rate unlimitedly due to leaves mutual shading caused by clumping effect. Lower layer of the bent shoots contributes negatively to the total canopy carbohydrate balance if its leaf area exceeds an optimal range (Pien et al., 2001). As a consequence, before adopting any type of training system or pruning strategy one should consider the results of *in situ* researches and try to optimize the canopy architecture and morphology based on environmental conditions and plant physiological and phonological characteristics.


Table 1. Means comparison of the measured properties within different leaf layers of *Rosa hybrida* 'Habari'

Training systems or pruning methods may influence canopy photosynthetic rate by altering source-sink relationship. This alteration may lead to negative feedback control of leaf photosynthesis capacity. In *citrus unshiu*, girldling and defruiting induced leaf starch accumulation and reduced photosynthesis, whereas partial defoliation induced the opposite effect (Iglesias et al., 2002). Partial defoliated apple trees have shown similar results (Zhou & Quebedeaux, 2003). Matloobi et al. (2008) indicated that in cut roses the leaf attached to the bud immediately below the harvesting place, actively contributes in assimilate supply to the new growing shoot. Photosynthetic rate of the leaf attached to the bud, above which the shoot was pruned, was more or less constant from time of harvest until the growth of axillary bud. After the bud started to grow, the photosynthetic rate began to decline sharply, showing that the leaf had been degrading photosynthesis-related enzymes and other chloroplast proteins in order to support the growing young shoot (strong sink). This reduction in carbon fixation may arise from N depletion due to remobilization of N towards the growing point. Surprisingly, removing flower bud (another strong sink) did not significantly affect carbon assimilation rate of the leaf nearest to the flower bud over one week of gas exchange measurements. This implies that sink removal might have contrasting responses regarding plant species, type of the sink organ to be removed and its spatial position in relation to the sources.

#### **6. References**

252 Advances in Photosynthesis – Fundamental Aspects

amount of light incident on the canopy. In other words, interaction between light intensity, light quality, leaf age, leaf area and many other intrinsic factors related to the leaf photosynthetic capacity have determined the canopy entire photosynthetic rate (Fig. 6 & Fig. 7). However, it was clearly deduced that canopy training system affected plant leaf area distribution between different leaf layers and accordingly impacted the rate of photosynthesis in each leaf layer. Plants developed by T.S. 1-1 method showed the lowest rate of canopy photosynthesis because they produced much less leaf area among the other training systems (Fig. 6). Although photosynthetic rate of the bent-shoots layer per unit leaf area was lower owing to the lower incident PAR, this layer accounted for about 40% of the whole plant photosynthetic capacity as a result of increased leaf area. Increasing leaf area does not enhance canopy assimilation rate unlimitedly due to leaves mutual shading caused by clumping effect. Lower layer of the bent shoots contributes negatively to the total canopy carbohydrate balance if its leaf area exceeds an optimal range (Pien et al., 2001). As a consequence, before adopting any type of training system or pruning strategy one should consider the results of *in situ* researches and try to optimize the canopy architecture and morphology based on

environmental conditions and plant physiological and phonological characteristics.

*hybrida* 'Habari'

position in relation to the sources.

 Incident PAR Photosynthesis Leaf area Leaf layers μmol m-2 s-1 μmol m-2leaf s-1 μmol m-2layer s-1 cm2 Top layer 630.1 a 18.25 a 0.979 c 534.8 c Middle layer 502.6 b 16.91 a 1.495 b 874.4 b Bottom layer 343.6 d 10.78 b 0.747 c 664.6 c Bent layer 411.1 c 11.90 b 2.102 a 1769.5 a Table 1. Means comparison of the measured properties within different leaf layers of *Rosa* 

Training systems or pruning methods may influence canopy photosynthetic rate by altering source-sink relationship. This alteration may lead to negative feedback control of leaf photosynthesis capacity. In *citrus unshiu*, girldling and defruiting induced leaf starch accumulation and reduced photosynthesis, whereas partial defoliation induced the opposite effect (Iglesias et al., 2002). Partial defoliated apple trees have shown similar results (Zhou & Quebedeaux, 2003). Matloobi et al. (2008) indicated that in cut roses the leaf attached to the bud immediately below the harvesting place, actively contributes in assimilate supply to the new growing shoot. Photosynthetic rate of the leaf attached to the bud, above which the shoot was pruned, was more or less constant from time of harvest until the growth of axillary bud. After the bud started to grow, the photosynthetic rate began to decline sharply, showing that the leaf had been degrading photosynthesis-related enzymes and other chloroplast proteins in order to support the growing young shoot (strong sink). This reduction in carbon fixation may arise from N depletion due to remobilization of N towards the growing point. Surprisingly, removing flower bud (another strong sink) did not significantly affect carbon assimilation rate of the leaf nearest to the flower bud over one week of gas exchange measurements. This implies that sink removal might have contrasting responses regarding plant species, type of the sink organ to be removed and its spatial


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**Part 3** 

**The Path of Carbon in Photosynthesis** 

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### **The Path of Carbon in Photosynthesis**

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Tissue D.T., Thomas, R.B. & Strain B.R. (1997) Atmospheric CO2 enrichment Increases

Vogelmann, T.C. & Martin, G. (1993). The Functional Significance of Palisade Tissue:

Walters, R. G. (2005). Towards an Understandign of Photosynthetic Acclimation*, J. Exp. Bot*,

Weiss, M., Baret, F., Smith, G.J., Joncheere, I. & Coppin, P. (2004). Review of Methods for *in* 

Wullschleger, S. D. (1993). Biochemical Limitations to Carbon Assimilation in C3 Plants – A

Xu, L. & Baldocchi, D. (2003). Seasonal Trends in Photosynthetic Parameters and Stomatal

Zhou, R. & Quebedeaux, B. (2003). Changes in Photosynthesis and Carbohydrate

Sampling. *Agricultural and Forest Meteorology*, Vol. 121, pp. 37-53

and High Temperature, *Tree Physiology*, Vol. 23, pp. 865-877

Manipulation, J. Amer. Soc. Hort. Sci. Vol. 128, 113-119

Chloroplasts Dependent on Intraleaf Light Environment. *Plant Cell Physiology*, Vol.

Growth and Photosynthesis of *Pinus taeda*: a 4 Year Experiment in the Field. *Plant,* 

Penetration of Directional *versus* Diffuse Light, *Plant, Cell and Environment*, Vol. 16,

*situ* Leaf Area Index (LAI) Determination Part II. Estimation of LAI, Errors and

Retrospective Analysis of the A/Ci Curves from 109 Species, *Journal of Experimental* 

Conductance of Blue Oak (*Quercus douglasii*) under Prolonged Summer Drought

Metabolism in Mature Apple Leaves in Response to Whole Plant Source-sink

Photosynthesis, *Plant Cell Environment*, Vol. 18, pp. 1111-1128

*Annu. Rev. Ecol. Syst*. Vol. 25, pp. 629-660

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*Cell and Environment*, Vol. 20, pp. 1123-1134

pp. 815-823

its Implications for

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*Botany*, Vol. 44, pp. 907-920

pp. 65-72

Assimilation Rate, and Plant Nitrogen Nutrition: A global Ecology Scaling Exercise.

Conductance of *Eucalyptus globulus* ssp*. Globulus* Leaves, *Tree Physiology*, Vol. 20,

**13** 

*USA* 

**The Path of Carbon in Photosynthesis – XXVIII –** 

A series of discoveries began 63 years ago through the collaboration of Melvin Calvin and Andrew Benson 1948. Paving the path (Benson 2002a) has since included the publications that have described the initial products of carbon fixation, from phosphoglycerate onward. From its inception, the program has followed a design that has been based on far-reaching interdisciplinary discourse, quoting the philosophy of Alexander Graham Bell, that, "Great discoveries and improvements invariably involve the cooperation of many minds." For example, investigations three decades ago by Wolf, Nonomura & Bassham 1985, established characteristics of the alga, "Showa," that accumulated the highest *in vitro* concentrations of hydrocarbons, 40% botryococcenes; from which gasoline and aviation fuels may be derived by catalytic hydrocracking in a conventional petroleum refinery. Studies of algal metabolism were as critical to the development of concepts of the Prize from 1948 through 1961, as they are today--special reference given to the lollipop (Nobelprize, 2011), a flat panel glass chamber that was designed by Benson and built by Harry Powell for controlled culture of *Chlorella* to track radiolabeled carbon metabolism in the laboratory --and thereby, paths converged, as Nonomura & Benson 1992 developed experimental methods for the feeding of single-carbon (C1) fragments to "Showa." That led to foliar delivery into angiosperms of up to 15 M C1 formulations supplemented with normally phytotoxic levels of ammoniacal nitrogen. When these applications were undertaken in sunlit fields, adjacent controls wilted by mid-afternoon, but rows treated with C1 formulations remained fully turgid, showing no signs of wilt. Moreover, Benson & Nonomura 1992 discovered that treatments with these C1 formulations inhibited glycolate formation, confirming their observations of long durations of reduced photorespiration. In addition to replicating increased yields, Ligocka et al. 2003 discovered corresponding increases of nitrate reductase and alkaline phosphatase by C1 formulations. In the meantime, Gout et al. 2000 followed metabolism of the C1 formulations by NMR to the identification of methyl--Dglucopyranoside; be that the case, little is known about glycosylation although it is a natural process in the metabolism of C1 fragments. Thus, in our programs of experimental biology, we investigated responses of plants to substituted glycopyranosides (Benson, 2002a); and we recently showed that not only do substituted glycopyranosides improve productivity, they are transported in plants and metabolized (Benson et al., 2009; Biel et al., 2010;

**1. Introduction** 

**Response of Plants to Polyalkylglucopyranose** 

Arthur M. Nonomura, Barry A. Cullen and Andrew A. Benson *Scripps Institution of Oceanography, University of California San Diego* 

**and Polyacylglucopyranose** 

### **The Path of Carbon in Photosynthesis – XXVIII – Response of Plants to Polyalkylglucopyranose and Polyacylglucopyranose**

Arthur M. Nonomura, Barry A. Cullen and Andrew A. Benson *Scripps Institution of Oceanography, University of California San Diego USA* 

#### **1. Introduction**

A series of discoveries began 63 years ago through the collaboration of Melvin Calvin and Andrew Benson 1948. Paving the path (Benson 2002a) has since included the publications that have described the initial products of carbon fixation, from phosphoglycerate onward. From its inception, the program has followed a design that has been based on far-reaching interdisciplinary discourse, quoting the philosophy of Alexander Graham Bell, that, "Great discoveries and improvements invariably involve the cooperation of many minds." For example, investigations three decades ago by Wolf, Nonomura & Bassham 1985, established characteristics of the alga, "Showa," that accumulated the highest *in vitro* concentrations of hydrocarbons, 40% botryococcenes; from which gasoline and aviation fuels may be derived by catalytic hydrocracking in a conventional petroleum refinery. Studies of algal metabolism were as critical to the development of concepts of the Prize from 1948 through 1961, as they are today--special reference given to the lollipop (Nobelprize, 2011), a flat panel glass chamber that was designed by Benson and built by Harry Powell for controlled culture of *Chlorella* to track radiolabeled carbon metabolism in the laboratory --and thereby, paths converged, as Nonomura & Benson 1992 developed experimental methods for the feeding of single-carbon (C1) fragments to "Showa." That led to foliar delivery into angiosperms of up to 15 M C1 formulations supplemented with normally phytotoxic levels of ammoniacal nitrogen. When these applications were undertaken in sunlit fields, adjacent controls wilted by mid-afternoon, but rows treated with C1 formulations remained fully turgid, showing no signs of wilt. Moreover, Benson & Nonomura 1992 discovered that treatments with these C1 formulations inhibited glycolate formation, confirming their observations of long durations of reduced photorespiration. In addition to replicating increased yields, Ligocka et al. 2003 discovered corresponding increases of nitrate reductase and alkaline phosphatase by C1 formulations. In the meantime, Gout et al. 2000 followed metabolism of the C1 formulations by NMR to the identification of methyl--Dglucopyranoside; be that the case, little is known about glycosylation although it is a natural process in the metabolism of C1 fragments. Thus, in our programs of experimental biology, we investigated responses of plants to substituted glycopyranosides (Benson, 2002a); and we recently showed that not only do substituted glycopyranosides improve productivity, they are transported in plants and metabolized (Benson et al., 2009; Biel et al., 2010;

The Path of Carbon in Photosynthesis – XXVIII – Response of Plants

**3. Results** 

Control.

tetramethylglucopyranose.

to Polyalkylglucopyranose and Polyacylglucopyranose 261

the concentrate from 25% methanol:5% formic aqueous eluants. Isolates showed a chromatographic metabolite, Rf 0.34, identified by staining with ninhydrin. The isolated metabolite was dissolved in 100 ml of aqueous solution for treatment of roots. For all populations, means of different treatment groups were compared using Student's t-test with significance at the 95% probability level. Confidence intervals of the population means are "p" values, counts of population numbers are "n" values, and ± standard error is denoted, "SE." Specialty chemicals were from Sigma (St. Louis, MO), including the following: N6 benzyladenine glucoside; tetramethylglucopyranose (TMG); tetraacetylglucopyranose (TAG); and methylglucopyranosides (MeG). A mixture of polyacetylglucopyranoses (MPG) was made by the authors with a modified chemical synthesis method of Hyatt & Tindall 1993, in which the extent of reaction was controlled by heating cycles. Vascular plants included Canola Nexera 500, *Brassica napus* L., a shoot crop; radish 'Cherry Bell' *Raphanus sativus* L., a root crop; rice, *Oryza sativa* L., a cereal crop; and corn TMF 114, *Zea mays* L. ssp. *Mays;* and these species were maintained as previously described (Benson et al., 2009).

We initiated this investigation by surveying polysubstituted glycopyranoses formulated with nutrients to establish discernable trends of growth responses without deficiency. We started with tetramethylglucopyranose because of a consistency of response that we had experienced with methylglucopyranoside. Manual spray-to-drip foliar treatments were applied to even stands of 5 cm tall radish, formulated as follows: Nutrient Control 15 mM (NH4)2SO4, 1 ppm Fe, 1 ppm Mn, 1 g/L surfactants; and 0.3 mM tetramethylglucopyranose and 1 mM tetramethylglucopyranose supplemented with 15 mM (NH4)2SO4, 1 ppm Fe, 1 ppm Mn, and 1 g/L surfactants. The growth of radish shoots was not affected within the two week trial period and radish roots treated with foliar applications of formulations of Nutrient Control or 0.3 mM tetramethylglucopyranose showed no significant difference (n=36; SE 0.05; p=0.8) from controls. However, foliar applications of 1 mM tetramethylglucopyranose to radish shoots resulted in a significant (n=36; SE 0.07; p=0.05) 27% enhancement of mean weights of roots

over those of Nutrient Control. Results of root analyses are displayed in Figure 1.

Following our establishment of an effective foliar dose that improved root yields, we developed a formulation for row crops that delivered 186 L/ha, less than a quarter the volume of manual applications to shoots. Foliar treatments to even stands of corn were of the following: Nutrient Control, 15 mM (NH4)2SO4, 1 ppm Fe, 1 ppm Mn, 1 g/L surfactants; and 3 mM tetramethylglucopyranose, identically supplemented with the above nutrients. The results of this experiment can be seen in Figure 2, where the application of foliar 3 mM tetramethylglucopyranose resulted in a significant (n=18; p=0.03) increase over the Nutrient

Tetraacetylglucopyranose (TAG) is similar to tetramethylglucopyranose except that the sugar is substituted around the pyranose-ring with four acyl-groups instead of alkylgroups. The range of activity for polyacylglycopyranoses was unknown, therefore, 10 mM TAG was explored in these trials. Foliar 10 mM TAG, formulated in the same nutrient solution as Nutrient Control, was applied to shoots of radish and harvested a week later. Results of foliar application (Fig. 3) showed a significant (n=11, p=0.004) 27% increase of root mean dry weight as compared to Nutrient Control. The growth response of the roots of radish to foliar tetraacetylglucopyranose, therefore, was similar to that of

Nonomura et al., 2011). For our current studies, we selected a polyalkylglycopyranose and we manufactured polyacylglycopyranoses as candidate compounds. Plant responses to formulations of 0.5 to 10 mM polysubstituted glucopyranoses were similar to those of growth enhancements from treatment with foliar formulations of 0.3 M methylglucopyranosides. Therefore, we conclude that when highly substituted sugars are appropriately formulated for application at optimal dosages, visibly discernible enhancement of vegetative productivity may be achieved that is statistically significant and highly consistent. Treatments with polyacylglucopyranoses resulted in significant enhancements of root and shoot growth without toxicity and we tracked putative metabolites of polyacylglucopyranoses and ammoniacal nitrogen. Finally, we gather evidence of mechanisms in which substituted glycopyranoses compete with sugars to release them from lectins.

#### **2. Materials and methods**

Plants were cultured in research facilities according to previously described methods (Benson et al., 2009). General supplementation of foliar formulations included the following: 10 - 50 mM ammonium salt; 1 - 6 ppm manganese, Mn-EDTA; and 1 - 6 ppm iron, Fe-HEDTA. For example, foliar solutions of 1,2,3,4-tetramethyl-β-D-glucopyranose, hereafter referred to as tetramethylglucopyranose, were formulated as follow: 23 mM ammonium sulfate, (NH4)2SO4, 6 ppm Mn and 6 ppm Fe; and Nutrient Control contained 23 mM (NH4)2SO4, 6 ppm Mn and 6 ppm Fe. We found the above supplements to be effective with polyalkylglucopyranoses and polyacylglucopyranoses, particularly, with Mn adding consistency of response to treatments. Moreover, our preliminary tests showed that without ammoniacal and Mn supplementation, the compounds were inactive. Solutions for foliar applications included phytobland surfactant blends applied at a concentration of 1 g/L made from a random block copolymer (Pluronic® L-62, BASF) with a polysiloxane wetter (Q2-5211, Dow Corning®) compounded at a 3:1 ratio. Controls were placed in the same location and all plants were given identical irrigation, fertigation, and handling. Plants were cultured in trays containing soil-less MetroMix® 560 media. Grasses were cultured in MetroMix® 560 blended with 25% calcined clay (Turface®). Plants were matched to control populations, treated after emergence of cotyledon and true leaves, and later harvested within one or two weeks for analysis. For biomass, plants were dried completely in ovens heated to 80**°**C and weighed. The performance of compounds was evaluated by comparing statistical means of individual weights of shoots and roots. Over the course of taking replicated corresponding measurements, we found that dry biomass gains were proportional to fresh wet gains, allowing us to exclude cell enlargement responses such as to gibberellin and to undertake exploratory surveys and dose response curves with wet weights. When the dosage was sufficiently narrowed, we obtained dry weights, as well. Individual plantings were cultured in plastic flats of identical volumes as needed for optimal growth for size and age within the same planting cycle. All potted plants were regularly given water-culture nutrients (Hoagland & Arnon, 1950). Foliar spray applications of identical volumes, either 100 or 186 liters/hectare (L/ha), were mechanically applied. Manual sprays were spray-to-drip volumes of approximately 800 L/ha. Isolation of metabolites of mixed polyacetylglucopyranoses was undertaken by methods of Biel et al. 2010 and modified by collecting numerous two dimensional chromatography strips, followed by C18 reverse phase column chromatography, and yielding 10 milligrams (mg) of the concentrate from 25% methanol:5% formic aqueous eluants. Isolates showed a chromatographic metabolite, Rf 0.34, identified by staining with ninhydrin. The isolated metabolite was dissolved in 100 ml of aqueous solution for treatment of roots. For all populations, means of different treatment groups were compared using Student's t-test with significance at the 95% probability level. Confidence intervals of the population means are "p" values, counts of population numbers are "n" values, and ± standard error is denoted, "SE." Specialty chemicals were from Sigma (St. Louis, MO), including the following: N6 benzyladenine glucoside; tetramethylglucopyranose (TMG); tetraacetylglucopyranose (TAG); and methylglucopyranosides (MeG). A mixture of polyacetylglucopyranoses (MPG) was made by the authors with a modified chemical synthesis method of Hyatt & Tindall 1993, in which the extent of reaction was controlled by heating cycles. Vascular plants included Canola Nexera 500, *Brassica napus* L., a shoot crop; radish 'Cherry Bell' *Raphanus sativus* L., a root crop; rice, *Oryza sativa* L., a cereal crop; and corn TMF 114, *Zea mays* L. ssp. *Mays;* and these species were maintained as previously described (Benson et al., 2009).

#### **3. Results**

260 Advances in Photosynthesis – Fundamental Aspects

Nonomura et al., 2011). For our current studies, we selected a polyalkylglycopyranose and we manufactured polyacylglycopyranoses as candidate compounds. Plant responses to formulations of 0.5 to 10 mM polysubstituted glucopyranoses were similar to those of growth enhancements from treatment with foliar formulations of 0.3 M methylglucopyranosides. Therefore, we conclude that when highly substituted sugars are appropriately formulated for application at optimal dosages, visibly discernible enhancement of vegetative productivity may be achieved that is statistically significant and highly consistent. Treatments with polyacylglucopyranoses resulted in significant enhancements of root and shoot growth without toxicity and we tracked putative metabolites of polyacylglucopyranoses and ammoniacal nitrogen. Finally, we gather evidence of mechanisms in which substituted glycopyranoses compete with sugars to

Plants were cultured in research facilities according to previously described methods (Benson et al., 2009). General supplementation of foliar formulations included the following: 10 - 50 mM ammonium salt; 1 - 6 ppm manganese, Mn-EDTA; and 1 - 6 ppm iron, Fe-HEDTA. For example, foliar solutions of 1,2,3,4-tetramethyl-β-D-glucopyranose, hereafter referred to as tetramethylglucopyranose, were formulated as follow: 23 mM ammonium sulfate, (NH4)2SO4, 6 ppm Mn and 6 ppm Fe; and Nutrient Control contained 23 mM (NH4)2SO4, 6 ppm Mn and 6 ppm Fe. We found the above supplements to be effective with polyalkylglucopyranoses and polyacylglucopyranoses, particularly, with Mn adding consistency of response to treatments. Moreover, our preliminary tests showed that without ammoniacal and Mn supplementation, the compounds were inactive. Solutions for foliar applications included phytobland surfactant blends applied at a concentration of 1 g/L made from a random block copolymer (Pluronic® L-62, BASF) with a polysiloxane wetter (Q2-5211, Dow Corning®) compounded at a 3:1 ratio. Controls were placed in the same location and all plants were given identical irrigation, fertigation, and handling. Plants were cultured in trays containing soil-less MetroMix® 560 media. Grasses were cultured in MetroMix® 560 blended with 25% calcined clay (Turface®). Plants were matched to control populations, treated after emergence of cotyledon and true leaves, and later harvested within one or two weeks for analysis. For biomass, plants were dried completely in ovens heated to 80**°**C and weighed. The performance of compounds was evaluated by comparing statistical means of individual weights of shoots and roots. Over the course of taking replicated corresponding measurements, we found that dry biomass gains were proportional to fresh wet gains, allowing us to exclude cell enlargement responses such as to gibberellin and to undertake exploratory surveys and dose response curves with wet weights. When the dosage was sufficiently narrowed, we obtained dry weights, as well. Individual plantings were cultured in plastic flats of identical volumes as needed for optimal growth for size and age within the same planting cycle. All potted plants were regularly given water-culture nutrients (Hoagland & Arnon, 1950). Foliar spray applications of identical volumes, either 100 or 186 liters/hectare (L/ha), were mechanically applied. Manual sprays were spray-to-drip volumes of approximately 800 L/ha. Isolation of metabolites of mixed polyacetylglucopyranoses was undertaken by methods of Biel et al. 2010 and modified by collecting numerous two dimensional chromatography strips, followed by C18 reverse phase column chromatography, and yielding 10 milligrams (mg) of

release them from lectins.

**2. Materials and methods** 

We initiated this investigation by surveying polysubstituted glycopyranoses formulated with nutrients to establish discernable trends of growth responses without deficiency. We started with tetramethylglucopyranose because of a consistency of response that we had experienced with methylglucopyranoside. Manual spray-to-drip foliar treatments were applied to even stands of 5 cm tall radish, formulated as follows: Nutrient Control 15 mM (NH4)2SO4, 1 ppm Fe, 1 ppm Mn, 1 g/L surfactants; and 0.3 mM tetramethylglucopyranose and 1 mM tetramethylglucopyranose supplemented with 15 mM (NH4)2SO4, 1 ppm Fe, 1 ppm Mn, and 1 g/L surfactants. The growth of radish shoots was not affected within the two week trial period and radish roots treated with foliar applications of formulations of Nutrient Control or 0.3 mM tetramethylglucopyranose showed no significant difference (n=36; SE 0.05; p=0.8) from controls. However, foliar applications of 1 mM tetramethylglucopyranose to radish shoots resulted in a significant (n=36; SE 0.07; p=0.05) 27% enhancement of mean weights of roots over those of Nutrient Control. Results of root analyses are displayed in Figure 1.

Following our establishment of an effective foliar dose that improved root yields, we developed a formulation for row crops that delivered 186 L/ha, less than a quarter the volume of manual applications to shoots. Foliar treatments to even stands of corn were of the following: Nutrient Control, 15 mM (NH4)2SO4, 1 ppm Fe, 1 ppm Mn, 1 g/L surfactants; and 3 mM tetramethylglucopyranose, identically supplemented with the above nutrients. The results of this experiment can be seen in Figure 2, where the application of foliar 3 mM tetramethylglucopyranose resulted in a significant (n=18; p=0.03) increase over the Nutrient Control.

Tetraacetylglucopyranose (TAG) is similar to tetramethylglucopyranose except that the sugar is substituted around the pyranose-ring with four acyl-groups instead of alkylgroups. The range of activity for polyacylglycopyranoses was unknown, therefore, 10 mM TAG was explored in these trials. Foliar 10 mM TAG, formulated in the same nutrient solution as Nutrient Control, was applied to shoots of radish and harvested a week later. Results of foliar application (Fig. 3) showed a significant (n=11, p=0.004) 27% increase of root mean dry weight as compared to Nutrient Control. The growth response of the roots of radish to foliar tetraacetylglucopyranose, therefore, was similar to that of tetramethylglucopyranose.

The Path of Carbon in Photosynthesis – XXVIII – Response of Plants

to Polyalkylglucopyranose and Polyacylglucopyranose 263

Fig. 3. Foliar application of tetraacetylglucopyranose, 10 mM TAG, to shoots of radish resulted in a significant (n=11; p=0.004) increase of root mean dry weight as compared to

We investigated a process for the chemical synthesis of our mixed polyacylglucopyranoses (MPG) at different temperatures to control the reaction; and results are summarized in Figure 4. Between 60º and 72° C, we succeeded in stocking supplies that we manufactured in 500 gram batches. We incorporated 4 mM and 8 mM tetraacetylglucopyranose (TAG) into our tests as positive controls to compare against the different 60º and 72° C batches of 4 mM, 8 mM and 12 mM MPG. Treatments with untreated Control, Nutrient Control, and 12 mM MPG resulted in no difference of growth. In contrast, 4 mM and 8 mM concentrations, showed significantly (n=36; p<0.05) higher vegetative yields than controls. With similar results from these two different batches comparable to tetraacetylglucopyranose, we

On Canola, responses to foliar applications of 3 mM mixed polyacetylglucopyranoses, 4 mM tetraacetylglucopyranose and 309 mM methylglucopyranoside were compared. All treatments contained 40 mM ammonium nitrate and surfactant blend, including Nutrient Control, and results are graphically depicted in Figure 5. Three treated populations each showed significant (p=0.000) shoot wet weight increases over Nutrient Control, as follow: 3 mM mixed polyacetylglucopyranoses, n=37, 18% increase; 4 mM tetraacetylglucopyranose, n=35, 20% increase; and 309 mM methylglucopyranoside, n=36, 14% increase. We have clearly demonstrated that the far lower concentrations of 3 - 4 mM polyacetylglucopyranoses than the ~100-fold higher dose of 309 mM methylglucopyranoside resulted in comparable growth

Nutrient Control. Error bars indicate ±SE.

increase responses from Canola.

extended tests to concentrations on various species of plants.

Fig. 1. Initial surveys of radish showed that applications of high volumes of 1 mM tetramethylglucopyranose formulations, 1 mM TMG, to shoots, significantly (n=36; p=0.05) enhanced root mean dry weights as compared to Nutrient Control. Means are marked as small open circles at the midpoint of error bars that indicate ±SE.

Fig. 2. As a result of foliar applications of 3 mM tetramethylglucopyranose, 3 mM TMG applied at 186 L/ha, a volume typical for row crops, shoot mean dry weights of corn improved significantly (n=11; p=0.004) as compared to Nutrient Control. Error bars indicate ±SE.

Fig. 1. Initial surveys of radish showed that applications of high volumes of 1 mM

small open circles at the midpoint of error bars that indicate ±SE.

tetramethylglucopyranose formulations, 1 mM TMG, to shoots, significantly (n=36; p=0.05) enhanced root mean dry weights as compared to Nutrient Control. Means are marked as

Fig. 2. As a result of foliar applications of 3 mM tetramethylglucopyranose, 3 mM TMG applied at 186 L/ha, a volume typical for row crops, shoot mean dry weights of corn

improved significantly (n=11; p=0.004) as compared to Nutrient Control.

Error bars indicate ±SE.

Fig. 3. Foliar application of tetraacetylglucopyranose, 10 mM TAG, to shoots of radish resulted in a significant (n=11; p=0.004) increase of root mean dry weight as compared to Nutrient Control. Error bars indicate ±SE.

We investigated a process for the chemical synthesis of our mixed polyacylglucopyranoses (MPG) at different temperatures to control the reaction; and results are summarized in Figure 4. Between 60º and 72° C, we succeeded in stocking supplies that we manufactured in 500 gram batches. We incorporated 4 mM and 8 mM tetraacetylglucopyranose (TAG) into our tests as positive controls to compare against the different 60º and 72° C batches of 4 mM, 8 mM and 12 mM MPG. Treatments with untreated Control, Nutrient Control, and 12 mM MPG resulted in no difference of growth. In contrast, 4 mM and 8 mM concentrations, showed significantly (n=36; p<0.05) higher vegetative yields than controls. With similar results from these two different batches comparable to tetraacetylglucopyranose, we extended tests to concentrations on various species of plants.

On Canola, responses to foliar applications of 3 mM mixed polyacetylglucopyranoses, 4 mM tetraacetylglucopyranose and 309 mM methylglucopyranoside were compared. All treatments contained 40 mM ammonium nitrate and surfactant blend, including Nutrient Control, and results are graphically depicted in Figure 5. Three treated populations each showed significant (p=0.000) shoot wet weight increases over Nutrient Control, as follow: 3 mM mixed polyacetylglucopyranoses, n=37, 18% increase; 4 mM tetraacetylglucopyranose, n=35, 20% increase; and 309 mM methylglucopyranoside, n=36, 14% increase. We have clearly demonstrated that the far lower concentrations of 3 - 4 mM polyacetylglucopyranoses than the ~100-fold higher dose of 309 mM methylglucopyranoside resulted in comparable growth increase responses from Canola.

The Path of Carbon in Photosynthesis – XXVIII – Response of Plants

to Polyalkylglucopyranose and Polyacylglucopyranose 265

Fig. 5. Foliar applications with low concentrations of polyacetylglucopyranoses, 3 mM MPG and 4 mM TAG, were comparable to treatments with high methylglucopyranosides, 309

mM MeG, and resulted in significant shoot enhancements over Nutrient Control.

Fig. 6. Dose response of corn roots exposed to various concentrations of mixed polyacetylglucopyranoses (MPG) showed best results at 1 mM MPG with significant (n=2; p=0.006) shoot enhancement over Nutrient Control. Error bars indicate ±SE.

Fig. 4. Similar to nutrient-supplemented formulations of 4 mM and 8 mM tetraacetylglucopyranose (4 mM and 8 mM TAG), responses of populations of Canola to foliar applications of two different batches of mixed polyacetylglucopyranoses (4 mM MPG 72 C & 4 mM MPG 60 C) showed significantly higher (n=36; p<0.05) mean shoot weights than the untreated Control and the Nutrient Control. Error bars indicate ±SE.

A dose response curve of 1 – 3 mM mixed polyacetylglucopyranoses (MPG) was applied to roots of corn with treatments that were supplemented with identical nutrients to the control. Roots were saturated *in situ* with 5 ml of respective formulations per plant. Two weeks later, shoots were harvested and weighed. Statistical analyses showed trends in Figure 6, as follow: 1 mM MPG showed a signficant (n=21; p=0.006) increase in yield over Nutrient Control; 2 mM MPG showed a positive trend that was not significant (n=21; p=0.07); and 3 mM MPG showed a negative trend that may have resulted from exposure of roots to a critical concentration of the acidic mixed polyacetylglucopyranoses.

Treatments of roots of rice with mixed polyacetylglucopyranoses was compared to a treatment with a high concentration of methylglucopyranosides. All solutions were supplemented with identical nutrients including the following: 17 mM (NH4)2SO4, 5 mM (NH4)2HPO4 and 3 ppm Mn. Roots were saturated *in situ* with 10 ml of respective formulations per 100 cc culture vessel of each individual plant for two weeks. Roots exposed to formulations of 0.5 mM mixed polyacetylglucopyranoses and 50 mM methylglucopyranosides showed significant (n=27; p=0.000) increases in shoot yields of approximately 15% over controls. Results are graphically summarized in Figure 7.

Fig. 4. Similar to nutrient-supplemented formulations of 4 mM and 8 mM

than the untreated Control and the Nutrient Control. Error bars indicate ±SE.

critical concentration of the acidic mixed polyacetylglucopyranoses.

tetraacetylglucopyranose (4 mM and 8 mM TAG), responses of populations of Canola to foliar applications of two different batches of mixed polyacetylglucopyranoses (4 mM MPG 72 C & 4 mM MPG 60 C) showed significantly higher (n=36; p<0.05) mean shoot weights

A dose response curve of 1 – 3 mM mixed polyacetylglucopyranoses (MPG) was applied to roots of corn with treatments that were supplemented with identical nutrients to the control. Roots were saturated *in situ* with 5 ml of respective formulations per plant. Two weeks later, shoots were harvested and weighed. Statistical analyses showed trends in Figure 6, as follow: 1 mM MPG showed a signficant (n=21; p=0.006) increase in yield over Nutrient Control; 2 mM MPG showed a positive trend that was not significant (n=21; p=0.07); and 3 mM MPG showed a negative trend that may have resulted from exposure of roots to a

Treatments of roots of rice with mixed polyacetylglucopyranoses was compared to a treatment with a high concentration of methylglucopyranosides. All solutions were supplemented with identical nutrients including the following: 17 mM (NH4)2SO4, 5 mM (NH4)2HPO4 and 3 ppm Mn. Roots were saturated *in situ* with 10 ml of respective formulations per 100 cc culture vessel of each individual plant for two weeks. Roots exposed to formulations of 0.5 mM mixed polyacetylglucopyranoses and 50 mM methylglucopyranosides showed significant (n=27; p=0.000) increases in shoot yields of

approximately 15% over controls. Results are graphically summarized in Figure 7.

Fig. 5. Foliar applications with low concentrations of polyacetylglucopyranoses, 3 mM MPG and 4 mM TAG, were comparable to treatments with high methylglucopyranosides, 309 mM MeG, and resulted in significant shoot enhancements over Nutrient Control.

Fig. 6. Dose response of corn roots exposed to various concentrations of mixed polyacetylglucopyranoses (MPG) showed best results at 1 mM MPG with significant (n=2; p=0.006) shoot enhancement over Nutrient Control. Error bars indicate ±SE.

The Path of Carbon in Photosynthesis – XXVIII – Response of Plants

to Polyalkylglucopyranose and Polyacylglucopyranose 267

Fig. 8. Top: Roots of corn exposed to 1 mM mixed polyacetylglucopyranoses (1 mm MPG) showed significantly (n=21; p=0.000) increased mean shoot wet weights. Bottom: Dry weights corresponded to wet weights, showing a significant (n=21; p=0.006) 12% increase of

We collected sufficient samples of chromatographic isolates of putative metabolites (Isolate) of mixed polyacetylglucopyranoses (MPG) from aqueous extracts of host plants to undertake a comparison of activities. Mixed polyacetylglucopyranoses, 5 mM MPG, were positive controls. All aqueous solutions for the treatment of roots were adjusted to pH 6.5 and were made up with the following nutrients: 23 mM (NH4)2SO4; 5 mM

mean dry weight over Nutrient Control. Error bars indicate ±SE.

Fig. 7. Roots of whole rice plants exposed to 0.5 mM mixed polyacetylglucopyranoses (0.5 mM MPG) or 50 mM methylglucopyranosides (50 mM MeG), showed significant (n=27; p=0.000) shoot increases over Nutrient Control. Error bars indicate ±SE.

Roots of corn immersed in 1 mM mixed polyacetylglucopyranoses formulated with phosphate buffer to avoid artifacts associated with acidity, were additionally supplemented with the following: 23 mM (NH4)2SO4, 5 mM K2HPO4 , 3 mM KH2PO4 and 3 ppm Mn. Roots were saturated with 5 ml of respective formulations per 100 cc soil-less media per plant. The individual shoots of each of the rice plants were harvested after two weeks. Results are displayed in Figure 8 and show significant (n=21; Mean Wet Weight p=0.000; Mean Dry Weight p=0.006) increases of 12% in vegetative yields of shoots over the population of the Nutrient Control. Buffering the solution with phosphates may have nutritionally contributed to the rapid growths of the Nutrient Control and the active treatments while safening the solutions at the same time.

Treatment of roots with mixed polyacetylglucopyranoses resulted in enhancement of shoots, therefore, our hypothesis was that 100 L/ha foliar applications would similarly accelerate development of shoots. Foliar 1 mM and 7 mM mixed polyacetylglucopyranoses were compared to Nutrient Control, both solutions containing, 1 g/L surfactant blend, 23 mM (NH4)2SO4 and 3 ppm Mn. Populations included the following: untreated control, n=20; Nutrient Control, n=20; 1 mM MPG, n=20; and 7 mM MPG, n=19; and with no significant difference between the untreated and Nutrient Controls. Foliar treatments of 1 mM and 7 mM mixed polyacetylglucopyranoses resulted in a significant (p=0.003) increase of shoot mean wet weight and a significant (p=0.002) 20% increase of mean dry weight as compared to the Nutrient Control. Shoot dry weights are summarized in Figure 9.

Fig. 7. Roots of whole rice plants exposed to 0.5 mM mixed polyacetylglucopyranoses (0.5 mM MPG) or 50 mM methylglucopyranosides (50 mM MeG), showed significant (n=27;

Roots of corn immersed in 1 mM mixed polyacetylglucopyranoses formulated with phosphate buffer to avoid artifacts associated with acidity, were additionally supplemented with the following: 23 mM (NH4)2SO4, 5 mM K2HPO4 , 3 mM KH2PO4 and 3 ppm Mn. Roots were saturated with 5 ml of respective formulations per 100 cc soil-less media per plant. The individual shoots of each of the rice plants were harvested after two weeks. Results are displayed in Figure 8 and show significant (n=21; Mean Wet Weight p=0.000; Mean Dry Weight p=0.006) increases of 12% in vegetative yields of shoots over the population of the Nutrient Control. Buffering the solution with phosphates may have nutritionally contributed to the rapid growths of the Nutrient Control and the active treatments while

Treatment of roots with mixed polyacetylglucopyranoses resulted in enhancement of shoots, therefore, our hypothesis was that 100 L/ha foliar applications would similarly accelerate development of shoots. Foliar 1 mM and 7 mM mixed polyacetylglucopyranoses were compared to Nutrient Control, both solutions containing, 1 g/L surfactant blend, 23 mM (NH4)2SO4 and 3 ppm Mn. Populations included the following: untreated control, n=20; Nutrient Control, n=20; 1 mM MPG, n=20; and 7 mM MPG, n=19; and with no significant difference between the untreated and Nutrient Controls. Foliar treatments of 1 mM and 7 mM mixed polyacetylglucopyranoses resulted in a significant (p=0.003) increase of shoot mean wet weight and a significant (p=0.002) 20% increase of mean dry weight as compared to the Nutrient Control. Shoot dry weights

p=0.000) shoot increases over Nutrient Control. Error bars indicate ±SE.

safening the solutions at the same time.

are summarized in Figure 9.

Fig. 8. Top: Roots of corn exposed to 1 mM mixed polyacetylglucopyranoses (1 mm MPG) showed significantly (n=21; p=0.000) increased mean shoot wet weights. Bottom: Dry weights corresponded to wet weights, showing a significant (n=21; p=0.006) 12% increase of mean dry weight over Nutrient Control. Error bars indicate ±SE.

We collected sufficient samples of chromatographic isolates of putative metabolites (Isolate) of mixed polyacetylglucopyranoses (MPG) from aqueous extracts of host plants to undertake a comparison of activities. Mixed polyacetylglucopyranoses, 5 mM MPG, were positive controls. All aqueous solutions for the treatment of roots were adjusted to pH 6.5 and were made up with the following nutrients: 23 mM (NH4)2SO4; 5 mM

The Path of Carbon in Photosynthesis – XXVIII – Response of Plants

to Polyalkylglucopyranose and Polyacylglucopyranose 269

Fig. 10. Treatment of roots of corn with a metabolite of mixed polyacetylglucopyranoses, 0.5 mg Isolate, resulted in significant increases comparable to effects of treatments with ten times the quantity of mixed polyacetylglucopyranoses, 5 mg MPG. Error bars indicate ±SE.

Fig. 11. A chromatographically isolated metabolite of mixed polyacylglycopyranoses (Isolate) was applied to roots of young corn plants and, after a week, the experiment was photographed to show that the enhanced growth of plants treated with the Isolate was visibly discernable from Nutrient Controls. The population of 0.5 mg Isolate, right, is also

Whether to shoots or roots, nutrient applications of relatively low concentrations of polyalkylglycopyranose or polyacylglycopyranoses enhanced vegetative productivity of

tagged "Isol8," and Nutrient Control is separated, to the left. Scale = 1 cm.

**4. Discussion** 

(NH4)2HPO4; and chelated 3 ppm Mn. For each corn plant, 5 ml of test solution was applied to saturate roots for 6 hours, according to the following doses: 5 mg mixed polyacetylglucopyranoses per plant; and 0.5 mg Isolate per plant. Entire plants were harvested after one week for whole weights of shoots with roots. A summary of data in Figure 10 shows treatment with 0.5 mg Isolate resulted in significant (n=21; p=0.005) 11% increase and treatment with mixed polyacetylglucopyranoses (MPG) resulted in significant (n=21; p=0.000) 15% increase of whole plant mean wet weight over Nutrient Control.

Fig. 9. Foliar applications of formulations of 1 mM and 7 mM mixed polyacetylglucopyranoses (1 mM MPG & 7 mM MPG) resulted in significant (p=0.002) increased shoot mean dry weights of approximately 20% over the population of the Nutrient Control. Error bars indicate ±SE.

The photograph of corn plants exhibited in Figure 11, was taken immediately prior to harvest, one week after treatment with the metabolite of mixed polyacetylglucopyranoses, 0.5 mg Isolate. The Isolate, right, in which the leaf tip reaches the top of the photograph, is visibly distinguishable from the Nutrient Control, left, of which the shoots reach approximately 1 cm below the top border of the photograph. This visual comparison of the generally larger plants of the Isolate, corroborates the 15% increase in the total mean weight of the plant as compared to the Nutrient Control.

Finally, N6-benzyladenine glucoside chromatographed similarly to our metabolic Isolate of mixed polyacetylglucopyranoses. Therefore, these chromatographic results may suggest that supplementation of formulations with ammoniacal nitrogen may be suited to the incorporation of a nitrogen moiety into polyalkylglycopyranoses and polyacylglycopyranoses.

(NH4)2HPO4; and chelated 3 ppm Mn. For each corn plant, 5 ml of test solution was applied to saturate roots for 6 hours, according to the following doses: 5 mg mixed polyacetylglucopyranoses per plant; and 0.5 mg Isolate per plant. Entire plants were harvested after one week for whole weights of shoots with roots. A summary of data in Figure 10 shows treatment with 0.5 mg Isolate resulted in significant (n=21; p=0.005) 11% increase and treatment with mixed polyacetylglucopyranoses (MPG) resulted in significant (n=21; p=0.000) 15% increase of whole plant mean wet weight over Nutrient

Fig. 9. Foliar applications of formulations of 1 mM and 7 mM mixed

Control. Error bars indicate ±SE.

of the plant as compared to the Nutrient Control.

polyacetylglucopyranoses (1 mM MPG & 7 mM MPG) resulted in significant (p=0.002) increased shoot mean dry weights of approximately 20% over the population of the Nutrient

The photograph of corn plants exhibited in Figure 11, was taken immediately prior to harvest, one week after treatment with the metabolite of mixed polyacetylglucopyranoses, 0.5 mg Isolate. The Isolate, right, in which the leaf tip reaches the top of the photograph, is visibly distinguishable from the Nutrient Control, left, of which the shoots reach approximately 1 cm below the top border of the photograph. This visual comparison of the generally larger plants of the Isolate, corroborates the 15% increase in the total mean weight

Finally, N6-benzyladenine glucoside chromatographed similarly to our metabolic Isolate of mixed polyacetylglucopyranoses. Therefore, these chromatographic results may suggest that supplementation of formulations with ammoniacal nitrogen may be suited to the incorporation of a nitrogen moiety into polyalkylglycopyranoses and polyacylglycopyranoses.

Control.

Fig. 10. Treatment of roots of corn with a metabolite of mixed polyacetylglucopyranoses, 0.5 mg Isolate, resulted in significant increases comparable to effects of treatments with ten times the quantity of mixed polyacetylglucopyranoses, 5 mg MPG. Error bars indicate ±SE.

Fig. 11. A chromatographically isolated metabolite of mixed polyacylglycopyranoses (Isolate) was applied to roots of young corn plants and, after a week, the experiment was photographed to show that the enhanced growth of plants treated with the Isolate was visibly discernable from Nutrient Controls. The population of 0.5 mg Isolate, right, is also tagged "Isol8," and Nutrient Control is separated, to the left. Scale = 1 cm.

#### **4. Discussion**

Whether to shoots or roots, nutrient applications of relatively low concentrations of polyalkylglycopyranose or polyacylglycopyranoses enhanced vegetative productivity of

The Path of Carbon in Photosynthesis – XXVIII – Response of Plants

carbon in photosynthesis (Benson, 2002a & b).

**5. References** 

to Polyalkylglucopyranose and Polyacylglucopyranose 271

to a shoot (Fig. 5) should be sufficient to saturate the system. Therefore, we propose that under conditions in which the cellular sugar concentration of a plant is diminished, chemical competition against substituted sugars acts to release sugar from lectin—and this is an essential process to sustain viability. Also, this act of competitive binding by lectins may naturally displace sugars on a regular basis allowing energy to be reapportioned for growth resulting from metabolism of the freed sugars. For example, we may assume that in the field, the concentration of methyl--D-glucopyranoside remains nearly constant in the plant (Aubert et al., 2004) and as a result of midday photorespiratory depletion of the concentration of glucose, competition for binding to lectin by the methyl--D-glucopyranoside arises and glucose is released. To an extent, the timely release of this free glucose may mitigate the effects of any impoverishment of glucose. Afterwards, under conditions more conducive to photosynthesis, perhaps later in the afternoon and morning, critical concentrations of glucose are rebuilt to sufficiently high levels that a surfeit of glucose outcompetes methyl--Dglucopyranoside, causing the substituted sugar to be displaced, while glucose wins by binding to the lectin. This cycle repeats itself on a daily basis, releasing sugar at each lengthy deprivation event, followed by the capture of fresh sugar upon resuming photosynthesis. Indeed, Nature's response to major environmental stimuli by means of chemical competition is well known. For example, photosynthesis turns to photorespiration strictly as a result of oxygen outcompeting carbon dioxide for binding to Rubisco. In our case, the higher the quantity of lectins residing in the plant, the more capable it may be of capturing and releasing sugars to endure prolonged periods of photorespiration. In contrast, when exogenous chemical competitors for binding sites on lectins are applied to plants, especially by the input of substrates, such as methyl--D-glucopyranoside, that do not naturally occur in plants, the duration of the effect may be substantially extended precisely because such foreign compounds may be selected for competitive advantages. Therefore, responses to treatments with substituted sugars must be carefully measured against the conformation of binding sites, biochemical structure, and their orders of preferences for prospective sugars. From another perspective, empirically formulated dosages of crops may possibly reflect the content and binding determinations of major lectins in a cultivar. For example, in the present investigation, our hypothesis is that the relatively low concentrations of tetraacetylglucopyranose required for the improved growth response of treated plants over controls evidently suggests a proportionally higher order of binding to lectins than methyl--D-glucopyranoside. Therefore, our search in the future will be focused on the details of descriptions of the functions of substituted sugars in relation to defining suitability to our proposed actions of lectins in the path of carbon, from which we are looking forward to elucidation of the most appropriate competitors, their quantities of application, and when to bind them. Across the broad field of photosynthetic ecosystem management, there exist numerous lectins, each variant with an array of binding characteristics that, hereafter, will further elucidate a competitive path of

Aubert, S., Choler, P., Pratt, J., Douzet, R., Gout, E. & Bligny, R. 2004. Methyl-β-D-

 resonance. *Journal of Experimental Botany* 55(406): 2179–2189 Benson, A. A. 2002a. Paving the Path. *Annual Review of Plant Biology* 53: 1-25.

 glucopyranoside in higher plants: accumulation and intracellular localization in *Geum montanum* L. leaves and in model systems studied by 13C nuclear magnetic

plants as compared to control populations. For example, applications of polyalkylglucopyranose to shoots of radish resulted in significant root enhancements over controls; and, furthermore, applications of polyacylglucopyranoses to roots of corn resulted in significant increases of shoots as compared to controls, all of which were supplemented with identical carrier solutions. Similar to findings of our previous experiments with alkylglycopyranosides and C1 fragments (Nonomura & Benson, 1992; Benson & Nonomura, 2009), polyalkylglycopyranose and polyacylglycopyranose required supplementation with nitrogen for active improvements of growth and our discovery of a ninhydrin-stained product of polyacylglycopyranose indicated incorporation of nitrogen into a highly active metabolite. To our knowledge, little is understood of the structure or functions of the metabolites of polyalkylglycopyranoses and polyacylglycopyranoses, however, a metabolized fraction of alkylglycopyranosides similarly stained with ninhydrin (Biel et al., 2010). These nitrogenous metabolites may be related to sugar-conjugated plant growth regulators (SPGRs) and in consideration of known pathways to conjugation and content of as much as 100 mM SPGRs in plants, Nonomura et al. 2011 reported significant growth enhancements from treatments with SPGRs, including 0.3 mM N6-benzyladenine glucoside. Therefore, it may be useful to undertake similar investigations of polyacylglycopyranoses in the presence of various soluble nutrients to further elucidate roles of their metabolites (Nonomura and Benson, 1992), understand involvements of molecular networks regulated by carbon and nitrogen (Nonomura et al., 2011), and determine the nature of manganese requirement (Benson et al., 2009) for their potential and for their capacity as vehicles of crop improvement to be realized. Ammoniacal nitrogen sources, such as for example, ammonium sulfate, are substrates for NADPH:Cytochromes P450 reductase, and as such, may suggest a potential direction for investigation of the involvements of the Cytochromes P450, an offering of a tantalizing area for future research. Additionally, other pathways, such as those of site-directed alkylation and transmembrane transporters, have been shown to involve glycopyranosides (Nonomura et al., 2011) and, as it pertains throughout the eukaryotes, we suggest looking into similar models for photosynthetic organisms that are expanded to include polyalkylglucopyranoses (Kaback et al., 2007).

Inasmuch as the development and tracking of appropriate probes in our future investigations will most certainly shed light on the mode of activity of substituted sugars, the determination of their functions in diverse complexes and the elucidation of their mechanisms remain a goal. Thus, upon consideration of the accumulated results of our recent series of experiments, we have sought a system that involves competitive binding of sugars that would be both abundant and ubiquitous. That is, the system should occur in C3 and C4 plants because we have found that Canola and corn respond to treatments of MeG and TAG. Moreover, we have been looking for a system that could bind -D-glycopyranosides and β-D-glycopyranosides. Upon careful examination, we found that plant lectins possess just these suitable features. In plants, up to a quarter of the protein content of seeds may be attributable to lectins as well as up to ten percent of the protein content of leaves, but even with such prominence, it had been held that vacuolar lectins served no endogenous role in plants (Lannoo et al., 2007). Notably, a number of lectins have been structurally defined to the extent that it has been generally established that manganese is required for competitive binding of methyl--Dglucopyranoside and glucose to occur (see, for example, Brewer et al., 1973), fulfilling yet another requirement of the system. Furthermore, based on Biel et al. 2010 showing that radiolabeled methylglucopyranoside is transported into leaves intact with a large portion of label in the protein fraction, we expect that our application of 309 mM methylglucopyranoside

plants as compared to control populations. For example, applications of polyalkylglucopyranose to shoots of radish resulted in significant root enhancements over controls; and, furthermore, applications of polyacylglucopyranoses to roots of corn resulted in significant increases of shoots as compared to controls, all of which were supplemented with identical carrier solutions. Similar to findings of our previous experiments with alkylglycopyranosides and C1 fragments (Nonomura & Benson, 1992; Benson & Nonomura, 2009), polyalkylglycopyranose and polyacylglycopyranose required supplementation with nitrogen for active improvements of growth and our discovery of a ninhydrin-stained product of polyacylglycopyranose indicated incorporation of nitrogen into a highly active metabolite. To our knowledge, little is understood of the structure or functions of the metabolites of polyalkylglycopyranoses and polyacylglycopyranoses, however, a metabolized fraction of alkylglycopyranosides similarly stained with ninhydrin (Biel et al., 2010). These nitrogenous metabolites may be related to sugar-conjugated plant growth regulators (SPGRs) and in consideration of known pathways to conjugation and content of as much as 100 mM SPGRs in plants, Nonomura et al. 2011 reported significant growth enhancements from treatments with SPGRs, including 0.3 mM N6-benzyladenine glucoside. Therefore, it may be useful to undertake similar investigations of polyacylglycopyranoses in the presence of various soluble nutrients to further elucidate roles of their metabolites (Nonomura and Benson, 1992), understand involvements of molecular networks regulated by carbon and nitrogen (Nonomura et al., 2011), and determine the nature of manganese requirement (Benson et al., 2009) for their potential and for their capacity as vehicles of crop improvement to be realized. Ammoniacal nitrogen sources, such as for example, ammonium sulfate, are substrates for NADPH:Cytochromes P450 reductase, and as such, may suggest a potential direction for investigation of the involvements of the Cytochromes P450, an offering of a tantalizing area for future research. Additionally, other pathways, such as those of site-directed alkylation and transmembrane transporters, have been shown to involve glycopyranosides (Nonomura et al., 2011) and, as it pertains throughout the eukaryotes, we suggest looking into similar models for photosynthetic organisms that are expanded to

Inasmuch as the development and tracking of appropriate probes in our future investigations will most certainly shed light on the mode of activity of substituted sugars, the determination of their functions in diverse complexes and the elucidation of their mechanisms remain a goal. Thus, upon consideration of the accumulated results of our recent series of experiments, we have sought a system that involves competitive binding of sugars that would be both abundant and ubiquitous. That is, the system should occur in C3 and C4 plants because we have found that Canola and corn respond to treatments of MeG and TAG. Moreover, we have been looking for a system that could bind -D-glycopyranosides and β-D-glycopyranosides. Upon careful examination, we found that plant lectins possess just these suitable features. In plants, up to a quarter of the protein content of seeds may be attributable to lectins as well as up to ten percent of the protein content of leaves, but even with such prominence, it had been held that vacuolar lectins served no endogenous role in plants (Lannoo et al., 2007). Notably, a number of lectins have been structurally defined to the extent that it has been generally established that manganese is required for competitive binding of methyl--Dglucopyranoside and glucose to occur (see, for example, Brewer et al., 1973), fulfilling yet another requirement of the system. Furthermore, based on Biel et al. 2010 showing that radiolabeled methylglucopyranoside is transported into leaves intact with a large portion of label in the protein fraction, we expect that our application of 309 mM methylglucopyranoside

include polyalkylglucopyranoses (Kaback et al., 2007).

to a shoot (Fig. 5) should be sufficient to saturate the system. Therefore, we propose that under conditions in which the cellular sugar concentration of a plant is diminished, chemical competition against substituted sugars acts to release sugar from lectin—and this is an essential process to sustain viability. Also, this act of competitive binding by lectins may naturally displace sugars on a regular basis allowing energy to be reapportioned for growth resulting from metabolism of the freed sugars. For example, we may assume that in the field, the concentration of methyl--D-glucopyranoside remains nearly constant in the plant (Aubert et al., 2004) and as a result of midday photorespiratory depletion of the concentration of glucose, competition for binding to lectin by the methyl--D-glucopyranoside arises and glucose is released. To an extent, the timely release of this free glucose may mitigate the effects of any impoverishment of glucose. Afterwards, under conditions more conducive to photosynthesis, perhaps later in the afternoon and morning, critical concentrations of glucose are rebuilt to sufficiently high levels that a surfeit of glucose outcompetes methyl--Dglucopyranoside, causing the substituted sugar to be displaced, while glucose wins by binding to the lectin. This cycle repeats itself on a daily basis, releasing sugar at each lengthy deprivation event, followed by the capture of fresh sugar upon resuming photosynthesis. Indeed, Nature's response to major environmental stimuli by means of chemical competition is well known. For example, photosynthesis turns to photorespiration strictly as a result of oxygen outcompeting carbon dioxide for binding to Rubisco. In our case, the higher the quantity of lectins residing in the plant, the more capable it may be of capturing and releasing sugars to endure prolonged periods of photorespiration. In contrast, when exogenous chemical competitors for binding sites on lectins are applied to plants, especially by the input of substrates, such as methyl--D-glucopyranoside, that do not naturally occur in plants, the duration of the effect may be substantially extended precisely because such foreign compounds may be selected for competitive advantages. Therefore, responses to treatments with substituted sugars must be carefully measured against the conformation of binding sites, biochemical structure, and their orders of preferences for prospective sugars. From another perspective, empirically formulated dosages of crops may possibly reflect the content and binding determinations of major lectins in a cultivar. For example, in the present investigation, our hypothesis is that the relatively low concentrations of tetraacetylglucopyranose required for the improved growth response of treated plants over controls evidently suggests a proportionally higher order of binding to lectins than methyl--D-glucopyranoside. Therefore, our search in the future will be focused on the details of descriptions of the functions of substituted sugars in relation to defining suitability to our proposed actions of lectins in the path of carbon, from which we are looking forward to elucidation of the most appropriate competitors, their quantities of application, and when to bind them. Across the broad field of photosynthetic ecosystem management, there exist numerous lectins, each variant with an array of binding characteristics that, hereafter, will further elucidate a competitive path of carbon in photosynthesis (Benson, 2002a & b).

#### **5. References**

Aubert, S., Choler, P., Pratt, J., Douzet, R., Gout, E. & Bligny, R. 2004. Methyl-β-D glucopyranoside in higher plants: accumulation and intracellular localization in *Geum montanum* L. leaves and in model systems studied by 13C nuclear magnetic resonance. *Journal of Experimental Botany* 55(406): 2179–2189

Benson, A. A. 2002a. Paving the Path. *Annual Review of Plant Biology* 53: 1-25.

**14** 

*Russia* 

**The Role of C to N Balance in the** 

Vladimir I. Chikov and Svetlana N. Batasheva

**Regulation of Photosynthetic Function** 

*Kazan Institute of Biochemistry and Biophysics of the Russian Academy of Sciences* 

Among the numerous factors affecting plant growth and development one of the most important ones is mineral nutrition. Here, phosphorous and potassium sustain energetics and metabolite transport in the cell. The efficiency of their using can be elevated by repeated circulation of the atoms in conjugated processes. But nitrogen is expended in building plant cell mass and the growth is impossible without continues inflow of new portions of the element. That is why it has a special impact on all physiological processes, including

Data accumulated so far about the interaction between carbon and nitrogen metabolisms in plants indicate its key role in regulation of plant vital functions. C to N balance in a whole plant was shown to take part in regulation of photosynthesis, germination, senescence, morphogenesis (Malamy and Ryan, 2001; Martin et al., 2002; Paul and Foyer, 2001; Paul and

In order to find out the regulatory mechanisms it is necessary to consider all points of contact between carbon and nitrogen metabolisms in plant, and one point that is often overlooked is a significant influence of nitrogen on photo-assimilate transport in plants.

At the first glance, the literature data on the action of nitrogen nutrition on assimilate transport is controversial. For instance, in some publications it is noted that additional nitrogen nutrition delays assimilate export from leaves (Kudryavtsev & Roktanen, 1965; Marty, 1969; Vaklinova et al., 1958; Zav'yalova, 1976), while in others the opposite effect is

Very important here is a period of the plant ontogenetic development, in which plant nitrogen nutrition level changes. In starving for the element juvenile plants, in which the sink-source system consists only of leaves and roots, nitrogen fertilization leads to intensified inflow of photo-assimilates to roots for active metabolization of mineral nitrogen, and these results will probably be interpreted as activation of transport processes by nitrogen. On the other hand, nitrogen supply of older plants with formed sink-source

Pellny, 2003). Nevertheless, mechanisms underlying this regulation are still elusive.

**2. The influence of nitrogen nutrition level on photosynthesis and** 

noted (Anisimov, 1959; Grinenko, 1964; Hartt, 1970; Pristupa & Kursanov, 1957).

**2.1 The influence of nitrogen nutrition on photoassimilate transport** 

**1. Introduction** 

photosynthesis and assimilate transport.

**photo-assimilate partitioning** 


### **The Role of C to N Balance in the Regulation of Photosynthetic Function**

Vladimir I. Chikov and Svetlana N. Batasheva *Kazan Institute of Biochemistry and Biophysics of the Russian Academy of Sciences Russia* 

#### **1. Introduction**

272 Advances in Photosynthesis – Fundamental Aspects

Benson, A. A. 2002b. Following the Path of Carbon in Photosynthesis: A personal story.

Benson, A. A. & Nonomura, A. M. (1992) The Path of Carbon in Photosynthesis: Methanol

 *Photosynthesis 1, Proceedings of the IX International Congress on Photosynthesis,*

Benson, A. A., Nonomura, A. M. & Gerard, V. A. (2009) The Path of Carbon in

Biel, K. Y., Nonomura, A. M., Benson, A. A. & Nishio, J. N. (2010) The Path of Carbon in

Brewer, C. F., Sternlicht, H. , Marcus, D. M. & Grollman, A. P. (1973) Binding of 13C-

Calvin, M. and Benson, A. A. (1948) The Path of Carbon in Photosynthesis, Science, 107: 476. Gout, E., Aubert, S., Bligny, R., Rebeille, F., Nonomura, A., Benson, A. A. & Douce, R.

Hoagland, D. R. and D. I. Arnon. 1950. The Water-Culture Method for Growing Plants

Hyatt, J. A. & Tindall, G. W. (1993) The Intermediacy of Sulfate Esters in Sulfuric Acid Catalyzed Acetylation of Carbohydrates. *Heterocycles* 35(1): 227-234. Kaback, H. R., Dunten, R., Frillingos, S., Venkatesan, P., Kwaw, I. , Zhang, W. & Ermolova,

Ligocka, A., Zbiec, I, Karczmarczyk, S. & Podsialdo, C. (2003) Response of some cultivated

Lannoo, N., Vandenborre, G., Miersch, O., Smagghe, G., Wasternack, C., Peumans, W. J.

Nonomura, A. M. & Benson, A. A. (1992) The Path of Carbon in Photosynthesis: Improved

Nonomura, A. M., Benson, A. A. & Biel, K. Y. (2011) The Path of Carbon in

Wolf, F. R., Nonomura, A. M. & Bassham, J. A. (1985) Growth and branched hydrocarbon

inhibition of glycolic acid accumulation, *in* Murata, N. (ed.), *Research in* 

Photosynthesis. XXV. Plant and Algal Growth Responses to Glycopyranosides,

Photosynthesis. XXVI. Uptake and transport of methylglucopyranoside throughout

Enriched Methyl-D-Glucopyranoside to Concanavalin A as Studied by Carbon Magnetic Resonance. *Proceedings of the National Academy of Sciences, USA* 70 (4):

(2000) Metabolism of methanol in plant cells. Carbon-13 nuclear magnetic

without Soil. California Agricultural Experiment Station Circular 347, The College

http://plantbio.berkeley.edu/newpmb/faculty/arnon/Hoagland\_Arnon\_Solution

N. (2007) Site-directed alkylation and the alternating access model for LacY.

plants to methanol as compared to supplemental irrigation. *Electronic Journal of* 

& Van Damme, Els J. M. (2007) The Jasmonate-Induced Expression of the

crop yields with methanol. *Proceedings of the National Academy of Sciences, USA*

Photosynthesis. XXVII. Sugar-conjugated plant growth regulators enhance

production in a strain of *Botryococcus braunii* (Chlorophyta). *Journal of Phycology*

*Photosynthesis Research* 73: 29-49.

Kluver, Nagoya, Japan*,* P-522.

1007-1011.

.pdf

URL: http://www.ejpau.media.pl

89(20): 9794-9798.

21(3): 388-396.

 URL: http://www.pcp.oxfordjournals.org Nobelprize.org. (2011) The Nobel Prize in Chemistry 1961.

*Journal of Plant Nutrition* 32(7): 1185-1200.

plants. *Journal of Plant Nutrition* 33(6): 902–913.

resonance studies. *Plant Physiology* 123(1): 287-296.

of Agriculture, University of California, Berkeley: 32 pp. URL:

*Proceedings of the National Academy of Sciences, USA* 104: 491-494.

*Polish Agricultural Universities,* Agronomy, Volume 6, Issue 1.

URL: http://nobelprize.org/nobel\_prizes/chemistry/laureates/1961/

general productivity. *Journal of Plant Nutrition* 34:(5): 653—664.

*Nicotiana tabacum* Leaf Lectin. *Plant Cell Physiology* 1–12

Among the numerous factors affecting plant growth and development one of the most important ones is mineral nutrition. Here, phosphorous and potassium sustain energetics and metabolite transport in the cell. The efficiency of their using can be elevated by repeated circulation of the atoms in conjugated processes. But nitrogen is expended in building plant cell mass and the growth is impossible without continues inflow of new portions of the element. That is why it has a special impact on all physiological processes, including photosynthesis and assimilate transport.

Data accumulated so far about the interaction between carbon and nitrogen metabolisms in plants indicate its key role in regulation of plant vital functions. C to N balance in a whole plant was shown to take part in regulation of photosynthesis, germination, senescence, morphogenesis (Malamy and Ryan, 2001; Martin et al., 2002; Paul and Foyer, 2001; Paul and Pellny, 2003). Nevertheless, mechanisms underlying this regulation are still elusive.

In order to find out the regulatory mechanisms it is necessary to consider all points of contact between carbon and nitrogen metabolisms in plant, and one point that is often overlooked is a significant influence of nitrogen on photo-assimilate transport in plants.

#### **2. The influence of nitrogen nutrition level on photosynthesis and photo-assimilate partitioning**

#### **2.1 The influence of nitrogen nutrition on photoassimilate transport**

At the first glance, the literature data on the action of nitrogen nutrition on assimilate transport is controversial. For instance, in some publications it is noted that additional nitrogen nutrition delays assimilate export from leaves (Kudryavtsev & Roktanen, 1965; Marty, 1969; Vaklinova et al., 1958; Zav'yalova, 1976), while in others the opposite effect is noted (Anisimov, 1959; Grinenko, 1964; Hartt, 1970; Pristupa & Kursanov, 1957).

Very important here is a period of the plant ontogenetic development, in which plant nitrogen nutrition level changes. In starving for the element juvenile plants, in which the sink-source system consists only of leaves and roots, nitrogen fertilization leads to intensified inflow of photo-assimilates to roots for active metabolization of mineral nitrogen, and these results will probably be interpreted as activation of transport processes by nitrogen. On the other hand, nitrogen supply of older plants with formed sink-source

The Role of C to N Balance in the Regulation of Photosynthetic Function 275

nitrogen form) the next day after plant fertilization. The experiments were performed with the upper leaf of wheat plants, grown at moderate level of full mineral nutrition till the stage of kariopsides formation. At this time the export function of the upper leaf is most expressed. On the eve of the experiment, plants were watered with solutions of calcium nitrate or urea with concentrations calculated so to be equal to 2 grams of N per pot. Next day from 10 to 12 a.m. the flag leaf was exposed to 14CO2 for 2 min and fixed to study PCM. To establish specialities of the influence of nitrogen fertilization on photorespiratory glycolate pathway PCM was investigated under two CO2 concentrations (0.03 and 0.3%) and two O2 concentration (21% and 1%). Notably, the gas composition in the leaf photosynthetic chamber was altered only for the period of 14CO2 assimilation by

The experiment showed significant differences in the action of oxidized and reduced N on PCM (Table 3). The influence of urea and nitrates on PCM had some common features as well as distinct ones. Irrespective of the form of N used, the introduction of 14C into phosphorous esters of sugars decreased and into malate, aspartate and alanine increased, that implied diminished phosphoglyceric acid (PGA) reduction to sugar phosphates and its enhanced non-reductive metabolism. Additionally, in plants fertilized with nitrates, the

> Phosphorus esters of sugars

Serine, glycine, glycolate

Alanine, malate, aspartate

formation of glycolate pathway products (serine, glycine, glycolate) increased.

Free sugars

Control 680 42.2 ± 1.0 27.6 ± 1.1 11.8 ± 0.1 7.7 ± 0.4 Urea 740 44.5 ± 0.8 24.1 ± 0.9 11.3 ± 1.0 12.2 ± 1.1 Nitrate 260 40.1 ± 1.5 16.3 ± 2.6 18.9 ± 2.2 11.8 ± 0.4

Control 990 44.9 ± 1.0 28.9 ± 1.4 4.0 ± 0.6 13.2 ± 0.6 Urea 1430 47.3 ± 1.1 14.1 ± 2.5 2.4 ± 0.4 22.0 ± 0.8 Nitrate 1030 46.8 ± 2.0 14.2 ± 1.6 9.6 ± 0.5 22.5 ± 1.0

distribution among some labeled products after 2 min 14CO2 assimilation (% radioactivity of

According to common knowledge, glycolate is synthesized from ribulose-1,5-bisphosphate in the RuBP-oxygenase reaction of photosynthesis which requires oxygen, and the oxygenase reaction competes with carboxylase one for RuBP. All this occurs in the joint active centre of the Rubisco enzyme. Thus, O2 and CO2 compete for the binding of RuBP in the reaction center, and to lower the activity of oxygenase reaction of Rubisco one needs to decrease the concentration of O2 and increase the concentration of CO2. Such a situation was created in the experiment: in the period of 14CO2 assimilation a gas mixture of oxygen (1%) and carbon dioxide (0.3%) was delivered into the photosynthetic leaf chamber containing a

As a result, 14C incorporation into the products of glycolate metabolism relatively (%) reduced in all plants; however, in nitrate plants it was the least expressed. If one calculates the formation of glycolate pathway products in unit mass of fixed carbon dioxide (µg CO2

Table 3. The influence of different CO2 and O2 concentrations and N forms on 14C

water-ethanol soluble fraction) in wheat (Chikov & Bakirova, 1999)

Photosynthesis intensity (µg СО2m -2s-1)

treated leaf at the same concentration of 14CO2.

the leaf.

Treatment

21% О2; 0.03% СО<sup>2</sup>

1% О2; 0.3% СО<sup>2</sup>


relationships usually results in relative inhibition of assimilate export from source leaves to sink organs (Table 1).

Table 1. The influence of pre-planting nitrogen fertilization of soft wheat cv. Saratovskaya 29 on 14C-photoassimilate export from leaves and their inflow into ears (% radioactivity of above ground plant part) in the milky stage of grain development (Tarchevsky et al., 1973)

The characteristic feature of photosynthetic carbon metabolism in plants grown on increased nitrogen background is a lowered ratio of labeled sucrose to hexoses (Table 2). Using our own method of extraction of the labeled photosynthetic products from the apoplast (Chikov et al., 2001), it was established that at increasing nitrate nutrition level the ratio of labeled sucrose to hexoses decreases in the apoplast rather than in mesophyll cells (Table 2). Thus, the enhanced sucrose hydrolysis is a property of the apoplastic compartment.


Table 2. The influence of nitrate nutrition on the ratio of labeled sucrose to hexoses in the leaves of flax plants (Chikov et al., 2001)

Delayed assimilate export from leaves of plants fertilized with nitrogen is believed to be linked with intensified synthesis of nitrogen containing compounds and diverting to the process carbon fixed in photosynthesis with lesser formation of transport photosynthetic products, i.e. sugars (Champigny & Foyer, 1992). The data on increased sucrose hydrolysis in the apoplast which is an intermediate in the sucrose transfer to the phloem suggest that the reason of lowered export lies not in the shortage of sugars but in the mechanism itself of their transport from leaves.

#### **2.2 The influence of nitrogen nutrition on plant photosynthetic carbon metabolism (PCM)**

As a rule, plants grown at various levels of nitrogen differ dramatically in their morphological features such as sizes and densities of leaf blades, photosynthetic pigment contents, the ratios of above-ground part to root weights, etc.; and all this confuses interpretation of data on PCM. That is why to identify differences between the influence of different nitrogen forms it is desirable to assess physiological and biochemical characteristics before pronounced visual changes of plants become obvious. Bearing all this in mind, we compared the influence of nitrate on PCM to that of urea (as a reduced

relationships usually results in relative inhibition of assimilate export from source leaves to

Treatment Radioactivity of a plant part, %

Control 36.1 ± 4.1 51.8 ± 6.2 Nitrogen 50.6 ± 3.8 28.2 ± 1.7 % to control 140 54

Table 1. The influence of pre-planting nitrogen fertilization of soft wheat cv. Saratovskaya 29 on 14C-photoassimilate export from leaves and their inflow into ears (% radioactivity of above ground plant part) in the milky stage of grain development (Tarchevsky et al., 1973) The characteristic feature of photosynthetic carbon metabolism in plants grown on increased nitrogen background is a lowered ratio of labeled sucrose to hexoses (Table 2). Using our own method of extraction of the labeled photosynthetic products from the apoplast (Chikov et al., 2001), it was established that at increasing nitrate nutrition level the ratio of labeled sucrose to hexoses decreases in the apoplast rather than in mesophyll cells (Table 2). Thus,

the enhanced sucrose hydrolysis is a property of the apoplastic compartment.

Treatment Upper part 14C-donor part

(Non-fertilized) 15.0 ± 0.15 149.5 ± 25.0 16.9 ± 7.0 128.0 ± 20.0 NO3-fertilized 11.6 ± 3.4 36.7 ± 6.7 13.6 ± 0.7 38.1 ± 3.8 control/ NO3 1.29 4.07 1.24 3.36 Table 2. The influence of nitrate nutrition on the ratio of labeled sucrose to hexoses in the

Delayed assimilate export from leaves of plants fertilized with nitrogen is believed to be linked with intensified synthesis of nitrogen containing compounds and diverting to the process carbon fixed in photosynthesis with lesser formation of transport photosynthetic products, i.e. sugars (Champigny & Foyer, 1992). The data on increased sucrose hydrolysis in the apoplast which is an intermediate in the sucrose transfer to the phloem suggest that the reason of lowered export lies not in the shortage of sugars but in the mechanism itself of

**2.2 The influence of nitrogen nutrition on plant photosynthetic carbon metabolism** 

As a rule, plants grown at various levels of nitrogen differ dramatically in their morphological features such as sizes and densities of leaf blades, photosynthetic pigment contents, the ratios of above-ground part to root weights, etc.; and all this confuses interpretation of data on PCM. That is why to identify differences between the influence of different nitrogen forms it is desirable to assess physiological and biochemical characteristics before pronounced visual changes of plants become obvious. Bearing all this in mind, we compared the influence of nitrate on PCM to that of urea (as a reduced

Leaf Ear

leaves apoplast leaves apoplast

sink organs (Table 1).

Control

their transport from leaves.

**(PCM)** 

leaves of flax plants (Chikov et al., 2001)

nitrogen form) the next day after plant fertilization. The experiments were performed with the upper leaf of wheat plants, grown at moderate level of full mineral nutrition till the stage of kariopsides formation. At this time the export function of the upper leaf is most expressed. On the eve of the experiment, plants were watered with solutions of calcium nitrate or urea with concentrations calculated so to be equal to 2 grams of N per pot. Next day from 10 to 12 a.m. the flag leaf was exposed to 14CO2 for 2 min and fixed to study PCM. To establish specialities of the influence of nitrogen fertilization on photorespiratory glycolate pathway PCM was investigated under two CO2 concentrations (0.03 and 0.3%) and two O2 concentration (21% and 1%). Notably, the gas composition in the leaf photosynthetic chamber was altered only for the period of 14CO2 assimilation by the leaf.

The experiment showed significant differences in the action of oxidized and reduced N on PCM (Table 3). The influence of urea and nitrates on PCM had some common features as well as distinct ones. Irrespective of the form of N used, the introduction of 14C into phosphorous esters of sugars decreased and into malate, aspartate and alanine increased, that implied diminished phosphoglyceric acid (PGA) reduction to sugar phosphates and its enhanced non-reductive metabolism. Additionally, in plants fertilized with nitrates, the formation of glycolate pathway products (serine, glycine, glycolate) increased.


Table 3. The influence of different CO2 and O2 concentrations and N forms on 14C distribution among some labeled products after 2 min 14CO2 assimilation (% radioactivity of water-ethanol soluble fraction) in wheat (Chikov & Bakirova, 1999)

According to common knowledge, glycolate is synthesized from ribulose-1,5-bisphosphate in the RuBP-oxygenase reaction of photosynthesis which requires oxygen, and the oxygenase reaction competes with carboxylase one for RuBP. All this occurs in the joint active centre of the Rubisco enzyme. Thus, O2 and CO2 compete for the binding of RuBP in the reaction center, and to lower the activity of oxygenase reaction of Rubisco one needs to decrease the concentration of O2 and increase the concentration of CO2. Such a situation was created in the experiment: in the period of 14CO2 assimilation a gas mixture of oxygen (1%) and carbon dioxide (0.3%) was delivered into the photosynthetic leaf chamber containing a treated leaf at the same concentration of 14CO2.

As a result, 14C incorporation into the products of glycolate metabolism relatively (%) reduced in all plants; however, in nitrate plants it was the least expressed. If one calculates the formation of glycolate pathway products in unit mass of fixed carbon dioxide (µg CO2

The Role of C to N Balance in the Regulation of Photosynthetic Function 277

also, alanine and aspartate, resulting from non-reducing PGA metabolism, can be used for the protein synthesis when the leaf recommences its growth through expansion. In our experiments with the removal of fruit elements from a cotton plant a leaf which was a source of assimilates could augment its size as many as 1.5-2 times in 10-15 days after the

The return of the carbon of glycolate into the Calvin cycle is carried along the chain 2 glycolate → 2 glyoxylate + NH2 → 2 glycine → serine + NH2 → oxyglycerate + NH2 →

If the products of the glycolate pathway do not return to the Calvin cycle but accumulate and used in synthetic processes then the carbon will come to glycerate in smaller quantities. In this case the ratio of radioactivities of glycolate+glycine+serine to that of glycerte must

This conclusion was confirmed in studies of the kinetics of 14C introduction into those compounds. In the fertilized plants labeled carbon entered such compounds as glycolate, glycine, serine and alanine to a greater extent than in the controls, while glycerate, conversely, to a lesser extent (Fig. 2). As a result, the ratio of glycolate+glycine+serine/glycerte increased several-fold. The kinetic curves show that these differences need certain time (not less than 30 s) to reveal oneselves, that supports the metabolism of these substances in the direction of

It is interesting that under conditions of hindered assimilate export from leaves after removal of sink organs a similar kinetics of 14C introduction from 14CO2 into glycolate and glycine was found at ambient CO2 concentration (Chikov, 1987). The results were in good agreement with the idea of transketolase mechanism of glycolate formation, because in experimental plants labeled carbon from 14CO2 appeared in glycolate earlier than that occurred in control ones. Characteristically, at saturable CO2 concentration the kinetics curves for control and experimental plants were the same and both resembled the curve for experimental plants under ambient CO2 (Chikov, 1987). The data indicate again that under the circumstances of slowed assimilate export and saturable CO2 concentration glycolate and the products of its

Thus, inhibition of assimilate export from the leaf and an increase of the Warburg effect in leaf photosynthetic gas-exchange, observed in some cases (Chikov, 1987), are accompanied by enhanced CO2 metabolism through the glycolate pathway, but with increasing potion of glycolate formed in reactions not related with RuBP-oxidase one, most likely, in transketolase reaction of the Calvin cycle. This mechanism probably works also at delay of assimilate export from leaves under elevated level of plant nitrate nutrition. In the case of nitrate presence in the leaf, an oxidizer required to perform the transketolase reaction could

In both cases growth processes become enhanced in the source leaf, for which early photosynthetic products in the form of amino acids alanine, serine, glycine and aspartate are used. It should be mentioned that these four amino acids (of 20 proteinogenic ones) represent over 30% (by number, not by weight) of fraction 1 protein (the main chloroplastic protein). Furthermore, the suppression of sugar export can enhance the metabolism of phosphoerythroses through the shikimate pathway with the formation of aromatic (see Fig. 1) amino acids (tyrosine, phenylalanine, tryptophan) and then hormonal substances (auxins), creating an additional substrate base for metabolism rearrangement in the leaf (and whole plant). So, a key factor for triggering metabolism readjustment in the leaf (the plant)

metabolism are derived from sugar phosphates that are predecessors of RuBP.

appear from NO2- reduction in the chloroplast ETC (see Fig. 1).

is inhibition of sugar outflow from leaves.

exposure (Chikov, 1987).

glycerate + ATP → PGA.

glycolate → glycine → serine → glycerate.

increase.

m-2 s-1) he will find that in control and urea fed plants the formation of these products lessened twofold while in nitrate plants it augmented twofold.

Fig. 1. The scheme of photosynthetic carbon metabolism regulation. ETC – electron transport chain, FD – ferredoxin, PES – phosphorous esters of sugars, PGA – phosphoglyceric acid, PSI – photosystem I, PSII – photosystem II, RuBP – ribulose 1,5-bisphosphate, TKR – transketolase reaction, X – unknown oxidizer, double line – intensification of a process

Based on this data the following conclusions can be made. Firstly, the suppression of RuBPoxigenase activity by low O2 and elevated CO2 occurs only without nitrates. When nitrates are present, the formation of glycolate and its metabolites even enhances. Secondly, as the amount of CO2 fixation product (PGA) increases at the saturable CO2 concentration its nonreductive metabolism with appearance of alanine, malate and aspartate rises. Apparently, the latter is partly associated with rising CO2 fixation in dark type-reaction catalyzed by phosphoenolpyruvate (PEP) carboxylaze (see the scheme in fig. 1).

The active production of glycolate under conditions suppressing RuBP-oxydenase reaction suggests that in the presence of nitrates glycolate is formed from other (not RuBP) phosphorous esters of sugars (PES) in transketolase reactions. Transketolase reaction requires superoxide radical (Asami & Akasava, 1977), produced in the Mehler reaction (Takabe et al., 1980).

The enhanced oxidation of PES may account for their decreased radioactivity and overall elevated non-carbohydrate tendency of photosynthesis in nitrate fed plants. The possibility of such a mechanism was declared long ago (Asami, Akasava, 1977). One of the probable mechanisms of transketolaze reaction (TKR) activation and glycolate formation from sugar di-phosphates upstream of RuBP might be an inhibited activity of phosphatases of fructoseand sedoheptulose-diphosphates (Heldt et al., 1978).

For fully grown leaves, which are typical exporters of assimilates, glycolate metabolism mainly terminates with formation of PGA (see the scheme in Fig. 1), that returns back to the Calvin cycle and is reduced to sugars. In the case of hindered sugar export the glycolate pathway becomes less closed. Glycine and serine, amino acids derived from glycolate, and

m-2 s-1) he will find that in control and urea fed plants the formation of these products

Fig. 1. The scheme of photosynthetic carbon metabolism regulation. ETC – electron transport chain, FD – ferredoxin, PES – phosphorous esters of sugars, PGA – phosphoglyceric acid, PSI – photosystem I, PSII – photosystem II, RuBP – ribulose 1,5-bisphosphate, TKR – transketolase reaction, X – unknown oxidizer, double line – intensification of a process

Based on this data the following conclusions can be made. Firstly, the suppression of RuBPoxigenase activity by low O2 and elevated CO2 occurs only without nitrates. When nitrates are present, the formation of glycolate and its metabolites even enhances. Secondly, as the amount of CO2 fixation product (PGA) increases at the saturable CO2 concentration its nonreductive metabolism with appearance of alanine, malate and aspartate rises. Apparently, the latter is partly associated with rising CO2 fixation in dark type-reaction catalyzed by

The active production of glycolate under conditions suppressing RuBP-oxydenase reaction suggests that in the presence of nitrates glycolate is formed from other (not RuBP) phosphorous esters of sugars (PES) in transketolase reactions. Transketolase reaction requires superoxide radical (Asami & Akasava, 1977), produced in the Mehler reaction

The enhanced oxidation of PES may account for their decreased radioactivity and overall elevated non-carbohydrate tendency of photosynthesis in nitrate fed plants. The possibility of such a mechanism was declared long ago (Asami, Akasava, 1977). One of the probable mechanisms of transketolaze reaction (TKR) activation and glycolate formation from sugar di-phosphates upstream of RuBP might be an inhibited activity of phosphatases of fructose-

For fully grown leaves, which are typical exporters of assimilates, glycolate metabolism mainly terminates with formation of PGA (see the scheme in Fig. 1), that returns back to the Calvin cycle and is reduced to sugars. In the case of hindered sugar export the glycolate pathway becomes less closed. Glycine and serine, amino acids derived from glycolate, and

phosphoenolpyruvate (PEP) carboxylaze (see the scheme in fig. 1).

and sedoheptulose-diphosphates (Heldt et al., 1978).

(Takabe et al., 1980).

lessened twofold while in nitrate plants it augmented twofold.

also, alanine and aspartate, resulting from non-reducing PGA metabolism, can be used for the protein synthesis when the leaf recommences its growth through expansion. In our experiments with the removal of fruit elements from a cotton plant a leaf which was a source of assimilates could augment its size as many as 1.5-2 times in 10-15 days after the exposure (Chikov, 1987).

The return of the carbon of glycolate into the Calvin cycle is carried along the chain 2 glycolate → 2 glyoxylate + NH2 → 2 glycine → serine + NH2 → oxyglycerate + NH2 → glycerate + ATP → PGA.

If the products of the glycolate pathway do not return to the Calvin cycle but accumulate and used in synthetic processes then the carbon will come to glycerate in smaller quantities. In this case the ratio of radioactivities of glycolate+glycine+serine to that of glycerte must increase.

This conclusion was confirmed in studies of the kinetics of 14C introduction into those compounds. In the fertilized plants labeled carbon entered such compounds as glycolate, glycine, serine and alanine to a greater extent than in the controls, while glycerate, conversely, to a lesser extent (Fig. 2). As a result, the ratio of glycolate+glycine+serine/glycerte increased several-fold. The kinetic curves show that these differences need certain time (not less than 30 s) to reveal oneselves, that supports the metabolism of these substances in the direction of glycolate → glycine → serine → glycerate.

It is interesting that under conditions of hindered assimilate export from leaves after removal of sink organs a similar kinetics of 14C introduction from 14CO2 into glycolate and glycine was found at ambient CO2 concentration (Chikov, 1987). The results were in good agreement with the idea of transketolase mechanism of glycolate formation, because in experimental plants labeled carbon from 14CO2 appeared in glycolate earlier than that occurred in control ones. Characteristically, at saturable CO2 concentration the kinetics curves for control and experimental plants were the same and both resembled the curve for experimental plants under ambient CO2 (Chikov, 1987). The data indicate again that under the circumstances of slowed assimilate export and saturable CO2 concentration glycolate and the products of its metabolism are derived from sugar phosphates that are predecessors of RuBP.

Thus, inhibition of assimilate export from the leaf and an increase of the Warburg effect in leaf photosynthetic gas-exchange, observed in some cases (Chikov, 1987), are accompanied by enhanced CO2 metabolism through the glycolate pathway, but with increasing potion of glycolate formed in reactions not related with RuBP-oxidase one, most likely, in transketolase reaction of the Calvin cycle. This mechanism probably works also at delay of assimilate export from leaves under elevated level of plant nitrate nutrition. In the case of nitrate presence in the leaf, an oxidizer required to perform the transketolase reaction could appear from NO2- reduction in the chloroplast ETC (see Fig. 1).

In both cases growth processes become enhanced in the source leaf, for which early photosynthetic products in the form of amino acids alanine, serine, glycine and aspartate are used. It should be mentioned that these four amino acids (of 20 proteinogenic ones) represent over 30% (by number, not by weight) of fraction 1 protein (the main chloroplastic protein).

Furthermore, the suppression of sugar export can enhance the metabolism of phosphoerythroses through the shikimate pathway with the formation of aromatic (see Fig. 1) amino acids (tyrosine, phenylalanine, tryptophan) and then hormonal substances (auxins), creating an additional substrate base for metabolism rearrangement in the leaf (and whole plant). So, a key factor for triggering metabolism readjustment in the leaf (the plant) is inhibition of sugar outflow from leaves.

The Role of C to N Balance in the Regulation of Photosynthetic Function 279

Fig. 3. The influence of nitrate or urea fertilization of wheat plants on the dynamics of 14C

**3. The influence of different N forms in the transpiration water stream on photosynthetic carbon metabolism and assimilate transport from leaves** 

conditions of direct sunlight without demonstrating any visible lesions.

To study the immediate influence of increased nitrogen nutrition level on photosynthesis and assimilate transport we have designed a special device (Fig. 4) for introduction of solutions or water into a shoot under the pressure of 104 Pa, corresponding to a normal root pressure. Preliminary studies had shown that a shoot could survive for several days under

1 – a mandrel for shoot fixation; 2 – a silicone tube with a solution fed into the apoplast; 3 – a bath with water and a monostat (4) drowned in it to the depth of 1 m to imitate root pressure; C –a compressor

For photosynthesis investigations a central shoot part was exposed to 14CO2 using a photosynthetic chamber. This allowed not only to determine 14C distribution among the labeled products of 2-3 min photosynthesis, but also to assess the character of allocation of

Fig. 4. The scheme of solution introduction into an isolated flax shoot

content in sucrose after 1 min 14CO2 assimilation by leaves

**3.1 Methodical peculiarities of the procedure** 

Fig. 2. The kinetics of 14C introduction into some products of photosynthesis (% radioactivity of water-ethanol soluble fraction) in leaves of wheat plants cv. Moskovskaya-35. 1 – control; 2 - fertilized with 2 g of nitrogen as Ca(NO3)2

#### **2.3 The influence of nitrogen supply on the dynamics of post-photosynthetic conversion of 14C-products of photosynthesis**

The actions of oxidized and reduced N vary not only in primary photosynthetic products formation but in their subsequent metabolisation to the end transport compound, sucrose.

After short exposure of leaves to 14CO2, 14C content in sucrose was reduced under N treatments, irrespectively of the N form, which seemed to be due to intensive formation of non-carbohydrate substances (organic and amino acids). Then in the next 30 min 14C accumulated in the end photosynthetic product, sucrose, in all plant variants (Fig. 3). However in plants fed with nitrogen this accumulation occurred at higher rates than in control, and as a result, 14C content in sucrose in both N fed variants almost equaled and approached the control level.

From this moment sucrose radioactivity began to decrease in all variants. During 1.5 h it reduced by 75% in control and by 45% in plants fed with urea. In the next 20 h sucrose radioactivity in the plants was running down steadily to the level of 4-5% of the maximal value. Unlike these two variants, nitrate plants exhibited even descent of sucrose radioactivity from 2 h point and during the next 20 h not reaching 30% of the maximal level. All this evidenced significant distinction of the sucrose transport in nitrate plants from those both in control and urea fed ones.

In summary, at water or urea feeding labeled primary photosynthetic products are quite successfully converted into sucrose with its subsequent export in the post-photosynthetic period, while in nitrate fed plants labeled assimilates are piled up as sucrose which remains in the source leaves for a long time period.

To reveal a mechanism of assimilate export delay from leaves special model experiments were performed, where nitrate or urea solutions were injected into isolated plant shoots.

Fig. 2. The kinetics of 14C introduction into some products of photosynthesis (% radioactivity of water-ethanol soluble fraction) in leaves of wheat plants cv. Moskovskaya-35. 1 – control;

The actions of oxidized and reduced N vary not only in primary photosynthetic products formation but in their subsequent metabolisation to the end transport compound, sucrose. After short exposure of leaves to 14CO2, 14C content in sucrose was reduced under N treatments, irrespectively of the N form, which seemed to be due to intensive formation of non-carbohydrate substances (organic and amino acids). Then in the next 30 min 14C accumulated in the end photosynthetic product, sucrose, in all plant variants (Fig. 3). However in plants fed with nitrogen this accumulation occurred at higher rates than in control, and as a result, 14C content in sucrose in both N fed variants almost equaled and

From this moment sucrose radioactivity began to decrease in all variants. During 1.5 h it reduced by 75% in control and by 45% in plants fed with urea. In the next 20 h sucrose radioactivity in the plants was running down steadily to the level of 4-5% of the maximal value. Unlike these two variants, nitrate plants exhibited even descent of sucrose radioactivity from 2 h point and during the next 20 h not reaching 30% of the maximal level. All this evidenced significant distinction of the sucrose transport in nitrate plants from those

In summary, at water or urea feeding labeled primary photosynthetic products are quite successfully converted into sucrose with its subsequent export in the post-photosynthetic period, while in nitrate fed plants labeled assimilates are piled up as sucrose which remains

To reveal a mechanism of assimilate export delay from leaves special model experiments were performed, where nitrate or urea solutions were injected into isolated plant shoots.

**2.3 The influence of nitrogen supply on the dynamics of post-photosynthetic** 

2 - fertilized with 2 g of nitrogen as Ca(NO3)2

approached the control level.

both in control and urea fed ones.

in the source leaves for a long time period.

**conversion of 14C-products of photosynthesis** 

Fig. 3. The influence of nitrate or urea fertilization of wheat plants on the dynamics of 14C content in sucrose after 1 min 14CO2 assimilation by leaves

#### **3. The influence of different N forms in the transpiration water stream on photosynthetic carbon metabolism and assimilate transport from leaves**

#### **3.1 Methodical peculiarities of the procedure**

To study the immediate influence of increased nitrogen nutrition level on photosynthesis and assimilate transport we have designed a special device (Fig. 4) for introduction of solutions or water into a shoot under the pressure of 104 Pa, corresponding to a normal root pressure. Preliminary studies had shown that a shoot could survive for several days under conditions of direct sunlight without demonstrating any visible lesions.

1 – a mandrel for shoot fixation; 2 – a silicone tube with a solution fed into the apoplast; 3 – a bath with water and a monostat (4) drowned in it to the depth of 1 m to imitate root pressure; C –a compressor

Fig. 4. The scheme of solution introduction into an isolated flax shoot

For photosynthesis investigations a central shoot part was exposed to 14CO2 using a photosynthetic chamber. This allowed not only to determine 14C distribution among the labeled products of 2-3 min photosynthesis, but also to assess the character of allocation of

The Role of C to N Balance in the Regulation of Photosynthetic Function 281

the radioactivity of serine was higher (Table 4). Watering of plants grown in the soil with Ca(NO3)2 solution on the eve of the day of experiment apparently leaves the plants enough time for activation of protein synthesizing systems, utilizing serine. The protein synthesizing systems of plants supplied with nitrates through the transpiration water stream were probably not ready for utilization of ample amounts of newly-formed amino acids and this resulted in 14C accumulation in amino acids (first of all, serine). The speed of nitrate reduction exceeds the flow through the GOGAT-pathway approximately by 25%, leading to reduced nitrogen piling up in the immediate products such as ammonium and glutamine as well as photorespiration metabolites, glycine and serine (Stitt et al., 2002). Why does 14C distribution into sucrose immediately after 14CO2 assimilation decrease under nitrate nutrition of plants? The common explanation of reduction in sucrose synthesis after nitrogen nutrition of plants is the following: at enhanced inflow of nitrogen into the plant photosynthetic products and energy are diverted from sucrose synthesis to the formation of nitrogen-containing compounds (Champigny & Foyer, 1992). However, this explanation does not accord with different actions of oxidized and reduced nitrogen on sucrose production (Batasheva et al., 2007). Undoubtedly, the reduction of nitrates requires more energy spending, but the electron transport in chloroplast is known to have significant plasticity and different types of mitochondrial electron transport are probably take part in the maintenance of necessary ATP/NAD(P)H ratio in the cell (Noctor & Foyer, 1998; 2000). Another reason of decreased sucrose synthesis under nitrate nutrition may be a feedback regulation of photosynthesis resulted from slowdown of sucrose transport from leaves. Injection of nitrate solution into a flax shoot resulted in pronounced response of photosynthetic rate, photosynthetic carbon metabolism and assimilate transport measured in 3 h after 14CO2 assimilation (Batasheva et al., 2007). That is why we decided to study those

Studies of the dynamics of 14C distribution along the shoot have revealed that feeding nitrates to the apoplast resulted in inhibition of assimilate export. In 30 min after 14CO2 assimilation in shoots fed with KNO3, relative content and distribution of 14C outside the 14C-donor part did not virtually differ from those in control (Table 5). In additional 2.5 h of post-photosynthesis, in control plants, 14C relative contents outside the donor part increased with significant rising of 14C content in the lower part (Table 5). In shoots fed with nitrates, 14C content outside the donor part in 2.5 h also increased but in lesser degree than in control, and the overall 14C relative content in the lower shoot part became smaller whereas in the

In our previous experiments it was found that feeding urea solution (0.15% (w/v)) containing the same amount of nitrogen as potassium nitrate solution (0.5% (w/v)), led to almost the same distribution of 14C through the shoot as feeding water (Batasheva et al., 2007). NH4NO3 solution feeding led to lesser inhibition of assimilate export and slightly changed the pattern of 14C partitioning along the shoot compared to those under KNO3

In flax, most 14C-sucrose after being loaded into the phloem terminals in source leaves moves downwards within stem phloem vessels, and 14C content in the plant lower parts gradually raises (Chikov & Bakirova, 2004). During their movement along the stem assimilates can partly escape into the stem apoplast, and long distant transport is controlled by retention and retrieval mechanisms in the phloem (Ayre et al., 2003). The assimilates lost to the apoplast can either be re-loaded back into the phloem (Kühn et al., 1997) or be

changes in more detail.

upper part – greater compared to control.

solution feeding (Batasheva et al., 2007).

labeled photosynthetic products along the shoot to different shoot parts (14C-source leaves, the apex, parts above and below the 14C-source part) in the post-photosynthetic period.

There are two main types of plants differing in phloem loading strategy and transportable photosynthetic products (Lohaus & Fischer, 2002). These are plants with the apoplastic phloem loading strategy in which photoassimilates move outside the plasma membrane of parenchyma cells and are loaded from the apoplast into the companion cells or sieve elements using energy-dependent transporters. In other plants photoassimilates move from assimilating to conducting cells along the system of plasmodesmata without membrane crossing. For our experiments we have chosen flax (*Linum usitatissimum* L.) as a plant with apoplastic phloem loading and a willow-herb (*Chamerion angustifolium* (L.) Holub.) as a symplastic plant.

#### **3.2 The introduction of nitrate solution into an apoplastic plant**

The study of 14CO2 distribution among the labeled products of photosynthesis in flax leaves has shown that nitrate injection into the apoplast had the same effect on metabolism as fertilization of plants with nitrogen via soil. The introduction of nitrates resulted in decreased 14C incorporation into sucrose and the ratio of labeled sucrose to hexoses, and increased 14C distribution into the glycolate pathway products (Table 4).


Table 4. The influence of nitrate and urea feeding into the apoplast on 14C distribution among labeled products in flax source leaves immediately after exposure to 14CO2 (% radioactivity of water-soluble fraction)

As it was indicated above, increased label incorporation into the glycolate pathway products is also characteristic of enhanced soil nitrogen nutrition, however at soil fertilization 14C was mostly incorporated into glycine and glycolate while at direct nitrate feeding into the shoot

labeled photosynthetic products along the shoot to different shoot parts (14C-source leaves, the apex, parts above and below the 14C-source part) in the post-photosynthetic

There are two main types of plants differing in phloem loading strategy and transportable photosynthetic products (Lohaus & Fischer, 2002). These are plants with the apoplastic phloem loading strategy in which photoassimilates move outside the plasma membrane of parenchyma cells and are loaded from the apoplast into the companion cells or sieve elements using energy-dependent transporters. In other plants photoassimilates move from assimilating to conducting cells along the system of plasmodesmata without membrane crossing. For our experiments we have chosen flax (*Linum usitatissimum* L.) as a plant with apoplastic phloem loading and a willow-herb (*Chamerion angustifolium* (L.) Holub.) as a

The study of 14CO2 distribution among the labeled products of photosynthesis in flax leaves has shown that nitrate injection into the apoplast had the same effect on metabolism as fertilization of plants with nitrogen via soil. The introduction of nitrates resulted in decreased 14C incorporation into sucrose and the ratio of labeled sucrose to hexoses, and

(0.5%)

Sucrose 59.2 1.6 52.6 2.5 48.1 2.8 47.0 1.8 56.4 0.9

of sugars 3.2 0.3 2.1 0.8 3.5 0.3 0.3 0.0 2.9 0.8 Hexoses 4.3 0.6 4.0 0.5 6.0 0.5 5.0 0.8 3.5 0.5 Sucrose/hexoses 13.8 13.2 8.0 9.4 16.1 Amino acids 21.1 1.1 29.2 3.1 32.1 3.2 29.4 3.3 24.5 0.8

glycine 1.6 0.2 2.1 0.5 2.6 0.7 3.7 0.4 1.8 0.1 serine 5.7 0.3 14.9 2.5 15.3 1.6 11.8 2.3 5.3 0.3 alanine 11.7 0.7 9.4 0.3 10.8 1.0 10.1 1.0 13.5 0.7 Organic acids 3.6 0.3 6.5 0.9 4.3 0.4 5.9 0.7 6.4 0.2

glycerate 0.8 0.1 4.5 0.9 1.6 0.1 1.7 0.2 0.7 0.1 malate 1.8 0.1 0.9 0.1 1.5 0.3 3.1 0.5 4.8 0.1 Pigments 2.7 0.2 1.2 0.1 2.0 0.2 3.0 0.4 1.5 0.1 Others 5.9 4.4 4 9.4 4.8 Table 4. The influence of nitrate and urea feeding into the apoplast on 14C distribution among labeled products in flax source leaves immediately after exposure to 14CO2

As it was indicated above, increased label incorporation into the glycolate pathway products is also characteristic of enhanced soil nitrogen nutrition, however at soil fertilization 14C was mostly incorporated into glycine and glycolate while at direct nitrate feeding into the shoot

KNO3 (1.5%) Ca(NO3)2 (0.5%)

Urea (2.5%)

**3.2 The introduction of nitrate solution into an apoplastic plant** 

increased 14C distribution into the glycolate pathway products (Table 4).

Compounds Water KNO3

(% radioactivity of water-soluble fraction)

period.

symplastic plant.

Phosphorous esters

including:

including:

the radioactivity of serine was higher (Table 4). Watering of plants grown in the soil with Ca(NO3)2 solution on the eve of the day of experiment apparently leaves the plants enough time for activation of protein synthesizing systems, utilizing serine. The protein synthesizing systems of plants supplied with nitrates through the transpiration water stream were probably not ready for utilization of ample amounts of newly-formed amino acids and this resulted in 14C accumulation in amino acids (first of all, serine). The speed of nitrate reduction exceeds the flow through the GOGAT-pathway approximately by 25%, leading to reduced nitrogen piling up in the immediate products such as ammonium and glutamine as well as photorespiration metabolites, glycine and serine (Stitt et al., 2002).

Why does 14C distribution into sucrose immediately after 14CO2 assimilation decrease under nitrate nutrition of plants? The common explanation of reduction in sucrose synthesis after nitrogen nutrition of plants is the following: at enhanced inflow of nitrogen into the plant photosynthetic products and energy are diverted from sucrose synthesis to the formation of nitrogen-containing compounds (Champigny & Foyer, 1992). However, this explanation does not accord with different actions of oxidized and reduced nitrogen on sucrose production (Batasheva et al., 2007). Undoubtedly, the reduction of nitrates requires more energy spending, but the electron transport in chloroplast is known to have significant plasticity and different types of mitochondrial electron transport are probably take part in the maintenance of necessary ATP/NAD(P)H ratio in the cell (Noctor & Foyer, 1998; 2000).

Another reason of decreased sucrose synthesis under nitrate nutrition may be a feedback regulation of photosynthesis resulted from slowdown of sucrose transport from leaves.

Injection of nitrate solution into a flax shoot resulted in pronounced response of photosynthetic rate, photosynthetic carbon metabolism and assimilate transport measured in 3 h after 14CO2 assimilation (Batasheva et al., 2007). That is why we decided to study those changes in more detail.

Studies of the dynamics of 14C distribution along the shoot have revealed that feeding nitrates to the apoplast resulted in inhibition of assimilate export. In 30 min after 14CO2 assimilation in shoots fed with KNO3, relative content and distribution of 14C outside the 14C-donor part did not virtually differ from those in control (Table 5). In additional 2.5 h of post-photosynthesis, in control plants, 14C relative contents outside the donor part increased with significant rising of 14C content in the lower part (Table 5). In shoots fed with nitrates, 14C content outside the donor part in 2.5 h also increased but in lesser degree than in control, and the overall 14C relative content in the lower shoot part became smaller whereas in the upper part – greater compared to control.

In our previous experiments it was found that feeding urea solution (0.15% (w/v)) containing the same amount of nitrogen as potassium nitrate solution (0.5% (w/v)), led to almost the same distribution of 14C through the shoot as feeding water (Batasheva et al., 2007). NH4NO3 solution feeding led to lesser inhibition of assimilate export and slightly changed the pattern of 14C partitioning along the shoot compared to those under KNO3 solution feeding (Batasheva et al., 2007).

In flax, most 14C-sucrose after being loaded into the phloem terminals in source leaves moves downwards within stem phloem vessels, and 14C content in the plant lower parts gradually raises (Chikov & Bakirova, 2004). During their movement along the stem assimilates can partly escape into the stem apoplast, and long distant transport is controlled by retention and retrieval mechanisms in the phloem (Ayre et al., 2003). The assimilates lost to the apoplast can either be re-loaded back into the phloem (Kühn et al., 1997) or be

The Role of C to N Balance in the Regulation of Photosynthetic Function 283

Sucrose 71.6 1.7 50.7 1.6 67.7 0.5 75.0 1.6 Glucose 9.3 1.7 17.2 1.7 7.6 0.3 4.8 0.6 Fructose 4.9 0.5 15.0 1.9 3.3 0.6 4.2 0.4 Glycine 0.9 0.5 1.9 0.6 2.4 0.3 1.0 0.3 Serine 1.7 1.3 2.2 0.2 5.3 1.3 1.4 0.5 Aspartate 0.2 0.0 0.2 0.1 0.2 0.1 0.2 0.1 Glutamate 0.9 0.1 0.5 0.1 0.9 0.2 0.5 0.0 Malate 1.3 0.1 2.5 0.5 1.4 0.3 1.0 0.2 Alanine 1.3 0.1 1.0 0.3 1.2 0.1 1.4 0.1 Pigments 1.9 0.3 2.2 0.5 1.5 0.1 1.6 0.1 Others 6.0 6.6 8.5 8.9 Table 6. The incorporation of 14C into the products of photosynthesis in 30 min and in 3 h

In 3 h of post-photosynthesis in control leaves 14C mostly disappeared from large bundles. In nitrate fed leaves the differences between 14C contents inside and outsides the large bundles became even more contrasty. It seemed likely that in control shoots 14C-sucrose export exceeded its synthesis while in nitrate fed shoots 14C-sucrose synthesis was not compensated by its removing from the leaf. Earlier it was shown that feeding urea (0.15% (w/v)) into the apoplast led to the same picture of 14C-sucrose dynamics as feeding water

Thus, nitrate feeding resulted in graduate accumulation of sucrose in source leaves, which was observed on the background of assimilate export suppression. It is interesting that similar dynamics of sucrose radioactivity changes in mature leaves was observed by Möller and Beck (1992), who studied metabolism of labeled sucrose when unlabelled sucrose was constantly inflowing into the apoplast. In the work high sucrose content in the apoplast led to labeled sucrose accumulation within cells. A question arises why labelled sucrose was not hydrolyzed especially under the conditions of inhibited export appearing when sucrose is

Fig. 6. The influence of water or KNO3 solution (0.5%) feeding on 14С distribution within the donor leaf in 30 min (A – water; B – nitrate) and 3 h (C – water; D – nitrate) after exposure to

fed into the apoplast or observed in our experiments upon nitrate feeding.

14CO2. Darker places correspond to greater 14C contents

Н2О KNO3 30 min 3 h 30 min 3 h

Labelled compounds

after 2.5 min 14CO2 assimilation

(Batasheva et al., 2007).

transported upwards with the transpiration water stream. If assimilates leaked in the apoplast cannot be quickly loaded back into the phloem due to some reason then the portion of the assimilates inflowing into the upper plant part with the transpiration water stream would increase. It seems likely that this is the reason of increased 14C content in the upper parts of the shoots fed with nitrates. Enhancement of sucrose hydrolysis in the apoplast in the presence of nitrates must result in appearing large amounts of labeled hexoses, which can not be loaded into the phloem (Turkina et al., 1999) and are easily carried away upwards with the transpiration stream.


Table 5. The influence of nitrate feeding through the transpiration water stream on 14C distribution among the organs of flax in 30 min and 3 h after 14CO2 assimilation by the middle shoot part (% of whole shoot radioactivity)

As we have shown previously (Batasheva et al., 2007), increased 14C content in the lower part of plants fed with water is not connected with label accumulation due to synthesis of any high-molecular weight substances in this period.

In 30 min after 14CO2 assimilation relative content of 14C-sucrose in source leaves increased both in control and in nitrate fed plants (Table 6). In the following 2.5 h in water fed shoots 14C-sucrose content decreased to lower values compared to those observed immediately after 14CO2 fixation whereas in nitrate fed shoots it continued growing. Thus, in 3 h after 14CO2 assimilation relative 14C content in sucrose in source leaves of the shoots fed with KNO3 rose significantly up to 75%. Relative radioactivity of hexoses practically did not change which resulted in increase of the labeled sucrose/hexoses. The similar picture of 14C-sucrose dynamics was described above for wheat plants fertilized with nitrates (Fig. 3).

Localization of labeled sucrose in leaves was determined by autoradioagraphy of whole leaves taken from 14C-donor part in 30 min and 3 h after 14CO2 assimilation. It turned out that in 30 min after 14CO2 assimilation in water fed shoots the label was concentrated mainly in leaf large veins whereas in nitrate fed shoots – outside them (Fig. 6). It indicates that upon nitrate feeding the accumulation of the labeled assimilates occurred either in mesophyll cells or in minor vein cells from which they were not transported to large veins.

transported upwards with the transpiration water stream. If assimilates leaked in the apoplast cannot be quickly loaded back into the phloem due to some reason then the portion of the assimilates inflowing into the upper plant part with the transpiration water stream would increase. It seems likely that this is the reason of increased 14C content in the upper parts of the shoots fed with nitrates. Enhancement of sucrose hydrolysis in the apoplast in the presence of nitrates must result in appearing large amounts of labeled hexoses, which can not be loaded into the phloem (Turkina et al., 1999) and are easily carried away upwards

30 min 3 h 30 min 3 h

Shoot part Н2О KNO3

Above 14C-donor part 3.5 0.6 5.2 0.9 3.1 0.4 9.8 2.0

top 0.2 0.0 1.0 0.3 0.2 0.0 3.3 1.2 leaves 2.0 0. 1.7 0.4 1.3 0,5 1.4 0.3 cortex 0.9 0.0 1.5 0.4 1.1 0,1 3.1 0.6 wood 0.4 0.05 1.0 0.30 0.5 0,1 2.0 0.5 14C-donor part 82.2 2.8 67.5 0.9 82.0 1.3 76.3 2.5 Below 14C-donor part 14.3 0.9 27.3 1.1 14.9 1.3 13.9 3.8

leaves 9.9 1.1 13.5 1.5 7.3 2.1 9.0 1.4 cortex 3.0 0.3 6.7 0.8 5.8 1.7 2.2 0.6 wood 1.4 0.2 7.1 1.6 1.8 0.7 2.7 0.8 Above/below 4.1 5.25 4.8 1.4 Table 5. The influence of nitrate feeding through the transpiration water stream on 14C distribution among the organs of flax in 30 min and 3 h after 14CO2 assimilation by the

As we have shown previously (Batasheva et al., 2007), increased 14C content in the lower part of plants fed with water is not connected with label accumulation due to synthesis of

In 30 min after 14CO2 assimilation relative content of 14C-sucrose in source leaves increased both in control and in nitrate fed plants (Table 6). In the following 2.5 h in water fed shoots 14C-sucrose content decreased to lower values compared to those observed immediately after 14CO2 fixation whereas in nitrate fed shoots it continued growing. Thus, in 3 h after 14CO2 assimilation relative 14C content in sucrose in source leaves of the shoots fed with KNO3 rose significantly up to 75%. Relative radioactivity of hexoses practically did not change which resulted in increase of the labeled sucrose/hexoses. The similar picture of 14C-sucrose dynamics was described above for wheat plants fertilized with

Localization of labeled sucrose in leaves was determined by autoradioagraphy of whole leaves taken from 14C-donor part in 30 min and 3 h after 14CO2 assimilation. It turned out that in 30 min after 14CO2 assimilation in water fed shoots the label was concentrated mainly in leaf large veins whereas in nitrate fed shoots – outside them (Fig. 6). It indicates that upon nitrate feeding the accumulation of the labeled assimilates occurred either in mesophyll cells

or in minor vein cells from which they were not transported to large veins.

with the transpiration stream.

middle shoot part (% of whole shoot radioactivity)

any high-molecular weight substances in this period.

including:

including:

nitrates (Fig. 3).


Table 6. The incorporation of 14C into the products of photosynthesis in 30 min and in 3 h after 2.5 min 14CO2 assimilation

In 3 h of post-photosynthesis in control leaves 14C mostly disappeared from large bundles. In nitrate fed leaves the differences between 14C contents inside and outsides the large bundles became even more contrasty. It seemed likely that in control shoots 14C-sucrose export exceeded its synthesis while in nitrate fed shoots 14C-sucrose synthesis was not compensated by its removing from the leaf. Earlier it was shown that feeding urea (0.15% (w/v)) into the apoplast led to the same picture of 14C-sucrose dynamics as feeding water (Batasheva et al., 2007).

Thus, nitrate feeding resulted in graduate accumulation of sucrose in source leaves, which was observed on the background of assimilate export suppression. It is interesting that similar dynamics of sucrose radioactivity changes in mature leaves was observed by Möller and Beck (1992), who studied metabolism of labeled sucrose when unlabelled sucrose was constantly inflowing into the apoplast. In the work high sucrose content in the apoplast led to labeled sucrose accumulation within cells. A question arises why labelled sucrose was not hydrolyzed especially under the conditions of inhibited export appearing when sucrose is fed into the apoplast or observed in our experiments upon nitrate feeding.

Fig. 6. The influence of water or KNO3 solution (0.5%) feeding on 14С distribution within the donor leaf in 30 min (A – water; B – nitrate) and 3 h (C – water; D – nitrate) after exposure to 14CO2. Darker places correspond to greater 14C contents

The Role of C to N Balance in the Regulation of Photosynthetic Function 285

When the dynamics of changes in ultrastructure of leaves in the presence of nitrate in the apoplast was studied it turned out that in mesophyll, bundle sheath and phloem parenchyma cells the mitochondria matrices clarified and dictyosomes curled in the first 30 min, but repaired their structure by 1 h after beginning of nitrate feeding (Abdrakhimov et al., 2008). We supposed that such a two-phase response could consist of a quick direct action of nitric oxide, formed from nitrate, on leaf ultrastructure and slower changes related with

Fig. 7. Ultrastructure of terminal vein cells in the leaves of common flax 1 h after supplying

Thus, analysis of flax leaf terminal vein ultrastructure revealed vacuole formation in companion cells in response to nitrate feeding into the apoplast. One can guess that, similarly to putting a cold collar on the petiole, nitrate feeding into the apoplast initially creates hindrances to assimilate transport within the sieve elements or assimilate transport from companion cells to sieve elements. Augmentation of callose synthesis is known to happen very quickly in response to low temperature, within several minutes (Kursanov, 1976). It is tempting to speculate that when nitrates are excessive NO is generated, which, as

The introduction of nitrate solution into the plant apoplast of willow-herb shoots (control – water) caused an inhibition of 14CO2 fixation by leaves (Khamidullina et al., 2011). Analysis of distribution of labeled photosynthetic products among the plant organs in 3 h after 14CO2 assimilation has shown that the intensity of assimilate export from willow-herb leaves (both in control and treated plants) was lower than that from flax leaves. In willow-herb plants it was 8.1 % in control and 4.5% in nitrate plants, while under the same conditions in flax plants more than 40% of 14C-assimilates formed in leaves were exported (Khamidullina et al., 2011). Such a difference in the export rate in plants with various types of phloem loading could be linked with synthesis of transport products represented by tri- and tetra-saccharides in symplastic plants. Probably it is due to variable mechanisms of transport, longer time required for oligosaccaride synthesis, and different diurnal dynamics of assimilate export (Gamalei, 2004)

the transfer of labeled photosynthetic products was slower in willow-herb than in flax.

water (a) or 0.5% KNO3 (b) to the apoplast. CC - companion cell; SE - sieve element

**3.3 The influence of nitrates fed into the apoplast of a symplastic plant** 

inhibited photoassimilate export (Abdrakhimov et al., 2008)

(Batasheva et al., 2007)

a stress signal, can trigger synthesis of callose.

It is probable that labeled sucrose was in the leaf conducting system where sucrose hydrolyzing activity is negligible (Dubinina et al., 1984). On the other side, we can not rule out the possibility that at least a part of labeled sucrose was accumulated in mesophyll cells. For instance, in barley shoots, suppression of assimilate export resulted in sucrose accumulation in bundle sheath parenchyma cells and in mesophyll cells and probably in the bundles themselves. In the mesophyll and bundle sheath cells an increase in glucose contents was also observed (Koroleva et al., 1998; Pollock et al., 2003).

Thus, it turned out that nitrates fed into the cut shoots with the transpiration water stream exerted the same action on assimilate outflow and 14C distribution among the products of photosynthesis as nitrate fertilization.

The similar action of nitrate fertilization (Chikov et al., 2001) and artificial nitrate infusion into the apoplast on 14C distribution among the products of photosynthesis, primarily, the ratio of labeled sucrose to hexoses, allowed us to conclude that enhanced sucrose hydrolysis under increased nitrate fertilization of plants was in some way connected with the presence of nitrate anion in the apoplast. Urea did not exert such an effect (Batasheva et al., 2007).

Thus, a possible reason of changes in metabolism at soil fertilization of plants with nitrates may be an enhancement of sucrose hydrolysis in the apoplast at nitrate income therein. Because monoses formed in the process of sucrose hydrolysis cannot be loaded into the phloem terminals and, consequently, take part in assimilate transport, assimilate outflow from leaves becomes lowered. The monoses have to return back to mesophyll cells and then, decreased photosynthesis rate and its non-carbohydrate tendency can be the consequences of feedback inhibition of photosynthesis and sucrose formation.

The possible influence of nitrates through activation of sucrose hydrolysis by cell wall invertase is supported by numerous data on the similar action of nitrates and sugars on certain gene expressions, primarily those connected with nitrogen metabolism (Stitt et al., 2002). Furthermore, it was shown that one of nitrate carrier genes is induced by NO3 and decrease of its transcript abundance in the dark could be prevented by addition of sucrose. The gene could also be induced in the absence of external NO3 - by a sharp decline of medium pH from 6.5 to 5.5 (Forde, 2000), which could cause an activation of the apoplastic invertase, because its pH optimum lies in the acidic pH range (Brovchenko, 1970).

In plants with the apoplastic type of phloem loading inhibition of photoassimilate transport by putting a cold collar on the petiole was associated with appearance of large central vacuoles in phloem companion cells (Gamalei & Pakhomova, 2000). So, it could be possible that in our experiments in the post-photosynthetic period sucrose accumulated not in the transport path itself but in vacuoles formed in companion cells.

Significant changes in minor vein cell structure as a response to increase in nitrate concentration in the apoplast were found (Fig. 7). In control leaves, companion cells were characterized by well developed system of cell wall invaginations and very slight vacuolization of their protoplasts (Fig. 7A). In 30 min after beginning of KNO3 (0.5%) solution feeding sieve elements filled up with electron-transparent vesicles, and a large central vacuole was formed in companion cells (Fig. 7B). Some indication of endocytosis allows to guess that the vacuolation was a result of seizure of extracellular milieu containing sugar in high concentration. This could be the way by which companion cells protected themselves from osmotic stress. The vacuole was growing in size for the next 2 h (Abdrakhimov et al., 2008).

It is probable that labeled sucrose was in the leaf conducting system where sucrose hydrolyzing activity is negligible (Dubinina et al., 1984). On the other side, we can not rule out the possibility that at least a part of labeled sucrose was accumulated in mesophyll cells. For instance, in barley shoots, suppression of assimilate export resulted in sucrose accumulation in bundle sheath parenchyma cells and in mesophyll cells and probably in the bundles themselves. In the mesophyll and bundle sheath cells an increase in glucose

Thus, it turned out that nitrates fed into the cut shoots with the transpiration water stream exerted the same action on assimilate outflow and 14C distribution among the products of

The similar action of nitrate fertilization (Chikov et al., 2001) and artificial nitrate infusion into the apoplast on 14C distribution among the products of photosynthesis, primarily, the ratio of labeled sucrose to hexoses, allowed us to conclude that enhanced sucrose hydrolysis under increased nitrate fertilization of plants was in some way connected with the presence of nitrate anion in the apoplast. Urea did not exert such an effect (Batasheva et al., 2007). Thus, a possible reason of changes in metabolism at soil fertilization of plants with nitrates may be an enhancement of sucrose hydrolysis in the apoplast at nitrate income therein. Because monoses formed in the process of sucrose hydrolysis cannot be loaded into the phloem terminals and, consequently, take part in assimilate transport, assimilate outflow from leaves becomes lowered. The monoses have to return back to mesophyll cells and then, decreased photosynthesis rate and its non-carbohydrate tendency can be the consequences

The possible influence of nitrates through activation of sucrose hydrolysis by cell wall invertase is supported by numerous data on the similar action of nitrates and sugars on certain gene expressions, primarily those connected with nitrogen metabolism (Stitt et al., 2002). Furthermore, it was shown that one of nitrate carrier genes is induced by NO3-

decrease of its transcript abundance in the dark could be prevented by addition of sucrose. The gene could also be induced in the absence of external NO3- by a sharp decline of medium pH from 6.5 to 5.5 (Forde, 2000), which could cause an activation of the apoplastic

In plants with the apoplastic type of phloem loading inhibition of photoassimilate transport by putting a cold collar on the petiole was associated with appearance of large central vacuoles in phloem companion cells (Gamalei & Pakhomova, 2000). So, it could be possible that in our experiments in the post-photosynthetic period sucrose accumulated not in the

Significant changes in minor vein cell structure as a response to increase in nitrate concentration in the apoplast were found (Fig. 7). In control leaves, companion cells were characterized by well developed system of cell wall invaginations and very slight vacuolization of their protoplasts (Fig. 7A). In 30 min after beginning of KNO3 (0.5%) solution feeding sieve elements filled up with electron-transparent vesicles, and a large central vacuole was formed in companion cells (Fig. 7B). Some indication of endocytosis allows to guess that the vacuolation was a result of seizure of extracellular milieu containing sugar in high concentration. This could be the way by which companion cells protected themselves from osmotic stress. The vacuole was growing in size for the next 2 h

invertase, because its pH optimum lies in the acidic pH range (Brovchenko, 1970).

and

contents was also observed (Koroleva et al., 1998; Pollock et al., 2003).

of feedback inhibition of photosynthesis and sucrose formation.

transport path itself but in vacuoles formed in companion cells.

(Abdrakhimov et al., 2008).

photosynthesis as nitrate fertilization.

When the dynamics of changes in ultrastructure of leaves in the presence of nitrate in the apoplast was studied it turned out that in mesophyll, bundle sheath and phloem parenchyma cells the mitochondria matrices clarified and dictyosomes curled in the first 30 min, but repaired their structure by 1 h after beginning of nitrate feeding (Abdrakhimov et al., 2008). We supposed that such a two-phase response could consist of a quick direct action of nitric oxide, formed from nitrate, on leaf ultrastructure and slower changes related with inhibited photoassimilate export (Abdrakhimov et al., 2008)

Fig. 7. Ultrastructure of terminal vein cells in the leaves of common flax 1 h after supplying water (a) or 0.5% KNO3 (b) to the apoplast. CC - companion cell; SE - sieve element (Batasheva et al., 2007)

Thus, analysis of flax leaf terminal vein ultrastructure revealed vacuole formation in companion cells in response to nitrate feeding into the apoplast. One can guess that, similarly to putting a cold collar on the petiole, nitrate feeding into the apoplast initially creates hindrances to assimilate transport within the sieve elements or assimilate transport from companion cells to sieve elements. Augmentation of callose synthesis is known to happen very quickly in response to low temperature, within several minutes (Kursanov, 1976). It is tempting to speculate that when nitrates are excessive NO is generated, which, as a stress signal, can trigger synthesis of callose.

#### **3.3 The influence of nitrates fed into the apoplast of a symplastic plant**

The introduction of nitrate solution into the plant apoplast of willow-herb shoots (control – water) caused an inhibition of 14CO2 fixation by leaves (Khamidullina et al., 2011). Analysis of distribution of labeled photosynthetic products among the plant organs in 3 h after 14CO2 assimilation has shown that the intensity of assimilate export from willow-herb leaves (both in control and treated plants) was lower than that from flax leaves. In willow-herb plants it was 8.1 % in control and 4.5% in nitrate plants, while under the same conditions in flax plants more than 40% of 14C-assimilates formed in leaves were exported (Khamidullina et al., 2011). Such a difference in the export rate in plants with various types of phloem loading could be linked with synthesis of transport products represented by tri- and tetra-saccharides in symplastic plants. Probably it is due to variable mechanisms of transport, longer time required for oligosaccaride synthesis, and different diurnal dynamics of assimilate export (Gamalei, 2004) the transfer of labeled photosynthetic products was slower in willow-herb than in flax.

The Role of C to N Balance in the Regulation of Photosynthetic Function 287

In 3 h after beginning of nitrate feeding into the apoplast the most evident changes of ultrastructure were observed in the mitochondrion and vacuolar system of cells of conducting system (Khamidullina et al., 2011). The electron density of mitochondrial matrix increased and christa lumens swelled indicating increased osmotic pressure in the cytosol of cells in conducting system. This was paralleled by accumulation of fibrillar inclusions represented by polymer substance that generated a homogenous network evenly distributed

Fig. 8. Companion cells in the leaves of fireweed plants in 3 h after water or potassium nitrate (50 mM) feeding into the apoplast. BSC – bundle sheath cells, CC – companion cells

**4. The influence of NO donor, sodium nitroprusside on photosynthetic** 

As it was shown above in model experiments, under direct feeding of salts, containing nitrate anion, in the plant apoplast, decreased 14CO2 assimilation rate and photoassimilate export from leaves with changes in photosynthetic carbon metabolism and ultrastructure of phloem companion cells were observed. Elevation of nitrate content in a plant is a condition advantageous for nitric oxide generation from nitrite by both enzymatic and non-enzymatic ways (Neill et al., 2003). From whence a participation of NO signal system in triggering a mechanism, arresting assimilate export from leaves under increased nitrate nutrition level

The nature of the inclusions is unknown, but, bearing in mind the abundance of the polymer and the fact that analogous formations were revealed also in the sieve elements we have guessed that the observed structures is either photosynthetic transport product itself or the result of its polymerization. But irregardless of the way of formation its accumulation must also express a blockage of transfer of carbohydrate transport product in willow-herb plants. The organelles covered with semi-permeable membranes, mitochondria and plastids are known to be ideal intracellular osmometers (Gamalei, 2004). Obviously, hindered transport of low molecular weight compounds from leaf blades must result in their piling up in the places of phloem loading and osmotic contraction of the organelles. That is just what was observed in our experiments (Fig. 8). Similar changes we have earlier seen in an apoplastic plant, flax, in 3 h after beginning of nitrate feeding through the transpiration stream (see above). However, oligosaccarides rather than sucrose are accumulated in companion cell

throughout the organelle interior on the cross-sections (Fig. 8).

(Khamidullina et al., 2011)

**carbon metabolism** 

vacuoles, diagnosable by fibrillar inclusions.

As in the flax plants, in the willow-herb plants nitrates stimulated the transfer of labeled assimilates to the upper shoot part. The difference will become larger if the content of labeled assimilates in the upper shoot part is assessed relatively to that exported from the 14C-donor leaf. As a result the ratio of exported labeled assimilates in the upper part to that in the lower part was 4.8 in control and 2.4 in nitrate plants. Because the labeled assimilates are likely to get into the upper shoot part with the transpiration water stream after their leakage from the phloem into the stem apoplast the data evidences the enhancement by nitrates of the permeability of phloem tubes to transport photosynthetic products. The latter could be due to the slowdown of phloem transport and increased concentration of products in the stem phloem or reduced return of sugars from the apoplast back into the phloem (Kühn, et al., 1999).

The analysis of 14C distribution among the labeled products of low molecular weight fraction revealed both common traits and differences in the action of nitrates on photosynthetic metabolism in flax and willow herb. In both cases 14C introduction into sucrose decreased under nitrates, that indicates the similarity of nitrate influence on photosynthetic carbon metabolism in symplastic and apoplastic plants. The influence on photosynthetic carbon metabolism is not connected with the presence of potassium cation, because the same changers were also observed under calcium nitrate nutrition of plants (Chikov et al., 1998).

After comparing the dynamics of 14C acquirement by sucrose in flax and willow herb the similarity was also found. After short expositions to 14CO2 mirroring the intensity of sucrose synthesis a decrease in sucrose radioactivity was observed. Later in treated plants the label piled up in sucrose as a result of sucrose export arrest. Thus, the characters of postphotosynthetic changes of sucrose radioactivity in both plant types were resemblant.

As it is known in symplastic plants a significant fraction of exported sugars is represented by oligosaccharides (Pristupa, 1959). Probably that is why the dynamics of changes in 14C content in this group of compounds is similar to that of sucrose. After analysis of the data on oligosaccharide and sucrose content in the willow-herb it was found that in nitrate plants 14C was gradually accumulated with time in sucrose and oligosaccharides. In control, this parameter changed insignificantly (within the error).

Thus, nitrate anion hinders assimilate export irregardless of the way of sugar transfer from leaf mesophyll cells to the phloem, suggesting that the mechanisms of nitrate action on assimilate transport in both plant types have much in common.

The radioactivity of phosphorous esters of sugars (PES) in nitrate fed willow-herb plants immediately after 3 min exposition to 14CO2 was increased apparently indicating their hampered conversion to export sugars (Khamidullina et al., 2011). The general elevation of radioactivity in the glycolate pathway products (serine, glycine and glycolate) suggests that photooxidation processes become activated under nitrate feeding. As it was shown by Chikov and Bakirova (1999) the enhanced production of glycolate pathway products under increased nitrate nutrition may be connected not with atmospheric oxygen but with reactive oxygen species, generated in the course of nitrate reduction in the chloroplast electron transfer chain, because their formation only slightly declined after a decrease in oxygen concentration down to 1%. A possible existence of other sources of glycolate and its metabolites under delayed assimilate export from the leaf was demonstrated previously in the kinetic experiments (Chikov et al., 1985).

As in the flax plants, in the willow-herb plants nitrates stimulated the transfer of labeled assimilates to the upper shoot part. The difference will become larger if the content of labeled assimilates in the upper shoot part is assessed relatively to that exported from the 14C-donor leaf. As a result the ratio of exported labeled assimilates in the upper part to that in the lower part was 4.8 in control and 2.4 in nitrate plants. Because the labeled assimilates are likely to get into the upper shoot part with the transpiration water stream after their leakage from the phloem into the stem apoplast the data evidences the enhancement by nitrates of the permeability of phloem tubes to transport photosynthetic products. The latter could be due to the slowdown of phloem transport and increased concentration of products in the stem phloem or reduced return of sugars from the apoplast back into the phloem

The analysis of 14C distribution among the labeled products of low molecular weight fraction revealed both common traits and differences in the action of nitrates on photosynthetic metabolism in flax and willow herb. In both cases 14C introduction into sucrose decreased under nitrates, that indicates the similarity of nitrate influence on photosynthetic carbon metabolism in symplastic and apoplastic plants. The influence on photosynthetic carbon metabolism is not connected with the presence of potassium cation, because the same changers were also observed under calcium nitrate nutrition of plants

After comparing the dynamics of 14C acquirement by sucrose in flax and willow herb the similarity was also found. After short expositions to 14CO2 mirroring the intensity of sucrose synthesis a decrease in sucrose radioactivity was observed. Later in treated plants the label piled up in sucrose as a result of sucrose export arrest. Thus, the characters of post-

As it is known in symplastic plants a significant fraction of exported sugars is represented by oligosaccharides (Pristupa, 1959). Probably that is why the dynamics of changes in 14C content in this group of compounds is similar to that of sucrose. After analysis of the data on oligosaccharide and sucrose content in the willow-herb it was found that in nitrate plants 14C was gradually accumulated with time in sucrose and oligosaccharides. In control, this

Thus, nitrate anion hinders assimilate export irregardless of the way of sugar transfer from leaf mesophyll cells to the phloem, suggesting that the mechanisms of nitrate action on

The radioactivity of phosphorous esters of sugars (PES) in nitrate fed willow-herb plants immediately after 3 min exposition to 14CO2 was increased apparently indicating their hampered conversion to export sugars (Khamidullina et al., 2011). The general elevation of radioactivity in the glycolate pathway products (serine, glycine and glycolate) suggests that photooxidation processes become activated under nitrate feeding. As it was shown by Chikov and Bakirova (1999) the enhanced production of glycolate pathway products under increased nitrate nutrition may be connected not with atmospheric oxygen but with reactive oxygen species, generated in the course of nitrate reduction in the chloroplast electron transfer chain, because their formation only slightly declined after a decrease in oxygen concentration down to 1%. A possible existence of other sources of glycolate and its metabolites under delayed assimilate export from the leaf was demonstrated previously in

photosynthetic changes of sucrose radioactivity in both plant types were resemblant.

parameter changed insignificantly (within the error).

the kinetic experiments (Chikov et al., 1985).

assimilate transport in both plant types have much in common.

(Kühn, et al., 1999).

(Chikov et al., 1998).

In 3 h after beginning of nitrate feeding into the apoplast the most evident changes of ultrastructure were observed in the mitochondrion and vacuolar system of cells of conducting system (Khamidullina et al., 2011). The electron density of mitochondrial matrix increased and christa lumens swelled indicating increased osmotic pressure in the cytosol of cells in conducting system. This was paralleled by accumulation of fibrillar inclusions represented by polymer substance that generated a homogenous network evenly distributed throughout the organelle interior on the cross-sections (Fig. 8).

Fig. 8. Companion cells in the leaves of fireweed plants in 3 h after water or potassium nitrate (50 mM) feeding into the apoplast. BSC – bundle sheath cells, CC – companion cells (Khamidullina et al., 2011)

The nature of the inclusions is unknown, but, bearing in mind the abundance of the polymer and the fact that analogous formations were revealed also in the sieve elements we have guessed that the observed structures is either photosynthetic transport product itself or the result of its polymerization. But irregardless of the way of formation its accumulation must also express a blockage of transfer of carbohydrate transport product in willow-herb plants. The organelles covered with semi-permeable membranes, mitochondria and plastids are known to be ideal intracellular osmometers (Gamalei, 2004). Obviously, hindered transport of low molecular weight compounds from leaf blades must result in their piling up in the places of phloem loading and osmotic contraction of the organelles. That is just what was observed in our experiments (Fig. 8). Similar changes we have earlier seen in an apoplastic plant, flax, in 3 h after beginning of nitrate feeding through the transpiration stream (see above). However, oligosaccarides rather than sucrose are accumulated in companion cell vacuoles, diagnosable by fibrillar inclusions.

#### **4. The influence of NO donor, sodium nitroprusside on photosynthetic carbon metabolism**

As it was shown above in model experiments, under direct feeding of salts, containing nitrate anion, in the plant apoplast, decreased 14CO2 assimilation rate and photoassimilate export from leaves with changes in photosynthetic carbon metabolism and ultrastructure of phloem companion cells were observed. Elevation of nitrate content in a plant is a condition advantageous for nitric oxide generation from nitrite by both enzymatic and non-enzymatic ways (Neill et al., 2003). From whence a participation of NO signal system in triggering a mechanism, arresting assimilate export from leaves under increased nitrate nutrition level

The Role of C to N Balance in the Regulation of Photosynthetic Function 289

Compounds Control (Н2О) Sodium nitroprusside (1 mM)

Table 8. The influence of sodium nitroprusside solution (1 mM) infused into the flax shoot with the transpiration water stream on 14C distribution among the labeled compounds of 2.5 min 14CO2 assimilation by leaves (% water-ethanol soluble fraction) (Batasheva et al., 2010) On the background of lower 14C content in sucrose the formation of the glycolate pathway products substantially increased, which is also a characteristic of metabolism under increased nitrate concentration in the plant. However, in distinction from nitrate treatment under nitroprusside injection 14C income into amino acids did not increase compared to control. This was probably related with the absent of additional nitrogen as a substrate for

Infiltration of plants with NO donor lead to appreciable alterations in the organization of both assimilating and conducting system cells (Fig. 9). In 30 min after beginning of feeding sodium nitroprusside into the apoplast the structural changes became obvious (Fig. 9 В, C). They were expressed in vacuolization of companion cells with appearing of a large central vacuole (Fig. 9B). The peaks of the crests of cell wall labyrinth became osmophilic and often reached the vacuole interior. Structural changes of the assimilating cell-bundle sheath cellphloem parenchyma domain expressed in clarification of mitochondrion matrices (a place of glycine decarboxylation) and dictyosome curling into ring-shaped structures (Batasheva et

Fig. 9. The influence of sodium nitroprusside solution (50 µM) on ultrastructure of flax leaf companion cells. A – control, B, C –after 30 min of introduction of sodium nitroprusside with the transpiration water stream; CC – companion cell, SE – sieve element (Batasheva et

NO induced formation of numerous multimembrane and multivesicular structures in assimilating cells (Fig. 9C) in many ways analogous to those observed in 30 min after

Because the effects of NO and nitrates on cell ultrastructure were similar, one can suppose that a product of incomplete reduction of nitrate, NO, is capable of inhibiting assimilate

beginning of nitrate salts feeding into the apoplast (Abdrakhimov et al., 2008).

Sucrose 48.0 ± 3.9 33.6 ± 1.1 Hexoses 5.3 ± 0.9 10.1 ± 1.9 Serine+glycine+glycolate 0.7 ± 0.2 4.1 ± 0.4 Amino acids 22.0 ±1.7 23.3 ± 1.8 Oligosaccharides 3.3 ± 0.9 6.2 ± 1.2 Pigments 2.6 ± 0.5 3.1 ± 0.7 Others 20.7 22.7

amino acid synthesis.

al., 2010).

al., 2010)

may be proposed. To test this proposition we investigated the influence of NO donor, sodium nitroprusside, on photosynthetic metabolism and ultrastructure of cells of flax leaf blades.

As in experiments with injection of potassium nitrate solution into the shoot, sodium nitroprusside solutions (SNP) with concentrations of 50 µM, 100 µM and 1mM were introduced into cut flax shoots. In 30 min a middle shoot part containing 8-10 leaves was placed into a photosynthetic chamber, where 14CO2 (0.03%) was delivered at natural sunlight (14C-donor part of a shoot).

Feeding SNP solution into the apoplast resulted in reduced 14CO2 assimilation, with the inhibition of 14CO2 fixation by SNP (50 µM, 100 µM) being almost equal to that by potassium nitrate (50 mM). Further elevation of nitroprusside concentration to 1 mM enhanced the inhibition of photosynthesis up to 75% (Table 7, Batasheva et al., 2010). Such a sharp drop of carbon dioxide uptake after increasing SNP concentration up to 1 mM can partially be explained by NO participating in stomatal closure (Mata & Lamattina, 2001). NO involved in stomatal movements can be generated from nitrate anion by nitrate reductase (Desikan et al., 2002).

SNP introduction into the shoot affected 14C-assimilate distribution throughout the plant similarly to nitrate feeding. In the treated plants assimilate outflow from the donor part slowed down and their transfer into the upper part relatively increased (Table 7). Whereas in control the main part of 14C-products of photosynthesis exported from the donor-part appeared in the lower shoot part, after feeding SNP the relatively higher content of exported 14C-assimilates was found in the upper shoot part (Batasheva et al., 2010).


Table 7. The influence of sodium nitroprusside solution introduced into the flax shoot with the transpiration water stream on 14C distribution among different plant parts in 3 h after 14CO2 assimilation by the middle shoot part (% total shoot radioactivity) (Batasheva et al., 2010)

SNP treatment resulted in altered photosynthetic carbon metabolism. Analysis of 14C distribution among the labeled products of photosynthesis has shown that the largest relative changes occurred in the glycolate pathway compounds (serine, glycine, glycolate) and sugars (Table 8). As in the case of nitrate feeding into the apoplast (Batasheva et al., 2007) the portion of 14C in sucrose decreased, leading to the lowered ratio of labelled sucrose to hexoses. Elevated monosaccharide availability relatively enhanced production of other storage sugars (oligosaccharides). As a result the ratio of sucrose to oligosaccharides reduced almost three times.

may be proposed. To test this proposition we investigated the influence of NO donor, sodium nitroprusside, on photosynthetic metabolism and ultrastructure of cells of flax leaf

As in experiments with injection of potassium nitrate solution into the shoot, sodium nitroprusside solutions (SNP) with concentrations of 50 µM, 100 µM and 1mM were introduced into cut flax shoots. In 30 min a middle shoot part containing 8-10 leaves was placed into a photosynthetic chamber, where 14CO2 (0.03%) was delivered at natural

Feeding SNP solution into the apoplast resulted in reduced 14CO2 assimilation, with the inhibition of 14CO2 fixation by SNP (50 µM, 100 µM) being almost equal to that by potassium nitrate (50 mM). Further elevation of nitroprusside concentration to 1 mM enhanced the inhibition of photosynthesis up to 75% (Table 7, Batasheva et al., 2010). Such a sharp drop of carbon dioxide uptake after increasing SNP concentration up to 1 mM can partially be explained by NO participating in stomatal closure (Mata & Lamattina, 2001). NO involved in stomatal movements can be generated from nitrate anion by nitrate reductase (Desikan et al., 2002). SNP introduction into the shoot affected 14C-assimilate distribution throughout the plant similarly to nitrate feeding. In the treated plants assimilate outflow from the donor part slowed down and their transfer into the upper part relatively increased (Table 7). Whereas in control the main part of 14C-products of photosynthesis exported from the donor-part appeared in the lower shoot part, after feeding SNP the relatively higher content of exported

14C-assimilates was found in the upper shoot part (Batasheva et al., 2010).

(water)

Top 2.5 ± 0.5 3.0 ± 1.4 6.0 ± 0.8 1.8 ± 0.2 Upper shoot part 5.3 ± 1.3 6.3 ± 2.3 9.1 ± 0.5 11.5 ± 6.2 Above 14C-source part 7.8 9.8 15.1 13.3 14C-source leaves 42.3 ± 4,5 49.2 ± 4,4 46.0 ± 1,8 21.4 ± 2.8 14C-stem 24.2 ± 3.4 31.5 ± 8.1 28.5 ± 4.2 57.1 ± 13.2 14C-source part summarized 66.5 80.6 74.5 78.5 Below 14C-source part 25.7 ± 4.8 9.5 ± 3.1 10.4 ± 2.0 8.0 ± 4.4 Above/Below part 3.29 0.97 0.69 0.60

(% control) 100 88 86 25

Table 7. The influence of sodium nitroprusside solution introduced into the flax shoot with the transpiration water stream on 14C distribution among different plant parts in 3 h after 14CO2 assimilation by the middle shoot part (% total shoot radioactivity) (Batasheva et al., 2010)

SNP treatment resulted in altered photosynthetic carbon metabolism. Analysis of 14C distribution among the labeled products of photosynthesis has shown that the largest relative changes occurred in the glycolate pathway compounds (serine, glycine, glycolate) and sugars (Table 8). As in the case of nitrate feeding into the apoplast (Batasheva et al., 2007) the portion of 14C in sucrose decreased, leading to the lowered ratio of labelled sucrose to hexoses. Elevated monosaccharide availability relatively enhanced production of other storage sugars (oligosaccharides). As a result the ratio of sucrose to oligosaccharides

SNP (50 µM)

SNP (100 µM)

SNP (1mM)

blades.

sunlight (14C-donor part of a shoot).

Shoot parts Control

Uptake of 14СО<sup>2</sup>

reduced almost three times.


Table 8. The influence of sodium nitroprusside solution (1 mM) infused into the flax shoot with the transpiration water stream on 14C distribution among the labeled compounds of 2.5 min 14CO2 assimilation by leaves (% water-ethanol soluble fraction) (Batasheva et al., 2010)

On the background of lower 14C content in sucrose the formation of the glycolate pathway products substantially increased, which is also a characteristic of metabolism under increased nitrate concentration in the plant. However, in distinction from nitrate treatment under nitroprusside injection 14C income into amino acids did not increase compared to control. This was probably related with the absent of additional nitrogen as a substrate for amino acid synthesis.

Infiltration of plants with NO donor lead to appreciable alterations in the organization of both assimilating and conducting system cells (Fig. 9). In 30 min after beginning of feeding sodium nitroprusside into the apoplast the structural changes became obvious (Fig. 9 В, C). They were expressed in vacuolization of companion cells with appearing of a large central vacuole (Fig. 9B). The peaks of the crests of cell wall labyrinth became osmophilic and often reached the vacuole interior. Structural changes of the assimilating cell-bundle sheath cellphloem parenchyma domain expressed in clarification of mitochondrion matrices (a place of glycine decarboxylation) and dictyosome curling into ring-shaped structures (Batasheva et al., 2010).

Fig. 9. The influence of sodium nitroprusside solution (50 µM) on ultrastructure of flax leaf companion cells. A – control, B, C –after 30 min of introduction of sodium nitroprusside with the transpiration water stream; CC – companion cell, SE – sieve element (Batasheva et al., 2010)

NO induced formation of numerous multimembrane and multivesicular structures in assimilating cells (Fig. 9C) in many ways analogous to those observed in 30 min after beginning of nitrate salts feeding into the apoplast (Abdrakhimov et al., 2008).

Because the effects of NO and nitrates on cell ultrastructure were similar, one can suppose that a product of incomplete reduction of nitrate, NO, is capable of inhibiting assimilate

The Role of C to N Balance in the Regulation of Photosynthetic Function 291

Depending on the character of such a disequilibrium the meeting of the two disturbed mass flows may occur either in the aboveground plant part or in the root system. According to the place of the meeting, generation of NO and triggering of NO signal system may take place in the root area or in the shoot. Both are also influenced by the intensity of the transpiration,

**Excess of nitrates in roots** 

**NO formation**

**NO signalling**

**Excess of PES (erythrosephosphate)** 

> **Activation of the shikimic acid pathway**

**Formation of arginine and phytohormones** 

**Intensification of nitrate flow into the above-ground part of the plant** 

**Gene expression**

**Initiation of new sink organs** 

Fig. 10. Scheme of NO-signal system participation in the rearrangement of metabolism in the

**Activation of growth processes** 

When excessive nitrates appear and the available nitrate reducing enzymes and their activity are not enough to utilize this massive flow of nitrates, the latters will surge into the upper plant part without having been reduced, where they will interact with the sugar flow, and NO signal system will become triggered. Oppositely, when sugars are in excess, they

In the first case, growth processes will be initiated in the above-ground plant part, and gradual nitrate utilization in leaves (shoots) will lead to a step-down in root nitrates, and augmented photosynthetic apparatus will send extensively increased sugar flow to the

whole plant under increased nitrate

**Sucrose signalling** 

**Glucose signalling** 

will be transported to roots and interact with nitrates therein.

that carries mineral nutrients from roots to leaves with the water stream.

**Callose accumulation**

**Inhibition of photosynthate export** 

**Sucrose hydrolysis** 

**Sucrose accumulation** 

**Increase in glucose** 

**Abscisic acid conjugation** 

transport directly in phloem cells. This suggestion is supported by the observation that NO is able to elevate callose content in the leaf, β-1,3-glucane, taking part, inter alia, in plugging sieve plate pores (Paris et al., 2007). Exhibitive of this mechanism is also data on increased NO synthesis (Zottini et al., 2007) and inhibited callose destruction (Serova et al., 2006) in the presence of salicylic acid, implying that the suppression of callose breakdown may be mediated by nitric oxide.

Appearing of vesicles inside the vacuole owing to endocytosis can be indicative of rising osmolarity of cell environment, probably as a consequence of sugar accumulation in the leaf apoplast due to slowdown of sugar export along the phloem.

The discovered facts allow us to propose that a likely reason of assimilate transport suppression by nitrate is generation of nitric oxide under increased nitrate concentration in cells and point to involvement of NO signal system in regulation of assimilate movement in the whole plant system.

#### **5. A possible regulatory interaction of nitrates and sugars at alterations of other external factors**

In the plant there is a regulatory link between the two main mass flows – sugars of photosynthetic origin and nitrates. The flows are heading towards each other and their interaction reacts to a change in sink-source relationships between assimilating and photosynthate consuming organs.

Because an influx of nitrates into a plant is determined by root system activity, this assimilate consumer holds a special place compared to other sink organs, competing with the latters for photosynthates. Different sink organs are uneven in their functions. Unlike other acceptors, such a sink as the root system, could provide feed-back to photosynthetic apparatus not only through sugar consumption but also through export of mineral nutrients (primarily nitrates) and triggering NO signal system (Fig. 10).

The results of the works (Batasheva et al., 2007; Abdrakhimov et al., 2008) on ultrastructure and radioautography of leaves after NO3- intrusion into the apoplast evidence a block to the sugar flow at the level of long-distance transport, most likely, either on the stage of transition of primary sieve tubes to the vascular bundles or when sucrose is moving along the phloem. NO donor, sodium nitroprusside, is known to induce callose accumulation (Paris et al., 2007), involved in clogging the pores in the sieve elements of phloem.

The degree of nitrate reduction is directly related to the sugar availability (Stitt & Krapp, 1999). The process of nitrate uptake has long been known to require photosynthetic energy. At low doses of nitrates and intense photosynthesis they are almost completely reduced in the roots with nitrogen coming to aboveground organs in the form of amides and amino acids. As nitrate concentration in the roots rises, in substances exported from roots to leaves a fraction of amino acids increases at first, and then also NO3 -.

A disproportion of the two main mass flows (nitrate and sugars) is likely to be of a great importance. Here, two modes of events are possible:

1) a sudden elevation of nitrate income into the plant relatively to the existing (established) small amount of synthesized photosynthetic products (sugars);

2) sugar availability increases at unchanged (or decreased) nitrate influx into the plant.

The first may occur after additional input of mineral (nitrate) fertilizers, shortage of irradiance or partial loss of plant leaf blades, while the second, conversely, under raise of lighting, damage or cutting of root system by machinery in the course of planting treatment.

transport directly in phloem cells. This suggestion is supported by the observation that NO is able to elevate callose content in the leaf, β-1,3-glucane, taking part, inter alia, in plugging sieve plate pores (Paris et al., 2007). Exhibitive of this mechanism is also data on increased NO synthesis (Zottini et al., 2007) and inhibited callose destruction (Serova et al., 2006) in the presence of salicylic acid, implying that the suppression of callose breakdown may be

Appearing of vesicles inside the vacuole owing to endocytosis can be indicative of rising osmolarity of cell environment, probably as a consequence of sugar accumulation in the leaf

The discovered facts allow us to propose that a likely reason of assimilate transport suppression by nitrate is generation of nitric oxide under increased nitrate concentration in cells and point to involvement of NO signal system in regulation of assimilate movement in

**5. A possible regulatory interaction of nitrates and sugars at alterations of** 

In the plant there is a regulatory link between the two main mass flows – sugars of photosynthetic origin and nitrates. The flows are heading towards each other and their interaction reacts to a change in sink-source relationships between assimilating and

Because an influx of nitrates into a plant is determined by root system activity, this assimilate consumer holds a special place compared to other sink organs, competing with the latters for photosynthates. Different sink organs are uneven in their functions. Unlike other acceptors, such a sink as the root system, could provide feed-back to photosynthetic apparatus not only through sugar consumption but also through export of mineral nutrients

The results of the works (Batasheva et al., 2007; Abdrakhimov et al., 2008) on ultrastructure and radioautography of leaves after NO3- intrusion into the apoplast evidence a block to the sugar flow at the level of long-distance transport, most likely, either on the stage of transition of primary sieve tubes to the vascular bundles or when sucrose is moving along the phloem. NO donor, sodium nitroprusside, is known to induce callose accumulation

The degree of nitrate reduction is directly related to the sugar availability (Stitt & Krapp, 1999). The process of nitrate uptake has long been known to require photosynthetic energy. At low doses of nitrates and intense photosynthesis they are almost completely reduced in the roots with nitrogen coming to aboveground organs in the form of amides and amino acids. As nitrate concentration in the roots rises, in substances exported from roots to leaves

A disproportion of the two main mass flows (nitrate and sugars) is likely to be of a great

1) a sudden elevation of nitrate income into the plant relatively to the existing (established)

2) sugar availability increases at unchanged (or decreased) nitrate influx into the plant. The first may occur after additional input of mineral (nitrate) fertilizers, shortage of irradiance or partial loss of plant leaf blades, while the second, conversely, under raise of lighting, damage or cutting of root system by machinery in the course of planting treatment.

(Paris et al., 2007), involved in clogging the pores in the sieve elements of phloem.

apoplast due to slowdown of sugar export along the phloem.

(primarily nitrates) and triggering NO signal system (Fig. 10).

a fraction of amino acids increases at first, and then also NO3-.

small amount of synthesized photosynthetic products (sugars);

importance. Here, two modes of events are possible:

mediated by nitric oxide.

the whole plant system.

**other external factors** 

photosynthate consuming organs.

Depending on the character of such a disequilibrium the meeting of the two disturbed mass flows may occur either in the aboveground plant part or in the root system. According to the place of the meeting, generation of NO and triggering of NO signal system may take place in the root area or in the shoot. Both are also influenced by the intensity of the transpiration, that carries mineral nutrients from roots to leaves with the water stream.

Fig. 10. Scheme of NO-signal system participation in the rearrangement of metabolism in the whole plant under increased nitrate

When excessive nitrates appear and the available nitrate reducing enzymes and their activity are not enough to utilize this massive flow of nitrates, the latters will surge into the upper plant part without having been reduced, where they will interact with the sugar flow, and NO signal system will become triggered. Oppositely, when sugars are in excess, they will be transported to roots and interact with nitrates therein.

In the first case, growth processes will be initiated in the above-ground plant part, and gradual nitrate utilization in leaves (shoots) will lead to a step-down in root nitrates, and augmented photosynthetic apparatus will send extensively increased sugar flow to the

The Role of C to N Balance in the Regulation of Photosynthetic Function 293

We believe that the link between nitrates and sugars is to be sought not at the molecular level, i.e. at the level of their metabolism in the cell or their influence on gene expression, but at a higher level - at the level of transport of these substances within the plant. This view is supported by observation that information on the nitrogen and carbon status of the plant is transmitted over long distances, revealed by the well known effect of root nitrate on the metabolism of the above-ground plant part and on shoot to root weight ratio (Scheible et al., 1997). In this connection, there is now a large group of studies devoted to the search of a "signal" coordinating shoot and root responses to nitrogen availability (Walch-Liu et al.,

Activation of the hydrolysis of sucrose in the apoplast in the presence of nitrates is in good agreement with a similar effect of nitrate and sugars on the expression of several genes (Stitt et al., 2002), as well as with discerned differences in systemic and local effect of nitrate on the morphogenesis of the roots (Zhang et al., 2007 ). And the systemic action of nitrate is associated with its negative influence on the flow of assimilates to roots

Currently, signaling functions are ascribed not only to nitrate but also to products of nitrate reduction. Depending on the ratio of available carbon and nitrogen in the plant the ratio of oxidized and reduced nitrogen will vary. The influence of the products of nitrate reduction was noted to be opposite to nitrate influence, though the mechanism of their action is also as yet unknown, but supposed to involve glutamine content or glutamine/2-oxoglutarate ratio

Since an increase in amount of nitrates in the plant creates conditions favorable for the generation of nitric oxide from nitrite in both enzymatic and non-enzymatic ways (Neill et al., 2003), we can assume that the signaling effects of nitrate are partially realized through the formation of nitric oxide and triggering of NO-signaling system. This is confirmed by found similarities in actions of nitrate and nitric oxide generator, sodium nitroprusside, on assimilate transport and metabolism. However, in contrast to nitrate, nitric oxide preferably

Actually, the difference of nitrate and nitric oxide actions may be due to differences in the activity of amino acid synthesis, that, as was mentioned above, can also perform signaling roles. Study of the dynamics of gene expression activation under the influence of nitrate showed that many genes induced by nitrate in the first 0-5 and 5-10 minutes are subjected to

Thus, there remains a lot to be elucidated in the signaling mechanism of nitrate and the study of mechanisms of nitrate influence on the transport of sugars can be very promising, not only for this area of research, but also to discovering how the different processes in the

Abdrakhimov, F.A., Batasheva, S.N, Bakirova, G.G. & Chikov, V.I. (2008). Dynamics of

ultrastructural changes in common flax leaf blades during assimilate transport inhibition with nitrate anion. *Tsitologiya*, Vol. 50, pp. 700-710, ISSN 0041-3771

2005).

(Scheible et al., 1997).

plant are interrelated.

**7. References** 

(Foyer and Noctor, 2002; Stitt et al., 2002).

activates genes involved in plant defense (Grün et al., 2006).

negative regulation by as early as 20 minutes (Castaings et al., 2011).

roots. In the second case, interaction of nitrates with sugars and NO signal system triggering will occur in roots and the process of new secondary root formation will become activated.

This thesis is well illustrated by experiments of L. B. Vysotskaya (Vysotskaya, 2001). Removal of the largest part of roots from seven-days old wheat seedlings suppressed shoot growth and activated biomass growth of remaining roots in as soon as 2 h. In the remaining roots auxins and cytokinins were accumulated while in the growing part of the shoot a rapid decline of auxin content compared to intact plants was found. This suggests that excessive sugar flow to the reduced root system creates prerequisites for interaction of the changed nitrate to sugar ratio and NO signal system triggering (alike an analogue of apical dominance alleviation) and synthesis of cytokinines that activate new root formation. Initially, the prerequisites are most probably the immediate fueling of the nitrate uptake process by better sugar supply of roots. Additional nitrates, in their turn, will trigger NO signal system.

The proposed concept on the role of NO signaling in the regulation of plant metabolism is supported by split-root experiments where plant roots were exposed to culture mediums of different concentrations (Trapeznikov et al., 1999). By placing one part of roots of an individual potato plant into a medium of high concentration and the other part into low-salt one, the authors have found that in the concentrated medium a massive formation of small (absorbing) roots occurred while in the low-salt one numerous tubers appeared (Fig. 11).

Fig. 11. Root system of an individual potato plant at local nutrition. HS – high-salt culture medium, LS – low-salt culture medium (Trapeznikov et al., 1999)

#### **6. Conclusion**

Nitrate has been shown to act as a signal molecule, inducing expression of genes, primarily related with nitrogen metabolism and organic acid synthesis. However, low sugar level in the plant inhibits nitrate assimilation, overriding signals from nitrogen metabolism (Stitt et al., 2002). In this regard a concept has appeared that for regulation of various processes in the plant not sugar and nitrate concentrations are important but a certain ratio between them which was called a C/N-balance (Coruzzi & Bush, 2001).

roots. In the second case, interaction of nitrates with sugars and NO signal system triggering will occur in roots and the process of new secondary root formation will become activated. This thesis is well illustrated by experiments of L. B. Vysotskaya (Vysotskaya, 2001). Removal of the largest part of roots from seven-days old wheat seedlings suppressed shoot growth and activated biomass growth of remaining roots in as soon as 2 h. In the remaining roots auxins and cytokinins were accumulated while in the growing part of the shoot a rapid decline of auxin content compared to intact plants was found. This suggests that excessive sugar flow to the reduced root system creates prerequisites for interaction of the changed nitrate to sugar ratio and NO signal system triggering (alike an analogue of apical dominance alleviation) and synthesis of cytokinines that activate new root formation. Initially, the prerequisites are most probably the immediate fueling of the nitrate uptake process by better sugar supply of roots. Additional nitrates, in their turn, will trigger NO

The proposed concept on the role of NO signaling in the regulation of plant metabolism is supported by split-root experiments where plant roots were exposed to culture mediums of different concentrations (Trapeznikov et al., 1999). By placing one part of roots of an individual potato plant into a medium of high concentration and the other part into low-salt one, the authors have found that in the concentrated medium a massive formation of small (absorbing) roots occurred while in the low-salt one numerous tubers appeared (Fig. 11).

**Н С В С**

**LS HS** 

Fig. 11. Root system of an individual potato plant at local nutrition. HS – high-salt culture

Nitrate has been shown to act as a signal molecule, inducing expression of genes, primarily related with nitrogen metabolism and organic acid synthesis. However, low sugar level in the plant inhibits nitrate assimilation, overriding signals from nitrogen metabolism (Stitt et al., 2002). In this regard a concept has appeared that for regulation of various processes in the plant not sugar and nitrate concentrations are important but a certain ratio between

medium, LS – low-salt culture medium (Trapeznikov et al., 1999)

them which was called a C/N-balance (Coruzzi & Bush, 2001).

signal system.

**6. Conclusion** 

We believe that the link between nitrates and sugars is to be sought not at the molecular level, i.e. at the level of their metabolism in the cell or their influence on gene expression, but at a higher level - at the level of transport of these substances within the plant. This view is supported by observation that information on the nitrogen and carbon status of the plant is transmitted over long distances, revealed by the well known effect of root nitrate on the metabolism of the above-ground plant part and on shoot to root weight ratio (Scheible et al., 1997). In this connection, there is now a large group of studies devoted to the search of a "signal" coordinating shoot and root responses to nitrogen availability (Walch-Liu et al., 2005).

Activation of the hydrolysis of sucrose in the apoplast in the presence of nitrates is in good agreement with a similar effect of nitrate and sugars on the expression of several genes (Stitt et al., 2002), as well as with discerned differences in systemic and local effect of nitrate on the morphogenesis of the roots (Zhang et al., 2007 ). And the systemic action of nitrate is associated with its negative influence on the flow of assimilates to roots (Scheible et al., 1997).

Currently, signaling functions are ascribed not only to nitrate but also to products of nitrate reduction. Depending on the ratio of available carbon and nitrogen in the plant the ratio of oxidized and reduced nitrogen will vary. The influence of the products of nitrate reduction was noted to be opposite to nitrate influence, though the mechanism of their action is also as yet unknown, but supposed to involve glutamine content or glutamine/2-oxoglutarate ratio (Foyer and Noctor, 2002; Stitt et al., 2002).

Since an increase in amount of nitrates in the plant creates conditions favorable for the generation of nitric oxide from nitrite in both enzymatic and non-enzymatic ways (Neill et al., 2003), we can assume that the signaling effects of nitrate are partially realized through the formation of nitric oxide and triggering of NO-signaling system. This is confirmed by found similarities in actions of nitrate and nitric oxide generator, sodium nitroprusside, on assimilate transport and metabolism. However, in contrast to nitrate, nitric oxide preferably activates genes involved in plant defense (Grün et al., 2006).

Actually, the difference of nitrate and nitric oxide actions may be due to differences in the activity of amino acid synthesis, that, as was mentioned above, can also perform signaling roles. Study of the dynamics of gene expression activation under the influence of nitrate showed that many genes induced by nitrate in the first 0-5 and 5-10 minutes are subjected to negative regulation by as early as 20 minutes (Castaings et al., 2011).

Thus, there remains a lot to be elucidated in the signaling mechanism of nitrate and the study of mechanisms of nitrate influence on the transport of sugars can be very promising, not only for this area of research, but also to discovering how the different processes in the plant are interrelated.

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**15** 

*2CREST, JST,* 

*Japan* 

**High-CO2 Response Mechanisms in Microalgae** 

The concentrations of atmospheric CO2 and aquatic inorganic carbon have decreased over geologic time with minor fluctuations. In contrast, O2 concentration has increased through the actions of photosynthetic organisms. Therefore, photosynthetic organisms must adapt to such dramatic environmental change. Aquatic photosynthetic microorganisms, namely eukaryotic microalgae, cyanobacteria, and non-oxygen-evolving photosynthetic bacteria, have developed the ability to utilize CO2 efficiently for photosynthesis because CO2 is a substrate for the primary CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and its related metabolic pathways such as the Calvin– Benson cycle (C3 cycle). As the Rubisco carboxylase reaction is suppressed by elevated O2 concentrations via competition with CO2, photosynthetic organisms have developed special mechanisms for acclimating and adapting to changes in both CO2 and O2 concentrations. Examples of such mechanisms are the microalgal CO2-concentrating mechanisms (CCM), the facilitation of "indirect CO2 supply" with the aid of carbonic anhydrase and dissolved inorganic carbon (DIC)-transporters (see Section 3), and C4 photoysnthesis (for review, see Giordano et al., 2005; Raven, 2010). Many reports on low-CO2-acclimation/adaptation mechanisms have been published, particularly in relation to certain cyanobacteria and unicellular eukaryotes. However, knowledge of high-CO2 acclimation/adaptation mechanisms is very limited. We recently identified an acceptable high-CO2-inducible extracellular marker protein, H43/Fea1 (Hanawa et al., 2007) and a *cis*-element involved in high-CO2-inducible gene expression in the unicellular green alga *Chlamydomonas reinhardtii* (Baba et al., 2011a). We also identified other high-CO2-inducible proteins in the same alga using proteomic analysis (Baba et al. 2011b). In this chapter, we briefly introduce low-CO2-inducible phenomena and mechanisms as background and then review recent progress in elucidating the molecular mechanisms of the high-CO2

The CO2 concentration dissolved in aqueous solution (dCO2) is equilibrated with the partial pressure of atmospheric CO2 (pCO2) by Henry's law and depends on various environmental factors such as temperature, Ca2+ and Mg2+ levels, and salinity (e.g., Falkowski & Raven,

**1. Introduction** 

response in microalgae.

**2. Aquatic inorganic carbon system** 

Masato Baba1,2 and Yoshihiro Shiraiwa1,2 *1Graduate School of Life and Environmental Sciences,* 

*University of Tsukuba, Tsukuba, Ibaraki,* 

Zottini, M., Costa, A., De Michele, R., Ruzzene, M., Carimi, F. & Lo Schiavo, F. (2007). Salicylic acid activates nitric oxide synthesis in Arabidopsis, *J. Exp. Bot*., Vol. 58, pp. 1397-1405, ISSN 0022-0957

### **High-CO2 Response Mechanisms in Microalgae**

Masato Baba1,2 and Yoshihiro Shiraiwa1,2

*1Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 2CREST, JST, Japan* 

#### **1. Introduction**

298 Advances in Photosynthesis – Fundamental Aspects

Zottini, M., Costa, A., De Michele, R., Ruzzene, M., Carimi, F. & Lo Schiavo, F. (2007).

1397-1405, ISSN 0022-0957

Salicylic acid activates nitric oxide synthesis in Arabidopsis, *J. Exp. Bot*., Vol. 58, pp.

The concentrations of atmospheric CO2 and aquatic inorganic carbon have decreased over geologic time with minor fluctuations. In contrast, O2 concentration has increased through the actions of photosynthetic organisms. Therefore, photosynthetic organisms must adapt to such dramatic environmental change. Aquatic photosynthetic microorganisms, namely eukaryotic microalgae, cyanobacteria, and non-oxygen-evolving photosynthetic bacteria, have developed the ability to utilize CO2 efficiently for photosynthesis because CO2 is a substrate for the primary CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and its related metabolic pathways such as the Calvin– Benson cycle (C3 cycle). As the Rubisco carboxylase reaction is suppressed by elevated O2 concentrations via competition with CO2, photosynthetic organisms have developed special mechanisms for acclimating and adapting to changes in both CO2 and O2 concentrations. Examples of such mechanisms are the microalgal CO2-concentrating mechanisms (CCM), the facilitation of "indirect CO2 supply" with the aid of carbonic anhydrase and dissolved inorganic carbon (DIC)-transporters (see Section 3), and C4 photoysnthesis (for review, see Giordano et al., 2005; Raven, 2010). Many reports on low-CO2-acclimation/adaptation mechanisms have been published, particularly in relation to certain cyanobacteria and unicellular eukaryotes. However, knowledge of high-CO2 acclimation/adaptation mechanisms is very limited. We recently identified an acceptable high-CO2-inducible extracellular marker protein, H43/Fea1 (Hanawa et al., 2007) and a *cis*-element involved in high-CO2-inducible gene expression in the unicellular green alga *Chlamydomonas reinhardtii* (Baba et al., 2011a). We also identified other high-CO2-inducible proteins in the same alga using proteomic analysis (Baba et al. 2011b). In this chapter, we briefly introduce low-CO2-inducible phenomena and mechanisms as background and then review recent progress in elucidating the molecular mechanisms of the high-CO2 response in microalgae.

#### **2. Aquatic inorganic carbon system**

The CO2 concentration dissolved in aqueous solution (dCO2) is equilibrated with the partial pressure of atmospheric CO2 (pCO2) by Henry's law and depends on various environmental factors such as temperature, Ca2+ and Mg2+ levels, and salinity (e.g., Falkowski & Raven,

High-CO2 Response Mechanisms in Microalgae 301

The atmospheric CO2 level has gradually decreased over recent geological time with some fluctuations (Condie & Sloan, 1998; Falkowski & Raven, 2007; Giordano et al., 2005; Inoue, 2007), although it has been increasing rapidly due to CO2 emissions from fossil fuels since the industrial revolution. Thus, photosynthetic organisms have adapted to utilize CO2 efficiently for photosynthesis. Generally, eukaryotic microalgae and cyanobacteria have developed efficient CO2-utilization mechanisms and exhibit high photosynthetic affinity for CO2 when grown under CO2-limiting conditions. Under elevated CO2 conditions, they exhibit low affinity for CO2, as enough CO2 is available for photosynthesis. These properties can change over hours when photosynthetic microorganisms are grown under various CO2

Fig. 2. Relationship between photosynthetic rate and external dissolved inorganic carbon (DIC) concentration in microalgae grown under low-, high-, and extremely high-CO2 conditions. A, low-CO2-acclimated cells (grown in air with 0.04% CO2); B, low-CO2 acclimated cells treated with a carbonic anhydrase inhibitor (e.g., *Chlorella*); C, high-CO2 acclimated cells (grown in air containing 1–5% CO2) (e.g., *Chlamydomonas*); D, high-CO2 acclimated cells (e.g., *Chlorella*); D, extremely high-CO2-acclimated cells grown under >40%

High photosynthetic activity of low-CO2-grown cells under a CO2-limiting concentration is due to the CCM, which is induced by cellular acclimation to limiting CO2 (e.g., Aizawa and Miyachi, 1986). Two main factors are essential for CCM: inorganic carbon transporters that facilitate DIC membrane transport of CO2 and/or bicarbonate through the plasmalemma and the chloroplast envelope, and carbonic anhydrases (CAs), which facilitate diffusion by stimulating the indirect supply of CO2 from outside of cells to Rubisco. CA catalyzes the equilibration reaction of the hydration and dehydration of CO2 and bicarbonate, respectively. The rapid equilibration catalyzed by CA stimulates the increase in bicarbonate concentration at physiological pH and augments the contribution of bicarbonate for diffusion. Finally, the processes driven by CA induce increases in the amount of bicarbonate

CO2 conditions. This figure is modified from Miyachi et al. (2003).

**3. The CO2-concentrating mechanism and phenomena induced by** 

conditions (for review, see Miyachi et al., 2003) (Fig. 2).

**CO2 limitation** 

2007). The dCO2 dissociates into bicarbonate (HCO3 - ), and carbonate (CO32-) and these three species of DIC attain equilibrium at a certain ratio depending on pH, ion concentrations, and salinity (Fig. 1). HCO3- is the dominant species at physiological pH (around 8), which is similar to that in the chloroplast stroma where photosynthetic CO2 fixation is actively driven (for review, see Bartlett et al., 2007). However, Rubisco [E.C. 4.1.1.39] reacts only with dCO2, not bicarbonate or carbonate ions. At a pH of 8, the dCO2/HCO3- ratio becomes extremely small (approximately 1/100) resulting in a high bicarbonate concentration and an increase in the total DIC pool size. The dCO2 concentration equilibrates with atmospheric CO2 at approximately 10 μM, whereas the bicarbonate concentration is approximately 2 mM at the surface of the ocean (Falkowski & Raven, 2007).

Fig. 1. Equilibration of dissolved inorganic carbon species in freshwater and seawater. Parameters used were as follows (at 25°C): For freshwater, p*K*a1= 6.35, p*K*a2 = 10.33; for seawater, p*K*a1 = 6.00, p*K*a2 = 9.10 (Table 5.2, Falkowski & Raven, 2007). Filled symbols and solid line, freshwater; clear symbols and dotted line, seawater; diamonds, dCO2; squares, bicarbonate; triangles, carbonate.

CO2 must be supplied rapidly when it is actively fixed by Rubisco in the chloroplast stroma during photosynthesis. CO2 is supplied by both diffusion from outside of cells and the conversion of bicarbonate. However, these processes are very slow and become limiting for photosynthetic CO2 fixation. In the former case, CO2 must be continuously transported from outside of the cells via the cytoplasm through the plasmalemma and the chloroplast envelope. The diffusion rate of CO2 in water is approximately 10,000-fold lower than that in the atmosphere (Jones, 1992). In the latter case, bicarbonate accumulated in the stroma can be a substrate when the dehydration rate to convert bicarbonate to CO2 is comparable to Rubisco activity. However, the rate of chemical equilibration between CO2 and the bicarbonate ion is very slow relative to photosynthetic consumption of CO2 (Badger & Price, 1994; Raven, 2001); the first-order rate constants of hydration (CO2 to bicarbonate) and dehydration (bicarbonate to CO2) are 0.025–0.04 s-1 and 10–20 s-1, respectively, at 25°C (Ishii et al., 2000). Such CO2-limiting stress becomes a motive for photosynthetic organisms to develop unique CO2-response mechanisms.

species of DIC attain equilibrium at a certain ratio depending on pH, ion concentrations, and salinity (Fig. 1). HCO3- is the dominant species at physiological pH (around 8), which is similar to that in the chloroplast stroma where photosynthetic CO2 fixation is actively driven (for review, see Bartlett et al., 2007). However, Rubisco [E.C. 4.1.1.39] reacts only with dCO2, not bicarbonate or carbonate ions. At a pH of 8, the dCO2/HCO3- ratio becomes extremely small (approximately 1/100) resulting in a high bicarbonate concentration and an increase in the total DIC pool size. The dCO2 concentration equilibrates with atmospheric CO2 at approximately 10 μM, whereas the bicarbonate concentration is approximately 2 mM at the

Fig. 1. Equilibration of dissolved inorganic carbon species in freshwater and seawater. Parameters used were as follows (at 25°C): For freshwater, p*K*a1= 6.35, p*K*a2 = 10.33; for seawater, p*K*a1 = 6.00, p*K*a2 = 9.10 (Table 5.2, Falkowski & Raven, 2007). Filled symbols and solid line, freshwater; clear symbols and dotted line, seawater; diamonds, dCO2; squares,

CO2 must be supplied rapidly when it is actively fixed by Rubisco in the chloroplast stroma during photosynthesis. CO2 is supplied by both diffusion from outside of cells and the conversion of bicarbonate. However, these processes are very slow and become limiting for photosynthetic CO2 fixation. In the former case, CO2 must be continuously transported from outside of the cells via the cytoplasm through the plasmalemma and the chloroplast envelope. The diffusion rate of CO2 in water is approximately 10,000-fold lower than that in the atmosphere (Jones, 1992). In the latter case, bicarbonate accumulated in the stroma can be a substrate when the dehydration rate to convert bicarbonate to CO2 is comparable to Rubisco activity. However, the rate of chemical equilibration between CO2 and the bicarbonate ion is very slow relative to photosynthetic consumption of CO2 (Badger & Price, 1994; Raven, 2001); the first-order rate constants of hydration (CO2 to bicarbonate) and dehydration (bicarbonate to CO2) are 0.025–0.04 s-1 and 10–20 s-1, respectively, at 25°C (Ishii et al., 2000). Such CO2-limiting stress becomes a motive for photosynthetic organisms to

), and carbonate (CO32-) and these three

2007). The dCO2 dissociates into bicarbonate (HCO3-

surface of the ocean (Falkowski & Raven, 2007).

bicarbonate; triangles, carbonate.

develop unique CO2-response mechanisms.

#### **3. The CO2-concentrating mechanism and phenomena induced by CO2 limitation**

The atmospheric CO2 level has gradually decreased over recent geological time with some fluctuations (Condie & Sloan, 1998; Falkowski & Raven, 2007; Giordano et al., 2005; Inoue, 2007), although it has been increasing rapidly due to CO2 emissions from fossil fuels since the industrial revolution. Thus, photosynthetic organisms have adapted to utilize CO2 efficiently for photosynthesis. Generally, eukaryotic microalgae and cyanobacteria have developed efficient CO2-utilization mechanisms and exhibit high photosynthetic affinity for CO2 when grown under CO2-limiting conditions. Under elevated CO2 conditions, they exhibit low affinity for CO2, as enough CO2 is available for photosynthesis. These properties can change over hours when photosynthetic microorganisms are grown under various CO2 conditions (for review, see Miyachi et al., 2003) (Fig. 2).

Fig. 2. Relationship between photosynthetic rate and external dissolved inorganic carbon (DIC) concentration in microalgae grown under low-, high-, and extremely high-CO2 conditions. A, low-CO2-acclimated cells (grown in air with 0.04% CO2); B, low-CO2 acclimated cells treated with a carbonic anhydrase inhibitor (e.g., *Chlorella*); C, high-CO2 acclimated cells (grown in air containing 1–5% CO2) (e.g., *Chlamydomonas*); D, high-CO2 acclimated cells (e.g., *Chlorella*); D, extremely high-CO2-acclimated cells grown under >40% CO2 conditions. This figure is modified from Miyachi et al. (2003).

High photosynthetic activity of low-CO2-grown cells under a CO2-limiting concentration is due to the CCM, which is induced by cellular acclimation to limiting CO2 (e.g., Aizawa and Miyachi, 1986). Two main factors are essential for CCM: inorganic carbon transporters that facilitate DIC membrane transport of CO2 and/or bicarbonate through the plasmalemma and the chloroplast envelope, and carbonic anhydrases (CAs), which facilitate diffusion by stimulating the indirect supply of CO2 from outside of cells to Rubisco. CA catalyzes the equilibration reaction of the hydration and dehydration of CO2 and bicarbonate, respectively. The rapid equilibration catalyzed by CA stimulates the increase in bicarbonate concentration at physiological pH and augments the contribution of bicarbonate for diffusion. Finally, the processes driven by CA induce increases in the amount of bicarbonate

High-CO2 Response Mechanisms in Microalgae 303

specificity of Rubisco is a typical example. Even in the present environment, high-CO2 conditions occur in soil where CO2 concentration changes drastically between the atmospheric level and 10% (v/v) (for review, see Buyanovsky & Wagner, 1983; Stolzy, 1974). Accordingly, phenomena that are induced under high-CO2 conditions, such as high-CO2 acclimation, remain important for microalgae to survive in various environments. Among the various phenomena induced by high CO2 concentrations, keenly interesting topics are how to maximize inorganic CO2 fixation and organic production by microalgae for CO2 mitigation and mass cultivation. The most frequently used species for studies on fast growth and tolerance to high CO2 levels is *Chlorella sp.*, followed by *Scenedesmus sp*., *Nannochloropsis sp*., and *Chlorococcum sp*. The CO2 concentration used for such studies varies from atmospheric levels to 100% (Kurano et al., 1995; Maeda et al., 1995; Olaizola, 2003; Seckbach et al., 1970). Appropriate CO2 supply for saturation of microalgal growth is approximately 5% in the unicellular green alga *Chlorella* (Nielsen 1955). The growth of microalgae and cyanobacteria is generally inhibited under very high concentrations of CO2. Some species isolated from extreme environments can grow rapidly with tolerance to very high and extremely high CO2 conditions such as >40% (for review, see Miyachi et al., 2003). Even in a high-CO2-tolerant microalga, growth is suppressed at > 60% CO2 in air (Satoh et al., 2004). The rate of maximum photosynthesis per packed cell volume increases in some species, such as *Chlorella,* but not in other species, such as *Chlamydomonas*, even when cells are acclimated to high-CO2 conditions (Miyachi et al., 2003) (Fig. 2). However, the detailed

Many reports have focused on lipid biosynthesis for biofuel production, and response surface methodology (Box & Wilson, 1951) has been used very effectively to evaluate multiple factors associated with total biomass production. Excellent review articles on largescale cultivation for biofuel production by microalgae and cyanobacteria have focused on how to obtain the best productivity under high-CO2 conditions (Ho et al., 2011; Kumar et al., 2010; Lee J.S. & Lee J.P., 2003), but not on the underlying mechanisms of how cells provide

One of the best examples of sequential analysis was performed systematically in the high-CO2-tolerant unicellular green alga *Chlorococcum littorale* (for review, see Miyachi et al., 2003). *C. littorale* is a unicellular marine chlorophyte that was isolated from a saline pond in Kamaishi City, Japan; it grows rapidly under extremely high CO2 conditions (e.g., 40%, and even at 60% CO2; Chihara et al., 1994; Kodama et al., 1993; Satoh et al., 2004). Several experiments have revealed that cellular responses, namely the regulation of photosystem (PS) I and PS II, the production of ATP, and pH homeostasis are well maintained particularly in *C. littorale*, but not in high-CO2-sensitive species such as the green soil alga *Stichococcus bacillaris*, during a lag period when cells are transferred from low to extremely high levels of CO2 (Demidov et al., 2000; Iwasaki et al., 1996, 1998; Pescheva et al., 1994; Pronina et al., 1993; Sasaki et al., 1999; Satoh et al., 2001, 2002). However, many of the processes that make it possible for cells to grow under such extremely high-CO2 conditions

Photosynthesis in acidic environment, the influence by ocean acidification, and the effect of O2 on photorespiration are also deeply associated with high-CO2-induced phenomena. Some microalgal species have been isolated mainly from acidic environments where only CO2 is predominant and supplied to algal cells as a substrate for photosynthesis (Balkos & Colman, 2007; Colman & Balkos, 2005; Diaz & Maberly, 2009; Verma et al., 2009; for review see Raven, 2010). Three synurophyte algae, *Synura petersenii*, *Synura uvella,* and *Tessellaria* 

mechanism on such high CO2 tolerance needs to be clarified.

high productivity under fine regulation.

remain to be understood.

carried near Rubisco and then CO2 produced from bicarbonate is immediately supplied to Rubisco when CA is located near Rubisco (see also Fig. 4). The relative specificity to CO2/O2 and affinity to CO2 of Rubisco became more efficient over evolutionary time, indicating that Rubisco in eukaryotic microalgae is more efficient for CO2 fixation than that in cyanobacteria (Falkowski & Raven, 2007). Such species-specific properties remain unchanged in present living organisms. However, even in eukaryotic algae, the affinity of Rubisco for CO2 is insufficient to saturate activity at present atmospheric CO2 concentrations. Therefore, the cells continuously activate mechanisms such as CCM to increase their affinity for CO2. For further information on CCM, we recommend reading several previously published reviews (e.g., Aizawa & Miyachi, 1986; Badger et al., 2006; Giordano et al., 2005; Kaplan & Reinhold, 1999; Miyachi et al., 2003; Moroney & Ynalvez, 2007; Raven et al., 2008; Raven, 2010; Spalding, 2008; Yamano & Fukuzawa, 2009).

CCM is reversibly induced/suppressed by the decrease/increase in CO2 concentration, respectively, in cyanobacteria and eukaryotic microalgae when the duration of acclimation is on an hour- or day-order length. However, in the unicellular green alga *Chlamydomonas reinhardtii* in which CCM is the most characterized among eukaryotic microalgae, cells grown for 1000 generations under high-CO2 conditions are unable to re-acclimate to low-CO2 conditions, exhibiting low photosynthetic affinity for CO2 even when the cells are reexposed to low CO2 conditions (Collins & Bell, 2004; Collins et al., 2006). This suggests that CCM can be irreversibly lost when cells undergo prolonged acclimation/adaptation to high-CO2 conditions. Such adaptation has been suggested to occur in natural populations (Collins & Bell, 2006). Although CCM-deficient mutants of cyanobacteria and the green alga *C. reinhardtii* are lethal, such lethality is prevented by elevated CO2 concentration (e.g., Price & Badger, 1989; Spalding et al., 1983; Suzuki & Spalding, 1989). In *C. reinhardtii,* CCM is induced under either 1.2% CO2 in air at 1000 μmol photons m-2 s-1 or 0.04% CO2 in air at 120 μmol photons m-2 s-1, suggesting that CCM induction can be regulated by not only external CO2 concentration but also other signals derived from photorespiratory and/or excess photoenergy stresses, although the detailed mechanisms are not yet known (Yamano et al., 2008). CCM can be induced by an artificially produced strong limitation in CO2 supply in large-scale photobioreactors where dCO2 is consumed via photosynthesis (Yun & Park, 1997).

In some microalgae, the supply of CO2, not bicarbonate, is strongly limited at alkaline pHs in closed culture systems and such a limitation may be a factor or signal for inducing CCM (Colman & Balkos, 2005; Diaz & Maberly, 2009; Verma et al., 2009). The euglenophyte *Euglena mutabilis* and an acid-tolerant strain of *Chlamydomonas* do not induce CCM under any conditions (Balkos & Colman, 2007; Colman & Balkos, 2005), suggesting that photosynthetic carbon fixation is not limited by CO2 supply even under ambient atmospheric conditions. These results indicate that there is species-specific variation in the induction mechanism of CCM depending on physiological and ecological conditions (for review, see Giordano et al., 2005; Raven, 2010).

#### **4. High-CO2 response phenomena**

The atmospheric CO2 level is presumed to have been very high during the ancient geological era (Condie & Sloan, 1998; Falkowski & Raven, 2007; Giordano et al., 2005; Inoue, 2007), so microalgae are believed to have been high-CO2-adapted/acclimated cells. Microalgae preserve their ancient physiological properties at present, and the relative

carried near Rubisco and then CO2 produced from bicarbonate is immediately supplied to Rubisco when CA is located near Rubisco (see also Fig. 4). The relative specificity to CO2/O2 and affinity to CO2 of Rubisco became more efficient over evolutionary time, indicating that Rubisco in eukaryotic microalgae is more efficient for CO2 fixation than that in cyanobacteria (Falkowski & Raven, 2007). Such species-specific properties remain unchanged in present living organisms. However, even in eukaryotic algae, the affinity of Rubisco for CO2 is insufficient to saturate activity at present atmospheric CO2 concentrations. Therefore, the cells continuously activate mechanisms such as CCM to increase their affinity for CO2. For further information on CCM, we recommend reading several previously published reviews (e.g., Aizawa & Miyachi, 1986; Badger et al., 2006; Giordano et al., 2005; Kaplan & Reinhold, 1999; Miyachi et al., 2003; Moroney & Ynalvez,

2007; Raven et al., 2008; Raven, 2010; Spalding, 2008; Yamano & Fukuzawa, 2009).

1997).

review, see Giordano et al., 2005; Raven, 2010).

**4. High-CO2 response phenomena** 

CCM is reversibly induced/suppressed by the decrease/increase in CO2 concentration, respectively, in cyanobacteria and eukaryotic microalgae when the duration of acclimation is on an hour- or day-order length. However, in the unicellular green alga *Chlamydomonas reinhardtii* in which CCM is the most characterized among eukaryotic microalgae, cells grown for 1000 generations under high-CO2 conditions are unable to re-acclimate to low-CO2 conditions, exhibiting low photosynthetic affinity for CO2 even when the cells are reexposed to low CO2 conditions (Collins & Bell, 2004; Collins et al., 2006). This suggests that CCM can be irreversibly lost when cells undergo prolonged acclimation/adaptation to high-CO2 conditions. Such adaptation has been suggested to occur in natural populations (Collins & Bell, 2006). Although CCM-deficient mutants of cyanobacteria and the green alga *C. reinhardtii* are lethal, such lethality is prevented by elevated CO2 concentration (e.g., Price & Badger, 1989; Spalding et al., 1983; Suzuki & Spalding, 1989). In *C. reinhardtii,* CCM is induced under either 1.2% CO2 in air at 1000 μmol photons m-2 s-1 or 0.04% CO2 in air at 120 μmol photons m-2 s-1, suggesting that CCM induction can be regulated by not only external CO2 concentration but also other signals derived from photorespiratory and/or excess photoenergy stresses, although the detailed mechanisms are not yet known (Yamano et al., 2008). CCM can be induced by an artificially produced strong limitation in CO2 supply in large-scale photobioreactors where dCO2 is consumed via photosynthesis (Yun & Park,

In some microalgae, the supply of CO2, not bicarbonate, is strongly limited at alkaline pHs in closed culture systems and such a limitation may be a factor or signal for inducing CCM (Colman & Balkos, 2005; Diaz & Maberly, 2009; Verma et al., 2009). The euglenophyte *Euglena mutabilis* and an acid-tolerant strain of *Chlamydomonas* do not induce CCM under any conditions (Balkos & Colman, 2007; Colman & Balkos, 2005), suggesting that photosynthetic carbon fixation is not limited by CO2 supply even under ambient atmospheric conditions. These results indicate that there is species-specific variation in the induction mechanism of CCM depending on physiological and ecological conditions (for

The atmospheric CO2 level is presumed to have been very high during the ancient geological era (Condie & Sloan, 1998; Falkowski & Raven, 2007; Giordano et al., 2005; Inoue, 2007), so microalgae are believed to have been high-CO2-adapted/acclimated cells. Microalgae preserve their ancient physiological properties at present, and the relative specificity of Rubisco is a typical example. Even in the present environment, high-CO2 conditions occur in soil where CO2 concentration changes drastically between the atmospheric level and 10% (v/v) (for review, see Buyanovsky & Wagner, 1983; Stolzy, 1974). Accordingly, phenomena that are induced under high-CO2 conditions, such as high-CO2 acclimation, remain important for microalgae to survive in various environments.

Among the various phenomena induced by high CO2 concentrations, keenly interesting topics are how to maximize inorganic CO2 fixation and organic production by microalgae for CO2 mitigation and mass cultivation. The most frequently used species for studies on fast growth and tolerance to high CO2 levels is *Chlorella sp.*, followed by *Scenedesmus sp*., *Nannochloropsis sp*., and *Chlorococcum sp*. The CO2 concentration used for such studies varies from atmospheric levels to 100% (Kurano et al., 1995; Maeda et al., 1995; Olaizola, 2003; Seckbach et al., 1970). Appropriate CO2 supply for saturation of microalgal growth is approximately 5% in the unicellular green alga *Chlorella* (Nielsen 1955). The growth of microalgae and cyanobacteria is generally inhibited under very high concentrations of CO2. Some species isolated from extreme environments can grow rapidly with tolerance to very high and extremely high CO2 conditions such as >40% (for review, see Miyachi et al., 2003). Even in a high-CO2-tolerant microalga, growth is suppressed at > 60% CO2 in air (Satoh et al., 2004). The rate of maximum photosynthesis per packed cell volume increases in some species, such as *Chlorella,* but not in other species, such as *Chlamydomonas*, even when cells are acclimated to high-CO2 conditions (Miyachi et al., 2003) (Fig. 2). However, the detailed mechanism on such high CO2 tolerance needs to be clarified.

Many reports have focused on lipid biosynthesis for biofuel production, and response surface methodology (Box & Wilson, 1951) has been used very effectively to evaluate multiple factors associated with total biomass production. Excellent review articles on largescale cultivation for biofuel production by microalgae and cyanobacteria have focused on how to obtain the best productivity under high-CO2 conditions (Ho et al., 2011; Kumar et al., 2010; Lee J.S. & Lee J.P., 2003), but not on the underlying mechanisms of how cells provide high productivity under fine regulation.

One of the best examples of sequential analysis was performed systematically in the high-CO2-tolerant unicellular green alga *Chlorococcum littorale* (for review, see Miyachi et al., 2003). *C. littorale* is a unicellular marine chlorophyte that was isolated from a saline pond in Kamaishi City, Japan; it grows rapidly under extremely high CO2 conditions (e.g., 40%, and even at 60% CO2; Chihara et al., 1994; Kodama et al., 1993; Satoh et al., 2004). Several experiments have revealed that cellular responses, namely the regulation of photosystem (PS) I and PS II, the production of ATP, and pH homeostasis are well maintained particularly in *C. littorale*, but not in high-CO2-sensitive species such as the green soil alga *Stichococcus bacillaris*, during a lag period when cells are transferred from low to extremely high levels of CO2 (Demidov et al., 2000; Iwasaki et al., 1996, 1998; Pescheva et al., 1994; Pronina et al., 1993; Sasaki et al., 1999; Satoh et al., 2001, 2002). However, many of the processes that make it possible for cells to grow under such extremely high-CO2 conditions remain to be understood.

Photosynthesis in acidic environment, the influence by ocean acidification, and the effect of O2 on photorespiration are also deeply associated with high-CO2-induced phenomena. Some microalgal species have been isolated mainly from acidic environments where only CO2 is predominant and supplied to algal cells as a substrate for photosynthesis (Balkos & Colman, 2007; Colman & Balkos, 2005; Diaz & Maberly, 2009; Verma et al., 2009; for review see Raven, 2010). Three synurophyte algae, *Synura petersenii*, *Synura uvella,* and *Tessellaria* 

High-CO2 Response Mechanisms in Microalgae 305

rates even under atmospheric CO2 levels, so we need to carefully optimize growth conditions when we want to obtain high algal growth and production using CO2 enrichment

Microalgae can acclimate to high-CO2 conditions by changing their photosynthetic properties such as CCM. The half-saturation concentration of CO2 for changing cellular photosynthetic characteristics, i.e., CO2 affinity, is 0.5% in the unicellular green alga *Chlorella kessleri* 211-11h (formerly *C. vulgaris* 11h; Shiraiwa & Miyachi, 1985). CCM-related proteins are also degraded simultaneously when cells are transferred from low- to high-CO2 conditions (see references in section 3). Yang et al. (1985) found that, during acclimation to high-CO2 conditions, CA, an essential component of CCM, was passively degraded and

*C. reinhardtii* cells in freshwater and in soil are exposed to drastically fluctuating concentrations of CO2 between atmospheric level and 10% (v/v) (for review, see Stolzy, 1974; Buyanovsky & Wagner, 1983). To grow in such habitats and maintain optimum growth, the alga needs to rapidly change its physiology. Such rapid acclimation was in fact observed in *C. reinhardtii* cells that were successfully acclimated to 20% CO2 within a few days (Hanawa, 2007). The specific growth rate (μ) of *C. reinhardtii* was 0.176 in ordinary air containing 0.04% CO2 where dCO2 and total DIC were 1.62 and 6.19 μM, respectively, at pH 6.8 (Hanawa, 2007) (Fig. 3). Although dCO2 and total DIC concentrations in the culture media, which were equilibrated with 0.3, 1.0, and 3.0% CO2 (v/v) in air, were 28-, 121-, and 489-fold higher than that in ordinary air, respectively, alga-specific growth rates under the respective conditions were only 1.3-, 1.8-, and 1.7-fold higher than that in air (Hanawa, 2007) (Fig. 3). In a wall-less mutant of *C. reinhardtii* CC-400 (same as *CW-15*), the growth rate and the amount of total proteins increased only 1.5-fold even when the CO2 concentration was increased from atmospheric level to 3% (Baba et al., 2011b). These results clearly indicate that, in *C. reinhardtii*, CO2 enrichment is not advantageous to increase in growth rate, as the fully low-CO2-acclimated cells acquire CCM and grow quickly with a near-maximum growth rate even under atmospheric levels of CO2. These results are true when cells are growing logarithmically at low cell density to prevent self-shading. However, when cell density is quite high, the ratio of growth at high to low CO2 is usually quite high. This is probably due to the decrease in growth under air conditions. Under such conditions, CO2 supply is strongly limited resulting in very low growth rates under air-level CO2. Nevertheless, the growth rate does not exceed the specific growth rate obtained at the

High-CO2-grown *C. reinhardtii* declines CCM physiologically by losing CA and active DIC transport systems in order to avoid secondary inhibitory effects caused by excess DIC accumulation (for review, see Miyachi et al., 2003; Spalding, 2008; Yamano & Fukuzawa, 2009) but no other significant responses have been reported until recently. Recently, we found drastic changes in extracellular protein composition (Baba et al., 2011b) including

The wall-less mutant of *C. reinhardtii, CW-15*, releases a large amount of extracellular matrix, including periplasm-locating proteins, named as extracellular proteins, into the medium (Hanawa et al., 2007; Baba et al., 2011b). Our previous studies clearly showed that the extracellular protein composition changes drastically when *C. reinhardtii* cells are transferred

induction of the H43/Fea1 protein (Hanawa et al., 2004, 2007; Kobayashi et al., 1997).

**5. Molecular mechanisms for high-CO2 responses** 

(see also section 5).

thus the process took almost 1 week.

logarithmic growth stage.

*volvocina*, have been studied in detail for the DIC uptake mechanism and show unique photosynthetic properties (Bhatti & Coleman, 2008). These species have no external carbonic anhydrase on the cell surface, no bicarbonate uptake ability, and exhibit a low affinity for DIC during photosynthesis, indicating a lack of CCM as in high-CO2-grown/acclimated cells. However, their Rubisco shows a relatively high affinity for CO2, and cells such as *S. petersenii* accumulate large amounts of internal DIC via diffusive uptake of CO2 facilitated by a pH gradient across the cell membranes, as reported previously in spinach chloroplasts (Heldt et al., 1973). These data suggest that the affinity of Rubisco for CO2 and the homeostasis of the pH gradient play key roles in the whole-cell affinity for CO2 and the pHtolerance of microalgae. Under high-CO2 conditions, Rubisco can get enough CO2 supply although CCM is usually lost in high-CO2 cells. The physiological status of synurophyte algae living at acidic pH may be similar to cells that are exposed to high-CO2 conditions even under low-CO2 conditions.

Increasing pCO2 induces a decrease in oceanic pH and causes gradual equilibrium shifts from bicarbonate ions to CO2 in seawater. Therefore, ocean acidification is said to be another high-CO2 problem (for review, see Doney et al., 2008). Coccolithophorids, marine phytoplankton that form cells covered with CaCO3, are very sensitive to calcium carbonate saturation and pH shifts in seawater. The effects of ocean acidification on algal physiology have been studied in several coccolithophorid species such as *Emiliania huxleyi* and *Pleurochrysis carterae,* although some conflicting results have been reported (Fukuda et al., 2011; Igresiaz-Rhodorigez et al., 2008; Riebesell et al., 2000). Hurd et al. (2009) indicated the importance of maitaining pH in experiments and demonstrated that doing so via high-CO2 bubbling creates conditions that are much closer to actual ocean acidification than acidification by adding HCl. The effects of high-CO2 conditions on calcification and photosynthesis would be closed up in later analyses. Fukuda et al. (2011) reported that the coccolithophorid *E. huxleyi* possesses alkalization activity, which helps compensate for acidification when photosynthesis is actively driven. Furthermore, when oceanic acidification is caused by the bubbling of air with elevated CO2, coccolithophorid cells increase both photosynthetic activity and growth and are not damaged because of the stimulation of photosynthesis (unpublished data by S. Fukuda, Y. Suzuki & Y. Shiraiwa). These results suggest that ocean acidification will not immediately harm coccolithophorids. However, long-term experimental evidence is strongly required on this topic.

Badger et al. (2000) described how low-CO2-grown microalgae tend to have low photorespiratory activity, as determined by photosynthetic O2 uptake in C4 plants because of the function of CCM. O2 uptake under illumination is relatively insensitive to changes in CO2 concentration, because the activity depends predominantly on the activity of nonphotorespiratory reactions probably such as the Mehler reaction and oxidizing reaction in the mitochondria (Badger et al., 2000). CO2 insensitivity is also observed in *C. reinhardtii*  (Sültemeyer et al., 1987) although photosynthetic O2 uptake increases considerably with increasing light intensity (Sültemeyer et al., 1986). Accordingly, the photosynthetic productivity of microalgae may not be significantly enhanced by suppressing photorespiration. The rate of maximum photosynthesis, calculated on a cell volume, increases clearly in *Chlorella* but not so in *Chlamydomonas* when cells are acclimated to high-CO2 conditions (Miyachi et al., 2003) (Fig. 2). In *C. reinhardtii*, growth rate is only slightly higher (1.3–1.8-fold) in cells grown under high-CO2 than in those grown under ordinary air (Baba et al., 2011b; Hanawa, 2007). These results suggest that low-CO2-acclimated/grown cells have a very highly efficient carbon-fixation mechanism for maintaining high growth

*volvocina*, have been studied in detail for the DIC uptake mechanism and show unique photosynthetic properties (Bhatti & Coleman, 2008). These species have no external carbonic anhydrase on the cell surface, no bicarbonate uptake ability, and exhibit a low affinity for DIC during photosynthesis, indicating a lack of CCM as in high-CO2-grown/acclimated cells. However, their Rubisco shows a relatively high affinity for CO2, and cells such as *S. petersenii* accumulate large amounts of internal DIC via diffusive uptake of CO2 facilitated by a pH gradient across the cell membranes, as reported previously in spinach chloroplasts (Heldt et al., 1973). These data suggest that the affinity of Rubisco for CO2 and the homeostasis of the pH gradient play key roles in the whole-cell affinity for CO2 and the pHtolerance of microalgae. Under high-CO2 conditions, Rubisco can get enough CO2 supply although CCM is usually lost in high-CO2 cells. The physiological status of synurophyte algae living at acidic pH may be similar to cells that are exposed to high-CO2 conditions

Increasing pCO2 induces a decrease in oceanic pH and causes gradual equilibrium shifts from bicarbonate ions to CO2 in seawater. Therefore, ocean acidification is said to be another high-CO2 problem (for review, see Doney et al., 2008). Coccolithophorids, marine phytoplankton that form cells covered with CaCO3, are very sensitive to calcium carbonate saturation and pH shifts in seawater. The effects of ocean acidification on algal physiology have been studied in several coccolithophorid species such as *Emiliania huxleyi* and *Pleurochrysis carterae,* although some conflicting results have been reported (Fukuda et al., 2011; Igresiaz-Rhodorigez et al., 2008; Riebesell et al., 2000). Hurd et al. (2009) indicated the importance of maitaining pH in experiments and demonstrated that doing so via high-CO2 bubbling creates conditions that are much closer to actual ocean acidification than acidification by adding HCl. The effects of high-CO2 conditions on calcification and photosynthesis would be closed up in later analyses. Fukuda et al. (2011) reported that the coccolithophorid *E. huxleyi* possesses alkalization activity, which helps compensate for acidification when photosynthesis is actively driven. Furthermore, when oceanic acidification is caused by the bubbling of air with elevated CO2, coccolithophorid cells increase both photosynthetic activity and growth and are not damaged because of the stimulation of photosynthesis (unpublished data by S. Fukuda, Y. Suzuki & Y. Shiraiwa). These results suggest that ocean acidification will not immediately harm coccolithophorids.

However, long-term experimental evidence is strongly required on this topic.

Badger et al. (2000) described how low-CO2-grown microalgae tend to have low photorespiratory activity, as determined by photosynthetic O2 uptake in C4 plants because of the function of CCM. O2 uptake under illumination is relatively insensitive to changes in CO2 concentration, because the activity depends predominantly on the activity of nonphotorespiratory reactions probably such as the Mehler reaction and oxidizing reaction in the mitochondria (Badger et al., 2000). CO2 insensitivity is also observed in *C. reinhardtii*  (Sültemeyer et al., 1987) although photosynthetic O2 uptake increases considerably with increasing light intensity (Sültemeyer et al., 1986). Accordingly, the photosynthetic productivity of microalgae may not be significantly enhanced by suppressing photorespiration. The rate of maximum photosynthesis, calculated on a cell volume, increases clearly in *Chlorella* but not so in *Chlamydomonas* when cells are acclimated to high-CO2 conditions (Miyachi et al., 2003) (Fig. 2). In *C. reinhardtii*, growth rate is only slightly higher (1.3–1.8-fold) in cells grown under high-CO2 than in those grown under ordinary air (Baba et al., 2011b; Hanawa, 2007). These results suggest that low-CO2-acclimated/grown cells have a very highly efficient carbon-fixation mechanism for maintaining high growth

even under low-CO2 conditions.

rates even under atmospheric CO2 levels, so we need to carefully optimize growth conditions when we want to obtain high algal growth and production using CO2 enrichment (see also section 5).

#### **5. Molecular mechanisms for high-CO2 responses**

Microalgae can acclimate to high-CO2 conditions by changing their photosynthetic properties such as CCM. The half-saturation concentration of CO2 for changing cellular photosynthetic characteristics, i.e., CO2 affinity, is 0.5% in the unicellular green alga *Chlorella kessleri* 211-11h (formerly *C. vulgaris* 11h; Shiraiwa & Miyachi, 1985). CCM-related proteins are also degraded simultaneously when cells are transferred from low- to high-CO2 conditions (see references in section 3). Yang et al. (1985) found that, during acclimation to high-CO2 conditions, CA, an essential component of CCM, was passively degraded and thus the process took almost 1 week.

*C. reinhardtii* cells in freshwater and in soil are exposed to drastically fluctuating concentrations of CO2 between atmospheric level and 10% (v/v) (for review, see Stolzy, 1974; Buyanovsky & Wagner, 1983). To grow in such habitats and maintain optimum growth, the alga needs to rapidly change its physiology. Such rapid acclimation was in fact observed in *C. reinhardtii* cells that were successfully acclimated to 20% CO2 within a few days (Hanawa, 2007). The specific growth rate (μ) of *C. reinhardtii* was 0.176 in ordinary air containing 0.04% CO2 where dCO2 and total DIC were 1.62 and 6.19 μM, respectively, at pH 6.8 (Hanawa, 2007) (Fig. 3). Although dCO2 and total DIC concentrations in the culture media, which were equilibrated with 0.3, 1.0, and 3.0% CO2 (v/v) in air, were 28-, 121-, and 489-fold higher than that in ordinary air, respectively, alga-specific growth rates under the respective conditions were only 1.3-, 1.8-, and 1.7-fold higher than that in air (Hanawa, 2007) (Fig. 3). In a wall-less mutant of *C. reinhardtii* CC-400 (same as *CW-15*), the growth rate and the amount of total proteins increased only 1.5-fold even when the CO2 concentration was increased from atmospheric level to 3% (Baba et al., 2011b). These results clearly indicate that, in *C. reinhardtii*, CO2 enrichment is not advantageous to increase in growth rate, as the fully low-CO2-acclimated cells acquire CCM and grow quickly with a near-maximum growth rate even under atmospheric levels of CO2. These results are true when cells are growing logarithmically at low cell density to prevent self-shading. However, when cell density is quite high, the ratio of growth at high to low CO2 is usually quite high. This is probably due to the decrease in growth under air conditions. Under such conditions, CO2 supply is strongly limited resulting in very low growth rates under air-level CO2. Nevertheless, the growth rate does not exceed the specific growth rate obtained at the logarithmic growth stage.

High-CO2-grown *C. reinhardtii* declines CCM physiologically by losing CA and active DIC transport systems in order to avoid secondary inhibitory effects caused by excess DIC accumulation (for review, see Miyachi et al., 2003; Spalding, 2008; Yamano & Fukuzawa, 2009) but no other significant responses have been reported until recently. Recently, we found drastic changes in extracellular protein composition (Baba et al., 2011b) including induction of the H43/Fea1 protein (Hanawa et al., 2004, 2007; Kobayashi et al., 1997).

The wall-less mutant of *C. reinhardtii, CW-15*, releases a large amount of extracellular matrix, including periplasm-locating proteins, named as extracellular proteins, into the medium (Hanawa et al., 2007; Baba et al., 2011b). Our previous studies clearly showed that the extracellular protein composition changes drastically when *C. reinhardtii* cells are transferred

High-CO2 Response Mechanisms in Microalgae 307

enough or was still missing some necessary factors to trigger mating. Otherwise, these gametogenesis-related protein families and/or HRGPs may have another function under

The biological meaning of the expression of gametogenesis-related proteins at the stage of vegetative growth is quite mysterious. CCM may be differentially regulated by changes in nitrogen availability, depending on the species (for review, see Giordano et al., 2005). In *C. reinhardtii*, mildly limited nitrogen availability suppresses CCM and mitochondrial β-CA

efficiency of photosynthetic CO2 utilization (Beardall & Giordano, 2002). From these results, Giordano et al. (2005) suggested that the induction of CCM and related phenomena induced by CO2 limitation is regulated to satisfy an adequate C/N ratio. Basically, cells growing under high-CO2 conditions may require more nitrogen, at least no less than low-CO2 acclimated cells, and tend to attain nitrogen-limitation status easily. In contrast, Giordano et al. (2005) suggested that activating CCM may reduce the loss of nitrogen through the photorespiratory nitrogen cycle. Namely, NH4+ produced by converting Gly to Ser through the C2 cycle in mitochondria is transported to and re-fixed in the chloroplasts by the GS2/GOGAT cycle where chloroplastic GS2 is induced in response to CO2 concentration in *C. reinhardtii* (Ramazanov & Cárdenas, 1994). In previous works, the NH4+ excretion rate from algal cells was lower in high-CO2 cells than in low-CO2 cells when monitored in the presence of 1 mM 1-methionine sulfoximine, a specific inhibitor of GS activity, to prevent refixation of NH4+ in *C. reinhardtii CW-15* (Ramazanov & Cárdenas, 1994) and similarly in *C. vulgaris* 211-11h (Shiraiwa & Schmid, 1986). A decrease in the intracellular NH4+ level was first reported to induce gametogenesis-related genes in *C. reinhardtii* (Matsuda et al., 1992). Thus, it is reasonable to hypothesize that gametogenesis is triggered by a decrease in intracellular NH4+ levels under high-CO2 conditions when photorespiration is suppressed. However, further study is required, as photorespiratory activity in *C. reinhardtii* is very low

Another report suggested the close participation of CO2 in inorganic nitrogen assimilation (for review, see Fernández et al., 2009). LCIA, or NAR1.2, is involved in the bicarbonate transport system in chloroplasts (Duanmu et al., 2009) but is not regulated by nitrogen availability, and has been identified as a low-CO2-inducible gene by expressed sequence tag (EST) analysis (Miura et al., 2004). However, NAR1 genes generally involve members of the formate/nitrite transporter family (Rexach et al., 2000). In fact, LCIA-expressing *Xenopus* oocytes display both low-affinity bicarbonate transport and high-affinity nitrite transport activities (Mariscal et al., 2006), suggesting that LCIA is involved in both bicarbonate uptake and nitrite uptake induced under low-CO2 conditions. In other words, the suppression of LCIA by high-CO2 conditions may reduce nitrogen availability in the chloroplast. Additionally, the molecular structure of the high-affinity-bicarbonate transporter *cmpABCD* is very similar to that of the nitrate/nitrite transporter *nrtABCD* in *Synechococcus* sp. PCC7942 (for review, see Badger & Price, 2003). The expression of highaffinity nitrate and nitrite transporter (HANT/HANiT) system IV is triggered by a sensing signal of low CO2 but not NH4+ (Galván et al., 1996; Rexach et al., 1999). These data suggest that changes in CO2 concentration may also affect intracellular nitrogen availability. Further study should be conducted to identify the cooperative effect of CO2 and nitrogen availability on the expression of CO2, nitrogen, and gametogenesis-

+ concentration promotes the

expression (Giordano et al., 2003) and the increase in NH4

high-CO2 conditions.

(Badger et al., 2000).

responsive proteins.

Fig. 3. Relationship between the specific growth rate and dCO2 concentration in an airbubbled culture of *Chlamydomonas reinhardtii* (A), and the concentrations of three dissolve inorganic carbon (DIC) species in the culture (B). The concentration of dCO2 was experimentally determined. Each DIC species was calculated by Henley's law and the Henderson–Hasselbalch equation, respectively. The parameters were as follows (for freshwater at 25°C): pKa1 = 6.35, pka2 = 10.33. The culture medium used was a high salt medium supplemented with 30 mM MOPS (pH 6.8). Crosses, specific growth rate; diamonds, dCO2; circles, total DIC; squares, bicarbonate; triangles, carbonate. Fig. 3A is modified from Hanawa, 2007.

from atmospheric air to 3% CO2 in air (Hanawa et al., 2004, 2007; Kobayashi et al., 1997), whereas an SDS-PAGE profile of intracellular-soluble and -insoluble proteins showed no clear difference (Baba et al., 2011b). Recently, we analyzed 129 proteins by proteomic analysis and identified 22 high-CO2-inducible proteins from *C. reinhardtii* cells transferred from low- to high-CO2 conditions (Baba et al., 2011b). These high-CO2-inducible proteins are multiple extracellular hydroxyproline-rich glycoproteins (HRGPs), such as nitrogen-starved gametogenesis (NSG) protein (Abe et al., 2004), inversion-specific glycoprotein (ISG) (Ertl et al., 1992), and cell wall glycoprotein (GP) (Goodenough et al., 1986), together with sexual pherophorin (PHC) (Hallmann, 2006), gamete-specific (GAS) protein (Hoffmann & Beck, 2005), and gamete-lytic enzymes (Buchanan & Snell, 1988; Kinoshita et al., 1992; Kubo et al., 2001). Both GP and ISG are classified as HRGPs together with PHC, GAS, and sexual agglutinin with a shared origin (Adair, 1985). HRGPs are generally involved in sexual recognition of mating-type, plus or minus gametes, in the *Chlamydomonas* lineage (Lee et al., 2007). Among these proteins, NSG, GAS, and gamete-lytic enzymes are generally known to be induced during the gametogenetic process. The sexual program, including gametogenesis in *Chlamydomonas*, is strictly regulated by nitrogen availability (for review, see Goodenough et al., 2007). Drastic changes in the expression of gametogenesis-related extracellular proteins were clearly observed in *C. reinhardtii* cells in response to high-CO2 but not to environmental nitrogen concentrations, because the experiment was performed under nitrogen-sufficient conditions (Baba et al., 2011b). No visible effect of high-CO2 signal alone was observed on mating (Baba et al., 2011b). From these results, we concluded that the high-CO2 signal induced gametogenesis-related proteins but that the signal was not strong

Fig. 3. Relationship between the specific growth rate and dCO2 concentration in an airbubbled culture of *Chlamydomonas reinhardtii* (A), and the concentrations of three dissolve

(for freshwater at 25°C): pKa1 = 6.35, pka2 = 10.33. The culture medium used was a high salt

from atmospheric air to 3% CO2 in air (Hanawa et al., 2004, 2007; Kobayashi et al., 1997), whereas an SDS-PAGE profile of intracellular-soluble and -insoluble proteins showed no clear difference (Baba et al., 2011b). Recently, we analyzed 129 proteins by proteomic analysis and identified 22 high-CO2-inducible proteins from *C. reinhardtii* cells transferred from low- to high-CO2 conditions (Baba et al., 2011b). These high-CO2-inducible proteins are multiple extracellular hydroxyproline-rich glycoproteins (HRGPs), such as nitrogen-starved gametogenesis (NSG) protein (Abe et al., 2004), inversion-specific glycoprotein (ISG) (Ertl et al., 1992), and cell wall glycoprotein (GP) (Goodenough et al., 1986), together with sexual pherophorin (PHC) (Hallmann, 2006), gamete-specific (GAS) protein (Hoffmann & Beck, 2005), and gamete-lytic enzymes (Buchanan & Snell, 1988; Kinoshita et al., 1992; Kubo et al., 2001). Both GP and ISG are classified as HRGPs together with PHC, GAS, and sexual agglutinin with a shared origin (Adair, 1985). HRGPs are generally involved in sexual recognition of mating-type, plus or minus gametes, in the *Chlamydomonas* lineage (Lee et al., 2007). Among these proteins, NSG, GAS, and gamete-lytic enzymes are generally known to be induced during the gametogenetic process. The sexual program, including gametogenesis in *Chlamydomonas*, is strictly regulated by nitrogen availability (for review, see Goodenough et al., 2007). Drastic changes in the expression of gametogenesis-related extracellular proteins were clearly observed in *C. reinhardtii* cells in response to high-CO2 but not to environmental nitrogen concentrations, because the experiment was performed under nitrogen-sufficient conditions (Baba et al., 2011b). No visible effect of high-CO2 signal alone was observed on mating (Baba et al., 2011b). From these results, we concluded that the high-CO2 signal induced gametogenesis-related proteins but that the signal was not strong

inorganic carbon (DIC) species in the culture (B). The concentration of dCO2 was experimentally determined. Each DIC species was calculated by Henley's law and the Henderson–Hasselbalch equation, respectively. The parameters were as follows

medium supplemented with 30 mM MOPS (pH 6.8). Crosses, specific growth rate; diamonds, dCO2; circles, total DIC; squares, bicarbonate; triangles, carbonate.

Fig. 3A is modified from Hanawa, 2007.

enough or was still missing some necessary factors to trigger mating. Otherwise, these gametogenesis-related protein families and/or HRGPs may have another function under high-CO2 conditions.

The biological meaning of the expression of gametogenesis-related proteins at the stage of vegetative growth is quite mysterious. CCM may be differentially regulated by changes in nitrogen availability, depending on the species (for review, see Giordano et al., 2005). In *C. reinhardtii*, mildly limited nitrogen availability suppresses CCM and mitochondrial β-CA expression (Giordano et al., 2003) and the increase in NH4 + concentration promotes the efficiency of photosynthetic CO2 utilization (Beardall & Giordano, 2002). From these results, Giordano et al. (2005) suggested that the induction of CCM and related phenomena induced by CO2 limitation is regulated to satisfy an adequate C/N ratio. Basically, cells growing under high-CO2 conditions may require more nitrogen, at least no less than low-CO2 acclimated cells, and tend to attain nitrogen-limitation status easily. In contrast, Giordano et al. (2005) suggested that activating CCM may reduce the loss of nitrogen through the photorespiratory nitrogen cycle. Namely, NH4+ produced by converting Gly to Ser through the C2 cycle in mitochondria is transported to and re-fixed in the chloroplasts by the GS2/GOGAT cycle where chloroplastic GS2 is induced in response to CO2 concentration in *C. reinhardtii* (Ramazanov & Cárdenas, 1994). In previous works, the NH4 + excretion rate from algal cells was lower in high-CO2 cells than in low-CO2 cells when monitored in the presence of 1 mM 1-methionine sulfoximine, a specific inhibitor of GS activity, to prevent refixation of NH4+ in *C. reinhardtii CW-15* (Ramazanov & Cárdenas, 1994) and similarly in *C. vulgaris* 211-11h (Shiraiwa & Schmid, 1986). A decrease in the intracellular NH4+ level was first reported to induce gametogenesis-related genes in *C. reinhardtii* (Matsuda et al., 1992). Thus, it is reasonable to hypothesize that gametogenesis is triggered by a decrease in intracellular NH4+ levels under high-CO2 conditions when photorespiration is suppressed. However, further study is required, as photorespiratory activity in *C. reinhardtii* is very low (Badger et al., 2000).

Another report suggested the close participation of CO2 in inorganic nitrogen assimilation (for review, see Fernández et al., 2009). LCIA, or NAR1.2, is involved in the bicarbonate transport system in chloroplasts (Duanmu et al., 2009) but is not regulated by nitrogen availability, and has been identified as a low-CO2-inducible gene by expressed sequence tag (EST) analysis (Miura et al., 2004). However, NAR1 genes generally involve members of the formate/nitrite transporter family (Rexach et al., 2000). In fact, LCIA-expressing *Xenopus* oocytes display both low-affinity bicarbonate transport and high-affinity nitrite transport activities (Mariscal et al., 2006), suggesting that LCIA is involved in both bicarbonate uptake and nitrite uptake induced under low-CO2 conditions. In other words, the suppression of LCIA by high-CO2 conditions may reduce nitrogen availability in the chloroplast. Additionally, the molecular structure of the high-affinity-bicarbonate transporter *cmpABCD* is very similar to that of the nitrate/nitrite transporter *nrtABCD* in *Synechococcus* sp. PCC7942 (for review, see Badger & Price, 2003). The expression of highaffinity nitrate and nitrite transporter (HANT/HANiT) system IV is triggered by a sensing signal of low CO2 but not NH4+ (Galván et al., 1996; Rexach et al., 1999). These data suggest that changes in CO2 concentration may also affect intracellular nitrogen availability. Further study should be conducted to identify the cooperative effect of CO2 and nitrogen availability on the expression of CO2, nitrogen, and gametogenesisresponsive proteins.

High-CO2 Response Mechanisms in Microalgae 309

human Rhesus protein in a homology search and is a paralog of the ammonium and/or CO2 channels (Soupene et al., 2002). The lack of Rh1 impairs cell growth in *C. reinhardtii* under high-CO2 conditions (Soupene et al., 2004). Fong et al. (2007) proposed that Rh proteins served as H2CO3 transporters in *Escherichia coli* under high-CO2 conditions. Rh1 was originally expected to be located on the chloroplast envelope *in silico* but the Rh1-GFP fusion protein is located in the plasma membrane in transgenic *C. reinhardtii* cells (Yoshihara et al.,

Some mechanisms of CCM, the photorespiratory nitrogen cycle, and the nitrate/nitrite transport system, and the interactions among them, are summarized in relation to high- and

How can microalgal cells sense the CO2 signal and respond to changes in CO2 concentration? The most abundant extracellular carbonic anhydrase, CAH1, in low-CO2 cells is replaced by high-CO2-inducible extracellular 43 kDa protein/Fe-assimilation 1 (H43/FEA1) when low-CO2-cells are transferred to high-CO2 conditions (Allen et al., 2007; Baba et al., 2011a; Hanawa et al., 2004, 2007; Kobayashi et al., 1997). We found that H43/FEA1 was the most abundant extracellular soluble protein, which occupied about 26% of the total extracellular proteins of high (3%)-CO2-grown cells for 3 days (Baba et al., 2011b). *H43/FEA1* homologous genes are found in the genomic sequences of the chlorophytes *Scenedesmus obliquus*, *Chlorococcum littorale*, and *Volvox carteri*, and the dinoflagellate *Heterocapsa triquerta* (Allen et al., 2007). This suggests that the *H43/FEA1* 

The function of H43/FEA1 is not completely understood but one possible role may be in iron assimilation (Allen et al., 2007; Rubinelli et al., 2002). Allen et al. (2007) identified *FEA1*, *FEA2*, and a candidate ferrireductase (*FRE1*) are expressed coordinately with iron assimilation components, and it was hypothesized that the proteins may facilitate iron uptake with high affinity by concentrating iron in the vicinity of the cells (Allen et al., 2007). *FEA1* and *FRE1* homologs were previously identified as the high-CO2-responsive genes *HCR1* and *HCR2* in the marine chlorophyte *C. littorale*, suggesting that the components of the iron-assimilation pathway are responsive to changes in CO2 concentration (Sasaki et al., 1998). A homology search of DNA sequences showed that *H43, FEA1,* and *HCR1* are identical (Allen et al., 2007; Hanawa et al., 2007), indicating that *H43/FEA1* expression was also induced by iron deficiency with transcriptional regulation. Therefore, we proposed that

In *C. reinhardtii*, 0.3% (v/v) CO2 in air is sufficient to trigger the expression of the high-CO2 inducible *H43/FEA1* and expression is correlated linearly between 0.04% and 0.3% (Hanawa et al., 2007). *H43/FEA1* can also be induced under heterotrophic conditions in the presence of acetate as an organic carbon source even under low-CO2 conditions (Hanawa et al., 2007). In a previous study, the dCO2 concentration in a cell suspension increased about 28 times from 1 to approximately 28 μM, which was identical to that equilibrated under the bubbling of 0.22% CO2 in light, when cells were incubated in the presence of acetate and 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) (Hanawa et al., 2007). From these data, the authors concluded that the induction of *H43/FEA1* is triggered by the CO2 signal, even CO2 generated from respiration, but not acetate itself or the change in carbon metabolite

orthologs may be widely distributed among at least chlorophyte algae.

the gene is expressed as *H43/FEA1* (Baba et al., 2011a, 2011b*)*.

2008).

low-CO2-acclimated cells in Figure 4.

**6. High-CO2 signaling** 

Fig. 4. Schematic illustration of a C/N-status model in low- (A) and high-CO2-acclimated cells (B) under respective CO2 conditions produced during acclimation in *C. reinhardtii*. Dissolved inorganic carbon and nitrogen species drawn in bold dominate. CA, carbonic anhydrases; CA2, CAH2 (Fujiwara et al., 1990; Rawat & Moroney, 1991; Tachiki et al., 1992); NiR, nitrite reductase; NR, nitrate reductase; PG, 2-phosphoglycolate; PGA, 3 phosphoglycerate; PSII, photosystem II; Rh, Rh1 (Soupene et al., 2002; Yoshihara et al., 2008); T, (putative) transporters; Ta, LCIA (Duanmu et al., 2009; Mariscal et al., 2006); Tb, HANT/HANiT system IV (Galván et al., 1996; Rexach et al., 1999). CCM models of WT/LC cells, inorganic nitrogen assimilation, and photorespiratory carbon oxidation in *C. reinhardtii* are modified from Yamano et al. (2010), Fernández et al. (2009), and Spalding (2009), respectively.

CAH2 was first reported as an active α-type carbonic anhydrase induced under high-CO2 conditions and light (Fujiwara et al., 1990; Rawat & Moroney, 1991; Tachiki et al., 1992), but it is poorly expressed and located in the periplasmic space (Rawat & Moroney, 1991). However, the physiological roles and expressional regulation of high-CO2-inducible CAH2 are not well understood. Another high-CO2-inducible protein, Rh1, has been identified as a human Rhesus protein in a homology search and is a paralog of the ammonium and/or CO2 channels (Soupene et al., 2002). The lack of Rh1 impairs cell growth in *C. reinhardtii* under high-CO2 conditions (Soupene et al., 2004). Fong et al. (2007) proposed that Rh proteins served as H2CO3 transporters in *Escherichia coli* under high-CO2 conditions. Rh1 was originally expected to be located on the chloroplast envelope *in silico* but the Rh1-GFP fusion protein is located in the plasma membrane in transgenic *C. reinhardtii* cells (Yoshihara et al., 2008).

Some mechanisms of CCM, the photorespiratory nitrogen cycle, and the nitrate/nitrite transport system, and the interactions among them, are summarized in relation to high- and low-CO2-acclimated cells in Figure 4.

#### **6. High-CO2 signaling**

308 Advances in Photosynthesis – Fundamental Aspects

Fig. 4. Schematic illustration of a C/N-status model in low- (A) and high-CO2-acclimated cells (B) under respective CO2 conditions produced during acclimation in *C. reinhardtii*. Dissolved inorganic carbon and nitrogen species drawn in bold dominate. CA, carbonic anhydrases; CA2, CAH2 (Fujiwara et al., 1990; Rawat & Moroney, 1991; Tachiki et al., 1992);

phosphoglycerate; PSII, photosystem II; Rh, Rh1 (Soupene et al., 2002; Yoshihara et al., 2008); T, (putative) transporters; Ta, LCIA (Duanmu et al., 2009; Mariscal et al., 2006); Tb, HANT/HANiT system IV (Galván et al., 1996; Rexach et al., 1999). CCM models of WT/LC cells, inorganic nitrogen assimilation, and photorespiratory carbon oxidation in *C. reinhardtii* are modified from Yamano et al. (2010), Fernández et al. (2009), and Spalding (2009),

CAH2 was first reported as an active α-type carbonic anhydrase induced under high-CO2 conditions and light (Fujiwara et al., 1990; Rawat & Moroney, 1991; Tachiki et al., 1992), but it is poorly expressed and located in the periplasmic space (Rawat & Moroney, 1991). However, the physiological roles and expressional regulation of high-CO2-inducible CAH2 are not well understood. Another high-CO2-inducible protein, Rh1, has been identified as a

NiR, nitrite reductase; NR, nitrate reductase; PG, 2-phosphoglycolate; PGA, 3-

respectively.

How can microalgal cells sense the CO2 signal and respond to changes in CO2 concentration? The most abundant extracellular carbonic anhydrase, CAH1, in low-CO2 cells is replaced by high-CO2-inducible extracellular 43 kDa protein/Fe-assimilation 1 (H43/FEA1) when low-CO2-cells are transferred to high-CO2 conditions (Allen et al., 2007; Baba et al., 2011a; Hanawa et al., 2004, 2007; Kobayashi et al., 1997). We found that H43/FEA1 was the most abundant extracellular soluble protein, which occupied about 26% of the total extracellular proteins of high (3%)-CO2-grown cells for 3 days (Baba et al., 2011b). *H43/FEA1* homologous genes are found in the genomic sequences of the chlorophytes *Scenedesmus obliquus*, *Chlorococcum littorale*, and *Volvox carteri*, and the dinoflagellate *Heterocapsa triquerta* (Allen et al., 2007). This suggests that the *H43/FEA1*  orthologs may be widely distributed among at least chlorophyte algae.

The function of H43/FEA1 is not completely understood but one possible role may be in iron assimilation (Allen et al., 2007; Rubinelli et al., 2002). Allen et al. (2007) identified *FEA1*, *FEA2*, and a candidate ferrireductase (*FRE1*) are expressed coordinately with iron assimilation components, and it was hypothesized that the proteins may facilitate iron uptake with high affinity by concentrating iron in the vicinity of the cells (Allen et al., 2007). *FEA1* and *FRE1* homologs were previously identified as the high-CO2-responsive genes *HCR1* and *HCR2* in the marine chlorophyte *C. littorale*, suggesting that the components of the iron-assimilation pathway are responsive to changes in CO2 concentration (Sasaki et al., 1998). A homology search of DNA sequences showed that *H43, FEA1,* and *HCR1* are identical (Allen et al., 2007; Hanawa et al., 2007), indicating that *H43/FEA1* expression was also induced by iron deficiency with transcriptional regulation. Therefore, we proposed that the gene is expressed as *H43/FEA1* (Baba et al., 2011a, 2011b*)*.

In *C. reinhardtii*, 0.3% (v/v) CO2 in air is sufficient to trigger the expression of the high-CO2 inducible *H43/FEA1* and expression is correlated linearly between 0.04% and 0.3% (Hanawa et al., 2007). *H43/FEA1* can also be induced under heterotrophic conditions in the presence of acetate as an organic carbon source even under low-CO2 conditions (Hanawa et al., 2007). In a previous study, the dCO2 concentration in a cell suspension increased about 28 times from 1 to approximately 28 μM, which was identical to that equilibrated under the bubbling of 0.22% CO2 in light, when cells were incubated in the presence of acetate and 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) (Hanawa et al., 2007). From these data, the authors concluded that the induction of *H43/FEA1* is triggered by the CO2 signal, even CO2 generated from respiration, but not acetate itself or the change in carbon metabolite

High-CO2 Response Mechanisms in Microalgae 311

(Rawat & Moroney, 1991), suggesting that CAH2 expression is regulated by CCM1/CIA5 in the wild type. However, another high-CO2-inducible protein, Rh1, is not likely regulated by

Fig. 5. Schematic model of high-CO2 signaling for *H43/FEA1* induction. Solid and broken lines are expective and putative signaling flows, respectively. Low-CO2 signaling is modified from Miura et al. (2004) and Yamano and Fukuzawa (2009). Iron-deficientinducible genes are according to Allen et al. (2007). Cd signaling on *H43/FEA1* induction,

Compared to low-CO2-inducible mechanisms that are well understood, analyses of high-CO2-responsive mechanisms in microalgae at the molecular level have just started using the unicellular green alga *C. reinhardtii*. An accurate characterization of the acclimation mechanisms to high-CO2 conditions will be important for both a detailed understanding of sensing and responding to environmental CO2 changes and maximizing algal biomass productivity in mass cultivation. H43/FEA1, the most abundant extracellular protein in high-CO2-acclimated cells, is expressed in response to multiple signals, including high-CO2, iron-deficiency, or Cd-stress conditions. This suggests that, in addition to the high-CO2 signal itself, abnormally stressful conditions such as strong nutrient depletion caused by rapid growth under high-CO2 conditions may trigger expression of the gene. Targeted proteomics of whole *C. reinhardtii* established by Wienkoop et al. (2010) and a cDNA array (Yamano et al., 2008) or transcriptomics (Yamano & Fukuzawa, 2009), which has been applied to an expression analysis of CCM-associated genes,, would be useful for further detailed analysis of high-CO2 response phenomena. Our recent data indicate that the expression of gametogenesis-related proteins, which are strictly regulated by nitrogen availability, is triggered by high-CO2 signals with a drastic change in extracellular proteins. These gametogenesis-related proteins in the periplasmic space of *C. reinhardtii* cells may

play novel and crucial roles when *C. reinhardtii* is grown under high-CO2 conditions.

proposed by Rubinelli et al. (2002), is not drawn because little about it is known.

CCM1/CIA5 (Wang et al., 2005).

**7. Conclusion** 

abundance. Thus, *H43/FEA1* expression can be regulated by a high-CO2 signal at the transcriptional level, irrespective of high-CO2 conditions. H43/FEA1 is highly reliable as a high-CO2 response marker. The signal for *H43/FEA1* expression might be sensed by putative proteins localized on the cell membrane, which are influenced by protein modifiers and send the signal for *H43/FEA1* expression (Hanawa et al., 2007).

*H43/FEA1* expression is induced under excessive levels of Cd (>25 μM) or iron-deficient conditions (<1 μM) (Allen et al., 2007; Rubinelli et al., 2002). Fei et al. (2009) reported two transcriptional *cis*-elements that are responsive to the Fe-deficient signal (FeREs) for *H43/FEA1* expression, namely FeRE1 and FeRE2, which are located at -273/-259 and -106/- 85 upstream from the *H43/FEA1* transcriptional initiation site. The conserved sequence motif was identified from some iron-deficiency-inducible genes (Fei et al., 2009). However, according to our recent study, the two *cis*-elements are not necessary for the high-CO2 induced expression of the *H43/FEA1* gene (Baba et al., 2011a). The high-CO2-responsive *cis*element (HCRE) was located at a -537/-370 upstream region from the *H43/FEA1* transcriptional initiation site, although the precise location has not yet been determined (Baba et al., 2011a). These results show that *H43/FEA1* expression is regulated by the high-CO2 signal alone via the HCRE, which is located distantly from the iron-deficient-responsive element. This observation indicates that *H43/FEA1* is a multi-signal-regulated gene (Fig. 5). We have not yet determined whether all of these signals may affect the expression of other high-CO2-inducible proteins (Baba et al., 2011b). Allen et al. (2007) reported some proteins that are iron-deficient-responsive but not CO2-responsive, so those proteins are considered components of the iron-assimilation system. In addition, an iron-assimilation component was not found among high-CO2-inducible extracellular proteins analyzed experimentally (Baba et al., 2011b). The expression by either high-CO2 or iron-deficient signals is a unique feature of *H43/FEA1*.

The regulation of CCM-related gene expression, which is positively induced by a low-CO2 signal and negatively induced by a high-CO2 signal, has been well characterized in *C. reinhardtii*. A zinc-finger protein named CCM1/CIA5 has been identified as a candidate of the CCM master regulator (Fukuzawa et al., 2001; Miura et al., 2004; Xiang et al., 2001). CCM1/CIA5 is a protein complex with a molecular mass of approximately 290–580 kDa that is induced independently by DIC availability; Zn is necessary for its enzymatic function (Kohinata et al., 2008). One of the CCM1/CIA5-mediated signaling systems functions in the expression of *CAH1,* which encodes a low-CO2-inducible periplasmic carbonic anhydrase (Fukuzawa et al., 1990) and the signaling is mediated by a Myb-type transcriptional regulator named LCR1 (Yoshioka et al., 2004). CCM1/CIA5 may possibly function as an amplifier for the CO2 signaling cascade (Yamano et al., 2008). A direct signaling factor for CCM induction has not been identified, although some candidates have been reported (Giordano et al., 2005; Kaplan & Reinhold, 1999; Yamano et al., 2008). The *CCM1/CIA5* mutant lacks suppression of *H43/FEA1* expression under both low-CO2 and iron-sufficient conditions (Allen et al., 2007), suggesting that *H43/FEA1* expression is regulated by the CCM1/CIA5-dependent signaling cascade. However, the regulatory mechanism seems to be complex. The responses of CAH1 and H43/FEA1 expression are not an all-or-none type to the signals for a change in environmental CO2 concentration, acetate concentration, and light intensity (Hanawa et al., 2007). Signaling for *H43/FEA1* expression may be partially associated with CCM1/CIA5 signaling, although additional signals may also exist (Fig. 5). CAH2 is continuously expressed in a *CCM1/CIA5* mutant independent of CO2 concentration

abundance. Thus, *H43/FEA1* expression can be regulated by a high-CO2 signal at the transcriptional level, irrespective of high-CO2 conditions. H43/FEA1 is highly reliable as a high-CO2 response marker. The signal for *H43/FEA1* expression might be sensed by putative proteins localized on the cell membrane, which are influenced by protein modifiers and

*H43/FEA1* expression is induced under excessive levels of Cd (>25 μM) or iron-deficient conditions (<1 μM) (Allen et al., 2007; Rubinelli et al., 2002). Fei et al. (2009) reported two transcriptional *cis*-elements that are responsive to the Fe-deficient signal (FeREs) for *H43/FEA1* expression, namely FeRE1 and FeRE2, which are located at -273/-259 and -106/- 85 upstream from the *H43/FEA1* transcriptional initiation site. The conserved sequence motif was identified from some iron-deficiency-inducible genes (Fei et al., 2009). However, according to our recent study, the two *cis*-elements are not necessary for the high-CO2 induced expression of the *H43/FEA1* gene (Baba et al., 2011a). The high-CO2-responsive *cis*element (HCRE) was located at a -537/-370 upstream region from the *H43/FEA1* transcriptional initiation site, although the precise location has not yet been determined (Baba et al., 2011a). These results show that *H43/FEA1* expression is regulated by the high-CO2 signal alone via the HCRE, which is located distantly from the iron-deficient-responsive element. This observation indicates that *H43/FEA1* is a multi-signal-regulated gene (Fig. 5). We have not yet determined whether all of these signals may affect the expression of other high-CO2-inducible proteins (Baba et al., 2011b). Allen et al. (2007) reported some proteins that are iron-deficient-responsive but not CO2-responsive, so those proteins are considered components of the iron-assimilation system. In addition, an iron-assimilation component was not found among high-CO2-inducible extracellular proteins analyzed experimentally (Baba et al., 2011b). The expression by either high-CO2 or iron-deficient signals is a unique

The regulation of CCM-related gene expression, which is positively induced by a low-CO2 signal and negatively induced by a high-CO2 signal, has been well characterized in *C. reinhardtii*. A zinc-finger protein named CCM1/CIA5 has been identified as a candidate of the CCM master regulator (Fukuzawa et al., 2001; Miura et al., 2004; Xiang et al., 2001). CCM1/CIA5 is a protein complex with a molecular mass of approximately 290–580 kDa that is induced independently by DIC availability; Zn is necessary for its enzymatic function (Kohinata et al., 2008). One of the CCM1/CIA5-mediated signaling systems functions in the expression of *CAH1,* which encodes a low-CO2-inducible periplasmic carbonic anhydrase (Fukuzawa et al., 1990) and the signaling is mediated by a Myb-type transcriptional regulator named LCR1 (Yoshioka et al., 2004). CCM1/CIA5 may possibly function as an amplifier for the CO2 signaling cascade (Yamano et al., 2008). A direct signaling factor for CCM induction has not been identified, although some candidates have been reported (Giordano et al., 2005; Kaplan & Reinhold, 1999; Yamano et al., 2008). The *CCM1/CIA5* mutant lacks suppression of *H43/FEA1* expression under both low-CO2 and iron-sufficient conditions (Allen et al., 2007), suggesting that *H43/FEA1* expression is regulated by the CCM1/CIA5-dependent signaling cascade. However, the regulatory mechanism seems to be complex. The responses of CAH1 and H43/FEA1 expression are not an all-or-none type to the signals for a change in environmental CO2 concentration, acetate concentration, and light intensity (Hanawa et al., 2007). Signaling for *H43/FEA1* expression may be partially associated with CCM1/CIA5 signaling, although additional signals may also exist (Fig. 5). CAH2 is continuously expressed in a *CCM1/CIA5* mutant independent of CO2 concentration

send the signal for *H43/FEA1* expression (Hanawa et al., 2007).

feature of *H43/FEA1*.

(Rawat & Moroney, 1991), suggesting that CAH2 expression is regulated by CCM1/CIA5 in the wild type. However, another high-CO2-inducible protein, Rh1, is not likely regulated by CCM1/CIA5 (Wang et al., 2005).

Fig. 5. Schematic model of high-CO2 signaling for *H43/FEA1* induction. Solid and broken lines are expective and putative signaling flows, respectively. Low-CO2 signaling is modified from Miura et al. (2004) and Yamano and Fukuzawa (2009). Iron-deficientinducible genes are according to Allen et al. (2007). Cd signaling on *H43/FEA1* induction, proposed by Rubinelli et al. (2002), is not drawn because little about it is known.

#### **7. Conclusion**

Compared to low-CO2-inducible mechanisms that are well understood, analyses of high-CO2-responsive mechanisms in microalgae at the molecular level have just started using the unicellular green alga *C. reinhardtii*. An accurate characterization of the acclimation mechanisms to high-CO2 conditions will be important for both a detailed understanding of sensing and responding to environmental CO2 changes and maximizing algal biomass productivity in mass cultivation. H43/FEA1, the most abundant extracellular protein in high-CO2-acclimated cells, is expressed in response to multiple signals, including high-CO2, iron-deficiency, or Cd-stress conditions. This suggests that, in addition to the high-CO2 signal itself, abnormally stressful conditions such as strong nutrient depletion caused by rapid growth under high-CO2 conditions may trigger expression of the gene. Targeted proteomics of whole *C. reinhardtii* established by Wienkoop et al. (2010) and a cDNA array (Yamano et al., 2008) or transcriptomics (Yamano & Fukuzawa, 2009), which has been applied to an expression analysis of CCM-associated genes,, would be useful for further detailed analysis of high-CO2 response phenomena. Our recent data indicate that the expression of gametogenesis-related proteins, which are strictly regulated by nitrogen availability, is triggered by high-CO2 signals with a drastic change in extracellular proteins. These gametogenesis-related proteins in the periplasmic space of *C. reinhardtii* cells may play novel and crucial roles when *C. reinhardtii* is grown under high-CO2 conditions.

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#### **8. Acknowledgments**

This work was financially supported, in part, by a Grant-in-Aid for Scientific Research (Basic Research Area (S), No. 22221003 to YS) from the Japan Society for the Promotion of Science, the Core Research of Evolutional Science & Technology program (CREST) from the Japan Science and Technology Agency (JST) (to MB & YS), another fund from the Japan Science and Technology Agency (CREST/JST, to YS), and by the Global Environment Research Fund from the Japanese Ministry of Environment (FY2008-2010) to YS.

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**16** 

*Malaysia* 

**Photosynthesis and Quantum Yield of Oil** 

H.Z.E. Jaafar and Mohd Hafiz Ibrahim

*Universiti Putra Malaysia (UPM)* 

**Palm Seedlings to Elevated Carbon Dioxide** 

Photosynthesis is a metabolic process through which green plants synthesize organic compounds from inorganic raw materials in the presence of sunlight. This process can be regarded as a procedure of converting radiant energy of the sun into chemical energy of plant tissues in the form of organic molecules. Photosynthesis increases the total free energy available to organism and provides energy to the world, directly or indirectly, necessary for sustaining all forms of life on earth. Farming is basically a system of exploiting solar energy to synthesize organic matter through photosynthesis. The yield of crop plants ultimately depends on the size and efficiency of their photosynthetic system (Anderson, 2000). The most important factors of biomass production of any crop are the amount of radiation intercepted by the crop and the effectiveness of using the radiation in dry matter production. All organisms on earth need energy for growth and maintenance. As a result, higher plants, algae and certain types of bacteria capture this energy directly from the sunlight and utilise it for the biosynthesis of essential food materials for dry matter increase. The plant photosynthetic apparatus contain the necessary pigments in leaf able to absorb light and channel the energy of the excited pigment molecules into a series of photochemical and enzymatic reactions. Light energy is absorbed by protein-bound chlorophylls that are located in light-harvesting complexes and the energy migration to photosynthetic reaction centres results in electron excitation and transfer to other components of the electron

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**1. Introduction** 

transfer chain (Hall and Rao, 1999).


### **Photosynthesis and Quantum Yield of Oil Palm Seedlings to Elevated Carbon Dioxide**

H.Z.E. Jaafar and Mohd Hafiz Ibrahim *Universiti Putra Malaysia (UPM)* 

*Malaysia* 

#### **1. Introduction**

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Photosynthesis is a metabolic process through which green plants synthesize organic compounds from inorganic raw materials in the presence of sunlight. This process can be regarded as a procedure of converting radiant energy of the sun into chemical energy of plant tissues in the form of organic molecules. Photosynthesis increases the total free energy available to organism and provides energy to the world, directly or indirectly, necessary for sustaining all forms of life on earth. Farming is basically a system of exploiting solar energy to synthesize organic matter through photosynthesis. The yield of crop plants ultimately depends on the size and efficiency of their photosynthetic system (Anderson, 2000). The most important factors of biomass production of any crop are the amount of radiation intercepted by the crop and the effectiveness of using the radiation in dry matter production. All organisms on earth need energy for growth and maintenance. As a result, higher plants, algae and certain types of bacteria capture this energy directly from the sunlight and utilise it for the biosynthesis of essential food materials for dry matter increase. The plant photosynthetic apparatus contain the necessary pigments in leaf able to absorb light and channel the energy of the excited pigment molecules into a series of photochemical and enzymatic reactions. Light energy is absorbed by protein-bound chlorophylls that are located in light-harvesting complexes and the energy migration to photosynthetic reaction centres results in electron excitation and transfer to other components of the electron transfer chain (Hall and Rao, 1999).

Carbon dioxide is a trace gas in the atmosphere, presently accounting for about 0.037%, or 370 parts per million (ppm), of air. The partial pressure of ambient CO2 (Ca) varies with atmospheric pressure and is approximately 36 pascals (Pa) at sea level. The current atmospheric concentration of CO2 is almost twice the concentration that has prevailed during most of the last 160,00 years, as measured from air bubbles trapped in glacial ice in Antarctica. For the last 200 years, CO2 concentrations during the recent geological past have been low, fluctuating between 180 and 260 ppm. These low concentrations were typical of times extending back to the Cretaceous, when Earth was much warmer and the CO2 concentration may have been as high as 1200 to 2800 ppm (Ehleringer et al., 1991). However, with the rapid increases in world population and economic activity, a doubling of the present atmospheric [CO2], assuming a mean annual increase rate of 1.5 ppm, which was observed over the past decade 1984–1993 (Stoskoptf, 1981), could be expected before the end

Photosynthesis and Quantum Yield of Oil Palm Seedlings to Elevated Carbon Dioxide 323

be low or high temperature, deficiency or over supply of water or nutrient, low CO2 or high O2 concentration, and low light intensity. In the meantime, many plant internal factors including developmental hormones and respiration, etc. may also have a significant effect on net photosynthetic rate. The main limitation site of net photosynthetic rate in C3 plants, however, is often in the reaction centre catalyzed by the enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Therefore, reducing or eliminating its oxygenase function or photorespiration or increasing the affinity of the enzyme for CO2 is a long-term

The quantum yield of a process in which molecules give up their excitation energy (known as "decay") is the fraction of excited molecules that decay via that pathway (Wells et al., 1982; Taiz and Zieger, 1991). The quantum yield of a process, such as photochemistry, has been defined (Clayton 1971; 1980) mathematically as the yield of photochemical products divided by total number of quanta absorbed. For a particular process, the value of quantum yield can range between 0 (when the process does not involve any decay of the excited state) and 1.0 (when the process involves deactivation of the excited state). Taiz and Zieger (1991) explained that all possible processes would ultimately contribute to a sum 1.0 of the quantum yields. Basically, in functional chloroplast that is kept in dim light, the quantum yield of photochemistry is approximately 0.95, the quantum yield of fluorescence is 0.05 or lower, and the quantum yields of other processes are negligible. The vast majority of excited

The reciprocal of the quantum yield is called the quantum requirement. For a high yielding crop, it is not only a high photosynthetic rate in strong light is important but a high quantum yield in weak light is also crucial. Therefore, Ort and Baker (1988) believed that the improvement of future crop production should aim at increasing their quantum yield. It is well known that in photosynthesis of the Calvin-Benson cycle, the assimilation of one molecule of CO2 into carbohydrate requires 2NADPH and 3ATP. The production of 2NADPH is the result of transporting four electrons from 2H2O to 2NADP along an electron transport chain. Because the chain includes two photosystems in series, two photons are needed for one electron transport. Thus, at least eight photons are required for the production of 2NADPH. Therefore, the maximal or theoretical quantum yield for photosynthetic carbon assimilation is 0.125 mole CO2/mole photons. In field studies Xu (1988) found that the apparent quantum yield of photosynthetic carbon assimilation often displayed a significant midday decline in many C3 plants such as soybean and wheat but not in C4 plants such as maize and sorghum. It was deduced that photoinhibition may be a cause of the midday decline of the photosynthetic efficiency (Xu et al. 1990). The molecular mechanism of photoinhibition, however, is still not fully understood. For more than a decade photoinhibition has been considered almost synonymous with photodamage to the photosynthetic apparatus (Nigoyi, 1999). In addition to photoinhibition, enhanced photorespiration is another cause of the midday decline in the photosynthetic efficiency of C3 plants (Guo et al., 1994). For a long time photorespiration has been considered a wasteful process. Many efforts have been made to eliminate or reduce photorespiration but no success has yet been reported. Extensive screening programs involving several species (wheat, barley, oats, soybean, potato, tall fescue) failed to identify genotypes with low CO2 compensation point (Hay and Walker, 1989). Attempts to select C3 plants with low rates of

goal to increase productivity of the plant (Xu and Shen, 2000).

chlorophyll molecules, therefore, lead to photochemistry.

**3. Quantum yield** 

of the 21st century (Baker and Ort, 1992). Rising atmospheric [CO2] could benefit many economically important crops, especially the C3; however, gains may or may not be realized in long-term growth because of the interaction of various environmental factors that complicate the issue (Farquhar and Sharkley, 1982). The current CO2 concentration of the atmosphere is increasing by about 1 ppm each year, primarily because of the burning of fossil fuels. Since 1958, when systematic measurements of CO2 began at Mauna Loa, Hawaii, atmospheric CO2 concentrations have increased by more than 17% (Keeling et al. 1995), and by 2020 the atmospheric CO2 concentration could reach up to 600 ppm.

With the increase in [CO2], many crops may be affected either positively or negatively. Oil palm is an industrial perennial plant widely cultivated in Southeast Asia where it plays a major role in the economics of the regional income. Claimed to be the most productive oil bearing plant as compared to coconut, olive, rapeseed and soybean, the crop has contributed about 8.2% of Malaysian gross domestic products (GDP) and the second largest economic contributor after exported goods and petroleum. In 2009, large area of about 4.6 million ha had been cultivated with oil palm. As the concentration of CO2 is expected to increase to 600 ppm by 2020, the productivity of oil palm could also be increased. However, the research on oil palm acclimation to increased CO2 issue is still lacking especially in the leaf gas exchange aspects.

Net photosynthesis and quantum yield are good indicators of plant acclimation to elevated CO2. The notion of photosynthetic efficiency in the literature involves some different terms including photosynthetic rate, quantum yield of carbon assimilation and photochemical efficiency of PSII, which is often expressed as a ratio of variable to maximal fluorescence (Xu and Shen, 2000). These terms are different but they linked to each other. Both photosynthetic rate and quantum yield are related to characteristics of the leaf, cell, and chloroplast itself and the environmental conditions. Photosynthetic rate is often expressed as number of molecules of CO2 fixed or O2 evolved per unit leaf area per unit time while quantum yield is expressed as number of molecules of CO2 fixed or O2 evolved per photon absorbed. The efficiency of photosynthesis of the whole plant is crucial to agriculture, forestry, ecology, etc. when it comes to analyzing productivity for food and fuels and many other product users. The quality and quantity of photosynthetic incident light (or photosynthetic active radiation, PAR), temperature and water availability, mineral nutrients availability and utilization, photorespiratory losses, presence of pollutants in the atmosphere and in the soil (heavy metals), etc., are some of the factors that affect plant productivity. How these factors interact with the changing environment is now the subject of much practical and basic research.

This chapter focuses on the photosynthetic responses, particularly net photosynthesis and apparent quantum yield, of oil palm to enhanced growth [CO2]. The leaf gas exchange and apparent quantum yield response of oil palm seedling to elevated [CO2] will be discussed. The net photosynthesis and apparent quantum yield data are directly collected from LICOR 6400 using light response curve analysis. As the photosynthetic mechanism of a plant species is the major determinant of how it will respond to rising atmospheric [CO2], understanding the mechanisms of photosynthesis acclimation to rising [CO2] could potentially be translated into a basic framework for improving the efficiency of crop production in a future climate-changed world.

#### **2. Factors limiting photosynthetic rates**

A number of external environmental factors can influence the rate of photosynthesis, leading to up-regulation or down-regulation of photosynthetic capacity. These factors might be low or high temperature, deficiency or over supply of water or nutrient, low CO2 or high O2 concentration, and low light intensity. In the meantime, many plant internal factors including developmental hormones and respiration, etc. may also have a significant effect on net photosynthetic rate. The main limitation site of net photosynthetic rate in C3 plants, however, is often in the reaction centre catalyzed by the enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Therefore, reducing or eliminating its oxygenase function or photorespiration or increasing the affinity of the enzyme for CO2 is a long-term goal to increase productivity of the plant (Xu and Shen, 2000).

#### **3. Quantum yield**

322 Advances in Photosynthesis – Fundamental Aspects

of the 21st century (Baker and Ort, 1992). Rising atmospheric [CO2] could benefit many economically important crops, especially the C3; however, gains may or may not be realized in long-term growth because of the interaction of various environmental factors that complicate the issue (Farquhar and Sharkley, 1982). The current CO2 concentration of the atmosphere is increasing by about 1 ppm each year, primarily because of the burning of fossil fuels. Since 1958, when systematic measurements of CO2 began at Mauna Loa, Hawaii, atmospheric CO2 concentrations have increased by more than 17% (Keeling et al. 1995), and

With the increase in [CO2], many crops may be affected either positively or negatively. Oil palm is an industrial perennial plant widely cultivated in Southeast Asia where it plays a major role in the economics of the regional income. Claimed to be the most productive oil bearing plant as compared to coconut, olive, rapeseed and soybean, the crop has contributed about 8.2% of Malaysian gross domestic products (GDP) and the second largest economic contributor after exported goods and petroleum. In 2009, large area of about 4.6 million ha had been cultivated with oil palm. As the concentration of CO2 is expected to increase to 600 ppm by 2020, the productivity of oil palm could also be increased. However, the research on oil palm acclimation to increased CO2 issue is still lacking especially in the leaf gas exchange

Net photosynthesis and quantum yield are good indicators of plant acclimation to elevated CO2. The notion of photosynthetic efficiency in the literature involves some different terms including photosynthetic rate, quantum yield of carbon assimilation and photochemical efficiency of PSII, which is often expressed as a ratio of variable to maximal fluorescence (Xu and Shen, 2000). These terms are different but they linked to each other. Both photosynthetic rate and quantum yield are related to characteristics of the leaf, cell, and chloroplast itself and the environmental conditions. Photosynthetic rate is often expressed as number of molecules of CO2 fixed or O2 evolved per unit leaf area per unit time while quantum yield is expressed as number of molecules of CO2 fixed or O2 evolved per photon absorbed. The efficiency of photosynthesis of the whole plant is crucial to agriculture, forestry, ecology, etc. when it comes to analyzing productivity for food and fuels and many other product users. The quality and quantity of photosynthetic incident light (or photosynthetic active radiation, PAR), temperature and water availability, mineral nutrients availability and utilization, photorespiratory losses, presence of pollutants in the atmosphere and in the soil (heavy metals), etc., are some of the factors that affect plant productivity. How these factors interact with the changing environment is now the subject of much practical and basic research. This chapter focuses on the photosynthetic responses, particularly net photosynthesis and apparent quantum yield, of oil palm to enhanced growth [CO2]. The leaf gas exchange and apparent quantum yield response of oil palm seedling to elevated [CO2] will be discussed. The net photosynthesis and apparent quantum yield data are directly collected from LICOR 6400 using light response curve analysis. As the photosynthetic mechanism of a plant species is the major determinant of how it will respond to rising atmospheric [CO2], understanding the mechanisms of photosynthesis acclimation to rising [CO2] could potentially be translated into a basic framework for improving the efficiency of crop

A number of external environmental factors can influence the rate of photosynthesis, leading to up-regulation or down-regulation of photosynthetic capacity. These factors might

by 2020 the atmospheric CO2 concentration could reach up to 600 ppm.

production in a future climate-changed world.

**2. Factors limiting photosynthetic rates** 

aspects.

The quantum yield of a process in which molecules give up their excitation energy (known as "decay") is the fraction of excited molecules that decay via that pathway (Wells et al., 1982; Taiz and Zieger, 1991). The quantum yield of a process, such as photochemistry, has been defined (Clayton 1971; 1980) mathematically as the yield of photochemical products divided by total number of quanta absorbed. For a particular process, the value of quantum yield can range between 0 (when the process does not involve any decay of the excited state) and 1.0 (when the process involves deactivation of the excited state). Taiz and Zieger (1991) explained that all possible processes would ultimately contribute to a sum 1.0 of the quantum yields. Basically, in functional chloroplast that is kept in dim light, the quantum yield of photochemistry is approximately 0.95, the quantum yield of fluorescence is 0.05 or lower, and the quantum yields of other processes are negligible. The vast majority of excited chlorophyll molecules, therefore, lead to photochemistry.

The reciprocal of the quantum yield is called the quantum requirement. For a high yielding crop, it is not only a high photosynthetic rate in strong light is important but a high quantum yield in weak light is also crucial. Therefore, Ort and Baker (1988) believed that the improvement of future crop production should aim at increasing their quantum yield. It is well known that in photosynthesis of the Calvin-Benson cycle, the assimilation of one molecule of CO2 into carbohydrate requires 2NADPH and 3ATP. The production of 2NADPH is the result of transporting four electrons from 2H2O to 2NADP along an electron transport chain. Because the chain includes two photosystems in series, two photons are needed for one electron transport. Thus, at least eight photons are required for the production of 2NADPH. Therefore, the maximal or theoretical quantum yield for photosynthetic carbon assimilation is 0.125 mole CO2/mole photons. In field studies Xu (1988) found that the apparent quantum yield of photosynthetic carbon assimilation often displayed a significant midday decline in many C3 plants such as soybean and wheat but not in C4 plants such as maize and sorghum. It was deduced that photoinhibition may be a cause of the midday decline of the photosynthetic efficiency (Xu et al. 1990). The molecular mechanism of photoinhibition, however, is still not fully understood. For more than a decade photoinhibition has been considered almost synonymous with photodamage to the photosynthetic apparatus (Nigoyi, 1999). In addition to photoinhibition, enhanced photorespiration is another cause of the midday decline in the photosynthetic efficiency of C3 plants (Guo et al., 1994). For a long time photorespiration has been considered a wasteful process. Many efforts have been made to eliminate or reduce photorespiration but no success has yet been reported. Extensive screening programs involving several species (wheat, barley, oats, soybean, potato, tall fescue) failed to identify genotypes with low CO2 compensation point (Hay and Walker, 1989). Attempts to select C3 plants with low rates of

Photosynthesis and Quantum Yield of Oil Palm Seedlings to Elevated Carbon Dioxide 325

in net CO2 exchange rates up to 63% and growth up to 58% with a doubling of the present atmospheric [CO2] (Brinkman and Frey, 1978). Long-term exposure to elevated [CO2] leads to a variety of acclimation effects, which include changes in the photosynthetic biochemistry and stomatal physiology and alterations in the morphology, anatomy, branching, tillering, biomass, and timing of developmental events as well as life cycle completion (Evan and Dunstone, 1970). A greater number of mesophyll cells and chloroplasts have been reported for plants grown under elevated [CO2] (Poskuta and Nelson, 1986). In terms of dark respiration, the exposure of plants to elevated [CO2] usually results in lowering the dark respiration rate, which can be explained by both direct and indirect effects (Pettigrew and Meredith, 1994). The mechanism for the direct effect appears to be an inhibition of the enzymes in the mitochondrial electron transport system, and for the indirect (acclimation) effect of elevated [CO2] on dark respiration may be related to changes in tissue composition

Many C3 species grown for long periods at elevated [CO2] show a down-regulation of leaf photosynthesis (Zelith, 1982); and carbohydrate source-sink balance is believed to have a major role in the regulation of photosynthesis through the feedback inhibition (Wells et al., 1986). Source-sink imbalances may occur during exposure to elevated [CO2] when photosynthetic rate exceeds the export capacity or the capacity of sinks to use photosynthates for growth resulting in an accumulation of carbohydrates in photosynthetically active source leaves (Dong, 1991). Although growth is enhanced under elevated [CO2] the extent to which starch and soluble sugars accumulate depends largely on the species differences. In many plants, the increase in starch also seems to be greater than that of soluble sugars. More frequently observed is the correlation between starch accumulation and inhibition of leaf photosynthesis (Wells et al., 1986) implying that high starch content may be responsible for down regulation of photosynthesis under elevated [CO2]. For many plant species, the long exposure to elevated [CO2] has also resulted in a down-regulation of Rubisco (Mohanty and Boyer, 1976). Zhang et al. (1992) observed the down-regulation in cotton, cucumber, parsley, pea, radish, soybean, spinach, tobacco and wheat exposed to elevated [CO2] due to increased leaf acid invertase activities, an indication of starch accumulation in the leaf; conversely, an up-regulation of photosynthesis in bean, plantain and sunflower was also detected suggesting variations in responses by species

Levels of soluble sugars in plant cells have been shown to influence the regulation of expression of several genes coding for key photosynthetic enzymes (Osmond et al., 1980; Xu and Shen, 2000). The buildup in carbohydrates may signal the repression but does not directly inhibit the expression of Rubisco and other proteins that are required for photosynthesis (Oquist et al., 1982). Although the signal transduction pathway for regulation of the sugar-sensing genes may involve phosphorylation of hexoses, derived from sucrose hydrolysis by acid invertase via hexokinase (Guo et al., 1996), unknown gaps still exist between hexose metabolism and repression of gene expression at elevated growth

**5. Photosynthetic and quantum yield up-regulation under elevated CO2**

Carbon dioxide is the substrate that through the light and dark reactions of photosynthesis, are combined into dry mass (Pinkard et al., 2010). Thus [CO2] can be a major factor limiting photosynthesis (Hall and Rao, 1992). Stomata regulate the diffusion of CO2 into leaves;

(Yin et al., 1956; Xu and Shen, 2000).

differences to elevated [CO2].

[CO2] (Hong and Xu, 1998; Xu and Shen, 2000).

photorespiration and high rates of net photosynthesis also have had little success (Xu and Shen, 2000).

#### **3.1 Factors affecting quantum yield 3.1.1 External factors**

Emerson and Lewis (1943) showed that the values of quantum yield were related to the quality of light. A high quantum yield was measured at red light around 680 nm. The quantum yields of sun and shade leaves grown under different light intensities were similar, although there was a significant difference in light saturated photosynthetic rate between them (Oquist and Hallgren, 1982). At 21% O2 and a temperature range of 15–35°C the quantum yield decreased gradually with temperature increase in C3 plants but not in C4 plants (Ku and Edwards, 1978; Xu and Shen, 2000). Water deficiency and excessive water or flooding could lead to a decline in quantum yield (Mohanty and Boyer, 1976). After several rainy days, the photosynthetic quantum efficiency became lower in spinach leaves (Li et al. 1991). The reason may be that the reduction of NADP is severely hindered in swollen chloroplasts under hypotonic conditions (Ye et al., 1995). Decreasing O2 concentration or increasing CO2 concentration in air could increase quantum yield in C3 plants but not in C4 plants (Monson et al., 1982). This may be due to decreased excitation energy transport from antenna pigments to PSII reaction centers and enhanced excitation energy dissipation as heat under phosphate deficiency conditions (Jacob, 1995; Xu and Shen, 2000).

#### **3.1.2 Internal factors**

Among all internal factors, photorespiration has the most significant effect on quantum yield. The effects of air temperature and CO2 or O2 concentration on quantum yield mentioned earlier, in fact, are related to the changes in photorespiratory rate caused by these factors. In normal air and at 20–25°C, the quantum yields of C3 and C4 plants were similar. However, when the air temperature was over 30°C, the quantum yield in C4 plants was slightly higher than that in C3 plants (Ehleringer and Pearcy, 1983). When photorespiration was inhibited by high CO2 and/or low O2, C4 plants had about 30% lower quantum yields than C3 plants because they used two additional ATP molecules in the C4 pathway for fixation of one molecule of CO2 to form carbohydrate (Osmond et al. 1980; Xu and Shen, 2000).

#### **4. C3 species response to elevated CO2**

The present atmospheric [CO2] limits the photosynthetic capability, growth, and yield of many agricultural crop plants, among which the C3 species show the greatest potential for response to rising [CO2] (Xu et al., 1984). Current atmospheric CO2 and O2 levels and C3 Rubisco specificity factors translate into photorespiratory losses of 25% or more for C3 species (Farquhar and Sharkey, 1982). The projection that a rise in atmospheric [CO2] will reduce the deleterious effect of O2 on C3 photosynthesis but that it has a negligible effect on C4 photosynthesis is indeed supported by experimental growth data. Exposure of C3 plants to elevated [CO2] generally results in stimulated photosynthesis and enhanced growth and yield (Sharkawy et al., 1990).

A compilation of the existing data available from the literature for C3 agricultural crops, including agronomic, horticultural, and forest tree species, shows an average enhancement

photorespiration and high rates of net photosynthesis also have had little success (Xu and

Emerson and Lewis (1943) showed that the values of quantum yield were related to the quality of light. A high quantum yield was measured at red light around 680 nm. The quantum yields of sun and shade leaves grown under different light intensities were similar, although there was a significant difference in light saturated photosynthetic rate between them (Oquist and Hallgren, 1982). At 21% O2 and a temperature range of 15–35°C the quantum yield decreased gradually with temperature increase in C3 plants but not in C4 plants (Ku and Edwards, 1978; Xu and Shen, 2000). Water deficiency and excessive water or flooding could lead to a decline in quantum yield (Mohanty and Boyer, 1976). After several rainy days, the photosynthetic quantum efficiency became lower in spinach leaves (Li et al. 1991). The reason may be that the reduction of NADP is severely hindered in swollen chloroplasts under hypotonic conditions (Ye et al., 1995). Decreasing O2 concentration or increasing CO2 concentration in air could increase quantum yield in C3 plants but not in C4 plants (Monson et al., 1982). This may be due to decreased excitation energy transport from antenna pigments to PSII reaction centers and enhanced excitation energy dissipation as

heat under phosphate deficiency conditions (Jacob, 1995; Xu and Shen, 2000).

Among all internal factors, photorespiration has the most significant effect on quantum yield. The effects of air temperature and CO2 or O2 concentration on quantum yield mentioned earlier, in fact, are related to the changes in photorespiratory rate caused by these factors. In normal air and at 20–25°C, the quantum yields of C3 and C4 plants were similar. However, when the air temperature was over 30°C, the quantum yield in C4 plants was slightly higher than that in C3 plants (Ehleringer and Pearcy, 1983). When photorespiration was inhibited by high CO2 and/or low O2, C4 plants had about 30% lower quantum yields than C3 plants because they used two additional ATP molecules in the C4 pathway for fixation of one molecule of CO2 to form carbohydrate (Osmond et al. 1980; Xu and Shen,

The present atmospheric [CO2] limits the photosynthetic capability, growth, and yield of many agricultural crop plants, among which the C3 species show the greatest potential for response to rising [CO2] (Xu et al., 1984). Current atmospheric CO2 and O2 levels and C3 Rubisco specificity factors translate into photorespiratory losses of 25% or more for C3 species (Farquhar and Sharkey, 1982). The projection that a rise in atmospheric [CO2] will reduce the deleterious effect of O2 on C3 photosynthesis but that it has a negligible effect on C4 photosynthesis is indeed supported by experimental growth data. Exposure of C3 plants to elevated [CO2] generally results in stimulated photosynthesis and enhanced growth and

A compilation of the existing data available from the literature for C3 agricultural crops, including agronomic, horticultural, and forest tree species, shows an average enhancement

Shen, 2000).

**3.1.1 External factors** 

**3.1.2 Internal factors** 

**4. C3 species response to elevated CO2**

yield (Sharkawy et al., 1990).

2000).

**3.1 Factors affecting quantum yield** 

in net CO2 exchange rates up to 63% and growth up to 58% with a doubling of the present atmospheric [CO2] (Brinkman and Frey, 1978). Long-term exposure to elevated [CO2] leads to a variety of acclimation effects, which include changes in the photosynthetic biochemistry and stomatal physiology and alterations in the morphology, anatomy, branching, tillering, biomass, and timing of developmental events as well as life cycle completion (Evan and Dunstone, 1970). A greater number of mesophyll cells and chloroplasts have been reported for plants grown under elevated [CO2] (Poskuta and Nelson, 1986). In terms of dark respiration, the exposure of plants to elevated [CO2] usually results in lowering the dark respiration rate, which can be explained by both direct and indirect effects (Pettigrew and Meredith, 1994). The mechanism for the direct effect appears to be an inhibition of the enzymes in the mitochondrial electron transport system, and for the indirect (acclimation) effect of elevated [CO2] on dark respiration may be related to changes in tissue composition (Yin et al., 1956; Xu and Shen, 2000).

Many C3 species grown for long periods at elevated [CO2] show a down-regulation of leaf photosynthesis (Zelith, 1982); and carbohydrate source-sink balance is believed to have a major role in the regulation of photosynthesis through the feedback inhibition (Wells et al., 1986). Source-sink imbalances may occur during exposure to elevated [CO2] when photosynthetic rate exceeds the export capacity or the capacity of sinks to use photosynthates for growth resulting in an accumulation of carbohydrates in photosynthetically active source leaves (Dong, 1991). Although growth is enhanced under elevated [CO2] the extent to which starch and soluble sugars accumulate depends largely on the species differences. In many plants, the increase in starch also seems to be greater than that of soluble sugars. More frequently observed is the correlation between starch accumulation and inhibition of leaf photosynthesis (Wells et al., 1986) implying that high starch content may be responsible for down regulation of photosynthesis under elevated [CO2]. For many plant species, the long exposure to elevated [CO2] has also resulted in a down-regulation of Rubisco (Mohanty and Boyer, 1976). Zhang et al. (1992) observed the down-regulation in cotton, cucumber, parsley, pea, radish, soybean, spinach, tobacco and wheat exposed to elevated [CO2] due to increased leaf acid invertase activities, an indication of starch accumulation in the leaf; conversely, an up-regulation of photosynthesis in bean, plantain and sunflower was also detected suggesting variations in responses by species differences to elevated [CO2].

Levels of soluble sugars in plant cells have been shown to influence the regulation of expression of several genes coding for key photosynthetic enzymes (Osmond et al., 1980; Xu and Shen, 2000). The buildup in carbohydrates may signal the repression but does not directly inhibit the expression of Rubisco and other proteins that are required for photosynthesis (Oquist et al., 1982). Although the signal transduction pathway for regulation of the sugar-sensing genes may involve phosphorylation of hexoses, derived from sucrose hydrolysis by acid invertase via hexokinase (Guo et al., 1996), unknown gaps still exist between hexose metabolism and repression of gene expression at elevated growth [CO2] (Hong and Xu, 1998; Xu and Shen, 2000).

#### **5. Photosynthetic and quantum yield up-regulation under elevated CO2**

Carbon dioxide is the substrate that through the light and dark reactions of photosynthesis, are combined into dry mass (Pinkard et al., 2010). Thus [CO2] can be a major factor limiting photosynthesis (Hall and Rao, 1992). Stomata regulate the diffusion of CO2 into leaves;

Photosynthesis and Quantum Yield of Oil Palm Seedlings to Elevated Carbon Dioxide 327

area index to elevated [CO2] have been observed in trees than other functional types (Ainsworth and Long, 2005). Nevertheless, there is often a poor correlation between photosynthetic capacity measured as Amax and total biomass production under elevated

At elevated [CO2] condition, when plant photosynthesis becomes RubP limited, Rubisco will be in excess of requirements (Ainsworth and Rogers, 2007). The excess capacity for carboxylation could be reduced through a reduction in the activation state of Rubisco (Cen and Sage, 2005). Alternatively, because less Rubisco is required by these plants at elevated [CO2], redistribution of the excess N invested in Rubisco could further increase N use efciency at elevated [CO2] without negatively impacting potential C acquisition (Parry et al., 2003; Ainsworth and Rogers, 2007). However, there is only benet in reducing the amount of N invested in Rubisco at elevated [CO2] when the resources invested in it can be usefully deployed elsewhere (Parry et al. 2003). Ainsworth and Long (2005) reported that the stimulation in Asat at elevated [CO2] was 23% lower in plants grown with a low N supply. Meanwhile, under elevated [CO2] the Vcmax was decreased at both high and low N, with greater reduction of 85% in low N condition. This result is in agreement with summaries of earlier studies conducted in controlled environments and eld enclosures by Ainsworth and Rogers (2007), Drake et al. (1997), Moore et al. (1999) and Stitt and Krapp (1999), and is consistent with current understanding of the mechanism underlying acclimation under elevated [CO2]. When plants are N limited, sink development is restricted, C supply is in excess of demand, and the sugar feedback mechanism as outlined earlier can operate to reduce Rubisco content and increase N use efciency. As N supply increases, the limitation imposed by sink capacity decreases and the sugar linked signal for down-regulating Rubisco content is reduced (Ainsworth and Rogers, 2007; Drake et al. 1997;

Dened here as the capacity to utilize photosynthate, sink strength can be a major constraint on carbon acquisition (Ainsworth and Rogers, 2007). A reduced or insufcient sink capacity may be the result of many potentially limiting processes e.g. N supply (Rogers et al., 1998), genetic constraints (Ainsworth et al., 2004), temperature (Ainsworth et al., 2003b) or developmental changes (Bernacchi et al., 2005; Rogers and Ainsworth, 2006). However, the net result is the same, i.e. the appearance of a carbohydrate-derived signal that can lead to the subsequent down-regulation (acclimation) of photosynthetic machinery, principally Rubisco (Stitt and Krapp, 1999; Long et al., 2004). Davey et al. (2006) showed that poplar grown at elevated [CO2] had a large sink capacity. Poplar was able to export >90% of its photosynthate during the day; it also had a large capacity for the temporary storage of overow photosynthate as starch (Ainsworth and Rogers, 2007; Stitt and Quick 1989; Davey et al., 2006). These two traits enabled poplar to maintain high photosynthetic rates at elevated [CO2] and avoid a major source–sink imbalance that could lead to a reduction in the potential for C acquisition (Ainsworth and Rogers, 2007). In contrast, *L. perenne* can

**6. Environmental factors determining the response of photosynthesis to** 

[CO2] enrichment (Oren et al., 2001).

Rogers et al. 1998; Long et al., 2004).

**6.2 Sink strength** 

**elevated [CO2] 6.1 Nitrogen supply** 

stomata can respond sensitively to [CO2] as part of a proportionate response to the CO2 requirement for photosynthesis; increasing concentrations are, therefore, associated with a closing response and vice-versa (Pinkard et al., 2010). Hence, elevated [CO2] is anticipated to increase or up-regulate photosynthesis, decrease stomatal conductance and increase intrinsic water-use efciency i.e. the ratio of leaf photosynthesis to stomatal conductance (Long et al., 2004). Many factors other than [CO2] determine photosynthetic rate, and the law of limiting factors (von Liebig, 1840) will ultimately determine photosynthetic responses to [CO2] where more than one limiting factors may be involved. Sala and Hoch (2009) suggested that elevated CO2 would improve carbon balance in light-limited as well as highlight environments through the CO2 enhancement of quantum yield. The present CO2:O2 ratio of the air constrains photosynthesis by 30-40% because of O2 inhibition of carboxylation and associated photorespiration (Booth and Jayanovic, 2005). As CO2 concentration increases, quantum yield is increased because the ratio of carboxylation to oxygenation by Rubisco increases and photorespiration decreases (Pinkard et al., 2010). Several studies have shown that CO2 enrichment enhances photosynthesis and growth under limiting irradiance condition, and in some cases the relative enhancement was greater at low rather than at high irradiances (Gifford et al., 1981). In C3 plants, elevated [CO2] increases the quantum yield of photosynthesis by reducing photorespiration caused by the oxygenase activity of Rubisco. Maximum, single-leaf quantum yield was increased from 0·065 to 0·080 (Long and Drake, 1992).

Up-regulation of photosynthesis refers to a signicant increase in the light-saturated rate of photosynthesis (Asat), the rate of photosynthesis under ambient light (A), and/or diurnal photosynthesis (A). Elevated [CO2] up-regulates photosynthesis by increasing the carboxylation rate (Vc) of ribulose bisphosphate carboxylase (Rubisco) and competitively inhibiting the oxygenation of ribulose bisphosphate (RuBP), thereby reducing photorespiration (Luo and Reynold, 1999). Elevated [CO2] is also associated with the expression of several other changes that affect photosynthesis. The common observation of reduced stomatal conductance, gs, will tend to dampen the extent to which any upregulation is expressed at a leaf-scale, but may conserve water such that stand-scale responses are positive (Ainsworth and Rogers, 2007).

Photosynthetic acclimation refers to longer-term adaptive changes in the photosynthetic responses to external stimuli that reduce the net level of the initial response; acclimation is also referred to as down-regulation (Pinkard et al., 2010). Acclimation is commonly observed and arises from the plant's need to balance all resources that are allocated for photosynthetic processes, including the external [CO2] (Gunderson and Wullschleger, 1994). For elevated [CO2], acclimation is mechanistically linked to decreased maximum apparent carboxylation velocity (Vcmax) and reduced investment in Rubisco (Rogers and Humphries, 2000). It is also associated with reduction in N content. These changes are linked to a decrease in control of Amax by Vcmax but an increase in the regeneration of RuBP, Jmax (Long and Drake, 1992). There is also an increase in starch and sugar content.

In the short term, rising [CO2] increases photosynthesis in many of the woody species as have been studied by Ainsworth and Long (2005). These species have the potential to yield signicantly with increases in the rates of biomass accumulation. The allocation of dry mass to the above-ground parts in forest free air carbon dioxide enrichment (FACE) experiment was also found to increase by 28% on the average; this includes a greater allocation to woody components. In general, larger responses in growth, biomass production and leaf area index to elevated [CO2] have been observed in trees than other functional types (Ainsworth and Long, 2005). Nevertheless, there is often a poor correlation between photosynthetic capacity measured as Amax and total biomass production under elevated [CO2] enrichment (Oren et al., 2001).

#### **6. Environmental factors determining the response of photosynthesis to elevated [CO2]**

#### **6.1 Nitrogen supply**

326 Advances in Photosynthesis – Fundamental Aspects

stomata can respond sensitively to [CO2] as part of a proportionate response to the CO2 requirement for photosynthesis; increasing concentrations are, therefore, associated with a closing response and vice-versa (Pinkard et al., 2010). Hence, elevated [CO2] is anticipated to increase or up-regulate photosynthesis, decrease stomatal conductance and increase intrinsic water-use efciency i.e. the ratio of leaf photosynthesis to stomatal conductance (Long et al., 2004). Many factors other than [CO2] determine photosynthetic rate, and the law of limiting factors (von Liebig, 1840) will ultimately determine photosynthetic responses to [CO2] where more than one limiting factors may be involved. Sala and Hoch (2009) suggested that elevated CO2 would improve carbon balance in light-limited as well as highlight environments through the CO2 enhancement of quantum yield. The present CO2:O2 ratio of the air constrains photosynthesis by 30-40% because of O2 inhibition of carboxylation and associated photorespiration (Booth and Jayanovic, 2005). As CO2 concentration increases, quantum yield is increased because the ratio of carboxylation to oxygenation by Rubisco increases and photorespiration decreases (Pinkard et al., 2010). Several studies have shown that CO2 enrichment enhances photosynthesis and growth under limiting irradiance condition, and in some cases the relative enhancement was greater at low rather than at high irradiances (Gifford et al., 1981). In C3 plants, elevated [CO2] increases the quantum yield of photosynthesis by reducing photorespiration caused by the oxygenase activity of Rubisco. Maximum, single-leaf quantum yield was increased from

Up-regulation of photosynthesis refers to a signicant increase in the light-saturated rate of photosynthesis (Asat), the rate of photosynthesis under ambient light (A), and/or diurnal photosynthesis (A). Elevated [CO2] up-regulates photosynthesis by increasing the carboxylation rate (Vc) of ribulose bisphosphate carboxylase (Rubisco) and competitively inhibiting the oxygenation of ribulose bisphosphate (RuBP), thereby reducing photorespiration (Luo and Reynold, 1999). Elevated [CO2] is also associated with the expression of several other changes that affect photosynthesis. The common observation of reduced stomatal conductance, gs, will tend to dampen the extent to which any upregulation is expressed at a leaf-scale, but may conserve water such that stand-scale

Photosynthetic acclimation refers to longer-term adaptive changes in the photosynthetic responses to external stimuli that reduce the net level of the initial response; acclimation is also referred to as down-regulation (Pinkard et al., 2010). Acclimation is commonly observed and arises from the plant's need to balance all resources that are allocated for photosynthetic processes, including the external [CO2] (Gunderson and Wullschleger, 1994). For elevated [CO2], acclimation is mechanistically linked to decreased maximum apparent carboxylation velocity (Vcmax) and reduced investment in Rubisco (Rogers and Humphries, 2000). It is also associated with reduction in N content. These changes are linked to a decrease in control of Amax by Vcmax but an increase in the regeneration of RuBP, Jmax (Long

In the short term, rising [CO2] increases photosynthesis in many of the woody species as have been studied by Ainsworth and Long (2005). These species have the potential to yield signicantly with increases in the rates of biomass accumulation. The allocation of dry mass to the above-ground parts in forest free air carbon dioxide enrichment (FACE) experiment was also found to increase by 28% on the average; this includes a greater allocation to woody components. In general, larger responses in growth, biomass production and leaf

0·065 to 0·080 (Long and Drake, 1992).

responses are positive (Ainsworth and Rogers, 2007).

and Drake, 1992). There is also an increase in starch and sugar content.

At elevated [CO2] condition, when plant photosynthesis becomes RubP limited, Rubisco will be in excess of requirements (Ainsworth and Rogers, 2007). The excess capacity for carboxylation could be reduced through a reduction in the activation state of Rubisco (Cen and Sage, 2005). Alternatively, because less Rubisco is required by these plants at elevated [CO2], redistribution of the excess N invested in Rubisco could further increase N use efciency at elevated [CO2] without negatively impacting potential C acquisition (Parry et al., 2003; Ainsworth and Rogers, 2007). However, there is only benet in reducing the amount of N invested in Rubisco at elevated [CO2] when the resources invested in it can be usefully deployed elsewhere (Parry et al. 2003). Ainsworth and Long (2005) reported that the stimulation in Asat at elevated [CO2] was 23% lower in plants grown with a low N supply. Meanwhile, under elevated [CO2] the Vcmax was decreased at both high and low N, with greater reduction of 85% in low N condition. This result is in agreement with summaries of earlier studies conducted in controlled environments and eld enclosures by Ainsworth and Rogers (2007), Drake et al. (1997), Moore et al. (1999) and Stitt and Krapp (1999), and is consistent with current understanding of the mechanism underlying acclimation under elevated [CO2]. When plants are N limited, sink development is restricted, C supply is in excess of demand, and the sugar feedback mechanism as outlined earlier can operate to reduce Rubisco content and increase N use efciency. As N supply increases, the limitation imposed by sink capacity decreases and the sugar linked signal for down-regulating Rubisco content is reduced (Ainsworth and Rogers, 2007; Drake et al. 1997; Rogers et al. 1998; Long et al., 2004).

#### **6.2 Sink strength**

Dened here as the capacity to utilize photosynthate, sink strength can be a major constraint on carbon acquisition (Ainsworth and Rogers, 2007). A reduced or insufcient sink capacity may be the result of many potentially limiting processes e.g. N supply (Rogers et al., 1998), genetic constraints (Ainsworth et al., 2004), temperature (Ainsworth et al., 2003b) or developmental changes (Bernacchi et al., 2005; Rogers and Ainsworth, 2006). However, the net result is the same, i.e. the appearance of a carbohydrate-derived signal that can lead to the subsequent down-regulation (acclimation) of photosynthetic machinery, principally Rubisco (Stitt and Krapp, 1999; Long et al., 2004). Davey et al. (2006) showed that poplar grown at elevated [CO2] had a large sink capacity. Poplar was able to export >90% of its photosynthate during the day; it also had a large capacity for the temporary storage of overow photosynthate as starch (Ainsworth and Rogers, 2007; Stitt and Quick 1989; Davey et al., 2006). These two traits enabled poplar to maintain high photosynthetic rates at elevated [CO2] and avoid a major source–sink imbalance that could lead to a reduction in the potential for C acquisition (Ainsworth and Rogers, 2007). In contrast, *L. perenne* can

Photosynthesis and Quantum Yield of Oil Palm Seedlings to Elevated Carbon Dioxide 329

followed by 800 µmol mol-1 and 400 µmol mol-1 CO2. During the experiment, elevated CO2 had caused the Asat of oil palm to increase by 52 to 78% compared to the ambient. Apparent quantum yield, calculated from the initial slope of the light response curves, was slightly lower in ambient treatment compared to the elevated CO2 treatments (800 and 1200 µmol mol-1 CO2; Figure 2b). During measurements, the elevated treatment exhibited a higher quantum yield than the ambient leaves (P < 0.05). Quantum yield was enhanced by 2 fold and 3 fold respectively in the 800 and 1200 µmol mol-1 CO2 treatments compared to 400 µmol

0 200 400 600 800 1000

**Photosynthetic photon flux density, PPFD (µmol m-2s**

400 800 1200 Poly. (400 ) Poly. (800 ) Poly. (1200 )

Elevated CO2 leaves had a lower light compensation point (Γ) and dark respiration rate (Rd) than the ambient leaves (Figure 2c; 2d). Estimated from the photosynthetic light response curves, it was also demonstrated a significant effect of elevated CO2 on Γ or Rd (P≤0.01). Using the light response curves, the light compensation point for ambient CO2 was recorded at 78.21 followed by 66.21 for 400 and 800 µmol mol-1 CO2 respectively and 30.24 for 1200 µmol mol-1 CO2 treatment. It was also observed that the dark respiration was reduced by

Many studies have suggested that the enriched CO2 leaves respond to atmospheric CO2 enrichment to a greater extent than the ambient- CO2 leaves as a result of increased quantum yields (Hanstein & Felle, 2002). A small increase in quantum yield may increase daily carbon gain under low light conditions (Kiirats et al., 2002). In our study, elevated CO2 increased apparent quantum yields in the 800 and 1200 µmol mol-1 CO2 treatments. Light response curve analysis of oil palm seedling had showed that CO2 enriched seedlings had reduced their dark respiration rate by 43 to 70% through enhancement of their Asat and apparent quantum yield (φ) by 52 to 78% and 15 to 62%, respectively. The enhancement of Г and φ signify direct inhibition of the activity of key respiratory enzymes under elevated CO2 (Drake et al., 1997). This result has been supported by Henson and Haniff (2005) who reported productivity or dry matter production of plants would increase if respiration could

Fig. 1. Light response curve as affected by different CO2 levels in oil palm seedlings

1.72 to 3.21 µmol m-2s-1 compared to ambient levels that recorded 5.71 µmol m-2s-1.

**-1)**

mol-1 CO2 one.



0

5

10

**Net assimilation rate, A sat (µmol m-2 s-1)**

15

20

25

become extremely sink limited at elevated [CO2] (Rogers and Ainsworth, 2006). As reported, large accumulations of carbohydrate which build up in grasses over several days and weeks are common (Fischer et al., 1997; Isopp et al., 2000; Rogers & Ainsworth 2006). The most likely explanation for the sink limitation observed in *L. perenne* is an insufcient N supply (Fischer et al., 1997; Rogers et al., 1998).

The excess of C and shortage of N may explain why grasses reduced their Rubisco content at elevated [CO2] despite the negative impact on potential carbon gain (Rogers et al., 1998). Therefore, a reduction in carboxylation capacity would be expected. However, legumes can trade photosynthate for reduced forms of N with their bacterial symbionts (Rogers et al., 2006b). The benet of an increase in N use efciency resulting from the reduction of Rubisco content and the sugar-derived signal required for a reduction in carboxylation capacity would not be expected (Ainsworth & Rogers, 2007). It follows that acclimation in legumes is likely to occur through reduction in Rubisco activity rather than through a loss of Rubisco protein content. This occurs in order to maintain the balance between the supply and demand for the products of the light reactions (Ainsworth & Rogers, 2007). Alternatively, other nutrient limitations may also impact N-xation and sink capacity at elevated [CO2] (Almeida et al., 2000; Hungate et al., 2004).

#### **7. Oil palm responses to elevated CO2**

An experiment was carried out using three levels of [CO2] (400, control; 800 and 1200 µmol mol-1 CO2) to demonstrate the responses of oil palm seedlings on photosynthesis and quantum yield to elevated [CO2]. Photosynthetic light response curves of oil palm were measured at growth CO2 concentrations on with an open flow infrared gas analyzer with an attached red LED light source (LI-6400, Li-Cor, Inc., Lincoln, NE). Measurements began with approximately 5 minutes of saturating light (1500 µmol m−2s−1) followed by nine incremental reductions until the irradiance was 0 µmol m−2s−1. Decreasing light was used rather than increasing light to reduce the equilibrium time required for stomatal opening and photosynthetic induction (Kubiske & Pregitzer, 1996). Preliminary trials indicated that photosynthetic rates reached steady state within 2 minutes following each incremental decrease in light. Measurements were made on fully expanded leaves. Gas exchange measurements were restricted to the hours between 0800 and 1200 hours on sunny days to minimize diurnal effects on photosynthesis. Leaf temperatures averaged 28.89 ± 0.89°C within each measurement period. The differences in light response curves due to CO2 concentration were examined by calculating and statistically comparing light-saturated photosynthesis (Asat), dark respiration (Rd), light compensation point (Γ) (where A = Rd), and apparent quantum yield (φ). Apparent quantum yield (φ) and Rd were estimated from the measured data. Values of φ were calculated as the slope of photosynthesis (A) versus the incident irradiance. Light compensation points (Γ) were estimated by extrapolating between measured data. The shape of the average light response curve in each CO2 concentration and canopy position was modeled by fitting data to a non-rectangular hyperbola (Leverenz, 1987; Leverenz, 1995) by means of a nonlinear least squares curve-fitting program (JMP, SAS Institute, Inc., Cary, NC)

Photosynthetic light response curves of oil palm are shown in Figure 1. Light-saturated net photosynthesis (Asat) was greater in elevated treatment than in ambient CO2. The elevated [CO2] exposure of oil palm seedling resulted in higher rates of Asat (P < 0.001; Figure 2a). The enhancement of Asat by [CO2] enrichment was significantly greater for 1200 µmol mol-1 CO2

become extremely sink limited at elevated [CO2] (Rogers and Ainsworth, 2006). As reported, large accumulations of carbohydrate which build up in grasses over several days and weeks are common (Fischer et al., 1997; Isopp et al., 2000; Rogers & Ainsworth 2006). The most likely explanation for the sink limitation observed in *L. perenne* is an insufcient N supply

The excess of C and shortage of N may explain why grasses reduced their Rubisco content at elevated [CO2] despite the negative impact on potential carbon gain (Rogers et al., 1998). Therefore, a reduction in carboxylation capacity would be expected. However, legumes can trade photosynthate for reduced forms of N with their bacterial symbionts (Rogers et al., 2006b). The benet of an increase in N use efciency resulting from the reduction of Rubisco content and the sugar-derived signal required for a reduction in carboxylation capacity would not be expected (Ainsworth & Rogers, 2007). It follows that acclimation in legumes is likely to occur through reduction in Rubisco activity rather than through a loss of Rubisco protein content. This occurs in order to maintain the balance between the supply and demand for the products of the light reactions (Ainsworth & Rogers, 2007). Alternatively, other nutrient limitations may also impact N-xation and sink capacity at elevated [CO2]

An experiment was carried out using three levels of [CO2] (400, control; 800 and 1200 µmol mol-1 CO2) to demonstrate the responses of oil palm seedlings on photosynthesis and quantum yield to elevated [CO2]. Photosynthetic light response curves of oil palm were measured at growth CO2 concentrations on with an open flow infrared gas analyzer with an attached red LED light source (LI-6400, Li-Cor, Inc., Lincoln, NE). Measurements began with approximately 5 minutes of saturating light (1500 µmol m−2s−1) followed by nine incremental reductions until the irradiance was 0 µmol m−2s−1. Decreasing light was used rather than increasing light to reduce the equilibrium time required for stomatal opening and photosynthetic induction (Kubiske & Pregitzer, 1996). Preliminary trials indicated that photosynthetic rates reached steady state within 2 minutes following each incremental decrease in light. Measurements were made on fully expanded leaves. Gas exchange measurements were restricted to the hours between 0800 and 1200 hours on sunny days to minimize diurnal effects on photosynthesis. Leaf temperatures averaged 28.89 ± 0.89°C within each measurement period. The differences in light response curves due to CO2 concentration were examined by calculating and statistically comparing light-saturated photosynthesis (Asat), dark respiration (Rd), light compensation point (Γ) (where A = Rd), and apparent quantum yield (φ). Apparent quantum yield (φ) and Rd were estimated from the measured data. Values of φ were calculated as the slope of photosynthesis (A) versus the incident irradiance. Light compensation points (Γ) were estimated by extrapolating between measured data. The shape of the average light response curve in each CO2 concentration and canopy position was modeled by fitting data to a non-rectangular hyperbola (Leverenz, 1987; Leverenz, 1995) by means of a nonlinear least squares curve-fitting program (JMP, SAS

Photosynthetic light response curves of oil palm are shown in Figure 1. Light-saturated net photosynthesis (Asat) was greater in elevated treatment than in ambient CO2. The elevated [CO2] exposure of oil palm seedling resulted in higher rates of Asat (P < 0.001; Figure 2a). The enhancement of Asat by [CO2] enrichment was significantly greater for 1200 µmol mol-1 CO2

(Fischer et al., 1997; Rogers et al., 1998).

(Almeida et al., 2000; Hungate et al., 2004).

**7. Oil palm responses to elevated CO2**

Institute, Inc., Cary, NC)

followed by 800 µmol mol-1 and 400 µmol mol-1 CO2. During the experiment, elevated CO2 had caused the Asat of oil palm to increase by 52 to 78% compared to the ambient. Apparent quantum yield, calculated from the initial slope of the light response curves, was slightly lower in ambient treatment compared to the elevated CO2 treatments (800 and 1200 µmol mol-1 CO2; Figure 2b). During measurements, the elevated treatment exhibited a higher quantum yield than the ambient leaves (P < 0.05). Quantum yield was enhanced by 2 fold and 3 fold respectively in the 800 and 1200 µmol mol-1 CO2 treatments compared to 400 µmol mol-1 CO2 one.

**Photosynthetic photon flux density, PPFD (µmol m-2s -1)**

400 800 1200 Poly. (400 ) Poly. (800 ) Poly. (1200 )

Fig. 1. Light response curve as affected by different CO2 levels in oil palm seedlings

Elevated CO2 leaves had a lower light compensation point (Γ) and dark respiration rate (Rd) than the ambient leaves (Figure 2c; 2d). Estimated from the photosynthetic light response curves, it was also demonstrated a significant effect of elevated CO2 on Γ or Rd (P≤0.01). Using the light response curves, the light compensation point for ambient CO2 was recorded at 78.21 followed by 66.21 for 400 and 800 µmol mol-1 CO2 respectively and 30.24 for 1200 µmol mol-1 CO2 treatment. It was also observed that the dark respiration was reduced by 1.72 to 3.21 µmol m-2s-1 compared to ambient levels that recorded 5.71 µmol m-2s-1.

Many studies have suggested that the enriched CO2 leaves respond to atmospheric CO2 enrichment to a greater extent than the ambient- CO2 leaves as a result of increased quantum yields (Hanstein & Felle, 2002). A small increase in quantum yield may increase daily carbon gain under low light conditions (Kiirats et al., 2002). In our study, elevated CO2 increased apparent quantum yields in the 800 and 1200 µmol mol-1 CO2 treatments. Light response curve analysis of oil palm seedling had showed that CO2 enriched seedlings had reduced their dark respiration rate by 43 to 70% through enhancement of their Asat and apparent quantum yield (φ) by 52 to 78% and 15 to 62%, respectively. The enhancement of Г and φ signify direct inhibition of the activity of key respiratory enzymes under elevated CO2 (Drake et al., 1997). This result has been supported by Henson and Haniff (2005) who reported productivity or dry matter production of plants would increase if respiration could

Photosynthesis and Quantum Yield of Oil Palm Seedlings to Elevated Carbon Dioxide 331

especially Rubisco (Ramachandra & Das, 1986). The latter might also up–regulate several enzymes related to carbon metabolism which simultaneously increase the Ci (Anderson et al., 2001). This data imply that high A under elevated CO2 could be due to more efficient net assimilation resulting from extra carbon fixation as exhibited by high Ci per unit area which is related to increased thickness of mesophyll layer, mainly due to increased palisade layer (Lawson et al., 2002). Up-regulation of A may as represented by increases in leaf intercellular CO2 concentration (Ci) may also be related to increase in the thickness of the leaves under elevated CO2 that contain high photosynthetic protein especially Rubisco (Ramachandra and Das, 1986). The latter might up–regulate several enzyme related to carbon metabolism that simultaneously increase the Ci (Anderson et al., 2001). This data implied that high A under elevated CO2 could be due to more efficient net assimilation due to extra carbon fixation exhibited by high Ci per unit area which is related to increased thickness of mesophyll layer, mainly due to increased palisade layer (Lawson et al., 2002).

400 800 1200

400 800 1200

Fig. 3. Net photosynthesis as affected by CO2 levels in oil palm seedlings

Fig. 4. Water use efficiency as affected by CO2 levels in oil palm seedlings

0

0

1 2

**W** 

**ater use efficiency, W**

3 4

5 6

 **U E** 7

2

**N** 

**et photosynthesis, A**

 **(µmol m-2 s-1 )**

4

6

8

10

12

14

be minimized without affecting gross assimilation, or if gross assimilation could be increased without increasing respiration. This shows that increase in CO2 would enhance gross assimilation and reduce respiration by compensating respiration rate with high carbon gain. Usually, compensation irradiance is reduced while quantum efficiency is increased in plant under elevated CO2 (Vavin et al., 1995). The same result was also observed by Kubiske and Preigitzer (1996) with red oak seedlings grown at elevated CO2 in shaded open top chamber.

Fig. 2. The maximum net assimilation rate Asat (a), apparent quantum yield (b), Light compensation point (c) and dark respiration rate (d) as affected by different CO2 levels in oil palm seedlings

In oil palm, it was found that enrichment with high levels of [CO2] have enhanced the leaf gas exchange of oil palm seedlings. As CO2 levels increased from 400 to 800 and 1200 µmol mol-1 CO2 the net photosynthesis (Figure 3) and water use efficiency (Figure 4) were also improved. Net photosynthesis (A) and water use efficiency (WUE) were been enhanced by respective 211 to 278% and 158 to 224% when enriched with [CO2] (800 and 1200 µmol mol-1 CO2). As CO2 levels increased, it was observed that the intercellular CO2 (Ci) increased higher in oil palm seedling treated with high levels of [CO2] (Figure 5). The Ci for 1200 µmol mol-1 CO2 recorded the highest (361.11 µmol mol-1 CO2) value followed by that of 800 µmol mol-1 CO2 (311.11 µmol mol-1 CO2) with the lowest at 400 µmol mol-1 CO2 that recorded 289.12 µmol mol-1. Up-regulation of A may as represented by increases in leaf intercellular CO2 concentration (Ci) that could also be related to increase in the thickness of the leaves (high SLA) achieved under elevated [CO2] that contains high photosynthetic protein

be minimized without affecting gross assimilation, or if gross assimilation could be increased without increasing respiration. This shows that increase in CO2 would enhance gross assimilation and reduce respiration by compensating respiration rate with high carbon gain. Usually, compensation irradiance is reduced while quantum efficiency is increased in plant under elevated CO2 (Vavin et al., 1995). The same result was also observed by Kubiske and Preigitzer (1996) with red oak seedlings grown at elevated CO2 in shaded open top

> 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

**D** 

In oil palm, it was found that enrichment with high levels of [CO2] have enhanced the leaf gas exchange of oil palm seedlings. As CO2 levels increased from 400 to 800 and 1200 µmol mol-1 CO2 the net photosynthesis (Figure 3) and water use efficiency (Figure 4) were also improved. Net photosynthesis (A) and water use efficiency (WUE) were been enhanced by respective 211 to 278% and 158 to 224% when enriched with [CO2] (800 and 1200 µmol mol-1 CO2). As CO2 levels increased, it was observed that the intercellular CO2 (Ci) increased higher in oil palm seedling treated with high levels of [CO2] (Figure 5). The Ci for 1200 µmol mol-1 CO2 recorded the highest (361.11 µmol mol-1 CO2) value followed by that of 800 µmol mol-1 CO2 (311.11 µmol mol-1 CO2) with the lowest at 400 µmol mol-1 CO2 that recorded 289.12 µmol mol-1. Up-regulation of A may as represented by increases in leaf intercellular CO2 concentration (Ci) that could also be related to increase in the thickness of the leaves (high SLA) achieved under elevated [CO2] that contains high photosynthetic protein

Fig. 2. The maximum net assimilation rate Asat (a), apparent quantum yield (b), Light compensation point (c) and dark respiration rate (d) as affected by different CO2 levels in oil

**ark respiration, Rd (µm**

 **ol m-2 s- 1 )**

**A** 

**pparent quantum**

 **yield, ϕ (m ol m ol - 1 )**

400 800 1200

c) d)

400 800 1200

chamber.

palm seedlings

**L ig h t com p en sa tio n p o in t, ┌ (µ m ol m- 2<sup>s</sup> - 1 )**

**A sat (µmol m -2 s-1)**

400 800 1200

a) b)

400 800 1200

especially Rubisco (Ramachandra & Das, 1986). The latter might also up–regulate several enzymes related to carbon metabolism which simultaneously increase the Ci (Anderson et al., 2001). This data imply that high A under elevated CO2 could be due to more efficient net assimilation resulting from extra carbon fixation as exhibited by high Ci per unit area which is related to increased thickness of mesophyll layer, mainly due to increased palisade layer (Lawson et al., 2002). Up-regulation of A may as represented by increases in leaf intercellular CO2 concentration (Ci) may also be related to increase in the thickness of the leaves under elevated CO2 that contain high photosynthetic protein especially Rubisco (Ramachandra and Das, 1986). The latter might up–regulate several enzyme related to carbon metabolism that simultaneously increase the Ci (Anderson et al., 2001). This data implied that high A under elevated CO2 could be due to more efficient net assimilation due to extra carbon fixation exhibited by high Ci per unit area which is related to increased thickness of mesophyll layer, mainly due to increased palisade layer (Lawson et al., 2002).

Fig. 3. Net photosynthesis as affected by CO2 levels in oil palm seedlings

Fig. 4. Water use efficiency as affected by CO2 levels in oil palm seedlings

Photosynthesis and Quantum Yield of Oil Palm Seedlings to Elevated Carbon Dioxide 333

justified by reduced light compensation point (┌) and dark respiration rate (Rd), with the plant enriched with high CO2 having enhanced apparent quantum yield and net

Despite increases in A and WUE, stomatal conductance of oil palm seedlings enriched with high levels of CO2 decreased as levels of CO2 increases (Figure 6). In 400 µmol mol-1 CO2, stomatal conductance recorded a value at 42.3 mmol m-2 s-1; with increasing [CO2] to 800 and 1200 µmol mol-1 the stomatal conductance documented lower values (27.1 to 32.3 mmol m-2 s-1). Further enhancing the plants to 800 and 1200 µmol mol-1 had shown to reduce stomatal conductance (gs) of the CO2-enriched seedlings versus the ambient CO2 treated plants with lowest gs. The decreased gs simultaneously reduced the transpiration rate (E) of plant under elevated CO2. This phenomenon is usually reported in plant treated with high than ambient CO2 (Rashke, 1986; Lodge et al., 2001; Lawson et al., 2002). It was believed that reduced gs might contribute to plant acclimation to high intercellular

It was also found that nitrogen levels were influenced by CO2 levels applied to the oil palm seedlings. From Figure 7 it is observed that the nitrogen levels were highest in leaves followed by stems and lowest in the roots. As [CO2] levels increased from 400 to 1200 µmol mol-1 CO2 the nitrogen content decreased highly in 1200 followed by 800 and lowest in 400 µmol mol-1 CO2. This implies that plant enriched with high levels of [CO2] have high dilution of nitrogen content in the plant tissues. Nitrogen content was influenced by the application of CO2 levels to the seedlings. As the levels of CO2 increased from 400 to 1200 µmol mol-1 CO2, nitrogen content were found to be reduced. The decrease in nitrogen content with increasing CO2 levels has been reported by Porteus et al. (2009). Several researchers attributed this phenomenon to decreasing uptake of nitrogen as transpiration rate (E) was decreased due to reduction in stomata conductance (gs) under elevated CO2

> Leaf Stem Root **Plant parts**

Fig. 7. Nitrogen levels as affected by CO2 levels in different part of oil palm seedlings

400 800 1200

assimilation rates (Kubiske & Preigitzer, 1996).

CO2 (Ci) (Morrison & Jarvis, 1980).

level (Conroy and Hawking, 1993).

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

**Nitrogen contents (%)**

Fig. 5. Intercelluar CO2 as affected by CO2 levels in oil palm seedlings

Fig. 6. Stomatal conductance, gs as affected by CO2 levels in oil palm seedlings

Statistically, higher A was observed over the controlled plants as the levels of enrichment increased. Similar result was obtained by Downtown (1990) and Van and Megonigal (2001). Theoretically, exposure to higher [CO2] would increase A by increasing the availability of the substrate (CO2; Downtown, 1990). In the present study, the increase in A might be

400 800 1200

400 800 1200

Statistically, higher A was observed over the controlled plants as the levels of enrichment increased. Similar result was obtained by Downtown (1990) and Van and Megonigal (2001). Theoretically, exposure to higher [CO2] would increase A by increasing the availability of the substrate (CO2; Downtown, 1990). In the present study, the increase in A might be

Fig. 6. Stomatal conductance, gs as affected by CO2 levels in oil palm seedlings

Fig. 5. Intercelluar CO2 as affected by CO2 levels in oil palm seedlings

0

**Stomatal conductance, gs (mol m-2s-1)**

50

100

150

200

**Intercellular CO2, Ci (µmol mol-1)**

250

300

350

400

justified by reduced light compensation point (┌) and dark respiration rate (Rd), with the plant enriched with high CO2 having enhanced apparent quantum yield and net assimilation rates (Kubiske & Preigitzer, 1996).

Despite increases in A and WUE, stomatal conductance of oil palm seedlings enriched with high levels of CO2 decreased as levels of CO2 increases (Figure 6). In 400 µmol mol-1 CO2, stomatal conductance recorded a value at 42.3 mmol m-2 s-1; with increasing [CO2] to 800 and 1200 µmol mol-1 the stomatal conductance documented lower values (27.1 to 32.3 mmol m-2 s-1). Further enhancing the plants to 800 and 1200 µmol mol-1 had shown to reduce stomatal conductance (gs) of the CO2-enriched seedlings versus the ambient CO2 treated plants with lowest gs. The decreased gs simultaneously reduced the transpiration rate (E) of plant under elevated CO2. This phenomenon is usually reported in plant treated with high than ambient CO2 (Rashke, 1986; Lodge et al., 2001; Lawson et al., 2002). It was believed that reduced gs might contribute to plant acclimation to high intercellular CO2 (Ci) (Morrison & Jarvis, 1980).

It was also found that nitrogen levels were influenced by CO2 levels applied to the oil palm seedlings. From Figure 7 it is observed that the nitrogen levels were highest in leaves followed by stems and lowest in the roots. As [CO2] levels increased from 400 to 1200 µmol mol-1 CO2 the nitrogen content decreased highly in 1200 followed by 800 and lowest in 400 µmol mol-1 CO2. This implies that plant enriched with high levels of [CO2] have high dilution of nitrogen content in the plant tissues. Nitrogen content was influenced by the application of CO2 levels to the seedlings. As the levels of CO2 increased from 400 to 1200 µmol mol-1 CO2, nitrogen content were found to be reduced. The decrease in nitrogen content with increasing CO2 levels has been reported by Porteus et al. (2009). Several researchers attributed this phenomenon to decreasing uptake of nitrogen as transpiration rate (E) was decreased due to reduction in stomata conductance (gs) under elevated CO2 level (Conroy and Hawking, 1993).

Fig. 7. Nitrogen levels as affected by CO2 levels in different part of oil palm seedlings

Photosynthesis and Quantum Yield of Oil Palm Seedlings to Elevated Carbon Dioxide 335

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Brinkman, M.A. & Frey, K.J.(1978). Flag leaf physiological analysis of oat isolines that differ in grain yield from their recurrent parents. *Crop Science* Vol 18, pp.69–73 Cen Y.P. & Sage R.F. (2005) The regulation of rubisco activity in response to variation in

Clayton, R.K. (1971) Light and living matter: A guide to the study of photobiology. New

Clayton, R.K. (1980) Photosynthesis: Physical mechanism and chemical patterns.

Conroy, J. and Hocking, P. (1993). Nitrogen nutritions of C3 plants at elevated carbon

Davey P.A., Olcer H., Zakhleniuk O., Bernacchi C.J., Calfapietra C., Long S.P. & Raines C.A.

Dong, S.T. (1991). Studies on the relationship between canopy apparent photosynthesis and

Downton, W.J.S., Grant, W.J.R. and Chacko, E.K. (1990). Effect of elevated carbon dioxide on

Drake B.G., Gonzalez-Meler M.A. & Long S.P. (1997) More efcient plants: a consequence of

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#### **8. Conclusion**

The results demonstrated by the oil palm seedlings indicated a positive response of oil palm seedling to elevated CO2 enrichment in term of enhanced photosynthesis rate and quantum yield as compared with ambient CO2 condition. The positive impact of oil palm seedlings to CO2 enrichment was shown by enhancement of the leaf gas exchange characteristics of oil palm. The positive responses have been shown to cause increases in net photosynthesis, Asat, apparent quantum yield and reduction of dark respiration rate and light compensation point. The findings suggest that in the next 22nd century, it would be expected that oil palm to benefit from changes in the climate as long as temperature does not increase beyond the palm optimum level. Further research in the future needs to be conducted to confirm these effects especially involving many environmental conditions under elevated [CO2]. Producing crops under climate change conditions, then, would be a growing challenge in the new agriculture of the world.

#### **9. Acknowledgment**

The authors are grateful to the Ministry of Higher Education Malaysia and the Research Management Centre of Universiti Putra Malaysia for supporting this work under the Graduate Research Assistant Grant Scheme.

#### **10. References**


The results demonstrated by the oil palm seedlings indicated a positive response of oil palm seedling to elevated CO2 enrichment in term of enhanced photosynthesis rate and quantum yield as compared with ambient CO2 condition. The positive impact of oil palm seedlings to CO2 enrichment was shown by enhancement of the leaf gas exchange characteristics of oil palm. The positive responses have been shown to cause increases in net photosynthesis, Asat, apparent quantum yield and reduction of dark respiration rate and light compensation point. The findings suggest that in the next 22nd century, it would be expected that oil palm to benefit from changes in the climate as long as temperature does not increase beyond the palm optimum level. Further research in the future needs to be conducted to confirm these effects especially involving many environmental conditions under elevated [CO2]. Producing crops under climate change conditions, then, would be a growing challenge in

The authors are grateful to the Ministry of Higher Education Malaysia and the Research Management Centre of Universiti Putra Malaysia for supporting this work under the

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**17** 

Alexander A. Ivlev

*Russian Federation* 

**Oscillatory Nature of Metabolism** 

**and Carbon Isotope Distribution** 

*Russian Agrarian State University – "MSKHA of K.A.Timirjazev"* 

A study of carbon isotopic characteristics of plants and animals, such as, shifts in carbon isotope ratio of plant biomass relative to environmental CO2, 13C values of biochemical fractions and individual metabolites, different isotopic patterns of biomolecules and diurnal isotopic changes of respired CO2, evidences that in a living cell carbon isotope fractionation

The above characteristics might be the source of valuable information on cell metabolism and regulation of metabolic processes, on assimilate transport, and different aspects of "organism – habitat" interactions. The efficiency of the involving this information in living organism studies greatly depends on the validity of carbon isotope fractionation model used for the interpretation. The validity of the model first of all is determined by the adopted

Two alternative points of view have been suggested in the literature. One of them asserts (Galimov, 1985; Schmidt, 2003) that carbon isotope effect and isotope distribution in biomolecules are of thermodynamic order. It means that isotope distribution of metabolites doesn't depend on biosynthesis pathway but is determined by the properties of the molecules themselves, i.e. by their structure and energy characteristics. According to the second point, supported by most of the researchers, the metabolic isotope effects are of the kinetic nature and carbon isotope distributions in metabolites are determined by

A lot of facts accumulated till now allow saying with confidence that thermodynamic concept is erroneous and the rare casual coincidences only simulate thermodynamic equilibrium (O'Leary & Yapp, 1978; Monson & Hayes, 1982; Ivlev, 2004). In some publications it was shown that the "thermodynamic" idea is "incompatible with the concept of life as a fundamental phenomenon" (Varshavski, 1988; Buchachenko, 2003). So we'll

Within the frame of the "kinetic" concept two different approaches have been developed. The first is the steady-state model assumes that all the processes in a living cell during photosynthesis proceed simultaneously in stationary conditions. It also means that carbon isotope fractionation proceeds in stationary conditions too. The approach was put forward by Park and Epstein (Park & Epstein, 1960, 1961) and was developed by Farquhar et al.

**1. Introduction** 

takes place.

view on the nature of isotope effect origin.

mechanisms and pathways of their formation.

concentrate on the kinetic concept.

**in Photosynthesizing Cells** 

Zhang, S.Y.; Lu, G.Y. ;Wu, H.; Shen, Z.X.; Zhong, H.M.; Shen,Y.G.; Xu, D.Y.; Ding, H,G. &, Hu, W.X. (1992). Photosynthesis of major C3 plants on Qinghai plateau. Acta Botany Singapore Vol 34, pp 176–184

### **Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells**

Alexander A. Ivlev *Russian Agrarian State University – "MSKHA of K.A.Timirjazev" Russian Federation* 

#### **1. Introduction**

340 Advances in Photosynthesis – Fundamental Aspects

Zhang, S.Y.; Lu, G.Y. ;Wu, H.; Shen, Z.X.; Zhong, H.M.; Shen,Y.G.; Xu, D.Y.; Ding, H,G. &,

Botany Singapore Vol 34, pp 176–184

Hu, W.X. (1992). Photosynthesis of major C3 plants on Qinghai plateau. Acta

A study of carbon isotopic characteristics of plants and animals, such as, shifts in carbon isotope ratio of plant biomass relative to environmental CO2, 13C values of biochemical fractions and individual metabolites, different isotopic patterns of biomolecules and diurnal isotopic changes of respired CO2, evidences that in a living cell carbon isotope fractionation takes place.

The above characteristics might be the source of valuable information on cell metabolism and regulation of metabolic processes, on assimilate transport, and different aspects of "organism – habitat" interactions. The efficiency of the involving this information in living organism studies greatly depends on the validity of carbon isotope fractionation model used for the interpretation. The validity of the model first of all is determined by the adopted view on the nature of isotope effect origin.

Two alternative points of view have been suggested in the literature. One of them asserts (Galimov, 1985; Schmidt, 2003) that carbon isotope effect and isotope distribution in biomolecules are of thermodynamic order. It means that isotope distribution of metabolites doesn't depend on biosynthesis pathway but is determined by the properties of the molecules themselves, i.e. by their structure and energy characteristics. According to the second point, supported by most of the researchers, the metabolic isotope effects are of the kinetic nature and carbon isotope distributions in metabolites are determined by mechanisms and pathways of their formation.

A lot of facts accumulated till now allow saying with confidence that thermodynamic concept is erroneous and the rare casual coincidences only simulate thermodynamic equilibrium (O'Leary & Yapp, 1978; Monson & Hayes, 1982; Ivlev, 2004). In some publications it was shown that the "thermodynamic" idea is "incompatible with the concept of life as a fundamental phenomenon" (Varshavski, 1988; Buchachenko, 2003). So we'll concentrate on the kinetic concept.

Within the frame of the "kinetic" concept two different approaches have been developed. The first is the steady-state model assumes that all the processes in a living cell during photosynthesis proceed simultaneously in stationary conditions. It also means that carbon isotope fractionation proceeds in stationary conditions too. The approach was put forward by Park and Epstein (Park & Epstein, 1960, 1961) and was developed by Farquhar et al.

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 343

carbon fluxes at the down-stream cross-points. Thus the integrative steady-state approach is insufficient for the explanation of short-term or intramolecular carbon isotope fractionation

Another approach of the kinetic concept is presented in the works of Ivlev and colleagues (Ivlev, 1989, 1993, 2008; Igamberdiev et al., 2001; Ivlev et al. 2004; Roussel et al, 2007). Opposite to steady-state idea, the authors put forward and developed the idea that metabolic processes are discrete and periodic ones. Periodicity of metabolic processes allows concluding that substrate pools in cells are periodically filled and depleted. It is well known fact that isotope fractionation accompanying the metabolic processes in case of depletion is followed by Raleigh effect (Melander&Sauders,1983). This effect establishes the dependence between isotope ratio

), reaction product ( <sup>13</sup>*Сproduct*

. <sup>1</sup> [ 10 1] [ 10 1] [1 (1 ) ] *С С product init substrate <sup>F</sup>*

 *= 12k/13k* , *12k* and *13k* are the rate constants of isotopic species of the molecules. The Raleigh effect is the most essential feature of carbon metabolism in a living cell. It closely relates to filling/depletion regime of cell functioning and to oscillatory character of metabolic reactions. Another fundamental feature related to Raleigh effect is the strict temporal sequence of metabolic reactions, i.e. temporal organization in a cell. Using equation (3) and carbon isotopic composition of metabolites, it is possible to distinguish the

Kinetic nature of carbon isotope effect and participation of polyatomic carbon molecules in metabolic reactions give the evidences that most of the biomolecules in metabolic chains inherit their isotope distributions from the precursors thus proving there is no isotope exchange between carbon atoms within the carbon skeletons. Most frequent cases where isotopic shifts emerge are linked with C – C bond cleavage, especially at the cross-points of metabolic pathways. The kinetic nature of isotope effect is manifested by the fact that only those carbon atoms of skeleton disposed at the ends of broken bonds undergo isotopic shifts. These and specificity of enzymatic interactions determine individual isotopic pattern of the biomolecules. Taking into account the above factors in combination with Raleigh effect and the putative pathways of the metabolite synthesis allows reconstructing of

Finally, the known regularities of inter- and intramolecular carbon isotope distribution in a cell indicate that metabolic oscillations are undamped and in-phase. Otherwise these isotopic regularities couldn't exist. The existence of the regularities on account of the Raleigh effect means, that at a given functional state of a cell, the metabolite syntheses within the repeated cell cycles occur at a certain level of substrate pool depletion. Moreover the

The first step to the oscillation model was the emergence of the discrete model based on the experimentally observed data on 12C enrichment of plant and photosynthesizing

coefficient () and the extent of pool depletion *F* in accordance with the equation:

<sup>13</sup> <sup>3</sup> <sup>13</sup> <sup>3</sup> 1/

isotopic patterns of the molecules and gives a fine tool for metabolism study.

**2. Carbon isotope fractionation in photosynthesis and photosynthetic** 

  , (3)

*F*

), isotope fractionation

processes.

where 

of initial substrate ( <sup>13</sup>*Сinit substrate* .

temporal sequence of many metabolic events.

functioning of different cells is synchronized.

**oscillation concept** 

(1982), Vogel (1993) and others (Gillon & Griffiths, 1997). Hayes (2001) has extended this approach to the common case, including secondary metabolism (metabolism in glycolytic chain).

According to the steady-state model, carbon isotope fractionation in photosynthesis can be presented as follows:

$$
\Delta = a + (b - a) \, p/p\_a \tag{1}
$$

where Δ is a carbon isotope discrimination, equal to the difference between 13C of environmental CO2 and that of biomass carbon; *a* is a carbon isotope effect of CO2 diffusion from the space into a photosynthesizing cell; *b* is a carbon isotope effect of ribuloso-1,5 bisphosphate (RuBP) carboxylation appearing in CO2 fixation; *pa* and *pi* are the CO2 partial pressures in the atmosphere and in the leaf space.

This simple steady-state balance model was rather convenient to explain the coherence between physiological response of plants to changing environmental conditions that impact stomatal conductance and net photosynthesis. Especially positive results were obtained in the field of carbon – water relations (Farquhar et al., 1989). Nevertheless even such a simple expression (1) turned to be contradicting. According to (1), isotope discrimination Δ approaches *a* or *b* values dependent on what is rate controlling stage – diffusion either biochemical. Direct measurements of activation energy of mesophyll cell conductance (Laisk, 1977) showed that diffusion is a rate-limiting stage in CO2 assimilation. Hence, according to model, most of C3-plants should be "heavier" than they are and Δ values should approach 4 - 5‰, i.e. *a*, whereas in fact they are close to 29‰, i.e. *b*. Other discrepancies were described in (Ivlev, 2003). The more the equation (1) was used, the more inconsistencies were found. Numerous corrections were introduced into expression (1) to take into account other processes, where carbon isotope fractionation might be, and to remove inconsistencies. Entirely the expression (1) was transformed into expression like the following (Farquhar & Lloid, 1993):

$$\Delta = \mathbf{a} \, \frac{p\_a - p\_i}{p\_a} + \mathbf{a}\_i \, \frac{p\_i - p\_c}{p\_a} + \mathbf{b} \, \frac{p\_c}{p\_a} - \frac{1}{p\_a} (\frac{eR\_d}{k} + f \cdot \Gamma^\*) \tag{2}$$

where *pa, pi* and *pc* refer to the partial pressure of CO2 in the atmosphere, substomatal cavity and chloroplast, respectively, *a* is the fractionation during the diffusion in air, *ai* is the combined fractionation during dissolution and diffusion through the liquid phase , *b* is the assumed net fractionation during carboxylation by ribuloso-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and by phosphoenolpyruvate carboxylase (PEPC), *k* is the carboxylation efficiency, *Rd* is the day respiration rate, Г\* is the CO2 compensation point in the absence of day respiration, *e* and *f* are the fractionation during dark respiration and photorespiration, respectively.

The expression (2), unlike to (1), is inconvenient for isotope fractionation analysis in photosynthesis since contains too many parameters to be determined. Even more complex expressions are obtained when it is required to describe intramolecular isotope distribution (Tcherkez et al., 2004). Using theoretical analysis of carbon isotope fractionation in metabolic chain under stationary conditions Hayes (2001) have shown that it was impossible to predict isotope composition of metabolites and their isotopic patterns since they depend not only on the isotope characteristics of the prior metabolites in the chain, but on the partitioning of

(1982), Vogel (1993) and others (Gillon & Griffiths, 1997). Hayes (2001) has extended this approach to the common case, including secondary metabolism (metabolism in glycolytic

According to the steady-state model, carbon isotope fractionation in photosynthesis can be

where Δ is a carbon isotope discrimination, equal to the difference between 13C of environmental CO2 and that of biomass carbon; *a* is a carbon isotope effect of CO2 diffusion from the space into a photosynthesizing cell; *b* is a carbon isotope effect of ribuloso-1,5 bisphosphate (RuBP) carboxylation appearing in CO2 fixation; *pa* and *pi* are the CO2 partial

This simple steady-state balance model was rather convenient to explain the coherence between physiological response of plants to changing environmental conditions that impact stomatal conductance and net photosynthesis. Especially positive results were obtained in the field of carbon – water relations (Farquhar et al., 1989). Nevertheless even such a simple expression (1) turned to be contradicting. According to (1), isotope discrimination Δ approaches *a* or *b* values dependent on what is rate controlling stage – diffusion either biochemical. Direct measurements of activation energy of mesophyll cell conductance (Laisk, 1977) showed that diffusion is a rate-limiting stage in CO2 assimilation. Hence, according to model, most of C3-plants should be "heavier" than they are and Δ values should approach 4 - 5‰, i.e. *a*, whereas in fact they are close to 29‰, i.e. *b*. Other discrepancies were described in (Ivlev, 2003). The more the equation (1) was used, the more inconsistencies were found. Numerous corrections were introduced into expression (1) to take into account other processes, where carbon isotope fractionation might be, and to remove inconsistencies. Entirely the expression (1) was transformed into expression like the

Δ = *a + ( b – a) pi/ pa* (1)

chain).

presented as follows:

pressures in the atmosphere and in the leaf space.

following (Farquhar & Lloid, 1993):

photorespiration, respectively.

Δ = a *a i a p p p*

+ ai *i c*

*a p p p*

+ b *<sup>c</sup>*

where *pa, pi* and *pc* refer to the partial pressure of CO2 in the atmosphere, substomatal cavity and chloroplast, respectively, *a* is the fractionation during the diffusion in air, *ai* is the combined fractionation during dissolution and diffusion through the liquid phase , *b* is the assumed net fractionation during carboxylation by ribuloso-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and by phosphoenolpyruvate carboxylase (PEPC), *k* is the carboxylation efficiency, *Rd* is the day respiration rate, Г\* is the CO2 compensation point in the absence of day respiration, *e* and *f* are the fractionation during dark respiration and

The expression (2), unlike to (1), is inconvenient for isotope fractionation analysis in photosynthesis since contains too many parameters to be determined. Even more complex expressions are obtained when it is required to describe intramolecular isotope distribution (Tcherkez et al., 2004). Using theoretical analysis of carbon isotope fractionation in metabolic chain under stationary conditions Hayes (2001) have shown that it was impossible to predict isotope composition of metabolites and their isotopic patterns since they depend not only on the isotope characteristics of the prior metabolites in the chain, but on the partitioning of

*a p*

*p* – <sup>1</sup> \* ( ) *<sup>d</sup> a eR <sup>f</sup> p k*

(2)

carbon fluxes at the down-stream cross-points. Thus the integrative steady-state approach is insufficient for the explanation of short-term or intramolecular carbon isotope fractionation processes.

Another approach of the kinetic concept is presented in the works of Ivlev and colleagues (Ivlev, 1989, 1993, 2008; Igamberdiev et al., 2001; Ivlev et al. 2004; Roussel et al, 2007). Opposite to steady-state idea, the authors put forward and developed the idea that metabolic processes are discrete and periodic ones. Periodicity of metabolic processes allows concluding that substrate pools in cells are periodically filled and depleted. It is well known fact that isotope fractionation accompanying the metabolic processes in case of depletion is followed by Raleigh effect (Melander&Sauders,1983). This effect establishes the dependence between isotope ratio of initial substrate ( <sup>13</sup>*Сinit substrate* . ), reaction product ( <sup>13</sup>*Сproduct* ), isotope fractionation coefficient () and the extent of pool depletion *F* in accordance with the equation:

$$[\delta^{13}\mathcal{C}\_{product}\cdot 10^{-3} + 1] = [\delta^{13}\mathcal{C}\_{init\,\,subtimes\,\text{finite}}\cdot 10^{-3} + 1]\frac{1}{F}[1 - (1 - F)^{1/a}]\,\,\prime\tag{3}$$

where *= 12k/13k* , *12k* and *13k* are the rate constants of isotopic species of the molecules.

The Raleigh effect is the most essential feature of carbon metabolism in a living cell. It closely relates to filling/depletion regime of cell functioning and to oscillatory character of metabolic reactions. Another fundamental feature related to Raleigh effect is the strict temporal sequence of metabolic reactions, i.e. temporal organization in a cell. Using equation (3) and carbon isotopic composition of metabolites, it is possible to distinguish the temporal sequence of many metabolic events.

Kinetic nature of carbon isotope effect and participation of polyatomic carbon molecules in metabolic reactions give the evidences that most of the biomolecules in metabolic chains inherit their isotope distributions from the precursors thus proving there is no isotope exchange between carbon atoms within the carbon skeletons. Most frequent cases where isotopic shifts emerge are linked with C – C bond cleavage, especially at the cross-points of metabolic pathways. The kinetic nature of isotope effect is manifested by the fact that only those carbon atoms of skeleton disposed at the ends of broken bonds undergo isotopic shifts. These and specificity of enzymatic interactions determine individual isotopic pattern of the biomolecules. Taking into account the above factors in combination with Raleigh effect and the putative pathways of the metabolite synthesis allows reconstructing of isotopic patterns of the molecules and gives a fine tool for metabolism study.

Finally, the known regularities of inter- and intramolecular carbon isotope distribution in a cell indicate that metabolic oscillations are undamped and in-phase. Otherwise these isotopic regularities couldn't exist. The existence of the regularities on account of the Raleigh effect means, that at a given functional state of a cell, the metabolite syntheses within the repeated cell cycles occur at a certain level of substrate pool depletion. Moreover the functioning of different cells is synchronized.

#### **2. Carbon isotope fractionation in photosynthesis and photosynthetic oscillation concept**

The first step to the oscillation model was the emergence of the discrete model based on the experimentally observed data on 12C enrichment of plant and photosynthesizing

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 345

glycine decarboxylation produces CO2 enriched in 12C evolving from the cell, whereas carbon substrates spinning in the loop are enriched in 13C and result in corresponding enrichment of photorespiratory products and biomass. The level of 13C enrichment depends on what how many turns carbon substrate flux makes in the loop, or what the extent of

1 – a point of carbon isotope fractionation in CO2 assimilation (carbon isotope effect in RuBP

Fig. 1. Oscillating model of carbon isotope fractionation in photosynthesis.

**2.1 Experimental facts support the presence of photosynthetic oscillations** 

glycine decarboxylation)

photorespiratory loop.

carboxylation), 11 – a point of carbon isotope fractionation in photorespiration (carbon isotope effect in

The experimental data presented in Tables 1-3 show distinct differences in isotope composition of metabolites derived in the both phases of Calvin cycle oscillations. Table 1 illustrates 13C enrichment of leaf oxalates of different C3 and CAM-plants (Rivera & Smith, 1979; Raven et al., 1982). Their synthesis is mainly bound to glycolate cycle of

photorespiratory pool depletion is achieved (Raleigh effect).

microorganism biomass relative to ambient CO2 at different conditions. The model assumed that CO2 assimilation is a discrete process and CO2 enters the cells by separate batches (Ivlev, 1989), but not by continuous flow like in a steady-state model (Farquhar et al., 1982). On account of isotope effect in RuBP carboxylation discrete model explained different levels of 12C enrichment of photosynthetic biomass by the Raliegh effect accepting that only part of the CO2 batches is fixed. The observed isotopic difference between C3 and C4 plants (Smith & Epstein, 1971) was explained by the same manner. Indeed, due to anatomical peculiarities of C4- plants (Edwards & Walker, 1983) they are capable to re-assimilate almost all respired CO2 thus increasing the extent of CO2 batches depletion (*F* in expression 3)

The question was - what's the reason making CO2 flux to be discrete? It was assumed that CO2 assimilation flux periodically is interrupted by the reverse flux of the respired CO2 directed from the cell to the environments. It was also assumed that such a "ping -pong" mechanism is due to double function of the key photosynthetic enzyme – Rubisco, which is capable to function as carboxylase or oxygenase depending on CO2/O2 concentration ratio in a cell (Ivlev, 1992). Switching mechanism splits CO2 flux entering and leaving the cell into separate batches. This hypothesis got strong support when new carbon isotope effect of photorespiration has been discovered (Ivlev, 1993).

Some facts known from the literature the traditional steady-state model failed to explain. In gas exchange experiments with the use of CO2 enriched in 13C the advantageous fixation of "heavy" molecules instead of "light" ones by leaves of different plants was observed (Sanadze et al., 1978). The similar results with the use of 14CO2 were obtained in the experiments with alga (Voznesenskii et al., 1982). Moreover the primary assimilates turned to be isotopically "heavier" relative to the ambient CO2. In the experiments with photosynthesizing bacteria *Ectothiorhodospira shaposhnikovii* there was a change in the sign of isotope discrimination linked with the growth of 13C content in the ambient CO2 (Ivanov et al.,1978). To explain these facts it was assumed an existence of the new isotope effect related to photorespiration, and having the opposite sign to that in CO2 assimilation. The analysis of the tentative points in photorespiration loop, where such an effect might emerge, showed that the most plausible point for its origin was glycine dehydrogenase reaction (Ivlev, 1993) (Fig.1), where decarboxylation of glycine occurs.

The following study of Calvin cycle and photorespiration biochemistry in virtue of carbon isotope composition of the primary assimilates allowed concluding on two phases of Calvin cycle functioning. In the first phase Calvin cycle produces glucose-6-phosphate (G6P) and other products from the fixed CO2. During this phase the derived products are used to accumulate the reserve pool of starch to feed glycolytic chain in the dark and provide substrates for lipid, protein and lignin components syntheses. Carbon isotope fractionation in RuBP carboxylation results in 12C enrichment of the cycle metabolites and finally the biomass as a whole relative to the ambient CO2.

It takes some time to substantiate the experimental validity of the hypothesis and to prove that glycine decarboxylation is the very point *in vivo* where carbon isotope fractionation results in 13C enrichment of biomass (Ivlev et al., 1996; 1999; Igamberdiev et al., 2001; 2004).

In the second phase Calvin cycle forms in combination with glycolate cycle the photorespiratory loop. The residual part of G6P produced in the previous phase converts into pentoses and then in form of phosphoglycolate leaves Calvin cycle and enters glycolate cycle where oxidative glycine decarboxylation occurs (Fig.1). After some transformations carbon flux in form of trioses returns back to Calvin cycle. Carbon isotope fractionation in

microorganism biomass relative to ambient CO2 at different conditions. The model assumed that CO2 assimilation is a discrete process and CO2 enters the cells by separate batches (Ivlev, 1989), but not by continuous flow like in a steady-state model (Farquhar et al., 1982). On account of isotope effect in RuBP carboxylation discrete model explained different levels of 12C enrichment of photosynthetic biomass by the Raliegh effect accepting that only part of the CO2 batches is fixed. The observed isotopic difference between C3 and C4 plants (Smith & Epstein, 1971) was explained by the same manner. Indeed, due to anatomical peculiarities of C4- plants (Edwards & Walker, 1983) they are capable to re-assimilate almost all respired

The question was - what's the reason making CO2 flux to be discrete? It was assumed that CO2 assimilation flux periodically is interrupted by the reverse flux of the respired CO2 directed from the cell to the environments. It was also assumed that such a "ping -pong" mechanism is due to double function of the key photosynthetic enzyme – Rubisco, which is capable to function as carboxylase or oxygenase depending on CO2/O2 concentration ratio in a cell (Ivlev, 1992). Switching mechanism splits CO2 flux entering and leaving the cell into separate batches. This hypothesis got strong support when new carbon isotope effect of

Some facts known from the literature the traditional steady-state model failed to explain. In gas exchange experiments with the use of CO2 enriched in 13C the advantageous fixation of "heavy" molecules instead of "light" ones by leaves of different plants was observed (Sanadze et al., 1978). The similar results with the use of 14CO2 were obtained in the experiments with alga (Voznesenskii et al., 1982). Moreover the primary assimilates turned to be isotopically "heavier" relative to the ambient CO2. In the experiments with photosynthesizing bacteria *Ectothiorhodospira shaposhnikovii* there was a change in the sign of isotope discrimination linked with the growth of 13C content in the ambient CO2 (Ivanov et al.,1978). To explain these facts it was assumed an existence of the new isotope effect related to photorespiration, and having the opposite sign to that in CO2 assimilation. The analysis of the tentative points in photorespiration loop, where such an effect might emerge, showed that the most plausible point for its origin was glycine dehydrogenase reaction (Ivlev, 1993)

The following study of Calvin cycle and photorespiration biochemistry in virtue of carbon isotope composition of the primary assimilates allowed concluding on two phases of Calvin cycle functioning. In the first phase Calvin cycle produces glucose-6-phosphate (G6P) and other products from the fixed CO2. During this phase the derived products are used to accumulate the reserve pool of starch to feed glycolytic chain in the dark and provide substrates for lipid, protein and lignin components syntheses. Carbon isotope fractionation in RuBP carboxylation results in 12C enrichment of the cycle metabolites and finally the

It takes some time to substantiate the experimental validity of the hypothesis and to prove that glycine decarboxylation is the very point *in vivo* where carbon isotope fractionation results in 13C enrichment of biomass (Ivlev et al., 1996; 1999; Igamberdiev et al., 2001; 2004). In the second phase Calvin cycle forms in combination with glycolate cycle the photorespiratory loop. The residual part of G6P produced in the previous phase converts into pentoses and then in form of phosphoglycolate leaves Calvin cycle and enters glycolate cycle where oxidative glycine decarboxylation occurs (Fig.1). After some transformations carbon flux in form of trioses returns back to Calvin cycle. Carbon isotope fractionation in

CO2 thus increasing the extent of CO2 batches depletion (*F* in expression 3)

photorespiration has been discovered (Ivlev, 1993).

(Fig.1), where decarboxylation of glycine occurs.

biomass as a whole relative to the ambient CO2.

glycine decarboxylation produces CO2 enriched in 12C evolving from the cell, whereas carbon substrates spinning in the loop are enriched in 13C and result in corresponding enrichment of photorespiratory products and biomass. The level of 13C enrichment depends on what how many turns carbon substrate flux makes in the loop, or what the extent of photorespiratory pool depletion is achieved (Raleigh effect).

1 – a point of carbon isotope fractionation in CO2 assimilation (carbon isotope effect in RuBP carboxylation), 11 – a point of carbon isotope fractionation in photorespiration (carbon isotope effect in glycine decarboxylation)

Fig. 1. Oscillating model of carbon isotope fractionation in photosynthesis.

#### **2.1 Experimental facts support the presence of photosynthetic oscillations**

The experimental data presented in Tables 1-3 show distinct differences in isotope composition of metabolites derived in the both phases of Calvin cycle oscillations. Table 1 illustrates 13C enrichment of leaf oxalates of different C3 and CAM-plants (Rivera & Smith, 1979; Raven et al., 1982). Their synthesis is mainly bound to glycolate cycle of photorespiratory loop.

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 347

The same picture illustrated by the data on 13C distribution in biomass of marine alga *Chlorella stigmatophora* grown under different environmental conditions on the CO2 of the

Lipids and proteins distinctly differ in carbon isotope ratio as compared with labile sugars and organic acids. In special experimental studies (Kalinkina &Udel'nova, 1991; Kalinkina & Naumova, 1992) the authors have proven that these components were the products of photorespiration pathway whereas most of lipids and proteins are synthesized via

Quite another object, C3-CAM tropical plant *Clusia minor,* grown under different environmental conditions is given in Table 4. Soluble sugars and organic acids, whose origin is linked with photorespiratory carbon flux, are enriched in 13C as compared with amino acid and lipid fractions. Notably the latter fraction, besides lipids, contains pigments some of which, like chlorophyll (Ivlev, 1993), at least partially are formed at the expense of photorespiratory flux. It makes lipid fraction isotopically "heavier" than amino acid fraction is. Thus all the presented data confirmed the idea on two phases of Calvin cycle functioning and drew to the conclusion that the phases are alternating, i.e. are separated in time. In fact, if the processes proceeded simultaneously the isotopically different carbon fluxes couldn't arise. Passing the same pieces of Calvin cycle they would inevitably mix. Bearing in mind that the first phase of cycle functioning corresponds to carboxylase function of Rubisco, while the second – to oxygenase one, we called the first as carboxylase phase and the second as oxygenase. To confirm the oscillating idea we tried to get more independent arguments and examined isotopic patterns of metabolites derived in different phases of Calvin cycle.

1 Total carbon -25.7 -30.3 -24.6 -29.1

3 Amino acids -31.7 -32.6 -31.3 -32.7 4 Soluble sugars -21.2 -29.2 -17.9 -21.9

Table 4. Carbon isotope composition of biochemical fractions isolated from leaves of *Clusia minor* under different environmental conditions (Borland et al., 1994). Samples were taken at

**2.2 Intramolecular isotopic patterns of glucose, anomalous isotope composition of CO2 evolved in light enhanced dark respiration, and some non-isotopic arguments** 

As noted above, kinetic nature of isotope effects and specificity of enzymatic interactions provide specific carbon isotope distribution of many metabolites. Having compared isotopic patterns of G6P, formed in carboxylase and oxygenase phases of Calvin cycle we found they should be quite different. According to the theoretical estimates the synthesis of G6P in carboxylase phase results in practically uniform 13C distribution along the molecule skeleton due to transaldolase and transketolase cycle reactions which randomize atoms with cycle

dawn and dusk. 13C Values are given in per mille relative to PDB standard.

pigments -28.7 -32.2 -27.7 -30.8

acids -22.3 -27.7 -21.1 -24.5

WET SEASON DRY SEASON Exposed leaf Shaded leaf Exposed leaf Shaded leaf Dawn Dusk Dawn Dusk

known carbon isotope ratio is presented in Table 3.

N FRACTION

<sup>2</sup>Lipids and

<sup>5</sup>Organic Organic

**support the oscillation hypothesis** 

glycolytic chain and Krebs cycle (Metun, 1963; Strikland, 1963).


Table 1. Carbon isotope ratio of leaf biomass and oxalates of some oxalate accumulating plants.

13C Distribution in protein fraction of some photosynthesizing microorganisms (Abelson & Hoering, 1961) gives more evidences in favor of the oscillation model (Table 2). Amino acids like serine, glycine, alanine and aspartic acid, whose pools, at least, in part are supplied from photorespiratory loop, have appeared more enriched in 13C as compared with those whose synthesis predominantly bound to glycolytic chain and Krebs cycle, like glutamic acid, leucine and lysine (Igambediev, 1988; 1991).


Table 2. Carbon isotope distribution in amino acids from protein fraction from biomass of some photosynthesizing microorganisms. Isotopic shifts are given relative to nutrient CO2 having 13C = 0‰. Extract from Table 3 in (Abelson & Hoering, 1961).


Table 3. Distribution of 13C in biomass and biochemical fractions of marine alga *Chorella stigmatophora*, grown under effect of different environmental factors. 13C of ambient CO2 is - 21%o (Ivlev & Kalinkina, 2001; Kalinkina & Udel'nova, 1990)

13C Distribution in protein fraction of some photosynthesizing microorganisms (Abelson & Hoering, 1961) gives more evidences in favor of the oscillation model (Table 2). Amino acids like serine, glycine, alanine and aspartic acid, whose pools, at least, in part are supplied from photorespiratory loop, have appeared more enriched in 13C as compared with those whose synthesis predominantly bound to glycolytic chain and Krebs cycle, like glutamic

> *Anacystis*  total carbon

Serine - 5,7 - - 14,1 - 8,3 Glycine - 14.3 - 10,0 - 10,2 - 10,0 Alanin - 10,3 - 9,8 - 15,2 -14,3 Aspartic acid - 6,6 - 9,7 - 14,4 -9,7 Glytamic acid - 18,7 - 11,1 - 17,2 -17,3 Leucine - 22,7 - 17,3 - 22,5 -23,5 Lysine - 17,0 - -22,8 Table 2. Carbon isotope distribution in amino acids from protein fraction from biomass of some photosynthesizing microorganisms. Isotopic shifts are given relative to nutrient CO2

Index Concentration of NaCl in medium, mM

13C of dry matter, ‰ - 61.6 - 59.0 - 64.5 13C of lipids, ‰ - 66.0 - 65.0 - 63.8 13C of proteins, ‰ - 42.1 - 40.9 - 47.3 3C of labile sugars‰ - 30.0 - - 30.5 13C of proline, ‰ - 29.0 - - 31.5 Table 3. Distribution of 13C in biomass and biochemical fractions of marine alga *Chorella stigmatophora*, grown under effect of different environmental factors. 13C of ambient CO2 is -

*Gracilaria*  Total carbon

0 425 595

*Euglena*  total carbon

acid, leucine and lysine (Igambediev, 1988; 1991).

*Chlorella*  total carbon

having 13C = 0‰. Extract from Table 3 in (Abelson & Hoering, 1961).

21%o (Ivlev & Kalinkina, 2001; Kalinkina & Udel'nova, 1990)

Plant species Plant type Whole leaf Oxalates Reference *Spinaceae oleracea* C3 - 27.5 - 11.9 Rivera&Smith, 1979 *Pelagronium* C3 - 31.0 - 12.4 Rivera&Smith, 1979 *Mereurialis perennis* C3 - 27.9 - 13.7 Rivera&Smith, 1979 *Spinaceae oleracea* C3 - 25.7 - 19.9 Raven et al., 1982 *Echinomastus intertextus* CAM - 13.4 - 7.3 Raven et al., 1982 *Echinomastus horizonthalomus* CAM - 13.0 - 7.8 Raven et al., 1982 *Escobaria ruberoulosa* CAM - 12.3 - 8.3 Raven et al., 1982 *Opuntia euglemannii* CAM - 13.3 - 8,5 Raven et al., 1982 *Opuntia imbricata* CAM - 14.1 - 8.7 Raven et al., 1982 Table 1. Carbon isotope ratio of leaf biomass and oxalates of some oxalate accumulating

plants.

Microorganism Amino acid

The same picture illustrated by the data on 13C distribution in biomass of marine alga *Chlorella stigmatophora* grown under different environmental conditions on the CO2 of the known carbon isotope ratio is presented in Table 3.

Lipids and proteins distinctly differ in carbon isotope ratio as compared with labile sugars and organic acids. In special experimental studies (Kalinkina &Udel'nova, 1991; Kalinkina & Naumova, 1992) the authors have proven that these components were the products of photorespiration pathway whereas most of lipids and proteins are synthesized via glycolytic chain and Krebs cycle (Metun, 1963; Strikland, 1963).

Quite another object, C3-CAM tropical plant *Clusia minor,* grown under different environmental conditions is given in Table 4. Soluble sugars and organic acids, whose origin is linked with photorespiratory carbon flux, are enriched in 13C as compared with amino acid and lipid fractions. Notably the latter fraction, besides lipids, contains pigments some of which, like chlorophyll (Ivlev, 1993), at least partially are formed at the expense of photorespiratory flux. It makes lipid fraction isotopically "heavier" than amino acid fraction is. Thus all the presented data confirmed the idea on two phases of Calvin cycle functioning and drew to the conclusion that the phases are alternating, i.e. are separated in time. In fact, if the processes proceeded simultaneously the isotopically different carbon fluxes couldn't arise. Passing the same pieces of Calvin cycle they would inevitably mix. Bearing in mind that the first phase of cycle functioning corresponds to carboxylase function of Rubisco, while the second – to oxygenase one, we called the first as carboxylase phase and the second as oxygenase. To confirm the oscillating idea we tried to get more independent arguments and examined isotopic patterns of metabolites derived in different phases of Calvin cycle.


Table 4. Carbon isotope composition of biochemical fractions isolated from leaves of *Clusia minor* under different environmental conditions (Borland et al., 1994). Samples were taken at dawn and dusk. 13C Values are given in per mille relative to PDB standard.

#### **2.2 Intramolecular isotopic patterns of glucose, anomalous isotope composition of CO2 evolved in light enhanced dark respiration, and some non-isotopic arguments support the oscillation hypothesis**

As noted above, kinetic nature of isotope effects and specificity of enzymatic interactions provide specific carbon isotope distribution of many metabolites. Having compared isotopic patterns of G6P, formed in carboxylase and oxygenase phases of Calvin cycle we found they should be quite different. According to the theoretical estimates the synthesis of G6P in carboxylase phase results in practically uniform 13C distribution along the molecule skeleton due to transaldolase and transketolase cycle reactions which randomize atoms with cycle

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 349

compared with the carbon isotopic composition of other plant organs (leaf, stem) (Lerman et

(Rossman et al., 1991) -25.2 -1.6 -0.4 +2.1 +6.3 -1.7 -5.1

(Rossman et al., 1991) -10.8 -1.7 -0.1 + 1.1 +3.6 -0.2 -3.6

(Ivlev et al., 1987) -12.5 -3.1 + 1.9 -1.9

(Galimov et al., 1977) -23.1 -7.1 +3.5\* -7.1

(Galimov et al., 1977) -26.1 -6.9 +3.5\* -6.9

(Galimov et al., 1977) -24.9 -4.1 +2.1\* -4.1 Note: The isotopic shifts of the carbon atoms Δ13C are given relative to total glucose carbon. 13C values of glucose are given in PDB units. \* The 13C values of C-3 and C-4 atoms were calculated according to Galimov et al. (1977) assuming that the isotopic composition of the other carbon atoms equals to that of

Table 5. Intramolecular carbon isotope distribution in the starch glucose of storage organs of

The uneven carbon isotope distribution in oxygenase G6P explains the recently established fact of anomalous 13C enrichment of light enhanced dark respiration CO2 relative to labile carbohydrates from phloem sup, the supposed respiratory substrate. Indeed, the consideration of labile carbohydrates (oxygenase G6P), accumulated in the light, as substrate for dark synthesis of organic acids (Borland et al, 1994), allows concluding the following way of the conversion (Ivlev & Dubinsky, 2011). At first glucose splits into two triose molecules. Then the latter are subjected to decarboxylation and derived C2-fragments are used to form organic acid skeletons while the evolved CO2 forms LEDR CO2 which inherits atoms from the C-3 and C-4 positions. The atoms as shown above are enriched in 13C. The level of 13C enrichment depends on light intensity and confirms the existence of Raliegh effect in photorespiration. The increase in illumination intensifies photorespiration, thus implying the increase in number of turns of carbon flux in photorespiratory loop. This in turn leads to photorespiratory pool depletion and to 13C enrichment of the respired CO2. Barbour et al. (2007) have noticed the relationship of

The oscillatory model suggests a coherent explanation of the relative 13С enrichment of heterotrophic tissue of plants (seeds, stem, roots) comparing with autothrophic ones (leaves)

light intensity and LEDR CO2 13C enrichment in the experiment.

Δ13C = 13C - 13Cglucose , i - atom number OCH(1)-HC(2)OH-OHC(3)H-HC(4)OH-HC(5)OH-C(6)H2OH

C1 C2 C3 C4 C5 C6


al., 1974; White, 1993; Saranga et al., 1999; Ivlev et al., 1999).

13C of glucose

Object

*Beta vulgaris,* tuber

*Zea mays,* seeds

*Zea mays,* seeds

*Triticum aestivum,* seeds

*Solanum tuberosum,*  tuber (Galimov et al., 1977)

*Oryza sativum,* seeds

*Pisum sativum,* seeds

the C – 1 and C – 6.

various plants.

turns growth. Carbon isotope distribution of G6P synthesized in photorespiration loop is characterized by 13C enrichment of carbon atoms in C-3 and C-4 positions of glucose skeleton, slight 13C enrichment in C-2 and C-5 positions, while atoms C-1 and C-6 are enriched in 12C (Fig. 2). To understand this specific distribution let's follow what isotope fractionation in glycine dehydrogenase complex (GDC) occurs.

Empty circles (1) denote isotope composition of carbon atoms in the initial substrate, filled circles (2) denote atoms get enriched in 12C, asterisks (3)

Fig. 2. The emergence of the isotope inhomogeneity in G6P as a result of kinetic carbon isotope effect in GDC.

As shown on Fig. 2, isotope distribution in G6P is determined by isotope distributions in glycine and C2-fragment derived in decarboxylation. In glycine decarboxylation both atoms of residual glycine get enriched in 13C while methylene carbon atom as well as CO2 located at the ends of the cleaved C – C bond relative to the atoms in the initial substrate get enriched in 12C (Melander & Saunders, 1983). In GDC the methylene fragment linked with the cofactor, tetrahydrofolic acid (THFA), is transferred to the residual glycine molecule thereby forming the serine (Oliver et al., 1990). Following transformations result in the specific isotopic pattern of G6P shown on Fig.2. Moreover at each turn of the carbon flux, spinning in photorespiratory loop, isotope distribution not only retains, but is reproduced again and again. So 13C enrichment of G6P as well as intramolecular isotopic discrepancies increase with the number of turns (with the growth of photorespiration intensity) (Ivlev et al., 2010). Isotope pattern of G6P synthesized in carboxylase phase is not studied yet. But glucose from the starch of storage organs of some plants has been investigated (Table 5).

Bearing in mind that G6P is the main structural unit used for glucose synthesis and comparing data in Table 5 with the results of G6P modeling (Ivlev, 2005; Ivlev et al., 2010), it is easy to conclude they are strongly resembled. Hence the starch glucose is of photorespiratory origin. The assertion is supported by the fact that storage organs are formed in the period of ontogenesis when oxidative processes related to intensification of photorespiration sharply increase (Abdurachmanova et al., 1990; Igamberdiev, 1991). This fact correlates with the observed 13C enrichment of seeds, fruits, and edible roots of plants as

turns growth. Carbon isotope distribution of G6P synthesized in photorespiration loop is characterized by 13C enrichment of carbon atoms in C-3 and C-4 positions of glucose skeleton, slight 13C enrichment in C-2 and C-5 positions, while atoms C-1 and C-6 are enriched in 12C (Fig. 2). To understand this specific distribution let's follow what isotope

Empty circles (1) denote isotope composition of carbon atoms in the initial substrate, filled circles (2)

Fig. 2. The emergence of the isotope inhomogeneity in G6P as a result of kinetic carbon

As shown on Fig. 2, isotope distribution in G6P is determined by isotope distributions in glycine and C2-fragment derived in decarboxylation. In glycine decarboxylation both atoms of residual glycine get enriched in 13C while methylene carbon atom as well as CO2 located at the ends of the cleaved C – C bond relative to the atoms in the initial substrate get enriched in 12C (Melander & Saunders, 1983). In GDC the methylene fragment linked with the cofactor, tetrahydrofolic acid (THFA), is transferred to the residual glycine molecule thereby forming the serine (Oliver et al., 1990). Following transformations result in the specific isotopic pattern of G6P shown on Fig.2. Moreover at each turn of the carbon flux, spinning in photorespiratory loop, isotope distribution not only retains, but is reproduced again and again. So 13C enrichment of G6P as well as intramolecular isotopic discrepancies increase with the number of turns (with the growth of photorespiration intensity) (Ivlev et al., 2010). Isotope pattern of G6P synthesized in carboxylase phase is not studied yet. But glucose from the starch of storage organs of some plants has been investigated (Table 5). Bearing in mind that G6P is the main structural unit used for glucose synthesis and comparing data in Table 5 with the results of G6P modeling (Ivlev, 2005; Ivlev et al., 2010), it is easy to conclude they are strongly resembled. Hence the starch glucose is of photorespiratory origin. The assertion is supported by the fact that storage organs are formed in the period of ontogenesis when oxidative processes related to intensification of photorespiration sharply increase (Abdurachmanova et al., 1990; Igamberdiev, 1991). This fact correlates with the observed 13C enrichment of seeds, fruits, and edible roots of plants as

fractionation in glycine dehydrogenase complex (GDC) occurs.

denote atoms get enriched in 12C, asterisks (3)

isotope effect in GDC.


compared with the carbon isotopic composition of other plant organs (leaf, stem) (Lerman et al., 1974; White, 1993; Saranga et al., 1999; Ivlev et al., 1999).

Note: The isotopic shifts of the carbon atoms Δ13C are given relative to total glucose carbon. 13C values of glucose are given in PDB units. \* The 13C values of C-3 and C-4 atoms were calculated according to Galimov et al. (1977) assuming that the isotopic composition of the other carbon atoms equals to that of the C – 1 and C – 6.

Table 5. Intramolecular carbon isotope distribution in the starch glucose of storage organs of various plants.

The uneven carbon isotope distribution in oxygenase G6P explains the recently established fact of anomalous 13C enrichment of light enhanced dark respiration CO2 relative to labile carbohydrates from phloem sup, the supposed respiratory substrate. Indeed, the consideration of labile carbohydrates (oxygenase G6P), accumulated in the light, as substrate for dark synthesis of organic acids (Borland et al, 1994), allows concluding the following way of the conversion (Ivlev & Dubinsky, 2011). At first glucose splits into two triose molecules. Then the latter are subjected to decarboxylation and derived C2-fragments are used to form organic acid skeletons while the evolved CO2 forms LEDR CO2 which inherits atoms from the C-3 and C-4 positions. The atoms as shown above are enriched in 13C. The level of 13C enrichment depends on light intensity and confirms the existence of Raliegh effect in photorespiration. The increase in illumination intensifies photorespiration, thus implying the increase in number of turns of carbon flux in photorespiratory loop. This in turn leads to photorespiratory pool depletion and to 13C enrichment of the respired CO2. Barbour et al. (2007) have noticed the relationship of light intensity and LEDR CO2 13C enrichment in the experiment.

The oscillatory model suggests a coherent explanation of the relative 13С enrichment of heterotrophic tissue of plants (seeds, stem, roots) comparing with autothrophic ones (leaves)

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 351

interior is well ventilated, leading to a reasonably uniform distribution of oxygen. Oxygen may participate in photorespiration (4), eventually leading to the appearance of glycine in the mitochondria and thus to photorespiration (5). The carbon dioxide produced by photorespiration is free to diffuse through the cytoplasm to the chloroplasts (6). Also shown is the inhibition of photorespiration by carbon dioxide and of carbon fixation by oxygen. To check up this possibility we carried out the computational analysis of the scheme (Dubinsky & Ivlev, 2011). It can be presented as follows (Fig. 4). According to the scheme (Fig. 4), RuBP binds to the enzyme which is activated by Mg2+ and CO2 (this is not considered here for simplification) and a quasi-equilibrium of the RuBP with the enzyme E is attained first (Tapia et al., 1995; Mauser et al., 2001). Then RuBP–enzyme complex reacts either with CO2 or O2 and the formation of the assimilation products occurs. The products are used either for further transformations in the cycle (the carboxylase phase) or for utilization in the photorespiration loop, comprising the Calvin cycle coupled with the

Fig. 4. The principal scheme of photosynthesis considering carboxylase and oxygenase

The scheme on Fig. 4 is convenient for mathematical description and computational

glycolate cycle (initiated by the oxygenase phase).

analysis. It was described by three differential equations:

functions of Rubisco.

(Cernusak et al, 2009). In fact, labile carbohydrates are the main carbon source for heterotrophic growth (Kursanov, 1976). On the other hand, labile carbohydrates, being photorespiratory products, are enriched with 13C. Gessler et. al. (2008) has confirmed this assertion experimentally. The authors found that water soluble fraction of leaf organic matter mainly consisting of the labile carbohydrates is enriched with 13C unlike to the insoluble fraction mainly consisting of proteins and lipids whose origin relates to starch formed in carboxylase phase. Similarly, the model explains the resemblance in δ13C values of the leaf water soluble organic matter and that of the phloem sap. Hence the above isotopic data firmly support the oscillation hypothesis.

There was an endeavor to find a direct evidence of the photosynthetic oscillations (Roussel et al., 2007). By using a fast response CO2 gas exchange system the authors measured CO2 concentration fluctuations in the subcellular space in tobacco leaves at low CO2 concentrations nearby the compensation point. The chosen condition provided an easier way to discover the assumed oscillations. Because of a background noise, a special mathematical procedure was required to isolate the periodic component in the temporal sequence and to build an attractor proving the existence of the real oscillatory regime. The CO2 concentration pulses with a period of the order of a few seconds were explained by the feedback interactions between CO2 assimilation and photorespiration.

Fig.3 shows the principal interactions between the main participants of photosynthesis process and key enzyme Rubisco having dual function. Since the process occurs in different compartments: CO2 assimilation in chloroplasts, photorespiratory CO2 release in mitochondria, a certain time interval is needed for the CO2 depletion near Rubisco. The delay in CO2 release, following RuBP oxygenation, and competition between CO2 and O2 provide the conditions for oscillations (Roussel & Igamberdiev, 2011).

Fig. 3. Simplified scheme for carbon assimilation and photorespiration.

Dashed lines indicate compartmental boundaries. Carbon dioxide in the atmosphere passes through the stomata and enters the substomatal space (step 1). Eventually, it reaches the chloroplasts (transport step 2) where carbon is fixed (3). Under normal conditions, the leaf

(Cernusak et al, 2009). In fact, labile carbohydrates are the main carbon source for heterotrophic growth (Kursanov, 1976). On the other hand, labile carbohydrates, being photorespiratory products, are enriched with 13C. Gessler et. al. (2008) has confirmed this assertion experimentally. The authors found that water soluble fraction of leaf organic matter mainly consisting of the labile carbohydrates is enriched with 13C unlike to the insoluble fraction mainly consisting of proteins and lipids whose origin relates to starch formed in carboxylase phase. Similarly, the model explains the resemblance in δ13C values of the leaf water soluble organic matter and that of the phloem sap. Hence the above

There was an endeavor to find a direct evidence of the photosynthetic oscillations (Roussel et al., 2007). By using a fast response CO2 gas exchange system the authors measured CO2 concentration fluctuations in the subcellular space in tobacco leaves at low CO2 concentrations nearby the compensation point. The chosen condition provided an easier way to discover the assumed oscillations. Because of a background noise, a special mathematical procedure was required to isolate the periodic component in the temporal sequence and to build an attractor proving the existence of the real oscillatory regime. The CO2 concentration pulses with a period of the order of a few seconds were explained by the

Fig.3 shows the principal interactions between the main participants of photosynthesis process and key enzyme Rubisco having dual function. Since the process occurs in different compartments: CO2 assimilation in chloroplasts, photorespiratory CO2 release in mitochondria, a certain time interval is needed for the CO2 depletion near Rubisco. The delay in CO2 release, following RuBP oxygenation, and competition between CO2 and O2

isotopic data firmly support the oscillation hypothesis.

feedback interactions between CO2 assimilation and photorespiration.

provide the conditions for oscillations (Roussel & Igamberdiev, 2011).

Fig. 3. Simplified scheme for carbon assimilation and photorespiration.

Dashed lines indicate compartmental boundaries. Carbon dioxide in the atmosphere passes through the stomata and enters the substomatal space (step 1). Eventually, it reaches the chloroplasts (transport step 2) where carbon is fixed (3). Under normal conditions, the leaf interior is well ventilated, leading to a reasonably uniform distribution of oxygen. Oxygen may participate in photorespiration (4), eventually leading to the appearance of glycine in the mitochondria and thus to photorespiration (5). The carbon dioxide produced by photorespiration is free to diffuse through the cytoplasm to the chloroplasts (6). Also shown is the inhibition of photorespiration by carbon dioxide and of carbon fixation by oxygen.

To check up this possibility we carried out the computational analysis of the scheme (Dubinsky & Ivlev, 2011). It can be presented as follows (Fig. 4). According to the scheme (Fig. 4), RuBP binds to the enzyme which is activated by Mg2+ and CO2 (this is not considered here for simplification) and a quasi-equilibrium of the RuBP with the enzyme E is attained first (Tapia et al., 1995; Mauser et al., 2001). Then RuBP–enzyme complex reacts either with CO2 or O2 and the formation of the assimilation products occurs. The products are used either for further transformations in the cycle (the carboxylase phase) or for utilization in the photorespiration loop, comprising the Calvin cycle coupled with the glycolate cycle (initiated by the oxygenase phase).

Fig. 4. The principal scheme of photosynthesis considering carboxylase and oxygenase functions of Rubisco.

The scheme on Fig. 4 is convenient for mathematical description and computational analysis. It was described by three differential equations:

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 353

distributions within metabolites. The pools of metabolites accumulated in this phase and utilized further in secondary metabolism to provide glycolytic chain, lignin synthesis and other metabolic needs with carbon source form so-called "light" (enriched in 12C) carbon

Fig. 6. Calvin cycle in carboxylase phase of Rubisco functioning.

The figures on the arrows and before the molecular formulas denote the number of the molecules involved in transformations of the cycle and formed in them; the figures before the atoms denote the number of carbon atoms in the PGA molecule; asterisks \* are the exogenous carbon atoms attached to the carboxyl group of 2-carboxy-3-ketopentite and then

In oxygenase phase Calvin cycle works as shown on Fig. 7. Due to the isotope effect in GDC all metabolites formed in cycle transformations get enriched in 13C relative to G6P, left after carboxylase phase. At that the specific intramolecular 13C distributions determined by kinetic nature of the effect, by the specificity of enzymatic interactions and by the Raleigh effect appear. 13C-Enrichment and heterogeneity of isotope distribution of metabolites becomes greater with the photorespiration intensity. The pools of metabolites mainly labile

to the C-3 position of PGA; P in a circle is the phosphate group in the molecules.

flux (see below).

where following notations are used: x, y and z are the RuBP, CO2 and O2 concentrations respectively, *K*CO2 , *K*O2 and *K*RuBP are the equilibrium constants of the reactions E · RuBP ·CO2→E · RuBP + CO2, E · RuBP ·O2→E · RuBP + O2 and E · RuBP→E + RuBP, respectively. Two first equations in their simplified form (Dubinsky et al., 2010) describe sugar (x) and CO2 (y) concentration variations. The third describes variations of O2 (z) concentration. In the set of equations *V*c is the maximum rate of RuBP carboxylation, *V*ox is the maximum rate of RuBP oxygenation. *V*out is the maximum rate of sugar efflux, *K*out is the Michaelis constant of the pseudoenzyme by means of which sugars are removed from the system (the real mechanism of sugars removal is certainly more complicated but it is the simplest way to describe the effect of sugar efflux saturation). *k*CO2 is the CO2 diffusion coefficient from the surrounding medium into a cell, CO2out is the CO2 concentration in the medium, kO2 is the O2 diffusion coefficient from the medium into the cell, O2out is the O2 concentration in the medium.

The solution of the system with cell parameters, taken from the literature (Dubinsky & Ivlev, 2011), results in establishing of counter-phase undamped oscillations with the period of 1 – 3 sec for CO2 and O2 and in respective oscillations of CO2/O2 ratio (Fig. 5). The oscillations could switch over Rubisco from carboxylase function to oxygenase and back.

Fig. 5. The calculated photosynthetic oscillations of CO2/O2 concentration ratio according to the model described in the text.

Thus the theoretical calculations proved the principal possibility of the existence of sustained oscillations in carbon metabolism of a photosynthesizing cell.

Let's consider now how Calvin cycle works in different phases of photosynthetic oscillations from the point of 13C isotope distribution in metabolites. In carboxylase phase of oscillations Calvin cycle works, as shown on Fig. 6. Due to carbon isotope effect in RuBP carboxylase complex all carbon atoms fixed happened to be enriched in 12C relative to ambient CO2. Transketolase and transaldolase reactions of the cycle randomize carbon atoms along carbon skeletons, i.e. Calvin cycle works as a mixer. It results in practically uniform 13C

where following notations are used: x, y and z are the RuBP, CO2 and O2 concentrations respectively, *K*CO2 , *K*O2 and *K*RuBP are the equilibrium constants of the reactions E · RuBP ·CO2→E · RuBP + CO2, E · RuBP ·O2→E · RuBP + O2 and E · RuBP→E + RuBP, respectively. Two first equations in their simplified form (Dubinsky et al., 2010) describe sugar (x) and CO2 (y) concentration variations. The third describes variations of O2 (z) concentration. In the set of equations *V*c is the maximum rate of RuBP carboxylation, *V*ox is the maximum rate of RuBP oxygenation. *V*out is the maximum rate of sugar efflux, *K*out is the Michaelis constant of the pseudoenzyme by means of which sugars are removed from the system (the real mechanism of sugars removal is certainly more complicated but it is the simplest way to describe the effect of sugar efflux saturation). *k*CO2 is the CO2 diffusion coefficient from the surrounding medium into a cell, CO2out is the CO2 concentration in the medium, kO2 is the O2 diffusion coefficient from the medium into the cell, O2out is the O2 concentration in the

The solution of the system with cell parameters, taken from the literature (Dubinsky & Ivlev, 2011), results in establishing of counter-phase undamped oscillations with the period of 1 – 3 sec for CO2 and O2 and in respective oscillations of CO2/O2 ratio (Fig. 5). The oscillations

Fig. 5. The calculated photosynthetic oscillations of CO2/O2 concentration ratio according to

Thus the theoretical calculations proved the principal possibility of the existence of

Let's consider now how Calvin cycle works in different phases of photosynthetic oscillations from the point of 13C isotope distribution in metabolites. In carboxylase phase of oscillations Calvin cycle works, as shown on Fig. 6. Due to carbon isotope effect in RuBP carboxylase complex all carbon atoms fixed happened to be enriched in 12C relative to ambient CO2. Transketolase and transaldolase reactions of the cycle randomize carbon atoms along carbon skeletons, i.e. Calvin cycle works as a mixer. It results in practically uniform 13C

sustained oscillations in carbon metabolism of a photosynthesizing cell.

could switch over Rubisco from carboxylase function to oxygenase and back.

medium.

the model described in the text.

distributions within metabolites. The pools of metabolites accumulated in this phase and utilized further in secondary metabolism to provide glycolytic chain, lignin synthesis and other metabolic needs with carbon source form so-called "light" (enriched in 12C) carbon flux (see below).

Fig. 6. Calvin cycle in carboxylase phase of Rubisco functioning.

The figures on the arrows and before the molecular formulas denote the number of the molecules involved in transformations of the cycle and formed in them; the figures before the atoms denote the number of carbon atoms in the PGA molecule; asterisks \* are the exogenous carbon atoms attached to the carboxyl group of 2-carboxy-3-ketopentite and then to the C-3 position of PGA; P in a circle is the phosphate group in the molecules.

In oxygenase phase Calvin cycle works as shown on Fig. 7. Due to the isotope effect in GDC all metabolites formed in cycle transformations get enriched in 13C relative to G6P, left after carboxylase phase. At that the specific intramolecular 13C distributions determined by kinetic nature of the effect, by the specificity of enzymatic interactions and by the Raleigh effect appear. 13C-Enrichment and heterogeneity of isotope distribution of metabolites becomes greater with the photorespiration intensity. The pools of metabolites mainly labile

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 355

The idea on the existence of energy and carbon oscillations in glycolytic chain was firstly proved in respect to heterotrophic organisms (Sel'kov, 1975, 1978). We have accepted this

Fig. 8. The simplified diagram depicting temporal organization of secondary metabolism in glycolytic chain (see the text). Dotted lines denote the enzymatic pyruvate decarboxylase complex where carbon isotope fractionation occurs. Abbreviations: X5P, xylose-5 phosphate; R5P, ribose-5-phophate; RuBP, ribulose-1,5-bisphosphate; 6PG, 6 phosphogluconate; F6P, fructose 6-phosphate; FBP, fructose-1,5-bisphosphate; PGA,

phosphoglyceric acid; DHA-P, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; ,

carbon isotope fractionation coefficient; F, the extent of pyruvate pool depletion.

**3. Carbon isotope fractionation in secondary metabolism of** 

**photosynthesizing cell** 

carbohydrates, some amino acids (glycine, serine, and related compounds) accumulated in oxygenase phase, like those formed in carboxylase phase, are utilized in secondary metabolism syntheses (organic acid, some parts of complex molecules, etc.)

Fig. 7. Calvin cycle in oxygenase phase of Rubisco functioning.

All symbols denote the same as on Fig. 6.

Strict temporal organization of metabolism in a cell prevent from complete mixing of the above carbon fluxes (see below) and allows to use isotopic characteristics to investigate metabolic relations, pathways, assimilate transport, etc. More arguments evidencing in favor of photosynthetic oscillations were given in the work of Ivlev (2010).

#### **3. Carbon isotope fractionation in secondary metabolism of photosynthesizing cell**

354 Advances in Photosynthesis – Fundamental Aspects

carbohydrates, some amino acids (glycine, serine, and related compounds) accumulated in oxygenase phase, like those formed in carboxylase phase, are utilized in secondary

metabolism syntheses (organic acid, some parts of complex molecules, etc.)

Fig. 7. Calvin cycle in oxygenase phase of Rubisco functioning.

of photosynthetic oscillations were given in the work of Ivlev (2010).

Strict temporal organization of metabolism in a cell prevent from complete mixing of the above carbon fluxes (see below) and allows to use isotopic characteristics to investigate metabolic relations, pathways, assimilate transport, etc. More arguments evidencing in favor

All symbols denote the same as on Fig. 6.

The idea on the existence of energy and carbon oscillations in glycolytic chain was firstly proved in respect to heterotrophic organisms (Sel'kov, 1975, 1978). We have accepted this

Fig. 8. The simplified diagram depicting temporal organization of secondary metabolism in glycolytic chain (see the text). Dotted lines denote the enzymatic pyruvate decarboxylase complex where carbon isotope fractionation occurs. Abbreviations: X5P, xylose-5 phosphate; R5P, ribose-5-phophate; RuBP, ribulose-1,5-bisphosphate; 6PG, 6 phosphogluconate; F6P, fructose 6-phosphate; FBP, fructose-1,5-bisphosphate; PGA, phosphoglyceric acid; DHA-P, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; , carbon isotope fractionation coefficient; F, the extent of pyruvate pool depletion.

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 357

it is necessary to find out 13C distribution in the structural units produced in pyruvate

Three structural units are produced in the above reaction. They are CO2, evolved in decarboxylation, (C1-fragments), acetyl-KoA (C2-fragments), and residual pyruvate (C3 fragments). According to the isotope effect theory (Melander & Saunders, 1983) only the atoms located at the ends of the broken bonds are subjected to kinetic isotope effect (Fig.9).

Empty circles denote atoms with non-changeable isotopic composition in the reaction; filled circles denote atoms getting enriched in 12C relative to the respective atoms of the pyruvate molecules subjected to decarboxylation; asterisks denote atoms getting enriched in 13C to the

Given the isotope composition of the initial pyruvate as that of G6P, derived in carboxylase phase, and taking it as reference level, it is convenient to divide all the atoms in the above fragments into three types. At that one should consider kinetic nature of pyruvate

1. Methyl atoms of C2-fragments and C3-fragments. Their isotopic compositions during carboxylation remain unchanged and are inherited from the corresponding G6P carbon

2. Carbonyl carbon atoms of C2-fragments and CO2 disposed at the ends of the cleaved C-C bonds. Depending on the extent of pyruvate pool depletion F, their isotope composition can be both enriched in 12C, if F is less than 0,5, or depleted in 12C, if F is

3. Carboxyl and the neighboring carbonyl atoms of C3-fragments. Their isotope composition at any F is enriched in 13C relative to atoms of the first and second type. The 13C distributions in metabolites were analyzed by means of their skeleton reconstruction with allowance of the known pathway, and the specificity of the enzymatic reactions and mixing of atoms in metabolic cycles. The comparison of the theoretically expected and experimentally observed isotope distributions gives the strong arguments in favor of the

Isotope distributions in lipid components, made of C2-fragments, are the easiest objects for the isotopic pattern analysis. With allowance for the known fatty acids synthesis pathway (Strickland, 1963), namely the condensation of C2-fragments according to the head-to-tail principle, there are only odd carbon atoms of skeleton that change their isotope ratios (atoms of the second type). The even atoms (atoms of the first type) remain their isotope composition inherited from the atoms of nutrient carbohydrate. In Table 6 carbon isotope

**3.1 Some examples of 13C distribution in secondary metabolites affirming the** 

It means that the effect results in heterogeneous intramolecular isotope distribution

Fig. 9. Three types of carbon atoms resulting in pyruvate decarboxylation.

decarboxylation carbon isotope effect and Raleigh effect of pool depletion.

respective atoms of the initial pyruvate molecules.

atoms. It was accepted as an internal standard;

**oscillatory character of glycolytic chain metabolism** 

more than 0,5.

glycolytic oscillations

decarboxylation and their dependence on the Raleigh effect.

idea suitable for autotrophic organisms taking into consideration that heterotrophic organisms have originated first in the course of evolution while autotrophs emerged later adding pentose phosphate reductive cycle (Calvin cycle) to glycolytic chain to feed it with substrates (Ivlev, 2009).

Hence photosynthesizing organisms have inherited glycolytic chain from the precursors with all its functions, regulation and temporal organization and with carbon isotope fractionation mechanism as well. Following studies of 13C distribution regularities in autotrophic and heterotrophic biomass showed they have much in common and confirmed this assumption. Unlike to photosynthetic oscillations, glycolytic ones were found to be long-term. According to Sel'kov (1975, 1978), glycolytic oscillations consist of two phases: glycolysis and gluconeogenesis. In glycolysis, which correlates with dark period of photosynthesis, carbon flux goes "down" the chain (Fig. 8). It means carbohydrates (starch) accumulated in carboxylase phase of photosynthetic oscillations transform into lipids and proteins. In gluconeogenesis, which correlates with light period of photosynthesis, carbon flux goes "up". It means that pools of lipids and proteins accumulated in the dark partly destroy and form the reverse substrate flux directed to carbohydrates. "Up" and "down" indicate only general direction of transformations, since glycolytic and gluconeogenetic pathways do not coincide entirely. The fructose-1,6-bisphosphate futile cycle, is the main regulator of glycolytic oscillations, capable to work in opposite directions (hydrolysis/phosphorilation) depending on concentration ratio of hexosomonophosphates to fructose-1,6-bisphophate (Sel'kov, 1975, 1978).

Carbon isotope fractionation occurs in phase of glycolysis and relates to pyruvate dehydrogenase complex, which is the main cross-point in the chain. Due to pyruvate decarboxylation occurring in this point the pyruvate pool is depleted followed by the Raleigh isotope effect. Glycolysis is organized in such a way that metabolites derived of C2 fragments (fatty acids, carotenoids, steroids, etc.) referred to lipids, emerge when the extent of the pool depletion is less than a half (F1< 0,5). It causes lipids in general are enriched in 12C relative to ambient carbohydrates. This piece of glycolysis phase is depicted on the Fig.8 as dotted circle with isotope characteristics and F1. The second period of glycolysis mainly corresponds to Krebs cycle functioning and protein synthesis. This piece is depicted on the Fig.8 as dotted circle with isotope characteristics and F2. The glycolysis proceeds when the extent of pyruvate pool depletion is more than a half (F2 > 0.5). That is why total proteins are enriched in 13C relative to lipids and to ambient carbohydrates as well. It was adopted that 13C patterns of metabolites related to glycolytic chain are determined solely by isotope fractionation in pyruvate decarboxylation and by the specificity of the following enzymatic interactions since no proofs are available evidencing for carbon isotope fractionation in gluconeogenesis phase.

Now let's see carbon isotope fractionation in pyruvate decarboxylation in a more detail. The important role of the reaction is conditioned at least by two reasons. First, the reaction is located at the cross-point of central metabolic pathways. Hence carbon isotope fractionation is typical to all photosynthesizing organisms. Second, the products of the reaction are used as structural units for the synthesis practically for all secondary metabolites. Taking into account the kinetic nature of isotope effect, metabolic pathways and specificity of enzymatic interactions the intramolecular carbon isotope distributions of many metabolites can be easily predicted to be compared with the experimental data (see below). To get this objective

idea suitable for autotrophic organisms taking into consideration that heterotrophic organisms have originated first in the course of evolution while autotrophs emerged later adding pentose phosphate reductive cycle (Calvin cycle) to glycolytic chain to feed it with

Hence photosynthesizing organisms have inherited glycolytic chain from the precursors with all its functions, regulation and temporal organization and with carbon isotope fractionation mechanism as well. Following studies of 13C distribution regularities in autotrophic and heterotrophic biomass showed they have much in common and confirmed this assumption. Unlike to photosynthetic oscillations, glycolytic ones were found to be long-term. According to Sel'kov (1975, 1978), glycolytic oscillations consist of two phases: glycolysis and gluconeogenesis. In glycolysis, which correlates with dark period of photosynthesis, carbon flux goes "down" the chain (Fig. 8). It means carbohydrates (starch) accumulated in carboxylase phase of photosynthetic oscillations transform into lipids and proteins. In gluconeogenesis, which correlates with light period of photosynthesis, carbon flux goes "up". It means that pools of lipids and proteins accumulated in the dark partly destroy and form the reverse substrate flux directed to carbohydrates. "Up" and "down" indicate only general direction of transformations, since glycolytic and gluconeogenetic pathways do not coincide entirely. The fructose-1,6-bisphosphate futile cycle, is the main regulator of glycolytic oscillations, capable to work in opposite directions (hydrolysis/phosphorilation) depending on concentration ratio of hexosomonophosphates

Carbon isotope fractionation occurs in phase of glycolysis and relates to pyruvate dehydrogenase complex, which is the main cross-point in the chain. Due to pyruvate decarboxylation occurring in this point the pyruvate pool is depleted followed by the Raleigh isotope effect. Glycolysis is organized in such a way that metabolites derived of C2 fragments (fatty acids, carotenoids, steroids, etc.) referred to lipids, emerge when the extent of the pool depletion is less than a half (F1< 0,5). It causes lipids in general are enriched in 12C relative to ambient carbohydrates. This piece of glycolysis phase is depicted on the Fig.8 as dotted circle with isotope characteristics and F1. The second period of glycolysis mainly corresponds to Krebs cycle functioning and protein synthesis. This piece is depicted on the Fig.8 as dotted circle with isotope characteristics and F2. The glycolysis proceeds when the extent of pyruvate pool depletion is more than a half (F2 > 0.5). That is why total proteins are enriched in 13C relative to lipids and to ambient carbohydrates as well. It was adopted that 13C patterns of metabolites related to glycolytic chain are determined solely by isotope fractionation in pyruvate decarboxylation and by the specificity of the following enzymatic interactions since no proofs are available evidencing for carbon isotope fractionation in

Now let's see carbon isotope fractionation in pyruvate decarboxylation in a more detail. The important role of the reaction is conditioned at least by two reasons. First, the reaction is located at the cross-point of central metabolic pathways. Hence carbon isotope fractionation is typical to all photosynthesizing organisms. Second, the products of the reaction are used as structural units for the synthesis practically for all secondary metabolites. Taking into account the kinetic nature of isotope effect, metabolic pathways and specificity of enzymatic interactions the intramolecular carbon isotope distributions of many metabolites can be easily predicted to be compared with the experimental data (see below). To get this objective

substrates (Ivlev, 2009).

gluconeogenesis phase.

to fructose-1,6-bisphophate (Sel'kov, 1975, 1978).

it is necessary to find out 13C distribution in the structural units produced in pyruvate decarboxylation and their dependence on the Raleigh effect.

Three structural units are produced in the above reaction. They are CO2, evolved in decarboxylation, (C1-fragments), acetyl-KoA (C2-fragments), and residual pyruvate (C3 fragments). According to the isotope effect theory (Melander & Saunders, 1983) only the atoms located at the ends of the broken bonds are subjected to kinetic isotope effect (Fig.9). It means that the effect results in heterogeneous intramolecular isotope distribution

Fig. 9. Three types of carbon atoms resulting in pyruvate decarboxylation.

Empty circles denote atoms with non-changeable isotopic composition in the reaction; filled circles denote atoms getting enriched in 12C relative to the respective atoms of the pyruvate molecules subjected to decarboxylation; asterisks denote atoms getting enriched in 13C to the respective atoms of the initial pyruvate molecules.

Given the isotope composition of the initial pyruvate as that of G6P, derived in carboxylase phase, and taking it as reference level, it is convenient to divide all the atoms in the above fragments into three types. At that one should consider kinetic nature of pyruvate decarboxylation carbon isotope effect and Raleigh effect of pool depletion.


The 13C distributions in metabolites were analyzed by means of their skeleton reconstruction with allowance of the known pathway, and the specificity of the enzymatic reactions and mixing of atoms in metabolic cycles. The comparison of the theoretically expected and experimentally observed isotope distributions gives the strong arguments in favor of the glycolytic oscillations

#### **3.1 Some examples of 13C distribution in secondary metabolites affirming the oscillatory character of glycolytic chain metabolism**

Isotope distributions in lipid components, made of C2-fragments, are the easiest objects for the isotopic pattern analysis. With allowance for the known fatty acids synthesis pathway (Strickland, 1963), namely the condensation of C2-fragments according to the head-to-tail principle, there are only odd carbon atoms of skeleton that change their isotope ratios (atoms of the second type). The even atoms (atoms of the first type) remain their isotope composition inherited from the atoms of nutrient carbohydrate. In Table 6 carbon isotope

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 359

Fig.10 the known synthesis pathway of monoterpenes from C2-fragments is shown (Nicolas, 1963). As before methyl atoms denoted by empty circles (first type atoms) have approximately equal isotope composition and affirm that isotope effect in CO2 assimilation was about -25‰ for all studied plants. Isotope ratio of carboxyl atoms (second type atoms) denoted by filled circles vary in a wide range what is expected for them and affirm that fatty

acids synthesis in cell cycle have some time length.

The empty and filled circles indicate carbon atoms of the first and second type

plant compounds (Schmidt et al, 1995).

Fig. 10. Biosynthesis pathway of monoterpenes (Nicolas, 1963) and 13С distribution in some


distributions of some fatty acids isolated from lipid fraction of *E. coli*, grown on glucose with the known isotope ratio are presented.

Note: 1' <sup>3</sup>С of nutrient glucose is equal to 9,96‰

Table 6. 13С distribution in some fatty acids from lipid fraction of E*. coli* grown on glucose of the known carbon isotope composition (Monson & Hayes 1982).

As follows from Table 6, isotopic data completely correspond to the known fatty acid synthesis pathway. It confirms that the 13С pattern is determined by isotope effect in pyruvate decarboxylation. Isotope composition of the even atoms (C-10 and C-12) is close to that of the carbon atoms of nutrient glucose, while 13С values of the odd atoms (C-1, C-9 and C-11) vary from -13,9 to -27,1. The variations in 13С of odd atoms prove they belong to the second type and indirectly evidence on the existence of Raleigh effect accompanying pyruvate pool depletion. The latter in turn indicates the existence of the oscillations. The odd atoms of the fatty acids in all cases are enriched in 12C relative to nutrient glucose (13С= -9,96). In the frame of the model, it means the fatty acids are derived at the extent of pool depletion less than 0,5.


Table 7. 13С distribution in acetate evolved by *E. coli* into the medium in the fermentation of the microorganisms on glucose of the known isotope composition (Blair et al, 1985)

The similar results one can see from Table 7. Acetate, like fatty acids, is made of C2 units, what is confirmed by its 13С pattern. Carboxyl atom has unusual "heavy" isotope composition (1' <sup>3</sup>С = + 15‰), while methyl (even) atom has carbon isotope composition close to the nutrient glucose. Such unusual the 13С enrichment of carboxyl atoms supports again its relation to the Raleigh effect and evidences the acetate is formed at high level of pyruvate pool depletion. On contrary, fatty acids have "light" carbon isotope composition (13С = - 12,2‰) evidencing that their carboxyl atom is enriched in 12C relative to glucose and the fatty acid molecules are derived at extent of pool depletion less than 0,5, as in previous example.

Similar conclusions may be done from the analysis of 13С distribution in quite different class of compounds, plant monoterpenes which also made of C2 structures (Fig. 10). On the top of

distributions of some fatty acids isolated from lipid fraction of *E. coli*, grown on glucose with

Table 6. 13С distribution in some fatty acids from lipid fraction of E*. coli* grown on glucose of

As follows from Table 6, isotopic data completely correspond to the known fatty acid synthesis pathway. It confirms that the 13С pattern is determined by isotope effect in pyruvate decarboxylation. Isotope composition of the even atoms (C-10 and C-12) is close to that of the carbon atoms of nutrient glucose, while 13С values of the odd atoms (C-1, C-9 and C-11) vary from -13,9 to -27,1. The variations in 13С of odd atoms prove they belong to the second type and indirectly evidence on the existence of Raleigh effect accompanying pyruvate pool depletion. The latter in turn indicates the existence of the oscillations. The odd atoms of the fatty acids in all cases are enriched in 12C relative to nutrient glucose (13С= -9,96). In the frame of the model, it means the fatty acids are derived at the extent of pool

Fatty acids 13С distribution in acetate

Table 7. 13С distribution in acetate evolved by *E. coli* into the medium in the fermentation of

The similar results one can see from Table 7. Acetate, like fatty acids, is made of C2 units, what is confirmed by its 13С pattern. Carboxyl atom has unusual "heavy" isotope

close to the nutrient glucose. Such unusual the 13С enrichment of carboxyl atoms supports again its relation to the Raleigh effect and evidences the acetate is formed at high level of pyruvate pool depletion. On contrary, fatty acids have "light" carbon isotope composition (13С = - 12,2‰) evidencing that their carboxyl atom is enriched in 12C relative to glucose and the fatty acid molecules are derived at extent of pool depletion less than 0,5, as in

Similar conclusions may be done from the analysis of 13С distribution in quite different class of compounds, plant monoterpenes which also made of C2 structures (Fig. 10). On the top of

<sup>3</sup>С = + 15‰), while methyl (even) atom has carbon isotope composition


the microorganisms on glucose of the known isotope composition (Blair et al, 1985)




the known carbon isotope composition (Monson & Hayes 1982).

<sup>3</sup>С of nutrient glucose is equal to 9,96‰

Total C N 1'

1 11

<sup>3</sup>С, ‰ Odd atoms Even atoms



Total carbon Carboxyl atom Methyl atom

<sup>3</sup>С, ‰ N 1'

10

12

<sup>3</sup>С, ‰

...



the known isotope ratio are presented.

Fatty acids 1'

Myristic 14:0 Palmitic 16:0 Palmitoleic 16:1

17: cycle Vaccenic 18:1

Note: 1'

9,10-Methylenepalmitic

depletion less than 0,5.

Nutrient glucose

composition (1'

previous example.

Fig.10 the known synthesis pathway of monoterpenes from C2-fragments is shown (Nicolas, 1963). As before methyl atoms denoted by empty circles (first type atoms) have approximately equal isotope composition and affirm that isotope effect in CO2 assimilation was about -25‰ for all studied plants. Isotope ratio of carboxyl atoms (second type atoms) denoted by filled circles vary in a wide range what is expected for them and affirm that fatty acids synthesis in cell cycle have some time length.

The empty and filled circles indicate carbon atoms of the first and second type

Fig. 10. Biosynthesis pathway of monoterpenes (Nicolas, 1963) and 13С distribution in some plant compounds (Schmidt et al, 1995).

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 361

13C-Distribution in sinigrin is shown on Fig. 12. It is a glucosinolate that belongs to the family of glucosides found in some plants of the *Brassicaceae* family. At the top of the figure the scheme of its biosynthesis pathway is drawn. The same very good coincidence of the

Fig. 12. Biosynthesis pathway and 13С distribution of sinigrin, isolated from plant Angelica

According to the scheme, all three types of isotopic atoms are involved in sinigrin skeleton formation. There are two atoms of the first type which are most enriched in 12C, one atom relating to the third type which was expectedly most enriched in 13C, and one atom of the second type with intermediate isotope composition. The full coincidence of the observed sinigrin isotopic pattern to that expected from the above pathway and the Raliegh law gives one more strong argument in favor of oscillatory character of glycolytic metabolism. Some difference in 13С values for the first type atoms are the result of two different measurement techniques. Carbon atom of the first type adjacent to double C=C bond was measured by

Archangelica (Schmidt et al, 1995).

predicted 13C distribution with that of observed takes place.

Fig. 11. Biosynthesis pathway of branched aminoacids (Metun, 1963) and 13С distribution in angelic acid precursor of isoleucine, isolated from plant *Angelica Archangelica* (Schmidt et al, 1995).

Two possible synthesis pathways for branched amino acids in photosynthesizing organisms, known from the literature, are shown on Fig.11. One of them leads to leucine (left), another to isoleucine (right). C2 and C3 – fragments are the structural units used for their synthesis. Isotopic atoms of all three types, denoted as before, are included in the molecules. Angelic acid, the precursor of isoleucine, was experimentally studied. The entire coincidence with the expected 13С distribution is observed in spite of the internal regrouping of the molecule which occurs at the step of -aceto--hydroxybuterate formation.

Fig. 11. Biosynthesis pathway of branched aminoacids (Metun, 1963) and 13С distribution in

Two possible synthesis pathways for branched amino acids in photosynthesizing organisms, known from the literature, are shown on Fig.11. One of them leads to leucine (left), another to isoleucine (right). C2 and C3 – fragments are the structural units used for their synthesis. Isotopic atoms of all three types, denoted as before, are included in the molecules. Angelic acid, the precursor of isoleucine, was experimentally studied. The entire coincidence with the expected 13С distribution is observed in spite of the internal regrouping of the molecule

angelic acid precursor of isoleucine, isolated from plant *Angelica Archangelica*

which occurs at the step of -aceto--hydroxybuterate formation.

(Schmidt et al, 1995).

13C-Distribution in sinigrin is shown on Fig. 12. It is a glucosinolate that belongs to the family of glucosides found in some plants of the *Brassicaceae* family. At the top of the figure the scheme of its biosynthesis pathway is drawn. The same very good coincidence of the predicted 13C distribution with that of observed takes place.

Fig. 12. Biosynthesis pathway and 13С distribution of sinigrin, isolated from plant Angelica Archangelica (Schmidt et al, 1995).

According to the scheme, all three types of isotopic atoms are involved in sinigrin skeleton formation. There are two atoms of the first type which are most enriched in 12C, one atom relating to the third type which was expectedly most enriched in 13C, and one atom of the second type with intermediate isotope composition. The full coincidence of the observed sinigrin isotopic pattern to that expected from the above pathway and the Raliegh law gives one more strong argument in favor of oscillatory character of glycolytic metabolism. Some difference in 13С values for the first type atoms are the result of two different measurement techniques. Carbon atom of the first type adjacent to double C=C bond was measured by

Oscillatory Nature of Metabolism and Carbon Isotope Distribution in Photosynthesizing Cells 363

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means of NMR technique which is less précised than mass-spectrometric technique. Nevertheless both atoms are considerably "lighter" than atoms of other types and characterize isotope effect in RuBP carboxylation (or carbon isotope ratio of carbohydrates synthesized in carboxylase phase). Notably, glucose in carbohydrate part of sinigrin (-25.2‰), as compared with atoms of the first type, is much more "heavier". It means that it is originated from the labile carbohydrates formed in oxygenase phase of photosynthetic oscillations. Some other arguments proving the oscillation concept are given in the recent publications (Ivlev, 2010, 2011)

#### **4. Conclusions**

The performed analysis of isotopic data proves that primary carbon metabolism in photosynthesis and secondary metabolism in glycolytic chain are of oscillatory character. They discover the existence of Raliegh effect that in turn evidences that cells work in filling/depletion regime and there is strict temporal organization of metabolic events. The examination of isotopic patterns of metabolites allows establishing the sequence of metabolites biosyntheses in cell cycle. It is a very important since it changes the fundamental view on the mechanisms underlying all cell processes. This means that besides metabolic pathways one should consider the parameter of temporal organization (Lloid, 2009).

The existence of regularities in 13С distribution proves the following assertions. 1) cell cycles are rather stable, by other words, cell oscillations are in-phase, i.e. at given functional state conditions temporal sequence of metabolic events weakly depends on the environmental factors; 2) cycle oscillations in different cells are synchronized. This fact is in compliance with the known independence of metabolic clocks on the same factors (Shnol', 1996). It means that oscillatory characteristics are determined by the internal properties of the system itself. By the other words the stable temporal sequence of metabolites syntheses which determine isotopic regularities of 13С distribution in metabolites is formed in the course of evolution (Ivlev, 2009). The changes in the environmental conditions can partly change the sequence of events in metabolic organization to better adaptation of organisms to the environments.

#### **5. References**


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The performed analysis of isotopic data proves that primary carbon metabolism in photosynthesis and secondary metabolism in glycolytic chain are of oscillatory character. They discover the existence of Raliegh effect that in turn evidences that cells work in filling/depletion regime and there is strict temporal organization of metabolic events. The examination of isotopic patterns of metabolites allows establishing the sequence of metabolites biosyntheses in cell cycle. It is a very important since it changes the fundamental view on the mechanisms underlying all cell processes. This means that besides metabolic

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**18** 

*Iran*

**Photosynthetic Carbon Metabolism:** 

Carbon metabolism is the important part of the photosynthetic process in that plant green cells convert physical and chemical sources of energy into carbohydrates. Over the last 50 years, knowledge and understanding of carbon metabolism has improved considerably. Photosynthetic carbon metabolism can no longer be explained by a single, invariable cycle. It is no longer restricted to just the chloroplast or even to a single cell. In addition to carbon reduction, nitrogen assimilation, sulphate reduction and other aspects of intermediary

Like other physiological processes, photosynthesis differs greatly among various plant species and under different environmental conditions. Over the evolutionary history of land plants, selection pressures led to evolution of variants of photosynthetic carbon metabolism namely C4 and crassulacean acid metabolism (CAM) pathways. In this chapter we will focus on the effect of environmental factors on photosynthetic carbon metabolism in order to show the considerable plasticity of this process. In addition, the processes in the evolution of main types of photosynthetic carbon metabolisms will be discussed regarding anatomical,

The pathway by which all photosynthetic eukaryotic organisms incorporate CO2 into carbohydrates is known Calvin cycle or photosynthetic carbon reduction (PCR) cycle. The PCR cycle can be divided into three primary stages: (1) carboxylation which fixes the CO2 in the presence of the five-carbon acceptor molecule, ribulose bisphosphate (RuBP), and converts it into two molecules of a three-carbon acid. The carboxylation reaction is catalyzed by the enzyme ribulose-1,5-bisphospahate carboxylase-oxygenase (Rubisco). (2) reduction, which consumes the ATP and NADPH produced by photosynthetic electron transport to convert the three-carbon acid to trios phosphate, and (3) regeneration, which consumes additional ATP to convert some of the triose phosphate back into RuBP to ensure the

The first stable intermediate of Calvin cycle is a three-carbon acid, 3-phosphoglycerate.

**2. Photosynthetic carbon metabolism: A general description** 

**1. Introduction** 

metabolism are tightly connected with this process.

capacity for the continuous fixation of CO2 (Fig. 1).

Therefore, the PCR cycle is commonly referred to as the C3 cycle.

physiological and molecular evidences.

**Plasticity and Evolution** 

Roghieh Hajiboland *Plant Science Department, University of Tabriz* 


### **Photosynthetic Carbon Metabolism: Plasticity and Evolution**

Roghieh Hajiboland

*Plant Science Department, University of Tabriz Iran*

#### **1. Introduction**

366 Advances in Photosynthesis – Fundamental Aspects

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Carbon metabolism is the important part of the photosynthetic process in that plant green cells convert physical and chemical sources of energy into carbohydrates. Over the last 50 years, knowledge and understanding of carbon metabolism has improved considerably. Photosynthetic carbon metabolism can no longer be explained by a single, invariable cycle. It is no longer restricted to just the chloroplast or even to a single cell. In addition to carbon reduction, nitrogen assimilation, sulphate reduction and other aspects of intermediary metabolism are tightly connected with this process.

Like other physiological processes, photosynthesis differs greatly among various plant species and under different environmental conditions. Over the evolutionary history of land plants, selection pressures led to evolution of variants of photosynthetic carbon metabolism namely C4 and crassulacean acid metabolism (CAM) pathways. In this chapter we will focus on the effect of environmental factors on photosynthetic carbon metabolism in order to show the considerable plasticity of this process. In addition, the processes in the evolution of main types of photosynthetic carbon metabolisms will be discussed regarding anatomical, physiological and molecular evidences.

#### **2. Photosynthetic carbon metabolism: A general description**

The pathway by which all photosynthetic eukaryotic organisms incorporate CO2 into carbohydrates is known Calvin cycle or photosynthetic carbon reduction (PCR) cycle. The PCR cycle can be divided into three primary stages: (1) carboxylation which fixes the CO2 in the presence of the five-carbon acceptor molecule, ribulose bisphosphate (RuBP), and converts it into two molecules of a three-carbon acid. The carboxylation reaction is catalyzed by the enzyme ribulose-1,5-bisphospahate carboxylase-oxygenase (Rubisco). (2) reduction, which consumes the ATP and NADPH produced by photosynthetic electron transport to convert the three-carbon acid to trios phosphate, and (3) regeneration, which consumes additional ATP to convert some of the triose phosphate back into RuBP to ensure the capacity for the continuous fixation of CO2 (Fig. 1).

The first stable intermediate of Calvin cycle is a three-carbon acid, 3-phosphoglycerate. Therefore, the PCR cycle is commonly referred to as the C3 cycle.

Photosynthetic Carbon Metabolism: Plasticity and Evolution 369

have had little significance to the survival of the organism. It is believed that oxygen began to accumulate in the atmosphere primarily due to photosynthetic activity and the atmospheric content of O2 had increased to significant proportions during the following stages of evolution of land plants. By this view, then, the oxygenase function is an

However, this is an oversimplified view of photorespiration since photorespiratory mutants of *Arabidopsis* proved to be lethal under certain growth conditions, indicating the essential nature of the photorespiratory pathway in C3 plants. There is no evidence that selection

**2.1.2 Plants have turned this apparent evolutionary deficiency into a useful metabolic** 

The glycolate pathway, undoubtedly serves as a scavenger function. For each two turns of the cycle, two molecules of phosphoglycolate are formed by oxygenation. Of these four carbon atoms, one is lost as CO2 and three are returned to the chloroplast. The glycolate pathway thus recovers 75% of the carbon that would otherwise be lost as glycolate. There is also the possibility that some of the intermediates, serine and glycine, for example, are of use in other biosynthetic pathways, although this possibility is still subject of some debate.

**2.1.3 Photorespiration functions as a safety valve for dissipation of excess excitation** 

A significant decline in the photosynthetic capacity of leaves irradiated in the absence of CO2 and O2 has been reported. Injury is prevented, however, if sufficient O2 is present to permit photorespiration to occur. Apparently, the O2 consumed by photorespiration is sufficient to protect the plant from photo-oxidative damage by permitting continued operation of the electron transport system. This could be of considerable ecological value under conditions of high light and limited CO2 supply, for example, when the stomata are closed due to water stress. Photorespiratory mutants of *Arabidopsis* are more sensitive to

In order to increase crop productivity efforts have been made on the inhibition or genetically eliminating photorespiration. Effort has been expended in the search for chemicals that inhibit the glycolate pathway or selective breeding for low-photorespiratory strains through finding a Rubisco with lower affinity for oxygen. All of these efforts have

pressures have caused evolution of a form of Rubsico with lower affinity to O2.

evolutionary "hangover" that has no useful role.

Fig. 2. The photorespiratory glycolate pathway.

photoinhibition than their wild type counterparts.

**sequence** 

**energy** 

#### **2.1 Photorespiration, principles and significance**

An important property of Rubisco is its ability to catalyze both the carboxylation and the oxygenation of RuBP. Oxygenation is the primary reaction in a process known as photorespiration. Photosynthesis and photorespiration work in opposite directions, photorespiration results in loss of CO2 from cells that are simultaneously fixing CO2 by the Calvin cycle. The C2 glycolate cycle, also known as the photosynthetic carbon oxidation cycle, begins with the oxidation of RuBP to 3P-glycerate and P-glycolate (Fig. 2).

Fig. 1. The three stages of the photosynthetic carbon reduction (PCR) cycle or Calvin cycle.

In normal air (21% O2), the rate of photorespiration in sunflower leaves is about 17% of gross photosynthesis. Every photorespired CO2, however, requires an input of two molecules of O2 and the true rate of oxygenation is about 34% and the ratio of carboxylation to oxygenation is about 3 to 1. The ratio of carboxylation to oxygenation depends, however, on the relative levels of O2 and CO2 since both gases compete for binding at the active site on Rubisco. Increase in the relative level of O2 (or decrease in CO2) shifts the balance in favor of oxygenation. An increase in temperature will also favor oxygenation. Increase in temperature declines the solubility of gases in water, but O2 solubility is less affected than CO2. Thus O2 will inhibit photosynthesis, measured by net CO2 reduction. There is also an energy cost associated with photorespiration and the glycolate pathway. Not only is the amount of ATP and NAD(P)H expended in the glycolate pathway following oxygenation (5ATP+3NADPH) greater than that expended for the reduction of one CO2 in the PCR cycle (3ATP+2NADPH), but there is also a net loss of carbon. Photorespiration appears to be a costly energy and carbon acquisition. It is logical to ask why should the plants indulge in such an apparently wasteful process? Several ideas have been proposed (Hopkins and Hüner, 2004; Foyer et al., 2009; Bauwe, 2011).

#### **2.1.1 Oxygenation is an unavoidable consequence of evolution**

It has been proposed that the oxygenase function of Rubisco is an inescapable process. Rubisco evolved at a time when the atmosphere contained large amounts of CO2 but little oxygen. Under these conditions, an inability to discriminate between the two gases would

An important property of Rubisco is its ability to catalyze both the carboxylation and the oxygenation of RuBP. Oxygenation is the primary reaction in a process known as photorespiration. Photosynthesis and photorespiration work in opposite directions, photorespiration results in loss of CO2 from cells that are simultaneously fixing CO2 by the Calvin cycle. The C2 glycolate cycle, also known as the photosynthetic carbon oxidation

Fig. 1. The three stages of the photosynthetic carbon reduction (PCR) cycle or Calvin cycle. In normal air (21% O2), the rate of photorespiration in sunflower leaves is about 17% of gross photosynthesis. Every photorespired CO2, however, requires an input of two molecules of O2 and the true rate of oxygenation is about 34% and the ratio of carboxylation to oxygenation is about 3 to 1. The ratio of carboxylation to oxygenation depends, however, on the relative levels of O2 and CO2 since both gases compete for binding at the active site on Rubisco. Increase in the relative level of O2 (or decrease in CO2) shifts the balance in favor of oxygenation. An increase in temperature will also favor oxygenation. Increase in temperature declines the solubility of gases in water, but O2 solubility is less affected than CO2. Thus O2 will inhibit photosynthesis, measured by net CO2 reduction. There is also an energy cost associated with photorespiration and the glycolate pathway. Not only is the amount of ATP and NAD(P)H expended in the glycolate pathway following oxygenation (5ATP+3NADPH) greater than that expended for the reduction of one CO2 in the PCR cycle (3ATP+2NADPH), but there is also a net loss of carbon. Photorespiration appears to be a costly energy and carbon acquisition. It is logical to ask why should the plants indulge in such an apparently wasteful process? Several ideas have been proposed (Hopkins and

It has been proposed that the oxygenase function of Rubisco is an inescapable process. Rubisco evolved at a time when the atmosphere contained large amounts of CO2 but little oxygen. Under these conditions, an inability to discriminate between the two gases would

cycle, begins with the oxidation of RuBP to 3P-glycerate and P-glycolate (Fig. 2).

**2.1 Photorespiration, principles and significance** 

Hüner, 2004; Foyer et al., 2009; Bauwe, 2011).

**2.1.1 Oxygenation is an unavoidable consequence of evolution** 

have had little significance to the survival of the organism. It is believed that oxygen began to accumulate in the atmosphere primarily due to photosynthetic activity and the atmospheric content of O2 had increased to significant proportions during the following stages of evolution of land plants. By this view, then, the oxygenase function is an evolutionary "hangover" that has no useful role.

However, this is an oversimplified view of photorespiration since photorespiratory mutants of *Arabidopsis* proved to be lethal under certain growth conditions, indicating the essential nature of the photorespiratory pathway in C3 plants. There is no evidence that selection pressures have caused evolution of a form of Rubsico with lower affinity to O2.

Fig. 2. The photorespiratory glycolate pathway.

#### **2.1.2 Plants have turned this apparent evolutionary deficiency into a useful metabolic sequence**

The glycolate pathway, undoubtedly serves as a scavenger function. For each two turns of the cycle, two molecules of phosphoglycolate are formed by oxygenation. Of these four carbon atoms, one is lost as CO2 and three are returned to the chloroplast. The glycolate pathway thus recovers 75% of the carbon that would otherwise be lost as glycolate. There is also the possibility that some of the intermediates, serine and glycine, for example, are of use in other biosynthetic pathways, although this possibility is still subject of some debate.

#### **2.1.3 Photorespiration functions as a safety valve for dissipation of excess excitation energy**

A significant decline in the photosynthetic capacity of leaves irradiated in the absence of CO2 and O2 has been reported. Injury is prevented, however, if sufficient O2 is present to permit photorespiration to occur. Apparently, the O2 consumed by photorespiration is sufficient to protect the plant from photo-oxidative damage by permitting continued operation of the electron transport system. This could be of considerable ecological value under conditions of high light and limited CO2 supply, for example, when the stomata are closed due to water stress. Photorespiratory mutants of *Arabidopsis* are more sensitive to photoinhibition than their wild type counterparts.

In order to increase crop productivity efforts have been made on the inhibition or genetically eliminating photorespiration. Effort has been expended in the search for chemicals that inhibit the glycolate pathway or selective breeding for low-photorespiratory strains through finding a Rubisco with lower affinity for oxygen. All of these efforts have

Photosynthetic Carbon Metabolism: Plasticity and Evolution 371

any net carbon reduction. The plant relies ultimately on the operation of the PCR cycle in

Within the general pattern of the C4 cycle described above there are three variations include NADP-malic enzyme type, NAD-malic enzyme and PEP carboxykinase types. Regardless of these variations, the principal effect of the C4 cycle remains to concentrate CO2 in the bundlesheath cells where the enzymes of the PCR cycle are located. By shuttling the CO2 in the form of organic acids it is possible to build much higher CO2 concentrations in the bundle-sheath cells than would be possible relying on the diffusion of CO2 alone. The concentration of CO2 in bundle-sheath cells may reach 60 mM about tenfold higher than that in C3 plants. Higher CO2 concentrations would suppress photorespiration and support higher rates of photosynthesis. Under optimal conditions, C4 crop species can assimilate CO2 at rates two to three times that of C3 species. All this productivity, however, has an energy cost to building the CO2 concentration in the bundle-sheath cells. For every CO2 assimilated, two ATP must be expended in the regeneration of PEP. This is in addition to the ATP and NADPH required in the PCR cycle. Thus the net energy requirement for assimilation of CO2 by the C4 cycle is five

Another CO2 concentrating mechanism is CAM. This specialized pattern of photosynthesis was originally studied in the family Crassulaceae. One of the most striking features of CAM plants is an inverted stomatal cycle i.e. the stomata open mainly during the nighttime hours and are usually closed during the day. This means that CO2 uptake also occurs mainly at night. In addition, CAM plants are characterized by an accumulation of malate at night and its subsequent depletion during hours and storage carbohydrate levels. Nocturnal stomatal opening supports a carboxylation reaction producing C4 acids that are stored in the large vacuoles. Accumulation of the organic acids leads to a marked acidification of these cells at night. The acids are subsequently decarboxylated during daylight hours and the resulting CO2 is fixed by the PCR cycle. As in C4 plants, the enzyme PEPC is central to CAM operation (Fig. 4). CAM species may be distinguished by the enzymes which catalyse organic acid decarboxylation, NAD-malic enzyme, NADP-malic enzyme and PEP-

the bundle sheath chloroplast for the synthesis of triose phosphates.

ATP and two NADPH (Bhagwat, 2005; Edwards and Voznesenskaya, 2011).

carboxykinase types (Bhagwat, 2005; Dodd et al., 2002; Holtum et al., 2005).

Fig. 4. Schematic of crassulacean acid metabolism (CAM).

**4. CAM mode of carbon assimilation** 

been unsuccessful, presumably because the basic premise that photorespiration is detrimental to the plant and counterproductive is incorrect.

Clearly, success in increasing photosynthesis and improving productivity lies in other directions. A mechanism for concentrating CO2 in the photosynthetic cells could be one way to suppress photorespiratory loss and improve the overall efficiency of carbon assimilation. That is exactly what has been achieved by C4 and CAM plants. A limited extent of photorespiration in C4 and CAM plants is a consequence of mechanisms that concentrate CO2 in the Rubisco environment and thereby suppress the oxygenation reaction (Hopkins and Hüner, 2004; Foyer et al., 2009; Bauwe, 2011)

#### **3. C4 mode of carbon assimilation**

C4 plants are distinguished by the fact that the first product is a four-carbon acid oxaloacetate (OAA). The key to the C4 cycle is the enzyme phosphoenol pyruvate carboxylase (PEPC), which catalyzes the carboxylation of PEP using the bicarbonate ion (HCO3−) as the substrate (rather than CO2). C4 plants also exhibit a number of specific anatomical, physiological and biochemical characteristics that constitute the "C4 syndrome". One particular anatomical feature characteristic of most C4 leaves is the presence of two distinct photosynthetic tissues. In C4 leaves the vascular bundles are quite close together and each bundle is surrounded by a tightly fitted layer of cells called the bundle sheath. Between the vascular bundles and adjacent to the air spaces of the leaf are the more loosely arranged mesophyll cells (Fig. 3). This distinction between mesophyll and bundle sheath photosynthetic cells called Kranz anatomy plays a major role in the C4 syndrome (Bhagwat, 2005; Edwards and Voznesenskaya, 2011).

Fig. 3. Leaf anatomy of a C4 plant (left). Note the tightly fitted bundle sheath cells (ovals) surrounded by a concentric layer of mesophyll cells (hexagons). Schematic of the C4 photosynthesis carbon assimilation cycle (right).

There are certain similarities between the PCR cycle and C4 metabolism. Like Rubisco, the PEPC carboxylation reaction is virtually irreversible and, consequently, energetically very favorable. Reducing potential is required at some point to remove the product and ATP is required to regenerate the acceptor molecule, PEP (Fig. 3). A very significant difference between the PCR cycle and C4 metabolism, however, is that once in the bundle sheath cell, the C4 acid is decarboxylated, giving up the CO2 originally assimilated in the mesophyll cell. This decarboxylation means that, unlike the C3-cycle, the C4 cycle does not of itself result in

been unsuccessful, presumably because the basic premise that photorespiration is

Clearly, success in increasing photosynthesis and improving productivity lies in other directions. A mechanism for concentrating CO2 in the photosynthetic cells could be one way to suppress photorespiratory loss and improve the overall efficiency of carbon assimilation. That is exactly what has been achieved by C4 and CAM plants. A limited extent of photorespiration in C4 and CAM plants is a consequence of mechanisms that concentrate CO2 in the Rubisco environment and thereby suppress the oxygenation reaction (Hopkins

C4 plants are distinguished by the fact that the first product is a four-carbon acid oxaloacetate (OAA). The key to the C4 cycle is the enzyme phosphoenol pyruvate carboxylase (PEPC), which catalyzes the carboxylation of PEP using the bicarbonate ion (HCO3−) as the substrate (rather than CO2). C4 plants also exhibit a number of specific anatomical, physiological and biochemical characteristics that constitute the "C4 syndrome". One particular anatomical feature characteristic of most C4 leaves is the presence of two distinct photosynthetic tissues. In C4 leaves the vascular bundles are quite close together and each bundle is surrounded by a tightly fitted layer of cells called the bundle sheath. Between the vascular bundles and adjacent to the air spaces of the leaf are the more loosely arranged mesophyll cells (Fig. 3). This distinction between mesophyll and bundle sheath photosynthetic cells called Kranz anatomy plays a major role in the C4 syndrome (Bhagwat,

Fig. 3. Leaf anatomy of a C4 plant (left). Note the tightly fitted bundle sheath cells (ovals) surrounded by a concentric layer of mesophyll cells (hexagons). Schematic of the C4

There are certain similarities between the PCR cycle and C4 metabolism. Like Rubisco, the PEPC carboxylation reaction is virtually irreversible and, consequently, energetically very favorable. Reducing potential is required at some point to remove the product and ATP is required to regenerate the acceptor molecule, PEP (Fig. 3). A very significant difference between the PCR cycle and C4 metabolism, however, is that once in the bundle sheath cell, the C4 acid is decarboxylated, giving up the CO2 originally assimilated in the mesophyll cell. This decarboxylation means that, unlike the C3-cycle, the C4 cycle does not of itself result in

detrimental to the plant and counterproductive is incorrect.

and Hüner, 2004; Foyer et al., 2009; Bauwe, 2011)

**3. C4 mode of carbon assimilation** 

2005; Edwards and Voznesenskaya, 2011).

photosynthesis carbon assimilation cycle (right).

any net carbon reduction. The plant relies ultimately on the operation of the PCR cycle in the bundle sheath chloroplast for the synthesis of triose phosphates.

Within the general pattern of the C4 cycle described above there are three variations include NADP-malic enzyme type, NAD-malic enzyme and PEP carboxykinase types. Regardless of these variations, the principal effect of the C4 cycle remains to concentrate CO2 in the bundlesheath cells where the enzymes of the PCR cycle are located. By shuttling the CO2 in the form of organic acids it is possible to build much higher CO2 concentrations in the bundle-sheath cells than would be possible relying on the diffusion of CO2 alone. The concentration of CO2 in bundle-sheath cells may reach 60 mM about tenfold higher than that in C3 plants. Higher CO2 concentrations would suppress photorespiration and support higher rates of photosynthesis. Under optimal conditions, C4 crop species can assimilate CO2 at rates two to three times that of C3 species. All this productivity, however, has an energy cost to building the CO2 concentration in the bundle-sheath cells. For every CO2 assimilated, two ATP must be expended in the regeneration of PEP. This is in addition to the ATP and NADPH required in the PCR cycle. Thus the net energy requirement for assimilation of CO2 by the C4 cycle is five ATP and two NADPH (Bhagwat, 2005; Edwards and Voznesenskaya, 2011).

#### **4. CAM mode of carbon assimilation**

Another CO2 concentrating mechanism is CAM. This specialized pattern of photosynthesis was originally studied in the family Crassulaceae. One of the most striking features of CAM plants is an inverted stomatal cycle i.e. the stomata open mainly during the nighttime hours and are usually closed during the day. This means that CO2 uptake also occurs mainly at night. In addition, CAM plants are characterized by an accumulation of malate at night and its subsequent depletion during hours and storage carbohydrate levels. Nocturnal stomatal opening supports a carboxylation reaction producing C4 acids that are stored in the large vacuoles. Accumulation of the organic acids leads to a marked acidification of these cells at night. The acids are subsequently decarboxylated during daylight hours and the resulting CO2 is fixed by the PCR cycle. As in C4 plants, the enzyme PEPC is central to CAM operation (Fig. 4). CAM species may be distinguished by the enzymes which catalyse organic acid decarboxylation, NAD-malic enzyme, NADP-malic enzyme and PEPcarboxykinase types (Bhagwat, 2005; Dodd et al., 2002; Holtum et al., 2005).

Fig. 4. Schematic of crassulacean acid metabolism (CAM).

Photosynthetic Carbon Metabolism: Plasticity and Evolution 373

 RWC%=(fresh mass – dry mass)/(water saturated mass – dry mass)×100 (4) Low cell turgor and RWC slow growth and decrease the stomatal conductance for H2O (gs). Water deficiency covers the range from fully hydrated cells (100% RWC), as the control state with metabolism functioning at the potential rate to very dehydrated cells (50% RWC or

Progressive decrease in RWC decreases *A* (net CO2 assimilation rate) of leaves. *A* depends on the activity of Rubisco per unit leaf, the rate of RuBP synthesis (hence on capture of photosynthetically active radiation, PAR) and on the CO2 supply, determined by *gs* and the ambient CO2 concentration (*Ca*) (Lawlor, 2001). CO2 supply to the PCR cycle in the chloroplast is determined by *Ca* and conductance of the pathway for diffusion between air and enzyme active sites, principally *gs* in the gas phase and gm in the liquid phase, which includes all physicochemical and biochemical factors (von Caemmerer 2000). The CO2

 *A* = *gs* (*Ca* – *Ci*) (5)

 *A* = *gm* (*Ci* – *Cc*). (6) By measuring *A* as a function of *Ci* (*A*/*Ci* response curve) under standard PAR flux, the limitations to *A* could be assessed. The maximum rate of *A* under saturating CO2 (*Ca*, *Ci* and *Cc*) and light in fully hydrated leaves is defined as *Apot*. To achieve the same *Apot* at small RWC as at large RWC, *Cc* must saturate Rubisco and so Ca must be sufficient to overcome the limitation of *gs*. If *Apot* at small RWC does not attain the value of *Apot* at large RWC,

Experimental studies on CO2 assimilation of C3 plants under decreasing RWC show that there are fundamental differences between species in the relative roles of *gs* limitation of CO2 supply and metabolic limitation of *Apot*. Basically data obtained from various species fall into

*Type 1:* With RWC=90% –75%, increasing *Ca* to 5% restores *A* fully to the *Apot* of control leaves (Cornic and Massacci 1996). At RWC<75% restoration of *Apot* to the value at 100% RWC is not achieved and the response to CO2 becomes progressively smaller. Therefore, in Type 1 response, there are two main, relatively distinct phases with a transition between them. The stomatal limitation phase occurs at RWC between 100 and 75%, without effect on *Apot*, so that *A* may be restored to *Apot* by large concentration of CO2. The metabolic phase of limitation is at

*Type 2:* Elevated CO2 increases *A* to *Apot* in unstressed leaves (RWC 100 to 75%), but *A* is progressively less stimulated as RWC decreases, i.e. *A* is not restored to the unstressed *Apot*, so the potential rate of CO2 assimilation is decreased (Tezara *et al*. 1999). Inhibition of *A* with 10% or greater *Ca*, rather than restoration, shows that metabolism is affected by elevated CO2, but as *A* is not restored to *Apot*, photosynthetic metabolism must be impaired. The evidence is therefore of partial metabolic inhibition of *A* by moderate stress and substantial inhibition at more severe stress. In Type 2, the phases are not distinct but progressive, lacking the two clearly distinguished phases of Type 1. Stomatal regulation, i.e. decreased *gs*, dominates at relatively large RWC, leading to a lower *Ci* and *Cc*. *gs* becomes progressively less important and metabolic limitations more important as RWC falls (Lawlor and Cornic,

The CO2 concentration at the active sites of Rubisco in the chloroplasts (Cc) is given by:

despite CO2 saturation, then metabolism is inhibited (Lawlor and Cornic, 2002).

lower RWC <75% where *Apot* is limited by metabolism (Lawlor and Cornic, 2002).

2002).

two groups, which have been called Type 1 and Type 2 responses (Fig. 5).

less) at which the cell will not recover when rehydrated.

concentration within the leaf, *Ci*, depends on *A*, *gs* and *Ca*:

#### **5. Carbon isotope discrimination in carbon assimilation pathways**

There are two naturally occurring stable isotopes of carbon, 12C and 13C. Most of the carbon is 12C (98.9%), with 1.1% being 13C. The overall abundance of 13C relative to 12C in plant tissue is commonly less than in the carbon of atmospheric carbon dioxide, indicating that carbon isotope discrimination occurs in the incorporation of CO2 into plant biomass. Variation in the 13C /12C ratio is the consequence of so called "isotope effects," which are expressed during the formation and destruction of bonds involving a carbon atom, or because of other processes that are affected by mass, such as gaseous diffusion (Farquhar et al., 1989). Isotope effect, denoted by α, is defined as the ratio of carbon isotope ratios in reactant and product:

$$\mathbf{u} = \mathbf{R}\_r / \mathbf{R}\_p \tag{1}$$

Rr is the 13C /12C molar ratio of reactant and Rp is that of the product. For plants Ra (Rr) is isotopic abundance in the air and Rp is defined isotopic abundance in the plant. For numerical convenience, instead of using the isotope effect (α=Ra/Rp), it has been proposed to use the Δ, the deviation of α from unity, as the measure of the carbon isotope discrimination by the plant:

$$
\Delta = \mathbf{q} - \mathbf{1} = \mathbf{R}\_{\mathbf{a}} / \mathbf{R}\_{\mathbf{P}} - \mathbf{1} \tag{2}
$$

Isotopic composition is another parameter is specified as 13C values, Rs is the 13C/12C molar ratio of the standard:

$$\delta^{13} = \mathbf{R\_p}/\mathbf{R\_s} - \mathbf{1} \tag{3}$$

Isotopic composition of plants (13) is negative, whereas the process of CO2 diffusion and carboxylation by Rubisco have positive discrimination (i.e. against 13CO2). Therefore, Δ values are usually positive while those of 13 are usually negative. Typically, Δ and <sup>13</sup> values are ~10-35×10-3, which is normally presented as 10-35‰ ("per mil").

Because Rubisco preferentially fixes the light 12C isotope over the heavier 13C, plant tissues are enriched in 12C relative to the bulk atmosphere. However, C4 plants exhibit much lower rates of Δ (and higher rates of 13) than C3 plants. Plants exhibiting CAM have intermediate values which appear to be related to the relative proportions of C3 and C4 fixation by these species (Farquhar et al., 1989). The evolutionary modifications that lead to the enhancement of fixation by the C4-cycle in C3-C4 intermediates are also associated with reduction in Δ values and increase in 13 values from C3 to near C4 values (Hobbie and Werner, 2004)(See below).

#### **6. Effect of environmental factors on photosynthetic carbon metabolism**

#### **6.1 Effect of water availability**

Plant and cell water balance is determined by water lost in transpiration and water absorption from the soil. When transpiration exceeds absorption, cell turgor and relative water content (RWC) decrease, while the concentration of cellular contents increases. Under these conditions, osmotic potential and water potential fall.

RWC normalizes water content by expressing it relative to the fully turgid state and is an easily measured indicator of water status:

There are two naturally occurring stable isotopes of carbon, 12C and 13C. Most of the carbon is 12C (98.9%), with 1.1% being 13C. The overall abundance of 13C relative to 12C in plant tissue is commonly less than in the carbon of atmospheric carbon dioxide, indicating that carbon isotope discrimination occurs in the incorporation of CO2 into plant biomass. Variation in the 13C /12C ratio is the consequence of so called "isotope effects," which are expressed during the formation and destruction of bonds involving a carbon atom, or because of other processes that are affected by mass, such as gaseous diffusion (Farquhar et al., 1989). Isotope effect, denoted by α, is defined as the ratio of carbon isotope ratios in

Rr is the 13C /12C molar ratio of reactant and Rp is that of the product. For plants Ra (Rr) is isotopic abundance in the air and Rp is defined isotopic abundance in the plant. For numerical convenience, instead of using the isotope effect (α=Ra/Rp), it has been proposed to use the Δ, the deviation of α from unity, as the measure of the carbon isotope

Isotopic composition is another parameter is specified as 13C values, Rs is the 13C/12C molar

Isotopic composition of plants (13) is negative, whereas the process of CO2 diffusion and carboxylation by Rubisco have positive discrimination (i.e. against 13CO2). Therefore, Δ values are usually positive while those of 13 are usually negative. Typically, Δ and <sup>13</sup>

Because Rubisco preferentially fixes the light 12C isotope over the heavier 13C, plant tissues are enriched in 12C relative to the bulk atmosphere. However, C4 plants exhibit much lower rates of Δ (and higher rates of 13) than C3 plants. Plants exhibiting CAM have intermediate values which appear to be related to the relative proportions of C3 and C4 fixation by these species (Farquhar et al., 1989). The evolutionary modifications that lead to the enhancement of fixation by the C4-cycle in C3-C4 intermediates are also associated with reduction in Δ values and increase in 13 values from C3 to near C4 values (Hobbie and Werner, 2004)(See

**6. Effect of environmental factors on photosynthetic carbon metabolism** 

Plant and cell water balance is determined by water lost in transpiration and water absorption from the soil. When transpiration exceeds absorption, cell turgor and relative water content (RWC) decrease, while the concentration of cellular contents increases. Under

RWC normalizes water content by expressing it relative to the fully turgid state and is an

values are ~10-35×10-3, which is normally presented as 10-35‰ ("per mil").

α=Rr/Rp (1)

Δ= α–1= Ra/Rp –1 (2)

13= Rp/Rs –1 (3)

**5. Carbon isotope discrimination in carbon assimilation pathways** 

reactant and product:

discrimination by the plant:

ratio of the standard:

below).

**6.1 Effect of water availability** 

easily measured indicator of water status:

these conditions, osmotic potential and water potential fall.

#### RWC%=(fresh mass – dry mass)/(water saturated mass – dry mass)×100 (4)

Low cell turgor and RWC slow growth and decrease the stomatal conductance for H2O (gs). Water deficiency covers the range from fully hydrated cells (100% RWC), as the control state with metabolism functioning at the potential rate to very dehydrated cells (50% RWC or less) at which the cell will not recover when rehydrated.

Progressive decrease in RWC decreases *A* (net CO2 assimilation rate) of leaves. *A* depends on the activity of Rubisco per unit leaf, the rate of RuBP synthesis (hence on capture of photosynthetically active radiation, PAR) and on the CO2 supply, determined by *gs* and the ambient CO2 concentration (*Ca*) (Lawlor, 2001). CO2 supply to the PCR cycle in the chloroplast is determined by *Ca* and conductance of the pathway for diffusion between air and enzyme active sites, principally *gs* in the gas phase and gm in the liquid phase, which includes all physicochemical and biochemical factors (von Caemmerer 2000). The CO2 concentration within the leaf, *Ci*, depends on *A*, *gs* and *Ca*:

$$A = g\_s \left(\mathbf{C}\_a - \mathbf{C}\_i\right) \tag{5}$$

The CO2 concentration at the active sites of Rubisco in the chloroplasts (Cc) is given by:

$$A = g\_m \left(\mathbf{C}\_i - \mathbf{C}\_c\right). \tag{6}$$

By measuring *A* as a function of *Ci* (*A*/*Ci* response curve) under standard PAR flux, the limitations to *A* could be assessed. The maximum rate of *A* under saturating CO2 (*Ca*, *Ci* and *Cc*) and light in fully hydrated leaves is defined as *Apot*. To achieve the same *Apot* at small RWC as at large RWC, *Cc* must saturate Rubisco and so Ca must be sufficient to overcome the limitation of *gs*. If *Apot* at small RWC does not attain the value of *Apot* at large RWC, despite CO2 saturation, then metabolism is inhibited (Lawlor and Cornic, 2002).

Experimental studies on CO2 assimilation of C3 plants under decreasing RWC show that there are fundamental differences between species in the relative roles of *gs* limitation of CO2 supply and metabolic limitation of *Apot*. Basically data obtained from various species fall into two groups, which have been called Type 1 and Type 2 responses (Fig. 5).

*Type 1:* With RWC=90% –75%, increasing *Ca* to 5% restores *A* fully to the *Apot* of control leaves (Cornic and Massacci 1996). At RWC<75% restoration of *Apot* to the value at 100% RWC is not achieved and the response to CO2 becomes progressively smaller. Therefore, in Type 1 response, there are two main, relatively distinct phases with a transition between them. The stomatal limitation phase occurs at RWC between 100 and 75%, without effect on *Apot*, so that *A* may be restored to *Apot* by large concentration of CO2. The metabolic phase of limitation is at lower RWC <75% where *Apot* is limited by metabolism (Lawlor and Cornic, 2002).

*Type 2:* Elevated CO2 increases *A* to *Apot* in unstressed leaves (RWC 100 to 75%), but *A* is progressively less stimulated as RWC decreases, i.e. *A* is not restored to the unstressed *Apot*, so the potential rate of CO2 assimilation is decreased (Tezara *et al*. 1999). Inhibition of *A* with 10% or greater *Ca*, rather than restoration, shows that metabolism is affected by elevated CO2, but as *A* is not restored to *Apot*, photosynthetic metabolism must be impaired. The evidence is therefore of partial metabolic inhibition of *A* by moderate stress and substantial inhibition at more severe stress. In Type 2, the phases are not distinct but progressive, lacking the two clearly distinguished phases of Type 1. Stomatal regulation, i.e. decreased *gs*, dominates at relatively large RWC, leading to a lower *Ci* and *Cc*. *gs* becomes progressively less important and metabolic limitations more important as RWC falls (Lawlor and Cornic, 2002).

Photosynthetic Carbon Metabolism: Plasticity and Evolution 375

modulation by glucose 6-phosphate and phosphorylation by a protein kinase using ATP (S.C. Huber and J.L. Huber, 1996). The latter activates SPS under osmotic stress, suggesting that inactivation may be related to decreased ATP content (Lawlor and Cornic, 2002).

Water deficiency changes the proportion of different carbohydrates. Starch, glucose and fructose concentrations increase with mild drought but sucrose change little. Such changes may be adaptive i.e. osmoregulation, and is associated with increased soluble (vacuolar)

Low temperature is one of the most important factors affecting plant performance and distribution (Stitt and Hurry, 2002). Photosynthetic carbon metabolism is greatly influenced by low temperature, directly via the modulation of enzymes activity and indirectly via

Ecological and physiological studies have uncovered a strong correlation between sugar concentrations and frost resistance (Guy et al., 1992). Sugars either act as osmotica or protect specific macromolecules during dehydration. Changes in the subcellular concentration and distribution of sugars might also provide a mechanism to protect specific compartments. For example, although sucrose is largely restricted to the cytosol of leaves at high temperatures, there are reports that it accumulates in the chloroplast in cold-acclimated cabbage (Fowler et

In recent years, molecular studies unravel mechanisms underlying responses of photosynthetic metabolism under low temperatures. The constitutive increases in the frost tolerance was observed in an *Arabidopsis* mutant over-expressing one dehydrationresponsive element gene that is correlated with increased sugar contents (Gilmour et al., 2000). A freezing sensitive mutant with impaired cold acclimation has sugar levels that are

Decreased temperatures lead to an acute Pi-limitation of photosynthesis. Indeed, some of the changes in photosynthetic metabolism that occur during cold acclimation are reminiscent of the response to low Pi (Nielsen et al., 1998). Evidence that changes in Pi concentration or availability to metabolism contribute to cold acclimation has been provided by studies of *pho1* and *pho2* mutants. *pho1* mutant with decreased shoot Pi concentration shows accentuation of the low-temperature-induced increase of sucrose phosphate synthase activity and of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase gene expression, abolishment of the decrease in the transcript levels of genes encoding for Rubsico and light harvesting chlorophyll a/b binding protein after chilling, accentuation of the cold-induced shift in carbon allocation from starch to sucrose and increase in the proline accumulation after chilling (Hurry et al., 2000). The opposite of these metabolic characteristics was observed in *pho2* mutant with higher shoot Pi concentration compared with control (Hurry et al., 2000). These results reveal that signals relating to altered Pi concentration or availability to metabolism lead to the activation and increased expression of enzymes in the sucrose synthesis pathway. They also lead to changes in the relative

Functional importance of sugar metabolism has also been demonstrated during cold acclimation. Optimal rates of photosynthesis require an appropriate balance between the rates of carbon fixation and sucrose synthesis (Stitt, 1996). Excessive sucrose synthesis

**6.2.1 Carbon metabolism under low temperature and during cold acclimation** 

changes in sink demand of plants experiencing low temperature stress.

lower than those of wild type *Arabidopsis* plants (McKown et al., 1996).

activities of enzymes in the Calvin cycle (Hurry et al., 2000).

acid invertase activity in C3 species (Pelleschi *et al*. 1997).

**6.2 Effect of temperature** 

al., 2001).

Fig. 5. There are two types of response of *Apot* to decreasing RWC: Type 1 (left) with no decrease as RWC falls from 100 to~75% and Type 2 with progressive decrease over this range (right). Redrawn according to Lawlor and Cornic (2002) with permission.

Although effect of different experimental approaches could not be excluded in different response curve of species as described by type 1 and 2, this difference is most likely related to differences in the particular characteristics of metabolism in different species. They may reflect differences in the cell water balance and differences in cell elastic modulus. Alternatively, they could reflect differences in sensitivity of a basic process to changing cellular conditions in different species or under different conditions, e.g. if ionic concentration in chloroplasts differed and thus resulted in different rates of ATP synthesis (Lawlor and Cornic, 2002).

The potential capacity for light harvesting, energy transduction, electron transfer in reaction centers of the photosystems (PSII and PSI) and electron transport in thylakoids are unaffected by a wide range of RWC, only severe loss of RWC decreases photosystem activity and alters the structure of PSII (Giardi *et al*. 1996). The rate of electron transport at saturating PAR is determined by sink capacity for electrons, principally *A* at large RWC. Decreased sink capacity for electrons results in increased non-photochemical energy dissipation in Type 1 and 2 responses (Müller et al., 2001). However, maintenance of *Apot* in the Type 1 response will enable greater CO2 recycling and energy use within the tissue than if *Apot* decreases as in Type 2. At low RWC, where *Apot* decreases in both responses, electron transport is decreased because of biochemical, as opposed to biophysical, limitations (Lawlor and Cornic, 2002).

As *A* falls with decreasing RWC, the amount of assimilate available for export as triosephosphate from chloroplast to cytosol and sucrose synthesis diminishes. Sucrose content in leaves falls in rapidly stressed leaves at RWC<80%, caused by low *A* and continued respiration. Thus, it is very unlikely that accumulation of assimilates would result in feedback inhibition of *A* or that the capacity of the triose-phosphate-Pi transporter in the chloroplast envelope is affected by low RWC. The rate of sucrose synthesis also depends on the activity of sucrose phosphate synthase, which is greatly decreased by even small loss of RWC. Sucrose phosphate synthase is subjected to complex control, including allosteric modulation by glucose 6-phosphate and phosphorylation by a protein kinase using ATP (S.C. Huber and J.L. Huber, 1996). The latter activates SPS under osmotic stress, suggesting that inactivation may be related to decreased ATP content (Lawlor and Cornic, 2002).

Water deficiency changes the proportion of different carbohydrates. Starch, glucose and fructose concentrations increase with mild drought but sucrose change little. Such changes may be adaptive i.e. osmoregulation, and is associated with increased soluble (vacuolar) acid invertase activity in C3 species (Pelleschi *et al*. 1997).

#### **6.2 Effect of temperature**

374 Advances in Photosynthesis – Fundamental Aspects

Fig. 5. There are two types of response of *Apot* to decreasing RWC: Type 1 (left) with no decrease as RWC falls from 100 to~75% and Type 2 with progressive decrease over this

Although effect of different experimental approaches could not be excluded in different response curve of species as described by type 1 and 2, this difference is most likely related to differences in the particular characteristics of metabolism in different species. They may reflect differences in the cell water balance and differences in cell elastic modulus. Alternatively, they could reflect differences in sensitivity of a basic process to changing cellular conditions in different species or under different conditions, e.g. if ionic concentration in chloroplasts differed and thus resulted in different rates of ATP synthesis

The potential capacity for light harvesting, energy transduction, electron transfer in reaction centers of the photosystems (PSII and PSI) and electron transport in thylakoids are unaffected by a wide range of RWC, only severe loss of RWC decreases photosystem activity and alters the structure of PSII (Giardi *et al*. 1996). The rate of electron transport at saturating PAR is determined by sink capacity for electrons, principally *A* at large RWC. Decreased sink capacity for electrons results in increased non-photochemical energy dissipation in Type 1 and 2 responses (Müller et al., 2001). However, maintenance of *Apot* in the Type 1 response will enable greater CO2 recycling and energy use within the tissue than if *Apot* decreases as in Type 2. At low RWC, where *Apot* decreases in both responses, electron transport is decreased because of biochemical, as opposed to biophysical, limitations

As *A* falls with decreasing RWC, the amount of assimilate available for export as triosephosphate from chloroplast to cytosol and sucrose synthesis diminishes. Sucrose content in leaves falls in rapidly stressed leaves at RWC<80%, caused by low *A* and continued respiration. Thus, it is very unlikely that accumulation of assimilates would result in feedback inhibition of *A* or that the capacity of the triose-phosphate-Pi transporter in the chloroplast envelope is affected by low RWC. The rate of sucrose synthesis also depends on the activity of sucrose phosphate synthase, which is greatly decreased by even small loss of RWC. Sucrose phosphate synthase is subjected to complex control, including allosteric

range (right). Redrawn according to Lawlor and Cornic (2002) with permission.

(Lawlor and Cornic, 2002).

(Lawlor and Cornic, 2002).

#### **6.2.1 Carbon metabolism under low temperature and during cold acclimation**

Low temperature is one of the most important factors affecting plant performance and distribution (Stitt and Hurry, 2002). Photosynthetic carbon metabolism is greatly influenced by low temperature, directly via the modulation of enzymes activity and indirectly via changes in sink demand of plants experiencing low temperature stress.

Ecological and physiological studies have uncovered a strong correlation between sugar concentrations and frost resistance (Guy et al., 1992). Sugars either act as osmotica or protect specific macromolecules during dehydration. Changes in the subcellular concentration and distribution of sugars might also provide a mechanism to protect specific compartments. For example, although sucrose is largely restricted to the cytosol of leaves at high temperatures, there are reports that it accumulates in the chloroplast in cold-acclimated cabbage (Fowler et al., 2001).

In recent years, molecular studies unravel mechanisms underlying responses of photosynthetic metabolism under low temperatures. The constitutive increases in the frost tolerance was observed in an *Arabidopsis* mutant over-expressing one dehydrationresponsive element gene that is correlated with increased sugar contents (Gilmour et al., 2000). A freezing sensitive mutant with impaired cold acclimation has sugar levels that are lower than those of wild type *Arabidopsis* plants (McKown et al., 1996).

Decreased temperatures lead to an acute Pi-limitation of photosynthesis. Indeed, some of the changes in photosynthetic metabolism that occur during cold acclimation are reminiscent of the response to low Pi (Nielsen et al., 1998). Evidence that changes in Pi concentration or availability to metabolism contribute to cold acclimation has been provided by studies of *pho1* and *pho2* mutants. *pho1* mutant with decreased shoot Pi concentration shows accentuation of the low-temperature-induced increase of sucrose phosphate synthase activity and of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase gene expression, abolishment of the decrease in the transcript levels of genes encoding for Rubsico and light harvesting chlorophyll a/b binding protein after chilling, accentuation of the cold-induced shift in carbon allocation from starch to sucrose and increase in the proline accumulation after chilling (Hurry et al., 2000). The opposite of these metabolic characteristics was observed in *pho2* mutant with higher shoot Pi concentration compared with control (Hurry et al., 2000). These results reveal that signals relating to altered Pi concentration or availability to metabolism lead to the activation and increased expression of enzymes in the sucrose synthesis pathway. They also lead to changes in the relative activities of enzymes in the Calvin cycle (Hurry et al., 2000).

Functional importance of sugar metabolism has also been demonstrated during cold acclimation. Optimal rates of photosynthesis require an appropriate balance between the rates of carbon fixation and sucrose synthesis (Stitt, 1996). Excessive sucrose synthesis

Photosynthetic Carbon Metabolism: Plasticity and Evolution 377

Human activities have caused the concentration of atmospheric CO2 to increase continuously from about 280 parts per million (ppm) at the beginning of the 19th century to 369 ppm at the beginning of the 21st century (Prentice et al., 2001). Future projections of atmospheric CO2 concentration range between about 450 and 600 ppm by the year 2050 and

Long-term studies on the effects of CO2 enrichment on plants have provided a rich suite of data and understanding about a wide variety of plant responses (McLeod and Long, 1999). Initial short-term experiments demonstrated that elevated CO2 concentrations partially alleviated the limitation of C3 (but not C4) photosynthesis by CO2 supply and acted as a negative feedback on transpiration in both C3 and C4 species (Long, 1991). Subsequent and often longer-term experiments have shown that photosynthesis could acclimate downwards in response to CO2 enrichment, and there is now some evidence to suggest that photosynthesis is stimulated in C4 species in response to CO2 enrichment (Ghannoum et al 2000). In species with the C3 photosynthetic pathway, high irradiance can lead to photoinhibition. Field studies have now demonstrated that CO2 enrichment can reduce the severity of photoinhibition, although this effect is dependent on rubisco activity (Hymus et

Leaf thickness generally increases whereas specific leaf area decreases as a result of CO2 enrichment. A detailed analysis of leaf development in Scots pine (*Pinus sylvestris*) after four years of exposure to CO2 enrichment confirmed that leaf thickness was increased but also indicated reductions in stomatal density (Lin et al., 2001). This stomatal-density response confirms observations of reduced stomatal conductance in response to CO2 enrichment. Recently, a gene *HIC* (*HI*gh *C*arbon dioxide) has been identified whose disruption leads to large increases in the number of stomata initiated in response to CO2 enrichment (Gray et al., 2000). The *HIC* gene encodes an enzyme involved in the synthesis of those long-chain fatty acids that are typically found in the cuticle of leaves. Changes in these fatty acids may influence the cell-to-cell signaling of stomatal development. The short-distance cell-to-cell signaling of stomatal development is complimented by longer distance systemic signaling of stomatal development. The systemic signal allows the development of stomata in immature leaves to be controlled after CO2 concentration is detected by mature leaves (Lake et al.,

Early experiments demonstrated significant reductions in stomatal conductance under CO2 enrichment. Analysis of 13 long-term (i.e. duration of more than one year) field-based studies on tree species demonstrated an overall reduction of 21% in stomatal conductance (Medlyn et al., 2001). The observation of reduced stomatal conductance was much more consistent in the longer-term than in the shorter-term studies. In combination with the partial down regulation of photosynthetic rate as the plants acclimatized to elevated CO2, the reduction in stomatal conductance led to a 40% increase in instantaneous water use

Carbon allocation to reproduction is strongly stimulated after a long-term of CO2 enrichment. In a long-term study, trees growing in the enriched CO2 were twice as likely to

are strongly dependent on future scenarios of anthropogenic emissions.

**6.3.1 Effect on stomatal conductance and water use efficiency** 

**6.3 Effect of CO2 concentration** 

al., 2000).

2001).

efficiency (Woodward, 2002).

**6.3.2 Assimilates allocation** 

depletes the phosphorylated Calvin cycle intermediates and inhibits the regeneration of ribulose-1,5-bisphosphate. Conversely, inadequate sucrose synthesis leads to accumulation of phosphorylated intermediates and depletion of Pi, resulting in inhibition of ATP synthesis, accumulation of 3-phosphoglycerate and inactivation of Rubisco. A sequence of events reverses the inhibition of sucrose synthesis and photosynthesis as the plants acclimate to low temperatures (Hurry et al., 2000). Short- and mid-term adjustments act primarily on sucrose synthesis but also stimulate photosynthesis by relieving the acute Pilimitation. Longer-term adjustments affect photosynthesis directly. The recovery has two important functions: increased sucrose production (Strand et al., 1999) and protection against photoinhibition by allowing increased turnover of the photosynthetic electron chain (Hurry et al., 2000). The transfer of warm-grown *Arabidopsis* plants to 4°C leads to the posttranslational activation of sucrose phosphate synthase within 30 min. Over the next few days, sucrose synthesis is stimulated by two further adjustments. One is a selective increase in the expression of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase genes, the two key regulated enzymes in the pathway of sucrose synthesis (Strand et al., 1999). The second is a shift in the subcellular distribution of Pi. In the leaves, most of the Pi is in the vacuole. Indirect evidence indicates that the Pi distribution shifts towards the cytoplasm at low temperatures allowing phosphorylated metabolites to increase without depleting the free Pi (Hurry et al., 2000).

Full acclimation occurs in leaves that develop at low temperature. They retain the selective increase in the expression of sucrose synthesis enzymes, and also have higher activities of all of the Calvin cycle enzymes on a fresh weight or leaf area basis. Two factors contribute to this increase. First, whereas transcripts for light harvesting chlorophyll a/b binding protein and Rubisco genes decrease after transfer to low temperature, they recover in leaves that develop at low temperature. This recovery occurs even though leaf sugars rise, indicating that sugar-repression of these genes is overridden at low temperature in acclimated leaves. Second, leaves that mature at low temperature have reduced water and increased protein contents due to an increase in the volume of the cytoplasm relative to that of the vacuole (Strand et al., 1997; 1999) .

#### **6.2.2 Carbon metabolism under higher temperatures**

Photosynthesis is very responsive to high temperatures (Knight and Ackerly, 2003). In semiarid regions, temperature and precipitation are often negatively correlated, with lower rainfall in warmer environments. Therefore, studies on photosynthetic thermal tolerance of plants are complicated by the fact that a variety of environmental factors can affect photosynthesis, including plant water status, soil salinity and light levels. Photosynthetic acclimation can occur on the scale of minutes to hours in response to moderately elevated temperatures. During long-term effects of various co-existing factors, however, morphological processes may also contribute to the plastic acclimation responses of photosynthesis (Knight and Ackerly, 2003). Reduction of specific leaf area and increased expression levels of small heat shock proteins are form adaptations of plants carbon metabolism to high temperatures. Small heat shock proteins dominate protein synthesis during and after high temperature stress. Variation between species for expression levels of the chloroplast small heat shock proteins following heat stress is positively correlated with the maintenance of PSII electron transport (Preczewskiet at al., 2000).

#### **6.3 Effect of CO2 concentration**

376 Advances in Photosynthesis – Fundamental Aspects

depletes the phosphorylated Calvin cycle intermediates and inhibits the regeneration of ribulose-1,5-bisphosphate. Conversely, inadequate sucrose synthesis leads to accumulation of phosphorylated intermediates and depletion of Pi, resulting in inhibition of ATP synthesis, accumulation of 3-phosphoglycerate and inactivation of Rubisco. A sequence of events reverses the inhibition of sucrose synthesis and photosynthesis as the plants acclimate to low temperatures (Hurry et al., 2000). Short- and mid-term adjustments act primarily on sucrose synthesis but also stimulate photosynthesis by relieving the acute Pilimitation. Longer-term adjustments affect photosynthesis directly. The recovery has two important functions: increased sucrose production (Strand et al., 1999) and protection against photoinhibition by allowing increased turnover of the photosynthetic electron chain (Hurry et al., 2000). The transfer of warm-grown *Arabidopsis* plants to 4°C leads to the posttranslational activation of sucrose phosphate synthase within 30 min. Over the next few days, sucrose synthesis is stimulated by two further adjustments. One is a selective increase in the expression of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase genes, the two key regulated enzymes in the pathway of sucrose synthesis (Strand et al., 1999). The second is a shift in the subcellular distribution of Pi. In the leaves, most of the Pi is in the vacuole. Indirect evidence indicates that the Pi distribution shifts towards the cytoplasm at low temperatures allowing phosphorylated metabolites to increase without

Full acclimation occurs in leaves that develop at low temperature. They retain the selective increase in the expression of sucrose synthesis enzymes, and also have higher activities of all of the Calvin cycle enzymes on a fresh weight or leaf area basis. Two factors contribute to this increase. First, whereas transcripts for light harvesting chlorophyll a/b binding protein and Rubisco genes decrease after transfer to low temperature, they recover in leaves that develop at low temperature. This recovery occurs even though leaf sugars rise, indicating that sugar-repression of these genes is overridden at low temperature in acclimated leaves. Second, leaves that mature at low temperature have reduced water and increased protein contents due to an increase in the volume of the cytoplasm relative to that of the vacuole

Photosynthesis is very responsive to high temperatures (Knight and Ackerly, 2003). In semiarid regions, temperature and precipitation are often negatively correlated, with lower rainfall in warmer environments. Therefore, studies on photosynthetic thermal tolerance of plants are complicated by the fact that a variety of environmental factors can affect photosynthesis, including plant water status, soil salinity and light levels. Photosynthetic acclimation can occur on the scale of minutes to hours in response to moderately elevated temperatures. During long-term effects of various co-existing factors, however, morphological processes may also contribute to the plastic acclimation responses of photosynthesis (Knight and Ackerly, 2003). Reduction of specific leaf area and increased expression levels of small heat shock proteins are form adaptations of plants carbon metabolism to high temperatures. Small heat shock proteins dominate protein synthesis during and after high temperature stress. Variation between species for expression levels of the chloroplast small heat shock proteins following heat stress is positively correlated with

depleting the free Pi (Hurry et al., 2000).

**6.2.2 Carbon metabolism under higher temperatures** 

the maintenance of PSII electron transport (Preczewskiet at al., 2000).

(Strand et al., 1997; 1999) .

Human activities have caused the concentration of atmospheric CO2 to increase continuously from about 280 parts per million (ppm) at the beginning of the 19th century to 369 ppm at the beginning of the 21st century (Prentice et al., 2001). Future projections of atmospheric CO2 concentration range between about 450 and 600 ppm by the year 2050 and are strongly dependent on future scenarios of anthropogenic emissions.

Long-term studies on the effects of CO2 enrichment on plants have provided a rich suite of data and understanding about a wide variety of plant responses (McLeod and Long, 1999). Initial short-term experiments demonstrated that elevated CO2 concentrations partially alleviated the limitation of C3 (but not C4) photosynthesis by CO2 supply and acted as a negative feedback on transpiration in both C3 and C4 species (Long, 1991). Subsequent and often longer-term experiments have shown that photosynthesis could acclimate downwards in response to CO2 enrichment, and there is now some evidence to suggest that photosynthesis is stimulated in C4 species in response to CO2 enrichment (Ghannoum et al 2000). In species with the C3 photosynthetic pathway, high irradiance can lead to photoinhibition. Field studies have now demonstrated that CO2 enrichment can reduce the severity of photoinhibition, although this effect is dependent on rubisco activity (Hymus et al., 2000).

#### **6.3.1 Effect on stomatal conductance and water use efficiency**

Leaf thickness generally increases whereas specific leaf area decreases as a result of CO2 enrichment. A detailed analysis of leaf development in Scots pine (*Pinus sylvestris*) after four years of exposure to CO2 enrichment confirmed that leaf thickness was increased but also indicated reductions in stomatal density (Lin et al., 2001). This stomatal-density response confirms observations of reduced stomatal conductance in response to CO2 enrichment.

Recently, a gene *HIC* (*HI*gh *C*arbon dioxide) has been identified whose disruption leads to large increases in the number of stomata initiated in response to CO2 enrichment (Gray et al., 2000). The *HIC* gene encodes an enzyme involved in the synthesis of those long-chain fatty acids that are typically found in the cuticle of leaves. Changes in these fatty acids may influence the cell-to-cell signaling of stomatal development. The short-distance cell-to-cell signaling of stomatal development is complimented by longer distance systemic signaling of stomatal development. The systemic signal allows the development of stomata in immature leaves to be controlled after CO2 concentration is detected by mature leaves (Lake et al., 2001).

Early experiments demonstrated significant reductions in stomatal conductance under CO2 enrichment. Analysis of 13 long-term (i.e. duration of more than one year) field-based studies on tree species demonstrated an overall reduction of 21% in stomatal conductance (Medlyn et al., 2001). The observation of reduced stomatal conductance was much more consistent in the longer-term than in the shorter-term studies. In combination with the partial down regulation of photosynthetic rate as the plants acclimatized to elevated CO2, the reduction in stomatal conductance led to a 40% increase in instantaneous water use efficiency (Woodward, 2002).

#### **6.3.2 Assimilates allocation**

Carbon allocation to reproduction is strongly stimulated after a long-term of CO2 enrichment. In a long-term study, trees growing in the enriched CO2 were twice as likely to

Photosynthetic Carbon Metabolism: Plasticity and Evolution 379

Photosynthetic gas exchange pattern of CAM consists of four phases (Osmond 1978). Phase I consisted of nocturnal uptake of CO2 via open stomata, fixation by PEPC and vacuolar storage of CO2 in the form of organic acids, mainly malic acid. Daytime remobilization of vacuolar organic acids, decarboxylation and refixation plus assimilation of CO2 behind closed stomata in the Calvin-cycle were named phase III. Between these two phases there are transitions when stomata remain open for CO2 uptake for a short time during the very early light period (phase II) and reopen again during the late light period for CO2 uptake with direct assimilation to carbohydrate when vacuolar organic acid is exhausted (phase IV)

Fig. 6. Diurnal course of net CO2 assimilation rate, malate accumulation and stomatal

High internal CO2-concentrations in phase III resulting from malate decarboxylation repress photorespiration, due to a generally low O2/CO2-ratio in CAM plants. The main advantage of CAM, in comparison to C3 photosynthesis, is that it offers much higher water use efficiency, due to stomatal closure during the day. Such an adaptation is beneficial to plants

Plasticity in the expression of various CAM phases described above is a ubiquitous feature of the majority of CAM plants. CAM is intimately linked with the environment and can be perturbed by temperature, light level and water status. Plasticity in expression of CAM has been shown in the members of Crassulaceae. Thinner-leaved species of Crassulaceae are highly plastic in photosynthetic expression and behave like C3 species, both in terms of the duration of diurnal atmospheric CO2 uptake and light use efficiency. In contrast, thickerleaved, more succulent species suffer from extreme CO2 -diffusion limitation and are more strongly bound to nocturnal CO2 fixation for the daytime supply of carbon (Winter, 1985). Carbon isotope discrimination studies showed also that CAM photosynthesis is inducible (facultative) or constitutive (obligate) (Kluge et al., 1995). In addition, the stage of plant

**7. Plasticity in CAM** 

resistance in a CAM plant.

living in dry and saline environments.

(Fig. 6).

be reproductively mature, and produced three times more seeds than control plants growing in ambient CO2 concentrations (LaDeau and Clark 2001). This result indicates that CO2 enrichment hastens significantly the onset of seed production, a feature that may prove to be effective in tracking climatic change. In contrast, flowering and seed set in grasslands, where species may have deterministic life cycles, were unaffected, reduced or stimulated under CO2 enrichment. The species-specific nature of these responses indicates a strong potential for CO2 enrichment to change the composition of plant communities (Woodward, 2002).

#### **6.3.3 Acclimation of plants to CO2 enrichment**

Long-term field experiments indicate that leaf photosynthesis is stimulated by CO2 enrichment in C3 species from 7% for legume herbs to 98% for *Pinus radiata* (Long et al., 2004). However, in the longer term when photosynthesis exceeds the capacity for carbohydrate export and utilization, a down regulation process is expected. This response is exacerbated by genetic limitations, such as determinate growth patterns, and environmental limitations, such as N deficiency or low temperature (Ainsworth et al., 2004).

The causes of photosynthetic downregulation have been variously ascribed to a reduction in carbohydrate sink strength, a limited capacity to sequester carbon in a storage form, changes in nitrogen allocation and a reduction of rubisco concentration (Woodward, 2002). These responses indicate not only a decreased expression of photosynthetic genes but also a coordination of the carbon to nitrogen balance (Paul and Foyer 2001). For example, nitrate and ammonium uptake, and nitrate reductase activity are sensitive to CO2. These co-ordinating activities match photosynthetic capacity with the capacities for growth and carbon storage (Walch-Liu et al., 2001).

Respiration rates have been observed to decline, or remain unchanged with CO2 enrichment, depending on the species. Rates of dark respiration are directly correlated with leaf nitrogen content (Hamilton et al., 2001). Therefore, when CO2 enrichment leads to a reduction in leaf nitrogen concentration, respiration also declines. Surprisingly, CO2 enrichment increases the average number of mitochondria in each cell, even though leaf respiration rate decreases in response to elevated CO2 across a diverse selection of plant species (Griffin et al., 2001).

At elevated CO2 concentrations, Rubisco content was decreased by about 20%, but in contrast there was little change in capacity for Ribulose-1,5-bisphosphate regeneration and little or no effect on photosynthetic rate. In long-term CO2 enrichments, the loss of Rubisco cannot be explained as the result of an overall decline in leaf N, but instead appears specific and accounts for most of the decrease in N per unit of leaf area. These results suggest that loss of Rubisco is more appropriately described as an acclimatory change benefiting N use efficiency rather than as down-regulation (Long et al., 2004). Both genetic and experimental modifications of source-sink balance provide results consistent with current models of carbohydrate feedback on Rubisco expression. There is no evidence of acclimation in C4 species under long term CO2 enrichment, and increases in photosynthesis and production are consistent with the hypothesis that this results from improved water use efficiency. The findings have important implications both for predicting the future terrestrial biosphere and understanding how crops may need to be adapted to the changed and changing atmosphere.

#### **7. Plasticity in CAM**

378 Advances in Photosynthesis – Fundamental Aspects

be reproductively mature, and produced three times more seeds than control plants growing in ambient CO2 concentrations (LaDeau and Clark 2001). This result indicates that CO2 enrichment hastens significantly the onset of seed production, a feature that may prove to be effective in tracking climatic change. In contrast, flowering and seed set in grasslands, where species may have deterministic life cycles, were unaffected, reduced or stimulated under CO2 enrichment. The species-specific nature of these responses indicates a strong potential for CO2 enrichment to change the composition of plant communities (Woodward,

Long-term field experiments indicate that leaf photosynthesis is stimulated by CO2 enrichment in C3 species from 7% for legume herbs to 98% for *Pinus radiata* (Long et al., 2004). However, in the longer term when photosynthesis exceeds the capacity for carbohydrate export and utilization, a down regulation process is expected. This response is exacerbated by genetic limitations, such as determinate growth patterns, and environmental

The causes of photosynthetic downregulation have been variously ascribed to a reduction in carbohydrate sink strength, a limited capacity to sequester carbon in a storage form, changes in nitrogen allocation and a reduction of rubisco concentration (Woodward, 2002). These responses indicate not only a decreased expression of photosynthetic genes but also a coordination of the carbon to nitrogen balance (Paul and Foyer 2001). For example, nitrate and ammonium uptake, and nitrate reductase activity are sensitive to CO2. These co-ordinating activities match photosynthetic capacity with the capacities for growth and carbon storage

Respiration rates have been observed to decline, or remain unchanged with CO2 enrichment, depending on the species. Rates of dark respiration are directly correlated with leaf nitrogen content (Hamilton et al., 2001). Therefore, when CO2 enrichment leads to a reduction in leaf nitrogen concentration, respiration also declines. Surprisingly, CO2 enrichment increases the average number of mitochondria in each cell, even though leaf respiration rate decreases in response to elevated CO2 across a diverse selection of plant

At elevated CO2 concentrations, Rubisco content was decreased by about 20%, but in contrast there was little change in capacity for Ribulose-1,5-bisphosphate regeneration and little or no effect on photosynthetic rate. In long-term CO2 enrichments, the loss of Rubisco cannot be explained as the result of an overall decline in leaf N, but instead appears specific and accounts for most of the decrease in N per unit of leaf area. These results suggest that loss of Rubisco is more appropriately described as an acclimatory change benefiting N use efficiency rather than as down-regulation (Long et al., 2004). Both genetic and experimental modifications of source-sink balance provide results consistent with current models of carbohydrate feedback on Rubisco expression. There is no evidence of acclimation in C4 species under long term CO2 enrichment, and increases in photosynthesis and production are consistent with the hypothesis that this results from improved water use efficiency. The findings have important implications both for predicting the future terrestrial biosphere and understanding how crops may need to be adapted to the changed and changing

limitations, such as N deficiency or low temperature (Ainsworth et al., 2004).

2002).

(Walch-Liu et al., 2001).

species (Griffin et al., 2001).

atmosphere.

**6.3.3 Acclimation of plants to CO2 enrichment** 

Photosynthetic gas exchange pattern of CAM consists of four phases (Osmond 1978). Phase I consisted of nocturnal uptake of CO2 via open stomata, fixation by PEPC and vacuolar storage of CO2 in the form of organic acids, mainly malic acid. Daytime remobilization of vacuolar organic acids, decarboxylation and refixation plus assimilation of CO2 behind closed stomata in the Calvin-cycle were named phase III. Between these two phases there are transitions when stomata remain open for CO2 uptake for a short time during the very early light period (phase II) and reopen again during the late light period for CO2 uptake with direct assimilation to carbohydrate when vacuolar organic acid is exhausted (phase IV) (Fig. 6).

Fig. 6. Diurnal course of net CO2 assimilation rate, malate accumulation and stomatal resistance in a CAM plant.

High internal CO2-concentrations in phase III resulting from malate decarboxylation repress photorespiration, due to a generally low O2/CO2-ratio in CAM plants. The main advantage of CAM, in comparison to C3 photosynthesis, is that it offers much higher water use efficiency, due to stomatal closure during the day. Such an adaptation is beneficial to plants living in dry and saline environments.

Plasticity in the expression of various CAM phases described above is a ubiquitous feature of the majority of CAM plants. CAM is intimately linked with the environment and can be perturbed by temperature, light level and water status. Plasticity in expression of CAM has been shown in the members of Crassulaceae. Thinner-leaved species of Crassulaceae are highly plastic in photosynthetic expression and behave like C3 species, both in terms of the duration of diurnal atmospheric CO2 uptake and light use efficiency. In contrast, thickerleaved, more succulent species suffer from extreme CO2 -diffusion limitation and are more strongly bound to nocturnal CO2 fixation for the daytime supply of carbon (Winter, 1985). Carbon isotope discrimination studies showed also that CAM photosynthesis is inducible (facultative) or constitutive (obligate) (Kluge et al., 1995). In addition, the stage of plant

Photosynthetic Carbon Metabolism: Plasticity and Evolution 381

moist, tropical forests with dew formation occurring mainly during the late dark period. During acid remobilization in phase III, osmotic and turgor pressures decline again but the water gained is available to the plants (Lüttge, 2004). CAM also occurs in some resurrection plants such as *Haberla rhodopensis* and *Ramonda serbica* (Gesneriaceae) that are desiccationtolerant and can shift between biosis and anabiosis as they dry out and are rewatered,

Light quality and intensity affects CAM in different ways. Intensity of photosynthetically active radiation during the day (phase III) determines the rate of organic acid mobilization from the vacuole. A signaling function of light is also obvious i.e. long-day dependent induction of CAM. Phytochrome, the red-light receptor involved in photoperiodism, elicits CAM expression (Brulfert et al., 1985). In C3/CAM intermediate species, light responses of stomata change dramatically when CAM is induced. In *Portulacaria afra*, blue-light and redlight responses of stomata in the C3-state are lost in the CAM-state. In *M. crystallinum* after the C3-CAM transition, the opening response of guard cells to blue and white light is lost in parallel with light-dependent xanthophyll formation. The xanthophyll zeaxanthin is involved in the signal transduction chain from light to stomatal opening (Tallman et al.,

One of the major effects of salinity is osmotic stress, and hence there are intimate relationships to drought stress. Therefore, considering CAM as a major photosynthetic accommodation to water stress, CAM might be expected to be a prominent trait among halophytes. Moreover, halophytes are often succulent as they sequester NaCl in large central vacuoles, which is called salt succulence (Ellenberg, 1981). However, observations do not support this expectation as, in general, halophytes are not CAM plants and CAM plants are not halophytes. Generally CAM plants, including desert succulents, are highly salt sensitive (Lüttge, 2004). CAM plants inhabiting highly saline ecosystems are either effectively functional salt excluders at the root level, such as some cacti or complete escape from the saline substrate by retreat to epiphytic niches (Lüttge, 2004). The single exception is the annual facultative halophyte and facultative CAM species *Mesembryanthemum crystallinum* (Cushman and Bohnert, 2002). This plant can grow well in the absence of NaCl but has its growth optimum at several hundred mM NaCl in the medium and can complete its life

There are some photosynthetic physiotypes for the metabolic cycle of CAM include full CAM, CAM idling, CAM cycling, C3/CAM and C4/CAM (Table 1). In CAM idling stomata remain closed day and night and the day/night organic acid cycle is fed by internal recycling of nocturnally re-fixed respiratory CO2. In CAM cycling, stomata remain closed during the dark period but some nocturnal synthesis of organic acid fed by respiratory CO2 occurs, and stomata are open during the light period with uptake of atmospheric CO2 and direct Calvin-cycle CO2 reduction (C3-photosynthesis) in addition to assimilation of CO2 remobilized from nocturnally stored organic acid. CAM idling is considered as a form of very strong CAM, while CAM cycling is weak CAM (Sipes and Ting, 1985). In the epiphytic

respectively (Markovska et al., 1997).

cycle at 500 mM NaCl (Lüttge, 2002).

**7.2 CAM physiotypes** 

**7.1.2 Light** 

1997).

**7.1.3 Salinity** 

development affects CAM expression in plants. Several C3/CAM intermediate species change their mode of metabolism in response to stress conditions (Dodd et al. 2002).

Facultative or inducible CAM species use the C3 pathway to maximize growth at times of sufficient water supply but switch to CAM as a means of reducing water loss while maintaining photosynthetic integrity during periods of limited water supply. This C3/CAM intermediate pathway is predominantly found among the Aizoaceae, Crassulaceae, Portulacaceae and Vitaceae (Smith and Winter 1996). CAM expression in facultative species is primarily determined by genotype and the severity of water limitation, but may also be induced by ontogenetic and other environmental factors. Although no unique enzymes are required to facilitate the C3–CAM transition, the imposition of water stress has a profound influence on the abundance and regulation of enzymes involved in organic acid and carbohydrate formation, turnover and intracellular transport functions (Cushman and Borland, 2002). Salt-induced C3–CAM transition is linked with an increased activity of antioxidative enzymes. It has been suggested that, the redox status in the proximity of PSII in the C3/CAM intermediate plants controls the expression of key genes encoding scavengers of reactive oxygen species such as superoxide dismutase, ascorbate peroxidase and activity of NADP-malic enzyme (Ślesak et al., 2002).

#### **7.1 Effect of environmental factors on CAM**

Generally, water is considered to be the most important factor and CAM to be an adaptation to water-shortage stress. However, CAM is also observed in submerged freshwater plants (Keeley, 1996). CO2 has been considered as the central factor and most important driving force for CAM. It has been assumed that early CAM evolution occurred during geological times when atmospheric CO2 concentration was low (Raven and Spicer, 1996). CAM is a CO2-concentrating mechanism due to the much higher substrate affinity of PEPC for HCO3<sup>−</sup> than of the C3-photosynthesis/Calvin cycle carboxylase Rubisco for CO2. Thus, during the dark period a concentrated CO2 pool is built up in the form of vacuolar malic acid accumulation, and during phase III its remobilization in the light leads to internal CO2 concentrations that may be 2-60 times more than atmospheric CO2 concentration. For aquatic plants, this CO2-concentrating mechanism provides a benefit for CO2 acquisition. For terrestrial plants, the benefit of CO2-concentrating by CAM is considered to be related to water use as will be discussed below (Lüttge et al., 2004).

#### **7.1.1 Water availability**

For terrestrial plants, the greatest benefit of CAM is considered to be increased water use efficiency because stomatal opening during the dark period causes much less transpirational loss of water than opening during the light period. With this high water use efficiency, CAM plants not only inhabit arid habitats e.g. cacti, agaves and euphorbs, but also inhabit tropical rainforests. These CAM species are mainly epiphytic and subjected also to the particular problems of water supply in this habitat (Zotz and Hietz, 2001). In addition to CAM phasedependent stomatal responses affecting WUE, CAM plants have other structural and functional ways for water storage. The large vacuolar concentrations of nocturnally accumulated organic acids are osmotically active. The increased osmotic pressure drives water uptake into the cells, which is associated with increased turgor pressure. This allows CAM plants extra acquisition of water, particularly towards the end of the dark period when vacuolar organic acid levels become rather high. It may be a particular advantage in moist, tropical forests with dew formation occurring mainly during the late dark period. During acid remobilization in phase III, osmotic and turgor pressures decline again but the water gained is available to the plants (Lüttge, 2004). CAM also occurs in some resurrection plants such as *Haberla rhodopensis* and *Ramonda serbica* (Gesneriaceae) that are desiccationtolerant and can shift between biosis and anabiosis as they dry out and are rewatered, respectively (Markovska et al., 1997).

#### **7.1.2 Light**

380 Advances in Photosynthesis – Fundamental Aspects

development affects CAM expression in plants. Several C3/CAM intermediate species

Facultative or inducible CAM species use the C3 pathway to maximize growth at times of sufficient water supply but switch to CAM as a means of reducing water loss while maintaining photosynthetic integrity during periods of limited water supply. This C3/CAM intermediate pathway is predominantly found among the Aizoaceae, Crassulaceae, Portulacaceae and Vitaceae (Smith and Winter 1996). CAM expression in facultative species is primarily determined by genotype and the severity of water limitation, but may also be induced by ontogenetic and other environmental factors. Although no unique enzymes are required to facilitate the C3–CAM transition, the imposition of water stress has a profound influence on the abundance and regulation of enzymes involved in organic acid and carbohydrate formation, turnover and intracellular transport functions (Cushman and Borland, 2002). Salt-induced C3–CAM transition is linked with an increased activity of antioxidative enzymes. It has been suggested that, the redox status in the proximity of PSII in the C3/CAM intermediate plants controls the expression of key genes encoding scavengers of reactive oxygen species such as superoxide dismutase, ascorbate peroxidase

Generally, water is considered to be the most important factor and CAM to be an adaptation to water-shortage stress. However, CAM is also observed in submerged freshwater plants (Keeley, 1996). CO2 has been considered as the central factor and most important driving force for CAM. It has been assumed that early CAM evolution occurred during geological times when atmospheric CO2 concentration was low (Raven and Spicer, 1996). CAM is a CO2-concentrating mechanism due to the much higher substrate affinity of PEPC for HCO3<sup>−</sup> than of the C3-photosynthesis/Calvin cycle carboxylase Rubisco for CO2. Thus, during the dark period a concentrated CO2 pool is built up in the form of vacuolar malic acid accumulation, and during phase III its remobilization in the light leads to internal CO2 concentrations that may be 2-60 times more than atmospheric CO2 concentration. For aquatic plants, this CO2-concentrating mechanism provides a benefit for CO2 acquisition. For terrestrial plants, the benefit of CO2-concentrating by CAM is considered to be related to

For terrestrial plants, the greatest benefit of CAM is considered to be increased water use efficiency because stomatal opening during the dark period causes much less transpirational loss of water than opening during the light period. With this high water use efficiency, CAM plants not only inhabit arid habitats e.g. cacti, agaves and euphorbs, but also inhabit tropical rainforests. These CAM species are mainly epiphytic and subjected also to the particular problems of water supply in this habitat (Zotz and Hietz, 2001). In addition to CAM phasedependent stomatal responses affecting WUE, CAM plants have other structural and functional ways for water storage. The large vacuolar concentrations of nocturnally accumulated organic acids are osmotically active. The increased osmotic pressure drives water uptake into the cells, which is associated with increased turgor pressure. This allows CAM plants extra acquisition of water, particularly towards the end of the dark period when vacuolar organic acid levels become rather high. It may be a particular advantage in

change their mode of metabolism in response to stress conditions (Dodd et al. 2002).

and activity of NADP-malic enzyme (Ślesak et al., 2002).

water use as will be discussed below (Lüttge et al., 2004).

**7.1.1 Water availability** 

**7.1 Effect of environmental factors on CAM** 

Light quality and intensity affects CAM in different ways. Intensity of photosynthetically active radiation during the day (phase III) determines the rate of organic acid mobilization from the vacuole. A signaling function of light is also obvious i.e. long-day dependent induction of CAM. Phytochrome, the red-light receptor involved in photoperiodism, elicits CAM expression (Brulfert et al., 1985). In C3/CAM intermediate species, light responses of stomata change dramatically when CAM is induced. In *Portulacaria afra*, blue-light and redlight responses of stomata in the C3-state are lost in the CAM-state. In *M. crystallinum* after the C3-CAM transition, the opening response of guard cells to blue and white light is lost in parallel with light-dependent xanthophyll formation. The xanthophyll zeaxanthin is involved in the signal transduction chain from light to stomatal opening (Tallman et al., 1997).

#### **7.1.3 Salinity**

One of the major effects of salinity is osmotic stress, and hence there are intimate relationships to drought stress. Therefore, considering CAM as a major photosynthetic accommodation to water stress, CAM might be expected to be a prominent trait among halophytes. Moreover, halophytes are often succulent as they sequester NaCl in large central vacuoles, which is called salt succulence (Ellenberg, 1981). However, observations do not support this expectation as, in general, halophytes are not CAM plants and CAM plants are not halophytes. Generally CAM plants, including desert succulents, are highly salt sensitive (Lüttge, 2004). CAM plants inhabiting highly saline ecosystems are either effectively functional salt excluders at the root level, such as some cacti or complete escape from the saline substrate by retreat to epiphytic niches (Lüttge, 2004). The single exception is the annual facultative halophyte and facultative CAM species *Mesembryanthemum crystallinum* (Cushman and Bohnert, 2002). This plant can grow well in the absence of NaCl but has its growth optimum at several hundred mM NaCl in the medium and can complete its life cycle at 500 mM NaCl (Lüttge, 2002).

#### **7.2 CAM physiotypes**

There are some photosynthetic physiotypes for the metabolic cycle of CAM include full CAM, CAM idling, CAM cycling, C3/CAM and C4/CAM (Table 1). In CAM idling stomata remain closed day and night and the day/night organic acid cycle is fed by internal recycling of nocturnally re-fixed respiratory CO2. In CAM cycling, stomata remain closed during the dark period but some nocturnal synthesis of organic acid fed by respiratory CO2 occurs, and stomata are open during the light period with uptake of atmospheric CO2 and direct Calvin-cycle CO2 reduction (C3-photosynthesis) in addition to assimilation of CO2 remobilized from nocturnally stored organic acid. CAM idling is considered as a form of very strong CAM, while CAM cycling is weak CAM (Sipes and Ting, 1985). In the epiphytic

Photosynthetic Carbon Metabolism: Plasticity and Evolution 383

are no unique enzymes and metabolic reactions specifically required for CAM. CAM in the terrestrial angiosperms is thought to have diversified polyphyletically from C3 ancestors sometime during the Miocene, possibly as a consequence of reduced atmospheric CO2 concentration (Raven and Spicer, 1996). There is strong evidence that the evolutionary direction has been from C3/CAM intermediates to full CAM, paralleled by specialization to and colonization of new, increasingly arid habitats (Kluge et al., 2001). A rearrangement and appropriately regulated complement of enzyme reactions present for basic functions in any green plant tissue are sufficient for performing CAM (Lüttge 2004). However, CAM-specific isoforms of key enzymes have evolved. Analysis of PEPC gene families from facultative and obligate CAM species led to the conclusion that during the induction of CAM, in addition to the existing housekeeping isoform, a CAM-specific PEPC isoform is expressed, which is responsible for primary CO2 fixation of this photosynthetic pathway (Cushman and Bohnert 1999). A single family member of a small gene family (e.g. four to six isogenes) is recruited to fulfill the increased carbon flux demand of CAM. The recruited family member typically shows enhanced expression in CAM-performing leaves. Remaining isoforms, which presumably fulfill anapleurotic 'housekeeping' or tissue-specific functional roles, generally have lower transcript abundance and show little change in expression following water deficit. This 'gene recruitment' paradigm is likely to apply to other gene families as well (Cushman and Borland, 2002). In addition to enzymes involved in malate synthesis and mobilization, CAM induction involves large increases in carbohydrate-forming and degrading enzymes (Häusler *et al*. 2000). Such activity changes are matched by corresponding changes in gene expression of at least one gene family member of glyceraldehyde-3-phosphate dehydrogenase, enolase and phosphoglyceromutase (Cushman and Borland, 2002). CAM induction causes a dramatic increase in transcripts encoding PEP-Pi and glucose-6-phosphate-Pi translocators, with expression peaking in the light period, whereas transcripts for a chloroplast glucose transporter and a triose-phosphate transporter

Duplication events appear to be the source of CAM-specific genes recruited from multigene families during CAM evolution (Cushman and Bohnert 1999). Enzyme isoforms with different subcellular locations are also thought to have evolved through gene duplication of pre-existing. Following gene duplication, modification of multipartite cis-regulatory elements within non-coding 5′� and 3′ flanking regions is likely to have occurred, conferring water-deficit-inducible or enhanced expression patterns for CAM-specific isogenes

Transcriptional activation appears to be the primary mechanism responsible for increased or enhanced expression of CAM-specific genes following water-deficit stress. Most changes in transcript abundance correlate with changes in protein amounts arising from *de novo* protein synthesis. Alterations in the translational efficiency of specific mRNA populations may also contribute significantly to the expression of key CAM enzymes (Cushman and Borland,

Evolution of C4 species undoubtedly involved steps in which anatomical characteristics

Evidences suggest that C4 plants have evolved from ancestors possessing the C3 pathway of photosynthesis and this has occurred independently over 45 times in taxonomically diverse

remain largely unchanged (Häusler *et al*. 2000).

(Cushman and Borland, 2002).

**8. C3-C4 intermediate species** 

were between those of C3 and C4 species.

2002).

*Codonanthe crassifolia* (Gesneriaceae), CAM cycling was observed in well-watered plants and CAM idling in drought-stressed plants. CAM cycling that scavenges respiratory CO2 appears to be a starting point for CAM evolution (Guralnick et al., 2002). The various forms of weak and strong CAM may be restricted to different individual species or may also be expressed temporarily in one given species. For example, *Sedum telephium* has the potential to exhibit pure C3 characteristics when well-watered and a transition to CAM when droughted, including a continuum of different stages of CAM expression which are repeatedly reversible under changing drought and watering regimes (Lee and Griffiths, 1987).


Table 1. Various CAM physiotypes with different degrees of CAM expression.

There are true intermediate species (C3/CAM) that can switch between full C3 photosynthesis and full CAM. The large genus *Clusia*, comprises three photosynthetic physiotypes, i.e. C3, C3/CAM and CAM. There are also some C4/CAM intermediate species, e.g. *Peperomia camptotricha*, *Portulaca oleracea* and *Portulaca grandiflora* (Guralnick et al., 2002). Only succulent C4 dicotyledons are capable of diurnal fluctuations of organic acids, where dark-respiratory CO2 is trapped in bundle sheaths by PEPC and the water storage tissue in the succulent leaves may also participate in the fixation of internally released CO2. In *Portulaca*, this may be a form of CAM cycling in leaves with C4 photosynthesis, while stems perform CAM idling (Guralnick et al., 2002). However, although C4 photosynthesis and weak CAM occur in the same leaves, they are separated in space and do not occur in the same cells.

Compatibility of CAM and C4 photosynthesis has been questioned (Sage, 2002a). Incompatibility of C4 photosynthesis and CAM may be due to anatomical, biochemical and evolutionary incompatibilities. The separation of malate synthesis and decarboxylation in space in C4 photosynthesis and in time in CAM, respectively, and the primary evolution of C4 photosynthesis for scavenging photorespiratory CO2 and of CAM for scavenging respiratory CO2 (CAM cycling) may be the most important backgrounds of these incompatibilities. Although single cells may perform C4 photosynthesis, there is intracellular compartmentation of carboxylation and decarboxylation, and these cells never perform CAM. Unlike C3-CAM coupling, there is never C4-CAM coupling and both pathways only occur side by side in C4/CAM intermediate species (Sage, 2002a).

#### **7.3 CAM evolution**

CAM occurs in approximately 6% of plants, comprising monocots and dicots, encompassing 33 families and 328 genera including terrestrial and aquatic angiosperms, gymnosperms and *Welwitschia mirabilis* (Sayed, 2001). Its polyphyletic evolution was facilitated because there

*Codonanthe crassifolia* (Gesneriaceae), CAM cycling was observed in well-watered plants and CAM idling in drought-stressed plants. CAM cycling that scavenges respiratory CO2 appears to be a starting point for CAM evolution (Guralnick et al., 2002). The various forms of weak and strong CAM may be restricted to different individual species or may also be expressed temporarily in one given species. For example, *Sedum telephium* has the potential to exhibit pure C3 characteristics when well-watered and a transition to CAM when droughted, including a continuum of different stages of CAM expression which are repeatedly reversible under changing drought and watering regimes (Lee and Griffiths,

> Phase of stomatal closure

Full CAM I II, III, IV >15 High CAM idling --- I, II, III, IV >15 High CAM cycling II, III, IV I >5 Low C3/CAM Intermediate C4/CAM Intermediate

There are true intermediate species (C3/CAM) that can switch between full C3 photosynthesis and full CAM. The large genus *Clusia*, comprises three photosynthetic physiotypes, i.e. C3, C3/CAM and CAM. There are also some C4/CAM intermediate species, e.g. *Peperomia camptotricha*, *Portulaca oleracea* and *Portulaca grandiflora* (Guralnick et al., 2002). Only succulent C4 dicotyledons are capable of diurnal fluctuations of organic acids, where dark-respiratory CO2 is trapped in bundle sheaths by PEPC and the water storage tissue in the succulent leaves may also participate in the fixation of internally released CO2. In *Portulaca*, this may be a form of CAM cycling in leaves with C4 photosynthesis, while stems perform CAM idling (Guralnick et al., 2002). However, although C4 photosynthesis and weak CAM occur in the same leaves, they are separated in space and do not occur in the

Compatibility of CAM and C4 photosynthesis has been questioned (Sage, 2002a). Incompatibility of C4 photosynthesis and CAM may be due to anatomical, biochemical and evolutionary incompatibilities. The separation of malate synthesis and decarboxylation in space in C4 photosynthesis and in time in CAM, respectively, and the primary evolution of C4 photosynthesis for scavenging photorespiratory CO2 and of CAM for scavenging respiratory CO2 (CAM cycling) may be the most important backgrounds of these incompatibilities. Although single cells may perform C4 photosynthesis, there is intracellular compartmentation of carboxylation and decarboxylation, and these cells never perform CAM. Unlike C3-CAM coupling, there is never C4-CAM coupling and both pathways only

CAM occurs in approximately 6% of plants, comprising monocots and dicots, encompassing 33 families and 328 genera including terrestrial and aquatic angiosperms, gymnosperms and *Welwitschia mirabilis* (Sayed, 2001). Its polyphyletic evolution was facilitated because there

occur side by side in C4/CAM intermediate species (Sage, 2002a).

Table 1. Various CAM physiotypes with different degrees of CAM expression.

Diel Fluctuation of malate concentration Diel pH Fluctuation

1987).

CAM physiotypes

same cells.

**7.3 CAM evolution** 

Phase of CO2 fixation

are no unique enzymes and metabolic reactions specifically required for CAM. CAM in the terrestrial angiosperms is thought to have diversified polyphyletically from C3 ancestors sometime during the Miocene, possibly as a consequence of reduced atmospheric CO2 concentration (Raven and Spicer, 1996). There is strong evidence that the evolutionary direction has been from C3/CAM intermediates to full CAM, paralleled by specialization to and colonization of new, increasingly arid habitats (Kluge et al., 2001). A rearrangement and appropriately regulated complement of enzyme reactions present for basic functions in any green plant tissue are sufficient for performing CAM (Lüttge 2004). However, CAM-specific isoforms of key enzymes have evolved. Analysis of PEPC gene families from facultative and obligate CAM species led to the conclusion that during the induction of CAM, in addition to the existing housekeeping isoform, a CAM-specific PEPC isoform is expressed, which is responsible for primary CO2 fixation of this photosynthetic pathway (Cushman and Bohnert 1999). A single family member of a small gene family (e.g. four to six isogenes) is recruited to fulfill the increased carbon flux demand of CAM. The recruited family member typically shows enhanced expression in CAM-performing leaves. Remaining isoforms, which presumably fulfill anapleurotic 'housekeeping' or tissue-specific functional roles, generally have lower transcript abundance and show little change in expression following water deficit. This 'gene recruitment' paradigm is likely to apply to other gene families as well (Cushman and Borland, 2002). In addition to enzymes involved in malate synthesis and mobilization, CAM induction involves large increases in carbohydrate-forming and degrading enzymes (Häusler *et al*. 2000). Such activity changes are matched by corresponding changes in gene expression of at least one gene family member of glyceraldehyde-3-phosphate dehydrogenase, enolase and phosphoglyceromutase (Cushman and Borland, 2002). CAM induction causes a dramatic increase in transcripts encoding PEP-Pi and glucose-6-phosphate-Pi translocators, with expression peaking in the light period, whereas transcripts for a chloroplast glucose transporter and a triose-phosphate transporter remain largely unchanged (Häusler *et al*. 2000).

Duplication events appear to be the source of CAM-specific genes recruited from multigene families during CAM evolution (Cushman and Bohnert 1999). Enzyme isoforms with different subcellular locations are also thought to have evolved through gene duplication of pre-existing. Following gene duplication, modification of multipartite cis-regulatory elements within non-coding 5′� and 3′ flanking regions is likely to have occurred, conferring water-deficit-inducible or enhanced expression patterns for CAM-specific isogenes (Cushman and Borland, 2002).

Transcriptional activation appears to be the primary mechanism responsible for increased or enhanced expression of CAM-specific genes following water-deficit stress. Most changes in transcript abundance correlate with changes in protein amounts arising from *de novo* protein synthesis. Alterations in the translational efficiency of specific mRNA populations may also contribute significantly to the expression of key CAM enzymes (Cushman and Borland, 2002).

#### **8. C3-C4 intermediate species**

Evolution of C4 species undoubtedly involved steps in which anatomical characteristics were between those of C3 and C4 species.

Evidences suggest that C4 plants have evolved from ancestors possessing the C3 pathway of photosynthesis and this has occurred independently over 45 times in taxonomically diverse

Photosynthetic Carbon Metabolism: Plasticity and Evolution 385

ring around the bundle sheath cells as in a C4 leaf, but are arranged as in leaves of C3 species where interveinal distances are also much greater. In all intermediate species, the bundle sheath cells contain large numbers of organelles. Numerous mitochondria, the peroxisomes and many of the chloroplasts are located centripetally in the bundle sheath cells. The mitochondria are found along the cell wall adjacent to the vascular tissue and are overlain by the chloroplasts. Quantitative studies have shown that the mitochondria and peroxisomes are four times more abundant per unit cell area than in adjacent mesophyll cells and that these mitochondria have twice the profile area of those in the mesophyll

Fig. 8. Leaf anatomy in a C3-C4 intermediate species. Note the concentric layer of not welldeveloped bundle sheath cells (large hexagons) surrounded by not concentrically-arranged

Although some of the C3-C4 species, notably in *Flaveria* and *Moricandia*, do not have very well developed Kranz anatomy, they all exhibit a tendency to partition more cells to the bundle sheath and to concentrate organelles in bundle sheath cells. The tendency to partition organelles to the bundle sheath was not accomplished in a parallel way in the various C3-C4 species. The small bundle sheath cells in *Neurachne minor*, for example, resulted in only 5% of the total cell profile area being in the bundle sheath. But the high concentration of organelles in bundle sheath cells compensated for their small size. In other C3-C4 species, increased partitioning of organelles in bundle sheath cells compared to C3 species resulted from both higher organelle concentrations and increased bundle sheath cells size and/or number relative to mesophyll cells (Brown and Hattersley, 1989; McKown and Dengler, 2007, 2009). In addition, C3-C4 intermediate species plasmodesmatal densities at the bundle sheath/mesophyll interface approach those of C4 species and are much greater

Photosynthetic rates of C3 and C3-C4 intermediate species are comparable in a range of light and atmospheric gas compositions, but the responses of gas exchange parameters which

(Brown and Hattersley, 1989; McKown and Dengler, 2007, 2009).

mesophyll cells (small hexagons).

than those of the C3 species studied (Brown et al, 1983).

**8.2 Leaf gas exchange in C3-C4 intermediate species** 

groups (Sage, 2004). Naturally occurring species with photosynthetic characteristics intermediate between C3 and C4 plants have been identified in the genera *Eleucharis*  (Cyperaceae), *Panicum* (Poaceae), *Neurachne* (Poaceae), *Mollugo* (Aizoaceae), *Moricandia* (Brassicaceae), *Flaveria*, (Asteraceae) *Partheniurn* (Asteraceae), Salsola (Chenopodiaceae), Heliotropium (Boraginaceae) and *Alternanthera* (Amaranthaceae) (Brown and Hattersley 1989; Rawsthorne, 1992; Voznesenskaya et al., 2001; Muhaidat, 2007). All of these genera include C3 species and most also include C4 species.

The intermediate nature of these species is reflected in the isotopic composition (13), CO2 compensation point () as well as in the differential distribution of organelles in the bundle sheath cells (Table 2).


Table 2. Main characteristics of C3-C4 species from various genera showing the intermediate nature of these species.

Intermediate species are also recognized in their CO2 net assimilation rate as a function of intercellular CO2 concentration and in the CO2 compensation point as a function of O2 concentration in the medium (Fig. 7).

Fig. 7. Generalized curves for net assimilation rate (left) and compensation point (right) of CO2 in C3, C4 and C3-C4 intermediate species.

#### **8.1 Leaf anatomy**

C3-C4 species have anatomical characteristics between those of C3 and C4. The vascular bundles are surrounded by chlorenchymatous bundle sheath cells reminiscent of the Kranz anatomy of leaves of C4 plants (Fig. 8). However, the mesophyll cells are not in a concentric

groups (Sage, 2004). Naturally occurring species with photosynthetic characteristics intermediate between C3 and C4 plants have been identified in the genera *Eleucharis*  (Cyperaceae), *Panicum* (Poaceae), *Neurachne* (Poaceae), *Mollugo* (Aizoaceae), *Moricandia* (Brassicaceae), *Flaveria*, (Asteraceae) *Partheniurn* (Asteraceae), Salsola (Chenopodiaceae), Heliotropium (Boraginaceae) and *Alternanthera* (Amaranthaceae) (Brown and Hattersley 1989; Rawsthorne, 1992; Voznesenskaya et al., 2001; Muhaidat, 2007). All of these genera

The intermediate nature of these species is reflected in the isotopic composition (13), CO2 compensation point () as well as in the differential distribution of organelles in the bundle

> Organelles in bundle sheath cells (%) Chloroplasts Mitochondria

+Peroxisomes

Γ (µmol mol-1)

C3 ~ −30 48−62 9-11 8-19 C3−C4 ~ −28 9−17 13-25 25-52 C4 ~ −15 3−5 28-53 30-74

Table 2. Main characteristics of C3-C4 species from various genera showing the intermediate

Intermediate species are also recognized in their CO2 net assimilation rate as a function of intercellular CO2 concentration and in the CO2 compensation point as a function of O2

Fig. 7. Generalized curves for net assimilation rate (left) and compensation point (right) of

C3-C4 species have anatomical characteristics between those of C3 and C4. The vascular bundles are surrounded by chlorenchymatous bundle sheath cells reminiscent of the Kranz anatomy of leaves of C4 plants (Fig. 8). However, the mesophyll cells are not in a concentric

include C3 species and most also include C4 species.

δ 13Value (‰)

sheath cells (Table 2).

nature of these species.

concentration in the medium (Fig. 7).

CO2 in C3, C4 and C3-C4 intermediate species.

**8.1 Leaf anatomy** 

Photosynthetic

type

ring around the bundle sheath cells as in a C4 leaf, but are arranged as in leaves of C3 species where interveinal distances are also much greater. In all intermediate species, the bundle sheath cells contain large numbers of organelles. Numerous mitochondria, the peroxisomes and many of the chloroplasts are located centripetally in the bundle sheath cells. The mitochondria are found along the cell wall adjacent to the vascular tissue and are overlain by the chloroplasts. Quantitative studies have shown that the mitochondria and peroxisomes are four times more abundant per unit cell area than in adjacent mesophyll cells and that these mitochondria have twice the profile area of those in the mesophyll (Brown and Hattersley, 1989; McKown and Dengler, 2007, 2009).

Fig. 8. Leaf anatomy in a C3-C4 intermediate species. Note the concentric layer of not welldeveloped bundle sheath cells (large hexagons) surrounded by not concentrically-arranged mesophyll cells (small hexagons).

Although some of the C3-C4 species, notably in *Flaveria* and *Moricandia*, do not have very well developed Kranz anatomy, they all exhibit a tendency to partition more cells to the bundle sheath and to concentrate organelles in bundle sheath cells. The tendency to partition organelles to the bundle sheath was not accomplished in a parallel way in the various C3-C4 species. The small bundle sheath cells in *Neurachne minor*, for example, resulted in only 5% of the total cell profile area being in the bundle sheath. But the high concentration of organelles in bundle sheath cells compensated for their small size. In other C3-C4 species, increased partitioning of organelles in bundle sheath cells compared to C3 species resulted from both higher organelle concentrations and increased bundle sheath cells size and/or number relative to mesophyll cells (Brown and Hattersley, 1989; McKown and Dengler, 2007, 2009). In addition, C3-C4 intermediate species plasmodesmatal densities at the bundle sheath/mesophyll interface approach those of C4 species and are much greater than those of the C3 species studied (Brown et al, 1983).

#### **8.2 Leaf gas exchange in C3-C4 intermediate species**

Photosynthetic rates of C3 and C3-C4 intermediate species are comparable in a range of light and atmospheric gas compositions, but the responses of gas exchange parameters which

Photosynthetic Carbon Metabolism: Plasticity and Evolution 387

evolution in response to environmental change (Furbank et al., 2000; Sage, 2001; Keeley and

Molecular phylogenies indicate that grasses were the first C4 plants, arising about 24–34 million yr ago. Chenopods were probably the first C4 dicots, appearing 15 –20 million yr ago. By 12–14 million yr ago, C4 grasses were abundant enough to leave detectable fossil and isotopic signatures. By the end of the Miocene, C4-dominated grasslands expanded across many of the low latitude regions of the globe, and temperate C4 grasslands were

Rubisco and the C3 mode of photosynthesis evolved early in the history of life and apparently were so successful that competing forms of net photosynthetic carbon fixation have gone extinct. In high CO2 atmospheres, Rubisco operates relatively efficiently. However, the active site chemistry that carboxylates RuBP can also oxygenate i.e. photorespiration. In the current atmosphere, photorespiration can inhibit photosynthesis by over 30% at warmer temperatures (> 30°C). Evolving a Rubisco that is free of oxygenase activity also appears unlikely because the active site biochemistry is constrained by similarities in the oxygenase and carboxylase reactions. In the absence of further improvements to Rubisco, the other solution to the photorespiratory problem is to enhance the stromal concentration of CO2 or to reduce O2. Reducing O2 is unlikely due to unfavorable energetics. Increasing CO2 around Rubisco by 1000 ppm would nearly eliminate oxygenase activity, and under circumstances of high photorespiration could justify the additional energy costs required to operate a CO2 pump (von Caemmerer and

PEPC is the other major carboxylase in C3 plants. In its current configuration, however, PEP carboxylation does not allow for net CO2 fixation into carbohydrate, because the carbon added to PEP is lost as CO2 in the Krebs cycle. For PEPC to evolve into a net carboxylating enzyme, fundamental rearrangements in carbon flow would also be required, while the existing role of PEPC would have to be protected or replaced in some manner (Sage, 2004). Instead of evolving novel enzymes, CO2 concentration requires changes in the kinetics, regulatory set points, and tissue specificity of existing enzymes. This pattern of exploiting existing biochemistry rather than inventing new enzymes is the general rule in complex trait evolution. Given these considerations, it is no surprise that the primary means of compensating for photorespiration in land plants has been the layering of C4 metabolism over existing C3 metabolism. All C4 plants operate a complete C3 cycle, so in this sense the C4 pathway supplements, rather than replaces, C3 photosynthesis. Because it uses existing biochemistry, the evolutionary trough that must be crossed to produce a C4 plant is relatively shallow, and could be bridged by a modest series of incremental steps (Furbank et

C4 photosynthesis has been described as an adaptation to hot and dry environments or to CO2 deficiency. These views, however, have been challenged in recent publications. C4 plants do not appear to be any more drought-adapted than C3 species from arid zones and a diverse flora of C4 grasses occurs in the tropical wetland habitats. In addition, there is a disparity between the timing of C4 expansion across the earth and the appearance of low atmospheric CO2. C4-dominated ecosystems expanded 5 and 10 million yr ago, but no obvious shift in CO2 has been documented for this period (Cerling, 1999). Indeed, C4

al., 2000; Sage, 2001; Keeley and Rundel 2003; Sage, 2004; Sage et al., 2011).

**9.1 Effect of environmental factors on C4** 

Rundel 2003; Sage, 2004; Sage et al., 2011).

present by 5 million yr ago (Cerling et al., 1999).

Furbank, 2003).

provide a measure of photorespiratory activity differ widely between these two photosynthetic groups. In contrast to C3 plants where Γ is essentially unaffected by light intensity, Γ is strongly light-dependent in C3-C4 intermediate species. There is no evidence that the oxygenation reaction of Rubisco was itself being suppressed to any major extent by a C4-like mechanism. Whereas about 50% of the photorespiratory CO2 of a C3 leaf is recaptured before it escapes from the leaf, it was estimated that up to 73% is recaptured in a C3-C4 leaf. Clearly, the improved recapture of CO2 could account for a low Γ in C3-C4 species but a mechanism was required to explain how this improvement occurred (Hunt et al., 1987; Sudderth et al., 2007).

#### **8.3 Biochemical mechanisms in C3-C4 intermediate species**

Because of the intermediate nature of Γ and the somewhat C4-like leaf anatomy of the C3-C4 species, many researchers attempted to show that these species had a partially functional C4 cycle which accounted for their low rates of photorespiration and hence Γ. However, there is now good evidence that C3-C4 intermediates in the genera *Alternanthera, Moricandia, Panicurn* and *Parthenium* do not have a C4 cycle which could account for their low rates of photorespiration. Activities of PEPC and the C4 cycle decarboxylases are far lower than in C4 leaves, and Rubisco and PEPC are both present in mesophyll and bundle sheath cells. Label from 14CO2 is not transferred from C4 compounds to Calvin cycle intermediates during photosynthesis. There was clearly another explanation for low apparent photorespiration in these species. Since gas exchange measurements indicated that CO2 was being extensively recaptured via photosynthesis, and the unusual leaf anatomy was at least in part consistent with this mechanism, the location of the photorespiratory pathway in leaves of the C3-C4 species has been examined (Rawsthorne, 1992)*.* 

It was shown that, the differential distribution of glycine decarboxylase is a major key to the unusual photorespiratory metabolism and Γ of C3-C4 intermediate species. This enzyme is abundant in the mitochondria of leaves of higher plants but is only detected at very low levels in mitochondria from other tissues. Glycine decarboxylase has four heterologous subunits (P, H, T, and L) which catalyse, in association with serine hydroxymethyltransferase, the metabolism of glycine to serine, CO2 and ammonia. The P, H, T, and L subunits are all required for activity of gdc but the P subunit catalyses the decarboxylation of glycine. Immunocytological and in-situ hybridization studies have shown that the P subunit, is absent from the mesophyll mitochondria and the expression of the P subunit gene in the mesophyll is specifically prevented in the leaves of C3-C4 intermediate species. It seems likely, therefore, that the differential distribution of glycine decarboxylase must contribute to the observed reduction in apparent photorespiration in the C3-C4 species (Rawsthorne, 1992; Yoshimura et al., 2004).

#### **9. Evolution of C4 photosynthesis**

C4 photosynthesis is a series of biochemical and anatomical modifications that concentrate CO2 around the carboxylating enzyme Rubisco. Many variations of C4 photosynthesis exist, reflecting at least 45 independent origins in 19 families of higher plants. C4 photosynthesis is present in about 7500 species of flowering plants, or some 3% of the estimated 250 000 land plant species. Most C4 plants are grasses (4500 species), followed by sedges (1500 species) and dicots (1200 species). C4 photosynthesis is an excellent model for complex trait

provide a measure of photorespiratory activity differ widely between these two photosynthetic groups. In contrast to C3 plants where Γ is essentially unaffected by light intensity, Γ is strongly light-dependent in C3-C4 intermediate species. There is no evidence that the oxygenation reaction of Rubisco was itself being suppressed to any major extent by a C4-like mechanism. Whereas about 50% of the photorespiratory CO2 of a C3 leaf is recaptured before it escapes from the leaf, it was estimated that up to 73% is recaptured in a C3-C4 leaf. Clearly, the improved recapture of CO2 could account for a low Γ in C3-C4 species but a mechanism was required to explain how this improvement occurred (Hunt et al., 1987;

Because of the intermediate nature of Γ and the somewhat C4-like leaf anatomy of the C3-C4 species, many researchers attempted to show that these species had a partially functional C4 cycle which accounted for their low rates of photorespiration and hence Γ. However, there is now good evidence that C3-C4 intermediates in the genera *Alternanthera, Moricandia, Panicurn* and *Parthenium* do not have a C4 cycle which could account for their low rates of photorespiration. Activities of PEPC and the C4 cycle decarboxylases are far lower than in C4 leaves, and Rubisco and PEPC are both present in mesophyll and bundle sheath cells. Label from 14CO2 is not transferred from C4 compounds to Calvin cycle intermediates during photosynthesis. There was clearly another explanation for low apparent photorespiration in these species. Since gas exchange measurements indicated that CO2 was being extensively recaptured via photosynthesis, and the unusual leaf anatomy was at least in part consistent with this mechanism, the location of the photorespiratory pathway in leaves of the C3-C4

It was shown that, the differential distribution of glycine decarboxylase is a major key to the unusual photorespiratory metabolism and Γ of C3-C4 intermediate species. This enzyme is abundant in the mitochondria of leaves of higher plants but is only detected at very low levels in mitochondria from other tissues. Glycine decarboxylase has four heterologous subunits (P, H, T, and L) which catalyse, in association with serine hydroxymethyltransferase, the metabolism of glycine to serine, CO2 and ammonia. The P, H, T, and L subunits are all required for activity of gdc but the P subunit catalyses the decarboxylation of glycine. Immunocytological and in-situ hybridization studies have shown that the P subunit, is absent from the mesophyll mitochondria and the expression of the P subunit gene in the mesophyll is specifically prevented in the leaves of C3-C4 intermediate species. It seems likely, therefore, that the differential distribution of glycine decarboxylase must contribute to the observed reduction in apparent photorespiration in

C4 photosynthesis is a series of biochemical and anatomical modifications that concentrate CO2 around the carboxylating enzyme Rubisco. Many variations of C4 photosynthesis exist, reflecting at least 45 independent origins in 19 families of higher plants. C4 photosynthesis is present in about 7500 species of flowering plants, or some 3% of the estimated 250 000 land plant species. Most C4 plants are grasses (4500 species), followed by sedges (1500 species) and dicots (1200 species). C4 photosynthesis is an excellent model for complex trait

**8.3 Biochemical mechanisms in C3-C4 intermediate species** 

species has been examined (Rawsthorne, 1992)*.* 

the C3-C4 species (Rawsthorne, 1992; Yoshimura et al., 2004).

**9. Evolution of C4 photosynthesis** 

Sudderth et al., 2007).

evolution in response to environmental change (Furbank et al., 2000; Sage, 2001; Keeley and Rundel 2003; Sage, 2004; Sage et al., 2011).

Molecular phylogenies indicate that grasses were the first C4 plants, arising about 24–34 million yr ago. Chenopods were probably the first C4 dicots, appearing 15 –20 million yr ago. By 12–14 million yr ago, C4 grasses were abundant enough to leave detectable fossil and isotopic signatures. By the end of the Miocene, C4-dominated grasslands expanded across many of the low latitude regions of the globe, and temperate C4 grasslands were present by 5 million yr ago (Cerling et al., 1999).

Rubisco and the C3 mode of photosynthesis evolved early in the history of life and apparently were so successful that competing forms of net photosynthetic carbon fixation have gone extinct. In high CO2 atmospheres, Rubisco operates relatively efficiently. However, the active site chemistry that carboxylates RuBP can also oxygenate i.e. photorespiration. In the current atmosphere, photorespiration can inhibit photosynthesis by over 30% at warmer temperatures (> 30°C). Evolving a Rubisco that is free of oxygenase activity also appears unlikely because the active site biochemistry is constrained by similarities in the oxygenase and carboxylase reactions. In the absence of further improvements to Rubisco, the other solution to the photorespiratory problem is to enhance the stromal concentration of CO2 or to reduce O2. Reducing O2 is unlikely due to unfavorable energetics. Increasing CO2 around Rubisco by 1000 ppm would nearly eliminate oxygenase activity, and under circumstances of high photorespiration could justify the additional energy costs required to operate a CO2 pump (von Caemmerer and Furbank, 2003).

PEPC is the other major carboxylase in C3 plants. In its current configuration, however, PEP carboxylation does not allow for net CO2 fixation into carbohydrate, because the carbon added to PEP is lost as CO2 in the Krebs cycle. For PEPC to evolve into a net carboxylating enzyme, fundamental rearrangements in carbon flow would also be required, while the existing role of PEPC would have to be protected or replaced in some manner (Sage, 2004).

Instead of evolving novel enzymes, CO2 concentration requires changes in the kinetics, regulatory set points, and tissue specificity of existing enzymes. This pattern of exploiting existing biochemistry rather than inventing new enzymes is the general rule in complex trait evolution. Given these considerations, it is no surprise that the primary means of compensating for photorespiration in land plants has been the layering of C4 metabolism over existing C3 metabolism. All C4 plants operate a complete C3 cycle, so in this sense the C4 pathway supplements, rather than replaces, C3 photosynthesis. Because it uses existing biochemistry, the evolutionary trough that must be crossed to produce a C4 plant is relatively shallow, and could be bridged by a modest series of incremental steps (Furbank et al., 2000; Sage, 2001; Keeley and Rundel 2003; Sage, 2004; Sage et al., 2011).

#### **9.1 Effect of environmental factors on C4**

C4 photosynthesis has been described as an adaptation to hot and dry environments or to CO2 deficiency. These views, however, have been challenged in recent publications. C4 plants do not appear to be any more drought-adapted than C3 species from arid zones and a diverse flora of C4 grasses occurs in the tropical wetland habitats. In addition, there is a disparity between the timing of C4 expansion across the earth and the appearance of low atmospheric CO2. C4-dominated ecosystems expanded 5 and 10 million yr ago, but no obvious shift in CO2 has been documented for this period (Cerling, 1999). Indeed, C4

Photosynthetic Carbon Metabolism: Plasticity and Evolution 389

photosynthesis as a functional type should not be threatened by CO2 rise in the near term

Evolution was not directed towards C4 photosynthesis, and each step had to be stable, either by improving fitness or at a minimum by having little negative effect on survival of the genotype. The predominant mechanisms in the evolution of C4 genes are proposed to be gene duplication followed by nonfunctionalization and neofunctionalization (Monson, 1999, 2003), and alteration of *cis*-regulatory elements in single copy genes to change expression patterns (Rosche and Westhoff, 1995). Major targets for non- and neofunctionalization are the promoter and enhancer region of genes to allow for altered expression and compartmentalization, and the coding region to alter regulatory and catalytic properties. Both non- and neofunctionalization can come about through mutations, crossover events, and insertions of mobile elements (Kloeckener-Gruissem and Freeling, 1995; Lynch & Conery, 2000). A model for C4 evolution has been presented that recognizes seven

The term Kranz anatomy is commonly used to describe the dual-cell system associated with C4 photosynthesis, consisting of mesophyll cells containing PEPC and initial reactions of C4 biochemistry, and bundle sheath cells containing enzymes for generating CO2 from C4 acids and the C3 carbon reduction pathway, including Rubisco. Kranz anatomy is an elegant evolutionary solution to separating the processes, and for more than three decades it was considered a requirement for the function of C4 photosynthesis in terrestrial plants

This paradigm was broken when two species, *Borszczowia aralocaspica* and *Bienertia cycloptera*, both representing monotypic genera of the family Chenopodiaceae, were shown to have C4 photosynthesis within a single cell without the presence of Kranz anatomy (Voznesenskaya et al., 2001; Sage, 2002b; Edwards and Voznesenskaya, 2011). *Borszczowia* grows in central Asia from northeast of the Caspian lowland east to Mongolia and western China, whereas *Bienertia* grows from east Anatolia eastward to Turkmenistan and Pakistani

Single-cell C4 plants can capture CO2 effectively from Rubisco without Kranz anatomy and the bundle sheath cell wall barrier. Photosynthesis in the single-cell systems is not inhibited by O2, even under low atmospheric levels of CO2, and their carbon isotope values are the same as in Kranz-type C4 plants, whereas the values would be more negative if there were leakage of CO2 and overcycling through the C4 pathway (Voznesenskaya et al., 2001;

*Borszczowia* has a single layer of elongate, cylindrical chlorenchyma cells below the epidermal and hypodermal layers, which surround the veins and internal water storage tissue. The cells are tightly packed together with intercellular space restricted to the end of the cells closest to the epidermis. The anatomy of *Bienertia* leaves with respect to photosynthetic tissue is very different in that there are two to three layers of shorter chlorenchyma cells that surround the centrally located water-storage and vascular tissue in the leaf. The cells are loosely arranged, with considerable intercellular space around them

(Sage, 2004).

**9.2 Evolutionary pathways to C4 photosynthesis** 

significant phases (Sage, 2004) (Table 3).

**10. Single cell C4 photosynthesis** 

(Edwards et al., 2001).

Baluchestan (Akhani et al., 2003).

Edwards and Voznesenskaya, 2011).

(Edwards et al., 2004).

photosynthesis is not a specific drought, salinity or low-CO2 adaptation, but it as an adaptation that compensates for high rates of photorespiration and carbon deficiency. In this context, all environmental factors that enhance photorespiration and reduce carbon balance are responsible for evolution of C4 photosynthesis. Heat, drought, salinity and low CO2 are the most important factors, but others, such as flooding, could also stimulate photorespiration under certain conditions (Sage, 2004).

#### **9.1.1 Heat. Salinity and drought**

High temperature is a major environmental requirement for C4 evolution because it directly stimulates photorespiration and dark respiration in C3 plants. The availability of CO2 as a substrate also declines at elevated temperature due to reduced solubility of CO2 relative to O2. Aridity and salinity are important because they promote stomatal closure and thus reduce intercellular CO2 level, again stimulating photorespiration and aggravating a CO2 substrate deficiency. Relative humidity is particularly low in hot, arid regions, which will further reduce stomatal conductance, particularly if the plant is drought stressed. The combination of drought, salinity, low humidity and high temperature produces the greatest potential for photorespiration and CO2 deficiency (Ehleringer and Monson, 1993), so it is not surprising that these environments are where C4 photosynthesis would most frequently arise. Many C3-C4 intermediates are from arid or saline zones, for example intermediate species of *Heliotropium, Salsola*, *Neurachne*, *Alternanthera* and a number of the *Flaveria*  intermediates (Sage, 2004).

C4 photosynthesis may have evolved in moist environments as well, which can be consistent with the carbon-balance hypothesis if environmental conditions are hot enough to promote photorespiration. The sedge lineages largely occur in low-latitude wetlands, indicating they may have evolved on flooded soils and the aquatic C4 species certainly evolved in wet environments (Bowes et al., 2002). In the case of the aquatic, single-celled C4 species, warm shallow ponds typically become depleted in CO2 during the day when photosynthetic activity from algae and macrophytes is high. Many of the C3-C4 intermediates such as *Flaveria linearis, Mollugo verticillata* also occur in moist, disturbed habitats such as riverbanks, roadsides and abandoned fields indicate that disturbance is also an important factor in C4 evolution, particularly for lineages that may have arisen in wetter locations (Monson 1989).

#### **9.1.2 Low CO2 concentration**

In recent geological time, low CO2 prevailed in the earth's atmosphere. For about a fifth of the period of past 400 000 yr, CO2 was below 200 ppm. Because low CO2 prevailed in recent geological time, discussions of C4 evolution must consider selection pressures in atmospheres with less CO2 than today. In low CO2, C3 photosynthesis is impaired by the lack of CO2 as a substrate in addition to enhanced photorespiration (Ehleringer, 2005). As a result, water and nitrogen-use efficiencies and growth rates are low, competitive ability and fecundity is reduced and recovery from disturbance is slow (Ward, 2005). There is a strong additive effect between heat, drought and salinity and CO2 depletion, so that, the inhibitory effects of heat, drought and salinity increase considerably in low CO2.

Manipulation of the biosphere by human and increases in atmospheric CO2 could halt the rise of new C4 life forms and may lead to the reduction of existing ones (Edwards *et al*., 2001). However, certain C4 species are favored by other global change variables such as climate warming and deforestation. Hence, while many C4 species may be at risk, C4

photosynthesis is not a specific drought, salinity or low-CO2 adaptation, but it as an adaptation that compensates for high rates of photorespiration and carbon deficiency. In this context, all environmental factors that enhance photorespiration and reduce carbon balance are responsible for evolution of C4 photosynthesis. Heat, drought, salinity and low CO2 are the most important factors, but others, such as flooding, could also stimulate

High temperature is a major environmental requirement for C4 evolution because it directly stimulates photorespiration and dark respiration in C3 plants. The availability of CO2 as a substrate also declines at elevated temperature due to reduced solubility of CO2 relative to O2. Aridity and salinity are important because they promote stomatal closure and thus reduce intercellular CO2 level, again stimulating photorespiration and aggravating a CO2 substrate deficiency. Relative humidity is particularly low in hot, arid regions, which will further reduce stomatal conductance, particularly if the plant is drought stressed. The combination of drought, salinity, low humidity and high temperature produces the greatest potential for photorespiration and CO2 deficiency (Ehleringer and Monson, 1993), so it is not surprising that these environments are where C4 photosynthesis would most frequently arise. Many C3-C4 intermediates are from arid or saline zones, for example intermediate species of *Heliotropium, Salsola*, *Neurachne*, *Alternanthera* and a number of the *Flaveria* 

C4 photosynthesis may have evolved in moist environments as well, which can be consistent with the carbon-balance hypothesis if environmental conditions are hot enough to promote photorespiration. The sedge lineages largely occur in low-latitude wetlands, indicating they may have evolved on flooded soils and the aquatic C4 species certainly evolved in wet environments (Bowes et al., 2002). In the case of the aquatic, single-celled C4 species, warm shallow ponds typically become depleted in CO2 during the day when photosynthetic activity from algae and macrophytes is high. Many of the C3-C4 intermediates such as *Flaveria linearis, Mollugo verticillata* also occur in moist, disturbed habitats such as riverbanks, roadsides and abandoned fields indicate that disturbance is also an important factor in C4 evolution, particularly for lineages that may have arisen in wetter locations (Monson 1989).

In recent geological time, low CO2 prevailed in the earth's atmosphere. For about a fifth of the period of past 400 000 yr, CO2 was below 200 ppm. Because low CO2 prevailed in recent geological time, discussions of C4 evolution must consider selection pressures in atmospheres with less CO2 than today. In low CO2, C3 photosynthesis is impaired by the lack of CO2 as a substrate in addition to enhanced photorespiration (Ehleringer, 2005). As a result, water and nitrogen-use efficiencies and growth rates are low, competitive ability and fecundity is reduced and recovery from disturbance is slow (Ward, 2005). There is a strong additive effect between heat, drought and salinity and CO2 depletion, so that, the inhibitory

Manipulation of the biosphere by human and increases in atmospheric CO2 could halt the rise of new C4 life forms and may lead to the reduction of existing ones (Edwards *et al*., 2001). However, certain C4 species are favored by other global change variables such as climate warming and deforestation. Hence, while many C4 species may be at risk, C4

effects of heat, drought and salinity increase considerably in low CO2.

photorespiration under certain conditions (Sage, 2004).

**9.1.1 Heat. Salinity and drought** 

intermediates (Sage, 2004).

**9.1.2 Low CO2 concentration** 

photosynthesis as a functional type should not be threatened by CO2 rise in the near term (Sage, 2004).

#### **9.2 Evolutionary pathways to C4 photosynthesis**

Evolution was not directed towards C4 photosynthesis, and each step had to be stable, either by improving fitness or at a minimum by having little negative effect on survival of the genotype. The predominant mechanisms in the evolution of C4 genes are proposed to be gene duplication followed by nonfunctionalization and neofunctionalization (Monson, 1999, 2003), and alteration of *cis*-regulatory elements in single copy genes to change expression patterns (Rosche and Westhoff, 1995). Major targets for non- and neofunctionalization are the promoter and enhancer region of genes to allow for altered expression and compartmentalization, and the coding region to alter regulatory and catalytic properties. Both non- and neofunctionalization can come about through mutations, crossover events, and insertions of mobile elements (Kloeckener-Gruissem and Freeling, 1995; Lynch & Conery, 2000). A model for C4 evolution has been presented that recognizes seven significant phases (Sage, 2004) (Table 3).

#### **10. Single cell C4 photosynthesis**

The term Kranz anatomy is commonly used to describe the dual-cell system associated with C4 photosynthesis, consisting of mesophyll cells containing PEPC and initial reactions of C4 biochemistry, and bundle sheath cells containing enzymes for generating CO2 from C4 acids and the C3 carbon reduction pathway, including Rubisco. Kranz anatomy is an elegant evolutionary solution to separating the processes, and for more than three decades it was considered a requirement for the function of C4 photosynthesis in terrestrial plants (Edwards et al., 2001).

This paradigm was broken when two species, *Borszczowia aralocaspica* and *Bienertia cycloptera*, both representing monotypic genera of the family Chenopodiaceae, were shown to have C4 photosynthesis within a single cell without the presence of Kranz anatomy (Voznesenskaya et al., 2001; Sage, 2002b; Edwards and Voznesenskaya, 2011). *Borszczowia* grows in central Asia from northeast of the Caspian lowland east to Mongolia and western China, whereas *Bienertia* grows from east Anatolia eastward to Turkmenistan and Pakistani Baluchestan (Akhani et al., 2003).

Single-cell C4 plants can capture CO2 effectively from Rubisco without Kranz anatomy and the bundle sheath cell wall barrier. Photosynthesis in the single-cell systems is not inhibited by O2, even under low atmospheric levels of CO2, and their carbon isotope values are the same as in Kranz-type C4 plants, whereas the values would be more negative if there were leakage of CO2 and overcycling through the C4 pathway (Voznesenskaya et al., 2001; Edwards and Voznesenskaya, 2011).

*Borszczowia* has a single layer of elongate, cylindrical chlorenchyma cells below the epidermal and hypodermal layers, which surround the veins and internal water storage tissue. The cells are tightly packed together with intercellular space restricted to the end of the cells closest to the epidermis. The anatomy of *Bienertia* leaves with respect to photosynthetic tissue is very different in that there are two to three layers of shorter chlorenchyma cells that surround the centrally located water-storage and vascular tissue in the leaf. The cells are loosely arranged, with considerable intercellular space around them (Edwards et al., 2004).

Photosynthetic Carbon Metabolism: Plasticity and Evolution 391

Fig. 9. Model of proposed function of C4 photosynthesis in the two types of single cell systems in *Borszczowia* (A) and *Bienertia* (B). Note that chloroplasts are in two distinct

**A CO B 2**

A model has been proposed for the operation of C4 photosynthesis in a single chlorenchyma cell in *Borszczowia* and *Bienertia* (Edwards et al., 2004; Edwards and Voznesenskaya, 2011). In *Borszczowia*, atmospheric CO2 enters the chlorenchyma cell at the distal end, which is surrounded by intercellular air space. Here, the carboxylation phase of the C4 pathway assimilates atmospheric CO2 into C4 acids. Two key enzymes in the process are pyruvate-Pi dikinase (PPDK), located in chloroplasts at the proximal part and PEPC, located in the cytosol. The C4 acids diffuse to the proximal part of the cell through a thin, cytoplasmic space at the periphery of the middle of the cell, which is devoid of organelles. In the proximal end, the C4 acids are decarboxylated by NAD-malic enzyme (NAD-ME) in mitochondria that appear to be localized exclusively in this part of the cell. The CO2 is captured by Rubisco that is localized exclusively in chloroplasts surrounding the

In *Bienertia* there is a similar concept of organelle partitioning in a single cell to operate the C4 process. However, it has a very different compartmentation scheme (Fig. 9B). Atmospheric CO2 enters the cell around the periphery, which is exposed to considerable intercellular air space, and here the carboxylation phase of the C4 pathway functions to convert pyruvate and CO2 into OAA through the combined action of PPDK in the chloroplast and PEPC in the cytosol. C4 acids diffuse to the central cytoplasmic compartment through cytoplasmic channels and are decarboxylated by NAD-ME in mitochondria, which are specifically and abundantly located there. Chloroplasts in the central cytoplasmic compartment surround the mitochondria and fix the CO2 by Rubisco,

cytoplasmic compartments.

mitochondria in the proximal part of the cell (Fig. 9A).


Table 3. The main evolutionary pathways towards C4 photosynthesis (Adapted from Sage, 2004).

first arose in grasses and is prolific in this family.

genetically isolated populations.

in CO2 compensation points.

**Modification of the gene copies without losing the original function:** multiplication of genes by duplication → selection and screen for adaptive functions in the short-lived annuals and perennials → reproductive barriers →

**Increase in bundle sheath organelles:** the number of chloroplasts and mitochondria in the bundle sheath increases in order to maintain

photosynthetic capacity in leaves with enlarged BSC→ increased capacity of BSC to process glycine from the mesophyll → subsequent development of a photorespiratory CO2 pump → further increase in organelle number → greater growth and fecundity in high photorespiratory environments → maintaining incremental rise in BSC organelle content → significant reduction

**Changes in the glycine decarboxylase (GDC) genes:** duplication of GDC genes, production of distinct operations with separate promoters in the MC and BSC → loss of function mutation in the MC GDC → movement of glycine from MC to the BSC to prevent lethal accumulation of photorespiratory

**Enhancement of PEPC activity in the MC**: reorganization of expression pattern of enzymes: specific expression of C4 cycle enzymes in the MC and localization of Rubisco in BSC, increase in the activity of carboxylating enzymes: NADP-ME, NAD-ME through increasing transcriptional intensity,

**Avoidance of competition between PEPC and Rubisco in the MC for CO2 and ATP increase in the phases of C4 cycle:** further reorganization of the expression pattern of enzymes: reduction in the carbonic anhydrase activity in chloroplasts of BSC for preventing its conversion to bicarbonate and its diffusion out of the cell without being fixed by Rubisco, increase in the cytosol of MC to support high PEPC activity → large gradient of CO2 between BSC

**Selection for traits that allow plants to exploit the productive potential of** 

(1) Optimization of NADP-ME regulation in the earlier phases of C4 evolution: increase in the specific activity of NADP-ME and reduction of Km for malate. (2) Optimization of PEPC in the final stages of C4 evolution: reduction of sensitivity of PEPC to malate, increased sensitivity to the activator glucose-6-

(3) Optimization of Rubisco: evolving into a higher catalytic capacity but lower

(4) Improvement of water-use efficiency: increased stomatal sensitivity to CO2 and light→ enhancing the ability of stomata to respond to environmental variation at relatively low conductances, reduction of leaf specific hydraulic

products → subsequent selection for efficient glycine shuttle.

and MC, reduction of MC Rubisco activity in the later stages.

**the C4 pathway to the maximum:** adjustment and optimization of photosynthetic efficiency, kinetic properties and regulatory set-points of enzymes to compensate for changes in the metabolic environment:

phosphate, increased affinity for bicarbonate and reduced for PEP.

conductivity by increasing leaf area per unit of conducting tissue.

increased PPDK activity in the later stages.

specificity with no negative consequences.

Table 3. The main evolutionary pathways towards C4 photosynthesis (Adapted from Sage,

**Decline of distance between mesophyll (MC) and bundle sheath cells (BSC) for rapid diffusion of metabolites:** reduction of interveinal distance and enhancement of BSC layer size → adaptive traits without relationship with photosynthesis: improvement of structural integrity in windy locations and enhancement of water status of the leaf in hot environments → selection. Easier reduction of MC and BSC distance in species with parallel venation (grasses) than in species with reticulate venation (dicots) → C4 photosynthesis

**Stage Events**

**General Preconditioning**

**Anatomical Preconditioning**

**Creating Metabolic Sink for Glycine Metabolism and C4 Acids** 

**Glycine Shuttles and Photorespiratory CO2 Pumps**

**Efficient Scavenging of CO2 Escaping from** 

**Integration of C3 and** 

**Optimization and Whole-Plant Coordination**

**the BSC** 

**C4 Cycles**

2004).

Fig. 9. Model of proposed function of C4 photosynthesis in the two types of single cell systems in *Borszczowia* (A) and *Bienertia* (B). Note that chloroplasts are in two distinct cytoplasmic compartments.

A model has been proposed for the operation of C4 photosynthesis in a single chlorenchyma cell in *Borszczowia* and *Bienertia* (Edwards et al., 2004; Edwards and Voznesenskaya, 2011). In *Borszczowia*, atmospheric CO2 enters the chlorenchyma cell at the distal end, which is surrounded by intercellular air space. Here, the carboxylation phase of the C4 pathway assimilates atmospheric CO2 into C4 acids. Two key enzymes in the process are pyruvate-Pi dikinase (PPDK), located in chloroplasts at the proximal part and PEPC, located in the cytosol. The C4 acids diffuse to the proximal part of the cell through a thin, cytoplasmic space at the periphery of the middle of the cell, which is devoid of organelles. In the proximal end, the C4 acids are decarboxylated by NAD-malic enzyme (NAD-ME) in mitochondria that appear to be localized exclusively in this part of the cell. The CO2 is captured by Rubisco that is localized exclusively in chloroplasts surrounding the mitochondria in the proximal part of the cell (Fig. 9A).

In *Bienertia* there is a similar concept of organelle partitioning in a single cell to operate the C4 process. However, it has a very different compartmentation scheme (Fig. 9B). Atmospheric CO2 enters the cell around the periphery, which is exposed to considerable intercellular air space, and here the carboxylation phase of the C4 pathway functions to convert pyruvate and CO2 into OAA through the combined action of PPDK in the chloroplast and PEPC in the cytosol. C4 acids diffuse to the central cytoplasmic compartment through cytoplasmic channels and are decarboxylated by NAD-ME in mitochondria, which are specifically and abundantly located there. Chloroplasts in the central cytoplasmic compartment surround the mitochondria and fix the CO2 by Rubisco,

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which is only present in the chloroplasts of this compartment, through the C3 cycle (Edwards et al., 2004; Edwards and Voznesenskaya, 2011).

Single-cell C4 photosynthesis could simply be an alternative mechanism to Kranz type C4 photosynthesis. Although it may be equally complex in its control of compartmentation of functions, is less complex in that it does not require the cooperative function of two cell types, nor does it require development of Kranz anatomy. Single-cell C4 allows more flexibility in mode of photosynthesis than Kranz-type C4 plants by, for example, shifting from C3 to C4 depending on environmental conditions (Edwards et al., 2004; Edwards and Voznesenskaya, 2011).

#### **11. Conclusion**

Life on earth largely depends on the photosynthetic carbon fixation using light energy. Energy-rich sugar molecules are the basis of many growth and developmental processes in plants. Reduced carbon products in the leaves, however, are used not only for synthesis of carbohydrates but also in a number of primary and secondary metabolic pathways in plants including nitrogen assimilation, fatty acid synthesis and phenolic metabolism.

Photosynthetic carbon assimilation is an investment of resources and the extent of this investment responds to the economy of the whole plant. Maintenance of energy homeostasis requires sophisticated and flexible regulatory mechanisms to account for the physiological and developmental plasticity observed in plants. It this regard, sugars not only are the prime carbon and energy sources for plants, but also play a pivotal role as a signaling molecule that control metabolism, stress response, growth, and development of plants.

Environmental factors determine the distribution and abundance of plants and evolutionary adaptation is an inevitable response to environmental change. Throughout the course of geological time, the environments in which plants grew have been changing, often radically and irreversibly. Physiological adaptation to environmental variables cannot improve without associated changes in morphology and anatomy. Evolution of C4 plants is an excellent example of parallel evolution of leaf physiology and anatomy. Finally, any physiological evolution must be associated with changes at biochemical and molecular level. This chapter provides an introduction to theses area with a focus on plasticity in the carbon metabolism and evolution of variants of the carbon assimilation pathways.

#### **12. References**


which is only present in the chloroplasts of this compartment, through the C3 cycle

Single-cell C4 photosynthesis could simply be an alternative mechanism to Kranz type C4 photosynthesis. Although it may be equally complex in its control of compartmentation of functions, is less complex in that it does not require the cooperative function of two cell types, nor does it require development of Kranz anatomy. Single-cell C4 allows more flexibility in mode of photosynthesis than Kranz-type C4 plants by, for example, shifting from C3 to C4 depending on environmental conditions (Edwards et al., 2004; Edwards and

Life on earth largely depends on the photosynthetic carbon fixation using light energy. Energy-rich sugar molecules are the basis of many growth and developmental processes in plants. Reduced carbon products in the leaves, however, are used not only for synthesis of carbohydrates but also in a number of primary and secondary metabolic pathways in plants

Photosynthetic carbon assimilation is an investment of resources and the extent of this investment responds to the economy of the whole plant. Maintenance of energy homeostasis requires sophisticated and flexible regulatory mechanisms to account for the physiological and developmental plasticity observed in plants. It this regard, sugars not only are the prime carbon and energy sources for plants, but also play a pivotal role as a signaling molecule that control metabolism, stress response, growth, and development of plants. Environmental factors determine the distribution and abundance of plants and evolutionary adaptation is an inevitable response to environmental change. Throughout the course of geological time, the environments in which plants grew have been changing, often radically and irreversibly. Physiological adaptation to environmental variables cannot improve without associated changes in morphology and anatomy. Evolution of C4 plants is an excellent example of parallel evolution of leaf physiology and anatomy. Finally, any physiological evolution must be associated with changes at biochemical and molecular level. This chapter provides an introduction to theses area with a focus on plasticity in the carbon

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**Part 4** 

**Special Topics in Photosynthesis** 


## **Part 4**

**Special Topics in Photosynthesis** 

400 Advances in Photosynthesis – Fundamental Aspects

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ISSN 0022-0957

**19** 

**Photosynthetic Adaptive Strategies in** 

**Evergreen and Semi-Deciduous Species** 

**of Mediterranean Maquis During Winter** 

Mediterranean-type ecosystems are characterised by a particular temperature and rainfall regime that limits plant growth in both summer and winter seasons (Mitrakos, 1980; Larcher, 2000). Mediterranean plant community is very heterogeneous and include many evergreen and semi-deciduous species that present a complex mixture of elements, some deriving from *in situ* evolution, others having colonized the area from adjacent regions in different periods in the past (Blondel & Aronson 1999; Gratani & Varone, 2004). The result of this evolution is that the Mediterranean maquis species are well adapted to environmental stress conditions and successfully overcome them (Sànchez-Blanco et al., 2002; Varone &

Structural and physiological adaptations consist in a mixture of characteristics that make these species very resistant to stresses. High leaf consistency, leaf tissue density, leaf thickness, and reduced leaf area are traits improving drought resistance by decreasing photochemical damages to the photosynthetic system (Abril & Hanano 1998; Castro-Díez et

In this study we have focused our attention on photosynthetic adaptive strategies in Mediterranean evergreen and semi-deciduous species subjected to winter temperatures. Winter depression of photosynthetic activity, occurring between December and February, is the consequence of low temperatures which are responsible for slowing down metabolic

Under these conditions, photosynthetic performance may decline and may be restored when the environmental conditions become favourable for growth in spring (Larcher, 2000; Oliveira & Peñuelas, 2004). The combination of low temperatures and high light, may induce a reduction in photochemical efficiency, increasing the sensitivity of photosystems to photoinhibition (Powles, 1984). Mediterranean plant communities comprise many evergreen and semi-deciduous species that cope with winter cold through different strategies that include biochemical, physiological, anatomical and cytological modifications (Huner et al., 1981; Boese & Huner 1990; Long et al., 1994; Oliveira & Peñuelas, 2000; Tattini et al., 2000).

processes and cessation of growth (Rhizopoulou et al., 1989; Larcher, 2000).

**1. Introduction** 

Gratani, 2007).

al. 1998; Gratani & Ghia 2002).

Carmen Arena1 and Luca Vitale2

*University of Naples Federico II,* 

*(ISAFoM – CNR)* 

*Italy* 

*1Department of Structural and Functional Biology,* 

*2Istituto per I Sistemi Agricoli e Forestali del Mediterraneo,* 

### **Photosynthetic Adaptive Strategies in Evergreen and Semi-Deciduous Species of Mediterranean Maquis During Winter**

Carmen Arena1 and Luca Vitale2

*1Department of Structural and Functional Biology, University of Naples Federico II, 2Istituto per I Sistemi Agricoli e Forestali del Mediterraneo, (ISAFoM – CNR) Italy* 

#### **1. Introduction**

Mediterranean-type ecosystems are characterised by a particular temperature and rainfall regime that limits plant growth in both summer and winter seasons (Mitrakos, 1980; Larcher, 2000). Mediterranean plant community is very heterogeneous and include many evergreen and semi-deciduous species that present a complex mixture of elements, some deriving from *in situ* evolution, others having colonized the area from adjacent regions in different periods in the past (Blondel & Aronson 1999; Gratani & Varone, 2004). The result of this evolution is that the Mediterranean maquis species are well adapted to environmental stress conditions and successfully overcome them (Sànchez-Blanco et al., 2002; Varone & Gratani, 2007).

Structural and physiological adaptations consist in a mixture of characteristics that make these species very resistant to stresses. High leaf consistency, leaf tissue density, leaf thickness, and reduced leaf area are traits improving drought resistance by decreasing photochemical damages to the photosynthetic system (Abril & Hanano 1998; Castro-Díez et al. 1998; Gratani & Ghia 2002).

In this study we have focused our attention on photosynthetic adaptive strategies in Mediterranean evergreen and semi-deciduous species subjected to winter temperatures.

Winter depression of photosynthetic activity, occurring between December and February, is the consequence of low temperatures which are responsible for slowing down metabolic processes and cessation of growth (Rhizopoulou et al., 1989; Larcher, 2000).

Under these conditions, photosynthetic performance may decline and may be restored when the environmental conditions become favourable for growth in spring (Larcher, 2000; Oliveira & Peñuelas, 2004). The combination of low temperatures and high light, may induce a reduction in photochemical efficiency, increasing the sensitivity of photosystems to photoinhibition (Powles, 1984). Mediterranean plant communities comprise many evergreen and semi-deciduous species that cope with winter cold through different strategies that include biochemical, physiological, anatomical and cytological modifications (Huner et al., 1981; Boese & Huner 1990; Long et al., 1994; Oliveira & Peñuelas, 2000; Tattini et al., 2000).

Photosynthetic Adaptive Strategies in Evergreen and

**Evergreens** 

**2.1 The experimental planning schema** 

**2. Material and methods** 

**Spring (SO)** 

**Spring (SO)** 

**Winter (WO)** 

*C. incanus* **Chlorophyll** *a* **fluorescence** 

First experiment. Two years old plants of *Q. ilex*, *P. angustifolia* and *L. nobilis* coming from the garden centre of Corpo Forestale dello Stato of Sabaudia (Latina, Italy) were transplanted in 15 L pots in January 2004 and placed outdoor in the Botanical Garden of Naples University for one year. Pots were large enough to avoid limitations in root growth and were filled with a mixture of peat and soil in the proportion 50:50. The temperature conditions at the experimental site during plant growth were typical of the Mediterranean region with cold winters and warm summers (Fig. 2A). Gas exchange and chlorophyll *a* fluorescence measurements were performed in winter (early March 2005) and in spring (during May 2005); for winter measurements, 8 mature leaves of one year old and 8 young

**Winter (WO)** 

examined. The experimental planning of the work is reported in Fig. 1.

*Q. ilex P. angustifolia L. nobilis*

**Semideciduous** 

Fig. 1. The experimental planning of the work.

**2.2 Plant material and growth conditions** 

Semi-Deciduous Species of Mediterranean Maquis During Winter 405

Two different experiments have been considered in this study; the first experiment has been carried out on evergreens *L. nobilis*, *P. angustifolia* and *Q. ilex* and analyzes the photosynthetic and the photochemical performance of young and mature leaves during the winter and of mature leaves during the winter and following spring. The second experiment is focused on the photochemical behaviour of young and mature leaves of the semideciduous species *C. incanus* during winter and of mature leaves during the winter and the following spring. It is well know that the *C. incanus* species produces two different typologies of leaves: winter leaves and summer leaves with dissimilar morpho-anatomical traits (Aronne & De Micco, 2001). In the present study only winter leaves have been

> **Gas exchange and chlorophyll**  *a* **fluorescence measurements on mature leaves**

**measurements, pigment content, leaf traits determination on mature leaves** 

 **Chlorophyll** *a* **fluorescence measurements, pigment content, leaf traits determination on young and mature leaves**

**Gas exchange and chlorophyll**  *a* **fluorescence measurements on young and mature leaves** 

The chilling-induced photosynthetic decline can be attributed both to a reduced activity of enzymes involved in the photosynthetic carbon reduction cycle (Sassenrath et al., 1990; Hutchinson et al., 2000), or to a photoinhibitory process. In fact, when chilling is protracted for a long time, the reduction of carbon assimilation can lead to an increase of excitation energy to reaction centres, that if not safely dissipated, induces damages at photosystems level compromising the whole photosynthetic apparatus (Baker, 1994; Tjus et al., 1998). However, in nature, the photosynthetic decrease as well as the reduction of photochemical activity at low temperatures, often represent a regulatory mechanism associated with photoprotective strategies that promote the dissipation of excess excitation energy avoiding irreversible damages to photosystems (Long et al., 1994; D'Ambrosio et al., 2006). Several mechanisms have evolved in plants in order to protect photosystems against photodamages; they include thermal dissipation, chloroplasts movements, chlorophyll concentration changes, increases in the capacity for scavenging the active oxygen species and the PSII ability to transfer electrons to acceptors different from CO2 (Niyogi, 2000).

It has been reported that the resistance of Mediterranean maquis evergreen species to photoinhibition is associated mainly to the increase in scavenging capacity and thermal dissipation processess, as well as to the increment of carotenoids pool or reduction in chlorophyll content (Garcìa-Plazaola et al., 1999, 2000; Arena et al., 2008). On the other hand, the semi-deciduous species such as *Cistus* rely on pheno-morphological features such as short lifetime of leaves and leaf pubescence to protect leaves from the excess of light and, thus, reduce the investment in other physiological mechanisms (Werner et al., 1999; Oliveira & Peñuelas, 2001, 2002, 2004). Previous studies have demonstrated that the resistance to environmental constraints such as low temperature or high irradiance can depend on leaf age (Shirke, 2001; Bertamini & Nedunchezhian, 2003). Young and mature leaves may differ both in photosynthetic performance and some leaf functional traits such as the sclerophylly index LMA (leaf mass per area) and its opposite leaf specific area (SLA), leaf dry matter content (LDMC) and relative water content (RWC). These properties affect significantly the whole plant physiology. More specifically, LMA variations are linked to biomass allocation strategies (Wilson et al., 1999) and to photosynthetic acclimation under different conditions, RWC is a good indicator to evaluate the plant water status (Cornelissen et al., 2003; Teulat et al., 1997) and LDMC represent an index of resource use by plant (Garnier et al., 2001). LDMC is related to leaf lifespan and it is involved in the trade-off between the quick production of biomass and the efficient conservation of nutrients (Poorter & Garnier, 1999; Ryser & Urbas, 2000). Generally young leaves appears more vulnerable than mature leaves to stress, since have a reduced degree of xeromorphism (lower LMA). In this chapter has been examined the photosynthetic and photochemical behaviour of young and mature leaves of different species of the Mediterranean maquis, grown during the winter, in response to low temperatures. In particular our attention has been focused on the evergreen species *Laurus nobilis* L., *Phillyrea angustifolia* L. and *Quercus ilex* L. and on the semi-deciduous species *Cistus incanus* L. that are widespread in Southern Italy area. Our specific purposes were: 1) to focus on eco-physiological strategies adopted by the different species to optimize the carbon gain during winter and minimize the photoinhibitory damage risks; 2) to compare the behaviour of young and mature leaves under low winter temperature in order to elucidate if the photoprotective mechanisms may be influenced by the leaf age.

#### **2. Material and methods**

404 Advances in Photosynthesis – Fundamental Aspects

The chilling-induced photosynthetic decline can be attributed both to a reduced activity of enzymes involved in the photosynthetic carbon reduction cycle (Sassenrath et al., 1990; Hutchinson et al., 2000), or to a photoinhibitory process. In fact, when chilling is protracted for a long time, the reduction of carbon assimilation can lead to an increase of excitation energy to reaction centres, that if not safely dissipated, induces damages at photosystems level compromising the whole photosynthetic apparatus (Baker, 1994; Tjus et al., 1998). However, in nature, the photosynthetic decrease as well as the reduction of photochemical activity at low temperatures, often represent a regulatory mechanism associated with photoprotective strategies that promote the dissipation of excess excitation energy avoiding irreversible damages to photosystems (Long et al., 1994; D'Ambrosio et al., 2006). Several mechanisms have evolved in plants in order to protect photosystems against photodamages; they include thermal dissipation, chloroplasts movements, chlorophyll concentration changes, increases in the capacity for scavenging the active oxygen species and the PSII

It has been reported that the resistance of Mediterranean maquis evergreen species to photoinhibition is associated mainly to the increase in scavenging capacity and thermal dissipation processess, as well as to the increment of carotenoids pool or reduction in chlorophyll content (Garcìa-Plazaola et al., 1999, 2000; Arena et al., 2008). On the other hand, the semi-deciduous species such as *Cistus* rely on pheno-morphological features such as short lifetime of leaves and leaf pubescence to protect leaves from the excess of light and, thus, reduce the investment in other physiological mechanisms (Werner et al., 1999; Oliveira & Peñuelas, 2001, 2002, 2004). Previous studies have demonstrated that the resistance to environmental constraints such as low temperature or high irradiance can depend on leaf age (Shirke, 2001; Bertamini & Nedunchezhian, 2003). Young and mature leaves may differ both in photosynthetic performance and some leaf functional traits such as the sclerophylly index LMA (leaf mass per area) and its opposite leaf specific area (SLA), leaf dry matter content (LDMC) and relative water content (RWC). These properties affect significantly the whole plant physiology. More specifically, LMA variations are linked to biomass allocation strategies (Wilson et al., 1999) and to photosynthetic acclimation under different conditions, RWC is a good indicator to evaluate the plant water status (Cornelissen et al., 2003; Teulat et al., 1997) and LDMC represent an index of resource use by plant (Garnier et al., 2001). LDMC is related to leaf lifespan and it is involved in the trade-off between the quick production of biomass and the efficient conservation of nutrients (Poorter & Garnier, 1999; Ryser & Urbas, 2000). Generally young leaves appears more vulnerable than mature leaves to stress, since have a reduced degree of xeromorphism (lower LMA). In this chapter has been examined the photosynthetic and photochemical behaviour of young and mature leaves of different species of the Mediterranean maquis, grown during the winter, in response to low temperatures. In particular our attention has been focused on the evergreen species *Laurus nobilis* L., *Phillyrea angustifolia* L. and *Quercus ilex* L. and on the semi-deciduous species *Cistus incanus* L. that are widespread in Southern Italy area. Our specific purposes were: 1) to focus on eco-physiological strategies adopted by the different species to optimize the carbon gain during winter and minimize the photoinhibitory damage risks; 2) to compare the behaviour of young and mature leaves under low winter temperature in order to elucidate if the photoprotective mechanisms may be influenced by the leaf age.

ability to transfer electrons to acceptors different from CO2 (Niyogi, 2000).

Two different experiments have been considered in this study; the first experiment has been carried out on evergreens *L. nobilis*, *P. angustifolia* and *Q. ilex* and analyzes the photosynthetic and the photochemical performance of young and mature leaves during the winter and of mature leaves during the winter and following spring. The second experiment is focused on the photochemical behaviour of young and mature leaves of the semideciduous species *C. incanus* during winter and of mature leaves during the winter and the following spring. It is well know that the *C. incanus* species produces two different typologies of leaves: winter leaves and summer leaves with dissimilar morpho-anatomical traits (Aronne & De Micco, 2001). In the present study only winter leaves have been examined. The experimental planning of the work is reported in Fig. 1.

#### **2.1 The experimental planning schema**

Fig. 1. The experimental planning of the work.

#### **2.2 Plant material and growth conditions**

First experiment. Two years old plants of *Q. ilex*, *P. angustifolia* and *L. nobilis* coming from the garden centre of Corpo Forestale dello Stato of Sabaudia (Latina, Italy) were transplanted in 15 L pots in January 2004 and placed outdoor in the Botanical Garden of Naples University for one year. Pots were large enough to avoid limitations in root growth and were filled with a mixture of peat and soil in the proportion 50:50. The temperature conditions at the experimental site during plant growth were typical of the Mediterranean region with cold winters and warm summers (Fig. 2A). Gas exchange and chlorophyll *a* fluorescence measurements were performed in winter (early March 2005) and in spring (during May 2005); for winter measurements, 8 mature leaves of one year old and 8 young

Photosynthetic Adaptive Strategies in Evergreen and

in spring only mature leaves were considered.

from 3 different specimens.

in Table 1.

Semi-Deciduous Species of Mediterranean Maquis During Winter 407

chosen for photochemical measurements and photosynthetic pigments and leaf functional traits determinations. At the end of April, mature leaves were analysed again and compared to mature leaves in winter on basis of chlorophyll *a* fluorescence measurements, chlorophyll content and leaf functional traits determinations. All analyses were carried out on six leaves

In the first experiment, on the three evergreen species, gas exchange and chlorophyll *a* fluorescence measurements were performed simultaneously in winter (March 2008) and in spring (May 2005) by a portable gas exchange system (HCM-1000, Walz, Germany) in a climatized cuvette equipped with a fiber optic connected with a portable pulse amplitude modulated fluorometer (Mini-PAM, Walz, Germany). All measurements were performed at midday under clear-sky conditions. In winter both young and mature leaves were analysed,

In the second experiment, on the semi-deciduous species *C. incanus* L. measurements of chlorophyll *a* fluorescence were performed by a portable pulse amplitude modulated fluorometer (Mini-PAM, Walz, Germany) equipped with a leaf-clip holder (Leaf-Clip Holder 2030-B, Walz, Germany), which allows the simultaneous recording of the incident photosynthetic photon flux density on the leaf and abaxial leaf temperature. Measurements were performed at midday, under natural light and temperature conditions, on young and mature leaves, during winter (February 2008), and on mature leaves during spring (April 2008). The air temperature (Tair) and the Photosynthetic Photon Flux Densities (PPFD) experienced by *C. incanus* leaves at midday, during the days of measurements, are reported

**measurements Young leaves Mature leaves** 

**T air** 2 Feb 2008 12 ± 0.12 11 ± 0.22 29 Apr 2008 - 22 ± 0.05

**PPFD** 2 Feb 2008 693 ± 28 712 ± 29

Table 1. Air temperature (Tair, °C) and Photosynthetic photon flux density (PPFD, mol photons m-2 s-1) measured at midday on *C. incanus* plants at the experimental site in the days

29 Apr 2008 - 1074 ± 28

For gas exchange measurements, each leaf has been kept in cuvette for 5-6 min. The acquisition of data was made when steady-state rate of net assimilation was achieved. A constant photosynthetic photon flux density (PPFD) of 1000 mol photons m-2 s-1 was provided to the leaves by an external light source (1050-H, Walz, Germany) positioned on the cuvette plane. The PPFD of 1000 mol photons m-2 s-1 was selected in order to obtain the

Net photosynthetic rate (AN), stomatal conductance to water (gH2O) and intercellular CO2 concentration (*C*i) were calculated by the software operating in HCM-1000 using the von

**2.3 Gas exchange and chlorophyll a fluorescence measurements** 

**Days of** 

of measurements. Data reported are means ± SE (n=6).

values of light-saturated net photosynthetic rate for each species.

leaves sprouted in late October of the previous year, were selected randomly for each species from 4 different plants. The photosynthetic behaviour of one-year old leaves in winter was compared with that of one-year old leaves in spring.

Second experiment. In November 2007, eight plants of *C. incanus*, of three years old, were collected in the field in the Castel Volturno Natural reserve (Naples, Italy). The climate of the reserve is typically Mediterranean, with dry summers and rainy autumns and winters. The main vegetation type is maquis often opening into garrigue formations dominated by evergreen sclerophylls and seasonally dimorphic species.

The collected plants were excavated *in situ* and quickly transplanted in 15 L pots filled with native soil, then were carried to the Department of Structural and Functional Biology of Naples University and placed outdoors in a open area of the Department. The temperature conditions experienced by plants during growth are shown in Fig. 2B. Outdoor temperatures at the experimental site, during the experimental period, ranged between minimum values of 2 °C and maximum values of 16 °C.

Fig. 2. Monthly mean air temperature (T °C) at the two experimental sites during the evergreens growth (A) and *C. incanus* growth (B). Data have been collected from Naples Largo San Marcellino weather station.

At the beginning of February, three healthy plants were selected for eco-physiological analyses; young leaves of about 15 days old and mature leaves of about 30 days old were

leaves sprouted in late October of the previous year, were selected randomly for each species from 4 different plants. The photosynthetic behaviour of one-year old leaves in

Second experiment. In November 2007, eight plants of *C. incanus*, of three years old, were collected in the field in the Castel Volturno Natural reserve (Naples, Italy). The climate of the reserve is typically Mediterranean, with dry summers and rainy autumns and winters. The main vegetation type is maquis often opening into garrigue formations dominated by

The collected plants were excavated *in situ* and quickly transplanted in 15 L pots filled with native soil, then were carried to the Department of Structural and Functional Biology of Naples University and placed outdoors in a open area of the Department. The temperature conditions experienced by plants during growth are shown in Fig. 2B. Outdoor temperatures at the experimental site, during the experimental period, ranged between

Fig. 2. Monthly mean air temperature (T °C) at the two experimental sites during the evergreens growth (A) and *C. incanus* growth (B). Data have been collected from Naples

**Jan- 08**

**Dec**

At the beginning of February, three healthy plants were selected for eco-physiological analyses; young leaves of about 15 days old and mature leaves of about 30 days old were

**Feb**

**Mar**

**Apr**

Largo San Marcellino weather station.

**T °C**

winter was compared with that of one-year old leaves in spring.

evergreen sclerophylls and seasonally dimorphic species.

minimum values of 2 °C and maximum values of 16 °C.

A

**Jan- 04Feb**

B

**Nov- 07**

**0**

**5**

**10**

**15**

**20**

**T °C**

**Mar**

**Apr**

**MayJun**

**Jul**

**Aug**

**SepOct**

**Nov**

**Dec Jan- 05**

**Feb Mar**

**Apr**

**May** 

chosen for photochemical measurements and photosynthetic pigments and leaf functional traits determinations. At the end of April, mature leaves were analysed again and compared to mature leaves in winter on basis of chlorophyll *a* fluorescence measurements, chlorophyll content and leaf functional traits determinations. All analyses were carried out on six leaves from 3 different specimens.

#### **2.3 Gas exchange and chlorophyll a fluorescence measurements**

In the first experiment, on the three evergreen species, gas exchange and chlorophyll *a* fluorescence measurements were performed simultaneously in winter (March 2008) and in spring (May 2005) by a portable gas exchange system (HCM-1000, Walz, Germany) in a climatized cuvette equipped with a fiber optic connected with a portable pulse amplitude modulated fluorometer (Mini-PAM, Walz, Germany). All measurements were performed at midday under clear-sky conditions. In winter both young and mature leaves were analysed, in spring only mature leaves were considered.

In the second experiment, on the semi-deciduous species *C. incanus* L. measurements of chlorophyll *a* fluorescence were performed by a portable pulse amplitude modulated fluorometer (Mini-PAM, Walz, Germany) equipped with a leaf-clip holder (Leaf-Clip Holder 2030-B, Walz, Germany), which allows the simultaneous recording of the incident photosynthetic photon flux density on the leaf and abaxial leaf temperature. Measurements were performed at midday, under natural light and temperature conditions, on young and mature leaves, during winter (February 2008), and on mature leaves during spring (April 2008). The air temperature (Tair) and the Photosynthetic Photon Flux Densities (PPFD) experienced by *C. incanus* leaves at midday, during the days of measurements, are reported in Table 1.


Table 1. Air temperature (Tair, °C) and Photosynthetic photon flux density (PPFD, mol photons m-2 s-1) measured at midday on *C. incanus* plants at the experimental site in the days of measurements. Data reported are means ± SE (n=6).

For gas exchange measurements, each leaf has been kept in cuvette for 5-6 min. The acquisition of data was made when steady-state rate of net assimilation was achieved. A constant photosynthetic photon flux density (PPFD) of 1000 mol photons m-2 s-1 was provided to the leaves by an external light source (1050-H, Walz, Germany) positioned on the cuvette plane. The PPFD of 1000 mol photons m-2 s-1 was selected in order to obtain the values of light-saturated net photosynthetic rate for each species.

Net photosynthetic rate (AN), stomatal conductance to water (gH2O) and intercellular CO2 concentration (*C*i) were calculated by the software operating in HCM-1000 using the von

Photosynthetic Adaptive Strategies in Evergreen and

tissues (Cornelissen et al., 2003).

**gH2O (mmol m-2 s-1)**

**AN (mol m-2 s-1)**

**Ci/Ca**

**a,A**

**0**

**2**

**a,A**

B

**a,A a,A**

**b,A**

**4**

**6**

**8**

A

**0.6**

**0.0**

**0.2**

**0.4**

**3. Results** 

**during winter** 

Semi-Deciduous Species of Mediterranean Maquis During Winter 409

(LDMC) measurements. Specific leaf area was calculed as the ratio of leaf area to leaf dry mass and expressed as cm2 g -1 dw (dry weight). For dry mass determination, leaves were dried at 70 °C for 48 h. Leaf dry matter content (LDMC) was measured as the oven-dry mass of a leaf divided by its water-saturated fresh mass and expressed as g g -1 wslm (water saturated leaf mass). Leaf dry matter content is related to the average density of the leaf

**3.1 Young and mature leaves of** *L. nobilis* **L.,** *P. angustifolia* **L. and** *Quercus ilex* **L.**

*L. nobilis P. angustifolia Q. ilex*

D

**c,A**

**a,A**

**a,A a,A a,A**

C F

Fig. 3. Net photosynthetic rate (AN), stomatal conductance to water (gH2O) and ratio of intercellular to ambient CO2 concentration (Ci/Ca) in young and mature leaves of *Laurus nobilis*, *Phillyrea angustifolia* and *Quercus ilex*, during winter. Different letters indicate statistical differences between young and mature leaves (small letters) and among species

(capital letters). Values are means ± SD (n=8).

During winter, mature leaves of all species showed an higher (P<0.001) net photosynthetic rate (AN) compared to young leaves. In both young and mature leaves the highest (P<0.001) AN values were measured in *Q. ilex* whereas the lowest (P<0.001) in *L. nobilis* (Fig. 3A, D).

**Young Mature**

**b,B**

**b,A b,A**

**b,A b,A**

**a,B**

**a,A**

E

**0**

**0.6**

**0.0**

**0.2**

**0.4**

**Ci/Ca**

**2**

**4**

**AN (mol m-2 s-1)**

**gH2O (mmol m-2 s-1)**

**6**

**c,B**

**8**

Caemmerer and Farquhar equations (1981). The ratio of intercellular to ambient CO2 concentration, Ci/Ca, was used to calculate the apparent carboxylation efficiency.

As concerns chlorophyll *a* fluorescence measurements, in the early morning, on 30 min dark-adapted leaves, the background fluorescence signal, Fo, was induced by light of about 0.5 mol photons m-2 s-1 at the frequency of 0.6 kHz. In order to determine the maximal fluorescence level in the dark-adapted state, Fm, a 1s saturating light pulse (10000 mol photons m-2 s-1) was applied by previously setting the frequency at 20 kHz; the maximum PSII photochemical efficiency (Fv/Fm) was calculated as:

$$\left[\mathbf{F}\_{\mathrm{v}}/\mathrm{F}\_{\mathrm{m}} = (\mathbf{F}\_{\mathrm{m}}\mathrm{-F}\_{\mathrm{o}})/\mathrm{F}\_{\mathrm{m}}\right]$$

The saturating pulse intensity was chosen in order to saturate the fluorescence yield but avoiding photoinhibition during the pulse.

At midday, the steady-state fluorescence signal (Ft) and the maximal fluorescence (Fm') under illumination were measured, setting the light measure at a frequency of 20 kHz. Fm' was determined by a 1s saturating light pulse (10000 mol photons m-2 s-1). The partitioning of absorbed light energy was calculated following the model of Kramer et al. (2004). The quantum yield of PSII linear electron transport (PSII) was estimated following Genty et al. (1989) as:

$$
\Phi\_{\rm FSI} = (\rm F\_m \rm - \rm F\_t) / \rm F\_m \hat{\bf }
$$

The yields of regulated energy dissipation was calculated as:

$$\left(\bullet\right)\_{\mathrm{NPQ}} = 1 \,\mathrm{-}\,\Phi\_{\mathrm{PSII}} \,\mathrm{-}1/\,\mathrm{(NPQ} + 1 + \mathrm{q}\_{\mathrm{L}} \times \mathrm{(F\_m/F\_o-1)}\right)$$

whereas the non-regulated energy dissipation in PSII was calculated as:

$$\mathbf{Q\_{NO}} = \mathbf{1}/(\mathbf{NPQ} + \mathbf{1} + \mathbf{q\_L} \times (\mathbf{F\_m}/\mathbf{F\_o} \mathbf{-1})) $$

The coefficient of photochemical quenching, qL, was defined and calculated following Kramer et al. (2004) as:

$$\left(\mathbf{F\_m}' - \mathbf{F\_t}\right) / \left(\mathbf{F\_m}' - \mathbf{F\_o}'\right) \times \mathbf{F\_o}' / \mathbf{F\_t} = \mathbf{q\_p} \times \mathbf{F\_o}' / \mathbf{F\_t}$$

The value of Fo' was estimated as: Fo' = Fo/(Fv/Fm + Fo/Fm') (Oxborough & Baker, 1997). Non-photochemical quenching was expressed according to Bilger & Björkman (1990) as:

$$\left[\mathrm{NIPQ} = (\mathrm{F\_{m^{-}}F\_{m}}')/\mathrm{F\_{m}}'\right]$$

The statistical analysis of the data was performed by one-way ANOVA followed by Student-Newman-Keuls test (*Sigma-Stat 3.1*) based on a significance level of P < 0.05. Data are means ± SE (at least n = 6).

#### **2.4 Photosynthetic pigment content and functional leaf traits determination**

After fluorescence measurements, leaves were detached from *C. incanus* plants and carried to the laboratory for the photosynthetic pigment content determination. Pigments were extracted with a mortar and pestle in ice-cold 100% acetone and quantified by a spectrophotometer according to Lichtenthaler (1987). A different group of leaves of comparable age to those used for fluorescence measurements and pigment determinations, was collected and utilized for the specific leaf area (SLA) and leaf dry matter content (LDMC) measurements. Specific leaf area was calculed as the ratio of leaf area to leaf dry mass and expressed as cm2 g -1 dw (dry weight). For dry mass determination, leaves were dried at 70 °C for 48 h. Leaf dry matter content (LDMC) was measured as the oven-dry mass of a leaf divided by its water-saturated fresh mass and expressed as g g -1 wslm (water saturated leaf mass). Leaf dry matter content is related to the average density of the leaf tissues (Cornelissen et al., 2003).

#### **3. Results**

408 Advances in Photosynthesis – Fundamental Aspects

Caemmerer and Farquhar equations (1981). The ratio of intercellular to ambient CO2

As concerns chlorophyll *a* fluorescence measurements, in the early morning, on 30 min dark-adapted leaves, the background fluorescence signal, Fo, was induced by light of about 0.5 mol photons m-2 s-1 at the frequency of 0.6 kHz. In order to determine the maximal fluorescence level in the dark-adapted state, Fm, a 1s saturating light pulse (10000 mol photons m-2 s-1) was applied by previously setting the frequency at 20 kHz; the maximum

[Fv/Fm=(Fm-Fo)/Fm] The saturating pulse intensity was chosen in order to saturate the fluorescence yield but

At midday, the steady-state fluorescence signal (Ft) and the maximal fluorescence (Fm') under illumination were measured, setting the light measure at a frequency of 20 kHz. Fm' was determined by a 1s saturating light pulse (10000 mol photons m-2 s-1). The partitioning of absorbed light energy was calculated following the model of Kramer et al. (2004). The quantum yield of PSII linear electron transport (PSII) was estimated following Genty et al.

ΦNPQ = 1 - ΦPSII - 1/(NPQ + 1 + qL x (Fm/Fo-1)

ΦNO = 1/(NPQ + 1 + qL x (Fm/Fo-1)) The coefficient of photochemical quenching, qL, was defined and calculated following

(Fm' – Ft)/( Fm' – Fo') x Fo'/Ft = qp x Fo'/Ft The value of Fo' was estimated as: Fo' = Fo/(Fv/Fm + Fo/Fm') (Oxborough & Baker, 1997). Non-photochemical quenching was expressed according to Bilger & Björkman (1990) as:

[NPQ = (Fm-Fm')/Fm'] The statistical analysis of the data was performed by one-way ANOVA followed by Student-Newman-Keuls test (*Sigma-Stat 3.1*) based on a significance level of P < 0.05. Data

After fluorescence measurements, leaves were detached from *C. incanus* plants and carried to the laboratory for the photosynthetic pigment content determination. Pigments were extracted with a mortar and pestle in ice-cold 100% acetone and quantified by a spectrophotometer according to Lichtenthaler (1987). A different group of leaves of comparable age to those used for fluorescence measurements and pigment determinations, was collected and utilized for the specific leaf area (SLA) and leaf dry matter content

**2.4 Photosynthetic pigment content and functional leaf traits determination** 

– Ft) Fm'

PSII = (Fm'

concentration, Ci/Ca, was used to calculate the apparent carboxylation efficiency.

PSII photochemical efficiency (Fv/Fm) was calculated as:

The yields of regulated energy dissipation was calculated as:

whereas the non-regulated energy dissipation in PSII was calculated as:

avoiding photoinhibition during the pulse.

(1989) as:

Kramer et al. (2004) as:

are means ± SE (at least n = 6).

#### **3.1 Young and mature leaves of** *L. nobilis* **L.,** *P. angustifolia* **L. and** *Quercus ilex* **L. during winter**

During winter, mature leaves of all species showed an higher (P<0.001) net photosynthetic rate (AN) compared to young leaves. In both young and mature leaves the highest (P<0.001) AN values were measured in *Q. ilex* whereas the lowest (P<0.001) in *L. nobilis* (Fig. 3A, D).

Fig. 3. Net photosynthetic rate (AN), stomatal conductance to water (gH2O) and ratio of intercellular to ambient CO2 concentration (Ci/Ca) in young and mature leaves of *Laurus nobilis*, *Phillyrea angustifolia* and *Quercus ilex*, during winter. Different letters indicate statistical differences between young and mature leaves (small letters) and among species (capital letters). Values are means ± SD (n=8).

Photosynthetic Adaptive Strategies in Evergreen and

same species was observed in Ci/Ca ratio (Fig. 3 C, F).

values of NPQ and NO, respectively, in mature leaves.

F), but similar values of gH2O (Fig. 5E).

observed among species (Fig. 6 D).

Fv/Fm, were observed among species (Fig. 6H).

spring (P<0.005) for all species (Fig. 6D, H).

**and spring** 

Semi-Deciduous Species of Mediterranean Maquis During Winter 411

The ratio of intercellular to ambient CO2 concentration (Ci/Ca) was similar for young leaves of all species, conversely in mature leaves was lower (P<0.001) in *L. nobilis* compared to *P. angustifolia* and *Q. ilex*. No significant difference within young and mature leaves of the

The analysis of photochemistry showed that, among young leaves of different species, the quantum yield of PSII linear electron transport (PSII) was higher (P<0.005) in *P. angustifolia*  and *Q. ilex* compared to *L. nobilis* (Fig. 4A) on the contrary *L. nobilis* showed the highest regulated energy dissipation, NPQ, (P<0.05) and the lowest (P<0.005) non-regulated energy dissipation, NO, compared to other species. No difference was detected in NPQ and NO between *P. angustifolia* and *Q. ilex* (Fig. 4B, C). All mature leaves exhibited no significant difference in PSII (Fig. 4E) but leaves of *L. nobilis* showed again the highest NPQ (P<0.05); the highest (P<0.005) NO was found in *P. angustifolia* (Fig. 4 F, G). No variation in maximum PSII photochemical efficiency (Fv/Fm) among different species and between young and mature leaves were found (Fig. 4 D, H). The comparison between young and mature leaves evidenced no difference in PSII and lower (P<0.001) and higher (P<0.005)

**3.2 Mature leaves of** *L. nobilis* **L.,** *P. angustifolia* **L. and** *Quercus ilex* **L. during winter** 

During winter, within different species, *Q. ilex* showed higher net photosynthetic rate (AN) (P<0.001) and stomatal conductance to water (gH2O) (P<0.05) as well as a lower (P<0.005) intercellular to ambient CO2 concentration ratio (Ci/Ca) compared to *L. nobilis* and *P. angustifolia* (Fig. 5A, B, C). The lowest values of AN and gH2O was found in *L. nobilis*. No significant difference between *L. nobilis* and *P. angustifolia* in Ci/Ca ratio was found. During spring, among species, *Q. ilex* exhibited again the highest (P<0.001) net photosynthetic rate (AN) and the lowest Ci/Ca ratio (P<0.05) compared to *L. nobilis* and *P. angustifolia* (Fig. 5 D,

The comparison between winter and spring showed that, during spring, an increase in AN (P<0.001) and gH2O (P<0.05) were observed in all species compared to winter (Fig. 5D, E); on

In particular, *L. nobilis* showed the lowest (P<0.001) quantum yield of PSII linear electron transport (FPSII) and non-regulated energy dissipation (NO), as well as the highest (P<0.01) regulated energy dissipation (NPQ) (Fig. 6A, B, C). No difference in Fv/Fm values was

During spring, *Q. ilex* and *P. angustifolia* showed an higher (P<0.001) PSII than *L. nobilis* (Fig. 6E). The lowest (P<0.01) NPQ was detected in *Q. ilex*, whereas the highest (P<0.01) FNO was found in *L. nobilis* (Fig. 6F, G). Similar values of maximum PSII photochemical efficiency,

The comparison between the two campaign of measurements has evidenced that in all species FPSII and NPQ were respectively higher and lower (P<0.001) in spring than in winter (Fig. 6A, E, B, F). In spring compared to winter, NO increased (P<0.01) only in *L. nobilis*, whereas decreased (P<0.05) in *P. angustifolia* and remained unvaried in *Q. ilex* (Fig. 6C, G). The maximum PSII photochemical efficiency Fv/Fm was lower in winter as compared to

the other hand, no significant difference in Ci/Ca ratio was found (Fig. 5F). During winter the photochemical performance varied among species (Fig. 6).

Fig. 4. Quantum yield of linear PSII electron transport (PSII), regulated energy dissipation (NPQ), non-regulated energy dissipation (NO) and maximum PSII photochemical efficiency (Fv/Fm) in young and mature leaves of *Laurus nobilis*, *Phillyrea angustifolia* and *Quercus ilex*, during winter. Different letters indicate statistical differences between young and mature leaves (small letters) and among species (capital letters). Values are means ± SD (n=8).

In young leaves, stomatal conductance to water (gH2O) did not differ among the species; on the contrary, in mature leaves gH2O was significantly lower (P<0.01) in *L. nobilis* than *P. angustifolia* and *Q. ilex*. No significant difference in gH2O between young and mature leaves of the same species was measured (Fig. 3B, E).

*L. nobilis P. angustifolia Q. ilex*

**b,A b,A**

A E

**b,A b,A**

B F

**a,A b,A b,A**

D H

C G

**Young Mature**

**a,A a,A a,A**

**a,B b,B b,B**

**a,B a,B b,B**

**PSII**

**NPQ**

**NO**

**Fv/F**

**m**

**0.00**

**0.0**

**0.4**

**0.0**

**0.8**

**0.0**

**0.2**

**0.4**

**0.6**

**0.1**

**0.2**

**0.3**

**0.2**

**0.4**

**0.6**

**0.05**

**0.10**

**0.15**

**0.20**

Fig. 4. Quantum yield of linear PSII electron transport (PSII), regulated energy dissipation (NPQ), non-regulated energy dissipation (NO) and maximum PSII photochemical efficiency (Fv/Fm) in young and mature leaves of *Laurus nobilis*, *Phillyrea angustifolia* and *Quercus ilex*, during winter. Different letters indicate statistical differences between young and mature leaves (small letters) and among species (capital letters). Values are means ± SD (n=8).

**a,A a,A a,A a,A a,A a,A**

In young leaves, stomatal conductance to water (gH2O) did not differ among the species; on the contrary, in mature leaves gH2O was significantly lower (P<0.01) in *L. nobilis* than *P. angustifolia* and *Q. ilex*. No significant difference in gH2O between young and mature leaves

of the same species was measured (Fig. 3B, E).

**NPQ**

**PSII**

**NO**

**Fv/F**

**m**

**0.0**

**0.4**

**0.0**

**0.8**

**0.0**

**0.2**

**0.4**

**0.6**

**0.1**

**0.2**

**0.3**

**0.2**

**0.4**

**0.6**

**0.00**

**0.05**

**0.10**

**a,A**

**a,A**

**0.15**

**0.20**

The ratio of intercellular to ambient CO2 concentration (Ci/Ca) was similar for young leaves of all species, conversely in mature leaves was lower (P<0.001) in *L. nobilis* compared to *P. angustifolia* and *Q. ilex*. No significant difference within young and mature leaves of the same species was observed in Ci/Ca ratio (Fig. 3 C, F).

The analysis of photochemistry showed that, among young leaves of different species, the quantum yield of PSII linear electron transport (PSII) was higher (P<0.005) in *P. angustifolia*  and *Q. ilex* compared to *L. nobilis* (Fig. 4A) on the contrary *L. nobilis* showed the highest regulated energy dissipation, NPQ, (P<0.05) and the lowest (P<0.005) non-regulated energy dissipation, NO, compared to other species. No difference was detected in NPQ and NO between *P. angustifolia* and *Q. ilex* (Fig. 4B, C). All mature leaves exhibited no significant difference in PSII (Fig. 4E) but leaves of *L. nobilis* showed again the highest NPQ (P<0.05); the highest (P<0.005) NO was found in *P. angustifolia* (Fig. 4 F, G). No variation in maximum PSII photochemical efficiency (Fv/Fm) among different species and between young and mature leaves were found (Fig. 4 D, H). The comparison between young and mature leaves evidenced no difference in PSII and lower (P<0.001) and higher (P<0.005) values of NPQ and NO, respectively, in mature leaves.

#### **3.2 Mature leaves of** *L. nobilis* **L.,** *P. angustifolia* **L. and** *Quercus ilex* **L. during winter and spring**

During winter, within different species, *Q. ilex* showed higher net photosynthetic rate (AN) (P<0.001) and stomatal conductance to water (gH2O) (P<0.05) as well as a lower (P<0.005) intercellular to ambient CO2 concentration ratio (Ci/Ca) compared to *L. nobilis* and *P. angustifolia* (Fig. 5A, B, C). The lowest values of AN and gH2O was found in *L. nobilis*. No significant difference between *L. nobilis* and *P. angustifolia* in Ci/Ca ratio was found. During spring, among species, *Q. ilex* exhibited again the highest (P<0.001) net photosynthetic rate (AN) and the lowest Ci/Ca ratio (P<0.05) compared to *L. nobilis* and *P. angustifolia* (Fig. 5 D, F), but similar values of gH2O (Fig. 5E).

The comparison between winter and spring showed that, during spring, an increase in AN (P<0.001) and gH2O (P<0.05) were observed in all species compared to winter (Fig. 5D, E); on the other hand, no significant difference in Ci/Ca ratio was found (Fig. 5F).

During winter the photochemical performance varied among species (Fig. 6).

In particular, *L. nobilis* showed the lowest (P<0.001) quantum yield of PSII linear electron transport (FPSII) and non-regulated energy dissipation (NO), as well as the highest (P<0.01) regulated energy dissipation (NPQ) (Fig. 6A, B, C). No difference in Fv/Fm values was observed among species (Fig. 6 D).

During spring, *Q. ilex* and *P. angustifolia* showed an higher (P<0.001) PSII than *L. nobilis* (Fig. 6E). The lowest (P<0.01) NPQ was detected in *Q. ilex*, whereas the highest (P<0.01) FNO was found in *L. nobilis* (Fig. 6F, G). Similar values of maximum PSII photochemical efficiency, Fv/Fm, were observed among species (Fig. 6H).

The comparison between the two campaign of measurements has evidenced that in all species FPSII and NPQ were respectively higher and lower (P<0.001) in spring than in winter (Fig. 6A, E, B, F). In spring compared to winter, NO increased (P<0.01) only in *L. nobilis*, whereas decreased (P<0.05) in *P. angustifolia* and remained unvaried in *Q. ilex* (Fig. 6C, G). The maximum PSII photochemical efficiency Fv/Fm was lower in winter as compared to spring (P<0.005) for all species (Fig. 6D, H).

Photosynthetic Adaptive Strategies in Evergreen and

**NPQ**

**PSII**

**NO**

**Fv/F**

**m**

**0.0**

**0.4**

**0.0**

**0.8**

**0.0**

**0.2**

**0.4**

**0.6**

**0.1**

**0.2**

**0.3**

**0.2**

**0.4**

**0.6**

**0.0**

**0.1**

**a,A**

**a,A**

**a,A**

**0.2**

**0.3**

**0.4**

Semi-Deciduous Species of Mediterranean Maquis During Winter 413

*L. nobilis P. angustifolia Q. ilex*

**b,A c,A a,B**

A E

B F

C G

D H

**Winter Spring**

**b,A b,A a,B a,B b,B**

**b,A b,A a,B b,B b,A**

**b,B c,B**

**PSII**

**NPQ**

**NO**

**Fv/F**

**m**

**0.0**

**0.6**

**0.0**

**0.4**

**0.0**

**0.8**

**0.0**

**0.2**

**0.4**

**0.6**

**0.1**

**0.2**

**0.3**

**0.2**

**0.4**

**0.1**

**0.2**

**0.3**

**0.4**

Fig. 6. Quantum yield of linear PSII electron transport (PSII), regulated energy dissipation (NPQ), non-regulated energy dissipation (NO) and maximum PSII photochemical efficiency (Fv/Fm) in mature leaves of *Laurus nobilis*, *Phillyrea angustifolia* and *Quercus ilex*, during winter and spring. Different letters indicate statistical differences among species (small

**a,A a,A a,A a,B a,B a,B**

The results relative to leaf functional traits and photosynthetic pigment content are reported in the table 2. The analysis of functional leaf traits has evidenced that, as compared to mature leaves, young leaves showed lower values (P<0.05) of leaf area (LA), but no difference in specific leaf area (SLA) and leaf dry matter content (LDMC). Functional leaf

letters) and between seasons (capital letters). Values are means ± SD (n=8).

Fig. 5. Net photosynthetic rate (AN), stomatal conductance to water (gH2O) and ratio of intercellular to ambient CO2 concentration (Ci/Ca) in mature leaves of *Laurus nobilis*, *Phillyrea angustifolia* and *Quercus ilex*, during winter and spring. Different letters indicate statistical differences among species (small letters) and between seasons (capital letters). Values are means ± SD (n=8).

#### **3.3 The semi-deciduous species** *Cistus incanus* **L.**

The comparison between young and mature leaves of the semi-deciduous species *C. incanus* evidenced that the quantum yield of PSII linear electron transport (PSII) was lower in mature as compared to young leaves (P<0.001) whereas the quantum yield of regulated energy dissipation (NPQ) showed an opposite tendency (P<0.05) (Fig. 7A, B). No significant difference in non regulated energy dissipation (NO) and maximum photochemical efficiency (Fv/Fm) was detected (P<0.05) between the two leaf typologies (Fig. 7C, D).

The photochemical behavior of mature *C. incanus* leaves was different during winter and the following spring. More specifically, in spring leaves showed higher values of PSII (P<0.001) and lower values of NPQ and NO (P<0.005) compared to winter (Fig. 7E, F, G), whereas no significant difference in Fv/Fm between the two seasons was observed (Fig. 7H).

*L. nobilis P. angustifolia Q. ilex*

**c,A a,B**

**c,A a,B a,B**

**a,A**

**b,A**

**Winter Spring**

**b,B**

**0**

**80**

**0**

**0.0**

**0.2**

**0.4**

**Ci/Ca**

**0.6**

**20**

**40**

**gH2O (mmol m-2 s-1)**

**60**

**2**

**4**

**6**

**AN (mol m-2 s-1)**

**8**

**c,B**

**a,A**

**c,A**

**10**

Fig. 5. Net photosynthetic rate (AN), stomatal conductance to water (gH2O) and ratio of intercellular to ambient CO2 concentration (Ci/Ca) in mature leaves of *Laurus nobilis*, *Phillyrea angustifolia* and *Quercus ilex*, during winter and spring. Different letters indicate statistical differences among species (small letters) and between seasons (capital letters).

**b,A**

The comparison between young and mature leaves of the semi-deciduous species *C. incanus* evidenced that the quantum yield of PSII linear electron transport (PSII) was lower in mature as compared to young leaves (P<0.001) whereas the quantum yield of regulated energy dissipation (NPQ) showed an opposite tendency (P<0.05) (Fig. 7A, B). No significant difference in non regulated energy dissipation (NO) and maximum photochemical

The photochemical behavior of mature *C. incanus* leaves was different during winter and the following spring. More specifically, in spring leaves showed higher values of PSII (P<0.001) and lower values of NPQ and NO (P<0.005) compared to winter (Fig. 7E, F, G), whereas no

efficiency (Fv/Fm) was detected (P<0.05) between the two leaf typologies (Fig. 7C, D).

significant difference in Fv/Fm between the two seasons was observed (Fig. 7H).

Values are means ± SD (n=8).

**0.0**

**0.2**

**0.4**

**0.6**

**gH2O (mmol m-2 s-1)**

**AN (mol m-2 s-1)**

**Ci/Ca**

**0**

**20**

**40**

**60**

**80**

**0**

**2**

**a,A**

**a,A**

**a,A a,A**

**b,A**

A D

**b,A**

B E

C F

**4**

**6**

**8**

**10**

**3.3 The semi-deciduous species** *Cistus incanus* **L.** 

Fig. 6. Quantum yield of linear PSII electron transport (PSII), regulated energy dissipation (NPQ), non-regulated energy dissipation (NO) and maximum PSII photochemical efficiency (Fv/Fm) in mature leaves of *Laurus nobilis*, *Phillyrea angustifolia* and *Quercus ilex*, during winter and spring. Different letters indicate statistical differences among species (small letters) and between seasons (capital letters). Values are means ± SD (n=8).

The results relative to leaf functional traits and photosynthetic pigment content are reported in the table 2. The analysis of functional leaf traits has evidenced that, as compared to mature leaves, young leaves showed lower values (P<0.05) of leaf area (LA), but no difference in specific leaf area (SLA) and leaf dry matter content (LDMC). Functional leaf

Photosynthetic Adaptive Strategies in Evergreen and

**4. Discussion** 

*Quercus ilex* **L. in winter** 

circumstance that favors the production of ROS.

Semi-Deciduous Species of Mediterranean Maquis During Winter 415

LA (cm2) 3.02±0.14 a 8.01±0.28 b 8.32±0.44 b SLA (cm2 g-1 dw) 127.13±11.68 a 120.40±4.96 a 134.03±8.88 a LDMC (g g-1 wslm) 0.22±0.01 a 0.20±0.02 a 0.21±0.01 a chl (a+b) (g cm-2) 57.90±1.18 a 76.61±5.8 b 88,01±6 b car (x+c) (g cm-2) 11.09±0.29 a 14.22±1.05 b 16±2.32 b Chl a/b 3.03±0.01 a 2.37±0.23 b 2.5±0.34 b Table 2. Leaf Area (LA), Specific Leaf Area (SLA), Leaf Dry Matter Content (LDMC), total chlorophyll (chl a+b), total carotenoids (car x+c) and chlorophyll a/b ratio in *C. incanus* young and mature leaves during winter and in mature leaves during spring. Data reported

are means ± SE (n=6). Different letters indicate statistically significant differences.

**4.1 Young and mature leaves of** *Laurus nobilis* **L.,** *Phillyrea angustifolia* **L. and** 

In disagreement with data reported in literature for other species (Urban et al., 2008), young leaves of all species showed lower AN values compared to mature ones, indicating a marked sensitivity to winter temperatures. It is likely to hypothesize that this could be attributable to a reduced capacity of the mesophyll to assimilate CO2 because no difference in apparent carboxilation efficiency (Ci/Ca) between young and mature leaves was found. The significant differences between the two leaf populations, indicate the higher resistance of mature leaves photosynthetic machinery to low temperature. However, despite photosynthesis reduction, no variation in PSII between young and mature leaves was detected; thus the lower AN values in young leaves may be due either to limitations in photosynthetic dark reactions or to additional dissipative processes, other than CO2 assimilation, active in consuming the reductive power of the electron transport chain (*e.g.* photorespiration and/or Mehler reaction). The fluorescence analysis has evidenced that in young leaves the excess of absorbed light was dissipated more by photochemical processes than by thermal dissipation associated to xanthophylls cycle, as indicated by lower NPQ values compared to mature leaves. Although such photochemical processes are useful to protect the photosynthetic apparatus by photoinhibitory damage risks, it is well known that they can lead to an overproduction of reactive oxygen species (ROS). Even if ROS are continuously produced and removed during normal physiological events, when plants experience severe stress conditions, more O2 molecules are expected to be used as alternative electron acceptors disturbing the ROS production-removal balance and promoting the accumulation of ROS (Osório et al., 2011). Our results indicate that, in young leaves, under winter temperature, a large part of absorbed energy was diverted to nonregulated energy conversion processes (increase in ΦNO) than in mature leaves, a

On the contrary, in mature leaves, more absorbed light was dissipated by thermal dissipation processes associated to xanthophylls cycle (higher NPQ). This result is in contrast with data reported by other authors who found a reduction in thermal dissipation by xanthophylls cycle as the leaves expanded (Choinski & Eamus, 2003; Jiang et al., 2005). Our data suggest that leaf age influences the photoprotection mechanisms. More

**Winter Spring** 

**Young leaves Mature leaves Mature leaves** 

traits did not show any difference between mature leaves in both winter and spring campaigns. The total chlorophyll content, chl (a+b), as well as the total carotenoid content, car (x+c), were higher in mature than in (P<0.01) young leaves, that showed a lower (P<0.05) chl a/b ratio. No difference in total chlorophyll and carotenoid content, between winter and spring, in mature leaves was detected.

Fig. 7. Quantum yield of linear PSII electron transport (PSII), regulated energy dissipation (NPQ), non-regulated energy dissipation (NO) and maximum PSII photochemical efficiency (Fv/Fm) in *C. incanus* young and mature leaves during winter and in mature leaves during spring. Different letters indicate statistical differences between young and mature leaves (small letters) and between seasons (capital letters). Values are means ± SD (n=6).


Table 2. Leaf Area (LA), Specific Leaf Area (SLA), Leaf Dry Matter Content (LDMC), total chlorophyll (chl a+b), total carotenoids (car x+c) and chlorophyll a/b ratio in *C. incanus* young and mature leaves during winter and in mature leaves during spring. Data reported are means ± SE (n=6). Different letters indicate statistically significant differences.

#### **4. Discussion**

414 Advances in Photosynthesis – Fundamental Aspects

traits did not show any difference between mature leaves in both winter and spring campaigns. The total chlorophyll content, chl (a+b), as well as the total carotenoid content, car (x+c), were higher in mature than in (P<0.01) young leaves, that showed a lower (P<0.05) chl a/b ratio. No difference in total chlorophyll and carotenoid content, between winter and

**Winter Spring**

**B**

**B**

**B**

**PSII**

**NPQ**

**NO**

**Fv/F**

**m**

**0.0**

**0.6**

**0.0**

**0.0 0.1 0.2 0.3 0.4**

**0.0 0.2 0.4 0.6 0.8**

**0.2**

**0.4**

**0.2**

**0.4**

**0.6**

**0.8**

Fig. 7. Quantum yield of linear PSII electron transport (PSII), regulated energy dissipation (NPQ), non-regulated energy dissipation (NO) and maximum PSII photochemical efficiency (Fv/Fm) in *C. incanus* young and mature leaves during winter and in mature leaves during spring. Different letters indicate statistical differences between young and mature leaves (small letters) and between seasons (capital letters). Values are means ± SD (n=6).

**<sup>a</sup> a,A a,A**

spring, in mature leaves was detected.

**PSII**

**NPQ**

**NO**

**Fv/F**

**m**

**0.0**

**0.6**

**0.0**

**0.0 0.1 0.2 0.3 0.4**

**0.0 0.2 0.4 0.6 0.8**

**0.2**

**0.4**

**0.2**

**0.4**

**a**

**a**

**a**

**b,A**

**Young Mature**

A E

**b,A**

B F

C G

D H

**a,A**

**0.6**

**0.8**

#### **4.1 Young and mature leaves of** *Laurus nobilis* **L.,** *Phillyrea angustifolia* **L. and**  *Quercus ilex* **L. in winter**

In disagreement with data reported in literature for other species (Urban et al., 2008), young leaves of all species showed lower AN values compared to mature ones, indicating a marked sensitivity to winter temperatures. It is likely to hypothesize that this could be attributable to a reduced capacity of the mesophyll to assimilate CO2 because no difference in apparent carboxilation efficiency (Ci/Ca) between young and mature leaves was found. The significant differences between the two leaf populations, indicate the higher resistance of mature leaves photosynthetic machinery to low temperature. However, despite photosynthesis reduction, no variation in PSII between young and mature leaves was detected; thus the lower AN values in young leaves may be due either to limitations in photosynthetic dark reactions or to additional dissipative processes, other than CO2 assimilation, active in consuming the reductive power of the electron transport chain (*e.g.* photorespiration and/or Mehler reaction). The fluorescence analysis has evidenced that in young leaves the excess of absorbed light was dissipated more by photochemical processes than by thermal dissipation associated to xanthophylls cycle, as indicated by lower NPQ values compared to mature leaves. Although such photochemical processes are useful to protect the photosynthetic apparatus by photoinhibitory damage risks, it is well known that they can lead to an overproduction of reactive oxygen species (ROS). Even if ROS are continuously produced and removed during normal physiological events, when plants experience severe stress conditions, more O2 molecules are expected to be used as alternative electron acceptors disturbing the ROS production-removal balance and promoting the accumulation of ROS (Osório et al., 2011). Our results indicate that, in young leaves, under winter temperature, a large part of absorbed energy was diverted to nonregulated energy conversion processes (increase in ΦNO) than in mature leaves, a circumstance that favors the production of ROS.

On the contrary, in mature leaves, more absorbed light was dissipated by thermal dissipation processes associated to xanthophylls cycle (higher NPQ). This result is in contrast with data reported by other authors who found a reduction in thermal dissipation by xanthophylls cycle as the leaves expanded (Choinski & Eamus, 2003; Jiang et al., 2005). Our data suggest that leaf age influences the photoprotection mechanisms. More

Photosynthetic Adaptive Strategies in Evergreen and

**4.4** *Cistus incanus* **L. mature leaves in winter and spring** 

temperatures.

pigment adjustments.

photochemistry.

photochemical efficiency (Fv/Fm).

(*i.e.* 22 °C), an increase of (PSII) was observed.

Semi-Deciduous Species of Mediterranean Maquis During Winter 417

photochemistry reduces the need for the thermal dissipative process, in particular the fraction of the regulated thermal energy dissipation (low NPQ values). Mature leaves showed an opposite tendency. However in both leaf typologies no variation of nonregulated energy dissipation component (ΦNO) was found. High values of NPQ are indicative of a high photoprotective capacity, whereas high values of ΦNO may reflect the inability of a plant to protect itself against photodamage (Klughammer & Schreiber, 2008; Osório et al., 2011). In our opinion, as maximum PSII photochemical efficiency (Fv/Fm) and ΦNO are similar in the two leaf populations, we suppose that the different strategies adopted by young and mature leaves are equally helpful in leaf photoprotection under winter

The acclimation of plants in relation to the environmental conditions is expressed, among other factors, also by their leaf characteristics (Bussotti et al., 2008) and photosynthetic

Functional leaf traits analyses indicate that, even if specific leaf area (SLA) as well as the leaf dry matter content (LDMC) do not vary between young and mature *C. incanus* leaves, mature leaves present a greater leaf blade and have a higher total chlorophyll and carotenoid contents per unit leaf area. The adjustment of photosynthetic pigment composition in mature leaves could be interpreted as further strategy in order to enhance the light harvest and thus compensate for the reduction in allocation of absorbed light in

The behaviour of *C. incanus* mature leaves differ in winter and spring. The analysis of photochemistry showed that temperatures of 11 °C does not injure the photosynthetic apparatus, but affects significantly its efficiency. Indeed, the low values of PSII evidenced a decline in photochemical activity that may lead to an increase of excitation pressure in photosystem II with important consequence for the plant cells in terms of decrease of intracellular ATP and NADP production. On the other hand, the fraction of the regulated energy dissipation (NPQ) higher in leaves during winter compared to spring, indicates that the regulated thermal dissipation for winter leaves was enhanced under low temperature to compensate for reduced photochemistry. Nevertheless during winter, leaves show also an higher non-regulated energy dissipation in PSII (ΦNO), indicating the occurrence of a stress condition for photosynthetic apparatus (Osòrio et al., 2011). It is reasonable to hypothesize that leaves during winter cope with low temperature by means of flexible component of thermal energy dissipation and the alternative electron sink and/or additional quenching mechanism(s). These factors may contribute to the high stress resistance of *C. incanus* leaves and allow photosynthetic apparatus to maintain during winter a high maximal PSII

The Fv/Fm values found in leaves during winter were close to those reported for winter leaves of other *Cistus* species as well as to those of unstressed plants of other Mediterranean species (Oliveira & Peñuelas, 2001, 2004). In spring, after the return to mild temperatures

These results suggest that during February the reduction in photochemistry found at temperatures of 11 °C and at PPFD of about 700 mol photons m-2 s-1 (table 1) was due to a downregulation of PSII reaction centres, rather than to an impairment of photosynthetic apparatus. This strategy may represent a safety mechanism against the photoinhibitory

specifically, young and mature leaves regulate in a different way the dissipation of absorbed light energy in order to maintain high the photochemical efficiency. The absence of significant differences in Fv/Fm ratio between the two leaf population indicates that both thermal dissipation and the alternative electron sink and/or additional quenching mechanism(s) are suitable for photoprotection, assuming a similar weight in photoprotection.

Among species, the higher AN rates in *Q. ilex* compared to *P. angustifolia* and *L. nobilis* in both young and mature leaves indicates *Q. ilex* as the species with more efficient photosynthetic process at low temperature (Ogaya & Peñuelas, 2003). This is likely due to the highest utilization of reductive power of electron transport chain in C fixation rather than in dissipative processes under low temperature. Our data demonstrate that under low temperatures, the strategies utilized to dissipate the excess of absorbed light vary among species. In particular in both young and mature leaves, *L. nobilis*, as compared to *P. angustifolia* and *Q. ilex*, diverts more excitation energy to regulated energy dissipation processes than to non-regulated energy dissipation processes (higher NPQ, lower NO). These different mechanisms seem equally important in maintaining an elevated maximum PSII photochemical efficiency, as confirmed by comparable Fv/Fm ratio in all species.

#### **4.2 Mature leaves of** *L. nobilis* **L.,** *P. angustifolia* **L. and** *Quercus ilex* **L. during winter and spring**

Equinoctial periods, characterized by the absence of drought and cold stress, are the most favorable seasons for the photosynthetic activity of Mediterranean vegetation (Savè et al., 1999). Data presented in this section are consistent with literature, indeed in spring, compared to winter, high rates of gas exchanges and a better photochemical efficiency were measured for all species. The highest values of AN and gH2O measured during winter in *Q. ilex*, suggest for this species a better resistance to low temperature (Ogaya & Peñuelas, 2003), differently from *L. nobilis* that showed the lowest photosynthetic activity and stomatal conductance and the highest Ci/Ca ratio. This latter constitutes a proxy tool to evaluate the occurrence of non-stomatal limitations to photosynthesis. In *L. nobilis*, the similar Ci/Ca values found in winter compared to spring, despite the low photosynthetic activity, denote the presence of non-stomatal limitation to photosynthetic process likely due to a reduced activity of Rubisco (Sage & Sharkey, 1987), and/or of other carbon assimilation enzymes (Sassenrath et al., 1990) at low temperatures. The analysis of photosynthetic energy partitioning evidenced that in winter, when net CO2 assimilation was limited by low temperatures, more absorbed energy was converted into regulated energy dissipation (higher NPQ) compared to spring. On the contrary, in spring when air temperature became favourable for photosynthesis, the absorbed energy was diverted mainly to net CO2 assimilation (higher PSII) and only a little in non-regulated energy dissipation (low NO). The higher thermal dissipation and the low Fv/Fm values in winter compared to spring were likely the result of a photoprotective mechanisms by which plants cope with winter stress. This strategy is probably based on maintaining PSII primed for energy dissipation and engaged in diurnal energy dissipation throughout the night (Adams et al., 2001).

#### **4.3** *Cistus incanus* **L. young and mature leaves in winter**

Under winter temperature, *C. incanus* young leaves exhibit a higher photochemical activity than mature leaves. The utilization of reductive power of electron transport in

specifically, young and mature leaves regulate in a different way the dissipation of absorbed light energy in order to maintain high the photochemical efficiency. The absence of significant differences in Fv/Fm ratio between the two leaf population indicates that both thermal dissipation and the alternative electron sink and/or additional quenching mechanism(s) are suitable for photoprotection, assuming a similar weight in

Among species, the higher AN rates in *Q. ilex* compared to *P. angustifolia* and *L. nobilis* in both young and mature leaves indicates *Q. ilex* as the species with more efficient photosynthetic process at low temperature (Ogaya & Peñuelas, 2003). This is likely due to the highest utilization of reductive power of electron transport chain in C fixation rather than in dissipative processes under low temperature. Our data demonstrate that under low temperatures, the strategies utilized to dissipate the excess of absorbed light vary among species. In particular in both young and mature leaves, *L. nobilis*, as compared to *P. angustifolia* and *Q. ilex*, diverts more excitation energy to regulated energy dissipation processes than to non-regulated energy dissipation processes (higher NPQ, lower NO). These different mechanisms seem equally important in maintaining an elevated maximum

PSII photochemical efficiency, as confirmed by comparable Fv/Fm ratio in all species.

engaged in diurnal energy dissipation throughout the night (Adams et al., 2001).

Under winter temperature, *C. incanus* young leaves exhibit a higher photochemical activity than mature leaves. The utilization of reductive power of electron transport in

**4.3** *Cistus incanus* **L. young and mature leaves in winter** 

**4.2 Mature leaves of** *L. nobilis* **L.,** *P. angustifolia* **L. and** *Quercus ilex* **L. during winter** 

Equinoctial periods, characterized by the absence of drought and cold stress, are the most favorable seasons for the photosynthetic activity of Mediterranean vegetation (Savè et al., 1999). Data presented in this section are consistent with literature, indeed in spring, compared to winter, high rates of gas exchanges and a better photochemical efficiency were measured for all species. The highest values of AN and gH2O measured during winter in *Q. ilex*, suggest for this species a better resistance to low temperature (Ogaya & Peñuelas, 2003), differently from *L. nobilis* that showed the lowest photosynthetic activity and stomatal conductance and the highest Ci/Ca ratio. This latter constitutes a proxy tool to evaluate the occurrence of non-stomatal limitations to photosynthesis. In *L. nobilis*, the similar Ci/Ca values found in winter compared to spring, despite the low photosynthetic activity, denote the presence of non-stomatal limitation to photosynthetic process likely due to a reduced activity of Rubisco (Sage & Sharkey, 1987), and/or of other carbon assimilation enzymes (Sassenrath et al., 1990) at low temperatures. The analysis of photosynthetic energy partitioning evidenced that in winter, when net CO2 assimilation was limited by low temperatures, more absorbed energy was converted into regulated energy dissipation (higher NPQ) compared to spring. On the contrary, in spring when air temperature became favourable for photosynthesis, the absorbed energy was diverted mainly to net CO2 assimilation (higher PSII) and only a little in non-regulated energy dissipation (low NO). The higher thermal dissipation and the low Fv/Fm values in winter compared to spring were likely the result of a photoprotective mechanisms by which plants cope with winter stress. This strategy is probably based on maintaining PSII primed for energy dissipation and

photoprotection.

**and spring** 

photochemistry reduces the need for the thermal dissipative process, in particular the fraction of the regulated thermal energy dissipation (low NPQ values). Mature leaves showed an opposite tendency. However in both leaf typologies no variation of nonregulated energy dissipation component (ΦNO) was found. High values of NPQ are indicative of a high photoprotective capacity, whereas high values of ΦNO may reflect the inability of a plant to protect itself against photodamage (Klughammer & Schreiber, 2008; Osório et al., 2011). In our opinion, as maximum PSII photochemical efficiency (Fv/Fm) and ΦNO are similar in the two leaf populations, we suppose that the different strategies adopted by young and mature leaves are equally helpful in leaf photoprotection under winter temperatures.

The acclimation of plants in relation to the environmental conditions is expressed, among other factors, also by their leaf characteristics (Bussotti et al., 2008) and photosynthetic pigment adjustments.

Functional leaf traits analyses indicate that, even if specific leaf area (SLA) as well as the leaf dry matter content (LDMC) do not vary between young and mature *C. incanus* leaves, mature leaves present a greater leaf blade and have a higher total chlorophyll and carotenoid contents per unit leaf area. The adjustment of photosynthetic pigment composition in mature leaves could be interpreted as further strategy in order to enhance the light harvest and thus compensate for the reduction in allocation of absorbed light in photochemistry.

#### **4.4** *Cistus incanus* **L. mature leaves in winter and spring**

The behaviour of *C. incanus* mature leaves differ in winter and spring. The analysis of photochemistry showed that temperatures of 11 °C does not injure the photosynthetic apparatus, but affects significantly its efficiency. Indeed, the low values of PSII evidenced a decline in photochemical activity that may lead to an increase of excitation pressure in photosystem II with important consequence for the plant cells in terms of decrease of intracellular ATP and NADP production. On the other hand, the fraction of the regulated energy dissipation (NPQ) higher in leaves during winter compared to spring, indicates that the regulated thermal dissipation for winter leaves was enhanced under low temperature to compensate for reduced photochemistry. Nevertheless during winter, leaves show also an higher non-regulated energy dissipation in PSII (ΦNO), indicating the occurrence of a stress condition for photosynthetic apparatus (Osòrio et al., 2011). It is reasonable to hypothesize that leaves during winter cope with low temperature by means of flexible component of thermal energy dissipation and the alternative electron sink and/or additional quenching mechanism(s). These factors may contribute to the high stress resistance of *C. incanus* leaves and allow photosynthetic apparatus to maintain during winter a high maximal PSII photochemical efficiency (Fv/Fm).

The Fv/Fm values found in leaves during winter were close to those reported for winter leaves of other *Cistus* species as well as to those of unstressed plants of other Mediterranean species (Oliveira & Peñuelas, 2001, 2004). In spring, after the return to mild temperatures (*i.e.* 22 °C), an increase of (PSII) was observed.

These results suggest that during February the reduction in photochemistry found at temperatures of 11 °C and at PPFD of about 700 mol photons m-2 s-1 (table 1) was due to a downregulation of PSII reaction centres, rather than to an impairment of photosynthetic apparatus. This strategy may represent a safety mechanism against the photoinhibitory

Photosynthetic Adaptive Strategies in Evergreen and

experiments.

**7. References** 

1146-609X

(printed) 0166-8595

644, ISSN 0168-9452

(printed) 0032-0935

0067-1924

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Abril, M. & Hanano, R. (1998). Ecophysiological responses of three evergreen woody

Adams, W.W.; Demmig-Adams, B., Rosentiel, T.N., & Ebbert, V. (2001). Dependence of

Arena, C.; Vitale, L. & Virzo De Santo A. (2008). Photosynthesis and photoprotective

Aronne, G. & De Micco, V. (2001). Seasonal dimorphism in the Mediterranean *Cistus incanus* L. subsp. *incanus*. *Annals of Botany*, Vol. 87, pp. 789-794, ISSN 0305-7364 Baker, N.R. (1994). Chilling stress and photosynthesis. *Causes of Photooxidative Stress and* 

Bertamini, M. & Nedunchezhian, N. (2003). Photoinhibition of photosynthesis in mature and

Bilger, W. & Björkman, O. (1990). Role of the xanthophyll cycle in photoprotection

Blondel, J. & Aronson, J. (1999). Biology and Wildlife of the Mediterranean Region. ISBN 0

Boese, S.R. & Huner, N.P.A. (1990). Effect of growth temperature and temperature shift on

Bussotti, F. (2008). Functional leaf traits, plant communities and acclimation processes in

Caemmerer, S. von & Farquhar, G.D. (1981). Some relationship between the biochemistry of

Castro-Díez, P.; Villar-Salvador, P., Pérez-Rontomé, C., Maestro-Martínez, M. & Montserrat-

Choinski, Jr. & Eamus, D. (2003) Changes in photosynthesis during leaf expansion in

Cornelissen, J.H.C.; Lavorel, S., Garnier, E., Díaz, S., Buchmann, N., Gurvich, D.E., Reich,

cold. *Plant Biosystems*, Vol. 142, pp. 472-479, ISSN 1126-3504

127-154. CRC Press, Boca Raton, Florida.

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*Amelioration of Defences Systems in Plants* (eds C.H Foyer & P.M. Mullineaux), pp

young leaves of grapevine (*Vitis vinifera* L.). *Plant Science*, Vol. 164, No. 4, pp. 635-

elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of *Hedera canariensis*. *Photosynthesis Research*, Vol. 25,

spinach leaf morphology and photosynthesis. *Plant Physiology*, Vol. 94, pp. 1830-

relation to oxidative stress in trees: a critical overview. *Global Change Biology*, Vol.

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Martí, G. (1998) Leaf morphology, leaf chemical composition and stem xylem characteristics in two *Pistacia* (Anarcardiaceae) species along climatic gradient.

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P.B., ter Steege, H., Morgan, H.D., van der Heijden, M.G.A., Pausas, J.G. & Poorter

damage risk as a consequence of combined effect of low temperature and moderately high irradiances on photosystems. In this view, the lack of significant differences in maximum PSII photochemical efficiency (Fv/Fm), as well as in total chlorophylls and carotenoids content between mature leaves in winter and spring supports this hypothesis, confirming that photochemical apparatus of *C. incanus* remained stable and effective at winter temperatures.

#### **5. Conclusions**

The results of the present study indicate that leaf age influences the photoprotection mechanisms. Under saturating irradiance and low winter temperature mature leaves of all evergreen species, by higher CO2 assimilation rates and higher thermal energy dissipation linked to the flexible component, cope more efficiently with the excess of absorbed light and result to be less sensitive to photoinhibition. On the other hand young leaves utilize the reducing power mainly in processes other than photosynthesis and show higher values of non-regulated energy dissipation in PSII. However both different mechanisms are useful in maintain the maximum PSII photochemical efficiency at comparable values in young and mature leaves.

Among species both young and mature leaves of *Q. ilex* exhibited the highest photosynthetic performance indicating a better resistance to low temperatures.

The comparison between mature leaves in winter and spring shows higher values of net photosynthesis and photochemical efficiency in all evergreen species during spring and a lower contribute of flexible and sustained thermal dissipation in winter. At low temperature, the significant increase of thermal and photochemical processes other than photosynthesis allow mature leaves of evergreen species to maintain an elevated photochemical efficiency, despite the strong reduction of carbon assimilation. Among species, *Q. ilex* showed the best photosynthetic performance under winter, indicating a better acclimation capability of photosynthetic apparatus.

In *C. incanus* species, during winter, young leaves showed a higher photochemical efficiency than mature leaves. The increase in photochemistry leads to a reduction of thermal dissipative processes. On the other hand, the mature leaves exhibited an opposite tendency. However, both strategies are useful in leaf photoprotection under winter since maximum PSII photochemical efficiency is high and similar in the two leaf populations.

The comparison between mature leaves in winter and spring has evidenced a lower quantum yield of PSII linear electron transport and an increase of regulated thermal dissipation processes during winter. The recovery of photochemical activity in spring under mild temperature, indicates that the drop in photochemistry in winter was due to the balance between energy absorbed and dissipated at PSII level rather than to an impairment of photosynthetic apparatus. In this context, the higher thermal dissipation in winter compensate for the reduced photochemistry, allowing maximum PSII photochemical efficiency to remain unchanged compared to spring. This may be interpreted as a dynamic regulatory process protecting the photosynthetic apparatus from severe damage by excess light at low temperature.

#### **6. Acknowledgments**

The authors are grateful to Prof. Mazzarella of the Department of Geophysic and Vulcanology (University Federico II Naples) for providing meteorological data and to Corpo Forestale dello Stato of Sabaudia (Latina, Italy) for supplying the plants used in the experiments.

#### **7. References**

418 Advances in Photosynthesis – Fundamental Aspects

damage risk as a consequence of combined effect of low temperature and moderately high irradiances on photosystems. In this view, the lack of significant differences in maximum PSII photochemical efficiency (Fv/Fm), as well as in total chlorophylls and carotenoids content between mature leaves in winter and spring supports this hypothesis, confirming that photochemical apparatus of *C. incanus* remained stable and effective at winter temperatures.

The results of the present study indicate that leaf age influences the photoprotection mechanisms. Under saturating irradiance and low winter temperature mature leaves of all evergreen species, by higher CO2 assimilation rates and higher thermal energy dissipation linked to the flexible component, cope more efficiently with the excess of absorbed light and result to be less sensitive to photoinhibition. On the other hand young leaves utilize the reducing power mainly in processes other than photosynthesis and show higher values of non-regulated energy dissipation in PSII. However both different mechanisms are useful in maintain the maximum PSII photochemical efficiency at comparable values in young and

Among species both young and mature leaves of *Q. ilex* exhibited the highest photosynthetic

The comparison between mature leaves in winter and spring shows higher values of net photosynthesis and photochemical efficiency in all evergreen species during spring and a lower contribute of flexible and sustained thermal dissipation in winter. At low temperature, the significant increase of thermal and photochemical processes other than photosynthesis allow mature leaves of evergreen species to maintain an elevated photochemical efficiency, despite the strong reduction of carbon assimilation. Among species, *Q. ilex* showed the best photosynthetic performance under winter, indicating a

In *C. incanus* species, during winter, young leaves showed a higher photochemical efficiency than mature leaves. The increase in photochemistry leads to a reduction of thermal dissipative processes. On the other hand, the mature leaves exhibited an opposite tendency. However, both strategies are useful in leaf photoprotection under winter since maximum

The comparison between mature leaves in winter and spring has evidenced a lower quantum yield of PSII linear electron transport and an increase of regulated thermal dissipation processes during winter. The recovery of photochemical activity in spring under mild temperature, indicates that the drop in photochemistry in winter was due to the balance between energy absorbed and dissipated at PSII level rather than to an impairment of photosynthetic apparatus. In this context, the higher thermal dissipation in winter compensate for the reduced photochemistry, allowing maximum PSII photochemical efficiency to remain unchanged compared to spring. This may be interpreted as a dynamic regulatory process protecting the photosynthetic apparatus from severe damage by excess

The authors are grateful to Prof. Mazzarella of the Department of Geophysic and Vulcanology (University Federico II Naples) for providing meteorological data and to Corpo

PSII photochemical efficiency is high and similar in the two leaf populations.

performance indicating a better resistance to low temperatures.

better acclimation capability of photosynthetic apparatus.

**5. Conclusions** 

mature leaves.

light at low temperature.

**6. Acknowledgments** 


Photosynthetic Adaptive Strategies in Evergreen and

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**20** 

*USA* 

**The Core- and Pan-Genomes** 

Jeffrey W. Touchman and Yih-Kuang Lu

*Arizona State University* 

**of Photosynthetic Prokaryotes** 

Genome sequencing projects are revealing new information about the distribution and evolution of photosynthesis and phototrophy, particularly in prokaryotes. Although coverage of the five phyla containing photosynthetic prokaryotes (Chlorobi, Chloroflexi, Cyanobacteria, Proteobacteria and Firmicutes) is limited and uneven, full genome sequences are now available for 82 strains from these phyla. In this chapter, we present data and comparisons that reflect recent advances in phototroph biology as a result of insights from genome sequencing. By performing a comprehensive analysis of the core-genome (the pool of genes shared by all phototrophic prokaryotes) and pan-genome (the global gene repertoire of all phototrophic prokaryotes: core genome + dispensable genome) along with available biological data for each organism, we address the following key questions: 1) what are the principal drivers behind the evolution and distribution of phototrophy and 2) how do environmental parameters correlate with genomic content to define niche partitioning

Over a decade has passed since the first phototrophic prokaryote, the cyanobacterium *Synechocystis* sp. PCC 6803, was completely sequenced (Kaneko et al., 1996). Since then, availability of an increasing diversity of newly sequenced species is accumulating in public databases at a sustained pace and there is little indication that this trend will level off in the near future (Raymond & Swingley, 2008). A deepening archive of complete genomes has enabled comparative genomic analyses, which has heavily influenced our views of genome evolution and uncovered the extent of gene sharing between organisms (Pallen & Wren, 2007). The analysis of pan-and core-genomes in particular allows us to link genome content to the relationship of organisms to one another and to their physical surroundings. For example, a low pan-genome diversity due to extensive overlap of metabolic function among groups of bacteria could reflect shared environmental habitats and resource utilization, while distinctive species that adapt to disparate environments would be expected to have a high pan-genome diversity. This approach was first developed by Tettelin et al. (2005) and Hogg et al. (2007) for tracking the number of unique genes among multiple strains of *Streptococcus agalactiae* and *Haemophilus influenzae*, respectively. Such analysis resulted in the determination of core-genes that encode functions related to the basic metabolism and phenotype of the species, and a pan-genome that consists of dispensable or unique genes

**1. Introduction** 

and ecotype distributions in photic environments?

that impart specific functionalities to individual strains.


### **The Core- and Pan-Genomes of Photosynthetic Prokaryotes**

Jeffrey W. Touchman and Yih-Kuang Lu *Arizona State University USA* 

#### **1. Introduction**

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macchia plants to avoid photoinhibitory damage by excessive radiation levels

content as alternative predictors of plant strategies. *New Phytologist*, Vol. 143, pp.

Genome sequencing projects are revealing new information about the distribution and evolution of photosynthesis and phototrophy, particularly in prokaryotes. Although coverage of the five phyla containing photosynthetic prokaryotes (Chlorobi, Chloroflexi, Cyanobacteria, Proteobacteria and Firmicutes) is limited and uneven, full genome sequences are now available for 82 strains from these phyla. In this chapter, we present data and comparisons that reflect recent advances in phototroph biology as a result of insights from genome sequencing. By performing a comprehensive analysis of the core-genome (the pool of genes shared by all phototrophic prokaryotes) and pan-genome (the global gene repertoire of all phototrophic prokaryotes: core genome + dispensable genome) along with available biological data for each organism, we address the following key questions: 1) what are the principal drivers behind the evolution and distribution of phototrophy and 2) how do environmental parameters correlate with genomic content to define niche partitioning and ecotype distributions in photic environments?

Over a decade has passed since the first phototrophic prokaryote, the cyanobacterium *Synechocystis* sp. PCC 6803, was completely sequenced (Kaneko et al., 1996). Since then, availability of an increasing diversity of newly sequenced species is accumulating in public databases at a sustained pace and there is little indication that this trend will level off in the near future (Raymond & Swingley, 2008). A deepening archive of complete genomes has enabled comparative genomic analyses, which has heavily influenced our views of genome evolution and uncovered the extent of gene sharing between organisms (Pallen & Wren, 2007). The analysis of pan-and core-genomes in particular allows us to link genome content to the relationship of organisms to one another and to their physical surroundings. For example, a low pan-genome diversity due to extensive overlap of metabolic function among groups of bacteria could reflect shared environmental habitats and resource utilization, while distinctive species that adapt to disparate environments would be expected to have a high pan-genome diversity. This approach was first developed by Tettelin et al. (2005) and Hogg et al. (2007) for tracking the number of unique genes among multiple strains of *Streptococcus agalactiae* and *Haemophilus influenzae*, respectively. Such analysis resulted in the determination of core-genes that encode functions related to the basic metabolism and phenotype of the species, and a pan-genome that consists of dispensable or unique genes that impart specific functionalities to individual strains.

The Core- and Pan-Genomes of Photosynthetic Prokaryotes 425

Table 1. Summary of 82 photosynthetic prokaryotes with whole-genome sequences

Within prokaryotes, photosynthetic capability is present within five major groups, which include heliobacteria, green filamentous bacteria (*Chloroflexus sp.*), green sulfur bacteria (*Chlorobium sp.*), *Proteobacteria*, and *Cyanobacteria* (Blankenship, 1992; Gest & Favinger, 1983; Olson & Pierson, 1987; Vermaas, 1994). While only *Cyanobacteria,* which contain two distinct reaction centers linked to each other, are capable of oxygenic photosynthesis, other photosynthetic bacteria primarily carry out anoxygenic photosynthesis with a single reaction center. Traditionally, the phylogenetic relationship of these five distinct photosynthetic groups has been constructed by comparing sequences of the small subunit 16S rRNA gene (Ludwig & Klenk, 2001). But the use of the 16S rRNA gene is unable to resolve the relationships among these phototrophs with confidence, which is central to understanding their evolution. For example, phylogenetic trees based on a comparison of different combinations of 527 shared genes amongst all five photosynthetic prokaryote groups shows that no less than 15 different tree topologies can be constructed depending on the subset of genes used in the analysis, only one of which matches the traditional 16S rDNA tree (Raymond et al., 2002). In fact, comparing just those genes involved in photosynthesis supports no coherent relationship among the different photosynthetic bacteria either, indicating that such genes may have been subjects of lateral gene transfers (ibid).

Recent genome sequencing efforts have made whole genome data available for many more representatives of each of the five phyla of bacteria with photosynthetic members. To resolve the complicated relationship between bacterial phototrophy and evolutionary history, we describe an analysis of the 82 fully-sequenced photosynthetic prokaryotes to construct the pan- and core-genomes across all available strains. We present results showing various gene-based indicators of the relationship between genome and phenotype among these organisms. Not surprisingly, our findings describe new relationships between gene content and environmental habitat. These results add to a complete gene-based functional annotation of the phototrophic prokaryotes, and set the groundwork for continuing studies on genetic and evolutionary dynamics of this important photosynthetic community.

#### **2. Whole-genome analysis of phototrophic prokaryotes**

The list and summary details of 82 fully-sequenced photosynthetic species used in this study are shown in Table 1 (Liolios et al., 2006). Every species exhibits common characteristics with other relatives in the same phylum. For example, the *Chlorobia* and heliobacteria (*Firmicutes*) are strictly anaerobic while the *Chloroflexia* and *Proteobacteria* are facultatively anaerobic. The *Chloroflexia* are alkali-trophic thermophiles whereas other phylyl members are neutral pH mesophiles. Genome size is generally uniform among the *Chlorobia* and *Chloroflexia*, but varies widly among the *Cyanobacteria* and *Proteobacteria*. Furthermore, both C*hloroflexia* and *Proteobacteria* possess a pheophytin-quinone reaction center, while *Heliobacteria* and C*hlorobia* use an iron-sulfur reaction center. *Cyanobacteria* exclusively possesses two types of reaction centers. Both *Chlorobia* and *Cyanobacteria* are two phyla comprised entirely of photosynthetic representatives. Although most of the photosynthetic species are free-living organisms, *Nostoc sp.* PCC 7120, *Nostoc punctiforme* PCC 73102, and *Acaryochloris marina* MBIC11017 in the *Cyanobacteria* and *Bradyrhizobium* 

BTAi1, ORS278 and some *Methylobacterium* strains in the *Proteobacteria* form a mutual relationship with terrestrial plants and coral. The *Heliobacteria* (e.g., *Heliobacterium modesticaldum)* are the only photosynthetic members of the *Firmicutes*. The genome of *Heliobacillus mobilis*, the strain most studied biochemically, still remains proprietary and was not included in our analysis.

Within prokaryotes, photosynthetic capability is present within five major groups, which include heliobacteria, green filamentous bacteria (*Chloroflexus sp.*), green sulfur bacteria (*Chlorobium sp.*), *Proteobacteria*, and *Cyanobacteria* (Blankenship, 1992; Gest & Favinger, 1983; Olson & Pierson, 1987; Vermaas, 1994). While only *Cyanobacteria,* which contain two distinct reaction centers linked to each other, are capable of oxygenic photosynthesis, other photosynthetic bacteria primarily carry out anoxygenic photosynthesis with a single reaction center. Traditionally, the phylogenetic relationship of these five distinct photosynthetic groups has been constructed by comparing sequences of the small subunit 16S rRNA gene (Ludwig & Klenk, 2001). But the use of the 16S rRNA gene is unable to resolve the relationships among these phototrophs with confidence, which is central to understanding their evolution. For example, phylogenetic trees based on a comparison of different combinations of 527 shared genes amongst all five photosynthetic prokaryote groups shows that no less than 15 different tree topologies can be constructed depending on the subset of genes used in the analysis, only one of which matches the traditional 16S rDNA tree (Raymond et al., 2002). In fact, comparing just those genes involved in photosynthesis supports no coherent relationship among the different photosynthetic bacteria either, indicating that such genes may have been subjects of lateral gene transfers

Recent genome sequencing efforts have made whole genome data available for many more representatives of each of the five phyla of bacteria with photosynthetic members. To resolve the complicated relationship between bacterial phototrophy and evolutionary history, we describe an analysis of the 82 fully-sequenced photosynthetic prokaryotes to construct the pan- and core-genomes across all available strains. We present results showing various gene-based indicators of the relationship between genome and phenotype among these organisms. Not surprisingly, our findings describe new relationships between gene content and environmental habitat. These results add to a complete gene-based functional annotation of the phototrophic prokaryotes, and set the groundwork for continuing studies

on genetic and evolutionary dynamics of this important photosynthetic community.

The list and summary details of 82 fully-sequenced photosynthetic species used in this study are shown in Table 1 (Liolios et al., 2006). Every species exhibits common characteristics with other relatives in the same phylum. For example, the *Chlorobia* and heliobacteria (*Firmicutes*) are strictly anaerobic while the *Chloroflexia* and *Proteobacteria* are facultatively anaerobic. The *Chloroflexia* are alkali-trophic thermophiles whereas other phylyl members are neutral pH mesophiles. Genome size is generally uniform among the *Chlorobia* and *Chloroflexia*, but varies widly among the *Cyanobacteria* and *Proteobacteria*. Furthermore, both C*hloroflexia* and *Proteobacteria* possess a pheophytin-quinone reaction center, while *Heliobacteria* and C*hlorobia* use an iron-sulfur reaction center. *Cyanobacteria* exclusively possesses two types of reaction centers. Both *Chlorobia* and *Cyanobacteria* are two phyla comprised entirely of photosynthetic representatives. Although most of the photosynthetic species are free-living organisms, *Nostoc sp.* PCC 7120, *Nostoc punctiforme* PCC 73102, and

BTAi1, ORS278 and some *Methylobacterium* strains in the *Proteobacteria* form a mutual relationship with terrestrial plants and coral. The *Heliobacteria* (e.g., *Heliobacterium modesticaldum)* are the only photosynthetic members of the *Firmicutes*. The genome of *Heliobacillus mobilis*, the strain most studied biochemically, still remains proprietary and was

**2. Whole-genome analysis of phototrophic prokaryotes** 

*Acaryochloris marina* MBIC11017 in the *Cyanobacteria* and *Bradyrhizobium* 

not included in our analysis.

(ibid).


Table 1. Summary of 82 photosynthetic prokaryotes with whole-genome sequences

The Core- and Pan-Genomes of Photosynthetic Prokaryotes 427

The number of gene clusters specific to a particular phylum is much smaller. Both *Cyanobacteria* and *Proteobacteria* possess 32,316 (11.8%) and 30,717 (11.3%) phylyl-specific gene clusters, respectively, whereas both *Chlorobi* and *Chloroflexi* have 2,290 (0.8%) and 3,123 (1.1%) gene clusters, respectively. Additionally, 16,665 genes of all species (6.1%) are in common (that is, are contained in the phototrophic prokaryote core-genome). On the surface, this result suggests a remarkable degree of overlap in the gene composition across

Fig. 2. Distribution of clustered genes within the pan-genome of the five photosynthetic

Fig. 3. Plot of the contraction of the core-genome (left) or expansion of the pan-genome (right) as the number of photosynthetic prokaryote genomes analyzed is increased.

bacterial phyla. Numbers indicate the number of genes specific to each group.

all five major phyla of photosynthetic prokaryotes.

#### **2.1 Clustering of ortholog groups of photosynthetic prokaryotes**

All of the 312,254 protein sequences from 82 photosynthetic prokaryote genomes were collected and clustered with the Markov clustering algorithm Ortho-MCL (Chen et al., 2006). Ortho-MCL is a graph-clustering algorithm designed to identify homologous proteins based on sequence similarity and distinguish true orthologs from paralogous relationships without computationally intensive phylogenetic analysis. Upon clustering, 41,824 proteins (13.3%) were removed due to the absence of detectable sequence similarities (BLASTP; E=10-5) and 272,686 (86.7%) were assigned to clusters. To assess clustering performance we modified a method described by Frech and Chen (2010) whereby both false-positive (the number of proteins that are found in two or more separate clusters) and false-negative (number of proteins that are found in wrong clusters) results were calculated using both the KEGG (Kanehisa & Goto, 2000) and COG (Natale et al., 2000) databases as a reference. An inflation index is then calculated that controls cluster granularity and gene family size while limiting error (Huerta-Cepas et al., 2008). The inflation parameter impacts the calculation of the number of shared orthologss in each phylum.

As Figure 1 shows, an increasing false-positive rate is anti-parallel to decreasing falsenegative rate in the inflation parameter. In order to obtain an adequate clustering result, we adjusted the Ortho-MCL program parameters such that reference ortholog clusters compared to both KEGG and COG are classified correctly to minimize erroneous clustering of orthologous groups (inflation index of 15). In our analysis, each predicted orthologous group was evaluated and corrected based on information from both KEGG and COG databases.

#### **2.2 The assembly of core- and pan-genomes**

The pan-genome of all 82 species contains 312,254 genes that form 23,362 ortholog clusters. Based on the clustering results, we observed that every photosynthetic prokaryote shares large portions of its genes with others. 204,074 genes that represent 74.8% of the entire data were found to co-exist in at least two organisms from any phyla ("multi-shares"; Figure 2).

All of the 312,254 protein sequences from 82 photosynthetic prokaryote genomes were collected and clustered with the Markov clustering algorithm Ortho-MCL (Chen et al., 2006). Ortho-MCL is a graph-clustering algorithm designed to identify homologous proteins based on sequence similarity and distinguish true orthologs from paralogous relationships without computationally intensive phylogenetic analysis. Upon clustering, 41,824 proteins (13.3%) were removed due to the absence of detectable sequence similarities (BLASTP; E=10-5) and 272,686 (86.7%) were assigned to clusters. To assess clustering performance we modified a method described by Frech and Chen (2010) whereby both false-positive (the number of proteins that are found in two or more separate clusters) and false-negative (number of proteins that are found in wrong clusters) results were calculated using both the KEGG (Kanehisa & Goto, 2000) and COG (Natale et al., 2000) databases as a reference. An inflation index is then calculated that controls cluster granularity and gene family size while limiting error (Huerta-Cepas et al., 2008). The inflation parameter impacts the calculation of

Fig. 1. Estimate of false positive and false negative error rate during ortholog clustering

As Figure 1 shows, an increasing false-positive rate is anti-parallel to decreasing falsenegative rate in the inflation parameter. In order to obtain an adequate clustering result, we adjusted the Ortho-MCL program parameters such that reference ortholog clusters compared to both KEGG and COG are classified correctly to minimize erroneous clustering of orthologous groups (inflation index of 15). In our analysis, each predicted orthologous group was evaluated and corrected based on information from both KEGG and COG

The pan-genome of all 82 species contains 312,254 genes that form 23,362 ortholog clusters. Based on the clustering results, we observed that every photosynthetic prokaryote shares large portions of its genes with others. 204,074 genes that represent 74.8% of the entire data were found to co-exist in at least two organisms from any phyla ("multi-shares"; Figure 2).

**2.1 Clustering of ortholog groups of photosynthetic prokaryotes** 

the number of shared orthologss in each phylum.

**2.2 The assembly of core- and pan-genomes** 

databases.

The number of gene clusters specific to a particular phylum is much smaller. Both *Cyanobacteria* and *Proteobacteria* possess 32,316 (11.8%) and 30,717 (11.3%) phylyl-specific gene clusters, respectively, whereas both *Chlorobi* and *Chloroflexi* have 2,290 (0.8%) and 3,123 (1.1%) gene clusters, respectively. Additionally, 16,665 genes of all species (6.1%) are in common (that is, are contained in the phototrophic prokaryote core-genome). On the surface, this result suggests a remarkable degree of overlap in the gene composition across all five major phyla of photosynthetic prokaryotes.

Fig. 2. Distribution of clustered genes within the pan-genome of the five photosynthetic bacterial phyla. Numbers indicate the number of genes specific to each group.

Fig. 3. Plot of the contraction of the core-genome (left) or expansion of the pan-genome (right) as the number of photosynthetic prokaryote genomes analyzed is increased.

The Core- and Pan-Genomes of Photosynthetic Prokaryotes 429

Fig. 4. Both core- and pan-genomes present in all five photosynthetic phyla. Each colored circle represents a phylum: *Firmicutes* (grey), *Chlorobi* (green), *Chloroflexi* (dark-green), *Proteobacteria* (purple), *Cyanobacteria* (light-green), and all phyla together (red). Numbers represent the ortholog clusters contained within the core genome or pan genome of each phyla. \* The Firmicutes, with only a single sequenced genome, lack a pan genome.

Given that there was only a single fully-sequenced genome from the phototrophic *Heliobacteria (Firmicutes)* available for study, the core genome of *Heliobacteria* was provisionally constructed by excluding those genes that are homologous to any known genes from the other sequence-available *Firmicutes*. It is worth noting that although there are four heliobacteria genera containing a total of ten species that have been formally described: *Heliobacterium*, *Heliobacillus*, *Heliophilum*, and *Heliorestis*, the phototrophic *Heliobacterium modesticaldum* is the only sequenced bacteria representing them (Sattley et al., 2008). When *H. modesticaldum* was compared with the available bacterial genomes of the *Firmicutes*, we identified 123 ortholog clusters tentatively assigned to the core-genome of this organism. Genes encoding proteins involved in major genetic, cellular, and environmental processes and metabolism are very limited. This may be partly due to their mutualistic relationship with plants. Other major ortholog groups in this core-genome are involved in sporulation. The previous examination of several other *Heliobacteria* species for sporulating genes has

**2.3.1** *Heliobacteria (Firmictues)*

To estimate the change of the core-genome size within a particular phyla upon sequential addition of each new genome sequence, a plot was extrapolated by fitting a power law function to the data (Figure 3). As more genomes are compared, there is an asymptotic decline in the number of core orthologs in every phyla, similar to observations for *Streptococcus* (Lefebure & Stanhope, 2007) and *Prochlorococcus* genomes (Kettler et al., 2007). The pan-genome, in contrast, was determined by the plot of the numbers of new orthologs, which fit a decaying exponential curve (Figure 3). The gene accumulation curve for each phyla is clearly far from saturated.

#### **2.3 The core- and pan-genomes of phototrophic prokaryotes**

The results of the clustering analysis to determine the core- and pan-genome sizes for each phylum and all phyla together is shown in Figure 4. The size of the phylum coregenomes are: 819 genes in the *Chlorobi*, 1,392 in the *Chloroflexi*, 619 in the *Cyanobacteria*, and 644 in the *Proteobacteria.* The core-genome of all 82 phototrophs considered together consists of 268 genes shared by all organisms. This overall core-genome encompasses a large number of housekeeping genes involved in genetic processes and metabolism and a small number of genes involved in cellular and environmental processes. The housekeeping genes involved in genetic processes include DNA polymerase, ligase, and helicase for DNA replication; RNA polymerase, ribosomal proteins, and tRNA synthetases for translation; and chaperones and signal peptidase for post-translational processes. The housekeeping genes involved in metabolism are mainly involved in the biosynthesis of amino acids, nucleotides, and coenzymes, and a few key enzymes such as transketolase, phosphoglycerate mutase, phosphoglycerate kinase of the glycolysis, acetyl-CoA carboxylase of the tricarbxylic acid (TCA) cycle, H+-transporting ATPase, acyl carrier protein, and UDP-N-acetylmuramate-L-alanine ligase for the biosynthesis of bacterial cell wall are preserved. Moreover, we identified the chlorophyll-synthesizing enzymes that include porphobilinogen synthase, oxygen-independent coproporphyrinogen III oxidase, magnesium chelatase, chlorophyll synthase, magnesiumprotoporphyrin O-methyltransferase, and light-independent protochlorophyllide reductase. For both cellular and environmental processes, glycosyltransferase for cell membrane biogenesis, phosphate transport system proteins, signal recognition SRP54, and sec-independent protein TatC for membrane transport were identified, suggesting that transferring phosphate and translocating membrane proteins are universal in photosynthetic organisms. The large proportion of housekeeping genes responsible for nearly all major genetic functions and the biosynthesis for both amino acids and nucleotides is understandable since these genes are essential for basic life functions. We observed a paucity of genes involved in both cellular and environmental processes in the overall core-genome. This observation supports the view in which essential life functions are unchanging while nonessential or environment-specific functions are found in a flexible genome (Kettler et al., 2007).

Core-genomes were also calculated in a pairwise fashion between photosynthetic phyla to gauge the number of shared orthologs in a given pair of phylyl pan-genomes (Figure 5). Each circle in the figure is proportional to the size (number in the circle) of the shared orthologs. Although these results are heavily influenced by the size of the dataset for an individual phyla, it provides a provisional measure of shared genes between phyla.

To estimate the change of the core-genome size within a particular phyla upon sequential addition of each new genome sequence, a plot was extrapolated by fitting a power law function to the data (Figure 3). As more genomes are compared, there is an asymptotic decline in the number of core orthologs in every phyla, similar to observations for *Streptococcus* (Lefebure & Stanhope, 2007) and *Prochlorococcus* genomes (Kettler et al., 2007). The pan-genome, in contrast, was determined by the plot of the numbers of new orthologs, which fit a decaying exponential curve (Figure 3). The gene accumulation curve for each

The results of the clustering analysis to determine the core- and pan-genome sizes for each phylum and all phyla together is shown in Figure 4. The size of the phylum coregenomes are: 819 genes in the *Chlorobi*, 1,392 in the *Chloroflexi*, 619 in the *Cyanobacteria*, and 644 in the *Proteobacteria.* The core-genome of all 82 phototrophs considered together consists of 268 genes shared by all organisms. This overall core-genome encompasses a large number of housekeeping genes involved in genetic processes and metabolism and a small number of genes involved in cellular and environmental processes. The housekeeping genes involved in genetic processes include DNA polymerase, ligase, and helicase for DNA replication; RNA polymerase, ribosomal proteins, and tRNA synthetases for translation; and chaperones and signal peptidase for post-translational processes. The housekeeping genes involved in metabolism are mainly involved in the biosynthesis of amino acids, nucleotides, and coenzymes, and a few key enzymes such as transketolase, phosphoglycerate mutase, phosphoglycerate kinase of the glycolysis, acetyl-CoA carboxylase of the tricarbxylic acid (TCA) cycle, H+-transporting ATPase, acyl carrier protein, and UDP-N-acetylmuramate-L-alanine ligase for the biosynthesis of bacterial cell wall are preserved. Moreover, we identified the chlorophyll-synthesizing enzymes that include porphobilinogen synthase, oxygen-independent coproporphyrinogen III oxidase, magnesium chelatase, chlorophyll synthase, magnesiumprotoporphyrin O-methyltransferase, and light-independent protochlorophyllide reductase. For both cellular and environmental processes, glycosyltransferase for cell membrane biogenesis, phosphate transport system proteins, signal recognition SRP54, and sec-independent protein TatC for membrane transport were identified, suggesting that transferring phosphate and translocating membrane proteins are universal in photosynthetic organisms. The large proportion of housekeeping genes responsible for nearly all major genetic functions and the biosynthesis for both amino acids and nucleotides is understandable since these genes are essential for basic life functions. We observed a paucity of genes involved in both cellular and environmental processes in the overall core-genome. This observation supports the view in which essential life functions are unchanging while nonessential or environment-specific functions are found in a

Core-genomes were also calculated in a pairwise fashion between photosynthetic phyla to gauge the number of shared orthologs in a given pair of phylyl pan-genomes (Figure 5). Each circle in the figure is proportional to the size (number in the circle) of the shared orthologs. Although these results are heavily influenced by the size of the dataset for an

individual phyla, it provides a provisional measure of shared genes between phyla.

phyla is clearly far from saturated.

flexible genome (Kettler et al., 2007).

**2.3 The core- and pan-genomes of phototrophic prokaryotes** 

Fig. 4. Both core- and pan-genomes present in all five photosynthetic phyla. Each colored circle represents a phylum: *Firmicutes* (grey), *Chlorobi* (green), *Chloroflexi* (dark-green), *Proteobacteria* (purple), *Cyanobacteria* (light-green), and all phyla together (red). Numbers represent the ortholog clusters contained within the core genome or pan genome of each phyla. \* The Firmicutes, with only a single sequenced genome, lack a pan genome.

#### **2.3.1** *Heliobacteria (Firmictues)*

Given that there was only a single fully-sequenced genome from the phototrophic *Heliobacteria (Firmicutes)* available for study, the core genome of *Heliobacteria* was provisionally constructed by excluding those genes that are homologous to any known genes from the other sequence-available *Firmicutes*. It is worth noting that although there are four heliobacteria genera containing a total of ten species that have been formally described: *Heliobacterium*, *Heliobacillus*, *Heliophilum*, and *Heliorestis*, the phototrophic *Heliobacterium modesticaldum* is the only sequenced bacteria representing them (Sattley et al., 2008). When *H. modesticaldum* was compared with the available bacterial genomes of the *Firmicutes*, we identified 123 ortholog clusters tentatively assigned to the core-genome of this organism. Genes encoding proteins involved in major genetic, cellular, and environmental processes and metabolism are very limited. This may be partly due to their mutualistic relationship with plants. Other major ortholog groups in this core-genome are involved in sporulation. The previous examination of several other *Heliobacteria* species for sporulating genes has

The Core- and Pan-Genomes of Photosynthetic Prokaryotes 431

superfamily (MFS) transporter, were identified. Since the *Chlorobi* species are capable of fixing nitrogen, preserving these transport systems is necessary to support this process. A total of 1,774 ortholog clusters are assigned to the pan-genome of *Chlorobia*. Most of these are associated with phylogenetically close species and have functions such as secretion, extracellular constituents, and cell wall biogenesis. These are conspicuous features of the genuses *Chlorobium* and *Prosthecochloris*. Although the *Chlorobia* have been wellcharacterized biochemically and microbiologically (Frigaard & Bryant, 2004), our finding that the *Chlorobia* possess a relatively uniform core-genome complemented by a relatively limited set of accessory genes enhancing cellular activities provides further insight into their anoxygenic phototrophic lifestyle. The core-and pan-genome pattern suggests a largely vertical inheritance that has preserved the core-genome needed for major cellular activities--

The *Chloroflexi* core-genome contains 1,392 ortholog clusters, the largest size among the five phototrophic groups. It reflects roughly 35% of the genes of a *Chloroflexus* specie's genome. The functional composition of this core-genome is somewhat similar to that of the *Chlorobi* core-genome, since many core genes involved in both genetic and cellular processes were cross-identified. However, the core set also contains type II reaction centers, NADH dehydrogenase, and cytochrome *c* oxidase, similar to the *Proteobacteria* but different from the *Chlorobi*. Moreover, many transporters for metal ions, inorganic and organic compounds, as well as two-component histidine kinases for signal transduction were identified in the *Chloroflexi* but not seen in the *Chlorobi*. The conservation of functionally diverse transporters with signal-transduction histidine kinases may be related to a more dynamic life-style. Generally, *Chloroflexus* is a photoheterotroph and usually found in the lower layers of microbial mats with cyanobacteria growing above it that provide organic byproducts. The *Chloroflexi* core-genome possesses numerous heat shock proteins, chaperones, and signal peptidases involved in protein folding and translocating processes that likely serve to reinforce protein structures in the thermophilic *Chloroflexus* species. For genes involved in major metabolic pathways, the core-genes appear to be largely conserved across all photosynthetic phyla. Although the *Chloroflexi* are found to be distinctive from the *Chlorobi*, they do have some common characteristics such as the absence of intra-cytoplasmic membrane structures and chlorosomes on their plasma membranes. They also use the same

The *Chloroflexi* pan-genome contains 4,348 genes, and in contrast to the *Chlorobi* pan-genome it is comprised of more putative genes for extracelluar constituents, inter-cellular communication, and other physiological and biochemical activities. For example, genes involved in the 3-hydroxypropionate pathway for carbon fixation were found. But generally, the *Chloroflexi* core-genome equips most of the major functional genes for a wide range of metabolisms such as synthesis of organic compounds, energy production, transport, genetic processing, etc. Such coverage throughout most cellular activities makes

Representing the largest sampled phylogenetic clade of the phototrophic prokaryotes, the *Cyanobacteria* have 37 completely sequenced genomes available for analysis, resulting in the

the core-genome of the *Chloroflexi* similar in character to that of *Chlorobia*.

a result of living in environmentally stable niches.

**2.3.3** *Chloroflexi*

Bchl *a* and *c* biosythesis pathways.

**2.3.4** *Cyanobacteria*

indicated that sporulation gene presence may be universal within the heliobacteria (Kimble-Long & Madigan, 2001). It should be noted that a set of genes involved in bacteriochlorophyll (Bchl) *g* biosynthesis, not found in other phototrophs, were frequently reported in other heliobacteria species (reviewed in Asao & Madigan, 2010). These enzymes were not clustered into the core-genome due to their absence in the non-phototrophic *Firmicutes*. Additional genome sequences from the heliobacteria group will aid in our understanding of their specific core-genome.

Fig. 5. Pairwise comparison of shared ortholog groups between phyla

#### **2.3.2** *Chlorobi*

The *Chlorobia* core-genome contains 819 genes representing 30-40% of the total genes in a given *Chlorobia* genome. As a phyla, they are very similar with respect to gene content compared to the other phototrophic prokaryotes. In addition to the components of the coregenome for all species, the *Chlorobia* core orthologs are composed of major metabolism genes such as the electron transport chain that supports photosynthesis and sulfur oxidation, the reductive TCA cycle supporting carbon fixation and transport, and others for the biosynthesis of amino acids, lipids, and coenzymes. The core-genome also contains the type I reaction center unique to the *Chlorobi*. Our findings are similar to other recent reports (Davenport et al., 2010). In addition to those identified orthologs for central metabolism, we also identified genes involved in the biosynthesis of Bchl, carotenoids, and the photosynthetic "chlorosome" apparatus. Most pigment-synthesizing enzymes operate downstream of the metabolic pathways for final products like Bchl *c*, *d*, chlorobactene, and γ-carotene, which are located on the chlorosome to harvest light. A few metal and inorganic compound transporters for iron, nickel, and molybdate as well as the major facilitator superfamily (MFS) transporter, were identified. Since the *Chlorobi* species are capable of fixing nitrogen, preserving these transport systems is necessary to support this process. A total of 1,774 ortholog clusters are assigned to the pan-genome of *Chlorobia*. Most of these are associated with phylogenetically close species and have functions such as secretion, extracellular constituents, and cell wall biogenesis. These are conspicuous features of the genuses *Chlorobium* and *Prosthecochloris*. Although the *Chlorobia* have been wellcharacterized biochemically and microbiologically (Frigaard & Bryant, 2004), our finding that the *Chlorobia* possess a relatively uniform core-genome complemented by a relatively limited set of accessory genes enhancing cellular activities provides further insight into their anoxygenic phototrophic lifestyle. The core-and pan-genome pattern suggests a largely vertical inheritance that has preserved the core-genome needed for major cellular activities- a result of living in environmentally stable niches.

#### **2.3.3** *Chloroflexi*

430 Advances in Photosynthesis – Fundamental Aspects

indicated that sporulation gene presence may be universal within the heliobacteria (Kimble-Long & Madigan, 2001). It should be noted that a set of genes involved in bacteriochlorophyll (Bchl) *g* biosynthesis, not found in other phototrophs, were frequently reported in other heliobacteria species (reviewed in Asao & Madigan, 2010). These enzymes were not clustered into the core-genome due to their absence in the non-phototrophic *Firmicutes*. Additional genome sequences from the heliobacteria group will aid in our

understanding of their specific core-genome.

Fig. 5. Pairwise comparison of shared ortholog groups between phyla

The *Chlorobia* core-genome contains 819 genes representing 30-40% of the total genes in a given *Chlorobia* genome. As a phyla, they are very similar with respect to gene content compared to the other phototrophic prokaryotes. In addition to the components of the coregenome for all species, the *Chlorobia* core orthologs are composed of major metabolism genes such as the electron transport chain that supports photosynthesis and sulfur oxidation, the reductive TCA cycle supporting carbon fixation and transport, and others for the biosynthesis of amino acids, lipids, and coenzymes. The core-genome also contains the type I reaction center unique to the *Chlorobi*. Our findings are similar to other recent reports (Davenport et al., 2010). In addition to those identified orthologs for central metabolism, we also identified genes involved in the biosynthesis of Bchl, carotenoids, and the photosynthetic "chlorosome" apparatus. Most pigment-synthesizing enzymes operate downstream of the metabolic pathways for final products like Bchl *c*, *d*, chlorobactene, and γ-carotene, which are located on the chlorosome to harvest light. A few metal and inorganic compound transporters for iron, nickel, and molybdate as well as the major facilitator

**2.3.2** *Chlorobi*

The *Chloroflexi* core-genome contains 1,392 ortholog clusters, the largest size among the five phototrophic groups. It reflects roughly 35% of the genes of a *Chloroflexus* specie's genome. The functional composition of this core-genome is somewhat similar to that of the *Chlorobi* core-genome, since many core genes involved in both genetic and cellular processes were cross-identified. However, the core set also contains type II reaction centers, NADH dehydrogenase, and cytochrome *c* oxidase, similar to the *Proteobacteria* but different from the *Chlorobi*. Moreover, many transporters for metal ions, inorganic and organic compounds, as well as two-component histidine kinases for signal transduction were identified in the *Chloroflexi* but not seen in the *Chlorobi*. The conservation of functionally diverse transporters with signal-transduction histidine kinases may be related to a more dynamic life-style. Generally, *Chloroflexus* is a photoheterotroph and usually found in the lower layers of microbial mats with cyanobacteria growing above it that provide organic byproducts. The *Chloroflexi* core-genome possesses numerous heat shock proteins, chaperones, and signal peptidases involved in protein folding and translocating processes that likely serve to reinforce protein structures in the thermophilic *Chloroflexus* species. For genes involved in major metabolic pathways, the core-genes appear to be largely conserved across all photosynthetic phyla. Although the *Chloroflexi* are found to be distinctive from the *Chlorobi*, they do have some common characteristics such as the absence of intra-cytoplasmic membrane structures and chlorosomes on their plasma membranes. They also use the same Bchl *a* and *c* biosythesis pathways.

The *Chloroflexi* pan-genome contains 4,348 genes, and in contrast to the *Chlorobi* pan-genome it is comprised of more putative genes for extracelluar constituents, inter-cellular communication, and other physiological and biochemical activities. For example, genes involved in the 3-hydroxypropionate pathway for carbon fixation were found. But generally, the *Chloroflexi* core-genome equips most of the major functional genes for a wide range of metabolisms such as synthesis of organic compounds, energy production, transport, genetic processing, etc. Such coverage throughout most cellular activities makes the core-genome of the *Chloroflexi* similar in character to that of *Chlorobia*.

#### **2.3.4** *Cyanobacteria*

Representing the largest sampled phylogenetic clade of the phototrophic prokaryotes, the *Cyanobacteria* have 37 completely sequenced genomes available for analysis, resulting in the

The Core- and Pan-Genomes of Photosynthetic Prokaryotes 433

were identified in the diazotrophic *Bradyrhizobiaceae* and *Rhizobiaceae*. Most photosynthetic bacteria in these two orders are capable of forming mutualistic symbiosis with terrestrial plants by fixing nitrogen inside special structures called legumes. Genes for hydrogen production or metabolizing C1-compounds such as methane were identified in *Rhodopseudomonas*, some *Rhodobacteraceae*, and *Methylobacterium*. These traits have garned much-warranted attention for their potential ability to reduce CH4 (greenhouse gas) emission (Eller & Frenzel, 2001; Lidstrom & Chistoserdova, 2002). Based on the construction of both core- and pan-genomes, photosynthetic members in the *Proteobacteria* exhibit the greatest gene diversity amongst all phyla studied. This diversity reflects their ability to grow chemoheterophically as well as phototrophically, which makes them better at living in a

Taken together, we have identified core-genes responsible for phylum-specific reactions. We have also observed a wide variety of accessory functions supporting smaller groups of bacteria. The core-genome assembled from a group of closely related bacteria represents a backbone of essential components regulating the adaptability to specific niches. Our results indicate that the gene content of each phylum-specific core is distinctive and can exemplify the very different evolutionary histories of the major photosynthetic groups, where the accessory components comprising the pan-genomes provide fitness advantages in distinct

Construction of both core- and pan-genomes of all photosynthetic bacteria provides a novel opportunity to determine the phylogenetic relationship among these prokaryotes. Several methods have been used to evaluate the phylogenies of different bacterial groups such as single-gene phylogenies (e.g., 16S rDNA), concatenated sequences of photosynthesis-related proteins (Rokas et al., 2003), and signature sequences of house-keeping proteins (Gupta, 2003). The sequences compared in these methods are necessarily present in all analyzed species. Here, we present a phylogeny that is formulated using the clustered pan-genome that does not rely on a universally shared collection of genes. Hierarchical clustering with resampling 100 times was performed based on a relative Manhattan distance calculated on the presence/absence of an ortholog between a given pair of genomes (Snipen & Ussery, 2010). It in essence generates a tree based on shared gene content. Figure 6 shows the resulting tree. There is broad agreement with this tree and traditional single-gene phylogenies. But surprisingly, the topology of the tree shows that both *A. vinosum* DSM 180 and *H. halophila* SL1, both belonging to the γ-*Proteobacteria* class, are situated outside of the *Proteobacteria* clade and positioned between the *Chlorobi* and *Firmicutes*. We investigated in detail the gene content of these two organisms and found that *A. vinosum* DSM 180 has lost most of the *Proteobacteria*-specific orthologs, while *H. halophila* SL1 contains more shared orthologs with *A. vinosum* DSM 180 than between the other purple bacteria species. Another unusual topology is found in the *Proteobacteria* clade where both *Rhodobacter* and *Rhodospirillum* families are closer to the *Rhodopesudomonas* genus, which belongs phylogenetically to the

We further utilized the pan-genome to reveal a three-dimensional relationship between individual species and the major photosynthetic lineages. By performing a multidimensional scaling analysis of the ortholog distribution across all 82 species, we found that related species were clustered in groups reflecting their phyla (Figure 7). While the *Heliobacteria*, *Chlorobi*, and *Chloroflexi* species occupied a central space, the *Proteobacteria* and *Cyanobacteria* were greatly

**2.4 Phylogeny of photosynthetic prokaryotes using the pan-genome** 

*Rhizobia* within the *Brydorhizobia* and *Methylobacteria* families.

broader range of environments than the *Cyanobacteria*.

habitats.

smallest core- (619 genes) and largest pan-genome (13,072 genes) of all five phyla. The proportion of genes designated "core" with respect to any cyanobacterial genome varies from less than 10% (in the the non-*Prochlorococcus*/*Synechococcus* genomes) to nearly 38% for *Prochlorococcus* and *Synechococcus* strains. The core orthologs are responsible for several major reactions such as the Calvin cycle, glycolysis, the incomplete TCA cycle, and pathways to synthesize amino acids and cofactors. Two types of photosystems (PS I and PS II) and the participating electron transport chain for oxygenic photosynthesis are also included.

The large pan-genome of cyanobacteria appears to support diverse abilities and processes. There are many genes found in the pan-genome that carry out metabolic activities unrelated to photosynthesis. For example, *M. aeruginosa* NIES-843 produces a diverse range of toxins with the non-ribosomal peptide synthetases (Kaneko et al., 2007). These enzymes produce neurotoxins and hepatotoxins that cause a variety of human illnesses, and are responsible for deaths in native and domestic animals. *T. erythraeum* IMS101 can perform nitrogen fixation in the presence of oxygen (Sandh et al., 2011). *Nostocaceae* species generally have an unbranched filamentous cell type, develop heterocysts, and possess multiple plasmids. In contrast, *Prochlorococcus* and *Synechococcus* species have a small round shape with no plasmids. Finally, a large number of genes identified in members of the *Nostocaceae* and *A. marina* MBIC11017 have unknown functions. Judging by the life style of these cyanobacterial species, which have a mutualistic relationship with terrestrial plants (Baker et al., 2003) and coral (Marquardt et al., 1997), it is possible that these genes are involved in supporting intercommunication and mutualism with their host.

#### **2.3.5** *Proteobacteria*

The *Proteobacteria* contain 644 core gene clusters and 13,207 non-redundant genes in the pan genome. The percentage of core genes in any of the *Proteobacteria* genomes varies from 10% to 25%, similar to the results obtained for the *Cyanobacteria*. This is because the *Proteobacteria* is the second major photosynthetic group with 28 completely sequenced genomes from phylogenetically distinct clades. The *Proteobacteria* core-genome preserves most of the key enzymes essential to major cellular activities, similar to other core-genomes. The type II reaction center and light-harvesting proteins are in the core genome, the former of which was also identified in the *Chloroflexi*. Nevertheless, the additional orthologs coding for the bacterial flagella, chemotaxis, and respiratory electron transfer chain proteins unique to the *Proteobacteria* were also identified. Both flagella and chemotaxis help cells move either toward nutrients or away from unfavourable living conditions and both anaerobic and aerobic respiration supports chemo-heterotrophic growth when phototrophic growth is not possible. Thus, integrating both cell mobility and respiration to the *Proteobacteria* core genome suggests an ecological advantage of adaptation to a broader range of living environments than other phototrophic phyla.

In contrast to the core-genome, the characteristics of the pan-genome are widely diverse- from variant types of nitrogen assimilation, carbon assimilation, and hydrogen metabolism to inter-cellular communications and nodulation. Such vast variety in the functional repertoire associated with the pan-genome can give the *proteobacteria*, such as *Rhodobacter*, *Rhodopseudomonas*, and *Rhodospillium* genera, a broad range of growth conditions for anaerobic phototrophy and aerobic chemoheterotrophy in the absence of light (Larimer et al., 2004; Lu et al., 2010; Mackenzie et al., 2007). Over 50 genes associated with nodulation

smallest core- (619 genes) and largest pan-genome (13,072 genes) of all five phyla. The proportion of genes designated "core" with respect to any cyanobacterial genome varies from less than 10% (in the the non-*Prochlorococcus*/*Synechococcus* genomes) to nearly 38% for *Prochlorococcus* and *Synechococcus* strains. The core orthologs are responsible for several major reactions such as the Calvin cycle, glycolysis, the incomplete TCA cycle, and pathways to synthesize amino acids and cofactors. Two types of photosystems (PS I and PS II) and the participating electron transport chain for oxygenic photosynthesis are also

The large pan-genome of cyanobacteria appears to support diverse abilities and processes. There are many genes found in the pan-genome that carry out metabolic activities unrelated to photosynthesis. For example, *M. aeruginosa* NIES-843 produces a diverse range of toxins with the non-ribosomal peptide synthetases (Kaneko et al., 2007). These enzymes produce neurotoxins and hepatotoxins that cause a variety of human illnesses, and are responsible for deaths in native and domestic animals. *T. erythraeum* IMS101 can perform nitrogen fixation in the presence of oxygen (Sandh et al., 2011). *Nostocaceae* species generally have an unbranched filamentous cell type, develop heterocysts, and possess multiple plasmids. In contrast, *Prochlorococcus* and *Synechococcus* species have a small round shape with no plasmids. Finally, a large number of genes identified in members of the *Nostocaceae* and *A. marina* MBIC11017 have unknown functions. Judging by the life style of these cyanobacterial species, which have a mutualistic relationship with terrestrial plants (Baker et al., 2003) and coral (Marquardt et al., 1997), it is possible that these genes are involved in supporting inter-

The *Proteobacteria* contain 644 core gene clusters and 13,207 non-redundant genes in the pan genome. The percentage of core genes in any of the *Proteobacteria* genomes varies from 10% to 25%, similar to the results obtained for the *Cyanobacteria*. This is because the *Proteobacteria* is the second major photosynthetic group with 28 completely sequenced genomes from phylogenetically distinct clades. The *Proteobacteria* core-genome preserves most of the key enzymes essential to major cellular activities, similar to other core-genomes. The type II reaction center and light-harvesting proteins are in the core genome, the former of which was also identified in the *Chloroflexi*. Nevertheless, the additional orthologs coding for the bacterial flagella, chemotaxis, and respiratory electron transfer chain proteins unique to the *Proteobacteria* were also identified. Both flagella and chemotaxis help cells move either toward nutrients or away from unfavourable living conditions and both anaerobic and aerobic respiration supports chemo-heterotrophic growth when phototrophic growth is not possible. Thus, integrating both cell mobility and respiration to the *Proteobacteria* core genome suggests an ecological advantage of adaptation to a broader range of living

In contrast to the core-genome, the characteristics of the pan-genome are widely diverse- from variant types of nitrogen assimilation, carbon assimilation, and hydrogen metabolism to inter-cellular communications and nodulation. Such vast variety in the functional repertoire associated with the pan-genome can give the *proteobacteria*, such as *Rhodobacter*, *Rhodopseudomonas*, and *Rhodospillium* genera, a broad range of growth conditions for anaerobic phototrophy and aerobic chemoheterotrophy in the absence of light (Larimer et al., 2004; Lu et al., 2010; Mackenzie et al., 2007). Over 50 genes associated with nodulation

included.

communication and mutualism with their host.

environments than other phototrophic phyla.

**2.3.5** *Proteobacteria* 

were identified in the diazotrophic *Bradyrhizobiaceae* and *Rhizobiaceae*. Most photosynthetic bacteria in these two orders are capable of forming mutualistic symbiosis with terrestrial plants by fixing nitrogen inside special structures called legumes. Genes for hydrogen production or metabolizing C1-compounds such as methane were identified in *Rhodopseudomonas*, some *Rhodobacteraceae*, and *Methylobacterium*. These traits have garned much-warranted attention for their potential ability to reduce CH4 (greenhouse gas) emission (Eller & Frenzel, 2001; Lidstrom & Chistoserdova, 2002). Based on the construction of both core- and pan-genomes, photosynthetic members in the *Proteobacteria* exhibit the greatest gene diversity amongst all phyla studied. This diversity reflects their ability to grow chemoheterophically as well as phototrophically, which makes them better at living in a broader range of environments than the *Cyanobacteria*.

Taken together, we have identified core-genes responsible for phylum-specific reactions. We have also observed a wide variety of accessory functions supporting smaller groups of bacteria. The core-genome assembled from a group of closely related bacteria represents a backbone of essential components regulating the adaptability to specific niches. Our results indicate that the gene content of each phylum-specific core is distinctive and can exemplify the very different evolutionary histories of the major photosynthetic groups, where the accessory components comprising the pan-genomes provide fitness advantages in distinct habitats.

#### **2.4 Phylogeny of photosynthetic prokaryotes using the pan-genome**

Construction of both core- and pan-genomes of all photosynthetic bacteria provides a novel opportunity to determine the phylogenetic relationship among these prokaryotes. Several methods have been used to evaluate the phylogenies of different bacterial groups such as single-gene phylogenies (e.g., 16S rDNA), concatenated sequences of photosynthesis-related proteins (Rokas et al., 2003), and signature sequences of house-keeping proteins (Gupta, 2003). The sequences compared in these methods are necessarily present in all analyzed species. Here, we present a phylogeny that is formulated using the clustered pan-genome that does not rely on a universally shared collection of genes. Hierarchical clustering with resampling 100 times was performed based on a relative Manhattan distance calculated on the presence/absence of an ortholog between a given pair of genomes (Snipen & Ussery, 2010). It in essence generates a tree based on shared gene content. Figure 6 shows the resulting tree.

There is broad agreement with this tree and traditional single-gene phylogenies. But surprisingly, the topology of the tree shows that both *A. vinosum* DSM 180 and *H. halophila* SL1, both belonging to the γ-*Proteobacteria* class, are situated outside of the *Proteobacteria* clade and positioned between the *Chlorobi* and *Firmicutes*. We investigated in detail the gene content of these two organisms and found that *A. vinosum* DSM 180 has lost most of the *Proteobacteria*-specific orthologs, while *H. halophila* SL1 contains more shared orthologs with *A. vinosum* DSM 180 than between the other purple bacteria species. Another unusual topology is found in the *Proteobacteria* clade where both *Rhodobacter* and *Rhodospirillum* families are closer to the *Rhodopesudomonas* genus, which belongs phylogenetically to the *Rhizobia* within the *Brydorhizobia* and *Methylobacteria* families.

We further utilized the pan-genome to reveal a three-dimensional relationship between individual species and the major photosynthetic lineages. By performing a multidimensional scaling analysis of the ortholog distribution across all 82 species, we found that related species were clustered in groups reflecting their phyla (Figure 7). While the *Heliobacteria*, *Chlorobi*, and *Chloroflexi* species occupied a central space, the *Proteobacteria* and *Cyanobacteria* were greatly

The Core- and Pan-Genomes of Photosynthetic Prokaryotes 435

Fig. 7. Multidimensional scaling analysis showing the organization of 82 photosynthetic

prokaryotes in a pan-genomic distribution.

separated and located on opposing poles. The relative position of the *Proteobacteria* and *Cyanobacteria* groups apart from each other indicate that their ortholog profiles have diverged substantially. Additionally, the distribution of both *Cyanobacteria* and *Proteobacteria* species is also consistent with their phylogenetic positions in Figure 6. However, the γ*-Proteobacterial* species, *A. vinosum* DSM 180 and *H. halophila* SL1 were exceptionally close to both *Chlorobi* and *Chloroflexi*, a result similar to the two-dimensional pan-genome-based phylogenetic tree. Clustering organisms by determining the occurrence of the specific patterns of orthologs shared by a group of species reveals an overall pattern consistent with both 16s rDNA- and pan-genome-based phylogenies. Yet, the observation of shared orthologs in one or a group of species can highlight functional divergence or convergence in groups that can be quantified by gene analysis but missed by single-gene-based phylogenies.

Fig. 6. Phylogenetic relationship of 82 photosynthetic prokaryotes reconstructed with the pan-genome

separated and located on opposing poles. The relative position of the *Proteobacteria* and *Cyanobacteria* groups apart from each other indicate that their ortholog profiles have diverged substantially. Additionally, the distribution of both *Cyanobacteria* and *Proteobacteria* species is also consistent with their phylogenetic positions in Figure 6. However, the γ*-Proteobacterial* species, *A. vinosum* DSM 180 and *H. halophila* SL1 were exceptionally close to both *Chlorobi* and *Chloroflexi*, a result similar to the two-dimensional pan-genome-based phylogenetic tree. Clustering organisms by determining the occurrence of the specific patterns of orthologs shared by a group of species reveals an overall pattern consistent with both 16s rDNA- and pan-genome-based phylogenies. Yet, the observation of shared orthologs in one or a group of species can highlight functional divergence or convergence in groups that can be quantified by

Fig. 6. Phylogenetic relationship of 82 photosynthetic prokaryotes reconstructed with the

pan-genome

gene analysis but missed by single-gene-based phylogenies.

Fig. 7. Multidimensional scaling analysis showing the organization of 82 photosynthetic prokaryotes in a pan-genomic distribution.

The Core- and Pan-Genomes of Photosynthetic Prokaryotes 437

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#### **3. Acknowledgements**

This work was supported by the U.S. National Science Foundation Phototrophic Prokaryotes Sequencing Project, grant number 0950550.

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**21** 

*China* 

**A Review: Polyamines and Photosynthesis** 

*Vegetable Crop Genetic Improvement, Ministry of Agriculture, Nanjing* 

*College of Horticulture, Nanjing Agricultural University, Key Laboratory of Southern* 

Polyamines (PAs) are low molecular weight ubiquitous nitrogenous compounds found in all living organisms (Kaur-Sawhney et al., 2003). In higher plants, the most common polyamines are spermidine (Spd), spermine (Spm) and their diamine obligate precursor putrescine (Put). They are formed by aliphatic hydrocarbons substituted with two or more amino groups (Figure.1). Because of the polycationic nature at physiological pH, PAs are present in the free form or as conjugates bound to phenolic acids and other low molecular weight compounds or to proteins and nucleic acids (Childs et al., 2003). Like hormones, PAs displaying high biological activity are involved in a wide array of fundamental processes in plants, such as replication and gene expression, growth and development, senescence, membrane stabilization, enzyme activity modulation and adaptation to abiotic and biotic stresses (Galston et al., 1997; Bais and Ravishankar, 2002; Zapata et al., 2008). Although, according to these reports, PAs seem to be important growth regulators, their precise

It has been shown that chloroplasts and photosynthetic subcomplexes including thylakoids, LHCII complex and PSII membranes are enriched with three major polyamines, while PSII core and the reaction center of PSII are exclusively rich in Spm (Kotzabasis et al., 1993; Navakoudis et al., 2003). The potential role of polyamines in maintaining the photochemical efficiency of plants has become a research focus. These studies mainly focused on the effect PAs exert a positive role in the photosynthesis of plants in response to various environmental stresses. In green alga, it was shown that the bound Put content of the thylakoid membrane was increased in environments with high CO2 concentrations, which caused an increase in reaction center density and led to an increased photosynthetic rate (Logothetis et al., 2004). An increase in conjugated Put content can stabilize the thylakoid membrane, thus enhancing resistance of tobacco plants to ozone pollution (Navakoudis et al., 2003) and UV-B radiation (Lütz et al., 2005). Low temperature stress reduced the content of Put as well as the Put/Spm ratio in thylakoids and the light-harvesting complexes LHCII in *Phaseolus vulgaris* L., leading to a decrease in photosynthetic electron transport rate and inactivation of the PSII reaction center (Sfakianaki et al., 2006). Put is also involved in the induction of a photosynthetic apparatus owning high concentration of reaction center with a small functional antenna that leads to enhance photochemical quenching of the absorbed light energy (Kotzabasis et al., 1999). PAs biosynthesis is controlled by light and the Spm/Put ratio is correlated to the structure and function of the photosynthetic apparatus

physiological function and mechanism of action still remain unclear.

**1. Introduction** 

Sheng Shu, Shi-Rong Guo and Ling-Yun Yuan


### **A Review: Polyamines and Photosynthesis**

Sheng Shu, Shi-Rong Guo and Ling-Yun Yuan

*College of Horticulture, Nanjing Agricultural University, Key Laboratory of Southern Vegetable Crop Genetic Improvement, Ministry of Agriculture, Nanjing China* 

#### **1. Introduction**

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Polyamines (PAs) are low molecular weight ubiquitous nitrogenous compounds found in all living organisms (Kaur-Sawhney et al., 2003). In higher plants, the most common polyamines are spermidine (Spd), spermine (Spm) and their diamine obligate precursor putrescine (Put). They are formed by aliphatic hydrocarbons substituted with two or more amino groups (Figure.1). Because of the polycationic nature at physiological pH, PAs are present in the free form or as conjugates bound to phenolic acids and other low molecular weight compounds or to proteins and nucleic acids (Childs et al., 2003). Like hormones, PAs displaying high biological activity are involved in a wide array of fundamental processes in plants, such as replication and gene expression, growth and development, senescence, membrane stabilization, enzyme activity modulation and adaptation to abiotic and biotic stresses (Galston et al., 1997; Bais and Ravishankar, 2002; Zapata et al., 2008). Although, according to these reports, PAs seem to be important growth regulators, their precise physiological function and mechanism of action still remain unclear.

It has been shown that chloroplasts and photosynthetic subcomplexes including thylakoids, LHCII complex and PSII membranes are enriched with three major polyamines, while PSII core and the reaction center of PSII are exclusively rich in Spm (Kotzabasis et al., 1993; Navakoudis et al., 2003). The potential role of polyamines in maintaining the photochemical efficiency of plants has become a research focus. These studies mainly focused on the effect PAs exert a positive role in the photosynthesis of plants in response to various environmental stresses. In green alga, it was shown that the bound Put content of the thylakoid membrane was increased in environments with high CO2 concentrations, which caused an increase in reaction center density and led to an increased photosynthetic rate (Logothetis et al., 2004). An increase in conjugated Put content can stabilize the thylakoid membrane, thus enhancing resistance of tobacco plants to ozone pollution (Navakoudis et al., 2003) and UV-B radiation (Lütz et al., 2005). Low temperature stress reduced the content of Put as well as the Put/Spm ratio in thylakoids and the light-harvesting complexes LHCII in *Phaseolus vulgaris* L., leading to a decrease in photosynthetic electron transport rate and inactivation of the PSII reaction center (Sfakianaki et al., 2006). Put is also involved in the induction of a photosynthetic apparatus owning high concentration of reaction center with a small functional antenna that leads to enhance photochemical quenching of the absorbed light energy (Kotzabasis et al., 1999). PAs biosynthesis is controlled by light and the Spm/Put ratio is correlated to the structure and function of the photosynthetic apparatus

A Review: Polyamines and Photosynthesis 441

chloroplasts seemed to induce an imbalance between capture and utilization of light in photosynthesis. Although these changes were accompanied by thylakoid scattering, membrane degradation and reduction of thylakoid interconnections, transplastomic plants

At present, the roles of PAs in the structure and functions of the photosynthetic apparatus are widely investigated. Most of researchers consider the PAs and their related-metabolic enzymes be positive regulators of plant photosynthesis in response to various environmental stresses. However, the specific mechanism of polyamine on the protection of photochemical efficiency of stressed-plants remains until today largely unknown. Thus, we need use advanced molecular biology and proteomic approaches to further understanding

Stomatal is defined by two guard cells and is responsible for gas exchange between plants and the atmosphere (Mansfield et al., 1990). Stomatal plays a key role in signal transduction, sensing and adaptive responses to abiotic stresses like drought, heat, chilling and high salinity (Hetherington and Woodward, 2003). Polyamines regulated stomatal closely correlated with the improvement of photosynthesis, which could be caused by the greater amount of CO2 available for its fixation by photosynthetic enzymes. However, most of the roles of polyamines in regulating stomatal research are focused on environmental stress

All natural polyamines, including cadaverine (Cad) and putrescine (Put), spermidine (Spd), spermine (Spm) at a given concentration, strongly inhibited opening of stomata. Liu et al. (2000) found that 1 mM Spd and Spm completely prevented light-induced stomatal opening, whereas Cad and Put inhibited this opening by 88% and 63%, respectively. Although all polyamines significantly reduced the stomatal aperture, Spd and Spm appeared to be more effective than Put at 1 mM. Çavuşoğlu et al. (2007) showed that polyamine-pretreaments could decrease stomata number and length in the upper surface under saline conditions by reducing the transpiration. In the Spm-pretreated Citrus Reticulata samples were observed in smaller stomatal aperture size than the control at a given time point or during the whole

Many environmental factors regulated stomatal aperture through modulation of ion channel activity in guard cells (MacRobbie, 1997). Changes in guard cell turgor that instigate stomatal movements are controlled by a number of ion channels and pumps (Raschke et al.,

could be maintained and reproduced in vitro.

the role of PAs in the regulation of photosynthetic processes.

Fig. 1. The chemical structure of three major polyamines

conditions, and the specific mechanisms are still unclear.

**2. Effect of polyamines on photosynthesis** 

**2.1 Stomatal opening/ closure** 

experiment (Shi et al., 2010).

during photoadaptation. These studies indicated that changes of endogenous polyamines might be involved in an important protective role in the photosynthetic apparatus.

A lot of researches started to pay attention to the effects of application of exogenous polyamines on photosynthesis under various stresses. It has been demonstrated that exogenously applied polyamines can rapidly enter the intact chloroplast (He et al., 2002) and play a role in protecting the photosynthetic apparatus from adverse effects of environmental stresses (Navakoudis et al., 2003). However, the effect of polyamines on the photosynthetic efficiency of stressed plants depends on the stress level and the type of exogenous polyamines. Exogenous polyamines improved the photosynthetic capacity of salt-stressed cucumber plants by increasing the level of the photochemical efficiency of PSII (Zhang et al., 2009). In green alga *Scenedesmus obliquus* cultures, exogenously added Put was used to adjust the increase in the functional size of the antenna and the reduction in the density of active photosystem II reaction centers, so that to confer some kind of tolerance to the photosynthetic apparatus against enhanced NaCl-salinity and permit cell growth even in NaCl concentrations that under natural conditions would be toxic (Demetriou et al., 2007). Investigations into restoration of the maximum photochemical efficiency (Fv/Fm) by adding Put, Spd and Spm to low salt thylakoid showed that Spd are the most efficient ones in Fv/Fm restoration, but higher amounts of Spm and/or Spd reverse the effect and lead to a decline of the Fv/Fm (Ioannidis and Kotzabasis, 2007). When *Physcia semipinnata* was exposed to UV-A radiation, it was also found that exogenously Spd added samples had higher Chl a content and photosystem II activity than Spm and Put added samples (Unal et al., 2008). In addition, analysis of PSII particles isolated from leaf fragments floated in the presence of Put, Spd and Spm solutions under the dark conditions was conducted. It was observed that Spd could interact directly with thylakoid membranes, which was effective in the retardation of the loss of LHCII observed in water-treated detached leaves, so that they become more stable to degradation during senescence (Legocka and Zajchert, 1999).

Several studies have shown that chloroplasts contain high activities of polyamine biosynthetic enzymes and transglutaminase (TGase) catalyzing the covalent binding of polyamines to proteins (Del Duca et al., 1994; Andreadakis and Kotzabasis, 1996; Della Mea et al., 2004) (Table 1). These enzymes are also involved in regulation of photosynthesis in response to stress conditions (Wang et al., 2010). Arginine decarboxylase (ADC) has been shown to be mainly localised in the chloroplasts of leaves and nuclei of roots (Borrell et al., 1995). It was established that spinach ADC was associated with LHC of photosystem II (Legocka and Zaichert, 1999). PAs synthesised in chloroplasts evidently stabilized photosynthetic complexes of thylakoid membranes under stress conditions (Borrell et al., 1995). An evidence is supported by salt treatment induced a decreased chlorophyll content and photosynthetic efficiency in the lower arginine decarboxylase activity of mutant plants, which leads to reduced salt tolerance in Arabidopsis thaliana (Kasinathan and Wingler, 2004). TGase is present in the chloroplasts of higher plants, where its activity is modulated by the presence of light. Its substrates are Rubisco and some antenna complexes of thylakoids, such as LHCII, CP29, CP26 and CP24 (Del Duca et al., 1994). In *Dunaliella salina* whole cells, TGase seems to play a role in the acclimimation to high salt concentrations under light condition, and the content of chlorophylla and b of chloroplast were enhanced, the amount of 68kD and 55kDa polypeptides was particularly high in algae already acclimated cells (Dondini et al., 2001). Recently, Ortigosa et al. (2009) showed that the overexpression of maize plastidial transglutaminas (chlTGZ) in the young leaves of tobacco

during photoadaptation. These studies indicated that changes of endogenous polyamines

A lot of researches started to pay attention to the effects of application of exogenous polyamines on photosynthesis under various stresses. It has been demonstrated that exogenously applied polyamines can rapidly enter the intact chloroplast (He et al., 2002) and play a role in protecting the photosynthetic apparatus from adverse effects of environmental stresses (Navakoudis et al., 2003). However, the effect of polyamines on the photosynthetic efficiency of stressed plants depends on the stress level and the type of exogenous polyamines. Exogenous polyamines improved the photosynthetic capacity of salt-stressed cucumber plants by increasing the level of the photochemical efficiency of PSII (Zhang et al., 2009). In green alga *Scenedesmus obliquus* cultures, exogenously added Put was used to adjust the increase in the functional size of the antenna and the reduction in the density of active photosystem II reaction centers, so that to confer some kind of tolerance to the photosynthetic apparatus against enhanced NaCl-salinity and permit cell growth even in NaCl concentrations that under natural conditions would be toxic (Demetriou et al., 2007). Investigations into restoration of the maximum photochemical efficiency (Fv/Fm) by adding Put, Spd and Spm to low salt thylakoid showed that Spd are the most efficient ones in Fv/Fm restoration, but higher amounts of Spm and/or Spd reverse the effect and lead to a decline of the Fv/Fm (Ioannidis and Kotzabasis, 2007). When *Physcia semipinnata* was exposed to UV-A radiation, it was also found that exogenously Spd added samples had higher Chl a content and photosystem II activity than Spm and Put added samples (Unal et al., 2008). In addition, analysis of PSII particles isolated from leaf fragments floated in the presence of Put, Spd and Spm solutions under the dark conditions was conducted. It was observed that Spd could interact directly with thylakoid membranes, which was effective in the retardation of the loss of LHCII observed in water-treated detached leaves, so that they

might be involved in an important protective role in the photosynthetic apparatus.

become more stable to degradation during senescence (Legocka and Zajchert, 1999).

Several studies have shown that chloroplasts contain high activities of polyamine biosynthetic enzymes and transglutaminase (TGase) catalyzing the covalent binding of polyamines to proteins (Del Duca et al., 1994; Andreadakis and Kotzabasis, 1996; Della Mea et al., 2004) (Table 1). These enzymes are also involved in regulation of photosynthesis in response to stress conditions (Wang et al., 2010). Arginine decarboxylase (ADC) has been shown to be mainly localised in the chloroplasts of leaves and nuclei of roots (Borrell et al., 1995). It was established that spinach ADC was associated with LHC of photosystem II (Legocka and Zaichert, 1999). PAs synthesised in chloroplasts evidently stabilized photosynthetic complexes of thylakoid membranes under stress conditions (Borrell et al., 1995). An evidence is supported by salt treatment induced a decreased chlorophyll content and photosynthetic efficiency in the lower arginine decarboxylase activity of mutant plants, which leads to reduced salt tolerance in Arabidopsis thaliana (Kasinathan and Wingler, 2004). TGase is present in the chloroplasts of higher plants, where its activity is modulated by the presence of light. Its substrates are Rubisco and some antenna complexes of thylakoids, such as LHCII, CP29, CP26 and CP24 (Del Duca et al., 1994). In *Dunaliella salina* whole cells, TGase seems to play a role in the acclimimation to high salt concentrations under light condition, and the content of chlorophylla and b of chloroplast were enhanced, the amount of 68kD and 55kDa polypeptides was particularly high in algae already acclimated cells (Dondini et al., 2001). Recently, Ortigosa et al. (2009) showed that the overexpression of maize plastidial transglutaminas (chlTGZ) in the young leaves of tobacco chloroplasts seemed to induce an imbalance between capture and utilization of light in photosynthesis. Although these changes were accompanied by thylakoid scattering, membrane degradation and reduction of thylakoid interconnections, transplastomic plants could be maintained and reproduced in vitro.

At present, the roles of PAs in the structure and functions of the photosynthetic apparatus are widely investigated. Most of researchers consider the PAs and their related-metabolic enzymes be positive regulators of plant photosynthesis in response to various environmental stresses. However, the specific mechanism of polyamine on the protection of photochemical efficiency of stressed-plants remains until today largely unknown. Thus, we need use advanced molecular biology and proteomic approaches to further understanding the role of PAs in the regulation of photosynthetic processes.

Fig. 1. The chemical structure of three major polyamines

#### **2. Effect of polyamines on photosynthesis**

#### **2.1 Stomatal opening/ closure**

Stomatal is defined by two guard cells and is responsible for gas exchange between plants and the atmosphere (Mansfield et al., 1990). Stomatal plays a key role in signal transduction, sensing and adaptive responses to abiotic stresses like drought, heat, chilling and high salinity (Hetherington and Woodward, 2003). Polyamines regulated stomatal closely correlated with the improvement of photosynthesis, which could be caused by the greater amount of CO2 available for its fixation by photosynthetic enzymes. However, most of the roles of polyamines in regulating stomatal research are focused on environmental stress conditions, and the specific mechanisms are still unclear.

All natural polyamines, including cadaverine (Cad) and putrescine (Put), spermidine (Spd), spermine (Spm) at a given concentration, strongly inhibited opening of stomata. Liu et al. (2000) found that 1 mM Spd and Spm completely prevented light-induced stomatal opening, whereas Cad and Put inhibited this opening by 88% and 63%, respectively. Although all polyamines significantly reduced the stomatal aperture, Spd and Spm appeared to be more effective than Put at 1 mM. Çavuşoğlu et al. (2007) showed that polyamine-pretreaments could decrease stomata number and length in the upper surface under saline conditions by reducing the transpiration. In the Spm-pretreated Citrus Reticulata samples were observed in smaller stomatal aperture size than the control at a given time point or during the whole experiment (Shi et al., 2010).

Many environmental factors regulated stomatal aperture through modulation of ion channel activity in guard cells (MacRobbie, 1997). Changes in guard cell turgor that instigate stomatal movements are controlled by a number of ion channels and pumps (Raschke et al.,

A Review: Polyamines and Photosynthesis 443

stabilisation could be due to their modification of chlorophyll-bound proteins, catalysed by TGase. In senescing leaves, foliar spray with 0.2 mM Spm treatment prevented degradation of Chla and Chlb, and increased TGase activity, producing more PA-protein conjugates. Spm was translocated to chloroplasts and bound mainly onto fractions enriched in PSII, whose light-harvesting complexes (LHC) sub-fractions contained TGase (Serafini-Fracassini

CO2 assimilation is the process of carbohydrates formation which utilized the ATP and NADPH produced by light. The capacity of CO2 assimilation is connected with the plant growth, biomass and productivity. However, the effect of polyamines on CO2 assimilation has been investigated very rarely and only a few information concerning the relationship

Iqbal and Ashraf (2005) analyzed the influence of pre-sowing seed treatment with PAs on growth and photosynthetic capacity in two spring wheat (*Triticum aestivum* L.) cultivars MH-97(salt intolerant) and Inqlab-91(salt tolerant). The results showed that different priming agent did not affect the net CO2 assimilation rate. The role of three PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)] in improving drought tolerance in fine grain aromatic rice (*Oryza Sativa* L.) has been appraised by Farooq et al. (2009). Three of them were used each at 10 µM as seed priming (by soaking seeds in solution) and foliar spray. Drought stress significantly reduced maximum leaf CO2 assimilation rate, while application of PAs significantly improved the leaf CO2 assimilation rate but decreased Gs and transpiration rate (Tr) under drought stress. The resultes suggested that PAs enhanced drought tolerance in rice was due to improved CO2 assimilation by Rubisco in producing photosyntheate and their partitioning in dry matter yield. Huang et al. (2010) found 0.1 mM CA treatment decreased P*N*, but it did not affect the photosynthetic apparatus, which suggest that the decline is at least partially attributed to a lowered RuBPC activity. However, the exogenous application of 1 mM Spd partially restored RuBPC activity in leaves, the key enzyme of carbon reduction cycle (dark reaction), and thus improving the photosynthetic rate. Chen et al. (2010) reported that exogenous Spd application decreased the carbohydrate accumulation in leaves and total sugar, sucrose content in roots under salt stress, thus reduced negative feedback inhibition to photosynthesis caused by carbohydrate accumulation. Exogenous application of Spd can alleviate the damage caused by hypoxia stress and enhance the conversion and use of carbohydrate in roots, which can promote the formation of new metabolic balance seedlings attributed to tolerance of hypoxia stress

It is necessary to maintain structural integrity and orderliness of chloroplast that plays a role in conversion of light energy for photosynthesis (Li et al., 2009). The chloroplasts are usually 5-10 micrometer long and consist of circular DNA molecules. In higher plants, the photosynthetic machinery is mainly localized in thylakoid membranes of the chloroplasts (Kirchhoff et al., 2007). The membrane has no homogenous structure, but is subdivided into two domains: the strictly stacked grana thylakoids and the unstacked stroma lamellae (Kirchhoff et al., 2003). The structure of thylakoids is a major factor that affects functionality and performance of the photosynthetic apparatus (Ioannidis et al., 2009). It was reported

et al., 2010).

**2.3 CO2 assimilation** 

(Zhou et al., 2007)

**2.4 Chloroplast ultrastructure** 

between PAs and carbon assimilation.

1988; Ward et al., 1995). As an important player in stomatal regulation, the I Kin is an indirect target of polyamine action. A number of studies have shown that I Kin-inhibiting processes or factors often inhibit stomatal opening (Assmann, 1993). Liu et al. (2000) using patch-clamp analysis demonstrated that intracellular application of polyamines inhibited the inward K1 current across the plasma membrane of Vicia faba guard cells and modulated stomatal movement. Changes of free Ca2+ in the cytoplasm of guard cells are involved in stomatal aperture/closure. In the Spm-deficient mutant Arabidopsis seems to be impaired in Ca2+ homeostasis, which affects the stomatal movement, and that this inhibited effect was restored by application of exogenous Spd in the mutant plants (Yamaguchi et al., 2007). In addition, it has been reported that polyamine-induced ROS scavenging is an essential effect and stimulated stomatal closure (lower water loss) upon dehydration, which may function collectively to enhance dehydration tolerance (Pei et al., 2000; Bright et al., 2006). These findings suggest that polyamines target KAT1-like inward K1 channels in guard cells and modulate stomatal movements, providing a link between stress conditions, polyamine levels, and stomatal regulation.

#### **2.2 Photosynthetic pigment**

Chlorophyll (Chl) is a molecule substance that plays an important role in photosynthesis for the plant growth process, such as light absorption, and combination with protein complex, transfer the energy into carbohydrate (Meskauskiene et al., 2001). A variety of reports indicate that changes in chlorophyll levels of plants may decrease in response to environmental factors or leaf senescence (Sfichi-Duke et al., 2008; Munzi et al., 2009). Aliphatic polyamines (PAs) are involved in the delay loss of chlorophyll and lead to an increased efficiency of light capture resulting in the improvement of net photosynthetic rate, but the molecular mechanism is not clarified.

Positive effects of exogenous supplied polyamines on the content of Chla and total Chl in leaves were observed for various stresses, but there were distinct differences in the effect of three main polyamines. Unal et al. (2008) found that Chla content was significantly increased in *Physcia semipinnata* by exogenously added polyamines during exposure to UV-A radiation, and exogenously Spd added samples had higher Chla content than Spm and Put added samples. Spd delayed the loss of Chl more than Spd or Put in detached wheat leaves during dark incubation implying the importance of valency of organic cations (Subhan and Murthy, 2001). The result is in agreement with those of Aldesuquy et al. (2000) who reported that using detached wheat leaves infected with the yellow rust *Puccinia striiformis*. Among of exogenous supplied polyamines, in contrast with Put and Spd, Spm has been shown to regulate the in vivo amount of protochlorophyllide (PChlide) and Chl both in darkness and in light (Beigbeder and Kotzabasis, 1994). Beigbeder et al. (1995) suggested that the intracellular level of Put was decreased by the use of 1, 4-diamino-2 butanone inhibitor (1,4DB) dramatically increasing the PChlide levels with parallel reduction of chlorophyll. Many workers have reported retention of chlorophyll induced by exogenously supplied polyamines during the normal developmental senescence of leaves. Cheng and Kao (1983) demonstrated that Spd and Spm were effective in retarding loss of chlorophyll from detached leaves of rice, wheat and soybean. They describe the effect of locally applied polyamines as being similar to that of cytokinins. In plants of *Heliotropium* sp., leaf senescence is associated with low endogenous concentrations of polyamines (Birecka et al., 1984). One of the mechanisms by which PAs modulate chlorophyll stabilisation could be due to their modification of chlorophyll-bound proteins, catalysed by TGase. In senescing leaves, foliar spray with 0.2 mM Spm treatment prevented degradation of Chla and Chlb, and increased TGase activity, producing more PA-protein conjugates. Spm was translocated to chloroplasts and bound mainly onto fractions enriched in PSII, whose light-harvesting complexes (LHC) sub-fractions contained TGase (Serafini-Fracassini et al., 2010).

#### **2.3 CO2 assimilation**

442 Advances in Photosynthesis – Fundamental Aspects

1988; Ward et al., 1995). As an important player in stomatal regulation, the I Kin is an indirect target of polyamine action. A number of studies have shown that I Kin-inhibiting processes or factors often inhibit stomatal opening (Assmann, 1993). Liu et al. (2000) using patch-clamp analysis demonstrated that intracellular application of polyamines inhibited the inward K1 current across the plasma membrane of Vicia faba guard cells and modulated stomatal movement. Changes of free Ca2+ in the cytoplasm of guard cells are involved in stomatal aperture/closure. In the Spm-deficient mutant Arabidopsis seems to be impaired in Ca2+ homeostasis, which affects the stomatal movement, and that this inhibited effect was restored by application of exogenous Spd in the mutant plants (Yamaguchi et al., 2007). In addition, it has been reported that polyamine-induced ROS scavenging is an essential effect and stimulated stomatal closure (lower water loss) upon dehydration, which may function collectively to enhance dehydration tolerance (Pei et al., 2000; Bright et al., 2006). These findings suggest that polyamines target KAT1-like inward K1 channels in guard cells and modulate stomatal movements, providing a link between stress conditions, polyamine

Chlorophyll (Chl) is a molecule substance that plays an important role in photosynthesis for the plant growth process, such as light absorption, and combination with protein complex, transfer the energy into carbohydrate (Meskauskiene et al., 2001). A variety of reports indicate that changes in chlorophyll levels of plants may decrease in response to environmental factors or leaf senescence (Sfichi-Duke et al., 2008; Munzi et al., 2009). Aliphatic polyamines (PAs) are involved in the delay loss of chlorophyll and lead to an increased efficiency of light capture resulting in the improvement of net photosynthetic rate,

Positive effects of exogenous supplied polyamines on the content of Chla and total Chl in leaves were observed for various stresses, but there were distinct differences in the effect of three main polyamines. Unal et al. (2008) found that Chla content was significantly increased in *Physcia semipinnata* by exogenously added polyamines during exposure to UV-A radiation, and exogenously Spd added samples had higher Chla content than Spm and Put added samples. Spd delayed the loss of Chl more than Spd or Put in detached wheat leaves during dark incubation implying the importance of valency of organic cations (Subhan and Murthy, 2001). The result is in agreement with those of Aldesuquy et al. (2000) who reported that using detached wheat leaves infected with the yellow rust *Puccinia striiformis*. Among of exogenous supplied polyamines, in contrast with Put and Spd, Spm has been shown to regulate the in vivo amount of protochlorophyllide (PChlide) and Chl both in darkness and in light (Beigbeder and Kotzabasis, 1994). Beigbeder et al. (1995) suggested that the intracellular level of Put was decreased by the use of 1, 4-diamino-2 butanone inhibitor (1,4DB) dramatically increasing the PChlide levels with parallel reduction of chlorophyll. Many workers have reported retention of chlorophyll induced by exogenously supplied polyamines during the normal developmental senescence of leaves. Cheng and Kao (1983) demonstrated that Spd and Spm were effective in retarding loss of chlorophyll from detached leaves of rice, wheat and soybean. They describe the effect of locally applied polyamines as being similar to that of cytokinins. In plants of *Heliotropium* sp., leaf senescence is associated with low endogenous concentrations of polyamines (Birecka et al., 1984). One of the mechanisms by which PAs modulate chlorophyll

levels, and stomatal regulation.

**2.2 Photosynthetic pigment** 

but the molecular mechanism is not clarified.

CO2 assimilation is the process of carbohydrates formation which utilized the ATP and NADPH produced by light. The capacity of CO2 assimilation is connected with the plant growth, biomass and productivity. However, the effect of polyamines on CO2 assimilation has been investigated very rarely and only a few information concerning the relationship between PAs and carbon assimilation.

Iqbal and Ashraf (2005) analyzed the influence of pre-sowing seed treatment with PAs on growth and photosynthetic capacity in two spring wheat (*Triticum aestivum* L.) cultivars MH-97(salt intolerant) and Inqlab-91(salt tolerant). The results showed that different priming agent did not affect the net CO2 assimilation rate. The role of three PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)] in improving drought tolerance in fine grain aromatic rice (*Oryza Sativa* L.) has been appraised by Farooq et al. (2009). Three of them were used each at 10 µM as seed priming (by soaking seeds in solution) and foliar spray. Drought stress significantly reduced maximum leaf CO2 assimilation rate, while application of PAs significantly improved the leaf CO2 assimilation rate but decreased Gs and transpiration rate (Tr) under drought stress. The resultes suggested that PAs enhanced drought tolerance in rice was due to improved CO2 assimilation by Rubisco in producing photosyntheate and their partitioning in dry matter yield. Huang et al. (2010) found 0.1 mM CA treatment decreased P*N*, but it did not affect the photosynthetic apparatus, which suggest that the decline is at least partially attributed to a lowered RuBPC activity. However, the exogenous application of 1 mM Spd partially restored RuBPC activity in leaves, the key enzyme of carbon reduction cycle (dark reaction), and thus improving the photosynthetic rate. Chen et al. (2010) reported that exogenous Spd application decreased the carbohydrate accumulation in leaves and total sugar, sucrose content in roots under salt stress, thus reduced negative feedback inhibition to photosynthesis caused by carbohydrate accumulation. Exogenous application of Spd can alleviate the damage caused by hypoxia stress and enhance the conversion and use of carbohydrate in roots, which can promote the formation of new metabolic balance seedlings attributed to tolerance of hypoxia stress (Zhou et al., 2007)

#### **2.4 Chloroplast ultrastructure**

It is necessary to maintain structural integrity and orderliness of chloroplast that plays a role in conversion of light energy for photosynthesis (Li et al., 2009). The chloroplasts are usually 5-10 micrometer long and consist of circular DNA molecules. In higher plants, the photosynthetic machinery is mainly localized in thylakoid membranes of the chloroplasts (Kirchhoff et al., 2007). The membrane has no homogenous structure, but is subdivided into two domains: the strictly stacked grana thylakoids and the unstacked stroma lamellae (Kirchhoff et al., 2003). The structure of thylakoids is a major factor that affects functionality and performance of the photosynthetic apparatus (Ioannidis et al., 2009). It was reported

A Review: Polyamines and Photosynthesis 445

subcomplexes Put and Spm levels fluctuated depending on the light adaptational status of the photosynthetic apparatus. Put and Spm are bound to the photosynthetic complexes, mainly to the LHCII oligomeric and monomeric forms (Navakoudis et al., 2007). It is well documented that the LHCII protein is abundant in thylakoids and its surface is negatively charged (Standfuss et al., 2005). Kirchhoff et al. (2007) have recently shown that incubation of thylakoids under unstacking conditions leads to intermixing and randomization of the protein complexes, accompanied by disconnection of LHCII trimers from PSII and a decreased connectivity between PSIIα centers. Interestingly, exogenously added polyamines can reverse those damaging effects. Thus, there was strong indication that polyamines

Chloroplasts also contain high activities of several polyamine biosynthetic enzymes (Borrell et al.1995; Andreadakis and Kotzabasis 1996) and transglutaminase (TGase) catalyzing the covalent binding of polyamines to proteins (Del-Duca et al., 1994; Dondini et al., 2001; Della-Mea et al., 2004). Polyamines can covalently bind with thylakoid membrane proteins under the TGase form of protein- glutamyl-polyamine or protein-glutamyl-polyamines-glutamylprotein (Dondini et al., 2003), such as D1, D2 protein and cytochrome b6/f, together with the involvement of polyamine, could be important for stabilisation of molecular complexes in the thylakoid membranes of osmotically stressed oat leaves (Besford et al., 1993). Immunodetection of TGase in thylakoid fraction revealed that formation of covalent bonds between PAs and proteins by TGase is involved in regulating the process of chloroplast

However, high concentration of polyamines added to submembrane fractions of photosynthetic apparatus causes a strong inhibition of PSII activity (Hamdani et al., 2010). FTIR spectroscopy analysis Spd and Spm at higher cation concentrations (5 and 10 mM), the result showed that the polyamine significant alterations of the thylakoid protein secondary structure with a decrease of the α-helical domains from 47% (uncomplexed PSII) up to 37% (cation complexes) and an increase in the β-sheet structure from 18% up to 29% (Bograh et al., 1997). So far, this specific inhibition mechanism of polyamines is not clear, but it is likely that the proteins were affected by these polycations re either extrinsic polypeptides associated with the oxygen evolving complex or portions of integral polypeptides

Salt stress causes an initial water-deficit and ion-specific stresses resulting from changes in K+/Na+ ratios. Thus, it leads to an increased Na+ and Cl− concentrations that decrease plant growth and productivity by disrupting physiological processes, especially photosynthesis (Shu et al., 2010). Salt stress affects photosynthetic efficiency of plant through stomatal limitation and non-stomatal limitations, such as stomatal closure (Meloni et al., 2003), chlorophyll content loss (Sudhir and Murthy, 2004), inhibition of Rubisco activity (Brugnoli and Björkman, 1992; Ziska et al., 1990), and degradation of membrane proteins in

It has been suggested that exogenous application of polyamines can to some extent alleviate salinity-induced decline in photosynthetic efficiency, but this effect strongly depended both on PAs concentrations or types and stress levels (Duan et al., 2008). The maximum quantum efficiency of PSII (Fv/Fm) measured in leaves of salt-stressed cucumber seedlings was not

protruding at the surface of the PSII membranes (Beauchemin et al., 2007).

possessed a pivotal role in photosynthesis.

senescence (Sobieszczuk-Nowicka et al., 2009).

**3. Polyamines and stress photosynthesis** 

photosynthetic apparatus (Khan and Ungar, 1997).

**3.1 Salinity** 

that some stresses led to the decrease in the photochemical efficiency and electron transport activity might be associated with the changes of the structure of photosynthetic apparatus (Parida et al., 2003).

Several studies have showed that polyamines are involved in stabilization the structure and function of photosynthetic apparatus in response to unfavourable environment factors (Demetriou et al., 2007). Under Nacl stress, Put as organic cations dramatically enhanced lipid accumulation in the chloroplasts and prevented the membrane degradation in the granal and stromal thylakoids by interacting with the negatively charged membrane sites (Tiburcio et al., 1994). The increase in the number of plastoglobules in the chloroplasts affected by Put can result from the redirection of cell metabolism towards the products of higher reduction potential (Paramonova et al., 2003). In our study, we observed that Put can alleviate the degradation of thylakoid membrane proteins induced by salt stress, and thus making a normal stacking order in the adjacent grana thylakoids (data not published). In addition, exogenous Spd have been reported to protect the structure of chloroplasts by keeping an orderly arrangement of the thylakoids membrane and also have an ability to maintain a higher photosynthetic efficiency of *Nymphoides peltatum* under Cadmium stress (Li et al., 2009).

However, few earlier studies showed that high concentrations of polyamines may destroy the structure of chloroplasts which depended on different light conditions. Spd treatment with chloroplast for about 10 s displayed the envelope and the typical dense network of the thylakoid lamellae interspersed with numerous areas of stacked grana (Pjon et al., 1990). After 72 h, the chloroplast envelopes of spermidine-treated leaf disks incubated in the dark and under light conditions, the chloroplast envelope was destroyed by polyamine treatment, but there were distinct differences in the appearance of the chloroplast ultrastructures of dark and light-incubated leaves (Cohen et al., 1979).

#### **2.5 Thylakoid membrane protein complexes**

Photosynthetic apparatus in higher plants is a membrane bound protein complex composed of chlorophyll and carotenoid pigments that function in the conversion of light energy to chemical energy. It has been suggested that a large number of these proteins are related to photosynthesis. The thylakoid membranes within the chloroplast are the subcompartment in which the primary reactions of photosynthesis occur. These reactions are organized in the four major multisubunit protein complexes, photosystem I (PSI), PSII, the ATP-synthase complex and cytochrome b6/f complex (Hippler et al., 2001).

Polyamines such as putrescine, spermidine, spermine, and methylamine interact with protein (H-bonding) through polypeptide C=O, C-N and N-H groups with major perturbations of protein secondary structure as the concentration of amines was raised. It has been shown the chloroplasts and various photosynthetic subcomplexes including thylakoids, LHCII complex and PSII membranes are enriched with polyamines, especially are exclusively rich in PSII core and the reaction center of PSII (Kotzabasis et al. 1993; Navakoudis et al. 2003). Several studies have reported the interaction of polyamines with proteins of photosynthetic apparatus under various environmental factors. However, the action site of polyamines at photosynthetic proteins may vary with polyamine concentration and stress levels. In the alga *S. obliquus* the bound polyamines were found to be associated with both the oligomers and the monomers of LHCII, as well as with the CPs (Kotzabasis et al., 1993). However, the distribution does not reflect a constant pattern: in LHCII

that some stresses led to the decrease in the photochemical efficiency and electron transport activity might be associated with the changes of the structure of photosynthetic apparatus

Several studies have showed that polyamines are involved in stabilization the structure and function of photosynthetic apparatus in response to unfavourable environment factors (Demetriou et al., 2007). Under Nacl stress, Put as organic cations dramatically enhanced lipid accumulation in the chloroplasts and prevented the membrane degradation in the granal and stromal thylakoids by interacting with the negatively charged membrane sites (Tiburcio et al., 1994). The increase in the number of plastoglobules in the chloroplasts affected by Put can result from the redirection of cell metabolism towards the products of higher reduction potential (Paramonova et al., 2003). In our study, we observed that Put can alleviate the degradation of thylakoid membrane proteins induced by salt stress, and thus making a normal stacking order in the adjacent grana thylakoids (data not published). In addition, exogenous Spd have been reported to protect the structure of chloroplasts by keeping an orderly arrangement of the thylakoids membrane and also have an ability to maintain a higher photosynthetic efficiency of *Nymphoides peltatum* under Cadmium stress

However, few earlier studies showed that high concentrations of polyamines may destroy the structure of chloroplasts which depended on different light conditions. Spd treatment with chloroplast for about 10 s displayed the envelope and the typical dense network of the thylakoid lamellae interspersed with numerous areas of stacked grana (Pjon et al., 1990). After 72 h, the chloroplast envelopes of spermidine-treated leaf disks incubated in the dark and under light conditions, the chloroplast envelope was destroyed by polyamine treatment, but there were distinct differences in the appearance of the chloroplast

Photosynthetic apparatus in higher plants is a membrane bound protein complex composed of chlorophyll and carotenoid pigments that function in the conversion of light energy to chemical energy. It has been suggested that a large number of these proteins are related to photosynthesis. The thylakoid membranes within the chloroplast are the subcompartment in which the primary reactions of photosynthesis occur. These reactions are organized in the four major multisubunit protein complexes, photosystem I (PSI), PSII, the ATP-synthase

Polyamines such as putrescine, spermidine, spermine, and methylamine interact with protein (H-bonding) through polypeptide C=O, C-N and N-H groups with major perturbations of protein secondary structure as the concentration of amines was raised. It has been shown the chloroplasts and various photosynthetic subcomplexes including thylakoids, LHCII complex and PSII membranes are enriched with polyamines, especially are exclusively rich in PSII core and the reaction center of PSII (Kotzabasis et al. 1993; Navakoudis et al. 2003). Several studies have reported the interaction of polyamines with proteins of photosynthetic apparatus under various environmental factors. However, the action site of polyamines at photosynthetic proteins may vary with polyamine concentration and stress levels. In the alga *S. obliquus* the bound polyamines were found to be associated with both the oligomers and the monomers of LHCII, as well as with the CPs (Kotzabasis et al., 1993). However, the distribution does not reflect a constant pattern: in LHCII

ultrastructures of dark and light-incubated leaves (Cohen et al., 1979).

complex and cytochrome b6/f complex (Hippler et al., 2001).

**2.5 Thylakoid membrane protein complexes** 

(Parida et al., 2003).

(Li et al., 2009).

subcomplexes Put and Spm levels fluctuated depending on the light adaptational status of the photosynthetic apparatus. Put and Spm are bound to the photosynthetic complexes, mainly to the LHCII oligomeric and monomeric forms (Navakoudis et al., 2007). It is well documented that the LHCII protein is abundant in thylakoids and its surface is negatively charged (Standfuss et al., 2005). Kirchhoff et al. (2007) have recently shown that incubation of thylakoids under unstacking conditions leads to intermixing and randomization of the protein complexes, accompanied by disconnection of LHCII trimers from PSII and a decreased connectivity between PSIIα centers. Interestingly, exogenously added polyamines can reverse those damaging effects. Thus, there was strong indication that polyamines possessed a pivotal role in photosynthesis.

Chloroplasts also contain high activities of several polyamine biosynthetic enzymes (Borrell et al.1995; Andreadakis and Kotzabasis 1996) and transglutaminase (TGase) catalyzing the covalent binding of polyamines to proteins (Del-Duca et al., 1994; Dondini et al., 2001; Della-Mea et al., 2004). Polyamines can covalently bind with thylakoid membrane proteins under the TGase form of protein- glutamyl-polyamine or protein-glutamyl-polyamines-glutamylprotein (Dondini et al., 2003), such as D1, D2 protein and cytochrome b6/f, together with the involvement of polyamine, could be important for stabilisation of molecular complexes in the thylakoid membranes of osmotically stressed oat leaves (Besford et al., 1993). Immunodetection of TGase in thylakoid fraction revealed that formation of covalent bonds between PAs and proteins by TGase is involved in regulating the process of chloroplast senescence (Sobieszczuk-Nowicka et al., 2009).

However, high concentration of polyamines added to submembrane fractions of photosynthetic apparatus causes a strong inhibition of PSII activity (Hamdani et al., 2010). FTIR spectroscopy analysis Spd and Spm at higher cation concentrations (5 and 10 mM), the result showed that the polyamine significant alterations of the thylakoid protein secondary structure with a decrease of the α-helical domains from 47% (uncomplexed PSII) up to 37% (cation complexes) and an increase in the β-sheet structure from 18% up to 29% (Bograh et al., 1997). So far, this specific inhibition mechanism of polyamines is not clear, but it is likely that the proteins were affected by these polycations re either extrinsic polypeptides associated with the oxygen evolving complex or portions of integral polypeptides protruding at the surface of the PSII membranes (Beauchemin et al., 2007).

#### **3. Polyamines and stress photosynthesis**

#### **3.1 Salinity**

Salt stress causes an initial water-deficit and ion-specific stresses resulting from changes in K+/Na+ ratios. Thus, it leads to an increased Na+ and Cl− concentrations that decrease plant growth and productivity by disrupting physiological processes, especially photosynthesis (Shu et al., 2010). Salt stress affects photosynthetic efficiency of plant through stomatal limitation and non-stomatal limitations, such as stomatal closure (Meloni et al., 2003), chlorophyll content loss (Sudhir and Murthy, 2004), inhibition of Rubisco activity (Brugnoli and Björkman, 1992; Ziska et al., 1990), and degradation of membrane proteins in photosynthetic apparatus (Khan and Ungar, 1997).

It has been suggested that exogenous application of polyamines can to some extent alleviate salinity-induced decline in photosynthetic efficiency, but this effect strongly depended both on PAs concentrations or types and stress levels (Duan et al., 2008). The maximum quantum efficiency of PSII (Fv/Fm) measured in leaves of salt-stressed cucumber seedlings was not

A Review: Polyamines and Photosynthesis 447

mM Put also alleviated the reduction of salt stress on P*N*. However, Put had no effect on Gs and transpiration rate (Tr), and aggravated the reduction of salt stress on Ci. The result suggested that Put strongly affects photosynthetic apparatus involving in enhancement of photochemical quenching rather than regulation of stomatal closure or opening (Zhang et al., 2009). It was also found that exogenous Put improved the photoadaptability of Scenedesmus by altering the content of LHCII monomers and oligomers and PSI and PSII core proteins in the thylakoid membranes (Navakoudis et al., 2007). In green alga Scenedesmus under high salinity, exogenously applied Put effectively decreased the functional size of the antenna and increased the density of active PSII reaction centers, thereby reducing the salt-induced increase in excitation pressure that may cause oxidative damage to the photosynthetic apparatus (Demetriou et al., 2007). Application of Put, Spd and Spm through the root was also effective in alleviating the salt damage to PSI and PSII activities (Chattopadhayay et al., 2002). However, the effects of polyamines on the salt damage to the structure and functions of the photosynthetic apparatus of higher plants

Several publications have reported that changes of endogenous polyamine level and forms are involved in regulating the photochemical efficiency of salt-stressed plants, and polyamines metabolism-related enzymes are closely correlated with photosynthesis. Exogenous polyamines increased bound Spd contents in chloroplasts to enhance the photosynthetic capacities of corn suffering salt stress (Liu et al., 2006). Exogenously supplied Put in the salt stressed of green alga cultures was shown to increase the Put/Spm ratio in thylakoids and lead to a decrease of the functional antenna size, both by decreasing the size of LHCII and increasing the quantum yield of PSII primary photochemistry, so that the damaging effects induced by salinity are diminished (Demetriou et al., 2007). In our study, applications of 8 mM Put to salt-stressed plant leaves increased content of endogenous polyamines in the thylakoid membranes and overcame the damaging effects of salt stress on the structure and function of the photosynthetic apparatus, which was associated with an

Drought is the major abiotic stress factor limiting crop productivity in the worldwide (Wang et al., 2003; Sharp et al., 2004). Under drought stress, plants rapidly close stomata with decrease in leaf water potential, thus leading to a significant inhibition of photosynthesis (Zlatev & Yordanov, 2004). To cope with drought, plants initiate a reprogramming of transcriptional, post-transcriptional and metabolite processes that restricts water loss. Several studies have reported that exogenous PAs application is involved in improving drought tolerance against the perturbation of biochemical processes (Yang et al., 2007; Alcázar et al., 2010), but mechanisms of their action during exogenous application in modulating physiological phenomena especially in photosynthesis is not fully understood (Bae et al., 2008). Both net photosynthetic net (P*N*) and water use efficiency (WUE) in leaves of rice subjected to drought stress for 7 days were significantly improved by spraying of plants with 10 µM Put, Spd and Spm solutions, while amongst the PAs, Spm was the most effective (Farooq et al., 2009). Drought resistance of two different tomato cultivars, application of 0.1mM exogenous Spd increased the P*N*, Gs and Tr in the tomato seedling leaves of the two tomato cultivars and decreased Ci by preventing stoma closure and stimulating CO2 uptake during the later period under drought stress (Zhang et al., 2010).

remain until today largely unknown.

**3.2 Drought stress** 

improvement in the actual PSⅡ efficiency (data unpublished).


Table 1. High activities of arginine decarboxylase (ADC), adenosylmethionine decarboxylase (SAMDC) and trantaminase (TGase) are involed in regulation of photosynthesis in response to environmental stress. SAMDC is an enzyme that catalyzes the conversion of S-adenosyl methionine to S-adenosylmethioninamine. ADC is an important enzyme responsible for putrscine synthesis. TGase is present in the chloroplasts of higher plants and its activity is modulated by the presence of light.

much influenced by 1mM Spd application, although Spd could ameliorate plant growth and increase net photosynthetic rate (P*N*), stomatal conductance (Gs), intercellular CO2 concentration (Ci), actual efficiency of photosystem II (ФPSⅡ) and the coefficient of photochemical quenching (qP) of cucumber seedlings subjected to salinity (Li et al., 2007). 10

Measured photosynthetic

Chlorophyll-a/b antenna complex (LHCII, CP24, CP26 and CP29);large subunit of ribulose-l,5-

carboxylase/oxygenase.

complexes and Rubisco

photosynthetic efficiency

transformation; Grana stacking; PSII and PSI

stomata conductance and

Thylakoid remodelling chloroplast ultrastructure

Light-harvesting complex II thylakoid activity

Chloroplast

aperture

Chl contents stomatal density

ADC activity in the thylakoids membrane Author

Del-Duca et al. (1994)

Borrell et al. (1995)

Dondini et al. (2001)

et al.(2002)

Della-Mea et al. (2004)

Kasinathan, and Wingler (2004)

Nowicka et al. (2009)

Ioannidis et al. (2009)

Alcázar et al. (2010)

Ortigosa et al. (2010)

Wang et al. (2011)

parameters

bisphosphate,

Stress reponse

stimulate

stress

Spinacia oleracea SAMDC Chilling Photoinhibition He

condition

condition

stress

stresses

photosynthesis in response to environmental stress. SAMDC is an enzyme that catalyzes the conversion of S-adenosyl methionine to S-adenosylmethioninamine. ADC is an important enzyme responsible for putrscine synthesis. TGase is present in the chloroplasts of higher

much influenced by 1mM Spd application, although Spd could ameliorate plant growth and increase net photosynthetic rate (P*N*), stomatal conductance (Gs), intercellular CO2 concentration (Ci), actual efficiency of photosystem II (ФPSⅡ) and the coefficient of photochemical quenching (qP) of cucumber seedlings subjected to salinity (Li et al., 2007). 10

Table 1. High activities of arginine decarboxylase (ADC), adenosylmethionine decarboxylase (SAMDC) and trantaminase (TGase) are involed in regulation of

Arabidopsis ADC Drought Transpiration rate

TGase Oxidative

ADC Multiple

plants and its activity is modulated by the presence of light.

Barley TGase Senescence Thylakoid fraction Sobieszczuk-

ADC Salt stress Chl contents

Dunaliella salina TGase Salt stress Thylakoid photosynthetic

TGase Light

TGase Ca and light

Species Types of

Helianthus tuberosus

Helianthus tuberosus

Arabidopsis thaliana

Nicotiana tabacum

Poncirus trifoliata

polyaminerelated enzyme

*Avena sativa* ADC Osmotic

Zea mays TGase Natural

mM Put also alleviated the reduction of salt stress on P*N*. However, Put had no effect on Gs and transpiration rate (Tr), and aggravated the reduction of salt stress on Ci. The result suggested that Put strongly affects photosynthetic apparatus involving in enhancement of photochemical quenching rather than regulation of stomatal closure or opening (Zhang et al., 2009). It was also found that exogenous Put improved the photoadaptability of Scenedesmus by altering the content of LHCII monomers and oligomers and PSI and PSII core proteins in the thylakoid membranes (Navakoudis et al., 2007). In green alga Scenedesmus under high salinity, exogenously applied Put effectively decreased the functional size of the antenna and increased the density of active PSII reaction centers, thereby reducing the salt-induced increase in excitation pressure that may cause oxidative damage to the photosynthetic apparatus (Demetriou et al., 2007). Application of Put, Spd and Spm through the root was also effective in alleviating the salt damage to PSI and PSII activities (Chattopadhayay et al., 2002). However, the effects of polyamines on the salt damage to the structure and functions of the photosynthetic apparatus of higher plants remain until today largely unknown.

Several publications have reported that changes of endogenous polyamine level and forms are involved in regulating the photochemical efficiency of salt-stressed plants, and polyamines metabolism-related enzymes are closely correlated with photosynthesis. Exogenous polyamines increased bound Spd contents in chloroplasts to enhance the photosynthetic capacities of corn suffering salt stress (Liu et al., 2006). Exogenously supplied Put in the salt stressed of green alga cultures was shown to increase the Put/Spm ratio in thylakoids and lead to a decrease of the functional antenna size, both by decreasing the size of LHCII and increasing the quantum yield of PSII primary photochemistry, so that the damaging effects induced by salinity are diminished (Demetriou et al., 2007). In our study, applications of 8 mM Put to salt-stressed plant leaves increased content of endogenous polyamines in the thylakoid membranes and overcame the damaging effects of salt stress on the structure and function of the photosynthetic apparatus, which was associated with an improvement in the actual PSⅡ efficiency (data unpublished).

#### **3.2 Drought stress**

Drought is the major abiotic stress factor limiting crop productivity in the worldwide (Wang et al., 2003; Sharp et al., 2004). Under drought stress, plants rapidly close stomata with decrease in leaf water potential, thus leading to a significant inhibition of photosynthesis (Zlatev & Yordanov, 2004). To cope with drought, plants initiate a reprogramming of transcriptional, post-transcriptional and metabolite processes that restricts water loss. Several studies have reported that exogenous PAs application is involved in improving drought tolerance against the perturbation of biochemical processes (Yang et al., 2007; Alcázar et al., 2010), but mechanisms of their action during exogenous application in modulating physiological phenomena especially in photosynthesis is not fully understood (Bae et al., 2008). Both net photosynthetic net (P*N*) and water use efficiency (WUE) in leaves of rice subjected to drought stress for 7 days were significantly improved by spraying of plants with 10 µM Put, Spd and Spm solutions, while amongst the PAs, Spm was the most effective (Farooq et al., 2009). Drought resistance of two different tomato cultivars, application of 0.1mM exogenous Spd increased the P*N*, Gs and Tr in the tomato seedling leaves of the two tomato cultivars and decreased Ci by preventing stoma closure and stimulating CO2 uptake during the later period under drought stress (Zhang et al., 2010).

A Review: Polyamines and Photosynthesis 449

al., 2009). He et al. (2002) reported that Spd-pretreated cucumber plants had a high Spd content in both leaves and thylakoid membranes during chilling. They also found Spd pretreatment no effect of stomatal conductance in chilled leaves, but alleviated the decline of the maximum efficiency of PSII photochemistry (Fv/Fm), photosynthetic electron transport activity of thylakoids and activity of enzymes in carbon metabolism. Szalai et al. (1997) observed a marked increase of Put and Spd contents together with a significant decline of Fv/Fm in maize leaves during chilling at 5℃. Based on these results, we conclude that the increase of polyamine contents might be important in plant defense to the photosynthetic apparatus against the low-temperature photoinhibition. Biochemical and physico-chemical measurements showed that the response of the photosynthetic apparatus to low temperature is affected by the changes occurring in the pattern of LHCII-associated Put and Spm which adjust the size of LHCII. The decrease of Put/Spm ratio, mainly due to the reduction in the quantity of LHCII-associated Put led to an increase of the LHCII, especially of the oligomeric forms (Sfakianaki et al., 2006), which is consistent with a data obtained for spinach showed that the low temperature induced a decrease in the Put associated to the thylakoid membranes (He et al., 2002). These results suggest the Put/Spm ratio in the structure of the photosynthetic apparatus associated with the photosynthetic efficiency and the maximal photosynthetic rate, although the mechanism by which polyamines contribute

An important consequence of stratospheric ozone depletion is the increased transmission of solar ultraviolet radiation (UV) to the Earth's lower atmosphere and surface. Effects of enhanced UV radiation (200-320 nm) on plants have been studied on plant morphology, growth and development, and physiology aspects, which have potential adverse effects on agricultural production and natural plant ecosystems (Caldwell et al., 1998; Madronich et al., 1998; An et al., 2000; Zhang et al., 2003; Bjorn et al., 1996). UV radiation may also induce the formation of reactive oxygen species (ROS) in plants, leading to the damage of photosynthetic apparatus, lipid peroxidation and the changes of PAs levels. The potential role of polyamines in maintaining the photochemical efficiency of plants in response to UV

In the leaves of six genotypes of silver birch (Betula pendula Roth) seedlings, PAs analyses showed that the concentrations of Put were increased and Spd decreased with elevated temperature. Therefore, there was a change in polyamine accumulation towards more conjugated forms. It has been suggested that the conformation of antenna proteins is regulated by polyamines, affecting the efficiency of light harvesting. The changes in Put and Spm metabolism implied that the moderately elevated temperature increased photosynthetic antenna size in silve birch leaves (Tegelberg et al., 2008). The functional and biochemiacal aspects of the photosynthetic apparatus in response to UV-B radiation were examined in unicellular oxygenic algae *Scenedesmus obliquus*, which is the first comparative characterization of the photosynthetic responses exhibited by the Wt (wild type) and Wt-lhc (a chlorophyll b-less mutant) mutant to UV-B irradiation with emphasis on the response of polyamines and xanthophylls (Sfichi-Duke et al., 2008). The results showed that light stress led to an 204% increased of Spm (which could positively regulate antenna size) and a threefold decrease in Put/Spm ratio. The attachment of Spm to thylakoids could be of structural or functional importance. Along with xanthophylls, polyamines at the LHCII level

to the increasing tolerance to low temperatures is not yet understood.

**3.4 UV radiation stress** 

stress has become a research focus.

They also observed that the mitigative effects of exogenous Spd on photosynthesis in drought-sensitive cultivars were greater than those in high drought-resistant cultivars. In addition, exogenous Spm to pines under drought caused a decline in transpiration rates, enhanced photosynthesis and promoted osmotic adjustment, which would help to maintain turgor (Anisul et al., 2003; Pang et al., 2007). Base on the above studies, we proposed a model describing the role of PAs on photosynthesis during drought-stress: PAs may modulate the activities of certain ion channels at the plasma membrane and stimulate stomatal closure, which would help to enhance photosynthetic efficiency under drought.

#### **3.3 Temperature stress**

Temperature is an important ecological factor for plant growth and development, when the temperature is too high or too low, plant growth will stop (Berry and Raison, 1981). It has been demonstrated that high temperature or low temperature adversely affects plant growth and survival, but the impact of temperature stress on the photosynthetic apparatus is considered to be of particular significance because photosynthesis is often inhibited before other cell functions are impaired (Haldimann and Feller, 2004). Extensive research over the last years has focused on the organization and structure of photosynthetic complexes in response to high temperature or low temperature stress (Santamaria and Vierssen, 1997). There are at least three major stress-sensitive sites in the photosynthetic machinery, the PSII, the ATPase and the carbon assimilation process (Allakhverdiev et al., 2008; Mathur et al., 2011).

Heat-shock often induced changes in polyamine content which can be ascribed as protective responses aimed at structural integrity of membrane and cell walls (Edreva et al., 1998), and tolerant plants are able to increase total spermidine and spermine pools under heat stress (Bouchereau et al., 1999). Increased contents of polyamines can stabilize the structure of thylakoid membranes and prevent chlorophyll loss, playing an important role in the protective response of plants to heat stress. Because of the polycationic nature at a physiological pH, polyamines can bind strongly to cellular constituents such as nucleic acids, proteins and membranes (Childs et al., 2003). Several reports have indicated the involvement of polyamines in regulating heat stress-induced the inhibition of photosynthetic efficiency. Exogenous application of 4 mM Spd improved the plant heatresistance in two tomato cultivars, and especially in tolerant cultivars have higher ability to hardening and higher resistance to thermal damage of the pigment-protein complexes structure and the activity of PSII than sensitive cultivars (Murkowski, 2001). At filling stage of rice, high temperature stress caused a decline in photosynthetic capacity, chlorophyll content and RuBPc activity of two different resistance cultivars. However this effect was more severe in heat-sensitive variety than in heat-resistant variety, because it is closely related to the heat-resistant cultivars have a high content of endogenous polyamines, especially Put accumulation (Huang et al., 1999). In vivo and in vitro experiment showed that exogenous Spm was effective in alleviating heat damage to the photosynthetic apparatus of cucumber, suggesting that protein complexes in thylakoids were made more stable to heat due to their binding to Spm (Li et al., 2003).

In addition, published data showed that PAs levels increased the tolerance degree of the photosynthetic apparatus to low temperatures in different plant species (Hummel et al., 2004), comparison within species has revealed that cold-tolerant varieties/lines show higher endogenous PA contents in response to low temperature than non-tolerant ones (Zhang et

They also observed that the mitigative effects of exogenous Spd on photosynthesis in drought-sensitive cultivars were greater than those in high drought-resistant cultivars. In addition, exogenous Spm to pines under drought caused a decline in transpiration rates, enhanced photosynthesis and promoted osmotic adjustment, which would help to maintain turgor (Anisul et al., 2003; Pang et al., 2007). Base on the above studies, we proposed a model describing the role of PAs on photosynthesis during drought-stress: PAs may modulate the activities of certain ion channels at the plasma membrane and stimulate stomatal closure, which would help to enhance photosynthetic efficiency under drought.

Temperature is an important ecological factor for plant growth and development, when the temperature is too high or too low, plant growth will stop (Berry and Raison, 1981). It has been demonstrated that high temperature or low temperature adversely affects plant growth and survival, but the impact of temperature stress on the photosynthetic apparatus is considered to be of particular significance because photosynthesis is often inhibited before other cell functions are impaired (Haldimann and Feller, 2004). Extensive research over the last years has focused on the organization and structure of photosynthetic complexes in response to high temperature or low temperature stress (Santamaria and Vierssen, 1997). There are at least three major stress-sensitive sites in the photosynthetic machinery, the PSII, the ATPase and the carbon assimilation process (Allakhverdiev et al., 2008; Mathur et al.,

Heat-shock often induced changes in polyamine content which can be ascribed as protective responses aimed at structural integrity of membrane and cell walls (Edreva et al., 1998), and tolerant plants are able to increase total spermidine and spermine pools under heat stress (Bouchereau et al., 1999). Increased contents of polyamines can stabilize the structure of thylakoid membranes and prevent chlorophyll loss, playing an important role in the protective response of plants to heat stress. Because of the polycationic nature at a physiological pH, polyamines can bind strongly to cellular constituents such as nucleic acids, proteins and membranes (Childs et al., 2003). Several reports have indicated the involvement of polyamines in regulating heat stress-induced the inhibition of photosynthetic efficiency. Exogenous application of 4 mM Spd improved the plant heatresistance in two tomato cultivars, and especially in tolerant cultivars have higher ability to hardening and higher resistance to thermal damage of the pigment-protein complexes structure and the activity of PSII than sensitive cultivars (Murkowski, 2001). At filling stage of rice, high temperature stress caused a decline in photosynthetic capacity, chlorophyll content and RuBPc activity of two different resistance cultivars. However this effect was more severe in heat-sensitive variety than in heat-resistant variety, because it is closely related to the heat-resistant cultivars have a high content of endogenous polyamines, especially Put accumulation (Huang et al., 1999). In vivo and in vitro experiment showed that exogenous Spm was effective in alleviating heat damage to the photosynthetic apparatus of cucumber, suggesting that protein complexes in thylakoids were made more

In addition, published data showed that PAs levels increased the tolerance degree of the photosynthetic apparatus to low temperatures in different plant species (Hummel et al., 2004), comparison within species has revealed that cold-tolerant varieties/lines show higher endogenous PA contents in response to low temperature than non-tolerant ones (Zhang et

stable to heat due to their binding to Spm (Li et al., 2003).

**3.3 Temperature stress** 

2011).

al., 2009). He et al. (2002) reported that Spd-pretreated cucumber plants had a high Spd content in both leaves and thylakoid membranes during chilling. They also found Spd pretreatment no effect of stomatal conductance in chilled leaves, but alleviated the decline of the maximum efficiency of PSII photochemistry (Fv/Fm), photosynthetic electron transport activity of thylakoids and activity of enzymes in carbon metabolism. Szalai et al. (1997) observed a marked increase of Put and Spd contents together with a significant decline of Fv/Fm in maize leaves during chilling at 5℃. Based on these results, we conclude that the increase of polyamine contents might be important in plant defense to the photosynthetic apparatus against the low-temperature photoinhibition. Biochemical and physico-chemical measurements showed that the response of the photosynthetic apparatus to low temperature is affected by the changes occurring in the pattern of LHCII-associated Put and Spm which adjust the size of LHCII. The decrease of Put/Spm ratio, mainly due to the reduction in the quantity of LHCII-associated Put led to an increase of the LHCII, especially of the oligomeric forms (Sfakianaki et al., 2006), which is consistent with a data obtained for spinach showed that the low temperature induced a decrease in the Put associated to the thylakoid membranes (He et al., 2002). These results suggest the Put/Spm ratio in the structure of the photosynthetic apparatus associated with the photosynthetic efficiency and the maximal photosynthetic rate, although the mechanism by which polyamines contribute to the increasing tolerance to low temperatures is not yet understood.

#### **3.4 UV radiation stress**

An important consequence of stratospheric ozone depletion is the increased transmission of solar ultraviolet radiation (UV) to the Earth's lower atmosphere and surface. Effects of enhanced UV radiation (200-320 nm) on plants have been studied on plant morphology, growth and development, and physiology aspects, which have potential adverse effects on agricultural production and natural plant ecosystems (Caldwell et al., 1998; Madronich et al., 1998; An et al., 2000; Zhang et al., 2003; Bjorn et al., 1996). UV radiation may also induce the formation of reactive oxygen species (ROS) in plants, leading to the damage of photosynthetic apparatus, lipid peroxidation and the changes of PAs levels. The potential role of polyamines in maintaining the photochemical efficiency of plants in response to UV stress has become a research focus.

In the leaves of six genotypes of silver birch (Betula pendula Roth) seedlings, PAs analyses showed that the concentrations of Put were increased and Spd decreased with elevated temperature. Therefore, there was a change in polyamine accumulation towards more conjugated forms. It has been suggested that the conformation of antenna proteins is regulated by polyamines, affecting the efficiency of light harvesting. The changes in Put and Spm metabolism implied that the moderately elevated temperature increased photosynthetic antenna size in silve birch leaves (Tegelberg et al., 2008). The functional and biochemiacal aspects of the photosynthetic apparatus in response to UV-B radiation were examined in unicellular oxygenic algae *Scenedesmus obliquus*, which is the first comparative characterization of the photosynthetic responses exhibited by the Wt (wild type) and Wt-lhc (a chlorophyll b-less mutant) mutant to UV-B irradiation with emphasis on the response of polyamines and xanthophylls (Sfichi-Duke et al., 2008). The results showed that light stress led to an 204% increased of Spm (which could positively regulate antenna size) and a threefold decrease in Put/Spm ratio. The attachment of Spm to thylakoids could be of structural or functional importance. Along with xanthophylls, polyamines at the LHCII level

A Review: Polyamines and Photosynthesis 451

No differences between control and UV-irradiated calli were detected in lower layers.

In natural conditions, flooding, excessive irrigation and soil compaction extremely lead to oxygen deficiency in the root-zone of plant, thus causing to hypoxia stress (Drew, 1997). Hypoxia stress is one of an important environmental factor inhibits plant growth and yield (Avijie et al., 2002). Previous several studies have been shown that suitable concentration of exogenous polyamines could alleviate hypoxia stress-induced physiological damage and improve photochemical efficiency of plants (Vigne and Frelin, 2008). Zhou et al. (2006) demonstrated application of exogenous polyamines to some extent increased net photosynthetic rate (P*N*) and water use efficiency (WUE) in cucumber leaves. Shi et al. (2009) also observed that exogenous Put alleviated the reduction of P*N*, Gs, Ci of cucumber subjected to root-zone hypoxia through enhancing the actual and maximal nitrate reductase activities. Application of exogenous 0.05 mM Spd added to the hypoxia nutrient solutions significantly suppressed the accumulation and insoluble bound Put in roots and leaves of cucumber seedlings, which was associated with a decreased in dissipation energy (NPQ) and xanthophylls de-epoxidation state (DEPS) during hypoxia stress, while Spd enhanced maximal photochemical efficiency (Fv/Fm), PSII photochemistry rate (ΦPSII) and P*N* (Jia, 2009). These results implied that exogenous polyamines increased photosynthetic capacity might be involved in regulating changes of endogenous polyamine contents in the

Oxidative stress has been postulated, years ago, to be a causal factor in responses to environmental stresses (Velikova et al., 2000; Patil et al., 2007). The basic tenet of this hypothesis is that the stress-associated decline in the functional capacity of biological systems is primarily due to the accumulation of irreparable oxidative molecular damage (Sohal et al., 1995). Adverse environmental stresses induced a decrease in photosynthetic activity which often associated with the oxidative stress (Krause, 1994). It has been shown that plant could overcome the effect of the oxidative stress and sustain photosynthetic efficiency that may be related to the scavenging of stress-induced toxic oxygen species, such

Polyamines (PAs) are regarded as a new class of growth substances and are also well known for their positive effects on the photosynthetic efficiency under various stress conditions due to their acid neutralizing and antioxidant properties, as well as to their membrane and cell wall stabilizing abilities (He et al., 2002; Mapelli et al., 2008; Zhao and Yang, 2008). Exogenous application of spermidine (Spd) could alleviate salt-induced membrane injury of chloroplast by increasing the active oxygen scavenging ability. The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), the contents of ascorbic acid (AsA), Car and glutathione reduced form (GSH), and the ratios of GSH/GSSG in chloroplasts were increased, which increased the salinity tolerance of the photosynthetic apparatus in cucumber seedlings (Duan et al., 2008a; Duan et al., 2009b). Pretreatment with Spd markedly reduced lipid peroxidation and membrane relative permeability in wheat leaves under water stress (Duan et al., 2006). They found that Spd

· -

) (Sopory

as hydrogen peroxide (H2O2), hydroxyl radical (OH·) and superoxide radical (O2

Spermidine and spermine were not affected by UV treatment.

chloroplasts, thus enhancement of root-zone hypoxia tolerance.

**3.5 Hypoxia stress** 

**3.6 Oxidative stress** 

et al., 1990).

are more sensitive to UV-B stress than xanthophylls and their responsiveness is abolished in Chl b-less mutants. The tobacco cultivars Bel B and Bel W3 were employed to describe possible protective functions of polyamines against UV-B radiation in sun light simulators (GSF/Munich) with natural diurnal fluctuations of simulated UV-B (Lütz et al., 2005). The results indicated that an increase of PAs, especially of Put level, in thylakoid membranes upon elevated UV-B exposure comprises one of the primary protective mechanisms in the photosynthetic apparatus of the tobacco variety Bel B against UV-B radiation. In addition, the sensitivity to UV-B of Bel W3 (sensitive to ozone) is attributed to its incapability to enhance Put level in thylakoid membranes. After prolongation of UV-B exposure, when endogenous plant balances are being gradually restored, due to secondary responses, (e.g., biosynthesis of carotenoids and of additional flavonoids) and the plant is adapting to the altered environmental conditions, the the PAs level is being reduced. Unal et al.(2007) repoted that under exposure to UV-A for 24 and 48h, the photosynthetic quantum yield ratio of Physcia semipinnata decreased, while that of 1mM polyamine treated were not influenced. It was also found that exogenously added Spd had higher Chla content than Spm and Put added samples. By exposure of the samples with polyamines before the UV-A treatment for 48h, MDA contents was found lower than control group and other groups. These data supported that polyamines, especially Spd and Spm, would play a role in protecting Chl a content, protein content and decreasing lipid peioxidation. This is in agreement with the report of Kramer et al. (1991) whose found membrane lipid may be a target of UV-B damage and polyamine accumulation in response to UV-B radiation stress is consistent with similar reaponses to other environmental stressors. Smith et al. (2001) investigated the influence of UV-B radiation on the UV-B sensitive legume *Phaseolus vulgaris* L. "Top Crop" over a two-week period. Total free polyamines showed marked decreases in response to UV-B radiation, primarily due to a decrease in Put, which was correlated with UV-B induced chlorophyll loss. Kramer et al. (1992) reported that photosynthetically active radiation (PAR) had a large effect on polyamine levels in leaves, with higher levels of Put and Spd observed at 600 than at 300 µmol m-2 s-1 in both cultivars (UV-B-sensitive and– insensitive cultivars). The results indicated that the inhibition of UV-B stress by high PAR may involve polyamine accumulation.

The sensitivity of the photosynthetic apparatus to UV-B irradiation was studied in cultures of unicellular green alga *Scenedesmus obliquus* incubated in low light (LL) and high light (HL) conditions, treated or not with exogenous polyamines. UV-B radiation induces a decrease in the thylakoid-associated Put and an increase in Spm, so that the reduction of Put/Spm ratio leads to the increase of light-harvesting complex II (LHCII) size per active reaction center and, consequently, the amplification of UV-B effects of the photosynthetic apparatus. The separation of oligomeric and monomeric froms of LHCII from isolated thylakoids showed that UV-B induces an increase in the oligomeric forms of LHCII, which was more intense in LL than in HL. By manipulating the LHCII size with exogenous polyamines, the sensitivity degree of the photosynthetic apparatus to UV-B changed significantly. Specifically, the addition of Put decreased highly the sensitivity of LL culture to UV-B because of the inhibitory effect of Put on the LHCII size increasing, wheres the addition of Spm enhanced the UV-B injury induced in HL culture because of the increasing of LHCII size (Sfichi et al., 2004). Zacchini et al. (2004) reported that the put content in tobacco was enhanced, compared with control, in upper layers of calli 6 h after UV-C high dose stress and decreased 24 and 48 h after irradiation, though remained statistically higher than control. No differences between control and UV-irradiated calli were detected in lower layers. Spermidine and spermine were not affected by UV treatment.

#### **3.5 Hypoxia stress**

450 Advances in Photosynthesis – Fundamental Aspects

are more sensitive to UV-B stress than xanthophylls and their responsiveness is abolished in Chl b-less mutants. The tobacco cultivars Bel B and Bel W3 were employed to describe possible protective functions of polyamines against UV-B radiation in sun light simulators (GSF/Munich) with natural diurnal fluctuations of simulated UV-B (Lütz et al., 2005). The results indicated that an increase of PAs, especially of Put level, in thylakoid membranes upon elevated UV-B exposure comprises one of the primary protective mechanisms in the photosynthetic apparatus of the tobacco variety Bel B against UV-B radiation. In addition, the sensitivity to UV-B of Bel W3 (sensitive to ozone) is attributed to its incapability to enhance Put level in thylakoid membranes. After prolongation of UV-B exposure, when endogenous plant balances are being gradually restored, due to secondary responses, (e.g., biosynthesis of carotenoids and of additional flavonoids) and the plant is adapting to the altered environmental conditions, the the PAs level is being reduced. Unal et al.(2007) repoted that under exposure to UV-A for 24 and 48h, the photosynthetic quantum yield ratio of Physcia semipinnata decreased, while that of 1mM polyamine treated were not influenced. It was also found that exogenously added Spd had higher Chla content than Spm and Put added samples. By exposure of the samples with polyamines before the UV-A treatment for 48h, MDA contents was found lower than control group and other groups. These data supported that polyamines, especially Spd and Spm, would play a role in protecting Chl a content, protein content and decreasing lipid peioxidation. This is in agreement with the report of Kramer et al. (1991) whose found membrane lipid may be a target of UV-B damage and polyamine accumulation in response to UV-B radiation stress is consistent with similar reaponses to other environmental stressors. Smith et al. (2001) investigated the influence of UV-B radiation on the UV-B sensitive legume *Phaseolus vulgaris* L. "Top Crop" over a two-week period. Total free polyamines showed marked decreases in response to UV-B radiation, primarily due to a decrease in Put, which was correlated with UV-B induced chlorophyll loss. Kramer et al. (1992) reported that photosynthetically active radiation (PAR) had a large effect on polyamine levels in leaves, with higher levels of Put and Spd observed at 600 than at 300 µmol m-2 s-1 in both cultivars (UV-B-sensitive and– insensitive cultivars). The results indicated that the inhibition of UV-B stress by high PAR

The sensitivity of the photosynthetic apparatus to UV-B irradiation was studied in cultures of unicellular green alga *Scenedesmus obliquus* incubated in low light (LL) and high light (HL) conditions, treated or not with exogenous polyamines. UV-B radiation induces a decrease in the thylakoid-associated Put and an increase in Spm, so that the reduction of Put/Spm ratio leads to the increase of light-harvesting complex II (LHCII) size per active reaction center and, consequently, the amplification of UV-B effects of the photosynthetic apparatus. The separation of oligomeric and monomeric froms of LHCII from isolated thylakoids showed that UV-B induces an increase in the oligomeric forms of LHCII, which was more intense in LL than in HL. By manipulating the LHCII size with exogenous polyamines, the sensitivity degree of the photosynthetic apparatus to UV-B changed significantly. Specifically, the addition of Put decreased highly the sensitivity of LL culture to UV-B because of the inhibitory effect of Put on the LHCII size increasing, wheres the addition of Spm enhanced the UV-B injury induced in HL culture because of the increasing of LHCII size (Sfichi et al., 2004). Zacchini et al. (2004) reported that the put content in tobacco was enhanced, compared with control, in upper layers of calli 6 h after UV-C high dose stress and decreased 24 and 48 h after irradiation, though remained statistically higher than control.

may involve polyamine accumulation.

In natural conditions, flooding, excessive irrigation and soil compaction extremely lead to oxygen deficiency in the root-zone of plant, thus causing to hypoxia stress (Drew, 1997). Hypoxia stress is one of an important environmental factor inhibits plant growth and yield (Avijie et al., 2002). Previous several studies have been shown that suitable concentration of exogenous polyamines could alleviate hypoxia stress-induced physiological damage and improve photochemical efficiency of plants (Vigne and Frelin, 2008). Zhou et al. (2006) demonstrated application of exogenous polyamines to some extent increased net photosynthetic rate (P*N*) and water use efficiency (WUE) in cucumber leaves. Shi et al. (2009) also observed that exogenous Put alleviated the reduction of P*N*, Gs, Ci of cucumber subjected to root-zone hypoxia through enhancing the actual and maximal nitrate reductase activities. Application of exogenous 0.05 mM Spd added to the hypoxia nutrient solutions significantly suppressed the accumulation and insoluble bound Put in roots and leaves of cucumber seedlings, which was associated with a decreased in dissipation energy (NPQ) and xanthophylls de-epoxidation state (DEPS) during hypoxia stress, while Spd enhanced maximal photochemical efficiency (Fv/Fm), PSII photochemistry rate (ΦPSII) and P*N* (Jia, 2009). These results implied that exogenous polyamines increased photosynthetic capacity might be involved in regulating changes of endogenous polyamine contents in the chloroplasts, thus enhancement of root-zone hypoxia tolerance.

#### **3.6 Oxidative stress**

Oxidative stress has been postulated, years ago, to be a causal factor in responses to environmental stresses (Velikova et al., 2000; Patil et al., 2007). The basic tenet of this hypothesis is that the stress-associated decline in the functional capacity of biological systems is primarily due to the accumulation of irreparable oxidative molecular damage (Sohal et al., 1995). Adverse environmental stresses induced a decrease in photosynthetic activity which often associated with the oxidative stress (Krause, 1994). It has been shown that plant could overcome the effect of the oxidative stress and sustain photosynthetic efficiency that may be related to the scavenging of stress-induced toxic oxygen species, such as hydrogen peroxide (H2O2), hydroxyl radical (OH·) and superoxide radical (O2 · - ) (Sopory et al., 1990).

Polyamines (PAs) are regarded as a new class of growth substances and are also well known for their positive effects on the photosynthetic efficiency under various stress conditions due to their acid neutralizing and antioxidant properties, as well as to their membrane and cell wall stabilizing abilities (He et al., 2002; Mapelli et al., 2008; Zhao and Yang, 2008). Exogenous application of spermidine (Spd) could alleviate salt-induced membrane injury of chloroplast by increasing the active oxygen scavenging ability. The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), the contents of ascorbic acid (AsA), Car and glutathione reduced form (GSH), and the ratios of GSH/GSSG in chloroplasts were increased, which increased the salinity tolerance of the photosynthetic apparatus in cucumber seedlings (Duan et al., 2008a; Duan et al., 2009b). Pretreatment with Spd markedly reduced lipid peroxidation and membrane relative permeability in wheat leaves under water stress (Duan et al., 2006). They found that Spd

A Review: Polyamines and Photosynthesis 453

PSII Incubation Natural

spraying

Leaves Incubation Normal

Hoagland nutrient solution

Leaves Incubation Dark

medium

in the solution (pretreatment)

Plants Whole plant spraying Stress Measured

condition

Yellow rust

conditions

conditions

Spm 10ug/L Roots Soaking Water P*N*, WUE, Tr Iqbal et al.

Put 1 mM Plants Spraying Salt Ultrastructure of

Put 1 mM Plants Spraying Salt Ultrastructure of

photosynthetic parameters

FTIR, secondary structure of PSII

Chl contents, chloroplast ultrastructure

Heat Fv/Fm, Amax, Rfd Murkowski

Stomatal movements

Chl contents PSI, PSII activities

PSI activity, PSII

Fv/Fm, qP, qN

Chl contents, chloroplast structure

leaf mesophyll

chloroplasts

Salt Total Chl content,

activity

Chilling Chl contents

Ozone P*N*, Fv/Fm,

Author

Bograh et al.(1997)

Aldesuquy et al.(2000)

Liu et al. (2000)

(2001)

Subhan et al. (2001)

Chattopadhayay et al. (2002)

He et al. (2002)

(2003)

Navakoudis et al. (2003)

Paramonova et al. (2003)

Paramonova et al. (2004)

Organ Mode of PAs treatment

Leaves Foliar

to

Leaves Root

Spd 0.5 mM Leaves Incubation

Species PAs

Spinacea oleracea

Triticum aestivum

Lycopersicon esculentum

Triticum aestivum

Cucumis sativus

Pinus strobus

Nicotiana tabacum

Mesembryan themum crystallinum

Mesembryan themum crystallinum

Oryza sativa Spd

type

Spd

Spm

Put Spd Spm

Put Spd Spm

Spm

Put Spd Spm 1 mM 1 mM 1 mM

Vicia faba Spd 0.1, 0.5,

PAs dosage /concentration

10 μM 0.1mM 1 mM 5 mM 10 mM

10 μM 0.1 mM 0.5 mM

1.0, 3.0, 6.0 mM

20 μM 20 μM 20 μM

1.0 mM 1.0 mM

Spd 4 mM Roots Addition

also improved the transcription of PSII genes and translation of the corresponding proteins, which sustains a higher activity in PSII during water stress. Yiu et al. (2009) showed that 2 mM putrescine (Put) pre-treatment confers flooding tolerance to the photosynthetic efficiency of welsh onion plants, probably through inducing the activities of various antioxidative systems. It has been shown that lipid peroxidation levels were significantly decreased by PAs treated with Physcia semipinnata during the exposure to UV-A (Unal et al., 2008). Among the three polyamines, Spm-treated samples had lower concentration of MDA and higher in the amount of chlorophyll a levels than spd-and put-treated samples. These results indicate that polyamines may protect photosystem II from oxidative stress.

The mechanistic details of PAs effects have not been clarified, although a proposed mechanism is based on PAs neutralizing harmful ROS in tissues or cells and inducing the reorganization of the photosynthetic apparatus. Several results have confirmed that the increase in intra-cellular polyamine content or polyamine metabolism related enzyme activity played an important role in photosynthesis of plants and in oxidative stress resistance (Dondini et al., 2001; Cochόn et al., 2007; Demetriou et al., 2007). Polyamines and especially the thylakoid-associated polyamines play a decisive role in protecting photosynthetic apparatus against oxidative stress. He et al. (2008) found that Put induced the changes of endogenous polyamines in the photosynthetic apparatus to some extent, might be involved in the reduction of H2O2 contents and membrane lipid peroxidation under salt stress. This result is supported by our experiments that Put reduced the number of plastoglobuli resulting from gradual thylakoid membrane degradation induced by salinity (Unpublished data). It has been suggested that transglutaminase (chlTGZ) plays an important functional role in the formation of grana stacks, probably due to antenna protein polyaminylation (Ioannidis et al., 2009). Recently, in the transplastomic tobacco young leaves, oxidative stress induced the over-expression of chlTGZ in the tobacco chloroplasts seems to be related to polyaminylation of antenna proteins and thylakoid remodelling, the extended effect of this over-expression seems to induce an imbalance between capture and utilization of light in photosynthesis, changes in the photosynthetic electron transport chain and increasing oxidative risk (Ortigosa et al., 2010).


also improved the transcription of PSII genes and translation of the corresponding proteins, which sustains a higher activity in PSII during water stress. Yiu et al. (2009) showed that 2 mM putrescine (Put) pre-treatment confers flooding tolerance to the photosynthetic efficiency of welsh onion plants, probably through inducing the activities of various antioxidative systems. It has been shown that lipid peroxidation levels were significantly decreased by PAs treated with Physcia semipinnata during the exposure to UV-A (Unal et al., 2008). Among the three polyamines, Spm-treated samples had lower concentration of MDA and higher in the amount of chlorophyll a levels than spd-and put-treated samples. These results indicate that polyamines may protect photosystem II from oxidative stress. The mechanistic details of PAs effects have not been clarified, although a proposed mechanism is based on PAs neutralizing harmful ROS in tissues or cells and inducing the reorganization of the photosynthetic apparatus. Several results have confirmed that the increase in intra-cellular polyamine content or polyamine metabolism related enzyme activity played an important role in photosynthesis of plants and in oxidative stress resistance (Dondini et al., 2001; Cochόn et al., 2007; Demetriou et al., 2007). Polyamines and especially the thylakoid-associated polyamines play a decisive role in protecting photosynthetic apparatus against oxidative stress. He et al. (2008) found that Put induced the changes of endogenous polyamines in the photosynthetic apparatus to some extent, might be involved in the reduction of H2O2 contents and membrane lipid peroxidation under salt stress. This result is supported by our experiments that Put reduced the number of plastoglobuli resulting from gradual thylakoid membrane degradation induced by salinity (Unpublished data). It has been suggested that transglutaminase (chlTGZ) plays an important functional role in the formation of grana stacks, probably due to antenna protein polyaminylation (Ioannidis et al., 2009). Recently, in the transplastomic tobacco young leaves, oxidative stress induced the over-expression of chlTGZ in the tobacco chloroplasts seems to be related to polyaminylation of antenna proteins and thylakoid remodelling, the extended effect of this over-expression seems to induce an imbalance between capture and utilization of light in photosynthesis, changes in the photosynthetic electron transport chain

and increasing oxidative risk (Ortigosa et al., 2010).

Organ Mode of PAs treatment

Leaves Incubation Normal

spraying

Incubation Dark and

Stress Measured

conditions

light conditions

Natural condition photosynthetic parameters

Chl content photosynthetic activity chloroplast ultrastructure

chlorophyll

Protochlorophyllide

Chl content Cheng

Author

and Kao (1983)

Pjon et al. (1990)

Beigbeder and Kotzabasis. (1994)

PAs dosage /concentration

10 mM 10 mM

Spd 1 mM Leaf

discs

Spm 320 μM Leaves Foliar

Species PAs

Oryza sativa Triticumae stivum

Dicotyledonous komatsuna

Scenedesmus obliquus

type

Spd Spm


A Review: Polyamines and Photosynthesis 455

Stress Measured

Drought Chl contents

Incubation UV-A Chla content,

Fv/Fm

photosynthetic parameters

Stomatal status

Hypoxia P*N*, Gs, Tr, Ci Zhou

Salt P*N*, Fv/Fm Duan et al.

Salt P*<sup>N</sup>* Duan JJ

Salt P*<sup>N</sup>* Duan JJ

Salt P*<sup>N</sup>* Duan JJ

Fv/Fm, qP, qN ФPSII, AQY

Drought P*N*,Gs, Tr, WUE,

Hypoxia P*N*, Ci, Gs, Tr

Hypoxia P*N*, Ci, Gs, Tr Vcyt, Valt

Author

Yamaguchi et al. (2007)

(2007)

(2008)

Unal et al. (2008)

(2009)

(2009)

(2009)

Farooq et al. (2009)

Jia (2009)

Shi et al. (2009)

Organ Mode of PAs treatment

Plants Addition to wet lter papers

spraying

Hoagland nutrient solution

to

to nutrient solution

to nutrient solution

to

Seed soaking

Roots Addition to

> to nutrient solution

Hoagland nutrient solution

Hoagland nutrient solution

Species PAs

Arabidopsis Put

Cucumis sativus

Cucumis sativus

Physcia semipinnata

Cucumis sativus

Cucumis sativus

Cucumis sativus

Cucumis sativus

Cucumis sativus

Oryza sativa Put

Spd Spm

Spd 0.05 mM

10 µM 10 µM 10 µM

type

Spd Spm

Put Spd Spm PAs dosage /concentration

1 Mm 1 mM 1 mM

1 Mm 1 mM 1 mM

Spd 0.5mM Leaves Foliar

Spd 0.1 mM Roots Addition

Lichen thalli

Spd 0.1 mM Roots Addition

Spd 0.1 mM Roots Addition

Spd 0.1 mM Roots Addition

Whole plants

Put 0.5 mM Roots Addition


Stress Measured

Water stress

condition

Medium Salt Fluorescence

Normal conditions

conditions

photosynthetic parameters

Salt P*N*, Ci, Gs, Tr Iqbal and

Chlcontents, PSII phothochemical

Salt P*N*, Fv/Fm Liu et al.

Hypoxia P*N*, Gs, Tr, Ci Zhou et al.

Oxygen evolution activity, Fv/Fo,Chl fluorescence decay

Thermoluminescen

number, stomata index, stomata length, the epidermis cell number, stomata

induction, oxygen evolving activity, chlorophyll content

Fv/Fm, PSIIα and

Fv/Fm, qP,ФPSⅡ

activity

kinetics

ce, OJIP

width

PSIIβ photophosphorylation oxygen evolution

JIP-test parameters LHCIIoligomeric monomeric forms

Salt P*N*, Ci, Gs

Salt The stomata

Author

Ashraf (2005)

(2006)

(2006)

Beauchemie et al. (2007)

Çavusoglu et al. (2007)

Demetriou et al. (2007)

Ioannidis and Kotzabasis (2007)

Li et al. (2007)

Navakoudis et al. (2007)

Duan et al. (2006)

Organ Mode of PAs treatment

Seed Soaking (presowing)

> or spraying

> spraying

spraying

PSII Incubation Natural

Spraying Whole plants

Floated in the solutions

spraying

Leaves Foliar

Leaves Foliar

Spd 0.2 mM PSII Irrigating

Species PAs

Zea mays Cad,

Berley Cad,

Scenedesmus obliquus

Nicotiana tabacum

Cucumis sativus

Triticum aestivum

Triticum aestivum

Cucumis sativus

Spinacea oleracea

type

Put Spd Spm

Put Spd, Spm

Put Spd Spm

Spd Spm

Put Spd, Spm

Put 100 mM

> 1 mM 0.1 mM 0.05 mM

Put Spd Spm PAs dosage /concentration

2.5 mM 5 mM 2.5 mM

1 mM 1 mM 1 mM 1 mM

1 mM 0.5 mM 0.5 mM

Millim olar range

10 µM 10 µM 10 µM The whole plant

Isolated thylakoids

Leaf discs

Spd 1 mM Leaves Foliar

Green alga Put 1 mM Cell Incubation Normal


A Review: Polyamines and Photosynthesis 457

germination of seeds, stimulation, and development of flower buds and fruits, somatic embryogenesis, differentiation and plant morphogenesis, signal transduction and in the protection of the photosynthetic apparatus. Although considerable evidence indicates that PAs exhibits various positive effects on photosynthetic processes of plant in response to abiotic stresses (Table 2), their precise role in these specific processes is still far from being complete. So far, it seems that one of the modes of PAs action in the regulation of photosynthesis under environmental stresses is probably by reducing the production of free radicals, scavenging the free radicals and/or involved in activation of expression of genes encoding antioxidant enzymes. On the other hand, we can also speculate that conjugated PAs play an important role in the protection of related proteins in the photosynthetic apparatus. The mechanism of their action probably involves direct binding of PAs to the extrinsic proteins and the hydrophilic portions of intrinsic polypeptides of PSII through electrostatic interaction due to their poly-cationic properties, and the poly-cationic selectivity effect in decreasing order is Spm4+>Spd3+>Put2+. This electrostatic interaction could provide some stability to the conformation of thylakoid proteins against various stresses and consequently help in maintaining the photosynthetic activity. In addition, the biological functions of PAs on photosynthesis may be also as a signal component to cascade with other growth regulator or hormone. In order to better elucidate the role of PAs in the photosynthesis of plants response to abiotic stress, application of advanced molecular biology and proteomic approaches will help elucidate the mechanisms of PAs in particular improvement of photochemical efficiency in plant processes involved in stress tolerance.

This work was funded by National Basic Research Program of China (973 Program, No.2009CB119000) and National Natural Science Foundation of China (No. 30900995; No. 31071831; No. 30871736) and A Project Fund by the Priority Academic Program Development of Jiangsu Higher Education Institutions and Supported by the China earmarked fund for Modern Agro-industry Technology Research System (CARS-25-C-03). I may not fully understand all reseachers working in field of polyamines and photosynthesis under stress

Alcázar, R.; Planas, J. Saxena, T. Zarza, X. Bortolotti, C. Cuevas, J. Bitrián, M. Tiburcio, AF. &

Aldesuquy, HS.; Abdel-Fattah, GM. & Baka, ZA. (2000). Changes in chlorophyll,

Allakhverdiev, S.; Kreslavski, V. Klimov, V. Los, D. Carpentier, R. & Mohanty, P. (2008).

Altabella, T. (2010). Putrescine accumulation confers drought tolerance in transgenic arabidopsis plants over-expressing the homologous *Arginine* 

polyamines and chloroplast ultrastructure of puccinia striiformis induced 'green islands' on detached leaves of *Triticum aestivum*. *Plant Physiologyand Biochemistry,* 

Heat stress: an overview of molecular responses in photosynthesis, *Photosynthesis* 

conditions during writing this chapter, here I express my most sincere apologies.

*decarboxylase* 2 gene. *Plant Physiology and Biochemistry,* 48: 547–552.

**5. Acknowledgement** 

**6. References** 

38: 613−620.

*Research,* 98: 541–550.


Table 2. Effects of exogenous polyamines (Cad, Put, Spd, Spm) on various photosynthetic parameters of plants in response to stress and non-stress conditions. Chl, chlorophyll; P*N*, net photosynthetic rate; Ci, intercellular CO2; Gs, stomatal conductance; Tr, transpiration rate; WUE, water use efficiency; Fv/Fm, maximum quantum efficiency of photosystem II; Amax, area above the fluorescence induction curve; Rfd, informs about the interaction and equilibrium between primary photosynthetic reactions and dark enzymatic reactions; qP, photochemical quenching; qN, non-photochemical quenching; PSI, photosystem I; PSII, photosystem II; ФPSII, actual photochemical efficiency; AQY, apparent quantum efficiency; Vcyt, cytochrome; Valt, alternative respiration; Rubisco, ribulose-1,5-bisphospate carboxylase/oxygenase

#### **4. Conclusion**

From the published literature, it can be deduced that PAs play an important role in wide spectrum of physiological processes such as cell division, dormancy breaking of tubers and

Stress Measured

Dehydration

Cinnamic acid

Senescence photosynthetic parameters

Stomatal closure/

opening

Flood Chl contents Fv/Fm

Salt Chl contents, P*<sup>N</sup>*

NPQ

Salt Fv/Fm, qP, NPQ ФPSII, rETR

Salt P*N*, Gs, Ci, Tr, total soluble sugar, sucrose, starch

,Gs, Tr, Ci, qP Fv/Fm, ФPSII,

Fv/Fm, ФPSⅡ Rubisco activity

Chl contents light-harvesting complexes

Drought P*N*, Gs, Tr, Ci Zhang

Author

Shi et al. (2010)

Yiu et al. (2009)

Zhang et al. (2009)

Huang and Bie (2010)

Serafini-Fracassini et al. (2010)

Shu et al. (2010)

et al.(2010)

Chen (2011)

Organ Mode of PAs treatment

> spraying (pretreatment)

spraying

spraying

Spraying/ Incubation

spraying

spraying

Hoagland nutrient solution

Table 2. Effects of exogenous polyamines (Cad, Put, Spd, Spm) on various photosynthetic parameters of plants in response to stress and non-stress conditions. Chl, chlorophyll; P*N*, net photosynthetic rate; Ci, intercellular CO2; Gs, stomatal conductance; Tr, transpiration rate; WUE, water use efficiency; Fv/Fm, maximum quantum efficiency of photosystem II; Amax, area above the fluorescence induction curve; Rfd, informs about the interaction and equilibrium between primary photosynthetic reactions and dark enzymatic reactions; qP, photochemical quenching; qN, non-photochemical quenching; PSI, photosystem I; PSII, photosystem II; ФPSII, actual photochemical efficiency; AQY, apparent quantum efficiency;

to

Vcyt, cytochrome; Valt, alternative respiration; Rubisco, ribulose-1,5-bisphospate

From the published literature, it can be deduced that PAs play an important role in wide spectrum of physiological processes such as cell division, dormancy breaking of tubers and

Plants Applied to the substrate surface

Species PAs

Welsh onion Put 1,2,

Cucumis sativus

Cowpea cultivar

Lactuca sativus

Cucumis sativus

Cucumis Sativus

Lycopersicon esculentum

carboxylase/oxygenase

**4. Conclusion** 

type

PAs dosage /concentration

Citrus Spm 1 mM Leaves Foliar

3mM

Spm 0.2~2 mM

Put 10mM Leaves Foliar

Spd 1 mM Leaves Foliar

Put 8 mM Leaves Foliar

Spd 0.1mM Leaves Foliar

Spd 0.1mM Roots Addition

Leaves /Leaf discs

germination of seeds, stimulation, and development of flower buds and fruits, somatic embryogenesis, differentiation and plant morphogenesis, signal transduction and in the protection of the photosynthetic apparatus. Although considerable evidence indicates that PAs exhibits various positive effects on photosynthetic processes of plant in response to abiotic stresses (Table 2), their precise role in these specific processes is still far from being complete. So far, it seems that one of the modes of PAs action in the regulation of photosynthesis under environmental stresses is probably by reducing the production of free radicals, scavenging the free radicals and/or involved in activation of expression of genes encoding antioxidant enzymes. On the other hand, we can also speculate that conjugated PAs play an important role in the protection of related proteins in the photosynthetic apparatus. The mechanism of their action probably involves direct binding of PAs to the extrinsic proteins and the hydrophilic portions of intrinsic polypeptides of PSII through electrostatic interaction due to their poly-cationic properties, and the poly-cationic selectivity effect in decreasing order is Spm4+>Spd3+>Put2+. This electrostatic interaction could provide some stability to the conformation of thylakoid proteins against various stresses and consequently help in maintaining the photosynthetic activity. In addition, the biological functions of PAs on photosynthesis may be also as a signal component to cascade with other growth regulator or hormone. In order to better elucidate the role of PAs in the photosynthesis of plants response to abiotic stress, application of advanced molecular biology and proteomic approaches will help elucidate the mechanisms of PAs in particular improvement of photochemical efficiency in plant processes involved in stress tolerance.

#### **5. Acknowledgement**

This work was funded by National Basic Research Program of China (973 Program, No.2009CB119000) and National Natural Science Foundation of China (No. 30900995; No. 31071831; No. 30871736) and A Project Fund by the Priority Academic Program Development of Jiangsu Higher Education Institutions and Supported by the China earmarked fund for Modern Agro-industry Technology Research System (CARS-25-C-03). I may not fully understand all reseachers working in field of polyamines and photosynthesis under stress conditions during writing this chapter, here I express my most sincere apologies.

#### **6. References**


A Review: Polyamines and Photosynthesis 459

Chattopadhayay, MK.; Tiwari, BS. Chattopadhayay, G. Bose, A. Sengupta, DN. & Ghosh, B.

Chen, LF.; Lu, W. Sun, J. Guo, SR. Zhang, ZX. & Yang, YJ. (2011). Effects of exogenous

Cheng, SH. & Kao, CH. (1983). Localized effect of polyamines on chlorophyll loss. *Plant and* 

Childs, AC.; Mehta, DJ. & Gerner, EW. (2003). Polyamine-dependent gene expression.

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**22** 

 *Charlottetown, PE,* 

*Canada* 

**An Overview of Plant Photosynthesis** 

*1Crops and Livestock Research Centre, Agriculture and Agri-Food Canada,* 

*2Department of Biology, University of Prince Edward Island, Charlottetown, PE,* 

In 1893, Charles Barnes (1858-1910) proposed to designate the biological process for "synthesis of complex carbon compounds out of carbonic acid, in the presence of chlorophyll, under the influence of light" as "photosyntax" or "photosynthesis". His preference went for the word "photosyntax", but "photosynthesis" came into common usage as the term of choice (Gest, 2002). Although this definition is still widely used today, the Oxford English Dictionary (OED, 1989) has defined the biological photosynthesis as "the process by which carbon dioxide is converted into organic matter in the presence of the chlorophyll of plants under the influence of light, which in all plants except some bacteria involves the production of oxygen from water". By considering the photosynthetic bacteria that do not produce oxygen and do not necessarily require carbonic acid as carbon source, Gest (Gest, 1993) has refined these definitions as followed: "photosynthesis is a series of processes in which electromagnetic energy is converted to chemical energy used for biosynthesis of organic cell materials; a photosynthetic organism is one in which a major fraction of the energy required for cellular syntheses is supplied by light". Photosynthesis is therefore a unique source for carbon sequestration and allows the aerobic organisms to survive based upon not only on its released oxygen but also its synthesized organic compounds. As such, plants are an ideal host for pathogenic microorganisms such as fungi, bacteria and viruses and also the only food source for herbivores. Theoretically, the photosynthetic capacity of plants is unlimited when the optimal growing conditions are met. Unfortunately, terrestrial plants are constantly challenged by abiotic (UV, water, salinity, temperature) (Baker et al., 1988, Baldry et al., 1966, Barhoumi et al., 2007, Barrow and Cockburn, 1982, Bassham, 1977, Batista-Santos et al., 2011, Bauerle et al., 2007, Berry, 1975, Bischof et al., 2000, Ripley et al., 2008, Ripley et al., 2007, Roberntz and Stockfors, 1998) and biotic (pathogens, pests, animal and human) stresses that reduce their productivity and even threaten their survival (Bilgin et al., 2010, Bonfig et al., 2006, Erickson and Hawkins, 1980, Garavaglia et al., 2010, Kocal et al., 2008, Tang et al., 2009). While the regulation of plant defence responses has been extensively investigated, the effects of pathogen infection on primary metabolism, including photosynthesis, are however less known. Currently, interest in this research area is growing and some aspects of photosynthesis, assimilate

**1. Introduction** 

**Modulation by Pathogen Attacks** 

Kumarakurubaran Selvaraj1,2 and Bourlaye Fofana1

primary root growth in Arabidopsis. *Journal of Experimental Botany,* doi:10.1093/jxb/erq463.


### **An Overview of Plant Photosynthesis Modulation by Pathogen Attacks**

Kumarakurubaran Selvaraj1,2 and Bourlaye Fofana1

*1Crops and Livestock Research Centre, Agriculture and Agri-Food Canada, Charlottetown, PE, 2Department of Biology, University of Prince Edward Island, Charlottetown, PE, Canada* 

#### **1. Introduction**

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*the Arid Areas,* 3: 182–187.

*Horticulturae,* 116: 442–447.

primary root growth in Arabidopsis. *Journal of Experimental Botany,*

T. (2007). A protective role for the polyamine spermine against drought stress in arabidopsis. *Biochemical and Biophysical Research Communications,* 352: 486–490. Yang, JC.; Zhang, JH. Liu, K. Wang, ZQ. Liu, LJ. (2007). Involvement of polyamines in the drought resistance of rice. *Journal of Experimental Botany,* 58: 1545–1555. Yiu, JC.; Juang, LD. Fang, DYT. Liu, CW. & Wu, SJ. (2009). Exogenous putrescine reduces

flooding-induced oxidative damage by increasing the antioxidant properties of

photosynthesis of tomato seedlings under drought stress. *Agricultural Research in* 

exchange characteristics and chlorophyll fluorescence of NaCl-stressed cucumber

chilling tolerance of cucumber (*Cucumis sativus* L.) through modulating

induced by cadmium chloride stress in *Malus hupehensis* Rehd. *Scientia* 

carbohydrate metabolism in cucumber seedlings under hypoxia stress.[Degree].

characteristics and membrane lipid peroxidation of cucumis sativas seedlings

In 1893, Charles Barnes (1858-1910) proposed to designate the biological process for "synthesis of complex carbon compounds out of carbonic acid, in the presence of chlorophyll, under the influence of light" as "photosyntax" or "photosynthesis". His preference went for the word "photosyntax", but "photosynthesis" came into common usage as the term of choice (Gest, 2002). Although this definition is still widely used today, the Oxford English Dictionary (OED, 1989) has defined the biological photosynthesis as "the process by which carbon dioxide is converted into organic matter in the presence of the chlorophyll of plants under the influence of light, which in all plants except some bacteria involves the production of oxygen from water". By considering the photosynthetic bacteria that do not produce oxygen and do not necessarily require carbonic acid as carbon source, Gest (Gest, 1993) has refined these definitions as followed: "photosynthesis is a series of processes in which electromagnetic energy is converted to chemical energy used for biosynthesis of organic cell materials; a photosynthetic organism is one in which a major fraction of the energy required for cellular syntheses is supplied by light". Photosynthesis is therefore a unique source for carbon sequestration and allows the aerobic organisms to survive based upon not only on its released oxygen but also its synthesized organic compounds. As such, plants are an ideal host for pathogenic microorganisms such as fungi, bacteria and viruses and also the only food source for herbivores. Theoretically, the photosynthetic capacity of plants is unlimited when the optimal growing conditions are met. Unfortunately, terrestrial plants are constantly challenged by abiotic (UV, water, salinity, temperature) (Baker et al., 1988, Baldry et al., 1966, Barhoumi et al., 2007, Barrow and Cockburn, 1982, Bassham, 1977, Batista-Santos et al., 2011, Bauerle et al., 2007, Berry, 1975, Bischof et al., 2000, Ripley et al., 2008, Ripley et al., 2007, Roberntz and Stockfors, 1998) and biotic (pathogens, pests, animal and human) stresses that reduce their productivity and even threaten their survival (Bilgin et al., 2010, Bonfig et al., 2006, Erickson and Hawkins, 1980, Garavaglia et al., 2010, Kocal et al., 2008, Tang et al., 2009). While the regulation of plant defence responses has been extensively investigated, the effects of pathogen infection on primary metabolism, including photosynthesis, are however less known. Currently, interest in this research area is growing and some aspects of photosynthesis, assimilate

An Overview of Plant Photosynthesis Modulation by Pathogen Attacks 467

Fig. 1. Calvin-Benson cycle adapted from Taiz and Zeiger (Taiz and Zeiger, 2010)

The outcome of CO2 fixation by higher photosynthetic plants is the production of carbohydrates. As a result of photosynthetic CO2 reduction during the day, starch granules accumulate in the chloroplast while an excess of assimilates are continuously allocated, mostly in the form of sucrose, to sink tissues such as developing leaves, roots, meristems, fruits, and flowers, that are unable to produce sufficient amounts of assimilates by themselves and therefore require their net import via the phloem (Kocal et al., 2008). Sucrose is loaded into the phloem in the minor veins of leaves before export (Zhang and Turgeon, 2009). Recently, Zhang and Turgeon (Zhang and Turgeon, 2009) have proposed two active, species-specific loading mechanisms. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the second putative mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the

**2.1.1.2 Outcome** 

partitioning, and source–sink regulation in different types of plant–pathogen interactions have been investigated. Berger et al (Berger et al., 2007) have recently reviewed how plant physiology meets phytopathology. The reader can also get more detail on this topic in previous study (Trotta et al., 2011, Essmann et al., 2008, Scharte et al., 2009). Here, we will focus our review on current knowledge on the process of higher plant photosynthesis, its outcome for both plants and fungal pathogens, the roles for some of the metabolites and transduction pathways that are implicated in this twined inter-relationship as well as the potential targets as future strategy.

#### **2. Photosynthesis and pathogen**

Plants and pathogens have developed dynamic interactions. Whereas plants tend to survive through different mechanisms following pathogen attack, the later looks for maximizing feed intake to insure its reproduction and dissemination (Korves and Bergelson, 2003, Berger et al., 2007). In this context, the photosynthetate – the energy source for both the plant and pathogens – synthesis and its availability is the focus of a struggle to death. The next section will discuss how photosynthesis proceeds in these challenging conditions.

#### **2.1 Photosynthesis**

Chloroplast is the factory for photosynthesis in higher plants. However, new evidences suggest a contribution for mitochondrial functions in the maintenance of efficient photosynthesis (Nunes-Nesi et al., 2008). The general process of photosynthesis, its outcome and limiting factors will be briefly described in the next sections.

#### **2.1.1 Process and outcome**

#### **2.1.1.1 Process**

In higher plants, the photosynthetic CO2 fixation occurs in the green leaves, considered as source organs, with the absorption of light by chlorophyll, much of which is located in the light-harvesting complexes (LHCs) of PSII and PSI within the thylakoid membrane of chloroplasts (Murchie and Niyogi, 2011). The mesophyll cell of higher plants, due to its higher chloroplast content, is the most active photosynthetic tissue. In general photosynthesis proceeds through 2 major phases: a) a light phase that produces ATP and NADPH in the chloroplast thylakoids and released in the stroma and b) the CO2 reduction phase in presence of water in the stroma and that consumes the ATP and NADPH generated in phase a) to produce a triose phosphate through the Calvin-Benson cycle which comprises three stages (Figure 1).

Briefly, the carboxylation of 3 molecules of ribulose 1,5 biphosphate fixes 3 molecules of CO2 and H2O under the ribulose 1,5 biphosphate carboxylase/oxygenase (rubisco) catalysis in the Calvin-Benson cycle and leads to 6 molecules of 3-phosphoglycerate. The 3 phosphoglycerates are then phosphorylated in presence of ATP produced during the light reaction by the catalytic action of 3-phosphoglycerate kinase into 1,3 bisphosphoglycerate which is further reduced by NADPH and NADP-glyceraldehyde 3-phosphate dehydrogenase into 6 molecules of glyceraldehyde 3-phosphate (6 triose phosphates). Of these six triose phosphates, one represents the net synthesis from CO2 fixation and, 9 ATP and 6 NADPH are utilized. The remaining five triose phosphates are used to regenerate the ribulose 1,5 biphosphate to insure continuous CO2 fixation (Taiz and Zeiger, 2010).

Fig. 1. Calvin-Benson cycle adapted from Taiz and Zeiger (Taiz and Zeiger, 2010)

#### **2.1.1.2 Outcome**

466 Advances in Photosynthesis – Fundamental Aspects

partitioning, and source–sink regulation in different types of plant–pathogen interactions have been investigated. Berger et al (Berger et al., 2007) have recently reviewed how plant physiology meets phytopathology. The reader can also get more detail on this topic in previous study (Trotta et al., 2011, Essmann et al., 2008, Scharte et al., 2009). Here, we will focus our review on current knowledge on the process of higher plant photosynthesis, its outcome for both plants and fungal pathogens, the roles for some of the metabolites and transduction pathways that are implicated in this twined inter-relationship as well as the

Plants and pathogens have developed dynamic interactions. Whereas plants tend to survive through different mechanisms following pathogen attack, the later looks for maximizing feed intake to insure its reproduction and dissemination (Korves and Bergelson, 2003, Berger et al., 2007). In this context, the photosynthetate – the energy source for both the plant and pathogens – synthesis and its availability is the focus of a struggle to death. The next section will discuss how photosynthesis proceeds in these challenging conditions.

Chloroplast is the factory for photosynthesis in higher plants. However, new evidences suggest a contribution for mitochondrial functions in the maintenance of efficient photosynthesis (Nunes-Nesi et al., 2008). The general process of photosynthesis, its outcome

In higher plants, the photosynthetic CO2 fixation occurs in the green leaves, considered as source organs, with the absorption of light by chlorophyll, much of which is located in the light-harvesting complexes (LHCs) of PSII and PSI within the thylakoid membrane of chloroplasts (Murchie and Niyogi, 2011). The mesophyll cell of higher plants, due to its higher chloroplast content, is the most active photosynthetic tissue. In general photosynthesis proceeds through 2 major phases: a) a light phase that produces ATP and NADPH in the chloroplast thylakoids and released in the stroma and b) the CO2 reduction phase in presence of water in the stroma and that consumes the ATP and NADPH generated in phase a) to produce a triose phosphate through the Calvin-Benson cycle which

Briefly, the carboxylation of 3 molecules of ribulose 1,5 biphosphate fixes 3 molecules of CO2 and H2O under the ribulose 1,5 biphosphate carboxylase/oxygenase (rubisco) catalysis in the Calvin-Benson cycle and leads to 6 molecules of 3-phosphoglycerate. The 3 phosphoglycerates are then phosphorylated in presence of ATP produced during the light reaction by the catalytic action of 3-phosphoglycerate kinase into 1,3 bisphosphoglycerate which is further reduced by NADPH and NADP-glyceraldehyde 3-phosphate dehydrogenase into 6 molecules of glyceraldehyde 3-phosphate (6 triose phosphates). Of these six triose phosphates, one represents the net synthesis from CO2 fixation and, 9 ATP and 6 NADPH are utilized. The remaining five triose phosphates are used to regenerate the

ribulose 1,5 biphosphate to insure continuous CO2 fixation (Taiz and Zeiger, 2010).

and limiting factors will be briefly described in the next sections.

potential targets as future strategy.

**2.1 Photosynthesis** 

**2.1.1.1 Process** 

**2.1.1 Process and outcome** 

comprises three stages (Figure 1).

**2. Photosynthesis and pathogen** 

The outcome of CO2 fixation by higher photosynthetic plants is the production of carbohydrates. As a result of photosynthetic CO2 reduction during the day, starch granules accumulate in the chloroplast while an excess of assimilates are continuously allocated, mostly in the form of sucrose, to sink tissues such as developing leaves, roots, meristems, fruits, and flowers, that are unable to produce sufficient amounts of assimilates by themselves and therefore require their net import via the phloem (Kocal et al., 2008). Sucrose is loaded into the phloem in the minor veins of leaves before export (Zhang and Turgeon, 2009). Recently, Zhang and Turgeon (Zhang and Turgeon, 2009) have proposed two active, species-specific loading mechanisms. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the second putative mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the

An Overview of Plant Photosynthesis Modulation by Pathogen Attacks 469

Plants pathogens are of diverse nature and include pathogenic fungi, virus, and bacteria. Whereas all of these pathogens are of great interests for plant biologists, the sake of this

Plant infection by pathogenic fungi and bacteria can occur in multiple ways. It could be passive, i.e. accidental, by suction into the plant through natural plant openings such as stomata, hydathodes or lenticels, entrance through abrasions or wounds on leaves, stems or roots (Hu and Rijkenberg, 1998b, Vidaver and Lambrecht, 2004). Infection of host plants by biotrophic plant pathogens generally involves the sequential development of specialized host-parasite interfaces, exemplified by those of haustoria, which are maintained over an extended period of time without causing significant cytological damage to host tissue in the infected region (Tariq and Jeffries, 1984, Tariq and Jeffries, 1986). Using scanning electron microscopy, Hu and Rijkenberg (Hu and Rijkenberg, 1998b) identified key time points in the formation of infection structures by *P. triticinia* on susceptible and resistant lines of hexaploid wheat. Six hours after infection, the fungus forms appressoria over stomata openings. After 12 hours, the fungus has successfully penetrated into the stroma, formed substomatal vesicles (SSV), and primary infection hyphae are visible. After SSV formation, the primary infection hypha grows and attaches to a mesophyll or epidermal cell. At 24 hours postinoculation, a septum appears separating the haustorial mother cell from the infection hypha after which the fungus forms haustorium and penetrates the cell (Hu and Rijkenberg, 1998b). Recently, Garg et al (Garg et al., 2010) described the infection process in susceptible and resistant genotypes of *Brassica napus* against *Sclerotinia sclerotiorum*. They demonstrated at the cellular level that resistance to *S. sclerotiorum* in *B. napus* is a result of retardation of pathogen development, both on the plant surface and within host tissues. There are some indications that the infection process is dependent on the nutritional status of the inoculums (Garg et al., 2010). Indeed, previous studies suggested that the presence of nutrients is essential for hyphal development, penetration and for subsequent establishment of a successful invasion of a susceptible host by the pathogen (Tariq and Jeffries, 1984, Tariq

Plant pathogens like viruses, fungi, oomycetes, and bacteria are known to interfere with the source-sink balance (Berger et al., 2007, Biemelt and Sonnewald, 2006, Seo et al., 2007), and in the case of a successful interaction, pathogens are believed to reprogram a plant's metabolism to their own benefit (Biemelt and Sonnewald, 2006). This comprises the suppression of plant defence responses and the reallocation of photoassimilates to sufficiently supply the pathogen with nutrients (Kocal et al., 2008). In accordance with this, the infected leaf is assumed to undergo a source to sink transition or retains its sink character. For example, infection of maize leaves with *Ustilago maydis* prevents establishment of C4 photosynthesis because *U. maydis*-induced leaf galls exhibited carbon dioxide response curves, CO2 compensation points and enzymatic activities that are characteristic of C3 photosynthesis (Horst et al., 2008). An indication for this is provided by a stimulation of cell wall-bound invertase (cw-Inv) that mobilizes hexoses at the infection site

contribution will be limited to the pathogenic fungi with some reference to bacteria.

**2.2 Pathogens in plants** 

**2.2.1 Invasion process** 

and Jeffries, 1986, Garg et al., 2010). **2.2.1.1 Outcomes of pathogen invasion** 

and a decreased rate of photosynthesis (Kocal et al., 2008).

*2.2.1.1.1 For the pathogen* 

phloem, not by transporter activity. Thus, a coordinated sequence of assimilate production, allocation, and utilization is essential for normal plant growth and development (Kocal et al., 2008). Indeed, carbohydrate accumulation in the leaves can lead to decreased expression of photosynthetic genes and accelerated leaf senescence when there is an imbalance between source and sink at the whole plant level (Paul and Foyer, 2001). Generally, when sink activity is decreased by removing active sinks or introducing nutrient deficiency, carbohydrates accumulate in leaves and photosynthesis becomes inhibited (Ainsworth and Bush, 2011, Paul and Pellny, 2003). Similarly, when sucrose export from source leaves is restricted, for example by cold girdling of petioles or down-regulation of sucrose transporter abundance, photosynthesis is inhibited. It has also been reported that a downregulation of sucrose transporter 1 (SUT1) in several sucrose-transporting plants, shown to be apoplastic loaders, led to an accumulation of sugars and leaf chlorosis (Zhang and Turgeon, 2009). In contrast, no such phenotype developed when a symplastic loading plant such as *Verbascum phoeniceum* was downregulated, emphasizing the importance of either active or passive assimilates exports.

#### **2.1.1.3 Limiting factors**

Although the Calvin-Benson cycling capacity seems unlimited in the presence of light, CO2 and H2O, many limiting factors counteract this highly regulated biological process. First, if the presence of CO2 and light are required, their levels are of great importance. As light (photon flux and intensity) increases (in constant optimal CO2), the rate of photosynthesis rises until it is saturated. An excess of light can lead to photoinhibition (Bertamini and Nedunchezhian, 2004, Murchie and Niyogi, 2011). A correlation between the *in vivo* rates of net CO2 assimilation and the atmospheric CO2 concentrations was observed when intact C3 and C4 plants were exposed to different atmospheric CO2 concentrations (Aguera et al., 2006, Ainsworth and Rogers, 2007, Bhatt et al., 2010). In general, current atmospheric CO2 concentration is adequate for both C3 and C4 plants. However, with global warming and its related rising temperature and CO2 level, a higher biomass production and a change in C3 and C4 plants distribution are expected (depending on rainfall) because of their differential photosynthetic and water use efficiency. Nontheless, many temperate plant species may not adapt as rate of photosynthesis was found to decline at moderately high temperature in a temperate species such as *Arabidopsis thaliana* (Kumar et al., 2009, Kurek et al., 2007). If CO2 generally favours photosynthesis, other environmental clues such as UV-B (Albert et al., 2008), heat shock and water deficit (Abrams et al., 1990, Ackerson and Hebert, 1981, Allakhverdiev et al., 2008, ZhangWollenweber et al., 2008), Cold (Batista-Santos et al., 2011, Bilska and Sowinski, 2010), herbivore and pathogen attacks have detrimental effects on photosynthesis (Halitschke et al., 2011, Horst et al., 2008, King and Caylor, 2010, Nabity et al., 2009). The primary targets of thermal damage in plants are the oxygen evolving complex along with the associated cofactors in photosystem II (PSII), carbon fixation by Rubisco and the ATP generating system (Allakhverdiev et al., 2008). Recent studies on the combined effect of moderate light intensity and heat stress suggest that moderately high temperatures do not cause serious PSII damage but inhibit the repair of PSII. Repair of PSII involves *de novo* synthesis of proteins, particularly the D1 protein of the photosynthetic machinery that is damaged due to generation of reactive oxygen species (ROS). Attacks by ROS during moderate heat stress principally affects the repair system of PSII, resulting in the reduction of carbon fixation and oxygen evolution, as well as the disruption of the linear electron flow (Allakhverdiev et al., 2008).

#### **2.2 Pathogens in plants**

468 Advances in Photosynthesis – Fundamental Aspects

phloem, not by transporter activity. Thus, a coordinated sequence of assimilate production, allocation, and utilization is essential for normal plant growth and development (Kocal et al., 2008). Indeed, carbohydrate accumulation in the leaves can lead to decreased expression of photosynthetic genes and accelerated leaf senescence when there is an imbalance between source and sink at the whole plant level (Paul and Foyer, 2001). Generally, when sink activity is decreased by removing active sinks or introducing nutrient deficiency, carbohydrates accumulate in leaves and photosynthesis becomes inhibited (Ainsworth and Bush, 2011, Paul and Pellny, 2003). Similarly, when sucrose export from source leaves is restricted, for example by cold girdling of petioles or down-regulation of sucrose transporter abundance, photosynthesis is inhibited. It has also been reported that a downregulation of sucrose transporter 1 (SUT1) in several sucrose-transporting plants, shown to be apoplastic loaders, led to an accumulation of sugars and leaf chlorosis (Zhang and Turgeon, 2009). In contrast, no such phenotype developed when a symplastic loading plant such as *Verbascum phoeniceum* was downregulated, emphasizing the importance of either active or passive

Although the Calvin-Benson cycling capacity seems unlimited in the presence of light, CO2 and H2O, many limiting factors counteract this highly regulated biological process. First, if the presence of CO2 and light are required, their levels are of great importance. As light (photon flux and intensity) increases (in constant optimal CO2), the rate of photosynthesis rises until it is saturated. An excess of light can lead to photoinhibition (Bertamini and Nedunchezhian, 2004, Murchie and Niyogi, 2011). A correlation between the *in vivo* rates of net CO2 assimilation and the atmospheric CO2 concentrations was observed when intact C3 and C4 plants were exposed to different atmospheric CO2 concentrations (Aguera et al., 2006, Ainsworth and Rogers, 2007, Bhatt et al., 2010). In general, current atmospheric CO2 concentration is adequate for both C3 and C4 plants. However, with global warming and its related rising temperature and CO2 level, a higher biomass production and a change in C3 and C4 plants distribution are expected (depending on rainfall) because of their differential photosynthetic and water use efficiency. Nontheless, many temperate plant species may not adapt as rate of photosynthesis was found to decline at moderately high temperature in a temperate species such as *Arabidopsis thaliana* (Kumar et al., 2009, Kurek et al., 2007). If CO2 generally favours photosynthesis, other environmental clues such as UV-B (Albert et al., 2008), heat shock and water deficit (Abrams et al., 1990, Ackerson and Hebert, 1981, Allakhverdiev et al., 2008, ZhangWollenweber et al., 2008), Cold (Batista-Santos et al., 2011, Bilska and Sowinski, 2010), herbivore and pathogen attacks have detrimental effects on photosynthesis (Halitschke et al., 2011, Horst et al., 2008, King and Caylor, 2010, Nabity et al., 2009). The primary targets of thermal damage in plants are the oxygen evolving complex along with the associated cofactors in photosystem II (PSII), carbon fixation by Rubisco and the ATP generating system (Allakhverdiev et al., 2008). Recent studies on the combined effect of moderate light intensity and heat stress suggest that moderately high temperatures do not cause serious PSII damage but inhibit the repair of PSII. Repair of PSII involves *de novo* synthesis of proteins, particularly the D1 protein of the photosynthetic machinery that is damaged due to generation of reactive oxygen species (ROS). Attacks by ROS during moderate heat stress principally affects the repair system of PSII, resulting in the reduction of carbon fixation and oxygen evolution, as well as the disruption of the linear electron flow

assimilates exports. **2.1.1.3 Limiting factors** 

(Allakhverdiev et al., 2008).

Plants pathogens are of diverse nature and include pathogenic fungi, virus, and bacteria. Whereas all of these pathogens are of great interests for plant biologists, the sake of this contribution will be limited to the pathogenic fungi with some reference to bacteria.

#### **2.2.1 Invasion process**

Plant infection by pathogenic fungi and bacteria can occur in multiple ways. It could be passive, i.e. accidental, by suction into the plant through natural plant openings such as stomata, hydathodes or lenticels, entrance through abrasions or wounds on leaves, stems or roots (Hu and Rijkenberg, 1998b, Vidaver and Lambrecht, 2004). Infection of host plants by biotrophic plant pathogens generally involves the sequential development of specialized host-parasite interfaces, exemplified by those of haustoria, which are maintained over an extended period of time without causing significant cytological damage to host tissue in the infected region (Tariq and Jeffries, 1984, Tariq and Jeffries, 1986). Using scanning electron microscopy, Hu and Rijkenberg (Hu and Rijkenberg, 1998b) identified key time points in the formation of infection structures by *P. triticinia* on susceptible and resistant lines of hexaploid wheat. Six hours after infection, the fungus forms appressoria over stomata openings. After 12 hours, the fungus has successfully penetrated into the stroma, formed substomatal vesicles (SSV), and primary infection hyphae are visible. After SSV formation, the primary infection hypha grows and attaches to a mesophyll or epidermal cell. At 24 hours postinoculation, a septum appears separating the haustorial mother cell from the infection hypha after which the fungus forms haustorium and penetrates the cell (Hu and Rijkenberg, 1998b). Recently, Garg et al (Garg et al., 2010) described the infection process in susceptible and resistant genotypes of *Brassica napus* against *Sclerotinia sclerotiorum*. They demonstrated at the cellular level that resistance to *S. sclerotiorum* in *B. napus* is a result of retardation of pathogen development, both on the plant surface and within host tissues. There are some indications that the infection process is dependent on the nutritional status of the inoculums (Garg et al., 2010). Indeed, previous studies suggested that the presence of nutrients is essential for hyphal development, penetration and for subsequent establishment of a successful invasion of a susceptible host by the pathogen (Tariq and Jeffries, 1984, Tariq and Jeffries, 1986, Garg et al., 2010).

#### **2.2.1.1 Outcomes of pathogen invasion**

#### *2.2.1.1.1 For the pathogen*

Plant pathogens like viruses, fungi, oomycetes, and bacteria are known to interfere with the source-sink balance (Berger et al., 2007, Biemelt and Sonnewald, 2006, Seo et al., 2007), and in the case of a successful interaction, pathogens are believed to reprogram a plant's metabolism to their own benefit (Biemelt and Sonnewald, 2006). This comprises the suppression of plant defence responses and the reallocation of photoassimilates to sufficiently supply the pathogen with nutrients (Kocal et al., 2008). In accordance with this, the infected leaf is assumed to undergo a source to sink transition or retains its sink character. For example, infection of maize leaves with *Ustilago maydis* prevents establishment of C4 photosynthesis because *U. maydis*-induced leaf galls exhibited carbon dioxide response curves, CO2 compensation points and enzymatic activities that are characteristic of C3 photosynthesis (Horst et al., 2008). An indication for this is provided by a stimulation of cell wall-bound invertase (cw-Inv) that mobilizes hexoses at the infection site and a decreased rate of photosynthesis (Kocal et al., 2008).

An Overview of Plant Photosynthesis Modulation by Pathogen Attacks 471

Pathogen attacks result in a decreased rate of plant photosynthesis (Kocal et al., 2008), and as a consequence yield loss (Berger et al., 2007). Pathogen infection often leads to plant death, the development of chlorotic and necrotic (Kim et al., 2010) lesions and to a decrease in photosynthetic assimilate production. Using chlorophyll fluorescence imaging, it has been reported that the changes in photosynthesis upon infection are local. In *Arabidopsis* leaves infected with *A. candida* and in tomato plants infected with *B. cinerea*, a ring of enhanced photosynthesis was detectable surrounding the area with decreased photosynthesis at the infection site. At present, it is not clear if this stimulation of photosynthesis is due to the defence strategy of the plant (Berger et al., 2007). A decrease in photosynthesis has also been reported in incompatible interactions (Bonfig et al., 2006). The decrease in photosynthesis was detectable earlier with the avirulent strain than with the virulent strain. It is suggested that plants switch off photosynthesis and other assimilatory metabolism to initiate respiration and other processes required for defence (Berger et al., 2007). Recently, Petit et al (Petit et al., 2006) characterized the photosynthetic apparatus of grape leaves infected with esca disease. Foliar symptoms were associated with stomatal closure and alteration of the photosynthetic apparatus. A decrease in CO2 assimilation, transpiration, a significant increase in intercellular CO2 concentration, a strong drop in the maximum fluorescence yield and the effective Photosystem II quantum yields, and a reduction of total chlorophyll but a stable carotenoid

Several mechanisms have been described to explain the suppression of plant defence responses and the reprogramming of the plant's metabolism to the pathogen own benefit (Garavaglia et al., 2010). The pathogens *Stagonospora nodorum* and *Pyrenophora tritici-repentis*, the causal agents of *Stagonospora nodorum* blotch (SNB) and tan spot, respectively, produce multiple effectors (Ptr ToxA, Ptr ToxB, and Ptr ToxC), also known as host-selective toxins (HSTs), that interact with corresponding host sensitivity genes in an inverse gene-for-gene manner to cause the diseases in wheat. A compatible interaction requires both the effector (HST) and the host gene and results in susceptibility as opposed to host resistance (R) genes. R-genes lead to a resistance response known as effector-triggered immunity (ETI) which includes localized programmed cell death (PCD) or hypersensitive response (HR), to restrict pathogen growth. The absence of either the effector or the host gene results in an incompatible interaction (Zhang et al., 2011, Faris et al., 2010). *Pyrenophora tritici-repentis* produces oval or diamond-shaped to elongated irregular spots that enlarge and turn tan with a yellow border and a small dark brown spot near the center causing necrotic and/or chlorotic lesions on infected leaves, which can significantly reduce total photosynthetic area and yield loss (Kim et al., 2010). The development of chlorosis in response to Ptr ToxB results from an inhibition of photosynthesis in the host, leading to the photooxidation of chlorophyll molecules as illuminated thylakoid membranes become unable to dissipate excess excitation energy (Strelkov et al., 1998). Recently, Kim et al (Kim et al., 2010) showed that treatment of wheat leaves with Ptr ToxB results in significant changes in the abundance of more than 100 proteins, including proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defence response. These authors also examined the direct effect of Ptr ToxB on photosynthesis and found a net decline of photosynthesis within 12 h of toxin-treatment, long before chlorosis develops at 48–72 h. A

**2.3 Photosynthesis and pathogens invasions** 

content were reported (Petit et al., 2006).

**2.3.2 Mechanistic alteration of the photosynthetic capacity** 

**2.3.1 Photosynthesis efficiency under pathogen attacks** 

#### *2.2.1.1.2 For the plant*

Pathogen attacks result in the development of symptoms that include leaf and fruit wilt, stem and root rot (Rekah et al., 1999), coverage of leaf surface with pustule, chlorosis and necrosis (Fofana et al., 2007, Kocal et al., 2008) (Figure 2), a decreased rate of plant photosynthesis (Kocal et al., 2008), and as a consequence plant death or yield loss ensues (Berger et al., 2007).

Fig. 2. Symptoms of wheat leaf rust (*Pucinia triticina)* on wheat near isogenic RL6003 line inoculated with a) avirulent race 1 (BBB), and b) virulent race 7-2 (TJB) of *Pucinia triticina*. An incompatible interaction showing ; 1- infection types with very small pustules, no sporulation and hypersensitive reaction (BBB) and compatible interaction showing a 3+4 infection type with large pustules, abundant sporulation and chlorotic reaction around sporulations (TJB).

#### **2.3 Photosynthesis and pathogens invasions**

470 Advances in Photosynthesis – Fundamental Aspects

Pathogen attacks result in the development of symptoms that include leaf and fruit wilt, stem and root rot (Rekah et al., 1999), coverage of leaf surface with pustule, chlorosis and necrosis (Fofana et al., 2007, Kocal et al., 2008) (Figure 2), a decreased rate of plant photosynthesis (Kocal et al., 2008), and as a consequence plant death or yield loss ensues

Fig. 2. Symptoms of wheat leaf rust (*Pucinia triticina)* on wheat near isogenic RL6003 line inoculated with a) avirulent race 1 (BBB), and b) virulent race 7-2 (TJB) of *Pucinia triticina*. An incompatible interaction showing ; 1- infection types with very small pustules, no sporulation and hypersensitive reaction (BBB) and compatible interaction showing a 3+4 infection type with large pustules, abundant sporulation and chlorotic reaction around

*2.2.1.1.2 For the plant* 

(Berger et al., 2007).

sporulations (TJB).

#### **2.3.1 Photosynthesis efficiency under pathogen attacks**

Pathogen attacks result in a decreased rate of plant photosynthesis (Kocal et al., 2008), and as a consequence yield loss (Berger et al., 2007). Pathogen infection often leads to plant death, the development of chlorotic and necrotic (Kim et al., 2010) lesions and to a decrease in photosynthetic assimilate production. Using chlorophyll fluorescence imaging, it has been reported that the changes in photosynthesis upon infection are local. In *Arabidopsis* leaves infected with *A. candida* and in tomato plants infected with *B. cinerea*, a ring of enhanced photosynthesis was detectable surrounding the area with decreased photosynthesis at the infection site. At present, it is not clear if this stimulation of photosynthesis is due to the defence strategy of the plant (Berger et al., 2007). A decrease in photosynthesis has also been reported in incompatible interactions (Bonfig et al., 2006). The decrease in photosynthesis was detectable earlier with the avirulent strain than with the virulent strain. It is suggested that plants switch off photosynthesis and other assimilatory metabolism to initiate respiration and other processes required for defence (Berger et al., 2007). Recently, Petit et al (Petit et al., 2006) characterized the photosynthetic apparatus of grape leaves infected with esca disease. Foliar symptoms were associated with stomatal closure and alteration of the photosynthetic apparatus. A decrease in CO2 assimilation, transpiration, a significant increase in intercellular CO2 concentration, a strong drop in the maximum fluorescence yield and the effective Photosystem II quantum yields, and a reduction of total chlorophyll but a stable carotenoid content were reported (Petit et al., 2006).

#### **2.3.2 Mechanistic alteration of the photosynthetic capacity**

Several mechanisms have been described to explain the suppression of plant defence responses and the reprogramming of the plant's metabolism to the pathogen own benefit (Garavaglia et al., 2010). The pathogens *Stagonospora nodorum* and *Pyrenophora tritici-repentis*, the causal agents of *Stagonospora nodorum* blotch (SNB) and tan spot, respectively, produce multiple effectors (Ptr ToxA, Ptr ToxB, and Ptr ToxC), also known as host-selective toxins (HSTs), that interact with corresponding host sensitivity genes in an inverse gene-for-gene manner to cause the diseases in wheat. A compatible interaction requires both the effector (HST) and the host gene and results in susceptibility as opposed to host resistance (R) genes. R-genes lead to a resistance response known as effector-triggered immunity (ETI) which includes localized programmed cell death (PCD) or hypersensitive response (HR), to restrict pathogen growth. The absence of either the effector or the host gene results in an incompatible interaction (Zhang et al., 2011, Faris et al., 2010). *Pyrenophora tritici-repentis* produces oval or diamond-shaped to elongated irregular spots that enlarge and turn tan with a yellow border and a small dark brown spot near the center causing necrotic and/or chlorotic lesions on infected leaves, which can significantly reduce total photosynthetic area and yield loss (Kim et al., 2010). The development of chlorosis in response to Ptr ToxB results from an inhibition of photosynthesis in the host, leading to the photooxidation of chlorophyll molecules as illuminated thylakoid membranes become unable to dissipate excess excitation energy (Strelkov et al., 1998). Recently, Kim et al (Kim et al., 2010) showed that treatment of wheat leaves with Ptr ToxB results in significant changes in the abundance of more than 100 proteins, including proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defence response. These authors also examined the direct effect of Ptr ToxB on photosynthesis and found a net decline of photosynthesis within 12 h of toxin-treatment, long before chlorosis develops at 48–72 h. A

An Overview of Plant Photosynthesis Modulation by Pathogen Attacks 473

induced resistance observed for elicited plants. Moreover, chitin labelling revealed that the walls and lobes of fungal haustoria within both treatments were undisturbed, suggesting that PR proteins, such as chitinases and β-1,3-glucanases, may not play a major role in the early events of induced resistance for cucumber (Fofana et al., 2005). However, Gonzalez-Teuber et al. (Gonzalez-Teuber et al., 2010) recently reported glucanases and chitinases as causal agents in the protection of *acacia* extrafloral nectar from infestation by phytopathogens. Nectars are rich in primary metabolites and as protective strategy, floral nectar of ornamental tobacco (*Nicotiana langsdorffii x Nicotiana sanderae*) contains "nectarins," proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, *Acacia* extrafloral nectar contains pathogenesis-related (PR) proteins. This nectar is secreted in the context of defensive reactions. Gonzalez-Teuber et al (Gonzalez-Teuber et al., 2010) showed that PR proteins causally underlie the protection of *Acacia* extrafloral nectar from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function. Salicylic acid (SA) and Jasmonic acid have long been considered as signal molecules in disease resistance (Ward et al., 1991). Recently, *Arabidopsis* GH3-type proteins functioning in auxin signaling, in association with a salicylic acid (SA)-dependent pathway, was reported to positively regulate resistance to *Pseudomonas syringae* (Jagadeeswaran et al., 2007). Accordingly, Fu et al (Fu et al., 2011) suggested that GH3-2 encodes an IAA-amido synthetase and positively regulates rice disease resistance by suppressing pathogen-induced accumulation of IAA in rice. Activation of GH3-2 confers to rice a broad spectrum and partial resistance against *Xanthomonas oryzae pv oryzae and Xanthomonas oryzae pv oryzicola* and the

The role for secondary metabolites in the plant's interaction with its environment is widely recognized (Rhodes, 1994). The primary metabolites deriving from photosynthesis are channeled into different metabolite pathways for the synthesis, storage, and modification (hydroxylation, glycosylation, acetylation, etc) of myriads of compounds, and for use to cope abiotic and biotic clues. Within each of the major groups of secondary metabolites such as alkaloids, phenylpropanoids and terpenoids, several thousand individual compounds accumulating in plants have been characterised and their role in plant-pathogen interactions studied (Ishihara et al., 2011). For example, induction of phenolic compounds, flavonoid phytoalexins, (Daayf et al., 1997, Fawe et al., 1998, Fofana et al., 2002, McNally et al., 2003b, McNally et al., 2003) was reported in cucumber plants following pathogen attacks and elicitor treatments. Synthesis of phytoalexins involves the rapid transcriptional activation of genes encoding a number of key biosynthetic enzymes that include anthranilate synthase (AS) (IshiharaHashimotoTanaka et al., 2008) phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) which is the early committed key enzyme of the flavonoid/isoflavonoid pathway, chalcone isomerase (CHI) and isoflavone reductase (IFR) (Dixon et al., 1995, Baldridge et al., 1998, Fofana et al., 2002, Fofana et al., 2005). The chemical nature of some of these compounds is now well elucidated (Ibanez et al., 2010, Ishihara et al., 2011, McNally et al., 2003). McNally et al (McNally et al., 2003b, McNally et al., 2003) reported the synthesis of complex C-glycosyl flavonoid phytoalexins, referred to as vitexin-6-(4-hydroxy-1 ethylbenzene) (cucumerin A) and isovitexin-8-(4-hydroxy-1-ethylbenzene) (cucumerin B), as a site-specific response to fungal penetration in cucumber. In a recent study, Ishihara et al (IshiharaHashimotoTanaka et al., 2008) reported on an induced accumulation of Trp-

fungal *Magnaporthe grisea* in rice.

**3.1.2 Secondary metabolites** 

role for ROS generation and disruptions of the photosynthetic electron transport shortly after pathogen attack or toxin treatment have been suggested as potential mechanism (Kim et al., 2010). Similar mechanism has been proposed by Allakhverdiev et al (Allakhverdiev et al., 2008) following heat stress that targets the oxygen evolving complex along with the associated cofactors in photosystem II (PSII), carbon fixation by rubisco and the ATP generating system. In another system, Kocal et al (Kocal et al., 2008) studied the role of cell wall invertase (cw-Inv) in transgenic tomato (*Solanum lycopersicum*) plants silenced for the major leaf cw-Inv isoforms during normal growth and during the compatible interaction with *Xanthomonas campestris pv vesicatoria*. Cw-Inv expression was found to be induced upon microbial infection and was most likely associated with an apoplastic hexose accumulation during the infection process. The hexoses formed are thought to aid the pathogen's nutrition (Berger et al., 2007, Biemelt and Sonnewald, 2006, Seo et al., 2007). Fungal pathogens also produce their own invertases to ensure their nutritional supply (Chou et al., 2000, Voegele et al., 2006). One of the most sophisticated mechanisms that divert plant metabolites to pathogen is the role of auxin in host-pathogen interactions (Fu et al., 2011, Navarro et al., 2006). Indole-3-acetic acid (IAA) is the major form of auxin in most plants and induces the loosening of plant cell wall, the natural protective barrier to invaders. *X. oryzae pv oryzae*, *X. oryzae pv oryzicola*, and *M. grisea* secrete IAA, which, in turn, may induce rice to synthesize its own IAA at the infection site. IAA induces the production of expansins, the cell wallloosening proteins, and makes rice vulnerable to pathogens (Fu et al., 2011). Similarly, Garavaglia et al (Garavaglia et al., 2010) have reported an eukaryotic-acquired gene by a biotrophic phytopathogen that allows its prolonged survival on the host by counteracting the shut-down of plant photosynthesis.

#### **3. Plant reactions to pathogen attack**

During plant-pathogen interactions, the host develops a variety of defence reactions. Nonhost resistance against a biotrophic fungal pathogen is often manifested as the ability of the attacked plant to prevent fungal penetration, or the ability to terminate the development and/or functioning of the fungal feeding structure such as the intracellular hypha or the haustorium before it extracts enough nutrition from the plant cells (Wen et al., 2011). These reactions may involved development of physical barriers such as exocytosis, cell wall modifications and de novo metabolites synthesis (IshiharaHashimotoMiyagawa et al., 2008).

#### **3.1 Primary metabolites and secondary metabolite production 3.1.1 Primary metabolites**

Cell wall strengthening by callosic (Wen et al., 2011) and papillae formation, cell wall apposition (Fofana et al., 2005, Wurms et al., 1999), lignin deposition (Hammerschmidt and Kuc, 1982) as well as hydrolytic PR proteins (Hu and Rijkenberg, 1998a) have been reported as first line of defence mechanism developed by plants. Components of these cell wall makeups are of primary metabolite origin. It is worth noting that papillae were at times observed as an initial response to fungal penetration. Generally, papillae relate to powdery mildew fungal penetration in two ways: in some instances, penetration fails when papillae are present and alternatively, penetration may succeed and the papilla becomes a collar for the haustorial neck (Hammerschmidt and Yang-Cashman, 1995). Using transmission electron microscopy, Fofana et al (Fofana et al., 2005) observed both outcomes for elicited and nonelicited cucumber plants, strongly suggesting that papillae formation alone may not be sufficient to explain the level of induced resistance observed for elicited plants. Moreover, chitin labelling revealed that the walls and lobes of fungal haustoria within both treatments were undisturbed, suggesting that PR proteins, such as chitinases and β-1,3-glucanases, may not play a major role in the early events of induced resistance for cucumber (Fofana et al., 2005). However, Gonzalez-Teuber et al. (Gonzalez-Teuber et al., 2010) recently reported glucanases and chitinases as causal agents in the protection of *acacia* extrafloral nectar from infestation by phytopathogens. Nectars are rich in primary metabolites and as protective strategy, floral nectar of ornamental tobacco (*Nicotiana langsdorffii x Nicotiana sanderae*) contains "nectarins," proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, *Acacia* extrafloral nectar contains pathogenesis-related (PR) proteins. This nectar is secreted in the context of defensive reactions. Gonzalez-Teuber et al (Gonzalez-Teuber et al., 2010) showed that PR proteins causally underlie the protection of *Acacia* extrafloral nectar from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function. Salicylic acid (SA) and Jasmonic acid have long been considered as signal molecules in disease resistance (Ward et al., 1991). Recently, *Arabidopsis* GH3-type proteins functioning in auxin signaling, in association with a salicylic acid (SA)-dependent pathway, was reported to positively regulate resistance to *Pseudomonas syringae* (Jagadeeswaran et al., 2007). Accordingly, Fu et al (Fu et al., 2011) suggested that GH3-2 encodes an IAA-amido synthetase and positively regulates rice disease resistance by suppressing pathogen-induced accumulation of IAA in rice. Activation of GH3-2 confers to rice a broad spectrum and partial resistance against *Xanthomonas oryzae pv oryzae and Xanthomonas oryzae pv oryzicola* and the fungal *Magnaporthe grisea* in rice.

#### **3.1.2 Secondary metabolites**

472 Advances in Photosynthesis – Fundamental Aspects

role for ROS generation and disruptions of the photosynthetic electron transport shortly after pathogen attack or toxin treatment have been suggested as potential mechanism (Kim et al., 2010). Similar mechanism has been proposed by Allakhverdiev et al (Allakhverdiev et al., 2008) following heat stress that targets the oxygen evolving complex along with the associated cofactors in photosystem II (PSII), carbon fixation by rubisco and the ATP generating system. In another system, Kocal et al (Kocal et al., 2008) studied the role of cell wall invertase (cw-Inv) in transgenic tomato (*Solanum lycopersicum*) plants silenced for the major leaf cw-Inv isoforms during normal growth and during the compatible interaction with *Xanthomonas campestris pv vesicatoria*. Cw-Inv expression was found to be induced upon microbial infection and was most likely associated with an apoplastic hexose accumulation during the infection process. The hexoses formed are thought to aid the pathogen's nutrition (Berger et al., 2007, Biemelt and Sonnewald, 2006, Seo et al., 2007). Fungal pathogens also produce their own invertases to ensure their nutritional supply (Chou et al., 2000, Voegele et al., 2006). One of the most sophisticated mechanisms that divert plant metabolites to pathogen is the role of auxin in host-pathogen interactions (Fu et al., 2011, Navarro et al., 2006). Indole-3-acetic acid (IAA) is the major form of auxin in most plants and induces the loosening of plant cell wall, the natural protective barrier to invaders. *X. oryzae pv oryzae*, *X. oryzae pv oryzicola*, and *M. grisea* secrete IAA, which, in turn, may induce rice to synthesize its own IAA at the infection site. IAA induces the production of expansins, the cell wallloosening proteins, and makes rice vulnerable to pathogens (Fu et al., 2011). Similarly, Garavaglia et al (Garavaglia et al., 2010) have reported an eukaryotic-acquired gene by a biotrophic phytopathogen that allows its prolonged survival on the host by counteracting

During plant-pathogen interactions, the host develops a variety of defence reactions. Nonhost resistance against a biotrophic fungal pathogen is often manifested as the ability of the attacked plant to prevent fungal penetration, or the ability to terminate the development and/or functioning of the fungal feeding structure such as the intracellular hypha or the haustorium before it extracts enough nutrition from the plant cells (Wen et al., 2011). These reactions may involved development of physical barriers such as exocytosis, cell wall modifications and de novo metabolites synthesis (IshiharaHashimotoMiyagawa et al., 2008).

Cell wall strengthening by callosic (Wen et al., 2011) and papillae formation, cell wall apposition (Fofana et al., 2005, Wurms et al., 1999), lignin deposition (Hammerschmidt and Kuc, 1982) as well as hydrolytic PR proteins (Hu and Rijkenberg, 1998a) have been reported as first line of defence mechanism developed by plants. Components of these cell wall makeups are of primary metabolite origin. It is worth noting that papillae were at times observed as an initial response to fungal penetration. Generally, papillae relate to powdery mildew fungal penetration in two ways: in some instances, penetration fails when papillae are present and alternatively, penetration may succeed and the papilla becomes a collar for the haustorial neck (Hammerschmidt and Yang-Cashman, 1995). Using transmission electron microscopy, Fofana et al (Fofana et al., 2005) observed both outcomes for elicited and nonelicited cucumber plants, strongly suggesting that papillae formation alone may not be sufficient to explain the level of

the shut-down of plant photosynthesis.

**3.1.1 Primary metabolites** 

**3. Plant reactions to pathogen attack** 

**3.1 Primary metabolites and secondary metabolite production** 

The role for secondary metabolites in the plant's interaction with its environment is widely recognized (Rhodes, 1994). The primary metabolites deriving from photosynthesis are channeled into different metabolite pathways for the synthesis, storage, and modification (hydroxylation, glycosylation, acetylation, etc) of myriads of compounds, and for use to cope abiotic and biotic clues. Within each of the major groups of secondary metabolites such as alkaloids, phenylpropanoids and terpenoids, several thousand individual compounds accumulating in plants have been characterised and their role in plant-pathogen interactions studied (Ishihara et al., 2011). For example, induction of phenolic compounds, flavonoid phytoalexins, (Daayf et al., 1997, Fawe et al., 1998, Fofana et al., 2002, McNally et al., 2003b, McNally et al., 2003) was reported in cucumber plants following pathogen attacks and elicitor treatments. Synthesis of phytoalexins involves the rapid transcriptional activation of genes encoding a number of key biosynthetic enzymes that include anthranilate synthase (AS) (IshiharaHashimotoTanaka et al., 2008) phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) which is the early committed key enzyme of the flavonoid/isoflavonoid pathway, chalcone isomerase (CHI) and isoflavone reductase (IFR) (Dixon et al., 1995, Baldridge et al., 1998, Fofana et al., 2002, Fofana et al., 2005). The chemical nature of some of these compounds is now well elucidated (Ibanez et al., 2010, Ishihara et al., 2011, McNally et al., 2003). McNally et al (McNally et al., 2003b, McNally et al., 2003) reported the synthesis of complex C-glycosyl flavonoid phytoalexins, referred to as vitexin-6-(4-hydroxy-1 ethylbenzene) (cucumerin A) and isovitexin-8-(4-hydroxy-1-ethylbenzene) (cucumerin B), as a site-specific response to fungal penetration in cucumber. In a recent study, Ishihara et al (IshiharaHashimotoTanaka et al., 2008) reported on an induced accumulation of Trp-

An Overview of Plant Photosynthesis Modulation by Pathogen Attacks 475

culture media under different nutritional regimes and in comparison with fungal growth in infected barley (GuldenerSeong et al., 2006). Guldener et al. (GuldenerSeong et al., 2006) were able to detect the fungal gene expression during plant infection, test for sensitivity limits for detecting fungal RNA *in planta* and the potential for cross-hybridization between fungal probe sets and plant RNAs. A total of 11,994 of a possible 17,809 *Fusarium* probe sets (67.35%) were detected under various conditions of the fungus grown in culture and a total of 7132 probe sets (40.05%) were detected from the fungus during infection of barley. As of July 5, 2011 however, only 96 pathogenic genes from *F. graminearum* curated for lab experimental, molecular and biological information on genes proven to affect the outcome pathogen-host interactions in cereals (oat, wheat, barley, rye, maize), *Arabidopsis*, and tomato were reported in PHI-base database (http://www.phi-base.org/query.php) and 4000 proteins that have been annotated in MIPS *F.graminearum* Genome Database (FGDB), (GuldenerMannhaupt et al., 2006) which is far from complete. To speed up the process, a computational network approach to predict pathogenic genes for *Fusarium graminearum* was proposed by Liu et al (Liu et al., 2010). With a small number of known pathogenic genes as seed genes, the authors were able to identify a subnetwork that consists of potential pathogenic genes from the protein-protein interaction network (PPIN) of *F. graminearum*, where the genes in the subnetwork generally share similar functions and are involved in similar biological processes. The genes that interact with at least two seed genes can be identified because these genes are more likely to be pathogenic genes due to their tight interactions with the seed genes. The protein-protein interactions that connect networks with each other are thought to be the signalling pathways between biological processes. On the basis of our current understanding of pathogenicity of model pathogens, *F. graminearum*  is thought to organize a complex network of proteins and other molecules, including those that might be secreted into host cells, to adapt the life inside its host plant. Hence, Zhao et al (Zhao et al., 2009) showed that *F. graminearum* protein-protein interactions (FPPI ) contains 223,166 interactions among 7406 proteins which represent about 52 % of the whole *F. graminearum* proteome. Although these computational predictions are fascinating, system biology based on experiment data is also making considerable progress. Song et al (Song et al., 2011) reported the first proteome of infection structures from parasitized wheat leaves, enriched for *Puccinia triticina* (Pt) haustoria using 2-D PAGE MS/MS and gel-based LC-MS (GeLC-MS) to separate proteins. They compared the generated spectra with a partial proteome predicted from a preliminary *Pt* genome and ESTs, with a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, *Puccinia graminis* f. sp. *tritici (Pgt),* and with various plant resources. The authors identified over 260 fungal proteins, 16 of which matched peptides from *Pgt.* Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six had effector protein signatures, some were related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. By measuring the gene expression over several life cycle stages of ten representative candidates using quantitative RT-PCR, all tested genes were shown to be strongly upregulated and of which four were expressed solely upon infection (Song et al., 2011). Similarly, El-Bebany et al (El-Bebany et al., 2010) identified potential pathogenicity factors including isochorismate hydrolase, a potential plant-defence suppressor that may inhibit the production of salicylic acid, which is

derived secondary metabolites, including tryptamine, serotonin, and hydroxycinnamic acid amides of serotonin in rice leaves by infection with *Bipolaris oryzae*. Using enantiomers of α- (fluoromethyl)tryptophan (αFMT – R- and S- αFMT), S-αFMT but not R-αFMT effectively inhibited tryptophan decarboxylase activity extracted from rice leaves infected by *Bipolaris oryzae*, suppressed accumulation of serotonin, tryptamine, and hydroxycinnamic acid amides of serotonin in a dose-dependent manner, and lead to were severely damaged leaves showing lesions that lacked deposition of brown materials, compared to control without SαFMT. Administration of tryptamine to S-αFMT-treated leaves restored accumulation of tryptophan-derived secondary metabolites as well as deposition of brown material and reduced damage caused by fungal infection (Ishihara et al., 2011).

#### **3.2 Transduction pathways**

Plants have developed a sophisticated innate immune surveillance system to recognize pathogens (Dodds and Rathjen, 2010, Liu et al., 2011). This surveillance system consists of an integral plasma membrane proteins with extracellular receptor domains to perceive conserved pathogen associated molecular patterns (PAMPs) presented by pathogens during infection, and an intra- cellular Resistance (R) proteins to recognize the presence of specific pathogen effector proteins in host cells (Elmore et al., 2011). Two recognition models have been reported for non-host resistance (non-race specific elicitor as signal) and gene-for-gene resistance (race-specific elicitor/ avirulence gene products as signal) interactions, with a receptor and a resistance gene product as signal perception, respectively (Romeis, 2001). Upon perception, takes place a signal transduction cascade involving protein kinases and cellular responses to the intruders ensue. Either the disease develops or resistance phenotypes are observed. For review, please see more details in previous reports (Romeis et al., 2001, Romeis, 2001, Elmore et al., 2011, Elmore and Coaker, 2011a, Liu et al., 2011, Elmore and Coaker, 2011b). The plant reaction to the outcome of signalling has a dramatic consequence on the plant's ability to photosynthesize even in incompatible interactions where HR responses lead to a localized cell death at infection sites and restrict the pathogen progression. In these conditions, with patchy leaf area (no chlorophyll for light inception), the photosynthetic capacity is reduced compared to non-infected plants.

#### **4. New insights from the genomics and proteomics era**

The genomics and proteomics era, with its high-throughput capability, has enabled the expression profiling analysis of thousands of genes and proteins simultaneously (Lee et al., 2004). Hence, the global analysis of many plant processes, including the response to pathogen attack, their interlinked regulatory networks and signalling pathways have been made possible (Duggan et al., 1999, Eulgem, 2005, GuldenerSeong et al., 2006, Schenk et al., 2000).

#### **4.1 Gene and protein networks in plant-pathogen interactions 4.1.1 In the pathogen**

One of the challenges faced by biologists in plant-pathogen interactions was their ability to differentiate plant genes from the pathogen genes. This has become feasible with the release of the genome sequences for several fungus and plant species. Exploiting these genomic resources it has been possible to design and perform the wide-genome microarray transcription profiling of the plant pathogenic fungus, *Fusarium graminearum,* grown in

derived secondary metabolites, including tryptamine, serotonin, and hydroxycinnamic acid amides of serotonin in rice leaves by infection with *Bipolaris oryzae*. Using enantiomers of α- (fluoromethyl)tryptophan (αFMT – R- and S- αFMT), S-αFMT but not R-αFMT effectively inhibited tryptophan decarboxylase activity extracted from rice leaves infected by *Bipolaris oryzae*, suppressed accumulation of serotonin, tryptamine, and hydroxycinnamic acid amides of serotonin in a dose-dependent manner, and lead to were severely damaged leaves showing lesions that lacked deposition of brown materials, compared to control without SαFMT. Administration of tryptamine to S-αFMT-treated leaves restored accumulation of tryptophan-derived secondary metabolites as well as deposition of brown material and

Plants have developed a sophisticated innate immune surveillance system to recognize pathogens (Dodds and Rathjen, 2010, Liu et al., 2011). This surveillance system consists of an integral plasma membrane proteins with extracellular receptor domains to perceive conserved pathogen associated molecular patterns (PAMPs) presented by pathogens during infection, and an intra- cellular Resistance (R) proteins to recognize the presence of specific pathogen effector proteins in host cells (Elmore et al., 2011). Two recognition models have been reported for non-host resistance (non-race specific elicitor as signal) and gene-for-gene resistance (race-specific elicitor/ avirulence gene products as signal) interactions, with a receptor and a resistance gene product as signal perception, respectively (Romeis, 2001). Upon perception, takes place a signal transduction cascade involving protein kinases and cellular responses to the intruders ensue. Either the disease develops or resistance phenotypes are observed. For review, please see more details in previous reports (Romeis et al., 2001, Romeis, 2001, Elmore et al., 2011, Elmore and Coaker, 2011a, Liu et al., 2011, Elmore and Coaker, 2011b). The plant reaction to the outcome of signalling has a dramatic consequence on the plant's ability to photosynthesize even in incompatible interactions where HR responses lead to a localized cell death at infection sites and restrict the pathogen progression. In these conditions, with patchy leaf area (no chlorophyll for light inception),

The genomics and proteomics era, with its high-throughput capability, has enabled the expression profiling analysis of thousands of genes and proteins simultaneously (Lee et al., 2004). Hence, the global analysis of many plant processes, including the response to pathogen attack, their interlinked regulatory networks and signalling pathways have been made possible (Duggan et al., 1999, Eulgem, 2005, GuldenerSeong et al., 2006, Schenk et al., 2000).

One of the challenges faced by biologists in plant-pathogen interactions was their ability to differentiate plant genes from the pathogen genes. This has become feasible with the release of the genome sequences for several fungus and plant species. Exploiting these genomic resources it has been possible to design and perform the wide-genome microarray transcription profiling of the plant pathogenic fungus, *Fusarium graminearum,* grown in

reduced damage caused by fungal infection (Ishihara et al., 2011).

the photosynthetic capacity is reduced compared to non-infected plants.

**4. New insights from the genomics and proteomics era** 

**4.1 Gene and protein networks in plant-pathogen interactions** 

**3.2 Transduction pathways** 

**4.1.1 In the pathogen** 

culture media under different nutritional regimes and in comparison with fungal growth in infected barley (GuldenerSeong et al., 2006). Guldener et al. (GuldenerSeong et al., 2006) were able to detect the fungal gene expression during plant infection, test for sensitivity limits for detecting fungal RNA *in planta* and the potential for cross-hybridization between fungal probe sets and plant RNAs. A total of 11,994 of a possible 17,809 *Fusarium* probe sets (67.35%) were detected under various conditions of the fungus grown in culture and a total of 7132 probe sets (40.05%) were detected from the fungus during infection of barley. As of July 5, 2011 however, only 96 pathogenic genes from *F. graminearum* curated for lab experimental, molecular and biological information on genes proven to affect the outcome pathogen-host interactions in cereals (oat, wheat, barley, rye, maize), *Arabidopsis*, and tomato were reported in PHI-base database (http://www.phi-base.org/query.php) and 4000 proteins that have been annotated in MIPS *F.graminearum* Genome Database (FGDB), (GuldenerMannhaupt et al., 2006) which is far from complete. To speed up the process, a computational network approach to predict pathogenic genes for *Fusarium graminearum* was proposed by Liu et al (Liu et al., 2010). With a small number of known pathogenic genes as seed genes, the authors were able to identify a subnetwork that consists of potential pathogenic genes from the protein-protein interaction network (PPIN) of *F. graminearum*, where the genes in the subnetwork generally share similar functions and are involved in similar biological processes. The genes that interact with at least two seed genes can be identified because these genes are more likely to be pathogenic genes due to their tight interactions with the seed genes. The protein-protein interactions that connect networks with each other are thought to be the signalling pathways between biological processes. On the basis of our current understanding of pathogenicity of model pathogens, *F. graminearum*  is thought to organize a complex network of proteins and other molecules, including those that might be secreted into host cells, to adapt the life inside its host plant. Hence, Zhao et al (Zhao et al., 2009) showed that *F. graminearum* protein-protein interactions (FPPI ) contains 223,166 interactions among 7406 proteins which represent about 52 % of the whole *F. graminearum* proteome. Although these computational predictions are fascinating, system biology based on experiment data is also making considerable progress. Song et al (Song et al., 2011) reported the first proteome of infection structures from parasitized wheat leaves, enriched for *Puccinia triticina* (Pt) haustoria using 2-D PAGE MS/MS and gel-based LC-MS (GeLC-MS) to separate proteins. They compared the generated spectra with a partial proteome predicted from a preliminary *Pt* genome and ESTs, with a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, *Puccinia graminis* f. sp. *tritici (Pgt),* and with various plant resources. The authors identified over 260 fungal proteins, 16 of which matched peptides from *Pgt.* Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six had effector protein signatures, some were related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. By measuring the gene expression over several life cycle stages of ten representative candidates using quantitative RT-PCR, all tested genes were shown to be strongly upregulated and of which four were expressed solely upon infection (Song et al., 2011). Similarly, El-Bebany et al (El-Bebany et al., 2010) identified potential pathogenicity factors including isochorismate hydrolase, a potential plant-defence suppressor that may inhibit the production of salicylic acid, which is

An Overview of Plant Photosynthesis Modulation by Pathogen Attacks 477

Genes coding for the synthesis of jasmonic acid and those involved in the responses to salicylic acid and ethylene were upregulated. The upregulation of JA and SA genes suggest that the downregulation of photosynthesis-related genes was part of a defence response. Analysis of gene clusters revealed that the transcript levels of 84% of the genes that carry a chloroplast targeting peptide sequence were decreased (Bilgin et al., 2010). The concept of computational network analysis (Liu et al., 2010) appears to be of good relevance as it could assist in identifying not only networks specific to the plant, to the pathogen but also genes that interact

between the plant and the pathogen.

important for plant defence response signaling. Much progress is still needed not only in the identification but in the mechanistic action of the genes and proteins identified.

#### **4.1.2 In the plant**

The role of photosynthesis in plant defence is a fundamental question awaiting further molecular and physiological elucidation. Different pathogens, based on life cycle (biotroph vs necrotroph), develop different pathogenesis mechanisms that impact differently on the plant's photosynthesis efficiency as well as on its gene and proteome profiling. *Xanthomonas axonopodis pv*. *citri*, the bacterial pathogen responsible for citrus canker encodes a plant-like natriuretic peptide (XacPNP) that is expressed specifically during the infection process and prevents deterioration of the physiological condition of the infected tissue to the benefit of the invaders (Nembaware et al., 2004, Gehring and Irving, 2003). The wild pathogen expressing the XacPNP peptide maintains the plant in a condition that prevents chlorosis and no significant drop of photosynthesis. In contrast, citrus leaves infected with a XacPNP deletion mutant (DeltaXacPNP) resulted significant reduction of photosynthesis efficiency, and proteomic assays revealed a major reduction in photosynthetic proteins such as Rubisco, Rubisco activase and ATP synthase as a compared with infection with wild type bacteria (Garavaglia et al., 2010). Similarly, *Pyrenophora tritici-repentis*, is an important foliar disease of wheat. The fungus produces the host-specific, chlorosis-inducing toxin Ptr ToxB. Kim et al (Kim et al., 2010) examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis was significantly reduced within 12 h of toxin treatment, prior to the development of chlorosis at 48-72 h. Proteomics analysis by 2-DE revealed a total of 102 protein spots with significantly altered intensities 12-36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer-treated control. In the last decade, an abundant literature has treated large dataset gene expression profiling of plant-pathogen interactions (Bilgin et al., 2010, Eichmann et al., 2006, Fofana et al., 2007, Lee et al., 2004, Zou et al., 2005) among many others. Of interest was the study by Fofana et al (Fofana et al., 2007) where difference in temporal gene expression profiling of the wheat leaf rust pathosystem was reported in compatible and incompatible defence pathways using cDNA microarray. Gene ontology assignment of differentially expressed genes showed alterations in gene expression for different molecular functions, cellular location and biological process for genes (Figure 3). The authors observed changes in the expression of genes involved in different biological processes such as photosynthesis, redox control, resistance and resistance-related genes (NBS-LRR, cyclophilin-like protein, MLo4-like gene, MRP1), components of the shikimatephenylpropanoid pathway as well as genes involved in signal transduction (Myb-like transcription factors, calmodulin MAPKK, PI4PK), heat shock proteins, osmotic control genes and metabolisms (Fofana et al., 2007). Six hours after inoculation, a coordinated decrease in transcription of photosynthesis genes (photosystemmII phosphoprotein, ribulose-1,5 biphosphate carboxylase/oxygenase small unit, Type III LHCII CAB precursor protein, photosystem II type II chlorophyll A/B-binding protein, ribulose-1,5 biphosphate carboxylase activase) in the resistant but not susceptible interactions was observed in agreement with the general trends of photosynthesis inhibition. Biotic stress globally downregulates photosynthesis genes (Bilgin et al., 2010). By comparing transcriptomic data from microarray experiments after 22 different forms of biotic damage on eight different plant species, Bilgin et al (Bilgin et al., 2010) reported that transcript levels of photosynthesis light reaction, carbon reduction cycle and pigment synthesis genes decreased regardless of the type of biotic attack.

important for plant defence response signaling. Much progress is still needed not only in the

The role of photosynthesis in plant defence is a fundamental question awaiting further molecular and physiological elucidation. Different pathogens, based on life cycle (biotroph vs necrotroph), develop different pathogenesis mechanisms that impact differently on the plant's photosynthesis efficiency as well as on its gene and proteome profiling. *Xanthomonas axonopodis pv*. *citri*, the bacterial pathogen responsible for citrus canker encodes a plant-like natriuretic peptide (XacPNP) that is expressed specifically during the infection process and prevents deterioration of the physiological condition of the infected tissue to the benefit of the invaders (Nembaware et al., 2004, Gehring and Irving, 2003). The wild pathogen expressing the XacPNP peptide maintains the plant in a condition that prevents chlorosis and no significant drop of photosynthesis. In contrast, citrus leaves infected with a XacPNP deletion mutant (DeltaXacPNP) resulted significant reduction of photosynthesis efficiency, and proteomic assays revealed a major reduction in photosynthetic proteins such as Rubisco, Rubisco activase and ATP synthase as a compared with infection with wild type bacteria (Garavaglia et al., 2010). Similarly, *Pyrenophora tritici-repentis*, is an important foliar disease of wheat. The fungus produces the host-specific, chlorosis-inducing toxin Ptr ToxB. Kim et al (Kim et al., 2010) examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis was significantly reduced within 12 h of toxin treatment, prior to the development of chlorosis at 48-72 h. Proteomics analysis by 2-DE revealed a total of 102 protein spots with significantly altered intensities 12-36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer-treated control. In the last decade, an abundant literature has treated large dataset gene expression profiling of plant-pathogen interactions (Bilgin et al., 2010, Eichmann et al., 2006, Fofana et al., 2007, Lee et al., 2004, Zou et al., 2005) among many others. Of interest was the study by Fofana et al (Fofana et al., 2007) where difference in temporal gene expression profiling of the wheat leaf rust pathosystem was reported in compatible and incompatible defence pathways using cDNA microarray. Gene ontology assignment of differentially expressed genes showed alterations in gene expression for different molecular functions, cellular location and biological process for genes (Figure 3). The authors observed changes in the expression of genes involved in different biological processes such as photosynthesis, redox control, resistance and resistance-related genes (NBS-LRR, cyclophilin-like protein, MLo4-like gene, MRP1), components of the shikimatephenylpropanoid pathway as well as genes involved in signal transduction (Myb-like transcription factors, calmodulin MAPKK, PI4PK), heat shock proteins, osmotic control genes and metabolisms (Fofana et al., 2007). Six hours after inoculation, a coordinated decrease in transcription of photosynthesis genes (photosystemmII phosphoprotein, ribulose-1,5 biphosphate carboxylase/oxygenase small unit, Type III LHCII CAB precursor protein, photosystem II type II chlorophyll A/B-binding protein, ribulose-1,5 biphosphate carboxylase activase) in the resistant but not susceptible interactions was observed in agreement with the general trends of photosynthesis inhibition. Biotic stress globally downregulates photosynthesis genes (Bilgin et al., 2010). By comparing transcriptomic data from microarray experiments after 22 different forms of biotic damage on eight different plant species, Bilgin et al (Bilgin et al., 2010) reported that transcript levels of photosynthesis light reaction, carbon reduction cycle and pigment synthesis genes decreased regardless of the type of biotic attack.

identification but in the mechanistic action of the genes and proteins identified.

**4.1.2 In the plant** 

Genes coding for the synthesis of jasmonic acid and those involved in the responses to salicylic acid and ethylene were upregulated. The upregulation of JA and SA genes suggest that the downregulation of photosynthesis-related genes was part of a defence response. Analysis of gene clusters revealed that the transcript levels of 84% of the genes that carry a chloroplast targeting peptide sequence were decreased (Bilgin et al., 2010). The concept of computational network analysis (Liu et al., 2010) appears to be of good relevance as it could assist in identifying not only networks specific to the plant, to the pathogen but also genes that interact between the plant and the pathogen.

An Overview of Plant Photosynthesis Modulation by Pathogen Attacks 479

allow the design of strategies to suicide specifically pathogen vital interactome (such as a key component of virulence interactome or life cycle) and to dismantle any genes linking plant–pathogens network clusters through which the invader diverts the photosynthetates for its own. A second avenue could consist of combining system biology approach and agronomic practices that can contribute to increased plant photosynthetic capacity. Recently, Zhang et al (ZhangXie et al., 2008) described a soil symbiotic bacteria that augments photosynthesis in *Arabidopsis* by decreasing glucose sensing and abscisic acid levels *in planta*. Would such symbiotic system be applicable in a field system? Would there be any such symbiont that could antagonistically interfere with plant pathogenetic soil born diseases and reduce their impact on plant productivity? Those are some of the questions, we

Photosynthesis is a process that converts solar energy to chemical energy in many different organisms, ranging from plants to bacteria. It provides all the food we eat and all the fossil fuel we use. Photosynthesis of terrestrial higher plants is however constantly challenged by abiotic and biotic stresses. In this review, we described briefly the general process of photosynthesis, its outcome and limiting factors; the complex plant-pathogen interrelationships and their effects on photosynthesis; and the insights the genomics and proteomics era can shed into the elucidation of the many genes and protein clusters and networks that sustain the plant-pathogen interactions in general, and photosynthesis, in particular. Photosynthesis feeds the globe and pathogen threats are increasing. A system biology approach, using both computational gene network discovery and gene and proteome analysis in living plants challenged with pathogens, was proposed as one of the pivotal player in identifying key gene and protein network clusters and their interactomes in both the plant and pathogens towards the design of strategies to suicide specifically pathogen vital interactome and to dismantle any genes linking plant – pathogens network clusters. This approach could be complemented with agronomic practices contributing to

The authors warmly thank Dr. Kaushik Ghose (University of Prince Edwards Island) for his kind willingness to proof read this Manuscript. We also wish to acknowledge Dr. Cloutier and her lab (Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Manitoba), the lab from which Dr. Fofana has performed his work on the leaf-rust pathosystem. This chapter is in recognition and gratitude to Professor Patrick du Jardin (Gembloux Agro-Bio Tech, Universite de Liege, Belgium) from whom I received most of my

Abrams, M. D., Kubiske, M. E. & Steiner, K. C. (1990) Drought adaptations and responses in

five genotypes of *Fraxinus pennsylvanica* Marsh.: photosynthesis, water relations

believe, could be the focus for further investigations.

increased plant photosynthetic capacity.

taste and flavour for molecular plant physiology.

and leaf morphology. *Tree physiology,* 6, 305-15.

**6. Acknowledgments** 

**7. References** 

**5. Conclusion**

Fig. 3. Gene ontology assignment of differentially expressed genes (Fofana et al., 2007). A BLASTX search of the differentially expressed sequences against the set of predicted *Arabidopsis thaliana* proteins was used to assign gene ontology. The first hit with an *E* value less than or equal to 1 × 10-5 was used as a functional assignment and the TAIR GO annotation tool was used to bin the genes into the ontology groupings; a) cellular location b) molecular function and c) biological process.

#### **4.2 What could be the future strategies?**

Plant productivity depends on the plant's ability to produce higher biomass and seed, which relies on its photosynthetic capacity. However, as mentioned above, this inherent potential is constantly challenged and compromised by phytopathogens. The main question facing plant biologists remains our ability to improve plant productivity under increasing biotic pressure. One of the avenues could consist of emphasizing on gene networks discovery through both computational network discovery strategy (Liu et al., 2010) and gene and proteome analysis in living plants challenged with pathogens. Recently, Zhu et al (Zhu et al., 2010) proposed C4 rice as an ideal arena for systems biology research. This group raised the possibility of engineering C4 photosynthetic machinery into C3 plant such as rice. However, the pivotal role to be played by system biology in identifying key regulatory elements controlling development of C4 features, identifying essential biochemical and anatomical features required to achieve high photosynthetic efficiency, elucidating the genetic mechanisms underlining C4 differentiation and ultimately identifying viable routes to engineer C4 rice has been emphasized to decipher the complexity of such engineering (Zhu et al., 2010). A second level complexity comes from the interaction between two organisms as is the case in plant-pathogen interactions. It will be of great interest to put emphasis on the identification of a) more plant gene and protein network clusters and their interactomes, b) more pathogen gene and protein network clusters and their interactomes, c) more plant-pathogen gene and protein network clusters and the interacting genes and proteins linking both organisms, for a better understanding of key target points. This would allow the design of strategies to suicide specifically pathogen vital interactome (such as a key component of virulence interactome or life cycle) and to dismantle any genes linking plant–pathogens network clusters through which the invader diverts the photosynthetates for its own. A second avenue could consist of combining system biology approach and agronomic practices that can contribute to increased plant photosynthetic capacity. Recently, Zhang et al (ZhangXie et al., 2008) described a soil symbiotic bacteria that augments photosynthesis in *Arabidopsis* by decreasing glucose sensing and abscisic acid levels *in planta*. Would such symbiotic system be applicable in a field system? Would there be any such symbiont that could antagonistically interfere with plant pathogenetic soil born diseases and reduce their impact on plant productivity? Those are some of the questions, we believe, could be the focus for further investigations.

#### **5. Conclusion**

478 Advances in Photosynthesis – Fundamental Aspects

Fig. 3. Gene ontology assignment of differentially expressed genes (Fofana et al., 2007). A BLASTX search of the differentially expressed sequences against the set of predicted *Arabidopsis thaliana* proteins was used to assign gene ontology. The first hit with an *E* value

annotation tool was used to bin the genes into the ontology groupings; a) cellular location b)

Plant productivity depends on the plant's ability to produce higher biomass and seed, which relies on its photosynthetic capacity. However, as mentioned above, this inherent potential is constantly challenged and compromised by phytopathogens. The main question facing plant biologists remains our ability to improve plant productivity under increasing biotic pressure. One of the avenues could consist of emphasizing on gene networks discovery through both computational network discovery strategy (Liu et al., 2010) and gene and proteome analysis in living plants challenged with pathogens. Recently, Zhu et al (Zhu et al., 2010) proposed C4 rice as an ideal arena for systems biology research. This group raised the possibility of engineering C4 photosynthetic machinery into C3 plant such as rice. However, the pivotal role to be played by system biology in identifying key regulatory elements controlling development of C4 features, identifying essential biochemical and anatomical features required to achieve high photosynthetic efficiency, elucidating the genetic mechanisms underlining C4 differentiation and ultimately identifying viable routes to engineer C4 rice has been emphasized to decipher the complexity of such engineering (Zhu et al., 2010). A second level complexity comes from the interaction between two organisms as is the case in plant-pathogen interactions. It will be of great interest to put emphasis on the identification of a) more plant gene and protein network clusters and their interactomes, b) more pathogen gene and protein network clusters and their interactomes, c) more plant-pathogen gene and protein network clusters and the interacting genes and proteins linking both organisms, for a better understanding of key target points. This would

less than or equal to 1 × 10-5 was used as a functional assignment and the TAIR GO

molecular function and c) biological process.

**4.2 What could be the future strategies?** 

Photosynthesis is a process that converts solar energy to chemical energy in many different organisms, ranging from plants to bacteria. It provides all the food we eat and all the fossil fuel we use. Photosynthesis of terrestrial higher plants is however constantly challenged by abiotic and biotic stresses. In this review, we described briefly the general process of photosynthesis, its outcome and limiting factors; the complex plant-pathogen interrelationships and their effects on photosynthesis; and the insights the genomics and proteomics era can shed into the elucidation of the many genes and protein clusters and networks that sustain the plant-pathogen interactions in general, and photosynthesis, in particular. Photosynthesis feeds the globe and pathogen threats are increasing. A system biology approach, using both computational gene network discovery and gene and proteome analysis in living plants challenged with pathogens, was proposed as one of the pivotal player in identifying key gene and protein network clusters and their interactomes in both the plant and pathogens towards the design of strategies to suicide specifically pathogen vital interactome and to dismantle any genes linking plant – pathogens network clusters. This approach could be complemented with agronomic practices contributing to increased plant photosynthetic capacity.

#### **6. Acknowledgments**

The authors warmly thank Dr. Kaushik Ghose (University of Prince Edwards Island) for his kind willingness to proof read this Manuscript. We also wish to acknowledge Dr. Cloutier and her lab (Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Manitoba), the lab from which Dr. Fofana has performed his work on the leaf-rust pathosystem. This chapter is in recognition and gratitude to Professor Patrick du Jardin (Gembloux Agro-Bio Tech, Universite de Liege, Belgium) from whom I received most of my taste and flavour for molecular plant physiology.

#### **7. References**

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**23** 

*Japan* 

Shinji Masuda

**The Stringent Response in Phototrophs** 

*Center for Biological Resources & Informatics, Tokyo Institute of Technology* 

Organisms must respond to environmental changes if they are to survive. As a result**,** species have evolved numerous intracellular and intercellular regulatory systems that often reflect an organism's environment. In bacteria, one of the most important regulatory systems is the stringent response (Cashel et al., 1996). Signaling *via* this response is mediated by guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3' diphosphate (ppGpp), which function as second messengers. The stringent response was first discovered over 40 years ago in *Escherichia coli*. When *E. coli* cells are grown under nutrient-rich conditions but then transferred to a nutrient-limited environment, intracellular levels of pppGpp and ppGpp ((p)ppGpp) rapidly increase (Cashel et al., 1996). (p)ppGpp controls many vital cellular processes, including transcription and translation. For example, (p)ppGpp directly binds RNA polymerase and alters its promoter-binding affinity (Chatterji et al., 1998; Toulokhonov et al., 2001; Artsimovitch et al., 2004). When nutrient availability changes, therefore, the stringent response simultaneously adjusts the level of transcription for many genes. In *E. coli*, synthesis and degradation of (p)ppGpp are catalyzed by two

Deficiencies in iron, phosphate, nitrogen, or carbon each represent environmental stresses that trigger (p)ppGpp accumulation (Cashel et al., 1996). For photosynthetic bacteria, sunlight is also an important "nutrient". Characterization of a SpoT homolog in the purple photosynthetic bacterium, *Rhodobacter capsulatus*, showed that the stringent response also regulates photosynthesis (Masuda & Bauer, 2004). Genes that encode (p)ppGpp synthases and hydrolases are highly conserved in plants (van der Biezen et al., 2000; Kasai et al., 2002; Yamada et al., 2003; Givens et al., 2004; Tozawa et al., 2007; Masuda et al., 2008a; Kim et al., 2009) and are called RSHs (RelA/SpoT homologs). All known plant RSHs are targeted to chloroplasts, suggesting that they may control chloroplast function. Here we summarize our current understanding of the stringent response in phototrophs. For details concerning the mechanisms of the stringent response itself, several recent reviews are available (Magnusson et al., 2005; Braeken et al., 2006; Jain et al., 2006; Ochi, 2007; Potrykus & Cashel, 2008;

In *E. coli*, the level of (p)ppGpp is controlled by two enzymes, RelA and SpoT (Cashel et al., 1996). Both enzymes synthesize (p)ppGpp by transferring the pyrophosphate of ATP to the

**1. Introduction** 

enzymes RelA and SpoT (Cashel et al., 1996).

**2. (p)ppGpp synthases and hydrolases in bacteria** 

Srivatsan & Wang, 2008).

phosphatase 2A prevents unnecessary defense reactions under low light in *Arabidopsis thaliana*. *Plant physiology,* 156, 1464-1480.


### **The Stringent Response in Phototrophs**

#### Shinji Masuda

*Center for Biological Resources & Informatics, Tokyo Institute of Technology Japan* 

#### **1. Introduction**

486 Advances in Photosynthesis – Fundamental Aspects

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DeLucia, E. & Clough, S. J. (2005) Expression profiling soybean response to *Pseudomonas syringae* reveals new defense-related genes and rapid HR-specific downregulation of photosynthesis. *Molecular plant-microbe interactions : MPMI,* 18, Organisms must respond to environmental changes if they are to survive. As a result**,** species have evolved numerous intracellular and intercellular regulatory systems that often reflect an organism's environment. In bacteria, one of the most important regulatory systems is the stringent response (Cashel et al., 1996). Signaling *via* this response is mediated by guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3' diphosphate (ppGpp), which function as second messengers. The stringent response was first discovered over 40 years ago in *Escherichia coli*. When *E. coli* cells are grown under nutrient-rich conditions but then transferred to a nutrient-limited environment, intracellular levels of pppGpp and ppGpp ((p)ppGpp) rapidly increase (Cashel et al., 1996). (p)ppGpp controls many vital cellular processes, including transcription and translation. For example, (p)ppGpp directly binds RNA polymerase and alters its promoter-binding affinity (Chatterji et al., 1998; Toulokhonov et al., 2001; Artsimovitch et al., 2004). When nutrient availability changes, therefore, the stringent response simultaneously adjusts the level of transcription for many genes. In *E. coli*, synthesis and degradation of (p)ppGpp are catalyzed by two enzymes RelA and SpoT (Cashel et al., 1996).

Deficiencies in iron, phosphate, nitrogen, or carbon each represent environmental stresses that trigger (p)ppGpp accumulation (Cashel et al., 1996). For photosynthetic bacteria, sunlight is also an important "nutrient". Characterization of a SpoT homolog in the purple photosynthetic bacterium, *Rhodobacter capsulatus*, showed that the stringent response also regulates photosynthesis (Masuda & Bauer, 2004). Genes that encode (p)ppGpp synthases and hydrolases are highly conserved in plants (van der Biezen et al., 2000; Kasai et al., 2002; Yamada et al., 2003; Givens et al., 2004; Tozawa et al., 2007; Masuda et al., 2008a; Kim et al., 2009) and are called RSHs (RelA/SpoT homologs). All known plant RSHs are targeted to chloroplasts, suggesting that they may control chloroplast function. Here we summarize our current understanding of the stringent response in phototrophs. For details concerning the mechanisms of the stringent response itself, several recent reviews are available (Magnusson et al., 2005; Braeken et al., 2006; Jain et al., 2006; Ochi, 2007; Potrykus & Cashel, 2008; Srivatsan & Wang, 2008).

#### **2. (p)ppGpp synthases and hydrolases in bacteria**

In *E. coli*, the level of (p)ppGpp is controlled by two enzymes, RelA and SpoT (Cashel et al., 1996). Both enzymes synthesize (p)ppGpp by transferring the pyrophosphate of ATP to the

The Stringent Response in Phototrophs 489

To date, six bacterial phyla have been shown to contain species capable of chlorophyll-based photosynthesis: *Cyanobacteria*, *Proteobacteria* (purple bacteria), *Chlorobi* (green sulfur bacteria), *Chloroflexi* (anoxygenic filamentous bacteria), *Firmicutes* (heliobacteria), and *Acidobacteria* (Bryant & Frigaard, 2006; Bryant et al., 2007). All of these photosynthetic bacteria produce SpoT and/or RelA proteins, but a careful analysis of these enzymes has only been done for two species: *Rhodobacter capsulatus* (a purple bacterium) (Masuda & Bauer, 2004), and *Anabaena* sp. PCC7120 (a cyanobacterium) (Ning et al., 2011). Genomic analysis has indicated that both of these bacterial species encode a single SpoT-like protein, but not a RelA-like protein. Based on complementation tests using *E. coli relA* and *relA*/*spoT* mutants, the SpoT-like enzyme of each of these bacteria likely have (p)ppGpp synthase activity. In wild-type strains of both bacteria, cellular levels of (p)ppGpp increase upon amino-acid starvation (caused by addition of serine hydroxamate), suggesting that these SpoT-like proteins induce the stringent response upon amino-acid starvation. In addition, these *spoT*-like genes are essential for cell viability, as loss-of-function mutations are lethal

*R. capsulatus* is one of the most extensively studied photosynthetic purple bacteria (Bauer, 2004). It exhibits remarkable bio-energetic versatility and is capable of aerobic respiratory growth, anaerobic respiratory growth (using dimethyl sulfoxide as an electron donor), and photosynthetic growth. As such, this bacterium can adjust its mode of growth in response to environmental conditions (e.g., oxygen concentration, light intensity). In fact, sophisticated regulatory systems that respond to changes in redox and light have been identified (Bauer et al., 2003). Masuda et al. (2004) asked whether the stringent response affects growth-mode control in *R. capsulatus* by functionally characterizing *spoT* of this organism. They found that the lethality associated with a *spoT* mutation could be rescued by loss of *hvrA*, a gene that encodes a nucleoid protein (Masuda & Bauer, 2004). HvrA was originally identified as a *trans*-acting factor that represses transcription of photosynthesis genes under intense light conditions (Buggy et al., 1994). In intense light, *R. capsulatus* down-regulates components of the photosynthetic apparatus (e.g., the light-harvesting complexes, and the photosynthetic reaction center) to avoid photo-damage. *hvrA* mutants, however, cannot reduce photopigment synthesis under intense light. HvrA is a typical bacterial nucleoid protein that resembles *E. coli* H-NS and StpA (Bertin et al., 1999; Masuda & Bauer, 2004). Nucleoid proteins bind curved DNA with low sequence specificity and affect a large number of genes involved in multiple physiological processes (McLeod & Johnson, 2001; Dorman & Deighan, 2003). In fact, *R. capsulatus* HvrA transcriptionally regulates (positively and negatively) genes involved in photosynthesis, nitrogen fixation, and electron transfer, for example (Buggy et al., 1994; Kern et al., 1998; Swem & Bauer, 2002). Although it is not entirely clear why loss of *hvrA* rescues the lethality associated with *spoT*-like loss-of-function, this result suggests a functional link between the (p)ppGpp-dependent stringent response and the nucleoid protein HvrA. A similar phenomenon exists in *E. coli* (Johansson et al., 2000); strains lacking the nucleoid proteins H-NS and StpA have a slow-growth phenotype, which can be partially suppressed by mutations in *spoT* and *relA*. These results suggest that genes regulated by (p)ppGpp are also regulated by the nucleoid structure. Notably, *hvrA* expression itself is controlled by RegA and RegB, a redox-sensitive, two-component system in *R. capsulatus* (Du et al., 1999). Specifically, *hvrA* transcription is repressed or activated under aerobic or anaerobic conditions, respectively. Taken together, these results suggest

**3. (p)ppGpp synthases and hydrolases in photosynthetic bacteria** 

(Masuda & Bauer, 2004; Ning et al., 2011).

ribose of GTP or GDP at the 3' hydroxyl position. (p)ppGpp is hydrolyzed by SpoT, but not RelA, as RelA has only (p)ppGpp synthase activity. Biochemical and crystallographic studies have revealed two distinct domains in SpoT that mediate either synthesis or hydrolysis of (p)ppGpp (Fig. 1) (Cashel et al., 1996). As expected, the (p)ppGpp hydrolysis domain (HD) is not conserved in RelA. The (p)ppGpp HD is found in a superfamily of metal-dependent phosphohydrolases (Aravind & Koonin, 1998; Hogg et al., 2004). In fact, SpoT-like proteins require a divalent cation such a Mn**2+** for their hydrolase activity (Wendrich et al., 2000).

A large number of bacterial genomes have been sequenced. These data have revealed that SpoT-like proteins, which contain both (p)ppGpp synthase and (p)ppGpp hydrolase domains, are generally conserved among bacterial species (Mittenhuber, 2001a). In contrast, RelA-like proteins, which contain only a (p)ppGpp synthase domain, have only been found in - and - *Proteobacteria*. Phylogenetic analyses suggest that RelA branched off from a SpoT-like protein following the divergence of - and - *Proteobacteria* (Mittenhuber, 2001a).

Fig. 1. Schematic depiction of the domain structure of SpoT and RelA from *E. coli* and of Arabidopsis RSHs. The region used to construct the phylogenetic tree (Fig. 3) is indicated by a dashed line. TM: putative transmembrane region; cTP: chloroplast transit peptide; EFhand: Ca2+-binding domain; HD: HD domain responsible for (p)ppGpp degradation. RelA and SpoT do not conserve several critical amino acids in the HD domain. RSH1 does not conserve the critical Gly residue (changed to Ser) that is necessary for (p)ppGpp synthase activity of RelA. For more detals, see text.

A small (p)ppGpp synthase protein, which lacks an HD domain, has also been found in some *Firmicutes* bacteria (Lemos et al., 2007; Nanakiya et al., 2008). Biochemical analyses indicate that these small enzymes have (p)ppGpp synthase activity.

ribose of GTP or GDP at the 3' hydroxyl position. (p)ppGpp is hydrolyzed by SpoT, but not RelA, as RelA has only (p)ppGpp synthase activity. Biochemical and crystallographic studies have revealed two distinct domains in SpoT that mediate either synthesis or hydrolysis of (p)ppGpp (Fig. 1) (Cashel et al., 1996). As expected, the (p)ppGpp hydrolysis domain (HD) is not conserved in RelA. The (p)ppGpp HD is found in a superfamily of metal-dependent phosphohydrolases (Aravind & Koonin, 1998; Hogg et al., 2004). In fact, SpoT-like proteins require a divalent cation such a Mn**2+** for their hydrolase activity

A large number of bacterial genomes have been sequenced. These data have revealed that SpoT-like proteins, which contain both (p)ppGpp synthase and (p)ppGpp hydrolase domains, are generally conserved among bacterial species (Mittenhuber, 2001a). In contrast, RelA-like proteins, which contain only a (p)ppGpp synthase domain, have only been found in - and - *Proteobacteria*. Phylogenetic analyses suggest that RelA branched off from a SpoT-like protein following the divergence of - and - *Proteobacteria* (Mittenhuber, 2001a).

Fig. 1. Schematic depiction of the domain structure of SpoT and RelA from *E. coli* and of Arabidopsis RSHs. The region used to construct the phylogenetic tree (Fig. 3) is indicated by a dashed line. TM: putative transmembrane region; cTP: chloroplast transit peptide; EFhand: Ca2+-binding domain; HD: HD domain responsible for (p)ppGpp degradation. RelA and SpoT do not conserve several critical amino acids in the HD domain. RSH1 does not conserve the critical Gly residue (changed to Ser) that is necessary for (p)ppGpp synthase

A small (p)ppGpp synthase protein, which lacks an HD domain, has also been found in some *Firmicutes* bacteria (Lemos et al., 2007; Nanakiya et al., 2008). Biochemical analyses

indicate that these small enzymes have (p)ppGpp synthase activity.

activity of RelA. For more detals, see text.

(Wendrich et al., 2000).

#### **3. (p)ppGpp synthases and hydrolases in photosynthetic bacteria**

To date, six bacterial phyla have been shown to contain species capable of chlorophyll-based photosynthesis: *Cyanobacteria*, *Proteobacteria* (purple bacteria), *Chlorobi* (green sulfur bacteria), *Chloroflexi* (anoxygenic filamentous bacteria), *Firmicutes* (heliobacteria), and *Acidobacteria* (Bryant & Frigaard, 2006; Bryant et al., 2007). All of these photosynthetic bacteria produce SpoT and/or RelA proteins, but a careful analysis of these enzymes has only been done for two species: *Rhodobacter capsulatus* (a purple bacterium) (Masuda & Bauer, 2004), and *Anabaena* sp. PCC7120 (a cyanobacterium) (Ning et al., 2011). Genomic analysis has indicated that both of these bacterial species encode a single SpoT-like protein, but not a RelA-like protein. Based on complementation tests using *E. coli relA* and *relA*/*spoT* mutants, the SpoT-like enzyme of each of these bacteria likely have (p)ppGpp synthase activity. In wild-type strains of both bacteria, cellular levels of (p)ppGpp increase upon amino-acid starvation (caused by addition of serine hydroxamate), suggesting that these SpoT-like proteins induce the stringent response upon amino-acid starvation. In addition, these *spoT*-like genes are essential for cell viability, as loss-of-function mutations are lethal (Masuda & Bauer, 2004; Ning et al., 2011).

*R. capsulatus* is one of the most extensively studied photosynthetic purple bacteria (Bauer, 2004). It exhibits remarkable bio-energetic versatility and is capable of aerobic respiratory growth, anaerobic respiratory growth (using dimethyl sulfoxide as an electron donor), and photosynthetic growth. As such, this bacterium can adjust its mode of growth in response to environmental conditions (e.g., oxygen concentration, light intensity). In fact, sophisticated regulatory systems that respond to changes in redox and light have been identified (Bauer et al., 2003). Masuda et al. (2004) asked whether the stringent response affects growth-mode control in *R. capsulatus* by functionally characterizing *spoT* of this organism. They found that the lethality associated with a *spoT* mutation could be rescued by loss of *hvrA*, a gene that encodes a nucleoid protein (Masuda & Bauer, 2004). HvrA was originally identified as a *trans*-acting factor that represses transcription of photosynthesis genes under intense light conditions (Buggy et al., 1994). In intense light, *R. capsulatus* down-regulates components of the photosynthetic apparatus (e.g., the light-harvesting complexes, and the photosynthetic reaction center) to avoid photo-damage. *hvrA* mutants, however, cannot reduce photopigment synthesis under intense light. HvrA is a typical bacterial nucleoid protein that resembles *E. coli* H-NS and StpA (Bertin et al., 1999; Masuda & Bauer, 2004). Nucleoid proteins bind curved DNA with low sequence specificity and affect a large number of genes involved in multiple physiological processes (McLeod & Johnson, 2001; Dorman & Deighan, 2003). In fact, *R. capsulatus* HvrA transcriptionally regulates (positively and negatively) genes involved in photosynthesis, nitrogen fixation, and electron transfer, for example (Buggy et al., 1994; Kern et al., 1998; Swem & Bauer, 2002). Although it is not entirely clear why loss of *hvrA* rescues the lethality associated with *spoT*-like loss-of-function, this result suggests a functional link between the (p)ppGpp-dependent stringent response and the nucleoid protein HvrA. A similar phenomenon exists in *E. coli* (Johansson et al., 2000); strains lacking the nucleoid proteins H-NS and StpA have a slow-growth phenotype, which can be partially suppressed by mutations in *spoT* and *relA*. These results suggest that genes regulated by (p)ppGpp are also regulated by the nucleoid structure. Notably, *hvrA* expression itself is controlled by RegA and RegB, a redox-sensitive, two-component system in *R. capsulatus* (Du et al., 1999). Specifically, *hvrA* transcription is repressed or activated under aerobic or anaerobic conditions, respectively. Taken together, these results suggest

The Stringent Response in Phototrophs 491

(p)ppGpp hydrolase domains of SpoT-like and RSH proteins. In this tree, bacterial SpoT-like proteins and plant RSHs are clearly separated, although SpoT-like proteins of the bacterial phyla *Deinococcus-Thermus* form a branch with plant RSH families (RSH1). It has been suggested that plant RSHs were introduced into a proto-plant cell by endosymbiosis of an ancestral cyanobacterium (Givens et al., 2004). Our phylogenetic analysis, however, does not clearly support this hypothesis, although cyanobacterial SpoT-like proteins are relatively similar to plant RSHs. Our results may agree with a previous phylogenetic analysis, which suggested that plant RSHs were introduced into a proto-plant cell by lateral gene transfer from a pathogenic bacterium (van der Biezen, 2000). Additional experiments are necessary

Arabidopsis has four RSHs, RSH1, RSH2, RSH3, and CRSH (Ca2+-activated RSH) (van der Biezen et al., 2000; Masuda et al., 2008a). Primary structures of these RSHs are shown in Fig. 1. Each of these four proteins has a putative chloroplast transit peptide at the N-terminus, suggesting that each functions in plastids. CRSH has two Ca2+-binding domains (EF-hand motifs) at the C-terminus. Sequences similar to the (p)ppGpp synthase and hydrolase domains of *E. coli* SpoT are found in the central region of RSHs. However, the conserved Gly residue, which is necessary for RelA (p)ppGpp synthase activity, is not conserved in RSH1 (changed to Ser). In addition, the HD domain that mediates (p)ppGpp hydrolase activity in SpoT is not conserved in CRSH (Masuda et al., 2008a). Both the Gly residue and the HD domain are conserved in RSH2 and RSH3. These results suggest that RSH1 and CRSH may have only (p)ppGpp hydrolase and synthase activity, respectively, whereas RSH2 and RSH3

Protein domain structures (Fig. 1) and the phylogenetic tree (Fig. 3) clearly show that Arabidopsis RSHs can be classified into three distinct families: RSH1, RSH2/3, and CRSH. Mining existing databases, all three types of RSHs can be found in another dicotyledon plant (*Nicotiana tabacum*), a monocotyledon plant (*Oryza sativa*), and a moss (*Physcomitrella patens*) (Fig. 3). RSH amino acid sequence alignments reveal that the RSH1, RSH2/3, and CRSH families each have a conserved linear arrangement of domains (Fig. 1). In addition, all RSH1-family members lack the conserved Gly residue that is critical for (p)ppGpp synthesis, all CRSH-family members lack the HD domain required for (p)ppGpp hydrolysis but have two EF-hand motifs instead, and all RSH2/3-family members contain both the Gly residue and the HD domain. These results suggest that the functional roles for these three RSH families may have been conserved between plant species. The green algae *Chlamydomonas reinhardtii* has a single *RSH* gene that does not cluster within any other plant RSH (Fig. 3), suggesting that the three plant RSH families (RSH1, RSH2/3 and CRSH) diverged after the separation of algae and mosses but before the separation of mosses and seed plants. Perhaps these three families were established when plant species adapted to terrestrial growth. Importantly, the *Chlamydomonas* RSH is not similar to bacterial SpoT-like proteins (Fig. 3),

suggesting that RSH genes were not introduced into plant cells by endosymbiosis.

Recently, a small family of proteins that contain a domain resembling the SpoT HD domain was identified in metazoa, including *Drosophila melanogaster, Caenorhabditis elegans* and human (Sun et al., 2010). The protein was named Mesh1 (metazoan SpoT homolog 1). Biochemical analyses indicated that *Drosophila* Mesh1 has (p)ppGpp hydrolase activity. Arabidopsis also has a Mesh1 homolog (Sun et al., 2010), found from a cDNA clone submitted to GenBank as part of the Arabidopsis full-length cDNA cloning project (accession no. BAF00616). However, I found that the nucleotide sequence of Arabidopsis *Mesh1* is identical, at least in part, to *RSH2*. Compared to full-length *RSH2*, *Mesh1* lacks ~200

to clarify the origin of plant RSHs.

may have both activities.

that HvrA and SpoT-like proteins are functionally linked in *R. capsulatus* to efficiently utilize energy sources (e.g., oxygen, light, amino acids, nitrogen, and carbon) in response to changing environmental conditions (Fig. 2). In support of this hypothesis, *hvrA* and *spoT*like double mutants produce significantly lower levels of photopigments (Masuda & Bauer, 2004). Importantly, exogenously added carbon compensates for pigmentation loss in these double mutants (Masuda & Bauer, 2004), suggesting that the stringent response (induced by the SpoT-like protein) promotes photopigment synthesis in *R. capsulatus* specifically during starvation.

Fig. 2. A model for the coordinated regulation of photosynthesis (PS) gene expression in response to redox, light, and nutrient conditions in the purple bacterium *R. capsulatus*. Putative signaling pathways are indicated by dashed arrows. Solid arrows represent signaling pathways supported by experimental evidence.

*Anabaena* sp. PCC7120 is a filamentous cyanobacterium that is frequently used as a model organism to characterize nitrogen fixation in heterocysts, which are specialized cells that fix nitrogen. It is well established that nitrogen depletion induces heterocyst formation. In *Anabaena* sp. PCC7120, however, the stringent response is not likely involved in heterocyst formation, as neither SpoT-like proteins nor (p)ppGpp levels increase upon nitrogen depletion (Ning et al., 2011). In these experiments, however, bulk levels of (p)ppGpp were measured, so it is possible that (p)ppGpp levels rise specifically in heterocyst-forming cells. In fact, (p)ppGpp accumulation was previously observed upon nitrogen depletion in other cyanobacteria (Akinyanju & Amith, 1979; Friga et al., 1981). If this is true, then the activity of the SpoT-like protein would be controlled post-translationally (as in *E. coli*) because nitrogen depletion does not affect *spoT*-like expression. To understand the stringent response in cyanobacteria, therefore, genetic analysis of the *spoT*-like gene is necessary.

#### **4. (p)ppGpp synthases and hydrolases in plants and algae**

van der Biezen et al. (2000) identified RelA/SpoT-like proteins in the model plant *Arabidopsis thaliana* and designated them as RSHs. Since then, genes encoding RSHs have been found in many plant and algal species (Kasai et al., 2002; Yamada et al., 2003; Givens et al., 2004; Tozawa et al., 2007; Masuda et al., 2008a; Kim et al., 2009). Figure 3 shows a phylogenetic tree that is based on amino-acid sequences of (p)ppGpp synthase and

that HvrA and SpoT-like proteins are functionally linked in *R. capsulatus* to efficiently utilize energy sources (e.g., oxygen, light, amino acids, nitrogen, and carbon) in response to changing environmental conditions (Fig. 2). In support of this hypothesis, *hvrA* and *spoT*like double mutants produce significantly lower levels of photopigments (Masuda & Bauer, 2004). Importantly, exogenously added carbon compensates for pigmentation loss in these double mutants (Masuda & Bauer, 2004), suggesting that the stringent response (induced by the SpoT-like protein) promotes photopigment synthesis in *R. capsulatus* specifically during

Fig. 2. A model for the coordinated regulation of photosynthesis (PS) gene expression in response to redox, light, and nutrient conditions in the purple bacterium *R. capsulatus*. Putative signaling pathways are indicated by dashed arrows. Solid arrows represent

*Anabaena* sp. PCC7120 is a filamentous cyanobacterium that is frequently used as a model organism to characterize nitrogen fixation in heterocysts, which are specialized cells that fix nitrogen. It is well established that nitrogen depletion induces heterocyst formation. In *Anabaena* sp. PCC7120, however, the stringent response is not likely involved in heterocyst formation, as neither SpoT-like proteins nor (p)ppGpp levels increase upon nitrogen depletion (Ning et al., 2011). In these experiments, however, bulk levels of (p)ppGpp were measured, so it is possible that (p)ppGpp levels rise specifically in heterocyst-forming cells. In fact, (p)ppGpp accumulation was previously observed upon nitrogen depletion in other cyanobacteria (Akinyanju & Amith, 1979; Friga et al., 1981). If this is true, then the activity of the SpoT-like protein would be controlled post-translationally (as in *E. coli*) because nitrogen depletion does not affect *spoT*-like expression. To understand the stringent response in

van der Biezen et al. (2000) identified RelA/SpoT-like proteins in the model plant *Arabidopsis thaliana* and designated them as RSHs. Since then, genes encoding RSHs have been found in many plant and algal species (Kasai et al., 2002; Yamada et al., 2003; Givens et al., 2004; Tozawa et al., 2007; Masuda et al., 2008a; Kim et al., 2009). Figure 3 shows a phylogenetic tree that is based on amino-acid sequences of (p)ppGpp synthase and

cyanobacteria, therefore, genetic analysis of the *spoT*-like gene is necessary.

**4. (p)ppGpp synthases and hydrolases in plants and algae** 

signaling pathways supported by experimental evidence.

starvation.

(p)ppGpp hydrolase domains of SpoT-like and RSH proteins. In this tree, bacterial SpoT-like proteins and plant RSHs are clearly separated, although SpoT-like proteins of the bacterial phyla *Deinococcus-Thermus* form a branch with plant RSH families (RSH1). It has been suggested that plant RSHs were introduced into a proto-plant cell by endosymbiosis of an ancestral cyanobacterium (Givens et al., 2004). Our phylogenetic analysis, however, does not clearly support this hypothesis, although cyanobacterial SpoT-like proteins are relatively similar to plant RSHs. Our results may agree with a previous phylogenetic analysis, which suggested that plant RSHs were introduced into a proto-plant cell by lateral gene transfer from a pathogenic bacterium (van der Biezen, 2000). Additional experiments are necessary to clarify the origin of plant RSHs.

Arabidopsis has four RSHs, RSH1, RSH2, RSH3, and CRSH (Ca2+-activated RSH) (van der Biezen et al., 2000; Masuda et al., 2008a). Primary structures of these RSHs are shown in Fig. 1. Each of these four proteins has a putative chloroplast transit peptide at the N-terminus, suggesting that each functions in plastids. CRSH has two Ca2+-binding domains (EF-hand motifs) at the C-terminus. Sequences similar to the (p)ppGpp synthase and hydrolase domains of *E. coli* SpoT are found in the central region of RSHs. However, the conserved Gly residue, which is necessary for RelA (p)ppGpp synthase activity, is not conserved in RSH1 (changed to Ser). In addition, the HD domain that mediates (p)ppGpp hydrolase activity in SpoT is not conserved in CRSH (Masuda et al., 2008a). Both the Gly residue and the HD domain are conserved in RSH2 and RSH3. These results suggest that RSH1 and CRSH may have only (p)ppGpp hydrolase and synthase activity, respectively, whereas RSH2 and RSH3 may have both activities.

Protein domain structures (Fig. 1) and the phylogenetic tree (Fig. 3) clearly show that Arabidopsis RSHs can be classified into three distinct families: RSH1, RSH2/3, and CRSH. Mining existing databases, all three types of RSHs can be found in another dicotyledon plant (*Nicotiana tabacum*), a monocotyledon plant (*Oryza sativa*), and a moss (*Physcomitrella patens*) (Fig. 3). RSH amino acid sequence alignments reveal that the RSH1, RSH2/3, and CRSH families each have a conserved linear arrangement of domains (Fig. 1). In addition, all RSH1-family members lack the conserved Gly residue that is critical for (p)ppGpp synthesis, all CRSH-family members lack the HD domain required for (p)ppGpp hydrolysis but have two EF-hand motifs instead, and all RSH2/3-family members contain both the Gly residue and the HD domain. These results suggest that the functional roles for these three RSH families may have been conserved between plant species. The green algae *Chlamydomonas reinhardtii* has a single *RSH* gene that does not cluster within any other plant RSH (Fig. 3), suggesting that the three plant RSH families (RSH1, RSH2/3 and CRSH) diverged after the separation of algae and mosses but before the separation of mosses and seed plants. Perhaps these three families were established when plant species adapted to terrestrial growth. Importantly, the *Chlamydomonas* RSH is not similar to bacterial SpoT-like proteins (Fig. 3), suggesting that RSH genes were not introduced into plant cells by endosymbiosis.

Recently, a small family of proteins that contain a domain resembling the SpoT HD domain was identified in metazoa, including *Drosophila melanogaster, Caenorhabditis elegans* and human (Sun et al., 2010). The protein was named Mesh1 (metazoan SpoT homolog 1). Biochemical analyses indicated that *Drosophila* Mesh1 has (p)ppGpp hydrolase activity. Arabidopsis also has a Mesh1 homolog (Sun et al., 2010), found from a cDNA clone submitted to GenBank as part of the Arabidopsis full-length cDNA cloning project (accession no. BAF00616). However, I found that the nucleotide sequence of Arabidopsis *Mesh1* is identical, at least in part, to *RSH2*. Compared to full-length *RSH2*, *Mesh1* lacks ~200

The Stringent Response in Phototrophs 493

Recently, a novel (p)ppGpp degradation system was found in the bacterium *Thermus thermophilus* (Ooga et al., 2009). Specifically, a Nudix (nucleoside diphosphates linked to some moiety X) pyrophosphatase degrades ppGpp (both *in vivo* and *in vitro*) to maintain proper levels of ppGpp. Over 20 genes that encode Nudix pyrophosphatases have been identified in plants (Ogawa et al., 2005; Gunawardana et al., 2009). These proteins have pyrophosphatase activity and degrade a variety of substrates that include (d)NTPs, nucleotide sugars, NADH, and NADPH. Given that some Nudix pyrophosphatases localize to chloroplasts (Ogawa et al., 2008), these proteins could be involved in (p)ppGpp degradation by cooperating with the RSH1 and RSH2/3 families of enzymes. If this is the case, Nudix proteins could degrade (p)ppGpp to ppGp or pGpp or pGp, which are strong inhibitors of purine biosynthesis (Pao et al., 1980; Pao and Dyess, 1981). This suggests that the degradation products of (p)ppGpp may participate in the stringent response in plastids.

Complementation analyses using *relA* and *relA*/*spoT* mutants in *E. coli* indicate that plant RSH2/3 and CRSH family members can functionally replace these bacterial enzymes. This suggests that plant RSHs have (p)ppGpp synthase activity (Givens et al., 2004; Tozawa et al., 2007; Masuda et al., 2008a; Mizusawa et al., 2008). This result was confirmed biochemically, clearly demonstrating that plant RSHs synthesize (p)ppGpp *in vitro* (Givens et al., 2004; Tozawa et al., 2007; Masuda et al., 2008a). These *in vitro* studies also showed that the (p)ppGpp synthase activity of CRSH is activated by Ca2+ (Tozawa et al., 2007; Masuda et al., 2008a). As mentioned previously, RSH1 may not have (p)ppGpp synthase activity, as it lacks the critical Gly residue found in bacterial RelA proteins. Mizusawa et al. (2008) showed that expression of Arabidopsis RSH1 does not rescue the *E. coli relA* and *relA*/*spoT* mutants, supporting this hypothesis. van der Biezen et al. (2000), however, reported the opposite result, concluding that RSH1 can complement the *relA* mutation. In addition, the (p)ppGpp hydrolase activities of RSH1, RSH2/3, and CRSH have not been confirmed.

Protein import experiments indicate that the *Chlamydomonas* RSH is targeted to isolated chloroplasts, suggesting that the protein primarily localizes to plastids (Kasai et al., 2002). In addition, when Arabidopsis RSH1 and CRSH are fused to green fluorescent protein, they also localize to chloroplasts (Masuda et al., 2008a; Mizusawa et al., 2008). Finally, the localization of *N. tabacum* RSH2 has been studied by western blotting, which indicated that the enzyme is highly enriched in the chloroplast fraction (Givens et al., 2004). These results suggest that proteins from each RSH family in plants localize to plastids. For each plant RSH, however, the exact localization within chloroplasts seems to differ. *N. tabacum* RSH2 is in the insoluble fraction of chloroplasts (Givens et al., 2004), whereas Arabidopsis CRSH is in the soluble fraction (Masuda et al., 2008a). These localization differences may reflect different modes of enzymatic regulation. It has been suggested that membrane-associated ribosomes interact with *N. tabacum* RSH2 and regulate its activity, as is the case with bacterial RelA proteins (Givens et al., 2004). There is evidence to suggest that CRSH activity depends on Ca2+ levels in the chloroplast stroma (Tozawa et al., 2007; Masuda et al., 2008a). Finally, RSH1 contains two putative transmembrane helices in its C-terminal region (Fig. 1) (van der Biezen et al., 2000), suggesting that it localizes to the thylakoid and/or envelope

membrane of plastids. This, however, has not been demonstrated experimentally.

**5. Physiological functions of RSHs in plants** 

Clearly, these two issues require further investigation.

bp from the 5' end, and it contains the first and third intron sequences. These results indicate that *Mesh1*, if it represents a functional transcript, is likely a splice variant of *RSH2*. Given that no *Mesh1* homologs have been identified in other plant species, and no data concerning Mesh1 function have been reported, the putative (p)ppGpp hydrolase, Mesh1, will not be discussed here.

Fig. 3. A phylogenetic tree based on (p)ppGpp synthase and (p)ppGpp hydrolase domains of bacterial SpoT-like proteins and plant RSHs. The region used to construct the phylogenetic tree corresponds to amino acid residues 25–325 of *E. coli* SpoT (indicated by the dashed line in Fig. 1). All gaps in the sequence alignment were omitted, and the tree was constructed using the neighbor-joining method.

bp from the 5' end, and it contains the first and third intron sequences. These results indicate that *Mesh1*, if it represents a functional transcript, is likely a splice variant of *RSH2*. Given that no *Mesh1* homologs have been identified in other plant species, and no data concerning Mesh1 function have been reported, the putative (p)ppGpp hydrolase, Mesh1, will not be

Fig. 3. A phylogenetic tree based on (p)ppGpp synthase and (p)ppGpp hydrolase domains

phylogenetic tree corresponds to amino acid residues 25–325 of *E. coli* SpoT (indicated by the dashed line in Fig. 1). All gaps in the sequence alignment were omitted, and the tree was

of bacterial SpoT-like proteins and plant RSHs. The region used to construct the

constructed using the neighbor-joining method.

discussed here.

Recently, a novel (p)ppGpp degradation system was found in the bacterium *Thermus thermophilus* (Ooga et al., 2009). Specifically, a Nudix (nucleoside diphosphates linked to some moiety X) pyrophosphatase degrades ppGpp (both *in vivo* and *in vitro*) to maintain proper levels of ppGpp. Over 20 genes that encode Nudix pyrophosphatases have been identified in plants (Ogawa et al., 2005; Gunawardana et al., 2009). These proteins have pyrophosphatase activity and degrade a variety of substrates that include (d)NTPs, nucleotide sugars, NADH, and NADPH. Given that some Nudix pyrophosphatases localize to chloroplasts (Ogawa et al., 2008), these proteins could be involved in (p)ppGpp degradation by cooperating with the RSH1 and RSH2/3 families of enzymes. If this is the case, Nudix proteins could degrade (p)ppGpp to ppGp or pGpp or pGp, which are strong inhibitors of purine biosynthesis (Pao et al., 1980; Pao and Dyess, 1981). This suggests that the degradation products of (p)ppGpp may participate in the stringent response in plastids.

#### **5. Physiological functions of RSHs in plants**

Complementation analyses using *relA* and *relA*/*spoT* mutants in *E. coli* indicate that plant RSH2/3 and CRSH family members can functionally replace these bacterial enzymes. This suggests that plant RSHs have (p)ppGpp synthase activity (Givens et al., 2004; Tozawa et al., 2007; Masuda et al., 2008a; Mizusawa et al., 2008). This result was confirmed biochemically, clearly demonstrating that plant RSHs synthesize (p)ppGpp *in vitro* (Givens et al., 2004; Tozawa et al., 2007; Masuda et al., 2008a). These *in vitro* studies also showed that the (p)ppGpp synthase activity of CRSH is activated by Ca2+ (Tozawa et al., 2007; Masuda et al., 2008a). As mentioned previously, RSH1 may not have (p)ppGpp synthase activity, as it lacks the critical Gly residue found in bacterial RelA proteins. Mizusawa et al. (2008) showed that expression of Arabidopsis RSH1 does not rescue the *E. coli relA* and *relA*/*spoT* mutants, supporting this hypothesis. van der Biezen et al. (2000), however, reported the opposite result, concluding that RSH1 can complement the *relA* mutation. In addition, the (p)ppGpp hydrolase activities of RSH1, RSH2/3, and CRSH have not been confirmed. Clearly, these two issues require further investigation.

Protein import experiments indicate that the *Chlamydomonas* RSH is targeted to isolated chloroplasts, suggesting that the protein primarily localizes to plastids (Kasai et al., 2002). In addition, when Arabidopsis RSH1 and CRSH are fused to green fluorescent protein, they also localize to chloroplasts (Masuda et al., 2008a; Mizusawa et al., 2008). Finally, the localization of *N. tabacum* RSH2 has been studied by western blotting, which indicated that the enzyme is highly enriched in the chloroplast fraction (Givens et al., 2004). These results suggest that proteins from each RSH family in plants localize to plastids. For each plant RSH, however, the exact localization within chloroplasts seems to differ. *N. tabacum* RSH2 is in the insoluble fraction of chloroplasts (Givens et al., 2004), whereas Arabidopsis CRSH is in the soluble fraction (Masuda et al., 2008a). These localization differences may reflect different modes of enzymatic regulation. It has been suggested that membrane-associated ribosomes interact with *N. tabacum* RSH2 and regulate its activity, as is the case with bacterial RelA proteins (Givens et al., 2004). There is evidence to suggest that CRSH activity depends on Ca2+ levels in the chloroplast stroma (Tozawa et al., 2007; Masuda et al., 2008a). Finally, RSH1 contains two putative transmembrane helices in its C-terminal region (Fig. 1) (van der Biezen et al., 2000), suggesting that it localizes to the thylakoid and/or envelope membrane of plastids. This, however, has not been demonstrated experimentally.

The Stringent Response in Phototrophs 495

inhibit PEP- mediated transcription when exogenously added *in vitro* (Sato et al., 2009). This suggests that (p)ppGpp directly controls PEP activity. The second "indirect" control of plastidial RNA polymerases needs to be studied in more detail. If this mechanism is real, both PEP and NEP activities should be affected. It has recently been reported that some plant hormones, including jasmonates and auxin, affect transcription of chloroplast genes (Zubo et al., 2011). Because *RSH2* expression is induced by jasmonates (Givens et al., 2004; Mizusawa et al., 2008), it is possible that RSH2-dependent (p)ppGpp synthesis mediates the

In addition to regulating transcription, (p)ppGpp also controls translation in bacteria (Milon et al., 2006). The translation initiation factor, IF2, binds and hydrolyzes GTP to initiate translation. (p)ppGpp binds to the GTP-binding pocket of IF2, thereby inhibiting translation initiation (Milon et al., 2006). Given that a bacterial IF2 homolog is found in chloroplasts (Miura et al., 2007), (p)ppGpp may also control translation of plastid genes; the chloroplast genome encodes genes involved in photosynthesis, electron transfer, and fatty-acid biosynthesis, for example. These various plastidial functions, therefore, should be regulated

Bacteria produce several GTP-binding proteins, some of which are conserved in plants and function in chloroplasts (Mittenhuber, 2001b; Masuda et al., 2008b). Because (p)ppGpp interacts with the GTP-binding pocket of IF2, chloroplast GTP-binding proteins may also be regulated by (p)ppGpp. Several enzymes involved in nucleotide biosynthesis are regulated by (p)ppGpp in an allosteric manner (Gallant et al., 1971; Hou et al., 1999). It is thought that nucleotide biosynthesis can take place in plastids because one of the enzymes that catalyzes phosphoribosyl diphosphate synthesis (the first step in purine and pyrimidine biosynthesis) localizes to plastids (Krath & Hove-Jensen, 1999). As a result, nucleotide biosynthesis in plastids may be directly regulated by (p)ppGpp. Furthermore, consumption of GTP (GDP) and ATP during (p)ppGpp synthesis may also indirectly influence nucleotide metabolism in

effects of plant hormones on plastidial gene expression.

Fig. 4. A model for the stringent response in higher plants.

by the (p)ppGpp-dependent stringent response.

plastids.

The expression patterns of Arabidopsis *RSH* genes also suggest that there are functional differences between RSHs (Mizusawa et al., 2008). Microarray data indicate that *RSH* genes have diurnal rhythms of expression. Peak levels of expression are reached at noon, evening, and midnight for Arabidopsis *RSH2/3*, *RSH1*, and *CRSH*, respectively (Mizusawa et al., 2008). Given that RSH2/3 enzymes have both synthase and hydrolase activities, they likely maintain plastidial (p)ppGpp levels during daylight hours. At evening, RSH1 may then degrade (p)ppGpp, as it seems to have only (p)ppGpp hydrolase activity. At night, (p)ppGpp levels are likely kept low because keeping plants in the dark reduces cellular (p)ppGpp levels (Takahashi et al., 2004). These data suggest that (p)ppGpp is required to control light-dependent plastidial activities, such as photosynthesis. At night, CRSH likely maintains (p)ppGpp levels based on Ca2+ levels. Plastidial Ca2+ concentrations change in response to environmental stimuli, such as light conditions (Johnson et al., 1995; Sai & Johnson, 2002), allowing CRSH to translate these environmental changes into (p)ppGpp synthesis.

Expression of *RSH2* genes in Arabidopsis, rice and *N. tabacum* is elevated in response to cold and/or exogenous application of the plant hormone jasmonic acid (or its precursor 12-oxophytodienoic acid) (Xiong et al., 2001; Givens et al., 2004; Lee et al., 2005; Mizusawa et al., 2008). (p)ppGpp levels similarly increase in response to jasmonic acid (Takahashi et al., 2004). These observations suggest that the RSH2/3 family synthesizes (p)ppGpp in response to abiotic stresses. The plastidial stringent response may play a role in these types of plant defense responses, although the mechanisms remain unclear.

Histochemical analyses indicate that Arabidopsis *RSH2* and *RSH3* are expressed in all green tissue (Mizusawa et al., 2008), suggesting that the RSH2/3 family controls light-dependent plastidial function. Interestingly, all *RSH* genes are expressed at high levels in flower tissues, such as the pistil and stamen, suggesting an important role for (p)ppGpp in flower development (Mizusawa et al., 2008). Genetic knockdown of *CRSH* disrupts flower morphology, as pistil and stamen development are not coordinated. This defect results in infertility (Masuda et al., 2008a), indicating that RSH-dependent (p)ppGpp production is required for plant reproduction. As discussed below, (p)ppGpp may control the biosynthesis of amino acids, fatty acids, and nucleotides in plastids, as has been observed in bacteria. Because these compounds serve as precursors for plant hormones (e.g., jasmonates, cytokinin, and auxin), the plastidial stringent response may control host plant development by regulating hormone biosynthesis.

#### **6. Which plastidial functions are regulated by (p)ppGpp?**

In bacteria, (p)ppGpp regulates the transcription of a large number of genes *via* two distinct mechanisms. First, (p)ppGpp allosterically controls RNA polymerase activity through direct association with the - or '- subunit of the polymerase (Chatterji et al., 1998; Toulokhonov et al., 2001; Artsimovitch et al., 2004). Second, RelA- and SpoT-dependent (p)ppGpp synthesis uses ATP and GTP (or GDP) as substrates. This significantly reduces the amount of NTPs available for RNA synthesis, thereby indirectly decreasing RNA polymerase activity (Krasny & Gourse, 2004). In chloroplasts, two RNA polymerases transcribe the plastid genome (Shiina et al., 2005; Liere et al., 2011). One is the bacterial type of RNA polymerase called plastid-encoded plastid RNA polymerase (PEP). The other is the T7 phase type of RNA polymerase called nuclear-encoded plastid RNA polymerase (NEP). As with *E. coli* RNA polymerase, (p)ppGpp directly binds to PEP (Sato et al., 2009) and can 494 Advances in Photosynthesis – Fundamental Aspects

The expression patterns of Arabidopsis *RSH* genes also suggest that there are functional differences between RSHs (Mizusawa et al., 2008). Microarray data indicate that *RSH* genes have diurnal rhythms of expression. Peak levels of expression are reached at noon, evening, and midnight for Arabidopsis *RSH2/3*, *RSH1*, and *CRSH*, respectively (Mizusawa et al., 2008). Given that RSH2/3 enzymes have both synthase and hydrolase activities, they likely maintain plastidial (p)ppGpp levels during daylight hours. At evening, RSH1 may then degrade (p)ppGpp, as it seems to have only (p)ppGpp hydrolase activity. At night, (p)ppGpp levels are likely kept low because keeping plants in the dark reduces cellular (p)ppGpp levels (Takahashi et al., 2004). These data suggest that (p)ppGpp is required to control light-dependent plastidial activities, such as photosynthesis. At night, CRSH likely maintains (p)ppGpp levels based on Ca2+ levels. Plastidial Ca2+ concentrations change in response to environmental stimuli, such as light conditions (Johnson et al., 1995; Sai & Johnson, 2002), allowing CRSH to translate these environmental changes into (p)ppGpp

Expression of *RSH2* genes in Arabidopsis, rice and *N. tabacum* is elevated in response to cold and/or exogenous application of the plant hormone jasmonic acid (or its precursor 12-oxophytodienoic acid) (Xiong et al., 2001; Givens et al., 2004; Lee et al., 2005; Mizusawa et al., 2008). (p)ppGpp levels similarly increase in response to jasmonic acid (Takahashi et al., 2004). These observations suggest that the RSH2/3 family synthesizes (p)ppGpp in response to abiotic stresses. The plastidial stringent response may play a role in these types of plant

Histochemical analyses indicate that Arabidopsis *RSH2* and *RSH3* are expressed in all green tissue (Mizusawa et al., 2008), suggesting that the RSH2/3 family controls light-dependent plastidial function. Interestingly, all *RSH* genes are expressed at high levels in flower tissues, such as the pistil and stamen, suggesting an important role for (p)ppGpp in flower development (Mizusawa et al., 2008). Genetic knockdown of *CRSH* disrupts flower morphology, as pistil and stamen development are not coordinated. This defect results in infertility (Masuda et al., 2008a), indicating that RSH-dependent (p)ppGpp production is required for plant reproduction. As discussed below, (p)ppGpp may control the biosynthesis of amino acids, fatty acids, and nucleotides in plastids, as has been observed in bacteria. Because these compounds serve as precursors for plant hormones (e.g., jasmonates, cytokinin, and auxin), the plastidial stringent response may control host plant development

In bacteria, (p)ppGpp regulates the transcription of a large number of genes *via* two distinct mechanisms. First, (p)ppGpp allosterically controls RNA polymerase activity through direct association with the - or '- subunit of the polymerase (Chatterji et al., 1998; Toulokhonov et al., 2001; Artsimovitch et al., 2004). Second, RelA- and SpoT-dependent (p)ppGpp synthesis uses ATP and GTP (or GDP) as substrates. This significantly reduces the amount of NTPs available for RNA synthesis, thereby indirectly decreasing RNA polymerase activity (Krasny & Gourse, 2004). In chloroplasts, two RNA polymerases transcribe the plastid genome (Shiina et al., 2005; Liere et al., 2011). One is the bacterial type of RNA polymerase called plastid-encoded plastid RNA polymerase (PEP). The other is the T7 phase type of RNA polymerase called nuclear-encoded plastid RNA polymerase (NEP). As with *E. coli* RNA polymerase, (p)ppGpp directly binds to PEP (Sato et al., 2009) and can

defense responses, although the mechanisms remain unclear.

**6. Which plastidial functions are regulated by (p)ppGpp?** 

by regulating hormone biosynthesis.

synthesis.

inhibit PEP- mediated transcription when exogenously added *in vitro* (Sato et al., 2009). This suggests that (p)ppGpp directly controls PEP activity. The second "indirect" control of plastidial RNA polymerases needs to be studied in more detail. If this mechanism is real, both PEP and NEP activities should be affected. It has recently been reported that some plant hormones, including jasmonates and auxin, affect transcription of chloroplast genes (Zubo et al., 2011). Because *RSH2* expression is induced by jasmonates (Givens et al., 2004; Mizusawa et al., 2008), it is possible that RSH2-dependent (p)ppGpp synthesis mediates the effects of plant hormones on plastidial gene expression.

Fig. 4. A model for the stringent response in higher plants.

In addition to regulating transcription, (p)ppGpp also controls translation in bacteria (Milon et al., 2006). The translation initiation factor, IF2, binds and hydrolyzes GTP to initiate translation. (p)ppGpp binds to the GTP-binding pocket of IF2, thereby inhibiting translation initiation (Milon et al., 2006). Given that a bacterial IF2 homolog is found in chloroplasts (Miura et al., 2007), (p)ppGpp may also control translation of plastid genes; the chloroplast genome encodes genes involved in photosynthesis, electron transfer, and fatty-acid biosynthesis, for example. These various plastidial functions, therefore, should be regulated by the (p)ppGpp-dependent stringent response.

Bacteria produce several GTP-binding proteins, some of which are conserved in plants and function in chloroplasts (Mittenhuber, 2001b; Masuda et al., 2008b). Because (p)ppGpp interacts with the GTP-binding pocket of IF2, chloroplast GTP-binding proteins may also be regulated by (p)ppGpp. Several enzymes involved in nucleotide biosynthesis are regulated by (p)ppGpp in an allosteric manner (Gallant et al., 1971; Hou et al., 1999). It is thought that nucleotide biosynthesis can take place in plastids because one of the enzymes that catalyzes phosphoribosyl diphosphate synthesis (the first step in purine and pyrimidine biosynthesis) localizes to plastids (Krath & Hove-Jensen, 1999). As a result, nucleotide biosynthesis in plastids may be directly regulated by (p)ppGpp. Furthermore, consumption of GTP (GDP) and ATP during (p)ppGpp synthesis may also indirectly influence nucleotide metabolism in plastids.

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#### **7. Concluding remarks**

It has been almost one-half century since (p)ppGpp was first discovered in *E. coli*. Since then, the physiological roles for (p)ppGpp in controlling bacterial cell metabolism have been well documented. The role of the stringent response in photosynthetic bacteria, however, remains unclear, even though photosynthesis is one of the most important anabolic reactions in biology. Future studies are needed if we are to understand how the stringent response controls different types of photosynthesis.

Many plant and algal species produce (p)ppGpp synthases and hydrolases called RSHs. Studies in Arabidopsis indicate that RSHs can be classified into three distinct families, RSH1, RSH2/3 and CRSH, all of which function in plastids. *RSH* gene expression profiles and the domain structures of RSHs suggest that RSH families are functionally diverse. Furthermore, these functional differences are likely necessary to properly regulate plastidial (p)ppGpp levels. Although the specific target proteins of (p)ppGpp remain largely unknown in plastids, the RSH-dependent stringent response regulates many aspects of plastidial function, including transcription, translation, nucleotide metabolism, and biosynthesis of amino acids and fatty acids. As a result, the stringent response may also regulate plant hormone biosynthesis, which is required for host plant development. A model of the stringent response in plastids is shown in Fig. 4. Additional genetic and physiological experiments are needed if we are to understand the precise roles of the (p)ppGpp-mediated stringent response in higher plants.

#### **8. Acknowledgment**

The work cited from the author's laboratory is currently funded by the Ministry of Education, Culture, Science and Technology of Japan.

#### **9. References**


It has been almost one-half century since (p)ppGpp was first discovered in *E. coli*. Since then, the physiological roles for (p)ppGpp in controlling bacterial cell metabolism have been well documented. The role of the stringent response in photosynthetic bacteria, however, remains unclear, even though photosynthesis is one of the most important anabolic reactions in biology. Future studies are needed if we are to understand how the stringent

Many plant and algal species produce (p)ppGpp synthases and hydrolases called RSHs. Studies in Arabidopsis indicate that RSHs can be classified into three distinct families, RSH1, RSH2/3 and CRSH, all of which function in plastids. *RSH* gene expression profiles and the domain structures of RSHs suggest that RSH families are functionally diverse. Furthermore, these functional differences are likely necessary to properly regulate plastidial (p)ppGpp levels. Although the specific target proteins of (p)ppGpp remain largely unknown in plastids, the RSH-dependent stringent response regulates many aspects of plastidial function, including transcription, translation, nucleotide metabolism, and biosynthesis of amino acids and fatty acids. As a result, the stringent response may also regulate plant hormone biosynthesis, which is required for host plant development. A model of the stringent response in plastids is shown in Fig. 4. Additional genetic and physiological experiments are needed if we are to understand the precise roles of the (p)ppGpp-mediated

The work cited from the author's laboratory is currently funded by the Ministry of

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**24** 

*Brasil* 

**Morphological and Physiological** 

*Universidade Federal do Espírito Santo* 

**Adjustments in Juvenile Tropical Trees** 

Geraldo Rogério Faustini Cuzzuol and Camilla Rozindo Dias Milanez

Luminosity is considered one of the most relevant environmental factors in plant growth, and it is closely associated to forest succession. It controls from morphogenetic processes of germination to morphological and physiological patterns of plant growth in different classes

There are several proposals to define succession classes of tree based on the placement of the species in the forest. Overall, two extreme successional groups are distinguished: a) species of the early-succession category (pioneers), which germinate, survive, and grow only in glades; and b) species of the final or late-succession category (climax), which require shady environments in the understory to grow. However, a large number of species that occupy intermediate status between these two succession classes has already

In the 1980s, early species began to be categorized into sun plants; and late species into shade plants. In the beginning of this century, the concept of sun-requiring species and shade-requiring species has been adopted for plants that need high irradiance and intense shading, respectively, in order to develop. Few species suit these limitations. Studies show that most species are able to tolerate intermediate irradiance conditions. They are, therefore,

Other terms that have often been used are shade-tolerant plants and sun-tolerant plants. Shade-tolerant plants correspond to shade-requiring plants, or simply shade plants. Sun-

Since the term *tolerance* suggests better performance under optimum environmental conditions, but able to acclimatize under conditions that are less favorable to growth, in this chapter we adopt the term *sun plants* (pioneer, sun-requiring or sun-tolerant plants), and *shade plants* (non-pioneer, shade-requiring or shade tolerant). The species in-between these two categories we call: facultative sun plants (early intermediate plants), which develop under full sunlight but tolerate moderate irradiance; and facultative shade plants (late intermediate

The initiative of categorizing the species into different status of succession is based on quantitative and qualitative criteria of the luminous spectrum occurring in natural environments, which varies significantly from the edge to the interior forest. However,

plants), which prefer intense shading but are able to grow under moderate shading.

categorized as facultative sun and facultative shade species.

tolerant plants are the sun-requiring plants, also called sun plants.

**1. Introduction** 

of forest succession.

been acknowledged.

**Under Contrasting Sunlight Irradiance** 


### **Morphological and Physiological Adjustments in Juvenile Tropical Trees Under Contrasting Sunlight Irradiance**

Geraldo Rogério Faustini Cuzzuol and Camilla Rozindo Dias Milanez *Universidade Federal do Espírito Santo Brasil* 

#### **1. Introduction**

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ppGpp to *Escherichia coli* RNA polymerase is allosteric, modular, and occurs near

(2007). Calcium-activated (p)ppGpp synthetase in chloroplasts of land plants. *J.* 

RelA/SpoT homologs implicate (p)ppGpp in plant signaling. *Proc. Natl. Acad. Sci.* 

suppression subtractive hybridization and differential screening. *Mol. Plant Micro.* 

confers salt tolerance in *Escherichia coli* and *Saccharomyces cerevisiae*. *Plant Cell*

gibberellic acid, and auxin affect transcription and transcript accumulation of

Luminosity is considered one of the most relevant environmental factors in plant growth, and it is closely associated to forest succession. It controls from morphogenetic processes of germination to morphological and physiological patterns of plant growth in different classes of forest succession.

There are several proposals to define succession classes of tree based on the placement of the species in the forest. Overall, two extreme successional groups are distinguished: a) species of the early-succession category (pioneers), which germinate, survive, and grow only in glades; and b) species of the final or late-succession category (climax), which require shady environments in the understory to grow. However, a large number of species that occupy intermediate status between these two succession classes has already been acknowledged.

In the 1980s, early species began to be categorized into sun plants; and late species into shade plants. In the beginning of this century, the concept of sun-requiring species and shade-requiring species has been adopted for plants that need high irradiance and intense shading, respectively, in order to develop. Few species suit these limitations. Studies show that most species are able to tolerate intermediate irradiance conditions. They are, therefore, categorized as facultative sun and facultative shade species.

Other terms that have often been used are shade-tolerant plants and sun-tolerant plants. Shade-tolerant plants correspond to shade-requiring plants, or simply shade plants. Suntolerant plants are the sun-requiring plants, also called sun plants.

Since the term *tolerance* suggests better performance under optimum environmental conditions, but able to acclimatize under conditions that are less favorable to growth, in this chapter we adopt the term *sun plants* (pioneer, sun-requiring or sun-tolerant plants), and *shade plants* (non-pioneer, shade-requiring or shade tolerant). The species in-between these two categories we call: facultative sun plants (early intermediate plants), which develop under full sunlight but tolerate moderate irradiance; and facultative shade plants (late intermediate plants), which prefer intense shading but are able to grow under moderate shading.

The initiative of categorizing the species into different status of succession is based on quantitative and qualitative criteria of the luminous spectrum occurring in natural environments, which varies significantly from the edge to the interior forest. However,

Morphological and Physiological Adjustments in Juvenile

1

3

by Paradyzo (2011) and Mengarda (2011).

plants (shade or climax plants).

Tropical Trees Under Contrasting Sunlight Irradiance 503

2

Days before abscission Days after abscission

Fig. 1. Morphological features of leaves of Brazilian tropical species grown under full sunlight and shading (20% of photosynthetically active radiation). *Cariniana estrellensis* 

 *Caesalpinia echinata* (Fabaceae) moderately shade-tolerant. Notice the little difference in coloration of *C. estrellensis* leaves under the two luminosity conditions. The leaves of *P. peroba* under full sunlight, however, presented chlorosis and burns at the veins. Notice the burn at the *C. echinata* pinnules before abscission. In the new pinnules sprouted after abscission, the reduced leaf area and lighter color can be noticed. Photographs provided

For tropical forest species in the early succession stage or sun plants, RGR ranged between 40 and 60 mg.g-1.day-1 under full sunlight, whereas for late or shade plants RGR was around 20 mg.g-1.day-1 under high irradiance (Figure 2). Nevertheless, the species *Caesalpinia echinata,* considered moderately shade-tolerant (facultative shade or early intermediate plant) tropical tree, showed higher RGR under full sunlight than under shading. This shows that RGR does not always follow the value decreasing from early species (sun plants) to late

As far as net assimilation rate (NAR) is concerned, the early species showed values under full sunlight in which NAR ranged from 0.3 to 0.6mg.cm2.day-1 (Figure 2). When under shading, the early and late species almost did not present differences regarding NAR, which was under 0.2 mg.cm2.day-1. In some cases, NAR can reach very low values

(Lecythidaceae), sun-tolerant; *Paratecoma peroba* (Bignoniaceae), shade-tolerant;

Sun Shade Sun Shade

1 cm

this categorization does not always correspond to the results obtained under controlled irradiance conditions. Some species considered ombrophytes of tropical forest have shown high phenotypic plasticity at the juvenile stage. They have been able to survive and grow under full sunlight. Nevertheless, the best performance occurred under moderate shading — typical characteristic of facultative shade species. Another aspect to be taken into account is phenology, because there could be different responses from juvenile to adult stage.

With so many environmental and ontogenetic variables influencing the morphological and physiological responses, it is difficult to find a scale for growth, biochemical, and physiological patterns which is able to characterize sun and shade requiring and facultative species.

Due to their high sensitivity to luminosity, the shade species have received special attention. Studies carried out with shade plants have shown that this kind of plant has lower plasticity under contrasting irradiance, which, in some cases, can compromise its growth and survival under full sunlight. When exposed to high sun irradiance, shade species suffer immediate and irreversible damages such as chlorosis, burns, and necrosis (Figure 1), followed by leaf abscission. If they are not capable of adapting to the new environment, they can collapse because of photoinhibition.

Morphological and physiological responses to variations in light intensity are well documented regarding leaves of arboreal vegetation in temperate areas. Studies on tropical tree have increasingly focused on medium term responses, leaving a gap concerning short term responses to light stress. Especially, regarding shade tolerant, semidecidual species. Based on the few tropical shade tolerant species in this study, we understand that the damages appear in the first seven days of exposure to direct solar radiation. In this period, there are photoinhibition and photo-oxidation followed by partial or complete abscission of leaves. Even so, they are able to sprout new leaves with new morphological and physiological characteristics without compromising survival.

This chapter aims at presenting up-to-date and unpublished results about the morphological, biochemical, and physiological adjustment of tropical shade arboreal vegetation after exposure to full sunlight. These data may encourage revisions to the status in forest succession of tropical species, because the descriptions of their ecological preferences concerning luminosity are quite contradictory.

#### **2. Morphology and growth measurement**

#### **2.1 Growth**

Species of the same succession group and even ecotypes of the same species have different reactions to irradiance alterations. In general, shade species of temperate climates do not survive or have low survival rates when exposed to full sunlight.

Regardless of their status in the forest succession, tropical forest plants under limiting irradiance have low root: shoot ratio (R:S); and higher leaf area ratio (LAR), leaf mass ratio (LMR), and specific leaf area (SLA). These responses provide higher photosynthetic activity in relation to breathing, allowing these species to be established inside the forest, where luminosity represents only between 2 to 8% of sunlight irradiance in the canopy.

Aiming at relating the succession status of 15 semideciduous tropical trees (Table 1) to growth measurements (Figure 2, 3 e 4), Souza & Válio (2003) verified that early-succession species (pioneer or sun plant) kept higher relative growth rate (RGR), even in the shade.

this categorization does not always correspond to the results obtained under controlled irradiance conditions. Some species considered ombrophytes of tropical forest have shown high phenotypic plasticity at the juvenile stage. They have been able to survive and grow under full sunlight. Nevertheless, the best performance occurred under moderate shading — typical characteristic of facultative shade species. Another aspect to be taken into account is phenology, because there could be different responses from

With so many environmental and ontogenetic variables influencing the morphological and physiological responses, it is difficult to find a scale for growth, biochemical, and physiological patterns which is able to characterize sun and shade requiring and facultative

Due to their high sensitivity to luminosity, the shade species have received special attention. Studies carried out with shade plants have shown that this kind of plant has lower plasticity under contrasting irradiance, which, in some cases, can compromise its growth and survival under full sunlight. When exposed to high sun irradiance, shade species suffer immediate and irreversible damages such as chlorosis, burns, and necrosis (Figure 1), followed by leaf abscission. If they are not capable of adapting to the new environment, they can collapse

Morphological and physiological responses to variations in light intensity are well documented regarding leaves of arboreal vegetation in temperate areas. Studies on tropical tree have increasingly focused on medium term responses, leaving a gap concerning short term responses to light stress. Especially, regarding shade tolerant, semidecidual species. Based on the few tropical shade tolerant species in this study, we understand that the damages appear in the first seven days of exposure to direct solar radiation. In this period, there are photoinhibition and photo-oxidation followed by partial or complete abscission of leaves. Even so, they are able to sprout new leaves with new morphological and

This chapter aims at presenting up-to-date and unpublished results about the morphological, biochemical, and physiological adjustment of tropical shade arboreal vegetation after exposure to full sunlight. These data may encourage revisions to the status in forest succession of tropical species, because the descriptions of their ecological

Species of the same succession group and even ecotypes of the same species have different reactions to irradiance alterations. In general, shade species of temperate climates do not

Regardless of their status in the forest succession, tropical forest plants under limiting irradiance have low root: shoot ratio (R:S); and higher leaf area ratio (LAR), leaf mass ratio (LMR), and specific leaf area (SLA). These responses provide higher photosynthetic activity in relation to breathing, allowing these species to be established inside the forest, where

Aiming at relating the succession status of 15 semideciduous tropical trees (Table 1) to growth measurements (Figure 2, 3 e 4), Souza & Válio (2003) verified that early-succession species (pioneer or sun plant) kept higher relative growth rate (RGR), even in the shade.

luminosity represents only between 2 to 8% of sunlight irradiance in the canopy.

physiological characteristics without compromising survival.

preferences concerning luminosity are quite contradictory.

survive or have low survival rates when exposed to full sunlight.

**2. Morphology and growth measurement** 

juvenile to adult stage.

because of photoinhibition.

species.

**2.1 Growth** 

Days before abscission Days after abscission

Fig. 1. Morphological features of leaves of Brazilian tropical species grown under full sunlight and shading (20% of photosynthetically active radiation). *Cariniana estrellensis*  (Lecythidaceae), sun-tolerant; *Paratecoma peroba* (Bignoniaceae), shade-tolerant; *Caesalpinia echinata* (Fabaceae) moderately shade-tolerant. Notice the little difference in coloration of *C. estrellensis* leaves under the two luminosity conditions. The leaves of *P. peroba* under full sunlight, however, presented chlorosis and burns at the veins. Notice the burn at the *C. echinata* pinnules before abscission. In the new pinnules sprouted after abscission, the reduced leaf area and lighter color can be noticed. Photographs provided by Paradyzo (2011) and Mengarda (2011).

For tropical forest species in the early succession stage or sun plants, RGR ranged between 40 and 60 mg.g-1.day-1 under full sunlight, whereas for late or shade plants RGR was around 20 mg.g-1.day-1 under high irradiance (Figure 2). Nevertheless, the species *Caesalpinia echinata,* considered moderately shade-tolerant (facultative shade or early intermediate plant) tropical tree, showed higher RGR under full sunlight than under shading. This shows that RGR does not always follow the value decreasing from early species (sun plants) to late plants (shade or climax plants).

As far as net assimilation rate (NAR) is concerned, the early species showed values under full sunlight in which NAR ranged from 0.3 to 0.6mg.cm2.day-1 (Figure 2). When under shading, the early and late species almost did not present differences regarding NAR, which was under 0.2 mg.cm2.day-1. In some cases, NAR can reach very low values

Morphological and Physiological Adjustments in Juvenile

Souza & Válio (2003).

are formed.

Tropical Trees Under Contrasting Sunlight Irradiance 505

Fig. 2. A. Relative growth rate (RGR) and B. net assimilation rate (NAR) of the studied tree species under full sun (FS), artificial shade (AS) and natural shade (NS) treatments. Measurements for 0-100 days time interval. Sol = *Solanum*, Tre = *Trema*, Cec = *Cecropia*, Bau = *Bauhinia*, Sen = *Senna*, Sch = *Schizolobium*, Pip = *Piptadenia*, Cho = *Chorisia*, Pse = *Pseudobombax*, Fic = *Ficus*, Ese = *Esenbeckia*, Pac = *Pachystroma*, Myr = *Myroxylon*, Hym = *Hymenaea*. Values followed by the same letter are not significantly different.

No difference in the R:S ratio has been noticed among early and late species, both under full sunlight and artificial shading (Figure 3). In general, R:S ratio ranged between 0.25 and 0.5. LMR showed higher plasticity for early species whose value ranged between 0.3 and 0.7 g.g-1, especially under effect of shading (Figure 3). These results can be confirmed by the higher SLA values of early species under artificial shading (6 dm2.g-1). Under full sunlight, almost no difference has been found in terms of SLA of early and late plants. Early species under shading tended to present increased LAR values; around 3.4 dm2.g-1. Under full sunlight,

Although there are data maintaining that late species or shade plants show better performance than pioneer or sun plants under low luminous intensity, it does not always happen. Some species that are considered sun plants can show low RGR; typical of shade plants. The opposite can also happen, as seen in *C. echinata,* a moderately shade-tolerant

Overall, the results have shown that morphological variations of tropical arboreal plants

Tropical tree shade species are able to develop in long periods of shading, keeping low growth rate, which favors the formation of a seedling bank. Due to their tolerance to higher irradiance, these plants show to be able to develop under increased luminosity, when glades

Therefore, the task of establishing a relation between growth measurement and successional status of tropical arboreal plants is complex. There is a paucity of more consistent data that

have higher influence on RGR when sun-tolerant species are under effect of shading.

the early and late species did not show significant LAR differences.

species that showed higher RGR under full sunlight than in the shade.

(around 0.01 mg.cm2.day-1), as seen in *C. echinata,* a species moderately shade-tolerant, under high shade.

This inconsistent pattern in terms of growth and forest succession status can be attributed to ontogenesis. Therefore, one should be careful not to extrapolate results obtained in the juvenile stage to adult stage. There is also the climatic factor in which the experiments were carried out. Overall, the experiments with tropical tree plants have been carried out in areas that differ in terms of irradiance intensity, precipitation, humidity, altitude, and average temperature. Another aspect that hinders comparisons in the analysis of results regards the lack of standard growth measurements, especially for growth rates expressed using different units of measurement.


Table 1. Species studied, classification according to the successional status (E = early-uccessional; I = intermediate, L = late-successional). Souza & Válio (2003).

For tropical trees, low RGR values for early species under low irradiance have been associated to reduction in photosynthetic activity, as indicated by low NAR (Figure 2). However, RGR is not always related to NAR (physiological component of RGR). In some cases, RGR can be related to LAR (morphological component of RGR). These relations between RGR, NAR, and LAR depend much on the intensity of solar radiation. Not taking succession status into account, the 15 species analyzed by Souza & Válio (2003) showed that RGR of plants under full sunlight and under natural shading is related to NAR, but not to LAR (Figure 4). This shows higher influence of the physiological component on growth rate. However, under artificial shading, the RGR was correlated to LAR, but not to NAR (Figure 4). In this case, the morphological component, particularly leaf area, had greater effect on growth rate. These differences in the correlation between RGR and NAR or LAR concerning artificial and natural shading have also been attributed to the luminous spectrum quality. Under natural shading, the red light: distant red light ratio is low, suggesting the involvement of phytochrome in the increase in SLA, component of LAR, and in LMR. These results indicate that leaf thickness and allocation of biomass to the leaves are the most pronounced morphological alterations, regardless of the species' forest succession status.

(around 0.01 mg.cm2.day-1), as seen in *C. echinata,* a species moderately shade-tolerant,

This inconsistent pattern in terms of growth and forest succession status can be attributed to ontogenesis. Therefore, one should be careful not to extrapolate results obtained in the juvenile stage to adult stage. There is also the climatic factor in which the experiments were carried out. Overall, the experiments with tropical tree plants have been carried out in areas that differ in terms of irradiance intensity, precipitation, humidity, altitude, and average temperature. Another aspect that hinders comparisons in the analysis of results regards the lack of standard growth measurements, especially for growth rates expressed using

Species Successional

*Solanum granuloso-leprosum* (Solanaceae) E *Trema micrantha* (Ulmaceae) E *Cecropia pachystachya* (Cecropiaceae) E *Bauhinia forficata* (Caesalpiniaceae) E *Senna macranthera* (Caesalpiniaceae) E *Schizolobium parahyba* (Caesalpiniaceae) E *Piptadenia gonoacantha* (Mimosaceae) E *Chorisia speciosa* (Bombacaceae) I *Pseudobombax grandiflorum* (Bombacaceae) I *Ficus guaranitica* (Moraceae) L *Esenbeckia leiocarpa* (Rutaceae) L *Pachystroma longifolium* (Euphorbiaceae) L *Myroxylon peruiferum* (Fabaceae) L *Hymenaea courbaril* (Caesalpiniaceae) L

Table 1. Species studied, classification according to the successional status

(E = early-uccessional; I = intermediate, L = late-successional). Souza & Válio (2003).

For tropical trees, low RGR values for early species under low irradiance have been associated to reduction in photosynthetic activity, as indicated by low NAR (Figure 2). However, RGR is not always related to NAR (physiological component of RGR). In some cases, RGR can be related to LAR (morphological component of RGR). These relations between RGR, NAR, and LAR depend much on the intensity of solar radiation. Not taking succession status into account, the 15 species analyzed by Souza & Válio (2003) showed that RGR of plants under full sunlight and under natural shading is related to NAR, but not to LAR (Figure 4). This shows higher influence of the physiological component on growth rate. However, under artificial shading, the RGR was correlated to LAR, but not to NAR (Figure 4). In this case, the morphological component, particularly leaf area, had greater effect on growth rate. These differences in the correlation between RGR and NAR or LAR concerning artificial and natural shading have also been attributed to the luminous spectrum quality. Under natural shading, the red light: distant red light ratio is low, suggesting the involvement of phytochrome in the increase in SLA, component of LAR, and in LMR. These results indicate that leaf thickness and allocation of biomass to the leaves are the most pronounced morphological alterations, regardless of the species' forest succession status.

status

under high shade.

different units of measurement.

Fig. 2. A. Relative growth rate (RGR) and B. net assimilation rate (NAR) of the studied tree species under full sun (FS), artificial shade (AS) and natural shade (NS) treatments. Measurements for 0-100 days time interval. Sol = *Solanum*, Tre = *Trema*, Cec = *Cecropia*, Bau = *Bauhinia*, Sen = *Senna*, Sch = *Schizolobium*, Pip = *Piptadenia*, Cho = *Chorisia*, Pse = *Pseudobombax*, Fic = *Ficus*, Ese = *Esenbeckia*, Pac = *Pachystroma*, Myr = *Myroxylon*, Hym = *Hymenaea*. Values followed by the same letter are not significantly different. Souza & Válio (2003).

No difference in the R:S ratio has been noticed among early and late species, both under full sunlight and artificial shading (Figure 3). In general, R:S ratio ranged between 0.25 and 0.5. LMR showed higher plasticity for early species whose value ranged between 0.3 and 0.7 g.g-1, especially under effect of shading (Figure 3). These results can be confirmed by the higher SLA values of early species under artificial shading (6 dm2.g-1). Under full sunlight, almost no difference has been found in terms of SLA of early and late plants. Early species under shading tended to present increased LAR values; around 3.4 dm2.g-1. Under full sunlight, the early and late species did not show significant LAR differences.

Although there are data maintaining that late species or shade plants show better performance than pioneer or sun plants under low luminous intensity, it does not always happen. Some species that are considered sun plants can show low RGR; typical of shade plants. The opposite can also happen, as seen in *C. echinata,* a moderately shade-tolerant species that showed higher RGR under full sunlight than in the shade.

Overall, the results have shown that morphological variations of tropical arboreal plants have higher influence on RGR when sun-tolerant species are under effect of shading.

Tropical tree shade species are able to develop in long periods of shading, keeping low growth rate, which favors the formation of a seedling bank. Due to their tolerance to higher irradiance, these plants show to be able to develop under increased luminosity, when glades are formed.

Therefore, the task of establishing a relation between growth measurement and successional status of tropical arboreal plants is complex. There is a paucity of more consistent data that

Morphological and Physiological Adjustments in Juvenile

Tropical Trees Under Contrasting Sunlight Irradiance 507

Fig. 4. Correlation between RGR and NAR (A); RGR and LAR (B); LAR and SLA (C); and LAR and LMR (D). Pooled data of all species under each one of the treatments (FS = full sun;

The ability a plant has to overcome the alarming stage is a result of physiological adjustments combined with morphological adaptations. This interaction has been considered the most relevant factor to acclimatization and survival of shade plants, when exposed to high irradiance. The morphological adjustment can start in existing plants. However, they are most

In shade species, the damage caused by intense irradiance takes place already on the first days, resulting in leaf abscission. In *C. echinata*, total leaf abscission took place on the first seven days. However, in other tropical species this effect can come later, as observed in *Minquartia guianensis,* a shade species of the Amazon forest, in which 30% of its leaves

AS = artificial shade, NS = natural shade). RGR = relative growth rate, NAR = net assimilation rate, LAR = leaf area ratio, LMR = leaf mass ratio. Souza & Válio (2003).

pronounced in young leaves sprouted after high irradiance exposure.

collapsed before the end of the second week under full sunlight.

**2.2 Leaf morphology** 

allow defining sun and shade plants, as well as characterizing facultative sun and shade plants (intermediate plants in forest succession).

Fig. 3. A. Root:shoot ratio (R:S); B. leaf mass ratio (LMR); C. specific leaf area (SLA); and D. leaf area ratio (LAR) of the studied tree species under full sun (FS), artificial shade (AS), and natural shade (NS) treatments. Measurements after 100 days. Sol = *Solanum*, Tre = *Trema*, Cec = *Cecropia*, Bau = *Bauhinia*, Sen = *Senna*, Sch = *Schizolobium*, Pip = *Piptadenia*, Cho = *Chorisia*, Pse = *Pseudobombax*, Fic = *Ficus*, Ese = *Esenbeckia*, Pac = *Pachystroma*, Myr = *Myroxylon*, Hym = *Hymenaea*. Values followed by the same letter are not significantly different. Souza & Válio (2003).

allow defining sun and shade plants, as well as characterizing facultative sun and shade

Fig. 3. A. Root:shoot ratio (R:S); B. leaf mass ratio (LMR); C. specific leaf area (SLA); and D. leaf area ratio (LAR) of the studied tree species under full sun (FS), artificial shade (AS),

Tre = *Trema*, Cec = *Cecropia*, Bau = *Bauhinia*, Sen = *Senna*, Sch = *Schizolobium*, Pip = *Piptadenia*,

Myr = *Myroxylon*, Hym = *Hymenaea*. Values followed by the same letter are not significantly

and natural shade (NS) treatments. Measurements after 100 days. Sol = *Solanum*,

different. Souza & Válio (2003).

Cho = *Chorisia*, Pse = *Pseudobombax*, Fic = *Ficus*, Ese = *Esenbeckia*, Pac = *Pachystroma*,

plants (intermediate plants in forest succession).

Fig. 4. Correlation between RGR and NAR (A); RGR and LAR (B); LAR and SLA (C); and LAR and LMR (D). Pooled data of all species under each one of the treatments (FS = full sun; AS = artificial shade, NS = natural shade). RGR = relative growth rate, NAR = net assimilation rate, LAR = leaf area ratio, LMR = leaf mass ratio. Souza & Válio (2003).

#### **2.2 Leaf morphology**

The ability a plant has to overcome the alarming stage is a result of physiological adjustments combined with morphological adaptations. This interaction has been considered the most relevant factor to acclimatization and survival of shade plants, when exposed to high irradiance. The morphological adjustment can start in existing plants. However, they are most pronounced in young leaves sprouted after high irradiance exposure.

In shade species, the damage caused by intense irradiance takes place already on the first days, resulting in leaf abscission. In *C. echinata*, total leaf abscission took place on the first seven days. However, in other tropical species this effect can come later, as observed in *Minquartia guianensis,* a shade species of the Amazon forest, in which 30% of its leaves collapsed before the end of the second week under full sunlight.

Morphological and Physiological Adjustments in Juvenile

UV wavelength.

Mengarda (2011).

**3. Photosynthesis** 

through non-photochemical processes.

Tropical Trees Under Contrasting Sunlight Irradiance 509

Besides the anatomic alterations, variations in secondary metabolite content may take place in plants under intense solar radiation. Phenolic and flavonoid compounds tend to accumulate in the epidermis and mesophyll of tropical tree shade plants under higher solar intensity. Leaves of *C. echinata* under shading have shown accumulation of phenols only in the epidermis, whereas under full sunlight, they also accumulated these compounds in mesophyll cells (Figure 5). Phenol accumulation indicates that the existence of an efficient antioxidative defense system working on the sequestration of several reactive oxygen species (ROS) and O2 singlet in chroloplasts of plants under intense solar radiation. The stress caused by excessive solar radiation also induces biosynthesis of polyphenols, among them, flavonoids. Probably, using ROS as molecular signals. Also, an increase in flavonoid concentration in leaves of arboreal plants lessens the penetration of

Shade Sun

Fig. 5. Cross section of *Caesalpinia echinata* pinnules in the shade (a and c) and under full sunlight (b and d). The mesophyll of leaves under full sunlight showed chrolophyll

parenchyma and adaxial epidermis thickening. Notice the higher accumulation of phenolic compounds in the limb of plants under full sunlight (d). Bar = 50m. Data provided by

The acclimatization strategy to high irradiance varies among species, and even among ecotypes of the same species. The physiological adjustments of shade plants exposed to high irradiance involve decrease in total chrolophyll concentration (Chltot) or increase in ratio between violaxanthin cycle pigments and Chltot. Violaxanthin and carotenoids reduce photoinhibition risks, oxidative damage, and increase dissipation of excessive energy

In the stage of light stress signalization of tropical shade tree species, the photoinhibition signals can be seen already in the first 24 hours of exposure to full sunlight. In *C. echinata*, a photosynthetic carbon assimilation (A), maximum quantum yield of photosystem II (Fv/FM),

a b

c d

Differently from what had been speculated about understory species, the results have shown different degrees of sensitivity after these plants were exposed to high irradiance. Some understory species of humid tropical forest such as *Ouratea lucens* showed moderate photoinhibition, preserving most of their leaves*.* For *Hybanthus prunifolius*, however, there was severe photoinhibition and almost total loss of leaves.

The phenotypic plasticity of tropical arboreal plants to luminosity involves characteristics that are related to higher efficiency in capture or dissipation of light through the leaves. This essentially depends on the adjustments of morphological and anatomical components.

Among the most significant anatomical adjustments observed in shad species under high sun irradiance, we can highlight the thickening of cuticle, palisade parenchyma, and increase in stomatic density, and trichomes. For *C. echinata,* the new leaves sprouted after abscission showed thickening of palisade parenchyma (Table 2), which suggests an efficient morphological strategy to reduce photo-oxidative damage. In general, the highest stomatal density is associated to reduction in the stomatal opening area and, consequently, resistance to water loss through transpiration. Cuticle and adaxial epidermal cell thickening is also one of the adjustments often seen in tropical shade species, when exposed to full sunlight. These adaptations minimize leave surface heating by promoting of light reflection.

It is important to high light that the intensity of these responses may vary significantly among the leaves before and after abscission. In *C. echinata,* exposure to full sunlight induced limb thinning because of thickness reduction in adaxial epidermis and palisade parenchyma during the first seven days of exposure preceding leaf abscission (Table 2). However, the new leaves sprouted after abscission showed increased thickness in palisade and spongy parenchyma, which were the main contributors to limb thickening (Table 2). In this aspect, the palisade parenchyma increased 142% under full sunlight, whereas the spongy parenchyma increased 58.3% and the adaxial epidermis 12.5% compared to plants under shading. The higher elongation of chlorophyllian tissue in the new leaves reflected the higher water content; 50% higher compared to plants under shading. These data suggest that *C. echinata* is a species that uses water effectively under full sunlight.

The reduction in SLA after solar radiation exposure is common among tropical arboreal plants. This response was observed, for example, in *M. guianensis* and *C. echinata (*Table 2). Reduction in SLA means smaller solar radiation interception area, contributing to water loss reduction and improvement of photosynthetic performance, growth, and survival of the plants under full sunlight.


Table 2. SLA values, limb thickness, palisade parenchyma, lacunary parenchyma, and water content in leaves of *Caesalpinia echinata* after 7 and 60 days of transfer of plants from shade to full sun. ± represents standard error of the mean (n=6). Data provided by Mengarda (2010).

Differently from what had been speculated about understory species, the results have shown different degrees of sensitivity after these plants were exposed to high irradiance. Some understory species of humid tropical forest such as *Ouratea lucens* showed moderate photoinhibition, preserving most of their leaves*.* For *Hybanthus prunifolius*, however, there

The phenotypic plasticity of tropical arboreal plants to luminosity involves characteristics that are related to higher efficiency in capture or dissipation of light through the leaves. This essentially depends on the adjustments of morphological and anatomical components. Among the most significant anatomical adjustments observed in shad species under high sun irradiance, we can highlight the thickening of cuticle, palisade parenchyma, and increase in stomatic density, and trichomes. For *C. echinata,* the new leaves sprouted after abscission showed thickening of palisade parenchyma (Table 2), which suggests an efficient morphological strategy to reduce photo-oxidative damage. In general, the highest stomatal density is associated to reduction in the stomatal opening area and, consequently, resistance to water loss through transpiration. Cuticle and adaxial epidermal cell thickening is also one of the adjustments often seen in tropical shade species, when exposed to full sunlight. These

It is important to high light that the intensity of these responses may vary significantly among the leaves before and after abscission. In *C. echinata,* exposure to full sunlight induced limb thinning because of thickness reduction in adaxial epidermis and palisade parenchyma during the first seven days of exposure preceding leaf abscission (Table 2). However, the new leaves sprouted after abscission showed increased thickness in palisade and spongy parenchyma, which were the main contributors to limb thickening (Table 2). In this aspect, the palisade parenchyma increased 142% under full sunlight, whereas the spongy parenchyma increased 58.3% and the adaxial epidermis 12.5% compared to plants under shading. The higher elongation of chlorophyllian tissue in the new leaves reflected the higher water content; 50% higher compared to plants under shading. These data suggest

The reduction in SLA after solar radiation exposure is common among tropical arboreal plants. This response was observed, for example, in *M. guianensis* and *C. echinata (*Table 2). Reduction in SLA means smaller solar radiation interception area, contributing to water loss reduction and improvement of photosynthetic performance, growth, and survival of the

Shade Sun Shade Sun

Variables 7 days 60 days

SLA (mg.cm2) 250±19 170±16 210±21 150±14 Limb (mm) 120±14 135±18 137±11 195±18 Palisade parenchyma (mm) 32±06 44±08 40±08 80±10 Lacunary parenchyma (mm) 60±10 60±13 67±14 88±09 H2O (mg.cm2) 18±02 19±03 18±02 27±04 Table 2. SLA values, limb thickness, palisade parenchyma, lacunary parenchyma, and water content in leaves of *Caesalpinia echinata* after 7 and 60 days of transfer of plants from shade to full sun. ± represents standard error of the mean (n=6). Data provided by Mengarda (2010).

adaptations minimize leave surface heating by promoting of light reflection.

that *C. echinata* is a species that uses water effectively under full sunlight.

plants under full sunlight.

was severe photoinhibition and almost total loss of leaves.

Besides the anatomic alterations, variations in secondary metabolite content may take place in plants under intense solar radiation. Phenolic and flavonoid compounds tend to accumulate in the epidermis and mesophyll of tropical tree shade plants under higher solar intensity. Leaves of *C. echinata* under shading have shown accumulation of phenols only in the epidermis, whereas under full sunlight, they also accumulated these compounds in mesophyll cells (Figure 5). Phenol accumulation indicates that the existence of an efficient antioxidative defense system working on the sequestration of several reactive oxygen species (ROS) and O2 singlet in chroloplasts of plants under intense solar radiation. The stress caused by excessive solar radiation also induces biosynthesis of polyphenols, among them, flavonoids. Probably, using ROS as molecular signals. Also, an increase in flavonoid concentration in leaves of arboreal plants lessens the penetration of UV wavelength.

Fig. 5. Cross section of *Caesalpinia echinata* pinnules in the shade (a and c) and under full sunlight (b and d). The mesophyll of leaves under full sunlight showed chrolophyll parenchyma and adaxial epidermis thickening. Notice the higher accumulation of phenolic compounds in the limb of plants under full sunlight (d). Bar = 50m. Data provided by Mengarda (2011).

#### **3. Photosynthesis**

The acclimatization strategy to high irradiance varies among species, and even among ecotypes of the same species. The physiological adjustments of shade plants exposed to high irradiance involve decrease in total chrolophyll concentration (Chltot) or increase in ratio between violaxanthin cycle pigments and Chltot. Violaxanthin and carotenoids reduce photoinhibition risks, oxidative damage, and increase dissipation of excessive energy through non-photochemical processes.

In the stage of light stress signalization of tropical shade tree species, the photoinhibition signals can be seen already in the first 24 hours of exposure to full sunlight. In *C. echinata*, a photosynthetic carbon assimilation (A), maximum quantum yield of photosystem II (Fv/FM),

Morphological and Physiological Adjustments in Juvenile

bars indicate stardad error. Mengarda et al. (2009).

exposure for *C. echinata* plants (Figure 9).

Tropical Trees Under Contrasting Sunlight Irradiance 511

Fig. 8. Stomatic conductance (A) and transpiration (B) of *C. echinata* plants subjected to constant artificial shade of 50% () and transferred from shade to full sunlight (). Vertical

The chrolophyll fluorescence parameters have been widely used to analyze responses from shade plants exposed to intense irradiance. Especially to detect alarming and resistance stages. Overall, tropical shade tree plants show higher density of absorbed photons per reaction center of PSII (ABS/RC) when exposed to high irradiance, as seen in *C. echinata* and *Aniba roseodora*. During the first three days of exposure to full sunlight, *C. echinata* plants (Figure 9) showed an increase in energy dissipated in the form of heat or fluorescence (DI0/RC), in absorbed energy (ABS/RC), and in energy captured and converted into redox energy for electron transport (ET0/TR0). These responses can indicate inactivation of the reaction center (RC). On the second day of transfer of *C. echinata* plants from the shaded environment to full sunlight, there was a significant increase in the probability that the electron captured by the reaction center of PSII remain in the transport chain beyond QA- (ET0/TR0). Nevertheless, the significant reduction in the density of active reaction centers of PSII (RC/ABS) on the first seven days may have influenced the reduction in effective quantum efficiency of radiant energy conversion (FV/F0), and in the performance index (PIABS). The inactivation of 58 to 78% of RC in *C. echinata* (Figure 9), along with reduction in maximum quantum yield of photosystem II (Fv/FM), indicate an increase in energy dissipation in the form of heat and fluorescence for some tropical tree plants exposed to intense irradiance. Although parameters such as RC/ABS, FV/F0 and PIABS have shown an increase after 20 days, the effective recovery of the photosynthetic apparatus was not observed during the 180 days of full sunlight

water-use efficiency (WUE), stomatic conductance (g) e transpiration (E) decreased in the first three hours (Figure 6, 7 e 8) until they reached the lowest values in 48 hours, in a 192 hour period. During this period, it was not possible to identify the restitution stage that precedes the resistance stage.

Fig. 6. Photosynthetic carbon assimilation (A) and maximum quantum yield of photosystem II (B) of *C. echinata* plants subjected to constant artificial shade of 50% () and transferred from shade to full sunlight () at 0, 3, 24, 48 and 192 h. after the start of the experiment. Vertical bars indicate standard error. Mengarda et al. (2009).

Fig. 7. Water-use efficiency of *C. echinata* plants subjected to constant artificial shade of 50% () and transferred from shade to full sunlight (). Vertical bars indicate stardad error. Mengarda et al. (2009).

The factors that limit photosynthesis vary according to irradiance intensity. Plants developing in shaded environments invest more in light-capturing complexes, whereas plants developing in the sun invest in Calvin cycle and electron transport proteins. Thus, irradiance variations cause alterations in A because of the differences in maximum velocity of Rubisco carboxylation (Vc-max.) and in the maximum rate of ribulose biphosphate regeneration. The results obtained from tropical shade species show limited capacity to increase A in environments under high irradiance, due to inability to increase Vc-max.

water-use efficiency (WUE), stomatic conductance (g) e transpiration (E) decreased in the first three hours (Figure 6, 7 e 8) until they reached the lowest values in 48 hours, in a 192 hour period. During this period, it was not possible to identify the restitution stage that

Fig. 6. Photosynthetic carbon assimilation (A) and maximum quantum yield of photosystem II (B) of *C. echinata* plants subjected to constant artificial shade of 50% () and transferred from shade to full sunlight () at 0, 3, 24, 48 and 192 h. after the start of the experiment.

Fig. 7. Water-use efficiency of *C. echinata* plants subjected to constant artificial shade of 50% () and transferred from shade to full sunlight (). Vertical bars indicate stardad

The factors that limit photosynthesis vary according to irradiance intensity. Plants developing in shaded environments invest more in light-capturing complexes, whereas plants developing in the sun invest in Calvin cycle and electron transport proteins. Thus, irradiance variations cause alterations in A because of the differences in maximum velocity of Rubisco carboxylation (Vc-max.) and in the maximum rate of ribulose biphosphate regeneration. The results obtained from tropical shade species show limited capacity to increase A in environments under high irradiance, due to inability to increase

Vertical bars indicate standard error. Mengarda et al. (2009).

precedes the resistance stage.

error. Mengarda et al. (2009).

Vc-max.

Fig. 8. Stomatic conductance (A) and transpiration (B) of *C. echinata* plants subjected to constant artificial shade of 50% () and transferred from shade to full sunlight (). Vertical bars indicate stardad error. Mengarda et al. (2009).

The chrolophyll fluorescence parameters have been widely used to analyze responses from shade plants exposed to intense irradiance. Especially to detect alarming and resistance stages. Overall, tropical shade tree plants show higher density of absorbed photons per reaction center of PSII (ABS/RC) when exposed to high irradiance, as seen in *C. echinata* and *Aniba roseodora*. During the first three days of exposure to full sunlight, *C. echinata* plants (Figure 9) showed an increase in energy dissipated in the form of heat or fluorescence (DI0/RC), in absorbed energy (ABS/RC), and in energy captured and converted into redox energy for electron transport (ET0/TR0). These responses can indicate inactivation of the reaction center (RC). On the second day of transfer of *C. echinata* plants from the shaded environment to full sunlight, there was a significant increase in the probability that the electron captured by the reaction center of PSII remain in the transport chain beyond QA- (ET0/TR0). Nevertheless, the significant reduction in the density of active reaction centers of PSII (RC/ABS) on the first seven days may have influenced the reduction in effective quantum efficiency of radiant energy conversion (FV/F0), and in the performance index (PIABS). The inactivation of 58 to 78% of RC in *C. echinata* (Figure 9), along with reduction in maximum quantum yield of photosystem II (Fv/FM), indicate an increase in energy dissipation in the form of heat and fluorescence for some tropical tree plants exposed to intense irradiance. Although parameters such as RC/ABS, FV/F0 and PIABS have shown an increase after 20 days, the effective recovery of the photosynthetic apparatus was not observed during the 180 days of full sunlight exposure for *C. echinata* plants (Figure 9).

Morphological and Physiological Adjustments in Juvenile

adjustment and survival of the plant.

shading.

**4. Soluble carbohydrates** 

morphological processes.

this species' tolerance to saline stress (Figure 10).

Tropical Trees Under Contrasting Sunlight Irradiance 513

The stress condition is caused by excessive input of energy in the system, which hinders the use of this energy in the photosynthetic process. Under ideal conditions, the plants show low free energy in the system and optimum thermodynamic status, without compromising the photochemical stage in PSII. However, the photoinhibition observed in *C. echinata* plants subjected to direct solar radiation can represent a photoprotection mechanism, working on the balance between effective non-photochemical dissipation of excessive energy and the photosynthesis itself. This does nor decharacterizes a stress condition, but allows the

Chrolophyll fluorescence *a* can be considered a potential tool to differ species of particular successional status, which is useful to select species to be used for recovering degraded areas. In general, sun-requiring or facultative sun species (pioneer and early intermediate) show higher photochemical ability and increasing tendency to dissipate excessive energy as luminous intensity grows. A study carried out on three tropical tree leguminous plants (*C. echinata, C. ferrea and Machaerium obovatum*) observed higher electron transport rates and higher FV/FM with higher intensity of light saturation for *C. ferrea*, which suggests that this species is best adapted to environments with high irradiance, whereas *C. echinata*  and *M. obovatum* do not show these responses. Therefore, *C. echinata* was characterized as late intermediate species, because it does not present photosynthetic acclimatization that is effective to high irradiance conditions. However, partial photosynthetic acclimatization and sufficient photoprotection mechanisms have been observed, allowing young plants to survive and grow under full sunlight. Thus, planting these species in reforestation has been suggested, adopting the intercropping system, in which plants receive moderate

One of the immediate metabolic signs in response to abrupt increase in irradiance is in the variation of soluble carbohydrates. These compounds as recognized as important molecular signals in the plant-environment relation and modeling agents of physiological and

Higher concentrations of glucose (Glu), fructose (Fru) and sucrose (Suc) were related to tolerance to water deficit during the winter for some tropical forest leguminous and tree plants. Polyols such as mannitol (Man) were also associated to water stress in *Fraxinus excelsior.* For *Olea europaea,* a Mediterranean tree, the accumulation of Man was associated to

The accumulation of *monosaccharides* and Suc in leaf tissues of tree plants under water deficiency, increased salinity and intense irradiance has often been associated to osmoregulation. The relevant few studies published showed reduction in osmotic potential in leaf cells of Mediterranean tree plants such as *Ilex aquifolium* and *O. europaeae* under high irradiance. For *C. echinata,* a tropical forest specie, the leaves of plants under full sunlight showed higher water content (Table 2). This result was associated to higher content of total

More recently, special attention has been paid to raffinose (Raf), which despite being found in low levels in leaves, has a significant role in the osmotic adjustment in herbaceous plants under stressing conditions, as observed in *Arabidopsis thaliana.* Raf is a trisaccharide that, along with stachyose and verbascose, is part of the oligosaccharides family of raffinose (RFO), which are synthesized from sucrose. Nevertheless, few are the publication on the

soluble carbohydrates (TCS), which possibly led to the reduction in water potential.

Fig. 9. Chrolophyll fluorescence emission *a* of *C. echinata* plants transferred from shade to full sunlight. Energy dissipation per reaction center (DI0/RC), energy absorption per reaction center (ABS/RC), and probability that an excitron captured by the RC of FSII move an electron in the transport chain to beyond QA- (ET0/TR0); (B) Ratio between number of active reaction centers of FSII and the amount of light absorbed by the antenna system (RC/ABS), maximum quantum yield of PSII (FV/FM), and performance index based on absorption (PIABS). Values expressed in percentage in relation to control (n=5). Data provided by Mengarda (2011).

The joint analysis of FV/FM and PIABS can be related to the ability plants have to transform luminous energy into metabolic reactions of the photosynthesis biochemical processes. Thus, taking into account the reduced FV/FM values and the significant drop in PIABS observed in plants under full sunlight, it can be concluded that the exposure of young *C. echinata* plants to full sunlight caused photoinhibition and reduction in primary photochemical efficiency, which can compromise photosynthesis.

In the first acclimatization weeks under high irradiance, the tropical tree plant *Minguartia guianensis* showed an increase in initial fluorescence (F0), and reduction in maximum fluorescence (FM) and FV/FM. Recovery only took place after four months of exposure to full sunlight, when FV/FM values reached 93% of the values observed for plants kept in the shade. For the tree plant *A. rosaeodora* of the Brazilian Amazon forest, reduction in FV/FM took place at 2 days of exposure to high irradiance, and recovery took place only at 60 days, when the values approached those observed for shaded plants.

For other tropical forest arboreal plants, the physiological adjustment process can slower. The transfer of Amazonian *M. guianensis* plants from a shaded environment to high irradiance caused a sharp decrease in FV/FM during the first three days, followed by gradual recovery until maximum values were reached on the 120th day, reaching the control group.

Fig. 9. Chrolophyll fluorescence emission *a* of *C. echinata* plants transferred from shade to full sunlight. Energy dissipation per reaction center (DI0/RC), energy absorption per reaction center (ABS/RC), and probability that an excitron captured by the RC of FSII move an electron in the transport chain to beyond QA- (ET0/TR0); (B) Ratio between number of active reaction centers of FSII and the amount of light absorbed by the antenna system (RC/ABS), maximum quantum yield of PSII (FV/FM), and performance index based on absorption (PIABS). Values expressed in percentage in relation to control (n=5). Data

The joint analysis of FV/FM and PIABS can be related to the ability plants have to transform luminous energy into metabolic reactions of the photosynthesis biochemical processes. Thus, taking into account the reduced FV/FM values and the significant drop in PIABS observed in plants under full sunlight, it can be concluded that the exposure of young *C. echinata* plants to full sunlight caused photoinhibition and reduction in primary

In the first acclimatization weeks under high irradiance, the tropical tree plant *Minguartia guianensis* showed an increase in initial fluorescence (F0), and reduction in maximum fluorescence (FM) and FV/FM. Recovery only took place after four months of exposure to full sunlight, when FV/FM values reached 93% of the values observed for plants kept in the shade. For the tree plant *A. rosaeodora* of the Brazilian Amazon forest, reduction in FV/FM took place at 2 days of exposure to high irradiance, and recovery took place only at 60 days,

For other tropical forest arboreal plants, the physiological adjustment process can slower. The transfer of Amazonian *M. guianensis* plants from a shaded environment to high irradiance caused a sharp decrease in FV/FM during the first three days, followed by gradual recovery until maximum values were reached on the 120th day, reaching the control group.

photochemical efficiency, which can compromise photosynthesis.

when the values approached those observed for shaded plants.

provided by Mengarda (2011).

The stress condition is caused by excessive input of energy in the system, which hinders the use of this energy in the photosynthetic process. Under ideal conditions, the plants show low free energy in the system and optimum thermodynamic status, without compromising the photochemical stage in PSII. However, the photoinhibition observed in *C. echinata* plants subjected to direct solar radiation can represent a photoprotection mechanism, working on the balance between effective non-photochemical dissipation of excessive energy and the photosynthesis itself. This does nor decharacterizes a stress condition, but allows the adjustment and survival of the plant.

Chrolophyll fluorescence *a* can be considered a potential tool to differ species of particular successional status, which is useful to select species to be used for recovering degraded areas. In general, sun-requiring or facultative sun species (pioneer and early intermediate) show higher photochemical ability and increasing tendency to dissipate excessive energy as luminous intensity grows. A study carried out on three tropical tree leguminous plants (*C. echinata, C. ferrea and Machaerium obovatum*) observed higher electron transport rates and higher FV/FM with higher intensity of light saturation for *C. ferrea*, which suggests that this species is best adapted to environments with high irradiance, whereas *C. echinata*  and *M. obovatum* do not show these responses. Therefore, *C. echinata* was characterized as late intermediate species, because it does not present photosynthetic acclimatization that is effective to high irradiance conditions. However, partial photosynthetic acclimatization and sufficient photoprotection mechanisms have been observed, allowing young plants to survive and grow under full sunlight. Thus, planting these species in reforestation has been suggested, adopting the intercropping system, in which plants receive moderate shading.

#### **4. Soluble carbohydrates**

One of the immediate metabolic signs in response to abrupt increase in irradiance is in the variation of soluble carbohydrates. These compounds as recognized as important molecular signals in the plant-environment relation and modeling agents of physiological and morphological processes.

Higher concentrations of glucose (Glu), fructose (Fru) and sucrose (Suc) were related to tolerance to water deficit during the winter for some tropical forest leguminous and tree plants. Polyols such as mannitol (Man) were also associated to water stress in *Fraxinus excelsior.* For *Olea europaea,* a Mediterranean tree, the accumulation of Man was associated to this species' tolerance to saline stress (Figure 10).

The accumulation of *monosaccharides* and Suc in leaf tissues of tree plants under water deficiency, increased salinity and intense irradiance has often been associated to osmoregulation. The relevant few studies published showed reduction in osmotic potential in leaf cells of Mediterranean tree plants such as *Ilex aquifolium* and *O. europaeae* under high irradiance. For *C. echinata,* a tropical forest specie, the leaves of plants under full sunlight showed higher water content (Table 2). This result was associated to higher content of total soluble carbohydrates (TCS), which possibly led to the reduction in water potential.

More recently, special attention has been paid to raffinose (Raf), which despite being found in low levels in leaves, has a significant role in the osmotic adjustment in herbaceous plants under stressing conditions, as observed in *Arabidopsis thaliana.* Raf is a trisaccharide that, along with stachyose and verbascose, is part of the oligosaccharides family of raffinose (RFO), which are synthesized from sucrose. Nevertheless, few are the publication on the

Morphological and Physiological Adjustments in Juvenile

mean (n=6). Data provided by Mengarda (2010).

**5. Conclusion** 

**6. Acknowledgment** 

pp. 1245-1253

**7. References** 

Tropical Trees Under Contrasting Sunlight Irradiance 515

Glu 1.2±0.68 6.0±0.27 1.0±0.65 1.0±0.25 Fru 1.1±0.41 7.0±0.15 0.7±0.09 0.5±0.05 Suc 10.3±2.53 17.0±2.16 7.0±1.25 14.0±1.36 Raf 0.26±0.07 0.26±0.08 0.04±0.00 0.7±0.06 TSC 11.2±1.58 30.0±3.86 8.0±2.95 14.0±3.25 Table 3. Leaf concentration of soluble carbohydrates in *C. echinata* plants after 7 and 60 days

of transfer from shade to full sunlight. Glucose (Glu); Fructose (Fru); Sucrose (Suc); Raffinose (Raf) and total soluble carbohydrates (TSC). ± represents standard error of the

proposal of a new forest succession classification for some tropical tree plants.

*do Espírito Santo* (FAPES-Process 39044823/2007) for the financial support.

Vol.15, (September, No.6), pp. 358-364, ISSN 1432-2285

Thanks are owed to *Fundação Biodiversitas*, *Fundação de Apoio Científico e Tecnológico da Prefeitura Municipal de Vitória* (FACITEC-Process 38/2007) and *Fundação de Apoio à Pesquisa* 

Aranda, I.; L. Gil & J. Pardos. (2001). Effects of thinning in a *Pinus sylvestris* L. stand on foliar

Cao, K.F. 2000. Leaf anatomy and clorophyll contento f 12 woody species in contrasting

water relations of *Fagus sylvatia* L. seedlings planted within the pinewood. *Trees*,

light conditions in a Bornean heath forest. *Canadian Journal of Botany*, Vol.78, No.10,

Some tropical tree plants considered shade plants or late intermediate in forest succession are able to survive under high irradiance. Even if at the alarming stage of light stress they show partial or total abscission of leaves, survival rate is high. This is due to these plants' ability to sprout new morphologically and physiologically adjusted leaves in environments with higher irradiance. Some tropical species considered shade plants show high plasticity to contrasting sunlight, at least in their early stages of growth. In part, physiological variables contribute more to plasticity index at the alarming stage of luminous stress. At the resistance stage, the new leaves sprouted show morphological alterations, such as SLA reduction and limb thickening. The most frequent morphological adjustment such as elongation and/or increase in the number of layers of palisade parenchyma, and reduction in SLA seem to be under the control of Suc. Photo-oxidative damage such as reduction in the *Chla:b* and *Chltotal:carotenoids* ratio, maximum photochemical yield of PSII, and PIABS. did not compromise the survival of shade plants when they were exposed to high solar radiation. The results point to the need of increasing the number of tropical species under controlled conditions, and assess their physiological and morphological mechanisms in field conditions at different stages of development; from seedling to adult stage. These data can support the

Shade Sun Shade Sun

Carbohydrates 7 days 60 days

involvement of Raf in tree plants' tolerance to high irradiance. Studies on *C. echinata* showed higher concentration of Raf in leaves after 60 days of exposure to full sunlight, suggesting an antioxidant action. Besides being osmoregulators, the monosaccharides, disaccharides such as Suc and RFOs have also been related to antioxidant actions in plants that are tolerant to water deficit and high irradiance. Therefore, investigating oxidative stress and its relation to carbohydrates could provide data to better understand the mechanisms of tolerance to light stress in tropical tree plants.

Fig. 10. Regulation of mannitol transport and metabolism as a mechanism providing salt tolerance in *O. europaea*. Conde et al. 2007.

Carbohydrates can also modify the morphological components of leaves. There are indications that the increase in palisade parenchyma thickness through elongation and periclinal cell divisions in tree plants under high irradiance is related to Suc concentration in leaves. *Chenopodium album L.* leaves*,* an annual herbaceous plant, showed increase in the number of layers of palisade parenchyma cells when exposed to high irradiance. This response is associated to concentration of Suc. In *C. echinata*, limb thickening because of palisade and lacunary parenchyma elongation has also been associated to higher concentration of leaf sucrose of plants under full sunlight.

There are indications that carbohydrates are also able to model plant growth, modifying biomass allocation patterns, growth rates, and R:S ratio. Under full sunlight, tree plants tend to invest more in root growth to the detriment of leaf area, resulting in RGR and NAR reduction.

In general, physiological and morphological responses associated to carbohydrates were obtained after exposing the plants to high irradiance for several days, leaving a gap concerning understanding the adjustment mechanisms at early stages of light stress. Studies on *C. echinata* have been carried out in order to categorize the alarming and resistance stages to luminous stress in a 180-day period. In this regard, carbohydrate contents and their relation to morphological and physiological adjustment have been analyzed at short periods of time (0, 2, 4, 7, 15, 30, 40, 50, 60, 120 and 180 days). The results show that the alarming stage was characterized by increased Glu and Fru contents with maximum peak at seven days. The resistance stage started with new leaves sprouting (at 15 days, Table 3), which stood out because of their higher concentrations of Suc and Raf. However, gradual reduction in Suc contents was observed by the 180th day (10 mg.g-1 DW), equal to the control group (shaded plants).


Table 3. Leaf concentration of soluble carbohydrates in *C. echinata* plants after 7 and 60 days of transfer from shade to full sunlight. Glucose (Glu); Fructose (Fru); Sucrose (Suc); Raffinose (Raf) and total soluble carbohydrates (TSC). ± represents standard error of the mean (n=6). Data provided by Mengarda (2010).

#### **5. Conclusion**

514 Advances in Photosynthesis – Fundamental Aspects

involvement of Raf in tree plants' tolerance to high irradiance. Studies on *C. echinata* showed higher concentration of Raf in leaves after 60 days of exposure to full sunlight, suggesting an antioxidant action. Besides being osmoregulators, the monosaccharides, disaccharides such as Suc and RFOs have also been related to antioxidant actions in plants that are tolerant to water deficit and high irradiance. Therefore, investigating oxidative stress and its relation to carbohydrates could provide data to better understand the mechanisms of tolerance to light

Fig. 10. Regulation of mannitol transport and metabolism as a mechanism providing salt

Carbohydrates can also modify the morphological components of leaves. There are indications that the increase in palisade parenchyma thickness through elongation and periclinal cell divisions in tree plants under high irradiance is related to Suc concentration in leaves. *Chenopodium album L.* leaves*,* an annual herbaceous plant, showed increase in the number of layers of palisade parenchyma cells when exposed to high irradiance. This response is associated to concentration of Suc. In *C. echinata*, limb thickening because of palisade and lacunary parenchyma elongation has also been associated to higher

There are indications that carbohydrates are also able to model plant growth, modifying biomass allocation patterns, growth rates, and R:S ratio. Under full sunlight, tree plants tend to invest more in root growth to the detriment of leaf area, resulting in RGR and NAR

In general, physiological and morphological responses associated to carbohydrates were obtained after exposing the plants to high irradiance for several days, leaving a gap concerning understanding the adjustment mechanisms at early stages of light stress. Studies on *C. echinata* have been carried out in order to categorize the alarming and resistance stages to luminous stress in a 180-day period. In this regard, carbohydrate contents and their relation to morphological and physiological adjustment have been analyzed at short periods of time (0, 2, 4, 7, 15, 30, 40, 50, 60, 120 and 180 days). The results show that the alarming stage was characterized by increased Glu and Fru contents with maximum peak at seven days. The resistance stage started with new leaves sprouting (at 15 days, Table 3), which stood out because of their higher concentrations of Suc and Raf. However, gradual reduction in Suc contents was observed by the 180th day (10 mg.g-1 DW), equal to the

stress in tropical tree plants.

tolerance in *O. europaea*. Conde et al. 2007.

reduction.

control group (shaded plants).

concentration of leaf sucrose of plants under full sunlight.

Some tropical tree plants considered shade plants or late intermediate in forest succession are able to survive under high irradiance. Even if at the alarming stage of light stress they show partial or total abscission of leaves, survival rate is high. This is due to these plants' ability to sprout new morphologically and physiologically adjusted leaves in environments with higher irradiance. Some tropical species considered shade plants show high plasticity to contrasting sunlight, at least in their early stages of growth. In part, physiological variables contribute more to plasticity index at the alarming stage of luminous stress. At the resistance stage, the new leaves sprouted show morphological alterations, such as SLA reduction and limb thickening. The most frequent morphological adjustment such as elongation and/or increase in the number of layers of palisade parenchyma, and reduction in SLA seem to be under the control of Suc. Photo-oxidative damage such as reduction in the *Chla:b* and *Chltotal:carotenoids* ratio, maximum photochemical yield of PSII, and PIABS. did not compromise the survival of shade plants when they were exposed to high solar radiation. The results point to the need of increasing the number of tropical species under controlled conditions, and assess their physiological and morphological mechanisms in field conditions at different stages of development; from seedling to adult stage. These data can support the proposal of a new forest succession classification for some tropical tree plants.

#### **6. Acknowledgment**

Thanks are owed to *Fundação Biodiversitas*, *Fundação de Apoio Científico e Tecnológico da Prefeitura Municipal de Vitória* (FACITEC-Process 38/2007) and *Fundação de Apoio à Pesquisa do Espírito Santo* (FAPES-Process 39044823/2007) for the financial support.

#### **7. References**


Morphological and Physiological Adjustments in Juvenile

pp. 117-127, ISSN 1432-2285

*Botany,* Vol.45, pp. 165-178

three species. *Árvore*, Vol.31, pp. 959-966

63, ISSN 1677-0420

681-696, ISNN 1758-4469

1365-3040.

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Kitajima, K. & Poorter, L. (2010). Tissue-level leaf toughness, but not lamina thickness,

Kitao, M.; Lei T. T.; Koike T.; Tobita H. & Maruyama Y. (2000). Susceptibility to

Kuptz, D.; Grams, T.E.E. & Günter, S. (2009). Light acclimation of four native tree species in

Laisk, A.; Eichelmann, H.; Oja, V.; Rasulov, B.; Padu, E.; Bichele, I.; Pettai, H. & Kull, O.

Marenco, R.A.; Gonçalves, J.F. & Vieira, G. (2001). Leaf gas exchange and carbohydrates in

Marenco, R.A. & Vieira, G. (2005). Specific leaf area and photosynthetic parameters of tree

Central Amazonia. *Journal of Tropical Forest Science*, Vol.17, No.2, pp. 265-278 Matsuki, S.; Ogawa, K.; Tanaka, A. & Hara, T. (2003). Morphological and photosynthetic

Melgar, J.C.; Guidi, L.; Remorini, D.; Agati, G.; Degl'Innocenti, E.; Castelli, S.; Baratto, M.C.;

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predicts sapling leaf lifespan and shade tolerance of tropical tree species. *New* 

photoinhibition of three deciduous broadleaf tree species with different successional traits raised under various light regimes. Plant *Cell and Environment*,

felling gaps within a tropical mountain rainforest. *Trees*, Vol.24, No1. (February),

2005. Adjustment of leaf photosynthesis to shade in a natural canopy: rate parameters. *Plant Cell and Environment*, Vol.28, No.3 (March), pp. 375-388, ISSN

tropical trees differing in successional status in two light environments in central Amazonia. *Tree Physiology*, Vol.21, No.18 (December), pp. 1311-1318, ISNN 1758-

species in the forest understorey as a function of the microsite light environment in

responses of *Quercus crispula* seedlings to high-light conditions. *Tree Physiology*,

Faraloni, C. & Tattini, M. (2009). Antioxidant defences and oxidative damage in salt-treated olive plants under contrasting sunlight irradiance. *Tree Physiology*,

contrasting Mediterranean light environments – effects on structure and chemical composition of foliage and plant water relations. *Environmental and Experimental* 

G.R.F. (2009). Ligth as an indicator of ecological succession in brazilwood (*Caesalpinia echinata* Lam.) *Brazilian Journal of Plant Physiology*, Vol.21, No.1, pp. 55-

Chloroplastid pigment contents and chlorophyll *a* fluorescence in Amazon tropical

morphology, physiology and light interception in temperate deciduous woody species of contrasting shade tolerance. *Tree Physiology*, Vol.18, No.10 (October), pp.

conductance limits photosynthesis more strongly in older leaves of Mediterranea


Conde, C., Silva, P., Agasse, A., Lemoine, R., Delrot, S., Tavares, R. & Gerós, H. (2007).

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Gibson, S.I. 2004. Sugar and phytohormone response pathways: navigating a signalling

Gleason, S.M. & Ares, A. 2004. Photosynthesis, carbohydrates storage and survival of a

Gonçalves, J.F.C. & Santos-Jr, U.M. 2005. Utilization of the chlorophyll a fluorescence

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*vulgare*. *Tree Physiology*, Vol.28, No.6, (June), pp. 873-883, ISSN 1758-4469 Hanba, Y.T., Kogami H. and Terashima I. 2002. The effect of growth irradiance on leaf

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savanna trees. *Tree Physiology*, Vol.27, (May), pp. 717-725, ISSN1758-4469 Geßler, A., Duarte, H.M., Franco, A.C., Lüttge, U., Mattos, E.A., Nahm, M., Rodrigues,

*of Experimental Botany*, Vol.57, (Februray), pp. 449-459, ISSN 1460-2431 Demmig-Adams, B. & Adams, W.W. 2006. Photoprotection in an ecological context: the

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1460-2431

Utilization and transporto f mannitol in *Olea europaea* and implications for salt stress tolerance. *Plant and Cell Physiol*ogy, Vol.48, (January, No.1) No.1, pp. 42-53,

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tropical woody seedlings to nitrogen limitation and high light. *Plant Cell and* 


**25** 

*1Greece 2Spain* 

**Transglutaminase is Involved in** 

*1Department of Biology, University of Crete, Heraklion, Crete,* 

Nikolaos E. Ioannidis1, Josep Maria Torné2, Kiriakos Kotzabasis1 and Mireya Santos2

*CRAG-CSIC-IRTA-UAB, Barcelona,* 

**the Remodeling of Tobacco Thylakoids** 

*2Departament de Genètica Molecular, Centre for Research in Agricultural Genomics,* 

Photosynthesis light reactions are among the more fast, complex and important processes in the ecosystem. They take place in specific membranes the so-called thylakoids and they produce O2, energy (ATP) and reducing equivalents (NADPH). In this chapter we will discuss recent findings that shed light in important aspects of thylakoid architecture and functional organization. Key role for the remodeling of thylakoids plays a plastidal transglutaminase that was recently cloned from maize. Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines. Transglutaminase (TGase) activity is present in chloroplasts of higher plants being PSII antenna proteins the enzyme's natural substrates. Although the functionality of this plastidial enzyme is not clear, a role in antenna regulation has been hypothesized. The isolation, for the first time in plants, of two related complementary maize DNA clones, *tgz15*  and *tgz21,* encoding active maize (*Zea mays L*) chloroplastic TGase (chlTGZ) has contributed to deep on the role of this enzyme in plants (Torné et al. 2002; Villalobos et al 2004). In addition, the main polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm) are normally produced and oxidized in chloroplasts. Thus, all types of post-translational modifications (i.e. mono-Put, mono-Spd, mono-Spm, bis-Put, bis-Spd and bis-Spm) are in theory probable for the target proteins. These modifications may alter charge and/or conformation of the target protein as well as their linking with other proteins (Kotzabasis et

A strong tool for a deeper study of gene functionality is the effect of its over-expression in an heterologous plant system. Here we will discuss in detail the information about the recent chlTGZ over-expression in tobacco (*Nicotiana tabacum* var. Petit Havana) chloroplasts (Ioannidis et al. 2009) and its characterization. After chloroplast transformation, transglutaminase activity in TGZ-over-expressers was up-regulated 4-fold with respect to the wild-type plants, which in turn rised its thylakoid-associated polyamine content about 90%. A major increase in the granum size (i.e. increase in the number of stacked layers)

**1. Introduction** 

al. 1993; Del Duca et al. 1994; Della Mea et al. 2004).

evergreen broad-leaved species. *Plant Cell and Environment*, Vol.28, No.12 (December), pp. 1552-1567, ISSN 1365-3040


### **Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids**

Nikolaos E. Ioannidis1, Josep Maria Torné2, Kiriakos Kotzabasis1 and Mireya Santos2 *1Department of Biology, University of Crete, Heraklion, Crete, 2Departament de Genètica Molecular, Centre for Research in Agricultural Genomics, CRAG-CSIC-IRTA-UAB, Barcelona, 1Greece 2Spain* 

#### **1. Introduction**

518 Advances in Photosynthesis – Fundamental Aspects

Nishizawa, A.; Y. Yabuta & S. Shigeoka. (2008). Galactinol and rafnose constitute a novel

Oguchi, R.; Hirosaka, K. & Hisose, T. (2003). Does the photosynthetic light-acclimation need

Oguchi, R.; Hirosaka, K. & Hisose, T. (2005). Leaf anatomy as a constraint for photosynthetic

Parida, A.K.; Das, A.B. & Mittra, B. (2004). Effects of salt on growth, ion accumulation,

Peters, S.; Mundree, J.A.; Thomson, J.M.; Farrant & F. Keller. (2007). Protection mechanisms

*Experimental Botany*, Vol.58, No.8, (June), pp. 1947–1956, ISSN 1460-2431 Reyes-Díaz, M.; Ivanov, A.G.; Huner, N.P.A.; Alberdi, M.; Corcuera, L.J. & Bravo, L.A.

Souza, R.P. & Válio, I. F.M. (2003). Seedling growth of fifteen Brazilian tropical tree species

Terashima, I.; Hanba, Y.T.; Tazoe, Y.; Vyas, P. & Yano, S. (2006). Irradiance and phenotype:

Urban, O.; Kosvancová, M.; Marek, M.V. & Lichtenthaler, H.K. (2007). Induction of

zone. *Tree Physiolgy*, Vol.27, No.8 (August), pp. 1207-1215, ISNN 1758-4469 Valladares, F.; Arrieta, S.; Aranda, I.; Lorenzo, D.; Sánchez-Gómez, D.; Tena, D.; Suárez, F. &

Wyka, T.; Robakowski, P. & Zytkowiak, R. (2007). Acclimation of leaves to contrasting

Yano, S. & I. Terashima. 2004. Developmental process of sun and shade leaves in

Vol.25, No.8 (August), pp. 1041-1052, ISSN 1758-4469

(September), pp. 1293-1306, ISSN 1758-4469

(December), pp. 1552-1567, ISSN 1365-3040

Vol.18, No.2 (March), pp. 167-174, ISSN1432-2285

(May), pp. 1251–126, ISSN

512, ISSN 1365-3040

927, ISSN 1365-3040

pp. 35-47, ISNN 0100-8404.

ISSN 1460-2431

ISSN 1365-3040

evergreen broad-leaved species. *Plant Cell and Environment*, Vol.28, No.12

function to protect plants from oxidative damage. *Plant Physiology*, Vol.147, No.3

change in leaf anatomy? *Plant Cell and Environment*, Vol.26, No.4 (April), pp. 505-

acclimation: differential responses in leaf anatomy to increasing growth irradiance among three deciduos trees. *Plant Cell and Environment*, Vol.28, No.7 (July), pp. 916-

photosynthesis and leaf anatomy of the mangrove, *Bruguiera parviflora*. *Trees*,

in the resurrection plant *Xerophyta viscosa* (Baker): both sucrose and rafnose family oligosaccharides (RFOs) accumulate in leaves in response to water decit. *Journal of* 

(2009). Thermal energy dissipation and its components in two developmental stages of shade-tolerants species, *Nothofagus nitida*, and a shade-intolerant species, *Nothofagus dombeyi*. *Tree Physiology*, Vol.29, No.5 (May), pp. 651-662, ISSN 1758-4469

differing in sucessional status. *Revista Brasileira de Botânica*, Vol.26, No.1 (March),

comparative eco-development of sun and shade leaves in relation to photosynthetic CO2 diffusion. *Journal of Experimental Botany*, Vol.57, No.2, (January), pp. 343–354,

photosynthesis and importance of limitations during the induction phase in sun and shade leaves of five ecologically contrasting tree species from the temperate

Pardos, J.A. (2005). Shade tolerance, photoinhibition sensitivity and phenotypic plasticity of *Ilex aquifolium* in continental Mediterranean sites. *Tree Physiology*,

irradiance in juvenile trees differing in shade tolerance. *Tree Physiology*, Vol.27, No.9

*Chenopidium album* L. *Plant, Cell and Environment*, Vol.27, No.6 (June), pp. 7181-793,

Photosynthesis light reactions are among the more fast, complex and important processes in the ecosystem. They take place in specific membranes the so-called thylakoids and they produce O2, energy (ATP) and reducing equivalents (NADPH). In this chapter we will discuss recent findings that shed light in important aspects of thylakoid architecture and functional organization. Key role for the remodeling of thylakoids plays a plastidal transglutaminase that was recently cloned from maize. Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines. Transglutaminase (TGase) activity is present in chloroplasts of higher plants being PSII antenna proteins the enzyme's natural substrates. Although the functionality of this plastidial enzyme is not clear, a role in antenna regulation has been hypothesized. The isolation, for the first time in plants, of two related complementary maize DNA clones, *tgz15*  and *tgz21,* encoding active maize (*Zea mays L*) chloroplastic TGase (chlTGZ) has contributed to deep on the role of this enzyme in plants (Torné et al. 2002; Villalobos et al 2004). In addition, the main polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm) are normally produced and oxidized in chloroplasts. Thus, all types of post-translational modifications (i.e. mono-Put, mono-Spd, mono-Spm, bis-Put, bis-Spd and bis-Spm) are in theory probable for the target proteins. These modifications may alter charge and/or conformation of the target protein as well as their linking with other proteins (Kotzabasis et al. 1993; Del Duca et al. 1994; Della Mea et al. 2004).

A strong tool for a deeper study of gene functionality is the effect of its over-expression in an heterologous plant system. Here we will discuss in detail the information about the recent chlTGZ over-expression in tobacco (*Nicotiana tabacum* var. Petit Havana) chloroplasts (Ioannidis et al. 2009) and its characterization. After chloroplast transformation, transglutaminase activity in TGZ-over-expressers was up-regulated 4-fold with respect to the wild-type plants, which in turn rised its thylakoid-associated polyamine content about 90%. A major increase in the granum size (i.e. increase in the number of stacked layers)

Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids 521

this effect plays the stromal surface of the LHCII trimer which is mainly flat and negatively charged as demonstrated by recent structural studies in higher plants (Standfuss et al. 2005). This complex collects excitation energy and transfers it to the reaction centres of PSII and PSI (van Amerongen & Dekker 2003). Also, LHCII prevents damage to the photosynthetic system by several different mechanisms when there is too much light. Potentially harmful chlorophyll (Chl) triplets are quenched by carotenoids in the complex while a special mechanism, referred to as nonphotochemical quenching (NPQ), has evolved in plants to

The interplay between grana and stroma lamellae regions is of exceptional importance because it defines the available space for photosystems and the other supercomplexes of the photosynthetic apparatus such as ATPase. It is well established that "sun" and "shade" plants show distinct differences in the organization of their thylakoid system (Staehelin & van der Staay 1996). In turn, this affect the efficiency with which light is harvested and

With respect to thylakoid membrane biogenesis, Wang et al. 2004 showed that the *Thf1* gene product played a crucial role in a dynamic process of vesicle-mediated thylakoid membrane biogenesis in *Arabidopsis.* Recently, Chi et al. 2008 have reported that a rice thioredoxin *m* isoform (*Ostrxm)* seems to be required for chloroplast biogenesis and differentiation. However, the factors that determine grana formation are not yet fully

Transglutaminases (TGases) are intracellular and extra cellular enzymes that catalyse post-translational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines (Lorand & Graham 2003). However, the role of TGases in chloroplast is not fully understood yet. Maize (*Zea mays L.)* TGase was immunodetected in meristematic calli and their isolated chloroplasts, as a unique 58 kDa band. The activity was shown to be light sensitive, affected by hormone deprivation and with a light/dark rhythm (Bernet 1997; Bernet et al 1999). Subcellular localization studies showed that, in adult plants, the enzyme was specifically localized in the chloroplast grana-appressed thylakoids and close to LHCII and its abundance depended on the degree of grana development (Villalobos et al 2001; Villalobos 2007; Santos et al. 2007). An important step for the elucidation of the plastidal TGase role in plants was the isolation for the first time in plants of two related complementary maize DNA clones, *tgz15* and *tgz21,* encoding active maize TGase (Torné et al. 2002; Villalobos et al. 2004). Interestingly, their expression is dependent on the duration of light exposure, indicating a role for adaptation in different light environmental conditions including natural habitats (Pintó-Marijuan et al 2007; Carvajal et al. 2007-2011). Proteomic studies indicates that plastidial maize TGase is a peripheral thylakoid protein forming part of a specific PSII protein complex which includes LHCII, ATPase and PsbS proteins, its expression pattern changing according to chloroplast developmental stage and light regime (Campos et al. 2010). Tacking into account all the described results, it has been hypothesized that TGases are implicated in the photosynthetic process (Villalobos et al 2004; Pintó-Marijuan et al

dissipate excess energy as heat (Pascal et al. 2005).

**3. Transglutaminases and polyamines** 

2007; Serafini-Fracassini & Del Duca, 2008).

utilized.

understood.

accompanied by a concomitant decrease of stroma thylakoids in the TGase overexpressers was observed. Functional comparison between wild type tobacco and chlTGZ over-expressers was according to these observations, and illustrated in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both *in vivo* probing and extensive electron microscopy studies indicated thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIα) at the expense of centers located mainly in stroma thylakoids (PSIIβ). Finally, late stages of plant development present alterations in the photosynthetic apparatus, chloroplast ultrastructure, and, particularly, oxidative and antioxidative metabolism pathways are induced (Ortigosa et al 2010). At the same time, the over-expressed TGZ protein, accumulated progressively in chloroplast inclusion bodies (Villar-Piqué et al. 2010). These results are discussed in line with chlTGZ involvement in chloroplast functionality.

#### **2. Thylakoids and photosynthesis**

The chloroplasts of higher plants are bounded by two envelope membranes that surround an aqueous matrix, the stroma, and the internal photosynthetic membranes, the thylakoids (Staehelin & van der Staay 1996). Chloroplasts have an apparently periodic ultrastructure: cylindrical grana stacks of about 10–20 layers with a diameter of 300–600 nm, interconnected by lamellae of several hundred nm in length (Mustardy & Garab 2003). Although our understanding regarding architecture of thylakoids is advanced, many issues such as self-assembly and structural flexibility, still remain to be explored (Mustardy & Garab 2003).

The two photosystems are spatially separated in thylakoids in vivo : photosystem II and its main chlorophyll a/b light-harvesting complex, (LHCII), are found predominantly in the stacked membranes; this region is largely deficient in photosystem I (PSI), LHCI and ATPase, which are enriched in the stroma membranes (Andersson & Andersson 1980). Separation of the two pigment systems is probably important in preventing unregulated excitation energy flow between the two photosystems (Andersson & Andersson 1988). Without this, PSI, which is much faster than PSII, would disturb the balance of the energy distribution between the two photosystems (Trissl & Wilhelm 1993). Also PSII exhibits a heterogeneity in terms of antenna size with centers of large chlorophyll antenna size termed PSIIα (occur in grana) and of smaller antenna termed PSIIβ (occur in stroma lamellae) (Melis & Homann 1976; Melis 1989; Kirschhoff et al 2007; Kaftan et al. 1999).

The abundance of LHCII in the granum suggests that these antenna complexes also play a structural role. Indeed, LHCII has been shown to stabilize the granum ultrastructure, and to participate in the cation-mediated stacking of the membranes (Staehelin & van der Staay 1996; Kirchhoff et al. 2007; Arnzten 1978; Duniec et al 1981; Barber 1982). These lightharvesting complexes have also been shown to be involved, via electrostatic and osmotic forces, in the lateral organization of the membranes (Garab et al. 1991). Previous studies showed that the strength of stacking is affected by the phosphorylation of LHCII and of several other phosphoproteins (Allen et al. 1981). LHCII is largely responsible for the organization of the plant photosynthetic system by maintaining the tight appression of thylakoid membranes in chloroplast grana (Allen & Forsberg 2001). An important role for

accompanied by a concomitant decrease of stroma thylakoids in the TGase overexpressers was observed. Functional comparison between wild type tobacco and chlTGZ over-expressers was according to these observations, and illustrated in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both *in vivo* probing and extensive electron microscopy studies indicated thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIα) at the expense of centers located mainly in stroma thylakoids (PSIIβ). Finally, late stages of plant development present alterations in the photosynthetic apparatus, chloroplast ultrastructure, and, particularly, oxidative and antioxidative metabolism pathways are induced (Ortigosa et al 2010). At the same time, the over-expressed TGZ protein, accumulated progressively in chloroplast inclusion bodies (Villar-Piqué et al. 2010). These results are discussed in line with chlTGZ

The chloroplasts of higher plants are bounded by two envelope membranes that surround an aqueous matrix, the stroma, and the internal photosynthetic membranes, the thylakoids (Staehelin & van der Staay 1996). Chloroplasts have an apparently periodic ultrastructure: cylindrical grana stacks of about 10–20 layers with a diameter of 300–600 nm, interconnected by lamellae of several hundred nm in length (Mustardy & Garab 2003). Although our understanding regarding architecture of thylakoids is advanced, many issues such as self-assembly and structural flexibility, still remain to be explored

The two photosystems are spatially separated in thylakoids in vivo : photosystem II and its main chlorophyll a/b light-harvesting complex, (LHCII), are found predominantly in the stacked membranes; this region is largely deficient in photosystem I (PSI), LHCI and ATPase, which are enriched in the stroma membranes (Andersson & Andersson 1980). Separation of the two pigment systems is probably important in preventing unregulated excitation energy flow between the two photosystems (Andersson & Andersson 1988). Without this, PSI, which is much faster than PSII, would disturb the balance of the energy distribution between the two photosystems (Trissl & Wilhelm 1993). Also PSII exhibits a heterogeneity in terms of antenna size with centers of large chlorophyll antenna size termed PSIIα (occur in grana) and of smaller antenna termed PSIIβ (occur in stroma lamellae) (Melis

The abundance of LHCII in the granum suggests that these antenna complexes also play a structural role. Indeed, LHCII has been shown to stabilize the granum ultrastructure, and to participate in the cation-mediated stacking of the membranes (Staehelin & van der Staay 1996; Kirchhoff et al. 2007; Arnzten 1978; Duniec et al 1981; Barber 1982). These lightharvesting complexes have also been shown to be involved, via electrostatic and osmotic forces, in the lateral organization of the membranes (Garab et al. 1991). Previous studies showed that the strength of stacking is affected by the phosphorylation of LHCII and of several other phosphoproteins (Allen et al. 1981). LHCII is largely responsible for the organization of the plant photosynthetic system by maintaining the tight appression of thylakoid membranes in chloroplast grana (Allen & Forsberg 2001). An important role for

& Homann 1976; Melis 1989; Kirschhoff et al 2007; Kaftan et al. 1999).

involvement in chloroplast functionality.

**2. Thylakoids and photosynthesis** 

(Mustardy & Garab 2003).

this effect plays the stromal surface of the LHCII trimer which is mainly flat and negatively charged as demonstrated by recent structural studies in higher plants (Standfuss et al. 2005). This complex collects excitation energy and transfers it to the reaction centres of PSII and PSI (van Amerongen & Dekker 2003). Also, LHCII prevents damage to the photosynthetic system by several different mechanisms when there is too much light. Potentially harmful chlorophyll (Chl) triplets are quenched by carotenoids in the complex while a special mechanism, referred to as nonphotochemical quenching (NPQ), has evolved in plants to dissipate excess energy as heat (Pascal et al. 2005).

The interplay between grana and stroma lamellae regions is of exceptional importance because it defines the available space for photosystems and the other supercomplexes of the photosynthetic apparatus such as ATPase. It is well established that "sun" and "shade" plants show distinct differences in the organization of their thylakoid system (Staehelin & van der Staay 1996). In turn, this affect the efficiency with which light is harvested and utilized.

With respect to thylakoid membrane biogenesis, Wang et al. 2004 showed that the *Thf1* gene product played a crucial role in a dynamic process of vesicle-mediated thylakoid membrane biogenesis in *Arabidopsis.* Recently, Chi et al. 2008 have reported that a rice thioredoxin *m* isoform (*Ostrxm)* seems to be required for chloroplast biogenesis and differentiation. However, the factors that determine grana formation are not yet fully understood.

#### **3. Transglutaminases and polyamines**

Transglutaminases (TGases) are intracellular and extra cellular enzymes that catalyse post-translational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines (Lorand & Graham 2003). However, the role of TGases in chloroplast is not fully understood yet. Maize (*Zea mays L.)* TGase was immunodetected in meristematic calli and their isolated chloroplasts, as a unique 58 kDa band. The activity was shown to be light sensitive, affected by hormone deprivation and with a light/dark rhythm (Bernet 1997; Bernet et al 1999). Subcellular localization studies showed that, in adult plants, the enzyme was specifically localized in the chloroplast grana-appressed thylakoids and close to LHCII and its abundance depended on the degree of grana development (Villalobos et al 2001; Villalobos 2007; Santos et al. 2007). An important step for the elucidation of the plastidal TGase role in plants was the isolation for the first time in plants of two related complementary maize DNA clones, *tgz15* and *tgz21,* encoding active maize TGase (Torné et al. 2002; Villalobos et al. 2004). Interestingly, their expression is dependent on the duration of light exposure, indicating a role for adaptation in different light environmental conditions including natural habitats (Pintó-Marijuan et al 2007; Carvajal et al. 2007-2011). Proteomic studies indicates that plastidial maize TGase is a peripheral thylakoid protein forming part of a specific PSII protein complex which includes LHCII, ATPase and PsbS proteins, its expression pattern changing according to chloroplast developmental stage and light regime (Campos et al. 2010). Tacking into account all the described results, it has been hypothesized that TGases are implicated in the photosynthetic process (Villalobos et al 2004; Pintó-Marijuan et al 2007; Serafini-Fracassini & Del Duca, 2008).

Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids 523

Fig. 1. Schematic representation of tobacco plastid genome transformation using the maize transglutaminase *tgz* gene. A, map of the wild-type and *tgz-*transformed genomes. Regions for homologous recombination are underlined in the native chloroplast genome; B, the 0.81 kb fragment (P1) of the targeting region for homologous recombination and the 0.95 kb *tgz*  sequence (P2) were used as probes for Southern blot analysis; C, D, Southern blot analysis of five independent transgenic lines is shown. Blots were probed with P1 (C) and P2 (D). ORF131, trnV, 16S rRNA, trnI, trnA, 23S rRNA: original sequences of the chloroplast genome; aadA: aminoglycoside 3´- adenylyltransferase; Prrn: 16S rRNA promoter; PpsbA:

psbA promoter; TpsbA: terminator region of the psbA gene; WT: wild-type plant.

(PG) and wild-type tobacco (WT). From Ioannidis et al. 2009.

Phenotype of typical leaves used for this study from plants of TGZ-transplastomic tobacco

A rather overlooked post-translational modification of LHCII that might be important for stacking of thylakoids is its polyaminylation. Polyamines (PAs) are low molecular weight aliphatic amines that are almost fully protonated under normal pH values and thus possess a net charge of up to +4. The main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) are normally found in the LHCII of higher plants (Kotzabasis et al. 1993a). Plastidal Transglutaminases might attach covalently polyamines of all thylakoid proteins specifically in LHCII, CP29, CP26 and CP24 (Del Duca et al 1994). Recently, it was demonstrated that a plastidial TGase activity in maize polyaminylates purified LHCII catalyzing the production of mono and bis glutamyl PAs in a light dependent way (Della Mea et al 2004). As commented elsewhere, authors indicated that the additional positive charges inserted on proteins by the protein-bound PAs might induce conformational changes by conjugation of the two terminal amino-groups of PAs to one or two glutamine residues of LHCII and they discussed if light sensitivity is due to the enzyme or to the substrate. In the work of Carvajal et al. (2011), when purified plastidial maize TGase (TGZ) was added to maize thylakoid protein extracts,TGase activity was significantly higher (in a light dependent manner) than that of the same extract without TGZ addition, indicating that thylakoid proteins are the specific substrate of TGZ. However, in the same work it is demonstrated that, if a non-plant protein was used as TGZ substrate, TGase activity was not light-dependent. These last results indicate that light dependence of plastidial TGase activity is probably related to its specific substrate (thylakoid proteins) and not to the enzyme itself.

First evidence for a role of plastidial TGase in the thylakoids 3D architecture comes from tobacco chloroplasts over expressing maize TGase (TGZ) (Ioannidis et al 2009). In that work, we hypothesized that TGase is implicated in the ratio regulation of grana to stroma thylakoids (Villalobos et al. 2004). A combination of genetic engineering and *in vivo* probing approach was used to test this hypothesis. Here we discuss in detail the information about the effect of maize *tgz* gene over-expression and its characterization in tobacco chloroplasts via plastid transformation, where the transgene is integrated in the plastid genome by homologous recombination (Maliga 2004; Fernández-San Millán et al 2007, 2008).

#### **4. Maize transglutaminase over-expressed in tobacco chloroplasts**

#### **4.1 Vector construction, chloroplast transformation and plant regeneration**

To introduce the *tgz13* gene into tobacco Wt chloroplasts, the *tgz* gene was PCR amplified, fused to the promoter and 5'untranslated region of the *psbA* gene and finally introduced into the multiple cloning site of the pAF vector, rendering the final vector, pAF*-tgz13* (Fig. 1A). The pAF vector was specifically constructed for tobacco plastid transformation and includes the *trnI* and *trnA* border sequences, homologous to the inverted repeat regions of the tobacco plastid genome (Fernández-San Millán et al 2008). The regulatory sequences of the *psbA* gene were chosen due to the high levels of heterologous gene expression they confer in transplastomic plants (Fernández-San Millán et al 2003; Molina et al 2004). After that, leaves of tobacco Wt plants were bombarded with gold microprojectils coated with plasmid DNA containing the *tgz* gene and plants were regenerated in the selective spectinomycin medium. Southern blot analysis performed on shoots developed after the second round of selection with spectinomycin revealed some plants that were homoplasmic for the *tgz* gene (Fig. 1) (Ioannidis et al. 2009). These experiments were carried out in J. Veramendi laboratory (Public Univ. Navarra. Spain).

A rather overlooked post-translational modification of LHCII that might be important for stacking of thylakoids is its polyaminylation. Polyamines (PAs) are low molecular weight aliphatic amines that are almost fully protonated under normal pH values and thus possess a net charge of up to +4. The main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) are normally found in the LHCII of higher plants (Kotzabasis et al. 1993a). Plastidal Transglutaminases might attach covalently polyamines of all thylakoid proteins specifically in LHCII, CP29, CP26 and CP24 (Del Duca et al 1994). Recently, it was demonstrated that a plastidial TGase activity in maize polyaminylates purified LHCII catalyzing the production of mono and bis glutamyl PAs in a light dependent way (Della Mea et al 2004). As commented elsewhere, authors indicated that the additional positive charges inserted on proteins by the protein-bound PAs might induce conformational changes by conjugation of the two terminal amino-groups of PAs to one or two glutamine residues of LHCII and they discussed if light sensitivity is due to the enzyme or to the substrate. In the work of Carvajal et al. (2011), when purified plastidial maize TGase (TGZ) was added to maize thylakoid protein extracts,TGase activity was significantly higher (in a light dependent manner) than that of the same extract without TGZ addition, indicating that thylakoid proteins are the specific substrate of TGZ. However, in the same work it is demonstrated that, if a non-plant protein was used as TGZ substrate, TGase activity was not light-dependent. These last results indicate that light dependence of plastidial TGase activity is probably related to its specific substrate (thylakoid proteins)

First evidence for a role of plastidial TGase in the thylakoids 3D architecture comes from tobacco chloroplasts over expressing maize TGase (TGZ) (Ioannidis et al 2009). In that work, we hypothesized that TGase is implicated in the ratio regulation of grana to stroma thylakoids (Villalobos et al. 2004). A combination of genetic engineering and *in vivo* probing approach was used to test this hypothesis. Here we discuss in detail the information about the effect of maize *tgz* gene over-expression and its characterization in tobacco chloroplasts via plastid transformation, where the transgene is integrated in the plastid genome by

To introduce the *tgz13* gene into tobacco Wt chloroplasts, the *tgz* gene was PCR amplified, fused to the promoter and 5'untranslated region of the *psbA* gene and finally introduced into the multiple cloning site of the pAF vector, rendering the final vector, pAF*-tgz13* (Fig. 1A). The pAF vector was specifically constructed for tobacco plastid transformation and includes the *trnI* and *trnA* border sequences, homologous to the inverted repeat regions of the tobacco plastid genome (Fernández-San Millán et al 2008). The regulatory sequences of the *psbA* gene were chosen due to the high levels of heterologous gene expression they confer in transplastomic plants (Fernández-San Millán et al 2003; Molina et al 2004). After that, leaves of tobacco Wt plants were bombarded with gold microprojectils coated with plasmid DNA containing the *tgz* gene and plants were regenerated in the selective spectinomycin medium. Southern blot analysis performed on shoots developed after the second round of selection with spectinomycin revealed some plants that were homoplasmic for the *tgz* gene (Fig. 1) (Ioannidis et al. 2009). These experiments were carried out in J. Veramendi laboratory

homologous recombination (Maliga 2004; Fernández-San Millán et al 2007, 2008).

**4. Maize transglutaminase over-expressed in tobacco chloroplasts 4.1 Vector construction, chloroplast transformation and plant regeneration** 

and not to the enzyme itself.

(Public Univ. Navarra. Spain).

Fig. 1. Schematic representation of tobacco plastid genome transformation using the maize transglutaminase *tgz* gene. A, map of the wild-type and *tgz-*transformed genomes. Regions for homologous recombination are underlined in the native chloroplast genome; B, the 0.81 kb fragment (P1) of the targeting region for homologous recombination and the 0.95 kb *tgz*  sequence (P2) were used as probes for Southern blot analysis; C, D, Southern blot analysis of five independent transgenic lines is shown. Blots were probed with P1 (C) and P2 (D). ORF131, trnV, 16S rRNA, trnI, trnA, 23S rRNA: original sequences of the chloroplast genome; aadA: aminoglycoside 3´- adenylyltransferase; Prrn: 16S rRNA promoter; PpsbA: psbA promoter; TpsbA: terminator region of the psbA gene; WT: wild-type plant. Phenotype of typical leaves used for this study from plants of TGZ-transplastomic tobacco (PG) and wild-type tobacco (WT). From Ioannidis et al. 2009.

Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids 525

Fig. 3. Ultrastructure of chloroplasts from tobacco Wt and over-expressing TGZ.

2009.

**4.4 Fluorescence induction kinetics** 

values of about 0.81 (Table 1).

Transformed tobacco (B, D) shows an increased stacking of thylakoids and a reduced stroma thylakoid network. Large grana appear with a size many hundreds nm bigger than Wt plants (A, C). G= grana; NT= non-appressed thylakoids; p= plastoglobuli; pbl= prolamellar body lattice. Arrow in C= 0.35 µm approx.; arrow in D= 0.9 µm approx. From Ioannidis et al.

Over-expression of maize *tgz* in tobacco has a small effect (about 13% decrease) in the structure and functionality of PSII, as judged by the FV/FM values. Maximum quantum efficiency of PSII in the transformants is about 0.7, whereas Wt tobacco exhibits optimal

#### **4.2 Transglutaminase activity and thylakoid associated polyamines**

The TGase activity in tobacco leaves over-expressing maize *TGZ* was nearly four times higher than that of the Wt plants (Table 1). This result was corroborated by the presence of TGZ protein in the over-expressers detected by western blot and analyzed by mass spectrometry (data not presented). By a sensitive HPLC method we have estimated the amount of associated polyamines in thylakoids (Kotzabasis et al 1993b). Plants overexpressing TGZ showed a total increase of 90% in the titer of thylakoid associated polyamines (Put, Spd and Spm) on a Chl basis (Fig. 2). Bound Put was increased about 3 times and the higher polyamines about 60% in comparison to the Wt.

Fig. 2. Thylakoid associated polyamines of *Nicotiana tabacum* Wt and tobacco overexpressing *tgz* from maize. Data are presented on a chlorophyll basis because protein titer was substantially higher in transformed tobacco due to the over-expression of *tgz*. Vertical bars denote standard deviation (n=3). From Ioannidis et al. 2009.

#### **4.3 Thylakoid ultrastructure and pigment content**

Transmission electronic microscopy revealed important differences between Wt and TGZ over-expressing chloroplasts. Wt chloroplasts exhibit a normal thylakoid network architecture (Fig. 3, A and C), exhibiting grana and stroma lamellae in a normal proportion. Over\_expression of *tgz* resulted in a severe depletion of chloroplast stroma lamellae and, interestingly, a grana dominance (Fig. 3, B and D), the granum size (number of stacked layers) being increased up to nearly 1000 nm, the double that of the Wt granum size (Table 1 and Fig. 3D). Furthermore, a reduction in the total Chl content was evident from 1.86 (mg·g-1FW) in Wt to 0.6 (mg·g-1FW) in over-*tgz* with a parallel decrease of the Chla/Chlb ratio (Table 1). The total carotenoid titer was also reduced (Table 1). In fact, as commented in the next paragraph, at later stages of development TGZ-plants are severely chlorotic (Ortigosa et al. 2010).

The TGase activity in tobacco leaves over-expressing maize *TGZ* was nearly four times higher than that of the Wt plants (Table 1). This result was corroborated by the presence of TGZ protein in the over-expressers detected by western blot and analyzed by mass spectrometry (data not presented). By a sensitive HPLC method we have estimated the amount of associated polyamines in thylakoids (Kotzabasis et al 1993b). Plants overexpressing TGZ showed a total increase of 90% in the titer of thylakoid associated polyamines (Put, Spd and Spm) on a Chl basis (Fig. 2). Bound Put was increased about 3

**4.2 Transglutaminase activity and thylakoid associated polyamines** 

times and the higher polyamines about 60% in comparison to the Wt.

Fig. 2. Thylakoid associated polyamines of *Nicotiana tabacum* Wt and tobacco over-

bars denote standard deviation (n=3). From Ioannidis et al. 2009.

**4.3 Thylakoid ultrastructure and pigment content** 

et al. 2010).

expressing *tgz* from maize. Data are presented on a chlorophyll basis because protein titer was substantially higher in transformed tobacco due to the over-expression of *tgz*. Vertical

Transmission electronic microscopy revealed important differences between Wt and TGZ over-expressing chloroplasts. Wt chloroplasts exhibit a normal thylakoid network architecture (Fig. 3, A and C), exhibiting grana and stroma lamellae in a normal proportion. Over\_expression of *tgz* resulted in a severe depletion of chloroplast stroma lamellae and, interestingly, a grana dominance (Fig. 3, B and D), the granum size (number of stacked layers) being increased up to nearly 1000 nm, the double that of the Wt granum size (Table 1 and Fig. 3D). Furthermore, a reduction in the total Chl content was evident from 1.86 (mg·g-1FW) in Wt to 0.6 (mg·g-1FW) in over-*tgz* with a parallel decrease of the Chla/Chlb ratio (Table 1). The total carotenoid titer was also reduced (Table 1). In fact, as commented in the next paragraph, at later stages of development TGZ-plants are severely chlorotic (Ortigosa

Fig. 3. Ultrastructure of chloroplasts from tobacco Wt and over-expressing TGZ. Transformed tobacco (B, D) shows an increased stacking of thylakoids and a reduced stroma thylakoid network. Large grana appear with a size many hundreds nm bigger than Wt plants (A, C). G= grana; NT= non-appressed thylakoids; p= plastoglobuli; pbl= prolamellar body lattice. Arrow in C= 0.35 µm approx.; arrow in D= 0.9 µm approx. From Ioannidis et al. 2009.

#### **4.4 Fluorescence induction kinetics**

Over-expression of maize *tgz* in tobacco has a small effect (about 13% decrease) in the structure and functionality of PSII, as judged by the FV/FM values. Maximum quantum efficiency of PSII in the transformants is about 0.7, whereas Wt tobacco exhibits optimal values of about 0.81 (Table 1).

Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids 527

More pronounced differences appear at later stages of development. A detailed transient kinetics of fluorescence induction shows that there is a major difference both in the shape and in the amplitude between Wt and over-TGZ tobacco plants (Fig. 4). The maximal difference in FV during fluorescence induction is at 10 ms (about 70% higher values for Wt in comparison to the transformed) and there are also large differences in the FM values (about 50% higher for the wild type). The effective PSII antenna size increased also in the overexpressers as indicated by the shortest closure time of their reaction centers (see Table 1 parameter t1/2DCMU ) in comparison to that of the Wt. The value of the energy-dependent component of the non-photochemical quenching (qE) in the case of the transformed tobacco

The results obtained with later stages of leaf development revealed that photochemistry impairment and oxidative stress increased with transplastomic leaf age. These alterations included decrease in pigment levels, changes in the photosynthetic apparatus, in the

Fig. 5. Ultrastructure of tobacco chloroplasts over-expressing TGZ and TGZ

t, thylakoids. From Ortigosa et al. Planta 2010

immunolocalization. **a** A dividing chloroplast, showing increased grana appression and a reduced stroma thylakoid network. **b** Two oldest-leaf-chloroplasts containing large inclusion bodies. In the upper chloroplast an over-appressed granum is still visible. In the lower chloroplast the thylakoid network is disorganized. IBs larger than 1 µm are usually present. **c** Subcellular immunolocalization of TGZ-protein in the IB, using an anti-TGZ4 antibody. IB, inclusion body; G, grana; m, mitochondria; p, plastoglobuli; SG, starch grains;

is about 6 times higher than that of the Wt (Table 1).

**4.5 Oxidative stress symptoms and leaf aging** 


\*measured from 50 chloroplasts

Table 1**.** Comparison of fluorescence parameters, pigment content, maximum granum size and transglutaminase activity in *Nicotiana tabacum* Wt and overexpressing *tgz* (over TGZ and oldest TGZ) leaves. Numbers in parenthesis denote standard deviation (n=3). Transformed tobacco values (right column) were statistically different in comparison to the Wt values (left column) at p<0.05. From Ioannidis et al. 2009.

Fig. 4. Fluorescence induction curves of *Nicotiana tabacum* Wt and tobacco over-expressing *tgz* from maize (TGZ). Samples were dark adapted for 20 min and were illuminated with 3000 μmol [photons] m-2s-1. The time axis is semi-logarithmic for clarity and data are normalized to Fo.

Table 1**.** Comparison of fluorescence parameters, pigment content, maximum granum size and transglutaminase activity in *Nicotiana tabacum* Wt and overexpressing *tgz* (over TGZ and oldest TGZ) leaves. Numbers in parenthesis denote standard deviation (n=3).

Transformed tobacco values (right column) were statistically different in comparison to the

Fig. 4. Fluorescence induction curves of *Nicotiana tabacum* Wt and tobacco over-expressing *tgz* from maize (TGZ). Samples were dark adapted for 20 min and were illuminated with 3000 μmol [photons] m-2s-1. The time axis is semi-logarithmic for clarity and data are

Wt values (left column) at p<0.05. From Ioannidis et al. 2009.

FV/FM 0.812 (0.031) 0.702 (0.070) 0,122 (0.02) qE 0.16 (0.02) 1.008 (0.08) -ND t1/2DCMU (ms) 166 (9) 110 (12) -ND Chl a (mg·g-1FW) 1.38 (0.11) 0.42 (0.08) 0.1 (0.01) Chl b (mg·g-1FW) 0.48 (0.04) 0.18 (0.05) 0.06 (0.01). Total Chls (mg·g-1FW) 1.86 (0.15) 0.60 (0.13) 0.16 (0.02) Chla/Chlb 2.87 (0.06) 2.33 (0.17) 1.73 (0.25) Carotenoids (mg·g-1FW) 0.29 (0.02) 0.13 (0.02) 0.03 Maximum granum size (nm) 400 (\*) 1000 (\*) ----

Transglutaminase activity pmol Put ·mg protein·h-1

\*measured from 50 chloroplasts

normalized to Fo.

**Wt tobacco over TGZ Oldest over TGZ** 

758.9 (89.2) 3067.3 (661) 1636.6 (172.4)

More pronounced differences appear at later stages of development. A detailed transient kinetics of fluorescence induction shows that there is a major difference both in the shape and in the amplitude between Wt and over-TGZ tobacco plants (Fig. 4). The maximal difference in FV during fluorescence induction is at 10 ms (about 70% higher values for Wt in comparison to the transformed) and there are also large differences in the FM values (about 50% higher for the wild type). The effective PSII antenna size increased also in the overexpressers as indicated by the shortest closure time of their reaction centers (see Table 1 parameter t1/2DCMU ) in comparison to that of the Wt. The value of the energy-dependent component of the non-photochemical quenching (qE) in the case of the transformed tobacco is about 6 times higher than that of the Wt (Table 1).

#### **4.5 Oxidative stress symptoms and leaf aging**

The results obtained with later stages of leaf development revealed that photochemistry impairment and oxidative stress increased with transplastomic leaf age. These alterations included decrease in pigment levels, changes in the photosynthetic apparatus, in the

Fig. 5. Ultrastructure of tobacco chloroplasts over-expressing TGZ and TGZ immunolocalization. **a** A dividing chloroplast, showing increased grana appression and a reduced stroma thylakoid network. **b** Two oldest-leaf-chloroplasts containing large inclusion bodies. In the upper chloroplast an over-appressed granum is still visible. In the lower chloroplast the thylakoid network is disorganized. IBs larger than 1 µm are usually present. **c** Subcellular immunolocalization of TGZ-protein in the IB, using an anti-TGZ4 antibody. IB, inclusion body; G, grana; m, mitochondria; p, plastoglobuli; SG, starch grains; t, thylakoids. From Ortigosa et al. Planta 2010

Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids 529

& Melis 1983), we have *in vivo* estimated the poise between PSIIα and PSIIβ centers. The results indicate that PSIIα centers are accumulating in over-TGZ plants and, simultaneously, the number of PSIIβ centers is declining. PSIIα centers are of large antenna size and are considered to occur in grana regions (Melis 1989; Kirchhoff et al. 2007). PSIIβ centers possess a smaller antenna size and are considered to occur in stroma lamellae (Melis 1989; Kirchhoff et al. 2007). As the phenotype of the transformed plants is getting more intense, the portion of PSIIα increases at the expense of PSIIβ centers approaching 100% (Fig. 6). Furthermore, the remarkable increase in PSIIα/PSIIβ ratio indicates diminishing of stroma thylakoids. In order to crosscheck this hypothesis we studied the ultra-thin structure of the chloroplast. Transmission electron microscopy revealed that *tgz* over-expression resulted in an increase of grana stacking and a decrease of stroma lamellae. Remarkably, the size of the granum (number of stacked layers) in TGZ- chloroplasts is up to 1000 nm, whereas in Wt chloroplasts it was not larger than 400 nm. On the ground that in higher plants granum diameter is up to 600 nm (Mustardy and Garab 2003) the over-expression of *tgz* caused a significant and relative uncommon

Fig. 6. The comparison of PSII antenna heterogeneity of *Nicotiana tabacum* Wt and tobacco overexpressing *tgz* from maize, as estimated by the fraction of PSIIα (white) and PSIIβ (black). Relative amount of PSIIα and PSIIβ for early and later stages of development of

TGZ-transplastomic tobacco. From Ioannidis et al. 2009.

increase in granum size.

chloroplast ultrastructure, and, particularly, the activation of oxidative and antioxidative metabolism pathways (see Tables 2 and 3). At the same time, the over-expressed TGZ protein accumulated progressively in chloroplast inclusion bodies. These traits were accompanied by thylakoid scattering, membrane degradation and reduction of thylakoid interconnections (Fig. 5). Consequently, the electron transport between photosystems decrease dramatically in the old leaves. In spite of these alterations, transplastomic plants can be maintained and reproduced *in vitro* (Ortigosa et al. 2010). These experiments were carried out in J. A. Hernandez laboratory (CEBAS-CSIC, Murcia, Spain).

#### **5. Current and future developments**

The over-expression of a heterologous gene could be a valuable tool for the understanding of the corresponding protein functionality. As mentioned above, the over-expression of TGZ resulted in a 4-fold increase of plastidial TGase activity, causing a significant increase in grana size and about 90% increase in thylakoid-associated polyamines (Ioannidis et al 2009). Interestingly, transformed plants exhibit increased ability to induce NPQ, a small decrease in maximal quantum yield of PSII and about 6 times higher qE, in comparison to the Wt. These results are in line with recent studies showing that elevation of Spd and Spm titers could lead to an increase in NPQ in tobacco (Ioannidis et al. 2007). Also, the effect of TGZ over PSII antenna is showed in the decrease of Chl a/Chl b ratio, which is an indicator for changes in the stoichiometry of the photosystems (in particular their LHCs) (Table 1) and the effective PSII antenna size increase. These results are in line with accumulating data showing that a plastidial TGase activity specifically polyaminylate PSII antenna proteins such as LHCII, CP29, CP26 and CP24 (Del Duca et al. 1994; Della Mea et al 2004). Chl b is found in LHCII and, consequently, a decrease in the Chl a/Chl b ratio suggests an increase in the abundance of LHCs of PSII relative to PSI similar to that suggested for a hyperstacking mutant of *Arabidopsis* (Häussler et al. 2009). Also noteworthy is the fact that OJIP transients indicate an increase in the connectivity of PSII centers in the transformed tobacco. *In vitro* investigations such us microscopy of thylakoids at high resolution hopefully will shed light on this matter in the near future.


Table 2. Effect of TGZ over-expression on H2O2 content (nmol g-1 FW) and lipid peroxidation (TBARS) (nmol g-1 FW) in tobacco plant leaves. Different letters indicate significant differences according to Tukey's test (*P* ≤ 0.05). From Ortigosa et al. 2010.

Regarding the apparent PSII antenna increase, two possible explanations can be hypothesized: either both PSIIα and PSIIβ increase their antenna size or the portion of the large antenna centers (PSIIα) is increased. By using a non destructive method (Andersson

chloroplast ultrastructure, and, particularly, the activation of oxidative and antioxidative metabolism pathways (see Tables 2 and 3). At the same time, the over-expressed TGZ protein accumulated progressively in chloroplast inclusion bodies. These traits were accompanied by thylakoid scattering, membrane degradation and reduction of thylakoid interconnections (Fig. 5). Consequently, the electron transport between photosystems decrease dramatically in the old leaves. In spite of these alterations, transplastomic plants can be maintained and reproduced *in vitro* (Ortigosa et al. 2010). These experiments were

The over-expression of a heterologous gene could be a valuable tool for the understanding of the corresponding protein functionality. As mentioned above, the over-expression of TGZ resulted in a 4-fold increase of plastidial TGase activity, causing a significant increase in grana size and about 90% increase in thylakoid-associated polyamines (Ioannidis et al 2009). Interestingly, transformed plants exhibit increased ability to induce NPQ, a small decrease in maximal quantum yield of PSII and about 6 times higher qE, in comparison to the Wt. These results are in line with recent studies showing that elevation of Spd and Spm titers could lead to an increase in NPQ in tobacco (Ioannidis et al. 2007). Also, the effect of TGZ over PSII antenna is showed in the decrease of Chl a/Chl b ratio, which is an indicator for changes in the stoichiometry of the photosystems (in particular their LHCs) (Table 1) and the effective PSII antenna size increase. These results are in line with accumulating data showing that a plastidial TGase activity specifically polyaminylate PSII antenna proteins such as LHCII, CP29, CP26 and CP24 (Del Duca et al. 1994; Della Mea et al 2004). Chl b is found in LHCII and, consequently, a decrease in the Chl a/Chl b ratio suggests an increase in the abundance of LHCs of PSII relative to PSI similar to that suggested for a hyperstacking mutant of *Arabidopsis* (Häussler et al. 2009). Also noteworthy is the fact that OJIP transients indicate an increase in the connectivity of PSII centers in the transformed tobacco. *In vitro* investigations such us microscopy of thylakoids at high resolution

> **H2O2 nmol g-1 FW**

**Upper leaves** 2.57c 0.108bc **Middle leaves** 2.30 c 0.331b

**PG leaves** 4.37a 1.11a **Y leaves** 3.42 b 0.97a Table 2. Effect of TGZ over-expression on H2O2 content (nmol g-1 FW) and lipid peroxidation

Regarding the apparent PSII antenna increase, two possible explanations can be hypothesized: either both PSIIα and PSIIβ increase their antenna size or the portion of the large antenna centers (PSIIα) is increased. By using a non destructive method (Andersson

(TBARS) (nmol g-1 FW) in tobacco plant leaves. Different letters indicate significant

Transformed Plants

differences according to Tukey's test (*P* ≤ 0.05). From Ortigosa et al. 2010.

**TBARS nmol g-1 FW** 

carried out in J. A. Hernandez laboratory (CEBAS-CSIC, Murcia, Spain).

**5. Current and future developments** 

hopefully will shed light on this matter in the near future.

Wt

& Melis 1983), we have *in vivo* estimated the poise between PSIIα and PSIIβ centers. The results indicate that PSIIα centers are accumulating in over-TGZ plants and, simultaneously, the number of PSIIβ centers is declining. PSIIα centers are of large antenna size and are considered to occur in grana regions (Melis 1989; Kirchhoff et al. 2007). PSIIβ centers possess a smaller antenna size and are considered to occur in stroma lamellae (Melis 1989; Kirchhoff et al. 2007). As the phenotype of the transformed plants is getting more intense, the portion of PSIIα increases at the expense of PSIIβ centers approaching 100% (Fig. 6). Furthermore, the remarkable increase in PSIIα/PSIIβ ratio indicates diminishing of stroma thylakoids. In order to crosscheck this hypothesis we studied the ultra-thin structure of the chloroplast. Transmission electron microscopy revealed that *tgz* over-expression resulted in an increase of grana stacking and a decrease of stroma lamellae. Remarkably, the size of the granum (number of stacked layers) in TGZ- chloroplasts is up to 1000 nm, whereas in Wt chloroplasts it was not larger than 400 nm. On the ground that in higher plants granum diameter is up to 600 nm (Mustardy and Garab 2003) the over-expression of *tgz* caused a significant and relative uncommon increase in granum size.

Fig. 6. The comparison of PSII antenna heterogeneity of *Nicotiana tabacum* Wt and tobacco overexpressing *tgz* from maize, as estimated by the fraction of PSIIα (white) and PSIIβ (black). Relative amount of PSIIα and PSIIβ for early and later stages of development of TGZ-transplastomic tobacco. From Ioannidis et al. 2009.

Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids 531

**Enzymatic activity Upper leaves Middle leaves PG leaves Y leaves** 

**nmol min-1 mg-1 prot** 1053b 961b 1557a 1479a

**nmol min-1 mg-1 prot** 57.98c 60.18c 75.48b 93.62a

**nmol min-1 mg-1 prot** 15.11b 4.74c 26.67a 20.22b

**nmol min-1 mg-1 prot** 54.37a 57.73a 62.83a 57.25a

**µmol min-1 mg-1 prot** 172.1a 178.2a 88.3b 74.9c

**nmol min-1 mg-1 prot** 118.6c 164.2c 423.0b 822.4a

**nmol min-1 mg-1 prot** 13.64c 28.02c 211.6b 330.3a

**nmol min-1 mg-1 prot** 6.17a 6.13a 5.47ab 4.25b

**nmol min-1 mg-1 prot** nd nd nd nd

**nmol min-1 mg-1 prot** 10.55b 6.12c 16.15a 16.74a

**U mg-1 prot** 31.0b 39.7b 57.6a 54.9a

Table 3. Effect of chlTGZ over-expression on antioxidative enzyme activities in tobacco plant leaves. Different letters indicate statistical significance according to Tukey's test (*P* ≤ 0.05);

Thylakoid architecture is a major factor which affects functionality and efficiency of the photosynthetic apparatus. Light conditions in terms of quality and intensity define thylakoid architecture, but the details of the molecular mechanism which is responsible for this regulation is largely unknown (Anderson 1999; Mullineaux 2005). We provide evidence that the remodeling of the grana could be feasible through over-expression of a single enzyme. Therefore, we suggest that *tgz* has an important functional role in the formation of the grana stacks. Moreover TGZ over-expression, due to the enormous and stable granum size, may provide a powerful tool for the study and understanding of grana function that

**APX** 

**MDHAR** 

**DHAR** 

**CATALASE** 

**NADH-POX** 

**GR** 

**POX** 

**GST** 

**GPX** 

**G6PDH** 

nd, not detectable. From Ortigosa et al. 2010.

has long been debated (Mullineaux 2005).

**5.1 Implications of the work** 

**SOD** 

Wt Transformed plants

On the other hand, the reduction in the amount of stroma thylakoids leads to a number of problems regarding the functionality of the photosynthetic apparatus. Stroma lamellae are among others the major site of ATPase and a chloroplast with severely reduced stroma lamellae would not accommodate as many ATPases as Wt. Given that ATPases allow lumen protons to escape in stroma less "proton channels" means higher ΔpH between stroma and lumen during illumination (Kramer et al. 2003). Consistent with this view, the light induced energization of the thylakoid was higher (i.e. higher qE for *tgz*). At this point it should be noted that the high NPQ of the over-expressers is not fully understood at the moment. Perhaps the increased stacking (Goss et al. 2007) or the increased antenna of PSII (Pascal et al. 2005) are also contributing factors to the high NPQ values since total carotenoids in transplastomic plants are less than in Wt. First evidence i.e. the elevated qE (Table 1) and the FM′ value (end of light phase) that is close to F0 value (Fig 4B open triangles Ioannidis et al. 2009 for transformed tobacco, indicate that the lumen-pH induced dissipative conformation of antenna and/or PSII reaction center is more efficiently formed in TGZ than in Wt. A possible interpretation -which still needs experimental verification- on the ground that LHCII, CP29, CP26 and CP24 are normal substrates of the plastidal TGase (Del Duca et al. 1994; Della Mea et al. 2004) and putative sites of qE (Pascal et al. 2005; Kovacs et al 2006 and refs therein) is that they are changing their conformation upon polyaminylation which in turn promotes dissipation. However, further research is on going for the elucidation of this phenomenon. In addition, structural and biochemical changes that appeared only sparsely in early phases (Fig 3D) and progressively appear more frequently in the latest phases of plant development are indicative of oxidative stress (Austin et al 2006; Ortigosa et al. 2010), due to impairment of photochemistry, as indicated also by the decreased FV/FM (Table 1).

On the contrary the underlying causes of increased stacking are better understood. Polyaminylation of proteins result in significant change in the charge of the target protein (Della Mea et al 2004). It is well established that negative charges of chlorophyll binding proteins must be neutralized by positive cations in order adjacent membranes to stack and in turn grana formation to occur (Standfuss et al. 2005). This kind of charge neutralization is feasible with monovalent or divalent inorganic cations (Kirchhoff et al. 2007; Barber 1982) or with organic cations such as polyamines (Ioannidis et al. 2007). Noteworthy, fluorescence transients of tobacco thylakoids indicate that the higher polyamines are much more efficient in stacking than Mg+2 (Ioannidis et al. 2007). Although the later works quantified the coulombic effects of non-covalently bound polyamines it seems that bound polyamines can also cause stacking (Ioannidis et al. 2009). The self-assembly of the thylakoids into grana was suggested to occur upon *in vitro* cation addition, and migration of minor LHCIIs from PSIIβ to PSIIα (Kirchhoff et al. 2007). Our *in vivo* results showing a PSIIβ reduction and a thylakoid-stacking increase in the *tgz*-transformants are in line with this view, Recent electron microscope tomography results and proposed models for the three-dimensional organisation of thylakoids are also in agreement with our results (Shimoni et al 2005). In addition, lower chlorophyll content and lower Chl a/Chl b ratio was also the case for a mutant of Arabidopsis (*adg1-1/tpt-1*) that exhibit increased stacking (Häusler et al. 2009). This phenomenon is also present in our tgz-transformants that presented less chlorophyll content per leaf basis than the Wt and, at later stages of development, this phenomenon is more intense (Ortigosa et al. 2010).

On the other hand, the reduction in the amount of stroma thylakoids leads to a number of problems regarding the functionality of the photosynthetic apparatus. Stroma lamellae are among others the major site of ATPase and a chloroplast with severely reduced stroma lamellae would not accommodate as many ATPases as Wt. Given that ATPases allow lumen protons to escape in stroma less "proton channels" means higher ΔpH between stroma and lumen during illumination (Kramer et al. 2003). Consistent with this view, the light induced energization of the thylakoid was higher (i.e. higher qE for *tgz*). At this point it should be noted that the high NPQ of the over-expressers is not fully understood at the moment. Perhaps the increased stacking (Goss et al. 2007) or the increased antenna of PSII (Pascal et al. 2005) are also contributing factors to the high NPQ values since total carotenoids in transplastomic plants are less than in Wt. First evidence i.e. the elevated qE (Table 1) and the FM′ value (end of light phase) that is close to F0 value (Fig 4B open triangles Ioannidis et al. 2009 for transformed tobacco, indicate that the lumen-pH induced dissipative conformation of antenna and/or PSII reaction center is more efficiently formed in TGZ than in Wt. A possible interpretation -which still needs experimental verification- on the ground that LHCII, CP29, CP26 and CP24 are normal substrates of the plastidal TGase (Del Duca et al. 1994; Della Mea et al. 2004) and putative sites of qE (Pascal et al. 2005; Kovacs et al 2006 and refs therein) is that they are changing their conformation upon polyaminylation which in turn promotes dissipation. However, further research is on going for the elucidation of this phenomenon. In addition, structural and biochemical changes that appeared only sparsely in early phases (Fig 3D) and progressively appear more frequently in the latest phases of plant development are indicative of oxidative stress (Austin et al 2006; Ortigosa et al. 2010), due to impairment of

photochemistry, as indicated also by the decreased FV/FM (Table 1).

more intense (Ortigosa et al. 2010).

On the contrary the underlying causes of increased stacking are better understood. Polyaminylation of proteins result in significant change in the charge of the target protein (Della Mea et al 2004). It is well established that negative charges of chlorophyll binding proteins must be neutralized by positive cations in order adjacent membranes to stack and in turn grana formation to occur (Standfuss et al. 2005). This kind of charge neutralization is feasible with monovalent or divalent inorganic cations (Kirchhoff et al. 2007; Barber 1982) or with organic cations such as polyamines (Ioannidis et al. 2007). Noteworthy, fluorescence transients of tobacco thylakoids indicate that the higher polyamines are much more efficient in stacking than Mg+2 (Ioannidis et al. 2007). Although the later works quantified the coulombic effects of non-covalently bound polyamines it seems that bound polyamines can also cause stacking (Ioannidis et al. 2009). The self-assembly of the thylakoids into grana was suggested to occur upon *in vitro* cation addition, and migration of minor LHCIIs from PSIIβ to PSIIα (Kirchhoff et al. 2007). Our *in vivo* results showing a PSIIβ reduction and a thylakoid-stacking increase in the *tgz*-transformants are in line with this view, Recent electron microscope tomography results and proposed models for the three-dimensional organisation of thylakoids are also in agreement with our results (Shimoni et al 2005). In addition, lower chlorophyll content and lower Chl a/Chl b ratio was also the case for a mutant of Arabidopsis (*adg1-1/tpt-1*) that exhibit increased stacking (Häusler et al. 2009). This phenomenon is also present in our tgz-transformants that presented less chlorophyll content per leaf basis than the Wt and, at later stages of development, this phenomenon is


Table 3. Effect of chlTGZ over-expression on antioxidative enzyme activities in tobacco plant leaves. Different letters indicate statistical significance according to Tukey's test (*P* ≤ 0.05); nd, not detectable. From Ortigosa et al. 2010.

#### **5.1 Implications of the work**

Thylakoid architecture is a major factor which affects functionality and efficiency of the photosynthetic apparatus. Light conditions in terms of quality and intensity define thylakoid architecture, but the details of the molecular mechanism which is responsible for this regulation is largely unknown (Anderson 1999; Mullineaux 2005). We provide evidence that the remodeling of the grana could be feasible through over-expression of a single enzyme. Therefore, we suggest that *tgz* has an important functional role in the formation of the grana stacks. Moreover TGZ over-expression, due to the enormous and stable granum size, may provide a powerful tool for the study and understanding of grana function that has long been debated (Mullineaux 2005).

Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids 533

enzyme and increased granum size more than 100%. PSIIα centers increased, and , concomitantly, stroma thylakoids were depleted. At the same time, thylakoid associated

On the grounds that TGases have LHCbs as a natural substrate it is plausible that polyamines increase in thylakoids were due to LHCII modification. In future works, we will test whether LHCII has a different profile of bound polyamines due to TGZ over-expression. If this is the case (if more polyamines are LHCII-attached), then, PSIIα centers increase could be the direct outcome of LHCII polyaminylation. First results show a 80% Spd and Spm increase in isolated LHCII antenna proteins from tobacco TGZ over-expressers (Ioannidis et al. in preparation). TGases may affect, not only the thylakoid structure, but also the architecture of the thylakoid network. This enzyme could alter the function of photosynthetic complexes and affect photosynthesis in multiple ways. Given that LHCII has a key role in light harvesting, photoprotective qE and state transitions, a highly polyaminylated LHCII *in vivo* should be tested for every one of these processes. First results show that antenna down regulation is much more sensitive under these conditions. Future experiments should also reveal the exact residue(s) of polyaminylation and increase further our understanding regarding the structure and plasticity of the thylakoid network. Last but not least, TGases may cross link the complexes of PSII outer antenna with the core. Newly

Overexpression of chlTGZ in tobacco increased the activity of plastidal transglutaminase, the thylakoid associated polyamines, the fraction of PSIIα centers and thylakoid stacking. We suggest that chlTGZ has an important role in the remodeling of the thylakoid

NEI thanks Greek Fellowship Foundation for funding (UOC). Authors thanks all the groups that contributed to a part of the revised results: J. Veramendi (Publ. Univ. Navarra), J.A. Hernandez (CEBAS-CSIC, Murcia), I. Fleck (Fac. Biology, Univ. Barcelona), A. V. Coelho (ITQB, Univ. Lisboa). This study was supported by the Spanish projects MEC BFU2006- 15115-01/BMC, BFU 2009-08575, CGL2005-03998/BOS and BIO2005-00155. Also, CERBA

Allen JF, Bennett J, Steinback KE & Arntzen CJ, (1981) Chloroplast protein phosphorylation

Allen JF & Forsberg J, (2001) Molecular recognition in thylakoid structure and function.

Anderson J. M (1999) Insights into the consequences of grana stacking of thylakoid

couples plastoquinone redox state to distribution of excitation energy between

membranes in vascular plants: a personal perspective. *Aust J. Plant Physiol* 26: 625-

polyamines increased 90%.

**6. Conclusion** 

**8. References** 

639

**7. Acknowledgements** 

network.

engineered plants will help to elucidate these issues.

(Generalitat de Catalunya) supported partially this work.

photosystems*, Nature* 291: 25–29.

*Trends Plant Sci* 6: 317–326.

#### **i. Insight into the role of thylakoid bound polyamines**

Polyamines are ubiquitous molecules with an ill defined mode of action. Although thousands of papers appeared the last decades concerning their effects, their role remains obscure. The interest is still high because polyamines are essential for cell growth and important for plant tolerance to stress. The fact that, in plants, free, bound and phenolicconjugated polyamine forms are present, make their role more puzzling. This work significantly improve our understanding by sheding light mainly on the role of bound polyamines that will facilitate to understand the implication of the other polyamine forms.

#### **ii. Transglutaminases in thylakoids and photosynthetic implications**

Transglutaminase activity depends on Ca2+, GTP and light (Villalobos et al. 2004; Del Duca & Fracassini 2008), which are key factors for chloroplast energetics. Transglutaminase activity was shown to be light sensitive, affected by hormone deprivation and with a light/dark rhythm (Bernet 1997; Bernet et al. 1999). Subcellular localization studies showed that, the enzyme was specifically localized in the chloroplast grana-appressed thylakoids and close to LHCII (Villalobos et al. 2001; Villalobos 2007; Santos et al. 2007). Finally, proteomic studies indicates that maize chloroplastic TGase is a peripheral thylakoid protein forming part of a specific PSII protein complex which includes LHCII, ATPase and PsbS proteins (Campos et al. 2010). With the presented results, we give important *in vivo* and *in vitro* data that reinforce the idea that the role of TGase in thylakoids is the modification of LHCII antenna proteins by polyaminilation, giving new properties to the complex, in particular under low light or stress conditions.

Why the photosynthetic apparatus has enzymes with TGase action near the reaction centers of PSII? A plausible hypothesis is that biological glues such as transglutaminases have a "polymerizing" and/or a "stabilizing" role. More particularly, crosslink of LHCIIs could increase the absorption cross section of PSII which in turn will increase photon harvesting by PSII. The latter could account for the significant increase of PSIIα centers in TGZ overexpressers. On the other hand, attachment of polyamines increase the positive charge of the protein as well as the connections intra and inter molecularly, stabilizing more firmly loosely aggregated complexes. This stabilization may be of importance during stress conditions conferring tolerance to the photosynthetic apparatus (Lütz et al 2005; Navakoudis et al 2007; Demetriou et al 2007; Sfichi et al 2008). In consequence, a possible role for polyamines on LHCII could be the activation of the dissipative antenna conformation (Ioannidis et al 2011). Furthermore, although thylakoid localization of ADC (arginine decarboxylase, Put producer) was long ago reported (Borrell et al 1995) and its importance for stress tolerance acknowledged (Galston 2001), only recently it becomes apparent that Put, and higher polyamines derived from Put, could modulate the photosynthesis protonic circuit, which is central for plant life and stress tolerance (Ioannidis et al 2011 submitted). An enzyme as TGase, that modulates the poise between free and bound polyamine forms in the following equilibrium may have a key role for the fine tuning of these processes.

$$\mathsf{Pas} + \mathsf{L} \mathsf{H} \mathsf{C} \mathsf{U} \xrightleftharpoons[]{\mathsf{T} \mathsf{Gas} \mathsf{o}} \quad \mathsf{Pas} \mathsf{-} \mathsf{L} \mathsf{H} \mathsf{C} \mathsf{U} \tag{1}$$

#### **5.2 Future experiments**

This chapter summarized recent results showing that over-expression of TGZ in tobacco, dramatically alter the organization of the thylakoid network. TGZ acted as a grana making enzyme and increased granum size more than 100%. PSIIα centers increased, and , concomitantly, stroma thylakoids were depleted. At the same time, thylakoid associated polyamines increased 90%.

On the grounds that TGases have LHCbs as a natural substrate it is plausible that polyamines increase in thylakoids were due to LHCII modification. In future works, we will test whether LHCII has a different profile of bound polyamines due to TGZ over-expression. If this is the case (if more polyamines are LHCII-attached), then, PSIIα centers increase could be the direct outcome of LHCII polyaminylation. First results show a 80% Spd and Spm increase in isolated LHCII antenna proteins from tobacco TGZ over-expressers (Ioannidis et al. in preparation). TGases may affect, not only the thylakoid structure, but also the architecture of the thylakoid network. This enzyme could alter the function of photosynthetic complexes and affect photosynthesis in multiple ways. Given that LHCII has a key role in light harvesting, photoprotective qE and state transitions, a highly polyaminylated LHCII *in vivo* should be tested for every one of these processes. First results show that antenna down regulation is much more sensitive under these conditions. Future experiments should also reveal the exact residue(s) of polyaminylation and increase further our understanding regarding the structure and plasticity of the thylakoid network. Last but not least, TGases may cross link the complexes of PSII outer antenna with the core. Newly engineered plants will help to elucidate these issues.

#### **6. Conclusion**

532 Advances in Photosynthesis – Fundamental Aspects

Polyamines are ubiquitous molecules with an ill defined mode of action. Although thousands of papers appeared the last decades concerning their effects, their role remains obscure. The interest is still high because polyamines are essential for cell growth and important for plant tolerance to stress. The fact that, in plants, free, bound and phenolicconjugated polyamine forms are present, make their role more puzzling. This work significantly improve our understanding by sheding light mainly on the role of bound polyamines that will facilitate to understand the implication of the other polyamine forms.

Transglutaminase activity depends on Ca2+, GTP and light (Villalobos et al. 2004; Del Duca & Fracassini 2008), which are key factors for chloroplast energetics. Transglutaminase activity was shown to be light sensitive, affected by hormone deprivation and with a light/dark rhythm (Bernet 1997; Bernet et al. 1999). Subcellular localization studies showed that, the enzyme was specifically localized in the chloroplast grana-appressed thylakoids and close to LHCII (Villalobos et al. 2001; Villalobos 2007; Santos et al. 2007). Finally, proteomic studies indicates that maize chloroplastic TGase is a peripheral thylakoid protein forming part of a specific PSII protein complex which includes LHCII, ATPase and PsbS proteins (Campos et al. 2010). With the presented results, we give important *in vivo* and *in vitro* data that reinforce the idea that the role of TGase in thylakoids is the modification of LHCII antenna proteins by polyaminilation, giving new properties to the complex, in

Why the photosynthetic apparatus has enzymes with TGase action near the reaction centers of PSII? A plausible hypothesis is that biological glues such as transglutaminases have a "polymerizing" and/or a "stabilizing" role. More particularly, crosslink of LHCIIs could increase the absorption cross section of PSII which in turn will increase photon harvesting by PSII. The latter could account for the significant increase of PSIIα centers in TGZ overexpressers. On the other hand, attachment of polyamines increase the positive charge of the protein as well as the connections intra and inter molecularly, stabilizing more firmly loosely aggregated complexes. This stabilization may be of importance during stress conditions conferring tolerance to the photosynthetic apparatus (Lütz et al 2005; Navakoudis et al 2007; Demetriou et al 2007; Sfichi et al 2008). In consequence, a possible role for polyamines on LHCII could be the activation of the dissipative antenna conformation (Ioannidis et al 2011). Furthermore, although thylakoid localization of ADC (arginine decarboxylase, Put producer) was long ago reported (Borrell et al 1995) and its importance for stress tolerance acknowledged (Galston 2001), only recently it becomes apparent that Put, and higher polyamines derived from Put, could modulate the photosynthesis protonic circuit, which is central for plant life and stress tolerance (Ioannidis et al 2011 submitted). An enzyme as TGase, that modulates the poise between free and bound polyamine forms in the following equilibrium may have a key role for the fine tuning

(1)

This chapter summarized recent results showing that over-expression of TGZ in tobacco, dramatically alter the organization of the thylakoid network. TGZ acted as a grana making

**i. Insight into the role of thylakoid bound polyamines** 

particular under low light or stress conditions.

of these processes.

**5.2 Future experiments** 

**ii. Transglutaminases in thylakoids and photosynthetic implications** 

Overexpression of chlTGZ in tobacco increased the activity of plastidal transglutaminase, the thylakoid associated polyamines, the fraction of PSIIα centers and thylakoid stacking. We suggest that chlTGZ has an important role in the remodeling of the thylakoid network.

#### **7. Acknowledgements**

NEI thanks Greek Fellowship Foundation for funding (UOC). Authors thanks all the groups that contributed to a part of the revised results: J. Veramendi (Publ. Univ. Navarra), J.A. Hernandez (CEBAS-CSIC, Murcia), I. Fleck (Fac. Biology, Univ. Barcelona), A. V. Coelho (ITQB, Univ. Lisboa). This study was supported by the Spanish projects MEC BFU2006- 15115-01/BMC, BFU 2009-08575, CGL2005-03998/BOS and BIO2005-00155. Also, CERBA (Generalitat de Catalunya) supported partially this work.

#### **8. References**


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Kotzabasis K, Fotinou C, Roubelakis-Angelakis KA, & Ghanotakis D (1993) Polyamines in

Kotzabasis K., M.D. Christakis-Hampsas & K.A. Roubelakis-Angelakis (1993). A narrow

Kramer DM, Cruz JA, & Kanazawa A, (2003) Balancing the central roles of the thylakoid

Kovacs L, Damkjaer J, Kereiche S, Ilioaia C, Ruban AV, Boekema EJ, Jansson S, & Horton

Lorand L & Graham RM, (2003) Transglutaminases: crosslinking enzymes with pleiotropic

Lütz C., Navakoudis, H.Seidlitz K, & Kotzabasis K (2005). Simulated solar irradiation with

Melis A, & Homann PH (1976) Heterogeneity of the photochemical centers in system II of

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Mustardy L, & Garab G, (2003) Granum revisited. A three-dimensional model - where

Navakoudis E, Vrentzou K, & Kotzabasis K (2007) A polyamine- and LHCII protease

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Pascal AA, Liu Z, Broess K, van Oort B, van Amerongen H, Wang C, Horton P, Robert B,

Mullineaux C.W. (2005) Function and evolution of grana *Trends Plant Sci* 10: 521-525

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photosynthetic light-harvesting, *Nature* 436: 134–137.

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Chang W, Ruban A, (2005) Molecular basis of photoprotection and control of


**26** 

*Argentina* 

 **Reductases** 

Matías A. Musumeci, Eduardo A. Ceccarelli

*Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Facultad de* 

Ferredoxin-NADP+ reductases (FNRs, EC 1.18.1.2) constitute a family of hydrophilic, monomeric enzymes that contain non-covalently bound FAD as prosthetic group. These flavoenzymes deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. The main physiological role of the chloroplast FNR is to catalyze the final step of photosynthetic electron transport, namely, the electron transfer from the ferredoxin (Fd), reduced by photosystem I, to NADP+ (Eqn. 1) (Shin & Arnon, 1965). This reaction provides the NADPH

FNRs also participate in others electron transfer metabolic processes as nitrogen fixation, isoprenoid biosynthesis, steroid metabolism, xenobiotic detoxification, oxidative-stress response and iron-sulfur cluster biogenesis (Carrillo & Ceccarelli, 2003, Ceccarelli et al., 2004, Medina & Gomez-Moreno, 2004, Rohrich et al., 2005, Seeber et al., 2005). Eqn. 1 represents the electron flow through FNR as it occurs in the photosynthetic electron chain. However, the physiological direction of the reaction catalyzed by FNRs involved in the other pathways is opposite, i.e. toward the production of reduced Fd. On this basis, FNRs are sometimes classified as autotrophic (photosynthetic FNRs) and heterotrophic (all other

Some bacteria and algae posses the FMN-containing flavodoxin (Fld), that is able to efficiently replace Fd as the electron partner of FNR in different metabolic routes, including photosynthesis (Razquin et al., 1996). In cyanobacteria, Fld expression is induced under condition of iron deficit, when the [2Fe-2S] cluster of Fd cannot be assembled (Razquin et al., 1996). In other prokaryotes, flavodoxins are constitutively expressed, or induced by oxidants (Zheng et al., 1999). Both Fld and FNR participate in the detoxification of reactive oxygen

The FNR displays strong preference for NADP(H) and is a very poor NAD(H) oxidoreductase. In contrast, various redox compounds, including complexed metals and aromatic molecules, can replace Fd or Fld as electron acceptors *in vitro*, in the so-called diaphorase activity (Avron & Jagendorf, 1956). All FNR-mediated reactions can thus be

species in aerobic and facultative bacteria (Krapp et al., 2002, Zheng et al., 1999).

*Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario* 

2 Fd(Fe2+) + NADP+ + H+ ↔ 2 Fd (Fe3+) + NADPH (1)

and Daniela L. Catalano-Dupuy

**The Plant–Type Ferredoxin-NADP<sup>+</sup>**

necessary for CO2 assimilation in plants and cyanobacteria.

FNRs) (Aliverti et al., 2008, Arakaki et al., 1997).

**1. Introduction** 

Wang Q, Sullivan RW, Kight A, Henry HJ, Huang J, & Jones AM, (2004) Deletion of the chloroplast-localized Thylakoid Formation1 gene product in Arabidopsis leads to deficient thylakoid formation and variegated leaves, *Plant Physiol* 136: 3594- 3604.

#### **The Plant–Type Ferredoxin-NADP<sup>+</sup> Reductases**

Matías A. Musumeci, Eduardo A. Ceccarelli

and Daniela L. Catalano-Dupuy *Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario Argentina* 

#### **1. Introduction**

538 Advances in Photosynthesis – Fundamental Aspects

Wang Q, Sullivan RW, Kight A, Henry HJ, Huang J, & Jones AM, (2004) Deletion of the

3604.

chloroplast-localized Thylakoid Formation1 gene product in Arabidopsis leads to deficient thylakoid formation and variegated leaves, *Plant Physiol* 136: 3594-

> Ferredoxin-NADP+ reductases (FNRs, EC 1.18.1.2) constitute a family of hydrophilic, monomeric enzymes that contain non-covalently bound FAD as prosthetic group. These flavoenzymes deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. The main physiological role of the chloroplast FNR is to catalyze the final step of photosynthetic electron transport, namely, the electron transfer from the ferredoxin (Fd), reduced by photosystem I, to NADP+ (Eqn. 1) (Shin & Arnon, 1965). This reaction provides the NADPH necessary for CO2 assimilation in plants and cyanobacteria.

> FNRs also participate in others electron transfer metabolic processes as nitrogen fixation, isoprenoid biosynthesis, steroid metabolism, xenobiotic detoxification, oxidative-stress response and iron-sulfur cluster biogenesis (Carrillo & Ceccarelli, 2003, Ceccarelli et al., 2004, Medina & Gomez-Moreno, 2004, Rohrich et al., 2005, Seeber et al., 2005). Eqn. 1 represents the electron flow through FNR as it occurs in the photosynthetic electron chain. However, the physiological direction of the reaction catalyzed by FNRs involved in the other pathways is opposite, i.e. toward the production of reduced Fd. On this basis, FNRs are sometimes classified as autotrophic (photosynthetic FNRs) and heterotrophic (all other FNRs) (Aliverti et al., 2008, Arakaki et al., 1997).

$$2\,\mathrm{Fd(Fe^{2+})} + \mathrm{NADP^{\*}} + \mathrm{H^{\*}} \quad \leftrightarrow \,\, 2\,\mathrm{Fd} \,\mathrm{(Fe^{3+})} + \mathrm{NADPH} \tag{1}$$

Some bacteria and algae posses the FMN-containing flavodoxin (Fld), that is able to efficiently replace Fd as the electron partner of FNR in different metabolic routes, including photosynthesis (Razquin et al., 1996). In cyanobacteria, Fld expression is induced under condition of iron deficit, when the [2Fe-2S] cluster of Fd cannot be assembled (Razquin et al., 1996). In other prokaryotes, flavodoxins are constitutively expressed, or induced by oxidants (Zheng et al., 1999). Both Fld and FNR participate in the detoxification of reactive oxygen species in aerobic and facultative bacteria (Krapp et al., 2002, Zheng et al., 1999).

The FNR displays strong preference for NADP(H) and is a very poor NAD(H) oxidoreductase. In contrast, various redox compounds, including complexed metals and aromatic molecules, can replace Fd or Fld as electron acceptors *in vitro*, in the so-called diaphorase activity (Avron & Jagendorf, 1956). All FNR-mediated reactions can thus be

The Plant–Type Ferredoxin-NADP<sup>+</sup>

assimilation (Rochaix, 2011).

simultaneously to NADP+ (Eqn. 5) (Medina, 2009).

Reductases 541

to the NADP(H) region (Arakaki et al., 1997, Bruns & Karplus, 1995, Ceccarelli et al., 2004, Karplus et al., 1991). This group can be further classified into a plastidic and a bacterial class, which differ not only in their sequences, but also in the environment of the active site, FAD conformations and catalytic efficiencies (Carrillo & Ceccarelli, 2003). Plastidic FNRs display high catalytic efficiencies (turnover numbers in the range 100-600 s-1), whereas bacterial reductases are much less active (Ceccarelli et al., 2004). In plants and cyanobacteria, optimization for FNR catalytic efficiency might be related to the demands of the photosynthetic process that requires a very fast electron flow to sustain CO2 fixation rates. In organisms growing on heterotrophic metabolisms or anoxygenic photosynthesis, FNR is involved in pathways that proceed at a much lower pace, acting as a shuttle between the

This chapter will focus on structural and functional aspects of the ferredoxin-NADP(H) reductase in association with the metabolic process of photosynthesis. Special attention will be pay to techniques and approaches that could help to appreciate the importance of the

The generation of reducing power is crucial for all biosynthetic processes within chloroplasts. The main source of reduction equivalents is the light-driven photosynthesis. The FNR is a key enzyme of photosynthetic electron transport. FNR transfers electrons between the one-electron carrier ferredoxin and the two-electron carrier NADP(H) at the end of the photosynthetic electron transport chain. FNR also participates in others relevant processes as the electron cyclic flow around the photosystem I and in the control of the

Photosynthetic electron flow is driven by two photochemical reactions catalyzed by photosystem II (PSII) and photosystem I (PSI), which are linked by the electron transport chain (Figure 2). Linear electron transport starts with the photo-induced water oxidation catalyzed by PSII. Electrons are transferred from PSII through the plastoquinone pool to cytochrome *b*6*f*. The electron transport is coupled to proton translocation into the thylakoid lumen, and the resulting pH gradient drives the ATP synthase to produce ATP. Next, electrons move from cytochrome *b*6*f* to the soluble electron carrier plastocyanin and then to PSI, which acts as light-driven plastocyanin ferredoxin oxidoreductase. Ultimately these electrons can be used by ferredoxin-NADP+ reductase to produce NADPH that together with the ATP generated by the ATP synthase will drive the Calvin cycle for CO2

The primary function of PSI is to reduce NADP+ to NADPH, which is then used in the assimilation of CO2 (Setif, 2006, Vishniac & Ochoa, 1951). In plants, it occurs via reduction of the soluble [2Fe-2S] ferredoxin (Fd) by PSI. Subsequent reduction of NADP+ by Fdrd is catalyzed by FNR (Arakaki et al., 1997). In most cyanobacteria, and algae under low iron conditions, Fld, in particular Fldsq/Fldhq, substitutes for the Fdox/Fdrd pair in this reaction (Eqn. 4) (Bottin & Lagoutte, 1992, Medina & Gomez-Moreno, 2004). Two Fldsq molecules transfer two electrons from two PSI molecules to one FNR. FNR becomes fully reduced through formation of the intermediate, FNRsq, and later transfers both electrons

It was demonstrated that FNR is one of the rate-limiting steps in photosynthesis, and controls the balance between the demand for redox equivalents and photosynthetic activity

abundant NAD(P)H pool and the low potential electron carriers.

**2. FNR in the photosynthetic electron transport** 

enzyme and to understand, conceive and/or execute research on FNR.

NADPH/NADP+ homeostasis of stressed chloroplasts (Palatnik et al., 1997).

interpreted consisting of two-steps: hydride exchange with pyridine nucleotide (Eqn. 2) and electron transfer to and from the other partner (Eqn. 3).

$$\text{NADPH} + \text{H}^\* + \text{FNR}\_{\text{ox}} \quad \leftrightarrow \text{NADP\*} + \text{FNRH}\_2 \tag{2}$$

$$\text{FNRH}\_2 + n\text{A}\_{\text{ox}} \rightarrow \text{FNR}\_{\text{ox}} + n\text{A}\_{\text{red}} \tag{3}$$

A, electron acceptor *n* = 1, 2

The capability of FNRs to exchange electrons between mono and bi-electronic substrates is due to the prosthetic group FAD, which can exist in three redox states: oxidised, as radical reduced by one electron (semiquinone) and completely reduced (hydroquinone) by two electrons (Dudley et al., 1964). The active chemically moiety of the FAD is the isoalloxazine (Figure 1). Besides, the isoalloxazine ring can be protonated, providing a wide opportunity for tautomers (Heelis P.F., 1982).

Fig. 1. Main structural traits of FAD. A) View of FAD as found in the pea FNR. B) Redox states adopted by the FAD. In blue, red and white are showed nitrogen, oxygen and carbon atoms respectively; the yellow colour depicts double bonds.

The protein environment modulates the redox potential of the isoalloxazine in such manner that in photosynthetic tissues it promotes the production of NADPH from reduced ferredoxin (Aliverti et al., 2008).

Two great families of FAD containing proteins displaying FNR activity have evolved from different and independent origins. The enzymes from mitochondria and some bacterial genera are members of the structural superfamily of disulfide oxidoreductases whose prototype is glutathione reductase. A second group, comprising the FNRs from plastids and most eubacteria, constitutes a unique family, the plant-type FNRs, totally unrelated in sequence with the former (Aliverti et al., 2008, Ceccarelli et al., 2004). In spite of their different origins, flavoproteins of the two FNR families display similar modes of NADP(H) docking and catalysis and can exchange electron partners (ferredoxin, flavodoxin, adrenodoxin) *in vitro* (Faro et al., 2003, Jenkins et al., 1997, Ziegler & Schulz, 2000). Members of the plant-type group can be readily identified by the presence of clusters of highly conserved residues, three of them belonging to the FAD (FMN) domain, and the remaining

interpreted consisting of two-steps: hydride exchange with pyridine nucleotide (Eqn. 2) and

 A, electron acceptor *n* = 1, 2 The capability of FNRs to exchange electrons between mono and bi-electronic substrates is due to the prosthetic group FAD, which can exist in three redox states: oxidised, as radical reduced by one electron (semiquinone) and completely reduced (hydroquinone) by two electrons (Dudley et al., 1964). The active chemically moiety of the FAD is the isoalloxazine (Figure 1). Besides, the isoalloxazine ring can be protonated, providing a wide opportunity

Fig. 1. Main structural traits of FAD. A) View of FAD as found in the pea FNR. B) Redox states adopted by the FAD. In blue, red and white are showed nitrogen, oxygen and carbon

The protein environment modulates the redox potential of the isoalloxazine in such manner that in photosynthetic tissues it promotes the production of NADPH from reduced

Two great families of FAD containing proteins displaying FNR activity have evolved from different and independent origins. The enzymes from mitochondria and some bacterial genera are members of the structural superfamily of disulfide oxidoreductases whose prototype is glutathione reductase. A second group, comprising the FNRs from plastids and most eubacteria, constitutes a unique family, the plant-type FNRs, totally unrelated in sequence with the former (Aliverti et al., 2008, Ceccarelli et al., 2004). In spite of their different origins, flavoproteins of the two FNR families display similar modes of NADP(H) docking and catalysis and can exchange electron partners (ferredoxin, flavodoxin, adrenodoxin) *in vitro* (Faro et al., 2003, Jenkins et al., 1997, Ziegler & Schulz, 2000). Members of the plant-type group can be readily identified by the presence of clusters of highly conserved residues, three of them belonging to the FAD (FMN) domain, and the remaining

atoms respectively; the yellow colour depicts double bonds.

NADPH + H+ + FNRox ↔ NADP+ + FNRH2 (2)

FNRH2 + *n*Aox → FNRox + *n*Ared (3)

electron transfer to and from the other partner (Eqn. 3).

for tautomers (Heelis P.F., 1982).

ferredoxin (Aliverti et al., 2008).

to the NADP(H) region (Arakaki et al., 1997, Bruns & Karplus, 1995, Ceccarelli et al., 2004, Karplus et al., 1991). This group can be further classified into a plastidic and a bacterial class, which differ not only in their sequences, but also in the environment of the active site, FAD conformations and catalytic efficiencies (Carrillo & Ceccarelli, 2003). Plastidic FNRs display high catalytic efficiencies (turnover numbers in the range 100-600 s-1), whereas bacterial reductases are much less active (Ceccarelli et al., 2004). In plants and cyanobacteria, optimization for FNR catalytic efficiency might be related to the demands of the photosynthetic process that requires a very fast electron flow to sustain CO2 fixation rates. In organisms growing on heterotrophic metabolisms or anoxygenic photosynthesis, FNR is involved in pathways that proceed at a much lower pace, acting as a shuttle between the abundant NAD(P)H pool and the low potential electron carriers.

This chapter will focus on structural and functional aspects of the ferredoxin-NADP(H) reductase in association with the metabolic process of photosynthesis. Special attention will be pay to techniques and approaches that could help to appreciate the importance of the enzyme and to understand, conceive and/or execute research on FNR.

#### **2. FNR in the photosynthetic electron transport**

The generation of reducing power is crucial for all biosynthetic processes within chloroplasts. The main source of reduction equivalents is the light-driven photosynthesis. The FNR is a key enzyme of photosynthetic electron transport. FNR transfers electrons between the one-electron carrier ferredoxin and the two-electron carrier NADP(H) at the end of the photosynthetic electron transport chain. FNR also participates in others relevant processes as the electron cyclic flow around the photosystem I and in the control of the NADPH/NADP+ homeostasis of stressed chloroplasts (Palatnik et al., 1997).

Photosynthetic electron flow is driven by two photochemical reactions catalyzed by photosystem II (PSII) and photosystem I (PSI), which are linked by the electron transport chain (Figure 2). Linear electron transport starts with the photo-induced water oxidation catalyzed by PSII. Electrons are transferred from PSII through the plastoquinone pool to cytochrome *b*6*f*. The electron transport is coupled to proton translocation into the thylakoid lumen, and the resulting pH gradient drives the ATP synthase to produce ATP. Next, electrons move from cytochrome *b*6*f* to the soluble electron carrier plastocyanin and then to PSI, which acts as light-driven plastocyanin ferredoxin oxidoreductase. Ultimately these electrons can be used by ferredoxin-NADP+ reductase to produce NADPH that together with the ATP generated by the ATP synthase will drive the Calvin cycle for CO2 assimilation (Rochaix, 2011).

The primary function of PSI is to reduce NADP+ to NADPH, which is then used in the assimilation of CO2 (Setif, 2006, Vishniac & Ochoa, 1951). In plants, it occurs via reduction of the soluble [2Fe-2S] ferredoxin (Fd) by PSI. Subsequent reduction of NADP+ by Fdrd is catalyzed by FNR (Arakaki et al., 1997). In most cyanobacteria, and algae under low iron conditions, Fld, in particular Fldsq/Fldhq, substitutes for the Fdox/Fdrd pair in this reaction (Eqn. 4) (Bottin & Lagoutte, 1992, Medina & Gomez-Moreno, 2004). Two Fldsq molecules transfer two electrons from two PSI molecules to one FNR. FNR becomes fully reduced through formation of the intermediate, FNRsq, and later transfers both electrons simultaneously to NADP+ (Eqn. 5) (Medina, 2009).

It was demonstrated that FNR is one of the rate-limiting steps in photosynthesis, and controls the balance between the demand for redox equivalents and photosynthetic activity

The Plant–Type Ferredoxin-NADP<sup>+</sup>

Joliot & Joliot, 2006, Shikanai, 2007).

**3.1 Purification procedures** 

*Escherichia coli*

**3. Purification and characterization of FNR** 

different procedures to obtain pure FNR.

Reductases 543

which is required for inducing ATP synthesis and for triggering non-photochemical quenching, which in turn down-regulates PSII. Cyclic electron flow is also important when photorespiration is active because more ATP is required for CO2 fixation under these conditions (Osmond, 1981). Several routes for cyclic electron flow have been proposed. In the first, ferredoxin transfers its electrons through FNR to NADP+ and the NAD(P)H dehydrogenase (Ndh) complex and ultimately to the plastoquinone pool (Figure 2). However, in plants, the level of this complex is probably too low for mediating sufficient cyclic electron flow required for ATP production under steady state conditions (Burrows et al., 1998). FNR, which is found both in the stroma and associated with thylakoid membranes (through TROL or the FNR binding protein, see below), has been proposed to modulate partitioning between the cyclic and linear electron pathways (Rochaix, 2011). In the second pathway, ferredoxin is thought to transfer electrons to the plastoquinone pool through a ferredoxin–plastoquinone oxidoreductase (Cleland & Bendall, 1992), an enzyme that has however not yet been identified. This function has been recently assigned to the FNR (Szymanska et al., 2011). In the third, ferredoxin may interact directly with cytochrome *b*6*f* and transfer its electrons to cytochrome c′, a new component identified in the crystal structure of the cytochrome *b*6*f* complex, using a Q-cycle derived mechanism (reviewed in

It has been proposed that the transhydrogenase activity of the FNR may function *in vivo* as an intra-chloroplastic source of NADH (Chopowick & Israelstam, 1971, Krawetz & Israelstam, 1978). However, the presence of NADH in chloroplasts is most likely to be related to the malate/oxalacetate shuttle (Carrillo & Vallejos, 1987, Krause & Heber, 1976).

FNRs can be obtained using transgenic expression in *Escherichia coli* cells or from biological samples such as plant leaves, plant roots or cyanobacteria. Nevertheless, the purification procedures further applied to obtain FNR are similar in any case. In this section we will discuss how to obtain the soluble protein extracts from diverse sources, and how to apply

**3.1.1 Preparation of soluble protein extracts from FNR transgenic expression in** 

High throughput preparations of FNRs can be obtained from recombinant expression in *E. coli* cells by using vectors of the pET (Novagen, USA) or pQE (QIAGEN, USA) series. For the culture of FNR-expressing *E. coli* cells, Luria-Bertani or 2YT (16 g/l tryptone, 10 g/l yeast extract, 5.0 g/l NaCl) media are recommended. The expression conditions may vary depending on the features of the FNR being studied. Usually, for the wild-type FNR from plants cloned in vectors under the control of the *Lac* promoter 0.2–1 mM IPTG is used for the induction of protein expression during 2 to 5 h at 25-37ºC (Aliverti et al., 1990, Catalano-Dupuy et al., 2006, Onda et al., 2000). For the *Anabaena variabilis* FNR no inductor was applied and the culture maintained overnight at 30ºC (Tejero et al., 2003). For unstable FNR mutants, longer induction periods (10-14 h) at low temperature (18–20ºC) are recommended. In these cases, lower inductor concentration (0.1 mM IPTG) gives better yields (Musumeci et al., 2008, Musumeci et al., 2011). Nevertheless, extremely long induction periods (20 h or

under a wide range of environmental conditions (Hajirezaei et al., 2002). It gives to this enzyme relevance as a possible target for crop improvement and treatment of weeds.

Fig. 2. Photosynthetic electron transport. The thylakoid membrane with the different component of the electron transport chain is shown. Electron transport pathways are shown by dotted lines with arrows to indicate the direction of electron flow. P680 is the PSII primary electron donor; QA and QB are the primary electron acceptors of PSII. Once QB has accepted two electrons, is released into the plastoquinone pool (PQ). PQH2, reduced plastoquinone pool; PC, plastocyanin; Ndh, NADH/NADPH dehydrogenase complex; Ox, oxidase. P700 is the PSI primary electron donor. FX, FA, and FB are internal [4Fe-4S] centers of the PSI. Adapted from (Rochaix, 2011).

In addition, ferredoxin donates electrons to other pathways such as sulfur and nitrogen assimilation, to thioredoxins, which regulate carbon assimilation and to the cyclic electron transfer process (Rochaix, 2011, Yamamoto et al., 2006). Many studies have suggested the involvement of FNR also in cyclic electron flow around PSI (Figure 2) besides its role in the linear electron transport of photosynthesis. In cyclic electron flow, electrons are transferred from PSI to cytochrome *b*6*f* complex via Fd, with a concomitant formation of proton gradient. Consequently, cyclic electron transfer produces ATP without accumulation of NADPH (Mulo, 2011, Shikanai, 2007).

$$\text{PSI}\_{\text{rd}} + \text{Fld}\_{\text{sd}} \rightarrow \text{PSI} + \text{Fld}\_{\text{ld}} \tag{4}$$

$$\text{NADP\*} + 2\text{ Fld}\_{\text{hg}} \xrightarrow[\text{FNR}]{} \text{NADPH} + 2\text{ Fld}\_{\text{sq}} \tag{5}$$

While cyclic electron flow is well documented in cyanobacteria, unicellular algae, and C4 plants, it is only recently that its importance has been recognized and studied in C3 plants (Joliot & Joliot, 2006). In C3 plants, cyclic electron flow is especially important under specific stress conditions such as low CO2, high light, drought, or during dark to light transitions. Under these conditions, cyclic electron flow allows for acidification of the thylakoid lumen,

under a wide range of environmental conditions (Hajirezaei et al., 2002). It gives to this

enzyme relevance as a possible target for crop improvement and treatment of weeds.

Fig. 2. Photosynthetic electron transport. The thylakoid membrane with the different component of the electron transport chain is shown. Electron transport pathways are shown by dotted lines with arrows to indicate the direction of electron flow. P680 is the PSII primary electron donor; QA and QB are the primary electron acceptors of PSII. Once QB has accepted two electrons, is released into the plastoquinone pool (PQ). PQH2, reduced plastoquinone pool; PC, plastocyanin; Ndh, NADH/NADPH dehydrogenase complex; Ox, oxidase. P700 is the PSI primary electron donor. FX, FA, and FB are internal [4Fe-4S] centers

In addition, ferredoxin donates electrons to other pathways such as sulfur and nitrogen assimilation, to thioredoxins, which regulate carbon assimilation and to the cyclic electron transfer process (Rochaix, 2011, Yamamoto et al., 2006). Many studies have suggested the involvement of FNR also in cyclic electron flow around PSI (Figure 2) besides its role in the linear electron transport of photosynthesis. In cyclic electron flow, electrons are transferred from PSI to cytochrome *b*6*f* complex via Fd, with a concomitant formation of proton gradient. Consequently, cyclic electron transfer produces ATP without accumulation of

PSIrd + Fldsq → PSI + Fldhq (4)

While cyclic electron flow is well documented in cyanobacteria, unicellular algae, and C4 plants, it is only recently that its importance has been recognized and studied in C3 plants (Joliot & Joliot, 2006). In C3 plants, cyclic electron flow is especially important under specific stress conditions such as low CO2, high light, drought, or during dark to light transitions. Under these conditions, cyclic electron flow allows for acidification of the thylakoid lumen,

NADPH + 2 Fldsq (5)

of the PSI. Adapted from (Rochaix, 2011).

NADPH (Mulo, 2011, Shikanai, 2007).

NADP+ + 2 Fldhq FNR

which is required for inducing ATP synthesis and for triggering non-photochemical quenching, which in turn down-regulates PSII. Cyclic electron flow is also important when photorespiration is active because more ATP is required for CO2 fixation under these conditions (Osmond, 1981). Several routes for cyclic electron flow have been proposed. In the first, ferredoxin transfers its electrons through FNR to NADP+ and the NAD(P)H dehydrogenase (Ndh) complex and ultimately to the plastoquinone pool (Figure 2). However, in plants, the level of this complex is probably too low for mediating sufficient cyclic electron flow required for ATP production under steady state conditions (Burrows et al., 1998). FNR, which is found both in the stroma and associated with thylakoid membranes (through TROL or the FNR binding protein, see below), has been proposed to modulate partitioning between the cyclic and linear electron pathways (Rochaix, 2011). In the second pathway, ferredoxin is thought to transfer electrons to the plastoquinone pool through a ferredoxin–plastoquinone oxidoreductase (Cleland & Bendall, 1992), an enzyme that has however not yet been identified. This function has been recently assigned to the FNR (Szymanska et al., 2011). In the third, ferredoxin may interact directly with cytochrome *b*6*f* and transfer its electrons to cytochrome c′, a new component identified in the crystal structure of the cytochrome *b*6*f* complex, using a Q-cycle derived mechanism (reviewed in Joliot & Joliot, 2006, Shikanai, 2007).

It has been proposed that the transhydrogenase activity of the FNR may function *in vivo* as an intra-chloroplastic source of NADH (Chopowick & Israelstam, 1971, Krawetz & Israelstam, 1978). However, the presence of NADH in chloroplasts is most likely to be related to the malate/oxalacetate shuttle (Carrillo & Vallejos, 1987, Krause & Heber, 1976).

#### **3. Purification and characterization of FNR**

#### **3.1 Purification procedures**

FNRs can be obtained using transgenic expression in *Escherichia coli* cells or from biological samples such as plant leaves, plant roots or cyanobacteria. Nevertheless, the purification procedures further applied to obtain FNR are similar in any case. In this section we will discuss how to obtain the soluble protein extracts from diverse sources, and how to apply different procedures to obtain pure FNR.

#### **3.1.1 Preparation of soluble protein extracts from FNR transgenic expression in**  *Escherichia coli*

High throughput preparations of FNRs can be obtained from recombinant expression in *E. coli* cells by using vectors of the pET (Novagen, USA) or pQE (QIAGEN, USA) series. For the culture of FNR-expressing *E. coli* cells, Luria-Bertani or 2YT (16 g/l tryptone, 10 g/l yeast extract, 5.0 g/l NaCl) media are recommended. The expression conditions may vary depending on the features of the FNR being studied. Usually, for the wild-type FNR from plants cloned in vectors under the control of the *Lac* promoter 0.2–1 mM IPTG is used for the induction of protein expression during 2 to 5 h at 25-37ºC (Aliverti et al., 1990, Catalano-Dupuy et al., 2006, Onda et al., 2000). For the *Anabaena variabilis* FNR no inductor was applied and the culture maintained overnight at 30ºC (Tejero et al., 2003). For unstable FNR mutants, longer induction periods (10-14 h) at low temperature (18–20ºC) are recommended. In these cases, lower inductor concentration (0.1 mM IPTG) gives better yields (Musumeci et al., 2008, Musumeci et al., 2011). Nevertheless, extremely long induction periods (20 h or

The Plant–Type Ferredoxin-NADP<sup>+</sup>

**3.1.4 Long term storage conditions** 

**3.2 Properties of the purified FNR 3.2.1 FNR spectroscopic properties** 

enzyme (Piubelli et al., 2000).

performance of the prosthetic group and the flavoenzyme.

indicate depletion of the flavin during purification and handling.

**3.2.2 Analysis of FNR by UV-visible spectroscopy** 

(Nakajima et al., 2002).

Reductases 545

fusion protein is eluted with 100-200 mM imidazole in the same buffer. Then, the histidine tag can be removed by digestion with the appropriate protease and an additional Ni-NTA chromatography (Catalano-Dupuy et al., 2006). Further purification steps have been applied to improve enzyme quality after the Ni-NTA affinity chromatography (Tejero et al., 2003).

FNR frozen at -20ºC retain the activity for several months (Zanetti et al., 1984). FNR can be stored for longer periods (years) at -70ºC in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl. Addition of 1 mM 2-mercaptoethanol or DTT avoid covalent aggregation of some FNRs

The transition energies of the isoalloxazine portion of FAD are susceptible to variations by the near environment. These perturbations are detectable by different spectroscopic techniques, which provide value information about the structure and the functional

The isoalloxazine, the structural component of the flavin involved in electron transfer, absorbs light in the UV and visible spectral range giving rise to the yellow appearance of flavin and flavoproteins (Latin: *flavus* = yellow) (Macheroux, 1999). In solution, the FAD shows a spectrum with maxima at 380 and 450 nm. The typical plant FNR spectrum displays peaks at 385 and 456 nm (Figure 3A, green line). The interaction of the protein scaffold with the isoalloxazine induces a decrease of the maximum energy levels of the isoalloxazine orbitals that results in spectral shifts to higher wavelengths. The presence of typical peaks or shoulders in the spectrum may indicate chemical variations in the flavin or in its protein environment (Figure 3A, red and blue lines), or the persistence of a bound nucleotide to the

The UV-visible absorption spectroscopy can be used with different purposes. Thus, a simple UV-visible spectrum provides information about the quality of the FNR enzyme. Aggregates or contaminants that cause light dispersion may result in an absorbance increase at wavelengths below 600 nm (Figure 3A, grey line). Knowledge of the enzyme extinction coefficient can be helpful to assess the stoichiometry of the flavin and protein moiety. In general, one flavin per polypeptide is found. Substoichiometric amounts of flavin may

The UV-visible spectroscopy can be used to study the binding of substrates, substrate analogues and inhibitors (Aliverti et al., 1995, Medina et al., 2001, Paladini et al., 2009, Piubelli et al., 2000). Titration with the compound of interest yields the dissociation constant and information about changes that occur in the active site environment (Figure 3B) (Paladini et al., 2009). Besides, the spectrum shape can provide useful information about the nature of the interaction with substrates. Experimental data indicate that when NADP+ is present at saturating concentrations, the degree of nicotinamide ring occupancy of the binding site is 14-15% for pea FNR, as revealed by the extinction coefficient value of the peak near 510 nm in differential spectra elicited by pyridine nucleotide binding to the various pea FNR forms (Piubelli et al., 2000). This value has been used to analyse

more) are detrimental for the quality of the obtained FNR due to the incorporation of a modified flavin or an improper protein folding (Figure 3A).

After the induction, *E. coli* cells that over-expressed FNR are harvested by centrifugation and resuspended in an appropriate buffer solution (25-50 mM Tris-HCl, pH 7.5–8.0, 100–150 mM NaCl). Addition of 5 mM benzamidine hydrochloride or 1 mM phenylmethylsulfonyl fluoride (PMSF) can be used to inhibit unwanted FNR proteolytic degradation. Cells are disrupted by sonication or French Press at 60 MPa. After 20 min incubation with DNase and RNase, centrifugation at 40,000 X *g* (60 min, 4 °C) is applied in order to remove cell debris and to obtain the soluble protein extract.

#### **3.1.2 Preparation of soluble protein extracts from photosynthetic tissues**

The biochemical purification of FNR from biological tissues was neglected with the advent of the recombinant DNA technology, which ensures the production of high amounts of enzyme in lesser times. However, the biochemical purification from photosynthetic tissues can be applied for some specific purposes. Several methodologies have been described in the literature for the purification of the FNR from paprika leaves (Dorowski et al., 2000), spinach leaves (Grzyb et al., 2004), soluble extracts of wheat chloroplasts (Grzyb et al., 2008) and from different plant types roots (Green et al., 1991, Morigasaki et al., 1990, Onda et al., 2000). FNR can be also purified from cyanobacteria **c**ell cultures grown in BG 11 medium as described (Sancho et al., 1988).

#### **3.1.3 Purification of FNR from soluble protein extract**

All purification protocol steps should be carried out at 4ºC to ensure good quality preparations of FNR. Soluble protein extract can be subjected to different purification procedures including precipitation with ammonium sulphate, affinity-, anion exchange- and hydrophobic-chromatography.

A typical purification protocol for FNR is as follows: The soluble extract is load onto a dye (reactive red or cibacron blue) affinity chromatography matrix. The FNR remains bound to the resin by interaction with the adenosine-like structure of the dye. After washing off the unabsorbed material, bound FNR is eluted with a linear gradient of NaCl or NADP+ (Aliverti et al., 1990, Carrillo & Vallejos, 1983, Dorowski et al., 2000). The FNR containing fractions are dialyzed and applied to an ionic exchange chromatography, such as DEAE Sepharose column. After extensive washing, a gradient of NaCl in the same buffer is applied. The FNR containing fractions are pooled and dialyzed. If a further purification is needed, the sample can be applied to a phenyl-Sepharose column. The FNR is eluted with a linear decreasing gradient of (NH4)2SO4 (Dorowski et al., 2000). Affinity chromatography also can be applied by using immobilized ferredoxin. This methodology has been used for purification purposes (Sakihama et al., 1992) and also for FNR analysis (Onda & Hase, 2004). Purification procedures using specific tags as glutathion *S*-transferase (Serra et al., 1993) or polihystidines (Catalano-Dupuy et al., 2006) have been reported. In these cases, a recognition site for specific proteases (as factor Xa, thrombin, TEV protease) is inserted between the FNR sequence and the tag to allow removal of the carrier protein if necessary. Amino terminal extensions are preferred to the carboxy-terminal due to the involvement of this latter region in substrate binding and catalysis.

For Ni-NTA affinity chromatography the protein extract containing the His-tagged FNR is loaded onto a column containing the resin. After intensive washing with Tris-HCl buffer the

more) are detrimental for the quality of the obtained FNR due to the incorporation of a

After the induction, *E. coli* cells that over-expressed FNR are harvested by centrifugation and resuspended in an appropriate buffer solution (25-50 mM Tris-HCl, pH 7.5–8.0, 100–150 mM NaCl). Addition of 5 mM benzamidine hydrochloride or 1 mM phenylmethylsulfonyl fluoride (PMSF) can be used to inhibit unwanted FNR proteolytic degradation. Cells are disrupted by sonication or French Press at 60 MPa. After 20 min incubation with DNase and RNase, centrifugation at 40,000 X *g* (60 min, 4 °C) is applied in order to remove cell debris

The biochemical purification of FNR from biological tissues was neglected with the advent of the recombinant DNA technology, which ensures the production of high amounts of enzyme in lesser times. However, the biochemical purification from photosynthetic tissues can be applied for some specific purposes. Several methodologies have been described in the literature for the purification of the FNR from paprika leaves (Dorowski et al., 2000), spinach leaves (Grzyb et al., 2004), soluble extracts of wheat chloroplasts (Grzyb et al., 2008) and from different plant types roots (Green et al., 1991, Morigasaki et al., 1990, Onda et al., 2000). FNR can be also purified from cyanobacteria **c**ell cultures grown in BG 11 medium as

All purification protocol steps should be carried out at 4ºC to ensure good quality preparations of FNR. Soluble protein extract can be subjected to different purification procedures including precipitation with ammonium sulphate, affinity-, anion exchange- and

A typical purification protocol for FNR is as follows: The soluble extract is load onto a dye (reactive red or cibacron blue) affinity chromatography matrix. The FNR remains bound to the resin by interaction with the adenosine-like structure of the dye. After washing off the unabsorbed material, bound FNR is eluted with a linear gradient of NaCl or NADP+ (Aliverti et al., 1990, Carrillo & Vallejos, 1983, Dorowski et al., 2000). The FNR containing fractions are dialyzed and applied to an ionic exchange chromatography, such as DEAE Sepharose column. After extensive washing, a gradient of NaCl in the same buffer is applied. The FNR containing fractions are pooled and dialyzed. If a further purification is needed, the sample can be applied to a phenyl-Sepharose column. The FNR is eluted with a linear decreasing gradient of (NH4)2SO4 (Dorowski et al., 2000). Affinity chromatography also can be applied by using immobilized ferredoxin. This methodology has been used for purification purposes (Sakihama et al., 1992) and also for FNR analysis (Onda & Hase, 2004). Purification procedures using specific tags as glutathion *S*-transferase (Serra et al., 1993) or polihystidines (Catalano-Dupuy et al., 2006) have been reported. In these cases, a recognition site for specific proteases (as factor Xa, thrombin, TEV protease) is inserted between the FNR sequence and the tag to allow removal of the carrier protein if necessary. Amino terminal extensions are preferred to the carboxy-terminal due to the involvement of

For Ni-NTA affinity chromatography the protein extract containing the His-tagged FNR is loaded onto a column containing the resin. After intensive washing with Tris-HCl buffer the

**3.1.2 Preparation of soluble protein extracts from photosynthetic tissues** 

modified flavin or an improper protein folding (Figure 3A).

**3.1.3 Purification of FNR from soluble protein extract** 

this latter region in substrate binding and catalysis.

and to obtain the soluble protein extract.

described (Sancho et al., 1988).

hydrophobic-chromatography.

fusion protein is eluted with 100-200 mM imidazole in the same buffer. Then, the histidine tag can be removed by digestion with the appropriate protease and an additional Ni-NTA chromatography (Catalano-Dupuy et al., 2006). Further purification steps have been applied to improve enzyme quality after the Ni-NTA affinity chromatography (Tejero et al., 2003).

#### **3.1.4 Long term storage conditions**

FNR frozen at -20ºC retain the activity for several months (Zanetti et al., 1984). FNR can be stored for longer periods (years) at -70ºC in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl. Addition of 1 mM 2-mercaptoethanol or DTT avoid covalent aggregation of some FNRs (Nakajima et al., 2002).

#### **3.2 Properties of the purified FNR**

#### **3.2.1 FNR spectroscopic properties**

The transition energies of the isoalloxazine portion of FAD are susceptible to variations by the near environment. These perturbations are detectable by different spectroscopic techniques, which provide value information about the structure and the functional performance of the prosthetic group and the flavoenzyme.

#### **3.2.2 Analysis of FNR by UV-visible spectroscopy**

The isoalloxazine, the structural component of the flavin involved in electron transfer, absorbs light in the UV and visible spectral range giving rise to the yellow appearance of flavin and flavoproteins (Latin: *flavus* = yellow) (Macheroux, 1999). In solution, the FAD shows a spectrum with maxima at 380 and 450 nm. The typical plant FNR spectrum displays peaks at 385 and 456 nm (Figure 3A, green line). The interaction of the protein scaffold with the isoalloxazine induces a decrease of the maximum energy levels of the isoalloxazine orbitals that results in spectral shifts to higher wavelengths. The presence of typical peaks or shoulders in the spectrum may indicate chemical variations in the flavin or in its protein environment (Figure 3A, red and blue lines), or the persistence of a bound nucleotide to the enzyme (Piubelli et al., 2000).

The UV-visible absorption spectroscopy can be used with different purposes. Thus, a simple UV-visible spectrum provides information about the quality of the FNR enzyme. Aggregates or contaminants that cause light dispersion may result in an absorbance increase at wavelengths below 600 nm (Figure 3A, grey line). Knowledge of the enzyme extinction coefficient can be helpful to assess the stoichiometry of the flavin and protein moiety. In general, one flavin per polypeptide is found. Substoichiometric amounts of flavin may indicate depletion of the flavin during purification and handling.

The UV-visible spectroscopy can be used to study the binding of substrates, substrate analogues and inhibitors (Aliverti et al., 1995, Medina et al., 2001, Paladini et al., 2009, Piubelli et al., 2000). Titration with the compound of interest yields the dissociation constant and information about changes that occur in the active site environment (Figure 3B) (Paladini et al., 2009). Besides, the spectrum shape can provide useful information about the nature of the interaction with substrates. Experimental data indicate that when NADP+ is present at saturating concentrations, the degree of nicotinamide ring occupancy of the binding site is 14-15% for pea FNR, as revealed by the extinction coefficient value of the peak near 510 nm in differential spectra elicited by pyridine nucleotide binding to the various pea FNR forms (Piubelli et al., 2000). This value has been used to analyse

The Plant–Type Ferredoxin-NADP<sup>+</sup>

obtained for wild-type enzyme.

**3.2.4 Use of CD spectroscopy** 

1993, Whitmore & Wallace, 2004).

2011).

group environment (Musumeci et al., 2011).

Reductases 547

the FAD in solution. Thus, it is possible follow the process of FAD dissociation by measuring the increase in FAD fluorescence of a FNR sample at different times. The obtained data can be used to calculate the FNR:FAD complex dissociation constant or the

Fig. 4. Application of FAD fluorescence to study structural features of FNR. A) Fluorescence spectra obtained upon titration with NADP+. Inset: The increase of fluorescence at 525 nm was plotted against NADP+ concentration, and then *Kd* value can be obtained by adjusting the plot to the sigmoidal function. B) Study of FAD accessibility by titration with a dynamic quencher (KI). Pea Y308S mutant in which the tyrosine facing the isoalloxazine was replaced by a serine shows an increase in the flavin exposure (red) with respect to wild-type FNR (green) (Paladini et al., 2009). C) Fluorescence in solution as a function of time of wild-type FNR (green) and C266M mutant (blue). The *koff* for FAD of the mutant is higher than the one

CD spectroscopy can be used for the structural analysis of plant FNRs. The far UV spectral region (240–190 nm or lower) correspond to the peptide bond chromospheres. The absorption consists of a weak but broad *n*→π\* transition centred around 210 nm and an intense π→π\* transition around 190 nm (Munro A.W. et al., 1999). Figure 5A shows a typical CD spectrum in the far-UV of a plant FNR properly folded. The spectrum can be used to assess the conservation of secondary structure of FNR and how it is modified by mutations or external factors such as temperature, salts or solvents. Besides, the far-UV region allows to calculate the content of different secondary structure elements (-helix, -sheet and random coil), which can be done using available software and servers (Deleage & Geourjon,

On the other hand, the aromatic residues are the main source of signals in the near UV region. These spectra can be used to obtain relevant information on perturbations in the tertiary structure (Figure 5B). Peaks in the near-UV and visible region (350–600 nm) are assigned to the bound FAD and can be applied to analyze perturbations of the prosthetic

The denaturation process of FNR can be followed by monitoring the CD signal at 220 nm whilst the temperature of the sample or the denaturant concentration is gradually increased (Figure 5C). A sigmoidal curve is obtained from which parameters associated to the transition and the stability of FNR can be calculated (Musumeci et al., 2008, Musumeci et al.,

By measuring CD spectra it is possible to assess whether the cofactor binding site in a FNR is more sensitive to the denaturant than the overall enzyme structure. The decrease of the

FAD dissociation rate constant (*koff*) (Musumeci et al., 2011, Paladini et al., 2009).

nicotinamide occupancy when NADP+ is bound to different mutant FNR enzymes (Figure 3C).

On the other hand, the FNR spectral UV-visible properties also depend on the redox state of the flavin. These spectral data are very useful to analyse the flavoprotein function (Macheroux, 1999, Nogues et al., 2004).

Fig. 3. Analysis of FNR by UV-visible spectroscopy. A) Absorption spectra of pea FNR obtained after 6 (green, good quality), 19 (blue) and 25 (red) hours of induction in *E. coli*. FNR preparation containing insoluble aggregates (grey line). B) Differential spectra obtained upon titration of wild-type FNR with NADP+. Inset: absorbance change at 515 nm plotted as function of NADP+ concentration. C) Differential spectra elicited by NADP+ binding to different FNR mutants (grey, yellow, cyan, red and blue corresponds to the spectra of wildtype, C266A, C266AL266A and C266M FNR forms respectively (Musumeci et al., 2008)).

#### **3.2.3 Application of fluorescence spectroscopy**

Plant FNRs display typical fluorescence spectra with emissions at 525-535 nm, which are obtained by exciting the sample at 450-460 nm (Figure 4A). The fluorescence of bound FAD is known to be largely quenched in the native oxidised form of wild-type FNR being 0.5- 0.75% of that of free FAD in solution (Forti, 1966; Shin, 1973). For fluorescence measurements, the FNR sample should be of the highest purity and diluted into a buffer solution of desired pH (e.g., Tris-HCl or sodium phosphate). Particulate matter should be removed by brief centrifugation at high speed. Passing the sample/buffer solution through a gel filtration column may also remove particles and improve the quality of collected data (Munro A.W. & Noble, 1999). Besides, this procedure is needed to remove the excess of free FAD that can interfere in the measurements.

It is possible to examine the interaction between FNR and substrates such as simple molecules as NADP+ or proteins as ferredoxin or flavodoxin by analysis of the FAD fluorescence. NADP+ binding induces an increase of FAD fluorescence which can be employed to study the interaction of FNR with its substrate (Figure 4A) (Musumeci et al., 2008). Similarly, quenching of FNR tryptophan fluorescence by ferredoxin or flavodoxin is used to investigate the Fd:FNR complex formation (Davis, 1990). The main advantage of this procedure is that samples with low concentration can be used.

The degree of flavin accessibility to the solvent molecules can be estimated by titration of the FNR sample with a dynamic quencher, as KI (Figure 4B). The steeper graph of FAD fluorescence quenching versus quencher concentration indicates higher accessibility of the prosthetic group in the FNR enzyme (Paladini et al., 2009).

Fluorescence also can be applied to study the FNR affinity for FAD (Figure 4C). Although FNRs have high affinity for FAD, they can slowly exchange the bound prosthetic group with

nicotinamide occupancy when NADP+ is bound to different mutant FNR enzymes (Figure

On the other hand, the FNR spectral UV-visible properties also depend on the redox state of the flavin. These spectral data are very useful to analyse the flavoprotein function

Fig. 3. Analysis of FNR by UV-visible spectroscopy. A) Absorption spectra of pea FNR obtained after 6 (green, good quality), 19 (blue) and 25 (red) hours of induction in *E. coli*. FNR preparation containing insoluble aggregates (grey line). B) Differential spectra obtained upon titration of wild-type FNR with NADP+. Inset: absorbance change at 515 nm plotted as function of NADP+ concentration. C) Differential spectra elicited by NADP+ binding to different FNR mutants (grey, yellow, cyan, red and blue corresponds to the spectra of wildtype, C266A, C266AL266A and C266M FNR forms respectively (Musumeci et al., 2008)).

Plant FNRs display typical fluorescence spectra with emissions at 525-535 nm, which are obtained by exciting the sample at 450-460 nm (Figure 4A). The fluorescence of bound FAD is known to be largely quenched in the native oxidised form of wild-type FNR being 0.5- 0.75% of that of free FAD in solution (Forti, 1966; Shin, 1973). For fluorescence measurements, the FNR sample should be of the highest purity and diluted into a buffer solution of desired pH (e.g., Tris-HCl or sodium phosphate). Particulate matter should be removed by brief centrifugation at high speed. Passing the sample/buffer solution through a gel filtration column may also remove particles and improve the quality of collected data (Munro A.W. & Noble, 1999). Besides, this procedure is needed to remove the excess of free

It is possible to examine the interaction between FNR and substrates such as simple molecules as NADP+ or proteins as ferredoxin or flavodoxin by analysis of the FAD fluorescence. NADP+ binding induces an increase of FAD fluorescence which can be employed to study the interaction of FNR with its substrate (Figure 4A) (Musumeci et al., 2008). Similarly, quenching of FNR tryptophan fluorescence by ferredoxin or flavodoxin is used to investigate the Fd:FNR complex formation (Davis, 1990). The main advantage of this

The degree of flavin accessibility to the solvent molecules can be estimated by titration of the FNR sample with a dynamic quencher, as KI (Figure 4B). The steeper graph of FAD fluorescence quenching versus quencher concentration indicates higher accessibility of the

Fluorescence also can be applied to study the FNR affinity for FAD (Figure 4C). Although FNRs have high affinity for FAD, they can slowly exchange the bound prosthetic group with

3C).

(Macheroux, 1999, Nogues et al., 2004).

**3.2.3 Application of fluorescence spectroscopy** 

FAD that can interfere in the measurements.

procedure is that samples with low concentration can be used.

prosthetic group in the FNR enzyme (Paladini et al., 2009).

the FAD in solution. Thus, it is possible follow the process of FAD dissociation by measuring the increase in FAD fluorescence of a FNR sample at different times. The obtained data can be used to calculate the FNR:FAD complex dissociation constant or the FAD dissociation rate constant (*koff*) (Musumeci et al., 2011, Paladini et al., 2009).

Fig. 4. Application of FAD fluorescence to study structural features of FNR. A) Fluorescence spectra obtained upon titration with NADP+. Inset: The increase of fluorescence at 525 nm was plotted against NADP+ concentration, and then *Kd* value can be obtained by adjusting the plot to the sigmoidal function. B) Study of FAD accessibility by titration with a dynamic quencher (KI). Pea Y308S mutant in which the tyrosine facing the isoalloxazine was replaced by a serine shows an increase in the flavin exposure (red) with respect to wild-type FNR (green) (Paladini et al., 2009). C) Fluorescence in solution as a function of time of wild-type FNR (green) and C266M mutant (blue). The *koff* for FAD of the mutant is higher than the one obtained for wild-type enzyme.

#### **3.2.4 Use of CD spectroscopy**

CD spectroscopy can be used for the structural analysis of plant FNRs. The far UV spectral region (240–190 nm or lower) correspond to the peptide bond chromospheres. The absorption consists of a weak but broad *n*→π\* transition centred around 210 nm and an intense π→π\* transition around 190 nm (Munro A.W. et al., 1999). Figure 5A shows a typical CD spectrum in the far-UV of a plant FNR properly folded. The spectrum can be used to assess the conservation of secondary structure of FNR and how it is modified by mutations or external factors such as temperature, salts or solvents. Besides, the far-UV region allows to calculate the content of different secondary structure elements (-helix, -sheet and random coil), which can be done using available software and servers (Deleage & Geourjon, 1993, Whitmore & Wallace, 2004).

On the other hand, the aromatic residues are the main source of signals in the near UV region. These spectra can be used to obtain relevant information on perturbations in the tertiary structure (Figure 5B). Peaks in the near-UV and visible region (350–600 nm) are assigned to the bound FAD and can be applied to analyze perturbations of the prosthetic group environment (Musumeci et al., 2011).

The denaturation process of FNR can be followed by monitoring the CD signal at 220 nm whilst the temperature of the sample or the denaturant concentration is gradually increased (Figure 5C). A sigmoidal curve is obtained from which parameters associated to the transition and the stability of FNR can be calculated (Musumeci et al., 2008, Musumeci et al., 2011).

By measuring CD spectra it is possible to assess whether the cofactor binding site in a FNR is more sensitive to the denaturant than the overall enzyme structure. The decrease of the

The Plant–Type Ferredoxin-NADP<sup>+</sup>

as cytochrome *c* reductase activity.

Cenas et al., 2001, Tognetti et al., 2007).

Potassium ferricyanide

Table 1. Activities catalyzed by Ferredoxin-NADP+ reductase

indophenols viologens tetrazolium salts

Electron

NADPH

Reductases 549

NADP+ to the catalytic site, followed by the binding of reduced ferredoxin, which reduces FNR to the semiquinone state. Then, the oxidised ferredoxin is released and the binding of a second ferredoxin molecule carrying one electron proceeds in order to completely reduce the FNR. Finally, the FNR transfers two electrons to NADP+ and produces NADPH. The reverse reaction, which involves the electron transfer from NADPH to ferredoxin can be followed *in vitro* by using cytochrome *c* as final electron acceptor in a coupled assay known

Different activities have been associated with both soluble and membrane-bound FNR. Besides Fd, several other electron acceptors can participate in the reductase-mediated oxidation of NADPH, according to the Eqn. 3 (see the Introduction section), in which Aox and Ared are the oxidised and reduced forms of the electron acceptor, respectively. The term *n* equals one or two depending whether the oxidant behaves as a one- (ferricyanide, cytochromes) or two-electron carriers (NAD+, 2,6-dichlorophenol indophenol). A summary of the different reactions catalyzed by FNR is given in Table 1. To measure these reactions *in vitro* the commercially available reduced nucleotide can be employed. An alternative source of NADPH can be obtained using a regenerating system containing NADP+, glucose- 6-

Steady-state measurements have demonstrated that the diaphorase reactions proceeds through a two-step transfer "ping-pong" mechanism for either ferricyanide, indophenols dyes and tetrazolium salts (Forti & Sturani, 1968, Masaki et al., 1979, Nakamura & Kimura, 1971, Zanetti & Curti, 1981, Zanetti & Forti, 1966). The same kinetic mechanism holds for the transhydrogenase activity (Böger, 1971a, Shin & Pietro, 1968). Diaphorase activity is probably devoid of physiological meaning in most cases, but it has paid an enormous service to the understanding of FNR function and catalytic mechanism. Recently, emerging functions of these activities have been proposed for bacterial FNRs (Takeda et al., 2010, Yeom et al., 2009). Moreover, some of these artificial reactions might have technological relevance for bioremediation and pharmaceutical industry (Carrillo & Ceccarelli, 2003,

phosphate (0.3 mM) and glucose-6-phosphate dehydrogenase (1 unit/ml).

donor Electron acceptor Activity *kcat* (s-1) Ferredoxin NADP+ Fd-NADP(H) reductase 500

NADPH Fd-cytochrome *c* cytochrome *c* reductase 50-100 NADPH NAD+ Transhydrogenase 5-10 NADPH O2, Fd-O2, Fld-O2 Oxidase 0.5

Reduction of cytochrome *c* shows a strict requirement for ferredoxin. The reaction is most often described as consisting of two hemi-reactions: FNR-catalyzed reduction of ferredoxin by NADPH, and the subsequent reoxidation of the iron-sulfur protein by cytochrome *c*. It has been also proposed that the electrostatic complex Fd-cytochrome *c* is the true substrate for the reductase. Significant rates of oxygen uptake have been found to occur along with

Diaphorase

100–500 (depending on the electron acceptor)

ellipticity in the far UV region measures the disruption of secondary structure, while a similar change at the visible region can be used to assess the integrity of the binding site of the flavin cofactor (Munro A.W. et al., 1999).

Fig. 5. Structural study of FNRs by CD spectroscopy. A and B) Typical CD spectrum in the far-UV (A) and near-UV (B) regions of plant FNR properly folded (pea FNR). C) Thermal unfolding of different FNRs. From these curves it is possible to estimate the parameters associated to FNR stability. Green: pea FNR; red: *E. coli* FNR; blue: *Xanthomonas axonopodis* FNR.

For CD spectroscopy the FNR sample concentration should be in the range of 0.5–3 µM for the far UV and 20-30 µM for the near UV. It is essential to minimize the absorption due to other components in the mixture (buffers, electrolytes, detergents, etc.), which can be achieved using 0.1 cm path length cuvettes. For the analysis of the far UV region of the FNR CD spectrum (190 nm or lower) suitable buffer systems as phosphate or borate, should be chosen and high chloride concentrations (> 50 mM) should be avoided (Munro A.W. et al., 1999).

#### **3.2.5 Application of RMN to study FNRs**

al., 2007)

RMN studies can be employed to assess the mobility of different FNR regions and to identify the amino acids residues that interact with nucleotide and protein substrates. Using this technique the formation of the catalytic competent complex of FNR and substrates was analysed, providing evidences of the movement of the carboxy-terminal region of the enzyme (Maeda et al., 2005). On the other hand, analysis of H/D exchange has provided relevant information about how FNR is modulated by pH, and that the NADP+ binding domain is made of two subdomains with different dynamic behaviours. The flexible subdomain may be important for controlling the binding affinity of FNR for NADP+ (Lee et

### **3.3 Natural and artificial substrates and Kinetic properties**

The optimum pH for FNR activity is in the range of 7.5-8.0 (Masaki et al., 1982, Melamed-Harel et al., 1985) which is coincident value with the pH of the chloroplast stroma during the light step of photosynthesis (Heldt et al., 1973, Werdan et al., 1975). The FNR isolectric point varies in the range of 5.5–6.8 (Grzyb et al., 2008), thus FNRs would be negatively charged at the stromal pH which would enable it to interact with redox partners. With respect to the photosynthetic reaction, the mechanism of electron transfer from reduced ferredoxin to NADP+ was characterized *in vitro* and it was postulated to occur as a twosubstrate process (Carrillo & Ceccarelli, 2003). The first step involves the access of the

ellipticity in the far UV region measures the disruption of secondary structure, while a similar change at the visible region can be used to assess the integrity of the binding site of

Fig. 5. Structural study of FNRs by CD spectroscopy. A and B) Typical CD spectrum in the far-UV (A) and near-UV (B) regions of plant FNR properly folded (pea FNR). C) Thermal unfolding of different FNRs. From these curves it is possible to estimate the parameters associated to FNR stability. Green: pea FNR; red: *E. coli* FNR; blue: *Xanthomonas axonopodis*

For CD spectroscopy the FNR sample concentration should be in the range of 0.5–3 µM for the far UV and 20-30 µM for the near UV. It is essential to minimize the absorption due to other components in the mixture (buffers, electrolytes, detergents, etc.), which can be achieved using 0.1 cm path length cuvettes. For the analysis of the far UV region of the FNR CD spectrum (190 nm or lower) suitable buffer systems as phosphate or borate, should be chosen and high chloride concentrations (> 50 mM) should be avoided (Munro A.W. et al.,

RMN studies can be employed to assess the mobility of different FNR regions and to identify the amino acids residues that interact with nucleotide and protein substrates. Using this technique the formation of the catalytic competent complex of FNR and substrates was analysed, providing evidences of the movement of the carboxy-terminal region of the enzyme (Maeda et al., 2005). On the other hand, analysis of H/D exchange has provided relevant information about how FNR is modulated by pH, and that the NADP+ binding domain is made of two subdomains with different dynamic behaviours. The flexible subdomain may be important for controlling the binding affinity of FNR for NADP+ (Lee et

The optimum pH for FNR activity is in the range of 7.5-8.0 (Masaki et al., 1982, Melamed-Harel et al., 1985) which is coincident value with the pH of the chloroplast stroma during the light step of photosynthesis (Heldt et al., 1973, Werdan et al., 1975). The FNR isolectric point varies in the range of 5.5–6.8 (Grzyb et al., 2008), thus FNRs would be negatively charged at the stromal pH which would enable it to interact with redox partners. With respect to the photosynthetic reaction, the mechanism of electron transfer from reduced ferredoxin to NADP+ was characterized *in vitro* and it was postulated to occur as a twosubstrate process (Carrillo & Ceccarelli, 2003). The first step involves the access of the

the flavin cofactor (Munro A.W. et al., 1999).

**3.2.5 Application of RMN to study FNRs** 

**3.3 Natural and artificial substrates and Kinetic properties** 

FNR.

1999).

al., 2007)

NADP+ to the catalytic site, followed by the binding of reduced ferredoxin, which reduces FNR to the semiquinone state. Then, the oxidised ferredoxin is released and the binding of a second ferredoxin molecule carrying one electron proceeds in order to completely reduce the FNR. Finally, the FNR transfers two electrons to NADP+ and produces NADPH. The reverse reaction, which involves the electron transfer from NADPH to ferredoxin can be followed *in vitro* by using cytochrome *c* as final electron acceptor in a coupled assay known as cytochrome *c* reductase activity.

Different activities have been associated with both soluble and membrane-bound FNR. Besides Fd, several other electron acceptors can participate in the reductase-mediated oxidation of NADPH, according to the Eqn. 3 (see the Introduction section), in which Aox and Ared are the oxidised and reduced forms of the electron acceptor, respectively. The term *n* equals one or two depending whether the oxidant behaves as a one- (ferricyanide, cytochromes) or two-electron carriers (NAD+, 2,6-dichlorophenol indophenol). A summary of the different reactions catalyzed by FNR is given in Table 1. To measure these reactions *in vitro* the commercially available reduced nucleotide can be employed. An alternative source of NADPH can be obtained using a regenerating system containing NADP+, glucose- 6 phosphate (0.3 mM) and glucose-6-phosphate dehydrogenase (1 unit/ml).

Steady-state measurements have demonstrated that the diaphorase reactions proceeds through a two-step transfer "ping-pong" mechanism for either ferricyanide, indophenols dyes and tetrazolium salts (Forti & Sturani, 1968, Masaki et al., 1979, Nakamura & Kimura, 1971, Zanetti & Curti, 1981, Zanetti & Forti, 1966). The same kinetic mechanism holds for the transhydrogenase activity (Böger, 1971a, Shin & Pietro, 1968). Diaphorase activity is probably devoid of physiological meaning in most cases, but it has paid an enormous service to the understanding of FNR function and catalytic mechanism. Recently, emerging functions of these activities have been proposed for bacterial FNRs (Takeda et al., 2010, Yeom et al., 2009). Moreover, some of these artificial reactions might have technological relevance for bioremediation and pharmaceutical industry (Carrillo & Ceccarelli, 2003, Cenas et al., 2001, Tognetti et al., 2007).


Table 1. Activities catalyzed by Ferredoxin-NADP+ reductase

Reduction of cytochrome *c* shows a strict requirement for ferredoxin. The reaction is most often described as consisting of two hemi-reactions: FNR-catalyzed reduction of ferredoxin by NADPH, and the subsequent reoxidation of the iron-sulfur protein by cytochrome *c*. It has been also proposed that the electrostatic complex Fd-cytochrome *c* is the true substrate for the reductase. Significant rates of oxygen uptake have been found to occur along with

The Plant–Type Ferredoxin-NADP<sup>+</sup>

other protein substrates (Bruns & Karplus, 1995).

in blue and Fd in orange. See text for details.

**5. Interaction of FNR with biological membranes** 

(Kurisu et al., 2001).

Reductases 551

but the edge of the dimethyl-benzyl ring is exposed and participates in electron transfer to

Fig. 6. Computer graphic based on X-ray diffraction data for FNR. A) Pea FNR. B) Detailed view of the isoalloxazine ring system and the NADP+ binding in the FNR-Y308S mutant C) Representation of the superposition of the maize leaf bipartite FNR:Fd complex and the pea FNR:NADP(H) complex. The FAD binding domain of FNR is shown in pink, the NADP(H) binding domain in light blue, the FAD prosthetic group in yellow, the NADP+

Other crystal structures of wild-type FNRs with NADP+ bound have been obtained. In these structures the P-AMP portion of NADP+ is properly located and the nicotinamide is far from the catalytic site (Bruns & Karplus, 1995, Nascimento et al., 2007, Serre et al., 1996). It has been suggested that this NADP+ conformations may represent different intermediate steps that occur during substrate binding and catalysis (Serre et al., 1996, Tejero et al., 2005). Structural studies of binary complexes of oxidised FNR and Fd were resolved by X-ray crystallography for both the *Anabaena* and maize pairs (Kurisu et al., 2001, Morales et al., 2000). The [2S–2Fe] cluster of Fd and the FAD of FNR are in the appropriate proximity (6.0 Å) for electron transfer. Fd binds to a concave region of the FAD domain of maize FNR burying about 5% and 15% of the total surface areas of FNR and Fd, respectively, using in maize and *Anabaena* complexes the same FNR surface region for the interaction with Fd

During the early research on FNR, it was observed that the photosynthetic enzyme was mainly located on the outer surface of the thylakoid membrane (Berzborn, 1968, Berzborn, 1969), in agreement with its important roles in the photosynthetic electron transport. For years, the nature of the interaction of the enzyme with the membrane has been matters of research and debate. Three pools of FNR, soluble, loosely- and tightly- bound to the membrane are dynamically interchanged and may have a metabolic relevance (Carrillo & Vallejos, 1982, Matthijs et al., 1986, Mulo, 2011). In *Arabidopsis* and maize instead of a unique FNR which equilibrates between the membrane and the stroma, distinct FNRs have been identified (Hanke et al., 2005). These FNR isoforms display differential partitioning between the stroma and the membrane in response to their tissue localization and metabolic state of

citochrome *c* reductase activity under aerobic conditions (Carrillo & Vallejos, 1987). The oxidase activity of FNR is very low (Carrillo & Vallejos, 1987, Gomez-Moreno et al., 1994). This reaction is enhanced several-fold by different electronic acceptors, including oneelectron reduced ferredoxin or flavodoxin, viologens, nitroderivates and quinones, that can readily engage in oxygen-dependent redox cycling leading to superoxide formation (Gomez-Moreno et al., 1994, Shah & Spain, 1996). In addition, FNR catalyses the transhydrogenation between NADPH and NAD+ (Böger, 1971b).

The stopped-flow technique allows to study fast enzyme kinetic events in the range of milliseconds. This approach has been employed to characterize pre-steady state processes which involve the interaction and electron transfer between oxidised FNR and NADPH, reduced FNR and NADP+ and reduced ferredoxin or flavodoxin and oxidised FNR (Anusevicius et al., 2005, Hurley et al., 1995, Martinez-Julvez et al., 1998, Medina et al., 2001, Paladini et al., 2009, Tejero et al., 2007).

The first reduction of pea FNR by Fd produces a semiquinone form too fast to be measured by rapid mixing techniques (Carrillo & Ceccarelli, 2003) and references therein. Fortunately, this step was characterized for *Anabaena* FNR (Hurley et al., 1995, Martinez-Julvez et al., 1998). The electron transfer processes that involve the dissociation of oxidised ferredoxin, the binding of reduced ferredoxin and flavin reduction occurs more slowly and have been characterized in wild-type and mutant enzymes (Medina, 2009, Nogues et al., 2004).

#### **4. Crystal structure of FNR and its complex with natural substrates**

The understanding of the FNR structure–function relationships available nowadays derives from a combination of extensive biochemical studies with the structural analysis of wildtype and mutant FNR enzymes from a variety of sources.

Three-dimensional models of oxidised and fully reduced forms of different FNRs have been reported (Bruns & Karplus, 1995, Correll et al., 1993, Deng et al., 1999, Dorowski et al., 2000, Karplus et al., 1991, Kurisu et al., 2001). A resolution of up to 1.05 Å has been recently obtained for the enzyme from maize (Tronrud et al., 2010). A close related enzyme from the photosynthetic organism *Anabaena variabilis* has been crystallized and their structure resolved (Serre et al., 1996).

All FNRs are similar at the topology level. They are made up of two structural domains, each containing approximately 150 amino acids (Fig. 6A). The amino terminal of ca. 150 residues form a -barrel FAD-binding domain (Fig. 6A, pink) whereas the carboxy-terminal region includes most of the residues involved in NADP(H) binding and displays a characteristic -helix/-strand fold (Fig. 6A, light blue). The FAD in plastidic FNRs is bound in an extended conformation through hydrogen bonds and van der Waals contacts (Bruns & Karplus, 1995, Serre et al., 1996), which is primarily obtained by the interaction of the AMP moiety of FAD with a strand-loop-strand -hairpin motif of the protein that is partially absent in bacterial FNRs. The isoalloxazine stacks between the aromatic side chains of two tyrosine residues, Tyr89 on the *si*-face and Tyr308 on the *re*-face (Fig. 6B, numbers as in pea FNR). The phenol ring of Tyr308 is the carboxy terminus in pea FNR as in all plastidic type FNRs and is coplanar to the flavin in such a way as to maximize -orbital overlap. Consequently, the NADP(H) nicotinamide should displace the terminal tyrosine for productive binding and catalysis (Fig. 6B) (Deng et al., 1999, Karplus et al., 1991, Musumeci et al., 2008, Tejero et al., 2005). Most of the isoalloxazine moiety is shielded from the solution

citochrome *c* reductase activity under aerobic conditions (Carrillo & Vallejos, 1987). The oxidase activity of FNR is very low (Carrillo & Vallejos, 1987, Gomez-Moreno et al., 1994). This reaction is enhanced several-fold by different electronic acceptors, including oneelectron reduced ferredoxin or flavodoxin, viologens, nitroderivates and quinones, that can readily engage in oxygen-dependent redox cycling leading to superoxide formation (Gomez-Moreno et al., 1994, Shah & Spain, 1996). In addition, FNR catalyses the

The stopped-flow technique allows to study fast enzyme kinetic events in the range of milliseconds. This approach has been employed to characterize pre-steady state processes which involve the interaction and electron transfer between oxidised FNR and NADPH, reduced FNR and NADP+ and reduced ferredoxin or flavodoxin and oxidised FNR (Anusevicius et al., 2005, Hurley et al., 1995, Martinez-Julvez et al., 1998, Medina et al., 2001,

The first reduction of pea FNR by Fd produces a semiquinone form too fast to be measured by rapid mixing techniques (Carrillo & Ceccarelli, 2003) and references therein. Fortunately, this step was characterized for *Anabaena* FNR (Hurley et al., 1995, Martinez-Julvez et al., 1998). The electron transfer processes that involve the dissociation of oxidised ferredoxin, the binding of reduced ferredoxin and flavin reduction occurs more slowly and have been

The understanding of the FNR structure–function relationships available nowadays derives from a combination of extensive biochemical studies with the structural analysis of wild-

Three-dimensional models of oxidised and fully reduced forms of different FNRs have been reported (Bruns & Karplus, 1995, Correll et al., 1993, Deng et al., 1999, Dorowski et al., 2000, Karplus et al., 1991, Kurisu et al., 2001). A resolution of up to 1.05 Å has been recently obtained for the enzyme from maize (Tronrud et al., 2010). A close related enzyme from the photosynthetic organism *Anabaena variabilis* has been crystallized and their structure

All FNRs are similar at the topology level. They are made up of two structural domains, each containing approximately 150 amino acids (Fig. 6A). The amino terminal of ca. 150 residues form a -barrel FAD-binding domain (Fig. 6A, pink) whereas the carboxy-terminal region includes most of the residues involved in NADP(H) binding and displays a characteristic -helix/-strand fold (Fig. 6A, light blue). The FAD in plastidic FNRs is bound in an extended conformation through hydrogen bonds and van der Waals contacts (Bruns & Karplus, 1995, Serre et al., 1996), which is primarily obtained by the interaction of the AMP moiety of FAD with a strand-loop-strand -hairpin motif of the protein that is partially absent in bacterial FNRs. The isoalloxazine stacks between the aromatic side chains of two tyrosine residues, Tyr89 on the *si*-face and Tyr308 on the *re*-face (Fig. 6B, numbers as in pea FNR). The phenol ring of Tyr308 is the carboxy terminus in pea FNR as in all plastidic type FNRs and is coplanar to the flavin in such a way as to maximize -orbital overlap. Consequently, the NADP(H) nicotinamide should displace the terminal tyrosine for productive binding and catalysis (Fig. 6B) (Deng et al., 1999, Karplus et al., 1991, Musumeci et al., 2008, Tejero et al., 2005). Most of the isoalloxazine moiety is shielded from the solution

characterized in wild-type and mutant enzymes (Medina, 2009, Nogues et al., 2004).

**4. Crystal structure of FNR and its complex with natural substrates** 

type and mutant FNR enzymes from a variety of sources.

transhydrogenation between NADPH and NAD+ (Böger, 1971b).

Paladini et al., 2009, Tejero et al., 2007).

resolved (Serre et al., 1996).

but the edge of the dimethyl-benzyl ring is exposed and participates in electron transfer to other protein substrates (Bruns & Karplus, 1995).

Fig. 6. Computer graphic based on X-ray diffraction data for FNR. A) Pea FNR. B) Detailed view of the isoalloxazine ring system and the NADP+ binding in the FNR-Y308S mutant C) Representation of the superposition of the maize leaf bipartite FNR:Fd complex and the pea FNR:NADP(H) complex. The FAD binding domain of FNR is shown in pink, the NADP(H) binding domain in light blue, the FAD prosthetic group in yellow, the NADP+ in blue and Fd in orange. See text for details.

Other crystal structures of wild-type FNRs with NADP+ bound have been obtained. In these structures the P-AMP portion of NADP+ is properly located and the nicotinamide is far from the catalytic site (Bruns & Karplus, 1995, Nascimento et al., 2007, Serre et al., 1996). It has been suggested that this NADP+ conformations may represent different intermediate steps that occur during substrate binding and catalysis (Serre et al., 1996, Tejero et al., 2005). Structural studies of binary complexes of oxidised FNR and Fd were resolved by X-ray crystallography for both the *Anabaena* and maize pairs (Kurisu et al., 2001, Morales et al., 2000). The [2S–2Fe] cluster of Fd and the FAD of FNR are in the appropriate proximity (6.0 Å) for electron transfer. Fd binds to a concave region of the FAD domain of maize FNR burying about 5% and 15% of the total surface areas of FNR and Fd, respectively, using in maize and *Anabaena* complexes the same FNR surface region for the interaction with Fd (Kurisu et al., 2001).

#### **5. Interaction of FNR with biological membranes**

During the early research on FNR, it was observed that the photosynthetic enzyme was mainly located on the outer surface of the thylakoid membrane (Berzborn, 1968, Berzborn, 1969), in agreement with its important roles in the photosynthetic electron transport. For years, the nature of the interaction of the enzyme with the membrane has been matters of research and debate. Three pools of FNR, soluble, loosely- and tightly- bound to the membrane are dynamically interchanged and may have a metabolic relevance (Carrillo & Vallejos, 1982, Matthijs et al., 1986, Mulo, 2011). In *Arabidopsis* and maize instead of a unique FNR which equilibrates between the membrane and the stroma, distinct FNRs have been identified (Hanke et al., 2005). These FNR isoforms display differential partitioning between the stroma and the membrane in response to their tissue localization and metabolic state of

The Plant–Type Ferredoxin-NADP<sup>+</sup>

al., 2004).

**6. Concluding remarks** 

Reductases 553

the crystal structure of the PsbQ (the 17,5 kDa identified as the reductase binding protein) on residues Pro9-Pro10-Pro11-Pro12, which forms a typical left-handed helix (or a polyproline type II structure) (Balsera et al., 2005). These structural elements have been implicated in protein–protein interactions in various cytosolic signal transduction pathways and eucariotic proteins. In addition, the carboxy terminus of TROL may have a similar structure as observed by sequence comparison (Juric et al., 2009). Thus, all proteins that

FNR interacts with different integral components of the thylakoid membrane. Several year ago Wagner et al by using flash kinetic spectroscopy demonstrated that the enzyme undergoes a very rapid rotational diffusion on the thylakoid membrane surface (Wagner et al., 1982). This movement was significantly reduced upon addition of Fd due to the formation of a ternary complex between the reductase and the photosystem I protein complex. Direct interaction with subunit of PSI was also detected (Andersen et al., 1992), but the relevance or significance of this interaction is unknown. As in the case of PSI, FNR has also been detected in citochrome *b*6*f* preparations (Clark et al., 1984, Zhang et al., 2001). FNR associated with the cytochrome *b*₆*f* complex can participate in the cyclic electron transport as PSI-plastoquinone or NADPH-plastoquinone oxidoreductases, which can explain the

Some authors have identified FNR in the Ndh complex. The NAD(P)H dehydrogenase (Ndh) complex in chloroplast thylakoid membranes functions in cyclic electron transfer and in chlororespiration. Ndh is composed of at least 15 subunits, including both chloroplastand nuclear-encoded proteins (Suorsa et al., 2009). As already mentioned, among the detected subunits of the Ndh complex is the PsbQ which was found associated with FNR. However, the amount of Ndh complex is a minor component of the chloroplast and it is present at ~1.5% of the level of PSII on a molar basis (Burrows et al., 1998) and probably makes a small contribution to the total cyclic electron transport measured *in vivo* (Joliot et

FNR is an abundant protein in chloroplast. Moreover, protein concentration in the chloroplast stroma was determined to be within the range of 520 to 730 g/dm3 for spinach and pea (Lilley, 1983). FNR structure is suitable for interaction with membrane and proteins. It is therefore likely to detect many interactions of the FNR with other structures inside the

For over six decades much effort has been devoted to the elucidation of the structure, catalytic mechanism and regulation of the FNR. Similarly, enormous progress has been made in understanding the insertion and importance of this enzyme on the various metabolic processes in plants and other organisms. The ability to produce FNR by transgenic expression and the modern techniques available nowadays have provided the means to analyze more deeply this flavoprotein. However, multiple aspects of this enzyme are still unknown as the structural bases for the increase of its catalytic competence and the ways to change the nucleotide substrate specificity. Furthermore, the enzyme may be an attractive target for the development of new herbicides and for the design of bactericides. Likewise, the ability of FNR to metabolize xenobiotics and environmental pollutants remain to be exploited. The widespread distribution of FNR will allow in the near future to increase the knowledge of the enzyme by comparative analysis of FNRs from different backgrounds

have been identified up to date to firmly interact with FNR contain this structure.

presence of FNR in citochrome *b*6*f* preparations (Szymanska et al., 2011).

chloroplast, many of which may have metabolic relevance.

the chloroplast (Grzyb et al., 2008, Hanke et al., 2005, Lintala et al., 2007, Okutani et al., 2005, Zanetti & Arosio, 1980).

FNR solubilisation from the membrane plays a role in maintaining the NADPH/NADP+ homeostasis of the stressed plastid. The enzyme changes its function from a membranebound NADPH producer to a soluble NADPH consumer to overcome the accumulation of NADPH. The excess of reducing power might otherwise increase the risk of oxidative damage through the production of hydroxyl radicals (Palatnik et al., 1997).

The persistence of a fraction that remains tightly bound to the membrane has drive to several research groups to search for the existence of an internal membrane protein that acts as binding site for the reductase. The soluble and membrane-bound forms of FNR show different allotropic properties which may reflect conformational changes of the enzyme upon binding to the membrane (Carrillo et al., 1981, Schneeman & Krogmann, 1975). In this way, the membrane component may serve to transmit internal thylakoid conditions as pH changes and membrane fluidity to the bound reductase, which then changes its kinetics behaviour. Several years ago a polypeptide of 17.5 kDa was identified as the binding protein for the reductase (Ceccarelli et al., 1985, Chan et al., 1987, Vallejos et al., 1984). The 17.5-FNR complex was detected in different higher plants, including C3, C4, and Crassulacean acid metabolism species (Soncini & Vallejos, 1989). During solubilisation of FNR under stress condition the reductase-binding protein was released together with FNR, suggesting that it might be the target of some regulation of the membrane bound state (Palatnik et al., 1997). The reductase-binding protein was identified to be the same than the PsbQ like protein (Soncini & Vallejos, 1989). This localization challenges its participation as a FNR binding protein. It has been proposed that the PsbQ may also contribute to the integrity of grana thylakoids stacks (Anderson et al., 2008). Moreover, PsbQ-like homologs have been identified as essential members of the chloroplast NAD(P)H dehydrogenase complex in *Arabidopsis* (Yabuta et al., 2010). The reductase-binding protein (PsbQ like protein) may probably has a ubiquitous function on the thylakoid membrane structure. That makes this topic an interesting issue to pursue.

More recently, a thylakoidial transmembrane protein with a rhodanase like structure was identified as binding site for FNR (Juric et al., 2009). This integral membrane protein contains a conserved carboxy-terminal domain that interacts with high affinity with the FNR enzyme. Three imperfect of such domain were found in one of the proteins of the translocon at the inner envelope membrane of chloroplasts Tic62. Tic62 was proposed as a redox sensor of the translocon and may possibly act as a regulator during the translocation process and serves as a docking site for FNR. Moreover, Tic62 was found bound to thylakoids, soluble in the chloroplast stroma and attached to the inner membrane of the organelle envelope (Benz et al., 2009, Peltier et al., 2004).

An unquestionable evidence of the role of TROL protein in FNR binding is that its absence in knockout plants disables FNR from being tethered to the membrane. In these plants, a decrease of the electron transfer rates under high light intensity was detected (Juric et al., 2009). Nevertheless, the authors suggest that TROL is an important, but not exclusive site for FNR since the enzyme was found in a number of other protein complexes.

It was recently observed by crystallographic studies that the FNR-binding motif is a polyproline type II helix, which is located in the carboxy terminus of Tic62 (Alte et al., 2010). The polyproline type II helix mediates self-assembly of the FNR dimer without influence on the active sites of the enzyme. Interestingly, this type of secondary structure was detected on

the chloroplast (Grzyb et al., 2008, Hanke et al., 2005, Lintala et al., 2007, Okutani et al., 2005,

FNR solubilisation from the membrane plays a role in maintaining the NADPH/NADP+ homeostasis of the stressed plastid. The enzyme changes its function from a membranebound NADPH producer to a soluble NADPH consumer to overcome the accumulation of NADPH. The excess of reducing power might otherwise increase the risk of oxidative

The persistence of a fraction that remains tightly bound to the membrane has drive to several research groups to search for the existence of an internal membrane protein that acts as binding site for the reductase. The soluble and membrane-bound forms of FNR show different allotropic properties which may reflect conformational changes of the enzyme upon binding to the membrane (Carrillo et al., 1981, Schneeman & Krogmann, 1975). In this way, the membrane component may serve to transmit internal thylakoid conditions as pH changes and membrane fluidity to the bound reductase, which then changes its kinetics behaviour. Several years ago a polypeptide of 17.5 kDa was identified as the binding protein for the reductase (Ceccarelli et al., 1985, Chan et al., 1987, Vallejos et al., 1984). The 17.5-FNR complex was detected in different higher plants, including C3, C4, and Crassulacean acid metabolism species (Soncini & Vallejos, 1989). During solubilisation of FNR under stress condition the reductase-binding protein was released together with FNR, suggesting that it might be the target of some regulation of the membrane bound state (Palatnik et al., 1997). The reductase-binding protein was identified to be the same than the PsbQ like protein (Soncini & Vallejos, 1989). This localization challenges its participation as a FNR binding protein. It has been proposed that the PsbQ may also contribute to the integrity of grana thylakoids stacks (Anderson et al., 2008). Moreover, PsbQ-like homologs have been identified as essential members of the chloroplast NAD(P)H dehydrogenase complex in *Arabidopsis* (Yabuta et al., 2010). The reductase-binding protein (PsbQ like protein) may probably has a ubiquitous function on the thylakoid membrane structure. That makes this

More recently, a thylakoidial transmembrane protein with a rhodanase like structure was identified as binding site for FNR (Juric et al., 2009). This integral membrane protein contains a conserved carboxy-terminal domain that interacts with high affinity with the FNR enzyme. Three imperfect of such domain were found in one of the proteins of the translocon at the inner envelope membrane of chloroplasts Tic62. Tic62 was proposed as a redox sensor of the translocon and may possibly act as a regulator during the translocation process and serves as a docking site for FNR. Moreover, Tic62 was found bound to thylakoids, soluble in the chloroplast stroma and attached to the inner membrane of the

An unquestionable evidence of the role of TROL protein in FNR binding is that its absence in knockout plants disables FNR from being tethered to the membrane. In these plants, a decrease of the electron transfer rates under high light intensity was detected (Juric et al., 2009). Nevertheless, the authors suggest that TROL is an important, but not exclusive site for

It was recently observed by crystallographic studies that the FNR-binding motif is a polyproline type II helix, which is located in the carboxy terminus of Tic62 (Alte et al., 2010). The polyproline type II helix mediates self-assembly of the FNR dimer without influence on the active sites of the enzyme. Interestingly, this type of secondary structure was detected on

FNR since the enzyme was found in a number of other protein complexes.

damage through the production of hydroxyl radicals (Palatnik et al., 1997).

Zanetti & Arosio, 1980).

topic an interesting issue to pursue.

organelle envelope (Benz et al., 2009, Peltier et al., 2004).

the crystal structure of the PsbQ (the 17,5 kDa identified as the reductase binding protein) on residues Pro9-Pro10-Pro11-Pro12, which forms a typical left-handed helix (or a polyproline type II structure) (Balsera et al., 2005). These structural elements have been implicated in protein–protein interactions in various cytosolic signal transduction pathways and eucariotic proteins. In addition, the carboxy terminus of TROL may have a similar structure as observed by sequence comparison (Juric et al., 2009). Thus, all proteins that have been identified up to date to firmly interact with FNR contain this structure.

FNR interacts with different integral components of the thylakoid membrane. Several year ago Wagner et al by using flash kinetic spectroscopy demonstrated that the enzyme undergoes a very rapid rotational diffusion on the thylakoid membrane surface (Wagner et al., 1982). This movement was significantly reduced upon addition of Fd due to the formation of a ternary complex between the reductase and the photosystem I protein complex. Direct interaction with subunit of PSI was also detected (Andersen et al., 1992), but the relevance or significance of this interaction is unknown. As in the case of PSI, FNR has also been detected in citochrome *b*6*f* preparations (Clark et al., 1984, Zhang et al., 2001). FNR associated with the cytochrome *b*₆*f* complex can participate in the cyclic electron transport as PSI-plastoquinone or NADPH-plastoquinone oxidoreductases, which can explain the presence of FNR in citochrome *b*6*f* preparations (Szymanska et al., 2011).

Some authors have identified FNR in the Ndh complex. The NAD(P)H dehydrogenase (Ndh) complex in chloroplast thylakoid membranes functions in cyclic electron transfer and in chlororespiration. Ndh is composed of at least 15 subunits, including both chloroplastand nuclear-encoded proteins (Suorsa et al., 2009). As already mentioned, among the detected subunits of the Ndh complex is the PsbQ which was found associated with FNR. However, the amount of Ndh complex is a minor component of the chloroplast and it is present at ~1.5% of the level of PSII on a molar basis (Burrows et al., 1998) and probably makes a small contribution to the total cyclic electron transport measured *in vivo* (Joliot et al., 2004).

FNR is an abundant protein in chloroplast. Moreover, protein concentration in the chloroplast stroma was determined to be within the range of 520 to 730 g/dm3 for spinach and pea (Lilley, 1983). FNR structure is suitable for interaction with membrane and proteins. It is therefore likely to detect many interactions of the FNR with other structures inside the chloroplast, many of which may have metabolic relevance.

#### **6. Concluding remarks**

For over six decades much effort has been devoted to the elucidation of the structure, catalytic mechanism and regulation of the FNR. Similarly, enormous progress has been made in understanding the insertion and importance of this enzyme on the various metabolic processes in plants and other organisms. The ability to produce FNR by transgenic expression and the modern techniques available nowadays have provided the means to analyze more deeply this flavoprotein. However, multiple aspects of this enzyme are still unknown as the structural bases for the increase of its catalytic competence and the ways to change the nucleotide substrate specificity. Furthermore, the enzyme may be an attractive target for the development of new herbicides and for the design of bactericides. Likewise, the ability of FNR to metabolize xenobiotics and environmental pollutants remain to be exploited. The widespread distribution of FNR will allow in the near future to increase the knowledge of the enzyme by comparative analysis of FNRs from different backgrounds

The Plant–Type Ferredoxin-NADP<sup>+</sup>

*24*, 436-446.

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*Biophys. Acta 1698*, 155-165.

61.

815.

338.

Reductases 555

Berzborn, R. (1968). On soluble and insoluble chloroplast antigens. Demonstration of

Berzborn, R. J. (1969). Studies on the surface structure of the thylakoid system of

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This work was supported by grants from CONICET, Agencia de Promoción Científica y Tecnológica (ANPCyT) and Fundación Bunge y Born, Argentina.

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studies of NADP+ binding at 2.25 A resolution. *J. Mol. Biol. 263*, 20-39. Setif, P. (2006). Electron Transfer from the Bound Iron–Sulfur Clusters to

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trinitrophenylmethylnitramine (tetryl) catalyzed by ferredoxin NADP+


**27** 

*USA* 

**Primary Production in the Ocean** 

*Department of Oceanography, Texas A & M University, College Station, Texas,* 

Primary productivity is the process by which inorganic forms of carbon are synthesized by living organisms into simple organic compounds. Most carbon on Earth is in inorganic

Inorganic carbon must be chemically reduced to form the organic molecules which are the building blocks of life and the mechanism by which energy is stored in living organisms. The reduction of inorganic carbon requires an investment of energy and this can come from light or from energy stored in some reduced inorganic compounds. Autotrophs are organisms capable of fixing inorganic carbon. Photoautotrophs use light energy to fix carbon, whereas chemoautotrophs use the energy released through the oxidation of reduced

Both photosynthesis and chemosynthesis contribute to the primary production of the oceans, however oxygenic photosynthesis is by far the dominant process in terms of the amount of carbon fixed and energy stored in organic compounds. Photosynthesis occurs in all parts of the ocean where there is sufficient light, whereas chemosynthesis is limited to locations where there are sufficient concentrations of reduced chemical substrates. Although the vast majority of the ocean's volume is too dark to support photosynthesis, organic carbon and energy is transferred to the dark waters via processes such as particle sinking and the vertical migrations of organisms. Almost all ecosystems in the ocean are fueled by organic carbon and energy which was initially fixed by oxygenic photosynthesis. Anoxygenic photosynthesis does occur in the ocean, however it is confined to anaerobic environments in which there is sufficient light or associated with aerobic anoxygenic photosynthesis (Kolber et al., 2000), the global significance of which is yet to be determined. Consequently, in this overview of primary production in the ocean I will focus on oxygenic

Blankenship (2002) defined photosynthesis as: 'a process in which light energy is captured and stored by an organism, and the stored energy is used to drive cellular processes.' Oxygenic photosynthesis may be expressed as an oxidation-reduction reaction in the form:

 2H2O + CO2 + light → (CH2O) + H2O + O2 (Falkowski & Raven, 2007) (1) In this reaction carbohydrate is formed from carbon dioxide and water with light providing the energy for the reduction of carbon dioxide. Equation 1 is an empirical summary of the overall reaction, which in reality occurs in a number of steps. The light energy for the

oxidized forms such as carbon dioxide (CO2), bicarbonate (HCO3-

inorganic substrates to fix carbon into organic compounds.

reaction is primarily absorbed by the green pigment chlorophyll.

**1. Introduction** 

photosynthesis.

Daniel Conrad Ogilvie Thornton

), and carbonate (CO32-).


### **Primary Production in the Ocean**

Daniel Conrad Ogilvie Thornton

*Department of Oceanography, Texas A & M University, College Station, Texas, USA* 

#### **1. Introduction**

562 Advances in Photosynthesis – Fundamental Aspects

Yabuta, S., Ifuku, K., Takabayashi, A., Ishihara, S., Ido, K., Ishikawa, N., Endo, T., & Sato, F.

Yeom, J., Jeon, C. O., Madsen, E. L., & Park, W. (2009). Ferredoxin-NADP+ reductase from *Pseudomonas putida* functions as a ferric reductase. *J. Bacteriol. 191*, 1472-1479. Zanetti, G., Aliverti, A., & Curti, B. (1984). A cross-linked complex between ferredoxin and

Zanetti, G. & Arosio, P. (1980). Solubilization from spinach thylakoids of a higher molecular weight form of ferredoxin-NADP+ reductase. *FEBS Lett 111*, 373-376. Zanetti, G. & Curti, B. (1981). Interactions between ferredoxin-NADP+ reductase and ferredoxin at different reduction levels of the two proteins. *FEBS Lett 129*, 201-204.

Zanetti, G. & Forti, G. (1966). Studies on the triphosphopyridine nucleotide-cytochrome *f*

Zhang, H., Whitelegge, J. P., & Cramer, W. A. (2001). Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome *b*6*f* complex. *J. Biol. Chem. 276*, 38159-38165. Zheng, M., Doan, B., Schneider, T. D., & Storz, G. (1999). OxyR and SoxRS regulation of fur.

Ziegler, G. A. & Schulz, G. E. (2000). Crystal structures of adrenodoxin reductase in complex

with NADP+ and NADPH suggesting a mechanism for the electron transfer of an

ferredoxin-NADP+ reductase. *J. Biol. Chem. 259*, 6153-6157.

reductase of chloroplasts. *J. Biol. Chem. 241*, 279-285.

enzyme family. *Biochemistry 39*, 10986-10995.

tobacco. *Plant Cell Physiol 47*, 1355-1371.

*J. Bacteriol. 181*, 4639-4643.

(2010). Three PsbQ-like proteins are required for the function of the chloroplast NAD(P)H dehydrogenase complex in *Arabidopsis*. *Plant Cell Physiol 51*, 866-876. Yamamoto, H., Kato, H., Shinzaki, Y., Horiguchi, S., Shikanai, T., Hase, T., Endo, T.,

Nishioka, M., Makino, A., Tomizawa, K., & Miyake, C. (2006). Ferredoxin limits cyclic electron flow around PSI (CEF-PSI) in higher plants-stimulation of CEF-PSI enhances non-photochemical quenching of Chl fluorescence in transplastomic

> Primary productivity is the process by which inorganic forms of carbon are synthesized by living organisms into simple organic compounds. Most carbon on Earth is in inorganic oxidized forms such as carbon dioxide (CO2), bicarbonate (HCO3 -), and carbonate (CO32-). Inorganic carbon must be chemically reduced to form the organic molecules which are the building blocks of life and the mechanism by which energy is stored in living organisms. The reduction of inorganic carbon requires an investment of energy and this can come from light or from energy stored in some reduced inorganic compounds. Autotrophs are organisms capable of fixing inorganic carbon. Photoautotrophs use light energy to fix carbon, whereas chemoautotrophs use the energy released through the oxidation of reduced inorganic substrates to fix carbon into organic compounds.

> Both photosynthesis and chemosynthesis contribute to the primary production of the oceans, however oxygenic photosynthesis is by far the dominant process in terms of the amount of carbon fixed and energy stored in organic compounds. Photosynthesis occurs in all parts of the ocean where there is sufficient light, whereas chemosynthesis is limited to locations where there are sufficient concentrations of reduced chemical substrates. Although the vast majority of the ocean's volume is too dark to support photosynthesis, organic carbon and energy is transferred to the dark waters via processes such as particle sinking and the vertical migrations of organisms. Almost all ecosystems in the ocean are fueled by organic carbon and energy which was initially fixed by oxygenic photosynthesis. Anoxygenic photosynthesis does occur in the ocean, however it is confined to anaerobic environments in which there is sufficient light or associated with aerobic anoxygenic photosynthesis (Kolber et al., 2000), the global significance of which is yet to be determined. Consequently, in this overview of primary production in the ocean I will focus on oxygenic photosynthesis.

> Blankenship (2002) defined photosynthesis as: 'a process in which light energy is captured and stored by an organism, and the stored energy is used to drive cellular processes.' Oxygenic photosynthesis may be expressed as an oxidation-reduction reaction in the form:

$$\text{2H}\_2\text{O} + \text{CO}\_2 + \text{light} \rightarrow \text{(CH=O)} + \text{H}\_2\text{O} + \text{O}\_2 \text{ (Falkowski é Raven, 2007)}\tag{1}$$

In this reaction carbohydrate is formed from carbon dioxide and water with light providing the energy for the reduction of carbon dioxide. Equation 1 is an empirical summary of the overall reaction, which in reality occurs in a number of steps. The light energy for the reaction is primarily absorbed by the green pigment chlorophyll.

Primary Production in the Ocean 565

in the western equatorial Pacific and up to 82 % in the subtropical north Pacific (Liu et al., 1997). *Prochlorococcus* is probably the most abundant photosynthetic organism on Earth. Important cyanophyceae include the coccoid *Synechococcus*, which makes a significant contribution to biomass and photosynthesis of the open ocean. For example, Morán et al. (2004) found that picophytoplankton dominated primary production in the North Atlantic subtropical gyre in 2001. *Synechococus* spp. contributed 3 and 10 % of the picophytoplankton biomass, respectively, in the subtropical and tropical domains. However, although *Synechococcus* spp. was significant, *Prochlorococcus* spp. dominated, contributing 69 % of

Diatoms (Heterokontophyta, Bacillariophyceae) are characterized by a cell wall composed of silica. Estimates of extant diatom species vary between 10,000 (Falkowski & Raven, 2007) and 100,000 (Falciatore & Bowler, 2002). They are found in a wide range of freshwater and marine environments, both in the water column and attached to surfaces. Diatoms make a significant contribution to global primary production on both a local and global scale. It is estimated that diatoms account for 40 to 45% of net oceanic productivity (approximately 20 Pg C yr-1; 1 Pg = 1015 g) or almost a quarter of the carbon fixed annually on Earth by photosynthesis (Mann 1999; Falciatore & Bowler 2002; Sarthou et al. 2005), though in my opinion, this is probably an overestimate. Phytoplankton populations in relatively cool, well mixed waters are often dominated by diatoms in terms of productivity and biomass. In addition, diatoms often dominate the microphytobenthos (Thornton et al., 2002), which are populations of microalgae inhabiting the surface layers of sediments in shallow, coastal waters where there is sufficient light reaching the seabed to support photosynthesis. Diatoms may form monospecific blooms of rapidly growing populations; for example, *Skeletonema costatum* frequently forms blooms in coastal waters (Gallagher, 1980; Han et al., 1992; Thornton et al., 1999). Diatom blooms often terminate with aggregate formation, which in addition to the fecal pellets produced by grazers, can lead to the rapid flux of carbon and other nutrients from surface waters to deeper water and the seafloor (Thornton,

Prymnesiophytes (Prymnesiophyta) are motile, unicellular phytoplankton with two flagella. Most genera also have a filamentous appendage located between the flagella called a haptonema, the function of which is unknown. The cell surface of most prymnesiophytes is covered in elliptical organic scales, which are calcified in many genera. These scales of calcium carbonate are called coccoliths and the prymnesiophytes which possess them are coccolithophores. Coccolithophores are common in warm tropical waters characterized by a low partial pressure of carbon dioxide and saturated or supersaturated with calcium carbonate (Lee, 2008). The importance of the coccolithophores as primary producers during Earth's history is exemplified by the thick chalk deposits found in many parts of the world, such as the white cliffs along the coast of southern England. These deposits were formed from coccoliths that sank to the bottom of warm, shallow seas during the Cretaceous geological period. Moreover, calcium carbonate is the largest reservoir of carbon on Earth. Blooms of coccolithophores such as *Emiliania huxleyi* may be extensive and have been observed on satellite images as milky patches covering large areas of ocean (Balch et al.

biomass to the subtropical and 52 % to the tropical domain.

**2.2.2 Diatoms** 

2002) (see section 7.1).

**2.2.3 Prymnesiophytes** 

#### **2. Which organisms are important primary producers in the ocean?**

In terms of number of species, phylogenetic diversity and contribution to total global primary production, the unicellular phytoplankton dominate primary production in the ocean (Falkowski et al*.*, 2004). Almost all oxygenic photosynthetic primary producers in the ocean are either cyanobacteria (Cyanophyta) or eukarytotic algae. The eukaryotic algae are a diverse polyphyletic group, including both unicellular and multicellular organisms.

#### **2.1 Multicellular primary producers**

Most multicellular primary producers grow attached to substrates, therefore they are usually restricted to the coastal margins of the ocean in shallow waters where there are both attachment sites and sufficient light for photosynthesis. Important primary producers include seagrasses that form beds that are rooted in sediments in shallow water in tropical and temperate latitudes. Seagrasses (e.g *Zostera*) are flowering plants, unlike the macroalgae, which are not flowering plants and are phylogenetically diverse. Kelps, such as *Macrocystis* and *Laminaria* are locally significant macroalgae in shallow temperate and subpolar waters where there are suitable hard substrates for attachment. Geider et al. (2001) estimated that the net annual primary production by saltmarshes, estuaries and macrophytes was 1.2 Pg C, which is a relatively small proportion of total annual marine production (see section 6).

Some macroalgae are found in the open ocean; *Sargassum* is planktonic and forms rafts at the sea surface in tropical waters (Barnes & Hughes, 1988), mainly in the Gulf of Mexico and Sargasso Sea. The biomass of rafts rival phytoplankton biomass in the mixed layer in the Gulf of Mexico (on an areal basis) with a total standing stock of 2 - 11 million metric tons (Lapointe, 1995; Gower & King, 2008).

#### **2.2 Phytoplankton**

There are several groups of eukaryotic phytoplankton which make a significant contribution to global primary production. The most significant of these groups are the diatoms, dinoflagelletes and prymnesiophytes. Diatoms and dinoflagellates are usually found in the microphytoplankton (20 – 200 µm), whereas the prymnesiophytes are nanophytoplankton (2 – 20 µm). Photosynthetic bacteria contribute to the picophytoplankton and are < 2 µm in diameter. Oxygenic photosynthetic bacteria in the oceans belong to the division Cyanophyta, which contains the cyanobacteria (cyanophyceae) and the prochlorophytes (prochlorophyceae).

#### **2.2.1 Photosynthetic bacteria**

Arguably, the most important discovery of 20th century biological oceanography was the major role that prokaryotes have in nutrient cycling and production in the water column. As detection and enumeration methods improved it became apparent that photosynthetic bacteria are ubiquitous and make a significant contribution to biomass and primary productivity. The prochlorophytes possess the photosynthetic pigment divinyl chlorophyll *a*, but not chlorophyll *a* which is found in all other Cyanophyta and eukaryotic algae in the ocean. The prochlorophyte *Prochlorococcus marinus* is an abundant and significant primary producer in the open ocean (Chisolm et al., 1988; Karl, 2002) and can be found in concentrations in excess of 105 cells ml-1 (Chisolm et al., 1988). *Prochlorococcus* has been shown to contribute 9 % of gross primary production in the eastern equatorial Pacific, 39 % in the western equatorial Pacific and up to 82 % in the subtropical north Pacific (Liu et al., 1997). *Prochlorococcus* is probably the most abundant photosynthetic organism on Earth. Important cyanophyceae include the coccoid *Synechococcus*, which makes a significant contribution to biomass and photosynthesis of the open ocean. For example, Morán et al. (2004) found that picophytoplankton dominated primary production in the North Atlantic subtropical gyre in 2001. *Synechococus* spp. contributed 3 and 10 % of the picophytoplankton biomass, respectively, in the subtropical and tropical domains. However, although *Synechococcus* spp. was significant, *Prochlorococcus* spp. dominated, contributing 69 % of biomass to the subtropical and 52 % to the tropical domain.

#### **2.2.2 Diatoms**

564 Advances in Photosynthesis – Fundamental Aspects

In terms of number of species, phylogenetic diversity and contribution to total global primary production, the unicellular phytoplankton dominate primary production in the ocean (Falkowski et al*.*, 2004). Almost all oxygenic photosynthetic primary producers in the ocean are either cyanobacteria (Cyanophyta) or eukarytotic algae. The eukaryotic algae are a

Most multicellular primary producers grow attached to substrates, therefore they are usually restricted to the coastal margins of the ocean in shallow waters where there are both attachment sites and sufficient light for photosynthesis. Important primary producers include seagrasses that form beds that are rooted in sediments in shallow water in tropical and temperate latitudes. Seagrasses (e.g *Zostera*) are flowering plants, unlike the macroalgae, which are not flowering plants and are phylogenetically diverse. Kelps, such as *Macrocystis* and *Laminaria* are locally significant macroalgae in shallow temperate and subpolar waters where there are suitable hard substrates for attachment. Geider et al. (2001) estimated that the net annual primary production by saltmarshes, estuaries and macrophytes was 1.2 Pg C, which is

Some macroalgae are found in the open ocean; *Sargassum* is planktonic and forms rafts at the sea surface in tropical waters (Barnes & Hughes, 1988), mainly in the Gulf of Mexico and Sargasso Sea. The biomass of rafts rival phytoplankton biomass in the mixed layer in the Gulf of Mexico (on an areal basis) with a total standing stock of 2 - 11 million metric tons

There are several groups of eukaryotic phytoplankton which make a significant contribution to global primary production. The most significant of these groups are the diatoms, dinoflagelletes and prymnesiophytes. Diatoms and dinoflagellates are usually found in the microphytoplankton (20 – 200 µm), whereas the prymnesiophytes are nanophytoplankton (2 – 20 µm). Photosynthetic bacteria contribute to the picophytoplankton and are < 2 µm in diameter. Oxygenic photosynthetic bacteria in the oceans belong to the division Cyanophyta, which contains the cyanobacteria (cyanophyceae) and the prochlorophytes

Arguably, the most important discovery of 20th century biological oceanography was the major role that prokaryotes have in nutrient cycling and production in the water column. As detection and enumeration methods improved it became apparent that photosynthetic bacteria are ubiquitous and make a significant contribution to biomass and primary productivity. The prochlorophytes possess the photosynthetic pigment divinyl chlorophyll *a*, but not chlorophyll *a* which is found in all other Cyanophyta and eukaryotic algae in the ocean. The prochlorophyte *Prochlorococcus marinus* is an abundant and significant primary producer in the open ocean (Chisolm et al., 1988; Karl, 2002) and can be found in concentrations in excess of 105 cells ml-1 (Chisolm et al., 1988). *Prochlorococcus* has been shown to contribute 9 % of gross primary production in the eastern equatorial Pacific, 39 %

**2. Which organisms are important primary producers in the ocean?** 

diverse polyphyletic group, including both unicellular and multicellular organisms.

a relatively small proportion of total annual marine production (see section 6).

**2.1 Multicellular primary producers** 

(Lapointe, 1995; Gower & King, 2008).

**2.2 Phytoplankton** 

(prochlorophyceae).

**2.2.1 Photosynthetic bacteria** 

Diatoms (Heterokontophyta, Bacillariophyceae) are characterized by a cell wall composed of silica. Estimates of extant diatom species vary between 10,000 (Falkowski & Raven, 2007) and 100,000 (Falciatore & Bowler, 2002). They are found in a wide range of freshwater and marine environments, both in the water column and attached to surfaces. Diatoms make a significant contribution to global primary production on both a local and global scale. It is estimated that diatoms account for 40 to 45% of net oceanic productivity (approximately 20 Pg C yr-1; 1 Pg = 1015 g) or almost a quarter of the carbon fixed annually on Earth by photosynthesis (Mann 1999; Falciatore & Bowler 2002; Sarthou et al. 2005), though in my opinion, this is probably an overestimate. Phytoplankton populations in relatively cool, well mixed waters are often dominated by diatoms in terms of productivity and biomass. In addition, diatoms often dominate the microphytobenthos (Thornton et al., 2002), which are populations of microalgae inhabiting the surface layers of sediments in shallow, coastal waters where there is sufficient light reaching the seabed to support photosynthesis. Diatoms may form monospecific blooms of rapidly growing populations; for example, *Skeletonema costatum* frequently forms blooms in coastal waters (Gallagher, 1980; Han et al., 1992; Thornton et al., 1999). Diatom blooms often terminate with aggregate formation, which in addition to the fecal pellets produced by grazers, can lead to the rapid flux of carbon and other nutrients from surface waters to deeper water and the seafloor (Thornton, 2002) (see section 7.1).

#### **2.2.3 Prymnesiophytes**

Prymnesiophytes (Prymnesiophyta) are motile, unicellular phytoplankton with two flagella. Most genera also have a filamentous appendage located between the flagella called a haptonema, the function of which is unknown. The cell surface of most prymnesiophytes is covered in elliptical organic scales, which are calcified in many genera. These scales of calcium carbonate are called coccoliths and the prymnesiophytes which possess them are coccolithophores. Coccolithophores are common in warm tropical waters characterized by a low partial pressure of carbon dioxide and saturated or supersaturated with calcium carbonate (Lee, 2008). The importance of the coccolithophores as primary producers during Earth's history is exemplified by the thick chalk deposits found in many parts of the world, such as the white cliffs along the coast of southern England. These deposits were formed from coccoliths that sank to the bottom of warm, shallow seas during the Cretaceous geological period. Moreover, calcium carbonate is the largest reservoir of carbon on Earth. Blooms of coccolithophores such as *Emiliania huxleyi* may be extensive and have been observed on satellite images as milky patches covering large areas of ocean (Balch et al.

Primary Production in the Ocean 567

by chlorophyll; light energy is either lost through fluorescence, heat dissipation or used in photochemistry (Maxwell & Johnson, 2000) and the balance between these processes changes depending on the physiological status of the phytoplankton, including rate of

A more accurate estimate of photosynthetic biomass than *in vivo* chlorophyll fluorescence may be obtained if the photosynthetic pigments are extracted from the organism. For water samples containing phytoplankton, a known volume is filtered onto a glass fiber filter and the photosynthetic pigments are extracted using a known volume of organic solvent such as acetone or methanol. The concentration of chlorophyll *a* is then measured in the extract by spectrophotometry or fluorescence (Parsons et al.*,* 1984; Jeffrey et al.*,* 1997). While still widely used, these relatively simple techniques have a number of drawbacks. Firstly, chlorophyll degradation products may absorb light at the same wavelengths as chlorophyll, leading to an overestimation of chlorophyll concentration (Wiltshire, 2009). Secondly, the emission spectra of chlorophyll *a* and *b* overlap, which will result in inaccurate measurement of chlorophyll *a* in water containing chlorophyll *b* containing organisms (Wiltshire, 2009). For the accurate measurement of chlorophyll *a*, other photosynthetic pigments, and their derivatives, high performance liquid chromatography (HPLC) methods should be used (see Wiltshire (2009) for description). HPLC enables the pigments to be separated by chromatography and therefore relatively pure pigments pass through a fluorescence or spectrophotometric detector. In addition to chlorophyll *a*, algae contain multiple pigments. These accessory pigments are often diagnostic for major taxonomic groups (see Table 10.1; Wiltshire, 2009). CHEMTAX (Mackay et al.*,* 1996) is a method by which the total amount of chlorophyll *a* can be allocated to the major taxonomic groups of algae based on the concentrations and ratios of accessory pigments. Thus, HPLC can be used to estimate phytoplankton biomass in terms of chlorophyll *a* and potentially determine the

The Coastal Zone Color Scanner (1978-1986) was the first satellite mission that measured chlorophyll *a* concentrations using top of the atmosphere radiances (McClain, 2009). The success of this mission led to a number of missions to measure ocean color at either global or regional spatial scales by Japanese, European and United States space agencies. As a result of these missions, we now have over 30 years of ocean color measurements. The color of the ocean is affected by particulates and dissolved substances in the water and the absorption of light by water itself. Water is transparent at blue and green wavelengths, but strongly absorbs light at longer wavelengths (McLain, 2009). Chlorophyll *a* has a primary absorption peak near 440 nm and chromophoric dissolved organic matter (CDOM) absorbs in the UV (McLain, 2009). Thus, there is a shift from blue to brown water as pigment and particulate

Measurements of ocean color enabled oceanographers to infer the spatial and temporal distribution of phytoplankton on ocean basin and global scales for the first time. The Seaviewing Wide Field-of-view Sensor (SeaWIFS) and Moderate Resolution Imaging Spectroradiometers (MODIS) are currently active and have been collecting data since 1997 and 2002 (Aqua MODIS), respectively. MODIS collects data from 36 spectral bands from the entire Earth's surface very 1 to 2 days (http://modis.gsfc.nasa.gov). SeaWIFS also produces complete global coverage every two days (Miller, 2004). Goals for accuracy of satellite

photosynthesis and prior light history (Kromkamp & Forster, 2003).

dominant groups of phytoplankton in the sample.

concentrations increase (McLain, 2009).

**3.2 Ocean color** 

1991). *Phaeocystis* is an important primary producer in coastal waters. This genus does not have coccoliths, it is characterized by a colonial life stage in which the cells are embedded in a hollow sphere of gelatinous polysaccharide. Colonies may be large enough to be seen by the naked eye. *Phaeocystis pouchetii* forms extensive blooms in the North Sea (Bätje & Michaelis, 1986) and *Phaeocystis antarctica* is an important primary producer in the Ross Sea (DiTullo et al., 2000).

#### **2.2.4 Dinoflagellates**

Dinoflagelletes (Dinophyta) are a largely planktonic division of motile unicellular microalgae that have two flagella. They can be found in both freshwater and marine environments. Generally, dinoflagellates are a more significant component of the phytoplankton in warmer waters. Some photosynthetic dinoflagellates form symbiotic relationships with other organisms, such as the zooxanthellae found in the tissues of tropical corals. Other dinoflagellates do not contribute to primary production as they are nonphotosynthetic heterotrophs which are predatory, parasitic or saprophytic (Lee, 2008). Dinoflagellates often dominate surface stratified waters; in temperate zones there may be a succession from diatoms to dinoflagellates as the relatively nutrient rich, well mixed water column of spring stabilizes to form a stratified water column with relatively warm, nutrient poor surface waters. Dinoflagellates have a patchy distribution and may bloom to form 'red tides.' Some dinoflagellates are toxic and form harmful algal blooms; *Karenia brevis* blooms in the Gulf of Mexico and on the Atlantic coast of the USA, resulting in fish kills and human health problems (Magaña et al., 2003).

#### **3. Measuring phytoplankton biomass**

The best measure of biomass would be to determine the amount of organic carbon in the phytoplankton cells. However, such a measure is almost impossible in a natural seawater sample due to the presence of other organisms, detritus and dissolved organic matter. Consequently, photosynthetic pigments (usually chlorophyll *a*) are used as a proxy for the biomass of phytoplankton. There are a number of techniques for measuring the concentration of chlorophyll *a* and other photosynthetic pigments in water samples. These methods provide information that is relevant to that particular time and location, but these 'snapshots' have limited use at the regional or ocean basin scale. Over the last 30 years our understanding of the spatial and temporal distribution of phytoplankton biomass has been revolutionized by the measurement of ocean color from satellites orbiting the Earth. These instruments provide measurements over a short period of time of a large area, which is not possible from platforms such as ships.

#### **3.1 Pigment analysis**

Chlorophyll fluorescence has been used as tool to determine the distribution of phytoplankton biomass in the ocean since the development of flow-through flourometers (Lorenzen, 1966; Platt, 1972). Most oceanographic research vessels are fitted with flowthrough chlorophyll fluorometers that provide continuous chlorophyll fluorescence data. However, there is not a truly linear relationship between *in vivo* flourescence and phytoplankton chlorophyll concentrations (Falkowski & Raven, 2007). The lack of linearity between *in vivo* fluorescence and chlorophyll is related to the fate of light energy absorbed

1991). *Phaeocystis* is an important primary producer in coastal waters. This genus does not have coccoliths, it is characterized by a colonial life stage in which the cells are embedded in a hollow sphere of gelatinous polysaccharide. Colonies may be large enough to be seen by the naked eye. *Phaeocystis pouchetii* forms extensive blooms in the North Sea (Bätje & Michaelis, 1986) and *Phaeocystis antarctica* is an important primary producer in the Ross Sea

Dinoflagelletes (Dinophyta) are a largely planktonic division of motile unicellular microalgae that have two flagella. They can be found in both freshwater and marine environments. Generally, dinoflagellates are a more significant component of the phytoplankton in warmer waters. Some photosynthetic dinoflagellates form symbiotic relationships with other organisms, such as the zooxanthellae found in the tissues of tropical corals. Other dinoflagellates do not contribute to primary production as they are nonphotosynthetic heterotrophs which are predatory, parasitic or saprophytic (Lee, 2008). Dinoflagellates often dominate surface stratified waters; in temperate zones there may be a succession from diatoms to dinoflagellates as the relatively nutrient rich, well mixed water column of spring stabilizes to form a stratified water column with relatively warm, nutrient poor surface waters. Dinoflagellates have a patchy distribution and may bloom to form 'red tides.' Some dinoflagellates are toxic and form harmful algal blooms; *Karenia brevis* blooms in the Gulf of Mexico and on the Atlantic coast of the USA, resulting in fish kills and human

The best measure of biomass would be to determine the amount of organic carbon in the phytoplankton cells. However, such a measure is almost impossible in a natural seawater sample due to the presence of other organisms, detritus and dissolved organic matter. Consequently, photosynthetic pigments (usually chlorophyll *a*) are used as a proxy for the biomass of phytoplankton. There are a number of techniques for measuring the concentration of chlorophyll *a* and other photosynthetic pigments in water samples. These methods provide information that is relevant to that particular time and location, but these 'snapshots' have limited use at the regional or ocean basin scale. Over the last 30 years our understanding of the spatial and temporal distribution of phytoplankton biomass has been revolutionized by the measurement of ocean color from satellites orbiting the Earth. These instruments provide measurements over a short period of time of a large area, which is not

Chlorophyll fluorescence has been used as tool to determine the distribution of phytoplankton biomass in the ocean since the development of flow-through flourometers (Lorenzen, 1966; Platt, 1972). Most oceanographic research vessels are fitted with flowthrough chlorophyll fluorometers that provide continuous chlorophyll fluorescence data. However, there is not a truly linear relationship between *in vivo* flourescence and phytoplankton chlorophyll concentrations (Falkowski & Raven, 2007). The lack of linearity between *in vivo* fluorescence and chlorophyll is related to the fate of light energy absorbed

(DiTullo et al., 2000).

**2.2.4 Dinoflagellates** 

health problems (Magaña et al., 2003).

possible from platforms such as ships.

**3.1 Pigment analysis** 

**3. Measuring phytoplankton biomass** 

by chlorophyll; light energy is either lost through fluorescence, heat dissipation or used in photochemistry (Maxwell & Johnson, 2000) and the balance between these processes changes depending on the physiological status of the phytoplankton, including rate of photosynthesis and prior light history (Kromkamp & Forster, 2003).

A more accurate estimate of photosynthetic biomass than *in vivo* chlorophyll fluorescence may be obtained if the photosynthetic pigments are extracted from the organism. For water samples containing phytoplankton, a known volume is filtered onto a glass fiber filter and the photosynthetic pigments are extracted using a known volume of organic solvent such as acetone or methanol. The concentration of chlorophyll *a* is then measured in the extract by spectrophotometry or fluorescence (Parsons et al.*,* 1984; Jeffrey et al.*,* 1997). While still widely used, these relatively simple techniques have a number of drawbacks. Firstly, chlorophyll degradation products may absorb light at the same wavelengths as chlorophyll, leading to an overestimation of chlorophyll concentration (Wiltshire, 2009). Secondly, the emission spectra of chlorophyll *a* and *b* overlap, which will result in inaccurate measurement of chlorophyll *a* in water containing chlorophyll *b* containing organisms (Wiltshire, 2009). For the accurate measurement of chlorophyll *a*, other photosynthetic pigments, and their derivatives, high performance liquid chromatography (HPLC) methods should be used (see Wiltshire (2009) for description). HPLC enables the pigments to be separated by chromatography and therefore relatively pure pigments pass through a fluorescence or spectrophotometric detector. In addition to chlorophyll *a*, algae contain multiple pigments. These accessory pigments are often diagnostic for major taxonomic groups (see Table 10.1; Wiltshire, 2009). CHEMTAX (Mackay et al.*,* 1996) is a method by which the total amount of chlorophyll *a* can be allocated to the major taxonomic groups of algae based on the concentrations and ratios of accessory pigments. Thus, HPLC can be used to estimate phytoplankton biomass in terms of chlorophyll *a* and potentially determine the dominant groups of phytoplankton in the sample.

#### **3.2 Ocean color**

The Coastal Zone Color Scanner (1978-1986) was the first satellite mission that measured chlorophyll *a* concentrations using top of the atmosphere radiances (McClain, 2009). The success of this mission led to a number of missions to measure ocean color at either global or regional spatial scales by Japanese, European and United States space agencies. As a result of these missions, we now have over 30 years of ocean color measurements. The color of the ocean is affected by particulates and dissolved substances in the water and the absorption of light by water itself. Water is transparent at blue and green wavelengths, but strongly absorbs light at longer wavelengths (McLain, 2009). Chlorophyll *a* has a primary absorption peak near 440 nm and chromophoric dissolved organic matter (CDOM) absorbs in the UV (McLain, 2009). Thus, there is a shift from blue to brown water as pigment and particulate concentrations increase (McLain, 2009).

Measurements of ocean color enabled oceanographers to infer the spatial and temporal distribution of phytoplankton on ocean basin and global scales for the first time. The Seaviewing Wide Field-of-view Sensor (SeaWIFS) and Moderate Resolution Imaging Spectroradiometers (MODIS) are currently active and have been collecting data since 1997 and 2002 (Aqua MODIS), respectively. MODIS collects data from 36 spectral bands from the entire Earth's surface very 1 to 2 days (http://modis.gsfc.nasa.gov). SeaWIFS also produces complete global coverage every two days (Miller, 2004). Goals for accuracy of satellite

Primary Production in the Ocean 569

line *in situ*; bottles deployed at different depths will be exposed to the ambient light and temperature at that depth. Changes in oxygen concentration can be monitored with oxygen electrodes or by taking water samples that are fixed and oxygen concentration is subsequently measured by Winkler titration (Parsons et al.*,* 1984). In laboratory studies using pure cultures of phytoplankton oxygen electrode chambers have been used extensively in photosynthesis research (e.g. Colman & Rotatore, 1995; Johnston & Raven, 1996). These systems comprise of a small, optically clear, chamber (usually a few ml) which has a Clark-type oxygen electrode set in the base (see Allen and Holmes (1986) for a full description). Carbon assimilation rates based on oxygen production often assume a ratio of moles of O2 produced for every mole of CO2 assimilated, called the photosynthetic quotient,

In sediments, profiles and changes in oxygen concentration over time may be made using oxygen microelectrodes (Revsbech & Jørgensen, 1983). The microphytobenthos is usually limited to the surface 2 or 3 mm of sediment, therefore high resolution measurements are required; photosynthesis is measured to a resolution of 100 m and the sensing tips of the microelectrode have diameters of only 2 – 10 m (Revsbech et al.*,* 1989). While oxygen microelectrodes just measure oxygen concentration, it is possible to measure gross photosynthesis rates using the light-dark shift method (Revsbech & Jørgensen, 1983; Glud et al.*,* 1992; Lassen et al.*,* 1998; Hancke & Glud, 2004). Moreover, oxygen concentration profiles can be used to calculate respiration and net photosynthesis rates according to Kühl et al. (1996) and Hancke & Glud (2004) based on Fick's first law of diffusion. Estimates of benthic primary productivity are also made using oxygen exchange across the sediment-water

Optodes have recently been used to measure changes in oxygen concentrations associated with photosynthesis. Optodes work by using fluorescence quenching by oxygen of a luminophore. The intensity of fluorescence is inversely proportional to the O2 partial pressure at the luminophore (Glud et al.*,* 1999). For example, Glud et al. (1999) used the luminophore ruthenium (III)-Tris-4,7-diphenyl-1,10-phena-throline, which absorbs blue light (450 nm), with the intensity of the emitted red light (650 nm) decreasing with increasing O2 partial pressure. Unlike Clark-type oxygen electrodes, optodes do not consume oxygen. Two designs of optodes are used in photosynthesis measurements: optodes that are used in a similar way to oxygen microelectrodes (Miller & Dunton 2007), and planer optodes that produce a two-dimensional image of oxygen concentrations (Glud et al.*,* 1999, 2001). Miller & Dunton (2007) used a micro-optode to measure photosynthesisirradiance curves for the kelp *Laminaria hyperborea*. Planar optodes have been used to produce images of oxygen concentrations across the sediment-water interface in sediments colonized by photosynthetic biofilms (Glud et al.*,* 1999, 2001). As planar optodes produce a two dimensional image, multiple oxygen profiles can be extracted from a single measurement (Glud et al.*,* 2001). Moreover, the light-dark shift method can be used to

Carbon exists in three isotopes in nature. The most common isotope is 12C, which makes up 98.9% of the natural carbon on Earth. Carbon also exists in another stable form as 13C (1.1 %) and an insignificant amount of the radioactive isotope 14C (< 0.0001 %) (Falkowski & Raven, 2007). The relatively low abundance of 14C and 13C means that these isotopes can potentially

which usually deviates from the 1:1 ratio indicated by equation 1.

interface using benthic chambers or sediment cores (Thornton et al.*,* 2002).

measure gross photosynthesis rates (Glud et al.*,* 1999).

**4.2 Isotopes as tracers of aquatic photosynthesis** 

products are ± 5% for water-leaving radiances and ± 35 % for open-ocean chlorophyll *a*  (McLain, 2009). In addition to rapid regional or global estimates of chlorophyll *a*, algorithms have been developed to estimate net primary productivity (NPP) based on ocean color (Behrenfield & Falkowski, 1997. See section 6 of this chapter).

#### **4. Measuring marine photosynthesis**

Photosynthesis results in primary productivity. The terms production and productivity are often used interchangeably and there no generally accepted definition of primary production (Underwood & Kromkamp, 1999). Falkowski & Raven (2007) define primary productivity as a time dependent process which is a rate with dimensions of mass per unit time; whereas primary production is defined as a quantity with dimensions of mass. In contrast, Underwood & Kromkamp (1999) define primary production as a *rate* of assimilation of inorganic carbon into organic matter by autotrophs. For the purposes of this review, I will use the definitions of Falkowski & Raven (2007).

There are a number of methods that are regularly used to measure rates of primary productivity. Techniques are based around gas exchange, the use of isotope tracers, or chlorophyll fluorescence. Primary productivity is usually expressed as the production of oxygen or the assimilation of inorganic carbon into organic carbon over time (equation 1). Carbon assimilation is a more useful measure as it can be directly converted into biomass and used to calculate growth. Common units for primary productivity in marine environment are mg C m-2 day-1 or g C m-2 year-1. Primary productivity is often normalized to biomass, as it is useful to know how much biomass is responsible for the observed rates of productivity.

Different techniques will produce slightly different rates of productivity (Bender et al.*,* 1987) as a result of the biases associated with each method. No single technique provides a 'true' measurement of primary productivity. Consequently, researchers should select their methodology based on what factors they want to relate their measurements to, time available to make the measurements, and which assumptions and sources of error are tolerable to answer their particular research questions. Most methods for measuring primary productivity in the ocean require that a sample of water is enclosed in a container, this in itself effects the primary producers. Phytoplankton may be killed on contact with the container or there may be an exchange of solutes between the walls of the container and the sample (Fogg & Thake, 1987). When working in oligotrophic waters contamination of the samples onboard ship is a serious problem. Williams & Robertson (1989) found that the rubber tubing associated with a Niskin sampling bottle severely inhibited primary productivity in samples taken in the oligotrophic Indian Ocean. Moreover, large areas of the ocean are iron limited (Boyd et al., 2000) and it is a challenge to prevent iron contamination in these areas given that oceanographers generally work from ships fabricated from steel.

#### **4.1 Gas exchange methods**

Changes in oxygen concentration over time in water samples can be used to calculate rates of photosynthesis and therefore primary productivity. This method involves enclosing water samples and incubating them in the dark and light either onboard ship or *in situ*. Bottles incubated in the dark are used to measure dark respiration rates. To ensure that the rates of photosynthesis are representative, the bottles should be incubated at *in situ*  temperature and under ambient light. One way of doing this is to deploy the bottles on a

products are ± 5% for water-leaving radiances and ± 35 % for open-ocean chlorophyll *a*  (McLain, 2009). In addition to rapid regional or global estimates of chlorophyll *a*, algorithms have been developed to estimate net primary productivity (NPP) based on ocean color

Photosynthesis results in primary productivity. The terms production and productivity are often used interchangeably and there no generally accepted definition of primary production (Underwood & Kromkamp, 1999). Falkowski & Raven (2007) define primary productivity as a time dependent process which is a rate with dimensions of mass per unit time; whereas primary production is defined as a quantity with dimensions of mass. In contrast, Underwood & Kromkamp (1999) define primary production as a *rate* of assimilation of inorganic carbon into organic matter by autotrophs. For the purposes of this

There are a number of methods that are regularly used to measure rates of primary productivity. Techniques are based around gas exchange, the use of isotope tracers, or chlorophyll fluorescence. Primary productivity is usually expressed as the production of oxygen or the assimilation of inorganic carbon into organic carbon over time (equation 1). Carbon assimilation is a more useful measure as it can be directly converted into biomass and used to calculate growth. Common units for primary productivity in marine environment are mg C m-2 day-1 or g C m-2 year-1. Primary productivity is often normalized to biomass, as it is useful to know how much biomass is responsible for the observed rates

Different techniques will produce slightly different rates of productivity (Bender et al.*,* 1987) as a result of the biases associated with each method. No single technique provides a 'true' measurement of primary productivity. Consequently, researchers should select their methodology based on what factors they want to relate their measurements to, time available to make the measurements, and which assumptions and sources of error are tolerable to answer their particular research questions. Most methods for measuring primary productivity in the ocean require that a sample of water is enclosed in a container, this in itself effects the primary producers. Phytoplankton may be killed on contact with the container or there may be an exchange of solutes between the walls of the container and the sample (Fogg & Thake, 1987). When working in oligotrophic waters contamination of the samples onboard ship is a serious problem. Williams & Robertson (1989) found that the rubber tubing associated with a Niskin sampling bottle severely inhibited primary productivity in samples taken in the oligotrophic Indian Ocean. Moreover, large areas of the ocean are iron limited (Boyd et al., 2000) and it is a challenge to prevent iron contamination in these areas given that oceanographers generally work from ships fabricated from steel.

Changes in oxygen concentration over time in water samples can be used to calculate rates of photosynthesis and therefore primary productivity. This method involves enclosing water samples and incubating them in the dark and light either onboard ship or *in situ*. Bottles incubated in the dark are used to measure dark respiration rates. To ensure that the rates of photosynthesis are representative, the bottles should be incubated at *in situ*  temperature and under ambient light. One way of doing this is to deploy the bottles on a

(Behrenfield & Falkowski, 1997. See section 6 of this chapter).

review, I will use the definitions of Falkowski & Raven (2007).

**4. Measuring marine photosynthesis** 

of productivity.

**4.1 Gas exchange methods** 

line *in situ*; bottles deployed at different depths will be exposed to the ambient light and temperature at that depth. Changes in oxygen concentration can be monitored with oxygen electrodes or by taking water samples that are fixed and oxygen concentration is subsequently measured by Winkler titration (Parsons et al.*,* 1984). In laboratory studies using pure cultures of phytoplankton oxygen electrode chambers have been used extensively in photosynthesis research (e.g. Colman & Rotatore, 1995; Johnston & Raven, 1996). These systems comprise of a small, optically clear, chamber (usually a few ml) which has a Clark-type oxygen electrode set in the base (see Allen and Holmes (1986) for a full description). Carbon assimilation rates based on oxygen production often assume a ratio of moles of O2 produced for every mole of CO2 assimilated, called the photosynthetic quotient, which usually deviates from the 1:1 ratio indicated by equation 1.

In sediments, profiles and changes in oxygen concentration over time may be made using oxygen microelectrodes (Revsbech & Jørgensen, 1983). The microphytobenthos is usually limited to the surface 2 or 3 mm of sediment, therefore high resolution measurements are required; photosynthesis is measured to a resolution of 100 m and the sensing tips of the microelectrode have diameters of only 2 – 10 m (Revsbech et al.*,* 1989). While oxygen microelectrodes just measure oxygen concentration, it is possible to measure gross photosynthesis rates using the light-dark shift method (Revsbech & Jørgensen, 1983; Glud et al.*,* 1992; Lassen et al.*,* 1998; Hancke & Glud, 2004). Moreover, oxygen concentration profiles can be used to calculate respiration and net photosynthesis rates according to Kühl et al. (1996) and Hancke & Glud (2004) based on Fick's first law of diffusion. Estimates of benthic primary productivity are also made using oxygen exchange across the sediment-water interface using benthic chambers or sediment cores (Thornton et al.*,* 2002).

Optodes have recently been used to measure changes in oxygen concentrations associated with photosynthesis. Optodes work by using fluorescence quenching by oxygen of a luminophore. The intensity of fluorescence is inversely proportional to the O2 partial pressure at the luminophore (Glud et al.*,* 1999). For example, Glud et al. (1999) used the luminophore ruthenium (III)-Tris-4,7-diphenyl-1,10-phena-throline, which absorbs blue light (450 nm), with the intensity of the emitted red light (650 nm) decreasing with increasing O2 partial pressure. Unlike Clark-type oxygen electrodes, optodes do not consume oxygen. Two designs of optodes are used in photosynthesis measurements: optodes that are used in a similar way to oxygen microelectrodes (Miller & Dunton 2007), and planer optodes that produce a two-dimensional image of oxygen concentrations (Glud et al.*,* 1999, 2001). Miller & Dunton (2007) used a micro-optode to measure photosynthesisirradiance curves for the kelp *Laminaria hyperborea*. Planar optodes have been used to produce images of oxygen concentrations across the sediment-water interface in sediments colonized by photosynthetic biofilms (Glud et al.*,* 1999, 2001). As planar optodes produce a two dimensional image, multiple oxygen profiles can be extracted from a single measurement (Glud et al.*,* 2001). Moreover, the light-dark shift method can be used to measure gross photosynthesis rates (Glud et al.*,* 1999).

#### **4.2 Isotopes as tracers of aquatic photosynthesis**

Carbon exists in three isotopes in nature. The most common isotope is 12C, which makes up 98.9% of the natural carbon on Earth. Carbon also exists in another stable form as 13C (1.1 %) and an insignificant amount of the radioactive isotope 14C (< 0.0001 %) (Falkowski & Raven, 2007). The relatively low abundance of 14C and 13C means that these isotopes can potentially

Primary Production in the Ocean 571

of weeks (Luz & Barkin, 2000). Luz & Barkin (2009) showed that combining 17∆ with O2/Ar

Chlorophyll *a* fluorescence can be used for more than estimating phytoplankton biomass (see 3.1) and there has been a wealth of research over the last 20 years on the application of variable chlorophyll *a* fluorescence to the measurement of photosynthesis and the physiological status of photosynthetic organisms. Energy absorbed by chlorophyll *a* may be used in photochemistry and stored in photosynthetic products, dissipated as heat, or lost as fluorescence. Chlorophyll *a* fluorescence is largely derived from the chlorophyll *a* associated with photosystem II (PSII); changes in the quantum yield of fluorescence directly relate to O2 evolving capability as PSII is the oxygen evolving complex within the photosynthetic apparatus (Suggett, 2011). There are two main types of fluorometers that are used to measure variable chlorophyll *a* fluorescence; Pulse Amplitude Modulation (PAM) fluorometers (Schreiber et al.*,* 1986) and Fast Repetition Rate (FRR) fluorometers (Kolber et al.*,* 1998). These instruments use a modulated light source that allows measurements to be made in the presence of background light or under field light conditions (Maxwell & Johnson, 2000). The PAM approach is not sensitive enough to use in open ocean conditions (Suggett et al., 2003), although it is increasingly being used to measure photosynthetic parameters associated with the microphytobenthos (Underwood, 2002; Perkins et al., 2002; 2011; Serôdio, 2004), macrophytes (Enríquez & Borowitzka, 2011), and has been used with cultures of phytoplankton (Suggett et al.*,* 2003; Thornton, 2009). FRR has been used in the open ocean (Babin et al.*,* 1996; Suggett et al.*,* 2001). The difference between PAM and FRR is

ratios enables gross and net oxygen production to be estimated.

beyond the scope of this review; for an overview see Huot & Babin (2011).

in terms of taxonomic composition and physiological status.

Modulated chlorophyll *a* fluorometers cannot be used to measure photosynthesis directly. One of the primary measurements made with modulated chlorophyll *a* fluorometers is the quantum yield of PSII photochemistry (ФPSII). Genty et al. (1989) demonstrated that ФPSII correlated with CO2 assimilation in maize and barley, raising the possibility that variable chlorophyll *a* fluorescence could be used to estimate photosynthesis rates. ФPSII multiplied by the rate of light absorption by PSII is used to calculate electron transfer rate (ETRPSII) through PSII (Enríquez & Borowitzka, 2011; Suggett et al.*,* 2011; White & Critchley, 1999). ETRPSII has been used as a proxy for photosynthesis. However, there are several reasons why the relationship between ФPSII (and therefore ETRPSII) and CO2 assimilation or O2 production may not be constant (see Suggett, 2011). This effect may be compounded in the algae due to their taxonomic and resultant physiological diversity (Suggett, 2011). Suggett et al.*,* (2009) used an FRR fluorometer to measure ETRPSII and examined the relationship between ETRPSII and photosynthesis measured by either gross O2 production or 14CO2 fixation. Measurements were made using six species of eukaryotic phytoplankton, representing a diversity of taxonomic groups. ETRPSII was linearly related to the rate of gross O2 production in all species; however, the slope of the relationship was significantly different for different species. ETRPSII was also linearly related to 14CO2 fixation; however, both the slope and intercept of the relationship was different for different species. These results highlight some of the challenges involved in using ETRPSII to estimate photosynthesis rates, especially in natural populations of phytoplankton which are likely to be diverse both

**4.3 Chlorophyll fluorescence** 

be used to measure photosynthesis rates and follow the passage of carbon through photosynthetic organisms when added as tracers. Uptake and assimilation of inorganic carbon into acid-stable organic carbon (Falkowski & Raven, 2007) is the most commonly employed method for measuring photosynthesis using the radioactive tracer 14C (Steeman-Nielson, 1952). The rationale for the 14C method is that the incorporation of radioactively labeled carbon is quantitatively proportional to the rate of incorporation of non-labeled inorganic carbon. Over relatively short incubations the results are a good approximation of gross photosynthesis and an approximation of net photosynthesis over longer time periods (Falkowski & Raven, 2007). This technique (described in Parsons et al., 1984) has been the primary method for measuring the primary productivity of phytoplankton for over fifty years. The method has the advantage of being relatively simple and sensitive. Although widely used, the technique is not without drawbacks and ambiguities. For example, there is an isotopic discrimination between 14C and the natural isotope 12C; less 14C is fixed as it is heavier than 12C, and a discrimination factor of 5 % is usually incorporated into the calculation of inorganic carbon fixation rates (Falkowski & Raven, 2007). Furthermore, the organic carbon, including the 14C which has been fixed during the incubation, is usually separated from the sample by filtration. This can lead to a loss of 14C labeled organic carbon due to rupture of cells on contact with the filter (Sharp, 1977) or exudation of photosynthetic products. There is also a continuing debate as to whether primary productivity measured with the 14C method represents gross or net rates, or something in between the two (Underwood & Kromkamp, 1999).

The advantage of using 13C as a tracer for photosynthesis is that it is not radioactive. This means that it is logistically simpler to use if one has access to an isotope ratio mass spectrometer. Moreover, unlike 14C, 13C can be added as tracer to natural ecosystems and used to trace the assimilation of carbon and transfer to higher trophic levels. Miller & Dunton (2007) used 13C to measure the photosynthesis of the macroalga *Laminaria hyperborea.* Middelburg et al. (2000) and Bellinger et al. (2009) used 13C as a tracer to trace carbon flow through intertidal benthic biofilms dominated by diatoms and cyanobacteria. The tracer was added to the sediment at low tide and followed through the ecosystem over a period of hours to days. Middelburg et al. (2000) showed that carbon fixed through photosynthesis was transferred to bacteria and nematodes within hours. Bellinger et al. (2009) examined the incorporation of the tracer into important biomolecules, including exopolymers (EPS) and phospholipid fatty acids (PLFAs).

Photosynthesis rates have also been measured with the stable isotope 18O by adding labeled water as a tracer and measuring the production of 18O labeled oxygen with a mass spectrometer (Bender et al.*,* 1987; Suggett et al., 2003). The method produces a relatively precise measurement of gross photosynthesis (Falkowski & Raven, 2007). However, this technique has not been used extensively.

Oxygen exists in nature in the form of three isotopes; 16O (99.76 % of the oxygen on Earth), 18O (0.20 %), and 17O (0.04 %) (Falkowski & Raven, 2007). Luz & Barken (2000) developed the triple isotope method using natural abundances of oxygen isotopes to estimate the production of photosynthetic oxygen using the isotopic composition of dissolved oxygen in seawater. The method was based on the 17O anomaly (17∆), which is calculated from 17O/16O and 18O/16O (Luz & Barkin, 2000, 2009). This innovative technique does not require water to be enclosed in bottles and therefore avoids bottle effects. The method is used to determine gross photosynthesis rates, enabling integrated productivity to be estimated on a time scale

be used to measure photosynthesis rates and follow the passage of carbon through photosynthetic organisms when added as tracers. Uptake and assimilation of inorganic carbon into acid-stable organic carbon (Falkowski & Raven, 2007) is the most commonly employed method for measuring photosynthesis using the radioactive tracer 14C (Steeman-Nielson, 1952). The rationale for the 14C method is that the incorporation of radioactively labeled carbon is quantitatively proportional to the rate of incorporation of non-labeled inorganic carbon. Over relatively short incubations the results are a good approximation of gross photosynthesis and an approximation of net photosynthesis over longer time periods (Falkowski & Raven, 2007). This technique (described in Parsons et al., 1984) has been the primary method for measuring the primary productivity of phytoplankton for over fifty years. The method has the advantage of being relatively simple and sensitive. Although widely used, the technique is not without drawbacks and ambiguities. For example, there is an isotopic discrimination between 14C and the natural isotope 12C; less 14C is fixed as it is heavier than 12C, and a discrimination factor of 5 % is usually incorporated into the calculation of inorganic carbon fixation rates (Falkowski & Raven, 2007). Furthermore, the organic carbon, including the 14C which has been fixed during the incubation, is usually separated from the sample by filtration. This can lead to a loss of 14C labeled organic carbon due to rupture of cells on contact with the filter (Sharp, 1977) or exudation of photosynthetic products. There is also a continuing debate as to whether primary productivity measured with the 14C method represents gross or net rates, or something in between the two

The advantage of using 13C as a tracer for photosynthesis is that it is not radioactive. This means that it is logistically simpler to use if one has access to an isotope ratio mass spectrometer. Moreover, unlike 14C, 13C can be added as tracer to natural ecosystems and used to trace the assimilation of carbon and transfer to higher trophic levels. Miller & Dunton (2007) used 13C to measure the photosynthesis of the macroalga *Laminaria hyperborea.* Middelburg et al. (2000) and Bellinger et al. (2009) used 13C as a tracer to trace carbon flow through intertidal benthic biofilms dominated by diatoms and cyanobacteria. The tracer was added to the sediment at low tide and followed through the ecosystem over a period of hours to days. Middelburg et al. (2000) showed that carbon fixed through photosynthesis was transferred to bacteria and nematodes within hours. Bellinger et al. (2009) examined the incorporation of the tracer into important biomolecules, including

Photosynthesis rates have also been measured with the stable isotope 18O by adding labeled water as a tracer and measuring the production of 18O labeled oxygen with a mass spectrometer (Bender et al.*,* 1987; Suggett et al., 2003). The method produces a relatively precise measurement of gross photosynthesis (Falkowski & Raven, 2007). However, this

Oxygen exists in nature in the form of three isotopes; 16O (99.76 % of the oxygen on Earth), 18O (0.20 %), and 17O (0.04 %) (Falkowski & Raven, 2007). Luz & Barken (2000) developed the triple isotope method using natural abundances of oxygen isotopes to estimate the production of photosynthetic oxygen using the isotopic composition of dissolved oxygen in seawater. The method was based on the 17O anomaly (17∆), which is calculated from 17O/16O and 18O/16O (Luz & Barkin, 2000, 2009). This innovative technique does not require water to be enclosed in bottles and therefore avoids bottle effects. The method is used to determine gross photosynthesis rates, enabling integrated productivity to be estimated on a time scale

(Underwood & Kromkamp, 1999).

exopolymers (EPS) and phospholipid fatty acids (PLFAs).

technique has not been used extensively.

of weeks (Luz & Barkin, 2000). Luz & Barkin (2009) showed that combining 17∆ with O2/Ar ratios enables gross and net oxygen production to be estimated.

#### **4.3 Chlorophyll fluorescence**

Chlorophyll *a* fluorescence can be used for more than estimating phytoplankton biomass (see 3.1) and there has been a wealth of research over the last 20 years on the application of variable chlorophyll *a* fluorescence to the measurement of photosynthesis and the physiological status of photosynthetic organisms. Energy absorbed by chlorophyll *a* may be used in photochemistry and stored in photosynthetic products, dissipated as heat, or lost as fluorescence. Chlorophyll *a* fluorescence is largely derived from the chlorophyll *a* associated with photosystem II (PSII); changes in the quantum yield of fluorescence directly relate to O2 evolving capability as PSII is the oxygen evolving complex within the photosynthetic apparatus (Suggett, 2011). There are two main types of fluorometers that are used to measure variable chlorophyll *a* fluorescence; Pulse Amplitude Modulation (PAM) fluorometers (Schreiber et al.*,* 1986) and Fast Repetition Rate (FRR) fluorometers (Kolber et al.*,* 1998). These instruments use a modulated light source that allows measurements to be made in the presence of background light or under field light conditions (Maxwell & Johnson, 2000). The PAM approach is not sensitive enough to use in open ocean conditions (Suggett et al., 2003), although it is increasingly being used to measure photosynthetic parameters associated with the microphytobenthos (Underwood, 2002; Perkins et al., 2002; 2011; Serôdio, 2004), macrophytes (Enríquez & Borowitzka, 2011), and has been used with cultures of phytoplankton (Suggett et al.*,* 2003; Thornton, 2009). FRR has been used in the open ocean (Babin et al.*,* 1996; Suggett et al.*,* 2001). The difference between PAM and FRR is beyond the scope of this review; for an overview see Huot & Babin (2011).

Modulated chlorophyll *a* fluorometers cannot be used to measure photosynthesis directly. One of the primary measurements made with modulated chlorophyll *a* fluorometers is the quantum yield of PSII photochemistry (ФPSII). Genty et al. (1989) demonstrated that ФPSII correlated with CO2 assimilation in maize and barley, raising the possibility that variable chlorophyll *a* fluorescence could be used to estimate photosynthesis rates. ФPSII multiplied by the rate of light absorption by PSII is used to calculate electron transfer rate (ETRPSII) through PSII (Enríquez & Borowitzka, 2011; Suggett et al.*,* 2011; White & Critchley, 1999). ETRPSII has been used as a proxy for photosynthesis. However, there are several reasons why the relationship between ФPSII (and therefore ETRPSII) and CO2 assimilation or O2 production may not be constant (see Suggett, 2011). This effect may be compounded in the algae due to their taxonomic and resultant physiological diversity (Suggett, 2011). Suggett et al.*,* (2009) used an FRR fluorometer to measure ETRPSII and examined the relationship between ETRPSII and photosynthesis measured by either gross O2 production or 14CO2 fixation. Measurements were made using six species of eukaryotic phytoplankton, representing a diversity of taxonomic groups. ETRPSII was linearly related to the rate of gross O2 production in all species; however, the slope of the relationship was significantly different for different species. ETRPSII was also linearly related to 14CO2 fixation; however, both the slope and intercept of the relationship was different for different species. These results highlight some of the challenges involved in using ETRPSII to estimate photosynthesis rates, especially in natural populations of phytoplankton which are likely to be diverse both in terms of taxonomic composition and physiological status.

Primary Production in the Ocean 573

and there is evidence of phosphorus limitation in coastal ecosystems (Sundareshwar et al., 2003). In over 20 % of the ocean there are excess nutrients (nitrate, phosphate, silicate) and light, but the biomass of phytoplankton is relatively low (Martin et al., 1994). These areas are known as the high-nitrate, low-chlorophyll (HNLC) areas. They are located in the equatorial Pacific, the subarctic Pacific, and the Southern Ocean (Falkowski & Raven, 2007). Iron is an essential component of the nitrogenase enzyme, consequently iron limitation limits nitrogen fixation by cyanobacteria over large areas of the ocean (Falkowski et al.*,* 1998) (see 7.3). Iron is supplied to the open ocean via wind blown dust from arid areas of the continents (Duce & Tindale, 1991). The upwelling of deep waters containing nitrate and phosphate produced from the remineralization of organic matter is important in maintaining high primary productivity in many areas of the ocean, such as along the western margins of Africa and South America. Conversely, thermal stratification and downwelling will limits primary production in the subtropical gyres as the sunlit surface waters are largely isolated from

Light (solar radiation) provides the energy that drives photosynthesis. Light is variable on a number of spatial and temporal scales. Low latitudes receive more solar radiation than high latitudes and have less variation in solar radiation over the course of one year. At high latitudes there are pronounced seasons and variations in day length. Imposed on these month to month or season to season variations in solar radiation are short term fluctuations. The angle of the sun above the horizon affects how much light is reflected off the surface of the ocean. At midday, when the sun is at an angle of 90º to the sea surface, 2 % of the incoming solar radiation is reflected; this value increases to 40 % during the evening and early morning when the sun is at an angle of 5 º (Trujillo & Thurman, 2005). Reflection off cloud cover also significantly reduces the input of solar radiation into the ocean. Conversely, net primary productivity may be inhibited by too much light, which can lead to

The depth of the euphotic zone (surface layer in which there is enough light to support photosynthesis) is often less than 10 m and rarely greater than 100 m (Fogg & Thake, 1987), whereas the mean depth of the ocean is approximately 3,700 m. Therefore, photosynthesis and primary production is limited to a thin layer at the ocean surface and whether phytoplankton cells are mixed into the dark waters below will effect primary productivity. As the mixed layer of a water column increases the average photon flux density (i.e. light) to which the cells are exposed will decrease as the circulating cell will spend longer in darkness. Therefore the total gross productivity of the phytoplankton population will decrease (Fig. 1). However, the respiration rate of the population will be relatively constant, whatever the depth of mixing. This results in a *critical depth* in a mixed water column; if the cells are mixed below the critical depth then there will be no net productivity as the respiratory loss of carbon will exceed photosynthetic carbon gain. Net photosynthesis and the resulting net primary productivity will only occur in mixed water where the mixing is

Grazing also effects production; in a heavily grazed population of phytoplankton individual cells may show high rates of productivity, but there may be a low biomass of primary producers as a large proportion of the primary production is transferred to other trophic levels. In recent years there has been a realization that phytoplankton are subject to lytic viral infection (Suttle et al.*,* 1990; Nagasaki et al., 2004), which will have an effect similar as

grazing by reducing the biomass of primary producers in the water column.

photoinhibition or conditions conducive to photorespiration (Fig. 1).

less than the critical depth (Sverdrup, 1953; Kirk, 1983).

nutrient rich waters below the thermocline.

There are several advantages to variable fluorescence techniques; the techniques are not intrusive and do not harm the organisms, measurements can be made at high spatial and temporal resolution (Suggett et al.*,* 2003), measurements do not require any wet chemistry, and the water sample does not have to be enclosed in a bottle (Kolber et al.*,* 1998). Consequently, variable fluorescence instruments are suited to ocean observing programs;

Yoshikawa & Furuya (2004) used a fluorometer moored *in situ* to monitor photosynthesis in coastal waters. Some of the disadvantages to variable fluorescence stem from the fact that this is a relatively young and rapidly evolving field. In the 1970s and 1980s technology was the limiting factor to the development of the field. Since the 1990s there has been a rapid evolution of the technology leading to a large number of commercially available instruments. However, an understanding of the physiology and development of theory associated with variable chlorophyll *a* fluorescence has arguably lagged behind instrument development in recent years. For new users, the large number of fluorescence parameters and their definitions can be confusing (Cosgrove & Borowitza, 2011). This is compounded by the fact that there is no standardized terminology and many fluorescence parameters have several synonyms in the literature. Attempts have been made to standardize terminology (Kromkamp & Forster, 2003).

Photoacoustics has also been used to study phytoplankton photosynthesis (Grinblat & Dubinsky, 2011). This is not a fluorescence technique, however it is based on the same principle that only a small and variable fraction of the energy absorbed by photosynthetic pigment is stored in photosynthetic products. While the preceding discussion has focused on fluorescence, only a few percent of the absorbed light energy is actually lost as fluorescence. The major loss of energy is through heat, which may account for over 60 % of the energy absorbed (Grinblat & Dubinsky, 2011). The photoacoustic method is based on the conversion of light energy to heat energy that results in a rise in temperature and an increase in pressure (photothermal effect) (Grinblat & Dubinsky, 2011). In practice, a suspension of phytoplankton is exposed to a laser pulse, some of the energy from the laser pulse is stored in the photochemical products of photosynthesis and the remainder is dissipated as heat, resulting in an acoustic wave which is measured by a detector (Grinblat & Dubinsky, 2011). This technique has not been used extensively; for further details see Grinblat & Dubinsky (2011).

#### **5. Temporal and spatial variation in oceanic primary production**

The mean chlorophyll *a* concentration in the global ocean is 0.32 mg m-3 (Falkowski & Raven, 2007). However, this is not evenly distributed throughout the ocean. Primary production at any one location will vary in space and time in response to factors limiting or stimulating photosynthesis and phytoplankton growth. Photosynthesis and growth in the sea are limited by nutrients, light or temperature. In the dynamic environment of a water column resources are patchy both in time and space. Consequently, phytoplankton may receive nutrients and light in pulses rather than a continuous supply. Generally, it is the interplay between nutrient and light availability that affects phytoplankton photosynthesis and primary production.

The traditional paradigm of biological oceanography was that bioavailable nitrogen is the nutrient limiting primary production in the ocean (Ryther & Dunstan, 1971; Howarth, 1988). This is an over simplification and is increasingly being challenged. For example, the Mediterranean Sea appears to be phosphorus limited (Thingstad & Rassoulzadegan, 1995)

There are several advantages to variable fluorescence techniques; the techniques are not intrusive and do not harm the organisms, measurements can be made at high spatial and temporal resolution (Suggett et al.*,* 2003), measurements do not require any wet chemistry, and the water sample does not have to be enclosed in a bottle (Kolber et al.*,* 1998). Consequently, variable fluorescence instruments are suited to ocean observing programs; Yoshikawa & Furuya (2004) used a fluorometer moored *in situ* to monitor photosynthesis in coastal waters. Some of the disadvantages to variable fluorescence stem from the fact that this is a relatively young and rapidly evolving field. In the 1970s and 1980s technology was the limiting factor to the development of the field. Since the 1990s there has been a rapid evolution of the technology leading to a large number of commercially available instruments. However, an understanding of the physiology and development of theory associated with variable chlorophyll *a* fluorescence has arguably lagged behind instrument development in recent years. For new users, the large number of fluorescence parameters and their definitions can be confusing (Cosgrove & Borowitza, 2011). This is compounded by the fact that there is no standardized terminology and many fluorescence parameters have several synonyms in the literature. Attempts have been made to standardize

Photoacoustics has also been used to study phytoplankton photosynthesis (Grinblat & Dubinsky, 2011). This is not a fluorescence technique, however it is based on the same principle that only a small and variable fraction of the energy absorbed by photosynthetic pigment is stored in photosynthetic products. While the preceding discussion has focused on fluorescence, only a few percent of the absorbed light energy is actually lost as fluorescence. The major loss of energy is through heat, which may account for over 60 % of the energy absorbed (Grinblat & Dubinsky, 2011). The photoacoustic method is based on the conversion of light energy to heat energy that results in a rise in temperature and an increase in pressure (photothermal effect) (Grinblat & Dubinsky, 2011). In practice, a suspension of phytoplankton is exposed to a laser pulse, some of the energy from the laser pulse is stored in the photochemical products of photosynthesis and the remainder is dissipated as heat, resulting in an acoustic wave which is measured by a detector (Grinblat & Dubinsky, 2011). This technique has not been used extensively; for further details see

**5. Temporal and spatial variation in oceanic primary production** 

The mean chlorophyll *a* concentration in the global ocean is 0.32 mg m-3 (Falkowski & Raven, 2007). However, this is not evenly distributed throughout the ocean. Primary production at any one location will vary in space and time in response to factors limiting or stimulating photosynthesis and phytoplankton growth. Photosynthesis and growth in the sea are limited by nutrients, light or temperature. In the dynamic environment of a water column resources are patchy both in time and space. Consequently, phytoplankton may receive nutrients and light in pulses rather than a continuous supply. Generally, it is the interplay between nutrient and light availability that affects phytoplankton photosynthesis

The traditional paradigm of biological oceanography was that bioavailable nitrogen is the nutrient limiting primary production in the ocean (Ryther & Dunstan, 1971; Howarth, 1988). This is an over simplification and is increasingly being challenged. For example, the Mediterranean Sea appears to be phosphorus limited (Thingstad & Rassoulzadegan, 1995)

terminology (Kromkamp & Forster, 2003).

Grinblat & Dubinsky (2011).

and primary production.

and there is evidence of phosphorus limitation in coastal ecosystems (Sundareshwar et al., 2003). In over 20 % of the ocean there are excess nutrients (nitrate, phosphate, silicate) and light, but the biomass of phytoplankton is relatively low (Martin et al., 1994). These areas are known as the high-nitrate, low-chlorophyll (HNLC) areas. They are located in the equatorial Pacific, the subarctic Pacific, and the Southern Ocean (Falkowski & Raven, 2007). Iron is an essential component of the nitrogenase enzyme, consequently iron limitation limits nitrogen fixation by cyanobacteria over large areas of the ocean (Falkowski et al.*,* 1998) (see 7.3). Iron is supplied to the open ocean via wind blown dust from arid areas of the continents (Duce & Tindale, 1991). The upwelling of deep waters containing nitrate and phosphate produced from the remineralization of organic matter is important in maintaining high primary productivity in many areas of the ocean, such as along the western margins of Africa and South America. Conversely, thermal stratification and downwelling will limits primary production in the subtropical gyres as the sunlit surface waters are largely isolated from nutrient rich waters below the thermocline.

Light (solar radiation) provides the energy that drives photosynthesis. Light is variable on a number of spatial and temporal scales. Low latitudes receive more solar radiation than high latitudes and have less variation in solar radiation over the course of one year. At high latitudes there are pronounced seasons and variations in day length. Imposed on these month to month or season to season variations in solar radiation are short term fluctuations. The angle of the sun above the horizon affects how much light is reflected off the surface of the ocean. At midday, when the sun is at an angle of 90º to the sea surface, 2 % of the incoming solar radiation is reflected; this value increases to 40 % during the evening and early morning when the sun is at an angle of 5 º (Trujillo & Thurman, 2005). Reflection off cloud cover also significantly reduces the input of solar radiation into the ocean. Conversely, net primary productivity may be inhibited by too much light, which can lead to photoinhibition or conditions conducive to photorespiration (Fig. 1).

The depth of the euphotic zone (surface layer in which there is enough light to support photosynthesis) is often less than 10 m and rarely greater than 100 m (Fogg & Thake, 1987), whereas the mean depth of the ocean is approximately 3,700 m. Therefore, photosynthesis and primary production is limited to a thin layer at the ocean surface and whether phytoplankton cells are mixed into the dark waters below will effect primary productivity. As the mixed layer of a water column increases the average photon flux density (i.e. light) to which the cells are exposed will decrease as the circulating cell will spend longer in darkness. Therefore the total gross productivity of the phytoplankton population will decrease (Fig. 1). However, the respiration rate of the population will be relatively constant, whatever the depth of mixing. This results in a *critical depth* in a mixed water column; if the cells are mixed below the critical depth then there will be no net productivity as the respiratory loss of carbon will exceed photosynthetic carbon gain. Net photosynthesis and the resulting net primary productivity will only occur in mixed water where the mixing is less than the critical depth (Sverdrup, 1953; Kirk, 1983).

Grazing also effects production; in a heavily grazed population of phytoplankton individual cells may show high rates of productivity, but there may be a low biomass of primary producers as a large proportion of the primary production is transferred to other trophic levels. In recent years there has been a realization that phytoplankton are subject to lytic viral infection (Suttle et al.*,* 1990; Nagasaki et al., 2004), which will have an effect similar as grazing by reducing the biomass of primary producers in the water column.

Primary Production in the Ocean 575

shown that phytoplankton respond to changes in light, nutrients and temperature by adjusting cellular pigment content to meet requirements for photosynthesis. This physiological response can be seen in changes in the carbon: chlorophyll *a* ratio of the phytoplankton and therefore a remote sensing measure of the carbon: chlorophyll *a* ratio may provide an index of the physiological status of the phytoplankton. Westberry et al.*,* (2008) recently refined this model and estimated a global ocean NPP of approximately

Geider et al. (2001), based on the data of Longhurst et al. (1995) and Field et al. (1998), estimated NPP for different ocean environments, with values of: 1.2 Pg C yr-1 for salt marshes, macrophytes and estuaries; 0.7 Pg C yr-1 for coral reefs; 10.7 Pg C yr-1 for the coastal domain; 6.4 Pg C yr-1 for the polar regions; 29.3 Pg C yr-1 for the remaining ocean. The average NPP of the land (excluding areas permanently covered in ice) is 426 g C m-2 yr-1, compared to 140 g C m-2 yr-1 for the ocean (Field et al.*,* 1998). NPP per unit area of the land is three times greater than that of the ocean; however, the ocean contributes almost half of net global primary production as it covers approximately three quarters (70.8 %) of the surface

**7. Oceanic primary production and the biogeochemical cycling of elements**  Biogeochemical cycles occur as a result of the interplay between physical, geological, chemical and biological processes, which affect the distribution of elements between living and non-living reservoirs on Earth. Living organisms both react to changes in the environment and affect environmental change. This concept has led to 'Earth system science' in which the biosphere, chemosphere and geosphere are not regarded in isolation, but as an integrated whole. Phytoplankton play a crucial role in biogeochemical processes on the Earth. Phytoplankton and other marine primary producers are composed of elements in addition to carbon, oxygen, and hydrogen (equation 1). A 'typical' phytoplankton cell is composed of 25 to 50 % protein, 5 to 50 % polysaccharide, 5 to 20 % lipids, 3 to 20 % pigments, and 20 % nucleic acids (Emerson & Hedges, 2008). Therefore, the elements nitrogen, phosphorus and sulphur are required in relatively large quantities to build a functioning phytoplankton cell. Other elements are required in trace amounts, such as many metals. Consequently, phytoplankton photosynthesis and subsequent phytoplankton growth directly affect the cycling of biologically important elements on Earth. For example, cyanobacteria have been extant on Earth for at least 2.7 billion years (Knoll, 2003) and have been the dominant oxygenic photosynthetic organisms on Earth for most of that time. Photosynthesis by cyanobacteria led to an accumulation of oxygen in the atmosphere, replacing an anoxic atmosphere with something approaching the contemporary atmosphere

Stoichiometric variation in the elemental composition of photosynthetic organisms profoundly affects biological productivity, food web dynamics and the biogeochemical cycling of elements across all spatial and temporal scales (Sterner & Elser, 2002). One of the earliest ecological stoichiometry paradigms was the Redfield ratio. Redfield showed that the ratio of C:N:P in organic material collected with a plankton net (i.e. mainly phytoplankton and zooplankton) was conservative and 106:16:1 (Redfield, 1958). Redfield proposed that the ratio of nitrate: phosphate in the ocean was determined by the requirements of

by 2.2 billion years ago (Falkowski et al.*,* 1998; Knoll, 2003).

**7.1 Ecological stoichiometry of phytoplankton** 

52 Pg C yr-1.

area of Earth.

Fig. 1. Schematic of photosynthesis and respiration rates with depth in the ocean. The green line shows gross photosynthesis rate, which declines from a maximum just below the surface to zero in response to the availability of light. Phytoplankton respiration rate is constant with depth and is shown in blue; net photosynthesis is gross photosynthesis minus respiration. The compensation depth occurs where the net photosynthesis rate is zero as the respiration rate is equal to the gross photosynthesis rate. The critical depth occurs deeper in the water column and it is where depth integrated gross photosynthesis equals depth integrated respiration.

#### **6. Global estimates of primary productivity**

Estimates of net primary productivity in the oceans are on the order of 40 -55 Pg C yr-1 (1 Pg = 1 gigaton = 1015 g), which is approaching half of global annual net primary productivity. Falkowski et al. (1998), citing data from a number of papers, estimated that marine phytoplankton fix approximately 45 Pg C yr-1. Falkowski et al. (1998) estimated that 16 of the 45 Pg C yr-1 are exported from the surface to the ocean interior (see 7.2).

A key goal of satellite observations of ocean color has been to convert ocean color data to net primary productivity (NPP) and a variety of NPP algorithms exist (McCLain, 2009). Longhurst et al. (1995) estimated global net primary productivity of the oceans to be 45 – 50 Pg C yr-1 and Field et al. (1998) estimated a value of 48.5 Pg C yr-1. Since this initial work, many different algorithms have been developed to estimate NPP from ocean color data. Carr et al. (2006) compared 24 algorithms and found that the mean NPP estimate for the ocean was 51 Pg C yr-1, with the range of the estimates spanning 32 Pg C yr-1. Most of these algorithms have an empirical physiological parameter to account for phytoplankton physiological status (McClain, 2009), which is difficult to determine from space based observations (Behrenfeld et al.*,* 2005).

Behrenfeld et al., (2005) developed a carbon-based model that does not require a physiological parameter. The logic of their approach was that laboratory experiments have

Fig. 1. Schematic of photosynthesis and respiration rates with depth in the ocean. The green line shows gross photosynthesis rate, which declines from a maximum just below the surface to zero in response to the availability of light. Phytoplankton respiration rate is constant with depth and is shown in blue; net photosynthesis is gross photosynthesis minus respiration. The compensation depth occurs where the net photosynthesis rate is zero as the respiration rate is equal to the gross photosynthesis rate. The critical depth occurs deeper in the water column and it is where depth integrated gross photosynthesis equals depth

Estimates of net primary productivity in the oceans are on the order of 40 -55 Pg C yr-1 (1 Pg = 1 gigaton = 1015 g), which is approaching half of global annual net primary productivity. Falkowski et al. (1998), citing data from a number of papers, estimated that marine phytoplankton fix approximately 45 Pg C yr-1. Falkowski et al. (1998) estimated that

A key goal of satellite observations of ocean color has been to convert ocean color data to net primary productivity (NPP) and a variety of NPP algorithms exist (McCLain, 2009). Longhurst et al. (1995) estimated global net primary productivity of the oceans to be 45 – 50 Pg C yr-1 and Field et al. (1998) estimated a value of 48.5 Pg C yr-1. Since this initial work, many different algorithms have been developed to estimate NPP from ocean color data. Carr et al. (2006) compared 24 algorithms and found that the mean NPP estimate for the ocean was 51 Pg C yr-1, with the range of the estimates spanning 32 Pg C yr-1. Most of these algorithms have an empirical physiological parameter to account for phytoplankton physiological status (McClain, 2009), which is difficult to determine from space based

Behrenfeld et al., (2005) developed a carbon-based model that does not require a physiological parameter. The logic of their approach was that laboratory experiments have

16 of the 45 Pg C yr-1 are exported from the surface to the ocean interior (see 7.2).

integrated respiration.

**6. Global estimates of primary productivity** 

observations (Behrenfeld et al.*,* 2005).

shown that phytoplankton respond to changes in light, nutrients and temperature by adjusting cellular pigment content to meet requirements for photosynthesis. This physiological response can be seen in changes in the carbon: chlorophyll *a* ratio of the phytoplankton and therefore a remote sensing measure of the carbon: chlorophyll *a* ratio may provide an index of the physiological status of the phytoplankton. Westberry et al.*,* (2008) recently refined this model and estimated a global ocean NPP of approximately 52 Pg C yr-1.

Geider et al. (2001), based on the data of Longhurst et al. (1995) and Field et al. (1998), estimated NPP for different ocean environments, with values of: 1.2 Pg C yr-1 for salt marshes, macrophytes and estuaries; 0.7 Pg C yr-1 for coral reefs; 10.7 Pg C yr-1 for the coastal domain; 6.4 Pg C yr-1 for the polar regions; 29.3 Pg C yr-1 for the remaining ocean. The average NPP of the land (excluding areas permanently covered in ice) is 426 g C m-2 yr-1, compared to 140 g C m-2 yr-1 for the ocean (Field et al.*,* 1998). NPP per unit area of the land is three times greater than that of the ocean; however, the ocean contributes almost half of net global primary production as it covers approximately three quarters (70.8 %) of the surface area of Earth.

#### **7. Oceanic primary production and the biogeochemical cycling of elements**

Biogeochemical cycles occur as a result of the interplay between physical, geological, chemical and biological processes, which affect the distribution of elements between living and non-living reservoirs on Earth. Living organisms both react to changes in the environment and affect environmental change. This concept has led to 'Earth system science' in which the biosphere, chemosphere and geosphere are not regarded in isolation, but as an integrated whole. Phytoplankton play a crucial role in biogeochemical processes on the Earth. Phytoplankton and other marine primary producers are composed of elements in addition to carbon, oxygen, and hydrogen (equation 1). A 'typical' phytoplankton cell is composed of 25 to 50 % protein, 5 to 50 % polysaccharide, 5 to 20 % lipids, 3 to 20 % pigments, and 20 % nucleic acids (Emerson & Hedges, 2008). Therefore, the elements nitrogen, phosphorus and sulphur are required in relatively large quantities to build a functioning phytoplankton cell. Other elements are required in trace amounts, such as many metals. Consequently, phytoplankton photosynthesis and subsequent phytoplankton growth directly affect the cycling of biologically important elements on Earth. For example, cyanobacteria have been extant on Earth for at least 2.7 billion years (Knoll, 2003) and have been the dominant oxygenic photosynthetic organisms on Earth for most of that time. Photosynthesis by cyanobacteria led to an accumulation of oxygen in the atmosphere, replacing an anoxic atmosphere with something approaching the contemporary atmosphere by 2.2 billion years ago (Falkowski et al.*,* 1998; Knoll, 2003).

#### **7.1 Ecological stoichiometry of phytoplankton**

Stoichiometric variation in the elemental composition of photosynthetic organisms profoundly affects biological productivity, food web dynamics and the biogeochemical cycling of elements across all spatial and temporal scales (Sterner & Elser, 2002). One of the earliest ecological stoichiometry paradigms was the Redfield ratio. Redfield showed that the ratio of C:N:P in organic material collected with a plankton net (i.e. mainly phytoplankton and zooplankton) was conservative and 106:16:1 (Redfield, 1958). Redfield proposed that the ratio of nitrate: phosphate in the ocean was determined by the requirements of

Primary Production in the Ocean 577

content of carbon dioxide. For example, if there is a significant change in either marine photosynthesis or respiration, or the balance between the two processes, then the mixing ratio of carbon dioxide in the atmosphere will change. Consequently, determining the fate of primary production in the ocean is important in understanding the global carbon cycle and global climate change. An important question is how much organic carbon is exported from surface waters and into the deep ocean. Most of the carbon fixed through photosynthesis is remineralized back to carbon dioxide in surface waters. However, some organic carbon sinks into deeper water and a very small fraction of organic carbon will make it all the way to the seafloor. Even if the organic matter is remineralized at depth, it may take thousands of years for it to be returned to the atmosphere as CO2 and carbon buried in the seafloor may be potentially sequestered for millions of years. The carbon pump refers to processes that affect a vertical gradient in dissolved inorganic carbon concentration in the ocean (Emerson & Hedges, 2008). These processes collectively remove carbon from the surface ocean and atmosphere and into the deep ocean. The carbon pump has two components, the solubility pump and biological carbon pump. The biological carbon pump is the biologically mediated processes that transport carbon from the surface ocean into the deep ocean, such as the sinking of phytoplankton aggregates (Thornton, 2002), fecal pellets, dead organisms,

Cyanobacteria have an important role in the nitrogen cycle as many species are nitrogen fixing and consequently sustain primary productivity in nitrogen-limited areas of the ocean (Capone, 2000). The availability of nitrogen generally limits phytoplankton photosynthesis and growth in the ocean (Ryther & Dunstan, 1971; Howarth, 1988; Tyrell 1999). Although 78 % of the atmosphere is in the form of N2, this can only be used by organisms possessing nitrogense enzymes to reduce N2 to biologically available NH4+; this nitrogen fixing ability is limited to certain groups of prokaryotes, including many cyanophyceae. In the vast oligotrophic tropical and subtropical areas of ocean the colonial, filamentous cyanobacteria of the genus *Trichodesmium* are important primary producers as they can overcome nitrogen limitation by fixation (Capone et al.*,* 1997). *Trichodesmium* may form extensive surface blooms (it is buoyant due to gas vesicles) in stable, clear, low nutrient water columns. It has a cosmopolitan distribution and is probably a significant contributor to global primary productivity (Capone et al.*,* 1997). The remineralization of carbon associated with the cyanobacteria will release bioavailable nitrogen into the water column which may support the growth of other groups of primary producers. See Zehr & Paerl (2008) for a recent

The discussion above implies that cyanobacteria are constantly fixing nitrogen from the atmosphere and enriching the ocean with bioavailable nitrogen, which would accumulate in the ocean over time. This is not the case as there are processes that remove bioavailable nitrogen from ecosystems. A description of the marine nitrogen cycle is beyond the scope of this review (see Capone 2000; Thamdrup & Dalsgaard, 2008). However, nitrogen exists in a number of oxidation states in the ocean with the transformation between oxidation states largely conducted by prokaryotes. There are two major microbial processes that remove bioavailable nitrogen from the ocean and return it to the sink of the atmosphere: denitrification and anaerobic ammonium oxidation (anammox) (Thamdrup & Dalsgaard,

and DOC.

2008).

**7.3 Primary production and nitrogen cycling** 

review of nitrogen fixation in the ocean.

phytoplankton, which release nitrogen and phosphorus into the environment as they are remineralized (Arrigo, 2005).

In the last few years there has been a resurgence of interest in measuring the elemental stoichiometry of marine phytoplankton (Quigg et al.*,* 2003; Ho et al.*,* 2003; Leonardos & Geider 2004; Bertilsson et al.*,* 2003; Klausmeier et al.*,* 2004), as determining the degree to which C:N:P stoichiometry can deviate from the Redfield ratio of 106:16:1 is seen as critical in the understanding of the role of phytoplankton in biogeochemistry (Falkowski, 2000; Geider & La Roche, 2002). For example, as we have seen, the Redfield ration of C:N:P of 106:16:1 represents a mean value for marine phytoplankton; there are actually significant differences in C:N, C:P and N:P ratios in different phyla of phytoplankton (Quigg et al.*,* 2003). Klausmeier et al. (2004) used a modeling approach to show that N:P ratios depend on ecological conditions and that optimum N:P ratios vary between 8.2 and 45.0. Stoichiometric variation may occur between taxa or even within one species depending on growth conditions. A source of variation comes from the fact that different components of the cell's structure have different stoichiometric ratios (Arrigo, 2005). Cell machinery for acquiring resources (light and nutrients), such as chlorophyll and protein, are rich in N and contain low P. On the other hand, growth machinery such as ribosomal RNA are rich in both N and P. Consequently, changes in the relative proportions of key cell components will affect the stoichiometry of an individual phytoplankton (Arrigo, 2005). Different growth strategies will result in different cell stoichiometries; exponentially growing bloom-forming phytoplankton have an optimal N:P ratio of 8 as they are optimized for growth. Whereas the N:P ratio of phytoplankton in a resource scarce environmental is optimal at 36 to 45 as they contain more machinery for resource acquisition (Klausmeier et al.*,* 2004; Arrigo, 2005).

#### **7.2 Primary production and carbon cycling**

As we have seen, marine photosynthesis supports approximately half of global primary production and therefore marine primary producers have a profound effect on the global carbon cycle. While there are some non-photosynthetic primary producers in the ocean, their contribution to carbon fixation in the ocean is negligible. Therefore, ultimately all the organic matter was originally fixed by oxygenic photosynthetic organisms. The pool of organic carbon associated with living organisms in the ocean is relatively small. Falkowski and Raven (2007) estimate that the amount of carbon in phytoplankton in the global ocean is between 0.25 and 0.65 Pg. To support an annual primary production of 50 Pg C yr-1, the biomass of phytoplankton must turn over between 60 and 150 times per year, or every 2 to 6 days. This is very different from terrestrial primary productivity, which is dominated by multicellular woody organisms and has a mean turnover time of 12 to 16 years (Falkowski & Raven, 2007). The pool of non-living organic carbon in the ocean is much larger than the carbon associated with living organisms, with an estimate of 1000 Pg C (Falkowski et al. 2000). Most of the non-living carbon in the ocean is in the form of dissolved organic carbon (DOC) rather than particulate organic carbon (POC). Hansell et al. (2009) estimated that the oceans contain 662 Pg C as dissolved organic carbon (DOC). To put this into context, the atmosphere contained 612 Pg C in 1850 and 784 Pg C in 2000 (Emerson & Hedges 2008). However, the largest pool of carbon in the ocean is dissolved inorganic carbon, which contains 38,000 Pg C (Emerson & Hedges, 2008).

Given that the annual rate of marine primary productivity is around 50 Pg C year-1, it can be seen that primary productivity in the ocean has the potential to affect the atmospheric

phytoplankton, which release nitrogen and phosphorus into the environment as they are

In the last few years there has been a resurgence of interest in measuring the elemental stoichiometry of marine phytoplankton (Quigg et al.*,* 2003; Ho et al.*,* 2003; Leonardos & Geider 2004; Bertilsson et al.*,* 2003; Klausmeier et al.*,* 2004), as determining the degree to which C:N:P stoichiometry can deviate from the Redfield ratio of 106:16:1 is seen as critical in the understanding of the role of phytoplankton in biogeochemistry (Falkowski, 2000; Geider & La Roche, 2002). For example, as we have seen, the Redfield ration of C:N:P of 106:16:1 represents a mean value for marine phytoplankton; there are actually significant differences in C:N, C:P and N:P ratios in different phyla of phytoplankton (Quigg et al.*,* 2003). Klausmeier et al. (2004) used a modeling approach to show that N:P ratios depend on ecological conditions and that optimum N:P ratios vary between 8.2 and 45.0. Stoichiometric variation may occur between taxa or even within one species depending on growth conditions. A source of variation comes from the fact that different components of the cell's structure have different stoichiometric ratios (Arrigo, 2005). Cell machinery for acquiring resources (light and nutrients), such as chlorophyll and protein, are rich in N and contain low P. On the other hand, growth machinery such as ribosomal RNA are rich in both N and P. Consequently, changes in the relative proportions of key cell components will affect the stoichiometry of an individual phytoplankton (Arrigo, 2005). Different growth strategies will result in different cell stoichiometries; exponentially growing bloom-forming phytoplankton have an optimal N:P ratio of 8 as they are optimized for growth. Whereas the N:P ratio of phytoplankton in a resource scarce environmental is optimal at 36 to 45 as they contain more machinery for resource acquisition (Klausmeier et al.*,* 2004; Arrigo, 2005).

As we have seen, marine photosynthesis supports approximately half of global primary production and therefore marine primary producers have a profound effect on the global carbon cycle. While there are some non-photosynthetic primary producers in the ocean, their contribution to carbon fixation in the ocean is negligible. Therefore, ultimately all the organic matter was originally fixed by oxygenic photosynthetic organisms. The pool of organic carbon associated with living organisms in the ocean is relatively small. Falkowski and Raven (2007) estimate that the amount of carbon in phytoplankton in the global ocean is between 0.25 and 0.65 Pg. To support an annual primary production of 50 Pg C yr-1, the biomass of phytoplankton must turn over between 60 and 150 times per year, or every 2 to 6 days. This is very different from terrestrial primary productivity, which is dominated by multicellular woody organisms and has a mean turnover time of 12 to 16 years (Falkowski & Raven, 2007). The pool of non-living organic carbon in the ocean is much larger than the carbon associated with living organisms, with an estimate of 1000 Pg C (Falkowski et al. 2000). Most of the non-living carbon in the ocean is in the form of dissolved organic carbon (DOC) rather than particulate organic carbon (POC). Hansell et al. (2009) estimated that the oceans contain 662 Pg C as dissolved organic carbon (DOC). To put this into context, the atmosphere contained 612 Pg C in 1850 and 784 Pg C in 2000 (Emerson & Hedges 2008). However, the largest pool of carbon in the ocean is dissolved inorganic carbon, which

Given that the annual rate of marine primary productivity is around 50 Pg C year-1, it can be seen that primary productivity in the ocean has the potential to affect the atmospheric

remineralized (Arrigo, 2005).

**7.2 Primary production and carbon cycling** 

contains 38,000 Pg C (Emerson & Hedges, 2008).

content of carbon dioxide. For example, if there is a significant change in either marine photosynthesis or respiration, or the balance between the two processes, then the mixing ratio of carbon dioxide in the atmosphere will change. Consequently, determining the fate of primary production in the ocean is important in understanding the global carbon cycle and global climate change. An important question is how much organic carbon is exported from surface waters and into the deep ocean. Most of the carbon fixed through photosynthesis is remineralized back to carbon dioxide in surface waters. However, some organic carbon sinks into deeper water and a very small fraction of organic carbon will make it all the way to the seafloor. Even if the organic matter is remineralized at depth, it may take thousands of years for it to be returned to the atmosphere as CO2 and carbon buried in the seafloor may be potentially sequestered for millions of years. The carbon pump refers to processes that affect a vertical gradient in dissolved inorganic carbon concentration in the ocean (Emerson & Hedges, 2008). These processes collectively remove carbon from the surface ocean and atmosphere and into the deep ocean. The carbon pump has two components, the solubility pump and biological carbon pump. The biological carbon pump is the biologically mediated processes that transport carbon from the surface ocean into the deep ocean, such as the sinking of phytoplankton aggregates (Thornton, 2002), fecal pellets, dead organisms, and DOC.

#### **7.3 Primary production and nitrogen cycling**

Cyanobacteria have an important role in the nitrogen cycle as many species are nitrogen fixing and consequently sustain primary productivity in nitrogen-limited areas of the ocean (Capone, 2000). The availability of nitrogen generally limits phytoplankton photosynthesis and growth in the ocean (Ryther & Dunstan, 1971; Howarth, 1988; Tyrell 1999). Although 78 % of the atmosphere is in the form of N2, this can only be used by organisms possessing nitrogense enzymes to reduce N2 to biologically available NH4+; this nitrogen fixing ability is limited to certain groups of prokaryotes, including many cyanophyceae. In the vast oligotrophic tropical and subtropical areas of ocean the colonial, filamentous cyanobacteria of the genus *Trichodesmium* are important primary producers as they can overcome nitrogen limitation by fixation (Capone et al.*,* 1997). *Trichodesmium* may form extensive surface blooms (it is buoyant due to gas vesicles) in stable, clear, low nutrient water columns. It has a cosmopolitan distribution and is probably a significant contributor to global primary productivity (Capone et al.*,* 1997). The remineralization of carbon associated with the cyanobacteria will release bioavailable nitrogen into the water column which may support the growth of other groups of primary producers. See Zehr & Paerl (2008) for a recent review of nitrogen fixation in the ocean.

The discussion above implies that cyanobacteria are constantly fixing nitrogen from the atmosphere and enriching the ocean with bioavailable nitrogen, which would accumulate in the ocean over time. This is not the case as there are processes that remove bioavailable nitrogen from ecosystems. A description of the marine nitrogen cycle is beyond the scope of this review (see Capone 2000; Thamdrup & Dalsgaard, 2008). However, nitrogen exists in a number of oxidation states in the ocean with the transformation between oxidation states largely conducted by prokaryotes. There are two major microbial processes that remove bioavailable nitrogen from the ocean and return it to the sink of the atmosphere: denitrification and anaerobic ammonium oxidation (anammox) (Thamdrup & Dalsgaard, 2008).

Primary Production in the Ocean 579

data of ocean color, NPP in the global ocean is estimated to be around 50 Pg C year-1, which is similar to terrestrial NPP. Primary production in the ocean affects the cycling of biologically significant elements, such as carbon, nitrogen, and phosphorus. A key question is how phytoplankton production affects the biogeochemical cycling of elements (e.g.

This material is based upon work supported by the National Science Foundation under Grant No. OCE 0726369 to Daniel C. O. Thornton. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author and do not

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carbon, nitrogen, phosphorus, and iron) and climate change.

necessarily reflect the views of the National Science Foundation.

No.7057, pp. 349-355, ISSN 0028-0836

**9. Acknowledgment** 

**10. References** 

0025-3162

20, ISSN 0024-3590

No.2, pp.169-187, ISSN 0948-3055

#### **7.4 Primary production and phosphorus cycling**

Concentrations of phosphorus in the ocean are controlled by geological processes; phosphorus is added to the ocean from the weathering of rocks and removed by burial in marine sediments. Unlike nitrogen, phosphorus has no atmospheric source. On geological time scales phosphorus is the *ultimate limiting nutrient* for primary production in the ocean (Tyrell, 1999). However, on shorter timescales bioavailable nitrogen is the *proximate limiting nutrient* in the sense of Liebig's law (Tyrell, 1999). The addition of just nitrogen (i.e. nitrate or ammonium) to surface waters from most of the ocean would result in an enhancement of phytoplankton production. Moreover, there is a slight excess of phosphate in the ocean compared to nitrate in surface waters as nitrate is depleted before phosphate (Tyrell, 1999).

#### **7.5 Primary production and iron cycling**

There was a vigorous debate as to what limited primary production in the HNLC areas of the global ocean (see section 5). John Martin (1992) proposed the hypothesis that iron availability limits the growth of phytoplankton in the HNLC ocean. The implications of this hypothesis are that the uptake of other nutrients is limited by iron availability and that the amount of carbon dioxide in the atmosphere varies as a function of iron transport to the surface of the ocean (Millero, 2006). This has implications for the global carbon cycle as it implies that iron supply to iron limited phytoplankton plays a role in determining how much carbon is sequestered in the deep ocean and underlying sediments. The iron hypothesis was tested in the laboratory and during ship board experiments. While these experiments showed iron limitation, the results do not necessary reflect the *in situ* response to iron fertilization as small samples enclosed in bottles are subject to bottle effects and do not capture the complexity of natural systems. Consequently, a number of experiments have been conducted in which large areas of ocean have been fertilized with iron (Martin et al.*,* 1994; Coale et al.*,* 1996; 2004; Boyd et al.*,* 2000; Tsuda et al.*,* 2003). These experiments have shown that HNLC are iron limited. There was a significant increase in phytoplankton biomass over the course of a few days in the iron fertilized patches resulting in a significant draw down of carbon dioxide due to photosynthetic carbon fixation. Moreover, the alleviation of iron limitation affected the efficiency of photosynthesis as there was an increase in ФPSII (Kolber et al.*,* 1994; Coale et al.*,* 2004). The addition of iron to HNLC areas has been proposed as a geoengineering approach to sequester atmospheric carbon dioxide in the deep ocean and thereby partially offset anthropogenic inputs of carbon dioxide from fossil fuel burning and land use change. This is controversial, as although the addition of iron to HNLC areas stimulates primary production and the drawdown of carbon dioxide, there is relatively little evidence that a significant proportion of the resulting organic carbon sinks into the deep ocean and is sequestered (Buesseler et al.*,* 2004; Boyd et al.*,* 2004).

#### **8. Conclusion**

Photosynthesis and primary production in the ocean is dominated by a phylogenetically diverse group of microalgae that make up the phytoplankton. A number of techniques are used to measure phytoplankton photosynthesis. These techniques are based on gas exchange, the use of radioactive or stable isotopes as tracers, and variable chlorophyll *a* fluorescence. The techniques are not equivalent, and different techniques will produce different rates of photosynthesis dependent on the biases and assumptions intrinsic in the methodology. Phytoplankton photosynthesis is a globally significant process. Using satellite

Concentrations of phosphorus in the ocean are controlled by geological processes; phosphorus is added to the ocean from the weathering of rocks and removed by burial in marine sediments. Unlike nitrogen, phosphorus has no atmospheric source. On geological time scales phosphorus is the *ultimate limiting nutrient* for primary production in the ocean (Tyrell, 1999). However, on shorter timescales bioavailable nitrogen is the *proximate limiting nutrient* in the sense of Liebig's law (Tyrell, 1999). The addition of just nitrogen (i.e. nitrate or ammonium) to surface waters from most of the ocean would result in an enhancement of phytoplankton production. Moreover, there is a slight excess of phosphate in the ocean compared to nitrate in surface waters as nitrate is depleted before phosphate (Tyrell, 1999).

There was a vigorous debate as to what limited primary production in the HNLC areas of the global ocean (see section 5). John Martin (1992) proposed the hypothesis that iron availability limits the growth of phytoplankton in the HNLC ocean. The implications of this hypothesis are that the uptake of other nutrients is limited by iron availability and that the amount of carbon dioxide in the atmosphere varies as a function of iron transport to the surface of the ocean (Millero, 2006). This has implications for the global carbon cycle as it implies that iron supply to iron limited phytoplankton plays a role in determining how much carbon is sequestered in the deep ocean and underlying sediments. The iron hypothesis was tested in the laboratory and during ship board experiments. While these experiments showed iron limitation, the results do not necessary reflect the *in situ* response to iron fertilization as small samples enclosed in bottles are subject to bottle effects and do not capture the complexity of natural systems. Consequently, a number of experiments have been conducted in which large areas of ocean have been fertilized with iron (Martin et al.*,* 1994; Coale et al.*,* 1996; 2004; Boyd et al.*,* 2000; Tsuda et al.*,* 2003). These experiments have shown that HNLC are iron limited. There was a significant increase in phytoplankton biomass over the course of a few days in the iron fertilized patches resulting in a significant draw down of carbon dioxide due to photosynthetic carbon fixation. Moreover, the alleviation of iron limitation affected the efficiency of photosynthesis as there was an increase in ФPSII (Kolber et al.*,* 1994; Coale et al.*,* 2004). The addition of iron to HNLC areas has been proposed as a geoengineering approach to sequester atmospheric carbon dioxide in the deep ocean and thereby partially offset anthropogenic inputs of carbon dioxide from fossil fuel burning and land use change. This is controversial, as although the addition of iron to HNLC areas stimulates primary production and the drawdown of carbon dioxide, there is relatively little evidence that a significant proportion of the resulting organic carbon

sinks into the deep ocean and is sequestered (Buesseler et al.*,* 2004; Boyd et al.*,* 2004).

Photosynthesis and primary production in the ocean is dominated by a phylogenetically diverse group of microalgae that make up the phytoplankton. A number of techniques are used to measure phytoplankton photosynthesis. These techniques are based on gas exchange, the use of radioactive or stable isotopes as tracers, and variable chlorophyll *a* fluorescence. The techniques are not equivalent, and different techniques will produce different rates of photosynthesis dependent on the biases and assumptions intrinsic in the methodology. Phytoplankton photosynthesis is a globally significant process. Using satellite

**7.4 Primary production and phosphorus cycling** 

**7.5 Primary production and iron cycling** 

**8. Conclusion** 

data of ocean color, NPP in the global ocean is estimated to be around 50 Pg C year-1, which is similar to terrestrial NPP. Primary production in the ocean affects the cycling of biologically significant elements, such as carbon, nitrogen, and phosphorus. A key question is how phytoplankton production affects the biogeochemical cycling of elements (e.g. carbon, nitrogen, phosphorus, and iron) and climate change.

#### **9. Acknowledgment**

This material is based upon work supported by the National Science Foundation under Grant No. OCE 0726369 to Daniel C. O. Thornton. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author and do not necessarily reflect the views of the National Science Foundation.

#### **10. References**


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### *Edited by Mohammad Mahdi Najafpour*

Photosynthesis is one of the most important reactions on Earth. It is a scientific field that is the topic of many research groups. This book is aimed at providing the fundamental aspects of photosynthesis, and the results collected from different research groups. There are three sections in this book: light and photosynthesis, the path of carbon in photosynthesis, and special topics in photosynthesis. In each section important topics in the subject are discussed and (or) reviewed by experts in each book chapter.

Advances in Photosynthesis - Fundamental Aspects

Advances in Photosynthesis

Fundamental Aspects

*Edited by Mohammad Mahdi Najafpour*

Photo by coleong / iStock