**1. Introduction**

206 Artificial Photosynthesis

Yamaguchi-Shinozaki, K. & Shinozaki, K. (2006). Responses and tolerance to dehydration

Yamazaki, J-Y., Suzuki, T., Maruta, E. & Kamimura, Y. (2005). The stoichiometry and

Yan, Y., Stolz, S., Chetelat, A., Reymond, P., Pagni, M., Dubugnon, L. & Farmer, E.E. (2007).

Yokthongwattana, K. & Melis, A. (2006). Photoinhibition and recovery in oxygenic

Yuan, H.Y., Chen, X.P., Zhu, L.L. & He, G.C. (2005). Identification of genes responsive to

Zangerl, A.R. & Bazzaz, F.A. (1993). Theory and pattern in plant defense allocation, in Fritz,

Zangerl, A.R., Hamilton, J.G., Miller, T.J., Crofts, A.R., Oxborough, K., Berenbaum, M.R. &

Zhang, Z., Huang, L., Shulmeister, V.M., Chi, Y.I., Kim, K.K., Hung, L.W., Crofts, A.R.,

Zhang, S. & Scheller, H.V. (2004). Light-harvesting complex II binds to several small subunits of photosystem I, *Journal of Biological Chemistry* 279: 3180-3187. Zhu-Salzman, K., Salzman, R.A., Ahn, J.E. & Koiwa H. (2004) Transcriptional regulation of

Zou, J., Rodriguez-Zas, S., Aldea, M., Li, M., Zhu, J., Gonzalez, D.O., Vodkin, L.O., DeLucia,

of photosynthesis, *Molecular Plant-Microbe Interactions* 18: 1161-1174. Zvereva, E.L., Lanta, V. & Kozlov, M.V. (2010). Effects of sap-feeding insect herbivores on

antenna size of the two photosystems in marine green algae, *Bryopsis maxima* and *Ulva pertusa*, in relation to the light environment of their natural habitat, *Journal of* 

A downstream mediator in the growth repression limb of the jasmonate pathway,

photosynthesis: mechanism of a photosystem II damage and repair cycle, in Demmig-Adams, B., Adams, W.W. III & Mattoo, A.K. (eds) *Photoprotection, photoinhibition, gene regulation, and environment*. Springer, The Netherlands, pp 175-

brown planthopper *Nilaparvata lugens* Stal (Homoptera, Delphacidae) feeding in

S.R. & Simms, E.L. (eds) *Plant Resistance to Herbivores and Pathogens*, University of

DeLucia, E.H. (2002). Impact of folivory on photosynthesis is greater than the sum of its holes, *Proceedings of the National Academy of Sciences of the USA* 99: 1088-1091. Zavala, J.A. & Baldwin, I.T. (2006). Jasmonic acid signaling and herbivore resistance traits

constrain regrowth after herbivore attack in *Nicotiana attenuata*, *Plant Cell &* 

Berry, E.A. & Kim, S.H. (1998). Electron transfer by domain movement in

sorghum defense determinants against a phloem-feeding aphid, *Plant Physiology*

E. & Clough, S.J. (2005). Expression profiling soybean response to *Pseudomonas syringae* reveals new defense-related genes and rapid HR-specific down regulation

growth and reproduction of woody plants: a meta-analysis of experimental studies,

and cold stresses, *Annual Review of Plant Biology* 57: 781-803.

*Experimental Botany* 56: 1517-1523.

*The Plant Cell* 19: 2470-2483.

rice, *Planta* 221: 105-112.

*Environment* 29: 1751-1760.

134: 420-431.

*Oecologia* 163: 949-960.

Chicago Press, Chicago pp. 363-391.

cytochrome bc1, *Nature* 392: 677-684.

191.

Plants are frequently exposed to environmental stress both in natural and agricultural conditions and it is common for more than one abiotic stress to occur at a given time; for example, drought, heat and high illumination. The concept of stress is intimately associated with that of stress tolerance, which is the plant's ability to cope with an unfavourable environment. Plants exhibit great variations in their tolerance to stress. Some plants show sufficient developmental plasticity to respond to a range of light regimes, growing as sun plants in sunny areas and as shade plants in shady habitats. However, other species of plants are adapted to sunny environments or to shaded environments, and they show different levels of tolerance to high illumination. Generally, sun plants support exposure to high light better than shade plants, which experience photoinhibition (Bray et al., 2000; Levitt, 1980; Osmond, 1994; Saura & Quiles, 2008; Wentworth et al., 2006).

Abiotic stress limits crop productivity and plays a major role in determining the distribution of plant species across different types of environments. Thus, understanding the physiological processes that underlie stress injury and the tolerance mechanisms of plants to environmental stress is of immense importance for both agriculture and the environment. Tolerance to environmental stresses results from integrated events occurring at all organization levels, from the anatomical and morphological to the cellular, biochemical and molecular level. At the biochemical level, plants alter their metabolism in various ways to accommodate environmental stress, with photosynthesis being the most important of these ways.

The photosynthetic apparatus in the plants absorbs large amounts of light energy and processes it into chemical energy. The absorption of photons excites the pigment molecules and this excitation energy is used in the photochemical reactions of photosynthesis, though part of the excitation energy is dissipated by fluorescence (emission of photons by chlorophyll molecules) and heat emission, principally in the antenna system. These three processes (photochemistry, fluorescence and thermal energy dissipation) compete in the dissipation of the excitation energy, while the total energy dissipated is the sum of all three (Long et al., 1994). Estimation of these processes in different conditions will permit us

Using Chlorophyll Fluorescence Imaging for Early Assessment of

in response to these adverse conditions.

Photosynthesis Tolerance to Drought, Heat and High Illumination 209

photochemical reaction is the transfer of one electron from pigment P680 in the first excited singlet state (P680\*) to pheophytin *a*. From there, the electron is transferred to the primary quinone-type acceptor, QA. When all the reaction centers are in an active state, with the quinones totally or partially oxidized ("open" state), the fluorescence yield is minimal (F0). However, when QA is fully reduced, the excitation of P680 cannot result in stable charge separation and all the reaction centers are in a "closed" state; in this situation, maximum fluorescence yield (FM) is obtained. The variable fluorescence emission (FV), is the difference between FM and F0 (FV = FM - F0). In a dark-adapted leaf, the plastoquinone pool is fully oxidized, the reaction centers are open and the fluorescence emitted under a weak measuring light is minimal (F0). When a saturating pulse of white light is given the plastoquinone pool is reduced, the rate of QA reduction being faster than the rate of reoxidation, the reaction centers are closed and FM is reached; at that moment the maximal quantum yield of PS II can be estimated as FV / FM. This ratio is an important and easily measurable parameter of the physiological state of the photosynthetic apparatus in intact plant leaves. Additionally, the kinetic of the increase in fluorescence during the saturation pulse can be displayed as the fluorescence induction curve. Most fluorescence is emitted by the PS II antenna, and PS I only contributes around 1-2 % of the total fluorescence; for this

reason, the changes in this radiation reflect the state of PS II (Krause and Weis, 1991).

In recent years, the versatility of the chlorophyll fluorescence measurement technique has increased significantly with the development of fluorescence imaging systems, these provides a powerful tool for investigating leaf photosynthesis in a variety of conditions and reveal a wide range of internal leaf characteristics, including spatial variations due to differences in physiology and development (for a review see Papageorgiou & Govindjee, 2004). This technique may also represent a simple and effective tool for the early detection of the effects caused by adverse factors (Oxborough, 2004a), which affect photosynthesis and cause an imbalance in the processes of excitation energy dissipation. Fluorescence imaging permits us to compare the variation in these processes and to study any damage caused in the same leaf as time progresses. However, not all fluorescence parameters are suitable for the early detection of plant stress. Usually, changes in the maximum quantum yield of PS II are used as an indicator of the functional state of the photosynthetic apparatus (Barbagallo et al., 2003; Oxborough, 2004b), since this parameter, which has a value between 0.70 and 0.85 in unstressed leaves, falls under the influence of adverse factors (Ehlert & Hincha, 2008; Havaux & Lannoye, 1985; Joshi & Mohantly, 2004; Quiles & López, 2004; Teicher et al., 2000). However, in the present paper we show that in both sun (*Chrysanthemum morifolium*) and shade (*Spathiphyllum wallisii)* plants exposed to drought, high illumination and heat and showing no visible damage, the images of the maximal quantum yield of PS II (Fv/Fm) in dark-adapted leaves vary little from those in control plants, and, in all cases, the values are quite normal (above 0.74). Therefore, other fluorescence parameters are required to assess the tolerance of plants to those adverse factors. In this respect, we show that images of fluorescence yield, the effective PSII quantum yield and the non-photochemical quenching of fluorescence in illuminated leaves clearly showed variations in the energy dissipation processes between sun and shade plants exposed to drought, high illumination and heat. As a consequence, the measurement of these fluorescence parameters can be considered a better and earlier indicator of functional alterations of the photosynthetic apparatus than maximal quantum yield of PS II (Fv/Fm), which, as we have mentioned, shows only small variations

compare the competition that exists among the three of them and to evaluate possible alterations in the functioning of the photosynthetic apparatus. When plants are exposed for long periods of time to more light than they can use, photosynthesis is inhibited in a phenomenon known as photoinhibition. If the excess of absorbed light energy is not dissipated safely toxic species may be produced which can damage the photosynthetic apparatus. Photosynthetic organisms therefore contain a complex set of regulatory and repair mechanisms to avoid this situation. However, even with all these protective mechanisms, the photosynthetic apparatus is still sometimes damaged. PS II is the most sensitive site to photoinhibition, whereas PS I is more stable, probably because it plays a photoprotective role through cyclic electron flow (Quiles and López, 2004). In high-light conditions, the xanthophylls cycle operates, of which violaxanthin together with antheraxanthin and zeaxanthin are components (Demmig-Adams & Adams, 1993; Lichtenthaler et al., 1992; Schindler & Lichtenthaler, 1994, 1996). The xanthophylls cycle is essential to prevent the rapid photoinhibition of PS II (Havaux & Gruszecki, 1993; Lichtenthaler & Babani, 2004; Ruban & Horton, 1995). Sun plants accumulate zeaxanthin during high-light stress of several hours to photoprotect their photosynthetic apparatus against photoinhibition and photooxidation, whereas shade plants do not possess zeaxanthin but only its oxidized form violaxanthin with some traces of antheraxanthin, and these plants are more sensitive to photoinhibition (Lichtenthaler & Babani, 2004 ).

The classic methods used to determine the damage induced by adverse factors in leaves (such as the measurement of transpiration, respiration and photosynthesis rates; stomatal conductance; water potential; the concentration of photosynthetic pigments, stress metabolites and heat shock proteins) are all quite slow and require considerable effort. Moreover, many of these methods only provide one datum per leaf and measurement, and involve the destruction of tissues, so that subsequent measurements are not possible in the same leaf. In many cases, these methods are effective only for assessing the damage caused by stress to the plant in advanced situations, when the damage is visible, but do not allow early detection of alterations caused by adverse conditions, before the damage becomes visible. This is unfortunate because early detection is important and, in many cases, would make it possible to prevent the onset of irreversible damages. For these reasons, it would be of great interest to develop rapid, non-destructive and quantitative techniques for the early detection of stress in plants. One non-intrusive method for monitoring photosynthetic events and for judging the physiological state of the plant is to measure the chlorophyll fluorescence emitted by intact plant leaves, using a fluorometer (Sayed, 2003). Based on pulse amplitude modulation (PAM) and the saturation pulse method (Schreiber, 2004), chlorophyll fluorometry provides quantitative information concerning the maximal quantum yield of PS II in dark-adapted leaves, the fluorescence yield, the effective PSII quantum yield or photochemical efficiency and the non-photochemic quenching of fluorescence, which represents the heat dissipation in the antenna system (Müller et al., 2001). Three major components of non-photochemical quenching have been identified in plants, namely, energy-dependent quenching, photoinhibitory quenching and statetransition quenching, which are related to trans-thylakoid proton gradient, photoinhibition and energy redistribution, respectively (Allen, 1992; Krause, 1988; Krause & Weis, 1991).

The rate of fluorescence emission depends on the absorbed light flux and on all the competing reactions that result in a return of the excited chlorophyll molecule to the ground state. The most important of those reactions are the photochemical reactions, thermal deactivation and the excitation energy transfer. In the PS II reaction center, the primary

compare the competition that exists among the three of them and to evaluate possible alterations in the functioning of the photosynthetic apparatus. When plants are exposed for long periods of time to more light than they can use, photosynthesis is inhibited in a phenomenon known as photoinhibition. If the excess of absorbed light energy is not dissipated safely toxic species may be produced which can damage the photosynthetic apparatus. Photosynthetic organisms therefore contain a complex set of regulatory and repair mechanisms to avoid this situation. However, even with all these protective mechanisms, the photosynthetic apparatus is still sometimes damaged. PS II is the most sensitive site to photoinhibition, whereas PS I is more stable, probably because it plays a photoprotective role through cyclic electron flow (Quiles and López, 2004). In high-light conditions, the xanthophylls cycle operates, of which violaxanthin together with antheraxanthin and zeaxanthin are components (Demmig-Adams & Adams, 1993; Lichtenthaler et al., 1992; Schindler & Lichtenthaler, 1994, 1996). The xanthophylls cycle is essential to prevent the rapid photoinhibition of PS II (Havaux & Gruszecki, 1993; Lichtenthaler & Babani, 2004; Ruban & Horton, 1995). Sun plants accumulate zeaxanthin during high-light stress of several hours to photoprotect their photosynthetic apparatus against photoinhibition and photooxidation, whereas shade plants do not possess zeaxanthin but only its oxidized form violaxanthin with some traces of antheraxanthin, and

these plants are more sensitive to photoinhibition (Lichtenthaler & Babani, 2004 ).

The classic methods used to determine the damage induced by adverse factors in leaves (such as the measurement of transpiration, respiration and photosynthesis rates; stomatal conductance; water potential; the concentration of photosynthetic pigments, stress metabolites and heat shock proteins) are all quite slow and require considerable effort. Moreover, many of these methods only provide one datum per leaf and measurement, and involve the destruction of tissues, so that subsequent measurements are not possible in the same leaf. In many cases, these methods are effective only for assessing the damage caused by stress to the plant in advanced situations, when the damage is visible, but do not allow early detection of alterations caused by adverse conditions, before the damage becomes visible. This is unfortunate because early detection is important and, in many cases, would make it possible to prevent the onset of irreversible damages. For these reasons, it would be of great interest to develop rapid, non-destructive and quantitative techniques for the early detection of stress in plants. One non-intrusive method for monitoring photosynthetic events and for judging the physiological state of the plant is to measure the chlorophyll fluorescence emitted by intact plant leaves, using a fluorometer (Sayed, 2003). Based on pulse amplitude modulation (PAM) and the saturation pulse method (Schreiber, 2004), chlorophyll fluorometry provides quantitative information concerning the maximal quantum yield of PS II in dark-adapted leaves, the fluorescence yield, the effective PSII quantum yield or photochemical efficiency and the non-photochemic quenching of fluorescence, which represents the heat dissipation in the antenna system (Müller et al., 2001). Three major components of non-photochemical quenching have been identified in plants, namely, energy-dependent quenching, photoinhibitory quenching and statetransition quenching, which are related to trans-thylakoid proton gradient, photoinhibition and energy redistribution, respectively (Allen, 1992; Krause, 1988; Krause & Weis, 1991). The rate of fluorescence emission depends on the absorbed light flux and on all the competing reactions that result in a return of the excited chlorophyll molecule to the ground state. The most important of those reactions are the photochemical reactions, thermal deactivation and the excitation energy transfer. In the PS II reaction center, the primary photochemical reaction is the transfer of one electron from pigment P680 in the first excited singlet state (P680\*) to pheophytin *a*. From there, the electron is transferred to the primary quinone-type acceptor, QA. When all the reaction centers are in an active state, with the quinones totally or partially oxidized ("open" state), the fluorescence yield is minimal (F0). However, when QA is fully reduced, the excitation of P680 cannot result in stable charge separation and all the reaction centers are in a "closed" state; in this situation, maximum fluorescence yield (FM) is obtained. The variable fluorescence emission (FV), is the difference between FM and F0 (FV = FM - F0). In a dark-adapted leaf, the plastoquinone pool is fully oxidized, the reaction centers are open and the fluorescence emitted under a weak measuring light is minimal (F0). When a saturating pulse of white light is given the plastoquinone pool is reduced, the rate of QA reduction being faster than the rate of reoxidation, the reaction centers are closed and FM is reached; at that moment the maximal quantum yield of PS II can be estimated as FV / FM. This ratio is an important and easily measurable parameter of the physiological state of the photosynthetic apparatus in intact plant leaves. Additionally, the kinetic of the increase in fluorescence during the saturation pulse can be displayed as the fluorescence induction curve. Most fluorescence is emitted by the PS II antenna, and PS I only contributes around 1-2 % of the total fluorescence; for this reason, the changes in this radiation reflect the state of PS II (Krause and Weis, 1991). In recent years, the versatility of the chlorophyll fluorescence measurement technique has

increased significantly with the development of fluorescence imaging systems, these provides a powerful tool for investigating leaf photosynthesis in a variety of conditions and reveal a wide range of internal leaf characteristics, including spatial variations due to differences in physiology and development (for a review see Papageorgiou & Govindjee, 2004). This technique may also represent a simple and effective tool for the early detection of the effects caused by adverse factors (Oxborough, 2004a), which affect photosynthesis and cause an imbalance in the processes of excitation energy dissipation. Fluorescence imaging permits us to compare the variation in these processes and to study any damage caused in the same leaf as time progresses. However, not all fluorescence parameters are suitable for the early detection of plant stress. Usually, changes in the maximum quantum yield of PS II are used as an indicator of the functional state of the photosynthetic apparatus (Barbagallo et al., 2003; Oxborough, 2004b), since this parameter, which has a value between 0.70 and 0.85 in unstressed leaves, falls under the influence of adverse factors (Ehlert & Hincha, 2008; Havaux & Lannoye, 1985; Joshi & Mohantly, 2004; Quiles & López, 2004; Teicher et al., 2000). However, in the present paper we show that in both sun (*Chrysanthemum morifolium*) and shade (*Spathiphyllum wallisii)* plants exposed to drought, high illumination and heat and showing no visible damage, the images of the maximal quantum yield of PS II (Fv/Fm) in dark-adapted leaves vary little from those in control plants, and, in all cases, the values are quite normal (above 0.74). Therefore, other fluorescence parameters are required to assess the tolerance of plants to those adverse factors. In this respect, we show that images of fluorescence yield, the effective PSII quantum yield and the non-photochemical quenching of fluorescence in illuminated leaves clearly showed variations in the energy dissipation processes between sun and shade plants exposed to drought, high illumination and heat. As a consequence, the measurement of these fluorescence parameters can be considered a better and earlier indicator of functional alterations of the photosynthetic apparatus than maximal quantum yield of PS II (Fv/Fm), which, as we have mentioned, shows only small variations in response to these adverse conditions.

Using Chlorophyll Fluorescence Imaging for Early Assessment of

0

protected by mechanisms that dissipate excess excitation energy.

exposed to stress photoperiods in either sun or shade plants.

20

40

60

RWC (%)

four independent experiments.

80

100

Photosynthesis Tolerance to Drought, Heat and High Illumination 211

decreased in both species to around 60 % after the two stress photoperiods with low

C 1S 2S

Fig. 1. The relative water content (RWC) of leaves from *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants in control conditions (C) and after exposure to one (1S) and two (2S) stress photoperiods (18 h, 1060 mol·m-2·s-1 PPFD, 35 ºC and low watering). The stress photoperiods were separated by 6 h night-periods at 24 ºC. The values are means SE from

The fluorescence imaging technique was used to assess photosynthesis in sun and shade plants. Figure 2 shows the images of the maximal quantum yield of PS II (Fv/Fm) from a typical leaf, and the means values SE of *C. morifolium* and *S. wallisii* plants in control conditions and exposed to one and two stress photoperiods. The results are shown as colour-coded images according to the pattern shown below the images. All the leaves provided similar images with a homogeneous colour throughout the leaf. The mean Fv/Fm values in all cases were higher than 0.74, indicating that maximal quantum yield of PS II in leaves from *C. morifolium* and *S. wallisii* plants, in control conditions and exposed to one and two stress photoperiods, was quite normal (Krause & Weis, 1991; Schereiber et al., 1997) and that the maximal photosynthetic capacity of PS II in these species was probably unaffected by the stress condition used here; furthermore it seems that the photosynthetic apparatus is

Figure 3 shows the amounts of total chlorophylls and carotenoids in leaves from *C. morifolium* and *S. wallisii* plants in control conditions and exposed to one and two stress photoperiods. No significant difference was observed between the control plants and those

*C. morifolium S. wallisii*

watering, indicating that the plants were subjected to moderate water deficit.
