**Meet the editor**

Tetsuji Nagata, M.D, Ph.D, Professor Emeritus (Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto, and Department of Anatomy, Shinshu Institute of Alternative Medicine and Welfare, Nagano) was Born in Nagano, Japan, February 5, 1931. After high school he finished premedical course at Shinshu University College of Liberal Arts and

Sciences; medical education at Shinshu University School of Medicine, Matsumoto, Japan, 1951-1955. He received his PhD in Anatomy 1961., also at Shinshu University Graduate School of Medicine, Matsumoto, Japan. Dr. Nagata received several awards and honors. He is author of 10 single-authored books and 112 co-authored books, 586 original and review papers, 713 contributed papers at national and international conferences in Asian-Pacific, European and American countries.

Contents

**Preface XI** 

Chapter 1 **Photo- and Free Radical-Mediated** 

Jean-François Rontani

Chapter 4 **Functional Approaches to** 

Chapter 5 **Plant Ageing,** 

**Oxidation of Lipid Components** 

Chapter 2 **Role of Intracellular Hydrogen Peroxide** 

Chapter 3 **Metabolic Regulation of Leaf Senescence** 

**During the Senescence of Phototrophic Organisms 3** 

**as Signalling Molecule for Plant Senescence 31**  Ulrike Zentgraf, Petra Zimmermann and Anja Smykowski

**in Sunflower (***Helianthus annuus* **L.) Plants 51**  Eloísa Agüera, Purificación Cabello, Lourdes de la Mata,

**a Counteracting Agent to Xenobiotic Stress 89** 

Matt Humphry and Juan Pablo Sanchez-Tamburrino

Laurence Dupont, Geneviève Alloing, Olivier Pierre,

**From Symbiotic Nitrogen Fixation to Senescence 137** 

Sarra El Msehli, Julie Hopkins, Didier Hérouart and Pierre Frendo

Estefanía Molina and Purificación de la Haba

**Study Leaf Senescence in Sunflower 69**  Paula Fernandez, Sebastián Moschen, Norma Paniego and Ruth A. Heinz

David Delmail and Pascal Labrousse

Chapter 6 **Some Aspects of Leaf Senescence 107**  Hafsi Miloud and Guendouz Ali

Chapter 7 **Advances in Plant Senescence 117**  Kieron D. Edwards,

Chapter 8 **The Legume Root Nodule:** 

**Part 1 Plant 1** 

## Contents



Contents VII

Chapter 20 **Alternative Splicing in** 

Chapter 21 **Quantification of Elastin,** 

Chapter 23 **All Your Eggs in One Basket:** 

**Part 3 Human 585**

Chapter 24 **Parkinson's Disease:** 

Chapter 25 **The Emerging Role** 

Kayoko Maehara

**Endothelial Senescence:** 

Chapter 22 **Calcium Regulation in Neuronal Function**

**Mechanisms of Xenobiotic**

Erik J. Behringer and Sue P. Duckles

Jing-ye Zhou, Yong Yu, Xian-Lun Zhu, Chi-Ping Ng, Gang Lu and Wai-Sang Poon

**Proteins in Cellular Senescence 617**

Chapter 26 **The Functioning of "Aged" Heterochromatin 631**

**of Centromere/Kinetochore** 

Chapter 27 **New Targets for the Identification of** 

Chapter 28 **Molecular Biomarkers of Aging 667** 

Chapter 29 **Female Vascular Senescence 681**

Rita Ostan and Claudio Franceschi

Davide Zella and Giovanni Scapagnini

Carlos Hermenegildo and Pascual Medina

**Role of the TGF-β Co-Receptor Endoglin 499** 

**Products as Functions of Age and Hypertension 519**  Milena Atanasova, Aneliya Dimitrova, Boryana Ruseva, Angelina Stoyanova, Miglena Georgieva and Emiliana Konova

**with Advancing Age: Limits of Homeostasis 531**  John N. Buchholz, William J. Pottorf, Conwin K. Vanterpool,

**Induced Female Reproductive Senescence 559** 

Alexander P. Sobinoff, Ilana R. Bernstein and Eileen A. McLaughlin

**Insights from the Laboratory and Clinical Therapeutics 587**

Teimuraz A. Lezhava, Tinatin A. Jokhadze and Jamlet R. Monaselidze

**an Anti-Inflammatory Anti-Senescence Activity 647**

Patrizia d'Alessio, Annelise Bennaceur-Griscelli,

Sergio Davinelli, Sonya Vasto, Calogero Caruso,

Susana Novella, Ana Paula Dantas, Gloria Segarra,

Francisco J. Blanco and Carmelo Bernabéu

**Collagen and Advanced Glycation End**

	- **Part 3 Human 585**

VI Contents

**Part 2 Animal 169**

Chapter 9 **The Nucleolus and** 

Chapter 10 **Senescence in Animals:**

Chapter 12 **Programming and** 

Jaba Tkemaladze,

Chapter 14 **Cell Senescence as Observed** 

Tetsuji Nagata

Chapter 15 **Macromolecular Synthesis**

Tetsuji Nagata

Chapter 16 **Macromolecular Synthesis**

Tetsuji Nagata

Chapter 17 **Macromolecular Synthesis**

Tetsuji Nagata

Keith Wheaton

Diego Julio Arenas-Aranda,

Chapter 19 **Caveolar Vesicles in Cellular Senescence 463**

Chapter 13 **Cellular Degradation Machineries in** 

Mikael Altun, Max Grönholdt-Klein, Lingzhan Wang and Brun Ulfhake

**Ribosomal Genes in Aging and Senescence 171** 

Katherine M. Hannan, Austen Ganley and Ross D. Hannan

Thiago Monaco, Daniel Silvestre and Paulo S. P. Silveira

Paula Medone, Jorge Rabinovich, Eliana Nieves, Soledad Ceccarelli,

Nadine Hein, Elaine Sanij, Jaclyn Quin,

Chapter 11 **The Quest for Immortality in Triatomines:** 

**Why Evolutionary Theories Matter 209**

**A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 225** 

Delmi Canale, Raúl L. Stariolo and Frédéric Menu

**Implementation of Age-Related Changes 251** 

Alexander Tavartkiladze and Konstantin Chichinadze

**by Electron Microscopic Radioautography 287** 

**in the Digestive and Respiratory Systems 315**

**in the Urinary and Reproductive Systems 359**

Chapter 18 **Cellular Senescence and Its Relation with Telomere 439** 

**in the Endocrine, Nervous and Sensory Systems 387**

Elena Hernández-Caballero and Fabio Salamanca-Gómez

**Age-Related Loss of Muscle Mass (Sarcopenia) 269**



Hulya Yucel

## Preface

This book is aimed to describe all the phenomena related to aging and senescence of all forms of life on Earth, i.e. plants, animals and the human beings. The book is comprised of 36 chapters written by diverse authors, including botanists, zoologists and physicians who study the aging and senescence of plants, animals and humans from structural and functional viewpoints.

Aging is the time frame during which a person, animal or a plant has lived, or a thing has existed. On the contrary, senescence are the signs of old age that a person, animal or a plant shows once they get old. This book aims to describe all the phenomena appearing in plants, animals and humans after they got old and became senescent.

The book contains 36 carefully reviewed chapters written by different authors, aiming to describe the aging and senescent changes of living creatures, i.e. plants and animals.

In each section, the chapters are arranged from lower plants or animals to higher creatures, as well as from the organ of movement to cardiovascular, visceral and neuro-sensory systems, according to the order of anatomy and histology. Thus, the readers will be able to carry each volume easily.

The Editor hopes that this book will have an interactive role in various fields of biology and medicine necessary to conduct further studies on aging and senescence. It should recount the background and current status of our knowledge in this field as well.

Finally, the Editor would like to express sincere gratitude to all the contributing authors and the staff of InTech – Open Access Publisher, especially Ms. Iva Simcic who regularly communicated with the editor and the respective authors, for their expertise and cooperation in the publication of this book.

> **Dr. Tetsuji Nagata**  Shinshu University School of Medicine Japan

**Part 1** 

**Plant** 

**Part 1** 

**Plant** 

**1** 

*France*

Jean-François Rontani

**Photo- and Free Radical-Mediated** 

**Oxidation of Lipid Components During the** 

**Senescence of Phototrophic Organisms** 

*Laboratory of Microbiology, Geochemistry and Marine Ecology (UMR 6117),* 

*Center of Oceanology of Marseille, Aix-Marseille University, Campus of Luminy, Marseille,* 

Recently, the role played by photochemical and free radical-mediated processes in the degradation of lipid components during the senescence of phototrophic organisms was investigated. The present paper reviews the results obtained in the course of these studies. In a first part, visible and UV light-induced photooxidation of the main lipid cell components (chlorophylls, carotenoids, sterols, unsaturated fatty acids, highly branched isoprenoid and linear alkenes, alkenones, cuticular waxes …) in senescent phototrophic organisms (phytoplankton, cyanobacteria, higher plants, purple sulfur bacteria and aerobic anoxygenic phototrophic bacteria) is examined. Probably due to its long lifetime in hydrophobic micro-environments and thus in senescent cells, singlet oxygen plays a key

The second part of this paper describes the free radical oxidation (autoxidation) of lipid components during the senescence of phototrophic organisms, which have been virtually ignored until now in the literature. In senescent phototrophic organisms**,** the mechanism of initiation of free-radical oxidation seems to be the homolytic cleavage (catalyzed by some metal ions) of photochemically produced hydroperoxides. It was also demonstrated recently that viral infection and autocatalytic programmed cell death could also lead to elevated production of

Several works suggested photo-oxidation as an important sink of organic matter in the photic layer of oceans (Zafiriou, 1977; Zafiriou et al., 1984). However, due to the lack of suitable markers this phenomenon has never been fully addressed. Owing to the problem of stratospheric ozone depletion, some studies have recently examined the degradative effects of enhanced UV-B doses on phytoplanktonic lipids (He and Häder, 2002). However, photochemical damages in phytoplanktonic cells are not a monopoly of UV radiation. In fact, due to the presence of chlorophylls (which are very efficient photosensitizers (Foote, 1976; Knox and Dodge, 1985)), numerous organic components of phytoplankton are susceptible to

being photodegraded during senescence by photosynthetically active radiation (PAR).

reactive oxygen species (ROS) able to induce the degradation of cell components.

**2. Photodegradation processes in phototrophic organisms**

role in the photodegradation of most of the lipid components.

**1. Introduction** 

## **Chapter 1**

## **Photo- and Free Radical-Mediated Oxidation of Lipid Components During the Senescence of Phototrophic Organisms**

Jean-François Rontani

*Laboratory of Microbiology, Geochemistry and Marine Ecology (UMR 6117), Center of Oceanology of Marseille, Aix-Marseille University, Campus of Luminy, Marseille, France* 

## **1. Introduction**

Recently, the role played by photochemical and free radical-mediated processes in the degradation of lipid components during the senescence of phototrophic organisms was investigated. The present paper reviews the results obtained in the course of these studies.

In a first part, visible and UV light-induced photooxidation of the main lipid cell components (chlorophylls, carotenoids, sterols, unsaturated fatty acids, highly branched isoprenoid and linear alkenes, alkenones, cuticular waxes …) in senescent phototrophic organisms (phytoplankton, cyanobacteria, higher plants, purple sulfur bacteria and aerobic anoxygenic phototrophic bacteria) is examined. Probably due to its long lifetime in hydrophobic micro-environments and thus in senescent cells, singlet oxygen plays a key role in the photodegradation of most of the lipid components.

The second part of this paper describes the free radical oxidation (autoxidation) of lipid components during the senescence of phototrophic organisms, which have been virtually ignored until now in the literature. In senescent phototrophic organisms**,** the mechanism of initiation of free-radical oxidation seems to be the homolytic cleavage (catalyzed by some metal ions) of photochemically produced hydroperoxides. It was also demonstrated recently that viral infection and autocatalytic programmed cell death could also lead to elevated production of reactive oxygen species (ROS) able to induce the degradation of cell components.

## **2. Photodegradation processes in phototrophic organisms**

Several works suggested photo-oxidation as an important sink of organic matter in the photic layer of oceans (Zafiriou, 1977; Zafiriou et al., 1984). However, due to the lack of suitable markers this phenomenon has never been fully addressed. Owing to the problem of stratospheric ozone depletion, some studies have recently examined the degradative effects of enhanced UV-B doses on phytoplanktonic lipids (He and Häder, 2002). However, photochemical damages in phytoplanktonic cells are not a monopoly of UV radiation. In fact, due to the presence of chlorophylls (which are very efficient photosensitizers (Foote, 1976; Knox and Dodge, 1985)), numerous organic components of phytoplankton are susceptible to being photodegraded during senescence by photosynthetically active radiation (PAR).

Photo- and Free Radical-Mediated

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 5

1O2. The type II (i.e. involving 1O2) photosensitized oxidation of the phytol moiety of chlorophylls leads to the production of photoproducts of structures **a** and **b** (Fig. 1), quantifiable after NaBH4-reduction and alkaline hydrolysis respectively in the form of 6,10,14-trimethylpentadecan-2-one (**1**) (phytone) and 3-methylidene-7,11,15-trimethyl-

**CH2OR'**

**CH2OR'**

**Photosensitized isomerization UVR**

> **Type II photosensitized degradation**

> > **CH2OR'**

 **Type II photosensitized degradation**

**PAR UVR**

**R**

**R**

**R**

 **Alkaline hydrolysis**

 **NaBH**<sup>4</sup> **reduction**

**CH2OR'**

**OOH**

**c**

**CH2OH**

**CH2OR'**

**OH**

**OH**

**OOH**

**CH2OH**

**C (CH2)2**

**O**

**R" = More or less oxidized tetrapyrrolic structure**

**R"**

**2 3 and 4**

**CH2OR'**

**OH**

**R =**

**R' =**

Fig. 1. Photooxidation of chlorophyll phytyl side-chain and reactions of oxidation products

**OH**

**R**

**OR'**

**a b**

**OR'**

**R**

**R**

 **Alkaline hydrolysis**

**CHO**

**O**

**1**

during alkaline hydrolysis.

hexadecan-1,2-diol (phytyldiol) (**2**) (Fig. 1) (Rontani et al., 1994).

**R**

**R**

**PAR UVR**

**OOH**

**PAR UVR**

**OH**

 **NaBH**<sup>4</sup> **reduction NaBH**<sup>4</sup> **reduction**

**OH**

**R**

 **Type II photosensitized degradation**

**R**

**Retroaldol**

**R**

 **Alkaline hydrolysis**

**R**

#### **2.1 Photodegradation of the main lipidic components of phytoplankton during senescence**

When a chlorophyll molecule absorbs a quantum of light energy, an excited singlet state (1Chl) is formed which, in healthy cells, leads predominantly to the characteristic fast reactions of photosynthesis (Foote, 1976). However, a small proportion (<0.1%) undergoes intersystem crossing (ISC) to form the longer lived triplet state (3Chl; Knox and Dodge, 1985). 3Chl is not only itself potentially damaging in type I reactions (hydrogen atom or electron abstraction) (Knox and Dodge, 1985), but can also generate highly reactive oxygen species (ROS) and, in particular, singlet oxygen (1O2), by reaction with ground state oxygen (3O2) via Type II processes. In order to avoid oxidative damage, there are many antioxidant protective mechanisms in chloroplasts. Carotenoids quench 3Chl and 1O2 by energy transfer mechanisms at very high rates (Foote, 1976) and tocopherols can remove 1O2, O2 -, HOO and HO by acting as sacrificial scavengers (Halliwell, 1987). Superoxide dismutase enzyme (SOD) and ascorbic acid may also scavenge O2 - (Halliwell, 1987), while catalase activity decreases H2O2 levels.

In senescent phototrophic organisms, the fast reactions of photosynthesis clearly do not operate, so an accelerated rate of formation of 3Chl and 1O2 would be expected (Nelson, 1993). The rate of formation of these potentially damaging species can then exceed the quenching capacity of the photoprotective system and photodegradation can occur (photodynamic effect; Merzlyak and Hendry, 1994). In phytodetritus, when the ordered structure of the thylakoid membranes has been disrupted, pigments tend to remain associated with other hydrophobic cellular components such as membrane lipids (Nelson, 1993). As a result, the photooxidative effect of chlorophyll sensitization might be strongly amplified within such a hydrophobic micro-environment. Moreover, the lifetime of 1O2 produced from sensitizers in a lipid-rich hydrophobic environment could be longer, and its potential diffusive distance greater, than its behaviour in aqueous solution (Suwa et al., 1977). It is not surprising, therefore, that photodegradation processes act on the majority of unsaturated lipid components of senescent phytoplankton.

## **2.1.1 Chlorophylls**

Irradiation of dead phytoplankton cells by PAR and UVR radiations results in rapid degradation of chlorophylls (Nelson, 1993; Rontani et al., 1995; Christodoulou et al., 2010). Photodegradation of chlorophyll-*a* and -*c* in killed cells of *E. huxleyi* appeared to be induced by both PAR and UVR (Christodoulou et al., 2010). The photochemical degradation of chlorophylls has so far been studied almost exclusively with respect to the macrocycle moiety of the molecule, which is the more reactive. Despite some progress regarding intermediary photoproducts (Engel et al., 1991; Iturraspe et al., 1994), no stable and specific markers for the chlorophyll macrocycle photodegradation have been characterised.

The isoprenoid phytyl side-chain of chlorophylls is also sensitive to photochemical processes. In fact, in phytodetritus, the photodegradation rates were only 3 to 5 times higher for the chlorophyll tetrapyrrolic structure than for the phytyl side-chain (Cuny et al., 1999; Christodoulou et al., 2010). Analysis of isoprenoid photoproducts of chlorophylls after irradiation of different dead phytoplanktonic cells by visible light clearly established that the photodegradation of the chlorophyll phytyl side-chain in phytodetritus involved mainly

When a chlorophyll molecule absorbs a quantum of light energy, an excited singlet state (1Chl) is formed which, in healthy cells, leads predominantly to the characteristic fast reactions of photosynthesis (Foote, 1976). However, a small proportion (<0.1%) undergoes intersystem crossing (ISC) to form the longer lived triplet state (3Chl; Knox and Dodge, 1985). 3Chl is not only itself potentially damaging in type I reactions (hydrogen atom or electron abstraction) (Knox and Dodge, 1985), but can also generate highly reactive oxygen species (ROS) and, in particular, singlet oxygen (1O2), by reaction with ground state oxygen (3O2) via Type II processes. In order to avoid oxidative damage, there are many antioxidant protective mechanisms in chloroplasts. Carotenoids quench 3Chl and 1O2 by energy transfer

**2.1 Photodegradation of the main lipidic components of phytoplankton during** 

mechanisms at very high rates (Foote, 1976) and tocopherols can remove 1O2, O2

(SOD) and ascorbic acid may also scavenge O2

unsaturated lipid components of senescent phytoplankton.

by acting as sacrificial scavengers (Halliwell, 1987). Superoxide dismutase enzyme

In senescent phototrophic organisms, the fast reactions of photosynthesis clearly do not operate, so an accelerated rate of formation of 3Chl and 1O2 would be expected (Nelson, 1993). The rate of formation of these potentially damaging species can then exceed the quenching capacity of the photoprotective system and photodegradation can occur (photodynamic effect; Merzlyak and Hendry, 1994). In phytodetritus, when the ordered structure of the thylakoid membranes has been disrupted, pigments tend to remain associated with other hydrophobic cellular components such as membrane lipids (Nelson, 1993). As a result, the photooxidative effect of chlorophyll sensitization might be strongly amplified within such a hydrophobic micro-environment. Moreover, the lifetime of 1O2 produced from sensitizers in a lipid-rich hydrophobic environment could be longer, and its potential diffusive distance greater, than its behaviour in aqueous solution (Suwa et al., 1977). It is not surprising, therefore, that photodegradation processes act on the majority of

Irradiation of dead phytoplankton cells by PAR and UVR radiations results in rapid degradation of chlorophylls (Nelson, 1993; Rontani et al., 1995; Christodoulou et al., 2010). Photodegradation of chlorophyll-*a* and -*c* in killed cells of *E. huxleyi* appeared to be induced by both PAR and UVR (Christodoulou et al., 2010). The photochemical degradation of chlorophylls has so far been studied almost exclusively with respect to the macrocycle moiety of the molecule, which is the more reactive. Despite some progress regarding intermediary photoproducts (Engel et al., 1991; Iturraspe et al., 1994), no stable and specific

The isoprenoid phytyl side-chain of chlorophylls is also sensitive to photochemical processes. In fact, in phytodetritus, the photodegradation rates were only 3 to 5 times higher for the chlorophyll tetrapyrrolic structure than for the phytyl side-chain (Cuny et al., 1999; Christodoulou et al., 2010). Analysis of isoprenoid photoproducts of chlorophylls after irradiation of different dead phytoplanktonic cells by visible light clearly established that the photodegradation of the chlorophyll phytyl side-chain in phytodetritus involved mainly

markers for the chlorophyll macrocycle photodegradation have been characterised.



**senescence** 

and HO

decreases H2O2 levels.

**2.1.1 Chlorophylls** 

1O2. The type II (i.e. involving 1O2) photosensitized oxidation of the phytol moiety of chlorophylls leads to the production of photoproducts of structures **a** and **b** (Fig. 1), quantifiable after NaBH4-reduction and alkaline hydrolysis respectively in the form of 6,10,14-trimethylpentadecan-2-one (**1**) (phytone) and 3-methylidene-7,11,15-trimethylhexadecan-1,2-diol (phytyldiol) (**2**) (Fig. 1) (Rontani et al., 1994).

**R" = More or less oxidized tetrapyrrolic structure**

Fig. 1. Photooxidation of chlorophyll phytyl side-chain and reactions of oxidation products during alkaline hydrolysis.

Photo- and Free Radical-Mediated

ranging from 0.30 to 0.35 instead of 0.1).

**HO HO**

**Allylic rearrangement**

**OOH**

**OOH**

Fig. 3. Type II photosensitized oxidation of 5 sterols.

**11**

**12**

**HO**

**Epimerization**

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 7

*Phaeodactylum tricornutum* and *Emiliania huxleyi* (Rontani et al., 1997a; 1997b; 1998) resulted in a quick photodegradation of the sterol components of these algae. The results obtained clearly established that the photooxidation of sterols in senescent cells of phytoplankton involves type II photoprocesses. These processes mainly produce6-5-hydroperoxides (**8**) and to a lesser extent 4-6/6-hydroperoxides (**9** and **10**) (Fig. 3) (Nickon and Bagli, 1961; Kulig and Smith, 1973). 6-5-hydroperoxysterols (**8**) are relatively unstable and may undergo allylic rearrangement to 5-7-hydroperoxysterols (**11**), which in turn epimerize to the corresponding 7-hydroperoxides (**12**) (Fig. 3) (Smith, 1981). It was previously demonstrated that during singlet oxygen-mediated photooxidation of sterols in biological membranes (Korytowski *et al*., 1992) and senescent phytoplanktonic cells (Rontani *et al*., 1997a) the photogeneration of Δ4-6α/6β-hydroperoxides (**9** and **10**) was more favourable than in homogeneous solution (ratio Δ4-6α/6β-hydroperoxides/Δ6-5α-hydroperoxysterols

**HO**

**HO**

**8 9 and 10**

**Type II photoprocesses**

**OOH**

**OOH**

**<sup>7</sup> <sup>5</sup> <sup>6</sup>**

Allylic rearrangement of 6-5-hydroperoxides (**8**) appeared to take place very weakly in senescent phytoplanktonic cells (Rontani et al., 1997a; 1997b; 1998). This surprising stability was attributed by Korytowski et al. (1992) either to hydrogen bonding between the unsaturated fatty acyl chain of phospholipids and 6-5-hydroperoxides (**8**) which could hinder the allylic rearrangement, or to differences of polarity in the carbon 7-10 zone of the fatty acyl chain (where sterols tend to localize in phospholipid/sterol bilayers (MacIntosch, 1978)). It is also interesting to note that the reduction of hydroperoxysterols to the corresponding diols weakly operates in killed phytoplanktonic cells (Rontani et al., 1997a).

**1 O**2

Irradiation with UVR resulted in the additional production of small amounts of *Z*-phytol and *Z* and *E*-3,7,11,15-tetramethylhexadec-3-en-1,2-diols (**3,4**) (Christodoulou et al., 2010). The detection of *Z*-phytol allowed to demonstrate the induction of *cis-trans* photosensitized isomerization by UVR. These reactions probably involve triplet states of ketones as sensitizers. Type II photosensitized oxidation of the *Z* configuration of phytol, which should lead to the production of photoproducts of structures **a**, **b** and **c** (Fig. 1) (Schulte-Elte et al., 1979), explains the detection of small amounts of *Z* and *E*-3,7,11,15-tetramethylhexadec-3-en-1,2-diols (**3,4**) after irradiation with UVR. Irradiation with UVR also resulted in a faster degradation of chlorophyll phytyl side-chain oxidation products (Christodoulou et al., 2010). This higher reactivity was attributed to UVR-induced homolysis of the peroxyl group of photoproducts of structures **a**, **b** and **c** (Fig. 1).

Phytyldiol (**2**) is ubiquitous in the marine environment and has been proposed as tracer for photodegradation of chlorophyll's phytyl side chain (Rontani et al. 1994; 1996a; Cuny and Rontani 1999). Further, the molar ratio phytyldiol:phytol (Chlorophyll Phytyl side-chain Photodegradation Index, CPPI) was employed to estimate the extent of chlorophyll photodegraded in natural marine samples by the empirical equation: chlorophyll photodegradation % = (1-(CPPI + 1)-18.5) x 100 (Cuny et al. 2002).

#### **2.1.2 Carotenoids**

In phytodetritus, chlorophylls and carotenoids remain in a close molecular-scale association at relatively high localized concentrations, even though the structure of the thylakoid membrane has been disrupted (Nelson, 1993). Thus, the sensitized photooxidation of carotenoids is enhanced. The photosensitized oxidation (involving 1O2) of carotenoids in solvents has been studied (Iseo et al., 1972) and loliolide (**5**), *iso*-loliolide (**6**) and dihydroactinidiolide (**7**) (Fig. 2) were identified as major photoproducts, depending on the functionality of carotenoids at C-3. Loliolide (**5**) and *iso*-loliolide (**6**) have been detected in killed cells of *Dunaliella* sp. irradiated by visible light (Rontani et al., 1998). However, due to their apparent production by anaerobic bacteria (Repeta, 1989) and during dark incubations of killed phytoplanktonic cells (Rontani et al., 1998), these compounds cannot constitute unequivocal indicators of photooxidative processes.

Fig. 2. Structure of the main carotenoid oxidation products.

#### **2.1.3 <sup>5</sup> -sterols**

As important unsaturated components of biological membranes, 5-sterols are highly susceptible to photooxidative degradation during the senescence of phytoplankton. Irradiation by visible light of killed cells of *Skeletonema costatum*, *Dunaliella* sp.,

Irradiation with UVR resulted in the additional production of small amounts of *Z*-phytol and *Z* and *E*-3,7,11,15-tetramethylhexadec-3-en-1,2-diols (**3,4**) (Christodoulou et al., 2010). The detection of *Z*-phytol allowed to demonstrate the induction of *cis-trans* photosensitized isomerization by UVR. These reactions probably involve triplet states of ketones as sensitizers. Type II photosensitized oxidation of the *Z* configuration of phytol, which should lead to the production of photoproducts of structures **a**, **b** and **c** (Fig. 1) (Schulte-Elte et al., 1979), explains the detection of small amounts of *Z* and *E*-3,7,11,15-tetramethylhexadec-3-en-1,2-diols (**3,4**) after irradiation with UVR. Irradiation with UVR also resulted in a faster degradation of chlorophyll phytyl side-chain oxidation products (Christodoulou et al., 2010). This higher reactivity was attributed to UVR-induced homolysis of the peroxyl group

Phytyldiol (**2**) is ubiquitous in the marine environment and has been proposed as tracer for photodegradation of chlorophyll's phytyl side chain (Rontani et al. 1994; 1996a; Cuny and Rontani 1999). Further, the molar ratio phytyldiol:phytol (Chlorophyll Phytyl side-chain Photodegradation Index, CPPI) was employed to estimate the extent of chlorophyll photodegraded in natural marine samples by the empirical equation: chlorophyll

In phytodetritus, chlorophylls and carotenoids remain in a close molecular-scale association at relatively high localized concentrations, even though the structure of the thylakoid membrane has been disrupted (Nelson, 1993). Thus, the sensitized photooxidation of carotenoids is enhanced. The photosensitized oxidation (involving 1O2) of carotenoids in solvents has been studied (Iseo et al., 1972) and loliolide (**5**), *iso*-loliolide (**6**) and dihydroactinidiolide (**7**) (Fig. 2) were identified as major photoproducts, depending on the functionality of carotenoids at C-3. Loliolide (**5**) and *iso*-loliolide (**6**) have been detected in killed cells of *Dunaliella* sp. irradiated by visible light (Rontani et al., 1998). However, due to their apparent production by anaerobic bacteria (Repeta, 1989) and during dark incubations of killed phytoplanktonic cells (Rontani et al., 1998), these compounds cannot constitute

**<sup>O</sup> <sup>O</sup> <sup>O</sup> HO HO**

**5 6 7**

As important unsaturated components of biological membranes, 5-sterols are highly susceptible to photooxidative degradation during the senescence of phytoplankton. Irradiation by visible light of killed cells of *Skeletonema costatum*, *Dunaliella* sp.,

**O O O**

of photoproducts of structures **a**, **b** and **c** (Fig. 1).

unequivocal indicators of photooxidative processes.

Fig. 2. Structure of the main carotenoid oxidation products.

**2.1.2 Carotenoids** 

1

**-sterols** 

2

3

**2.1.3 <sup>5</sup>**

photodegradation % = (1-(CPPI + 1)-18.5) x 100 (Cuny et al. 2002).

*Phaeodactylum tricornutum* and *Emiliania huxleyi* (Rontani et al., 1997a; 1997b; 1998) resulted in a quick photodegradation of the sterol components of these algae. The results obtained clearly established that the photooxidation of sterols in senescent cells of phytoplankton involves type II photoprocesses. These processes mainly produce6-5-hydroperoxides (**8**) and to a lesser extent 4-6/6-hydroperoxides (**9** and **10**) (Fig. 3) (Nickon and Bagli, 1961; Kulig and Smith, 1973). 6-5-hydroperoxysterols (**8**) are relatively unstable and may undergo allylic rearrangement to 5-7-hydroperoxysterols (**11**), which in turn epimerize to the corresponding 7-hydroperoxides (**12**) (Fig. 3) (Smith, 1981). It was previously demonstrated that during singlet oxygen-mediated photooxidation of sterols in biological membranes (Korytowski *et al*., 1992) and senescent phytoplanktonic cells (Rontani *et al*., 1997a) the photogeneration of Δ4-6α/6β-hydroperoxides (**9** and **10**) was more favourable than in homogeneous solution (ratio Δ4-6α/6β-hydroperoxides/Δ6-5α-hydroperoxysterols ranging from 0.30 to 0.35 instead of 0.1).

Fig. 3. Type II photosensitized oxidation of 5 sterols.

Allylic rearrangement of 6-5-hydroperoxides (**8**) appeared to take place very weakly in senescent phytoplanktonic cells (Rontani et al., 1997a; 1997b; 1998). This surprising stability was attributed by Korytowski et al. (1992) either to hydrogen bonding between the unsaturated fatty acyl chain of phospholipids and 6-5-hydroperoxides (**8**) which could hinder the allylic rearrangement, or to differences of polarity in the carbon 7-10 zone of the fatty acyl chain (where sterols tend to localize in phospholipid/sterol bilayers (MacIntosch, 1978)). It is also interesting to note that the reduction of hydroperoxysterols to the corresponding diols weakly operates in killed phytoplanktonic cells (Rontani et al., 1997a).

Photo- and Free Radical-Mediated

**<sup>R</sup> <sup>11</sup> 10 9 <sup>8</sup> R'**

**R' = -(CH**2**)**6**-COOH**

**O**

**O**

**O HO**

**+ H**2**O**

**CHO OHC COOH**

**ROOH ROOH**

**HOOC COOH COOH**

**R = -(CH**2**)**6**-CH**<sup>3</sup>

**OOH**

Fig. 4. Type II photosensitized oxidation of oleic acid.

**COOH**

**Heterolytic cleavage**

**H+**

**COOH**

**COOH**

acid) (RH = hydrogen donors, e.g. lipids or reduced sensitizers).

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 9

**R'**

**<sup>10</sup> <sup>R</sup> <sup>11</sup>**

**Radical allylic rearrangement**

**Radical allylic rearrangement**

**COOH**

**O**

**RH R**

Fig. 5. Degradation of allylic hydroperoxides resulting from Type II photosensitized oxidation of monounsaturated fatty acids (the example given is this of 9-hydroperoxyoctadec-10-enoic

**hv or Homolytic cleavage**

**10 9 8**

**COOH**

**Disproportionation**

**COOH**

**Retroaldolisation**

**OH COOH**

**O2 HOO**

**O**

**+ H2O**

**OH O**

**COOH**

**HOO R'**

**9 8 OOH**

**11**

**OOH**

**R**

**<sup>R</sup> R' <sup>11</sup> 10 9 8**

> **h** <sup>1</sup> **O**2

> > **R'**

**8**

**11**

**10**

**R OOH 9**

6-5-Hydroperoxysterols (**8**) are potential type II photodegradation markers, not only because they are the major products of singlet oxygen attack on the steroidal 5-3- system, but also because biological functionalization of steroids at C-5 is rare. Unfortunately, if these compounds are particularly stable in phytodetritus, they decay slowly in the sediment to their corresponding 5-7/-derivatives (**11** and **12**) (Rontani and Marchand, 2000), which are not selective markers (see chapter 3.3). Moreover, according to the stability of the alkyl radicals formed during β-scission of the corresponding alkoxyl radicals, the following order of stability was proposed: Δ4-6-hydroperoxysterols (**9** and **10**) > Δ5-7-hydroperoxysterols (**11** and **12**) > Δ6-5-hydroperoxy-sterols (**8**) (Christodoulou et al., 2009). Consequently, 4-6 hydroperoxysterols (**9** and **10**) (or their degradative products 4-6/-hydroxysterols and 4-6/-oxosterols) may be considered as more reliable *in situ* markers of type II photodegradation processes than 6-5-hydroperoxides (**8**).

### **2.1.4 Unsaturated fatty acids**

Chloroplast membrane components are particularly susceptible to type II photooxidation (Heath and Packer, 1968). This is the case notably for unsaturated fatty acids, which generally predominate in algal lipids, particularly in the photosynthetic membranes (Woods, 1974). In killed phytoplanktonic cells, the photodegradation rates of unsaturated fatty acids logically increase with their unsaturation degree (Rontani et al., 1998). Singlet oxygen-mediated photooxidation of monounsaturated fatty acids involves a direct reaction of 1O2 with the carbon–carbon double bond by a concerted 'ene' addition (Frimer 1979) and leads to formation of hydroperoxides at each carbon of the original double bond. Thus, photooxidation of oleic acid produces a mixture of 9- and 10-hydroperoxides with an allylic *trans*-double bond (Frankel et al. 1979; Frankel, 1998), which can subsequently undergo stereoselective radical allylic rearrangement to 11-*trans* and 8-*trans* hydroperoxides, respectively (Porter et al. 1995) (Fig. 4).

The free radical nature of the allylic hydroperoxide rearrangement is supported by the observation that the rearrangement is catalysed by free radical initiators or light and inhibited by phenolic antioxidants (Porter et al., 1995). This allylic rearrangement weakly intervenes in most of the killed phytoplanktonic cells examined (Rontani et al., 1998). This was attributed to the relatively high localized fatty acid concentrations present in phytodetritus (Nelson, 1993), which favoured the dimerisation of hydroperoxides. Hydrogen atom abstraction to form allylperoxyl radicals does indeed occur readily from hydroperoxide monomers but not from hydroperoxide dimers (Porter et al., 1995).

During early diagenesis, isomeric hydroperoxyacids undergo heterolytic cleavage to aldehydes and -oxocarboxylic acids (Frimer, 1979) or homolytic cleavage and subsequent transformation to the corresponding alcohols or ketones (Fig. 5).

Taking into account the high amounts of photoproducts of mono-unsaturated fatty acids detected in the particulate matter samples (Marchand and Rontani, 2001; Christodoulou et al., 2009; Rontani et al., 2011a), and the well known increasing photooxidation rates of fatty acids with their degree of unsaturation (Frankel., 1998), it can be concluded that considerable amounts of poly-unsaturated fatty acids must be photooxidized during the senescence of phytoplankton in the marine environment. However, at this time photooxidation products of this kind of fatty acids could not be detected in natural samples.

**Radical allylic rearrangement**

### **R = -(CH**2**)**6**-CH**<sup>3</sup>

8 Senescence

6-5-Hydroperoxysterols (**8**) are potential type II photodegradation markers, not only because they are the major products of singlet oxygen attack on the steroidal 5-3- system, but also because biological functionalization of steroids at C-5 is rare. Unfortunately, if these compounds are particularly stable in phytodetritus, they decay slowly in the sediment to their corresponding 5-7/-derivatives (**11** and **12**) (Rontani and Marchand, 2000), which are not selective markers (see chapter 3.3). Moreover, according to the stability of the alkyl radicals formed during β-scission of the corresponding alkoxyl radicals, the following order of stability was proposed: Δ4-6-hydroperoxysterols (**9** and **10**) > Δ5-7-hydroperoxysterols (**11** and **12**) > Δ6-5-hydroperoxy-sterols (**8**) (Christodoulou et al., 2009). Consequently, 4-6 hydroperoxysterols (**9** and **10**) (or their degradative products 4-6/-hydroxysterols and 4-6/-oxosterols) may be considered as more reliable *in situ* markers of type II

Chloroplast membrane components are particularly susceptible to type II photooxidation (Heath and Packer, 1968). This is the case notably for unsaturated fatty acids, which generally predominate in algal lipids, particularly in the photosynthetic membranes (Woods, 1974). In killed phytoplanktonic cells, the photodegradation rates of unsaturated fatty acids logically increase with their unsaturation degree (Rontani et al., 1998). Singlet oxygen-mediated photooxidation of monounsaturated fatty acids involves a direct reaction of 1O2 with the carbon–carbon double bond by a concerted 'ene' addition (Frimer 1979) and leads to formation of hydroperoxides at each carbon of the original double bond. Thus, photooxidation of oleic acid produces a mixture of 9- and 10-hydroperoxides with an allylic *trans*-double bond (Frankel et al. 1979; Frankel, 1998), which can subsequently undergo stereoselective radical allylic rearrangement to 11-*trans* and 8-*trans* hydroperoxides,

The free radical nature of the allylic hydroperoxide rearrangement is supported by the observation that the rearrangement is catalysed by free radical initiators or light and inhibited by phenolic antioxidants (Porter et al., 1995). This allylic rearrangement weakly intervenes in most of the killed phytoplanktonic cells examined (Rontani et al., 1998). This was attributed to the relatively high localized fatty acid concentrations present in phytodetritus (Nelson, 1993), which favoured the dimerisation of hydroperoxides. Hydrogen atom abstraction to form allylperoxyl radicals does indeed occur readily from

During early diagenesis, isomeric hydroperoxyacids undergo heterolytic cleavage to aldehydes and -oxocarboxylic acids (Frimer, 1979) or homolytic cleavage and subsequent

Taking into account the high amounts of photoproducts of mono-unsaturated fatty acids detected in the particulate matter samples (Marchand and Rontani, 2001; Christodoulou et al., 2009; Rontani et al., 2011a), and the well known increasing photooxidation rates of fatty acids with their degree of unsaturation (Frankel., 1998), it can be concluded that considerable amounts of poly-unsaturated fatty acids must be photooxidized during the senescence of phytoplankton in the marine environment. However, at this time photooxidation products of this kind of fatty acids could not be detected in natural samples.

hydroperoxide monomers but not from hydroperoxide dimers (Porter et al., 1995).

transformation to the corresponding alcohols or ketones (Fig. 5).

photodegradation processes than 6-5-hydroperoxides (**8**).

**2.1.4 Unsaturated fatty acids** 

respectively (Porter et al. 1995) (Fig. 4).

## **R' = -(CH**2**)**6**-COOH**

Fig. 4. Type II photosensitized oxidation of oleic acid.

Fig. 5. Degradation of allylic hydroperoxides resulting from Type II photosensitized oxidation of monounsaturated fatty acids (the example given is this of 9-hydroperoxyoctadec-10-enoic acid) (RH = hydrogen donors, e.g. lipids or reduced sensitizers).

Photo- and Free Radical-Mediated

**2.1.6 n-Alkenes** 

(Mouzdahir et al., 2001).

2010; Massé et al., 2008).

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 11

The visible light-induced degradation of *n*-alkenes was previously investigated in killed cells of the Prymnesiophycea *E. huxleyi* and the Eustigmatophycea *Nannochloropsis salina*

In *E. huxleyi* killed cells, minor C31 and C33 *n*-alkenes were strongly photodegraded, while the major C37 and C38 *n*-alkenes appeared particularly recalcitrant towards photochemical processes. These strong differences of photoreactivity imply distinct biological syntheses and/or functions for these two groups of hydrocarbons in *E. huxleyi* cells. Interestingly, the stereochemistry of the internal double bonds in C31 and C33 n-alkenes has been established to be *cis*, while C37 and C38 alkenes internal double bonds exhibit a *trans* geometry (Rieley et al., 1998; Grossi et al., 2000). The photochemical recalcitrance of C37 and C38 n-alkenes could

Irradiation of dead cells of *N. salina* resulted in a strong modification of the hydrocarbon fraction. It did not provide evidence of a significant light-dependent degradation of monounsaturated hydrocarbons; this result was attributed to the terminal position of the double bond in these compounds (Gelin et al., 1997), which is poorly reactive towards singlet oxygen (Hurst et al., 1985). In contrast, di-, tri-, and tetraenes were strongly photodegraded during irradiation. The visible light-dependent degradation of phytoplanktonic *n*-alkenes showed apparent second-order kinetics with respect to light exposure and the half-life doses obtained logically decrease with increasing number of

HBI alkenes are widely distributed in aquatic environments (Rowland and Robson, 1990; Sinninghe-Damsté et al., 2004), although they appear to originate from a relatively small number of diatomaceous algae including *Haslea* spp., *Rhizosolenia* spp., *Pleurosigma* spp. and *Navicula* spp. (Volkman et al., 1994; Sinninghe-Damsté et al., 2004; Belt et al., 2000, 2001; Allard et al., 2001; Grossi et al., 2004). Despite this, they have been commonly reported in marine sediments worldwide and provide some insight into the deposition of organic matter from the water column. One HBI alkene, a mono-unsaturated isomer termed IP25, has been used as a proxy for the occurrence of spring sea ice in the Arctic (e.g. Belt et al., 2007,

Examination of the photoreactivity of several mono-, di-, tri- and tetra-unsaturated HBI alkenes in the presence of a photosensitizer solution and in dead cells of *H. ostrearia* allowed to show that HBI alkenes possessing at least one tri-substituted double bond may be photooxidized at similar or higher rates compared to other highly reactive lipids (e.g. PUFAs, vitamin E and chlorophyll *a*) during the senescence of diatom cells (Rontani et al., 2011b). As a consequence, it is proposed that HBI alkenes possessing trisubstituted double bonds are likely to be susceptible to photodegradation within the euphotic zone. In contrast, HBIs containing only mono- and di-substituted double bonds were found to be significantly less reactive towards 1O2 and should, therefore, be relatively preserved during sedimentation through the water column (Rontani et al., 2011b). The kinetic experiments are supported by product analysis, which revealed that the main reaction with 1O2 primarily occurs with the trisubstituted double bonds of HBI alkenes affording tertiary and secondary allylic hydroperoxides (Fig. 6). In contrast, the extremely low photoreactivity of the HBI monoene

thus be partly attributed to the *trans* geometry of their internal double bonds.

double bonds in these compounds (Mouzdahir et al., 2001).

**2.1.7 Highly branched isoprenoid (HBI) alkenes** 

This is possibly due to: (i) the instability of the hydroperoxides formed, or (ii) the involvement of cross-linking reactions leading to the formation of macromolecular structures (Neff et al., 1988) non-amenable by gas chromatography.

#### **2.1.5 Alkenones**

Alkenones are a class of mono-, di-, tri-, tetra- and penta-unsaturated C35-C40 methyl and ethyl ketones (Boon et al., 1978; Volkman et al., 1980; de Leeuw et al., 1980; Marlowe et al., 1984; Prahl et al., 2006; Jaraula et al., 2010), which are produced by certain marine haptophytes. *Emiliania huxleyi* and *Gephyrocapsa oceanica* are the major sources of alkenones in the open ocean (Volkman et al., 1980; 1995; Conte et al., 1994). The unsaturation ratio of C37 alkenones, defined as 37 *<sup>K</sup> U* = [C37:2] / ([C37:2] + [C37:3]) where [C37:2] and [C37:3] are the concentrations of diand tri-unsaturated C37 alkenones respectively, varies positively with the growth temperature of the alga (Prahl and Wakeham, 1987; Prahl et al., 1988). The 37 *<sup>K</sup> U* - growth temperature relationship in haptophyte algae and transferred to sinking marine particulate matter leads to a linear relationship between sedimentary C37 alkenone composition and mean annual SST records throughout the oceans (Rosell-Melé et al., 1995; Müller et al., 1998). The 37 *<sup>K</sup> U* index is now routinely used for paleotemperature reconstruction.

For alkenones to be useful as measures of sea surface temperature in the geological record, it is essential that any effects of degradation in the water column and in sediments either do not affect the temperature signal established during their initial biosynthesis by the alga (Harvey, 2000; Grimalt et al., 2000), or if there is a change its extent can be reasonably estimated.

Visible light-induced photodegradation of these compounds was thus previously investigated in order to determine if photochemical processes could appreciably modify 37 *<sup>K</sup> U* ratios during algal senescence (Rontani et al., 1997b; Mouzdahir et al., 2001; Christodoulou et al., 2010). Though potentially selective, photochemical degradation of alkenones is not fast enough in killed cells of *E. huxleyi* to induce strong modifications of the 37 *<sup>K</sup> U* ratio before the photodestruction of the photosensitizing substances (Rontani et al., 1997b; Mouzdahir et al., 2001). UVR also appeared to be inefficient to alter the 37 *<sup>K</sup> U* ratio (Christodoulou et al., 2010).

This stability was attributed to the *trans* configuration of alkenone double bonds (Rechka and Maxwell, 1988) that is 7 to 10 times less sensitive against singlet oxygen-mediated oxidation than the classical *cis* configuration of fatty acids (Hurst et al., 1985). This may explain the difference of photoreactivity observed between the alkenones and fatty acids with the same number of unsaturations. We also previously attributed the poor photoreactivity of alkenones to a localisation of these compounds elsewhere than in cell membranes (Rontani et al., 1997b; Mouzdahir et al., 2001), which could significantly decrease the likelihood of interaction between singlet oxygen and alkenones. Although this hypothesis is well supported by the recent results of Eltgroth et al. (2005), who demonstrated that alkenones are mainly localized into cytoplasmic vesicles, the migration of singlet oxygen from phytodetritus to attached heterotrophic bacteria previously observed (Rontani et al., 2003a; Christodoulou et al., 2010) strongly suggests a diffusion of this excited form of oxygen also in these cytoplasmic vesicles.

## **2.1.6 n-Alkenes**

10 Senescence

This is possibly due to: (i) the instability of the hydroperoxides formed, or (ii) the involvement of cross-linking reactions leading to the formation of macromolecular

Alkenones are a class of mono-, di-, tri-, tetra- and penta-unsaturated C35-C40 methyl and ethyl ketones (Boon et al., 1978; Volkman et al., 1980; de Leeuw et al., 1980; Marlowe et al., 1984; Prahl et al., 2006; Jaraula et al., 2010), which are produced by certain marine haptophytes. *Emiliania huxleyi* and *Gephyrocapsa oceanica* are the major sources of alkenones in the open ocean (Volkman et al., 1980; 1995; Conte et al., 1994). The unsaturation ratio of C37 alkenones,

and tri-unsaturated C37 alkenones respectively, varies positively with the growth temperature

relationship in haptophyte algae and transferred to sinking marine particulate matter leads to a linear relationship between sedimentary C37 alkenone composition and mean annual SST

For alkenones to be useful as measures of sea surface temperature in the geological record, it is essential that any effects of degradation in the water column and in sediments either do not affect the temperature signal established during their initial biosynthesis by the alga (Harvey, 2000; Grimalt et al., 2000), or if there is a change its extent can be reasonably

Visible light-induced photodegradation of these compounds was thus previously investigated in order to determine if photochemical processes could appreciably modify

 ratios during algal senescence (Rontani et al., 1997b; Mouzdahir et al., 2001; Christodoulou et al., 2010). Though potentially selective, photochemical degradation of alkenones is not fast enough in killed cells of *E. huxleyi* to induce strong modifications of the

ratio before the photodestruction of the photosensitizing substances (Rontani et al.,

This stability was attributed to the *trans* configuration of alkenone double bonds (Rechka and Maxwell, 1988) that is 7 to 10 times less sensitive against singlet oxygen-mediated oxidation than the classical *cis* configuration of fatty acids (Hurst et al., 1985). This may explain the difference of photoreactivity observed between the alkenones and fatty acids with the same number of unsaturations. We also previously attributed the poor photoreactivity of alkenones to a localisation of these compounds elsewhere than in cell membranes (Rontani et al., 1997b; Mouzdahir et al., 2001), which could significantly decrease the likelihood of interaction between singlet oxygen and alkenones. Although this hypothesis is well supported by the recent results of Eltgroth et al. (2005), who demonstrated that alkenones are mainly localized into cytoplasmic vesicles, the migration of singlet oxygen from phytodetritus to attached heterotrophic bacteria previously observed (Rontani et al., 2003a; Christodoulou et al., 2010) strongly suggests a diffusion of this excited

1997b; Mouzdahir et al., 2001). UVR also appeared to be inefficient to alter the 37

records throughout the oceans (Rosell-Melé et al., 1995; Müller et al., 1998). The 37

= [C37:2] / ([C37:2] + [C37:3]) where [C37:2] and [C37:3] are the concentrations of di-

*<sup>K</sup> U*


*<sup>K</sup> U*

*<sup>K</sup> U*

ratio

index is

structures (Neff et al., 1988) non-amenable by gas chromatography.

of the alga (Prahl and Wakeham, 1987; Prahl et al., 1988). The 37

now routinely used for paleotemperature reconstruction.

**2.1.5 Alkenones** 

defined as 37

estimated.

37 *<sup>K</sup> U*

37 *<sup>K</sup> U*

(Christodoulou et al., 2010).

form of oxygen also in these cytoplasmic vesicles.

*<sup>K</sup> U*

The visible light-induced degradation of *n*-alkenes was previously investigated in killed cells of the Prymnesiophycea *E. huxleyi* and the Eustigmatophycea *Nannochloropsis salina* (Mouzdahir et al., 2001).

In *E. huxleyi* killed cells, minor C31 and C33 *n*-alkenes were strongly photodegraded, while the major C37 and C38 *n*-alkenes appeared particularly recalcitrant towards photochemical processes. These strong differences of photoreactivity imply distinct biological syntheses and/or functions for these two groups of hydrocarbons in *E. huxleyi* cells. Interestingly, the stereochemistry of the internal double bonds in C31 and C33 n-alkenes has been established to be *cis*, while C37 and C38 alkenes internal double bonds exhibit a *trans* geometry (Rieley et al., 1998; Grossi et al., 2000). The photochemical recalcitrance of C37 and C38 n-alkenes could thus be partly attributed to the *trans* geometry of their internal double bonds.

Irradiation of dead cells of *N. salina* resulted in a strong modification of the hydrocarbon fraction. It did not provide evidence of a significant light-dependent degradation of monounsaturated hydrocarbons; this result was attributed to the terminal position of the double bond in these compounds (Gelin et al., 1997), which is poorly reactive towards singlet oxygen (Hurst et al., 1985). In contrast, di-, tri-, and tetraenes were strongly photodegraded during irradiation. The visible light-dependent degradation of phytoplanktonic *n*-alkenes showed apparent second-order kinetics with respect to light exposure and the half-life doses obtained logically decrease with increasing number of double bonds in these compounds (Mouzdahir et al., 2001).

## **2.1.7 Highly branched isoprenoid (HBI) alkenes**

HBI alkenes are widely distributed in aquatic environments (Rowland and Robson, 1990; Sinninghe-Damsté et al., 2004), although they appear to originate from a relatively small number of diatomaceous algae including *Haslea* spp., *Rhizosolenia* spp., *Pleurosigma* spp. and *Navicula* spp. (Volkman et al., 1994; Sinninghe-Damsté et al., 2004; Belt et al., 2000, 2001; Allard et al., 2001; Grossi et al., 2004). Despite this, they have been commonly reported in marine sediments worldwide and provide some insight into the deposition of organic matter from the water column. One HBI alkene, a mono-unsaturated isomer termed IP25, has been used as a proxy for the occurrence of spring sea ice in the Arctic (e.g. Belt et al., 2007, 2010; Massé et al., 2008).

Examination of the photoreactivity of several mono-, di-, tri- and tetra-unsaturated HBI alkenes in the presence of a photosensitizer solution and in dead cells of *H. ostrearia* allowed to show that HBI alkenes possessing at least one tri-substituted double bond may be photooxidized at similar or higher rates compared to other highly reactive lipids (e.g. PUFAs, vitamin E and chlorophyll *a*) during the senescence of diatom cells (Rontani et al., 2011b). As a consequence, it is proposed that HBI alkenes possessing trisubstituted double bonds are likely to be susceptible to photodegradation within the euphotic zone. In contrast, HBIs containing only mono- and di-substituted double bonds were found to be significantly less reactive towards 1O2 and should, therefore, be relatively preserved during sedimentation through the water column (Rontani et al., 2011b). The kinetic experiments are supported by product analysis, which revealed that the main reaction with 1O2 primarily occurs with the trisubstituted double bonds of HBI alkenes affording tertiary and secondary allylic hydroperoxides (Fig. 6). In contrast, the extremely low photoreactivity of the HBI monoene

Photo- and Free Radical-Mediated

irradiation (Rontani et al., 2003a).

**R1OH2C**

**R1OH2C**

the natural environment.

**organisms** 

subsequent cutin depolymerisation (Rontani et al., 2005a).

**R1OH2C**

**HOO**

**HO**

**RH R**

**Homolytic cleavage**

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 13

major unsaturated fatty acid of these organisms (*cis*-vaccenic acid) could be detected after

As in the case of phytoplankton and cyanobacteria, visible light-dependent degradation processes act significantly on the chlorophyll phytyl side-chain (Rontani et al., 1996b), unsaturated fatty acids and sterols (Rontani, Unpublished results) during terrestrial higher plant senescence affording similar photoproducts. 9-Hydroperoxy-18-hydroxyoctadec-10(*trans*)-enoic (**13**) and 10-hydroperoxy-18-hydroxyoctadec-8(*trans*)-enoic (**14**) acids deriving from type II photooxidation of 18-hydroxyoleic acid (**15**) (Fig. 7) were detected after visible light-induced senescence experiments carried out with *Petroselinum sativum* and

**COOR2 R1OH2C**

**9 10**

**13 14**

1 **O**<sup>2</sup> **h** **15**

**COOR2**

Fig. 7. Type II photosensitized oxidation of 18-hydroxyoleic acid in cutin polymers.

**3. Free radical degradation (autoxidation) processes in phototrophic** 

**R1OH2C**

**16 17**

These results showed that in senescent plants, where the 1O2 formation rate exceeds the quenching capacity of the photoprotective system, 1O2 can migrate outside the chloroplasts and affect the unsaturated components of cutins. Significant amounts of 9,18 dihydroxyoctadec-10(*trans*)-enoic (**16**) and 10,18-dihydroxyoctadec-8(*trans*)-enoic (**17**) acids resulting from the reduction of these photoproducts of 18-hydroxyoleic acid were also detected in different natural samples (Rontani et al., 2005a). These results well support the significance of the photooxidation of the unsaturated components of higher plant cutins in

Autoxidation is the direct reaction of molecular oxygen with organic compounds under mild conditions. The autoxidation of organic compounds (in particular, lipids) involves free

**COOR2**

**COOR2**

**OOH**

**OH**

**RH R**

**Homolytic cleavage**

**COOR2**

IP25, can be attributed to its containing only the least photochemically reactive double bond. This lack of reactivity supports (in part) the good preservation of IP25 generally observed in sediments (Belt et al., 2007, 2010; Massé et al., 2008).

Fig. 6. Type II photosensitized oxidation of HBI alkenes (RH = hydrogen donors)

#### **2.2 Photodegradation processes in other phototrophic organisms**

Visible light-dependent degradation processes have been also studied in senescent cells of two purple sulfur bacteria (*Thiohalocapsa halophila* and *Halochromatium salexigens*) isolated from microbial mats from Camargue (France) (Marchand and Rontani, 2003). These reactions act intensively on the phytyl side chain of bacteriochlorophyll-*a* and lead to the production of phytone (**1**) and phytyldiol (**2**) as in the case of chlorophylls (Fig. 1). Palmitoleic and *cis*-vaccenic acids also undergo strong photodegradation, affording mainly isomeric allylic oxo-, hydroxy- and hydroperoxyacids.

These processes were also investigated in aerobic anoxygenic phototrophic bacteria (AAPs) (Rontani et al., 2003a). These organisms constitute a relatively recently discovered bacterial group (Yurkov and Beatty, 1998) and seem to be widespread in the open ocean (Kolber et al., 2000). They perform photoheterotrophic metabolism, requiring organic carbon for growth, but they are capable to use photosynthesis as an auxiliary source of energy (Kolber et al., 2001). Though sensitive to photochemical processes in senescent purple sulfur bacteria (Marchand and Rontani., 2003), the isoprenoid phytyl side-chain of bacteriochlorophyll -*a* is not significantly photodegraded in senescent cells of AAPs (Rontani et al., 2003a). In contrast, significant amounts of allylic hydroxyacids arising from the photo-oxidation of the

IP25, can be attributed to its containing only the least photochemically reactive double bond. This lack of reactivity supports (in part) the good preservation of IP25 generally observed in

<sup>1</sup> **<sup>h</sup> <sup>O</sup>**<sup>2</sup>

16 17 18 19 20 21 22 23 24 25

**Homolytic cleavage Homolytic cleavage**

**OOH**

**O**

**RH R**

**OH O**

**R RH**

**Disproportionation**

sediments (Belt et al., 2007, 2010; Massé et al., 2008).

**O O**

**-cleavage**

**OH**

**NaBH**4 **reduction**

**HOO**

1 2 3 4 <sup>5</sup> <sup>6</sup> <sup>7</sup> <sup>8</sup> 9 10 11 12 13 14 15

**HO**

**RH R**

**2.2 Photodegradation processes in other phototrophic organisms** 

isomeric allylic oxo-, hydroxy- and hydroperoxyacids.

Fig. 6. Type II photosensitized oxidation of HBI alkenes (RH = hydrogen donors)

Visible light-dependent degradation processes have been also studied in senescent cells of two purple sulfur bacteria (*Thiohalocapsa halophila* and *Halochromatium salexigens*) isolated from microbial mats from Camargue (France) (Marchand and Rontani, 2003). These reactions act intensively on the phytyl side chain of bacteriochlorophyll-*a* and lead to the production of phytone (**1**) and phytyldiol (**2**) as in the case of chlorophylls (Fig. 1). Palmitoleic and *cis*-vaccenic acids also undergo strong photodegradation, affording mainly

These processes were also investigated in aerobic anoxygenic phototrophic bacteria (AAPs) (Rontani et al., 2003a). These organisms constitute a relatively recently discovered bacterial group (Yurkov and Beatty, 1998) and seem to be widespread in the open ocean (Kolber et al., 2000). They perform photoheterotrophic metabolism, requiring organic carbon for growth, but they are capable to use photosynthesis as an auxiliary source of energy (Kolber et al., 2001). Though sensitive to photochemical processes in senescent purple sulfur bacteria (Marchand and Rontani., 2003), the isoprenoid phytyl side-chain of bacteriochlorophyll -*a* is not significantly photodegraded in senescent cells of AAPs (Rontani et al., 2003a). In contrast, significant amounts of allylic hydroxyacids arising from the photo-oxidation of the major unsaturated fatty acid of these organisms (*cis*-vaccenic acid) could be detected after irradiation (Rontani et al., 2003a).

As in the case of phytoplankton and cyanobacteria, visible light-dependent degradation processes act significantly on the chlorophyll phytyl side-chain (Rontani et al., 1996b), unsaturated fatty acids and sterols (Rontani, Unpublished results) during terrestrial higher plant senescence affording similar photoproducts. 9-Hydroperoxy-18-hydroxyoctadec-10(*trans*)-enoic (**13**) and 10-hydroperoxy-18-hydroxyoctadec-8(*trans*)-enoic (**14**) acids deriving from type II photooxidation of 18-hydroxyoleic acid (**15**) (Fig. 7) were detected after visible light-induced senescence experiments carried out with *Petroselinum sativum* and subsequent cutin depolymerisation (Rontani et al., 2005a).

Fig. 7. Type II photosensitized oxidation of 18-hydroxyoleic acid in cutin polymers.

These results showed that in senescent plants, where the 1O2 formation rate exceeds the quenching capacity of the photoprotective system, 1O2 can migrate outside the chloroplasts and affect the unsaturated components of cutins. Significant amounts of 9,18 dihydroxyoctadec-10(*trans*)-enoic (**16**) and 10,18-dihydroxyoctadec-8(*trans*)-enoic (**17**) acids resulting from the reduction of these photoproducts of 18-hydroxyoleic acid were also detected in different natural samples (Rontani et al., 2005a). These results well support the significance of the photooxidation of the unsaturated components of higher plant cutins in the natural environment.

## **3. Free radical degradation (autoxidation) processes in phototrophic organisms**

Autoxidation is the direct reaction of molecular oxygen with organic compounds under mild conditions. The autoxidation of organic compounds (in particular, lipids) involves free

Photo- and Free Radical-Mediated

**R1 CH2OR2 OOR**

**- HO**

*Z* **and** *E*

**18 and 19**

**R1 CH2OR2 <sup>O</sup> R1 CH2OR2**

**1. + O**<sup>2</sup> **2. +H**

**1. NaBH**4 **reduction 2. Alkaline hydrolysis**

**3.2 Unsaturated fatty acids** 

(Fig. 9) (Frankel, 1998).

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 15

be detected in particulate matter samples (Marchand et al., 2005) and *E. huxleyi* cells (Rontani et al., 2007a) attesting to the involvement of such processes in senescent phytoplanktonic cells.

**ROO ROOH**

**+ ROO**

**OOR**

**20**

**R1 CH2OR2 OH**

**21**

detriment of addition reactions (Huyser and Johnson, 1968).

Fig. 8. Free radical-mediated oxidation of chlorophyll phytyl side-chain.

Free radical oxidation of chlorophyll phytyl chain appeared to be different in senescent cells of *S. costatum* (Rontani et al., 2003b). The differences observed were attributed to the well documented high chlorophyllase activity of this strain (Jeffrey and Hallegraeff, 1987) catalysing the hydrolysis of chlorophyll to free phytol and chlorophyllide. Indeed, in the case of free allylic alcohols hydrogen abstraction at carbon 1 is strongly favoured to the

Free radical oxidation of isolated classical 1,2-disubstituted double bonds generally involved mainly allylic hydrogen abstraction. Addition of peroxyl or alkoxyl radicals to the double bond becomes competitive only in the case of conjugated, terminal, or trisubstituted double bonds (Schaich, 2005). Effectively, autoxidation of mono-unsaturated fatty acids appears to mainly involve allylic hydrogen abstraction and subsequent oxidation of the allylic radical thus formed. For example, autoxidation of oleic acid mainly results in the formation of 9-hydroperoxyoctadec-*trans-*10-enoic (**24**), 10-hydroperoxyoctadec-*trans-*8-enoic (**25**), 11-hydroperoxyoctadec-*trans-*9-enoic (**26**), 11-hydroperoxyoctadec-*cis-*9-enoic (**27**), 8-hydroperoxyoctadec-*trans*-9-enoic (**28**) and 8-hydroperoxyoctadec-*cis*-9-enoic (**29**) acids

**OH**

**OOH**

**R1 CH2OR2**

**C (CH2)2 O**

**R**1 **= R**2 **=**

**R1 CH2OR2 R1 CH2OR2**

**R1 CH2OR2**

**1. + O**<sup>2</sup> **2. +H**

**R1 CH2OH R1 CH2OH OH OH**

*Z* **and** *E Z* **and** *E*

**22 and 23 3 and 4**

**HOO OOH**

**1. NaBH**4 **reduction 2. Alkaline hydrolysis** **Pyr**

**Pyr = More or less oxidized tetrapyrrolic structure**

radical reaction chains and thus includes an initiation, a propagation and a termination phase. Mechanisms of initiation for the free radical processes have been the subject of many studies. In senescent phytoplanktonic cells, initiation seems to result from the decomposition of hydroperoxides produced during photodegradation of cellular organic matter (Rontani et al., 2003b). Until now, autoxidative degradation in the marine environment has been largely ignored. Specific markers of these reactions have been highlighted by *in vitro* studies (Frankel, 1998; Rontani et al., 2003b; Rontani and Aubert, 2005). Using these markers, it was demonstrated *in situ* that autoxidation plays a very significant role in the degradation of particulate organic matter (Marchand et al., 2005; Rontani et al., 2006; Christodoulou et al., 2009; Rontani et al., 2011a).

Although the occurrence of autoxidation processes was clearly demonstrated *in situ*, it is not easy to induce these processes in laboratory cultures. Indeed, the mechanism of initiation of lipid radical oxidation, which has been debated for many years, seems to be the homolytic cleavage of photochemically produced hydroperoxides in phytodetritus (Rontani et al., 2003b). Redox-active metal ions are generally considered as the initiators of perhaps greatest importance for lipid oxidation in biological systems (Pokorny, 1987; Schaich, 1992). They may direct the cleavage of hydroperoxides either through alkoxyl or peroxyl radicals. In classical culture media (such as f/2) the metal chelator EDTA, which is present in high amounts, tightly binds free catalytic metal ions and thus renders them unavailable. EDTA thus acts in the culture media as an antioxidant and strongly limits radical oxidation processes.

Recently, autoxidative damages in cells of *E. huxleyi* strain CS-57 could be induced after incubation of this strain under an atmosphere of air + 0.5% CO2 (Rontani et al., 2007a). The presence of additional CO2 allowed: (i) to induce a stress that favoured oxidative damage and (ii) to decrease the pH of the culture medium releasing metal ions from EDTA complexes, which can act as catalysts of hydroperoxide homolysis.

It was also demonstrated recently that viral infection (Evans et al., 2006) and autocatalytic programmed cell death (Bidle and Falkowski, 2004) of phytoplanktonic cells could also lead to elevated production of reactive oxygen species (ROS) able to induce the degradation of cell components.

#### **3.1 Chlorophyll phytyl side-chain**

Autoxidation of the esterified chlorophyll phytyl chain involves either addition of peroxyl radicals to the double bond or hydrogen abstraction at the allylic carbon 4 (Rontani and Aubert, 1994; Rontani and Aubert, 2005). Classical addition of peroxyl radical to the double bond gives a tertiary radical (Fig. 8). This radical can then: (i) lead to *Z* and *E* epoxides (**18** and **19**) by fast intramolecular homolytic substitution (Fossey et al., 1995), or (ii) react with molecular oxygen affording (after hydrogen abstraction on another molecule of substrate) a diperoxide (**20**) (Fig. 8). Subsequent NaBH4-reduction and alkaline hydrolysis of these compounds gives 3,7,11,15-tetramethylhexadecan-1,2,3-triol (**21**) (Fig. 8). In contrast, abstraction (by photochemically-produced peroxyl radicals) of a hydrogen atom at the allylic carbon 4 of the phytyl chain and subsequent oxidation of the allylic radicals thus formed affords (after NaBH4-reduction and alkaline hydrolysis) *Z* and *E* 3,7,11,15-tetramethylhexadec-3-en-1,2-diols (**3** and **4**) and *Z* and *E* 3,7,11,15-tetramethyl-hexadec-2-en-1,4-diols (**22** and **23**) (Fig. 8). Compounds **22 and 23** (which are well specific markers of free radical oxidation) could be detected in particulate matter samples (Marchand et al., 2005) and *E. huxleyi* cells (Rontani et al., 2007a) attesting to the involvement of such processes in senescent phytoplanktonic cells.

Fig. 8. Free radical-mediated oxidation of chlorophyll phytyl side-chain.

Free radical oxidation of chlorophyll phytyl chain appeared to be different in senescent cells of *S. costatum* (Rontani et al., 2003b). The differences observed were attributed to the well documented high chlorophyllase activity of this strain (Jeffrey and Hallegraeff, 1987) catalysing the hydrolysis of chlorophyll to free phytol and chlorophyllide. Indeed, in the case of free allylic alcohols hydrogen abstraction at carbon 1 is strongly favoured to the detriment of addition reactions (Huyser and Johnson, 1968).

## **3.2 Unsaturated fatty acids**

14 Senescence

radical reaction chains and thus includes an initiation, a propagation and a termination phase. Mechanisms of initiation for the free radical processes have been the subject of many studies. In senescent phytoplanktonic cells, initiation seems to result from the decomposition of hydroperoxides produced during photodegradation of cellular organic matter (Rontani et al., 2003b). Until now, autoxidative degradation in the marine environment has been largely ignored. Specific markers of these reactions have been highlighted by *in vitro* studies (Frankel, 1998; Rontani et al., 2003b; Rontani and Aubert, 2005). Using these markers, it was demonstrated *in situ* that autoxidation plays a very significant role in the degradation of particulate organic matter (Marchand et al., 2005;

Although the occurrence of autoxidation processes was clearly demonstrated *in situ*, it is not easy to induce these processes in laboratory cultures. Indeed, the mechanism of initiation of lipid radical oxidation, which has been debated for many years, seems to be the homolytic cleavage of photochemically produced hydroperoxides in phytodetritus (Rontani et al., 2003b). Redox-active metal ions are generally considered as the initiators of perhaps greatest importance for lipid oxidation in biological systems (Pokorny, 1987; Schaich, 1992). They may direct the cleavage of hydroperoxides either through alkoxyl or peroxyl radicals. In classical culture media (such as f/2) the metal chelator EDTA, which is present in high amounts, tightly binds free catalytic metal ions and thus renders them unavailable. EDTA thus acts in the culture media as an antioxidant and strongly limits radical oxidation

Recently, autoxidative damages in cells of *E. huxleyi* strain CS-57 could be induced after incubation of this strain under an atmosphere of air + 0.5% CO2 (Rontani et al., 2007a). The presence of additional CO2 allowed: (i) to induce a stress that favoured oxidative damage and (ii) to decrease the pH of the culture medium releasing metal ions from EDTA

It was also demonstrated recently that viral infection (Evans et al., 2006) and autocatalytic programmed cell death (Bidle and Falkowski, 2004) of phytoplanktonic cells could also lead to elevated production of reactive oxygen species (ROS) able to induce the degradation of

Autoxidation of the esterified chlorophyll phytyl chain involves either addition of peroxyl radicals to the double bond or hydrogen abstraction at the allylic carbon 4 (Rontani and Aubert, 1994; Rontani and Aubert, 2005). Classical addition of peroxyl radical to the double bond gives a tertiary radical (Fig. 8). This radical can then: (i) lead to *Z* and *E* epoxides (**18** and **19**) by fast intramolecular homolytic substitution (Fossey et al., 1995), or (ii) react with molecular oxygen affording (after hydrogen abstraction on another molecule of substrate) a diperoxide (**20**) (Fig. 8). Subsequent NaBH4-reduction and alkaline hydrolysis of these compounds gives 3,7,11,15-tetramethylhexadecan-1,2,3-triol (**21**) (Fig. 8). In contrast, abstraction (by photochemically-produced peroxyl radicals) of a hydrogen atom at the allylic carbon 4 of the phytyl chain and subsequent oxidation of the allylic radicals thus formed affords (after NaBH4-reduction and alkaline hydrolysis) *Z* and *E* 3,7,11,15-tetramethylhexadec-3-en-1,2-diols (**3** and **4**) and *Z* and *E* 3,7,11,15-tetramethyl-hexadec-2-en-1,4-diols (**22** and **23**) (Fig. 8). Compounds **22 and 23** (which are well specific markers of free radical oxidation) could

Rontani et al., 2006; Christodoulou et al., 2009; Rontani et al., 2011a).

complexes, which can act as catalysts of hydroperoxide homolysis.

processes.

cell components.

**3.1 Chlorophyll phytyl side-chain** 

Free radical oxidation of isolated classical 1,2-disubstituted double bonds generally involved mainly allylic hydrogen abstraction. Addition of peroxyl or alkoxyl radicals to the double bond becomes competitive only in the case of conjugated, terminal, or trisubstituted double bonds (Schaich, 2005). Effectively, autoxidation of mono-unsaturated fatty acids appears to mainly involve allylic hydrogen abstraction and subsequent oxidation of the allylic radical thus formed. For example, autoxidation of oleic acid mainly results in the formation of 9-hydroperoxyoctadec-*trans-*10-enoic (**24**), 10-hydroperoxyoctadec-*trans-*8-enoic (**25**), 11-hydroperoxyoctadec-*trans-*9-enoic (**26**), 11-hydroperoxyoctadec-*cis-*9-enoic (**27**), 8-hydroperoxyoctadec-*trans*-9-enoic (**28**) and 8-hydroperoxyoctadec-*cis*-9-enoic (**29**) acids (Fig. 9) (Frankel, 1998).

Photo- and Free Radical-Mediated

**OTMS**

*m/z* **241**

*m/z* **241**

*m/z* **343**

**OTMS**

under an atmosphere of air + 0.5% CO2.

autoxidation..

**OTMS**

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 17

**COOTMS**

**COOTMS**

**COOTMS**

**29.5 30.0 30.5 31.0 31.5**

*m/z* **227** *m/z* **329**

*m/z* **343**

**OTMS**

**OTMS**

*m/z* **227**

**OTMS**

*m/z* **329**

**COOTMS**

**COOTMS**

**COOTMS**

*m/z* **343** *m/z* **241**

**Retention time (min) -->**

Fig. 10. Partial mass chromatogram of *m/z* 227, 329, 241 and 343 revealing the presence of oxidation products of oleic acid in the saponified fraction of *E. huxleyi* strain CS-57 grown

Owing to: their lack of specificity (possible formation by allylic rearrangement of photochemically-produced 5-hydroperoxides (see chapter 2.1.3), 7-hydroperoxides cannot be employed as tracers of autoxidation processes in phytodetritus. In contrast, it is generally considered that 5*α/β*,6*α/β*-epoxysterols arise mainly from peroxidation processes (Breuer and Björkhem, 1995; Giuffrida et al., 2004). Unfortunately, these compounds are not very stable and may be easily hydrolysed to the corresponding triol in seawater and during the treatment of the samples. 5*α/β*,6*α/β*-Epoxysterols and the corresponding 3*β*,5*α*,6*β*-trihydroxysterols were thus finally selected as tracers of sterol

5*α/β*,6*α/β*-Epoxysterols and 3*β*,5*α*,6*β*-trihydroxysterols corresponding to sitosterol, stigmasterol and campesterol were previously detected in young and old cell cultures of *Chenopodium rubrum* (Meyer and Spiteller, 1997). The results showed that the increase of

these oxidation products well correlated with the age of the culture.

**R' = -(CH**2**)**6**-COOH R = -(CH**2**)**6**-CH**<sup>3</sup>

Fig. 9. Free radical-mediated oxidation of oleic acid.

Free radical oxidative processes can be easily characterised based on the presence of *cis* allylic hydroperoxyacids, which cannot be produced photochemically (see Fig. 4) and are specific products of these degradation processes (Porter et al., 1995; Frankel, 1998).

Large amounts of oxidation products of oleic acid could be detected in cells of *E. huxleyi* grown under an atmosphere of air + 0.5% CO2 for 10 days (Rontani et al., 2007a). The presence (after NaBH4-reduction) of a high proportion of 11-hydroxyoctadec-*cis-*9-enoic (**27**) and 8-hydroxyoctadec-*cis*-9-enoic (**29**) acids (Fig. 10) showed that under these conditions the degradation of oleic acid mainly involved free radical oxidation processes.

#### **3.3 <sup>5</sup> -sterols**

Free radical autoxidation of Δ5-stenols yields mainly 7*α*- and 7*β*-hydroperoxides and, to a lesser extent, 5*α/β*,*6α/β*-epoxysterols and 3*β*,5*α*,6*β*-trihydroxysterols (Smith, 1981; Morrissey and Kiely, 2006) (Fig. 11).

**<sup>R</sup> R' <sup>11</sup> 10 9 8**

**Free radical oxidation**

**OOH R' <sup>8</sup>**

**Radical allylic rearrangement**

Free radical oxidative processes can be easily characterised based on the presence of *cis* allylic hydroperoxyacids, which cannot be produced photochemically (see Fig. 4) and are

Large amounts of oxidation products of oleic acid could be detected in cells of *E. huxleyi* grown under an atmosphere of air + 0.5% CO2 for 10 days (Rontani et al., 2007a). The presence (after NaBH4-reduction) of a high proportion of 11-hydroxyoctadec-*cis-*9-enoic (**27**) and 8-hydroxyoctadec-*cis*-9-enoic (**29**) acids (Fig. 10) showed that under these conditions the

Free radical autoxidation of Δ5-stenols yields mainly 7*α*- and 7*β*-hydroperoxides and, to a lesser extent, 5*α/β*,*6α/β*-epoxysterols and 3*β*,5*α*,6*β*-trihydroxysterols (Smith, 1981; Morrissey

specific products of these degradation processes (Porter et al., 1995; Frankel, 1998).

degradation of oleic acid mainly involved free radical oxidation processes.

**R**

**27 26 29 28 25 24**

**OOH**

**Radical allylic rearrangement**

**R**

**10**

**HOO R'**

**R'**

**R OOH 9**

**<sup>R</sup> <sup>11</sup>**

**<sup>R</sup> R' <sup>11</sup> OOH**

> **R' = -(CH**2**)**6**-COOH R = -(CH**2**)**6**-CH**<sup>3</sup>

**3.3 <sup>5</sup>**

**-sterols** 

and Kiely, 2006) (Fig. 11).

**OOH**

**R'**

Fig. 9. Free radical-mediated oxidation of oleic acid.

**<sup>R</sup> R' <sup>8</sup>**

**Retention time (min) -->**

Fig. 10. Partial mass chromatogram of *m/z* 227, 329, 241 and 343 revealing the presence of oxidation products of oleic acid in the saponified fraction of *E. huxleyi* strain CS-57 grown under an atmosphere of air + 0.5% CO2.

Owing to: their lack of specificity (possible formation by allylic rearrangement of photochemically-produced 5-hydroperoxides (see chapter 2.1.3), 7-hydroperoxides cannot be employed as tracers of autoxidation processes in phytodetritus. In contrast, it is generally considered that 5*α/β*,6*α/β*-epoxysterols arise mainly from peroxidation processes (Breuer and Björkhem, 1995; Giuffrida et al., 2004). Unfortunately, these compounds are not very stable and may be easily hydrolysed to the corresponding triol in seawater and during the treatment of the samples. 5*α/β*,6*α/β*-Epoxysterols and the corresponding 3*β*,5*α*,6*β*-trihydroxysterols were thus finally selected as tracers of sterol autoxidation..

5*α/β*,6*α/β*-Epoxysterols and 3*β*,5*α*,6*β*-trihydroxysterols corresponding to sitosterol, stigmasterol and campesterol were previously detected in young and old cell cultures of *Chenopodium rubrum* (Meyer and Spiteller, 1997). The results showed that the increase of these oxidation products well correlated with the age of the culture.

Photo- and Free Radical-Mediated

**Other minor oxidation products**

**O**

**R**

**O**

**30**

**R**

**HO**

**MeO**

**O**

**R**

**O**

**HO**

**O**

 **Spiro-trimers (two stereoisomers)**

**Methanolysis**

**R =**

Fig. 12. Autoxidation of vitamin E and methanolysis of the foregoing trimers.

**O**

**R**

**O**

**O**

**O**

**O**

**Spiro-dimers Ethano-dimers**

**R**

**O**

**R**

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 19

**R**

**Free radical oxidation**

**O**

Fig. 11. Free radical-mediated oxidation of 5 sterols.

#### **3.4 Vitamin E**

Vitamin E is relatively abundant in most photosynthetic organisms, such as higher plants (Rise et al., 1988; Schultz, 1990), cyanobacteria (Dasilva and Jensen, 1971), microalgae (Brown et al., 1999) and macroalgae (Sanchez-Machado et al., 2002), where it plays an essential role in the removal of toxic forms of oxygen (singlet oxygen, superoxide anion, hydroxyl and peroxyl radicals), by acting as sacrificial chemical scavenger (Halliwell, 1987); the process results in the irreversible oxidation of the tocopherol molecule. Vitamin E reacts rapidly with peroxyl radicals, affording small amounts of phytone (**1**), 4,8,12,16 tetramethylheptadecan-4-olide, -tocopherylquinone and epoxy--tocopherylquinones, and dimers and trimers as major oxidation products (Liebler, 1994; Frankel, 1998; Rontani et al., 2007b) (Fig. 12).

 **Free radical oxidation**

**7**

**HO HO OOH**

Vitamin E is relatively abundant in most photosynthetic organisms, such as higher plants (Rise et al., 1988; Schultz, 1990), cyanobacteria (Dasilva and Jensen, 1971), microalgae (Brown et al., 1999) and macroalgae (Sanchez-Machado et al., 2002), where it plays an essential role in the removal of toxic forms of oxygen (singlet oxygen, superoxide anion, hydroxyl and peroxyl radicals), by acting as sacrificial chemical scavenger (Halliwell, 1987); the process results in the irreversible oxidation of the tocopherol molecule. Vitamin E reacts rapidly with peroxyl radicals, affording small amounts of phytone (**1**), 4,8,12,16 tetramethylheptadecan-4-olide, -tocopherylquinone and epoxy--tocopherylquinones, and dimers and trimers as major oxidation products (Liebler, 1994; Frankel, 1998; Rontani et

**HO**

**HO**

**3.4 Vitamin E** 

al., 2007b) (Fig. 12).

**O**

**5 6**

**+ H**2**O**

**HO OH**

Fig. 11. Free radical-mediated oxidation of 5 sterols.

Fig. 12. Autoxidation of vitamin E and methanolysis of the foregoing trimers.

Photo- and Free Radical-Mediated

**O**

**O**

**O**

**O**

**O**

**OTMS**

**OTMS**

**OTMS**

**OTMS**

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 21

**OOH**

**OOH**

**OOH**

**OTMS**

**OOH**

**1. NaBH4 reduction**

**Free radical oxidation**

**2. Silylation**

**OTMS**

**OTMS**

double bond of the C37:3 alkenone (TMS = trimethylsilyl).

Fig. 13. Characterization of oxidation products derived from the autoxidation of the 22

**OTMS**

Isomeric trimers have been previously observed as products in numerous oxidations of vitamin E (e.g. Suarna et al., 1988; Krol et al., 2001). Such compounds cannot be easily detected since they are too heavy to be amenable by gas chromatography. However, methanolysis of the residues obtained resulted to the formation of high amounts of 5amethoxytocopherol (**30**) arising from the methanolysis of the ketal group of trimers (Fig. 12) (Yamauchi et al., 1988). ESI-TOF MS analyses of oxidation products were also carried out in order to confirm the presence of high proportions of trimers (Nassiry et al., 2009).

Despite the intensive study of vitamin E oxidation since several decades, trimeric oxidation products could be detected in plants only very recently by Row et al. (2007). These authors detected these trimers in seeds of *Euryale ferox* containing extraordinarily high content of tocopherols. It is interesting to note that trimers were previously obtained as the major reaction products of vitamin E autoxidized under mild conditions in solution (1%) in methyl linoleate (Yamauchi et al., 1988). In plastoglobules, which are lipid monolayer subcompartments of the thylakoid membranes of chloroplasts (Maeda and Dellapenna, 2007), the concentration of tocopherols can reach 10% of the total fatty acids (Vidi et al., 2006). At such a concentration, the formation of a high proportion of trimers during photodynamic damages is thus very likely. In order to check this hypothesis, we searched for the presence of 5a-methoxytocopherol (**30**) after methanolysis of NaBH4-reduced and non-reduced lipid extracts obtained from cells of *Emiliania huxleyi* strain TWP1 and *Chrysotila lamellosa* strain HAP17. The detection of significant amounts of this methanolysis product of trimers (Yamauchi et al., 1988) in these extracts (Nassiry et al., 2009) well supported the presence of such trimeric oxidation products of vitamin E in these algae.

#### **3.5 Alkenones**

The autoxidative reactivity of alkenones was studied in the laboratory in the presence of a radical initiator (di-*tert*-butyl nitroxide) and a radical enhancer (*tert*-butyl hydroperoxide) (Rontani et al., 2006). Alkenones appeared to be more sensitive towards oxidative free radical processes than analogues of other common marine lipids such as phytyl acetate, methyl oleate and cholesteryl acetate, and their oxidation rates increase in proportion with their number of double bonds. As the result of this increasing reactivity with degree of unsaturation, the 37 *<sup>K</sup> U* ratio increased significantly (up to 0.20) during the incubation.

Autoxidation of alkenones appears to mainly involve allylic hydrogen abstraction and subsequent oxidation of the allylic radical thus formed (Fig. 13). According to these processes, oxidation of each double bond of alkenones and subsequent NaBH4 reduction affords four positional isomeric alkenediols. These compounds could be very useful indicators of autoxidation of alkenones but, unfortunately, they did not accumulate during the incubation. Indeed, due to the presence of additional reactive double bonds, hydroperoxyalkenones may undergo subsequent oxidation reactions affording, di-, tri- and tetrahydroperoxyalkenones according to the degree of unsaturation of the starting alkenone. In seawater, these different hydroperoxides may undergo two main degradative processes: (i) homolysis of the O-O bond leading to carbonyl (dehydration), alcoholic (reduction) and fragmentation (-scission) products (Rontani et al., 2007c) and (ii) heterolysis of the O-O bond leading to the formation of two carbonyl fragments (Hock cleavage), this protoncatalysed cleavage being initiated by migration of groups to positive oxygen (Frimer, 1979). Dimeric and oligomeric compounds cross-linked through either peroxide or ether linkages (Frankel, 1998) may also be formed during autoxidation of alkenones.

Isomeric trimers have been previously observed as products in numerous oxidations of vitamin E (e.g. Suarna et al., 1988; Krol et al., 2001). Such compounds cannot be easily detected since they are too heavy to be amenable by gas chromatography. However, methanolysis of the residues obtained resulted to the formation of high amounts of 5amethoxytocopherol (**30**) arising from the methanolysis of the ketal group of trimers (Fig. 12) (Yamauchi et al., 1988). ESI-TOF MS analyses of oxidation products were also carried out in

Despite the intensive study of vitamin E oxidation since several decades, trimeric oxidation products could be detected in plants only very recently by Row et al. (2007). These authors detected these trimers in seeds of *Euryale ferox* containing extraordinarily high content of tocopherols. It is interesting to note that trimers were previously obtained as the major reaction products of vitamin E autoxidized under mild conditions in solution (1%) in methyl linoleate (Yamauchi et al., 1988). In plastoglobules, which are lipid monolayer subcompartments of the thylakoid membranes of chloroplasts (Maeda and Dellapenna, 2007), the concentration of tocopherols can reach 10% of the total fatty acids (Vidi et al., 2006). At such a concentration, the formation of a high proportion of trimers during photodynamic damages is thus very likely. In order to check this hypothesis, we searched for the presence of 5a-methoxytocopherol (**30**) after methanolysis of NaBH4-reduced and non-reduced lipid extracts obtained from cells of *Emiliania huxleyi* strain TWP1 and *Chrysotila lamellosa* strain HAP17. The detection of significant amounts of this methanolysis product of trimers (Yamauchi et al., 1988) in these extracts (Nassiry et al., 2009) well supported the presence of such trimeric oxidation products of vitamin E in these algae.

The autoxidative reactivity of alkenones was studied in the laboratory in the presence of a radical initiator (di-*tert*-butyl nitroxide) and a radical enhancer (*tert*-butyl hydroperoxide) (Rontani et al., 2006). Alkenones appeared to be more sensitive towards oxidative free radical processes than analogues of other common marine lipids such as phytyl acetate, methyl oleate and cholesteryl acetate, and their oxidation rates increase in proportion with their number of double bonds. As the result of this increasing reactivity with degree of

Autoxidation of alkenones appears to mainly involve allylic hydrogen abstraction and subsequent oxidation of the allylic radical thus formed (Fig. 13). According to these processes, oxidation of each double bond of alkenones and subsequent NaBH4 reduction affords four positional isomeric alkenediols. These compounds could be very useful indicators of autoxidation of alkenones but, unfortunately, they did not accumulate during the incubation. Indeed, due to the presence of additional reactive double bonds, hydroperoxyalkenones may undergo subsequent oxidation reactions affording, di-, tri- and tetrahydroperoxyalkenones according to the degree of unsaturation of the starting alkenone. In seawater, these different hydroperoxides may undergo two main degradative processes: (i) homolysis of the O-O bond leading to carbonyl (dehydration), alcoholic (reduction) and fragmentation (-scission) products (Rontani et al., 2007c) and (ii) heterolysis of the O-O bond leading to the formation of two carbonyl fragments (Hock cleavage), this protoncatalysed cleavage being initiated by migration of groups to positive oxygen (Frimer, 1979). Dimeric and oligomeric compounds cross-linked through either peroxide or ether linkages

(Frankel, 1998) may also be formed during autoxidation of alkenones.

ratio increased significantly (up to 0.20) during the incubation.

order to confirm the presence of high proportions of trimers (Nassiry et al., 2009).

**3.5 Alkenones** 

unsaturation, the 37

*<sup>K</sup> U*

Fig. 13. Characterization of oxidation products derived from the autoxidation of the 22 double bond of the C37:3 alkenone (TMS = trimethylsilyl).

Photo- and Free Radical-Mediated

biases during paleotemperature reconstruction.

*Phytochemistry* 56, 795-800.

*Chemistry* 270, 20278-20284.

**5. Acknowledgements** 

**6. References** 

Oxidation of Lipid Components During the Senescence of Phototrophic Organisms 23

It was recently demonstrated that most of the unsaturated lipid components of these organisms (chlorophylls, carotenoids, unsaturated fatty acids, sterols, *n*-alkenes and HBI alkenes) could be photodegraded by visible and UV radiations during the senescence. This degradation mainly involves type II (i.e. involving 1O2) photoprocesses. Singlet oxygen appeared to be sufficiently stable in this hydrophobic micro-environment to migrate outside

Free radical-mediated oxidation (autoxidation) processes also intervene intensively during the senescence of phototrophic organisms. Induction of these processes seems to mainly result from the homolytic cleavage (catalyzed by some metal ions) of photochemically produced hydroperoxides. Unsaturated fatty acids, chlorophyll phytyl side-chain, vitamin E, sterols and alkenones appeared to be strongly affected by these degradative processes. In the case of alkenones, it is very important to note that autoxidative degradation processes may alter significantly their unsaturation ratio and thus constitute a potential source of

Financial support over many years from the Centre National de la Recherche Scientifique

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isomers. *Geochimica & Cosmochimica Acta* 64, 3839-3851.

years. *Quaternary Science Reviews* 29, 3489-3504.

microorganisms. *Nature Review 2*, 643-655.

the chloroplasts and affect the unsaturated components of cutins of higher plants.

These results were corroborated by the further finding of significant amounts of alkenediols arising from NaBH4-reduction of the corresponding hydroperoxyalkenones in cultures of *E. huxleyi* strain CS-57 grown under an atmosphere of air + 0.5% CO2 (Rontani et al., 2007a) and more recently after incubation of a culture of the strain *E. huxleyi* TWP1 under darkness (Rontani, Unpublished results) (Fig. 14) both exhibiting an anomalously high unsaturation ratio It seems thus that autoxidation processes have the potential to affect alkenone distributions leading to a warm bias in estimates of palaeotemperatures derived from alkenone ratios in sediments.

Fig. 14. Partial mass fragmentograms of *m/z* 311 and 325 revealing the presence of silylated C37 and C38 alkenediols after NaBH4-reduction and silylation of the total lipid extract of *E. huxleyi* cells incubated under darkness (A) and standard autoxidation products of alkenones (B).

#### **4. Conclusions**

Due to the lack of adequate tracers, the role played by light-induced photochemical and free radical-mediated (autoxidative) processes during the degradation of lipid components of phototrophic organisms has been virtually ignored until now.

It was recently demonstrated that most of the unsaturated lipid components of these organisms (chlorophylls, carotenoids, unsaturated fatty acids, sterols, *n*-alkenes and HBI alkenes) could be photodegraded by visible and UV radiations during the senescence. This degradation mainly involves type II (i.e. involving 1O2) photoprocesses. Singlet oxygen appeared to be sufficiently stable in this hydrophobic micro-environment to migrate outside the chloroplasts and affect the unsaturated components of cutins of higher plants.

Free radical-mediated oxidation (autoxidation) processes also intervene intensively during the senescence of phototrophic organisms. Induction of these processes seems to mainly result from the homolytic cleavage (catalyzed by some metal ions) of photochemically produced hydroperoxides. Unsaturated fatty acids, chlorophyll phytyl side-chain, vitamin E, sterols and alkenones appeared to be strongly affected by these degradative processes. In the case of alkenones, it is very important to note that autoxidative degradation processes may alter significantly their unsaturation ratio and thus constitute a potential source of biases during paleotemperature reconstruction.

## **5. Acknowledgements**

Financial support over many years from the Centre National de la Recherche Scientifique (CNRS) and the Université de la Méditerranée is gratefully acknowledged.

## **6. References**

22 Senescence

These results were corroborated by the further finding of significant amounts of alkenediols arising from NaBH4-reduction of the corresponding hydroperoxyalkenones in cultures of *E. huxleyi* strain CS-57 grown under an atmosphere of air + 0.5% CO2 (Rontani et al., 2007a) and more recently after incubation of a culture of the strain *E. huxleyi* TWP1 under darkness (Rontani, Unpublished results) (Fig. 14) both exhibiting an anomalously high unsaturation ratio It seems thus that autoxidation processes have the potential to affect alkenone distributions leading to a warm bias in estimates of palaeotemperatures derived from

**Retention time (min) -->**

**61.0 63.0 65.0 67.0 69.0 71.0**

*m/z***<sup>325</sup> OTMS**

**61.0 63.0 65.0 67.0 69.0 71.0 Retention time (min) -->**

Fig. 14. Partial mass fragmentograms of *m/z* 311 and 325 revealing the presence of silylated C37 and C38 alkenediols after NaBH4-reduction and silylation of the total lipid extract of *E. huxleyi* cells incubated under darkness (A) and standard autoxidation products of

Due to the lack of adequate tracers, the role played by light-induced photochemical and free radical-mediated (autoxidative) processes during the degradation of lipid components of

*m/z* **311**

*m/z* **311**

*m/z* **325**

**OTMS**

**OTMS**

*m/z* **325**

*m/z* **325**

**OTMS OTMS**

*m/z* **311**

*m/z* **311**

**OTMS OTMS**

**OTMS**

**OTMS**

**OTMS**

*m/z* **325**

*m/z* **325**

alkenone ratios in sediments.

**OTMS OTMS**

**OTMS**

alkenones (B).

**4. Conclusions** 

**B**

**A**

**OTMS OTMS**

*m/z* **311**

**OTMS**

*m/z* **311**

phototrophic organisms has been virtually ignored until now.


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**2** 

*Germany* 

**Role of Intracellular Hydrogen Peroxide** 

Ulrike Zentgraf, Petra Zimmermann and Anja Smykowski

), hydroperoxylradical (HO2·-), hydrogen peroxide

*ZMBP, University of Tübingen* 

 **as Signalling Molecule for Plant Senescence** 

All aerobic organisms use molecular oxygen as terminal oxidant during respiration. Oxygen is neither very reactive nor harmful, but it has the potential to be only partially reduced, leading to the formation of very reactive and therefore toxic intermediates, like singlet

(H2O2) and hydroxylradical (·OH). These forms are called "reactive oxygen species" (ROS). All ROS are extremely reactive and may oxidize biological molecules, such as DNA, proteins and lipids. However, these reactive molecules are unavoidable by-products of an aerobic metabolism. It is known that reactive oxygen species may have a dual role in plant stress response (Dat et al. 2000). Whereas high concentrations of hydrogen peroxide are toxic for the cell, low concentrations may act as signal which triggers the plant response upon a variety of biotic and abiotic stresses (Dat et al., 2000; Grant & Loake, 2000). It has been known for many years that common signal transduction molecules like MAPKs and calmodulin play an important role in some of these ROS signal transduction pathways.

Mitochondria are an important origin of ROS. During respiration, the ubiquinone pool is the main source for superoxide production. The alternative oxidase (AOX) could be identified in plants and protists, e.g. Trypanosoma, fungi, like *Neurospora crassa* and *Hansenula anomala* and in green algae, e.g. in Chlamydomonas (McIntosh, 1994). It acts as a quinoloxidase by transferring electrons from the reduced ubiquinone directly to molecular oxygen forming water (Siedow & Moore, 1993). AOX mediates an energy-wasteful form of respiration, but its physiological significance is still a matter of intense debate (Rasmusson et al., 2009; Vanlerberghe et al., 2009; Millar et al., 2011). The plant alternative oxidases form homodimers (Moore et al., 2002) and are encoded by a small gene family. In *Arabidopsis thaliana* five genes are known, *AOX1a*, *AOX1b*, *AOX1c, AOX1d* and *AOX2*, each exhibiting organ specific expression (Saisho et al., 1997; https:/www.genevestigator.com). Among these five *AOX* genes in *Arabidopsis thaliana*, *AOX1a* is the major isoform expressed in leaves (Clifton et al., 2006). One important function of the alternative oxidase is to prevent the formation of excess of reactive oxygen molecules (Maxwell et al., 1999). AOX ensures a low reduction status of the ubiquinone pool by oxidizing ubiquinol. Thus, the electron flow is guaranteed (Millenaar & Lambers, 2003). This reaction is necessary, if the cytochrome *c* dependent pathway is restricted by naturally occurring cyanide, NO, sulphide, high concentrations of CO2, low temperatures or phosphorus deprivation (Millenaar & Lambers, 2003) as well as wounding, drought, osmotic stress, ripening and pathogen infection

**1. Introduction** 

oxygen (1O2), superoxide radical (O2·-


## **Role of Intracellular Hydrogen Peroxide as Signalling Molecule for Plant Senescence**

Ulrike Zentgraf, Petra Zimmermann and Anja Smykowski *ZMBP, University of Tübingen Germany* 

## **1. Introduction**

30 Senescence

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*oceanica* - implications for studies of paleoclimate. *Geochimica & Cosmochimica Acta*

model compound, 2,2,5,7,8-pentamethylchroman-6-ol, in autoxidizing methyl

All aerobic organisms use molecular oxygen as terminal oxidant during respiration. Oxygen is neither very reactive nor harmful, but it has the potential to be only partially reduced, leading to the formation of very reactive and therefore toxic intermediates, like singlet oxygen (1O2), superoxide radical (O2·- ), hydroperoxylradical (HO2·-), hydrogen peroxide (H2O2) and hydroxylradical (·OH). These forms are called "reactive oxygen species" (ROS). All ROS are extremely reactive and may oxidize biological molecules, such as DNA, proteins and lipids. However, these reactive molecules are unavoidable by-products of an aerobic metabolism. It is known that reactive oxygen species may have a dual role in plant stress response (Dat et al. 2000). Whereas high concentrations of hydrogen peroxide are toxic for the cell, low concentrations may act as signal which triggers the plant response upon a variety of biotic and abiotic stresses (Dat et al., 2000; Grant & Loake, 2000). It has been known for many years that common signal transduction molecules like MAPKs and calmodulin play an important role in some of these ROS signal transduction pathways.

Mitochondria are an important origin of ROS. During respiration, the ubiquinone pool is the main source for superoxide production. The alternative oxidase (AOX) could be identified in plants and protists, e.g. Trypanosoma, fungi, like *Neurospora crassa* and *Hansenula anomala* and in green algae, e.g. in Chlamydomonas (McIntosh, 1994). It acts as a quinoloxidase by transferring electrons from the reduced ubiquinone directly to molecular oxygen forming water (Siedow & Moore, 1993). AOX mediates an energy-wasteful form of respiration, but its physiological significance is still a matter of intense debate (Rasmusson et al., 2009; Vanlerberghe et al., 2009; Millar et al., 2011). The plant alternative oxidases form homodimers (Moore et al., 2002) and are encoded by a small gene family. In *Arabidopsis thaliana* five genes are known, *AOX1a*, *AOX1b*, *AOX1c, AOX1d* and *AOX2*, each exhibiting organ specific expression (Saisho et al., 1997; https:/www.genevestigator.com). Among these five *AOX* genes in *Arabidopsis thaliana*, *AOX1a* is the major isoform expressed in leaves (Clifton et al., 2006). One important function of the alternative oxidase is to prevent the formation of excess of reactive oxygen molecules (Maxwell et al., 1999). AOX ensures a low reduction status of the ubiquinone pool by oxidizing ubiquinol. Thus, the electron flow is guaranteed (Millenaar & Lambers, 2003). This reaction is necessary, if the cytochrome *c* dependent pathway is restricted by naturally occurring cyanide, NO, sulphide, high concentrations of CO2, low temperatures or phosphorus deprivation (Millenaar & Lambers, 2003) as well as wounding, drought, osmotic stress, ripening and pathogen infection

Role of Intracellular Hydrogen Peroxide as Signalling Molecule for Plant Senescence 33

might be used as a signal to promote senescence. In Arabidopsis the coordinate regulation of the hydrogen peroxide scavenging enzymes catalase (CAT) and ascorbate peroxidase (APX) leads to a defined increase of hydrogen peroxide content during bolting time (Ye et al., 2000; Zimmermann et al., 2006). Removing the bolt and thereby delaying the decrease in APX activity led to a delay in chlorophyll degradation and senescence (Ye et al., 2000). Since APX enzyme activity appears to be regulated on the posttranscriptional level (Panchuk et al., 2005; Zimmermann et al., 2006) and appears to be inhibited by hydrogen peroxide itself in this developmental stage, the initial event to create the hydrogen peroxide peak during bolting time at the onset of senescence is the transcriptional down-regulation of *CAT2*. The transcription factor responsible for this down-regulation was isolated by a yeast-one hybrid screen and turned out to be a member of the bZIP transcription factor family, namely GBF1. If *GBF1* is knocked out by a T-DNA insertion, the down-regulation of *CAT2* during bolting time is abolished, the hydrogen peroxide peak during bolting time disappears and senescence is delayed (Smykowski et al., 2010). This hydrogen peroxide peak is discussed to trigger senescence induction by activating the systemic expression of the senescence-related

In order to understand the correlation between mitochondrial ROS production and senescence in *Arabidopsis thaliana*, we treated cell cultures and whole Arabidopsis plants with antimycin A, an inhibitor of cytochrom *c* oxidase, and measured hydrogen peroxide production and senescence parameters. In addition, two different genes encoding the peroxisomal enzyme catalase have been knocked-out and the single knock-out plants *cat2* and *cat3* have been crossed to produce double knock-out plants *cat2/3*. In these plants also

Dufour and others (2000) characterized an almost immortal mutant of the fungus *Podospora anserina* carrying a mutation in the gene encoding subunit V of the cytochrom *c* oxidase complex. These mutants exclusively used the alternative respiration pathway thus clearly leading to a lower content of reactive oxygen species than in normal growing fungi. In Arabidopsis resistance to oxidative stress and longevity are also tightly correlated (Kurepa et al., 1998; Woo et al., 2004). Therefore, we wanted to analyse Arabidopsis plants and cells with increased alternative respiration for mitochondrial ROS production and a delay in senescence.

The alternative respiration in plants can be induced by application of antimycin A (Vanlerberghe & McIntosh, 1992), which was isolated from *Streptomyces* sp. and inhibits specifically the electron transport between cytochrome *b* and *c1*. To investigate the influence of antimycin A on the production of ROS, we analysed antimycin A treated *Arabidopsis thaliana* cell cultures for their hydrogen peroxide contents. Two hours after treatment with 5 µM antimycin A or 0.02 % ethanol as control the H2O2 concentration slightly increased, whereas further incubation clearly lowered H2O2 content in antimycin A treated cells in comparison to control cells (Fig. 1 A). The transient increase in H2O2 levels might be a result of the inhibition of cytochrome *c* oxidase as it was already shown by Maxwell and

the consequences on hydrogen peroxide levels and leaf senescence were analysed.

**2.1 Changes in mitochondrial hydrogen peroxide production** 

**2.1.1 Antimycin A treatment of cell cultures and whole plants** 

transcription factors e.g. *WRKY53* (Miao et al., 2004).

**2. Results and discussion** 

(McIntosh, 1994; Moore et al., 2002). Photo-oxidative stress of chloroplasts is also involved in *AOX* up-regulation (Yoshida et al. 2008). Moreover, ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV (Bartoli et al., 2000) and leaves of the *AOX*-overexpressing lines accumulate more ascorbic acid than wild-type leaves (Bartoli et al., 2006). A lack of AOX can lead to an up-regulation of transcripts of the antioxidant defense system at low temperature (Watanabe et al, 2008). Therefore, it is likely that AOX is an important component in antioxidant defense mechanisms.

In addition, it is proposed that AOX also has important functions outside the mitochondria (Arnholdt-Schmitt et al., 2006; Clifton et al., 2006; Van Aken et al., 2009). Furthermore, a beneficial role for AOX in illuminated leaves has been suggested and AOX-deficient *aox1a* mutant showed a lowered operating efficiency of photosystem II and an enhanced activity of cyclic electron transport around photosystem I (CET-PSI) at high irradiance (Yoshida et al., 2011). However, in most cases, transgenic plants with altered levels of AOX exhibited no obvious variation in plant growth phenotype (Vanlerberghe et al., 2009), implying that AOX does not severely affect photosynthetic carbon gain and biomass productivity**.** In addition**,**  AOX also has an effect on the control of NO levels in plant cells (Wulff et al., 2009).

There is some evidence that alternative respiration is correlated with senescence and longevity. Aging potato slides showed a decline in the capacity of cytochrome *c* dependent respiration whereas the alternative respiration as well as the protein content of AOX increased (Hiser & McIntosh, 1990). Expression of *AOX1a* of Arabidopsis is highest in rosette leaves at the onset of senescence (https:/www.genevestigator.com). Interestingly, the inactivation of subunit V of the cytochrome *c* oxidase complex in the fungus *Podospora anserina* led to the exclusive use of the alternative respiration pathway and to a decline in ROS formation in these mutants. This inactivation of the cytochrome *c* oxidase resulted in an extraordinary longevity of this fungus (Dufour et al. 2000). There are several lines of evidence that beside mitochondria also chloroplasts and peroxisomes trigger leaf senescence. For peroxisomes a ROS-mediated function in leaf senescence has been described (del Río et al. 1998). Tobacco deficient in the thylacoid Ndh complex showed a delay in leaf senescence. It was discussed that the senescence delay was achieved by lower ROS production (Zapater et al., 2005).

In different Arabidopsis mutants a tight correlation between extended longevity and tolerance against oxidative stress has been observed (Kurepa et al., 1998). The most extended longevity mutant of this collection which also showed the highest tolerance against paraquat treatment was *gigantea3*. GIGANTEA acts in blue light signaling and has biochemically separable roles in circadian clock and flowering time regulation (Martin-Tryon et al., 2007). However, the link between this nuclear localized protein and resistance to oxidative stress is still unclear. CATALASE2 (CAT2) and CATALASE3 (CAT3) enzymes, which are expressed under the control of the circadian clock, might be good candidates. They exhibit a higher activity in the *gigantea3* mutant which might be responsible for the elevated oxidative stress tolerance (Zentgraf & Hemleben, 2007). In contrast, the delayed leaf senescence mutants of Arabidopsis *ore1, ore3,* and *ore9* also exhibit increased tolerance to various types of oxidative stress but the activities of antioxidant enzymes were similar or lower in the mutants, as compared to wild type providing evidence that oxidative stress tolerance is also genetically linked to control of leaf longevity in plants (Woo et al., 2004).

In addition, the expression of many SAGs is enhanced by increased levels of reactive oxygen species (Miller et al., 1999; Navabpour et al., 2003) indicating that elevated levels of ROS might be used as a signal to promote senescence. In Arabidopsis the coordinate regulation of the hydrogen peroxide scavenging enzymes catalase (CAT) and ascorbate peroxidase (APX) leads to a defined increase of hydrogen peroxide content during bolting time (Ye et al., 2000; Zimmermann et al., 2006). Removing the bolt and thereby delaying the decrease in APX activity led to a delay in chlorophyll degradation and senescence (Ye et al., 2000). Since APX enzyme activity appears to be regulated on the posttranscriptional level (Panchuk et al., 2005; Zimmermann et al., 2006) and appears to be inhibited by hydrogen peroxide itself in this developmental stage, the initial event to create the hydrogen peroxide peak during bolting time at the onset of senescence is the transcriptional down-regulation of *CAT2*. The transcription factor responsible for this down-regulation was isolated by a yeast-one hybrid screen and turned out to be a member of the bZIP transcription factor family, namely GBF1. If *GBF1* is knocked out by a T-DNA insertion, the down-regulation of *CAT2* during bolting time is abolished, the hydrogen peroxide peak during bolting time disappears and senescence is delayed (Smykowski et al., 2010). This hydrogen peroxide peak is discussed to trigger senescence induction by activating the systemic expression of the senescence-related transcription factors e.g. *WRKY53* (Miao et al., 2004).

In order to understand the correlation between mitochondrial ROS production and senescence in *Arabidopsis thaliana*, we treated cell cultures and whole Arabidopsis plants with antimycin A, an inhibitor of cytochrom *c* oxidase, and measured hydrogen peroxide production and senescence parameters. In addition, two different genes encoding the peroxisomal enzyme catalase have been knocked-out and the single knock-out plants *cat2* and *cat3* have been crossed to produce double knock-out plants *cat2/3*. In these plants also the consequences on hydrogen peroxide levels and leaf senescence were analysed.

## **2. Results and discussion**

32 Senescence

(McIntosh, 1994; Moore et al., 2002). Photo-oxidative stress of chloroplasts is also involved in *AOX* up-regulation (Yoshida et al. 2008). Moreover, ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV (Bartoli et al., 2000) and leaves of the *AOX*-overexpressing lines accumulate more ascorbic acid than wild-type leaves (Bartoli et al., 2006). A lack of AOX can lead to an up-regulation of transcripts of the antioxidant defense system at low temperature (Watanabe et al, 2008). Therefore, it is likely

In addition, it is proposed that AOX also has important functions outside the mitochondria (Arnholdt-Schmitt et al., 2006; Clifton et al., 2006; Van Aken et al., 2009). Furthermore, a beneficial role for AOX in illuminated leaves has been suggested and AOX-deficient *aox1a* mutant showed a lowered operating efficiency of photosystem II and an enhanced activity of cyclic electron transport around photosystem I (CET-PSI) at high irradiance (Yoshida et al., 2011). However, in most cases, transgenic plants with altered levels of AOX exhibited no obvious variation in plant growth phenotype (Vanlerberghe et al., 2009), implying that AOX does not severely affect photosynthetic carbon gain and biomass productivity**.** In addition**,** 

There is some evidence that alternative respiration is correlated with senescence and longevity. Aging potato slides showed a decline in the capacity of cytochrome *c* dependent respiration whereas the alternative respiration as well as the protein content of AOX increased (Hiser & McIntosh, 1990). Expression of *AOX1a* of Arabidopsis is highest in rosette leaves at the onset of senescence (https:/www.genevestigator.com). Interestingly, the inactivation of subunit V of the cytochrome *c* oxidase complex in the fungus *Podospora anserina* led to the exclusive use of the alternative respiration pathway and to a decline in ROS formation in these mutants. This inactivation of the cytochrome *c* oxidase resulted in an extraordinary longevity of this fungus (Dufour et al. 2000). There are several lines of evidence that beside mitochondria also chloroplasts and peroxisomes trigger leaf senescence. For peroxisomes a ROS-mediated function in leaf senescence has been described (del Río et al. 1998). Tobacco deficient in the thylacoid Ndh complex showed a delay in leaf senescence. It was discussed that the

In different Arabidopsis mutants a tight correlation between extended longevity and tolerance against oxidative stress has been observed (Kurepa et al., 1998). The most extended longevity mutant of this collection which also showed the highest tolerance against paraquat treatment was *gigantea3*. GIGANTEA acts in blue light signaling and has biochemically separable roles in circadian clock and flowering time regulation (Martin-Tryon et al., 2007). However, the link between this nuclear localized protein and resistance to oxidative stress is still unclear. CATALASE2 (CAT2) and CATALASE3 (CAT3) enzymes, which are expressed under the control of the circadian clock, might be good candidates. They exhibit a higher activity in the *gigantea3* mutant which might be responsible for the elevated oxidative stress tolerance (Zentgraf & Hemleben, 2007). In contrast, the delayed leaf senescence mutants of Arabidopsis *ore1, ore3,* and *ore9* also exhibit increased tolerance to various types of oxidative stress but the activities of antioxidant enzymes were similar or lower in the mutants, as compared to wild type providing evidence that oxidative stress tolerance is also genetically linked to control of leaf longevity in plants (Woo et al., 2004).

In addition, the expression of many SAGs is enhanced by increased levels of reactive oxygen species (Miller et al., 1999; Navabpour et al., 2003) indicating that elevated levels of ROS

AOX also has an effect on the control of NO levels in plant cells (Wulff et al., 2009).

senescence delay was achieved by lower ROS production (Zapater et al., 2005).

that AOX is an important component in antioxidant defense mechanisms.

## **2.1 Changes in mitochondrial hydrogen peroxide production**

Dufour and others (2000) characterized an almost immortal mutant of the fungus *Podospora anserina* carrying a mutation in the gene encoding subunit V of the cytochrom *c* oxidase complex. These mutants exclusively used the alternative respiration pathway thus clearly leading to a lower content of reactive oxygen species than in normal growing fungi. In Arabidopsis resistance to oxidative stress and longevity are also tightly correlated (Kurepa et al., 1998; Woo et al., 2004). Therefore, we wanted to analyse Arabidopsis plants and cells with increased alternative respiration for mitochondrial ROS production and a delay in senescence.

## **2.1.1 Antimycin A treatment of cell cultures and whole plants**

The alternative respiration in plants can be induced by application of antimycin A (Vanlerberghe & McIntosh, 1992), which was isolated from *Streptomyces* sp. and inhibits specifically the electron transport between cytochrome *b* and *c1*. To investigate the influence of antimycin A on the production of ROS, we analysed antimycin A treated *Arabidopsis thaliana* cell cultures for their hydrogen peroxide contents. Two hours after treatment with 5 µM antimycin A or 0.02 % ethanol as control the H2O2 concentration slightly increased, whereas further incubation clearly lowered H2O2 content in antimycin A treated cells in comparison to control cells (Fig. 1 A). The transient increase in H2O2 levels might be a result of the inhibition of cytochrome *c* oxidase as it was already shown by Maxwell and

Role of Intracellular Hydrogen Peroxide as Signalling Molecule for Plant Senescence 35

Since we were interested in analyzing the induction of senescence in whole Arabidopsis plants, we watered plants of different developmental stages with antimycin A and measured the hydrogen peroxide content 24 h after the treatment. In all developmental stages the hydrogen peroxide content was significantly lower in leaves of antimycin A treated plants (Fig. 1 C) indicating that in all developmental stages AOX and alternative

In order to elucidate the long term effects of alternative respiration on plant development and senescence, soil grown Arabidopsis plants were watered over a time period of five weeks with 10 ml 20 µmol antimycin A solution every second day beginning with 5-weekold plants. Control plants were treated with 0.8 % ethanol in which antimycin A was dissolved. Since it was possible to reduce the hydrogen peroxide levels by the induction of the alternative pathways in all developmental stages, we assume that these plants grew under conditions favouring the alternative respiration from week 5 on. We have chosen this experimental design in order to guarantee that plant growth and development is not impaired in early stages by the lack of a functional cytochrome *c* pathway and the ATP it generates. Therefore, we did not use cytochrome *c* oxidase knock-out mutants, which

appear to be impaired in growth and development from early on (data not shown).

The hydrogen peroxide content of antimycin A watered and control plants was measured weekly and the H2O2 level at the beginning of the experiment was set as 100 % (Fig. 2A). H2O2 concentrations of the control plants exhibit a peak in 7-week-old plants during the time of bolting and an increase in late stages of development as it was already shown before

In contrast, the antimycin A treated plants showed a slight decrease up to 8 weeks and recovered in older stages to the starting level (Fig. 2A). This coincides with the results of Dufour and coworkers (2000) for the fungus *Podospora anserine*, where long term activated alternative respiration led to lower hydrogen peroxide contents and strongly increased longevity. However, in Arabidopsis no obvious differences in the development and the progression of senescence could be detected phenotypically in antimycin A treated plants (Fig. 2B). In contrast, a transgenic Arabidopsis line overexpressing the senescence-associated transcription factor *WRKY53* exhibited an accelerated senescence phenotype (Fig. 2B; Miao et al., 2004). In accordance with the phenotype, chlorophyll and total protein content differed only slightly between antimycin A and ethanol treated plants, but were reduced earlier in 35S:*WRKY53* plants (Fig. 2C). Northern blot analyses revealed that the senescencespecific cystein protease gene *SAG12* was induced earlier and stronger in the antimycin A treated plants (Fig. 2D). This implies that even though less reactive oxygen species are produced in plants with favoured alternative respiration, development and senescence are not impaired or even slightly accelerated. Maxwell et al. (2002) presented evidence that, besides AOX, different senescence associated genes of tobacco (e.g. ACC, GST and Cystein protease precursor) can rapidly be induced by antimycin A treatment and this rapid induction can be prevented by ROS scavengers (Maxwell et al., 2002). In addition, overexpression of AOX in tobacco culture cells led to a decline in ROS concentration and a reduced expression of antioxidative enzymes, like superoxide dismutase or glutathione

respiration was induced to reduce mitochondrial ROS production.

**2.1.2 Long term treatment of plants with antimycin A** 

(Miao et al. 2004; Zimmermann et al. 2006).

peroxidase (Maxwell et al., 1999).

coworkers (1999) for tobacco cells. Here an initial hydrogen peroxide production after antimycin A treatment could be localized almost exclusively to the mitochondria using laser scanning microscopy of H2DCF-DA and mitotracker double-labelled cells. Dot blot analyses of 10 µg total RNA isolated from Arabidopsis culture cells and subsequent hybridization revealed that alternative oxidase 1a (AOX 1a) was induced by antimycin A as well as by hydrogen peroxide treatment (Fig. 1 B). This was also observed in tobacco cells, where antimycin A led to a more efficient alternative respiration capacity (Maxwell et al., 2002) and subsequently to a reduced mitochondrial ROS formation (Maxwell et al., 1999).

**A)** *Arabidopsis thaliana* cell cultures were treated with 5 µM antimycin A or 0.02 % ethanol as control and were analysed for their hydrogen peroxide content. The 0 h value was referred to as 100%. The error bars indicate the standard deviation of 4 independent experiments. **B)** Hybridization of 10µg of total RNA isolated from antimycin A or H2O2 treated culture cells dotted on a nylon filter with an *AOX1a* specific probe. **C)** *Arabidopsis thaliana* plants of different developmental stages were watered with 10 ml of a 20 µmol antimycin A solution whereas control plants were treated with 0.8 % ethanol and were analysed for their hydrogen peroxide content after 24 hours. The values of ethanol treated plants were referred to as 100%. The error bars indicate the standard deviation of 4 independent experiments.

Since we were interested in analyzing the induction of senescence in whole Arabidopsis plants, we watered plants of different developmental stages with antimycin A and measured the hydrogen peroxide content 24 h after the treatment. In all developmental stages the hydrogen peroxide content was significantly lower in leaves of antimycin A treated plants (Fig. 1 C) indicating that in all developmental stages AOX and alternative respiration was induced to reduce mitochondrial ROS production.

## **2.1.2 Long term treatment of plants with antimycin A**

34 Senescence

coworkers (1999) for tobacco cells. Here an initial hydrogen peroxide production after antimycin A treatment could be localized almost exclusively to the mitochondria using laser scanning microscopy of H2DCF-DA and mitotracker double-labelled cells. Dot blot analyses of 10 µg total RNA isolated from Arabidopsis culture cells and subsequent hybridization revealed that alternative oxidase 1a (AOX 1a) was induced by antimycin A as well as by hydrogen peroxide treatment (Fig. 1 B). This was also observed in tobacco cells, where antimycin A led to a more efficient alternative respiration capacity (Maxwell et al., 2002) and

subsequently to a reduced mitochondrial ROS formation (Maxwell et al., 1999).

Fig. 1. *Short term treatment of cell cultures and whole plants with antimycin A*.

**A)** *Arabidopsis thaliana* cell cultures were treated with 5 µM antimycin A or 0.02 % ethanol as control and were analysed for their hydrogen peroxide content. The 0 h value was referred to as 100%. The error bars indicate the standard deviation of 4 independent experiments. **B)** Hybridization of 10µg of total RNA isolated from antimycin A or H2O2 treated culture cells dotted on a nylon filter with an *AOX1a* specific probe. **C)** *Arabidopsis thaliana* plants of different developmental stages were watered with 10 ml of a 20 µmol antimycin A solution

hydrogen peroxide content after 24 hours. The values of ethanol treated plants were referred to as 100%. The error bars indicate the standard deviation of 4 independent experiments.

whereas control plants were treated with 0.8 % ethanol and were analysed for their

In order to elucidate the long term effects of alternative respiration on plant development and senescence, soil grown Arabidopsis plants were watered over a time period of five weeks with 10 ml 20 µmol antimycin A solution every second day beginning with 5-weekold plants. Control plants were treated with 0.8 % ethanol in which antimycin A was dissolved. Since it was possible to reduce the hydrogen peroxide levels by the induction of the alternative pathways in all developmental stages, we assume that these plants grew under conditions favouring the alternative respiration from week 5 on. We have chosen this experimental design in order to guarantee that plant growth and development is not impaired in early stages by the lack of a functional cytochrome *c* pathway and the ATP it generates. Therefore, we did not use cytochrome *c* oxidase knock-out mutants, which appear to be impaired in growth and development from early on (data not shown).

The hydrogen peroxide content of antimycin A watered and control plants was measured weekly and the H2O2 level at the beginning of the experiment was set as 100 % (Fig. 2A). H2O2 concentrations of the control plants exhibit a peak in 7-week-old plants during the time of bolting and an increase in late stages of development as it was already shown before (Miao et al. 2004; Zimmermann et al. 2006).

In contrast, the antimycin A treated plants showed a slight decrease up to 8 weeks and recovered in older stages to the starting level (Fig. 2A). This coincides with the results of Dufour and coworkers (2000) for the fungus *Podospora anserine*, where long term activated alternative respiration led to lower hydrogen peroxide contents and strongly increased longevity. However, in Arabidopsis no obvious differences in the development and the progression of senescence could be detected phenotypically in antimycin A treated plants (Fig. 2B). In contrast, a transgenic Arabidopsis line overexpressing the senescence-associated transcription factor *WRKY53* exhibited an accelerated senescence phenotype (Fig. 2B; Miao et al., 2004). In accordance with the phenotype, chlorophyll and total protein content differed only slightly between antimycin A and ethanol treated plants, but were reduced earlier in 35S:*WRKY53* plants (Fig. 2C). Northern blot analyses revealed that the senescencespecific cystein protease gene *SAG12* was induced earlier and stronger in the antimycin A treated plants (Fig. 2D). This implies that even though less reactive oxygen species are produced in plants with favoured alternative respiration, development and senescence are not impaired or even slightly accelerated. Maxwell et al. (2002) presented evidence that, besides AOX, different senescence associated genes of tobacco (e.g. ACC, GST and Cystein protease precursor) can rapidly be induced by antimycin A treatment and this rapid induction can be prevented by ROS scavengers (Maxwell et al., 2002). In addition, overexpression of AOX in tobacco culture cells led to a decline in ROS concentration and a reduced expression of antioxidative enzymes, like superoxide dismutase or glutathione peroxidase (Maxwell et al., 1999).

Role of Intracellular Hydrogen Peroxide as Signalling Molecule for Plant Senescence 37

were not altered. This indicates that in Arabidopsis the lower production of ROS does not lead to compensatory reduction of oxidative stress enzymes. A senescence phenotype was

The analysis of transgenic plants is helpful to gain more information about the function of a gene. For this reason, plants expressing the genes *AOX1a* under the constitutive 35S promoter were generated. This isoform was selected since it is strongly expressed in leaves. Plants of the T2 generation of these overexpressing lines were tested for *AOX1a* expression and three lines were obtained which overexpressed the transgene about 20-fold. The H2O2 content of these lines was analysed and a clear reduction in the hydrogen peroxide content in the transgenic lines could be measured (Fig.3B). Again, no obvious senescence phenotype could be detected (Fig. 3A). If at all, a slight acceleration of leaf senescence can be observed in the 35S:*AOX1a* lines. Fiorani et al. (2005) could observe a phenotype in 35S:*AOX1a* lines under low temperature conditions (12°C) with increased leaf area and larger rosettes. This could not be observed in our 35S:*AOX1a* lines under normal growth conditions. However, the cytochrom *c* dependent respiration is still functional in these plants probably masking

Millenaar and Lambers (2003) describe that there is no clear positive correlation between the concentration of AOX protein and its activity *in vivo*, since an increase in protein formation does not change pyruvate concentration and the reduction state of ubiquinone, which are necessary for the activation of the AOX protein. For example, tobacco leaves infected with tobacco mosaic virus showed an increased AOX protein level but no change in activity of the alternative respiration (Lennon et al., 1997). In the transgenic plants overexpressing AOX, the capacity of the alternative respiration pathway appears to be elevated, but this does not necessarily reflect its activation. In the same line of evidence neither overexpression nor inactivation of AOX caused a change in ROS formation in the fungus *Podospora anserina* (Lorin et al., 2001). There was no effect on lifespan or senescence in the transgenic fungi either. However, in our transgenic lines the ROS production is clearly reduced indicating an activation of the alternative respiration pathway but nevertheless no effect on senescence

Overexpression of *AOX* in tobacco culture cells leads to a decline in ROS concentration and a reduced expression of other antioxidative enzymes, like superoxide dismutase or glutathione peroxidase (Maxwell et al., 1999) whereas transgenic tobacco culture cells carrying an antisense construct for *AOX* show an increased ROS formation and an elevated transcript abundance of catalase (Maxwell et al., 1999). This would suggest that the plants would be either more sensitive or more resistant to oxidative stress. In contrast, in transgenic Arabidopsis lines either overexpressing *AOX* or an *AOX* antisense construct transcript levels of the antioxidative enzymes MnSOD, organellar APX, cytosolic and organellar glutathione reductase and peroxiredoxins were not altered (Umbach et al., 2005). In consistence with these findings, no altered resistance against oxidative stress could be observed in the transgenic 35S:*AOX1a* transgenic plants, which we germinated on MS plates and applied oxidative stress by spraying the seedlings with hydrogen

also not observed in these lines.

the effect of increased levels of AOX.

could be observed.

peroxide (Fig. 3C).

**2.1.3 Transgenic plants overexpressing** *AOX1a*

#### Fig. 2. *Long term treatment of whole plants with antimycin A*.

**A)** *Arabidopsis thaliana* plants were watered with 10 ml of a 20 µmol antimycin A solution every second day over a time period of five weeks beginning with 5-week-old plants. Control plants were treated with 0.8 % of ethanol. These plants were analysed for their hydrogen peroxide content every week. The values of the 5-week-old plants were referred to as 100%. The error bars indicate the standard deviation of 3 independent experiments. **B)** Phenotypic analyses of antimycin A or ethanol treated 8-week-old wildtype plants and ethanol treated transgenic *WRKY53* overexpressing line (35S:*WRKY53*). Whole plants are shown upside down to visualize older leaves of the rosette. In addition, the leaves were sorted according to their age using a specific colour code. **C)** Chlorophyll (left) and total protein (right) were measured in ethanol treated wildtype plants (Col-0), antimycin A treated wildtype plants and ethanol treated *WRKY53* overexpessing plants (35S:*WRKY53*). The values of 5-week-old plants were referred to as 100%. The error bars indicate the standard deviation of 3 independent experiments. **D)** Northern blot analyses of 15 µg of total RNA isolated from antimycin A (A) or ethanol treated (E) plants. The nylon filters were hybridized with a *SAG12* specific probe. Rehybridization with a 25S rRNA probe was used as loading control.

Transgenic tobacco culture cells carrying an antisense construct for *AOX* show an increased ROS formation and an elevated transcript abundance of catalase (Maxwell et al., 1999). In contrast, Umbach and coworkers (2005) observed that in *AOX* overexpressing or *AOX* antisense transgenic Arabidopsis lines transcript levels of the antioxidative enzymes MnSOD, organellar APX, cytosolic and organellar glutathione reductase and peroxiredoxins were not altered. This indicates that in Arabidopsis the lower production of ROS does not lead to compensatory reduction of oxidative stress enzymes. A senescence phenotype was also not observed in these lines.

#### **2.1.3 Transgenic plants overexpressing** *AOX1a*

36 Senescence

Fig. 2. *Long term treatment of whole plants with antimycin A*.

as loading control.

**A)** *Arabidopsis thaliana* plants were watered with 10 ml of a 20 µmol antimycin A solution every second day over a time period of five weeks beginning with 5-week-old plants. Control plants were treated with 0.8 % of ethanol. These plants were analysed for their hydrogen peroxide content every week. The values of the 5-week-old plants were referred to as 100%. The error bars indicate the standard deviation of 3 independent experiments. **B)** Phenotypic analyses of antimycin A or ethanol treated 8-week-old wildtype plants and ethanol treated transgenic *WRKY53* overexpressing line (35S:*WRKY53*). Whole plants are shown upside down to visualize older leaves of the rosette. In addition, the leaves were sorted according to their age using a specific colour code. **C)** Chlorophyll (left) and total protein (right) were measured in ethanol treated wildtype plants (Col-0), antimycin A treated wildtype plants and ethanol treated *WRKY53* overexpessing plants (35S:*WRKY53*). The values of 5-week-old plants were referred to as 100%. The error bars indicate the standard deviation of 3 independent experiments. **D)** Northern blot analyses of 15 µg of total RNA isolated from antimycin A (A) or ethanol treated (E) plants. The nylon filters were hybridized with a *SAG12* specific probe. Rehybridization with a 25S rRNA probe was used

Transgenic tobacco culture cells carrying an antisense construct for *AOX* show an increased ROS formation and an elevated transcript abundance of catalase (Maxwell et al., 1999). In contrast, Umbach and coworkers (2005) observed that in *AOX* overexpressing or *AOX* antisense transgenic Arabidopsis lines transcript levels of the antioxidative enzymes MnSOD, organellar APX, cytosolic and organellar glutathione reductase and peroxiredoxins The analysis of transgenic plants is helpful to gain more information about the function of a gene. For this reason, plants expressing the genes *AOX1a* under the constitutive 35S promoter were generated. This isoform was selected since it is strongly expressed in leaves. Plants of the T2 generation of these overexpressing lines were tested for *AOX1a* expression and three lines were obtained which overexpressed the transgene about 20-fold. The H2O2 content of these lines was analysed and a clear reduction in the hydrogen peroxide content in the transgenic lines could be measured (Fig.3B). Again, no obvious senescence phenotype could be detected (Fig. 3A). If at all, a slight acceleration of leaf senescence can be observed in the 35S:*AOX1a* lines. Fiorani et al. (2005) could observe a phenotype in 35S:*AOX1a* lines under low temperature conditions (12°C) with increased leaf area and larger rosettes. This could not be observed in our 35S:*AOX1a* lines under normal growth conditions. However, the cytochrom *c* dependent respiration is still functional in these plants probably masking the effect of increased levels of AOX.

Millenaar and Lambers (2003) describe that there is no clear positive correlation between the concentration of AOX protein and its activity *in vivo*, since an increase in protein formation does not change pyruvate concentration and the reduction state of ubiquinone, which are necessary for the activation of the AOX protein. For example, tobacco leaves infected with tobacco mosaic virus showed an increased AOX protein level but no change in activity of the alternative respiration (Lennon et al., 1997). In the transgenic plants overexpressing AOX, the capacity of the alternative respiration pathway appears to be elevated, but this does not necessarily reflect its activation. In the same line of evidence neither overexpression nor inactivation of AOX caused a change in ROS formation in the fungus *Podospora anserina* (Lorin et al., 2001). There was no effect on lifespan or senescence in the transgenic fungi either. However, in our transgenic lines the ROS production is clearly reduced indicating an activation of the alternative respiration pathway but nevertheless no effect on senescence could be observed.

Overexpression of *AOX* in tobacco culture cells leads to a decline in ROS concentration and a reduced expression of other antioxidative enzymes, like superoxide dismutase or glutathione peroxidase (Maxwell et al., 1999) whereas transgenic tobacco culture cells carrying an antisense construct for *AOX* show an increased ROS formation and an elevated transcript abundance of catalase (Maxwell et al., 1999). This would suggest that the plants would be either more sensitive or more resistant to oxidative stress. In contrast, in transgenic Arabidopsis lines either overexpressing *AOX* or an *AOX* antisense construct transcript levels of the antioxidative enzymes MnSOD, organellar APX, cytosolic and organellar glutathione reductase and peroxiredoxins were not altered (Umbach et al., 2005). In consistence with these findings, no altered resistance against oxidative stress could be observed in the transgenic 35S:*AOX1a* transgenic plants, which we germinated on MS plates and applied oxidative stress by spraying the seedlings with hydrogen peroxide (Fig. 3C).

Role of Intracellular Hydrogen Peroxide as Signalling Molecule for Plant Senescence 39

observed with the highest transcript abundance in old plants. In young, up to 7-week-old plants, no expression could be detected by Northern blot analyses (Fig. 4A). This coincides with genevestigator data and with the *AOX* expression in different stages of the leaf development in potatoes, where an increase in AOX protein from young to mature leaves could be observed (Svensson & Rasmusson, 2001; https:/www.genevestigator.com). Furthermore, there is a *de novo* synthesis of alternative oxidase in aging potato slides (Hiser & McIntosh, 1990). Our *in silico* analysis of about 1500 bp upstream the coding region of the *AOX1a* gene revealed, amongst others, several W-box core elements and one sequence for a circadian element. The W-boxes indicate a regulation by WRKY transcription factors which are involved in senescence or pathogen dependent regulation (Eulgem et al., 2000; Miao et al., 2004) whereas the circadian element points out a clock dependent regulation. Based on these results, we used 8.5-week-old plants to harvest leaf material every three hours over 27 h. A circadian regulation of *AOX1a* could be detected with the maximum of expression in the early morning hours with the beginning of illumination (Fig. 4B). This corresponds to

Northern blot analyses of 15 µg of total RNA isolated from plants of **A)** 5-week-old to 9 week-old plants and **B)** 8-week-old plants at different day times. The nylon filters were hybridized with an *AOX1a* specific probe. Equal loading was controlled by Toluidin blue

Peroxisomes are organelles encircled by only a single membrane layer embedding an extensive oxidative metabolism. These organelles are found in all eukaryotic organisms. In plants, peroxisomes participate in many physiological processes like seed germination, leaf senescence, fruit maturation, response to abiotic and biotic stress, photomorphogenesis, biosynthesis of the plant hormones jasmonic acid and auxin, and in cell signaling by reactive oxygen and nitrogen species. A specific feature of peroxisomes is their dynamic metabolism meaning that the enzymatic constitution of peroxisomes is adjusted to the organism, cell or tissue-type, and also to a variety of environmental conditions (Palma et al., 2009). One important source for ROS formation, especially for H2O2, is photorespiration. During CO2 fixation, ribulose-1,5-bisphosphate-carboxylase (RubisCO) can use CO2 to carboxylate ribulose-1,5-bisphosphate but also molecular oxygen to oxygenate ribulose-1,5-bisphosphate forming glycolate. The glycolate is then transported from the chloroplasts into the peroxisomes where it is oxidized generating H2O2 as a by-product. Peroxisomes and ROS generated in these organelles were shown to play a central role in natural and dark induced senescence in pea (del Rio et al., 1998) and appear to play an important role as a supplier of

the expression of *AOX* in tobacco (Dutilleul et al., 2003).

Fig. 4. *Senescence-associated expression of AOX1a* 

**2.2 Changes in peroxisomal hydrogen peroxide production** 

staining of the membranes.

#### Fig. 3. *Transgenic plants overexperessing AOX1a*

**A)** Phenotypic analyses of wildtype (Col-0) and 35S:*AOX1a* transgenic plants. Whole plants are shown upside down to visualize older leaves of the rosette. In addition, the leaves were sorted according to their age using a specific colour code. **B)** 4-6 wildtype (Col-0) and 35S:*AOX1a* transgenic plants were pooled and analysed for their hydrogen peroxide content. The values of the wild type plants were referred to as 100%. The error bars indicate the standard deviation of 2 independently collected plant pools. **C)** Phenotypic analyses of hydrogen peroxide treated seedlings of wild type (Col-0) and 35S:*AOX1a* transgenic plants.

#### **2.1.4 Senescence-associated and circadian expression of** *AOX1a*

The family of alternative oxidases comprises five genes with an organ specific expression (Saisho et al., 1997; https://www.genevestigator.com). In general, *AOX* is expressed only at a very low level under normal conditions. By using leaf material of plants of different age harvested in the morning hours, a senescence dependent expression of *AOX1a* could be

**A)** Phenotypic analyses of wildtype (Col-0) and 35S:*AOX1a* transgenic plants. Whole plants are shown upside down to visualize older leaves of the rosette. In addition, the leaves were sorted according to their age using a specific colour code. **B)** 4-6 wildtype (Col-0) and 35S:*AOX1a* transgenic plants were pooled and analysed for their hydrogen peroxide content. The values of the wild type plants were referred to as 100%. The error bars indicate the standard deviation of 2 independently collected plant pools. **C)** Phenotypic analyses of hydrogen peroxide treated seedlings of wild type (Col-0) and 35S:*AOX1a* transgenic plants.

The family of alternative oxidases comprises five genes with an organ specific expression (Saisho et al., 1997; https://www.genevestigator.com). In general, *AOX* is expressed only at a very low level under normal conditions. By using leaf material of plants of different age harvested in the morning hours, a senescence dependent expression of *AOX1a* could be

**2.1.4 Senescence-associated and circadian expression of** *AOX1a*

Fig. 3. *Transgenic plants overexperessing AOX1a* 

observed with the highest transcript abundance in old plants. In young, up to 7-week-old plants, no expression could be detected by Northern blot analyses (Fig. 4A). This coincides with genevestigator data and with the *AOX* expression in different stages of the leaf development in potatoes, where an increase in AOX protein from young to mature leaves could be observed (Svensson & Rasmusson, 2001; https:/www.genevestigator.com). Furthermore, there is a *de novo* synthesis of alternative oxidase in aging potato slides (Hiser & McIntosh, 1990). Our *in silico* analysis of about 1500 bp upstream the coding region of the *AOX1a* gene revealed, amongst others, several W-box core elements and one sequence for a circadian element. The W-boxes indicate a regulation by WRKY transcription factors which are involved in senescence or pathogen dependent regulation (Eulgem et al., 2000; Miao et al., 2004) whereas the circadian element points out a clock dependent regulation. Based on these results, we used 8.5-week-old plants to harvest leaf material every three hours over 27 h. A circadian regulation of *AOX1a* could be detected with the maximum of expression in the early morning hours with the beginning of illumination (Fig. 4B). This corresponds to the expression of *AOX* in tobacco (Dutilleul et al., 2003).

#### Fig. 4. *Senescence-associated expression of AOX1a*

Northern blot analyses of 15 µg of total RNA isolated from plants of **A)** 5-week-old to 9 week-old plants and **B)** 8-week-old plants at different day times. The nylon filters were hybridized with an *AOX1a* specific probe. Equal loading was controlled by Toluidin blue staining of the membranes.

#### **2.2 Changes in peroxisomal hydrogen peroxide production**

Peroxisomes are organelles encircled by only a single membrane layer embedding an extensive oxidative metabolism. These organelles are found in all eukaryotic organisms. In plants, peroxisomes participate in many physiological processes like seed germination, leaf senescence, fruit maturation, response to abiotic and biotic stress, photomorphogenesis, biosynthesis of the plant hormones jasmonic acid and auxin, and in cell signaling by reactive oxygen and nitrogen species. A specific feature of peroxisomes is their dynamic metabolism meaning that the enzymatic constitution of peroxisomes is adjusted to the organism, cell or tissue-type, and also to a variety of environmental conditions (Palma et al., 2009). One important source for ROS formation, especially for H2O2, is photorespiration. During CO2 fixation, ribulose-1,5-bisphosphate-carboxylase (RubisCO) can use CO2 to carboxylate ribulose-1,5-bisphosphate but also molecular oxygen to oxygenate ribulose-1,5-bisphosphate forming glycolate. The glycolate is then transported from the chloroplasts into the peroxisomes where it is oxidized generating H2O2 as a by-product. Peroxisomes and ROS generated in these organelles were shown to play a central role in natural and dark induced senescence in pea (del Rio et al., 1998) and appear to play an important role as a supplier of

Role of Intracellular Hydrogen Peroxide as Signalling Molecule for Plant Senescence 41

would be expected that these knock-out plants are more sensitive against all stresses implying an increased ROS production and that they most likely show a senescence phenotype. The lack of peroxisomal catalase CTL-2 in *Caenorhabditis elegans* causes a progeric phenotype whereas the lack of the cytosolic catalase CTL-1 has no effect on nematode aging (Petriv & Rachubinski, 2004). In yeast, catalase T activity but not catalase A activity was necessary to assure longevity under repressing conditions on glucose media. However, under derepressing conditions, on ethanol media, both catalases were required for longevity assurance (Van Zandycke et al., 2002) indicating a correlation between CAT

activity and longevity in animal systems.

Fig. 5. *Phenotypic analyses of catalase mutants* 

**A)** Plant development, **B)** Percantage of phenotypical appearance of the leaves of ten rosettes, **C)** Germination rate of wild type (Col-0), *cat2*, *cat3*, and *cat2/3* mutant plants.

Surprisingly, *cat2* knock-out plants appear to be more or less inconspicuous. They are slightly impaired in germination (Fig. 5C) but once germinated the plants developed relatively normaly (Queval et al., 2007; Fig. 5A). Photoperiod and CO2 levels have a high impact on the phenotypic appearance of the plants and on the ascorbate and glutathione contents and their balances of the oxidized and reduced form, respectively. Under high CO2

signal molecules like NO. (nitric oxide), O2 -, H2O2 and possibly S-nitrosoglutathione (del Rio et al., 1998; 2002; 2003). These signaling molecules can trigger specific gene expression by so far largely unknown signal transduction pathways (Corpas et al., 2001; 2004; del Rio et al., 2002). However, the concentration of these molecules is tightly regulated by a sensitive balance between production and decomposition by different specific scavenging systems. Catalases are the most abundant enzymes in peroxisomes and convert hydrogen peroxide into water and oxygen without the consumption of reducing equivalents. Besides catalases all enzymes of the antioxidant ascorbate-glutathione cycle, also called Foyer-Halliwell-Asada cycle, are present in peroxisomes to detoxify H2O2 through the oxidation of ascorbate and glutathione in an NADPH-dependent manner, thus complementing the action of catalase in peroxisomes. If the mitochondrial and the peroxisomal ascorbate-glutathione cycles are compared during progression of senescence, it can be speculated that peroxisomes may participate longer in the cellular oxidative mechanism of leaf senescence than mitochondria, since mitochondria appear to be affected by oxidative damage earlier than peroxisomes (Jiménez et al. , 1998; del Rio et al., 2003).

Catalases are tetrameric heme containing enzymes and are present in all aerobic organisms. Due to a very high apparent Michaelis constant catalases are not easily saturated with substrate and can act over a wide range of H2O2 concentrations maintaining a controlled intracellular H2O2 concentration. Whereas animals have only one form of catalase, plants have evolved small gene families encoding catalases. The plant catalases can be grouped into three classes depending on their expression and physiological parameters. In *Arabidopsis*, the small catalase gene family has been characterized to consist of three members, the class III catalase *CAT1*, class I catalase *CAT2* and class II catalase *CAT3.* All three Arabidopsis catalases show a senescence-specific alteration in expression and activity (Zimmermann et al., 2006*). CAT2* expression and activity is down-regulated at an early time point when plants are bolting. Subsequently, expression and activity of *CAT3* is upregulated during progression of senescence. In contrast to *CAT2* expression, which is predominantly located in mesophyll cells, *CAT3* expression is mainly expressed in vascular tissue indicating that the vascular system appears to be protected against oxidative stress during senescence to guarantee the transport of nutrients and minerals out of the senescing tissue into developing parts of the plant like e.g. the seeds (Zimmermann et al., 2006). CAT1 expression and activity is very low during plant development and only increases significantly during germination and in very late stages of senescence. Due to this expression pattern, its activity is discussed to be related to fatty acid degradation which takes place when peroxisomes are converted into glyoxisomes.

Especially the transcriptional down-regulation of *CAT2* appears to be involved in the regulation of the onset of senescence. This down-regulation is executed by the bZIP transcription factor GBF1. Insertion of a T-DNA into the *GBF1* gene revealed a loss of *CAT2* down-regulation and resulted in the loss of a hydrogen peroxide increase during bolting time. These *gbf1* mutant plants exhibit a delayed onset of senescence (Smykowski et al., 2010). Consequently, the idea suggests itself that *CAT2* knock-out plants also have a senescence phenotype. Taking into consideration that *CAT2* is expressed not only in leaves but also in roots, stems and flowers contributing substantially to the regulation of intracellular hydrogen peroxide contents and the protection of the cells against ROS in stress situations, the knock-out of this gene would have severe effects on the plants. The loss of such an important enzyme has to be compensated somehow during development but it

et al., 1998; 2002; 2003). These signaling molecules can trigger specific gene expression by so far largely unknown signal transduction pathways (Corpas et al., 2001; 2004; del Rio et al., 2002). However, the concentration of these molecules is tightly regulated by a sensitive balance between production and decomposition by different specific scavenging systems. Catalases are the most abundant enzymes in peroxisomes and convert hydrogen peroxide into water and oxygen without the consumption of reducing equivalents. Besides catalases all enzymes of the antioxidant ascorbate-glutathione cycle, also called Foyer-Halliwell-Asada cycle, are present in peroxisomes to detoxify H2O2 through the oxidation of ascorbate and glutathione in an NADPH-dependent manner, thus complementing the action of catalase in peroxisomes. If the mitochondrial and the peroxisomal ascorbate-glutathione cycles are compared during progression of senescence, it can be speculated that peroxisomes may participate longer in the cellular oxidative mechanism of leaf senescence than mitochondria, since mitochondria appear to be affected by oxidative damage earlier than

Catalases are tetrameric heme containing enzymes and are present in all aerobic organisms. Due to a very high apparent Michaelis constant catalases are not easily saturated with substrate and can act over a wide range of H2O2 concentrations maintaining a controlled intracellular H2O2 concentration. Whereas animals have only one form of catalase, plants have evolved small gene families encoding catalases. The plant catalases can be grouped into three classes depending on their expression and physiological parameters. In *Arabidopsis*, the small catalase gene family has been characterized to consist of three members, the class III catalase *CAT1*, class I catalase *CAT2* and class II catalase *CAT3.* All three Arabidopsis catalases show a senescence-specific alteration in expression and activity (Zimmermann et al., 2006*). CAT2* expression and activity is down-regulated at an early time point when plants are bolting. Subsequently, expression and activity of *CAT3* is upregulated during progression of senescence. In contrast to *CAT2* expression, which is predominantly located in mesophyll cells, *CAT3* expression is mainly expressed in vascular tissue indicating that the vascular system appears to be protected against oxidative stress during senescence to guarantee the transport of nutrients and minerals out of the senescing tissue into developing parts of the plant like e.g. the seeds (Zimmermann et al., 2006). CAT1 expression and activity is very low during plant development and only increases significantly during germination and in very late stages of senescence. Due to this expression pattern, its activity is discussed to be related to fatty acid degradation which

Especially the transcriptional down-regulation of *CAT2* appears to be involved in the regulation of the onset of senescence. This down-regulation is executed by the bZIP transcription factor GBF1. Insertion of a T-DNA into the *GBF1* gene revealed a loss of *CAT2* down-regulation and resulted in the loss of a hydrogen peroxide increase during bolting time. These *gbf1* mutant plants exhibit a delayed onset of senescence (Smykowski et al., 2010). Consequently, the idea suggests itself that *CAT2* knock-out plants also have a senescence phenotype. Taking into consideration that *CAT2* is expressed not only in leaves but also in roots, stems and flowers contributing substantially to the regulation of intracellular hydrogen peroxide contents and the protection of the cells against ROS in stress situations, the knock-out of this gene would have severe effects on the plants. The loss of such an important enzyme has to be compensated somehow during development but it


signal molecules like NO. (nitric oxide), O2

peroxisomes (Jiménez et al. , 1998; del Rio et al., 2003).

takes place when peroxisomes are converted into glyoxisomes.

would be expected that these knock-out plants are more sensitive against all stresses implying an increased ROS production and that they most likely show a senescence phenotype. The lack of peroxisomal catalase CTL-2 in *Caenorhabditis elegans* causes a progeric phenotype whereas the lack of the cytosolic catalase CTL-1 has no effect on nematode aging (Petriv & Rachubinski, 2004). In yeast, catalase T activity but not catalase A activity was necessary to assure longevity under repressing conditions on glucose media. However, under derepressing conditions, on ethanol media, both catalases were required for longevity assurance (Van Zandycke et al., 2002) indicating a correlation between CAT activity and longevity in animal systems.

**A)** Plant development, **B)** Percantage of phenotypical appearance of the leaves of ten rosettes, **C)** Germination rate of wild type (Col-0), *cat2*, *cat3*, and *cat2/3* mutant plants.

Surprisingly, *cat2* knock-out plants appear to be more or less inconspicuous. They are slightly impaired in germination (Fig. 5C) but once germinated the plants developed relatively normaly (Queval et al., 2007; Fig. 5A). Photoperiod and CO2 levels have a high impact on the phenotypic appearance of the plants and on the ascorbate and glutathione contents and their balances of the oxidized and reduced form, respectively. Under high CO2

fails.

**3. Conclusion** 

important for its signalling function.

Role of Intracellular Hydrogen Peroxide as Signalling Molecule for Plant Senescence 43

However, the hydrogen peroxide content between different leaves and plants varied remarkably so that the standard deviation was quite high in the measurements of the mutants. How the CAT2 or CAT3 activity losses were compensated is not yet clear but APX activity appeared to be not elevated (Fig 6B); in contrast, it appeared to be even slightly reduced in the double mutant. Hydrogen peroxide levels in these plants clearly indicated that the loss of the CAT activity must have been compensated. This is consistent with the finding of Rizhky and co-workers (2002), who claimed that there appears to be a sensitive balance between the antioxidant enzymes with compensating mechanisms, since they observed that double antisense plants for CAT or APX are more tolerant to oxidative stress than single antisense plants (Rizhsky et al., 2002). A slight activation of CAT1 can be observed in all our catalase mutants, especially in the *cat3* and *cat2/3* mutant plants. This is also indicated by the heterodimer formation between CAT2 and CAT1 in the *cat3* mutant. However, the activity of this isoform appears to be only low compared to the loss of catalase activity which would be present in wild type plants (Fig. 6B). Remarkably, glutathione levels are increased and shifted towards the more oxidized form in *cat2* plants under long day conditions (Queval et al., 2007). Taken together, the ROS levels appear to be very tightly regulated on many levels with the possibility of compensation if one detoxifying system

Antimycin A treatment leads to the inhibition of the cytochrom *c* dependent electron transport lowering the production of hydrogen peroxide in mitochondria. Conversely, it is assumed that if stress occurs in a cellular compartment and increasing amounts of hydrogen peroxide are formed, these hydrogen peroxide molecules also can pass membranes and can be transported into the cytosol. This signal can then be transduced into the nucleus, where it induces the expression of many genes including *AOX*. As soon as the newly synthesized AOX protein is active, it minimizes the formation of ROS in the mitochondria by preventing the overreduction of the electron transport chain. Therefore, alternative oxidase might be regarded as mechanism to protect the plant from oxidative stress. Even though oxidative stress tolerance and longevity in Arabidopsis are tightly correlated (Kurepa et al. 1998, Woo et al., 2004) and hydrogen peroxide is discussed as signalling molecule to induce leaf senescence in Arabidopsis (Navabpour et al., 2003; Miao et al. 2004; Zimmermann et al., 2006), minimizing hydrogen peroxide production in the mitochondria by long-term antimycin A treatment did not delay senescence. In contrast, if down-regulation of *CAT2* expression and activity is abolished in *gbf1* mutants, the onset of senescence is delayed. On the other hand, if *CAT2* gene expression is prevented from early on in development in *cat2*  T-DNA insertion lines, also no effect on senescence could be observed and hydrogen peroxide contents are not significantly altered. Therefore, we can assume that the intracellular origin but also the developmental time point of the hydrogen peroxide production might have an impact on its signalling function. In addition, the loss of one detoxifying system can be compensated by the cells and there seems to be a very sensitive balance between the different antioxidative protection systems. Remarkably, hydrogen peroxide plays a role in many different signal transduction pathways but how specificity is mediated is still an open question. Compartment-specific hydrogen peroxide fluorescent sensor molecules like roGFP or Hyper will help to clarify whether the intracellular origin of the hydrogen peroxide and changes during specific developmental time points might be

conditions no obvious phenotype could be observed whereas growth under ambient air, which favours photorespiration, led to a lower biomass production of the rosette and an altered leaf shape (Queval et al., 2007; Fig 5). We characterized SALK T-DNA insertion lines for *CAT2* and *CAT3* for homozygous insertion of the T-DNA and crossed the homozygous *cat2* and *cat3* mutants and selected the offsprings for a homozygous double knock-out line *cat2/3*. After separation of leaf protein extracts of these lines on native PAGEs, we could confirm that according to the gene knock-out the activity of the respective isoform disappeared (Fig. 6 A). When we analyzed plant development under long day conditions, leaf or plant senescence does not seem to be impaired (Fig. 5A, B); only leaf shape and biomass production were slightly altered in *cat2 and cat2/3* plants. However, the mutant plant populations did not senesce as homogenously as the wildtype populations. If the hydrogen peroxide content was measured, the profiles appeared to be not much different indicating that a very efficient compensation of the loss of CAT activity has been activated (Fig 6A).

**A)** Catalase activity, **B)** Ascorbate peroxidase activity. **C)** Hydrogen peroxide content of wild type (Col-0), cat2, cat3, and cat2/3 mutant plants. The value of the 4–week-old wildtype plants was referred to as 100%. The error bars indicate the standard deviation of 4 independent experiments.

However, the hydrogen peroxide content between different leaves and plants varied remarkably so that the standard deviation was quite high in the measurements of the mutants. How the CAT2 or CAT3 activity losses were compensated is not yet clear but APX activity appeared to be not elevated (Fig 6B); in contrast, it appeared to be even slightly reduced in the double mutant. Hydrogen peroxide levels in these plants clearly indicated that the loss of the CAT activity must have been compensated. This is consistent with the finding of Rizhky and co-workers (2002), who claimed that there appears to be a sensitive balance between the antioxidant enzymes with compensating mechanisms, since they observed that double antisense plants for CAT or APX are more tolerant to oxidative stress than single antisense plants (Rizhsky et al., 2002). A slight activation of CAT1 can be observed in all our catalase mutants, especially in the *cat3* and *cat2/3* mutant plants. This is

also indicated by the heterodimer formation between CAT2 and CAT1 in the *cat3* mutant. However, the activity of this isoform appears to be only low compared to the loss of catalase activity which would be present in wild type plants (Fig. 6B). Remarkably, glutathione levels are increased and shifted towards the more oxidized form in *cat2* plants under long day conditions (Queval et al., 2007). Taken together, the ROS levels appear to be very tightly regulated on many levels with the possibility of compensation if one detoxifying system fails.

## **3. Conclusion**

42 Senescence

conditions no obvious phenotype could be observed whereas growth under ambient air, which favours photorespiration, led to a lower biomass production of the rosette and an altered leaf shape (Queval et al., 2007; Fig 5). We characterized SALK T-DNA insertion lines for *CAT2* and *CAT3* for homozygous insertion of the T-DNA and crossed the homozygous *cat2* and *cat3* mutants and selected the offsprings for a homozygous double knock-out line *cat2/3*. After separation of leaf protein extracts of these lines on native PAGEs, we could confirm that according to the gene knock-out the activity of the respective isoform disappeared (Fig. 6 A). When we analyzed plant development under long day conditions, leaf or plant senescence does not seem to be impaired (Fig. 5A, B); only leaf shape and biomass production were slightly altered in *cat2 and cat2/3* plants. However, the mutant plant populations did not senesce as homogenously as the wildtype populations. If the hydrogen peroxide content was measured, the profiles appeared to be not much different indicating that a very efficient compensation of the loss of CAT activity has been activated (Fig 6A).

Fig. 6. *Physiological analyses of catalase mutants* 

independent experiments.

**A)** Catalase activity, **B)** Ascorbate peroxidase activity. **C)** Hydrogen peroxide content of wild type (Col-0), cat2, cat3, and cat2/3 mutant plants. The value of the 4–week-old wildtype plants was referred to as 100%. The error bars indicate the standard deviation of 4

Antimycin A treatment leads to the inhibition of the cytochrom *c* dependent electron transport lowering the production of hydrogen peroxide in mitochondria. Conversely, it is assumed that if stress occurs in a cellular compartment and increasing amounts of hydrogen peroxide are formed, these hydrogen peroxide molecules also can pass membranes and can be transported into the cytosol. This signal can then be transduced into the nucleus, where it induces the expression of many genes including *AOX*. As soon as the newly synthesized AOX protein is active, it minimizes the formation of ROS in the mitochondria by preventing the overreduction of the electron transport chain. Therefore, alternative oxidase might be regarded as mechanism to protect the plant from oxidative stress. Even though oxidative stress tolerance and longevity in Arabidopsis are tightly correlated (Kurepa et al. 1998, Woo et al., 2004) and hydrogen peroxide is discussed as signalling molecule to induce leaf senescence in Arabidopsis (Navabpour et al., 2003; Miao et al. 2004; Zimmermann et al., 2006), minimizing hydrogen peroxide production in the mitochondria by long-term antimycin A treatment did not delay senescence. In contrast, if down-regulation of *CAT2* expression and activity is abolished in *gbf1* mutants, the onset of senescence is delayed. On the other hand, if *CAT2* gene expression is prevented from early on in development in *cat2*  T-DNA insertion lines, also no effect on senescence could be observed and hydrogen peroxide contents are not significantly altered. Therefore, we can assume that the intracellular origin but also the developmental time point of the hydrogen peroxide production might have an impact on its signalling function. In addition, the loss of one detoxifying system can be compensated by the cells and there seems to be a very sensitive balance between the different antioxidative protection systems. Remarkably, hydrogen peroxide plays a role in many different signal transduction pathways but how specificity is mediated is still an open question. Compartment-specific hydrogen peroxide fluorescent sensor molecules like roGFP or Hyper will help to clarify whether the intracellular origin of the hydrogen peroxide and changes during specific developmental time points might be important for its signalling function.

Role of Intracellular Hydrogen Peroxide as Signalling Molecule for Plant Senescence 45

were incubated for 2 h in 2 ml reagent mixture containing 50 mM potassium phosphate buffer pH 7.0, 0.05 % guaiacol (Sigma) and horseradish peroxidase (2.5 u/ml, Serva) at room temperature in the dark. Four moles of hydrogen peroxide are required to form 1 mole of tetraguaiacol, which has an extinction coefficient of = 26.6 cm-1mM-1 at 470 nm. The

Leaf discs were homogenized in 0.2 ml 25 mM potassium phosphate buffer, pH 7.0, containing 2 mM EDTA. Subsequently, 0.8 ml acetone was added, and the samples were shaken vigorously for 1 h at room temperature. After centrifugation at 14000 g for 30 min at room temperature, the total chlorophyll content of the supernatant was measured and calculated following the method described by Arnon (1949). To determine total protein content, leaf discs were ground in 100 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA at 4°C. After centrifugation at 14000 g for 30 min at 4°C, the supernatant was directly used for protein quantification according to the method of Bradford (1976) using

For analyses of APX isozymes, crude protein extracts were separated on 10% native polyacrylamide gels (0.375 M Tris-HCl, pH 8.8, as gel buffer) with a 5% stacking gel (0.125 M Tris-HCl, pH 6.8, as gel buffer) for 16 h (120V) at 4°C using 2 mM ascorbate, 250 mM glycine, and 25 mM Tris-HCl, pH 8.3, as electrophoresis buffer. After electrophoresis, the gels were soaked in 50 mM potassium phosphate buffer, pH 7.0, containing 2 mM ascorbate for 10 min (3x) and, subsequently, in 50 mM potassium phosphate buffer, pH 7.0, containing 4 mM ascorbate, and 1 mM H2O2 for 20 min. After rinsing in water, the gels were stained in 50 mM potassium phosphate buffer, pH 7.8, containing 14 mM TEMED (*N,N,N´,N´* tetramethylethylenediamine) and 2.45 mM NBT (nitro blue tetrazolium) for 10-30 min. For the analyses of CAT isozymes the protein extracts were separated on 7.5% native polyacrylamide gels (0.375 M Tris-HCl, pH 8.8, as gel buffer) with a 3.5% stacking gel (0.125 M Tris-HCl, pH 6.8, as gel buffer) for 16 h (70-80V) at 18°C using 250 mM glycine and 25 mM Tris-HCl, pH 8.3, as electrophoresis buffer. Subsequently, the gels were stained for the activity of catalases as follows: The gels were soaked in 0.01% of hydrogen peroxide solution for 5 min, washed twice in water and incubated for 5 min in 1% FeCl3 and 1% K3[Fe(CN)6].

We thank the Nottingham Arabidopsis Stock Centre (NASC) for providing seeds of the T-DNA insertion lines for *CAT2* (SALK\_057998) and *CAT3* (SALK\_092911). This work was

Arnholdt-Schmitt, B.; Costa, J.H. & de Melo, D.F. (2006). AOX- a functional marker for

efficient cell reprogramming under stress? *Trends Plant Sci*e*nces* 11(6), pp. 281-287,

absorbance in the reaction mixture was measured immediately at 470 nm.

**4.4 Chlorophyll and total protein content** 

After staining, the gels were washed once more in water.

supported by the Deutsche Forschungsgemeinschaft (SFB 446).

BSA as standard.

**4.5 CAT and APX acitivities** 

**5. Acknowledgment** 

ISSN 1360-1385

**6. References** 

## **4. Experimental procedures**

## **4.1 Plant material**

Seeds from *Arabidopsis thaliana,* ecotype Columbia, were grown in a climatic chamber at 22°C under 16 h of illumination under low light conditions (60 µmol s-1m-2). Under these conditions plants developed flowers within 7 weeks, mature seeds could be harvested after 12 weeks. For long term treatment, plants were watered every second day with 5 ml of 40 µM antimycin A or 0.8 % ethanol as a control in addition to normal watering.

Suspension cells of *Arabidopsis thaliana*, ecotype Landsberg erecta*,* were grown under constant light on a rotary shaker (120 rpm) at 20°C and were subcultured every 7 days by 30-fold dilution in fresh growth medium (100 ml culture in 250 ml flasks). The Murashige and Skoog growth medium contains 3 % (w/v) sucrose, 0.5 mg/l -naphthaleneacetic acid and 0.05 mg/l kinetin; pH was adjusted to 5.8 with KOH. Cell cultures with a density of about 100 mg/ml medium were treated with 5 µM antimycin A (Sigma) or 5 mM hydrogen peroxide.

The full length cDNA of AOX1a (At3g22370) was amplified by PCR form reverse transcripted poly A+ RNA isolated from mature leaf material. The cDNA was cloned into the vector PY01 adjacent to a CaMV35S promoter. The construct was verified by sequencing. Arabidopsis transformation was performed by the vacuum infiltration procedure (Bechthold & Pelletier, 1998). The seeds of the transgenic plants were selected by spraying with 0.1% Basta. *WRKY53* overexpressing plants were constructed as described before (Miao et al., 2004)

T-DNA insertion lines in *CAT2* (SALK\_057998) and *CAT3* (SALK\_092911) were obtained from the Nottingham Arabidopsis Stock Centre (NASC). Homozygous lines were characterized by PCR using gene specific and T-DNA left border primers (LBb1 5'GCGTGG ACC GCT TGC TGC AAC T 3'; CAT2-LP2 5' TCG CAT GAC TGT GGT TGG TTC 3'; CAT2- RP2 5' ACC ACC AAC TCT GGT GCT CCT 3'; CAT3-LP 5' CAC CTG AGT AAT CAA ATC TAC ACG 3'; CAT3-RP 5' TCA GGG ATC CTC TCT CTG GTG AA 3'). Homozygous plants were crossed and homozygous double knock-out lines were selected by PCR screening using the same primers. Knock-out was verified by native PAGE and subsequent CAT activity staining. Since *CAT2* and *CAT3* are under circadian regulation, leaves were always harvested 3 h after the beginning of illumination. Leaves were pooled in all experiments.

### **4.2 RNA isolation and Northern and dot blot analyses**

Total RNA was isolated from leaves according to the protocol of PURESCRIPT RNA isolation kit (Gentra). Total RNA was either denatured 15 min at 55°C and spotted on nylon membranes or separated on MOPS-formaldehyde (6.2 %) agarose gels (1.5 %) and transferred to nylon membranes using 10 x SSC as transfer buffer. The membranes were hybridized at 65°C, washed twice at room temperature for 20 min with 2 x SSPE, 0.1 % SDS and once at 65°C for 30 min with 0.2 x SSPE, 0.1 % SDS. A fragment of the 5' UTR of the *AOX1a* gene or of the 3´UTR of the *SAG12* gene (At5g45890) was used as radioactive labeled hybridization probe.

#### **4.3 Measurement and detection of hydrogen peroxide**

Hydrogen peroxide was measured according to the method described by Kuźniak and others (1999). Ten leaf discs (diameter 1 cm) or pelleted suspension cells (approx. 100 mg) were incubated for 2 h in 2 ml reagent mixture containing 50 mM potassium phosphate buffer pH 7.0, 0.05 % guaiacol (Sigma) and horseradish peroxidase (2.5 u/ml, Serva) at room temperature in the dark. Four moles of hydrogen peroxide are required to form 1 mole of tetraguaiacol, which has an extinction coefficient of = 26.6 cm-1mM-1 at 470 nm. The absorbance in the reaction mixture was measured immediately at 470 nm.

### **4.4 Chlorophyll and total protein content**

Leaf discs were homogenized in 0.2 ml 25 mM potassium phosphate buffer, pH 7.0, containing 2 mM EDTA. Subsequently, 0.8 ml acetone was added, and the samples were shaken vigorously for 1 h at room temperature. After centrifugation at 14000 g for 30 min at room temperature, the total chlorophyll content of the supernatant was measured and calculated following the method described by Arnon (1949). To determine total protein content, leaf discs were ground in 100 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA at 4°C. After centrifugation at 14000 g for 30 min at 4°C, the supernatant was directly used for protein quantification according to the method of Bradford (1976) using BSA as standard.

## **4.5 CAT and APX acitivities**

44 Senescence

Seeds from *Arabidopsis thaliana,* ecotype Columbia, were grown in a climatic chamber at 22°C under 16 h of illumination under low light conditions (60 µmol s-1m-2). Under these conditions plants developed flowers within 7 weeks, mature seeds could be harvested after 12 weeks. For long term treatment, plants were watered every second day with 5 ml of 40

Suspension cells of *Arabidopsis thaliana*, ecotype Landsberg erecta*,* were grown under constant light on a rotary shaker (120 rpm) at 20°C and were subcultured every 7 days by 30-fold dilution in fresh growth medium (100 ml culture in 250 ml flasks). The Murashige and Skoog growth medium contains 3 % (w/v) sucrose, 0.5 mg/l -naphthaleneacetic acid and 0.05 mg/l kinetin; pH was adjusted to 5.8 with KOH. Cell cultures with a density of about 100 mg/ml

The full length cDNA of AOX1a (At3g22370) was amplified by PCR form reverse transcripted poly A+ RNA isolated from mature leaf material. The cDNA was cloned into the vector PY01 adjacent to a CaMV35S promoter. The construct was verified by sequencing. Arabidopsis transformation was performed by the vacuum infiltration procedure (Bechthold & Pelletier, 1998). The seeds of the transgenic plants were selected by spraying with 0.1% Basta. *WRKY53*

T-DNA insertion lines in *CAT2* (SALK\_057998) and *CAT3* (SALK\_092911) were obtained from the Nottingham Arabidopsis Stock Centre (NASC). Homozygous lines were characterized by PCR using gene specific and T-DNA left border primers (LBb1 5'GCGTGG ACC GCT TGC TGC AAC T 3'; CAT2-LP2 5' TCG CAT GAC TGT GGT TGG TTC 3'; CAT2- RP2 5' ACC ACC AAC TCT GGT GCT CCT 3'; CAT3-LP 5' CAC CTG AGT AAT CAA ATC TAC ACG 3'; CAT3-RP 5' TCA GGG ATC CTC TCT CTG GTG AA 3'). Homozygous plants were crossed and homozygous double knock-out lines were selected by PCR screening using the same primers. Knock-out was verified by native PAGE and subsequent CAT activity staining. Since *CAT2* and *CAT3* are under circadian regulation, leaves were always harvested 3 h after the beginning of illumination. Leaves were pooled in all experiments.

Total RNA was isolated from leaves according to the protocol of PURESCRIPT RNA isolation kit (Gentra). Total RNA was either denatured 15 min at 55°C and spotted on nylon membranes or separated on MOPS-formaldehyde (6.2 %) agarose gels (1.5 %) and transferred to nylon membranes using 10 x SSC as transfer buffer. The membranes were hybridized at 65°C, washed twice at room temperature for 20 min with 2 x SSPE, 0.1 % SDS and once at 65°C for 30 min with 0.2 x SSPE, 0.1 % SDS. A fragment of the 5' UTR of the *AOX1a* gene or of the 3´UTR of the *SAG12* gene (At5g45890) was used as radioactive labeled

Hydrogen peroxide was measured according to the method described by Kuźniak and others (1999). Ten leaf discs (diameter 1 cm) or pelleted suspension cells (approx. 100 mg)

µM antimycin A or 0.8 % ethanol as a control in addition to normal watering.

medium were treated with 5 µM antimycin A (Sigma) or 5 mM hydrogen peroxide.

overexpressing plants were constructed as described before (Miao et al., 2004)

**4.2 RNA isolation and Northern and dot blot analyses** 

**4.3 Measurement and detection of hydrogen peroxide** 

hybridization probe.

**4. Experimental procedures** 

**4.1 Plant material** 

For analyses of APX isozymes, crude protein extracts were separated on 10% native polyacrylamide gels (0.375 M Tris-HCl, pH 8.8, as gel buffer) with a 5% stacking gel (0.125 M Tris-HCl, pH 6.8, as gel buffer) for 16 h (120V) at 4°C using 2 mM ascorbate, 250 mM glycine, and 25 mM Tris-HCl, pH 8.3, as electrophoresis buffer. After electrophoresis, the gels were soaked in 50 mM potassium phosphate buffer, pH 7.0, containing 2 mM ascorbate for 10 min (3x) and, subsequently, in 50 mM potassium phosphate buffer, pH 7.0, containing 4 mM ascorbate, and 1 mM H2O2 for 20 min. After rinsing in water, the gels were stained in 50 mM potassium phosphate buffer, pH 7.8, containing 14 mM TEMED (*N,N,N´,N´* tetramethylethylenediamine) and 2.45 mM NBT (nitro blue tetrazolium) for 10-30 min. For the analyses of CAT isozymes the protein extracts were separated on 7.5% native polyacrylamide gels (0.375 M Tris-HCl, pH 8.8, as gel buffer) with a 3.5% stacking gel (0.125 M Tris-HCl, pH 6.8, as gel buffer) for 16 h (70-80V) at 18°C using 250 mM glycine and 25 mM Tris-HCl, pH 8.3, as electrophoresis buffer. Subsequently, the gels were stained for the activity of catalases as follows: The gels were soaked in 0.01% of hydrogen peroxide solution for 5 min, washed twice in water and incubated for 5 min in 1% FeCl3 and 1% K3[Fe(CN)6]. After staining, the gels were washed once more in water.

## **5. Acknowledgment**

We thank the Nottingham Arabidopsis Stock Centre (NASC) for providing seeds of the T-DNA insertion lines for *CAT2* (SALK\_057998) and *CAT3* (SALK\_092911). This work was supported by the Deutsche Forschungsgemeinschaft (SFB 446).

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**3** 

**Metabolic Regulation of Leaf Senescence** 

Eloísa Agüera, Purificación Cabello,

*University of Córdoba* 

 *Spain* 

*Department of Botany, Ecology and Plant Physiology,* 

**in Sunflower (***Helianthus annuus* **L.) Plants** 

Lourdes de la Mata, Estefanía Molina and Purificación de la Haba

The leaf is the main photosynthetic organ of plants and its development a complex process governed by a combination of environmental factors and intrinsic and genetically regulated signals (Van Lijsebettens & Clarke, 1998). Usually, leaf ontogeny includes an early phase of increasing photosynthetic rates while the leaf is actively expanding, a mature phase where such rates peak and a senescence phase where they decline (Gepstein, 1988; Miller et al., 2000). During early development, the leaf is a sink receiving nutrients from the rest of the plant; however, as soon as it reaches full photosynthetic capacity, it becomes the main source organ of the plant. After this productive period, the leaf enters the senescence phase, during which most compounds present in it are removed and reused (Hörtensteiner & Feller, 2002; Buchanan-Wollaston et al., 2003a). Leaf senescence, which is last stage in leaf development, is a highly regulated and programmed degeneration process governed by a variety of developmental and environmental signals (Lim et al., 2003). This important phase in the leaf lifespan period may last as long as leaf maturation and involves a shift from nutrient assimilation to nutrient remobilization and recycling (Guiboileau et al., 2010). In senescent leaf metabolism, carbon and nitrogen assimilation are replaced by catabolism of chlorophyll and macromolecules such as proteins, RNA and membrane lipids, the degradation of which marks the senescence phase. Unsurprisingly, senescence alters the expression of many genes. These senescence-associated genes include regulatory genes encoding transcription factors; genes involved in degradative processes that code for hydrolytic enzymes such as proteases, lipases and ribonucleases; and genes with secondary functions in senescence that code for proteins involved in nutrient remobilization (e.g. glutamine synthetase, which catalyses the conversion of ammonium into glutamine to enable nitrogen recycling in senescing cells) (Taiz & Zeiger, 2010). Environmental cues such as day length and temperature, and various biotic and abiotic sources of stress, can also

affect the initiation and progress of such a high complex as leaf senescence.

During senescence, some metabolic pathways are triggered and others turned off. These dramatic metabolic changes result in orderly degradation of cellular structures, starting with chloroplasts (Wiedemuth et al., 2005), and also in the subsequent remobilization of the resulting materials. Chloroplasts play a dual role; thus, they are the main source of nitrogen

**1. Introduction** 


## **Metabolic Regulation of Leaf Senescence in Sunflower (***Helianthus annuus* **L.) Plants**

Eloísa Agüera, Purificación Cabello, Lourdes de la Mata, Estefanía Molina and Purificación de la Haba *Department of Botany, Ecology and Plant Physiology, University of Córdoba Spain* 

## **1. Introduction**

50 Senescence

Zapata, J.M.; Guera, A.; Esteban-Carrasco, A.; Martin, M. & Sabater, B. (2005). Chloroplasts

Zentgraf; U. & Hemleben, V. (2007). Molecular cell biology: Are reactive oxygen species

Zimmermann, P.; Orendi, G.; Heinlein, C. & Zentgraf, U. (2006). Senescence specific

*Cell Death and Differentiation* 12 (10), pp. 1277-1284, ISSN 1350-9047

29 (6), pp. 1049-1060, ISSN 0140-7791, online ISSN1365-3040

ISBN 978-3-540-72953-2, ISSN 0340-4773

regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco.

regulators of leaf senescence? In: Lüttge, U.; Beyschlag, W. & Murata, J. (Eds), Progress in Botany, Vol. 69, Springer, Berlin, Heidelberg, New York, pp. 117-138,

regulation of catalases in *Arabidopsis thaliana* (L.) Heynh. *Plant Cell & Environment*

The leaf is the main photosynthetic organ of plants and its development a complex process governed by a combination of environmental factors and intrinsic and genetically regulated signals (Van Lijsebettens & Clarke, 1998). Usually, leaf ontogeny includes an early phase of increasing photosynthetic rates while the leaf is actively expanding, a mature phase where such rates peak and a senescence phase where they decline (Gepstein, 1988; Miller et al., 2000). During early development, the leaf is a sink receiving nutrients from the rest of the plant; however, as soon as it reaches full photosynthetic capacity, it becomes the main source organ of the plant. After this productive period, the leaf enters the senescence phase, during which most compounds present in it are removed and reused (Hörtensteiner & Feller, 2002; Buchanan-Wollaston et al., 2003a). Leaf senescence, which is last stage in leaf development, is a highly regulated and programmed degeneration process governed by a variety of developmental and environmental signals (Lim et al., 2003). This important phase in the leaf lifespan period may last as long as leaf maturation and involves a shift from nutrient assimilation to nutrient remobilization and recycling (Guiboileau et al., 2010). In senescent leaf metabolism, carbon and nitrogen assimilation are replaced by catabolism of chlorophyll and macromolecules such as proteins, RNA and membrane lipids, the degradation of which marks the senescence phase. Unsurprisingly, senescence alters the expression of many genes. These senescence-associated genes include regulatory genes encoding transcription factors; genes involved in degradative processes that code for hydrolytic enzymes such as proteases, lipases and ribonucleases; and genes with secondary functions in senescence that code for proteins involved in nutrient remobilization (e.g. glutamine synthetase, which catalyses the conversion of ammonium into glutamine to enable nitrogen recycling in senescing cells) (Taiz & Zeiger, 2010). Environmental cues such as day length and temperature, and various biotic and abiotic sources of stress, can also affect the initiation and progress of such a high complex as leaf senescence.

During senescence, some metabolic pathways are triggered and others turned off. These dramatic metabolic changes result in orderly degradation of cellular structures, starting with chloroplasts (Wiedemuth et al., 2005), and also in the subsequent remobilization of the resulting materials. Chloroplasts play a dual role; thus, they are the main source of nitrogen

Metabolic Regulation of Leaf Senescence in Sunflower (*Helianthus annuus* L.) Plants 53

We examined various markers widely used to monitor leaf development (viz. photosynthetic pigment level, protein content and CO2 fixation rate) in primary leaves of sunflower plants grown for 42 days. The start of senescence in sunflower plants was associated with a considerable decrease in protein content and specific leaf masses referred

> Specific leaf mass (mg DW cm-2)

> > 2.2 0.11 3.1 0.28 3.0 0.27 2.5 0.23 2.2 0.29

**2. Growth-related parameters and photosynthetic activity during sunflower** 

Soluble protein (mg g-1 DW)

> 152.3 9.4 178.5 7.7 108.1 4.6 89.6 1.9 62.2 1.4

Table 1. Changes in soluble protein and specific leaf mass during sunflower primary leaf ageing. Data are means SD for duplicate determinations in three separated experiments.

These changes may reflect alterations in N and C compound distributions as a consequence of N remobilization, the efficiency of which is related to the ratio between biomass in the sink and source organs (Wiedemuth et al., 2005; Diaz et al., 2008). Since chloroplasts contain the largest amounts of protein in leaves, their breakdown releases most of the nitrogen that is reused by other plant organs. The mechanisms behind chloroplast degradation in senescing leaves are poorly understood (especially those for the degradation of Rubisco and chlorophyll-binding light-harvesting proteins, which are the most abundant chloroplastic proteins) (Martínez et al., 2008). Chloroplasts contain a large number of proteases, some of which are encoded by senescence-associated genes, which are up-regulated during senescence. Degradation of some thylakoid proteins such as LHCII seemingly occurs exclusively within chloroplasts and requires the prior release and breakdown of pigments (Hörtensteiner & Feller, 2002; Buchanan-Wollaston et al., 2003a). CND41 protease is believed to be involved in Rubisco degradation and in the translocation of nitrogen during senescence in tobacco leaves (Kato et al., 2004, 2005). However, the central vacuole and SAVs also play a role here, as they help complete the degradation of Rubisco and other stromal proteins (Martínez et al., 2008). The relative rates of degradation of some photosynthetic components may be altered by the environmental conditions. Thus, LHCII degradation in rice is delayed by low irradiances (Hidema et al., 1991). Also, the protein content in senescing sunflower leaves was found to drop earlier in nitrogen-deficient plants than in high-nitrogen plants (Agüera et al., 2010). Changes in photosynthetic pigment contents also indicate progress of leaf senescence (Yoo et al., 2003; Guo & Gan, 2005; Ougham et al., 2008). The chlorophyll breakdown pathways operating during leaf senescence are well-known and require pigment degradation and avoiding photodamage in order to maintain the ability to export released nutrients to other plant parts (Hörtensteiner,

**leaf senescence** 

as weight (Table 1).

Leaf age (days)

and also the regulators of their own degradation during senescence (Zapata et al., 2005). Most of the protein in green cells is located in chloroplasts, which thus constitute their main reserves of organic nitrogen. Efficient recycling of nitrogen from the photosynthetic apparatus during early senescence requires the presence of intact mitochondrial, nuclear and cellular membranes (Gan & Amasino, 1997; Nam, 1997; Noodén et al., 1997; Hörtensteiner & Feller, 2002; Cabello et al*.*, 2006). Leaf proteins (particularly photosynthetic proteins) are extensively degraded during senescence (Martínez et al., 2008), which confirms that one of the primary functions of leaf senescence is to recycle nutrients (especially through nitrogen remobilization) (Himelblau & Amasino, 2001). Protein breakdown starts early in senescence and proteolysis is believed to start within chloroplasts. Some proteins (e.g. chlorophyll-binding light-harvesting proteins LHCII) seem to be entirely degraded within chloroplasts, whereas Rubisco and other chloroplastic proteins may be broken down via a hybrid pathway involving both chloroplasts and extraplastidic compartments such as the central vacuole and small senescence-associated vacuoles (SAVs), which are absent from mature, non-senescing leaves but present in large numbers during senescence (Otegui et al., 2005; Martínez et al., 2008). Degradation of chloroplastic proteins releases potentially phototoxic chlorophylls that necessitate degradation. Therefore, leaf senescence is characterized by a decline in photosynthetic activity and chlorophyll content, and the rapid chlorophyll loss associated with chloroplast degeneration is frequently used as a biomarker for the start of senescence. Although chlorophyll degradation is an early senescence signal, leaf yellowing is not an appropriate marker of early senescence because it is observed when senescence has progressed to a great extent (Diaz et al., 2005). Nitrogen and carbon metabolism plays a crucial role in the senescence process, which is seemingly governed by both external and internal factors. Thus, leaf senescence induction involves the joint action of external (nitrogen availability, light) and internal signals (regulating metabolites, C/N ratio) (Wingler et al., 2006; Wingler & Roitsch, 2008).

Other important signals for induction or progression of senescence include the redox status of leaf cells and the production of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide radical (Kukavica & Veljovic-Jovanovic, 2004; Zimmermann & Zentgraf, 2005). There are many sources of reactive oxygen species, which are produced during aerobic metabolism in chloroplasts, mitochondria and peroxisomes in both photosynthetically active and senescent cells. The toxicity of these reactive species is dictated by various enzymatic and non-enzymatic protective antioxidant defences. Superoxide dismutases, catalases, peroxidases and the ascorbate–glutathione cycle enzymes are the primary antioxidant enzymes. Plant ageing increases oxidative stress and the levels of reactive oxygen species, which may additionally diminish antioxidant protection (Buchanan-Wollaston et al., 2003b; Zimmermann & Zentgraf, 2005). Chloroplasts are probably the main target of age-associated oxidative stress in plants (Munné-Bosch & Alegre, 2002). Therefore, a plausible model for regulation of leaf senescence is a shifted balance between the production of reactive oxygen species and their removal by antioxidant systems.

In this chapter, we describe various aspects of leaf senescence in sunflower plants, with special emphasis on changes in the contents of some nitrogen and carbon metabolites potentially acting as regulators or markers of senescence during sunflower leaf development, and also on the role of oxidative stress in this process and the influence of external factors such nitrogen supply and irradiance exposition on it.

and also the regulators of their own degradation during senescence (Zapata et al., 2005). Most of the protein in green cells is located in chloroplasts, which thus constitute their main reserves of organic nitrogen. Efficient recycling of nitrogen from the photosynthetic apparatus during early senescence requires the presence of intact mitochondrial, nuclear and cellular membranes (Gan & Amasino, 1997; Nam, 1997; Noodén et al., 1997; Hörtensteiner & Feller, 2002; Cabello et al*.*, 2006). Leaf proteins (particularly photosynthetic proteins) are extensively degraded during senescence (Martínez et al., 2008), which confirms that one of the primary functions of leaf senescence is to recycle nutrients (especially through nitrogen remobilization) (Himelblau & Amasino, 2001). Protein breakdown starts early in senescence and proteolysis is believed to start within chloroplasts. Some proteins (e.g. chlorophyll-binding light-harvesting proteins LHCII) seem to be entirely degraded within chloroplasts, whereas Rubisco and other chloroplastic proteins may be broken down via a hybrid pathway involving both chloroplasts and extraplastidic compartments such as the central vacuole and small senescence-associated vacuoles (SAVs), which are absent from mature, non-senescing leaves but present in large numbers during senescence (Otegui et al., 2005; Martínez et al., 2008). Degradation of chloroplastic proteins releases potentially phototoxic chlorophylls that necessitate degradation. Therefore, leaf senescence is characterized by a decline in photosynthetic activity and chlorophyll content, and the rapid chlorophyll loss associated with chloroplast degeneration is frequently used as a biomarker for the start of senescence. Although chlorophyll degradation is an early senescence signal, leaf yellowing is not an appropriate marker of early senescence because it is observed when senescence has progressed to a great extent (Diaz et al., 2005). Nitrogen and carbon metabolism plays a crucial role in the senescence process, which is seemingly governed by both external and internal factors. Thus, leaf senescence induction involves the joint action of external (nitrogen availability, light) and internal signals (regulating metabolites, C/N ratio)

Other important signals for induction or progression of senescence include the redox status of leaf cells and the production of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide radical (Kukavica & Veljovic-Jovanovic, 2004; Zimmermann & Zentgraf, 2005). There are many sources of reactive oxygen species, which are produced during aerobic metabolism in chloroplasts, mitochondria and peroxisomes in both photosynthetically active and senescent cells. The toxicity of these reactive species is dictated by various enzymatic and non-enzymatic protective antioxidant defences. Superoxide dismutases, catalases, peroxidases and the ascorbate–glutathione cycle enzymes are the primary antioxidant enzymes. Plant ageing increases oxidative stress and the levels of reactive oxygen species, which may additionally diminish antioxidant protection (Buchanan-Wollaston et al., 2003b; Zimmermann & Zentgraf, 2005). Chloroplasts are probably the main target of age-associated oxidative stress in plants (Munné-Bosch & Alegre, 2002). Therefore, a plausible model for regulation of leaf senescence is a shifted balance between the production of reactive oxygen species and their removal by antioxidant

In this chapter, we describe various aspects of leaf senescence in sunflower plants, with special emphasis on changes in the contents of some nitrogen and carbon metabolites potentially acting as regulators or markers of senescence during sunflower leaf development, and also on the role of oxidative stress in this process and the influence of

external factors such nitrogen supply and irradiance exposition on it.

(Wingler et al., 2006; Wingler & Roitsch, 2008).

systems.

## **2. Growth-related parameters and photosynthetic activity during sunflower leaf senescence**

We examined various markers widely used to monitor leaf development (viz. photosynthetic pigment level, protein content and CO2 fixation rate) in primary leaves of sunflower plants grown for 42 days. The start of senescence in sunflower plants was associated with a considerable decrease in protein content and specific leaf masses referred as weight (Table 1).


Table 1. Changes in soluble protein and specific leaf mass during sunflower primary leaf ageing. Data are means SD for duplicate determinations in three separated experiments.

These changes may reflect alterations in N and C compound distributions as a consequence of N remobilization, the efficiency of which is related to the ratio between biomass in the sink and source organs (Wiedemuth et al., 2005; Diaz et al., 2008). Since chloroplasts contain the largest amounts of protein in leaves, their breakdown releases most of the nitrogen that is reused by other plant organs. The mechanisms behind chloroplast degradation in senescing leaves are poorly understood (especially those for the degradation of Rubisco and chlorophyll-binding light-harvesting proteins, which are the most abundant chloroplastic proteins) (Martínez et al., 2008). Chloroplasts contain a large number of proteases, some of which are encoded by senescence-associated genes, which are up-regulated during senescence. Degradation of some thylakoid proteins such as LHCII seemingly occurs exclusively within chloroplasts and requires the prior release and breakdown of pigments (Hörtensteiner & Feller, 2002; Buchanan-Wollaston et al., 2003a). CND41 protease is believed to be involved in Rubisco degradation and in the translocation of nitrogen during senescence in tobacco leaves (Kato et al., 2004, 2005). However, the central vacuole and SAVs also play a role here, as they help complete the degradation of Rubisco and other stromal proteins (Martínez et al., 2008). The relative rates of degradation of some photosynthetic components may be altered by the environmental conditions. Thus, LHCII degradation in rice is delayed by low irradiances (Hidema et al., 1991). Also, the protein content in senescing sunflower leaves was found to drop earlier in nitrogen-deficient plants than in high-nitrogen plants (Agüera et al., 2010). Changes in photosynthetic pigment contents also indicate progress of leaf senescence (Yoo et al., 2003; Guo & Gan, 2005; Ougham et al., 2008). The chlorophyll breakdown pathways operating during leaf senescence are well-known and require pigment degradation and avoiding photodamage in order to maintain the ability to export released nutrients to other plant parts (Hörtensteiner,

Metabolic Regulation of Leaf Senescence in Sunflower (*Helianthus annuus* L.) Plants 55

irradiance than in others grown at a low photon flux density, also indicating that an

Fig. 2. Carbon dioxide fixation rates and transpiration in sunflower primary leaves of different age. Data are means SD of measured values on primary leaves of ten plants

**3. Carbon and nitrogen metabolites as regulators of leaf senescence in** 

The contents in soluble sugars of sunflower plants increase with leaf ageing, and the opposite holds for the starch content. Our results show that accumulation of soluble sugars in plants grown at high irradiance is not much greater than in plants grown at low irradiance, although a substantial increase in the monosaccharide-to-sucrose ratio is observed at the start of senescence (especially at high irradiance levels) (Fig. 3). The accumulation of soluble sugars is associated to leaf age but unrelated to photosynthetic activity because CO2 fixation rates decrease during ageing; rather, it is due to starch hydrolysis. The increase in soluble sugars may also be ascribed to senescence causing a loss of functional and structural integrity in cell membranes, thereby boosting membrane lipid catabolism and hence sugar production by gluconeogenesis (Buchanan-Wollaston et al., 2003b; Lim et al., 2007). Leaf senescence is a plastic process triggered by a variety of external and internal factors (Weaver & Amasino, 2001; Buchanan-Wollaston et al., 2003a; Balibrea-Lara et al., 2004; Wingler et al., 2006). Senescence reduces photosynthetic carbon fixation, but is important for the recycling of nitrogen and other nutrients (Díaz et al., 2005; Wingler et al., 2005). By virtue of its lying at the crossroads of carbon and nitrogen metabolism, senescence is regulated by carbon and nitrogen signals. Increasing evidence suggests a role for hexose accumulation in ageing leaves as a signal for either senescence initiation or acceleration in annual plants (Masclaux et al., 2000; Moore et al., 2003; Díaz et al., 2005; Masclaux-Daubresse et al., 2005; Parrott et al., 2005; Pourtau et al., 2006; Wingler & Roitsch, 2008; Agüera et al., 2010). Recently, the role of sugar accumulation or starvation in leaf senescence was critically evaluated by van Doorn (2008), who pointed out that little is known about sugar concentrations and senescence regulation in different

increased irradiance may accelerate leaf senescence.

randomly selected for each age.

**sunflower plants** 

tissues and cells.

2006; Ougham et al., 2008). Chlorophylls in sunflower plants are more susceptible to degradation than are carotenoids during leaf senescence, and both total chlorophyll and carotenoid contents are high in young and mature leaves, their levels peaking at 22 days and decreasing afterwards during senescence (Fig. 1). Carotenoid degradation is usually slower than chlorophyll breakdown and can be especially complex depending on the particular pigment species (Suzuki & Shioi, 2004).

Fig. 1. Changes in pigment levels during ageing of sunflower primary leaves. Data are means SD for duplicate determinations in three separated experiments.

Chlorophyll loss in sunflower plants is also a typical phenomenon of leaf senescence of potential use as an indicator. The marked decrease in total chlorophyll observed after 28 days is mainly due to the loss of chlorophyll *a*, which is the form most strongly affected by leaf ageing as revealed by a significant decrease in Chl *a*/Chl *b* ratio in senescent leaves (Cabello et al., 2006). In radish cotyledons, however, the ratio of Chl *a* to Chl *b* increases slightly during senescence, which suggests that Chl *b* is degraded faster than is Chl *a* (Suzuki & Shioi, 2004).

Other typical changes observed during senescence are a rapid decline in photosynthetic activity, which may be a senescence-inducing signal (Bleecker & Patterson, 1997; Quirino et al., 2000), and a reduction in transpiration rate, which is probably due to an increase in abscisic acid levels inducing stomatal closure, although this is not a direct induction factor for senescence (Weaver & Amasino, 2001). A marked decrease in CO2 fixation rate and transpiration in sunflower plants was observed during natural leaf senescence, a process that starts and develops in plants aged 28–42 days (Fig. 2).

Although natural senescence is the final stage of leaf development, it may start prematurely by effect of exposure to environmental stress or nutrient deprivation (Quirino et al., 2000; Lim et al., 2003, 2007, Wingler et al., 2009). In fact, poor nitrogen nutrition and exposure to high irradiance are known to lead to early senescence in sunflower leaves (Agüera et al., 2010). Thus, the decrease in chlorophyll content associated to leaf senescence starts earlier in sunflower plants grown with low nitrogen, which suggests that leaf senescence is accelerated under these conditions. In addition, the decline in photosynthetic activity is more apparent with nitrogen deficiency (Agüera et al., 2010). Similarly, the loss of photosynthetic activity is more marked in leaves of sunflower plants grown at high

2006; Ougham et al., 2008). Chlorophylls in sunflower plants are more susceptible to degradation than are carotenoids during leaf senescence, and both total chlorophyll and carotenoid contents are high in young and mature leaves, their levels peaking at 22 days and decreasing afterwards during senescence (Fig. 1). Carotenoid degradation is usually slower than chlorophyll breakdown and can be especially complex depending on the particular

Fig. 1. Changes in pigment levels during ageing of sunflower primary leaves. Data are

Chlorophyll loss in sunflower plants is also a typical phenomenon of leaf senescence of potential use as an indicator. The marked decrease in total chlorophyll observed after 28 days is mainly due to the loss of chlorophyll *a*, which is the form most strongly affected by leaf ageing as revealed by a significant decrease in Chl *a*/Chl *b* ratio in senescent leaves (Cabello et al., 2006). In radish cotyledons, however, the ratio of Chl *a* to Chl *b* increases slightly during senescence, which suggests that Chl *b* is degraded faster than is Chl *a*

Other typical changes observed during senescence are a rapid decline in photosynthetic activity, which may be a senescence-inducing signal (Bleecker & Patterson, 1997; Quirino et al., 2000), and a reduction in transpiration rate, which is probably due to an increase in abscisic acid levels inducing stomatal closure, although this is not a direct induction factor for senescence (Weaver & Amasino, 2001). A marked decrease in CO2 fixation rate and transpiration in sunflower plants was observed during natural leaf senescence, a process

Although natural senescence is the final stage of leaf development, it may start prematurely by effect of exposure to environmental stress or nutrient deprivation (Quirino et al., 2000; Lim et al., 2003, 2007, Wingler et al., 2009). In fact, poor nitrogen nutrition and exposure to high irradiance are known to lead to early senescence in sunflower leaves (Agüera et al., 2010). Thus, the decrease in chlorophyll content associated to leaf senescence starts earlier in sunflower plants grown with low nitrogen, which suggests that leaf senescence is accelerated under these conditions. In addition, the decline in photosynthetic activity is more apparent with nitrogen deficiency (Agüera et al., 2010). Similarly, the loss of photosynthetic activity is more marked in leaves of sunflower plants grown at high

means SD for duplicate determinations in three separated experiments.

that starts and develops in plants aged 28–42 days (Fig. 2).

pigment species (Suzuki & Shioi, 2004).

(Suzuki & Shioi, 2004).

irradiance than in others grown at a low photon flux density, also indicating that an increased irradiance may accelerate leaf senescence.

Fig. 2. Carbon dioxide fixation rates and transpiration in sunflower primary leaves of different age. Data are means SD of measured values on primary leaves of ten plants randomly selected for each age.

#### **3. Carbon and nitrogen metabolites as regulators of leaf senescence in sunflower plants**

The contents in soluble sugars of sunflower plants increase with leaf ageing, and the opposite holds for the starch content. Our results show that accumulation of soluble sugars in plants grown at high irradiance is not much greater than in plants grown at low irradiance, although a substantial increase in the monosaccharide-to-sucrose ratio is observed at the start of senescence (especially at high irradiance levels) (Fig. 3). The accumulation of soluble sugars is associated to leaf age but unrelated to photosynthetic activity because CO2 fixation rates decrease during ageing; rather, it is due to starch hydrolysis. The increase in soluble sugars may also be ascribed to senescence causing a loss of functional and structural integrity in cell membranes, thereby boosting membrane lipid catabolism and hence sugar production by gluconeogenesis (Buchanan-Wollaston et al., 2003b; Lim et al., 2007). Leaf senescence is a plastic process triggered by a variety of external and internal factors (Weaver & Amasino, 2001; Buchanan-Wollaston et al., 2003a; Balibrea-Lara et al., 2004; Wingler et al., 2006). Senescence reduces photosynthetic carbon fixation, but is important for the recycling of nitrogen and other nutrients (Díaz et al., 2005; Wingler et al., 2005). By virtue of its lying at the crossroads of carbon and nitrogen metabolism, senescence is regulated by carbon and nitrogen signals. Increasing evidence suggests a role for hexose accumulation in ageing leaves as a signal for either senescence initiation or acceleration in annual plants (Masclaux et al., 2000; Moore et al., 2003; Díaz et al., 2005; Masclaux-Daubresse et al., 2005; Parrott et al., 2005; Pourtau et al., 2006; Wingler & Roitsch, 2008; Agüera et al., 2010). Recently, the role of sugar accumulation or starvation in leaf senescence was critically evaluated by van Doorn (2008), who pointed out that little is known about sugar concentrations and senescence regulation in different tissues and cells.

Metabolic Regulation of Leaf Senescence in Sunflower (*Helianthus annuus* L.) Plants 57

cytokinins and light is known to increase cytokinin oxidase/dehydrogenase activity during

Some results also suggest that leaf senescence is regulated by the carbon–nitrogen balance (Masclaux et al., 2000). However, in spite of the drastic changes in leaf metabolism occurring during senescence, carbon and nitrogen metabolite contents have scarcely been determined (Diaz et al., 2005). Cabello et al. (2006) found sunflower leaf senescence to be associated with significant changes in the contents of carbon and nitrogen metabolites. The highest ammonium concentrations were found in young and senescent leaves, as reported in tobacco (Masclaux et al., 2000). Our results indicate that sunflower plants exhibit their peak ammonium contents in young and late senescing leaves (Table 2). The high ammonium contents of young leaves are probably a result of strong photosynthetic nitrate reduction activity and photorespiration. In addition, young leaves have low contents in soluble carbohydrates, and sugar availability is known to be a limiting factor for ammonium assimilation (Morcuende et al., 1998). The high ammonium contents of senescent leaves are mainly due to protein degradation, amino acid deamination and nucleic acid catabolism, but

Senescent leaves contain low levels of free amino acids, probably because their remobilization is essential with a view to supplying developing organs in the plant (Buchanan-Wollaston, 1997). The concentrations of glutamate (a precursor of other amino acids) and aspartate (a direct product of glutamate transamination) decrease in the final stages of senescence in *Arabidopsis*. Glutamine and asparagine, the major amino acids translocated in the phloem sap, are mobilized more efficiently during late senescence (Diaz et al., 2005). As suggested by a genome array study (Lin & Wu, 2004), the synthesis of asparagine for nitrogen remobilization during dark-induced leaf senescence in *Arabidopsis* seems to occur via a novel biochemical pathway. Cabello et al. (2006) found glutamate to be the most abundant free amino acid in sunflower leaves as previously also found in rice (Kamachi et al., 1991), tobacco (Masclaux et al., 2000; Tercé-Laforgue et al., 2004) and *Arabidopsis* (Diaz et al., 2005). The ratio (Glu + Asp)/(Gln + Asn) peaked in sunflower leaves of 22 days, but decreased gradually in leaves of 28, 36 and 42 days (Table 2), which suggests that N-rich amino acids (specially Asn, which has a lower C to N ratio) are produced for efficient export from leaves in late senescence, as proposed for *Arabidopsis* (Diaz et al., 2005).

> Ammonium (µmol g-1 DW)

> > 11.40 1.0 8.57 0.9 7.29 0.7 8.94 0.5 10.91 0.7

ratio during sunflower primary leaf ageing. Data are means SD for duplicate

determinations in three separated experiments

Table 2. Changes in ammonium content and glutamate + aspartate /glutamine + asparagine

(Glu + Asp/Gln + Asn) Ratio

> 2.11 2.29 1.77 1.51 1.44

senescence of barley leaf segments (Schlüter et al., 2011).

also to photorespiration.

Leaf age (days)

Fig. 3. Changes in glucose, fructose, sucrose and starch contents, and in hexoses-to-sucrose ratio, during development of sunflower primary leaves. Plants were grown at 125 µmol photons m-2 s-1 (grey bars) or 350 µmol photons m-2 s-1 (black bars). Data are means SD for duplicate determinations in three separate experiments.

Although sugars may not always be the direct cause of leaf senescence, there is enough evidence suggesting that sugar signalling plays a role in senescence regulation in a complex network involving a variety of other signals (Masclaux-Daubresse et al., 2007; Wingler & Roitsch, 2008; Wingler et al., 2009). Thus, cytokinin oxidase/dehydrogenase activity and senescence are positively correlated. The enzyme probably boosts senescence by destroying

Fig. 3. Changes in glucose, fructose, sucrose and starch contents, and in hexoses-to-sucrose ratio, during development of sunflower primary leaves. Plants were grown at 125 µmol photons m-2 s-1 (grey bars) or 350 µmol photons m-2 s-1 (black bars). Data are means SD for

Although sugars may not always be the direct cause of leaf senescence, there is enough evidence suggesting that sugar signalling plays a role in senescence regulation in a complex network involving a variety of other signals (Masclaux-Daubresse et al., 2007; Wingler & Roitsch, 2008; Wingler et al., 2009). Thus, cytokinin oxidase/dehydrogenase activity and senescence are positively correlated. The enzyme probably boosts senescence by destroying

duplicate determinations in three separate experiments.

cytokinins and light is known to increase cytokinin oxidase/dehydrogenase activity during senescence of barley leaf segments (Schlüter et al., 2011).

Some results also suggest that leaf senescence is regulated by the carbon–nitrogen balance (Masclaux et al., 2000). However, in spite of the drastic changes in leaf metabolism occurring during senescence, carbon and nitrogen metabolite contents have scarcely been determined (Diaz et al., 2005). Cabello et al. (2006) found sunflower leaf senescence to be associated with significant changes in the contents of carbon and nitrogen metabolites. The highest ammonium concentrations were found in young and senescent leaves, as reported in tobacco (Masclaux et al., 2000). Our results indicate that sunflower plants exhibit their peak ammonium contents in young and late senescing leaves (Table 2). The high ammonium contents of young leaves are probably a result of strong photosynthetic nitrate reduction activity and photorespiration. In addition, young leaves have low contents in soluble carbohydrates, and sugar availability is known to be a limiting factor for ammonium assimilation (Morcuende et al., 1998). The high ammonium contents of senescent leaves are mainly due to protein degradation, amino acid deamination and nucleic acid catabolism, but also to photorespiration.

Senescent leaves contain low levels of free amino acids, probably because their remobilization is essential with a view to supplying developing organs in the plant (Buchanan-Wollaston, 1997). The concentrations of glutamate (a precursor of other amino acids) and aspartate (a direct product of glutamate transamination) decrease in the final stages of senescence in *Arabidopsis*. Glutamine and asparagine, the major amino acids translocated in the phloem sap, are mobilized more efficiently during late senescence (Diaz et al., 2005). As suggested by a genome array study (Lin & Wu, 2004), the synthesis of asparagine for nitrogen remobilization during dark-induced leaf senescence in *Arabidopsis* seems to occur via a novel biochemical pathway. Cabello et al. (2006) found glutamate to be the most abundant free amino acid in sunflower leaves as previously also found in rice (Kamachi et al., 1991), tobacco (Masclaux et al., 2000; Tercé-Laforgue et al., 2004) and *Arabidopsis* (Diaz et al., 2005). The ratio (Glu + Asp)/(Gln + Asn) peaked in sunflower leaves of 22 days, but decreased gradually in leaves of 28, 36 and 42 days (Table 2), which suggests that N-rich amino acids (specially Asn, which has a lower C to N ratio) are produced for efficient export from leaves in late senescence, as proposed for *Arabidopsis* (Diaz et al., 2005).


Table 2. Changes in ammonium content and glutamate + aspartate /glutamine + asparagine ratio during sunflower primary leaf ageing. Data are means SD for duplicate determinations in three separated experiments

Metabolic Regulation of Leaf Senescence in Sunflower (*Helianthus annuus* L.) Plants 59

Ageing affects glutamine synthetase activity but plays a direct role in the regulation of GS gene expression (Cabello et al., 2006). A Northern blot test using a probe corresponding to an internal fragment from *Helianthus annuus* GS2 cDNA revealed that the levels of GS2 transcripts decreased during leaf development and were very low in the late stage of senescence (42 days) (Fig. 5). Glutamine synthetase activity has been found to decrease during natural leaf senescence in a wide variety of plants including cereals, tomato and tobacco (Streit & Feller, 1983; Kamachi et al., 1991; Pérez-Rodríguez & Valpuesta, 1996; Masclaux et al., 2000). This loss of activity is mainly due to a gradual decrease in the major plastidial GS2 isoform since the cytosolic GS1 isoform remains constant or increases during

Northern blots and immunological analyses indicate that both GS transcripts and polypeptides are affected (Pérez-Rodríguez & Valpuesta 1996). GS1 plays a major role in the synthesis of glutamine for transport and remobilization of leaf organic nitrogen (Tercé-Laforgue et al., 2004), whereas GS2 takes part in the reassimilation of ammonium from photorespiration in photosynthetic tissues (Kamachi et al., 1992). The stimulation of the cytosolic GS1 isoform during senescence can be ascribed to the need for toxic ammonium to be reassimilated in order to produce glutamine for export to sink organs; this has led some authors to assume a shift in ammonia assimilation from the chloroplast to the cytosol of leaf cells during senescence (Brugière et al., 2000). Total GS activity was found to drop by a effect of a strong decrease in GS2 activity was found during sunflower leaf ageing despite the simultaneous increase in GS1 activity. GS2 transcript levels also diminished during ageing. Our results (Figs. 4 and 5) are therefore consistent with others previously reported for

Time (days)

16 22 28 36 42

leaf ageing (Pérez-Rodríguez & Valpuesta, 1996; Masclaux et al., 2000).

Fig. 5. Effect of ageing on GS2 mRNA accumulation in sunflower leaves.

Amino acids and other metabolites related to N metabolism deficit may act as signals to induce senescence in combination with hexose accumulation. Thus, leaf senescence in sunflower plants is induced by high sugar levels and accelerated by a low nitrogen supply, which supports the view that high sugar/low nitrogen conditions trigger senescence and facilitate its development (Wingler et al., 2009). Our results suggest that leaf senescence in sunflower plants is accelerated by nitrogen deficiency and high irradiance, and also that

tomato and tobacco.

We examined changes in glutamine synthetase (GS) expression and activity during leaf development (Cabello et al., 2006). GS, which is the key enzyme in ammonia assimilation, is present as chloroplastic (GS2) and cytosolic (GS1) isoforms in sunflower leaves (Cabello et al., 1991). In order to confirm whether these isoforms are differently affected by senescence in sunflower leaves, we determined their specific activity during plant development. As shown in Figure 4, total GS activity decreased with the leaf age. The decrease was consequence of a strong decline in chloroplastic GS2 activity. On the other hand, cytosolic GS1 activity increased with ageing. It should be noted that GS1 was the predominant isoform in senescent leaves of 42 days, but accounted for only 7 % of total GS activity in young leaves (16 days). As a result, the GS2/GS1 ratio decreased from 13.3 in young leaves (16 days) to 0.9 in senescent leaves (42 days) (Fig. 4). These results indicate that leaf senescence has an adverse effect on the activity of chloroplastic GS2 (the main glutamine synthetase isoform) and reduces total GS activity despite its boosting GS1 activity.

Fig. 4. Effect of ageing on total GS activity and on the activities of GS1 and GS2 isoforms in sunflower leaves. Data are means SD of duplicate determinations from three separated experiments.

We examined changes in glutamine synthetase (GS) expression and activity during leaf development (Cabello et al., 2006). GS, which is the key enzyme in ammonia assimilation, is present as chloroplastic (GS2) and cytosolic (GS1) isoforms in sunflower leaves (Cabello et al., 1991). In order to confirm whether these isoforms are differently affected by senescence in sunflower leaves, we determined their specific activity during plant development. As shown in Figure 4, total GS activity decreased with the leaf age. The decrease was consequence of a strong decline in chloroplastic GS2 activity. On the other hand, cytosolic GS1 activity increased with ageing. It should be noted that GS1 was the predominant isoform in senescent leaves of 42 days, but accounted for only 7 % of total GS activity in young leaves (16 days). As a result, the GS2/GS1 ratio decreased from 13.3 in young leaves (16 days) to 0.9 in senescent leaves (42 days) (Fig. 4). These results indicate that leaf senescence has an adverse effect on the activity of chloroplastic GS2 (the main glutamine synthetase isoform) and reduces total GS activity despite its boosting GS1 activity.

Fig. 4. Effect of ageing on total GS activity and on the activities of GS1 and GS2 isoforms in sunflower leaves. Data are means SD of duplicate determinations from three separated

experiments.

Ageing affects glutamine synthetase activity but plays a direct role in the regulation of GS gene expression (Cabello et al., 2006). A Northern blot test using a probe corresponding to an internal fragment from *Helianthus annuus* GS2 cDNA revealed that the levels of GS2 transcripts decreased during leaf development and were very low in the late stage of senescence (42 days) (Fig. 5). Glutamine synthetase activity has been found to decrease during natural leaf senescence in a wide variety of plants including cereals, tomato and tobacco (Streit & Feller, 1983; Kamachi et al., 1991; Pérez-Rodríguez & Valpuesta, 1996; Masclaux et al., 2000). This loss of activity is mainly due to a gradual decrease in the major plastidial GS2 isoform since the cytosolic GS1 isoform remains constant or increases during leaf ageing (Pérez-Rodríguez & Valpuesta, 1996; Masclaux et al., 2000).

Northern blots and immunological analyses indicate that both GS transcripts and polypeptides are affected (Pérez-Rodríguez & Valpuesta 1996). GS1 plays a major role in the synthesis of glutamine for transport and remobilization of leaf organic nitrogen (Tercé-Laforgue et al., 2004), whereas GS2 takes part in the reassimilation of ammonium from photorespiration in photosynthetic tissues (Kamachi et al., 1992). The stimulation of the cytosolic GS1 isoform during senescence can be ascribed to the need for toxic ammonium to be reassimilated in order to produce glutamine for export to sink organs; this has led some authors to assume a shift in ammonia assimilation from the chloroplast to the cytosol of leaf cells during senescence (Brugière et al., 2000). Total GS activity was found to drop by a effect of a strong decrease in GS2 activity was found during sunflower leaf ageing despite the simultaneous increase in GS1 activity. GS2 transcript levels also diminished during ageing. Our results (Figs. 4 and 5) are therefore consistent with others previously reported for tomato and tobacco.

Amino acids and other metabolites related to N metabolism deficit may act as signals to induce senescence in combination with hexose accumulation. Thus, leaf senescence in sunflower plants is induced by high sugar levels and accelerated by a low nitrogen supply, which supports the view that high sugar/low nitrogen conditions trigger senescence and facilitate its development (Wingler et al., 2009). Our results suggest that leaf senescence in sunflower plants is accelerated by nitrogen deficiency and high irradiance, and also that

Metabolic Regulation of Leaf Senescence in Sunflower (*Helianthus annuus* L.) Plants 61

increases ROS potentially regulating the accumulation of mRNA encoding antioxidant

peroxidase

(days) (mol g-1 DW) (U g-1 DW) (nmol MDA g-1 DW)

The activity and expression of antioxidant enzymes are seemingly sensitive to high

We found H2O2 accumulation in senescent sunflower to be slightly more marked in plants grown under a nitrogen deficiency; the differences, however, were not large enough to assume that H2O2 is a major factor regulating the induction of leaf senescence in N-deficient plants (Table 3). Interestingly, catalase and ascorbate peroxidase activity decreased steadily in plants grown with low nitrogen, but increased during early leaf development and then declined during senescence in plants grown with high nitrogen (Agüera et al., 2010). Production of ROS during leaf senescence is essentially governed by chloroplasts, which have a strong photooxidative potential (Zapata et al., 2005). A simultaneous increase in lipid peroxidation was observed. Mutations in the *Arabidopsis CPR5/OLD1* gene may cause early senescence through deregulation of the cellular redox balance (Jing et al., 2008). Also, there is evidence suggesting that inadequate oxidant and carbonyl group production are intrinsically related to plant ageing, and that low mitochondrial, superoxide dismutase and ascorbate peroxidase activities may contribute to extensive protein carbonylation (Vanacker

In conclusion, during sunflower leaf development some coordinated metabolic and physiological changes are produced, and the senescence process induces significant alterations in the levels of carbon and nitrogen metabolites. Glutamine synthetase of sunflower leaves is regulated both at transcriptional and enzyme levels during leaf ontogeny. Post-translational regulation of the GS2 isoform could be due, at least partially, to oxidative processes. GS activity may be used as a biochemical marker of leaf ageing, since the beginning of senescence at about 28 days is accompanied by a drastic drop in the GS2/GS1 ratio due to the increase of the cytosolic GS1 activity and the decline of the chloroplastic GS2 activity. Our results suggest that both high irradiance and nitrogen deficiency accelerates senescence of the primary leaf, probably for maintaining the functionality of the young leaves, and that one of the reasons for this accelerated senescence

16 1.22 0.15 1.12 0.10 17.21 1.22 336.4 28 87.6 8.2 22 1.38 0.14 1.70 0.12 17.99 2.12 356.2 39 85.6 7.4 28 3.84 0.42 2.25 0.26 28.22 3.22 538.5 42 155.5 12.3 36 4.76 0.30 1.94 0.17 21.34 2.19 1450.5 112 171.5 14.5 42 5.28 0.51 1.24 0.15 14.53 1.17 985.2 92 188.4 12.8 Table 3. Hydrogen peroxide accumulation, catalase, ascorbate peroxidase and superoxide

dismutase activities, and lipid peroxidation levels during sunflower primary leaf development. Data are means SD for duplicate determinations in three separated

irradiance stress (Yoshimura et al., 2000; Hernández et al., 2004).

et al., 2006; Srivalli & Khanna-Chopra, 2009).

Superoxide

dismutase Lipid peroxidation

enzymes (Hernández et al., 2006).

experiments.

Age H2O2 Catalase Ascorbate

some factors such the levels of soluble sugars and amino acids may interact in a complex network to promote this process.

## **4. Oxidative stress in sunflower plants**

Leaf senescence is an oxidative process that involves degradation of cellular and subcellular structures and macromolecules, and mobilization of the degradation products to other parts of the plants (Vanacker et al., 2006). Oxidative stress during senescence may be caused or increased by a loss of antioxidant enzymatic activities (Zimmermann & Zentgraf, 2005; Zimmermann et al., 2006; Procházkova & Wilhelmova, 2007). Senescence is also accompanied by an increase in ROS, one of the origins of which is an imbalance between the production and consumption of electrons in the photosynthetic electron transport chain caused by preferential inhibition of stromal reactions in contrast with photosystem II photochemistry (Špundová et al., 2003). The inhibition of stromal reactions increases the electron flow to molecular oxygen and causes ROS to accumulate and chloroplast components to be damaged as a result (Špundová et al., 2005; Couée et al., 2006). Chloroplasts are the main source of ROS in plants (Zimmermann & Zentgraf, 2005) and also the major target of oxidative damage (Munné-Bosch & Alegre, 2002). Stromal protein degradation during leaf senescence may be initiated by oxidative processes associated with the generation of free radicals and reactive species (Procházkova et al., 2001). Like Rubisco and other chloroplastic proteins, GS2 is susceptible to degradation initiated by reactive oxygen species (Ishida et al., 2002). The chloroplastic GS2 isoform is one of the first targets of oxidative damage at high irradiation levels (Palatnik et al., 1999). Oxidized GS becomes more susceptible to proteolysis (Ortega et al., 1999); under photo-oxidative stress, GS2 cleavage occurs preferentially around the catalytic site (Ishida et al., 2002). Senescence may therefore have a direct impact on GS2 activity through enzyme degradation initiated by reactive oxygen species as reported in Rubisco (Ishida et al., 1997; Roulin & Feller, 1998). Our results indicate that the decrease in GS2/GS1 ratio during sunflower leaf ageing may be partly due to a different sensitivity to oxidative stress of the two isoforms; in fact, chloroplastic GS2 is much more sensitive to oxidative modification *in vitro* than is cytosolic GS1 (Cabello et al., 2006). Therefore, ageing induces oxidative stress in sunflower leaves and can thus have an adverse effect on chloroplastic GS2, as well as on photosynthetic pigments. Antioxidant enzyme activities in sunflower leaves were found to decline during late senescence (42 days). Similar results have been reported for tobacco (Dhindsa et al., 1981), *Arabidopsis* (Ye et al., 2000), pea (Olsson, 1995) and maize (Procházkova et al., 2001). Oxidative stress during late senescence may be caused or increased by the loss of antioxidant enzymatic activities (Zimmermann & Zentgraf, 2005). Also, the decline in antioxidant activities is believed to be a consequence rather than the origin of senescence (Dertinger et al., 2003).

Susceptibility to oxidative stress depends on the overall balance between production of oxidants and cell antioxidant capability. In sunflower plants, considerable oxidative stress has been observed *in vivo* during leaf senescence, as revealed by lipid peroxidation, H2O2 accumulation and a decrease in the levels of antioxidant enzymes such as catalase, ascorbate peroxidase and superoxide dismutase (Table 3). Lipid peroxidation only occurs during the late stage of senescence (Berger et al., 2001; Jongebloed et al., 2004; Wingler et al., 2005). High irradiance causes reversible photoinhibition of photosynthesis in pea chloroplasts and

some factors such the levels of soluble sugars and amino acids may interact in a complex

Leaf senescence is an oxidative process that involves degradation of cellular and subcellular structures and macromolecules, and mobilization of the degradation products to other parts of the plants (Vanacker et al., 2006). Oxidative stress during senescence may be caused or increased by a loss of antioxidant enzymatic activities (Zimmermann & Zentgraf, 2005; Zimmermann et al., 2006; Procházkova & Wilhelmova, 2007). Senescence is also accompanied by an increase in ROS, one of the origins of which is an imbalance between the production and consumption of electrons in the photosynthetic electron transport chain caused by preferential inhibition of stromal reactions in contrast with photosystem II photochemistry (Špundová et al., 2003). The inhibition of stromal reactions increases the electron flow to molecular oxygen and causes ROS to accumulate and chloroplast components to be damaged as a result (Špundová et al., 2005; Couée et al., 2006). Chloroplasts are the main source of ROS in plants (Zimmermann & Zentgraf, 2005) and also the major target of oxidative damage (Munné-Bosch & Alegre, 2002). Stromal protein degradation during leaf senescence may be initiated by oxidative processes associated with the generation of free radicals and reactive species (Procházkova et al., 2001). Like Rubisco and other chloroplastic proteins, GS2 is susceptible to degradation initiated by reactive oxygen species (Ishida et al., 2002). The chloroplastic GS2 isoform is one of the first targets of oxidative damage at high irradiation levels (Palatnik et al., 1999). Oxidized GS becomes more susceptible to proteolysis (Ortega et al., 1999); under photo-oxidative stress, GS2 cleavage occurs preferentially around the catalytic site (Ishida et al., 2002). Senescence may therefore have a direct impact on GS2 activity through enzyme degradation initiated by reactive oxygen species as reported in Rubisco (Ishida et al., 1997; Roulin & Feller, 1998). Our results indicate that the decrease in GS2/GS1 ratio during sunflower leaf ageing may be partly due to a different sensitivity to oxidative stress of the two isoforms; in fact, chloroplastic GS2 is much more sensitive to oxidative modification *in vitro* than is cytosolic GS1 (Cabello et al., 2006). Therefore, ageing induces oxidative stress in sunflower leaves and can thus have an adverse effect on chloroplastic GS2, as well as on photosynthetic pigments. Antioxidant enzyme activities in sunflower leaves were found to decline during late senescence (42 days). Similar results have been reported for tobacco (Dhindsa et al., 1981), *Arabidopsis* (Ye et al., 2000), pea (Olsson, 1995) and maize (Procházkova et al., 2001). Oxidative stress during late senescence may be caused or increased by the loss of antioxidant enzymatic activities (Zimmermann & Zentgraf, 2005). Also, the decline in antioxidant activities is believed to be a consequence rather than the origin of senescence

Susceptibility to oxidative stress depends on the overall balance between production of oxidants and cell antioxidant capability. In sunflower plants, considerable oxidative stress has been observed *in vivo* during leaf senescence, as revealed by lipid peroxidation, H2O2 accumulation and a decrease in the levels of antioxidant enzymes such as catalase, ascorbate peroxidase and superoxide dismutase (Table 3). Lipid peroxidation only occurs during the late stage of senescence (Berger et al., 2001; Jongebloed et al., 2004; Wingler et al., 2005). High irradiance causes reversible photoinhibition of photosynthesis in pea chloroplasts and

network to promote this process.

(Dertinger et al., 2003).

**4. Oxidative stress in sunflower plants** 


increases ROS potentially regulating the accumulation of mRNA encoding antioxidant enzymes (Hernández et al., 2006).

Table 3. Hydrogen peroxide accumulation, catalase, ascorbate peroxidase and superoxide dismutase activities, and lipid peroxidation levels during sunflower primary leaf development. Data are means SD for duplicate determinations in three separated experiments.

The activity and expression of antioxidant enzymes are seemingly sensitive to high irradiance stress (Yoshimura et al., 2000; Hernández et al., 2004).

We found H2O2 accumulation in senescent sunflower to be slightly more marked in plants grown under a nitrogen deficiency; the differences, however, were not large enough to assume that H2O2 is a major factor regulating the induction of leaf senescence in N-deficient plants (Table 3). Interestingly, catalase and ascorbate peroxidase activity decreased steadily in plants grown with low nitrogen, but increased during early leaf development and then declined during senescence in plants grown with high nitrogen (Agüera et al., 2010). Production of ROS during leaf senescence is essentially governed by chloroplasts, which have a strong photooxidative potential (Zapata et al., 2005). A simultaneous increase in lipid peroxidation was observed. Mutations in the *Arabidopsis CPR5/OLD1* gene may cause early senescence through deregulation of the cellular redox balance (Jing et al., 2008). Also, there is evidence suggesting that inadequate oxidant and carbonyl group production are intrinsically related to plant ageing, and that low mitochondrial, superoxide dismutase and ascorbate peroxidase activities may contribute to extensive protein carbonylation (Vanacker et al., 2006; Srivalli & Khanna-Chopra, 2009).

In conclusion, during sunflower leaf development some coordinated metabolic and physiological changes are produced, and the senescence process induces significant alterations in the levels of carbon and nitrogen metabolites. Glutamine synthetase of sunflower leaves is regulated both at transcriptional and enzyme levels during leaf ontogeny. Post-translational regulation of the GS2 isoform could be due, at least partially, to oxidative processes. GS activity may be used as a biochemical marker of leaf ageing, since the beginning of senescence at about 28 days is accompanied by a drastic drop in the GS2/GS1 ratio due to the increase of the cytosolic GS1 activity and the decline of the chloroplastic GS2 activity. Our results suggest that both high irradiance and nitrogen deficiency accelerates senescence of the primary leaf, probably for maintaining the functionality of the young leaves, and that one of the reasons for this accelerated senescence

Metabolic Regulation of Leaf Senescence in Sunflower (*Helianthus annuus* L.) Plants 63

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may be the high cellular oxidation and oxidative damage caused by the earlier decline of the activity of the antioxidant enzymes in these plants (Pompelli et al., 2010).

#### **5. Acknowledgment**

This work was supported by Junta de Andalucía (grant P07-CVI-02627 and PAI group BIO-0159) and DGICYT (AGL2009-11290).

#### **6. References**


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**4** 

*Argentina* 

**Functional Approaches to** 

*Biotechnology Institute - CICVyA- INTA Castelar* 

**Study Leaf Senescence in Sunflower** 

Paula Fernandez, Sebastián Moschen, Norma Paniego and Ruth A. Heinz

Senescence is an age-dependent process at the cellular, tissue, organ or organism level, leading to death at the end of the life span (Noodén 1988). Annual plants as grain and oil crops undergo a visual process towards the end of the reproductive stage that is accompanied by nutrient remobilization from leaf to developing seeds (Buchanan-Wollaston et al. 2003). The final stage of this process is leaf death but this is actively delayed until all nutrients have been removed and recycle through the process of developmental senescence. It have been documented that a delay in leaf senescence has an important impact on grain yield trough the maintenance of the photosynthetic leaf area during the reproductive stage in different crops (Ewing & Claverie 2000), including sunflower (Sadras et al. 2000; De la Vega et al. 2011). The potential yields of sunflower crop are far from the real ones in all Argentina productive regions. In Balcarce, for example, while the potential yields are estimated in 5,000 kg.ha-1, those obtained by the best producers only reach 3,000 kg.ha-1, and the average in the region ranges in 1,800 kg.ha-1 (Dosio & Aguirrezábal 2004). These differences could possibly be due to the inability of current hybrids to keep their green leaf area for long periods, which would allow greater use of the incident radiation during the grain filling period which plays an important role in determining the yield and oil

Besides autonomous (internal) factors as age, reproductive stage and phytohormone levels, leaf senescence is hardly affected by environmental factors. Among these environmental factors, including extreme temperature, drought, shading, nutrient deficient and pathogen infection, the most limiting ones are water and nutrient availability (Gan & Amasino 1997; Sadras et al. 2000; Sadras et al. 2000; Dosio et al. 2003; Lim et al. 2003; Aguera et al. 2010).

During leaf senescence, critical and dramatic changes occurred in a highly regulated manner following a genetically programmed process of high complexity. Chlorophyll degradation, nutrient recycling and remobilization are preceded or paralleled by RNA and protein degradation. Even though leaf senescence has been widely recognized and accepted as a type of Programmed Cell Death (PCD) (Noodén & Leopold 1987), the onset and progression of senescence is accompanied by global changes in gene expression. Thus, deep extensive efforts have been achieved to reveal relevant molecular process by identifying and analysing

concentration in sunflower (Dosio et al. 2000; Aguirrezábal et al. 2003).

**1. Introduction** 


## **Functional Approaches to Study Leaf Senescence in Sunflower**

Paula Fernandez, Sebastián Moschen, Norma Paniego and Ruth A. Heinz *Biotechnology Institute - CICVyA- INTA Castelar Argentina* 

## **1. Introduction**

68 Senescence

Zimmermann, P., & Zentgraf, U. (2005). The correlation between oxidative stress and leaf

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pp. 515-534, ISSN 1425-8153

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senescence during plant development. *Cellular & Molecular Biology Letters*, Vol.10,

regulation of catalases in *Arabidopsis thaliana* (L.) Heynh. *Plant Cell and Environment*,

Senescence is an age-dependent process at the cellular, tissue, organ or organism level, leading to death at the end of the life span (Noodén 1988). Annual plants as grain and oil crops undergo a visual process towards the end of the reproductive stage that is accompanied by nutrient remobilization from leaf to developing seeds (Buchanan-Wollaston et al. 2003). The final stage of this process is leaf death but this is actively delayed until all nutrients have been removed and recycle through the process of developmental senescence. It have been documented that a delay in leaf senescence has an important impact on grain yield trough the maintenance of the photosynthetic leaf area during the reproductive stage in different crops (Ewing & Claverie 2000), including sunflower (Sadras et al. 2000; De la Vega et al. 2011). The potential yields of sunflower crop are far from the real ones in all Argentina productive regions. In Balcarce, for example, while the potential yields are estimated in 5,000 kg.ha-1, those obtained by the best producers only reach 3,000 kg.ha-1, and the average in the region ranges in 1,800 kg.ha-1 (Dosio & Aguirrezábal 2004). These differences could possibly be due to the inability of current hybrids to keep their green leaf area for long periods, which would allow greater use of the incident radiation during the grain filling period which plays an important role in determining the yield and oil concentration in sunflower (Dosio et al. 2000; Aguirrezábal et al. 2003).

Besides autonomous (internal) factors as age, reproductive stage and phytohormone levels, leaf senescence is hardly affected by environmental factors. Among these environmental factors, including extreme temperature, drought, shading, nutrient deficient and pathogen infection, the most limiting ones are water and nutrient availability (Gan & Amasino 1997; Sadras et al. 2000; Sadras et al. 2000; Dosio et al. 2003; Lim et al. 2003; Aguera et al. 2010).

During leaf senescence, critical and dramatic changes occurred in a highly regulated manner following a genetically programmed process of high complexity. Chlorophyll degradation, nutrient recycling and remobilization are preceded or paralleled by RNA and protein degradation. Even though leaf senescence has been widely recognized and accepted as a type of Programmed Cell Death (PCD) (Noodén & Leopold 1987), the onset and progression of senescence is accompanied by global changes in gene expression. Thus, deep extensive efforts have been achieved to reveal relevant molecular process by identifying and analysing

Functional Approaches to Study Leaf Senescence in Sunflower 71

Senescence studies are generally based on the accumulation of messenger RNA coding for enzymes involved in degradation of structures, however, this process has a high degree of interaction between endogenous and environmental signals, involving different genes whose expression is induced or inhibited in different stages of the process (Gan & Amasino 1997). On the other hand, there are relevant studies that inversely correlate senescence with a high level of nitrogen in soil. According to these evidences a high nutritional nitrogen performance along soil profile should lead to a delay leaf senescence in sunflower, avoiding

Senescence Associated Genes (SAGs) refer to genes whose expression level is up-regulated during senescence, in contrast with Senescence Down-regulated Genes (SDGs). These genes could be classified into two classes depending on their expression patterns: Class I genes are those whose expression is only activated during senescence (senescence-specific) whereas class II are those that maintain a basal level of expression during early leaf development, but this level increases when senescence begins (Gan & Amasino 1997). The expression patterns of these genes may change in response to different conditions of plant growth. Many of these genes can be shared by different regulatory pathways whereas others may belong to a particular pathway. Thus, the inactivation or overexpression of many SAGs may not exhibits significant effect, suggesting a complex regulatory network in leaf senescence process. SAGs can be grouped into several categories based on their predictive function, including macromolecular degradation and recycling, amino acid transport, metabolism,

The main objective in sunflower to open new insights into the early leaf senescence process focuses in the identification and characterization of genetic sequences and metabolic pathways involved in the onset and evolution of the leaf senescence process. This aim involved the analysis of transcriptional and metabolic profiles in leaves from plants growing under different conditions that may alter the senescence rate, concomitant with studies of physiological and biochemical aspects. The specific items involved in this work include:

1. Study of the evolution of leaf area, chlorophyll and sugar content in leaf of different ages in a traditional sunflower hybrid subjected to treatments that alter the senescence

2. Identification in public sunflower databases of gene sequences orthologous to Senescence Associated Genes (SAG) or Senescence Down-regulated Genes (SDG). 3. Identification of new candidate genes through a sunflower microarray expression

4. Verification and quantification of the expression profiles of these genes under

6. Integration of metabolic and transcriptional profile analysis and physiological variables for the detection of useful biomarkers for application in sunflower breeding.

Following a candidate genes strategy, a preliminary assay to detect putative SAGs in sunflower was achieved by selecting few candidates previously described for *Arabidopsis thaliana*, due to the fact that this was the very first model plant for which a large-scale SAG

the pronounced symptoms occurred for chlorophyll content (Aguera et al. 2010).

**2. Candidate gene approach to identify SAGs in sunflower** 

detoxification, regulatory genes, among others (Gepstein et al. 2003).

conditions that accelerate or delay the senescence process.

5. Study of metabolic changes that occurred during the senescence process.

under both field and greenhouse conditions.

analysis.

Senescence Associated Genes (SAGs) as prior tags to disclosure the core of this complex process (Kim et al. 2007). SAGs genes have been extensively studied in model plant species (Audic & Claverie 1997; Gepstein et al. 2003; Balazadeh et al. 2008; Hu et al. 2010) and in some agronomical relevant crops (Andersen et al. 2004; Conesa et al. 2005; Espinoza et al. 2007). Yet, although senescence and ageing might be considered synonyms, a distinct reference was previously discussed because the former comprises all those degenerative changes and cellular degradation occurring with little or non-reference to death, whereas the latter is considered the final developmental stage culminating in death (Nooden & Leopold 1988; Shahri 2011). In the last year, considering this limitation, many efforts are being achieved to disclosure and obtain genomic information for this oil crop (Kane et al. 2011) but complete sequence information are still no available.

Sunflower (*Helianthus annuus L*.) is one of the most relevant crops as source of edible oil and many efforts have been achieved to build up useful functional genomics tools for cultivated sunflower involving transcriptional and metabolic profiles (Fernandez et al. 2003; Cabello et al. 2006; Paniego et al. 2007; Fernandez et al. 2008; Peluffo et al. 2010). Although, molecular studies focused on the onset of the senescence process in sunflower leaf are scarce (Fernandez et al. 2003; Dezar et al. 2005; Manavella et al. 2006; Jobit et al. 2007; Paniego et al. 2007; Fernandez et al. 2008; Manavella et al. 2008; Peluffo et al. 2010; Fernandez et al. 2011). Thus, two different approaches are envisage for studying molecular events occurring during leaf senescence: the first strategy relays on the identification of sunflower SAGs based on a candidate gene approach while the second approach involves concerted gene expression studies based on high density oligonucleotide microarrays, whole transcriptome shotgun sequencing and microRNA detection by RNA-seq (Buermans et al. 2010; Dhahbi et al. 2011).

Leaf senescence is a complex and highly coordinated process (Noodén et al. 1997). Although symptoms have been explored, the involved processes and the mechanisms that control it have not been characterized yet (Buchanan-Wollaston et al. 2003). The distinctive symptom of leaf senescence is the breakdown of chloroplasts, therefore the decrease in chlorophyll content becomes a key indicator of the process (Hörtensteiner 2006). Both, the beginning and the rate of senescence may be affected by autonomous and environmental signals.

Environmental factors such as light (Weaver & Amasino 2001), nutrient availability, concentration of CO2, abiotic and biotic stresses caused by disease (Sadras et al. 2000) may affect the rate of senescence. A previous work (Pic et al. 2002) showed that the sequence of certain events at macroscopic, biochemical and molecular level in pea leaf senescence were not modified in leaves of different age, or under conditions of moderate water stress. Since some of the environmental conditions that affect senescence have important effects on carbon metabolism, previous works assigned to sugar content in leaves an integrating role of environmental signals, regulating leaf senescence (Wingler et al. 2006). Reproductive growth is mentioned as a factor that usually impacts on leaf senescence, and particularly in sunflower, the lack of sinks delays the onset of senescence (Sadras et al. 2000). Control of senescence by growth of reproductive structures was not observed in *Arabidopsis thaliana* (Noodén & Penny 2001). Moreover, determining the onset of senescence is complex because there is no a "symptom" indicating this moment. Visual parameters are often used to assess these processes, but both the variation in chlorophyll content and yellowing or necrosis of leaves, are detectable long after the signalling cascade of senescence process is activated.

Senescence Associated Genes (SAGs) as prior tags to disclosure the core of this complex process (Kim et al. 2007). SAGs genes have been extensively studied in model plant species (Audic & Claverie 1997; Gepstein et al. 2003; Balazadeh et al. 2008; Hu et al. 2010) and in some agronomical relevant crops (Andersen et al. 2004; Conesa et al. 2005; Espinoza et al. 2007). Yet, although senescence and ageing might be considered synonyms, a distinct reference was previously discussed because the former comprises all those degenerative changes and cellular degradation occurring with little or non-reference to death, whereas the latter is considered the final developmental stage culminating in death (Nooden & Leopold 1988; Shahri 2011). In the last year, considering this limitation, many efforts are being achieved to disclosure and obtain genomic information for this oil crop (Kane et al.

Sunflower (*Helianthus annuus L*.) is one of the most relevant crops as source of edible oil and many efforts have been achieved to build up useful functional genomics tools for cultivated sunflower involving transcriptional and metabolic profiles (Fernandez et al. 2003; Cabello et al. 2006; Paniego et al. 2007; Fernandez et al. 2008; Peluffo et al. 2010). Although, molecular studies focused on the onset of the senescence process in sunflower leaf are scarce (Fernandez et al. 2003; Dezar et al. 2005; Manavella et al. 2006; Jobit et al. 2007; Paniego et al. 2007; Fernandez et al. 2008; Manavella et al. 2008; Peluffo et al. 2010; Fernandez et al. 2011). Thus, two different approaches are envisage for studying molecular events occurring during leaf senescence: the first strategy relays on the identification of sunflower SAGs based on a candidate gene approach while the second approach involves concerted gene expression studies based on high density oligonucleotide microarrays, whole transcriptome shotgun sequencing and microRNA detection by RNA-seq (Buermans et al. 2010; Dhahbi et al. 2011). Leaf senescence is a complex and highly coordinated process (Noodén et al. 1997). Although symptoms have been explored, the involved processes and the mechanisms that control it have not been characterized yet (Buchanan-Wollaston et al. 2003). The distinctive symptom of leaf senescence is the breakdown of chloroplasts, therefore the decrease in chlorophyll content becomes a key indicator of the process (Hörtensteiner 2006). Both, the beginning and

the rate of senescence may be affected by autonomous and environmental signals.

Environmental factors such as light (Weaver & Amasino 2001), nutrient availability, concentration of CO2, abiotic and biotic stresses caused by disease (Sadras et al. 2000) may affect the rate of senescence. A previous work (Pic et al. 2002) showed that the sequence of certain events at macroscopic, biochemical and molecular level in pea leaf senescence were not modified in leaves of different age, or under conditions of moderate water stress. Since some of the environmental conditions that affect senescence have important effects on carbon metabolism, previous works assigned to sugar content in leaves an integrating role of environmental signals, regulating leaf senescence (Wingler et al. 2006). Reproductive growth is mentioned as a factor that usually impacts on leaf senescence, and particularly in sunflower, the lack of sinks delays the onset of senescence (Sadras et al. 2000). Control of senescence by growth of reproductive structures was not observed in *Arabidopsis thaliana* (Noodén & Penny 2001). Moreover, determining the onset of senescence is complex because there is no a "symptom" indicating this moment. Visual parameters are often used to assess these processes, but both the variation in chlorophyll content and yellowing or necrosis of leaves, are detectable long after the signalling cascade of senescence process is activated.

2011) but complete sequence information are still no available.

Senescence studies are generally based on the accumulation of messenger RNA coding for enzymes involved in degradation of structures, however, this process has a high degree of interaction between endogenous and environmental signals, involving different genes whose expression is induced or inhibited in different stages of the process (Gan & Amasino 1997). On the other hand, there are relevant studies that inversely correlate senescence with a high level of nitrogen in soil. According to these evidences a high nutritional nitrogen performance along soil profile should lead to a delay leaf senescence in sunflower, avoiding the pronounced symptoms occurred for chlorophyll content (Aguera et al. 2010).

## **2. Candidate gene approach to identify SAGs in sunflower**

Senescence Associated Genes (SAGs) refer to genes whose expression level is up-regulated during senescence, in contrast with Senescence Down-regulated Genes (SDGs). These genes could be classified into two classes depending on their expression patterns: Class I genes are those whose expression is only activated during senescence (senescence-specific) whereas class II are those that maintain a basal level of expression during early leaf development, but this level increases when senescence begins (Gan & Amasino 1997). The expression patterns of these genes may change in response to different conditions of plant growth. Many of these genes can be shared by different regulatory pathways whereas others may belong to a particular pathway. Thus, the inactivation or overexpression of many SAGs may not exhibits significant effect, suggesting a complex regulatory network in leaf senescence process. SAGs can be grouped into several categories based on their predictive function, including macromolecular degradation and recycling, amino acid transport, metabolism, detoxification, regulatory genes, among others (Gepstein et al. 2003).

The main objective in sunflower to open new insights into the early leaf senescence process focuses in the identification and characterization of genetic sequences and metabolic pathways involved in the onset and evolution of the leaf senescence process. This aim involved the analysis of transcriptional and metabolic profiles in leaves from plants growing under different conditions that may alter the senescence rate, concomitant with studies of physiological and biochemical aspects. The specific items involved in this work include:


Following a candidate genes strategy, a preliminary assay to detect putative SAGs in sunflower was achieved by selecting few candidates previously described for *Arabidopsis thaliana*, due to the fact that this was the very first model plant for which a large-scale SAG

Functional Approaches to Study Leaf Senescence in Sunflower 73

The three selected genes did not show significant differences between the evaluated conditions at the sampling times tested (63 days post-emergence) (Table 1). It is worth noting that the target genes showed high expression levels even in controls plants with values close to the water stressed samples. Thus, these genes were probably induced by internal plant factors at an early time point, prior to the tested time in that assay. On the other hand, sampling for the incidence of head excision assessment on senescence could be consistent with an early stage of bud development in which there would be no evident differences between the two conditions (Zavaleta-Mancera et al. 1999a; Zavaleta-Mancera et

**SAGs genes (Gepstein et al. 2003) RGs genes (Fernandez et al. 2011)** 

**Treatment Samples Cq CV Cq CV Cq CV Cq CV Cq CV** 

C.L1 3 30.49 2.5 31.06 5.0 28.74 3.9 33.69 1.7 30.08 2.6

C.L2 3 30.00 0.8 30.19 3.8 27.42 4.5 34.20 1.9 25.57 7.2

FE.L1 3 30.46 3.9 28.96 4.3 26.42 6.6 33.52 2.9 27.16 1.7

FE.L2 3 29.28 0.6 29.13 1.1 24.84 2.7 33.32 2.6 26.73 12.9

D.L1 3 30.45 2.1 30.31 1.7 26.10 5.1 33.67 0.2 27.80 6.7

D.L2 3 29.75 2.0 29.12 5.5 24.98 1.5 33.38 4.5 30.07 5.7

three biological replicates per treatment (Fernandez et al. 2011).

Table 1. Average Cq and CV value for R2, D3 and D4 genes and the two best ranked RGs for

As a result from these analyses, the adjustment of the sampling time and frequency turns out as a highly critical point in studying gene expression profiling of candidate genes, according to the treatments on evaluation. Earlier samplings are necessary to detect the trigger moment of different candidate genes for leaf senescence process in sunflower. Considering Table 1, it is worth mentioning that relative quantification of a putative SAG would be overestimated if EF-1 (AN CAA37212.1) would have been used as a single reference gene, which reinforces the importance of normalizing against two or more experimentally validated RG when quantifying transcripts (Fernandez et al. 2011). In order to reach a wider search of new candidate genes, an additional set of new published genes were considered and their predicted functionality was evaluated with the aim to give new insights into this process. For a preliminary detection of potential SAGs, classical macromolecular degradation SAGs were discarded of our analysis because they are probably not associated with early leaf senescence, but with induced changes later in the time course of the process. In this sense, Chlorophyll-Binding Proteins (CBP) were first isolated in soybean (Guiamet et al. 1991) whereas SAGs N4 and SAG12 were detected by differential screen of *Arabidopsis* leaf senescence cDNA libraries (Gan & Amasino 1995; Park

**D4 (AN At1g18210)**

**α-TUB (AN AF401481.1)** 

**EF-1 (AN CAA37212.1)** 

**D3 (AN At5g60360)**

al. 1999b; Thomas & Donnisson 2000).

**(AN At4g32940)**

**R2** 

transcriptome was available (Gepstein et al. 2003). For this purpose six candidate SAGs were selected from this plant model (Moschen 2009) to search for orthologous genes in the sunflower EST database using the tblastx algorithm (Altschul et al. 1990), employing bioinformatics tools locally installed and developed. Sequences showing significant similarity parameters were selected and confirmed. Specific oligonucleotides were designed to amplify fragments of approximately 150 bp for further evaluation by quantitative PCR. In a previous study, we have reported the evaluation and identification of a panel of eight reference genes for their application to transcriptional analysis of the leaf senescence process, thus enabling the use of genuine reference genes in ongoing expression studies (Fernandez et al. 2011). Exploratory studies of senescence by qPCR comparing two treatments which affect the rate of leaf senescence were performed: water stress and head excision, relative to a control condition. Samples were taken from two leaves of different ages, leaf 15 and 25 in order to identify functional markers for this process. Two of the selected genes, a gamma vacuolar processing enzyme (AN At5g60360) (D3 gene) involved in the maturation and activation of vacuolar proteins and an aleurain protease AALP, (AN At1g18210) (D4 gene), belonging to the cystein-protease family are classified in the group of macromolecular degradation and recycling; the third gene, a calcium binding protein (AN At4g32940) (R2 gene) belongs to the group of regulatory genes (Gepstein et al. 2003). Furthermore two reference genes were evaluated against these conditions for relative expression studies, Elongation Factor 1-α (AN) and α-Tubuline, selected from a previous study of the performance of different reference genes against these experimental conditions in sunflower (Fernandez et al. 2011). Alfa tubuline (α-Tubuline) showed the most stable behavior; therefore, it was selected as internal control in further analysis of expression of these SAGs (Figure 1).

Fig. 1. Average Cq of analyzed SAGs genes normalizing against a-TUB as RG. Error bars show standard deviation (Fernandez et al. 2011).

transcriptome was available (Gepstein et al. 2003). For this purpose six candidate SAGs were selected from this plant model (Moschen 2009) to search for orthologous genes in the sunflower EST database using the tblastx algorithm (Altschul et al. 1990), employing bioinformatics tools locally installed and developed. Sequences showing significant similarity parameters were selected and confirmed. Specific oligonucleotides were designed to amplify fragments of approximately 150 bp for further evaluation by quantitative PCR. In a previous study, we have reported the evaluation and identification of a panel of eight reference genes for their application to transcriptional analysis of the leaf senescence process, thus enabling the use of genuine reference genes in ongoing expression studies (Fernandez et al. 2011). Exploratory studies of senescence by qPCR comparing two treatments which affect the rate of leaf senescence were performed: water stress and head excision, relative to a control condition. Samples were taken from two leaves of different ages, leaf 15 and 25 in order to identify functional markers for this process. Two of the selected genes, a gamma vacuolar processing enzyme (AN At5g60360) (D3 gene) involved in the maturation and activation of vacuolar proteins and an aleurain protease AALP, (AN At1g18210) (D4 gene), belonging to the cystein-protease family are classified in the group of macromolecular degradation and recycling; the third gene, a calcium binding protein (AN At4g32940) (R2 gene) belongs to the group of regulatory genes (Gepstein et al. 2003). Furthermore two reference genes were evaluated against these conditions for relative expression studies, Elongation Factor 1-α (AN) and α-Tubuline, selected from a previous study of the performance of different reference genes against these experimental conditions in sunflower (Fernandez et al. 2011). Alfa tubuline (α-Tubuline) showed the most stable behavior; therefore, it was selected as internal control in further analysis of expression of

Fig. 1. Average Cq of analyzed SAGs genes normalizing against a-TUB as RG. Error bars

show standard deviation (Fernandez et al. 2011).

these SAGs (Figure 1).

The three selected genes did not show significant differences between the evaluated conditions at the sampling times tested (63 days post-emergence) (Table 1). It is worth noting that the target genes showed high expression levels even in controls plants with values close to the water stressed samples. Thus, these genes were probably induced by internal plant factors at an early time point, prior to the tested time in that assay. On the other hand, sampling for the incidence of head excision assessment on senescence could be consistent with an early stage of bud development in which there would be no evident differences between the two conditions (Zavaleta-Mancera et al. 1999a; Zavaleta-Mancera et al. 1999b; Thomas & Donnisson 2000).


Table 1. Average Cq and CV value for R2, D3 and D4 genes and the two best ranked RGs for three biological replicates per treatment (Fernandez et al. 2011).

As a result from these analyses, the adjustment of the sampling time and frequency turns out as a highly critical point in studying gene expression profiling of candidate genes, according to the treatments on evaluation. Earlier samplings are necessary to detect the trigger moment of different candidate genes for leaf senescence process in sunflower. Considering Table 1, it is worth mentioning that relative quantification of a putative SAG would be overestimated if EF-1 (AN CAA37212.1) would have been used as a single reference gene, which reinforces the importance of normalizing against two or more experimentally validated RG when quantifying transcripts (Fernandez et al. 2011). In order to reach a wider search of new candidate genes, an additional set of new published genes were considered and their predicted functionality was evaluated with the aim to give new insights into this process. For a preliminary detection of potential SAGs, classical macromolecular degradation SAGs were discarded of our analysis because they are probably not associated with early leaf senescence, but with induced changes later in the time course of the process. In this sense, Chlorophyll-Binding Proteins (CBP) were first isolated in soybean (Guiamet et al. 1991) whereas SAGs N4 and SAG12 were detected by differential screen of *Arabidopsis* leaf senescence cDNA libraries (Gan & Amasino 1995; Park

Functional Approaches to Study Leaf Senescence in Sunflower 75

of grain filling has already begun (Figure 2). These results are consistent with those observed in *Arabidopsis*, and turn this gene a potential functional marker of the progress of senescence, representing an important tool for future implications in the sunflower crop improvement (Moschen et al. 2010). In order to confirm *in-situ* the functionality of this putative ORE1 gene in sunflower, a comparative bioinformatics analysis has been performed using the Blastx algorithm (Altschul et al. 1990), searching for proteins in the database at the National Center for Biotechnology Information NCBI (http://www.ncbi.nlm.nih.gov/), using as query the nucleotide sequence of putative sunflower ORE1. These results showed a high similarity with ORE *Arabidopsis* protein (GI 15241819) suggesting a possible role of this gene as NAC transcription factor. Moreover, searches for functional protein domains in Pfam (http://pfam.sanger.ac.uk/) revealed that main protein domain in sunflower ORE1-like gene sequence corresponds to the family of NAM transcription factors (No Apical Meristem) (pfam02365), as well as the *Arabidopsis ORE1 sequence* pfam02365. Figure 3 shows *Arabidopsis* alignments and putative sunflower ORE1 proteins against Pfam NAC domain. Others relevant *in-silico* candidates for a putative sunflower SAG are: RAV1 gene, a transcription factor whose expression is closely associated with leaf maturation and senescence (Woo et al. 2010), which has been detected with a high score level and statistically low E-value, and CAT2, a member of a small gene H2O2 detoxifying enzyme family, widely characterized in *Arabidopsis* (Gergoff et al. 2010;

Fig. 2. Differential expression of putative sunflower *ORE1* gene in subsequent samplings, taking as control condition sampling number 1 and referred to α-TUB expression level

(Moschen et al. 2010).

Smykowski et al. 2010), although not yet tested in sunflower.

et al. 1998). They encode an apparent cysteine proteinase and their expression is highly senescence specific (Lohman et al. 1994; Gan & Amasino 1995; Martinez et al. 2007) mainly localized in small senescence associated vacuoles (Saeed et al. 2003; Otegui et al. 2005). However, neither SAG12 nor SEN4 match any full sequence in sunflower with a high identity score level. For this reason, a second set of candidate SAGs (OsNAC5, WRKY6, ORS1 YUCCA6, among others) (Ülker & Somssich 2004; Balazadeh et al. 2011; Kim et al. 2011; Song et al. 2011) was compared against *Helianthus annuus* unigene collection but a low score level to *Helianthus annuus* sequences was detected. Therefore, other candidate genes were added to be functionally tested for early leaf senescence in sunflower. The special case of transcription factors (TFs) as crucial regulators of gene expression by binding to distinct cis-elements, generally located in the 5' upstream regulatory regions of target genes, were specially considered to detect early senescence leaf makers (Balazadeh et al. 2008). NAC transcription factors related to senescence have been recently identified in model species and they play a relevant role in the regulation of development of leaf senescence related to programmed cell death (Olsen et al. 2005; Kim et al. 2009; Balazadeh et al. 2010; Hu et al. 2010; Nuruzzaman et al. 2010; Balazadeh et al. 2011). A single one NAC gene (AtNAP), also called NAC2 or ANAC029 (Guo & Gan 2006), has been the main one identified to control leaf senescence, although approximately 20 NAC genes in *Arabidopsis* shown high expression in senescing leaves (Guo et al. 2004; Lin & Wu 2004). ROS reagents acting as senescence stimulus were also reported within a narrow cross talk involving hormones and TFs both in natural and stress-related senescence (Rivero et al. 2007; Khanna-Chopra 2011), indicating that elevated ROS levels might be detected as a potential signal of senescence induction. Under this assumption *ORE1*, a NAC transcription factor that has been extensively studied in recent years, has been described as strongly related to leaf senescence, probably coevolving genes with ORS1 (Ooka et al. 2003). This TF can be considered a new further positive regulator of senescence in conjunction with AtNAP (Balazadeh et al. 2011), controlling leaf senescence in *Brassicacea*e. In *Arabidopsis*, *ORE1* mutants show a delay in leaf senescence whereas overexpression through an inductive promoter, accelerates senescence in relation to wild type plants (Balazadeh et al. 2010) and the forest tree *Populus trichocarpa* in which approximately 2,900 TFs were reported (Hu et al. 2010) and will be soon tested for sunflower candidate SAG detection. Microarray studies showed that 46% of up regulated genes in *Arabidopsis ORE1* overexpression lines, are known as senescence-associated genes, including many genes previously reported as senescence regulated, suggesting an important role in the development of the senescence process (Balazadeh et al. 2010). In wheat, it was reported that NAC TFs not only accelerate senescence but also improve nutrient remobilization by increasing protein, iron and zinc content (Uauy et al. 2006). ORE1 expression is under control of the ethylene signaling pathway and is subjected to regulation by miRNA164, being negatively regulated. When the leaf is young, miR164 transcripts remain at high levels regulating the expression of ORE1 but during the leaf aging process, its expression gradually decreases, thus increasing the expression of ORE1 (Kim et al. 2009).

In sunflower, a sequence similar ORE1 has been detected in the *Helianthus annuus* unigene collection developed at INTA (ATGC Sunflower Database: http://bioinformatica.inta.gov.ar/ATGC) with a Blast score of 96 and E-value of e-10 (Altschul et al. 1990). Expression profiles studies at different sunflower developmental stages showed a significant increase of putative ORE1 transcripts in samples close to anthesis stage, prior to the start of the first symptoms of senescence, when the critical period

et al. 1998). They encode an apparent cysteine proteinase and their expression is highly senescence specific (Lohman et al. 1994; Gan & Amasino 1995; Martinez et al. 2007) mainly localized in small senescence associated vacuoles (Saeed et al. 2003; Otegui et al. 2005). However, neither SAG12 nor SEN4 match any full sequence in sunflower with a high identity score level. For this reason, a second set of candidate SAGs (OsNAC5, WRKY6, ORS1 YUCCA6, among others) (Ülker & Somssich 2004; Balazadeh et al. 2011; Kim et al. 2011; Song et al. 2011) was compared against *Helianthus annuus* unigene collection but a low score level to *Helianthus annuus* sequences was detected. Therefore, other candidate genes were added to be functionally tested for early leaf senescence in sunflower. The special case of transcription factors (TFs) as crucial regulators of gene expression by binding to distinct cis-elements, generally located in the 5' upstream regulatory regions of target genes, were specially considered to detect early senescence leaf makers (Balazadeh et al. 2008). NAC transcription factors related to senescence have been recently identified in model species and they play a relevant role in the regulation of development of leaf senescence related to programmed cell death (Olsen et al. 2005; Kim et al. 2009; Balazadeh et al. 2010; Hu et al. 2010; Nuruzzaman et al. 2010; Balazadeh et al. 2011). A single one NAC gene (AtNAP), also called NAC2 or ANAC029 (Guo & Gan 2006), has been the main one identified to control leaf senescence, although approximately 20 NAC genes in *Arabidopsis* shown high expression in senescing leaves (Guo et al. 2004; Lin & Wu 2004). ROS reagents acting as senescence stimulus were also reported within a narrow cross talk involving hormones and TFs both in natural and stress-related senescence (Rivero et al. 2007; Khanna-Chopra 2011), indicating that elevated ROS levels might be detected as a potential signal of senescence induction. Under this assumption *ORE1*, a NAC transcription factor that has been extensively studied in recent years, has been described as strongly related to leaf senescence, probably coevolving genes with ORS1 (Ooka et al. 2003). This TF can be considered a new further positive regulator of senescence in conjunction with AtNAP (Balazadeh et al. 2011), controlling leaf senescence in *Brassicacea*e. In *Arabidopsis*, *ORE1* mutants show a delay in leaf senescence whereas overexpression through an inductive promoter, accelerates senescence in relation to wild type plants (Balazadeh et al. 2010) and the forest tree *Populus trichocarpa* in which approximately 2,900 TFs were reported (Hu et al. 2010) and will be soon tested for sunflower candidate SAG detection. Microarray studies showed that 46% of up regulated genes in *Arabidopsis ORE1* overexpression lines, are known as senescence-associated genes, including many genes previously reported as senescence regulated, suggesting an important role in the development of the senescence process (Balazadeh et al. 2010). In wheat, it was reported that NAC TFs not only accelerate senescence but also improve nutrient remobilization by increasing protein, iron and zinc content (Uauy et al. 2006). ORE1 expression is under control of the ethylene signaling pathway and is subjected to regulation by miRNA164, being negatively regulated. When the leaf is young, miR164 transcripts remain at high levels regulating the expression of ORE1 but during the leaf aging process, its expression gradually decreases, thus increasing the expression of ORE1 (Kim et al. 2009). In sunflower, a sequence similar ORE1 has been detected in the *Helianthus annuus* unigene collection developed at INTA (ATGC Sunflower Database: http://bioinformatica.inta.gov.ar/ATGC) with a Blast score of 96 and E-value of e-10 (Altschul et al. 1990). Expression profiles studies at different sunflower developmental stages showed a significant increase of putative ORE1 transcripts in samples close to anthesis stage, prior to the start of the first symptoms of senescence, when the critical period of grain filling has already begun (Figure 2). These results are consistent with those observed in *Arabidopsis*, and turn this gene a potential functional marker of the progress of senescence, representing an important tool for future implications in the sunflower crop improvement (Moschen et al. 2010). In order to confirm *in-situ* the functionality of this putative ORE1 gene in sunflower, a comparative bioinformatics analysis has been performed using the Blastx algorithm (Altschul et al. 1990), searching for proteins in the database at the National Center for Biotechnology Information NCBI (http://www.ncbi.nlm.nih.gov/), using as query the nucleotide sequence of putative sunflower ORE1. These results showed a high similarity with ORE *Arabidopsis* protein (GI 15241819) suggesting a possible role of this gene as NAC transcription factor. Moreover, searches for functional protein domains in Pfam (http://pfam.sanger.ac.uk/) revealed that main protein domain in sunflower ORE1-like gene sequence corresponds to the family of NAM transcription factors (No Apical Meristem) (pfam02365), as well as the *Arabidopsis ORE1 sequence* pfam02365. Figure 3 shows *Arabidopsis* alignments and putative sunflower ORE1 proteins against Pfam NAC domain. Others relevant *in-silico* candidates for a putative sunflower SAG are: RAV1 gene, a transcription factor whose expression is closely associated with leaf maturation and senescence (Woo et al. 2010), which has been detected with a high score level and statistically low E-value, and CAT2, a member of a small gene H2O2 detoxifying enzyme family, widely characterized in *Arabidopsis* (Gergoff et al. 2010; Smykowski et al. 2010), although not yet tested in sunflower.

Fig. 2. Differential expression of putative sunflower *ORE1* gene in subsequent samplings, taking as control condition sampling number 1 and referred to α-TUB expression level (Moschen et al. 2010).

Functional Approaches to Study Leaf Senescence in Sunflower 77

Microarrays using ESTs and full length gene sequences allowed SAGs identification during leaf senescence at the genome-wide scale in *Arabidopsis* and other plants (Lim et al. 2007). In parallel, other high-throughput system has been assayed in other species: cDNA macro and microarray were developed for sunflower to study sunflower seed development (Hewezi et al. 2006) and the response to biotic (Alignan et al. 2006), and abiotic stresses (Hewezi et al. 2006; Roche et al. 2007; Fernandez et al. 2008). This last work reported for the first time, a concerted study on gene expression in early responses to chilling and salinity using a fluorescence microarray assay based on organ-specific unigenes in sunflower. These two strategies, although useful, are limited to the analysis of a limited set of genes. Currently, the shortage of candidate genes underlying agronomically important traits represents one of the main drawbacks in sunflower molecular breeding. In this context, functional tools which allow concerted transcriptional studies, as high density oligonucleotide microarray, strongly support the discovery and characterization of novel genes. Oligonucleotide-based chips not only allow the analysis for a whole transcriptome but they are also considered more accurate than cDNA-based chips due to the reduction of manipulation steps (Larkin et al. 2005; Lai et al. 2006). The possibility to implement this technology on any custom array system like Agilent, Nimblegen, and others, has the potential to create a very useful tool for gene discovery in orphan crops (Nazar et al. 2010; Ophir et al. 2010). In addition, the use of longer probe format represents a major advantage of Agilent oligonucleotide microarrays over others technologies based on a higher stability in the presence of sequence mismatches, being consequently, more suitable for the analysis of highly polymorphic regions

In general, the analysis of complex biological processes based on a gene by gene approach seldom leads to limited or erroneous conclusions requiring an alternative approach based on systemic association studies. Under this assumption, new insights into molecular senescence events might be cleared up by high-resolution microarray data, for example, considering different points of leaf development (Breeze et al. 2011) or predicting putative SAGs by tissue and functional categories (Thomas et al. 2009). In our lab, a public and proprietary datasets of *H. annuus L.* ESTs have been used to create a comprehensive sunflower unigene collection. This dataset comprises 34 cDNAs libraries available from different cultivars, various tissues and anatomical parts, from plants grown at different

Figure 4 describes the routines applied for the *H. annuus L* unigene collection design.

A Digital Gene Expression Profile (Audic & Claverie 1997) was assayed with the EST public data in order to detect any bias that would be pseudo-enriching the gene index by full representation of one library over another considering full public ESTs derived from public collections (Table 2). This analysis ("digi-Northern") detected that ESTs were equally represented among differential cDNA libraries, showing that the *H. annuus* unigene collection generated would be fully represented by different transcripts, lacking of a potential enrichment or overestimation among organ-specific ESTs libraries. This unigene collection was used to design the first custom sunflower oligonucleotide-based microarray based on Agilent technology as a main goal for functional genomics approaches, generated within the frame of a collaborative project involving Argentinean research sunflower groups (Sunflower PAE Consortium), Facultad de Agronomía (UBA) and the Bioinformatics facility at the Principe Felipe Institute, Valencia , España. A Chado-based database (Mungall et al.

(Hardiman 2004).

physiological conditions.


Fig. 3. *Arabidopsis* NAM domain and putative *sunflower* ORE1 protein alignment (pfam02365) (http://www.ncbi.nlm.nih.gov/cdd/).

As mentioned above, the execution of the senescence process consists of multiple interconnecting pathways which regulate and/or modulate this series of orderly steps; therefore different transcription factors play an important role as regulators of these pathways. Recently, a list of transcription factors that regulate leaf senescence in *Arabidopsis* has been published (Balazadeh et al. 2008). The search for tentative orthologous genes in the *Helianthus annuus* unigene collection, using Blast algorithm, led to the identification of 42 genes with a significant score value to transcription factors like NAC, MYB, WRKY, ARP among others, some of these genes are being studied their expression patterns by qPCR.

## **3. Concerted gene expression studies to elucidate sunflower senescence process**

Although microarray technology started a new era of high-throughput transcriptomic analysis approximately ten years ago, starting with 8,000 printed genes by Affymetrix in *Arabidopsis thaliana* (Zhu & Wang 2000) and later on scaling up to 45,000 printed genes in rice (Jung et al. 2008) and 90,000 in *Brassica* (Trick et al. 2009), next generation sequencing (NGS) technologies are nowadays opening a new era of even deeper understanding of genomics and transcriptomics in different species . However, for the foreseeable future both technologies will coexist each focusing on different tasks, or by complementing biological and value information (Fenart et al. 2010) or by designing dedicated oligonucleotide arrays to support functional studies on a specified pathway/developmental stage (Kusnierczyk et al. 2008; Cosio & Dunand 2010; Ott et al. 2010). One obvious application of microarray technology is the transcriptional profiling in species that have neither their own genome sequenced nor a reference genome from a closely related species. For some of these species a commercial microarray based on an existing own-design are available (Agilent, Affimetrix, Nimblegen, etc) (Close et al. 2004; Li et al. 2008; Martinez-Godoy et al. 2008; Mascarrell-Creus et al. 2009; Trick et al. 2009; Booman et al. 2010; Curtiss et al. 2011). Sunflower is a species that fits into this framework, even though a genome sequence initiative is in progress (Kane et al. 2011), there is no reference genome available. In this case, the only source of functional information is limited to ESTs databases, which in the case of cultivated sunflower is rather extensive, more than 133,000 ESTs are publicly available (http://ncbi.nlm.nih.gov/dbEST/dbEST\_summary.html) covering libraries prepared from several lines and cultivars (Table2). However, it should also be noted that ESTs libraries tend to be significantly contaminated with vector sequences and chimeras, and have relatively low quality DNA information derived from the library sequencing strategy which prioritizes obtaining a large number of single pass sequences, being necessary to standardize a set of bioinformatics routines in order to clean and decontaminate public raw sequences (Figure 4).

As mentioned above, the execution of the senescence process consists of multiple interconnecting pathways which regulate and/or modulate this series of orderly steps; therefore different transcription factors play an important role as regulators of these pathways. Recently, a list of transcription factors that regulate leaf senescence in *Arabidopsis* has been published (Balazadeh et al. 2008). The search for tentative orthologous genes in the *Helianthus annuus* unigene collection, using Blast algorithm, led to the identification of 42 genes with a significant score value to transcription factors like NAC, MYB, WRKY, ARP among others, some of these genes are being studied their expression patterns by qPCR.

**3. Concerted gene expression studies to elucidate sunflower senescence** 

Although microarray technology started a new era of high-throughput transcriptomic analysis approximately ten years ago, starting with 8,000 printed genes by Affymetrix in *Arabidopsis thaliana* (Zhu & Wang 2000) and later on scaling up to 45,000 printed genes in rice (Jung et al. 2008) and 90,000 in *Brassica* (Trick et al. 2009), next generation sequencing (NGS) technologies are nowadays opening a new era of even deeper understanding of genomics and transcriptomics in different species . However, for the foreseeable future both technologies will coexist each focusing on different tasks, or by complementing biological and value information (Fenart et al. 2010) or by designing dedicated oligonucleotide arrays to support functional studies on a specified pathway/developmental stage (Kusnierczyk et al. 2008; Cosio & Dunand 2010; Ott et al. 2010). One obvious application of microarray technology is the transcriptional profiling in species that have neither their own genome sequenced nor a reference genome from a closely related species. For some of these species a commercial microarray based on an existing own-design are available (Agilent, Affimetrix, Nimblegen, etc) (Close et al. 2004; Li et al. 2008; Martinez-Godoy et al. 2008; Mascarrell-Creus et al. 2009; Trick et al. 2009; Booman et al. 2010; Curtiss et al. 2011). Sunflower is a species that fits into this framework, even though a genome sequence initiative is in progress (Kane et al. 2011), there is no reference genome available. In this case, the only source of functional information is limited to ESTs databases, which in the case of cultivated sunflower is rather extensive, more than 133,000 ESTs are publicly available (http://ncbi.nlm.nih.gov/dbEST/dbEST\_summary.html) covering libraries prepared from several lines and cultivars (Table2). However, it should also be noted that ESTs libraries tend to be significantly contaminated with vector sequences and chimeras, and have relatively low quality DNA information derived from the library sequencing strategy which prioritizes obtaining a large number of single pass sequences, being necessary to standardize a set of bioinformatics routines in order to clean and decontaminate

Fig. 3. *Arabidopsis* NAM domain and putative *sunflower* ORE1 protein alignment

(pfam02365) (http://www.ncbi.nlm.nih.gov/cdd/).

**process** 

public raw sequences (Figure 4).

Microarrays using ESTs and full length gene sequences allowed SAGs identification during leaf senescence at the genome-wide scale in *Arabidopsis* and other plants (Lim et al. 2007). In parallel, other high-throughput system has been assayed in other species: cDNA macro and microarray were developed for sunflower to study sunflower seed development (Hewezi et al. 2006) and the response to biotic (Alignan et al. 2006), and abiotic stresses (Hewezi et al. 2006; Roche et al. 2007; Fernandez et al. 2008). This last work reported for the first time, a concerted study on gene expression in early responses to chilling and salinity using a fluorescence microarray assay based on organ-specific unigenes in sunflower. These two strategies, although useful, are limited to the analysis of a limited set of genes. Currently, the shortage of candidate genes underlying agronomically important traits represents one of the main drawbacks in sunflower molecular breeding. In this context, functional tools which allow concerted transcriptional studies, as high density oligonucleotide microarray, strongly support the discovery and characterization of novel genes. Oligonucleotide-based chips not only allow the analysis for a whole transcriptome but they are also considered more accurate than cDNA-based chips due to the reduction of manipulation steps (Larkin et al. 2005; Lai et al. 2006). The possibility to implement this technology on any custom array system like Agilent, Nimblegen, and others, has the potential to create a very useful tool for gene discovery in orphan crops (Nazar et al. 2010; Ophir et al. 2010). In addition, the use of longer probe format represents a major advantage of Agilent oligonucleotide microarrays over others technologies based on a higher stability in the presence of sequence mismatches, being consequently, more suitable for the analysis of highly polymorphic regions (Hardiman 2004).

In general, the analysis of complex biological processes based on a gene by gene approach seldom leads to limited or erroneous conclusions requiring an alternative approach based on systemic association studies. Under this assumption, new insights into molecular senescence events might be cleared up by high-resolution microarray data, for example, considering different points of leaf development (Breeze et al. 2011) or predicting putative SAGs by tissue and functional categories (Thomas et al. 2009). In our lab, a public and proprietary datasets of *H. annuus L.* ESTs have been used to create a comprehensive sunflower unigene collection. This dataset comprises 34 cDNAs libraries available from different cultivars, various tissues and anatomical parts, from plants grown at different physiological conditions.

Figure 4 describes the routines applied for the *H. annuus L* unigene collection design.

A Digital Gene Expression Profile (Audic & Claverie 1997) was assayed with the EST public data in order to detect any bias that would be pseudo-enriching the gene index by full representation of one library over another considering full public ESTs derived from public collections (Table 2). This analysis ("digi-Northern") detected that ESTs were equally represented among differential cDNA libraries, showing that the *H. annuus* unigene collection generated would be fully represented by different transcripts, lacking of a potential enrichment or overestimation among organ-specific ESTs libraries. This unigene collection was used to design the first custom sunflower oligonucleotide-based microarray based on Agilent technology as a main goal for functional genomics approaches, generated within the frame of a collaborative project involving Argentinean research sunflower groups (Sunflower PAE Consortium), Facultad de Agronomía (UBA) and the Bioinformatics facility at the Principe Felipe Institute, Valencia , España. A Chado-based database (Mungall et al.

Functional Approaches to Study Leaf Senescence in Sunflower 79

methodology. Differential gene expression was also carried out using the limma package (Smyth 2004). Multiple testing adjustments of p-values was done according to Benjamini and Hochberg methodology (Benjamini & Hochberg 1995). Gene set analysis was carried out according to the Gene Ontology terms using FatiScan (Al-Shahrour et al. 2007)

**Library ID Developmental stage**

strains/cultivars

responsive genes in sunflower

stress/chemical induction

shoots/hulls/flowers environmental

HaSSH Molecular characterization of phosphorus-

HaHeaS heart-shaped embryo vs cotyledonary embryo

CCF (STU) EST sequences from several different

sunflower girasol silvestre (wild sunflower)

HaDevS1 4 days after self-pollination embryo HaDevS2 7 days after self-pollination embryo

HaDevR5 4 days after self-pollination embryo HaDevR8 15 days after self-pollination embryo HaDis unknown/cotyledons/ (Genoplante)

HaR INTA: organ-specific cDNA libraries (root) HaT INTA: organ-specific cDNA libraries (stem)

HaEF INTA: organ-specific cDNA libraries (early flower) HaF INTA: organ-specific cDNA libraries (flower) HaH INTA: organ-specific cDNA libraries (leaf)

Table 2. Public cDNA libraries deposited in GenBank for which *H. annuus* unigene collection

HaHeaR heart-shaped embryo HaCotR cotyledonary embryo HaGlbR globular embryo

HaDevR1 leaves HaDevR2 terminal bud

HaDevR3 stem HaDevR6 embryo

HaSemS4 hypocotyl HaDpsR1 hypocotyl

HaDplR protoplast HaERF embryo HaERS embryo

HaDplR2 hypocotyl 1-5 days

integrated in Babelomics suite (Al-Shahrour et al. 2005).

QH-RHA 280/QH\_ABCDI

CHA(XYZ) common wild

sunflower RHA801

was designed.

2007) and a visualization tool call ATGC (Clavijo et al., unpublished) was developed to integrate and browse sunflower transcriptome information. Figure 5 shows the output of the ATGC interface for one functional annotated sunflower unigene.

Fig. 4. Bioinformatics routines applied to design *Helianthus annuus* unigene collection (http://bionformatica.inta.gov.ar/ ATGC/).

Sunflower gene expression chip probes were designed using eArray® web application (Agilent Technologies). For this instance, two probe sets were designed: one including noncontrol specific probes for the sequences of sunflower unigene collection and a second control probe set consisting in 74 probes derived from 80 differentially expressed sunflower genes identified in a previously work (Fernandez et al. 2008). The latest group was used as 'Replicate Controls' with 10 replicates each. To utilize the full capacity of the microarray, probes were randomly selected to be represented in duplicate in the final design, which also included Agilent Technologies' standard panel of quality control and spike-in probes. This design was then used to manufacture microarrays using Agilent SurePrint™ Technology in the 4 x 44 format. Agilent's microarrays include the Spike-In Kit that consists of a set of 10 positive control transcripts optimized to anneal to complementary probes on the microarray, minimizing self-hybridization or cross-hybridization. This work contemplates the microarray validation through diverse differential expression analysis in order to analyze early senescence in sunflower through a classical approach and a pipeline-based

2007) and a visualization tool call ATGC (Clavijo et al., unpublished) was developed to integrate and browse sunflower transcriptome information. Figure 5 shows the output of the

Fig. 4. Bioinformatics routines applied to design *Helianthus annuus* unigene collection

Sunflower gene expression chip probes were designed using eArray® web application (Agilent Technologies). For this instance, two probe sets were designed: one including noncontrol specific probes for the sequences of sunflower unigene collection and a second control probe set consisting in 74 probes derived from 80 differentially expressed sunflower genes identified in a previously work (Fernandez et al. 2008). The latest group was used as 'Replicate Controls' with 10 replicates each. To utilize the full capacity of the microarray, probes were randomly selected to be represented in duplicate in the final design, which also included Agilent Technologies' standard panel of quality control and spike-in probes. This design was then used to manufacture microarrays using Agilent SurePrint™ Technology in the 4 x 44 format. Agilent's microarrays include the Spike-In Kit that consists of a set of 10 positive control transcripts optimized to anneal to complementary probes on the microarray, minimizing self-hybridization or cross-hybridization. This work contemplates the microarray validation through diverse differential expression analysis in order to analyze early senescence in sunflower through a classical approach and a pipeline-based

(http://bionformatica.inta.gov.ar/ ATGC/).

ATGC interface for one functional annotated sunflower unigene.

methodology. Differential gene expression was also carried out using the limma package (Smyth 2004). Multiple testing adjustments of p-values was done according to Benjamini and Hochberg methodology (Benjamini & Hochberg 1995). Gene set analysis was carried out according to the Gene Ontology terms using FatiScan (Al-Shahrour et al. 2007) integrated in Babelomics suite (Al-Shahrour et al. 2005).


Table 2. Public cDNA libraries deposited in GenBank for which *H. annuus* unigene collection was designed.

Functional Approaches to Study Leaf Senescence in Sunflower 81

Knowing the time of onset the the cascade of events that trigger senescence could determine the causes of this process and generate molecular tools to facilitate future interventions on it, useful for application in assisted breeding of this crop with major growing oil impact in the

The sunflower chip, designed within a PAE Consortium made up of six laboratories and one private company working in different areas of research and development, was validated by means of the analysis of global changes in gene expression profiles in response to water deficit as a physiological event which induces senescence, taken as a model experiment, for which reference genes have also been previously identified (Fernandez et al. 2011). This high-throughput transcriptome tool will allow the discovery, identification and analysis of a new set of putative SAGs for sunflower which would bring novel insights for this process. The integrated analysis of transcriptional and metabolic profiles will allow the identification of concerted regulation of distinct metabolic pathways facilitating the discovery of robust candidate genes and key metabolic pathways involved in the outbreak of the early senescence process in sunflower leaves. We expect that the integration of the information generated by this project will allow the construction of the quantitative predictive model of senescence in sunflower, under field and greenhouse conditions, which is required to interpret the regulation of the underlying complex biological processes. There will also be practical applications in directed gene discovery for other important agronomic traits involving plant responses to biotic and abiotic stresses. Finally, this project will have impact based in the establishment of microarray technologies and metabolic analysis, as well as on the knowledge of appropriated statistical and bioinformatics procedures supporting

functional genomics ranging from the transcriptome to the metabolome.

Científicas y Técnicas (CONICET, Argentina) and INTA researchers.

systems." *BMC Bioinformatics* 8(1): 114.

This research was supported by CONICET PIP 5788, ANPCyT/FONCYT, Préstamo BID PICT 15-32905 and PICT 0960, INTA-PE AEBIO 241001and 245001, INTA-PE AEBIO 245732, INTA-AEBI0 243532, INTA PN CER 1336 and UNMdP, AGR212, AGR260. Lic. Sebastián Moschen holds a fellowship from ANPCyT to support his PhD studies whereas Dr. PdCF, Dr. RAH, Dr. NBP are career members of the Consejo Nacional de Investigaciones

Aguera, E., P. Cabello and P. de la Haba (2010). "Induction of leaf senescence by low

Aguirrezábal, L. A. N. A., Y. Lavaud, G. A. A. Dosio, N. Izquierdo, F. Andrade and L.

sunflower weight per seed and oil concentration." *Crop Science* 43: 152-161. Al-Shahrour, F., L. Arbiza, H. Dopazo, J. Huerta-Cepas, P. Minguez, D. Montaner and J.

nitrogen nutrition in sunflower (*Helianthus annuus)* plants." *Physiol Plant* 138(3):

González (2003). "Intercepted solar radiation during seed filling determines

Dopazo (2007). "From genes to functional classes in the study of biological

**4. Conclusions and perspectives** 

world.

**5. Acknowledgment** 

**6. References** 

256-267.

Fig. 5. ATGC view for an annotated sunflower unigene.

## **4. Conclusions and perspectives**

80 Senescence

Fig. 5. ATGC view for an annotated sunflower unigene.

Knowing the time of onset the the cascade of events that trigger senescence could determine the causes of this process and generate molecular tools to facilitate future interventions on it, useful for application in assisted breeding of this crop with major growing oil impact in the world.

The sunflower chip, designed within a PAE Consortium made up of six laboratories and one private company working in different areas of research and development, was validated by means of the analysis of global changes in gene expression profiles in response to water deficit as a physiological event which induces senescence, taken as a model experiment, for which reference genes have also been previously identified (Fernandez et al. 2011). This high-throughput transcriptome tool will allow the discovery, identification and analysis of a new set of putative SAGs for sunflower which would bring novel insights for this process. The integrated analysis of transcriptional and metabolic profiles will allow the identification of concerted regulation of distinct metabolic pathways facilitating the discovery of robust candidate genes and key metabolic pathways involved in the outbreak of the early senescence process in sunflower leaves. We expect that the integration of the information generated by this project will allow the construction of the quantitative predictive model of senescence in sunflower, under field and greenhouse conditions, which is required to interpret the regulation of the underlying complex biological processes. There will also be practical applications in directed gene discovery for other important agronomic traits involving plant responses to biotic and abiotic stresses. Finally, this project will have impact based in the establishment of microarray technologies and metabolic analysis, as well as on the knowledge of appropriated statistical and bioinformatics procedures supporting functional genomics ranging from the transcriptome to the metabolome.

## **5. Acknowledgment**

This research was supported by CONICET PIP 5788, ANPCyT/FONCYT, Préstamo BID PICT 15-32905 and PICT 0960, INTA-PE AEBIO 241001and 245001, INTA-PE AEBIO 245732, INTA-AEBI0 243532, INTA PN CER 1336 and UNMdP, AGR212, AGR260. Lic. Sebastián Moschen holds a fellowship from ANPCyT to support his PhD studies whereas Dr. PdCF, Dr. RAH, Dr. NBP are career members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and INTA researchers.

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**5** 

*France* 

**Plant Ageing,** 

*GRESE EA 4330, Limoges* 

David Delmail1,2 and Pascal Labrousse2

*UMR CNRS 6226 SCR/PNSCM, Rennes* 

*1University of Rennes 1, Lab. of Pharmacognosy & Mycology,* 

*2University of Limoges, Lab. of Botany & Cryptogamy,* 

**a Counteracting Agent to Xenobiotic Stress** 

A xenobiotic can be defined as any chemical or other substance that is not normally found in the ecosystems or that is present at concentrations harmful to all biological organisms. This general definition could be applied to anthropogenic and naturally occurring constituents. Organic contaminants can include pesticides, solvents and petroleum products. Inorganic xenobiotics include heavy metals, nonmetals, metalloids, radionuclides and simple soluble

Indeed, after absorption in plant cell, these toxics induce a broad range of disturbances like competition between elements. But the main effect remains the oxidative stress which disrupts many physiological pathways. Reactive oxygen species which initiate the oxidation are produced through several mechanisms in all cell compartments (Delmail et al., 2009; Thompson et al., 1987). Some reactive oxygen species are less deleterious to the plant cell than others but they can act as initiator of the production of more toxic compounds (Delmail

To prevent from the production of reactive oxygen species, plants can use the senescence process to eliminate the xenobiotics from their organisms. Toxic compounds like radionuclides can be sequestrated by metallothioneins preferentially in vacuoles of specific organs like trichomes and old leaves. Indeed, morphological structures as non-glandular trichomes which are not implied in any physiological process, could store many xenobiotics. Moreover, potentially abscised organs as mature leaves are used to eliminate toxics from the living parts to protect the young organs from any disturbance of the photosynthetic

This chapter will focus on xenobiotics having anthropogenic origins and will address organic and inorganic xenobiotics. Moreover, the origin of the oxidative stress induced by the xenobiotic assimilation, its consequence on the ageing of morphological, physiological and cellular patterns, as well as the functioning of antioxidant pathways, the implication of scavengers and the role of the senescence in reducing the oxidative disturbance, will be

**1. Introduction** 

salts (Schwab, 2005).

et al., 2011c, 2011d).

pathways (Delmail et al., 2011c, 2011d).

discussed in this chapter.


## **Plant Ageing, a Counteracting Agent to Xenobiotic Stress**

David Delmail1,2 and Pascal Labrousse2

*1University of Rennes 1, Lab. of Pharmacognosy & Mycology, UMR CNRS 6226 SCR/PNSCM, Rennes 2University of Limoges, Lab. of Botany & Cryptogamy, GRESE EA 4330, Limoges France* 

## **1. Introduction**

88 Senescence

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integrating environmental signals during the regulation of leaf senescence." *Journal* 

"The RAV1 transcription factor positively regulates leaf senescence in Arabidopsis."

senescent Nicotiana leaves: II Redifferentiation of plastids." *Journal of Experimental* 

senescent Nicotiana leaves: I Reappearance of NADPH-protochlorophyllide oxidoreductase and light-harvesting chlorophyll a/b-binding protein." *Journal of*  A xenobiotic can be defined as any chemical or other substance that is not normally found in the ecosystems or that is present at concentrations harmful to all biological organisms. This general definition could be applied to anthropogenic and naturally occurring constituents. Organic contaminants can include pesticides, solvents and petroleum products. Inorganic xenobiotics include heavy metals, nonmetals, metalloids, radionuclides and simple soluble salts (Schwab, 2005).

Indeed, after absorption in plant cell, these toxics induce a broad range of disturbances like competition between elements. But the main effect remains the oxidative stress which disrupts many physiological pathways. Reactive oxygen species which initiate the oxidation are produced through several mechanisms in all cell compartments (Delmail et al., 2009; Thompson et al., 1987). Some reactive oxygen species are less deleterious to the plant cell than others but they can act as initiator of the production of more toxic compounds (Delmail et al., 2011c, 2011d).

To prevent from the production of reactive oxygen species, plants can use the senescence process to eliminate the xenobiotics from their organisms. Toxic compounds like radionuclides can be sequestrated by metallothioneins preferentially in vacuoles of specific organs like trichomes and old leaves. Indeed, morphological structures as non-glandular trichomes which are not implied in any physiological process, could store many xenobiotics. Moreover, potentially abscised organs as mature leaves are used to eliminate toxics from the living parts to protect the young organs from any disturbance of the photosynthetic pathways (Delmail et al., 2011c, 2011d).

This chapter will focus on xenobiotics having anthropogenic origins and will address organic and inorganic xenobiotics. Moreover, the origin of the oxidative stress induced by the xenobiotic assimilation, its consequence on the ageing of morphological, physiological and cellular patterns, as well as the functioning of antioxidant pathways, the implication of scavengers and the role of the senescence in reducing the oxidative disturbance, will be discussed in this chapter.

Plant Ageing, a Counteracting Agent to Xenobiotic Stress 91

developed from primary ones after oxidation and hydrolysis processes. Moreover, they may be adsorbed on the organic matter in soils and waters, or on microorganisms (which could also absorb and accumulate them). Associations between colloids and xenobiotics could be observed in all natural compartments as a colloidal system may be solid, liquid or gaseous.

Fig. 1. Overview of selective environmental stress factors which could threaten plants. Biotic

and abiotic environmental selective factors are considered.

## **2. The xenobiotics**

A xenobiotic (from the Greek *xenos* "stranger" and *biotic* "related to living beings") is a biological (Qiu et al., 2002), physical (Sacco et al., 2004) or chemical disturbance which above a certain degree, and in certain environmental conditions, could lead to toxic effects on a part or the whole ecosystem. It implies that a xenobiotic acts as a pollutant or a contaminant of one or several compartments of the natural environments (atmosphere, lithosphere and hydrosphere) and of biological organisms among the biosphere. This compound disrupts the ecosystem functioning above the limit of tolerance.

The pollution introduced directly or indirectly by humans in all natural compartments, could have prejudicial consequences on its own species and others, on biological resources, on climates and on infrastructures (Delmail, 2007). This impact depends on the type of pollution as it could be distinguished the pollution of proximity and the regional/global pollution (Delmail et al., 2011a; Ritter et al., 2002). The first one is constituted by factory smokes, fumes, sewer gas, etc. and it is directly produced by an anthropogenic source. The second one results from more complex and diverse physicochemical phenomenon (e.g. ozone synthesis in troposphere, acid rains, greenhouse effect).

The xenobiotics could be classified according to their nature (solid, liquid, gas, mineral, organic), their radiation (X, ultraviolet, infrared, radioactivity) and their origin (natural, synecological, autoecological, chemical, industrial) (Fig. 1). They may be also distinguished depending on their environmental targets (air, soil, and water), their biological targets (e.g. plants, fungi, mammals, invertebrates) and their cytotoxicity (e.g. cell types, organites). Their mode of action brings also information as some xenobiotics have an acute (death) or chronic toxicity (e.g. carcinogenesis, mutagenesis), or synergistic effect on organisms. They could be toxic at infinitesimal concentrations (micropollutants) or at a more concentrated range (macropollutants) (Delmail et al., 2010; Delmail et al., 2011b). Moreover, their effects have different duration of action: they could be degradable or persistent, or have a half-life like radioelements from several microseconds to many thousands of years.

Ecological exposure to environmental stressors occurs when a xenobiotic in a form that is bioavailable, reaches an organism. In order to be bioavailable, a xenobiotic must reach a location on or in an organism where it can cause an effect. The notion of phytoavailability defines the fraction of a bioavailable compound which could be absorbed by roots (Hinsinger et al., 2005).

The phytoavailability of xenobiotics is strongly correlated to the concentrations of contaminant species that occurred in the natural environments (Kabata-Pendias & Pendias, 2000). It is also linked to the physicochemical properties of the environment, the plant taxon and the xenobiotic considered. Thus, the phytoavailability is dependent from several parameters allowing the transfer from aerial, solid or aqueous phase to the plant: the availability (or chemical mobility), the accessibility (or physical mobility) and the assimilation (or biological mobility) (Hinsinger et al., 2005).

The xenobiotics could be observed under free forms depending on environmental conditions, but in many cases they may interact with different elements from the environments which will have an influence on its behavior. They could be included in primary minerals originated from the rock crystallization, or in secondary minerals

A xenobiotic (from the Greek *xenos* "stranger" and *biotic* "related to living beings") is a biological (Qiu et al., 2002), physical (Sacco et al., 2004) or chemical disturbance which above a certain degree, and in certain environmental conditions, could lead to toxic effects on a part or the whole ecosystem. It implies that a xenobiotic acts as a pollutant or a contaminant of one or several compartments of the natural environments (atmosphere, lithosphere and hydrosphere) and of biological organisms among the biosphere. This compound disrupts

The pollution introduced directly or indirectly by humans in all natural compartments, could have prejudicial consequences on its own species and others, on biological resources, on climates and on infrastructures (Delmail, 2007). This impact depends on the type of pollution as it could be distinguished the pollution of proximity and the regional/global pollution (Delmail et al., 2011a; Ritter et al., 2002). The first one is constituted by factory smokes, fumes, sewer gas, etc. and it is directly produced by an anthropogenic source. The second one results from more complex and diverse physicochemical phenomenon (e.g.

The xenobiotics could be classified according to their nature (solid, liquid, gas, mineral, organic), their radiation (X, ultraviolet, infrared, radioactivity) and their origin (natural, synecological, autoecological, chemical, industrial) (Fig. 1). They may be also distinguished depending on their environmental targets (air, soil, and water), their biological targets (e.g. plants, fungi, mammals, invertebrates) and their cytotoxicity (e.g. cell types, organites). Their mode of action brings also information as some xenobiotics have an acute (death) or chronic toxicity (e.g. carcinogenesis, mutagenesis), or synergistic effect on organisms. They could be toxic at infinitesimal concentrations (micropollutants) or at a more concentrated range (macropollutants) (Delmail et al., 2010; Delmail et al., 2011b). Moreover, their effects have different duration of action: they could be degradable or persistent, or have a half-life

Ecological exposure to environmental stressors occurs when a xenobiotic in a form that is bioavailable, reaches an organism. In order to be bioavailable, a xenobiotic must reach a location on or in an organism where it can cause an effect. The notion of phytoavailability defines the fraction of a bioavailable compound which could be absorbed by roots

The phytoavailability of xenobiotics is strongly correlated to the concentrations of contaminant species that occurred in the natural environments (Kabata-Pendias & Pendias, 2000). It is also linked to the physicochemical properties of the environment, the plant taxon and the xenobiotic considered. Thus, the phytoavailability is dependent from several parameters allowing the transfer from aerial, solid or aqueous phase to the plant: the availability (or chemical mobility), the accessibility (or physical mobility) and the

The xenobiotics could be observed under free forms depending on environmental conditions, but in many cases they may interact with different elements from the environments which will have an influence on its behavior. They could be included in primary minerals originated from the rock crystallization, or in secondary minerals

**2. The xenobiotics** 

(Hinsinger et al., 2005).

the ecosystem functioning above the limit of tolerance.

ozone synthesis in troposphere, acid rains, greenhouse effect).

like radioelements from several microseconds to many thousands of years.

assimilation (or biological mobility) (Hinsinger et al., 2005).

developed from primary ones after oxidation and hydrolysis processes. Moreover, they may be adsorbed on the organic matter in soils and waters, or on microorganisms (which could also absorb and accumulate them). Associations between colloids and xenobiotics could be observed in all natural compartments as a colloidal system may be solid, liquid or gaseous.

Fig. 1. Overview of selective environmental stress factors which could threaten plants. Biotic and abiotic environmental selective factors are considered.

Plant Ageing, a Counteracting Agent to Xenobiotic Stress 93

1997). The produced fatty-acid radical then reacts with molecular oxygen, thereby creating a peroxyl fatty acid radical. This last one reacts with another phospholipid, producing a new radical and a lipid peroxide, or a cyclic peroxide if it reacts with itself. This cycle continues as a chain reaction mechanism (Schaich, 2005). This process ends up when two radicals react and produce a non-radical compound. It happens when the concentrations of radicals is high enough. Living organisms have evolved different molecules that speed up termination by catching the reactive oxygen species (Paramesha et al., 2011). Among such antioxidants, the most important are the scavengers mainly constituted with α-tocopherol (or vitamin E)

and carotenoids (β-caroten, xantophylls) (Figs. 2 and 3) (Delmail et al., 2011c, 2011d).

Fig. 2. Main plant antioxidant pathways including enzymes and scavengers

(based on Delmail (2011)).

#### **3. The reactive oxygen species**

All plants use the dioxygen as a source of energy for their development. However, this aerobic process could lead to the production of reactive oxygen species which are diversified chemically reactive molecules containing oxygen. Reactive oxygen species are a natural byproduct of the metabolism and play important roles in homeostasis and cell signaling. However, under environmental stress, their levels can increase dramatically which lead to disruptions and damages in cell compartments.

#### **3.1 Diversity and toxicity**

An uncompleted reduction of the dioxygen through cytochroms from the respiratory chain implies the production of reactive oxygen species as singlet oxygen (1O2) and superoxide radical (O2 •−) which leads to the synthesis of hydroxyl radical (•OH), hydroperoxyl radical (•O2H) and hydrogen peroxide (H2O2) (Fig. 2). The radicals alkoxyl (RO•) and peroxyl (RO2•) are the consequence of the peroxidation of membrane phospholipids (or lipoperoxidation) by the previous reactive oxygen species (Fig. 3) (Edreva, 2005; Lagadic et al., 1997; Li et al., 1994; Thompson et al., 1987).

At the same time, the photosynthetic electron transport chains could product high concentrations of reactive oxygen species. Indeed, the electrons tetravalently reduce the intracellular oxygen to water. But, some electrons could leak from many sites along the electron transport chain, resulting in a univalent reduction of dioxygen to form the extremely reactive superoxide radical which can dismutate to form hydrogen peroxide (Alscher et al., 2002). This last reaction is spontaneous or catalyzed by one of the superoxide dismutases (Fig. 2) depending on the cell compartment where the reaction occurs: manganese-superoxide dismutase (mitochondria, peroxisome), iron-superoxide dismutase (chloroplast) or copper/zinc-superoxide dismutase (chloroplast, cytosol) (Fornazier et al., 2002; Gill & Tuteja, 2010; Pereira et al., 2002).

The hydrogen peroxide is not a free radical due to all its matched electrons. However, it has a strong toxicity potential: it has a long lifespan and a high diffusibility far from its synthesis site. Indeed, it could pass through biological membranes via aquaporins as it presents a chemical structure close to water (Bienert et al., 2006, 2007; Parent et al., 2008). The concentration of this oxidative compound is regulated by antioxidant enzymes like the ascorbate peroxidase, the catalase or the glutathione peroxidase (Fig. 2). These proteins use the nicotinamide adenine dinucleotide phosphate (NADPH) produced during the photosynthesis in chloroplasts for their functioning (Fig. 2). However, the reactive oxygen species could disrupt the photosynthetic electron transport chains in thylakoid membranes and some electrons are deflected. Without a normal synthesis of NADPH, plants use a cytosolic secondary catabolism pathway to produce it, the pentose phosphate pathway (Fig. 4) (Delmail, 2011; Kruger & von Schaewen, 2003). The hydrogen peroxide could be also produced through the bivalent reduction of the oxygen in presence of oxidases like the peroxisomal glycolate oxidase or the amine oxidase (Parent et al., 2008). The toxicity of hydrogen peroxide is also linked to its implication in the synthesis of the hydroxyl and hydroperoxyl radicals through the Haber-Weiss and Fenton reactions (Fig. 2). Like their reactive-oxygen-species mother, these short-lifespan radicals are very diffusive through biological membranes and they could disturb and affect all organites and cell compartments. They are also mainly implied in the lipoperoxidation (Fig. 3) (Edreva, 2005 ; Lagadic et al.,

All plants use the dioxygen as a source of energy for their development. However, this aerobic process could lead to the production of reactive oxygen species which are diversified chemically reactive molecules containing oxygen. Reactive oxygen species are a natural byproduct of the metabolism and play important roles in homeostasis and cell signaling. However, under environmental stress, their levels can increase dramatically

An uncompleted reduction of the dioxygen through cytochroms from the respiratory chain implies the production of reactive oxygen species as singlet oxygen (1O2) and superoxide

(•O2H) and hydrogen peroxide (H2O2) (Fig. 2). The radicals alkoxyl (RO•) and peroxyl (RO2•) are the consequence of the peroxidation of membrane phospholipids (or lipoperoxidation) by the previous reactive oxygen species (Fig. 3) (Edreva, 2005; Lagadic et

At the same time, the photosynthetic electron transport chains could product high concentrations of reactive oxygen species. Indeed, the electrons tetravalently reduce the intracellular oxygen to water. But, some electrons could leak from many sites along the electron transport chain, resulting in a univalent reduction of dioxygen to form the extremely reactive superoxide radical which can dismutate to form hydrogen peroxide (Alscher et al., 2002). This last reaction is spontaneous or catalyzed by one of the superoxide dismutases (Fig. 2) depending on the cell compartment where the reaction occurs: manganese-superoxide dismutase (mitochondria, peroxisome), iron-superoxide dismutase (chloroplast) or copper/zinc-superoxide dismutase (chloroplast, cytosol) (Fornazier et al.,

The hydrogen peroxide is not a free radical due to all its matched electrons. However, it has a strong toxicity potential: it has a long lifespan and a high diffusibility far from its synthesis site. Indeed, it could pass through biological membranes via aquaporins as it presents a chemical structure close to water (Bienert et al., 2006, 2007; Parent et al., 2008). The concentration of this oxidative compound is regulated by antioxidant enzymes like the ascorbate peroxidase, the catalase or the glutathione peroxidase (Fig. 2). These proteins use the nicotinamide adenine dinucleotide phosphate (NADPH) produced during the photosynthesis in chloroplasts for their functioning (Fig. 2). However, the reactive oxygen species could disrupt the photosynthetic electron transport chains in thylakoid membranes and some electrons are deflected. Without a normal synthesis of NADPH, plants use a cytosolic secondary catabolism pathway to produce it, the pentose phosphate pathway (Fig. 4) (Delmail, 2011; Kruger & von Schaewen, 2003). The hydrogen peroxide could be also produced through the bivalent reduction of the oxygen in presence of oxidases like the peroxisomal glycolate oxidase or the amine oxidase (Parent et al., 2008). The toxicity of hydrogen peroxide is also linked to its implication in the synthesis of the hydroxyl and hydroperoxyl radicals through the Haber-Weiss and Fenton reactions (Fig. 2). Like their reactive-oxygen-species mother, these short-lifespan radicals are very diffusive through biological membranes and they could disturb and affect all organites and cell compartments. They are also mainly implied in the lipoperoxidation (Fig. 3) (Edreva, 2005 ; Lagadic et al.,

•−) which leads to the synthesis of hydroxyl radical (•OH), hydroperoxyl radical

**3. The reactive oxygen species** 

**3.1 Diversity and toxicity** 

radical (O2

which lead to disruptions and damages in cell compartments.

al., 1997; Li et al., 1994; Thompson et al., 1987).

2002; Gill & Tuteja, 2010; Pereira et al., 2002).

1997). The produced fatty-acid radical then reacts with molecular oxygen, thereby creating a peroxyl fatty acid radical. This last one reacts with another phospholipid, producing a new radical and a lipid peroxide, or a cyclic peroxide if it reacts with itself. This cycle continues as a chain reaction mechanism (Schaich, 2005). This process ends up when two radicals react and produce a non-radical compound. It happens when the concentrations of radicals is high enough. Living organisms have evolved different molecules that speed up termination by catching the reactive oxygen species (Paramesha et al., 2011). Among such antioxidants, the most important are the scavengers mainly constituted with α-tocopherol (or vitamin E) and carotenoids (β-caroten, xantophylls) (Figs. 2 and 3) (Delmail et al., 2011c, 2011d).

Fig. 2. Main plant antioxidant pathways including enzymes and scavengers (based on Delmail (2011)).

2008).

Plant Ageing, a Counteracting Agent to Xenobiotic Stress 95

Considering all these elements, the reactive oxygen species are considered as phytotoxic compounds. However, it is currently admitted that their synthesis, in relation to the respiratory and photosynthetic metabolisms, plays an essential role in the life and the death of plant cells. Indeed, they could play an alternative role and act as cell signalization molecules to establish some defense mechanisms towards a xenobiotic stress (Parent et al.,

The reactive oxygen species are known for their importance in the plant responses towards environmental disturbances. Several symptoms like necrosis (Fig. 5), are the consequences

of a high oxidative-compound accumulation and a disturbance of cell homeostasis.

Fig. 5. Leaf of *Tradescantia sp.* (A) and *Begonia sp.* (B) with symptoms of photosynthetic-

pigment oxidation and cell necrosis.

**3.2 Role in cell death and protection of living parts** 

Fig. 3. Mechanisms of lipid peroxidation in biological membranes. The produced peroxyl radicals could react either with another lipid to supply the lipoperoxidative chain reaction mechanism or with a scavenger like the vitamin E which disrupts and stops the oxidative process.

Fig. 4. Reactions of NADPH synthesis through the oxidative phase of the pentose phosphate pathway of plants (based on Delmail (2011)).

Fig. 3. Mechanisms of lipid peroxidation in biological membranes. The produced peroxyl radicals could react either with another lipid to supply the lipoperoxidative chain reaction mechanism or with a scavenger like the vitamin E which disrupts and stops the oxidative

Fig. 4. Reactions of NADPH synthesis through the oxidative phase of the pentose phosphate

pathway of plants (based on Delmail (2011)).

process.

Considering all these elements, the reactive oxygen species are considered as phytotoxic compounds. However, it is currently admitted that their synthesis, in relation to the respiratory and photosynthetic metabolisms, plays an essential role in the life and the death of plant cells. Indeed, they could play an alternative role and act as cell signalization molecules to establish some defense mechanisms towards a xenobiotic stress (Parent et al., 2008).

## **3.2 Role in cell death and protection of living parts**

The reactive oxygen species are known for their importance in the plant responses towards environmental disturbances. Several symptoms like necrosis (Fig. 5), are the consequences of a high oxidative-compound accumulation and a disturbance of cell homeostasis.

Fig. 5. Leaf of *Tradescantia sp.* (A) and *Begonia sp.* (B) with symptoms of photosyntheticpigment oxidation and cell necrosis.

Plant Ageing, a Counteracting Agent to Xenobiotic Stress 97

This interaction becomes incompatible several days later and it leads to an oxidative burst, with the appearance of necrotic lesions due to reactive oxygen species, which isolates the pathogen from the living parts. This conducts to the inhibition of the pathogen growth. These necrotic lesions are due to hypersensitive cell death in the host and the resistance phenotype was due to the action of a gene known to confer a hypersensitive response, Rpt1

Fig. 6. Schematic structure and organization of phytochelatins implied in the sequestration mechanism of cadmium (Cd) through thiol function (SH) and constituted each of 2 γ-

Leaf senescence is a highly regulated process particularly well studied in crop plants and *Arabidopsis* (Balazadeh et al., 2008). Nowadays it is conspicuous that environmental stresses can induce precocious senescence (Balazadeh et al., 2008) as hypothesized since 1997 by Ouzounidou et al. during the observation of the effect of cadmium on wheat; but the effect of heavy metal ions on this phenomenon is still poorly documented. However, it was demonstrated that protein functioning as metal chelator like metallothionein may be needed to protect normal cell functions from the toxic effects of metal ions released during senescence. In that sense, metallothioneins may be involved in chaperoning released metal ions to avoid metal toxicity or metal induced-oxidative stress in plant cell during the senescence process. Guo et al. (2003) indicated that all the *Arabidopsis* metallothionein genes expressed in vegetative tissues were upregulated in senescing leaves thus protecting cells from metal ions toxicity during senescence. A similar observation of the implication of some metallothioneins

in leaf senescence and in heavy metal stress was done in barley by Heise et al. (2007).

Another important molecule involved during senescence is the yellow stripe-like transporter family (YSL). Curie et al. (2009) indicated that five out the eight Arabidopsis YSL genes are most strongly expressed in senescent leaves. Indeed, the expression of AtYSL1 and

glutamylcysteine parts (PC2) (based on Delmail (2011)).

**4. Senescence and abscission** 

(Heist et al., 2004).

This phenomenon is due to an oxidation of photosynthetic pigments in chlorophyllian organs and to the death of isolated cells or groups of cells in many plant tissues. Despite that reactive oxygen species could be produced in normal conditions, the increase of their concentrations in plants is often linked to xenobiotics (Parent et al., 2008). For example, an increase of hydrogen peroxide is observed in peroxisomes of the aquatic macrophyte *Myriophyllum alterniflorum* after an exposition to cadmium chloride from 0.5 to 10 µg.l-1 (Delmail, 2011). Moreover, the oxidative stress generated by this reactive oxygen species is all the more important during the 2-3 weeks of contamination that the heavy-metal concentration is high. Indeed, the activity of the catalase is higher during a longer period when the toxicity increases (Delmail, 2011).

It could be also noted that many of the symptoms due to the xenobiotics stress are amplified by the presence of reactive oxygen species. In the same species, when *M. alterniflorum* is contaminated with copper sulphate from 5 to 100 µg.l-1, an increase of the catalase activity is observed up to 25 µg.l-1 to reduce this reactive oxygen species into water (Delmail, 2011). Beyond this toxicity limit, the intensity of the enzymatic activity decreases due to a disruption of the antioxidant pathways. The catalase activity of plants is known to be sensitive to oxidative stress when a lack of iron (or sometimes magnesium) occurs (Esfandiari et al., 2010; Iturbe-Ormaetxe et al., 1995; Tewari et al., 2005) as this protein needs an iron ion in its constitutive heme (Arménia Carrondo et al., 2007). A competitive effect between the excess of copper and the other elements during the adsorption/absorption (Bernal et al., 2007) could lead to a disturbance during the catalase synthesis (Delmail, 2011).

Despite of their extremely toxic nature, the reactive oxygen species are also implied in cascades of signalization which induce the expression and the regulation of many genes. These genes could be involved in the defense mechanisms, like the phytochelatine synthase which allows the synthesis of heavy-metal binding peptides, the phytochelatins. These compounds play important roles in the detoxification of toxic heavy metals and the regulation of intracellular concentrations of essential metals in plants (Hirata et al., 2005). The primary structure of phytochelatins generally have the form (γ-glutamate-cysteine)nglycine and these peptides could form complexes with heavy metals such as cadmium (Fig. 6), copper, zinc, mercury, silver and arsenic, which are stored as inactive in the cell vacuoles. The expression of phytochelatin synthase in *Populus tremula x tremuloides* cv. Etrepole transgenic lines expressing the wheat phytochelatin synthase TaPCS1 is stimulated by the presence of heavy metal and this protein aimed at increasing metal tolerance and metal accumulation through overproduction of phytochelatins (Couselo et al., 2010).

The reactive oxygen species are also implied in the apoptosis (or programmed cell death) of plants as regulating agents (Dat et al., 2003; Van Breusegem et al., 2006). This event occurs during all the life of organisms and selected cells or organs are eliminated from the living parts through senescence or abscission to maintain the optimal development of the organisms. During the growth, the apoptosis is involved in several phenomena like the triggering of the aleurone cells to release amylase during the germination of caryopsis, the differentiation of xylem and phloem elements, the development of the root cap and the abscission of leaves (Parent et al., 2008). But plants may use this apoptosis to adapt and to resist towards environmental stress like pathogens. For example, the infection of *Nicotiana obtusifolia* by the downy mildew pathogen *Peronospora tabacina* resulted in a compatible interaction, in which *P. tabacina* penetrated and colonized host leaf tissue (Heist et al., 2004).

This phenomenon is due to an oxidation of photosynthetic pigments in chlorophyllian organs and to the death of isolated cells or groups of cells in many plant tissues. Despite that reactive oxygen species could be produced in normal conditions, the increase of their concentrations in plants is often linked to xenobiotics (Parent et al., 2008). For example, an increase of hydrogen peroxide is observed in peroxisomes of the aquatic macrophyte *Myriophyllum alterniflorum* after an exposition to cadmium chloride from 0.5 to 10 µg.l-1 (Delmail, 2011). Moreover, the oxidative stress generated by this reactive oxygen species is all the more important during the 2-3 weeks of contamination that the heavy-metal concentration is high. Indeed, the activity of the catalase is higher during a longer period

It could be also noted that many of the symptoms due to the xenobiotics stress are amplified by the presence of reactive oxygen species. In the same species, when *M. alterniflorum* is contaminated with copper sulphate from 5 to 100 µg.l-1, an increase of the catalase activity is observed up to 25 µg.l-1 to reduce this reactive oxygen species into water (Delmail, 2011). Beyond this toxicity limit, the intensity of the enzymatic activity decreases due to a disruption of the antioxidant pathways. The catalase activity of plants is known to be sensitive to oxidative stress when a lack of iron (or sometimes magnesium) occurs (Esfandiari et al., 2010; Iturbe-Ormaetxe et al., 1995; Tewari et al., 2005) as this protein needs an iron ion in its constitutive heme (Arménia Carrondo et al., 2007). A competitive effect between the excess of copper and the other elements during the adsorption/absorption (Bernal et al., 2007) could lead to a disturbance during the catalase synthesis (Delmail, 2011). Despite of their extremely toxic nature, the reactive oxygen species are also implied in cascades of signalization which induce the expression and the regulation of many genes. These genes could be involved in the defense mechanisms, like the phytochelatine synthase which allows the synthesis of heavy-metal binding peptides, the phytochelatins. These compounds play important roles in the detoxification of toxic heavy metals and the regulation of intracellular concentrations of essential metals in plants (Hirata et al., 2005). The primary structure of phytochelatins generally have the form (γ-glutamate-cysteine)nglycine and these peptides could form complexes with heavy metals such as cadmium (Fig. 6), copper, zinc, mercury, silver and arsenic, which are stored as inactive in the cell vacuoles. The expression of phytochelatin synthase in *Populus tremula x tremuloides* cv. Etrepole transgenic lines expressing the wheat phytochelatin synthase TaPCS1 is stimulated by the presence of heavy metal and this protein aimed at increasing metal tolerance and

metal accumulation through overproduction of phytochelatins (Couselo et al., 2010).

The reactive oxygen species are also implied in the apoptosis (or programmed cell death) of plants as regulating agents (Dat et al., 2003; Van Breusegem et al., 2006). This event occurs during all the life of organisms and selected cells or organs are eliminated from the living parts through senescence or abscission to maintain the optimal development of the organisms. During the growth, the apoptosis is involved in several phenomena like the triggering of the aleurone cells to release amylase during the germination of caryopsis, the differentiation of xylem and phloem elements, the development of the root cap and the abscission of leaves (Parent et al., 2008). But plants may use this apoptosis to adapt and to resist towards environmental stress like pathogens. For example, the infection of *Nicotiana obtusifolia* by the downy mildew pathogen *Peronospora tabacina* resulted in a compatible interaction, in which *P. tabacina* penetrated and colonized host leaf tissue (Heist et al., 2004).

when the toxicity increases (Delmail, 2011).

This interaction becomes incompatible several days later and it leads to an oxidative burst, with the appearance of necrotic lesions due to reactive oxygen species, which isolates the pathogen from the living parts. This conducts to the inhibition of the pathogen growth. These necrotic lesions are due to hypersensitive cell death in the host and the resistance phenotype was due to the action of a gene known to confer a hypersensitive response, Rpt1 (Heist et al., 2004).

Fig. 6. Schematic structure and organization of phytochelatins implied in the sequestration mechanism of cadmium (Cd) through thiol function (SH) and constituted each of 2 γglutamylcysteine parts (PC2) (based on Delmail (2011)).

## **4. Senescence and abscission**

Leaf senescence is a highly regulated process particularly well studied in crop plants and *Arabidopsis* (Balazadeh et al., 2008). Nowadays it is conspicuous that environmental stresses can induce precocious senescence (Balazadeh et al., 2008) as hypothesized since 1997 by Ouzounidou et al. during the observation of the effect of cadmium on wheat; but the effect of heavy metal ions on this phenomenon is still poorly documented. However, it was demonstrated that protein functioning as metal chelator like metallothionein may be needed to protect normal cell functions from the toxic effects of metal ions released during senescence. In that sense, metallothioneins may be involved in chaperoning released metal ions to avoid metal toxicity or metal induced-oxidative stress in plant cell during the senescence process. Guo et al. (2003) indicated that all the *Arabidopsis* metallothionein genes expressed in vegetative tissues were upregulated in senescing leaves thus protecting cells from metal ions toxicity during senescence. A similar observation of the implication of some metallothioneins in leaf senescence and in heavy metal stress was done in barley by Heise et al. (2007).

Another important molecule involved during senescence is the yellow stripe-like transporter family (YSL). Curie et al. (2009) indicated that five out the eight Arabidopsis YSL genes are most strongly expressed in senescent leaves. Indeed, the expression of AtYSL1 and

Plant Ageing, a Counteracting Agent to Xenobiotic Stress 99

The role of plant growth regulator in senescence and in heavy-metal resistance is quite complex but cytokinins for their senescence delaying action (for a review see Werner & Schmulling, 2009) and brassinosteroids for their role in responses to various environmental stress (for a review see Bajguz & Hayat, 2009) appeared as major candidates for further studies to understand the heavy-metal induced senescence processes. Indeed, Arora et al. (2010) demonstrated that the brassinosteroid 24-epibrassinolide present stress-ameliorative properties in *Brassica juncea* plant during chromium stress as an improved growth and antioxidant enzymes activities. Similar conclusion was highlighted by Anuradha & Rao (2007) on *Raphanus sativus* plant were brassinosteroids supplementation alleviated the toxic effect of cadmium. More recently, Bajguz (2011) noted on *Chlorella vulgaris* that the brassinosteroid application to the culture prevents chlorophyll, sugar and protein loss and increases phytochelatin synthesis during heavy metal stress (cadmium, copper and lead). These reactions call to mind the delayed senescence process observed previously in aquatic macrophytes when treated with cytokinins during a heavy-metal stress. In the same way, brassinosteroid treatment improves sunflower (genotype 2603) and turnip (var. rave du Limousin) resistance to cadmium stress in terms of photosynthesis activities (Figs. 7 and 8,

Fig. 7. Photosynthetic activities and respiration rate of one-month old sunflower specimens after 48h of cadmium exposure (1 mM) combined or not with 3 µM 24-epibrassinolid

(Delmail et al. unpublished data). FW, fresh weight.

Delmail et al. unpublished data).

AtYSL3 is increased during senescence and although the leave of the double ysl1ysl3 mutant loses only 10% of copper content between the 4th and the 5th week of growth, the wild type *Arabidopsis* loses almost 60%. More recently Xiao & Chye (2011) evidenced new roles for acyl-CoA-binding proteins (ACPBs). Indeed, in *Arabidopsis* the expression of AtACPB3 was upregulated during senescence and AtACPB3-KOs *Arabidopsis* displayed delayed leaf senescence whereas AtACPB3-overexpressors *Arabidopsis* present an accelerated leaf senescence phenotype. On the other hand these authors indicated that *Arabidopsis* AtACPB2 overexpressors were more tolerant to cadmium in the growing media.

Among the different mechanisms adopted by plants to cope with metallic stress (phenological escape, exclusion, amelioration and tolerance), the amelioration one implies that the ion must be removed from the circulation or tolerated within the cytoplasm. These amelioration processes include excretion either actively - through glands on aerial part or by roots - or passively by accumulation in old leaves followed by abscission (Adams & Lamoureux, 2005). The simplest form of excretion is the loss of an organ which has accumulated the toxic compounds. This is generally true for the old leaves that present higher content of toxics than the young leaves and buds. For example, Yasar et al. (2006) noticed that the toxic sodium ion was stored in old leaves of the salt-tolerant Gevas Sirik 57 (GS57) green bean genotype acting as a protection mechanism from the detrimental effect of sodium for young leaves. In the same way, Szarek-Lukaszewska et al. (2004) indicated that an *Armeria maritima* population from metalliferous soil directed to the oldest leaves a part of the metal transported to aboveground plant organs. For these authors the ability to accumulate metals in withering leaves characterizes plants growing under strong environmental pressure from metal contamination. Detoxification mechanism by leaf fall was a strategy previously suggested by Dahmani-Mueller et al. (2000) in *Armeria maritima spp. halleri* where metal content (cadmium, copper, lead and zinc) in ageing leaves (brown leaves) were 3-8 times higher than in green leaves. A similar observation was done by Monni et al. (2001) on a shrub (*Empetrum nigrum*) which accumulates metals (cadmium, copper, iron, lead, nickel and zinc) in older tissues, mainly leaves and bark, by both accumulation and surface contamination. For tree species, Pahalawattaarachchi et al. (2009) shown that in *Rhizophora mucronata* chromium, cadmium and lead were accumulated in leaves before abscission and thus eliminated. A major disadvantage of the excretion strategy for plant is that they are stationary so the excreted substance will remain in the root zone and may eventually lead to a build-up of the xenobiotic (Adams & Lamoureux, 2005).

Only few data were available on aquatic macrophyte, a case where this major disadvantage did not apply. For example in *Spirodela polyrrhiza*, the excess of iron and copper induces plant necrosis, colony disintegration and root abscission (Xing et al., 2010). It should be noted that in another aquatic macrophyte, *Lemna minor*, the frond abscission could be used to test water toxicity induced by metal and other compounds (Henke et al., 2011). Our previous data (Delmail et al., 2011d) suggest that as in terrestrial plants a similar excretion strategy could occur in aquatic plants. Indeed, *Myriophyllum alterniflorum* old leaves are much more affected by heavy-metal pollution than younger ones. Previous study of Jana & Chouduri (1982) on three submerged aquatic macrophytes (*Potamogeton pectinatus*, *Vallisneria spiralis* and *Hydrilla verticillata*) demonstrated that all the heavy metals tested (cadmium, copper, lead and mercury) hastened the senescence process. These authors evidenced the role of the plant growth regulator kinetin in the reduction of the senescence induced by heavy metals.

AtYSL3 is increased during senescence and although the leave of the double ysl1ysl3 mutant loses only 10% of copper content between the 4th and the 5th week of growth, the wild type *Arabidopsis* loses almost 60%. More recently Xiao & Chye (2011) evidenced new roles for acyl-CoA-binding proteins (ACPBs). Indeed, in *Arabidopsis* the expression of AtACPB3 was upregulated during senescence and AtACPB3-KOs *Arabidopsis* displayed delayed leaf senescence whereas AtACPB3-overexpressors *Arabidopsis* present an accelerated leaf senescence phenotype. On the other hand these authors indicated that *Arabidopsis* AtACPB2-

Among the different mechanisms adopted by plants to cope with metallic stress (phenological escape, exclusion, amelioration and tolerance), the amelioration one implies that the ion must be removed from the circulation or tolerated within the cytoplasm. These amelioration processes include excretion either actively - through glands on aerial part or by roots - or passively by accumulation in old leaves followed by abscission (Adams & Lamoureux, 2005). The simplest form of excretion is the loss of an organ which has accumulated the toxic compounds. This is generally true for the old leaves that present higher content of toxics than the young leaves and buds. For example, Yasar et al. (2006) noticed that the toxic sodium ion was stored in old leaves of the salt-tolerant Gevas Sirik 57 (GS57) green bean genotype acting as a protection mechanism from the detrimental effect of sodium for young leaves. In the same way, Szarek-Lukaszewska et al. (2004) indicated that an *Armeria maritima* population from metalliferous soil directed to the oldest leaves a part of the metal transported to aboveground plant organs. For these authors the ability to accumulate metals in withering leaves characterizes plants growing under strong environmental pressure from metal contamination. Detoxification mechanism by leaf fall was a strategy previously suggested by Dahmani-Mueller et al. (2000) in *Armeria maritima spp. halleri* where metal content (cadmium, copper, lead and zinc) in ageing leaves (brown leaves) were 3-8 times higher than in green leaves. A similar observation was done by Monni et al. (2001) on a shrub (*Empetrum nigrum*) which accumulates metals (cadmium, copper, iron, lead, nickel and zinc) in older tissues, mainly leaves and bark, by both accumulation and surface contamination. For tree species, Pahalawattaarachchi et al. (2009) shown that in *Rhizophora mucronata* chromium, cadmium and lead were accumulated in leaves before abscission and thus eliminated. A major disadvantage of the excretion strategy for plant is that they are stationary so the excreted substance will remain in the root zone and may eventually lead to a build-up of the xenobiotic (Adams & Lamoureux, 2005).

Only few data were available on aquatic macrophyte, a case where this major disadvantage did not apply. For example in *Spirodela polyrrhiza*, the excess of iron and copper induces plant necrosis, colony disintegration and root abscission (Xing et al., 2010). It should be noted that in another aquatic macrophyte, *Lemna minor*, the frond abscission could be used to test water toxicity induced by metal and other compounds (Henke et al., 2011). Our previous data (Delmail et al., 2011d) suggest that as in terrestrial plants a similar excretion strategy could occur in aquatic plants. Indeed, *Myriophyllum alterniflorum* old leaves are much more affected by heavy-metal pollution than younger ones. Previous study of Jana & Chouduri (1982) on three submerged aquatic macrophytes (*Potamogeton pectinatus*, *Vallisneria spiralis* and *Hydrilla verticillata*) demonstrated that all the heavy metals tested (cadmium, copper, lead and mercury) hastened the senescence process. These authors evidenced the role of the plant growth regulator kinetin in the reduction of the senescence

induced by heavy metals.

overexpressors were more tolerant to cadmium in the growing media.

The role of plant growth regulator in senescence and in heavy-metal resistance is quite complex but cytokinins for their senescence delaying action (for a review see Werner & Schmulling, 2009) and brassinosteroids for their role in responses to various environmental stress (for a review see Bajguz & Hayat, 2009) appeared as major candidates for further studies to understand the heavy-metal induced senescence processes. Indeed, Arora et al. (2010) demonstrated that the brassinosteroid 24-epibrassinolide present stress-ameliorative properties in *Brassica juncea* plant during chromium stress as an improved growth and antioxidant enzymes activities. Similar conclusion was highlighted by Anuradha & Rao (2007) on *Raphanus sativus* plant were brassinosteroids supplementation alleviated the toxic effect of cadmium. More recently, Bajguz (2011) noted on *Chlorella vulgaris* that the brassinosteroid application to the culture prevents chlorophyll, sugar and protein loss and increases phytochelatin synthesis during heavy metal stress (cadmium, copper and lead). These reactions call to mind the delayed senescence process observed previously in aquatic macrophytes when treated with cytokinins during a heavy-metal stress. In the same way, brassinosteroid treatment improves sunflower (genotype 2603) and turnip (var. rave du Limousin) resistance to cadmium stress in terms of photosynthesis activities (Figs. 7 and 8, Delmail et al. unpublished data).

Fig. 7. Photosynthetic activities and respiration rate of one-month old sunflower specimens after 48h of cadmium exposure (1 mM) combined or not with 3 µM 24-epibrassinolid (Delmail et al. unpublished data). FW, fresh weight.

Plant Ageing, a Counteracting Agent to Xenobiotic Stress 101

concerns the die-back symptom of *Phragmites communis* where the premature senescence of shoot appears to result at least in part from phytotoxin action (acetic, propionic, n- and iso-

Fig. 9. Superoxide dismutase activity of one-month old sunflower specimens after 48h of cadmium exposure (1 mM) combined or not with 3 µM 24-epibrassinolid (Delmail et al.

Fig. 10. Catalase activity of one-month old turnip specimens after 72h of cadmium exposure (1 mM) combined or not with 3 µM 24-epibrassinolid (Delmail et al. unpublished data).

unpublished data).

butyric and n-caproic acids and sulphide) (Armstrong & Armstrong, 2001).

Fig. 8. Photosynthetic activities and respiration rate of one-month old turnip specimens after 72h of cadmium exposure (1 mM) combined or not with 3 µM 24-epibrassinolid (Delmail et al. unpublished data). FW, fresh weight.

Moreover, the effect of brassinosteroids on antioxidant enzymatic activities during cadmium stress could be similar in young and old leaves as shown in Fig. 9 for a decrease in catalase activity of sunflower plants treated with phytohormons during a heavy-metal stress. On the other hand, as demonstrated in turnip (Fig. 10) a differential effect between old and young leaves could appear with an increase in superoxide-dismutase activity in young leaves and a decrease in old leaves (Delmail et al. unpublished data). In these two plants, brassinosteroid application clearly has a protective effect on the raw photosynthesis activity, probably indicating a delayed heavy-metal senescence. These protective actions probably also occur on the enzymatic antioxidant system even if the complexity of the involved cascade reactions lead to a more unclear landscape inducing pattern variations between studied enzymes, age of plant parts and plant species. It appears clearly that much more studies are needed to understand the complex interwoven relationship existing between plant physiology under heavy-metal stress, senescence and plant growth regulators.

Concerning the organic xenobiotics effect on plant senescence even much less data are available. For example, Cape et al. (2003) noted that in *Lotus corniculatus* exposes to a mixture of six volatile organic compounds (acetone, acetonitrile, dichloromethane, ethanol, methyl t-butyl ether and toluene), a premature senescence occurs but in this case a premature senescence refers to advanced timing of seed pot production. Another example

Fig. 8. Photosynthetic activities and respiration rate of one-month old turnip specimens after 72h of cadmium exposure (1 mM) combined or not with 3 µM 24-epibrassinolid (Delmail et

Moreover, the effect of brassinosteroids on antioxidant enzymatic activities during cadmium stress could be similar in young and old leaves as shown in Fig. 9 for a decrease in catalase activity of sunflower plants treated with phytohormons during a heavy-metal stress. On the other hand, as demonstrated in turnip (Fig. 10) a differential effect between old and young leaves could appear with an increase in superoxide-dismutase activity in young leaves and a decrease in old leaves (Delmail et al. unpublished data). In these two plants, brassinosteroid application clearly has a protective effect on the raw photosynthesis activity, probably indicating a delayed heavy-metal senescence. These protective actions probably also occur on the enzymatic antioxidant system even if the complexity of the involved cascade reactions lead to a more unclear landscape inducing pattern variations between studied enzymes, age of plant parts and plant species. It appears clearly that much more studies are needed to understand the complex interwoven relationship existing between plant

Concerning the organic xenobiotics effect on plant senescence even much less data are available. For example, Cape et al. (2003) noted that in *Lotus corniculatus* exposes to a mixture of six volatile organic compounds (acetone, acetonitrile, dichloromethane, ethanol, methyl t-butyl ether and toluene), a premature senescence occurs but in this case a premature senescence refers to advanced timing of seed pot production. Another example

physiology under heavy-metal stress, senescence and plant growth regulators.

al. unpublished data). FW, fresh weight.

concerns the die-back symptom of *Phragmites communis* where the premature senescence of shoot appears to result at least in part from phytotoxin action (acetic, propionic, n- and isobutyric and n-caproic acids and sulphide) (Armstrong & Armstrong, 2001).

Fig. 9. Superoxide dismutase activity of one-month old sunflower specimens after 48h of cadmium exposure (1 mM) combined or not with 3 µM 24-epibrassinolid (Delmail et al. unpublished data).

Fig. 10. Catalase activity of one-month old turnip specimens after 72h of cadmium exposure (1 mM) combined or not with 3 µM 24-epibrassinolid (Delmail et al. unpublished data).

Plant Ageing, a Counteracting Agent to Xenobiotic Stress 103

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## **5. Conclusion**

Senescence implies a succession of physiological events integrated with developmental program which lead to the loss of several organs from the plant. This biological process constitutes an integral part of the normal plant developmental cycle which can be observed at different organization levels (cell, tissue and organ). The senescence is the final event in the life of many plant tissues and it is a highly regulated process that involves structural, biochemical and molecular changes.

Organic and inorganic xenobiotics could hasten the senescence processes and they may lead to a premature death of the plants. At the opposite, the senescence occurring in the plant organs could isolate the stressors and/or eliminate the toxics from the living parts through induced abscission.

## **6. Acknowledgment**

This research was partially supported by the University of Limoges, the GRESE (Research Group on Water, Soil and the Environment) EA 4330, and the Conseil Régional du Limousin.

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Senescence implies a succession of physiological events integrated with developmental program which lead to the loss of several organs from the plant. This biological process constitutes an integral part of the normal plant developmental cycle which can be observed at different organization levels (cell, tissue and organ). The senescence is the final event in the life of many plant tissues and it is a highly regulated process that involves structural,

Organic and inorganic xenobiotics could hasten the senescence processes and they may lead to a premature death of the plants. At the opposite, the senescence occurring in the plant organs could isolate the stressors and/or eliminate the toxics from the living parts through

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**1. Introduction** 

remobilization during leaf senescence.

very obvious in some other cases.

**quiescence** 

**2. Terminology and types of senescence** 

**6** 

 *Algeria* 

**Some Aspects** 

**of Leaf Senescence** 

Hafsi Miloud and Guendouz Ali

*Faculty of Natural Sciences and Life, Ferhat ABBAS University, Setif* 

*Crop Production, Department of Agronomy,* 

*Laboratory of Improvement and Development of Livestock and* 

The word *senescence* derives from two Latin words: *senex* and *senescere*. *Senex* means 'old'; this Latin root is shared by 'senile', 'senior', and even 'senate'. In ancient Rome the 'Senatus' was a 'council of elders' that was composed of the heads of patrician families. *Senescere*  means 'to grow old'. The Merriam-Webster online dictionary defines *senescence* as 'the state of being old or the process of becoming old'. Aging is also the process of getting older. Therefore, aging has been regarded as a synonym of senescence, and the two words have often been used interchangeably, which, in some cases, is fine but in some other cases causes confusion. This paper will first briefly discuss the terminology of senescence, and then will review the literature related to mitotic senescence, a topic that has not been well discussed in the plant senescence research area and discuss some results relating to nutrient

Senescence is a universal phenomenon in living organisms, and the word *senescence* has been used by scientists working on a variety of systems, such as yeast, fruit fly, worm, human being and plants. However, the meaning of the word *senescence* to scientists working on different organisms can be different, and the difference can be subtle in some cases and

Plants exhibit both types of senescence. An example of mitotic senescence in plants is the arrest of apical meristem; the meristem consists of non differentiated, germ line-like cells that can divide finite times to produce cells that will be then differentiated to form new organs such as leaves and flowers. The arrest of apical meristem is also called proliferative senescence in plant literature .This is similar to replicative senescence in yeast and animal

**3. Plants exhibit mitotic senescence, post mitotic senescence and cell** 


## **Some Aspects of Leaf Senescence**

Hafsi Miloud and Guendouz Ali

*Laboratory of Improvement and Development of Livestock and Crop Production, Department of Agronomy, Faculty of Natural Sciences and Life, Ferhat ABBAS University, Setif Algeria* 

## **1. Introduction**

106 Senescence

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The word *senescence* derives from two Latin words: *senex* and *senescere*. *Senex* means 'old'; this Latin root is shared by 'senile', 'senior', and even 'senate'. In ancient Rome the 'Senatus' was a 'council of elders' that was composed of the heads of patrician families. *Senescere*  means 'to grow old'. The Merriam-Webster online dictionary defines *senescence* as 'the state of being old or the process of becoming old'. Aging is also the process of getting older. Therefore, aging has been regarded as a synonym of senescence, and the two words have often been used interchangeably, which, in some cases, is fine but in some other cases causes confusion. This paper will first briefly discuss the terminology of senescence, and then will review the literature related to mitotic senescence, a topic that has not been well discussed in the plant senescence research area and discuss some results relating to nutrient remobilization during leaf senescence.

## **2. Terminology and types of senescence**

Senescence is a universal phenomenon in living organisms, and the word *senescence* has been used by scientists working on a variety of systems, such as yeast, fruit fly, worm, human being and plants. However, the meaning of the word *senescence* to scientists working on different organisms can be different, and the difference can be subtle in some cases and very obvious in some other cases.

## **3. Plants exhibit mitotic senescence, post mitotic senescence and cell quiescence**

Plants exhibit both types of senescence. An example of mitotic senescence in plants is the arrest of apical meristem; the meristem consists of non differentiated, germ line-like cells that can divide finite times to produce cells that will be then differentiated to form new organs such as leaves and flowers. The arrest of apical meristem is also called proliferative senescence in plant literature .This is similar to replicative senescence in yeast and animal

Some Aspects of Leaf Senescence 109

remobilization from senescing plant parts to surviving structures is a hallmark of the 'execution' of the senescence process in both annual plants, in which nutrients are retranslocated to the seeds, and perennial species, in which nutrients are transported to

Plants need a number of elements in higher quantities or concentrations to complete their life cycle (macronutrients, including C, O, H, N, P, S, K, Mg and Ca), while a number of additional elements (micronutrients, including Fe, Mn, Zn, Cu, B, Mo, Cl and Ni) are needed in comparatively small quantities (Marschner, 1995). Some elements are essential only for specific taxonomic groups (e.g. Na, Si) and/or are considered beneficial

Quantitatively, nitrogen is the most important mineral nutrient in plants (Marschner, 1995). It is often a limiting factor for plant growth, yield and/or quality (Gastal & Lemaire, 2002; Good *et al.*, 2004). Additionally, as for carbon, the principal form in which many plants acquire nitrogen from the environment (nitrate) is more oxidized than the form in which it can be integrated into metabolites and macro molecules, demanding substantial energy input for the synthesis of nitrogen compounds. Although the biochemistry involved is different, the establishment and maintenance of a symbiosis with N2-fixing microorganisms (e.g. in legumes) is also costly (Crawford *et al.*, 2000; Lodwig & Poole, 2003). For these reasons, efficient N remobilization increases the competitiveness of wild plants. Additionally, due to the economic and ecological (N runoff from agricultural soils) cost of N

In most plant tissues, the largest fraction of organic nitrogen, which is potentially available for remobilization during senescence, is contained in proteins. In photosynthetically active tissues of C3 species, over 50% of this nitrogen is found in soluble (Calvin cycle) and insoluble (thylakoid) chloroplast proteins (Peoples and Dalling, 1988; Feller and Fischer, 1994). Intriguingly, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) alone

All other cellular nitrogen fractions, including cytosolic and other proteins, nucleic acids, chlorophylls and free amino acids, while not negligible, represent relatively minor stores of organic nitrogen. Efforts at understanding nitrogen remobilization during leaf senescence have therefore focused on the biochemistry of plastidial protein degradation. Mae *et al.* (1983), using elegant 15 N-labeling techniques, have demonstrated that the synthesis and degradation phases of Rubisco are surprisingly clearly separated during leaf development. High rates of synthesis were observed until full leaf expansion; after this point, synthesis was minimal, but degradation rates started to increase. In this context, it is well known that the photosynthetic capacity of a leaf declines early during leaf senescence, while mitochondrial integrity and respiration are maintained longer (Gepstein, 1988; Feller and Fischer, 1994). That efficient N remobilization is associated with (early) loss of CO2 assimilation represents a formidable problem in annual crops. In this context, agronomists are well aware of the negative correlation between seed protein

surviving structures such as bulbs and roots.

fertilization, this trait is of considerable importance to farmers.

represents 50% of the total plastidial nitrogen.

(Marschner, 1995).

and yield.

**5.1 Nitrogen remobilization** 

cells in culture. Another example of mitotic senescence is the arrest of mitotic cell division at early stages of fruit development. Fruit size is a function of cell number, cell size and intercellular space, and cell number is the major factor.

Cell number is determined at the very early stage of fruit development and remains unchanged thereafter. Post mitotic senescence occurs in some plant organs, such as leaves and floral petals. Once formed, cells in these organs rarely undergo cell division; their growth is mainly contributed by cell expansion; thus, their senescence, unlike mitotic senescence, is not due to an inability to divide. This type of senescence involving predominantly somatic tissues is very similar to that.

## **4. Physiological regulation**

Reproductive development appears to play an important role in regulating proliferative senescence in plants, which is especially true in many monocarpic plants. Hensel *et al.*  (1994) found that meristems of all inflorescence branches in the wild-type *Arabidopsis*  ecotype Landsberg *erecta* (L*er*) ceased to produce flowers coordinately, but such a coordinated proliferative arrest did not occur in the wild-type L*er* plants with their fruits surgically removed. Similarly, meristem arrest was not observed in a male-sterile line that never sets seeds. This result suggests that the arrest of inflorescence meristems is regulated by developing fruits/seeds (Hensel *et al.*, 1994). Hensel *et al.* further proposed two models to explain the effect of developing fruits on the mitotic activity of meristems. One model is that a factor necessary for sustaining mitotic activity at the SAM is gradually taken and eventually depleted by developing fruits, resulting in arrest. The other model is that developing fruits produce a negative regulator of mitotic activities and that the negative regulator is transferred to and accumulated in the SAM to a threshold level so that the SAM is arrested. The factor, either positive or negative, is unknown.

## **5. Nutrient remobilization during leaf senescence**

Senescence is the last stage in the development of leaves and other plant organs. While many plants are perennial (barring adverse conditions leading to premature death), and some species even very long-lived (at least from a human perspective), senescence and death of organs such as leaves is often an annual event. Due to its importance for agriculture, the senescence of annual crops (e.g. corn, rice, wheat, barley and some legumes) has been most intensely studied (Feller & Fischer, 1994; Hayati *et al.*, 1995; Crafts-Brandner *et al.*, 1998; Yang *et al.*, 2003; Robson *et al.*, 2004; Parrott *et al.*, 2005;Weng *et al.*, 2005). Additionally, as in other areas of plant science research,*Arabidopsis* has emerged as an important model system (Diaz *et al.*, 2005; Levey & Wingler, 2005; Otegui *et al.*, 2005). These plants show monocarpic senescence, i.e. fruit set and maturation are directly associated with whole-plant senescence and death. Other types of senescence, such as top senescence (in species with bulbs, tubers, tap roots or rhizomes), deciduous senescence (in some trees and shrubs of temperate climate zones) and progressive senescence (e.g. in evergreen trees) have received less attention. In contrast to annuals, leaf (or whole-shoot) senescence is often not directly associated with seed filling in perennial plants (Feller & Fischer 1994; Nood´en *et al.*, 2004). However, nutrient

cells in culture. Another example of mitotic senescence is the arrest of mitotic cell division at early stages of fruit development. Fruit size is a function of cell number, cell size and

Cell number is determined at the very early stage of fruit development and remains unchanged thereafter. Post mitotic senescence occurs in some plant organs, such as leaves and floral petals. Once formed, cells in these organs rarely undergo cell division; their growth is mainly contributed by cell expansion; thus, their senescence, unlike mitotic senescence, is not due to an inability to divide. This type of senescence involving

Reproductive development appears to play an important role in regulating proliferative senescence in plants, which is especially true in many monocarpic plants. Hensel *et al.*  (1994) found that meristems of all inflorescence branches in the wild-type *Arabidopsis*  ecotype Landsberg *erecta* (L*er*) ceased to produce flowers coordinately, but such a coordinated proliferative arrest did not occur in the wild-type L*er* plants with their fruits surgically removed. Similarly, meristem arrest was not observed in a male-sterile line that never sets seeds. This result suggests that the arrest of inflorescence meristems is regulated by developing fruits/seeds (Hensel *et al.*, 1994). Hensel *et al.* further proposed two models to explain the effect of developing fruits on the mitotic activity of meristems. One model is that a factor necessary for sustaining mitotic activity at the SAM is gradually taken and eventually depleted by developing fruits, resulting in arrest. The other model is that developing fruits produce a negative regulator of mitotic activities and that the negative regulator is transferred to and accumulated in the SAM to a threshold level so

that the SAM is arrested. The factor, either positive or negative, is unknown.

Senescence is the last stage in the development of leaves and other plant organs. While many plants are perennial (barring adverse conditions leading to premature death), and some species even very long-lived (at least from a human perspective), senescence and death of organs such as leaves is often an annual event. Due to its importance for agriculture, the senescence of annual crops (e.g. corn, rice, wheat, barley and some legumes) has been most intensely studied (Feller & Fischer, 1994; Hayati *et al.*, 1995; Crafts-Brandner *et al.*, 1998; Yang *et al.*, 2003; Robson *et al.*, 2004; Parrott *et al.*, 2005;Weng *et al.*, 2005). Additionally, as in other areas of plant science research,*Arabidopsis* has emerged as an important model system (Diaz *et al.*, 2005; Levey & Wingler, 2005; Otegui *et al.*, 2005). These plants show monocarpic senescence, i.e. fruit set and maturation are directly associated with whole-plant senescence and death. Other types of senescence, such as top senescence (in species with bulbs, tubers, tap roots or rhizomes), deciduous senescence (in some trees and shrubs of temperate climate zones) and progressive senescence (e.g. in evergreen trees) have received less attention. In contrast to annuals, leaf (or whole-shoot) senescence is often not directly associated with seed filling in perennial plants (Feller & Fischer 1994; Nood´en *et al.*, 2004). However, nutrient

**5. Nutrient remobilization during leaf senescence** 

intercellular space, and cell number is the major factor.

predominantly somatic tissues is very similar to that.

**4. Physiological regulation** 

remobilization from senescing plant parts to surviving structures is a hallmark of the 'execution' of the senescence process in both annual plants, in which nutrients are retranslocated to the seeds, and perennial species, in which nutrients are transported to surviving structures such as bulbs and roots.

Plants need a number of elements in higher quantities or concentrations to complete their life cycle (macronutrients, including C, O, H, N, P, S, K, Mg and Ca), while a number of additional elements (micronutrients, including Fe, Mn, Zn, Cu, B, Mo, Cl and Ni) are needed in comparatively small quantities (Marschner, 1995). Some elements are essential only for specific taxonomic groups (e.g. Na, Si) and/or are considered beneficial (Marschner, 1995).

## **5.1 Nitrogen remobilization**

Quantitatively, nitrogen is the most important mineral nutrient in plants (Marschner, 1995). It is often a limiting factor for plant growth, yield and/or quality (Gastal & Lemaire, 2002; Good *et al.*, 2004). Additionally, as for carbon, the principal form in which many plants acquire nitrogen from the environment (nitrate) is more oxidized than the form in which it can be integrated into metabolites and macro molecules, demanding substantial energy input for the synthesis of nitrogen compounds. Although the biochemistry involved is different, the establishment and maintenance of a symbiosis with N2-fixing microorganisms (e.g. in legumes) is also costly (Crawford *et al.*, 2000; Lodwig & Poole, 2003). For these reasons, efficient N remobilization increases the competitiveness of wild plants. Additionally, due to the economic and ecological (N runoff from agricultural soils) cost of N fertilization, this trait is of considerable importance to farmers.

In most plant tissues, the largest fraction of organic nitrogen, which is potentially available for remobilization during senescence, is contained in proteins. In photosynthetically active tissues of C3 species, over 50% of this nitrogen is found in soluble (Calvin cycle) and insoluble (thylakoid) chloroplast proteins (Peoples and Dalling, 1988; Feller and Fischer, 1994). Intriguingly, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) alone represents 50% of the total plastidial nitrogen.

All other cellular nitrogen fractions, including cytosolic and other proteins, nucleic acids, chlorophylls and free amino acids, while not negligible, represent relatively minor stores of organic nitrogen. Efforts at understanding nitrogen remobilization during leaf senescence have therefore focused on the biochemistry of plastidial protein degradation. Mae *et al.* (1983), using elegant 15 N-labeling techniques, have demonstrated that the synthesis and degradation phases of Rubisco are surprisingly clearly separated during leaf development. High rates of synthesis were observed until full leaf expansion; after this point, synthesis was minimal, but degradation rates started to increase. In this context, it is well known that the photosynthetic capacity of a leaf declines early during leaf senescence, while mitochondrial integrity and respiration are maintained longer (Gepstein, 1988; Feller and Fischer, 1994). That efficient N remobilization is associated with (early) loss of CO2 assimilation represents a formidable problem in annual crops. In this context, agronomists are well aware of the negative correlation between seed protein and yield.

Some Aspects of Leaf Senescence 111

reutilization of plastidial (thylakoid) lipids via *β*-oxidation, glyoxylate cycle and gluconeogenesis, allowing export of at least some of the carbon 'stored' in plastidial lipids from the senescing leaf. These observations have since been confirmed and extended (Pistelli *et al.*, 1991; Graham *et al.*, 1992; McLaughlin & Smith, 1994). He and Gan (2002) have shown an essential role for an *Arabidopsis* lipase in leaf senescence; however, it is not yet clear if this or other lipases are involved in preparing substrates (free fatty acids) for *β*-oxidation and gluconeogenesis. Roulin *et al.* (2002) have found an induction of (1→3, 1→4)-*β*-d-glucan hydrolases during dark-induced senescence of barley seedlings,

Using radioactive labeling studies,Yang *et al.* (2003) demonstrated considerable remobilization of pre-fixed 14C from vegetative tissues to grains in senescent wheat plants. Interestingly, this process was enhanced under drought conditions, when leaf photosynthetic rates declined faster. Together, these data suggest that while C remobilization during leaf senescence has received less attention than N remobilization, it probably makes important contributions to seed development, at least in annual crops.

Besides carbon and nitrogen, sulfur is the third nutrient, which (relative to its main form of uptake, sulfate) is reduced by plants prior to its incorporation into certain metabolites and macromolecules. It is noteworthy, however, that plants also contain oxidized ('sulfated') sulfur metabolites (Crawford et al., 2000). Identically to carbon and nitrogen, sulfur is an essential element of both low-molecular weight compounds (including the protein amino acids cysteine and methionine) and macromolecules (proteins). Glutathione (γ -glutamylcysteinyl-glycine) represents the quantitatively most important reduced sulfur metabolite; it can reach millimolar concentrations in chloroplasts (Rennenberg and Lamoureux, 1990). Sulfur remobilization from older leaves has been shown; however, the extent of its retranslocation appears to depend on the nitrogen status, at least in some systems (Marschner, 1995). Sunarpi & Anderson (1997) demonstrated the remobilization of both soluble (non protein) and insoluble (protein) sulfur from senescing leaves. This study also indicated that homoglutathione (containing β-alanine instead of glycine) is the principal

Next to nitrogen, potassium is the mineral nutrient required in the largest amount by plants. It is highly mobile within individual cells, within tissues and in long-distance transport via the xylem and phloem (Marschner, 1995). In contrast to the nutrients discussed above, potassium is not metabolized, and it forms only weak complexes, in which it is easily exchangeable. Next to the transport of carbohydrates and nitrogen compounds, potassium transport has been studied most intensely, using both physiological and molecular approaches (Kochian, 2000). Many plant genes encoding K+ transporters have been identified, and some of them have been studied in detail in heterologous systems, such as K+-transport-deficient yeast mutants. Similarly to the situation discussed for nitrogen transport, analysis of K+ transport is complicated by the

suggesting a remobilization of cell wall glucans under these conditions.

export form of metabolized protein sulfur from senescing soybean leaves.

**5.4 Sulfur** 

**5.5 Potassium** 

#### **5.2 Macro- and micronutrient remobilization**

Developing (young) leaves constitute significant net importers ('sinks') for all nutrients, which are utilized to build the organ's cellular and molecular components. After the socalled sink–source transition (Ishimaru *et al.*, 2004; Jeong *et al.*, 2004), leaves become net exporters ('sources') of carbohydrates from photosynthesis, while import (through the xylem) and export (through the phloem) of phloem-mobile nutrients are (roughly) at an equilibrium in mature leaves (Marschner, 1995). The onset of leaf senescence is associated with a transition to net export of 'mobile' (see below) compounds, i.e. total (per leaf) content of some nutrients starts to decrease (Marschner, 1995). The literature often refers to this situation as 'redistribution', 'retranslocation', 'resorption' or 'remobilization' (Marschner, 1995; Killingbeck, 2004).

The main transport route from senescing leaves to nutrient sinks is the phloem (Atkins, 2000; Tilsner *et al.*, 2005). Using various approaches, including sampling and analysis of phloem sap and (radioactive) tracer studies, it has been established that macronutrients with the exception of calcium (i.e. N, P, S, K and Mg) are generally highly mobile in the phloem, while micronutrients with the exception of manganese (i.e. Fe, Zn, Cu, B, Mo, Cl and Ni) show at least moderate mobility (Marschner, 1995). As a consequence, while some mobile nutrients decrease during leaf senescence, this is not true for calcium, which continues to accumulate throughout a leaf's life span. The molecular form, in which nutrients fulfill their biological functions, determines the biochemical steps necessary to make them phloem mobile. A certain percentage of many nutrients is biochemically inert, and cannot be remobilized (Marschner, 1995; Killingbeck, 2004). Cell wall components are a good example, and explain why fully senesced (dead) leaves are usually rich in carbon as compared to nitrogen. Some macronutrients, including carbon, nitrogen, phosphorus and sulfur, are covalently bound in myriads of both lowmolecular-weight metabolites and macromolecules. Proteins and nucleic acids are important stores of nitrogen, phosphorus (nucleic acids) and sulfur (proteins); these macromolecules have to be degraded by specific hydrolases prior to phloem loading and transport. Metals (both macro- and micronutrients) can also be tightly bound, mostly by macromolecules, e.g. cell wall compounds or proteins. Their release is therefore often linked with the degradation of the functional complexes/macromolecules, to which they belong.

#### **5.3 Carbon**

Because it is taken up in gaseous form and a large amount of energy is needed for its reduction prior to its incorporation into metabolites, carbon occupies a special position in plant metabolism. Additionally, as discussed obove, degradation of the photosynthetic apparatus is an early event during leaf senescence, leading to a decrease of photoassimilate production and export to sinks, and to an increasing dependence of senescing tissues on respiratory metabolism (Gepstein, 1988; Feller & Fischer, 1994). Metabolization and, to some degree, remobilization of reduced carbon are therefore important for senescing leaves. In this context, Gut and Matile (1988, 1989) observed an induction of key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, in senescent barley leaves. Based on these data, and based on low respiratory quotients (0.6), these authors suggested a

Developing (young) leaves constitute significant net importers ('sinks') for all nutrients, which are utilized to build the organ's cellular and molecular components. After the socalled sink–source transition (Ishimaru *et al.*, 2004; Jeong *et al.*, 2004), leaves become net exporters ('sources') of carbohydrates from photosynthesis, while import (through the xylem) and export (through the phloem) of phloem-mobile nutrients are (roughly) at an equilibrium in mature leaves (Marschner, 1995). The onset of leaf senescence is associated with a transition to net export of 'mobile' (see below) compounds, i.e. total (per leaf) content of some nutrients starts to decrease (Marschner, 1995). The literature often refers to this situation as 'redistribution', 'retranslocation', 'resorption' or 'remobilization' (Marschner,

The main transport route from senescing leaves to nutrient sinks is the phloem (Atkins, 2000; Tilsner *et al.*, 2005). Using various approaches, including sampling and analysis of phloem sap and (radioactive) tracer studies, it has been established that macronutrients with the exception of calcium (i.e. N, P, S, K and Mg) are generally highly mobile in the phloem, while micronutrients with the exception of manganese (i.e. Fe, Zn, Cu, B, Mo, Cl and Ni) show at least moderate mobility (Marschner, 1995). As a consequence, while some mobile nutrients decrease during leaf senescence, this is not true for calcium, which continues to accumulate throughout a leaf's life span. The molecular form, in which nutrients fulfill their biological functions, determines the biochemical steps necessary to make them phloem mobile. A certain percentage of many nutrients is biochemically inert, and cannot be remobilized (Marschner, 1995; Killingbeck, 2004). Cell wall components are a good example, and explain why fully senesced (dead) leaves are usually rich in carbon as compared to nitrogen. Some macronutrients, including carbon, nitrogen, phosphorus and sulfur, are covalently bound in myriads of both lowmolecular-weight metabolites and macromolecules. Proteins and nucleic acids are important stores of nitrogen, phosphorus (nucleic acids) and sulfur (proteins); these macromolecules have to be degraded by specific hydrolases prior to phloem loading and transport. Metals (both macro- and micronutrients) can also be tightly bound, mostly by macromolecules, e.g. cell wall compounds or proteins. Their release is therefore often linked with the degradation of the functional complexes/macromolecules, to which they

Because it is taken up in gaseous form and a large amount of energy is needed for its reduction prior to its incorporation into metabolites, carbon occupies a special position in plant metabolism. Additionally, as discussed obove, degradation of the photosynthetic apparatus is an early event during leaf senescence, leading to a decrease of photoassimilate production and export to sinks, and to an increasing dependence of senescing tissues on respiratory metabolism (Gepstein, 1988; Feller & Fischer, 1994). Metabolization and, to some degree, remobilization of reduced carbon are therefore important for senescing leaves. In this context, Gut and Matile (1988, 1989) observed an induction of key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, in senescent barley leaves. Based on these data, and based on low respiratory quotients (0.6), these authors suggested a

**5.2 Macro- and micronutrient remobilization** 

1995; Killingbeck, 2004).

belong.

**5.3 Carbon** 

reutilization of plastidial (thylakoid) lipids via *β*-oxidation, glyoxylate cycle and gluconeogenesis, allowing export of at least some of the carbon 'stored' in plastidial lipids from the senescing leaf. These observations have since been confirmed and extended (Pistelli *et al.*, 1991; Graham *et al.*, 1992; McLaughlin & Smith, 1994). He and Gan (2002) have shown an essential role for an *Arabidopsis* lipase in leaf senescence; however, it is not yet clear if this or other lipases are involved in preparing substrates (free fatty acids) for *β*-oxidation and gluconeogenesis. Roulin *et al.* (2002) have found an induction of (1→3, 1→4)-*β*-d-glucan hydrolases during dark-induced senescence of barley seedlings, suggesting a remobilization of cell wall glucans under these conditions.

Using radioactive labeling studies,Yang *et al.* (2003) demonstrated considerable remobilization of pre-fixed 14C from vegetative tissues to grains in senescent wheat plants. Interestingly, this process was enhanced under drought conditions, when leaf photosynthetic rates declined faster. Together, these data suggest that while C remobilization during leaf senescence has received less attention than N remobilization, it probably makes important contributions to seed development, at least in annual crops.

## **5.4 Sulfur**

Besides carbon and nitrogen, sulfur is the third nutrient, which (relative to its main form of uptake, sulfate) is reduced by plants prior to its incorporation into certain metabolites and macromolecules. It is noteworthy, however, that plants also contain oxidized ('sulfated') sulfur metabolites (Crawford et al., 2000). Identically to carbon and nitrogen, sulfur is an essential element of both low-molecular weight compounds (including the protein amino acids cysteine and methionine) and macromolecules (proteins). Glutathione (γ -glutamylcysteinyl-glycine) represents the quantitatively most important reduced sulfur metabolite; it can reach millimolar concentrations in chloroplasts (Rennenberg and Lamoureux, 1990). Sulfur remobilization from older leaves has been shown; however, the extent of its retranslocation appears to depend on the nitrogen status, at least in some systems (Marschner, 1995). Sunarpi & Anderson (1997) demonstrated the remobilization of both soluble (non protein) and insoluble (protein) sulfur from senescing leaves. This study also indicated that homoglutathione (containing β-alanine instead of glycine) is the principal export form of metabolized protein sulfur from senescing soybean leaves.

## **5.5 Potassium**

Next to nitrogen, potassium is the mineral nutrient required in the largest amount by plants. It is highly mobile within individual cells, within tissues and in long-distance transport via the xylem and phloem (Marschner, 1995). In contrast to the nutrients discussed above, potassium is not metabolized, and it forms only weak complexes, in which it is easily exchangeable. Next to the transport of carbohydrates and nitrogen compounds, potassium transport has been studied most intensely, using both physiological and molecular approaches (Kochian, 2000). Many plant genes encoding K+ transporters have been identified, and some of them have been studied in detail in heterologous systems, such as K+-transport-deficient yeast mutants. Similarly to the situation discussed for nitrogen transport, analysis of K+ transport is complicated by the

Some Aspects of Leaf Senescence 113

This paper discussed some results relating to nutrient remobilization during leaf senescence.Complex regulatory network controlling senescence in plants may be the result of selection pressure driven by different environmental stresses for the development of senescence.Focus on limited number of model plant systems studied by plant senescence scientists may be required for more efcient research, and is likely to be highly relevant to agriculture as well as to our basic understanding of the senescence

[1] Crafts-Brandner, S.J. (1992).Phosphorus nutrition inuence on leaf senescence in

[2] Crafts-Brandner, S.J., Holzer, R. & Feller, U. (1998).Inuence of nitrogen deciency on

[3] Crawford, N.M., Kahn, M.L., Leustek, T. & Long, S.R. (2000). Nitrogen and sulfur in

[4] Diaz, C., Purdy, S., Christ, A., Morot-Gaudry, J.-F., Wingler, A. & Masclaux Daubresse, C.

[5] Debrunner, N. & Feller, U. (1995).Solute leakage from detached plant parts of winter

[6] Feller, U. & Fischer, A. (1994). Nitrogen metabolism in senescing leaves. Crit Rev Plant

[7] Gastal, F. & Lemaire, G.(2002). N uptake and distribution in crops: an agronomical and

[8] Gepstein, S. (1988).Photosynthesis. In: Senescence and Aging in Plants (edsNood´ en,

[9] Good, A.G., Shrawat, A. K. & Muench, D.G. (2004).Can less yield more? Is reducing

[10] Graham, I.A., Leaver, C.J. & Smiths. (1992).Induction of malate synthase gene expression in Senescent and detached organs of cucumber. Plant Cell 4,349–357. [11] Gut, H. & Matile, P. (1989).Break down of galactolipids in senescent barley leaves. Bot

[12] Gut, H.& Matile, P. (1988). Apparent induction of key enzymes of the glyoxylic acid

nutrient in put into the environment compatible with maintaining crop

L.D.and Leopold, A.C.).Academic Press, SanDiego, CA, pp.85–109.

ecophysio- Logical perspective. J Exp Bot 53(370), 789–799.

production? Trends Plant Sci 9 (12), 597–605.

cycle in senescent barley leaves. Planta 176,548–550.

senescence and the amounts of RNA and proteins in wheat leaves. Physiol

Biochemistry and Molecular Biology of Plants (Eds Buchanan, B., Gruissem, W.and Jones, R.).American Society of Plant Physiologists, Rockville, MD, pp.786–

(2005). Characterization of markers to determine the extent and variability of leaf senescence in Arabidopsis. A metabolic proling approach. Plant Physiol 138,898–

wheat: Inuence of maturation stage and incubation temperature. J Plant Physiol

soybean. Plant Physiol 98, 1128–1132.

Plantarum 102,192–200.

**6. Conclusions** 

process in plants.

**7. References** 

849.

908.

145,257–260.

Sci 13(3), 241–273.

Acta 102, 31–36.

fact that these transporters are organized in multigene families with (partially?) redundant functions (Kochian, 2000). Potassium was repeatedly reported to be remobilized in significant quantities from senescing tissues (Hill *et al.*, 1979; Scott *et al.*, 1992; Tyler, 2005). However, it has to be considered that this element easily leaches from tissues, especially senescing tissues (Tukey, 1970; Debrunner & Feller, 1995). Therefore, actually remobilized potassium quantities may be smaller than those reported in the literature.

## **5.6 Phosphorus**

Unlike carbon dioxide, nitrate and sulfate, phosphate (main form of P uptake) is not reduced, but utilized in its oxidized form by plants (Marschner, 1995), both in lowmolecular- weight metabolites and in macromolecules (nucleic acids). Studies on P remobilization from senescing leaves are scarce. Snapp and Lynch (1996) concluded that in maturing common bean plants, leaf P remobilization supplied more than half of the pod plus seed phosphorus. In contrast, Crafts-Brandner (1992) observed no net leaf P remobilization during reproductive growth of soybeans cultivated at three different P regimes. Therefore, while P is a mobile nutrient, its remobilization may be influenced by a number of exogenous and endogenous/genetic factors, making generalizations on the importance of its remobilization difficult. Nucleic acids (especially RNA) constitute a major phosphorus store but, depending on the species and growth condition investigated, considerable P amounts are also present in lipids, in esterified (organic) form, and as inorganic phosphate (Hart & Jessop, 1984; Valenzuela *et al.*, 1996). Similarly to the situation with nitrogen 'bound' in proteins, release of phosphorus from nucleic acids depends on the activities of hydrolytic enzymes. A decrease in nucleic acid levels is typical for senescing tissues, and increases in nuclease activities have also been observed (Feller and Fischer, 1994; Lers *et al.*, 2001), indicating that if P is remobilized from senescing tissues, at least part of it is derived from the degradation of RNA and DNA.

#### **5.7 Magnesium, calcium and micronutrients**

Magnesium has not often been considered in studies on nutrient remobilization. However, despite the fact that this element is considered phloem mobile (Marschner, 1995), available results indicate a tendency of continued accumulation during leaf senescence (Killingbeck, 2004). Unsurprisingly, calcium, which is the least mobile of all macronutrients (Marschner, 1995), has repeatedly been found to increase in senescing leaves (Killingbeck, 2004).

Information on remobilization of micronutrients does not allow a generalized picture. For several of them, including Fe, Cu, Mn (which is the least phloem mobile among the micronutrients) and Zn, both remobilization from and accumulation in senescing leaves have been reported (Killingbeck, 2004, and references cited therein). Tyler (2005) gives a broad overview of the fate of numerous elements (including the micronutrients Fe, B, Mn, Zn, Cu, Mo and Ni) during senescence and decomposition of *Fagus sylvatica* leaves; however, in view of the results cited above, it is probably not possible to generalize conclusions from this study, e.g. with regard to the situation in annual crops.

#### **6. Conclusions**

112 Senescence

fact that these transporters are organized in multigene families with (partially?) redundant functions (Kochian, 2000). Potassium was repeatedly reported to be remobilized in significant quantities from senescing tissues (Hill *et al.*, 1979; Scott *et al.*, 1992; Tyler, 2005). However, it has to be considered that this element easily leaches from tissues, especially senescing tissues (Tukey, 1970; Debrunner & Feller, 1995). Therefore, actually remobilized potassium quantities may be smaller than those reported in the

Unlike carbon dioxide, nitrate and sulfate, phosphate (main form of P uptake) is not reduced, but utilized in its oxidized form by plants (Marschner, 1995), both in lowmolecular- weight metabolites and in macromolecules (nucleic acids). Studies on P remobilization from senescing leaves are scarce. Snapp and Lynch (1996) concluded that in maturing common bean plants, leaf P remobilization supplied more than half of the pod plus seed phosphorus. In contrast, Crafts-Brandner (1992) observed no net leaf P remobilization during reproductive growth of soybeans cultivated at three different P regimes. Therefore, while P is a mobile nutrient, its remobilization may be influenced by a number of exogenous and endogenous/genetic factors, making generalizations on the importance of its remobilization difficult. Nucleic acids (especially RNA) constitute a major phosphorus store but, depending on the species and growth condition investigated, considerable P amounts are also present in lipids, in esterified (organic) form, and as inorganic phosphate (Hart & Jessop, 1984; Valenzuela *et al.*, 1996). Similarly to the situation with nitrogen 'bound' in proteins, release of phosphorus from nucleic acids depends on the activities of hydrolytic enzymes. A decrease in nucleic acid levels is typical for senescing tissues, and increases in nuclease activities have also been observed (Feller and Fischer, 1994; Lers *et al.*, 2001), indicating that if P is remobilized from senescing tissues, at least part of it is derived from the degradation of RNA and

Magnesium has not often been considered in studies on nutrient remobilization. However, despite the fact that this element is considered phloem mobile (Marschner, 1995), available results indicate a tendency of continued accumulation during leaf senescence (Killingbeck, 2004). Unsurprisingly, calcium, which is the least mobile of all macronutrients (Marschner,

Information on remobilization of micronutrients does not allow a generalized picture. For several of them, including Fe, Cu, Mn (which is the least phloem mobile among the micronutrients) and Zn, both remobilization from and accumulation in senescing leaves have been reported (Killingbeck, 2004, and references cited therein). Tyler (2005) gives a broad overview of the fate of numerous elements (including the micronutrients Fe, B, Mn, Zn, Cu, Mo and Ni) during senescence and decomposition of *Fagus sylvatica* leaves; however, in view of the results cited above, it is probably not possible to generalize

1995), has repeatedly been found to increase in senescing leaves (Killingbeck, 2004).

conclusions from this study, e.g. with regard to the situation in annual crops.

literature.

DNA.

**5.7 Magnesium, calcium and micronutrients** 

**5.6 Phosphorus** 

This paper discussed some results relating to nutrient remobilization during leaf senescence.Complex regulatory network controlling senescence in plants may be the result of selection pressure driven by different environmental stresses for the development of senescence.Focus on limited number of model plant systems studied by plant senescence scientists may be required for more efcient research, and is likely to be highly relevant to agriculture as well as to our basic understanding of the senescence process in plants.

#### **7. References**


Some Aspects of Leaf Senescence 115

[30] Otegui, M.S., Noh, Y.S., Martinez, D.E., et al. (2005)Senescence-associated vacuoles

[31] Parrott, D., Yang, L., Shama, L. & Fischer, A.M. (2005) .Senescence is accelerated, and

[32] Peoples, M.B. & Dalling, M.J. (1988).The interplay between proteolysis and amino acid

[33] Pistelli, L., DeBellis, L. & Alpi, A. (1991) Peroxisomal enzyme activities in attached

[34] Rennenberg, H. & Lamoureux, G.L. (1990). Physiological processes that modulate the

[35] Robson, P.R.H., Donnison, I.S. & Wang., et al. (2004).Leaf senescence is delayed in

[35] Roulin, S., Buchala, A.J.and Fincher, G.B. (2002). Induction of (1→3, 1→4)-β-D-glucan hydrolases in leave sofdark-incubated barley seedlings. Planta 215, 51–59. [36] Scott, D.A., Proctor. & Thompson. (1992). Ecological studies on a low land ever green

[37] Snapp, S.S. & Lynch, J.P. (1996). Phosphorus distribution and remobilization in bean

[38] Sunarpi and Anderson, J.W. (1997) .Effect of nitrogen nutrition on remobilization of

[39] Tukey, H.B. (1970). The leaching of substances from plants. Annu Rev Plant Physiol

[40] Tyler, G.(2005) . Changes in the concentrations of major, minor and rare-earth elements

[41] Yang,J.C.,Zhang,J.H.,Wang,Z.Q.,Zhu,Q.S.& Liu,L.J.(2003). Involvement of abscisic

[42] Valenzuela, J.L., Ruz, J.M., Belakbir, A. & Romero, L. (1996). Effects of nitrogen,

plants as inuenced by phosphorus nutrition. Crop Sci 36,929–935.

sulfur metabolites. Plant Physiol 115, 1671–1680.

Commun Soil Sci Plant Anal 27(5–8), 1417–1425.

senescence-enhanced promoter. Plant Biotechnol J 2,101–112.

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senescing leaves. Planta 184,151–153.

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several proteases are induced by carbon 'feast' conditions in barley (*Hordeum* 

metabolism during senescence and nitrogen reallocation. In: Senescence and Aging in Plants (Eds Nooden, L.D. & Leopold, A.C.).Academic Press, San Diego, CA,

concentration of glutathione in plant cells. In: Sulfur Nutrition and Sulfur Assimilation in Higher Plants (ed. Rennenberg, H.). XPB Academic PublishersB.V.,

maize expressing the Agrobacterium IPT gene under the control of a novel maize

rainforest on Maraca island, Brazil.II: Litter and nutrient recycling. J Ecol 80,705–

protein sulfur in the leaves of vegetative soybean and associated changes insoluble

during leaf Senescence and decomposition in a *Fagus sylvatica* forest. Forest Ecol

acid and Cytokinins in the senescence and remobilization of carbon reserves in wheat subjected to water Stress during grain lling. Plant Cell Environ 26,1621–

phosphorus and potassium treatments on phosphorus fractions in melon plants.


[13] Hart, A. L. & Jessop, D.(1984).Leaf phosphorus fractionation and growth responses to

[14] Hayati, R., Egli, D.B. & Crafts-Brandner, S.J. (1995).Carbon and nitrogen supply during

[15] He, Y. & Gan, S. (2002).Agene encoding anacyl hydrolase is involved in leaf senescence

[16] Hensel, L.L., Nelson, M.A., Richmond, T.A. & Bleecker, A.B. (1994). The fate of

[17] Hill, J., Robson, A.D.and Loneragan, J.F. (1979) .The effect of copper supply on the

[18] Ishimaru, K., Kosone, M.,Sasaki, H.and Kashiwagi,T.(2004).Leaf contents differ

[19] Jeong,M.L.,Jiang,H.,Chen,H.-S.,Tsai,C.-J.andHarding,S.A.(2004)Metabolic proling of

[20] Kilian, A., Stiff, C. & Kleinhofs,A.(1995) . Barley telomerees shorten during differentiation but grow in callus culture. Proc Natl Acad Sci U SA 92, 9555–9559. [21] Killing beck, K.T. (2004) .Nutrient resorption.In: Plant Cell Death Processes (ed.Nood´

[22] Kochian, L.V. (2000) .Molecular physiology of mineral nutrient acquisition, transport,

[23] Lers, A., Lomaniec, E., Burd, S.& Khalchitski,A.(2001).The characterization of LeNUC1,a

[24] Levey, S. & Wingler, A. (2005).Natural variation in the regulation of leaf senescence and relation to other traits in Arabidopsis. Plant Cell Environ 28,223–231. [25] Lodwig , E.& Poole, P. (2003) Metabolism of Rhizobium bacteroids. Crit Rev Plant Sci

[26] Mae, T., Makino, A. & Ohira, K. (1983). Changes in the amounts of ribulose

[27] Marschner, H.(1995) Mineral Nutrition of Higher Plants. Academic Press, London. [28] Mc Laughlin, J.C. & Smith, S. M.(1994)Metabolic regulation of glyoxylate-cycle enzyme synthesis in detached cucumber cotyledons and protoplasts. Planta 195, 22–28. [29] Nood´ en, L .D. Guiam, J.L. & John, I. (2004) .Whole plant senescence. In: Plant Cell

(Oryzasativa L.). Plant Cell Physiol 24(6), 1079–1086.

en, L.D.). Elsevier Academic Press, Amsterdam, pp.215–226.

seed lling and leaf senescence in soybean. Crop Sci 35, 1063–1069.

*pedunculatus*. Physiol Plant 61, 435–440.

in Arabidopsis. Plant Cell 14,805–815.

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phosphorus of the forage legumes *Trifolium repens*, *T.dubium* and *Lotus* 

inorescence meristems is controlled by developing fruits in Arabidopsis. Plant

senescence and the retranslocation of nutrients of the oldest leaf of wheat. Ann Bot

depending on the position in a rice leaf sheath during sink–source transition. Plant

the sink-to-source transition in developing leaves of quaking aspen. Plant Physiol

and utilization. In: Biochemistry and Molecular Biology of Plants (Eds Buchanan, B., Gruissem, W. & Jones, R.).American Society of Plant Physiologists, Rockville,

Nuclease associated with leaf senescence of tomato. Physiol Plantarum, 112,176–

bisphosphate carboxylase synthesized and degraded during the life spanofrice leaf

Death Processes (Ed .Nood´ en, L.D.).Elsevier Academic Press, Amsterdam, pp


**1. Introduction** 

of this chapter.

for crop growth and food production.

**7** 

*UK* 

Kieron D. Edwards,

*Advanced Technologies (Cambridge) Ltd.* 

**Advances in Plant Senescence** 

Matt Humphry and Juan Pablo Sanchez-Tamburrino

Senescence is an integral component of a plant's lifecycle, which refers to changes that take place as the plant matures. A general distinction between plant senescence and animal senescence is the events observed in the animal kingdom typically steer growth while plant senescence orchestrates a massive shutdown or coordinated cell death in response to

In order to assist in the survival of the plant species, a sequence of tightly regulated genetic events efficiently governs a plant's death. These events are observable in a variety of plant models and in the different plant parts such as leaves, petals, reproductive organs (stamens and style), root cap, cortex and germinating seed. Leaf senescence will be the primary focus

A popular aspect of leaf senescence is the bright hues that can be observed on trees and plants during Autumn. The brilliant burst of colour that precedes the browning of leaves is an indication of active metabolic changes that result in the recycling or redistribution of nutrients to other parts of the plant. Evidence indicates the primary purpose of senescence in plants is for mobilization and recycling, a phenomenon that has tremendous implications

Senescence marks the final phase of a leaf's development thereby launching degradation processes integral to the recycling and redistribution of the leaf's nutrients. Plant growth regulators, reproduction, cellular differentiation and hormone levels are internal factors that influence senescence (Thomas and Stoddart 1980; Smart 1994). Environmental stress also influences growth and can promote premature senescence. Certain parts of the plant may be sacrificed to enhance the chances of survival for the rest of the plant. Environmental cues include stress factors that adversely affect plant development and productivity; such as: drought, waterlogging, high or low solar radiation, extreme temperatures, ozone and other air pollutants, excessive soil salinity and inadequate mineral nutrition in the soil (Thomas and Stoddart 1980; Smart 1994). These environmental cues may accelerate leaf senescence by affecting the endogenous factors previously mentioned (Alegre and Munné-Bosch, 2004). Regardless of the trigger, the endogenous and exogenous signals that induce senescence appear to be coordinated through a common signalling network (Hopkins, 2007) involving the signalling molecules ethylene, jasmonic acid (JA), salicylic acid (SA) and Abscisic Acid

(ABA) (Smart, 1994; Buchanan-Wollaston et al., 2005; van der Graaff et al., 2006)

various stimuli designed to facilitate survival of the plant itself or the plant species.

[43] Weng, X.-Y., Xu, H.-X. & Jiang, D.-A. (2005). Characteristics of gas exchange, chlorophyll uorescence and expression of key enzymes in photosynthesis during leaf senescence in rice plants. J Integr Plant Biol 47(5), 560–566.

## **Advances in Plant Senescence**

Kieron D. Edwards,

Matt Humphry and Juan Pablo Sanchez-Tamburrino *Advanced Technologies (Cambridge) Ltd. UK* 

## **1. Introduction**

116 Senescence

[43] Weng, X.-Y., Xu, H.-X. & Jiang, D.-A. (2005). Characteristics of gas exchange,

leaf senescence in rice plants. J Integr Plant Biol 47(5), 560–566.

chlorophyll uorescence and expression of key enzymes in photosynthesis during

Senescence is an integral component of a plant's lifecycle, which refers to changes that take place as the plant matures. A general distinction between plant senescence and animal senescence is the events observed in the animal kingdom typically steer growth while plant senescence orchestrates a massive shutdown or coordinated cell death in response to various stimuli designed to facilitate survival of the plant itself or the plant species.

In order to assist in the survival of the plant species, a sequence of tightly regulated genetic events efficiently governs a plant's death. These events are observable in a variety of plant models and in the different plant parts such as leaves, petals, reproductive organs (stamens and style), root cap, cortex and germinating seed. Leaf senescence will be the primary focus of this chapter.

A popular aspect of leaf senescence is the bright hues that can be observed on trees and plants during Autumn. The brilliant burst of colour that precedes the browning of leaves is an indication of active metabolic changes that result in the recycling or redistribution of nutrients to other parts of the plant. Evidence indicates the primary purpose of senescence in plants is for mobilization and recycling, a phenomenon that has tremendous implications for crop growth and food production.

Senescence marks the final phase of a leaf's development thereby launching degradation processes integral to the recycling and redistribution of the leaf's nutrients. Plant growth regulators, reproduction, cellular differentiation and hormone levels are internal factors that influence senescence (Thomas and Stoddart 1980; Smart 1994). Environmental stress also influences growth and can promote premature senescence. Certain parts of the plant may be sacrificed to enhance the chances of survival for the rest of the plant. Environmental cues include stress factors that adversely affect plant development and productivity; such as: drought, waterlogging, high or low solar radiation, extreme temperatures, ozone and other air pollutants, excessive soil salinity and inadequate mineral nutrition in the soil (Thomas and Stoddart 1980; Smart 1994). These environmental cues may accelerate leaf senescence by affecting the endogenous factors previously mentioned (Alegre and Munné-Bosch, 2004). Regardless of the trigger, the endogenous and exogenous signals that induce senescence appear to be coordinated through a common signalling network (Hopkins, 2007) involving the signalling molecules ethylene, jasmonic acid (JA), salicylic acid (SA) and Abscisic Acid (ABA) (Smart, 1994; Buchanan-Wollaston et al., 2005; van der Graaff et al., 2006)

Advances in Plant Senescence 119

genes responsible for synthesis of the enzymes involved in gluconeogenesis are reported to be significantly expressed during this time (Buchanan-Wollaston and Ainsworth 1997; Kim

Leaf senescence also results in the breakdown of nucleic acids to purines and pyrimidines, which ultimately degrade to small and transportable carbon and nitrogenous compounds that are transported to growing parts of the plant (Buchanan-Wollaston and Ainsworth, 1997). In addition to mobilization of carbon and nitrogen, other nutrients like sulphur and metallic ions are also known to be transported from senescing leaves. Sugar content can also be modified at the onset of senescence. Generally crops under a limited nitrogen nutrition and high light regimen undergo early senescence and this is usually accompanied by an incremental rise of sugar levels in the leaves. Sugar has been suggested to trigger a senescence response based on gain or loss function experiments with hexokinase genes,

Before the advent of modern biotechnology, which enabled scientists to commence deciphering the relationship between genes and life, senescence was perceived as an uncoordinated collection of events resulting in the metabolic and physiological changes to plant organs described above. The study of plant genetics, genomics, proteomics and more recently metabolomics have altered this perception and demonstrated that the process is dynamic and well organised. Below, a few examples are provided to emphasize the importance of a better understanding of plant senescence and the consequent potential of

Several techniques and different plant models have been employed in the pursuit of understanding the genetic mechanisms underlying the changes in gene expression associated with senescence. The process of senescence is initiated in source tissues prompting dramatic changes in gene expression, during which genes involved in basic metabolism, including photosynthesis and protein biosynthesis, are down-regulated while those involved in programmed cell death and stress response and/or encoding various hydrolytic enzymes are up-regulated (Hopkins et al., 2007; Lim et al., 2007). Not surprisingly the initial discoveries involving Senescence Associated Genes (SAGs) were made in the model plant Arabidopsis (*Arabidopsis thaliana*) by methods including differential display (Lohman et al. 1994), senescence-specific enhancer trap line screening using a range of senescence promoting factors (He et al., 2001), subtractive hybridization (Gepstein et al., 2003) and microarray experiments (Andersson et al., 2004). Many of the genes expressed during senescence of tissues encode hydrolytic enzymes that are capable of disassembling the ultra-structure of the cell and the breakdown of macromolecules (Smart, 1994; Griffith et al., 1997; Watanabe et al., 1994). In addition, a large number of transcription factors, as well as genes encoding carbohydrate and nitrogen-mobilising enzymes, nucleases and stressresponsive proteins, have been found to exhibit increased expression in senescing leaves (Buchanan-Wollaston and Ainsworth, 1997; Comai et al., 1989; Kim and Smith, 1994). The gene expression changes and biological processes that are up- and down-regulated during senescence as indicated by such studies, have been reviewed elsewhere (Guo and Gan, 2005), so will only be touched on in this review. What is more in the scope of this review are

the potential implications that senescence has for plant biotechnological applications.

principal regulators of a glucose signalling pathway (van Doorn, 2008).

**3. Regulation of senescence and potential for biotechnology** 

applications derived from that understanding.

and Smith, 1994).

### **2. Progress of senescence in plants**

The general purpose of a leaf is to gather and generate nutrients for the plant. As a green leaf grows and develops, it creates an organ packed with nutrients. When the plant no longer requires the leaf, the senescence process is induced and recycling of all the nutrients that can be remobilized occurs. Leaf death is the final stage in the process; however, death is actively delayed until all nutrients have been removed.

The dismantling of the leaf begins with the chloroplasts, the energy-generating, photosynthetic powerhouse of the plant. Not unlike another energy generating organelle (ie, the mitochondria), chloroplasts are semiautonomous and they possess their own genome with its inherent transcriptional and translational machinery. Gradually the chloroplasts shrink and transform into gerontoplasts, an artefact characterised by the disintegration of the thylakoid membranes and accumulation of the plastoglobulin (Friedrich and Huffaker, 1980; Mae et al., 1984). The process of breaking down the chlorophyll is so pronounced that chlorophyll loss and the associated yellowing of the leaves are commonly used as indicators of plant senescence (Noodén et al., 1997). Control of the process is so tightly regulated that experiments demonstrating the reversibility of senescence have shown that the chloroplasts can recover structural features, re-synthesize chloroplast proteins and re-commence photosynthesis **(**Thomas and Donnison, 2000; Zavaleta-Mancera et al., 1999**).** 

Degradation and remobilization of the chloroplast proteins and RNA contribute nitrogen and other nutrients for seed growth (Wittenbach, 1978). The mechanisms governing degradation of the chloroplast are not completely understood and there are competing theories about where proteins are degraded; for example they may be degraded locally within the chloroplast or in a centralized vacuole for degradation (Hortensteiner and Feller, 2002). Findings that support the possibility that the photosynthetic machinery is degraded *in situ* by the chloroplast include the presence of chloroplast enzymes, which are localized hydrolases that catalyze the initial steps of chlorophyll breakdown. (Hortensteiner, 2006). Proteases of the Clp, FtsH and DegP families are also expressed in chloroplasts and representative genes for these proteases are up-regulated in senescing leaves (Sokolenko et al., 1998; Nakabayashi et al., 1999; Itzhaki et al., 1998; Haussühl et al., 2001). Despite this observation, chloroplastic proteases are unlikely to account for the degradation of most photosynthetic proteins (eg, Rubisco) during senescence. Senescence-associated vacuoles, with strong proteolytic activity, have been identified in senescing tissue and likely also contribute towards the degradation of soluble photosynthetic proteins (Hensell et al., 1993; Comai et al., 1989)**.** 

Chloroplast degradation is followed by lipid, protein and nucleic acid degradation. Membrane integrity and cellular compartmentalisation are maintained until the latter stages of leaf senescence (Lohman et al., 1994; Smart, 1994; Pruzinska et al., 2005). A decline in photosynthesis during senescence may result in sugar starvation leading to the activation of conversion of lipids to sugars. Thylakoid breakdown leads to release of lipids, which are known to be converted to sugars through the glycoxylate cycle (Buchanan-Wollaston and Ainsworth, 1997; Kim and Smith, 1994). The sugars produced by conversion of large amounts of lipids may be in excess to that required for respiration of the senescing leaves and this excess may be exported to other growing and demanding parts of the plant. It appears that the expression of genes for the enzymes participating in the process of gluconeogenesis for production of sucrose play an important role during senescence as the

The general purpose of a leaf is to gather and generate nutrients for the plant. As a green leaf grows and develops, it creates an organ packed with nutrients. When the plant no longer requires the leaf, the senescence process is induced and recycling of all the nutrients that can be remobilized occurs. Leaf death is the final stage in the process; however, death is

The dismantling of the leaf begins with the chloroplasts, the energy-generating, photosynthetic powerhouse of the plant. Not unlike another energy generating organelle (ie, the mitochondria), chloroplasts are semiautonomous and they possess their own genome with its inherent transcriptional and translational machinery. Gradually the chloroplasts shrink and transform into gerontoplasts, an artefact characterised by the disintegration of the thylakoid membranes and accumulation of the plastoglobulin (Friedrich and Huffaker, 1980; Mae et al., 1984). The process of breaking down the chlorophyll is so pronounced that chlorophyll loss and the associated yellowing of the leaves are commonly used as indicators of plant senescence (Noodén et al., 1997). Control of the process is so tightly regulated that experiments demonstrating the reversibility of senescence have shown that the chloroplasts can recover structural features, re-synthesize chloroplast proteins and re-commence

Degradation and remobilization of the chloroplast proteins and RNA contribute nitrogen and other nutrients for seed growth (Wittenbach, 1978). The mechanisms governing degradation of the chloroplast are not completely understood and there are competing theories about where proteins are degraded; for example they may be degraded locally within the chloroplast or in a centralized vacuole for degradation (Hortensteiner and Feller, 2002). Findings that support the possibility that the photosynthetic machinery is degraded *in situ* by the chloroplast include the presence of chloroplast enzymes, which are localized hydrolases that catalyze the initial steps of chlorophyll breakdown. (Hortensteiner, 2006). Proteases of the Clp, FtsH and DegP families are also expressed in chloroplasts and representative genes for these proteases are up-regulated in senescing leaves (Sokolenko et al., 1998; Nakabayashi et al., 1999; Itzhaki et al., 1998; Haussühl et al., 2001). Despite this observation, chloroplastic proteases are unlikely to account for the degradation of most photosynthetic proteins (eg, Rubisco) during senescence. Senescence-associated vacuoles, with strong proteolytic activity, have been identified in senescing tissue and likely also contribute towards the degradation of soluble photosynthetic proteins (Hensell et al., 1993;

Chloroplast degradation is followed by lipid, protein and nucleic acid degradation. Membrane integrity and cellular compartmentalisation are maintained until the latter stages of leaf senescence (Lohman et al., 1994; Smart, 1994; Pruzinska et al., 2005). A decline in photosynthesis during senescence may result in sugar starvation leading to the activation of conversion of lipids to sugars. Thylakoid breakdown leads to release of lipids, which are known to be converted to sugars through the glycoxylate cycle (Buchanan-Wollaston and Ainsworth, 1997; Kim and Smith, 1994). The sugars produced by conversion of large amounts of lipids may be in excess to that required for respiration of the senescing leaves and this excess may be exported to other growing and demanding parts of the plant. It appears that the expression of genes for the enzymes participating in the process of gluconeogenesis for production of sucrose play an important role during senescence as the

photosynthesis **(**Thomas and Donnison, 2000; Zavaleta-Mancera et al., 1999**).** 

**2. Progress of senescence in plants** 

Comai et al., 1989)**.** 

actively delayed until all nutrients have been removed.

genes responsible for synthesis of the enzymes involved in gluconeogenesis are reported to be significantly expressed during this time (Buchanan-Wollaston and Ainsworth 1997; Kim and Smith, 1994).

Leaf senescence also results in the breakdown of nucleic acids to purines and pyrimidines, which ultimately degrade to small and transportable carbon and nitrogenous compounds that are transported to growing parts of the plant (Buchanan-Wollaston and Ainsworth, 1997). In addition to mobilization of carbon and nitrogen, other nutrients like sulphur and metallic ions are also known to be transported from senescing leaves. Sugar content can also be modified at the onset of senescence. Generally crops under a limited nitrogen nutrition and high light regimen undergo early senescence and this is usually accompanied by an incremental rise of sugar levels in the leaves. Sugar has been suggested to trigger a senescence response based on gain or loss function experiments with hexokinase genes, principal regulators of a glucose signalling pathway (van Doorn, 2008).

## **3. Regulation of senescence and potential for biotechnology**

Before the advent of modern biotechnology, which enabled scientists to commence deciphering the relationship between genes and life, senescence was perceived as an uncoordinated collection of events resulting in the metabolic and physiological changes to plant organs described above. The study of plant genetics, genomics, proteomics and more recently metabolomics have altered this perception and demonstrated that the process is dynamic and well organised. Below, a few examples are provided to emphasize the importance of a better understanding of plant senescence and the consequent potential of applications derived from that understanding.

Several techniques and different plant models have been employed in the pursuit of understanding the genetic mechanisms underlying the changes in gene expression associated with senescence. The process of senescence is initiated in source tissues prompting dramatic changes in gene expression, during which genes involved in basic metabolism, including photosynthesis and protein biosynthesis, are down-regulated while those involved in programmed cell death and stress response and/or encoding various hydrolytic enzymes are up-regulated (Hopkins et al., 2007; Lim et al., 2007). Not surprisingly the initial discoveries involving Senescence Associated Genes (SAGs) were made in the model plant Arabidopsis (*Arabidopsis thaliana*) by methods including differential display (Lohman et al. 1994), senescence-specific enhancer trap line screening using a range of senescence promoting factors (He et al., 2001), subtractive hybridization (Gepstein et al., 2003) and microarray experiments (Andersson et al., 2004). Many of the genes expressed during senescence of tissues encode hydrolytic enzymes that are capable of disassembling the ultra-structure of the cell and the breakdown of macromolecules (Smart, 1994; Griffith et al., 1997; Watanabe et al., 1994). In addition, a large number of transcription factors, as well as genes encoding carbohydrate and nitrogen-mobilising enzymes, nucleases and stressresponsive proteins, have been found to exhibit increased expression in senescing leaves (Buchanan-Wollaston and Ainsworth, 1997; Comai et al., 1989; Kim and Smith, 1994). The gene expression changes and biological processes that are up- and down-regulated during senescence as indicated by such studies, have been reviewed elsewhere (Guo and Gan, 2005), so will only be touched on in this review. What is more in the scope of this review are the potential implications that senescence has for plant biotechnological applications.

Advances in Plant Senescence 121

During whole plant senescence, fixed carbon and nitrogen are mobilized to reproductive or storage organs, which are harvested for human consumption (Vierstra, 1996; Hopkins et al., 2007; Lim et al., 2007). The process of senescence impacts all crop species and so the increased understanding of the tight regulatory mechanisms that control the process could potentially have an immeasurable impact on the world's agricultural production. Whole plant senescence plays a key role in remobilizing and transferring nutrients into the vegetative tissue and eventually to grain. The grain filling period is a critical period because many processes can influence the final grain yield (Yang and Zhang, 2006). For example, delaying whole plant senescence can be achieved by heavy use of fertilizer or development of a stay-green phenotype produced using a genetic or transgenic strategy. Extending or delaying senescence is believed to augment the grain filling stage thereby increasing grain yield. Contrarily, stresses, such as drought, induce early senescence, prompting the reduction of photosynthesis and shortening the grain filling period (Gregerson et al., 2008) and thus having the opposite affect on yield. Ectopic expression of SAG101, a protein with acyl hydrolase activity, has been shown to cause precocious senescence in both attached and detached leaves of transgenic Arabidopsis plants (He and Gan, 2002). Antisense expression of the gene, resulting in repression of the endogenous genes expression, was shown to cause a delay in the onset of senescence (He and Gan, 2002). Utilising genes such as SAG 101 to induce a stay-green/delayed senescence phenotype could potentially be employed in

Effective recycling of nutrients could have a massive impact on crop yields. Recycling of carbon and nitrogen during senescence involves the sequestering of cytoplasm and organelles into special autophagic vesicles**.** These vesicles deliver their contents to the vacuole (or lysosome) for breakdown by localized hydrolases (Thompson and Vierstra, 2005; Bassham, 2007). The breakdown products are either consumed by the host cell or transported to other tissues and organs. Under normal growth conditions, autophagy takes place at a basal level. The process ramps up in response to nutritional demand, biotic or abiotic stresses, and senescence. Autophagy plays an important role in the proper recycling of nutrients especially as a plant scavenges available nutrients from storage tissues and

When a pathway has been highly conserved evolutionarily, other organisms can provide the reference point for understanding a system in plants. The genes associated with autophagy discovered in yeast, enabled investigators to identify homologous genes in Arabidopsis and, subsequently, in rice and maize. Genome searches of Arabidopsis identified a collection of proteins structurally and functionally related to many of the ATG components present in yeast (Thompson and Vierstra, 2005; Bassham, 2007). In an effort to determine the importance of autophagy to crop plants, investigators at the University of Wisconsin, using the Arabidopsis as a reference, described a collection of components that participate in the ATG8/12 conjugation cascades in both rice (*Oryza sativa*) and maize (*Zea mays*). Remarkably, all components required for ATG8/12 conjugation in yeast and Arabidopsis (Ohsumi, 2001; Thompson and Vierstra, 2005) were identified in both rice and maize suggesting that the pathway is highly conserved. The group went on to greater characterize the expression of the maize ATG genes (Chung et al., 2009). The investigators observed an increase in ATG transcripts during leaf senescence and under nitrogen and fixed-carbon limiting conditions. The results indicate that the highly conserved process of autophagy plays a key role in

biotechnological strategies to increase yields in crops such as wheat.

**3.2 Impact on yield** 

older senescing leaves.

In addition to the conventional use of crops as food sources, innovations continue to expand the role of crop species in society. With these changes, the importance of understanding senescence becomes even more significant. Crops and trees are being developed as an alternative fuel source. Plants are also being integrated into the production of pharmaceutical ingredients and complex protein therapies such as vaccines (Lossl and Waheed, 2011). These and other innovative uses for plants make obtaining a greater understanding of senescence a necessary step for harnessing the influence of senescence on the plant lifecycle and reducing the impact this has on product yields and stability. The SAGs found through Arabidopsis investigations have provided a reference point for studies in other plant species, providing the potential to translate fundamental understanding into applied tools. Delaying the onset of senescence could increase the production of the desired plant product. This may be of particular interest in plastid expression systems (reviewed in Day and Goldschmidt., 2010), given that the chloroplast degradation occurs at a relatively early stage of senescence.

#### **3.1** *Populus tremula* **and bio fuel**

As suggested above, crops are being developed for alternative applications including bio fuels and paper production. The deciduous Aspen tree species *Populus tremula* is one such plant being developed for alternative fuel production. By comparing expressed sequence tag (EST) libraries generated from young fully-expanded leaves to leaves harvested immediately prior to visible signs of senescence, Bhalerao et al., (2003) identified *P. tremula* homologs for many known Arabidopsis SAGs. Altering the expression of these SAGs may have an effect on dormancy in this species with possible implications on the wood yield from these trees.

The onset of growth cessation and dormancy represents a critical ecological and evolutionary trade-off between survival and growth in most forest trees. Without this dormant stage nutrients stored in green leaves would be lost to frost, which would impact growth in the spring. Tight regulation over the timing of senescence is thus important. Latitudinal clines influence the critical photoperiod for onset of bud set (dormancy) and leaf senescence in Aspen (Fracheboud et al., 2009). This cline in dormancy was associated with multiple alleles of *PHYTOCHROME B2* (*PHYB2*), a photoreceptor that is related to light perception and light input to the circadian clock (the internal timing mechanism of the plant). The circadian clock enables the plant to co-ordinate its endogenous activities with the external environment to maximise the effectiveness of its activity. These activities occur on a daily basis, such as the timing of photosynthetic gene expression (Harmer et al., 2000, Edwards et al., 2006), and on an annual basis when measuring photoperiod and coordinating activities such as transitions to flowering, senescence or dormancy (reviewed in Jackson, 2009). Indeed, previous experiments suggest more accurate timing by the clock in relation to the external environmental cycles also has the potential to improve crop yields (Dodd et al., 2005). Such regulation governing the timing of critical events is relevant to all crop species grown in temperate climates. In the case of senescence, utilising regulatory mechanisms such as the circadian clock has the potential to alter the timing of this process with benefits to both wood production (reduced loss of nutrients to frost) as well as, for example, increasing the length of the grain filling period in other crops.

#### **3.2 Impact on yield**

120 Senescence

In addition to the conventional use of crops as food sources, innovations continue to expand the role of crop species in society. With these changes, the importance of understanding senescence becomes even more significant. Crops and trees are being developed as an alternative fuel source. Plants are also being integrated into the production of pharmaceutical ingredients and complex protein therapies such as vaccines (Lossl and Waheed, 2011). These and other innovative uses for plants make obtaining a greater understanding of senescence a necessary step for harnessing the influence of senescence on the plant lifecycle and reducing the impact this has on product yields and stability. The SAGs found through Arabidopsis investigations have provided a reference point for studies in other plant species, providing the potential to translate fundamental understanding into applied tools. Delaying the onset of senescence could increase the production of the desired plant product. This may be of particular interest in plastid expression systems (reviewed in Day and Goldschmidt., 2010), given that the chloroplast degradation occurs at a relatively

As suggested above, crops are being developed for alternative applications including bio fuels and paper production. The deciduous Aspen tree species *Populus tremula* is one such plant being developed for alternative fuel production. By comparing expressed sequence tag (EST) libraries generated from young fully-expanded leaves to leaves harvested immediately prior to visible signs of senescence, Bhalerao et al., (2003) identified *P. tremula* homologs for many known Arabidopsis SAGs. Altering the expression of these SAGs may have an effect on dormancy in this species with possible implications on the wood yield

The onset of growth cessation and dormancy represents a critical ecological and evolutionary trade-off between survival and growth in most forest trees. Without this dormant stage nutrients stored in green leaves would be lost to frost, which would impact growth in the spring. Tight regulation over the timing of senescence is thus important. Latitudinal clines influence the critical photoperiod for onset of bud set (dormancy) and leaf senescence in Aspen (Fracheboud et al., 2009). This cline in dormancy was associated with multiple alleles of *PHYTOCHROME B2* (*PHYB2*), a photoreceptor that is related to light perception and light input to the circadian clock (the internal timing mechanism of the plant). The circadian clock enables the plant to co-ordinate its endogenous activities with the external environment to maximise the effectiveness of its activity. These activities occur on a daily basis, such as the timing of photosynthetic gene expression (Harmer et al., 2000, Edwards et al., 2006), and on an annual basis when measuring photoperiod and coordinating activities such as transitions to flowering, senescence or dormancy (reviewed in Jackson, 2009). Indeed, previous experiments suggest more accurate timing by the clock in relation to the external environmental cycles also has the potential to improve crop yields (Dodd et al., 2005). Such regulation governing the timing of critical events is relevant to all crop species grown in temperate climates. In the case of senescence, utilising regulatory mechanisms such as the circadian clock has the potential to alter the timing of this process with benefits to both wood production (reduced loss of nutrients to frost) as well as, for

example, increasing the length of the grain filling period in other crops.

early stage of senescence.

from these trees.

**3.1** *Populus tremula* **and bio fuel** 

During whole plant senescence, fixed carbon and nitrogen are mobilized to reproductive or storage organs, which are harvested for human consumption (Vierstra, 1996; Hopkins et al., 2007; Lim et al., 2007). The process of senescence impacts all crop species and so the increased understanding of the tight regulatory mechanisms that control the process could potentially have an immeasurable impact on the world's agricultural production. Whole plant senescence plays a key role in remobilizing and transferring nutrients into the vegetative tissue and eventually to grain. The grain filling period is a critical period because many processes can influence the final grain yield (Yang and Zhang, 2006). For example, delaying whole plant senescence can be achieved by heavy use of fertilizer or development of a stay-green phenotype produced using a genetic or transgenic strategy. Extending or delaying senescence is believed to augment the grain filling stage thereby increasing grain yield. Contrarily, stresses, such as drought, induce early senescence, prompting the reduction of photosynthesis and shortening the grain filling period (Gregerson et al., 2008) and thus having the opposite affect on yield. Ectopic expression of SAG101, a protein with acyl hydrolase activity, has been shown to cause precocious senescence in both attached and detached leaves of transgenic Arabidopsis plants (He and Gan, 2002). Antisense expression of the gene, resulting in repression of the endogenous genes expression, was shown to cause a delay in the onset of senescence (He and Gan, 2002). Utilising genes such as SAG 101 to induce a stay-green/delayed senescence phenotype could potentially be employed in biotechnological strategies to increase yields in crops such as wheat.

Effective recycling of nutrients could have a massive impact on crop yields. Recycling of carbon and nitrogen during senescence involves the sequestering of cytoplasm and organelles into special autophagic vesicles**.** These vesicles deliver their contents to the vacuole (or lysosome) for breakdown by localized hydrolases (Thompson and Vierstra, 2005; Bassham, 2007). The breakdown products are either consumed by the host cell or transported to other tissues and organs. Under normal growth conditions, autophagy takes place at a basal level. The process ramps up in response to nutritional demand, biotic or abiotic stresses, and senescence. Autophagy plays an important role in the proper recycling of nutrients especially as a plant scavenges available nutrients from storage tissues and older senescing leaves.

When a pathway has been highly conserved evolutionarily, other organisms can provide the reference point for understanding a system in plants. The genes associated with autophagy discovered in yeast, enabled investigators to identify homologous genes in Arabidopsis and, subsequently, in rice and maize. Genome searches of Arabidopsis identified a collection of proteins structurally and functionally related to many of the ATG components present in yeast (Thompson and Vierstra, 2005; Bassham, 2007). In an effort to determine the importance of autophagy to crop plants, investigators at the University of Wisconsin, using the Arabidopsis as a reference, described a collection of components that participate in the ATG8/12 conjugation cascades in both rice (*Oryza sativa*) and maize (*Zea mays*). Remarkably, all components required for ATG8/12 conjugation in yeast and Arabidopsis (Ohsumi, 2001; Thompson and Vierstra, 2005) were identified in both rice and maize suggesting that the pathway is highly conserved. The group went on to greater characterize the expression of the maize ATG genes (Chung et al., 2009). The investigators observed an increase in ATG transcripts during leaf senescence and under nitrogen and fixed-carbon limiting conditions. The results indicate that the highly conserved process of autophagy plays a key role in

Advances in Plant Senescence 123

present in tobacco smoke is ongoing, with lists such as the 44 Hoffmann analytes, being produced by researchers and public health organisations (Hoffmann and Wynder 1967; Baker, 1999; Norman, 1999; Borgerding and Klus, 2005). A major focus of tobacco research is related to lowering the levels of these chemicals from smoke in an effort to reduce the harmful effects associated with tobacco use (for an overview of such research the scientific website of British American Tobacco [BAT] n.d.). Understanding the regulation and effect of senescence on tobacco leaf chemistry could be of particular importance to these traits since it

The availability of microarrays has considerably increased the extent to which differentially expressed genes can be identified and this line of research provides valuable insights into the identification of senescence related genes. High throughput analysis makes it possible to monitor changes in gene expression throughout the lifecycle of a plant. Researchers from Advanced Technologies (Cambridge) have recently described the generation of a tobacco (*Nicotiana tabacum*) custom expression array (Edwards et al., 2010). This array was used to develop the Tobacco Expression Atlas (TobEA), a map of gene expression from multiple tissues sampled throughout the life cycle of the tobacco plant which can be used as a reference data set for plant researchers. The expression data is freely available via the Solanaceae Genomics Network (SGN), a web based genomic resource for plants of the Solanaceae family (Mueller et al., 2005). Studying the changes in gene expression has the potential to identify targets that enable modifications or changes to leaf constituents in

is following the onset of senescence that 'ripe' tobacco leaves are harvested.

tobacco using transgenic or non-transgenic (e.g. molecular breeding) approaches.

SA-mediated defence (Figures 1H and I; Feys et al., 2005; Morris et al., 2000).

oxygen species and cell death (Yoshida, 2003).

Included in the TobEA study was a set of leaves from different positions that were categorised into either green (sink) or four distinct senescent (source) leaves, based on the average amount of yellowing and chlorosis across the leaf (Figure 1A; data not shown). Analysis of the gene expression changes in the tobacco leaf series suggested that tobacco showed similar changes during the progression of senescence as Arabidopsis leaves. For example the defence-associated phytohormone SA is known to play a role in developmental leaf senescence in Arabidopsis, with mutants and transgenic lines defective in the SAmediated signalling pathway exhibiting delayed senescence (Buchanan-Wollaston et al., 2005; Morris et al., 2000). A significant over-representation of genes associated with systemic acquired resistance and the SA-mediated signalling pathway was observed in the upregulated genes in the TobEA dataset, including presumptive orthologues of *ENHANCED SUSCEPTIBILITY 1* (*EDS1*) and *PHYTOALEXIN DEFICIENT 4* (*PAD4*), central regulators of

There was also significant over-representation of Gene Ontology categories associated with defence against fungal pathogens as well as cell death and innate immune responses in leaves at more advanced stages of senescence. These included a homologue of the Arabidopsis basic chitinase *PR3* (Verburg and Huynh 1991) in addition to other components of plant immunity/defence. This supports the growing evidence that pathogen defence and senescence share common components (Quirino et al., 1999; Feys et al., 2005), presumably largely via the use of similar signalling pathways leading to accumulation of reactive

**4.1 Gene expression changes in senescing tobacco leaves** 

nutrient remobilization with some variations unique to maize. The description of the maize ATG system provides a set of molecular and biochemical tools to study autophagy in this crop under field conditions (Chung et al., 2009). The same is true for rice (Ohsumi, 2001; Thompson and Vierstra, 2005). This type of knowledge may help to reveal important control points in autophagy that could be manipulated in both food and bio fuel crops to enhance nutrient use efficiency or to better allocate carbon and nitrogen to specific organs for improved yield.

In addition to highly conserved genes, specific genes or gene families that can also be employed to influence grain quality and yield. Uauy et. al., (2006) cloned a Quantitative Trait Locus (QTL) associated with increased grain protein, zinc, and iron content known as Gpc-B1. The ancestral wild wheat allele encodes a functional NAC transcription factor (NAM-B1) that accelerates senescence and increases nutrient remobilization from leaves to developing grains. In contrast, modern wheat varieties carry a non-functional NAM-B1 allele. Reduction in RNA levels of the multiple NAM homologues by RNA interference delayed senescence by more than three weeks and reduced wheat grain protein, zinc, and iron content by more than 30%. Other examples of specific genes having an effect in senescence include the cytokinin synthesis gene IPT, which has been shown to delay leaf senescence (Gan and Amasino, 1995), thereby providing the potential to increase seed setting time and yield, but the affect this has on nutritional value must also be considered.

#### **3.3 Ripening**

Although the main focus of this review relates to leaf senescence, fruit ripening is an aspect of plant senescence that is also of global significance. The timing of ripening is a key consideration when harvesting and transporting fruit to market. Successful efforts to control fruit ripening are based on either reducing the biosynthesis of the plant hormone ethylene or slowing down the rate of fruit softening by targeting the genes involved in cell wall modification (Causier et al., 2002). The Flavr Savr tomato is an example of an early attempt to slow ripening using the latter strategy. Researchers at Calgene hoped to slow the ripening process of the tomato by engineering in an antisense gene to interfere with production of the enzyme polygalacturonase (Weasel, 2009). The enzyme normally degrades pectin in the cell walls and results in softening.

More recently, investigators have attempted to characterize the N-glycan processing enzymes and their role in during non-climacteric fruit softening. The plant hormone ethylene does not influence ripening of non climacteric fruits and different genes need to be targeted for the different categories of fruits (Causier et al., 2002). Two ripening-specific Nglycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase were identified in the fruit capsicum (*Capsicum annuum*, Ghosh et al., 2010). Using RNA interference to suppress production of such enzymes has the potential to improve the shelf life of fruits, with obvious implications for improved food stability/storage.

#### **4. A view on tobacco biotechnology and senescence**

Tobacco is different from many of the crops discussed above because the organ harvested for human use is the leaf rather than reproductive organs (i.e. seed and fruit). The smoke generated during the burning of tobacco is a complex mixture of thousands of chemicals (Rodgman and Perfetti, 2008). Research to identify and characterise the harmful components

nutrient remobilization with some variations unique to maize. The description of the maize ATG system provides a set of molecular and biochemical tools to study autophagy in this crop under field conditions (Chung et al., 2009). The same is true for rice (Ohsumi, 2001; Thompson and Vierstra, 2005). This type of knowledge may help to reveal important control points in autophagy that could be manipulated in both food and bio fuel crops to enhance nutrient use efficiency or to better allocate carbon and nitrogen to specific organs for

In addition to highly conserved genes, specific genes or gene families that can also be employed to influence grain quality and yield. Uauy et. al., (2006) cloned a Quantitative Trait Locus (QTL) associated with increased grain protein, zinc, and iron content known as Gpc-B1. The ancestral wild wheat allele encodes a functional NAC transcription factor (NAM-B1) that accelerates senescence and increases nutrient remobilization from leaves to developing grains. In contrast, modern wheat varieties carry a non-functional NAM-B1 allele. Reduction in RNA levels of the multiple NAM homologues by RNA interference delayed senescence by more than three weeks and reduced wheat grain protein, zinc, and iron content by more than 30%. Other examples of specific genes having an effect in senescence include the cytokinin synthesis gene IPT, which has been shown to delay leaf senescence (Gan and Amasino, 1995), thereby providing the potential to increase seed setting time and yield, but the affect this has on nutritional value must also be considered.

Although the main focus of this review relates to leaf senescence, fruit ripening is an aspect of plant senescence that is also of global significance. The timing of ripening is a key consideration when harvesting and transporting fruit to market. Successful efforts to control fruit ripening are based on either reducing the biosynthesis of the plant hormone ethylene or slowing down the rate of fruit softening by targeting the genes involved in cell wall modification (Causier et al., 2002). The Flavr Savr tomato is an example of an early attempt to slow ripening using the latter strategy. Researchers at Calgene hoped to slow the ripening process of the tomato by engineering in an antisense gene to interfere with production of the enzyme polygalacturonase (Weasel, 2009). The enzyme normally degrades pectin in the cell

More recently, investigators have attempted to characterize the N-glycan processing enzymes and their role in during non-climacteric fruit softening. The plant hormone ethylene does not influence ripening of non climacteric fruits and different genes need to be targeted for the different categories of fruits (Causier et al., 2002). Two ripening-specific Nglycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase were identified in the fruit capsicum (*Capsicum annuum*, Ghosh et al., 2010). Using RNA interference to suppress production of such enzymes has the potential to improve the shelf

Tobacco is different from many of the crops discussed above because the organ harvested for human use is the leaf rather than reproductive organs (i.e. seed and fruit). The smoke generated during the burning of tobacco is a complex mixture of thousands of chemicals (Rodgman and Perfetti, 2008). Research to identify and characterise the harmful components

life of fruits, with obvious implications for improved food stability/storage.

**4. A view on tobacco biotechnology and senescence** 

improved yield.

**3.3 Ripening** 

walls and results in softening.

present in tobacco smoke is ongoing, with lists such as the 44 Hoffmann analytes, being produced by researchers and public health organisations (Hoffmann and Wynder 1967; Baker, 1999; Norman, 1999; Borgerding and Klus, 2005). A major focus of tobacco research is related to lowering the levels of these chemicals from smoke in an effort to reduce the harmful effects associated with tobacco use (for an overview of such research the scientific website of British American Tobacco [BAT] n.d.). Understanding the regulation and effect of senescence on tobacco leaf chemistry could be of particular importance to these traits since it is following the onset of senescence that 'ripe' tobacco leaves are harvested.

## **4.1 Gene expression changes in senescing tobacco leaves**

The availability of microarrays has considerably increased the extent to which differentially expressed genes can be identified and this line of research provides valuable insights into the identification of senescence related genes. High throughput analysis makes it possible to monitor changes in gene expression throughout the lifecycle of a plant. Researchers from Advanced Technologies (Cambridge) have recently described the generation of a tobacco (*Nicotiana tabacum*) custom expression array (Edwards et al., 2010). This array was used to develop the Tobacco Expression Atlas (TobEA), a map of gene expression from multiple tissues sampled throughout the life cycle of the tobacco plant which can be used as a reference data set for plant researchers. The expression data is freely available via the Solanaceae Genomics Network (SGN), a web based genomic resource for plants of the Solanaceae family (Mueller et al., 2005). Studying the changes in gene expression has the potential to identify targets that enable modifications or changes to leaf constituents in tobacco using transgenic or non-transgenic (e.g. molecular breeding) approaches.

Included in the TobEA study was a set of leaves from different positions that were categorised into either green (sink) or four distinct senescent (source) leaves, based on the average amount of yellowing and chlorosis across the leaf (Figure 1A; data not shown). Analysis of the gene expression changes in the tobacco leaf series suggested that tobacco showed similar changes during the progression of senescence as Arabidopsis leaves. For example the defence-associated phytohormone SA is known to play a role in developmental leaf senescence in Arabidopsis, with mutants and transgenic lines defective in the SAmediated signalling pathway exhibiting delayed senescence (Buchanan-Wollaston et al., 2005; Morris et al., 2000). A significant over-representation of genes associated with systemic acquired resistance and the SA-mediated signalling pathway was observed in the upregulated genes in the TobEA dataset, including presumptive orthologues of *ENHANCED SUSCEPTIBILITY 1* (*EDS1*) and *PHYTOALEXIN DEFICIENT 4* (*PAD4*), central regulators of SA-mediated defence (Figures 1H and I; Feys et al., 2005; Morris et al., 2000).

There was also significant over-representation of Gene Ontology categories associated with defence against fungal pathogens as well as cell death and innate immune responses in leaves at more advanced stages of senescence. These included a homologue of the Arabidopsis basic chitinase *PR3* (Verburg and Huynh 1991) in addition to other components of plant immunity/defence. This supports the growing evidence that pathogen defence and senescence share common components (Quirino et al., 1999; Feys et al., 2005), presumably largely via the use of similar signalling pathways leading to accumulation of reactive oxygen species and cell death (Yoshida, 2003).

Advances in Plant Senescence 125

In addition to SA, several other hormone pathways were identified as being overrepresented in the TobEA leaf senescence dataset including Jasmonic acid (JA). JA is known to have a role in developmental senescence, with both levels of JA itself and a number of JA biosynthetic genes found to increase during senescence (He et al., 2002). A similar response was observed in the developmental senescence dataset, with *12-OXOPHYTODIENOATE REDUCTASE* (*OPR*) family members being induced and an overall over-representation of genes involved in JA-meditated induced systemic resistance (Figure 1J; data not shown).

Ethylene is also known to play a role in promoting the onset of senescence (Grbic and Bleecker, 1995). Interestingly however, ethylene–mediated responses were not significantly over-represented in the TobEA data, suggesting that either ethylene is not as important in senescence of tobacco or that changes in this pathway are occurring post-transcriptionally. Stress can induce a senescence response in plants and one of the principal mediators of the stress response is the phyto-hormone Abscisic Acid or ABA (Smart 1994). ABA is a participant in drought (water) and cold stress responses (Wingler and Roitsch, 2008) and directly influences the sugar accumulation in response to stress. Interestingly, ABA will induce senescence during drought stress whereas it will delay senescence during cold stress (Xue –Xuan et al., 2010). In the TobEA data the ABA metabolic processes were significantly

Cytokinin levels in senescing leaves are though to play a key role in developmental leaf senescence, with both external and endogenous application resulting in delayed senescence (Smart 1994). This is largely reflected in the transcriptional responses to developmental senescence in Arabidopsis (Buchanan-Wollaston et al., 2005), as well as in the TobEA data, where cytokinin response processes were significantly down-regulated compared to controls. Interestingly, phenylpropanoid biosynthesis was identified as a significantly down-regulated process the TobEA data. It would be expected that increased production of photo-protective phenylpropanoids, flavonoids in particular, would be observed during developmental leaf senescence, due to increased light stress during the degradation of chlorophyll (Buchanan-Wollaston 2005). Indeed, Buchanan-Wollaston et al., (2005) found a number of flavonoid

Consistent with the phenotypic observations of the leaves themselves (Figure 1A), there was an enrichment of genes associated with photosynthesis being down-regulated in the TobEA data. This was accompanied by significant number of down-regulated genes associated with chloroplast components as well as responses to red, far-red and ultraviolet light stimuli. Over all the data suggest that similar processes occur during leaf senescence in Tobacco as in Arabidopsis and highlights the potential to translate findings in model species to

Environmental and economic issues combined have increased the need to better understand the role and fate of nitrogen in crop production systems. Nitrogen is one of the most important nutrients recycled by the plant during senescence, with up to 90% recovered from the leaf during this process (reviewed in Liu et al., 2008). Adding nitrogen to the soil increases crop yields and delays senescence, whereas a reduced fertilizer regimen generally triggers early whole plant senescence in crops due to low nitrogen. A strong coordination of

reduced late in senescence, the reason for which is unknown (data not shown).

biosynthesis genes had increased expression during developmental senescence.

biotechnological applications in other crops including Tobacco.

**4.2 Nitrogen metabolism and harm reduction** 

#### **Leaf Type**

Fig. 1. Gene expression changes in tobacco source and sink leaves (A) A sink to source series of leaves harvested from different positions on tobacco plants included in the Tobacco Expression Atlas and categorised as early- (E), mid-early- (M-E), mid-late- (M-L) and late- (L) senescent leaves based on level of Chlorosis (TobEA; Edwards et al., 2010). (B-J) Log expression data for selected transcripts associated with Nitrogen metabolism and plant hormone responses shown (See top right of each plot for transcript identification). Expression data was pre-processed with RMA and normalised against mature leaf (ML) samples showing no visible signs of senescence (also included in the TobEA data set). Differentially expressed genes from the sink-source series versus the mature leaf control were identified by one-way analysis of variance with Tukey HSD post hoc testing in GeneSpring GX 10 (P < 0.05). Gene ontology analysis of the up- and downregulated genes in each condition (described in main text) were analysed by a custom script as described previously (Edwards et. al., 2010).

**A**

2

0


0

**Relative Expression**


2

0


ML

E

M-E

as described previously (Edwards et. al., 2010).

M-L

L

Fig. 1. Gene expression changes in tobacco source and sink leaves

ML

**B CD**

**EFG**

**HI J**

E

(A) A sink to source series of leaves harvested from different positions on tobacco plants included in the Tobacco Expression Atlas and categorised as early- (E), mid-early- (M-E), mid-late- (M-L) and late- (L) senescent leaves based on level of Chlorosis (TobEA; Edwards et al., 2010). (B-J) Log expression data for selected transcripts associated with Nitrogen metabolism and plant hormone responses shown (See top right of each plot for transcript identification). Expression data was pre-processed with RMA and normalised against mature leaf (ML) samples showing no visible signs of senescence (also included in the TobEA data set). Differentially expressed genes from the sink-source series versus the mature leaf control were identified by one-way analysis of variance with Tukey HSD post hoc testing in GeneSpring GX 10 (P < 0.05). Gene ontology analysis of the up- and downregulated genes in each condition (described in main text) were analysed by a custom script

M-E

**Leaf Type**

M-L

*EDS1 PAD4 OPR1*

L

ML

E

M-E

M-L

L

early mid-early mid-late late

*NADH-GOGAT Fd-GOGAT GS2*

*NiR GDH1 GDH2*

In addition to SA, several other hormone pathways were identified as being overrepresented in the TobEA leaf senescence dataset including Jasmonic acid (JA). JA is known to have a role in developmental senescence, with both levels of JA itself and a number of JA biosynthetic genes found to increase during senescence (He et al., 2002). A similar response was observed in the developmental senescence dataset, with *12-OXOPHYTODIENOATE REDUCTASE* (*OPR*) family members being induced and an overall over-representation of genes involved in JA-meditated induced systemic resistance (Figure 1J; data not shown).

Ethylene is also known to play a role in promoting the onset of senescence (Grbic and Bleecker, 1995). Interestingly however, ethylene–mediated responses were not significantly over-represented in the TobEA data, suggesting that either ethylene is not as important in senescence of tobacco or that changes in this pathway are occurring post-transcriptionally. Stress can induce a senescence response in plants and one of the principal mediators of the stress response is the phyto-hormone Abscisic Acid or ABA (Smart 1994). ABA is a participant in drought (water) and cold stress responses (Wingler and Roitsch, 2008) and directly influences the sugar accumulation in response to stress. Interestingly, ABA will induce senescence during drought stress whereas it will delay senescence during cold stress (Xue –Xuan et al., 2010). In the TobEA data the ABA metabolic processes were significantly reduced late in senescence, the reason for which is unknown (data not shown).

Cytokinin levels in senescing leaves are though to play a key role in developmental leaf senescence, with both external and endogenous application resulting in delayed senescence (Smart 1994). This is largely reflected in the transcriptional responses to developmental senescence in Arabidopsis (Buchanan-Wollaston et al., 2005), as well as in the TobEA data, where cytokinin response processes were significantly down-regulated compared to controls.

Interestingly, phenylpropanoid biosynthesis was identified as a significantly down-regulated process the TobEA data. It would be expected that increased production of photo-protective phenylpropanoids, flavonoids in particular, would be observed during developmental leaf senescence, due to increased light stress during the degradation of chlorophyll (Buchanan-Wollaston 2005). Indeed, Buchanan-Wollaston et al., (2005) found a number of flavonoid biosynthesis genes had increased expression during developmental senescence.

Consistent with the phenotypic observations of the leaves themselves (Figure 1A), there was an enrichment of genes associated with photosynthesis being down-regulated in the TobEA data. This was accompanied by significant number of down-regulated genes associated with chloroplast components as well as responses to red, far-red and ultraviolet light stimuli. Over all the data suggest that similar processes occur during leaf senescence in Tobacco as in Arabidopsis and highlights the potential to translate findings in model species to biotechnological applications in other crops including Tobacco.

#### **4.2 Nitrogen metabolism and harm reduction**

Environmental and economic issues combined have increased the need to better understand the role and fate of nitrogen in crop production systems. Nitrogen is one of the most important nutrients recycled by the plant during senescence, with up to 90% recovered from the leaf during this process (reviewed in Liu et al., 2008). Adding nitrogen to the soil increases crop yields and delays senescence, whereas a reduced fertilizer regimen generally triggers early whole plant senescence in crops due to low nitrogen. A strong coordination of

Advances in Plant Senescence 127

**NR**

cytoplasm


Fig. 2. Primary nitrogen assimilation in plants.

2OG Glu


and glu by the cyclic activity of the enzymes GS and GOGAT.

NO2 -

Representation of the primary nitrogen assimilation pathway in a mesophyll cell

(modified from Mohr and Schopfer, 1994), showing reduction of Nitrate to Nitrite and then Ammonia by NR and NiR respectively and subsequent incorporation of nitrogen into gln

No probe sets for NR could be identified on the tobacco array, so the reduction in expression of this gene shown by Masclaux et al., (2000) could not be confirmed in the TobEA data. However, a decrease in NiR expression over the leaf series was shown, supporting a reduction in nitrogen fixation activity in the older leaves (Figure 1B). The ammonia generated by NR and NiR activity is incorporated into amino acids by the GS-GOGAT cycle. Plants have two types of GOGAT; ferredoxin dependent (Fd-GOGAT) and NADH dependent (NADH-GOGAT). Similarly *GS* genes can be subdivided into cytosolic and plastidic forms (*GS1* and *GS2* respectively). Fd-GOGAT functions in concert with GS2 and NADH-GOGAT is associated with GS1. In previous studies (Buchanan Wollaston 2005, Lin and Wu 2004), GS1 and NADH-GOGAT have demonstrated a co-ordinated increase in expression in Arabidopsis. *GS1* expression was previously shown to be induced in tobacco source leaves, whereas *GS2* transcripts were shown to be down regulated (Masclaux et al., 2000). No tobacco orthologues for *GS1* were identified on the tobacco microarray and transcripts for *NADH-GOGAT* did not demonstrate changes in expression over the TobEA

NH4 +

Gln

2Glu

**GS**

Glu

ATP

**NiR**

**GOGAT**

ADP + Pi + H2O

NO3

H2O

½O2

½O2

stroma

6ferredoxinreduced

chloroplast thylakoid

2ferredoxinreduced

2ferredoxinoxidised

6ferredoxinoxidised H2O

nitrogen-uptake, assimilation and remobilization is required for a beneficial grain filling stage (Hortensteiner and Feller 2002). The period that follows flowering can be critical in this process. Some crops, such as maize (C4 photosynthesis), use Nitrogen sourced both from the root's uptake and assimilation of NO3- as well as nitrogen remobilized during leaf senescence. Other crops, such as oil seed rape, primarily rely on the remobilization of nitrogen from leaves, making these crops more dependent on the senescence process (Coque et. al., 2008). When nitrogen inputs to the soil system exceed crop needs, there is a possibility that excessive amounts of nitrate (NO3- ) may enter either ground or surface water causing a detrimental effect on the environment.

In the case of tobacco, a greater understanding of the metabolism of nitrogen could also be applicable in an attempt to reduce the harmful constituents contained in cigarettes. One class of chemicals likely to feature in any future legislation of the tobacco industry is the Tobacco Specific Nitrosamines (TSNAs); 4-(N-methlynitrosamino)-1-(3-pyridyl)-1-butanone (NNK), Nnitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT).

TSNAs are principally formed by the nitrosation of tobacco alkaloids during the curing (drying) of tobacco leaf (Burton et al., 1989; Burton et al., 1994; Hoffmann et al., 1994; Spiegelhalder and Bartsch, 1996). Several studies have demonstrated a significant correlation between nitrite (formed by the microbial reduction of nitrate during curing) and TSNA levels in tobacco leaf, leading to the proposal of nitrite as the key nitrosating agent for TSNA formation (Burton et al., 1989; Fischer et al., 1989; Burton et al., 1994; Spiegelhalder and Bartsch, 1996; Wu et al., 2005). Curing conditions (including airflow, temperature and humidity) and their affect on microbial activity have been shown to affect the levels of TSNAs formed (Burton et al., 1989; Burton et al., 1994). Further understanding the nitrogen metabolism of tobacco could aid in reducing the potential for accumulating nitrosating agents during the curing process helping to limit the formation of TSNAs, and potentially reducing the levels of these toxicants in tobacco smoke.

Figure 2 shows a summary of the nitrogen assimilation pathway in plants. Plant nitrogen assimilation primarily occurs in mesophyll cells and involves the reduction of nitrate (taken up by the root) into ammonia by the enzymes—Nitrate Reductase (NR) and Nitrite Reductase (NiR; Figure 2). The ammonia is subsequently assimilated into the amino acids glutamine (gln) and glutamate (glu) via the cyclic action of Glutamine Synthetase (GS) and Glutamine-2-oxoglutarate aminotransferase (GOGAT; Figure 2; Lea and Miflin 1974).

Nitrogen assimilation is regulated by many factors, including the availability of sugars and other metabolites and also shows significant variation over the diurnal cycle (reviewed in Stitt et. al., 2002). The expression and activity of genes involved in nitrogen reduction and assimilation have previously been shown to be down-regulated in tobacco leaves at more advanced stages of senescence (Mascluax et. al., 2000). Masclaux et. al, compared leaves from different positions on mature tobacco plants and showed that there was a switch between nitrogen assimilation and nitrogen recycling from sink to source leaves at more advanced stages of senescence. The leaf series included in the TobEA microarray data set described above is similar to the leaf series tested by Masclaux et. al (Masclaux et. al 2000). Expression of nitrogen metabolism genes in the TobEA leaves compared with fully expanded mature leaves showing no visible signs of senescence (also included in the TobEA data), demonstrated consistent results with Masclaux et al., (2000; Figure 1).

nitrogen-uptake, assimilation and remobilization is required for a beneficial grain filling stage (Hortensteiner and Feller 2002). The period that follows flowering can be critical in this process. Some crops, such as maize (C4 photosynthesis), use Nitrogen sourced both from the root's uptake and assimilation of NO3- as well as nitrogen remobilized during leaf senescence. Other crops, such as oil seed rape, primarily rely on the remobilization of nitrogen from leaves, making these crops more dependent on the senescence process (Coque et. al., 2008). When nitrogen inputs to the soil system exceed crop needs, there is a possibility

In the case of tobacco, a greater understanding of the metabolism of nitrogen could also be applicable in an attempt to reduce the harmful constituents contained in cigarettes. One class of chemicals likely to feature in any future legislation of the tobacco industry is the Tobacco Specific Nitrosamines (TSNAs); 4-(N-methlynitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-

TSNAs are principally formed by the nitrosation of tobacco alkaloids during the curing (drying) of tobacco leaf (Burton et al., 1989; Burton et al., 1994; Hoffmann et al., 1994; Spiegelhalder and Bartsch, 1996). Several studies have demonstrated a significant correlation between nitrite (formed by the microbial reduction of nitrate during curing) and TSNA levels in tobacco leaf, leading to the proposal of nitrite as the key nitrosating agent for TSNA formation (Burton et al., 1989; Fischer et al., 1989; Burton et al., 1994; Spiegelhalder and Bartsch, 1996; Wu et al., 2005). Curing conditions (including airflow, temperature and humidity) and their affect on microbial activity have been shown to affect the levels of TSNAs formed (Burton et al., 1989; Burton et al., 1994). Further understanding the nitrogen metabolism of tobacco could aid in reducing the potential for accumulating nitrosating agents during the curing process helping to limit the formation of TSNAs, and potentially

Figure 2 shows a summary of the nitrogen assimilation pathway in plants. Plant nitrogen assimilation primarily occurs in mesophyll cells and involves the reduction of nitrate (taken up by the root) into ammonia by the enzymes—Nitrate Reductase (NR) and Nitrite Reductase (NiR; Figure 2). The ammonia is subsequently assimilated into the amino acids glutamine (gln) and glutamate (glu) via the cyclic action of Glutamine Synthetase (GS) and Glutamine-2-oxoglutarate aminotransferase (GOGAT; Figure 2; Lea and Miflin 1974).

Nitrogen assimilation is regulated by many factors, including the availability of sugars and other metabolites and also shows significant variation over the diurnal cycle (reviewed in Stitt et. al., 2002). The expression and activity of genes involved in nitrogen reduction and assimilation have previously been shown to be down-regulated in tobacco leaves at more advanced stages of senescence (Mascluax et. al., 2000). Masclaux et. al, compared leaves from different positions on mature tobacco plants and showed that there was a switch between nitrogen assimilation and nitrogen recycling from sink to source leaves at more advanced stages of senescence. The leaf series included in the TobEA microarray data set described above is similar to the leaf series tested by Masclaux et. al (Masclaux et. al 2000). Expression of nitrogen metabolism genes in the TobEA leaves compared with fully expanded mature leaves showing no visible signs of senescence (also included in the TobEA data), demonstrated consistent results with Masclaux et al., (2000;

nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT).

) may enter either ground or surface water causing a

that excessive amounts of nitrate (NO3-

detrimental effect on the environment.

reducing the levels of these toxicants in tobacco smoke.

Figure 1).

Fig. 2. Primary nitrogen assimilation in plants.

Representation of the primary nitrogen assimilation pathway in a mesophyll cell (modified from Mohr and Schopfer, 1994), showing reduction of Nitrate to Nitrite and then Ammonia by NR and NiR respectively and subsequent incorporation of nitrogen into gln and glu by the cyclic activity of the enzymes GS and GOGAT.

No probe sets for NR could be identified on the tobacco array, so the reduction in expression of this gene shown by Masclaux et al., (2000) could not be confirmed in the TobEA data. However, a decrease in NiR expression over the leaf series was shown, supporting a reduction in nitrogen fixation activity in the older leaves (Figure 1B). The ammonia generated by NR and NiR activity is incorporated into amino acids by the GS-GOGAT cycle. Plants have two types of GOGAT; ferredoxin dependent (Fd-GOGAT) and NADH dependent (NADH-GOGAT). Similarly *GS* genes can be subdivided into cytosolic and plastidic forms (*GS1* and *GS2* respectively). Fd-GOGAT functions in concert with GS2 and NADH-GOGAT is associated with GS1. In previous studies (Buchanan Wollaston 2005, Lin and Wu 2004), GS1 and NADH-GOGAT have demonstrated a co-ordinated increase in expression in Arabidopsis. *GS1* expression was previously shown to be induced in tobacco source leaves, whereas *GS2* transcripts were shown to be down regulated (Masclaux et al., 2000). No tobacco orthologues for *GS1* were identified on the tobacco microarray and transcripts for *NADH-GOGAT* did not demonstrate changes in expression over the TobEA

Advances in Plant Senescence 129

Harvest 1 Harvest 2 Harvest 3

**\***

**\*\***

A

B

C

Fig. 3. Increased threonine production in tobacco leaves

transgenic plants compared to (C) wild type plants.

Levels of threonine (nano moles per gram of cured leaf) for wild type tobacco and four independent transgenic tobacco lines expressing a mutated form of the Arabidopsis Aspartate Kinase AK:HSD (See inset key for line identification). Bars show mean threonine levels and error bars represent Standard Error of the mean. Leaves were taken from three harvest positions from the bottom to the top of the plant (Harvest 1 – 3). Asterisks represent significant difference between transgenic lines and WT for each harvest position based on one way analysis of variance with Tukey HSD post hoc testing (\* *P* < 0.005, \*\* *P* < 0.001). Constitutive expression of the same gene results in increased threonine levels in tobacco leaves (data not shown), but results in reduced growth and altered morphology in (B)

**\* \*\* \*\* \***

AK1 AK2 AK3 AK4 WT

Thr content (nmol/g)

dataset (Figure 1D). However, consistent with previous results, *GS2* transcripts were down regulated over the series of leaves (Figure 1C). A similar pattern of expression was also shown by tobacco *Fd-GOGAT* transcripts, supporting the proposed coordinated regulation and activity for these genes and a reduction of chloroplastic nitrogen assimilation in source leaves (Figures 1C and E).

Glutamate dehydrogenase (GDH) catalyses a reversible reaction adding or removing amino groups from glutamate. It has been proposed that the principal role of GDH is the deamination of glutamate in order to maintain a homeostatic balance of this amino acid that is thought to play a key role in the cross talk between the carbon and nitrogen assimilation pathways (Labboun et al., 2009). It has also been suggested that GDH amination may play a role in replacing glutamine synthetase (GS) activity in nitrogen assimilation within source leaves, which is lost during senescence (Masclaux et al., 2000). Previous studies have shown an increase in *GDH* expression in source leaves (Masclaux et al., 2000). Consistent with this, tobacco *GDH2* orthologs did show an increase in expression over the series; however, little change was shown by *GDH1* (Figures 1F and G).

Changes in the expression of genes involved in nitrogen assimilation shown by the leaves suggested that nitrogen metabolism was altered in source leaves towards remobilisation of the nitrogen resources to sink leaves. Consistent with this, gene ontology analysis of clusters of genes showing up-regulation in leaves demonstrating more advanced senescence revealed over representation of genes with functions related to proteolysis, the proteosome and endoplasmic reticulum associated protein catabolism. Increased understanding of the regulation of senescence in tobacco leaves could potentially help to limit the content of nitrate (and other nitrosonating agents) in harvested leaves prior to curing. This may augment efforts to reduce the levels of TSNAs in tobacco smoke; however, the study of senescence also provides other tools to facilitate TSNA reduction.

The main focus of agricultural research has been towards increased yield along with other agronomic traits. It is apparent in some crops that this has led towards an ignorance of flavour and texture components (as well as the associated nutritional value). Research is currently ongoing to understand and ultimately adjust the metabolic content of crops, such as those found in tomato, that contribute towards flavour and nutrition. This orientation toward flavour highlights the realisation that a perceived consumer benefit and consumer acceptance is becoming a more important driver in the development of new crops (Klee 2010). As indicated above, tobacco crops can be cured by multiple methods and the resulting leaf, or grades are blended together to produce the constituents of a cigarette. Dependent on the design, air cured tobacco typically only constitutes up to 30% of the blend in a cigarette, the rest being mainly made up of flue cured leaf (see Davis and Nielsen 1999 for a description of tobacco agronomy and chemistry). The conditions during air curing can lead to the formation of high levels of TSNAs. Thus, removing such grades from the blend could have beneficial affect on the overall level of TSNAs in the product. However, air cured grades make a significant contribution to the overall flavour of the cigarette, so the resulting product may not be consumer relevant and thus have no impact on harm reduction efforts. Replacing air cured grades with other grades that replace the flavour characteristics of these grades, but without the inherent higher levels of TSNAs provides one potential solution.

Thr content (nmol/g)

128 Senescence

dataset (Figure 1D). However, consistent with previous results, *GS2* transcripts were down regulated over the series of leaves (Figure 1C). A similar pattern of expression was also shown by tobacco *Fd-GOGAT* transcripts, supporting the proposed coordinated regulation and activity for these genes and a reduction of chloroplastic nitrogen assimilation in source

Glutamate dehydrogenase (GDH) catalyses a reversible reaction adding or removing amino groups from glutamate. It has been proposed that the principal role of GDH is the deamination of glutamate in order to maintain a homeostatic balance of this amino acid that is thought to play a key role in the cross talk between the carbon and nitrogen assimilation pathways (Labboun et al., 2009). It has also been suggested that GDH amination may play a role in replacing glutamine synthetase (GS) activity in nitrogen assimilation within source leaves, which is lost during senescence (Masclaux et al., 2000). Previous studies have shown an increase in *GDH* expression in source leaves (Masclaux et al., 2000). Consistent with this, tobacco *GDH2* orthologs did show an increase in expression over the series; however, little

Changes in the expression of genes involved in nitrogen assimilation shown by the leaves suggested that nitrogen metabolism was altered in source leaves towards remobilisation of the nitrogen resources to sink leaves. Consistent with this, gene ontology analysis of clusters of genes showing up-regulation in leaves demonstrating more advanced senescence revealed over representation of genes with functions related to proteolysis, the proteosome and endoplasmic reticulum associated protein catabolism. Increased understanding of the regulation of senescence in tobacco leaves could potentially help to limit the content of nitrate (and other nitrosonating agents) in harvested leaves prior to curing. This may augment efforts to reduce the levels of TSNAs in tobacco smoke; however, the study of

The main focus of agricultural research has been towards increased yield along with other agronomic traits. It is apparent in some crops that this has led towards an ignorance of flavour and texture components (as well as the associated nutritional value). Research is currently ongoing to understand and ultimately adjust the metabolic content of crops, such as those found in tomato, that contribute towards flavour and nutrition. This orientation toward flavour highlights the realisation that a perceived consumer benefit and consumer acceptance is becoming a more important driver in the development of new crops (Klee 2010). As indicated above, tobacco crops can be cured by multiple methods and the resulting leaf, or grades are blended together to produce the constituents of a cigarette. Dependent on the design, air cured tobacco typically only constitutes up to 30% of the blend in a cigarette, the rest being mainly made up of flue cured leaf (see Davis and Nielsen 1999 for a description of tobacco agronomy and chemistry). The conditions during air curing can lead to the formation of high levels of TSNAs. Thus, removing such grades from the blend could have beneficial affect on the overall level of TSNAs in the product. However, air cured grades make a significant contribution to the overall flavour of the cigarette, so the resulting product may not be consumer relevant and thus have no impact on harm reduction efforts. Replacing air cured grades with other grades that replace the flavour characteristics of these grades, but without the inherent higher levels

leaves (Figures 1C and E).

change was shown by *GDH1* (Figures 1F and G).

of TSNAs provides one potential solution.

senescence also provides other tools to facilitate TSNA reduction.

Fig. 3. Increased threonine production in tobacco leaves

Levels of threonine (nano moles per gram of cured leaf) for wild type tobacco and four independent transgenic tobacco lines expressing a mutated form of the Arabidopsis Aspartate Kinase AK:HSD (See inset key for line identification). Bars show mean threonine levels and error bars represent Standard Error of the mean. Leaves were taken from three harvest positions from the bottom to the top of the plant (Harvest 1 – 3). Asterisks represent significant difference between transgenic lines and WT for each harvest position based on one way analysis of variance with Tukey HSD post hoc testing (\* *P* < 0.005, \*\* *P* < 0.001). Constitutive expression of the same gene results in increased threonine levels in tobacco leaves (data not shown), but results in reduced growth and altered morphology in (B) transgenic plants compared to (C) wild type plants.

Advances in Plant Senescence 131

Alternatively, Biotechnological tools (i.e. gene vectors) and progressive strategies, such as molecular breeding, make it possible to apply research findings to addressing modern challenges. For example, an understanding of the tight regulation of senescence can be applied to modify the grain filling stage in an appropriate plant organism in order to increase the grain yield (harvest index). Altering the senescence stage to enhance remobilization or delay senescence through stay-green strategies (the most successful approaches being enhancing endogenous cytokine pathways and reducing ethylene production or perception) has become a routine approach to increasing productivity (Gan and Amasino 1995). Similarly senescence promoters and pathways have already been used to augment the flavour and deter the spoilage of products such as tomatoes and augment

Development of transcriptional data-sets such as the one we describe in tobacco will continue to facilitate discovery and drive innovation. Understanding how plants use nitrogen could potentially lead to improving nitrogen strategies that increase productivity of the plant and enhance the sustainability of farming. In the case of tobacco, knowledge of senescence and nitrogen metabolism is being applied to altering the leaf to decrease the level of target chemicals found in tobacco smoke. The extent to which plant genome initiatives are being undertaken by governments, academics and industrial partners will serve to ensure that genomics and the related branches of research will continue to contribute new tools, including genes and pathways that can regulate senescence and applications that promise to

We would like to acknowledge Susie Davenport and Gwendoline Leach for the provision of

Alegre, H., & Munné-Bosch S. (2004). Drought-induced changes in flavonoids and other low

Andersson, A., Keskitalo, J., Sjödin, A., Bhalerao, R., Sterky, F., Wissel, K., Tandre, K.,

Baker, R. R. (1999) Smoke chemistry, In: *Tobacco Producton, Chemistry and Technology*, Davis

Bassham, D. C. (2007) Plant autophagy--more than a starvation response, *Current Opinion in* 

Borgerding, M., & Klus, H. (2005) Analysis of complex mixtures--cigarette smoke. *Exp* 

Bhalerao, R., Keskitalo, J., Sterky, F., Erlandsson, R., Björkbacka, H., Birve, S. J., Karlsson, J.,

molecular weight antioxidants in *Cistus clusii* grown under Mediterranean field

Aspeborg, H., Moyle, R., Ohmiya, Y., Bhalerao, R., Brunner, A., Gustafsson, P., Karlsson, J., Lundeberg, J., Nilsson, O., Sandberg, G., Strauss, S., Sundberg, B., Uhlen, M., Jansson, S., & Nilsson, P. (2004) A transcriptional timetable of autumn

D L & Nielsen M. T., pp. 398-439, Blackwell Science Ltd., ISBN 0-632-04791-7,

Gardeström, P., Gustafsson, P., Lundeberg, J. & Jansson, S. (2003) Gene expression

data and Barbara Nasto for assistance with the writing/editing of this manuscript.

conditions, *Tree Physiology,* 24(11) pp 1303-1311

in autumn leaves. *Plant Physiology,* 131(2) pp 430-442.

senescence, *Genome Biology,* 5: R24

*Plant Biology*, 10(6) pp 587-593

*Toxicol Pathol,* 57(1) pp 43-73.

the nutrition of wheat.

have an impact on modern society.

**6. Acknowledgement** 

Oxford

**7. References** 

Amongst other differences, air cured tobaccos tend to have lower levels of sugars and an altered balance of free amino acids compared to flue cured tobacco leaf (Davis and Nielsen 1999). Threonine (thr) is one of the amino acids observed in higher levels in air cured leaf compared to flue cured, indicating it may contribute towards the flavour and aroma of the tobacco. Within the leaf, the biosynthetic pathway leading to production of thr is tightly regulated by a negative feedback control loop. In the case of feedback inhibition the endproduct, in this case thr, competitively inhibits the activity of the bifunctional enzyme ASPARTATE KINASE (AK) (EC 2.7.2.4) -HOMOSEREINE DESATURATE (HSD) (EC 1.1.1.3) and consequently blocks the enzymatic processes leading to its own synthesis (Shaul and Galili,1993). Disabling the enzyme that switches off thr production would prompt a greater accumulation of the compound, but, if the accumulation takes place too early in the plant's life cycle, the fitness of the plant is severely compromised (Fig 3A: data not shown). To overcome this obstacle, the promoter of the senescence associated gene SAG12, identified in Arabidopsis (Lohman 1984), was used to drive expression of mutated forms of AK:HSD gene from Arabidopsis in tobacco. Elevated leaf thr levels were achieved in the modified plants without compromising the plant's fitness (Fig 3 B and C). If the increase in thr results in an increase of the air cured 'flavour' in the tobacco, then such an approach could provide the potential to reduce the amount of this tobacco in the blend resulting in an associated reduction in the TSNA levels.

Optimising the timing and absolute level of expression by selecting other senescence associated promoters from tobacco could help to increase the yield of thr present in harvested leaves. Such promoters, could also be used to up, or down regulate the synthesis of other target flavour or toxicant precursors at the correct stage in the plant's life cycle to maximise the target phenotype, with limited effect on the growth of the plant.

## **5. The translational nature of innovation**

Nature has long been an infinite resource for the purpose of scientific discovery. Consequently, the study of plant senescence possesses immeasurable potential for increasing the understanding of the plant kingdom and the technological application of that knowledge. Genomics and the subsequent disciplines of proteomics and metabolomics have provided a complete reorientation toward the ways in which plants are designed to facilitate preservation of their own species. Thousands of genes that increase expression during leaf senescence have been isolated from a number of crop varieties; such as: Arabidopsis, wheat, tomato, maize, rice, and tobacco; these are just the tip of the iceberg.

Many of the recent advances in the understanding of plants (and organisms in general) can be attributed to the exponential increase in the sequencing and bioinformatics capacity of the world's research communities, coupled with numerous initiatives being driven by governments, academics and the private sector. Advances in gene sequencing techniques have made it possible to decipher entire genomes and high throughput microarray analysis and other techniques make it possible to monitor changes in a plant over time. By comparing what genes are switched on and off as a plant senesces, a collection of SAGs have already been discovered. Tracing the homology of conserved sequences through the evolutionary line, not only has facilitated the discovery of more SAGs, it has helped to elucidate the dynamics of an entire senescence-associated biosynthetic pathways such as ATG8/12. Comparative studies between species not only reveal similarities, researchers inevitably find unique differences specific to the plant variety and species contributing even more information to the pool.

Amongst other differences, air cured tobaccos tend to have lower levels of sugars and an altered balance of free amino acids compared to flue cured tobacco leaf (Davis and Nielsen 1999). Threonine (thr) is one of the amino acids observed in higher levels in air cured leaf compared to flue cured, indicating it may contribute towards the flavour and aroma of the tobacco. Within the leaf, the biosynthetic pathway leading to production of thr is tightly regulated by a negative feedback control loop. In the case of feedback inhibition the endproduct, in this case thr, competitively inhibits the activity of the bifunctional enzyme ASPARTATE KINASE (AK) (EC 2.7.2.4) -HOMOSEREINE DESATURATE (HSD) (EC 1.1.1.3) and consequently blocks the enzymatic processes leading to its own synthesis (Shaul and Galili,1993). Disabling the enzyme that switches off thr production would prompt a greater accumulation of the compound, but, if the accumulation takes place too early in the plant's life cycle, the fitness of the plant is severely compromised (Fig 3A: data not shown). To overcome this obstacle, the promoter of the senescence associated gene SAG12, identified in Arabidopsis (Lohman 1984), was used to drive expression of mutated forms of AK:HSD gene from Arabidopsis in tobacco. Elevated leaf thr levels were achieved in the modified plants without compromising the plant's fitness (Fig 3 B and C). If the increase in thr results in an increase of the air cured 'flavour' in the tobacco, then such an approach could provide the potential to reduce the amount of this tobacco in the blend resulting in an associated reduction in the TSNA levels. Optimising the timing and absolute level of expression by selecting other senescence associated promoters from tobacco could help to increase the yield of thr present in harvested leaves. Such promoters, could also be used to up, or down regulate the synthesis of other target flavour or toxicant precursors at the correct stage in the plant's life cycle to

maximise the target phenotype, with limited effect on the growth of the plant.

tomato, maize, rice, and tobacco; these are just the tip of the iceberg.

Nature has long been an infinite resource for the purpose of scientific discovery. Consequently, the study of plant senescence possesses immeasurable potential for increasing the understanding of the plant kingdom and the technological application of that knowledge. Genomics and the subsequent disciplines of proteomics and metabolomics have provided a complete reorientation toward the ways in which plants are designed to facilitate preservation of their own species. Thousands of genes that increase expression during leaf senescence have been isolated from a number of crop varieties; such as: Arabidopsis, wheat,

Many of the recent advances in the understanding of plants (and organisms in general) can be attributed to the exponential increase in the sequencing and bioinformatics capacity of the world's research communities, coupled with numerous initiatives being driven by governments, academics and the private sector. Advances in gene sequencing techniques have made it possible to decipher entire genomes and high throughput microarray analysis and other techniques make it possible to monitor changes in a plant over time. By comparing what genes are switched on and off as a plant senesces, a collection of SAGs have already been discovered. Tracing the homology of conserved sequences through the evolutionary line, not only has facilitated the discovery of more SAGs, it has helped to elucidate the dynamics of an entire senescence-associated biosynthetic pathways such as ATG8/12. Comparative studies between species not only reveal similarities, researchers inevitably find unique differences specific to the plant variety and species contributing even more information to the pool.

**5. The translational nature of innovation** 

Alternatively, Biotechnological tools (i.e. gene vectors) and progressive strategies, such as molecular breeding, make it possible to apply research findings to addressing modern challenges. For example, an understanding of the tight regulation of senescence can be applied to modify the grain filling stage in an appropriate plant organism in order to increase the grain yield (harvest index). Altering the senescence stage to enhance remobilization or delay senescence through stay-green strategies (the most successful approaches being enhancing endogenous cytokine pathways and reducing ethylene production or perception) has become a routine approach to increasing productivity (Gan and Amasino 1995). Similarly senescence promoters and pathways have already been used to augment the flavour and deter the spoilage of products such as tomatoes and augment the nutrition of wheat.

Development of transcriptional data-sets such as the one we describe in tobacco will continue to facilitate discovery and drive innovation. Understanding how plants use nitrogen could potentially lead to improving nitrogen strategies that increase productivity of the plant and enhance the sustainability of farming. In the case of tobacco, knowledge of senescence and nitrogen metabolism is being applied to altering the leaf to decrease the level of target chemicals found in tobacco smoke. The extent to which plant genome initiatives are being undertaken by governments, academics and industrial partners will serve to ensure that genomics and the related branches of research will continue to contribute new tools, including genes and pathways that can regulate senescence and applications that promise to have an impact on modern society.

## **6. Acknowledgement**

We would like to acknowledge Susie Davenport and Gwendoline Leach for the provision of data and Barbara Nasto for assistance with the writing/editing of this manuscript.

## **7. References**


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**8** 

*1France 2Tunisia* 

**The Legume Root Nodule: From** 

Laurence Dupont, Geneviève Alloing, Olivier Pierre,

*2Laboratory of Plant Physiology, Science University, Tunis,* 

**Symbiotic Nitrogen Fixation to Senescence** 

Sarra El Msehli, Julie Hopkins, Didier Hérouart and Pierre Frendo

Biological nitrogen fixation (BNF) is the biological process by which the atmospheric nitrogen (N2) is converted to ammonia by an enzyme called nitrogenase. It is the major source of the biosphere nitrogen and as such has an important ecological and agronomical role, accounting for 65 % of the nitrogen used in agriculture worldwide. The most important source of fixed nitrogen is the symbiotic association between rhizobia and legumes. The nitrogen fixation is achieved by bacteria inside the cells of *de novo* formed organs, the nodules, which usually develop on roots, and more occasionally on stems. This mutualistic relationship is beneficial for both partners, the plant supplying dicarboxylic acids as a carbon source to bacteria and receiving, in return, ammonium. Legume symbioses have an important role in environment-friendly agriculture. They allow plants to grow on nitrogen poor soils and reduce the need for nitrogen inputs for leguminous crops, and thus soil pollution. Nitrogen-fixing legumes also contribute to nitrogen enrichment of the soil and have been used from Antiquity as crop-rotation species to improve soil fertility. They produce high protein-containing leaves and seeds, and legumes such as soybeans, groundnuts, peas, beans, lentils, alfalfa and clover are a major source of protein for human and animal consumption. Most research concentrates on the two legume-rhizobium model systems *Lotus-Mesorhizobium loti* and *Medicago-Sinorhizobium meliloti,* with another focus on the economically-important *Glycine max* (soybean) *-Bradyrhizobium japonicum* association. The legume genetic models *Medicago truncatula* and *Lotus japonicus* have a small genome size of *ca*. 450 Mbp while *Glycine max* has a genome size of 1,115 Mbp, and all are currently targets of large-scale *genome sequencing* projects (He et al., 2009; Sato et al., 2008; Schmutz et al., 2010). The complete genome sequence of their bacterial partners has been established (Galibert et al., 2001; Kaneko et al., 2000; Kaneko et al., 2002; Schneiker-Bekel et al., 2011).

Symbiotic interaction begins with the infection process, which is initiated by a reciprocal exchange of signals between plant and the compatible bacteria. Aromatic compounds -

**1. Introduction** 

**1.1 Early interaction and nodule development** 

*1UMR "Biotic Interactions and Plant Health" INRA 1301-CNRS 6243 University of Nice-Sophia Antipolis, F-06903 Sophia-Antipolis Cedex,* 


## **The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence**

Laurence Dupont, Geneviève Alloing, Olivier Pierre, Sarra El Msehli, Julie Hopkins, Didier Hérouart and Pierre Frendo *1UMR "Biotic Interactions and Plant Health" INRA 1301-CNRS 6243 University of Nice-Sophia Antipolis, F-06903 Sophia-Antipolis Cedex, 2Laboratory of Plant Physiology, Science University, Tunis, 1France* 

*2Tunisia* 

## **1. Introduction**

136 Senescence

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ISBN-13: 978-0814401644, New York

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443-451

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pp 79-84

Biological nitrogen fixation (BNF) is the biological process by which the atmospheric nitrogen (N2) is converted to ammonia by an enzyme called nitrogenase. It is the major source of the biosphere nitrogen and as such has an important ecological and agronomical role, accounting for 65 % of the nitrogen used in agriculture worldwide. The most important source of fixed nitrogen is the symbiotic association between rhizobia and legumes. The nitrogen fixation is achieved by bacteria inside the cells of *de novo* formed organs, the nodules, which usually develop on roots, and more occasionally on stems. This mutualistic relationship is beneficial for both partners, the plant supplying dicarboxylic acids as a carbon source to bacteria and receiving, in return, ammonium. Legume symbioses have an important role in environment-friendly agriculture. They allow plants to grow on nitrogen poor soils and reduce the need for nitrogen inputs for leguminous crops, and thus soil pollution. Nitrogen-fixing legumes also contribute to nitrogen enrichment of the soil and have been used from Antiquity as crop-rotation species to improve soil fertility. They produce high protein-containing leaves and seeds, and legumes such as soybeans, groundnuts, peas, beans, lentils, alfalfa and clover are a major source of protein for human and animal consumption. Most research concentrates on the two legume-rhizobium model systems *Lotus-Mesorhizobium loti* and *Medicago-Sinorhizobium meliloti,* with another focus on the economically-important *Glycine max* (soybean) *-Bradyrhizobium japonicum* association. The legume genetic models *Medicago truncatula* and *Lotus japonicus* have a small genome size of *ca*. 450 Mbp while *Glycine max* has a genome size of 1,115 Mbp, and all are currently targets of large-scale *genome sequencing* projects (He et al., 2009; Sato et al., 2008; Schmutz et al., 2010). The complete genome sequence of their bacterial partners has been established (Galibert et al., 2001; Kaneko et al., 2000; Kaneko et al., 2002; Schneiker-Bekel et al., 2011).

#### **1.1 Early interaction and nodule development**

Symbiotic interaction begins with the infection process, which is initiated by a reciprocal exchange of signals between plant and the compatible bacteria. Aromatic compounds -

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 139

which are able to revert to a free-living lifestyle. Conversely, legumes such as *Medicago truncatula*, *Pisum sativum* (pea) or *Trifolium* (clover) form indeterminate nodules that possess a permanent meristem and elongate, to become cylindrical. In mature nodule of this type, several histological zones of consecutive developmental states can be distinguished (Vasse et al., 1990). The apical meristem, free of bacteria, is the zone I. Zone II is the infection zone in which post-mitotic cells enter the nodule differentiation programme and where infection threads penetrate the plant cells and release rhizobia. In zone III, the bacteroids are able to fix N2. A root proximal senescence zone (zone IV) can be observed in older nodules, where the bacteroids, together with the plant cells, are degraded. Upon aging this zone gradually extends to reach the apical part and the nodule degenerates. Proximal to the zone IV is a region (zone V) containing undifferentiated bacteria, which appear to proliferate in the decaying plant tissue (Timmers et al., 2000). In contrast to bacteroids housed in determinate nodules, those from undeterminate nodules have lost their capacity to reproduce. Thus at the end of symbiosis, essentially bacteria that are released from infection threads can return

to a free-living lifestyle and recolonize the rhizosphere (Mergaert et al., 2006).

Fig. 1. Schematic representation of indeterminate (A) and determinate mature nodules (B) I, meristemic zone ; II, infection zone ; III, nitrogen fixing zone ; IV, senescence zone ; V,

Nitrogen fixing bacteroids in determinate and indeterminate nodules originate from distinct differentiation processes. Bacteroids in legume species forming determinate type nodules present the same cell size, genomic DNA content and reproductive capacity as the freeliving bacteria. Conversely, differentiation of bacteroids in indeterminate nodules is linked to drastic morphological and cytological changes, such as cell elongation coupled to genome amplification, membrane permeabilisation and loss of reproductive capacity (Mergaert et al., 2006). This terminal differentiation is mediated by plant-host factors identified as the nodule-specific cysteine-rich (NCR) peptides (Kereszt et al., 2011; Van de Velde et al., 2010). In *M. truncatula* the NCR gene family encodes more than 300 different peptides, which

resemble defensine-type antimicrobial peptides (AMPs) (Mergaert et al., 2003).

saprophytic zone.

mostly flavonoids - are secreted by the plants into the rhizosphere and activate the bacterial NodD proteins that are members of the LysR family of transcriptional activators, which in turn induce the expression of the *nod* genes (Long, 2001). This results in the secretion by the bacteria of lipo-chitin oligosaccharide molecules called Nod factors, which are recognized by epidermal cells via specific receptor kinases containing extracellular LysM domains. The spectrum of flavonoids exuded by a legume, as well as the strain-specific chemical structures of the Nod factors, are primary determinants of host specificity (Broughton et al., 2000). Additional bacterial components such as exopolysaccharides, type III and type IV secretion systems are also required for an effective infection (Perret et al., 2000; Saeki, 2011).

Nod factor perception initiates a complex signalling pathway essential for bacterial invasion of the host plant and formation of the nodule. Nod factor signal transduction requires a calcium signalling pathway, which includes the activation of a calcium and calmodulin dependent protein kinase in response to nuclear calcium oscillations. The ensuing induction of gene expression results in rearrangements of the root hair cytoskeleton and initiation of bacterial infection at the epidermis. The root hairs curl and trap the rhizobia which enter the root hair through tubular structures called infection threads. Simultaneously Nod factors induce root cortex cells dedifferentiation and division, leading to the formation of nodule primordia which then differentiate into N2-fixing nodules (Crespi & Frugier, 2008; Oldroyd et al. 2011). The growing infection threads traverse the root epidermis and cortex, penetrate primordial cells, and then invading bacteria are released into the host cells by an endocytosis-like mechanism (Ivanov et al., 2010). Each bacterium is surrounded by a plant cell membrane, the peribacteroid membrane (PBM), the whole forming an organelle-like structure called the symbiosome where bacteria differentiate into nitrogen-fixing bacteroids. These symbiosomes ultimately completely fill the cytoplasm of infected cells. As the bacteria differentiate, infected cells undergo enlargement coupled to repeated endoreduplication cycles - genomic DNA replication without mitosis or cytokinesis - and become large polyploid cells housing thousands of bacteroids (Jones et al., 2007; Kondorosi et al., 2000). Mature nodules actively fix nitrogen until they enter senescence upon aging or stress.

Nodule organogenesis is accompanied by major changes in plant gene expression. Several hundred of genes were found to be strongly and specifically up- or -down regulated during the nodulation process (Benedito et al., 2008; El Yahyaoui et al., 2004). In *M. truncatula*, two distinct waves of gene expression reprogramming accompany the differentiation of both the plant infected cell and bacteroids (Maunoury et al., 2010). Genes exclusively expressed or strongly up-regulated in nodules have been termed "nodulins". The early nodulins are involved in signal transduction and nodule development and the late nodulins are induced when N2 fixation begins. Different expression profiling tools relying on genome and highthroughput EST-sequencing have been developed to identify nodulin genes on a large scale (Kuster et al., 2007; Schauser et al., 2008).

Nodules can be classified into two main groups according to their mode of development (Franssen et al., 1992; Maunoury et al., 2008) (Figure 1). Legumes such as *Phaseolus vulgaris* (bean), *Lotus japonicus* or *Glycine max* (soybean) form determinate nodules that have no permanent meristem and adopt a globular shape. The mature nodules contain a homogenous central tissue composed of infected cells fully packed with nitrogen-fixing bacteroids and some uninfected cells. Senescence in these nodules occurs radially, beginning at the center and extending to the periphery. Decaying nodules release bacteroids most of

mostly flavonoids - are secreted by the plants into the rhizosphere and activate the bacterial NodD proteins that are members of the LysR family of transcriptional activators, which in turn induce the expression of the *nod* genes (Long, 2001). This results in the secretion by the bacteria of lipo-chitin oligosaccharide molecules called Nod factors, which are recognized by epidermal cells via specific receptor kinases containing extracellular LysM domains. The spectrum of flavonoids exuded by a legume, as well as the strain-specific chemical structures of the Nod factors, are primary determinants of host specificity (Broughton et al., 2000). Additional bacterial components such as exopolysaccharides, type III and type IV secretion systems are also required for an effective infection (Perret et al., 2000; Saeki, 2011). Nod factor perception initiates a complex signalling pathway essential for bacterial invasion of the host plant and formation of the nodule. Nod factor signal transduction requires a calcium signalling pathway, which includes the activation of a calcium and calmodulin dependent protein kinase in response to nuclear calcium oscillations. The ensuing induction of gene expression results in rearrangements of the root hair cytoskeleton and initiation of bacterial infection at the epidermis. The root hairs curl and trap the rhizobia which enter the root hair through tubular structures called infection threads. Simultaneously Nod factors induce root cortex cells dedifferentiation and division, leading to the formation of nodule primordia which then differentiate into N2-fixing nodules (Crespi & Frugier, 2008; Oldroyd et al. 2011). The growing infection threads traverse the root epidermis and cortex, penetrate primordial cells, and then invading bacteria are released into the host cells by an endocytosis-like mechanism (Ivanov et al., 2010). Each bacterium is surrounded by a plant cell membrane, the peribacteroid membrane (PBM), the whole forming an organelle-like structure called the symbiosome where bacteria differentiate into nitrogen-fixing bacteroids. These symbiosomes ultimately completely fill the cytoplasm of infected cells. As the bacteria differentiate, infected cells undergo enlargement coupled to repeated endoreduplication cycles - genomic DNA replication without mitosis or cytokinesis - and become large polyploid cells housing thousands of bacteroids (Jones et al., 2007; Kondorosi et al., 2000). Mature nodules actively fix nitrogen until they enter senescence upon aging or stress.

Nodule organogenesis is accompanied by major changes in plant gene expression. Several hundred of genes were found to be strongly and specifically up- or -down regulated during the nodulation process (Benedito et al., 2008; El Yahyaoui et al., 2004). In *M. truncatula*, two distinct waves of gene expression reprogramming accompany the differentiation of both the plant infected cell and bacteroids (Maunoury et al., 2010). Genes exclusively expressed or strongly up-regulated in nodules have been termed "nodulins". The early nodulins are involved in signal transduction and nodule development and the late nodulins are induced when N2 fixation begins. Different expression profiling tools relying on genome and highthroughput EST-sequencing have been developed to identify nodulin genes on a large scale

Nodules can be classified into two main groups according to their mode of development (Franssen et al., 1992; Maunoury et al., 2008) (Figure 1). Legumes such as *Phaseolus vulgaris* (bean), *Lotus japonicus* or *Glycine max* (soybean) form determinate nodules that have no permanent meristem and adopt a globular shape. The mature nodules contain a homogenous central tissue composed of infected cells fully packed with nitrogen-fixing bacteroids and some uninfected cells. Senescence in these nodules occurs radially, beginning at the center and extending to the periphery. Decaying nodules release bacteroids most of

(Kuster et al., 2007; Schauser et al., 2008).

which are able to revert to a free-living lifestyle. Conversely, legumes such as *Medicago truncatula*, *Pisum sativum* (pea) or *Trifolium* (clover) form indeterminate nodules that possess a permanent meristem and elongate, to become cylindrical. In mature nodule of this type, several histological zones of consecutive developmental states can be distinguished (Vasse et al., 1990). The apical meristem, free of bacteria, is the zone I. Zone II is the infection zone in which post-mitotic cells enter the nodule differentiation programme and where infection threads penetrate the plant cells and release rhizobia. In zone III, the bacteroids are able to fix N2. A root proximal senescence zone (zone IV) can be observed in older nodules, where the bacteroids, together with the plant cells, are degraded. Upon aging this zone gradually extends to reach the apical part and the nodule degenerates. Proximal to the zone IV is a region (zone V) containing undifferentiated bacteria, which appear to proliferate in the decaying plant tissue (Timmers et al., 2000). In contrast to bacteroids housed in determinate nodules, those from undeterminate nodules have lost their capacity to reproduce. Thus at the end of symbiosis, essentially bacteria that are released from infection threads can return to a free-living lifestyle and recolonize the rhizosphere (Mergaert et al., 2006).

Fig. 1. Schematic representation of indeterminate (A) and determinate mature nodules (B) I, meristemic zone ; II, infection zone ; III, nitrogen fixing zone ; IV, senescence zone ; V, saprophytic zone.

Nitrogen fixing bacteroids in determinate and indeterminate nodules originate from distinct differentiation processes. Bacteroids in legume species forming determinate type nodules present the same cell size, genomic DNA content and reproductive capacity as the freeliving bacteria. Conversely, differentiation of bacteroids in indeterminate nodules is linked to drastic morphological and cytological changes, such as cell elongation coupled to genome amplification, membrane permeabilisation and loss of reproductive capacity (Mergaert et al., 2006). This terminal differentiation is mediated by plant-host factors identified as the nodule-specific cysteine-rich (NCR) peptides (Kereszt et al., 2011; Van de Velde et al., 2010). In *M. truncatula* the NCR gene family encodes more than 300 different peptides, which resemble defensine-type antimicrobial peptides (AMPs) (Mergaert et al., 2003).

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 141

reductions of N2-fixing bacteroid capacity are detectable and a senescence process occurs in the N -fixing nodule zone. This phenomena is related to the onset of pod filling in grain legumes like soybean, pea and common bean (Bethlenfalvay & Phillips, 1977; Lawn & Brun, 1977). Thus, the lifespan of the rhizobia-plant symbiotic relationship is relatively short and

The spatial dynamics of the senescence process in nodules is nodule type dependent. The pink N2-fixing tissues of the zone III become green in color in zone IV due to leghemoglobin breakdown (Lehtovaara & Perttila, 1978). In determinate nodules, histological analyses of cross-sections using this simple visible change revealed that senescence develops radially, starting from the center and gradually spreading toward the outside (Puppo et al., 2005). In contrast, in undeterminate nodules, histological analyses of longitudinal sections of nodules based on pink-to-green color changes or based on the expression pattern of bacteroid genes involved in the N2-fixing process using promoter-lacZ fusions (i.e. NifH) have led many authors to consider the front of senescence as a planar structure (Puppo et al., 2005). Recently, using toluidine blue staining to discriminate senescent from healthy cells and studying the gene expression pattern of a small family of plant cysteine proteases as early markers of nodule senescence on serial transversal sections of *M. truncatula* nodules, Pérez Guerra and collaborators proposed a conical organization of the developmental senescence zone: the earliest signs of senescence in a few infected cells in the center of the N-fixing zone occurred similar to determined nodules, and this phenomena progressively extended toward the nodule periphery in subsequent proximal cell layers of the nodule (Pérez Guerra

Comparison between N2-fixing and senescent cells in soybean nodule showed a decrease of density of plant cytoplasm, the apparition of vesicles associated with the deterioration of symbiosomes and modifications in organelles like peroxisomes, mitochondria and plastids (Lucas et al., 1998; Puppo et al., 2005). Ultrastructural analysis of *M. truncatula* mature nodule cells has revealed at least two stages during the developmental senescence of N2 fixing cells: first, a disintegration of bacteroids and symbiosomes revealed by the presence of numerous membranes in the plant cytoplasm associated with the formation of lytic symbiosome compartments probably involved in reabsorption processes and second, the decay of plant infected cells associated with collapse phenomena and that of the plant uninfected cells (Perez Guerra et al., 2010; Timmers et al., 2000; Van de Velde et al., 2006; Vasse et al., 1990). The fusion of symbiosomes to form lytic compartments resembles vacuole formation. Analysis of the relation between the symbiosome formation and the endocytic pathway showed that the lifespan of bacteria in individual symbiosomes compartments during the N2-fixing stage is achieved by delaying the acquisition of vacuolar identity such as vacuolar SYP22 and VTI11 SNAREs (Limpens et al., 2009). The acquisition of vacuolar identity by symbiosomes upon senescence likely allows the delivery of newly formed proteases to facilitate nutrient remobilization and a sink-to-source transition. Indeed, nodule senescence is accompanied by increased plant proteolytic activities that might cause large-scale protein degradation in soybean (Malik et al., 1981), French bean

the disruption of this symbiosis affects the yield of the culture.

**2.1 Structural analysis of developmental senescence** 

(Pladys et al., 1991) and alfalfa (Pladys & Vance, 1993).

et al., 2010).

#### **1.2 Nodule functioning**

Bacteria that have completed the bacteroid differentiation program express the enzymes of the nitrogenase complex and begin to fix nitrogen. The reduction by nitrogenase of 1 molecule of N2 to 2 molecules of NH4+ requires 16 molecules of ATP and 8 electrons (Jones et al., 2007). Thus, bacteroids require high rate flux of O2 to enable high rates of ATP synthesis, but this must be achieved whilst maintaining a very low concentration of free O2 to avoid inactivation of O2-labile nitrogenase. These conditions exist due to the presence of an O2 diffusion barrier and the synthesis of nodule-specific leghemoglobins, which accumulate to millimolar concentrations in the cytoplasm of infected cells prior to nitrogen fixation and buffer the free O2 concentration at around 7-11 nM, while maintaining high O2 flux for respiration (Appleby, 1984; Downie, 2005; Ott et al., 2005). The unique low-O2 environment provided for the bacteroid is a key signal in bacteroid metabolism, inducing a regulatory cascade controlling gene expression of the nitrogenase complex and the microaerobic respiratory enzymes of the bacteroid. The O2-sensing two-component regulatory system FixL-FixJ activates the transcription of the two intermediate regulators *nifA* and *fixK* genes, which induce the expression of *nif* and *fix* genes involved in nitrogen fixation and respiration (Reyrat et al., 1993). More generally, bacteroid differentiation is accompanied by a global change in gene expression compared with free-living bacteria. There is down-regulation of many genes such as most housekeeping genes and genes involved in synthesis of membrane proteins and peptidoglycan in favour of symbiosis specific processes (Becker et al., 2004; Bobik et al., 2006; Capela et al., 2006; Karunakaran et al., 2009; Pessi et al., 2007).

The reduction of N2 to ammonium is accompanied, in bacteroids, by the switching-off of ammonium assimilation into amino acids. Ammonium is secreted to the plant cytosol, for assimilation into the amides glutamine and asparagine or into ureides. In return, the plant provides carbon and energy sources to bacteroids in the form of dicarboxylic acids, particularly malate and succinate, which are produced from sucrose via sucrose synthase and glycolytic enzymes. Their metabolization by the TCA cycle provides bacteroids with reducing equivalents, ATP and metabolites for amino acid synthesis and other biosynthetic pathways (White et al., 2007; Lodwig & Poole, 2003). Pea bacteroids also depend on plant for branched-chain amino-acid (LIV) supply, as the bacteroids become symbiotic auxotrophs for these amino-acids (Prell et al., 2009).

The nodule functioning has many peculiarities, involving a plant-microbe crosstalk associated to a metabolism which needs a high energy level under micro-oxic conditions. Nodule development and senescence also have specific features. Whereas multiple review articles have described the early steps of nodule formation and functioning, the rupture of the interaction has not been reviewed recently. In this context, this review focuses on the different characteristics of root nodule senescence.

#### **2. Developmental senescence**

For many years, the majority of the research concerning N-fixing symbioses focused on understanding the mechanisms leading to the establishment of this symbiotic relationship, from the invasion of plant cells to the N-fixing bacteroid state. In all nodule types, the N2 fixation period is optimal between 4 and 5 weeks after infection. Beyond this period, first reductions of N2-fixing bacteroid capacity are detectable and a senescence process occurs in the N -fixing nodule zone. This phenomena is related to the onset of pod filling in grain legumes like soybean, pea and common bean (Bethlenfalvay & Phillips, 1977; Lawn & Brun, 1977). Thus, the lifespan of the rhizobia-plant symbiotic relationship is relatively short and the disruption of this symbiosis affects the yield of the culture.

### **2.1 Structural analysis of developmental senescence**

140 Senescence

Bacteria that have completed the bacteroid differentiation program express the enzymes of the nitrogenase complex and begin to fix nitrogen. The reduction by nitrogenase of 1 molecule of N2 to 2 molecules of NH4+ requires 16 molecules of ATP and 8 electrons (Jones et al., 2007). Thus, bacteroids require high rate flux of O2 to enable high rates of ATP synthesis, but this must be achieved whilst maintaining a very low concentration of free O2 to avoid inactivation of O2-labile nitrogenase. These conditions exist due to the presence of an O2 diffusion barrier and the synthesis of nodule-specific leghemoglobins, which accumulate to millimolar concentrations in the cytoplasm of infected cells prior to nitrogen fixation and buffer the free O2 concentration at around 7-11 nM, while maintaining high O2 flux for respiration (Appleby, 1984; Downie, 2005; Ott et al., 2005). The unique low-O2 environment provided for the bacteroid is a key signal in bacteroid metabolism, inducing a regulatory cascade controlling gene expression of the nitrogenase complex and the microaerobic respiratory enzymes of the bacteroid. The O2-sensing two-component regulatory system FixL-FixJ activates the transcription of the two intermediate regulators *nifA* and *fixK* genes, which induce the expression of *nif* and *fix* genes involved in nitrogen fixation and respiration (Reyrat et al., 1993). More generally, bacteroid differentiation is accompanied by a global change in gene expression compared with free-living bacteria. There is down-regulation of many genes such as most housekeeping genes and genes involved in synthesis of membrane proteins and peptidoglycan in favour of symbiosis specific processes (Becker et al., 2004; Bobik et al., 2006; Capela et al., 2006; Karunakaran et

The reduction of N2 to ammonium is accompanied, in bacteroids, by the switching-off of ammonium assimilation into amino acids. Ammonium is secreted to the plant cytosol, for assimilation into the amides glutamine and asparagine or into ureides. In return, the plant provides carbon and energy sources to bacteroids in the form of dicarboxylic acids, particularly malate and succinate, which are produced from sucrose via sucrose synthase and glycolytic enzymes. Their metabolization by the TCA cycle provides bacteroids with reducing equivalents, ATP and metabolites for amino acid synthesis and other biosynthetic pathways (White et al., 2007; Lodwig & Poole, 2003). Pea bacteroids also depend on plant for branched-chain amino-acid (LIV) supply, as the bacteroids become symbiotic auxotrophs for

The nodule functioning has many peculiarities, involving a plant-microbe crosstalk associated to a metabolism which needs a high energy level under micro-oxic conditions. Nodule development and senescence also have specific features. Whereas multiple review articles have described the early steps of nodule formation and functioning, the rupture of the interaction has not been reviewed recently. In this context, this review focuses on the

For many years, the majority of the research concerning N-fixing symbioses focused on understanding the mechanisms leading to the establishment of this symbiotic relationship, from the invasion of plant cells to the N-fixing bacteroid state. In all nodule types, the N2 fixation period is optimal between 4 and 5 weeks after infection. Beyond this period, first

**1.2 Nodule functioning** 

al., 2009; Pessi et al., 2007).

these amino-acids (Prell et al., 2009).

**2. Developmental senescence** 

different characteristics of root nodule senescence.

The spatial dynamics of the senescence process in nodules is nodule type dependent. The pink N2-fixing tissues of the zone III become green in color in zone IV due to leghemoglobin breakdown (Lehtovaara & Perttila, 1978). In determinate nodules, histological analyses of cross-sections using this simple visible change revealed that senescence develops radially, starting from the center and gradually spreading toward the outside (Puppo et al., 2005). In contrast, in undeterminate nodules, histological analyses of longitudinal sections of nodules based on pink-to-green color changes or based on the expression pattern of bacteroid genes involved in the N2-fixing process using promoter-lacZ fusions (i.e. NifH) have led many authors to consider the front of senescence as a planar structure (Puppo et al., 2005). Recently, using toluidine blue staining to discriminate senescent from healthy cells and studying the gene expression pattern of a small family of plant cysteine proteases as early markers of nodule senescence on serial transversal sections of *M. truncatula* nodules, Pérez Guerra and collaborators proposed a conical organization of the developmental senescence zone: the earliest signs of senescence in a few infected cells in the center of the N-fixing zone occurred similar to determined nodules, and this phenomena progressively extended toward the nodule periphery in subsequent proximal cell layers of the nodule (Pérez Guerra et al., 2010).

Comparison between N2-fixing and senescent cells in soybean nodule showed a decrease of density of plant cytoplasm, the apparition of vesicles associated with the deterioration of symbiosomes and modifications in organelles like peroxisomes, mitochondria and plastids (Lucas et al., 1998; Puppo et al., 2005). Ultrastructural analysis of *M. truncatula* mature nodule cells has revealed at least two stages during the developmental senescence of N2 fixing cells: first, a disintegration of bacteroids and symbiosomes revealed by the presence of numerous membranes in the plant cytoplasm associated with the formation of lytic symbiosome compartments probably involved in reabsorption processes and second, the decay of plant infected cells associated with collapse phenomena and that of the plant uninfected cells (Perez Guerra et al., 2010; Timmers et al., 2000; Van de Velde et al., 2006; Vasse et al., 1990). The fusion of symbiosomes to form lytic compartments resembles vacuole formation. Analysis of the relation between the symbiosome formation and the endocytic pathway showed that the lifespan of bacteria in individual symbiosomes compartments during the N2-fixing stage is achieved by delaying the acquisition of vacuolar identity such as vacuolar SYP22 and VTI11 SNAREs (Limpens et al., 2009). The acquisition of vacuolar identity by symbiosomes upon senescence likely allows the delivery of newly formed proteases to facilitate nutrient remobilization and a sink-to-source transition. Indeed, nodule senescence is accompanied by increased plant proteolytic activities that might cause large-scale protein degradation in soybean (Malik et al., 1981), French bean (Pladys et al., 1991) and alfalfa (Pladys & Vance, 1993).

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 143

Large modifications of the redox balance occur upon natural nodule senescence. Redox balance is defined by the equilibrium between the production of ROS and their degradation by the antioxidant defence system (Apel & Hirt, 2004). Ascorbate (Asc), homoglutathione (hGSH) and glutathione (GSH) are major antioxidants and redox buffers in plant nodule cells (Becana et al., 2010). The regulation of Asc and hGSH biosynthesis has been studied in common bean (*Phaseolus vulgaris*) nodules during aging (Loscos et al., 2008). The expression of five genes of the major Asc biosynthetic pathway was analyzed in nodules, and evidence was found that L-galactono-1,4-lactone dehydrogenase (GalLDH) , the last committed step of the pathway, is post-transcriptionally regulated. Large differences of Asc concentrations and redox states were observed in *P. vulgaris* nodules at different senescence stages suggesting that the lifespan of nodules is in part controlled by endogenous factors like Asc. Biochemical assays on alfalfa dissected nodules revealed that the senescent zone had lower GalLDH activity and ascorbate concentration compared to the infected zone (Matamoros et al., 2006). A strong positive correlation between N2-fixing activity and nodule Asc and GSH contents was also observed during pea nodule development and senescence (Groten et al., 2005). Peroxiredoxins (Prx) have also been described in N2-fixing nodules (Groten et al., 2006). Pea nodules contain at least two isoforms of Prx, located potentially in the cytosol (PrxIIB C) and mitochondria (PrxIIF). The levels of PrxIIB C declined with nodule senescence, but those of PrxIIF remained unaffected (Groten et al., 2006). The progressive decrease of antioxidant content during pea nodule senescence is not accompanied by an increase in ROS such as O2.- and H2O2 (Groten et al., 2005). In contrast, in aging soybean nodules, an oxidative stress has been detected including an increase of ROS, oxidized hGSH, catalytic Fe and oxidatively modified proteins and DNA bases, but no changes in Asc or tocopherol (Evans et al., 1999). The imbalance in redox state leading to oxidative stress induces the oxidation of lipids and proteins and the degradation of membranes. Lipid peroxidation was found to be elevated in senescent nodules of pigeon pea (*Cajanus cajan*) and bean (Loscos et al., 2008; Swaraj et al., 1995). In senescent soybean nodules, the presence of large amounts of H2O2 in the cytoplasmic and apoplastic compartments of the central infected tissue was detected and associated with a widespread expression of a cysteine protease gene (Alesandrini et al., 2003), suggesting a link between oxidative stress and

Various proteases, including those of the acid, serine, aspartic and cysteine types, have been isolated from senescing nodule tissue of soybean, alfalfa, French bean, and pea (Kardailsky & Brewin, 1996; Malik et al., 1981; Pfeiffer et al., 1983; Pladys & Vance, 1993; Pladys & Rigaud, 1985). The induction of cysteine protease genes during nodule senescence has been shown in soybean (Alesandrini et al., 2003), Chinese milk vetch (Naito et al., 2000), pea (Kardailsky & Brewin, 1996) and *M. truncatula* (Fedorova et al., 2002). The general transcriptomic analysis of senescent nodules in *M. truncatula* using cDNA-AFLP (Van de Velde et al., 2006) confirmed the predominant presence of genes encoding representatives of cysteine proteases that are highly homologous to one of the prominent markers of leaf senescence, *Sag12* (Lohman et al., 1994), indicating that these proteinases play an important role in the regulation of developmental nodule senescence. This hypothesis was confirmed in *Astragalus sinicus* since the silencing by RNA interference of the Asnodf32 gene, encoding a nodule-specific cysteine proteinase delayed root nodule senescence with a significant extention of the period of bacteroid active nitrogen fixation. Interestingly, elongated nodules

proteolitic activities detected upon nodule senescence.

were also observed on Asnodf32-silenced hairy roots (Li et al., 2008).

#### **2.2 Physiological and biochemical modifications during developmental senescence**

Developmental nodule senescence is a complex and programmed process which induces a decrease of N2-fixing activity and leghemoglobin content, modifications in the nodule redox state components and an increase of proteolytic activity, ultimately leading to the death of infected cells.

Leghemoglobin (Lb), which has a fundamental role in nodule functioning, is an important physiological marker for following the progression of nodule senescence (Figure 2). Lb content progressively decreases with the onset of senescence. This diminution of Lb impacts not only general metabolism of nodule by decreasing the O2 availability to bacteroids with a low free O2 content but also by potentially releasing free iron to produce reactive oxygen species (ROS) via the Fenton reaction. Indeed, the auto-oxidation of the active form of Lb, ferro-Lb-O2 (Lb-Fe2+- O2), is associated with superoxide anion (O2 .-) production (Fridovich, 1986; Puppo et al., 1981) and the degradation of the heme group of Lb by H2O2 likely allows the release of the catalytic Fe which enhances the production of OH● through the Fenton and the Haberweiss reactions (Becana & Klucas, 1992; Puppo & Halliwell, 1988). The importance of Lb in nodule ROS production has been shown in a transgenic *Lotus japonicus* line in which diminution of the Lb content is correlated with diminution of the H2O2 production (Gunther et al., 2007).

Fig. 2. Metabolic pathways involving Lb and formation of ROS.

A, Reversible oxygenation of Lb-Fe2+; B, Autoxidation of Lb-Fe2+-O2 to Lb-Fe3+ with release of O2**.**-; C, Lb-Fe3+ reduction by ferric Lb reductase (LbR); D, H2O2 reaction with Lb-Fe3+ to generate the inactive Lb-FeIV (ferryl) form; E, Lb-FeIV reduction to Lb-Fe3+ by ascorbate or thiols; other ROS can also be generated from O2 **.**-, by its dismutation to H2O2; F, and by H2O2 reduction to OH through Fenton reaction (G).

**2.2 Physiological and biochemical modifications during developmental senescence**  Developmental nodule senescence is a complex and programmed process which induces a decrease of N2-fixing activity and leghemoglobin content, modifications in the nodule redox state components and an increase of proteolytic activity, ultimately leading to the death of

Leghemoglobin (Lb), which has a fundamental role in nodule functioning, is an important physiological marker for following the progression of nodule senescence (Figure 2). Lb content progressively decreases with the onset of senescence. This diminution of Lb impacts not only general metabolism of nodule by decreasing the O2 availability to bacteroids with a low free O2 content but also by potentially releasing free iron to produce reactive oxygen species (ROS) via the Fenton reaction. Indeed, the auto-oxidation of the active form of Lb, ferro-Lb-O2 (Lb-Fe2+-

and the degradation of the heme group of Lb by H2O2 likely allows the release of the catalytic Fe which enhances the production of OH● through the Fenton and the Haberweiss reactions (Becana & Klucas, 1992; Puppo & Halliwell, 1988). The importance of Lb in nodule ROS production has been shown in a transgenic *Lotus japonicus* line in which diminution of the Lb

content is correlated with diminution of the H2O2 production (Gunther et al., 2007).

Fig. 2. Metabolic pathways involving Lb and formation of ROS.

reduction to OH through Fenton reaction (G).

A, Reversible oxygenation of Lb-Fe2+; B, Autoxidation of Lb-Fe2+-O2 to Lb-Fe3+ with release of O2**.**-; C, Lb-Fe3+ reduction by ferric Lb reductase (LbR); D, H2O2 reaction with Lb-Fe3+ to generate the inactive Lb-FeIV (ferryl) form; E, Lb-FeIV reduction to Lb-Fe3+ by ascorbate or thiols; other ROS can also be generated from O2**.**-, by its dismutation to H2O2; F, and by H2O2

.-) production (Fridovich, 1986; Puppo et al., 1981)

infected cells.

O2), is associated with superoxide anion (O2

Large modifications of the redox balance occur upon natural nodule senescence. Redox balance is defined by the equilibrium between the production of ROS and their degradation by the antioxidant defence system (Apel & Hirt, 2004). Ascorbate (Asc), homoglutathione (hGSH) and glutathione (GSH) are major antioxidants and redox buffers in plant nodule cells (Becana et al., 2010). The regulation of Asc and hGSH biosynthesis has been studied in common bean (*Phaseolus vulgaris*) nodules during aging (Loscos et al., 2008). The expression of five genes of the major Asc biosynthetic pathway was analyzed in nodules, and evidence was found that L-galactono-1,4-lactone dehydrogenase (GalLDH) , the last committed step of the pathway, is post-transcriptionally regulated. Large differences of Asc concentrations and redox states were observed in *P. vulgaris* nodules at different senescence stages suggesting that the lifespan of nodules is in part controlled by endogenous factors like Asc. Biochemical assays on alfalfa dissected nodules revealed that the senescent zone had lower GalLDH activity and ascorbate concentration compared to the infected zone (Matamoros et al., 2006). A strong positive correlation between N2-fixing activity and nodule Asc and GSH contents was also observed during pea nodule development and senescence (Groten et al., 2005). Peroxiredoxins (Prx) have also been described in N2-fixing nodules (Groten et al., 2006). Pea nodules contain at least two isoforms of Prx, located potentially in the cytosol (PrxIIB C) and mitochondria (PrxIIF). The levels of PrxIIB C declined with nodule senescence, but those of PrxIIF remained unaffected (Groten et al., 2006). The progressive decrease of antioxidant content during pea nodule senescence is not accompanied by an increase in ROS such as O2.- and H2O2 (Groten et al., 2005). In contrast, in aging soybean nodules, an oxidative stress has been detected including an increase of ROS, oxidized hGSH, catalytic Fe and oxidatively modified proteins and DNA bases, but no changes in Asc or tocopherol (Evans et al., 1999). The imbalance in redox state leading to oxidative stress induces the oxidation of lipids and proteins and the degradation of membranes. Lipid peroxidation was found to be elevated in senescent nodules of pigeon pea (*Cajanus cajan*) and bean (Loscos et al., 2008; Swaraj et al., 1995). In senescent soybean nodules, the presence of large amounts of H2O2 in the cytoplasmic and apoplastic compartments of the central infected tissue was detected and associated with a widespread expression of a cysteine protease gene (Alesandrini et al., 2003), suggesting a link between oxidative stress and proteolitic activities detected upon nodule senescence.

Various proteases, including those of the acid, serine, aspartic and cysteine types, have been isolated from senescing nodule tissue of soybean, alfalfa, French bean, and pea (Kardailsky & Brewin, 1996; Malik et al., 1981; Pfeiffer et al., 1983; Pladys & Vance, 1993; Pladys & Rigaud, 1985). The induction of cysteine protease genes during nodule senescence has been shown in soybean (Alesandrini et al., 2003), Chinese milk vetch (Naito et al., 2000), pea (Kardailsky & Brewin, 1996) and *M. truncatula* (Fedorova et al., 2002). The general transcriptomic analysis of senescent nodules in *M. truncatula* using cDNA-AFLP (Van de Velde et al., 2006) confirmed the predominant presence of genes encoding representatives of cysteine proteases that are highly homologous to one of the prominent markers of leaf senescence, *Sag12* (Lohman et al., 1994), indicating that these proteinases play an important role in the regulation of developmental nodule senescence. This hypothesis was confirmed in *Astragalus sinicus* since the silencing by RNA interference of the Asnodf32 gene, encoding a nodule-specific cysteine proteinase delayed root nodule senescence with a significant extention of the period of bacteroid active nitrogen fixation. Interestingly, elongated nodules were also observed on Asnodf32-silenced hairy roots (Li et al., 2008).

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 145

Legume BNF is particularly sensitive and perturbed by environmental stress conditions such as drought, salt stress, defoliation, continuous darkness and cold stress. Adverse environmental conditions affect nodule structure, impair nodule functioning and induce drastic metabolic and molecular modifications leading ultimately to a stress-induced

**3.1 Stress induced senescence has typical features when compared to developmental** 

As stated earlier, developmental induced senescence occurs typically in 5 to 11 week old nodules with a slow diminution of BNF during this time period (Evans et al., 1999; Puppo et al., 2005). In contrast, BNF declines drastically and quickly under environmental stress conditions. In less than a week, drought (Gonzalez et al., 1995; Larrainzar et al., 2007; Serraj et al., 1999), salt stress (Soussi et al., 1998; Swaraj and Bishnoi, 1999), dark stress (Matamoros et al., 1999; Gogorcena et al., 1997) and cold stress (van Heerden et al., 2008) decrease dramatically BNF. Thus, SIS is a much faster process than developmental senescence. Moreover, whereas developmental senescence is associated with the establishment of the nodule senescent zone which increases over time, SIS induces the degeneration of the whole nodule in a short time period (Matamoros et al., 1999; Perez Guerra et al., 2010; Vauclare et al., 2010). At the structural level, microscopic analyses also show that developmental senescence and dark stress-induced senescence present different features in *M. truncatula.* Dark-induced senescence leads to the condensation of the bacteroid content whereas the PBM remains intact even though most of the bacteroid content had disappeared (Perez Guerra et al., 2010). In contrast, developmental senescence induces a pronounced vesicle mobilisation in the host cytoplasm which is correlated with the degeneration of the PBM and the mixing of the symbiosome content with the cytoplasm (Perez Guerra et al., 2010; Van de Velde et al., 2006). However, structural analyses of the SIS have not been extensively performed on different nodule types for all the different environmental stress. Thus, it is possible that the different SIS do not develop similarly. Moreover, as for developmental senescence, SIS may process differently in determinate and indeterminate nodules. Indeed, nitrate induced senescence has been shown to induce bacteroid degradation in pea (indeterminate nodule) after two days of treatment in contrast to bean (determinate nodule), in which nitrate has little effect on the shape of bacteroids even after four days of 10mM

**3.2 Stress induced senescence is characterized by modifications in nodule carbon** 

SIS is characterized by multiple early modifications of nodule physiology (Figure 2). Amongst them, modifications in carbon metabolism play a major role. As stated before, BNF is a highly energetic process which requires a constant energy supply. Modification of nodule sucrose content has been observed during drought stress (Galvez et al., 2005; Gordon et al., 1997), salt stress (Gordon et al., 1997; Lopez et al., 2008; Sanchez et al., 2011; Lopez et al., 2009; Ben Salah et al., 2009), dark stress (Gogorcena et al., 1997; Matamoros et al., 1999; Vauclare et al., 2010) and cold stress (Walsh & Layzell, 1986; van Heerden et al., 2008). However, whereas dark stress induces a diminution of sucrose concentration,

**3. Stress-induced senescence in legume root nodule** 

senescence (SIS).

nitrate treatment (Matamoros et al., 1999).

**metabolism and respiration** 

**senescence** 

#### **2.3 Transcriptomic analysis of developmental senescence**

The onset of senescence involves the expression of genes whose products are required to carry out senescence-related processes (Gepstein, 2004). In order to isolate genes up- or down-regulated during nodule senescence, several genetic analyses including cDNA libraries and differential screening, mRNA differential display or cDNA-AFLP, have been performed in soybean (Alesandrini et al., 2003; Webb et al., 2008), and *M. truncatula* (Fedorova et al., 2002; Van de Velde et al., 2006). Using a mixture of effective nodules from 7 week-old plants, the first database specific to *M. truncatula* nodule senescence was obtained by isolating 140 000 Expressed Sequence Tags which are available in the J. Craig Venter Institute (http://www.jcvi.org/cms/research/groups/plant-genomics/resources/). To enrich plant material in senescent tissue, recent analyses in *M. truncatula* were performed from cross sections of nodules of 5 and 9 weeks by isolating the zones I, II and III from zones IV and V based on pink-to-green color changes (Van de Velde et al., 2006). This analysis using a modified cDNA-AFLP protocol has resulted in a collection of 508 gene tags that were expressed differentially. Functional clustering of these data has revealed a clear transition from carbon sink to nutriment source for the nodule by up-regulation of genes representative of several different proteases, genes involved in proteasome pathway and degradation of nucleic acids, membrane-derived lipids, and sugars. Moreover, this analysis suggests that three major hormones, ethylene, jasmonic acid and gibberellin, play an important role in nodule senescence (Van de Velde et al., 2006). From a more general point of view, it was been found that a significant overlap exists between genes expressed during leaf senescence in *Arabidopsis thaliana* and nodule senescence in *M. truncatula* (Van de Velde et al., 2006). However, more recent transcriptomic analysis of *M. truncatula* leaf senescence showed that only a minority of common genes are regulated during leaf and nodule senescence (De Michele et al., 2009).

#### **2.4 Hormonal regulation of developmental senescence**

Abscisic acid has been proposed to be an important signal in nodule senescence (Puppo et al., 2005), but no direct abscisic acid-responsive genes were present in the cDNA-AFLP dataset from *M. truncatula* nodules (Van de Velde et al., 2006). This analysis revealed that ethylene and jasmonic acid may play a positive role in nodule senescence, just as they do in the senescence of other plant tissues. The positive role of ethylene is illustrated by the upregulation of ERF transcription factors and ethylene biosynthetic genes, such as S-adenosyl-Met (SAM) synthetase and 1-aminocyclopropane-1-carboxylate oxidase. Involvement of jasmonic acid is suggested by the induction of lipoxygenase genes during different stages of nodule senescence. Moreover, a strong induction of a gene coding for the GA 2-oxidase, that converts active gibberellins to inactive forms (Thomas et al., 1999), was observed in senescent nodule suggesting that gibberellins might repress the senescence process. Finally, the induction of genes encoding a SAM synthase and a spermidine synthethase suggests the involvement of polyamine biosynthetic pathways in nodule senescence. Concerning the potential implication of the two major hormones, auxin and cytokinin, in nodule senescence, only a small amount of data is available. In lupin (*Lupinus albus*), an elevated accumulation of the LaHK1 transcripts, encoding a cytokinin receptor homologue, was detected during nodule developmental senescence suggesting a putative role for this cytokinin receptor homologue in nodule senescence (Coba de la Pena et al., 2008).

## **3. Stress-induced senescence in legume root nodule**

144 Senescence

The onset of senescence involves the expression of genes whose products are required to carry out senescence-related processes (Gepstein, 2004). In order to isolate genes up- or down-regulated during nodule senescence, several genetic analyses including cDNA libraries and differential screening, mRNA differential display or cDNA-AFLP, have been performed in soybean (Alesandrini et al., 2003; Webb et al., 2008), and *M. truncatula* (Fedorova et al., 2002; Van de Velde et al., 2006). Using a mixture of effective nodules from 7 week-old plants, the first database specific to *M. truncatula* nodule senescence was obtained by isolating 140 000 Expressed Sequence Tags which are available in the J. Craig Venter Institute (http://www.jcvi.org/cms/research/groups/plant-genomics/resources/). To enrich plant material in senescent tissue, recent analyses in *M. truncatula* were performed from cross sections of nodules of 5 and 9 weeks by isolating the zones I, II and III from zones IV and V based on pink-to-green color changes (Van de Velde et al., 2006). This analysis using a modified cDNA-AFLP protocol has resulted in a collection of 508 gene tags that were expressed differentially. Functional clustering of these data has revealed a clear transition from carbon sink to nutriment source for the nodule by up-regulation of genes representative of several different proteases, genes involved in proteasome pathway and degradation of nucleic acids, membrane-derived lipids, and sugars. Moreover, this analysis suggests that three major hormones, ethylene, jasmonic acid and gibberellin, play an important role in nodule senescence (Van de Velde et al., 2006). From a more general point of view, it was been found that a significant overlap exists between genes expressed during leaf senescence in *Arabidopsis thaliana* and nodule senescence in *M. truncatula* (Van de Velde et al., 2006). However, more recent transcriptomic analysis of *M. truncatula* leaf senescence showed that only a minority of common genes are regulated during leaf and nodule

Abscisic acid has been proposed to be an important signal in nodule senescence (Puppo et al., 2005), but no direct abscisic acid-responsive genes were present in the cDNA-AFLP dataset from *M. truncatula* nodules (Van de Velde et al., 2006). This analysis revealed that ethylene and jasmonic acid may play a positive role in nodule senescence, just as they do in the senescence of other plant tissues. The positive role of ethylene is illustrated by the upregulation of ERF transcription factors and ethylene biosynthetic genes, such as S-adenosyl-Met (SAM) synthetase and 1-aminocyclopropane-1-carboxylate oxidase. Involvement of jasmonic acid is suggested by the induction of lipoxygenase genes during different stages of nodule senescence. Moreover, a strong induction of a gene coding for the GA 2-oxidase, that converts active gibberellins to inactive forms (Thomas et al., 1999), was observed in senescent nodule suggesting that gibberellins might repress the senescence process. Finally, the induction of genes encoding a SAM synthase and a spermidine synthethase suggests the involvement of polyamine biosynthetic pathways in nodule senescence. Concerning the potential implication of the two major hormones, auxin and cytokinin, in nodule senescence, only a small amount of data is available. In lupin (*Lupinus albus*), an elevated accumulation of the LaHK1 transcripts, encoding a cytokinin receptor homologue, was detected during nodule developmental senescence suggesting a putative role for this cytokinin receptor

**2.3 Transcriptomic analysis of developmental senescence** 

senescence (De Michele et al., 2009).

**2.4 Hormonal regulation of developmental senescence** 

homologue in nodule senescence (Coba de la Pena et al., 2008).

Legume BNF is particularly sensitive and perturbed by environmental stress conditions such as drought, salt stress, defoliation, continuous darkness and cold stress. Adverse environmental conditions affect nodule structure, impair nodule functioning and induce drastic metabolic and molecular modifications leading ultimately to a stress-induced senescence (SIS).

#### **3.1 Stress induced senescence has typical features when compared to developmental senescence**

As stated earlier, developmental induced senescence occurs typically in 5 to 11 week old nodules with a slow diminution of BNF during this time period (Evans et al., 1999; Puppo et al., 2005). In contrast, BNF declines drastically and quickly under environmental stress conditions. In less than a week, drought (Gonzalez et al., 1995; Larrainzar et al., 2007; Serraj et al., 1999), salt stress (Soussi et al., 1998; Swaraj and Bishnoi, 1999), dark stress (Matamoros et al., 1999; Gogorcena et al., 1997) and cold stress (van Heerden et al., 2008) decrease dramatically BNF. Thus, SIS is a much faster process than developmental senescence. Moreover, whereas developmental senescence is associated with the establishment of the nodule senescent zone which increases over time, SIS induces the degeneration of the whole nodule in a short time period (Matamoros et al., 1999; Perez Guerra et al., 2010; Vauclare et al., 2010). At the structural level, microscopic analyses also show that developmental senescence and dark stress-induced senescence present different features in *M. truncatula.* Dark-induced senescence leads to the condensation of the bacteroid content whereas the PBM remains intact even though most of the bacteroid content had disappeared (Perez Guerra et al., 2010). In contrast, developmental senescence induces a pronounced vesicle mobilisation in the host cytoplasm which is correlated with the degeneration of the PBM and the mixing of the symbiosome content with the cytoplasm (Perez Guerra et al., 2010; Van de Velde et al., 2006). However, structural analyses of the SIS have not been extensively performed on different nodule types for all the different environmental stress. Thus, it is possible that the different SIS do not develop similarly. Moreover, as for developmental senescence, SIS may process differently in determinate and indeterminate nodules. Indeed, nitrate induced senescence has been shown to induce bacteroid degradation in pea (indeterminate nodule) after two days of treatment in contrast to bean (determinate nodule), in which nitrate has little effect on the shape of bacteroids even after four days of 10mM nitrate treatment (Matamoros et al., 1999).

#### **3.2 Stress induced senescence is characterized by modifications in nodule carbon metabolism and respiration**

SIS is characterized by multiple early modifications of nodule physiology (Figure 2). Amongst them, modifications in carbon metabolism play a major role. As stated before, BNF is a highly energetic process which requires a constant energy supply. Modification of nodule sucrose content has been observed during drought stress (Galvez et al., 2005; Gordon et al., 1997), salt stress (Gordon et al., 1997; Lopez et al., 2008; Sanchez et al., 2011; Lopez et al., 2009; Ben Salah et al., 2009), dark stress (Gogorcena et al., 1997; Matamoros et al., 1999; Vauclare et al., 2010) and cold stress (Walsh & Layzell, 1986; van Heerden et al., 2008). However, whereas dark stress induces a diminution of sucrose concentration,

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 147

However, nodule O2 metabolic modifications are not always similar. Some types of stress decrease nodule permeability to O2, lowering the O2 availability to bacteroids, which in turn inhibits nitrogenase activity through a lower nodule respiration rate and lower energetic supply. In this context, a nitrate-nitric oxide respiration process has been identified in nodule which may play a role in the maintenance of energetic status under low oxygen conditions (Horchani et al., 2011). In contrast to stress which reduces oxygen availability, some stress increase nodule O2 concentration (increased nodule permeability and/or lower respiration rate) which inhibits nitrogenase activity through a direct O2-induced

Leghemoglobin (Lb) is also a general physiological marker of SIS. As mentioned before, Lb has a crucial role in nodule functioning (Ott et al., 2009; Ott et al., 2005). Decrease of Lb content has been shown during drought stress (Gogorcena et al., 1995; Gordon et al., 1997; Guerin et al., 1990), salt stress (Gordon et al., 1997; Mhadhbi et al., 2011) and dark stress (Gogorcena et al., 1997; Matamoros et al., 1999). This diminution of Lb will impact the general metabolism of nodule by decreasing the O2 availability to bacteroid and by potentially releasing free iron which may be a co-factor of the Fenton reaction to produce

In conclusion, the general production of the high energy level needed for the efficient

**3.3 Stress induced senescence is characterized by modifications in the nodule redox** 

As during developmental senescence, modifications of the redox balance are involved in nodule SIS. As discussed above, the high respiration rates, the important Lb concentration and the release of the catalytic Fe may be major ROS production systems. The accumulation of catalytic Fe has been detected during dark stress (Gogorcena et al., 1997; Becana & Klucas, 1992) and drought stress (Gogorcena et al., 1995) and participates in OH production during dark stress (Becana & Klucas, 1992). The modification of iron metabolism during SIS is also noticeable through the up regulation of ferritin and metallothionein during drought stress (Clement et al., 2008) and dark stress (Perez Guerra et al., 2010). These proteins sequester

free Fe to decrease the Fenton reaction and protect the cellular primary components.

ROS accumulation is also regulated by antioxidant defence which participates in their degradation (Figure 4). Nodule antioxidant defence and the importance of the regulation of the redox balance has been extensively studied in root nodule (for review: Becana et al., 2010; Chang et al., 2009; Marino et al., 2009). Modifications of the antioxidant defence parameters have been used extensively as a marker for nodule SIS. Content and redox state of GSH and Asc, two antioxidant molecules, are modified during drought stress (Gogorcena et al., 1995; Marino et al., 2007), salt stress (Swaraj and Bishnoi, 1999) and dark stress (Matamoros et al., 1999; Gogorcena et al., 1997). Superoxide dismutase and catalase, two enzyme families involved in ROS degradation, are down regulated during dark stress (Matamoros et al., 1999; Gogorcena et al., 1997), salt stress (Jebara et al., 2005) and drought stress (Gogorcena et al., 1995; Rubio et al., 2002). Similarly, enzymes of the Asc-GSH cycle are modulated during nodule SIS (Gogorcena et al., 1995; Jebara et al., 2005; Matamoros et al., 1999; Mhadhbi et al., 2011). Nevertheless, whereas the majority of the reports suggest that SIS is associated with a decrease of the antioxidant defence, other articles have shown

inactivation.

reactive oxygen species.

**state components** 

nitrogen fixation is generally altered at the onset of SIS.

drought stress, salt stress and cold stress lead in general to its accumulation in nodules. The diminution of sucrose concentration linked to a shortage in photosynthate feeding result in a deficiency in energy production. In contrast, the accumulation of sucrose during stress suggests that nodule glycolytic enzymes are affected. Sucrose synthase, which is involved in the degradation of sucrose into glucose and fructose in the root nodule, is inhibited during drought stress and salt stress (Ben Salah et al., 2009; Gordon et al., 1997) and malate content, which is one of the preferred substrates for bacteroid respiration (Prell and Poole, 2006), decreases under these two stress conditions (Marino et al., 2007; Galvez et al., 2005; Ben Salah et al., 2009). A fine-tuning of O2 concentration is also important for nodule functioning, since a good supply of O2 is determinant for nodule respiration and energy requirement while a low O2 pressure must be maintained in the nitrogen fixing zone to prevent nitrogenase inhibition. The availability of O2 through the nodule diffusion barrier and the rate of nodule respiration are thus important parameters of the nodule fitness and they have been shown to be modified during various SIS such as drought stress (Del Castillo et al., 1994; Guerin et al., 1990; Naya et al., 2007; Serraj & Sinclair, 1996; Vessey et al., 1988), salt stress (Bekki et al., 1987; Serraj et al., 1994; Aydi et al., 2004; L'Taief et al., 2007), dark stress (Gogorcena et al., 1997) and cold stress (van Heerden et al., 2008; Kuzma et al., 1995).

Fig. 3. Scheme showing the major processes modified during stress induced senescence. A, modification of the redox balance; B, alteration of the bacteroid nutrition; C, alteration of O2 homeostasis.

drought stress, salt stress and cold stress lead in general to its accumulation in nodules. The diminution of sucrose concentration linked to a shortage in photosynthate feeding result in a deficiency in energy production. In contrast, the accumulation of sucrose during stress suggests that nodule glycolytic enzymes are affected. Sucrose synthase, which is involved in the degradation of sucrose into glucose and fructose in the root nodule, is inhibited during drought stress and salt stress (Ben Salah et al., 2009; Gordon et al., 1997) and malate content, which is one of the preferred substrates for bacteroid respiration (Prell and Poole, 2006), decreases under these two stress conditions (Marino et al., 2007; Galvez et al., 2005; Ben Salah et al., 2009). A fine-tuning of O2 concentration is also important for nodule functioning, since a good supply of O2 is determinant for nodule respiration and energy requirement while a low O2 pressure must be maintained in the nitrogen fixing zone to prevent nitrogenase inhibition. The availability of O2 through the nodule diffusion barrier and the rate of nodule respiration are thus important parameters of the nodule fitness and they have been shown to be modified during various SIS such as drought stress (Del Castillo et al., 1994; Guerin et al., 1990; Naya et al., 2007; Serraj & Sinclair, 1996; Vessey et al., 1988), salt stress (Bekki et al., 1987; Serraj et al., 1994; Aydi et al., 2004; L'Taief et al., 2007), dark stress (Gogorcena et al., 1997) and cold stress (van Heerden et al., 2008; Kuzma et al., 1995).

Fig. 3. Scheme showing the major processes modified during stress induced senescence. A, modification of the redox balance; B, alteration of the bacteroid nutrition; C, alteration of

O2 homeostasis.

However, nodule O2 metabolic modifications are not always similar. Some types of stress decrease nodule permeability to O2, lowering the O2 availability to bacteroids, which in turn inhibits nitrogenase activity through a lower nodule respiration rate and lower energetic supply. In this context, a nitrate-nitric oxide respiration process has been identified in nodule which may play a role in the maintenance of energetic status under low oxygen conditions (Horchani et al., 2011). In contrast to stress which reduces oxygen availability, some stress increase nodule O2 concentration (increased nodule permeability and/or lower respiration rate) which inhibits nitrogenase activity through a direct O2-induced inactivation.

Leghemoglobin (Lb) is also a general physiological marker of SIS. As mentioned before, Lb has a crucial role in nodule functioning (Ott et al., 2009; Ott et al., 2005). Decrease of Lb content has been shown during drought stress (Gogorcena et al., 1995; Gordon et al., 1997; Guerin et al., 1990), salt stress (Gordon et al., 1997; Mhadhbi et al., 2011) and dark stress (Gogorcena et al., 1997; Matamoros et al., 1999). This diminution of Lb will impact the general metabolism of nodule by decreasing the O2 availability to bacteroid and by potentially releasing free iron which may be a co-factor of the Fenton reaction to produce reactive oxygen species.

In conclusion, the general production of the high energy level needed for the efficient nitrogen fixation is generally altered at the onset of SIS.

#### **3.3 Stress induced senescence is characterized by modifications in the nodule redox state components**

As during developmental senescence, modifications of the redox balance are involved in nodule SIS. As discussed above, the high respiration rates, the important Lb concentration and the release of the catalytic Fe may be major ROS production systems. The accumulation of catalytic Fe has been detected during dark stress (Gogorcena et al., 1997; Becana & Klucas, 1992) and drought stress (Gogorcena et al., 1995) and participates in OH production during dark stress (Becana & Klucas, 1992). The modification of iron metabolism during SIS is also noticeable through the up regulation of ferritin and metallothionein during drought stress (Clement et al., 2008) and dark stress (Perez Guerra et al., 2010). These proteins sequester free Fe to decrease the Fenton reaction and protect the cellular primary components.

ROS accumulation is also regulated by antioxidant defence which participates in their degradation (Figure 4). Nodule antioxidant defence and the importance of the regulation of the redox balance has been extensively studied in root nodule (for review: Becana et al., 2010; Chang et al., 2009; Marino et al., 2009). Modifications of the antioxidant defence parameters have been used extensively as a marker for nodule SIS. Content and redox state of GSH and Asc, two antioxidant molecules, are modified during drought stress (Gogorcena et al., 1995; Marino et al., 2007), salt stress (Swaraj and Bishnoi, 1999) and dark stress (Matamoros et al., 1999; Gogorcena et al., 1997). Superoxide dismutase and catalase, two enzyme families involved in ROS degradation, are down regulated during dark stress (Matamoros et al., 1999; Gogorcena et al., 1997), salt stress (Jebara et al., 2005) and drought stress (Gogorcena et al., 1995; Rubio et al., 2002). Similarly, enzymes of the Asc-GSH cycle are modulated during nodule SIS (Gogorcena et al., 1995; Jebara et al., 2005; Matamoros et al., 1999; Mhadhbi et al., 2011). Nevertheless, whereas the majority of the reports suggest that SIS is associated with a decrease of the antioxidant defence, other articles have shown

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 149

increased during dark stress (Matamoros et al., 1999; Gogorcena et al., 1997) and drought stress (Gogorcena et al., 1995). The differential regulation in lipid peroxidation suggests that different degradation mechanisms may partially occur during SIS. This raises the question

One of the specificities of root nodule is the rapid onset of senescence occurring under nitrate treatment. Nitrate concentrations above 2 to 3 mM have strong detrimental effects on the NFS as there is inhibition of several developmental steps ranging from the infection process to the nitrogen fixation in mature nodule (Mortier et al., 2011; Streeter & Wong, 1988). As for other SIS, the inhibition of nitrogenase is correlated with an increase in O2 diffusion and supply (Escuredo et al., 1996; Matamoros et al., 1999; Minchin et al., 1986; Minchin et al., 1989). Nitrate application also reduces carbon supply from leaves to nodules as measured by plant treatment with 11C and 14C-labelled CO2 (Fujikake et al., 2003). This reduction in carbon supply is sometimes correlated with the diminution of the sucrose pool (Matamoros et al., 1999) and the down expression of sucrose synthase (Gordon et al., 2002). Nitrate treatment also decreases the antioxidant defence of the nodule with a diminution of the ascorbate pool and of the activities of ascorbate peroxidases and catalases (Escuredo et al., 1996; Matamoros et al., 1999). At the ultrastructural level, the symbiosome membrane seems to be affected by the senescence process before the bacteroid (Matamoros et al., 1999). Nitrate effect results in both local and systemic regulation of the nodulation process (Jeudy et al., 2009). Systemic regulation has been described in numerous leguminous plants. Indeed, shoot-determined supernodulators with a nitrate-tolerant nodulation process have been described in soybean (Searle et al., 2003), pea (Duc & Messager, 1989), *L. japonicus* (Krusell et al., 2002) and *M. truncatula* (Penmetsa et al., 2003). Gene analyses have led to the identification of orthologous Leucine Rich Repeat-Receptor Like Kinases (LRR-RLKs) which play a crucial role in the autoregulation of nodulation. The systemic regulation may occur via the induction of specific CLV3/ESR (CLE) peptides produced after nitrate treatment (Okamoto et al., 2009; Reid et al., 2011). In this context, NOD3 is involved in the production or in the transport of the root signal molecule involved in the systemic regulation (Novak,

Finally, nitrogen limitation regulates nodule growth and stimulates BNF activity via a LRR-RLKs independent response suggesting that a local nodule adaptation may also be involved

The last review analyzing root nodule senescence presented oxidative stress and hormones as potential key players of the senescence process (Puppo et al., 2005). The concentration of abscissic acid (ABA), a hormone involved in plant response to abiotic and biotic stress (Cutler et al., 2010; Raghavendra et al., 2010), is strongly increased in soybean nodules under drought stress (Clement et al., 2008). The five-fold accumulation of ABA in stressed nodules compared to stressed roots shows that ABA accumulation is much higher in nodules than in roots. The effect of ABA on nodule functioning has been shown by exogenous treatment of pea nodules (Gonzalez et al., 2001). ABA treatment decreases the BNF and Lb content

**3.5 Molecular modifications occurring during stress induced senescence** 

in the nitrate regulation of the BNF (Jeudy et al., 2009).

of SIS regulation and the specificity of the plant response to the different stress.

**3.4 Nodule senescence induced by nitrate** 

2010).

that some elements of the antioxidant defence are stable or even up regulated during stress. This discrepancy may be linked to the stress intensity, the plant adaptation to the treatment and to the growth conditions which modify the responses of plant to stress.

Fig. 4. Scheme showing processes for production and removal of Reactive Oxygen Species. CAT, catalase; SOD, superoxide dismutase; APX, ascorbate peroxidase; MR, monodehydroascorbate reductase; DR, dihydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; ASC, reduced ascorbate; MDHA, monodehydroascorbate; DHA, dehydroascorbate.

The imbalance in redox state leading to oxidative stress is characterized by the oxidation of major cellular components such as lipids and proteins. One of the major targets is the membrane. Lipid peroxidation, measured as the reaction of thiobarbituric acid with malondialdehyde, is significantly attenuated during dark stress (Matamoros et al., 1999; Gogorcena et al., 1997) in contrast to drought stress and salt stress during which lipid peroxidation increases (Gogorcena et al., 1995; Mhadhbi et al., 2011). Protein oxidation is increased during dark stress (Matamoros et al., 1999; Gogorcena et al., 1997) and drought stress (Gogorcena et al., 1995). The differential regulation in lipid peroxidation suggests that different degradation mechanisms may partially occur during SIS. This raises the question of SIS regulation and the specificity of the plant response to the different stress.

## **3.4 Nodule senescence induced by nitrate**

148 Senescence

that some elements of the antioxidant defence are stable or even up regulated during stress. This discrepancy may be linked to the stress intensity, the plant adaptation to the treatment

Fig. 4. Scheme showing processes for production and removal of Reactive Oxygen Species.

The imbalance in redox state leading to oxidative stress is characterized by the oxidation of major cellular components such as lipids and proteins. One of the major targets is the membrane. Lipid peroxidation, measured as the reaction of thiobarbituric acid with malondialdehyde, is significantly attenuated during dark stress (Matamoros et al., 1999; Gogorcena et al., 1997) in contrast to drought stress and salt stress during which lipid peroxidation increases (Gogorcena et al., 1995; Mhadhbi et al., 2011). Protein oxidation is

monodehydroascorbate reductase; DR, dihydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; ASC, reduced ascorbate;

CAT, catalase; SOD, superoxide dismutase; APX, ascorbate peroxidase; MR,

MDHA, monodehydroascorbate; DHA, dehydroascorbate.

and to the growth conditions which modify the responses of plant to stress.

One of the specificities of root nodule is the rapid onset of senescence occurring under nitrate treatment. Nitrate concentrations above 2 to 3 mM have strong detrimental effects on the NFS as there is inhibition of several developmental steps ranging from the infection process to the nitrogen fixation in mature nodule (Mortier et al., 2011; Streeter & Wong, 1988). As for other SIS, the inhibition of nitrogenase is correlated with an increase in O2 diffusion and supply (Escuredo et al., 1996; Matamoros et al., 1999; Minchin et al., 1986; Minchin et al., 1989). Nitrate application also reduces carbon supply from leaves to nodules as measured by plant treatment with 11C and 14C-labelled CO2 (Fujikake et al., 2003). This reduction in carbon supply is sometimes correlated with the diminution of the sucrose pool (Matamoros et al., 1999) and the down expression of sucrose synthase (Gordon et al., 2002). Nitrate treatment also decreases the antioxidant defence of the nodule with a diminution of the ascorbate pool and of the activities of ascorbate peroxidases and catalases (Escuredo et al., 1996; Matamoros et al., 1999). At the ultrastructural level, the symbiosome membrane seems to be affected by the senescence process before the bacteroid (Matamoros et al., 1999).

Nitrate effect results in both local and systemic regulation of the nodulation process (Jeudy et al., 2009). Systemic regulation has been described in numerous leguminous plants. Indeed, shoot-determined supernodulators with a nitrate-tolerant nodulation process have been described in soybean (Searle et al., 2003), pea (Duc & Messager, 1989), *L. japonicus* (Krusell et al., 2002) and *M. truncatula* (Penmetsa et al., 2003). Gene analyses have led to the identification of orthologous Leucine Rich Repeat-Receptor Like Kinases (LRR-RLKs) which play a crucial role in the autoregulation of nodulation. The systemic regulation may occur via the induction of specific CLV3/ESR (CLE) peptides produced after nitrate treatment (Okamoto et al., 2009; Reid et al., 2011). In this context, NOD3 is involved in the production or in the transport of the root signal molecule involved in the systemic regulation (Novak, 2010).

Finally, nitrogen limitation regulates nodule growth and stimulates BNF activity via a LRR-RLKs independent response suggesting that a local nodule adaptation may also be involved in the nitrate regulation of the BNF (Jeudy et al., 2009).

#### **3.5 Molecular modifications occurring during stress induced senescence**

The last review analyzing root nodule senescence presented oxidative stress and hormones as potential key players of the senescence process (Puppo et al., 2005). The concentration of abscissic acid (ABA), a hormone involved in plant response to abiotic and biotic stress (Cutler et al., 2010; Raghavendra et al., 2010), is strongly increased in soybean nodules under drought stress (Clement et al., 2008). The five-fold accumulation of ABA in stressed nodules compared to stressed roots shows that ABA accumulation is much higher in nodules than in roots. The effect of ABA on nodule functioning has been shown by exogenous treatment of pea nodules (Gonzalez et al., 2001). ABA treatment decreases the BNF and Lb content

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 151

Mutations in some bacteroid genes have an incidence on its lifespan *in planta* and thus on the nodule integrity. However, it is difficult to assess the role of bacteroid genes on nodule

> elongated differentiated bacteroids

reduced number of intracellular bacteria, mixture of normal elongated and abnormal bacteroids

fix+/- (30%/WT), fewer smaller bacteroids with a lower DNA content

fix+/- (25%/WT), abnormal bacteroids, with an increased size

fix+/- (25%/WT), mixture of white, slightly pink and pink

fix+/- (30%/WT), differentiated bacteroids irregular in

fix+/- (25%/WT), senescence before or after differentiation into bacteroid

fix+/- (30%/WT), mixture of pink and white nodules

fix+/- (50%/WT), mixture of pink and white nodules

nodules

shape

fix-,

Maunoury et al.,

Mitsui, (2004)

Prell et al., (2009)

Moris et al., (2005)

Davies et al., (2007)

Jamet et al., (2003)

Harrison et al., (2005)

Luo et al., (2005)

Torres-Quesada et al., (2010)

(2008)

(2005)

**affected Species Function Symbiotic phenotype** Reference

(2010) *lspB S. meliloti* Lipopolysaccharide


acids transporters

*gltA S. meliloti* Citrate synthase fix+/- (20%/WT) Grzemski et al.,

*gshB R. tropici* Glutathione synthetase fix+/- Muglia et al.,

Until now, there is no global transcriptomic study which could give information on the bacteroid gene expression profile when nitrogen fixing bacteroids turn off to become senescent. Moreover, due to the difficulty in finding a reliable and efficient screen to isolate bacteroid senescent mutants *in planta*, a library of such mutants is still not available. The data described below mostly come from the analysis of the symbiotic phenotype of rhizobial

biosynthesis

senescence due to a lack of genetic tools to investigate this question.

*fixJ and fixK S. meliloti* Nitrogen fixation fix-,

*aap bra R. leguminosarum* Branched-chain amino

*relA R. etli* Stringent response

*sitA S. meliloti* Manganese uptake

*gshB S. meliloti* Glutathione synthetase

*lsrB1 S. meliloti* LysR-type transcriptional

*hfq S. meliloti* RNA chaperone

regulator

Table 1. Bacterial genes in which mutations cause early nodule senescence

*katA katC S. meliloti* Catalases

**Gene** 

*nifH, fixA,* 

*rpoH1 S. meliloti* 

declines in parallel with the BNF. However, sucrose synthase activity, another parameter of drought stress effect, is not affected by this treatment suggesting that ABA is not the only player of nodule response to stress. Jasmonic acid (JA), another hormone involved in plant stress response (Reinbothe et al., 2009), has also been shown to be involved in the regulation of nodule functioning (Hause & Schaarschmidt, 2009). Exogenous JA treatment induces an accumulation of lipid peroxides and modifies ascorbate metabolism suggesting that JA could influence nodule senescence (Loscos et al., 2008).

Redox state modifications seem to be a regulatory element of the nodule SIS (Marino et al., 2006). Exogenous treatment with paraquat, which generates ROS, induces the alteration of the GSH and ASC pools toward a more oxidized state. This alteration of the redox state is associated with a diminution of BNF and decrease in Lb content. Moreover, an early decrease in sucrose synthase activity is also detected during the treatment. These results suggest that oxidative stress is involved in the signalling pathway leading to nodule SIS. Interestingly, sucrose synthase seems to be regulated at both the transcriptional and posttranslational levels by oxidizing agents such as paraquat (Marino et al., 2008). Finally, genetic modifications allowing decrease and increase of GSH content in the nitrogen fixing zone has shown that BNF and Lb expression level are correlated with GSH content (El Msehli et al., 2011). These results strengthen the idea that cellular redox state plays a crucial role in the regulation of nodule functioning.

Developmental senescence and SIS present different structural and temporal features. At the transcriptomic level, analysis of the expression of 58 genes up regulated during developmental senescence has been performed during dark stress (Perez Guerra et al., 2010). 21 genes are induced during both types of senescence. Amongst these genes, some serine/threonine kinase and some genes involved in metal metabolism (metal transporters and metallothionein) have similar profiles of induction. Nine are up-regulated during both senescence types with different induction levels or transient induction during dark stress. Amongst these genes, cysteine and aspartic proteases are well represented. Finally, 28 genes are up regulated during developmental senescence and not by dark stress. Amongst these genes, proteins associated to proteasome function and vesicular trafficking are not induced during dark stress suggesting partial different regulatory processes between the two types of senescence. In soybean, a screen for genes involved in root nodule senescence has led to the isolation of the senescence-associated nodulin 1 (SAN1) multigene family showing a high homology with plant 2-oxoglutarate-dependent dioxygenases and including two functional genes *SAN1A* and *SAN1B* and a pseudogene *SAN1C* (Webb et al., 2008). Analyses of the steady-state mRNA levels of *SAN1A* and *SAN1B* during developmental senescence showed no significant differences for both genes. In contrast, during induced senescence by treatment with nitrate or darkness, *SAN1A* is down-regulated and *SAN1B* is up-regulated by both treatments.

Nevertheless, dark stress, drought stress and salt stress may induce specific senescence cascades and transcriptomic analyses will have to be realized to define the similarities and the differences in gene expression patterns in nodules subjected to the different stress.

## **4. Bacterial mutants and nodule senescence**

The microsymbiont, differentiated into bacteroids inside the symbiosome, is not only dependent on "senescent" signals coming from the cytosolic environment of its host plant.

declines in parallel with the BNF. However, sucrose synthase activity, another parameter of drought stress effect, is not affected by this treatment suggesting that ABA is not the only player of nodule response to stress. Jasmonic acid (JA), another hormone involved in plant stress response (Reinbothe et al., 2009), has also been shown to be involved in the regulation of nodule functioning (Hause & Schaarschmidt, 2009). Exogenous JA treatment induces an accumulation of lipid peroxides and modifies ascorbate metabolism suggesting that JA

Redox state modifications seem to be a regulatory element of the nodule SIS (Marino et al., 2006). Exogenous treatment with paraquat, which generates ROS, induces the alteration of the GSH and ASC pools toward a more oxidized state. This alteration of the redox state is associated with a diminution of BNF and decrease in Lb content. Moreover, an early decrease in sucrose synthase activity is also detected during the treatment. These results suggest that oxidative stress is involved in the signalling pathway leading to nodule SIS. Interestingly, sucrose synthase seems to be regulated at both the transcriptional and posttranslational levels by oxidizing agents such as paraquat (Marino et al., 2008). Finally, genetic modifications allowing decrease and increase of GSH content in the nitrogen fixing zone has shown that BNF and Lb expression level are correlated with GSH content (El Msehli et al., 2011). These results strengthen the idea that cellular redox state plays a crucial

Developmental senescence and SIS present different structural and temporal features. At the transcriptomic level, analysis of the expression of 58 genes up regulated during developmental senescence has been performed during dark stress (Perez Guerra et al., 2010). 21 genes are induced during both types of senescence. Amongst these genes, some serine/threonine kinase and some genes involved in metal metabolism (metal transporters and metallothionein) have similar profiles of induction. Nine are up-regulated during both senescence types with different induction levels or transient induction during dark stress. Amongst these genes, cysteine and aspartic proteases are well represented. Finally, 28 genes are up regulated during developmental senescence and not by dark stress. Amongst these genes, proteins associated to proteasome function and vesicular trafficking are not induced during dark stress suggesting partial different regulatory processes between the two types of senescence. In soybean, a screen for genes involved in root nodule senescence has led to the isolation of the senescence-associated nodulin 1 (SAN1) multigene family showing a high homology with plant 2-oxoglutarate-dependent dioxygenases and including two functional genes *SAN1A* and *SAN1B* and a pseudogene *SAN1C* (Webb et al., 2008). Analyses of the steady-state mRNA levels of *SAN1A* and *SAN1B* during developmental senescence showed no significant differences for both genes. In contrast, during induced senescence by treatment with nitrate or darkness, *SAN1A* is down-regulated and *SAN1B* is up-regulated

Nevertheless, dark stress, drought stress and salt stress may induce specific senescence cascades and transcriptomic analyses will have to be realized to define the similarities and the differences in gene expression patterns in nodules subjected to the different stress.

The microsymbiont, differentiated into bacteroids inside the symbiosome, is not only dependent on "senescent" signals coming from the cytosolic environment of its host plant.

could influence nodule senescence (Loscos et al., 2008).

role in the regulation of nodule functioning.

**4. Bacterial mutants and nodule senescence** 

by both treatments.

Mutations in some bacteroid genes have an incidence on its lifespan *in planta* and thus on the nodule integrity. However, it is difficult to assess the role of bacteroid genes on nodule senescence due to a lack of genetic tools to investigate this question.


Table 1. Bacterial genes in which mutations cause early nodule senescence

Until now, there is no global transcriptomic study which could give information on the bacteroid gene expression profile when nitrogen fixing bacteroids turn off to become senescent. Moreover, due to the difficulty in finding a reliable and efficient screen to isolate bacteroid senescent mutants *in planta*, a library of such mutants is still not available. The data described below mostly come from the analysis of the symbiotic phenotype of rhizobial

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 153

expression. When alfalfa plants infected with the ts mutants were transferred to 30°C, the nodules lost the ability to fix nitrogen. Microscopic examination of the nodules revealed the loss of bacteroids in infected cells and morphological changes that resembled changes seen

These experiments with CS ts mutants showed that CS activity is needed in mature nodules to maintain bacteroid integrity and that removing CS activity via a temperature shift converts an effective nodule into an empty nodule. This implies that CS is essential for

Another example of early nodule senescence associated to a nutrient defect in *Rhizobium* has been described recently. Prell and collaborators (2009) have shown that *Rhizobium leguminosarum*, the bacterial partner of peas and broad beans (biovar *viciae*), becomes symbiotic auxotroph for the branched-chain amino acids Leucine, Isoleucine, Valine (LIV) when differentiated into bacteroids in root nodules (Prell et al., 2009). While these bacteria are prototrophs for LIV amino acids as free-living bacteria, they become dependent on the plant as nitrogen-fixing bacteroids, due to a major reduction in gene expression and activity of LIV biosynthetic enzymes. Peas inoculated with bacterial mutants impaired in their capacity to transport LIV, have an early senescent phenotype (nod+, fix+/-). Peas are yellow, have small, pale pink nodules and a dry weight similar to un-inoculated plants. This is correlated with a 70% decrease of the nitrogen fixation capacity for plants inoculated with these mutants compared to the plants inoculated with the *R. leguminosarum* wild-type strain. Thus, plants not only provide a carbon source (dicarboxylic acids) to the bacteroid but also precursors of proteins. The authors have shown that a defect in bacteroid LIV nutrition leads to a reduction in its persistence in plant infected cells, which in turn induces senescence. This means that the plant cell might receive information from the bacteroid in order to sense the fitness of the microsymbiont to maintain or interrupt the symbiotic

Concerning nutriment stress perceived by the bacteria *in planta*, a mutation in the *relA* gene of *Rhizobium etli*, induces symbiotic defects at the intermediate and/or late stages of the interaction with *Phaseolus vulgaris* (Moris et al., 2005). RelA allows the production of the alarmone (p)ppGpp, which mediates the stringent response in bacteria. This response results in transcriptional down regulation of ribosomal and tRNA genes, upon conditions of amino acid starvation. Despite this role, RelA has been reported to be important for biofilm formation and for interaction of bacteria, pathogenic or beneficial, with their eukaryotic host. Interaction of a *relA* mutant with common bean plants strongly reduces the nitrogen fixation efficiency by 75% and the plant yield. Microscopic studies showed that bacteria differentiated into bacteroids in the symbiosomes were larger in size than the wild-type ones. Thus, in the *R. etli* bacteroids, *relA* plays a role in physiology adaptation and regulation of gene expression. However, the step impaired in the nitrogen fixation defect of

It is generally accepted that symbiotic bacteria are submitted to an oxidative burst released by the host plant during the first steps of infection (Pauly et al., 2006). In addition to this role as a general plant defence mechanism against bacterial invasion, oxidative burst might also

**4.1.2 Genes encoding function involved in oxidative stress response** 

nodule maintenance as well as in the early stages of plant cell invasion.

during nodule senescence.

interaction.

a *relA* mutant has not been investigated.

strains affected in one specific gene. The characteristics associated to these analyses include: plant yield, nodule morphology, nitrogen fixation efficiency, and sometimes, ultrastructure of the nodule and of the bacteroid. More recently, some symbiotic phenotype studies also include bacterial genome endoreduplication and transcriptome analyses. This section will mainly focus on the impact of a mutation in the bacterial genome on early nodule senescence and on delayed nodule senescence (Table 1).

#### **4.1 Bacterial mutants and early nodule senescence**

The rhizobial mutants that present a symbiotic phenotype are divided into four groups: the nodule deficient mutants impaired in the first steps of infection (nod fix- ), the bacterial mutants which induce nodules that present an early nodule senescence phenotype i) blocked in their bacteroid differentiation process (nod+ fix- ), ii) fully differentiated but unable to reduce N2 (nod+ fix-) and iii) differentiated into bacteroids less efficient in N2 fixation compared to the wild-type strain (nod+ fix+/-). Bacterial mutants leading to nodule development abortion due to a defect in bacteroid differentiation such as *bacA* (Saeki, 2011) or *parA* (Liu et al., 2011) mutants will not be presented here. The rhizobial nod+ fix- mutants that could differentiate into bacteroids and pass the two transcriptome switch-points encountered during the differentiation of the wild-type bacteroids have been described recently (Maunoury et al., 2010). They are affected in genes encoding for symbiotic function and nitrogen fixation machinery (*nifH*, *fixA*, *fixJ* and *fixK*) or in lipopolysaccharide biosynthesis (*lspB*). The *rpoH1* mutant of *S. meliloti*, impaired in the synthesis of the 32-like protein, involved in bacterial protection against environmental stresses has also a nod+ fixsymbiotic phenotype in interaction with alfalfa (Mitsui et al., 2004). The bacterial *rpoH1* mutant is still able to elicit nodule formation, efficient plant cell invasion and differentiation into bacteroids. But, the degeneration of bacteroids rapidly occurred in the proximal zone, adjacent to the infection zone, leading to ineffective white nodules associated to an early nodule senescence phenotype. Only the latter group of early nodule senescence mutants (nod+ fix+/-) will be described below. The genes affected in these mutants fall mainly into two categories: genes encoding function involved in carbon and nitrogen metabolism, and genes important for stress adaptation. Genes with other function will also be presented.

#### **4.1.1 Genes encoding function involved in carbon and nitrogen metabolism of the bacteroid and in the nutriment stress response**

The host plant supplies bacteroids with metabolites, including dicarboxylic and amino acids, used by the bacteroids to support the reduction of N2 into ammonia in amounts sufficient for plant growth. To better understand the role of the decarboxylating part of the *S. meliloti* TCA cycle in a nitrogen fixing nodule, Gremski and collaborators (2005) have used an elegant approach (Grzemski et al., 2005). In *S. meliloti*, a mutant in the TCA cycle *gltA* gene encoding citrate synthase forms empty nodules devoid of intracellular bacteria. The *gltA* mutants are clearly unsuitable for experiments to determine whether citrate synthase (CS) is essential during nitrogen fixation in a mature nodule because they have a defect in development that prevents them from forming normal bacteroids. So, the authors constructed temperature-sensitive (ts) mutants in the *S. meliloti* citrate synthase (*gltA*) gene. This allows the formation of nitrogen-fixing nodules at the permissive temperature but, once nodule development was complete, an elevation of root temperature prevents CS

strains affected in one specific gene. The characteristics associated to these analyses include: plant yield, nodule morphology, nitrogen fixation efficiency, and sometimes, ultrastructure of the nodule and of the bacteroid. More recently, some symbiotic phenotype studies also include bacterial genome endoreduplication and transcriptome analyses. This section will mainly focus on the impact of a mutation in the bacterial genome on early nodule

The rhizobial mutants that present a symbiotic phenotype are divided into four groups: the

mutants which induce nodules that present an early nodule senescence phenotype i)

unable to reduce N2 (nod+ fix-) and iii) differentiated into bacteroids less efficient in N2 fixation compared to the wild-type strain (nod+ fix+/-). Bacterial mutants leading to nodule development abortion due to a defect in bacteroid differentiation such as *bacA* (Saeki, 2011)

that could differentiate into bacteroids and pass the two transcriptome switch-points encountered during the differentiation of the wild-type bacteroids have been described recently (Maunoury et al., 2010). They are affected in genes encoding for symbiotic function and nitrogen fixation machinery (*nifH*, *fixA*, *fixJ* and *fixK*) or in lipopolysaccharide biosynthesis (*lspB*). The *rpoH1* mutant of *S. meliloti*, impaired in the synthesis of the 32-like protein, involved in bacterial protection against environmental stresses has also a nod+ fixsymbiotic phenotype in interaction with alfalfa (Mitsui et al., 2004). The bacterial *rpoH1* mutant is still able to elicit nodule formation, efficient plant cell invasion and differentiation into bacteroids. But, the degeneration of bacteroids rapidly occurred in the proximal zone, adjacent to the infection zone, leading to ineffective white nodules associated to an early nodule senescence phenotype. Only the latter group of early nodule senescence mutants (nod+ fix+/-) will be described below. The genes affected in these mutants fall mainly into two categories: genes encoding function involved in carbon and nitrogen metabolism, and genes important for stress adaptation. Genes with other function will also be presented.

**4.1.1 Genes encoding function involved in carbon and nitrogen metabolism of the** 

The host plant supplies bacteroids with metabolites, including dicarboxylic and amino acids, used by the bacteroids to support the reduction of N2 into ammonia in amounts sufficient for plant growth. To better understand the role of the decarboxylating part of the *S. meliloti* TCA cycle in a nitrogen fixing nodule, Gremski and collaborators (2005) have used an elegant approach (Grzemski et al., 2005). In *S. meliloti*, a mutant in the TCA cycle *gltA* gene encoding citrate synthase forms empty nodules devoid of intracellular bacteria. The *gltA* mutants are clearly unsuitable for experiments to determine whether citrate synthase (CS) is essential during nitrogen fixation in a mature nodule because they have a defect in development that prevents them from forming normal bacteroids. So, the authors constructed temperature-sensitive (ts) mutants in the *S. meliloti* citrate synthase (*gltA*) gene. This allows the formation of nitrogen-fixing nodules at the permissive temperature but, once nodule development was complete, an elevation of root temperature prevents CS

or *parA* (Liu et al., 2011) mutants will not be presented here. The rhizobial nod+ fix-

fix-

), ii) fully differentiated but

), the bacterial

mutants

senescence and on delayed nodule senescence (Table 1).

**4.1 Bacterial mutants and early nodule senescence** 

**bacteroid and in the nutriment stress response** 

blocked in their bacteroid differentiation process (nod+ fix-

nodule deficient mutants impaired in the first steps of infection (nod-

expression. When alfalfa plants infected with the ts mutants were transferred to 30°C, the nodules lost the ability to fix nitrogen. Microscopic examination of the nodules revealed the loss of bacteroids in infected cells and morphological changes that resembled changes seen during nodule senescence.

These experiments with CS ts mutants showed that CS activity is needed in mature nodules to maintain bacteroid integrity and that removing CS activity via a temperature shift converts an effective nodule into an empty nodule. This implies that CS is essential for nodule maintenance as well as in the early stages of plant cell invasion.

Another example of early nodule senescence associated to a nutrient defect in *Rhizobium* has been described recently. Prell and collaborators (2009) have shown that *Rhizobium leguminosarum*, the bacterial partner of peas and broad beans (biovar *viciae*), becomes symbiotic auxotroph for the branched-chain amino acids Leucine, Isoleucine, Valine (LIV) when differentiated into bacteroids in root nodules (Prell et al., 2009). While these bacteria are prototrophs for LIV amino acids as free-living bacteria, they become dependent on the plant as nitrogen-fixing bacteroids, due to a major reduction in gene expression and activity of LIV biosynthetic enzymes. Peas inoculated with bacterial mutants impaired in their capacity to transport LIV, have an early senescent phenotype (nod+, fix+/-). Peas are yellow, have small, pale pink nodules and a dry weight similar to un-inoculated plants. This is correlated with a 70% decrease of the nitrogen fixation capacity for plants inoculated with these mutants compared to the plants inoculated with the *R. leguminosarum* wild-type strain. Thus, plants not only provide a carbon source (dicarboxylic acids) to the bacteroid but also precursors of proteins. The authors have shown that a defect in bacteroid LIV nutrition leads to a reduction in its persistence in plant infected cells, which in turn induces senescence. This means that the plant cell might receive information from the bacteroid in order to sense the fitness of the microsymbiont to maintain or interrupt the symbiotic interaction.

Concerning nutriment stress perceived by the bacteria *in planta*, a mutation in the *relA* gene of *Rhizobium etli*, induces symbiotic defects at the intermediate and/or late stages of the interaction with *Phaseolus vulgaris* (Moris et al., 2005). RelA allows the production of the alarmone (p)ppGpp, which mediates the stringent response in bacteria. This response results in transcriptional down regulation of ribosomal and tRNA genes, upon conditions of amino acid starvation. Despite this role, RelA has been reported to be important for biofilm formation and for interaction of bacteria, pathogenic or beneficial, with their eukaryotic host. Interaction of a *relA* mutant with common bean plants strongly reduces the nitrogen fixation efficiency by 75% and the plant yield. Microscopic studies showed that bacteria differentiated into bacteroids in the symbiosomes were larger in size than the wild-type ones. Thus, in the *R. etli* bacteroids, *relA* plays a role in physiology adaptation and regulation of gene expression. However, the step impaired in the nitrogen fixation defect of a *relA* mutant has not been investigated.

#### **4.1.2 Genes encoding function involved in oxidative stress response**

It is generally accepted that symbiotic bacteria are submitted to an oxidative burst released by the host plant during the first steps of infection (Pauly et al., 2006). In addition to this role as a general plant defence mechanism against bacterial invasion, oxidative burst might also

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 155

accumulation. Expression of this gene in a wild-type background is enhanced at late stage of nodule development, suggesting its antioxidant role against ROS accumulation during nodule senescence. In these species, GSH is important to keep nodules functional over time. In contrast, this does not hold true for *Bradyrhizobium* sp. where disruption of the *gshA* gene does not affect the ability to form effective nodules (Sobrevals et al., 2006). In this latter case, it is possible that the defect in intracellular GSH was compensated for by other compounds

**4.1.3 Genes encoding bacterial function involved in the regulation of gene expression**  Ninety putative genes encoding LysR-type transcriptional regulators were identified in the *S. meliloti* genome. These regulators are typically 300 amino acids long with an N-terminal DNA binding domain and a C-terminal sensing domain for signal molecules and function as activators or repressors. LysR regulated genes have promoters which contain at least one TN11A motif and are usually divergently transcribed from the LysR regulator (Schell, 1993). To determine the role of LysR regulators in symbiosis, a mutagenesis analysis of all 90 putative *lysR* genes was realized (Luo et al., 2005). This allowed the isolation of the *lsrB1*  mutant that presents a symbiotic phenotype. An *lsrB1* mutant was deficient in symbiosis and elicited a mixture of pink (45%) and white (55%) nodules on alfalfa plants. These plants exhibited lower overall nitrogenase activity (30%) than plants inoculated with the wild-type strain. This is consistent with the fact that most of the alfalfa plants inoculated with the *lsrB1*  mutant were short (50 to 80% shorter than the plants inoculated with the WT strain) and light green. Cells of the *lsrB1* mutant were recovered from both pink and white nodules, suggesting that *lsrB1* mutants could be blocked either early or late during nodulation. Similar numbers of bacterial cells were recovered from the pink nodules of plants inoculated with the wild-type strain Rm1021 and pink and white nodules from the plants inoculated with the mutants. These findings suggest that the *lsrB1* mutants were able to invade plant cells. The *lsrB* gene is located downstream from the *trxB* gene for thioredoxin reductase, which also participates in the bacterial antioxidant defence. The *trxB* gene is transcribed from its own promoter in the same direction as the *lsrB* gene. The *trxB* promoter contains a nearly perfect recognition site (TN11A) for a LysR regulator so it is possible that LsrB regulates the expression of both the *trxB* and *lsrB* genes. The authors suggest that the early senescence phenotype observed *in planta* could be linked to a defect in detoxification of ROS

It has been shown recently that the *S. meliloti* RNA chaperone Hfq plays a role in the survival of the microsymbiont within the alfalfa nodule cells (Torres-Quesada et al., 2010). Hfq is considered to act as a global post-transcriptional regulator of gene expression since it interacts with diverse RNA molecules and small non-coding RNAs (sRNA). In free living bacteria, an *hfq* mutant down-regulates 91 genes mostly involved in central carbon metabolism (uptake and utilization of carbon substrates) and up-regulates genes involved in the uptake and catabolism of diverse N compounds. During late interaction with alfalfa (30 days post-infection), plants inoculated with the *hfq* mutant strain are composed of 60% of white non fixing nodules and 40% of pink fixing nodules. Thus, the plant yield was 64% of that of the wild-type-inoculated plants. Histological analysis of the white nodules revealed that the bacteroid differentiation was efficient but the bacteroid-infected tissues were restricted to the interzone II-III since the zone III was replaced by a large senescent zone IV. Indeed, an Hfq impaired mutant showed a premature senescent phenotype. The authors

acting as antioxidants.

in *S. meliloti*. However, this has not been demonstrated.

play a role in the lifespan of the bacteroid. The high rate of bacteroid respiration necessary to supply energy required for the nitrogen reduction process generates high levels of ROS in the nodule. In legume root nodules, a large amount of H2O2 surrounding disintegrating bacteroids in senescent zone IV is detected (Rubio et al, 2004). This reflects the close relationship between oxidative stress and nodule senescence. Indeed, most of the bacterial mutant strains that show a symbiotic nod+ fix+/- phenotype (early senescence) are affected in their antioxidant defence. To escape the stress generated by H2O2 and O2 .-, bacteria encode a set of enzymes such as superoxide dismutases, catalases and alkylhydroperoxidases, and also antioxidant molecules such as GSH.

In *S. meliloti*, disruption of the *sitA* gene induces a decrease in Mn/Fe SodB activity and a higher sensitivity to ROS (Davies and Walker, 2007). The *sitA* gene encodes a periplasmic protein involved in manganese uptake. During alfalfa interaction, a *sitA* mutant is either affected in its infection efficiency leading to small white nodules or is possibly altered in the survival of the differentiated form leading to intermediate nodules, with a slight pink fixing zone, smaller in size than the wild-type nodules. As a consequence, the nitrogenase activity and the plant yield are greatly reduced in these mutant infected plants compared to the plants inoculated with the wild-type bacteria. It is difficult to assess the role of the other superoxide dismutase of *S. meliloti (*SodA) in the natural senescence process since a *sodA* mutant failed to differentiate into bacteroids after release into plant cells (Santos et al., 2000).

To cope with H2O2 production, *S. meliloti* possesses three catalases. In free living bacteria, KatA and KatC are encoded by genes mainly transcribed in oxidative stress conditions while the *katB* gene is constitutively expressed. In 6 week-old nodules of M*edicago sativa*, KatA is the predominant catalase present in the bacteroids. The *katB* gene is also expressed in the nitrogen fixation zone III while *katC* is only transcribed in the infection zone II (Jamet et al., 2003). Single mutant strains of *katA*, *katB* or *katC* genes have no significant impact on nitrogen fixation efficiency of alfalfa nodules containing these mutants compared to those infected with wild-type bacteria. However a *katA katC* double mutant presents a dramatic decrease of nitrogen fixation capacity (Sigaud et al., 1999), associated with an early senescence of the nodule. These nodules were devoid of a clear zone III, instead the senescent zone IV was adjacent to interzone II-III (Jamet et al., 2003). In most plant cells, bacteria were correctly released from infection threads and were able to differentiate into bacteroids. This shows that efficient detoxification of H2O2 by the microsymbiont is essential in the latter steps of bacteroid differentiation leading to nitrogen fixing bacteria.

The antioxidant GSH plays an important role during symbiosis and nodule senescence. This tripeptide is synthesized by a two-step process. In bacteria, GshA catalyses the conjugation of glutamate and cysteine to form EC and, in a second enzymatic step, GshB completes GSH synthesis by addition of glycine. In *S. meliloti*, while a *gshA* mutant is unable to form nodules (nod fix- phenotype), a *gshB* mutant has a nod+ phenotype coupled to a 75% reduction in the nitrogen fixation capacity (Harrison et al., 2005). In these nodules, bacteria are correctly released from the infection thread into host plant cells and enter into early senescence after differentiation into bacteroids. These data show that, in *S. meliloti*, GSH is important to maintain bacteroid during symbiotic interaction with alfalfa. This is also true in some determinate-type nodules as the survival of the common bean (*P. vulgaris)*  microsymbiont, *Rhizobium tropicii*, is dependent on GSH production (Muglia et al., 2008). A *gshB* mutant has an early senescent pattern associated with increased levels of superoxide

play a role in the lifespan of the bacteroid. The high rate of bacteroid respiration necessary to supply energy required for the nitrogen reduction process generates high levels of ROS in the nodule. In legume root nodules, a large amount of H2O2 surrounding disintegrating bacteroids in senescent zone IV is detected (Rubio et al, 2004). This reflects the close relationship between oxidative stress and nodule senescence. Indeed, most of the bacterial mutant strains that show a symbiotic nod+ fix+/- phenotype (early senescence) are affected in

set of enzymes such as superoxide dismutases, catalases and alkylhydroperoxidases, and

In *S. meliloti*, disruption of the *sitA* gene induces a decrease in Mn/Fe SodB activity and a higher sensitivity to ROS (Davies and Walker, 2007). The *sitA* gene encodes a periplasmic protein involved in manganese uptake. During alfalfa interaction, a *sitA* mutant is either affected in its infection efficiency leading to small white nodules or is possibly altered in the survival of the differentiated form leading to intermediate nodules, with a slight pink fixing zone, smaller in size than the wild-type nodules. As a consequence, the nitrogenase activity and the plant yield are greatly reduced in these mutant infected plants compared to the plants inoculated with the wild-type bacteria. It is difficult to assess the role of the other superoxide dismutase of *S. meliloti (*SodA) in the natural senescence process since a *sodA* mutant failed to differentiate into bacteroids after release into plant cells (Santos et al., 2000). To cope with H2O2 production, *S. meliloti* possesses three catalases. In free living bacteria, KatA and KatC are encoded by genes mainly transcribed in oxidative stress conditions while the *katB* gene is constitutively expressed. In 6 week-old nodules of M*edicago sativa*, KatA is the predominant catalase present in the bacteroids. The *katB* gene is also expressed in the nitrogen fixation zone III while *katC* is only transcribed in the infection zone II (Jamet et al., 2003). Single mutant strains of *katA*, *katB* or *katC* genes have no significant impact on nitrogen fixation efficiency of alfalfa nodules containing these mutants compared to those infected with wild-type bacteria. However a *katA katC* double mutant presents a dramatic decrease of nitrogen fixation capacity (Sigaud et al., 1999), associated with an early senescence of the nodule. These nodules were devoid of a clear zone III, instead the senescent zone IV was adjacent to interzone II-III (Jamet et al., 2003). In most plant cells, bacteria were correctly released from infection threads and were able to differentiate into bacteroids. This shows that efficient detoxification of H2O2 by the microsymbiont is essential

.-, bacteria encode a

their antioxidant defence. To escape the stress generated by H2O2 and O2

in the latter steps of bacteroid differentiation leading to nitrogen fixing bacteria.

The antioxidant GSH plays an important role during symbiosis and nodule senescence. This tripeptide is synthesized by a two-step process. In bacteria, GshA catalyses the conjugation of glutamate and cysteine to form EC and, in a second enzymatic step, GshB completes GSH synthesis by addition of glycine. In *S. meliloti*, while a *gshA* mutant is unable to form

reduction in the nitrogen fixation capacity (Harrison et al., 2005). In these nodules, bacteria are correctly released from the infection thread into host plant cells and enter into early senescence after differentiation into bacteroids. These data show that, in *S. meliloti*, GSH is important to maintain bacteroid during symbiotic interaction with alfalfa. This is also true in some determinate-type nodules as the survival of the common bean (*P. vulgaris)*  microsymbiont, *Rhizobium tropicii*, is dependent on GSH production (Muglia et al., 2008). A *gshB* mutant has an early senescent pattern associated with increased levels of superoxide

fix- phenotype), a *gshB* mutant has a nod+ phenotype coupled to a 75%

also antioxidant molecules such as GSH.

nodules (nod-

accumulation. Expression of this gene in a wild-type background is enhanced at late stage of nodule development, suggesting its antioxidant role against ROS accumulation during nodule senescence. In these species, GSH is important to keep nodules functional over time. In contrast, this does not hold true for *Bradyrhizobium* sp. where disruption of the *gshA* gene does not affect the ability to form effective nodules (Sobrevals et al., 2006). In this latter case, it is possible that the defect in intracellular GSH was compensated for by other compounds acting as antioxidants.

## **4.1.3 Genes encoding bacterial function involved in the regulation of gene expression**

Ninety putative genes encoding LysR-type transcriptional regulators were identified in the *S. meliloti* genome. These regulators are typically 300 amino acids long with an N-terminal DNA binding domain and a C-terminal sensing domain for signal molecules and function as activators or repressors. LysR regulated genes have promoters which contain at least one TN11A motif and are usually divergently transcribed from the LysR regulator (Schell, 1993). To determine the role of LysR regulators in symbiosis, a mutagenesis analysis of all 90 putative *lysR* genes was realized (Luo et al., 2005). This allowed the isolation of the *lsrB1*  mutant that presents a symbiotic phenotype. An *lsrB1* mutant was deficient in symbiosis and elicited a mixture of pink (45%) and white (55%) nodules on alfalfa plants. These plants exhibited lower overall nitrogenase activity (30%) than plants inoculated with the wild-type strain. This is consistent with the fact that most of the alfalfa plants inoculated with the *lsrB1*  mutant were short (50 to 80% shorter than the plants inoculated with the WT strain) and light green. Cells of the *lsrB1* mutant were recovered from both pink and white nodules, suggesting that *lsrB1* mutants could be blocked either early or late during nodulation. Similar numbers of bacterial cells were recovered from the pink nodules of plants inoculated with the wild-type strain Rm1021 and pink and white nodules from the plants inoculated with the mutants. These findings suggest that the *lsrB1* mutants were able to invade plant cells. The *lsrB* gene is located downstream from the *trxB* gene for thioredoxin reductase, which also participates in the bacterial antioxidant defence. The *trxB* gene is transcribed from its own promoter in the same direction as the *lsrB* gene. The *trxB* promoter contains a nearly perfect recognition site (TN11A) for a LysR regulator so it is possible that LsrB regulates the expression of both the *trxB* and *lsrB* genes. The authors suggest that the early senescence phenotype observed *in planta* could be linked to a defect in detoxification of ROS in *S. meliloti*. However, this has not been demonstrated.

It has been shown recently that the *S. meliloti* RNA chaperone Hfq plays a role in the survival of the microsymbiont within the alfalfa nodule cells (Torres-Quesada et al., 2010). Hfq is considered to act as a global post-transcriptional regulator of gene expression since it interacts with diverse RNA molecules and small non-coding RNAs (sRNA). In free living bacteria, an *hfq* mutant down-regulates 91 genes mostly involved in central carbon metabolism (uptake and utilization of carbon substrates) and up-regulates genes involved in the uptake and catabolism of diverse N compounds. During late interaction with alfalfa (30 days post-infection), plants inoculated with the *hfq* mutant strain are composed of 60% of white non fixing nodules and 40% of pink fixing nodules. Thus, the plant yield was 64% of that of the wild-type-inoculated plants. Histological analysis of the white nodules revealed that the bacteroid differentiation was efficient but the bacteroid-infected tissues were restricted to the interzone II-III since the zone III was replaced by a large senescent zone IV. Indeed, an Hfq impaired mutant showed a premature senescent phenotype. The authors

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence 157

nodule senescence, the progression of senescence symptoms seems to be differentially controlled. Indeed, even if common general features have been described during nodule senescence, the few available microscopic and transcriptomic analyses show that nodule senescence may occur differently in developmental senescence and SIS. Moreover, the senescence occurring under different environmental stress conditions such as dark stress, drought stress or nitrate treatment may also involve different genetic and physiological programs. In this context, more detailed spatiotemporal analysis of the multiple senescence processes will have to be performed to determine the similarities and the differences between the various senescence processes. Microarray analysis or "Whole Transcriptome Shotgun Sequencing" will be valuable tools to analyse the transcriptome modifications occurring under the different senescence processes (Lister et al., 2009). In conditions in which the senescence process does not appear to be a homogenous process such as in developmental senescence, laser capture microdissection (Barcala et al., 2009) will allow the analysis of transcriptomic patterns of senescent infected cells. The development of two legume model systems, *M. truncatula* (http://www.medicago.org/) and *L. japonicus*  (http://www.lotusjaponicus.org/) will facilitate an efficient analysis of the senescence process by developing tools dedicated to cell biology, genetic and transcriptomic analyses. The role of the bacterial partner needs also to be clarified. Indeed, whereas senescence phenotypes are observed in nodules obtained with bacterial mutants affected in their nitrogen fixation efficiency, the regulation of this bacterial-induced senescence has not yet been studied. The abortion of nodule development when using such bacterial mutants suggests that the interaction may switch from a compatible to an incompatible interaction. In this context, the molecular events which trigger this switch still need to be defined. Nevertheless, the work with the flavodoxin overexpressing-bacterial strain showed that the symbiotic interaction may also be improved to resist to the various endogenous and environmental stress conditions. The construction of plant and bacteria with higher resistance to environmental stress (Zurbriggen et al., 2008) may be an interesting

opportunity to increase the benefit from an efficient BNF in agronomy.

senescence. *New Phytol,* Vol. 158, No. 1, pp. 131-138

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**6. References** 

pp. 698-712

proposed that this phenotype could be linked to a defect in intracellular survival under prolonged stress present in the plant cell environment.

## **4.2 Bacterial mutants and delayed senescence**

The delayed senescent bacterial mutants might have a nod+ phenotype associated with a fix+ phenotype for a period longer than the natural fixing period associated with the wild type bacteria in interaction with its host plant. Thus, such mutants are obligate differentiated bacteroids.

Compared to the data connected with the consequences of bacterial gene inactivation on early nodule senescence, little information on the role of bacterial mutants on delayed nodule senescence are available. Knowing that most of the mutations that induce an acceleration of senescence affect genes involved in ROS detoxification, in bacterial fitness, in import and/or processing of carbon skeletons, amino acids and antioxidants, we might suspect that a delayed senescence bacterial mutant should have a gain of function rather than an invalidated one. In that sense, it is possible that an increase in synthesis and/or activity of molecules involved in stress resistance, especially to ROS, should improve the bacteroid lifetime in the symbiosome and thus should enhance the nitrogen fixing period. This aspect of the role of bacterial genes in the functional life of symbiotic fixing nodules remains to be explored. However, one encouraging study sustains this postulate (Redondo et al., 2009). In fact, the authors of this work have overexpressed a cyanobacteria *Anabaena variabilis* gene encoding flavodoxin in *S. meliloti*. Knowing that natural senescence-inducing signals from the plant leads to a decrease in antioxidant content and thus an increase in ROS accumulation in an irreversible manner, they analyse the consequences of the overexpression of this flavodoxin protein involved in the response to oxidative stress. They have shown that the decline of nitrogenase activity was delayed and that the structural and ultrastructural modifications associated with nodule senescence had a later onset in flavodoxin-expressing nodules. Lipid peroxidation, a marker of senescence, was significantly reduced and the oxidative balance was improved in comparison to the control nodules. In conclusion, flavodoxin over-expression had an impact on bacteroid antioxidant metabolism, leading to delayed senescence.

In conclusion, we can propose that bacteroids inside the nodule infected cells are not only tributary from the plant to initiate nodule senescence. Genes encoding proteins implicated in bacterial nutrition and stress response are also essential since mutations in these genes alter the fitness of the differentiated bacteroids. In turn, this leads to the death of the microsymbiont followed by the senescence of the plant cells and nodule. Future aspects on the role of bacteroid genes on senescence should include the development of bacteroid genetic tools. The over-expression of pertinent genes specifically in the bacteroid or the conditional invalidation of rhizobial genes after bacteroid differentiation will be important to define senescence-related genes.

## **5. Perspectives**

The data summarized in this review indicate that one of the general physiological features of nodule senescence is the decrease in nitrogen fixation efficiency. This diminution may be related to plant and/or bacterial-dependent factors. However whereas this diminution of the nitrogen fixation efficiency is observed during both developmental and stress inducednodule senescence, the progression of senescence symptoms seems to be differentially controlled. Indeed, even if common general features have been described during nodule senescence, the few available microscopic and transcriptomic analyses show that nodule senescence may occur differently in developmental senescence and SIS. Moreover, the senescence occurring under different environmental stress conditions such as dark stress, drought stress or nitrate treatment may also involve different genetic and physiological programs. In this context, more detailed spatiotemporal analysis of the multiple senescence processes will have to be performed to determine the similarities and the differences between the various senescence processes. Microarray analysis or "Whole Transcriptome Shotgun Sequencing" will be valuable tools to analyse the transcriptome modifications occurring under the different senescence processes (Lister et al., 2009). In conditions in which the senescence process does not appear to be a homogenous process such as in developmental senescence, laser capture microdissection (Barcala et al., 2009) will allow the analysis of transcriptomic patterns of senescent infected cells. The development of two legume model systems, *M. truncatula* (http://www.medicago.org/) and *L. japonicus*  (http://www.lotusjaponicus.org/) will facilitate an efficient analysis of the senescence process by developing tools dedicated to cell biology, genetic and transcriptomic analyses.

The role of the bacterial partner needs also to be clarified. Indeed, whereas senescence phenotypes are observed in nodules obtained with bacterial mutants affected in their nitrogen fixation efficiency, the regulation of this bacterial-induced senescence has not yet been studied. The abortion of nodule development when using such bacterial mutants suggests that the interaction may switch from a compatible to an incompatible interaction. In this context, the molecular events which trigger this switch still need to be defined. Nevertheless, the work with the flavodoxin overexpressing-bacterial strain showed that the symbiotic interaction may also be improved to resist to the various endogenous and environmental stress conditions. The construction of plant and bacteria with higher resistance to environmental stress (Zurbriggen et al., 2008) may be an interesting opportunity to increase the benefit from an efficient BNF in agronomy.

#### **6. References**

156 Senescence

proposed that this phenotype could be linked to a defect in intracellular survival under

The delayed senescent bacterial mutants might have a nod+ phenotype associated with a fix+ phenotype for a period longer than the natural fixing period associated with the wild type bacteria in interaction with its host plant. Thus, such mutants are obligate differentiated

Compared to the data connected with the consequences of bacterial gene inactivation on early nodule senescence, little information on the role of bacterial mutants on delayed nodule senescence are available. Knowing that most of the mutations that induce an acceleration of senescence affect genes involved in ROS detoxification, in bacterial fitness, in import and/or processing of carbon skeletons, amino acids and antioxidants, we might suspect that a delayed senescence bacterial mutant should have a gain of function rather than an invalidated one. In that sense, it is possible that an increase in synthesis and/or activity of molecules involved in stress resistance, especially to ROS, should improve the bacteroid lifetime in the symbiosome and thus should enhance the nitrogen fixing period. This aspect of the role of bacterial genes in the functional life of symbiotic fixing nodules remains to be explored. However, one encouraging study sustains this postulate (Redondo et al., 2009). In fact, the authors of this work have overexpressed a cyanobacteria *Anabaena variabilis* gene encoding flavodoxin in *S. meliloti*. Knowing that natural senescence-inducing signals from the plant leads to a decrease in antioxidant content and thus an increase in ROS accumulation in an irreversible manner, they analyse the consequences of the overexpression of this flavodoxin protein involved in the response to oxidative stress. They have shown that the decline of nitrogenase activity was delayed and that the structural and ultrastructural modifications associated with nodule senescence had a later onset in flavodoxin-expressing nodules. Lipid peroxidation, a marker of senescence, was significantly reduced and the oxidative balance was improved in comparison to the control nodules. In conclusion, flavodoxin over-expression had an impact on bacteroid antioxidant

In conclusion, we can propose that bacteroids inside the nodule infected cells are not only tributary from the plant to initiate nodule senescence. Genes encoding proteins implicated in bacterial nutrition and stress response are also essential since mutations in these genes alter the fitness of the differentiated bacteroids. In turn, this leads to the death of the microsymbiont followed by the senescence of the plant cells and nodule. Future aspects on the role of bacteroid genes on senescence should include the development of bacteroid genetic tools. The over-expression of pertinent genes specifically in the bacteroid or the conditional invalidation of rhizobial genes after bacteroid differentiation will be important

The data summarized in this review indicate that one of the general physiological features of nodule senescence is the decrease in nitrogen fixation efficiency. This diminution may be related to plant and/or bacterial-dependent factors. However whereas this diminution of the nitrogen fixation efficiency is observed during both developmental and stress induced-

prolonged stress present in the plant cell environment.

**4.2 Bacterial mutants and delayed senescence** 

metabolism, leading to delayed senescence.

to define senescence-related genes.

**5. Perspectives** 

bacteroids.


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**Animal** 

**9** 

**The Nucleolus and Ribosomal** 

Nadine Hein1,\*, Elaine Sanij1,2,\*,

*University of Melbourne, Melbourne,* 

*Monash University, Melbourne,* 

*1,2,3,5,6Australia 4New Zealand* 

Jaclyn Quin1,3, Katherine M. Hannan1,

Austen Ganley4,# and Ross D. Hannan1,3,5,6,#

*3Department of Biochemistry and Molecular Biology,* 

*5Department of Biochemistry and Molecular Biology,* 

**Genes in Aging and Senescence** 

*1Division of Cancer Research, Peter MacCallum Cancer Centre, Melbourne,* 

*6School of Biomedical Sciences, The University of Queensland, Melbourne,* 

The nucleolus forms around the tandem repeats of the ribosomal RNA (rRNA) genes (rDNA) that are transcribed by RNA polymerase I (Pol I), giving rise to the production of rRNAs. These represent the nucleic acid backbone of the functional ribosomes in the cytoplasm, and as such rDNA transcription dictates the cells' protein translational capacity. More recently it has become apparent that the epigenetic status of these rDNA repeats and the integrity of the nucleolus can modulate cellular homeostasis beyond ribosome biogenesis. Such roles include mediating the titration of tumor suppressors and oncogenes, modulating the heterochromatic state of many RNA Polymerase II (Pol II) transcribed genes, and importantly, regulating the process of aging and senescence. This chapter will focus on the molecular and cellular evidence that the nucleolus and the rDNA repeats play critical

This section will provide a brief overview of the regulation of rDNA transcription, however,

roles in the control of aging and cellular senescence in yeast and mammals.

for more details refer to (Tschochner & Hurt, 2003; McStay & Grummt, 2008).

**2. Introduction to rDNA transcription and the nucleolus** 

\* These authors contributed equally to this work.
