Photosynthesis and the Environment (Advances in Photosynthesis and Respiration)

Photosynthesis and the Environment

Advances in Photosynthesis VOLUME 5

Series Editor: GOVINDJEE Department of Plant Biology University of Illinois, Urbana, Illinois, U.S.A. Consulting Editors: Jan AMESZ, Leiden, The Netherlands Eva-Mari ARO, Turku, Finland James BARBER, London, United Kingdom Robert E. BLANKENSHIP, Tempe, Arizona, U.S.A. Norio MURATA, Okazaki, Japan Donald R. ORT, Urbana, Illinois, U.S.A. Advances in Photosynthesis is an ambitious new book series seeking to provide a comprehensive and state-of-the-art account of photosynthesis research. Photosynthesis is the process by which higher plants, algae and certain species of bacteria transform and store solar energy in the form of energy-rich organic molecules. These compounds are in turn used as the energy source for all growth and reproduction in these organisms. As such, virtually all life on the planet ultimately depends on photosynthetic energy conversion. This series of multiauthored books spans topics from physics to agronomy, from femtosecond reactions to season long production, from the photophysics of reaction centers to the physiology of whole organisms, and from X-ray crystallography of proteins to the morphology of intact plants. The intent of this new series of publications is to offer beginning researchers, graduate students, and even research specialists a comprehensive current picture of the remarkable advances across the full scope of photosynthesis research.

Photosynthesis and the Environment Edited by

Neil R. Baker Department of Biological and Chemical Sciences, University of Essex, Colchester, United Kingdom

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

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Contents xi

Preface 1

Processing of Excitation Energy by Antenna Pigments Thomas G. Owens

1–23

Summary I. Introduction II. Structure and Composition of Photosynthetic Antennae III. Role of the Antenna in Photosynthesis IV. Light-Harvesting Function of Antenna Pigments V. Non-Photochemical Quenching and Regulation of Light Energy Utilization VI. Concluding Remarks Acknowledgments References

2

Control and Measurement of Photosynthetic Electron Transport in Vivo David Mark Kramer and Antony Richard Crofts

25–66

Summary I. Introduction II. Control of the Photosynthetic Electron Transfer Chain III. What Reactions Can We Measure? IV. Instrumentation and Measurement V. The Future of Instrumentation for Intact Plants Acknowledgments References

3

Regulation of Light Utilization for Photosynthetic Electron Transport B. Genty and J. Harbinson

26 27 28 31 32 58 59 60

67–99

Summary I. Introduction II. Operation of Light-driven Energy Transduction in Leaves III. Significance of Structural Acclimation on the Operation of Light-driven Energy Transduction. A Case Study: Acclimation to Growth Irradiance IV. Conclusions V. Appendix: The Use of Light-Induced Absorbance Changes Around 820 nm to Measure P700 Oxidation Acknowledgments References

4

Mechanisms of Photodamage and Protein Degradation During Photoinhibition of Photosystem II B. Andersson and J. Barber Summary I. Introduction II. Photosystem II: Structure and Function III. Photochemical Processes Giving Rise to Damage

1 2 4 5 9 12 21 21 21

68 68 69 86 90

91 92 92

101–121 101 102 104 106

IV. Does Triggering for D1 Protein Degradation Require a Conformational Change? V. Degradation of Reaction Center Subunits in Photosystem II VI. Repair of Photodamaged Photosystem II Requires Co-ordination Between Degradation and Biosynthesis Acknowledgments References

5

Radical Production and Scavenging in the Chloroplasts Kozi Asado

Metabolic Regulation of Photosynthesis Mark Stitt

Carbon Metabolism and Photorespiration: Temperature Dependence in Relation to Other Environmental Factors Richard C. Leegood and Gerald E. Edwards Summary I. General Philosophy II. Stomatal Versus Biochemical/Photochemical Limitations III. Changes in Biochemical Versus Photochemical Efficiency IV. Effects of Temperature on Metabolism V. Effects of Temperature on Photosynthesis in Plants VI. Effects of Temperature on Photosynthesis VII. Effects of Temperature on Crassulacean Acid Metabolism VIII. Temperature Compensation in Photosynthetic Metabolism IX. Effects of Temperature on Carbon Partitioning to Starch and Sucrose X. Acclimation of Photosynthesis to Temperature Shifts References

vi

124 124 125 127 130 141 142 144 145 145

151–190

Summary I. Introduction II. Pathways and Metabolite Measurements: Evidence for Highly Coordinated Regulation of Many Reactions III. Regulatory Properties of Calvin Cycle Enzymes IV. Coarse Regulatability V. How can the Regulatory Capacity of a Protein be Evaluated? VI. Distribution of Control in Photosynthetic Carbon Metabolism Acknowledgments References

7

116 117 117

123–150

Summary I. Introduction II. Radicals and Dioxygen III. The Primary Target Molecules and Sites IV. Production of Reactive Oxygens and Radicals and their Scavenging Enzymes V. Microcompartmentation of the Scavenging Systems of Superoxide and Hydrogen Peroxide in Chloroplasts VI. Dioxygen Protects from Photoinhibition VII. Concluding Remarks Acknowledgments References

6

110 111

152 153 154 155 166 167 173 183 183

191–221 192 192 193 193 193 194 200 205 206 207 211 215

8

Gas Exchange: Models and Measurements John M. Cheeseman and Matej Lexa

223–240

Summary I. Introduction II. The Biochemical Model III. Beyond the Biochemical Model IV. The Feedback Loop: Consequences for Field Studies V. Conclusion Acknowledgments References

9

223 224 226 228 235 237 237 237

Stomata: Biophysical and Biochemical Aspects William H. Outlaw Jr., Shuqiu Zhang, Daniel R. C. Hite and Anne B. Thistle

241–259

Summary I. Introduction II. Plasmalemma Guard Cell Proton Pump III. Plasmalemma Potassium Channels IV. Plasmalemma Anion Channels V. Tonoplast Transport Processes VI. Abscisic Acid, Calcium, and the Phosphoinositide Messenger Systems VII. Integrating Role of Abscisic Acid in the Plant’s Physiology VIII. Carbon Metabolism IX. Concluding Remarks References

10 Source-Sink Relations: The Role of Sucrose C. J. Pollock and J. F. Farrar Summary I. Introduction II. Sucrose As a Regulator III. Changes in Source Leaf Metabolism IV. Sinks V. Potential Mechanisms of Gene Regulation by Sugars VI. Conclusion References

11 Developmental Constraints on Photosynthesis: Effects of Light and Nutrition John Richard Evans Summary I. Introduction II. Effects of Light III. Effects of Nutrition IV. Conclusions Acknowledgments References

241 242 242 244 245 246 247 249 249 253 253

261–279 262 262 263 266 271 274 275 276

281–304 281 282 283 295 299 300 300

vii

12 Molecular Biological Approaches to Environmental Effects on Photosynthesis Christine A. Raines and Julie C. Lloyd

305–319

Summary I. Introduction II. Genetics and Biogenesis of the Photosynthetic Apparatus III. Molecular Approaches to Environmental Stress IV. Environmental Stress in Photosynthetic Systems V. Conclusions References

305 306 307 308 315 317 317

13 Photosynthesis in Fluctuating Light Environments 321–346 Robert W. Pearcy, John P. Krall and Gretchen F. Sassenrath-Cole Summary I. Introduction II. The Nature of Sunfleck Light Regimes III. Factors Regulating the Photosynthetic Utilization of Sunflecks IV. Regulation of the Transient Responses to Individual Lightflecks V. Are There Specific Adaptations in Shade Leaves for the Utilization of Sunflecks? VI. Sunfleck Utilization in Natural Light Regimes Acknowledgments References

14 Leaf Photosynthesis Under Drought Stress Gabriel Cornic and Angelo Massacci Summary I. Introduction II. The Resistance of Photosynthetic Mechanisms to Drought Concentration Inside the Chloroplast During Drought is Low III. IV. Changes in Metabolic and Whole Leaf Photosynthetic Responses Induced by Water Deficits V. Maintenance of Plant Water Content During Soil Drying VI. Light Utilization by Plants Under Drought VII. Conclusions Acknowledgments References

15 Photosynthetic Adjustment to Temperature Stefan Falk, Denis P. Maxwell, David E. Laudenbach and Norman P. A. Huner Summary I. Introduction II. Short-Term Temperature Response of Photosynthesis III. Long-Term Temperature Response of Photosynthesis IV. Thylakoid Membrane Lipids V. Temperature and Chloroplast Development VI. Interaction of Light and Temperature VII. Photosynthetic Adaptation, Acclimation and Stress Acknowledgments References

viii

321 322 323 324 334 340 341 343 343

347–366 347 348 351 354 356 358 359 362 363 363

367–385 367 368 369 372 375 377 378 380 380 380

16 Photosynthetic Responses to Changing Atmospheric Carbon Dioxide Concentration George Bowes Summary in Perspective I. Rising in Plants II. Sites of Action of III. Adaptation to Changes in Atmospheric IV. Diversity in Photosynthetic Responses to V. Concluding Comments Acknowledgments References

387–407 387 388 389 390 393 402 402 402

Enrichment

17 The Modification of Photosynthetic Capacity Induced by Ozone Exposure Robert L. Heath

409–433

Summary I. Introduction and Background II. Model Studies III. Whole Plant Studies IV. Photosynthesis or Stomates? V. Conclusions References

18 Ultraviolet-B Radiation and Photosynthesis Alan H. Teramura and Lewis H. Ziska

409 410 414 418 420 429 429

435–450

Summary I. Introduction II. Penetration of UV-B Radiation III. Direct Effects of UV-B Radiation on the Light Reaction of Photosynthesis IV. Direct Effects of UV-B Radiation on Carbon Reduction V. Direct Effects of UV-B Radiation on Carbon Oxidation VI. UV-B Induced Changes in Leaf Development VII. Changes in Plant Growth and Development with UV-B Radiation VIII. Protection and Repair of Photosynthesis IX. Future Research Priorities Acknowledgments References

19 Evaluation and Integration of Environmental Stress Using Stable Isotopes H. Griffiths Summary I. Introduction II. Background to Stable Isotope Studies III. Applications of Stable Isotope Techniques IV. Future Potential Acknowledgments References Summary

ix

435 436 437 437 440 441 442 443 444 446 446 446

451–468 451 452 453 459 464 465 465 469

20 Environmental Constraints on Photosynthesis: An Overview of Some Future Prospects Neil R. Baker I. Introduction II. Light Energy Transduction by Thylakoids III. Carbon Metabolism IV. Leaf Gas Exchange V. Scaling from the Chloroplast and Leaf to the Canopy Acknowledgments References

Index

469–476 469 470 472 472 475 475 475

477

X

Preface Over the past decade there has been increasing concern about the potential future impact of global climate changes on crop production and the ability to feed an increasing world population. Accurate prediction of the effects of changing climatic variables on plant productivity will almost certainly be dependent upon the development of robust dynamic mechanistic models, which are built upon a sound understanding of the mechanisms by which environmental factors can influence photosynthetic processes. Although a detailed understanding of the molecular mechanisms and systems involved in photosynthesis has been achieved over the past decade, our knowledge of the intrinsic biological factors determining photosynthetic capacity and efficiency and how these factors can be modified by edaphic and climatic variables is not as well advanced. This is primarily due to the complexities of the dynamic interactions between components of the photosynthetic apparatus and the modifications of these interactions by extrinsic factors. Such complexities are only likely to be properly understood from integrated multidisciplinary studies involving analyses of structural, functional and developmental aspects of the photosynthetic systems. This volume was conceived with a view to providing an up to date reference text for advanced students and scientists who want to understand how photosynthetic performance may be influenced by environmental change. Consequently, the book contains contributions from authors drawn from a wide range of disciplines, all of whom have interests in the responses of the photosynthetic system to environmental challenges. The first part of the book examines structural and functional aspects of the photosynthetic apparatus in the context of responses to environmental stimuli and deals specifically with the processing of light energy by thylakoids, metabolic regulation, gas exchange and source-sink relations. Consideration is then given to development and genetic responses to environmental change. This is followed by a number of chapters which examine the effects of specific environmental variables (light, temperature, water,

concentration, ozone and UV-B) on photosynthetic performance and illustrate the complexities of the responses and need for multidisciplinary approaches. Recent developments in the methodology for studying photosynthetic performance of leaves have been important in advancing knowledge of the regulation of photosynthetic processes and the effects of the environment on photosynthesis. Details of new methods and their applications are presented at appropriate places throughout the book. It was with great sadness that I recently learnt of the death of Harold W. Woolhouse. Harold was a polymath with interests in a wide range of biological issues and who made many important contributions to plant biology, particularly in the area of environmental physiology. For many years prior to environmental biology becoming a fashionable subject Harold had held the view that an understanding of the responses of the photosynthetic apparatus to environmental factors would require an integration of knowledge across the biological disciplines. For as long as I can remember he had also always advocated vigorously the application of new and advanced biophysical and biochemical techniques to resolve problems in whole plant physiology. Many may consider that his major contribution to plant biology occurred towards the end of his illustrious career when he played a major role in encouraging and facilitating the use of molecular biological and molecular genetic approaches. There is no doubt that Harold had a great influence on the development of many plant biologists, not least myself. I would like to think that Harold would have supported strongly the philosophy underlying the production of this volume. I dedicate this volume to the memory of Harold, a very good friend and excellent colleague of many. The production of this volume has involved the efforts of a number of people who I would like to thank. Firstly, I thank the authors for their contributions and patience in dealing with editorial changes. Secondly, Larry Orr deserves special thanks for the production of the page layout of the book, which at times was less than straightforward; it was

xi

a pleasure to work with Larry and experience his friendly and efficient manner in dealing with problems. Finally, I must thank my wife, Maxine

Baker, for her patience during the preparation of the volume and her help with checking references and construction of the index. –Neil R. Baker

xii

Chapter 1 Processing of Excitation Energy by Antenna Pigments Thomas G. Owens Section of Plant Biology, Cornell University, Ithaca, NY 14853, USA

Summary I. Introduction II. Structure and Composition of Photosynthetic Antennae III. Role of the Antenna in Photosynthesis A. Light-Harvesting Function B. Protection Against Active Oxygen Species C. Regulation of Light Energy Utilization IV. Light-Harvesting Function of Antenna Pigments A. Energy Transfer And Excited State Dynamics B. Spectral Equilibration and the Transfer Equilibrium State V. Non-Photochemical Quenching and Regulation of Light Energy Utilization A. Components of Non-photochemical Quenching B. and the Role of C. Proposed Mechanisms for Quenching D. Reaction Center Versus Antenna Quenching Sites E. Role of Transfer Equilibrium in q E Quenching F. Light State Transitions and Regulation in PS I VI. Concluding Remarks Acknowledgments References

1 2 4 5 5 6 7 9 9 10 12 12 14 14 18 18 20 21 21

21

Summary Absorption and transduction of light by photosynthetic organisms provides the principal energy source for all living organisms. At the same time, absorption of excess light (light in excess of the capacity of the organism to use the energy to drive photosynthesis) represents a primary site of environmental injury. Recent studies have shown that photosynthetic organisms have the ability to regulate the utilization of absorbed light energy through a group of related processes commonly called non-photochemical quenching. These process dissipate excess absorbed energy as heat. In order to remain competitive, photosynthetic organisms must seek out the delicate balance between efficient light-harvesting under limiting light conditions and regulated dissipation of energy under excess light conditions. Excess light absorption may occur as the result of increased incident intensity or a decrease in the rate of photosynthesis due to other environmental stresses. The underlying reactions of nonphotochemical quenching may occur in the antennae, the reaction centers, or both, and are not well understood. Independent of the quenching site, the reactions of non-photochemical quenching must cooperate and compete with those of normal light-harvesting. Here, the proposed mechanisms of non-photochemical quenching and the common energy transfer reactions affecting both light-harvesting and non-photochemical quenching are examined in order to provide a more general framework in which the utilization of light energy can be described. Neil R. Baker (ed): Photosynthesis and the Environment, pp. 1–23. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

2 I. Introduction The process of photosynthesis involves the coordination of a large number of reactions which are separated both spatially and temporally. These reactions begin in the thylakoid membrane with the processes of light absorption by antenna pigments and primary photochemistry by the reaction center pigments, and terminate in the partitioning of photosynthate in the cytoplasm with the ultimate distribution of fixed carbon to the various metabolic sinks of the organism. Essentially photosynthesis can be viewed as a network of tightly interconnected reactions which cooperate in energy transduction, carbon fixation, and allocation and distribution of ATP, reducing potential and fixed carbon (Fig. 1). In addition, numerous other physiological processes, such as inorganic nitrogen and sulfur assimilation, transpiration and water potential, and respiration indirectly impact on photosynthesis via metabolic coupling of common intermediates and transport phenomena. Although it is traditional to study each of these component reactions independently of the others, it is essential to recognize that in living organisms, coupling among these reactions often dictates the complex responses of plants to variations in environmental parameters. Absorption of light energy and conservation of this energy in the form of Chl excited states represents the starting point for the reactions of photosynthesis. Conversion of the excited state energy into chemical redox energy by the reaction centers provides the driving force for photosynthetic electron transport, the production of ATP and NADPH, and the ultimate utilization of these products in inorganic carbon fixation and other anabolic reactions. At the same time, absorption of light energy represents one of the primary sites at which environmentally induced damage occurs in plants (Krause, 1988; Long et al., Abbreviations: Chl – chlorophyll; CPxx – chlorophyll-protein complex with apparent molecular weight of xx kDa; DCCD – dicyclohexylcarbodiimide; –maximum fluorescence level; – initial fluorescence level; – variable fluorescence, – rate constant for photochemistry; LHC I, LHCII – lightharvesting complexes of PS I and PS II; PQ – plastoquinone; – primary quinone acceptor of PS II; – secondary quinone acceptor of Photosystem II; – energy-dependent component of non-photochemical quenching; – photoinhibition-related component of non-photochemical quenching; – nonphotochemical quenching; – photochemical quenching; – state transition-related component of non-photochemical quenching; TE – transfer equilibrium

Thomas G. Owens 1994). Absorbed energy which is in excess of the capacity of the organism to utilize the energy in photosynthesis leads to the formation of reactive oxygen species (singlet superoxide and associated free radicals; Chapter 5) and damage to the PS II reaction center complex (Krause, 1988; Chapter 4). Excess light absorption may result from an increase in ambient light intensity, but is more frequently the consequence of a decrease in the capacity for photosynthesis due to environmental stress (Long et al., 1994). In a sense, dealing with excess absorbed light energy can be seen as a common thread which links the physiological responses of plants to changes in a number of different environmental conditions, even when environmental perturbation does not directly involve an increase in ambient light. Beginning with the experiments of Emerson and Arnold (1932a,b), which demonstrated the cooperation of hundreds of pigments in the evolution of oxygen in photosynthesis, the function of antenna pigments in the light-harvesting processes of photosynthesis has been widely studied (van Grondelle et al., 1994). Until recently, our understanding of how plants deal with excess light absorption has concentrated on processes outside the antennae. Photosynthetic organisms have the ability to deal indirectly with the consequences of excess light absorption by limiting singlet accumulation through quenching of singlet by carotenoids and through quenching of triplet Chl in the antenna (triplet Chl is the sensitizer of singlet formation; Cogdell and Frank, 1987), by removing superoxide and the resulting free radicals through the action of superoxide dismutase and a variety of free radical scavengers (Pell and Steffen, 1991; Chapter 5), and by repair of damaged PS II through synthesis and insertion of new polypeptides (Melis, 1991; Chapter 4). Each of these processes involves an interaction with the products of excess light absorption rather than limiting the formation of these products. There is increasing evidence that the antenna pigments also serve a photoprotective function in photosynthesis, dealing directly with excess absorbed light energy by dissipating excess energy in the antenna as heat in competition with normal photochemistry in the reaction center, as well as the detrimental processes of singlet and superoxide formation, and damage to the PS II reaction center (Demmig-Adams, 1990; Horton and Ruban, 1992). Thus, antenna pigments and their associated pigment-

Chapter 1

Processing of Excitation Energy

3

4 protein complexes play essential roles in both lightharvesting and photoprotection by regulating the absorption and utilization of light energy in photosynthesis. Their function is to seek the delicate balance between optimal light-harvesting efficiency under light-limiting conditions and precise energy dissipation under excess light conditions. Considering the natural variability of light and other environmental factors, regulation of light energy utilization between these two extremes must be tightly controlled in order to maintain efficient and competitive growth under variable conditions. At the molecular level, any attempt to draw relationships between structure and function in the antenna must recognize that the transition between the two extremes is rapid (minutes or less) and appears to require only small changes in the structure and composition of the antenna. From the perspective of evolution, there must have been strong selection for organisms that could minimize light-induced damage while at the same time maximizing photosynthetic efficiency. Understanding the efficiency of photosynthesis under both optimal and stress conditions thus requires a description of the common features of light-harvesting and photoprotective processes in the antenna, and of the processes which control the utilization of absorbed light energy in photosynthesis.

II. Structure and Composition of Photosynthetic Antennae With only a few minor exceptions, all functional pigments in photosynthetic membranes are thought to be specifically bound in a variety of pigmentprotein complexes. This binding environment alters the in vitro properties of the pigments and provides new pathways for decay of the excited state which are essential to determining and distinguishing the in vivo functions of the pigments. For example, the observation of excited state energy transfer or photochemical charge separation between pigments in a dilute solution of Chl a is rare because both of these processes require a specific spatial arrangement among pairs or larger aggregates of pigments within the lifetime of the excited state. The binding environment in pigment-protein complexes provides these spatial requirements by fixing the pigments at specific intermolecular distances and orientations which optimize either energy or electron transfer. In addition, pigment-pigment and pigment-protein

Thomas G. Owens interactions within the protein complexes modify some intrinsic properties of the pigments, particularly the excited state energy levels (absorption maxima) and redox properties, leading to further specialization of function. In general, photosynthetic pigments and pigmentprotein complexes serve one of two primary functions: reaction centers or antennae. There is good evidence that the PS I and PS II reaction center complexes are highly conserved in structure, composition and function among all oxygenic photosynthetic organisms (Nitschke and Rutherford, 1991). In contrast, there is tremendous diversity in the antenna pigments and pigment-protein complexes, especially among the algal classes (Owens, 1988). The antenna systems of most photosynthetic organisms are heterogeneous with respect to pigment and protein composition, structure and function. In higher plants and all algae examined to date, PS I and PS II have unique antenna systems that differ in both pigment and protein composition. The antennae of both photosystems can be divided into two general groups: the core and peripheral antenna complexes. In the core antenna complexes, the pigments occur in a fixed stoichiometry with respect to the reaction center pigment, and the pigment and protein components are largely conserved among all oxygenic photosynthetic organisms (Bassi et al., 1990). In PS I, the core antenna consists of approximately 100– 120 Chl a and 15 carotene molecules bound in the same complex that contains the P700 reaction center. The PS II core antenna is composed of two pigmentprotein complexes, CP43 and CP47, which are separate from the PS II reaction center complex. Each binds 20–25 Chl a and several carotene molecules (Bassi et al., 1990). The spectral compositions of the PS II and PS I core antennae also differ. The PS II core Chl a molecules have their main red absorptions between 678–680 nm range while the PS I core Chl a population spans the region from 664 to 705 nm with the bulk of the absorption at wavelengths >680 nm. The diversity among antenna systems occurs in the peripheral antenna complexes. The peripheral antenna complexes are the variable component of the antenna; the size and composition of the peripheral antenna can be adjusted in response to environmental conditions. Such changes represent long-term (days) adaptation to average conditions rather than shortterm (minutes-hours) responses to changing conditions (Chapters 3 and 11). In higher plants and

Chapter 1

Processing of Excitation Energy

green algae, the peripheral antennae of PS I and PS II are composed of two classes of Chl a/b-binding proteins, LHC I and LHC II respectively. Under any given growth conditions, the peripheral antennae are composed of several different polypeptides which represent a subset of their respective gene families. Altered growth conditions often induce a change in the subsets of apoproteins that are expressed (Laroche et al, 1991; Levy et al., 1993). Presumably, each apoprotein provides different pigment-binding environments, altering the type and number of pigments and their in vivo properties in order to optimize growth under a range of physiological conditions. The unique properties of the individual peripheral antenna proteins can lead to heterogeneous function, particularly in PS II. The main LHC II complexes fall into two groups: those that are tightly associated with PS II and those that can reversibly dissociate from PS II upon phosphorylation (Bassi et al., 1990). In addition, PS II contains three minor peripheral antenna complexes, CP29, CP26 and CP24, which are thought to provide specific connections between the main LHC II antenna and the PS II core (Bassi and Dainese, 1992). Heterogeneity within the peripheral antennae is likely to extend to processes other than light-harvesting , with variations in the pigment and protein composition of the antenna being one way in which photosynthesis can be regulated. This heterogeneity also extends to the structure and composition of the thylakoid membrane. In higher plants and green algae, thylakoids are divided into regions of stacked vesicles (grana) interconnected by single vesicular regions (the stroma lamellae). From both a structural and functional perspective, the appropriate distinction is between the stromaexposed thylakoid regions versus appressed regions where two thylakoid membranes are in direct contact (Anderson and Andersson, 1988). Associated with these different membrane regions is a lateral heterogeneity in the distribution of thylakoid membrane proteins (Anderson and Andersson, 1988). PS I appears to be almost completely restricted to the stroma-exposed regions while PS II is largely found in the appressed regions. Although theoretical and experimental studies of thylakoid protein interactions have provided plausible explanations for the structural aspects of thylakoid stacking and lateral heterogeneity, the overall function of these features in photosynthesis remains controversial (Anderson and Andersson,

5 1988). Of particular interest is the role of heterogeneity among PS II units heterogeneity; Melis, 1991). Functional PS II units are located in the appressed regions of the thylakoids and participate in linear electron transport from water to In contrast, units have a reduced antenna size, are incapable of electron transport beyond and are located in the stroma–exposed thylakoid region. These units may be intermediates in the repair of reaction centers damaged by photoinhibition (Melis, 1991; Chapter 4). Evolution has determined the composition and structure of pigment-protein complexes in order to facilitate excited state energy transfer and photochemical reactions and optimize the yield of photosynthesis. These same types of reactions are likely to be involved in regulating the utilization of absorbed light energy under excess light conditions, a process that is equally essential to the survival of photosynthetic organisms. Thus both the lightharvesting and photoprotective function of antenna pigments probably arise from a common structural origin. However, the properties of the pigments, both intrinsic or as modified by the protein-binding environment, must be unique in order to distinguish the light-harvesting and photoprotective functions. In the end, an understanding of the regulation of light energy utilization in photosynthesis requires a description of the function of the antenna pigments at both the physiological and molecular levels.

III. Role of the Antenna in Photosynthesis

A. Light-Harvesting Function All photosynthetic pigments, including the reaction center pigments, are capable of direct absorption of sunlight. One might reasonably ask why photosynthetic organisms typically have several hundred antenna pigments per reaction center. The ability of a pigment to absorb light is determined by its wavelength-dependent optical absorption crosssection. This is a quantum mechanical property of the molecule which can be thought of as the effective cross-sectional area that a pigment presents for absorption of light at a specific wavelength. The cross-section for absorption of visible light by Chl a, weighted by the solar emission spectrum and averaged over visible wavelengths (400–700 nm), is about 0.67 which means that at peak physiological light

6 intensity (~2000 photons the rate of light absorption per Chl a is about 8 photons per second. Thus, for more typical light intensities, the rate of light absorption by a reaction center pigment alone is far below the capacity for photosynthetic electron transport and would not provide sufficient energy to drive autotrophic metabolism. Evolution has overcome this limitation by coupling the absorption of hundreds of antenna pigments to each reaction center. The nature of this coupling is such that an excited state, which is formed by light absorption by any arbitrary antenna pigment, will be transferred to the reaction center with high efficiency. For PS II in higher plants and green algae, the maximum efficiency of photochemistry (including energy transfer in the antenna) is about 85%; in PS I the maximum photochemical efficiency is >95% (van Grondelle et al., 1994). The coupling of many antenna pigments to the reaction center increases the effective cross-section of the reaction center in two ways. First, it increases the total number of pigments whose cross-sections contribute to absorption. Typical antenna sizes are on the order of 200–300 Chls per reaction center, which for an average light intensity of photons makes the rate of light absorption per reaction center commensurate with the capacity for electron transport. The incorporation of accessory pigments such as Chl b and carotenoids, whose peak absorptions occur at wavelengths where Chl a absorption is weak, also broadens the spectral range over which light absorption can occur.

B. Protection Against Active Oxygen Species The excited singlet state of Chl a which forms upon absorption of light is unstable and will decay back to the lowest energy (ground) state by one of several competing processes. For Chl a molecules in a dilute solution, the lifetime of the excited state is about 5 ns and three intrinsic processes contribute to the decay of the excited state. These are intersystem crossing to the triplet state, radiative decay (fluorescence), and thermal emission (heat), accounting for about 65%, 30% and 5% of the in vitro decay, respectively (Fig. 2). When the same Chl a is incorporated into the photosynthetic apparatus, energy transfer to the reaction center and its ultimate utilization in photochemistry accounts for >85% of the decay of antenna excited states under optimal conditions. Binding of the pigment in a protein environment

Thomas G. Owens does not significantly alter the intrinsic properties that govern triplet formation, fluorescence and thermal emission. Thus, the decay of the remaining excited states not used in photochemistry is partitioned among these three processes in proportions similar to those measured in vitro. In PS II under optimal conditions, these remaining losses are approximately 10% in triplet formation, 4.5% in fluorescence and 0.5% in heat. These losses are an unavoidable consequence of the molecular properties of Chl a and the construction of photosynthetic antennae. However, these losses provide powerful links to the competition for absorbed light energy between photochemistry and other processes can be monitored by in vivo Chl fluorescence (Krause and Weis, 1991; Chapter 2) or thermal emission (Malkin and Cananni, 1994; Chapter 2). The triplet state of Chl a, like many porphyrins, can sensitize the formation of an excited singlet state of oxygen, via triplet energy transfer:

The singlet produced is highly reactive and can oxidize many important biological molecules, particularly lipids. Without some mechanism of dissipating triplet Chl states in the antenna, photosynthetic organisms even under optimal conditions would be faced with continuous and significant production of singlet Note that triplet Chl can also be formed by charge recombination in the reaction centers. However, under physiological conditions, the yield of triplet Chl in the reaction center is insignificant compared to that produced in the antenna. In all oxygenic photosynthetic organisms, this problem is overcome by the presence, in the antenna complexes, of carotenoids which rapidly quench Chl triplet states (Cogdell and Frank, 1987). The quenching reaction involves the triplet energy transfer from Chl a to the carotenoid followed by nondestructive thermal dissipation of the triplet energy on the carotenoid:

In addition, carotenoids can quench singlet directly through a similar triplet transfer reaction:

Chapter 1 Processing of Excitation Energy

7

Thus, the essential role of carotenoids in photosynthetic antennae is that of photoprotection by preventing the formation and accumulation of singlet This accounts for the inability of carotenoidless plants to grow in anything but minimal light conditions.

C. Regulation of Light Energy Utilization The rate of photosynthesis, like all enzymatically mediated reactions, exhibits a saturation phenomenon with respect to its principle substrate, light (Fig. 3). At limiting light intensities, the rate of photosynthesis is linear with the incident light intensity and unless other external factors (e.g. temperature or water stress) are affecting photosynthesis, the optimal light utilization described in the previous section is achieved. As light intensity increases, the rate of photosynthesis increases non-linearly and eventually becomes independent of light intensity. Under optimal growth conditions, saturation of photosynthesis is thought to result from a limitation in the capacity of the dark reactions and not in photosynthetic electron transport (Foyer et al., 1990). Over the same physiological range of light intensity, the rate of photon absorption remains linear with light intensity. As a consequence, exposure of plants to intensities

which approach or exceed saturation results in the absorption of light energy in excess of the capacity of photosynthesis to utilize the energy for fixation and other anabolic reactions (Fig. 3). It is also critical to recognize that an increase in light intensity is not the only environmental change

8 that can lead to excess light absorption. Any perturbation that depresses the rate of photosynthesis at constant light intensity will also cause an increase in excess light absorption (Fig. 3). For example, water stress frequently results in partial or complete closing of stomates, with consequent reductions in leaf internal and the rate of photosynthesis (see Chapters 9 and 14). The natural variation in light, temperature, water availability and other factors in most environments suggests that excess light absorption is a problem that is frequently encountered by plants in the field (Long et al, 1994). Understanding the consequences of excess light absorption requires consideration of the coupling between the light and dark reactions of photosynthesis. For any environmental factor (e.g. light or temperature) which results in a limitation of photosynthesis in the dark reactions, there is an inhibition of electron transport due to slower regeneration of ADP or inorganic phosphate (Fig. 1). This leads to a reduction of electron carriers on the acceptor sides of both PS I and PS II, closing of PS II reaction centers and increased acidification of the thylakoid lumen. The decrease in PS II photochemistry due to closing of the traps results in higher yields of fluorescence and triplet formation. If the rate of triplet formation exceeds the capacity of carotenoids to quench triplets, net production of singlet results with associated oxidative damage. At the same time, limited availability of allows molecular to compete with ferredoxin as an acceptor from PS I, resulting in the production of superoxide and other free radicals which also cause oxidative damage (Pell and Steffen, 1991; Chapter 5). Finally, extensive reduction of PS II acceptors is one of the major causes of photoinhibitory damage to the PS II reaction center complex (Krause, 1988; Chapter 4). Chloroplasts contain metabolic machinery to deal with the consequences of excess light absorption by destruction of singlet and free radicals (Pell and Steffen, 1991; Chapter 5) and by a complex process for repair of damaged PS II reaction centers (Melis, 1991; Chapter 4). However, these processes do not deal directly with excess absorbed light energy, only with its consequences. Recently, a more general regulatory process has been described in which excess absorbed light energy is directly dissipated as heat in competition with reactions that cause light-induced damage. This process of non-photochemical quenching of Chl excited states competes with

Thomas G. Owens photochemical quenching in the reaction center and other intrinsic processes by creating a new pathway for decay of excited states (Fig. 2). In order for an organism to maintain optimal efficiency under a variety of growth conditions, the process of nonphotochemical quenching must allow for maximal light energy utilization under limiting light conditions and regulated quenching under a range of excess light conditions. That is, the processes that regulate must be able to quantitatively sense the imbalance between the rate of light absorption and the capacity for light utilization in photosynthesis and adjust to dissipate only the excess fraction of total absorbed light. Responses of photosynthetic organisms to changes in excess light conditions due to environmental fluctuations occur on a continuum of time scales from less than a second (leaf flutter or focusing by surface waves) to seasonal changes (see also Chapter 13). However, two general time scales can be identified based on the biological level at which the primary response occurs: times which are shorter than that required to trigger the synthesis of new proteins (seconds to tens of minutes), and those which permit the synthesis of new proteins (>30–60 minutes). In addition, adaptation on the time scale of evolution is also important, because it has resulted in the diversification of antenna systems, especially among the algae (Owens, 1988). Although the processes of photoprotection and regulation of light energy utilization have not been extensively studied among the algal classes, the fact that light-harvesting and photoprotective processes are shared functions of the antenna suggests that a similar diversity of these functions can be expected among the algae (Ting and Owens, 1994). Among higher plants, which share a common family of antenna pigment-protein complexes, there remains a (smaller) diversity within the gene family encoding antenna proteins which may contribute to the dominance of certain species under differing environmental conditions (e.g. sun versus shade species) through regulation of gene expression. This is particularly evident in green algal antenna systems where stable changes in growth conditions on time scales of hours to days trigger the synthesis of different components of the antenna protein family (Laroche et al., 1991; Levy et al., 1993). Such changes are likely to constitutively optimize light-harvesting efficiency under low light conditions or light energy dissipation under chronic environmental stress. On

Chapter 1

Processing of Excitation Energy

shorter time scales, the regulatory processes of nonphotochemical quenching and light state transitions are of primary importance. In the face of rapid (seconds to minutes) variations in light intensity and slower (hours) changes in other environmental conditions, these processes represent the first line of defense against the consequences of excess light absorption. Because improvements in the ability to balance efficient light-harvesting and dissipation of excess absorbed energy should be important on an evolutionary time scale, it is possible that there may be multiple mechanisms that contribute to regulating the utilization of absorbed light energy. The overlapping functions of photosynthetic antennae including light-harvesting, protection against active oxygen species, and regulation of light energy utilization are each dependent upon excited state energy transfer among the antenna pigments. The dynamics of energy transfer are, in turn, determined by structure and composition of the antenna. Once an excited state is formed by light absorption, there is a competition between photochemistry in the reaction center and non-photochemical processes for the excited state energy. In order to optimize light-harvesting efficiency while at the same time minimizing damage induced by excess light absorption, photosynthetic organisms must regulate the competition between energy transfer to functional reaction centers (photochemical quenching) and dissipation of excess light energy (nonphotochemical quenching). Thus, the regulation of light energy utilization in photosynthesis depends to a large extent on the common energy transfer features of these two processes.

IV. Light-Harvesting Function of Antenna Pigments

A. Energy Transfer And Excited State Dynamics The light-harvesting function of photosynthetic antenna systems requires the cooperation of hundreds of pigments in light absorption and efficient transfer of the resulting excited state energy to the reaction center. An essential feature of an efficient lightharvesting system is rapid energy transfer among all coupled pigments in the photosynthetic unit. This requirement is a consequence of the fact that the ‘internal’ decay processes of triplet formation, fluorescence and thermal emission on each pigment

9 can lead to wasteful loss of the excited state in the antenna if any step of energy transfer to the reaction center is slow. The physical mechanism which dictates the rate of pairwise energy transfer among most pigments relies on a resonance coupling between the dipole transitions of the donor and acceptor pigments. Strong coupling (exciton states) and electron exchange interactions also contribute to energy transfer but are thought to be spatially and temporally localized (van Grondelle et al., 1994). Thus, the network of excited state transfers can be adequately modeled as a sequence of pairwise transfers in which the excited state hops between adjacent antenna pigments until its energy is utilized in the reaction center or lost via one of the many competing processes in the antenna. There are several lines of evidence which suggest that in all antenna systems pairwise transfer rates between nearest neighbor pigments are extremely rapid, occurring on time scales of a few picoseconds to a few hundred femtoseconds (van Grondelle et al., 1994). In the two higher plant pigment-protein complexes whose structures are known to near-atomic resolution (LHC II and the PS I reaction center/core antenna complex), the mean spacing between pigments is in the range of 0.7 to 1.4 nm (Kühlbrandt and Wang, 1991; Krauss et al., 1993). With reasonable spectral overlap and pigment orientation, these distances translate into pairwise transfer times of a picosecond or less. At the same time, there are substantial homologies among antenna complexes with respect to pigment composition, protein sequence (Zuber, 1985) and predicted pigmentbinding volumes (Laible et al., 1994), indicating that close packing of pigments is a common feature of most antenna complexes. In addition, direct experimental evidence of sub-picosecond transfer has been obtained for a variety of pigments including carotenoid to Chl a (Shreve et al., 1992), Chl b to Chl a (Eads et al., 1989) and Chl a to Chl a (Du et al., 1993). In all photosynthetic organisms, photochemical quenching by the reaction center is the primary process that limits the lifetime of the excited state. However, the dynamics of excited state motion among the antenna and reaction center pigments prior to photochemical quenching is the major factor which determines the efficiency of the antenna. Except in the algal classes which have phycobilin-based peripheral antennae, these dynamics are dominated by pairwise transfers among neighboring Chl a

10 pigments. Energy transfer from accessory pigments (Chls b, c and carotenoids) to Chl a in the peripheral antenna complexes is rapid and only weakly reversible because poor spectral overlap limits the rate of back transfer. Nearly all subsequent transfer steps occur among Chl a pigments in the peripheral and core antenna complexes. Once the excited state reaches the reaction center, its fate is determined by the competition between photochemical quenching (stable charge separation) and back transfer of the excited state from the reaction center to the antenna (detrapping). This competition also has a major influence on the overall dynamics of excited state motion in the antenna. Consider two limiting cases. In the diffusion-limited case, the rate of photochemical quenching on the reaction center greatly exceeds the rate of detrapping. Here, the excited state is quenched as soon as it reaches the reaction center and its lifetime is limited by the time required for the excited state to diffuse through the antenna to the reaction center. In the other extreme, the rate of detrapping exceeds the rate of photochemical quenching on the reaction center. In this case, the excited state makes many visits to the reaction center before photochemistry occurs. The system is said to be trap-limited because the lifetime of the excited state is limited by the ability of the reaction center to ‘trap’ or capture the excited state from the antenna. In principle, any intermediate description between these two extremes is also possible.

B. Spectral Equilibration and theTransfer Equilibrium State At first glance, it might appear that a trap-limited system would be less efficient than a diffusionlimited system because back transfer to the antenna would allow greater losses in the antenna through fluorescence, triplet formation and thermal emission. However, this need not be the case because in both limits the overall photochemical efficiency is determined by a balance of antenna size, average transfer rates, and the competition between photochemistry and detrapping at the reaction center. In fact, there is strong experimental and theoretical evidence that both PS I and PS II are trap-limited despite their high photochemical efficiencies (Holzwarth, 1991; Laible et al, 1994; van Grondelle et al., 1994). An important consequence of this traplimited description of excited state dynamics is that,

Thomas G. Owens on average, the excited state distribution among antenna spectral forms approaches that predicted by the Boltzmann distribution on time scales which are short compared to the photochemically-limited lifetime (Jennings et al., 1993). This equilibration of the excited state has been observed experimentally in both PS I (Owens et al., 1988) and PS II (Schatz et al., 1988 ). Simulations of excited state dynamics in models of PS I (Jean et al., 1989) and PS II (Beauregard et al., 1991) also indicate the rapid formation of equilibrium-like excited state distributions. These studies concluded that high photochemical efficiency and rapid spectral equilibration among the antenna spectral forms depend on (i) rapid single step transfer times among all antenna pigments, and (ii) significant detrapping of the excited state from the reaction center consistent with the trap-limited description of excited state dynamics (Holzwarth, 1991; Laible et al., 1994). The spectrally equilibrated distribution of the excited state in trap-limited systems does not represent a true equilibrium condition because there is a continuous loss of the excited state via photochemistry on the reaction center. Laible et al. (1994) have recently characterized this condition as a transfer equilibrium (TE) state in which the fractional distribution of the excited state on any pigment i, where

remains constant with time. Here, is the time dependent distribution on pigment i and the sum in the denominator is over all N coupled pigments in the antenna. Recognizing that the energy transfer processes that partition the excited state among the coupled antenna pigments depend on the energies of the antenna pigments (through the requirement for overlap of donor emission with absorption of the acceptor; the Förster overlap integral), the equilibrium distribution among the antenna pigments in the absence of all decay processes is precisely the Boltzmann distribution for the system. However, photochemical quenching on the reaction center perturbs the TE distribution from that predicted by the Boltzmann distribution, with the resulting TE distribution being smaller than the Boltzmann value on the reaction center and its neighboring pigments

Chapter 1

Processing of Excitation Energy

and larger on all other antenna pigments (Laible et al., 1994). The greater the spectral diversity of the antenna, the larger the deviations of the TE distribution from the Boltzmann distribution. The occurrence of TE states in photosynthetic systems indicates that the overall dynamics of excited state motion and decay in the Chl-based antennae of PS I and PS II can be attributed to two principal kinetic phases (Fig. 4). In the first equilibration phase, following the initial absorption of light by an antenna pigment (which depends on the wavelength distribution of incident light), there is a redistribution of the excited state among the antenna pigments through rapid energy transfer and detrapping. After a short time, this redistribution has relaxed to the TE state. Experimentally, the equilibration phase results in short lifetime decay components with both positive and negative amplitudes, indicative of transfer between groups of spectrally distinct antenna pigments. In the second photochemical phase of the decay, photochemical quenching on the reaction

11

center depletes the TE state in such a manner that the fractional distribution of the excited state remains constant for the remainder of the decay (Fig. 4). Because the TE state dominates the excited state decay in PS I and PS II, the actual distribution of the excited state among the reaction center and antenna spectral forms is central to determining many important features of antenna function. In particular, the restriction of photochemistry to the reaction center means that the fractional distribution on the reaction center will determine the excited state lifetime and thus photochemical efficiency under optimal conditions. The excited state lifetime is given by

where is the fractional distribution of the excited state on the reaction center at TE, and is the rate constant for photochemistry.

12 The occurrence of TE states in antenna systems has important implications for other photosynthetic phenomena as well. Throughout its lifetime, the excited state spends most of its time among the antenna pigments. During this time, the ‘internal’ decay processes of triplet formation, fluorescence and thermal emission on each antenna pigment are competing with energy transfer and photochemical quenching on the reaction center. Thus, most of the losses which occur under optimal conditions are a fundamental consequence of antenna composition and structure. The fluorescence level (the initial fluorescence level measured under conditions of maximum photochemical efficiency and a parameter which is widely used to evaluate photosynthetic physiology; Krause and Weis, 1991) is largely a consequence of the TE state. Most importantly, any process in the antenna or reaction center which regulates the utilization of absorbed light energy must directly compete with the rapid energy transfer reactions that lead to the formation of the TE state. At the same time, because this regulatory process is likely to be restricted to specific pigments, the yield of the process will be determined in part by the TE distribution at the quenching site. V. Non-Photochemical Quenching and Regulation of Light Energy Utilization Regulation of light energy utilization in photosynthesis is accomplished by a class of reactions that are collectively called non-photochemical quenching, or These reactions dissipate absorbed light energy by competing with photochemical quenching in the reaction centers for Chl excited states in the antenna (Fig. 2). The distinction between and is not strictly defined by either the site at which the quenching occurs (reaction center versus antenna pigments) or by the participation of photochemical charge separation, because at least two components of may utilize some form of charge separation in the PS II reaction center as a part of the quenching mechanism. Rather, the distinction should be made at the level of whether or not the quenching reaction leads to stable storage of the excited state energy in biological oxidants and reductants or dissipation of the energy as heat The phenomenology of and is closely tied with measurement of Chl fluorescence yield because the reactions of and compete with the‘internal’

Thomas G. Owens decay processes of fluorescence, thermal emission and triplet formation. The identification and quantitation of and is most easily accomplished by measurements of room temperature fluorescence emission (Schreiber et al., 1986; Krause and Weis, 1991). As a result, is commonly referred to as ‘non-photochemical quenching of Chl fluorescence’. This term is misleading because the organism is not regulating fluorescence emission, rather it is regulating the availability of excited states for photosynthesis by introducing new processes which compete with all other processes, including photochemistry and fluorescence, for excited states (Fig. 2). In addition, because room temperature fluorescence emission is dominated by PS II antenna pigments, the phenomenology of and is largely restricted to processes in PS II. It is now well established that the competition between fluorescence and photochemistry in PS II is the origin of variable fluorescence and (Krause and Weis, 1991; Govindjee, 1995). Fluorescence quenching that is independent of photochemistry was first reported by Murata and Sugahara (1969). Subsequent work by Wraight and Crofts (1970) demonstrated that this quenching depended on the extent of the pH gradient across the thylakoid membrane. Bradbury and Baker (1981) and Krause et al. (1982) introduced the first techniques to separate and quantify and Most recently, the availability of commercial modulated Chl fluorometers (Schreiber et al., 1986; Bolhar-Nordenkampf et al., 1989) has greatly simplified the measurement of and and their correlation with other physiological phenomena. Using these techniques, the complexity of processes that contribute to is slowly being revealed (Chapter 2). These processes range between those that serve to protect the photosynthetic apparatus against the effects of excess light (Chapters 2 and 3) to those that are consequences of damage induced by excess light (Chapter 4). The common feature of these processes is that they represent new pathways for decay of Chl excited states that compete with fluorescence and with stable charge separation in the PS II reaction center (Fig. 2).

A. Components of Non-photochemical Quenching The results of numerous studies indicate that the physiological processes that contribute to total nonphotochemical quenching are both complex and

Chapter 1

Processing of Excitation Energy

heterogeneous. In general, these processes fall into three categories: (i) energy-dependent quenching which is regulated to a large extent by the pH of the thylakoid lumen (Demmig-Adams, 1990; Horton et al., 1994), (ii) photoinhibitory quenching which is related to the slowly reversible, light-dependent depression in the light-saturated rate of photosynthesis (Krause, 1988; Ruban and Horton, 1995), and (iii) light-state transitions which quench PS II fluorescence by physically altering the antenna size of PS II units and/or spillover of energy to PS I (Williams and Allen, 1987; McCormac et al., 1994). While each of these categories contain processes that contribute to protection of the photosynthetic apparatus against the effects of excess light, also contains contributions from the damage to PS II that results from excess light. Under normal physiological growth conditions, is thought to be the major component of total and is thus the dominant process regulating light energy utilization in PS II (Horton et al., 1994). The term energy-dependent quenching arises from the observation that the extent of is regulated by the size of the across the thylakoid membrane; that is, by the extent that the membrane is energized for ATP synthesis. Increases in correlate with decreases in the quantum yield of photochemistry in open PS II reaction centers (Weis and Berry, 1987) clearly indicating its primary role in regulating the utilization of absorbed light energy. The amount of increases most rapidly when the rate of light absorption exceeds the capacity of the dark reactions to use the products of electron transport either at saturating light or when environmental stress has depressed the capacity of reactions that limit the rate of photosynthesis. As a result, has been widely used as an indicator of the responses of plants to environmental stress. Simultaneous measurements of and may be sufficient to estimate the rate of linear electron transport (Weis and Berry, 1987) although it is important to note that the relationship between and electron transport is empirical and will likely be dependent on the species and previous growth conditions. Under more severe stress (excess light) conditions, the photoinhibitory component of may become dominant due to increases in both the range of photoprotective processes that the plant calls upon to deal with excess light and to the accumulation of damaged PS II reaction center complexes resulting from excess light absorption. The photoprotective

13 component of like serves to reduce excess light-induced damage in PS II by dissipating excess absorbed light energy in competition with photochemistry and fluorescence in PS II. Unlike does not readily relax in the dark, nor is it sensitive to the action of uncouplers (Ruban and Horton, 1995). Thus, although the two quenching components may utilize common constituents of the thylakoid membrane, the regulation of the underlying processes must be distinct. The photodamage component of is the result of the well characterized loss of variable fluorescence that occurs upon photoinhibitory damage to the PS II reaction center complex (Krause, 1988). The damaged reaction center remains an efficient quencher of excited states from the PS II antenna, accounting for the loss of variable fluorescence and the observation that the reaction center appears to be ‘stuck’ at The mechanism of this quenching is poorly understood, however it does not result in stable charge separation at PS II reaction centers. Distinguishing between the protective and damage-related components of is essential because the principal role of the protective component is to limit the extent of damage to PS II (Ting and Owens, 1994). Light state transitions were originally observed in algae exposed to light that was preferentially absorbed by PS I (State I) or PS II (State II). More recently, state transitions in higher plants and green algae have been attributed to a phosphorylation-induced redistribution of a portion of the LHC II antenna between the appressed and stroma-exposed regions of the thylakoid membrane (Williams and Allen, 1987). The extent of LHC II phosphorylation is under control of the redox state of the plastoquinone pool such that overexcitation of PS II leads to a reduction of the plastoquinone pool, phosphorylation of LHC II and subsequent movement of the phosphoLHC II to the stroma-exposed membranes (Allen, 1992). The fate of excited states in the phospho-LHC II remains controversial, but the net result is a decrease in the antenna size of PS II in state II with a corresponding decrease in fluorescence yield (McCormac et al., 1994). Thus the effect of LHC II phosphorylation associated with light state transitions is to reduce the excitation density in PS II at any incident light intensity. Although it is generally believed that is a minor component of (Krause and Weis, 1991; Andrews et al., 1993), the overall significance of light state transitions on the regulation of light energy utilization remains poorly understood.

14

B.

Thomas G. Owens

and the Role of

Because of the competition between photosynthetic organisms in either terrestrial or aquatic environments, it is reasonable to assume that there has been strong evolutionary selection for organisms that are capable of precise regulation of light energy utilization via This precision occurs at the level of dissipating only that fraction of absorbed light energy that is in excess of the capacity of photosynthesis. While dissipating too little energy can lead to increased light-induced damage, dissipating too large a fraction of absorbed energy results in wasting energy that could be used in photosynthesis. Such precise regulation requires a tight feedback control mechanisms that link the extent of with specific intermediates that are sensitive to the imbalance between light absorption and the capacity for photosynthesis. Logical sensing points for detecting an imbalance between light absorption and the capacity for photosynthesis lie in the metabolic intermediates that link the light-driven reactions of electron transport to the dark reactions of carbon fixation and other metabolic processes in the cell (Fig. 1; see also Chapter 3). Although the ratios of ATP/ADP and in chloroplasts are sensitive to the balance of the light and dark reactions of photosynthesis, there is little evidence of direct regulation of by these intermediates (see, however, Gilmore and Björkman, 1994). Rather, regulation of light energy utilization seems to be by the degree of acidification of the thylakoid lumen and by the redox state of the plastoquinone pool Thus, energetic regulation in chloroplasts is analogous to respiratory control in mitochondria (Foyer et al., 1990). Under conditions where the rate of light absorption exceeds the ability of the dark reactions to utilize ATP and NADPH produced by electron transport, synthesis of ATP and NADPH are limited by slow recycling of ADP, and from the dark reactions. This subsequently leads to a decrease in the lumen pH, reduction of the electron acceptors of PS I and PS II, direct feedback regulation of electron transport, and increased and (see also Chapters 2 and 3). Strong relationships exist between lumen pH and the extent of in vivo (Noctor et al., 1993) and in isolated LHC II (Ruban and Horton, 1992). At the same time, Laasch (1987) has shown that the correlation between and depends on species

and previous growth conditions. It is particularly important to note that a large fraction of can be inhibited with dicyclohexylcarbodiimide (DCCD), a compound that specifically binds to acidic residues buried in hydrophobic regions of proteins. Walters et al. (1994) have shown that DCCD binds to LHC II polypeptides, particularly the minor CP29, CP26 and C24 complexes. This suggests that a major regulatory site for is in a localized proton domain in the minor PS II antenna complexes rather than in a domain directly exposed to the lumen phase (see also Pfundel et al., 1994). This is also supported by the observation that dibucaine collapses the bulk phase pH gradient across the thylakoid without diminishing (Noctor et al. 1993).

C. Proposed Mechanisms for

Quenching

There is wide agreement that the principal role of is dissipation of excited state energy in competition with photochemistry, fluorescence and other decay processes as a means of regulating the utilization of absorbed light energy and minimizing damage from excess light absorption. Because the yields of stable energy storage in the reaction center and fluorescence emission from the antenna both decrease with increasing it is assumed that arises from a new process which ultimately dissipates the excited state energy as heat. This assumption is quite realistic, but the actual mechanism(s) of remain a matter of conjecture. A number of mechanisms have been proposed, differing in both the components of the quenching reaction and in the site at which the quenching occurs. Demmig-Adams and coworkers were the first to demonstrate a correlation between the extent or capacity for and the accumulation of the carotenoid zeaxanthin in thylakoid membranes (DemmigAdams, 1990). Although zeaxanthin-independent quenching has been observed by several authors, it is clear that under physiological conditions, a major proportion of the rapidly (within minutes) reversible correlates with zeaxanthin content. Zeaxanthin is formed in thylakoids by reversible de-epoxidation of violaxanthin (a diepoxide) via the mono-epoxide antheraxanthin (Demmig-Adams, 1990) in a process called the ‘xanthophyll cycle’ (Fig. 5). Regulation of the xanthophyll cycle remains an active area of research, but it is known that the de-epoxidation reaction forming zeaxanthin is favored by low lumenal pH

Chapter 1

Processing of Excitation Energy

(Pfundel et al., 1994). However, the role of in is not solely through activation of zeaxanthin formation because uncouplers can collapse the pH gradient and much more rapidly than zeaxanthin is converted back to violaxanthin (Gilmore and Yamamoto, 1993). The correlation of with zeaxanthin content was one indication that has led to the suggestion that the site of the quenching reaction was in the peripheral antenna because zeaxanthin and other xanthophylls are not known to be components of the PS II reaction center or core antenna complexes (see also Chapter 2). Several other lines of evidence, including the DCCD binding data described above, implicate CP29, CP26 and CP24 as a major site of quenching (Horton et al., 1994; but also see Chapter 2). In particular, the observation that quenches the initial fluorescence as well as the variable fluorescence is a strong indication that the antenna is the site of quenching. This interpretation is based on the models of Butler (1978), and although quantitative aspects of these models have been questioned, quenching of is still seen as an indication of that the quenching

15

site is in the antenna rather than the reaction center. Demmig-Adams (1990) suggested two possible roles for the direct participation of zeaxanthin in (i) transient charge separation between an excited antenna Chl and zeaxanthin followed by rapid charge recombination to the ground state, and (ii) singlet energy transfer from Chl to zeaxanthin. Transient charge separation between covalently linked tetrapyrroles and carotenoids has been observed in model compounds (Hermant et al., 1993) but differences between the abilities of violaxanthin and zeaxanthin to participate in this reaction have not been examined. In contrast, Owens (1994) has proposed a quenching mechanism involving singlet energy transfer that is based on the assumption that differences between the photophysical properties of violaxanthin and zeaxanthin are central to the mechanism of Carotenoids differ from Chls in that they have low energy excited states that do not appear in the absorption spectrum (Fig. 6). That is, absorption of a photon cannot promote the transition from the ground state to the lowest excited state. The

16

absorption of carotenoids in the blue-green part of the spectrum is due to a strongly allowed transition to a higher excited state. The hypothesis that this low lying state of carotenoids might be involved in energy transfer from carotenoids to Chl (the lightharvesting function) was recently confirmed by Shreve et al. (1990). In addition, it has been shown that the energy of the carotenoid state decreases with increasing number of conjugated carbon-carbon double bonds. This led DeCoster et al. (1992) to suggest that the state of more highly conjugated carotenoids might lie below that of Chl, precluding their function in light-harvesting. This being the case then a mechanism that distinguishes between the abilities of violaxanthin and zeaxanthin to participate in the quenching reaction can be suggested. Violaxanthin is a di-epoxide with 9 conjugated double bonds. Its state is thought to lie well above that of Chl a (Frank et al., 1994; Owens, 1994). This permits efficient energy transfer from violaxanthin to Chl a (Fig. 7) but prevents energy transfer from Chl to violaxanthin. Conversion of violaxanthin to zeaxanthin removes the epoxides and increases the conjugation length to 11 double bonds. This lowers the energy of the zeaxanthin state so that it is at or

Thomas G. Owens

slightly below that of Chl a (Frank et al., 1994; Owens, 1994) and permits reversible energy transfer between the two pigments (Fig. 7). The lifetime of the carotenoid state is very short (10 ps) compared to that of Chl a (5 ns) due to rapid vibrational relaxation (thermal emission) to the ground state. Thus, reversible energy transfer between Chl a and zeaxanthin creates a weak quenching center in the antenna. The quenching efficiency of each zeaxanthin will be a complex function of energy transfer and thermal emission rates and in general will also depend on the overall dynamics of excited state motion and trapping in PS II. The role of in this mechanism is directly related to the effect of acidification of the thylakoid lumen on the local electric field in LHC II. With decreasing pH in the lumen, protonation of amino acids with pKa <7 will occur (Pfundel et al., 1994). These protonation events will alter the local field in the vicinity of the protonation site. A change in the local electric field could increase the rate of energy transfer between Chl and zeaxanthin by either shifting the Chl’s emission to provide better spectral overlap with the zeaxanthin state, or by increasing the dipole character of the zeaxanthin to transition

Chapter 1 Processing of Excitation Energy

(Owens, 1994). In either case, this mechanism requires both the presence of zeaxanthin and for quenching to occur. These roles for violaxanthin-zeaxanthin interconversion and in rapidly reversible also provide a simple explanation for the occurrence of constitutive (zeaxanthin- and/or -independent) that is frequently observed under chronic stress conditions. Constitutive quenching could result from modification of the protein environment in the region of the zeaxanthin binding site such that (i) zeaxanthin is permanently bound to the complex (not converted to violaxanthin in the dark) or zeaxanthin is replaced by another carotenoid with eleven or more conjugated double bonds and (ii) the amino acid(s) that is responsible for proton binding and regulation of reversible in the minor antenna complexes is replaced by a different amino acid with a higher such that it remains protonated under the full physiological range of lumen pH. These conditions seem to be met in the green alga Dunaliella where a unique, zeaxanthin-binding protein accumulates in

17

the thylakoid membranes under conditions of prolonged salt or nutrient stress (Levy et al., 1993). Horton and coworkers (Horton et al., 1994) have proposed an alternative mechanism for quenching in the PS II antenna. Their hypothesis is based on the assumption that zeaxanthin does not play a direct role in the quenching mechanism, but that it facilitates -induced structural changes in LHC II. These structural changes are proposed to cause aggregation of LHC II subunits leading to altered interactions among specific LHC II Chls. These interactions may be analogous to the observation of concentrationdependent quenching of excited states in solutions of pure Chl (Beddard and Porter, 1976), although the mechanism of this quenching is not known. Based on comparisons of amino acid sequence data from the major and minor LHC II complexes, Crofts and Yerkes (1994) have proposed a specific site at which this quenching might be regulated in the minor complexes (see Chapter 2). There is considerable evidence in support of this mechanism based upon studies of LHC II aggregation in vitro and comparison

18 to in vivo experiments (Horton et al., 1994). A major difference between the LHC II aggregation model and the model of Owens (1994) is that zeaxanthin is not required for quenching in the former; it only increases the extent of quenching by facilitating LHC II aggregation. However, both models require protonation of LHC II at specific sites. Weis and coworkers (Krieger and Weis, 1993) have proposed a very different mechanism for in which the quenching site is in the PS II reaction center rather than in the antenna. Their model proposes a pH-dependent release of bound on the donor side of the PS II reaction center. This was shown to inhibit electron donation to and increase the redox potential of the primary quinone acceptor of PS II, and it was proposed that the rate of charge recombination between and increased. The net result is that excited state energy is dissipated as heat rather than being stored in stable redox components in the electron transport chain. The role of zeaxanthin, if any, in such a mechanism is not obvious.

D. Reaction Center Versus Antenna Quenching Sites Each of these proposed mechanisms for has its own strengths and weaknesses and there are presently no experimental results that preclude the possibility that any of these mechanisms function in vivo. It is well known that terrestrial and aquatic environments are highly variable, both in terms of incident light intensity and other environmental factors that influence the rate of photosynthesis. Considering the importance of in regulation of light energy utilization in photosynthesis and that natural selection may work on very small increases in efficiency, it is possible that each of the mechanisms outlined above may contribute to total under various physiological conditions. A redundancy of processes is one way that plants could optimize growth and minimize losses due to irreversible photooxidative reactions and damage to the PS II reaction center. Thus, one must consider the possibility that is heterogeneous and that these mechanisms are not mutually exclusive. While one may dominate in one species under a give set of conditions, another may be important under different conditions. At the same time, it is important to be able to distinguish between quenching action at competing sites as a component of an overall description of non-photochemical quenching.

Thomas G. Owens One of the most convenient methods to distinguish between antenna versus reaction center sites of is Chl fluorescence (Krause and Weis, 1991). The models of photosynthesis described by Butler (1978) have been widely used to evaluate changes in rates of photosynthesis and Chl fluorescence yield in response to environmental perturbation. In particular, these models predict that non-photochemical quenching of excited states in the PS II antenna would lead to a reduction in both the and components of the fluorescence rise, while quenching in the reaction center would affect but not While these criteria have been applied by many authors, interpretation of the results is far from ambiguous. A decrease in while indicative of antenna quenching, does not preclude quenching in the reaction center as well. Comparisons of the decreases in and accompanying can be made, but assigning unique values to the extent of quenching in the antenna and reaction center is difficult. The situation is further complicated by the fact that any photoinhibitory damage to PS II will cause independent changes in and that may complicate or completely mask the dependent changes (Franklin et al., 1992). One technique that can distinguish between antenna and reaction center quenching is photoacoustic spectroscopy (Malkin and Cananni, 1994). This technique directly measures light-induced heat release from samples (Chapter 2). While thermal emission derived from quenching in the antenna should occur on a time scale comparable with the excited, state lifetime (a few hundred ps), quenching in the reaction center should occur on the time scale of and recombination Recently, Mullineaux et al. (1994) demonstrated a correlation in thermal emission from samples with increasing Because the apparatus measured only emission that occurred within a few of the laser excitation, the authors concluded that their observations were consistent with antenna rather than reaction center quenching. However, the authors were not able to make quantitative comparisons between fluorescence quenching, thermal emission and energy storage, which means that they could not completely eliminate the possibility that quenching in the reaction center was occurring simultaneously. Clearly this is an area that merits further investigation.

E. Role of Transfer Equilibrium in

Quenching

The phenomenon of rapid excited state equilibration

Chapter 1

Processing of Excitation Energy

among antenna spectral forms and formation of the transfer equilibrium (TE) state has important implications for any proposed mechanism of nonphotochemical quenching. Independent of whether the quenching reaction is localized in the antenna, the reaction center, or both, the quenching reactions must compete with the rapid energy transfer that occurs among all Chl a pigments (antenna and reaction center). If the reactions leading to are localized in the PS II reaction center (as proposed by Weis and coworkers), then the role of the TE state in is identical to that in photochemical quenching. Assuming that conversion of a PS II reaction center from a photochemical to a non-photochemical quencher does not involve a change in but only a change in the probability of charge recombination (Krieger and Weis, 1993), then the principal reaction that competes with remains detrapping to the antenna. Under these conditions, the yield of in each reaction center is determined by the fractional distribution of the excited state on the reaction center at TE and by (see Eq. 5) and is identical to the yield of photochemistry in the absence of This explains why is not affected by quenching in the reaction center. Similarly, the lifetime and dynamics of excited state motion and trapping will be unaffected by the quenching reaction. Interestingly, the proposed mechanism for in the reaction center is consistent with a simple two state model of PS II in which photochemistry in the reaction center is either followed by normal electron transfer or by charge recombination, as originally suggested by Weis and Berry (1987). Thus the overall yield of is determined by the fraction of reaction centers converted to the quenching state and must be equal to the fractional loss of On the other hand, any mechanism of that is localized in the antenna must be profoundly influenced by rapid spectral equilibration and the formation of the TE state. Again, two limiting cases must be considered. If the quenching reaction is very fast compared to the time constant for energy transfer from the quencher back to the antenna (the strong quencher case), formation of the quencher effectively converts PS II from a trap-limited to a diffusionlimited system. In this case, formation of a single, strong quencher per PS II leads to complete quenching in that PS II unit. This can also be modeled as a simple two state system (each PS II unit is either quenched or normal). However, unlike the proposed mechanism of in the reaction center, formation of

19 a strong quencher in the antenna is predicted to decrease the excited state lifetime in PS II by a factor of five or more due to the diffusion-limited dynamics. The high quenching efficiency is not a consequence of the quencher being located on a specific path of energy transfer. The path from any arbitrary pigment on which the excited state forms to the quencher is still largely a random one (and determined by the dynamics in the equilibration phase, Fig. 4). Rather the high efficiency results from the fact that detrapping dominates photochemistry at the reaction center while there is no detrapping from the quencher. Formation of strong quenchers is not consistent with the zeaxanthin- and -dependent antenna quenching proposed by Owens (1994) because the quenching reaction (internal conversion from to of zeaxanthin) is probably slow (10 ps) compared to energy transfer from zeaxanthin to Chl a (probably in the range of a few hundred fs to a few ps; Fig. 7). It may be consistent with the LHC II aggregation hypothesis of Horton and coworkers, but no detailed mechanism has yet been proposed for this hypothesis. The other possible scenario for antenna quenching is that the quenching reaction is slow compared to the time constant for energy transfer from the quencher to the antenna (the weak quencher case). In this case, PS II remains trap-limited and the efficiency of each quenching center is determined by the competition between the quenching reaction and transfer to the antenna, and by the TE distribution of the excited state on the quencher (by analogy to Eq. 5). To account for the high levels of measured under high light conditions the weak quenching case requires the presence of multiple quenchers per PS II. This is consistent with the formation of about ten zeaxanthins per PS II (Bassi et al., 1993; Owens, 1994) and may also be consistent with the LHC II aggregation hypothesis. In contrast to the strong quencher model, the weak quencher cannot be described by a simple two state model. Rather, there is a graded response in each PS II depending on the number and efficiency of the quenchers. The excited state lifetime in PS II also decreases gradually with the number of quenchers. Because back transfer from the quencher to the antenna competes with the quenching reaction, location of the quencher on a specific energy transfer pathway cannot contribute to the mechanism or efficiency of the reaction. These considerations of excited state dynamics also suggest that the three proposed mechanisms of

20 may be distinguished by examining the lifetime of the excited state in PS II (with all PS II traps open) as a function of the extent of In the reaction center quenching model, the lifetime should not be expected to decrease with increasing because remains the principal reaction that quenches the excited state in both normal and quenched conditions. The strong antenna quencher case predicts a bimodal distribution of PS II lifetimes, the amplitudes of the two components depending on the fraction of PS II units that contain quenchers. Finally, the weak antenna quenching case predicts a gradual decrease in PS II lifetime with an increasing number of quenchers per PS II unit. Using picosecond fluorescence lifetime measurements, both Mullineaux et al. (1993) and Gilmore et al. (1995) have observed a significant decrease in fluorescence lifetime correlating with increases in These data are consistent with a quenching site(s) in the antenna. At the same time, the data of Gilmore et al. (1995) also seem to preclude any simultaneous non-photochemical quenching in the reaction center as the amplitudes of the major lifetime components exhibit a linear relationship with the amount of in the sample. However, it is important to note that protection against the effects of excess light are largely independent of the choice of mechanism of because each of the mechanisms discussed adjusts the overall rate of electron transport so that it does not exceed the capacity of the dark reactions to utilize the products of electron transport, either by completely quenching a fraction of the PS II units (reaction center or strong antenna quenching) or by partial quenching of all PS II units (weak antenna quenching).

F. Light State Transitions and Regulation in PS I Non-photochemical quenching in PS II associated with light state transitions represents another mechanism of regulating the utilization of light energy in photosynthesis. When becomes limiting due to high light or limitations in the dark reactions, the rate of oxidation by PS I decreases because of slower electron transport through PS I. Reduction of the PQ pool activates a protein kinase that phosphorylates a fraction of LHC II, inducing the movement of the phospho-LHC II from PS II in the appressed regions to the stroma-exposed regions of the thylakoid membrane (Allen, 1992). When the PQ pool becomes oxidized, the kinase is deactivated and

Thomas G. Owens the process is reversed via dephosphorylation of LHC II by a constitutive phosphatase. In this manner, light state transitions regulate the rate of light absorption in PS II, decreasing absorption when the rate of electron transport exceeds the capacity of the dark reactions to regenerate as a PS I acceptor. The quenching of Chl fluorescence observed with light state transitions is primarily a result of decreasing the PS II antenna size (the number of pigments over which the excited state is delocalized). Under physiological conditions, light state transitions are thought to account for at most about 15% of total (Horton et al., 1994; Andrews et al., 1993). The fate of excited states which form in the phospho-LHC II in the stroma-exposed thylakoids raises an interesting issue about regulation of excited state utilization in PS I. It is clear that the presence of phospho-LHC II does not result in a large increase in fluorescence, indicating that excited states are somehow quenched in these complexes. The two most likely scenarios are that the phospho-LHC II is coupled to either units or to PS I (Melis, 1991). Because are incapable of electron transport beyond these traps are closed even at very low light intensity (Cao and Govindjee, 1990). Increasing the antenna size of by coupling to phosphoLHC II would result in only a minor fluorescence increase. In addition, is much less susceptible to photoinhibitory damage than (Melis, 1991), so that coupling to is a relatively safe process. On the other hand, coupling of phospho-LHC II to PS I increases the rate of light absorption in PS I and, by analogy to PS II, potentially increases the rate of damage in that photosystem. This would argue for a similar regulation of light energy utilization in PS I by Detection of in PS I is not possible by standard fluorescence techniques since PS I contributes very little to total room temperature fluorescence. However, it has also been suggested that direct regulation in PS I is not necessary because energy dissipation in PS I is automatically regulated by the supply of electrons from PS II (Harbinson et al., 1989; also see Chapters 2 and 3). The explanation for this hypothesis is as follows. PS I differs from PS II in that the rate at which the reaction center reopens following charge separation is determined by the supply of electrons on the donor side (reduction of by PS II via plastocyanin) rather than by the demand for electrons on the acceptor side (oxidation of by oxidants generated in PS I). In the absence of electron donors, is stable for

Chapter 1

Processing of Excitation Energy

hours or longer. It is also well documented that the lifetime of excited states in PS I is independent of the redox state of P700 (Owens et al., 1987). This means that is as effective a quencher of excited states from the PS I antenna as the open reaction center, P700. However, in the state photochemistry is not possible and the excited state energy is dissipated as heat (Owens et al., 1990). Thus, PS I has a built-in mechanism of dissipating excess absorbed light energy. As long as the rate of electron transport from water to is appropriately regulated by in PS II, energy absorbed by PS I which is in excess of the photochemical turnover of PS II is automatically dissipated as heat. To a large extent, this probably explains the insensitivity of PS I to excess light under most physiological conditions. However, it does not preclude the possibility of direct regulation of energy utilization in PS I by any direct mechanism, including something analogous to in PS II.

VI. Concluding Remarks The ability of photosynthetic organisms to regulate the utilization of absorbed light energy is an essential component of growth in environments in which many factors adversely affect the rate and capacity of photosynthesis. Absorption of light energy that is in excess of the capacity of photosynthesis can lead to a range of damaging photooxidative reactions and probably represents a common response to many environmental stresses. Recent studies have demonstrated that a group of reactions collectively referred to as non-photochemical quenching, or can dissipate excess absorbed light energy in PS II in competition with photochemistry in the reaction center and prevent excess light-induced damage. Although the molecular mechanism(s) of remain a matter of conjecture, physiological studies indicate that is tightly regulated in vivo. Thus, the antenna and reaction center of PS II must strike a delicate balance between optimizing light absorption under limiting light conditions and energy dissipation under excess light conditions. Regardless of whether the quenching reaction occurs in the reaction center or the antenna, the dynamics of excited state motion and photochemical trapping will have profound effects on because these reactions occur in the same pigment bed.

21 Acknowledgments The contributions of J. K. Trautman, A. P. Shreve, J. B. van Beek, C. S. Ting, P. D. Laible and A. C. Albrecht to the theory and experiments described here are gratefully acknowledged. Financial support for this work was provided by the National Science Foundation (DMB-9005574), the US Department of Agriculture (93-373060-9042) and HATCH funds (NYS-185405).

References Allen JF (1992) How does protein phosphorylation regulate photosynthesis? TIBS 17: 12–17 Anderson JM and Andersson P-A (1988) The dynamic photosynthetic membrane and regulation of solar energy conversion. TIBS 13: 351–355 Andrews JR, Brendenkamp GJ and Baker NR (1993) Evaluation of the role of state transitions in determining the efficiency of light utiliza tion for assimilation in leaves. Photosynth Res 38: 15–26 Bassi R and Dainese P (1992) A supramolecular light-harvesting complex from chloroplast Photosystem II membranes. Eur J Biochem 204: 317–326 Bassi R, Rigoni F and Giacometti GM (1990) Chlorophyll binding proteins with antenna function in higher plants and green algae. Photochem Photobiol 52: 1187–1206 Bassi R, Pineau B, Dainese P and Marquardt J (1993) Carotenoidbinding proteins of Photosystem II. Eur J Biochem 212: 297– 303 Beauregard M, Martin I and Holzwarth AR (1991) Kinetic modelling of exciton migration in photosynthetic systems. (1) Effects of pigment heterogeneity and antenna topography on exciton kinetics and separation yields. Biochim Biophys Acta 1060: 271–283 Beddard GS and Porter G (1976) Concentration quenching in chlorophyll. Nature 260: 366–367 Bolhar-Nordenkampf HR, Long SP, Baker NR, Öquist G, Schreiber U and Lechner EG (1989) Chlorophyll fluorescence as a probe of photosynthetic competence of leaves in the field: A review of current instrumentation. Funct Ecol 3: 497–514 Bradbury M and Baker NR (1981) Analysis of the slow phase of in vivo Chl fluorescence induction curve. Biochim Biophys Acta 63 5: 542–551 Butler WR (1978) Energy distribution in the photochemical apparatus of Photosynthesis. Ann Rev Plant Physiol 29: 345– 378 Cao J and Govindjee (1990) Chlorophyll a fluorescence an in indicator of active and inactive Photosystem II in thylakoid membranes. Biochim Biophys Acta 1015: 180–188 Cogdell RJ and Frank HA (1987) How carotenoids function in photosynthetic bacteria. Biochim Biophys Acta 895: 63–79 Crofts AR and Yerkes CT (1994) A molecular mechanism for -quenching. FEBS Let 352: 265– 270 DeCoster B, Christensen RL, Gebhard R, Lugtenburg J, Farhoosh R and Frank HA (1992) Low lying electronic states of carotenoids. Biochim Biophys Acta 1102: 107–119

22 Demmig-Adams B (1990) Carotenoids and photoprotection in plants: a role for the carotenoid zeaxanthin. Biochim Biophys Acta 1020: 1–24 Du M, Xie X, Mets L and Fleming GR (1993) Direct observation of ultrafast energy transfer in PS I core. Chem Phys Lett 210: 535–542 Eads DD, Castner EW, Alberte RS, Mets L and Fleming GR (1989) Direct observation of energy transfer in a photosynthetic membrane: Chl b to Chl a transfer in LHC. J Phys Chem 93: 8271–8275 Emerson R and Arnold W (1932a) A separation of the reactions in photosynthesis by means of intermittent light. J Gen Physiol 15: 391–420 Emerson R and Arnold W (1932b) The photochemical reaction in photosynthesis. J Gen Physiol 16: 191–205 Foyer C, Furbank R, Harbinson J and Horton P (1990) The mechanisms contributing to the photosynthetic control of electron transport by carbon assimilation in leaves. Photosynth Res 25: 83–100 Frank HA, Cua A, Chyhwat V, Young A, Gosztola D and Wasielewski MR (1994) Photophysics of the carotenoids associated with the xanthophyll cycle in photosynthesis. Photosynth Res 41: 387–395 Franklin LA, Levavasseur G, Osmond CB, Henley WJ and Ramus J (1992) Two components of onset and recovery during photoinhibition of Ulva rotunda. Planta 186: 399–408 Gilmore AM and Björkman O (1994) Adenine nucleotides and the xanthophyll cycle in leaves: II. Comparison of the effects of a n d temperature-limited photosynthesis on Photosystem II fluorescence quenching, the adenylate energy charge and violaxanthin deepoxidation in cotton. Planta 192: 537–544 Gilmore AM and Yamamoto HY (1993) Linear models relating x a n t h o p h y l l s and lumen acidity to non-photochemical fluorescence quenching. Evidence that antheraxanthin explains zeaxanthin- independent quenching. Photosynth Res 35: 67– 78 Gilmore AM, Hazlett TL and Govindjee (1995) Xanthophyll cycle-dependent quenching of Photosystem II chlorophyll a fluorescence: Formation of a quenching complex with a short fluorescence lifetime. Proc Nat Acad Sci USA 92: 2273–2277 Govindjee (1995) Sixty–three years since Kautsky: Chlorophyll a fluorescence. Aust J Plant Physiol 22: 131–160 Harbinson J, Genty B and Baker NR (1989) Relationship between the quantum efficiencies of photosystems I and II in pea leaves. Plant Physiol 90: 1029–1034 Hermant RM, Liddell PA, Lin S, Alden RG, Kang HK, Moore AL, Moore TA and Gust D (1993) Mimicking carotenoid quenching of Chl fluorescence. J Am Chem Soc 1 1 5 : 2080– 2081 Holzwarth AR (1991) Excited state kinetics in Chl systems and its r e l a t i o n s h i p to the functional organization of the photosystems. In: Schemer H (ed) The Chlorophylls, pp 1125– 1151. CRC Press, Boca Raton Horton P and Ruban AV (1992) Regulation of Photosystem II. Photosynth Res 34: 375–385 Horton P and Ruban AV (1994) The role of LHC II in energy quenching. In: Baker NR and Bowyer JR (eds) Photoinhibition of Photosynthesis—From Molecular Mechanisms to the Field, pp 111–128. Bios Scientific Publishers, Oxford Horton P, Ruban A V and Walters RG (1994) Regulation of light-

Thomas G. Owens harvesting in green plants. Plant Physiol 106: 415–420 Jean JM, Chan C-K, Fleming GR and TG Owens (1989) Excitation transport and trapping on spectrally disordered lattices. Biophys J 56: 1203–1215 Jennings RC, Bassi R, Gorlaschi FM , Dainese P and Zuccheli G (1993) Distribution of the chlorophyll spectral forms in the chlorophyll-protein complexes of Photosystem II antenna. Biochem 32: 3203–3210 Krause GH (1988) Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms. Physiol Plant 74: 566–574 Krause GH and Weis E (1991) Chlorophyll fluorescence and photosynthesis: The Basics. Ann Rev Plant Physiol Plant Mol Biol 42: 313–349 Krause GH, Vernotte C and Briantais J-M (1982) Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution of two components. Biochim Biophys Acta 679: 1 16–124 Krauss N, Hinrichs W, Witt I, Fromme P, Pritzkow W, Dauter Z, Betzel C, Wilson KS, Witt HS and Saenger W (1993) Three dimensional structure of system I of photosynthesis at 6 Å resolution. Nature 361: 326–33 1 Krieger A and Weis E (1993) The role or calcium in the pHdependent control of Photosystem I I . Photosynth Res 37: 117– 130 Kühlbrandt W and Wang DN ( 1 9 9 1 ) Three-dimensional structure of plant light-harvesting complex determined by electron crystallography. Nature 350: 130–134 Laasch H (1987) Non-photochemical quenching of chlorophyll fluorescence in isolated chloroplasts under conditions of stressed photosynthesis. Planta 171:220–226 Laible PD, Zipfel W and Owens TG (1994) Excited state dynamics in chlorophyll-based antennae: The role of transfer equilibrium. Biophys J 66: 844–860 Laroche J, Mortain-Bertrand A and Falkowski PG (1991) Light intensity-induced changes in cab messenger RNA and lightharvesting complex II apoprotein levels in the unicellular chlophyte Diunaliella tertiolecta. Plant Physiol 97: 147–153 Levy H, Tal T, Shaish A and Zamir A (1993) Cbr, an algal homolog of plant early light-induced proteins, is a putative zeaxanthin binding protein. J Biol Chem 268: 20892–20896 Long SP, Humphries S and Falkowski PG (1994) Photoinhibition of photosynthesis in nature. Ann Rev Plant Physiol Plant Mol Biol 45: 633–661 Malkin S and Cananni O (1994) The use and characteristics of the photoacoustic method in the study of photosynthesis. Ann Rev Plant Physiol Plant Mol Biol 45: 493–526 McCormac DJ, Bruce D and Greenberg BM (1994) State transitions, light-harvesting antenna phosphorylation and lightharvesting antenna migration in vivo in the higher plant Spriodela oligorrhiza. Biochim Biophys Acta 1 187: 301–312 Melis A ( 1 9 9 1 ) Dynamics of photosynthetic membrane composition and function. Biochim Biophys Acta 1058: 87– 106 Mullineaux CW, Pascal AA, Horton P and Holzwarth AR (1993) Excitation-energy quenching n aggregates of the LHC II chlorophyll-protein complex. A time-resolved fluorescence study. Biochim Biophys Acta 1 1 4 1 : 23–28 Mullineaux CW, Ruban AV and Horton P (1994) Prompt heat release associated with -dependent quenching in spinach thylakoid membranes. Biochim Biophys Acta 1185: 119–123

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Murata N and Sugahara K (1969) Control of excitation transfer in photosynthesis. III. Light-induced decrease of chlorophyll a fluorescence related to photophosphorylation system in spinach chloroplasts. Biochim Biophys Acta 189: 182–189 Nitschke W and Rutherford W (1991) Photosynthetic reaction centers-variations on a theme. TIBS 16: 241–245 Noctor G, Ruban AV and Horton P (1993) Modulation of dependent non-photochemical quenching of chlorophyll fluorescence in spinach chloroplasts. Biochim Biophys Acta 1183: 339–344 Owens TG (1988) Light-harvesting antenna systems in the chlorophyll a/c-containing algae. In: Stevens SE and Bryant DA (eds) Light-Energy Transduction in Photosynthesis: Higher Plant and Bacterial Models, pp 122–136. American Society of Plant Physiologists, Rockville, MD Owens TG (1994) Excitation energy transfer between chlorophylls and carotenoids. A proposed molecular mechanism for nonphotochemical quenching. In: Baker NR and Bowyer JR (eds) Photoinhibition of Photosynthesis – From Molecular Mechanisms to the Field, pp 95–109. Bios Scientific Publishers, Oxford Owens TG, Webb SP, Alberte RS, Mets L and Fleming GR (1988) A n t e n n a structure and excitation dynamics in Photosystem I.I. Studies of detergent isolated Photosystem I preparations using time-resolved fluorescence analysis. Biophys J 53: 733–745 Owens TG, Carpentier R and Leblanc RM (1990) Detection of photosynthetic energy storage in a Photosystem 1 reaction center preparation by photoacoustic spectroscopy. Photosynth Res 24: 201–208 Pell EJ and Steffen KL (1991) Active Oxygen/Oxidative Stress and Plant Metabolism. American Society of Plant Physiology, Rockville, MD. Pfundel EE, Renganathan M, Gilmore AM, Yamamoto HY and Dilley RA (1994) Intrathylakoid pH in isolated pea chloroplasts as probed by violaxanthin deepoxidation. Plant Physiol 106: 1647– 1658 Ruban AV and Hoiton P (1992) Mechanism of -dependent dissipation of absorbed excitation energy by photosynthetic membranes. I. Spectroscopic analysis of isolated lightharvesting complexes. Biochim Biophys Acta 1102: 30–38 Ruban AV and Horton P (1995) An investigation of the sustained

23 component of nonphotochemical quenching of chlorophyll fluorescence in isolated chloroplasts and spinach leaves. Plant Physiol 108: 71–726 Schatz GH, Brock H and Holzwarth AR (1988) Kinetics and energetic model for the primary processes in Photosystem II. Biophys J 54: 397–405 Schreiber U, Schliwa U and Bilger W (1986) Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth Res 10: 51–62 Shreve AP, Trautman JK, Owens TG and Albrecht AC (1990) Two-photon excitation spectroscopy of thylakoid membranes form Phaeodactylum tricornutum: Evidence for an in vivo two-photon-allowed carotenoid state. Chem Phys Lett 170: 51–56 Shreve AP, Trautman JK, Frank HA, Owens TG, van Beek JB and Albrecht AC (1992) On subpicosecond excitation energy transfer in light-harvesting complexes (LHC): the B800-850 LHC of Rhodobacter sphaeroides 2.4.1. J Luminesc 53: 179– 186 Ting CS and Owens TG (1994) The effects of excess irradiance on photosynthesis in the marine diatom Phaeodactylm tricornutum. Plant Physiol 106: 763–770 van Grondelle R, Dekker JP, Gillbro T and Sundstrom V (1994) Energy transfer and trapping in photosynthesis. Biochim Biophys Acta 1187: 1–65 Walters RG, Ruban AV and Horton P (1994) Higher plant light harvesting complexes LHC IIa and LHC IIc are bound by dicyclohexylcarbodiimide during inhibition of energy dissipation. Eur J Biochem 226: 1063–1069 Weis E and Berry JA (1987) Quantum efficiency of Photosystem II in relation to ‘energy’-dependent quenching of chlorophyll fluorescence. Biochim Biophys Acta 894: 198–208 Williams WP and Allen JF (1987) State I/State 2 changes in higher plants and algae. Photosynth Res 13:19–45 Wraight CA and Crofts AR (1970) Energy-dependent quenching of chlorophyll a fluorescence in isolated chloroplasts. Eur J Biochem 17: 319–323 Zuber H (1985) Structure and function of light-harvesting complexes and their polypeptides. Photochem Photobiol 42: 821–844

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Chapter 2 Control and Measurement of Photosynthetic Electron Transport in Vivo David Mark Kramer Institute of Biological Chemistry, Washington State University, 467 Clark Hall, Pullman WA 99164, USA

Antony Richard Crofts Center of Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 338 Morrill Hall, 505 South Goodwin Avenue, Urbana, IL 61801, USA

Summary I. Introduction A. What Would Make the Perfect Instrument? B. Working with Intact Plants II. Control of the Photosynthetic Electron Transfer Chain A. Role of Lumenal pH B. What Do We Need to Measure? C. The Steady-state 1. Flux 2. Poise 3. Perturbation of the Steady-state D. Measurement of Mechanism through Transient Kinetics III. What Reactions Can We Measure? A. Optical Techniques 1. Fluorescence 2. Delayed Fluorescence 3. Thermoluminescence 4. Absorbance IV. Instrumentation and Measurement A. Fluorescence Yield Changes in Intact Plants B. Deconvolution of Components Contributing to Fluorescence Yield Changes C. Steady-state Fluorescence Measurements 1. Down Regulation of Photosynthetic Electron Transport 2. Quenching of Fluorescence Associated with the Proton Gradient (qE-quenching) 3. Role of Donor-side Oxidation in Photoinhibition 4. Where is the Antenna Quencher Located? 5. Evidence for a Role for the Minor Light-harvesting Complexes a. Quenchina Associated with Formation of Zeaxanthin b. qE-quenching in Strains Depleted in the Bulk LHCII c. Inhibition of qE-quencning by Dicyclonexylcarbodiimide 6. Mechanism of qE-quenching—General Conclusions 7. A Hypothetical Molecular Mechanism

Neil R. Baker (ed): Photosynthesis and the Environment, pp. 25–66. ©1996 Kluwer Academic Publishers. Printed in The Netherlands.

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8. Is there a Similar Protective Mechanism to Down-regulate PS I? D. Measurement of Fluorescence 1. Modulated Fluorimeters 2. Pulsed Kinetic Fluorimeters 3. Interpretation of Data from Pulsed Kinetic Fluorimeters 4. Use of Pulsed Kinetic Fluorimetry on Intact Plants 5. Fluorescence Video Imaging E. Delayed Luminescence in Intact Leaves 1. Delayed Luminescence as a General Indicator of Photosynthetic Energy Storage 2. Delayed Luminescence as an Indicator of the ‘Energization’ of the Thylakoid Membrane 3. Measurement of Delayed Luminescence F. Absorbance Measurements in Intact Plants 1. Kinetic Spectrophotometers 2. Measurements in the Near Infrared (NIR) a. Species Showing NIR Transitions b. Deconvolution c. Artifacts from Enhanced Path-length and Internal Absorption d. Estimation of Quantum Efficiency of PS I and PS II e. Donor-pool Size for PS I f. The Rate of Intersystem Electron Transfer Reactions in the Steady-state 3. Flash Measuring-beam Kinetic Spectrophotometer a. Proton Flux Measured through the 515 nm Electrochromic Change b. Redox Poise 4. Multi-wavelength Modulated Spectrophotometer a. Deconvolution of Absorbance Changes on Continuous Illumination 5. Measurement of pH Changes by Light-scattering G. Thermal Radiometry 1. Photothermal Radiometry 2. Photoacoustic Spectroscopy 3. Photothermal Beam Deflection H. Blue Fluorescence 1. What Can Be Measured with Blue Fluorescence? 2. Prospects and Problems Applying Blue Fluorescence Techniques to Intact Plants V. The Future of Instrumentation for Intact Plants A. A Principle of Sufficient Determination B. The Need for More Specific Measurements Acknowledgments References

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Summary We review the literature on the control of photosynthetic electron transport in intact plants, and the instrumentation currently available for exploring these reactions. Under conditions of full ambient sunlight, the rate of photosynthetic electron transport is determined by three main parameters: the availability of substrate, the flux of excitation to the photosystems, and the sensitivity of the electron transfer reactions to low lumenal pH. Control of these three is finely tuned so that delivery of excitation is matched to substrate availability, and the lumenal pH is maintained above inhibitory levels. We discuss the reactions of the electron transfer chain which might be effected by the proton gradient, and suggest that an important function of the diversion of excitation away from photochemistry is to prevent the lumenal pH from dropping into an inhibitory range. As substrate (usually ) is depleted, the metabolite pools back up, and the proton gradient builds up. The main mechanism for control of photosynthetic electron transport is through a change of function of the antenna apparatus under these conditions from light-harvesting to exciton dumping. The switch results in a lowering of fluorescence (nonphotochemical quenching), as one or more dissipative pathways are activated which compete with both photochemistry and fluorescence. The

Chapter 2 Measurement of Photosynthetic Electron Transport

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switching signal is the lowering of the lumenal pH on generation of the proton gradient, and the evidence suggests that the mechanism reflects changes in the minor light-harvesting complexes (CP29, CP26 and CP24), possibly affecting ligation of chromophores. The amplitude of the fluorescence lowering is determined by the extent of de-epoxidation of violaxanthin in these complexes. In order to explore these processes we must be able to assay the flux and poise of the partial reactions, including those of excitation delivery, electron transfer, the proton gradient and the metabolic acceptor pools, under the steady state conditions at maximal photosynthetic rates. We discuss the instrumentation available, and the limitations of methods based on measurement of parameters (fluorescence, delayed light emission, thermal radiometry) which reflect the interplay of many processes. These limitations can to some extent be overcome by selection of conditions in which only one or a few processes dominate. In the case of steady state fluorescence measurements, the saturation pulse technique has been used to distinguish photochemical and non-photochemical quenching. Since under physiological conditions the latter is dominated by fluorescence lowering, this has proved a useful practical approach, but at the expense of a simplification which precludes any detailed exploration of partial reactions. Deconvolution of fluorescence yield changes in the sub-ms time domain has proved a useful tool for exploring partial reactions close to PS II. Over the last five years, several groups using different technical approaches have developed spectrophotometers for measurement of absorbance changes in intact leaves, which have brought a new dimension to the analysis of photosynthesis in whole plants. Many of the reactions of the electron transfer chain, the xanthophyll cycle and the proton circuit can be measured directly. With the further development of a database of reliable spectra to aid deconvolution of overlapping changes, absorbance spectrophotometry can provide the specificity and sensitivity needed for analysis of partial reactions. We suggest that protocols based on perturbation of the steady state, and the simultaneous measurement of several parameters will be most rewarding. We note that future development in this field is hampered by the restricted availability of suitable instruments.

I. Introduction

A. What Would Make the Perfect Instrument? ‘Get me a TriCorder reading on that spaghetti tree, Spock.’ Science fiction has presented the ideal future of biological instrumentation: small electronic boxes that a technician can simply wave vaguely some distance from the subject of interest, and accumulate a wealth of critical information with no discernible effect on the subject. These imaginary instruments

have ‘achieved’ all of the goals of instrumentation: it is not necessary to dissect the sample to obtain information, the samples are not subjected to harmful chemicals or radiation, the information is perfectly specific, with each datum uncontaminated by interfering signals, and the specific information requested by the technician is always supplied. Though these ideal objectives have not been approached with instruments in use today, a number of interesting developments have occurred. In this chapter, we discuss our understanding of the control of photosynthetic electron transport at

Abbreviations: A – absorbance; ADRY reagent – reagents that accelerate the decay of the oxidized tyrosine ATPase – the chloropast coupling factor, or synthase; bf complex – the plastoquinol:plastocyanin oxidoreductase (the cytochrome complex); CCCP – carbonyl cyanide-m-chlorophenylhydrazone; CPX (X = 24, 26, 29...) – chlorophyll binding proteins with apparent molecular weights of X kDa; DCCD – dicyclohexylcarbodiimide; DCMU – 3-(3,4-dichlorophenyl)-1,1 -dimethylurea; DL – delayed luminescence; DTT – dithiothreitol; IR – infrared; LED – light-emitting diode; LHCII – Light-harvesting complex II; NIR – near infrared; OEC – oxygen-evolving complex; P700, – reduced and oxidized forms of the primary electron donor of PS I; P680, –reduced and oxidized forms of the primary electron donor of PS II; PC – plastocyanin; Pheo – pheophytin; pmf – protonmotive force; PQ – plastoquinone; – the primary quinone electron acceptor of PS II; – the secondary quinone electron acceptor of PS II; – the part of non-photochemical chlorophyll fluorescence quenching due to the formation of a proton gradient across the thylakoid membrane (or to membrane energization); – non-reversible chlorophyll fluorescence quenching associated with photoinhibition; – nonphotochemical chlorophyll fluorescence quenching; – photochemical quenching of chlorophyll fluorescence due to the presence of oxidized – the S-states of the oxygen-evolving complex; TL – thermoluminescence UV – ultraviolet; –tyrosine Z or the first electron donor to – absorbance difference; – standard free-energy change under defined conditions; proton motive force across thylakoid membrane; – difference across the thylakoid membrane; – electrical potential difference across the thylakoid membrane; – the quantum yield of PS I turnover; – the quantum yield of PS II turnover.

28 high light, and aspects of instrumentation which relate to measurement of critical reactions of photosynthetic electron transport in intact plants. A substantial part of the text will introduce recent instruments and discuss their application to specific problems. We will also examine our understanding of fluorescence quenching processes, and advances in the interpretation of data from instruments that have been available for some time. It is impossible in a limited space to give an indepth review of all aspects of the extensive literature covering the discoveries made with the instruments discussed here. We chose instead to introduce those papers that have advanced new types of instruments, novel ways of deriving interesting data from existing instruments, and important background material that affects the way data collected using these techniques are interpreted. Somewhat less emphasis has been placed on the commercially available modulated fluorimeters, in part because various aspects of these instruments, including performance and interpretation of data, have been previously reviewed several times in the past few years. We feel it would be more useful to explore alternative instrumental methods which promise to be useful, but are, in general, less disseminated in the photosynthesis community. The main obstacle to the spread of these alternative instruments appears to lie in the technical difficulty and amount of time involved in constructing more complex electronics and optics systems in individual laboratories. Progress in the field is hindered by a vicious circle; commercialization of a particular instrument can only be profitable when a sufficient number of laboratories express an interest in it, and this wider acceptance is inhibited by the slow diffusion of the technology. We hope that by discussing possible areas of application for advanced instrumentation we will accelerate the diffusion of these technologies and eventually expand the repertoire of technology available to the plant physiologist.

B. Working with Intact Plants Most fundamental discoveries on the photosynthetic electron transfer chain have been made using chloroplasts, or more purified sub-fractions (down to isolated enzymes) prepared from them. This attention to simpler systems is not surprising considering the relative ease with which isolated materials can be introduced into conventional instruments, manipulated by addition of chemicals that modify or inhibit

David M. Kramer and Antony R. Crofts certain partial reactions, separated and purified by biochemical techniques, concentrated to improve signal intensities, and so forth. The techniques of isolation necessarily disrupt or modify the natural state of the material, and the specificity of assays is partly dependent on the availability of isolated or purified materials, and ipso facto on the invasive techniques required for their preparation. In contrast, the study of photosynthesis in its physiological context requires that the living tissue be disrupted as little as possible. This requires the use of non-invasive techniques. To date, such biophysical approaches have depended mainly on use of photometric methods. Living organisms are complex, and parameters available for measurement often reflect the contributions from overlapping or interacting processes, making the results difficult to interpret. Apart from making a measurement with the required sensitivity, the experimentalist’s main concern is to deconvolute the signal of interest. Because of the complexity of the system, it is often more efficient to use a method of measurement which can assay a specific single phenomenon rather than a ‘rich’ signal reflecting many processes. As long as the measurement is specific and its interpretation certain, it can be related to other specific measurements of limited but different scope. However, such measurements generally call for special equipment which is not available commercially, and this has limited their application. In addition, certain phenomena are not readily accessible using this approach. The most obvious example is the control of light distribution through photon dumping in the pigment bed ( and its enhancement through formation of antheraxanthin and zeaxanthin) where the underlying mechanism is expressed through changes in fluorescence. Here, assay requires use of a relatively non-specific measurement (chlorophyll fluorescence), and the user should ensure that experiments are made under carefully controlled conditions which must eliminate, or allow deconvolution of, the potentially overlapping contributions from extraneous processes.

II. Control of the Photosynthetic Electron Transfer Chain Plants in the field have to contend with illumination intensities which vary by several orders of magnitude over the day, and over the depth of the canopy (see

Chapter 2 Measurement of Photosynthetic Electron Transport Chapter 13), and with variations in water (see Chapter 14), nutrient availability (see Chapter 11) and temperature (see Chapter 15). They cope with this variation through a photosynthetic apparatus which is basically the same in all leaves, and have evolved a range of physiological mechanisms which are tuned so as to match the delivery of excitation energy to the availability of substrates (most obviously in the metabolic pools (Weis and Berry, 1987; Genty et al., 1989; Foyer et al., 1990b; Krause and Weis, 1991; Demmig-Adams and Adams, 1992). At light levels below saturation, the light-harvesting apparatus acts to funnel excitation energy to the reaction centers, fulfilling its classical text-book role; photosynthesis is light-limited, and the apparatus must be tuned to maximize the throughput of energy (reduced cofactors and ATP) to the reactions. However, saturated rates of photosynthesis can often be reached at levels of illumination several fold less than full sunlight. At higher intensities, the excess excitation is potentially harmful, and has to be contained. What limits photosynthesis underthese conditions? How is the apparatus designed to ensure that the delivery of metabolic energy to the reactions is matched to the availability of nutrients, water, ? The turn-over of the photochemical and electron transfer reactions must be fine-tuned to allow adequate rates while at the same time protecting the photochemical reaction centers from photooxidative damage from the excess photon flux, and the intermediate chain from inhibition due to backpressure from the proton gradient as ATP utilization becomes limited.

A. Role of Lumenal pH It is clear that a major role in control of flux through the photosynthetic apparatus comes from lumenal pH. The modulating effects of lumenal pH have been well established for several important reactions, (i) Fluorescence lowering by (Murata and Sugahara, 1969; Wraight and Crofts, 1970; Briantais et al., 1979; Oxborough and Horton, 1988), and violaxanthin de-epoxidation (Hager, 1969; Yamamoto and Kamite, 1972; Demmig-Adams, 1990) which are both turned on as the lumenal pH drops, (ii) The electron transfer reactions associated with S-state transitions, cytochrome bf complex and plastocyanin. The reactions of the oxygen-evolving complex associated with the S-state transitions are inhibited (Wraight and Crofts, 1971; Wraight et al. 1972;

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Bowes and Crofts, 1981) or destroyed (Ono et al., 1992; Krieger and Weis, 1993) by low pH. Also, the turn-over of the cytochrome bf complex (BougesBocquet, 1981; Rich et al, 1987; Kramer and Crofts, 1993; Nisjhio and Whitmarsh, 1993) is slowed as the lumenal pH drops; we have demonstrated how the poise of the plastoquinol oxidizing reactions is controlled by lumenal pH (Kramer and Crofts, 1993). Lastly, the mid-point potential of plastocyanin (PC) is sensitive to pH, showing a pK on the reduced form at 5.5 in higher plants (Sykes, 1985) (or at about 6.5 in Chlamydomonas reinhardtii, D. Kramer, unpublished). This lowers the equilibrium constant for forward electron transfer to as the lumenal pH falls below these values. A related effect is the instability of plastocyanin at low pH (e.g. Gross et al., 1994; D. Kramer, unpublished).

B. What Do We Need to Measure? The effects of lumenal pH modulate excitation delivery and photosynthetic electron transfer so as to prevent build up of states which are inhibitory or sensitive to photodamage. Just how the control is achieved is one of the major unanswered questions of plant physiology. To understand the system we must be able to assay the reactions, and one of the main factors to be addressed is the adequacy of instrumentation for making the necessary measurements. It is in this context that several factors pertinent to the present chapter need to be stressed, (i) In order to determine control features, it will be necessary to measure the fluxes of the partial reactions, and the poise of reactants, under steady-state conditions in intact plants. This is possible with existing instruments, but these are available in only a few laboratories, (ii) Experience with chloroplasts has led to the idea that, under conditions of coupled steady-state electron flow and phosphorylation, the proton motive force is contributed largely or entirely by However, several workers had earlier questioned whether this was true in more intact systems. It is clear from the relatively high rates achieved in the controlled steady-state at high light that none of the reactions controlling flux is severely inhibited. The same conclusion can be drawn from measurement of rereduction of from the poise of the couple, and the stability of plastocyanin (see below). It is difficult to see how this could be true if the lumenal pH falls too low. It will be important to establish values for the

30 components of and the poise of the ATP synthase reactions in the steady state in intact systems to test the textbook dogma, (iii) Measurement of changes in through the electrochromic change are straightforward, but we do not have a good method for measurement of the gradients in dark adapted materials which provide the base-line data to which changes must be referred (the and maintained in the dark); nor do we have any good way to compensate steady-state measurements for overlapping light-scattering changes (see below). (iv) Measurement of lumenal pH in intact systems will be difficult. One approach is to use the pH dependence of the partial reactions as assayed in vitro, and then to determine the lumenal pH from measurement of rates in vivo, (v) There are numerous other parameters related to control of the metabolic pathways, some of which (the redox poise of ferredoxin and the NADP system, the poise of the thioredoxin system and the degree of activation of the enzymes under its control) may be accessible by photometric measurements.

C. The Steady-state 1. Flux It is axiomatic for a linear system of reactions that the concentrations of intermediates do not change in the steady-state, and that the stoichiometrically normalized rate of all partial reaction is the same, reflecting the input and output fluxes. To a first approximation, these considerations apply to photosynthesis. For practical purposes, the flux should be measured by the most convenient method. For terrestrial systems, this has usually involved measurement of oxygen production or using electrodes or infrared gas analysis. The instrumentation and methodology are well established, but fall outside the scope of this chapter (but see Chapter 8). The simplification assumed in treating photosynthesis as a linear system ignores many complicating features. Several cycles are tacked onto the linear chain (the Q-cycle around the bf-complex, the cycle around PS I, the fixation cycle, the glycolate cycle) each kinetically matched to the linear flux through appropriate stoichiometric factors. In addition, the pathways for N, P and S assimilation must also be concerted with C-pathway to sustain net photosynthesis. Nevertheless, for a constant nutrient

David M. Kramer and Antony R. Crofts input, the subsidiary cycles and pathways will maintain a flux in constant proportion with the main flux. Much work has gone into identifying the fluxes through these coupled processes by using alternative methods to assay the steady state flux through partial reactions. These methods have depended on perturbation of the steady state (see below). The rates of individual reactions are controlled by the concentrations of reactants, and the rate constants.

2. Poise The poise of intermediate reactions in the steady state provides useful information about the energetics and the control mechanisms of the system. If the poise of reactants differs from the value expected from the equilibrium constant (or value), either the rate constants are not large enough to keep up with the flux (usually indicating an allosteric control on the enzyme(s) involved), or additional workterms are contributing to the poise (classical ‘crossover’ points). Both conditions are of interest from the point of view of control. For photosynthetic reactions in intact plants, assay requires measurement of the redox poise of individual couples, of the separate components of the proton gradient against which the redox loops work, and of the poise of the ATP-synthase reactants. The limited attempts which have been made to measure these parameters will be discussed below. The major difficulties come from imprecision in measurement of individual reactions, (i) In measurements of redox poise using spectrophotometric methods, it is necessary to deconvolute the overlap of absorbance changes from concurrent processes, (ii) In fluorescence measurements, the many processes contributing to changes in fluorescence yield have to be sorted out. (iii) In measurements of metabolites, problems relate to the difficulties of separating different cellular types, and intracellular compartments, and of distinguishing between free and bound forms of metabolites and ions. These latter problems are outside the scope of this review, and will not be discussed further.

3. Perturbation of the Steady-state If a system in the steady state is perturbed, the poise of intermediates adjusts to reflect the change in flux. In photosynthetic systems perturbation is readily achieved by changing the level of illumination, and much useful information about mechanism has been

Chapter 2

Measurement of Photosynthetic Electron Transport

obtained by using this approach. The system can be maintained as near to the true steady state as desired by varying the ratio of light to dark periods in a repetitive illumination regime. Although the transient kinetics will be smaller as the dark period is made shorter, they can still be measured accurately because the approach is well adapted to averaging techniques. As with measurements of poise in the steady-state, the major difficulties come from imprecision in measurement of individual reactions because of the overlap of absorbance changes from concurrent processes, or in fluorescence measurements from the many processes contributing to changes in fluorescence yield. Inherent complexities make simplistic interpretation dangerous. For reactions close to the driving process (the photochemical reactions), the change in concentration of reactants on turning off the light reflects the steady-state rate. However, for processes removed from the driving process changes of reactant concentrations might be misleading because of the buffering by intermediate pools.

D. Measurement of Mechanism through Transient Kinetics While measurement of photosynthetic flux, and the interactions with metabolism through which flux is controlled, require probing of the steady state, more mechanistic problems are best tackled through measurement of transient kinetics. The main advantage lies in the fact that processes can be deconvoluted in the time domain, making possible a finer discrimination than measurements which rely on specific absorbance changes or fluorescence in the steady state or perturbed steady state. By appropriate choice of protocol, it is often possible to explore interactions with physiological process occurring over a different time scale than the reaction under assay, so that the utility of the approach can be extended beyond the exploration of the mechanism of partial reactions.

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thermoluminescence. Although in many systems optical methods are non-invasive, in photosynthetic systems the measuring beam may prove to be sufficiently actinic to perturb the system, and special care must be exercised to minimize such effects.

1. Fluorescence Fluorescence changes are associated with changes in photochemical flux as reaction centers open and close, and with various quenching states. The difficulties associated with use of fluorescence reflect the large number of processes which, either directly or indirectly, can affect the fluorescence yield, and are discussed extensively below.

2. Delayed Fluorescence Delayed fluorescence depends on the repopulation of the singlet state through back-reaction from stored electrons and holes close to PS II. The probability that back reactions will populate the singlet level depends on the energy conserved in the secondary donor and acceptor pools, the extent to which the poise of reactants is displaced by and the stimulating effect of in reversing the electrogenic reactions of the photosystem. The potential utility of such a direct relation to stored free-energy is diminished by the fact that the fate of excitons in the pigment bed depends on the fluorescence yield, so the yield of delayed fluorescence is subject to the same vagaries that limit the usefulness of fluorescence.

3. Thermoluminescence

III. What Reactions Can We Measure?

Thermoluminescence (TL) also reflects the energy levels of stored electron-hole pairs, but these are stimulated to recombine by providing thermal energy (by heating the system) to push the traps over the activation barriers separating them. At any particular rate of heating, the temperature at which the traps recombine is characteristic for different depths of trap, allowing for identification of specific processes (see review in this series by Inoue, 1996).

A. Optical Techniques

4. Absorbance

Most measurements have been made using optical techniques, either visible or near IR absorbance spectroscopy, fluorescence, delayed fluorescence or

Absorbance measurements in intact systems have been used to follow the specific absorbance changes due to redox reactions of couples in the electron

32 transfer chain (P700, P680, or both in the 800 nm region, cytochrome f, high and low potential forms of cytochrome plastocyanin), zeaxanthin formation associated with the xanthophyll cycle, electrochromic changes reflecting the electrogenic events of the photosynthetic chain or turn-over of the ATPsynthase, and light-scattering changes associated with development of a low lumenal pH. Thermal radiometry techniques including photoacoustic spectrometry have been used to probe the fate of excitation energy as an alternative approach to measurement of flux. Another approach has been the measurement of light-induced changes in blue fluorescence, in part due to redox changes on the acceptor side of PSI (mainly the reduction of ).

IV. Instrumentation and Measurement In the past several years, development of new instruments has progressed along several lines. The present state of the art in instrumentation is summarized in Table 1, which indicates which tools are available to probe individual photosynthetic partial reactions or the total photosynthetic flux. Several issues are emphasized by depicting the instrumentation this way. (i) The widely used, commercially-available instruments are most useful for measuring flux. Although these are useful instruments, a full understanding of what they measure will require a finer dissection of the electron transfer chain into individual partial reactions, (ii) A much wider range of techniques is available for measurement of flash-induced transient, or of initial rate, in isolated materials, and these techniques are generally more specific (i.e. they measure specific partial reactions). Some of these have been applied to work with intact plants, (iii) Few of these transient methods have been applied to the measurement of the steadystate in intact plants. However, many could inprinciple be applied to the interrupted steady state, and we can expect the introduction of new techniques which exploit this potential.

A. Fluorescence Yield Changes in Intact Plants Fluorescence induction in green plants is a seductive technique; the relative ease of measurement, and the obvious richness of information have resulted in an extensive literature of patchy quality. It is clear that

David M. Kramer and Antony R. Crofts at least ten phenomena contribute directly or indirectly to changes in fluorescence yield, and overlap in time, (i) Fluorescence changes associated with photochemical quenching in open reaction centers. Reaction centers ‘close’ as the acceptor, becomes reduced (either on reduction of the plastoquinone pool or because the photon flux exceeds the capacity for reoxidation), and the fluorescence rises, (ii) Centers can also become ‘closed’ through oxidation of the primary donor, but in this case fluorescence remains low because acts as a static quencher. It has generally been assumed in measurements with intact plants that this process is negligible, but this has not been adequately tested. (iii) The oxidized plastoquinone of the pool, is a quencher, which has been largely ignored in most previous work, but has recently been shown to contribute significantly to the quenching under conditions where PS II efficiency is high. (iv) Formation of chlorophyll triplets causes a quenching, which can be significant at high light intensities. (v) Fluorescence lowering, or quenching associated with the dumping of excess excitation energy under conditions of high light, which follows the generation and decay of low lumenal pH. (vi) Fluorescence lowering is modulated by formation of zeaxanthin and/or antheraxanthin, and may require that these de-epoxidation products of violaxanthin are present. The de-epoxidase of the lumen shows a strong dependence on pH, with increased activity as the pH falls below ~5.5. (vii) Irreversible quenching associated with photoinhibition which occurs when PS II is damaged. (viii) The redox state of the acceptor pools in intact systems reflects the state of activation of the assimilatory pathways, and also the effect of the back pressure from the proton motive force (back pmf) on the differential rate of filling and emptying of the quinone pool. (ix) The redox state of the donor side reactions is also strongly dependent on the lumenal pH, since protons are generated in the lumen is a product of water oxidation, and their activity enters directly into the mass action equation. Because the redox potentials of the partial reactions are not much lower than that of the couple at neutral pH, the equilibrium constants of the donor side are modulated so as to favor oxidation of P680 as the internal pH drops. (x) State transitions lead to changes in fluorescence associated with changes in absorption cross-section distribution between the photosystems. If fluorescence techniques are to be used to probe

Chapter 2

Measurement of Photosynthetic Electron Transport

specific photosynthetic functions, it is necessary to disentangle the contributions from all these different process.

B. Deconvolution of Components Contributing to Fluorescence Yield Changes The main goal of much recent research has been to find techniques which allow the contributions of different processes to fluorescence yield changes to be separated. The last five processes are nonphotochemical; conventional protocols can be used to distinguish the quenching arising from their operation from the quenching associated with the redox state of which is the main contribution to photochemical quenching Weis and Berry (1987), Weis et al. (1987), Genty et al. (1989) and Edwards and Baker (1993), building on earlier work (Krause et al., 1982; Bradbury and Baker, 1984; Quick and Morton, 1984), have had some success in extracting quantitative information from fluorescence induction curves by using a pulse of strong light superimposed on the measuring beam to probe the fractional contribution of This allows a deconvolution of contributions from and and an estimation of yield, and many groups have adopted this or similar techniques. Nevertheless, since only one experimental variable (fluorescence) is measured, it is clear that deconvolution on the basis of two conditions is simplistic, and that resolution of the variables corresponding to all the phenomena above can only be achieved by separate independent conditions of measurement which select for dominance of one of these processes at a time. It is not always obvious that this has been done, and in some cases the simple analysis clearly breaks down (Rees and Morton, 1990). Kramer et al. (1995) have noted that the saturating pulse (~ 1 s at > 10,000 ) used to ensure reduction of also causes reduction of the plastoquinone pool, giving rise to an additional change in yield which is especially important under conditions (e.g. with dark adapted material, or in the steady-state at low light intensity) under which the pool is oxidized. This difficulty can be circumvented in part by using a short (<10 ) saturating flash to reduce and assaying the fluorescence yield during the flash, or by extrapolating from the decay over the ~ 100 range (Kramer et al., 1995). We discuss the problem of deconvolution in the context of instrumentation below.

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C. Steady-state Fluorescence Measurements 1. Down Regulation of Photosynthetic Electron Transport Under high light intensities, the energy input from photon flux exceeds the availability of and the plant responds by switching on processes, quenching and de-epoxidation of violaxanthin, which shunt a fraction of the excitation energy away from the photochemistry as heat. These processes give rise to a decrease in quantum yield of photosynthesis, and a loss of fluorescence yield, because the thermal pathways compete with photochemical and fluorescence pathways for the excitation energy. This loss of fluorescence is the main component of nonphotochemical quenching. When water availability dictates stomatal closure, the availability of is further reduced, and the fraction of excitation dumped as heat increases to match, giving rise to a downregulation of photosynthesis (reviewed by Foyer et al., 1990b). The quenching state follows the generation of a low lumenal pH (Wraight and Crofts, 1970), and is rapidly reversible on darkening, allowing a rapid response to changing illumination. The amplitude of the quenching depends on the extent of de-epoxidation of violaxanthin (Gilmore and Yamamoto, 1993; Bilger and Björkman, 1994); the de-epoxidation products are formed more slowly, and are lost by epoxidation only after prolonged darkening (Demmig-Adams, 1990; Bilger and Björkman, 1994), thus allowing a modulation of the quenching response to suit the illumination intensity and the state of metabolic sinks. Under natural conditions, the fluorescence quenching and substrate availability are well matched, excess excitation energy is shunted to dissipative pathways, and net damage to the photosynthetic apparatus is avoided. Under prolonged intense illumination, a response is observed in which damage to PS II can occur at a rate which exceeds the capacity for repair, and this gives rise to a loss of photosynthetic capacity which is not rapidly reversible. We will refer to this condition as photoinhibition (Jones and Kok, 1966a,b), although recognizing that this term is sometimes used more widely to include the reversible down-regulation. Most studies of photoinhibition have involved plants grown under controlled conditions in growth chambers; it is not clear that native plants acclimated to natural conditions suffer any significant loss of

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David M. Kramer and Antony R. Crofts

Only those techniques currently in use or with a reasonable likelihood of applicability in intact plant work are presented. Key: N, no; N/ A, not applicable; P, possible (in principle) but has not yet been demonstrated; Y – yes, has been shown to be applicable; ?, likely to be difficulties in applying to intact plants; a, measurements solely at one wavelength in the near IR most likely represent a mixture of contributions (e.g. from PC, P680, P700); b, commercial machine available; c, application to intact leaf work is problematic due to the significant absorption of UV radiation by the leaf surface; d, in the steady state, a mixed population of S-states is expected, making it difficult to distinguish between the individual S-state reactions, and complicating measurements of the kinetics of reduction; e, the existence of oxygen sinks and significant barriers to diffusion, hinders the application of oxygen polarography in intact plants - steadystate measurements are also made difficult by the requirement for a restricted cuvette volume; f, in intact leaves, it is not possible to

Chapter 2

Measurement of Photosynthetic Electron Transport

photosynthetic capacity from photoinhibitory damage, because they are protected by the physiological mechanisms above (Wise et al., 1990; Gilmore and Björkman, 1994). Photoinhibition may become important when additional stresses (chilling, drought) render the plant more susceptible to damage because of secondary inhibitory effects; this is certainly a problem for some crops, especially when these are grown outside their natural range (cf. Nie et al., 1995). In chloroplasts, photoinhibition is exacerbated by any treatment which either inhibits the donor-side reactions so that strongly oxidized intermediates (including ) accumulate (Chen et al., 1992; Yerkes and Crofts, 1992b), or blocks the acceptor side sufficiently to overreduce and pheophytin (cf. Kirilovsky et al., 1994). A similar condition follows UV irradiation (Jones and Kok, 1966a,b), possibly through damage to Y161 of D1 (Yerkes et al., 1990), or the Mn-cluster, giving rise to UV-photoinhibition. Damaged PS II centers appear to contain a fluorescence quenching species, since a second type of long-lived non-photochemical quenching develops (qI-quenching), which is associated with photoinhibitory damage. As long as these damaged centers are connected to the main antenna bed, will provide a last line of defense against further photodamage by introducing an additional pathway for dissipation of excitation through thermal

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pathways. By sacrificing some centers, the remaining apparatus is protected against further damage. When damage occurs, centers are recycled through a mechanism involving proteolysis of the D1 protein (and possibly others), and reassembly of an active PS II (Sundby et al., 1993; Leitsch et al., 1994; Chapter 4). Apparently, evolution has taken advantage of the repair pathway to allow damaged centers a ‘heroic’ protective role by incorporation of a quenching species in the residual complex. The molecular identity of this quenching species is not known.

2. Quenching of Fluorescence Associated with the Proton Gradient Fluorescence lowering was first reported by Murata and Sugahara (1969), and the relation to the proton gradient was characterized by Wraight and Crofts (1970), who showed that quenching depended on the pH gradient generated across the thylakoid membrane. Following the early work of Krause (1973), a similar quenching has since been implicated in the physiological protective mechanism by which plants cope with high light (Weis and Berry, 1987; Genty et al., 1989; Foyer et al, 1990b; Krause and Weis, 1991; Demmig-Adams and Adams, 1992). Quenching can also be induced by the pH gradient generated by ATP hydrolysis (Gilmore and Yamamoto, 1992). It seems

distinguish between and since approximately half of the sites is occupied by semiquinone even in the dark, but it may be possible to oxidize this semiquinone using far-red illumination (Kramer et al., 1990); g, in the steady state, a mixed population of redox states is expected, therefore it will probably not be possible to distinguish between and using these techniques; h, signals are small, requiring instrumentation of high sensitivity, but with low actinic effect, such as the double-flash kinetic spectrophotometers - steady-state measures of the redox state of the cytochromes would be difficult due to large interfering light scattering changes, but application of saturation pulses in the steady-state may prove interesting; i, measurements of the slow rise in the electrochromic shift due to the turnover of the cytochrome bf complex are only clearly resolvable when the decay of the electrochromic shift is slow, this precludes such measurements in many cases, such as when the chloroplast coupling factor is reduced or activated; j, significant ambiguities in interpretation of data; k, in intact chloroplasts only (not in thylakoid membranes); 1, there are significant ambiguities in the deconvolution of the oxygen signal from other pressure signals; m, the signal is small and difficult to distinguish from those of the electrochromic shift or cytochrome f, making application to intact systems, particularly in the steady-state, difficult, n; the largest, most easily accessible absorbance signal, yet requires instrumentation of high sensitivity—could be used as a probe of PS I and PS II flux in the steady state by application of saturation pulses; o, an indirect probe of the electrical field across the thylakoid membrane, and the largest, most easily accessible absorbance signal. However, it still requires instrumentation of high sensitivity. In the steady-state or other conditions where the coupling factor is activated, the rise of electrochromism due to the turnover of the cytochrome bf complex is masked by the decay. The fast rise phases could be used as a probe of PS I and PS II flux in the steady state by application of saturation pulses; p, cannot be ruled out for application to intact plant work, but probably too invasive; q, measurement easy to make, but very indirect and many outstanding issues remain about interpretation of data; r, indirect method. Causative relationship between scattering changes and remains unclear; s, direct measure, does not measure flux through the photorespiratory, or other fixing pathways; t, indirect measure, estimates flux only through the PS II centers; u, direct measure, present techniques using measurements at only one wavelength could give artifactual results due to interference from other species; v, indirect, highly sensitive measure of energy storage, but deconvolution of contributing factors is not yet clear - also, required sample volume and geometry restricts application to in vivo system; w, most direct measure of energy storage, but relatively low sensitivity - well suited for in vivo and field studies

36 likely that all these dependent quenching phenomena reflect the same process, but the mechanism of quenching is still unclear. Two separate but interrelated phenomena are involved: quenching itself, and a modulation or amplification of the quenching by zeaxanthin and antheraxanthin formation (Hager, 1969; Yamamoto and Kamite, 1972; Demmig et al., 1987). These processes are distinguishable on the basis of differential effects of inhibitors, different light intensity dependencies, and an ascorbate requirement for zeaxanthin formation (Yamamoto, 1979; Oxborough and Horton, 1987; Demmig-Adams, 1990; Ruban et al., 1994). Three main classes of mechanism have been proposed to account for the associated with the proton gradient, (i) donor-side mechanisms, (ii) acceptor-side mechanisms, and (iii) antenna mechanisms. In donor-side mechanisms, the quenching is thought to reflect an inhibition of the oxygen-evolving reactions (Crofts et al., 1971; Bowes and Crofts, 198 l; Weis and Berry, 1987; Schreiber and Neubauer, 1990; Krieger et al., 1992). Such an inhibition would lead to formation of which is a quencher. Accumulation of oxidized states in the donor-side complex would be consistent with the stimulation of delayed fluorescence at times <5 ms under similar conditions (Wraight and Crofts, 1971). Quenching associated with acceptor-side mechanisms is suggested to involve pheophytin reduction and triplet formation, which occurs when the pool is fully reduced and becomes over-reduced (Vass et al., 1992; Kirilovsky et al., 1994), or a rapid dissipative cycle around PS II in which electrons from the reduced acceptor complex ( or ) are passed back to the donor side through a pathway involving cytochrome b-559 (Nedbal et al., 1992). Since a fraction of is always present in the oxidized form, even under intense illumination, it seems unlikely that the acceptor side ever achieves the degree of reduction required by these mechanisms, and Meunier and Bendall (1993) have shown that a cycle round PS II does not seem to be a significant pathway. Nevertheless, a slow cycle around Photosystem II could provide relief from overreduction of the acceptor complex (Poulson et al., 1995). Yerkes and Crofts (1992a) showed that quenching does not require an active PS II. In these experiments PS II was prevented from turning-over by DCMU inhibition, and a was generated

David M. Kramer and Antony R. Crofts through PS I. Using a weak measuring flash to detect fluorescence yield, it was shown that the quenching on illumination was not significantly dependent on the state of the oxygen-evolving complex (OEC) at time of illumination, set by 0 or 1 flash before addition of DCMU (Bowes and Crofts, 1981). Also quenching occurred with a normal rate and amplitude when the photochemistry of PS II was blocked by treatment with and DCMU, or when the extrinsic proteins of the OEC had been removed by Tris-treatment. With the latter treatments, the fluorescence yield returned to the maximal level after illumination, indicating that the quenching was not due to a dissipative cycle. Thus neither the Sstate transitions nor the turn-over of PS II were required for and no significant part of the quenching could be attributed to formation, eliminating mechanisms of the sort discussed in (i) and (ii) above. In quenching associated with the antenna, the mechanism is suggested to operate at the level of the light-harvesting apparatus. Horton and colleagues have been early champions of this idea, and have suggested various mechanisms by which quenching could occur in the light-harvesting complexes (Rees et al., 1989; Foyer et al., 1990b; Horton et al., 1991; Ruban and Horton, 1992; Horton et al., 1994; see also Chapter 1). They showed that aggregation of isolated LHCIIb in detergent solution led to a quenching of fluorescence, and proposed this as the most likely mechanism. More recently, emphasis has shifted to the role of the minor light-harvesting complexes, CP29, CP26, and CP24 (LHCIIa, LHCIIc, LHCIId, respectively) which seem a more likely site (see below).

3. Role of Donor-side Oxidation in Photoinhibition Stimulation of delayed fluorescence by shows that the oxidation potential in the donor-side reactant pool is substantially increased as the lumenal pH falls. Lowering the lumenal pH will likely change the equilibrium constant between the S-state reactants and the couple (Bowes and Crofts, 1981), because the S-state transitions involve release of protons (Fowler, 1977; Saphon and Crofts, 1977). Experiments with -treated (Yerkes and Crofts, 1992b) or Tris-treated (Chen et al., 1992) chloroplasts, in which the Mn-center has been disrupted and the extrinsic proteins of the oxygen-evolving complex have been removed, are photoinhibited even in

Chapter 2

Measurement of Photosynthetic Electron Transport

relatively weak light, indicating that centers in which is stabilized are especially susceptible to photooxidative damage. Although quenching by does not seem to be involved in fluorescence lowering, it is clear that the possibility of forming on generation of a low pH local to donor side of PS II represents a potentially hazardous state. An exciton dumping mechanism in the antenna complex would provide protection against damage by intercepting the excess photon flux before it reached the reaction center.

4. Where is the Antenna Quencher Located? If -quenching reflects a mechanism for dumping excitation energy before it gets to the reaction center, then the location must be in the light-harvesting antenna. To be effective as a protective mechanism, the pathway must compete with the reaction center, with a rate for the deexcitation process in the low or sub-picosecond range. The antenna has three main components: the CP43 and CP47 subunits of the reaction center complex; the bulk antenna consisting of trimers of LHCII in up to 30-fold subunit excess over the PS II reaction center; and the minor chlorophyll-protein complexes, CP24, CP26 and CP29, (about one per reaction center), which can associate with either of the other complexes, and probably form an interface between them (Bratt and Åkerlund, 1992; Dainese et al., 1992; Bassi et al. 1993). Previous mechanisms have considered the quenching to reside in the bulk LHCII (LHCIIb) (Horton et al., 1991; Owens et al., 1992; Ruban and Horton, 1992; Mullineaux et al., 1993; but see Section IV.5.b). An efficient quencher would not need to be present at a concentration greater than one per PS II. If the mechanism involves a vibrational deactivation pathway (with sub-ps half-time), then we can be sure that the quencher is not present in all light-harvesting complexes, since quenching affects mainly the variable fluorescence (1–5 ns life-time), and components of (with life-times in the 30–500 ps range) are not markedly quenched (Krieger et al., 1992). Either an efficient quencher is formed with weak statistical probability in the main complexes, an inefficient quencher is formed in most complexes, or an efficient quencher is formed at a specific site in some component of the antenna apparatus with a stoichiometry of ~ 1 per reaction center. The most likely sites are the minor chlorophyll-

37

protein complexes, CP24, CP26, and CP29 (Bassi et al., 1993; Crofts and Yerkes, 1994).

5. Evidence for a Role for the Minor Lightharvesting Complexes a. Quenching Associated with Formation of Zeaxanthin Early studies showed formation of zeaxanthin from violaxanthin under conditions similar to those leading to qp quenching (Hager, 1969; Yamamoto and Kamite, 1972; Demmig-Adams, 1990). Although -quenching can occur in the absence of zeaxanthin (DemmigAdams, 1990), Horton and colleagues (Rees et al., 1989; Ruban et al., 1992) have shown that the presence of zeaxanthin in leaves preilluminated before rapid preparation of chloroplasts correlated strongly with an enhanced level of -quenching, which otherwise showed properties similar to those in the absence of zeaxanthin. Gilmore and Yamamoto (1993) have shown that the extent of -quenching correlates with the sum of zeaxanthin and antheraxanthin (the intermediate between violaxanthin and zeaxanthin), and have suggested that the quenching mechanism requires the presence of one or the other. Antheraxanthin levels do not decay to zero even after extended dark periods, and could account for the -quenching which is seen in the absence of zeaxanthin. Although the de-epoxidation products might be necessary for quenching, they are clearly not sufficient, since onset and decay of the quenching state on illumination follow the pH gradient, and show no correlation to changes in the xanthophyll cycle intermediates (Bilger and Björkman, 1994). A number of laboratories have looked at the distribution of carotenoid pigments among the different light-harvesting chlorophyll protein complexes (Dainese et al., 1992; Bassi et al., 1993; Gilmore and Yamamoto, 1993; Ruban et al., 1994). Violaxanthin, the precursor of zeaxanthin and antheraxanthin in the xanthophyll cycle, partitioned predominantly into the smaller complexes, CP29, CP26 and CP24. Authors differ on the distribution of violaxanthin in LHCII; Bassi et al. (1993) found little or no violaxanthin in LHCIIb or in the PS II reaction center complexes CP43, CP47, or the D1/ D2 cytochrome b-559 core, but Ruban et al. (1994) found that 10–15% of the carotenoid content of the main LHCII (LHCIIb) was contributed by the xanthophyll cycle components. Thayer and Björkman

38 (1992) noted that the xanthophyll cycle components were equally distributed between Photosystems I and II. From sequence studies (Green et al., 1992), the smaller complexes are clearly closely related to the major LHCII components; however, they are more closely associated with PS II, and can coisolate with either PS II or the bulk LHCII trimers on biochemical separation (Bassi et al., 1993). A mechani sm for -quenching has been proposed by Owens et al. (1992; see also Chapter 1) based on the change in singlet energy levels on de-epoxidation of violaxanthin to zeaxanthin, relative to the chlorophyll lowest singlet level, and modulation of the energy levels by low lumenal pH. They suggest that this would be a relatively inefficient pathway occurring in a main fraction of light-harvesting complexes. In support of this idea, they indicated that maximal non-photochemical quenching involves accumulation of 75–100 zeaxanthins per PS II reaction center. This is greatly in excess of the stoichiometry of the minor CPs, and would require that a large fraction of zeaxanthin must reside elsewhere, possibly in LHCII complexes. However, although zeaxanthin formation can occur in excess of the binding sites available in the minor CPs, under conditions leading to maximal -quenching, the formation of zeaxanthin is less than maximal, and is preferentially associated with the minor CPs (Bassi et al., 1993).

b. -quenching in Strains Depleted in the Bulk LHCII A location for the quenching mechanism in the minor CPs is strongly supported by experiments with barley chlorina mutants lacking chlorophyll b, and with intermittent-light grown plants, which make little or no LHCIIb, but both of which have a normal complement of minor CPs, and a near normal quenching (Briantais, 1994; Falbel et al., 1994; Falk et al., 1994; Jahns and Krause, 1994; Andrews et al., 1995; Hartel and Lokstein, 1995). Although these experiments preclude the bulk LHCIIb as the location of -quenching, they indicate another interesting feature of the protective mechanism. Although quenching does not require the bulk LHCII, protection against photoinhibition becomes most effective only as LHCIIb becomes available to provide a connected antenna bed (Briantais, 1994; Falk et al., 1994). In the absence of LHCIIb, the quenching in individual centers is efficient (and provides an effective local

David M. Kramer and Antony R. Crofts protection), but the protective function provided by efficient quenching in one center can be shared among many centers only when the LHCIIb complexes provide excitation connectivity between centers.

c. Inhibition of -quenching by Dicyclohexylcarbodiimide Incubation with dicyclohexylcarbodiimide (DCCD) leads to formation of covalent DCCD adducts which are located in the smaller light-harvesting complexes, CP24, CP26, CP29 (Jahns and Junge, 1988; Webber and Gray, 1989; Ruban et al., 1992), and inhibition of q E -quenching (Ruban et al., 1992). Carbodiimides react preferentially with carboxylic acid groups, forming a covalent bond susceptible to hydrolysis in an aqueous environment. DCCD is a lipid soluble reagent, able to react with groups buried in the hydrophobic phase, where the covalent bond is protected from hydrolysis. This has directed attention to potentially reactive groups in the minor CPs, where several acidic residues have been identified on the lumenal side of folding models. From the structure of the LHCIIb complex (Kühlbrandt et al., 1994), and homologous alignment (cf. Green et al., 1992), Crofts and Yerkes (1994) identified in each of the minor CPs a buried glutamate residue which substitutes for a glutamine chlorophyll-ligand in the LHCIIb structure. These specific changes from neutral to acidic side chains would provide a suitable DCCD binding site in each of the minor CPs, and account for the differential susceptibility of the minor CPs to DCCD binding, and DCCD inhibition of quenching (Crofts and Yerkes, 1994).

6. Mechanism of Conclusions

-quenching—General

The -quenching is the main mechanism by which plants regulate the delivery of excitation to match metabolic demand. Although formation of P680+ following donor-side inhibition is not the mechanism of quenching, it seems likely that plants will have evolved strategies to avoid this potentially hazardous state by preventing the generation of a low pH local to the donor-side of PS II, and that an important role of fluorescence lowering is to dump exciton energy as heat before it reaches the reaction center of PS II and causes damage. It is clear that -quenching reflects a down-regulation of photosynthesis; it is a

Chapter 2

Measurement of Photosynthetic Electron Transport

reversible, physiological process, and it is useful to distinguish it from the irreversible quenching associated with damage to PS II centers, which we have labeled photoinhibition in this review. Since control is required under those conditions in which flux is maximal, the system must be poised so that quenching comes in before the lumenal pH falls low enough to damage the donor-side reactions, and inhibit other reactions of the chain. At the same time, a proton gradient sufficient to drive phosphorylation at the rate needed to sustain the maximal flux must be maintained, requiring a delicate balance, and possibly a role for the component of the gradient. The mechanism involves a change in state of the antenna complex as the lumenal pH is lowered (with a pK in the range 5.5 in higher plant chloroplasts), leading to dissipation of the singlet state through a thermal pathway which competes with both photochemical and fluorescence pathways. Association of zeaxanthin and antheraxanthin with CP29 and the other minor LHCs, amplifies, and may be necessary for, the effect of low lumenal pH in quenching the

39

fluorescence. However, although the de-epoxidation products may be necessary for the quenching state, the onset of quenching follows the development of low internal pH as the proton gradient builds up and not the formation of antheraxanthin and zeaxanthin. De-epoxidation is also enhanced at low lumenal pH, and therefore responds to the same feedback mechanisms that lead to quenching, but has a slower onset and a much longer decay; modulation of the level of the de-epoxidation products thus represents a mechanism to cope with more extended exposure. Structural models of the interface between PS II and the antenna (Bassi et al., 1993), and fluorescence emission spectra (Bratt and Åkerlund, 1992) suggest that the minor complexes serve as ‘bridges’ between the bulk LHCIIb and the reaction center antenna proteins (Fig. 1). In the light of the experiments with LHCIIb depleted systems, it seems clear, as suggested by Bassi et al. (1993), that the main action in quenching is at this interface, and involves CP29 (or the other minor LHCs), and not aggregation of LHCIIb.

40 7. A Hypothetical Molecular Mechanism We have recently suggested that the mechanism of quenching could in principle be quite simple (Crofts and Yerkes, 1994). Physico-chemical studies show that chlorophylls in solution at concentrations comparable to those in the leaf show no fluorescence, and this is attributed to an interaction between ‘statistical dimers’ which introduce additional energy levels allowing thermal pathways for de-excitation (Beddard et al., 1976). The chlorophylls in LHCII are held apart by ligation, with a variety of groups providing ligands (Kühlbrandt et al., 1994). We assumed a similar structure for CP24, CP26 and CP29 based on sequence homology, and noted that in each of the minor CPs, a liganding glutamine residue was substituted by a glutamate (Fig. 2). If an acidic chlorophyll ligand were accessible to from the lumenal phase, the liganding properties would change on acidification. Crofts and Yerkes (1994) suggested that such a change might allow the affected chlorophyll to interact at short enough range to form exciton-coupled bands with a neighboring pigment, and thus form a quencher of fluorescence. A change in ligand properties could either lead to release of a

David M. Kramer and Antony R. Crofts chlorophyll previously bound, specific ligation of a previously unbound chlorophyll, or an exchange of ligands. The model accounts economically for the dependence on low lumenal pH, the ligand residue changes between LHCIIb and the minor CPs, the preferential distribution of components of the xanthophyll cycle in the minor CPs, the inhibition of -quenching by DCCD and the specific binding of DCCD to the minor CPs, and is supported by the recent work on -quenching in plants with a reduced complement of LHCIIb. Bassi and colleagues (R. Bassi, personal communication; Giuffra et al., 1995) have recently made CP29 by synthesis of the apoprotein in E. coli and reconstitution with added pigments, and shown that the artificial complex has the spectral properties of the native CP29. A mutant complex with glutamate changed to glutamine to provide the configuration seen in LHCIIb showed a specific increase in absorbance in the chlorophyll bands, indicating formation of an additional ligand. Further experiments along these lines will serve to clarify the role of the glutamate residues identified by Crofts and Yerkes (1994) as of possible importance in the mechanism of -quenching and DCCD binding.

Chapter 2

Measurement of Photosynthetic Electron Transport

8. Is there a Similar Protective Mechanism to Down-regulate PS I? Sequence alignment (Green et al., 1992) with LHCIIb shows the same change in LHCI of a potential chlorophyll ligand from glutamine to glutamate which Crofts and Yerkes (1994) suggested might be important in the switching mechanism. The xanthophyll cycle reactions are also active in LHCI (Thayer and Björkman, 1992). This suggests that the excitation delivery to PS I might be controlled by an antenna switch similar to that in PS II. In the past it has been assumed that PS I quantum yield is ‘autoregulated’ because is a quencher. This would lead to efficient exciton dissipation in centers closed by formation of but there are circumstances in which an alternative regulation would be needed. Damage to the FeS acceptor complex of PS I occurs even at low light under chilling conditions (Sonoike et al., 1995; Byrd et al., 1995). The mechanism is not known, but requires aerobic conditions, and may involve a chilling-dependent block of acceptor reactions leading to over-reduction of PS I acceptors and formation of oxygen radicals. Over-reduction of the metabolite sinks might produce a similar condition physiologically (see Section IV.F). Under these circumstances would not form, and autoregulation would not be available. This suggests that it might be worth looking for conditions in which control by exciton dumping in the LHCI antenna complex occurs, which would be expressed (under conditions in which P700 is reduced) as a lowered efficiency on generation of low lumenal pH.

D. Measurement of Fluorescence 1. Modulated Fluorimeters Measurements of steady-state fluorescence have relied mainly on use of instruments with weak modulated measuring beams in which phase and frequency decoding have been used to isolate the fluorescence yield changes from the large fluorescence transients induced by continuous illumination (Ögren and Baker, 1985; Schreiber, 1986; Weis and Berry, 1987; Genty et al., 1989). A successful portable version based on LEDs was described by Schreiber and colleagues (Schreiber, 1986; Schreiber and Schliwa, 1987), and further development has led to commercial instruments which are now widely used (Bolhàr-Nordenkampf et al., 1989; Schreiber et al.,

41

1989). The rationale for the methodology, and protocols for use of this class of machine in the separation of contributions from photochemical and non-photochemical quenching of fluorescence yield have been extensively discussed elsewhere (Weis and Berry, 1987; Genty et al., 1989; Foyer et al, 1990b; Krause and Weis, 1991; Edwards and Baker, 1993). Variants of the LED-based modulated fluorimeters are those that use a xenon flashlamp(s) in place of LEDs for the probe beams; these are essentially pulsed kinetic fluorimeters (see below) used with a constant actinic light source (Yerkes and Crofts, 1992a; Schreiber et al., 1993). Since light from xenon flashlamps is extremely bright and can be filtered to give a probe beam over a wide spectral range, these instruments are useful for the study of materials at low concentrations and of algae with fluorescence excitation spectra which fall outside the red region of the LED emission.

2. Pulsed Kinetic Fluorimeters Pulsed kinetic fluorimeters probe specific partial reactions of the PS II donor and acceptor side reactions by selecting only certain kinetic components of the fluorescence yield change. This is done by giving a short single-turnover actinic flashes (the pulse) to start the photosynthetic reactions, and following the reopening of PS II by monitoring the associated fluorescence yield changes with one or a series of weak probe pulses. Depending upon the sequence of pulses given and the experimental time range, kinetics attributable to the functions of the oxygen-evolving complex, the oxidation of and the turnover of the two-electron gate can be observed by this type of instrument (Delosme, 1971;Joliot, 1974; Diner and Joliot, 1976; Bowes and Crofts, 1980; Taoka et al., 1983; Robinson and Crofts 1987; Kramer et al., 1990). Though some questions remain about the interpretation of these fluorescence changes, e.g. the long-lived period four oscillations in fluorescence yield, where no satisfactory model has been proposed (Robinson and Crofts, 1987; Shinkarev and Wraight, 1993; Shinkarev et al., 1994), in general, the nature of these reactions and their effects on fluorescence yield are better understood than those that influence steady-state fluorescence, and they are therefore less prone to misinterpretation. In the original double-flash technique (Joliot, 1974; Diner and Joliot, 1976; Bowes and Crofts, 1980; Robinson and Crofts, 1987), pairs of light pulses

42 were given to a sample of photosynthetic material. After a saturating single-turnover actinic flash (usually from a xenon flashlamp or a laser), a weak, nonactinic, measuring pulse (from a xenon flashlamp) was given at a variable time. In order to measure a kinetic trace, the experiment is repeated after a sufficient dark-adaptation to allow the system to relax, and the time interval between the actinic and measuring flashes is varied. Robinson (1986) demonstrated the feasibility of a field-portable, double-flash kinetic fluorimeter which he used to probe to electron transfer reactions in intact plants in the field. However, certain limitations of these instruments hindered their useful application to the study of intact systems. Because a single time point was sampled in each experiment, a kinetic trace required several experiments, one for each submillisecond time point in the trace. Furthermore, the inherent instability of the xenon flashes used for the measuring pulse limited the signal-to-noise ratio of these instruments, so that signal averaging was often required to obtain reasonable sensitivity. For experiments with thylakoids or cells in suspension, these difficulties could be overcome by use of fresh samples from a dark-adapted reservoir, but this option was not possible with leaves, making impractical many types of experiments, e.g. in studies of the oxygen-evolving complex, where the samples must be set to a particular state by dark-adaptation before the experiment. Another limitation of the conventional instrument comes from the extended trailing edge of the xenon actinic flashlamp, which lasts with significant intensity in relation the measuring flash for about 70 and swamps the signal from the measuring flash during this time. Kinetic information during this trailing edge is of interest because it contains information about the turnover of the oxygen-evolving complex and may yield a method of observing turnover of the S-states intact leaves. Kramer et al. (1990) developed an improved version of the instrument that overcame these limitation. The new design incorporated a reference channel, allowing measurements with very low noise (about 0.05% of ), thus eliminating the need for signal averaging, and detection as soon as 20 after the actinic flash. The design also incorporated three measuring flashes, which allowed three measurements in the sub-millisecond domain after each actinic flash, followed by a series of measurements as close as 5 ms, so that a complete kinetic trace from

David M. Kramer and Antony R. Crofts microseconds to seconds could be recorded in one experiment. The improved machine could therefore resolve several flash-number dependent kinetic phenomena in a single train of actinic flashes. Recently, we have developed an advanced version of this type of instrument, where the measuring xenon flashes are replaced with pulsed LEDs with 650 nm peak emission (DM Kramer, T Miller and L Nedbal, unpublished). This has several advantages. Measuring points can be given even more rapidly, currently at 10 intervals. The instrument is far less costly to construct. It is also considerably lighter and more portable. By pulsing the LEDs at high voltages, we are able to achieve signal-to-noise levels on the same order as with the xenon-based instrument. Data from this instrument is presented in Fig. 3 (see next section).

3. Interpretation of Data from Pulsed Kinetic Fluorimeters Sample data taken with a pulsed kinetic fluorimeter is presented in Fig. 3. After a single-turnover flash, fluorescence yield shows a transient increase, and then declines. Both the rise and fall consist of several kinetic phases which have been extensively studied in vitro. The fastest rise phase occurs on the nanosecond time scale, i.e. during the xenon flash and immediately after it. This phase is unresolved in the measurements with pulsed kinetic fluorimeters discussed here, and reflects a rapid re-reduction of the which is a quencher (Mauzerall, 1972; Butler, et al., 1979; Sonneveld, et al., 1979; Deprez, et al., 1983). This is followed by a rise occurring over tens to hundreds of microseconds. This phase most probably reflects a relatively small equilibrium constant (K ~5–15) between and and the subsequent loss of in the fraction of centers in which the reaction was incomplete, as becomes reduced by the S-states on this time scale (Robinson and Crofts, 1987). The period four pattern of this phase (Fig. 3,10 inset) was correlated by Delosme (1971) and Joliot (1974) to the concentrations of and predicted by the four S-state model of oxygen evolution proposed by Kok et al. (1970). Therefore, the extent and rate of this slower rise phase depends upon the particular state of the oxygen-evolving complex and has been used to monitor the turnover of this system in intact plants (Robinson and Crofts, 1987; Kramer et al., 1990). A period-four oscillation in the fluorescence

Chapter 2

Measurement of Photosynthetic Electron Transport

measured at longer times after the actinic flash (4 ms to seconds; Fig. 3, 4000 inset) has also been observed (Delosme, 1971; Joliot et al., 1971; Bowes and Crofts, 1980; Robinson and Crofts, 1983; Kramer et al. 1990) and has been related to the period-four turnover of the S-states (Joliot, et al 1971, Robinson and Crofts, 1983), although the cause of this quenching is still unclear (but see Shinkarev and Wraight, 1993; Shinkarev et al., 1994). The turnover of the acceptor side of PS II has been extensively studied by fluorescence measurement in the sub-millisecond range (reviewed in Crofts and Wraight, 1983). Briefly, in a dark-adapted leaf, the two-electron gate of PS II is expected to be in the state (ignoring the state of the donor side). After a single-turnover actinic flash, the state is formed rapidly, after which, an electron transfer from to can be observed with a half time of 150–250 After a second actinic flash, the state is formed and, following this, electron transfer from to to form plastoquinol at the site occurs with a half time of between 300–500

43

(Bowes and Crofts, 1980). This is reflected in the somewhat longer decay seen after even numbers of flashes (Fig. 3, 200 inset). The plastoquinol can then diffuse away from the site allowing another plastoquinone to bind. Thus, upon the third flash (and subsequent odd-numbered flashes), most sites are again in the dark-adapted state, giving rise to a binary pattern of decay rates for the state The slower decay phases of the fluorescence yield have been suggested to be related to the plastoquinone occupancy of the site and the time required for release of plastoquinol and replacement by plastoquinone (Robinson and Crofts, 1983, 1987; Taoka and Crofts, 1990; Crofts et al. 1993). Measurement of the kinetics under suitable conditions and their appropriate deconvolution can elucidate the binding constants for plastoquinone and plastoquinol species to the and the equilibrium constants for electron transfer from to the in its various forms, as has been demonstrated in the characterization of mutants of algae with modified properties (e.g. Crofts et al., 1993).

44

4. Use of Pulsed Kinetic Fluorimetry on Intact Plants With the introduction of field portable double-flash (Robinson, 1986) and multi-flash kinetic fluorimeters (Kramer et al. 1990; D. M. Kramer, T. Miller and L. Nedbal, unpublished), it became possible to measure the kinetics of partial reactions of the PS II acceptor complex in intact plants. Data obtained with these instruments on intact plants grown in the laboratory has revealed some interesting differences between the function of the PS II acceptor complex in isolated and intact systems. Although the kinetics of oxidation in intact plants were similar to those found in isolated thylakoids, in most species investigated about one-half of appeared to be in the semiquinone (one-electron reduced) form, even upon extensive dark adaptation (Rutherford et al., 1984; Robinson, 1986; Kramer et al., 1990a). The binary oscillations in the level of fluorescence at 150–200 which indicate that is mostly in the fully oxidized form, were observed only after preillumination with far-red (PS I-active light) to oxidize the PQ pool. Furthermore, dark adaptation after illumination with far-red light led to a disappearance of the binary oscillations. This indicates that, in intact plants in the dark, can be partly reduced to the semiquinone form by an unknown process. When measurements were made on some types of intact plants grown under field conditions, more dramatic differences were observed (Groom et al., 1993). Shortly after a light to dark transition, the reoxidation of became extremely slow. This was shown to be due to a non-photochemical reduction of the PQ pool. Under some conditions, this reduction eventually proceeded to completion, even leading to a reduction of The significance of this phenomena is not yet known, but its species distribution and behavior suggest a relationship to mode of fixation. The multi-flash kinetic fluorimeter has also provided a rapid system with which to probe the damaging effect of excess heat (Kanazawa et al., 1992) and UV radiation (Yerkes et al., 1990) in intact leaves. In the past, the pulse-probe technique of fluorescence measurement has been employed in experiments with leaves mainly for measurement of flash-induced kinetic changes. More recently, similar techniques have been applied to steady-state measurements, and work is currently underway to

David M. Kramer and Antony R. Crofts test an instrument designed for experiments in steadystate light, where single-turnover pulses are superimposed on a constant background illumination, and in the punctuated steady-state, where a chopped illumination is given. We have developed a pulseprobe fluorimeter in which 5 measuring flashes are provided by a group of LEDs, allowing a more rapid repetition frequency for measuring points. The most immediate advantage of the apparatus has been in deconvoluting the separate contributions of the yield changes due to reduction, and to reduction of the plastoquinone pool. This was achieved by assay of reduction following a saturating short xenon flash (~3 ). Measurement of the fluorescence yield with reduced requires a rapid kinetic resolution, since the re-oxidation kinetics occur with half-times in the 100–300 range. The level before the decay can be estimated by extrapolation from points measured as early as possible in the decay range, or measured directly during the flash, or measured in the presence of DCMU to inhibit the decay (Kramer et al., 1995). The results demonstrated that the long saturating pulse used to ensure reduction of in conventional PAM-fluorimeters also causes reduction of the plastoquinone pool, giving rise to an additional change in yield which introduced a nonlinearity into the calculation of flux. Most experiments reported in the literature have been performed under steady-state conditions in which the pool was substantially reduced, and the effect of further reduction would be negligible. However, this departure from linearity is especially important under conditions in which the pool is oxidized, e.g. with dark adapted material, or in the steady-state at low light intensity. The superior kinetic resolution, and actinic flash facility, allow this difficulty to be circumvented when using the pulse-probe approach.

5. Fluorescence Video Imaging Most instruments for use with intact plants have measured average fluorescence yield changes in a selected area of the leaf. For some situations, more useful information can be obtained by using video imaging to generate data from a larger area which is spatially resolved. Earlier work on fluorescence video imaging required a major commitment of equipment (Omasa et al., 1987), but the advent of the multimedia age has brought mass-produced video boards to the personal computer, making it possible to construct relatively inexpensive video imaging

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Measurement of Photosynthetic Electron Transport

systems (Fenton and Crofts, 1990) or to couple video cassette recordings of leaf fluorescence to a personal computer for further analysis (Mott et al., 1993). In the former instrument, the choice of video board allowed image capture during the induction phase, and the collection of kinetic data at frame-rates (30 ms per frame) to record induction curves simultaneously from several selected areas of the image. The instrument has been used in many applications, including selection of mutant strains of photosynthetic bacteria (Fenton and Crofts, 1990), cyanobacteria, Chlamydomonas reinhardtii and Arabidopsis, to follow the uptake of herbicides into leaves (B. Wade, J. Fenton and A. R. Crofts, unpublished), for detecting UV-damage in leaves (Yerkes et al., 1990). Such instruments have also been useful for detecting viral infection of leaves before visible damage (Balachandran et al., 1994; J. M. Fenton, J. Widholm and A. R. Crofts, unpublished). The ability to take video images during saturation pulses superimposed onto continuous illumination has allowed the mapping of spatial variations in the efficiency of PS II or in over the leaf surface (Daley et al., 1989). The most important results of this technique have been visual demonstrations of patchy distributions of photosynthetic activities across the leaf surface (Daley et al., 1989; Raschke et al., 1990; Mott et al., 1993; Cardon, 1993; Genty and Meyer, 1994; Meyer and Genty, 1995; Bro et al., 1995), which can lead to overestimation of the internal CO2 concentrations based on gas exchange (see Chapter 8). During water stress or after application of abscisic acid, non-uniform closure of stomates

45

can lead to significant declines in net uptake (Terashima et al., 1988; Downton et al., 1988; Chapter 14). Examples of these experiments are shown in Fig. 4, where oscillations in across the surface of a Xanthium strumerium leaf were observed (Cardon, 1993). After dark adaptation, the leaf was exposed to continuous illumination to initiate photosynthesis. Oscillations in stomatal resistance were initiated by lowering the water vapor pressure at time zero. Periodically, saturation pulses were applied, during which fluorescence images were recorded. In Fig. 4 images are shown of the non-photochemical quenching, obtained by subtracting the fluorescence image taken at different times during the steady-state illumination from the image of the maximal fluorescence, taken after dark adaptation, and dividing this difference by 0.8 times the maximal fluorescence. The bright patches represent areas of relatively large non-photochemical quenching, indicative of low photochemical yield and are associated with regions in the leaf of low stomatal conductance. Roughly complementary images were obtained by viewing the image of (Genty and Meyer, 1994; Meyer and Genty, 1995; Bro et al., 1995). The images in Fig. 4 indicate that the intensities of different bright patches oscillate out of phase with each other, and thus any effect on the total leaf photosynthetic uptake are canceled. These results have two very important implications: (i) oscillations in stomatal conductance may significantly affect photosynthetic rates, even under conditions where oscillations are not observed in uptake or autoradiography assays, and (ii) steady-state fluorescence parameters of an

46 average over a large area or of a small selected area from a leaf surface can be quite misleading. Further development of this instrument has made it possible to combine pulse kinetic fluorimetry with video imaging. By using a xenon flash for excitation, synchronized with the data reset pulse of the camera, and a measuring flash given during the following frame-capture, we have been able to achieve a kinetic resolution in the range from ~40 to 15 ms, with spatial resolution down to a millimeter or less (J. M. Fenton and A. R. Crofts, unpublished). A portable version of the instrument is likely to prove useful for diagnostic work (plant disease, herbicide tolerance, pollution monitoring) under field conditions.

E. Delayed Luminescence in Intact Leaves The spectrum of delayed luminescence (DL, alternatively called delayed fluorescence or delayed light) is the same as that of prompt fluorescence, indicating that it comes from the antennae associated with PS II. DL is emitted at relatively long times after illumination, and persists longer (for many minutes) than the normal prompt fluorescence. The ‘delay’ occurs because DL arises from the recombination of charged pairs stored on the donor and acceptor sides of the reaction center so as to repopulate the excited singlet state of the antenna bed (see reviews by Crofts et al., 1971; Lavorel, 1975; Murakami, et al, 1975; Malkin, 1977; Amesz and van Gorkom, 1978; Govindjee and Jursinic, 1979; Jursinic, 1986; Schmidt, 1988a). During illumination, electrons are stored on the reducing side and positive charges are stored on the oxidizing side of the reaction center. In the dark, these charges can recombine, with some fraction of the recombination events having sufficient energy to generate the singlet state of the primary donor (P*). In these cases, excitation transfer from P* into the antenna matrix can lead to the emission of fluorescence, delayed by the time of the recombination. The fraction of recombination events with sufficient energy to repopulate the singlet state depends on the depth of traps, and the kinetics of recombination, giving rise to a complex decay curve, and an exponential dependence on terms contributing to stored free-energy, e.g. pmf. In isolated chloroplasts, under controlled conditions, the intensity and decay kinetics of DL can be used to probe the energetics and population of individual redox states of PS II and its reaction partners, and the state of the proton gradient across

David M. Kramer and Antony R. Crofts the thylakoid membrane, i.e. ApH and (Kraan et al., 1970, Wraight and Crofts, 1971, Evans and Crofts, 1973; Lavorel, 1975; Murakami, et al., 1975; Malkin, 1977). Unfortunately, in intact materials, and especially during steady-state illumination, DL is expected to reflect all of these factors together. The scrambling of individual donor and acceptor states will give rise to a distribution of trapped energy levels, these levels will be further modified by the proton gradient formed across the thylakoid membrane, and the yield of fluorescence will be modified by changes in the distribution of antenna chlorophylls, and by the various chlorophyll quenching mechanisms discussed above. In principle, independent measurement of fluorescence yield could be used to correct these latter contributions. Several groups have attempted to use DL to probe the state of the photosynthetic apparatus in intact plants. These attempts fall into two categories: (i) to use DL as a general indicator of the plants ability to store and utilize photosynthetic energy, and (ii) to use DL as a probe of the energization state of the thylakoid membrane.

1. Delayed Luminescence as a General Indicator of Photosynthetic Energy Storage Though the interpretation of changes in DL in the steady-state is complex, the amplitude of the millisecond decaying DL has been shown to be a sensitive general indicator of the physiological state of the plant. It is possible to interpret such data with little detailed understanding of the mechanism of photosynthetic energy storage or its limitations. When the photosynthetic apparatus is functioning normally, relatively little DL on the microsecond to millisecond time scale is observed because energy stored in PS II, in the form of positive charges accumulated on the oxygen-evolving complex and electrons on the acceptor side, is efficiently consumed by forward electron transfer reactions (the evolution of oxygen and the reduction of plastoquinone through the QB site) before recombination leading to DL can occur. However, if the normal pathways for utilization of this stored electrochemical energy are hindered, back reactions will occur and bright DL will be observed. On the other hand, if the PS II centers are completely blocked or damaged to such an extent that no initial charge separations can occur, or so that the back reactions occur very rapidly, before the luminescence measuring time begins, a decrease in DL will be

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observed. These extremes are clearly seen in intact plants in the data of Björn and Forsberg (1979; also see Blaich, 1988) where, upon treatment of leaves with water at temperatures above 53 °C, complete destruction of the plants ability to store energy on the millisecond time scale was observed by a loss of DL. After treatment at lower temperatures (<48.5 °C), a significant increase in DL intensity was observed, indicating that the plant was able to store photosynthetic energy in the form of charge-separated states, but was not able to utilize this energy in subsequent dark reactions. Of course, further information is required to specify which part of the energy storage and utilization machinery is limiting. One may imagine that such a use of DL photometry would be only a starting point in the identification and characterization of limiting factors to photosynthesis. Such gross changes in DL intensity have been associated with a number of environmental stresses, including high temperature stress (Sundbohm and Björn, 1977; Björn and Forsberg, 1979; Blaich etal., 1982), low temperature stress (Björn and Forsberg, 1979; Blaich etal., 1982; Ortoidze etal., 1988; Fork and Murata, 1990; Brzostowicz, 1990), UV radiation damage (Bordonova et al., 1988), oscillations in stomatal opening (Ellenson and Raba, 1983), chemical stress (Ellenson and Amundson, 1981; Blaich et al., 1982; Schmidt and Senger, 1987; Vallegaleti et al., 1990) fungal or viral infections (Björn and Forsberg, 1979; Blaich et al., 1982), nutritional condition (Schmidt and Senger, 1987; Schmidt, 1988a,b), and high salt stress (Vallegaleti, et al. 1990). There is little doubt that such DL assays are sensitive indicators of plant stress, and, in at least some circumstances, have been shown to be a much more sensitive indicator than fluorescence analysis (Björn and Forsberg, 1979). On the other hand, the usefulness of steady-state DL in pinpointing the particular partial reaction involved in the inhibition is limited because of the many factors affecting the DL amplitude.

2. Delayed Luminescence as an Indicator of the ‘Energization’of the Thylakoid Membrane As discussed above, there is a strong correlation between the energization state of the thylakoid membrane and the intensity and kinetics of DL. A mechanism for these changes was proposed (Wraight and Crofts, 1971; Crofts et al., 1971; Wraight et al.,

47

1972; Evans and Crofts, 1973; Bowes and Crofts, 1981), wherein the activation threshold for recombination via the first excited singlet (or also via other routes) is lowered by buildup of the proton electrochemical gradient. Since all intermediates preceding recombination are in Boltzman equilibrium with the activated intermediate, lowering the difference in energy between the lowest state and the excited intermediate will result in an exponential increase in the rate of back reaction. Thus, an exponential relationship between the energization state of the membrane and the rate of recombination is expected (Evans and Crofts, 1973). In contrast, we expect a linear relationship between the concentration of a particular charge-separated state and the intensity of DL arising from back reaction from that state. Theoretically, one could use such a relationship to determine the actual energy level stored in the proton gradient. An exponential relationship between lightscattering changes, an indicator of the proton gradient (Deamer et al., 1967) and millisecond DL intensity has been reported for intact leaves by Morgun and Doldzhikov (1990), whereas Schreiber and Neubauer (1989) reported a more linear relationship between (also shown to be an related to the across the thylakoid membrane; see Section IV.C) and the intensity of 50 DL. Such mixed results are not surprising, however, since in intact plants in the steady-state, we expect complex interactions between the magnitude of the proton gradient and the concentrations of intermediate states in the electron transfer chain. Since, with DL alone, it is not possible to simultaneously quantify the concentrations of all recombining species and the extent of the gradient, this technique will not yield unambiguous information unless combined with other techniques.

3. Measurement of Delayed Luminescence Early studies on DL in leaves employed a Becquereltype phosphoroscope. This type of device, though adequate in the laboratory, has limited application to field studies on intact plants because the mechanical disk and bright light source are rather cumbersome and its relatively slow response limits the time range over which the DL can be measured, i.e. a dead time between pulse and measuring window openings of usually > 1 ms, limitations to the speed of the rotating disk. Schreiber and Schliwa (1987) have described a DL photometer in which the light source and rotating

48 disk chopper have been replaced by light emitting diodes (LEDs). Since LEDs can be switched on and off very rapidly (usually far less than 1 ) by relatively simple driver circuitry, the instrument has a much wider kinetic range over which it can operate; it can capture data from as little as 50 after the light pulses. It is also much more portable and rugged than a mechanical shutter device. Systems have also been constructed to record images of delayed fluorescence emission from leaves, a technique also called phytoluminography (Sundbohm and Björn, 1977; Björn and Forsberg, 1979; Ellenson and Amundson, 1981; Blaich et al., 1982; Ellenson and Raba, 1983; Blaich, 1988; Vallegaleti et al., 1990). In these instruments, an image intensifier is placed at the exit port of a phosphoroscope and the intensified image of the delayed luminescence emission pattern is projected onto film or onto a video camera. Thus, spatially resolved information can be obtained about the function of photosynthesis. It turns out that this type of measurement is well suited to detection of leaf damage that may have a particular spatial pattern (e.g. Vallegaleti et al., 1990). Other techniques must be employed to further characterize the effects.

F. Absorbance Measurements in Intact Plants One of the most powerful techniques to be introduced for whole plant work is kinetic spectrophotometry. The most direct method of measuring light-induced electron transport in plants is to follow the kinetics of the absorbance changes due to the redox components of the electron transport chains. Through appropriate deconvolution of the visible, infrared, and ultraviolet spectra, the kinetics of cytochromes b, cytochrome f, plastocyanin, P700, and other components can be determined (e.g. Joliot and Joliot, 1984a; Rich, et al., 1987). Absorption spectroscopy can also be used for measurement of the kinetics of charge transfer across the thylakoid membrane through the absorbance changes resulting from the electrochromic shift of carotenoid and chlorophyll molecules of the bulk pigments (Witt, 1975).

1. Kinetic Spectrophotometers Conventional instruments employ a constant (or sometimes chopped) collimated light beam which passes through the sample to be measured. The amount of light transmitted through the sample is

David M. Kramer and Antony R. Crofts measured by a photodetector and compared to a reference level (either measured simultaneously or as a baseline before the experiment). These types of instruments have been extensively used in the past to elucidate much of what we know of the photosynthetic electron transfer chain. Specific application of these instruments to work on intact leaves required consideration of the effects of the scattering nature of living tissue, fluorescence and sample shape. An excellent account of the issues and theory involved in these adaptations is given by Butler (1964) where the state of the art in instrument development for in vivo work in plants 30 years ago is described. Although the theory and principles outlines in the Butler review are still valid today, new technologies have enhanced the application of absorbance spectroscopy to intact plant work. Useful kinetic absorbance measurements require instrumentation of high sensitivity and fast response time. These requirements are difficult to meet in conventional absorbance instruments since the measuring beam must be kept at low incident intensity to avoid actinic effects. As a consequence, the signalto-noise ratio is usually limited by the number of photons that reach the detector. Since the number of photons measured per time point decreases with increasing response times, the signal-to-noise ratio of measurements made on conventional instruments decreases as the resolution time of the measurement becomes faster. Higher signal-to-noise ratios can be achieved by signal averaging, but in many instances, this strategy is impractical due to the long time required, and difficulties in preparing and maintaining the samples needed. The difficulties arising from the need to sample many repetitions is especially acute for experiments where the condition of the sample is modified for long periods of time by the actinic light used in the experiment. This is true for many in situ experiments, where the physiological condition of the leaves can be altered by extended dark periods, or where measurements are needed of processes like the two-electron gate, or the S-states, where a prolonged dark adaptation is needed between periods of actinic illumination to allow the system to reset to a known state. These difficulties are compounded in intact plants by the strong light-scattering of leaves which further reduces the light throughput and lowers the signal-to-noise ratios. During continuous illumination in steady-state experiments, a large overlap of contributions from changing species is observed, leading to ambiguity in interpretation.

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Measurement of Photosynthetic Electron Transport

Most importantly, the continuous light-induced changes are, in most cases, dominated by large changes in light scattering. Though the light scattering changes have been used as a probe of the energization state of the thylakoid membrane (Crofts et al., 1967; Deamer et al., 1967), interference from these scattering changes and difficulties in deconvolution of overlapping species are perhaps the most difficult hurdles to overcome in development of absorbance spectroscopy techniques applicable to steady-state studies in intact plant. Recent developments in absorbance spectrophotometers for intact plant work have focused on addressing these difficulties. There have been four basic approaches: (i) restricting measurements to wavelengths where very high light intensities can be used without actinic effects, i.e. the near IR; (ii) restricting measurements to kinetic conditions where various contributing species can be kinetically distinguished, e.g. flash-induced kinetics and ‘punctuated’ steady-state measurements; (iii) the use of pulsed measuring beams; (iv) and the use of multiple wavelength probe beams. We will discuss here three types of instruments developed in the past several years, each of which incorporates one or more of these design elements. We will attempt to give some idea of the utility of each instrument, a discussion of any unresolved issues that could impede the interpretation of data obtained with them, and possible future directions for development of these techniques.

2. Measurements in the Near Infrared (NIR) As discussed above, one way to overcome problems of low signal to noise is to work with wavelengths that do not induce photosynthetic electron transfer reactions. Very intense measuring beams can then be used without disturbing the photosynthetic reactions. Several electron transfer species contribute to absorbance changes in the NIR, and measurements of at least one of these species, P700, appears to be quite useful for elucidating steady-state phenomena (but see below for caveats). In response to this opportunity, simple but effective instruments have been developed to measure NIR changes. These use modulated or pulsed measuring beams from very efficient and intense light-emitting diodes (LEDs) and a matched frequency or phase-sensitive detection systems, and can give quite a good signal-to-noise levels for steady-state experiments (Harbinson and

49

Woodward, 1987; Schreiber and Neubauer, 1990). There are some differences in the details of the optics and electronics in these instruments but, judging from published traces, they give similar performances. The instruments work by giving out pulsed beams of NIR light which are directed to the surface of the leaf. The detector is usually placed on the opposite side of the leaf to measure transmitted light. Light not absorbed or scattered is measured by a photodiode detector amplified by frequency-sensitive electronics. The frequency-selectivity of the amplifier has two benefits. It increases the signal to noise ratio by narrowing the frequency response of the detecting circuitry to just that of the measuring beam, i.e. it eliminates contributions from all noise sources with frequencies different from that of the measuring beam (for discussion of these issues, see Butler, 1964). Secondly it eliminates interference from fluorescence or other changes associated with the continuous measuring beam, so that even intense background illumination can be employed. At present, more rapid flash-induced changes, on the microseconds time scale, can probably be best measured using a double-flash kinetic spectrophotometer (see Section IV.F.3), but there is no theoretical reason why even very rapid changes could not be adequately measured with LED machines provided the pulses are sufficiently intense and of short duration.

a. Species Showing NIR Transitions The absorbance changes in the NIR have received much less attention than those in the visible, and although much work has been done to explore the kinetics of P700 and P680 in thylakoids and isolated photosystem preparations, only a few detailed studies on NIR changes in leaves or more intact chloroplasts have been published (Inoue, et al. 1973; Schreiber, et al, 1988; Harbinson and Hedley, 1989; Klughammer and Schreiber, 1991; see also Chapter 3). For the time being, the available studies, mostly on isolated chloroplasts, will have to serve as the framework for intact plant studies, though further detailed spectroscopy in intact leaves would significantly advance the applicability of this technique. The lightinduced difference spectra in the NIR in chloroplasts and leaves consist of a complex overlap of broad signals (Fig. 5): a relatively large change due to with a narrow bleaching at 703 nm and a broad absorbance increase between 750 to 850 nm (Inoue et al. 1973; Haveman and Mathis, 1976; Klughammer

50

and Schreiber, 1991), another broad absorbance increase from oxidation of plastocyanin between about 700 to 1000 nm (Katoh at al., 1962; Klughammer and Schreiber, 1991), a broad absorbance decrease due to reduction of PS I acceptor components, most likely ferredoxin (Klughammer and Schreiber, 1991), an apparent electrochromic shift, centered around 730 nm, possibly related to the electrical field across the thylakoid membrane (Klughammer and Schreiber, 1991); and, in thylakoids at low lumenal pH, as would be expected under steady-state illumination in intact plants, or under conditions where the donor side reactions of PS II are inhibited, significant contributions from have been demonstrated (Haveman and Mathis, 1976). In addition, during steady-state illumination, the signals recorded in the NIR are dominated by large scattering changes (Klughammer and Schreiber, 1991).

David M. Kramer and Antony R. Crofts

b. Deconvolution Deconvolution of these overlapping contributions would appear to be difficult. At least some of these changes have sufficiently different spectra or kinetics to allow adequate deconvolution. The most useful measurement to be made in this range would be a clear-cut indicator of the redox state of P700, and, this is by far the most commonly measured species in the NIR. However, it is still unclear how specific these measurements are. The most distinct absorbance change in the NIR is that of P700, with its narrow peak at 700 nm. Unfortunately, a probe beam around 700 nm will excite chlorophyll fluorescence and any changes in absorbance associated with P700 redox changes will likely be swamped by the normally very large fluorescence yield changes observed in plants. One technique to eliminate fluorescence contributions is to use a highly collimated measuring beam, with the detector placed some distance from the sample so

Chapter 2

Measurement of Photosynthetic Electron Transport

that the intensity of the fluorescence, which is nearly perfectly scattered, decreases with the square of the distance between the sample and the detector, while the intensity of the measuring beam remains relatively unchanged (Butler, 1964). However, the highly scattering nature of intact leaves precludes such a solution for in vivo studies. Because of these difficulties, most measurements have been made at longer wavelengths. In most experiments, only one wavelength (either 820 or 830 nm) has been used, and the measurements have therefore been unable to distinguish among the different contributing species. Most authors have used a kinetic distinction, by cutting the actinic illumination for short intervals and measuring the decay of the signal for hundreds of milliseconds (Harbinson and Hedley 1988, 1993; Klughammer and Schreiber, 1991). The light scattering changes appear to decay at a much slower rate than those due to redox-related absorbance changes. It can be argued that most of the other changes at 820 nm are sufficiently minor that they can reasonably be ignored. The exceptions are and possibly An accumulation of in low pH-treated chloroplasts has been shown (Haveman and Mathis, 1976), but there is currently no test of whether contributes to the 820 nm change in intact leaves under physiological conditions. One may reasonably expect at least some contribution, however, since when the lumenal pH is lowered as expected on continuous illumination in intact material, the turnover of the reactions of the oxygenevolving complex would become inhibited. This is likely to happen to some extent under normal illumination conditions, as suggested by the increased emission of delayed fluorescence, but under physiological conditions, the development of such a potentially hazardous state might be prevented by fluorescence lowering (see above). If we ignore for the time being the possible contamination by we must still consider the contribution of oxidized plastocyanin, which can represent more than 37% of the 820 nm signal (Harbinson and Hedley, 1989; Klughammer and Schreiber, 1991). The argument that the contribution of PC can be ignored (Harbinson and Hedley, 1993) because the equilibrium constant for electron transfer between PC and P700 under some kinetic conditions appears to be nearly 1 rather than the expected 10 to 100 in favor of P700 reduction (Joliot and Joliot, 1984b; Delosme, 1991) ignores the complexities of the issue (but see also Chapter 3

51

for further discussion of this issue). The low apparent equilibrium constant may reflect local kinetic heterogeneity or local compartmentation (Delosme, 1991) at least under some conditions. However, this has not been definitively shown, and under some other conditions, e.g. flash-induced changes in darkadapted thylakoids (Bouges-Bocquet, 1977; Olsen, 1982; Joliot and Joliot, 1984a,b; Hope et al., 1992), the expected equilibrium constant is indeed reflected by the kinetics. Also, it is well known that the redox midpoint potential of PC is sensitive to pH below a pK at about 5.5 to 6.5, depending upon species (Sykes, 1985). One might expect that, as the pH of the lumen decreases during continuous illumination, the observed transfer of electrons between PC and P700 would be significantly affected. We suggest that when measurements are made at a single wavelength (820 to 830 nm) authors should include a disclaimer stating that the involvement of other species, particularly PC, is expected, and cannot be ruled out (see also Chapter 3). Through redox titrations of light-induced absorbance changes, Klughammer and Schreiber (1991) showed that the broader, longer wavelength bands of P700, PC and the other less well defined species contribute in different proportions over the NIR spectral range, from those predicted by the spectra of isolated components. Therefore, a better approach to the problem of signal overlap in the NIR would be to take measurements at more than one wavelength and to deconvolute the individual contributions of PC and P700 through their respective extinction coefficients at each wavelength.

c. Artifacts from Enhanced Path-length and Internal Absorption Another concern about the measurement of in the NIR is the disproportionately large absorbance change observed at 820 nm, compared to that at 700 nm. In preparations of PS I particles the ratio of absorbance changes due to PS I oxidation at 703 and 815 nm was found to be about 8:1, whereas in an intact chloroplast preparation, Klughammer and Schreiber (1991) found a ratio of only 2.5:1; an even lower ratio was found in intact leaves. The authors concluded that these effects were due to differential contributions at the different wavelengths from two artifacts: (i) increases in the effective pathlength from scattering; and (ii) self-absorbance by

52 chlorophyll (Butler, 1964; Rühle and Wild 1979). The scattering-induced increase in pathlength would be expected to be less in the NIR, but contributions from internal absorbance would truncate the scattering path, and dominate in the chlorophyll region. Despite ambiguities in interpretation of the NIR signals, particularly when changes at only one wavelength are probed, many results have been obtained solely at 820 nm (discussed below) which have allowed internally-consistent interpretation. The assumption is made in interpreting these experiments that the 820 or 830 nm changes reflect primarily changes in the redox state of P700. The internal consistency of such measurements can be rationalized by the fact that most significant contributors to the 820 signal are functionally related and are expected to respond to physiological conditions in qualitatively the same direction. Thus, if a measurement of the fraction of PS I centers oxidized is made solely at 820 nm, the contribution from oxidized PC will introduce a relatively small error, since conditions that lead to oxidized P700 will also tend to produce an oxidized PC pool, which will lead to an absorbance change in the same direction. The error due to formation will be quantitative, but will not change the sign of the overall changes. On the other hand, as more sensitive measurements are made and finer and finer interpretations are placed upon the data, such quantitative errors will certainly affect interpretation and will eventually need to be dealt with.

d. Estimation of Quantum Efficiency of PS I and PS II Measurements reflecting P700 oxidation have been used primarily to estimate the fraction of PS I centers that are open at any particular time during steadystate illumination, usually termed quantum efficiency, or (Harbinson and Woodward, 1987; Weis et al., 1987; Schreiber et al., 1988; Harbinson and Hedley, 1989; Harbinson et al. 1989; Weis and Lechtenberg, 1989; Foyer et al., 1990a; Genty et al., 1990; Harbinson and Foyer, 1991; Peterson, 1991; see also Chapter 3). These studies are particularly interesting because they allow a comparison of the quantum efficiencies of the two photosystems under steadystate illumination. A strictly sequential linear electron transfer chain predicts that the quantum efficiencies of the two photosystems during steady-state illumination should match, whereas phenomena like

David M. Kramer and Antony R. Crofts cyclic or pseudo-cyclic electron flow will introduce mismatches in the electron flow rates (and therefore quantum efficiencies) of each type of center. In plants, under most physiological conditions, a nearly linear relationship between the quantum efficiencies of the two photosystems has been found (Foyer et al., 1990a, 1992; Genty et al., 1990; Klughammer and Schreiber, 1994), suggesting that either non-linear processes do not significantly participate, or that their rates are strictly coupled to the rate of linear electron flow. Under some conditions, such as during the initial induction period, or under low atmospheric large discrepancies have been observed between the quantum efficiencies of PS I and PS II. These discrepancies were traced to a systematic artifact involved in estimating the quantum efficiency of PS I from 820 nm absorbance changes (Foyer et al., 1990a, 1992; Harbinson and Foyer, 1991; Klughammer and Schreiber, 1994). Previously, was estimated simply by the fraction of P700 oxidized because PS I centers with oxidized P700 are not able to undergo charge separations. PS I centers can also be blocked by acceptor-side limitation which leads to a reduction of the normal electron acceptor. Thus, the quantum efficiency of PS I was overestimated under certain conditions. Klughammer and Schreiber (1994) have introduced a new measuring protocol that enables estimation of the fraction of PS I centers with oxidized P700 as well as over-reduced acceptors. With this new technique, a linear relationship between the quantum efficiencies of both PS I and PS II was observed for plants, even during induction. This finding is of particular interest with respect to the regulation of PS I turn-over. If control of excitation delivery to PS II through fluorescence lowering is a primary regulatory device, what mechanism controls PS I so as to match the flux? As we have suggested above, it seems possible that an exciton dumping mechanism operating at the antenna level, similar to that in the PS II antenna, might also regulate excitation delivery to PS I (see also Chapter 3).

e. Donor-pool Size for PS I Another use for the 820 to 830 nm measurement has been the estimation of the number of electrons stored in the donor pool to PS I. Asada et al. (1992, 1993) have suggested that stromal components donate electrons to through the intersystem chain in chloroplasts of and plants. In these experiments,

Chapter 2

Measurement of Photosynthetic Electron Transport

the areas under the kinetic curves obtained when P700 was oxidized in intact leaves, then reduced by singe-turnover flashes, or when the intersystem chain was fully reduced by a 50 ms multiple-turnover pulse, were compared. These values were used to calibrate the area bounded by the oxidation kinetics of P700, and then to estimate the size of the pool under a variety of conditions. The donor pool appeared much larger under anaerobiosis, suggesting that, under these reducing conditions, a pool much larger than that of the intersystem chain could donate to PS I. However, in order for these experiments to yield consistent, quantitative data, the efficiency of PS I centers with reduced P700 must be the same under all conditions. In the light of demonstrations that, under similar acceptor-limiting conditions, reduction of PS I acceptor components will reduce the quantum efficiency of PS I turnover (see above), we conclude that changes in the areas of oxidation kinetics are not likely to yield reliable information. The oxidation kinetics would be slowed by the fraction of PS I centers with reduced P700 and reduced acceptors, leading to an overestimation of the area. It may be possible to add an artificial PS I electron acceptor, e.g. methyl viologen, to eleviate any possible acceptor side restrictions, but this would also disturb the intactness of the system. It is probably best at this point to avoid this type of measurement unless it can be shown that acceptor side restrictions can be eliminated.

f. The Rate of Intersystem Electron Transfer Reactions in the Steady-state Another interesting application of NIR measurements of P700 redox state has been to probe the rate of turnover of the reactions leading to re-reduction during the steady-state (Harbinson and Hedley, 1989; Laisk and Oja, 1994). These measurements were made by rapidly switching off the actinic light for short intervals during steady-state illumination (punctuated steady-state). A striking finding has been the relative insensitivity of the half-time of rereduction to the intensity of continuous illumination (Laisk and Oja, 1994). Since the turnover of the cytochrome complex is pH-sensitive (Kramer and Crofts, 1993) the lack of electron transfer control upon illumination suggests that the lumenal pH does not become as low as has been previously thought. If these findings are further substantiated, they should greatly affect the way we view the control of

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photosynthetic electron transfer (see Chapter 3).

3. Flash Measuring-beam Kinetic Spectrophotometer As discussed above, the NIR region is convenient for absorbance measurements on intact plants because an intense measuring beam can be used without actinic effects. However, the most specific absorption bands are concentrated in the blue, green, and red, where the actinic effects of the measuring beam can be severe. To make measurements in these very useful ranges with intact plants, Kramer and Crofts (1990) have introduced a portable instrument for the measurement of the kinetics of flash-induced absorbance changes in intact leaves. The instrument uses the design concepts of a pulsed measuring beam and differential optics first introduced by Joliot et al. (1980) and Joliot and Joliot (1984a), allowing a high sensitivity while subjecting the plant material to much less actinic illumination than a conventional machine. The signal-to-noise levels (noise level of < A, with typical signals of 2–5 × ) achieved in these measurements are, in most cases, high enough that signal averaging is not needed. This approach has made possible the routine measurement of many photosynthetic reactions on intact leaves, even under field conditions. We have demonstrated its utility for the measurement of flashinduced redox changes of the cytochromes of the bf complex (Kramer and Crofts, 1990). It is also readily applicable to the measurement of flash or continuous light-induced changes in the redox state of PS I at 700 or 820 nm, the oxidation of plastocyanin in a cytochrome bf complex minus mutant of maize (unpublished), the formation of zeaxanthin by deepoxidation of violaxanthin at 505 nm (unpublished) and measurements of the electrical field-induced shifts in chlorophylls and carotenoids (see following section).

a. Proton Flux Measured through the 515 nm Electrochromic Change The electrochromic shift at 515 nm reflects the formation or decay of an electrical field across the thylakoid membrane, and provides a useful ‘membrane voltmeter’ (Witt, 1975). The electrical field is part of the proton motive force (pmf) in the chemiosmotic proton circuit leading to the formation of ATP (for review of the chemiosmotic mechanism

54 of ATP synthesis, see Ort and Oxborough, 1992). The field is driven by charge separations in PS I, PS II and the cytochrome bf complex and it is subsequently consumed by turnover of the ATPase or dissipated by leakage. Because these partial reactions are well separated in the time domain following flash excitation, they can be easily deconvoluted. The sensitive and rapid measurement of the electrical field makes possible real time measurements of the regulation of the ATPase in situ (Kramer and Crofts, 1989). From in vitro studies, it was shown that the ATPase was activated only when a pmf, either in the form of a proton concentration gradient or an electrical field or both, were imposed across the thylakoid membrane (Ort and Oxborough, 1992). The pmf needed to activate the ATPase is modified by the redox state of a pair of thiol groups on the regulatory subunit of the complex, being less when the thiols are reduced. The redox state of the regulatory thiols is in turn controlled by the thioredoxin shuttle (Buchanan, 1992; Maheshwari et al., 1992; Ort and Oxborough , 1992). The thiol groups are reduced in the light via ferredoxin and thioredoxin by turnover of linear electron flow. Upon dark adaptation, the thiols are re-oxidized and the amount of proton motive force required to activate the ATPase increases. We were able to show with the field double-flash kinetic spectrophotometer that this regulation occurs in much the same way in intact leaves (Kramer and Crofts, 1989; Kramer et al., 1990b). We further showed that, under field and laboratory conditions, the regulatory step leading to the activation of the ATPase occurs at very low light levels and is not likely ever to be rate-limiting for photosynthesis (Kramer et al., 1990b). Observations of the activation and turnover of the ATPase in the field under droughtstressed conditions, using these techniques, showed that, in contrast to previous suggestions, water stress had little effect on the activation or turn-over of this enzyme (Wise et al, 1990). One striking finding of these studies was that the reduction and re-oxidation kinetics of the coupling factor were quite distinct and differed greatly from those of other thioredoxinregulated enzymes (Kramer and Crofts, 1989; Kramer et al., 1990; Ort and Oxborough, 1992). From the different kinetic profiles, we were able to predict relative redox properties of the regulatory thiol groups of many of the thioredoxin-regulated enzymes and these have since been confirmed by direct measurement (Hutchison and Ort, 1995). Thus, these studies have offered a non-invasive probe of the physiological

David M. Kramer and Antony R. Crofts state of the thioredoxin system.

b. Redox Poise The flash measuring beam spectrophotometer has not been used extensively to explore the redox poise in the steady state, or kinetics on perturbing the steady state. There is no technical difficulty in making steady-state measurements using this type of instrument; the design has provision for a continuous actinic beam which can be used instead of the flash actinic beam, and a series of measuring flashes can be given to detect transient kinetics at the onset or cessation of steady-state illumination. The main difficulty in such measurements is likely to come from large absorbance ‘artifacts’ due to light scattering changes, which overlap. Three possible approaches to overcoming this difficulty are presently being pursued. One approach is to lessen the effects of light scattering by pre-scrambling (or randomizing) the light, as discussed in a review by Butler (1964). D. M. Kramer (unpublished) has developed a new ‘diffused beam spectrophotometer’ that uses a very efficient light scrambler (a type of integrating sphere) to completely randomize the measuring light before and after passing through the sample. It essentially eliminates the effects of differential light scattering while achieving high signal-to-noise ratios by using flashed measuring beam and differential optics and detection. Another approach is to set up a punctuated steady-state, where the actinic beam is interrupted so that rapidly decaying components can be distinguished from slowly-decaying ones. A third approach is the use of saturating, single-turnover actinic flashes superimposed upon continuous illumination. This is of particular interest for the measurement of the rapid rise phase of the flashinduced electrochromic shift, which should reflect the total number of open PS I and PS II centers, and thus the quantum efficiency of the entire electron transfer chain.

4. Multi-wavelength Modulated Spectrophotometer Another approach to constructing an absorption spectrophotometer was used by Klughammer et al. (1990). These authors used an array of 16 light emitting diodes, each filtered by an interference filter to provide a series of different wavelength probes. The LEDs were sequentially pulsed, the light from

Chapter 2

Measurement of Photosynthetic Electron Transport

each was blended and led to the sample by a fiber optics assembly, and the signals from each LED pulse were detected sequentially by a single detector. The signals at different wavelengths, separated in time, could then be independently measured, and stored. The advantage of this approach is that, during kinetic measurements, an entire spectrum of absorbance changes is obtained, allowing a more detailed spectral deconvolution. The instrument operated in the green spectral region, where the electrochromic shift is easily measured, and where the cytochromes display their most distinct signatures.

a. Deconvolution of Absorbance Changes on Continuous Illumination The proper deconvolution of continuous light-induced spectral changes remains a difficult problem, however, even for the relatively distinct spectral contributions in the green and blue regions, because contributions both from known and unknown sources will overlap. Klughammer and Schreiber (1990) provided a computer algorithm that fits the measured spectral changes to linear combinations of measured component spectra. The major weakness of the technique seems to be the rather crude deconvolution of the large, superimposed light-induced scattering changes and the electrochromic shift. These are roughly subtracted by assuming they approximate a linear ramp. Published spectra of the light-induced scattering (Deamer et al., 1967) and electrochromic (Witt, 1975) changes are not well approximated by a straight line. Given the spectral proximity of the contributing species, such a rough approximation will inevitably interfere with this type of analysis. However, with further refinement, this approach holds great promise for elucidating the redox states of electron carriers in the steady-state. In the demonstration paper the instrument appeared to perform well in the seconds time range (Klughammer et al., 1990). Of course, as the time resolution of this type of instrument is increased, the signal-tonoise ratio will necessarily decrease; signal averaging can be used to decrease noise components, but at the cost of greatly slowing the acquisition of data and reducing the flexibility of experimental protocol. The signal-to-noise ratio of the instrument is most likely limited by the current state of the art in LED technology, where the intensity of shorter wavelength emitters is severely restricted. One interesting possibility for further development

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of this type of instrument would be the simultaneous measurement of spectral changes in the NIR region, where the currently available techniques could greatly benefit from more specificity (see Section IV.F.2). In this region, very intense probe beams could be employed since LEDs that emit in this spectral region are many times more efficient, and because light in this spectral region is non-actinic to the sample.

5. Measurement of pH Changes by Lightscattering The relation between light-scattering changes and the development of was explored by Deamer et al. (1967), who suggested that they might reflect conformational changes or ‘precipitation’ of membrane proteins as the lumenal pH fell. In principle, light-scattering changes, or the absorbance changes which arise from them, could be used to follow and a substantial literature has attempted to take advantage of this (Krause, 1973, 1974; Horton et al., 1991; Brugnoli and Bjorkman, 1992; Ruban and Horton, 1992; Bilger and Bjorkman, 1994). However, in practice, as many of these authors have noted, several factors in addition to local pH changes contribute the amplitude and kinetics of the scattering changes (Crofts et al., 1967; Mohanty et al., 1995), and we have no easy way to deconvolute these in intact systems.

G. Thermal Radiometry There are several techniques to probe the amount of energy stored by plants by measuring the heat evolved upon illumination (Malkin and Canaani, 1994). In general, these techniques can be termed thermal radiometry, though the details of each techniques are quite different. The principles involved in assaying the energy storage from heat emission are very similar to those for the fluorescence measurements (see sections IV. A, B, C and D). When photosynthetic centers are in their active states, they can convert a fraction of the energy in absorbed photons into stored electrochemical work, but when they are blocked, the energy of the absorbed photons is lost via several competing pathways, e.g. fluorescence, non-radiative relaxation (heat), triplet formation, recombination. A general feature of thermal radiometry techniques is that they must discriminate heat production temporally. This is necessary because, eventually, all the energy emitted by the light source will end up as

56 heat. Indeed, the kinetics of heat production and volume changes after short light pulses have been used to study the energy levels of electron transfer states in isolated photosynthetic materials (Callis et al., 1972; Arata and Parson, 1981; Nitsch et al., 1988). For intact plant work, this type of measurement has not been employed. Instead, the heat production at a fixed time after pulses of light or in phase with a modulated beam of a fixed frequency is measured (Bults et al., 1982a; Buschmann et al, 1984; Havaux, et al., 1987; N’soukpoé-Kossi et al., 1990; Charland et al., 1992; Reising and Schreiber, 1992). If appropriate parameters are chosen, thermal radiometry can be used to estimate the quantum yield of photosynthesis, which is related to the fraction of centers in active states and the efficiency of processes that compete with photochemical centers for photon energy (e.g. fluorescence, non-radiative dissipation and specific mechanisms for energy quenching, such as -quenching) by measuring the complementary yield of heat production. Under steady-state conditions, this heat dissipation can give relative information about flux of energy through the photosynthetic electron transfer chain. To yield such information, it is necessary, as in the steady-state fluorescence measurements (see Section IV.C), to obtain a reference value of heat production in a disturbed condition, either where all centers are blocked, or all are in open states. In recent work, this reference state has been obtained by applying saturating pulses of light (see below). The outcome of such experiments can provide data complementary to that obtained by other flux measurements, such as the uptake of or the measurements made using pulse-saturation fluorescence techniques (Snell et al., 1990). However, unlike fluorescence measurements, which primarily reflect changes in PS II chemistry, thermal radiometry measures the energy storage of both PS I and PS II centers, and therefore gives information about the quantum yield for the entire electron transfer chain, e.g. see results on Emerson enhancement observed with photoacoustic spectroscopy (Malkin and Canaani, 1994). Three strategies for measuring the yield of heat production appear in the literature: photoacoustic spectroscopy, photothermal radiometry, and photothermal beam deflection.

David M. Kramer and Antony R. Crofts

1. Photothermal Radiometry Photothermal radiometry measures infrared radiation as an indicator of the temperature of the leaf (Nordal and Kanstad, 1981; Malkin et al., 1991; Driesemaar et al., 1994). A weak, modulated beam of light, usually red light from a light emitting diode, is given to the sample. The amount of heat given off in phase with the modulated beam is measured by an infrared detector and a phase-sensitive amplifier. This technique is by far the most straightforward to interpret. It could in principle be used in open or closed gas exchange cuvettes or in the field. The main difficulty in applying the technique seems to be the low sensitivity of the IR detector. New, highsensitivity detectors should allow more general application of this technique.

2. Photoacoustic Spectroscopy Photoacoustic spectroscopy measures photo-induced changes in ambient pressure caused by release of heat, the uptake or emission of gases, or other volume changes from a sample. The pressure changes are registered on a sensitive microphone enclosed in the same sealed chamber as the sample. It is by far the most widely used thermal radiometry technique for photosynthetic materials and has been used frequently in the past decade to measure energy storage both in isolated preparations of photosynthetic material and in intact plants. For reviews on theory and interpretation, see Rosencwaig and Gersho (1975), Malkin and Cahen (1979), Patel and Tam (1981), Buschmann et al. (1984), Braslavsky (1986), Tam (1986), Buschmann (1989), Crippa et al. (1994), Malkin (1994). For examples of instrument design and performance, see Bults et al. (1982a,b), Poulet et al. (1983), Buschmann et al. (1984), Buschmann and Kocsanyi (1989), Malkin et al. (1981), Reising and Schreiber (1992). Though well constructed photoacoustic spectrophotometers are exceedingly sensitive, there are several drawbacks to this technique. The measurement requires that samples be placed in a restricted volume, isolated from the outside atmosphere. Since leaves are constantly producing and consuming gases, particularly under steady-state illumination, it is very difficult, to maintain controlled atmospheric conditions required for comparison with other assays. The photoacoustic signals represent a confluence of at least three volume

Chapter 2 Measurement of Photosynthetic Electron Transport changes: the photothermal signal (i.e. heat production), and at least two photobaric signals (i.e. changes in volume of the atmosphere not directly by heating) due to oxygen release and uptake (Bults et al., 1982a,b; Charland et al., 1992; Malkin, et al., 1992; Reising and Schreiber, 1992). In aqueous samples, rapid changes in volume associated with conformational changes in the reaction centers are very important contributors to the photoacoustic signals (Callis et al., 1972; Arata and Parson, 1981; Delosme et al., 1994) but these are unlikely to contribute to the very slow pressure changes observed in intact plant work. Though attempts have been made to distinguish these contributions kinetically, particularly the evolution information from the heat dissipation signals, using differential phase detection (Poulet et al., 1983; Malkin et al., 1992) or pulsed light source and kinetic resolution of the resulting signals (Reising and Schreiber, 1992), the assignment of at least some of the photobaric components remains tentative (Reising and Schreiber, 1992). A photobaric detector of oxygen release would certainly be a welcome innovation in photosynthesis research. However, when Charland et al. (1992) deconvoluted the photobaric from the photothermal signals, based on their relative frequency responses, they found that the signals originally attributable to release were modulated by other, undetermined factors, possibly the modulation of sinks, or the opening or closing of stomata. Due to these complications, it seems unlikely at present that a photoacoustic cell could make a useful barometric oxygen probe.

3. Photothermal Beam Deflection Photothermal beam deflection measures the ‘mirage effect’ associated with heat gradients in the atmosphere close to the leaf surface. Havaux et al. (1990) and Lorrain et al. (1990) attempted to use this technique to measure the heat production during steady-state photosynthesis. A narrow beam of light is passed very close to the surface of the sample (in this case a leaf). If the leaf emits heat, a layer of relatively hot air will form between the bulk atmosphere and the leaf surface. Since hot air has a lower index of refraction than cool air, the laser beam will be deflected to a different extent depending upon how much heat the plant generates. The technique is surprisingly sensitive. Although adequate

57

signal-to-noise ratios and reasonably interpretable signals were obtained, practical application to whole plant work is highly doubtful. First of all, it is doubtful if the precise positioning of the laser beam close to the leaf surface would be routinely feasible. It is doubtful that the technique would work with all but very smooth leaves. Furthermore, any motions of the leaf induced by flowing gases (as in a gas exchange cuvette or in the field) would certainly disturb the measurement. It is unclear at present if photoacoustic spectroscopy or photothermal beam deflection for steady-state measurements can offer any advantages over other techniques to measure photosynthetic flux, in particular the more direct uptake or photothermal radiometry, or the simpler and more easily available modulated fluorescence yield or P700 measurements. One interesting application of the technique is the determination of the spectral dependence of the quantum yield of photosynthesis (Canaani, 1990; Delosme et al., 1994).

H. Blue Fluorescence When algae or green plants are illuminated with UV light they emit fluorescence not only in the red and NIR (from chlorophyll) but also in the blue (Duysens and Amesz, 1957; Olson and Amesz, l960; Chappelle and Williams, 1987; Goulas et al., 1990). These blue fluorescence signals can be similar in amplitude to those in the red but have not been as extensively studied. In intact chloroplasts, the blue fluorescence signals have been observed to change with photosynthetic illumination and are therefore of interest as a non-invasive probe of photosynthetic reactions.

1. What Can Be Measured with Blue Fluorescence? Many compounds present in leaves fluoresce in the blue. These include NADH, NADPH, carotenes, riboflavin, lignin, flavins, pteridines, quinones and quinone analogs (e.g. vitamin K, tocopherol), and other polyphenolic compounds (Lunquist et al., 1978; Chapelle and Williams, 1987; Goulas et al., 1990). Though such a complex mixture of fluorescence signals may appear intractable, compounds can be distinguished based on their fluorescence absorption and emission spectra or fluorescence decay kinetics

58 (Goulas et al., 1990). Furthermore, only a few compounds may be expected to rapidly change upon illumination, including the pyridine nucleotides, flavins and quinones. Of these, the pyridine nucleotide NADPH is likely to be of the most interest since it is a key intermediate in electron flow, linking the reducing side of PS I to carbon metabolism (see Chapter 3), and appears to have a large, distinguishable blue fluorescence signal. Indeed, unambiguous observations of NADPH redox changes upon i l l u m i n a t i o n of intact chloroplasts has been demonstrated (Cerovic et al., 1993) using a fluorimeter with a nitrogen laser excitation beam.

2. Prospects and Problems Applying Blue Fluorescence Techniques to Intact Plants

David M. Kramer and Antony R. Crofts circumvented by removing a layer of the epidermis, as in Cerovic et al. (1993), but this certainly constitutes an invasive procedure. A better solution may be to search for plant species, mutation, or growth conditions where epidermal pigmentation is minimized. If routine, unambiguous assay of the redox state of the NADPH pool were possible, it would help us to understand the restrictions to photosynthetic electron transfer (see sections IVF.2.D and E where the issue of PS I acceptor side limitation is important).

V. The Future of Instrumentation for Intact Plants

A. A Principle of Sufficient Determination The kinetics of changes in NADPH redox state will reflect a competition between its reduction from the acceptor side of PS I and its re-oxidation through turnover of the carbon fixation cycle. The usual difficulties in interpreting the steady-state redox level will apply: uncertainty in the magnitude of the baseline dark-adapted level, and the static nature of intermediate levels in a linear chain in the steadystate. However, the use of interrupted or disturbed steady-state conditions may allow the measurement of the kinetics to and from the steady-state levels. In order to fully interpret the NADPH blue fluorescence signals, several other problems must be overcome. It will be difficult to distinguish between NADH and NADPH fluorescence, though one may assume that contamination from NADH signals would be minimal since NADPH would represent the more rapidly changing light-induced signal, and because of the 10-fold excess of NADPH over NADH (Heber and Santarius, 1965; Heineke et al., 1986). Another potential problem is that NADPH is a 3–5 times more efficient fluorophore when it is bound to enzymes (Duysens and Kronenburg, 1957), so changes in its binding state may significantly affect the data. However, the available data suggests that most of the NADPH is bound (Cerovic et al., 1993) and consequently this may not pose a significant problem. By far the largest difficulty in applying this technique to intact plants is the high concentration of UV-absorbing fluorescent compounds in the epidermis of plants which completely obstruct observations of NADPH changes in intact leaves (Cerovic et al., 1993). This problem may be

A well know algebraic axiom can be paraphrased as follows. For any number, n, of variables, to we may want to determine one or more values, e.g. . We have techniques to measure a set of parameters ( to ) related to the variables, but only as linear combinations of two or more of them (e.g. ). In order to find a unique solution for (or any of the other variables), we need to make n measurements. We end up with a matrix of linear equations, one for each variable or measurement technique, that can be solved by well known techniques to yield the values of each contributing species. In the straight forward case of absorption measurements, if we have a system of five contributing species, each with overlapping but distinct spectral contributions, we need five or more measurements at different wavelengths to determine the true contribution of any particular species (Rich et al., 1987). If one could demonstrate, or reasonably assume, that for a particular species no change has occurred, or that the changes in a few species of interest are so large that other contributions can be ignored, fewer points may be needed to arrive at a satisfactory deconvolution. However, such assumptions are usually dangerous. This principle of sufficient determination should be our guide when considering whether measurements can yield precise information. Unfortunately, the usefulness of a method is not necessarily well correlated with the ease of measurement! For example, as is well recognized, measurement of the fluorescence yield is a particularly underdetermined

Chapter 2

Measurement of Photosynthetic Electron Transport

indicator of the state of the photosynthetic apparatus. This is because, as discussed above, we have only one parameter to measure, the yield, which could be influenced by at least ten different factors. Of necessity, workers in this area have attempted to simplify system by treating the reactions of photosynthesis in the steady-state as a ‘black box’ containing only a few components. By ignoring the complexities, it has been possible to determine the flux through this simplified system using fluorescence yield measurements. By our criteria of sufficient determination, we could need as many as ten fluorescence measurements, each under different conditions, which should reflect to different extents each of the contributing processes. In the presently accepted protocols, the use of saturation pulses (to determine the yield when all centers are closed, and assumed to be in the P. state) and measurement of yield during the approach to steady-state in lightdark cycles (see sections IV.A, B and D), allows the parsing of contributions into so-called photochemical and non-photochemical quenching effects. This twocomponent analysis has clearly been useful, but there are obvious dangers in this simplistic approach, which might become apparent if the details ignored in the simplification turn out to be important in control (Kramer et al., 1995).

B. The Need for More Specific Measurements Further quantitative characterization of what happens inside the black box will demand new fluorescence techniques, or the combination of fluorescence measurements with other techniques (Brugnoli and Björkman, 1992; Bilger and Björkman, 1994). The same generalization can be made for those other approaches which we may loosely term ‘general indicators of photosynthetic flux or status.’ These include steady-state photoacoustic measurements, uptake, photothermal radiometry, and delayed fluorescence techniques. It is noteworthy that, although all of these techniques are relatively straightforward and yield data of high signal to noise, the interpretation of results is, as expected from the principle of sufficient determination, problematic or of limited specificity. We believe that future instruments will need to provide greater specificity of measurement. Design and construction of such instruments will likely be more technically demanding because a number of

59

separate components must be measured with sufficient precision and discrimination to make the necessary deconvolutions. The most promising area of development is in absorption spectroscopy, both in the visible and near IR, where measurements at a sufficient number of wavelengths should, in principle, yield completely determined data. In order to deconvolute the contributions of different partial reactions, we need quantitative information on the spectra of all components contributing to the steady state (or to absorbance changes on perturbation of the steady state), and to be able to measure at wavelengths where the components have different contributions. The development of instruments and a spectral database will need to be matched by the development of appropriate protocols for their use, and in this respect, the techniques based on perturbation of the steady-state look most promising. In principle, armed with such information, and instruments of sufficient resolution, a spectral snapshot of leaves in the steady-state, or more realistically, during transitions following perturbation of the steady-state, should yield a more or less complete picture of the state of the electron transfer system. Further exploration of the control of photosynthesis through the fluorescence quenching mechanisms w i l l require the combination of fluorescence yield measurements, and spectrophotometric techniques to probe the poise of the proton gradient, the components of the xanthophyll cycle and the partial reactions of the chain; correlations between these parameters would allow us to analyze the mechanism(s) of control in greater detail.

Acknowledgments The authors thank B. Genty and J. Berry for access to unpublished data, and F. Loreto, W. Nitschke and Govindjee for important discussions. ARC gratefully acknowledges support from US Department of Energy (Grant DE-FG02-86ER13594) and US Department of Agriculture (Grant NIR 91-03273) for work pertinent to this chapter; DMK gratefully acknowledges support from US Department of Energy (Grant DE-FG02-86ER13594) and from the Integrative Photosynthesis Research Training Grant sponsored by DOE/USDA/NSF.

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Chapter 2

Measurement of Photosynthetic Electron Transport

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transfer in the cytochrome bf complex. In: Biggins J (ed) Progress in Photosynthesis, Vol II, pp 453–460. Martinus Nijhoff, Dordrecht Robinson HH (1986) Non-invasive measurements of Photosystem II reactions in the field using flash fluorescence. In Gensler WG (ed) Advanced Agricultural Instrumentation, pp 92–106. Springer-Verlag, New York Robinson HH and Crofts AR (1983) Kinetics of proton uptake and the oxidation-reduction reactions of the quinone acceptor complex of PS II from pea chloroplasts. In: Sybesma C (ed) Advances in Photosynthesis Research, pp 477–480. Nijhoff/ Junk Publishers, The Hague Robinson H and Crofts AR (1987) Kinetics of the changes in oxidation-reduction state of the acceptors and donors of Photosystem II in pea thylakoids measured by flash fluorescence. In: Biggins J (ed) Progress in Photosynthesis, Vol II, pp 429–432. Martinus Nijhoff Publishers, Dordrecht Rosencwaig A and Gersho A (1975) Photo acoustic effect with solids a theoretical treatment. Science 190: 556–557 Ruban AV and Horton P (1992) Mechanism of dissipation of absorbed excitation energy by photosynthetic membranes. 1. Spectroscopic analysis of isolated lightharvesting complexes. Biochim Biophys Acta 1102: 30–38 Ruban AV, Walters RG and Horton P (1992) The molecular mechanism of the control of excitation energy dissipation in chloroplast membranes. FEBS Lett 309: 175–179 Ruban AV, Young AJ, Pascal AA and Horton P (1994) The effects of illumination on the xanthophyll composition of the Photosystem II light-harvesting complexes of spinach thylakoid membranes. Plant Physiol 104: 227–234 Rühle W and Wild A (1979) Measurements of cytochrome f and P700 in intact leaves of Sinapis alba grown under high light and low light conditions. Planta 146: 377–386 Rutherford W. Govindjee and Inoue Y (1984) Charge accumulation and photochemistry in leaves studied by thermoluminescence and delayed light emission. Proc Natl Acad Sci USA 81: 1107–1111 Saphon S and Crofts AR ( 1 9 7 7 ) Proteolytic reactions in Photosystem I I . A new model for the release of protons accompanying the photooxidation of water. Z Naturforsch 32c: 617–626 Schmidt, W (1988a) Luminescence of organic molecules. Theory and analytical application. In: Lichtenthaler HK (ed), Applications of Chlorophyll Fluorescence in Photosynthesis Research, Stress Physiology, Hydrobiology and Remote, pp 211–216. Kluwer Academic Publishers, Dordrecht Schmidt, W (1988b) Long term delayed luminescence in green organisms. In: Lichtenthaler HK (ed) Applications of Chlorophyll Fluorescence in Photosynthesis Research, Stress Physiology, Hydrobiology and Remote, pp 217–221. Kluwer Academic Publishers, Dordrecht Schmidt W and Senger H (1987) Long-term delayed luminescence in Scenedesmus obliquus II. Influence of exogenous factors. Biochim Biophys Acta 891: 22–27 Schreiber U (1986) Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorimeter. Photosynth Res 9: 261–272 Schreiber, U and Neubauer, C (1989) Correlation between dissipative fluorescence quenching at Photosystem II and 50 recombination luminescence. FEBS Lett 258: 339–342 Schreiber U and Neubauer C (1990)

66 flow, membrane energization and the mechanism of nonphotochemical quenching of chlorophyll fluorescence. Photosynth Res 25: 279–293 Schreiber U and Schliwa U (1987) A solid-state portable instrument for measurement of chlorophyll luminescence induction in plants. Photosynth Res 11: 173–182 Schreiber U, Klughammer C and Neubauer C (1988) Measuring P700 absorbance changes around 830 nm with a new type of pulse modulation system. Z Naturforsch 43c: 686–698 Schreiber U, Neubauer C and Klughammer C (1989) Devices and methods for room temperature fluorescence and analysis. Philos Trans R Soc Lond B Biol 258: 339–342 Schreiber U, Neubauer C and Schliwa U (1993) PAM fluorimeter based on a medium-frequency flash measuring light—A highly sensitive new tool in basic and applied photosynthesis research. Photosynth Res 36: 65–72 Shinkarev VP, and Wraight CA (1993) Kinetic factors in the bicycle model of oxygen evolution by Photosystem II. Photosynth Res 38: 315–321 Shinkarev VP, Xu C, Govindjee and Wraight CA (1994) Kinetics of the oxygen evolution step in situ estimated from its quenching effect on flash-induced chlorophyll a fluorescence. Biophys J 66: A114 Snel JFH, Kouijman M and Vredenberg WJ (1990) Correlation between chlorophyll fluorescence and photoacoustic signal transients in spinach leaves. Photosynth Res 25: 259–268 Sonneveld A, Rodemaker H and Duysens LNM (1979) C h l o r o p h y l l a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor of Photosystem I I . Biochim Biophys Acta 548: 536–551 Sonoike K, Terashima I, Iwaki M and Itoh S (1995) Destruction of Photosystem I iron-sulfur in leaves of Cucumis sativus L by weak illumination at chilling temperatures. FEBS Lett 362: 235–238 Sundbohm E and Björn,CD(1977) Phytoluminography: imaging plants by delayed l i g h t emission. Physiol Plant 40: 39–41 Sundby C, McCaffery S and Anderson JM (1993) Turnover of the Photosystem II D1 protein in higher plants under photoinhibitory and nonphotoinhibitory irradiance. J Biol Chem 268: 25476–25482 Sykes AG (1985) Plastocyanin. Chem Soc (Lond) Rev 14: 283– 314 Tam AC (1986) Applications of photoacoustic sensing techniques. Rev Mod Phys 58: 381–431 Taoka S and Crofts AR (1990) Two-electron gate in triazine resistant and susceptible Amaranthus hybridus. In: Baltscheffsky M (ed) Current Research in Photosynthesis Vol I, pp 547– 550. Kluwer Academic Publishers, Dordrecht Taoka S, Robinson HH and Crofts AR (1983) Kinetics of the reactions of the two-electron gate of Photosystem II: Studies on the competition between plastoquinone and inhibitors. In: Inoue Y, Crofts AR, Govindjee, Murata N, Renger G and Satoh K (eds) The Oxygen-evolving System of Photosynthesis pp 369–381. Academic Press, Tokyo Terashima I, Wong S-C, Osmond CB and Farquhar GB (1988) Characterisation of non-uniform photosynthesis induced by abscisic acid in leaves having different mesophyll anatomies. Plant Cell Physiol. 29: 385–394 Thayer SS and Björkman O (1992) Carotenoid distribution and de-epoxidation in thylakoid pigment-protein complexes from

David M. Kramer and Antony R. Crofts cotton leaves and bundle-sheath cells of maize. Photosynth Res 33: 213–225 Vallegaleti R, Kramer DM, Marsh SS, Reichenbach NG, Fleischman D and Corbin J (1990) Some approaches to rapid and pre-symptom diagnosis of chemical stress in plants. In: Wang W, Gorsuch JW and Lower WR (eds) Plants for Toxicity Assessment, pp 333- 345. American Society for Testing and Materials, Philadelphia, Pa Vass I, Styring S, Hundal T, Koivuniemi A, Aro EM and Andersson B (1992) Reversible and irreversible intermediates during photoinhibition of Photosystem 2. Stable reduced species promote chlorophyll triplet formation. Proc Natl Acad Sci USA 89: 1408–1412 Webber A and Gray JC (1989) Detection of calcium binding by Photosystem II polypeptides immobilised onto nitrocellulose membrane. FEBS Lett 249: 79–82 Weis E and Berry J (1987) Quantum efficiency of PS2 in relation to ‘energy’ dependent quenching of chlorophyll fluorescence. Biochim Biophys Acta 894: 198–208 Weis E and Lechtenburg D (1989) Fluorescence analysis during steady-state photosynthesis. Phil Tran. R Soc Lond B Biol Sci 323: 253–268 Weis E, Ball JT and Berry J (1987) Photosynthetic control of electron transport in leaves of Phaseolus vulgaris: Evidence for regulation of Photosystem 2 by the proton gradient. In: Biggins J (ed) Progress in Photosynthesis Research, Vol 2, pp 553–556. Martinus Nijhoff Publisher, Dordrecht Wise R R , Frederick JR, Alm DM, Kramer DM, Hesketh JD, Crofts AR and Ort DR (1990) Investigations of the limitation to photosynthesis induced by leaf water deficit in field-grown sunflower (Helianthus annuus L.). Plant Cell Environ 13: 923–931 Witt H ( 1 9 7 5 ) Energy conservation in the functional membrane of photosynthesis. Analysis by light pulse and electric pulse methods. Biochim Biophys Acta 505: 355–427 Wraight CA and Crofts AR (1970) Energy-dependent quenching of chlorophyll a fluorescence in isolated chloroplasts. Eur J Biochem 17: 319–323 Wraight CA and Crofts AR ( 1 9 7 1 ) Delayed fluorescence and high-energy state of chloroplasts. Eur J Biochem 19: 386–397 Wraight CA, Kraan GPB and Gerrits NM (1972) The pH dependence of delayed and prompt fluorescence in uncoupled chloroplasts. Biochim Biophys Acta 283: 259–267 Yamamoto HY (1979) Biochemistry of the violaxanthin cycle in higher plants. Pure Appl Chem 5 1 : 639–648 Yamamoto HY and Kamite L (1972) The effects of dithiothreitol on violaxanthin de-epoxidation and absorbance changes in the 500 nanometer region. Biochim Biophys Acta 267: 538–543 Yerkes CTand Crofts AR (1992a) The quenching of fluorescence in PS II by Dependence on the state of the oxygen evolving complex, and effects of antimycin. In: Murata N (ed) Research on Photosynthesis Vol I I , pp 635–638. Kluwer Academic Publishers, Dordrecht Yerkes CT and Crofts AR (1992b) Quenching of fluorescence in PS II by Mechanism and effects of antimycin. EBEC Short Reports 7: 5 Yerkes CT, Kramer DM, Fenton JM and Crofts AR (1990) UVPhotoinhibition: Studies in vitro and in intact plants. In: Balscheffsky M (ed) Current Research in Photosynthesis, Vol 2, pp 381–384. Kluwer Academic Publishers, Dordrecht

Chapter 3 Regulation of Light Utilization for Photosynthetic Electron Transport B. Genty Groupe Photosynthèse et Environment, Laboratoire d’Ecologie Végétale, CNRS URA 1492, Université de Paris XI, 91405 Orsay, France

J. Harbinson ATO-DLO, Pb17, 6700AA Wageningen, Netherlands

Summary 68 I. Introduction 68 II. Operation of Light-driven Energy Transduction in Leaves 69 A. Evidence from Gas Exchange 69 1. Relationship between Energy Input and Sink Activity 69 a. Fixation of and at Ribulose 1,5-bisphosphate Carboxylase-Oxygenase 69 b. Alternative Sinks for Reducing Equivalents 71 2. The Quantum Efficiency for Photosynthesis under Limiting Light 72 3. The Quantum Inefficiency for Photosynthesis under Non-limiting Light 74 B. Evidence from Measurements of Photochemistry and Electron Transport in Vivo 74 1. Changes in the Quantum Efficiencies of Photosystems I and II 74 2. Localization of the Principal Site of Limitation of Electron Transport 75 3. Operation of the Limitation of Electron Transport 76 a. Introduction 76 b. Measurement of the Apparent Rate Constant for Electron Transport 77 c. General Consequences for the Operation of Electron Transport 78 4. Determinants of Photochemical Efficiency and Co-ordination of Photochemical Efficiency with Metabolism 79 a. Irradiance, Photosynthetic Efficiency and the Driving Force for Electron Transport 79 b. Co-ordination between Thylakoids and Stromal Metabolism 80 c. Feedforward Activation of Metabolic Sink Demand 80 d. Feedback Control of Electron Transport 81 5. Consequences for the Photochemical Activities of Photosystem I and Photosystem II 82 a. Dissipation of Excess Energy at Photosystem I 82 b. Dissipation of Excess Energy in Photosystem II 83 c. Long Term Modulation: Photoinhibition 85 III. Significance of Structural Acclimation on the Operation of Light-driven Energy Transduction. A Case Study: Acclimation to Growth Irradiance 86 87 A. Electron carriers and the ATPase 88 B. The Relative Absorbance Cross-Sections of the Photosystems 89 C. Consequences for the Operation of Electron Transport 90 IV. Conclusions 91 V. Appendix: The Use of Light-Induced Absorbance Changes Around 820 nm to Measure P700 Oxidation 92 Acknowledgments 92 References

Neil R. Baker (ed): Photosynthesis and the Environment, pp. 67–99. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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B. Genty and J. Harbinson

Summary In nature photosynthesis is required to perform efficiently and safely with an energy resource, light, whose supply is remarkably variable. A description of the operational responses of the energy transduction apparatus in leaves is presented in the context of limiting and non-limiting light conditions for photosynthesis. In leaves linear electron transport appears to be the principal route for reducing equivalents. Under natural lightlimiting conditions for assimilation, the quantum yield for linear electron transport at steady state has been found to be largely uniform in non-stressed leaves, and its high value indicates that plants are able to maintain optimal co-ordination of photosystems at near maximal photochemical efficiency. Under non-limiting light conditions, electron transport operates under a rate restriction occurring at the step which results in a co-ordinated decline of the photochemical quantum efficiencies of both photosystems. Modulation of the rate of electron transfer at the step is also the means by which metabolism exerts feedback control over electron transport. Limitation of electron transport on the donor side of PS I results in the operation of a safe dissipation mechanism for excess excitation energy at PS I. At PS II, only a fraction of excess excitation energy is dissipated safely through a mechanism that is subject to control and whose capacity appears to be dependent on species and growth conditions, in particular irradiance. These general responses are discussed in the framework of an effective co-ordination by feedforward and feedback mechanisms that balance the supply of NADPH and ATP from electron transport with the sink strength of stromal metabolism. The significance for these short term operational responses of the environmental dependency of the stoichiometries of the photosystems and electron transport components is also considered.

I. Introduction The driving force for photosynthesis is light, a resource whose supply is highly variable. Depending on the habitat, season and time of day both, the flux and spectrum of solar irradiance available for plant photosynthesis will vary. The time constant and amplitude of these fluctuations can vary by about one order of magnitude within a few seconds during, for example, the passage of a sun-fleck (Pearcy, 1994; Chapter 13) or a cloud, to over three orders of magnitude during the diurnal cycling of light. Irradiance gradients of one or two orders of magnitude are also frequently encountered going from a full sun habitat to one of deep shade. Photosynthesis must therefore be capable of functioning with a variable energy input. Within the leaf the absorbed light is used to generate free energy gradients that are coupled to numerous processes. These include fixation, photorespiration, other light driven metabolic pathways, and reactions, such as the Mehler reaction. The primary photochemical event of photosynthesis is charge separation in the reaction centers of both PS I and PS II. When successful, this results in the accumulation of reduced and oxidized molecular species that form the driving force for photosynthetic electron transport. The flux of reducing equivalents through the photosynthetic electron transport chain

is the sum of numerous diffusive and electron transfer processes that are each associated with their own driving forces. All these driving forces are interdependent, and the flux of reducing equivalents with which they are associated results in the establishment of the two key driving forces that link electron transport with stromal metabolism: the transthylakoid electrochemical potential difference of protons and the redox state of the ferredoxin pool. The drives ATP synthesis (Ort and Melandri, 1982; Schlodder et al., 1982) and modulates electron transport (Horton, 1985; Foyer et al., 1990). The redox state of the ferredoxin pool drives NADPH reduction and, via thioredoxin, the activation of key enzymes in the stroma (Scheibe, 1990; Foyer, 1993), and may be important in modulating (Hosler and Yocum, 1985, 1987; Robinson, 1988; Schreiber et al., 1991). However, in addition to electron transport, the redox gradients within the photosynthetic electron transport chain also have potentially negative consequences. First, they result in a loss of photochemical efficiency, and second, they can drive damaging side reactions, such as the photoinhibition of PS II. Photosynthetic energy transduction occurs within chloroplasts which are located within cells in leaves, and these structures require materials and energy for their construction and maintenance. This imposes a burden on the plant and, by implication, places a

Chapter 3

Regulation of Photosynthetic Light Utilization

premium on the optimal organization of the photosynthetic apparatus. This organization is dependent on the environment and physiology of the plant, and embodies many compromises that reflect the physical and biochemical operation of photosynthesis on the one hand, and the unpredictability of the physical environment on the other, especially irradiance. Understanding these compromises is crucial to understanding how photosynthesis works and why it responds as it does in different plants. The responses of fixation and plant productivity to the light environment, both in terms of acclimation and instantaneous responses, have been extensively investigated for many decades and have been extensively reviewed (Boardman, 1977; Björkman, 1981; Edwards and Walker, 1983; Long, 1985; Anderson and Osmond, 1987; Woodrow and Berry, 1988; Heber et al., 1990; Baker and Ort, 1992). By contrast, the responses of the light-driven energy transduction machinery in relation to the needs of photosynthetic metabolism, and vice versa, during short and long term fluctuations of irradiance have only recently been subjected to systematic analysis (Horton, 1985; Weis et al., 1987; Laisk and Walker, 1989; Weis and Lechtenberg, 1989; Foyer et al., 1990; Walker, 1992). In this review, we will first briefly describe the operation of the energy transduction apparatus under conditions of changing irradiance and metabolic demand in leaves. We will then present the current state of knowledge of the dynamic responses and controls of the operation of energy transduction in vivo, and discuss the acclimation of the energy transducing apparatus to growth irradiance. This analysis will be largely confined to results obtained from plants. The photochemistry and chemistry of electron transport and the detailed structure of thylakoid components

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has been the subject of several recent reviews and will not be considered in this article (Bendall, 1982; Schlodder et al., 1982; Haehnel, 1984; Rutherford and Heathcote, 1985; Golbeck, 1987; O’Keefe, 1988; Rutherford, 1989; Andersson and Styring, 1991; Cramer et al., 1991; Golbeck and Bryant, 1991; Anderson, 1992; Debus, 1992; Ort and Oxborough, 1992).

II. Operation of Light-driven Energy Transduction in Leaves

A. Evidence from Gas Exchange 1. Relationship between Energy Input and Sink Activity a. Fixation of and at Ribulose 1,5bisphosphate Carboxylase-Oxygenase In air photosynthesizing leaves have complex gas exchange fluxes. Of these the most significant are evolution from PS II, and the fixation of and by ribulose 1,5-bisphosphate (RuBP) at the active site of ribulose 1,5-bisphosphate carboxylaseoxygenase (Rubisco). The combination of these opposing fluxes and a lack of a convenient measuring system has resulted in photosynthetic exchange being much less well documented than fixation. Consequently, most of the insights into photosynthesis obtained from gas analysis have come from measurements of fixation in parallel with water vapor release. Combined with quantitative biochemical models based on the in vitro behavior of Rubisco (Laisk, 1970; Laing et al., 1974; Farquhar et al., 1980; Farquhar and von Caemmerer, 1982), these

Abbreviations: – primary electron acceptor chlorophyll of PS I; – secondary electron acceptor quinone of PS I; – intercellular concentration in leaves; – quaternary electron acceptor iron-sulphur complexes of PS I; FBPase – fructose 1,6-bisphosphatase; – maximum chlorophyll fluorescence yield obtained when is maximally reduced in either the dark or light-adapted state; – minimum chlorophyll yield obtained when is maximally oxidized in either the dark or light-adapted state; – tertiary electron acceptor iron-sulphur complex of PS I; –variable chlorophyll fluorescence yield, NADP-MDH – NADP-malate dehydrogenase; NPQ – non-photochemical quenching of excitation energy; P680 –primary chlorophyll a electron donor in the PS II reaction center; P700 – primary chlorophyll a electron donor in the PS I reaction center; PAR – photosynthetically-active radiation (between wavelengths 400 and 700 nm); Pi – inorganic phosphate; PQ – plastoquinone; – plastoquinol; – primary quinone electron acceptor of PS II; – secondary quinone electron acceptor of PS II; – non-photochemical quenching coefficient of chlorophyll fluorescence; – photochemical quenching of chlorophyll fluorescence; Rubisco – ribulose 1,5-bisphosphate carboxylase-oxygenase; RuBP – ribulose 1,5-bisphosphate; SBPase – sedohetptulose 1,7-bisphosphatase; – half-time; – light-induced absorbance change around 820 nm; – transthylakoid electrochemical potential difference of protons; – relative effective cross-section for the photochemistry of PS I; – relative effective cross-section for the photochemistry of PS II; – quantum efficiency of fixation; – quantum efficiency of PS I photochemistry; – quantum efficiency of PS II photochemistry

70 gas exchange investigations have nonetheless allowed the general relationship between fixation, photorespiration and electron transport to be described (von Caemmerer and Farquhar, 1981; Farquhar and von Caemmerer, 1982; Sharkey, 1985). With increasing irradiance in air, fixation moves from the region of light limitation to that of light saturation, where the rate of photosynthesis is limited by physiological factors (Farquhar et al., 1980; Sage, 1990). This change of limitation with increasing irradiance was first quantitatively described by Blackman (1905), and his proposal that there was a region of light limitation and one of light saturation was essentially a derivative of Liebig’s law of the minimum. This biphasic model of the photosynthesis-irradiance response implies that in the light-limited phase photosynthesis should proceed with maximum quantum efficiency and that once light saturation is reached the quantum efficiency of photosynthesis will progressively diminish with further increases in irradiance (Sage, 1990; Baker and Ort, 1992). Photosynthesis-irradiance relationships do not, however, generally show a sudden transition from the light-limited to light-saturated phases (as shown in Fig. 1), a problem that has been generally attributed to the optical density of chlorophyll containing photosynthetic cells, leaves and tissues (Oya and Laisk, 1976; Gutschick, 1984; Terashima and Saeki, 1985; Osborne and Raven, 1986). The analysis of fixation to changing intercellular concentration has yielded

B. Genty and J. Harbinson clearer information about the changing limitation of fixation in leaves (von Caemmerer and Farquhar, 1981; Sharkey, 1985, 1989; Sage, 1990; Chapter 8). With increasing concentrations at saturating irradiance a clear discontinuity can be resolved in the fixation response (von Caemmerer and Farquhar, 1981; Evans, 1987b; Sage and Sharkey, 1987; Sage et al., 1989). This result has been successfully described using a model based on the kinetic properties of Rubisco (von Caemmerer and Farquhar, 1981), and the discontinuity has been attributed to a change in the limitation of photosynthesis by Rubisco at low concentrations to RuBP regeneration at higher concentrations (von Caemmerer and Farquhar, 1981; Long, 1985; Evans, 1987b). This discontinuity often occurs at values similar to those of the gaseous phase in which the leaf developed, implying that the limitation of fixation is shared between Rubisco and RuBP regeneration (von Caemmerer and Farquhar, 1981; Evans, 1987b; Sage et al., 1989). There are, however, numerous exceptions to this generalization (von Caemmerer and Farquhar, 1981; Sage and Sharkey, 1987). Though the limitation of RuBP regeneration is often believed to be due to limiting electron transport (von Caemmerer and Farquhar, 1981; Long, 1985), it can also be due to limitation by sink activity or the RuBP regenerating reactions of the Calvin cycle (Stitt, 1991). In leaves of plants in air Rubisco will catalyze both the oxygenation and carboxylation of RuBP. The oxygenase function of Rubisco strongly

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Regulation of Photosynthetic Light Utilization

influences the quantitative relationship between the rates of fixation and electron transport. By competing with for RuBP and through the release during the metabolism of the phosphoglycolate of formed by this oxygenation reaction, acts as an inhibitor of fixation (Farquhar and von Caemmerer, 1982; Edwards and Walker, 1983). The relative rates of carboxylation and oxygenation are determined by many factors, of which the relative concentrations of and in the stroma and the kinetic properties of Rubisco are the most significant when considering the environmental modulation of photosynthesis. Drought-induced stomatal closure and temperature changes producing changes in the solubility of and and the temperature effect on the kinetic properties of Rubisco are obvious routes by which the relative rates of carboxylation and oxygenation can be altered. It is expected that the rate of fixation will increase when photorespiration is eliminated owing to the inhibitory effect of on fixation, (Ehleringer and Björkman, 1977; Farquhar and von Caemmerer, 1982; Stitt, 1991). The size of increase in the rate of fixation upon eliminating photorespiration depends on the limiting factor for fixation. This limitation can arise directly from photosynthetic processes in the chloroplast where ATP or NADPH supply or Rubisco activity could be limiting (Farquhar and von Caemmerer, 1982; Stitt, 1991). Alternatively fixation could be restricted by carbohydrate metabolism in the cytosol or by supracellular carbohydrate transport processes (Foyer, 1987, 1988; Geiger, 1987; Stitt, 1991; van Bel, 1992), producing the condition of sink limitation of photosynthesis. The overall energetic requirements of photorespiration (including refixation of evolved are only slightly higher than those of carboxylation, i.e. 3.3 ATP and 2 NADPH per RuBP cycling at the compensation point against 3 ATP and 2NADPH per RuBP cycling for carboxylation only (nonphotorespiratory conditions; calculated from Farquhar et al., 1980). In the absence of any sink limitation of photosynthesis an alteration in the relative rates of photorespiration and fixation will not therefore be expected to have a significant effect on the operation of electron transport on energetic grounds.

b. Alternative Sinks for Reducing Equivalents Photosynthesis includes processes other than photorespiration and fixation. Nitrate and nitrite

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reduction, sulfate reduction, and amino acid, lipid and other biosynthetic activities are all important light driven metabolic processes (ap Rees, 1987; Robinson, 1988; Bloom et al., 1989; Brunold, 1993; Hanning and Heldt, 1993). In fact, it appears that in developing leaves photosynthetic activity is predominantly coupled to processes other than fixation and photorespiration (Baker, 1985). The non-metabolic process of the Mehler reaction, in which molecular oxygen is directly reduced by photosynthetic electron transport, can occur at rates of 1–10% of the rate of fixation in vivo (Badger, 1985; Robinson, 1988). The detoxification of the superoxide produced by this process generates further demand for photosynthetic reducing power (Asada, 1994; Foyer and Harbinson, 1994). Owing to a potential contribution of sink activity from these other light-driven processes, it is unwise to presume that under all conditions predictions of electron transport based on biochemical models of and photorespiration will account for all in vivo electron transport activity, e.g. during photosynthetic induction when the Mehler reaction is known to be particularly active (Radmer and Kok, 1976; Malkin, 1987). Nonetheless estimates of the rate of linear electron transport can often be largely accounted for by the parallel processes of fixation and photorespiration (Nespoulos et al., 1989; Peterson, 1989; Cornic and Briantais, 1991; Kent et al., 1992; Laisk and Sumberg, 1994), even when the stomata are closed and photorespiration is the predominant sink for reducing equivalents (Cornic and Briantais, 1991). This implies that in mature leaves the rate of electron transport is strongly dependent on the action of fixation and photorespiration. Models of photosynthesis based on the kinetics of Rubisco and the biochemistry of photosynthesis have been a useful framework for describing the overall responses of fixation, both in the presence and absence of photorespiration. Using these models it is possible to calculate the rates of electron transport necessary to support the measured rate of fixation and the predicted rate of photorespiration (Farquhar et al., 1980; von Caemmerer and Farquhar, 1981; Farquhar and von Caemmerer, 1982). Based on certain assumptions about the energetics of photosynthetic electron transport, the changes in the quantum yield of fixation in the presence and absence of photorespiration under limiting light have been predicted by these models (Farquhar and von Caemmerer, 1982). However, further decreases in

72 the quantum efficiency of fixation that occur as the irradiance increases, and ceases to be wholly limiting, have not been quantitatively predicted by biochemical models. Models based on the kinetics of Rubisco cannot simulate photosynthetic responses when other metabolic processes, such as cytosolic sucrose synthesis, are limiting. Because of their biochemical basis, the commonly used models of photosynthetic gas exchange and metabolism cannot reflect limitations imposed on photosynthesis by the operation of photochemistry or electron transport (for example, limitations due to RuBP regeneration, von Caemmerer and Farquhar, 1981). These models are also unable to give us any definite information about the regulatory relationships between photochemistry, electron transport and metabolism, or provide any insight into the operation of photochemistry and electron transport, or their responses to regulation. They can, however, define the boundary conditions which the operation of photochemistry and electron transport must fulfill.

2. The Quantum Efficiency for Photosynthesis under Limiting Light The irradiance dependency of photosynthetic electron transport under limiting light is dependent on the absolute quantum yield for charge separation, the relative absorbance cross sections of the two photosystems and the absorbance of the leaf for photosynthetically active radiation (Baker and Ort, 1992). Leaf absorbance is generally high and quite constant from species to species so to a first approximation leaves are physically a good trap for radiation (Björkman and Demmig, 1987). The effects on quantum yield of an imbalance in the excitation of the two types of photosystem and the consequences of intrinsic inefficiencies in light-driven charge separation have been, however, less critically examined. As discussed above, in addition to fixation other processes are also photosynthetic and require reducing power or ATP and these would also be expected to affect the quantitative relationship between the quantum yields for electron transport and fixation. The maximum quantum efficiencies for both photosynthetic evolution and fixation have been the subject of many investigations. Within the framework of the generally accepted scheme based on two photosystems acting in series (the Z scheme), it has been commonly supposed that in vivo the

B. Genty and J. Harbinson quantum yield for evolution will have a theoretical upper limit of 0.125 (8 photons per evolved), assuming that fixation is limited by NADPH synthesis and not ATP synthesis. This upper limit assumes that photochemistry can have an absolute quantum efficiency of one, and requires that all quanta absorbed by photosynthetic pigments have only one fate, the production of irreversible charge separation that results exclusively in linear electron transport through both photosystems. This exclusive chain of events may not occur. For example, there is good experimental evidence that the quantum yield for PS II charge separation and stabilization as and is approximately 0.85–0.95 (Kramer and Mathis, 1980; Thielen and van Gorkom, 1981; Schatz et al., 1988; Mauzerall and Greenbaum, 1989;Trissl and Wilhelm, 1993). In PS I the quantum yield for charge separation is believed to be at least 0.95 (Trissl and Wilhelm, 1993). Other causes of inefficiency have been reported (Myers, 1971). In the framework of the Z scheme these inefficiencies make it unlikely that the upper limit for the quantum yield for either photosynthetic evolution or fixation can be as high as 0.125. Accurate calculations of the absolute quantum yield for fixation or evolution by leaves are difficult to make for technical reasons, e.g. uncertainties concerning the total amount of light absorbed by photosynthetic pigments when nonphotosynthetic pigments are present. Using a broadband (white light) irradiance of limiting intensity under non-photorespiratory conditions, measurements of the yield of both fixation and evolution by leaves on an absorbed light basis have produced a range of values. These lie between 0.073– 0.098 for fixation (Ehleringer and Björkman, 1977; Monson et al., 1982; Ehleringer and Pearcy, 1983; Evans, 1987a; Long et al., 1993) and 0.083– 0.115 for evolution (Walker and Osmond, 1986; Björkman and Demmig, 1987; Evans, 1987a). Notably, Björkman and Demmig (1987) found a very highly conserved yield of 0.106 for evolution from a wide range of plants from contrasting habitats. These data imply that whatever their origins and acclimatory history, leaves are capable of achieving a consistent balanced distribution of excitation energy between PS I and PS II when subjected to a broadband irradiance. Under light-limiting conditions, the optimization of photosynthetic energy transduction as depicted by the Z scheme requires that for maximum efficiency

Chapter 3

Regulation of Photosynthetic Light Utilization

observed the rates of excitation of the populations of both photosystems should be similar. In a study of the wavelength dependency of the absolute quantum yield of evolution and fixation in the absence of photorespiration, Evans (1987a) found a range of values between 0.06 and 0.11 with a maximum yield at 600 nm. This variability was explained in terms of wavelength-dependent changes in the distribution of excitation energy between the photosystems, coupled with increasing absorbance by non-photosynthetic pigments in the blue region of the spectrum. This result suggests that under optimal irradiance conditions the yield of linear electron transport can reach 0.11, which is close to the yield predicted from reported values of the yields of PS I and PS II (see above). Evans also found similar quantum yields for -dependent net evolution and fixation in white light (ca. 0.09); unfortunately data obtained under the optimal wavelength of around 600 nm were not presented. These results suggest that the linear electron flux under limiting irradiance is used largely to drive fixation (see also Long et al., 1993). If the cause of the loss of efficiency for evolution from 0.11 under optimal conditions to 0.09 under white light were to be paralleled by changes in the yield of fixation (which seems likely), then the yields of both evolution or fixation under strictly limiting light would be determined by the quantum efficiency of the photochemical event. The Mehler reaction and other metabolic processes that consume reducing power must be minor sinks for reducing equivalents under these conditions. This conclusion with respect to the non-photorespiratory metabolic demands for reductant is supported by simultaneous measurements of evolution and fixation of leaves in air (Bloom et al., 1989). Recently, however, yields of evolution near to or slightly in excess of 0.125 have been reported using an irradiance of 680 nm (Osborne, 1994). Osborne (1994) has also suggested that these high yields are consistent with others obtained using broad-band irradiance. These measurements of high yields of net evolution around 680 nm were, however, based on quantum flux measurements made using commercial quantum sensors and these devices will underestimate the flux in the irradiance band around 680 nm, for example, see typical spectral responses of such devices in Evans (1987a). This underestmation of the quantum flux makes the high yields for evolution reported by Osborne (1994) unreliable. The observed high quantum yields also make it

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unlikely that the cyclic and pseudo-cyclic electron transport fluxes, whose operation would result in a loss of quantum efficiency for linear transport, operate to any significant degree (Haehnel, 1984). This is supported by data obtained for leaves using photoacoustic spectroscopy that show insignificant energy storage by cyclic electron transport around PS I (Herbert et al., 1990). The operation of the cyclic or pseudocyclic pathways to a significant degree has been thought necessary to balance ATP and NADPH generation by the thylakoids for fixation and other photosynthetic metabolism, but it would appear that this can be achieved by linear electron transport on its own. A Q cycle could act to increase the stoichiometry (Wraight, 1982; Crofts and Wraight, 1983). As this mechanism appears to occur in chloroplasts (Rich, 1988; Kramer and Crofts, 1993) it is a convenient candidate to solve the problem of the apparent deficiency of ATP synthesis when linear electron transport is operating alone (Evans, 1987a; Furbank et al., 1990). In air the quantum yield for fixation is much lower than the yield for fixation under conditions where photorespiration is minimized; typical values are 0.07 or less depending on the rate of photorespiration (Ehleringer and Björkman, 1977). The competitive effect of photorespiration is dependent on those factors, such as temperature and stomatal conductance, which modify the relative rates of carboxylation and oxygenation of RuBP (see Section II.A.1; Björkman, 1981; Wang et al., 1992). Because of the complications imposed by the presence of photorespiration measurements of the quantum yield for fixation in air provide much less useful information about the operation of electron transport, though this data is much more relevant in understanding plant production processes (Baker and Ort, 1992; Long and Drake, 1992). Under light-limiting conditions the presence of photorespiration is not expected to have any significant effect on the operation of electron transport because the energetic requirements of photorespiration and fixation are very similar (see Section II.A. 1). Though the behavior of the quantum yield for fixation in the presence of photorespiration appears to be consistent with the responses predicted by photosynthetic models based on Rubisco kinetics, the possibility cannot be discounted that under high conditions there will be an increase in the Mehler reaction. This flux is expected to be strongly influenced by the metabolic demand for reducing equivalents.

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3. The Quantum Inefficiency for Photosynthesis under Non-limiting Light We have argued that under wholly light-limiting conditions the efficiency of photosynthesis is determined by the irradiance dependency of the supply of reducing equivalents. This in turn is limited by the maximum efficiencies of photochemistry and the distribution of excitation energy between the photosystems. As the irradiance increases and ceases to be limiting, the quantum yields of fixation, evolution and electron transport decline. This loss of efficiency ultimately results in the irradiance saturation of photosynthetic electron transport. In this context the questions that need to be addressed are (i) what is the site at which electron transport in limited and by what mechanism is this limitation modulated, (ii) how is the photochemistry of PS I and PS II adjusted in response to declining efficiency of electron transport, and (iii) how general are these responses?

B. Evidence from Measurements of Photochemistry and Electron Transport in Vivo To answer these basic questions information is needed about the rate of electron transport, the components of the thylakoid and stromal electron chains between which irradiance-driven redox gradients are established, and the photochemistry and operation of PS I and PS II in vivo under a range of conditions known to change the rates of fixation and photorespiration. The tools available with which to routinely measure these processes in leaves are very limited. Using chlorophyll fluorescence it is possible to monitor the fate of excitation energy in PS II, and to derive estimates of the relative quantum yield for PS II photochemistry and the activity of competitive non-photochemical excitation quenching, NPQ (Bradbury and Baker, 1983; Dietz et al., 1985; Ögren and Baker, 1985; Schreiber, 1986; Schreiber et al., 1986; Weis et al., 1987; Genty et al., 1989; Krause and Weis, 1991). The relative oxidation state of P700 can be quantified using measurements of the light induced absorbance change around 820 nm, (see Section V for a discussion of this technique and Harbinson and Woodward, 1987; Weis et al., 1987; Schreiber et al., 1988; Harbinson et al., 1989; but see also Chapter 2), From this measurement estimates of the photochemical efficiency of PS I can be made (Weis et al., 1987; Harbinson et al., 1989) provided

B. Genty and J. Harbinson there is no limitation of P700 oxidation by a shortage of electron acceptors. In most cases no significant limitation of this kind appears to exist (see below), and various strategies exist for its detection (Harbinson and Foyer, 1991; Harbinson and Hedley, 1993; Klughammer and Schreiber, 1994; Harbinson, 1994). In the unusual case where P700 oxidation is limited by a shortage of electron acceptors, its oxidation can be used to provide information about the oxidation state of the acceptor side of PS I (Harbinson and Foyer, 1991; Foyer et al., 1992; Heber et al., 1992; Harbinson and Hedley, 1993; Harbinson, 1994; Klughammer and Schreiber, 1994; Laisk and Oja, 1994). The convenience and reliability of both the chlorophyll fluorescence and 820 nm absorbance measurements allows them to be used routinely to monitor the photochemical efficiencies of both photosystems in vivo. Leaf photoacoustic spectroscopy and photothermal radiometry have also provided valuable information about photosynthetic energy storage and evolution (Canaani and Malkin, 1984; Malkin, 1987; Malkin et al., 1991; Driesenaar et al., 1994; Malkin and Canaani, 1994). Other important techniques commonly used in biophysical studies in vitro, such as absorbance changes associated with cytochrome f and the electrochromic shift, have also given valuable information about the operation of photosynthesis in leaves (e.g. Ruhle et al., 1987; Chylla and Whitmarsh, 1989; see also Chapter 2). These techniques, however, are more difficult to exploit for in vivo measurements of photosynthesis and so have been less widely used than chlorophyll fluorescence and 820 nm absorption changes.

1. Changes in the Quantum Efficiencies of Photosystems I and II Measurements of the quantum efficiencies of PS I and PS II photochemistry made in parallel with those of fixation under a range of gas compositions (air and a range of and concentrations) in both and leaves show a decline with increasing irradiance (Fig. 1; Weis et al., 1987; Genty et al., 1989, 1990b; Harbinson et al., 1989; Peterson, 1989, 1991; Öquist et al., 1992; Jacob and Lawlor, 1993; Johnson et al., 1993; Oberhuber et al., 1993). This decline of and with increasing irradiance is of general occurrence in photosynthetic tissues, cells or organelles.

Chapter 3 Regulation of Photosynthetic Light Utilization The irradiance dependence of and shows that the range of irradiances within which both photosystems are operating at maximum efficiency is limited. In leaves with further increases of irradiance in air, or in other situations where there is fixation and/or photorespiration, the loss of parallels that of and the relationship between them is predominantly linear with both concurrently tending to zero (Weis et al., 1987, 1990; Harbinson et al., 1989, 1990a; Genty et al., 1990b; Peterson, 1991; Harbinson, 1994). Only at low irradiances, when both and are high, is any non-linearity commonly observed. This is generally due to a relatively greater loss of compared to though the size and even the presence of this nonlinear phase is variable from leaf to leaf (Harbinson et al., 1989, 1990a; Genty et al., 1990b; Peterson, 1991). This loss of is associated with reduction, implying that there is overexcitation of PS II relative to PS I (see Section III.B.). A greater loss of compared to in low light conditions is rarely encountered, but can develop following photoinhibition (Genty et al., 1990a), in chlorophyll b deficient mutants (Andrews et al., 1995) or during irradiation with far-red enriched light (Andrews et al., 1993; J. Harbinson, unpublished). The factors that determine the decline in photochemical efficiency require an understanding of the dynamic operation of photosynthesis and its relationship to the sink demand of photosynthetic metabolism under conditions where the energy input is increasing. Regardless of the action of the regulatory mechanisms that link photochemistry, electron transport and photosynthetic metabolism, the decline of the efficiency of photosynthesis with increasing irradiance requires that more energy is safely dissipated. In this context a number of questions need to be addressed, (i) As electron transport becomes limited where does this limitation act? (ii) How is the photochemistry of PS I and PS II adjusted in response to declining efficiency of electron transport? (iii) By what means is absorbed light energy in excess of requirements dissipated? (iv) What is the balance between feedforward regulation of metabolism versus feedback regulation of electron transport, and in particular how important is it to control photosynthetic electron transport? (v) How do the driving forces for electron transport respond to limitation?

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2. Localization of the Principal Site of Limitation of Electron Transport With increasing irradiance electron transport ceases to be completely light-limited and becomes progressively limited by either the electron transport chain or photosynthetic metabolism. Ultimately this changing pattern of limitation results in electron transport becoming light-saturated and independent of irradiance. The increasing oxidation of P700 with increasing irradiance under steady state conditions indicates that a limitation of electron transport is exerted on the donor side of PS I. Normally no significant limitation of P700 oxidation can be detected on the acceptor side of PS I in vivo. is normally calculated on the assumption that there is no inhibition of P700 oxidation owing to a deficiency of electron acceptors (Harbinson, 1994). The fact that estimates of calculated in this way correlate well with the quantum efficiency of assimilation and the total efficiency of energy storage by leaves implies that there is little inhibition of P700 oxidation because of a lack of electron acceptors (Weis et al., 1987; Harbinson and Foyer, 1991; Malkin et al., 1991; Foyer and Harbinson, 1994; Harbinson, 1994; Laisk and Oja, 1994). This is also supported by measurements of total P700 oxidizability during saturating multiple turnover flashes (Klughammer and Schreiber, 1994). Under certain circumstances P700 oxidation is strongly limited by a lack of electron acceptors, e.g. for short periods during photosynthetic induction (Klughammer and Schreiber, 1991; Siebke et al., 1991; Foyer et al., 1992; Heber et al., 1992; Harbinson and Hedley, 1993) and possibly briefly during sudden large increases in irradiance, and persistently under conditions of low concentration in the absence of photorespiration (Heber et al., 1992; Laisk and Oja, 1994). Consequently, only transitory restrictions of electron transport on the acceptor side of PS I will occur in leaves in air, or other gaseous phases permitting metabolic turnover of photosynthesis. This inference of an open PS I acceptor side, drawn from biophysical measurements, is supported by biochemical measurements of the redox state of electron carriers in the stroma. The activity of chloroplast NADP-malate dehydrogenase (NADPMDH) is believed to be an indicator of the redox state of the stroma (Cseke and Buchanan, 1986; Scheibe and Stitt, 1988; Scheibe, 1990), with increasing activation correlating with increased

76 reduction of the NADP pool (Scheibe, 1990; Foyer et al., 1992). Measurements of NADP-MDH activity (Scheibe and Stitt, 1988; Harbinson et al., 1990b), direct measurements of the redox state of NADP in leaves during steady state photosynthesis in air (Dietz and Heber, 1984), and direct measurements of NADP in isolated intact chloroplasts supplied with bicarbonate, indicate that the stroma remains relatively oxidized when fixation is active (Takahama et al., 1981). With increasing irradiance the photochemical quenching of chlorophyll fluorescence declines (Dietz et al., 1985; Weis et al., 1987; Genty et al., 1989), and it can be inferred from this that the pool becomes more reduced as the irradiance increases. This requires that the limitation of electron transport lies on the acceptor side of PS II and not on its donor side. Cytochrome f has also been shown to be oxidized in illuminated leaves (Ruhle et al., 1987; Klughammer et al., 1990) and based on measurements of plastoquinone reduction state in chloroplasts and intact algae (Amesz et al., 1971, 1972; Böhme and Cramer, 1972; Siggel, 1976) it seems likely that the limiting step of electron transport lies between the plastoquinol pool and the cytochromes of the cytochrome complex (Haehnel, 1984). This is supported by measurements of the degree of control exerted by cytochrome f and PS II on electron transport over a range of irradiances which show that with increasing irradiance the control exerted by cytochrome f increases and that of PS II decreases (Heber et al., 1988). This limitation of electron transport on the donor side of PS I, at the to cytochrome f electron transfer step, is a general feature of the regulation of photosynthetic electron transport under steady-state conditions. It develops under any irradiance that is not wholly limiting for fixation and is sustained with increasing irradiance (Weis et al., 1987; Harbinson et al., 1989, 1990a, 1990b; Genty et al., 1990b; Peterson, 1991; Harbinson, 1994), with decreasing temperatures (Foyer and Harbinson, 1994) and to a large degree with decreasing concentrations in a non-photorespiratory atmosphere (Harbinson et al., 1990b; Harbinson, 1994; Laisk and Oja, 1994). Only when the metabolic demand for the products of electron transport is very low is there clear evidence for a limitation of electron transport on the acceptor side of PS I and a loss of the normal regulatory state (see Section II.B.2). This localization of the limitation is purely

B. Genty and J. Harbinson phenomenological; it is certainly an effect of regulation but not necessarily the cause of regulation. For example, under low temperature treatment or during sink limitation fixation appears to be limited by a restriction of cytosolic carbohydrate metabolism resulting in -insensitive photosynthesis (Leegood and Furbank, 1986; Sharkey et al., 1986; Sage and Sharkey, 1987; Labate and Leegood, 1988; Stitt, 1991). Under these conditions electron transport is still restricted between PS II and PS I (Harbinson, 1994) even though photosynthesis as a whole is being limited by processes in the cytosol.

3. Operation of the Limitation of Electron Transport a. Introduction Photosynthesis requires controlled fluxes of energy and materials, and every flow, whether it be water moving along a pipe or electricity within a wire, can be understood in terms of a driving force (or potential gradient) acting across a resistance. Ohm’s law is a simple expression of this general relationship. Therefore the operational limitation that exists between the two photosystems can be analyzed as a flux resistance across which a driving force is applied, and any change of flux can be attributed to either a change of resistance to electron transport, a change of the driving force, or both. Close to equilibrium the driving force is usefully expressed as a chemical potential difference, but further from equilibrium the rate of reaction or flux ceases to be linearly related to potential. Under these conditions rates of reactions or fluxes are best modeled using chemical kinetics where concentration (or more strictly activity) can be seen as a driving force and the resistance to flux will be the inversely proportional to the rate constant of the reaction. Only close to equilibrium do the thermodynamic and kinetic approaches converge (Katchalsky and Curran, 1967). In photosynthetic electron transport the limiting step is the reduction of the cytochrome complex by (Section II.B.2). The resistance of this step is a function of the rate constant for the reaction between and the cytochrome complex, and the driving force is a function of the activity gradient between the and cytochrome f couples, assuming that the oxidation of the by cytochrome complex is driven by cytochrome independently of the state of the cytochrome b pool,

Chapter 3

Regulation of Photosynthetic Light Utilization

which may not be the case (Kramer and Crofts, 1993).

b. Measurement of the Apparent Rate Constant for Electron Transport Measurement of the resistance and the driving force in vivo is difficult. The resistance to the electron transport flux can be gauged from the kinetics of electron transport with fast kinetics representing a low resistance. The kinetics of electron transfer can be most conveniently monitored by measuring the rate of reduction of or cytochrome following either a light to dark transition or the addition of a probe flash to a leaf in continuous irradiance. Using measurements, the reduction kinetics of in vivo are easier to measure than those of cytochrome reduction and so most of what is known about kinetics in vivo comes from these data. However these rate constants are really pseudo-rate constants because they are a function of the redox state of the plastoquinol pool (Bendall, 1982; Rich, 1982; Hope et al., 1988), which is a component of the driving force. The redox state of the plastoquinol pool is not yet measurable in vivo so the changes in the pseudo-rate constant due to changes in the driving force have not yet been separated from those due to changes in the rate constant. Hence the pseudo-rate constant derived from kinetic measurements is an amalgam of the driving force and the resistance. Estimating the kinetics of electron transport from reduction kinetics of or cytochrome is further complicated by results which imply lower than expected equilibrium constants between P700, plastocyanin and cytochrome f (Joliot and Joliot, 1984; Delosme, 1991; Murakami and Fujita, 1992). Low equilibrium constants between these components would require that the rate of electron transport estimated from the reduction of one component be adjusted for electrons equilibrating on the others. In the case of estimates obtained from reduction, these would underestimate the actual rate of electron transport from to cytochrome This problem needs further resolution, and here estimates of the rate constant for electron transport obtained from kinetics will simply be the rate constant for this absorbance change without any adjustment. The relationship between the pseudo-rate constants for and cytochrome reduction will also depend on the concentration ratio of P700 and cytochrome f (see Section III.B); if there were to be more

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cytochrome f than P700 then the measured rate constant for reduction of would be expected to be greater than the rate constant for electron transport through the cytochrome f pool. Nonetheless the pseudo-resistance is still a very useful measure of the kinetics of electron transport and degree of photosynthetic control. The apparent rate constant for electron transport, which is proportional to the inverse of the half-time for reduction, does not appear to change with increasing irradiance (Fig 1; Harbinson and Hedley, 1989; Harbinson and Foyer, 1991; Harbinson, 1994; Laisk and Oja, 1994) except under conditions where sink limitation develops (Harbinson, 1994). The case of sink limitation excepted, this irradiance independence of apparent rate constant for electron transport has been observed in leaves with high and low rates of electron transport (Harbinson and Hedley, 1989; Harbinson, 1994; Laisk and Oja, 1994). In these leaves there is therefore no change in the degree of control of electron transport over an irradiance range that extends from a partially lightlimited condition to one of light-saturation. In other circumstances, however, the apparent rate constant is clearly under control; it changes during photosynthetic induction (Harbinson and Hedley, 1989), with temperature (Laisk and Oja, 1994) and with concentration (Harbinson, 1994; Laisk and Oja, 1994) or sink limitation (Harbinson, 1994). The greater the decrease in the demand for NADPH and ATP then the greater is the increase in the degree of control of electron transport. The effectiveness of control at the cytochrome step in regulating electron transport can be seen when the concentration of an atmosphere containing approximately 2% and 98% is decreased under conditions of constant irradiance (Fig. 2; Harbinson, 1994; Laisk and Oja, 1994). The apparent rate constant for linear electron transport decreased from 59 at 370 ppm to a minimum of 8.5 at 0 ppm (equivalent to a change of 11.8 ms to 83 ms), with control of limitation of electron transport being retained on the donor side of PS I above 35 ppm As the rate constant for electron transport was when the concentration was 35 ppm, the effective control in this leaf, i.e. with the limitation of electron transport being retained on the donor side of PS I, could be sustained until the rate constant for electron transport had decreased to 27% of the value in 370 ppm A loss of control on the donor side can be identified

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by the increase in the pool of unoxidized P700 upon changing from 35 ppm to 0 ppm producing an increase in the estimate for even though the rate constant for electron transport decreased from at 35 ppm to at 0 ppm In 20% decreasing from normal, ambient concentration to the compensation point produces a 50% decrease in the rate of electron transport at high light (Harbinson and Foyer, 1991), well within the range of control in this leaf.

c. General Consequences for the Operation of Electron Transport An implication of the importance of the apparent rate constant for linear electron transport in controlling electron transport is that it is expected that the light-saturated rate of electron transport should be positively related to the apparent rate constant for linear electron transport (Fig. 3). An absolutely identical relationship is not expected for all leaves because the rate of electron transport depends on the total amount of P700 in the leaf as well as the rate constant for its reduction. The relationship does not go through the origin of the graph implying that even with no fixation there would still be some electron transport, possibly for residual photorespiration, Mehler reaction or other

B. Genty and J. Harbinson

metabolic requirements. The relationship between the apparent rate constant for linear electron transport, photochemical efficiency and the rate of electron transport under light saturated conditions emphasizes the importance of the rate constant for electron transport in determining the operation of electron transport. Clearly as both change with changing irradiance even when the apparent rate constant for electron transport remains constant, a change in photochemical efficiency in response to a change in irradiance does not require or imply a change in the degree of photosynthetic control. The regulation of the transfer of reducing equivalents from the pool to the cytochrome complex in vitro has been relatively well characterized, even though uncertainties remain concerning the precise mode of operation of the cytochrome complex. Two key factors determine the rate of reduction of the cytochrome complex by or reduced artificial quinones. First, an increase in the concentration of reduced quinone will increase the electron transport rate (Bendall, 1982; Rich, 1982; Hope et al., 1988). Second, a decrease the intrathylakoid pH results in a decrease of the electron transfer rate (Siggel, 1976; Bendall, 1982; Tikhonov et al., 1984; Nishio and Whitmarsh, 1993). The effective control of electron transport at the

Chapter 3

Regulation of Photosynthetic Light Utilization

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4. Determinants of Photochemical Efficiency and Co-ordination of Photochemical Efficiency with Metabolism In the preceding sections we have described the responses of photochemical efficiency and discussed the site and regulation of the limitation of photosynthetic electron transport in vivo. We have done this within the context of photosynthesis operating as a force/flow system (see Section II.B.3) within which driving forces and resistances are present. It is these driving forces that link efficiency, flux and resistance, and which couple electron transport to photosynthetic metabolism. In addition analyzing the flux in terms of resistance and driving force highlights many of the compromises that underlie the acclimation of photosynthesis to irradiance, and the development of stress avoidance mechanisms. The changes to the apparent rate constant for linear electron transport, and the oxidation of the P700 pool in response to depressed fixation, whether this is caused by low concentrations, low temperatures or sink limitation, show that electron transport is closely controlled by feedback from the stroma (in the first instance). The reverse relationship also occurs; the electron transport chain can modulate stromal metabolism by feedforward control. to cytochrome f electron transfer step has important consequences for the operation of electron transport. One effect of controlling the supply of reducing equivalents to the stroma will be to minimize the rate of superoxide production by the Mehler reaction. The rate of this reaction increases as ferredoxin (Hosler and Yocum, 1985) and other stromal electron carriers, such as nitrite reductase, become more reduced (Robinson, 1988). Superoxide radicals are considered to be a major source of stress in aerobic photosynthetic systems, and because of their toxicity scavenging systems must be continually active (Asada and Takahashi, 1987; Halliwell, 1987; Asada, 1994; Foyer and Harbinson, 1994; also see Chapter 5). PS I also appears to be vulnerable to photoinhibition when illuminated under conditions where reducing equivalents accumulate on its acceptor side (Inoue et al., 1989), so a limitation of electron transport on the donor side of PS I should reduce the risk of light-induced damage to PS I.

a. Irradiance, Photosynthetic Efficiency and the Driving Force for Electron Transport As described earlier, the decrease in photochemical efficiency of PS I is associated with the accumulation of and that of PS II is, to a variable degree, associated with the accumulation of The driving force for electron transport through the resistance represented by the electron transport chain is a function of the overall redox gradient between and and especially the gradient between cytochrome To produce an increase in flux requires an increase in the driving force if the resistance remains constant, or a decrease in the resistance if the driving force remains constant, or a combination of both. Typically the resistance for electron transfer remains constant with changing irradiance. So, the tendency for an increased irradiance to produce an increased rate of electron transport as result of increased photosystem exciton densities (also a driving force) results in a decline in photochemical efficiency as the gradient

80 increases. Note, however, that though the formation of the chlorophyll singlet state precedes charge separation in both PS I and PS II reaction centers and can thus be seen as a driving force, the response of the population of chlorophyll singlets (and by implication their lifetime) to a change in the openness of the reaction center populations is non-linear. In PS I the lifetime of the chlorophyll singlet state is the same whether P700 is oxidized or not (Nuijs et al., 1986). In PS II, though the chlorophyll singlet lifetime is increased upon closing the reaction center (Holzwarth, 1991), this increase is limited by the actions of other de-excitation processes in leaves (Genty et al., 1992; Chapter 1). Consequently, chlorophyll singlets do not accumulate in response to reaction center closure. The increasing driving forces for intersystem electron transport have implications for the development of damage in PS II. An increase in the pool or PS II exciton density will result in an increase in damage in or around PS II (Baker and Bowyer, 1994; Foyer and Harbinson, 1994; Ohad et al., 1994; Whitmarsh et al., 1994; Chapter 4). Thus, the establishment of a large driving force not only results in a loss of photochemical efficiency in both photosystems, but will also increase the risk of damage to PS II and adjacent membranes. The association between the size of the driving force, electron transport, efficiency and stress has clear implications for the attainment of high rates of electron transport and acclimation to different irradiance regimes. The reciprocal relationship between driving force and efficiency, and the problems of stress associated with high driving forces, sets limits for increasing the rate of electron transport via an increase in and although the accumulation of appears to have no stress penalty it does result in a loss of efficiency. An alternative response is to decrease the resistance so that a high flux can be achieved with a small driving force, and thus a smaller decrease in This should be considered when examining shade acclimation strategies.

b. Co-ordination between Thylakoids and Stromal Metabolism We have described the phenomenology of the regulation of electron transport in response to changing metabolic demand in the stroma (Section II.B.3). It is relevant to consider the means by which the driving forces established by photosynthetic

B. Genty and J. Harbinson electron transport can feedforward to stimulate metabolic activity, or feedback to reduce photosynthetic electron transport activity. A detailed description of the regulation of the Calvin cycle lies outside the scope of this review and has, in any case, been discussed in several recent, comprehensive articles (Edwards and Walker, 1983; Leegood et al., 1985; Woodrow and Berry, 1988; Leegood, 1989; Foyer et al., 1990; Heber et al., 1990; Scheibe, 1990; Foyer, 1993; see also Chapters 6 and 7). It is, however, worth making several general points about how photosynthetic electron transport and metabolism interact. The point has been amply made that the driving force for photosynthetic carbon reduction is a function of the [NADPH]/[NADP] and [ATP]/ [ADP][Pi] ratios, and their product, assimilatory ‘power’ (Dietz and Heber, 1986; Heber et al., 1986, 1990; Gerst et al., 1994). This driving force acting across the kinetic limitation imposed on photosynthetic carbon metabolism by the diffusive pathways and enzyme kinetics determines the rate of fixation. This metabolic driving force is coupled to the driving forces generated by photosynthetic electron transport; the and the electrochemical potential of the oxidized/reduced ferredoxin couple (Section II.B.3.a). Consequently increases in the metabolic driving force will, on the one hand, tend to produce decreases in and and, on the other, increase the activation state of the stromal enzymes. It is the balance between these feedback and feedforward processes that will determine the efficiency of photosynthesis.

c. Feedforward Activation of Metabolic Sink Demand The effectiveness of the feedforward activation of stromal enzymes can be seen in the stability of both the redox state of the NADP pool, the ATP/ADP ratio and assimilatory power at steady state under over a range of irradiances (Takahama et al., 1981; Dietz and Heber, 1986; Fredeen et al., 1990; Heber et al., 1990). In contrast to this stability, the activation state of key photosynthetic enzymes that are redox activated by reduced thioredoxin, such as fructose1,6-bisphosphatase (FBPase) or sedoheptulose-1,7bisphospatase (SBPase), increases with irradiance (Harbinson et al., 1990b; Scheibe, 1990), thus lowering the flux resistance of photosynthetic metabolism and permitting an increased rate of fixation with a relatively constant driving force. Under

Chapter 3 Regulation of Photosynthetic Light Utilization these conditions the rate constant for reduction remains constant, and the P700 acceptor pool remains oxidized (see Section II.B.3). The quantitative consequences for the two driving forces that link photosynthetic electron transport to metabolism, the transthylakoid and the redox state of the ferredoxin pool are not known with certainty, but it is possible to draw some general conclusions. In the absence of any kinetic control, the increased redox activation of stromal enzymes requires that the degree of reduction of ferredoxin must increase, but this increase is not so great that it limits electron transport into the ferredoxin pool. The redox potential of thioredoxin and the regulatory thiol groups of FBPase are 150 and 120 mV higher, respectively, than the redox potential of ferredoxin (Ort and Oxborough, 1992). Consequently under equilibrium conditions both thioredoxin and FBPase will become very reduced before the degree of reduction of ferredoxin increases significantly. The kinetic constraints that operate upon electron transfer processes in the stroma are not known, so it is not possible to say how large the redox gradients must be to drive the electron flux through the stromal pathway from ferredoxin to NADP to or how large they must be to drive reducing equivalents from ferredoxin via thioredoxin to redox activated enzymes. An increased rate of fixation also requires an increased rate of ATP synthesis. The redox activation of the chloroplast ATPase is complete and rapid even at very low irradiances (Kramer et al., 1990; Ort and Oxborough, 1992) so an increase in the rate of ATP synthesis can only be achieved by an increase in the transthylakoid or an increase in either phosphate (Pi) or ADP if either is limiting (Strelow et al., 1990). Once the exceeds the energetic threshold for ATP synthesis, the rate of synthesis increases strongly with increasing reaching a maximum within a further increase of the equivalent of one pH unit (Junesch and Gräber, 1984; Rumberg et al., 1990). In thylakoids in vivo at steady state the is believed to be due largely to a pH gradient (Ort and Melandri, 1982), so the increase in the rate of ATP synthesis will be produced by a fall in the intrathylakoid pH. As the rate of reaction is pH sensitive (Bendall, 1982; Tikhonov et al., 1984; Nishio and Whitmarsh, 1993) an increasing rate of fixation would be expected to cause a decrease in the rate constant for reduction, but no such effect is observed. It may be that the increase in the degree of reduction of the plastoquinol pool that would

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accompany a decrease in the rate of results in an increase in the rate of this reaction which partly compensates for the pH effect. It is clear that under conditions of increasing irradiance, and in the absence of sink limitation of fixation, the predominant regulatory mechanism that balances the sources and sinks of photosynthesis is the feedforward activation of stromal metabolism.

d. Feedback Control of Electron Transport When the limitation imposed by stromal metabolism is increased, for example following a decrease in the intracellular concentration, a decrease in temperature or an increase in sink limitation, the consequences for the balance between the feedforward and feedback controls are complex (Dietz and Heber, 1986; Heber et al., 1990). It is clear, however that feedback control of electron transport by metabolism does operate, producing decreases in the rate constant for reduction (Section II.B.3.b). Though it is widely considered that this feedback occurs largely through a decrease in the intrathylakoid pH which acts to reduce the rate constant of the reaction (Horton, 1985; Weis et al., 1987; Foyer et al., 1990), this mechanism has not been subjected to a critical, quantitative appraisal. The intrathylakoid pH in vivo has not been measured, nor is its exact nature understood. It is clear, for example, that the bulk phase pH can be varied independently of local pH domains that control non-photochemical quenching of excitation energy in PS II, or electron transport at the cytochrome (Westerhoff et al., 1984; Ohmori et al., 1985; Laasch and Weis, 1988; Renganathan et al., 1991). Consequently, though the feedback control of electron transport via control of the intrathylakoid pH is plausible and qualitatively consistent with the chemistry of the reaction, the effects of artificial pH changes on the rate of electron transfer and the pH changes measured in chloroplasts in vitro, it is not possible to say if it is the only mechanism acting to modulate the rate constant for electron transport. The exact mechanism(s) by which intrathylakoid pH is lowered has also not been demonstrated, though a number of possibilities exist. A fall in the intrathylakoid pH will parallel an increase in the driving force and this may result from: (i) a decrease in the stromal Pi, for example during sink limitation or sudden temperature decreases (Labate

82 and Leegood, 1988; Sharkey and Vanderveer, 1989); (ii) the increase in the that is needed to sustain a given rate of ATP synthesis when the ADP concentration decreases as the ATP/ADP increases (Strelow et al., 1990); or (iii) cyclic or pseudocyclic fluxes that will pump protons into the lumen independently of electron transport to the fixation or photorespiratory pathways (Robinson, 1988; Foyer et al., 1990; Schreiber et al., 1991). These options are not exclusive, and may operate to different degrees in different plants. Even very small cyclic or pseudocyclic fluxes could be effective in controlling electron transport. They will both act to decrease intrathylakoid pH which will slow down electron transport and increase Once a lower intrathylakoid pH has been established, the cyclic or pseudo-cyclic flux will only need to be sufficiently active to maintain the pH against the increased rate of passive proton diffusion across the thylakoid membrane. The regulation of both cyclic and pseudo-cyclic fluxes has been linked to the degree of reduction of stromal redox carriers (Hosler and Yocum, 1985, 1987; Robinson, 1988), with increased reduction producing increased fluxes, so both could offer an effective mechanism for modulating linear electron transport into the stroma. The increases of the ATP/ADP ratio as the concentration is decreased (Dietz and Heber, 1986; Gilmore and Björkman, 1994), as temperature falls (Gilmore and Björkman, 1994) or with the development of sink limitation (Sawada et al., 1992) are consistent with the control of electron transport by intrathylakoid pH. In other cases, decreasing fixation capacity may be accompanied by decreased ADP/ATP and symptoms of limitation of photosynthesis by Pi, and then the thylakoid ATPase may be limited by Pi availability which is also consistent with regulation of electron transport by intrathylakoid pH.

5. Consequences for the Photochemical Activities of Photosystem I and Photosystem II As the irradiance saturation of photosynthesis increases, the increased limitation of electron transport by the kinetics of intersystem electron transfer implies that light is not only less limiting but is in excess. One problem for the operation of photosynthesis is that the efficient process of light absorption by photosynthetic pigments is irreversible and cannot therefore be controlled by the plant at the pigment level, though the radiation intercepted by

B. Genty and J. Harbinson leaves can be regulated by other means. The action of pulvini (in, for example, the families Oxalidaceae, Marantaceae, Piperacae and Leguminosae) allows leaves to move relatively quickly and therefore regulate the amount of the intercepted radiation (Björkman and Powles, 1982). Chloroplast movement might serve a similar purpose (Brugnoli and Björkman, 1992). Leaf wilting in response to water stress will also act to reduce intercepted irradiance (Chiariello et al., 1987). Though some mechanisms exist that will modulate the radiation interception by leaves, they are either relatively specialized, slowly acting or irreversible. In general, therefore, an excess of irradiance must be accommodated within the photosynthetic machinery, and will result in the first instance in a decline in photochemical efficiency. In addition to developing under environments with changing irradiance, surfeits of irradiance may also develop or increase under other regimes that decrease the rate of stromal metabolism, for example, in response to decreased temperatures, stomatal closure, or sink limitation of photosynthesis. Light supply is also unpredictable and can increase faster than the rates of fixation or photorespiration. Consequently photosynthesis must be able to regulate the dissipation of excess energy during transients (Pearcy, 1994; Chapter 13). The problem for plants is that the reactive intermediates produced by light trapping and photochemistry in both photosystems can be potentially destructive if the physiological electron transport reactions are limited (Chapter 5). This excess energy must therefore be dissipated by some controlled means.

a. Dissipation of Excess Energy at Photosystem I In PS I the oxidized reaction center is as good a quencher of excitation energy as the non-oxidized center so the accumulation of produces a direct proportional decline in the quantum yield for electron transport within PS I (Nuijs et al., 1986; Weis et al., 1987; Harbinson et al., 1989). This is wholly consistent with the low fluorescence yield, and absence of modulation in the yield of PS I fluorescence by PS I photochemistry. In the state charge separation is obviously impossible, and in this case the trapped energy of the absorbed quantum is dissipated as heat. As stated above (Section II.B), is calculated from the accumulation of and this estimate agrees well with other

Chapter 3 Regulation of Photosynthetic Light Utilization estimates of photochemical efficiency (Weis et al., 1987, 1990; Harbinson et al., 1989; Genty et al., 1990b; Malkin et al., 1991). This implies that in vivo, dissipation of excess energy within PS I is achieved via thermal dissipation in the fraction of P700 that is oxidized. A dissipative pathway involving the transfer of electrons from P700 to plastocyanin via oxygen/ superoxide has been demonstrated in isolated chloroplasts (Asada, 1994; Chapter 5), but there is so far no evidence for this pathway being important in leaves, though it may have a minor role, or be active only under certain circumstances. In the unusual case where PS I electron transport is blocked on the acceptor side, thus preventing electron transport from PS I, the exact fate of the absorbed energy in vivo is not clear, but reactive species may be formed that could be important regarding photoinhibition of PS I. Within the reaction center of PS I, excited P700 is oxidized by the primary electron acceptor from which the electron is passed via the electron transport components and to ferredoxin (Rutherford and Heathcote, 1985; Golbeck and Bryant, 1991). In vitro measurements of PS I with prereduced electron acceptors show that charge separation can still occur, but as the electron cannot proceed to ferredoxin it can recombine with the hole on to produce the relatively long-lived triplet state of P700. Though triplet formation can occur as a result of a back reaction from either or (Rutherford and Heathcote, 1985; Nuijs et al., 1986; Golbeck and Bryant, 1991), it is not known if either process occurs in vivo under physiological irradiances. On the basis of kinetics measured in vitro it seems likely that , with a lifetime of 30 ms, could accumulate. could also accumulate, though to a lesser degree than because of its lifetime of only 0.25 ms (Golbeck and Bryant, 1991). As is thought to precede the accumu–lation of a population of would result in some backreaction from to and thus result in P700 triplet formation (Golbeck and Bryant, 1991). This is potentially dangerous. A triplet molecule can interact with the normal triplet ground state and convert it to a highly reactive singlet state. The problem of damaging side reactions developing following reaction center triplet state formation in an oxygen containing environment has been studied in PS II (Telfer and Barber, 1994; Chapter 4). It is clear from these studies that singlet oxygen is produced and that a photoinhibition type damage to PS II can result. Interestingly, PS I

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photoinhibition is known to develop whenever electron transport is blocked on the acceptor side and triplet formation may occur (Inoue et al., 1989). Recently light-induced damage to PS I has been demonstrated under low temperature conditions (Havaux and Davaud, 1994; Terashima et al., 1994) which may be related to a chilling-induced prolongation of the limitation of electron transport on the acceptor side of PS I.

b. Dissipation of Excess Energy in Photosystem II Whereas the means of dissipation of excess energy in PS I appears to be a universal process with no known adaptive responses, that of PS II is more complex and differs from leaf to leaf. The intersystem limitation of electron transport imposes a restriction on the acceptor side of PS II, resulting in an accumulation of The accumulation of results in a loss of quantum efficiency via the closure of PS II traps, but it has been demonstrated that in vivo this mechanism is not sufficient to account for all the observed decline of in parallel with the overall decline of photosynthetic efficiency (Weis et al., 1987). Another major, short term, modulated deexcitation phenomenon has been demonstrated in vivo, that of non-photochemical quenching of excitation energy, NPQ (Weis and Berry, 1987; Weis et al., 1987; Genty et al., 1989; Chapter 1). There is clear syndrome associated with the action of NPQ in vivo, even though its mechanism is still the subject of debate. A similar collection of de-excitation phenomena which operate in the long term are also well known and are collectively referred to as photoinhibition (Osmond, 1994); these will be discussed separately. Using chlorophyll fluorescence it has been possible to investigate the relative importance of efficiency changes due to reduction which are measured using the parameter and changes in the efficiency of PS II due to NPQ which are measured using (Genty et al., 1989). This last parameter allows the effect of changes in NPQ on efficiency to be simply appreciated. The non-photochemical quenching coefficient, is widely used to describe NPQ (Schreiber et al., 1986), and though it is useful in this respect, it does not allow the effect of NPQ on photochemical efficiency to be clearly quantified. Quantitatively, the relative loss of PS II efficiency via NPQ or reduction is variable depending on

84 the species and the growth irradiance. However, there has only been a limited amount of data published which describes the acclimatory or interspecific variation in the capacity for dissipation of excitation energy by NPQ (Demmig-Adams and Adams, 1992; Johnson et al., 1993). The typical relative NPQdependent loss of is between 60% and 35%. In plants that can adapt to high light conditions the capacity for the NPQ-dependent decrease of is closer to 60% and can account for a large part of the excess energy dissipated in PS II under normal irradiance conditions (Johnson et al., 1993; Ruban et al., 1993). The strong development of NPQ results in the steady state fluorescence yield remaining largely unchanged as declines with increasing irradiance (Genty et al., 1989; Foyer and Harbinson, 1994). In fact, the light-adapted steady-state fluorescence yield in some plants can even decline to below the dark (Bilger and Björkman, 1990; Ruban et al., 1993). Plants adapted to low light conditions, and ecologically obligate shade plants, have relatively less capacity for NPQ, and in these most of the inefficiency of PS II is due to reduction (Bilger and Björkman, 1990; Demmig-Adams and Adams, 1992; Ruban et al., 1993). In these plants the poor development of NPQ means that as declines the steady state fluorescence yield increases as reduction increases. In leaves, the relative fluorescence yield is a linear function of the mean exciton life-time of PS II (Genty et al., 1992). Therefore, owing to the differing environmental responses of NPQ and reduction, and thus the irradiance dependency of the fluorescence yield, the irradiance dependence of exciton lifetime in PS II is also variable depending on species and growth conditions. Under the irradiance levels found in natural environments, the product of the quantum flux absorbed by PS II and exciton lifetime gives the exciton density in PS II. This will determine the rate of chlorophyll triplet formation (Kramer and Mathis, 1980) and the risk of formation of reactive singlet oxygen and associated photodamage (Foyer and Harbinson, 1994). The action of NPQ, therefore, not only modulates the rate of charge separation, but will also serve to control the photodynamic damage associated with excited chlorophyll species. The accumulation of may trigger photoinhibition, so by limiting formation the modulation of PS II by NPQ also has consequences for the process of photoinhibition. Non-photochemical quenching is still the subject

B. Genty and J. Harbinson of a debate which centers on two key issues: (i) what is the mechanism(s) of quenching and (ii) how is it regulated in vivo? Explanations for the short term, rapidly reversible mechanism(s) that commonly represent the major fraction of NPQ fall into two categories. In one class of models NPQ is proposed to occur in the reaction center, and this requires the conversion of a variable fraction of PS II reaction centers to a photochemically inactive state with an increased ability to thermally dissipate excitation energy (Weis and Berry, 1987; Krause and Weis, 1991; Kreiger et al., 1992; Chapter 1). In the other class the quenching is proposed to occur in the pigment bed, and this requires the appearance of specific quenching centers or processes associated with the pigment bed of PS II that increase the rate of thermal de-excitation in PS II (Genty et al., 1989, 1990c; Horton and Ruban, 1992; Chapters 1 and 2). In both models NPQ is proposed to be at least partially modulated by intrathylakoid pH, with a low pH producing a large NPQ (Briantais et al., 1979). The active component of the intrathylakoid pH may not be the bulk phase pH but rather a localized domain (Laasch and Weis, 1988; Noctor et al., 1993). It has been proposed that inactivation of some PS II reaction centers occurs through a low pH-induced limitation on their donor side, and that this is combined with a non-radiative charge recombination between the donor and acceptor sides of PS II (Kreiger et al., 1992). Alternatively in the pigment bed model it has been proposed that intrathylakoid pH modulates the rate of de-excitation in the PS II pigment bed with a decreased intrathylakoid pH producing an increase in the rate of de-excitation (Genty et al., 1990c, 1992; Horton and Ruban, 1992). It is also clear that low intrathylakoid pH is not the only effector for NPQ; conversion of violaxanthin to zeaxanthin (and possibly antheraxanthin) may modulate the development of NPQ either directly or indirectly (Demmig-Adams et al., 1990; Gilmore and Yamamoto, 1991, 1992; Chapter 1). A larger pool size of zeaxanthin, antheraxanthin and violaxanthin has been reported to correlate with the capacity for NPQ (see Chapter 1). In the framework of the pigment bed quenching mechanism, it has been proposed that (i) zeaxanthin (and possibly antheraxanthin) quenches excitation directly (Owens et al., 1992; Chapter 1), or (ii) pH-induced structural changes in LHC II, or minor complexes associated with LHC II, result in NPQ and the increase in zeaxanthin and possibly antheraxanthin, or the

Chapter 3 Regulation of Photosynthetic Light Utilization decrease in violaxanthin (Rees et al., 1992; Crofts and Yerkes, 1994; Horton and Ruban, 1994; Chapters 1 and 2). There is good evidence that the pigment bed quenching mechanism predominates in intact leaves (Genty et al., 1990c, 1992; Horton and Ruban, 1992, 1994; see also Chapter 1). Using protoplasts from Hordeum vulgare, the capability of PS II centers to evolve was found to be largely unchanged in the presence and absence of non-photochemical quenching of fluorescence (B. Genty, unpublished). These data show that NPQ does not result from modification of primary photo–chemical events and charge recombination by PS II, and imply that it is the pigment bed mechanism that is acting in vivo. The role of pH in modulating both NPQ and electron transfer from to the cytochrome obviously raises the question as to what co-ordination exists between these facets of regulation. Regardless of the mechanism, we do not believe that the regulation of PS II by changes in NPQ represents the principal means of modulating linear electron transport under steady state conditions; rather alterations in NPQ act to limit the reduction of the pool. Despite the regulation of PS II efficiency by NPQ that is so clearly evident, the increase of NPQ that occurs with increasing irradiance is paralleled by an increase in this implies that the principal limitation of linear electron transport lies on the acceptor side of PS II. Nonetheless the changes in the efficiency of PS II photochemistry that result from changes in NPQ are also expected to influence the redox state of the plastoquinol pool. As the redox state of the plastoquinol pool can influence the rate of reduction of the cytochrome complex, the modulation of the redox state of in response to changes in NPQ is expected to play some role in the overall modulation of electron transport. Regrettably, there is no direct demonstration of this relationship.

c. Long Term Modulation: Photoinhibition In addition to the rapidly reversible loss of photochemical efficiency of PS II that has been discussed earlier, there are also a group of processes that are collectively known as photoinhibition and that are characterized by a long term, slowly reversible loss of photochemical efficiency. The characteristics and possible mechanisms of both the short and long term losses of photochemical efficiency have been comprehensively discussed in a recent series of reviews (Aro et al., 1993; Baker and Bowyer, 1994).

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It has been shown that even at low light the operation of PS II photochemistry is associated with the continuous rapid turnover of the D1 protein of the PS II reaction center (Mattoo et al., 1981; Aro et al., 1993; Chapter 4). This turnover increases with increasing irradiance up to the irradiance at which photosynthesis becomes light-saturated, and this turnover increase corresponds to an increase in the inactivation, breakdown and repair cycle of D1 with no net decline of (Aro et al., 1993). Under laboratory conditions when photosynthetic material is exposed to irradiances higher than that required to saturate photosynthesis and when the pool is highly reduced, photoinactivated PS II reaction centers, which retain their D1 protein and do not turnover, have been observed (Aro et al., 1993; Sundby et al., 1993; van Wijk and van Hasselt, 1993). This persistent photoinactivation of PS II reaction centers has been proposed to be the primary cause of the light-induced inhibition of photosynthesis and the loss of in higher plants (Aro et al., 1993). However, it has not been so well demonstrated that the sometimes substantial, slowly reversible loss of PS II efficiency that can be seen under the natural range of irradiance for any particular plant is consistently related to PS II inactivation processes of the kind associated with laboratory photoinhibition. This long term reduction of PS II photochemical efficiency, which may be exacerbated by a stress of some kind (Powles, 1984), has also been associated with violaxanthin to zeaxanthin conversion, and it has therefore been suggested that the short and long term regulation of PS II photochemical efficiency are related (Demmig-Adams and Adams, 1992). Whereas a long term loss of efficiency due to damage can be seen as a consequence of the exposure of the plant to conditions in excess of its physiological tolerance, understanding the role of a long term loss of efficiency due to a protective mechanism, which may be similar in its mode of action to NPQ, is more problematical. It is not clear why a long term protective process should substitute for a short term process, implying as it does a loss of potential efficiency under the light-limiting conditions that will inevitably occur under field conditions. One possible situation where a long term inefficiency would have no negative consequences for the plant would be under conditions of sink limitation or prolonged stomatal closure. In this case a prolonged loss of photochemical efficiency would not limit fixation. However, though this might explain

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why a chronic loss of efficiency might not be harmful in some cases, it does not explain why the long term mechanism is chosen over the short term one. In terms of the regulation of electron transport, the development of long term loss of will result in becoming rate limiting for electron transport. As a result of this, will decline under lightlimiting conditions because of the deficiency of reducing equivalents from PS II (Fig. 4). With increasing light saturation the electron transfer step will again become limiting.

III. Significance of Structural Acclimation on the Operation of Light-driven Energy Transduction. A Case Study: Acclimation to Growth Irradiance In the preceding sections we have concentrated on describing the character of short term adjustments of photosynthetic energy transduction. We have presented our evidence and arguments for the overall regulation or limitation of electron transport between PS II and PS I at the step. This limitation, in combination with the simple management of dissipation of excess energy at PS I and the more complex control of energy dissipation at PS II, appears to be a universal feature of the regulation of

B. Genty and J. Harbinson

steady state photosynthetic energy transduction in leaves under normal physiological conditions. These adjustments to the limitation for electron flow and the efficiencies of both photosystems are occurring continually in plants under natural conditions in response to environmental change. The continuous tuning of photosynthesis, however, operates upon a background of slower changes to the structure and stoichiometry of the photosynthetic apparatus that are driven by acclimatory and developmental responses of the plant. They may be local in nature, for example changes in Rubisco concentration per unit leaf area or cytochrome f/PS II ratio, or they may operate on a whole plant basis, such as changes in leaf number or increases in sink activity. The short term adjustments of photosynthesis must operate within the result of these long term responses. In the event that the long term adjustments of photosynthesis are insufficient or incapable of responding quickly enough, greater emphasis is placed on the short-term responses. If these are incapable of responding to a sufficient degree then stress and injury will result (e.g. when a shade plant is exposed to full sunlight). Most studies of the acclimation of the composition and structure of the thylakoid membrane have dealt with responses to sun/shade or the growth irradiance (Björkman, 1981; Anderson, 1986; Anderson and Osmond, 1987; Chapter 11). In general most attention

Chapter 3 Regulation of Photosynthetic Light Utilization has been paid to the maximum rate of electron transport, chlorophyll concentration, thylakoid stacking and the concentrations of the major thylakoid components (such as PS II, PS I, cytochrome f, The maximum capacity for photosynthesis, whether measured as fixation, evolution or partial electron transport reactions has been found to be highly responsive to changes in growth irradiance and differs tremendously between plants that have evolved to suit different radiation environments; rapidly growing sun plants have rates of fixation that are commonly 10–20 times greater than those of rain forest floor herbs (Björkman, 1981). In contrast the quantum yield of fixation or evolution in an unstressed leaf remains high and largely invariant, whatever the growth environment and origin (Björkman and Demmig, 1987; Long et al., 1993; see Section II.A.2). Large variations in the relative amounts and stoichiometries of the major thylakoid components have been detected in response to changes in growth irradiance. With increasing irradiance the major change in the organization of the thylakoids is a decrease in the number of chlorophylls per electron transport chain (Boardman, 1977; Björkman, 1981; Anderson, 1986; Evans, 1988; Terashima and Evans, 1988) which reveals a major change in the allocation of material resources between the light-harvesting and electron transport components. The range of pigment concentrations per unit leaf area found in unstressed, non-variegated leaves does vary, and there is no consistent correlation between growth irradiance and the photosynthetic pigment content for leaves of higher plants (Björkman, 1981; Chapter 11). This may be expected as in most leaves the concentration of pigments is high enough so that leaf absorbance is only weakly dependent on pigment concentration (Chapter 11). The relationship between leaf absorbance and pigment concentration is also complicated by the complex nature of leaf optics, for example by the path length changes due to differences in microanatomy and related light scattering (Osborne and Raven, 1986; Vogelman, 1993; Chapter 11).

A. Electron carriers and the ATPase In plants grown under a large range of irradiances, the thylakoid components that show the greatest changes in concentration, on a chlorophyll basis, upon going from low to high irradiance are typically cytochrome f (as an indicator of the cytochrome

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complex content) and the plastoquinol pool (Boardman, 1977; Wild, 1979; Björkman, 1981; Anderson, 1986; Anderson et al., 1988; Chapter 11). The more extensive results for cytochrome f show that the amount of this component is well correlated with changes in both the electron transport capacity of thylakoids in vivo and the light-saturated evolution of leaves (Björkman, 1981; Evans, 1987b, 1988; Terashima and Evans, 1988; Chapter 11). The greatest changes in cytochrome f concentration occur over the lowest irradiances commonly employed (Leong and Anderson, 1984b; Anderson, 1986; Lee and Whitmarsh, 1989). Although there is less data for the changes in plastoquinol concentration with changing growth irradiance, it is clear that the plastoquinol pool also increases substantially with increasing irradiance (Björkman, 1981; Anderson, 1986). These responses of the plastoquinol pool and the cytochrome complex are consistent with their key role in the limitation and regulation of photosynthetic electron transport. Notably the other thylakoid component that has been commonly reported to respond strongly to growth irradiance is the ATPase which increases strongly with increasing growth irradiance (Leong and Anderson, 1984b; Anderson, 1986; Chow and Hope, 1987; Evans, 1987b; Chow et al., 1991a). Unfortunately information on other photosynthetic electron transport carriers, such as ferredoxin and plastocyanin, is so restricted as to make generalizations impossible (but see Anderson, 1986). Less is known about the effect of ecophysiologically meaningful changes in light quality during growth. There are no data presently available on the change in electron transport components induced by typical sun or shade light quality environments in the absence of intensity changes. Interestingly several comparative studies of blue and red light grown plants subjected to equal quantum fluxes have shown that under blue light the photosynthetic pigments and thylakoid component concentrations resembled those of plants grown under high irradiance conditions, whereas under red light they resemble those from low light grown plants (Lichtenthaler et al., 1980; Wild and Holzapfel, 1980; Leong and Anderson, 1984c). Though this pattern of response has been consistently obtained from several sun plants, the opposite response was obtained from the shade tolerant fern Asplenium australasicum (Leong et al., 1985).

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B. The Relative Absorbance Cross-Sections of the Photosystems The responses to growth irradiance of populations of the reaction center complexes of both photosystems, their associated antenna complexes and the distribution of excitation energy between the photosystems have been widely reported. Several patterns of response emerge from these data. The data for reaction center stoichiometries are much clearer than those for the absorbance cross-sections of the photosystems, and show significant changes in the relative stoichiometries of the reaction centers of PS I and PS II, depending on the species and growth conditions. The total number of PS II reaction centers, on a total chlorophyll basis, generally increases with increasing growth irradiance, though in some exceptional species the PS II reaction center density is largely independent of irradiance (Leong and Anderson, 1984b; Anderson, 1986; Wild et al., 1986; Anderson and Osmond, 1987; Chow and Hope, 1987; Evans, 1987b; Anderson et al., 1988; Lee and Whitmarsh, 1989; Walters and Horton, 1994). In common with the responses of cytochrome f most of the changes in PS II density occur at the lowest irradiances commonly employed in this type of investigation (Leong and Anderson, 1984b; Anderson, 1986; Lee and Whitmarsh, 1989; Chow et al., 1991a). PS II reaction center densities are, however, generally less responsive than those of cytochrome f. The reaction center density of PS I responds much less strongly to growth irradiance than that of PS II or may be independent of irradiance (Leong and Anderson, 1984b; Anderson, 1986; Wild et al., 1986; Anderson and Osmond, 1987; Chow and Hope, 1987; Evans, 1987b; Anderson et al., 1988; Lee and Whitmarsh, 1989; Walters and Horton, 1994). In most cases there is more PS II than PS I, with the exceptions possessing similar amounts of both (Melis and Brown, 1980; Whitmarsh and Ort, 1984; Anderson et al., 1988; Jursinic and Dennenberg, 1989; Lee and Whitmarsh, 1989). Critical interpretation of these data is complicated by the range of techniques used, and the assumptions that are implicit in these techniques (Mauzerall and Greenbaum, 1989). For example, the interpretation of PS II population size is also complicated by the existence of slowly turning over reaction centers that do not contribute to linear electron transport and which are often termed inactive centers (Graan and Ort, 1986;

B. Genty and J. Harbinson Jursinic and Dennenberg, 1989; Lavergne and Leci, 1993). A low growth irradiance is commonly reported to result in a decreased chlorophyll a/b ratio. This is considered to result from an increase in the absorbance cross-section of PS II relative to PS I due to an increase in the antenna size of individual units since PS I content remains relatively constant and PS II content decreases with decreasing growth irradiance (Boardman, 1977; Wild, 1979; Anderson, 1986; Anderson et al., 1988). Analysis of chlorophyllprotein complexes separated using gel electrophoresis supports this possibility (Leong and Anderson, 1984a; Anderson, 1986; Walters and Horton, 1994). Employing certain critical assumptions about thylakoid and leaf optics, calculations of the expected distribution of excitation energy between both photosystems, based on the amounts of chlorophyllprotein complexes and their absorbance spectra, have indicated an excess excitation of PS II relative to PS I under natural light conditions (Evans, 1986, 1988). This imbalance in favor of PS II does not change much (<5%) between radiation environments typical of sun and shade conditions (Evans, 1986, 1988). However, interpretation on a whole leaf basis of changes of the relative stoichiometries of PS I and PS II and in their absorbance properties is made difficult by the complexities of light penetration and absorbance by leaves. The relative effective cross-section for photochemistry (which is given by the product of the yield of photochemistry and the relative absorbance crosssection of the photosystems) is a more relevant parameter than the relative absorbance cross-section to describe the balance between the photochemistries of PS I and PS II (Myers, 1971). Measurements of the Emerson enhancement effect offer a means of measuring the relative effective cross-sections for photochemistry of both photosystems. Using photoacoustics it is possible to apply this measurement to intact leaves (Canaani and Malkin, 1984; Malkin and Canaani, 1994; Chapter 2). The data of Canaani and Malkin (1984) show that for most wavelengths of photosynthetically active radiation the relative effective cross-section for PS II photochemistry exceeds that for PS I even in plants that have been irradiated with PS II enriched light to produce state II. The wavelength at which imbalance between the cross-sections of PS I and PS II photochemistry (given by is maximal

Chapter 3 Regulation of Photosynthetic Light Utilization of ca. 1.6.) is coincident with the absorbance peaks of chlorophyll b at 650 nm and 480 nm, but when integrated over the entire PAR spectrum the imbalance in favor of PS II is smaller. Using these data obtained from tobacco leaf in State 1, we have calculated a range of Emerson enhancements of between 1.25 for a typical sunlight spectrum and 1.10 for typical shadelight spectrum (400–740 nm in both cases and assuming for wavelengths between 700–740 nm). Other qualitative evidence for an excess photochemical conversion at PS II exists; under lightlimiting conditions in the absence of extra far-red, P700 and cytochrome f remain reduced and some PS II centers close (Joliot et al., 1968; Chow et al., 1991b; Section II.B. 1,2). All these data suggest that plants maintain a structural arrangement that favors an excess excitation and photochemical rate of PS II relative to PS I under natural light conditions. When light quality is changed markedly, the relative reaction center stoichiometries and absorbance cross sections are changed. In contrast to the response to low irradiance in the absence of spectral changes, a marked spectral imbalance in favor of PS I irradiance, even with a parallel decrease in irradiance, results in an increase in PS II reaction centers relative to PS I (Melis and Harvey, 1981; Melis, 1984; Chow et al., 1990; Walters and Horton, 1994). Importantly, parallel to the increase of PS II during growth in light that is preferentially absorbed by PS I, decreases in the PS I reaction concentration per unit chlorophyll have been reported (Melis and Harvey, 1981; Chow et al., 1990; Walters and Horton, 1994). As with a simple decrease in irradiance, simulated shade also produces a decrease in the chlorophyll a/b ratio. It has been suggested that in both photosystems the size and composition of individual photosynthetic units is unaffected by light quality and irradiance, and all changes arise through adjustments to the populations of both photosystems (Melis, 1984; McKiernan and Baker, 1991) rather than changes in the absorbance cross section of PS II (Leong and Anderson, 1984a; Anderson, 1986). The greater imbalance of excitation in favor of PS II that develops under such simulated shade growth conditions has been interpreted as a response to the relative impoverishment under shade conditions of light preferentially exciting PS II (Melis, 1984; McKiernan and Baker, 1991). However, as we suggested in the previous discussion, under natural conditions such large changes in spectral balance may not be frequently encountered.

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C. Consequences for the Operation of Electron Transport It is pertinent to ask what effect these changes in the relative absorbance cross-sections have on the quantum yield for photosynthesis under strictly lightlimiting conditions. On the one hand, the quantum yield for healthy leaves is high and remarkably uniform (Björkman and Demmig, 1987; Section II.A.2). On the other, the environmentally and genetically determined imbalance of excitation and photochemical conversion in favor of PS II would be expected to be associated with variable decreases in the quantum yield for photosynthesis under a natural light environment. Although the ratio of PS II excitation relative to PS I excitation for natural light can appear quite large, typically 1.2–1.3, the detection of the resulting loss of quantum yield of 30% will be made more difficult by State transitions that may act to reduce the imbalance. Using more extreme, unnatural spectral distributions it has been possible to demonstrate the expected small differences in quantum yield resulting from acclimation to simulated sun/shade light quality (Chow et al., 1990). It may be that the commonly reported range of redistributions of excitation energy of 15%–30% following of State transitions in leaves will be sufficient to rebalance the system (Canaani and Malkin, 1984; Malkin et al., 1986; Veeranjaneyulu et al., 1991). This result should be contrasted with the situation in algae, which grow in an environment where the spectrum of the light environment is very variable and in which large adjustments have been demonstrated. The slight imbalance of excitation in favor of PS II that is generally detected may be important in ensuring redox poising of the plastoquinol pool even at low irradiances to ensure, adequate electron transport through the cytochrome complex after dark adaptation (State I). This imbalance may, however, be only temporary because of State transitions. In the framework of our section on quantum yield, this imbalance may result in a small additional inefficiency of photosynthesis in limiting light (Section II.A.2). The stoichiometric changes of the reaction centers of both photosystems, and related changes in chlorophyll a/b ratio, in response to alterations in growth irradiance and quality can be understood in terms of the possible kinetic limitation on electron transport that can be imposed by the reaction centers, and on the dependency of photochemical yield on the absorbance cross-section. The size of antenna per

B. Genty and J. Harbinson

90 reaction center cannot be increased indefinitely. The photochemical efficiency depends on rate of charge separation and the size of the pigment bed. In any pigment the excited singlet state has a finite lifetime and will relax to the ground state by a variety of routes (Holzwarth, 1991; Chapter 1). Since the rate of charge separation is considered to be limiting compared to the equilibration time of the excitation between pigments (Schatz et al., 1988; Leibl et al., 1989; Holzwarth, 1991), then for a given rate constant of charge separation the photochemical yield will be determined by the probability of excitation decay processes that do not result in charge separation in the reaction center; this probability increases as the ratio of non-reaction center pigment to reaction center pigment increases. This imposes a limit on antenna development for efficient photochemistry and may explain why a large increase of PS II reaction center number relative to PS I (Chow et al., 1990; Walters and Horton, 1994) is necessary to support the increase in the absorbance cross-section of PS II in response to growth with an irradiance artificially enriched in far-red light. The increase in the number of PS II reaction centers with increasing growth irradiance may be more a reflection of the necessity of the electron transport capacity of PS II as a whole, and D1 turnover, to match the increased capacity of the electron transport chain. In contrast to PS I reaction centers, the turnover time of PS II reaction centers (from water to is relatively slow, i.e. 2 ms (Witt, 1979; Rutherford, 1989; Debus, 1992). Consequently if there was no increase in the number of PS II reaction centers in conjunction with large increases in the electron transport carriers, then with increasing growth irradiance more and more of the kinetic limitation of the electron transport chain under nonlight-limiting conditions would reside on PS II and further photoinhibition-linked impairments of electron transport could occur. To increase the number of PS II reaction centers with their associated core chlorophylls and at the same time maintain a balance of excitation energy between the two photosystems requires a decrease in the light-harvesting complexes associated with PS II cores, and thus a decrease in the chlorophyll a/b ratio of the leaf. The fact that the PS I reaction center has a very fast turnover time may be seen as an explanation for the relative unresponsiveness of PS I to growth irradiance. The half-time for electron transfer from P700 to is not known

at room temperature, but as the back reaction from to is 250 (Golbeck and Bryant, 1991) and the quantum yield for PS I electron transport is so high, the to transfer time must be

IV. Conclusions 1. The quantum yield for evolution in plants under optimal light-limiting conditions is close to the maximum value calculated for linear electron transport within the framework of the Z scheme, and is largely invariant between plants implying that the rate of photochemical conversion by PS I and PS II is balanced, the supply of reductant is limiting and alternative pathways for electron transport, such as cyclic and pseudocyclic, are of minor importance in vivo. 2. Under natural light environments, the distribution of excitation energy between PS I and PS II slightly favors PS II, but since the quantum yield for photosynthesis remains high under these conditions there must be capacity for some short term adjustment to accommodate typical small imbalances in excitation produced by changes in light quality. 3. The photochemical quantum efficiencies of both photosystems decline as photosynthesis becomes light-saturated and linear electron transport remains the predominant route for reducing equivalents. 4. The restriction of electron transport at steady state under non-limiting light conditions occurs at the step. 5. The restriction of electron transport at the cytochrome step appears to be largely independent of irradiance but can be modulated in response to fluctuations in the sink capacity of the stroma for reducing power and ATP. This modulation is achieved principally by changes to the intrathylakoid pH. 6. The decline of photochemical efficiency is associated with the occurrence of non-damaging deexcitation mechanisms. However whereas the mechanism used in PS I appears to be capable of dissipating all excess energy safely, this is not true for PS II. The restriction of electron transport between

Chapter 3

Regulation of Photosynthetic Light Utilization

PS II and PS I allows the development of the effective non-harmful dissipation mechanism in PS I. The management of the dissipation of excess excitation energy in PS II is complex and determined by several factors, including the intrathylakoid pH. 7. The responses of photochemical efficiency and the operation of electron transport appear to be very consistent with changing species and conditions, implying an effective co-ordination by feedforward and feedback mechanisms of the supply of NADPH and ATP from electron transport and the sink strength of stromal metabolism. 8. The environmental dependency of the stoichiometries of the photosystems and electron transfer components is consistent with the short term operational responses of photosynthesis.

V. Appendix: The Use of Light-Induced Absorbance Changes Around 820 nm to Measure P700 Oxidation Absorbance changes around 820 nm due to other thylakoid components than P700 are well known (Harbinson and Hedley, 1993), but in vivo under the range of irradiances normally employed in investigations of photosynthetic physiology (< 10 000 most of these components can make no measurable contribution to the (Harbinson and Hedley, 1993; but see Chapter 2). The components that may interfere with the under physiological conditions in vivo are ferredoxin (Klughammer and Schreiber, 1991), plastocyanin (Harbinson and Hedley, 1989; Klughammer and Schreiber, 1991) and P680 (Döring et al., 1967; Harbinson and Hedley, 1993). All of these except ferredoxin produce a lightinduced absorbance increase around 820 nm. Ferredoxin displays a light-induced absorbance decrease associated with its reduction, and can interfere during photosynthetic induction, immediately following abrupt large increases in irradiance, or when leaves are illuminated under conditions which prevent any reoxidation of the PS I electron acceptor pool (Klughammer and Schreiber, 1991; Harbinson and Hedley, 1993), It can produce an absorbance change equivalent to about 10% of the maximum absorbance change due to P700 and plastocyanin oxidation (Klughammer and Schreiber, 1991; Harbinson and Hedley, 1993). The lifetime of

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is normally very short (20–250 ns, Brettel et al., 1984), but in vitro a lifetime of up to 5 ms can be produced (Schlodder and Meyer, 1987), and this could contribute to the produced by which has a lifetime of milliseconds or tens of milliseconds in vivo (Harbinson and Hedley, 1989, 1993; Schreiber et al., 1989; Harbinson and Foyer, 1991; Harbinson, 1994; Laisk and Oja, 1994). The low pH treatments which cause the formation of long lived would, however, be expected to substantially slow down reduction (Tikhonov et al., 1984), so the presence or absence of two components could be verified by an analysis of the decay kinetics. The problems of interference from plastocyanin are more substantial, as about 35% of the may be due to plastocyanin (Harbinson and Hedley, 1989; Klughammer and Schreiber, 1991). Because of the equilibrium constant between P700 and plastocyanin (Klughammer and Schreiber, 1991) and the pH sensitivity of the redox potential plastocyanin (Katoh et al., 1962), it might be expected that such a large and variable contribution to the overall by changes due to plastocyanin would make this measurement suitable only for qualitative use. When chemically oxidized in vitro, both plastocyanin and P700 contribute to the as would be expected on the basis of their redox potentials, stoichiometries and extinction coefficients at 820 nm (Klughammer and Schreiber, 1991). It has been reported that the effective plastocyanin/ P700 equilibrium constant in vivo is lower than expected from the redox potential difference between P700 and plastocyanin (Joliot and Joliot, 1984), and the smaller the equilibrium constant the more closely will the redox changes of plastocyanin follow those of P700. Another, possibly related, explanation is that leaves are optically dense, and when the irradiance-dependent absorbance changes for P700 and plastocyanin are simulated for an optically dense sample, with the assumption of an equilibrium constant of 40, the apparent equilibrium constant estimated from the combined plastocyanin and P700 absorbance changes decreases as the absorption of the simulated sample increases (Harbinson and van Vliet, 1994). This goes some way to explaining why plastocyanin absorbance changes interfere less than would be expected with the use of the to measure P700 oxidation, but the analysis is still incomplete (cf. Chapter 2).

92 Regardless of the mechanism of the interaction between plastocyanin and P700 in vivo, the presence of a contribution from plastocyanin has consequences for the interpretation of data. First, the presence of plastocyanin changes make it difficult to quantify measurements in terms of absolute amounts of P700, though this is not a serious problem for whole leaf measurements because scattering of the measuring beam by leaves (Rühle and Wild, 1979; Klughammer and Schreiber, 1991; Harbinson and Hedley, 1993) makes the measurement useful for only relative changes of P700 oxidation. Second, the uncertain equilibrium constant between P700 and plastocyanin makes it difficult to interpret decay kinetics of the produced by the reduction of oxidized P700 and plastocyanin following a lightdark transition in terms of numbers of electrons. However, it has been widely demonstrated in leaves and chloroplasts that without any correction for plastocyanin absorbance changes, measurements can be used to estimate P700 oxidation within the limits of verification (see Section II.B.1). The question remains as to how this empirical fact can be reconciled with expectation.

Acknowledgments We are grateful to F. Baoleck and D. Epron for helpful discussions and comments on the manuscript.

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Radmer RJ and Kok B (1976) Photoreduction of primes and replaces assimilation. Plant Physiol 58: 336–340 Rees D, Noctor G, Ruban AV, Crofts J, Young A and Horton P (1992) pH dependent chlorophyll fluorescence quenching in spinach thylakoids from light treated or dark adapted leaves. Photosynth Res 31: 11–19 Renganathan M, Pan RS, Ewy RG, Theg SM, Allnut FCT and Dilley RA (1991) Evidence that localized energy coupling in thylakoids can continue beyond the energetic threshold onset into steady illumination. Biochim Biophys Acta 1059: 16–27 Rich P (1982) A physicochemical model of quinone-cytochrome bc complex electron transfers. In: Trumpower BL (ed) Function of Quinones in Energy Conserving Systems, pp 73–86. Academic Press, New York Rich PR (1988) A critical examination of the supposed variable proton stoichiometry of the chloroplast cytochrome bf complex. Biochim Biophys Acta 932: 33–42 Robinson JM (1988) Does photoreduction occur within chloroplasts in vivo? Physiol Plant 72: 666–680 Ruban A V, Young AJ and Horton P (1993) Induction of nonphotochemical energy dissipation and absorbance changes in leaves. Evidence for changes in the state of the lightharvesting system of Photosystem II in vivo. Plant Physiol 102: 741–750 Rühle W and Wild A (1979) The intensification of absorbance changes in leaves by light dispersion. Planta 146: 551–557 Ruhle W, Pschorn R and Wild A (1987) Regulation of the photosynthetic electron transport during dark-light transitions by activation of the oxidoreductase in higher plants. Photosynth Res 11: 161–171 Rumberg B, Schubert K, Strelow F and Tran-Anh T (1990) The of spinach chloroplasts is four. In: Baltscheffsky M (ed) Current Research in Photosynthesis, Vol 3, pp 125–128. Kluwer Academic Publishers, Dordrecht Rutherford AW (1989) Photosystem II, the water-splitting enzyme. TIBS 14: 227–230 Rutherford AW and Heathcote P (1985) Primary photochemistry in Photosystem I. Photosynth Res 6: 293–316 Sage RF (1990) A model describing the regulation of ribulose1,5-bisphosphate carboxylase, electron transport, and triose phosphate use in response to light intensity and in plants. Plant Physiol 94: 1728–1734 Sage RF and Sharkey TD (1987) The effect of temperature on the occurrence of and insensitive photosynthesis in field grown plants. Plant Physiol 84: 658–664 Sage RF, Sharkey TD and Seemann JR (1989) Acclimation of photosynthesis to elevated in five species. Plant Physiol 89: 590–596 Sawada S, Usuda H and Tsukui T (1992) Participation of inorganic orthophosphate in regulation of the ribulose-l ,5-bisphosphate carboxylase activity in response to changes in the photosynthetic source-sink balance. Plant Cell Physiol 33: 943–949 Schatz GH, Brock H and Holzwarth AR (1988) Kinetic and energetic model for the primary processes in Photosystem II. Biophys J 54: 397–405 Scheibe R (1990) Light/dark modulation: regulation of chloroplast metabolism in a new light. Bot Acta 103: 327–334 Scheibe R and Stitt M (1988) Comparison of NADP-malate dehydrogenase activation, reduction and evolution in spinach leaves. Plant Physiol Biochem 26: 473–482 Schlodder E and Meyer B (1987) pH dependence of oxygen

98 evolution and reduction kinetics of photooxidized chlorophyll in Photosystem II particles from Synechococcus sp. Biochim Biophys Acta 890: 23–31 Schlodder E, Gräber P and Witt HT (1982) Mechanism of phosphorylation in chloroplasts. In: Barber J (ed) Electron transport and photophosphorylation, pp 103–175. Elsevier Science Publishers, Amsterdam Schreiber U (1986) Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorimeter. Photosynth Res 9: 261–272 Schreiber U, Schliwa U and Bilger W (1986) Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth Res 10: 51–62 Schreiber U, Klughammer C and Neubauer C (1988) Measuring P700 absorbance changes around 830 nm with a new type of pulse modulation system. Z Naturforsch C 43: 686–698 Schreiber U, Neubauer C and Klughammer C (1989) Devices and methods for room-temperature fluorescence analysis. Phil Trans R Soc Lond B 323: 241–251 Schreiber U, Reising H and Neubauer C (1991) Contrasting pHoptima of light-driven and in spinach-chloroplasts as measured via chlorophyll fluorescence quenching. Z Naturforsch C 46: 635–643 Sharkey TD (1985) Photosynthesis in intact leaves of plants: physics, physiology and rate limitations. Bot Rev 51: 53–105 Sharkey TD (1989) Evaluating the role of Rubisco regulation in photosynthesis of plants. Phil Trans R Soc Lond B 323: 435–448 Sharkey TD and Vanderveer PJ (1989) Stromal phosphate concentration is low during feedback limited photosynthesis. Plant Physiol 91:679–684 Sharkey TD, Stitt M, Heineke D, Gerhardt R, Raschke K and Heldt HW (1986) Limitation of photosynthesis by carbon metabolism .2. uptake results from limitation of triose phosphate utilization. Plant Physiol 81: 1123–1129 Siebke K, Laisk A, Neimanis S and Heber U (1991) Regulation of chloroplast metabolism in leaves - evidence that NADPdependent glyceraldehydephosphate dehydrogenase, but not ferredoxin-NADP reductase, controls electron flow to phosphoglycerate in the dark-light transition. Planta 185:337– 343 Siggel U (1976) The function of plastoquinone as (an) electron and proton carrier in photosynthesis. Bioelectrochem Bioenerg 3:302–318 Stitt M (1991) Rising levels and their potential significance for carbon flow in photosynthetic cells. Plant Cell Environ 14: 741–762 Strelow F, Tran-Anh T and Rumberg B (1990) Functional studies on the reaction pattern of the synthase in spinach chloroplasts. In: Baltscheffsky M (ed) Current Research in Photosynthesis, Vol 3, pp 157–160. Kluwer Academic Publishers, Dordrecht Sundby C, McCaffery S and Anderson JM (1993) Turnover of the Photosystem II Dl protein in higher plants under photoinhibitory and nonphotoinhibitory irradiance. J Biol Chem 268: 25476–25482 Takahama U, Shimuzu-Takahama M and Heber U (1981) The redox state of the NADP system in illuminated chloroplasts. Biochim Biophys Acta 637: 530–539

B. Genty and J. Harbinson Telfer A and Barber J (1994) Elucidating the molecular mechanisms of photoinhibition by studying isolated Photosystem II reaction centres. In: Baker NR and Bowyer JR (eds) Photoinhibition of Photosynthesis from Molecular Mechanisms to the Field, pp 25–49. Bios Scientific Publishers, Oxford Terashima I and Evans JR (1988) Effects of light and nutrition on the organization of the photosynthetic apparatus in spinach. Plant Cell Physiol 29: 143–155 Terashima I and Saeki T (1985) A new model for leaf photosynthesis incorporating the gradients of light environment and of photosynthetic properties of chloroplasts within a leaf. Ann Bot 56: 489–499 Terashima I, Funayama S and Sonoike K (1994) The site of photoinhibition in leaves of Cucumis sativus (L) at low temperatures is Photosystem I, not Photosystem II. Planta 193: 300–306 Thielen APGM and van Gorkom H (1981) Energy transfer and quantum yield in Photosystem I I . Biochim Biophys Acta 637: 439–446 Tikhonov AN, Khomutov GB and Ruuge EK (1984) Electron transport control in chloroplasts. Effects of magnesium ions on the electron flow between two photosystems. Photobiochem Photobiophys 8: 261–269 Trissl H-W and W i l h e l m C (1993) Why do thylakoid membranes from higher plants form grana stacks? TIBS 18: 415–419 van Bel AJE (1992) Mechanisms of sugar transfer. In: Baker NR and Thomas H (eds) Crop Photosynthesis: Spatial and Temporal Determinants, pp 177–211. Elsevier Science Publishers, Amsterdam van Wijk KJ and van Hasselt PR (1993) Kinetic resolution of different recovery phases of photoinhibited Photosystem II in cold-acclimated and non-acclimated spinach leaves. Physiol Plant 87: 187–198 Veeranjaneyulu K, Charland M and Charlebois D (1991) Photoacoustic study of changes in the energy storage of Photosystems I and II during state 1-state 2 transitions. Plant Physiol 97: 330–334 Vogelman TC (1993) Plant tissue optics. Ann Rev Plant Physiol Plant Mol Biol 44: 231–251 von Caemmerer S and Farquhar GD (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376–387 Walker DA (1992) Excited leaves. New Phytol 121: 325–345 Walker DA and Osmond CB (1986) Measurement of photosynthesis in vivo with a leaf disc electrode: correlations between light dependence of steady-state photosynthetic evolution and chlorophyll a transients. Proc Roy Soc London B 227: 267–280 Walters RG and Horton P (1994) Acclimation of Arabidopsis thailana to the light environment: Changes in composition of the photosynthetic apparatus. Planta 195: 248–256 Wang Y-P, McMurtrie RE and Landsberg JJ (1992) Modelling canopy photosynthetic productivity. In: Baker NR and Thomas H (eds) Crop Photosynthesis: Spatial and Temporal Determinants, pp 43–67. Elsevier Science Publishers, Amsterdam Weis E and Berry J (1987) Quantum efficiency of Photosystem II in relation to ‘energy’-dependent quenching of chlorophyll fluorescence. Biochim Biophys Acta 894: 198–208 Weis E and Lechtenberg D (1989) Fluorescence analysis during

Chapter 3 Regulation of Photosynthetic Light Utilization steady state photosynthesis. Phil Trans Roy Soc London B 323: 253–268 . Weis E, Ball JT and Berry J (1987) Photosynthetic control of electron transport in leaves of Phaseolus vulgaris: evidence for regulation of Photosystem II by the proton gradient. In: Biggins J (ed) Progress in Photosynthesis Research, Vol 2, pp 553–556. Martinus Nijhoff, Dordrecht Weis E, Lechtenberg D and Krieger A (1990) Physiological control of primary photochemical energy in higher plants. In: Baltscheffsky M (ed) Current Research in Photosynthesis, Vol 4, pp 307–312. Kluwer Academic Publishers, Dordrecht Westerhoff HV, Melandri BA, Venturoli G, Azzone GF and Kell DB (1984) A minimal hypothesis for membrane-linked freeenergy transduction. The role of independent, small coupling units. Biochim Biophys Acta 768: 257–292 Whitmarsh J and Ort DR (1984) Quantitative determination of the electron transport complexes in the thylakoid membranes of spinach and several other plant species. In: Sybesma C (ed) Advances in Photosynthesis Research, Vol 3, pp 231–234. Martinus Nijhoff, The Hague Whitmarsh J, Samson G and Poulson M (1994) Photoprotection in Photosystem II—the role of cytochrome b559. In: Baker NR and Bowyer JR (eds) Photoinhibition of Photosynthesis from Molecular Mechanisms to the Field, pp 75–93. Bios Scientific Publishers, Oxford

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Wild A (1979) Physiologie der Photosynthese höherer Pflanzen. Die Anpassung an die Lichtbedingungen. Ber Deutsch Bot Ges 92: 341–364 Wild A and Holzapfel A (1980) The effect of blue and red light on the content of chlorophyll, cytochrome f, soluble reducing sugars, soluble proteins and the nitrate reductase activity during growth of the primary leaves of Sinapsis alba. In: Senger H (ed) The Blue Light Syndrome, pp 444–451. Springer Verlag, Berlin Wild A, Hopfner M, Ruhle W and Richter M (1986) Changes in the stoichiometry of Photosystem II components as an adaptative response to high light and low light conditions during growth. Z Naturforsch 41c: 597–603 Witt HT (1979) Energy conversion in the functional membrane of photosynthesis. Analysis by light pulse and electric pulse methods. The central role of the electric field. Biochim Biophys Acta 505: 355–427 Woodrow IE and Berry JA (1988) Enzymatic regulation of photosynthetic fixation in plants. Ann Rev Plant Physiol Plant Mol Biol 39: 533–594 Wraight CA (1982) Current attitudes in photosynthesis research. In: Govindjee (ed) Photosynthetic Energy Conversion by Plants and Bacteria, Vol 1, pp 17–61. Academic Press, New York

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Chapter 4 Mechanisms of Photodamage and Protein Degradation During Photoinhibition of Photosystem II B. Andersson Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, Stockholm, S-10691

J. Barber Photosynthesis Research Group, Biochemistry Department, Wolfson Laboratories, Imperial College of Science, Technology & Medicine, London SW7 2AY, UK

Summary 101 I. Introduction 102 II. Photosystem II: Structure and Function 104 III. Photochemical Processes Giving Rise to Damage 106 A. Acceptor Side-induced Photodamage 106 B. Donor Side-induced Photodamage 109 IV. Does Triggering for D1 Protein Degradation Require a Conformational Change? 110 V. Degradation of Reaction Center Subunits in Photosystem II 111 112 A. Identification of Proteolytic Fragments 112 1. D1 Protein Degradation 114 2. D2 Protein Degradation B. Enzymology of D1 and D2 Protein Degradation 115 VI. Repair of Photodamaged Photosystem II Requires Co-ordination Between Degradation and Biosynthesis 116 117 Acknowledgments 117 References

Summary The primary target of damage during photoinhibition of oxygenic photosynthesis is Photosystem II (PS II). The molecular processes which underlie this vulnerability almost certainly arise from the fact that PS II is unique in that it can generate the very strong oxidants necessary to split water. This presumption has received considerable support from studies using various isolated PS II complexes that offer experimental systems which are amenable for detailed photochemical and biochemical measurements. Such studies have identified two distinct routes for photoinduced damage; designated as acceptor and donor side mechanisms. The acceptor side mechanism involves recombination of the radical pair where P680 is the primary donor of PS II and Phe is pheophytin, the primary acceptor of PS II. The recombination occurs either when the plastoquinone acceptor is doubly reduced (e.g. in high light) or when back reactions are favored between the partially reduced plastoquinone acceptor and the or states of the water splitting system (e.g. in low light). The recombination leads to the production of the P680 triplet state which is not quenched by carotenoids but instead leads to the formation of highly toxic singlet oxygen. As a consequence, the D1 protein is modified in such a way as to be triggered for proteolytic degradation. This degradation process involves an initial cleavage in the Neil R. Baker (ed): Photosynthesis and the Environment, pp. 101–121. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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loop joining transmembrane segments D and E near to the binding site (C-terminal side of residue 238). Under some circumstances, the D2 protein also undergoes similar degradation with the primary cleavage being in an analogous position to that determined for D1 protein. The donor side mechanism, however, is not dependent on the presence of oxygen and results from damage due to long lived oxidation states (e.g. The high redox potential of these states means that oxidation of pigments and accessory chlorophyll) and amino acids, can occur. Extensive oxidation of this type destabilizes the D1 and D2 protein and the pattern of degradation products observed is different from that generated by the acceptor side mechanism. In the case of the D1 protein, the primary cleavage occurs on the donor side of the membrane in the loop joining transmembrane segments A and B. There is no evidence to suggest that either the acceptor or donor side induced degradation of the D1 and D2 proteins is due to direct photochemical cleavage. Rather, it seems that the detrimental photochemical processes give rise to conformational changes in the D1 and D2 proteins that signal proteolytic reactions. Studies in vivo indicate that in the case of the D1 protein the proteolytic step is normally synchronized with the availability of newly synthesized protein.

I. Introduction Photoinhibition is a physiological stress condition induced in plants and algae as a consequence of an imbalance between the number of photons captured and their utilization by photosynthesis. It is observed as a light-induced depression of photosynthetic activity. The most vulnerable part of the photosynthetic apparatus is Photosystem II (PS II) which powers the oxidation of water and evolution of oxygen. This vulnerability of PS II to light-induced damage is emphasized by the fact that a key component of its reaction center, the D1 protein, turns over far more rapidly than any other protein in the photosynthetic membrane (Mattoo et al., 1989). There is good evidence to suggest that this turnover is part of a repair system that functions to replace damaged reaction centers with newly synthesized D1 protein and thus restore normal PS II activity (Ohad et al., 1984). If the rate of repair of PS II does not keep pace with its rate of damage, then photoinhibition is observed as a decrease in photosynthetic capacity. When the repair system copes with the rate of damage, then no apparent loss of photosynthetic capacity is observed unless protein synthesis is experimentally Abbreviations: CAP – chloramphenicol; D1 and D2 proteins – products of the psbA and psbD genes, respectively; DBMIB – 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCMU – 3-(3,4-dichlorophenyl)-1,1-dimethyl urea; EPR – electron paramagnetic resonance; FTIR – Fourier transformed infra-red; P680 – primary electron donor of PS II; Phe – pheophytin a; – primary quinone electron acceptor of PS II; – secondary quinone acceptor of PS II; SDS-PAGE – sodium docecyl sulfate polyacrylamide gel electrophoresis; and –tyrosine residues of the D1 and D2 proteins, respectively, which act as electron donors to P680

blocked by inhibitors, such as chloramphenicol, or slowed down by lowering the temperature (Fig. 1). As expected, loss of photosynthetic activity in a photoinhibited organism is recoverable when light stress is reduced and the repair system is able to regenerate and maintain the normal population of functional PS II complexes. There is good evidence that photoinhibition is a common stress phenomenon affecting both plant growth and productivity (Aro et al., 1993; Long et al., 1994). It is often enhanced by the existence of other stress conditions, such as nutrient or water shortage and abnormally low or high temperatures. Since the turnover of the D1 protein occurs at nonsaturating as well as saturating light intensities, it follows that photoinactivation of PS II is an intrinsic phenomenon which has a probability of occurring over a wide range of irradiances. As the light intensity is increased the rate of D1 protein turnover is stimulated in order to compensate for the increased probability of damage. Ultimately the rate of photoinduced damage of PS II reaction centers outstrips the rate of repair and a lowering of photosynthetic activity in the leaf or alga is observed. It therefore follows from the above arguments that even at moderate light intensities, where no net reduction of the photosynthetic capacity can be seen, plants are suffering from light-induced stress since they have to carry the burden of maintaining and enhancing a repair system which is energy demanding and calls on their metabolic reserves (Fig. 1). There seems to be a wide variety of mechanisms by which plants and algae adapt to reduce the impact of excess radiation (Aro et al., 1993; Long et al., 1994; Chapters 1–3). Some of these are long term (e.g. changes in pigment levels, reduction in the

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surface area of thylakoid membranes), medium term (e.g. change in leaf angle, rotation of chloroplasts), while others are short term responses (e.g. quenching of excitons within the antenna systems by interconversion of carotenoids associated with the xanthophyll cycle, redistribution of excitation energy away from PS II; see Chapters 1–3). Despite these

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protective strategies photoinduced stress does occur during normal growth (Raven, 1994) and is likely to manifest itself as photoinhibition under certain conditions as defined by a decrease in photosynthetic activity. This, therefore, leads to the questions as to the nature of the photochemical reactions that underlie the vulnerability of PS II to photoinduced damage

104 and the series of events leading to the degradation and replacement of the D1 protein in order to reinstate full PS II activity. It simply is impossible to answer the above questions fully by conducting experiments with intact tissue. For this reason studies have been carried out using in vitro systems of various levels of complexity. Many studies have been done with isolated thylakoids which have shown that photoinactivation of PS II and degradation of the D1 protein occurs when they are subjected to elevated levels of illumination. Ohad et al. (1984, 1985) were the first to link these two phenomenon. Later Aro et al (1990) showed that two phenomena could be resolved into distinct stages during high light stress. The first stage involved temperature-insensitive photochemical inactivation which did not give rise to the cleavage of peptide bonds. Only in a second temperature-sensitive stage, was the D1 protein enzymatically degraded in the dark. In this chapter we will initially review our knowledge of the photochemical reactions of PS II, focusing on those processes which are potentially harmful. We will then discuss this knowledge in terms of possible triggering mechanisms and the subsequent enzymology of D1 protein degradation. Our discussions will be extended to take account of the fact that, under certain circumstances, the D2 protein can also be degraded as a consequence of photoinactivation of PS II. The reader is referred to other reviews on the subject (Prásil et al., 1992; Barber and Andersson, 1992; Aro et al., 1993,for the more molecular aspects; Powells, 1984, Chow 1994; and many chapters in a book edited by Baker and Bowyer, 1994).

II. Photosystem II: Structure and Function Photosystem II (PS II) performs the unique chemistry of water splitting. In so doing it supplies the biosphere with the reducing potential necessary to convert atmospheric carbon dioxide to organic molecules. The chemical reactions involved are intrinsically dangerous for the protein environment in which they occur on two counts. Firstly, to extract electrons from water requires oxidizing potentials sufficiently high that other detrimental oxidation reactions are possible. Secondly, the oxidation of water results in the formation of molecular oxygen as a by-product. Oxygen can readily form highly reactive states which

B. Andersson and J. Barber can attack proteins and other components of PS II (see Chapter 5). Our knowledge of the functioning of PS II has steadily advanced over the past few decades (Babcock, 1987; Barber, 1989; Andersson and Styring, 1991; Debus, 1992). The primary electron donor P680 is a special form of chlorophyll which, when oxidized by illumination, generates a redox potential of about +1.17 V (Klimov et al., 1980). The primary electron acceptor is a pheophytin a molecule (Phe) which, in its reduced state, has a redox potential of approximately –0.65 V The electron residing on Phe is then passed to two plastoquinone molecules, and The first quinone acceptor is reduced in about 200 ps while the electron transfer from to is in the millisecond time domain. The first quinone acceptor normally accepts only one electron and is not protonated, while is a two electron acceptor and can be fully protonated. On the oxidizing side, is reduced by a redox active tyrosine residue which in turn is reduced by manganese. Because the oxidation of water to dioxygen is a four electron process, four oxidation equivalents are stored before oxygen evolution occurs. The storage of these oxidizing equivalents takes place on manganese atoms of which there is probably a cluster of four per P680. Therefore the rate of reduction of depends on the oxidation state of the manganese cluster. Thus PS II acts as a water-plastoquinone oxidoreductase which has a four electron gate on its oxidizing side and a two electron gate on its reducing side. Despite the fact that PS II is a multipeptide complex with more than twenty subunits, the above redox reactions are generally believed to be restricted to only two polypeptides known as the D1 and D2 proteins (Fig. 2). These proteins form the reaction center of PS II and show many characteristics similar to those of the L and M subunits of purple photosynthetic bacteria (Trebst, 1986; Barber, 1987; Michel and Deisenhofer, 1986). Biochemical confirmation of this relationship came from the isolation of a reaction center complex of PS II containing the D1/D2 proteins (Nanba and Satoh, 1987; Barber et al., 1987). As in the case of the purple bacteria (Michel and Deisenhofer, 1988), it is likely that the primary donor, P680, is composed of two chlorophyll molecules that are ligated to, and sandwiched between, the D1 and D2 proteins. By analogy with the bacterial system it is believed that the active Phe

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is bound to the D1 protein and that an inactive Phe is located by two fold symmetry on the D2 protein. The secondary plastoquinone acceptors, and are also believed to be related by two fold symmetry with on the D2 protein and on the D1 protein. The axis for this two fold symmetry is expected to pass through P680 and a non-heme iron positioned equi-distant between and (Fig. 2). When fully reduced and protonated, the plastoquinone diffuses out of the binding site and is replaced by a fully oxidized plastoquinone molecule available from the plastoquinone pool in the lipid matrix of the thylakoid membrane. In this way reducing equivalents are removed from the PS II reaction center and made available to subsequent electron transfer processes powered by Photosystem I (PSI). The tyrosine that acts as intermediate between the manganese cluster and is residue 161 on the

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D1 protein (Debus et al., 1988; Metz et al., 1989). A symmetrically related tyrosine residue on the D2 protein, known as can also be oxidized by but is not involved directly in the water oxidation process. Although the precise location of the manganese cluster is not known there is a general belief that it is ligated to surfaces of the D1 and D2 proteins (Debus, 1992) even though the involvement of other PS II subunits can not be excluded at this stage. Indeed, a number of amino acid residues have been identified as possible ligands on the two reaction center proteins (Nixon and Diner, 1992). As indicated in Fig. 2, the D1 and D2 proteins probably consist of five transmembrane segments each, as in the case of the L and M subunits of purple bacteria. The manganese cluster, P680, and are located on the lumenal side of the thylakoid membrane while the reducing side of the reaction center

106 heterodimer is towards the stromal side of the membrane. Consequently charge separation occurs across the membrane in a way which is common to all types of photosynthetic reaction centers (Barber and Andersson, 1994). As yet no detailed information is available for the structure of the PS II reaction center but molecular models have been constructed (Svensson et al., 1990; Ruffle et al., 1992) based on analogy with its purple bacterial counterpart where the structure has been determined to atomic resolution by X-ray crystallography (Deisenhofer et al., 1985; Allen et al., 1987a,b). The D1/D2 heterodimer, free of other polypeptides, can be isolated in a photo-active state (Tang et al., 1990). But this minimal system is unstable and functionally limited to primary charge separation. In a more stable form, the reaction center is isolated as a complex having, in addition to the heterodimer, the and subunits of cytochrome and a low molecular weight protein which is the product of the psbl gene (Nanba and Satoh, 1987; Barber et al., 1987; Webber et al., 1989). Again, this complex has limited functional activity, being unable to split water and perform normal electron acceptor function. Under some conditions it is highly sensitive to damage by light (Seibert et al., 1988; Chapman et al., 1989; McTavish et al., 1989). To obtain full PS II function it seems necessary to have additional polypeptides. A range of oxygenevolving PS II core complexes of varying degrees of complexity have been isolated. All the various preparations have been useful for elucidating the processes of PS II. They allow studies with improved signal to noise ratios and are separated from events associated with other parts of the photosynthetic machinery. In addition, these preparations have proved to be very valuable for investigating the molecular basis of photoinhibition, both at the level of photochemical induced damage and the subsequent degradation of the D1 and D2 proteins (Barber and Andersson, 1992).

III. Photochemical Processes Giving Rise to Damage There are a number of observations indicating that the initial damage to PS II involves photochemical processes that inactivate its function and trigger the D1 and D2 proteins for enzymatic degradation. There is no evidence to suggest that these proteins are

B. Andersson and J. Barber normally directly cleaved by photochemical reactions. Understanding the nature of the events leading to photoinactivation and triggering of the D1 protein for degradation has greatly benefited from studies using in vitro systems. Such studies have revealed two distinct pathways by which photodamage may occur, one induced from the acceptor side of PS II and another from the donor side of PS II (Eckert et al., 1991; Andersson and Styring, 1991; Prásil et al., 1992; Barber and Andersson, 1992).

A. Acceptor Side-induced Photodamage The idea that photoinhibition involved the acceptor side of PS II stems from the original suggestion that there is a link between this stress phenomenon and the turnover of the D1 protein (Kyle et al., 1984; Ohad et al., 1984). At that time the D1 protein was known as the ‘32 kDa binding protein’ and it was reasonable to speculate that the damaging mechanism may involve a reactive form of plastoquinone bound to the site (Kyle, 1987). This idea gained support from the fact that certain herbicides, such as 3-(3,4dichlorophenyl)-1,1 -dimethyl urea (DCMU) and atrazine, which bind to the D1 protein and displace plastoquinone from the pocket, are protective and prevent its rapid turnover (Kyle et al., 1984; Mattoo et al., 1984; Trebst et al., 1988; Jansen et al., 1993). Consequently, it was postulated that photoinhibition was due to the interaction of molecular oxygen with the plastoquinone anion in the site and that the resulting oxygen radicals would in turn lead to protein damage, thereby inhibiting electron transport (Kyle et al., 1984; Kyle, 1987). Based on this reasoning, experiments were conducted in vitro in the presence and absence of oxygen. The advantage of anaerobic conditions is that there is normally no degradation of the D1 protein when oxygen is absent (Arntz and Trebst, 1986; Hundal et al., 1990a; Kuhn and Böger, 1990; Nedbal et al., 1990) and that the inactivation of PS II function is reversible in its early stages without de novo protein synthesis (Hundal et al., 1990a; Kirilovsky and Etienne, 1991). Because of this it is possible to trap intermediates in the photoinactivation processes and characterize the reversible stages. Using this approach Vass et al. (1992b) postulated a pathway for photoinactivation of PS II function which was attributed to the over reduction of This occurred when the plastoquinone pool was fully reduced by strong illumination and the probability of the occupation of the site is expected to be low

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Mechanisms of Photosystem II Photodamage

(Crofts and Wraight, 1983). In such a situation the lifetime of increases considerably as compared to a few hundreds of ms when the site is occupied by a plastoquinone molecule. EPR spectroscopy can detect the long lived signal due to the state. If becomes either doubly reduced and/or leaves its binding site on the D2 protein, the characteristic EPR signal is lost. By combining EPR spectroscopy and changes in fluorescence yield, Vass et al. (1992b) concluded that under anaerobic conditions, photoinactivation can be resolved into four consecutive phases. A fast phase due to the full reduction of the plastoquinone pool (half-time of 30 s), a semi-stable phase due to the long lived state of (half-time of 2 min) and a stable state due to the double reduction and possible protonation of which is EPR silent (half-time of 30 min). Unlike the previous phases, the fourth and final phase was found to be irreversible under anaerobic conditions and was characterized by a non-decaying fluorescence and the absence of an inducible EPR signal. This non-decaying state was attributed to reaction centers with empty sites (Styring et al., 1990; Vass et al., 1992b) due to the release of double-reduced plastoquinone analogous with that proposed to occur during chemical double reduction of (van Mieghem et al., 1989). Support for the idea that plastoquinol can be released from the site comes from chromatographic analyses of PS II core complexes which were isolated from membranes which had been subjected to photoinhibiting treatment under anaerobic conditions (Koivuniemi et al., 1993). An important finding by Vass et al. (1992b) was that the addition of DCMU prevented the reversion of the first three phases under anaerobic conditions suggesting the recovery process involves the reestablishment of the oxidation level of the plastoquinone pool and re-occupation of the site. They also observed that associated with the slower phases of photoinactivation was the appearance of an EPR signal characteristic of a spin-polarized chlorophyll triplet state. This signal arises from recombination of the primary radical pair and is clearly observed in isolated reaction centers of PS II which do not retain plastoquinone in their and sites (Okamura et al., 1987; Telfer et al., 1988). Optical spectroscopy has also detected the P680 triplet state in isolated reaction centers (Takahashi et al., 1987; Durrant et al., 1990). The lifetime of this state is 1 ms in the absence of oxygen but shortens to about when measured under aerobic conditions.

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This is because chlorophyll triplet states can readily react with oxygen to form singlet oxygen

It was not surprising, therefore, when Vass et al. (1992b) observed that the reversibility of the photoinactivation which they detected under anaerobic conditions was lost in the presence of oxygen and that D1 protein degradation was initiated. It has been shown that exposure of isolated thylakoids to photoinhibitory illumination is accompanied by singlet oxygen production (Hideg et al., 1994). Comparing the kinetics of the loss of PS II function and D1 protein degradation with the trapping of singlet oxygen suggest that singlet oxygen itself or its radical product is responsible for the protein damage. Moreover generation of singlet oxygen by isolated PS II reaction centers has been shown by the detection of its luminescence at 1270 nm (Macpherson et al., 1993; Telfer et al., 1994b) and by an indirect chemical trapping method (Telfer and Barber, 1994; Telfer et al., 1994a). Further study of this isolated system has also indicated that the target for singlet oxygen attack is the P680 chlorophylls (Telfer et al., 1990,1991) which may be at its conjugated ring systems. Alternatively it could ligate with histidine, an amino acid known to react with singlet oxygen (Halliwell and Gutteridge, 1989). Yet another possibility is the formation of bityrosine crosslinks (Davies, 1987), as suggested by Prásil et al. (1992). When azide was added to the illuminated reaction center suspension, singlet oxygen could not be detected (Fig. 3) in line with the fact that azide is an effective quencher of this reactive oxygen species. Interestingly, the presence of azide did not protect the reaction center from photoinhibitory damage observed as selective irreversible bleaching of the P680 chlorophylls (Macpherson et al., 1993; Telfer et al., 1994a,b). Neither did this singlet oxygen quencher prevent the oxygen dependent degradation of the D1 protein (De Las Rivas et al., 1993b). These results indicate that singlet oxygen is generated within the protein matrix of the reaction center where it brings about specific damage. The fact that some singlet oxygen diffuses into the aqueous phase of the reaction center suspension is interesting in that if this was to occur in vivo, this reactive species could cause extensive damage to other components of PS II and the thylakoid membrane in general. We therefore

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must assume that in vivo the generation of singlet oxygen on an extensive scale must be avoided and in this sense isolated reaction centers, without and function, represent an extreme situation compared to that occurring in vivo. The concept that acceptor side photoinhibition is due to damage by singlet oxygen generated when radical pair recombination occurs has to be reconciled with various observations from a number of laboratories (Setlik et al., 1990; Kirilovsky et al., 1990; Vass et al., 1992b). Aro et al. (1993) have discussed many of these observations within the framework of the acceptor side mechanism given above. The conclusion is that the situation is not fully clear since the detailed role of the primary and

B. Andersson and J. Barber secondary plastoquinone acceptors during photoinactivation is somewhat controversial and remains to be established, particularly with respect to the situation in vivo. However, Ohad et al. (1994) have investigated the effect of light flashes on the degradation of the D1 protein in Chlamydomonas cells. In this way the occupancy of the binding site by semiquinone or quinol can be controlled. It was found that when long dark periods were interspersed between single flashes so as to allow recombination of the charge between states, an enhanced degradation of the D1 protein was observed relative to a dark control. With a train of flashes it was possible to induce oscillation of the D1 protein degradation related to the level of induced by the flash train. The logical conclusion therefore is that the recombination occurs via a back reaction involving the radical pair and that singlet oxygen is generated due to P680 triplet formation. This important work also gives a possible explanation for the occurrence of photoinhibitory damage and D1 protein turnover at low light intensities when the frequency of exciting a particular PS II reaction center is low and therefore facilitates a higher probability of lengthening the lifetime of the semireduced plastoquinone in the site. The possible formation of singlet oxygen by chlorophyll triplets is not unique to the chlorophylls that constitute P680. However, as elegantly shown by Wolff and Witt (1969) chlorophyll triplets, generated in antenna complexes as the light intensity is increased above the saturation level of photosynthesis, are effectively quenched by carotenoids (Fig. 4) and thus the problem of singlet oxygen production is avoided (see Chapter 1). Why then are the P680 triplets not made harmless by this mechanism? The reason almost certainly lies in the fact that has a redox potential which would efficiently oxidize a carotenoid molecule placed sufficiently close to it to act as a triplet quencher. Using an intermediate monomeric chlorophyll to increase the distance between the primary donor and carotenoid, as is the case in the reaction centers of purple bacteria, will not help, since the monomeric chlorophyll will also be oxidized by Thus PS II has an intrinsic problem which it cannot avoid and which from time to time will have serious consequences for it. Despite the compelling arguments for singlet oxygen as a main damaging species of PS II, other toxic oxygen and hydroxyl radicals may be involved (Kyle et al., 1984;Richter et al., 1990b;Greenberg et

Chapter 4

Mechanisms of Photosystem II Photodamage

al., 1990; see also Chapter 5). For example, several experiments using various oxygen radical scavengers have been shown to partially protect the D1 protein from degradation (Barenyi and Krause, 1985; Richter et al., 1990a,b; Sopory et al., 1990). It remains to be established how specific and frequent such damage to the D1 protein would be in comparison to damage mediated via singlet oxygen formed in the reaction center.

B. Donor Side-induced Photodamage Some of the complications experienced in the interpretation of data in terms of the acceptor side/ singlet oxygen mechanism are due to the fact that there seems to be a second oxygen-independent route by which PS II can be photoinactivated (Barber and Andersson, 1992). This photoinactivation occurs when the rate of electron donation to PS II does not match the rate of electron removal to the acceptor side. This situation may statistically occur during normal steady state electron flow even at low light intensities, and most certainly under conditions which destabilize the water splitting system, such as low

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temperatures (Wang et al., 1992a). Such destabilization will result in an increased lifetime for the state. Given that has a high oxidizing potential, this species has the capacity to extract electrons from its surroundings and thus cause irreversible oxidative damage. Experimental evidence for donor side inactivation has come mainly from studies using photosynthetic samples where a non-functional donor side of PS II has been purposely induced prior to photoinhibitory treatment. For example, susceptibility of PS II to photoinduced inactivation and protein degradation has been shown in hydroxylamine-treated leaf segments (Callahan et al., 1986), in leaves subjected to cold stress (Wang et al., 1992a), in Tris- (Wang et al., 1992b) and hydroxylamine-treated thylakoids (Blubaugh et al., 1990, 1991), and in thylakoids (Critchley et al., 1984; Jegerschöld et al., 1990). Eckert et al. (1991) showed that the quantum yield of electron transport inactivation increases 1000-fold inTris-washed PS II enriched membranes inhibited reaction centers per quantum) as compared to normal oxygen evolving PS II membranes inhibited reaction centers

110 per quantum). The increased susceptibility of the LF-1 mutant of Scenedesmus (Gong and Ohad, 1991) and the psbO-less mutant of Synechocystis 6803 (Mayes et al., 1991; Philbrick et al, 1991) compared to their wild types is also probably due to donor side effects. This is because the LF-1 mutant does not have water splitting activity due to its inability to process the carboxy-terminus of the D1 protein (Bowyer et al., 1992), while the absence of the 33 kDa extrinsic protein of PS II in the psbO-less Synechocystis mutant perturbs its donor side activity (Vass et al., 1992a). It is generally agreed that the site of donor side inactivation is between the manganese cluster and P680 (Eckert et al., 1991; Jegerschöld et al., 1990; Blubaugh and Cheniae 1990; Blubaugh et al., 1991). Blubaugh and coworkers (1990,1991) resolved three kinetically different phases of inactivation in hydroxylamine treated PS II membranes. The first phase was proposed to be a decrease in the rate of electron transfer between ind followed by a loss of formation. The third phase was very slow and was observed as a loss of formation. That the primary site of damage was between and P680 was also concluded by Eckert et al. (1991). From the above studies it is quite reasonable to conclude that the observed impairment of electron transport is due to oxidative damage by and/or Studies with isolated reaction centers have clearly shown that when the lifetime of is long (i.e. on illumination in the presence of an added electron acceptor but with no donor) there is a selective irreversible bleaching of and chlorophyll 670 contained within the complex (Fig. 5). This selective degradation of pigments is not oxygen dependent but is prevented if a suitable exogenous electron donor is added (Fig. 5; Telfer et al., 1991; De Las Rivas et al., 1993b). From EPR studies, Blubaugh and colleagues (Blubaugh and Chenaie, 1990; Blubaugh et al., 1991) have also identified the formation of carotenoid and chlorophyll radicals under conditions of donor side induced photoinhibition. They found that the appearance of these signals was prevented if manganese was present (Chen et al., 1992b). In addition to the irreversible bleaching of pigments it is possible that donor side photoinhibition also involves the detrimental oxidation of amino acids. Cheniae and coworkers (Chen et al., 1992a,b) have argued that in addition to cation radical

B. Andersson and J. Barber

mechanisms (discussed above) for explaining donor side photoinhibition, there is also a rapid process which involves superoxide radicals. Since there is evidence that donor side photoinhibition can occur under anaerobic conditions (Jegerschöld et al., 1990; Telfer et al., 1991; Shipton and Barber, 1992), then the involvement of superoxide may not present a primary route of damage. Superoxide, however, is likely to be produced in chloroplasts since oxygen is a good electron acceptor and superoxide dismutase is present. Whether this radical is produced on the donor side of PS II is not certain.

IV. Does Triggering for D1 Protein Degradation Require a Conformational Change? Both acceptor and donor side mechanisms of photoinhibition involve oxidative damage of one form or another. The question is, how does irreversible oxidative damage turn the D1 protein into a substrate for degradation? The primary cleavage of the D1

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Mechanisms of Photosystem II Photodamage

protein must be determined by an interaction with the proteolytic active site and this may not necessarily be identical to the site of the damage. It could therefore be argued that the oxidative damage may induce a conformational change in the reaction center which could serve as a triggering or sensing signal for the primary proteolytic cleavage (Ohad et al., 1985). There are, in fact, several lines of evidence for conformational changes in PS II during photoinhibitory illumination. One is based upon the well known protective effect of urea and triazine type herbicides which are inhibitors of D1 protein degradation (Kyle et al., 1984; Mattoo et al., 1984; Trebst et al., 1988; Jansen et al., 1993). Since these herbicides bind tightly in the site of the D1 protein they may restrict a conformational change that is required to trigger the damaged protein for proteolysis. As will be discussed in section V, one primary cleavage site of the D1 protein occurs in the D-E loop which is close to the (Greenberg et al., 1987). Experimental support for the conformational change hypothesis has been obtained by studying the effect of DCMU on D1 protein degradation at different temperatures (Salter et al., 1992a, 1994). If thylakoid membranes are subjected to strong illumination in the cold there is virtually no loss of the D1 protein but the degradation takes place in complete darkness if the photo-inactivated sample is transferred to 20–25 °C (Aro et al., 1990). When DCMU is present at the start of such an experiment, or just prior to warming up the sample, the normal protective effect of the herbicide can be seen (Salter et al., 1992a, 1994). In contrast, if DCMU is added after transferring the sample to a higher temperature it no longer reduces the extent of D1 protein degradation. It is likely that upon increasing the temperature of the photoinactivated thylakoids there is an alteration of the conformation around the site which lowers its affinity for DCMU and that this conformational change may be essential for turning the D1 protein into a substrate for proteolysis. In addition, gross conformational changes of photodamaged, but not degraded, reaction centers are suggested by the release of manganese during photoinhibitory illumination in vitro (van Wijk et al., 1992). Conformational changes in PS II have also been measured by FTIR spectroscopy after treatment of reaction centers with photoinhibitory light (He et al., 1991).

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V. Degradation of Reaction Center Subunits in Photosystem II Once inactivated and damaged a PS II reaction center has to be repaired in order to regain its functional activity. The current experimental data shows that the replacement of damaged D1 protein plays a key role in this repair process (Prásil et al., 1992; Aro et al., 1993). This repair, as mentioned above, is manifested by a light dependent turnover of the D1 protein, which can be fifty to eighty fold higher than for any other protein in the thylakoid membrane (Fig. 7). However, before a newly translated copy of the D1 protein can be inserted into the membrane and assembled into the PS II complex the damaged copy has to be removed. This removal of D1 protein can be specifically followed by radioactive pulse chase experiments in vivo or in vitro by western blotting of various thylakoid and sub-thylakoid preparations subjected to high light treatment. The same procedures can also monitor degradation of the D2 protein although this is normally far less than that observed with the D1 protein (Prásil et al., 1992). The majority of the experimental data demonstrate that the two reaction center proteins are degraded through proteolysis (Aro et al., 1993). This may not be self-evident since, as discussed in section II, the chemistry of light-driven water-plastoquinone oxidoreductase activity involves highly reactive intermediates. These species could cause protein cleavage, for example, through modification of proline residues (Sies, 1986) or scission of peptide bonds by reactive oxygen species (Wolff et al., 1986; Davies, 1987). However, this appears not to be the case as judged by the following experimental observations, (i) Inactivation of electron transport and D1 protein degradation does not occur in parallel (Ohad et al., 1985; Virgin et al., 1988) and show different pH optima (Shipton and Barber, 1992). (ii) D1 protein degradation occurs in complete darkness at room temperature once isolated thylakoids have been subjected to strong illumination in the cold (Aro et al., 1990). The protein is stable during complete PS II photoinactivation of mutants impaired in plastoquinol oxidation (Gong and Ohad, 1991) but is rapidly degraded following reoxidation of the PQ pool in low light or in the dark (Zer and Ohad, 1994). (iii) Protease inhibitors reduce the photoinduced degradation of the D1 protein (Virgin et al., 1991; Shipton and Barber, 1992; De Las Rivas et al.,

112 1993a). (iv) Phosphorylation of both the Dl and D2 proteins reduces their rate of degradation in vitro (Andersson et al., 1994). Most of these observations apply both under conditions of acceptor side-induced and donor side-induced photoinactivation. However, this does not imply that reactive radicals are not involved in the photoinhibitory process but that their involvement is at the level of protein damage rather than protein degradation. A central problem to be addressed is what are the primary sites for cleavage of the proteins and how is complete proteolysis achieved considering that each of the two proteins possess five membrane spanning regions interspersed by hydrophilic loops exposed at both sides of the thylakoid membrane (Fig. 2; Michel and Deisenhofer, 1986; Trebst, 1986). Is it possible, for example, that the complete degradation involves a single protease? Another important aspect for understanding turnover of the two reaction center proteins is therefore the identity, properties and regulation of the protease(s) involved. In fact, very little is known about the mechanisms for endogenous proteolysis of integral membrane proteins in any biological system and information based on studies on the turnover of soluble proteins may have limited relevance in this case.

A. Identification of Proteolytic Fragments Identification and characterization of proteolytic fragments from the D1 and D2 proteins would offer a direct way of determining the primary cleavage sites during degradation. Proteolytic fragments can not easily be detected in vivo or in isolated thylakoid membranes despite a pronounced degree of protein degradation. The likely explanation is that the secondary proteolysis, i.e. degradation of the primary fragments, is faster than the initial proteolysis of the D1 and D2 proteins. In contrast, degradation fragments of both proteins can readily be identified in more purified systems, in particular PS II core particles (Virgin et al., 1990; De Las Rivas et al., 1992; Salter et al., 1992b; Andersson et al., 1994) and PS II reaction centers (Shipton and Barber, 1991, 1992; Barbato et al., 1991) where secondary, but not primary, proteolysis appears to be impaired. Studies on these PS II preparations have also revealed that the pattern of primary fragments is different if the preceding photoinactivation and damage is induced from the acceptor or donor side of PS II (De Las Rivas et al., 1992, 1993a).

B. Andersson and J. Barber

1. D1 Protein Degradation Fragments of the D1 protein having apparent molecular masses of 23, 16 and 10 kDa have been identified after subjecting isolated oxygen evolving PS II cores to treatment with high light (Barber and Andersson, 1992;Aro et al., 1993). The same pattern of D1 protein fragments was also identified when PS II reaction centers were treated with photoinhibitory illumination, both in the presence and absence of added manganese ions (De Las Rivas et al., 1993a; Ponticos et al., 1993). In both kinds of experiments the photoinactivation took place via the acceptor side-induced mechanism. So far it has not been possible to perform successful N-terminal sequencing of these fragments or any other D1 protein fragments obtained after photoinhibitory illumination in vitro. Whether this is due to methodological problems or N-terminal blockage, or conceptual problems, such as some kind of unconventional form of peptide cleavage, is not known. Unfortunately, this has hampered attempts to use the specific fragments to precisely locate proteolytic cleavage sites on the D1 protein. However, by the use of indirect analyses, such as exogenous lysine-specific proteolysis, radioactive phosphate labeling of the N-terminus and western blotting by site-specific antibodies it has been possible to obtain some information on the origin of these degradation products. It has been shown that the 16 kDa and 10 kDa fragments are of C-terminal origin (Barbato et al., 1992b, Salter et al., 1992b, De Las Rivas et al., 1993a) while the 23 kDa fragment is derived from the N-terminal portion of the D1 protein (Salter et al., 1992b, De Las Rivas et al., 1993a). It is likely that the N-terminal 23 kDa and the C-terminal 10 kDa fragments originate from a primary site of cleavage situated in the stromal loop between helices D and E ofthe Dl protein (Fig. 6). By means of lysine specific proteolysis it has been shown that this cleavage site is located on the Cterminal side of residue 238 (Shipton et al., 1990; De Las Rivas et al., 1992) in agreement with the conclusions of Greenberg et al. (1987) based on studies using an in vivo system. A 17 kDa fragment of N-terminal origin has been identified (Friso et al., 1993) which, combined with the C-terminal 16 kDa fragment, could represent primary digestion products from an additional cleavage in the lumenal loop between helices C and D (Fig. 6). There is also evidence for a third cleavage site located at the loop exposed at the inner thylakoid surface and inter-

Chapter 4

Mechanisms of Photosystem II Photodamage

spersed between helices A and B (Fig. 6). This cleavage is clearly observed under conditions of donor side-induced photoinactivation and was first discovered in purified PS II reaction centers through the identification of a C-terminal 24 kDa fragment and a corresponding N-terminal 9 kDa fragment (Barbato et al., 1991). These two fragments can also be induced by high light treatment of PS II core preparations with impaired water splitting capacity (De Las Rivas et al., 1992). These fragments are induced by donor side photoinhibition and their appearance is not dependent on oxygen as is the case

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with the fragments obtained as a consequence of the acceptor side mechanism (Jegerschöld and Styring, 1991; De Las Rivas et al., 1993a). It is likely that other D1 protein fragments reported from various experiments using in vitro systems originate from secondary proteolysis of primary fragments (Salter et al., 1992a; Andersson et al., 1994). Although the differences in fragment pattern are interesting, it is essential to realize that characteristic donor side-induced fragments are often seen after prolonged acceptor side photoinactivation and vice versa (Salter et al., 1992a; Friso et al.,

114 1993). It is therefore likely that the distinct fragment patterns initially observed are not the result of different proteolytic enzymes but rather due to different triggering mechanisms under the two conditions for photoinactivation. This, in turn, induces primary proteolytic cleavage at different sites, but subsequently all potential endoproteolytic cuts must come into play to achieve complete degradation of the damaged protein. How does the characterization of D1 protein degradation observed with in vitro systems relate to what is known about the photoinduced degradation of this protein in vivo? As mentioned above, the identification of fragments in more intact systems is not easy because secondary proteolysis is completed very quickly. However, in the pioneering study by Greenberg et al. (1987) an N-terminal 23.5 kDa fragment, in addition to fragments of 8–14 kDa, were identified and shown to originate from a cleavage between helices D and E on the stromal side of the membrane. More recently, a C-terminal 10 kDa fragment has been identified in vivo after photoinhibitory treatment of wheat and pea leaves (Cánovas and Barber, 1993; Shipton and Barber, 1994). It is therefore evident from both in vivo and in vitro experiments that there is a primary cleavage site between helices D and E of the D1 protein. Taken together these results support the concept that the acceptor side-induced photoinactivation mechanism is dominating under in vivo conditions.

2. D2 Protein Degradation Degradation of the D2 protein has been less well studied compared to that of the D1 protein (Prásil et al., 1992). However, D2 protein degradation shares most of the features of D1 protein degradation and can therefore be described as a totally proteolytic event (M. Ponticos, J. Barber and B. Andersson, unpublished). Normally degradation of the D2 protein is considerably slower than that of the D1 protein. It could therefore be argued that D2 protein degradation is a secondary process induced by destabilization of the PS II complex upon removal of the D1 protein. However, in the presence of DBMIB and DCMU, when the degradation of the D1 protein is reduced, the loss of the D2 protein is less affected and can therefore be more rapid than that of the D1 protein (M. Ponticos, J. Barber and B. Andersson, unpublished). This implies that the D2 protein itself is damaged under photoinhibitory conditions and

B. Andersson and J. Barber therefore triggered for degradation. The probability of damaging the D2 protein under normal physiological conditions may be considerably less than for the D1 protein thus explaining its relatively limited degree of degradation and turnover. Identification of D2 protein fragments has been possible after high light treatment of both PS II core complexes and purified reaction centers (Virgin et al., 1990; Shipton and Barber, 1991; Barbato et al., 1992a; Andersson et al., 1994). The D2 protein degradation pattern is also very dependent on the type of photoinactivation. During acceptor sideinduced photoinactivation there is a major 21 kDa fragment in addition to 25 and 5 kDa fragments which are present in minor amounts (Andersson et al., 1994). Preliminary mapping with site-specific antibodies suggests that the two larger fragments are of N-terminal origin while the 5 kDa fragment is C-terminal. Thus there appears to be a primary cleavage site of the D2 protein at the outer thylakoid surface that is analogous to the cleavage at the D-E loop of the D1 protein. During donor side photoinactivation there are fragments sized at 18.5, 12 and 5 kDa. In purified reaction centers, 24 and 14 kDa D2 protein fragments can also be detected (M. Ponticos, J. Barber and B. Andersson, unpublished). Although the D2 protein does not normally turnover as rapidly as the D1 protein and is more resistant to photoinduced damage, there are conditions when this is not true. As Jansen et al. (1993) have shown, turnover of the D2 protein is greatly enhanced if UVB is superimposed on visible light. Moreover, specific breakdown fragments of the D2 protein are induced in isolated PS II reaction centers when UV-B treatment is given (Friso et al., 1994a). UV-B light was shown to be an effective inhibitor of PS II activity by Jones and Kok (1966) and indeed it not only speeds up the turnover of the D2 but also the D1 protein (Greenberg et al., 1989a). Consequently it has also been possible to detect UV-B induced fragments of this protein in isolated thylakoids (Trebst and Depka, 1990; Friso et al., 1994b) as well as in vivo (Greenberg et al., 1989b). Recently Frizzo et al. (1994) and Vass et al. (1994) have suggested that a primary site for damage by UV-B light is the water splitting system while the acceptor side damage reported by Greenberg et al. (1989a) occurs only when visible light has generated the semiquinone radicals of and However, UV-B irradiation of isolated PS II reaction centers which do not have the capacity to split water also brought about the degradation of the D1 and D2

Chapter 4

Mechanisms of Photosystem II Photodamage

proteins to yield discrete breakdown products, the patterns being determined by the nature of the electron acceptor present (Friso et al., 1994a, 1995).

B. Enzymology of D1 and D2 Protein Degradation The enzymology for proteolysis of membrane spanning hydrophobic proteins is not very advanced. As discussed above, it is difficult to envisage that one single proteolytic enzyme would have the accessibility to all regions of such proteins to allow complete degradation. In the case of the D1 and D2 proteins it could be speculated that distinct enzymatic activities occur on the stromal and lumenal side of the thylakoid membrane to allow for endoproteolytic cleavages in each loop of the two proteins once they have become triggered for degradation (Aro et al., 1993). The membrane spanning fragments produced may then become accessible for exoproteolytic activity leading to the complete degradation of D1 protein. It is likely that the D1 and D2 proteins are degraded by the same proteolytic machinery but there is no direct data to support this so far. The protease or proteases responsible for the degradation of the two PS II reaction center proteins are not yet known and their identification remains a central topic of photoinhibition research. It is likely that the proteolytic system is associated with the photosynthetic membrane since degradation can easily be followed in isolated thylakoid preparations (Ohad et al., 1985; Virgin et al., 1988). The possibility of inducing proteolysis of the two proteins in isolated PS II preparations, such as core complexes (Virgin et al., 1990, 1991; De Las Rivas et al., 1992; Salter et al., 1992b) or reaction centers (Shipton and Barber, 1991, 1992; Barbato et al., 1992a; De Las Rivas et al., 1993a), suggests that at least the enzymatic activities responsible for the primary endoproteolytic cleavages are an integral part or are tightly associated with the PS II complex. Interestingly, recent studies on other biological systems have demonstrated intrinsic proteolytic activities. For example, the cytochrome bc complex of plant mitochondria contains its own proteolytic activity (Braun et al., 1992; Glaser et al., 1994) and removal of the heatshock protein hsp-90 also appears to be an autoproteolytic event (Schnaider et al., 1993). In the case of isolated PS II preparations it could be argued that the proteolytic activity observed is due to a contaminating protease. However, extensive puri-

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fication of the various preparations does not reduce the extent of proteolysis and there is a requirement for a 1:1 stoichiometric relationship between PS II reaction centers and an added protease inhibitor to achieve complete inhibition of D1 protein degradation (De Las Rivas et al., 1993a). Moreover, dilution of PS II preparations during a photoinhibitory illumination does not reduce the extent of degradation. Using radiolabeled di-isopropylfluoro-phosphate, which covalently reacts with the active amino acid of serine-proteases, affinity labeling of isolated PS II core complexes was attempted (Salter et al., 1992b). The labeled subunit was found to be CP43, one of the two chlorophyll a antenna proteins of PS II, suggesting that this protein could carry a proteolytic activity. So far there is no further experimental support for this intriguing possibility. It has also been suggested that D1 protein degradation could be an autoproteolytic process contained within the reaction center itself (Virgin et al., 1990; Shipton and Barber, 1991). The possibility of obtaining primary Dl protein degradation and fragment accumulation in purified PS II reaction centers would support such a possibility (Shipton and Barber, 1991, 1992; Barbato et al., 1991; Ponticos et al., 1993; De Las Rivas et al., 1993a). More recently it has been suggested that a 41 kDa adduct, formed by cross linking between the D1 protein and the of cytochrome during photoinhibitory illumination (Barbato et al., 1992c), may be involved in such an autoproteolytic process (De Las Rivas et al., 1993a). Support for this idea has emerged from experiments which have identified the cross linking site in the FGQEE region of the loop joining the D and E transmembrane segments of the D1 protein (Barbato et al., 1995). As indicated above, the D1 and D2 proteins are degraded by a proteolytic machinery that is sensitive to a variety of serine-type protease inhibitors (Virgin et al., 1991, Shipton and Barber, 1992; De Las Rivas et al., 1993a), including the inhibitor di-isopropyl fluorophosphate (Salter et al., 1992b). In most studies with protease inhibitors using isolated PS II preparations a typical inactivation has been found to be around 50% (Virgin et al., 1991), but complete inhibition has, for example, been obtained by the addition of soybean trypsin inhibitor to isolated reaction centers (De Las Rivas et al., 1993a). Unlike several other endoproteolytic activities, including some found to be present in chloroplasts, degradation of the D1 and D2 proteins does not require ATP. The proteolytic degradation of the D1 protein is

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stimulated, but not dependent, on the addition of (Salter et al., 1992b). The proteolysis of the D1 protein shows an optimum in the pH range of 7.5–8.0 (Salter et al., 1992b; Shipton and Barber, 1992).

VI. Repair of Photodamaged Photosystem II Requires Co-ordination Between Degradation and Biosynthesis There are increasing amounts of experimental evidence suggesting that D1 protein degradation occurring during repair of photodamaged PS II centers is highly regulated (Aro et al., 1993). At the physiological level the repair of PS II may not be a priority under high light conditions and it has even been suggested that photoinactivated PS II complexes may be important in protecting neighboring active centers against over-excitation (Krause, 1988). At the biochemical level it would seem essential that the rate of protein degradation is in balance with the insertion of newly synthesized protein copies, as first suggested in the case of D1 protein turnover by Adir et al., (1990). If such a synchronization did not occur and the D1 protein (and/or D2 protein) was removed without immediate availability of a new copy then the PS II complex would probably undergo disassembly. As is the case for disassembled membrane protein complexes in general, the PS II subunits would then be rapidly proteolysed probably leading to irreversible photoinhibition. It is therefore worthy of note that unlike the situation in vivo (Adir et al., 1990; Melis, 1991), there is a pronounced disassembly of the PS II complex in isolated thylakoid membranes after D1 protein degradation has occurred (Hundal et al., 1990b). The connection between D1 protein degradation and biosynthesis can be demonstrated experimentally by pulse/chase analyses after addition of chloramphenicol to cyanobacterial cells during photoinhibitory treatment (Komenda and Barber, 1995). In the absence of this protein synthesis inhibitor the removal of the D1 protein occurs quite rapidly (Fig. 7). In contrast, in the absence of protein synthesis, degradation proceeds at a very slow rate and becomes more comparable to the degradation rates seen in vitro (Fig. 7). Thus, it seems that efficient D1 protein degradation does not occur unless there is a new copy of the protein available for integration into the damaged PS II complex. The mechanisms for this co-ordination are not known but could involve

different levels of cellular regulation. For example, a ribosomal complex containing a nascent D1 protein chain might have to attach to the PS II complex in order to activate the proteolytic process. It has been suggested that protein phosphorylation could influence and regulate turnover of the D1 protein (Kettunen et al., 1991; Aro et al., 1992, 1993; Elich et al., 1992). Indeed, a distinct form of the D1 protein (designated D1*) detectable by SDS-PAGE has been found to appear as a consequence of photoinhibitory treatment (Callahan et al., 1990; Kettunen et al., 1991). There is evidence that D1* is a phosphorylated form of the D1 protein. In vitro it has been shown that phosphorylation retards the degradation of both the D1 and D2 proteins (Aro et al., 1992; Andersson et al., 1994). It could therefore be speculated that phosphorylation is used to set aside photodamaged PS II centers under conditions when protein biosynthesis is not operational or becomes limiting. However, it must be kept in mind that in many lower photosynthetic organisms there is no phosphorylation of the D1 protein (Aro et al., 1993) and yet light-induced turnover of this protein and repair of photodamaged PS II reaction centers occurs readily.

Chapter 4

Mechanisms of Photosystem II Photodamage

Acknowledgments We wish to thank the BBSRC, RITE and Swedish National Science Research Council for financial support. We are most grateful to Markella Ponticos for her help with diagrams in this article.

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Chapter 4

Mechanisms of Photosystem II Photodamage

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B. Andersson and J. Barber Shipton CA, Marder JB and Barber J (1990) Determination of catabolism of the Photosystem II D1 subunit by structural motifs in the polypeptide sequence. Z Naturforsch 45c: 388– 394 Sies H (1986) Biochemistry of Oxidative Stress, Angew. Chem. Int. 25: 1058–1071 Sopory SK, Greenberg BM, Mehta RA, Edelman M and Mattoo AK (1990) Free radical scavengers inhibit light-dependent degradation of the 32 kDa Photosystem II reaction centre protein. Z Naturforsch 45c: 412–417 Styring S, Virgin I, Ehrenberg A and Andersson B (1990) Strong light photoinhibition of electron transport in Photosystem II. Impairment of the function of the first quinone acceptor Biochim Biophys Acta 1015: 269–278 Svensson B, Vass I, Cedergren E and Styring S (1990) Structure of donor side components in Photosystem II predicted by computer modelling. EMBO J 9: 2051–2059 Takahashi Y, Hansson Ö, Mathis P and Satoh K (1987) Primary radical pair in the Photosystem II reaction centre. Biochim Biophys Acta 893: 49–59 Tang X-S, Fushimi K and Satoh K (1990) D1-D2 complex of the Photosystem II reaction centre from spinach; isolation and partial characterization. FEBS Lett 273: 257–260 Telfer A and Barber J (1994) Elucidating the molecular mechanisms of photoinhibition by studying isolated PS II reaction centres. In: Baker NR and Bowyer JR (eds) Photoinhibition of Photosynthesis from Molecular Mechanisms to the Field, pp 25–49, Bios Scientific Publishers, Oxford Telfer A, Barber J and Evans M (1988) Oxidation-reduction potential dependence of reaction centre triplet formation in the isolated D1/D2 cytochrome b559 Photosystem II complex. FEBS Lett 232: 209–213 Telfer A, He W-Z and Barber J (1990) Spectral resolution of more than one chlorophyll electron donor in the isolated Photosystem II reaction centre complex.. Biochim Biophys Acta 1017: 143–151 Telfer A, De Las Rivas J and Barber J (1991) Beta-carotene within the isolated Photosystem II reaction centre; photo– oxidation and irreversible bleaching of this chromophore by oxidized P680. Biochim Biophys Acta 1060: 106–114 Telfer A, Bishop SM, Phillips D and Barber J (1994a) The isolated photosynthetic reaction of PS II as a sensitiser for the formation of singlet oxygen; detection and quantum yield determination using a chemical trapping technique. J Biol Chem 269: 13244–13253 Telfer A, Dhami S, Bishop S, Phillips D and Barber J (1994b) quenches singlet oxygen formed by isolated Photosystem II reaction centers. Biochemistry 33: 14469– 14474 Trebst A (1986) The topology of the plastoquinone and herbicide binding peptides of Photosystem II in the thylakoid membrane. Z. Naturforsch. 41c: 240–245 Trebst A and Depka B (1990) Degradation of the D1 protein subunit of Photosystem II in isolated thylakoids by UV light. Z Naturforsch 45c: 765–771 Trebst A, Depka B, Kraft B and Johanningmeier U (1988) The site modulates the conformation of the Photosystem II reaction centre polypeptides. Photosynth Res 18: 163–177 van Mieghem FJE, Nitschke W, Mathis P and Rutherford AW (1989) The influence of the quinone-iron electronic acceptor complex on the reaction centre photochemistry of

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Chapter 5 Radical Production and Scavenging in the Chloroplasts Kozi Asada The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan

Summary I. Introduction II. Radicals and Dioxygen A. Reactivities of Dioxygen and Radicals 1. Triplet Excited Molecules 2. Electron Donors with Low Redox Potentials 3. Free Radicals 4. Transition Metal Ions 5. Enzymes B. Active Species of Oxygen III. The Primary Target Molecules and Sites A. Reaction Center of Photosystem II B. Reaction Center of Photosystem I C. Enzymes of the Fixation Cycle and Other Enzymes in the Stroma IV. Production of Reactive Oxygens and Radicals and their Scavenging Enzymes A. Production and Scavenging of Superoxide Radicals 1. Photoproduction of Superoxide in the Thylakoid 2. Superoxide Dismutase (SOD) a. Cellular Location of Three Isozymes of SOD b. Reaction Rate and Mechanism c. Microcompartmentation of CuZn-SOD in Chloroplasts B. Production and Scavenging of Hydrogen Peroxide 1. Production of Hydrogen Peroxide in Chloroplasts and Peroxisomes a. Production of Hydrogen Peroxide via Spontaneous Disproportionation of Superoxide b. Production of Hydrogen Peroxide in the Chloroplasts c. Production of Hydrogen Peroxide in Peroxisomes 2. Catalase 3. Scavenging of Hydrogen Peroxide in Chloroplasts 4. Three Isozymes of Ascorbate Peroxidase 5. Molecular and Enzymatic Properties of Ascorbate Peroxidase a. Amino Acid Sequence b. Reaction Mechanism and Kinetics c. Inactivation of Ascorbate Peroxidase in the Absence of Ascorbate d. Inhibitors C. Production and Reduction of Monodehydroascorbate Radical to Ascorbate 1. Production of Monodehydroascorbate Radical 2. Monodehydroascorbate Reductase a. Cellular Location and Amino Acid Sequence b. Kinetic Properties and Reaction Mechanism 3. Ferredoxin-dependent Photoreduction of Monodehydroascorbate in the Thylakoid

Neil R. Baker (ed): Photosynthesis and the Environment, pp. 123–150. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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D. Production and Scavenging of Dehydroascorbate 1. Production of Dehydroascorbate via the Spontaneous Disproportionation of Monodehydroascorbate 2. Molecular Properties of Dehydroascorbate Reductase E. Production and Scavenging of Lipid Peroxides 1. Production of Lipid Peroxides 2. Phospholipid Hydroperoxide Glutathione Peroxidase V. Microcompartmentation of the Scavenging Systems of Superoxide and Hydrogen Peroxide in Chloroplasts VI. Dioxygen Protects from Photoinhibition A. Photorespiration B. Photoreducing System of Dioxygen VII. Concluding Remarks Acknowledgments References

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Summary Under the conditions where leaves are exposed to high photon flux densities in excess of their photon-utilizing capacity, excess photons produce active oxygens, radicals and triplet excited pigments, which cause photoinhibition of photosynthesis as a result of oxidation of target molecules. After a brief discussion on the relationship between radicals and active oxygens, the primary target molecules of these reactive molecules are discussed in relation to photoinhibition. Subsequently, the photoproduction of active oxygens and radicals in chloroplasts is described. To avoid photoinhibition, prompt scavenging of active oxygens and radicals at the sites where they are produced is essential in the chloroplasts. The enzymes participating in this scavenging and their microcompartmentation in chloroplasts are reviewed.

I. Introduction Plant leaves are always exposed to fluctuation of many environmental factors that affect photosynthesis. The most important environmental factor for photosynthesis, the solar intensity, is continuously variable, and other environmental factors are not always favorable to photosynthesis when the sun is bright. Availability of to chloroplasts is suppressed by drought via stomata closure, and low temperatures suppress the rate of the fixation. Under such conditions, the photon-utilizing capacity of plants is lowered, and the photons absorbed by leaves exceed the utilizing capacity associated with the partial reactions of photosynthesis from the absorption of photon energy by chlorophyll to the reduction of . Deficiency of mineral nutrients such as magnesium and phosphate can also result in Abbreviations: APX – ascorbate peroxidase; AsA – ascorbate; DHA – dehydroascorbate; DTT – dithiothreitol; Fd – ferredoxin; GPX – guaiacol peroxidase; GSH – glutathione (reduced); GSHPX – glutathione peroxidase; MDA – monodehydroascorbate radical; Phe – pheophytin; SOD – superoxide dismutase;

a decrease in the photon-utilizing capacity. Reduction in the rate of phosphate translocation at the chloroplast envelope induced by phosphate deficiency would lower the export of triose phosphate to the cytosol and suppress the photon-utilizing capacity. In addition to environmental conditions, endogenous factors also affect the photon-utilizing capacity of leaf tissues. For example, a low sink capacity for photosynthetic products of plants lowers the photon-utilizing capacity of leaves. The photon-utilizing capacity of plants is affected by both the environmental and endogenous factors. This is shown to be the case in cyanobacteria grown under different combinations of light intensity and the concentration of where the cell growth is remarkably suppressed under a high light intensity when the concentration is low. However, under a high light intensity the growth is little affected when the concentration of is high, since the photon-utilizing capacity of the cells is high (Fig. 1). When the photon intensity exceeds the photonutilizing capacity, the excitation energy and reducing equivalents generated by the excess photons produce

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electron transports can adjust the photoproducing ratio of ATP/NADPH to maintain photon-utilizing capacity, in addition to generating a proton gradient for the down-regulation of PS II. In this overview, first the photoproduction of radicals and active oxygens by the excess photons in chloroplasts and the target molecules or sites of these reactive molecules for photoinhibition are described. The photoproduction of radicals and active oxygens is unavoidable in chloroplasts even under the favorable conditions for photosynthesis, and their prompt scavenging is essential to protect the target molecules or sites from the photoproduced reactive molecules. This is especially true when the enzymes are the target, since the producing site of superoxide production is the same as that of the generation site of the reducing equivalents required for the operation of the cycle. Consequently, the microcompartmentation of the scavenging system of radicals and active oxygens to avoid their interaction with the target molecules in the stroma will be described. reactive species of oxygen and radicals in chloroplasts, and these reactive molecules oxidize target molecules. Under such photodamaging conditions, all of the excess photons do not produce reactive molecules. Plants have many systems to dissipate the energy of excess photons for suppression of the photoproduction of reactive molecules, and these relaxation systems convert the energy of excess photons finally to heat. Protection from the photon-excess stress can be associated with the movement of chloroplasts in cells to adjust photon acceptance, photorespiration (Section VI.A), the down-regulation of PS II associated with the formation of a proton gradient across the thylakoid membranes (see Chapters 1–3), the photoreduction of dioxygen to water (Section VI.B), cyclic electron transport for the generation of the proton gradient (Heber and Walker, 1992), and state transitions for adjustment of the excitation ratio of PS I and PS II (Allen, 1992). Photorespiration consumes NAD(P)H and ATP and releases in mesophyll cells. Consequently it supplies the photon energy acceptors to chloroplasts ADP and and suppresses the production of radicals and active oxygens (Osmond, 1981). The Fd-mediated (Cleland and Bendall, 1992; Mano et al., 1995; Miyake et al., 1995) and NAD(P)H-mediated (Asada et al., 1993; Mi et al., 1992, 1994, 1995) cyclic

II. Radicals and Dioxygen

A. Reactivities of Dioxygen and Radicals Dioxygen in the atmosphere (ground state oxygen, is characterized by its triplet state with two unpaired electrons having parallel spins in the two antibonding molecular orbitals, and which participate in reactions. Thus, dioxygen in the ground state is a biradical, and simply represented by a formula of or . Because of the unusual electron configuration of its reactions with singlet cell components paired electrons with antiparallel spins) are severely restricted to maintain spin conservation (spin-prohibited reaction). A low reactivity of is represented by its very low redox potential for one-electron oxidation even though is a strong oxidant if it is utilized as a four-electron oxidant The reaction of is allowed only with the reactants discussed below.

1. Triplet Excited Molecules Energy transfer or electron transfer to from the triplet excited molecules is not prohibited because of the same spin state of the two reactants,

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For Reaction (1), the photosensitized production of by is the typical case (Section III.A). The light-requiring herbicide p-nitrodiphenyl ether causes the accumulation of protoporphyrin IX in leaf cells by blocking the biosynthetic pathway of chlorophyll. The protoporphyrin acts as a photosensitizer for the generation of which damages chloroplasts (Haworth and Hess, 1988). Reaction 2 has not been found to occur in chloroplasts, but the photogeneration of by cercosporin (a toxin produced by many Cercospora species) has been shown (Daub and Hangarter, 1983).

2. Electron Donors with Low Redox Potentials When the electron donation potential of reductants is high – – 160 mV), one-electron reduction of yielding is possible. Such a reaction in chloroplasts is the photoproduction of in the PS I complex by the primary electron acceptors whose potentials are low enough to reduce (Section IV.A.1).

3. Free Radicals The reaction of with free radicals is allowed, and radicals readily transfer one electron to yielding especially when the redox potential of radicals is low,

Therefore, whenever is produced, the production of is likely to occur. However, in chloroplasts, no direct production of by photogenerated radicals of endogenous components has been demonstrated. Only when paraquat ylium) is added to chloroplasts, is it univalently photoreduced in PS I yielding its cation radical. Because of its low redox potential (–440 mV), the paraquat radical rapidly donates electrons to producing (Farrington et al., 1973). In chloroplast thylakoids, the semiquinone radicals of and the plastoquinones of the intersystem chain (Pace et al., 1992) and the phenoxy radicals of the Tyr residues in the D1 and D2 proteins of the PS II complex are photogenerated. In the PS I complex, the semiquinone

radical of phylloquinone is photogenerated (Iwaki and Itoh, 1994) by the electron donation from the reduced chlorophyll However, these radicals have little chance to interact with dioxygen due to their rapid electron transfer to the next carriers and their structural protection from interaction with dioxygen. During the chain-oxidation of unsaturated lipids in the thylakoid membranes the carbon-centered radicals are generated by hydrogen abstraction, and their interaction with yields the peroxy radicals of lipids,

This reaction may compete with the reaction in the thylakoids where the tocopherol (Toc) forms its chromanoxy radical

Other lipid radicals such as peroxy radical and (alkoxy radical) also are trapped by tocopherol yielding its radical (Reaction (5)). Tocopherol is regenerated from its radical by ascorbate, which has been found at 10–30 mM in the chloroplast stroma, yielding the ascorbate radical (MDA). MDA is generated also by other reactions (Section IV.C.1), but is reduced to ascorbate by either reduced Fd or NAD(P)H catalyzed with MDA reductase (Section IV.C.2 and 3). Fortunately its reactivity with is very low (Bielski, 1982), and little production of by MDA is expected in chloroplasts.

4. Transition Metal Ions Transition metal ions are characterized by a lone pair electron on the 3d orbital and can be regarded as a radical in respect of unpaired electron. Therefore, the reaction of with transition metal ions is not restricted, and they readily interact to form oxygenated metal complexes or to transfer an electron These oxygenated complexes are singlet state, and hence have a high reactivity with singlet cell components. Thus, transition metal ions catalyze the oxidation by dioxygen which often causes cellular toxicity. In plants, excess administration of transition metals such as Fe and Cu induces oxidative damage, which can be accounted

Chapter 5 Production and Scavenging of Radicals for by dioxygen-dependent oxidation and enhanced oxidation by the metal-catalyzed Haber-Weiss reaction (Section II.B).

5. Enzymes In addition to the above spontaneous reactions, glycolate oxidase in the peroxisome and ribulose 1,5 -bisphosphate oxygenase in the chloroplast stroma can interact with yielding plus glyoxylate and phosphoglycolate plus 3-phosphoglycerate, respectively. Further, the production of by NADPH-dehydrogenase, similar to that in neutrophiles in mammals, has been shown in plant cells under various stress conditions (Doke et al., 1994).

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and are referred to as active oxygens since their reactivities are high as compared with In addition to these reactive species, the radicals which have a high reactivity with such as semiquinone and paraquat radicals, and triplet excited pigments are physiologically equivalent to the active oxygens. Further, oxidized lipids, (lipid carbon centered radical), (lipid peroxy radical), LOOH (lipid hydroperoxide), (lipid alkoxy radical) and the degradation carbonyl products of lipids, such as malondialdehyde, also are molecular species similar to active oxygens with respect to their damaging effects on cells.

III. The Primary Target Molecules and Sites

B. Active Species of Oxygen Although the reactivity of is limited to the above reactions, the reactivities of the reduced and excited species of oxygen are high and can interact with cellular components. In addition to and the most reactive species, the hydroxyl radical is generated by the interaction of and transition metal ions such as Fe and Cu ions as follows:

Even when is not directly produced, it is generated spontaneously or by SOD-catalyzed reaction from (Section IV.B.l), and the reduced metal ions generate (Reaction (7)). Reduced metal ions are regenerated by (Reaction (8)), thus, a catalytic amount of metal ions could continuously produce if is generated. Since Reaction (8) also is possible by cellular reductants such as ascorbate, the prompt scavenging of is most essential to suppress the production of Excess metal ions can give photooxidative stress to cells, which is attributable to the overproduction of In chloroplasts Fe is stored in a form of phytoferritin that does not catalyze Reactions (7) and (8).

When leaves are exposed to excess photons, active molecules are generated in chloroplasts, even though many mechanisms for the dissipation of excess photons are operative, as described above. These active molecular species can oxidize chloroplast components at the sites where they are produced because of their high reactivities at nearly diffusioncontrolled rates. The diffusion distances from their generating sites and life times of and have been estimated to be very short (Asada, 1994a,b). Proteins, lipids, pigments, DNA and all other chloroplast components would be indiscriminately oxidized by active oxygens, especially by and if they are not properly scavenged. The oxidation of chlorophyll by active oxygens causes a visible damage of leaves, and usually represents a final stage of photoinhibition. For assessment of action of active oxygens upon photoinhibition, it is important to know which molecule or site is the primary target. Functional inactivation of the primary target molecules further lowers the photon-utilizing capacity resulting in an amplified production of active oxygens. The reaction centers of both PS I and PS II and the enzymes of -fixation cycle in the stroma seem to be the primary target and are described below (Fig. 2). Oxidation by active oxygens of other sites and molecules such as chlorophyll and lipids would also lower the photon-utilizing capacity.

A. Reaction Center of Photosystem II Under photon-excess, bright light conditions, PS II is primarily inactivated (Aro et al.,1993; see Chapter 4). In most cases, the electron transfer from

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the reaction center chlorophyll (P680) to and via pheophytin (Phe) is suppressed due to overreduction of the acceptors resulting from single or double reduction of by either strong light or unavailability of the electron acceptors for the linear electron flow. This inhibition of PS II by the suppression of electron transfer from P680 to the acceptor has been referred to as the acceptor-side photoinhibition (see Chapter 4). Under such conditions, is generated via the chargerecombination of and (Vass et al., 1992; 1993),

In vitro dioxygen is not necessary for initiation of the inactivation. At an initiation stage when dioxygen is not required, inactivates the intra-electron transport in the reaction center of PS II, probably through the Type I process in which directly oxidizes the target molecules via hydrogen abstraction,

At this stage no breakdown of the D1 protein of the reaction center of PS II has been observed. Under aerobic conditions, transfers its excitation energy to producing (Hideg et al., 1994a,b; Macpherson et al., 1993; Mishra et al., 1994; Miyao, 1994),

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The participates in triggering the proteolytic degradation of D1 protein via the oxidation of amino acid residues. may oxidize not only D1 protein, but also chlorophyll and other PS II components, which enhances further the photoinhibition of the acceptor side (Mishra et al, 1994; Chapter 4). In addition to acceptor-side photoinhibition, when the rate of electron donation from the oxidizing side in PS II is slower than the rate of electron withdrawal from the reaction center, donor-side photoinhibition occurs (see Chapter 4). The suppression of the rate of water oxidation may occur when the water oxidase activity in PS II is lowered by a release of from the oxygen-evolving complex into the lumen by formation in the thylakoids under photon-excess conditions (Krieger et al., 1992), or by a release of the extrinsic proteins required for the oxidation of water in PS II resulting from exposure to freezing temperatures (Wang et al., 1992). Under such conditions, life times of and Tyr in PS II are prolonged, and these strong oxidative radicals have a chance to oxidize PS II components. Until this primary step dioxygen is not required, but under aerobic conditions (Ananyev et al., 1994; Chen et al, 1992) and (Hideg et al., 1994b) are photoproduced, which promotes further the photoinactivation. The interaction of (Chen et al., 1992), the hydroxyurea radicals (Kawamoto et al., 1994) or the azidyl radicals (Kawamoto et al., 1995) with

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Production and Scavenging of Radicals

Tyr modifies the Tyr Z residue and inhibits the electron transport to P680 from the water oxidase complex. In the case of the donor-side photoinhibition also, the proteolytic degradation of D1 protein is triggered by these oxidants (Miyao, 1994). In the case of either acceptor-side or donor-side photoinhibition, D1 protein is the target molecule and is oxidatively damaged by photogenerated oxidants including active oxygens. Following this oxidative damage, the D1 protein is then degraded by a thylakoid-bound protease (Chapter 4). It is not understood why the D1 protein is the target but not the D2 protein, which is another subunit of the reaction center of PS II. However, chloroplasts have a high capacity for the de novo synthesis of D1 protein which is encoded in the chloroplast DNA, and its biosynthesis is regulated by its translation, not by transcription. The de novo synthesized precursor of D1 protein is inserted into the damaged PS II reaction center complex and is processed at the carboxy terminal (Takahashi et al., 1988), and the PS II activity is then recovered. Because of this rapid repair mechanism of the PS II reaction center, the turnover rate of D1 protein is the highest among chloroplast proteins (Aro et al., 1993).

B. Reaction Center of Photosystem I In addition to PS II, PS I also is inactivated when isolated thylakoids are illuminated under strong light (Inoue et al., 1986, 1989; Satoh, 1970). The photoinhibition is remarkable when the primary electron acceptors of PS I are reduced, as in the case of PS II. Under such conditions the electron flow from (chlorophyll) and (phylloquinone) to and is suppressed, and the probability of recombination of with either or increases producing Unlike the situation for in PS II, cannot quench in PS I to produce (Sétif et al., 1981; Takahashi and Katoh, 1984). However, dioxygen is required for the initiation of photoinhibition in PS I, which is not the case for photoinhibition of PS II except in Bryopsis chloroplasts (Satoh and Fork, 1982). Therefore, even if participates in the inactivation of PS I components via the type I process without the mediation of oxygen, it would not be the primary event. Recently, it has been shown that when cucumber (Terashima et al., 1994; Sonoike and Terashima, 1994) and potato (Havaux and Davand, 1994) leaves

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are illuminated at low temperature, PS I is photoinactivated before PS II is. Furthermore, PS I of spinach thylakoids is also photoinactivated under weak light before PS II is (Sonoike, 1995). The primary photoinactivation site is assigned to be either (Sonoike and Terashima, 1994; Sonoike et al., 1995), and dioxygen is required, at least for the initiation. Thus, it is very likely that and photogenerated by autooxidation of or participate in the inactivation of the reducing side of the reaction center of PS I. The participation of reduced oxygens in the photoinhibition of PS I is supported also by the observation that synthesis of SOD, APX and MDA reductase is induced during cold acclimation of poplar twigs (Nakagawara and Sagisaka, 1984) and spinach leaves (Schöner and Krause, 1990). The induction of catalase also has been shown in the cold-acclimated maize (Prasad et al., 1994).

C. Enzymes of the Fixation Cycle and Other Enzymes in the Stroma Inhibition of fixation by was first found by Kaiser (1976), and fructose 1,6-bisphosphatase, -glyceraldehyde 3 -phosphate dehydrogenase, ribulose 5-phosphate kinase and sedoheptulose 1,7bisphosphatase have been assigned as the sensitive enzymes (Kaiser, 1979; Tanaka et al., 1982). These enzymes have thiol groups participating in the catalytic reactions, and their oxidation to the disulfide form by converts the enzymes to an inactive form. The Fd-thioredoxin system reduces the disulfide-forms of the enzymes to recover their activities. The operation of the fixation cycle is suppressed if even one of the enzymes participating in the cycle is inactivated, consequently, fixation is lost at low concentrations of with a 50% inhibition at only 10 1976). In illuminated chloroplasts can accumulate to this concentration in 0.5 s if its scavenging system is not functioning. The thiol enzymes are generally sensitive to and therefore, it is very likely that these enzymes are susceptible to active oxygens other than Inactivation of the enzymes of the fixation cycle by has also been demonstrated (Jung and Kim, 1991). In addition to the above enzymes for the fixation, it has been shown that the glutamine synthetase of the chloroplasts participating in photorespiration is sensitive to the generated in

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130 the vicinity of the targeting His residue (Fucci et al., 1983).

2. Superoxide Dismutase (SOD) a. Cellular Location of Three Isozymes of SOD

IV. Production of Reactive Oxygens and Radicals and their Scavenging Enzymes

A. Production and Scavenging of Superoxide Radicals 1. Photoproduction of Superoxide in the Thylakoid The primary product of the photoreduction of dioxygen in PS I is (Asada et al., 1974), and it is produced within the thylakoid membranes via the autooxidation of the membrane-bound primary electron acceptors in PS I, possibly center X or center A/B (Asada, 1994b). The observed rate of photoproduction of within the membranes can be regarded as the ejection rate of from the surface of the membranes, which is around 20 mg (Asada and Takahashi, 1987) and corresponds to 7.4 molecules assuming that the P700 content is one molecule per 430 molecules of chlorophyll. The apparent low ejection rate has been inferred by the electron donation to either plastocyanin or cytochrome f, from the photogenerated within the PS I complex, i. e. by the putative cyclic electron transport around PS I (Asada, 1994b). In PS I the autooxidation of the photoreduced Fd is another possible source of However, its contribution would be minor in intact chloroplasts because of a low autooxidation rate of reduced Fd, as compared with that of the membrane-bound photoreductants (Asada, 1994b). Furthermore, in addition to the electron donation from the reduced Fd to the cyclic electron pathway (Miyake et al., 1995), a high reactivity of reduced Fd with MDA radicals see Section IVC.3) reduces the possibility of interaction of the reduced Fd with dioxygen, even when is not available to PS I. The contributions of PS II and the intersystem electron carriers to the photogeneration of are minor, at least, in the intact chloroplasts and thylakoids. However, the production of has been shown in PS II preparations (Ananyev et al., 1994; Chen et al., 1992a), and is probably due to destruction of the supermolecular organization in the PS II complex and its connection to the intersystem electron transport chain.

In plant tissues three types of SOD have been found with respect to the prosthetic metals; CuZn-SOD, Mn-SOD and Fe-SOD. Fe-SOD and Mn-SOD were successively acquired during the evolution of photosynthetic bacteria and cyanobacteria, and CuZnSOD appeared at the latest stage of evolution of eukaryotic algae. CuZn-SOD is the major SOD in gymnosperms and angiosperms (Asada et al., 1980), and appears to have diverged to chloroplastic and cytosolic isozymes from the ancestor CuZn-SOD, which were probably acquired by the eukaryotic green algae of the phragmostic type (Kanematsu and Asada, 1989, 1990). The chloroplastic isozyme is exclusively localized in chloroplasts. The cytosolic enzyme is abundant in non-photosynthetic tissues, although in leaf cells it is localized in compartments other than chloroplasts. The chloroplastic and cytosolic isozymes show characteristic sequences (Kanematsu and Asada, 1990). Although the ligand residues of Cu and Zn, six His residues and one Asp residue, are conserved in both the isozymes, the sequence homology between the chloroplastic and cytosolic CuZn-SODs is below 68%. However, the homologies among chloroplastic CuZn-SODs and cytosolic CuZn-SODs in angiosperms are over 90% and 80–90%, respectively (Bowler et al., 1994). Only one isoform of the chloroplastic CuZn-SOD has been found, but several isoforms of the cytosolic CuZn-SOD occur in each species of plant surveyed so far (Kanematsu and Asada, 1990). Cytosolic CuZnSOD is localized in apoplastic region and nucleus of spinach leaf cells, as detected by immunogold labeling (K. Ogawa, personal communciation), probably different isoforms in different compartments. In plants Mn-SOD is localized in mitochondria. In most angiosperms CuZn-SOD is the major SOD in chloroplast and Fe-SOD, if present, is localized in the chloroplast stroma (Kanematsu and Asada, 1990). The expression of Fe-SOD is generally suppressed, but in some species (e. g. Nuphar luteum) Fe-SOD is the major chloroplastic SOD (Bowler et al., 1994).

b. Reaction Rate and Mechanism SOD catalyzes the disproportionation of through the redox cycle of the prosthetic metal ions at the diffusion controlled rate; the enzyme first oxidizes to and the resulting

Chapter 5

Production and Scavenging of Radicals

reduced enzyme reduces to with the proton from the His residue ligated to the prosthetic metal ions,

The rate constant of the two partial reactions is 2 × for CuZn-, Mn- and Fe-SODs, which is the highest, diffusion-controlled rate among enzymatic reactions (Rotilio et al., 1972; Kanematsu and Asada, 1994). The ionic interaction of the superoxide anion radicals with the positive charged amino acid residues around the reaction center of CuZn-SOD contributes largely to the rapidity of the reaction (Getzoff et al., 1992). The three dimensional structure of the chloroplastic CuZn-SOD is available (Kitagawa et al, 1991), and its whole structure, including the reaction center, conserved with that of CuZn-SOD from other organisms. In addition to the ionic interaction, the diffusion of to the SOD molecule in the medium is also a factor in determining the rate of reaction. The reaction rate between and SOD can be lowered by increasing the viscosity of reaction medium upon addition of glycerol (Rotilio et al., 1972). The protein concentration in the stroma is nearly 40% (w/v), and its viscosity is sixty-nine times higher relative to that of water. When the high viscosity in the stroma is taken into consideration the rate constants of reactions 13 and 14 are expected to be being one order of magnitude lower than that in water (Ogawa et al., 1995).

c. Microcompartmentation of CuZn-SOD in Chloroplasts In spinach chloroplasts, the chloroplastic CuZn-SOD occurs in the stroma in a soluble form. After osmotic rupture of isolated intact chloroplasts and centrifugation, little SOD activity is detected in the thylakoid membranes, and the activity is recovered in the supernatant. Only when the thylakoid membranes are treated by detergents can the latent CuZn-SOD in the lumen be found, and this accounts for 4% the total chloroplastic CuZn-SOD (Hayakawa et al., 1984).

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The content of CuZn-SOD in the stroma is around one molecule per P700 or the PS I complex (Table 1). Immunogold labeling using the antibody specific for the chloroplastic CuZn-SOD has indicated its location on the thylakoid membranes. Over 70% of the immunogold particles attach to the stroma-faced surface of the thylakoid membranes (the stroma thylakoids, and the margins and ends of the grana thylakoids) within a 5 nm layer (Fig. 3; Ogawa et al., 1995), where the PS I complexes are primarily located. The 5 nm layer on the stroma-face of the thylakoid membrane is similar in size to that of the dimeric CuZn-SOD from spinach (6.2 × 3.3 × 3.6 nm, Kitagawa et al., 1991), suggesting that about one molecule of CuZn-SOD attaches to the surface of the membrane in the vicinity of the PS I complex, probably accompanying with the thylakoid-bound APX (Section IV.B.4). From the volume of the 5 nm layer on the stroma-face of the thylakoid membranes, the local concentration of CuZn-SOD on the membranes is estimated to be around 1 mM, and that in the remaining stroma is only 20 (Table 2). If CuZn-SOD distributes uniformly in the stroma, as supposed previously, its average concentration is 50 Weak ionic and hydrophobic interactions with the thylakoid membranes seem to participate in the ‘targeting’ of soluble CuZn-SOD on to the membranes, since buffer-washed thylakoids retain no SOD activity. Therefore, the interaction of CuZnSOD not only with the thylakoid membranes but also with soluble enzymes in the stroma is supposed to contribute to ‘targeting’. It is not known, however, what domains or residues of CuZn-SOD participate in such an interaction. Recently it has been shown that a point mutation of CuZn-SOD causes a syndrome of amyotrophic lateral sclerosis (Deng et al., 1993). The mutation of CuZn-SOD does not occur in the active site region, and the mutant CuZnSOD retains the activity. The mutation in domains or residues other than the reaction center region probably disturbs the ‘targeting’ of this soluble enzyme in human cells. For effective photosynthesis, the soluble Calvin cycle enzymes should be organized or compartmented in the chloroplast stroma. It is not known yet, however, how they are compartmented and, if the stroma is organized, by what mechanism of interaction the ‘soluble’ enzymes are so well compartmented that many reactions of the cycle proceed effectively and rapidly for the fixation. Süss et al. (1993, 1995) have indicated that the NADPH-consuming enzymes

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of the Calvin cycle are compartmented on the thylakoid membranes. Microcompartmentation of CuZn-SOD on the stroma-face of thylakoid membranes contributes to the prompt scavenging of just after it ejected from the PS I complex and allows a supply of for APX. Thus, in cooperation with the thylakoid-bound APX and the Fd-dependent photoreduction of MDA, the microcompartmentation of SOD suppresses the diffusion of and to the stroma, and their interactions with the target enzymes of the fixation cycle (see Section V and Fig. 7).

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B. Production and Scavenging of Hydrogen Peroxide 1. Production of Hydrogen Peroxide in Chloroplasts and Peroxisomes

a. Production of Hydrogen Peroxide via Spontaneous Disproportionation of Superoxide Superoxide radicals generated by one-electron reduction of dioxygen occur in two forms depending on pH; superoxide anion radical and its protonated form sometimes referred to as the hydroperoxyl radical). Since the pKa of superoxide is 4.88 (Bielski, 1978), in the stromal pH most superoxide occurs in a form of However, in a

Chapter 5

Production and Scavenging of Radicals

limited compartment of chloroplasts such as in the lumen when is formed, part of it occurs in the form of as well as and permeates readily through the membrane, but does not (Takahashi and Asada, 1983). Reactivity of is higher than that of for example, can initiate lipid peroxidation and oxidize glyceraldehyde 3phosphate dehydrogenase-bound NADH, but cannot (Chan and Bielski, 1980). Furthermore, the life times of and are very different. The spontaneous disproportionation rates between (Reaction (16)), and (Reaction (17)), and (reaction 18) have been determined as follows (Bielski, 1978),

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disproportionation of superoxide is accounted for by reaction 17 at neutral pH. The low rate of disproportionation at high pH is due to an electrostatic repulsion of for the collision and also to a low concentration of protons. In aprotic environments a prolonged life time of superoxide is expected, as in the case of photoproduction of superoxide within thylakoid membranes. When superoxide is photoproduced within the thylakoids and ejected into the stroma (in part via diffusion through the membranes from the lumen), its steady state concentration in the stroma would be around M as estimated from its rate of spontaneous disproportionation (Asada, 1994a), if the ejection rate was 20 mg (Section IV. A. 1). Thus, the concentration of superoxide would appear to be dangerous to chloroplasts if it is not lowered promptly by several orders of magnitude.

b. Production of Hydrogen Peroxide in the Chloroplasts

Effect of pH on the spontaneous disproportionation rate of superoxide (Fig. 4) indicates its decrease by one order of magnitude per pH unit above pH 4.9 (pKa of superoxide), with an accompanying decrease in the concentration of Thus, most spontaneous

In chloroplasts is mainly produced from the catalyzed by SOD. Microcompartmentation of CuZnSOD at a site where is ejected from the thylakoid membranes suggests that there is little chance of its spontaneous disproportionation. Therefore, the reduction of by ascorbate, GSH or reduced ferredoxin does not contribute to the production of in the chloroplasts,

No two-electron oxidase producing directly has been found in the chloroplast, and the SODcatalyzed reaction is the sole source of No evidence has been presented for the production of superoxide in the stroma, but the autooxidation of the stromal components, especially carbonyl compounds including the intermediates of the fixation cycle, is a possible source of even though its production rate is lower by several orders of magnitude than that in the thylakoids.

c. Production of Hydrogen Peroxide in Peroxisomes In the photorespiratory pathway, the glycolate produced from ribulose 1,5-bisphosphate via phosphoglycolate is oxidized by the two-electron

Kozi Asada

134 oxidase, glycolate oxidase, yielding directly peroxisomes,

in

Under atmospheric concentrations of and photorespiration is unavoidable and dissipates excess photons (Section IV.A), and it is not unusual for photosynthetic fixation of to be increased twofold by suppression of photorespiration in 1 % Therefore, the production rate of accompanying photorespiration might be several fold greater than that in the chloroplast thylakoids.

2. Catalase In leaf cells catalase is almost exclusively localized in peroxisomes and scavenges the generated by the action of glycolate oxidase. In this respect, the peroxisomal catalase plays an important role in the process of photorespiration for the dissipation of excess photons. Although chloroplasts lack catalase, it is present in mitochondria in mesophyll cells of maize. For a review of the molecular properties and biosynthetic regulation of catalase see Scandalios (1994). Catalase catalyzes the disproportionation of via two step reactions of the redox cycle of the enzyme-heme,

Because of an apparent high value for due to its reaction mechanism, catalase is required at high concentrations to lower the concentration of and it occurs sometimes in a crystalline state in peroxisomes (4 mM) (Newcomb and Frederick, 1971). Although the scavenging of by a peroxidase reaction requires electron donors, the requirement for the amounts of the enzyme is far below that of catalase. The average concentration of the thylakoid-bound and stromal APXs in chloroplasts is estimated to be only 70 It has been shown that catalase is photoinactivated by a mechanism in which its prosthetic heme is a photosensitizer for generation of the triplet heme and (Cheng et al.,1981). This is a specific case of enzyme inactivation in which the prosthetic group itself is a photosensitizer. The peroxisomal catalase

is actually photoinactivated in leaf cells, as has been observed by its high turnover rate of 3–4 h in rye leaves) which is close to that of the D1 protein in thylakoids of 1.5 h) under a strong light (Hertwig et al., 1992). In chloroplasts, unlike in peroxisomes, catalase does not participate in the scavenging of derived from via the SOD-catalyzed reaction. Plant chloroplasts scavenge via a peroxidase reaction using a photoreductant as the electron donor prior to diffusion from the generating site. Cyanobacteria, however, are divided to two groups with respect to the scavenging of the catalase-scavenging species and the peroxidase-scavenging species (Miyake et al., 1991), indicating that the peroxidasescavenging system of was acquired during the evolution of cyanobacteria years ago).

3. Scavenging of Hydrogen Peroxide in Chloroplasts In chloroplast little catalase has been found, and the derived from the generated in PS I is scavenged by a peroxidase reaction using a photoreductant generated in PS I as the electron donor (Nakano and Asada, 1980). In intact chloroplasts, the photoreduction rate of is equal to that of the evolution of from water (Asada and Badger, 1984). This stoichiometry is accounted for by assuming the following reaction sequences for the photoreduction of dioxygen to water in PS I,

The reducing equivalents

are equally used for

Chapter 5

Production and Scavenging of Radicals

the reduction of two molecules of to in PS I (Reaction 23) and for the reduction of to water (Reaction 26). This stoichiometry is effected only when the rate-limiting step of the sequences of the reaction is the photoreduction of dioxygen. If the Reactions (25) and (26) are a rate-limiting step, the accumulation of in intact chloroplasts is expected, but actually this is not the case (Asada and Badger, 1984). Foyer and Halliwell (1977) found dehydroascorbate (DHA) reductase in chloroplasts and proposed ascorbate as the reductant of Subsequently ascorbate peroxidase (APX) was detected in spinach (Kelly and Latzko, 1979) and Euglena (Shigeoka et al., 1980a), and its localization in plant chloroplasts has been confirmed (Nakano and Asada, 1981; Groden and Beck, 1979). Subsequent findings on the occurrence of the regenerating enzymes of ascorbate, DHA reductase and MDA reductase in chloroplasts, indicate further that in Reaction (25) is ascorbate, and the key role of APX in the scavenging of in chloroplasts has been established.

4. Three Isozymes of Ascorbate Peroxidase Three isozymes of APX have been found in plants. One is the cytosolic isozyme (cAPX) and others are chloroplastic (Chen and Asada, 1989). The chloroplastic isozymes occur in the stroma in a soluble form (sAPX) (Nakano and Asada, 1987) and in the thylakoids in a membrane-bound form (tAPX). tAPX mainly binds to the stromal thylakoids where the PS I complexes are located (Miyake and Asada, 1992). Recently, a peroxisome-bound APX has been found (Yamaguchi et al., 1995). Similar molar ratios of tAPX and sAPX occur in chloroplasts. From the activities of tAPX and sAPX in spinach chloroplasts (Miyake and Asada, 1992), the molar ratio of tAPX and sAPX to P700 is calculated at about 0.5 each, assuming that the content of P700 is one molecule per 430 molecules of chlorophyll (Table 1). Little difference in molecular properties between tAPX and sAPX has been found, but the molecular size of tAPX is about 10 kDa larger than that of sAPX, probably due to the requirement for binding to the membranes. Cytosolic and chloroplastic APXs can be distinguished by their specificity for the electron donor, stability of the Compound I and amino acid sequences. The difference in molecular properties among cAPX, sAPX and tAPX can be used for the specific assay of

135

the respective APXs. According to the preliminary results, APX in eukaryotic algae is a type of cAPX (Amako and Asada, 1994), and actually, in Euglena, APX is localized in cytosol but not in chloroplasts (Shigeoka et al., 1980b; 1987).

5. Molecular and Enzymatic Properties of Ascorbate Peroxidase APX is a hemoprotein containing protoporphyrin IX similar to other peroxidases such as cytochrome c peroxidase in yeast and to guaiacol peroxidases (GPX) that are ubiquitous in plants and are represented by horseradish peroxidase (Asada, 1992b; 1993). The molecular size of APX is about 30 kDa, and it occurs as a monomer, but several cAPXs exist as homodimers. Reflecting the difference in physiological functions between APX and GPX, the molecular properties of the two peroxidases are very different.

a. Amino Acid Sequence Whole sequences of amino acids of cAPXs from pea (Mittler and Zilinskas, 1991), Arabidopsis (Kubo et al., 1992) and cucumber (K. Amako, personal communication) have been deduced from their cDNAs, and they show a high degree of homology with each other (77–85%). cAPXs so far sequenced have no transit peptide for targeting. Partial sequences of cAPXs from maize (Koshiba, 1993) and spinach (Tanaka et al, 1991) have been determined, as they have for sAPX from tea (Chen et al., 1992b) and tAPX from spinach (Miyake et al., 1993). Both APX and GPX conserve the amino acid sequences around the heme ligation, the proximal and distal His residues, but, as a whole, the sequence homology between cAPX and GPX is only 16–19%, in contrast to a high degree of homology between cAPX and cytochrome c peroxidase from yeast (31–33%). Cytochrome c peroxidase is localized in mitochondria, and participates in the scavenging of similar to APX in chloroplasts. Thus, in accordance with the shared physiological function of in cell organelles, APX shows a higher degree of sequence homology with cytochrome c peroxidase rather than those of the plant GPXs. Probably for targeting to the cell wall GPX is glycosylated, and the Asn residues for the glycosylation are conserved, but the corresponding Asn residues could not be found in APX. APXs purified so far do not contain

136 any carbohydrate as is the case for cytochrome c peroxidase (Chen and Asada, 1989). The proximal and distal His regions of sAPX show a high degree of sequence homology with those of cAPX, but in the amino terminal regions, cAPX and chloroplastic APX (sAPX and tAPX) each show characteristic sequence (Fig. 5). The Trp residue (Trp–191) around the proximal His residue is conserved both in APX and cytochrome c peroxidase, and also in bacterial catalase/peroxidase. In cytochrome c peroxidase the reaction intermediate Compound I is the Fe(IV)Trp+(191) radical, which is different from that of GPX (Fe(IV)-porphyrin cation radical). Another characteristic amino acid sequence of GPX is the conserved eight Cys residues which serve for the formation of four disulfide bridges. Thiol-modifying reagents have no effect on the GPX activity. However, the conserved Cys residues of GPX are not found in cAPX and sAPX, and thiol-modifying reagents inhibit all isoforms of APX. In cAPX sequenced so far, the Cys–31 is conserved, suggesting its participation in the peroxidatic reaction, but no corresponding Cys residue is found in tAPX. From these properties of APX, the ancestor of this protein was deduced to be the bacterial catalase/ peroxidase, which diverged to cytochrome c peroxidase in fungi and APX in plants. Since the cytosolic type of APX has been found in eukaryotic algae, it is reasonable to suggest that the ancestral c APX in algae further diverged to chloroplastic tAPX and sAPX, and cAPX in angiosperms.

b. Reaction Mechanism and Kinetics The reaction kinetics of APX are inferred by a

Kozi Asada peroxidase ping-pong mechanism, which is the same as that for GPX (Dunford, 1991). APX is first oxidized by to form the two-electron oxidized intermediate, Compound I, and the Compound I oxidizes successively two one-electron oxidations of AsA to produce two molecules of MDA (Fig. 6). The absorption spectrum of the Compound I of t APX is similar to that of cytochrome c peroxidase, but not to that of GPX. When the Cys residue of tAPX is modified, Compound I is not formed, suggesting the participation of the Cys residue in the oxidation of APX by possibly to form the (Miyake and Asada, 1996). In case of cAPX, however, no has been detected by EPR spectroscopy (Patterson et al., 1995).

c. Inactivation of Ascorbate Peroxidase in the Absence of Ascorbate One of the characteristic properties of APX is its inactivation at low concentrations of AsA below 20 and this is one of the reasons why APX had not been found as readi ly as GPX. The discovery that AsA stabilized APX (Hossain and Asada, 1984b) allowed the purification of APX, and it is absolutely essential to add AsA during its purification. tAPX and sAPX are very labile with half lives of around 20 s, as compared with cAPX whose half life is over 10 min (Chen and Asada, 1989). The different sensitivities to the depletion of AsA between cAPX and chloroplastic APX allow their separate assay (Amakoetal., 1994). Lability of APX below 20 AsA is inferred by decomposition of Compound I, which is formed by the interaction of APX with nanomolar levels of

Chapter 5

Production and Scavenging of Radicals

generated by autooxidation of AsA at micromolar levels. The characteristic spectrum of Compound I spontaneously decays with a similar half life to the inactivation in the absence of AsA if excess is present (Miyake and Asada, 1996). Therefore, when AsA is not available to Compound I, the heme of APX is decomposed by and APX is inactivated (Fig. 6). Although APX and cytochrome c peroxidase show a high degree of sequence homology, the stabilities of their reaction intermediates are very different from each other. The Compound I of cytochrome c peroxidase is so stable that it has been used for the microassay of

137

p-aminophenol, hydroxyurea and hydroxylamine. These compounds themselves do not inhibit APX, but inactivate in the presence of For example, hydroxyurea (HU), is univalently oxidized by catalyzed with APX forming its aminoxy radicals though its rate is not so high as compared with that of AsA,

The formed in the reaction center interacts with APX or its Compound I, resulting in its inactivation,

d. Inhibitors As a hemoprotein APX is inhibited by either cyanide or azide via its ligation to the sixth coordination position of the heme-iron. Further, thiol modifying reagents inactivate APX by blocking the oxidation of heme with This is a specific property of APX, different from that of GPX, which is sustained by respective characteristic amino acid sequences for Cys residues (see above). This property has been shown to be useful to enable the specific assay of APX and GPX (Amako et al., 1994). In addition to these inhibitors, more specific ones for APX are the suicide inhibitors such as thiols,

In the presence of AsA, the enzymatic production of is suppressed by competition with AsA, and the is trapped by AsA producing MDA. By such a mechanism AsA protects the inactivation of APX by the suicide inhibitors (Chen and Asada, 1990). Dithiothreitol (DTT) is the most effective suicide inhibitor among thiols. Its thiyl radical is formed via the APX-catalyzed reaction and works as the inhibitor of APX (Chen and Asada, 1992). Actually DTT has been used to inhibit APX in intact chloroplasts (Neubauer and Yamamoto, 1992; 1994). It should be

138 noted here that DTT inhibits also the deepoxidation of violaxanthin to zeaxanthin (Neubauer, 1993).

C. Production and Reduction of Monodehydroascorbate Radical to Ascorbate 1. Production of Monodehydroascorbate Radical In chloroplasts the major producing reaction of MDA is an APX-catalyzed reaction (Fig. 6), in which the primary product has been identified to be MDA (Hossain et al., 1984). In addition to the APX reaction, the following reactions may generate MDA in chloroplasts. It has been shown that AsA is required for the deepoxidation of violoxanthin to antheraxanthin and further to zeaxanthin. The deepoxidation is induced by formation of across the thylakoid membrane, generated under the conditions of excess photons for photosynthesis (Demmig-Adams and Adams, 1992). It has been shown that the APX reaction and the deepoxidation of violaxanthin compete for AsA (Neubauer and Yamamoto, 1994). The deepoxidase has a low pH optimum at around 5 (Brat et al., 1995), and MDA is produced in the reaction (C. Miyake and K. Asada, unpublished). Further, when the donor side of PS II is inactivated, AsA, in place of water, donates electrons to the reaction center, generating MDA (J. Mano and K. Asada). Thus, enhanced photoproduction of MDA is very likely to occur under conditions of excess photons. A high reactivity with radicals is one of the characteristic properties of AsA, and whenever AsA acts as an antioxidant MDA is produced. Reduced radicals of dioxygen oxidize AsA to MDA. The reaction rate with is not high and SOD lowers its level, therefore, the contribution of to the formation of MDA would be expected to be very low in chloroplasts. Only when the scavenging of either or fails and is produced, will the interaction of with AsA at a diffusioncontrolled rate at pH7, Bielski, 1982) generate MDA. When GSH acts as an antioxidant in chloroplasts, especially by the interaction with radicals, glutathionyl thiyl radical, is formed, and its interaction with AsA is a source of MDA. Other organic radicals, such as carbon-centered peroxy and phenoxy radicals, also

Kozi Asada show a high reactivity with AsA producing MDA (Bielski, 1982). Further, when tocopherol in the thylakoid membranes scavenges the lipid radicals (carbon centered peroxy and alkoxy radicals), its radical is generated. Tocopherol occurs in thylakoid membranes at a ratio of 1 mol for 50 mol of chlorophyll and protects from peroxidation of the membrane lipids. Thus, AsA regenerates tocopherol from its radical producing MDA and sustains the antioxidant activity of tocopherol for a long time. Consequently AsA acts as a ‘sink’ for the radicals generated in chloroplasts, and MDA is produced whenever AsA scavenges the photoproduced and radicals. MDA is a resonance stabilized tricarbonyl species and has a similar life time to that of superoxide radical at neutral pH (Section IV.D. 1). MDA is detectable by EPR spectroscopy at room temperature and can be used as a cellular probe for oxidative stress (Buettler and Jurkiewicz, 1993). By application of oxidative stress, MDA, as detected in intact leaves, has been shown to increase (Westphal et al., 1992).

2. Monodehydroascorbate Reductase MDA reductase, a monomeric FAD-enzyme with a molecular weight of 47,000, catalyzes the reduction of two molecules of MDA to AsA by NAD(P)H,

a. Cellular Location and Amino Acid Sequence MDA reductase has been found to be localized in chloroplasts (Hossain et al., 1984), but no thylakoidbound activity has been found (Miyake and Asada, 1992). Most MDA reductase occurs in a soluble form in the stroma at a molar ratio to P700 of 0.14. In addition to chloroplasts, MDA reductase has been found also in non-photosynthetic tissues (Arrigoni et al., 1981; Bowditch and Donaldson, 1990) and appears to be a ubiquitous enzyme in plants, including algae (Shigeoka et al., 1987; Miyake et al., 1991). MDA reductase has been purified from cucumber fruits (Hossain and Asada, 1985), potato tubers (Borracino et al., 1986) and soybean root nodules (Dalton et al, 1992). All of them are the cytosolic

Chapter 5

Production and Scavenging of Radicals

isozyme. The chloroplastic isozyme shows a higher molecular mass by 7 kDa than the cytosolic one (M. A. Hossain et al., unpublished), as is the case for the mitochondrial isozyme (Leonardis et al., 1995). The whole sequences of the cytosolic MDA reductase from cucumber (Sano and Asada, 1994) and pea (Murthy and Zilinskas, 1994) have been deduced from their cDNAs. MDA reductase has the FAD- and NAD(P)H-binding domains, which are common to flavoenzymes, but has only a low homology with the flavoenzymes from eukaryotes such as ferredoxinreductase. The highest degree of homology of MDA reductase shares is with those of NADHiron-sulfur protein reductase from prokaryotes, indicating that MDA reductase is a new family of flavoenzyme which has not been found in eukaryotes until now (Sano and Asada, 1994). MDA reductase is the first enzyme whose substrate (electron acceptor) is an organic radical.

b. Kinetic Properties and Reaction Mechanism The apparent for NADH is lower than that for NADPH, about 4.4 for NADH and 210 for NADPH, in the cytosolic isozyme (Sano et al., 1995). The chloroplast enzyme also prefers NADH as the electron donor (Hossain et al., 1985). The reaction kinetics of MDA reductase (MDAR) support the ping-pong mechanism, as follows (Hossain and Asada, 1985),

The FAD of MDA reductase (MDAR-FAD) is reduced by NAD(P)H to a form of a charge-transfer complex as characterized by an absorption at around 600 nm. Reaction (31) is a diffusion-controlled one and its rate is as determined by the stopped flow method (Sano et al., 1995). The reduced enzyme (I) is oxidized successively by two molecules of MDA via the semiquinone form (II) as the intermediate. The reaction rate of (I) or (II) with MDA has been

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determined to be by the pulseradiolysis method (Kobayashi et al., 1995), indicating that the reduction of MDA with reduced MDAR also is a diffusion-controlled reaction though it is one order of magnitude lower than that of SOD with (Section IV. A.2). The redox reaction rates of MDARFAD decrease with increase in ionic strength, an indication of the enhanced electrostatic guidance of either NADH or MDA to the FAD. The pKa values of NADH and MDA are 3.9 and –0.45, respectively, indicating that they exist in anionic forms at neutral pH. Basic amino acid residues around the enzymeFAD might facilitate the electrostatic guidance of the dissociated substrates.

3. Ferredoxin-dependent Photoreduction of Monodehydroascorbate in the Thylakoid MDA reductase prefers NADH rather than NADPH as the electron donor, even in chloroplasts. Therefore, it is unlikely that the MDA formed by the APXreaction, especially by the thylakoid-bound APXreaction, will be reduced to AsA by NADH, since is the preferred electron acceptor in PS I. Another photoreducing system of MDA operates in chloroplast thylakoids, which accounts for almost all of the reduction of the MDA formed by the photoproduced via the APX-reaction in the vicinity of the PS I complex. NADH-dependent reduction of MDA catalyzed by MDA reductase may participate in the scavenging of the MDA formed in the stroma, as discussed in Section V MDA generated by the ascorbate oxidase-reaction is photoreduced by thylakoids (Miyake and Asada, 1992), but its rate of photoreduction is enhanced at least by twenty-fold on addition of Fd (Miyake and Asada, 1994). The participation of Fd in the photoreduction of MDA has been suggested also by the oxygen-exchange assay (Forti and Ehrenheim, 1993). The apparent reaction rate constant between the Fd photoreduced in the thylakoids and MDA is about which is thirty-four times higher than that of the photoreduction of (Miyake and Asada, 1994). Thus, (via reductase) and MDA compete for the photoreduced Fd in PS I, and the photoreduction of MDA will be preferred to that of This photoreduction of MDA in PS I also supports the contention that the NAD(P)H-dependent reduction of MDA catalyzed by MDA reductase is not the major scavenging system

140 of MDA, at least, in the vicinity of the PS I complex.

D. Production and Scavenging of Dehydroascorbate

1. Production of Dehydroascorbate via the Spontaneous Disproportionate of Monodehydroascorbate When MDA radicals fail to be reduced directly to AsA by either the photoreduced Fd or by NAD(P)H catalyzed with MDA reductase, MDA disproportionates spontaneously yielding DMA and AsA,

The disproportionation rate constant of MDA is similar to that of superoxide at pH 7. Its pH dependency also is similar to that of superoxide; the rate is increased with a decrease in pH, and the maximum rate is found below pH 3 (Bielski, 1982). No direct production of DHA, other than the disproportionation of MDA, has been shown in chloroplasts. DHA is labile at high pHs, even at a pH around 8 for chloroplast stroma under illumination, and decomposes to oxalate and threonate via diketogulonate (Tolbert and Ward, 1982). Thus, if the chloroplasts fail to reduce DHA to AsA, the total content of AsA plus DHA is decreased, and the biosynthesis of AsA is necessary to recover the level of AsA.

2. Molecular Properties of Dehydroascorbate Reductase

Kozi Asada (Hossain and Asada, 1984a) and potato tubers (Dipierro and Borracino, 1991). Its molecular weight is 23,000, and the thiol group participates in the reaction. Activity of DHA reductase has been found in the chloroplast stroma (Foyer and Halliwell, 1976; Jablonski and Anderson, 1981; Nakano and Asada, 1981) at a molar ratio to P700 of 0.09, but not in the thylakoid-bound form (Miyake and Asada, 1992). Actually DHA works as a Hill oxidant in chloroplast, coupled to PS I via the NAD(P)-glutathione reductase system. As discussed previously almost all of MDA generated in chloroplasts is directly reduced to AsA by either Fd-mediated reaction in PS I or MDA reductase-catalyzed reaction in the stroma, but chloroplasts still have a good photoregeneration capacity of AsA from DHA mediated by GSH and DHA reductase. Recently, the amino acid sequence of the chloroplastic DHA reductase has been shown to have a high degree of homology with that of trypsin inhibitors, and the purified DHA reductase shows a trypsin inhibitor activity when its thiol groups are oxidized to the disulfide(Trümper et al., 1994). This seems to indicate dual functions of DHA reductase depending on the redox state of chloroplasts. Thus, DHA reductase in chloroplasts appears to have physiological functions other than the regeneration of AsA from DHA. Protein disulfide isomerase has been found to show a DHA-reducing activity (Wells et al., 1990; Ahn and Moss, 1992), therefore, it may have other physiological roles than that associated with the GSH-dependent reducing activity of DHA.

E. Production and Scavenging of Lipid Peroxides 1. Production of Lipid Peroxides

DHA is spontaneously reduced to AsA by GSH which is increased with an increase in pH. At pH 8.3 the spontaneous reduction rate of DHA by GSH at the similar concentrations in chloroplasts is only 0.1% of the catalyzed rate by the chloroplastic DHA reductase (Nakano and Asada, 1981). Thus, the spontaneous reduction by GSH plays only a minor role in the regeneration of AsA from DHA in chloroplasts. DHA reductase catalyzes the reduction of DHA to AsA using GSH as the electron donor:

The enzyme has been purified from spinach leaves

When the hydrogen of unsaturated phospholipids (LH) in the membranes is abstracted by either or other radicals, the carbon-centered radical is generated. If is once generated, it interacts with yielding which could abstract further the hydrogen from LH generating LOOH. Thus, the chain oxidation of LH is initiated. When LOOH is further reduced, is generated and it also could abstract the hydrogen from LH, as discussed in Section II.A. Interaction of LH with produces directly LOOH, and causes the chain oxidation of LH. The interaction of two molecules of LOOH generates (Russel’s mechanism), which also extends the chain reaction.

Chapter 5

Production and Scavenging of Radicals

Chain propagation of lipid peroxidation is suppressed by trapping lipid radicals and with tocopherol in thylakoid membranes, and the resulting tocopherol radical is reduced to tocopherol by ascorbate as discussed in Section IV.C.1. Molar ratio of tocopherol to chlorophyll in thylakoids is about 0.02 (Bucke, 1968). Plant polyphenols, flavonoids and other antioxidants also would participate in the suppression of lipid peroxidation.

2. Phospholipid Hydroperoxide Glutathione Peroxidase In plant tissues, unlike mammalian tissues, little or no activity of glutathione peroxidase (GSHPX) has been detected. In mammalian cells, the glutathione reductase-GSHPX couple mainly participates in the scavenging of in mitochondria and other cell compartments, but in plants the function of GSHPX is replaced by that of APX (Asada, 1993). The reaction center of GSHPX is the selenocysteine residue which is encoded by the termination codon TGA. In addition to scavenging seleno-GSHPX, phospholipid hydroperoxide GSHPX has been found in mammals. This peroxidase shows a high specificity to phospholipid hydroperoxide (Thomas et al., 1990), but is devoid of a selenocysteine residue though it retains a low sequence homology with seleno-GSHPX. Recently, cDNA has been isolated from tobacco (Criqui et al., 1992) and citrus (Holland et al., 1993), whose deduced amino acid sequences show a high degree of homology with that of mammalian phospholipid hydroperoxide GSHPX. Further, a no stop condon (TGA) has been found in the position corresponding to the selenocysteine residue of the seleno-GSHPX. In Euglena selenium-less GSHPX has been found (Overbaugh and Fall, 1985). Under stress conditions, such as low temperature (Edwards et al., 1994; Hausladen and Alscher, 1994), high oxygen concentration (Foster and Hess, 1982) and drought (Gamble and Burke, 1984; Rensburg and Kruger, 1994), the biosynthesis of GSH, and also of glutathione reductase, have been shown to be induced, and glutathione reductasetransgenic plants show a tolerance to several environmental stresses (Aono et al., 1991, 1993; Foyer et al., 1991, 1994). As discussed in Section V, a role of GSH in the regeneration of ascorbate is not so important, therefore, GSH and its reductase might

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play a role in the reduction of lipid hydroperoxide. Thus, it is very likely that the enzyme for the scavenging of phospholipid hydroperoxide occurs also in plants and suppresses the peroxidation of membrane lipids. Biochemical characterization of this class of peroxidase in plants is required to allow an understanding of its physiological function.

V. Microcompartmentation of the Scavenging Systems of Superoxide and Hydrogen Peroxide in Chloroplasts As described in Section IV, the enzymes participating in the scavenging of and the derived from and also in the regeneration of ascorbate are not uniformly distributed within chloroplasts. Table 1 summarizes the molar ratio of the enzymes to P700 with their localizations, and Table 2 shows their concentrations at the respective locations. The stromafaced thylakoid is a PS I-rich region, to which APX binds (Section IV.B.4) and Fd seems to bind peripherally. Electron microscopic observations indicate the attachment of CuZn-SOD to the same regions of the thylakoid membranes (Section IV. A. 2). Thus, in the vicinity of the PS I complex, there is, at least, a molecule of the scavenging enzymes for two molecules of P700, and the ejected from the PS I complex is promptly disproportionated to and by the SOD attached to the membranes. The is reduced to water by ascorbate catalyzed by the thylakoid-bound APX, and the produced MDA is reduced by the photoreduced Fd to ascorbate. This microcompartmentation of the thylakoid scavenging enzymes (Fig. 7) allows the prompt scavenging of the and prior to their diffusion to the stroma where the target enzymes are localized (Fig. 2). In addition to the thylakoid membrane-bound and peripheral scavenging enzymes, the stroma contains SOD, sAPX, MDA reductase, DHA reductase and glutathione reductase. The last three enzymes participate in the regeneration of ascorbate from MDA and DHA by NAD(P)H and GSH, and the photoreduced Fd would not directly play a role in the regeneration in the stroma. The contents and concentrations of the scavenging enzymes in the stroma (Tables 1 and 2) are not so high as those in the thylakoidal system, indicating that the scavenging capacity of the stromal system is lower than that of the thylakoidal system. Actually the added to

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intact chloroplasts through the envelope inactivates very effectively fixation as compared with the photoproduced in the thylakoids (Asada, 1992a). The stromal system probably scavenges the active oxygens which are photoproduced but are not scavenged in the thylakoidal system. The NADHdependent regeneration of ascorbate in the stromal system, which is different from that by the photoreduced Fd in the thylakoidal system, would operate for the scavenging of active oxygens generated in the stroma. Table 2 summarizes the second order reaction rate constants for the interactions of the scavenging enzymes with active oxygens and MDA. Based on these rates and the local concentrations of the enzymes, the pseudo-first order rate constants are estimated. From the values of the half-lives of and MDA on the thylakoid membranes in the vicinity of the PS I complex are deduced to be only 0.4, 70 and 22 and those in the stroma are 20 20 and 0.2 ms, respectively. The high, diffusioncontrolled reaction rates of the respective reactions and the compartmentation of the participating enzymes within chloroplasts allow the rapid scavenging of and and also the rapid regeneration of ascorbate for the reduction of These half-lives are shorter by several orders of

Kozi Asada

magnitude than that of the linear electron flow in the thylakoids of 10 ms), indicating the prompt scavenging of active species of oxygen at a site where they are photogenerated by the linear electron flow. This kinetic simulation on the production and scavenging of active oxygens satisfies the requirement of the in Section IV.B.3; the photoreduction of dioxygen (Reaction (23)) is the rate-limiting step of the whole sequences (Reactions (22–26)). By microcompartmentation of CuZn-SOD on the thylakoid membranes (Section IV.A.2), the steady state concentration of within 5 nm layer on the membranes is lowered to below 7 nM, as compared with 27 nM if CuZn-SOD distributes in the whole stroma (Ogawa et al., 1995).

VI. Dioxygen Protects from Photoinhibition As described in the previous sections, active oxygens and radicals are generated from dioxygen, but, under anaerobic conditions or low oxygen conditions, photoinactivation of leaf tissues is enhanced. This is due to the requirement of dioxygen in photorespiration as the electron acceptor, and to the photoreduction of dioxygen to superoxide and the subsequent ascorbatemediated reduction to water which is needed to allow

Chapter 5

Production and Scavenging of Radicals

effective, linear electron flow in the thylakoids.

A. Photorespiration Photorespiration may be one of the important mechanisms for the dissipation of excess photons. In the photorespiratory process dioxygen is required for the oxygenation of ribulose 1,5-bisphosphate and the oxidation of glycolate, catalyzed by ribulose 1,5bisphosphate oxygenase in chloroplasts and glycolate oxidase in peroxisomes, respectively. Because of their high values for dioxygen, air-level concentrations of dioxygen are necessary for photorespiration to occur, and the decrease in its concentration to 1% causes photoinhibition due to reduced photorespiratory dissipation of excess photons (Osmond, 1981). When the supply to chloroplasts is not limited, a 1% concentration does not cause photoinhibition, but rather results in an increase in the fixation rate due to the

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suppression of photorespiration. However, in limiting environments such as the normal atmosphere a low concentration of will reduce photorespiration and induce the photoinhibition (but see also arguments in Chapter 14). The chloroplastic glutamine synthetase participates in the assimilation of ammonium ions released by a reaction of glycine decarboxylase in mitochondria, which is an important step of the photorespiratory pathway for the exchange of ammonium ions between mitochondria and chloroplasts. Glutamine synthetase is, however, a sensitive enzyme to active oxygens (Fucci et al., 1983). Transgenic tobacco plants with enhanced levels of chloroplastic glutamine synthetase have been shown to have higher resistance than the wild type parent plants to high light intensity under low conditions (Takeba and Kozaki, 1995). These observations indicate that the glutamine synthetase is a limiting step in photorespiration under photooxidative stress, and the enhanced levels of the

144 enzyme sustain the operation of photorespiration. In the first step of the photorespiratory process, dioxygen is incorporated via the oxygenase reaction into phosphoglycolate. however, is a putative intermediate for the reaction with an carbanion of ribulose bisphosphate at C-2. This reaction would occur in a ‘cage’ of the reaction center of the oxygenase (Lorimer, 1981), and no release of from the enzyme during the reaction has been shown.

B. Photoreducing System of Dioxygen After the initial discovery by Trebst (1962), enhanced photoinactivation of the electron transport of the thylakoids by anaerobiosis has been repeatedly reported. Under these conditions, there is a limited electron acceptor supply available to thylakoids, that is, the photon-utilizing capacity is severely reduced. The intersystem electron carriers will be highly photoreduced resulting in an increase of charge recombination in the reaction centers. Further, because of the restricted electron flux, a across the thylakoid membranes cannot be formed, which further increases the reduced level of the intersystem electron carriers due to a lack of the down regulation of the PS II activity. The apparent for dioxygen in its photoreduction in thylakoids is low (2–3 Asada and Nakano, 1978; Takahashi and Asada, 1982), therefore, for the protection of the chloroplasts from photoinhibition by electron transport to dioxygen, a much lower concentration of oxygen is sufficient, as compared to that required for photorespiration. In most cases when the thylakoids are illuminated under anaerobic conditions the activity of PS II is impaired, and similar photoinhibition has been observed in intact chloroplasts and algal cells (Asada and Takahashi, 1987). In the absence of oxygen, no proceeds (Ziem-Hanck and Heber, 1980), which indicates the participation of the photoreducing system of oxygen in the adjustment of photoproduction ratio of ATP/NADPH for the operation of the cycle. In this way dioxygen protects from the photoinhibition by an increase in the proper operation of the fixation cycle and in the availability of an electron acceptor to chloroplasts. The photoreducing system of dioxygen, i.e. the photoreductions of dioxygen and MDA (Fig. 7), actually can generate (Neubauer, 1990; Schreiber and Neubauer, 1990; Horman et al., 1994) and induce the formation of zeaxanthin (Neubauer

Kozi Asada and Yamamoto, 1992). In the photoreducing system of dioxygen, the accompanying production of MDA and its photoreduction increase two-fold the flux of the linear electron flow at a high rate (Table 2). As discussed in Section IV.C.1, in addition to the production of MDA by the APX-reaction, the MDA is additionally photoproduced by the deepoxidation of violaxanthin, electron donation to PS II and the reduction of the tocopherol radical, and further increases the electron flux required for the generation of the proton gradient. Thus, the contribution of the Fd-dependent photoreduction of MDA to the formation of the proton gradient is larger than that of the photoreduction of dioxygen itself. As a whole, when the photoreducing system of dioxygen is properly operating, it not only guarantees the protection of enzymes and of the PS I reaction center from the reduced species of oxygen, but it also ensures the electron flux in thylakoids, which allows the down regulation of PS II via and zeaxanthin formation (see Chapters 1–3).

VII. Concluding Remarks The photoproduction of reactive molecules, including reduced and excited species of oxygen, radicals and triplet excited pigments, is unavoidable in chloroplasts under natural environments, since the solar intensity is not always in concert with other environmental conditions for optimal photosynthesis, and under such environmental combinations the excess photon state is generated. The energy of the excess photons absorbed by leaves is dissipated as a heat by many relaxation systems in order to suppress the transfer of the excess photons to oxygens and other molecules. Even so, it is impossible to suppress completely the photoproduction of the reactive molecules in chloroplasts where several target sites are located. It should be emphasized that the sites of production of the reactive molecules and their target molecules are close to each other in the chloroplasts (Fig. 2). Because of the existence of the target molecules in chloroplasts, and high reactivities and short life times of the reactive molecules, their prompt scavenging prior to their interactions with the target molecules is indispensable for the maintenance of the photosynthetic activity. As discussed in the case of and their prompt scavenging prior to the interaction with the target molecules is supported by very high reactivities

Chapter 5

Production and Scavenging of Radicals

and also by the microcompartmentation of the scavenging enzymes. Although SOD has been found in all photosynthetic organisms from photosynthetic bacteria to angiosperms (Asada et al., 1980), the scavenging system of by the peroxidase reaction was acquired during the evolution of cyanobacteria (Miyake and Asada, 1991). In eukaryotic algae, the peroxidase is not localized in the chloroplasts but in the cytosol, suggesting that the derived from the photoproduced in the thylakoids diffuses to the cytosol where it is scavenged by APX. Therefore, the chloroplastic enzymes encounter the but it has been shown that the algal target enzymes of the fixation cycle are resistant to as compared with the angiosperm enzymes (Takeda et al., 1995). It is not known at what evolutionary step APX appeared in chloroplasts and further diverged to the thylakoid-binding isoform, but the present system in angiosperms seems to be essential to protect the labile enzymes of the fixation cycle and also to protect from photoinhibition by the down regulation of PS II via the formation of proton gradient across the thylakoid membrane. To increase stress resistance, increasing the activity of the scavenging enzymes of active oxygens in transgenic plants has been attempted (Foyer et al, 1994; Allen, 1995; Inzé and Montague, 1995). For acquisition of the effective resistance to the photoinhibition, the balanced increase of the scavenging and regenerating enzymes, and also of the targeting of the enzymes to the cell organelles or to the cell compartments where the active oxygens are photogenerated, is required. Furthermore, the microcompartmentation of the scavenging and regenerating enzymes within chloroplasts acquired during evolution indicates an importance of the intraorganelle targeting of soluble enzymes at the site where the active oxygens are photoproduced.

Acknowledgments The present work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, by a grant from the Human Frontier Science Program and by the Alexander von Humboldt Award, which made it possible to prepare the manuscript at Universität Würzburg. The author wishes to thank Y. Okubo for her typing.

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Chapter 5 Production and Scavenging of Radicals study of superoxide dismutase. Biochim Biophys Acta 268: 605–609 Sano S and Asada K (1994) cDNA Cloning of monodehydroascorbate radical reductase from cucumber: a high degree of homology in terms of amino acid sequence between this enzyme and bacterial flavoenzymes. Plant Cell Physiol 35: 425–437 Sano S, Miyake C, Mikami B and Asada K (1995) Molecular characterization of monodehydroascorbate radical reductase from cucumber overproduced in Escherichia coli. J Biol Chem 270: 21354–21361 Satoh K and Fork DC (1982) Photoinhibition of reaction centers of Photosystems I and II in intact Bryopsis chloroplasts under anaerobic conditions. Plant Physiol 70: 1004–1008 Scandalios JG (1994) Regulation and properties of plant catalase. In: Foyer CH and Mullineaux PM (eds) Causes of Photooxidative Stress and Ameriolation of Defense Systems in Plants, pp 275–315. CRC Press, Boca Raton Schöner S and Krause GH (1990) Protective systems against active oxygen species in spinach: response to cold acclimation in excess light. Planta 180: 383–389 Schreiber U and Neubauer C (1990) electron flow, membrane energization, and the mechanism of nonphotochemical quenching of chlorophyll fluorescence. Photosynth Res 25: 279–289 Sétif P, Hervo G and Mathis P (1981) Flash-induced absorption changes in Photosysteml. Radical pair or triplet state formation? Biochim Biophys Acta 638: 257–267 Shigeoka S, Nakano Y and Kitaoka S (1980a) Purification and some properties of L-ascorbic acid specific peroxidase in Euglena gracilis Z. Arch Biochem Biophys 201: 121–127 Shigeoka S, Nakano Y and Kitaoka S (1980b) Metabolism of hydrogen peroxide in Euglena gracilis. Biochem J 186: 377– 380 Shigeoka S, Yasumoto R, Onishi T, Nakano Y and Kitaoka S (1987) Properties of monodehydroascorbate reductase and dehydroascorbate reductase and their participation in the regeneration of ascorbate in Euglena gracilis. J Gen Microbiol 133: 227–232 Sonoike K (1995) Selective photoinhibition of Photosystem I in isolated thylakoid membranes from cucumber and spinach. Plant Cell Physiol 36: 825–830 Sonoike K and Terashima I (1994) Mechanism of Photosystem-I photoinhibition in leaves of Cucumis sativus L. Planta 194: 287–293 Sonoike K, Terashima I, Iwaki M and Itoh S (1995) Destruction of Photosystem I iron-sulfur centers in leaves of Cucumis sativus L. by weak illumination at chilling temperature. FEBS Lett 362: 235–238 Süss KH, Arkona C, Manteuffel R and Adler K (1993) Calvin cycle multienzyme complexes are bound to chloroplast thylakoid membranes of higher plant in situ. Proc Natl Acad Sci US 90: 5514–5518 Süss KH, Prokhorenko I and Abler K (1995) In situ association of Calvin cycle enzymes, ribulose-1,5-bisphosphate carboxylase/ oxygenase activase, reductase and nitrite reductase with thylakoid and pyrenoid membranes of Chlamydomonas reinhardtii chloroplasts as revealed by immunoelectron microscopy. Plant Physiol 107: 1387–1397 Takahashi M and Asada K (1982) Dependence of oxygen affinity for Mehler reaction on photochemical activity of chloroplast

149 thylakoids. Plant Cell Physiol 23: 1457–1461 Takahashi M and Asada K (1983) Superoxide anion permeability of phospholipids membranes and chloropiast thylakoids. Arch Biochem Biophys 226: 558–566 Takahashi M, Shiraishi T and Asada K (1988) COOH-terminal residues of D1 and the 44–kDa CPa-2 at spinach Photosystem II core complex. FEBS Lett 240: 6–8 Takahashi Y and Katoh S (1984) Triplet states in a Photosystem I reaction center complex. Inhibition of radical pair recombination by bipyridinium dyes and naphthoquinones. Plant Cell Physiol 25: 785–794 Takeba G and Kozaki A (1995) Photorespiration is an important mechanism for photoprotection. Plant Cell Physiol 36: s136 Takeda T, Yokota A and Shigeoka S (1995) Insusceptibility of algal photosynthesis to hydrogen peroxide. Plant Cell Physiol 36:1089–1095 Tanaka K, Takeuchi E, Kubo A, Sasaki T, Haraguchi K and Kawamura Y. (1992) Two immunologically different isozymes of ascorbate peroxidase from spinach leaves. Arch Biochem Biophys 286: 371–375 Terashima I, Funayama S and Sonoike K (1994) The site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is Photosystem I, not Photosystem II. Planta 193: 300–366 Thomas JP, Maiorino M, Ursini F and Girotti AW (1990) Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. J Biol Chem 265: 454–461 Tolbert BM and Ward JB (1982) Dehydroascorbic acid. In: Seib PA and Tolbert BM (eds) Ascorbic Acid: Chemistry, Metabolism and Uses, pp 101–123. American Chem Soc, Washington Trebst A (1962) Lichtinaktivierung der in der photosynthese. Z Naturforsch B 17: 660–663 Trümper A, Follmann H and Haberlein I (1994) A novel dehydroascorbate reductase from spinach chloroplasts homologous to plant trypsin inhibitor. FEBS Lett 352: 159– 162 Vass I and Styring S (1993) Characterization of chlorophyll triplet promoting states in Photosystem II sequentially induced during photoinhibition. Biochemistry 32: 3334–3341 Vass I, Styring S, Hundal T, Koivuniemi A, Aro E-M and Andersson B (1992) Reversible and irreversible intermediates during inhibition of Photosystem II: Stable reduced species promote chlorophyll triplet formation. Proc Natl Acad Sci USA 89: 1408–1412 Wang WQ, Chapman DJ and Barber J (1992) Effect of cold treatments on the binding stability of Photosystem II extrinsic proteins and an associated increase in susceptibility to photoinhibition. Plant Physiol 99: 21–25 Wells WW, Xu DP, Yang Y and Rocqne P A (1990) Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity. J Biol Chem 265: 15361–15364 Westphal S, Wagner E, Kjiollmuller M, Loreth W, Schuler P and Stegmann HB (1992) Impact of aminotriazole and paraquat on the oxidative defence system of spruce monitored by monodehydroascorbic acid. A test assay for oxidative stress causing agents in forest decline. Z Naturforsch 47c: 567–572 Winterbourn CC (1993) Superoxide as an intercellular radical sink. Free Rad Biol Med 14: 85–90

150 Yamaguchi K, Mori H and Nishimura M (1995) A novel isoenzyme of ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal membranes in pumpkin. Plant Cell Physiol 36:1157–1162

Kozi Asada Ziem-Hanck U and Heber U (1980) Oxygen requirement of photosynthetic assimilation. Biochim Biophys Acta 591: 266–274

Chapter 6 Metabolic Regulation of Photosynthesis Mark Stitt Botanisches Institut, Universität Heidelberg, lm Neuenheimer Feld 360, 69120 Heidelberg, Germany

Summary 152 I. Introduction 153 II. Pathways and Metabolite Measurements: Evidence for Highly Coordinated Regulation of Many Reactions 154 III. Regulatory Properties of Calvin Cycle Enzymes 155 A. Light Drives Photosynthesis by Activating Several Calvin Cycle Enzymes via Thioredoxin and Changes of Stromal pH and Magnesium 155 B. Substrates Modulate Thioredoxin Activation to Allow a Feedforward Coordination of Enzymes in the Calvin Cycle 156 C. Product-Inhibition and Feedback Inhibition Provide Additional Coordination of Calvin Cycle Fluxes 156 D. Coordination of ATP and NADPH Production 158 E. Rubisco 158 1. The Unique Importance of Rubisco 158 2. Regulation via Ribulose 1,5-bisphosohate Availability 159 3. Competition of Other Phosphorylated Intermediates with Ribulose 1,5-bisphosphate 159 4. Carbamylation (Activation) State 160 5. Carboxyarabitinol 1-Phosphate 161 F. Coordination of the Calvin Cycle and Photorespiration 162 162 G. Regulatory Properties of Enzymes of Sucrose and Starch Synthesis 1. Feedforward Regulation Coordinates Sucrose Synthesis and Fixation 162 2. A ‘Threshold’ Triose-Phosphate Concentration Reconciles Sucrose Synthesis with Calvin 164 Cycle Turnover 3. Feedback Regulation Decreases Sucrose Synthesis and Increases Starch Synthesis When 165 Sucrose Accumulates in a Leaf 4. Allocation can be Altered Without Inhibiting Photosynthesis 165 5. Sucrose Synthesis is also Modulated in Response to Nitrogen Assimilation and Water 165 Stress 6. Carbon can Move Out of the Chloroplast via the Hexose Transporter During Starch Degradation 166 7. Further Factors could also Regulate Sucrose and Starch Metabolism 166 IV. Coarse Regulatability 166 167 V. How can the Regulatory Capacity of a Protein be Evaluated? 167 A. Comparison of Metabolites and Fluxes B. Mechanistic Models 168 168 C. Control Analysis 168 1, Regulatory Capacity is Revealed by the Flux Control Coefficient 2. Theory Predicts that Enzymes with Hiqh Regulatability Will Not Always Have High Flux Control Coefficients 169 D. How Can Flux Control Coefficients Be Measured? 170 170 1. Calculation From Models 170 2. From the and Response of Photosynthesis

Neil R. Baker (ed): Photosynthesis and the Environment, pp. 151–190. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

152 3. ‘Dual Modulation’ 4. Inhibitor Titration 5. Mutants or Transgenic Plants E. Some Recent Extensions of the Theory 1. Evaluation of Large Changes 2. Changes of Kinetic Properties VI. Distribution of Control in Photosynthetic Carbon Metabolism A. Entry of 1. Boundary Layer Conductance 2. Stomata Conductance of the Aqueous Phase of the Leaf 3. 4. Regulation and Constraints C. Rubisco 1. Effect of Short-Term Conditions 2. Contribution in Ambient Growth Conditions 3. Adaptation to Limiting Nitrogen Supply 4. Adaptation to Irradiance Regimes 5. Allocation to Rubisco Is Not Always ‘Optimal’ C. Other Enzymes of the Calvin Cycle D. Partitioning 1. Starch Synthesis 2. Sucrose Synthesis 3. Interaction Between Sucrose Synthesis, Starch Synthesis and Photosynthetic Rate E. Conclusions 1. What Controls Fluxes in Photosynthesis? 2. What Makes a Protein Important for Regulation? Acknowledgments References

Mark Stitt 170 171 171 171 171 172 173 173 173 173 174 174 174 175 175 175 175 177 177 178 179 180 181 182 182 182 183 183

Summary The regulation of photosynthetic carbon metabolism is reviewed, drawing a distinction between regulatability and regulatory capacity. The former describes whether the properties of an enzyme allow its activity to be changed in vivo by naturally occurring mechanisms. The latter describes whether a change in the activity of an enzyme will lead to a change in flux through the pathway. The regulatory properties of the enzymes of photosynthetic carbon metabolism are first reviewed. I argue that they allow us to understand how a coordinated change in flux is achieved in response to changes in the external conditions, or the physiological status of the plant. This is illustrated by the responses of the Calvin cycle to irradiance, and by the regulation of partitioning and its coordination with the operation of the Calvin cycle. Important areas requiring more research are indicated, in particular the fine regulation of Rubisco and its activation state, regulation of sucrose phosphate synthase, and gene expression in mature leaves in response to external and physiological signals. I then discuss how regulatory capacity can be assessed by measuring flux control coefficients. The flux control coefficients for the processes involved in entry into the leaf, Rubisco, several Calvin cycle enzymes, four enzymes in the pathway of starch synthesis, and several enzymes for sucrose synthesis are estimated. Control is usually shared, and the distribution depends on short-term and long-term conditions. It is pointed out that acclimation can lead to an adjustment in the balance between different processes, which allows a leaf to escape from a one-sided limitation. Unexpectedly, many enzymes that catalyze readily reversible reactions develop substantial flux control coefficients when they are decreased two- to three-fold below wildtype levels. On the other hand, many enzymes that catalyze irreversible reactions and possess regulatory properties do not exhibit control in the wildtype, and will not until a large decrease in enzymic amount is engineered.

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The consequences for our understanding of pathway structure and regulation are then discussed, in particular the influence of the kinetic properties of an enzyme on its flux control coefficients. It is shown by theoretical analysis and experimental examples that the flux control coefficients will usually increase when the sensitivity of an enzyme to regulatory effectors is decreased. As a consequence, most enzymes with high regulatability actually have a low regulatory capacity! Their sophisticated regulatory properties serve to coordinate their activity with the rest of the pathway, and allow their activity to respond to changes initiated elsewhere, but they do not themselves provide a site from which flux can be altered. Enzymes with a high regulatory capacity possess kinetic properties which allow them to be modulated via effectors that do not interact strongly with the rest of the pathway. Processes with no regulatability also sometimes exert control. It is proposed that selection to avoid the consequences of metabolic imbalance and inefficient allocation tend to prevent individual processes from exerting total control.

I. Introduction The aim of the chapter is to provide abroad overview of the regulation of photosynthetic carbon metabolism. The term ‘regulation’ is used in many different ways. I shall follow the suggestion of Hofmeyr and Cornish-Bowden (1991) and distinguish between the regulatability and the regulatory capacity of an enzyme. With respect to the regulatability of an enzyme, we need to know whether the enzyme protein exhibits regulatory properties which will allow its activity to be altered in vivo by naturally occurring mechanisms? ‘Fine’ regulatory properties include sigmoidal substrate saturation kinetics, effectors which interact with the catalytic site, allosteric effectors, and reversible covalent protein modification. ‘Coarse’ regulatability is also possible; in this case, expression of the encoding gene or genes is modulated to allow marked or selective changes in the amount of the protein present in the cell. With respect to the regulatory capacity of an enzyme, we need to know whether a local change in the amount and/or activity of a particular enzyme (produced by one or more of the above mechanisms) actually leads to a change in the flux through the pathway or system under consideration?

As I will stress in this review, in many cases, the regulatory properties of one enzyme serve to coordinate its activity with the activity of the other enzymes in the pathway. In this case, the enzyme will possess high regulatability, but a low regulatory capacity. In some cases, regulation will serve to alter flux through a pathway. This will only occur if the regulatory capacity is also high. As discussed in more detail later, regulatability and regulatory capacity can be formalised as elasticity coefficients, and the flux control coefficient, of an enzyme, respectively (Kacser and Porteous, 1987; Hofmeyr and Cornish-Bowden, 1991; Fell, 1992). This will sometimes require use of a suitably modified form of these coefficients (see below). This distinction between regulatability and regulatory capacity is important when interpreting the results of experiments on stress, adaptation or acclimation. Due to the highly integrated nature of metabolism, many of the changes in enzyme activities or metabolite levels which occur are likely to be indirect changes, rather than primary events. Even more important: just because a parameter changes, it is not necessarily determining the response of the system. I will first discuss some general aspects of carbon

Abbreviations: ABA – abscisic acid; AGPase – ADP-glucose pyrophosphorylase; BE – branching enzyme; C – flux control coefficient; – flux control coefficient for physical resistance to diffusion through aqueous phase of leaf; – flux control coefficient for boundary layer conductance to – intercellular concentration of – flux control coefficient of Rubisco for photosynthesis; – flux control coefficient for stomatal conductance to CA – carboxyarabatinol; CABP – carboxyarabatinol 1,5bisphosphate; CAP – carboxyarabatinol 1 -phosphate; CAPase – carboxyarabatinol 1 -phosphatase; cFBPase – cytosolic form of fructose 1,6-phosphatase; cPGI – cytosoloic form of phosphoglucose isomerase; F6P – fructose 6-phosphate; FBP – fructose 1,6-bisphosphate; G6P – glucose 6-phosphate; KABP – ketoarabitinol 1,5-bisphosphate; MDH – malate dehydrogenase; NADP-GAPDH – NADPglyceraldehyde 3-phosphate dehydrogenase; Pi – inorganic phosphate; pFBPase – plastid form of fructose 1,6-phosphatase; PG – 2phosphoglycollate; PGA – 3-phosphoglyceric acid; PGI – phosphoglucose isomerase; pPGI – plastid form of phosphoglucose isomerase; pPGM –plastid form of phosphoglucose mutase;PPi–pyrophosphate;PRK–phosphoribulokinase;Rubisco–ribulose 1,5-bisphosphate carboxylase-oxygenase; RuBP – ribulose 1,5-bisphosphate; Ru5P – ribulose 5-phosphate; S7P – seduheptulose 7-phosphate; SBP – seduheptulose 1,7-bisphosphate; SBPase – seduheptulose 1,7-bisphosphatase; SPS – sucrose phosphate synthase; TPT – triosephosphate phosphate translocator; XuBP – xylulose 1,5-bisphosphate; – elasticity coefficient.

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metabolism, and describe the regulatory properties of photosynthetic enzymes. I will then discuss how we can identify enzymes with a high regulatory capacity. By combining this information, we learn which enzymes have high regulatability and a high regulatory capacity. We will see that this question is not trivial, because one often excludes the other! Excellent background reviews are provided by Woodrow and Berry (1988) and Portis (1992). A more detailed treatment of the regulation of sucrose synthesis can be found in Stitt (1991). II. Pathways and Metabolite Measurements: Evidence for Highly Coordinated Regulation of Many Reactions

The Calvin cycle is usually divided into three sections (Fig. 1): (i) carboxylation, (ii) the reduction of 3phosphoglyceric acid (PGA) to triose-phosphate and (iii) the regeneration of the acceptor ribulose 1,5-bisphosphate (RuBP). If the light-reactions and Calvin cycle are summed, the net reaction is the conversion of water and inorganic phosphate to triose-phosphates. The fourth step in photosynthetic carbon metabolism is therefore (iv) the conversion of triose-phosphate to a carbohydrate endproduct. This releases phosphate (Pi) which can be used for further ATP synthesis, and is a prerequisite for further fixation. Starch is synthesized in the plastid from fructose 6-phosphate, which is withdrawn from the regenerative phase of the Calvin cycle between the plastid fructose 1,6-bisphosphatase (pFBPase) and seduheptulose l,7-bisphosphatase(SBPase). Sucrose is synthesized in the cytosol, from triose-phosphates which exit the Calvin cycle before the pFBPase. There is a strict coupling of triose-phosphate export and phosphate uptake because it is facilitated by the triose-phosphate translocator (TPT) which catalyses a strict counter exchange (Heldt and Flügge, 1992). In the past, most studies of regulation have differentiated (Rolleston, 1972; Stitt, 1989a,b; 1990c) between (i) reactions which are readily reversible in vivo and are considered to be unimportant for regulation because the enzyme is ‘in excess’ and therefore has a low regulatory capacity and (ii) reactions which are displaced from equilibrium in vivo. The latter are assumed to have a high regulatory capacity. This is not necessarily true, as will be discussed later. Nevertheless, for this historical reason, most studies have concentrated on the

Mark Stitt irreversible steps catalysed by Rubisco, the plastid fructose 1,6-phosphatase (pFBPase), seduheptulose 1,7-bisphosphatase (SBPase), and phosphoribulokinase (PRK) in the Calvin cycle (Bassham and Krause, 1969); ADP-glucose pyrophosphorylase (AGPase) in starch synthesis (Bassham and Krause, 1969); and the cytosolic fructose 1,6-bisphosphatase (cFBPase), sucrose-phosphate synthase (SPS), sucrose phosphatase and pyrophosphate hydrolysis in sucrose synthesis in the cytosol (Gerhardt et al., 1987; Stitt et al., 1987a,b). Photosynthesis is a process which is driven by, and requires, external inputs. Leaves are, of necessity, exposed to large and unpredictable changes in the availability of light and water (and thence, and fluctuating temperature. They export carbohydrate to service growth and storage in sink organs. These growth processes are also very susceptible to the environmental conditions. We might therefore expect one cardinal feature of photosynthetic regulation, to be the ability to achieve coordinated changes of flux through these complex metabolic pathways in response to changes in the external conditions affecting the leaf, or changes in the demand in the rest of the plant. A striking feature of many measurements of steadystate metabolite levels in the leaf is their relative constancy across a wide range of conditions. The levels of ATP, NADPH and Calvin cycle intermediates do not change greatly across a wide range of light intensities and concentrations (Badger et al., 1984; Von Caemmerer and Edmondson, 1986; Dietz and Heber, 1984, 1986; Fischer et al., 1986). The same holds for the metabolites involved in sucrose synthesis in the cytosol (Stitt et al., 1980, 1983; Neuhaus et al., 1990), Changes in temperature (Dietz and Heber, 1986; Stitt and Große, 1988b) and water content (Quick et al., 1989) alter overall metabolite levels, but the relations between metabolites are less affected. Large changes of metabolites are found, of course, in extreme conditions. Dramatic changes also occur during transients (Prinsley et al., 1986; Sharkey et al., 1986; Stitt and Grosse, 1988a; Sage, 1990; Pearcy, 1990; Chapter 13) because enzymes are out of step due to different regulation mechanisms having different time constants. These unbalanced states are extremely useful as experimental tools to analyze regulation mechanisms in photosynthesis. These extreme cases, however, do not invalidate the conclusion that a balance is maintained between different enzymes and processes in the steady-state

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over a quite wide range of conditions. In the next section, I will therefore describe the regulatory properties of the enzymes of each pathway in terms of grouped or common responses, rather than dealing with each enzyme seperately. The aim is to show how this coordinated response of metabolism is brought about. Rubisco is dealt with seperately, because our current understanding of its regulatory properties does not yet allow it to be fully integrated with the remainder of photosynthetic metabolism.

III. Regulatory Properties of Calvin Cycle Enzymes

A. Light Drives Photosynthesis by Activating Several Calvin Cycle Enzymes via Thioredoxin and Changes of Stromal pH and Magnesium Illumination of isolated chloroplasts (Werdan et al.,

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1975) or leaves (Heineke and Heldt, 1988; Yin et al., 1990) leads to alkalization and increased free in the stroma (Portis, 1981). Several enzymes in the Calvin cycle are strongly activated by increasing pH and including pFBPase (Baier and Latzko, 1975; Gardemann et al., 1986), SBPase (Woodrow et al., 1984) and Rubisco (Andrews and Lorimer, 1987). The pH and sensitivity of pFBPase is largely due to an increase in the proportion of the total fructose 1,6 bisphosphate (FBP) pool present as which is the real substrate (Gardemann et al., 1986). An analogous explanation is probable for SBPase. PRK also becomes strongly sensitive to pH in the presence of inhibitors like PGA and FBP. These inhibitors contain phosphate groups with a pK in the physiological range, and only the protonated form is inhibitory (Gardemann et al., 1986). Illumination of chloroplasts (Laing et al., 1981; Leegood, 1985a) or leaves (Wirtz et al., 1982; Leegood, 1985) also leads within minutes to a

156 dramatic increase in the activity of several enzymes, provided the extracts are prepared quickly and assayed in suitable conditions. Activated enzymes include pFBPase, SBPase, PRK and NADP-glyceraldehyde3-phosphate dehydrogenase (NADP-GAPDH) in the Calvin cycle. An enzyme termed ferredoxin:thioredoxin oxidoreductase transfers reducing equivalents from ferredoxin to a small soluble protein called thioredoxin. Thioredoxin then modifies cysteine groups on the target protein (Buchanan, 1984). Further enzymes outside the Calvin cycle are also activated by thioredoxin, including NADP-malate dehydrogenase (Scheibe, 1991), and the synthase in the thylakoids (Mills et al., 1980), whereas the plastid glucose 6-phosphate dehydrogenase is deactivated by thioredoxin (Scheibe, 1990, 1991). The precise affect on the kinetic properties depends on the target enzyme. For pFBPase and SBPase, thioredoxin activation leads to an increased substrate affinity (Laing et al., 1981; Leegood, 1985). As a consequence, the increase in real substrate concentration due to rising pH and in the light (see above) reinforces the activation by thioredoxin. For PRK, the increase involves a change in whereas the kinetic properties are unaltered (Laing et al., 1981; Scheibe, 1991).

B. Substrates Modulate Thioredoxin Activation to Allow a Feedforward Coordination of Enzymes in the Calvin Cycle The mechanisms discussed in the last section allow the Calvin cycle to be activated in the light, but do not, in themselves, guarantee that the various reactions run at synchronous rates. Since the half-time of metabolites in the Calvin cycle is of the order of 0.1–3 s (Stitt et al., 1980) a small discrepancy in the activity of two enzymes will result in a large accumulation of the interfering intermediates, unless it is rapidly corrected. The resulting sequestration of Pi in these metabolic pools would lead to inhibition of photosynthesis, analogous to that seen when mannose or glycerol are fed to trap Pi as mannose 6phosphate (Walker and Sivak, 1986; Stitt and Quick, 1989). Studies of the in vitro activation of FBPase and SBPase revealed that the thioredoxin or (as an analog) dithiothreitol-dependent activation is accelerated when FBP or SBP are included in the activation mix (Wolusiuk et al., 1980; Woodrow et al., 1984). The

Mark Stitt physiological relevance of these observations was documented by experiments showing that the rate and extent of the thioredoxin-dependent activation can be varied by adding triose-phosphates or Pi to isolated chloroplasts to alter the stromal pool of FBP and SBP (Laing et al., 1981; Leegood, 1985). The increased activation occurs because the substrate modifies the mid-redox point potential of the cysteine groups on the enzymes, thus shifting the extent to which they can be reduced at a given reduction state in the chloroplast. For the pFBPase, the mid-redox potential is shifted from 1.34 to 0.04V by adding FBP (R. Scheibe, personal communication). Activation of PRK is promoted by rising ATP, which shifts the mid-redox potential from 0.58 to 0.31 V This modulation of thioredoxin activation by substrate levels can be viewed as a feedforward regulation, which allows a sharp increase in enzyme activation and activity when the substrate concentration rises, and restricts activity when substrate falls (Fig. 2). The significance of the PRK activation by ATP rather than ribulose 5-phosphate (Ru5P) will be discussed later.

C. Product-Inhibition and Feedback Inhibition Provide Additional Coordination of Calvin Cycle Fluxes Each of these enzymes is also inhibited by its products, or by intermediates occurring later in the Calvin cycle. The pFBPase is competitively inhibited by physiological levels of fructose 6-phosphate (F6P) leading to a switch from hyperbolic to sigmoidal substrate saturation kinetics (Gardemann et al., 1986). SBPase is non-competitively inhibited by sedoheptulose 7-phosphate (S7P) (Schimkat et al., 1990). PRK is inhibited by physiological concentrations of both products. Ribulose 1,5-bisphosphate (RuBP) acts competitively to Ru5P, and ADP acts competitively to ATP. It is also inhibited by PGA acting competitively to Ru5P (Gardemann et al., 1983). These feedback inhibition loops complement the feedforward loops described in the last subsection, and act to counteract imbalances in the flows of carbon and esterified phosphate around the cycle. The feedback inhibition of PRK by ADP and Ru5P serves an additional important function, because it coordinates the use of ATP at PRK and phosphoglycerate-kinase. Overconsumption of ATP by the high affinity irreversible PRK reaction would result

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158 in an accumulation of PGA, because phosphoglycerate-kinase has a low affinity for ATP (Walker and Sivak, 1986; Fig. 2).

D. Coordination of ATP and NADPH Production Photosynthesis requires formation of NADPH and ATP in the required stoichiometry. The regulation of poising and photosynthetic electron transport lies outside the scope of this review (see Chapter 3). However, I will consider an additional mechanism in carbon metabolism which acts, to balance ATP and NADPH formation. A shortage of ATP will lead to an accumulation of NADPH. This switches on a series of events, via a mechanism which shares a common principle with the feedforward modulation of the Calvin cycle enzymes (Scheibe, 1990, 1991). The NADP-malate dehydrogenase (NADP-MDH) in the chloroplast stroma is inactive in darkness or low light, and the reaction is strongly displaced from equilibrium (this is unusual for malate dehydrogenases, which are usually freely reversible in vivo). The enzyme is inactive because high inhibits activation of NADP-MDH by thioredoxin, shifting the mid-redox potential of the target cysteine from 0.21 V to 0.61 V (R. Scheibe, personal communication). When NADPH accumulates and declines, NADPMDH is activated (Rebeille and Hatch, 1986; Scheibe et al., 1990, 1991). This allows NADPH oxidation in the stroma, which in turn will allow more linear electron transport and ATP synthesis in the thylakoids (Scheibe, 1990, 1991). The malate is exported to the cytosol via the dicarboxylate carrier (Heldt and Flügge, 1992) and is oxidized outside the chloroplast by NAD-MDH. Oxaloacetate then reenters the chloroplast via a specific high affinity oxaloacetate carrier (Hatch et al., 1984). The rate of redox transfer over this cycle is controlled by the activation of the NADP-MDH (Heineke et al., 1991). The modulated ‘malate valve’ provided by NADPH-MDH (Scheibe, 1991) also has important consequences for extrachloroplastic metabolism, because it is a source of NADH and, potentially, ATP (Heldt and Flügge, 1992; Fig. 3). NADH is required to drive nitrate reduction in the cytosol. Some of the malate may enter the peroxisomes, instead of being oxidized by malate dehydrogenase in the cytosol. Oxidation via NAD-MDH in the peroxisome provides NADH for hydroxypyruvate reductase, in the photorespiratory pathway (Heupel et al., 1991). This,

Mark Stitt in turn, will allow some of the NADH formed during glycine decarboxylation to be retained in the mitochondria, rather than shuttling it to the peroxisome to support hydroxypyruvate reduction. As a result, NADH can be oxidized in the mitochondria to provide additional ATP. Clear evidence that mitochondria provide ATP for extrachloroplastic processes has been provided in experiments with oligomycin which is a specific inhibitor of the mitochondrial (Krömer and Heldt, 1991 a,b). The ATP can be used for sucrose synthesis, for transport processes, and could even be used together with NADH to allow some of the PGA to be reduced in the cytosol, instead of in the chloroplast.

E. Rubisco 1. The Unique Importance of Rubisco Rubisco catalyses the key reaction in which RuBP acts as an acceptor for forming 2 molecules of PGA. The competing reaction with oxygen leads to formation of 2-phosphoglycollate, which has to be recycled via the photorespiratory pathway. Rubisco has a low turnover rate (Woodrow and Berry, 1988). Due to the relatively high for < (27 Pa) and the competing reaction with (Jordan and Ogren, 1984), Rubisco is limited in ambient conditions (Andrews and Lorimer, 1987; Woodrow and Berry, 1988). The relative inefficiency of the enzyme is partly counterbalanced by the extraordinarily high amount, accounting for up to 30% of the protein in the leaf and reaching an active site concentration in the stroma of 4 mM or more (Woodrow and Berry, 1988). This can be measured by determination of protein (Evans, 1989) and by binding of labeled carboxyarabitinol 1,5bisphosphate (CABP), which is a very tight binding reaction intermediate analog (Yokota and Canvin, 1985; Hall et al., 1981). The relatively poor affinity for also entails a high stomatal conductance during rapid photosynthesis, resulting in increased transpiration. Rubisco therefore represents a vital nexus linking carbon, nitrogen and water economy in the plants. Its regulation, and the way it is integrated with the remainder of carbon metabolism will be of crucial importance in determining how effectively this (unavoidably) large investment in a single protein can be exploited.

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2. Regulation via Ribulose 1,5-bisphosphate Availability Rubisco has a high affinity for RuBP (Woodrow and Berry, 1988). Leaves contain a large pool of RuBP, and comparison with the concentration of CABP-binding sites indicates that the active sites of Rubisco are likely to be saturated or near-saturated with RuBP in most conditions, except low light and during transients (Badger et al., 1984; Mott et al., 1984; Dietz and Heber, 1984, 1986; Seeman and Sharkey, 1986; von Caemmerer and Edmondson, 1986; Sage et al., 1988,1989). This is a key element in the model of photosynthesis developed by Farquhar and colleagues (von Caemmerer and Farquhar, 1981; see below). There has been considerable debate over precisely how much RuBP is required, in order to occupy all the catalytic sites. Based on a theoretical consideration of the ionization states of RuBP, von Caemmerer and Edmondson (1985) calculated that 1.5–2 RuBP per site is needed. This is broadly consistent with ratios between 1–2 found in saturating irradiances (see above references, and also Sharkey, 1990). However, other factors can also influence the relation between leaf RuBP content and active site saturation (discussed below), especially competition with PGA, changes

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in activation state and the tight binding of RuBP to the inactive (decarbamylated) catalytic site. A recent study of the relation between RuBP levels, Rubisco active sites and photosynthetic rates in a series of antisense plants with decreased expression of NADPGAPDH indicates that higher RuBP may indeed be needed to achieve full saturation. Compared to the wildtype, photosynthesis was inhibited once the RuBP/CABP-binding site ratio fell below 2–3 (Price et al., 1995).

3. Competition of Other Phosphorylated Intermediates with Ribulose 1,5-bisphosphate A large range of Calvin cycle intermediates including FBP, NADPH, 6-phosphogluconate, Pi and PGA (Badger and Lorimer, 1981) bind to the active site of Rubisco. However, only PGA is likely to be of physiological importance during photosynthesis. Although the for PGA (0.9 mM) is considerably higher than the for RuBP extremely high concentrations of PGA can develop in leaves (10–20 mM; Mott et al., 1984; Von Caemmerer and Edmondson, 1986; Sharkey et al., 1986; Stitt et al., 1988). Woodrow and Berry (1988) used a rate equation to calculate that physiological concentrations of PGA could significantly decrease Rubisco activity

160 in some conditions in vivo. A role for PGA inhibition was proposed during high-low irradiance transitions (Prinsley et al., 1986), and during the middle part of a simulated natural day with a sinusoidal irradiance regime (Servaites et al., 1991).

4. Carbamylation (Activation) State Rubisco is unique, in that the catalytic site of the active enzyme form contains a carbamylated lysine to which a is complexed (termed ECM form) (Andrews and Lorimer, 1987). The enzyme can be carbamylated and decarbamylated in vivo, leading to a corresponding change in the ‘activation state’ (ECM/[E + ECM]). The activation state can be readily estimated by comparing activity measured in a fresh extract with that after pre-incubation at high pH, and to allow full carbamylation (Andrews and Lorimer, 1987), or by exploiting the different binding affinities of CABP for the carbamylated and decarbamylated enzyme (Butz and Sharkey, 1989). The activation state of Rubisco increases as irradiance increases. Activation often decreases in conditions where phosphorylated metabolites accumulate and Pi is depleted (Sharkey, 1990; Portis, 1992). The isolation and characterization of the rca mutant of Arabidopsis thaliana (Somerville et al., 1982; Salvucci, 1989; Portis, 1990) revealed that a protein called Rubisco activase is needed to enable these changes. Activase is thought to operate by decreasing the affinity of the decarbamylated enzyme for various inhibitory sugar bisphosphates. These would otherwise inhibit carbamylation or catalysis. Release of the bound sugar-bisphosphate requires ATP hydrolysis (Wang and Portis, 1992). The inhibitory sugar-bisphosphates include RuBP itself which binds tightly to the decarbamylated form of Rubisco (Portis, 1992). A significant correlation was found between the estimated number of inactive (decarbamylated) Rubisco catalytic sites, and the amount of RuBP tightly bound to Rubisco as ERuBP complex (Brooks and Portis, 1988; Cardon and Mott, 1989). Further potentially important tight-binding inhibitors are formed via a low frequency catalytic event. Xylulose 1,5-bisphosphate (XuBP) is formed at a frequency of about 1/100 due to a stereochemically incorrect readdition of a proton to the third carbon of the ene-diolate intermediate (Edmondson et al., 1990a,b). This side reaction

Mark Stitt explains the long-standing problem of the rapid ‘dieoff which occurs within minutes when Rubisco is assayed in vitro. A similar problem may also arise due to readdition at carbon-2, leading to formation of 3-ketoarabitinol 1,5-bisphosphate (KABP; Edmondson et al., 1990a). In vitro studies show that activase protects against this ‘die-off’, probably by releasing the inhibitor (Robinson and Portis, 1989). The subsequent hydrolysis of XuBP occurs via a separate and specific phosphatase (Larsen and Portis, 1994). Elegant evidence for the importance of activase in preventing a gradual ‘die-off’ of Rubisco in vivo due to binding of RuBP, XuBP or KABP has been provided by studies on transgenic tobacco plants with 5–10 fold decreased expression of Rubisco activase (Mate et al., 1993). Activation in the dark was actually higher than in the wildtype. However, after illumination, the activation state in the transformants decreased gradually over 15–30 min, instead of increasing as in the wildtype. What is the role of the changes in activation state? Sage (1990) has proposed that Rubisco activation is regulated to achieve a balance between RuBP formation and consumption, such that the active Rubisco sites (ECM) are near-saturated by RuBP. There is considerable empirical evidence for this idea (Sage et al., 1988; Sharkey, 1989). However, the functional advantage is not clear. It could be speculated that it may lie in avoiding sequestration of Pi as E-RuBP. Direct proof is not available, but could be provided by detailed analysis of plants with a small decrease in activase expression (Mate et al., 1993). It is also not clear how this is achieved. In order to fully understand how Rubisco is integrated into the remainder of carbon metabolism, we need to know more about the mechanisms which regulate Rubisco activase. One mechanism could involve modulation of activase by the ATP/ADP ratio. Activase operation requires ATP hydrolysis (Robinson and Portis, 1989a; Wang and Portis, 1992), and is inhibited by ADP in vitro. A reasonable correlation between Rubisco activation and the ATP/ ADP ratio is found in vivo in some conditions. Activation increases dramatically when low concentrations of methyl viologen are added to allow more linear electron transport and ATP synthesis (Brooks et al., 1988; Neuhaus and Stitt, 1989). The ATP/ADP ratio and Rubisco activation both decline when free Pi is depleted in isolated chloroplasts (Heldt et al., 1978) and leaves (Sharkey et al., 1986; Sharkey, 1990). However, we cannot exclude the possibility

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that these correlations are fortuitous, and other mechanisms are also needed. Regulation of activase by ATP/ADP ratios cannot explain why Rubisco activates as irradiance increases, because the stromal ATP/ADP ratio often decreases as irradiance increases (Dietz and Heber, 1986; Brooks et al., 1988). Also, although there was an inverse relation between ATP/ADP ratios and Rubisco activation in wildtype and antisense rbcS tobacco plants grown in moderate light (Quick et al., 1991), this relation was not found in high light (Hudson et al., 1992; Masle et al., 1993; M. Stitt, unpublished). Portis (1992) has suggested that there may be a second mechanism to modulate activase, linked to the thylakoid gradient. Detailed analysis of induction in leaves by Mott, Woodrow and colleagues has provided evidence that activase is inactivated in the dark or low light. A reversal of this inactivation is a precondition for a subsequent activation of Rubisco (Jackson et al., 1991; Woodrow and Mott, 1992; Mott and Woodrow, 1993). The proposed mechanism via protein phosphorylation of the activase is controversial at present.

5. Carboxyarabitinol 1-Phosphate Leaves of some species contain substantial levels of an unusual sugar phosphate, carboxyarabatinol 1phosphate (CAP) (Gutteridge et al., 1986). This is a closely related structure to 2-carboxy-3-ketoarabinitol 1,5-bisphosphate, which is the transition state analog of the carboxylation reaction (Andrews and Lorimer, 1987). CAP binds with high affinity to the ECM form of Rubisco and depresses (Berry et al., 1987). Leaves of many species including Phaseolus, tobacco, soybean, and Alocasia contain enough CAP to titrate out most or all of the Rubisco active sites in the leaf (Seemann, 1989; Seemann et al., 1990). Other species, e.g. spinach, wheat, barley, pea, Arabidopsis and Chenopodium contain negligible CAP. The reason for this species variation is unclear. In species which contain large amounts of CAP, the concentration increases in the dark, and decreases in the light (Salvucci, 1989; Kobza and Seemann, 1989a; Seemann et al., 1990; Sage et al., 1993). There is evidence that this could involve a cycle between CAP and the free sugar, carboxyarabitinol (CA). Bean leaves contain a specific CAPphosphatase (Gutteridge and Julien, 1989), which is stimulated by RuBP, FBP and

161 NADPH, and inhibited by ATP and Pi (Salvucci and Holbrook, 1989; Holbrook and Salvucci, 1991). Leaves of many species contain considerable amounts of the free sugar carboxyarabitinol (CA), in the light as well as the dark (Moore et al, 1992). Labeling experiments indicate that CA can act as a precursor for CAP, and that CAP is converted back to CA after illumination (Moore and Seemann, 1992). The enzyme(s) responsible for CAP synthesis has not yet been identified or characterized. The pathway for net production of (CA + CAP) has also not yet been characterized. Moore and Seemann (1990) found no evidence for labeling from newly fixed photosynthate, whereas up to 8% the fixed has been detected in CAP in some conditions (M.A.J. Parry and S. Gutteridge, personal communication). The CAP binding constant on Rubisco (32 nM) is 1000-fold higher than the of CAP-phosphatase Additional mechanisms are therefore needed to release CAP from Rubisco in the light, otherwise it will be effectively inaccessible to CAPase. Activase releases CAP from the ECM-CAP complex in vitro (Robinson and Portis, 1988). Direct evidence for a similar mechanism in vivo is provided by the demonstration that CAP declines much more slowly following illumination in antisense tobacco leaves with decreased activase (Mate et al., 1993). What is the role of CAP? It is obviously not universally essential for the light regulation of Rubisco. Its significance in species which contain CAP is also controversial (Portis, 1992). Seemann et al. (1990) have shown that the CAP content depends on the steady-state irradiance level at which leaves are illuminated. This means that, even though high irradiance leads to a rapid removal of CAP (see above) in a natural regime there is a gradual decline of CAP during the morning hours. This contributes to the gradual rise in Rubisco activity in the morning (Kobza and Seemann, 1989a; Servaites et al., 1991; Sage et al., 1993). After a period of high illumination, CAP is low, and requires several hours to increase again when the irradiance is decreased (Sage et al., 1993). At dusk, CAP therefore plays little or no role in modulating Rubisco. Regulation by carbamylation makes a larger contribution to regulation of Rubisco when CAP is low (Sage, 1990; Sage et al., 1993). It is unclear what advantages or disadvantages result from regulation via CAP, as compared to carbamylation, except that the former is less effective when irradiance is decreased (see above).

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F. Coordination of the Calvin Cycle and Photorespiration The rate of 2-phosphoglycollate (PG) formation is about one tenth of the rate of PGA formation during photosynthesis at 20 °C and ambient It will be higher if the temperature rises, or intercellular concentration falls due to stomatal closure. The lethal phenotype of photorespiration pathwaydeficient Arabidopsis mutants obtained by Somerville and colleagues (Somerville, 1986) graphically illustrates the need for mechanisms which can modulate the rate of RuBP regeneration and consumption, in response to possible restrictions in the photorespiratory pathway. There have been few systematic studies of this problem, but two possible mechanisms have been proposed. Firstly, the activation of Rubisco which normally occurs in isolated chloroplasts can be potently inhibited by glyoxalate (apparent (Campbell and Ogren, 1990). Analysis of various photorespiration mutants indicates that accumulation of glyoxylate could be responsible for the suppression of Rubisco activation observed in these mutants (Chastain and Ogren, 1985, 1989). Portis( 1992) has suggested that glyoxylate might interact with the (as yet unidentified) light-dependent activase-activating factor. Secondly, Schimkat et al. (1990) have reported that glycerate is a powerful non-competitive inhibitor of pFBPase and SBPase. This explains earlier observations that glycerate inhibits pFBPase and SBPase activity in isolated chloroplasts. Halfmaximal inhibition occurs at about 0.2 mM glycerate, resembling the for glycerate uptake into the chloroplast (Robinson, 1982) and glycerate kinase (Husic et al., 1987).

G. Regulatory Properties of Enzymes of Sucrose and Starch Synthesis There are two overriding reasons why endproduct synthesis must be regulated. Firstly, to coordinate endproduct synthesis with the rate of photosynthesis. If triose phosphates are withdrawn too slowly, photosynthesis will be inhibited because phosphorylated intermediates accumulate in the Calvin cycle and Pi is depleted. If triose-phosphates are withdrawn too quickly, photosynthesis is inhibited because the Calvin cycle intermediates are depleted, and RuBP cannot be regenerated. Secondly, to allow

Mark Stitt the partitioning of photosynthate between sucrose, starch and amino acid synthesis to be adjusted, in response to the physiological state of the leaf or plant. I have reviewed this topic previously (Stitt et al., 1987a; Stitt 1990a,b, 1991).

1. Feedforward Regulation Coordinates Sucrose Synthesis and Fixation Rising rates of photosynthesis lead to (i) a stimulation of the cytosolic FBPase caused by a decrease in the concentration of the inhibitory regulator metabolite fructose 2,6-bisphosphate (Stitt, 1990a) and (ii) an activation of SPS caused by dephosphorylation of the SPS-protein (Huber and Huber, 1992a, 1992b) (Fig. 4A). In both cases, the result is an increased substrate affinity. Fructose 2,6-bisphosphate is a competitive inhibitor of the cytosolic FBPase (Stitt, 1990a), and activation of SPS leads to changes in the kinetic properties (Siegl and Stitt, 1990). Both enzymes have cooperative kinetics in vivo. The cytosolic FBPase has sigmoidal substrate saturation kinetics in the presence of fructose 2,6-bisphosphate (Stitt and Heldt, 1985b). SPS shows a sigmoidal response to rising F6P in vivo because the accompanying increase of glucose 6-phosphate (G6P) activates the enzyme (Doehlert and Huber, 1984). An increase in their substrate affinity therefore provides a very effective mechanism to alter the rate of sucrose synthesis, without requiring large changes in the steady state metabolite concentration (Stitt et al., 1983). Additional amplification is introduced because fructose 2,6-bisphosphate concentration and SPS phosphorylation state are regulated by a cycle. Fructose 2,6-bisphosphate is synthesized via fructose 2,6-bisphosphate-kinase and degraded via fructose 2,6-bisphosphate-phosphatase (Stitt, 1990a). As the rate of photosynthesis increases, fructose 2,6bisphosphate decreases because fructose 2,6bisphosphate-kinase is inhibited by a rising PGA/Pi ratio, which is a good indication of an imbalance between fixation and Pi recycling (Walker and Sivak, 1986). Important advances are being made in elucidating how the phosphorylation of SPS is regulated (Huber and Huber, 1992a). Structural gene sequences are available for maize (Worrell et al., 1991), spinach (Klein et al., 1993; Sonnewald et al., 1993) and potato SPS (U. Sonnewald unpublished). Peptide

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mapping (Huber and Huber, 1992b) followed by sequencing (McMichael et al., 1993) has identified the major phosphorylation site involved in the lightdark regulation of SPS. Other phosphorylation sites also exist (Huber and Huber, 1992a,b), and might be implicated in further modes of regulation (Huber and Huber, 1990). SPS phosphorylation is regulated at least two levels during dark-light transitions. One level involves

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modulation of SPS-kinase by rising G6P (Huber and Huber, 1992a) and stimulation of SPS-phosphatase by falling Pi (Weiner et al., 1992). This direct modulation by metabolites will allow SPS activation to be increased in response to an increased flux at the cytosolic FBPase. However, this cannot be the only mechanism because these metabolites do not always change during a dark-light transition (Stitt et al., 1980; Gerhardt et al., 1987). A second level of

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regulation involves modification of SPS-phosphatase. SPS-phosphatase activity shows a diurnal rhythm (Weiner et al., 1992) which is due to a shift in the Pisensitivity of the enzyme (Weiner et al., 1993). The changes which occur after illumination can be simulated by feeding mannose in the dark and are blocked by cycloheximide (Weiner et al., 1992, 1993). These results indicate that SPS-phosphatase can be modified via a protein-synthesis dependent process, and that this change is triggered by a factor related to the phosphate status, rather than light per se. SPSphosphatase is a member of the PP2A family (Huber and Huber, 1992a). Weiner et al. (1993) point out that PP2A usually occurs as a heterotrimer in mammalian systems, and are modulated via regulatory subunits.

2. A ‘Threshold’ Triose-Phosphate Concentration Reconciles Sucrose Synthesis with Calvin Cycle Turnover The relationship between the triose-phosphate concentration and the rate of sucrose synthesis has been described in an empirical model (Stitt and Heldt, 1985b; Stitt et al., 1987b). The model shows good agreement between the modeled response based on the in vitro properties of the cytosolic FBPase, and the measured relationship between metabolites, fructose 2,6-bisphosphate and fluxes in spinach leaves (Fig. 5). A central feature of the model is that sucrose synthesis is inhibited below a critical ‘threshold’ concentration of triose-phosphate. This will prevent sucrose synthesis from depleting the triose-phosphate pool, and inhibiting Calvin cycle turnover. The triosephosphate concentration must remain high enough to (i) support the multiple reactions of aldolase and transketolase and (ii) generate an adequate stromal FBP concentration to allow activation of the stromal FBPase (see Section IIIB). As pointed out by Woodrow et al. (1986), a correct relationship between the kinetic properties of the plastid and cytosolic FBPase will be important in allowing stable operation of the Calvin cycle. The ‘threshold’ for activation of sucrose synthesis can be changed, as an adaptation to special requirements. In maize leaves, sucrose is synthesized from triose-phosphates in the mesophyll. A very high concentration of triose phosphates must be maintained in these cells to drive diffusion back to the bundle sheath (Leegood, 1985b; Stitt and Heldt, 1985a). This is possible, because the substrate affinity

of the cytosolic FBPase from maize is much lower than in plants (Stitt and Heldt, 1985a), Another interesting change was found in leaves at low temperatures. Spinach and barley plants maintain high levels of stromal metabolites at low temperature (Dietz and Heber, 1986), possibly to compensate for the declining rate of catalysis. This can be explained by temperature dependent changes in the kinetic properties of the cytosolic FBPase (Stitt and Grosse, 1988b). When triose phosphates rise above this ‘threshold’ there is a strong activation of sucrose synthesis, in response to small increases in the triose-phosphate concentration. This will minimise the risk that

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phosphorylated intermediates accumulate to the point where photosynthesis becomes Pi-limited. An important detail might be noted here; the cytosolic and plastid FBPase will both be strongly stimulated in vivo when triose-phosphates rise (because FBP rises), but they show a qualitatively different response to a rising PGA/Pi ratio in vivo. This is an important difference, because a rising PGA/Pi ratio is a particularly sensitive indicator of an imbalance between fixation and endproduct synthesis (Stitt et al., 1987a; Sharkey, 1990). In these conditions there will be a strong stimulation of cytosolic FBPase activity, because the rising PGA/Pi ratio inhibits F6P-kinase, and decreases the FBP concentration (see last subsection). In contrast, a rising PGA/Pi ratio will not lead to a large change in the activity of the stromal FBPase. Any effect will be very indirect; for example, if stimulation of AGPase by a rising PGA/Pi ratio (see next section) were to lead to significant drop of the stromal F6P concentration.

3. Feedback Regulation Decreases Sucrose Synthesis and Increases Starch Synthesis When Sucrose Accumulates in a Leaf Accumulation of sucrose in the leaf, or feeding exogenous carbohydrate, leads to deactivation of SPS (Stitt et al., 1988) and an increase of fructose 2,6-bisphosphate (Stitt, 1990a). As a result, cytosolic metabolites rise slightly (Gerhardt et al., 1987), export from the chloroplast is decreased, and starch synthesis increases (Fig. 4B). The mechanism(s) which decrease SPS activation have not yet been elucidated. It appears unlikely that a direct effect of sucrose on SPS, SPS-kinase or SPS-phosphatase is involved (Huber and Huber, 1990). Fructose 2,6-bisphosphate increases because F6P activates F6P-kinase and inhibits fructose 2,6-bisphosphate-phosphatase (Stitt, 1990a). This feedback loop has been studied using Clarkia mutants with decreased expression of cytosolic phosphoglucose isomerase (PGI). These mutants have an increased cytosolic F6P level, compared to the wildtype. As a result, fructose 2,6bisphosphate is increased and partitioning is changed. These mutants provide direct proof for the in vivo operation of this feedback loop, and have allowed its effectiveness to be quantitated (Kruckeberg et al., 1989; Neuhaus et al., 1989). Starch synthesis increases because ADP-glucose pyrophosphorylase (AGPase) is activated by PGA and inhibited by Pi (Preiss, 1988, 1991; Stitt, 1991).

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Definitive genetic evidence for the importance of these properties of the enzyme have been provided by detailed studies of structurally-altered AGPase mutants in Escherichia coli (Preiss and Romeo, 1994). Ball et al. (1992) have shown that a low-starch Chlamydomonas mutant is a result of altered allosteric properties of AGPase.

4. Allocation can be Altered Without Inhibiting Photosynthesis The rate of sucrose synthesis depends on the interaction between feedforward and feedback mechanisms. This has been studied in decreased cytosolic PGI Clarkia mutants (Neuhaus et al., 1989), and by comparing the saturation response of sucrose synthesis and starch synthesis in sucrose-depleted and sucrose-loaded spinach leaf discs (Neuhaus et al., 1990). The feedback loop operates very effectively to generate reciprocal changes of sucrose and starch synthesis at low to moderate rates of photosynthesis. In contrast, in high light and feedback regulation is overridden. This minimizes the risk that photosynthesis will be inhibited as a side-effect when partitioning is changed (Stitt, 1989a, b, 1991). The flexible response can be explained in terms of the properties of the cytosolic FBPase and changes in the concentrations of metabolites and their in vivo effectiveness as modulators of the enzyme (Neuhaus et al., 1989; Stitt, 1991).

5. Sucrose Synthesis is also Modulated in Response to Nitrogen Assimilation and Water Stress Pioneering studies by Turpin and coworkers in the alga Selanastrum minutum have shown that assimilation of nitrate or ammonia leads to dramatic changes in the regulation of carbon metabolism. Carbon fluxes are directed towards production of oxoglutarate, which is the acceptor for reduced nitrate in the GOGAT pathway (Turpin and Weger, 1990). An analogous though less marked change of flux occurs in leaves of higher plants. Supplying nitrate to detached wheat leaves leads to a partial inhibition of sucrose synthesis (Champigny et al., 1992; Champigny and Foyer, 1992), and increased synthesis of amino acids. Sucrose synthesis is decreased due to rising FBP and decreased SPS activation (Champigny et al., 1992). The interaction between sucrose and nitrogen metabolism is underlined by the finding that

166 nitrate reductase is regulated via protein phosphorylation (Kaiser et al., 1992; Huber et al., 1992a, b). The regulatory properties of the kinases and phosphatases are currently being investigated (Spill and Kaiser, 1994). Sucrose synthesis also increases in water-stressed leaves. Indirect evidence indicates this involves activation of SPS (Quick et al., 1989; Zrenner and Stitt, 1991). Starch degradation is accelerated in water stress (Fox and Geiger, 1986). Interestingly, water stress resulted in decreased cytosolic FBPase activity in soybean leaves (Cheikh and Brenner, 1992) and increased FBP in spinach leaves (Quick et al., 1989). These results indicate that carbon may exit the chloroplast via the hexose transporter, rather than the TPT, during water stress, which is similar (see below) to the dark.

6. Carbon can Move Out of the Chloroplast via the Hexose Transporter During Starch Degradation In isolated chloroplasts starch can be degraded (i) hydrolytically to hexose and exported via the hexose transporter, or (ii) phosphorolytically, followed by glycolysis to triose-phosphate and PGA which then leave the chloroplast via the TPT (Stitt, 1990b). Leaves contain high fructose 2,6-bisphosphate and low triose-phosphate in leaves in the dark (Stitt et al., 1987a). This indicates that starch phosphorolysis is likely to service respiration in the dark, because the cytosolic FBPase is inactive in these conditions. Detailed studies of diurnal changes in sugar beet leaves provide further indirect evidence that starchsucrose interconversions involve export of glucose in the dark (Servaites et al., 1989; Li et al., 1992). A similar conclusion was reached from studies of antisense TPT potato plants (Riesmeier et al., 1993; Heineke et al., 1994). These plants compensated for decreased TPT by accumulating more starch, and converting the starch to sucrose, presumably via the hexose transporter.

7. Further Factors could also Regulate Sucrose and Starch Metabolism So far I have tried to provide an overview of how sucrose synthesis is regulated. However, this has required some oversimplification, and I will now briefly indicate additional factors which could be important, and require more research. First,

Mark Stitt illumination decreases cytosolic calcium in Nitella (Miller and Sanders, 1987) and increases cytosolic pH (Yin et al., 1990). Both of these factors have a strong influence on cytosolic FBPase activity in vitro (Stitt et al., 1987a; Brauer et al., 1990). There is a need for studies of metabolism in experimental systems in which pH and cytosolic can be measured. Secondly, AMP is an extremely sensitive inhibitor of the cytosolic FBPase (Stitt 1990a). The significance is difficult to assess because the free cytosolic concentrations are so low that they are difficult to measure, and distinguish them from bound AMP. Third, there is considerable species variation in SPS (Huber et al., 1989). Fourth, there is evidence that hormones may directly or indirectly modify SPS and cytosolic FBPase activity (Cheikh and Brenner, 1992; Cheikh et al., 1992). Fifthly, there is evidence that circadian rhythms may be involved in regulating sucrose synthesis in soybean (Kerr and Huber, 1987) and starch degradation in sugar beet (Li et al., 1992). Sixthly, the activity of invertase and the recycling of hexose sugars may be important in the feedback regulation of sucrose synthesis (Foyer, 1988; Goldschmidt and Huber, 1992). On the other hand, some potential complications appear less likely in the light of recent publications. Although sucrosephosphatase catalyses a irreversible reaction, no evidence exists at present that it possesses physiologically significant regulatory properties (Krause and Stitt, 1992). Although feeding pyrophosphatase analogs leads to an inhibition of sucrose synthesis, enhanced removal of pyrophosphate by expressing pyrophosphatase from E. coli in the cytosol of tobacco or potato leaves did not significantly increase sucrose synthesis (Jelitto et al., 1992).

IV. Coarse Regulatability Research on the regulation of photosynthetic metabolism has been dominated by studies of fine regulatability. Many studies of protein levels and gene expression have been carried out during leaf development, but less in the mature leaf following a change in growth conditions. Coarse regulatability (regulation of gene expression, protein turnover) nevertheless plays an important role in photosynthesis, especially in adaptation to different conditions. I will illustrate this with four examples. Firstly, the balance between the amounts of different proteins in a leaf depends on the irradiance (Evans,

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1989; Chapter 11). Indeed the balance even changes in parallel with the changing light between the top and bottom of the leaf. This implies that selective light-dependent effects on gene expression are not only relevant for development, but also for adaptation. Secondly, the nitrogen supply alters the balance between different proteins in the leaf (Evans, 1989; Chapter 11). Significant shifts in the balance between Rubisco and other proteins occur in tobacco grown at different nitrogen or irradiance (Quick et al., 1992; Lauerer et al., 1993). This presumably reflects selective regulation of gene expression, or protein turnover. Thirdly, there is some evidence that key enzymes of sucrose and starch synthesis are regulated via gene expression in mature leaves. Expression studies reveal decreased SPS transcript in low light (Klein et al., 1993). Indirect evidence indicates that the increase of SPS activity in the cold in spinach leaves (Guy et al., 1992) and the diurnal changes of SPS activity in soybean leaves (Kerr and Huber, 1987) could involve changes in protein amount, rather than phosphorylation. There is also evidence for modulation of cytosolic FBPase expression by light and water stress (Harn and Daie, 1992; Khayat et al., 1993). Interestingly, the cytosolic FBPase decreases in water stress, which is consistent with studies of enzyme activities (see Section III.G.5). Expression of AGPase is also modulated, being induced by high carbohydrate (Müller-Röber et al., 1990). This occurs with in hours of inhibiting export by cooling the petiole (A. Krapp and M. Stitt, unpublished). It will be interesting to investigate whether gene expression is involved in the gradual adjustment of partitioning to daylength and irradiance intensity observed by Chatterton and coworkers (Chatterton and Silvius, 1980). As a fourth example, regulation of gene expression may be important for the ‘sink’ regulation of photosynthesis. Research into the feedback regulation of fixation has been dominated by studies of photosynthetic metabolism, in particular the hypothesis that Pi becomes limiting for photosynthesis in a leaf (reviewed in Stitt, 1991). Although this may occur in some conditions (Socias et al., 1993), in many cases the changes in metabolism are the opposite of those expected for Pi-limitation (reviewed in Stitt, 1991). An alternative explanation was revealed in studies of transgenic plants in which phloem export is decreased (von Schaewen et al., 1990; Stitt et al., 1991b). The leaves of these plants contain lower activities of photosynthetic proteins.

Two lines of evidence show that high carbohydrate leads to a rapid decrease of the expression of the genes which encode proteins needed for photosynthesis. First, Sheen (1990) has demonstrated using a maize bundle sheath protoplast transient expression system that sugars inhibit the expression of reporter gene constructs containing a promoter from a photosynthesis gene. Second, Kossmann et al. (1992) reported that feeding sucrose through the petiole to detached potato leaves leads to a large decrease of rbcS and plastid FBPase transcript. Krapp et al. (1993) cooled the petiole to decrease phloem export from leaves in potato and tobacco. Transcripts for rbcS and atpD decreased within a few hours. These changes could be mimicked by adding low concentrations of glucose to autotrophic cell suspension cultures (Krapp et al., 1993). Rubisco protein decreases more slowly with a half-time of 3–4 days in both experimental systems. Photosynthesis is not inhibited until about 4 days after girdling spinach leaves, by which time Rubisco has decreased by 30– 40%, the residual protein is fully activated, and RuBP and the concentration in the leaf both increase (A. Krapp and M. Stitt, unpublished data). These results indicate that the decreased expression is actually responsible for the inhibition of photosynthesis when carbohydrate accumulates in spinach leaves. The central role of gene expression in regulating inorganic nitrogen assimilation in higher plants is well appreciated (Vincentz et al., 1992) The techniques of molecular genetics and expression studies are now making ‘coarse’ control amenable to experimental analysis. The four examples discussed above illustrate the potential importance of ‘coarse’ regulatability forphotosynthetic carbon metabolism. Studies of adaptation and acclimation will, in the future, be able to investigate ‘fine’ and ‘coarse’ control in parallel.

V. How can the Regulatory Capacity of a Protein be Evaluated?

A. Comparison of Metabolites and Fluxes I now turn to the problem of how we can decide which enzymes have a high regulatory capacity; is an enzyme a master or a slave? This question cannot be answered by just measuring metabolites, enzyme activities or protein levels and directly comparing

168 them with fluxes. Many parameters will change in parallel, and correlation does not prove causality. Even if large changes of a parameter are found, this does not mean that it is important in determining flux; it is just as likely that the parameter is so unimportant that it is allowed to vary freely. The theory for interpretation of metabolite levels and fluxes developed by Rolleston (1972) is also not applicable; reciprocal changes of substrate concentration and flux reveal that the subsequent enzyme has high regulatability (e.g. its activity can increase even though its substrate concentration has decreased, because it has been activated by some further effector), but do not provide information about regulatory capacity.

B. Mechanistic Models Farquhar and coworkers (von Caemmerer and Farquhar, 1981; Farquhar and von Caemmerer, 1982) have developed an important model in which they make the simplifying assumption that photosynthesis is limited by Rubisco, or by the regeneration of RuBP. Rubisco was proposed to limit in saturating irradiance or low whereas regeneration of RuBP (due to low rate of electron transport restricting ATP and NADPH production) was proposed to limit in low irradiance (see Chapter 8). The equations in their model allowed the response of photosynthesis to the leaf internal concentration and to be used to test these predictions: (i) If electron transport is limiting, a decrease of or an increase of will have a relatively small effect on the net rate of fixation, which can be calculated from the ATP and NADPH requirements of photorespiration. (ii) If Rubisco itself limits photosynthesis leaf gas exchange should mirror the kinetic properties of Rubisco. There is good evidence that this is the case in low (see review by Woodrow and Berry, 1988). The model was subsequently developed to include possible effects of endproduct synthesis (Sharkey et al, 1986; Sharkey, 1989, 1990). Additional features of leaf behavior in gas exchange measurements were proposed which would allow this limitation to be identified (in particular, insensitivity to Several metabolite parameters were also identified which could be used to support the interpretation of the gas exchange kinetics, in particular the RuBP/Rubisco active site ratio (von Caemmerer and Edmondson,

Mark Stitt 1986), Rubisco activation state (Sharkey, 1990), and ATP, ADP and PGA levels (Stitt and Quick, 1989). This model is useful because it allows gas exchange to be interpreted in terms of biochemical events. However, it only allows a preliminary analysis in conditions where Rubisco is not limiting. Even in conditions where Rubisco is ‘limiting’, it is not clear that Rubisco is the only factor that limits the rate of photosynthesis. For example, it has been a matter of debate how much RuBP is required to fully saturate all the Rubisco binding sites (see Section II.E.2), and it is difficult to rigorously exclude the possibility that other metabolic factors in addition to substrate availability affect Rubisco activity in vivo. Further, it is possible that diffusion of through the aqueous phase of the leaf contributes to the limitation of photosynthesis (see below). Also the model takes as a starting point and therefore excludes a possible limitation by stomata (Farquhar and Sharkey, 1982).

C. Control Analysis 1. Regulatory Capacity is Revealed by the Flux Control Coefficient A direct way to decide whether an enzyme contributes to the control of flux through a pathway is to experimentally alter the activity of one enzyme slightly (e.g. by altering the amount) and then investigate the resulting change in flux through the whole pathway. The results can be used to calculate (Kacser and Burns, 1973; Kacser and Porteous, 1987) the flux control coefficient C, where

and E and J are the enzyme amount and pathway flux in the original state, dE is a small change in enzyme a amount, and dJ is the resulting change in pathway flux (see also Stitt, 1989a,b, 1990c; Woodrow and Mott, 1993). The flux control coefficient is a system property: it describes how flux through the whole pathway responds to a change in one enzyme. It is equivalent to the term ‘regulatory capacity’ (Hofmeyr and Cornish-Bowden, 1991). In the simplest case of a linear pathway, C can take a value between 0 (no control) and 1 (total limitation). Partial control is represented by an intermediate value. Since the flux control coefficients of all the enzymes in a pathway sum to unity (Kacser and

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Burns, 1973; Kacser and Porteous, 1987), the flux control coefficient provides a simple insight into the contribution of an enzyme to control of pathway flux in such pathways. In a branched pathway, or in a cycle, the flux control coefficients still sum to unity. However, interpretation of individual values becomes more complex because enzymes in one branch can have negative control coefficients for the flux in the other branch (e.g. mutants with decreased cytosolic PGI have a higher rate of starch synthesis than the wildtype; Kruckeberg et al., 1989). It is therefore theoretically possible for some enzymes to have a control coefficient greater than unity. This is not intuitively obvious, and illustrates how useful it is to interpret results within the framework of a rigorous theory. A striking example of a flux control coefficient above one is provided by studies of antisense TPT potato plants. An estimated 20–30% decrease of TPT resulted in a 30–60% inhibition of sucrose synthesis, and a 50–80% increase in starch synthesis (Riesmeier et al., 1993; Heineke et al., 1994).

2. Theory Predicts that Enzymes with High Regulatability Will Not Always Have High Flux Control Coefficients The value of the flux control coefficient of an enzyme does not depend simply on the enzymes properties, nor on the reversibility or irreversibility of the reaction, nor the presence or absence of regulatory properties. It depends on the relationship between the enzyme and the remainder of the enzymes in the pathway. To understand these interactions, we first need to define a second term: the elasticity coefficient (Kacser and Burns, 1973),

where and are the activity and effector of an enzyme, ds is a change in the effector concentration, and dv is the resulting change in enzyme activity. The elasticity coefficient appears to superficially resemble the control coefficient, but is in fact fundamentally different. It describes a local property (i.e. the response of the enzyme independently of the remainder of the pathway). The flux control coefficient emerges from the interaction between the elasticity coefficients. For the simplest case

169

where are the flux control coefficients, and and are the elasticity coefficients of the two enzymes E1 and E2 for a shared metabolite This relationship is termed the connectivity theorem (Kacser and Burns, 1973). It reveals that, perhaps unexpectedly, the more sensitive an enzyme is to a shared effector, the less control it will exert over pathway flux. These equations can readily be extended for effectors which affect many enzymes, or enzymes with many effectors (Fell, 1992; Woodrow and Mott, 1993). Enzymes can have a high elasticity for an effector in vivo for more than one reason. If a reaction is readily reversible in vivo, a small change in the concentration of the substrate will lead to a large change in net activity. This is because its net activity will respond sensitively to small changes in the rate of the forward or reverse reaction (see Rolleston, 1972 for a mathematical analysis; Kruckeberg et al., 1989; Stitt, 1989b for the estimation of the effect on the elasticity coefficient). Another case, however, would be an enzyme which is very sensitive to feedback inhibition, or feedforward activation. Such enzymes will respond to changes in flux initiated elsewhere in the pathway. However, if we try to initiate a change at the enzyme, it will negated by relatively small changes in the substrates or effectors of the enzyme without any large change in flux through the pathway (Stitt, 1989a,b, 1995). The connectivity theorem therefore predicts that a high regulatability can lead to a low regulatory capacity. It also implies that removing the regulatory properties of an enzyme could actually increase its regulatory capacity. I will return to these important points later in the chapter. So far, we have considered factors internal to the metabolic system. However, many events which we intuitively understand as regulation occur because an external factor (e.g. light, hormones) acts on a system. The action of an external effector, is described by an additional term, termed the response coefficient (Kacser and Burns, 1973; Kacser and Porteous, 1987),

170 where is the special elasticity coefficient of the enzyme E for the effector Q, C is the flux control coefficient of E, and is the response coefficient of pathway flux. can be estimated by measuring the fractional change in flux (dJ/J) which results from a fractional change in the external effector (dQ/Q). It is important to note that the special elasticity coefficient does not enter into the connectivity theorem (Kacser and Burns, 1973; Fell, 1992; Woodrow and Mott, 1993). Enzymes which are very sensitive to an external effector can therefore have a high flux control coefficient (although this is not necessarily the case). An enzyme with a high flux control coefficient and a high special elasticity coefficient for an external effector will obviously be able to regulate flux through the pathway very effectively.

Mark Stitt low but finite (0.1–0.2) control coefficients for the plastid FBPase, and for the combined enzymes of sucrose synthesis. Slightly negative control coefficients emerge for PRK.

2. From the and Photosynthesis

Response of

Woodrow and colleagues (Woodrow and Mott, 1988; Woodrow et al., 1990; Woodrow, 1994) have developed an elegant method to estimate the flux control coefficients of the boundary layer the stomata diffusion of in the aqueous phase of the leaf and Rubisco for photosynthesis. It is based on measurements of the effect of small changes in the or concentration on photosynthesis. Theses gases are external effectors and so, taking as an example,

D. How Can Flux Control Coefficients Be Measured? 1. Calculation From Models Flux control coefficients can be estimated by building a model in which each enzyme activity is described via a rate equation based on the kinetic properties of the enzymes. The equations can be differentiated to obtain elasticity coefficients for the enzymes for each of their substrates (Pettersson and RydePettersson, 1989, 1990; Woodrow and Mott, 1993). These models require simplification to make them mathematically amenable, and are obviously based on current knowledge. Nevertheless, they provide interesting insights into the interplay of the regulatory networks. The model of Pettersson and Ryde-Pettersson (1989) is based on the conditions found in isolated chloroplasts in saturating but simplifies by ignoring possible changes in redox status and setting a fixed Pi concentration in the medium. The model predicts that most control is exerted by SBPase and the thylakoid ATPase during photosynthesis with optimal concentrations of Pi in the medium. The distribution of control in this model appears to be mainly due to the maximal activity of the enzymes entered into the model. Woodrow and Mott (1993) modeled leaf photosynthesis in ambient and high irradiance. They simplify the feedback regulation in the Calvin cycle, and their model has not yet been extended to cover sucrose synthesis. Rubisco has an estimated control coefficient of about 0.7, and their estimates indicate

where dJ/J is the measured fractional change in photosynthesis which results from the of the concentration and is the special elasticity coefficient of the process or enzyme E for The special elasticity terms for processes involved in entry and Rubisco activity can be calculated from the physical laws governing diffusion, and from the known kinetic properties of Rubisco for and respectively. It is therefore possible to calculate the flux control coefficient of the enzyme E. The results will be discussed later.

3. ‘Dual Modulation’ This approach was proposed by Kacser and Burns (1979). If metabolism is perturbed in two independent ways, and the resulting changes of steady-state flux and metabolite levels are measured, it is possible to estimate the elasticities of enzyme for their substrates. The connectivity theorem can then be used to calculate the flux control coefficients. Examples of this approach can be found in Fell (1992) and in Neuhaus et al. (1990). This approach requires simplifying assumptions about the regulatory properties of the enzymes, because the equations which are used to estimate elasticities are otherwise too complex to solve. It is sensitive to error because it requires very accurate measurements of small changes in metabolite levels (see discussion in Fell, 1992).

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4. Inhibitor Tit ration By supplying a range of concentrations of an inhibitor, it is possible to progressively inhibit the target enzyme, and measure the resulting change in flux. Provided that it is known to what degree the target enzyme has been inhibited, it is possible to calculate the control coefficient (Groen et al, 1983). This approach is relatively straightforward for irreversibly-binding non-competitive inhibitors, but involves an increasing number of assumptions and potential inaccuracies for weakly binding inhibitors, especially if they act competitively. It has been applied to investigate the distribution of control in the electron transport chain of rat liver mitochondria (Groen et al., 1983), turnip mitochondria (Padova et al., 1989), photosynthetic electron transport (Heber et al., 1988) and several metabolic pathways in mammals (see Fell, 1992).

5. Mutants or Transgenic Plants Perhaps the most striking way to obtain a flux control coefficient is to genetically manipulate the expression of an enzyme, and measure the resulting change in flux (Stitt, 1993). This has been achieved for an increasingly large number of enzymes (see below). Potential problems (Stitt and Schulze, 1994) need to be appreciated, however, in particular the decrease in expression must be relatively small. Large changes of expression produce large changes in metabolism but are unsuitable for estimating control coefficients, which by definition are the response to an infinitesimally small change. Large changes are also unsuitable because they are often accompanied by complex plieotropic changes in metabolism and growth (Müller-Röber et al., 1992; Stitt and Schulze, 1994). This approach depends on careful quantification of enzyme levels and fluxes. Enzyme levels should, if possible, be measured immunologically and checked via measurements of the enzyme’s maximum activity (Stitt and Schulze, 1994) in the same material in which the fluxes are measured. It is important to realize that changes in enzyme activity due to reversible post-translational covalent protein modification represent a response to the changed amount of enzyme. Study of these responses provide interesting insights into ‘fine’ regulatory mechanisms, but is not relevant for the initial assessment of the change in protein amount. Accurate measurements of in vivo fluxes is often

171 difficult. For example, the amount of sucrose or starch in a leaf does not provide reliable information about the rate of synthesis; the content also depends on the rate of degradation, and export. Experiments comparing net photosynthesis with carbohydrate levels and export rates, or experiments provide more accurate estimates.

E. Some Recent Extensions of the Theory As control analysis was applied to real pathways, several areas emerged where the theoretical analysis required extension. This included coping with conserved moieties, for example the total phosphate in a metabolic compartment (Hofmeyr et al., 1986; Small and Fell, 1990), the analysis of cascades and covalent protein modification (Small and Fell, 1990), interpreting the effect of large changes in enzyme activity, and the question of whether changes in kinetic properties are more important than changes in the amount of the protein. I will briefly consider the latter two because of their relevance for the results being obtained in plants.

1. Evaluation of Large Changes The flux control coefficient represents the response to an infinitesimally small change in enzyme amount (Kacser and Porteous, 1987). However, measurements of control coefficients using mutants and transgenic plants (see Section V.D.5) requires relatively large changes in enzyme levels (at least 20–30%), due to the scatter in experimental data. Most studies have used considerably larger changes. How large is the error likely to be? Equations were developed to estimate flux control coefficients from larger changes, assuming that there is a hyperbolic relation between flux control coefficient and enzyme amount. In a sophisticated analysis. Small and Kacser (1993a,b) proposed that the response of flux to large changes in enzyme amount should be termed a deviation index, D, where

and is the change in flux that results from a relatively large change of the enzyme amount. and are the original values for the enzyme amount and flux, and and are the corresponding values for the change. The control

172 coefficient before the change (e.g. in the wildtype) is approximately equal to the deviation index scaled on the enzyme and flux after the change (e.g. in a mutant). This relation between the control coefficient and an appropriately scaled deviation index is only valid if the enzyme shows linearity in vivo (i.e. the enzyme is strongly substrate saturated, or the saturation state is similar in both states). Small and Kacser (1993a,b ) propose several ways in which the validity of this assumption can be checked. When they applied their equations to data from Kruckeberg et al. (1989) for PGI-deficient mutants of Clarkia, their more sophisticated analysis confirmed the original interpretation and provided some evidence that the assumption of linearity was realistic for this enzyme (Small and Kacser, 1993a). Small and Kacser (1993b) also show that the theoretical analysis is much more complicated in branched pathways than in unbranched pathways. In branched pathways the actual flux control coefficients can be much smaller than the deviation index obtained from large perturbations of expression. This needs to be borne in mind in interpreting some of the results presented later.

2. Changes of Kinetic Properties Most experimental measurements of flux control coefficients involve changing the amount of the enzyme. It can be argued that regulation in vivo often involves changes in kinetic properties. There is, for example, convincing genetic evidence from studies of mutants of AGPase in E. coli (Preiss and Romeo, 1994) that changes in the kinetic properties are a very effective way to alter glycogen synthesis. A dramatic illustration of this point in higher plants is provided by Stark et al. (1992). They showed that overexpression wildtype AGPase does not increase the rate of starch synthesis in potato tubers, whereas overexpression of a mutated AGPase from E. coli (which no longer requires PGA for activity and is insensitive to Pi inhibition) does increase starch yield. Removal of a regulatory property (e.g. a feedback inhibition) will usually lead to an increased control coefficient, because the enzymes control coefficient is no longer constrained (decreased) by interactions with the remainder of the pathway (the connectivity theorem, see Section IVC.2). In general, the effect of

Mark Stitt a change in an enzyme’s kinetic properties on pathway flux can be described by a response coefficient (D. A. Fell, personal communication) which describes the response of pathway flux to a change in the binding constant, for an effector, This response coefficient is defined as

where is the control coefficient of the enzyme whose kinetic property has been changed, and is an elasticity coefficient,

which describes the change in enzyme activity (dv/ v) which results from a fractional change in the binding constant of the enzyme In most cases will be numerically identical with (the elasticity coefficient of the enzyme for the effector, This means the will often exceed unity; for example, an effector binding at the allosteric site of a positively cooperative enzyme, or an enzyme where the binding of a positive effector competes with a negative effector. When a kinetic property of an enzyme is altered by a finite amount, its flux control coefficient may also change. Firstly, the resulting change of fluxes and metabolite levels will usually lead to a redistribution of control within the pathway (see below). Secondly, if the altered binding site interacts with an effector which is shared with other enzymes in the pathway, the control coefficient will change in accordance with the connectivity theorem. For example, if the affinity of an enzyme E for a inhibitor increases, and this inhibitor is also a substrate or activator of other enzymes in the pathway, then will decrease. Depending on the precise interactions in the metabolic system, the response coefficient can be larger or smaller than the flux coefficient derived from changes in enzyme amount (D. A. Fell, personal communication). In general, changes in kinetic constants for external effectors will usually have a larger response coefficient than changes in kinetic constants for internal metabolites, which are shared with other enzymes.

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VI. Distribution of Control in Photosynthetic Carbon Metabolism In this section I will discuss the experimental values for flux control coefficients of enzymes of photosynthetic metabolism. Many studies in microbial and mammalian systems (Groen et al., 1983; Fell, 1992) show that the contribution of a particular enzyme to control depends on (i) the short-term conditions (i.e. those in which the measurements are carried out) and (ii) the long-term conditions (i.e. those under which the organism has grown). It will become clear that this is also true for photosynthetic metabolism (Woodrow and Mott, 1988; Kruckeberg et al., 1989; Woodrow et al., 1990; Stitt et al., 1991a ; Quick et al., 1992; Lauerer et al., 1993; Woodrow, 1994).This means it is very important to monitor and document the conditions in which the plants are grown, and in which photosynthesis is measured. Otherwise, controversies could develop because different studies have been measuring different things. Table 1 summarizes values for the flux control coefficients which I have estimated from available data. Many of these values are preliminary, and have

been obtained in different species or conditions. In the following sections I will discuss these values in more detail and indicate how they may vary.

A. Entry of

1. Boundary Layer Conductance Woodrow et al. (1990) estimated a flux control coefficient of about 0.03 for boundary layer conductance at high irradiance, 350 ppm and a windspeed of They predicted that increases at lower wind speed, and decreases at lower irradiance or higher concentrations.

2. Stomata Stomatal conductance has a flux control coefficient for photosynthesis of 0.1–0.2 at 350 ppm and a vapor pressure deficit of (Woodrow et al., 1990; Stitt et al., 199la). Both studies showed that is decreased when the irradiance decreased, or the concentration is increased. rises appreciably to values of

174 0.5 or higher when the vapor pressure deficit is large (Woodrow et al., 1990).

3. Conductance of the Aqueous Phase of the Leaf Several studies indicate that the physical resistance to diffusion through the aqueous phase of the leaf can have a small effect on the rate of photosynthesis in some conditions. Woodrow et al. (1990) calculated that is about 0.03 at high irradiance and 25 °C. Price et al. (1995) have shown that a 90–98% reduction in the expression of carbonic anhydrase in antisense tobacco plants leads to a small change in discrimination, from which they calculate that a small but finite (4%) inhibition of photosynthesis would have resulted. The contribution of may depend on the prehistory of the leaves. Lauerer et al. (1993) found that a higher apparent was needed to maintain a given rate of photosynthesis in leaves grown in high light compared to low light. This implies that the conductance of the aqueous phase is smaller in the high-irradiance leaves. Sharkey et al. (1991) investigated transgenic tobacco plants which had an altered leaf composition, due to overexpression of phytochrome. Even though leaves of phytochromeoverexpressing plants contained more protein and chlorophyll per unit leaf area, photosynthesis did not increase. They concluded that increased resistance to diffusion may constrain the rate of photosynthesis in these genetically manipulated leaves. ‘Sun’ leaves adapt to high irradiance by becoming thicker and by packing more and more protein and chlorophyll into a given leaf area. This allows them to increase the fluxes of However, these changes will also tend to decrease the effective conductance for The high leaf temperatures often associated with high irradiance will exacerbate the problem, because they decrease the concentration of dissolved in the aqueous phase. These trends may reach a point where physical diffusion of starts to exert a small but significant degree of control over the rate of photosynthesis.

4. Regulation and Constraints Comparison of the three processes involved in entry illustrates a potential confusion which can arise due to terminology. All three partial processes have a finite control coefficient for photosynthesis

Mark Stitt (‘regulatory capacity’), and are therefore potential sites at which photosynthesis could be regulated. However, ‘regulation’ also requires that the process can be modulated in vivo (‘regulatability’). Stomatal conductance is modulated in response to light, and water status (e.g. via ABA). Stomatal conductance will therefore contribute to the regulation of photosynthesis. It is important to note, though, that since is much less than unity, the stomata only exert partial control (e.g. if a 10% decrease in stomatal conductance will only lead to a 1% inhibition of photosynthesis). The flux control coefficient of stomatal conductance for water loss will be much higher. This regulation is important because it allows the potentially conflicting requirements of fixation and water relations to be balanced against each other. Boundary layer conductance and aqueous diffusion probably have no ‘regulatability’, and it would be misleading to say they regulate photosynthesis. However, since they can have a finite flux control coefficient they will sometimes under (un)suitable conditions exert a constraint on entry, and slightly lower the rate of photosynthesis. The fact that these physical constraints are relatively small is presumably due to the operation of natural selection. It can be speculated that selection acts on the genes that determine leaf structure and development, by discriminating against leaf forms in which ‘trivial’ physical constraints impair the efficient utilization of the biomass invested in leaf formation.

C. Rubisco As already emphasized, Rubisco occupies a central role in the economy of the plant. It is therefore appropriate that more information is available about the flux control coefficient of Rubisco for photosynthesis than for any other enzyme in plant metabolism. Experimental values for have been obtained (i) by investigating the rate of photosynthesis in two sets of antisense rbcS tobacco plants (Masle et al., 1993; Stitt and Schulze, 1994), and (ii) by analyzing the effect of small changes of and concentration on the rate of photosynthesis in sunflower and soybean (Woodrow and Mott, 1988; Woodrow et al., 1990; Woodrow, 1994). Table 2 summarizes a large number of experimental values of obtained in a range of different conditions. Values of in ambient growth conditions are shown in bold type. Values found

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during short-term changes in irradiance or shown in faint type.

175 are

1. Effect of Short-Term Conditions The changes of in response to short-term alterations of the conditions agree, qualitatively, with the models of Farquhar and coworkers. increases as the irradiance is increased, or the concentration is decreased (Fig. 6). Nevertheless, there are some quantitative discrepancies. Even though Rubisco is the major controlling factor in saturating irradiance, is nevertheless less than unity in most cases (Woodrow and Mott, 1988; Stitt et al., 1991a; Quick et al., 1992; Lauerer et al., 1993). This implies that Rubisco is not the sole controlling factor. Much of the remaining control may be located in the processes involved in entry into the leaves (see above). Small but finite contributions might also be made by other enzymes (see below). It should be noted that if twenty different enzymes or processes had a control factor of about 0.02 (which would be almost undetectable), their summed contribution to flux control would be about 0.4!

3. Adaptation to Limiting Nitrogen Supply A considerable portion (20–30%) of the organic nitrogen in plants is invested in Rubisco. Provided that the plant has access to ample nitrogen, surplus (‘luxury’) investment in Rubisco will not impair growth, indeed, it may even bring some advantages including marginally better water use efficiency and a more efficient use of short periods of increased irradiance (Quick et al., 1991, 1992; Lauerer et al., 1993). However, we would expect that allocation of nitrogen becomes more critical during growth on limiting nitrogen. A relatively small change in the amount of Rubisco would have a large impact on the amount of protein available for investment in minor proteins. Plants growing on limiting nitrogen have a decreased Rubisco content, not just in absolute terms but also relative to other proteins (Evans, 1989; Chapter 11). As a result, in ambient aerial conditions is much higher in tobacco growing on limiting (0.5–0.6) than in well-fertilized tobacco (0.1–0.3) (Fig. 7; Quick et al., 1992). These results show that tobacco indeed ‘cuts back’ on ‘luxury’ investment in Rubisco when it becomes nitrogen-limited.

2. Contribution in Ambient Growth Conditions 4. Adaptation to Irradiance Regimes When tobacco was grown in low irradiance, was low but not zero (0.03–0.1, Table 2). When tobacco, sunflower or soybean were grown in high irradiances values of 0.25–0.8 were found for Rubisco, depending on the species, temperature, fertilization and irradiance (see below). Rubisco is obviously an important, but not the only, determinant of ambient photosynthetic rate in high light growth regimes. The experimental values for in high light growth regimes are broadly consistent with earlier gas exchange studies (reviewed in Stitt, 1991). When photosynthesis is measured in growth conditions, the operating often lies in the curvilinear sector of curves. This curvilinear sector was the interpreted as an area of shared limitation in the model of Farquhar and von Caemmerer (1982). Shared control between Rubisco and other proteins is also predicted by theoretical considerations of how nitrogen could be optimally allocated between proteins (Cowan, 1986).

Adaptation to different irradiance regimes also involves small changes in the balance between different proteins in the leaf (Evans, 1988, 1989; Chapter 11). Table 2 reveals that plants which have grown in low or high irradiance have different values for at a given measuring irradiance. For example, whereas tobacco plants which have grown at irradiance are almost totally limited by Rubisco when they are suddenly exposed to plants which have grown at 750 are only slightly limited by Rubisco at this irradiance (Lauerer et al., 1993). Woodrow and Mott (1988) found that acclimation of low light-grown sunflower to high irradiance resulted in a decrease of from 0.8 (immediately after transfer to high irradiance) to under 0.4 (after several days at high irradiance). Small changes in the stoichiometry between Rubisco and other proteins allow the leaf to ‘escape’ from a one-sided limitation by Rubisco over a quite wide range of irradiance. This potential for adaptation means that it is

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extremely dangerous to use short-term experiments to predict what may limit or control photosynthesis under different growth conditions. Another example of adaptation is provided by studies of sunflower growing at different concentrations (Woodrow, 1994). in sunflower growing at. was about 0,7. decreased to under 0.2 when the concentration was suddenly increased to 90 Pa. However, was still 0.7 when the plants were grown at

to ten-fold excess of Rubisco (Lauerer et al., 1993). This overinvestment in Rubisco can sometimes impair photosynthesis; plants containing three-fold less Rubisco had more chlorophyll (Quick et al., 1992) and thylakoid protein (Lauerer et al., 1993), and higher rates of photosynthesis than the wildtype in low irradiance (Quick et al., 1992; Lauerer et al., 1993).

5. Allocation to Rubisco Is Not Always ‘Optimal’

Information about the flux control coefficients of other Calvin cycle enzymes is becoming available, but detailed studies in different conditions have not yet been carried out. NADP-GAPDH can be decreased about three-fold in tobacco before photosynthesis is significantly inhibited (Price et al., 1995). Smaller changes already resulted in shifted levels of PGA and RuBP. This enzyme is evidently not present in a large excess in vivo, despite the very high activity which is found when the enzyme is extracted and assayed in optimal conditions.

Although plants can adjust the balance between Rubisco and other proteins over a wide range of conditions, an ‘optimal’ response is not always achieved. When tobacco was grown in Portugal at irradiance and high temperature, Rubisco was almost totally limiting for photosynthesis, was 0.8–0.9 (Krapp et al., 1994). The opposite problem was found when tobacco was grown in low irradiance. The leaves contained a five-

C. Other Enzymes of the Calvin Cycle

178

Mark Stitt present ‘in excess’. These results show that chloroplasts do not contain a large excess of aldolase. It can be speculated that metabolic pathways may have problems in compensating for decreased expression of such ‘non-regulatable’ enzymes (Sonnewald et al., 1994), presumably because the only possible response is to allow the substrate/ product ratio to increase, and this eventually impairs the operation of other enzymes in the pathway (see below for more examples). Antisense potato plants with 40, 15 and 12% wildtype plastid FBPase showed a 10%, 60% and 80% inhibition of photosynthesis when it was measured in limiting irradiance and a 20%, 75% and 90% inhibition of photosynthesis when it was measured in saturating irradiance and (KoBmann et al., 1992; Sonnewald et al., 1994). The 60% change in enzyme is rather large, but a control coefficient of about 0.07 and 0.16 can be estimated for low and high irradiances using the equations developed by Small and Kacser (1993a); these could be overestimated because the assumption of linearity is probably not valid for this data set. Antisense plants with an 80–90% reduction in PRK activity do not show any significant inhibition of photosynthesis (Paul et al., 1995). The of PRK is 20 fold higher than the maximum rate of photosynthesis (Laing et al., 1981), and the enzyme is usually strongly inhibited by various metabolites in vivo (see Section III). A decreased amount of protein can obviously be compensated via fine regulation without this leading to dramatic consequences for the steady-state rate of photosynthesis.

D. Partitioning

Removal of just 40% of the plastid aldolase activity resulted in decreased growth of antisense potato plants. Genotypes with 10–15% of wildtype aldolase were severely stunted (Sonnewald et al., 1994; R. Zrenner, unpublished). These effects on growth imply that photosynthesis is remarkably sensitive to decreased expression of aldolase. Aldolase is a typical example of an enzyme which has no known regulatory properties and catalyses a readily reversible reaction. It would therefore generally be considered to be

Triose-phosphates represent an important branch point in metabolism. Some are converted to sucrose in the cytosol, and some to starch in the chloroplast. The fluxes in these two pathways interact. There is also an important and complex interaction with the Calvin cycle (i.e. the flux before the branch point), because most of the triose-phosphates must be recycled to regenerate RuBP, and because fixation depends on the Pi which is recycled during endproduct synthesis (see Sections II and III.G). I will first describe the distribution of control within the isolated segments of metabolism represented by the pathways of starch and sucrose synthesis, and then briefly consider how they interact with the Calvin cycle.

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1. Starch Synthesis The distribution of control in the pathway of photosynthetie starch synthesis has been investigated using reduced-activity mutants of branching enzyme (BE) in pea, plastid phosphoglucose isomerase (PGI) in Clarkia xanthiana, and AGPase and plastid phosphoglucose mutase (pPGM) in Arabidopsis. Flux control coefficients were measured in limiting light, and in saturating light and In the latter conditions pathway flux was increased two- to three-fold above that occurring under the growth conditions. The result are summarized in Fig. 8. In limiting irradiance, AGPase had a control coefficient of about 0.3 for starch synthesis (Neuhaus and Stitt, 1990). No other enzymes in the pathway exerted significant control. The residual control is presumably located in processes involved in the harvesting and use of light energy. Since enzymes of sucrose synthesis have negative flux control coefficients for starch synthesis (see below), the amount of control located in AGPase is relatively small, compared to that located elsewhere. In saturating light and the distribution of control in the pathway changes. The flux control coefficient of AGPase for starch synthesis increases (0.64) but significant control is also exerted by pPGI (0.34; Kruckeberg et al., 1989) and pPGM (0.21; Neuhaus and Stitt, 1990). These individual control coefficients now sum to more than unity. This is possible because some of the enzymes of sucrose synthesis in the cytosol have negative control coefficients for starch synthesis in these conditions (see below). These results were obtained with mutants from different species, but they provide a first glimpse into how changing conditions lead to a redistribution of control within a pathway, and indicate that more than one enzyme can contribute to control in a given situation. The results with pPGI and pPGM provide another two examples of non-regulated enzymes that catalyze readily-reversible reactions, but are not present in large excess. The contribution of AGPase has also been investigated in transgenic plants. Transformation of potato with an antisense sequence to the B-subunit of AGPase resulted in plants with 70–95% less AGPase and 50–90% less starch than the wildtype (Müller-Röber et al., 1992; Müller-Röber and Kossmann, 1994). The change in AGPase is rather large, but can be used to calculate a (probably

overestimated) flux control coefficient of about 0.4 using the equations in Small and Kacser (1993a). This is in broad agreement with the result in Arabidopsis mutants (see above), confirming that AGPase exerts considerable but not complete control over starch synthesis. Although AGPase is important, it is misleading to talk of it being the ‘limiting’ factor. Even though AGPase has a significant flux control

180 coefficient, overexpression of the gene encoding wildtype E. coli AGPase did not significantly increase starch levels in tomato leaves (Stark et al., 1992). This indicates that other factors may start to exert control if starch synthesis is increased much above its normal rate. The redistribution of control to enzymes like pPGI and pPGM (Fig. 8) in saturating and light is consistent with this possibility. It might appear strange that a key enzyme like AGPase does not have a higher control coefficient. The reason is illustrated by further experiments of Stark et al. (1992). The mutated E. coli gene glc-C16 encodes an AGPase that is constitutively active, does not need PGA or other metabolites for activation, and is not inhibited by Pi. Callus material expressing this gene contained more starch. However, expression of this gene disrupted photosynthetic metabolism, and plantlets could only be regenerated when they were supplied with glucose. These results remind us that regulation of AGPase by the PGA/Pi ratio has two functions: (i) to allow starch synthesis to be increased when the ratio rises, and (ii) to inhibit starch synthesis and prevent depletion of the Calvin cycle or inhibition of sucrose synthesis when the ratio decreases. The wildtype higher plant AGPase is ‘tied into’ metabolism via its allosteric regulatory properties. Its control coefficient is constrained, in accordance with the connectivity theorem, because it responds sensitively to effectors like PGA and Pi that are shared with other enzymes.

2. Sucrose Synthesis Antisense plants with a 20–30% decrease in TPT expression showed a 27–50% inhibition of sucrose synthesis over the photoperiod (Riesmeyer et al., 1993; Heineke et al., 1994), from which a flux control coefficient for sucrose of 1.4–1.7 can be estimated. The TPT catalyses a passive and readily reversible counterexchange of Pi, triose-phosphates and PGA (Heldt and Flügge, 1992). This is a surprisingly large coefficient for a ‘non-regulated, reversible’ step. Two factors could explain these results. First, it is likely that a large percentage of the carrier activity is catalyzing homologous exchanges or competing heterologous exchanges (Flügge et al., 1983). In agreement, metabolite measurements show that the triose-phosphates are not fully equilibrated between the chloroplast and cytosol during ambient photosynthesis (Gerhardt et al., 1987). Secondly, TPT is located at a branch point, where extremely high

Mark Stitt control coefficients are possible (see below for further discussion). Direct estimates of the flux control coefficient of the cytosolic FBPase for sucrose synthesis are not yet available. Indirect estimates using the ‘dual modulation’ approach indicate a value of about 0.5 (Neuhaus et al., 1989; 1990). Studies of cytosolic phosphoglucose isomerase (cPGI) deficient mutants showed that this enzyme has a low but finite flux control coefficient for sucrose synthesis in the wildtype (0.02–0.03; Kruckeberg et al., 1989; Small and Kacser, 1993b). When the cPGI was decreased three- to five-fold below the wildtype there was a large increase of the F6P/G6P ratio, and sucrose synthesis was inhibited by 20% in low irradiance, and by 9% in saturating irradiances and This provides yet another example of a nonregulated enzyme which catalyses a readily reversible reaction, but is not present in large excess. Estimates of in vivo elasticity coefficients revealed why PGI starts to control in these reduced activity mutants (Neuhaus et al., 1989). The elasticity of pPGI for F6P in the low-activity mutants is comparable to the sensitivity of the cytosolic FBPase to inhibition by F6P via the FBP regulator cycle (Stitt 1989a,b]; 1990c). Current evidence indicates that PPi-dependent enzymes do not exert much control on the rate of sucrose synthesis. UDP-glucose pyrophosphorylase can be reduced 10 fold without any significant effect on sucrose levels (Zrenner et al., 1993). Expression of E. coli inorganic pyrophosphatase to accelerate the removal of pyrophosphate in the cytosol (Sonnewald, 1992) led to a small but statistically non-significant increase in the rate of sucrose synthesis (Jelitto et al., 1992). Sucrose accumulated in the leaves, but this was caused by decreased export rather than increased synthesis (J. Lerchl, U. Sonnewald, P. Geigenberger and M. Stitt, unpublished). A 95% reduction in expression of pyrophosphate : fructose 6-phosphate phosphotransferase in tobacco leaves did not significantly alter photosynthate partitioning (M. Paul, U. Sonnewald, D. T. Dennis and M. Stitt, unpublished results). Sucrose-phosphate synthase is thought to be a key regulatory enzyme for sucrose synthesis (Stitt et al., 1987a,b; Huber and Huber, 1992a). Galthier et al. (1993) have overexpressed maize leaf SPS in tomato leaves. A five-fold increase in SPS activity led to a 5– 10% increase in photosynthetic rate, a doubling of the leaf sucrose content, and a two- to three-fold

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increase of the sucrose/starch ratio in leaves. Direct measurements of incorporation into sucrose revealed small (10–15%) stimulation of sucrose synthesis (N. Galthier, R. Alred, P. Quick, C. H. Foyer, personal communication). When spinach leaf SPS was overexpressed in tobacco leaves, a two- to three-fold increase in total activity led to a small (circa 10%) but non-significant stimulation of incorporation into sucrose (K.-P. Krause, U. Sonnewald and M. Stitt, unpublished). Indirect evidence indicated that the introduced spinach enzyme may be inactivated via protein phosphorylation. In a complementary approach, antisense potato plants with a 25–70% decrease in leaf SPS were investigated (K.-P. Krause, U. Sonnewald, E. Macrae and M. Stitt, unpublished). Their leaves contained 20–50% lower soluble sugars, and 50–200% higher starch-labeling experiments showed a 12–40% inhibition of sucrose synthesis, a compensating increase in starch synthesis and photosynthesis remained unaltered. The equations of Small and Kacser (1993a) allow flux control coefficient of 0.3 to 0.45 for sucrose synthesis, and about –0.4 for starch synthesis to be estimated. These are similar to the value estimated from dual modulation experiments (Stitt, 1989a,b; Neuhaus et al., 1990). Taken together, these results show that SPS is an important factor in the control of sucrose synthesis in the wildtype. However, as with AGPase, overexpression does not lead to the expected large gain in sucrose synthesis. This is presumably because control redistributes to other enzymes and processes.

3. Interaction Between Sucrose Synthesis, Starch Synthesis and Photosynthetic Rate Studies with reduced activity mutants of cytosolic PGI (Neuhaus et al., 1989; Stitt, 1990c, 1993) and antisense TPT (Heineke et al., 1994) and SPS (K.-P. Krause, U. Sonnewald, E. Macrae and M. Stitt, unpublished) plants show that (i) a decreased rate of sucrose synthesis leads to an increased rate of starch synthesis; (ii) this change of partitioning occurs without an inhibition of photosynthesis; and, indeed, (iii) a decrease of cytosolic PGI has less effect on partitioning in saturating irradiance and than in limiting irradiance. This demonstrates that the feedback regulation of sucrose synthesis can be overridden to minimise or avoid an inhibition of (see Section III.G.4, also Neuhaus et

181 al., 1989; Stitt, 1989b, 1990c, 1991, 1993). Decreased expression of enzymes for starch synthesis results in rather complicated changes in the fluxes in the other two pathway segments. In low irradiance low-AGPase mutants had a higher rate of sucrose synthesis, and photosynthesis was not inhibited. In high irradiance, decreased expression of pPGI, pPGM, AGPase or BE resulted in an inhibition of photosynthesis. Sucrose synthesis was not increased and in some cases was even inhibited (Kruckeberg et al., 1989; Neuhaus and Stitt, 1990; Stitt, 1993). Apparently chloroplast fluxes respond in a flexible and interactive manner to changes in the cytosol, but not vice versa. It has been proposed that this is because regulatory loops normally function to modulate starch synthesis in response to sucrose synthesis, but not in the reverse direction (Stitt, 1990c, 1993). Enzymes in the pathway of sucrose synthesis therefore have negative flux control coefficients for the other branch (starch synthesis) but have negligible flux control coefficients for photosynthesis. Enzymes of starch synthesis have positive flux control coefficients for photosynthesis in high irradiance and The existence of negative flux control coefficients in branched pathways allows other enzymes to sometimes have very high flux coefficients. For example, pPGI has an estimated control coefficient of 1.8 for starch synthesis in reducedactivity mutants (Kruckeberg et al., 1989), and TPT has an estimated control coefficient of about 1.5 for sucrose synthesis (Riesmeyer et al., 1993; Heineke, 1994). The asymmetrical interactions and numerically large control coefficients which occur in branched pathways hamper a purely intuitive approach to regulation. Once they have been experimentally determined, however, we can use them to gain insights into the in vivo effectiveness of known regulatory mechanisms. For example, the observation that feedback regulation of the cytosolic FBPase is overridden in conditions allowing rapid photosynthesis (see above) was explained by an analysis of elasticity coefficients. This revealed that the feedback inhibition by fructose 2,6-bisphosphate can be overcome by a relatively small increase of triosephosphates under in vivo conditions (Neuhaus et al., 1989, 1990; Stitt, 1989a,b, 1990c). The surprisingly large effect of decreased TPT on sucrose and starch synthesis can also be rationalized a posteriori. Accumulation of phosphorylated intermediates in

182 the stroma could have a large effect on starch synthesis due to activation of AGPase by increased PGA, and falling Pi; it could also have a massive dual effect on sucrose synthesis because falling triose-phosphate leads to a very sharp restriction of cytosolic FBPase activity (see Section III.G.2) and falling cytosolic PGA simultaneously leads to a rise of fructose 2,6bisphosphate (see Section III.G.l).

E. Conclusions 1. What Controls Fluxes in Photosynthesis? The control of fluxes in leaves is usually shared between several enzymes or processes, and the distribution of control depends on the conditions, both long-term and short-term. Leaves actually adjust to avoid one-sided limitations. This will be important in optimizing leaf performance, but it will also complicate interpretation of data sets obtained in different conditions. In low irradiance, the rate of photosynthesis is probably mainly controlled by processes involved in the harvesting and use of light energy. More studies are needed to pinpoint the crucial proteins. Highly effective regulation mechanisms driven directly or indirectly by light allow fluxes in the Calvin cycle to be increased in parallel with the availability of light energy (Section III. A and B). Rubisco can have a low but finite flux control coefficient in low irradiance, and more research is needed to understand how Rubisco activity is modulated in response to light and the remainder of the Calvin cycle. None of the other Calvin cycle enzymes yet investigated have a large control coefficient in low irradiance, because their high regulatability serves to balance their activities and prevent bottlenecks from emerging (Sections III.C and D). Partitioning can be altered via an interaction between several enzymes including SPS and AGPase, which have significant control coefficients for sucrose and starch synthesis, without any inhibition of photosynthesis. This presupposes a ‘suitable’ tuning of the amounts and kinetic properties of the enzymes involved. In saturating irradiance, Rubisco is a major factor controlling the rate of photosynthesis, but it is rarely the only factor. A small but significant degree of control is often exerted by processes involved in entry. It is also possible that a large number of other enzymes exert an individually small, but collectively

Mark Stitt large, control of flux. This is indicated by the large number of cases where a two- to four-fold decrease in enzyme produces a measurable inhibition of flux, at least in its own segment of metabolism (e.g. NADP-GAPDH, pFBPase, pAldolase, TPT, pPGM, pPGI, cytosolic PGI, SPS). Conclusive proof is lacking, however, because it is difficult to make measurements with enough precision to decide whether these enzymes have a small flux control coefficient in the wildtype. Extrapolation of the values measured in antisense plants or mutants to the situation in the wildtype requires assumptions of linearity, which may not always be justified.

2. What Makes a Protein Important for Regulation? It is becoming increasingly clear that the distinction between ‘reversible and non-regulatory’ and ‘irreversible and regulatory’ enzymes is incorrect. Many enzymes which exhibit high regulatability and catalyze non-reversible reactions in vivo have low or non-existent flux control coefficients (e.g. pFBPase, PRK). Such enzymes often operate in vivo at a fraction of their potential capacity, (e.g. pFBPase, PRK, also AGPase; Neuhaus et al., 1989) because they are subject to fine regulation. This fine regulation (i) allows their activity to be rapidly modulated to maintain a balance within the pathway as conditions change, and (ii) allows them to respond to a change initiated elsewhere in the pathway. This fine regulation also means they can (iii) readily compensate for a decreased protein level in mutants or transgenic plants. Indeed it might be speculated that the risk of an inhibition of pathway flux is decreased in enzymes with complex regulatory properties, because there are several different ways in which they can compensate. It is also becoming apparent that many enzymes that have no obvious regulatory properties and catalyze readily reversible reactions are not present in large excess, and sometimes exert a small amount of control over pathway flux, especially if they are reduced two- to three-fold below the wildtype level. For such enzymes, compensation will have to occur via a change in the substrate/product ratio. It might be speculated that this lack of flexibility means that there is an increased risk that the operation of other enzymes in the pathway is impaired. The finding that the enzymes are not present in large excess is, perhaps, not surprising. It is not easy to understand how

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natural selection could operate to maintain a large excess of most of the enzymes in a pathway. Why do enzymes that catalyze highly exergonic (irreversible) reactions usually have complex regulatory properties? The answer may have less to do with flux regulation, than with the ‘structural’ requirements of a metabolic pathway. If highly exergonic reactions were allowed to run to equilibrium in vivo, this would result in an extremely low substrate concentration, and an extremely high product concentration. It can be speculated that this would be incompatible with the effective operation of other enzymes in the pathway, with the coordination of fluxes in different pathways, and with the efficient operation of conserved moiety cycles. The regulatory properties of many enzymes might therefore be primarily viewed as a solution to a physicochemical problem: how can an efficient and operational metabolic pathway be formed out of a series of sequential reactions which have very different standard free energy change? The problem could also, of course, be solved by producing a very small amount of enzyme for the highly exergonic reactions. However, this strategy would be very inflexible and would not permit sudden changes in pathway flux. The experimental observation that many enzymes with high regulatability have a low regulatory capacity (control coefficient) is fully consistent with the connectivity theorem. High sensitivity to internal effectors which are shared with other enzymes in the pathway entails that such enzymes will respond to changes in flux, but will not be able to initiate a change themselves. Direct experimental corroboration of the connectivity theorem and its implications for metabolic regulation is being provided by in vivo investigations of elasticity coefficients. They are revealing that enzymes which catalyze in vivo irreversible reactions often have quite high elasticity coefficients for their substrates, activators and inhibitors. These values are not much smaller than the elasticity coefficient of enzymes which catalyze readily reversible reactions for their substrates and products. This leads to an apparent dilemma, because ‘regulation’ requires that an enzyme has a high regulatory capacity and a high regulatability. Such enzymes will need to exhibit kinetic properties in vivo such that they are (i) relatively insensitive to effectors which they share with other enzymes within their pathway but (ii) respond sensitively to effectors which they does not share with other enzymes in that

183 particular segment of metabolism. These effectors may derive from an adjacent sector of metabolism (e.g. PGA acting on AGPase), or from other physiological processes in the plant (e.g. ABA acting on stomata) or from outside the plant (e.g. light acting via thylakoid proteins and receptors). The relative independence of such enzymes carries the risk that they can disrupt metabolism, rather than improve it. This risk may often be reduced by not allowing single enzymes to exert ‘too much’ control over key processes.

Acknowledgments I am grateful to S. von Caemmerer, J. Andrews, M. Badger, U. Sonnewald, W. Frommer, R. Zrenner, J. Kossmann, J. Lechl, M. Paul, J. Gray, D. Heineke, A. Portis, R. Scheibe, I. Woodrow, J. Preiss and M. Parry for making unpublished results available to me, and for discussions I am especially grateful to D. Fell for his extremely valuable advice on control analysis. I thank Heike Weiner for preparing the manuscript, and Fr. I. Lange for the photography.

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Mark Stitt dependent inactivation of spinach leaf nitrate reductase. Planta 192: 183–188 Stark DM, Timmermann KP, Barry GF, Preiss J and Kishore GM (1992) Regulation of the amount of starch in plant tissues by ADP-glucose pyrophosphorylase. Science 258: 287–292 Stitt M (1989a) Control analysis of photosynthetic sucrose synthesis: assignment of elasticity coefficients and flux control coefficients to the cytosolic fructose 1,6-bisphosphatase and sucrose-phosphate synthase. Phil Trans Roy Soc Lond B 323: 327–338 Stitt M (1989b) Control of sucrose synthesis. Estimation of free energy charges, investigations of the contribution of equilibrium and non-equilibrium reactions and estimation of elasticities and flux control coefficients. In: Barber J (ed) Techniques and New Developments in Photosynthetic Research, pp 365–392. Plenum Press, London Stitt M (1990a) Fructose–2,6-bisphosphate as regulatory metabolite in plants. Ann Rev Plant Physiol Mol Biol 41: 153– 185 Stitt M (1990b) The flux carbon between the chloroplast and the cytoplasm. In: Dennis DT and Turpin DH (eds) Advanced Plant Physiology: Interaction and Control of Metabolism, pp 319–340, Pitman Publishing, London Stitt M (1990c) Application of control analysis to sucrose synthesis. In: Cornish-Bowden A and Cardenas ML (eds) Control of Metabolic Pathways, pp 363–376. Academic Press, London Stitt M (1991) Rising levels and their potential significance for carbon flow in photosynthetic cells. Plant Cell Environ 14: 741–762 Stitt M (1993) Control of photosynthetic carbon fixation and partitioning: How can use of genetically manipulated plants improve the nature and quality of information about regulat ion? Phil Trans Roy Soc Lond B 340: 225–233 Stitt M (1995) The use of transgenic plants to study the regulation of metabolism. Aust J Plant Physiol 22: 635–646 Stitt M and Grosse H (1988a) Interaction between sucrose synthesis and photosynthesis. I. Slow transients during a biphasic induction of photosynthesis are related to a delayed activation of sucrose synthesis. J Plant Physiol 133: 129–137 Stitt M and Grosse H (1988b) Interaction between sucrose synthesis and photosynthesis. IV. Temperature dependent adjustment of the relation between sucrose synthesis and fixation. J Plant Physiol 133: 392–400 Stitt M and Heldt HW (1985a) Control of photosynthetic sucrose synthesis by fructose 2,6-bisphosphate. IV. Intercellular metabolite distribution and properties of the cytosolic fructose 1,6-bisphosphatase in maize leaves. Planta 164: 179–188 Stitt M and Heldt HW (1985b) Control of photosynthetic sucrose synthesis. VI. Regulation of the cytosolic fructose 1,6bisphosphatase in spinach leaves by an interaction between metabolic intermediates and fructose 2,6-bisphosphate. Plant Physiol 79: 599–608 Stitt M and Quick WP (1989) Photosynthetic carbon partitioning its regulation and possibilities for manipulation. Physiol Plant 77: 633–641 Stitt M and Schulze E-D (1994) Does Rubisco control the rate of photosynthesis and plant growth? An exercise in molecular ecophysiology. Plant Cell Environ 17: 465–487 Stitt M, Wirtz W and Heldt HW (1980) Metabolite levels in the chloroplast and extrachloroplast compartments of spinach

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Metabolic Regulation

protoplasts. Biochim Biophys Acta 593: 85–102 Stitt M, Wirtz W and Heldt HE (1983) Regulation of sucrose synthesis by cytoplasmic fructose 1,6-bisphosphatase and sucrose phosphate synthase during photosynthesis in varying light and carbon dioxide. Plant Phyusiol 72: 767–774 Stitt M, Huber SC and Kerr P (1987a) Control of photosynthetic sucrose synthesis. In: Hatch MD and Boardman NK (eds) Biochemistry of Plants, Vol 10, pp 327–407. Academic Press, New York Stitt M, Gerhardt R, Wilke I and Heldt, HW (1987b) The contribution of fructose 2,6-bisphosphate to the regulation of sucrose synthesis during photosynthesis. Physiol Plant 69: 377–386 Stitt M, Wilke I, Feil R and Heldt HW (1988) Coarse control of sucrose-phosphate synthase in leaves: Alterations of the kinetic properties in response to the rate of photosynthesis and the accumulation of sucrose. Planta 174: 217–230 Stitt M, Quick WP, Schurr U, Schulze E-D, Rodermel SR and Bogorad L (1991 a) Decreased Rubisco in tobacco transformed with ‘antisense’ rbcS. II. Flux control coefficients for photosynthesis in varying light, and air humidity. Planta 183: 555–566 Stitt M, von Schaewen A and Willmitzer L (1991b) Sink regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast inveratse in their cell wall involves a decrease of the Calvin cycle enzymes and an increase of glycolytic enzymes. Planta 183: 40–50 Turpin DH and Wegner HG (1990) Interactions between nitrogen assimilation, photosynthesis and assimilation. In: Dennis DT and Turpin DH (eds) Plant Physiology, Biochemistry and Molecular Biology, pp 430–441. Longman, Singapore Vincentz M, Maireaux T, Leydecker M-T, Vaucheret H and Caboche M (1992) Regulation of nitrate and nitrate reductase expression in Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites. Plant J 3: 315–324 von Caemmerer S and Edmonson DL (1986) Relationship between steady-state gas exchange, in vitro ribulose bisphosphate carboxylase activity and some carbon reduction cycle intermediates in Raphanus sativus. Aust J Plant Physiol 13: 669–688 von Caemmerer S and Farquhar G (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376–387 von Schaewen A, Stitt M, Schmidt R, Sonnewald U and Willmitzer L (1990) Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate, inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants. EMBO J 9: 3033–3044 Walker DA and Sivak MN (1986) Photosynthesis and phosphate: A cellular affair. Trends Biochem Sci 11: 176–179 Wang ZY and Portis AR (1992) Dissociation of ribulose 1,5bisphosphate bound to ribulose 1,5-bisphosphate carboxylase/ oxygenase and its enhancement by ribulose 1,5-bisphosphate carboxylase/oxygenase. Activase-mediated hydrolysis of ATP. Plant Physiol 99: 1348–1353 Weiner H, McMichael RW and Huber SC (1992) Identification of factors regulating the phosphorylation status of sucrosephosphate synthase in vivo. Plant Physiol 99: 1435–1442 Weiner H, Weiner H and Stitt M (1993) Sucrose-phosphate synthase phosphatase, a type 2A protein phosphatase, changes

189 its sensitivity towards inhibition in inorganic phosphate in spinach leaves. FEBS Lett 333: 159–164 Werdan K, Heldt HW and Milovancev M (1975) The role of pH in the regulation of carbon fixation in the chloroplast. Biochim Biophys Acta 396: 276–292 Wirtz W, Stitt M and Heldt HW (1982) Light activation of Calvin cycle enzymes as measured in pea leaves. FEBS Lett 142: 223–226 Wolusiuk RA, Perelmuter ME and Chehebar C (1980) Enhancement of chloroplast fructose 1,6-bisphosphatase activity by fructose 1,6-bisphosphate and dithiol-reduced thioredoxin. FEBS Lett 109: 283–293 Woodrow IE (1986) Control of the rate of photosynthetic carbon dioxide fixation. Biochim Biophys Acta 851: 181–192 Woodrow IE (1994) Control of steady-state photosynthesis in sunflowers growing in enhanced Plant Cell Environ 17: 277–286 Woodrow IE and Berry JA (1988) Enzymatic regulation of photosynthetic carbon dioxide fixation. Ann Rev Plant Physiol Plant Mol Biol 39: 533–594 Woodrow IE and Mott KA (1988) Quantitative assessment of the degree to which ribulose 1,5-bisphosphate carboxylase/ oxygenase determines the steady-state rate of photosynthesis during sun-shade acclimation in Helianthus annus L. Aust J Plant Physiol 15: 253–262 Woodrow IE and Mott KA (1992) Biphasic activation of ribulose bisphosphate carboxylase in spinach leaves as determined from non steady-state exchange. Plant Physiol 99: 298– 303 Woodrow IE and Mott KA(1993) Modelling photosynthesis: A sensitivity analysis of the photosynthetic carbon-reduction cycle. Planta 191: 421–432 Woodrow IE and Walker DA (1983) Regulation of stromal seduheptulose 1,7-bisphosphatase activity and its role in controlling the reductive pentose phosphate pathway of photosynthesis. Biochim Biophys Acta 722: 508–516 Woodrow IE, Murphy JM and Latzko E (1984) Regulation of seduheptulose 1,7-bisphosphatase activity by pH and concentration. J Biol Chem 259: 3791–3795 Woodrow IE, Furbank RT, Brooks A and Murphy DJ (1985) The requirements for a steady-state in the reductive pentose phosphate pathway of photosynthesis. Biochim Biophys Acta 787: 263–271 Woodrow IE, Ball JT and Berry JA (1990) Control of photosynthetic carbon fixation by the boundary layer, stomata and ribulose 1,5-bisphosphate carboxylase/oxygenase. Plant Cell Environ 13: 339–347 Worrell AC, Bruneau JM, Sommerfelt K, Boersig M and Voelker TA (1991) Expression of a maize sucrose-phosphate synthase in tomato alters leaf partitioning. Plant Cell 3: 1121–1130 Yin Z-H, Neimanis S, Wagner S and Heber U (1990) Light dependent pH changes in leaves of plants. I. Recording pH changes in various cellular compartments by fluorescent probes. Planta 182: 244–252 Yokota A and Canvin DT (1985) Ribulose bisphosphate carboxylase/oxygenase activity determined with bisphosphate in plants and algae. Plant Physiol 77: 735–739 Yin Z-H, Neimanis S, Wagner U and Heber U (1990) Light dependent pH charges in leaves of plants. Planta 182: 244– 269

190 Zrenner R and Stitt M (1991) Comparison of the effect of rapidly and gradually developing water stress on carbohydrate metabolism in spinach leaves. Plant Cell Environ 14: 939–946 Zrenner R, Willmitzer L and Sonnewald U (1993) Analysis of the

Mark Stitt expression of potato uridine phosphate glucose pyrophosphorylase and its inhibition by ‘antisense’ RNA. Planta 190: 247–252

Chapter 7 Carbon Metabolism and Photorespiration: Temperature Dependence in Relation to Other Environmental Factors Richard C. Leegood Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2UQ, U.K.

Gerald E. Edwards Botany Department, Washington State University, Pullman, WA 99164-4238, USA

Summary

I. II. III IV.

General Philosophy Stomatal Versus Biochemical/Photochemical Limitations Changes in Biochemical Versus Photochemical Efficiency Effects of Temperature on Metabolism A. Immediate Responses of Metabolism to Changes in Temperature V. Effects of Temperature on Photosynthesis in Plants A. Temperature Dependence of Ribulose 1,5-bisphosphate Carboxylase-oxygenase Kinetics and Photorespiration B. Simplified Model for Predicting Temperature Dependence of Photosynthesis Under Saturating Light Based on Ribulose 1,5-bisphosphate Carboxylase-Oxygenase Kinetics C. Temperature Dependence of Electron Transport D. Sensitivity to Low Temperature 1. Low Temperature in the Light 2. Low Night Temperatures E. Effect of Superoptimal Temperature on Photosynthesis 1. Effects on Enzymes and Metabolite Pools 2. Effects on Photochemistry 3. Sensitivity of Chloroplast Ribosomes VI. Effects of Temperature on Photosynthesis A. Changes in Photosynthesis and Metabolite Pools B. Properties of Rubisco C. Models for Predicting the Temperature Dependence of Photosynthesis D. Sensitivity to Lower Temperature 1. Effects on Maximum Quantum Yield of Assimilation and Carboxylation Efficiency Labeling in Chilling Sensitive Species 2. Metabolite Pools and 3. Loss of Activity of Enzymes a. In Vitro Studies b. In Vivo Studies 4. State of Activation of Enzymes 5. Metabolite Transport 6. Limitations on Photosynthesis Following Cool Nights E. High Temperature Tolerance Limits VII. Effects of Temperature on Crassulacean Acid Metabolism VIII. Temperature Compensation in Photosynthetic Metabolism Neil R. Baker (ed): Photosynthesis and the Environment, pp. 191–221. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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IX. Effects of Temperature on Carbon Partitioning to Starch and Sucrose A. End Product Inhibition B. Role of Phosphate C. Temperature Dependence of Starch and Sucrose Synthesis D. Dark Metabolism E. Temperature Dependent Effects on Phloem Translocation and Sink Activity X. Acclimation of Photosynthesis to Temperature Shifts A. Examples of Occurrence B. Factors Underlying the increase in Photosyntnetic Capacity after Acclimation to Low Temperatures 1. Acclimation of Electron Transport 2. Changes in Rubisco 3. Changes in Other Enzymes 4. Changes in the Capacity to Utilize Triose-phosphate 5. Changes in Protein Synthesis C. Possible Constraints on the Capacity for Acclimation References

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Summary Photosynthetic carbon metabolism is affected by a range of environmental factors. In this chapter we focus on the effect of temperature on photosynthesis in relation to other environmental factors. Plants grow over a wide range of temperatures and, apart from encountering large seasonal variations in temperature (including freezing), the aerial parts of a plant may face temperature variations of tens of degrees centigrade in a single day and smaller temperature changes in a matter of minutes. These are often coupled with changes in photon flux density and require a variety of regulatory responses. In addition, the mechanisms of photosynthesis associated with different photosynthetic types, and CAM, enable plants to perform better at specific temperatures, although they do not enable plants to tolerate temperature extremes which cause irreversible damage. It is known that different processes relating to photosynthesis, such as photochemistry, carboxylation or oxygenation by ribulose 1,5-bisphosphate carboxylase-oxygenase (and hence photorespiration), carbohydrate synthesis and export, dark respiration and growth have intrinsically different temperature sensitivities in vivo. Similarly, in a metabolic sequence, it is unlikely that the temperature dependence of the kinetic constants and the substrate concentrations of a series of enzymes will all change in the same manner. Hence it is inevitable that control will shift between different enzymes and component processes as the temperature changes. The lack of ability to accommodate these shifts in control will result in a limitation by one process and predispose the system to stress. A further important point to make concerning responses to low temperature is that induction of freezing tolerance and responses to low temperature are part of a continuum. Thus accumulation of sugars at low temperature as a result of decreased export can also be viewed as a mechanism which leads to cryoprotectant soluble sugars. Changes in partitioning can therefore be considered adaptive. The purpose of this chapter is to identify what limitations or shifts in regulation arise after exposure to different temperatures, with an emphasis on photosynthetic metabolism, and then to examine mechanisms by which these are overcome, either by short-term regulation or by longer term acclimation to changed temperatures and the circumstances in which these mechanisms fail.

I. General Philosophy Stress occurs when regulation fails and one component part of the regulatory network becomes overloaded. We must therefore understand regulation

and the limits within which it operates if we are to understand stress. The purpose of the following is, therefore, to focus on temperature in relation to other environmental factors, to show how regulation of photosynthesis is achieved and then to identify

Chapter 7 Carbon Metabolism and Temperature specific points at which regulation breaks down under stress. Of course, it is in the nature of stress that once one part of the system has failed to function, then the rest of the network of control will start to collapse. It is, therefore, often difficult unambiguously to identify the point at which a stress is primarily sensed.

II. Stomatal Versus Biochemical/ Photochemical Limitations As photosynthesis changes in response to variation of an environmental factor stomatal, biochemical and/or photochemical limitations may be involved. As noted by Farquhar (1988), if stomatal conductance remained constant with varying temperature, then the draw down of intercellular would be proportional to the assimilation rate. Stomatal conductance is not usually the primary limitation at high temperature per se, as high temperature does not cause a reduction in intercellular concentration unless it is associated with a high vapor pressure deficit (VPD) or drought (Monson et al., 1982; Long, 1985; Dai et al., 1992). Under a constant, or relatively low VPD, increased leaf temperature can increase stomatal conductance (Farquhar, 1988; Dai et al., 1992). A high VPD could develop by an increase in leaf temperature under low humidity in the air, cause stomatal closure, and hence limit the supply of for photosynthesis. The interactions between temperature and VPD are complex, and may be not only species dependent, but dependent on growth conditions (Aphalo and Jarvis, 1991). Of course, stomatal conductance can decrease and become limiting for photosynthesis if low soil water content is associated with high temperatures (Davies and Pereira, 1992; Chapter 14). Water stress inhibits photosynthesis and decreases stomatal conductance at high temperature but not under low temperature in Phaseolus vulgaris (Cornic and Ghashghaie, 1991).

193 III. Changes in Biochemical Versus Photochemical Efficiency Measurements of the effects of temperature on the maximum light utilization efficiency and the maximum carboxylation efficiency can provide information on the degree of photochemical versus biochemical limitations, as discussed later in this chapter (see also Chapter 8). The maximum quantum yield of assimilation is measured under conditions where photosynthesis is limited by light (determined from the initial slope of the rate of assimilation, A, versus absorbed light response curves); the maximum carboxylation efficiency is measured under conditions where photosynthesis is limited by (determined from the initial slope of A versus [intercellular response curves). If stress causes a decrease in it indicates a reduction in efficiency of utilization of absorbed quanta for fixation. This could occur due to reduced efficiency in excitation transfer to reaction centers, in the synthesis of ATP and NADPH, and the efficiency of utilization of this assimilatory power for fixation (e.g. as affected by the degree of photorespiration). If stress causes a reduction in it indicates a reduction in the efficiency of the carbon assimilation by an impairment of reaction(s) of photosynthetic carbon metabolism.

IV. Effects of Temperature on Metabolism

A. Immediate Responses of Metabolism to Changes in Temperature Metabolic responses of organisms to changes in temperature (temperature compensation) follow two patterns (excluding evolutionary, adaptational, changes), depending upon the length of exposure to the change in temperature. First, there may be

Abbreviations: A – rate of assimilation; CAM – crassulacean acid metabolism; – intercellular concentration of in the air space of the leaf; – maximum carboxylation efficiency; – activation energy; FBP – fructose 1,5-bisphosphate; FBPase – fructose 1,5bisphosphatase; – Michaelis-Menten constant of rubisco for – Michaelis-Menten constant of Rubisco for MDH – malic dehydrogenase; ME – malic enzyme; NADP-MDH – NADP dependent-malic dehydrogenase; Pi – inorganic phosphate; PGA – 3-phoshoglyceric acid; PEPC – phosphoenolpyruvate carboxylase; PPDK – pyruvate orthophosphate dikinase; PPFD – photosynthetically-active photon flux density; – temperature coefficient; – rate of respiration in dark; Rubisco – ribulose 1,5bisphosphate carboxylase/oxygenase; RuBP – ribulose 1,5-bisphosphate; SBPase – sedoheptulose 1,7-bisphosphatase; SPS – sucrose phosphate synthase; – relative specificity of Rubisco to function as a carboxylase versus as an oxygenase; – velocity of RuBP carboxylase; – velocity of RuBP oxygenase; VPD – vapor pressure deficit; – quantum yield of fixation at a given light intensity; – maximum quantum yield of fixation; – quantum yield of Photosystem II photochemistry; –compensation point for photorespiration in absence of dark respiration

194 instantaneous temperature compensation in metabolic processes. The notion of instantaneous compensation for changes in temperature arose from the observation that the resting metabolism of a wide range of invertebrates and reptiles which experience fluctuations in their thermal environment varied only slightly with temperature, with a of around unity (Hazel and Prosser, 1974). This type of temperature response has scarcely been considered in the context of plant metabolism, but it could be of considerable importance if it results in an effective increase in capacity as the temperature changes. In leaves, of course, short-term changes in temperature will also often be associated with changes in photon flux density (Fig. 1). Second, acclimation may occur over a period of several days or weeks as a result of differential gene expression. Temperature compensation can be achieved in a variety of ways. Short-term responses to temperature are unlikely to occur by increases in the amounts of enzymes, because the modulation of enzyme concentration as a function of temperature is both metabolically expensive and is relatively inefficient if the organism is confronted by large fluctuations in temperature (Somero, 1978). However, temperature will have a variety of effects on the kinetic properties of enzymes which can serve to modulate metabolism. For many enzymes that have been studied in animals, increases in temperature result in decreases in substrate affinity, and there is a tendency for the substrate affinity and the concentration of the substrate to track one another, a phenomenon often termed positive thermal modulation (Hochachka and Somero, 1984). In most situations positive thermal modulation would be expected because of the adverse, and potentially disastrous, effects that rises in substrate affinity might have when combined with the effect of decreasing temperature on reaction velocity. This would lead to extremely high values at lower temperatures. In practice, however, there are examples where this so-called ‘negative thermal modulation’ (Hochachka and Somero, 1984) occurs because it provides a very sensitive way of switching off a pathway. Temperature will also affect enzyme-modulator interactions, and there are possibilities for exploring how light activation, phosphorylation etc., which alter the kinetic properties of enzymes (changes in etc.) are employed by organisms so as to compensate temperature changes. There is good evidence that intracellular pH changes as the

Richard C. Leegood and Gerald E. Edwards

temperature is changed and that changes in may also occur (Minorsky, 1989). There are also opportunities for misinterpretation caused by changes in the properties of metabolic systems as the temperature changes. There are numerous reports of breaks in Arrhenius plots (i.e. sudden increases in activation energy at low temperatures) for both membrane-bound processes, such as succinate oxidation by isolated mitochondria (Raison, 1980), and for reactions catalysed by soluble enzymes (Krall and Edwards, 1993), suggesting a sudden predisposition to stress at a particular temperature. It is quite likely that some (but not necessarily all) of these phenomena are apparent, rather than real changes in the activation energy (Silvius et al., 1978, Krall and Edwards, 1993). For example, if an enzyme shows an increase in for its substrate as the temperature is lowered, then, unless the substrate concentration is kept fully saturating at all temperatures, the activation energy will apparently increase and, under certain conditions, it could do so with a convincing break in the Arrhenius plot.

V. Effects of Temperature on Photosynthesis in Plants Observations on the temperature dependence of photosynthesis made early this century showed that it had very variable temperature dependencies, which tended to rise to very high values at low temperatures and to low, or negative, values at higher temperatures as inactivating processes set in (Blackman, 1905; Warburg, 1919; Emerson and Green, 1934). In retrospect, the variability of the temperature response

Chapter 7 Carbon Metabolism and Temperature of photosynthesis is less surprising because it is now understood much better as the combined effect of temperature on a number of underlying processes, the importance of each of which can be dramatically influenced by light and

A. Temperature Dependence of Ribulose 1,5bisphosphate Carboxylase-oxygenase Kinetics and Photorespiration In plants photosynthesis has a steep response to temperature, but at atmospheric concentrations of photosynthesis has an essentially flat response to temperature (Fig. 2). This occurs because both the and for carboxylation by Rubisco have a similar temperature dependence when ambient concentrations of are at or below i.e. values of about 2.2 (Farquhar and von Caemmerer, 1982; Hall and Keys, 1983; Jordan and Ogren, 1984) and because the rate of photorespiratory release increases with increasing temperature. Higher temperatures promote oxygenation, and hence photorespiration, in two ways. First, the solubility of in water declines more rapidly than that of as the temperature is increased. For example, at 10 °C, the ratio of the solubilities of

195 to in water is 20, whereas at 40 °C it is 28 (Edwards and Walker, 1983). Second, the specificity factor of Rubisco decreases with increasing temperature in the range 7 °C to 35 °C (Jordan and Ogren, 1984). This is because the reaction mechanism of Rubisco (Andrews and Lorimer, 1987) involves a 2,3-enediol intermediate and its reaction with has a higher free energy of activation than the reaction with This means that oxygenation is more sensitive to temperature and increases faster than carboxylation as the temperature rises (Chen and Spreitzer, 1992). These effects cause to decrease with increasing temperature due to waste of energy in photorespiration and the increased energy requirements per fixed, whereas the is constant with varying temperature under saturating However, increases with increasing temperature due to increased capacity of the enzymes of carbon assimilation (Ku and Edwards, 1977; Edwards and Walker, 1983). While under atmospheric conditions the temperature response for uptake of in plants is rather flat with a low optimum, interpretations from fluorescence analysis indicate that PS II activity and gross rates of evolution show a steeper rise, and higher temperature optimum (Fig. 3). Also, rates of

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whole chain electron flow with increasing temperature are similar under photorespiratory versus nonphotorespiratory conditions (Weis and Berry, 1988; Oberhuber and Edwards, 1993). Rubisco activity is the predominant sink for reductant derived from photochemistry with 2 NADPH required per fixed by carboxylation and ca. 2 NADPH required per fixed by the oxygenase (Krall and Edwards, 1992). These results suggest that shifts between levels of carboxylation/oxygenation under normal air versus high have little effect on total Rubisco activity (sum of carboxylase and oxygenase activities), PS II activity and their temperature dependence.

B. Simplified Model for Predicting Temperature Dependence of Photosynthesis Under Saturating Light Based on Ribulose 1,5bisphosphate Carboxylase-Oxygenase Kinetics A simplified approach to predicting the photosynthetic response of plants to varying temperature at a given level is based on the kinetic properties

Richard C. Leegood and Gerald E. Edwards

of Rubisco (Farquhar, 1988). The ratio of oxygenase to carboxylase activities is defined as

where is the relative specificity factor (Jordan and Ogren, 1984). If it is assumed that under light saturation, ribulose 1,5-bisphosphate (RuBP) is saturating and photosynthesis is limited by Rubisco, then according to the model of Farquhar ( Sage and Sharkey, 1987; Farquhar, 1988):

is the velocity of carboxylation at substrate saturation, and and are the Michaelis constants for and Rd, rate of respiration in the dark, is used as an estimate of mitochondrial respiration with a of ca. 2 (Long, 1991; Collate et al., 1992; Edwards and Baker, 1993). The temperature

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Chapter 7 Carbon Metabolism and Temperature dependence of respiration and respiratory costs are similar in and plants, and appear not to be significant when considering differences between the net carbon gain between the two photosynthetic groups (Byrd et al., 1992). With increasing temperature the ratio increases, as noted earlier, due to a decrease in and increase in the ratio as shown in the analysis of Jordan and Ogren (1984). With increasing temperature and increase in parallel. Taken together this results in plants having a somewhat flat and broad temperature response curve for photosynthesis. The ratio can be calculated using inputs of and at a given temperature (Jordan and Ogren, 1984). Alternatively, it can be calculated using the equation

where (in Pa) is the compensation point for photorespiration in the absence of dark respiration, with a temperature dependence as determined by Brooks and Farquhar (1985) of

where T is temperature in °C, and P is the atmospheric pressure in MPa. The model predicts a broad temperature response of photosynthesis at current ambient concentrations of and an increase in the temperature optimum with higher concentrations of which is consistent with changes in the kinetic properties of Rubisco (Edwards and Ku, 1990; Long, 1991). In a detailed analysis Long (1991) showed how interactions between changes in levels and global warming could affect the rate of photosynthesis in plants. The results are in agreement with various data which show that the temperature optimum rises with increasing and the degree of enhancement of photosynthesis by increases with increasing temperature (Fig. 3). Models based on the kinetic properties of Rubisco have also been applied to a range of temperatures which show that photosynthesis becomes insensitive to below a specific temperature which is species dependent (Sage and Sharkey, 1987). Likewise, the degree of increase in the ratio of quantum yield of PS II/quantum yield of fixation in species under photorespiratory versus

nonphotorespiratory conditions, over a temperature range of 15 to 45 °C, can be explained based on the temperature-dependent change in the kinetic properties of Rubisco and partitioning of carbon flux into photorespiration (Oberhuber and Edwards, 1993). Of course there are various ways in which temperature can have an effect on photosynthesis other than through a direct effect on Rubisco. Temperature effects at other sites can cause a change in the flux of carbon through Rubisco either by limiting the availability of RuBP or changing the state of activation of Rubisco, particularly at temperature extremes.

C. Temperature Dependence of Electron Transport In air at temperatures between 20 °C and 30 °C under low to moderate light, photosynthesis in plants is generally poised between limitation by Rubisco and limitation by the rate of RuBP regeneration, although as we shall see, Rubisco inactivation at higher temperatures may modify this and metabolism may similarly be limiting at lower temperatures (see below). The temperature dependence of electron transport has been measured in experiments with isolated thylakoids (Armond et al., 1978; Nolan, 1980; Badger et al., 1982; Stidhamet al., 1982) and calculated from gas-exchange studies on leaves (Kirschbaum and Farquhar, 1984). The results are very similar, in showing a of around 2 at lower temperatures, and an optimum for the rate of electron transport which approximately coincides with the temperature optimum for whole leaf photosynthesis (Björkman et al., 1976, 1978).

D. Sensitivity to Low Temperature 1. Low Temperature in the Light Evidence for the responses of various steps in metabolism to both low and high temperature comes from work on leaves and from isolated chloroplasts. The autocatalytic nature of the Benson-Calvin cycle means that, even though individual reactions may have a of about 2, feedback through the cycle raises the overall (Baldry et al., 1966; Selwyn, 1966). In pea chloroplasts, the values for fixation rose as high as 9 below 15 °C (Baldry et al., 1966). However, Leegood and Walker (1983) and

198 Mächler et al. (1984) showed that the phosphate optimum for isolated wheat chloroplasts is both raised and broadened as the temperature is lowered. Optimal rates of fixation are, therefore, achieved at a different phosphate status at different temperatures. Consequently, the for chloroplast fixation measured under an optimal supply of phosphate is considerably lower (Leegood et al., 1985). Low temperature can cause impairment of photosynthesis through effects on carbon metabolism rather than by photochemical damage due to excess light. The metabolic evidence shows differences in regulation depending upon the concentration. In barley leaves in high light and high the triosephosphate/RuBP ratio (an indicator of the ability of the system to regenerate RuBP) remained roughly constant between 30 °C and 10 °C but decreased at 5 °C (Stitt and Grosse, 1988; Labate et al., 1990). Similarly, the mass-action ratio employed by Dietz and Heber (1984) to describe the reactions of RuBP regeneration decreased when the temperature was decreased from 30 to 12 °C. Both indicate that RuBP regeneration is maintained, despite conditions in which the supply of ATP and NADPH may be restricted (Dietz and Heber, 1986). However, under conditions of light and saturation, if photosynthesis becomes limited by the rate of utilization of triose-phosphate it becomes insensitive (Leegood et al., 1985; Sharkey, 1985a,b). In this case RuBP generation can be affected in quite a different way, becoming limited by the supply of phosphate (Pi). In contrast to the above observations, in high light at low the ratio triose-phosphate/RuBP increased in barley leaves as the temperature was decreased, suggesting that regeneration of RuBP becomes restricted as the temperature is lowered. This occurred despite the continued availability of ATP and reductant, indicated by a large fall in the glycerate 3phosphate/triose-phosphate ratio (Stitt and Grosse, 1988; Labate et al., 1990). This decrease in the utilization of the products of electron transport within the Benson-Calvin cycle is also evidenced by the rise in fructose 1,6-bisphosphate (FBP) at low temperature (Stitt and Grosse, 1988; Labate et al., 1990) and suggests that enzymic regulation within the Calvin cycle rather than the supply of energy is responsible for the decreased capacity for RuBP regeneration at low temperature. Tomato is much more chilling sensitive than

Richard C. Leegood and Gerald E. Edwards spinach, e.g. when exposed to 4 °C under high light. Interestingly, impairment of photosynthesis by chilling of tomato is due to effects on carbon metabolism rather than due to photoinhibition and damage to photochemistry. Inhibition of photosynthesis has been studied under two conditions, one while under low temperature, and the other following rewarming of plants following chilling treatments. During photosynthesis under low temperature RuBP and pentose-phosphate accumulated while glycerate 3-phosphate decreased. It was suggested that the catalytic efficiency of Rubisco was reduced, but this was not accounted for by changes in the activation state (which was slightly higher at 4 °C than at 30 °C). Also, there was an inexplicable drop in under chilling temperatures (Sassenrath and Ort, 1990). After exposure to low temperature and rewarming to 30 °C, photosynthesis was impaired, but in this case there was no apparent Rubisco limitation as the RuBP pool was lower than in untreated plants. It was suggested that photosynthesis was limited by a decrease in activities of the stromal bisphosphatases as amounts of FBP and sedoheptulose 1,7-bisphosphate both increased (Sassenrath et al., 1990; Sassenrath and Ort, 1990). Finally, if plants are exposed to excess light under low temperature photoinhibition can occur and cause a subsequent limitation on RuBP regeneration, particularly under low light. It is well documented in both and species that this results in a decrease in the indicating a reduction in the efficiency of production of assimilatory power (Baker, 1994; Chapter 15). Treatments which cause photoinhibition my also cause a decrease in in species (e.g. cucumber; Baker et al., 1988).

2. Low Night Temperatures Low night temperatures, of limited duration, may have a greater effect on carbon metabolism than on photochemistry. Limited periods of chilling in the dark have little or no effect on whereas chilling in high light causes a substantial reduction in this parameter. In tomato and olive low night temperature causes a drop in the maximum rates of photosynthesis without affecting (Martin and Ort, 1982; Bongi and Long, 1987). This is probably due to a direct effect of low temperature on photosynthetic carbon metabolism. Thus, chilling in the dark does not affect the efficiency of photochemistry nearly so much as chilling in the light

Chapter 7 Carbon Metabolism and Temperature since excess light causes photoinhibition and decrease in However, prolonged exposures of plants to low temperatures in the dark can result in a decrease in indicating an effect on photochemical efficiency (Baker et al., 1988).

E. Effect of Superoptimal Temperature on Photosynthesis 1. Effects on Enzymes and Metabolite Pools High temperatures can cause both reversible and irreversible effects on photosynthesis. In both and plants the temperature optimum for photosynthesis is well below the thermal tolerance limit. Above temperatures of about 20 °C, Baldry et al. (1966) observed a decrease in for photosynthesis in isolated pea chloroplasts to values below 2. Similarly low values in this temperature range had been observed by Emerson and Green (1934) and by Warburg (1919). Baldry et al. (1966) attributed this decline in above 20 °C to ‘heat-inactivation.’ The rate of fixation therefore remains relatively constant over quite a wide temperature range. The primary effect of mild heating on photosynthesis is probably its effect on membrane structure (Weis, 1982). Weis (1981 a) investigated the inhibitory action of mild heating in spinach leaves and chloroplasts. In the region 20 °C to 40 °C, the inactivation of assimilation was readily reversible (see also Monson et al., 1982). Heat-treated samples showed a large increase in the ratio of RuBP to glycerate 3-phosphate and a large decrease in the ratio of glycerate 3phosphate to triose phosphates, indicating an inhibition of Rubisco but maintenance of reduction of glycerate 3-phosphate. There was no evidence of adverse effects of mild heating upon ATP/ADP ratios or stromal pH, although light-scattering and the fast electrochromic shift were both influenced by heating and changed in parallel with the changes in the rate of assimilation. Mild heating interfered with the mechanism of activation of Rubisco, perhaps by interfering with the Rubisco activase system (Robinson and Portis, 1989), but not of other lightregulated enzymes of the Calvin cycle (Weis, 1981 a,b). Studies with wheat (Kobza and Edwards, 1987) and with cotton (Weis and Berry, 1988) showed a substantial decline in the activation state of Rubisco with increasing temperature. Whether the inactivation of Rubisco at above optimum temperatures is due to a direct effect on Rubisco, or is a secondary response

199 to effects on photochemistry or other temperatureinduced imbalances in the cycle is not known. However, this, together with temperature-dependent increases in photorespiration, is a major reversible constraint on photosynthetic metabolism which limits photosynthesis at higher temperatures. In the desert evergreen Nerium oleander, thermal instability of enzymes appeared unlikely to account for inhibition of fixation by high temperatures in leaves (Badger et al., 1982). Stitt and Grosse (1988) have suggested that, at higher temperatures (30 °C), low metabolite pools may be insufficient to maintain RuBP regeneration in the Calvin cycle. However, the results of Labate et al. (1990) suggest that RuBP regeneration is not adversely affected by high temperatures. A degree of inactivation of Rubisco at higher temperatures may regulate the cycle, reducing the flux required for RuBP regeneration, and preventing depletion of metabolites. In summary, the main effects of high temperature on photosynthetic metabolism in plants, which cause a reversible decrease in photosynthesis above the temperature optimum, are the increase in photorespiration and the inactivation of Rubisco.

2. Effects on Photochemistry A reversible down-regulation of PS II efficiency under high temperature can be associated with a limitation on Rubisco activity, i.e. via inactivation of the enzyme or by limited Rubisco oxygenase activity and photorespiration under artificially low concentrations of High light intensity may increase thermal stability by increasing membrane energization and down regulation of PS II (Weis and Berry, 1988). Thermal tolerance limits exist between ca. 40– 50 °C where irreversible, or slowly reversible, changes occur due to effects on photochemistry (Weis and Berry, 1988). These effects at high temperature can account for the decline in and a disruption of thylakoid membrane integrity which could obviously limit RuBP regeneration (Berry and Björkman, 1980; Monson et al., 1982; Weis and Berry, 1988). This may occur via (i) a decrease in efficiency of photophosphorylation, possible due to a decline in the proton electrochemical potential across the thylakoid membrane, which is slowly reversible, and (ii) thermal inactivation of PS II. The latter could be due to (i) heat inactivation of PS II by disruption of electron transfer to reaction centers from water;

200 (ii) disturbance of the lateral distribution of pigments between the photosystems, (iii) restriction of PS II reaction center photochemistry, possibly due to a detachment of the center from the core antenna, resulting in a large rise in the minimal fluorescence level

3. Sensitivity of Chloroplast Ribosomes A separate consideration is the effect of high temperature on the development of the photosynthetic apparatus. In plants high temperature may inhibit chloroplast biogenesis by causing a deficiency in chloroplast ribosomes. This has provided a useful means of identifying translation products of 80S ribosomes and the role of the cytoplasm in chloroplast biogenesis through studies on the high temperature (32 °C)-induced ribosome deficient plastids in rye (Feierabend and Schrader-Reichhardt, 1976; Hoinghaus and Feierabend, 1985). A specific reduction of 70S ribosomes has been observed in several species grown under high temperature, but not with the species maize (Feierabend and Mikus, 1977). The activity of Rubisco is severely reduced in the high temperature-grown plants of rye, while there is only a moderate reduction or no effect on the activities of nuclear-encoded proteins (Feierabend and Schrader-Reichhardt, 1976).

VI. Effects of Temperature on Photosynthesis

A. Changes in Photosynthesis and Metabolite Pools plants normally operate at saturating, or near saturating, concentrations of As a consequence, Rubisco is much more sensitive to temperature than in plants. Photosynthesis, therefore, shows a strong temperature dependence under high light with a of around 2, reflecting the influence of temperature on the maximum velocity of carboxylation (Berry and Farquhar, 1977). Instantaneous temperature compensation in plants is, at first sight, less necessary than in plants, because photosynthesis shows a strong temperature dependence. However, in low light there is again a relatively flat response to temperature (Fig. 2; Ludlow and Wilson, 1971a; Long et al., 1983; Oberhuber and Edwards, 1993) and if changes in photosynthetic metabolites are also

Richard C. Leegood and Gerald E. Edwards considered, then a different picture emerges. Unlike plants there is a large fall in total phosphorylated metabolites, particularly in metabolites of the cycle, at low temperatures (Fig. 4; Labate et al., 1990). Labate et al. (1990) suggest that the reason for this decline in metabolite pools at low temperature is related to the role of these compounds in intercellular transport. In leaves of plants, the exceptionally large pools of photosynthetic intermediates such as glycerate 3-phosphate, triose-phosphate and pyruvate reflect the requirement for diffusion-driven intercellular transport between the bundle-sheath and the mesophyll (Leegood and Osmond, 1990). As photosynthetic fluxes decline, so metabolite gradients of acids, glycerate 3-phosphate, triose-phosphate and pyruvate between the bundle-sheath and mesophyll would decline. On the other hand, intermediates of the Calvin cycle or of sucrose synthesis, hexose-phosphate behave similarly in and in plants in that they rise or are maintained at low temperature (Labate et al., 1990). This is presumably because hexose-phosphates are involved only in the Calvin cycle and in product synthesis and are not involved in intercellular transport processes.

B. Properties of Rubisco As noted above, there is a marked rise in photosynthesis in plants with increasing temperature under high light. This raises the question of whether Rubisco is saturated with the substrates (via the cycle) and RuBP (via photochemical regeneration of RuBP), in which case the temperature response would reflect the activity and energy of activation of Rubisco, provided that there is not a high temperature inactivation of the enzyme as has been observed in plants. In early work by Björkman and Pearcy (1971), at temperatures below 18 °C, uptake by intact leaves of the plant Atriplex rosea was found to have an activation energy similar to that of Rubisco extracted from leaves of the same species and assayed in vitro. In this case, over a moderate temperature range (12 to 18 °C) Rubisco may be functioning under near saturating levels of and RuBP. As the temperature rises above 18 °C photosynthesis decreases well below rates which would be expected based on the of Rubisco (Björkman and Pearcy, 1971). This may be due to a decrease in the of Rubisco, the state of activation of Rubisco, or decreased levels of substrates (RuBP or There is some evidence that Rubisco in vitro

Chapter 7 Carbon Metabolism and Temperature has a lower at higher temperatures (Björkman and Pearcy, 1971; Badger and Collatz, 1977). Whether this reflects the properties of the enzyme in vivo is uncertain. It remains to be determined whether the size of the inorganic carbon pool in the bundle sheath cells is maintained at a sufficient level to saturate Rubisco and limit oxygenase activity at high temperature, although photorespiration is not apparent from gas exchange and fluorescence analysis. The size of the pool is determined by the rate of the cycle, the leakiness of the bundle sheath to inorganic carbon (which may increase at high temperature), and the rate of utilization of in the bundle sheath (Jenkins et al., 1989). As noted earlier, with Rubisco from plants, the and rise approximately in parallel as temperature is increased (Badger and Collatz 1977, Jordan and Ogren 1984), which limits the temperature response of photosynthesis at low If the of plant Rubisco increases likewise with increasing temperature, a higher pool may be required at higher temperatures for saturation (Edwards and Ku, 1990). The pool size of RuBP and inorganic carbon, the state of activation of Rubisco, and the of Rubisco need to be evaluated to understand how provision of via the cycle, regeneration of RuBP, and properties of Rubisco are related to the response to higher temperatures. Other research on the plant maize, has shown that growth temperature (19, 25 and 31 °C) had no significant effect on Rubisco activity while having a marked effect on the maximum carboxylation efficiency, The maximum capacity for photosynthesis (measured at 30 °C) was similar in the 19 and 25 °C-grown plants and about 20% lower in the 31 °C-grown plants (Ward, 1987). However, the dramatic decrease in with increasing growth temperature was highly correlated with a decrease in activity of pyruvate orthophosphate dikinase (PPDK). It was suggested that the capacity to deliver to the bundle sheath cells by the cycle decreases relative to the capacity for net assimilation with increasing growth temperature. Therefore, plants grown under high temperature may have a problem in providing Rubisco with saturating

C. Models for Predicting the Temperature Dependence of Photosynthesis As noted above, photosynthesis in plants is thought normally to function with Rubisco close to saturation

201 with At 30 °C and moderate to high levels of light the concentration in the bundle sheath compartment is estimated to be 25 to 70 (Jenkins et al., 1989; Dai et al., 1993). This would result in a ratio for Rubisco carboxylation/oxygenation of ca. 8:1 to 20:1, and a rate of production by photorespiration which is only 2 to 6% of (considering 1 released in photorespiration for each oxygen reacting with RuBP). Increasing temperature over a wide range (15 to 40 °C) has no effect on the quantum yield of fixation in plants (Ehleringer and Björkman, 1977; Ku and Edwards, 1978; Dai et al., 1993) and the net rate of uptake and PS II activity increase in a similar manner due to low photorespiration (Fig. 3, Oberhuber and Edwards, 1993). If Rubisco functions under near saturating levels of over a range of temperatures, then the temperature response of photosynthesis would be largely independent of the of Rubisco and unaffected by Rubisco oxygenase. Under saturating levels of RuBP at high light levels in the absence of photorespiration Eq. (2) used earlier for photosynthesis would simplify to :

If the concentration in the bundle sheath cells is high relative to then the value of will have limited effect on the rate of fixation. Whether the ratio of ever decreases in the bundle sheath compartment with increasing temperature such that is limiting for Rubisco is uncertain, but this is clearly a possibility under water stress. Under high RuBP and photosynthesis rates in plants would be controlled by the temperature dependence of of Rubisco, which accounts for the strong temperature dependence of photosynthesis under high light (Edwards and Ku, 1990; Collatz et al., 1992). Collatz et al. (1992) developed a model of photosynthesis (including inputs for the temperature dependence of Rd, and considering the absence of photorespiration and inhibitor functions for the upper and lower temperature limits of and Rd) which generates temperature response curves typical of plants. There are obvious limitations to predicting response curves of photosynthesis at the temperature extremes which severely impair photochemistry or carbon assimilation. Another model for predicting rates of fixation (A) in maize is to use two measured inputs, PPFD

202 absorbed by the leaf and quantum yield of PS II plus (which is a constant representing the relationship between (Edwards and Baker, 1993) where

The predicted rates obtained by this method are very similar to measured rates of fixation by gas exchange over a wide temperature range (15–45 °C) at two PPFDs, which supports evidence that PS II activity is closely linked to fixation in plants due to low levels of photorespiration. Finally, while photorespiration is relatively low in plants, it does occur (DeVeau and Burris, 1989; Edwards and Baker, 1993), and may be significant in considering the temperature-dependence of photosynthesis under water stress when the intercellular levels of are limiting. In maize under limiting photosynthesis does become sensitive to and under limiting the quantum yield of photosynthesis does decrease with increasing temperature (Dai et al., 1993).

D. Sensitivity to Lower Temperature plants are largely absent from cold-environments but they can experience low temperature during their life cycle. Although many plants are prone to chilling injury and dramatic drops occur in photosynthesis below ca. 12 °C (Long et al., 1983; Baker, 1994), studies suggest that there are no particularly cold-sensitive steps in the pathway in vivo in cold tolerant Atriplex species grown at low temperatures (Caldwell et al., 1977) or in the cold tolerant Spartina townsendii when evaluated at 10 °C versus 25 °C (Thomas and Long, 1978; Long, 1983). In considering temperature extremes, at chilling temperatures the pathway is not considered inherently detrimental, nor is it likely to have any properties which render it more resistant to high temperature damage (Berry and Björkman 1980; Edwards et al., 1985a). Also, while stomatal conductance may be a factor in some cases, the chilling sensitivity of photosynthesis is considered to be largely a result of an influence on photochemistry and biochemistry. For example, under chilling temperatures in maize the primary limitation is not an effect on stomatal conductance but a reduction in mesophyll conductance (Long et al., 1983). However,

Richard C. Leegood and Gerald E. Edwards we are very ignorant of the metabolic changes which occur when the leaves of plants are subjected to abrupt changes to low temperatures and during chilling stress. Whether the primary sensitivity to low temperature resides in the photochemistry, metabolite transport, or enzymes of carbon assimilation, or whether there are multiple, direct effects at all of these levels is uncertain.

1. Effects on Maximum Quantum Yield of Assimilation and Carboxylation Efficiency Ludlow and Wilson (1971b) found that a number of tropical grasses (Cenchrus ciliaris, Panicum coloratum, P. maximum, Melinis minutiflora, Brachiaria ruziziensis, and Setaria sphacelata) had lower rates of photosynthesis when plants were grown at 20 °C and photosynthesis measured at 30 °C, than when plants were grown at 30 °C. By comparison, there was little effect on legumes exposed to the same treatments. The grasses grown at the lower temperature had a lower mesophyll conductance whereas there was little effect on which suggests the restriction on photosynthesis is due to a biochemical rather than a photochemical limitation. It was suggested low photosynthesis rates in plants grown at 20 °C may be due to a feedback effect by accumulated photosynthate, or a deficiency in enzymes of carbon assimilation. Interestingly, when the 20 °C-grown plants were exposed to 30 °C for one night they showed near complete acclimation in photosynthetic performance at 30 °C. Although the basis for this limitation is unknown, the acclimation resulted in a large increase in mesophyll conductance, without affecting stomatal conductance, indicating that the restriction in photosynthesis in the plants grown under low temperature is at the chloroplast level. Chilling of maize plants under high light produces an irreversible loss of capacity to assimilate directly proportional to the light received during chilling, with effects on photochemistry and carbon metabolism (Long et al., 1983). Following a chilling treatment of maize for 6 h under high light (1500 conditions which cause photoinhibition, both and are severely reduced (Baker et al., 1988). However, recovered within 1 h after returning to 20 °C, whereas recovered more slowly. These results suggest that both photochemistry and carbon metabolism are effected by chilling injury, and that carbon metabolism

Chapter 7 Carbon Metabolism and Temperature recovers more rapidly than photochemistry once the stress is removed. The recover of within 1 h suggests a readily reversible process (e.g. reactivation of inactivated protein) rather than damage which requires protein synthesis. The architecture of the plant and density of the canopy will effect whether or not leaves are functioning at limiting light. In canopies where many leaves are exposed to light well below saturating levels, a major limitation on photosynthesis following chilling stress may be linked to a decrease in (Baker et al., 1988; Baker and Ort, 1993). However, in plants having leaves exposed to higher light intensities (e.g. in crops early in the growing season well before a full canopy develops), chilling induced decreases in photosynthesis may be linked to a reduction in A reduction in indicates a decline in efficiency in producing assimilatory power. Since levels of ATP in the leaves of species increase dramatically during chilling stress prior to obvious cellular damage (Taylor et al., 1972), the production of NADPH through noncyclic electron flow may be more restricted than production of ATP via cyclic photophosphorylation.

2. Metabolite Pools and Chilling Sensitive Species

Labeling in

In cold-sensitive Sorghum bicolor under low temperature restrictions appear to develop in the interconversion of pathway intermediates and/or transport of metabolites (Taylor et al., 1972; Brookings and Taylor, 1973; Long, 1983). S. bicolor is a NADP-ME type species with malate as the predominant product of the cycle. Treatment of S. bicolor at 10 °C for 10 min followed by pulse chase experiments at 10 °C caused retention of a high percentage of the label in malate compared to the controls at 25 °C. Longer exposures to 10 °C (6.5 to 30 h) resulted in aspartate becoming the predominant labeled acid, with a subsequent slow transfer of label from aspartate during the chase period. Thus, under extended cold stress the synthesis of malate appears to be impaired. When plants are transferred from 25 °C to 10 °C the pool size of alanine dramatically decreases while that of aspartate increases (Taylor et al., 1972). Thus, it appears low temperature has specific effects on the function of the cycle, although it is not possible from these studies to determine which steps are cold sensitive. Irreversible damage can occur under long exposures (Taylor et al., 1974).

203

3. Loss of Activity of Enzymes a. In Vitro Studies Dissociation of oligomeric proteins at low temperature, resulting in a loss of activity, is a common phenomena among highly regulated mammalian enzymes (Bock and Frieden, 1978). In higher plants there have also been reports of changes in a number of functional aspects of enzymes in relation to changes in temperature. These include cold lability of enzymes, such as phosphofructokinase in potatoes (Dixon et al., 1981), inactivation of the cytosolic FBPase at lower temperatures (Weeden and Buchanan, 1983), and enzymes of the (Edwards et al., 1985a,b). This occurs with two enzymes of the carboxylation phase of the pathway, PPDK and PEPC. These tetrameric enzymes isolated from some species are cold labile and dissociate into inactive dimers or monomers (Edwards et al., 1985b; Krall and Edwards, 1993). As with other cold labile enzymes, other factors such as enzyme dilution, the absence of substrates, or presence of high salt can also lead to dissociation. Although in a number of studies it has been shown via Arrhenius plots that there is a sharp transition in rates of catalysis at low temperature (around 12 °C) there is no evidence that this is due to a direct effect of temperature on enzyme catalysis. Rather it appears to reflect dissociation of a cold-labile enzyme which occurs during incubation under cold temperature prior to assay (Krall and Edwards, 1993). The species Panicum maximum has a cold labile PEPC which is inactivated by preincubation at low temperatures prior to assay at room temperature, whereas PEPC from P. miliaceum is cold tolerant. However, Arrhenius plots of catalysis at various temperatures (determined without preincubation under low temperature) show a similar slightly curvilinear response for the enzyme from these two species without any abrupt transitions (Krall and Edwards, 1993). The dissociation of enzymes under low temperatures may be due to a decreased stability of hydrophobic bonds and/or a temperature dependent change in the pK value of ionizable groups which control association-dissociation (Bock and Frieden, 1978). Although the bonding involved in the quaternary structure of PPDK and PEPC is not established, the following points can be made. PPDK is protected against cold inactivation by a number of inorganic ions whereas

204 PEPC is not (Krall et al., 1989; Krall and Edwards, 1993). The quaternary structure of PEPC has been suggested to be dependent on hydrophobia bonds. With the cold labile enzyme from Panicum maximum, which is known to stabilize hydrophobic bonds, prevents low temperature dissociation of PEPC, and Triton X-100, known to destabilize hydrophobic bonds, causes loss of activity of PEPC at room temperature (Krall and Edwards, 1993). The quaternary structure of PPDK may be dependent on both hydrophobic bonds and ionic interactions. Inactivation under low temperatures is pH dependent which is consistent with pK changes on ionizable groups, while protection by glycerol is consistent with hydrophobic interactions (Krall et al., 1989). PPDK and PEPC have been shown to be cold labile in some species and cold tolerant in other species in vitro (McWilliam and Ferrar 1974; Shirahashi et al. 1978; Krall and Edwards, 1993). McWilliam and Ferrar (1974) reported that species classified as cold sensitive have a cold labile PEPC in vitro, and species classified as cold tolerant have a PEPC which is cold tolerant in vitro. The cold sensitive PEP carboxylases had a sharp transition at low temperature around 12 °C, whereas the cold tolerant PEPC were without a transition in Arrhenius plots.

b. In Vivo Studies Although it is clear that PEPC and PPDK from some species are cold labile in vitro, whether this occurs in vivo, and whether it contributes to cold lability of photosynthesis is uncertain. It is possible that the enzyme is in a more protected environment in vivo considering the concentration of the protein and solutes which may have a protective effect (Krall and Edwards, 1993). At least under short term exposures, leaves of P. maximum having a cold labile PEPC and leaves of P. miliaceum having a cold tolerant PEPC, exhibited a similar temperature response curve for photosynthesis. Also, Taylor et al. (1974) observed no significant change in the extractable activity of PEPC from Sorghum bicolor or maize leaves after a 3-day chilling treatment at 10 °C, although chilling induced changes in chloroplast ultrastructure and decreased net uptake within 24 h after the low temperature treatment. On the other hand, there is some evidence that cold treatment of leaves of the plants sorghum, maize and Digitaria sanguinalis causes a loss of PPDK activity

Richard C. Leegood and Gerald E. Edwards which may be linked to dissociation (Taylor et al., 1974; Hatch, 1979; Sugiyama et al., 1979). Although chilling for 14h did not affect Rubisco activity in the grass Echinochloa crusgalli (Potvin et al., 1986), it did lead to reductions in the activities of enzymes of the pathway, particularly NADP-malate dehydrogenase (NADP-MDH) and PPDK. The reductions were significantly larger for plants from the warmer environment of Mississippi when compared with plants from the cooler environment of Quebec. As noted earlier, whether exposure of plants to low temperature leads to a limitation of the cycle and the concentrating mechanism is uncertain. If Rubisco functions under saturating or near saturating under low temperatures, then Rubisco itself may be limiting. plants have much lower levels of Rubisco compared to plants which could put at a disadvantage under low temperature (Edwards et al., 1985a). Besides the effect of exposure of mature leaves to chilling temperature, it is important to consider the consequences of chilling temperatures on chloroplast development. Substantial research by Baker and colleagues show that chilling conditions can severely impair development of the photosynthetic apparatus in maize (Baker, 1994; Baker and Nie, 1994). This includes Rubisco content and activities of photosynthetic enzymes (FBPase, PPDK, PEPC, NADP-MDH) as well as chlorophyll and thylakoid protein content. If PPDK and PEPC are cold labile in vivo, then it is possible that compatible solutes could function to protect the enzymes. Various compatible solutes, e.g. polyols, proline, betaine, and trimethylamine-Noxide, protect against cold inactivation of the enzymes in vitro (Selinioti et al., 1987; Krall et al., 1989; Krall and Edwards, 1993).

4. State of Activation of Enzymes It appears that limitations on photosynthesis under low temperature is not related to effects of temperarure on the state of activation of enzymes. Under approximately 50% full sunlight temperatures between 10 °C and 35 °C made no difference to the activation states of NADP-MDH and PPDK in maize leaves, but activation in low light (3% full sunlight) was highest under low temperature (Edwards et al., 1980). Low temperature also slowed modulation of these enzymes.

Chapter 7 Carbon Metabolism and Temperature

5. Metabolite Transport It is possible that transport of metabolites of photosynthesis across membranes of the chloroplast or mitochondria is cold sensitive, although there are few data (Edwards et al., 1985a). This could occur if there were limited fluidity of the membranes at low temperature due to structural properties which are associated with plants being of tropical or semitropical origin (e.g. relatively high degree of saturation of certain membrane lipids; see chapter 15).

6. Limitations on Cool Nights

Photosynthesis Following

There is a selective inhibition of mesophyll chloroplast development which occurs by exposure of chilling sensitive plants to night temperatures of 0 to 5 °C which results in the appearance of lateral chlorotic stripes (‘Faris banding’) across the emerging leaf blades (Slack et al., 1974). Enzymes of the pathway in mesophyll cells were strongly effected, while photosynthetic enzymesexamined in the bundle sheath were near normal activities in the striped bands. The selective effect was suggested to be at least in part due to a failure of plastid ribosome production in mesophyll cells. However, there is other evidence that deficiency in plastid ribosomes does not impair synthesis of cycle enzymes. There are mutants of maize which are very sensitive to lower temperature. When grown below a threshold temperature, the leaves are yellow, Rubisco content is particularly low, while levels of other photosynthetic enzymes, including the cycle, are normal. The selective effect on Rubisco protein is consistent with evidence that these mutants have a deficiency in chloroplast ribosomes when grown at low temperature (Edwards and Jenkins, 1988). Thus, the ‘Faris banding’ may be due to effects other than on plastid ribosome content since nuclear encoded photosynthetic enzymes of the pathway are affected. Finally, low night temperatures can also cause a reduction in photosynthesis without visual damage to leaves which is associated with an impairment of starch breakdown (see Section IX).

E. High Temperature Tolerance Limits High-temperature stress has been shown by Björkman and Badger (1977) to cause inactivation of certain

205 enzymes of the Benson-Calvin cycle in leaves of Atriplex sabulosa (a cool-coastal species intolerant of high temperatures) and in leaves of Tidestromia oblongifolia (a hot-desert species). A number of stromal and extrachloroplastic enzymes showed much greater thermal stability in T. oblongifolia, but for several enzymes their heat stability was too great to explain thermal inhibition of photosynthesis in the whole plant, while for NADP-dependent glyceraldehyde-phosphate dehydrogenase in A. sabulosa and possibly glycerate 3-phosphate kinase in T. oblongifolia, heat-inactivation occurred at around the same temperatures which caused inactivation of photosynthesis. Whether these losses of photosynthetic capacity at high temperatures are caused by inactivation of enzyme activity or whether, like photoinhibition, loss of activity results from impaired electron transport capacity and hence diminished light-activation is not clear (Berry and Björkman, 1980). PEPC and Rubisco have a higher thermal stability at 50 °C in heat tolerant sorghum and peanut compared to the less heat tolerant maize and soybean further evidence that heat tolerance is not linked to photosynthetic types (Ghosh et al., 1989). As thermal tolerance limits are exceeded (between 40 and 50 °C) plants are susceptible to irreversible damage to photochemistry, just as plants, including a severe drop in and increase in the minimal fluorescence level. However, there is some evidence that plants having evolved to tolerant higher temperatures have higher thermal breakpoints than species (Berry and Björkman, 1980; Seemann et al., 1984; Edwards et al., 1985a).

VII. Effects of Temperature on Crassulacean Acid Metabolism Two distinct thermal optima occur in CAM plants. There is a high optimum for daytime photosynthesis and a low optimum for nocturnal acid accumulation (Vickery, 1954; Smith and Nobel, 1986). In many facultative CAM plants which can function in a versus CAM mode, high day temperature/low night temperature favors the CAM mode (Haag-Kerwer et al., 1992). Studies of the temperature optima for PEPC/MDH and of NADP-ME in crude extracts of Bryophyllum tubiflorum showed that acid production was optimal at 35 °C while acid degradation did not

206 reach an optimum even at 53 °C. Overall the results showed that acid production predominates below 15 °C. There was also a shift to iso-citrate production at moderate temperatures (Brandon, 1967). The data suggest that temperature-dependent alterations in the properties of these enzymes were responsible. The temperature responses of the relative activities and substrate affinities of PEPC and NADP-malic enzyme alone could not account for this behavior (Osmond and Holtum, 1981), although PEPC has been shown to exhibit a three-fold increase in the (PEP) between 15 °C and 30 °C and a halving of the (malate) between 25 and 35 °C, both of which would tend to favor activation of the enzyme at lower temperatures (Buchanan-Bollig et al., 1984). However, it is an oversimplification to regard these simply as metabolic optima. Osmond (1978) pointed out that sufficient regard must be paid to the role of stomatal closure at higher temperatures in the reduction of acid accumulation and suggested that acid production may be relatively temperatureinsensitive in Opuntia inermis. In addition, an increase in dark respiration promoted by higher temperatures may lead to an increase in evolution and an apparent decline in dark fixation (Kaplan et al., 1976; Nobel, 1988). Essentially, therefore, interconversion between carbohydrate and malate must be relatively insensitive to temperature, since the rates of acidification and deacidification are comparable. Mechanisms for the immediate compensation of changes in temperature in the shared sequences of glycolysis and gluconeogenesis must, therefore, operate in these plants. Chardot and Wedding (1992) examined the effect of temperature on the regulatory properties of PEPC from Crassula argentea. Both and (MgPEP) increased between 11 and 35 °C such that there was a relatively small effect of temperature on the enzyme (cf. Rubisco). Glucose 6-phosphate rendered more responsive and the less responsive to temperature. Measurements of leaf metabolites after a 12h night period at different temperatures showed that glucose 6-phosphate remained relatively constant with temperature, and the sum of pyruvate and PEP rose at higher temperatures, perhaps ensuring maintenance of enzyme activity under warm nocturnal conditions (in which the affinity of PEPC for PEP falls). However, the content of malate at 35 °C was only a quarter of that at 10 °C. It was suggested that these low concentrations of malate at high temperatures may be inadequate to block PEP

Richard C. Leegood and Gerald E. Edwards carboxylation during the day and may thereby set a limit on the night-time temperature for the operation of CAM. Agave and cacti have the highest temperature tolerances of any terrestrial plants so far examined. Thermal tolerance limits for Agave americana and a number of cacti species is 55 °C compared with 47 °C for 35 other species. When acclimated to high temperature, a number of species of cacti can survive for 1 h at 70 °C, temperatures which are lethal for other species tested (Nobel, 1988).

VIII. Temperature Compensation in Photosynthetic Metabolism Instantaneous temperature compensation can be achieved by increasing the catalytic capacity of an enzyme or by increasing its substrate affinity as the temperature is lowered. Photosynthetic systems in particular possess the capacity to alter the properties and activities of enzymes in response to light. These include Rubisco, which is activated by and in turn catalysed by Rubisco activase (Portis, 1992) and, in a large number of plants, controlled by a tight-binding inhibitor, carboxyarabinitol 1phosphate (Seemann et al., 1990), a range of thioredoxin-modulated enzymes which include phosphoribulokinase, glyceraldehyde-phosphate dehydrogenase, sedoheptulose 1,7-bisphosphatase (SBPase) and the stromal FBPase, as well as cytosolic enzymes which are sensitive to light and which are modulated by protein phosphorylation, such as sucrose phosphate synthase (SPS), PEPC and nitrate reductase. Thus, temperature-dependent control of light modulation of certain enzymes is a means of regulating their capacity and/or substrate affinity. An increase in the activation states of enzymes at lower temperature would compensate for the strong temperature dependence of the Calvin cycle. Holaday et al. (1992) showed that very rapid increases occur in the activation states of Rubisco, the stromal FBPase and NADP-MDH in spinach leaves under light limited conditions when the temperature was lowered. This contrasted with an inability to activate these enzymes (except NADP-MDH) in bean, a chilling sensitive species, which resembles observations made in tomato (as discussed earlier; Sassenrath et al., 1990). In white clover, Mächler (1981) and Schnyder et al. (1984) showed that leaves which were preincubated (for up to an hour) at lower temperatures in the light

Chapter 7 Carbon Metabolism and Temperature or the dark had an increase in the activity of Rubisco. In wheat grown at 20 °C the activities of Rubisco and FBPase rose as the measurement temperature was reduced from 45 °C to 15 °C, indicating an increase in the activation state of these enzymes (Kobza and Edwards, 1987). Of course, these changes could result not simply from changes in temperature but from the fact that the light saturation of enzyme activation will occur at lower PFDs at lower temperatures. Many light activated enzymes are fully activated under high light, and this would allow little or no flexibility in the response to temperature. Thus in maize leaves at 30 °C, Rubisco appears to be fully activated (Usuda, 1985) and FBPase, NADP-MDH and PPDK reached maximum activation at a PPFD of about 700 (Usuda et al., 1984). The question then arises as to the temperature range at a given PPFD over which effective temperature compensation can occur. Weis (1981a) showed that low temperature treatment of intact chloroplasts in the dark promoted activation of Rubisco. Low temperatures also prevent dark inactivation of Rubisco (Bahr and Jensen, 1978) and slows modulation of enzymes. This then raises the further question as to how far the damping of regulatory responses occurs at low temperatures, whether they are within metabolism (e.g. light activation) or electron transport, and whether or not they adversely affect the leaf’s ability to respond to a fluctuating light environment, a feature which of great importance to the carbon economy of shade plants (Pearcy, 1988; Chapter 13) and which might also lead to greater susceptibility to photoinhibition. An alternative means of temperature compensation is to maintain or increase metabolite pools, while enzymes’ affinities for their substrates are maintained or increased as the temperature is lowered. Unfortunately data on the temperature dependence of the properties of enzymes and their substrates in vivo are particularly sparse in plants. However, maintained levels of stromal metabolites at low temperatures could partially compensate for the declining activity of the Calvin cycle enzymes and allow RuBP regeneration to be maintained over a wide range of leaf temperatures (Stitt and Grosse, 1988; Labate et al., 1990). Phosphorylated metabolites which are involved in metabolism and product formation have been shown to increase at low temperatures (Fig. 4; Leegood, 1985; Labate et al., 1990). In plants such as maize, although total

207 phosphorylated intermediates fall at low temperature (because they are involved in transport and thus are closely linked to photosynthetic fluxes) pools of intermediates which are not involved in intercellular transport, such as hexose phosphates, actually rise at low temperatures, as in plants (Fig. 4). In plants it is apparent that photosynthesis and photosynthetic sucrose synthesis at low temperatures require metabolite pools which are as high or higher than at higher temperatures (Leegood, 1985; Dietz and Heber, 1986; Stitt and Grosse, 1988).

IX. Effects of Temperature on Carbon Partitioning to Starch and Sucrose Low temperatures can potentially influence the carbohydrate content of leaves in a number of different ways both in the short- and in the long-term. In the intact plant, low temperatures will diminish growth, sink activity and export of carbohydrate from leaves, disrupting the balance between the supply of, and demand for, assimilated carbon. In the longer term, the increased ratios of source to sink activity which occur at low temperatures will also influence the partitioning of carbohydrate, resulting in an increase in the accumulation of alternative carbohydrate reserves such as starch (Chatterton et al., 1987) or fructans in grasses (Pollock, 1984; Chapter 10).

A. End Product Inhibition Temperature-dependent decreases in the export or utilization of photosynthate have been shown to lead to end-product inhibition of photosynthesis (AzconBieto, 1983; Azcon-Bieto and Osmond, l983; AzconBieto et al., l983; Bagnall et al., 1988; BlechschmidtSchneider et al., 1989 ). Under these conditions, it has been proposed that increased amounts of carbohydrate within the leaf could both modulate the rate of photosynthesis, by changing gene expression (Sheen, 1994) and also lead to an increase in the availability of respiratory substrates and thereby increase the rate of respiration (Azcon-Bieto et al., 1983; Lambers, 1985).

B. Role of Phosphate Phosphate appears to play a crucial role, in the shortterm, in the responses of photosynthesis to low temperature. This is not a nutritional limitation, but

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may arise because Pi is either not recycled to the chloroplast by adequate rates relative to rates of carbohydrate synthesis or because the concentration of Pi within the cytosol or chloroplast is temporarily sub-optimal. A number of pieces of evidence support the notion that in plants the system becomes phosphate-limited at low temperatures. These are: (i) an increase in oscillatory behavior in both gas exchange and chlorophyll fluorescence at low temperatures (Sivak and Walker, 1987; Leegood and Furbank, 1986); (ii) the glycerate 3-phosphate/triosephosphate ratio, an indicator of the availability of ATP and NADPH (Heber et al., 1986), increases at low temperatures if is above ambient concentrations, implying a decreased capacity for photophosphorylation (Dietz and Heber, 1986; Leegood and Furbank, 1986; Sharkey et al., 1986; Kobza and Edwards, 1987); (iii) there is an increased sensitivity of photosynthesis to Pi-sequestering agents such as glycerol at low temperatures (Leegood et al., 1988); (iv) the Pi optimum for photosynthesis in isolated intact chloroplasts increases and becomes broader at low temperatures (Leegood and Walker, 1983; Mächler et al., 1984); (v) phosphorylated metabolites

Richard C. Leegood and Gerald E. Edwards

which are involved in metabolism and product formation have been shown to increase at low temperatures and these would decrease free Pi in the cytosol and chloroplast in the short-term; (vi) abrupt transfer of leaves of many plants to a sufficiently low temperature results in a limitation of the rate of photosynthetic carbon assimilation by phosphate and feeding Pi then stimulates carbon assimilation (Labate and Leegood, 1988). In plants Pi limitation, at least in the short term, results in decreased partitioning into sucrose as reflected in increased ratios of starch/ sucrose whereas in plants Pi depletion results in a decrease in partitioning into starch (Herold et al., 1976; Usuda, 1991; Usuda and Shimogawara, 1991). There is evidence that the unexplained increased partitioning into starch in maize is a direct effect of low Pi, rather than as a consequence of reduction in photosynthetic capacity (Usuda, 1991; Usuda and Shimogawara, 1991). The causes of the Pi limitation of photosynthesis under low temperature may be mixed but the role of sucrose and starch synthesis in recycling Pi from triose-phosphate to support continued photophosphorylation is of crucial importance. This is revealed

Chapter 7 Carbon Metabolism and Temperature by the lack of and of photosynthetic carbon assimilation which occurs at low temperatures at ambient or higher concentrations of (Jolliffe and Tregunna, 1968, 1973; Canvin, 1978; Cornic and Louason, 1980; Arrabaca et al., 1981; McVetty and Canvin, 1981; Leegood et al., 1985; Sharkey, 1985a; Leegood and Furbank, 1986; Sharkey et al., 1986; Sage and Sharkey, 1987; Sharkey et al., 1995). insensitivity is revealed by a lack of stimulation of photosynthesis after changing from 21% to 2% In the case of photosynthesis no longer responds to increases in the concentration of in a region of the response curve where decreases in photorespiration would normally be expected to increase the rate of photosynthesis. Genetic modification of end-product synthesis into starch or sucrose alters the temperature at which photosynthesis becomes insensitive to (Sharkey et al., 1995). The situation at low temperatures, in particular, may be more complicated because reverse sensitivity is often observed, i.e. low actually inhibits the assimilation rate when compared with 21% (Harley and Sharkey, 1991). Reversed sensitivity to is also sometimes accompanied by an inhibition of the assimilation rate by at higher CO2 concentrations (Woo and Wong, 1983). Harley and Sharkey (1991) have developed a model which could explain this reversed sensitivity to by incomplete recycling of the products of photorespiration. In 21% release of Pi could occur at phosphoglycolate phosphatase and, providing it was not re-used to phosphorylate glycerate, Pi could be made available to remedy a circumstance in which Pi was limiting photosynthesis. Sharkey and Vanderveer (1989) have shown that the Pi concentration in the chloroplast stroma, but not the cytosol, falls during feedback limited photosynthesis. Under insensitive conditions, Sharkey and Vassey (1989) showed little change in sucrose synthesis in potato plants, but a decrease in starch synthesis, which was held to be responsible for a decreased rate of Pi release. This was ascribed to an inhibition of chloroplast phosphoglucose isomerase activity causing a rise in glycerate 3phosphate under low However, Harley and Sharkey (1991) suggest that it may not be a general phenomenon, especially in view of the stimulation of starch synthesis observed under low by Viil et al. (1977). It is also clear that a reduced capacity for sucrose synthesis can lead to in a

209 mutant of Flaveria linearis which has a decreased activity of the cytosolic FBPase (Sharkey et al., 1988, 1992). Other evidence suggests that the mechanism of insensitivity/reverse sensitivity at low temperatures might also involve changes in Pi in the cytosol. Pi feeding is known to restore sensitivity and to stimulate the assimilation rate dramatically at low temperatures (Leegood and Furbank, 1986). On the other hand, mannose feeding (which is thought to sequester Pi in the cytosol) induces (Harris et al., 1983). If we are to accept the widely held view that, at least in the short term, there can be no net flux of Pi across the chloroplast envelope (Heldt and Flügge, 1992), then the Pi concentration in the cytosol may be suboptimal at low temperature. It may then be insufficient to support triose-phosphate export from the chloroplast.

C. Temperature Dependence of Starch and Sucrose Synthesis There is evidence that both starch and sucrose synthesis are particularly sensitive to low temperature. For example, Pollock and Lloyd (1987) showed that the diurnal fluctuation in starch in a number of chilling-resistant temperate species was greatly reduced after transfer to low temperatures (8 °C) and that starch synthesis showed a particularly high (from 3.6 in Taraxacum officinale to 8.3 in pea), when measured between 5 °C and 20 °C, compared with values of 1.4 for sucrose synthesis in pea. When the temperature sensitivity of the enzymes of starch and sucrose synthesis was compared in Lolium temulentum, the enzymes of starch synthesis (ADPglucose pyrophosphorylase, unprimed ADP glucose starch synthase and primed ADP glucose starch synthase) had a lower residual activity at 5 °C than did enzymes involved in sucrose and fructan synthesis (sucrose phosphate synthase, cytoplasmic FBPase and sucrose-sucrose fructosyl transferase). It should be emphasized that the above observations are not inconsistent with the overall accumulation of starch at low temperatures. In the case of sucrose synthesis, there is good circumstantial evidence that sucrose synthesis limits photosynthesis at low temperatures (Stitt and Grosse, 1988) and that sucrose-phosphate synthase cannot be fully activated after transfer to low temperature unlike other enzymes in carbon metabolism in spinach (Holaday et al., 1992). In a chilling sensitive species

210 such as bean, the activation state of sucrose-phosphate synthase declines at low temperatures and key enzymes of sucrose synthesis are particularly sensitive to temperature. For example, there is an increase in the of the cytosolic FBPase at low temperature under physiological conditions, an example of negative thermal modulation. This is not a simple effect because the remains constant in the range 5 to 30 °C under normal assay conditions, but it rises at low temperature when assayed under conditions likely to occur in vivo (in the presence of AMP and fructose 2,6-bisphosphate) because the sensitivity of the enzyme to its effectors changes in a temperature-dependent manner (Stitt and Grosse, 1988). A specific inhibition of the cytosolic FBPase at low temperatures in spinach and pea has also been reported by Weeden and Buchanan (1983), although Pollock and Lloyd (1987) did not find this in L. temulentum. Another important factor regulating hexose-phosphate metabolism will be the high of sucrose-phosphate synthase at low temperatures (Pollock and Lloyd, 1987; Stitt and Grosse, 1988). However, Sage et al. (1990) suggest that the of sucrose synthesis is not as high in vivo and there is evidence for an acclimatory increase in the rate of sucrose synthesis after exposure to low temperature (W. Martindale and R. C. Leegood, unpublished).

D. Dark Metabolism In considering the effect of low temperature on the carbohydrate status of leaves, export of carbon from the leaves during the dark and its consequences on photosynthesis also need to be considered. This is especially important for plants of tropical origin which include many species. In the cold sensitive plant, ‘pangola’ digitgrass (Digitaria decumbens) mobilization of starch is restricted by low night temperatures of 10 °C which is followed by a severe reduction in photosynthesis the following day (Hilliard and West, 1970). A similar effect has been observed in Sorghum bicolor (Taylor et al., 1972). In chilling sensitive-species low night temperature results in a decreased mesophyll conductance suggesting the limitation is biochemical rather than stomatal (Ludlow and Wilson, 1971a). Increasing the sink capacity of the rest of the plant by warming adjacent leaves to 30 °C failed to mobilize the starch (Garrard and West, 1972). It has been proposed that the starch accumulation leads to impairment of

Richard C. Leegood and Gerald E. Edwards chloroplast function, such as a reduction in the capacity of electron transport (West, 1973). In S. bicolor, however, accumulated starch was mobilized in leaves which were strongly illuminated at 10 °C (Taylor et al., 1972). The relative activity of starch degrading enzymes decreased more at low temperature in extracts from tropical as opposed to temperate grasses (Carter et al., 1972; Karbassi et al., 1972). In Panicum virgatum, a cold-tolerant species, low night temperature (10 °C) did not effect remobilization of starch, and there was little effect on mesophyll conductance, photosynthesis and growth of the plants (Ku et al., 1978). There was a delay in stomatal opening, and some reduction in photosynthesis during the first hours of the light period, which may be associated with a temporary development of a water deficit in the plants.

E. Temperature Dependent Effects on Phloem Translocation and Sink Activity Temperature-dependent effects on phloem translocation, and sink activity may influence activity of the source by feedback but the mechanisms are poorly understood (Farrar, 1988; Geiger et al., 1992). With respect to carbohydrates, it is well known that the rate of starch synthesis and sucrose/starch content can change in response to temperature. For example, soluble sugar content commonly increases in sink tissue, e.g. potato tubers, under low temperature. High temperature can impair development of the sink as shown by limitations on starch synthesis in barley and wheat endosperm and potato tubers. In barley, although the supply of sucrose to the grain was maintained, temperatures above 30 °C reduced the activity of sucrose synthase (MacLeod and Duffus, 1988). In the developing wheat grain, impairment of starch synthesis at 35 °C may be related to the fact that soluble starch synthase is very sensitive to high temperatures (Keeling et al., 1993; Hawker and Jenner, 1993). Potato tubers have a temperature optimum of 21.5 °C for incorporation of sucrose into starch (Mohabir and John, 1988). When exposed to 30 °C for 6 days tubers ceased growing; even though imported soluble sugars were available, starch synthesis was impaired and the activity of ADP glucose pyrophosphorylase reduced (Kraus and Marschner, 1984).

Chapter 7 Carbon Metabolism and Temperature

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X. Acclimation of Photosynthesis to Temperature Shifts

A. Examples of Occurrence The term photosynthetic acclimation denotes ‘phenotypic adjustments of the functional properties of photosynthesis that can be modified by environmental factors (Berry and Björkman, 1980; Öquist, 1983). Clearly stress may ensue if this process is limited in any way. The purpose of this section is to draw attention to those properties which are known to change and the factors which might limit such change, particularly in metabolic adjustments in acclimation to low temperature. There is abundant evidence for photosynthetic acclimation in a wide range of plants. Acclimation is evidenced by altered temperature optima and by increases in photosynthetic rates at the growth temperature (Fig. 5; Berry and Björkman, 1980; Berry and Raison, 1981; Badger et al., 1982; Öquist, 1983; Nobel, 1988). It has been argued that this phenomenon is most likely to occur in evergreen woody species which occupy a wide range of thermal environments and which are subject to large seasonal variations (up to 30 °C) in temperature (Berry and Björkman, 1980). Modification in the temperature response then maximizes carbon gain at any particular growth temperature. In contrast, plants from environments in which growth is restricted to a single season of the year, or which are from habitats in which temperature changes are small, have a relatively limited capacity for acclimation, e.g. the prairie grass, Bouteloua gracilis (Kemp and Williams, 1980). There is, therefore, a spectrum of responses and differences in the capacity to acclimate to temperature which will presumably be reflected in different susceptibilities to stress. In the most comprehensive study of acclimation to date, Badger et al. (1982) studied the desert evergreen Nerium oleander, which is capable of growth between 10 °C and 46 °C. In this study plants were grown at 20 °C and at 45 °C. The characteristics of these plants are common to many studies of acclimation. First, the plants grown at 20 °C had photosynthetic rates in air at the lower temperatures which were about double those of the plants grown at 45 °C. Second, the optimum temperature for the plants grown at low temperature was higher than the growth temperature (between 24 and 31 °C) whereas the

optimum for the plants grown at high temperature was slightly lower than the growth temperature (between 35 and 40 °C).

B. Factors Underlying the increase in Photosynthetic Capacity after Acclimation to Low Temperatures 1. Acclimation of Electron Transport Estimates of the maximum rate of electron transport from gas exchange studies made by Ferrar et al. (1989) in Nerium oleander and a range of Eucalyptus species showed parallel changes in the rate of electron transport, Rubisco and photosynthetic capacity in plants acclimating to different temperature regimes. However there is evidence for some specific restrictions by low temperature on the synthesis of certain components of electron transport (see Chapter 15).

2. Changes in Rubisco In N. oleander the increased capacity of the leaves of plants grown at lower temperatures was not due to a general increase in the amount of photosynthetic

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machinery (Badger et al., 1982). There were no significant differences in soluble leaf protein, and in most enzymes, although electron transport capacity was slightly elevated. The amounts of FBPase and Rubisco increased markedly in the plants grown at low temperature, in plants grown at 20 °C, although the of Rubisco was only just in excess of the rate of photosynthesis at 20 °C, whereas in the plants grown at 45 °C there was a two- to three-fold excess of carboxylation capacity. There is a considerable amount of further evidence which shows that the amount of Rubisco increases after acclimation to low temperature, although not all of the measurements cited provide evidence for adequate activation by These studies include two laboratory-grown and two field-grown populations of the arctic-alpine species, Oxyria digyna (Chabot et al., 1972), the plant, Atriplex lentiformis (Pearcy, 1977), tomato (Markus et al., 1981), Dunaliella tertiolecta (Morris and Farrell, 1971), leaves of spinach, bean and tobacco (Holaday et al., 1992), wheat (Lawlor et al., 1987a,b), Dactylis glomerata (Treharne and Eagles, 1970) and cotton (Downton and Slatyer, 1972). Gas exchange studies also support the view that Rubisco increases after growth at lower temperatures. Ferrar et al. (1989) estimated Rubisco activity from measurements of Both Nerium oleander and a range of Eucalyptus species had a higher indicating parallel changes in Rubisco and photosynthetic capacity in plants acclimating to a lower temperature regime. A similar change occurs in the plant, Atriplex glabriuscula, grown at 16 °C and at 40 °C (Björkman et al., 1975). A very different picture emerges in rice, a chillingsensitive plant. Maruyama et al. (1990) showed that low temperature (15 °C) prevented the normal developmental increase in Rubisco. Hahn and Walbot (1989) found that at low temperatures there was a decrease in the synthesis of Rubisco and a loss of coordination of the synthesis of the large and small subunits, with a greater suppression of the synthesis of both the protein and the mRNA for the small subunit. A similar observation has been made in rapeseed germinated at 0 °C (Meza-Basso et al., 1986). There would, therefore, appear to be a step in the synthesis of Rubisco in these plants which is particularly sensitive to low temperature, although this is not generally true of chilling sensitive plants because in bean Rubisco activity doubled over the

Richard C. Leegood and Gerald E. Edwards

course of 10 days at 10 °C (Holaday et al., 1992). There are other reports of decreases in Rubisco in plants grown at lower temperature. Sawada et al. (1974) showed that the Rubisco activity in wheat grown at 5–7 °C was less than half that in leaves from plants grown at 20–25 °C, but the enzyme was extracted under conditions which would have led to its deactivation. Phillips and McWilliam (1970) showed decreases in Rubisco in the plants, Caltha intraloba and wheat and in the Atriplex nummularia grown at low temperature, but these were expressed on a protein basis, which is likely to have increased at low temperature (see below).

3. Changes in Other Enzymes Acclimation to very different temperatures also requires the ability to increase the maximum catalytic activities of a number of other enzymes besides Rubisco. In experiments in which leaves of Nerium oleander were transferred between 20 °C and 45 °C, the change in the rate of assimilation correlated well with the activities of FBPase and Rubisco, while activities of other enzymes, e.g. PGA kinase, phosphohexoseisomerase and phosphoglucomutase increased, but to a lesser extent, after transfer to low temperature (Badger et al., 1982). FBPase showed the greatest change in extractable activity with growth temperature and on transfer of plants between 45 °C and 20 °C, it showed an increase in activity which paralleled the change in photosynthetic capacity. Although coarse control may be suspected in such circumstances, it is not proven, since the extractable activity of FBPase (fully activated with dithiothreitol) prior to the transfer from 45 °C to 20 °C was still more than sufficient (by at least two-fold) to catalyze the higher rate of photosynthesis following transfer. Holaday et al. (1992) showed that the maximum activities of a wide range of enzymes increased following transfer of spinach or bean plants from 25 °C to 10 °C. These included Rubisco, the cytosolic and stromal FBPase, SBPase, hexokinase, phosphoglucose isomerase, sucrose-phosphate synthase, pyruvate kinase and PEPC. In contrast, in chillingsensitive rice low temperature (15 °C) largely prevented the normal developmental increases in Rubisco, the stromal FBPase, NADP-glyceraldehydephosphate dehydrogenase and catalase, but not the cytosolic FBPase or two the enzymes involved in starch synthesis, ADPglucose pyrophosphorylase and

Chapter 7 Carbon Metabolism and Temperature Q-enzyme, suggesting specific curtailment by chilling of the synthesis of a number of enzymes (Maruyama et al., 1990).

4. Changes in the Capacity to Utilize Triosephosphate Calderon and Pontis (1985) showed that, after transfer of young wheat plants from 23 °C to 4 °C, sucrose accumulated rapidly and was accompanied by an increase in the activity of sucrose synthase (see also Crespi et al., 1991), but not of the cytosolic FBPase, invertase, SPS or UDPglucose pyrophosphorylase. The photosynthetic rate was little affected. It was suggested that this increase may be important in mediating transport of sucrose into the vacuole, for storage. Holaday et al. (1992) showed that the increases in both the activity and activation state of SPS in spinach (a chilling-tolerant species) after transfer to low temperature was delayed relative to activity changes in other enzymes, suggesting that the capacity of this enzyme might limit sucrose synthesis. A similar increase in the capacity and activation state of SPS has also been observed in spinach by Guy et al. (1992). In the chilling-sensitive bean, by contrast, SPS activation state and activity fell drastically after transfer to low temperature (Holaday et al., 1992). This observation accords with the increase in the activity of SPS which occurs when leaves of soybean (another chilling-sensitive plant) are warmed (Rufty et al., 1985). Further evidence for increases in the capacity to synthesize carbohydrate under low temperature comes from gas-exchange studies. In Nerium oleander (Badger et al., 1982), in the desert evergreen, Larrea divaricata (Mooney et al., 1978) and in spinach (Holaday et al., 1992), growth at low temperature resulted in an increase in photosynthetic capacity at higher temperatures above that of plants grown at the higher temperature when measured in high (1.5%) (and sometimes low but not when measured in air (Fig. 5). Woledge and Jewiss (1969) also showed that transfer of plants of tall fescue (Festuca arundinacea) from low to high temperature led to a temporary enhancement of rate of photosynthesis at the higher temperature. This superior performance of the plants in high is likely to be attributable to increases in the activities of the enzymes of starch and sucrose synthesis at low

213 temperature which can then be utilized in a circumstance (high and high temperature) in which the capacities of these processes would normally limit the rate of photosynthesis. Acclimation of the capacity to synthesize carbohydrate, and particularly new forms of stored carbohydrate, such as fructan, would increase the rate of Pi recycling during carbohydrate synthesis. Relief of the symptoms of a suboptimal Pi status are observed following acclimation, e.g. by increased of photosynthesis after growth at low temperatures (Cornic and Louason, 1980; Sage and Sharkey, 1987). Exposures of even a few hours to low temperatures in the dark can lead to relief of the symptoms of sub-optimal Pi status in leaves (such as increased Labate and Leegood, 1988) and to significantly increased rates of carbon assimilation. In these circumstances, recovery from Pi limitation presumably depends upon the rate and extent of Pi movement out of the vacuole in response to lowered temperatures. It is not a nutritional limitation. This ability of Pi to move between cellular compartments, albeit relatively slowly, means that it is unlikely that such limitations persist in the longer term (days to weeks) and that changes in gene expression will be more important in modulating responses over such periods. However, Hurry et al. (1993) have shown that in winter rye, the frosthardening response (induced by growth at 5 °C), including an increased photosynthetic rate and decreased sensitivity to photoinhibition, could be at least partially mimicked by feeding Pi through the transpiration stream. The carbohydrate status of plants also appears to play a role in the ability to acclimate to low temperature. Primary leaves of barley plants which contained large carbohydrate reserves and high hexose-phosphate showed photosynthetic acclimation to low temperature. By contrast, leaves from plants which were low in carbohydrate and hexosephosphate showed no photosynthetic acclimation (Labate and Leegood, 1989). It was suggested that high carbohydrate reserves may potentiate the system for the achievement of high rates of photosynthesis at low temperatures by accumulation of photosynthetic intermediates, such as hexose-phosphate, and that this partially overcomes Pi limitation of photosynthesis (Labate and Leegood, 1989). This interaction between carbohydrate reserves and acclimation to low temperature may also be related

214 to observations that chilling sensitivity in plants such as tomato is partly dependent upon their carbohydrate status (King et al., 1988).

5. Changes in Protein Synthesis Increased enzyme capacity clearly requires increased protein synthesis at low temperature. In addition, stress is often accompanied by the synthesis of stress proteins, believed to facilitate tolerance. There are a large number of reports that gene expression, protein synthesis, amounts of enzymes and freezing tolerance are altered by exposure to low temperature (Guy, 1990; Cattivelli and Bartels, 1992). It is known that nucleolar activity increases with accumulation of polysomes (Perras and Sarhan, 1990). In spinach, transcription was increased after 2d at 5 °C and rates of protein synthesis, although initially depressed, eventually rose to the rates observed in spinach plants maintained at 20 °C (Guy et al., 1985). Acclimation and deacclimation between 25 °C and 5 °C in spinach results in the de novo synthesis of cold acclimation proteins and in the promotion of the synthesis of polypeptides associated with increased tolerance to freezing (Guy and Haskell, 1987). However, the physiological role of most of these proteins remains obscure. In wheat, leaves from plants grown at a lower temperature, especially those provided with more nitrate, contained more soluble protein (Lawlor et al., 1987c). Lawlor et al. (1988) suggested that although there is a decrease in the rate of protein synthesis at low temperature, its duration is increased in smaller leaves, so that the content of protein per unit leaf area is greater in cool conditions. Acclimation of photosynthesis to temperature in the planktonic alga, Skeletonema costatum, results in rates of photosynthesis and respiration which are virtually the same at 8 °C and at 20 °C (Steemann-Nielsen and Jørgensen, 1968), and which are accompanied by a doubling of protein per cell (Jørgensen, 1968). The price paid for this large increase in protein synthesis is a lower growth rate at the lower temperature (Steemann-Nielsen and Jørgensen, 1968), a feature which may well be of importance in higher plants.

C. Possible Constraints on the Capacity for Acclimation The notion that acclimation to low temperature involves both activation of enzymes and increases in

Richard C. Leegood and Gerald E. Edwards their amounts may set constraints on the thermal range which any plant can occupy. It may be that any one plant can only cope with a limited temperature range. For example, a range of 30 °C would require an eight-fold change in enzyme activities or activation in order to achieve temperature compensation if the values equaled 2 for all reactions. Many of the data on acclimation of photosynthesis emphasize that acclimation to low temperature, either under natural conditions, or in material grown in greenhouses or growth cabinets, does not necessarily result in higher rates at the lowest temperatures, but rather an increased ability to photosynthesize at higher rates over a range of lower temperatures. In many cases the optimum does not shift to the new growth temperature. Even arctic plants may have temperature optima which are around 20 °C (Billings and Mooney, 1968). In Eucalyptus pauciflora, for example, the temperature optimum shifted by 0.34 °C per degree shift in the day growth temperature (Slatyer and Morrow, 1977). Similarly, in Oxyria, a 3 °C change in growth temperature led to a change of only 1 °C in the temperature optimum. Berry and Björkman (1980) point out that an increase in photosynthetic capacity which occurs following acclimation may be sufficient to shift the optimum downwards, but not necessarily to very low growth temperatures, because this would probably involve an inordinately large investment in enzymes, and hence nitrogen, with only a marginal photosynthetic gain. Of course, in the field a leaf will be operating in a fluctuating temperature regime. Nevertheless, despite the fact that there is normally little turnover of Rubisco or other Calvin cycle enzymes in mature leaves, it appears that mature leaves are capable of acclimation to a changed thermal regime by increasing amounts of proteins. Since Rubisco comprises a large proportion of the nitrogen invested in the photosynthetic apparatus, an increase in the amount of Rubisco and other enzymes has important implications for resource use efficiency, as it then requires reallocation of nitrogen to Rubisco from other processes, e.g. light harvesting (see Chapter 11). An enhanced supply of nitrogen to the plant will allow an increase in the amount of Rubisco and will tend to offset the impact of reallocation of nitrogen, while a deficiency will restrict an increase in Rubisco. It is therefore highly likely that manipulating the nitrogen supply can be used as a means of manipulating responses to temperature and that this will also influence growth at lower

Chapter 7 Carbon Metabolism and Temperature temperatures, in much the same manner as it affects acclimation between sun and shade.

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Chapter 7 Carbon Metabolism and Temperature RuBP-carboxylase in intact leaves of white clover. In: Akoyunoglou, G (ed) Photosynthesis IV, pp 63–68. Balaban Intl Sci Services, Philadelphia Mächler F, Schnyder H and Nösberger J (1984) Influence of inorganic phosphate on photosynthesis of wheat chloroplasts. I. Photosynthesis and assimilate export at 5 °C and 25 °C. J Exp Bot l53: 81–487 MacLeod LC and Duffus CM (1988) Reduced starch content and sucrose synthase activity in developing endosperm of barley plants grown at elevated temperatures. Aust J Plant Physiol 15: 367–375 Markus V, Lurie S, Bravdo B, Stevens MA and Rudich J (1981) High temperature effects on RuBP carboxylase and carbonic anhydrase activity in two tomato cultivars. Physiol Plant 53: 407–412 Maruyama S, Yatomi M and Nakamura Y (1990) Response of rice leaves to low temperature. I. Changes in basic biochemical parameters. Plant Cell Physiol 31: 303–309 Martin B and Ort DR (1982) Insensitivity of water-oxidation and Photosystem II activity in tomato to chilling temperatures. Plant Physiol 70: 689–694 McVetty PBE and Canvin DT ( 1981) Inhibition of photosynthesis by low oxygen concentrations. Can J Bot 59: 21–725 McWilliam JR and PU Ferrar (1974) Photosynthetic adaptation of higher plants to thermal stress. In: Bieleski RL, Ferguson AR and Cresswell MM (eds) Mechanisms of Regulation of Plant Growth, pp 467–476. The Royal Soc New Zealand, Wellington Meza-Basso L, Alberdi M, Raynal M, Ferrero-Cadinanos M-L and Delseny M. (1986) Changes in protein synthesis in rapeseed (Brassica napus) seedlings during a low temperature treatment. Plant Physiol 82: 733–738 Minorsky PV (1989) Temperature sensing by plants: A review and hypothesis. Plant Cell Environ 12: 119–135 Mohabir G and John P (1988) Effect of temperature on starch synthesis in potato tuber tissue and in amyloplasts. Plant Physiol 88: 1222–1228 Monson RK, Stidham MA, Williams GJ, Edwards GE, and Uribe EG (1982) Temperature dependence of photosynthesis in Agropyron smithii Rydb. Plant Physiol 69: 921–928. Mooney HA, Björkman O and Collatz GJ (1978) Photosynthetic acclimation to temperature in the desert shrub, Larrea divaricata. I. Carbon dioxide exchange characteristics of intact leaves. Plant Physiol 61: 406–410 Morris I and Farrell K (1971) Photosynthetic rates, gross patterns of carbon dioxide assimilation and activities of ribulose diphosphate carboxylase in marine algae grown at different temperatures. Physiol Plant 25: 372–377 Nobel PS (1988) Principles underlying the prediction of temperature in plants, with special reference to desert succulents. In: Long SP andWoodward FI (eds) Plants and Temperature, pp 1–23. Company of Biologists, Cambridge Nolan WG (1980) Effect of temperature on electron transport activities of isolated chloroplasts. Plant Physiol. 66: 234–237 Oberhuber W and Edwards GE (1993) Temperature dependence of the linkage of quantum yield of Photosystem II to fixation in and plants. Plant Physiol 101: 507–512 Öquist G (1983) Effects of low temperature on photosynthesis. Plant Cell Environ 6: 281–300 Osmond CB (1978) Crassulacean acid metabolism: acuriosity in context. Ann Rev Plant Physiol 29: 379–414

219 Osmond CB and Holtum JAM (1981) Crassulacean acid metabolism. In: Hatch MD and Boardman NK (eds) The Biochemistry of Plants, Vol 8, pp 283–328. Academic Press, New York Pammenter NW, Loreto F and Sharkey TD (1993) End product feedback effects on photosynthetic electron transport. Photosynth Res 35: 5–14 Pearcy RW (1977) Acclimation of photosynthetic and respiratory carbon dioxide exchange to growth temperature in Atriplex lentiformis (Torr.) Wats. Plant Physiol 59: 795–799 Pearcy RW (1988) Photosynthetic utilization of lightflecks by understory plants. Aust J Plant Physiol. 15: 223–238 Perras M and Sarhan F (1990) Polysome metabolism during cold acclimation of wheat. Plant Cell Physiol 31: 1083–1089 Phillips PG and McWilliam JR (1970) Thermal responses of the primary carboxylating enzymes from and plants adapted to contrasting temperature environments. In: Hatch MD, Osmond CB and Slayter RO (eds) Photosynthesis and Photorespiration, pp 97–104. Wiley, New York Pollock CJ (1984) Sucrose accumulation and the initiation of fructan biosynthesis in Lolium temulentum [Darnel ryegrass]. New Phytol 96: 527–534 Pollock CJ and Lloyd EJ (1987) The effect of low temperature upon starch, sucrose and fructan synthesis in leaves. Ann Bot 60: 231–235 Pollock CJ, Lloyd EJ, Stoddart JL and Thomas H (1983) Growth, photosynthesis and assimilate partitioning in Lolium temulentum exposed to chilling temperatures (Darnel ryegrass). Physiol Plant 59: 257–262 Portis AR Jr (1992) Regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity. Annu Rev Plant Physiol Plant Mol Biol 43: 415–537 Potvin C, Simon J-P and Strain BR (1986) Effect of low temperature on the photosynthetic metabolism of the grass Echinochloa crus-galli. Oecologia 69: 499–506 Raison JK (1980) Membrane lipids: Structure and function. In: Conn E and Stumpf P (eds) The Biochemistry of Plants, Vol 4, p 57. Academic Press, New York Robinson SP and Portis AR Jr (1989) Adenosine triphosphate hydrolysis by purified rubisco activase. Arch Biochem Biophys 268: 93–99 Rufty TW, Huber SC and Kerr PS (1985) Association between sucrose-phosphate synthase activity in leaves and plant growth rate in response to altered aerial temperature. Plant Sci 39: 7– 12 Sage RF and Sharkey TD (1987) The effect of temperature on the occurrence of and photosynthesis in field grown plants. Plant Physiol 84: 658–664 Sage RF, Sharkey TD and Pearcy RW (1990) The effect of leaf nitrogen and temperature on the response of photosynthesis in the dicot Chenopodium album L. Aust J Plant Physiol 17: 135–148 Sassenrath GF and Ort D (1990) The relationship between inhibition of photosynthesis at low temperature and inhibition of photosynthesis after rewarming in chill-sensitive tomato. Plant Physiol Biochem 28: 457–465 Sassenrath GF, Ort DR and Portis AR Jr( 1990) Impaired reductive activation of stromal triose in tomato leaves following low temperature exposure at high light. Arch Biochem Biophys 282: 302–308. Sawada S, Matsuhima H and Miyachi S. (1974) Effects of

220 growth temperature on photosynthetic carbon metabolism in green plants. III. Differences in structure, photosynthetic activities and activities of ribulose diphosphate carboxylase and glycolate oxidase in leaves of wheat grown under varied temperatures. Plant Cell Physiol 15: 239–248 Schnyder H, Mächler F and Nösberger J (1984) Influence of temperature and concentration on photosynthesis and light activation of ribulosebisphosphate carboxylase oxygenase in intact leaves of white clover (Trifolium repens L.). J Exp Bot 151: 147–156 Seemann J R, Berry J A and Downton W JS (1984) Photosynthetic response and adaptation to high temperature in desert plants. Plant Physiol 75: 364–368 Seemann JR, Kobza J and Moore BD (1990) Metabolism of 2carboxyyarabinitol 1–phosphate and regulation of ribulose1,5-bisphosphate carboxylase activity. Photosynth Res 23: 119–130 Selinioti E, Nikolopolous D and Manetas Y (1987) Organic cosolutes as stabilizers of phosphoenolpyruvate carboxylase in storage: An interpretation of their action. Aust J Plant Physiol 14: 203–210 Selwyn MJ (1966) Temperature and photosynthesis. II. A mechanism for the effects of temperature on carbon dioxide fixation. Biochim Biophys Acta 126: 214–224 Sharkey TD (1985a) photosynthesis in plants. Its occurrence and a possible explanation. Plant Physiol 78: 71–75 Sharkey TD (1985b) Photosynthesis in intact leaves of plants: Physics, physiology and rate limitations. Bot Rev 51: 53–105 Sharkey TD (1988) Estimating the rate of photorespiration in leaves. Physiol Plant 73: 147–152 Sharkey TD and Vanderveer PJ (1989) Stromal phosphate concentration is low during feedback limited photosynthesis. Plant Physiol 91: 679–684 Sharkey TD and Vassey TL (1989) Low oxygen inhibition of photosynthesis is caused by inhibition of starch synthesis. Plant Physiol 90: 385–387 Sharkey TD, Stitt M, Gerhardt R, Heineke D, Raschke K and Heldt HW (1986) Limitation of photosynthesis by carbon metabolism. II. photosynthesis results from a limitation of triose phosphate utilization. Plant Physiol 81: 1123–1129. Sharkey TD, Kobza J, Seemann JR and Brown RH (1988) Reduced cytosolic fructose-1,6-bisphosphatase activity leads to loss of sensitivity in a Flaveria linearis mutant. Plant Physiol 86: 667–671 Sharkey TD, Savitch LV, Vanderveer PJ and Micallef BJ (1992) Carbon partitioning in a Flaveria linearis mutant with reduced cytosolic fructose bisphosphatase. Plant Physiol 100: 210–215 Sharkey TD, Laporte MM, M1callef BJ, Shewmaker CK and Oakes JV (1995) Sucrose synthesis, temperature and plant yhield. In: Mathis, M (ed) Photosynthesis: From Light to Biosphere, Vol V, pp 635–640. Kluwer Academic Publishers, Dordrecht Sheen J (1994) Feedback control of gene expression. Photosynth Res 39: 427–438 Shirahashi K, Hayakawa S and Sugiyama T (1978) Cold lability of pyruvate, Pi dikinase in the maize leaf. Plant Physiol 62: 826–830 Silvius JR, Read BD and McElhaney RN (1978) Membrane enzymes: Artifacts in Arrhenius plots due to temperature

Richard C. Leegood and Gerald E. Edwards dependence of substrate-binding affinity. Science 199: 902– 904 Sivak MN and Walker DA (1987) Oscillations and other symptoms of limitation of in vivo photosynthesis by inadequate phosphate supply to the chloroplast. Plant Physiol Biochem 25: 635–648. Slack CR, Roughan PG and Bassett HCM (1974) Selective inhibition of mesophyll chloroplast development in some pathway species by low night temperatures. In: Bieleski RL, Ferguson AR, Cresswell MM (eds) Mechanisms of Regulation of Plant Growth, pp 499–504. The Royal Soc New Zealand, Wellington Slatyer RO and Morrow PA (1977) Altitudinal variation in the photosynthetic characteristics of snow gum, Eucalyptus pauciflora Sieb et Spreng. I. Seasonal changes under field conditions in the snowy mountains area of south-eastern Australia. Aust J Plant Physiol 25: 1–20 Smith SD and Nobel P (1986) Deserts. In: Baker NR and Long SP (eds) Photosynthesis in Contrasting Environments, pp 13– 62. Elsevier Science Publishers, Amsterdam Somero GN (1978) Temperature adaptation of enzymes: biological optimization through structure-function compromises. Ann Rev Ecol Systematics 9: 1–29 Steemann-Nielsen E and Jørgensen EG (1968) The adaptation of planktonic algae. I. General part. Physiol Plant 21: 401–413 Stidham MA, Uribe EG and Williams GJ (1982) Temperature dependence of photosynthesis in Agropyron smithii Rybd. Plant Physiol 69: 929-934 Stitt M and Grosse H (1988) Interactions between sucrose synthesis and fixation. IV. Temperature-dependent adjustment of the relation between sucrose synthesis and fixation. J Plant Physiol 133: 392–400 Sugiyama T, Schmitt MR, Ku SB and Edwards GE (1979) Differences in cold lability of pyruvate, Pi dikinase among species. Plant Cell Physiol 2: 965–971 Taylor AO, Slack CR and McPherson HG (1974) Plants under climatic stress. VI. Chilling and light effects on photosynthetic enzymes of sorghum and maize. Plant Physiol 54: 696–701 Taylor AO, Jepsen NM and Christeller T (1972) Plants under climatic stress. III. Low temperature, high light effects on photosynthetic products. Plant Physiol 49: 798–802 Taylor AO, Slack CR and McPherson HG (1974) Plants under climatic stress. VI. Chilling and light effects on photosynthetic enzymes of sorghum and maize. Plant Physiol 54: 696–701 Thomas SM and SP Long (1978) photosynthesis in Spartina townsendii at low and high temperatures. Planta 142: 171–174 Thomas H and Stoddardt JL (1984) Kinetics of leaf growth in Lolium temulentum at optimal and chilling temperatures. Ann Bot 53: 341–347 Treharne KJ and Eagles CF (1970) Effect of temperature on the photosynthetic activity of climatic races of Dactylis glomerata. Photosynthetica 4: 107–117 Usuda H (1985) The activation state of ribulose 1,5-bisphosphate carboxylase in maize leaves in dark and light. Plant Cell Physiol 26: 1455–1463 Usuda H and Shimogawara K (1991) Phosphate deficiency in maize. I. Leaf phosphate status, growth, photosynthesis and carbon partitioning. Plant Cell Physiol 32: 497–504 Usuda H, Ku MSB and Edwards GE (1984) Activation of NADP-malate dehydrogenase, pyruvate, Pi dikinase, and fructose 1,6-bisphosphatase in relation to photosynthetic rate

Chapter 7 Carbon Metabolism and Temperature in maize. Plant Physiol 76: 238–243 Vickery HB (1954) The effect of temperature on the behavior of malic acid and starch in leaves of Brophyllum calycinum cultured in darkness. Plant Physiol 29: 385–392 Viil J, Laisk A and Parnik T (1977) Enhancement of photosynthesis caused by oxygen under saturating irradiance and high concentrations. Photosynthetica 11: 251–259 Walker DA and Osmond CB (1989) New vistas in measurements of photosynthesis. Phil Trans Soc Lond B 323: 225–448 Warburg O (1919) Über die Geschwindigkeit der photochemischen Kohlensäuresetzung in lebenden Zellen. Biochem Z 100: 230–270 Ward DA (1987) The temperature acclimation of photosynthetic responses to in Zea mays and its relationship to the activities of photosynthetic enzymes and the concentrating mechanism of photosynthesis. Plant Cell Environ 10: 407– 411 Weeden NF and Buchanan BB (1983) Leaf cytosolic fructose1,6-bisphosphatase. A potential target site in low temperature stress. Plant Physiol 72: 259–261 Weis E (198 la) The temperature sensitivity of dark-inactivation and light-activation of the ribulose-l,5-bisphosphate carboxylase in spinach chloroplasts. FEBS Lett 129: 197–200

221 Weis E (1981 b) Reversible heat-inactivation of the Calvin cycle: A possible mechanism of the temperature regulation of photosynthesis. Planta 151: 33–39 Weis E (1982) Influence of light on the heat sensitivity of the photosynthetic apparatus in isolated spinach chloroplasts. Plant Physiol 70: 1530–1534 Weis E and JA Berry (1987) Quantum efficiency of Photosystem II in relation to energy-dependent quenching of chlorophyll fluorescence. Biochim Biophys Acta 894: 198–208 Weis E and Berry JA (1988) Plants and high temperature stress. In: Long SP and Woodward FI (eds) Plants and Temperature, pp 329–346. Company of Biologists, Cambridge West SH (1973) Carbohydrate metabolism and photosynthesis of tropical grasses subjected to low temperatures. In: Slatyer RO (ed) Plant Response to Climatic Factors, pp 165–168. UNESCO, Paris Woledge J and Jewiss OR (1969) The effect of temperature during growth on the subsequent rate of photosynthesis in leaves of tall fescue (Festuca arundinacea Schreb.). Ann Bot 33: 897–913 Woo KC and Wong SC (1983) Inhibition of assimilation by supraoptimal Effect of light and temperature. Aust J Plant Physiol 10: 75–85

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Chapter 8 Gas Exchange: Models and Measurements John M. Cheeseman and Matej Lexa Department of Plant Biology, University of Illinois, 505 S. Goodwin Ave., Urbana, IL 61801, USA

Summary I. Introduction II. The Biochemical Model A. The Integrated Model B. The Complete Model C. The Laisk Alternative III. Beyond the Biochemical Model A. Stomata 1. General Response Models 2. Stomatal Optimization 3. Stomatal Limitations B. Mesophyll Conductance C. Rubisco Activation D. Ribulose 1,5-bisphosphate Regeneration E. Photoprotection IV. The Feedback Loop: Consequences for Field Studies V. Conclusion Acknowledgments References

223 224 226 226 226 228 228 228 228 230 231 231 231 233 233 235 237 237 237

Summary In this chapter, we use mathematical models of photosynthesis from the chloroplast to the leaf level to consider the relationships between photosynthetic capacity and performance. The differences must reflect regulation of chloroplast processes under the influence of environmental limitations. Thus, we examine the means by which models handle regulation of individual processes and identify five critical areas of uncertainty. These are: stomatal interactions, mesophyll conductance, Rubisco activation, ribulose 1,5-bisphosphate regeneration and photoprotection. Each is discussed both with regard to modeling efforts which have been made, and experimental results which are yet to be fully assimilated. In all five cases, control is clearly dynamic, not static, and we note several areas in which current methods of data interpretation insufficiently take this into account. Finally, we return to the problem of data collection and interpretation under field conditions. We note the essentiality of merging the goals of experimental science with those of modeling. In this way, critical data will be available and used in formalizing future models of photosynthesis, and its biochemical and environmental regulation.

Neil R. Baker (ed): Photosynthesis and the Environment, pp. 223–240. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

224 I. Introduction Under field conditions, photosynthetic performance can easily be measured using mobile or portable infra-red gas analyzers. Comparing the measurements with data obtained under the least stressful conditions in the field or under controlled laboratory or greenhouse conditions, it is clear that that performance is often less than photosynthetic potential; the differences must be due to perturbations of the photosynthetic apparatus or its functioning. These perturbations, in turn, must reflect environmental pressures. The overall focus of this chapter will be on questioning how such constraints can be imposed, with mathematical models as the guide to the discussion. Because of the predominance of considerations in the modeling literature, we will limit the discussion to this group of plants. Even then, we should emphasize that the number of continuously alterable parameters involved in the determination of an instantaneous photosynthetic rate is very large. Our unfulfilled goal remains, therefore, to recognize when changes in performance are more than simple adjustments. In this review process, whenever possible, the models we include will be mechanistic or ‘more mechanistic’ rather than ‘more empirical’. Always, our objective will be to interdigitate models and experimental data, the latter largely derived from gas analysis and fluorescence measurements. In doing so, we hope to bring together formal modelers, biochemists and environmental physiologists. In keeping with the scope of this book, the problem will be restricted to the leaf level, without consideration of canopies as a whole. Leaf gas exchange studies require at least three kinds of models. First, the measurements themselves

John M. Cheeseman and Matej Lexa can only be made if there is a formal, i.e. mathematical, model linking the disappearance of from (or the appearance of in) a chamber to the activity of the photosynthetic apparatus, because that model is the basis for the calculations. The equipment, methods and equations required for virtually all such measurements have been collected and discussed in detail in two excellent references (Field et al., 1991; Long and Hällgren, 1993). As commercially produced infra-red analyzers are readily available with the models pre-programmed, the models will not be dealt with here. The second model is that required to interpret ‘clean’ measurements, regardless of the degree to which it has been formalized. Figure 1 shows the elements that such models must ultimately encompass; the success of photosynthesis depends on their interdependent control. If we could understand the interdependencies, we could probably answer most of questions this volume sets out to address. Three elements in Fig. 1 have received widespread attention; the light-dependent reactions, Calvin cycle biochemistry, and stomatal control. The partitioning of carbon within chloroplasts to starch or to export to the cytosol has played a less prominent role with the exception of possible feedback control by phosphate limitations. Carbon removal from the cytoplasm (not shown), either for leaf growth, export or maintenance has received little attention with respect to overall control of fixation on the scale of minutes, though regulation of sucrose synthesis interacts through phosphate (Pi) directly with chloroplast functions (Stitt, 1985), and response lags of 15–20 min between triose phosphate export and sucrose control are one possible explanation for oscillatory fixation (Laisk and Walker, 1986; Horton and Nicholson, 1987). In longer times, dysfunction in carbon export machinery may also contribute to

Abbreviations: A–rate of assimilation; –maximal rate of assimilation; C– partial pressure at the site of carboxylation; – partial pressure of in the atmosphere outside a leaf; CAP – carboxyarabinitol 1-phosphate; – partial pressure of at the chloroplasts; CE – carboxylation efficiency; – partial pressure of in the airspaces of a leaf; – t h e mole fraction at a leaf surface; DHAP–dihydroxyacetone phosphate; FBP–fructose 1,5-bisphosphate; FBPase–fructose 1,5-bisphosphatase; Fd–ferredoxin; – maximal fluorescence; – variable fluorescence; – stomatal conductance; GSH – glutathione (reduced); – mesophyll transfer conductance; I – irradiance; J – linear electron transport rate; – maximum potential rate of linear electron transport; – MichaelisMenten constant for response of ribulose 1,5-bisphosphate carboxylase/oxygenase; – Michaelis constant for of ribulose 1,5bisphosphate carboxylase/oxygenase; O – partial pressure at the site of carboxylation; PGA – 3-phosphoglyceric acid; Pi – inorganic phosphate; PPFD – photosynthetically-active photon flux density; – dark respiration occurring in the light; Ru5P – ribulose 5phosphate; Rubisco – ribulose 1,5-bisphosphate carboxylase/oxygenase; RuBP – ribulose 1,5-bisphosphate; SPB – sedoheptulose 1,7bisphosphate; SPBase – sedoheptulose 1,7-bisphosphatase; – leaf temperature; TPU — triose phosphate utilization; — velocity of RuBP carboxylation; –maximal velocity of RuBP carboxylation; –velocity of RuBP oxygenation; VPD–vapor pressure deficit; – compensation point; – compensation point in the absence of dark respiration; – specificity of Rubisco for over

Chapter 8

Gas Exchange: Models and Measurements

stress related changes, including mid-day depression (Horton and Nicholson, 1987; Laisk and Eichelmann, 1989). Much of the discussion of that control has been in the context of ‘down-regulation’ and its ultimate invocation involves the coordination of electron transport and fixation during exposure to excess irradiance. Though there is some controversy over the definition of photoinhibition and its occurrence under natural conditions (Baker and Bowyer, 1994), it can broadly be divided into questions of lightinduced damage to chloroplasts and biochemical

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regulation to prevent that damage. In Fig. 1, all such damage control mechanisms are grouped as photoprotection. The challenge of any model, particularly when the emphasis is on field conditions, is that biological variability, as well as technical constraints and demands, are always involved. The third kind of model, therefore, is that required to extract meaningful information from ‘messy’ data sets. Figure 2, showing light and conductance relationships to assimilation in mangrove leaves under field conditions, illustrates the extent of the variability that might be expected

226 when photosynthetic performance is simply surveyed, even when data sets are restricted to relatively narrow environmental ranges. The primary problem to be faced here is the interpretation of results, and the conceptual model of Fig. 1 must be combined with statistical methods. Few studies have attempted this (Cheeseman et al., 1991); this approach will be discussed briefly in Section IV. Throughout biology, the interpretation of any study depends on what the investigator expects to occur under ‘normal,’ ‘control’ or ‘up-regulated’ conditions. To recognize and quantify perturbations of performance and environmental constraints, therefore, we must first examine what we think we know about normalcy.

John M. Cheeseman and Matej Lexa are the partial pressures of and at the sites of carboxylation; and are the Michaelis constants for and respectively, of Rubisco, is the rate of dark respiration in the light and is the compensation point in the absence of dark respiration. The third equation:

defines the RuBP- or regeneration-limited rate of assimilation. One additional equation was added later which allows for the limitation of assimilation by phosphate supply through the dihydroxyacetone phosphate/Pi translocator,

II. The Biochemical Model Circular as it might seem, the normal condition can be defined as the expected, or as conforming to a model accepted by the individual performing the study. Models of photosynthesis integrated to the leaf level have progressed from a collection of partial process models to an integrated mathematical form over the past 25 years, with an undeniable dominance of Farquhar, von Caemmerer and collaborators in the process.

A. The Integrated Model For better or for worse, Farquhar and von Caemmerer (1982) summarized their effort in a section entitled ‘Integrated Metabolism’. There, the rate of assimilation to be expected at a fixed temperature was reduced to a calculation using only three equations:

which defines the potential rate of electron transport as a function of irradiance (I) and the maximum potential electron transport

which defines the RuBP-saturated rate of assimilation (A) as a function of the maximum carboxylation capacity of Rubisco C and O

where T is the rate of utilization of triose-phosphate and denotes the specificity of Rubisco for over (Sharkey, 1985b). The modeled rate of assimilation is the minimum of the values calculated using Eqs. (2–4). On the one hand, this simplified model has been very useful in teaching the characteristics of photosynthesis as they appear in infrared gas analysis studies and the interpretation of plots of assimilation (A) versus intercellular partial pressure i.e. curves. On the other hand, its acceptance in lieu of more complicated versions has led to a number of problems. The more complete model, therefore, warrants discussion here.

B. The Complete Model Farquhar and von Caemmerer (Farquhar et al., 1980; Farquhar and von Caemmerer, 1982) clearly summarized more than 10 years of research, much of it concerned with Calvin cycle activities, and that alone should justify the continued frequent reference to their papers. Figure 1 illustrates a Stella-type model largely based on their papers. In addition to synthesizing the biochemistry into a general model, they added their contributions in the consideration of electron transport, the production of ATP and NADPH, and their coordination with consumption. They introduced the concept of a regeneration limitation on fixation, and in modeling electron transport as a function of light absorbed and potential transport, they introduced ‘photosynthetic control’

Chapter 8 Gas Exchange: Models and Measurements

based either on the availability of ADP for phosphorylation (Farquhar et al., 1980) or of NADP for reduction (process 2 in Fig. 1; Farquhar and von Caemmerer, 1982). The complete model included a number of important simplifications, appropriate at the time perhaps, that are lost to those using only the ‘integrated’ model. Regeneration of ribulose 1,5bisphosphate (RuBP), for example, was dealt with according to Hall (1971) as a single equation, first order in 3-phosphoglyceric acid (PGA) and NADPH, and using a ‘fictional composite’ term for its maximal

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rate (process 3 in Fig. 1; Farquhar and von Caemmerer, 1982). Also omitted was dependence of regeneration on potential regulatory factors such as fructose 1,6bisphosphatase (FBPase) or sedoheptulose 1,7bisphosphatase (SBPase) based on the argument of excess RuBP regeneration capacity (Farquhar and von Caemmerer, 1982). A second simplification was the inclusion of a system of tuning such that electron transport was perfectly matched to carbon reduction through the energy carriers; this feature has been continued in all modifications of the model, regardless of authorship. This effectively puts all responsibility for photoprotection either before electrons enter the electron transport chain, or on photorespiration. As the latter should be associated with a large reduction of that is not characteristic of plants with low A and stomatal conductance the burden falls on zeaxanthinrelated mechanisms for quenching of excitation energy (see Chapters 1–3). A third simplification is that which set into Eqs. (2 and 3) without any coefficient for Calvin cycle activation. In their discussion, Farquhar and von Caemmerer concluded that ‘under normal conditions... [Rubisco] is likely to be fully activated,’ thus, changes in its activity appeared to be ecophysiologically unimportant (Farquhar and von Caemmerer, 1982). This gave rise to the concept of carboxylation efficiency (CE) as the initial slope of the curve, and CE has become an important parameter for interpretation of experimental results. It is immediately obvious that if is adjusted by a variable or dynamic activation coefficient, then the interpretation of curves is severely complicated. Since publication of their ultimate synthesis (Farquhar and von Caemmerer, 1982), this model has been supplemented in two major ways, each prompted both by the successes and limitations of the published efforts. First, von Caemmerer and Edmondson (1986) discussed the activation of Rubisco and the modulation of activity through the chelation of RuBP and PGA by Mg. This has made it possible to include activation more specifically in modeling the overall behavior of photosynthesis. In particular, their results allow the model to produce curves with initial slopes which depend on irradiance. This advance has, unfortunately, not supplanted use of simplified model, and deviations from a unique behavior have been interpreted as the result of extra-chloroplastic events. This has contributed to (and confused) the controversy over

228 the effects of patchy stomatal closure (Cheeseman, 1991; Terashima, 1992). Sharkey (1985b) provided the second supplement as Eq. (4) for phosphate, or triose-phosphate utilization (TPU) limitations on the Calvin cycle. This derived partly from circumstances under which exchange becomes insensitive to and and incorporated the well-characterized Pi translocator (after Heldt and Rapley, 1970; process 7 in Fig. 1) and the observation that Pi was required for continued fixation by isolated, intact chloroplasts (Walker and Sivak, 1985). Farquhar and von Caemmerer (1982) had discussed this; they noted that removal of dihydroxyacetone phosphate (DHAP) is associated with the movement of Pi back across the envelope to maintain the Pi supply, and that if it becomes limiting, A becomes insensitive to and but did not model it specifically. Harley and Sharkey (1991) extended the TPU study to explain the reversed sensitivity of assimilation to and by limiting the recycling of glycerate produced through photorespiration.

C. The Laisk Alternative While it is impossible to overstate the impact of the Farquhar and von Caemmerer model on photosynthetic research over the last dozen years, it is not the only mechanistic attempt to integrate the processes at the cellular level. A significant alternative is the Laisk model (Laisk and Eichelmann, 1989; Laisk et al., 1991, 1992a,b,c; Laisk, 1993), and if the Farquhar and von Caemmerer model is ‘more mechanistic,’ Laisk’s effort is arguably ‘most mechanistic.’ As his general model has evolved, its single theme has been the treatment of individual reactions using their standard free-energy change at pH 7.0 and values for substrates and products. Both because of an interest in the phenomenon and because its understanding could be a key to understanding the control of photosynthesis overall, Laisk and his coworkers have emphasized the prediction of oscillations as a criterion for success in modeling (Laisk and Walker, 1986; Laisk and Eichelmann, 1989; Laisk et al., 1989; Laisk, 1993). Their efforts have been accompanied by a parallel program of experimentation along lines suggested by the simulations. Those results, in turn, have been used to modify the model (e.g. Laisk et al., 1992c; Laisk, 1993). One area in which the Laisk models are perhaps superior to the Farquhar/von Caemmerer effort is

John M. Cheeseman and Matej Lexa that they lack artificial forcing mechanisms to coordinate activities. For example, Laisk’s model accomplishes the balances and coordination embodied in Eqs. (2–4) by mass action effects, based on the more complete definitions of the biochemical pathways. By including alternative paths of electron flow, thylakoid and energy dissipation, it also enables the prediction of fluorescence characteristics, regulation of photophosphorylation and control of electron flow to the terminal acceptors (Laisk and Eichelmann, 1989; Laisk, 1993). Because of its structure, Laisk’s model should be able to respond to changing conditions such as light flecks or pulses without the addition of other undefinable factors or poorly defined time constants. The most recent version of the model shows this potential, but only if there are physically associated complexes, termed ‘supercomplexes,’ of cytochrome ferredoxin and NADP reductase; a parallel line of experiments supports that requirement (Laisk et al., 1992c; Laisk, 1993). The full promise of the approach has yet to be fully realized however; its major difficulty being that any reaction for which the parameters are unknown must be excluded. In this case, major omissions include photorespiration and respiratory release. The omissions are required by the fact that control of mitochondrial respiration under photosynthetic conditions is poorly understood and that the necessary biochemical data for the glycolate pathway are not available (A. Laisk, personal communication). Thus, the model is effectively limited to non-photorespiratory conditions (Laisk and Walker, 1986).

III. Beyond the Biochemical Model At this point, it is probably more useful to hope that both modeling approaches continue to make progress rather than that one proceed at the expense of the other. It is also worthwhile to look at some aspects that need attention, and at their import to the question of performing and interpreting gas exchange studies under field conditions.

A. Stomata 1. General Response Models Because the biochemical models were developed based on the operation of photosynthesis within

Chapter 8 Gas Exchange: Models and Measurements chloroplasts, it should not be surprising that some leaf-level factors were omitted. The most obvious of these is stomatal functioning. While there are very few differences in basic biochemistry between species, stomata may respond to changes within the mesophyll, they may respond indirectly to photosynthesis by directly perceiving their local and water vapor environment, and they may respond directly to the light environment (see Chapter 9). These responses should and do vary between species, and this has led to a variety of approaches, generally empirical, to modeling them. The diversity of stomatal models can be illustrated by considering three examples. Extending the basic biochemical model, Farquhar and Wong (1984) defined a parameter, T, ‘loosely related to the ATP content of the mesophyll chloroplasts;’ stomatal conductance was defined as directly proportional to T.T represents the condition in which ATP production by photophosphorylation balances ATP consumption through photosynthetic carbon reduction, photosynthetic carbon oxidation and RuBP regeneration and depends on whether electron transport or carboxylation limits assimilation. Overall, the steady state performance of this model is highly satisfactory. It reproduces the semiconservative behavior of and the relationship between and A, even at low light where the influence of dark respiration is important in the overall balance. An acknowledged difficulty, however, is that the mesophyll directly and completely, but nonmechanistically, controls the guard cells; stomata do not directly perceive anything (Farquhar and Wong, 1984). The definition of T also calls attention again to the assumption of perfect tuning between electron transport and Calvin cycle activities. Alternately, Kirschbaum et al. (1988) developed a model to complement the ongoing work on lightfleck utilization by Pearcy and co-workers. The model consists of three steps: a biochemical signal responding directly to light, subsequent osmotic changes in the guard cells, and conductance changes proportional to the resulting water fluxes. The direct light response is of the ‘extended Michaelis-Menten’ form (Thornley, 1976), also known as the nonrectangular hyperbola. This is currently the form most commonly adopted for most light responses:

where is the parameter of interest (the biochemical signal in this case), I is the absorbed irradiance, is

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the initial slope of the curve, and is a convexity or curvature factor. When is zero, Eq. (5) becomes the usual Michaelis-Menten equation, and when it is 1, the equation becomes linear to the point of saturation, and then horizontal. In this form, asymptotically approaches a maximum of 1. The response rates of the model were tuned to experimental observations by adjusting the values of two time constants, one for increasing and the other for decreasing light. A third time constant defined the rate of solute movement in response to the biochemical signal. Finally, stomatal conductance was defined:

where w is the relative amount of water in the guard cells (i.e. variable between 0 and 1) and is the maximum possible conductance at high light. Thus, the instantaneous conductance in Kirschbaum’s model is dependent on four parameters and three time constants. Results of efforts to adjust the model to actual stomatal responses with flashing light or darkflecks suggested that the time ‘constants’ for the biochemical signal could depend on the physiological state of the stomata. This model is unique and directly opposite to the Farquhar and Wong model in that conductance is determined only by direct responses of the guard cells to the light environment, with no influence of any photosynthetic activity. This is undoubtedly too restrictive. It has, however, been successfully coupled to a biochemical module broadly similar to Farquhar and von Caemmerer’s (Pearcy et al., 1994) for simulation of responses to light flecks. The third approach includes the direct influence of both mesophyll and environmental factors on conductance, though as Aphalo and Jarvis (1993) pointed out, all models of this type actually describe a functional rather than a causal relationship between gs and A. This approach is illustrated by Collatz et al. (1991) in which

where A is net uptake determined using the biochemical model, and and are the relative humidity and mole fractions at the leaf surface. The model includes an energy balance module such that leaf surface temperature varies with changes in k and are the slope and intercept (minimum

230 conductance) of the linear response determined by regression of the relationship between conductance and the combined quotient using experimental data; it is not clear when or if they can be considered constants. The influence of irradiance is only indirect with this approach, through its influence on A and leaf temperature and it is instantaneous. Still, good agreement between predicted and measured steady state values of A and for soybean leaves was attainable so long as A was not close to zero (Collatz et al., 1991). Leuning (1990) proposed a similar analysis for Eucalyptus grandis, but found a better fit using instead of ( is the compensation point). In both cases, the authors reported that was more appropriate, i.e. gave a better fit, than D (vapor pressure difference). With Macadamia integrifolia, however, Lloyd (1991) preferred 1/D. Aphalo and Jarvis (1993), analyzing primarily an ivy (Hedera helix) data set proposed yet another model:

where are constants. In this case, the more complex inclusion of both D and leaf temperature serve a modifying function similar to in Ball’s model. To date, the basic application of this approach has been to discern if, and hopefully how, humidity might influence photosynthesis. Any of the humidity relationships can be re-written as the ratio of assimilation to conductance in which case they have a similar general form:

where f(h,T) is some function of humidity and temperature and k is a constant. At any temperature, over a narrow range of humidities, and with a constant boundary layer conductance and atmospheric i.e. normal field-type conditions, this says that the slope of the A versus relationship is constant and equal to Bethenod et al., 1988; Cheeseman et al., 1991). Though the simulations presented by Collatz et al. (1991) for diurnal time courses would have contained the data to address that relationship, they were not shown. Stomatal relations are critical to understanding the results of photosynthesis measurements in the field.

John M. Cheeseman and Matej Lexa The tight linear relationships between A and have been observed many times as has the relatively narrow range of (Wong et al., 1979; Farquhar and Wong, 1984). A most important test of any integrated photosynthesis model, therefore, may be that it simulates an appropriately conservative rather than that it generates an expected A/Ci curve when is forced to vary. It must, of course, incorporate a mechanistic, biochemical model of assimilation. The ultimate, currently unattainable model would define the mechanistic connection between mesophyll and guard cell activities as well. With this, we have circled around to the Farquhar and Wong model with which we began this section.

2. Stomatal Optimization Throughout the studies discussed in the previous section, there has been a notable absence of consideration of the question of optimization of stomatal conductance, i.e. the maximization of carbon gain per unit transpirational water loss over some relevant time period (Cowan, 1982). Two approaches are possible. First, the authors might have addressed the question of whether the predicted stomatal conductances were optimal, particularly if their empirical choices reflected their concepts of mechanisms. Alternately, they might have designed the models to produce optimization. Recently, Friend (1991) presented such a model, incorporating the basic Farquhar-von Caemmerer biochemical model. In it, A responds to changes in based on effects on and water potential. Optimization occurs by comparing the calculated A at the calculated with values at slightly higher and lower if a change in would result in a higher assimilation, conductance is adjusted to do so and the comparisons are repeated. A similar model was presented by Givnish (1986) with a more rudimentary calculation of A. It should be noted that leaf water potential is a function of a number of factors such as root hydraulic conductivity, the fraction of the plant biomass which is allocated to roots, soil water potential and vapor pressure deficit (VPD). In turn, the dependence of A on water potential is empirically based. Thus, this approach requires modeling substantially more of the soilplant-atmosphere transport and physiological systems than the other approaches. In this model, photosynthesis is affected directly by leaf water potential and leaf temperature, and

Chapter 8 Gas Exchange: Models and Measurements indirectly through VPD. Conductance at any is solely a function of A, however, making it comparable to Farquhar and Wong (1984). On the other hand, the adjustment of to maximize A is tantamount to a direct stomatal response to intercellular this distinguishes Friend’s model from that of Collatz et al. (1991) in which the use of surface conditions was simply a ‘logical requirement’ for separating boundary layer transport processes from physiological ones (see also Mott, 1990).

3. Stomatal Limitations The effect of stomates on net assimilation can be visualized from steady state curves as the difference between A at some conductance and the rate it would be if were equal to i.e. at infinite conductance. This is misleading if any of the stomatal models is correct in which mesophyll activity influences Clearly, for any condition in which the steady state has not been reached or for any other steady state, the calculation of a stomatal limitation is invalid; it should probably be specifically justified whenever it is reported.

B. Mesophyll Conductance One complexity of any stomatal addition to the biochemical model is the effect of the diffusion pathway for between the substomatal cavity and the chloroplast, i.e. the mesophyll or transfer conductance (Lloyd, 1991; von Caemmerer and Evans, 1991; Evans et al., 1994). The transfer conductance is partly a function of leaf anatomy, including the percentage of the volume which is air spaces, and the exposure of mesophyll walls to those spaces. Likewise, it is partly a function of the resistance to movement across cell walls and into chloroplasts, with a positive correlation to the proximity of the chloroplast surfaces to exposed walls (Lloyd et al., 1992; Evans et al., 1994; Syvertsen et al., 1995), All considerations to date have been restricted to simple anatomies (with well differentiated palisades and mesophyll layers); other cases such as Typha in which leaf lacunal levels may reach 5% (Constable and Longstreth, 1992) and bilateral, hypostomatous mangroves, present additional interesting complexities which are beyond the scope of the present discussion. While the biochemical model uses, as its input, the concentration or partial pressure at the

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chloroplast, leaf models and gas analysis software calculate the concentration just inside the guard cells. The two are joined through the mesophyll air spaces and are equal only if that conductance is infinite; application of stable isotope techniques indicates that it is not. In mesophytic crop species with air space volumes of 40–50%, values are on the order of 3 to with the effect of reducing by ~30% from the sub-stomatal level (von Caemmerer and Evans, 1991; Lloyd et al., 1992; Evans et al., 1994). The variability of between species is marked as well, decreasing with increases in xerophytic character. Lloyd et al. (1992), for example, reported that in Prunus, Citrus and Macadamia species with relatively low mesophyll air volumes, transfer conductances were only 1.1 to 2.2 with a proportional decrease in In Fagus and Castanea, transfer conductances were similarly on the same order as stomatal conductance, with a resulting limitation of assimilation by as much as 30% from the infinite conductance level (Epron et al., 1995). Mesophyll conductance has, so far, been inadequately modeled and the estimates of its limiting effect are based largely either on one-dimensional analyses or on comparisons of electron transport (estimated by fluorescence) and fixation; both have substantial difficulties (Parkhurst, 1994). In addition, experimental results to date indicate the conductance to be sensitive to growth period light levels (von Caemmerer and Evans, 1991) and it is, as yet, unknown whether gt can change due to chloroplast movements in short times (minutes to hours) as might occur on bright days in the field, or with water stress. For the present, the major, obvious effect of will be to decrease the initial slope of curves from what they would be if plotted on the basis of If is relatively constant, this will be purely a quantitative effect, but for comparisons between species or conditions, the potential complications are significant. Methods for assessing (relative or absolute) rapidly and under field conditions are clearly needed.

C. Rubisco Activation Of all the enzymes in the Calvin cycle, Rubisco is the most complex in terms of its control as well as the most significant in determining the overall rate of fixation. Progress in understanding Rubisco

232 control has been substantial since publication of the original Farquhar-von Caemmerer model (Portis, 1992) including the discovery and characterization of Rubisco activase (Salvucci et al., 1985). Though the activase mechanism of action is still poorly understood, especially once leaves have been illuminated for some time, it is clear that no future model can be viable which fails to incorporate a role for this enzyme. One current informal model involves the light-related formation of a across the thylakoid, partly because activase itself is only slightly pH sensitive over the range which is likely within the stroma (Portis, 1992). It is not yet clear how the gradient might be transduced to influence a stromal enzyme, though the involvement of the cytochrome complex has been suggested (Portis, 1992). This suggests a point of interface for biochemical models, especially Laisk’s. Light regulated changes in Rubisco carbamylation are important to the overall control of photosynthesis, matching Rubisco activity to the rate of RuBP regeneration (Jackson et al., 1991; see also Chapter 6). Rubisco activity can be modulated in vivo over periods ranging from minutes to hours. The changes are not restricted to transitions between dark and light, but may occur over a PPFD range of at least 1000 and over ranges from the compensation point to well above ambient (von Caemmerer and Edmondson, 1986). Over these ranges, activation is not necessarily linear nor does it necessarily reach 100% (Perchorowicz et al., 1981; Sage et al., 1993). Modulation of Rubisco activity undoubtedly contributes in a major way to all environmentally interesting studies. For example, von Caemmerer and Farquhar (1984) reported that except under severe water stress, changes in the initial slope of the curve paralleled the in vitro activity changes in Rubisco. Similarly, Pons et al. (1992) showed a linear relationship between Rubisco activity and the ‘induction state’ of photosynthesis five minutes after the re-illumination of leaves held for some time at low light. Under such conditions, photosynthesis increases biphasically (Mott et al., 1984; Kirschbaum and Pearcy, 1988b;Woodrow and Mott, 1989; Jackson et al., 1991). The increase during the slow phase, in spinach, is directly proportional to the amount of inactive Rubisco present at the time of the change (Woodrow and Mott, 1989). The increase in photosynthetic capacity reported for Alocasia

John M. Cheeseman and Matej Lexa (Kirschbaum and Pearcy, 1988b) most likely reflects the same activation phenomenon. Sharkey et al. (1986) measured Rubisco activity in Phaseolus under conditions in which assimilation was insensitive to finding that under steady state conditions at elevated RuBP levels were higher and usage rates and Rubisco activation states were lower when was reduced; at normal activation was insensitive to Limiting TPU by binding Pi with deoxyglucose produced insensitivity of assimilation at moderate and a sizable reduction in Rubisco activity at low This implied that at low or with restricted Pi, assimilation approached the maximum capacity of starch and sucrose synthesis pathways for triose-phosphate utilization, leading to the down-regulation of Rubisco. The changes in Rubisco activity in these studies are not simply minor modifications. Between the compensation point and the operational for example, Rubisco activity may double (von Caemmerer and Edmondson, 1986). Thus, as it appears in Eq. (2) cannot be considered a constant. This severely compromises the interpretation of curves, as well as the use of the initial slope, CE, for comparing results between treatments or species, or for calculating The report of Küppers et al. (1986) on eucalypts reinforces this conclusion; through a day with mid-day depression of photosynthesis, variations in conductance were associated with sizeable variations in photosynthetic capacity, i.e. In some plants, an additional factor may be the binding and release of carboxyarabinitol 1 -phosphate (CAP). An increase in the total activity of Rubisco (Vu et al., 1983) occurs concomitant with the release of CAP, and both events may be influenced or mediated by Rubisco activase (Robinson and Portis, 1988; Mate et al., 1993). Release of the inhibitor at the beginning of a day with a normal increase in light may be significant for appreciable times. In soybeans, for example, full release did not occur in an hour at irradiances below about 150 (Pons et al., 1992). On the other hand, Moore and Seemann (1992) reported that CAP was released and metabolized to carboxyarabinitol (CA) with a half-time of about 1 minute when leaves of several species known for high inhibitor levels were transferred from dark to light at 500 . At the other end of the day, even in the champion CAP species, Phaseolus vulgaris, decreases in Rubisco activity are primarily

Chapter 8 Gas Exchange: Models and Measurements due to changes in carbamylation state rather than inhibitor synthesis and binding (Sage, 1993; Sage et al., 1993). Finally, we should note that the measurement of Rubisco activity, degree of carbamylation, catalytic constant and total amount present is now straightforward using a spectrophotometric assay coupled to NADH oxidation using PGA kinase and glyceraldehyde phosphate dehydrogenase (Sharkey et al., 1991). This should encourage routine incorporation of these measurements into field related studies. We will return to this in Section IV.

D. Ribulose 1,5-bisphosphate Regeneration As noted earlier (Section II. B), Farquhar and von Caemmerer used a highly simplified scheme for RuBP regeneration controlled through the supply of ATP and NADPH rather than through enzyme activities or intermediate pools, possibly excepting Pi (Farquhar and von Caemmerer, 1982). Laisk’s models treat the regeneration system more thoroughly though still without modulation of individual enzyme activities. The importance of regeneration pathways should not, however, be minimized. Two sensitivity analyses have been published, both showing a relatively high sensitivity of carboxylation to ribulose 5-phosphate (Ru5P) regeneration and particularly to stromal FBPase activity (Laisk and Walker, 1986; Woodrow and Mott, 1993). Also significant was the activity of the combined sucrose synthetic pathway. Experimental studies indicate that the importance of the regeneration pathways depends on the time scale of interest. As many of these have involved specific environmental stresses considered elsewhere in this volume, they will be dealt with only briefly here. In work complementary to the re-illumination studies discussed in the previous section, RuBP quickly became limiting, i.e. it fell well below the Rubisco active site concentration, when light was decreased from 500 to 10 quanta (Sassenrath-Cole and Pearcy, 1992; see Chapter 13). Recovery to levels sufficient to support potential carboxylation depended on the time at low light. Modulation of FBPase and SBPase was indicated by transients in triose-phosphate, fructose 1,6bisphosphate (FBP), sedoheptulose 1,7-bisphosphate (SBP) and fructose 6-phosphate concentrations. The activity of both enzymes is known to be thioredoxin

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mediated. With longer low-light treatment, there was an additional limitation by Ru5P kinase inactivation. Limitations by RuBP regeneration and associated enzyme activation have also been implied in some studies of water and chilling stresses (but see Vu et al., 1987; Sharkey and Seemann, 1989; see also Chapter 14). In sunflowers, albeit at restrictive growth chamber light levels, Rubisco activity and activation changed little with water stress, and were unable to explain the 80% decrease in assimilation (Gimenez et al., 1992). Conductance changes were also unable to explain the decreases, and stomatal patchiness could be specifically discounted. The reductions were, instead, attributed to sizable reductions in RuBP and restrictions on regeneration. During the drought treatment, RuBP declined from just slightly above the concentration required to support fixation, to a level at which it was rate limiting. With chilling stress, Sassenrath et al. (1990) found that FBP and SBP accumulated in tomatoes during room temperature labeling after chilling in the light. This was associated with a loss of bisphosphatase activities which tracked the loss of photosynthesis. FBPase could be re-activated with dithiothreitol, indicating that thioredoxin activation was disrupted. RuBP did not accumulate during the post-chill labeling, nor were there limitations on electron transport or ATP formation. Though Rubisco function was compromised while at low temperatures (Sassenrath and Ort, 1990), it recovered too rapidly on re-warming to limit photosynthesis. Most recently, regeneration limitations have been supported studies using transgenic tobacco plants containing antisense rbcS DNA (Gunasekera and Berkowitz, 1993). Rubisco levels in these plants were decreased 68%, but reductions in assimilation under water stress were proportionately the same in wild type and transformed plants. If Rubisco were the limitation, RuBP should have increase in the transgenics under stress, but instead, it decreased. The authors concluded, therefore, that the overall limitation was due to an enzymatic step in RuBP regeneration.

E. Photoprotection In Fig. 1, photoprotection is used to include all those functions which prevent damage to the photosynthetic machinery of the chloroplast under conditions that supply more photons than can be productively

234 transduced by mainstream photochemical and biochemical photosynthetic pathways. Photoprotective responses at the chloroplast level have been best studied with respect to fluorescence quenching and the xanthophyll cycle. The topic in general has been reviewed recently (Demmig-Adams and Adams, 1992; see also Chapters 1–3), so its treatment here will be brief. In models of photosynthesis, photoprotection has been dealt with only minimally (Laisk and Eichelmann, 1989; Laisk, 1993) though its ecological and physiological significance is clear. Like Rubisco activase (Portis, 1992), but by processes that are somewhat better understood, the de-epoxidation of violaxanthin to zeaxanthin is controlled by across the thylakoid membranes. Recently, it has been shown that these changes can occur rapidly in Alocasia in sunflecks (Watling et al., 1993) and in other canopy species in the tropics (Königer et al., 1993). The responses can be completed within minutes when illumination either increases or decreases, indicating their importance in regulation of photosynthesis under varying light conditions. The interaction of zeaxanthin with the chlorophyll antennae results in the diversion of photon energies from the photochemical apparatus before electrons are released from the reaction center of PS II (see Chapter 1). This results in a decrease in the quantum efficiency of PS II photochemistry, the ratio of variable to maximal fluorescence and photochemical quenching of excitation energy, but without necessarily sacrificing stoichiometric coupling between electron transport and carbon fixation. It is, thus, compatible with the perfect tuning restriction (see Section II.B). Pulse-amplitude modulated fluorescence measurements can indicate the extent of down-regulation, most likely directly associated with zeaxanthin quenching, by comparison of to the latter being the ratio in light acclimated leaves under conditions in which the primary acceptor in PS II is maximally oxidized (see also Chapter 3). The ratio of the two ratios indicates how much the quantum efficiency of PS II photochemistry has been decreased due to down regulation (K. Oxborough, personal communication). Even if mathematical models of leaf level processes do not adequately treat this phenomenon, the combined use of fluorescence and gas exchange methods must increase, especially in field level studies. In particular, the perfect tuning convenience

John M. Cheeseman and Matej Lexa must be more carefully examined. Indeed, studies in our laboratory and under harsh field conditions in Western Australia using simultaneous fluorescence and infra-red gas analysis (unpublished) with the Rhizophora mangroves, indicate the possibility of uncoupling of electron transport and fixation (Fig. 3). In this case, fixation saturated at a PPFD of 350 while apparent electron flow increased linearly with PPFD to at least 1000 In response to water stress, electron flow was lower, but increased farther beyond light saturation. When measured electron flow was compared with the flow required to support A and changes in related to were included (using the equations of Farquhar and von Caemmerer, 1982), only 27% of the electrons could be accounted for (Cheeseman, 1994). A discrepancy of this sort can not be simulated by either Farquhar/von Caemmerer or Laisk-type models. Though the actual discrepancy is unlikely to be that large, it is also unlikely that photorespiration and the limitations of fluorescence analysis will completely eliminate it. Though the fate of the putative excess electrons in these experiments is unclear, it is not unreasonable to expect that they will be flowing to molecular oxygen. Antioxidant protection has received increasing (but scattered) attention in recent years in association with drought (Burke et al., 1985; Irigoyen et al., 1992), chilling (Hodgson and Raison, 1991; Jahnke et al., 1991; Walker and McKersie, 1993) and oxidant stresses (Guri, 1983; Burke et al., 1985). Laisk (Laisk and Eichelmann, 1989; Laisk, 1993) has included electron flow to oxygen in his more recent models, though the treatment of the phenomenon is still rudimentary. Equation 3 (Section II. A), also includes a small Mehler component to balance the electron flow requirements for ATP and NADPH production (Farquhar and von Caemmerer, 1982). One modeling difficulty is that there are several possible pathways for protection of photosynthetic machinery after the generation of superoxide (Fig. 4; see also Chapter 5). At present, it appears that direct photoreduction of monodehydroascorbate radicals (the Mehler-ascorbate peroxidase, or MAP, cycle) would be the most efficient (Hormann et al., 1994; Miyake and Asada, 1994). Clearly, more work is necessary to resolve which (if any) mechanisms are actually involved, and their overall significance.

Chapter 8 Gas Exchange: Models and Measurements

IV. The Feedback Loop: Consequences for Field Studies At this point, we will focus again on the problems and goals of experimental science operating under field conditions. While we clearly can not attempt to define a universal experimental protocol, for any study based around gas exchange methods, the primary goal in many studies will be to quantify photosynthetic performance. This would include, minimally, A, and but also (with the addition of fluorescence instrumentation) quantum yield of PS II photochemistry and linear electron transport rate. These measurements are easy to make, at least if A is not low, environmental conditions are stable at the leaf level, and photosynthesis itself is at steady state. These restrictions are, of course, not trivial, nor is it always simple to identify when they are not satisfied. Further, once performance is reduced to a single number or set of numbers, the ‘so-what’ question arises; what do the numbers mean and in what context? First, to attack the problem of marginal conditions, the best approach is probably through leaf chamber design for higher effective gain of the signal and improved environmental control. For example, Oja (1983) and Pearcy (1993) have published designs suitable for the analysis of rapid light transients; similar principles of small (or variable) chamber volumes with fast mixing rates should be incorporated

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in field chambers. An extreme, but important, condition which challenges most equipment is the hot but variably cloudy day on which both light and may fluctuate rapidly. The ability to produce reliable measurements under such conditions should, arguably, be a primary chamber design criterion. With the new generation of commercial instruments currently coming to market, some of these design questions have been addressed. Then, however, even with stability, the irreproducibility of field environments can lead to data sets such as those presented in Fig. 2. Despite first reactions, these should not be dismissed as horrible messes; they contain the information, however well hidden, that we ultimately need in order to understand, i.e. adequately model, photosynthesis at this experimental level. Traditional data presentation methods, e.g. single factor responses such as these, also do as much to obscure as to clarify results; neither panel represents a response free from the influence of the other. Thus, if we think of ideal data as comprising a multidimensional response surface (in Fig. 2, the equivalent to this surface is the lines placed at the upper limit of each response data set), we can refine the performance question to ask whether each point falls on the surface. More generally, this asks what the relationship is between photosynthetic performance and our expectation of photosynthetic potential,

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i.e. the question identified in the first paragraph of this chapter. Even the apparently most straightforward survey of performance, therefore, directly demands the understanding and interpretation of the models we have discussed here. Even points which lie on the surface reflect the integrated solution to multiple limitations. Points below the surface simply reflect greater restrictions of one or more component processes. Our next, objective, therefore, is to identify the probable restrictions as simply as possible, and then to examine those processes more carefully. One approach is to limit data a posteriori to those taken under a more narrow range of conditions (e.g. or to ranges in which responses are linear (e.g. A versus or flat (e.g. light saturation). To these, multiple regression

John M. Cheeseman and Matej Lexa

and residual analysis methods can be applied (Cheeseman et al., 1991). While these approaches increase the information which can be extracted from any data set, the new insight they can generate is limited. Beyond that, new field approaches are needed. For any environmental conditions, the major system uncertainties which we have identified here are relatively few, even if they are all substantial. Primarily, they include the activation state of Rubisco, the coupling between electron transport and the Calvin cycle, and downstream limitations including those related to Pi cycling. For studies extending beyond single individuals, transfer conductance may be significant, as may be photoprotection. Thus, new field approaches means, minimally,

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incorporating appropriate Rubisco activation assays, and fluorescence measured along with exchange. Though biochemical assays add substantially to the effort required for data collection, they may still be the most rapid and least ambiguous method of relating performance and potential. In other instances capacity might better be estimated by relieving the condition thought to be most limiting, e.g. by re-illumination of leaves at low light (Kirschbaum and Pearcy, 1988a; Pons et al., 1992), or suddenly increasing (Sharkey, 1985a; Sivak and Walker, 1987; Oja et al., 1988) or changing With this approach, the time per sample to estimate increases, with associated demands on the environmental control system. Or, if Pi or regeneration limitations are suspected, capacity as indicated by evolution at saturating maybe appropriate, but again as a supplement to other methods. Finally, we should caution that we are advocating extension of current field methods, not substitutions for them. Far from being a shotgun approach, however, this is meant as an experimental recognition of the complexity of leaf level photosynthesis, especially in the field, and the need to gather data for which multiple interpretations, i.e. multiple models, can be considered once the season is over, the interesting stage of development or the period of stress is passed, or hundreds of intervening kilometers make just one more experiment an impossibility.

For supplying pre-prints and reprints of papers, source codes and running models, thoughtful discussions and criticisms, and much of the primary data behind this review, the authors would like to gratefully acknowledge Timothy Ball, John Boyer, John Evans, Graham Farquhar, Lou Gross, Agu Laisk, Don Ort, Kevin Oxborough, Robert Pearcy and Archie Portis. The background to Fig. 1 was provided by Alison Cheeseman.

V. Conclusion

References

A most important aspect for any study of photosynthesis, and particularly for those addressing field problems, is the continual recognition that all studies incorporate models, not just for data analysis and interpretation, but at the earliest stages of experimental planning. Awareness of that, awareness of the models themselves, and careful selection of those which are most appropriate, is essential. The deficiencies in formal models of photosynthesis, especially with respect to mechanisms of activation, the generalized inclusion of stomata, photoprotection, and empirical inclusions used to produce realistic transient responses, are clear. These should not, however, be allowed to detract from their strengths. One thing that even the most rudimentary or empirical model can do is to provide ‘students’ at all levels with a feel for the interactions of processes, and a priori meaningful expectations of their

Allen RD (1995) Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol 107: 1049–1054 Aphalo PJ and Jarvis PG (1993) An analysis of Ball’s empirical model of stomatal conductance. Ann Bot 72: 321–327 Baker NR and Bowyer JR (1994) Photoinhibition of Photosynthesis—From Molecular Mechanisms to the Field. Bios Scientific Publishers, Oxford Bethenod O, Katerji N, Quetin P and Bertolini JM (1988) Efficiencé de l’eau d’une culture de pomme de terre (Solanum tuberosum L. cv. Bintje) 1. Mise en évidence de la régulation du interne à l’échelle foliaire. Photosynthetica 22: 491– 501 Bowler C, van Montagu M and Inzé D (1992) Superoxide dismutase and stress tolerance. Ann Rev Plant Physiol Plant Mol Biol 43: 83–116 Burke JJ, Gamble PE, Hatfield JL and Quisenberry JE (1985) Plant morphological and biochemical responses to field water deficits. I. Responses of glutathione reductase activity and paraquat sensitivity. Plant Physiol 79; 415–419 Cheeseman JM (1991) PATCHY—Simulating and visualizing the effects of stomatal patchiness on photosynthetic

experimental results. As such, it behooves even the most vociferous anti-modelers to familiarize themselves with some computer model. At the same time, those who program should strive to make their efforts user friendly, the outputs comprehensible and graphical, and the programs widely available. Given that, one should expect field studies to be improved by desktop experimentation complementing the biological considerations. This is a prime use of modeling, and an essential element of the feedback loop linking models and experiments. Our objective here, therefore, has been to identify some of the kinds of data critically needed to improve existing models, to reconcile the differences between performance and potential, and to understand the constraints imposed by environmental pressures.

Acknowledgments

238 exchange studies. Plant Cell Environ 14: 593–599 Cheeseman JM (1994) Depressions of photosynthesis in mangrove canopies. In: Baker NR and Bowyer JR (eds) Photoinhibition of Photosynthesis—From Molecular Mechanisms to the Field, pp 379–391. Bios Scientific Publishers, Oxford Cheeseman JM, Clough BF, Carter DR, Lovelock CE, Eong OJ and Sim RG (1991) The analysis of photosynthetic performance in leaves under field conditions—a case study using Bruguiera mangroves. Photosynth Res 29: 11–22 Collatz GJ, Ball JT, Grivet C and Berry JA (1991) Physiological and environmental regulation of stomatal conductance, photosynthesis and transpiration—a model that includes a laminar boundary layer. Agric For Met 54: 107–136 Constable JVH and Longstreth DJ (1992) Photosynthetic gas exchange of cattail, a species with elevated internal concentrations. Plant Physiol 99S: 102 Cowan IR (1982) Regulation of water use in relation to carbon gain in higher plants. In: Lange OL, Nobel PS, Osmond CB and Ziegler H (eds) Physiological Plant Ecology II, pp 589– 613. Springer Verlag, Berlin Demmig-Adams B and Adams WW (1992) Photoprotection and other responses of plants to high light stress. Ann Rev Plant Physiol Plant Mol Biol 43: 599–626 Epron D, Godard D, Cornic G and Genty B (1995) Limitation of net assimilation rate by internal resistances to transfer in the leaves of two tree species (Fagus sylvatica L. and Castanea sativa Mill.). Plant Cell Environ 18: 43–51 Evans JR, von Caemmerer S, Setchell BA and Hudson GS (1994) The relationship between transfer conductance and leaf anatomy in transgenic tobacco with a reduced content of rubisco. Aust J Plant Physiol 21: 475–495 Farquhar GD and von Caemmerer S (1982) Modeling of photosynthetic response to environmental conditions. In: Lange OL, Nobel PS, Osmond CB and Ziegler H (eds) Physiological Plant Ecology I I , pp 549–587. Springer-Verlag, Berlin Farquhar GD and Wong SC (1984) An empirical model of stomatal conductance. Aust J Plant Physiol 11: 191–210 Farquhar GD, von Caemmerer S and Berry JA (1980) A biochemical model of photosynthetic assimilation in leaves of C3 species. Planta 149: 78–90 Field CB, Ball JT and Berry JA (1991) Photosynthesis: Principles and field techniques. In: Pearcy RW, Ehleringer J, Mooney HA and Rundel PW (eds) Plant Physiological Ecology: Field Methods and Instrumentation, pp 209–253. Chapman and Hall, London Friend AD (1991) Use of a model of photosynthesis and leaf microenvironment to predict optimal stomatal conductance and leaf nitrogen partitioning. Plant Cell Environ 14: 895–905 Gimenez C, Mitchell VJ and Lawlor DW (1992) Regulation of photosynthetic rate of two sunflower hybrids under water stress. Plant Physiol 98: 516–524 Givnish TJ (1986) Optimal stomatal conductance, allocation of energy between leaves and roots, and the marginal cost of transpiration. In: Givnish TJ (ed) On the Economy of Plant Form and Function, pp 171–213. Cambridge University Press, Cambridge Gunasekera D and Berkowitz GA (1993) Use of transgenic plants with ribulose-1,5-bisphosphate carboxylase oxygenase antisense DNA to evaluate the rate limitation of photosynthesis under water stress. Plant Physiol 103: 629–635

John M. Cheeseman and Matej Lexa Guri A (1983) Variation in glutathione and ascorbic acid content among selected cultivars of Phaseolus vulgaris prior to and after exposure to ozone. Can J Plant Sci 63: 733–737 Hall A (1971) A model of leaf photosynthesis and respiration. Carnegie Inst Washington Yearbook 70: 530–540 Harley PC and Sharkey TD (1991) An improved model of C3 photosynthesis a thigh —reversed sensitivity explained by lack of glycerate reentry into the chloroplast. Photosynth Res 27: 169–178 Heldt HW and Rapley L (1970) Specific transport of inorganic phosphate, 3-phosphoglycerate and dihydroxyacetonephosphate, and dicarboxylate across the inner membrane of spinach chloroplasts. FEBS Lett 10: 143–148 Hodgson RAJ and Raison JK (1991) Lipid peroxidation and superoxide dismutase activity in relation to photoinhibition induced by chilling in moderate light. Planta 185: 215–219 Hormann H, C. N and Schreiber U (1994) An active Mehlerperoxidase reaction sequence can prevent cyclic PS I electron transport in the presence of dioxygen in intact spinach chloroplasts. Photosynth Res 41: 429–437 Horton P and Nicholson H (1987) Generation of oscillatory behaviour in the Laisk model of photosynthetic carbon assimilation. Photosynth Res 12: 129–143 Irigoyen J, Emerich DW and Sánchez-Díaz M (1992) Alfalfa leaf senescence induced by drought stress: photosynthesis, hydrogen peroxide metabolism, lipid peroxidation and ethylene evolution. Physiol Plant 84: 67–72 Jackson RB, Woodrow IE and Mott KA ( 1 9 9 1 ) Nonsteady-state photosynthesis following an increase in photon flux density (PFD). Effects of magnitude and duration of initial PFD. Plant Physiol 95: 498–503 Jahnke LS, Hull MR and Long SP (1991) Chilling stress and oxygen m e t a b o l i z i n g enzymes in Zea mays and Zea diploperennis. Plant Cell Environ 14: 97–104 Kirschbaum M U F and Pearcy RW (1988a) Gas exchange analysis of the fast phase of photosynthetic induction in Alocasia macrorrhiza. Plant Physiol 87: 818–821 Kirschbaum M U F and Pearcy RW (1988b) Gas exchange analysis of the relative importance of stomatal and biochemical factors in photosynthetic induction in Alocasia macorrhiza. Plant Physiol 86: 782–785 Kirschbaum MUF, Gross LJ and Pearcy RW (1988) Observed and modelled stomatal responses to dynamic light environments in the shade plant, Alocasia macrorhiza. Plant Cell Environ 11: 1 1 1 – 1 2 1 Königer M, Virgo A, Harris G and Winter K (1993). Xanthophyll cycle pigments in tropical C3 and CAM plants (abstract 77). Abstracts of 41st Harden Conference. Biochemical Society, London Küppers M, Wheeler AM, Küppers BIL, Kirschbaum MUF and Farquhar GD (1986) Carbon fixation in eucalypts in the field. Analysis of diurnal variations in photosynthetic capacity. Oecologia 70: 273–282 Laisk A (1993) Mathematical modeling of free-pool and channelled electron transport in photosynthesis: evidence for a functional supercomplex around photosystem 1. Proc Roy Soc Lond B 251: 243–251 Laisk A and Eichelmann H (1989) Towards understanding oscillations: a mathematical model of the biochemistry of photosynthesis. Phil Trans R Soc Lond B 323: 369–384

Chapter 8 Gas Exchange: Models and Measurements Laisk A and Walker DA (1986) Control of phosphate turnover as a rate-limiting factor and possible cause of oscillations in photosynthesis: a mathematical model. Proc R Soc Lond B 227: 281–302 Laisk A, Eichelmann H, Oja V, Eatherall A and Walker DA (1989) A mathematical model of the carbon metabolism in photosynthesis. Difficulties in explaining oscillations by fructose 2,6-bisphosphate regulation. Proc R Soc Lond B 237: 389–415 Laisk A, Siebke K, Gerst U, Eichelmann H, Oja V and Heber U (1991) Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle. Planta 185: 554–562 Laisk A, Kiirats O, Oja V, Gerst U, Weis E and Heber U (1992a) Analysis of oxygen evolution during photosynthetic induction and in multiple-turnover flashes in sunflower leaves. Planta 186: 434–441 Laisk A, Oja V and Heber U (1992b) Steady-state and induction kinetics of the photosynthetic electron transport related to donor side oxidation and acceptorside reduction of photosystem 1 in sunflower leaves. Photosynthetica 27: 449–463 Laisk A, Oja V, Walker D and Heber U (1992c) Oscillations in photosynthesis and reduction of photosystem 1 acceptor side in sunflower leaves. Functional cytochrome 1 ferredoxin-NADP reductase supercomplexes. Photosyn– thetica 27: 465–479 Leuning R (1990) Modeling stomatal behaviour and photosynthesis in Eucalyptus grandis. Aust J Plant Physiol 17: 159– 175 Lloyd J (1991) Modeling stomatal responses to environment in Macadamia integrifolia. Aust J Plant Physiol 18: 649–660 Lloyd J, Syvertsen JP, Kriedemann PE and Farquhar GD (1992) Low conductances for diffusion from stomata to the sites of carboxylation in leaves of woody species. Plant Cell Environ 15: 873–899 Long SP and Hällgren J-E (1993) Measurement of assimilation by plants in the field and the laboratory. In: Hall DO, Scurlock JMO, Bohlàr-Nordenkampf HR, Leegood RC and Long SP (eds) Photosynthesis and Production in a Changing Environment: A Field and Laboratory Manual, pp 129–167. Chapman and Hall, London Mate CJ, Hudson GS, von Caemmerer S, Evans JR and Andrews TJ (1993) Reduction of ribulose bisphosphate carboxylase activase levels in tobacco (Nicotiana tabacum) by antisense RNA reduces ribulose bisphosphate carboxylase carbamylation and impairs photosynthesis. Plant Physiol 102: 1119–1128 Miyake C and Asada K (1994) Ferredoxin-dependent photoreduction of the monodehydroascorbate radical in spinach thylakoids. Plant Cell Physiol 35: 539–549 Moore R and Seemann JR (1992) Metabolism of 2'-carboxyarabinitol in leaves. Plant Physiol 99: 1551–1555 Mott KA (1990), Sensing of atmospheric by plants. Plant Cell Environ 13: 731–737 Mott KA, Jensen RG, O’Leary JW and Berry JA (1984) Photosynthesis and ribulose 1,5-bisphosphate concentrations in intact leaves of Xanthium strumarium L. Plant Physiol 76: 968–971 Oja VM (1983) Fast gasometric device for investigation of leaf photosynthesis kinetics. Sov Plant Physiol 30: 795–802 Oja VM, Rasulov BH and Laisk AH (1988) An analysis of the

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temperature dependence of photosynthesis considering the kinetics of RuP2 carboxylase and the pool of RuP2 in intact leaves. Aust J Plant Physiol 15: 737–748 Parkhurst DF (1994) Diffusion of and other gases inside leaves. New Phytol 126: 449–479 Pearcy RW (1993) Sunfleck utilization. In: Hendry GAF and Grime JP (eds) Methods in Comparative Plant Ecology, pp 68–72. Chapman and Hall, London Pearcy RW, Chazdon RL, Gross LJ and Mott KA (1994) Photosynthetic utilization of sunflecks: A temporally patchy resource on a time scale of seconds to minutes. In: Caldwell MM and Pearcy RW (eds) Exploitation of Environmental Heterogeneity by Plants: Ecophysiological Processes Above and Below Ground, pp 175–208. Academic Press, San Diego Perchorowicz JT, Raynes DA and Jensen RG (1981) Light limitation of photosynthesis and activation of ribulose bisphosphate carboxylase in wheat seedlings. Proc Natl Acad Sci USA 78: 2985–2989 Pons TL, Pearcy RW and Seemann JR (1992) Photosynthesis in flashing light in soybean leaves grown in different conditions. 1. Photosynthetic induction state and regulation of ribulose1,5-bisphosphate carboxylase activity. Plant Cell Environ 15: 569–576 Portis AR (1992) Regulation of ribulose 1,5-bisphosphate carboxylase oxygenase activity. Ann Rev Plant Physiol Plant Mol Biol 43: 415–437 Robinson SP and Portis AR (1988) Release of the nocturnal inhibitor, carboxyarabinitol-1-phosphate, from ribulose bisphosphate carboxylase/oxygenase by rubisco activase. FEBS Lett 233: 413–416 Sage RF (1993) Light-dependent modulation of ribulose-1,5bisphosphate carboxylase oxygenase activity in the genus Phaseolus. Photosynth Res 35: 219–226 Sage RF, Reid CD, Moore BD and Seemann JR (1993) Longterm kinetics of the light-dependent regulation of ribulose-1,5bisphosphate carboxylase oxygenase activity in plants with and without 1-carboxyarabinitol 1-phosphate. Planta 191:222– 230 Salvucci ME, Jr. ARP and Ogren WL (1985) A soluble chloroplast protein catalyzes activation of ribulose bisphosphate carboxylase/oxygenase in vivo. Photosynth Res 7: 193–201 Sassenrath GF and Ort DR (1990) The relationship between inhibition of photosynthesis at low temperature and the inhibition of photosynthesis after rewarming in chill-sensitive tomato. Plant Physiol Biochem 28: 457–465 Sassenrath SF, Ort DR and Portis Jr. AR (1990) Impaired reductive activation of stromal bisphosphatases in tomato leaves following low-temperature exposure to high light. Arch Biochem Biophys 282: 302–308 Sassenrath-Cole GF and Pearcy RW (1992) The role of ribulose1,5-bisphosphate regeneration in the induction requirement of photosynthetic exchange under transient light conditions. Plant Physiol 99: 227–234 Sharkey TD (1985a) photosynthesis in C3 plants. Its occurrence and a possible explanation. Plant Physiol 78: 71–75 Sharkey TD (1985b) Photosynthesis in intact leaves of C3 plants: Physics, physiology and rate limitations. Bot Rev 51: 53–341 Sharkey TC and Seemann JR (1989) Mild water stress effects on carbon-reduction-cycle intermediates, ribulose bisphosphate

240 carboxylase activity, and spatial homogeneity of photosynthesis in intact leaves. Plant Physiol 89: 1060–1065 Sharkey TD, Seemann JR and Berry JA (1986) Regulation of ribulose-1,5-bisphosphate carboxylase activity in response to changing partial pressure of and light in Phaseolus vulgaris. Plant Physiol 81: 788–791 Sharkey TD, Savitch LV and Butz ND (1991) Photometric method for routine determination of and carbamylation of rubisco. Photosynth Res 28: 41–48 Sivak MN and Walker DA (1987) Oscillations and other symptoms of limitation of in vivo photosynthesis by inadequate phosphate supply to the chloroplast. Plant Physiol Biochem 25: 635–648 Stitt MN (1985) Fine control of sucrose synthesis by fructose 2,6 bisphosphate. In: Heath RL and Preiss J (eds) Regulation of Carbon Partitioning in Photosynthetic Tissues, pp 109–126. American Society of Plant Physiologists, Rockville Syvertsen JP, Lloyd J, McConchie C, Kriedemann PE and Farquhar GD (1995) On the relationship between leaf anatomy and diffusion through the mesophyll of hypostomatous leaves. Plant Cell Environ 18: 149–157 Terashima I (1992) Anatomy of non-uniform leaf photosynthesis. Photosynth Res 31: 195–212 Thornley J H M (1976) Mathematical models in plant physiology. Academic Press, New York von Caemmerer S and Edmondson DL (1986) Relationship between steady-state gas exchange, in vivo ribulose bisphosphate carboxylase activity and some carbon reduction cycle intermediates in Rhaphanus sativus. Aust J Plant Physiol 13: 669–688 von Caemmerer S and Evans JR ( 1 9 9 1 ) Determination of the average partial pressure of in chloroplasts from leaves of

John M. Cheeseman and Matej Lexa several C3 plants. Aust J Plant Physiol 18: 287–305 von Caemmerer S and Farquhar GD (1984) Effects of partial defoliation, changes in irradiance during growth, short-term water stress and growth at enhanced p( ) on the photosynthetic capacity of leaves of Phaseolus vulgaris L. Planta 160: 320–329 Vu CV, Allen Jr. LH and Bowes G (1983) Effects of light and elevated atmospheric on the ribulose bisphosphate carboxylase activity and ribulose bisphosphate level of soybean leaves. Plant Physiol 73: 729–734 Vu JCV, Allen Jr. LH and Bowes G (1987) Drought stress and elevated effects on soybean ribulose bisphosphate carboxylase activity and canopy photosynthesis rates. Plant Physiol 83: 573–578 Walker DA and Sivak MN (1985) Can phosphate limit photosynthetic carbon assimilation in vivo? Physiol Veg 23: 829–841 Walker MA and McKersie BD (1993) Role of the ascorbateglutathione antioxidant system in chilling resistance of tomato. J Plant Physiol 141: 234–239 Walling JR, Woodrow IE and Osmond CB (1993). Fluorescence changes and xanthophyll conversion in Alocasia macrorrhiza grown under different irradiance conditions: significance for energy dissipation during sunflecks (abstract 83). Abstracts of 41st Harden Conference. Biochemical Society, London Woodrow IE and Mott KA (1989) Rate limitation of non-steadystate photosynthesis by ribulose-1,5-bisphosphate carboxylase in spinach. Aust J Plant Physiol 16: 487–500 Woodrow IE and Mott KA (1993) ModelingC3 photosynthesis— a sensitivity analysis of the photosynthetic carbon-reduction cycle. Planta 191: 421–432

Chapter 9 Stomata: Biophysical and Biochemical Aspects William H. Outlaw Jr., Shuqiu Zhang*, Daniel R. C. Hite and Anne B. Thistle Department of Biological Science, Florida State University, Tallahassee, FL 32306- 3050, USA

and

* College of Biology, Beijing Agricultural University, Beijing 100094, China

Summary I. Introduction II. Plasmalemma Guard Cell Proton Pump III. Plasmalemma Potassium Channels IV. Plasmalemma Anion Channels V. Tonoplast Transport Processes VI. Abscisic acid, Calcium, and the Phosphoinositide Messenger Systems VII. Integrating Role of Abscisic Acid in the Plant’s Physiology VIII. Carbon Metabolism IX. Concluding Remarks References

241 242 242 244 245 246 247 249 249 253 253

Summary Regulation of gas exchange by stomata of adjustable aperture size in the leaf epidermis is crucial to a plant’s physiology: sufficient must be admitted into the leaves for growth, but loss of water, usually the limiting resource for terrestrial plants, must be minimized. In essence, the study of stomatal movements is an inquiry into accumulation and dissipation of salts, which account for the bulk of the osmotic change associated with stomatal movements. The cardinal event in stomatal opening is the activation of the ATPase of the guard cell plasmalemma, which hyperpolarizes the membrane, strengthening the driving force for passive inward permeation and opening the inward-rectifying channel. Ultimately, is transported into the vacuole, implying the importance of less well studied ion traffic across the tonoplast. Depolarization, in order to develop an outward driving force for is required for stomatal closure and results from anion efflux, which is stimulated by , the role of which is not fully understood. Abscisic acid diminishes gas exchange by activating the -out channel in a fashion. In addition, by stimulating a plasmalemma channel or by activating a phosphoinositide signal transduction pathway, it elevates cytosolic concentration. In turn, elevated concentration shifts more negative the gating potential of the channel and activates the anion efflux channels. Long term effects of abscisic acid on guard cells have not been investigated, but its integrating function as a long distance messenger is currently under intense investigation. Neil R. Baker (ed): Photosynthesis and the Environment, pp. 241-259. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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Potassium concentration fluctuations do not occur in isolation. If stoichiometric, accumulation of by an unknown active transport process would balance , and the pH within the cell would be stabilized even as protons were pumped out, however, accumulation is insufficient. Synthesis of malate, during which two protons are released to solution,‘replenishes’ extruded protons not accounted for by uptake. These and other processes ancillary to fluxes are addressed here.

I. Introduction Stomatal movements result from osmotic water flow. The guard cell pair subtending a stoma accumulates solutes, especially salts (Outlaw, 1983; Zeiger, 1983) and, to a lesser and variable extent, sugars (Outlaw and Manchester, 1979; Outlaw, 1982; Poffenroth et al., 1992). Water influx causes asymmetric cell enlargement, which increases stomatal aperture. Closing, though effected by different mechanisms, is a reversal, as solutes are dissipated. Stomatal movements, which are primary responses to environmental parameters such as light and concentration, are ultimately mediated through activity of guard cell ion pumps, prototypically the ATPase, and associated ion permeation processes and carbon metabolism. Because stomata control the uptake of and the loss of water (usually the resource most limiting to plant growth), their function is crucial to the plant’s physiology. It is, therefore, of paramount importance to understand the molecular mechanisms that underlie stomatal movements and the physiological mechanisms that integrate the functioning of the whole plant. In this chapter, a current understanding will be outlined. Emphases will be on ion transport processes, carbon metabolism, and short term effects of abscisic acid (ABA), the endogenous antitranspirant, as it integrates disparate functions. Various reviews of stomatal function, not otherwise explicitly cited, are of interest: MacRobbie (1988), Raschke et al. (1988), Tallman (1992), and Kearns and Assmann (1993). General background reviews are those by Jan and Abbreviations: ; ABA – abscisic acid; DAG – diacylglycerol; DCMU –3-(3,4-dichlorophenyl)-1,1-dimethylurea; DIDS — diisothiocyanatostilbenedisulfcnic acid; FBPase – fructose 1,6-bisphosphatase; FV – fast vacuolar; IAA – indole acetic acid; 1,4,5-triphosphate; NPPB – 5nitro-2,3-phenylpropylaminobenzoic acid; PEPC–phosphoenolpyruvatecarboxylasc;PFK–phosphofructokinase; Pi–inorganic phosphate; PKC – protein kinase C; PLC – phospholipase C; PPase – pyrophosphatase; Rubisco – ribulose 1,5-bisphosphate carboxylase/oxygenase; SV – slow vacuolar; TCA–tricarboxylic acid cycle; VK – vacuolar potassium channel; electrical potential difference

Jan (1989, voltage-sensitive ion channels; 1992, potassium channels), Serrano and Zeiger (1989) and Trewavas and Gilroy (1991, plant signal transduction), Okazaki and Tazawa (1990, and turgor regulation), Miller (1992, voltage-sensitive channels), Neher (1992, ion channels), Briskin and Hanson (1992, ATPases) and Chanson (1993, ATPases), Palmgren (1991, regulation of plant plasmalemma activity), and the book edited by Davies and Jones (1991), which has particularly relevant chapters by MacRobbie (1991) and McAinsh et al. (1991). Finally, several articles in Randall and Blevins (1989) address integrating mechanisms of plant function.

II. Plasmalemma Guard Cell Proton Pump Several lines of evidence converge to show consistently that the primary event in stomatal opening is the activation of a plasmalemma H+ pump (Fig. 1). First, electrical recordings over the course of a decade (Gunar et al., 1975; Zeiger et al., 1977; Moody and Zeiger, 1978; Assmann et al., 1985; Serrano et al., 1988) show that light, a stimulus for stomatal opening, hyperpolarizes the guard cell plasmalemma. Darkness has the opposite effect. Protons are excreted from guard cells (Raschke and Humble, 1973) prior to stomatal opening (Edwards et al., 1988) and, as implied, have been identified as the current carrier (Ishikawa et al., 1983; Shimazaki et al., 1986; and subsequent references). Further evidence of the role of proton extrusion was provided by Assmann et al. (1985), who showed (i) that blue light-stimulated hyperpolarization occurred only after a delay to the stimulus and (ii) that the low fluence rates were inconsistent with a direct energization of the pump. Sufficiency of the pump current to support observed rates of stomatal movements was demonstrated with intracellular electrodes (Blatt, 1987) and the slow whole cell configuration (Schroeder, 1988). Importantly, Schroeder (1988), but not Lohse and Hedrich (1992), demonstrated that a cellular factor (not ATP) was required for sustained maximum

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pump activity. This activating factor, as yet unidentified, may be provided by photosynthetically functioning chloroplasts (Serrano et al., 1988); whether it is related to a mesophyll factor (Lee and Bowling, 1992) that is not lowering concentration (Aphalo and Jarvis, 1993) is unknown. Diacylglycerol (DAG) activates the pump (Lee and Assmann, 1991), which will be discussed below, and auxin deactivates extrusion (Lohse and Hedrich, 1992). Biophysical (Serrano et al., 1988) and biochemical (Lee and Bowling, 1992) observations are consistent with the known attenuation of red light stimulation of stomatal openings (Sharkey and Raschke, 1981) and associated carbon metabolism (Ogawa et al., 1978) by the photosynthesis inhibitor DCMU. Stomatal opening is inhibited by vanadate (Gepstein et al., 1982; Lohse and Hedrich, 1992), blue light-induced guard cell protoplast swelling is inhibited by vanadate (Amodeo et al., 1992), a guard cell ATPase in extracts of guard cell protoplasts is inhibited by vanadate (Fricker and Willmer, 1987), plasmalemma pump current is inhibited by vanadate (Serrano et al., 1988), and vanadate overrides the effect of fusicoccin (a fungal toxin that promotes stomatal opening and induces a voltage-independent hyperpolarizing current; Assmann and Schwartz, 1992). This sensitivity implies, as expected, a plasmalemma P-type ATPase, i.e. one that operates with a phosphoenzyme intermediate. Studies (Blum et al., 1988; Fricker and Willmer, 1990a; Becker et al., 1993) have not revealed unique attributes of this plasmalemma electroenzyme that would provide a particular explanation for stomatal function. Phosphorylation of the plasmalemma ATPase, as also shown in Fig. 1, is without empirical support, but we include that feature as one reported for other plant cells (Schaller and Sussman, 1988). It is possible that phosphorylation prevents the autoinhibitory domain from interacting with the active site (Serrano, 1993). Only preliminary work (Fricker and Willmer, 1990b) has been carried out with the guard cell tonoplast , and a guard cell has not been reported for guard cells as far as we are aware. The preceding discussion establishes that a pump, specifically a P-type ATPase, initiates and sustains stomatal opening by dual mechanisms, viz. creation of an electrochemical potential gradient for passive influx and hyperpolarization of the membrane to a permissive voltage for conductance by the voltage-regulated inward rectifier. There are

two objections to the unqualified acceptance of the role of the as the sole energy transduction mechanism for uptake across the guard cell plasmalemma. First, under some circumstances, uptake is against an electrochemical potential gradient (Clint and Blatt, 1989). This observation, predicated on low external concentration, implies that potassium must traverse the membrane by means of primary or secondary active processes, without the domain of channels. Second, several groups (Vani and Raghavendra, 1989; Raghavendra, 1990; Pantoja and Willmer, 1991; Gautier et al., 1992) have investigated the possibility that blue light effects are mediated by a plasmalemma redox system, which is well established in plants. In this model, the low fluence requirement and the requirement for energy (from photosynthesis provided by a strong red light background or respiration) imply that blue light is involved in signal transduction and not energy transduction. In brief, reduced pyridine nucleotide and comprise the overall redox couple, which has a stoichiometry of four extruded for one taken up (Gautier et al., 1992). As envisioned by Raghavendra (1990), the flavoprotein that is part of the electron transport chain would be sensitized by blue light. The primary support for this model comes from stimulation by flavins and inhibition by KI or phenyl acetate, both of which quench the excitation of flavins. An earlier failure by vanadate to block blue light responses was also used as support for the importance of the plasmalemma redox system, but that failure is now attributed to poor permeation by vanadate under certain conditions (Schwartz et al., 1991).

III. Plasmalemma Potassium Channels The protracted realization of the importance of to stomatal movements (Outlaw, 1983) is in sharp contrast to the recent robust interest in the mechanism of ion exchange across guard cell membranes. Indeed, less than a decade passed between the seminal report on ion channels (Schroeder et al., 1984) in plant cells and the cloning of inward- and outward-rectifying channels (Schachtman et al., 1992; Cao et al., 1992; respectively). Most of the results described here are from patch-clamp recordings; the ideas of ion channels and use of membrane patches are old, and a simple technical innovation, the Gigaseal, has permitted this progress (Takeda et al., 1985). We will

Chapter 9

Stomata

discuss only guard cell channels, as the ubiquity of ion channels in plants is documented by Hedrich and Schroeder (1989) and Tester (1990). As a note of caution to the following narrative, I will describe the channels as the original authors do, which is in terms of conducting properties. Please recognize that this terminology is for convenience; e.g. characterized slow vacuolar (SV) and fast vacuolar (FV) channels are relatively nonspecific and may allow the passage of both anions and cations (Taiz, 1992). A well documented description of properties of channels of various plants by Ping et al. (1992) argues against broad generalizations. Schroeder et al. (1987) reported: , slowactivating, voltage-dependent inward and outward currents that were selective for both currents are slow activating, which explains the lack of evidence for voltage regulation in the earlier report (Schroeder et al., 1984). The delayed high-conductance outward rectifier of Vicia faba was soon confirmed by Hosoi et al. (1988), and several groups have contributed further to detailed knowledge, as listed below. Both inward and outward currents are blocked by extracellular barium ion; the outward current (e.g. Blatt, 1988) and inward current (e.g. Blatt, 1992) are blocked by tetraethylammonium, which is thought to fit into a channel vestibule and mimic the ‘ball’ in the ‘ball and chain’ mechanism of voltage sensitivity. The outward rectifier is relatively insensitive to pH, but the inward rectifier has a high dependence on H+ concentration (steady-state conductance is seven fold higher at pH 5.5 than at pH 7.4; Blatt, 1992). Thus, pH regulation (Blatt and Thiel, 1994) is an additional mechanism of integrating the pump with the inward channel. Plasmalemma hyperpolarization by the pump and voltage dependence of the channel provide a first mechanism. The inward current, but not the outward current, is blocked by (Schroeder, 1988), which explains inhibition of opening, but not closing, of stomata (Schnabl and Ziegler, 1975). This differential response to and to pH and (as listed by Fairley-Grenot and Assmann,1993) differential cation selectivities, differential sensitivity to regulators of GTP-binding proteins, and different activation/deactivation kinetics imply that the inward current and the outward current pass through different channels. Current work on channels is on comparative biophysics (e.g. Fairley-Grenot and Assmann, 1992a, 1993), regulation (e.g. Cosgrove and Hedrich, 1991), resolution (e.g. Schroeder and

245 Fang, 1991), and mechanisms (e.g. Thiel and Blatt, 1991). Though clearly of importance, these studies are beyond the scope of this general overview. External inhibits stomatal opening and causes stomatal closure (summarized by Raschke, 1979; see also summary of more recent work by Mansfield et al., 1990). As shown in Fig. 1, several roles are ascribed to The major points of controversy (Lemtiri-Chlieh and MacRobbie, 1994) are (i) whether elevation of cytoplasmic concentration is always associated with the ABA signal transduction chain and (ii) whether the source of cytoplasmic is ‘internal stores,’ as opposed to required influx across the guard cell plasmalemma. These and associated questions are difficult to answer because itself may have several effects, e.g. by having a long resident time in a channel and thus blocking it (Fairley-Grenot and Assmann, 1992b) or by exerting an internal effect when it is supplied externally. In addition, an effect of may be exerted indirectly (Luan et al., 1993) through a signal transduction pathway. These important questions have been the subject of numerous investigations, but it is possible that no single simple answer will emerge (cf. Neuhaus et al., 1993). Here, effects of on channelswill be summarized, and, in a following section, the regulatory role of will be discussed. In brief, external inhibits the channel directly (Fairley-Grenot and Assmann, 1992b). Cytoplasmic also inhibits the channel, but by shifting the gating potential more negative (from about –100 to about –200 mV, Schroeder and Hagiwara, 1989, 1990b). The effects of on the channel have been the subject of numerous experiments; although some data (Hosoi et al., 1988) point to a lack of effect, 0.5–1 mM external markedly inhibits the channel of Vicia faba (LemtiriChlieh and MacRobbie, 1994). It is important to note that the foregoing selected citations establish an effect of on channels, but alone, an effect does not imply regulation.

IV. Plasmalemma Anion Channels To a variable extent is taken up by guard cells when stomata open (Outlaw, 1983). Given the polarity of the membrane potential and the high internal concentrations of Linder and Raschke, 1992), influx must be by means of an active mechanism such as a (depicted in

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Fig. 1) or its equivalent, an antiport. As far as we are aware, such a carrier has not been studied in guard cells. Anion channels that would function in the efflux of and malate have been identified in the guard cell plasmalemma (Hedrich et al., 1990; Schroeder and Hagiwara, 1990b). The R-type channel (rapid activation/deactivation) probably functions in short term efflux, as it is deactivated by stimulating depolarizing Keller et al., 1989; Schroeder and Hagiwara 1990a) potentials with a half-time of ca. 15 sec (Hedrich et al., 1990; Schroeder and Hagiwara, 1989). This channel is activated by (Hedrich et al., 1990), which, as mentioned, is associated with stomatal closure, and by nucleotides (Hedrich et al., 1990). The R-type channel, with malate and carries both inward and outward currents (Hedrich et al., 1990), and it can be regulated by auxin (Marten et al., 1991,1992), but not by ABA (Marten et al., 1991). A second channel, the Stype (slow activation/deactivation), has been identified (Linder and Raschke, 1992; Schroeder and Keller, 1992; Schroeder et al., 1993). It is also voltage dependent and activated by The S-type channel is open at membrane potentials much more negative than is the R-type channel (Linder and Raschke, 1992; Schroeder and Keller, 1992). As succinctly discussed by Linder and Raschke (1992), efflux via the channel, required for stomatal closure, can occur only at membrane potentials positive to about –40 mV (see Fig, 1), which is more positive than the (about –120 mV). Collapse of the membrane potential is, therefore, necessary for efflux. The rapid kinetics for closure of the R-type anion channel precludes its role in sustaining stomatal closure. On the other hand, the S-type channel (or SLow Anion Channel, ‘SLAC,’ in the terminology of Linder and Raschke, 1992) would be well suited for sustained anion efflux (to hold the membrane potential positive to Indeed, the central importance of the S-type channel was demonstrated by Schroeder et al. (1993). These authors induced stomatal closure in Vicia faba epidermal peels by exposure to ABA and malate. The R-type channel blocker diisothiocyanatostilbene disulfonic acid (DIDS) (Marten et al., 1992) caused only a 20% diminution of stomatal closure, whereas 20 5-nitro-2,3-phenylpropylaminobenzoic acid (NPPB), at this concentration specific for the S-type channel, completely inhibited stomatal closure. As further support for the central role of the plasmalemma anion channel in stomatal

function, Marten et al. (1992) have shown that mesophyll cells are deficient in plasmalemma anion channels on the basis of lack of anion current and cross reactivity with an antibody produced against the anion channel of kidney membranes. This antibody was positive for guard cell plasmalemma preparations.

V.Tonoplast Transport Processes Although ion channels have been studied in the tonoplast for several years (Hedrich et al., 1986; Hedrich and Neher, 1987), only recently (Hedrich et al., 1988; Allen and Sanders, 1994; Ward and Schroeder, 1994) have guard cells been the focus of these studies. Ward and Schroeder (1994) identified a voltage-independent channel of high selectivity for They postulate that this channel would conduct in response to the rise in cytoplasmic concentration that has often been associated with stomatal closure (see below). The efflux of from the vacuole would result in a tonoplast depolarization that activates a second class of channels that cause loss from the vacuole (Allen and Sanders, 1994; Ward and Schroeder, 1994). Two results ensue: the first, or VK, channel would remain in a conducting state because of the elevated concentration, and in turn, efflux would maintain the permissive voltage range for the channel. The response of the VK channel to cytosolic pH suggests paradoxically that it would be downregulated by the cytosolic alkalinization that has been reported to accompany stomatal closure (Irving et al., 1992). Obviously, the system must be delicately balanced. On the one hand, there must be a sufficient transmembrane potential to provide the driving force for inward (to cytosol) rectification by the VK channel; this potential is presumably supplied by the tonoplast On the other hand, a small or positive membrane potential is required for activation of the SV channels. In addition to being voltage regulated, the major channel identified by Allen and Sanders (1994) is reduced by low vacuolar pH, which implies that the normally prevailing vacuolar pH considerably attenuates conductance, avoiding the unregulated loss of from the vacuole. The overall simplicity of the foregoing explanation is, of course, attractive, but there is considerable precedent for the regulation of channels by metabolites in the

Chapter 9 Stomata absence of changes in intracellular concentration (Toro and Stefani, 1991). A new feature (cf. Hite and Outlaw, 1994) of the model (Fig. 1), although not shown in guard cells, is active transport of by the (PPase, Davies et al., 1991, 1992, interpreted by Taiz, 1992; Rea et al., 1992). Rea and Poole (1993) summarize the strong arguments that support transport by this PPase. Briefly, (i) the activity requires ‘cytoplasmic’ with intact vacuoles for current into the vacuole, and, conversely, on the vacuolar side of the membrane to support currents to the ‘cytoplasm.’ (ii) Manipulation of the electrochemical potential difference for across the membrane has a predictable effect on transport, viz. change of the reversal potential, whereas, if were only an activating ion, there should be no effect on the equilibrium. It is important to note, however, that a recent review (Wink, 1993) did not provide the foregoing interpretation and relied on an earlier conclusion that only stimulates the Other recent authors (Schmidt and Briskin, 1993) noted that direct evidence for transport by the PPase is currently lacking. Regardless of the final role ascribed to the PPase, the energetics imply that some mechanism of active uptake of must be responsible for accumulation in guard cell vacuoles during stomatal opening.

VI. Abscisic Acid, Calcium, and the Phosphoinositide Messenger Systems (Gilroy et al., 1987; Schroeder and Thuleau, 1991) and phosphoinositide (Drøbak, 1992) messenger systems are well established for plant cells, although differing in some respects from the well studied mammalian system. In brief, the main elements in mammals are described below. (i) Occupation of an external receptor site results in the activation of phospholipase C (PLC), which is mediated by activated heterotrimeric G-proteins. (ii) The activated PLC cleaves phosphotidylinositol 4,5bisphosphate to yield two messengers, cytosolic inositol 1,4,5-trisphosphate which is water soluble, and 1,2 diacylglyerol (DAG), which remains in the membrane, (iii) causes the release of from internal stores. In plant cells, these internal stores have been identified as vacuoles (Schumaker and Sze, 1987), and recent evidence implicates the endoplasmic reticulum (Bush et al., 1993). (iv) DAG

247 activates protein kinase C (PKC). The evidence for an analogous plant system is incomplete, e.g. unequivocal demonstration of agonist-induced production in higher plants is lacking, and PKC does not have a direct functional equivalent in higher plants (Drøbak, 1992; but see Lee and Assmann, 1991). Elevation of cytoplasmic calcium concentration may occur independently of the system, through several mechanisms, e.g. channels on internal membranes (Johannes et al., 1992) and ABA-activated channels on the plasmalemma (Schroeder and Hagiwara, 1990b). As discussed, itself has a direct effect on guard cell channels (Fairley-Grenot and Assmann, 1992b), but additional, and perhaps more important, effects (Luan et al., 1993) may result from a signal transduction pathway involving calmodulin, which is concentrated in guard cells (Ling and Assmann, 1992). Plasmalemma (Felle et al., 1992) and endomembrane (Sievers and Busch, 1992) ATPases and buffering provide a mechanism for homeostasis. Against this background, the role of and ABA in stomatal movements will be examined. Studies of effects on stomatal movements have a long history (Mansfield et al., 1990). In brief, exogenous depresses stomatal aperture size (Fischer, 1968); blockers lower the inhibition by ABA of stomatal opening (De Silva et al., 1985a, b); external EGTA prevents closure in darkness and speeds light-stimulated opening (Schwartz, 1985), probably by lowering cytosolic concentration (Gilroy et al., 1991) but in a nonNernstian way; ABA causes influx, which results in a transient increase in cytoplasmic concentration (Schroeder and Hagiwara, 1990b); the latter was also found by McAinsh et al. (1990). Others suggested that external calcium and ABA have independent effects (Smith and Willmer, 1988; MacRobbie, 1989; Brindley, 1990; Curvetto and Delmastro, 1990). Without diminishing the importance of the preceding pioneering studies, which provided the basis for the present work, some salient observations that introduce ambiguities must be made. First, Terry et al. (1992) have reported that the commonly used blockers actually have direct effects on other plasmalemma-cation channels at the concentrations used. Second, Reid and Smith (1992) concluded that, in whole cells of Chara corallina, influx cannot normally be distinguished from extracellular binding. Third, a formulation for artificial apoplast solution is

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problematical. As the internal and external concentrations of and all are interrelated, distinguishing physiological from pharmacological effects is only equivocally done. Fourth, values for internal concentration obtained with the dyebinding method should be considered nominal, as it appears that the dissociation constant shifts that result from differences in ionic strength, competing ions, or other complications are not considered. As discussed earlier, exposure to ABA can lead to an increase in cytosolic concentration (McAinsh et al., 1990; Schroeder and Hagiwara 1990b). or released from the photolabile ‘caged’ form into guard cell cytosol causes stomatal closure (Gilroy et al., 1990). Complementarily, released into guard cells reversibly inactivated channels and activated a depolarizing in-current, presumably carried by (Blatt et al., 1990). Consistently, the GTP analog inhibits inward current, an effect that is abolished by a calcium chelator (FairleyGrenot and Assmann, 1991). Finally, several inositolphosphates have been rigorously identified in guard cells (Parmar and Brearley, 1993). From these results, one infers that a G-protein stimulates a phosphoinositide-specific PLC that forms DAG and This simple explanation, however, belies the complexity of the system, as (in another species) Lee et al. (1993) found that causes stomatal opening, and Lee and Assmann (1991) report that DAG stimulates the guard cell proton pump, which, of course, drives stomatal opening. Assmann (1993) has noted that the DAG stimulation is not inconsistent with the effects of as the DAG produced from breakdown is minimal compared to those from other sources. Altogether, the apparently contradictory results suggest that regulation is by several G-proteins with opposing activities or by a G-protein that is somewhat different from those characterized in animals (Kaufman, 1994). Thus, whereas it is clear that ABA action may be mediated by an increase in cytoplasmic concentration, it has been contentious whether an ABA-invoked mechanism exists. Arguing for an additional mechanism, Gilroy et al. (1991) found that any of several methods to increase cytoplasmic concentration caused stomatal closure, and, consistently, treatments to decrease cytoplasmic concentration resulted in opening. However, exposure to ABA, which always caused closure, resulted in the

elevation of cytoplasmic concentration in only a minority of guard cells. In response, McAinsh et al. (1992) conducted a detailed study, in which they preinjected guard cells with the indicator. Then, the stomata were opened and exposed to ABA; these precautions were intended to minimize artifacts and to ensure the use of competent cells. ABA exposure of guard cells injected in the closed state resulted in a variable increase in 80% of guard cells. Documenting various reasons for failure to detect cytoplasmic concentration elevations in the other guard cells (e.g. localized changes), McAinsh et al. (1992) concluded that only a mechanism of ABA action was present. Finally, Irving et al. (1992) made several relevant observations that are interpreted (Schroeder, 1992) as evidence for a pathway: (i) cytoplasmic concentration increase was not observed in all guard cells exposed to ABA, (ii) stomatal closure in the presence of procaine was not accompanied by an increase in cytoplasmic concentration, and (iii) stomatal opening, which was caused by kinetin, was also accompanied by an increase in cytoplasmic concentration. In interpreting this work on Paphiopedilum tonsum, it is important to note that this genus is atypical (if not unique) in lacking guard cell chloroplasts; high ABA concentrations are required to induce closure; stomata function only sluggishly, and involvement of in stomatal movements is equivocal (Outlaw et al., 1982a). To our knowledge, only MacRobbie (1990) has attempted to distinguish between -dependent and mechanisms in the context of compartmentation. In her work, a fast response did not require (reminiscent of the direct ABA effect on the plasmalemma rectifier; Schauf and Wilson, 1987), and a slow response did require (reminiscent of the SV-type channel). LemtiriChlieh and MacRobbie (1994) have insightfully elucidated the signal transduction pathway involving ABA by studying inward and outward rectification in the presence and absence of external and by using an internal buffer. They found that external was ineffective in modulating the inward current, but that diminution of cytoplasmic concentration eliminated the ABA response. However, the channel activated normally with ABA even when cytoplasmic concentration was buffered to very low levels.

Chapter 9

Stomata

VII. Integrating Role of Abscisic Acid in the Plant’s Physiology ABA is not simply an intraorgan agent; it serves to coordinate the physiology of the plant (Davies et al., 1990,1994). This stark introductory statement belies our poor understanding, as will become apparent. Water stress of mature leaves results in ABA synthesis there; this ABA is mobilized in the phloem and transported to roots and shoot tips (Hoad, 1973, 1975,1978). The root also serves as a source of ABA and exports ABA of root and of shoot origin via the xylem(Zeevaart and Boyer, 1984; Wolf et al., 1990). Treatments such as soil drying that elicit ABA synthesis in roots, even in the absence of foliar water deficiency, decrease stomatal conductance (Munns and Sharp, 1993; Davies et al., 1994). There are three rather different direct explanations for root control of leaf conductance by chemical signals. The simplest is that ABA moves from the roots to the leaves (Zhang et al., 1987; Neales et al., 1989). The second is that ABA export from leaves is inhibited and that it thus accumulates (Jackson and Hall, 1987; Castonguay et al., 1993). The third is that the signal is some chemical factor in addition to ABA (Munns and Sharp, 1993). As mentioned earlier, loss of turgor is one signal that triggers ABA synthesis; this signal also triggers ABA release by leaf to the apoplast (Hartung et al., 1983; Cornish and Zeevaart, 1985). The idea, attractive in its simplicity, that a stressed cell synthesizes and releases ABA, which then results in the conservation of water, does not, however, accord with other facts. For example, the increase in apoplastic ABA has been reported to occur after stomatal closure (Ackerson, 1982), and there is not always a relationship between xylem ABA concentration and conductance (Wartinger et al., 1990; see also references in Atkinson et al., 1992), and it has been suggested that there may be no direct relationship between the concentrations of ABA in the xylem and in the guard cell apoplast (Munns and Sharp, 1993). Furthermore, there are apparently mechanisms in the leaf that prevent stomata from being affected by the high concentrations of in the xylem (Atkinson et al., 1992; Canny, 1993), and stomatal sensitivity to ABA is also not fixed (Tardieu and Davies, 1992; Trejo et al., 1993). A full understanding of how ABA integrates the functioning of the whole plant will require that we know the means by which the ABA signal is perceived (Allan and Trewavas, 1994;

249 Anderson et al., 1994; Gilroy and Jones, 1994; Schwartz et al., 1994), the ionic composition of the guard cell apoplast that influences the sensitivity of guard cells to ABA, the rate of delivery of ABA to the guard cell, and the interaction of ABA and other regulatory substances.

VIII. Carbon Metabolism Primary carbon metabolism in guard cells has captivated the interest of plant biologists for a century perhaps because of the facile and intriguing observation that guard cell starch is degraded when the plant is illuminated. Immediately, this observation suggested that the breakdown of starch yielded sugars that caused turgor changes that accompanied stomatal opening (Wiggans, 1921; Heath, 1949; Yemm and Willis, 1954). This starch to sugar interconversion (the ‘Classical Theory’) dominated explanations of stomatal movements until the late 1960’s (Fischer, 1968), despite the inexact relationship between stomatal aperture size and starch content (Williams, 1952, 1954). Once the central role of fluctuations in concentrations was established (Outlaw, 1983), metabolism of starch assumed a secondary role. As inorganic anion fluxes across the guard cell plasmalemma under many conditions do not balance fluxes (Raschke and Humble, 1973), the synthesis and accumulation of organic anions, prototypically malate (Outlaw and Lowry, 1977), using carbon skeletons provided by starch (Outlaw and Manchester, 1979), has been offered as the likely explanation for these reciprocal changes in pool sizes (Fig. 2). It is exceedingly important to point out, however, that only a single quantitative study of the starch content of guard cells has been conducted (Outlaw and Manchester, 1979) and that this study was limited to a single dark treatment (to obtain closed stomata) and a single light treatment (with low concentration, to obtain open stomata). Thus, whereas unquestionable and broadly obtained evidence supports the accumulation of organic anions in guard cells during stomatal opening (Outlaw, 1982), the stoichiometric relationship between the hydrolysis (or phosphorolysis?) of starch is somewhat conjectural (Fig. 2) and should be investigated quantitatively. The accumulation of malate (Fig. 2) represents a ‘proton debt’ as implied above. Given a neutral carbohydrate precursor, two protons are released to

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solution in the production of the divalent anion (one at the oxidation of the aldehyde to the acid in the glycolytic sequence and one during the hydration of to form which is the substrate for phosphoenolpyruvate carboxylase, PEPC). As maintenance of cytosolic pH is crucial during extrusion by the guard cell plasmalemma ATPase, regulation of organic anion accumulation has been the subject of many investigations; the focus has been on PEPC (Outlaw, 1990) because this enzyme catalyzes the first step in the reaction sequence.

Commensurate with its role, the specific activity (converted to protein basis, Outlaw et al., 1981b) of guard cell PEPC is approximately 8 –12 times the corollary activity in the mesophyll (Outlaw and Kennedy, 1978). In general, the reported kinetic properties of guard cell PEPC are similar to those of other PEPCs (Outlaw, 1990), i.e. the enzyme is inhibited by malate, is activated by glucose 6-P, and has higher affinity with substrate at alkaline pH (Denecke et al., 1993;Tarczynski and Outlaw, 1990, 1993; Wang et al., 1994). As discussed in detail

Chapter 9 Stomata elsewhere (Wang et al., 1994), the physiological implications of these properties can be assigned only equivocally. Given the PEP concentrations (Outlaw and Kennedy, 1978), the binding of PEP and (Du and Outlaw, unpublished) to form the presumed substrate and a general value for free cytosolic (Yazaki et al., 1988), it is possible that organic anion synthesis is speeded if the cytosolic pH is elevated as protons are extruded during stomatal opening, i.e. the pH-stat mechanism, (Davies, 1979). Although glucose 6-P is an activator of guard cell PEPC, as mentioned, a regulatory role of this metabolite cannot be ascribed as the concentration of glucose 6-P is essentially the same in guard cells of opening stomata as in guard cells of closed or open stomata (Tarczynski and Outlaw, 1993). Finally, the only in planta direct measurement of malate in plant cytosol places the value at approximately 8 mM (Bodson et al., 1991; see also Hampp et al., 1984; Chang and Roberts, 1989). Paradoxically, this level of malate is sufficient to inhibit PEPC severely in vitro, but concomitant measurements of malate accumulation in the studied system (Raphanus sativus root tips) revealed that PEPC was not apparently inhibited (Bodson et al., 1991). One explanation of these results is that structural interactions (Srere, 1993) of PEPC (Queiroz-Claret and Queiroz, 1992; Wu and Wedding, 1992) that influence the reaction rates are lost in in vitro assays. Another explanation is that malate sensitivity of guard cell PEPC is diminished if the guard cell are of stomata that are in an opening phase (Zhang et al., 1994). This loss of malate sensitivity of guard cell PEPC, a putative type PEPC, is consistent with recent studies (Wang and Chollet, 1993) that show phosphorylation, presumably regulatory, of PEPC and with a survey (Toh et al., 1994) that shows conservation of the seryl phosphorylation site across different plant PEPCs (but not bacterial PEPCs). Altogether, these results indicate that guard cell PEPC is reversibly modified posttranslationally, as has been so thoroughly studied with the and CAM versions of PEPC (Jiao and Chollet, 1991). Considerable effort is being put forth (Schulz et al., 1992; Schnabl et al., 1993) to purify guard cell PEPC, which should lead to a clarification of its kinetic properties with respect to the physiological state of the stomata. Anion accumulation in guard cells during stomatal opening is not regulated solely by guard cell PEPC or any one process, but data on other components of the starch to malate pathway are sparse. There is evidence

251 both for (Rother et al., 1988) and against (Hedrich et al., 1985) the presence of significant levels of fructose 1,6-bisphosphatase (FBPase). The absence of this activity is reminiscent of certain amyloplasts (Borchert et al., 1993), which must obligatorily import hexose-P (instead of triose-P) as a precursor to starch. Indeed, Overlach et al. (1993) showed that pea guard cell chloroplasts possess a Pi-translocator that has an affinity with glucose 6-P equal to the affinity that it has with P-glycerate. These results, if confirmed and extended to other species, provide new insight (cf. Robinson and Preiss, 1985) into how guard cells function and suggest a number of avenues of investigation. Guard cells possess both PPi- and ATP-dependent phosphofructokinases (PFK, tabulated data in Hite et al., 1992) and the guard cellspecific activity (protein basis) of each of these enzymes is several-fold higher than the corresponding activity in palisade cells (Hite et al., 1992). The guard cell PPi-PFK is strongly stimulated by the novel regulatorymetabolite fructose 2,6-bisphosphate (Hedrich et al., 1985), which increases when guard cells or epidermal strips are exposed to light. As discussed earlier, light is a signal for stomatal opening. Finally, there is evidence for other enzymes of the glycolytic sequence between fructose 1,6-bisphosphate and PEP (Hampp et al., 1982; Shimazaki et al., 1989) in guard cells, but how these activities may be specifically regulated in the context of stomatal function has not been investigated. Following a decade-long hiatus, low-mass carbohydrates, prototypically sucrose, have reemerged as a focal point in stomatal physiology research. Various roles for sucrose, especially its service as an osmolyte that is ‘compatible’ with the cytoplasm, have been put forth (Outlaw, 1983). In support of a specific role for sugars, historical studies (Outlaw, 1982) often revealed that the guard cell sugar content increased when stomata opened. Quantitative studies are corroborative, but the absolute levels reported vary, as might be expected as different experimental systems were employed. As examples, Talbott and Zeiger (1993) found that the elevated level of sucrose was ca. 85 fmol guard , Poffenroth et al. (1992) reported 500 fmol guard and Outlaw and Manchester (1979) obtained 350 fmol guard Taking a value of 200 fmol guard (Vicia fabd) for reference, the concentration of sucrose would be, if distributed homogeneously throughout the cell, in the range of 40-80 mM, certainly well below the nominal 500

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mM that salts contribute to the osmotic potential. These calculations imply that sucrose may serve as an osmolyte, but that, for maximum importance, it must be restricted to defined regions of the cell. Interestingly, Lu et al. (1995) have reported a very large increase in apoplastic sucrose concentration in guard cells of Vicia faba leaves that have open stomata. The implication is that sucrose accumulates at the distal point of the evapotranspiration pathway in this species, an apoplastic phloem loader. Thus, in addition to many other roles, sucrose may serve as a signal metabolite that provides a measurement of water flux through the leaf. The source of elevated sucrose may be attributed nonexclusively to guard cell photosynthetic carbon reduction (which will be discussed later), starch degradation (Poffenroth et al., 1992) or import from photosynthetic cells (Outlaw and Fisher, 1975; Outlaw et al., 1975; Rohrig and Raschke, 1991). Present evidence does not permit distinction of the various fates of the breakdown products of starch, and as mentioned above, quantitative studies of guard cell starch content have been reported only once (Outlaw and Manchester, 1979). Several different lines of evidence prove the importance of sucrose uptake by guard cells. First, the high level of the degradative enzyme sucrose synthase (Hite et al., 1993) in guard cells implies that these cells are sucrose sinks (Dali et al., 1992). Interestingly, guard cells also have very high levels of the synthetic enzyme, sucrose-P synthase (Hite et al., 1993), indicating that cellspecific carbohydrate interconversions occur in these cells. Second, the mesophyll sucrose of labeled leaves reaches a maximum specific activity 10-15 min after the pulse, whereas the sucrose specific activity of epidermal strips taken from these leaves reaches a maximum specific activity only after about 40 min (Outlaw et al., 1975). Although interpretation of these results is confounded by the presence of some nonruptured and nonphotosynthetic epidermal cells, the simplest interpretation is that newly synthesized sucrose is transported from the mesophyll to guard cells. The retrieval of apoplastic sucrose by guard cells was demonstrated by Rohrig and Raschke (1991). Questions of photosynthesis have occupied the forefront of inquiries about carbon metabolism in guard cells. It now seems clearly resolved that guard cell conduct linear electron transport (Outlaw et al., 198la; Zeiger et al., 1981) but there is considerable controversy whether the photosynthetic carbon-

reduction pathway is ‘present’ or ‘absent’ in guard cells. As implied, a part of the argument is semantic (Tarczynski et al., 1989). Our own studies have been guided by the setting of ‘maximum sensitivity limits, below which ribulose 1,5-bisphosphate carboxylase/ oxygenase (Rubisco) would not make a significant contribution to carbon metabolism’ (Outlaw et al., 1982b). Using this criterion, Outlaw (1989) concluded that guard cells did not have significant capability as proven in reliable quantitative studies. Since that essay was published, several other reports have appeared. Reckmann et al. (1990) concluded that Pisum sativum guard cells contained Rubisco sufficient to supply only 2% of the osmolyte requirement during stomatal opening. Gautier et al. (1991) reported an insignificant stimulation of uptake by light with Commelina communis guard cell protoplasts. Shimazaki (1989) suggested that guard cells utilize reducing equivalents, obtained from photosynthetic water oxidation, largely for reactions other that photosynthetic fixation. In making these comparisons, it is important to note that guard cells contain <2– 4% of mesophyll chlorophyll, which would place a low limit on photosynthetic capability in any case. Shimazaki's (1989) conclusion is corroborated by Mawson (1993), who showed that guard cell chloroplasts contribute to the energy demands of extrusion. The report by Ohya and Shimazaki (1989) is less straightforward to interpret because there was no protein staining by silver at the expected migration position of the large sub-unit of Rubisco after electrophoretic separation of proteins on polyacrylamide gels of guard cell extracts, but this polypeptide was immunodetected after electroelution to nitrocellulose. Cardon and Berry (1992) reported chlorophyll fluorescence transients indicative of the presence of Rubisco in guard cell of Tradescantia albiflora. Their studies were qualitative, and as they point out, their results do not alter the conclusion that photosynthesis is far too low to be the primary contributor of hexoses for production of malic acid during stomal opening. By way of comparison, our assay results (Outlaw et al., 1982b) on a related species, T. ohioensis, indicated that guard cells contain approximately 0.6% the total Rubisco of mesophyll cells on a cell basis; 3.5 ± 2.7% on a dry-mass basis. In contrast, Poffenroth et al. (1992) reported significant sugar production, which was inhibited by DCMU, and Talbott and Zeiger (1993) also favor photosynthetic sugar production as a means of accumulating osmolyte

Chapter 9

Stomata

required for stomatal opening. In conclusion, whereas our interpretation of guard cell function does not call for a role of photosynthetic carbon reduction by guard cells in the overall carbon economy of these cells, other investigators with a long history of substantial contributions to this literature disagree. Perhaps overlooked physiological, environmental or historical parameters control the expression of this pathway. IX. Concluding Remarks Water, typically the most limiting resource for a terrestrial plant, is mostly lost to the atmosphere through stomata in the leaf epidermis. Thus, although evapotranspiration often benefits the plant through cooling or nutrient relocalization, the result of excess water loss is deleterious. As water is a finite resource in an agronomic context, the considerable effort to understand stomatal function is justified. In addition, the remarkable ion transport capability of guard cells, their immediate reaction to many environmental stimuli, and their unique pattern of carbon metabolism have attracted many capable investigators from diverse backgrounds, who use guard cells as a model system. Remarkable progress has been made in the past decade on ion traffic, from the seminal reports on ion channels to their physiological, biophysical and molecular biological characterization. It is probable that the major facets of ion fluxes have been established although an understanding of the intracellular signals that integrate stomatal movements remains somewhat sketchy. We now understand that ABA serves as an integrating signal, produced not only in the leaves, but also transported there from roots that penetrate dry soil. Several different explanations, but not necessarily exclusive ones, have been proposed to describe the molecular mechanism of ABA function. The outline of guard cell biochemistry that has evolved to complement the large ion fluxes has been established, but large gaps in our knowledge are evident. References Ackerson RC (1982) Synthesis and movement of abscisic acid in water-stressed cotton leaves. Plant Physiol 69: 609–613 Allan CA and Trewavas AJ (1994) Abscisic acid and gibberellin perception: Inside or out? Plant Physiol 104: 1107–1108 Allen GJ and Sanders D (1994) Two voltage-gated, calcium

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Sharkey TD and Raschke K (1981) Effect of light quality on stomatal opening in leaves of Xanthium strumarium L. Plant Physiol 68: 1170–1174 Shimazaki KI (1989) Ribulosebisphosphate carboxylase activity and photosynthetic evolution rate in Vicia guard cell protoplasts. Plant Physiol 91: 459–463 Shimazaki KI, Iino M and Zeiger E (1986) Blue light-dependent proton extrusion by guard cell protoplasts of Vicia faba. Nature 319: 324–326 Shimazaki KI, Terada J, Tanaka K and Kondo N (1989) CalvinBenson cycle enzymes in guard cell protoplasts from Vicia faba L. Implications for the greater utilization of phosphoglycerate/dihydroxyacetone phosphate shuttle between chloroplasts and the cytosol. Plant Physiol 90: 1057–1064 Sievers A and Busch MB (1992) An inhibitor of the in the sarcoplasmic andendoplasmic reticula inhibits transduction of the gravity stimulus in cress roots. Planta 188: 619–622 Smith GN and Willmer CM (1988) Effects of calcium and abscisic acid on volume changes of guard cell protoplasts of Commelina. J Exp Bot 39: 1529–1539 Srere PA (1993) Wanderings (Wonderings) in metabolism. Biol Chem Hoppe-Seyler 374: 833–842 Taiz L (1992) The plant vacuole. J Exp Biol 172: 113–122 Takeda K, Kurkdjian AC, and Kado RT (1985) Ionic channels, ion transport and plant cell membranes: potential applications of the patch-clamp technique. Protoplasma 127: 147–162 Talbott LD and Zeiger E (1993) Sugar and organic acid accumulation in guard cells of Vicia faba in response to red and blue light. Plant Physiol 102: 1163–1169 Tallman G (1992) The chemiosmotic model of stomatal opening revisited. Crit Rev Plant Sci 1 1 : 35–57 Tarczynski MC and Outlaw WH Jr (1990) Partial characterization of guard cell phosphoenolpyruvate carboxylase: kinetic datum collection in real time from single-cell activities. Arch Biochem Biophys 280: 153–158 Tarczynski MC and Outlaw WH Jr (1993) The interactive effects of pH, L-malate, and glucose-6-phosphate on guard cell phosphoenolpyruvate carboxylase. Plant Physiol 103: 1189– 1194 Tarczynski MC, Outlaw WH Jr, Arold N, Neuhoff V and Hampp R (1989) Electrophoretic assay for ribulose 1,5-bisphosphate carboxylase/oxygenase in guard cells and other leaf cells of Vica faba L. Plant Physiol 89: 1088–1093 Tardieu F and Davies WJ (1992) Stomatal response to abscisic acid is a function of current plant water status. Plant Physiol 98: 540–545 Terry BR, Findley GP and Tyerman SD (1992) Direct effects of blockers on plasma membrane cation channels of Amaranthus tricolor protoplasts. J Exp Bot 43: 1457–1473 Tester M (1990) Plant ion channels: whole-cell and singlechannel studies. Tansley Review No. 21. New Phytol 114: 305–340 Thiel G and Blatt MR (1991) The mechanism of ion permeation through channels of stomatal guard cells: voltage-dependent block by J Plant Physiol 138: 326–334 Toh H, Kawamura T and Izui K (1994) Molecular evolution of phosphoenolpyruvatre carboxylase. Plant Cell Environ 17: 31–43 Toro L and Stefani E (1991) Calcium-activated channels:

Metabolic regulation. J Bioenerg and Biomembr 23: 561–576 Trejo CL, Davies WJ and Ruiz LP (1993) Sensitivity of stomata to abscisic acid. An effect of the mesophyll. Plant Physiol 102: 497–502 Trewavas A and Gilroy S (1991) Signal transduction in plant cells. Trends In Genetics 7: 356–361 Vani T and Raghavendra AS (1989) Tetrazolium reduction by guard cells in abaxial epidermis of Vicia faba: Blue light stimulation of a plasmalemma redox system. Plant Physiol 90: 59–62 Wang XC, Outlaw WH Jr, DeBedout J A and Du Z (1994) Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard cell protoplasts of Vicia faba L. with respect to tissue pre-illumination. Histochem J 26:152–160 Wang YH and Chollet R (1993) In vitro phosphorylation of purified tobacco-leaf phosphoenolpyruvate carboxylase. FEBS Lett 328: 215–218 Ward JM and Schroeder Jl (1994) Calcium-activated channels and calcium-induced calcium release by slow vacuolar ion channels in guard cell vacuoles implicated in the control of stomatal closure. Plant Cell 6: 669–683 Wartinger A, Heilmeier H, Hartung WandSchulze E-D(1990) Daily and seasonal courses of leaf conductance and abscisic acid in the xylem sap of almond trees [Prunus dulcis (Miller) D. A. Webb] under desert conditions. New Phytol 116: 581– 587 Wiggans RG (1921) Variations in the osmotic concentration of guard cells during the opening and closing of stomata. Amer J Bot 8: 30–40 Williams WT( 1952) Studies in stomatal behavior. II The role of starch in the light response of stomata. Part 3. Quantitative relationships in Pelargonium. J Exp Bot 3: 110–127 Williams WT (1954) A new theory of the mechanism of stomatal movement. J Exp Bot 5: 343–352 Wink M (1993) The plant vacuole: a multifunctional compartment. J Exp Bot 44: 231–246 Wolf O, Jeschke WD and Hartung W (1990) Long distance transport of abscisic acid in NaCl-treated intact plants of Lupinus albus. J Exp Bot 41: 593–600 Wu MX and Wedding RT (1992) Inactivation of maize leaf phosphoenolpyruvate carboxylase by the binding to chloroplast membranes. Plant Physiol 100: 382–387 Yazaki Y, Asukagawa N, Ishikawa, Ohta E and Sakata M (1988) Estimation of cytoplasmic free levels and phosphorylation potentials in mung bean root tips by in vivo NMR spectroscopy. Plant Cell Physiol 29: 919–924 Yemm EW and Willis AJ (1954) Stomatal movements and changes of carbohydrate in leaves of Chrysanthemum maximum. New Phytol 53: 373–397 Zeevaart JAD and Boyer GL (1984) Accumulation and transport of abscisic acid and its metabolites in Ricinus and Xanthium. Plant Physiol 74: 934–939 Zeiger E (1983) The biology of stomatal guard cells. Annu Rev Plant Physiol 34: 441–475 Zeiger E, Moody W, Hepler P and Varela F (1977) Lightsensitive membrane potentials in onion guard cells. Nature 270: 270–271 Zeiger E, Armond P and Melis A (1981) Fluorescence properties of guard cell chloroplasts: evidence for linear electron transport

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Stomata

and light-harvesting pigments of Photosystems I and II. Plant Physiol 67: 17–20 Zhang J, Schurr U and Davies WJ (1987) Control of stomatal behaviour by abscisic acid which apparently originates in the roots. J Exp Bot 38: 1174–1181

259 Zhang SQ, Outlaw WH Jr, Chollet R 1994 Lessened malate inhibition of guard-cell phosphenolpyruvate carboxylase during stomatal opening. FEBS Lett 352: 45–48

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Chapter 10 Source-Sink Relations: The Role of Sucrose C. J. Pollock IGER, Plas Gogerddan, Aberystwyth, Dyfed SY23 3EB, U.K.

J. F. Farrar School of Biological Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, U.K.

Summary I. Introduction II. Sucrose As a Regulator A. Sucrose As an Environmental Sensor B. Sucrose As a Signal Molecule C. Conclusion III. Changes in Source Leaf Metabolism A. Evidence for Sucrose as a Regulator of Photosynthesis in Leaves 1. Repartitioning into Chloroplast Starch 2. The Repartitioning of Sucrose into Fructan in Leaves of Temperate Grasses 3. Over-Expression of Sucrose Phosphate Synthase Reduces Starch Content B. Potential Mechanisms of Sucrose Regulation 1. Mass-Action Effects 2. Fine Control of Enzyme Activity 3. Coarse Control Mediated via Alterations in Gene Expression 4. Other Forms of Coarse Control C. Conclusion IV. Sinks A. Evidence for Regulation of Sink Metabolism by Assimilate from Source Leaves 1. Roots 2. Cell Suspension Culture 3. Shoot Apices, and Storage and Reproductive Sinks B. Sucrose as a Morphogen 1. Differentiation of Vascular Tissue 2. Transition to Flowering 3. Fruit Abortion C. Conclusion V. Potential Mechanisms of Gene Regulation by Sugars VI. Conclusion References

Neil R. Baker (ed): Photosynthesis and the Environment, pp. 261–279. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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Summary We examine the idea that sucrose plays a central role in control of plant growth beyond that of substrate and product. The extended hypothesis we discuss is that sucrose can regulate source metabolism by down-regulating genes encoding proteins involved with photosynthesis, and sink metabolism by up-regulating genes encoding proteins involved with sucrose hydrolysis and growth. The rates of turnover, and of changes of pool size, of sucrose render it suitable for temporally integrating changes in the environment, or within the plant itself, over a period of hours to a few days. While much evidence can be adduced to support the extended hypothesis, a great deal of it is essentially correlational and we still lack the details of a mechanism by which sucrose status might modulate gene expression.

I. Introduction Sucrose is the major product of photosynthesis in most higher plants. It is also the main translocate, and the main substrate for carbon metabolism in sinks. But it may be more than merely product and substrate: in this chapter we argue that it also has regulatory functions. Specifically we consider the hypothesis that an ample supply of sucrose feeds forward to stimulate the capacity of sinks to grow, and that an accumulation of sucrose in source leaves feeds back to reduce the rate of photosynthesis. It takes little imagination to see how a combination of feedback and feedforward control would provide the plant with a powerful means of matching the capacity of sources to provide reduced carbon with that of sinks to consume it. There is a long history of ideas implicating assimilates with controlling source and sink function. The classic review of Neales and Incoll (1968) summarized many years of work suggesting that accumulation of assimilate in source leaves leads to a reduction in photosynthetic rate; their conclusion was that the idea was attractive, supported by much correlative evidence, but unproven, and certainly in, 1968 there was no plausible mechanism to account for these effects. More recently there has been a body of elegant and persuasive work on the short-term regulation of photosynthetic carbon flux, summarized in Stitt et al. (1989). Does this type of mechanism account for the phenomena discussed by Neales and Incoll? Here we suggest that it does not. We prefer Abbreviations: ADPG – adenosine -diphosphate glucose; – rate of photosynthesis at non-limiting concentrations of cFBPase–cytoplasmic form of fructose 1,6-bisphosphatase; FBP –fructose 2,6-bisphosphate; PEP – phosphoenol pyruvate; Pi – inorganic phosphate; Rubisco – ribulose 1,5-bisphosphate carboxylase-oxygenase; RuBP–ribulose 1,5-bisphosphate; SPS – sucrose phosphate synthetase

the view that fine regulation of existing photosynthetic machinery is an essential, short-term, response to a constantly varying environment, necessary to ensure the resupply of Pi and RuBP to photosynthetic metabolism. We therefore consider that the critical hypothesis is: longer-term (hours to days) adjustment of photosynthetic rate to the environment is regulated via the expression of genes encoding for proteins constituting the photosynthetic machinery. In addition we address the hypothesis that control at the level of the gene also occurs in sinks, adjusting the capacity of those sinks for metabolism and growth to the time-integrated supply of assimilate to them. The extended hypothesis is illustrated in Fig. 1. In advocating these hypotheses we are attempting to place the details of photosynthetic metabolism into the context of a whole, growing plant. Just two examples will suffice to demonstrate the need for such a context. Many models of crop growth have a fairly sophisticated photosynthetic component, but allow organ growth to be driven by the size of a carbohydrate pool. Implicit is the assumption that carbohydrates act only as a substrate, but it is equally likely that the carbohydrates are a signal setting the capacity of the sinks to grow (Farrar, 1992). Much current work on the effects of elevated atmospheric on plant growth has shown that although net photosynthesis increases substantially immediately after doubling this increase is not sustained (Stitt, 1991; see also Chapter 16). The ensuing downregulation of photosynthesis is ascribed to assimilate build-up due to sinks being unable to use the extra assimilate, resulting in sink-limitation of growth. In both of these cases, the interaction between photosynthesis and growth is critical, and the extended hypothesis that we present in this chapter is an attempt to further our understanding of that interaction. First we discuss whether sucrose is a suitable

Chapter 10 Source, Sinks and Sucrose

263 control of plant growth.

II. Sucrose As a Regulator There are three component partial processes of a regulatory system: sensing change, whether environmental or internal; transmitting the signal of that change around the plant to regions remote from where it was sensed; and putting in train the metabolic and morphogenetic responses to that signal. If sucrose is to have a controlling role, it might be involved directly or indirectly in these three partial processes. As an initial test of our hypothesis we seek evidence for effects beyond those of sucrose acting merely as a substrate.

A. Sucrose As an Environmental Sensor

candidate for sensing the environment, internal or external, and for transmitting signals around the plant in the manner of a growth regulator. Then we examine evidence for non-substrate (non-nutritional) effects of sucrose on sources and sinks. Finally we attempt to assess our current understanding of the role of sucrose in higher plant growth. Our aim is to review the evidence for a regulatory role for sucrose, to identify gaps in that evidence, and hopefully to encourage research to both fill those gaps and to build on the concepts that are emerging about the

Plants possess many means of detecting specific variables in their environment, such as red:far red ratio, gravity and drought, and of responding to their instantaneous value (Smith, 1990). But a quite different type of detection is potentially of enormous value to a non-mobile autotroph, and that is detecting the availability of resources within its environment, for example the supply of photosynthetically fixed carbon, or of available nitrogen. Since light intensities and concentrations vary both spatially around a plant and temporally over each day and with weather changes, measuring the amount of photosynthetic resource means integrating in time and in space the results of photosynthesis: ideally, over (say) one to three days. What better integral of photosynthetic performance could there be than sucrose itself? Therefore we can ask whether sucrose content of source leaves or of sinks changes over such a time course in response to internal or environmental changes. Quantitatively, such changes are feasible. Typically, in a young seedling soluble sugars constitute about 50–100 mg dry weight while photosynthesis and respiration proceed at about 20 and 10 mg carbohydrate g respectively on a wholeplant basis; hence the sugar pools are equivalent to only 3–10 h of gas exchange. Many examples of changing sucrose content are given by Avigad (1982). The instantaneous rate of source leaf photosynthesis depends on both light and concentration: a sudden increase in light intensity causes enhanced accumulation of both sucrose and starch in barley

264 source leaf blades within 8 h of treatment (Farrar and Farrar, 1987), and very large changes in carbohydrate occur, even in plants in controlled environments, during light-dark cycles (Farrar, 1989). Increased accumulation of carbohydrates in source leaves of plants transferred from 350 to 700 ppm has been reported many times (Farrar and Williams, 1991b), and soluble sugars increase along with starch (Ho, 1978). The sugar content of source leaves can also respond to the environment of sinks. Thus cooling the roots of Lolium results in a large increase in soluble sugars and fructans in the source leaves within hours (Simpson et al., 1991). Source leaves are not independent: when all leaves but one on a soybean are shaded, the starch and sugar content of the unshaded leaf falls over several days (Thorne and Koller, 1974). Internal changes, such as experimental manipulations of source:sink ratios, can also change source leaf sugar contents. Removing all but one leaf blade from young barley seedlings results in a decrease in the sugar content of the remaining blade within 6 h (Williams and Farrar, 1987). Sinks are much less studied. Changes still occur but sugar pools are often better buffered than those in source leaves. The soluble sugar content of barley roots changes only slightly during a 16 h light/8 h dark cycle (Farrar and Farrar, 1985) but a little more on prolonged (24 or 48 h) darkening of the shoot (Farrar, 1981; Williams and Farrar, 1990). When the source:sink ratio of barley seedlings was altered by removing all seminal roots except one, ethanolsoluble sugar rose within 18 h and a greater increase was found a week later; conversely, removing all leaf blades except one caused a reduction in root soluble sugars within 18 h, but 7 d later soluble sugars were at the same concentration as in control plants (Farrar and Jones, 1986; Bingham and Farrar, 1988; Williams and Farrar, 1990). Cooling of sinks, and thus reducing their growth rate but not the supply of assimilate to them, commonly results in their carbohydrate content increasing (Farrar, 1989). Knowledge of changes in total sugar contents of sources and sinks is of limited use, since there is compartmentation of sugars within cells and between cell types, and it is presumably cytosolic sugar in selected cell types that is of most consequence for potential sensing of the environment. Source leaves contain about 15 cell types between which sucrose is distributed in an imperfectly known manner. Leaf epidermal cells may be effectively sucrose-free while phloem may contain nearly molar sucrose; other cell

C. J. Pollock and J. F. Farrar types are intermediate but we know too little about them. We know more about intracellular compartmentation, although most of the data, such as that from freeze-fractionation (Gerhardt and Heldt, 1984), is averaged across an ill-defined range of cell types. Sucrose is synthesized in the cytosol but there is still no good technique for estimating its concentration there: estimates vary between 10 and 100 mM, depending on species and conditions, with a halftime for export and turnover of about an hour (Farrar, 1989). The apoplast contains sucrose and hexoses at low millimolar concentrations (Tetlow and Farrar, 1993; Delrot, 1989). Estimation of sucrose in vacuoles is easier. In barley leaves, up to 80% of sucrose is vacuolar, with concentrations of 26–120 mM (Farrar, 1989). Current evidence suggest that sucrose transport at the tonoplast is not energized, at least in cereal leaves (Kaiser and Heber, 1984; Martinoia et al, 1987). An example of fluxes through a leaf, emphasizing the small size of the cytosolic pool of sucrose relative to the fluxes through it, is given in Fig. 2. As in source leaves, sucrose in sinks is compartmented between cell types and within each cell. Again the bulk of sugar (which is often mainly hexose in roots) is vacuolar with rather less than half being readily accessible (assumed to be cytosolic plus apoplastic) in roots and some fruits (Farrar, 1985a, 1989; Farrar et al., 1994). We know almost nothing of between-cell compartmentation. In the few systems which have been studied in detail, the half-times for readily accessed sucrose is about an hour (Farrar, 1989; Farrar et al., 1994). Vacuolar sugar pools have substantially longer half times and thus can only buffer the accessible pool to a limited extent (Kholodova, 1967; Farrar et al., 1994). The nature of transport across the tonoplast seems to vary between species; in those storing sucrose in vacuoles, it is energized; in growing sinks with low sucrose concentrations in vacuoles it may not be (Ho, 1988). The size of the accessible pool will therefore vary over hours depending on the balance between import into the sink and use for growth or storage. For example in barley roots the cytosolic sugar pool varies in size by about 50% in response to selective pruning of root or shoot 24 h earlier (Williams and Farrar, 1990) and is therefore sufficiently sensitive to detect changes in the external or internal environment (Farrar and Williams, 1991a). These examples demonstrate that the concentrations of soluble sugars change in both sources and

Chapter 10 Source, Sinks and Sucrose

sinks, in response to the environment of source or sink, or to the source:sink ratio, reflecting an altered balance between consumption of sugar in sinks and its generation in source leaves.

B. Sucrose As a Signal Molecule As root and shoot grow in a mutually coordinated manner, communication between widely separated parts of the plant must occur to coordinate growth at widely separated meristems. Evidence cited in Section IIA shows that changes in the internal or external environment in one part of the plant can result in altered carbohydrate status in another part, suggesting effective communication of sugar status between plant parts, as would be expected from the properties of phloem. Loading and unloading of, and control of transport in, phloem have been reviewed elsewhere (Delrot, 1989; van Bel, 1992; Farrar, 1992; Patrick, 1990) and only salient points will be mentioned here. Fluxes along the phloem of 10 mg sugar phloem (300 mmol ) are common (Baker and Milburn, 1989) and up to 5 times this value has been recorded (Passioura and Ashford, 1974; Farrar and Jones, 1986), The turnover time of sugars in the phloem has not been estimated but it is likely to be

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low, as with a speed of 60–100 cm a molecule can travel the length of a typical experimental plant in minutes, as demonstrated by work with (Minchin and MacNaughton, 1984; Williams et al., 1991). Just how the sugar status of source and sink affects phloem transport is not clear, although there is abundant evidence that it does. There is increasing evidence that the flux down phloem is driven by a gradient of turgor pressure along it (Farrar, 1992). Therefore the achieving of high fluxes will be exercised via the generation of turgor in source leaf phloem, and the reduction of turgor in phloem within sinks. Whilst the ultimate driving force for transport must be metabolism and consumption of sucrose within the sink, it is not certain how this is coupled to regulation of turgor in the phloem. Although it seems plausible that the rate of synthesis of sucrose in source leaves would promote increased phloem loading and thus high turgor, and consumption of sucrose in sinks would promote removal of sucrose from phloem and thus low turgor, evidence for these assertions is scanty and they may be hopelessly simplistic. Although it has been suggested that sucrose concentration in source leaves controls rate of export (Swanson and Christy, 1976; Ho and Thornley, 1978) this takes no account of sucrose compartmentation,

266 of the influence of sinks on export from source leaves, or the probable role of phloem water relations. In barley leaves, export rate correlates with the amount of non-vacuolar sucrose, most of which may be in vascular bundles and mesophyll (Farrar and Farrar, 1986). The effects of sinks on export from source leaves have been described by Moorby and Jarman (1976) and Minchin, Farrar and Thorpe (1994) and the interdependence of source, sink and transport system has been modeled by Minchin, Thorpe and Farrar (1993).

C. Conclusion Sucrose possesses the attributes necessary to act as both a sensor of the internal and external environment, and as a signal molecule which communicates messages between sources and sinks and vice versa. Over a period of hours to a few days, changes in its concentration can communicate imbalances between production of assimilate in sources and consumption in sinks. It seems that plants may possess an unusually parsimonious system for responding to changing circumstance. But an essential third feature of such a system is that a change in sucrose content will put in train alterations in metabolism. The next two sections examine the evidence that it can, and the mechanism by which it does.

III. Changes in Source Leaf Metabolism

A. Evidence for Sucrose as a Regulator of Photosynthesis in Leaves If the assimilate abundance in leaves reflects the current balance between supply and demand, we should now consider the relationship between altered assimilate abundance, the rates and capacities for photosynthesis in source leaves and the control of product partitioning therein. The suggestions that sink activity can regulate source leaf photosynthesis via assimilate abundance can be summarized easily (Neales and Incoll, 1968; Herold, 1980; Azcón-Bieto, 1983). Altered demand for fixed carbon by distant sinks is transduced into source leaves and causes changes which eventually affect the maximal rates of photosynthesis. An important sub-set of this hypothesis, and the one which is discussed most extensively in this section, is that build-up of unexported assimilates (usually as sucrose or

C. J. Pollock and J. F. Farrar component hexoses) is the agent by which photosynthesis or partitioning is regulated. The problem comes when the supporting evidence is examined critically. It is such a simple argument, redolent with images of Le Chatelier, that many experiments appear to have been performed with the aim of finding consistent data rather than of critically evaluating the basic hypothesis. We wish, therefore, to consider critically the evidence that alteration in sucrose concentration either regulates directly any of the reactions of photosynthetic carbon metabolism in leaves, or causes repartitioning of fixed carbon into different chemical forms or into a different physical location. We will then discuss what mechanistic hypotheses can be erected for the mode of action of sucrose in these processes and what the evidence is for their occurrence in leaves. There are many experiments in which accumulati on of photoassimilates have been correlated with declines in (Neales and Incoll, 1968; Herold and McNeil, 1979; Stitt, 1991). Studies which alter assimilation by defoliation or by surgical alteration of sink strength may cause problems because of hormonal, nutritional or other interactions. AzcónBieto (1983) used the interaction between temperature, photoperiod duration and concentration to vary assimilate abundance in wheat (measured as sucrose, hexose and starch, but not including fructan) and obtained a positive correlation between decline in and leaf carbohydrate content. There was some evidence for a rapid recovery of after carbohydrate levels fell following darkening, suggesting that the effects were probably not modulated by changes in enzyme synthesis or degradation. Reductions in were observed at carbohydrate values above 100 mmol (roughly equivalent to 8 mmol sucrose ). In other studies, however, leaf carbohydrate contents well in excess of these values appear to have little effect on net photosynthesis measured as dry weight gain (Natr, 1967) or as total carbohydrate accumulation (Housley and Pollock, 1985). These studies used excised leaves of temperate Gramineae. The differences in experimental treatment used to generate high carbohydrate levels may obscure the relationships under test. It is difficult to design experiments where the direct effects of high irradiance, increased concentration or increased photoperiod upon the synthesis and turnover of photosynthetic components can be unequivocally distinguished from the indirect effects (on osmotic

Chapter 10

Source, Sinks and Sucrose

relations for example) mediated by altered sugar concentrations. It is also possible that some species may show sucrose-driven inhibition of photosynthesis, whereas others may merely repartition the excess sucrose into other chemical forms under certain conditions (see Sections III.A. 1 and III.A.2). Recently, however, blockage of export and accumulation of photoassimilate has been induced into tobacco leaves by expressing the yeast invertase gene in the cell walls (von Schaewen et al., 1990). This expression is associated with reduced growth and, within the leaves, by increased respiration and reduced photosynthesis. In the affected areas, regeneration of ribulose bisphosphate and carboxylation declined and activities of the relevant enzymes were also lower (Stitt et al., 1990). Feeding exogenous carbohydrates into autotrophic suspension cultures of Chenopodium rubium and intact detached leaves of spinach, and inducing sugar accumulation by cold-girdling also produced similar evidence of reduced expression of specific photosynthetic genes (Schafer et al., 1992; Krapp et al., 1993). These experiments provide good evidence for an inhibitory effect of assimilates on photosynthesis but do not leave us any clearer about whether such effects function under normal conditions or whether the ‘normal’ plant response involves principally product repartitioning (Sections III.A, III.B). One of the problems involved in experimentation of this sort is the lack of specificity of many of the treatments which are used to perturb the balance between photosynthesis and assimilate demand. The tacit assumption is that treatments which lead to accumulation of assimilate will cause a rise in cytosolic sucrose concentration or of some closely related metabolite in photosynthetic mesophyll cells and that it is these rises which are transduced into a decline in the rate of photosynthesis. Such assumptions are not always justified. It has been suggested that product partitioning into fructan allows assimilates to accumulate without large changes in sucrose content (Labhart et al., 1983). Tissue repartitioning may also function in the same way. Preferential accumulation of recently fixed sucrose into minor veins can be observed (Borland and Farrar, 1989) and both the parenchymatous bundle sheath (Williams et al., 1989) and the epidermis (Tomos et al., 1992) have distinct patterns of carbon accumulation. Non-aqueous fractionation (Gerhardt and Heldt, 1984) can be used to obtain gross estimates of intercellular compartmentation but the technique is

267 difficult and averages different cell types. Sampling of vacuolar sap (Tomos et al., 1992) allows direct measurement of vacuolar carbohydrate concentrations from specific cell types but only allows indirect assessment of cytosolic concentration, based on the argument that sucrose movement into leaf vacuoles is passive in some species (Kaiser and Heber, 1984). Nevertheless, applying the most common techniques used to alter assimilate abundance and then sampling vacuolar sap would show whether or not they produce changes in specific carbohydrates within the vacuoles of mesophyll cells. In our view there is also a conceptual problem in interpreting data on changes in photosynthetic rate associated with changes in assimilate abundance. The prevailing approach is to compare rates in control and treated leaves at a fixed time after the treatment is initiated rather than to consider changes across the ontogeny of the leaf. Many of the treatments involving excision and external administration of sugars will also initiate senescence, and indeed senescence processes themselves may be sensitive to assimilate abundance (Azumi et al., 1993). If we consider the leaf ontogeny of a typical crop species as being similar to that observed by Catsky et al. (1976) then measurements of photosynthetic rate or abundance of key photosynthetic proteins at a single time after imposition of the experimental treatment cannot distinguish between accelerated induction of the normal senescence processes and a more specific down-regulation which is independent of the normal ontogenic development (Fig. 3). It is known that environmental changes can alter the timing and extent of loss of specific components of the photosynthetic systems in leaves of Lolium temulentum (Mae et al., 1993). This may appear merely to be a semantic argument but it has consequences for consideration of the mechanisms for down-regulation of photosynthesis. It is known that a range of signals can affect the rate of senescence (Thomas, 1984). If assimilate abundance is merely one of these then the transduction pathway between signal and altered patterns of gene expression is likely to be more complex and less direct than the linear model which is often considered and which is discussed in detail for microbial systems in Section V These reservations extend principally to studies on down-regulation of photosynthesis mediated at the level of gene expression. By contrast the evidence for repartitioning within source leaves being sensitive

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2. The Repartitioning of Sucrose into Fructan in Leaves of Temperate Grasses The time course of this process also matches the attainment of a threshold concentration of sucrose beyond which there is progressive synthesis of larger fructan polymers (Fig. 4). The overall rates of carbohydrate accumulation are linear with time during the photoperiod (Housley and Pollock, 1985; Cairns and Pollock, 1988a) but the partitioning ratio changes markedly. Exogenous sucrose produces a similar response (Wagner et al., 1986) and feeding glucose or fructose mixtures also results in fructan accumulation following the initial resynthesis of sucrose within the leaf. Reversal of carbohydrate accumulation leads to a rapid decline in sucrose followed by a progressive depolymerization of fructan and a transient accumulation of reducing sugars (Simpson et al., 1991). to sucrose concentration is much more compelling. Three examples will be cited but consideration of recent reviews (Stitt, 1991; Farrar, 1992) will provide others.

1. Repartitioning into Chloroplast Starch The diel cycle of photosynthesis under natural environments leads to a profile of starch accumulation which is consistent with the initial establishment of a threshold concentration of sucrose (Gordon et al., 1980). The perturbation of this process by reducing export levels stimulates starch storage (Table 1; Stitt et al., 1984) This effect can be mimicked by feeding exogenous sugars to detached leaves. The amount of transcript for ADPGpyrophosphorylase is increased in leaves exposed to sugar (Muller-Rober et al., 1991).

3. Over-Expression of Sucrose Phosphate Synthase Reduces Starch Content Worrell et al. (1991) produced transgenic tomato plants expressing maize sucrose phosphate synthase under the control of the light-regulated leaf-specific Rubisco small sub-unit promoter. In these transgenic plants, elevated activity of sucrose phosphate synthase could be detected by enzyme assay and this was associated with reductions of starch content of up to 50% and a doubling of sucrose content in those transgenics with the highest assayable activity. These studies provide compelling, but not conclusive, evidence that sucrose itself or a close metabolite is the a priori determinant of the reallocation of reserves within leaves. There is, however, physiological evidence that leaf partitioning:export ratios are also sensitive to sink demands. Transfer of plants to short days increases the proportion of

Chapter 10

Source, Sinks and Sucrose

269 on chloroplast carbon metabolism and hence on photosynthesis is small, simply because of the equilibrium position for at least one of the reactions involved in the subsequent metabolism of triose phosphates into sucrose. Cytoplasmic fructose 1,6bisphosphatase (cFBPase) is the obligatory link between chloroplast and cytosolic carbon metabolism. Measurements of free energy in vivo confirm that the equilibrium strongly favors the hydrolysis of FBP (Stitt et al., 1987), thus making it unlikely that accumulation of metabolic intermediates downstream of this point could directly affect flux through the reaction.

2. Fine Control of Enzyme Activity

photosynthate retained in the leaf during the day for subsequent mobilization and export. This is associated with a rise in leaf sucrose content (which is consistent with the observations discussed above) but also with a fall in root sucrose content, suggesting that some element of the transport pathway is also being regulated (Sicher et al., 1982).

B. Potential Mechanisms of Sucrose Regulation If we accept, therefore, that there is an argument for the involvement of soluble sugars (particularly sucrose) in the regulation both of photosynthetic activity and of partitioning of assimilates within the leaf, how might this regulation be achieved? It is possible to conceive of three types of mechanism. Firstly, flux through the pathway could be modulated purely via product accumulation (a mass-action effect). Secondly, the activity of pre-existing proteins can be modulated either via changes in the concentrations of regulatory metabolites or via covalent modification of the enzyme itself. Thirdly, the amount of enzyme present in the tissue could vary. This latter change could be associated with alterations in the rate of gene expression, of posttranscriptional modifications or of protein turnover.

1. Mass-Action Effects The potential for mass-action effects of assimilates

Fine control of photosynthetic carbon metabolism is thought to occur via the modulation of enzyme activity (see Chapter 6). In spinach leaves, the key elements involve regulation of cFBPase via the signal metabolite fructose 2,6-bisphosphate (Stitt, 1990) together with regulation of sucrose phosphate synthase (SPS) via reversible phosphorylation of the enzyme protein and/or the accumulation of sucrose, sucrose phosphate or glucose 6-phosphate (Huber et al., 1992; see also Chapter 6). The paradigm for operation of this regulatory sequence involves a reduction in carbon flow into sucrose, either under conditions where supply of photosynthetic triose phosphate is low, or when assimilate abundance is high (Table 1; Stitt, 1990). The exact nature of the signal which reduces SPS activity and stimulates formation of FBP under high assimilate abundance is still under discussion (Foyer, 1988), but the end result in the chloroplast involves an elevation of triose phosphate concentration and a reduction in the concentration of Pi. This has the effect of stimulating the enzyme ADPG pyrophosphorylase and thus altering partitioning in favor of chloroplast starch synthesis (Preiss, 1987). A range of physiological, enzymological and metabolic measurements (Huber et al., 1992) provide strong evidence for repartitioning into starch being controlled in this way. It is less clear whether such changes in chloroplast metabolites can or do directly inhibit photosynthesis. It has been suggested that Pi limitations within the chloroplast may reduce photosynthesis by interfering with the regeneration of ribulose 1,5-bisphosphate (Foyer, 1987; Huber, 1989) but the conditions used to demonstrate this are generally somewhat extreme. The evidence that it occurs under natural conditions

270 is less compelling (Stitt, 1991). One is left with the conclusion that this metabolic sequence elegantly permits fine control of carbon flow to optimize the balance between regeneration of Calvin cycle intermediates, the synthesis and export of sucrose and the accumulation of chloroplast starch but that its role in regulating photosynthesis is not proven. There is also, of course, significant diversity between species in the extent of this response. The proportion of recently fixed carbon stored as chloroplast starch varies widely, with temperate Gramineae and members of the Alliaceae accumulating sucrose and fructans in preference to starch (Pollock, 1986). Why increased assimilate abundance in these leaves does not lead to alterations in the product partitioning between sucrose and starch is not known, although significant rises in FBP contents have been observed in Lolium temulentum where export is blocked (Pollock et al., 1989) and cFBPase in Lolium is inhibited in vitro by this metabolite (Collis and Pollock, 1991).

3. Coarse Control Mediated via Alterations in Gene Expression There is now good evidence that alterations in sucrose concentration are correlated with changes in gene expression and that these changes alter enzyme activities involved in the repartitioning of carbon and possibly in the fixation of carbon. We have already discussed the balance between sucrose accumulation and fructan biosynthesis in leaves of temperate grasses. When leaves are grown under low irradiance, they do not possess the enzyme activities required for the biosynthesis of fructan (Cairns, 1989; Cairns, 1992; Pollock and Housley, 1993). Restriction of sucrose export leads to an increase in activity and a progressive repartitioning of carbon into fructan. This repartitioning can be prevented by treatment with inhibitors of transcription and translation without altering the total rate of carbon accumulation (Cairns and Pollock, 1988b; Table 2). Administration of cycloheximide at different times after suppression of export has shown that the inhibitory effects were observed only over the first 8 hours. Following that, conversion to fructan could not be prevented. Cell-free translation of mRNA during this period also indicated differences in abundance of a restricted number of transcripts (A.L. Winters and C. J. Pollock, unpublished). This is a useful model system because the response can be

C. J. Pollock and J. F. Farrar made qualitative. By contrast, the proportion of carbon retained in leaves as starch appears to reflect a complex interaction between the activities of the enzymes of synthesis and degradation (Steup, 1987). Experiments of a different kind have been used to support a role for sucrose in the regulation of Calvin cycle enzyme activity at the level of transcription. Sheen (1990) fused promoter sequences from several maize genes coding for enzymes and proteins involved in photosynthesis to reporter genes and studied transient expression in maize protoplasts. Expression was markedly down-regulated by sucrose, hexose and acetate when compared with osmolytes such as mannitol (Table 3). This presents convincing evidence that such transcriptional down-regulation may occur but the sugar concentrations required (300 mM) are higher than those estimated to occur normally in intact tissues (Cairns et al., 1989; Section II.B), particularly with regard to hexose concentrations, which are generally low in leaves. Comparison of the promoter sequences failed to identify more than one which contained a consensus sequence for the binding of CCAAT enhancer binding protein (McKnight et al., 1989). It was not, therefore, possible to suggest a common mechanism for the effects of sucrose. Similar responses to sucrose have been described for other higher plant genes expressed in different tissues (Section IV). However there is no generally accepted model for the action of sucrose or hexose in controlling transcription or translation (Section V).

4. Other Forms of Coarse Control Metabolic regulation can occur via post-translational modification (Gallie, 1993) or via alteration in the rates of protein turnover (Barber and Andersson, 1992). There is little evidence that either occur in response to altered sucrose concentrations, still less that they affect either photosynthesis or partitioning, but this may reflect lack of experimentation rather than lack of significance. One of the enzyme activities associated with the synthesis of fructans in leaves rises during the day when sucrose concentrations rise and then falls at night as the assimilate abundance declines. This fall can be prevented by administration of leupeptin, a protease inhibitor, suggesting that there is continuous degradation of the relevant protein and that activity is determined mainly by alterations in the rate of de novo protein synthesis (Obenland et al., 1991).

Chapter 10

Source, Sinks and Sucrose

C. Conclusion The evidence discussed above suggests that many forms of metabolic control in higher plants are sensitive to tissue concentrations of soluble carbohydrate, and that, in leaves, these represent an indication of the balance between photosynthesis export and storage. The most general consequence seems to be alterations in product partitioning rather than attenuation of gross flux through the photosynthetic carbon fixation pathway but the data are far from comprehensive

IV. Sinks Sinks can be defined as regions of the plant which are heterotrophic for photosynthetically fixed carbon. All meristems are sinks, and the organs produced by the action of some meristems remain sinks. Growing sinks include roots and vegetative and reproductive

271

shoot apices; storage sinks, tap roots such as in beet, or stem internodes such as in sugarcane. A sink such as a root may supply all or part of the plant with water or nutrients; here it is defined solely by its status with respect to reduced carbon. The total growth of all sinks on the plant cannot exceed the ability of the sources to supply them with carbon. Each sink could have the capacity to grow appreciably faster than its actual growth rate, which would be determined by limited substrate supply from source leaves. There are several reasons for believing that this is not so. First, it would mean that large amounts of metabolic machinery were present but inactive, with appreciable and unnecessary maintenance costs. Second, there is ample evidence that the size of root system relative to that of the source leaves is regulated (Bastow Wilson, 1988; Farrar, 1992) since roots capture resources such as water and nutrients, and the maximum rate at which the plant can take up (for example) nitrogen will be determined by the size of the root system. Such

272 regulation implies control of assimilate transport to the sink concerned. And third, there is evidence for coarse control of sink metabolism by the sources supplying them. To this evidence we now turn.

A. Evidence for Regulation of Sink Metabolism by Assimilate from Source Leaves 1. Roots The sugar supply to roots can be reduced or eliminated by darkening the shoot, or by partial or complete defoliation, or excision of the part of the root system of interest. Such treatments alter much more than sugar supply: the import of any substance mobile in the phloem, including reduced nitrogen compounds, and those for which the root is heterotrophic such as thiamin and pyridoxine (Street, 1969), will be reduced, and both the export of compounds in, and delivery of nutrients to the root surface by, the transpiration stream will fall. This set of experiments, which will be referred to generically as starvation experiments, must therefore be assessed critically. Root elongation, and associated processes such as respiration, fall following excision, shading or defoliation (Evans, 1972, Saglio and Pradet, 1980; Farrar, 1981, Williams and Farrar, 1990) while cell division in apical meristems ceases (Van’t Hof, Hoppin and Yagi, 1973). Much detail is available on individual processes within roots during starvation; some individual enzymes fall in activity (for example acid and neutral invertase, J. H. H. Williams and J. F. Farrar unpublished; fumarase, B. E. Collis and J. F. Farrar unpublished.) and mitochondrial properties change (Couée et al, 1992). When root tips of barley were sugar-starved by excision, or by darkening the shoot, there were falls in both the mitotic index of the meristematic cells and the amount of cdk-like protein (measured using antibodies to the PSTAIRE motif; B. E. Collis, C. J. Pollock and J. F. Farrar, unpublished). In root nodules of white clover, carbohydrate, protein content and leghemoglobin fall when the host plant is partially defoliated (Gordon et al, 1986). Nitrogen fixation, nitrogenase-linked respiration and the content and synthesis of specific proteins decline after defoliation, although only PEP carboxylase and glutamine synthetase declined more than overall protein content (Gordon et al., 1990; Gordon and Kessler, 1990). Darkening of the shoot causes similar reduction in rates of processes in nodules (Minchin and Pate, 1974; Schwietzer and Harper, 1980). When

C. J. Pollock and J. F. Farrar soybean plants were darkened for 3 d and then returned to their normal light/dark regime, the levels of mRNAs within nodules coding for leghemoglobin, sucrose synthase and glutamine synthetase fell (by up to 50% within 24 h of darkening) and the transcript levels rapidly returned to normal after return to light (Gordon, Ougham and James, 1993); the amount and activity of only sucrose synthase protein fell during the dark period. It is not yet possible to ascribe unequivocally cause and effect to the suite of changes following darkening, defoliation or excision, or even to arrange the changes in the order in which they begin. Because of the uncertainty surrounding the interpretation of starvation experiments (is it really sugar deprivation or rather loss of reduced nitrogen or some other metabolite which triggers the suite of changes?), they are perhaps best seen as the first part of a more critical type of experiment. The second and key part is the resupply of a single substance, any effects of which on metabolism can then be ascribed to it with some certainty. This may be as near as the plant scientist can come to the classical replacement therapy experiments which have been so powerful in mammalian endocrinology. The elongation of roots of Lolium perenne is little inhibited by defoliation if they are bathed in glucose up to 110 mM (Evans, 1972). When barley roots are starved for 24 h, their rates of respiration and elongation, and activities of acid invertase, cytochrome c oxidase, fumarase, and the amounts of cytochrome c oxidase subunit II protein, the ubiquitination of protein, the proteosome content and the levels of monoubiquitin transcript all fall. Conversely, endoprotease activity and levels of polyubiquintin transcript rise. All of these changes can be reversed by supplying sucrose for a further 24 h and several specific proteins increase or decrease in amount (Bingham and Farrar, 1988; Williams and Farrar, 1990; McDonnell and Farrar, 1992; Williams et al., 1992; B. E. Collis, C. J. Pollock and J. F. Farrar, unpublished). Specific proteins increase, and others decrease, in sucrose-fed roots of millet (Baysdorfer and VanDerWoude, 1988). The expression of the sus 1 gene, and the activity of the gene product sucrose synthase, increase with the concentration of glucose supplied to excised maize root tips (Koch et al, 1992), and amounts of mRNA for sucrose synthase are sensitive to sugar concentration in Vicia faba and potato (Hein et al., 1993; Salanboubat and Belliard, 1989). The cell cycle in bean and maize root tips,

Chapter 10 Source, Sinks and Sucrose arrested in G1 and G2 stages by starvation, restarts after sucrose or glucose is supplied to the root tips (Van’t Hof et al., 1973; Van’t Hof, 1985; J. Evans, D. Francis and J. F. Farrar, unpublished). Transcripts of the cdc2a gene, which encodes a key regulator of the cell cycle, are less abundant in cell suspension when sucrose supply is reduced to one-tenth (Hemerly et al., 1993). The equivalence of sucrose, fructose, glucose and mannose (all of which are metabolized by barley roots), but not of 3-O-methyl glucose, which is not metabolized, is consistent with the signal molecule being metabolically close to hexose phosphates. There is a widely reported relationship between soluble (or total non-structural) carbohydrate and respiration rate (Penning de Vries et al, 1979; Farrar, 1985b; Lambers, 1985; Lambers et al, 1991; Williams and Farrar, 1991) which, although hardly evidence of causality, is consistent with sugar control of root growth, since respiration and growth rates are stoichiometrically related and these observations come from long-term experiments where the relationship with sugar is as likely to be exercised via coarse control as by sucrose simply acting as a substrate.

2. Cell Suspension Culture Tissue culture provides an alternative vehicle for studying regulation of growth and metabolism by sugars. A population of cells which, in spite of genetic variability, is comparatively homogeneous, is bathed in a medium a selected component of which can be increased or decreased at will; batch culture has been used more than continuous (fermenter) culture. The results of depriving sycamore cells in batch suspension culture of sucrose have been interpreted mainly in terms of the controlled autophagy of plant cells (Douce et al, 1991). When resuspended in fresh, sucrose-free, medium, the sucrose pool within cells falls rapidly, although perhaps partly buffered by starch degradation; the rate of sucrose degradation initially is greatly in excess of the rate of respiration (Douce et al, 1991). Re-supply of sucrose may be accompanied by a rise in respiration, and the activities of fumarase and cytochrome c oxidase (Avelange et al, 1990) while numerous other non-nutritional effects of sugars on cultured plant cells have been reported. In cucumber callus, expression of the genes for malate synthase and isocitrate lyase is induced by a

273 fall in intracellular sugars and repressed by external sugars including mannose anhd 2-deoxyglucose (Graham et al., 1994).

3. Shoot Apices, and Storage and Reproductive Sinks Little or no relevant evidence is available for these sink types, in spite of the agreed importance of assimilate supply for the growth of fruits (but see Section IV.B.3). Experiments that claim to show the dependence of fruit growth on assimilate supply do not demonstrate that the effect is due to sugars, as other substances supplied by source leaves could also be responsible. There are a number of reports from apparently unrelated areas of metabolism, and so not profitably listed here, of sugar-regulated expression of genes such as those for invertase, nitrate reductase, and patatin.

B. Sucrose as a Morphogen So far we have considered how sucrose can act on sinks non-nutritionally but still in an essentially quantitative manner: the capacity for or rate of growth is affected. There is also evidence, of variable quality, for sucrose and other sugars acting qualitatively as a morphogen, switching developmental pathways of groups of cells. Possibly, a role of sugars is to alter the sensitivity of tissues to plant growth substances (Trewavas, 1991) as well as or instead of having a direct effect as a growth substance. No attempt has been made in any of the cases to be discussed to localize sugars within or between cells before attempting to correlate them with the process they are purported to modulate.

1. Differentiation of Vascular Tissue When applied to blocks of undifferentiated callus tissue in combination with an auxin, sucrose can substitute for an excised bud in inducing the differentiation of vascular tissue within the callus block and remote from the site of application; at constant auxin concentration, increasing concentrations of sucrose increased the proportion of sucrose in the vascular tissue (Wetmore and Rier, 1963). The effect of sucrose is specific for disaccharides (Jeffs and Northcote, 1967).

274

2. Transition to Flowering Reviews by Bernier (1988) and Bodson and Bernier (1985) and papers by Houssa et al. (1991) and McDaniel et al (1991) suggest that a sufficient supply of sugar is a necessary, but perhaps not a sufficient, condition for the transition to flowering, and that the nutrient diversion hypothesis is neither proven nor falsified. In some cases direct application of sucrose or glucose triggers flower initiation but has no effect on growth, showing a specificity for the flowering process but not excluding the possibility that the sugars are substrates, rather than morphogens. Flower buds only form on callus cultures of Plumbago if either sucrose, cellobiose or maltose are above a threshold concentration; the effect is not osmotic (Audus, 1972).

3. Fruit Abortion Commonly many more fruits are initiated on a plant than develop to maturity: a large number abort early in development. There are suggestions that abortion is a function of assimilate supply, as a means of adjusting the number of reproductive sinks to the amount of assimilate available to support their development (Atherton and Harris, 1986; Erner, 1989; Looney, 1989). Since the fruits are small at the time of abortion, any such effect is unlikley to be nutritional.

C. Conclusion Sucrose may have some morphogenetic effects in sink tissues; the evidence is generally weak, and surely here is a problem ripe for attack by molecular techniques. What evidence there is, is consistent with the idea that sucrose can have an effect at the level of coarse control. From studies of sink metabolism there is abundant evidence that sucrose, or glucose, can exercise coarse control. Next we consider the mechanisms by which sucrose might exercise such coarse control.

V. Potential Mechanisms of Gene Regulation by Sugars There are now cogent reasons for believing that at least some of the regulatory actions of sucrose or its immediate metabolites operate at the level of coarse

C. J. Pollock and J. F. Farrar control; that is they alter the amount of metabolic and transport machinery present in the cell. The question is how this alteration is achieved. The minimum system that needs to be in place for such a response is a signal molecule, a receptor for that molecule and a transduction pathway which is followed after the binding of signal molecule to receptor (see Chapter 12). There are several reasons for believing that sucrose itself is unlikely to be the sole signal molecule; roots contain relatively little sucrose, most soluble sugar being hexose, and it is possible that a close metabolite of sucrose or hexose is indeed the signal (Krapp et al., 1993; Jang and Sheen, 1994; Graham et al., 1994). However it is clear that sucrose can have quite different effects from those of related simple sugars, for example the unique effects on cultured roots and some of the morphogenetic effects discussed in Section IV. It is, however, premature to conclude that only one mechanism exists for transducing changes in assimilate abundance into altered patterns of gene expression. The most intensively studied eukaryotic system is probably the hexose-repression of invertase genes in yeast (Carlson, 1987). A number of genes have been identified on the basis of isolation of constitutive mutants. Two have been assigned biochemical functions, one coding for a hexokinase which appears to have a regulatory as well as a catalytic role in glucose utilization. The other (snf1) encodes a protein kinase which appears to be part of a complex regulatory system affecting transcription. A higher plant homologue of s n f 1 has been isolated from rye and this can be used to restore glucose repression in yeast via functional complementation (Alderson et al., 1991). This suggests, but does not prove, that similar systems operate in higher plants. Sucroseresponsive upstream regulatory elements have also been detected in fusions between reporter genes and the promoter regions for patatin. Deletion analysis and transformation of potato plants has shown that this region was close to, but discrete from, a sequence which determines tuber specificity of expression (Liu et al., 1990). No mechanism for the action of sucrose was proposed. In prokaryotic systems, sucrose induction of levansucrase has been studied intensively in Bacillus subtilis and a model produced which describes a specific role for sucrose (Cote and Ahlgren, 1993). The movement of sucrose through the cell membrane is associated with its phosphorylation and with the

Chapter 10

Source, Sinks and Sucrose

dephosphorylation of the product of the sac X gene. The sac X gene product inhibits production of the sac Y gene product which functions as an antiterminator. Phosphorylation removes this inhibition and allows the production of the anti-terminator. The anti-terminator is thought to destabilize the stem loop structure which normally inhibits transcription in the absence of sucrose and which is coded by a region downstream of the sac R constitutive promoter. Once this destabilization occurs, read-through by RNA polymerase is possible, resulting in production of transcripts, the synthesis and secretion of the enzyme (Crutz et al., 1990).

VI. Conclusion In the period since 1968, the hypothesis of Neales and Incoll (1968) has been revisited often, with an increasingly sophisticated range of techniques. That there is still uncertainty about the existence of the phenomenon, its importance in vivo and its mechanism probably indicates the complexity of the response and the metabolic diversity of the species which have been studied. Nevertheless, there is now enough evidence to implicate assimilate status in both coarse and fine control of elements of carbon metabolism in sources and sinks. The essential aims of research in this area should be to identify the components of the assimilate pool which initiate changes and to produce a convincing mechanism to explain them. These criteria have largely been met in terms of fine control of product repartitioning in leaves although there are still uncertainties concerning the behavior of low starch plants and elements of the response pathway which generate the rises in hexose phosphate concentrations observed when sucrose export is blocked (Stitt et al., 1987; Pollock and Cairns, 1991). There is little equivalent evidence in sink tissue. The link between carbohydrate contents and respiration rate is marked but the mechanism uncertain, combining as it seems elements of both coarse and fine control. At the levels of gene expression and control of development, the current state of our knowledge is even more fragmentary. In leaves a range of effects that have been described which are consistent with the mechanistic link between carbohydrate accumulation and down-regulation of genes involved in photosynthesis, but again the nature of the link remains largely speculative. The problems of distinguishing

275 between altered patterns of leaf ontogeny and specific changes in individual processes have been discussed (Section III) and we suggest that greater care should be taken than hitherto in standardizing the developmental state of the tissues and in measuring the direct effects of carbohydrate accumulation upon the rates of senescence. Thought might also be given to using long-lived leaves such as ivy (Hedera helix) where the duration of experimental treatments would be statistically insignificant in terms of the life of a leaf which habitually lasts over at least one winter. Nevertheless, consideration of the mechanisms by which assimilate-driven photosynthetic downregulation might occur must, we feel, embrace the likelihood that the transduction pathway is via the complex set of signals and responses which control the timing of senescence and thus may not involve simple linear systems responsive only to sucrose or some related metabolite as occurs in the regulation of the bacterial levansucrase (Crutz et al., 1990). Consideration of the role of carbohydrates in mediating the source-sink balance by coarse control at the sink level are even more speculative. Good physiological systems exist in which morphological and developmental changes can be induced by altered assimilate status and a number of mRNA species have been described whose abundance alters during such treatments, but there is no effective connection between the two types of experiments. Whilst not generally enthusiastic about the study of metabolic integration in disaggregated systems such as plant tissue culture, we would suggest that the ease in which changes in gene expression could be followed makes the developmentally sensitive explant systems of Jeffs and Northcote (1967) appropriate for reexamination. There are, however, other opportunities for further progress in the general area of sucrose-induced gene expression. Short-term coarse control of chemical repartitioning in leaves does occur rapidly and reversibly in a manner which is broadly independent of ontogeny (Pollock and Cairns, 1991). If we concentrate on identifying the ways in which these processes are regulated by sucrose or its metabolites and isolate and characterize the transduction systems involved, it is then feasible to search for homologous systems acting at the level of photosynthesis or sink metabolism. More and more proteins with affinity for sucrose or hexoses are being characterized, sequenced and the genes cloned. These tools will be of considerable value when used in physiologically

276 defined systems where the timing of the regulatory events is accepted to be at least as important as their extent.

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Chapter 10

Source, Sinks and Sucrose

of substrate. In Emes M (ed) Compartmentation of Plant Metabolism in Nono-photosynthetic Tissues, pp 167–188. Cambridge University Press, Cambridge Farrar JF and Williams ML (1991b) The effects of increased atmospheric carbon dioxide and temperature on carbon partitioning, source-sink relations and respiration. Plant Cell Environ 14: 819–830 Farrar JF, Minchin PEH and Thorpe MR (1994) Carbon import into barley roots: stimulation by galactose. J Exp Bot 45: 17– 22 Farrar SC and Farrar JF (1985) Fluxes of carbon compounds in leaves and roots of barley plants. In Jeffcoat B, Hawkins AF, Stead AD (ed) Regulation of Sources and Sinks in Crop Plants, pp 67–84. British Plant Growth regulator Group, Bristol Farrar SC and Farrar JF (1987) Effects of photon fluence rate on carbon partitioning in barley source leaves. Plant Physiol Biochem 25: 541–548 Foyer CH (1987) The basis of source-sink interactions in leaves. Plant Physiol Biochem 25: 649–657 Foyer CH (1988) Feedback inhibition of photosynthesis through source-sink regulation in leaves. Plant Physiol Biochem 26: 483–497 Gallie DR (1993) Post-transcriptional regulation of gene expression in plants. Annu Rev Plant Physiol Plant Mol Biol 44: 77–105 Gerhardt R and Heldt HW (1984) Measurement of subcellular metabolite levels in leaves by fractionation of freeze-stopped material in non-aqueous media. Plant Physiol 75: 542–547 Gordon AJ and Kessler W (1990) Defoliation-induced stress in nodules of white clover. II. Immunological and enzymic measurements of key proteins. J Exp Bot 41: 1255–1262 Gordon AJ, Ryle GJA and Webb, G (1980) The relationship between sucrose and starch during ‘dark’ export from leaves of Uniculm barley. J Exp Bot 31: 845–850 Gordon AJ, Ryle GJA, Mitchell DF, Lowry KH and Powell CE (1986) The effect of defoliation on carbohydrate, protein and leghaemoglobin content of white clover nodules. Ann Bot 58: 141–154 Gordon AJ, Kessler W and Minchin FR (1990) Defoliation induced stress in nodules of white clover I Changes in physiological parameters and protein synthesis. J Exp Bot 41: 1245–1253 Gordon AJ, Ougham HJ and James CL (1993) Changes in levels of gene transcripts and their corresponding proteins in nodules of soybean plants subjected to dark-induced stress. J Exp Bot 44: 1453–1460 Graham I A, Derby KJ and Leaver CJ (1994) Carbon catabolite repression regulates glyoxylate cycle gene expression in cucumber. Plant Cell 6: 761–772 Hemerly AS, Ferreira P, Engler Jde A, Montagu Mvan, Engler G and Inze D (1993) cdc2a expression in Arabidopsis is linked with competence for cell division. Plant Cell 5: 1711–1723 Herold A (1980) Regulation of photosynthesis by sink activity— the missing link. New Phytol 86: 131–144 Herold A and McNeil PH (1979) Restoration of photosynthesis in pot-bound tobacco plants. J Exp Bot 30: 1187–1194 Hiem U, Weber H, Baumleim H and Wobus U (1993) A sucrosesynthase gene of Vicia faba L.: Expression pattern in developing seeds in relation to starch synthesis and metabolic regulation. Planta 191: 394–401 Ho LC (1978) The regulation of carbon transport and the carbon

277 balance of mature tomato leaves. Ann Bot 42: 155–164 Ho LC (1988) Metabolism and compartmentation of sugars in sink organs. Ann Rev Plant Physiol Plant Mol Biol 39: 355– 378 Ho LC and Thornley JHM (1978) Energy requirements for assimilate translocation from mature tomato leaves. Ann Bot 42: 481–483 Houssa S, Bernier G and Kinet JM (1991) Qualitative and

quantitative analysis of carbohydrates in leaf exudate of the short-day-plantXanthium strumarium L. during floral transition. J Plant Physiol 138: 24–28 Housley TL and Pollock CJ (1985) Photosynthesis and carbohydrate metabolism in detached leaves of Lolium temulentum L. New Phytol 99: 499–507 Huber SC (1989) Biochemical mechanism for regulation of sucrose accumulation in leaves during photosynthesis. Plant Physiol 91: 656–662 Huber SC, Huber JLA and McMichael Jr RW (1992) The regulation of sucrose synthesis in leaves. In: Pollock CJ, Farrar JF and Gordon AJ (eds) Carbon Partitioning Within and Between Organisms, pp. 1–26. Bios Scientific Publishers, Oxford Jang JC and Sheen J (1994) Sugar sensing in higher plants. Plant Cell 6: 1665–1679 Jeffs RA and Northcote DH (1967) The influence of indol-3-yl acetic acid and sugar on the pattern of induced differentiation in plant tissue culture. J Cell Sci 2: 77–88 Kaiser G and Heber V (1984) Sucrose transport into vacuoles isolated from barley mesophyll protoplasts. Planta 161: 562– 568 Kholodova VP (1967) Localization of sucrose in tissues of the storage root of sugar beet. Fiziol Rast 14: 444–450 Koch KE, Nolte KD, Duke ER, McCarty DR and Avigne WT (1992) Sugar levels modulate differential expression of maize sucrose synthase genes. Plant Cell 4: 59–69 Krapp A, Hofmann B, Schäfer C and Stitt M (1993) Regulation of the expression of rbcS and other photosynthetic genes by carbohydrates: a mechanism for the ‘sink regulation’ of photosynthesis? Plant J 3: 817–828 Labhart C, Nösberger J and Nelson CJ (1983) Photosynthesis and degree of polymerisation of fructan during reproductive growth of meadow fescue at two temperatures and two photon flux densities. J Exp Bot 34: 1037–1046 Lambers H (1985) Respiration in intact plants and tissues. In: Douce R, Day DA (ed) Encyclopedia of Plant physiology, Vol 18, pp 418–473. Springer, Berlin Lambers H, van der Werf A and Konings H (1991) Respiratory patterns in roots in relation to their functioning. In: Waisel Y, Eshel A, Kafkafi U (ed) Plant Roots: The Hidden Half, pp 229– 263. Dekker, New York Liu XJ, Pratt S, Willmitzer L and Fromer WB (1990) Cis regulatory elements directing tuber-specific and sucroseinducible expression of a chimeric class I patatin promoter/ GUS-gene fusion. Mol Gen Genet 223: 401–406 Looney NE (1989) Effects of crop reduction, gibberellin sprays and summer pruning on vegetative growth, yield, and quality of sweet cherries. In: Wright CJ (ed) Manipulation of Fruiting, pp 39–50. Butterworths, London Mae T, Thomas H, Gay AP, Makino A and Hidema J (1993) Leaf development in Lolium temulentum: Photosynthesis and photosynthetic proteins in leaves senescing under different

278 irradiances. Plant Cell Physiol 34: 391–399 Martinoia E, Kaiser G, Schramm MJ and Heber U (1987) Sugar transport across the plasma lemma and tonoplast of barley meophyll protoplasts. J Plant Physiol 131: 467–478 McDaniel CN, King RW, Evans LT (1991) Floral determination and in-vitro floral differentiation in isolated shoot apices of Lolium temulentum L. Planta 185: 9–16 McDonnell E and Farrar JF (1992) Substrate supply and its effect on mitochondrial and whole-tissue respiration in barley roots. In: Lambers H and van der Plas L (ed) Molecular, Biochemical and Physiological Aspects of Plant Respiration, pp 455–62. SPB, The Hague McKnight SL, Lane MD and Glueckshon-Waelsch S (1989) Is CCAAT/enhancer-binding protein a central regulator of energy metabolism? Genes Dev 3: 2021–2024 Minchin FR and Pate JS (1974) Diurnal fluctuating of the legume root nodule. J Exp Bot 25: 295–308 Minchin PEH, Farrar JF and Thorpe MR (1994) Partitioning in split root systems of barley: Effect of temperature of the root. J Exp Bot 45:1103–1109 Minchin PEH and McNaughton G (1984) Exudation of recently fixed carbon by non-sterile roots. J Exp Bot 35: 74–82 Minchin PEH, Thorpe MR and Farrar JF (1993) A simple mechanistic model of phloem transport which explains sink priority. J Exp Bot 44: 947–955 Moorby J and Jarman PD (1976) The use of compartmental analysis in the study of the movement of carbon through leaves. Planta 122: 155–168 Muller-Rober BT, Kossman J, Hannah LC, Willmitzer L, Sonnewald U (1991) ADPG-pyrophosphorylase genes from potato: Mode of RNA expression and its relation to starch synthesis. In: Bonnemain J-L (ed) Phloem Transport and Assimilate Compartmentation, pp 204–208. Ouest, Nantes Natr L (1967) Time-course for photosynthesis and maximum figures for the accumulation of assimilates in barley leaf segments. Photosynthetica 1: 29–36 Neales TF and Incoll LD (1968) The control of leaf photosynthesis rate by the level of assimilate concentration in the leaf: a review of the hypothesis. Bot Rev 34: 107–124 Obenland DM, Simmen U, Boller T and Wiemken A (1991) Regulation of sucrose-sucrose-fructosyl transferase in barley leaves. Plant Physiol 97: 811–813 Passioura JB, Ashford AE (1974) Rapid translocation in the phloem of wheat roots. Aust J P1 Physiol 1: 521–527 Patrick JW (1990) Sieve-element unloading: cellular pathway, mechanism and control. Physiol Plant 78: 298–308 Penning de Vries FWT, Witlage JM and Kremer D (1979) Rates of respiration and of increase in structural dry matter in young wheat, ryegrass and maize plants in relation to temperature, water stress and to their sugar content. Ann Bot 44: 595–609 Pollock CJ (1986) Fructans and the metabolism of sucrose in vascular plants. New Phytol 104: 1–24 Pollock CJ and Cairns AJ (1991) Fructan metabolism in grasses and cereals. Ann Rev Plant Physiol Plant Mol Biol42: 77–101 Pollock CJ and Housley TL (1993) The extraction and assay of 1 kestose: Sucrose fructosyl transferase from leaves of wheat. Plant Physiol 102: 537–539 Pollock CJ, Cairns, AJ, Collis BE and Walker RP (1989) Direct effects of low temperature upon components of fructan metabolism in leaves of Lolium temulentum L. J Plant Physiol 134: 203–208

C. J. Pollock and J. F. Farrar Preiss J (1987) Biosynthesis of starch and its regulation. In: Preiss J (ed) Carbohydrates (The Biochemistry of Plants, Vol 14), pp 181–254. Academic Press, San Diego Saglio PH, Pradet A (1980) Soluble sugars, respiration and energy charge during ageing of excised maize root tips. Plant Physiol 66: 516–519 Salanoubat M, Belliard G (1989) The steady-state level of potato sucrose synthase m R N A is dependent on wounding, anaerobiosis and sucrose. Gene 84: 181–185 Schäfer C, Simper H and Hofmann B (1992) Glucose feeding results in co-ordinated changes of chlorophyll content, ribulose– 1,5-bisphosphate carboxylase-oxygenase activity and photosynthetic potential in photoautotropic suspension cultured cells of Chenopodium rubrum. Plant Cell Environ 15: 343– 350 Schweitzer LE and Harper JE (1980) Effect of light, dark and temperature on root nodule activity of soyabeans. Plant Physiol 65: 51–56 Sheen J (1990) Metabolic repression of transcription in higher plants. Plant Cell 2: 1027–1038 Sicher RC, Harris WG, Kremer DF and Chatterton NJ (1982) Effects of shortened day length upon translocation and starch accumulation by maize, wheat and pangola grass leaves. Can J Bot 60: 1304–1309 Simpson RJ, Walker RP and Pollock CJ ( 1 9 9 1 ) Fructan exohydrolase activity in leaves of Lolium temulentum L. New Phytol 119: 499–507 Smith H (1990) Introduction: Signal perception, differential expression w i t h i n multigene families and the molecular basis of phenotypic plasticity. Plant Cell Environ. 13: 585–595 Steup M ( 1 9 8 7 ) Starch degradation. I n : Preiss J (ed) Carbohydrates. The Biochemistry of Plants, Vol 14. pp 255– 296. Academic Press, San Diego Stitt M (1990) Fructose-2,6-bisphosphate as a regulatory molecule in plants. Ann Rev Plant Physiol Plant Mol Biol 4l: 153–185 Stitt M (1991) Rising levels and their potential significance for carbon flow in photosynthetic cells. Plant Cell Environ. 14: 741–762 Stitt M, Kürzel B and Heldt H W (1984) Control of photosynthetic sucrose synthesis by fructose 2,6-bisphosphate. II. Partitioning between sucrose and starch. Plant Physiol 75: 554–560 Stitt M, Huber S and Kerr S (1987) Control of photosynthetic sucrose formation. In: Hatch MD and Boardman NK(eds)The Biochemistry of Plants, a Comprehensive Treatise, Vol 10, Photosynthesis, pp. 327–409. Academic Press, London Stitt M, von Schaewen A and Willmitzer L (1990) ‘Sink’ regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in the cell wall involves a down-regulation of the Calvin cycle and an up-regulation of glycolysis. Planta 183: 40–50 Street HE (1969) Growth in organized and unorganized systems. In: Steward FC (ed) Plant Physiology VB, pp 3–223. Academic Press, New York Swanson CA and Christy AL (1976) Control of translocation by photosynthesis and carbohydrate concentration of the source leaf. In: Wardlaw IF, Passioura JB (ed) Transport and Transfer Processes in Plants, pp 329–338. Academic Press, New York Tetlow IJ and Farrar JF (1993) Apoplastic sugar concentration and pH in barley leaves infected with brown rust. J Exp Bot 44: 929–936 Thomas H (1984) Cell senescence and protein metabolism in the

Chapter 10 Source, Sinks and Sucrose photosynthetic tissue of leaves. In: Davies I and Sigee DC (eds) Cell Ageing and Cell Death, pp. 171–188. Cambridge University Press, Cambridge Thorne JH and Koller HR (1974) Influence of assimilate demand on photosynthesis, diffusive resistance, translocation and carbohydrate levels of soybean leaves. Plant Physiol 54: 201– 207 Tomos AD, Leigh RA, Palta, JA and Williams JHH (1992) Sucrose and cell water relations. In: Pollock CJ, Farrar JF and Gordon AJ (eds) Carbon Partitioning Within and Between Organisms, pp 71–89. Bios Scientific Publishers, Oxford Trewavas A (1991) How do plant growth substances work? II: Opinion. Plant Cell Environ. 14: 1–12 van Bel AJE (1992) Pathways and mechanisms of phloem loading. In: Pollock CJ, Farrar JF, Gordon AJ (ed) Carbon Partitioning, pp 53–70. Bios Scientific Publishers, Oxford van’t Hof J (1985) Control points within the cell cycle. In: Bryant J A, Francis D (ed) The Cell Division Cycle in Plants, pp 1–13. Cambridge University Press, Cambridge van’t Hof J, Hoppin DP, Yagi S (1973) Cell arrest in G1 and G2 of the mitotic cycle of Vicia faba root meristems. Am J Bot 60: 889–895 von Schaewen A, Stitt M, Schmidt R, Sonnewald U and Willmitzer L(I990) Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants. EMBO J 9: 3033–3044

279 Wagner W, Wiemken A and Matile P (1986) Regulation of fructan metabolism in leaves of barley (Hordeum vulgare L. cv. Gerbel). Plant Physiol 81: 444–447 Wetmore RH and Rier JP (1963) Experimental induction of vascular tissues in callus of angiosperms. Am J Bot 50, 418– 430 Williams JHH and Farrar JF (1987) Endogenous control of photosynthesis in leaf blades of barley. Plant Physiol Biochem. 26: 503–509 Williams JHH and Farrar JF (1990) Control of barley root respiration. Physiol Plant 79: 259–266 Williams JHH, Farrar JF and Minchin PEH (1991) Carbon partitioning in split root systems of barley: Effect of osmotica. J Exp Bot 42: 453–460 Williams JHH, Winters AL and Farrar JF (1992) Sucrose: A novel plant growth regulator. In: Lambers H, van der Plas LHW (ed) Molecular, Biochemical and Physiological Aspects of Plant Respiration, pp 463–469. SPB, The Hague Williams M, Farrar J and Pollock CJ (1989) Cell specialisation within the parenchymatous bundle sheath of barley. Plant Cell Environ. 12: 909–918 Worrell AC, Bruneau J, Summerfelt K, Boersig M and Voelker TA (1991) Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning. Plant Cell 3: 1121–1130

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Chapter 11 Developmental Constraints on Photosynthesis: Effects of Light and Nutrition John Richard Evans Environmental Biology, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia, and Department of Plant Ecology and Evolutionary Biology, University of Utrecht, PO Box 800.84, 3508 TB, Utrecht, The Netherlands

Summary Introduction I. II. Effects of Light A. Protein Costs B. Photosynthetic Acclimation Response Curves C. D. Quantum Yield E. Leaf Anatomy F. Intraleaf Acclimation F. Photosynthetic Capacity per Unit Nitrogen III. Effects of Nutrition A. Relative Sensitivity B. Hill and Rubisco Activities C. Stomatal Conductance IV. Conclusions Acknowledgments References

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Summary Leaves have a considerable flexibility that enables them to perform effectively in a range of light environments or where the supply of mineral nutrients is limiting. This requires coordination of the many processes associated with photosynthesis within the leaf. Leaves respond to the irradiance during growth by changing the allocation of nitrogen between proteins. An analysis is presented which allows determination of the allocation that maximizes daily photosynthesis for a given amount of nitrogen. The leaf is faced with a trade-off between increasing light absorption or photosynthetic capacity. Absorption of light can be increased by investing more in pigment-protein complexes, which increases the quantum yield of photosynthesis. Conversely, photosynthetic capacity can be increased by allocating more nitrogen to soluble proteins. The ratio of thylakoid to soluble protein is thus highly responsive to growth irradiance for most species. Photosynthetic capacity per unit leaf area can also be increased by increasing nitrogen content per unit leaf area. This is invariably associated with increased leaf mass per unit leaf area due to elongation and/or more layers of palisade cells, such that nitrogen content per unit dry weight is independent of growth irradiance.

Neil R. Baker (ed): Photosynthesis and the Environment, pp. 281–304. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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Plants cope with limiting nutrients primarily by altering the production of leaves such that leaves that are formed generally have a minimum content of the element. With nitrogen deficiency, both light and dark reactions are equally affected; Hill and ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) activities are reduced in parallel. For several other nutrients, including P, Fe, Cu and Mn, deficiency reduces both Hill and Rubisco activities. With P and Fe deficiency, Hill activity is specifically affected and the excess Rubisco capacity is masked by lower activation of the enzyme to bring its activity into line with Hill activity. Stomatal conductance changes in concert with changes in assimilation capacity brought about by nutrient deficiency. Consequently, the amount of fixed per unit water transpired is remarkably constant and independent of the plants nutritional status.

I. Introduction Leaves from different species have evolved to exploit and cope with a remarkably wide range of environments. They have been able to do this by specialization of their anatomy, biochemistry and physiology. However, diverse species also share common responses in terms of photosynthetic properties to variation in environmental features. Light and nutrition are the two major features of the environment affecting the development of photosynthesis that are the subjects of this chapter. Many species possess the ability to alter leaf properties during their development in response to environmental features. The phenotypic flexibility has been termed plasticity and varies between species. Photosynthetic properties can be further modified after the leaf reaches full expansion. For example, a leaf that expanded initially in full sunlight may subsequently be shaded by growth of the same or neighboring plant and will acclimate to that new light environment. Nutrients are also frequently remobilized from leaves as they age. In this chapter I am therefore taking a broad definition of developmental constraint that includes both the period during leaf expansion which defines the leaf anatomy, as well as subsequent development prior to senescence. The analysis presented relies on the

Abbreviations: Chl/N – ratio of chlorophyll to leaf nitrogen fraction of leaf nitrogen in thylakoid and soluble nitrogen; H/Chl – Hill activity per unit chlorophyll H/N – Hill activity per unit nitrogen Hill activity – rate of whole chain electron transport measured under saturating irradiance; LMA – leaf mass per unit area; RuBP – ribulose 1,5-bisphosphate; Rubisco – ribulose 1,5-bisphosphate carboxylase/oxygenase; S– soluble protein nitrogen; T/Chl – thylakoid nitrogen per unit chlorophyll T/N – thylakoid nitrogen per unit leaf nitrogen v–soluble protein nitrogen per unit Hill activity

biochemistry of photosynthesis that is common to all plants. While it could be extended to include and crassulacean acid metabolism (CAM) plants, there is as yet insufficient evidence on which to base the argument. Photosynthesis relies on the coordination of the activities of many different proteins, and this appears to be closely regulated. It is the amount and activity of these proteins that determines the photosynthetic potential of the leaf. By dividing leaf protein into four groups, we can examine changes in the photosynthetic properties that occur in response to growth irradiance. The first division separates the light and dark reactions of photosynthesis, which correspond to thylakoid and soluble proteins. When leaves are ground in aqueous buffer and the extract centrifuged, the protein complexes in thylakoids (the membranes within the chloroplast) are generally insoluble and form a pellet in the bottom of the centrifuge tube while soluble proteins are recovered in the aqueous supernatant. Each of these two fractions are subdivided again. Within the thylakoids are pigment-protein complexes involved with light capture and the remaining proteins are involved with photosynthetic electron transport and photophosphorylation. For the soluble protein, there are proteins directly or indirectly involved with photosynthesis and those not involved with photosynthesis. Soluble proteins involved with photosynthesis include primarily Rubisco and the other enzymes in the Calvin and photorespiratory cycles as well as proteins indirectly involved in processes such as in membrane transport, carbohydrate metabolism, nitrate reduction, protein and nucleic acid synthesis. This wide spectrum is included because we are concerned with the ability of the leaf to maintain its photosynthetic capacity which requires protein turnover and export of photosynthate from the leaf. What strategy should a leaf employ to maximize

Chapter 11 Effects of Light and Nutrition on Photosynthesis daily photosynthesis for a given amount of nitrogen in response to different light environments? Photosynthesis responds to irradiance in a curvilinear manner, with the light-saturated rate depending on the photosynthetic capacity of the leaf. Photosynthetic capacity is determined by the amount of protein (which is equivalent to organic nitrogen) per unit leaf area. At low irradiances, photosynthetic rate depends on the proportion of incident light absorbed by the leaf, the absorptance, which is closely related to the chlorophyll content of the leaf. For a given amount of nitrogen, the leaf is thus faced with a trade-off. Photosynthetic capacity can be increased by having a greater proportion of protein in the electron transport chain and soluble proteins, but this is at the expense of pigment-proteins. That is, the increase in photosynthesis at high irradiance is offset by less photosynthesis at low irradiance. The ratio of photosynthetic capacity to pigment-proteins that maximizes daily photosynthesis depends on the light environment. Considerable effort has been spent in characterizing the differences between chloroplasts isolated from leaves that have been grown in full sunlight or from leaves grown deep within leaf canopies in shadelight. Shadelight has both a reduction in daily quanta as well as a change in the spectral quality. However, since it is generally easier to simply alter daily quanta, much work has compared leaves that have been grown under high or low irradiance. Fortunately, the photosynthetic characteristics of plants that come from sunlit versus shaded habitats are often similar to those of leaves grown under high versus low irradiance. The many changes that occur have been given the unifying term: photosynthetic acclimation. There are four main features that characterize acclimation to high irradiance (Boardman, 1977). Firstly, the rate of electron transport per unit chlorophyll increases. Secondly, the ratio of chlorophyll to soluble protein decreases. Thirdly, with few exceptions, the chlorophyll a/b ratio is lower. Fourthly, the leaf morphology is altered with leaves becoming thicker and heavier. Each of these features will be examined in turn, pointing out the common responses between species and highlighting the differences. The second feature that constrains the development of photosynthesis is mineral nutrition. The focus of this chapter is primarily around nitrogen because it is a major element in proteins and nucleic acids and frequently determines productivity and species

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composition of vegetation. It is not surprising that photosynthesis is strongly related to leaf nitrogen, given that photosynthesis depends on the majority of leaf proteins. In contrast to growth irradiance, nitrogen deficiency does not alter the relative abundance of proteins, but leads to a general reduction in leaf protein. The declines are coordinated such that the balance between the light and dark reactions is maintained. Stomatal conductance is also reduced in synchrony with photosynthetic capacity so that carbon gain per water transpired is relatively stable. Finally, deficiencies of several other nutrients are discussed.

II. Effects of Light

A. Protein Costs In the introduction, leaf proteins were partitioned into four groups. To enable us to trade protein between the groups, it is necessary to define two units of currency. Since nitrogen represents 16% of the mass of the protein, it is sensible to use nitrogen as one of the currency units. The other is chlorophyll, for two reasons. Firstly, light capture is closely related to chlorophyll content (Section II.D). Secondly, when chloroplast thylakoids are isolated from leaves for biochemical characterization, chlorophyll is the most readily quantified component. One final definition is necessary. In this chapter, Hill activity is defined as the rate of whole-chain electron transport measured under saturating irradiance and thus representing the maximum potential photosynthetic rate. We first need to factorize Hill activity per unit nitrogen, H/N, into several components that were noted as features of acclimation to growth irradiance:

where H/Chl is the Hill activity per unit chlorophyll, T/Chl is the nitrogen cost of thylakoids per unit chlorophyll, and T/N is the ratio of thylakoid to leaf nitrogen. The first term, Hill activity per unit chlorophyll was noted to increase during acclimation to high irradiance. Hill activity per unit chlorophyll increases in direct proportion to the increase in the content of cytochrome and coupling factor (ATP synthetase) complexes per unit chlorophyll (Evans, 1987a). Few species have been extensively studied

284 with respect to growth irradiance, but the general nature of this dependence is expected for several reasons. Both complexes are highly conserved across diverse genera. The cytochrome f content is greater in all cases where thylakoids isolated from leaves that had been grown under high irradiance are compared to thylakoids isolated from leaves grown under low irradiance (Table 1). Hill activity per unit cytochrome f is similar for Scenedesmus (Fleischhacker and Senger, 1978), Chlorella (Wilhelm and Wild, 1984), Atriplex (Björkman et al., 1972), Pisum (Evans, 1987a), Spinacea (Terashima and Evans, 1988) and Tradescantia (Chow et al., 1991). The second term, T/Chl, the nitrogen cost of the thylakoids has two components; the pigment-protein complexes and the proteins involved with electron transport and photophosphorylation. While acclimation alters the chlorophyll a/b ratio, due to changes in relative abundance of the pigment-protein complexes (Section II.D), it does not significantly alter the overall nitrogen cost of the pigment-protein complexes. Since the nitrogen cost is defined per unit

John Richard Evans chlorophyll, the cost of the pigment-proteins is approximately constant and independent of acclimation. What varies is the cost of the other thylakoid proteins. As stated above, Hill activity is closely related to the amount of cytochrome and coupling factor complexes. The nitrogen cost of thylakoids per unit chlorophyll thus has a constant term for pigment-protein complexes and a term that is proportional to Hill activity for the non-pigment proteins associated with electron transport and photophosphorylation. Overall, the nitrogen cost of the thylakoids, is linearly related to Hill activity (Evans 1989a):

The dashed line (Fig. 1A) represents the nitrogen cost of pigment-protein complexes and the area above represents the nitrogen cost of other thylakoid proteins, mainly coupling factor and cytochrome

Chapter 11 Effects of Light and Nutrition on Photosynthesis complexes. The dashed line is slightly curved due to the changing ratio between pigment-protein complexes (see Section II.D). Similar values of the nitrogen cost of thylakoids have been obtained for Spinacea (Park and Pon, 1963) and Pisum (Makino and Osmond, 1991), although it is difficult to compare them on the basis of their Hill activity. For Nicotiana tabacum, Lauerer et al. (1993) also found that the ratio of insoluble protein to chlorophyll increased with increasing growth irradiance, but again an absolute comparison cannot be made between protein and Kjeldahl nitrogen determinations. The third term in Eq. (1), T/N, the ratio of thylakoid nitrogen to leaf nitrogen can be calculated as follows:

where T/Chl can be calculated from Eq. (2) for a given Hill activity per unit chlorophyll and Chl/N is chlorophyll content divided by leaf nitrogen content. To realize an increased Hill activity per unit chlorophyll requires greater capacity of the dark reactions, that is, an increase in soluble proteins per unit chlorophyll. This was noted as the second feature associated with acclimation to high irradiance. As yet, only data for plants with high Hill activities per unit nitrogen are available. With these plants, the majority of soluble nitrogen appears to be involved with photosynthesis, so we initially ignore the second pool of non-photosynthetic nitrogen. It is not surprising to find that soluble protein nitrogen ( S mol N increases proportionally with Hill activity (mmol (Fig. 1B):

While a single line is drawn, it is expected that the slope of this relationship varies between species, unlike the relationship for thylakoid nitrogen. This is because many more proteins and organelles are involved. Considering just Rubisco, which can constitute 50% of soluble protein, it is known the specific activity varies between species (Evans, 1989a). The relationship in Eq. (4) is further complicated by having a dependence on leaf nitrogen content, although to a first approximation, this can be disregarded. Unfortunately, no soluble protein data are available in the literature yet to illustrate substantial variation between species, although we

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286 now have evidence of at least two-fold variation (M. Poorter and J. R. Evans, unpublished). The non-zero intercept for thylakoid nitrogen contrasts with the proportional response for soluble protein (Fig. 1A and 1B). Consequently, the ratio of thylakoid to soluble nitrogen should decrease with increasing Hill activity. Dramatic reductions are seen for Alocasia and Cucumis (Fig. 1C). It was necessary to calculate the soluble protein for Alocasia, based on the assumption that soluble protein equals total leaf nitrogen minus thylakoid nitrogen. The solid lines were calculated using Eqs. (2) and (4), which suggested that Alocasia has a much higher soluble protein cost than Cucumis. A second type of response is seen for Pisum and Spinacea in which the ratio of thylakoid to soluble protein remains constant and independent of Hill activity (Fig 1C, dashed lines). This response is uncommon and is considered in more detail below. Given the nitrogen costs of thylakoid and soluble components, it is possible to estimate the nitrogen cost required to achieve a given Hill activity. By combining Eqs. (2) and (4), we can calculate how the ratio of chlorophyll to leaf nitrogen should vary as a function of Hill activity as follows:

where f = (T+S)/N, the fraction of leaf nitrogen in thylakoid and soluble proteins. The ratio Chl/N is related to light capture by the leaf. Increases in H/Chl are at the expense of light capture because they cause a decline in Chl/N. Combining Eqs. (1) and (5), we can redefine the Hill activity per unit nitrogen simply as a function of Hill activity per unit chlorophyll:

To the extent that f is independent of changes in Hill activity per unit chlorophyll, H/Chl, Hill activity per unit nitrogen, H/N, should increase as H/Chl increases.

B. Photosynthetic Acclimation The biochemical detail necessary to establish the

John Richard Evans equations relating protein costs to Hill activity are available for only a few species. However, Hill activity per unit chlorophyll (H/Chl) and chlorophyll content per unit leaf nitrogen (Chl/N) have been examined for a wide range of species growing in different light environments. When leaves are grown under high irradiances, there is a considerable decrease in the ratio of chlorophyll to nitrogen with increasing irradiance (Fig. 2A). This occurs across a broad range of species and both herbaceous and tree species are presented to emphasize the general nature of this change. Importantly, it occurs regardless of the plants normal habitat, for both ‘sun’ and ‘shade’ plants (Boardman, 1977). This decline is linked to the increase in Hill activity per unit chlorophyll with acclimation to high irradiance (Fig. 2B). To increase Hill activity requires more thylakoid proteins as well as more soluble proteins to maintain the balance between the light and dark reactions. Therefore, greater Hill activity per unit chlorophyll is equivalent to greater protein to chlorophyll ratios and therefore lower chlorophyll to nitrogen ratios (Eq. 5). Once again, large changes are seen for both herbaceous and tree species, regardless of their origin. There has only been one report of a plant species where Hill activity per unit chlorophyll did not change with growth irradiance (Solanum aviculare; Turnbull, 1991). Since a related species, Solanum dulcamara, does have great flexibility in Hill activity per unit chlorophyll when grown in different irradiances (Ferrar and Osmond 1986), this anomaly needs to be confirmed. Both the ratio of chlorophyll to nitrogen and Hill activity per unit chlorophyll show dramatic changes with respect to growth irradiance. However, their product, Hill activity per unit nitrogen is generally quite stable (Fig. 2C). There is a tendency towards slightly lower values when grown under low irradiance, as expected from Eq. (6). Note that while Hill activity per unit nitrogen is generally independent of growth irradiance, there is considerable variation in the absolute value between species. This is due to variation in the soluble protein cost per unit Hill activity (Eq. 4) and variation in the proportion of leaf nitrogen accounted for in soluble and thylakoid proteins (Eq. 6). This aspect is reexamined in Section II.G. For a few species, e.g. Cecropia (StraussDebenedetti and Bazzaz, 1991), Pisum and Spinacea (Evans, 1993a), there is a noticeable decline in Hill activity per unit nitrogen. Cecropia is an early successional tree that is intolerant to shade, yet it clearly acclimates to low irradiance through

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decreasing Hill activity per unit chlorophyll. The reason that these three species show a decline in Hill activity per unit nitrogen with declining growth irradiance is that they each appear to have a fixed proportion of nitrogen partitioned into thylakoids (Fig. 1C). It is possible to explain these contrasting responses by examining the flexibility of partitioning nitrogen between thylakoid and soluble protein which was shown in Fig. 1C. In Fig. 3, Hill activity per unit chlorophyll is plotted against total leaf nitrogen per unit chlorophyll so that lines radiating from the origin represent Hill activity per unit nitrogen. Two responses are modeled for each species. The solid lines represent what would be expected from the relationships presented in Section II.A with the proportion of leaf nitrogen involved with photosynthesis being independent of Hill activity per unit chlorophyll. Acclimation to low irradiance reduces the Hill activity which requires less soluble proteins, so more protein could be allocated to the thylakoids to increase the amount of chlorophyll. The result is a higher ratio of thylakoid to soluble protein at low growth irradiances. Measurements of Alocasia leaves grown at different irradiances closely follow the predicted line. This type of response is typical of the majority of species. The dashed lines represent the uncommon case where thylakoid nitrogen is held as a fixed proportion of leaf nitrogen. Acclimation can still occur, so that Hill activity per unit chlorophyll varies considerably. However, now increased investment in pigment-proteins can only occur in exchange for protein from the electron transport chain and coupling factor. The total nitrogen cost per unit chlorophyll can therefore decline only slightly. This response has been observed only for Pisum and Spinacea. Cecropia was noted for having a declining Hill activity per unit N (Fig. 2C) which suggests that this species is also unable to alter the ratio of thylakoid to total leaf nitrogen.

C.

Response Curves

In the preceding section, the coordination between the light and dark reactions was presented in terms of their protein costs. Attention will now focus on the relative capacities for RuBP regeneration and consumption. Using the Farquhar, von Caemmerer and Berry (1980) model of photosynthesis, it is possible to get in vivo estimates of both Rubisco and Hill activities when rate of assimilation is determined as a function of intercellular partial

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pressure (see Chapter 8). Under high irradiance and intercellular partial pressures near the compensation point, the rate of assimilation is determined by Rubisco activity. As the intercellular partial pressure increases, the limitation will eventually pass over to the rate of regeneration of RuBP which is set by the rate of electron transport. In this chapter, Hill activity has been defined as the rate of whole-chain electron transport measured under saturating irradiance and provides an estimate of the maximum potential photosynthetic rate.

John Richard Evans When response curves for leaves acclimated to different irradiances are compared, two factors stand out (Fig. 4). Firstly, leaves acclimated to high irradiance have higher rates of assimilation per unit leaf area. This reflects higher leaf nitrogen contents. Secondly, the ratio of Hill to Rubisco activities is generally unaffected (Fig. 4A). Hill activity determines the rate at high while Rubisco activity determines the slope of the response at low consequently the ratio of their activities is proportional to the ratio of the rates at, for example, 60 and 20 Pa. This ratio did not vary significantly with growth irradiance for Alocasia and Colocasia (Sims and Pearcy, 1989), Toona, Argyrodendron and Flindersia (Thompson et al., 1988,1992). It is this coordination between the two activities that was exploited to link soluble protein costs to Hill activity in Eq. (4). The coordination is also clear when nitrogen is the source of variation (Section III.B), but here the discussion is limited to the effects of growth irradiance. The dramatic changes in the ratio of thylakoid to soluble protein seen in Fig. 1C result in response curves that can be normalized to superimpose simply by multiplying the rates for one leaf by a scaling factor (Fig. 4A). Doubling the rates for the Alocasia leaf acclimated to low irradiance results in a curve that closely matches that observed for the leaf acclimated to high irradiance. An alternative pattern of acclimation, which is uncommon, is when the ratio of thylakoid to leaf nitrogen is independent of growth irradiance. The leaf is unable to change the proportion of nitrogen allocated to the thylakoids. This leads to a reduction in Hill activity relative to Rubisco for leaves acclimated to low irradiance, as illustrated for Pisum (Fig. 4B), see also data for Piper (Walters and Field, 1987). The patterns that emerged by considering the partitioning of nitrogen between thylakoid and soluble proteins with respect to Hill activity (Fig. 3C), are thus evident in the photosynthetic response curves. By maintaining a fixed proportion of nitrogen in the thylakoids, leaves acclimated to low irradiance have more soluble protein per unit Hill activity than those leaves which increase the ratio of thylakoid to soluble protein when grown at low irradiance. That is seen by the small decline in the initial slope of the response curve relative to the large decline in rate at high intercellular partial pressures which reflects the Hill activity. Acclimation reduced the Hill activity per unit chlorophyll by reducing the amount of cytochrome and coupling factor so

Chapter 11 Effects of Light and Nutrition on Photosynthesis

that for a given amount of thylakoid nitrogen, more was invested in pigment-proteins and less in electron transport/ photophosphorylation capacity. Consequently, the rate of assimilation at high declined while there was little change at low

D. Quantum Yield Photosynthetic acclimation involves altering the balance between the various pigment-protein complexes as well as between the pigment-protein complexes and other thylakoid protein complexes. There has been much speculation about the purpose of these multiple changes. Björkman (1981) argued that because shadelight at the base of a leaf canopy had a very high proportion of far-red light which preferentially excited PS I, the ratio of PS II:PS I increased to redress the imbalance in energy distribution between the two photosystems. Experimental support for the increase in the ratio of PS II:PS I reaction centers in leaves grown under far-red enriched light has since been obtained with Pisum (Chow et al., 1990b,c), Silene (McKiernan and Baker, 1991) and Hordeum (Kim et al., 1993). When Pisum was grown under a light that preferentially excited PS I, the ratio of PS II:PS I increased and the quantum yields (mol evolved per mol quanta absorbed) were higher in the far-red enriched growth light than when measured in light that preferentially excited PS II (Chow et al., 1990c). However, there are also examples where the ratio

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of PS II:PS I is lower in shade-grown shade species (Chow et al., 1990a) and other measurements of quantum yield confuse the issue. If quantum yields are measured in white light, then they are the same for sun and shade leaves and independent of the ratio of PS II:PS I (Björkman, 1968; Björkman et al., 1972; Öquist et al., 1982; Evans, 1987b; McKiernan and Baker, 1991). Quantum yields are lowered by any imbalance in the distribution of light between the two photosystems. Therefore, the fact that quantum yields are independent of PS II:PS I ratio suggests that the distribution of light between the two photosystems is not altered by the PS II:PS I ratio (see Chapter 3). It is only evident when a narrow band of wavelengths is used to determine quantum yield. While Chow et al. (1990c) only made measurements with light that preferentially excited PS II or PS I, the quantum yields averaged for the two light treatments (as an approximation to white light) are identical for the two leaf types, regardless of their PS II:PS I ratio. When plants are grown in an environment where irradiance changes without change to the spectral quality, the ratio of PS II:PS I still changes. Acclimation to low irradiance involves a decline in the ratio of PS II:PS I (Leong and Anderson, 1984a,b; Chow and Hope, 1987; Evans, 1987a). Anderson (1982) argued that increasing the content of the light-harvesting chlorophyll a/b proteins relative to the reaction centre complexes would improve the absorption of blue-green wavelengths by PS II in shadelight. Acclimation to low irradiances

290 is associated with lower chlorophyll a/b ratios. Since chlorophyll b is only present in the light-harvesting complexes and not the reaction centre complexes (Anderson, 1980, 1986), the lower chlorophyll a/b ratio reflects an increase in the proportion of chlorophyll in the light-harvesting complexes and a decrease in PS II complexes. PS I content per unit chlorophyll is generally insensitive to growth irradiance. Calculations have been made using the absorption spectra of the pigment-protein complexes with sunlight or shadelight (Evans, 1986a, 1988; Evans and Seemann, 1989). The calculations revealed that altering the chlorophyll a/b ratio and thereby changing the ratio of PS II:PS I, did not change the distribution of light between the two photosystems, which is principally determined by the proportion of chlorophyll assigned to each photosystem. The fraction of light that was absorbed was also not significantly changed by the redistribution of chlorophyll between the pigment-protein complexes. The quantum yield data with white light and the calculations with absorption spectra suggest that it is not simply an excitation energy imbalance that drives the rearrangement between pigment-protein complexes. An alternative explanation can be put forward on the basis of the nitrogen cost of complexing the pigments. When light is limiting in the environment, it is of paramount importance to minimize the cost of complexing pigments and maximize the absorption of light. The absorptance of a leaf is closely related to chlorophyll content (Fig. 5, see also Kirchhoff et al., 1989), in the absence of complications due to special surface structures such as hairs and wax (Ehleringer, 1981). The amount of light that can be gained by the leaf per unit nitrogen can therefore be increased by increased investment in pigment-protein complexes made possible by a decreased investment in other proteins. The ratios of pigment to protein weight vary between the different complexes (Evans, 1987a; Evans and Seemann, 1989). The light-harvesting complexes have three times as much pigment per protein weight as the PS II reaction centre complex. Thus for each PS II unit replaced by light-harvesting complexes, three times more chlorophyll can be bound. For Pisum and Spinacea leaves acclimated to low irradiance, the increased light-harvesting complex and decreased PS II content increase the chlorophyll content by 5 to 8% (Fig. 1A, dashed line). Under low irradiances, it is unnecessary to

John Richard Evans

have a large electron transport capacity, so further increases in chlorophyll are possible using protein released from the electron transport chain components and coupling factor. Leaves acclimated to low irradiance have about 40% less cytochrome complex and coupling factor. Including these proteins in the reallocation to pigment-protein complexes allows leaves acclimated to low irradiance to increase chlorophyll content by 20 to 30%. For most plants, soluble proteins are also reduced so that overall, the chlorophyll to nitrogen ratio of the leaf acclimated to low irradiance is double that for a leaf acclimated to high irradiance (Fig. 2A). The increase in chlorophyll content per unit nitrogen achieved by lowering the chlorophyll a/b ratio is small in comparison with the overall changes in the chlorophyll to nitrogen ratio in the leaf. This suggests that the nitrogen cost of complexing pigments cannot satisfactorily explain why the chlorophyll a/b ratio is lower in leaves acclimated to low irradiance. While chlorophyll a/b ratio changes in response to growth irradiance in most species, there are exceptions. Tradescantia albiflora (Chow et al., 1991) and Perilla (Ida et al., 1992) acclimate to high growth irradiance with a higher Hill activity per unit chlorophyll but with no change in chlorophyll a/b

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ratio. Perhaps it is not the change in chlorophyll a/b ratio but rather the change in PS II content that is important. To change PS II content requires a change in light-harvesting complex too, because chlorophyll is being exchanged between these two complexes as PS I shows little change. The reaction centre of PS II contains the D1 polypeptide that is the most rapidly turned over protein in thylakoid membranes (see Chapter 4). The turnover rate depends on the irradiance and not on the amount of PS II per unit chlorophyll for non-photoinhibitory irradiances (Aro et al., 1993). Having more PS II is therefore associated with greater maintenance turnover of the D1 polypeptide. One can calculate the proportion of incident quanta that the turnover of D1 consumes (at 50 as 0.63 or 0.89% for leaves acclimated to low or high irradiance, assuming that the D1 polypeptide has 353 amino acid residues each requiring 16 ATP for the peptide synthesis (De Visser et al., 1992) and that 3 ATP are synthesized per 8 quanta. While the turnover rate increases at higher irradiances, the proportion of light required for the turnover is less. It is not yet clear whether this small reduction in respiratory costs would be a selective advantage in low light environments. Most leaves show the same high quantum yield of photosynthesis , regardless of species or growth irradiance (Björkman and Demmig, 1987; Evans, 1987b; Long et al., 1993). Exceptions to this are leaves that are suddenly exposed to much higher irradiances than they normally receive or are exposed to the combination of high irradiance during low temperatures (Öquist and Ögren, 1985; Aoki, 1986; Ball et al., 1991; Farage and Long, 1991). The quantum yields of leaves acclimated to full sunlight may also be temporarily lowered following exposure to full sunlight (Ögren, 1988; Demmig-Adams et al., 1989; Ögren and Evans, 1992), but this is associated with photoprotection (see Chapters 1–3) and full recovery generally occurs overnight. There are few examples of leaves in their natural environment that are chronically photoinhibited, that is, where quantum yields at dawn are lower than the normal maximal value for leaves (Anderson and Osmond, 1987).

E. Leaf Anatomy Light has long been known to influence anatomy during leaf development. Leaves that develop under full sunlight have greater mass per unit leaf area (LMA) and are thicker than leaves that develop in

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shadier environments. The increase in LMA that occurs with increasing irradiance is found both for herbaceous and tree species (Fig. 6A). The proportional increase in mass is generally similar, regardless of the type of leaf. For example, compare the sclerophyllous Flindersia leaf with the thin Urtica leaf The large differences in absolute values reflect structural characteristics of the leaves such as more massive vascular bundles with sclerenchyma or bundle sheath extensions and thicker epidermal and cuticle layers. While LMA increases with growth irradiance for a given species, it is not true to say that plants that occupy shady habitats necessarily have thin leaves, or vice versa (Boardman, 1977). There are many other factors that are associated with LMA, such as growth rate, leaf longevity, herbivory and water availability. The increase in leaf thickness in monocots is due to elongation of mesophyll cells (Ludlow and Wilson, 1971). In dicots, there is also a proliferation of palisade tissue which may develop more cell layers (Haberlandt, 1914; Hanson, 1917; Chabot and Chabot, 1977;Lichtentahler et al., 1981). Associated with the increase in mesophyll tissue are larger vascular bundles and increases in the thickness of epidermal layers which contribute to the greater LMA. Nitrogen composition of leaf material is insensitive to growth irradiance, possibly as a result of these associated changes (Fig. 6B). Consequently, the nitrogen content per unit leaf area increases with increasing growth irradiance (Fig. 6C), similar to the increase in LMA. It is instructive to explore a functional explanation for this general anatomical trend. Laisk et al. (1970) and Nobel and coworkers (Nobel et al., 1975; Longstreth et al., 1981; Nobel and Walker, 1985) have argued that diffusion from the intercellular airspaces to the sites of carboxylation may limit the rate of assimilation. The chloroplast surface needs to be exposed to intercellular airspace for to be able to diffuse easily to Rubisco, as was so perceptively noticed by Haberlandt (1914). To achieve greater rates of assimilation in an environment with greater irradiance would therefore require a greater surface area exposed to intercellular airspace per unit leaf area. There must also be increases in the amounts of photosynthetic components that determine the rate of assimilation, such as Rubisco (Björkman, 1981). Suppose chloroplast surface area was proportional to Rubisco content, then any increase in Rubisco

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John Richard Evans content would require an increase in chloroplast surface area. For several species (e.g. Triticum aestivum, Nicotiana tabacum, Spinacea oleracea), chloroplasts cover a high proportion of the exposed mesophyll cell walls. So, as growth irradiance increases, exposed surface of mesophyll cells per unit leaf area should increase. However, this is not always the case. For example in shade species only 30% of exposed surface was covered by chloroplasts (Araus et al., 1986; Chow et al., 1988). The increase in mesophyll cell surface area can be achieved by cell elongation, lobing and increasing the number of cell layers. Increased photosynthetic rate can also be associated with proportional increases in transpiration. The hydraulic conductivity of the leaf would therefore need to be increased as well, by increasing the size and/or frequency of vascular bundles. Because the amount of Rubisco and other photosynthetic components remain approximately constant per unit cell wall area, leaf nitrogen content per unit leaf area would increase while on a dry weight basis there would be little change. While this appears to be an appealing argument, there are examples where LMA continues to increase as growth irradiance increases without concomitant increase in photosynthetic capacity per unit leaf area (e.g. Fragaria, Chabot and Chabot, 1977; Jurik et al., 1979). Often under very high irradiance regimes considerable accumulation of starch occurs and the LMA needs to be corrected to ensure that starch is not included. If the differences in LMA remained without changes in photosynthetic capacity, other explanations for the change in leaf structure must also be sought. There are other considerations relating to the high irradiance phenotype. Firstly, increasing the total surface area of chloroplasts requires that there be more chloroplasts per unit leaf area. Light could then be distributed more evenly between chloroplasts. Increasing the number of cell layers also enables greater specialization of the chloroplasts which improves the ratio of photosynthesis to light absorbed at intermediate irradiances (Section II.F). Secondly, the increased rate of assimilation requires an increased conductance to through the leaf surface. This is achieved by increased stomatal aperture and/or frequency. As well, leaves may develop stomata on the upper (adaxial) surface, becoming amphistomatous and thereby increasing supply to the most actively photosynthesizing regions of the leaf (Mott et al., 1982; Mott and Michaelson, 1991). Thirdly, by concentrating

Chapter 11 Effects of Light and Nutrition on Photosynthesis photosynthesis into a smaller projected area, the leaf can reduce the amount of heating from the absorbed irradiance. For example, halving the projected leaf area, but doubling the photosynthetic rate per unit area would increase the amount of cooling due to the latent heat of evaporation and increase the boundary layer conductance allowing a closer coupling of the leaf and air temperatures. These two factors would combine to reduce the amount of water transpired, allowing the plant to invest less in roots and vascular tissue. Considering the opposite situation where leaves are in a low irradiance environment, raises another issue. Increasing the surface area of mesophyll cells per unit leaf area reduces the amount of light absorbed per unit mesophyll surface. Thus the carbon cost of absorbing light is greater, which in turn requires a longer time to recoup the investment in the leaf. When light is limiting, it is better to maximize the light absorbed per unit leaf dry weight by expanding a thin leaf and minimize mesophyll surface area per unit leaf area.

F. Intraleaf Acclimation The features of acclimation observed at the leaf level arise through biochemical changes in the chloroplasts. These result in ultrastructural changes in the chloroplast such as the number of thylakoids per granum and the amount of thylakoids relative to stroma (Goodchild et al., 1972; Lichtentahler et al., 1982; Aro et al., 1986). There exist gradients in irradiance through the leaf which have been demonstrated by paradermal (parallel to the leaf epidermis) sectioning (Terashima and Saeki, 1983, 1985) and by the insertion of optical fibers through leaves (Vogelmann et al., 1989;Cui et al., 1991). The gradients in irradiance cause acclimation of the chloroplasts which results in a gradient in photosynthetic capacity down through the leaf. Sun-type chloroplasts are found near the surface which receives the light and shade-type chloroplasts near the shaded surface. Detailed biochemical analysis of thylakoids isolated from different paradermal layers has revealed changes in chlorophyll a/b ratio, cytochrome coupling factor and Rubisco content and electron transport rate per unit chlorophyll (Terashima and Inoue, 1984, 1985a,b). The specialization of the chloroplasts can also be seen from their ultrastructure with shade-type chloroplasts near the lower surface having more thylakoids per granum and more

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thylakoids relative to stroma. Chloroplasts were similar within a given cell in Spinacea and Glycine suggesting that the cell is the minimum unit of specialization (Terashima et al., 1986). This is not surprising as the composition of the thylakoids is heavily reliant on nuclear genes, the products of which would presumably gain equal access into all chloroplasts within a cell. The chloroplasts can also reorient within a cell in response to light (Haupt, 1982; Brugnoli and Björkman, 1992), although it is not known whether individual chloroplasts could completely move around the cell in higher plants. The more cell layers present in the leaf, the greater the specialization that can occur in the chloroplasts. The consequence of this specialization is that the capacity for photosynthesis of each chloroplast can more closely match the light absorbed by it. This results in a more abruptly saturating light response curve which increases potential daily photosynthesis. If the curves are measured using unilateral illumination to the leaf surface that is generally shaded, a lower photosynthetic rate at intermediate and high irradiances is found compared to when the curve is measured with the leaf receiving light to which it is acclimated (Moss, 1964; Oya and Laisk, 1976; De Lucia et al., 1991; Evans et al., 1993). The acclimation profile through the leaf is fixed neither by leaf anatomy nor time as inversion of the leaf leads to reacclimation which is synchronized to the light absorption profile through the leaf (Terashima, 1986; Terashima et al. 1986; Ögren and Evans, 1993). Leaves that have a high photosynthetic capacity have a greater ability to develop gradients in chloroplast properties through their leaves because they generally have more cell layers. There are probably limits to the dynamic range in H/Chl for a given species. Chloroplasts near the upper surface of a shaded leaf may already be close to the limit of a shade-type chloroplast. For example, the gradient in chloroplast ultrastructural features was much less evident in Spinacea leaves grown under 15% sunlight compared to leaves grown in full sunlight (Terashima and Evans, 1988). Acclimation that occurs between chloroplasts down through the leaf is equivalent to what happens between leaves within a canopy. As canopies grow, leaves that originally developed in full sunlight gradually find themselves in a progressively more shaded environment. Two things happen that ameliorate the disadvantage to the plant caused by this shading. Firstly, chloroplasts in the leaves acclimate by

294 becoming more shade-like, the plant being unable to alter the leaf anatomy. This can yield up to a 20% increase in potential daily photosynthesis of the leaf compared to a leaf remaining in the sun-phenotype (Evans, 1993a,b). Secondly, nitrogen is remobilized from leaves as they are shaded and relocated to leaves that intercept more light. This increases the canopy photosynthesis for a given amount of nitrogen, with the extent of the benefit increasing as leaf area index and/or canopy nitrogen content per unit ground area increases (Hirose and Werger, 1987). Nitrogen retranslocation between leaves has a greater impact on daily canopy photosynthesis than nitrogen reallocation within shaded leaves because it is leaves that intercept most light that contribute most to daily canopy photosynthesis.

F. Photosynthetic Capacity per Unit Nitrogen Hill activity per unit nitrogen is relatively insensitive to growth irradiance for most species because of the flexibility the leaf displays allocating nitrogen between various proteins. It was pointed out, however, that the absolute value differed between species. This variation is due to variability in Rubisco activity per unit soluble protein. Two of the most likely explanations for this are, firstly, that the specific activity of Rubisco (maximum velocity per gram of pure enzyme) differs between species and secondly, that Rubisco constitutes different proportions of the soluble protein in different species. The fraction of leaf nitrogen not involved with photosynthesis may also vary between species, although scant information on this exists. The nitrogen intercept in the relationship between nitrogen and photosynthesis has been suggested to represent this pool (Field and Mooney, 1986) and it is affected by growth conditions (e.g. Lysimachia, Pons et al., 1989). Considerable variation can be seen in the relationships between Hill activity and leaf nitrogen, both expressed per unit chlorophyll, for a broad range of species acclimated to different irradiances (Fig. 7A). The use of chlorophyll as the basis of expression separates out the effects of growth irradiance from those associated with nitrogen nutrition. Variation in the latter will not alter the positions of the data because both Hill activity and nitrogen per unit chlorophyll are generally independent of leaf nitrogen content. The species have been grouped together according to their Hill activity per unit nitrogen. It is difficult to generalize Hill

John Richard Evans activity per unit nitrogen with respect to either the normal habitat of the plant or the form of the leaf or plant. For example, Ficus, Cecropia and Eucalyptus trees all have very high values of Hill activity per unit nitrogen. By expressing the same data on a leaf area basis, the variation in Hill activity per unit nitrogen remains (Fig. 7B). On this basis, it is not possible to distinguish the variation in nitrogen content per unit leaf area that is due here to growth irradiance from that which could have been generated by nitrogen deficiency. There is a broad overlap between the three groups of species in terms of the nitrogen content and variation due to growth irradiance. The six-fold variation in Hill activity per unit nitrogen emphasizes that a considerable fraction of leaf nitrogen in many species is devoted to non-photosynthetic functions. This pool of nitrogen has yet to be characterized, but it seems to scale in proportion to the Hill activity of the leaf. Instead of looking at the photosynthetic capacity per unit nitrogen, which represents the light and saturated potential photosynthesis, the photosynthetic rate per unit nitrogen measured under the growth conditions may also be important. Growth irradiance strongly affects the nitrogen content per unit leaf area (Fig. 6C) such that the photosynthetic rate per unit leaf area increases substantially when leaves are grown under higher irradiance as seen with the classic experiments with Atriplex patula (Björkman et al., 1972) and Panicum maximum (Ludlow and Wilson, 1971). However leaves seldom, if ever, have the nitrogen content that maximizes daily photosynthesis when nitrogen is being distributed between leaves in a canopy. If leaf nitrogen contents are too high when growing under low irradiance, only a fraction of the photosynthetic capacity is utilized (Pons et al., 1993). It seems unrealistic to expect a plant confronted with an unrelenting constant irradiance to fully exploit it, especially since leaves in deep shade may receive a substantial fraction of light in the form of sunflecks. Leaves exposed to full sunlight also spend a large amount of time at low irradiances. To predict the leaf nitrogen content and partitioning within the leaf that maximizes daily photosynthesis, it is necessary to integrate on a daily basis to account for the variation in irradiance. Survival in deep shade probably depends on a suite of plant attributes, such as the ability to stay in positive carbon balance by having low rates of respiration and not only on the extent of photosynthetic acclimation.

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III. Effects of Nutrition A. Relative Sensitivity When plants are in a nutrient deficient environment, their growth rate is reduced. Growth is by far the most sensitive indicator of a deficiency. New leaves are generally produced, or can expand, only if a certain minimum nutrient content is achieved. Consequently, when experimental attempts are made to manipulate the nutrient content of a leaf to perturb photosynthesis, the plant opposes the treatment very effectively. Often the nutrient content of the plant material is quite variable which leads to difficulty in obtaining reproducible results. In contrast to growth irradiance, deficiency of N, P or K does not lead to any change in leaf anatomy (Longstreth and Nobel, 1980), although starch content of the leaf often increases, which is reflected in greater leaf mass per unit leaf area. The relative change in three plant attributes are shown as a function of the nitrate concentration in the nutrient solution that flooded the soil daily, using

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wheat as an example (Fig. 8). Leaf area per plant was reduced to 5% of that of the control plant supplied with 12 mM nitrate, mainly through the suppression of tillering. However, the size of each leaf was also smaller, the flag leaf having only 40% of the area of the flag leaf from plants grown with 12 mM nitrate. Ultimately, the nitrogen content per unit leaf area was reduced to 60% of that found in leaves supplied with 12 mM nitrate. These attempts by the plant to maintain the nutrient content above a certain minimum are frequently observed (e.g. Vicia faba, Andreeva et al. 1972; Helianthus, Radin and Boyer, 1981; Oryza, Cook and Evans, 1983; Beta vulgaris, Abadia et al., 1987).

B. Hill and Rubisco Activities Nitrogen is an essential macronutrient and important resource in the environment and the most heavily used fertilizer for increasing plant production. Plants have devised methods of reducing nitrogen losses by increasing leaf longevity and retrieving nitrogen from leaves before they are shed (Aerts, 1990; Pugnaire

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and Chapin, 1993) and translocating much of the plant nitrogen into the seed for the subsequent generation. Symbiotic relationships have also evolved that allow atmospheric nitrogen to be reduced and made available to the plant. Photosynthesis is closely related to leaf nitrogen content because its component processes require proteins that account for the majority of the leaf nitrogen. Both light and dark reactions of photosynthesis are strongly affected by nitrogen deficiency, whether induced by restricting supply during leaf development or during senescence when nitrogen is retranslocated out of the leaf. Hill activity varies in direct proportion to leaf nitrogen content (Fig. 9). It does not matter whether Hill activity is calculated from gas exchange measurements in vivo or assayed using thylakoid preparations. Whereas acclimation to different irradiance involves altering the partitioning of nitrogen between protein fractions, using the same approach outlined in Section II.A, it can be shown that the ratio of chlorophyll to nitrogen is nearly insensitive to leaf nitrogen content (Pons et al., 1993). For a given light environment, chlorophyll content varies in proportion to leaf nitrogen content for many species (Evans, 1989a) and H/Chl is insensitive to leaf nitrogen content in Spinacea (Evans and Terashima, 1987) and Cucumis (Evans, 1989b). Rubisco activity also varies in proportion to leaf

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nitrogen content, as shown by examples that have been obtained either from gas exchange measurements or from in vitro assay of crude extracts (Fig. 10). The comparisons set up in Figs. 9 and 10 were to illustrate that Hill and Rubisco activities were proportional to leaf nitrogen whether based on gas exchange or biochemical assay for a range of species. However, it should also be noted that the slope of the lines differs between species. While this could simply reflect that different methods are being used, differences between species remain when the slopes are all calculated from gas exchange (Table 2). For Rubisco, care must be taken to use the same kinetic constants and to bear in mind that it is only an approximation because the partial pressure at the sites of carboxylation is less than intercellular partial pressure (Caemmerer and Evans, 1991). If Hill activity per unit nitrogen is low for a given species, then the corresponding Rubisco activity per unit nitrogen is also low because the ratio of Hill to

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Rubisco activities is highly conserved between species. For example, Wullschleger (1993) surveyed response curves measured on 109 species from many laboratories and found an overall close coordination between Hill and Rubisco activities,

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irrespective of plant type. However, the extreme range in the ratio of the maximum rate of electron transport to the maximal velocity of RuBP carboxylation he calculated was from 1.11 to 3.94. This reflects poor response curves which yielded spurious values because if a response curve is limited purely by Hill activity or Rubisco activity, the apparent ratio can only vary between 1.15 and about 2.9, respectively. This is because if a response were limited by Hill activity over the entire response curve, the initial slope set by the Hill activity would yield a Rubisco activity that would be equivalent to a ratio of 1.15. Conversely, if always limited by Rubisco, the Hill activity limitation is not seen over the range of intercellular partial pressures that can be routinely measured and the Hill activity calculated at the highest point on the Rubisco curve would yield a ratio of about 2.9. Values near this or beyond suggest that the Hill activity or Rubisco activity has not been properly determined. As well as Hill and Rubisco activities being strongly related to leaf nitrogen content, this is also true for PS I, PS II and the other pigment-protein complexes, cytochrome and coupling factor (Evans and Terashima, 1987), other Calvin cycle enzymes such as phosphoribulokinase, glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase (Makino et al., 1983) and carbonic anhydrase (Makino et al., 1992). The only exception seems to be that mitochondrial proteins decline as a proportion of leaf nitrogen in leaves with very high nitrogen contents (Makino and Osmond, 1991) which may make these leaves more likely to encounter a limitation by phosphate regeneration (Sage et al., 1990). In summary, as leaf nitrogen declines, there is a coordinated decline in nearly all leaf proteins.

298 Hill activity correlates closely with Rubisco activity, whether nitrogen, leaf age or species is the source of the variation. The conservative nature of the relationship between Hill and Rubisco activities has been observed for several species when nitrogen nutrition has been manipulated (Phaseolus, Caemmerer and Farquhar, 1981; Triticum, Evans, 1983; Spinacea, Evans and Terashima, 1987,1988; Chenopodium, Sage et al., 1990; Oryza, Makino et al., 1992). Growth irradiance also does not alter the relationship between Hill and Rubisco activities for most species (Section II.D). The coordination between Hill and Rubisco activities is also not specific to nitrogen deficiency. Calculating Hill activity and mesophyll conductance (slope of the response curve near the compensation point, which is related to Rubisco activity) from response curves reveals that both covary whether nitrogen, Cu or Mn were the source of the variation (Fig. 11). The data were presented as mesophyll conductance rather than Rubisco activity because calculating the Rubisco activity requires assumptions about kinetic properties of Rubisco and the draw-down in partial pressure from substomatal cavities to the sites of carboxylation (Caemmerer and Evans, 1991; Caemmerer et al., 1994; Evans et al., 1994). The coordination between Hill activity and mesophyll conductance is also seen with P (Brooks, 1986) and Fe (Taylor and Terry, 1984) deficiencies. In contrast to nitrogen deficiency, which reduces the amount of thylakoid and Rubisco proteins so that in vivo activities truly reflect the capacities of the light and dark reactions, other deficiencies affect specific proteins. To maintain the coordination between in vivo activities of the light and dark reactions requires down-regulation in the leaf. For example, leaves with Fe deficiency have less chlorophyll and thylakoid protein but the same amount of Rubisco as control leaves. Hill activity per unit chlorophyll, chlorophyll a/b ratio, cytochrome and P700 contents per unit chlorophyll are unchanged (Spiller and Terry, 1980), while Rubisco per unit chlorophyll is much greater. The constant ratio of mesophyll conductance to Hill activity, irrespective of Fe status therefore requires deactivation of Rubisco (Taylor and Terry, 1984). With P deficiency, Hill activity in vitro is unaffected but is reduced in vivo, presumably because ATP regeneration is limited by the availability of inorganic phosphorus (Brooks,

John Richard Evans

1986; Abadia et al., 1987). In turn, lower Hill activity is matched by a lower mesophyll conductance, due to a combination of a slightly lower Rubisco content and a lower activation state (Brooks, 1986). In addition, to avoid photoinhibitory damage because of the lower photosynthetic capacity, P deficient soybean leaves change from solar-tracking to avoidance (paraheliotropic) movements (Lauer et al., 1989). Mn deficiency should affect the PS II reaction centre and leaves do have a lower chlorophyll a/b ratio consistent with the lack of PS II pigmentprotein complexes being replaced by light-harvesting complexes. Hill activity per unit chlorophyll is reduced by Mn deficiency (Kriedemann and Anderson, 1988) and Rubisco activation state is probably lowered to match this (Fig. 11) via the mechanism that operates in P deficient leaves. In summary, where a nutrient deficiency affects a specific protein, the in vivo balance between Hill activity, RuBP regeneration and Rubisco activity is maintained by down regulation of various reactions.

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C. Stomatal Conductance

IV. Conclusions

Nutrient deficiency not only causes specific lesions or more widespread protein loss, either of which can reduce photosynthetic capacity, but it also affects stomatal conductance. The coordination observed between Hill and Rubisco activities also extends to stomatal conductance. When photosynthetic capacity is reduced by nitrogen deficiency, there is a similar reduction in stomatal conductance (Oryza sativa, Yoshida and Coronel, 1976; Zea mays, Wong et al., 1979; Goudriaan and van Keulen, 1979; Gossypium hirsutum, Wong et al., 1985). The correlation between photosynthetic capacity and stomatal conductance has also been observed with P deficiency (Zea mays, Wong et al., 1985). This means that the ratio of intercellular to ambient partial pressure remains approximately constant and independent of the nutritional status of the plant. Similarly, when leaf nitrogen levels fell with leaf age, Field et al. (1983) observed no significant change in the ratio of intercellular to ambient partial pressure. The ratio tends to be constant over a considerable range of leaf nitrogen contents, rising only with severe nitrogen deficiency (Evans, 1983; Makino et al., 1988; Sheriff, 1992). As with the ratio of Hill to Rubisco activities, there is no consistent trend in the ratio of intercellular to ambient partial pressure between plant types (Yoshie, 1986). Maintaining a close relationship between photosynthesis and stomatal conductance means that the amount of carbon gained per unit water lost is unaltered by nutrient deficiency. However, there are occasional reports of the ratio of carbon gained to water lost decreasing with nitrogen stress (Fredeen et al., 1991). It is not clear by what mechanism stomatal conductance is scaled to match that of photosynthetic activity. It cannot be through sensing intercellular partial pressure or photosynthetic rate because several types of transgenic plants which express antisense genes for particular photosynthetic enzymes have reduced photosynthetic capacity without affecting stomatal conductance (Hudson et al., 1992). However, it is another striking example of the plants ability to cope with environmental stress without suffering damage, in this case through excessive transpiration. The photosynthetic system somehow maintains its usual carbon gain per unit water lost in spite of reductions in photosynthetic rate by reducing stomatal conductance.

The patterns that emerge from photosynthetic properties of leaves in different light environments reveal that protein allocation is regulated to increase daily photosynthesis per unit nitrogen. The changes in allocation are widespread, although a few species seem only able to reallocate protein within the thylakoids. Significant variation in absolute photosynthetic rate per unit nitrogen exists between species, although not clearly between plant types, e.g. herbs versus trees, sun versus shade species. This variation is due to variation in specific activity of Rubisco, the amount of Rubisco per unit soluble protein and the fraction of leaf nitrogen actively involved with photosynthesis. However, the evidence for this is scanty at present and requires greater attention. A continuing enigma is the reason for changes in the chlorophyll a/b and PS II:PS I ratios, which are so widespread during acclimation to different irradiances. There still appear no satisfying explanations, despite the vast number of papers that report changes in chlorophyll a/b ratio. Linking photosynthetic capacity to leaf anatomy has been attempted in the past, the success varying with species. However, sufficient attention has not been given to the chloroplasts. To increase photosynthetic capacity per unit leaf area requires both biochemical and anatomical changes and there are no examples yet where Rubisco content and chloroplast surface area exposed to intercellular airspace per unit leaf area have been examined for leaves grown under a range of irradiances. This type of measurement is laborious, but has the potential to shed new insight into the anatomical changes leaves undergo in response to growth irradiance. A feature of leaf photosynthesis is the conservative nature of the ratio of Hill to Rubisco activities, as judged by response curves. The ratio seems little affected by growth irradiance, leaf age, species or mineral deficiency because of coordination between the capacities of the two processes. This usually occurs at the level of coordinating protein contents but capacities can be down-regulated when mineral deficiencies are encountered. While it has been fashionable to probe the photosynthetic system through the use of mineral deficiencies, the advent of variable reduction in specific proteins and/or the introduction of foreign proteins by molecular

300 biological techniques promises to lead to a much better understanding of the constraints on photosynthesis in terms of coordination between the various components and reactions.

Acknowledgments I am grateful to Hans Lambers for providing such a nice environment where much of this work was completed and I would like to especially thank my colleagues Susanne von Caemmerer, Hendrik Poorter and Thijs Pons for stimulating discussions.

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Eucalyptus globulus seedlings. Aust J Plant Physiol 19: 637– 652 Sims DA and Pearcy RW (1989) Photosynthetic acclimation to sun and shade conditions by a tropical forest understorey herb, Alocasia macrorrhiza, and a related crop species Colocasia esculenta. Oecologia 79: 53–59 Spiller S and Terry N (1980) Limiting factors in photosynthesis. II. Iron stress diminishes photochemical capacity by reducing the number of photosynthetic units. Plant Physiol 65: 121–125 Strauss-Debenedetti S and Bazazz FA (1991) Plasticity and acclimation to light in tropical Moraceae of different successional positions. Oecologia 87: 377–387 Taylor SE and Terry N (1984) Limiting factors in photosynthesis. V. Photochemical energy supply colimits photosynthesis at low values of intercellular concentration. Plant Physiol 75: 82–86 Terashima I (1986) Dorsiventrality in photosynthetic light response curves of a leaf. J Exp Bot 37: 399–405 Terashima I and Evans JR (1988) Effects of light and nitrogen nutrition on the organization of the photosynthetic apparatus in spinach. Plant Cell Physiol 29: 143–155 Terashima I and Inoue Y (1984) Comparative photosynthetic properties of palisade tissue chloroplasts and spongy tissue chloroplasts of Camellia japonica L.: Functional adjustment of photosynthetic apparatus to light environment within a leaf. Plant Cell Physiol 25: 555–563 Terashima I and Inoue Y (1985a) Palisade tissue chloroplasts and spongy tissue chloroplasts in spinach: Biochemical and ultrastructural differences. Plant Cell Physiol 26: 781–785 Terashima I and Inoue Y (1985b) Vertical gradients in photosynthetic properties of spinach chloroplasts dependent on intraleaf light environment. Plant Cell Physiol 26: 781–785 Terashima I and Saeki T (1983) Light environment within a leaf. I. Optical properties of paradermal sections of Camellia leaves with special reference to differences in the optical properties of palisade and spongy tissues. Plant Cell Environ 24: 1493– 1501 Terashima I and Saeki T (1985) A new model for leaf photosynthesis incorporating the gradients of light environment and of leaf photosynthetic properties of chloroplasts within a leaf. Ann Bot 56: 489–499 Terashima I, Sakaguchi S and Hara N (1986) Intra-leaf and intracellular gradients in chloropiast ultrastructure of dorsiventral leaves illuminated from the adaxial or abaxial side during their development. Plant Cell Physiol 27: 1023–1031 Thompson WA, Stocker GC and Kriedemann PE (1988) Growth and photosynthetic response to light and nutrients in Flindersia brayleyana F. Muell., a rainforest tree with a broad tolerance to sun and shade. I n : Evans JR, von Caemmerer S and Adams WW III (eds) Ecology of Photosynthesis in Sun and Shade, pp. 299–315. CSIRO Melbourne Thompson WA, Huang LK and Kriedemann PE (1992) Photosynthetic response to light and nutrients in sun-tolerant and shade-tolerant rainforest trees. II. Leaf gas exchange and component processes of photosynthesis. Aust J Plant Physiol 19: 19–42 Turnbull MH (1991) The effect of light quantity and quality during development on the photosynthetic characteristics of six Australian rainforest tree species. Oecologia 87: 110–117 Vogelmann TC, Bornman JF and Josserand S (1989) Photosynthetic light gradients and spectral regime within leaves of

304 Medicago sativa. Phil Trans R Soc Lond B 323: 411–421 von Caemmerer S and Evans JR (1991) Determination of the average partial pressure of in chloroplasts from leaves of several plants. Aust J Plant Physiol 18: 287–305 von Caemmerer S and Farquhar GD (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376–87 von Caemmerer S, Evans JR, Hudson GS and Andrews TJ (1994) The kinetics of ribulose-l,5-bisphosphate carboxylase/ oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco. Planta 195: 88–97 Walters MB and Field CB (1987) Photosynthetic light acclimation in two rainforest Piper species with different ecological amplitudes. Oecologia 72: 449–456 Wild A (1979) Physiologie der photosythese hoherer pflanzen. Die anpassung an die lichtbedingungen. Ber Deutsch Bot Ges 92: 341–364 Wilhelm C and Wild A (1984) The variability of the photosynthetic unit in Chlorella. II. The effect of light intensity and cell development on photosynthesis, and cytochrome f in

John Richard Evans homocontinuous and synchronous cultures of Chlorella. J Plant Physiol 115: 125–135 Wong SC Cowan IR and Farquhar GD (1979) Stomatal conductance correlates with photosynthetic capacity. Nature 282: 424–426 Wong SC Cowan IR and Farquhar GD (1985) Leaf conductance in relation to rate of assimilation. I. Influence of nitrogen nutrition, phosphorus nutrition, photon flux density, and ambient partial pressure of during ontogeny. Plant Physiol 78: 821–825 Wullschleger SD (1993) Biochemical limitations to carbon assimilation in plants–a retrospective analysis of the curves from 109 species. J Exp Bot 44: 907–920 Yoshida S and Coronel SV (1976) Nitrogen nutrition, leaf resistance and leaf photosynthetic rate of the rice plant. Soil Sci Plant Nutr 22: 207–211 Yoshie F (1986) Intercellular concentration and water-use efficiency of temperate plants with different life forms and from different microhabitats. Oecologia 68: 370–374

Chapter 12 Molecular Biological Approaches to Environmental Effects on Photosynthesis Christine A. Raines and Julie C. Lloyd Department of Biological and Chemical Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK

Summary I. Introduction II. Genetics and Biogenesis of the Photosynthetic Apparatus III. Molecular Approaches to Environmental Stress A. Isolation of Environmentally-regulated cDNA Clones 1. Differential Screening of cDNA Libraries 2. Subtractive cDNA Libraries 3. Differential Display B. Manipulation of Plant Physiology Using Transgenic Plants C. Determining Environmentally-responsive cis-acting Elements Using Transgenic Plants D. Identification of trans-acting Factors Using in vitro Techniques E. Using Mutants to Investigate Signal Perception F. Signal Transduction IV. Environmental Stress in Photosynthetic Systems V.

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Summary The application of molecular techniques to problems in plant biology has yielded a wealth of important information. In the area of biotechnology, the past few years have seen molecular and transgenic technology applied to solving problems of reduced yield due to viral and insect attack. A natural progression from this is the utilization of these powerful techniques to the analysis of stress responses in plants. This chapter addresses the molecular biological approaches which are available to investigate the effects of environmental stress on the photosynthetic apparatus. Enormous progress has been made in understanding the molecular biology of chloroplast biogenesis but a full picture awaits the elucidation of the regulatory mechanisms which control this process. In order to illustrate how recombinant DNA methods can be used to investigate stress effects, examples have been taken from other systems. Transgenic plant technology and the application of molecular genetic analysis to mutants also have the potential to make an enormous impact on this area of research. Finally, some examples are given of situations in which stress effects on photosynthesis have begun to be investigated at the molecular level. It is only by understanding the molecular bases of these responses that we will be able to engineer crops which overcome negative effects of stress on yield. Neil R. Baker (ed): Photosynthesis and the Environment, pp. 305–319. © 1996 K/uwer Academic Publishers. Printed in The Netherlands.

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I. Introduction Many events in the plant life cycle, including the biogenesis of the photosynthetic apparatus, are dependent on environmental cues for their initiation. Once a fully functional photosynthetic system is in place the efficiency with which it functions is dependent on environmental conditions and plants have some ability to adapt to suit these conditions. This ability of plants to respond, and in some cases acclimate, to changes in their environment is variable among species and is under genetic control, dictated by the genotype or genetic make-up of the species. The response induced by the environment begins with perception of the environmental signal or stress and culminates in an alteration in the physical characteristics, or phenotype, of the plant. One way in which these phenotypic changes are brought about is by the differential expression of a constant genome, i.e. without any change in the genotype of the plant (Smith, 1990). In species able to tolerate a particular change in environmental conditions the changes brought about in gene expression result in an acclimation response, but in sensitive species stress responses ensue. Since these stress and acclimatory responses have a genetic basis, it therefore follows that to understand fully the fundamental mechanisms involved in regulating plant responses to the environment it is essential to take a genetic approach to the problem. In recent years the addition of the techniques of molecular biology to the science of genetics has created a new discipline, that of molecular genetics, and the impact of the application of molecular genetics to fundamental plant science is just beginning to be felt. In the area of plant development, studies on floral development in Antirrhinum majus and Arabidopsis thaliana have led to the characterization of the regulatory genes which control this process (Coen and Meyerowitz, 1991). Aside from fundamental processes, tremendous developments have also been made in plant biotechnology, where genetic engineering allied to the ability to introduce novel genes into a rapidly growing list of plant species, Abbreviations: ABA – abscisic acid; CAM – crassulacean acid metabolism; cDNA – complementary deoxyribonucleic acid; GUS – Kb – kilobase pairs; PEPcase – phosphoenolpyruvate carboxylase; Rubisco – ribulose 1,5bisphosphate carboxylase-oxygenase; SOD – superoxide dismutase; T-DNA – transfer DNA from Agrobacterium tumefaciens

Christine A. Raines and Julie C. Lloyd including the relatively recalcitrant monocots, is now being exploited (Flavell, 1995; Mazur, 1995). Although few examples have yet gone beyond the research and development stage it is now possible to produce crop plants with resistance to ‘environmentally-friendly’ herbicides with the potential for weed control with lower environmental impact. This technology has also been applied to engineering resistance to pests and pathogens. Plants engineered to produce a potent insecticidal toxin (Bt toxin) naturally found in the bacterium Bacillus thuringiensis can deter insect attack and have the potential to reduce dependence on chemical insecticides which persist in the environment. The Bt toxin has been introduced into several crop species, including tomato (Fischhoff et al., 1987). Viral resistance can also be engineered into plants, increasing yield and to some extent reducing the use of insecticides which destroy their insect vectors. This has most commonly been achieved by expressing viral coat proteins in the plant but recently a dramatically different approach has engineered resistance by expressing antibodies to the virus in the plants (Tavladoraki et al., 1993). This is an striking illustration of the absence of species barriers in genetic engineering. These examples give some idea of the scope and power of the new technologies available to plant biologists and the aim of this chapter is to consider the range of molecular biological techniques which are now available to study the way in which the photosynthetic apparatus is affected by and responds to the environment. Molecular biology has already made an impact on our understanding of chloroplast biogenesis (outlined in Section II) and many of the genes whose products are structural components of the photosynthetic apparatus have been isolated and characterized. The gaps that now exist in our understanding lie in the control of these processes, the identification of the regulatory genes and the elucidation of the steps between environmental signal recognition and the effects on these regulatory genes. As yet the literature on molecular aspects of environmental stress on photosynthesis is limited, however in the last few years a considerable amount of information has accumulated regarding the wider issue of molecular mechanisms regulating plant responses to the environment. These examples, which will form the largest part of this chapter, will be used to illustrate the scope and potential success of the molecular approach. This is followed by a review of selected examples where molecular techniques have

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been applied to problems of environmental stress and photosynthesis.

producing a single RNA which codes for several proteins (such polycistronic transcripts are also a common feature of prokaryotes). These complex transcripts are then subject to processing by cleavage to remove portions of RNA and divide the transcript before translation. The processing of chloroplast transcripts is undoubtedly a major point of control in the differential expression of plastid genes, and modifications to the processing steps have further regulatory implications in terms of the subsequent translatability and susceptibility to degradation of the processed transcript (Deng et al., 1989; Mullet, 1993). The majority of proteins found in plastids are actually encoded in the nuclear genome and there are significant differences in the way in which the expression of genes in this compartment are regulated in comparison with chloroplast genes. Nuclear encoded genes are transcribed in the nucleus, each primary transcript contains the coding information for a single polypeptide and the primary transcript is subject to several processing steps. A ‘cap’ is added to one end of the RNA molecule and a poly-A tail to the other, followed by splicing to remove non-coding

II. Genetics and Biogenesis of the Photosynthetic Apparatus The biogenesis of the photosynthetic apparatus is a complex process (Webber and Baker, 1996) which involves the cooperative interaction of two distinct sets of genetic information located in separate cell compartments; the chloroplast and the nuclear genomes (Fig. 1). Our understanding of the contribution that the chloroplast genome makes in terms of the genes it carries is well advanced (Mullet, 1988), with the entire DNA sequence of the chloroplast genome having been determined for several species, e.g. the liverwort Marchantia polymorpha (Ohyama et al., 1986), Nicotiana tabacum (Shinozaki et al., 1986). Over half the genes present in the chloroplast genome are involved in transcribing and translating the plastid DNA, whilst the remainder encode proteins with photosynthetic functions. Many plastid genes are co-transcribed,

308 introns which interrupt the sequence of codons specifying the amino acid sequence. The processed messenger RNA (mRNA) then leaves the nucleus and is translated on cytoplasmic ribosomes. The expression of nuclear genes can be subject to regulation at many of these stages but a major point of control of nuclear gene expression is at the level of transcription. It is at this stage that the promoter sequences associated with a particular gene determine the rate of transcript production from that gene. Processing events, mRNA stability and translatability are all receiving attention to determine what role they might have but at present these are much less well understood. Once nuclear encoded chloroplast proteins have been translated they still have to be imported from the cytoplasm into the organelle and this is accomplished by the presence of a short ‘transit’ peptide sequence located at the aminoterminus of the protein (Keegstra et al., 1989). This directs the protein to the organelle and facilitates its import after which it is cleaved from the mature protein by processing proteases. At this stage proteins may require refolding into active conformations and in many cases assembly into multiprotein complexes. A large number of the genes encoding abundant chloroplast proteins have been cloned and the expression of individual genes from both the chloroplast and nuclear genomes have been studied extensively (Thompson and White, 1991). In photosynthetic tissues there are two major states which have to be accommodated in terms of gene expression. During chloroplast development, which occurs in response to developmental and light signals, there is a rapid accumulation of chloroplast and nuclear encoded photosynthetic proteins (Tobin and Silverthorne, 1985, Kuhlemeier et al., 1987). Once chloroplast development has taken place then a maintenance state is reached where there must be sufficient production of new proteins to replace those which are turned over. Proteins of the photosynthetic reaction centers, for example D1, are particularly susceptible to damage and will continue to be synthesized at a high rate even once chloroplast development is complete (Nickelsen and Rochaix, 1994; see also Chapter 4). The activation of both chloroplast development and the expression of genes encoding chloroplast proteins have been shown to be dependent upon light, developmental state and tissue type, however the mechanisms involved in co-ordinating nuclear

Christine A. Raines and Julie C. Lloyd and plastid gene expression are not well understood (Taylor, 1989; Chory, 1993). The levels of expression clearly must be linked in some way in order for structural components to accumulate in the required stoichiometries. The holoenzyme of ribulose 1,5bisphosphate carboxylase (Rubisco) contains eight small subunits (encoded in the nuclear genome) and eight large subunits (encoded in the chloroplast), and there is some evidence that the final level of the holoenzyme may be determined by the amount of small subunits present with excess large subunits being degraded (Rodermel and Bogorad, 1988). We really know nothing of the regulatory components of the processes. It is clear that the chloroplast and nucleus must communicate, and whilst the signals are believed to be generated within the plastid, the regulatory genes involved are almost certain to be located in the nuclear genome (Taylor, 1989). The chloroplast genome has been fully characterized and no regulatory genes have been identified. Any comprehensive study of environmental effects on the photosynthetic apparatus at the molecular level should consider both genomes and their interactions, but it is to the nuclear genome that we must look to identify the genes which control the normal programs of gene expression and the changes occurring in photosynthetic gene expression in response to environmental stress, both during development and in the mature leaf.

III. Molecular Approaches to Environmental Stress The aim of this section is to describe the molecular techniques which could now be applied to the problem of understanding the mechanisms determining plant responses to stress effects on the photosynthetic apparatus, using examples from related areas to illustrate the information which can be obtained and the limitations. Changes in gene expression have been shown to occur in response to a number of environmental factors including salinity, temperature and light and result in the production of novel proteins or the loss of essential proteins. Taking a molecular genetic approach to elucidate the regulatory mechanism(s) underlying these events involves isolating and studying the unknown components of signal perception and transduction pathways and understanding how these lead to altered gene

Chapter 12 Molecular Biological Approaches expression by direct interaction with the regulatory DNA sequences of genes. It also involves the analysis of genes which encode proteins whose expression is modified in response to environmental signals which play some other part in the stress or acclimatory response which is invoked. It is important to note that gene expression includes transcriptional, post-transcriptional, translational and post-translational processes (described in Section II). It is necessary to determine the relative roles of these different steps in regulating environmentally-induced protein changes before a molecular strategy to study the underlying mechanism can be planned. Early studies using cell free systems to translate in vitro mRNA from plants grown under stress conditions have shown that different populations of transcripts (translating into different proteins) are present in plants grown under these conditions (e.g. Gulick and Dvorak, 1987; Ramagopol, 1987; Catavelli and Bartels, 1989; Perras and Sarhan, 1989). Many plant species can acclimate and tolerate freezing temperatures if they are first given a period of treatment at low, non-freezing temperatures. This so called cold-hardening response is now known to involve changes in the mRNA and protein populations in these plants. Complementary DNA (cDNA) clones representing DNA copies of mRNA transcribed from cold-responsive genes have been isolated from a wide number of plant species (see Section III A) including barley (Dunn et al., 1990), rice (Aguan et al., 1991),Arabidopsis thaliana (Hajela et al., 1990) and alfalfa (Mohapatra et al., 1989). In some cases this type of analysis has been taken a step further to show that a significant proportion of the regulation of novel protein production in response to environmental stimuli resides at the level of transcription (Mohapatra et al., 1989; Hajela et al., 1990). This is done by isolating nuclei from plants grown under the two conditions (normal and treated) and pulse labeling RNA transcripts to determine the rate of transcription from a particular gene, as illustrated in Fig. 2. This in vitro transcription assay can only be performed where a cloned copy of a gene is available. Experiments of this type have led to attention being focused on understanding transcriptional changes resulting from stress. However, post-transcriptional processes should not be ignored as there is evidence that some environmental responses occur at this level of gene expression/ regulation (Hajela et al., 1990; DeRocher and Bohnert, 1993; Sullivan and Green, 1993).

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A. Isolation of Environmentally-regulated cDNA Clones A number of different techniques have now been used in order to identify mRNA transcripts whose levels are increased in response to stress and these are outlined below.

1. Differential Screening of cDNA Libraries Differential screening of cDNA libraries has been widely used to isolate genes whose expression is specifically regulated by development, tissue-type or environmental changes. The rationale behind this technique, which is illustrated in Fig. 3, is that when mRNA is isolated from a sample of plant tissue all the genes which are actively being transcribed at that time will be represented in that RNA population. If

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sets of plants are subjected to two different environmental treatments then the mRNA species present may differ between the two, reflecting the genes whose transcription is activated or repressed in the experimental, as opposed to the control, conditions. These two populations of mRNA, from control and experimental plants, can be radioactively labeled and used as probes to ‘differentially’ screen a cDNA library. A cDNA library is constructed by synthesizing double-stranded DNA copies of each mRNA present in the plant tissue expressing the protein of interest, which depending on the

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circumstances might be either the control or the treated tissue. The resulting mixture of individual cDNA molecules are cloned into a suitable vector which allows them to be multiplied inside host cells and the resulting cloned cDNA population, termed a cDNA library, is screened in duplicate using the mRNA from the treated and control plants. This approach normally yields a set of clones which potentially represent RNA transcripts either produced or absent in response to the environmental treatment being studied. By careful selection of the treatments given to the plants before RNA isolation and choice

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of tissue from which the library is made, it is possible to isolate clones which are induced by a treatment (in this case the library is made from ‘treated’ samples) or repressed by a treatment (the library must be made from normal control RNA). A differential screening approach was used to isolate a number of cDNA clones from cold-treated winter barley (Dunn et al, 1990). Low temperature treatment increases tolerance of the plants to subsequent freezing temperatures and this had previously been shown to coincide with alterations in gene expression (Hughes and Dunn, 1990). Individual cDNA clones are then studied to confirm that they respond to the environmental factor under study. This is done by determining the level of the corresponding mRNA, by RNA (Northern) blot analysis, in samples from plants grown under a range of conditions and exposed to the environmental factor for different times. The clones positively identified by screening are likely to exhibit a range of responses in terms of the kinetics and level of response to the environmental change. Such studies can be extremely valuable in determining whether the gene expression changes involved are primary (and therefore possibly regulatory) or secondary in nature. In order to assign a function to the protein encoded by a cDNA clone isolated in this way the nucleotide sequence is determined and decoded to reveal the amino acid sequence of the protein whose sequence it specifies. This is then compared with other known protein sequences which are available on computer database systems and can lead to identification of the protein or to the assignment of possible functions if it is similar to proteins or protein domains with known functions. This type of analysis was carried out on three barley cDNA clones identified as described above. The clone BLT4, which is induced by cold, absicisic acid (ABA) and drought, was shown to encode a polypeptide with similarity to a maize phospholipid transfer protein. Other members of this gene family have now been isolated and are being studied (Dunn et al., 1991; Hughes et al., 1992). In contrast to this the clone BLT101 is low temperature-specific (ABA and drought have no effect on expression) but as the sequence had no similarity to any proteins in the databases the function of the protein encoded by this gene remains to be elucidated (Goddard et al., 1993). The third clone, BLT63, encodes a protein synthesis elongation factor (Dunn et al., 1993).

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2. Subtractive cDNA Libraries A variation of the differential cDNA library screening approach described above involves subtractive hybridization of mRNA (or cDNA) prior to constructing the library. The purpose of subtractive hybridization is to reduce the total size of the library to be screened and increase the relative abundance of the clones of interest. In this procedure hybridization between the library RNA and a control sample of mRNA is used to remove DNA sequences common to both. Since these common sequences are not differentially expressed they are not of interest. This removal is often achieved by passing the library sample through a column containing a solid cellulose matrix to which the other sample (RNA or cDNA) has been attached. An alternative strategy, utilized to isolate temperature sensitive genes from rice, involved a denaturation-renaturation step prior to insertion of the cDNA into the vector. Again this hybridization step removed sequences which were not coldregulated from the library pool. The resulting selective library was screened and three cDNA clones of interest were isolated; one, lip 19, was shown to respond to low temperature (Aguan et al., 1991). Sequence analysis of this clone revealed that it encodes a protein with a structural motif found in certain DNA-binding proteins (Aguan et al., 1993). This raises the interesting possibility that lip 19 is a transcription factor involved in temperature regulated gene expression.

3. Differential Display This technique, like the previous two, is based on the differential expression of mRNA species in response to an environmental stimulus. However, in this case, amplification of RNA transcripts is carried out using the polymerase chain reaction (PCR) (Liang & Pardee, 1992) and no cDNA library is required during the identification and preliminary analysis of the differentially expressed cDNA clones. In this procedure total RNA is isolated from tissue exposed to the environmental stress and from a control sample, and copied into cDNA using the enzyme reverse transcriptase. The cDNA products are then amplified using PCR. These amplified cDNAs from the control and treated tissues are then separated using polyacrylamide gel electrophoresis, and compared to identify any which are differentially amplified in the treated sample. cDNA clones of interest which

312 appear to be more highly expressed in the treated sample (or perhaps which appear to be absent in stress conditions) are then analyzed further by excising the bands from the gel. The isolated DNA may be used in RNA blot hybridization to confirm it’s differential expression, or sequenced. This technique has been used successfully to identify ozone-induced cDNA clones one of which, AtOZI1, may be induced in response to active oxygen species (Sharma & Davis, 1995). As yet, none of the proteins encoded by the cDNA clones described above have been shown to have role in conferring cold tolerance or in stress responses in a physiological context. However, they do represent important starting points to begin to address mechanisms of cold-hardening and there are a number of possible routes that can be taken to help determine the function of these proteins. Selected cDNA clones can be used to produce large amounts of pure protein using expression systems which allow the protein to be synthesized in bacterial cells. Purified proteins can in turn be used to raise antibodies enabling the cellular location and factors affecting accumulation of the protein to be studied using western blotting and in situ immunolocalization techniques (Robertson et al., 1993). These techniques can provide very useful correlative data linking the protein to the adaptive or stress response. The next section describes how the analysis can be taken a step further by beginning to address protein function.

B. Manipulation of Plant Physiology Using Transgenic Plants If the expression of particular proteins changes in response to the environment it is important to determine the function and the role that these proteins may play in the response of the plant. One approach which can be taken is to exploit the availability of transgenic plant technology. Novel synthetic combinations of coding and regulatory sequences, which control expression, are constructed using genetic engineering techniques. These synthetic genes are then integrated into the genomes of host plants following their introduction either as naked DNA molecules, by particle bombardment, or by using Agrobacterium tumefaciens. In the latter case the construct is put into a circular plasmid DNA molecule present in the A. tumefaciens cytoplasm that is capable of transfer to plant cells (Klee et al., 1987). The target plant cells must be capable of regeneration into

Christine A. Raines and Julie C. Lloyd whole plants and antibiotic resistance marker genes are usually included in the DNA constructs to allow the selection of transformed plants. Following integration the synthetic gene (transgene) is transmitted in a normal Mendelian manner to the offspring of the transformed plant. It is now possible to transform a wide range of different plant species and a number of more recent reports have extended this technology to previously recalcitrant monocotyledonous species, which include some of the most important crop plants such as maize (D’Halluin et al., 1992), rice (Christou et al., 1991) and wheat (Vasil et al., 1993). Levels of individual components in the plant can be manipulated using chimeric genes constructed from either cDNA or genomic DNA sequences. To increase levels of an existing protein, or introduce a new protein, the construct is prepared using the cDNA (or genomic) clone in the normal (sense) orientation. To reduce levels constructs with the coding sequence in the reverse (antisense) orientation are prepared, resulting in the formation of RNA which is complementary to the endogenous transcript. The presence of the antisense RNA interferes with the production of protein from the normal transcript, although the exact mechanism is not known. Some examples of transgenic sense and antisense approaches relating to the photosynthetic apparatus are described below. Plants are known to derive some protection from oxidative stress damage by producing the enzyme superoxide dismutase (SOD) which converts superoxide radicals to (see Chapter 5). This observation has led a number of researchers to produce transgenic plants expressing higher levels of this enzyme. One of the most recent of these reports (Sen Gupta et al., 1993) demonstrated an increased tolerance to photoinhibition at low temperatures in tobacco plants overexpressing pea SOD. Freezing tolerance in alfalfa has also been enhanced in transgenic plants with elevated levels of SOD (McKersie et al., 1993). Chilling sensitivity of plants has been correlated with the level of saturation of fatty acids in the phosphatidylglycerol of chloroplast membranes, so this has also attracted attention as a possible target for genetic manipulation. The nuclear-encoded chloroplast enzyme glycerol 3-phosphate acyl transferase has a role in determining the level of saturation of the phosphatidylglycerol fatty acid saturation in the thylakoid membrane. Murata and

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others (1992) created transgenic Arabidopsis plants expressing an antisense construct prepared using a cDNA clone for this enzyme. Analysis of these plants revealed that the level of fatty acid desaturation was altered and correlated with an increase in the chilling tolerance of the transgenic Arabidopsis plants. Not related to environmental stress, but of interest in terms of chloroplast development, is recent work by Zhang et al. (1992) who produced transgenic Arabidopsis plants using an antisense construct expressing an ankyrin-type protein. Ankyrin repeatcontaining proteins have been found to interact with cytoskeletal, signal and receptor proteins and for this reason are believed to be important regulatory components in a number of eukaryotic systems. However, ankyrin proteins have not been identified or studied in plants. The antisense plants displayed a chlorotic phenotype and lacked thylakoid granal stacks indicating that this protein may have a role in chloroplast differentiation. These examples demonstrate the way in which transgenic technology can be employed to address important physiological and developmental problems. However, not all problems in plants can be solved or investigated by simply changing the levels of one protein alone. In many cases a number of different gene products may be involved and it will be very difficult to assign roles to proteins on an individual basis. It may be necessary to introduce multiple gene constructs either simultaneously, sequentially or in combination with genetic crosses made between transformants.

only cDNA clones, as they include upstream regulatory sequences and transcription start sites that are not present in cDNA clones. Consequently a library containing fragments of nuclear DNA (a genomic library) must be screened. This library is prepared from the plant species of interest by isolating high molecular weight DNA and cloning it into one of many suitable vectors (Sambrook et al., 1989). A cDNA clone is then used as a probe to isolate the corresponding genomic clone, which may be some 20 Kb in length. The next step is to identify the gene within this clone and, having isolated and sequenced the gene, the upstream regions can then be used to investigate it’s regulation. Delineation of functionally important upstream cis-acting elements involves the analysis of chimeric gene constructs in transgenic plants (Willmitzer, 1988). Upstream regulatory sequences of the gene of interest are joined to a reporter gene, such as luciferase (de Wet et al., 1987) or glucuronidase (GUS) (Jefferson, 1987) which have easily assayed products not found in the untransformed plant. Analysis of the expression of these constructs in transgenic plants is a very powerful tool for studying qualitative control, for example cell specific or developmentally regulated expression (Nagy et al., 1988). On a cautionary note this type of analysis is more difficult to interpret quantitatively due to large variation in levels of expression of the same construct in different transformed plants. Some of the most extensively studied genes in this context are light-regulated nuclear encoded genes for chloroplast proteins, particularly the Rubisco small subunit (rbcS) genes and chlorophyll a/b binding protein (cab) genes of pea (Thompson and White, 1991). The pea rbcS-3A gene is something of a paradigm and the sequence elements which are conserved between the promoter of this gene and other light-regulated genes have been reviewed (Gilmartin et al. 1990). Some of these elements have been shown, using transgenic plants, to be important in controlling expression. However, functional analysis, whilst conceptually straightforward, is complicated by the fact that many elements are repeated several times in the upstream sequence and may interact or have overlapping roles. Many, such as the binding sites for the nuclear factor GT-1, were initially identified as conserved motifs and later shown to interact with sequence-specific DNAbinding proteins (Green et al., 1988). Deletion and mutation studies in transgenic plants can help to

C. Determining Environmentally-responsive cis-acting Elements Using Transgenic Plants A major control point for gene expression is at the level of transcription (discussed in Section II). The rate of transcript production from a gene is determined by the frequency with which RNA polymerase, and other protein components of the transcription complex, assemble at the promoter (the site of transcript initiation). For most eukaryotic nuclear genes this is determined by the presence of DNA motifs, cis-acting elements, which reside in the upstream, non-coding regions of the gene and interact with DNA-binding proteins termed trans-acting factors. From this it can be seen that in order to study the mechanism by which transcription is regulated by the environment it is essential to study genes, not

314 identify the location of important sequences, but in order to establish their real function it is then necessary to perform what are termed gain-of-function assays. In such experiments the role of a regulatory element can be investigated out of its normal promoter context. In the case of GT-1 it was shown that this binding site could mediate both light-regulated and tissue-specific expression of a reporter gene in transgenic plants. A very considerable literature has been produced on light-regulated gene expression and many sequences have been implicated in controlling this regulation. The picture that is emerging, but as yet is still incomplete, is of a very complex set of interactions, which may be necessary to provide the subtle range of control during development and in response to different light qualities and quantities, required by the plant. In relation to other environmentally regulated responses very few genes have been studied to the extent of the examples given for light. Genes responding to temperature (Kurkela and Franck, 1990), drought (Urao et al., 1993) and salt (Cushman et al., 1989) stress have been isolated. A gene encoding the protein osmotin was cloned and used to prepare chimeric constructs and inserted into transgenic plants where it was found to respond to drought stress (Kononowicz et al., 1992). Similarly, the upstream sequences of a cold-regulated gene, cor-78, conferred temperature responsiveness when fused to the GUS reporter gene and introduced into transgenic plants (Horvath et al., 1993). cor-78 was isolated using a cDNA clone obtained by differential screening of an Arabidopsis cDNA library made from cold-treated tissue but the function of its gene product is unknown. In both of these cases the initial experiments form the basis for more extensive analysis of the regulatory regions of these genes and may lead to elucidation of the environmentally responsive elements. More recently, extensive analysis of two environmentally regulated genes, rd29a and rd29b (YagamuchiShinozaki and Shinozaki, 1994), has led to the identification of cis-acting domains in these genes which may be responsible for their differential responses to drought, low-temperature, or high-salt stress.

D. Identification of trans-acting Factors Using in vitro Techniques The binding of a trans-acting factor to the upstream region of a gene is the terminal event of signal

Christine A. Raines and Julie C. Lloyd transduction and therefore represents another route into the elucidation of the pathway from signal perception through to gene transcription. Again extensive work has been done in relation to lightregulated genes and from a gene which responds to drought stress (Yagamuchi-Shinozaki and Shinozaki, 1994) but no other information is available from other environmental systems as yet. However, it is interesting to note that two groups have isolated clones using differential screening which may code for DNA-binding proteins and function in the pathway leading to the expression of cold-regulated genes (Aguan et al, 1993; Urao et al., 1993). The techniques employed most commonly to identify the cognate trans-acting proteins interacting with DNA motifs include gel retardation assays, where the altered mobility of a DNA fragment when it binds to a protein is detected, and footprinting, which directly localizes the nucleotide bases in a DNA molecule interacting with a protein. These techniques were used to identify a light-regulated DNA binding protein, LRF-1, in Lemna gibba (Buzby et al., 1990). By extracting nuclear proteins from plants grown under different conditions it was shown that LRF-1 was more abundant in light-grown than in dark-adapted leaves and most interestingly that it appeared to be phytochrome regulated. LRF-1 is induced in dark-adapted leaves given a short red light treatment but this induction showed the far-red light reversibility characteristic of phytochrome regulation. A nuclear protein which binds to a droughtand low temperature-responsive gene has been identified by Yagamuchi-Shinozaki and Shinozaki (1994).

E. Using Mutants to Investigate Signal Perception Our understanding of the way in which plants perceive and respond to their environment is still relatively poor. Even if we consider photomorphogenesis, where it is known that at least three different pigment systems are involved in the initial perception of the light stimulus and one of these, phytochrome, has been studied extensively, still many questions remain to be answered, particularly in relation to developmental and acclimatory responses of the plant in differing light environments. The receptor(s) involved in plant responses to temperature and drought are unknown. One approach to identify these environmental perception systems is to study mutants which have lost the ability to respond to the stimulus of

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interest (Kendrick and Nagatani, 1991). The power of a mutagenesis approach is that it allows the isolation of genes whose products are completely unknown. A number of different strategies have been developed to create mutants, including random mutagenesis using chemical mutagens, such as ethyl methane sulfonate (EMS), or irradiation and insertional mutagenesis by transposon or transfer DNA (T-DNA) tagging (Cook and Miles, 1988;Walbot, 1992). Once mutants have been created a screening strategy can be devised to isolate plants defective in the response which is being investigated. Recently a putative blue light receptor protein was identified by taking a molecular genetic approach and screening T-DNA tagged Arabidopsis thaliana (Ahmad and Cashmore, 1993). Many plant processes are known to involve a blue light receptor(s), for example hypocotyl elongation, stomatal opening and expression of genes encoding chloroplast proteins. The screening approach identified mutants insensitive to blue light-induced inhibition of hypocotyl elongation. The gene involved, HY4, was identified using its molecular ‘tag’ to isolate a clone and then by DNA sequence comparison. Although the protein encoded by this gene has not yet been shown to bind the blue-light chromophore it has significant homology with bacterial photolyases which are involved in blue-light mediated responses. Consequently, the application of molecular genetics has proved a powerful tool giving insight into the elusive blue light pigment system, and this type of approach will inevitably be used in the search for further environmental receptor systems. After a receptor for an environmental signal has been characterized, mutants can still play a valuable part in exploring its physiological role. This can be illustrated using the example of the phytochrome photoreceptor family which is of central importance in the light-controlled program of plant development. In Arabidopsis the phytochrome apoprotein is encoded by a gene family, PHYA, B, C, D and E (Sharrock and Quail, 1989), and this diversity of structure has led to the proposal that different phytochromes have distinct roles in plant light responses (Smith and Whitelam, 1990). Mutants have been used in a number of different ways to investigate this question. For example, studies on the photomorphogenic responses (hypocotyl elongation) of mutants lacking either PHYA or PHYB have provided direct genetic evidence for separate functions for these two phytochrome types (Parks

and Quail, 1993; Reed et al., 1993). Mutants have also been produced using sense constructs to overexpress specific phytochromes either in a wild type genetic background (Wagner et al., 1991; Whitelam et al., 1992; McCormac et al., 1993) or in plants which lack PHYB (Wester et al., 1994). Where mutagenesis might potentially fall down is in more complex situations where there is duplication of function amongst separate components leading to a failure to detect the mutants.

F. Signal Transduction Almost nothing is known about the way in which environmental signals are transduced into biological messages in plants and what is known is restricted to light responses. The strategies employed to isolate mutants in the light signal transduction pathways of plants in theory can potentially lead to the identification not just of receptors (as described above) but also components of the signal transduction pathway including trans-acting regulatory proteins. This is likely to be the case amongst the photomorphogenic mutants of Arabidopsis thaliana, and the picture that emerges from studying the phenotypes of these mutants is of potential convergence of red and blue light pathways through common signal transduction components (Chory, 1993). Biochemical approaches have involved searching for components of signal transduction pathways of other better studied systems, for example mammalian pathways (Trewavas and Gilroy, 1991). Recently there has been much more effort put into addressing these aspects of cellular processes in plants. Sheen (1993) used specific inhibitors to implicate phosphatases in the greening response and lightregulated gene expression in maize. Another approach involved microinjection of potential signaling intermediates into cells of the PHYA deficient tomato aurea mutant and this revealed the involvement of G-proteins and activated calmodulin in light responses (Neuhaus et al., 1993).

IV. Environmental Stress in Photosynthetic Systems In a number of cases environmental stress has been related to the inability of the plant to develop a fully functional photosynthetic system. However, this is not the only stage of the plant life cycle when the

316 photosynthetic apparatus is sensitive to the environment. Once the photosynthetic system is fully established it remains capable of acclimation to a range of environmental conditions. Preliminary molecular analysis of environmental effects on the photosynthetic apparatus has indicated that changes in both chloroplast and nuclear gene expression are involved. It is beyond the scope of this chapter to review all interesting environmental systems that offer themselves for molecular study but in the following section selected examples of molecular approaches to environmental stress in photosynthesis are presented. The photosynthetic apparatus in a number of plant species is able to acclimate to changing light environments by modulating the stoichiometry of the components in the thylakoid membrane and stroma. The photoreceptors involved in initiating this response are not known but changes in the red:far red ratios are sensed therefore obvious candidates are phytochrome and the thylakoid chlorophylls. In an attempt to overcome the problem posed by overlapping absorption spectra of these components a photomorphogenic mutant lacking PHYA was used, and indicated that phytochrome may have a minor role in this acclimation (Smith et al., 1993). Studies made on a chilling sensitive species, rice, by Hahn and Walbot (1989) revealed that coldtreatment resulted in a reduction in mRNA levels and that protein synthesis of the nuclear-encoded chloroplast proteins, Rubisco and chlorophyll a/b binding protein, were significantly reduced. Studies on another chilling sensitive species, maize, have shown that growth at low temperatures results in an inability of the thylakoid membranes to accumulate chloroplast-encoded polypeptides (Nie and Baker, 1991). Using feeding it was shown that these effects were not due to a general inhibition of protein synthesis in the chloroplast but may relate to changes in processing, insertion or stabilizing events occurring on completion of translation (Baker and Nie, 1994). Transfer of maize seedlings grown at 14°C to 25°C did not lead to full recovery and it has been proposed that this may be due to a developmental time window which if missed cannot be retrieved, and indicate an essential regulatory gene product is no longer available (Nie et al., 1995). These studies suggest that the effects of low temperature on these species are in part related to a failure of the photosynthetic genes to be expressed appropriately.

Christine A. Raines and Julie C. Lloyd In order to address this problem it would be possible to take a differential screening approach. Cold-tolerant cereals require the continuation of growth and development to attain maximum freezing tolerance. In these plants it has been observed that a higher flux of carbon through the Calvin cycle to sucrose synthesis is maintained when compared to sensitive varieties. The cold tolerant varieties appear to be able to modulate photosynthesis by regulating photosystem II activity and carbon metabolism (Huner et al., 1993). The mechanism controlling this regulation has not been investigated at the molecular level but an antisense approach has been suggested as a possible way of assessing the contribution of individual enzymes during low temperature acclimation. One indication that changes in the expression of genes encoding enzymes of sucrose synthesis might be involved in this acclimation process came from a study on cold tolerant spinach. This work showed that the activity and synthesis of the sucrose phosphate synthase protein was increased in cold tolerant plants which had undergone cold acclimation treatment (Guy et al., 1992). The induction of crassulacean acid metabolism (CAM) by salt stress in the facultative halophyte Mesembryanthum crystallinum (the common ice plant) requires the synthesis of several enzymes. Using antibodies and cDNA clones for the CAM enzymes phosphoenol pyruvate carboxylase (PEPcase) and pyruvate phosphate dikinase, it has been shown that the both the protein and transcript levels are increased in the ice plant in response to salt treatment (Michalowski et al., 1989, 1992). These results suggested that the pathway for CAM metabolism is switched on by high salt treatment and nuclear run on assays indicated this was initiated at the level of transcription (Vernon et al., 1993). Two PEPcase genes from this plant have been isolated and characterized and only one of the gene copies responds to salt treatment (Cushman et al., 1989); the upstream sequences of these genes should be useful tools for the elucidation of the mechanism of gene activation. This facultative halophyte is currently being used as a model in an attempt to elucidate the signal perception and transduction pathways involved in osmotic stress and the effects of ABA, cytokinins and NaCl indicate a complex network of pathways (Thomas et al., 1992; Thomas and Bohnert, 1993).

Chapter 12 Molecular Biological Approaches V. Conclusions Physiological and biochemical analyses have shown that photosynthetic capacity is reduced as a consequence of environmental stress. However, such approaches have often been unable to identify the mechanisms involved. It is clear that molecular biological techniques offer an exciting alternative and will be applied increasingly to these problems in plant biology. Consequently, in the next few years some dramatic leaps forward in our fundamental understanding of plant stress responses can be expected. Ultimately, using knowledge of the molecular bases of stress responses, it may be possible to genetically engineer more tolerant crop species or to use gene probes identified as encoding proteins with key roles in stress responses in breeding programs aimed at selecting desirable traits.

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Molecular Biological Approaches

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Chapter 13 Photosynthesis in Fluctuating Light Environments Robert W. Pearcy and John P. Krall Section of Plant Biology, University of California, Davis, CA 95616, USA

Gretchen F. Sassenrath-Cole USDA-ARS Crop Simulation Research Unit, Department of Plant and Soil Science, P.O. Box 5367, Mississippi State, MS 39762-5367, USA

Summary I. Introduction II. The Nature of Sunfleck Light Regimes III. Factors Regulating the Photosynthetic Utilization of Sunflecks A. The Fast Induction Phase B. The Slow Induction Phase C. Modulation of Induction State in Fluctuating Light 1. Responses of Stomata 2. Role of Photosynthetic Enzyme Regulation IV. Regulation of the Transient Responses to Individual Lightflecks A. Dynamics of Assimilation Rate During Lightflecks Assimilation B. The Contribution of Post-Lightfleck C. Assimilatory Charge and Lightfleck Utilization D. The Electron Acceptor During Lightflecks E. Electron Transport Capacity and Lightfleck Utilization F. Photosynthesis and Lightfleck Utilization V. Are There Specific Adaptations in Shade Leaves for the Utilization of Sunflecks? VI. Sunfleck Utilization in Natural Light Regimes A. The Total Contribution of Sunfleck Utilization to Daily Assimilation in Natural Light Regimes Assimilation Enhance B. How Much Does the Induction Requirement Limit, or Post-lightfleck Carbon Gain in Natural Sunfleck Regimes? Acknowledgments References

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Summary The light environment in plant canopies is characterized by rapid fluctuations in photon flux density (PFD) because of the occurrence of sunflecks. These sunflecks can contribute most of the PFD available for photosynthesis and thus the mechanisms that control their utilization can have a significant impact on the carbon gain within canopies or in understories. When sunflecks are infrequent, their utilization is constrained by the induction requirement of the photosynthetic apparatus. The induction requirement has been shown to involve three separate factors consisting of an increase in the capacity for regeneration of ribulose bisphosphate that is important in the first 1–2 min after a light increase, the light activation of ribulose 1,5-bisphosphate Neil R. Baker (ed): Photosynthesis and the Environment, pp. 321–346. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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carboxylase that occurs over the first 5–10 min of induction, and an increase in stomatal conductance. Under the conditions of multiple sunflecks occurring in varying succession characteristic of canopy light regimes, the induction state of a leaf is a function of the up and down regulation of these three factors. The induction state determines the readiness of a leaf to respond to a sunfleck in terms of the maximum assimilation rate that can be achieved during it. Post-lightfleck assimilation occurring because of the utilization of high-energy metabolite pools built up during the lightfleck can substantially enhance the utilization of short lightflecks. This buildup occurs because of a transient uncoupling of electron transport and carbon assimilation rates as 3phosphoglyceric acid pools are reduced allowing for initially elevated electron transport rates during a lightfleck. Simulation modeling and measurements have shown that under natural sunfleck regimes in forest understories the induction state of leaves may limit daily assimilation by 10 to 25%. Post-lightfleck fixation, on the other hand, does not significantly enhance sunfleck use in this environment because the short sunflecks for which it is most important make little contribution to the available sunfleck PFD. Within crop canopies, where the contribution of short-duration sunflecks is much greater, simulation modeling indicates a more important role for post-lightfleck fixation.

I. Introduction Leaves in forest understories or within plant canopies experience rapid fluctuations in the photon flux density (PFD) available for photosynthesis because of the changing patterns of sunfleck and shade. Sunflecks are the direct beams of sunlight that penetrate through small gaps in a canopy, and although present for only a small fraction of the day, they often contribute a large fraction of the PFD available for photosynthesis by understory plants. These sunflecks have long been recognized by ecologists as being important for the survival and growth of forest understory plants (Lundegarth, 1921; Evans et al., 1960). Sunflecks are hard to define precisely because they vary so much in both their temporal and spatial characteristics. Long duration sunflecks (>10 min) grade into gap light environments of the type created by treefalls. At the other end of the time scale, changes in PFD of an order of magnitude or more can occur in sunflecks as short as a second because of Abbreviations: –assimilation rate at time T; –steady-state assimilation rate; –assimilation rate corrected to a constant intercellular pressure; CAP–carboxyarabinitol 1-phosphate; – intercellular pressure; F6P – fructose 6-phosphate; FBP – fructose 1,5-bisphosphate; FBPase – fructose 1,6-bisphosphatase; GADPH –glyceraldehyde 3-phosphatedehydrogenase; – stomatal conductance; – induction state at time T; LUE – lightfleck use efficiency; PCRC – photosynthetic carbon reduction cycle; PFD – photon flux density; PGA – 3-phosphoglyceric acid; PQ – plastoquinone; R5P – ribose 5-phosphate; Ru5P – ribulose 5-phosphate; Ru5P kinase–ribulose 5-phosphate kinase; Rubisco – ribulose 1,5-bisphosphate carboxylase; RuBP– ribulose l,5-bisphosphate;SBP–sedoheptulose 1,7-bisphosphate; SBPase – sedoheptulose 1,7-bisphosphatase; TP – triose-phosphate; – time constant, time required for a process to change 0.63 of the final-steady state in response to a step change in the environment

canopy movements in the wind. Within canopies themselves, the transient nature of the light regime is often striking, with leaves in some canopies receiving more than 1000 sunflecks per day on days with just moderate breezes (Pearcy et al., 1990). The steadystate conditions usually applied in measurements of leaf gas exchange do not hold under these transient light conditions characterizing sunflecks and canopy light environments. Instead, it is necessary to understand, in addition to the steady state photosynthetic characteristics, the transient response characteristics of the leaves in order to gain an understanding of the mechanisms regulating the use of sunflecks. Only in the past 10 years or so has the necessary technology in terms of rapid response infra-red analyzers and computer-based data acquisition systems been available for this kind of characterization of transient responses. The environmental and physiological controls on assimilation rate that operate during sunflecks are quite different than those operating under steadystate conditions. In the steady-state, the fluxes of carbon can be understood in terms of the concentrations of substrates and the effective resistances of specific diffusional and metabolic steps, which combine to determine the overall rate of assimilation. For photosynthesis under transient light conditions, the dynamic elements of the system that give it time dependence come into play. The dynamic elements consist of the metabolite pools that are built up and depleted as the photosynthetic rate changes over a time scale of seconds, the light-regulated enzymes that activate and deactivate over a time scale of minutes, and the stomata which open and close over time scales of minutes. Because the light-regulation

Chapter 13 Photosynthesis in Fluctuating Light of enzyme activity and stomatal conductances are much slower processes than are the light fluctuations in the environment, the response to any given light change will in part be a function of the previous light environment. In this chapter, we review the current state of knowledge regarding the controls on the dynamic responses of photosynthesis to light transients of the kind characterizing sunflecks. We focus on understanding the functional basis of the transient responses as they relate to the dynamics of metabolite pools and fluxes and photosynthetic regulation in variable light. Chazdon (1988) reviewed the ecological aspects of sunfleck utilization and a previous review (Pearcy, 1990) covered the early progress in understanding the mechanisms governing sunfleck utilization. We focus on terrestrial plant communities since much of our research has been carried out with either understory plants or crop or tree canopies. However, aquatic communities often exhibit similar highly dynamic light fluctuations because of wave actions (Gerard, 1984) and studies with algal species confirm the importance of the dynamic responses to light fluctuations for photosynthesis in these environments as well (Dromgoole, 1987, 1988).

II. The Nature of Sunfleck Light Regimes Sunflecks create the continually changing pattern of sun and shade patches on the ground or within a canopy that are readily observed during a walk in the woods on a sunny day. At any given spot, sunflecks are usually present less than 10% of the time, but on clear days they typically contribute 40 to 80% of the photon flux density (PFD) available for photosynthesis (Björkman and Ludlow, 1972; Pearcy, 1983; Chazdon and Fetcher, 1984). It appears that there is as much or more variation within forests as there is between forest types. Chazdon (1988) has compiled values from the literature showing that on clear days temperate deciduous and evergreen forests understories receive about 50% of their available PFD as sunflecks. In evergreen tropical forests the mean is 50 to 70%. By contrast, microsites separated by only a few meters within a forest can receive vastly different amounts of sunfleck PFD on any given day. Over weeks or months, these differences probably even out to some extent as the solar path changes. The temporal nature of sunfleck light regimes on a time scale of seconds to hours is determined in part

323 by the Earth’s rotation and in part by wind-driven movements of the canopy. In addition, the temporal pattern of cloudcover adds another element to the variability. The swaying of canopies causes many short sunflecks in the understory with durations on the order of 1 to 10 s. Within canopies, leaf movements in the wind cause even more rapid sunfleck activity. High frequency PFD measurements in quaking aspen (Populus tremuloides) and a cottonwood (Populus fremontii) canopies, which are characterized by leaf fluttering even in light breezes due to the flattened petiole structure of these poplar species revealed the highly dynamic nature of the resulting light environment (Roden and Pearcy, 1993a). Spectral analysis showed that under light winds when the leaves were fluttering the fluctuations in PFD had peak frequency of 3 to 4 Hz. Comparisons between still and windy days showed that leaf fluttering caused an increased number of shorter sunflecks and that the leaf and canopy movements may have caused a more spatially uniform light environment, both in the horizontal and vertical directions. Longer sunflecks in forest understories and canopies are caused by the Earth’s rotation. Frequently a longer sunfleck is preceded and followed by many short sunflecks because of the combined effects of the Earth’s rotation and canopy movement. Moreover, sunflecks are often clustered with several to many occurring in rapid succession, but with these clusters separated by periods with few or no sunflecks. In total, leaves in the understory may receive 50 to 300 sunflecks per day with most being shorter than 10 s (Pearcy, 1988; Pfitsch and Pearcy, 1989a). However, the 5% or so of sunflecks that are longer than 2 min typically contribute >75 % of the sunfleck PFD. Consequently, in forest understories, the mechanisms controlling the photosynthetic utilization of infrequent, long sunflecks are likely to be of more overall importance than those involved with utilization of short sunflecks. Most sunflecks in forest understories have maximum PFDs that are considerably below the full, direct-beam solar irradiance. Indeed, under tall canopies, only 1 to 2% of the sunflecks may reach PFDs approaching those of full sunlight (>1000 with two thirds or more only reaching PFDs of 50 to 200 . The solar disk has an apparent angular diameter of 0.5°, so gaps smaller than this block part of the disk. Thus, the direct PFD received at a point below will be reduced according

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to the fraction of the disk that is blocked. Larger gaps create sunflecks with a numbra providing full, direct beam solar irradiance at the center surrounded by a penumbra where the irradiances are gradually reduced towards the full shadow (umbra). As the distance from the gap to the ground increases, the numbra decreases and finally disappears while the penumbra increases. Due to the slight divergence of the solar beam, a sunfleck, while dimmer, covers a larger area as the canopy height increases. The spreading of sunflecks in this manner increases the probability that an understory plant will receive light from a given sunfleck. Penumbral effects therefore can substantially enhance assimilation. Simulations indicate that the enhancement could be as much as 200 to 500% as compared to a condition of no penumbral effects (H. L. Tong and R. W. Pearcy, unpublished). This effect of canopy structure may be an important but poorly studied aspect of sunfleck light regimes. Only a few studies in crops or grasslands are available for comparison to forest understory sunfleck regimes. Small leaf sizes and flexible canopies create highly dynamic light environments characterized by frequent but brief sunflecks (Tang et al., 1988, 1989; Pearcy et al., 1990). Moreover, penumbral effects are minimized by the short canopy structure so that most sunflecks have maximum PFDs equal to the direct beam solar PFD. The minimal penumbral effects coupled with their high frequency results in short sunflecks making a much larger contribution to the total PFD in crop canopies as compared to the situation in forest understories. Thus, mechanisms controlling the utilization of short sunflecks may also assume considerable importance in crop canopies. Within a canopy, the temporal light regimes range from mostly full sunlight punctuated by brief shade periods (shade flecks) near the top to mostly shade light punctuated by sunflecks deeper in the canopy. Consequently, different limitations to assimilation may come into play at different depths in the canopy.

III. Factors Regulating the Photosynthetic Utilization of Sunflecks The photosynthetic responses to sunflecks or lightflecks (simulated sunflecks created with an artificial light source and shutter) are complex because several components of the photosynthetic apparatus

respond with very different time constants. The initial reactions involved in light harvesting and energy transfer respond nearly instantaneously to changes in PFD. The flux through the enzymatic reactions involved in carbon fixation then respond on a somewhat slower time scale of 1–2 s as metabolite concentrations change. Photorespiratory release lags behind the initial oxygenation reactions, which further tempers the dynamics of the assimilation rate during lightflecks. The short-term maximum rate achieved during the lightfleck depends on the activity status of photosynthetic enzymes and on the stomatal conductance. Both exhibit more gradual changes in response to the light history of the leaf (time scale of 1–20 min). They are part of the induction requirement of photosynthesis first described by Osterhout and Hass (1919) and studied extensively since then (Walker, 1973, 1981; Edwards and Walker, 1983). The induction requirement, which involves the slow increase in assimilation rate observed following long shade periods, causes a substantial loss of potential assimilation as compared to the rapid increase in assimilation rate when the leaf is already induced (Fig. 1). This latter condition holds after brief (<1 minute) shade periods when the assimilation can increase from low values in the shade to the lightsaturated rate within 5 s. As will be discussed later in this chapter, the activity status of the light-regulated enzymes important in induction seems to depend on the electron transport rate as part of the signal transduction pathway. Relationships between electron transport and carbon metabolism therefore play an important role both in the short term and long term responses that determine carbon gain during sunflecks. Still longer-term changes (time-scale of days) involving acclimation, aging and cumulative stress effects that modify the photosynthetic capabilities of the leaf also occur (see Chapter 11). These are obviously important in the response of any given leaf to sunflecks but will not be considered here. The gradual increase in assimilation during the induction response has been found to be dependent on three separate processes, each operating on different time scales. A fast phase is associated with limitations to ribulose 1,5-bisphosphate (RuBP) regeneration (Kirschbaum and Pearcy, 1988b; Sassenrath-Cole and Pearcy, 1992). These limitations develop and relax quickly and are most evident following relatively short low light periods when the

Chapter 13 Photosynthesis in Fluctuating Light

other limitations have not yet developed to a significant extent. Under these conditions, the restriction in RuBP regeneration limits the rate of increase in assimilation during the first 1–2 min of induction. After long periods in low PFD, this fast phase may be masked by other, slower limitations consisting of the light-activation requirement for Rubisco and stomatal opening. Rubisco activation is largely complete within 7 to 10 min but stomatal opening may cause a continuing, but generally small, further increase in assimilation rate for up to an hour. For most leaves, however, 90% of the final steadystate assimilation rate is reached within 10 min of the light increase. The relaxation kinetics of these three limitations combine to determine the time course of assimilation rate during induction. A useful measure of the overall limitations imposed by the induction requirement during transient light is the induction state of a leaf. The induction state at any given time is defined by x 100, where is the assimilation rate at time T, measured in seconds from the light increase, and is the steady-state, light-saturated assimilation rate after induction is complete. The induction state measured at 5 s is sensitive to any additional limitations

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imposed by the fast induction component while the induction state measured 60 or 120 s after the increase in PFD or serves as a measure of the induction state after the fast induction component has relaxed but the stomatal and Rubisco limitations are still potentially present. Thus, these values can be used to provide an indication of the importance of each component in limiting the assimilation rate. Because the time required for full relaxation of the fast induction component varies between species, different times for determining induction state are needed for different species. Initial experiments on induction usually attempted to correlate an increase in a specific factor such as stomatal conductance, metabolite concentrations or light activation of an enzyme and the increase in photosynthetic rate in order to determine the role of a specific factor. It is possible to use this approach to demonstrate that some factor such as stomatal conductance cannot account for the observed induction response under the particular conditions imposed (i.e. saturating but not to determine with any certainty the role any specific factor. Under conditions where there may be multiple limiting factors, determination of the role of each in induction

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requires a more sophisticated analysis of gas exchange kinetics. A paradigm in leaf gas exchange analysis is that stomatal conductance regulates the supply of to the mesophyll whereas it is the biochemical capacity for fixation that sets the demand (Raschke, 1979; Farquhar and Sharkey, 1982). Under steady-state, light-saturated conditions, the biochemical capacity for assimilation is given by the slope of the assimilation rate (A) to intercellular pressure curve (the curve). If the increase in assimilation rate during induction is due solely to the increase in then assimilation rate and will increase along this steady-state curve. If, on the other hand, a biochemical limitation is also involved, then the assimilation rate will fall below this curve, approaching it as the biochemical limitation relaxes. In effect, each value of assimilation rate falls on a dynamic curve whose slope increases during induction (Fig. 2). The changing slope (biochemical capacity) and changing (supply function) define a trajectory for assimilation rate and The assimilation rate corrected by interpolation or extrapolation to the value expected if were constant is proportional to the dynamic slope and provides a convenient way to assess changes (Woodrow and Mott, 1989). The difference between and gives the amount that dynamically limits assimilation (Tinoco-Ojanguren and Pearcy, 1993a). Similarly, the total induction limitation can be shown by ratio of to the steady-state, light-saturated rate achieved at the end of induction. These approaches must be applied judiciously since errors in calculation of caused by patchy stomatal behavior (Terashima et al., 1988) or by the cuticular conductance (Kirschbaum and Pearcy, 1988c) cause errors in the assessment of the relative imitations imposed by stomata. In both of these cases, the errors lead to an underestimation of the role of stomata and hence an overestimation of the role of biochemical limitations.

A. The Fast Induction Phase The possibility that the fast induction phase was due to an RuBP-regeneration limitation was first postulated on the basis of transient gas exchange measurements that revealed that the increase in assimilation rate during the first minute of induction did not behave as if it were due to activation of Rubisco (Kirschbaum and Pearcy, 1988b). Instead, the kinetics at different pressures were consistent with the presence of a limitation in RuBP regeneration

that relaxed over the first minute of induction. The fast induction limitation is especially prominent after fully induced leaves shaded for 5 min and then reilluminated with saturating PFD (Fig. 3 A). This is so because, following a short shade period, Rubisco activity and stomatal conductance remain high, shifting more of the limitation to the more rapidly developing RuBP-regeneration limitation. Thus after 5 min may only be 0.2 to 0.3 whereas is still 0.7 (Fig. 3B). The RuBP-regeneration limitation decreases in prominence with longer shade periods because further inactivation of Rubisco diminishes the requirement for a high RuBP-regeneration capacity. Thus, after 60 min in the shade, both IS5 and IS60 may be reduced to <0.15 (Fig. 3B). The limitation imposed by the fast induction component seems to depend on the species and on growth conditions. The fast induction response is a prominent feature in soybean leaves after short low light periods (Pons et al., 1992) but is less evident in the shade plant, Alocasia macrorrhiza. However, in another shade plant, Adenocaulon bicolor, it was a quite prominent feature and strongly limited sunfleck utilization after short shade periods (Pfitsch and

Chapter 13 Photosynthesis in Fluctuating Light

Pearcy, 1989b). Thus, no unique relationship to sun versus shade adaptation can be postulated. Growth of the high light adapted species, Piper auritum at high as compared to shade PFDs enhanced the role of the fast induction limitation (Tinoco-Ojanguren and Pearcy, 1993a). More research is needed to understand why this limitation is prominent in some species but not in others and why growth conditions alter its prominence. Direct evidence that the fast induction limitation was indeed an RuBP-regeneration limitation was obtained by Sassenrath-Cole and Pearcy (1992) by measuring RuBP concentrations relative to Rubisco active site concentrations in soybean leaves. A ratio of RuBP to Rubisco active sites less than about 1.5 to 2 is taken to indicate an RuBP limitation to Rubisco turnover rate (von Caemmerer and Edmondson, 1986; Sharkey, 1989). Leaves were first fully induced and then shaded for various times before being reilluminated with saturating PFD. With 1 minute shading, which resulted in little development of a fast induction limitation, the ratio of RuBP to Rubisco active site concentration increased from 0.4 in the shade, to 2 with 5s re-illumination, and to 4 with 60 s of re-illumination (Fig. 4). The latter was similar to that observed under steady-state light-saturated

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conditions. Thus even by 5 s re-illumination, the RuBP concentration had increased enough so that it was probably not limiting. After a 5 min shade period, however, ratios of RuBP to Rubisco active sites had only increased to about 0.76 with 5 s reillumination and only reached 1.6 with 60 s reillumination. These low ratios are consistent with an RuBP limitation to Rubisco that relaxed over the 60 s re-illumination period. After 1 hour in the shade, ratios of RuBP to Rubisco active sites were 1.5 in the shade and increased to 2.3 with 5 s re-illumination and to 5.1 with 60 s re-illumination. The higher values in the shade as well as the greater increase upon re-illumination are consistent with significant down regulation of Rubisco by this time, and therefore decreased RuBP utilization rates. The close agreement between the occurrence of a fast induction limitation as shown by low values and low RuBP to Rubisco active site ratios is strong evidence for the identity of the fast induction limitation as being an RuBPregeneration limitation. An RuBP-regeneration limitation could develop either because depletion of the total metabolite pools in the chloroplast limited the RuBP pool, or because inactivation of an enzyme or enzymes in the RuBP regeneration path limited the metabolite flux to RuBP.

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Metabolite measurements did not support the former possibility since there was only a gradual decline in the total carbon equivalents in the metabolite pools of the photosynthetic carbon reduction cycle (PCRC) following shading (Sassenrath-Cole and Pearcy, 1992). Depletion of metabolites occurred faster in the dark than at low PFD, primarily because of a greater depletion in 3-phosphoglyceric acid (PGA) pools. When both the dark and shade data were included, there was no correlation between the development of the fast induction limitation and depletion of total metabolite pools. Thus, it is more likely that a light-dependent increase in activity of enzymes in the RuBP regeneration path is responsible for relaxation of the fast induction phase. Four enzymes, NADP glyceraldehyde 3 -phosphate dehydrogenase (GADPH), fructose-1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and ribulose 5-phosphate kinase (Ru5P kinase), in the RuBP regeneration path are known to be light activated (Edwards and Walker, 1983). In addition, the chloroplast coupling factor, is light activated but appears to function more as an

on/off switch at low PFD (Ort and Oxborough, 1992). A change in flux through these enzymes brought about by an alteration in activation state would be evident as an increase in substrate and depletion of product. However, in the case of the PCRC, this analysis is complicated by the cyclic nature of RuBP regeneration. A limitation can therefore affect metabolite pools both preceding and following it in the cycle. Moreover, the branch points leading to starch synthesis or export of triose-phosphate (TP) further complicate the measurements because they remove carbon from the PCRC. To a certain extent, these analytical limitations can be overcome by comparing the changes in metabolites occurring in response to a light transient under conditions where the limitation could be expected to differ. Thus it has been possible to gain insight into the specific steps in the RuBP regeneration path that may be limiting by examining specific metabolite pool changes in response to 5 s of re-illumination following shade periods of different durations (Sassenrath-Cole and Pearcy, 1992). Following 2 min of shade, TP, fructose 1,5-bisphosphate (FBP), fructose 6-phosphate (F6P) and seduheptulose 1,7bisphosphate (SBP) increased to a greater extent than did ribose 5-phosphate (R5P) and ribulose 5phosphate (Ru5P) pools. This pattern is consistent with a faster development in the shade of a limitation by down regulation of FBPase and/or SBPase than by down regulation of Ru5P kinase. After 5 to 10 rain of shade, however, the total pool size of R5P plus Ru5P also increased. This pattern indicates that, by this time, Ru5P-kinase may have also inactivated sufficiently to impose some additional limitation on RuBP regeneration. FBPase has been shown to deactivate rapidly in soybean leaves following a high to low PFD transition (Sassenrath-Cole and Pearcy, 1994), which is consistent with it limiting RuBP regeneration upon a subsequent transition from low to high PFD. The stromal FBPase and SBPase belong to a group of enzymes that require reduction by the thioredoxin system for activation (Buchanan, 1980), specifically thioredoxin f. The thioredoxin system receives reducing equivalents from PS I via ferredoxinthioredoxin reductase, and subsequently reduces thiol groups on specific enzymes, inducing a more active form. This reductive activation is one mechanism through which the biochemical reactions of the PCRC are coordinated in their activity with the rate of electron transport and energy (ATP and

Chapter 13 Photosynthesis in Fluctuating Light supply (Harbinson et al., 1990b). FBPase has a 30 mV more negative midpoint potential than thioredoxin (Ort and Oxborough, 1992), indicating that under certain conditions, it may be more sensitive to chloroplast redox state than the other PCRC enzymes, and hence limiting to RuBP regeneration. The synthase, which has a midpoint redox potential nearly equipotential with thioredoxin f, is already fully activated at perhaps as low as 4 (Ort and Oxborough, 1992), indicating that very low PFD may be all that is necessary to reduce thioredoxin. Differences in activation of the lightregulated RuBP-regeneration enzymes as a function of PFD would thus depend on the thermodynamic equilibrium of the chloroplast redox state. Because of its lower midpoint potential FBPase may therefore remain more oxidized and thus, may be more limiting to RuBP regeneration when the PFD increases than other thioredoxin-mediated enzymes. Indeed, measurements in soybean leaves of the steady-state levels of FBPase activity at different PFDs show that below approximately 70 the activity of FBPase relative to the level required for light saturated assimilation is lower than the comparable activities for Rubisco and R5P kinase (Sassenrath-Cole et al., 1994). In summary, the thioredoxin system activates at lower PFD levels, but deactivates more rapidly than does Rubisco, making it more sensitive to fluctuating light conditions. Of the thioredoxinmediated enzymes, FBPase seems to be the most sensitive to chloroplast redox state, most likely because of its very negative midpoint redox potential.

B. The Slow Induction Phase The increase in assimilation rate during the slow phase of induction is due to the combined effects of stomatal opening and light activation of Rubisco. Although there is good documentation of differences in rates of stomatal opening among species, there have been no studies of their impact on the rates of relaxation of the slow induction phase. Some species, such as the plant, sugar cane, exhibit very fast stomatal opening (half time of 2–3 min) in response to a light increase whereas others such as soybean exhibit considerably slower responses (half time of 10–5 min) (Grantz and Zeiger, 1986). Also, shadetolerant trees have been reported to have faster stomatal responses than shade-intolerant species (Woods and Turner, 1972) which was suggested to perhaps allow better sunfleck utilization. It could be

329 expected that faster stomatal opening such as that exhibited by sugar cane and shade tolerant trees would shift more of the limitation during the slow phase of induction to the biochemical steps involving Rubisco activation. There are two lines of evidence for the important role of light activation of Rubisco in the slow induction phase. The first is the initial increase in RuBP pool size within 1–2 min to levels well above the final steady-state pool size, which is then followed by a decrease to the steady-state pool size as induction proceeds (Perchorowicz et al., 1981; Seemann et al., 1988). This behavior of the RuBP pool size is consistent with a removal of a limitation to RuBP utilization via an increase in Rubisco activity during induction. The second is the good correlation between the time course of Rubisco activation and the increase in assimilation during induction. The correlation is especially good if mesophyll conductance (Pearcy, 1988) or (Woodrow and Mott, 1989) is calculated to eliminate stomatal effects. Both mesophyll conductance or and the initial activity of Rubisco, measured rapidly after extraction and without activation by additional or in the medium, exhibit a logarithmic increase versus time, with time constants ( ) of 4 to 5 min (Pearcy, 1988; Woodrow and Mott, 1989). The initial activity should reflect the in vivo activity status of Rubisco. Upon shading, the loss of initial Rubisco activity and the decrease in induction state or as measured by gas exchange are similarly much slower than the rate of activation with of 20 to 28 min. The light regulation of Rubisco activity involves both activation by the covalent addition of and to a lysine residue (carbamylation) and by the binding sugar phosphates that inhibit the activity (Portis, 1992). Carbamylation appears to be the primary and universal mode of regulation, particularly in the short term, that serves to maintain a balance between the capacity of Rubisco to utilize RuBP and the capacity of the chloroplast for RuBP regeneration (Woodrow and Berry, 1988). At steady state, the fraction of the enzyme that is carbamylated is proportional to the photosynthetic rate at that PFD (Woodrow and Berry, 1988). The steady-state carbamylation is dependent on stromal pH, and levels. The and pH in the stroma in turn depend on electron transport activity. Binding of sugar phosphates, especially RuBP to the decarbamylated and hence inactive enzyme, and a tightbinding inhibitor, carboxyarabinitol 1-phosphate

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(CAP), may serve as a longer-term regulatory mechanism important primarily after long shade periods or in the dark (Sage et al., 1993). The binding of RuBP to the decarbamylated (inactive) form of the enzyme is also a universal feature of Rubisco regulation but CAP is synthesized in only some species (Kobza and Seemann, 1988). The light-dependent increase in Rubisco activity is facilitated by another protein, Rubisco activase (Salvucchi et al., 1986). This protein may form a complex with Rubisco and facilitate activation by removal of sugar phosphates (Portis, 1992). The activity of Rubisco activase also depends on PFD, in part because it requires ATP and is inhibited by ADP (Streusand and Portis, 1987). In addition, PS I electron transport and a transthylakoid pH gradient have been shown to enhance the rate of activase mediated Rubisco activation (Campbell and Ogren, 1990). Thus, there may be several factors involved in sensing the PFD and then coordinately modulating the activity of Rubisco activase and Rubisco. There is also evidence for variation in the rate of activation at different PFD levels since Rubisco activase must be activated first and has a slower than activasemediated Rubisco activation (Lan et al., 1992). Since activase activation is saturated at relatively low PFD (135 the overall activation of Rubisco from PFDs lower than this is slower than it is for activation from higher PFDs. These measurements have only been done in spinach and the possibility that activase regulation is saturated at much lower PFDs in shade plants needs to be explored (Watling and Woodrow, 1993). Given the varied mechanisms involved, it is surprising that to date there is so little evidence for differences in the time dependence of Rubisco regulation among species. The data of Kobza and Seemann (1989) suggest no differences between species utilizing or not utilizing CAP. Similar values of (4–6 min for the increase, 18–25 min for the decrease) are evident in time courses for the tropical forest understory herb, Alocasia macrorrhiza (Seemann et al., 1988), the pioneer tree species, Piper auritum (Tinoco-Ojanguren and Pearcy, 1993a), spinach (Woodrow and Mott, 1989), the high light adapted trees, Populus tremuloides and P. deltoides (Roden and Pearcy, 1993b) and soybean (SassenrathCole and Pearcy, 1994). The for deactivation is more variable but in all cases examined to date it is much longer than the for activation. The mechanistic reasons for the very hysteretic behavior are not clear,

although its ecological impact is unmistakable in holding the activation state of Rubisco higher during brief shade periods or between sunflecks. Recent reports of species differences in rates of induction (Kursar and Coley, 1993; Poorter and Oberbauer, 1993) need to be explored further to establish their causal basis. Tropical rainforest species with longlived leaves had considerably slower induction than those with short-lived leaves, with the difference being insensitive to whether the measurements were done at low or high (presumably saturating) (Kursar and Coley, 1993). The response is consistent with the induction time differences being due to a difference in the rate of Rubisco activation, but further measurements are needed to fully rule out differences in stomatal opening as a causal factor.

C. Modulation of Induction State in Fluctuating Light In natural sunfleck regimes such as those in forest understories or crop canopies, it is likely that leaves are rarely fully induced. Full induction should only occur during long (>10 min) sunflecks or when an equivalent period of short sunflecks occur in rapid succession. The remainder of the time, the induction state will be a function of the immediate past light environment. This function is clearly very complex because it depends on the changes in the signal that senses whether the induction state is too high or too low for the current light environment and the time constants for changes in stomatal conductance and light regulation of the photosynthetic enzymes. The current light environment provides a set point for which there is an optimum level of enzyme activation and stomatal conductance. Then as the light environment changes during sunflecks this setpoint, and hence the signal, changes. However, the time constants for activation/deactivation of the key enzymes and for stomatal conductance result in the induction state lagging well behind the set point. In this regard, the hysteretic responses of both stomatal conductance and Rubisco regulation can be important in maintaining a high induction state between sunflecks.

1. Responses of Stomata Stomata respond both directly and indirectly to light with the former being mediated by a light receptor in the guard cell, probably a blue light receptor (Zeiger,

Chapter 13 Photosynthesis in Fluctuating Light 1983) and the latter being mediated by the decrease in in the mesophyll. Responses to both factors give a steady-state dependence of stomatal conductance to PFD that is similar in form to that for assimilation rate, except that at very low PFD there is still a positive conductance. Thus, over a wide range of PFDs, tends to remain rather constant, increasing only at the lowest PFDs. In response to fluctuating light regimes, stomata exhibit a dynamic response that is strongly hysteretic, impacting the induction state established in a fluctuating light regime and consequently the response of assimilation to lightflecks. The response of stomatal conductance to a lightfleck is illustrated in Fig. 5 and involves an initial short lag followed by increase in gs that continues for approximately 20 min, even when the lightfleck is much shorter (Kirschbaum et al., 1988 Tinoco-Oganguren and Pearcy, 1992). A slow closing response is then initiated which may last for 90 min. For a short lightfleck, most of the stomatal opening will not occur until after the lightfleck has already passed. The increase in occurring after a 1ightfleck can strongly influence the use of subsequent lightflecks occurring within a reasonable time (< 1 h). Two lightflecks in succession gives an additive response. This ‘pulse’ response is probably driven by the blue light photoreceptor system in guard cells (Iino et al., 1985). Responses of the membrane potential of isolated guard cell protoplasts to blue light pulses show a hysteretic hyperpolarization with a faster increase than decrease (Assmann et al., 1985). Although the blue light pulse response of membrane potential is much faster than the final changes in stomatal conductances, coupling of these fast changes to slower buildup of osmotic potential and water uptake accounts for the pulse response of (Kirschbaum et al., 1988). The pulse response of to lightflecks is also influenced by other environmental factors. The rate of closing after a lightfleck, but not the initial rate of opening, has been shown to depend on relative humidity, resulting in faster closure and consequently less hysteresis in the overall response at low relative humidities (Tinoco-Ojanguren and Pearcy, 1993b). Consequently, at low relative humidity, there will be less carryover of the increased stomatal conductance from one lightfleck to the next and a greater stomatal limitation on lightfleck use. An obvious cost of the pulse response is higher transpiration than if the stomata responded immediately. However, at the high humidities characteristic of most forest

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understories, this cost will be small. There is also evidence of species and acclimation-induced differences in the stomatal response to sunflecks that are significant in regulating the photosynthetic carbon gain. Growth of the high-light species Piper auritum under shade conditions resulted in slower stomatal opening and closing and a much smaller increase in in response to a lightfleck (Tinoco-Ojanguren and Pearcy, 1992). A result of this rather small pulse response was a poorer utilization of subsequent lightflecks as compared to a shade species Piper aequale, which has a large pulse response to lightflecks. Knapp and Smith (1987, 1990a,b; Knapp et al., 1989) have extensively studied the stomatal and photosynthetic responses of plants to sun-shade transitions caused by intermittent cloud cover. The cumulus cloud cover characteristic of the Rocky Mountain subalpine zone where most of the research was carried out gives alternating sun and shade periods of 5 to 8 min duration. Some species exhibited little stomatal response to sun-shade transitions while others exhibited substantial cycles of opening and closing. Those that showed a significant reduction in during the shade period also had an induction response during the following sun period resulting in the loss of potential carbon gain during this period.

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However, these species had higher water use efficiencies than those with little reduction in during the shade period. There is as yet no consistent pattern leading to an explanation for the differences among species. Plants in habitats with ample water supply, and in some cases woody plants with large water capacitance, seem to maintain high during intermittent shade, leading to less induction limitation. However, Quercus macrocarpa, an oak species of the central United States, had rapid responses (Knapp, 1992), suggesting that other factors than growth form or water capacitance are involved. The stomatal response kinetics of Q. macrocarpa were similar to those of and grasses from the nearby prairie communities.

2. Role of Photosynthetic Enzyme Regulation Although the advantages provided by light regulation of photosynthetic enzymes is still a matter of some speculation rather than hard evidence, it appears to function in a manner that matches electron transport capacity and carbon assimilatory capacity. Consequently, as PFD changes, a balance between the capacity to supply ATP and via electron transport and the capacity to utilize it for carbon reduction is maintained in balance. Adjustments in capacity at different parts of the PCRC are necessary to maintain metabolite pools at appropriate levels throughout the cycle. Thus, as the PFD is increased and the control of the flux shifts from RuBP regeneration to Rubisco, metabolite pool sizes throughout the cycle remain within appropriate limits. If this regulation were not to occur, too high or too low metabolite concentrations could lead to inhibition of flux rates or to an imbalance with the starch and triose phosphate export pathways, possibly leading to a depletion of the overall carbon pools in the PCRC. Given the above scenario for the role of lightregulation of key elements in the photosynthetic apparatus, it is perhaps not surprising that the activation state of the key regulatory enzymes, Rubisco and FBPase, exhibit a similar dependence on PFD to that of assimilation Sassenrath-Cole et al., 1994). For shade leaves, the PFD required for full activation of Rubisco and FBPase is lower than it is in sun leaves (Krall et al., 1995). These behaviors of enzyme activation states lead to a similar lightdependence of the equilibrium induction state achieved after sufficient time has elapsed to allow a

steady-state level of enzyme activation. However, at PFDs below light saturation, the induction state is always higher than the assimilation rate at that PFD expressed relative to the maximum light-saturated assimilation rate. In other words, when the PFD is increased there is always scope for an immediate increase up to the assimilation rate allowed by the induction state, which is then followed by the slower induction increase as the enzymes activate. The difference between the induction state and the relative assimilation rate at a given PFD decreases as PFD is increased. This behavior of induction state relative to photosynthetic rate is consistent with a down regulation of enzyme activities until a shared control is achieved between substrate concentrations and enzyme activation status. Thus, as the flux in the PCRC increases when PFD is increased, substrate concentrations for these regulated steps immediately increase until enzyme activation status becomes limiting. Activation of these enzymes to the new equilibrium level at the new PFD then re-establishes shared control by both metabolite concentrations and the enzyme activation status at the new higher assimilation rate. The greater scope for an immediate increase in assimilation indicates that metabolite concentrations exert a greater proportion of the control at low PFD. Thus, even at low induction state established after long periods in low PFD, there will always be some capacity to respond immediately to a lightfleck. Comparisons between induction states and enzyme activation states in flashing light designed to simulate rapid sunfleck activity and continuous light show lower activation levels and induction states in the former (Pons et al., 1992; Sassenrath-Cole et al., 1994). These responses are consistent with the control mechanisms having a saturating response, which in the case of bright lightflecks would give a lower average signal than would occur under a similar mean but constant PFD. A common element of all responses with this characteristic is electron transport, which is involved both in regulation of thioredoxindependent enzymes in the PCRC and Rubisco regulation via Rubisco activase (Campbell and Ogren, 1990). To examine this possibility, Krall et al. (1995) determined the relationships between activation states of Rubisco and FBPase, induction state, and electron transport measured via chlorophyll fluorescence techniques (Genty et al., 1989) in soybean and Alocasia macrorrhiza leaves. For both Rubisco and FBPase, activities measured after 1 h in a given light

Chapter 13 Photosynthesis in Fluctuating Light

regime so that equilibrium activation and induction states were established were highly correlated with the mean in vivo electron transport rate for the light regime (Fig. 6). For a given species, neither sun shade acclimation, which resulted in different

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photosynthetic capacities, nor measurement in flashing (1 s lightflecks) versus constant PFD, giving different electron transport rates at the same mean PFD, caused a deviation from the same fundamental relationship between mean electron transport rate

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and enzyme activities. In turn, induction state measured 120 s after a light increase was closely related to Rubisco activation and expressed relative to the maximum values at light saturation, and independent of whether the leaves were sun or shade acclimated or measured in flashing or constant PFD (Fig. 6). These results reveal a remarkably coordinated down regulation of stomal enzyme activity coupled with a similar down regulation of gs that determine the equilibrium induction state in a given light environment. Moreover, the results support the hypothesis that electron transport rate established in a given light environment, or something closely related to it, is responsible for this coordinated down regulation. The responses of enzyme activation to fluctuating PFD have important consequences for the regulation of induction state in the light regimes characteristic of plant canopies. It is clear that neither the induction state nor the activity status of PCRC enzymes can be predicted from the mean PFD to which the leaf has been exposed in the past. Moreover, the lower activation status and induction states under flashing light as compared to continuous light under the same mean PFD implies an even greater limitation to sunfleck use in the variable light regimes in canopies than predicted by the mean PFD. The appropriate function to predict the set point is likely to be a saturating light response similar to that exhibited by assimilation itself. However, the appropriate integration time for this light response is still unknown. This must relate to time constants of the details of the activation mechanisms, which are poorly understood. Once the set point is established, then the immediate past activation states and the time constants for activation or deactivation that determine the dynamic responses come into play. Since the different light regulated enzymes as well as stomatal conductance have different time constants, different limitations will be established, depending on the past light history. The slow down regulation of Rubisco may be important in maintaining a high induction state during short shade periods, but as discussed earlier, it can also result in a shift of more of the limitation to RuBP regeneration.

IV. Regulation of the Transient Responses to Individual Lightflecks In this section, we consider the transient responses of

photosynthesis to changes in PFD that determine the dynamics of assimilation during a single short lightfleck. We focus at this point on the dynamics of assimilation that are primarily due to changes in metabolite concentrations and pools that drive the reactions but do not involve changes in activation state of PCRC enzymes. These metabolite pools can be measured simultaneously with gas exchange rates with a freeze-clamp chamber (Badger et al., 1984) that rapidly stops all metabolism as a leaf disk is punched directly thorough its plastic film windows. It is also possible to deduce the behavior of the metabolite pools from transient gas exchange analysis (Laisk et al., 1984), which provides a complementary technique. Since Rubisco is the ‘gatekeeper’ for uptake (Woodrow and Berry, 1988), changes in RuBP pools are of most immediate interest in determining the assimilation rate. However, other pools are also important the dynamics of assimilation rate because they influence the buildup of RuBP by serving as precursors or, in the case of PGA in particular, serve as the primary electron acceptor linking electron transport and carbon metabolism.

A. Dynamics of Assimilation Rate During Lightflecks The dynamics of assimilation during lightflecks of different durations and at different induction states are illustrated in Fig. 7. Resolution of lightfleck responses such as these requires a fast gas exchange system with minimum instrumentation response times. The methods for achieving these capabilities are discussed elsewhere (Küppers et al., 1993; Pearcy, 1993). When leaves are already at a high induction state (Fig. 7B and C), the assimilation rate rapidly accelerates at the beginning of a lightfleck as RuBP concentrations in the stroma increase. However a plateau is reached within 2 s, at which point RuBP is saturating for Rubisco and the assimilation rate depends on the activity status of Rubisco and the Thus, if the leaf is uninuced, the maximum rate achieved during a lightfleck will be rather low (Fig. 7A). If the leaf is only partially induced so that an RuBP regeneration (fast induction) limitation is present then the rate does not plateau sharply but continues to increase, though at a slower rate, during a lightfleck (Pfitsch and Pearcy, 1989b). Following a lightfleck, assimilation decreases at an exponentially declining rate until the steady-state rate in low PFD is reached. This decrease generally

Chapter 13 Photosynthesis in Fluctuating Light

occurs within a few seconds in induced leaves but can take many seconds in uninduced leaves (Fig. 7A). For long lightflecks, a postillumination burst due to photorespiration is also present and overlaps post-lightfleck assimilation (Fig 7C). This photorespiratory burst is not apparent after short lightflecks because the necessary pools of photorespiratory intermediates do not build up enough to support high rates of evolution. Moreover, in photorespiration, metabolites shuttle from the chloroplast to the peroxisome and ultimately to the

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mitochondria before evolution occurs. This could cause evolution to lag significantly behind the oxygenation reactions at Rubisco. Because of the lag between oxygenation and photorespiratory release, the observed apparent assimilation should be transiently higher at the beginning of a light increase, This gulp’ can sometimes be observed, but it is usually masked by other limitations. Bursts and gulps of due to changes in its solubility in the stroma as light-induced pH changes occur can also be observed under certain circumstances (Siebke

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et al., 1990). However, neither the changes in solubility nor the lag in photorespiratory release contribute directly to the net assimilation due to a lightfleck. They can, however, influence the dynamics of intercellular pressure during a lightfleck which then affects the rate of assimilation.

B. The Contribution of Post-Lightfleck Assimilation As shown above, a substantial part of the assimilation due to a lightfleck can occur after the lightfleck. Early experiments (Pollard, 1970; Kriedemann et al., 1973) established that the mean photosynthetic rate measured in intermittent (flashing) light could be significantly higher than the rate that would be expected if the rate were simply switching instantaneously between the steady state values for the high and low PFD. The same holds true for single short (1–5 s) lightflecks where the integrated assimilation can considerably exceed that predicted from the steady-state rates (Chazdon and Pearcy, 1986). The ratio, expressed as a percentage, of the integrated carbon gain due to a lightfleck to the ‘expected’ carbon gain calculated from the steadystate rates at the high and low PFDs is the lightfleck use efficiency (LUE), which been shown to be as high as 150 to 250% for short (1–5 s) lightflecks (Chazdon and Pearcy, 1986, Pons and Pearcy, 1992). The ‘expected’ carbon gain is the predicted amount if there were no dynamic elements in the photosynthetic apparatus and assimilation always behaved as if it instantaneously reached a new steady state. There are two possible reasons for a LUE greater than 100%. First, the maximum assimilation rates could be higher in a lightfleck than in steady state. Stitt (1986) demonstrated that this can occur at high pressures where flashing light (5 s high/5 s low) alleviated a phosphate limitation that constrained assimilation rates under steady-state. At 350 however, there is no evidence that the assimilation rates achieved are higher in lightflecks than in the steady-state. The second possibility is that significant energy storage occurs in the form of reducing power and phosphorylation potential during the lightfleck, which then allows assimilation to occur at an elevated rate over a longer time. Related to this is the fact that values remain on average higher during lightflecks than at steady-state because the assimilation is spread over a longer time. The higher values enhance carbon gain by reducing

oxygenation of RuBP and photorespiration. The second possibility requires both the capacity for transiently greater electron transport rates that provide for energy capture as well as a mechanism for energy storage. The second possibility appears to be the more common mechanism accounting for high LUEs, and the one that would function under current ecological conditions. The high LUEs are due to the presence of dynamic elements, namely to metabolite pools, which store significant energy and then lead to a substantial contribution of post-lightfleck assimilation. The storage of energy in these metabolite pools and their utilization is quantitatively more significant for short as compared to long lightflecks since post-lightfleck assimilation accounts for a higher proportion of the total carbon gain for short lightflecks.

C. Assimilatory Charge and Lightfleck Utilization The occurrence of significant post-lightfleck assimilation is consistent with the notion that metabolite pools act in a manner analogous to a capacitance. During a transient increase in PFD, the acceleration of assimilation rate is due to the buildup of metabolite pools that support a higher flux rate. In particular the acceleration of assimilation is related to the buildup of RuBP. The buildup of metabolites, including the reduced (‘high energy’) precursors to RuBP, however, also contributes to a pool of high energy intermediates that can support continued assimilation after the lightfleck. Laisk et al. (1987) has called the pool size of RuBP and its reduced precursors that support the post-lightfleck assimilation the assimilatory charge. The assimilatory charge can be measured either by determination of metabolite pool sizes (Sharkey et al., 1986; Osmond et al., 1988) or from gas exchange techniques by assuming an equivalence of the amount of postillumination fixation with assimilatory charge (Laisk et al., 1984). The latter assumption appears reasonable under low pressures where all RuBP produced should be consumed in carboxylation (Osmond et al., 1988). Laisk et al. (1987) have also shown that the kinetics of decline in the assimilation rate during the postillumination fixation phase are consistent with the expected kinetics of Rubisco as the assimilatory charge is depleted. An ‘effective’ assimilatory charge (the part of the assimilatory charge that contributes to assimilation) can also be estimated from the response to ‘darkflecks’, which

Chapter 13 Photosynthesis in Fluctuating Light are short periods of shade or darkness imposed on leaves at steady-state photosynthesis in saturating PFD (Roden and Pearcy, 1993b). No dip in assimilation rate will occur for short darkflecks since the assimilatory charge allows continued assimilation over this interval. For long darkflecks, however, the loss of assimilation is proportional to darkfleck duration. A plot of the loss of assimilation versus darkfleck length then yields an intercept on the ordinate equal to the effective assimilatory charge. This method provides an estimate of the effective assimilatory charge at steady-state in saturating PFD, which does not necessarily reflect its buildup during lightflecks. A similar approach can be applied for total assimilation due to lightflecks of different duration, but, in this case, the estimate is sensitive to any induction loss that might occur between lightflecks. In order for leaves to have LUEs above 100%, it is clear that they must be capable of building up an assimilatory charge during a lightfleck greater than that stored during an equivalent period under steadystate conditions. This is because the assimilatory charge must, via support of post-lightfleck assimilation, be sufficient to compensate for the assimilation lost during the acceleration phase and

337 also enough extra assimilation to account for the LUE. During steady-state photosynthesis, the rate of electron transport is coupled to the rate of carbon assimilation because of the need to regenerate as an electron acceptor and ADP for photophosporylation. During transient photosynthesis characteristic of a lightfleck, however, the rate of electron transport and carbon assimilation can become uncoupled due to the capacitive behavior of metabolite pools. This uncoupling can be directly observed in simultaneous measurements of and exchange carried out with a fast response zirconium-oxide ceramic cell oxygen analyzer used in combination with a analyzer (Kirschbaum and Pearcy, 1988a). As shown in Fig. 8, a large burst of release occurs at the beginning of a lightfleck. During this burst the rate of electron transport as indicated by the evolution rate greatly exceeds the rate of assimilation (Kiirats, 1985; Kirschbaum and Pearcy, 1988a). After a few seconds, the rate of electron transport becomes largely coupled to the rate of assimilation because of the need to regenerate acceptors. At this time, the rate of evolution is usually somewhat higher than the rate of assimilation because of electron donation to other processes such as nitrate reduction, sulfate reduction

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and reduction of other pools of acceptors such as ascorbate and glutathione. In an uninduced leaf, both the rates of assimilation and evolution are reduced at this time, but the reduction is greater for assimilation. Thus a larger relative difference exists between exchange and assimilation in the uninduced state. Also, low pressures have been shown to increase the difference between evolution and uptake (J. P. Krall and R. W. Pearcy, unpublished). At the end of the lightfleck, evolution decreases immediately because of the direct light limitation on PS II, while post-lightfleck assimilation continues. In an uninduced leaf, a small apparent uptake or gulp of occurs as evidenced by the dip in the evolution rate. The burst only occurs when the PFD during the lightfleck is greater than that required to saturate the assimilation rate at the current activity status of Rubisco and the current stomatal conductance. Thus for a fully-induced leaf, the PFD must be above that light saturation of assimilation under steady-state conditions but much lower-PFD lightflecks will cause an burst in uninduced leaves.

D. The Electron Acceptor During Lightflecks The considerable excess of electron transport during the burst requires that there be an acceptor readily available. Early experiments with thylakoids demonstrated an burst due to the rapid reduction of the plastoquinone (PQ) pool in the intersystem electron chain (Malkin, 1987). It is doubtful that this pool or other components of the electron transport system contribute significantly to the burst. First, the pool of PQ is sufficient to account for only about 15% of the total burst observed (Anderson, 1986) and this is by far the largest pool in the electron transport chain. Sometimes the burst can be resolved into two peaks with the initial, much smaller burst being of the right magnitude to correspond to reduction of PQ (Laisk et al., 1989). The total pool sizes of and are also too small for storage of significant reducing power (Takahama et al., 1981). ATP must also be generated, but the total adenylate pool is also relatively small. Some capacity for phosphorylation can be stored as a proton gradient across the thylakoid membrane. This capacity is presumably greater in shade chloroplasts because of their extensive thylakoid development. Given that the pool sizes of acceptors within the

electron transport system itself are insufficient, the acceptor of most significance must be in carbon metabolism or in other electron donation reactions within the chloroplast. It now seems clear that the primary acceptor is PGA. Following a high to low PFD transition, the PGA pool has been shown to build up to levels 6-fold greater than the steady-state level in the shade and 2-fold greater than the steadystate level in saturating PFD (Badger et al., 1984; von Caemmerer and Edmondson, 1986; SassenrathCole and Pearcy, 1992). Measurements of metabolite pool sizes before, during and after 5 s lightflecks show a rapid decline in PGA levels that corresponds to the buildup of the high energy intermediates, TP and RuBP (Sharkey et al., 1986). Comparison of the pool size changes of PGA, RuBP and TP during and after a 5 s lightfleck established that the changes were consistent with buildup of an assimilatory charge during the lightfleck and utilization of this assimilatory charge at a level sufficient to account for the observed 150% LUE. Use of TP during the post-lightfleck phase would require ATP, but this can readily be supplied via post-lightfleck ATP synthesis from the transthylakoid proton gradient and that which continues to be generated under low PFD. Measurements of postillumination ATP synthesis by thylakoids indicate a sufficient capacity to support the necessary amounts of ATP required (Hangarter and Good 1988). Some of the electron transport during the burst may involve alternative acceptors associated with processes such as nitrate and sulfate reduction, regeneration of ascorbate and glutathione, and thioredoxin reduction required for enzyme activation. In addition, some of the reduced carbon flows into photorespiration. With the exception of photorespiration where the competition is obvious, it is not known whether these alternative acceptors compete with carbon reduction during a lightfleck and therefore reduce the buildup of the assimilatory charge, or whether they simply utilize excess electron transport capacity. Only nitrate and sulfate reduction result in net exchange or reduction-state changes since, in the other cases, subsequent oxygen uptake reactions occur. Nevertheless, if there is a lag between the evolution and uptake, the non-oxygenic mechanisms may contribute to the dynamics of exchange. The importance of these lags is clearly evident for photorespiration in which the lag between evolution by PS II and uptake by the oxygenase

Chapter 13 Photosynthesis in Fluctuating Light reaction might be quite small, but the lag before evolution occurs is much greater. Whatever the pathway, it can contribute to the burst and to the difference between observed and exchange. Under steady-state conditions, exchange rates have been estimated to be about 15% higher than exchange rates because of the reduction of alternative acceptors, especially nitrate, in leaves at light saturation (Bloom et al., 1989). Measurements of exchange under 0.21 mol are difficult and cannot readily be applied to lightflecks where there is also a need for resolving rapid responses. However, electron transport rates can also be estimated from pulse-modulated chlorophyll fluorescence techniques, which can be applied with some limitations to resolve the dynamics of electron transport through PS II. At low (0.02 mol and for fully-induced leaves, there was good agreement between estimates of the total evolved and total electron transport through PS II (J. P. Krall and R. W. Pearcy, unpublished). Moreover, the total assimilation was between 80 and 100% of the total evolution or PS II electron transport (expressed as equivalents) during a lightfleck. At 0.21 mol and 350 pressures, PS II electron transport exceeded that required for net assimilation by about 40%, which would be expected from the effects of on oxygenation by Rubisco. However, fluorescence analysis gave a considerably higher transient maximum electron transport rate than indicated by evolution, especially in noninduced leaves. In non-induced leaves there was almost 50% more total electron transport than could be accounted for by evolution. Thus, at the beginning of a lightfleck in induced leaves, and even in total for uninduced leaves, there is substantial electron transport that is not visible as evolution. This could be the case if part of the flow went to as in the Mehler reaction or if cycling of electrons around PS II occurred. It should also be noted that glutathione and ascorbate represent large pools in the stroma that are reduced via electron transport (Asada, 1994; Chapter 5), and therefore might contribute to the dynamics of PS II electron transport but not to net evolution. The gulp of at the end of a lightfleck in an uninduced leaf (Fig 8B) may indicate a reoxidation of some pool reduced during the lightfleck.

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E. Electron Transport Capacity and Lightfleck Utilization It is clear that in order for there to be a burst of and for substantial energy storage to occur via rapid reduction of the PGA pool, an excess of electron transport over carboxylation capacity must be present. Transient measurements reveal that this condition is met (Stitt, 1986; Kirschbaum and Pearcy, 1988a). The peak rates of evolution observed during the burst have typically been 2- to 3-fold greater than the maximum assimilation rates (Kiirats, 1985). However, Laisk et al. (1992) reported maximum exchange rates for repeated 200 ms lightflecks that were five-fold greater than the steady-state rates. These high transient rates may reflect the local electron transport capacity around PS II to PQ rather than the maximum rate of storage of the assimilatory charge. The electron transport capacity in leaves is also known to be highly regulated in response to changes in PFD. Under continuous PFD above that required for saturation of carbon assimilation, PS II is known to be down regulated in a way that matches its capacity to that of the PCRC and also protects against photoinhibitory damage (Foyer et al., 1990; Harbinson et al., 1990a; Chapter 3). This down regulation reduces the quantum efficiency of PS II and also reduces the excess electron transport capacity required for efficient lightfleck utilization. The consequences of this down regulation can be seen under a reduction in PFD within the saturating range, where a transient reduction in evolution under saturating conditions occurs (Stitt et al., 1989). The transient reduction was shown to be due to the combination of lower PFD and lower PS II quantum efficiency while the recovery occurred as PS II quantum efficiency increased. Low PFD periods before lightflecks will therefore help promote high PS II quantum efficiencies and therefore the capacity for high electron transport rates necessary for high LUEs. Indeed, the quantum efficiency of PS II has been shown to be higher during a lightfleck at a given PFD as compared to constant light of the same PFD (J. P. Krall and R. W. Pearcy, unpublished ).

F.

Photosynthesis and Lightfleck Utilization

photosynthesis presents a special problem because of the more complex pathway and the spatial

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separation of the generation of reducing power and carbon reduction. This has been shown to lead to LUEs that are less than 100% during short duration lightflecks (Krall and Pearcy, 1993). In NADP-malic enzyme type plants such as maize, the majority of all PS II activity is considered to be in the mesophyll whereas carbon reduction in the PCRC is in the bundle sheath. In this case, part of the PGA is reduced following its diffusion from the bundle sheath to the mesophyll, and the resultant TP then diffuses back to the bundle sheath. The remaining PGA is reduced by NADP produced in the decarboxylation of malate in the bundle sheath. Measurements with maize have shown that for brief (<2 s) lightflecks, there is a burst that greatly reduces the LUE. This burst may occur because there is insufficient time for TP-PGA exchange to meet the requirements of the PCRC in the bundle sheath. Thus, part of the released in the bundle sheath during the lightfleck leaks back out. The burst does not occur during longer lightflecks where there is sufficient time for the TP-PGA shuttle to occur. Interestingly, several shade species, such as the Hawaiian understory tree, Euphorbia forbesii (Pearcy and Francheschi, 1986) and the grass Paspalum conjugatum are NADP-malic enzyme type plants and potentially subject to constraints similar to those observed in maize. It is possible that evolutionary adaptation has somehow minimized these constraints or that it is relatively unimportant given the relatively small contribution of short duration sunflecks in forest understories. It may be more important in crop canopies where short duration sunflecks do make a significant contribution. Further studies will be needed to understand its significance in canopies.

V. Are There Specific Adaptations in Shade Leaves for the Utilization of Sunflecks? Studies now conducted with a variety of species indicate that the dynamic properties of the photosynthetic apparatus that are important in sunfleck utilization are an inherent property for all plants and not just those for which sunfleck utilization is important. The question still remains, however, as to the extent that these dynamic properties can be modified, either by evolutionary adaptation or by acclimation, and therefore differentially contribute to sunfleck utilization. In the case of the buildup of assimilatory charge and post-lightfleck

assimilation, it seems clear that relative to their photosynthetic capacity all plants possess roughly the same capacity for buildup of an assimilatory charge. The LUEs reported for sun- and shadegrown soybeans and for shade-grown Alocasia macrorrhiza are all roughly the same for short (1– 5 s) lightflecks (Kirschbaum and Pearcy, 1988a; Pons and Pearcy, 1992). Similar values have also been found for the shade intolerant tree, Populus tremuloides (Roden and Pearcy, 1993b) and the shade tolerant tree, Fagus sylvativca (Küppers and Schneider, 1993). For longer lightflecks, the lower efficiencies often reported for sun plants seem to be due to the tendency for induction to be lost more rapidly when high-light adapted plants are shaded. A greater electron transport capacity relative to carboxylation capacity in leaves could be expected to favor high LUEs. However, shade-grown pea leaves were reported to have a lower ratio than sun-grown leaves (Evans, 1987). The ratios of electron transport to carboxylation capacity for Alocasia were independent of the growth light environment (Sims and Pearcy, 1989). However, some tropical forest tree and shrub species have been reported to have quite high ratios of electron transport to carboxylation capacity (Walters and Field, 1987; Thompson et al., 1988) but it is unknown how these species respond to lightflecks. The total assimilatory charge was greater in sun- than shade-grown sunflowers, but relative to the photosynthetic capacity, which was also greater in the sun-grown plants, they were the same (Osmond et al., 1988). This proportionality should generate equal LUEs. Metabolite pool sizes have been found to increase in sun grown relative to shade grown plants but again this is in proportion to the increase in photosynthetic capacity (Seemann et al., 1987). A higher photosynthetic capacity in itself should enhance sunfleck utilization provided that a greater induction limitation does not offset its effects. Thus, any differences in the capacity to utilize sunflecks must lie in the induction responses. For a given species, shade-grown leaves tend to lose induction slower than sun-grown leaves. Differences in stomatal behavior cause different induction limitations during sun-shade transitions (Knapp et al., 1989). Moreover, differences in the stomatal response to lighflecks among sun- and shade-grown plants can certainly cause different induction limitations to subsequent lightflecks. In addition, the RuBP regeneration limitation is more pronounced in shade- than sun-grown leaves, and varies between

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species. However, it is not yet understood what particular induction behaviors minimize the constraints in a given habitat.

minutes of sunflecks received in different microsites as derived from hemispherical canopy photographs and the growth rate of tree seedlings in these microsites (Pearcy, 1983; Oberbauer et al., 1988).

VI. Sunfleck Utilization in Natural Light Regimes

B. How Much Does the Induction Requirement Limit, or Post-lightfleck Assimilation Enhance Carbon Gain in Natural Sunfleck Regimes?

A. The Total Contribution of Sunfleck Utilization to Daily Assimilation in Natural Light Regimes Determination of the daily contribution of sunfleck utilization to the leaf carbon balance can be done by integration of the diurnal course of photosynthesis. Only a few data sets of this type are available and these show wide variation in the role of sunflecks. On clear days in tropical forest understories, 30 to 60% of the daily assimilation can be due to sunfleck utilization (Björkman et al., 1972; Pearcy and Calkin, 1983; Pearcy, 1987; Pfitsch and Pearcy, 1989a). In these cases the contribution of sunfleck utilization to the daily assimilation total was roughly in proportion to the contribution of sunflecks to the daily total PFD. On the other hand, results from deciduous forests reveal only a 10 to 20% contribution due to sunfleck utilization (Schulze, 1972; Weber et al., 1985). This difference may be due to the higher diffuse light level in deciduous forests and relatively low photosynthetic capacities of the deciduous forest species studied. Low photosynthetic capacity minimizes the potential role of sunflecks. Measurements of the daily carbon gain of Adenocaulon bicolor in different microsites in a redwood forest revealed utilization of sunflecks contributed 30 to 65% of the total on clear days. Daily carbon gain was most closely related to sunfleck PFD in the different microsites and was less closely related to differences in diffuse PFD (Pfitsch and Pearcy, 1989a). On an annual basis, the contribution should be less because in most environments there are many cloudy or at least partly cloudy days. Few studies have been carried out on a seasonal basis. For A. bicolor, sunfleck utilization was estimated to contribute from 9 to 44% of the annual carbon gain on the basis of the differences in ratios of plants from sites differing the amount of sunfleck PFD received (Pearcy and Pfitsch, 1991). The potentially important role of sunflecks therefore extends to the annual carbon gain but also differs substantially between sites. Further evidence of the important role for sunflecks comes from the correlations between estimates of the

While the measurements discussed above establish that sunfleck utilization is an important contribution to the total carbon gain for many forest understory plants, they do not address the extent to which induction or post-lightfleck assimilation influences this total. The role of these dynamic factors are still poorly understood because of the difficulty in quantifying them under complex natural light regimes. Moreover, in crop canopies in particular, individual leaves may be partially in sunflecks and partially in shade at any time. Measurements with a leaf in a gas exchange chamber may therefore reflect as much the spatial average of this pattern rather than the true response of the photosynthetic apparatus to the sunflecks. Consequently, in order to assess the role of the dynamic responses to sunflecks, modeling approaches must be adopted. One approach is to combine model and measurement by predicting daily carbon gain from a steady-state model, which assumes an instantaneous response to light changes and therefore has neither the induction limitations or the postlightfleck fixation enhancements, and then comparing the prediction to actual measurements which do have the influences of the dynamic responses. When this was done for A. bicolor, the measured daily assimilation values were typically 20 to 30 % lower than the modeled values (Pfitsch and Pearcy, 1989a). On a day without sunflecks, however, when assimilation should be near steady-state nearly all of the time, the prediction and measurement were within 3%. Thus the model appeared to predict accurately assimilation under steady-state conditions. The lower measured than predicted carbon gain on days when sunflecks were present can be interpreted as indicating a strong induction limitation. Given that most of the PFD in the redwood forest understories is contributed by the relatively infrequent long sunflecks, conditions that are conducive to an induction limitation with little post-lightfleck assimilation, this interpretation is not surprising. It

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should be noted that the reductions are for the total daily assimilation and that if only the fraction due to sunflecks was considered, the relative differences between the steady-state model and the measured carbon gain would be even larger. A related approach to assessing the role of the dynamic responses is to compare the output of a dynamic model and a steady-state photosynthesis model under different light regimes. Gross et al. (1991) developed a semi-mechanistic, dynamic model for photosynthesis based on the steady-state model of Farquhar and von Caemmerer (1982). The dynamic modifications included differential equations with time constants for the light regulation of Rubisco along with metabolite pools that provided an assimilatory charge utilized for postillumination fixation during lightflecks as well as development of photorespiratory burst after long lightflecks. It also incorporated a previously developed dynamic stomatal model (Kirschbaum et al., 1988) that predicted the pulse response of to lightflecks. Subsequent modifications (Pearcy et al., 1994) added time-dependent steps simulating light regulation of RuBP regeneration capacity to simulate the fast induction limitation. The resulting model gave a good agreement with measurements for induction responses and for the responses to single lightflecks of different duration, including prediction of the post-lightfleck fixation. The model is driven by PFD values recorded at 0.1 to 1 s intervals which gives an output at a similar time step. When parameterized for Alocasia macrorrhiza and applied to light regimes digitally recorded at different sites in a tropical forest where this species occurs, the dynamic model predicted a daily carbon gain 1 to 26% lower than the steady-state model. The difference between the steady-state and dynamic prediction was greatest when sunflecks contributed more than 50% of the daily PFD but otherwise no particular relationship between the nature of the sunfleck regimes and the difference was apparent. Changing parameters in the model to selectively eliminate specific dynamic components indicated that the greatest limitation was caused by stomata with light regulation of Rubisco being a secondary factor. By contrast, the predicted contribution of post-lightfleck assimilation was insignificant. When the model was parameterized for the photosynthetic characteristics of soybean leaves and run with light inputs measured in a soybean canopy, the dynamic model gave a predicted daily assimilation

that was 1% higher to 12% lower than that of the steady-state model. Measurements in a similar soybean canopy showed that leaves in the shade at any given instant had on average an of 0.52, indicating a strong induction limitation (Pearcy and Seemann, 1990). The values for individual leaves varied widely from 0.2 to 0.9), presumably reflecting variation in the history of exposure to sunflecks over the previous several to many minutes. Stomatal conductance was correlated with induction state and with initial Rubisco activities. Thus, the prediction of a general induction limitation in this canopy was in agreement with the observed induction responses. However, in this case, selective elimination of dynamic components indicated that the fast induction limitation was a more significant limitation than either Rubisco or stomatal dynamics. In soybean canopies, many leaves experience periods of high light or frequent sunflecks that give a high induction state, followed by shade periods. During these shade periods the fast induction limitations could be expected to develop rapidly. Because of the many frequent short sunflecks, post-lightfleck fixation could be expected to be significant, especially on windy days. Indeed, post-lightfleck fixation may have increased the carbon gain by as much as 10%, thereby offsetting some of the induction limitations in this canopy. Some studies have also been conducted in aspen canopies that are characterized by highly dynamic light regimes because of leaf flutter under even light breezes (Roden and Pearcy, 1993a). Aspen tends to maintain high even in the understory and consequently this potential limitation is relatively significant. Measurements with aspen revealed little stomatal limitation to induction. Paired light measurements for individual leaves on windy and still, but otherwise clear days were used to test the consequences of leaf flutter on carbon gain. The dynamic model predicted carbon gains 6 to 21% higher on the windy as compared to still days, which was due to both the greater post-lightfleck assimilation as well as increased light penetration to lower canopy levels (J. Roden and R. W. Pearcy, unpublished). In conclusion, the dynamic responses of photosynthesis of importance in sunfleck utilization clearly have different effects in different canopy situations. Taken together, the effects cause substantial deviations from steady-state predictions with induction limitations dominating in the understory, where long

Chapter 13 Photosynthesis in Fluctuating Light infrequent sunflecks make the greatest contribution, but with postillumination fixation making a substantial contribution in canopies such as soybean and aspen where there are an abundance of short frequent sunflecks. There are, of course, differences through canopies in the relative role of each of these dynamic factors that are not yet well understood. Further studies are needed to scale up to the consequences of sunfleck utilization for whole canopy carbon gain, so that the overall significance of the dynamic photosynthetic responses can be understood.

Acknowledgments This research was supported by grant 91-371006670 from the USDA competitive Grants Program. We thank Dr. Gary Harris for his constructive comments on the manuscript.

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behavior and its role in carbon gain during lightflecks of a gap phase and an understory Piper species acclimated to high and low light. Oecologia 92: 222–228 Tinoco-Ojanguren C and Pearcy RW (1993a) Stomatal dynamics and its importance to carbon gain in two rainforest Piper species. I I . Stomatal versus biochemical limitations during photosynthetic induction. Oecologia 94: 395–402 Tinoco-Ojanguren C and Pearcy RW (1993b) Stomatal dynamics and its importance to carbon gain in 2 rainforest Piper species. I. VPD effects on the transient stomatal response to lightflecks. Oecologia 94: 388–394 von Caemmerer S and Edmondson DL (1986) Relationship between steady-state gas exchange, in vivo ribulose bisphosphate carboxylase activity and some carbon reduction cycle intermediates in Raphanus sativus. Aust J Plant Physiol 13: 669–688 Walker DA (1973) Photosynthetic induction phenomena and the light activation of ribulose diphosphate carboxylase. New Phytol 72: 209–235 Walker DA (1981) Photosynthetic induction. In: Akoyonoglou G (ed) Proceedings of the 5th International Congress on Photosynthesis Vol IV, pp 189–202. Balaban International, Philadelphia

Walters MB and Field CB (1987) Photosynthetic light acclimation in two rainforest Piper species with different ecological amplitudes. Oecologia 72: 449–456 Watling J and Woodrow I (1993) A new kind of induction response found in two rainforest species. In: Murata N (ed) Research in Photosynthesis, Vol I, pp 189–192. Kluwer, Dordrecht Weber JA, Jurik TW, Tenhunen JD and Gates DM (1985) Analysis of gas exchange in seedlings of Acer saccharum: integration of field and laboratory studies. Oecologia 65: 338– 347 Woodrow IE and Berry JA (1988) Enzymatic regulation of photosynthetic fixation in plants. Annu Rev Plant Physiol Plant Mol Biol 39: 533–594 Woodrow IE and Mott KA (1989) Rate limitation of non-steadystate photosynthesis by ribulose 1,5-bisphosphate carboxylase in spinach. Aust J Plant Physiol 16: 487–500 Woods DB and Turner NC (1972) Stomatal response to changing light by four species of varying shade tolerance. New Phytol 70: 77–84 Zeiger E (1983) The biology of stomatal guard cells. A n n u Rev Plant Physiol 34: 441–475

Chapter 14 Leaf Photosynthesis Under Drought Stress Gabriel Cornic Laboratoire d’Ecologie Végétale, Groupe Photosynthèse et Environnement, URA CNRS 1492. Bât.362, Université de Paris XI, F-91405, Orsay, France

Angelo Massacci Istituto di Biochimica ed Ecofisiologia Vegetali, CNR. 00016 Monterotondo Scalo, Roma, Italy

Summary I. Introduction A. Stomatal Versus Non-stomatal Effects of Drought on Leaf Photosynthesis B. Stomatal Effects and the Use of Non-destructive Techniques II. The Resistance of Photosynthetic Mechanisms to Drought Molar Fraction A. Evidence from Measurements at High B. Evidence from Measurements of Leaf Chlorophyll Fluorescence Concentration Inside the Chloroplast during Drought is Low III. IV. Changes in Metabolic and Whole Leaf Photosynthetic Responses Induced by Water Deficits A. Effect on Carbohydrate Partitioning B. Decline in Nitrate Reductase Activity During Drought Uptake Response to Environmental Factors C. Modification of Net Leaf V. Maintenance of Plant Water Content During Soil Drying A. Reduction of Transpiration B. Maintenance of Water Uptake VI. Light Utilization by Plants Under Drought A. Leaf Movements and Orientation B. Light Utilization by PS II 1. Thermal Dissipation 2. Other Processes Dissipating Excitation Energy C. Photoinhibition on Dehydrated Leaves VII. Conclusions Acknowledgments References

347 348 348 349 351 351 353 354 356 356 357 357 358 358 359 359 359 360 360 361 361 362 363 363

Summary The photosynthetic apparatus is resistant to drought. Net uptake of a leaf submitted to a mild desiccation decreases because of stomatal closure. As a result, concentration in the chloroplast decreases in plants exposed to water shortage. This drop in the chloroplast concentration causes: (i) a decrease in photochemical yield of open PS II centers and, consequently, an increase of thermal dissipation of the excitons trapped in PS II units; (ii) a decline in the activity of some enzymes, e.g. sucrose phosphate synthase and nitrate reductase; (iii) an increase in the activity of ribulose 1,5-bisphosphate oxygenase. The water status of the plants can be maintained under fluctuating water supply through a regulation of water Neil R. Baker (ed): Photosynthesis and the Environment, pp. 347–366. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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loss and water uptake, in which abscissic acid plays a major role. The role of photorespiration in protecting the photosynthetic apparatus against high light damage is unclear. Leaf movements (paraheliotropism) and thermal dissipation of excitons trapped in PS II units are probably much more efficient mechanisms than photorespiration to protect photosystems against photoinhibition. Drought also causes large changes in carbon partitioning at the cellular and plant levels, and profound modifications in the composition of membrane proteins and lipids in the photosynthetic apparatus.

I. Introduction Drought has dramatic effects on plant growth and morphogenesis and is likely to be one of the major environmental factors determining plant productivity and species distribution (Woodward, 1987; Wright, 1992). Its impact in arid environments, where it can severely limit plant growth, is obvious. However, even when the annual rainfall averages 2600 mm, as in Barro Colorado island (Panama), the low soil water potentials (about–1 MPa) observed in the first ten cm of the soil during the dry season (from December to April) are sufficient to affect terrestrial herbs and also trees to a lesser extent. Limited root development relative to the shoot, caused by growth under low light, probably contributes to low drought tolerance in understory plants (Wright, 1992). Also in rain forests, because of the height of the trees and the high evaporative demand during the day, the upper part of the canopy can undergo temporary low water potentials that correlated with stomatal closure. Due to its economic importance, the negative impact of water shortage on plant productivity has been studied extensively. In many cases, as water deficit increases during a drought, the limitation to plant growth is first exerted through a decrease in the Abbreviations: A – net rate of assimilation; – gross rate of assimilation; ABA – abscisic acid; concentration in ambient air, intercellular spaces and chloroplast, respectively; – maximum, minimal and variable fluorescence emission, respectively; – relative photochemical efficiency of open PS II traps; – leaf and internal conductance to respectively; – flux of electrons used for carboxylation and for oxygenation, respectively; –rate constant for excitation energy dissipation in PS II antenna; LNU – light not used for photochemistry; LWD – leaf water deficit; NR – nitrate reductase; PCR – photosynthetic carbon reduction; PGA – 3-phosphoglyceric acid; PPFD – photosynthetically-active photon flux density; – photochemical quenching; Rubisco – ribulose 1,5-bisphosphate carboxylase-oxygenase; RuBP – ribulose 1,5-bisphosphate; RWC – relative water content; S – specificity factor of Rubisco; SPS – sucrose phosphate synthase; – relative quantum yield of PS II photochemistry; – quantum yield of leaf photosynthesis; – maximum quantum yield of leaf photosynthesis.

growth rate of the assimilatory surface, and then later through an inhibition of leaf photosynthesis (Boyer, 1970; Ephrath and Hesketh, 1991). Drought stress effects on different aspects of plant productivity (Hanson and Hitz, 1982; Lawlor and Uprety, 1993) and plant water relationships (Boyer, 1985) have been reviewed. Extensive measurements have been made on plants under limiting water supply. In particular the effect of water deficit on the mechanisms of leaf photosynthesis has been examined carefully in order to understand the processes which limit net uptake in drying plants, with a view to improving the effects of this constraint through plant breeding. Owing to the array of changes induced by drought in the photosynthetic apparatus, this view was perhaps somewhat naive, and the most obvious results of this belief have been a frequent cause of conflicts between plant breeders and plant physiologists.

A. Stomatal Versus Non-stomatal Effects of Drought on Leaf Photosynthesis It has long been known that stomatal closure and net assimilation rate of a leaf (A) decline occur in parallel during drought. Consequently stomatal closure was thought to be the primary cause of the depressed leaf photosynthesis in dehydrated plants. However, as leaf net photosynthesis rate can be simply expressed as:

where is the leaf conductance for and and are the concentrations in the ambient air and at the mesophyll cell wall respectively, it is obvious that decline in A while is decreasing can also be due to a direct effect of the constraint on photo– synthetic mechanisms. This will affect (the internal concentration) causing a decrease in The wide use of measurements to asses the effect of constraints on leaf photosynthesis followed the theoretical work of the Canberra group (Farquhar

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and Sharkey, 1982), the development of leaf gas exchange analysis and the commercialization of straightforward gas exchange systems. During a drought either remained constant or decreased to an extent which could not be explained solely by stomatal closure (Lawlor, 1995). Thus, it was postulated that some non-stomatal components of leaf photosynthesis were affected by desiccation. This conclusion was in very good agreement indeed with earlier work on chloroplasts isolated from wilted leaves showing that drought rapidly impaired PS II activity (Boyer, 1976), inhibited the thylakoid ATP synthase (Boyer and Younis, 1983) and effectively induced a whole set of responses within the photosynthetic apparatus (Hanson and Hitz, 1982). However, as early as 1983, Laisk pointed out that calculation of by conventional analysis of leaf net uptake and transpiration, in plants submitted to constraints, could be misleading if stomatal closure was not homogeneous. Moreover the following year, Hashimoto et al. (1984), using a thermal image processing system to analyze the development of water deficit from leaves of sunflower plants subjected to a drought in a controlled environment, showed significant differences in temperature and water status of different parts of the leaves. The water deficit, which developed first at the margin of the leaves, was accompanied by stomatal closure and an increase of the temperature. It has been demonstrated for leaves fed with ABA (Downton et al., 1988; Terashima et al., 1988) and desiccated plants (Terashima, 1992) that the calculated value of can be quite misleading. Part of the problem regarding calculation is likely to be due to the heterogeneity of leaf photosynthesis when plants are subjected to stress. This heterogeneity is presumably the consequence of patchy stomatal closure and/or collapse of parts of the mesophyll due to loss of turgor, associated with a low lateral diffusion capacity (Cornic et al., 1989; Daley et al., 1989). It is probable that this ‘patchy photosynthesis’ occurs mainly when dehydration is rapid, which is often the case when drought experiments are performed on potted plants by withholding water. However, it may not occur in the field where dehydration can be slower (Gunasekera and Berkowitz, 1992). The fact that ‘patchiness’ may occur during drought (or during any other constraint) makes it necessary to assess its extent if calculation of is used to quantify stomatal and non-stomatal effects of the constraint. As pointed out by Cheeseman

in a theoretical study (1991) the error in the estimation of is probably not large (see also Chapter 8). However, it can change the response of assimilation rate to ( curve) quite substantially (Gunasekera and Berkowitz, 1992) and introduce a bias when calculating stomatal limitation by one of the current methods (Jones, 1985) as in Kubiske and Abrams (1993). The occurrence of ‘patchiness’ in any situation will inevitably lead to an underestimation of the limitation imposed by stomatal closure on leaf photosynthesis. Another aspect which is not often taken into account when discussing calculation is the cuticular transpiration. When stomata closed during drought, or after feeding a leaf with ABA through the transpiration stream (Meyer and Genty, 1996) cuticular transpiration becomes relatively more important and could be then the main cause of overestimation. This effect could well explain why rises most of the time when the rate of leaf uptake (and the rate of transpiration) is low in plants with a low relative water content (Cornic et al., 1987). It should be noted that this problem was first discussed as early as 1971 in a review by Jarvis.

B. Stomatal Effects and the Use of Nondestructive Techniques The development of non-destructive techniques in the last decade has provided an alternative set of methods to study the effect of constraints on leaf photosynthesis. They allow measurement of leaf photosynthesis in terms of evolution and estimation of electron transport rate and related activities within the thylakoid membrane despite stomatal closure. The high molar fractions used in the leaf disc oxygen electrodes provide a gradient large enough between the ambient air and chloroplast to supply the photosynthetic apparatus with its substrate even when stomata are closed. Consequently this allows determination of whether the photosynthetic mechanisms are impaired by a given treatment (Kaiser, 1987). Measurements of modulated chlorophyll fluorescence yield of intact leaves makes it possible, with the use of a proper model (Weis and Berry, 1987; Genty et al., 1989), to estimate rates of photosynthetic whole chain electron transport and study the regulation of light utilization at the PS II level in intact leaves (Chapters 2 and 3). The changes in absorbance of a leaf at various wavelengths upon illumination with actinic light

350 allows measurement of physiological activities associated with light energy transduction by thylakoids. Absorption changes at 820 nm can be used to determine the oxidation state of PS I (Harbinson et al., 1989; Chapter 3). The kinetics of relaxation of flash-induced absorption changes at 518 nm can provide information about in situ rates of photophosphorylation (Ortiz-Lopez et al., 1991) and it has been suggested that light scattering changes at 535 nm can be used to assess thylakoid energization (Schwab et al., 1989). Studies that started in the early eighties, either on cells and chloroplasts isolated from hydrated leaves or on intact leaves, using these non-destructive techniques (reviewed by Kaiser, 1987) showed that the photosynthetic apparatus is very resistant to water deficit. It was concluded that the decrease in A observed in most of the natural conditions during drought was mainly due to a stomatal closure. In particular, it was demonstrated that photosystem activities, whole chain electron transport (Kaiser et al., 1981; Sharkey and Badger, 1982) and related processes, as well as the activities of the photosynthetic carbon reduction cycle, were not impaired by a mild drought (maximum leaf water deficit not higher than 30%; Kaiser, 1987; Cornic et al., 1989; Chaves, 1991). This new picture of the response of photosynthesis to drought was in contrast with the one described in the previous section, obtained using measurements and destructive techniques. The reported effects of dehydration on PS II and ATP synthase activities, and other effects on whole chain electron transport, were then believed to be either artifacts of the isolation procedures of the photosynthetic membranes from wilted leaves, or the result of photoinhibition occurring during the constraint. This does not mean, of course, that photosynthetic system function cannot be damaged by dehydration when water deficit is high enough (Kaiser, 1987; Cornic et al., 1992). After rehydration (except for resurrection plants) the photosynthetic activity of a leaf which has been severely dehydrated does not recover to the rate it showed before the drought. The nature of the inhibition of photosynthetic mechanisms during severe desiccation is not yet well understood. It could be, at least partly, related to a reduction of cell volume which occurs as plants dehydrate. In agreement with this idea, Kaiser (1982) showed that leaf slices from plants having a very different solute concentration (osmotic potential) at water saturation, exhibit during dehydration in the absence of stomatal

Gabriel Cornic and Angelo Massacci control (measurements made at saturating molar ratio) very different relations between leaf water potential and photosynthesis but the same relationship between photosynthesis and cell volume. The reduction of cell volume would cause an increase of concentration of some critical anions in the chloroplast stroma (phosphate and sulfate; Kaiser et al. 1986), which in turn would inhibit unspecifically the activities of photosynthetic enzymes. Membrane damage and increase in protein concentration, which can causes protein crystallization in the chloroplast stroma (Vapaavuori and Nurmi, 1982) at low relative water content (lower than 50%), could also be involved in drought induced inhibition of photosynthesis. The relation observed between photosynthetic capacity and cell volume by Kaiser (1982) suggests that this parameter should always be directly measured (Berkowitz and Kroll, 1988) or at least estimated by measuring the relative water content or the leaf water deficit when assessing the effect of drought on photosynthetic activity. This probably would help clarify the meaning of ‘mild stress’ (leaf water deficit lower than 30%) and provide a framework for discussing stomatal and non-stomatal effects. The resistance of the photosynthetic apparatus to mild drought implies that the molar fraction in the chloroplasts of an illuminated dehydrated leaf is low and that the light energy which cannot be eliminated through photosynthetic carbon dioxide reduction must be channeled to other dissipation processes. Dry environments or dry periods during a year are often associated with high solar irradiation and high temperatures, and this may impose additional pressures on the photosynthetic apparatus. Plants subjected to drought stress must cope with excess light and have developed mechanisms to prevent excess excitation energy from arriving at the reaction centers of the photosystems. These mechanisms exist both at whole plant and at molecular levels. They include leaf movements, non-radiative dissipation of light energy, and enhanced photosynthetic reduction of Plants also react rapidly to the changes in environmental conditions, receiving signals from either the drying soil or from the ambient atmosphere. Most of the mechanisms involved, which include stomatal closure, osmotic adjustment and a decrease of shoot growth relative to roots, tend to maintain water status (cell volume) of the leaves within limits where the photosynthetic apparatus is unlikely to be

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damaged (0 to 30% water deficit). It is important to identify and understand these signals if we are interested in resolving the factors that determine maximum plant productivity under limiting water supply. Osmotic adjustment is particularly important to consider since it is also related to leaf growth. The aim of this review is to outline briefly the major evidence which shows that the photosynthetic apparatus is resistant to dehydration, the consequences of such resistance, and how plants in dry environments maintain water content and cope with excess light energy.

II. The Resistance of Photosynthetic Mechanisms to Drought

A. Evidence from Measurements at High Molar Fraction The variation of photosynthetic capacity of plants (measured as evolution in a leaf disc oxygen electrode) as a function of leaf water deficit (LWD) is shown Fig. 1A. It has been calculated from the published results obtained on range of plants from contrasting habitats (see the legend of the Fig. 1 for the list of the plant species). Measurements were made at saturating molar fractions (ranging from 5 to 17%) when dehydration was achieved either rapidly by cutting leaves or slowly by withholding watering for up to three weeks (Kaiser, 1987; Cornic et al., 1989; M. Brestic, unpublished). It is striking to note that such a curve with data of such low standard deviation can be obtained when considering the response of hygro-, meso- and xerophytes. While the resistance to water loss and the capacity for osmotic adjustment differs in hygro-, meso- and xerophytes, the sensitivity of photosynthetic mechanisms to water loss is similar in all three types of plants. This ‘mean’ curve, probably represents the sensitivity of the photosynthetic capacity to drought. Photosynthetic biochemistry is significantly affected only at a leaf water deficit of about 30%. Net leaf assimilation measured in normal air on many plants declines rapidly with increasing water deficit, being negligible at around 30% LWD (Cornic et al., 1989; Schwab et al., 1989). Thus, the data shown in Fig. 1A strongly suggest that stomatal control explains most of the observed decrease in leaf photosynthesis in plants submitted to mild drought. They suggest also that drought affects photosynthetic mechanisms through

an effect on cell volume estimated here as (1 -LWD), which equates to relative water content. This parameter, as discussed in the introduction, is more suitable than leaf water potential when studying drought effects on photosynthetic activities (Kaiser et al., 1981). It is relevant to note that this mean response to water deficit obtained by measuring leaf photosynthesis at elevated molar fractions using an oxygen electrode bears great similarities with the response to desiccation of a Ramonda mykoni leaf stripped of its lower epidermis and maintained in

352 normal air molar fraction 350 Schwab et al., 1989). The mean variation of maximum apparent quantum yield measured at high molar fraction on three different plants does not vary much over a 30% LWD range (Fig. 1B) showing that whole chain electron transport and related processes are also very resistant to dehydration. This is in agreement with (i) the observation that maximum PS II photochemical efficiency, estimated by the ratio of variable to the maximum fluorescence measured either at liquid nitrogen temperature or at room temperature after a long period of dark adaptation, is not affected by dehydration sufficient to fully inhibit net uptake by leaves (Björkman and Powles, 1984; Ben et al., 1987; Cornic et al., 1987, 1989; Genty et al. 1987); (ii) a study made using the flash-induced electrochromic absorption change at 518 nm which showed that the coupling factor is reduced, even in severely dehydrated leaves of sunflower, almost immediately upon illumination (Ortiz-Lopez et al., 1991); and (iii) the increase of light scattering that is observed when stomatal closure decreases leaf photosynthesis (Dietz and Heber, 1983). Quick et al. (1989), in agreement with the above conclusion, have shown that the ratio 3-phosphoglyceric acid (PGA) to triose phosphate was decreased and that the ratio of triose phosphate to ribulose 1,5bisphosphate (RuBP) increased in osmostically shocked spinach leaf discs maintained at a high molar fraction in a leaf disc oxygen electrode only when protoplast volume was reduced by about 30%. Obviously the operation of the photosynthetic carbon reduction (PCR) cycle, including RuBP regeneration, is not impaired in spinach leaf discs submitted to a mild drought. Moreover, in the same experiment, the RuBP/PGA ratio remained unchanged although protoplast volume was reduced by about 70% and net evolution by about 90%, providing clear evidence that Rubisco is not a prime target of water deficit. Sharkey and Seemann (1989) came to a similar conclusion by analyzing metabolite pool size on French bean leaves maintained in normal air and submitted to a mild drought and ‘found no evidence for a lesion in the chloroplast biochemistry necessary for photosynthesis’. It has often been claimed that the high molar fractions, which are necessary when using an oxygen electrode, would cause cellular pH to decrease and thus somehow inhibit leaf activity (Ögren and Evans, 1993). However, measurements published so far show,

Gabriel Cornic and Angelo Massacci for hydrated leaves of plants, that the maximum rate of photosynthesis, currently reached at a molar ratio of 2–3%, is very similar (or even 10 to 15% higher) to that measured using a standard gas exchange system at a saturating molar fraction. Moreover, it is possible to show using fluorescence techniques (see below) that in dehydrated bean leaves (LWD of about 30%) a molar fraction as high as 10–12% is necessary to inhibit the oxygenase function of Rubisco (M. Brestic, B. Genty, G. Cornic and N. R. Baker, unpublished). The experiments reported in Fig. 1 are difficult to perform on plants because high molar ratio of (above 2%) inhibits net photosynthesis of well hydrated leaves (see Fig. 2). The reasons for this inhibition are not well understood. However, as shown for maize leaves in Fig. 2, it is possible to at least partially reverse the negative effect of water deficit on leaf photosynthesis by increasing molar fraction, (presumably because of the inhibitory effect of high concentrations). Similar results were also obtained on Eleusina indica, although the effect of high molar fraction was less marked than in maize leaves (K. Saccardy and G. Cornic, unpublished). Thus, there probably also exists in plants a strong stomatal effect of drought on leaf photosynthesis. However, following the arguments of Lawlor

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(1995), and assuming (i) that the high molar ratio which is used inside the oxygen electrode causes a complete stomatal closure on dehydrated leaves, so that leaf conductance for is close to the cuticular conductance (about 0.004 mol Meyer and Genty, 1996), (ii) that the boundary layer conductance inside the cuvette is about 0.015 mol (1 cm of still air at around 25 °C), and (iii) that the internal molar fraction which is enough to saturate RuBP oxygenase is approximately 500 we calculate a value of about 0.6% for A = 20 There obviously is a problem here since 5 to 15% ambient is needed to saturate photosynthesis of desiccated leaves. It could be that the boundary layer resistance around a leaf disc in the compartment of an oxygen electrode is much higher than has been calculated here due to the leaf disc adhering to the glass of the top part of the measuring compartment, or that, for an unknown reason, a much higher molar ratio is needed to saturate RuBP oxygenase.

B. Evidence from Measurements of Leaf Chlorophyll Fluorescence The Genty analysis of fluorescence yield (Genty et al., 1989) provides a simple tool to study PS II activity and its regulation. Three fluorescence parameters are considered from the measurement of and (maximum, steady state and minimum fluorescence emission, respectively) for a leaf exposed to actinic light: (i) which estimates the relative photochemical efficiency of open (oxidized) PS II traps; (ii) the photochemical fluorescence quenching parameter, which estimates the relative change of PS II photochemical yield due to changes in the concentration of open PS II reaction centers; (iii) calculated as and is related to and as follows:

and is a relative measure of the quantum yield of PS II photochemistry ( see also Chapters 2 and 3). The relationship between and the quantum yield of gross leaf uptake where R is leaf respiration) of bean leaves in various environmental conditions, measured in 1% to inhibit photorespiration, is shown Fig. 3A for both

dehydrated and non-dehydrated leaves (Cornic and Ghashghaie, 1991). In non-photorespiratory conditions, photosynthetic electron transfer mainly reduces and 4 estimates the photosynthetic whole chain electron transport assuming four electrons are consumed for each fixed. In agreement with Genty et al. (1989) a linear relationship that extrapolates to the origin was obtained between and the apparent quantum yield of assimilation

354 in non-photorespiratory conditions. Genty et al. (1992) found also a linear relationship between and the quantum yield of leaf whole chain electron transfer estimated by measuring both absorption and evolution in bean leaves maintained in normal atmospheric molar fraction (21%). A similar relation is obtained for maize leaves in 21% (Fig. 3B). It is striking that, for both bean and maize leaves, data from dehydrated plants and non-dehydrated plants exhibit the same relationship (Fig. 3). This indicates that dehydration does not change the functioning of PS II in the absence of photorespiration, and that can be used to estimate PS II photochemistry in dehydrated leaves. It also suggests that drought does not induce a significant increase in alternative pathways of electron transport particularly that involved in the direct reduction of (Fig. 3B). In the following parts of this text, will be used either as an estimate of leaf whole chain electron transfer under constant light, or to calculate a rate of oxygenation and a rate of carboxylation knowing the amount of light absorbed by a leaf together with its net uptake and using a ‘calibration line’ similar to that presented in Fig. 3A (for the details of the calculations, see Petersen, 1989; Ghashghaie and Cornic, 1993). Such a calculation of and allows an in vivo estimation of the specificity factor of Rubisco (Laing et al., 1974). Interestingly, such an estimation is in good agreement with the published in vitro values both at around 25 °C (Petersen, 1989; Cornic and Briantais, 1991) and at temperatures ranging between 30 and 12 °C (Ghashghaie and Cornic, 1994), thus strongly suggesting that most of the uptake of in a leaf is due to the oxygenase function of Rubisco. The relationships between and measured on a French bean leaf in an atmosphere containing 21 % at different molar fractions (Fig. 4A) are the same as those measured on the same leaves submitted to a rapid dehydration. The same relationships were obtained for a maize leaf (Fig. 4B). This shows that the regulation of PS II activity at normal oxygen molar fraction is the same when photosynthesis is changed either by changing leaf water status or ambient molar fraction and again suggests that stomatal closure during dehydration causes the observed decline in leaf photosynthesis.

Gabriel Cornic and Angelo Massacci

III. Concentration Inside the Chloroplast During Drought is Low The above results show that the photosynthetic apparatus is resistant to desiccation. As a result, the concentration inside the chloroplast during a mild drought would be expected to be low. This is also demonstrated when the relative variations of A and are plotted during dehydration of a cut leaf of French bean, under limiting light and in 21% and 2% oxygen (Fig. 5). Although the decline of A is

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355 and by dehydrating rapidly (within 8 hours) the same leaf at normal concentration is shown in Fig. 6 (Epron and Cornic, 1993). In a dehydrating leaf, A decreased with a small increase in As explained in the above section, it is possible, using parallel measurements of made during the two sets of measurements shown in Fig. 6, to calculate the rate of carboxylation and a rate of oxygenation by the leaf. As the oxygen uptake in this situation is mainly due to oxygenase activity. is related to as follows:

very similar in the two conditions, due to inhibition of photorespiration, A was always the greatest in 2% As expected from the data presented Fig. 3, in 2% declines in parallel to A while in 21% declines only by about 25% despite the large decrease in leaf photosynthesis. This indicates that the proportion of photosynthetic electrons allocated to the reduction of via the oxygenase activity of Rubisco increases during desiccation. This is only possible if the concentration in dehydrated leaves decreases with time. Brestic et al. (1995) showed that the rate of whole chain electron transport, estimated by and its variation with increasing PPFD were identical both in leaves from well watered French bean plants maintained at the compensation point and on leaves from desiccating plants which did not have any net uptake in normal air. They also showed, in both cases, that most of this electron transfer was actually linked to reduction. In this specific situation values are presumably very low in leaves from drying plants. This observation is very similar to that of Cornic and Briantais (1991) made also on French bean. The relationship between A and obtained by changing ambient concentration around the leaf

where O is the concentration of oxygen dissolved in the chloroplast and S the specificity factor of Rubisco. Clearly is linearly related to when S and O are constant. The relation between A and is the same either when A is measured at different molar ratios or at the same when the leaf is dehydrating (Fig. 7). As the oxygen concentration dissolved in the chloroplast should not be very different in these two cases, this observation strongly suggests that S is not changed by a mild drought. This is in agreement with observations showing that Rubisco activity is

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not limiting net uptake of leaves submitted to desiccation (Holaday et al., 1992). It also supports the contention that activation of Rubisco in vitro by high bicarbonate concentration and in vivo by light (Brestic et al., 1995) is not changed in this situation. Consequently it is likely that the decline of A during leaf dehydration is only due to the decline of caused by stomatal closure. The high values of observed during leaf dehydration could be due to a ‘patchy’ stomatal closure and/or to the increasing relative importance of cuticular transpiration (Meyer and Genty, 1996), as stomata close during the wilting of the leaf. Renou et al. (1990) and Tourneux and Pelletier (1995) measuring leaf uptake together with -dependent evolution on whole plants and leaf discs, respectively, and Stülfauth et al. (1990) measuring absorption of intact leaves came to a similar conclusion, that photorespiration was much larger in proportion to photosynthesis in dehydrated leaves compared to non-dehydrated leaves, which was in agreement with earlier studies by Lawlor (1976) and Dietz and Heber (1983). However, this is in contrast with the recent study of Lauer and Boyer (1992) in which a method for the direct measurement of was used to overcome the problems raised by the possible occurrence of non-uniformity of

Gabriel Cornic and Angelo Massacci photosynthesis. They showed in intact leaves of sunflower, bush bean and soybean that a rapid dehydration caused an increase in They concluded that photosynthetic biochemistry limited photosynthesis during drought. It should be stressed that they measured and not Therefore, the contradiction between their results and those obtained by using chlorophyll fluorescence measurements could also mean that, in their case, there was an increase in the resistance of the -diffusion pathway between the cell walls and the carboxylating site in the chloroplasts. The observation by Gimenez et al. (1992) and Gunasekera and Berkowitz (1993) that the RuBP pool decreases in dehydrated leaves of sunflower and tobacco is more difficult to understand if we accept that is low in this condition; a rise (not a decrease) of the RuBP pool is expected (von Caemmerer and Edmondson, 1986). As discussed by Gimenez et al. (1992), the RuBP concentration within the chloroplast of a drying leaf is very difficult to assess, and, as there were no reported measurements of other PCR cycle intermediates in their paper, it is difficult to know if RuBP regeneration is actually limiting reduction. Quick et al. (1989) showed that RuBP concentration remains largely unchanged and even increased with increasing dehydration of spinach leaf discs when protoplast volume was taken into account. This observation raised the question of the most pertinent basis to express pool size when studying the effect of drought on leaf photosynthesis.

IV. Changes in Metabolic and Whole Leaf Photosynthetic Responses Induced by Water Deficits Water deficit is known to cause an increase in the concentration of compounds of low molecular weight, including proline and betaïne. This increase has often be related to the capacity for osmotic adjustment (Hanson and Hitz, 1982; Morgan, 1984). The following discussion will be restricted to the effect of drought on (i) partitioning between starch and soluble sugar, and (ii) nitrate reduction in leaves. We will also try to relate the observed changes to the variations of in this condition.

A. Effect on Carbohydrate Partitioning Mild water deficit causes an increase in the

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partitioning of newly fixed photosynthate to sucrose synthesis and a decrease in that allocated to starch synthesis when measurements are made at saturating molar ratio (Quick et al, 1989, 1992; Vassey and Sharkey, 1989). At normal atmospheric molar ratio sucrose synthesis will decrease in absolute terms because of the inhibition of photosynthetic net leaf uptake. This decrease in sucrose synthesis is consistent with the observation that sucrose phosphate synthase (SPS) activity decreased in wilted bean leaves (–0.9 MPa measured 3 days after watering was withheld) maintained at normal molar ratio (Vassey and Sharkey, 1989; Quick et al., 1992) providing that the enzyme was assayed with saturating amounts of its substrates, fructose 6-phosphate and UDP-glucose. It is interesting to note that the effect of desiccation on SPS activity was reversed by placing the dehydrated leaves in an atmosphere containing a high (1%) molar ratio (Vassey et al. 1991). However it took 4–5 h to restore the SPS activity in wilted leaves to a control level by such a treatment. The decrease in SPS activity during drought and the effect of high molar ratio on its activity measured in wilted leaves is in agreement with the suggestion that concentration within the leaf is low in dehydrating plants. It is also consistent with the observation of Batistelli et al. (1991) that the activation state of this enzyme is determined by a product of carbon assimilation. Indeed modulation of SPS activity requires both the presence of light and (Stitt et al., 1988; Chapter 6). Fox and Geiger (1985) have shown that water deficit stimulates conversion of starch to sucrose in sugar beet leaves at the compensation point. Thus, starch hydrolysis can also contribute to increase the levels of soluble sugars, particularly that of sucrose, which has sometimes been observed in wilting leaves. This increase will presumably play a role in osmotic adjustment (Morgan, 1984). A decrease in the translocation rate of photosynthetic products from the leaf to the other parts of the plant, as discussed by Quick et al. (1992), could also contribute to the increase of sucrose concentration, though such a drought effect on photosynthate export from the leaf has been considered unlikely (Hanson and Hitz, 1982). This increase of starch hydrolysis due to drought could also contribute to maintaining the size of the metabolic pools of PCR cycle intermediates and allow the functioning of photosynthetic oxygenation cycle (photorespiration) despite a net export of carbon from the chloroplast. Obviously

much more work is needed before such regulation and the roles of starch and sucrose synthesis and hydrolysis can be understood in dehydrating plants.

B. Decline in Nitrate Reductase Activity During Drought Nitrate reductase (NR) activity has been shown to be reduced in leaves during water shortage (Heuer et al., 1979; Smirnoff et al., 1985). This inhibition is correlated with an increase of nitrate levels in the tissue of herbaceous plants. Kaiser and Förster (1989) have shown that low molar ratios and desiccation prevented nitrate reduction in spinach leaves and also that the inhibition of nitrate reduction induced by a mild water stress could be reversed by raising external molar ratios sufficiently (10–15%) to overcome stomatal resistance. This result is very similar to that obtained by measuring SPS activity on water stressed leaves kept either at normal or high (see above). Interestingly, these authors measured a half time of about 30 min to obtain a 50% change in NR activity when a leaf was illuminated in free air. Thus, in natural conditions NR activity can be rapidly modulated in response to a water stressinduced stomatal closure by a mechanism coupled to net photosynthesis.

C. Modification of Net Leaf Uptake Response to Environmental Factors The low molar ratios which occur in the photosynthesizing leaf cell under drought, and the associated metabolic changes, have been shown to alter the response of net leaf uptake to temperature and low oxygen molar ratios. Due to the kinetic properties of Rubisco, a low molar ratio inside the chloroplast is expected to change the response of net leaf uptake to temperature. It will favor the oxygenase function of Rubisco because of the increase of the solubility ratio and thus decrease the temperature for maximum uptake. Such drought-induced changes in the thermal optimum of leaf photosynthesis can be quite substantial, decreasing from 22 to 15 °C for French bean (Cornic and Ghashghaie, 1991; normal and limiting light) and from 34 to 28 °C for soybean (Kao and Forseth, 1992; normal and near saturating light). Such changes in leaf photosynthesis in response to temperature are correlated to similar changes in stomatal conductance;

358 decreasing leaf temperature on a wilted leaf causes stomatal opening. This is presumably due, at least in part, to a modulation of the effect of abscissic acid by temperature as described by Cornic and Ghashghaie (1991). Sometimes, in contrast to what is observed for non-drought stressed leaves, lowering molar ratio at normal atmospheric molar ratio (350 neither increases the net uptake by a leaf suffering a mild drought stress and which had been dehydrated slowly (Von Caemmerer and Farquahr, 1984; Vassey and Sharkey, 1989; Cornic and Ghashghaie, 1991), nor increases its thermal optimum (Cornic and Ghashghaie, 1991). Such effects would be expected if the molar ratio inside the mesophyll cells was decreasing, as indeed has been indicated by the observations discussed in Sections II and III. The lack of a stimulatory effects of low molar ratios on the photosynthetic rate of dehydrating leaves has been attributed to a feedback limitation of photosynthesis due to SPS inhibition induced by water shortage. However, the fact that high molar ratios can restore all or part of (depending on dehydration state) the rate of photosynthesis of the same dehydrated leaves (Cornic et al. 1989) suggests that other factors, as yet poorly understood, are involved with the lack of this response to low molar ratio. An increase of cellular resistance, which will also increase the reassimilation of the produced by respiratory processes, could also account for this lack of response to low Moreover, it should be emphasized that often dehydrated leaves maintained at normal molar ratio respond as expected to low molar ratios, showing a higher rate of uptake in 1 or 2% than in 21 % (Lawlor, 1976; M. Brestic and G. Cornic, unpublished).

V. Maintenance of Plant Water Content During Soil Drying

A. Reduction of Transpiration When subjected to drought, plants can rapidly regulate their water loss through stomatal closure in order to maintain their water status despite the decrease in water availability. Plants can respond to changes in soil humidity and air humidity.

Gabriel Cornic and Angelo Massacci The evidence that in a drying soil a root signal is transmitted to the leaf causing stomatal closure before the plant water deficit increases has been reviewed by Davies and Zhang (1991). This signal is produced in the root tips which presumably sense the drought when they lose turgor. Some controversy still exits about the nature of this signal. However, most of the evidence shows that ABA is produced in the roots tips and then transmitted to the leaf via the xylem. Xylem sap from drying wheat and barley plants has been shown to have a strong antitranspirant activity even in the absence of ABA. However, this antitranspirant activity only appears after the xylem sap was kept frozen for a week (Munns et al., 1993). As demonstrated by Zhang et al. (1987), total mass flow, not the actual ABA concentration within the sap, controls the stomatal aperture. This root signal can probably be modulated rapidly during a single day both by environmental parameters (other than soil water potential) and the physiological status of the plants. Sensitivity of stomata to ABA has been shown to reversibly increase with (i) leaf temperature (Cornic and Ghashghaie, 1991); (ii) the pH of the xylem sap, which can change because of a decrease in anion (nitrate and phosphate) uptake which may exceed the change in cation uptake as soil is desiccating (Shurr et al., 1992); (iii) leaf water potential (Tardieu and Davies, 1992); and (iv) degradation of translocated ABA within mesophyll cells as shown by the action of tetcyclis, an inhibitor of ABA catabolism (Trejo et al., 1993). Stomatal conductance also responds rapidly and reversibly to changes in vapor pressure difference between leaf and air. This phenomenon varies among species (Schulze, 1986) and also with the water status of the plants, being easily measurable on plants submitted to mild water deficits (Nonami et al., 1990). The physiological basis of this response is not yet well understood, however, it appears that peristomatal transpiration does not play a major role as was previously thought (Nonami et al., 1990) and that changes in ABA concentrations within the guard cells may also be involved (Brinckmann et al., 1990). As a result of these two stomatal aperture responses to decreases in the water content of the air and soil, leaf relative water content (RWC) shows little variation, and unless drought is persistent, the photosynthetic apparatus continues to operate at a near optimal water condition (a RWC of about 80%; see Fig. 1). In plants under these conditions, there

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will mainly be a photosynthetic oxygen fixation via the oxygenase activity of Rubisco. However, photosynthetic uptake will resume very rapidly upon soil rehydration when stomata reopen (Cornic et al., 1987). Leaf paraheliotropism and leaf curling, which will be examined in the section dealing with photoinhibition, are also responses which decrease plant transpiration.

This decrease in osmotic potential, which is related to a net decrease in leaf solutes, can be facilitated by either a thickening of cell walls (decrease in cell volume) or an increase in cell wall elasticity which allows a large decrease in cell volume without turgor loss.

VI. Light Utilization by Plants under Drought

B. Maintenance of Water Uptake Growth of plants is inhibited during drought and in many cases root growth is less affected than shoot growth. As a result, there is a larger decrease in transpiration rate than in water uptake. This response obviously contributes to maintaining plant water content despite soil drying. This difference in the response of roots and shoots under limited water supply is not well understood. However, as seen by the effect of fluridone, ABA could again play a major role, helping to maintain root growth in a drying soil while inhibiting shoot growth in the same condition (Saab et al., 1990; Sharp et al., 1994). ABA, in particular, could trigger an osmotic adjustment in root tips, maintaining turgor pressure and as a consequence, root growth; proline could be, at least in Zea mays, the accumulated osmoticum (Voetberg and Sharp, 1991). Osmotic adjustment also contributes to the maintenance of the water potential gradient between soil water and the transpiring leaves and of cell turgor. It has also been related to the maintenance of stomatal conductance, photosynthesis and growth during drought. In fact plants showing osmotic adjustment also exhibit a better productivity under limited water supply (Morgan, 1984). Obviously all mechanisms contributing to the maintenance of the water status of plants in a drying soil interact in a complex fashion which largely remains to be explored. For example, an increase in ABA concentration in plant tissue during drought can induce an osmotic adjustment, thus decreasing the osmotic potential in the leaf and possibly changing the effect it can have on stomatal closure (Ward and Drake, 1988; Tardieu and Davies, 1992). In some cases, as in Artemisia tridentata (Evans et al., 1992), decreases in osmotic potential during the growing season can be due to a decrease in cell volume which causes a ‘passive’ solute concentration and results in the maintenance of turgor pressure.

The photosynthetic apparatus can be damaged when leaves are exposed to high light during a drought, and the reported effect of desiccation on PS II photochemistry is more an effect of high light than a direct effect of water shortage. Drought and high light are very often associated in natural environments and water-stressed plants have consequently developed mechanisms to avoid photoinhibition. Three main strategies can prevent an overreduction of the primary quinone electron acceptors of PS II, (i) mechanisms which prevent the absorption of the light; (ii) mechanisms which prevent the absorbed light from being used for photochemistry; and (iii) mechanisms which are able to consume the reducing power generated by PS II. Leaf movement or orientation, including leaf curling which frequently occurs in Graminae, and reflective structures (waxes, hairs) associated with the leaf epidermis are examples of the first type of mechanism. In relation to drought, only heliotropism will be discussed in the next section because it has received the most attention. Thermal dissipation at the PS II level belongs to the second type of mechanism and photosynthesis, photorespiration, Mehler reactions and electron cycling around PS II to the third. They will be considered together in order to discuss their relative importance.

A. Leaf Movements and Orientation Heliotropism refers to rapid and reversible leaf movements induced by direct solar radiation. It obviously plays a role in the control of leaf energy budgets; change in leaf orientation relative to direct solar irradiance affects the amount of light absorbed by the leaf and thus its photosynthetic activity, transpiration rate and temperature. Heliotropism is particularly common in leguminous plants, but is also found in other families such as the Oxalidaceae, Malvaceae and Labiaceae. However, it is not observed in all plant species. The motor organ for leaf

360 movement is the pulvinus, though there can be some leaf movement in the absence of this organ as is the case in Xanthium strumarium and Chenopodium album. Paraheliotropism (the orientation of the leaf parallel to direct irradiance) is observed in some plants during drought (Forseth and Ehleringer, 1980) and a negative linear relationship between leaflet angle and leaflet water potential has been reported in soybean (Kao and Forseth, 1992). Such movements result in more favorable leaf temperature and water status during drought by minimizing the incident radiation. Ludlow and Björkman (1984) observed that under drought, paraheliotropic leaf movements in Macroptilium purpureum reduced incident light levels, allowing leaves to persist longer into a drought cycle; they proposed that paraheliotopism could be an important protective mechanism against photoinhibition during drought. Leaves of bean plants, similar to those which were used in the experiments illustrated in Fig. 1, incline downwards during a drying cycle as soon as LWD reaches about 15%, corresponding to a leaf water potential of about –0.8 MPa (Brestic et al., 1995). In this situation direct incident irradiance is about seven fold lower than that received by leaves of well watered bean plants. Such a decrease in incident light in natural conditions is enough to minimize or prevent the damaging effect of high light (Powles and Björkman, 1982). The importance of vertical positioning of photosynthetic tissue in dry environments has recently been emphasized by Ehleringer and Cooper (1992). They observed that the maximum quantum yield of evolution measured on leaves, which were always maintained in a horizontal position since they do not perform heliotropic movements, and twigs (always in vertical position) of two desert shrubs (Hymenoclea salsola and Senecio douglasii) remained constant over a wide range of predawn water potentials (from –0.5 to–3 MPa). For predawn water potentials lower than–3 MPa leaf abscission occurred and the quantum yield began to decline in the twigs; the extent of this quantum yield reduction was dependent on the incident photon flux density and was greatly accelerated when twigs were reoriented to the nearhorizontal inclination typical of leaves. In both species, reoriented twigs become chlorotic and died within the three days following the exposure treatment. As the authors suggested ‘the near vertical orientation (of the twigs) might serve in maintaining photosynthetic structures through a drought period’.

Gabriel Cornic and Angelo Massacci a Light Utilization by PS II

1. Thermal Dissipation decreases during dehydration by about 40% in bean leaves and by about 60% in maize leaves (Fig. 4). This decline of the photochemical yield of open PS II centers corresponds to a large non-photochemical quenching of (about 37% and 55% respectively), and suggests that less excitation energy is available for photochemistry as a result of thermal dissipation processes within PS II units (Genty et al., 1989). The fraction of the absorbed light energy which is not used for photochemistry (LNU, Cornic 1994) can be calculated by using Eq. (1). This relationship shows that when (i.e. all the PS II reaction centers oxidized) In this case tends towards which is measured after a long dark period. Consequently,

Since is related to the quantum yield of whole chain electron transfer (Fig. 3), this expression is equivalent to 1– where is the maximum quantum yield of leaf photosynthesis measured under strictly limiting light conditions. The variation of LNU as a function of measured on a French bean leaf receiving a PPFD of 340 both in 21% and in 2% is shown in Fig. 8. The relationships between LNU and were the same irrespective of whether declined as a result of a decrease in molar ratio or a leaf dehydration. LNU was the highest in 1% showing that these processes are dependent on Presumably photorespiration, as shown in Fig. 5, can use part of the absorbed light energy, thus maintaining in the oxidized state (high ). LNU increases during dehydration from about 55% to 80–90% while A decreases from 26 to 3 after LWD reaches about 28%. Similar data has been obtained on maize leaves (G. Cornic, unpublished). The fraction of absorbed light which is not used in photosynthetic processes will, of course, increase as PPFD increases since in this situation decreases. The fraction of LNU which depends on changes in the photochemical yield of open PS II centers, is calculated from the expression Its variations during drought are also shown in Fig. 8 both in 21 % and in 2% It is evident that

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361 a transthylakoidal proton gradient, however the question is still not resolved (see Demmig-Adams 1990; Chapters 1–3).

2. Other Processes Dissipating Excitation Energy

the variations of during drought explain most of the variations of LNU. Variation of thermal dissipation during a treatment has often been studied by calculating the rate constant for excitation energy dissipation in the PS II antenna as defined in the Butler model (Butler, 1978), with appropriate assumptions (see Demmig-Adams, 1990). It can be also studied using the Stern-Volmer equation which is an expression of relative change of and thus a relative measurement of thermal dissipation at the PS II level. Cornic (1994) showed that the relationships between SV and observed for bean leaves both in photorespiratory and non-photorespiratory conditions are nearly the same, indicating that thermal dissipation depends mainly on the size of the electron sink. The molecular mechanism of thermal dissipation of absorbed light in the chlorophyll pigment bed is still largely unknown. It has been suggested that it could be linked to the presence of a xanthophyll, the zeaxanthin in the light harvesting complex of PS II (Thayer and Björkman, 1992) and to the build up of

In plants, photosynthetic and fixation together contribute to the dissipation of the excitation energy. The relative contribution of these two processes to the dissipation of excitation energy during dehydration at atmospheric molar ratio is shown in Fig. 8. The contribution of net uptake ranges from about 35% before desiccation to about 6% after desiccation. In 2% the fraction of the light energy dissipated by photosynthetic fixation added to LNU always gives 1. As concluded from the data in Fig. 2, the Mehler reaction and the other potential processes for eliminating excess excitation energy, such as electron cycling around PS II, are of minor quantitative importance. In 21 % adding the photosynthetic fixation contribution to that of the LNU does not give 1 because photorespiration is also contributing to de-excitation. The contribution of photorespiration is calculated assuming that the sum of the three processes is 1. The fraction of absorbed light energy eliminated by photorespiratory activity remains low ranging from 10% when is high to 20% when has decreased by about 50% during dehydration. As a whole, the contribution of photosynthetic and fixation to energy dissipation decreases from about 45% before dehydration to about 20% after dehydration. As was stressed above, the fraction eliminated by these two processes should decrease as PPFD increases. Thus, in dehydrated leaves exposed to high light photosynthetic processes eliminate a relatively minor fraction of the light which is absorbed by the leaf. A similar conclusion can be drawn from analysis of maize leaves ( G. Cornic, unpublished).

C. Photoinhibition on Dehydrated Leaves The decrease in caused by stomatal closure in dehydrating leaves of plants induces an immediate increase of thermal dissipation of the excitation energy trapped in PS II and a proportional increase in photorespiration. Osmond and Björkman (1972) suggested that photorespiration contributes to the protection of the photosynthetic system against the

362 deleterious effects of high light in dehydrated plants which exhibit low or no uptake in normal conditions because of stomatal closure. This possibility was studied by changing and molar fractions around hydrated leaves (Powles 1984). More recently Katona et al. (1992) proposed that photorespiration turnover protects desiccating leaves against photoinhibition not only by relieving excessive reduction of the electron transfer chain but also by avoiding excessive electron accumulation between the two photosystems. Such a mechanism would provide the poising conditions necessary for the operation of cyclic electron transfer, which together with electron transport to oxygen will support a transthylakoid proton gradient large enough for regulating PS II activity. However, no conclusive data have been published to demonstrate that photorespiration actually protects wilted leaves from damage by high light. On the contrary the results obtained by Brestic et al. (1995) for French bean suggest that photorespiration does not protect the photosynthetic apparatus against high light damage. In their experiments, bean leaves show no exchange at 340 after three weeks without water, although they exhibit a high rate of linear electron transport (measured using the ratio, Fig. 3) mainly due to the presence of and they retain most of their photosynthetic capacity measured as evolution, at high molar ratios (Fig. 1). As the leaves incline downwards, the direct irradiance they receive decreases from 350 to about 50 The photoinhibition resulting from a 1 to 2 hour exposure to high light (PPFD from 1000 to 1700 ) at 340 was found to be the same irrespective of whether the treatment was done in 21 % or 1 % (Fig. 9). As noted by Sharp and Boyer (1986) and Corinc and Briantais (1991) photoinhibition was enhanced in dehydrated leaves only when, in addition to low molar ratio, there was also a low molar ratio during the high light treatments. As discussed above this result is not surprising; photorespiration in a leaf under high light can only consume a small fraction of the absorbed energy. Moreover, its role in the regulation of PS II activity, if it exists, does not appear quantitatively important. The capacity of the leaf for non-radiative dissipation of the absorbed energy is probably of crucial importance for a leaf which cannot escape high light conditions (see Section VI.A). In plants, the situation should be similar since in this case

Gabriel Cornic and Angelo Massacci

photosynthetic assimilation and processes of thermal dissipation are the only way of de-activating PS II reaction centers. The rate of the Mehler reaction is probably low in plants and does not increase during a drought as seen by results in Fig. 3B.

VII. Conclusions It is clear that the photosynthetic apparatus is resistant to drought. The decline in net leaf uptake of a plant submitted to a mild desiccation is likely to be due to stomatal closure. In natural condition plants experiencing drought show a regulation of water loss and uptake allowing maintenance of their leaf relative water content within the limits where photosynthetic capacity and maximum quantum yield show no or little change.

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It has been emphasized that the variation of photosynthetic capacity of plants originating from contrasting habitats as a function of LWD is very similar (Fig. 1). However, the capacity of photosynthetic activity to recover from drought is strongly related to the growth environment. For example, -dependent evolution at saturating bicarbonate concentration of protoplasts of the xerophyte Nerium oleander recovers almost completely from an acute osmotic stress, whereas this activity of protoplasts of the hygrophyte Zebrina pendula did not recover from the same stress, and even showed a further inhibition (Kaiser, 1982). This difference is likely to be due to disruption of cellular compartmentation during the constraint in the hygrophyte compared to the xerophyte. As is often mentioned, resistance to drought is enhanced in plants which dehydrate slowly or are submitted to several cycles of dehydration rehydration (Hanson and Hitz, 1982). As shown above, in this situation many changes occur in carbon partitioning at the cellular levels and plant levels. The ‘orchestration of metabolic responses’ (Hanson and Hitz, 1982) is complex and not yet precisely described. At least we now know from the work of the last decade that low during water shortage could be one of the crucial signals inducing metabolic changes. Water deficit induces expression of particular genes and this is associated with the adaptive responses of stressed plants. Particularly, there is an expression of genes encoding enzymes for steps in the synthesis of osmotica involved in osmotic adjustment and also of genes encoding proteins probably involved in sequestration of ions. ABA (although not ABA alone) plays a major role in expression of genes during drought (Bray, 1993). Changes in the complement of proteins and lipids also occur in thylakoid membranes of plants exposed to a mild drought. An increase of the ratio of hydrophilic to hydrophobic proteins from 0.8 to 4 has been reported by Sgherri et al. (1993) for Helianthus annuus in the field when exposed to mild drought. This increase is probably due to a preferential hydrolysis of hydrophobic proteins. Though these changes may not be reflected in functional modifications of the photosynthetic system, they may be associated with a hardening response. As shown by Havaux (1993) drought enhances the resistance of PS II to heat and high light damage at high temperature. The reduced nitrate absorption from soil and the decreased NR activity during drought are also accompanied by an increase in the breakdown

of chloroplast proteins which probably are, in this case, an important source of mobile forms of nitrogen. This breakdown could induce a decrease in leaf photosynthesis by limiting the amount of photosynthetic enzymes in plants suffering from a long term drought. Hydrolysis of thylakoid proteins would also favor the accumulation of osmotically active solutes such as amino acids (Sgherri et al., 1993). Studies of hardened organisms and organs will certainly be developed, as will investigations to determine the stress signal transduction pathway. As succinctly stated by Bray (1993) ‘pathways that are induced in the laboratory (short term stresses) may not be successfully induced in the field.’

Acknowledgments We are grateful to Jem Wood for critical reading and for correcting our English, to M. Brestic forproviding us with some of his unpublished data and also to F. Loreto and B. Genty for critical reading.

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limitations to photosynthesis. Plant Cell Environ 8: 95–104 Kaiser WM (1982) Correlation between changes in photosynthetic activity and changes in total protoplast volume in leaf tissue from hygro-, meso-, and xerophytes under osmotic stress. Planta 154: 538–545 Kaiser WM (1987) Effect of water deficit on photosynthetic capacity. Physiol. Plant 71: 142–149 Kaiser WM and Förster K (1989) Low prevents nitrate reduction in leaves. Plant Physiol 91: 970–974 Kaiser WM, Kaiser G, Prachuab PK, Wildman SG and Heber U (1981) Photosynthesis under osmotic stress. Inhibition of photosynthesis of intact chloroplasts, protoplasts and leaf slices at high osmotic potentials. Planta 153: 416–422 Kaiser WM, Schröppel-Meier G, With E (1986) Enzyme activities in an artificial stroma medium. An experimental model for studying effects of dehydration on photosynthesis. Planta 167: 292–299 Kao WY and Forseth IN (1992) Responses of gas exchange and phototropic leaf orientation in soybean to soil water availability, leaf water potential, air temperature, and photosynthetic photon flux. Environ Exp Bot 32, 153–161 Katona E, Neimais S, Schönknechst G and Heber U (1992) Photosystem I-dependent cyclic electron transport is important in controlling photosystem II activity in leaves under conditions of water stress. Photosynth Res 34: 449–469 Kubiske ME and Abrams MD (1993) Stomatal and non-stomatal limitations of photosynthesis in 19 temperate tree species on contrasting sites during wet and dry years. Plant Cell Environ 16: 1123–1129 Laing WA, Ogren DA and Hageman RH (1974) Regulation of soybean net photosynthetic fixation by the interaction of and ribulose 1,5-diphosphate carboxylase. Plant Physiol 54: 678–685 Laisk A (1983) Calculations of leaf photosynthetic parameters considering the statistical aperture distributions of stomatal apertures. J Exp Bot 34: 1627–1635 Lauer MJ and Boyer JS (1992) Internal measured directly in leaves. Abscisic acid and low leaf water potential case opposing effects. Plant Physiol 98: 1310–1316 Lawlor DW (1976) Water stress induced changes in photosynthesis, photorespiration, respiration and compensation concentration of wheat. Photosynthetica 10: 378–387 Lawlor DW (1995) The effects of water deficit on photosynthesis. In: Smirnoff N (ed) Environment and Plant Metabolism. Flexibility and Acclimation, pp. 129–160. Bios Scientific Publishers, Oxford Lawlor DW and Uprety DC (1993) Effects of water stress on photosynthesis of crops and the biochemical mechanism. In: Abrol YP, Mohanty P and Govindjee (eds) Photosynthesis, Photoreactions to Plant Productivity, pp. 421–445. Oxford and IBH publishing CO PVT LTD, New Delhi Ludlow MM and Björkman O (1984) Paraheliotropic leaf movements in Siratro as a protective mechanism against drought-induced damage to primary photosynthetic reactions: damage by excessive light and heat. Planta 61: 505–516 Meyer S and Genty B (1995) Mapping intercellular molar fraction in rosa leaf fed with ABA. Significance of estimated from leaf gas exchange. In: Mathis P (ed) Photosynthesis: From Light to Biosphere, Vol V, pp 603–606. Kluwer Acad. Publishers, Dordrecht Morgan JM (1984) Osmoregulation and water stress in higher

plants. Ann Rev Plant Physiol 35: 299–319 Munns R, Passioura JB, Milborrow RW, James RA and Close TJ (1993) Stored xylem sap from wheat and barley in drying soil contains a transpiration inhibitor with a large molecule size. Plant Cell Environ 16: 867–872 Nonami H, Schulze ED and Zeiger H (1990) Mechanisms of stomatal movement in response to air humidity, irradiance and xylem water potential. Planta 183: 57–64 Ögren E., Evans JR. (1993) Photosynthetic light-response curves. I The influence of partial pressure and leaf inversion. Planta 189: 182–190 Ortiz-Lopez A, Ort DR and Boyer JS (1991) Photophosphorylation in attached leaves of Helianthus annuus at low water potentials. Plant Physiol 96: 1018–1025 Osmond CB and Björkman Ö (1972) Simultaneous measurement of effects on net photosynthesis and glycolate metabolism in C3 and C4 species of Atriplex. Carnegie Inst Wash Yrbk 71: 141–148 Petersen RB (1989) Partitioning of noncyclic photosynthetic electron transport to -dependent dissipative processes as probed by fluorescence and exchange. Plant Physiol 90: 1322–1328 Powles (1984) Photoinhibition of photosynthesis induced by visible light. Ann Rev Plant Physiol 35: 14–44 Powles SB and Björkman O (1982) Leaf movements in the shade species Oxalis oregana. II. Role in protection against injury by intense light. Carnegie Institution of Washington Yearbook 81: 63–66 Quick P, Siegl G, Neuhaus E, Feil R and Stitt M (1989) Short term water stress leads to a stimulation of sucrose synthesis by activating sucrose-phosphate synthase. Planta 177: 535–546 Quick WP, Chaves MM, Wendler R, David M, Rodrigues ML, Passaharino JA, Pereira JS, Adcock MD, Leegood RC and Stitt M (1992) The effect of water stress on photosynthetic carbon metabolism in four species grown under field conditions. Plant Cell Environ 15: 25–35 Renou JL, Gerbaud A, Just D and André M (1990) Differing substomatal and chloroplastic concentrations in waterstressed wheat. Planta 182: 415–419 Saab IN, Sharp RE, Pritchard J and Voetberg GS (1990) Increased endogenous abscissic acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials. Plant Physiol 93: 1329–1336 Schulze ED (1986) Carbon dioxide and water vapor exchange in response to drought in the soil. Ann Rev Plant Physiol 37: 247– 274 Schurr U, Gollan T and Schulze ED (1992) Stomatal response to drying soil in relation to changes in the xylem sap composition of Helianthus annuus. II. Stomatal sensitivity to abscisic acid imported from the xylem sap. Plant Cell Environ 15: 561–567 Schwab KB, Schreiber U, Heber U (1989) Response of photosynthesis and respiration of resurrection plants to desiccation and rehydration. Planta 177, 217–227 Sgherri CLM, Pinzino C and Navari-Izzo F (1993) Chemical changes and production in thylakoid membranes under water stress. Physiol Plant 87: 211–216 Sharkey TD and Badger MR (1982) Effects of water stress on photosynthetic electron transport, photophosphorylation and metabolite levels of Xanthium strumarium mesophyll cell. Planta 156: 199–206 Sharkey TD and Seemann JR (1989) Mild water stress effects on

366 carbon-reduction-cycle intermediates, ribulose bisphosphate carboxylase activity, and spatial homogeneity of photosynthesis in intact leaves. Plant Physiol 89: 1060–1065 Sharp RE and Boyer JS (1986) Photosynthesis at low water potentials in sunflower: Lack of photoinhibitory effects. Plant Physiol. 82: 90–95 Sharp RE, Wu YJ, Voetberg GS, Saab IN and Lenoble ME (1994) Confirmation that abscisic acid accumulation is required for maize primary root elongation at low water potentials. J Exp Bot 45: 1743–1751 Smirnoff N, Windolow MD and Stewart GR (1985) Nitrate reductase activity in leaves of barley (Hordeum vulgare) and durum wheat (Triticum durum) during field and rapidly applied water deficits. J Exp Bot 36: 1200–1208 Stitt M, Wilke I, Feil R and Heldt HW (1988) Coarse control of sucrose phosphate synthase in leaves: Alterations of the kinetic properties in response to the rate of photosynthesis and the accumulation of sucrose. Planta 174: 217–230 Stülfauth T, Scheuermann R and Fock HP (1990) Light energy dissipation under water stress conditions. Contribution or reassimilation an evidence for additional processes. Plant Physiol 92: 1053–1061 Tardieu F and Davies WJ (1992) Stomatal response to ABA is a function of current plant water status. Plant Physiol 98: 540– 545 Terashima I (1992) Anatomy of non-uniform leaf photosynthesis. Photosynth Res 31: 195–212 Terashima I, Wong SC, Osmond CB and Farquhar GD (1988) Characterization of non-uniform photosynthesis induced by abscisic acid in the leaves having different mesophyll anatomies. Plant Cell Physiol 29: 385–395 Thayer SS and Björkman Ö (1992) Carotenoid distribution and deepoxidation in thylakoid pigment-protein complexes from cotton leaves and bundle-sheath cells of maize. Photosynth Res 33: 213–225 Tourneux C and Peltier G (1995) Effect of water deficit on photosynthetic oxygen exchange measured using and mass spectrometry in Solatium tuberosum leaf discs. Planta 195: 570–577 Trejo LC, Davies WJ and Lucero del mar Ruiz P (1993) Sensitivity of stomata to abscissic acid. An effect of the mesophyll. Plant Physiol 102: 497–502

Gabriel Cornic and Angelo Massacci Vapaavuori E and Nurmi A (1982) Chlorophyll-protein complexes in Salex sp. ‘Aquatica gigantea’ under strong and weak light II. Effect of water stress on the chlorophyll-protein complexes and chloroplast ultrastructure: Plant Cell Physiol 23: 791–801 Vassey TL and Sharkey TD (1989) Mild water stress of Phaseolus vulgaris plants leads to reduce starch synthesis and extractable sucrose phosphate synthase activity. Plant Physiol 89: 1066– 1070 Vassey TL, Quick WP, Sharkey TD and Stitt M (1991) Water stress, carbon dioxide and light effects on sucrose phosphate synthase activity in Phaseolus vulgaris. Physiol Plant 81: 37– 44 Voetberg GS and Sharp RE (1991) Growth of the primary root at low water potentials. III. Role of increased proline deposition in osmotic adjustment. Plant Physiol 96: 1125–1130 von Caemmerer S and Edmondson DL (1986) Relationship between steady-state gas exchange, in vivo ribulose bisphosphate carboxylase activity and some carbon reduction cycle intermediates in Raphanus sativus, Aust J Plant Physiol 13: 669–688 von Caemmerer S and Farquhar GD (1984) Effects of partial defoliation, changes in irradiance during growth, short-term water stress, and growth at enhanced on the photosynthetic capacity of leaves of Phaseolus vulgaris L. Planta 160: 320–329 Ward DA and Drake BG (1988) Osmotic stress temporarily reverses the inhibition of photosynthesis and stomatal conductance by abscissic acid—Evidence that abscissic acid induces a localised closure of stomata in intact, detached leaves. J Exp Bot 39: 147–155 Weis Band Berry J (1987) Quantum efficiency of photosystem II in relation to energy-dependent quenching of chlorophyll fluorescence. Biochim Biophys Acta 894: 198–208 Woodward FI (1987) Climate and Plant Distribution. Cambridge Studies in Ecology. Cambridge University Press, Cambridge Wright SJ (1992) Seasonal drought, soil fertility and the species density of tropical forest palant communities. Tree 8: 260–262 Zhang J, Schurr U and Davies WJ (1987) Control of stomatal behaviour by abscissic acid which apparently originates in roots. J Exp Bot 38: 2015–2023

Chapter 15 Photosynthetic Adjustment to Temperature Stefan Falk, Denis P. Maxwell, David E. Laudenbach and Norman P. A. Huner Department of Plant Sciences, University of Western Ontario, London, Ontario N6A 5B7, Canada

Summary I. Introduction II. Short-Term Temperature Response of Photosynthesis Uptake and Evolution A. B. Carbon Metabolism C. Electron Transport III. Long-Term Temperature Response of Photosynthesis Uptake and Evolution A. B. Carbon Metabolism C. Electron Transport IV. Thylakoid Membrane Lipids V. Temperature and Chloroplast Development VI. Interaction of Light and Temperature VII. Photosynthetic Adaptation, Acclimation and Stress Acknowledgments References

367 368 369 369 370 371 372 372 373 374 375 377 378 380 380 380

Summary The description of a general mechanism for photosynthetic adjustment to temperature that encompasses all autotrophic species is not possible for three principal reasons: (i) inherent genetic diversity, (ii) differential strategies in growth and development, and (iii) organisms respond to temperature changes rather than to absolute temperature. Thus, ‘high’ and ‘low’ temperature are relative terms and will differ for pyschrophilic, mesophilic and thermophilic organisms. However, given this complexity, some consensus regarding photosynthetic adjustment to temperature is emerging. At low temperature (0–10 °C), photosynthesis is constrained thermodynamically. This may be manifested by chloroplast phosphate limitation due to reduced rates of sucrose synthesis and/or source-sink limitations. In this case, rates of uptake and evolution are regulated directly through metabolite accumulation (feedback inhibition) and photosynthetic control. Alternatively, feedback inhibition may be regulated indirectly through catabolite repression of photosynthetic genes. Although light may exacerbate susceptibility to photoinhibition at low temperature in many species, cold grown, chilling-tolerant plants exhibit increased capacity for carbohydrate synthesis at low temperature which alleviates phosphate limitation, supplies a cryoprotectant and results in higher photosynthetic capacity than warm-grown plants. However, photosynthetic adjustment in cold-grown higher plants and algae does not reflect adjustment to low temperature per se, but rather, changes in excitation pressure on PS II. In contrast, photosynthesis in chilling-sensitive plants is not only constrained thermodynamically by low temperature but is also severely inhibited developmentally. Through a comprehensive molecular genetic study, a direct link between photosynthetic temperature acclimation and thylakoid lipid unsaturation has been established in cyanobacteria. However, the evidence for Neil R. Baker (ed): Photosynthesis and the Environment, pp. 367–385. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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such a link in algae and higher plants is still equivocal. PS I may be primary site for photoinhibition at low temperatures in some chilling-sensitive species. Furthermore, susceptibility to low temperature photoinhibition is reduced by altering the level of unsatruation of chloroplast lipids in chilling-sensitive transgenic tobacco plants. With respect to high temperature (35–50 °C ), the consensus is that thylakoid membrane stability limits photosynthetic performance. In contrast to low temperature, light protects against high temperature inhibition of photosynthesis.

I. Introduction Land plants can experience and, thus, must be able to adjust to wide daily and seasonal fluctuations in temperature. In contrast, aquatic plants generally experience a more constant daily and seasonal temperature regime. The most productive marine environments are the Arctic Ocean and waters contiguous with the Antarctic continent where algae experience an average temperature of about –1.8 °C (Greene et al., 1992). When the external environment changes, responses by the plant to these changes may be separated into two principal components (Berry and Björkman, 1980; Prosser, 1986;Davison, 1991). First, adaptation is a genotypic response to longterm changes. These alterations are stable and will remain in the population over generations. Second, acclimation, is a response induced by an environmental change which causes a phenotypic alteration over a single generation time without any corresponding compositional change in the genetic complement. However, acclimative responses can be Abbreviations: AMP – adenosine monophosphate; rate of assimilation; cab–chlorophyll a/b–binding proteins; DGDG–digalactosyldiacylglycerol; FBPase–fructose 1,6-bisphosphatase; fluorescence yield with all PS II traps closed in dark- and light-adapted plant material, respectively; fluorescence yield in dark- and light-adapted plant material, respectively; LHCI, LHCII – lightharvesting chlorophyll a/b-binding protein complexes of PS I and PS II, respectively; MGDG –monogalactosyldiacylglycerol; P700 – primary chlorophyll a electron donor in PS I; PFD – photon flux density; PG – phosphatidylglycerol; PGA – 3phosphoglyceric acid; Pi – inorganic phosphate; primarily located in grana stacks, having large LHCII-antenna; primarily located in stromal lamellae, having small LHCII-antenna; stable quinone electron acceptor of PS II; energy state-dependent fluorescence quenching coefficient; of non-photochemical quenching of fluorescence; of photochemical quenching of fluorescence; Rubisco – ribulose 1,5-bisphosphate carboxylase/ oxygenase; RuBP – ribulose 1,5-bisphosphate; SBPase – sedoheptulose 1,7-bisphosphatase; yield of linear electron transport in PS II.

differentiated further into: (i) transient physiological and biochemical adjustments induced by abrupt or short-term changes in the environment, that is, a stress response which subsequently may lead to damage and ultimately senescence, and (ii) stable, long-term adjustments which may reflect a developmental response to a new environmental condition. This response will prevail only as long as the new environmental condition persists. Frequently, it is difficult to distinguish between stress responses and developmental responses, especially since the stress response may be part of an adjustment leading to the developmental response. Regardless, the extent to which a plant can acclimate is ultimately under genetic control and the degree of plant plasticity will, in turn, be dependent upon the regulation and expression of many genes. Photosynthesis represents an integration of photochemical as well as biochemical processes. Thus, ambient temperature fluctuations will have a direct impact on photosynthesis through its effects on the thermally sensitive biochemical and physiological processes. These include: (i) photosynthetic carbon reduction, (ii) sucrose synthesis, (iii) carbon partitioning, (iv) intersystem electron transport which is thought to be diffusion limited at the level of the mobile electron transport carriers, plastoquinone and plastocyanin (Lawlor, 1987). However, the photochemical events of light absorption, energy transfer and charge separation associated with PS II and PS I are insensitive to temperature in the biologically relevant range of 0 °C to 50 °C (Mathis and Rutherford, 1987). The combined effects of light and the differential sensitivity to temperature exhibited by the photochemical and thermochemical processes of photosynthesis can lead to metabolic imbalances which, in turn, can result in a significant impairment of photosynthesis as a result of photoinhibition (Powles, 1984) In this chapter, we provide a general overview of recent advances regarding the effects of short- and long-term exposure to low or high temperature on

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photosynthetic carbon assimilation and electron transport in higher plants, algae and cyanobacteria. Subsequently, we address the influence of growth temperature on thylakoid lipid composition and chloroplast development. Finally, we discuss briefly the combined effects of light and temperature on photosynthetic performance and conclude with an overall assessment of photosynthetic acclimation to temperature. For further reviews of the effects of high or low temperature on photosynthesis the reader is referred to Berry and Björkman (1980), Graham and Patterson (1982), Öquist and Martin (1986), Baker (1991,1993), Huner et al. (1993) and a volume edited by Long and Woodward (1988).

of the leaves from either plant species to warm temperatures. The reversible temperature inhibition of photosynthesis in tomato and spinach was interpreted to indicate the thermodynamic constraints on thermochemical rate constants (Sassenrath and Ort, 1990). However, exposure of the cold-sensitive tomato to low temperature for more than 1 h resulted in an irreversible inhibition of photosynthesis which was not observed in the spinach. The precise site of this irreversible inhibition has not been identified. The photosynthetic response of plants to changes in temperature also appears to be dependent upon the developmental history of the leaves as well as the physiological age of the leaf tissue. Light-saturated rates of uptake, evolution and photosynthetic efficiency under light limiting conditions were inhibited by 20% to 50% when spinach and rape grown at 16 °C were abruptly transferred to 5 °C (Maciejewska et al., 1984; Somersalo and Krause, 1989; Boese and Huner, 1990). Young, developing leaves of herbaceous plants appear to be more resilient to temperature changes since they are plastic in their photosynthetic response to sudden changes in temperature whereas mature, fully-expanded leaves exhibit a reduced capacity to adjust (Krol et al., 1984; Krol and Huner, 1985; Rütten and Santarius, 1992). Generally, inhibition of photosynthesis by low temperature can not be accounted for by stomatal limitations under light-saturating conditions and ambient concentrations (Lundmark et al., 1988; Nie et al., 1992). Although exposure to low temperature does increase stomatal resistance it could only explain about 19% of the low temperature inhibition of photosynthesis in cold-sensitive Zea mays (Nie et al., 1992). Similar conclusions have been reported for cold-sensitive tomato (Martin and Ort, 1985), olive (Bongi and Long, 1987), cold tolerant rye (Huner et al., 1986) and wheat (Hurry and Huner, 1991). Thus, low temperature-induced inhibition of photosynthesis most likely reflects alterations at the chloroplast level rather than limitations on actual leaf gas exchange. Heat adapted plant species have much higher at high temperatures than cold adapted species and vice versa (Berry and Björkman, 1980). Heat tolerance limits are usually determined by the thermal stability of soluble enzymes and membrane structure. Beyond a critical temperature, heat effects on the stability of thylakoid membrane components, rather than denaturation of soluble enzymes, are the primary

II. Short-Term Temperature Response of Photosynthesis

A.

Uptake and

Evolution

The optimum measuring temperature for photosynthesis exhibited by a plant species is generally higher than the growth temperature (Li, 1980; Grace, 1988). This optimum reflects the environmental temperature range to which the species has adapted with desert species exhibiting higher measuring temperature optima than cool, temperate species (Berry and Björkman, 1980; Grace, 1988). However, autotrophic organisms exhibit a high degree of plasticity with respect to the short-term temperature response of photosynthesis. Photosynthetic acclimation to short-term temperature changes in higher plants as well as psychrophilic and thermophilic algae has been characterized generally by an altered temperature optimum which is biased towards the new temperature regime (Berry and Björkman, 1980; Li, 1980; Öquist and Martin, 1986; Davison, 1987). The temperature sensitivity of photosynthesis is dependent upon both plant species and the time of exposure to the new temperature regime. Immediate reduction in the measuring temperature from 30 °C to 4 °C resulted in a 75% decrease in the lightsaturated rate of assimilation in both the cold tolerant spinach and cold-sensitive tomato (Sassenrath and Ort, 1990). However, the coldsensitive tomato also exhibited a 50% decrease in the maximum quantum yield of assimilation whereas in spinach this parameter was unaffected by temperature. Low temperature inhibition of photosynthesis was completely reversible upon exposure

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cause of irreversible inhibition of photosynthesis (Schreiber and Berry, 1977; Santarius and Müller, 1979; Armond et al., 1980; Süss and Yordanov, 1986; Weis and Berry, 1988). For example, photophosphorylation is one process that is inhibited by high temperature due to thermal uncoupling (Weis and Berry, 1988). This will result in a decrease in the supply of ATP necessary for carbon assimilation. However, the precise mechanism of this uncoupling is unclear. In addition, damage to PS II at the level of the O2 evolving complex (Santarius, 1975) or the destabilization of the light-harvesting chlorophyll a/b-binding complex associated with PS II (LHCII) (Armond et al., 1980) will decrease the supply of NADPH consumed directly in fixation as well as decrease the efficiency of the thioredoxin system responsible for the light activation of key regulatory enzymes of the Calvin cycle (Cseke and Buchanan, 1986). However, the high temperature inhibition of photosynthesis may be reversible as long as the heat stress is sublethal. The reversibility of high temperature inhibition is dependent upon both the temperature and duration of the heat stress (Bauer and Senser, 1979). In summary, the photosynthetic response to shortterm temperature fluctuations is species dependent and strongly influenced by developmental history. Sublethal low temperatures can exert a reversible limitation on photosynthetic rate due to thermodynamic constraints on enzyme catalyzed reactions. In contrast, supraoptimal temperatures cause irreversible inhibition of uptake or evolution primarily due to thylakoid membrane destabilization. Stomatal conductance appears to play only a minor role in the temperature limitation of photosynthesis.

B. Carbon Metabolism Abrupt shifts of plants from high (20– 30 °C) to low measuring temperatures (2–10 °C) results in insenstivity of fixation (Joliffe and Tregunna, 1973; Cornic and Louason, 1980; Mächler et al., 1984; Leegood, 1985; Schnyder et al., 1986; Labate et al., 1990; Paul et al., 1990, 1992). This decreased stimulation of photosynthesis by 2% dampens the magnitude of the oscillations normally associated with photosynthetic and fluorescence induction due to a Pi limitation of photosynthesis (Leegood, 1985; Sharkey, 1985a,b; Sharkey et al., 1986; Stitt et al., 1987; see also Chapter 7). This may, in part, be due to the fact that optimal rates of photosynthesis at low

measuring temperature require higher Pi concentrations than photosynthesis at moderate to high temperatures (Leegood, 1985; Chapter7). In addition, phosphorylated metabolites such as hexose phosphates tend to accumulate to higher levels after exposure to low measuring temperatures compared to high measuring temperatures which reduces the available Pi in the stroma for photosynthesis at low temperatures (Mächler et al., 1984; Labate and Leegood 1989; Labate et al., 1990). The ratio of 3phosphoglyceric acid (PGA) to triose phosphate, which reflects the redox as well as the phosphorylation potential of the chloroplast, rises as leaf measuring temperature decreases in spinach and wheat under ambient concentrations (Kobza and Edwards, 1987; Stitt and Grosse, 1988; Labate and Leegood, 1989). This should favor starch accumulation within the chloroplast over sucrose synthesis in the cytoplasm (Stitt et al., 1987). The rise in PGA/triose phosphate may occur because of the low temperature restriction of electron transport and thus, a decreased capacity to generate the necessary ATP and NADPH. Such a decrease in phosphorylation and reducing potential would cause a significant limitation on fixation. Sucrose synthesis is inhibited to varying degrees at low temperature, dependent upon the plant species (Stitt et al., 1987). This appears to be the consequence of the increased sensitivity of the cytosolic fructose 1,5-bisphosphatase (FBPase) to the important regulatory metabolite, fructose 2,6-bisphosphate and AMP (Stitt et al., 1987; see also Chapters 6 and 7). This leads to an increase in phosphorylated intermediates and a decrease in cytosolic Pi (Stitt et al., 1987). Furthermore, it has been suggested that maximal rates of photosynthesis are ultimately limited by the capacity for sucrose synthesis through restriction of the rate at which cytosolic Pi can be exchanged for stromal triose phosphate by the chloroplastic phosphate translocator (Stitt, 1986). Thus, it is possible that low temperature limitation of photosynthesis occurs at the level of the Pi translocator rather than sucrose synthesis per se. This would eventually limit the consumption of photosynthetically-derived ATP and NADPH and reduce PS II photochemical efficiency and rates of electron transport through photosynthetic control (see Chapter 3). Alternatively, inhibition of photosynthesis at low measuring temperatures may be due to a combination of sink limitations and restricted translocation. Low temperatures typically result in the accumulation of

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starch and sucrose which would lead to a reduction in the utilization of triose phosphate and, ultimately, to feedback inhibition of photosynthesis (Paul et al., 1991; Warner and Burke, 1993). However, feedback inhibition of photosynthesis has also been shown to be indirectly controlled through the catabolite repression of photosynthetic genes by end-product accumulation. Thus, both direct and indirect mechanisms appear to be important in the regulation of feedback inhibition of photosynthesis (Jang and Sheen, 1994; Sheen 1994; see also Chapters 6 and 10). Species differences in the level of sucrose/starch accumulation in response to low temperature shifts may reflect genetic differences with respect to photosynthetic end product accumulation (Goldschmidt and Huber, 1992). At high temperatures the fraction of the total absorbed energy dissipated by high energy state quenching of excitation energy decreases while photorespiration increases, resulting in a decrease in the quantum yield of assimilation (Weis and Berry, 1988). This reversible heat-induced depression in photosynthesis appears to be due to a down regulation of Rubisco (Kobza and Edwards, 1987). The activation state of Rubisco remains high between 20 °C and 30 °C but decreases by about 50% between 30 °C and 45 °C in cotton leaves (Weis and Berry, 1988). Thus, the lowered activation state of Rubisco appears to negate any temperature-induced increase in reaction rates. It has been suggested that the decline in Rubisco activation may be due to an inhibition of Rubisco activase (Portis, 1992). In addition, the capacity of the light reactions become limited by feedback inhibition from carbon metabolism at supraoptimal temperatures. This may be due to depletion of Calvin cycle intermediates as a consequence of an increased rate of triose phosphate export from the chloroplast to the cytoplasm relative to triose phosphate synthesis in the stroma (Stitt, 1986; Labate and Leegood, 1988; see also Chapter 7). The contribution that each of these mechanisms have to high temperature inhibition of uptake is unclear at this time. In summary, short-term exposure to low-temperature can induce a Pi-limitation of photosynthesis due to reduced rates of sucrose synthesis and/or sourcesink limitations. Alternatively, feedback inhibition of photoinhibition may be the result of indirect regulation through changes in enzyme levels as a consequence of catabolite repression of gene expression. The relative contributions of the direct

(Pi-limitation) and indirect (gene expression) mechanisms for regulation of feedback inhibition of photosynthesis has yet to be elucidated (see Chapters 6 and 10). In contrast, the activation state of Rubisco can limit at high temperatures.

C. Electron Transport Pre-exposure of leaves of chilling sensitive pumpkin (Aro et al., 1990) and tomato (Ortiz-Lopez et al., 1990) to a combination of low temperature and high light caused a significant reduction in light saturated rates of whole chain and PS II electron transport when measured at room temperature. Furthermore, Kee et al. (1986) showed that chilling tomato leaves in the darkness also inhibited light saturated whole chain and PS II electron transport whereas PS Imediated activity appeared to be stable. PS II inhibition appeared to be localized to the evolving complex. In chilling-tolerant plants such as spinach, Stitt and Grosse (1988) showed that reducing leaf temperature from 30 °C to 15 °C more than doubled the level of reduced estimated by the photochemical quenching parameter under lightsaturated but not light limiting conditions. This was coupled to a concomitant increase in the level of energy-dependent quenching of chlorophyll a fluorescence and a decrease in the quantum yield of PS II electron transport in barley and spinach as leaf temperature decreased (Labate and Leegood 1989; Holaday et al., 1992). These results were interpreted to reflect the low temperature limitation of carbon metabolism which causes energization of the thylakoid due to a reduced ratio of ATP/ADP as a consequence of Pi limitation of photosynthesis. This causes the build up of indicative of an increase in non-photochemical dissipation of energy. However, as the temperature is decreased to progressively lower temperatures, the rate at which is reduced under saturating light overtakes the rate of non-photochemical dissipation of energy resulting in the accumulation of reduced This enhances the probability of damage to PS II Exposure of leaves or green algae to elevated temperatures (35–45 °C) generally inhibits evolution, assimilation and photophosphorylation (Berry and Björkman, 1980; Quinn and Williams, 1985; Yordanov et al., 1986). Thylakoidassociated activities appear to be more heat sensitive than those of the stroma (Santarius, 1975) with PS II

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being the most heat-labile component of thylakoid membranes. Deactivation of PS II by high temperature is associated with denaturation of PS II polypeptides (Thompson et al., 1989), dissociation of LHCII from the PS II core (Armond et al., 1980; Gounaris et al., 1984; Sundby et al., 1986) and the inhibition of the oxygen evolving complex as a result of the release of ions (Nash et al., 1985). However, pre-exposure to high temperatures (30-35 °C) or water stress rapidly increases the thermal stability of PS II (Havaux, 1992, 1993). Although the temperature required for 50% inactivation of P700 and iron-sulfur centers of PS I in temperate plants is 10 °C to 20 °C lower than in thermophilic cyanobacteria, PS I-components are more thermostable than the PS II-components. This indicates that the structure and hence the heat stability of PS I has been conserved through evolution (Sonoike et al., 1990). In contrast to PS II, PS I activity measured in vivo (Havaux et al., 1991) and in vitro (Thomas et al., 1986; Huner and Reynolds, 1989; Reynolds and Huner, 1990; Boucher et al., 1990; Boucher and Carpentier, 1993) is stimulated by exposure to elevated temperatures. Heat stimulation of PS I appears to be associated with an increased capacity for cyclic electron flow around PS I. Furthermore, this stimulation appears to be transient in nature and requires cations for stabilization (Huner and Reynolds, 1989; Boucher et al., 1990). Mild heat treatment has been shown to induce an increase the in excitation energy emanating from PS I relative to PS II (Weis, 1985). Since this is reversed by far-red illumination, the phenomena is considered to be a heat-induced state transition. The high temperature-induced conversion of PS to PS (Sundby et al., 1986) may be involved in this high temperature-induced spillover from PS II to PS I (Weis and Berry, 1988). The possible role of increased PS I activity in protection of PS II against heat damage has been discussed (Weis, 1985; Sundby et al., 1986). However, Huner and Reynolds (1989) reported that temperature only affected the rate of stimulation and decay of PS I activity but not the extent of PS I stimulation in vitro between 0 °C and 65 °C. Thus, stimulation of PS I activity may not have any protective role per se but may simply reflect a temperature-induced perturbation of thylakoid membrane function in vitro. In summary, PS II is generally less stable to either high or low temperature than PS I. However, a short pre-exposure to high, sub-lethal temperature rapidly

increases the thermal stability of PS II. In chillingsensitive plant species, electron transport appears to be inhibited at the level of the evolving complex. However, in chilling-tolerant plants photosynthetic electron transport rates are reduced as a consequence of photosynthetic control which reflects short-term temperature limitations on carbon metabolism.

III. Long-Term Temperature Response of Photosynthesis In this section, the emphasis is on the long-term response of photosynthesis to low, chilling temperatures only. This is a reflection of the most recent literature on temperature and photosynthesis. For a general discussion on the effects of growth and development at high temperatures on photosynthesis, we refer the reader to Berry and Björkman (1980) and the chapters in the volume edited by Long and Woodward (1988).

A.

Uptake and

Evolution

The effects of long term exposure to suboptimal temperatures on the algal carbon fixation rates are species specific (Li, 1980). fixation rates per cell remain unchanged or decrease with decreasing growth temperature (Levasseur et al., 1990; Maxwell et al., 1995). Since light and dark reactions of photosynthesis are tightly coupled (Hall, 1976; Lawlor, 1987), the temperature-induced variations in carbon fixation rate and, probably, requirement of chemical energy, should result in predictable variations in the structure and dynamics of the photosynthetic light reactions (Levasseur et al., 1990). For example, Maxwell et al. (1994) reported that growth of Chlorella vulgaris at 5 °C resulted in a marked increase in the chlorophyll a/b-ratio and a concomitant decrease in the content of LHC II polypeptides compared to Chlorella grown at 27 °C. Experimental data from Mortain-Bertrand et al. (1988) confirms earlier reports (Platt and Jassby, 1976; Harrison and Platt, 1980, 1986) that the initial slope of the light response curve (i.e. the maximum guantum efficiency of photosynthesis) is controlled predominantly by light history, whereas the lightsaturated rate of photosynthesis is controlled by temperature. The photochemical events of PS II and PS I are in principle not affected by temperatures as low as –30 °C (Mathis and Rutherford, 1987). It has

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been argued that since many aspects of photosynthetic electron transport (e.g. photophosphorylation and plastoquinone diffusion) are temperature-dependent (Öquist, 1983; Raven and Geider, 1988), the lightlimited part of the light response curve may vary with temperature (Davison, 1991). This appears to occur in Arctic sea-ice diatoms (Palmisano et al., 1987) and in Laminaria saccharina (Davison, 1987), where the initial slope of the light response curve decreased with increasing assay temperature. Laminaria also displayed a decrease in light-limited quantum efficiency when grown at 20 °C as compared to sporophytes grown at 5 °C in all assay temperatures between 5 and 20 °C (Davison, 1987). In contrast to this, the prediction by several earlier investigators that light-limited photosynthesis is independent of temperature (Mortain-Bertrand et al., 1988) has been supported by experiments on Skeletonema costatum (Mortain-Bertrand et al., 1988). This is in agreement with the empirical evidence that the convexity, a parameter of the curvature of the light response curve affected by changes in ratios of PS II turnover versus whole-chain electron transport, is independent of temperatures between–2 and 35 °C (Leverenz and Öquist, 1987). Furthermore, no significant change of modeled maximum quantum yield could be detected in Chlamydomonas reinhardtii acclimated to 12 °C (Falk et al., 1990) or Chlorella vulgaris to 5 °C (Maxwell et al., 1994) compared to control material grown at 27 °C. The estimate of the maximum quantum yield did not change upon shifting Chlorella grown at 5 °C to 27 °C. We believe that some of the ambiguity of reported results may reflect the methods used to estimate the initial slope of the light response curve (Leverenz et al., 1990). Acclimation of temperate terrestrial plants to lowered growth temperatures is accompanied by an increased capacity for carbon metabolism (Berry and Björkman, 1980). After exposure to growth and development at 5 °C, winter wheat and rye exhibit maximum rates of assimilation or evolution that are greater than the same cultivars grown at 20 °C regardless of measuring temperature between 5 °C and 25 °C (Huner et al., 1986; Hurry and Huner, 1991; Öquist and Huner, 1993; Öquist et al., 1993). Similar trends have been reported for 30 °C and 13 °C-grown Brassica napus (Paul et al., 1990).This is accompanied by a significantly greater maximum carboxylation rate for purified Rubisco regardless of measuring temperature (Huner and Macdowall, 1979b). However, growth temperature does not affect

the maximum quantum yield for photosynthesis (Huner et al., 1986; Hurry and Huner, 1991; Öquist and Huner, 1993). Thus, winter wheat and rye are able to modulate photosynthetic rates during growth at low temperatures such that photosynthetic capacity is increased with minimal change in photosynthetic efficiency. In contrast to cold tolerant herbaceous annuals, exposure of conifers such as Scots pine to 5 °C and short-day conditions in controlled environments causes a significant depression in the maximum rate of photosynthesis and electron transport (Öquist et al., 1980). Thus, the extent of a low growth temperature stimulation or depression of the maximum rate of photosynthesis is cultivar and species dependent (Boese and Huner, 1990; Hurry and Huner, 1991; Öquist and Huner, 1993). In summary, there are conflicting reports in the literature regarding the effect of culturing at low temperature on the maximum quantum yield of photosynthesis in algae. This may be due to methodological problems. However, the data on terrestrial plants is consistent and indicate that growth temperature has no effect on the maximum quantum yield.

B. Carbon Metabolism Photosynthetic carbon reduction is generally stimulated when plants are exposed to 2% compared to 21% due to an inhibition of photorespiration. However, photosynthetic rates of Brassica napus (Paul et al., 1990), clover (Mächler et al., 1984) and winter rye (Huner etal., 1986) exhibit reduced sensitivity after growth and development at low temperature. Paul et al. (1990) concluded that 13 °C-grown Brassica are less limited by triose phosphate utilization, and thus less Pi limited, than the 30 °C-grown plants. As expected, analyses of phosphorylated intermediates indicate that the 13 °Cgrown plants exhibit two- to three-fold higher levels of hexose phosphates and triose phosphate than the 30 °C-grown plants. However, PGA/triose phosphate is four-fold lower in the 13 °C than the 30 °C-grown plants which, in turn, is coupled to four-fold higher levels of sucrose in the former than the latter. The ratio of sucrose:starch was 2.5 for the 13 °C plants compared to 1.1 for the 30 °C plants (Paul et al., 1990). Thus, it appears that temperate plants such as Brassica napus grown at cool temperatures are able to overcome, to some extent, the limitations imposed by low temperature upon photosynthesis and carbon

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metabolism. This is not observed when plants are shifted from high to low temperature. Recently, Hurry et al. (1993) investigated the role of Pi in photosynthetic regulation that is associated with low growth temperature in cereals. Growth of winter rye at 5 °C results in an increased capacity for evolution, dampening of the fluorescence induction transients and a reduction in the lag time to reach steady state photosynthesis. However, uptake of exogenous Pi in rye grown at 20 °C mimicked the photosynthetic characteristics of rye grown at the low temperature. They concluded that the enhanced photosynthetic rates observed upon growth at low temperature is due to increased Piavailability which may reflect an increased flux through the sucrose synthesis pathway (Huner et al., 1993). Pollock and Lloyd (1987) have shown that starch synthesis is more sensitive to low temperature than sucrose and fructan synthesis in 8 cold tolerant species. They propose that the use of sucrose and fructans as storage carbohydrates may counteract the predisposition for Pi-limitation at low temperatures. This should result in a more effective utilization of absorbed light at low temperatures. In addition, conversion of sucrose to fructans in the vacuole partially overcomes the possible negative osmotic effects of high sucrose accumulation in the cytosol (see Chapter 10). Thus, cold tolerant plants grown at low temperature appear to be able to circumvent or escape the potential for Pi limitation of photosynthesis thought to be imposed by low temperature upon carbon metabolism (Sharkey, 1985b; Sharkey et al., 1986; Stitt et al., 1987). Antisense mutations to block various steps of carbon metabolism and sink activities or over-expression of the same genes in cold tolerant plants may prove to be a powerful approach to elucidate the contribution of carbon metabolism to photosynthetic acclimation to low temperature. It can be hypothesized that changes in activity and/or amount of enzymes and metabolites involved in photosynthetic carbon metabolism could, in part or completely, compensate for the reduction in activity of individual enzyme molecules at low temperature (Jørgensen, 1968; Davison, 1987). The basic metabolic pathways of fixation in Skeletonema costatum were not affected by low temperature or low irradiance, but both of these factors increased labeling of synthesized by the CalvinBenson cycle and decreased levels of phosphoenolpyruvate and other metabolites. This indicates

an enhancement of Rubisco-activity, which was confirmed by a seven-fold increase of Rubisco activity at low temperatures (Mortain-Bertrand et al., 1988). These results have been supported by experiments with Chlorella vulgaris, where total Rubisco activity of cells grown at 5 °C was 50% higher than for cells grown at 27 °C (D. P. Maxwell, L.V Savitch and N. P. A. Huner, unpublished). This was associated with an measured at 5 °C that was, on a chlorophyll basis, 4-fold higher for cells acclimated to 5 °C than for cells grown at 27 °C (Maxwell et al., 1994). Similar trends have been reported for higher plants. For example, prolonged exposure to low temperature results in an increased stability of Rubisco structure, increased activity and light activation of this enzyme (Huner and Macdowall, 1979a,b; Grafflage and Krause, 1993) which appears to be related to the low temperature-induced changes in solute composition as well as changes in the structure of Rubisco itself. Guy and Carter (1984) have come to similar conclusions regarding glutathione reductase. Furthermore, Descolas-Gros and de Billy (1987) found that the maximum substrate affinity for Rubisco occurred at 4.5 °C in the enzyme from Antarctic diatoms and at 20 °C in the enzyme from temperate species. These structural and functional changes appear to reflect low temperature adjustment of carbon metabolism (Huner and Macdowall, 1979b; Grafflage and Krause, 1993). However, further work is requi red to understand the nature and the mechanism involved in the stabilization of Rubisco at low temperature. In summary, the apparent increase in sucrose synthesis in herbaceous higher plants serves two important aspects of acclimation to low temperatures: Firstly, it alleviates the low temperature-induced Pilimitation on photosynthesis and secondly, it results in the production of a cryoprotectant. In contrast, algae appear to modulate photosynthetic capacity during growth at low temperature primarily by regulation of the amount and kinetic properties of Rubisco during acclimation to low temperature. The role of gene regulation in this photosynthetic response has yet to be elucidated.

C. Electron Transport While PS II is unable to utilize light energy for photochemistry under conditions where the first stable electron acceptor, is reduced, PS I reaction centers can dissipate excitation energy regardless of their redox state (Turconi et al., 1993; Chapters 1–3). The

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need to regulate the input of energy to avoid lightinduced stress to the reaction centre thus appears more important in PS II than in PS I. PS II is also believed to be the most thermolabile component of the photosynthetic apparatus, causing the reduction in photosynthesis at temperatures above the temperature optima (Fork et al., 1979). The quantum yield of linear electron transport in PS II as estimated by Genty et al. (1989), is the product of and Rye and wheat grown at 5 °C exhibit higher than 20 °C-grown plants regardless of irradiance and measuring temperature. This is primarily due to low growth temperature effects on rather than (Hurry et al., 1992; Öquist and Huner, 1993). Three-fold higher light levels were required to reduce the ratio of oxidized to reduced to 0.5 in 5 °C than 20 °C-grown rye. Plots of versus irradiance and versus according to Weis and Berry (1987) indicate that the response of open PS II reaction centers to irradiance and to regulation through non-photochemical quenching are identical for leaves developed at 5 or 20 °C. Thus, growth at low temperatures appears to alter the proportion of open reaction centers as opposed to the nature of the PS II reaction centers. This, in turn, is coupled to increased photosynthetic capacity and carbon utilization and results in an increased resistance to photoinhibition (Huner et al., 1993). It appears that conifers and algae are dependent upon alternative mechanisms to cope with temperature-induced changes in the requirements of light energy for photosynthesis. Dunaliella tertiolecta has been shown to parallel a temperature-induced decrease in carbon fixation rate by a decrease of energy transfer efficiency, presumably by disconnecting the antennae from the PS II reaction centers, and a decrease of quantum yield (Levasseur et al., 1990). On the other hand, growth at low temperature did not significantly change the amount of PS II or the light absorption efficiency per unit chlorophyll a, or the amount of chlorophyll a per cell. This is contrary to results with other algal species like Phaeodactylum tricornutum, Chlorella vulgaris and Nitzschia closterium, where the decrease in photosynthetic rate per cell was paralleled by a decrease in chlorophyll a per cell (Morris and Glover, 1974; Maxwell et al., 1994). Similarly, conifers exposed to long-term cold hardening conditions exhibit an inhibition of PS II electron transport which is coupled to decreased photosynthetic capacity

(Öquist and Martin, 1986). However, in contrast to conifers, alterations in PS II of algae does result in decreased susceptibility to photoinhibition (Öquist and Huner, 1991; Maxwell et al., 1995). It appears that the response to low temperature at the level of PS II is species dependent and correlates with the growth strategy of the species (Huner et al., 1993). With regard to PS I, growth at low temperature results in 50% to 100% higher rates of in vitro, lightsaturated electron transport with no effect on lightlimited rates using methyl viologen as the terminal electron acceptor (Huner, 1985; Krol et al., 1988; Huner and Reynolds, 1989; Reynolds and Huner, 1990). Low temperature stimulation of light-saturated PS I activity was also observed in periwinkle leaves during natural overwintering conditions (Huner et al., 1988). However, the higher PS I activity in thylakoids from 5 °C-grown plants could be completely eliminated by isolation of the thylakoids in the absence of and (Huner and Reynolds, 1989). This resulted in identical light response curves for PS I activity in isolated thylakoids from 5 °C and 20 °C-grown plants. Re-addition of or in vitro to final concentrations of 10 mM reestablished the higher rates of PS I activity in thylakoids from 5 °C-grown leaves. Monovalent cations could also restore this activity but 10-fold higher concentrations were required (Chapman and Huner, 1993). Thus, in contrast to the transient stimulation of PS I activity observed upon shifting plants from 20 °C to 5 °C, growth at 5 °C results in stable, higher light-saturated PS I activity. Clearly, cations are required to stabilize this higher activity. In summary, PS II electron transport is modulated positively or negatively by growth at low temperature depending upon the species and is related to the protection of PS II against photoinhibition. However, growth at low temperature results in a stable increase in light saturated PS I activity. This appears to be associated with a cation-mediated alteration in thylakoid organization. Further work is required to elucidate the role of stimulated PS I activity.

IV. Thylakoid Membrane Lipids Higher plant lipid synthesis is regulated to provide the array of lipids necessary for the proper assembly and function of the thylakoid membrane as well as other membranes within a plant cell (Browse and Somerville, 1991). It is thought that many organisms

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respond to changes in environmental temperature by modulating membrane viscosity (Quinn and Williams, 1985). This can be accomplished in several ways: (i) a change in the ratio of lipid/protein, (ii) a change in the proportion of various lipid classes, and (iii) a change in the lipid molecular species and /or the level of fatty acid unsaturation. For a detailed discussion of these topics, the reader is referred to Quinn and Williams (1985). The effects of growth temperature on lipid content and composition appears to be species dependent and dependent upon the temperature range to which the plant is exposed. For example, Raison et al. (1982) and Lynch and Thompson (1984) reported significant adjustments in thylakoid membrane viscosity and lipid composition as a function of growth temperature in Nerium oleander and Dunaliella respectively. At high growth temperatures, the level of fatty acid unsaturation and thylakoid membrane viscosity decreased. However, the comparative growth temperatures (20–45 °C) were significantly higher than generally employed for cold tolerant plants (0–25 °C). These discrepancies may not only reflect species differences but may also reflect the fact that there is a maximum level of unsaturation that can be tolerated physiologically in the thylakoid membrane. In many plant species, this maximum level of unsaturation may be attained upon exposure to 15–20 °C and thus growth at lower temperatures may not increase levels of unsaturation significantly. We suggest that the capacity to modulate membrane viscosity by alteration of lipid unsaturation may therefore be more important over a high temperature range (20– 40 °C) than a low temperature range (0–20 °C). Based on molecular species analyses of thylakoid phosphatidylglycerol (PG), it was proposed that chilling-sensitive plants exhibit a greater proportion of gel phase lipid than chilling resistant plants at temperatures less than 10 °C (Murata et al., 1982; Murata and Yamaya, 1984). However, Low et al. (1984) showed that there was no correlation between chilling sensitivity and the presence or absence of a thermotropic phase transition for bulk thylakoid lipids at temperatures above 5 °C. An extensive survey of the literature by Bishop (1986) revealed that that the content of high melting point fatty acids in PG was as strongly correlated to genetic differences between species as to differences in chilling sensitivity. In addition, the assumption by Murata’s group that hexadecenoic acid has the same physical

properties as a saturated fatty acid does not appear to be correct since the phase transition of PG( 16:0/ trans–16:l) is 10 °C lower than PG(16:0/16:0) (Bishop and Kenrick, 1987). White and Somero (1982) suggested that increased unsaturation of the membrane fatty acids is a normal response to lower temperatures for most organisms. Whether or not the homeoviscosity of membranes is really achieved through modifications of fatty acids has been debated (Quinn, 1988). For example, the fatty acids of marine phytoplankton are frequently more unsaturated than in terrestrial plants living in far colder environments (Thompson etal., 1992b). It was also shown by Thompson et al. (1992b) that when eight marine phytoplankton species were investigated for changes in fatty acid composition as a response to culturing temperatures between 10 to 25 °C, only in one case was it possible to correlate an increased percentage of polyunsaturated fatty acids to a decrease in growth temperature. Instead, the percent composition of polyunsaturated fatty acids was correlated with PFD or PFD-controlled growth rates in four of the cultures. Thus, marine phytoplankton as a group do not appear to increase fatty acid unsaturation in response to lower temperatures per se. Murata and colleagues have utilized cyanobacteria as a model system to study the role of phase transitions of membrane lipids in the tolerance of organisms to chilling injury. Since a phase transition depends, in part, on the degree of unsaturation of fatty acids of the membrane lipids (Chapman, 1975), these researchers have exploited the ease of genetic engineering in cyanobacteria to manipulate the extent of fatty acid desaturation. They initiated their molecular studies by utilizing conventional chemical mutagenesis techniques to select low-temperature sensitive mutants of the cyanobacterium Synechocystis PCC6803 (Wada and Murata, 1989). Analysis of the fatty acid composition indicated that two of these cyanobacterial mutants were defective in fatty acid desaturation at and position of fatty acids (designated Fad6 and Fad 12, respectively). Subsequently, Wada et al. (1990) used standard gene complementation techniques to clone the gene (designated desA) responsible for the low temperature growth phenotype exhibited by Fad 12. Expression of the desA gene product in E. coli, which does not contain any fatty acid desaturase, was used to demonstrate that desA encodes the structural gene for the desaturase (Wada et al., 1993). The

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abundance of the desA transcript is increased 10-fold when cells are shifted from growth at high to low temperature. Los et al. (1993) have provided some evidence which suggests that the accumulation of desA transcript is dependent on the extent of the temperature change rather than the absolute growth temperature. The most significant breakthrough in our understanding of the relationship between chilling tolerance and fatty acid desaturation came when Murata’s group introduced the desA into a cyanobacterial strain lacking desaturation at the position (Wada et al., 1990). Cells transformed with the desA gene exhibited a lower phase transition temperature in their cytoplasmic membrane presumably because the cells were now able to introduce a second double bond in the cis configuration at the position of fatty acids and thereby to synthesize 16:2(9, 12) and 18:2(9, 12) fatty acids. Most importantly, these cells were more tolerant to low temperature than the original wild-type strain. The desA gene has also been used to create mutant cyanobacterial strains that contain lipids with defined levels of unsaturated fatty acids and these strains have been used to further elucidate the role of membrane fluidity in growth and photosynthesis at low temperatures (22 °C vs. 34 °C) and photosynthesis. Growth rates and photosynthesis were unaffected by low temperatures in the Fad6 mutant which contains mono- and diunsaturated lipids, when compared to wild-type Synechocystis PCC6803 which contains mono-, di-, and triunsaturated lipids. However, a Fad6/desA double mutant, which contains no polyunsaturated fatty acids, exhibited increased sensitivity to photoinhibition (Combos et al., 1992) and an inhibition of growth only at low temperatures (Wada et al., 1992). The Fad6/desA mutant was also sensitive to photoinhibition at 30 °C but the degree was less severe than that seen at 10 °C and 20 °C. These results emphasize the importance of the position of fatty acids in tolerance to photoinhibition at both low and normal temperatures in cyanobacteria. Oxygen evolution and photosynthetic electron transport, although reduced at low temperature in all strains tested, did not appear to be affected by the level of fatty acid desaturation. This indicates that changes in the saturation of fatty acids is not responsible for the decrease in photosynthetic activity seen in cyanobacteria at lower growth temperatures. Gombos et al. (1994) have also presented preliminary results which suggest a role for fatty

acid unsaturation in the tolerance of the photosynthetic apparatus to heat stress. They found that the degree of unsaturation at the position of fatty acids was also important in determining the susceptibility of the photosynthetic machinery to heat. However, the physiological significance of this observation remains to be determined. Progress has also been made in elucidating the primary signal in cyanobacteria responsible for the biological perception of temperature. Using an experimental system involving the palladiumcatalyzed hydrogenation of membrane lipids, Vigh et al. (1993) have shown that a reduction in the double bonds of lipids located specifically in the cytoplasmic membrane results in the stimulation of expression of the desA gene. This change in desA expression was similar to that observed when cells were exposed to low temperature. These results support the hypothesis that a low temperature-induced change in the level of the desA transcript is also regulated by the fluidity of the membrane. In summary, the evidence for a link between increased membrane lipid unsaturation and low temperature acclimation is relatively strong in cyanobacteria, resulting in increased resistance to photoinhibition and improved growth at low temperatures. However, the evidence in algae and higher plants for such a correlation is equivocal. The establishment of such a correlation in higher plants and algae appears to be confounded by the fact that inherent genetic differences between species exerts as great or greater influence on fatty acid unsaturation than low temperature.

V.Temperature and Chloroplast Development Cold-tolerant plants developed at low temperature produce small, thick leaves that exhibit a 3-fold increase in dry weight per unit area, a 1.5 -fold increase in chlorophyll/leaf area with no apparent change in chlorophyll a/b, thylakoid protein/chlorophyll, thylakoid protein/lipid or polypeptide composition (Krol et al., 1988; Huner et al., 1993). Furthermore, these low temperature-grown plants exhibit an increased light-saturated capacity for photosynthesis compared to the same plants grown at 20 °C (Huner et al., 1986,1993). Although chloroplast development is not impaired, the organization of PS II and PS I is significantly altered in cold tolerant monocots such as winter rye and wheat during chloroplast biogenesis

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at low temperature (Krol et al., 1988; Griffith et al., 1989). Leheny and Theg (1994) have observed no functional adjustment in the protein import of pea chloroplasts grown at low temperature. Both warm and cold grown pea were able to import proteins at temperatures as low as 4 °C, provided that the chloroplasts were illuminated. They suggest that low temperature-induced changes in membrane properties do not limit the import process in chloroplasts of cold-tolerant plants In contrast to cold-tolerant plants, chilling-sensitive plants such as maize develop small, chlorotic leaves at low temperatures which exhibit a reduced level of photosynthetic apparatus per leaf area and a reduced photosynthetic efficiency (Baker, 1993; Baker and Nie, 1994). This is associated with an inhibition of leaf protein accumulation (Nie and Baker, 1991). It was demonstrated that the accumulation of chloroplast encoded thylakoid proteins such as the PS II and PS I reaction centre polypeptides, cytochrome f, and the of the coupling factor were selectively reduced compared to the nuclear encoded chloroplast proteins of LHCII and LHCI (Nie and Baker, 1991). Furthermore, immunocytochemical studies in maize showed that mesophyll and bundle sheath cells exhibit differential effects of the inhibition of chloroplast protein accumulation at low growth temperatures (Robertson et al., 1993), and also differences in the ability of the chloroplasts to develop normal thylakoid protein complements on transfer of leaves to higher temperature (Nie et al., 1995). Mesophyll cells were heterogeneous in their response to chloroplast protein accumulation at low temperature, whereas bundle sheath cells responded uniformly. In addition, post-translational modification of certain maize thylakoid proteins was altered during exposure to chilling temperatures (Hayden et al., 1988). The observed reduction in accumulation of these chloroplast proteins undoubtedly contributes directly to the decreased photosynthetic efficiency and capacity observed upon growth of maize at low temperature (Nie et al., 1995). Since low temperature did not appear to inhibit preferentially chloroplastic versus cytoplasmic protein synthesis, it was suggested that low temperature-induced changes in thylakoid lipid composition could destabilize the thylakoid supramolecular complexes which contain chloroplastencoded polypeptides. However, there was no evidence presented to indicate that maize thylakoid lipid composition was altered by low temperature exposure.

Chill-induced changes in the synthesis of chloroplast proteins and mRNA accumulation have also been reported for rice (Hahn and Walbot, 1989), Brassica napus and tomato (Cooper and Ort, 1988; Martino-Catt and Ort, 1992). In tomato, it has been reported that the inhibition of photosynthesis upon exposure to chilling in the dark may be the result of a low temperature perturbation of the temporal control of transcription of cab and Rubisco activase (MartinoCatt and Ort, 1992). Although exposure to low temperature increased the life-time of cab and Rubisco mRNA, these stabilized mRNAs were not translated upon rewarming. Thus, low temperatureinduced interruption of circadian control of transcription may represent a novel clue in unraveling the complex mechanism of low temperature inhibition of photosynthesis in tomato. Further work is required to determine if this is general phenomenon in chilling sensitive plants. In summary, it appears that temperature may influence the photosynthetic capacity and efficiency of a species directly through thermodynamic constraints and/or indirectly through limitations on chloroplast development. The sensitivity of chloroplast development to low temperature appears to be species specific with the accumulation of chloroplast gene products being more sensitive to temperature than the accumulation of nuclear gene products.

VI. Interaction of Light and Temperature Exposure of leaves to an irradiance in excess of that required for photosynthesis can result in photoinhibitory damage. Susceptibility to photoinhibition is exacerbated by light in combination with other stresses such as drought and low temperature (Powles, 1984). In contrast to drought and low temperature stress, light appears to provide protection during exposure to heat stress (Havaux and Strasser, 1990, 1992; Havaux et al., 1991). Under in vivo dark conditions, leaves of Pisum sativum exhibited a significant and irreversible decrease in the maximum quantum yield and light-saturated rate of evolution. In the presence of even low light, heat had no effect on these parameters. The protective mechanism has not been elucidated but may involve non-radiative dissipation of absorbed energy in PS II antennae or reaction centres (Havaux and Strasser, 1990), protein phosphorylation (Süss and Yordanov, 1986) or the accumulation of chloroplastic heat shock proteins

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(Vierling, 1991). Furthermore, Rikin et al. (1993) reported that this heat resistance is associated with the circadian rhythm of the plants. These observations indicate that PS II should be protected from heat under natural conditions since high temperatures are usually associated with high irradiance. Growth at sub-optimal conditions in cold tolerant plants can lead to increased tolerance to photoinhibition. Somersalo and Krause (1989,1990) were the first to report that cold-acclimated plants exhibit a unique capacity to increase tolerance to photoinhibition. This has been confirmed for cereals as well as spinach (Boese and Huner, 1990; Öquist and Huner, 1991; Hurry and Huner, 1992). A unique feature of resistance to photoinhibition is that growth at 5 °C and moderate irradiance results in a significant decrease in susceptibility to high irradiance that is, 6 times the growth irradiance. Usually resistance to photoinhibition is associated with preexposure to high light. For example, plants exposed to full sunlight are typically less sensitive to photoinhibition than the same plants exposed to a shade environment (Powles, 1984; Ögren and Rosenqvist, 1992). Development at low temperature is an absolute requirement for the acquisition of resistance to photoinhibition in rye (Öquist and Huner, 1991) and spinach (Boese and Huner, 1992; Gray et al., 1994). Nie et al. (1992) reported that even growth of chillingsensitive maize at 17 °C increased its tolerance to high light at low temperatures. A loss of chlorophyll is observed in some higher plants (Berry and Björkman, 1980), macroalgae (Lapointeetal., 1984;Davison, 1991) and microalgae (Geider, 1987) transferred to sub-optimal temperatures but at constant levels of irradiance. This lowtemperature chlorosis is accentuated by increasing the levels of irradiance (Berry and Björkman, 1980; Geider, 1987). However, the interpretation of these experiments must be treated with some caution. In studies that manipulate temperature, it is difficult to maintain a stable energy status for the algal cells, therefore it is difficult to avoid manipulating more than one important physiological parameter at a time. Thompson et al. (1992a) observed that attempts to maintain growth at one selected irradiance giving non-photoinhibited, energy-saturated cells were unsuccessful, since the cells became less energysaturated with increasing temperature. This important observation has been further strengthened by recent experiments on Chlorella vulgaris grown at 27 and

5 °C, where it was shown that the reported increase in chlorophyll a/b-ratios, decrease in LHCII polypeptides and increase in photoinhibitory resistance during growth at 5 °C was completely reversed by a reduction of the irradiance during growth from 150 to (Maxwell et al., 1994; Maxwell et al., 1995). Similar effects have been observed for higherplants (Gray et al., 1995). Thus, it appears that low temperature-induced changes in photosynthetic performance may reflect acclimation to light stress even though the irradiance at the high and low growth temperatures remained constant. In turn, this is probably triggered by the reduced enzymatic activities at low temperature, resulting in less demand for ATP and NADPH. Given equal irradiation at high and low temperature, low growth temperature may result in a situation of excess excitation and potential photoinhibitory damage to PS II, which in C. vulgaris is mainly counteracted by a reduction in the antenna size whereas higher plants adjust by an increased photochemical quenching of PS II. The current view is that the primary target for photoinhibition is PS II (Powles, 1984). However, recent work by Terishima and co-workers (Sonoike and Terishima, 1994; Terishima etal., 1994) indicate that, in leaves of the chilling-sensitive Cucumis sativus, PS I and not PS II is the primary site of low temperature photoinhibition. Furthermore, the irradiance required to induce this PS I photoinhibition is almost one order of magnitude lower than that required for photoinhibition of PS II in this plant species. In addition, Moon et al. (1995) reported that unsaturation of chloroplast membrane lipids is an important factor which stabilizes the photosynthetic apparatus of transgenic tobacco against low temperature-induced photoinhibition. Further work is required to assess the generality of these observations in chilling-sensitive and cold-tolerant plant species. In summary, we suggest that, at least in some cases, reported low temperature-induced changes in antenna size and photosynthetic performance may actually reflect the fact that low temperature creates a situation of high light stress even under constant irradiance. This may necessitate a re-evaluation of published reports purporting low temperatureinduced changes in leaf physiology and gene expression in order to separate changes due to low temperature per se and changes due to a low temperature-induced high light stress.

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VII. Photosynthetic Adaptation, Acclimation and Stress From the discussion above, there is no general mechanism for photosynthetic acclimation to high or low temperatures that encompasses all possible photoautotrophic species. The difficulty in identifying such a mechanism arises, in part, because high temperature and low temperature are relative terms. Low temperature for one plant species may represent high temperature for another species. For example, the thermophilic cyanobacterium Synechococcus lividus which photosynthesizes at temperatures as high as 75 °C exhibits a change to more unsaturated fatty acids when the growth temperature is lowered from 55 °C to 38 °C (Fork et al., 1979). In addition, Synechocystis PCC6803 shifted from growth at 38 °C to 22 °C exhibited increased unsaturation of fatty acids (Wada and Murata, 1989, 1990). These typical ‘chilling responses’ occur within a temperature range which would be considered high temperature stress for many higher plant species. Similarly, the thermolability of PS II, which in leaves occurs in the range of 35 °C to 45 °C, is considered to set the upper limit for photosynthesis during heat stress (Berry and Björkman 1980; Thompson et al., l989; Havaux, 1993). However, several arctic and Antarctic algal species are photosynthetically competent at temperatures as low –1.8 °C but are unable to survive temperatures above 10 °C to 15 °C (Sheridan and Ulik, 1976; Palmisano et al., 1987; Davison, 1991; Ling and Seppelt, 1993), indicating that high temperature stress in these algae may involve a mechanism other than PS II-denaturation. Berry and Björkman (1980) defined photosynthetic acclimation as an improved photosynthetic performance which is usually characterized by an altered temperature optimum biased towards the new temperature regime (Berry and Björkman, 1980; Graham and Patterson, 1982; Öquist and Martin, 1986). We maintain that improved photosynthetic performance as suggested by Berry and Björkman (1980) is not necessarily a prerequisite for photosynthetic temperature acclimation. Although many plant species exhibit improved photosynthetic performance when exposed to a new temperature regime, maintenance of active photosynthesis must be a prerequisite for survival. In contrast, evergreens must become dormant in order to survive winter conditions and concomitantly down regulate photosynthesis such that active photosynthesis is

significantly reduced (Strand and Öquist, 1985; Bolhar-Nordenkampf and Lechner, 1988; Oberhauer and Bauer, 1991; Groom et al., 1991). Both strategies are essential to ensure the survival of the plant species during adverse environmental conditions. Therefore, both improved as well as reduced photosynthetic performance can be considered as photosynthetic acclimation to low temperature. We conclude that the more general definition of Öquist (1983) is most appropriate to describe photosynthetic acclimation to temperature. Thus, we suggest the following definition for photosynthetic temperature acclimation: phenotypic adjustments of the functional and structural properties of the photosynthetic apparatus that can be modulated by environmental temperature to enhance the probability of survival and reproduction of the plant species.

Acknowledgments The authors wish to thank N. R. Baker, G. H. Krause, N. Murata and S. Theg for providing us with unpublished results and manuscripts prior to publication. Research support from the Natural Science and Engineering Research Council of Canada (NSERCC) is gratefully acknowledged by NPAH and DEL. SF was supported by a NSERCC International Postdoctoral Fellowship. DPM was supported by an Ontario Graduate Scholarship. This chapter is dedicated to the memory of Dave Laudenbach who died on June 16, 1995.

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57: 113–119 Vierling E (1991) The role of heatshock proteins in plants. Annu Rev Plant Physiol Plant Mol Biol 42: 579–620 Vigh L, Los DA, Horvath I and Murata N (1993) The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803. Proc Natl Acad Sci USA 90: 9090–9094 Wada H and Murata N (1989) Synechocystis PCC6803 mutants defective in desaturation of fatty acids. Plant Cell Physiol 30: 971–978 Wada H and Murata N (1990) Temperature-induced changes in the fatty acid composition of the cyanobacterium, Synechocystis PCC6803. Plant Physiol 92: 1062–1069 Wada H, Gombos Z and Murata N (1990) Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation. Nature 347: 200–203 Wada H, Gombos Z, Sakamoto T and Murata N (1992) Genetic manipulation of the extent of desaturation of fatty acids in membrane l i p i d s in the cyanobacterium Synechocystis PCC6803. Plant Cell Physiol 33: 535–540 Wada H, Avelange-Macharel M and Murata N (1993) The desA gene of the cyanobacterium Synechocystis sp. strain PCC6803 is the structural gene for desaturase. J Bacteriol 175: 6056– 6058 Warner DA and Burke JJ (1993) Cool night temperatures alter leaf starch and Photosystem II chlorophyll fluorescence in cotton. Agron. J 85: 836–840 Weis E (1985) Short term acclimation of spinach to high temperatures. Plant Physiol 74: 402–407 Weis E and Berry J A (1987) Quantum efficiency of Photosystem II in relation to ‘energy’-dependent quenching of chlorophyll fluorescence. Biochim Biophys Acta 894: 198–207 Weis E and Berry JA (1988) Plants and high temperature stress. In: Long SP and Woodward FI (eds) Plants and Temperature, pp 329–346. The Company of Biologists Ltd, Cambridge White FN and Somero G (1982) Acid-base regulation and phospholipid adaptations to temperature: time courses and physiological significance of modeling the milieu for protein function. Physiol Rev 62: 40–90 Yordanov I, Dilova S, Petkova R, Pangelova T, Goltsev V and Süss K-H (1986) Mechanisms of the temperature damage and acclimation of the photosynthetic apparatus. Photochem Photobiophys 12: 147–155

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Chapter 16 Photosynthetic Responses to Changing Atmospheric Carbon Dioxide Concentration George Bowes Department of Botany, University of Florida, Gainesville, Florida 32611, USA

Summary I. Rising in Perspective II. Sites of Action of in Plants III. Adaptation to Changes in Atmospheric A. Declining B. Rising IV. Diversity in Photosynthetic Responses to Enrichment A. Species Differences and Acclimation Mechanisms B. Environmental Constraints that Modify Enrichment Responses V. Concluding Comments Acknowledgments References

387 388 389 390 390 392 393 393 399 402 402 402

Summary When plants made the transition to land, atmospheric concentration was up to 16-fold higher than today; since then it has fluctuated, but with an overall decline to very low values during the last glacial maximum. Modern-day plants exhibit photosynthetic adaptations to cope with a low ratio. These include: high specificity and low for of ribulose bisphosphate carboxylase-oxygenase (Rubisco); pathways to recapture photorespiratory C and N; concentrating mechanisms in some terrestrial and aquatic species based on or -use systems; improvements in stomatal regulation; and possibly lower ecological compensation points. Since the last glacial maximum, atmospheric concentration has doubled to 360 but it is still relatively low, and does not saturate photosynthesis; the mainstay of some 95% of terrestrial species. Herbarium and fossil studies indicate plants may be readapting to this rise by decreases in stomatal density. A further doubling of concentration has the potential to reduce the inhibition of Rubisco and halve photorespiration; reduce stomatal conductance and enhance water use efficiency; increase the C/N ratio; lower dark respiration; exert growth modulator effects; and in aquatic species influence the photosynthetic affinity for dissolved inorganic carbon. These enhance the use of other resources in an ‘efficiency effect’, but it is not always achieved because of acclimation. After an initial 50% increase in assimilation, acclimation often entails downregulation of the Rubisco- and/or ribulose 1,5-bisphosphate-limited portions of the assimilation versus intercellular curve. Acclimation may be a stress response when carbohydrate accumulation causes chloroplast deformation, but in most instances it seems to be an optimization process to balance carbon Neil R. Baker (ed): Photosynthesis and the Environment, pp. 387–407. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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acquisition with a limited utilization capacity, in part by reallocation of N. It usually involves a reduction in Rubisco protein and/or activation, and possibly up-regulation of carbohydrate metabolism. Some field-grown plants, notably soybean, show no down-regulation of the assimilation versus intercellular curve. Plants with substantial sink capacity, such as crop and competitive-strategy species, have the greatest response to enrichment , with on average a 30–40% stimulation of biomass; while those with small sinks, such as stresstolerant species, have the least. Even species may show growth stimulation through greater water use efficiency and leaf area. Among submersed species, the photosynthesis and growth of users is enhanced, but show minimal response. Enrichment can stimulate photosynthesis and growth when water, nutrients or light are suboptimal, and temperatures are high, though extreme conditions can abolish the benefits. The photosynthetic response is not always the major factor influencing competitive interactions among species, but directly or indirectly rising concentration will likely alter the species distribution and composition of ecosystems.

I. Rising

in Perspective

The global carbon cycle involves massive exchanges of inorganic carbon between the atmosphere and the terrestrial and aquatic reservoirs, with some 100 Gt assimilated annually by photosynthesis, and a similar amount evolved by processes such as respiration and decomposition (Post et al., 1990). The aquatic environment is by far the greatest active reservoir of the planet’s carbon, with an estimated 38,000 Gt, as compared to only 748 Gt in the atmosphere (Post et al., 1990). The major exchange processes approximately balance one another, producing a dynamic atmospheric compensation point that as of 1994 is 360 but is rising at a rate of over 1.5 (Keeling and Whorf, 1992). Anthropogenic processes, which now transfer from terrestrial reservoirs to the atmosphere the equivalent of 5–8 Gt carbon perturb the balance and are the driving force for the rise. There is legitimate concern over how the predicted doubling of concentration during the next century will influence the earth’s climate, ecosystems, and agricultural production; Abbreviations: A – rate of assimilation; C – competitor strategy; – ambient atmospheric concentration; intracellular concentration; CAM – crassulacean acid metabolism; concentrating mechanism; DIG – dissolved inorganic carbon; Gt– Gigatonne; I – irradiance; MYA – million years ago; inorganic carbon concentration that drives photosynthesis at half its maximum rate; – Michaelis-Menten constant for substrate; PEPC – phosphoenolpyruvate carboxylase; PCO – photorespiratory carbon oxidation; Pi – inorganic phosphate; PCR – photosynthetic carbon reduction; R – ruderal strategy; Rubisco – ribulose 1,5-bisphosphate carboxylase-oxygenase; RuBP – ribulose 1,5-bisphosphate; S–stress strategy; SPAR–soil-plantair-research; SPS – sucrose phosphate synthase; WUE – water use efficiency; compensation point; compensation point.

but substantial fluctuations in have occurred in the past. When plants made the transition to land, some 420 million years ago (MYA), the atmosphere may have contained as much as 4–6,000 but only 0.02 to 0.10 mol (Böger, 1980; Budyko et al., 1987; Yapp and Poths, 1992; Berner, 1993). The rise of vascular plants, and their attendant photosynthesis, was a major factor in the decline of atmospheric concentration, which by 300 MYA may have dropped to near present values (Berner, 1993). Thereafter concentration appears to have increased to perhaps four- or five-times present values, before declining to relatively low concentrations during the last 25–30 million years. As regards recent geological time, the past 160,000 years have seen atypically low atmospheric concentrations with glacial lows of 180–220, and interglacial highs of 250–300 (Post et al., 1990). Thus, atmospheric concentration has fluctuated perhaps by as much as 20-fold over geologic time, so higher values are not a new phenomenon for plants. Also, as discussed later, terrestrial and aquatic habitats exist today where plants have been exposed to high concentration for many generations (Sand-Jensen et al., 1992; Miglietta et al., 1993; Woodward, 1993). The current Holocene interglacial period is not only characterized by relatively low concentration, but also by a stable climate (Thomson, 1993). However, there is evidence that climate can oscillate rapidly, and in the previous interglacial period changes on the order of 10 °C may have occurred within only a few decades (Dansgaard et al., 1993). Such rapid and extreme temperature fluctuations would have more impact on the photosynthesis and survival of plants than a doubling in atmospheric As

Chapter 16 Photosynthesis and Rising rises, general circulation models predict increases in mean global temperatures of between 1.5 and 4.5 °C, and increased, but variable, precipitation patterns. However, at regional levels the magnitude of these changes is uncertain. The scope of this chapter precludes an extended analysis of how future fluctuations in temperature and precipitation may impact photosynthesis.

II. Sites of Action of

in Plants

First and foremost, is the substrate for the primary carboxylase of autotrophic organisms, ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). This enzyme is the entry point for inorganic carbon into the photosynthetic carbon reduction (PCR) cycle, and the organic biosphere. Rubisco is a major component regulating assimilation (A) in plants, which constitute 95% of the plant species. Control analyses show that at high irradiance the flux control coefficient for Rubisco is as high as 0.9, where 1.0 indicates it alone is the limiting factor. Under most conditions, control is partially shared with other photosynthetic processes (Woodrow and Mott, 1988; Stitt and Schulze, 1994; Woodrow, 1994). The affinity of Rubisco for varies among organisms. Terrestrial species have values for of about 10 and 30 respectively; microalgae and submersed plants have higher values in the range of 30–70 while cyanobacteria have the highest values, around 80– 300 (Bowes, 1991). Consequently, Rubisco is not saturated by in the present atmosphere, which is equivalent to only 10–20 in solution at temperatures between 25 and 10 °C. Atmospheric also interacts with Rubisco; as a competitive inhibitor with respect to and as a substrate for the mono-oxygenase activity of this bifunctional enzyme to produce phosphoglycolate, which is partially metabolized to in the photorespiratory carbon oxidation (PCO) cycle (Bowes, 1991). Through inhibition and photorespiration, the present atmospheric ratio causes about a 35% reduction in the photosynthesis of plants at 25 °C, and higher temperatures amplify this inhibition. Because of the competitive interaction, as rises it will diminish the inhibitory effects of a doubling of the present concentration should more than halve photorespiration. Past atmospheres of 2,000 would have

389 essentially eliminated the effects of but under current conditions Rubisco has several strikes against it. Somewhat surprisingly, a doubling in concentration also reduces dark respiration in a number of species (Amthor, 1991), but not all (Thomas et al., 1993). The effect seems to have two components. One is a direct inhibition of respiratory metabolism that is rapidly relieved when the concentration is reduced, but the mode of action is obscure (Bunce, 1990; Amthor et al., 1992). The other is a long-term depression of specific respiration (respiration expressed on a mass basis). In some instances, this may reflect lower maintenance respiration, because the concentration of Ncompounds in the plant is reduced, and from an energetic viewpoint these compounds are more expensive to maintain than carbohydrate (Baker et al., 1992b). Stomatal conductance is reduced by enrichment, but because high often increases the leaf area of a plant, water use may not decline on a whole plant basis (Allen, 1990; Morison, 1993). However, water use efficiency (WUE) is usually improved due to enhanced carbon gain under enrichment (Wong, 1979; Allen, 1990; Morison, 1993). These results of enrichment, culminate in the ‘efficiency effect’. Thus, increasing the supply for species not only provides more of a limiting resource (carbon), but also has the potential to improve the use of other resources and raise the optimum temperature for photosynthesis (Bowes, 1993). This effect is analogous to the concentrating mechanism (CCM) of species, and the potential benefits it provides, but without the associated running costs. As a consequence, even when other resources are limiting, enrichment may still enhance growth. However, acclimation phenomena can override the efficiency effect so it is not fully realized, which makes it difficult to accurately predict plant responses. Another interesting facet of enrichment studies, but one that is mechanistically unresolved, is the apparent action of as a growth modulator. A doubling in can cause changes in anatomy, morphology, and phenology (Allen, 1990; Bazzaz, 1990). These indirectly influence photosynthesis by altering various plant characteristics, including branching, leaf area, duration of assimilation, and sink capacity. In the aquatic environment changes in the dissolved

390 inorganic carbon (DIC) concentration can have profound effects on the photosynthetic characteristics, especially the affinity for and Thus, when grown at concentrations, a number of unicellular algae and cyanobacteria have low values for the dissolved inorganic carbon concentration that drives photosynthesis at half its maximum rate and the capacity to concentrate intracellularly (Badger, 1987, Badger and Andrews, 1987). In essence, a low ratio for growth induces a CCM, which compensates for as much as a 20-fold difference between the low concentration in the environment and the high of Rubisco (Suzuki et al., 1990; Kaplan et al., 1991). In contrast, high (0.5 to 5%) increases the and suppresses this CCM. Several promoters have been detected in the cyanobacterium Synechococcus. These may contain regulatory regions that respond directly to concentration, or indirectly via metabolites in the PCO pathway whose concentrations change with the ratio (Suzuki et al., 1990; Kaplan et al., 1991). Changes in photosynthetic characteristics in response to varying DIC concentration for growth are not confined to microorganisms, but occur among submersed angiosperms, in laboratory and natural systems (Holaday et al., 1983; Sand-Jensen and Gordon, 1986; Bowes and Salvucci, 1989). The affinity of Elodea canadensis for is reduced in conditions where is low or is high (SandJensen and Gordon, 1986). For Hydrilla verticillata, DIC-poor conditions induce a system that concentrates intracellularly, thereby reducing the photosynthetic and the inhibitory effects of (Bowes and Salvucci, 1989). This occurs without recourse to the compartmentation afforded by the Kranz anatomy of terrestrial species. In contrast, DIC-sufficiency suppresses the CCM. The biochemical or molecular sensing systems that initiate these changes have not been identified.

III. Adaptation to Changes in Atmospheric

A. Declining The expansion of terrestrial plants in the mid to late Paleozoic Era, approximately 300 MYA, appears to have had a substantial influence in reducing atmospheric by photosynthesis and facilitating

George Bowes the chemical weathering of silicates (Berner, 1993). The subsequent burial of organic and carbonate materials removed carbon from rapid recycling. Similarly, by the Cretaceous Period (100 MYA), photosynthesis had probably established the present content of the atmosphere (Böger, 1980). After the major mid-Paleozoic decline, atmospheric concentration fluctuated; by the Miocene (25 MYA) it had fallen to the present low range (Ehleringer et al., 1991). Ironically, the photosynthetic success of terrestrial plants imposed and stresses, which had profound feedback effects on the planet’s vegetation and climate. This section discusses photosynthetic adaptations (genotypic changes) that may have arisen to accommodate the decline in and rise in The intercellular concentration at which the rates of photosynthetic uptake and photorespiratory release are the same is the compensation point, (Brooks and Farquhar, 1985). For a leaf at 25 °C and in the presence of 0.21 mol it is about 40 However, in addition to the PCR and PCO cycles, fixation supplies carbon to other vital components, including growth and maintenance respiration. Consequently, for a whole plant, a concentration greater than is required to sustain growth, and this higher value has been termed the ecological compensation point (Robinson, 1994), or As a present-day example, the extrapolated from dry weight growth rates of soybeans grown in a range of concentrations and otherwise optimal conditions, was about 85–95 (Allen et al., 1991). Under more natural conditions, with increased costs to counter stresses, this value would probably be higher (Robinson, 1994). In the past, as atmospheric concentration declined to where it approached the selection pressure to maximize fixation at low concentration would have increased. During glacial episodes of the last 160,000 years, plants that could not adapt, and had values close to the lows of 180 may have become extinct (Robinson, 1994). It is a reasonable assumption that modern plants possess adaptations which directly or indirectly facilitated survival in a atmosphere, and that low concentration was instrumental in their origination. Two components that directly impact the ability of Rubisco to fix in the presence of are the specificity factor, and the operation of a CCM. The

Chapter 16 Photosynthesis and Rising specificity factor is the ratio between the values for the carboxylase and oxygenase activities, and thus it describes the partitioning of carbon between these two functions (Jordan and Ogren, 1981). Rubisco shows a wide variation in specificity; photosynthetic bacteria and cyanobacteria tend to have low values, while higher plants, especially species, exhibit high values (Jordan and Ogren, 1981, 1983; Badger and Andrews, 1987). This is circumstantial evidence that the enzyme acquired greater specificity for as the ancient atmosphere became depleted in and rich in but it assumes that present-day prokaryotes are representative of their ancestral counterparts, and that low specificity is not a secondarily-derived feature associated with the operation of a CCM. Despite high specificity factors, deleterious effects are still apparent in modern species. By contrast, the CCMs of species reduce oxygenase activity to only a few percent of carboxylation, by providing Rubisco with a steady-state concentration of 27–70 in the bundle sheath cells (Dai et al., 1993). Phosphoenolpyruvate carboxylase (PEPC) with its high activity and lack of inhibition (Bowes and Ogren, 1972) initiates this process. The pathway may well be a photosynthetic modification developed in response to declining and warmer climates that exacerbated photorespiratory losses in species (Ehleringer et al., 1991; Ehleringer and Monson, 1993). Direct fossil evidence for the origin of plants is limited, but the presence of Kranz anatomy in some fossils indicates plants were present in the late Miocene about 7 MYA (Thomasson et al., 1986). This coincides with late Miocene changes in the carbon-isotope composition of both fossil soil carbonates and fossil tooth enamel, indicative of shifts from dominated floras. Furthermore, pollen data are consistent with major expansions in grasslands during this period (Ehleringer et al., 1991; Cerling et al., 1993). Earlier dates for the origin of terrestrial photosynthesis, for example at the end of the Cretaceous (60 MYA) when concentration also was low, cannot be ruled out, but lack evidence. The PCO cycle of plants, even though it releases still conserves 75% of the carbon that enters phosphoglycolate, and prevents lethal accumulation of this product. Thus, it too may have developed in response to declining Terrestrial intermediate species take this conservation one step further by recycling part of the remaining 25% of

391 carbon. The expression of glycine decarboxylase is limited to rudimentary bundle sheath cells, which increases the likelihood that released by this photorespiratory enzyme will be refixed by Rubisco before it can exit the leaf. This modification improves the carboxylation efficiency, and is advantageous when is low, or leaf temperatures are high (Ehleringer and Monson, 1993), both being situations conducive to photorespiration. Intermediate species could represent stages in the development of terrestrial photosynthesis, and possibly were the result of reductions in global (Ehleringer et al., 1991). The development of CCMs may have occurred in the aquatic environment before their advent on land, in response to localized depletion and enrichment (Bowes, 1993). Because gas diffusion is about times slower in water than air, the photosynthesis of submersed autotrophs can be severely restricted by the supply of (Bowes and Salvucci, 1989). It is conceivable that as far back as the late Paleozoic (300 MYA) aquatic photosynthesis created localized areas of water that were far from equilibrium with the atmosphere. This phenomenon is well documented for submersed vegetation and algal blooms, where daytime dissolved concentrations of can drop to near zero, while dissolved concentration may exceed 500 equivalent to 200% air-saturation (Van et al. 1976; Beer et al., 1986; Bowes and Salvucci, 1989). To survive under these conditions, aquatic autotrophs containing Rubisco with a high and low specificity would need an auxiliary acquisition system, such as the active uptake of The and CAM pathways are not confined to terrestrial species, but occur with modifications in some submersed plants; usually as a carbonconservation measure in DIC-depleted waters (Keeley and Bowes, 1982; Salvucci and Bowes, 1983; Ueno et al., 1988; Bowes and Salvucci, 1989). In fact, the most primitive organism with a pathway is aquatic: the macroalga Udotea flabellum, which grows in subtropical and tropical seas (Reiskind and Bowes, 1991) and belongs to an ancient order of Chlorophyta: Siphonales. Its discovery raises the intriguing possibility that the first examples of photosynthesis may be traced back to DIC-depletion in aquatic environments. For terrestrial plants, another consequence of the decline in is a reduced gradient from the atmosphere into the leaf. With an abundant water supply, such as wetland or floating species experience,

392 stomates might compensate for lower by remaining fully open. However, where water is limited, stomatal regulation could become critical. Robinson (1994) has postulated that rapid stomatal responses and improved stomatal efficiency were selected to maximize gain per water lost. A further contention is that fast-evolving taxa, especially angiosperms such as the grasses, adapted their stomatal efficiency more than gymnosperms and ferns, and thus better accommodated the stresses associated with depletion. Other factors that influence the gradient include canopy and leaf resistances, and turbulent diffusion (Legg, 1985; Drake and Leadley, 1991). Open canopies with non-rigid, narrow leaves, which allow movement in the wind, generally have greater conductance to and may have been favored (Robinson, 1994). Low canopies could take advantage of soil as organic soils contain high concentrations, and its upward flux contributes to the carbon budget of a canopy; as much as 35% in wheat (Legg, 1985). Because carbon allocation patterns also affect the of a species, species with fewer structural components should have a better chance of surviving depletion (Robinson, 1994). For example, abundant water and nutrients allow less investment in roots and root respiration. In most of these scenarios grasses seem to be favored, and it does appear they expanded, while gymnospermdominated ecosystems contracted, during the last glacial maximum when atmospheric was at its lowest (Adams et al., 1990; Robinson, 1994). Various lines of evidence indicate that many aquatic angiosperms were originally land plants that became adapted to an aquatic existence (Sculthorpe, 1967). Depletion of atmospheric may have been a driving force for this retreat into the water. The water sufficiency, buoyancy, and nutrients provided by the aquatic environment should permit lower values, and greater stomatal conductance of aerial shoots. Certainly, submersed and amphibious angiosperms have minimal investment in structural components; they also possess higher fresh to dry weight ratios, and less root biomass than their terrestrial counterparts (Bowes and Salvucci, 1989).

B. Rising For the past 20–30 million years terrestrial vegetation has had to cope with stresses associated with a poor atmosphere,and has become progressively more

George Bowes adapted to such conditions. In contrast, within just the last 200 years the planet’s flora has experienced about a 28% rise in concentration. This raises questions as to what extent readaptation is occurring, and whether an increase in concentration is stressful for plants adapted to conditions. A number of studies suggest that readaptation may be occurring in photosynthesis-related processes. By examining herbarium specimens of temperate arboreal species from 1787 AD to the present, Woodward (1987) first reported a 67% decline in stomatal density. Concomitantly he calculated WUE to be improved by two-fold during this time period. In growth experiments stomatal index varied linearly with concentration, suggesting that was affecting stomatal initiation and not just epidermal cell expansion. Other workers have found similar results with herbarium specimens of Mediterranean species (Peñuelas and Matamala, 1990). Particularly interesting are Olea europaea leaves from funeral wreaths in Tutankhamen’s tomb (1327 BC) and from Pre-Ptolemaic Egypt of 332 BC (Beerling and Chaloner, 1993). The data show a 50% decrease in stomatal density as has increased by 80 to the present. Temperature effects were discounted because the plant is an indicator of a Mediterranean climate, and the results correspond to experimental data obtained at controlled temperatures (Woodward and Bazzaz, 1988). Similar trends have been found with fossil leaves of Salix herbacea (Beerling et al., 1993). From such data it appears that stomatal conductance and atmospheric concentration are negatively correlated over the past 16,500 years (Beerling and Woodward, 1993). Along with decreases in stomatal density, lower biological discrimination for of fossil leaves and some herbarium specimens are suggestive of improvements in WUE with increasing concentration (Peñuelas and Azcón-Bieto, 1992; Beerling and Woodward, 1993; see also Chapter 19). Not all studies have reached the same conclusion. Körner (1988) was unable to detect significant differences in stomatal density for over 200 lowland and alpine species from literature measurements of the past 100 years. However, over the time frame of the most extensive data sets the differences would be small (Woodward, 1993), and the historical counts did not record the replications, while the modern counts showed large intraspecific variation. Herbarium specimens also show a general decline

Chapter 16 Photosynthesis and Rising in plant nutrient concentrations over the past 250 years, including the N/C ratio (Peñuelas and Matamala, 1990, 1993). Changes in mineral content have been linked to the rise in concentration, but other anthropogenic influences on growth conditions and soil nutrients cannot be excluded. Although differences between herbarium or fossil specimens and present-day species are suggestive, they are not unequivocal evidence that plants are undergoing genotypic adaptation as a result of the recent rise in because similar acclimation patterns can be observed in short-term growth experiments (Bowes, 1993). An interesting geological phenomenon that is being exploited to study the adaptation of plants to high concentration is geothermal gas vents, whose emissions contain as much as 96% These enrich the air, so plants in the vicinity can be exposed to 10,000 potentially over hundreds of years (Miglietta et al., 1993). Quercus pubescens growing among natural gas vents in central Italy showed no differences in stomatal density or index as a function of distance from the vents; however, near the vents mean guard cell sizes and pore lengths were reduced (Miglietta and Raschi, 1993; Miglietta et al., 1993). Other support for adaptation has been obtained from population studies with seeds collected from naturally-enriched environments (Woodward et al., 1991; Woodward, 1993). Underground limestone springs in Florida are supersaturated with dissolved concentrations of several hundred (Rosenau et al., 1977); where they exit the ground they become a source of for submersed and terrestrial vegetation in the vicinity. Genetically-isolated populations of the terrestrial plant, Boehmeria cylindrica, were found growing at concentrations ranging from 350 to 505 When grown regime, seeds collected from under a naturally enriched populations produced plants with significantly greater stem and root dry weights than seeds from a non-enriched population. In contrast, there were no differences when the seeds were grown at 350 More studies with naturally enriched populations, or with a ‘fast plant’ such as Arabidopsis thaliana, are required to establish unequivocally that photosynthetic adaptations to rising occur in a short period (decades). Differences in fecundity in response to in a population of wild radish do point to the existence of genetically-based

393 variation, upon which selection could operate (Curtis et al., 1994). There are other examples of species with inter- and intra-population differences in response to elevated (Bazzaz, 1990).

IV. Diversity in Photosynthetic Responses to Enrichment

A. Species Differences and Acclimation Mechanisms The potential for enrichment to stimulate photosynthesis and growth has been recognized for more than a century, but experimental results have varied widely. Technical problems such as contaminated gas caused some early discrepancies (cited in Bolas and Henderson, 1928). Present-day designs are not immune from problems which confound the outcome. For example, small pot sizes which restrict root growth can markedly alter the photosynthetic responses of a species to elevated as can the use of growth chambers rather than field conditions (DeLucia et al., 1985; Radin et al., 1987; Thomas and Strain, 1991; Bowes, 1993; Sage, 1994). Even methodologies to elevate the around plants under field conditions, including soil-plant-air research (SPAR) units, open-top chambers, and free-air enrichment systems, have drawbacks (Dahlman, 1993; Rogers and Dahlman, 1993). So possible artifacts have to be taken into account when evaluating responses to elevated In the 1970s an understanding of and characteristics provided a way to differentiate species responses to An early experiment to test the concept showed that of 30 species stimulated the dry weight gain of those with but not photosynthesis (Akita and Tanaka, 1973). Differences in photosynthetic pathways and Rubisco kinetics continue to provide a basis to differentiate among species with respect to the effects of rising Until recently the focus of rising research has been narrow. It has understandably concentrated on terrestrial species of economic importance, and on phenotypic, as opposed to adaptive or genotypic, responses. A compilation of data for eight major crop species subjected to an approximate doubling in atmospheric showed that net photosynthesis was initially increased on average by 52%, but then declined to 29% (Cure and Acock, 1986). Despite

394 the partial down-regulation of photosynthetic rate, plants averaged 30 and 41% more in biomass and yield, respectively. An earlier survey of 430 observations had come to a similar conclusion: that increased yields of crop species by 33% (Kimball, 1983). It has been argued that cultivated plants cannot be used to predict responses of natural species, because the later have a much greater physiological and ecological range (Hunt et al., 1991). So, Hunt and colleagues chose 25 ‘wild’ and 2 crop species for based on different ecological growth strategies. The herbaceous species represented a wide range of strategies (Grime, 1974), including: competitor (C), associated with low levels of both environmental stress and disturbance; stress (S), associated with high stress and low disturbance; and ruderal (R), associated with low stress and high disturbance; and combinations thereof. Whole plant dry weight ratios at 700 versus 350 ranged from 1.0 to 3.7, with the highest values being associated with the C-strategy (mean of 1.43), while little response was found among species of extreme R- or S-strategies (mean of 1.03). Apparently, the capacity of species to respond to does differ, and may be related to the species’ ecological niche. One caveat is the small (11) pot size used in the study. A recent literature analysis of 156 species is consistent with this concept of strategy-related differences (Poorter, 1993). For an approximate doubling of concentration, growth of species was stimulated on average by 41%. Crops, which tend to be C-strategy species, increased more in dry weight than wild species (58% versus 35%). Fastgrowing wild species were stimulated more than the slow-growing (54% and 23%). Webber et al. (1994) suggest that the differences may be related to sink capacity: C-types tend to be species that invest in reproductive effort and resource acquisition, and consequently have substantial sink capacity. In contrast S-types are limited by stress, not photosynthate supply, and thus are unlikely to invest heavily in sink capacity. The concept that sink capacity influences photosynthetic responses to enrichment is elaborated upon later in the chapter. Long-term exposure to a doubling in concentration leads to a variety of acclimation effects that directly or indirectly influence the photosynthetic capacity of the plant. In addition to changes in photosynthetic biochemistry and stomatal physiology, acclimation has been observed in leaf area, leaf area

George Bowes index, leaf area duration, leaf thickness, branching, tillering, stem and root dry weights, fruit size, the timing of developmental events and life cycle completion (Allen, 1990; Bazzaz, 1990; Bowes, 1993). Consequently, even if photosynthesis per unit leaf area declines, changes in parameters such as leaf area or duration can result in greater biomass and yield (Spencer and Bowes, 1986; Allen, 1990; Bowes, 1993; Stitt and Schulze, 1994). One well-studied acclimation phenomenon is that of the assimilation versus intercellular response curve. At light saturation, assimilation by a leaf is a hyperbolic function of the concentration in the bulk air and intercellular spaces. The curve has been modeled on the biochemical factors that influence the activity of Rubisco (Farquhar et al., 1980; Long et al., 1993; Sage, 1994). The initial, linear phase of the curve is a measure of carboxylation efficiency, as it describes photosynthesis that is limited by the amount of active Rubisco. This phase is followed at higher concentrations by an inflection above which A rises more gradually, and is limited by the rate at which ribulose 1,5-bisphosphate (RuBP) can be regenerated by the PCR cycle. Limitations on RuBP regeneration and supply can usually be traced back to rates of non-cyclic electron transport, which provides ATP for the PCR cycle, or to PCR cycle enzymes which convert triose phosphates to RuBP. In the RuBP-limited phase, increasing concentration may still cause increases in A because photorespiration is reduced, and a greater proportion of RuBP is used for carboxylation instead of oxygenation. Under some conditions, RuBP regeneration is limited by the availability of inorganic phosphate (Pi), which depends on the rate at which Pi in triose phosphates is recycled back to the chloroplast (Sharkey and Vanderveer, 1989, Harley et al., 1992; Socias et al, 1993). During photosynthesis leaves tend to maintain at close to the inflection point, such that Rubisco and RuBP-regeneration capacity are co-limiting, and stays around 0.7 (Wong, 1979; Stitt, the ratio of 1991; Long et al., 1993; Sage, 1994). With a doubling of concentration stomatal conductance declines, keeping the ratio constant. Even long-term exposures do not greatly change the ratio in species, indicating there is little independent stomatal acclimation (Sage, 1994). The doubling in concentration also reduces stomatal limitations because rises from about 245 to 490

Chapter 16 Photosynthesis and Rising This rise in causes the initial 50% increase in photosynthesis that is often reported, and moves photosynthesis into the RuBP-limited region of the curve (Stitt, 1991; Woodrow, 1994). During long-term enrichment (days to weeks) acclimation may occur in the curve, with changes in the initial slope and/or RuBP-limited region. Various responses have been observed, ranging from soybean, where both the slope and RuBP-limited region were substantially greater (Campbell et al., 1988), to cotton where neither was changed (Radin et al., 1987), to cabbage where both were lower (Sage et al., 1989); and responses intermediate to these have also been reported (Sage, 1994). Experimental conditions have a substantial impact. Small pots or a restricted nutrient supply generally reduce A at a given (Thomas and Strain, 1991; Sage, 1994). This down-regulation is a common enrichment response in the literature, though whether it would be so in nature is still an open question, because field growth conditions minimize acclimation in the curve. No down-regulation and even upregulation have been reported for species in these circumstances, including crop plants: cotton, soybean, and kidney bean (Phaseolus vulgaris); a salt marsh sedge: Scirpus olneyi; and deciduous hardwood trees: yellow poplar and white oak (Radin et al., 1987; Campbell et al., 1988; Arp and Drake, 1991; Ziska et al., 1991; Gunderson et al., 1993; Sage, 1994). But, in some field situations acclimation does seem to occur; there are reports of species that failed to maintain photosynthetic or growth enhancements under natural field conditions, notably the sedge Eriophorum vaginatum in an arctic tundra ecosystem, and Poa pratensis in a tallgrass prairie (Tissue and Oechel, 1987; Owensby et al., 1993b). Low temperature, and competition for light, water and nutrients, may have restricted the response. The underlying causes of acclimation in have been subject to experimentation and speculation, but are only partially resolved. Is down-regulation a stress response, indicating physiological dysfunction in plants that over millennia have become adapted to low Or, is it an optimization process in reaction to a change in resources? In some species or conditions, elevated produces substantial carbohydrate accumulation within the leaves, which could be stressful. The leaf morphology can be deformed; massive starch granules can distort chloroplasts, and possibly disrupt function by

395 distending the thylakoid membranes and imposing constraints on the diffusion of gases or metabolites (Bowes, 1991; Stitt, 1991; Sage, 1994). However, in most instances, down-regulation of assimilation probably reflects a restricted capacity to handle the extra carbon, because of either insufficiency in other environmental resources, or inherent metabolic limitations. Photosynthetic acclimation, rather than being a stress response, is then an optimization process. According to this concept, acclimation involves the reallocation of resources away from non-limiting components such as carbon acquisition, and into more limiting components such as light harvesting, electron transport, and carbohydrate handling, thereby minimizing single limitations (Sage et al., 1987; Bowes, 1991; Sage, 1994). The resource reallocation would predominantly involve N, because enrichment increases the C:N ratio of plants (Wong, 1979; Conroy and Hocking, 1993). Various biochemical components have been implicated in acclimation, with Rubisco having the leading role. Rubisco is modulated by growth at elevated with reports of reduced activity in a number of species (Wong, 1979; Porter and Grodzinski, 1984; Spencer and Bowes, 1986;Besford and Hand, 1989; Sage et al., 1989; Besford et al., 1990; Rowland-Bamford et al., 1991; Tissue et al., 1993). A decrease in Rubisco activity may be manifested by a decline in Rubisco protein content, a lowered activation state, an inhibition of the carbamylated enzyme, lower specific activity, or altered kinetics; but not all apply to (Bowes, 1991). Decreases in Rubisco protein content are observed, in some cases as much as 60%, indicating that N is being reallocated (Sage et al., 1989; Besford et al., 1990; Rowland-Bamford et al., 1991; Tissue et al., 1993). However, Rubisco protein may still decline in the presence of adequate N supplies, and the reduction is not always sufficient to account for lower N concentrations (Sage et al., 1989; Rowland-Bamford et al., 1991; Conroy and Hocking, 1993). Also, some species show no decline in Rubisco content (Campbell et al., 1988; Sage et al., 1989; Socias et al., 1993; Sage, 1994). Consequently, changes in Rubisco content alone cannot always account for the acclimation phenomena that have been observed. In some situations a lower Rubisco activation state occurs, though this is not always observed (Campbell et al., 1988; Sage et al., 1989; Yelle et al., 1989;

396 Rowland-Bamford et al., 1991; Socias et al., 1993; Tissue et al., 1993). In terms of N optimization, a lowered activation state should be the initial regulatory response, with a subsequent reduction in Rubisco content being the long-term consequence of elevated but this is not always observed. Enrichment does not change the kinetic constants of Rubisco, though an unexplained decrease in specific activity is found in rice (Bowes, 1991; Rowland-Bamford et al., 1991). Perhaps the most often cited rationale for acclimation and the down-regulation of Rubisco is that enrichment causes an imbalance in the source-sink capacities, especially insufficient sink capacity for the excess carbohydrate production (Arp, 1991; Farrar and Williams, 1991; Stitt, 1991; Sheen, 1994; Woodrow, 1994). Studies on plants with large sinks, and manipulations of sink capacity, have led to this conclusion, and there is much evidence to commend it. Accordingly, N would be reallocated to upgrade sink capacity, and/or reduce source capacity to bring the two into confluence. The mechanism by which the imbalance is sensed, at least in part, is likely to involve feedback effects via end-product accumulation (Stitt, 1991; Sheen, 1994). This was indicated by a number of sugarfeeding studies which resulted in reduced photosynthesis, Rubisco activity and content. Similarly, the over-expression of acid invertase in transgenic plants, and the resultant hexose accumulation, decreased photosynthesis and PCR cycle enzyme activities (Stitt et al., 1990; Sheen, 1994). How is the feedback exerted? In cotton and kidney bean, the presence of photosynthesis points to a Pi-limitation of the RuBPregeneration capacity because carbohydrate accumulation ties-up Pi (Harley et al., 1992; Socias et al., 1993). However, this cannot be the sole reason, as it does not explain changes in extractable enzyme activities and amounts. A molecular model invokes the metabolite regulation of gene expression (Stitt, 1991; Sheen, 1994; see also Chapters 6, 10 and 12). Glucose provides a regulatory signal that represses the transcription of photosynthetic genes, including those encoding the small and large subunits of Rubisco, rbcS and rbcL (Krapp et al., 1993; Sheen, 1994). In addition, genes involved directly with carbohydrate metabolism can be positively, or negatively, regulated by sugars (Sheen, 1994). This could be a means to up-regulate enzymes that process carbo-

George Bowes hydrate, and thereby assist in balancing the sink capacity with the source. This concept is consistent with the findings that in rice, with increased sucrose and starch, the activity of sucrose phosphate synthase (SPS) is up-regulated about 20%, while Rubisco activity and content are down-regulated (Rowland-Bamford et al., 1990,1991; Hussain et al., 1992). A similar situation occurs in the sink-limited regions of transgenic tobacco leaves which have invertase over-expressed in the cell walls; Rubisco and fructose bisphosphatase activities decline, but SPS increases (Stitt et al., 1990; see also Chapter 6). By way of contrast, of kidney bean causes some reduction in SPS activity (Socias et al., 1993). More work is required to determine how enrichment influences the enzymes and allocation of carbohydrates in plants that are predominantly starchor sucrose-accumulators. In species, elevated not only reduces the amount of carbon entering the PCO cycle, but concomitantly the flux of N through the associated photorespiratory N-cycle. Photorespiratory N flux is large, being up to ten-fold greater than N assimilation (Conroy and Hocking, 1993). A high reduction in this flux should have a substantial impact on N metabolism, but it has received minimal attention. Leaf N concentration is generally lowered by enrichment, as are nitrate reductase activity and critical nitrate concentrations (Hocking and Meyer, 1991a,b). It has been suggested that nitrate may act as a metabolic signal for protein kinases, to regulate the flow of carbon between sucrose and amino acids (Champigny and Foyer, 1992). If so, a lowering of leaf nitrate concentration by high could divert carbon from amino acids to sucrose biosynthesis, in a mechanism ancillary to the glucose repression of gene expression. In summary, among species the response to enrichment is variable. Restrictive growth conditions can be influential, but evidence also points to the existence of inherent interspecific and intraspecific differences, reflective of different RuBP regeneration and sink capacities. Limitations in these capacities can lower or negate the increases that might otherwise be anticipated from Rubisco kinetics alone. The possibility that enrichment causes growth regulatory effects beyond those associated with enhanced photosynthesis cannot be discounted (Bowes, 1993). In regard to species, the presence of a CCM would lead one to anticipate little or no increase in

Chapter 16 Photosynthesis and Rising photosynthesis or growth from a doubling of atmospheric However, reported responses are often positive, though less than for plants. In the survey by Poorter (1993) the average stimulation in dry weight for 19 species, both cultivated and wild, was 22%, as compared to 41% for the species. In a comparative study of four grasses, the weedy annual Eragrostis orcuttiana showed a greater stimulation of aboveground dry weight in elevated than three grasses, despite a 44% decrease in assimilation on a leaf area basis (Smith et al., 1987). At a community level, net assimilation by a monospecific stand of the salt marsh grass Spartina patens was stimulated by 40% in the second year of exposure to doubled concentration, though over a four-year period biomass production was not enhanced (Drake and Leadley, 1991; Arp et al., 1993). It should be pointed out that enhancement of growth is not ubiquitous among species; in some instances no stimulation, or even inhibition, occurs (Poorter, 1993). What causes stimulatory effects of species? Several factors, that indirectly impinge on Rubisco, may be involved. First, elevated reduces the stomatal conductance of as well as species. In the case of E. orcuttiana this resulted in a 50% improvement in WUE (Smith et al., 1987). In waterstressed environments, a improvement in WUE could enhance growth. This has been proposed as a factor in the increased production by species in a tallgrass prairie ecosystem, and for the improved photosynthesis of the salt marsh community (Drake and Leadley, 1991; Owensby et al., 1993b). Second, a rise in concentration can enhance tillering and increase the leaf area and its duration (Smith et al., 1987; Allen, 1990; Bowes, 1993), so that total plant photosynthesis is greater, even without an improvement in assimilation per unit leaf area. Third, low N or high salinity regimes seem to undermine the effectiveness of the CCM by increasing leakage from the bundle sheath, thereby making plants more responsive to a rise in (Bowman et al., 1989; Wong and Osmond, 1991). Fourth, in dense stands on calm days can be drawn down, and if this shifts photosynthesis into the linear portion of its curve then A should respond positively to elevated concentration (Drake and Leadley, 1991). There is concern as to how the different responses of and photosynthesis will affect competitive interactions in the ecosystems of a higher world

397 (Bazzaz, 1990; Field et al., 1992). In several studies the competitive abilities of species were enhanced relative to (Carter and Peterson, 1983; Bazzaz and Carlson, 1984; Arp et al., 1993), but this is not always the case (Bazzaz, 1990; Wong and Osmond, 1991; Bazzaz and McConnaughay, 1992; Owensby et al., 1993a,b). Competitive interactions are complex; they are influenced by environmental factors, of which is not necessarily the most important, and plants in a community do not always respond the same as individually-grown plants (Wong and Osmond, 1991; Bazzaz and McConnaughay, 1992). In air, the rapid diffusion of generally precludes plants competing for it, so that changes in concentration have indirect effects on plant-to-plant interactions, which are exerted through competition for other resources (Bazzaz and McConnaughay, 1992). Also, though photosynthesis researchers may be reluctant to admit it, natural systems can be governed by factors only indirectly related to this process, such as increased herbivore grazing to compensate for the lower N concentration in enriched plants (Bazzaz, 1990). Higher atmospheric concentrations will not just influence and interactions; species do not all respond alike, so competition among plants within this category may also be modified. The competition studies indicate that the rise in will alter the species composition of communities, and species distribution. The exact changes cannot be predicted, though it appears are more likely to be favored than species. The few enrichments studies of CAM plants that have been undertaken have yielded mixed results. During the day CAM plants close their stomates and raise to more than 10,000 by the decarboxylation of malate accumulated the previous night via the activity of PEPC. Thus a doubling in atmospheric concentration should have little effect, and this seems true for pineapple (Hogan et al., 1991). However, if stomates open in late afternoon and fixation by Rubisco occurs, this provides an opportunity for enrichment to stimulate Phase IV photosynthesis. It may explain the 36% stimulation of dry weight reported for one CAM plant, and a mean of 15% for six other species (Nobel and Hartsock, 1986; Hogan et al., 1991; Nobel and Garcia de Cortázar, 1991; Poorter, 1993). The possibility of greater nocturnal fixation via PEPC needs further examination, as does the photosynthesis of facultative CAM plants operating in the mode. Rising research has largely focused on

398 terrestrial vegetation. Aquatic species have received less attention, though on a global scale at least 35 Gt carbon is assimilated annually by the photosynthesis of submersed autotrophs (Raven, 1994). Also, wetlands are large and vital ecosystems, and aquatic plants have a substantial impact on the supply and utilization of an increasingly valuable planetary resource: freshwater. Even one of the world’s major food crops, rice, is often grown as an emergent aquatic plant, Emersed aquatic plants include floating species, all of which are such as water hyacinth (Eichhornia crassipes) and duckweeds (Lemna species). They also include emergent forms, most of which are plants, such as cattails (Typha species) and reed (Phragmites australis), but some species occur, including papyrus (Cyperus papyrus) and barnyard grass (Echinochloa crus-gali). Although their roots may be submersed, emersed plants photosynthesize in air, and thus resemble terrestrial species in their responses. Biomass enhancements as high as 270% have been reported for floating species exposed to a doubling of concentration (Spencer and Bowes, 1986; Idso et al., 1987, 1990). Water hyacinth exhibited an increase in leaf area, but a decrease in stomatal conductance and the response. The response curve of enriched duckweeds was also down-regulated, but it was not associated with increased starch (Smernoff et al., 1993). Among emergents, photosynthesis and biomass production by the salt marsh sedge Scirpus olneyi has been stimulated by exposure over several years to increased atmospheric but the grass Spartina patens at the same location has not maintained such enhancements (Arp and Drake, 1991; Drake and Leadley, 1991; Arp et al., 1993). The Scirpus rhizomes provide a large carbohydrate sink capacity that may enable the plant to maintain the growth enhancement. Furthermore, an increase in improves the drought and salt tolerance of Scirpus, so it can invade the Spartina community which grows at higher elevations on the marsh (Arp et al., 1993). Some emergent species contain derived from the sediment via the root system. It is transported by mass flow to the stem or leaf lacunae, where concentrations as high as 40,000 have been reported (Brix, 1990; Constable et al., 1992; Hwang and Morris, 1992). If this internal is used for photosynthesis it should offset the effects of a doubling in atmospheric For species with

George Bowes high concentrations in the stems, less than 1.0% of the photosynthetic carbon is sediment-derived (Brix, 1990; Hwang and Morris, 1992). However, in cattails the lacunal is in the leaves, and may be more accessible for use in photosynthesis, though in such a tall plant the rate of supply from the roots may not match the photosynthetic requirements. The high lacunal concentration, and the importance of cattail as a dominant species in many freshwater ecosystems, make it a prime candidate for investigations. For marine and freshwater environments, the approximately 50,000 submersed photosynthetic species are taxonomically far more diverse than the estimated 300,000 species of vascular plants which constitute the major photosynthetic organisms of terrestrial habitats (Raven, 1994). The latter are believed to be derived in the past 450 million years from just one division and one class (Chlorophyta: Charophyceae). In contrast, submersed species include cyanobacteria, several algal divisions, bryophytes, lower vascular plants, and angiosperms, and some have histories that date back 2–3 billion years in environments with very variable and. concentrations. The diversity and long history gives them a greater potential for variability in carbon acquisition mechanisms than terrestrial species (Bowes, 1993; Raven, 1994). This, together with the fact that little is known about many of the individual species, makes predictions about responses to rising tenuous at best. Further complicating the issue is the spatial and temporal variability in the forms of DIC and their concentrations, and the fact that many water bodies are not air-equilibrated (Bowes, 1991; Raven, 1994). Diel changes in pH of more than two units are not unusual for freshwaters, and this can result in over a hundred-fold change in free concentration; so submersed species are already exposed to fluctuations in concentrations far greater than the mere doubling that terrestrial species may experience in the next century. Factors such as these make it difficult to estimate the extent to which a doubling in atmospheric will alter the dissolved and concentrations of natural waters, but free is likely to rise proportionally far more than favoring species that only use free (Bowes, 1993). Current thinking is that the rise in atmospheric should impact the submersed floras of aquatic systems less than those of terrestrial ecosystems (Raven, 1994).

Chapter 16 Photosynthesis and Rising Phytoplankton are the most important submersed species in the global carbon cycle. Their net primary productivity exceeds by several fold that of all other submersed species combined, and they are a crucial component of carbon sequestration in the ocean (Falkowski, 1994; Raven, 1994). For most of those studied, photosynthesis and growth is saturated by the present seawater DIC concentration (ca. 2 mM DIC, and 10.2 at 25 °C); in addition they utilize ions for photosynthesis and operate a CCM (Raven, 1994). From the Redfield ratio for phytoplankton (106C:16N:lP:0.01Fe) and nutrient concentrations in most oceanic regions it seems that N is more limiting than DIC (Falkowski, 1994; Raven, 1994). Consequently, a doubling in atmospheric should have little impact on marine phytoplankton productivity. There are caveats, however. Substantial drawdowns of and have been recorded in surface seawater due to phytoplankton blooms, and under these conditions the cells compete for DIC. Also, not all phytoplankton species use or operate a CCM; some rely just on the diffusive entry of Thus, an increase in atmospheric may alter species composition in favor of the later. Although many marine macroalgae and seagrasses use for photosynthesis, this is not true of all (Beer, 1989; Maberly et al., 1992). From drift and C determinations it has been shown that many red algae (Rhodophyta) depend upon uptake of free as do some intertidal seaweeds when they become exposed to air at low tide. The DIC concentration in seawater does not saturate the photosynthesis of all marine macrophytes (Holbrook et al., 1988; Madsen and Sand-Jensen, 1991), and as a group they are more likely to be carbon-limited than phytoplankton (Raven, 1994). Consequently, some might benefit from an increase in atmospheric In this context, thallus growth of the red macroalga Porphyra yezoensis was enhanced by enrichment (Gao et al., 1991). In contrast, the green macroalga Ulva lactuca, which is an effective user of ions (Drechsler and Beer, 1991), showed no photosynthetic acclimation or growth stimulation when enriched with 800 (T. V Madsen and G. Bowes, unpublished). For submersed freshwater plants, the free concentration needed to saturate photosynthesis is 30-fold greater than that for terrestrial species (Bowes and Salvucci, 1989; Madsen and SandJensen, 1991). Thus a doubling of atmospheric could markedly increase photosynthesis and growth,

399 but not in waters where the free concentration is above air-equilibrium, for example in Florida springs or Danish streams where 400 can be attained (Rosenau et al., 1977, Sand-Jensen et al., 1992). In waters where predominates, species with a high capacity for use should be less affected by air enrichment than those using only free This hypothesis was recently tested using the submersed, freshwater angiosperms Callitriche cophocarpa, and Elodea canadensis, a user and a respectively (T. V. Madsen and G. Bowes, unpublished). They were grown in water with 0.2 or 1.0 mM and sparged with air containing 350 or 800 The dry weight gain of Callitriche was doubled by the elevated treatment, but that of Elodea was stimulated only about 20%, irrespective of the concentration in the water. Conversely, the higher concentrations substantially increased the growth of Elodea, but had only a minor effect on Callitriche. Elevated caused little photosynthetic acclimation of either species. These data raise the possibility that for waters low in free the species composition may change as atmospheric rises, with only users being favored.

B. Environmental Constraints that Modify Enrichment Responses Environmental conditions clearly influence the degree to which stimulates photosynthesis and growth. Because of the efficiency effect, enrichment can improve resource use, so even when other parameters are co-limiting or exerting stress, stimulation of photosynthesis and growth may still occur (Gifford, 1992, Bowes, 1993). However, interactive effects are complex, and may defy interpretation within the simple ‘efficiency effect’ framework. Also, the high costs associated with multiple facilities, especially outdoors, has limited the number of reliable growth studies combining enrichment with other environmental variables (Allen, 1990; Rawson, 1992). Consequently, to suggest unifying principles takes temerity. The fact that is a greenhouse gas makes higher global temperatures an important consideration in the rising debate. Temperature and have interactive effects because a rise in temperature lowers the ratio of in solution, shifts the specificity of Rubisco towards oxygenase, enhances

400 photorespiration and dark respiration, and increases the sink response relative to the source. Using the biochemical model of Farquhar et al. (1980) as a basis, Long (1991) calculated that with no downregulation of Rubisco a rise in to 650 could increase light-saturated assimilation by 20% at 10 °C but by 105% at 35 °C, and raise the temperature optimum by 5 °C. According to this simulation, leaves with even a 40% loss in Rubisco could still show greater net photosynthesis above 25 °C. These photosynthetic gains may or may not be realized in long-term growth and yield due to an interplay of factors that complicate the issue. For example, leaves compensate for increased air temperatures by greater transpiration, whereas enrichment tends to raise foliar temperatures by reducing transpiration (Allen, 1990; Campbell etal., 1990; Long, 1991). Temperature and can have greater interactive effects on net leaf area production than photosynthesis per se (Coleman and Bazzaz, 1992). Furthermore, species in the category differ markedly in the temperature regimes to which they are adapted, and also in their tolerance of the low and high extremes where temperature becomes stressful. Even with a single plant, temperature regimes that enhance vegetative growth can negatively impact reproductive growth. Thus, the grain yield of rice showed about a 10% decline for each 1 °C rise above 26 °C, and similar scenarios have been reported for soybean and wheat (Baker et al., 1989, 1992a; Mitchell et al., 1993). This is because growth and reproduction reflect the integrated temperature response of metabolism and developmental processes, not just photosynthesis. As a consequence, species, developmental stage, light regime, nutrient status, and the temperature range, all modify the interactive temperature and responses (Coleman and Bazzaz, 1992; Rawson, 1992). The relative stimulation of photosynthesis and vegetative growth by high concentration is enhanced by a rise in temperature, within speciesdependent temperature limits; this even applies to some plants, though to a lesser degree (Carter and Peterson, 1983; Cure and Acock, 1986; Idso et al., 1987; Idso and Kimball, 1989; Bazzaz, 1990; Farrar and Williams, 1991; Long, 1991;Gifford, 1992;Nijs et al., 1992; Rawson, 1992). Based on the ratio of biomass increment of to that of ambient plants, Idso and colleagues (1987,1989)

George Bowes derived ‘growth modification factors’ for five species over a range of temperatures. The slope of the mean response to enrichment was 8.7% per 1 °C rise, with the stimulation extrapolating to zero at around 12 °C; but a literature survey indicates the more common response is closer to 2% per 1 °C (Rawson, 1992). The and temperature interaction is species-related. At 25 °C radish and carrot differed in growth modification factor, with values of 1.5 and 2.0, respectively (Idso and Kimball, 1989). With winter wheat no interaction was found (Mitchell et al., 1993), while for sunflower and cowpea higher temperatures had a negative effect (Rawson, 1992). Some of the discrepancies may be the result of temperature altering the development period (Mitchell et al., 1993). Growth at temperatures beyond the optimum for the species could also add a confounding stress response, though some evidence indicates that enrichment can alleviate the adverse effects of temperature extremes on vegetative growth (Bazzaz, 1990; Hogan et al., 1991), but not on reproductive development (Ahmed et al., 1993). It can be seen that future increases in atmospheric and day temperatures have the potential for positive interactive effects on the photosynthesis and vegetative growth of many species. But, in some regions the photosynthetic gains may not translate into greater yields, because of temperature stress on reproductive processes (Allen, 1990; Bowes, 1993). If mean global night temperatures increase the outcome is even less predictable. A higher mean temperature at night could negate the lower respiration of some enriched species (Amthor, 1991), and increase damage to the reproductive system (Ahmed et al., 1993); but in heat-tolerant plants it might improve carbohydrate mobilization and ease sink limitations on photosynthesis (Ahmed et al., 1993). As the concentration in the atmosphere rises, precipitation is predicted to increase, but regional patterns and water availability will differ. The responses of plants to rising concentration in water stress and saline conditions have been reviewed recently (Ball and Munns, 1992; Morison, 1993). As alluded to earlier, a common response to enrichment is a 30–60% reduction in stomatal conductance, due to decreases in stomatal aperture and density, though some plants seem to have insensitive stomata (Bunce, 1992). Leaf water potentials may improve at elevated and fac ilitate leaf expansion; but this is not always the case, because higher transpiration rates, evoked by a rise in leaf

Chapter 16 Photosynthesis and Rising temperature, can offset the reduction in stomatal conductance, as can an increase in leaf area index (Allen, 1990; Ball and Munns, 1992; Gifford, 1992; Arp et al., 1993; Morison, 1993). Observations involving 20 and 9 plants indicate that a doubling of concentration from present values doubles the instantaneous WUE, though the data show substantial scatter (Morison, 1993). The relative improvement for the two categories was not statistically different, despite the fact that at ambient the WUE was approximately 50% higher than that of the species. Measurements of C and WUE of plants grown at 150 to 350 suggest that present-day WUE may already be 50 to 100% greater than at the time of the last glacial maximum (Polley et al., 1993). As the concentration is increased the improvements in WUE are the result of increased assimilation and decreased water loss, with the latter becoming more important under water-stressed conditions. As the supply of water becomes less the relative enhancement of photosynthesis and growth by enrichment tends to be greater, which can alleviate drought stress and delay its onset (Allen, 1990; Bazzaz, 1990; Arp et al., 1993; Morison, 1993). For most species, also reduces the inhibitory effects of moderate, but not severe, salinity-stress (Bazzaz, 1990; Ball and Munns, 1992; Arp et al., 1993). Salt concentrations in leaf tissues are similar in plants grown under ambient or enriched conditions, indicating that salt transport is tightly regulated despite higher growth and lower water usage rates at elevated (Ball and Munns, 1992). The extent to which improvements in instantaneous leaf WUE at elevated apply to canopies is debated. Factors such as leaf area indices, respiration from non-photosynthetic components, night-time respiration and transpiration, root to shoot ratios, soil evaporation, and coupling between the vegetation and atmosphere in terms of energy and gas transfers, all complicate the issue (Morison, 1993). The limited canopy- and ecosystem-level data do suggest that an improvement in WUE is the rule, though not to the degree seen at the instantaneous leaf-level (Allen, 1990; Baker et al., 1990; Drake and Leadley, 1991; Morison, 1993; Owensby et al., 1993b; Baker and Allen, 1994). However, if a rise in global temperature is factored in, then simulations of evapotranspiration predict increased, not decreased, canopy water use under a doubled atmospheric concentration regime (Baker and Allen, 1994).

401 When the supply of nutrients is restricted, enrichment still usually causes a relative stimulation of photosynthesis and growth, due to the efficiency effect, even though nutrient-poor conditions depress the absolute photosynthetic rates (Wong, 1979; Hocking and Meyer, 199la; Wong and Osmond, 1991; Gifford, 1992; Bowes, 1993; Conroy and Hocking, 1993). In many environments root-derived nutrients, along with supply, co-limit the growth of plants; though in some soils the N and/or P content is low enough to abolish enrichment responses (Conroy et al., 1986; Tissue et al., 1993). Declines of as much as 20% in plant mineral concentrations have been reported in experiments, though total mineral content may increase because of greater dry matter accumulation (Overdieck, 1993). The majority of and nutrient interactive studies have focused on N, because it is a common limitation in many natural and agroecosystems (Field et al., 1992). Total plant N and its uptake may increase with enrichment but not to the same degree as C, or less N may be taken up as transpiration is reduced. In either case, tissue N concentrations are less and nitrogen use efficiency is enhanced, even at high N-application rates (Curtis et al., 1989; Conroy and Hocking, 1993; Mitchell et al., 1993; Owensby et al., 1993a). In contrast, leaf P concentrations are generally less affected by enrichment (Conroy and Hocking, 1993). Another report presents evidence that enrichment of white clover (Trifolium repens) caused fixation to increase in concert with photosynthesis and biomass (Ryle and Powell, 1992). If widespread, this could minimize N limitations to the response of leguminous species. In nature, photosynthesis occurs in both high and low light environments, and in the latter situation the processes involved in RuBP regeneration are a greater limitation than supply or Rubisco capacity. Several growth studies demonstrate that enrichment enhances light-limited photosynthesis (Sionit et al., 1982; Valle et al., 1985; Campbell et al., 1990; Long and Drake, 1991; Nijs et al., 1992). Because it reduces inhibition, a rise in concentration results in a higher quantum yield. Thus, assimilation versus irradiance (A/I) response curves for leaves of soybeans grown and measured at 660 not only showed greater lightsaturated rates, but also had steeper initial slopes, i.e. higher apparent quantum yields, and lower light compensation points than those grown at 330

George Bowes

402 (Valle et al., 1985). Likewise, due to improvements in quantum yield, canopy photosynthesis of soybean increased linearly with growthfrom 160 to 990 even after the canopy closed and light interception was maximal (Campbell et al., 1990). For perennial rye grass (Lolium perenne), the conversion efficiency of light during the morning hours was doubled in ‘ canopies, being 37 and 80 mmol photons at 358 and 626 respectively (Nijs et al., 1992). The improvement in quantum yield has important consequences for canopies and understories: for Scirpus canopies it is calculated to increase assimilation by as much as 86% on overcast days (Long and Drake, 1991). In the literature little attention has been paid to photon acquisition and processing, even though is associated with PS II photochemical activity (Blubaugh and Govindjee, 1988). At ambient the stromal concentration is estimated to be 220 which is at least three-fold higher than the for the effect on PS II. Consequently, an increase in is unlikely to increase this process, except perhaps when stomatal closure causes to be depleted in the light. This is supported by measurements of the ratio of variable to maximum chlorophyll fluorescence, which indicated that the maximum quantum efficiency of PS II photochemistry of Scirpus was unchanged by enrichment (Long and Drake, 1991). Fluorescence measurements with pine needles also indicated no change in PS II efficiency with elevated concentration (Conroy et al., 1986). Loblolly pine seedlings grown at elevated showed a greater down-regulation in Rubisco than in chlorophyll content; N from Rubisco seemed to be redirected into chlorophyll, possibly to up-regulate photon acquisition and/or processing relative to assimilation (Tissue et al., 1993). Thus, enrichment does not seem to down-regulate photosynthetic electron transport capacity, and limiting light conditions do not eliminate positive enrichment responses.

However, not all species have the capacity to respond, and those that do can be constrained by environmental parameters. Consequently, debate centers around the degree to which this greater photosynthetic potential will be realized during long-term growth of natural and agro-ecosystems, whether these systems can continue to sequester carbon, and how adaptation, competitive interactions and survival will be influenced (Allen, 1990;Bazzaz, 1990; Badger, 1992; Field et al., 1992; Gifford, 1992). Much of the current data pertain to agricultural systems, and there can be little doubt that most crops will perform better in a world, and may even require fewer subsidies. Less favorable C/N ratios may diminish crop quality, and higher temperatures may reduce grain yields, but breeding or molecular manipulation should correct these problems. Major changes in precipitation patterns would seem to have the most potential to disrupt current agricultural systems. The situation is less clear with regard to ‘natural’ ecosystems. Examples can be cited that do, and do not, respond to enrichment. Where nutrient availability and/or temperatures are low, responses will probably be constrained. However, many terrestrial, and some aquatic, ecosystems are likely to encounter marked shifts in species composition and distribution. But, just as increases in atmospheric are not novel, so continual changes in vegetation patterns are a fact of life on this planet, irrespective of whether humans regard them as beneficial.

Acknowledgments Work in my laboratory was supported by the USDA/ SEA National Research Initiatives Competitive Grants Photosynthesis Program, Grants No. 9037130-5576 and 93-37306-9386. Travel for collaborative research was also supported by NATO, Division of Scientific and Environmental Affairs, Collaborative Research Grants Programme, Grant No. 930940.

References V. Concluding Comments The upward trend in atmospheric probably has already enhanced the photosynthesis, WUE and growth of many of the earth’s plants, especially species, and potentially will continue to do so.

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George Bowes Stitt M (1991) Rising levels and their potential significance for carbon flow in photosynthetic cells. Plant Cell Environ 14: 741–762 Stitt M and Schulze D (1994) Does Rubisco control the rate of photosynthesis and plant growth? An exercise in molecular ecophysiology. Plant Cell Environ 17: 465–487 Suzuki K, Marek LF and Spalding MH (1990) A photorespiratory mutant of Chlamydomonas reinhardtii. Plant Physiol 93: 231– 237 Thomas RB and Strain BR (1991) Root restriction as a factor in photosynthetic acclimation of cotton seedlings grown in elevated carbon dioxide. Plant Physiol 96: 627–634 Thomas RB, Reid CD, Ybema R and Strain BR (1993) Growth and maintenance components of leaf respiration of cotton grown in elevated carbon dioxide partial pressure. Plant Cell Environ 16: 539–546 Thomasson JR, Nelson ME and Zakrzewski RJ (1986) A fossil grass (Graminae: Chloridoideae) from the Miocene with Kranz anatomy. Science 233: 876–878 Thomson K.S (1993) Northern exposures. Am Sci 81: 522–525 Tissue DT and Oechel WC (1987) Response of Eriophorum vaginatum to elevated and temperature in the Alaskan tussock tundra. Ecology 68: 401–410 Tissue DT, Thomas RB and Strain BR (1993) Long-term effects of elevated and nutrients on photosynthesis and rubisco in loblolly pine seedlings. Plant Cell Environ 16: 859–865 Ueno O, Samejima M, Muto S and Miyachi S (1988) Photosynthetic characteristics of an amphibious plant, Eleocharis vivipara: expression of and modes in contrasting environments. Proc Natl Acad Sci USA 85: 6733– 6737 Valle R, Mishoe JW, Campbell WJ, Jones JW and Allen LH Jr (1985) Photosynthetic responses of ‘Bragg’ soybean leaves adapted to different environments. Crop Sci 25: 333–339 Van TK, Haller WT and Bowes G (1976) Comparison of the photosynthetic characteristics of three submersed aquatic plants. Plant Physiol 58: 761–768 Webber AN, Nie G-Y and Long SP (1994) Acclimation of photosynthetic proteins to rising atmospheric Photosynth Res 39: 413–425 Wong SC (1979) Elevated atmospheric partial pressure of and plant growth. I. Interactions of nitrogen nutrition and photosynthetic capacity in and plants. Oecologia 44: 68–74 Wong SC and Osmond CB (1991) Elevated atmospheric partial pressure of and plant growth. III. Interactions between Triticum aeslivum and Echinochloa frumentacea during growth in mixed culture under different N nutrition and irradiance treatments, with emphasis on below-ground responses estimated using the value of root biomass. Aust J Plant Physiol 18: 137–152 Woodrow IE (1994) Optimal acclimation of the photosynthetic system under enhanced Photosynth Res 39: 401–412 Woodrow IE and Mott KA (1988) A quantitative assessment of the degree to which RuBP carboxylase/oxygenase determines the steady-state rate of photosynthesis during sun-shade acclimation in Helianthus annuus. Aust J Plant Physiol 15: 253–262 Woodward FI (1987) Stomatal numbers are sensitive to increases in from pre-industrial levels. Nature 327: 617–618 Woodward FI (1993) Plant responses to past concentrations of

Chapter 16 Photosynthesis and Rising Vegetatio 104/105: 145–155 Woodward FI and Bazzaz FA (1988) The responses of stomatal density to partial pressures. J Exp Bot 39: 1771–1781 Woodward FI, Thompson GB and McKee JF (1991)The effects of elevated concentrations of carbon dioxide on individual plants, populations, communities and ecosystems. Ann Bot 67 (Suppl 1): 23–38 Yapp CJ and Poths H (1992) Ancient atmospheric pressures inferred from natural goethites. Nature 355: 342–344

407 Yelle S, Beeson RC Jr, Trudel MJ and Gosselin A (1989) Acclimation of two tomato species to high atmospheric II. Ribulose-l,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase. Plant Physiol 90: 1473– 1477 Ziska LH, Hogan KP, Smith AP and Drake BG (1991) Growth and photosynthetic response of nine tropical species with longterm exposure to elevated carbon dioxide. Oecologia 86:383– 389

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Chapter 17 The Modification of Photosynthetic Capacity Induced by Ozone Exposure Robert L. Heath Department of Botany and Plant Sciences‚ University of California‚ Riverside‚ CA 92521-0124‚ USA

Summary I. Introduction and Background A. Air Pollution Injury B. Mechanisms of Air Pollution Tolerance C. Sequence of Events following Exposure to Air Pollutants 1. Entry of the Pollutant into the Leaf 2. Reactions of the Gases at the Cell Surface within the Wall Region 3. Movement of Reaction Product(s) into the Cell and Enzymatic or Chemical Transformations within the Cell II. Model Studies A. Chloroplasts 1. Reactions within the Chloroplast B. Algae III. Whole Plant Studies A. Fumigation Technology B. What is the Real Flux of Ozone? IV. Photosynthesis or Stomates? A. Changes within the Stomata—Conductance Alterations 1. Ethylene—Interaction with Transpiration B. Chloroplast Structure C. Photosynthesis and Associated Enzymes D. Ionic Control of Photosynthesis E. Electron Transfer and Photoinhibition F. Non-invasive Measurements V. Conclusions References

409 410 410 411 411 411 412 413 414 414 415 416 418 418 419 420 422 422 423 424 425 426 428 429 429

Summary Man’s activities continue to increase the level of ozone in the atmosphere. In the absence of strict controls‚ plants will be forced to grow in a polluted atmosphere. In crops‚ this higher level of ozone induces a lowering of productivity‚ while in natural ecosystems the plants become more at risk to other stresses including freezing‚ pathogens and insects. Knowing the mechanisms by which ozone induces these effects can allow for breeding and horticultural practices that may minimize any injuries or losses. The entry of ozone is through the stomata‚ so control of the aperture becomes critical to excluding the oxidant. Once inside the tissue‚ the primary events seem to be within the cell wall which induce changes in membrane permeability and transport in addition to generating toxic compounds. Carbon assimilation is inhibited by ozone exposure‚ but the question remains of Neil R. Baker (ed): Photosynthesis and the Environment‚ pp. 409–433. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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how. A controversy exists about the role of stomatal closure in limiting photosynthesis rather than any other event within the chloroplasts. However‚ carbon fixation is altered‚ under some circumstances‚ by a decline in ribulose 1‚5-bisphosphate carboxylase/oxygenase. This decline is induced principally in more mature leaves by a decline in the messenger RNAs for the subunits of the enzyme. Confounding these events is a strong wounding response induced by ozone that includes the production of ethylene‚ which in turn can interfere with photosynthesis. Placing the multiplicity of events into any coherent scheme is currently very difficult‚ but research is slowly sorting out these processes.

I. Introduction and Background Tropospheric air pollution‚ especially that caused by ozone‚ continues to be a pressing problem throughout the world (Seinfeld‚ 1989). Ozone exposure has been linked to a decline in crop productivity and changes within ecosystems far removed from urban settings. In Europe it is believed that forest decline is tightly linked to ozone levels‚ with complications caused by other environmental factors. In the U.S.‚ the government has made a clear commitment to reduce the levels of polluted air‚ primarily by tight emission standards. While the primary standard in the U.S. is based upon human health considerations‚ the secondary standard is based upon injury to plants. Thus‚ world-wide effort is presently being expended to find the effects induced by ozone that are harming our ecosystems and what processes may counter those effects. Yet‚ it is particularly difficult to identify stress events specifically due to ozone exposure. Ozone causes injury to the plant’s tissues resulting in decreased photosynthesis and productivity‚ but the sequence of events is currently not fully elucidated.

A. Air Pollution Injury At the present time it is very difficult to assess the damage caused by air pollutants (Woodwell et al.‚ 1989). Often visible injury to foliage is the only method available to judge it‚ but the manifestation of foliar injury through chlorosis and necrosis are commonly observed in many types of stresses‚ and Abbreviations: A – assimilation; AVG – aminoethyoxyvinylglycine; concentration of gas; concentration of gas; CSTR – continuously stirred tank reactors; Dl – 32 kDa protein of PS II reaction center complex; chlorophyll fluorescence obtained when is maximally reduced; chlorophyll fluorescence obtained when is maximally oxidized; chlorophyll fluorescence ; g – conductance of gas flow; IR – infrared; J – flux of gas entry into the leaf; NCLAN – national crop loss assessment network; Rubisco – ribulose 1‚5-bisphosphate carboxylase/oxygenase; RuBP – ribulose 1‚5-bisphosphate

different environmental conditions may alter the development of those injury patterns. The visible injury patterns‚ such as ‘water-logging’‚ lower surface ‘bronzing’ and upper surface ‘silvering’‚ can be used to assess injury by persons who are well versed in identification‚ but only an educated guess can be made as to the stress causing the visible injury (Jacobson and Hill‚ 1970). Other physiological processes that can be measured quantitatively‚ such as photosynthesis or transpiration‚ also have been used to assess injury (Koziol and Whatley‚ 1984; Schulte-Hostede et al.‚ 1988; Taylor et al.‚ 1988; Darrell‚ 1989; Lefohn‚ 1991). In fact there have been many studies on photosynthesis (for reviews see Hogsett et al.‚ 1986; Saxe‚ 1991). There are three modes of air pollutant-induced injury patterns currently categorized (Heath‚ 1988‚ 1994b); (i) acute stress‚ (ii) chronic stress and (iii) accelerated senescence. To evaluate the cause of each injury pattern‚ the length of exposure and dose of air pollutant must be known. Acute stress is generated by high atmospheric concentrations of pollutants for short periods of time. Distinct visible injury occurs generally due to cellular and tissue death leading to a decline in total area of metabolically active tissue‚ loss of membrane integrity‚ loss of metabolites into the extracellular tissue space‚ and the formation of oxidative products. The somewhat localized damage seems to be too severe for physiological preventive or repair processes to occur. Chronic stress is generated by lower concentrations of pollutants for long periods of time. Often no visible injury is observed‚ but lowered rates of photosynthesis or plant productivity are used to indicate injury. Metabolism is altered and the pool sizes of many metabolites are changed. More importantly‚ the altered biochemical state within the tissue seems to lead to the inability of the plant to respond properly to existing environmental conditions and to other stresses (Davison et al.‚ 1988; Manning and Keane‚ 1988).

Chapter 17 Ozone and Photosynthesis

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Accelerated senescence is generated by very low concentrations of pollutants for very long periods of time‚ even over a full growing season. This type of exposure seems to accelerate natural aging‚ resulting in earlier leaf abscission and flowering. Currently‚ loss of measured productivity can be measured only by integration of growth over long periods of time. The events leading to senescence (Rodriquez et al.‚ 1990) would seem to be critical to understand this process. Currently the external level of ozone is used to give an indication of ‘dose’. Clearly this is incorrect (Heath‚ 1994a‚ 1994b). Ozone-induced changes in the plant’s cuticle are minimal (Kerstiens and Landzian‚ 1989)‚ thus the main effect of ozone exposure is on the leaf’s internal tissue. As ozone has no easily measured isotope‚ few measurements have been done on actual dose of ozone; that is‚ the amount of ozone that reacts with individual constituent molecules within the leaf. As will be argued later‚ the measurement of dose must be determined by the amount of ozone that penetrates into the tissue through the stomata. The expression of this dose is as a rate of delivery to a surface area The major question of whether dose or total accumulation (i.e. rate integrated over exposure time‚ mol remains most critical for the quantification of the development of injury. However‚ Saxe (1991) argues that there should be two measures of exposure: an external dose which is the concentration of ozone in the air near the plant‚ and an internal dose which is the concentration inside the leaf. The latter measure is more precise for a measure of injury. However‚ most studies only measure external concentration of ozone and consequently description of that is necessary for comparative purposes.

There is not a single response to ozone exposure (Guzy and Heath‚ 1993). However‚ there are three sequential processes which combine to trigger ozone stress from the movement of gases from the atmosphere into the sites of action within the leaf (Heath‚ 1980).

B. Mechanisms of Air Pollution Tolerance

1. Entry of the Pollutant into the Leaf

Plant strategies to survive ozone stress include exclusion of ozone and tolerance of ozone or its products (Levitt‚ 1980)‚ yet few specific details exist of how these general events occur. Air pollutants can be excluded from the tissues via stomatal closure or by impermeability of the membrane. To a certain extent‚ plants can tolerate pollutants by its storage in a different tissue or organelle (as for NOx decomposition into nitrate/nitrite) or through detoxification (such as conversion of to sulfate); such chemical modifications allow less-harmful

This first necessary event includes gaseous diffusion through the leaf boundary layer and stomates into the substomatal cavity. The entry of gases into a leaf is dependent upon physical and chemical processes of the gas phase and surfaces‚ and has been defined as a simplified path which follows a linear flux rule of:

compounds to enter the normal metabolic pathways (see Wellburn‚ 1990). Past observations of ozone injury have indicated some of the many physiological and metabolic changes that have occurred (reviewed by Heath‚ 1988‚ 1994a; Harris and Bailey-Serres‚ 1994; Pell et al.‚ 1994). Response to air pollutants may also represent adaptation to a stress. There is clear evidence that plants subjected to repeated exposures will ‘harden off’ and become more resistant to later exposures (McCool et al.‚ 1988)‚ thus indicating that changes in gene expression may play a role. However‚ it is not known at what expense to plant growth this hardening occurs. It is also not known how rapidly a plant will recover after a single exposure. It is believed‚ but not proven‚ that the longer the time period between high peaks of ozone‚ the more able the plant is to maintain normal growth and development. However‚ a permanent shift in energy allocation under continuous ozone exposure may be less detrimental than a long period between temporary shifts. In ascertaining injury to ecosystems it is critical to have an understanding of the changes induced by long term exposure. The question inevitably arises during the standard setting process‚ should the average level or the peaks of ozone concentration be measured and controlled (McCool et al.‚ 1988)?

C. Sequence of Events following Exposure to Air Pollutants

where the flux (J) into the internal space of a leaf is

412 related to the conductance (g) through the boundary layer and stomates and the gradient of concentration of gas from the outside to inside the leaf For both water vapor and this formulation has been used for years (Farquhar and Sharkey‚ 1982; Ball‚ 1987)‚ For ozone‚ internal concentration is‚ in fact‚ very close to zero (Laisk et al.‚ 1989)‚ most probably because ozone is extremely reactive with cellular chemicals. Thus‚ the effective delivery rate is very close to with stomatal conductance being the major regulatory control point (Taylor et al.‚ 1982; Amiro et al.‚ 1984). The flow of gaseous pollutants is from the substomatal cavity within the leaf into the cell‚ through the wall. An equilibrium between the gas and aqueous phase occurs at this wall interface where the gaseous species dissolve into the water‚ presumably according to Henry’s Law (Heath‚ 1980‚ 1987;Wellburn‚ 1990). It is useful to calculate what rate of ozone delivery to the internal areas might be expected. However‚ it must be realized that such calculated values are at best only estimates. Each case is separate and must be examined on its own merits‚ but these figures can suggest maximum levels which might be expected. A high stomatal conductance would be 0.5 for a single side of a leaf (Eguchi and Kitano‚ 1986). Naturally a low wind speed would increase the boundary layer and decrease the boundary conductance and so lower the rate of delivery. Also stomatal aperture is controlled by metabolic events within the leaf and roots and so the conductance can be much lower (see Chapter 9). None the less‚ for a high level of ozone (e.g. for severe pollutant episodes in the Los Angeles basin‚ 0.3 ppm for 2 h) the entry level on both sides of a leaf can be calculated as from the areas of leaf involved multiplied by conductance and the external concentration and divided by molar volume of a perfect gas. For a two hour exposure this is equivalent to an accumulation of . In terms of a concentration of an accumulated product‚ this exposure would result in a maximum of 9 mM ozone or its products being present over two h‚ assuming the water content of crop leaves to be

2. Reactions of the Gases at the Cell Surface within the Wall Region Once ozone enters the water phase within the wall region‚ it can react with some of the molecules

Robert L. Heath exposed within that phase. Ozone is highly reactive with many compounds. These chemical reactions are poorly understood although there are some fundamental facts known (Heath‚ 1987; 1988; Wellburn‚ 1990). Ozone can react with organic molecules at double bonds to form carbonyl groups and‚ under certain circumstances‚ peroxides. Sulfhydryls are particularly easy targets for ozone resulting in the formation of disulfide bridges or sulfones (Heath‚ 1975). Oxidative products which have been linked to metabolic changes are hydrogen peroxide hydroxyl radical (HO)‚ and superoxide . Effective detoxification reactions can occur on these compounds via antioxidant metabolites and enzymes‚ such as ascorbate‚ glutathione‚ and superoxide dismutase‚ if they are present at high enough concentrations in the correct compartment (Matters and Scandalios‚ 1987; Castillo et al.‚ 1987; see also Chapter 5). If the ozone level is low at the chronic level‚ it is believed that stimulation of production of the antioxidants is a cellular response to ozone‚ albeit a slow one (Harris and Bailey-Serres‚ 1994; Scandalios‚ 1994). Certainly it has been shown that chemical modification of wall-specific compounds is possible (Castillo et al.‚ 1987). Alterations of enzymes within the wall space‚ such as glucan synthase (Ordin et al.‚ 1969) and diamine oxidase (Peters et al.‚ 1988)‚ have also been shown to occur. On the basis of the maximum rate of delivery of ozone given above‚ the detoxification rate would demand rates of 13 pmoles for the antioxidant enzyme levels. Heath (1994a) has previously calculated the amount of some antioxidants within a typical cell. While the amounts are highly variable‚ it appears that the enzymes which detoxify superoxide and hydrogen peroxide are present in sufficient amounts to handle a moderate exposure. However‚ the cellular location of each is far from clear in many cases. Catalase is not believed to be within the wall space so its activity would not aid the initial detoxification step. Also‚ even though concentration of glutathione is often low‚ the ability to regenerate it is assured‚ at least in part‚ by the high activity of glutathione reductase. It is important to note that such calculations estimate only the probable levels of contaminants and their probable rates of detoxification in a given exposure for any particular species. One could make a reasonable guess‚ however‚ that for the very low levels of ozone which are typical in many environments‚ antioxidants are in sufficient levels in cells to

Chapter 17 Ozone and Photosynthesis prevent the accumulation of oxidative species.

3. Movement of Reaction Product(s) into the Cell and Enzymatic or Chemical Transformations within the Cell It has been argued‚ based upon organelle and chemical reactivity‚ that ozone can not move into the cytoplasm because of its extreme reactivity (Heath‚ 1987). Thus‚ it is believed that the initial site of ozone damage is the plasma membrane (Fig. 1). While this is a plausible hypothesis‚ it has not been proven. This hypothesis predicts that all the reactivity of ozone would be expected to be at the wall site. Certainly‚ membrane functions‚ such as membrane fluidity (Pauls and Thompson‚ 1980) and permeability (Elkiey and Ormrod‚ 1979)‚ via ATPase reactions (Dominy and Heath‚ 1985) and exclusion (Castillo and Heath‚ 1990)‚ are rapidly lost. If ozone reacts with components of the cell wall‚ those alterations may also connect to the cytoplasm by membrane-specific proteins not directly linked to transport‚ such as regulatory transduction elements. The similarity of wounding responses (Langebartels et al‚ 1991) and ozone-induced membrane disruption suggests that induction of normal wound-regulated genes must be involved in responses to ozone (Ecker and Davis‚ 1987; Mehlhorn et al.‚ 1991).

413 Ozone is soluble in water but once having entered the aqueous phase‚ it rapidly breaks down (Staehelin and Holgné‚ 1985). Thus‚ ozone interactions at the membrane level must produce long lived oxidative products‚ which can diffuse more readily into the cell and react with many internal metabolites and structural compounds. However‚ again the presence of internal antioxidants would serve to eliminate or decrease these oxidants. No toxic radicals have been discovered within the cell; unfortunately‚ there have been few attempts to identify such radicals in leaves (but see Grimes et al.‚ 1983‚ and Mehlhorn et al.‚ 1990). In the absence of positive tests for oxidative or radical products within the cell‚ the tentative conclusion must be drawn that there are none. It can be postulated that three possible events could trigger responses within the cell after ozone exposure. (i) A disruption of normal ionic balance is produced due to the inhibited transport channels/ pumps and an increased permeability of the membrane‚ which then alters normal metabolism. (ii) A false trigger is generated due to the alteration by ozone reactions of signal transducer molecules located on the plasma membrane. Such signals‚ e.g. an activation of a specific protein kinase‚ could lead to changes in metabolism‚ (iii) A toxic product‚ similar to a compound normally found in the cell‚ is produced by the ozone reactions and has a sufficiently

414 long half-life to diffuse into the cytoplasm and alter a wide variety of metabolic events. The internal site of alteration of normal plant processes and secondary disruption of normal metabolism within the cells and tissues has been well studied‚ but causes and effects have yet to be determined (Koziol and Whatley‚ 1984; SchulteHostede et al.‚ 1988; Alscher and Wellburn‚ 1994). The remainder of this review will focus on the mechanism by which photosynthesis is inhibited. One current interesting mechanism of the primary trigger is a loss of calcium homeostasis due to membrane dysfunction (Fig. 1). This spurious signal may affect other control processes or signal transduction pathways. However‚ the possibility of a toxic product of ozone being involved with perturbations of photosynthesis can not be excluded; toxic products may migrate through the cytoplast to react with photosynthetic processes. In this context the role of possible reactions of ethylene within the wounding response will be discussed. Although poorly understood‚ exposure of leaves to ozone results in changes in stomatal conductance (Heath‚ 1994a)‚ and in a decline in the activity of carboxylation activity which can be traced to a decline of the subunits of ribulose 1‚5-bisphosphate carboxylase/ oxygenase (Rubisco) (Reddy et al.‚ 1993; Pell et al‚ 1994). Many of the metabolic events in process (iii) above may be controlled by changes in gene expression. It seems reasonable to suggest that steps to alleviate the effects of air pollutant exposure must occur through modified gene expression to counter alterations in the level of specific proteins and/or activation of those proteins. Under chronic or accelerated senescence‚ growth and morphological changes occur (Masuch and Kettrup‚ 1985). The plant’s developmental sequence has been altered and it is not the same plant as that grown under unpolluted air. Therefore‚ one would predict that the appearance of certain proteins and the activation of some genes should be delayed‚ and even their order of appearance may be changed due to ozone exposure. To date little progress has been made on the understanding of chronic stress‚ but clearly this is important for long term‚ ecologically-relevant pollutant injury.

II. Model Studies Model systems have been used in biology often and

Robert L. Heath consist of simplified systems which will simulate the process being studied. Much of the early work on modeling ozone effects was done with air streams of ozonated hexenes‚ which were believed to the pollutants present in oxidative urban atmospheres (Erickson and Wedding‚ 1956). Later work‚ principally by Mudd and his coworkers‚ introduced a gas stream of ozone in oxygen into a water solution of compounds which were suspected to be altered by ozone. These were simple chemical systems‚ but even so‚ the products are complex due to the diverse reactions which ozone undergoes in water. Many interesting insights have resulted from these studies. Several amino acids are easily oxidized by ozone; in particular‚ the sulfhydryl amino acids and tryptophan and some cyclic residues (reviewed in Heath‚ 1975). While double bonds are oxidized quite easily by the Criegee mechanism (Heath‚ 1987)‚ it is thought that unsaturated fatty acids are relatively protected from attack (at least at reasonably low levels of ozone) by their position within a hydrophobic region of the membrane. Ozone is a somewhat polar molecule (due to its resonating zwitterion characteristics; Bailey‚ 1982) with a water solubility of about tenfold that of oxygen. Mudd has used these insights to scale many of his investigations up to studies of proteins (Mudd et al.‚ 1969; Mudd‚ 1996). Space does not allow a complete discussion‚ but in general exposed sulfhydryl and cyclic amino acids are at high risk.

A. Chloroplasts Studies of ozone’s interaction in water solution with organelles and single cell algae provide some interesting insights into possible reactions of ozone in water and how deep ozone would be expected to penetrate into the cell. There were problems in introducing ozone into water‚ however it was found that reproducible results could be obtained when the solution was stirred vigorously as bubbles of ozone were introduced into the solution via a very small diameter glass tube (a 10 micropipette). Data indicated that equilibrium of solubility of ozone was not reached‚ but a constant amount of ozone was made soluble since general breakdown of ozone on surfaces was compensated for the constant inflow of gaseous ozone. Determination of the dose of ozone delivered to a biological model sample remains a vexing problem. As discussed above‚ the stomatal conductance must

Chapter 17 Ozone and Photosynthesis be taken into account in leaves. When model systems are used‚ they generally are water suspensions. The dose used under these conditions is problematical. Ozone is generated in an oxygen gas stream (at a concentrations from 100 to 300 ppm). The gas stream is introduced onto the solution and the solution is vigorously stirred (Landry and Pell‚ 1993) or the stream is introduced directly into the stirred solution through a very small tube (Heath‚ 1987). If the gas is in contact for long enough with the solution‚ an equilibrium will be established in which the Bunsen coefficient for ozone can be used (Thorp‚ 1954). While the coefficient is approximately known‚ there is some scatter in the experimental points (±15%) with a temperature sensitivity For these calculations‚ ozone is assumed to behave as a perfect gas‚ which is another approximation. The Bunsen coefficient‚ which is 0.22 at 25 °C (Thorp‚ 1954)‚ is used to calculate the amount of ozone which enters the solution at equilibrium. That value is converted to the amount in moles by the molecular volume calculated by the perfect gas law after correction for temperature. For an ozone concentration of 300 ppm in the gas stream the ozone concentration in solution is determined to be 3.2 .This assumes perfect gas behavior and equilibrium. Equilibrium is hard to achieve and so for 300 ppm in the gas stream the concentration of ozone in the solution is probably actually less than 3 Unfortunately‚ ozone breaks down rapidly in solution depending on the contents of the solution (Heath‚ 1987)‚ and thus the level of ozone within the solution is most probably much less than 1 The rate of delivery (the product of flow rate and ozone concentration of the gas stream) has been used to give another indication of the amount of ozone which might be expect to react or the amount of ozone-derived products formed per unit time. For example‚ typically a gas flow of 50 ml would be used‚ giving a delivery rate of If this was introduced into 5.0 ml of solution‚ the rate of delivery would be . This value should be compared with the amount of ozone delivered within the leaf. As previously calculated (see Section I.C.1)‚ the rate of ozone delivery to a leaf is about . For a typical leaf‚ the rate that ozone enters the leaf water solution is about moles which is about 50% of the rate used for most model studies. It has been suggested that model systems are not useful since they require the use of very high

415 concentrations of ozone. The rate of delivery calculations would suggest that argument is incorrect; the rates of ozone delivery to the solutions of the biological organisms are similar. However‚ the concentration of ozone within the cell’s water‚ at equilibrium‚ would be much lower. For example using the Bunsen coefficient‚ a leaf exposed to 0.3 ppm would have a concentration of ozone within its cell water of about 3 nM! Countering this‚ ozone in the wall’s water space is only 1 to 3 away from reactive compounds. Also exposures of 10 min in model system is common (Heath‚ 1987) while plant exposures are generally several hours‚ i.e. 10 to 20 times as long. Since the reactivity of ozone is great‚ one is hard pressed to determine a real value for the concentration of ozone anywhere in biological solutions.

1. Reactions within the Chloroplast As summarized by Heath (1994a) both photosystems in grana preparations were inhibited by ozone exposure. A question remains of whether uncoupling was caused by disruption of electron flow causing a decline in the ability to make a high energy gradient or by ozone increasing the membrane permeability. Interestingly‚ Nobel et al. (1973) used intact plastids to test how organic and amino acids could penetrate the envelope. They found that the envelope seemed to be made more permeable by exposure to ozone‚ which was similar to the results found by Heath’s group studying the plasma membrane in alga (summarized in the next section). An important conclusion was reached by the experiments done by Coulson and Heath (1974) shown in Table 1. They showed that ozone inhibited oxygen evolution in intact plastids‚ but that upon rupturing them by osmotic shock‚ the electron transfer capacity of the isolated grana was unaffected. Yet the same dose of ozone given to isolated grana did induce a sizable reduction in capacity. These results attested that ozone altered the dark reactions in intact chloroplasts‚ perhaps by altering the permeability of the envelope‚ without altering the light reactions; thus the ozone pathway would appear to be short within biological systems. There have been many studies using chloroplasts isolated from tissues which have been fumigated with ozone (e.g. Rhoads and Brennan‚ 1978; Landry and Pell‚ 1993). Some have found that there are changes in the isolated organelles‚ other have not.

Robert L. Heath

416

There are potential problems with any studies of chloroplasts isolated after fumigation. A strong wounding response is produced by plants subjected to levels of ozone which generate visible injury (Wang et al.‚ 1990; Langebartels et al.‚ 1991)‚ including the production of potentially toxic compounds. One can imagine an inhibition of isolated chloroplast function due to these compounds rather than direct inhibition by the ozone fumigation. In other words‚ the measured effect is an artifact due to cell breakage and organelle release in the presence of these compounds. The data do not give any more knowledge of why ozone causes an inhibition of photosynthesis unless careful controls are carried out to show that the ‘post-grinding’ solution does not have any potential injurious compounds. For in vivo fumigation followed by organelle isolation‚ one should test the supernatant of the isolation mixture against a control organelle preparation to show no such inhibitor is present. Similarly‚ direct exposure of even the plasma membrane to ozone may give little information as to the direct ozone effect. Certainly‚ the bubbling of a fragile membrane preparation does not help the situation due to both mechanical agitation and fractionation of membranes pushed upon the sides of

the container by the bubble action. For in vitro fumigation of organelles‚ one must do a careful control (under the same bubbling conditions‚ but with ozone removed from the oxygen stream by an activated charcoal filter) to test for a loss of protein and/or organelles from the solution. Such a study on the inhibition of the plasma membrane -stimulated ATPase by ozone exposure has been previously reported (Heath and Castillo‚ 1988). What was not reported in that article is that the protein content of the solution is lowered by the bubbling itself (Fig. 2). Fortunately‚ the loss seems to be similar for both ozone-in-oxygen and oxygen; thus‚ the loss is due to the manner in which the gas is introduced rather than the ozone. The earlier report remains accurate as the activity was expressed as ATPase per mg protein in the solution.

B. Algae Algae have been used as a model system to test a plant-like organism’s respond to ozone. For the most part these studies are of a cellular system possessing a wall which allows a tolerance of bubbling. The most complete study of an alga’s response to ozone has been done in the author’s laboratory over the

Chapter 17

Ozone and Photosynthesis

course of several years. Most of the studies have been summarized in a review (Heath‚ 1987). Several critical points regarding metabolism and photosynthesis‚ in particular‚ should be made. The gassing system used for Chlorella sorokiniana was similar to that used for the chloroplast studies. Generally an air stream flowing at 20 to 100 ml containing 100 to 240 ppm in was introduced into 5 ml of solution (phosphate buffer with and salts) containing about cells The control used the same air stream but ozone was removed by an activated charcoal filter. The fumigation dose (amount of ozone introduced per unit time) was varied by time of exposure (up to 30 min). During this short time period few alga were carried up onto the glass sides of the tube. Much of the work focused upon the plasma membrane level in terms of transport processes. In summary‚ the membrane transport and permeability to a wide variety of compounds Chimilkis and Heath‚ 1975; ‚Heath and Frederick‚ 1979; deoxyglucose‚ mannitol‚ acetate‚ and glucose‚ Heath‚ 1984) was increased by ozone. Heath (1987) concluded that the membrane potential was being initially depolarized and that event began much of the subsequent membrane dysfunction. No studies were done on the individual transport proteins‚ on direct measurements of membrane potential‚ or of turgor pressure to indicate the loss of osmotically active molecules. Not surprisingly many metabolic

417 events were disrupted. The loss of energy‚ as high energy molecules‚ was indicated by changes in the ratio of glucose to glucose 6-phosphate and in ATP level measured directly (Heath‚ 1984b). Respiration seemed to be inhibited only under longer periods of ozone exposure. Frederick and Heath (1975) showed that a product of lipid peroxidation (malondialdehyde) was induced by long periods of ozone exposure concurrent with a loss of unsaturated fatty acids. The levels of ozone required to induce any obvious lipid oxidation was far in excess of those required to severely damage the membrane function. In fact‚ Heath (1987) concluded that‚ under extreme ozone dose‚ the membrane structure was becoming disorganized with much of the membrane lipids (or perhaps internal fatty acids) being exposed to the outside level of ozone. Ultrastructural studies‚ likewise‚ indicated this large scale disruption after longer periods of ozone exposure (see below; Swanson et al.‚ 1982). In essence‚ the evidence of lipid oxidation induced by ozone is observed only after very high levels or long periods of exposure to ozone. Most work with whole plants (Heath‚ 1984a; Sakaki et al.‚ 1990) show lipid oxidation at high levels (0.5 to 1 ppm) of ozone which severely damages the entire tissue structure. Lipid oxidation then occurs only after the normal cell structure is ruptured. Photosynthesis in alga was inhibited by ozone (Heath et al.‚ 1982). Oxygen evolution was completely inhibited by an exposure for 20 min (time required for 50% inhibition was about 10 min‚ for a total introduced ozone of or Chlorophyll fluorescence was used to study photochemical events. Cells were exposed to ozone in the dark and removed from the solution for examination of fluorescence kinetics in a separate chamber. Within 2 min of ozone exposure the initial peak (often called P and observed within several seconds after illumination) became higher than the initial value. This peak was nearly totally inhibited after 8 min of exposure. The minimal fluorescence level was increased by about 10% after the first 5 min of ozone exposure‚ but then fell to less than half of its initial value after 20 min of exposure. The decline in occurred at about the same time as the general disruption of the cell (Heath‚ 1987). The rise in the peak fluorescence suggested a change in electron flow or energy transduction and is consistent with a blockage in electron flow out of PS II and the rise in The subsequent decline in the peak

Robert L. Heath

418 fluorescence suggested that normal energy transduction was being inhibited. In this latter period the normal permeability and membrane properties of the Chlorella were being changed such that ion distribution may be no longer normal which would then affect the processes of photosynthesis (Heath‚ 1987). Thus‚ all the data collected using Chlorella argues that there are three phases of ozone disruption: (i) ion leakage‚ transport impairment‚ depolarization and slight inhibition of metabolism including photosynthesis; (ii) general loss of membrane permeability leading to a fall in variable fluorescence with major blockages occurring in other metabolisms; and ultimately (iii) a general disruption of the integrity of the cell. The algal studies gave some insight to events within whole plants. It can be argued that the wall and the turgor pressure within the plant cell induces changes in the reactivity of ozone due to the nature of the microenvironment. Modern techniques in molecular biology and electrophysiology could be now used with profit on an algal system‚ providing that the doses used are consistent with some of the observed subtle changes in membrane properties.

III. Whole Plant Studies In the final analysis all model systems merely give insights into investigations of the actual plant. They should not be carried too far in interpretations. The fundamental problem faced by plants upon long term exposure to low concentrations of ozone is lower productivity and the inability to resist other concurrent stresses. In the long run plant productivity is traced‚ one way or another‚ to photosynthesis. Thus‚ we must understand how photosynthesis can be altered by ozone in order to understand ecological events in air pollution. Unfortunately‚ most of the techniques used to measure photosynthesis rely on single leaf measurements or disrupt the plant during the measurement. Too often leaf photosynthesis is also used directly to link an air pollution episode to a decline in plant productivity. Productivity is‚ of course‚ directly related to canopy events and how the whole plant integrates a decline in the photosynthetic capacity of individual leaves. From most summaries (Saxe‚ 1991)‚ it is clear that there is no uniform response by even individual leaves. The generalities that can be drawn are that under low levels of external

ozone in some cases the stomata appear to open more fully (usually measured by an increase in transpiration)‚ but at higher levels of ozone the stomata appear to close (transpiration declines). However‚ many studies show little or no response to transpiration rate.

A. Fumigation Technology It is critical to generate and mix the ozone into any chamber in a well defined manner. It has been shown that the use of electrical discharge or high fields in the presence of air can generate NOx compounds which can confound the ozone induced effects for multiple gas exposures (Olsyzk et al.‚ 1990; Saxe‚ 1991). Thus‚ ozone should be generated by high electric fields in gas streams of pure oxygen and then mixed with air (Horváth et al.‚ 1985). Ultraviolet lights have been used to generate ozone for small volumes (such as solution exposure)‚ but generally they only produce low levels (approximately‚ 20 ml at 300 ppm; Heath‚ 1978)‚ useful for small chambers but not for multiple plant fumigation chambers. The introduction of ozone into a well-defined atmosphere is important for routine exposures of plants (Krizek‚ 1982); consistent exposures of plants require well monitored environments. In the past‚ continuously stirred tank reactors (CSTR) have been used for fumigations (Heck et al.‚ 1978). These chambers are constructed from Teflon and the ozone is introduced through a large pipe‚ with the air stream blowing into a stirring fan located at the top of the chamber. A CSTR facility‚ generally located in a green house‚ sequentially monitors the oxidant levels in the chambers while vigorous stirring continuously mixes the air in each. The light intensity within the CSTR is closer to field conditions than that found in most growth chambers modified for ozone exposure. Exposure of plants in CSTR is imperfect since the light source is continuously changing and atmosphere control is imprecise. In order to obtain more fieldlike conditions open top chambers were introduced. These large cylindrical chambers (a ground diameter of some 5 m with 3 m high walls) continuously blow air into the canopy‚ often below the canopy‚ at wind speeds of several The pollutant is introduced into the air stream and flows into the foliage at near ground level. Monitoring is done sequentially (over some tens of minutes) in each chamber. Ambient with or without supplemental ozone and carbon

Chapter 17 Ozone and Photosynthesis filtered air provides the ozone and control levels. A non-chambered plot provides another control for the actual field conditions. In general the chambers are warmer and the air flow is much more vigorous‚ both in direction and speed‚ than actual field conditions. None the less‚ they have been used with profit for several decades and in a major study (Heck et al.‚ 1988). Although they do not duplicate field conditions‚ their conditions are closer than those of CSTR or growth chambers. There are several other systems available for other types of fumigation. Some use normal atmospheric gradients in the field while others introduce the pollutant downwind of the field plot (McLeod and Baker‚ 1988). All have their uses‚ but a detailed description of these systems is not appropriate here.

B. What is the Real Flux of Ozone? We can not really measure the internal concentration of ozone‚ nor can we determine its pathway(s). As previously stated ozone is highly reactive‚ thus‚ it breaks down on surfaces very rapidly. Ozone moves through the stomata reacting along its total pathway. Thus‚ cells at different distances from the stomata would be expected to be exposed to different levels of ozone. The cells closest to the stomatal aperture are the guard cells and a short path to those cells would serve to minimize the breakdown of ozone and maximize their exposure. The cells next closest are generally the subsidiary cells and these would be expected to be subjected to the next highest dose of ozone. In fact‚ the mesophyll cells containing the chloroplasts‚ are often the furthest from ozone’s entry point and should be exposed to the lowest dose. This suggests that the shortest path from the stomates to the cell would be the most damaging due to the highest internal ozone concentration (Heath‚ 1994b). In terms of visible injury‚ the collapse of the mesophyll cells due to water loss under extreme concentrations was observed only near the stomata (Bobrov‚ 1952; Uhring‚ 1978; Miyake et al.‚ 1989). Traditionally the flow of ozone is governed by its total conductance and its gradient into the leaf‚ similar to (equation 1). The conductance for ozone has been calculated by measuring water vapor conductance and converting that value via molecular weight ratios to an ozone conductance (Rogers et al.‚ 1977; Ball‚ 1987). To determine the gradient one must know the external and internal concentration of ozone. Unfortunately‚ a measurement of internal concen-

419 tration of ozone is difficult‚ if not impossible. Many groups have attempted its measurement and while they obtain a value for the conductance which is reasonable for gas flows through the stomatal aperture‚ the value of is variable. It is low (FreerSmith and Dobson‚ 1989; Laiski et al.‚ 1989; Rowland-Bamford et al.‚ 1989;Moldau et al.‚ 1990)‚ but not zero (Taylor et al.‚ 1982; Amiro et al‚ 1984). The major problem in the measurements of internal ozone concentration and flux in general seems to be accounting for all the ozone decomposition within the chamber and on the leaf (Rogers et al.‚ 1977). It has been found that the equilibration times are long (Moldau et al.‚ 1990); thus‚ one can not do rapid experiments. In general‚ surfaces must be saturated with ozone before stable readings can be made. There is also a conditioning of the surface which seems to take place. Furthermore‚ many studies find a positive‚ non-zero intercept when plotting flux versus external concentration. This positive intercept is a good indication of non-equilibration of the gas with surrounding surfaces. These studies generally use high concentrations of ozone to assess the flux and so data at low (near ambient) concentrations are missing. Many compounds react non-linearly with ozone (Bailey‚ 1982) and so data at high concentrations can not easily be extrapolated back to low‚ ambient levels. Measurement of for ozone is critical because the arguments regarding which biochemical events are important‚ rely upon a knowledge the internal concentration of gas within the wall (Heath‚ 1987; Chameides‚ 1989). In fact‚ a single internal concentration is a myth‚ since it must represent an average internal concentration. At each individual cellular site of interaction‚ the ozone concentration is‚ most probably‚ different. This differential delivery of ozone to various parts of the leaf tissue may well cause the ‘spotting’ of ozone injury‚ where only small regions of the tissue are damaged. Parkhurst (1984) argued that mesophyll resistance is based upon a one-dimensional model‚ while real diffusion/sink system is three-dimensional. In particular‚ the sink strength for the is proportional to the light intensity and level of throughout the tissue. Thus‚ for a model in which the stomate is represented as a hole in a cylinder of the mesophyll cells‚ a change in the stomatal aperture alters not only the conductance but also how far the can move into the cylinder. This in turn‚ affects internal concentration which is often expressed as the

420 mesophyll conductance. This same argument is even stronger for the reactive ozone. Unfortunately‚ there is simply no current alternative to a simple linear model for gas flow. For ozone with a near zero internal concentration‚ this translates into the simple equation given in Eq. (1).

IV. Photosynthesis or Stomates? Under extreme exposure conditions both photosynthesis and stomatal conductance are affected. Saxe (1991) has reviewed many of the findings and Heath (1994a) has discussed the relative relation of stomatal conductance to photosynthesis. In essence‚ many of the necessary measurements have not been carried out such that one can tell whether one process is more affected than the other. Aben et al. (1990) exposed Vicia faba for two weeks to 0.072 ppm and found that there was no statistical difference between treated and control plants for photosynthesis‚ compensation point‚ or dark respiration. But the conductance was decreased by 27%‚ which resulted in a rise in water use efficiency. Similar results were

Robert L. Heath found in wheat by Lehnherr et al. (1987) in which the soluble protein and the grain dry weight declined by nearly 50% (exposed for 8 weeks at 0.1 ppm ozone)‚ matching the conductance decline of nearly 60%. The amount of Rubisco declined by nearly 40%‚ while the compensation point was unchanged. Much of the old data on assimilation and stomatal conductance should be reanalyzed because stomatal physiology is now better understood. Unless water loss becomes dominant‚ the internal concentration of seems to be maintained such that as assimilation declines‚ the stomata partially close (Farquhar and Sharkey‚ 1982; Assmann‚ 1988). The fundamental analysis of the relationship between assimilation rate and internal concentration (the curve) is shown in Fig. 3 (see also Chapter 8). The assimilation rate (A) is proportional to concentration at the site of carboxylation which is proportional in turn to the amount of Rubisco and the ability of the chloroplast to supply the necessary NADPH and ATP for regeneration of RuBP). Generally the shape of the curve is hyperbolic. For a given external concentration of and associated assimilation rate the conductance (g‚

Chapter 17 Ozone and Photosynthesis given by the slope of the line from to is maintained so that the internal concentration is fixed at The assimilation rate can fall to A' if the internal concentration falls to meaning that the conductance must also have declined to g'. On the other hand‚ if the conductance does not change but the assimilation rate declines to A" due to a change in the curve (e.g. a reduced Rubisco concentration)‚ then the internal concentration of would have to rise to The insert in the Fig. 3 is of use in determining which process is affected by ozone first. If assimilation was unaffected by ozone and ozone only partially closed stomata‚ then Ao would still fall to A'‚ a small decline given by The internal concentration then would have to change but the relative magnitude of that change would depend upon what portion of the curve was involved. Similarly if the process of assimilation was affected (such that a new A/Ci curve was generated with a decline of and fell to but the regulation by internal was so strong that the internal remained constant‚ then the change in conductance would be dependent upon its position on the A/ curve. According to this analysis‚ assimilation can not change without altering conductance and conductance can not change without altering internal concentrations. A complete study of the A/Ci curve must be done in order to state absolutely which process alone was being affected by ozone. One good example of such an analysis is given in the paper on photosynthetic changes in response to changes in water vapor deficit (Guehl and Aussenac‚ 1987). The transition along the A/Ci curve was observed as the water vapor deficit increased. The movement along the curve (as in Fig. 3) shifted the curve to a lower one‚ and resulted in a decline in . Interestingly‚ it is clear that if the two measurements of the curve were separated by a long time‚ the decline in A and would have been observed making it impossible to understand which may have occurred first. Many experiments on ozone interactions with photosynthesis are done in this manner. However‚ one such analysis was performed by Farage et al. (1991) with important conclusions. The data of Farage et al. (1991) strongly supports the transition shown in Fig. 3 in which a decline in A is matched by a decline in such that the remains nearly the same‚ similar to that observed for a decline in water vapor deficit Although Farage et al. (1991) did not attempt to correlate the dose of

421 ozone with the decline in the light-saturated photosynthesis‚ Fig. 4 shows that such a correlation is quite good if an accumulated amount of ozone is used as the independent variable. The rate of delivery of ozone does not correlate nearly as well. Heath (1994a) has argued that under low light the rate of delivery might be more important. Further‚ since at low light the curve is very shallow (i.e. is close to a large change in g would be observed before a significant concurrent change in A could be measured. We are concerned ultimately with a decline in productivity. This may be due to (i) stomatal closure to limit the availability of (ii) a loss of ability to fix carbon because of inactivation of enzymes or a reduced availability of energy; (iii) a major shift in regulation between carbon and energy flows; and/or (iv) a shift in the ability to move carbon from the leaves to the sinks. It is not yet clear which of these processes are most sensitive to ozone exposure. However‚ the evidence so far from studies using conditions similar to normal field environments (high light and reasonable water vapor deficit) would suggest that carbon processes of photosynthesis may be inhibited first followed by a decline in stomatal conductance to maintain the internal level. The fourth reason for a decline in productivity is perhaps the most attractive‚ but as the yet the one which has been least tested. As summarized by both Koziol et al. (1988) and McCool (1988)‚ the carbohydrate level in roots declines with ozone

422 exposure. In some cases this lowered carbohydrate level causes a decline in the root mycorrhizal growth. This suggests that translocation is being inhibited. McCool (1988) states that this picture is not completely clear since growth is governed by many events in the shoot and root environments. As summarized by Cooley and Manning (1987)‚ it is difficult to piece together a coherent model of what is really happening to translocation when photosynthesis declines. In many cases the starch reserves are mobilized and so the soluble carbohydrates appear to actually rise within the leaf after ozone depression of photosynthesis. The other potential problem which makes understanding so difficult revolves about the changes in the ionic levels within the cell. If ions‚ including phosphate‚ are lost from the cell‚ the exchange of triose phosphate of the chloroplast (produced by photosynthesis) with cytoplasmic phosphate would be altered (Heber and Heldt‚ 1981). Less cytoplasmic phosphate would decrease the exchange and so plastid phosphate would remain high‚ thus influencing the soluble carbohydrate levels. For the most part‚ it is believed that translocation is sensitive to ozone‚ based upon the decline in productivity and in carbohydrate levels in the translocation sinks. Yet most investigations have not measured enough parameters to clearly place each event into a proper sequence. The decline measured for individual leaf photosynthesis is not great enough to account for the measured declines in productivity. The rates of photosynthesis and levels of tissue carbohydrate must be measured at same time as translocation rates during exposure to ozone. If these sets of measurements could then be coupled with productivity measurements‚ we might be able to trace out the sequence of events leading from a decline in photosynthesis to a loss of productivity.

A. Changes within the Stomata—Conductance Alterations The action of stomata opening and closing depends upon many interactive processes but‚ in essence‚ much depends upon the relative water potential difference between the guard and subsidiary cells (MacRobbie‚ 1987; Hetherington and Quatrano‚ 1991; Tallman‚ 1992; Chapter 9). The large amounts of ionic flows concurrent with pH changes during the opening can be disrupted by the loss of membrane permeability (Bowling and Smith‚ 1990). There are

Robert L Heath also many regulatory events which signal and control these movements and change their kinetics (Chapter 9). However‚ reduced to the simplest concept‚ the relative balance of osmotically active compounds between the two cell types govern the aperture of the stomate. Heath (1994a) has postulated that under some conditions of low ozone concentration‚ only the subsidiary cells adjacent to the stomata may be susceptible to the ozone action upon membrane permeability. The thicker wall of the guard cell might be expected to protect the cell due to difficulties in ozone movement to the plasmalemma. However‚ ozone through any alterations of membrane function of subsidiary cells would disrupt normal ion flow and differential water potential between subsidiary and guard cells. Loss of osmotically active materials from the subsidiary cells would increase water potential and water would flow into the guard cells (which possesses more osmotically active materials and have a lower water potential). The guard cells would become more turgid‚ and if possible result in a further opening of the stomate (Sanders et al.‚ 1992). As the ozone concentration increases‚ the higher level of ozone within the cell wall would begin to attack the plasmalemma of the guard cell‚ resulting in a decrease in permeability and that cell would lose osmotically active materials and its water potential would rise. The water potential of the subsidiary cells‚ now falling compared to the guard cells‚ would cause those cells to gain water from the guard cells. The stomates would begin to close. Consequently the varied responses of transpiration (and stomatal aperture) to ozone dose may be simply explained by the differential action of gaseous ozone upon the membranes of each cell type.

1. Ethylene—Interaction with Transpiration Increased ethylene production‚ a known response to tissue wounding‚ has been also identified as a plant response to ozone exposure. Tingey et al. (1976) showed for a variety of plants that the observed foliar injury (induced by up to 0.75 ppm ozone for 4 h) correlated with the production of stress ethylene (measured by trapping the ethylene in a plastic bag over the leaves maintained in the dark for 22 h). The amount of ethylene released was exponentially related to the amount of ozone (in external ppm). The exponential coefficient was both species dependent and related linearly to the percentage of the foliar

Chapter 17

Ozone and Photosynthesis

injury. Further‚ the amount of ozone-induced ethylene release declined upon repeated exposure indicating an acclimatization to ozone. The release of ethylene induced by wounding is not linear with time‚ but declines after the initial response. Stan et al. (1981) and Stan and Schicker (1982) showed that this is also true with ozone exposure. They found that the stress-induced ethylene production correlates better with exposure level of ozone rather than with duration of exposure. In other words‚ peaks of high ozone rather than accumulated dose generate a higher rate of ethylene release‚ at least for a single exposure to ozone under an acute dose. The correlation of ethylene release with ozoneinduced visible injury was likewise shown in pea cultivars by Dijak and Ormrod (1982). With ozone exposure (generally 6 h at 0.3 ppm)‚ the stomates closed by approximately 50% in 3 h with a dose of mol (with an average rate of moles as calculated from their data). Both sensitive and insensitive cultivars with visible injury released ethylene; sensitive cultivars scored higher in both visible injury and ethylene release for a given exposure. The correlation of ethylene release with visible injury is shown in Fig. 5. Although the correlation is non-linear‚ this follows the data by Tingey et al. (1976). Another indication that ethylene release is required for visible injury (or vice versa) is shown by the work of Mehlhorn and Wellburn (1987) which is summarized by Wang et al. (1990). Aminoethoxyvinylglycine (AVG) is an inhibitor of ethylene production‚ and also inhibits the development of visible injury (Mehlhorn and Wellburn‚ 1987). While inhibitor experiments may be not specific‚ the accumulation of data suggests that ethylene release is present when visible injury is observed and prevention of ethylene release prevents the production of visible injury. Gunderson and Taylor (1988; 1991) used exogenous ethylene to alter the gas exchange of soybeans and found an exponential decline of both stomatal conductance and carbon assimilation with ethylene‚ but not simultaneously. Interestingly the exogenous ethylene caused a slight rise in indicative of a lowering of which is not what was observed in the experiments of Farage et al. (1991) during ozone exposure. Ethylene does inhibit both stomatal conductance and carbon assimilation to some extent. Thus‚ one

423

could postulate that ozone induces a change in internal concentration‚ which generates a wounding response. One portion of this wounding would be the production of ethylene‚ which would generate the change in stomatal conductance and photosynthesis. Clearly these multiple events could confounds some earlier studies.

B. Chloroplast Structure If photosynthesis is inhibited directly‚ an obvious place for injury is within the chloroplasts. Thomson et al. (1966) first noted that chloroplasts accumulated crystals (‘granulation and electron density increase’) within the stroma after heavy fumigation of bean or cotton (0.4 to 1.0 ppm for 0.5 to 1 hour; summarized by Thomson‚ 1975). Swanson et al. (1973) noted a breakdown of membranes under some circumstances but did not often see the formation of crystals in tobacco exposed to a much lower level of ozone (0.3 ppm for 2 h). Pell and Weissberger (1976) also noted a collapse of the protoplasts with a disruption of the membrane system in soybean using 0.3 ppm for 3 h. Further‚ they observed that the membranes had a higher affinity for stains indicating some changes in the membrane structure. Miyake et al. (1984)‚ using much lower concentration of ozone (0.1 ppm for 8 h/ day for 6 days) for radish‚ found an increase in plastoglobuli (lipid containing bodies) and changes in chloroplast dimensions (similar to Swanson et al.‚ 1973). Thus‚ it appears that very high levels of ozone induce a rapid water loss; one response to extreme

424 fumigation is a wilting of the leaves or water-logging. Thomson (1975) argued that such water loss caused the crystal formation within the stroma. At lower‚ but still acute‚ levels of ozone membrane alterations become apparent. Lipid synthesis is altered by high levels of ozone (Sakaki et al.‚ 1990) and this alteration may be causing the changes in membrane staining properties and in the number of plastoglobuli. At lower levels of ozone‚ there are few reports of major changes in chloroplast structure. The major difficulty in this type of research is that the visible injury pattern is patchy. One group of cells may be injured while a surrounding group can appear normal. We do not really understand what causes this patchiness‚ although it may be related to the concentration of ozone within the tissue; most of the injury is often near the stomata (Bobrov‚ 1952). To add to the problem‚ the visible injury seems not to develop rapidly but rather sequentially. Pell and Weissberger (1976) showed that paraveinal tissue collapsed immediately after the fumigation (see above)‚ but only later did the collapse spread to the palisade parenchyma and ultimately to the spongy parenchyma (24 h later). Yet many researchers (see Povilaitis‚ 1962‚ Bobrov‚ 1952) have shown that only the palisade parenchyma was involved with visible injury. Thus‚ care must be taken when and where sampling of tissue is done after the fumigation

C. Photosynthesis and Associated Enzymes The most complete story on Rubisco has been developed by Pell and her coworkers (summarized in Pell et al.‚ 1994). Pell and Pearson (1983) showed a sizable decline in total Rubisco in alfalfa plants (subjected to 0.25 ppm for 2 h)‚ measured by staining of the individual protein bands separated by gel electrophoresis. The assays were done 2 days after the fumigation and the two cultivars gave quite different results. More importantly‚ even without a visible injury symptom there was sizable loss of Rubisco content in the cultivar Moapa 69 from 91 to dry weight. Dann and Pell (1989) followed with experiments on potato using a complex fumigation protocol lasting 6 h per day over 5 days (four days with an average of about 0.07 ppm and a fifth day with an average of 0.095 ppm). They had three separate experiments in which the plants were at a slightly different developmental age. However‚ if the experiments were overlaid so that the assimilation rates were

Robert L. Heath similar‚ it was clear that both assimilation and level of Rubisco had an activity peak dependent upon leaf age. Further‚ only in the later stages of leaf development‚ after maximum activity had been achieved‚ did ozone have a pronounced effect on the level of Rubisco (measured by activity or amount). This observation has been duplicated in radish (Pell et al.‚ 1994) and poplar (Landry and Pell‚ 1993). Only after the leaf has reached maximum size and the assimilation rate begins to decline does the ozone at moderate levels cause a decline in the activity and amount of Rubisco. Dann and Pell (1989) found that in vivo activation of Rubisco was not a factor in ozone-induced effects on the enzyme activity; the inhibition of measured activity was approximately the same if the activated or non-activated enzyme was used. Yet the enzyme in vivo is highly regulated so that total amount or activity of the enzyme in vitro may not give a full picture of its operation within the cell. The dependence of visible injury formation upon developmental age has been previously noted (Heath‚ 1994b). However‚ few really realize how critical it is to determine the leaf’s physiological age in order to compare these studies. Often a consistent age is used in the experiments according to good scientific protocol‚ but it is not noted in the published paper. Any limitation in photosynthetic rate induced by ozone exposure may be traceable to critical changes in the balance between conductance and assimilation processes during the course of leaf development. More importantly‚ the data of Dann and Pell (1989) shows that there is a clear difference in when a process should be measured after the fumigation sequence ceases (Table 2). Immediately after fumigation‚ carbon assimilation is inhibited‚ an inhibition which was found to be greater 24 h later. Yet 5 days after the fumigation this inhibition of assimilation had disappeared. On the other hand‚ the loss of Rubisco activity was small and variable immediately after fumigation but rose to a sizable level even at 5 days post fumigation. The disappearance of any inhibition in assimilation at 5 days suggests that the loss of Rubisco was not a limitation to carbon fixation for these older leaves‚ but rather may be indicative of other events. Under a chronic (low level and continuous) exposure to ozone radish shows similar responses. As ageing of the leaf progresses‚ the activity of Rubisco declines while the percentage inhibition of stomatal conductance by ozone increases (Pell et al.‚

Chapter 17 Ozone and Photosynthesis

1993). On the other hand‚ the percentage loss of Rubisco and inhibition of assimilation remains high; rising to 100% only in the older leaves. Under these conditions assimilation seemed to be limited by Rubisco in only the older leaves. This is consistent with the observation on wheat leaves by Nie et al. (1993) that the older sections (near the tip) showed a loss of the large and small subunits of Rubisco‚ whereas ozone had no effect on Rubisco levels in younger leaf sections. Heath (1994a) has argued that different plants in different environments may be governed by quite different rate limitations induced by ozone exposure. While that is not an especially insightful comment‚ it leads to the conclusion that one can not state that only conductance or Rubisco is the major factor in ozone induced injury to plants. Both may play a role but at different developmental states of the plant. Reddy et al. (1993) found much clearer results with respect to Rubisco loss by examining the mRNA for the subunits of the enzyme. They found a sizable decline in mRNA for the small subunit of Rubisco‚ as well as a slightly less pronounced decline in the mRNA for the large subunit. Others have found a similar decline upon fumigation (Schlagnbaufer et al.‚ 1995). For example‚ R.L. Heath‚ E.A. Bray and R. Miller (unpublished) found a 60–70% decline in the steady state level of the small subunit mRNA after 1 to 2 h of exposure to 0.33 ppm of ozone in tomato. It seems that a decline in this particular mRNA species may be useful to standardize injury induced by ozone under conditions where visible injury does not occur. How large the inhibition of assimilation is and how long this inhibition lasts is important in judging the ultimate loss of long-term productivity. Clearly the data suggest that loss of assimilation for mature leaves is harmful while there is less of an effect in young tissue. This loss in activity inevitably results in less photosynthate being available for the newly

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developing parts of the plant and less energy reserves to fight other stress or pathogen attacks. Finally this approach might be a method to examine the onset of early senescence under very low chronic levels of ozone. The loss of Rubisco seems to occur in the absence of visible injury.

D. Ionic Control of Photosynthesis While ozone induces a decline in ion transport and an increase in general permeability of two ions‚ and at the plasma membrane‚ no one has yet measured any lowering or increasing of the pool sizes of each ion or coupled these events to rates of photosynthesis within the chloroplasts. It is known that balance of both ions is critical to metabolism but of particular note would be the events described above on (i) the formation of mRNA for chloroplast proteins‚ (ii) the activity of the carbon fixation cycle‚ and (iii) the induction of photooxidation of proteins and pigments due to inability to dissipate light energy. Kaiser et al. (1980) have shown that ions do affect the ability to assimilate carbon and transport metabolites across the chloroplast envelope. Although plays a role in electron transfer within PS II (Yocum‚ 1991) and is accumulated during photosynthesis (Miller and Sanders‚ 1987)‚ little has been done to observe any role of in linking photosystems to fixation. However‚ Brauer et al. (1990) have found three potential targets of sugar production to a change in : (i) fructose 1‚ 6bisphosphatase (a critical enzyme in glyconeogenesis) is inhibited by an increase; (ii) sucrose phosphate synthase is activated upon interference of transport; and (iii) the turnover of pyrophosphate may be increased by the same interference. The production and movement of sugar for other growing tissues within the plant is very complex (Fig. 6). Production can be altered at many steps other than simply the electron transport and fixation steps

426

(direct photosynthesis). Thus‚ we may have been mislead by small changes in direct photosynthesis; more critical alterations by ozone exposure may be within the subsequent metabolic steps. The role of fructose 1‚6-bisphosphatase has been noted before as critical to normal translocation (Gerhardt et al.‚ 1987; Stitt‚ 1990). Thus‚ any interference with the levels of this by metabolic alterations caused by ozone would have profound effects upon the productivity of the plant. As suggested from Fig. 6‚ the balance of carbon flow into starch and sucrose must be maintained through fructose 1‚6-bisphosphatase. Furthermore‚ the movement of triose-phosphate (in exchange with phosphate) out of the chloroplasts has to be regulated (Flügge and Heldt‚ 1991; Chapter 6) in order to maintain phosphate balance between the organelles and to move carbon out of the chloroplast. Finally the production of sucrose requires balanced functioning of the cytoplasmic fructose 1‚6-bisphosphatase and sucrose synthase‚ and maintenance of the pyrophosphate level (Huber‚ 1989; Stitt‚ 1990; Chapter 6). All the steps in photosynthesis including translocation and storage must be balanced but most

Robert L. Heath

investigations center upon how the plant responds to a particular step in photosynthesis upon a particular stress. Unfortunately‚ little progress has been made in this area due to the difficulties of observing small subtle events at certain steps in photosynthesis in whole plants which generate large change overall. Perhaps the development of a model in which attention to specific rate limitation could indicate potential limitations to carbon fixation is a more easily accomplished method of future studies.

E. Electron Transfer and Photoinhibition The data do seem to suggest that the light reactions are not altered if the dose is low enough not to cause the formation of any visible injury. In a study of wheat Nie et al. (1993) showed that while photosynthetic capacity was lowered by 30% and the quantum yield was lowered by 20% upon an exposure to 0.15 ppm for 7 h‚ there was no change in thylakoid proteins‚ when studied by gel electrophoresis‚ or in the maximum quantum yield of PS II photochemistry‚ estimated from ratio of variable to maximal fluorescence‚ (see below). The major change

Chapter 17

Ozone and Photosynthesis

observed was a decline of the subunits of Rubisco. The conclusion of the authors was that carbon metabolism was the site of ozone-induced inhibition and that there was no evidence for photoinhibition of PS II under these relatively mild conditions. Similarly‚ Farage et al. (1991) using higher ozone concentrations observed large decreases in the light-saturated rate of photosynthesis in the absence of decreases in and atrazine-binding capacity of the thylakoids‚ thus indicating that photoinhibition to PS II was not a factor in the initial depression of photosynthetic performance by ozone. Under higher levels of ozone and on prolonged exposures chlorosis does occur and is one of the visible injury symptoms. Thus‚ ozone-induced events can give rise to conditions which ultimately cause photooxidation and a decline in chlorophyll. The sequence of these changes has not been studied. It may well be that the loss of ions and metabolic control within the cell causes cell death and the chlorophyll is then bleached by physical causes because it is exposed to light and oxygen without its normal protectants. Such photooxidation is the most reasonable explanation at the present time‚ yet doubt remains that photoinhibition by inhibition of physiological processes induces events leading to the photooxidation. Most of these claims rest upon changes in chlorophyll fluorescence. The relatively meager evidence from Chlorella studies suggests‚ however‚ cell disruption plays a larger role. The concept for photoinhibition comes from a long line of literature first summarized by Osmond (1981). The phenomenon was associated with lightdependent damage to photochemical processes occurring within chloroplast thylakoids. A loss of chlorophyll and carotenoids was one of the features of prolonged photoinhibition. It was argued that an interruption in normal energy transduction within the chloroplasts forced an overload of the electron transport chain. If the interruption was with carbon fixation‚ oxygen could substitute for the acceptor and prevent‚ in part‚ the photoinhibition. If the interruption was within the electron transfer system‚ PS II would over-oxidize and cause an uncontrolled oxidation of the pigments‚ lipids and proteins associated with the PS II reaction center complex (see Chapters 4 and 5). One of the primary target molecules is known to be the D1 protein (a 32 kDa protein functioning at the oxidizing side of PS II). Under conditions in which photoinhibition could occur‚ D1 would be damaged and replaced to

427

prevent the spread of the injury (see below and Chapter 4). Baker and Horton (1987) pointed out that photoinhibition is often accompanied by severe modifications of the characteristics of chlorophyll fluorescence. Thus‚ fluorescence kinetics‚ primarily arising from PS II and influenced by the function of D1‚ are now often used as a indication of photoinhibition (see Chapters 1–3). There are many ramifications of chlorophyll fluorescence measurements (see Chapters 2 and 3)‚ which I will not discuss here‚ but there are two events which are frequently taken to indicate photoinhibition: a decline in and a decline in the minimal fluorescence level‚ Fo (i.e. the fluorescence level when all PS II reaction centers are maximally oxidized). As photoinhibition proceeds‚ photobleaching or a loss in pigments becomes apparent. A somewhat trivial‚ but important‚ paper by Knudson et al. (1977) shows a linear relation between chlorophyll reduction and observed visible injury (due to necrosis and chlorosis) under varied ozone fumigation levels in Pinto bean. Both chlorophylls a and b were lost equally. Robinson and Wellburn (1991) showed that summer exposure of Norway spruce to ozone induced a loss of pigments in older needles‚ but only after over-wintering long after the exposure. However‚ their conclusion was that the use of pigment analyses to determine field injury by ozone (especially at low levels) was not very useful in determining the nature of damage since there were too many variables to be able to sort out which process was the dominate effect. From these effects we can postulate that photobleaching probably only occurs with high (perhaps acute) ozone exposure. This is strongly supported by the study of Farage et al. (1991)‚ who observed that fluorescence characteristics in wheat leaves were only affected at high levels of ozone or slightly lower levels given for much longer duration (e.g. 16 h at 0.4 ppm). Atrazine binding‚ which is a marker of the functionality of the D1 protein‚ was not affected even at 16 h of 0.4 ppm external ozone. Similarly they detected no change in the D1 protein using a Western blot. Their conclusion that PS II photochemistry was not altered at the lower levels of ozone‚ more typical of the normally polluted atmosphere (e.g. less than 0.3 ppm for several hours)‚ seems inescapable. Carbon fixation machinery was much more ‘at risk’ than the photochemical processes associated with the thylakoids (Baker et al.‚ 1994). This important

428 conclusion is similar to those reached from fluorescence studies of alga (see above) and developing wheat leaves (Nie et al.‚ 1993). However‚ there is one study which demonstrates increased turnover rate of D1 after ozone exposure which counters the hypothesis that photoinhibition does not occur at low ozone levels and suggests that increased rates of photodamage to D1 may occur under relative severe exposures. Pino et al. (1995) showed that a 32 kDa protein was labeled with (fed to the blade) more rapidly after ozone exposure (0.2 ppm for only a few hours) in corn. Corn is a relatively ozone-tolerant species but Pino et al. (1995) found conditions where visible injury developed on the older sections of the blade. They too found changes in polypeptides in the molecular weight range of 18 to 22 kDa. The earlier work by Schreiber et al. (1978) showed decreases in but they used relatively higher levels of ozone (0.3 ppm and higher) for 6 h. At 2.5 ppm the effects were clearly seen within 40 min‚ but this level is so severe that it may not be at all relevant to any field studies. There was‚ however‚ a slight effect at 0.125 ppm for 2 h (perhaps 10–20% decline). Most importantly there was a sizable developmental age effect. The lowering of the height of the peak in the fluorescence induction curve was most pronounced for mid-aged leaves‚ which also have maximum sensitivity to ozone as measured by visible injury (Heath‚ 1994b). Other work has confirmed these results at very high ozone levels (Schmidt et al.‚ 1990) and at lower levels with subzero temperature (Davison et al.‚ 1988). DM Olsyzk and RL Heath (unpublished data) could not find any reproducible changes in the fluorescent induction patterns of corn‚ squash‚ radish‚ bean and spinach except for a slight lowering of at ozone levels (0.1–0.2 ppm for several hours) where little visible injury occurred and but a slight (20%) depression in whole plant weight (a measure of productivity) occurred. Thus‚ we must conclude that fluorescence may well be a good marker for ozone-damage in extreme exposures (perhaps even more sensitive than visible injury development)‚ but gross kinetics of fluorescence do not easily detect physiological changes associated with lower exposures. It must be noted‚ however‚ that comparative work identifying the relationships‚ if any‚ between loss of Rubisco mRNA and protein‚ chlorophyll fluorescence parameters‚ visible injury development and productivity has not been done.

Robert L. Heath

F. Non-invasive Measurements Most measurements of photosynthesis and transpiration require that the leaf be handled and placed into an instrument. Such measurement is invasive and gives only a single data point in time. Attempts have been made to carry out measurements continuously in real time without interfering with the leaf. These methods generally rely upon a camera which can image the leaf externally giving a physiological measurement. Infra-red (IR) cameras (which measure the surface temperature of the leaf at a 10 wavelength) can be used to measure its gas exchange capacity via transpiration-induced cooling. The leaf temperature is derived from an energy balance between evaporative cooling and radiation loading (Omasa et al.‚ 1980; Koutaki et al.‚ 1983; Omasa and Aiga‚ 1987). The evaporative cooling is due to water loss through the stomates of the leaf which limits pollutant entry and dose. The equations for energy balance of a leaf are well-known (Clark‚ 1975; Monteith and Unsworth‚ 1990). These cameras often use a mirror scanning system which focuses the radiation onto a cooled solid state detector and the real time image is displayed by a television monitor. The temperature and spatial resolution of the camera is often better than 0.1 °C and 1–2 mm. When a leaf is placed in a fixed position in a fumigation chamber and the IR camera is focused upon it‚ the leaf temperature can be easily measured (see Heath‚ 1994b). Before fumigation‚ the heat balance of the leaf causes a temperature gradient across the leaf indicative of differential gas exchange. This variability in stomatal conductance would lead to a differential dose of a pollutant reaching different interior portions of the leaf. After 2 h of fumigation at 0.25 ppm (which causes no visible injury)‚ the surface temperature distribution has changed indicating that the general leaf temperature has risen due to partial closure of the stomata. Yet the general distribution of the temperature gradient across the leaf remains approximately the same. Even 24 h after fumigation the temperature of the leaf remains elevated‚ indicating that stomata are still closed. Ellenson and Raba (1983) have used a similar technique to measure delayed light emission (which gives information similar to that from chlorophyll fluorescence; see Chapter 2) from leaves and found that a brief pulse of ozone at a relative low concentration generated an oscillation in the delayed

Chapter 17 Ozone and Photosynthesis light picture. They suggested‚ based upon uptake measurements‚ that the oscillating size of the stomata limited entry and hence restricted the sink for the electrons transferred from the photosystems and resulted in the altered delayed light signals. The use of both these camera techniques can measure changes in the gas exchange characteristics of leaves due to a pollutant exposure non-invasively and continuously. But also these techniques should aid in identifying and sampling regions of the leaf which have had equivalent pollutant doses‚ such as found by measuring transpirational cooling due to water loss through the stomates. Biochemical analyses of such tissue samples should allow the detection of small changes in metabolism that result from exposure to pollutants.

V. Conclusions What do we really know about inhibition of photosynthesis by ozone? In order to answer this we need to review the processes by which ozone may alter the plant’s metabolism. The best concept is that the primary site of ozone interaction with the plants is the plasma membrane and wall space. Ozone‚ being so reactive‚ can really not move much further into the cell‚ although its breakdown products may well do so. Unfortunately‚ we do not know what‚ if any‚ those products are. Yet alterations at the plasma membrane can have dramatic effects upon the metabolism‚ especially if calcium balance is being disturbed. Under these conditions‚ an inhibition of photosynthesis‚ at a site removed from the plasma membrane‚ is not surprising. While the stomata must be open for ozone to cause any perturbation‚ there is considerable evidence that ozone induces changes in stomatal function; opening in some conditions‚ but generally inducing closure. Is the primary event stomatal closure or does an inhibition of carbon fixation cause a decline in stomatal aperture? The answer seems to depends upon the state of the plant and we can not predict the events well from our current knowledge. Certainly the best measure of ozone dose is the product of external concentration and stomatal conductance (for ozone)‚ not just external concentration. Understanding of how influential peaks of ozone concentration and duration of ozone exposure are upon plant metabolism is critical to our minimizing ozone effects upon productivity‚ but their role has

429 not yet been fully clarified. Clearly ozone under some conditions can directly lower the amount of Rubisco present in the chloroplasts in mature leaves. The decline in the enzyme itself is not as severe as the fall in its mRNA‚ but after many days of exposure the Rubisco activity may well be low enough to cause a decline in overall plant productivity. The largest uncertainty in predicting ozone-induced depressions in plant productivity is associated with the increased rate of senescence in exposed plants. If x leaves are lost y days earlier‚ what is the loss of net productivity? That type of question has not been answered satisfactorily for ozone exposure. This issue is similar to the question of loss in productivity due to a loss in leaf area resulting from the formation of visible injury. How much area needs to be lost before productivity is modified? We know that ozone inhibits plant productivity and induces responses within the plant that make the plant unable to respond to concurrent and subsequent stresses. Studies based on such observations would benefit from a better understanding of the underlying mechanisms. In the forty plus years since the initial studies on oxidant injury to plants in the Los Angeles basin were made‚ we have made good progress in defining the problem. We now should devote more time and energy to elucidating the fundamental processes from the initial cellular events to ecological responses induced by tropospheric ozone. Unfortunately‚ higher levels of ozone seem to be an inevitable part of modern man’s existence‚ a part to which we must respond with more resistant cultivars and more appropriate horticultural and ecological practices.

References Aben JMM‚ Janssen-Jurkovicova M and Adema EH (1990) Effects of low level ozone exposure under ambient conditions on photosynthesis and stomatai control of Vicia faba L. Plant Cell Environ 13: 463–469 Alscher RG and Wellburn AR (eds) (1994) Gaseous Pollutants and Plant Metabolism. Chapman and Hall‚ London Amiro BD‚ Gillespie TJ and Thurtell GW (1984) Injury response of Phaseolus vulgaris to ozone flux density. Atmos Environ 18: 1207–1215 Assmann SM (1988) Stomatal and non-stomatal limitations to carbon assimilation: An evaluation of the path-dependent method. Plant Cell Environ 11: 577–582 Bailey PS (1982) Ozonation of nucleophiles. In: Bailey PS (ed) Ozonation in Organic Chemistry‚ Vol II. Nonolefinic

430 Compounds‚ pp 155– 232. Academic Press‚ New York Baker NR and Horton P (1987) Chlorophyll fluorescence quenching during photoinhibition. In: Kyle DJ‚ Osmond CB and Arntzen CJ (eds) Photoinhibition‚ pp 145–168. Elsevier Science Publishers‚ Amsterdam Baker NR‚ Nie G-Y and Tomasevic M (1994) Responses of photosynthetic light use efficiency and chloroplast development on exposure of leaves to ozone. In: Alscher R and Wellburn AR (eds) Gaseous Pollutants and Plant Metabolism‚ pp 219– 238. Chapman and Hall‚ London Ball JT (1987) Calculations related to gas exchange. In: Zeiger E‚ Farquhar GD and Cowan IR (eds.) Stomatal Function‚ pp 445– 476. Stanford Press‚ Stanford‚ CA Bobrov RA (1952) The effect of smog on the anatomy of oat leaves. Phytopath 42: 552–563 Bowling DJF and Smith GN (1990) Apoplastic transport in the leaf epidemis in relation to stomatal activity. Biochem Physiol Pflanzen 186:309–316 Brauer M‚ Sanders D and Stitt M (1990) Regulation of photosynthetic sucrose synthesis: a role for calcium. Planta 182: 236–243 Bush DS (1995) Calcium regulation in plant cells and its role in signaling. Annu Rev Plant Physiol Plant Mol Biol 46: 95–122 Castillo FJ and Heath RL (1990) transport in membrane vesicles from pinto bean leaves and its alteration after ozone exposure. Plant Physiol 94: 788–795 Castillo FJ‚ Miller PR and Greppin H (1987) Extracellular biochemical markers of photochemical oxidant air pollution damage to Norway spruce. Experientia 43: 1 1 1 – 1 1 5 Chameides WL (1989) The Chemistry of ozone deposition to plant leaves: Role of ascorbic acid. Environ Sci Tech 23: 595– 600 Chimilkis PE and Heath RL (1975) Ozone-induced loss of intracellular potassium ion from Chlorella sorokiniana. Plant Physiol 56: 723–727 Clark JA (1975) Heat and mass transfer from real and model leaves. In: deVries DA and Afgans NH (eds) Heat and Mass Transfer in the Biosphere I: Transfer Processes in the Plant Environment‚ pp 413–422. John Wiley and Sons‚ NY Cooley DR and Manning WJ (1987) The impact of ozone on assimilate partitioning in plants: A review. Environ Poll 47: 95–113 Coulson C and Heath RL (1974) Inhibition of the photosynthetic capacity of isolated chloroplasts by ozone. Plant Physiol 53: 32–38 Dann MS and Pell EJ (1989) Decline of activity and quantity of ribulose bisphosphate carboxylase/oxygenase and net photosynthesis in potato foliage. Plant Physiol 91: 427–432 Darrall NM (1989) The effect of air pollutants on physiological processes in plants. Plant Cell Environ 12: 1–30 Davison AW‚ Barnes JD and Renner CJ (1988) Interactions between air pollutants and cold stress. In: Schulte-Hosted S‚ Blank L‚ Darrall N and Wellburn AW (eds). Air Pollution and Plant Metabolism‚ pp 307–328. De Gruyter‚ Berlin DeKoning H W and Jegier Z (1968) Quantitative relation between ozone concentration and reduction of photosynthesis of Euglena gracilis. Atmos Environ 2: 615–616 Dijak M and Ormrod DP (1982) Some physiological and anatomical characteristics associated with differential ozone sensitivity among pea cultivars. Envir Exp Bot 22: 395–402

Robert L. Heath Dominy PJ and Heath RL (1985) Inhibition of the ATPase of the plasinalemma of pinto bean leaves by ozone. Plant Physiol 77: 43–45 Ecker JR and Davis RW (1987) Plant defense genes are regulated by ethylene. Proc Natl Acad Sci USA 84: 5202–5206 Eguchi H and Kitano M (1986) Transpiration responding to light conditions in controlled environments—Effects of infra red radiation. Biotronics 15: 37-14 Elkiey T and Ormrod DP (1979) Ozone and/or sulphur dioxide effects on tissue permeability of petunia leaves. Atmos Environ. 13: 1165–1168 Ellenson JL and Raba RM (1983) Gas exchange and phytoluminography of single red kidney bean leaves during periods of induced stomatal oscillation. Plant Physiol 72: 90–95 Erickson L and Wedding RT (1956) Effects of ozonated hexene on photosynthesis and respiration of Lemna minor. Am .J Bot 43:32–36 Farage PK‚ Long SP‚ Lechner EG and Baker NR (1991) The sequence of changes within the photosynthetic apparatus of wheat following short term exposure to ozone. Plant Physiol 95: 529–535 Farquhar GD and Sharkey TD (1982) Stomatal conductance and photosynthesis. Annu Rev Plant Physiol 33: 317–345 Flügge U-I and Heldt HW (1991) Metabolite translocators of the chloroplast envelope. A n n u Rev Plant Physiol Plant Mol Biol 42:129–144 Frederick PE and Heath R L ( 1975) Ozone-induced fatty acid and viability changes in Chlorella‚ Plant Physiol 55: 15–19 Freer-Smith PH and Dobson MC (1989) Ozone flux to Picea sitchensis (Bong) Carr and Picea abies (L) Karst during short episodes and the effect of these on transpiration and photosynthesis. Environ Poll 59: 161–176 Gerhardt R‚ Stitt M and Heldt H W (1987) Subcellular metabolite levels in spinach leaves: Regulation of sucrose synthesis during diurnal alterations in photosynthetic partitioning. Plant Physiol 83: 399–407 Grimes HD‚ Perkins KK and Boss WF (1983) Ozone degrades into hydroxyl radical under physiological conditions. Plant Physiol 72: 1016–1020 Guehl J-M and Aussenac G (1987) Photosynthesis decrease and stomatal control of gas exchange in Abies alba Mill. in response to vapor pressure difference. Plant Physiol. 83: 316–322 Gunderson CA and Taylor Jr GE (1988) Kinetics of inhibition of foliar gas exchange by exogenous ethylene: An ultrasensitive response. New Phytol. 110: 517–524 Gunderson CA and Taylor Jr GE (1991) Ethylene directly inhibits foliar exchange in Glycine max. Plant Physiol. 95: 337–339 Guzy MR and Heath RL (1993) Response to ozone of varieties of common bean (Phaseolus vulgaris L.) New Phytol. 124: 617– 625 Harris MJ and Bailcy-Scrres J (1994) Ozone effects on gene expression and molecular approaches to breeding for air pollution resistance. In: Basra AS (ed) Stress-Induced Gene Expression in Plants‚ pp 185–207. Harwood Academic Press‚ Switzerland Heagle AS (1989) Ozone and crop yield. Annu Rev Phytopath 27: 397–423 Heath RL (1975) Ozone. In: Mudd JB and Kozlowski TT (eds) Responses of Plants to Air Pollution‚ pp 23–96. Academic Press‚ NY Heath RL (1978) The reaction stoichiometry between ozone and

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432 McCool PM (1988) Effects of Air Pollutants on Mycorrhizae. In: Schulte-Hostede S.‚ Darrall NM‚ Blank LW and Wellburn AR (eds) Air Pollution and Plant Metabolism‚ pp 356–365. Elsevier Applied Science‚ London McCool PM‚ Musselmann RC‚ Younglove T and Teso RR (1988) Response of kidney bean to sequential ozone exposures. Environ Exp Bot 28: 307–313 McLeod AR and Baker CK (1988) The use of open field systems to assess yield response to gaseous pollutants. In: Heck WW Taylor OC and Tingey DT (eds) Assessment of Crop Loss from Air Pollutants‚ pp 181–10. Elsevier Applied‚ New York Mehlhorn H and Wellburn AR (1987) Stress ethylene formation determines plant sensitivity to ozone. Nature 327: 417–418 Mehlhorn H‚ O’Shea JM and Wellburn R (1991) Atmospheric ozone interacts with stress ethylene formation by plants to cause visible plant injury. J Exp Bot 42: 17–24 Mehlhorn H.‚ Tabnes BJ and Wellburn AR (1990) Electron spin resonance evidence for the formation of free radicals in plants exposed to ozone. Physiol Plant 79: 377–383 Miller AJ and Sanders D (1987) Depletion of cytosolic free calcium induced by photosynthesis. Nature 326: 397–400 Miyake H.‚ Furakawa A‚ Totsuka T and Maede E (1984) Differential effects of ozone and on the fine structure of spinach leaf cells. New Phytol 96: 215–228 Miyake H‚ Matsumura H‚ Fujinuma Y and Totsuka T (1989) Effects of low concentration of ozone on the fine structure of radish leaves. New Phytol 1 1 1 : 187–195 Moldau H‚ Sober J and Sober A (1990) Differential sensitivity of stomata and mesophyll to sudden exposure of bean shoots to ozone. Photosynthetica 24: 446–458 Monteith JL and Unsworth MH (1990) Principles of Environmental Physics. Edward Arnold‚ London Mudd JB (1973) Biochemical effects of some air Pollutants and Adv Chem 122: 31–47 Mudd JB‚ Leavitt R‚ Ongun A and McManus TT (1969) Reaction of ozone with amino acids and proteins. Atmos Environ 3: 669–682 Mudd‚ JB (1996) Biochemical basis for the toxicity of ozone. In: Iqbal M and Ynus M (eds) Plant Response to Air Pollution‚ pp 267–283. John Wiley & Sons Ltd‚ London Nie G-Y‚ Tomasevic M and Baker NR (1993) Effects of ozone on the photosynthetic apparatus and leaf proteins during leaf development in wheat. Plant Cell Environ 16: 643–651 Nobel PS‚ Wang C and Antenill F (1973) Ozone increased permeability of isolated pea chloroplasts. Arch Biochem Biophys 157: 388–394 Olszyk DM‚ Dawson PJ‚ Morrison CL and Takemoto BK (1990) Plant response to nonfiltered air vs added ozone generated from dry air or oxygen. J Air Waste Management Assoc 40: 77–81 Omasa K and Aiga I (1987) Environmental measurement: Image instrumentation for evaluating pollution effects on plants. In: Singh MG (ed) Systems and Control Encyclopedia: Theory‚ Technology‚ Applications‚ Vol 2‚ pp 1516–1522. Pergamon Press‚ Oxford Omasa K‚ Abo F‚ Hasimoto Y and Aiga I (1980) Measurement of the thermal pattern of plant leaves under fumigation with air pollutant. Res Report Natl Inst Environ Stud (Japan) 11: 239– 248 Ordin L‚ Hall MA and Kindinger JI (1969) Oxidant induced inhibition of enzymes involved in cell wall polysaccharide

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Chapter 18 Ultraviolet-B Radiation and Photosynthesis Alan H. Teramura College of Natural Sciences, University of Hawaii, Honolulu, HI 96822, USA

Lewis H. Ziska Climate Stress Laboratory, USDA/ARS, Beltsville, MD 20705, USA

Summary I. Introduction II. Penetration of UV-B Radiation III. Direct Effects of UV-B Radiation on the Light Reaction of Photosynthesis A. Photosystem I B. Photosystem II IV. Direct Effects of UV-B Radiation on Carbon Reduction A. Stomatal Limitation B. Ribulose 1,5-bisphosphate Carboxylase/Oxygenase Activity C. Ribulose 1,5-bisphosphate Carboxylase/Oxygenase Content V. Direct Effects of UV-B Radiation on Carbon Oxidation VI. UV-B Induced Changes in Leaf Development A. Photosynthetic Pigments B. Stomata C. Leaf Morphology VII. Changes in Plant Growth and Development with UV-B Radiation A. Hormonal Changes B. Morphology VIII. Protection and Repair of Photosynthesis A. Flavonoid Production B. Polyamines C. Oxygen Radicals and Metabolites D. Photolyase and Photoreactivation E. Photosystem II Repair IX. Future Research Priorities Acknowledgments References

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Summary It has long been recognized that declining levels of stratospheric ozone and the subsequent increase in ultraviolet (UV)-B radiation may deleteriously affect plant photosynthesis. However, there is a wide range of susceptibility in the photosynthetic apparatus with respect to increasing UV-B radiation. Differences in photosynthetic sensitivity to UV-B radiation may be based both upon biological factors related to leaf optical properties and repair and protective mechanisms as well as physical factors related to experimental conditions, especially the quantity and quality of background radiation. Potentially, there can be a number of direct and Neil R. Baker (ed): Photosynthesis and the Environment, pp. 435-450. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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indirect consequences of UV-B radiation for photosynthesis. Direct effects may include a decrease in the efficiency of the light harvesting reactions with the D1 protein of the Photosystem II reaction center being particularly sensitive. In addition, photosynthetic carbon reduction may also be sensitive, with UV-B radiation having a direct effect on ribulose 1,5-bisphosphate carboxylase/oxygenase activity and content. There are also a number of indirect effects of UV-B radiation on leaf and whole plant development which may indirectly influence photosynthesis. These include changes in leaf photosynthetic pigments, stomatal conductance and changes in leaf and canopy structure. Although our knowledge concerning the impact of UV-B radiation is increasing, several gaps exist in our present understanding of how photosynthesis is affected by UV-B radiation. Despite these gaps, data are sufficient to suggest that a reduction in photosynthetic capacity may be an important consideration in the carbon dynamics of plants as stratospheric ozone continues to decline. Suggestions for additional research that would help elucidate specific changes in the photosynthetic apparatus upon UV-B radiation exposure are presented.

I. Introduction Biotic and abiotic limitations to maximum photosynthetic capacity occur for all plants grown in situ at some stage in their life cycle. Perturbations of maximal photosynthesis usually are a result of natural environmental limitations imposed through changes in light, temperature, water, nutrient supply, herbivory, etc., which reduce potential productivity for a given plant in a specific environment. Recently, however, there have been anthropogenic increases in certain trace gases which may impose an additional limitation on photosynthetic capacity in plants. The rise in chlorofluorocarbons (CFCs), methane and nitrous oxide may lead to significant reductions in global stratospheric ozone. A decrease in stratospheric ozone would result in an increase in ultraviolet-B radiation (UV-B, 290–320 nm) reaching the surface of the earth. Plants by necessity require light for photosynthesis and therefore must be subject to UV-B radiation exposure. Although only a small fraction of the total electromagnetic spectrum, UV-B radiation is sufficiently actinic to evoke a range of direct and indirect limitations on photosynthetic capacity. These limitations can occur at a number of functional levels, involving changes in macroAbbreviations: CFCs–chlorofluorocarbons; DCPIP–2,6-dichlorophenolindophenol; IAA – indole acetic acid; P680 – primary chlorophyll a electron donor in the PS II reaction center; PAR – photosynthetically-active radiation between 400 and 700 nm; PEP – phosphoenolpyruvate; – primary quinone electron acceptor of PS II; – secondary quinone acceptor of PS II; Rubisco – ribulose 1,5-bisphosphate carboxylase/oxygenase; RuBP – ribulose 1,5-bisphosphate; SLW – specific leaf weight; UV-A – ultraviolet-A radiation between 320 and 400 nm; UV-B – ultraviolet-B radiation between 290 and 320 nm; UV-C – ultravioIet-C radiation between 200 and 290 nm

molecules such as protein and DNA to changes in leaf and canopy development. As a result, the entire photosynthetic process, i.e. the capturing of light energy in order to oxidize water and reduce to the level of carbohydrate, will be affected accordingly. It is also clear that there is a wide range of intraand interspecific sensitivity to photosynthesis with increases in UV-B radiation (Bogenrieder and Klein, 1982; Teramura and Murali, 1987; Reed et al., 1992; Ziska et al., 1992). Data on the potential sensitivity of photosynthesis and/or biomass to UV-B radiation over the last 20 years indicate that approximately one-half of all plant species are negatively affected by increased UV-B radiation (Caldwell et al., 1989; Tevini and Teramura, 1989; Teramura, 1990). However, some species demonstrate no effect of extremely high doses of UV-B radiation on photosynthesis, indicating that some plants are welladapted to high UV-B radiation environments. Generalizations regarding the consequences of ozone depletion/UV-B radiation upon global productivity are difficult to develop because representatives from only about half the major terrestrial ecosystems have been studied to date. In turn, most of these plants have been annual agricultural species, and we are lacking sufficient data to determine if native perennial plants will respond similarly to increased UV-B radiation. In addition, many studies are derived from experiments using low levels of photosynthetically-active radiation (PAR) in combination with unrealistically high levels of UV-B radiation. Although not useful for predicting the consequences of stratospheric ozone depletion on plant productivity, this work is still relevant with respect to identifying key targets in the photosynthetic process which may be affected by UV-B radiation. Additional field experiments using natural levels of

Chapter 18 Ultraviolet-B Radiation white light, as well as the UV-A (320–400 nm)/blue light necessary for photorepair (Ahmad and Cashmore, 1993; Christopher and Mullet, 1994) and for flavonoid biosynthesis (Beggs and Wellmann, 1994), may be required to determine a more ecologically relevant picture of the effects of UV-B radiation on photosynthetic capacity. Although UV-B radiation can affect a number of processes at the molecular level with respect to nucleic acids, proteins, lipids, pigments and phytohormones (Teramura et al., 1983; Tevini and Teramura, 1989; Bornman and Teramura, 1993), our focus in this current assessment is on those specific physiological changes which limit photosynthetic capacity with plant exposure to UV-B radiation. Changes in physiology that can occur with increased UV-B radiation include both direct mechanistic damage to the photosynthetic apparatus and indirect changes in growth and morphology which may reduce light interception and competitiveness. We are also concerned with identifying specific adaptive processes that can confer photosynthetic protection from UV-B radiation. Such an assessment of our understanding of photosynthetic sensitivity to UV-B radiation may provide insight into potential reductions in photosynthesis and global productivity with future decreases in stratospheric ozone.

II. Penetration of UV-B Radiation The epidermis forms the first effective barrier against the penetration of UV-B radiation into the leaf. In general, the leaf epidermis is very effective in transmitting a large portion of PAR while limiting the amount of UV-B radiation. This effectiveness varies depending upon leaf thickness, the presence of UV-B absorbing compounds and leaf surface properties. For example, transmittance of UV-B radiation is less than 10% in Peperomia obtusifolia and Yucca treculeana, but was over 90% in the epidermis of onion, Allium cepa (Gausman et al., 1975). Overall, in a wide range of species tested, it has been estimated that UV-B absorbing pigments within the leaf epidermis attenuate 20–75% of the incoming UV radiation (Robberecht and Caldwell, 1978). The use of fiber optic microprobes within plant tissue provides a useful tool for obtaining in situ spectral data on leaf tissue (Bornman and Vogelmann, 1988; Cen and Bornman, 1993). Once placed within

437 the leaf, the fiber can also be used as a directional sensor, measuring both collimated and diffuse radiation by varying the angle of insertion. Penetration and internal distribution of UV-B radiation varies among plant species, and is strongly affected not only by epidermal thickness but by leaf anatomy, pigments and other physiological changes which result from exposure to UV-B radiation. In a study by Day et al. (1992) in which twenty two plant species were examined, UV-B penetrated deepest into leaves of herbaceous dicotyledons, with intermediate penetration into woody monocotyledons and grasses and almost no penetration into conifer needles. In most plants, UV-B screening pigments would be predominately located toward the adaxial surface in horizontal leaves (Bornman and Teramura, 1993). Selective filtering of UV-B radiation in leaves suggests that most plants in their natural environments probably do not suffer reduced photosynthetic capacity as a result of UV-B radiation (Caldwell et al., 1983). However, selective filtration is imperfect as evidenced by the wide range of studies showing photosynthetic inhibition with increased UV-B radiation (Tevini and Teramura, 1989). Even in conifers where UV-B attenuation is high, UV-B radiation may reduce photosynthesis during the period shortly following needle elongation and emergence past the highly UV-B protective bud scales (DeLucia et al., 1992; Sullivan, 1994). In addition, it is also possible that despite protective mechanisms, microsites of relatively high levels of UV-B radiation occur because of uneven distribution of screening pigments. Hence, if additional UV-B radiation occurs as a result of ozone depletion this increased radiation may have a deleterious effect on a number of important targets relevant to the photosynthetic process. Some important targets of UV-B radiation would include nucleic acids, proteins, and membrane lipids. Damage to these key elements will reflect a direct alteration in a number of key photosynthetic reactions.

III. Direct Effects of UV-B Radiation on the Light Reaction of Photosynthesis Plants (and cyanobacteria) possess two reaction center complexes PS I and PS II connected in series. The purpose of these complexes is to capture light energy for ultimate conversion and storage in chemical bonds. The light energy drives non-cyclic electron transport and results in oxidation of water with the release of

438 molecular oxygen and the production of reductants, such as NADPH. ATP is generated as a consequence of photosynthetic electron transport as the proton electrochemical gradient, generated across the thylakoid membranes as a result of electron transport, is dissipated by a proton-translocating ATPase. The ATP and reductants are then used in carbon reduction. Overall effects of UV-B irradiation on the light harvesting and photochemical apparatus include ultrastructural damage to chloroplasts (Brandle et al., 1977; Allen et al., 1978) changes in the exciton transfer between the antenna pigments of the different reaction centers (Renger et al., 1986) and changes in photosynthetic pigments, especially chlorophyll (Teramura and Caldwell, 1981;Vu et al., 1981; 1982b;

Alan H. Teramura and Lewis H. Ziska Strid et al., 1990; Strid and Porra, 1992; Deckmyn and Impens, 1994). The end result is a reduction in the photosynthetic response to light, with wide differences among species, ecotypes and cultivars in their sensitivity to UV-B radiation (Fig. 1). The specific effects of UV-B radiation on each photosystem are discussed in the following sections.

A. Photosystem I The composition of PS I and PS II suggests that UV radiation would be equally effective in inducing changes in both systems; however, this appears not to be the case (Bornman and Teramura, 1993). Although PS I can be inhibited when chloroplasts are irradiated

Chapter 18 Ultraviolet-B Radiation with intensive UV-C radiation (200–290 nm), such radiation is not present in the natural environment and there appears to be only a weak effect of UV-B radiation on PS I, relative to PS II, in most cases. For example, in chloroplast preparations from pea (Pisum sativum), collard (Brassica oleracad) and peanut (Arachis hypogea), a UV-B dose which completely inhibited PS II only decreased cyclic photophosphorylation through PS I by 35% (Van et al., 1977). From this the authors concluded that the ability of ascorbate-reduced 2,6-dichlorophenolindophenol (DCPIP) to restore the electron transport capacity of UV-B irradiated materials indicates that inhibition of photosynthesis was more closely associated with PS II than PS I (Van et al., 1977). In a similar experiment, PS I and cytochrome content decreased by 58% while PS II activity was reduced by 80% on a leaf area basis in pea (Strid et al., 1990). Interestingly, for this same experiment, the contents of PS I and cytochrome were unaffected when expressed on a chlorophyll basis (Strid et al., 1990). This is not to say that UV-B radiation does not inhibit PS I activity. In isolated chloroplasts from pea, although most of the inhibition of electron transport was associated with PS II, electron transport was still reduced by 12% when DCPIP with ascorbate was added to the reaction vessel as an electron donor to PS I (Brandle et al., 1977). UV-B radiation can induce changes in membrane integrity (Bornman et al., 1983; Murphy 1983) which could potentially affect PS I activity. However, to date no direct evidence for such an effect has been demonstrated and the influence of UV-B radiation on PS I is currently thought to be minor.

B. Photosystem II The core of the reaction center of PS II contains two membrane proteins with molecular masses of 32 and 34 kDa known as D1 and D2, respectively. P680, chlorophyll a, pheophytin and plastoquinone are bound to these membrane proteins (Barber, 1987). In chloroplast studies, PS II is extremely vulnerable to UV-B radiation with the pigments, cofactors and proteins being modified (Noorudeen and Kulandaivelu, 1982; Renger et al., 1989; Greenberg et al., 1989a; Jansen et al., 1993; Wilson and Greenberg, 1993; Barbato et al., 1995). The reaction center itself appears to be damaged by enhanced UV-B radiation (Iwanzik et al., 1983), both with respect to its ability to oxidize water and reduce plastoquinone (see Chapter 4).

439 The oxidizing side of PS II, i.e. the water splitting reaction, has been shown to be inhibited in a number of studies (Noorudeen and Kulandaivelu, 1982; Renger et al., 1989). Modification of the tyrosine residue of the D1 protein which facilitates transfers of the electrons from water to P680 has been implicated as well as a direct damage to the D1 and D2 proteins within the reaction centers (Renger et al., 1989; Greenburg et al., 1989a; Melis et al., 1992; Barbato et al., 1995). Recently, Barbato et al. (1995) have found that the Mn ions associated with the water-splitting system are required for UV-B-induced cleavage of the D1 protein into a 20 kDa and a 13 kDa C-terminal and N-terminal fragment pair. Renger et al. (1989) concluded that the primary effect of UVB radiation on PS II was a severe deterioration in the water oxidation mechanism. The D1 protein of the reaction center complex appears particularly sensitive, with UV-B radiation inducing rapid D1 degradation (Greenberg et al., 1989a; Wilson and Greenberg, 1993; see Chapter 4). Degradation of the D1 protein induced by UV-B radiation produces a 23.5 kDa polypeptide primary breakdown product (Greenberg et al., 1989b) which resembles the breakdownproduct found to be associated with exposure to the visible and far-red spectrum. UV-B driven, D1 protein degradation is believed to occur via the plastosemiquinone anion a reactive species that is formed upon exposure to UV-B (Jansen et al., 1993). The primary D1 cleavage site resides near the plastoquinone binding niche. The acceptor or reducing side of the D1 and D2 proteins can also be modified by UV-B radiation with a subsequent change in the number and activity of the quinone binding sites (Renger et al., 1989). Specifically, it has been suggested that UV irradiation primarily modifies the binding sides of the primary and secondary quinone electron acceptors (QA, QB) on the PS II acceptor side with a simultaneous blocking of pheophytin, the primary electron acceptor (Renger et al., 1986). It has been suggested that this photoreceptor is plastoquinone bound to the quinone niche of the D1 polypeptide (Jansen et al., 1993) possibly a semiquinone anion, while chlorophyll and carotenoids are photoreceptors in the visible and farred regions (Greenberg et al., 1989a). In a confirming study, high levels of UV-B radiation also inhibited formation of the semiquinone anion and decreased the overall photoreduction of plastoquinone (Melis et al., 1992). It was concluded that UV-B damaged the ability to reduce both associated with PS II and also the free plastoquinone pool

440 (Melis et al., 1992). UV-B radiation also decreases fluorescence decay, with the fast components accelerated and the slow components retarded suggesting the formation of additional quenchers of exciton energy (Renger et al., 1991). It has been suggested that UV-B radiation also causes direct damage to the plastoquinone molecule itself (Bornman and Teramura 1993). Plastoquinone with its many forms and oxidized or reduced states, may act as a primary UV-B photosensitive molecule (Melis et al., 1992). This seems likely since quinone, semiquinone anion and the quinol (the three redox states of plastoquinone) all absorb to some extent in the UV region. It is not clear how much of the reduction in PS II activity is due to increased nucleic acid damage with supplemental UV-B radiation. In pea leaves exposed for 3 days to UV-B radiation a substantial (>50%) reduction in chloroplast encoded psbA transcript levels, i.e. the mRNA of chloroplast-encoded D1 protein of the PS II reaction center, was observed (Jordan et al., 1991). Results from this study suggest rapid changes in the regulation of gene expression with increased UV-B radiation. However, much of the detail concerning this regulation still remains unknown. Given that supplementary UV-B radiation may decrease the activity and content of the PS II complex with a resulting decrease in electron transport, and presumably ATP synthesis, there could be corresponding decreases in photosynthetic capacity and maximum quantum yield (e.g. Strid et al., 1990; Ziska et al., 1992). However, caution should be exercised in such an assumption since inactivation of a fraction of PS II complexes could reduce the maximum quantum yield without having a significant affect on photosynthetic capacity. Also it should be noted that pea leaves exposed to UV-B exhibited large decreases in photosynthetic capacity in the absence of any damage to PS II (Nogués and Baker, 1995). Although our knowledge concerning the impact of UV-B radiation on the capture and processing of light energy is increasing, much of the underlying mechanism is still unknown. Areas which deserve scrutiny include the impact of UV-B radiation on the functioning of the cytochrome complex and the establishment of a pH gradient across the thylakoid membrane. In addition, while plastoquinone may be sensitive to UV-B radiation, changes in the diffusible intermediates between PS I and II (presumably plastoquinone and plastocyanin, which

Alan H. Teramura and Lewis H. Ziska shuttle electrons between PS II and PS I), remain largely unknown.

IV. Direct Effects of UV-B Radiation on Carbon Reduction

A. Stomatal Limitation It might be expected that UV-B radiation could influence carbon reduction by controlling the diffusion of to the site of carboxylation. However, those studies which have examined the impact of UV-B radiation on photosynthesis when the stomatal limitation to diffusion is zero, i.e. at supersaturating concentrations of still demonstrate a reduction in photosynthetic capacity (Ziska et al., 1992; Middleton and Teramura, 1994). This indicates a direct inhibition to photosynthesis not related to stomatal limitation. Additional evidence for a direct effect of UV-B radiation is given by Naidu et al. (1993) and Sullivan (1994), who demonstrated through carbon isotope discrimination a higher internal concentration within UV-B irradiated loblolly pine (Pinus taeda) needles. This suggests that in the absence of direct changes in stomatal conductance, UV-B radiation induces a chronic reduction in photosynthetic capacity. These changes may occur at a number of different sites within the photosynthetic carbon reduction cycle (Fig. 2).

B. Ribulose 1,5-bisphosphate Carboxylase/ Oxygenase Activity Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in all plant leaves and usually constitutes 30–50% of the total soluble protein. A number of studies have demonstrated an inverse correlation between increasing UV-B radiation and Rubisco activity. Van et al. (1977) showed that UV-B radiation reduced Rubisco activity in irradiated leaf discs and chloroplast preparations. Later studies also demonstrated that increases in UV-B levels corresponding to a 6, 21 and 36% decrease in stratospheric ozone (at a latitude of 60°) reduced Rubisco activity in whole leaves of 4 week old soybean and pea, and 8 week old tomato (Lycopersicon esculentum) in shaded greenhouse experiments (Vu et al., 1982a,b). Similarly, the maximum Rubisco activity was reduced in mature leaves of pea after 8 days of

Chapter 18 Ultraviolet-B Radiation

UV-B exposure (Strid et al., 1990). Levels of PEP carboxylase were also reduced in corn (Zea mays) a C4 species, but only at very high UV-B radiation levels (Vu et al., 1982a). Decreases in Rubisco activity can also be implied from other studies which show a decrease in the initial slope of the relationship between assimilation and internal with supplemental UV-B radiation (Ziska and Teramura, 1992).

C. Ribulose 1,5-bisphosphate Carboxylase/ Oxygenase Content Ribulose 1,5-bisphosphate carboxylase/oxygenase requires ATP and NADPH produced from the light reactions in order to reduce to carbohydrate. Consequently, it is not surprising to observe reductions in Rubisco activity with supplemental UV-B radiation. Such reductions could arise because of the decrease in RuBP (substrate) regeneration capacity as ATP and NADPH become limited (Strid et al., 1990; Sullivan and Teramura, 1990), although large UV-B-induced depressions in photosynthesis in pea leaves were not accompanied by any decreases in the maximum photochemical efficiency of PS II (Nogués and Baker, 1995). It has been observed that UV-B radiation can reduce leaf protein content (Vu

441 et al., 1982a) suggesting that UV-B radiation can also directly reduce the concentration of Rubisco within the leaf. Direct confirmation of this has been shown in other studies. For example, Vu et al. (1984) found that UV-B irradiance corresponding to a 36% depletion in stratospheric ozone reduced both Rubisco activity and the amount of Rubisco present. Details concerning how UV-B radiation may directly decrease the content of Rubisco are emerging. In a study by Jordan et al. (1992), the decline in maximum Rubisco activity (71% relative to the controls) was accompanied by a corresponding decline in Rubisco polypeptide subunits (56%) after 3 days of exposure to UV-B radiation (simulating a 2.5-fold increase in current ambient levels). Interestingly, the level of mRNA transcripts which code for the small (rbcS) and large (rbcL) subunits of Rubisco declined dramatically within hours of UV-B exposure, with rbcS reduced to 20% of the control value. This decrease in rbcS mRNA can be partially ameliorated if higher PAR values are used, suggesting that the effect of UV-B radiation on mRNA transcripts is reversible (Jordan et al., 1992). As with the effects of UV-B radiation on the psbA gene encoding of the chloroplast D1 protein, it is clear that rapid changes in the regulation of gene expression can occur with UV-B radiation.

V. Direct Effects of UV-B Radiation on Carbon Oxidation Little information is available concerning the impact on how UV-B radiation may affect respiratory metabolism. It has been suggested that observed increases in dark respiration may represent additional energy needed for repair(Ziska et al., 1992). Increases in dark respiration have also been observed in other crops such as pea (Brandle et al., 1977) and Rumex patentia (Sisson and Caldwell, 1976). However, other data from soybean indicated that no changes in dark respiration were observed with increased UV-B radiation over a PAR range (Teramura, 1980). In addition, no conclusive data are available concerning the impact of UV-B radiation on photorespiration (Teramura, 1983). Given the scarcity of information it is difficult to determine what impact increased UV-B radiation levels will have on carbon loss via respiration. Clearly, additional research is required in this area.

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VI. UV-B Induced Changes in Leaf Development In addition to direct effects on the photosynthetic electron transport reactions and/or carbon reduction, there are a number of morphological/developmental changes which occur at the level of the leaf which may also indirectly affect photosynthetic capacity.

A. Photosynthetic Pigments UV-B radiation may affect photosynthesis indirectly by photobleaching and photodegradation of photosynthetic pigments (Strid and Porra, 1992). High levels of UV-B radiation in combination with low levels of PAR have significantly reduced chlorophyll content in bean (Garrard et al., 1976; Tevini et al., 1981) barley and corn (Tevini et al., 1981) pea (Vu et al., 1984) and soybean (Vu et al., 1981, 1982b, 1984). However, increases as well as decreases in photosynthetic pigments have been observed with increased UV-B radiation (Murali et al., 1988; Panagopoulos et al., 1992; Middleton and Teramura, 1994). Increases in the concentration of primary pigments, e.g. chlorophylls, as well as secondary pigments, e.g. carotenoids, may account for some reports of increased photosynthesis on a leaf area basis. Nevertheless, with respect to chlorophyll light harvesting and exciton transfer efficiency, UV-B radiation still can reduce photosynthetic capacity (Teramura et al., 199 l;Teramura and Sullivan, 1994). This suggests that leaves which show photosynthetic insensitivity to UV-B radiation may still be responding through changes in the synthesis of photosynthetic pigments.

B. Stomata Although the effect of UV-B radiation on carbon reduction is not directly mediated by the diffusion of through the leaf, supplemental UV-B can indirectly limit photosynthetic capacity by inducing stomatal closure. It has been proposed that different UV-B levels can induce stomatal closure directly by inhibiting accumulation by guard cells (Wright and Murphy, 1982). Teramura et al. (1980) demonstrated that stomatal conductance decreased after a 2 week exposure to relatively low levels of UV-B radiation. Similarly, UV-B radiation has been found to induce stomatal closure in bean, soybean and cucumber (Bennett, 1981). The response in

Alan H. Teramura and Lewis H. Ziska cucumber may be especially sensitive. After 8–9 days of UV-B exposure cucumber was observed to lose all stomatal function (Teramura et al., 1983). However, the response of stomata to UV-B radiation may be dependent upon prevailing environmental conditions. Stomata close upon direct exposure to UV-B, but if strong white light is used, stomata can re-open rapidly (Negash and Björn, 1986; Negash, 1987). UV-B radiation in combination with low PAR appears to have the greatest impact on stomatal closure (Mirecki and Teramura, 1984). In addition to light, water stress may confound the influence of UV-B radiation on stomatal function. Under wellwatered conditions, UV-B may induce stomatal closure but no effect was observed in soybean when leaves were drought-stressed (Sullivan and Teramura, 1990). Indirect effects of UV-B radiation on stomata may also involve changes in stomata number or density. For example, UV-B induced reductions in leaf area and increases in leaf thickness may also reduce the number of stomata per unit leaf area (Tevini et al., 1986).

C. Leaf Morphology UV-B irradiance can also indirectly reduce photosynthetic capacity by reducing leaf area with a subsequent decrease in light interception. Leaf area appears to be a particularly sensitive parameter to increases in UV-B radiation. Reductions in leaf area have been recorded for such diverse crops as rice (Teramura et al., 1991), sunflower (Tevini and Teramura, 1989), rhubarb (Rheum rhaponiticum) and brussels sprouts (Brassica oleracea) (Biggs and Kossuth, 1978). Supplemental UV-B radiation reduced leaf area in over 60% of the crops examined in a growth chamber study of 70 unrelated species and cultivars (Biggs and Kossuth, 1978). For the most sensitive plants, leaf expansion was reduced by as much as 70% (Biggs et al., 1981; Tevini et al., 1981). As with stomata however, the influence of UV-B levels varies in proportion to background PAR (Teramura, 1980). Growth at moderate levels of UVB irradiance and high PAR may have no effect on leaf area or may even stimulate leaf expansion (Teramura and Caldwell, 1981; Ziska et al., 1993). Nevertheless, even at moderate levels of UV-B radiation and high PAR, sensitive species still show a reduction in leaf area (Teramura et al., 1983). Leaves may also show increases in specific leaf

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Chapter 18 Ultraviolet-B Radiation weight (SLW, the ratio of leaf mass to area) in response to UV-B radiation. Leaf thickness could indirectly influence photosynthesis by increasing the path length for diffusion to the site of carboxylation. Changes in leaf thickness probably occur at the cellular level. For example, in bean leaves, pronounced elongation of the palisade parenchyma cells was observed with increased exposure to UV radiation (Cen and Bornman, 1990). However, increases in leaf thickness with UV-B radiation may also occur as a result of an increase in cell number. For example, leaves of Brassica carinata and Medicago sativa increased the number of spongy mesophyll cells while palisade cells increased in B. campestris (Bornman and Vogelmann, 1991). Changes in leaf thickness per se do not appear to be associated with additional photosynthetic resistance to UV-B radiation (Biggs and Kossuth, 1978). Surface characteristics of leaves may also change in response to UV-B radiation. Changes in these characteristics may enhance light reflectance and light scattering (Cameron, 1970) reducing the amount of light energy available for photosynthesis. UV-B radiation has been shown to alter the biosynthesis and composition of epicuticular waxes in a number of crop species (Steinmuller and Tevini, 1985; Tevini and Steinmuller, 1987). In these experiments, enhanced levels of UV-B radiation increased total wax approximately 25% in all species examined (Steinmuller and Tevini, 1985). In addition to effects on light scattering, changes in epicuticular wax may also reduce transpiration which may in turn alter the photosynthetic response to temperature and humidity. Leaf pubescence also alters light absorbance; however, to date we are unaware of any studies which have demonstrated a UV-B induced change in leaf pubescence.

VII. Changes in Plant Growth and Development with UV-B Radiation Morphological changes induced by UV-B radiation including growth reductions of some plant parts, internodes and stimulated growth of other parts, e.g. axillary leaves or shoots, can occur without any apparent inhibition of photosynthesis at the single leaf level. Yet, changes in these parameters can alter light interception and produce phenological change with respect to the whole plant. In so doing, they serve to indirectly limit photosynthetic capacity.

A. Hormonal Changes Changes in plant growth may be mediated, in part, by differential sensitivity to auxin as a result of exposure to enhanced UV-B radiation. Auxin, specifically indole acetic acid (IAA), is a plant hormone which can control apical dominance and leaf arrangement with subsequent effects on light interception by the plant. Changes in plant height and internode length may be attributable to UV-B induced photo-oxidation of IAA (Beggs et al., 1986). IAA absorbs in the UVB region and can be converted in vitro and in vivo to different photo-oxidation products (Tevini et al., 1989). Increased UV-B radiation has been shown to reduce the concentration of auxin in fronds of Spirodella oligorhiza (Wiztum et al., 1978) and in the hypocotyls of sunflower seedlings (Tevini and Teramura, 1989).

B. Morphology Changes in IAA as a result of UV-B radiation may produce a number of morphological changes in plant development. A decrease in internode length and subsequent plant stunting has been observed in many species, especially in seedlings (Ambler et al., 1975; Tevini et al., 1982). This stunting is accentuated at high UV-B irradiation and low PAR (Teramura, 1980). However, even at moderate UV-B levels (simulating a 20% decrease in stratospheric ozone at 40° N latitude), reductions in leaf blade and internode lengths and increased leaf and axillary shoot production have been observed in several plant species (Barnes et al., 1990). Similar levels of UV-B radiation also reduced the number of tillers in sensitive rice cultivars (Teramura et al., 1991). In field experiments, UV-B irradiation reduced needle elongation in loblolly pine, and these reductions in total needle area may contribute to a decrease in total carbon assimilation (Sullivan and Teramura, 1992). UV-B radiation under field conditions has also increased the number of axillary leaves produced in a tropical crop, cassava (Ziska et al., 1993). In model agroecosystems, subtle UV-induced changes in wheat and wild-oat significantly altered light interception on a whole plant basis (Ryel et al., 1990). One of the most common observations in response to increased UV-B radiation is a decrease in plant biomass (Teramura, 1983; Krupa and Kickert, 1989). Reductions in biomass often correspond to changes in assimilate partitioning between different plant

444 organs. Such changes have clear implications with respect to leaf development, light interception and whole plant photosynthesis. Generally, in dicotyledons, although absolute leaf area is reduced, a greater proportion of assimilate is partitioned into leaves and less in stems and roots (Teramura, 1983). In recent field studies with cassava, for example, total leaf area increased with UV-B radiation, but root biomass decreased 32% (Ziska et al., 1993). Monocotyledons may also show changes in growth form but changes in partitioning appear to be variable. For example, in greenhouse studies with rice, sensitive cultivars showed significant reductions in leaves, stems and roots with no one plant organ (e.g. leaves) having a greater sensitivity to UV-B radiation (Teramura et al., 1991).

VIII. Protection and Repair of Photosynthesis Plants absorb UV-B radiation as a consequence of utilizing PAR in photosynthesis. As a result plants have evolved a number of protective strategies which may minimize the impact of UV-B radiation. Variation in these protective mechanisms can help determine the degree of inter- and intraspecific differences in sensitivity of photosynthetic systems to UV-B radiation. In addition, knowledge of these protective mechanisms can be used as a physiological data base for improving tolerance to UV-B radiation with further depletions in stratospheric ozone. Changes in leaf anatomy (e.g. thickness) and pigmentation which have been previously discussed can certainly serve to protect the photosynthetic apparatus from excess UV-B radiation. However, these mechanisms usually limit the amount of PAR which is absorbed by the leaf and as a consequence can also limit the extent of photosynthesis. Here we wish to focus on repair mechanisms which limit UV-B induced damage without significantly affecting maximum photosynthetic capacity.

A. Flavonoid Production Flavonoids, a class of water-soluble phenolic derivatives, have been thought to be associated with protection from UV-B radiation for several decades (Jagger, 1967). Flavonoids absorb specifically in the UV-B region with maximum absorption at around 300 nm. The accumulation of epidermal flavonoids has been associated with reduced transmittance of

Alan H. Teramura and Lewis H. Ziska UV-B radiation in many plant species (Robberecht and Caldwell, 1978; Tevini et al., 1991). Anthocyanins, a special class of flavonoids, are thought to confer protection in young flushing shoots of tropical trees (Lee and Lowry, 1980) and have been induced specifically by UV-B radiation in parsley cell culture (Wellmann, 1974). Recent evidence by Li et al. (1993) has indicated that isolines of Arabidopsis mutants, which had reduced levels of flavonoids and monocyclic sinapic acid ester phenolic compounds, were highly sensitive to UV-B radiation damage in comparison to the wild genotypes. Flavonoids are present in leaves, pollen, petals, stems and bark, primarily within vacuoles and cell walls. Leaves usually form the first protective barrier against UV-B radiation and concentrations of flavonoids within the epidermis are correspondingly high, i.e. 1–10 mM (Vierstra et al., 1982). Induction of flavonoid biosynthesis with enhanced UV-B radiation may be regulated at the gene level (Kubasek et al., 1992; Beggs and Wellmann, 1994) since UV-B radiation increases the concentration of certain key enzymes of the flavonoid pathway (Schulze-Lefert et al., 1989). Principal enzymes of flavonoid biosynthesis induced by UV radiation are chalcone synthase, phenylalanine ammonia-lyase, chalcone-flavanone isomerase (Chappell and Hahlbrock, 1984) and 4-coumerate-CoA-ligase (Douglas et al., 1987). Available evidence indicates that a flavin may act as a possible photoreceptor (Ensminger and Schafer, 1992; Ahmad and Cashmore, 1993). In this study cells irradiated with UV-B radiation and visible light produced higher amounts of chalcone synthase and flavonoids than controls. The role of other compounds in flavonoid accumulation is unclear. Reduced glutathione as a signal for UV-induction of flavonoid biosynthesis has been suggested (Wingate et al. 1988), but additional investigation is required. Although UV-B radiation may induce flavonoid biosynthesis (Li et al., 1993; Beggs and Wellmann, 1994), several studies have shown that the damaging effects of UV-B radiation may not be entirely alleviated with a simple increase in the concentration of leaf flavonoids (Sisson, 1981; Mirecki and Teramura, 1984; Sullivan and Teramura, 1989). The photosynthetic apparatus of some plant species found in high elevations in tropical regions appears resistant to increased UV-B radiation (Barnes et al., 1987; Larson et al., 1990; Ziska et al., 1992) and this resistance does not appear to be always associated

Chapter 18 Ultraviolet-B Radiation with increases in flavonoid concentration (Barnes et al., 1987). However, for some species found in tropical locations, such as Oenothera stricta and Manihot esculentum, flavonoid concentrations may be intrinsically higher resulting in a subsequently greater degree of photoprotection, even if flavonoid levels are unaffected by UV-B radiation (Ziska et al., 1992). Currently, it is thought that flavonoid accumulation can contribute to UV-B tolerance and photosynthetic protection, but a simple cause and effect relationship may not always be present. Additional work focusing on qualitative as well as quantitative changes in flavonoids and other related compounds with supplemental UV-B radiation combined with photosynthesis measurements would be extremely useful in elucidating photo-protective mechanisms.

B. Polyamines The accumulation of polyamines is sometimes associated with a number of biotic and abiotic stresses, especially water stress. However, specific types of polyamines such as spermidine, spermine and putrescine may reduce lipid peroxidation and appear to be stimulated in plants exposed to increased UV-B radiation (Kramer et al., 1991). Consequently, polyamine levels may be a contributing factor with respect to photosynthetic integrity at high UV-B radiation (Kramer et al., 1992).

C. Oxygen Radicals and Metabolites Key components of the photosynthetic apparatus e.g. membrane lipids and proteins are susceptible to damage by oxygen species and free radicals. UV radiation, in turn, may increase the level of these free oxygen radicals. It is not surprising, therefore, that a number of different protective systems are present in plants which protect them from these various oxygen species. These include superoxide dismutase (SOD), (vitamin E), hydrogen peroxide, ascorbate, and glutathione (Bornman and Teramura, 1993; Chapter 5). Interestingly, flavonoids, in addition to being attenuators of UV-B radiation, may also play a key role as antioxidants, prohibiting oxygen-promoted redox reactions in the chloroplast (Takahama, 1983).

445 proposed by which cells cope with DNA damage produced by UV radiation exposure. These are photoreactivation, excision repair, recombinational filling of daughter-strand gaps and resynthesis of DNA past UV-induced lesions (Pang and Hays, 1991). Although there is some indirect evidence for recombinational repair in Chlamydomonas (Rosen et al., 1980), most research on the mechanism of DNA repair has focused on photoreactivation. It has been suggested that the most economic and errorfree repair mechanism of DNA damage is photoreactivation (Sutherland, 1981; McLennan, 1987). In photoreactivation a single enzyme, photolyase, uses energy obtained from light at short wavelengths (300–500 nm) to repair cyclobutane pyrimidine dimers produced by UV radiation (Sancan and Sancan, 1988; Pang and Hays, 1991). Photoreactivation appears to be the primary mechanism for dimer repair in Arabidopsis (Pang and Hays, 1991), and Scots pine (Pinus sylvestris) seedlings, but has also been observed in maize pollen (Ikenaga et al., 1974; Jackson, 1987), pinto bean sprouts (Saito and Werbin, 1969), wild carrots (Howland, 1975) and ginko cells (Trosko and Mansour, 1969). However, photoreactivation has not been detectable in all species studied (Saito and Werbin, 1969; McLennan, 1987). Part of the detectability of this protective mechanism may be dependent on temperature since photolyase activity appears to be extremely temperature sensitive (Pang and Hays, 1991). Recently, an Arabidopsis gene encoding a protein with characteristics of a photolyase and a blue light photoreceptor was cloned (Ahmad and Cashmore, 1993). Since photolyase depends on light energy in the blue and UV-A regions, this implies an important secondary role for blue light and UV-A light with respect to photorepair in terrestrial plants exposed to high UV-B radiation (Fernbach and Mohr, 1992). However, Quaite et al. (1992) have also shown that DNA dimerization can be induced by UV-A radiation. Although the entire solar spectrum may be important in influencing the degree of photosynthetic protection, we lack specific details concerning how the ratio of different fluences at specific wavebands alter the protective response.

E. Photosystem II Repair D. Photolyase and Photoreactivation There are four distinct mechanisms which have been

The D1 and D2 protein of the PS II reaction center are damaged by UV-B radiation (Greenberg et al.,

446 1989a; Melis et al., 1992). The resulting nonfunctional reaction centers undergo a complex repair mechanism to recover photosynthetic activity (Aro et al, 1993; Christopher and Mullet, 1994; Chapter 4). The repair process involves proteolytic cleavage and removal of the damaged Dl and D2 subunits, which are replaced with newly synthesized functional counterparts. Therefore, the subunits undergo enhanced turnover upon UV-B radiation exposure. The ability to increase the rate of turnover is considered to be an adaptive mechanism in response to damage; those plants that cannot efficiently replace damaged protein have reduced photosynthesis (Aro et al., 1993). Recently, it was shown that blue light// UV-A activate gene expression to sustain D2 protein synthesis which is damaged in plants exposed to high light and UV (Christopher and Mullet, 1994). Blue light-induced gene expression is proposed to ameliorate the damage caused by UV-B radiation. Hence, in addition to playing a role in activating DNA repair, blue light/UV-A could be involved in repair processes in general.

IX. Future Research Priorities In order to facilitate our understanding of how photosynthetic capacity is influenced by UV-B radiation, future research should focus on several key areas. For example, a detailed mechanistic understanding of UV-B induced changes at the molecular, biochemical and physiological level and their specific consequences in regard to the processing of light energy and the reduction of is still needed. Detailed action spectra in order to characterize photosynthetic response at different organization levels (e.g. gene, enzyme, membrane) would be very useful in this regard. Such spectra would prove invaluable as a tool for determining inter- and infra-specific variation in photosynthesis for a wide range of plant types. In addition, it would be extremely useful to obtain mechanistic details of the UV-B response in an ecologically relevant setting, i.e. a natural field condition under a full solar spectrum. It is quickly becoming apparent that the photosynthetic and growth response of plants to UV-B radiation is greatly altered as a function of background radiation (Adamse and Britz, 1992; Middleton and Teramura, 1994). A better understanding of the role of naturally occurring UVB radiation on photosynthesis could be achieved

Alan H. Teramura and Lewis H. Ziska through simple exclusion studies. Very little data exists in regard to UV-B radiation effects on natural plants or ecosystems (Gehrke et al., 1995; Johanson et al., 1995). Consequently, it is difficult to determine the full scope of the impact of UV-B radiation on global net photosynthesis. Lastly, future anthropogenic changes will not be confined solely to increases in UV-B radiation, but will also include increases in global and/or temperature. To date, few data exist on the interactions between UV-B radiation and temperature and while some initial work indicates that increased may reduce the extent of photosynthetic reduction at high UV-B radiation in rice, (Teramura et al., 1990; Ziska and Teramura, 1992) and Jack pine, Pinus banksiana (Stewart and Hoddinott, 1993), the mechanistic basis for this response is almost completely unknown. It is important to note that increases in seasonal ozone depletion and the corresponding rise in UV-B radiation may continue at least for several more decades. Although the Montreal Protocol limits the use of CFCs by industrialized and developing countries, the long atmospheric lifetimes of CFCs (50–100 years) and their continued use by countries not party to the protocol suggest that it is premature to consider the threat of ozone depletion to be ended.

Acknowledgments The authors wish to thank David Christopher, Jim Bunce, Irv Forseth, Herb Reed and Joe Sullivan for their useful comments and suggestions. This work was funded in part by USDA/NRI grant no. 9237100-7576 awarded to Alan H. Teramura.

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Alan H. Teramura and Lewis H. Ziska irradiance on soybean. V. The dependence of plant sensitivity on the photosynthetic photon flux density during and after leaf expansion. Plant Physiol 74: 475–180 Murali NS, Teramura AH and Randall SK (1988) Response differences between two soybean cultivars with contrasting UV-B radiation sensitivities. Photochem Photobiol 47: 1–5 Murphy TM (1983) Membranes as targets of ultraviolet radiation. Physiol Plant 58: 81–388 Naidu SL, Sullivan JH, Teramura AH and DeLucia EH (1993) The effects of ultraviolet-B radiation on photosynthesis of different needle age classes in field-grown loblolly pine. Tree Physiol 12: 151–162 Negash L (1987) Wavelength dependence of stomatal closure by ultraviolet radiation in attached leaves of Eragrostis tef: Action spectra under backgrounds of red and blue lights. Plant Physol Biochem 25: 753–760 Negash L and Björn LO (1986) Stomatal closure by UV-B radiation. Physiol Plant 66:360–364 Nogués S and Baker NR (1995) Evaluation of the role of damage to Photosystem II in the inhibition of assimilation in pea leaves on exposure to UV-B radiation. Plant Cell Environ 18: 781–787 Noorudeen AM and Kulandaivelu G (1982) On the possible site of inhibition of photosynthetic electron transport by ultraviolet (UV-B) radiation. Physiol Plant 55: 161–166 Panagopoulos I, Bornman JF and Björn LO (1992) Response of sugar beet plants to ultraviolet-B (280–320 nm) radiation and Cercospora leaf spot disease. Physiol Plant 84: 140–145 Pang Q and Hays JB (1991) UV-B inducible and temperaturesensitive photoreactivation of cyclobutane pyrimidine dimers in Arabidopsis thaliana. Plant Physiol 95: 536–543 Quaite FE, Sutherland BM and Sutherland JC (1992) Action spectrum for DNA damage in alfalfa lowers predicted impact of ozone depletion. Nature 358: 576–578 Reed HE, Teramura AH and Kenworthy WJ (1992) Ancestral US soybean cultivars characterized for tolerance to ultraviolet B radiation. Crop Sci 32: 1214–1219 Renger G, Voss M, Graber P and Schulze A (1986) Effect of UV irradiation on different partial reactions of the primary process of photosynthesis. In: Worrest RC and Caldwell MM (eds) Stratospheric Ozone Reductions. Solar Ultraviolet Radiation and Plant Life, NATO ASI Series G, Vol 8, pp 171–184. Springer-Verlag, Berlin Renger G, Volker M, Eckert HJ, Fromme R, Hohm-Veit S and Graber P (1989) On the mechanism of Photosystem II deterioration by UV-B irradiation. Photochem and Photobiol 49: 97–105 Renger G, Rettig W and Graber P (1991) The effect of UV-B irradiation on the lifetimes of singlet excitons in isolated Photosystem II membrane fragments from spinach. J Photobiochem Photobiol 9: 201–210 Robberecht R and Caldwell MM (1978) Leaf epidermal transmittance of ultraviolet radiation and its implications for plant sensitivity to ultraviolet-radiation induced injury. Oecologia 32: 277–287 Rosen H, Rehn MM and Johnson BA (1980) The effect of caffeine on repair in Chlamydomonas reinhardii. I. Enhancement of recombination repair. Mutat Res 70: 301–309 Ryel RJ, Parnes PW, Beyschlag W, Caldwell MM and Flint SD (1990) Plant Competition for light analyzed with a multispecies canopy model. I. Model development and influence of enhanced

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Chapter 19 Evaluation and Integration of Environmental Stress Using Stable Isotopes H. Griffiths Department of Agricultural and Environmental Science, Ridley Building, The University, Newcastle upon Tyne, NE1 7RU, U.K.

Summary I. Introduction II. Background to Stable Isotope Studies A. Carbon 1. Carbon Isotope Discrimination in Organic Material 2. On-line, Instantaneous Discrimination B. Oxygen Isotopes in Plants, Water and 1. Source Water and Fractionation in Organic Material 2. Leaf Water and Discrimination During Gas Exchange C. Deuterium 1. Source Water 2. Leaf Water and Organic Material III. Applications of Stable Isotope Techniques A. Integration of Photosynthetic Metabolism B. Evaluation of Phenotypic and Genotypic Differences 1. Crop Vegetation 2. Natural Vegetation C. Interactions Between Water Sources and Efficiency of Utilization D. Exchanges of and Water with the Atmosphere IV. Future Potential Acknowledgments References

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Summary Recent developments in the use of stable isotopes are set in the context of advances in theory and analytical procedures, with emphasis on potential applications for the future. Starting from initial requirements for biological markers which would allow the deconvolution of past climatic conditions, a mechanistic framework has been developed for the isotopes of carbon oxygen and hydrogen Understanding the interplay between fractionation against a heavy isotope during equilibrium (phase changes) and kinetic processes (diffusion and biochemical reactions) has led to the analysis of biological discrimination as distinct from simple source effects. For each isotope, the effect of environmental stress on discrimination is translated into the signal that would be carried by biochemical intermediates and plant organic material, together with the instantaneous effects measurable during gas exchange. Discrimination against provides either an instantaneous or a long term measure of the interaction between carboxylation and stomatal plus mesophyll diffusive limitation. Traditional uses for evaluating photosynthetic pathways are updated for concentrating mechanisms in lichens. Real-time, on-line discrimination, coupled Neil R. Baker (ed): Photosynthesis and the Environment, pp. 451-468. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

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with analysis of the isotope composition of biochemical intermediates, is used to illustrate the interaction between environmental stress and photosynthetic metabolism. The relationship between gas exchange characteristics, dry matter accumulation, organic material and water use can aid the selection of clones suitable for agroforestry in arid regions or be used to evaluate the extent of phenotypic plasticity in natural populations in response to elevated Analysis of and will complement and extend our understanding of the utilization of water sources, particularly in arid environments. The development of a quantitative basis to fractionation during metabolism, provides an additional means for integrating leaf water composition, organic material and environmental conditions. Thus, a powerful suite of stable isotope techniques now have the potential for scaling up exchanges between vegetation and the atmosphere. The use of isotopes to evaluate plant responses to environmental stress has widespread applications for the future, particularly when coupled to more facile sample preparation and rapid throughput in automated analytical systems.

I. Introduction The relative natural abundance of stable isotopes of carbon oxygen and hydrogen (D/H) act as biological markers, integrating cellular metabolism and individual plant performance through to processes coupling exchanges between vegetation and the atmosphere. Historically, many applications arose as a result of geological studies, whereby isotopes were used as tracers for the composition of atmospheric or sources of water and then used to infer climatic conditions during a particular era. Experimental studies have been used to investigate the relative rates of transformation of the heavy and light isotopes. Models were then developed to describe the effects on deuterium and fractionation Abbreviations: A – rate of assimilation; A'– rate of assimilation; CAM – crassulacean acid metabolism; CCM – concentrating mechanism; DW – dry weight; F – rate of photorespiration; – stomatal conductance to –stomatal conductance to IRMS – isotope ratio mass spectrometry; –carboxylation conductance; – partial pressure of with representing the ratio of internal:external and subscripts s, st, w and c designate partial pressures at the leaf surface, substomatal cavity, cell wall and site of carboxylation, respectively; PDB – calibration standard derived originally from Cretaceous belemnite; PEPC– phosphoenolpyruvate carboxylase; R – molar abundance ratio of heavy/light isotope, subscripts a, i and p designate in air, intercellular spaces and plant organic material, respectively; PFD – photon flux density; R – rate of dark respiration; Rubisco-ribulose 1,5-bisphosphatecarboxyIaseoxygenase; – velocity of ribulose 1,5-bisphosphate carboxylase; – velocity of ribulose 1,5-bisphosphate oxygenase; VPD – vapor pressure deficit; WUE – water use efficiency; – isotope effect; compensation point of photosynthesis; –isotope ratio, expressed as a differential against a defined standard in – biological discrimination independent of source effects; derived from organic material or measured directly during photosynthesis, subscripts obs and i designate observed and predicted; -ratio of uptake to bulk air partial pressure

during evaporation of water from an open surface (Craig and Gordon, 1965), or for the dissolution of or precipitation of carbonates (Mook et al., 1974). The fractionation occurring against during uptake and assimilation of was then used to distinguish the various photosynthetic pathways (O’Leary, 1981). Armed with a quantitative understanding of the interactions between equilibrium and kinetic reactions, the balance between diffusive limitation and fractionation expressed by ribulose 1,5bisphosphate carboxylase-oxygenase (Rubisco) was then used to describe the exchange of and between plants and the atmosphere (Vogel, 1980; Farquhar et al., 1982). These developments have been extensively reviewed, with the theory and practice of isotope discrimination leading to a number of general publications covering the suite of isotopes available (also including sulfur and nitrogen: Raven, 1987; Rundel et al., 1989; Griffiths, 1991), as well as those dedicated specifically to carbon and plantwater relations (Farquhar et al., 1989a,b; Ehleringer et al., 1993). At the same time, the models describing fractionation processes have been refined to account for additional features, with the use of discrimination nomenclature to simplify interpretation of the interaction between plant organic material and water use efficiency (Farquhar and Richards, 1984; Farquhar et al., 1989a,b; Farquhar and Lloyd, 1993). Additional applications now incorporate effects reflected in both and with leaf organic material related to realtime, on-line measurements of uptake, transpiration and leaf-water budgets (Farquhar et al., 1993; Flanagan et al., 1994; Yakir et al., 1994). Given this wealth of detail, the purpose of this chapter is to provide a basic introduction to discrimination processes in the context of plant responses

Chapter 19 Integration Using Isotope Discrimination to environmental stress. Rather than extensively reiterating theoretical models, the aim will be to draw attention to differences in existing approaches and scope for the future. The scale of applications ranges from the instantaneous isotopic signal associated with inter- and intra-cellular fluxes of and metabolites which are integrated into long-term leaf and stem organic material. At the next level, phenotypic responses to environmental variables translate into crop productivity and performance of natural ecotypes, in relation to water sources and photosynthetic efficiency. Finally, the potential to couple gas exchange by vegetation with the atmosphere brings the original geochemical applications full circle, to fulfill the need for modeling the global climate.

II. Background to Stable Isotope Studies The historical development of stable isotope analyses of biological systems (Ehleringer and Rundel, 1989; Ehleringer and Vogel, 1993), has traditionally relied on dual inlet isotope ratio mass spectrometers (IRMS) which provided high precision analyses on gases which have initially been generated and purified from the sample. The molar abundance ratio (R) for particular isotopes (e.g. measured as mass ratio of 45/44 for carbon) is compared to that of a defined standard, such as the PDB belemnite, with composition arbitrarily set to zero. Results are expressed using a differential notation ( ), with the fractionation in biological systems resulting in samples being depleted in by a few parts per thousand and hence the of samples is usually negative. Individual processes or reactions which lead to fractionation can each be defined by an isotope effect usually defined as the isotope ratio in reactant and product, such that It is now common to combine the isotope composition of sample, standard and source, with a single term, discrimination reflecting the overall isotope effect, such that In the case of the extent of depletion is directly proportional to the magnitude of the positive discrimination term. The use of allows the fractionation resulting from specific biological processes to be defined, particularly when used for growth in laboratory experiments has a different source signal compared to in bulk air, currently around versus PDB. However, it should be

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emphasized that variations in source or should be measured routinely as part of any study. A recent important technical development has been continuous-flow IRMS, which can reduce some of the conventional labor intensive sample preparation and analytical methods. Here, an automated C-N analyzer or gas autosampler is coupled, via a gas chromatograph, to a mass spectrometer operating in single inlet mode. Such a system requires that standards are interspersed regularly to recalibrate the mass spectrometer. However, any possible loss of precision is countered by the opportunity for improved replication, with up to 100 samples, standards and controls analyzed continuously.

A. Carbon Initially used to distinguish and photosynthetic pathways, the of organic material of plants provides a conserved signal of integrated gas exchange throughout growth. This realization offered the promise that could be used to select stresstolerant cultivars which demonstrate an elusive combination of productivity and high water use efficiency. Real-time collection of during photosynthesis, with the extent of enrichment corresponding directly to discrimination, has also been used as a probe for diffusion and carboxylation processes within individual leaves. However, just as the partial pressure of within the substomatal cavity must be inferred indirectly from gas exchange, so we must derive the isotope composition of both in the substomatal cavity and at the site of carboxylation. Before going on to consider the wider applications of these techniques, the underlying assumptions are first summarized.

1. Carbon Isotope Discrimination in Organic Material The discrimination expressed by plants is related to stomatal conductance (g) and hence to (internal: external partial pressures of . The molar abundance ratio of in plant organic material is related to the rate of assimilation of and (A' and A, respectively), such that For and for Knowing the fractionation factors for discrimination against during diffusion (a, or such that and carboxylation (where

454 and with and as the isotope ratios for in air and the intercellular spaces, respectively), the terms for can be substituted with equivalent expressions based on such that

H. Griffiths where is the velocity of carboxylation by Rubisco, F is the rate of photorespiration and R is the rate of respiration in the light. By substituting for the effects on but including fractionation during the diffusion and respiratory processes, the following expression has now been refined by Farquhar and Lloyd (1993) to consist of:

and by rearrangement,

and hence

or

This derivation shows the basis for the link with water use, since both transpiration and carbon isotope discrimination are independently regulated via stomata in higher plants. This simple relationship has been shown to hold for a variety of species and cultivars (Farquhar et al., 1989; Ehleringer et al., 1993), with greater for plants where diffusion is less limiting. In essence, for an average of (corresponding to of approximately and carboxylation is twice as limiting as diffusion, and for every three molecules entering the leaf, two will leave by back diffusion. It is this process which carries away from the leaf and allows the discrimination against to be expressed. When scaling water use from instantaneous measurements of gas exchange to crop productivity and yield, it is important to include respiratory losses (see Section III). The theory defining carbon isotope discrimination has also been refined to include respiratory and photorespiratory effects during net assimilation. Starting from the assumption that:

where, represents the carboxylation conductance, and the compensation point for photosynthesis derived from a response curve; and are defined as the partial pressures at the leaf surface, substomatal cavity and site of carboxylation, respectively, together with fractionation factors for during diffusion through boundary layers dissolution diffusion through the cell respiration and photorespiration However, this detailed expression does not include a term to describe the fractionation that will be expressed against (photo)respiratory should it be presented back to Rubisco during photosynthesis. While this may not be significant for crop plants with high rates of assimilation, it may have implications for real-time measurements of instantaneous discrimination in plants with relatively high rates of respiration or photorespiration in the light, as will be discussed below. Other theoretical complications, whereby changes in translate firstly into a daily, and thence to a seasonal, integration of plant performance have been considered in some detail (Farquhar and Richards, 1984; Farquhar et al., 1989a,b; Farquhar and Lloyd, 1993). One technique which can integrate photosynthesis on a daily basis is the extraction and analysis of starch, sugar (Brugnoli et al., 1988), as well as other soluble organic components (Borland et al., 1994). In terrestrial and CAM plants, the use of as substrate (enriched in compared to gaseous Mook et al., 1974) and low discrimination of phosphoenolpyruvate carboxylase (PEPC) results in lower than in plants (O’Leary 1981; Farquhar et al., 1989a). These values are tempered in part by environmental conditions Madhaven et al., 1991)

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or by the extent of fixation in CAM plants (Griffiths 1992), although higher than predicted theoretically for both pathways, suggests that leakage allows inherent Rubisco discrimination to be expressed. Lichens are a stress-tolerant, poikilohydric group of plants for which carbon isotope discrimination has recently provided an insight into the various associations between fungal (mycobiont), algal (phycobiont) and/or cyanobacterial (cyanobiont) partners. Analysis of organic material shows that is high in the associations in which the primary phycobiont do not possess pyrenoids in chloroplasts (Fig. 1). In addition to the primary phycobiont, lichens in this group often contain additional cyanobacteria in cephalodial pockets within the thallus (Máguas et al., 1993, 1995). The carbon isotope composition is related to the occurrence of a concentrating mechanism (CCM) in certain algae (with pyrenoids) and cyanobacteria (in the carboxysome), which leads to reduced in organic material (Beardall et al., 1982; Sharkey and Berry, 1985; Badger et al., 1993). Recently, we have extended our understanding of the CCM to show for the first time that such a mechanism is associated with terrestrial bryophytes, namely Anthoceros, which also contain pyrenoids (Smith and Griffiths, 1996).

2. On-line, Instantaneous Discrimination Analysis of the enrichment in which remains in the residual air following net uptake, has a similar range of applications as that seen for organic material. In principle, can be collected in a liquid nitrogen cold trap under a partial vacuum, with a needle valve used to interface the gas flow from the leaf cuvette to the vacuum system (Evans et al., 1986; Griffiths et al., 1990; von Caemmerer and Evans, 1991; Broadmeadow et al., 1992). Care must be taken to ensure that all is trapped to prevent any further fractionation, and to correct (orre-purify) the sample for content. It is important to maintain the ratio of net uptake where subscripts e and o refer to entering and leaving the cuvette) around 10 or below, with large leaf areas and high photosynthetic rates allowing shorter collection times. On-line discrimination is derived as:

which, for a difference in isotope composition

entering and leaving the cuvette of and a of 10, corresponds to an instantaneous of around At the whole plant level, the relationship between and in plants has been confirmed, and, as will be discussed below, used to determine internal mesophyll conductance (Evans et al., 1986; von Caemmerer and Evans, 1991; Lloyd et al., 1992). Stomatal conductance is more constant across a range of environmental conditions in plants (Evans et al., 1986), and instantaneous discrimination can then be used to estimate the extent of bundle sheath

456 leakiness (Henderson et al., 1992). Alternatively, refixation of photorespiratory accounts for the higher values of instantaneous under low partial pressures in intermediates (von Caemmerer, 1989; von Caemmerer and Hubick, 1989). Forplants with crassulacean acid metabolism (CAM), the shift between carboxylation mediated via PEPC (at night) and Rubisco (by day) provides a marked shift in instantaneous discrimination corresponding to the phases of CAM (Griffiths et al., 1990; Borland et al., 1993). Measurement of instantaneous has provided a means of distinguishing between the phycobiont (plus pyrenoid) and cyanobiont groups of lichens with similar organic carbon isotope composition (Fig. 1) with lowest, more -like’ values found in the cyanobacterial association. In contrast, organic and instantaneous values were similar for the phycobiont associations lacking a pyrenoid, which are also found to have the highest, most -like, compensation points (Fig. 1). The interrelationship between isotope discrimination characteristics and CCM activity in the phycobiont plus pyrenoid and cyanobiont associations is clear, although thallus water content will also impose diffusive limitations (Lange et al., 1993) which temper discrimination characteristics (Máguas et al., 1995). The compensation point for a lichen thallus represents the combined activities of the mycobiont and photosynthetic partner, with rates of mycobiont respiration sometimes up to 50% of net uptake (Cowan et al., 1992; Máguas et al., 1993, 1995).This highlights two technical problems which should be taken into consideration when measuring instantaneous discrimination characteristics: both are related to source composition. One is external, in that use of tank with a of could alter the measured on-line characteristics by up to (I. R. Cowan, personal communication). The second is internal, in that the respiratory produced within the thallus provides a considerable source for photosynthesis, and this flux must be included in addition to that measured directly during net uptake. By analogy with higher plant leaves, photorespiratory production at 20 °C (with 2.5) presents an internal flux of around 0.2 in addition to each net taken up. The internal, mesophyll conductance will determine the extent that this flux is presented to Rubisco, and the extent

H. Griffiths that it joins the outward flux of (see above). However, this is not accounted by current models which are derived in relation to net assimilation alone (see Eq. (5)). This is in contrast to a model for lichen gas exchange which includes these fluxes (Cowan et al., 1992), and can be modified to account for discrimination expressed by Rubisco against that portion of respiratory and photorespiratory which is refixed (I. R. Cowan, personal communication). It is often found that the measured, (or observed) instantaneous is 2 to lower than that predicted theoretically from when it is assumed that (Evans et al., 1986), with now redefined as The drop in partial pressure between and however, has been shown to be proportional to the magnitude of and can be used to estimate the transfer conductance, (von Caemmerer and Evans, 1991; Lloyd et al., 1992; Farquhar and Lloyd, 1993 ; Syvertsen et al., 1995). While mesophyll conductances obtained depend on leaf type (e.g. herbaceous versus evergreen), and compare with those estimated from chlorophyll fluorescence techniques (e.g. Loreto et al., 1992), more subtle techniques would need to be developed to distinguish between possible inter- and intracellular components (Parkhurst, 1994). It is evident that future studies should include additional anatomical details to account for variations in leaf thickness, porosity and stomatal density in the estimation of mesophyll conductance parameters (Parkhust, 1994; Syvertsen et al., 1995), with rates of assimilation corrected to account for refixation of (photo)respiratory

B. Oxygen Isotopes in Plants, Water and The use of for investigating plant responses to stress has a similar wide range of applications to carbon. The content of gaseous oxygen has been directly related to fractionation during respiratory or photosynthetic metabolism, (Guy et al., 1989, 1993; Robinson et al., 1992), but recent interest has focused more generally on the use of these isotopes as a proxy for leaf-air vapor pressure deficit (VPD) in relation to gas exchange, plantwater status and soil-water source. The signal of source or tissue water, following extraction, can be equilibrated with gaseous in the headspace of a closed vial. This process is both beneficial and detrimental. Firstly, equilibration is extremely rapid within the leaf (catalysed by carbonic anhydrase),

Chapter 19 Integration Using Isotope Discrimination allowing the chloroplast water signal to be identified from gas exchange by on-line trapping and IRMS analysis. This is problematic because care must be taken to ensure complete separation of and during on-line sample collection. The is then analyzed by IRMS, with the composition determined from the analysis of the mass to charge ratio 46, which is derived routinely during measurements from the mass to charge ratios of and the contribution from oxygen to mass 45 and in 46. The recent development of a simple equilibration technique for plant and soil water, in conjunction with analysis by continuos-flow IRMS (C. M. Scrimgeour, personal communication), should circumvent complex distillation or chemical conversion procedures (Ehleringer and Osmond, 1989) and allow more widespread application of these procedures.

1. Source Water and Fractionation in Organic Material When water is taken up from the soil, there is no fractionation and so the signal in water from root, stem and twig water represents unadulterated source water. In contrast, the climatic conditions during initial evaporation and subsequent precipitation alter isotope composition depending on temperature, latitude and altitude, with the subsequent mixture with existing ground water resulting in a distinct source signature for both and (Sternberg, 1989; Ehleringer and Dawson, 1992; Yakir, 1992; Dawson, 1993b). In the leaf, evaporation leads to the enrichment of in residual water, which under certain conditions can be used to model the mixing of water within leaves (Leaney et al., 1985; Flanagan, et al., 1991; Yakir 1992) and leaf gas exchange (Farquhar and Lloyd, 1993; Flanagan et al., 1994; see Section II.B.2). The incorporation of during photosynthesis also leads to a distinct signal, with organic material enriched by around compared to source water, independent of photosynthetic pathway (Sternberg, 1989; Yakir, 1992). Analysis of cellulose nitrate, which overcomes problems of re-equilibration and secondary fractionation following initial synthesis (particularly for analyses: see below), has been used as a means to estimate past ground water composition (Yakir, 1992; Yakir et al., 1994). However, further experimental

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work is required to unravel the biochemical steps leading to the fractionation found in organic material, so as to provide a mechanistic basis for palaeoclimatic reconstruction.

2. Leaf Water and Discrimination During Gas Exchange The impact of plant processes on atmospheric and budgets has resulted in several approaches attempting to couple leaf water and exchange with the atmosphere (Farquhar et al., 1993; Flanagan et al., 1994; Flanagan and Varney, 1995; cf. Yakir et al., 1994). In principle, leaf water becomes enriched in during transpiration, which is dependent partly on equilibrium fractionation during the liquid-gas phase change, and also on the kinetic fractionation during diffusion through stomata and the boundary layer (Yakir, 1992; Farquhar and Lloyd, 1993; Flanagan, 1993). One limitation to the use of leaf water as a model for gas exchange is the requirement that leaf water should be at isotopic steady state. Thus, the isotopic composition of transpiring water should be the same as source or stem water, which may be achieved after 1 to 2 h in a gas exchange cuvette (Flanagan et al., 1991; Yakir, 1992; Flanagan, 1993), but may be more variable under natural conditions. This problem has been confounded by studies suggesting the existence of various pools of isotopically distinct water, including that unfractionated in veins or that, enriched at the site of transpiration, diffusing back into the tissue (Leaney et al., 1985; Farquhar and Lloyd, 1993). While these effects can be corrected mathematically (Farquhar and Lloyd, 1993), and the various metabolic contributions to leaf water partitioned by a mass balance relationship (Yakir, 1992), the ultimate question is now the isotopic composition of metabolic leaf water. With transpiration and extent of fractionation controlled by the leaf-air VPD, and by knowing the isotope composition of water vapor in air, it should then be possible to use analysis of in leaf water or cellulose to estimate VPD and effects of environmental stress (Yakir, 1992; Flanagan, 1993). Finally, the ratio of in air provides a further means of coupling plant gas exchange and the atmosphere, mediated by rapid equilibration between leaf water and catalysed by carbonic anhydrase. Thus, air passing over a leaf becomes

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enriched in (see Section II.A.2) and content, the latter reflecting the of water in the chloroplast. Analogous to discrimination, a model has been formulated for the resulting discrimination, in terms of the fractionation factors for diffusion, extent of equilibration as well as effects of photorespiration and respiration (Farquhar and Lloyd, 1993). The approach also requires that the chloroplast water be in full equilibration with that at the sites of evaporation (Farquhar et al., 1993; Flanagan et al., 1994). In contrast, measurement of the isotopic composition of chloroplast water from photorespiratory at the compensation point, or gaseous evolved during photosynthesis, does not support this contention (Yakir et al., 1994). These more direct estimates of chloroplast water suggest that it is depleted in as compared to the evaporating water at the cell surface, by some 7 to (Yakir et al., 1994). Once the discrepancies outlined above have been resolved, both for gas exchange at the individual leaf level and for the likely differences in coupling which will occur at the canopy level, there is considerable potential for measurements to integrate and evaluate plant responses to stress.

C. Deuterium The range of applications for analyses is similar to although in analytical terms the IRMS requires specific ion optics and collectors. Having extracted leaf water (see Section II.B), gaseous hydrogen can be prepared by reduction over uranium or zinc (Ehleringer and Osmond, 1989) although there is potential for a simplified equilibration technique. By comparison with the other isotopes considered above, deuterium has a proportionally greater mass than the maj or ion so that the large isotope effects give rise to isotope ratios which are an order of magnitude greater than for carbon or oxygen. However, this is offset by the lower relative abundance of D/H, and so the precision of determinations tends to be lower (Sternberg, 1989). Organic material is prepared as cellulose nitrate so as to prevent contamination from exchangeable H, which may have re-equilibrated with source water subsequent to synthesis (Epstein et al., 1976)

1. Source Water The large range of D/H isotope ratios found in

H. Griffiths meteoric water, with summer and winter rains differing by as much as reflects the fractionation associated with evaporation (depleting D) and precipitation (enriching D), and is ultimately regulated by atmospheric temperature. Analysis of source and plant water has resulted in some astonishing insights into the responses of plants to varying water supply (White et al., 1985; Dawson and Ehleringer, 1991; Dawson, 1993a), and shows great promise for the integration of water deficits. In particular, the marshaling of water sources in arid environments has proved to be particularly revealing, owing to the large differences between meteoric water, groundwater and direct effects of evaporation on soil water (Ehleringer and Dawson, 1992; Dawson, 1993b). The only environment where fractionation has been reported during water uptake is for salt exclusion in intertidal habitats (Lin and Sternberg, 1993), which emphasizes the importance of measuring the isotope composition of source- and stem-water in all studies.

2. Leaf Water and Organic Material The effect of transpiration is to enrich leaf water with deuterium, similar to the effects described for (see Section II.B. 1). Indeed, the relationship between modeled and actual differences in the isotopic composition of leaf water holds for both and and has been related to the degree of attainment of isotopic steady state (Flanagan et al., 1991; Flanagan, 1993). Leaf water was originally reported to differ depending on photosynthetic pathway (Leaney et al., 1985), although subsequently this has been related to variations in stomatal and boundary layer conductance (Flanagan et al., 1991). However, it is the composition of organic material which, in contrast to shows more systematic variations in relation to the occurrence of and CAM pathways, with photosynthetic intermediates distinguishable when analyzed in combination with (Sternberg et al., 1984; Sternberg 1989; Smith and Ziegler, 1990). In particular, the carbohydrates of CAM plants are enriched in deuterium, compared to and plants, which has been variously related to rates of transpiration (i.e. source water effect) or carbohydrate cycling as part of CAM. A mechanistic explanation has now been presented, whereby photosynthesis results in carbohydrates initially depleted in D with respect to leaf water, which are then enriched stepwise

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during post-photosynthetic metabolism (Yakir, 1992). The extent of any heterotrophic cycling of carbon through secondary metabolism, and hence the degree of enrichment, enables the composition of carbon accumulated in sink organs to be determined as the balance between recent photosynthate and that derived from exported (metabolized) carbon (Yakir et al., 1991).

III. Applications of Stable Isotope Techniques The developments outlined above are relevant to the interpretation of plant responses to stress at a number of levels. In the following section, selected examples are used to illustrate the increasingly diverse uses of natural abundance studies, with stable isotopes acting as markers for the effects of stress and interactions with the environment.

A. Integration of Photosynthetic Metabolism The application of isotopic analyses can provide insight into carboxylation processes and leaf carbohydrate metabolism (Brugnoli et al., 1988; Lauteri et al., 1993), particularly when large changes in discrimination are associated with different carboxylation pathways as found in CAM (Griffiths et al., 1990; Borland et al., 1993; Borland and Griffiths, 1995). CAM intermediate plants induce CAM in response to a variety of environmental stimuli, such as water deficit, photon flux density (PFD), which are related to seasonal changes under natural conditions (Griffiths, 1992; Maxwell et al., 1992; Borland and Griffiths, 1996). The combination of non-invasive instantaneous discrimination, when coupled to the analysis of organic metabolites has been particularly revealing for the to CAM transition in the hemi-epiphytic strangling fig, Clusia minor, under natural conditions in Trinidad (Borland et al., 1993, 1994). The pattern of gas exchange is dependent on degree of exposure, whether exposed or shaded, with the mid-day depression of gas exchange enhanced in the dry season (Fig. 2). Instantaneous demonstrates activity throughout the day in shaded leaves, whereas the magnitude and extent of activity extends well into the light period following CAM induction in exposed leaves (Fig. 2). The extent of and activity in the rainy season can be integrated from

instantaneous measurements to provide a predicted value for organic material, corresponding to values of and for exposed and shaded leaves. These values correlate almost exactly with actual leaf organic carbon isotope signatures, (i.e. and respectively), showing that the relatively short dry season contributes little to overall carbon gain. Despite this observation, during the dry season higher rates of net uptake occur by day, in addition to regenerated from the high concentrations of malic and citric acids which accumulate overnight. This suggests that cycling of carbon helps to dissipate any excess PFD incident at this time, particularly as daily integrated PFD increases from around 30 to 40 mol (Borland et al., 1993; Borland and Griffiths, 1996). The contribution of PEPC activity to carbon cycled between organic acid and carbohydrate pools can then be quantitatively demonstrated from analysis of the isotope composition of each fraction at dawn and dusk (Borland et al., 1994).

H. Griffiths

460 The movement of the label between organic acids (at dawn) into soluble sugars and glucans (at dusk) can be seen when CAM is fully induced in the dry season for both exposed and shaded leaves (Table 1). Here, the use of the nomenclature is retained because we are not certain of the isotope composition of source with a large proportion being derived from respiration. This data can be used to infer the partitioning between carbohydrate pools for storage (i.e. CAM) and export, since the carbon isotope composition of mobile carbohydrate pools can be distinguished by mass balance against background carbon (Deleens and Garnier-Dardart, 1977). Photosynthate produced in the afternoon, mediated directly by Rubisco, is transported from the leaf, and hence maintains the signal of bulk leaf organic material (Borland et al., 1994). Finally, it has also been possible to use this data to calculate the discrimination expressed by PEPC at night from analysis of the organic acid pools. Discrimination was lower in exposed leaves, with found to be in contrast to for shaded leaves, suggesting that high CAM activity leads to diffusion limitation at night (Griffiths 1992; Borland et al., 1994). Analysis of in soluble sugars provides a reliable estimate of assimilation-weighted in plants, perhaps providing an easier and more reliable measure of crop responses to drought stress than the conductance-weighted value derived from gas exchange measurements (Farquhar et al 1989a,b; Lauteri et al., 1993). Combined stable isotopes studies can provide yet more detail of carbon fluxes. In

maize, 16% of the cellulose-carbon accumulated by the corn cob is derived directly from carboxylation, with the remainder imported from 4 leaves (Yakir et al., 1991). However, when measured in conjunction with composition, the extent of heterotophic cycling can also determine the likely source leaf of carbon (Yakir et al., 1991).

B. Evaluation of Phenotypic and Genotypic Differences While in principle stable isotope techniques should be applicable to crops and natural vegetation alike, in reality the faith is observed in subtly different ways. The agronomist, in thrall to the harvest index, uses stable isotopes as a means of evaluating varietal differences, with the hope of finding a slight improvement in the relationship between water use and carbon gain which may have eluded conventional breeding techniques in the last few hundred years (Passioura et al., 1993). Meanwhile, the ecologist has a much broader church available for study in terms of underlying genotype. This translates into greater potential range of isotopic composition, representing the gradation within or between ecotypes, or speciation, in relation to environmental stress under natural conditions.

1. Crop Vegetation A wealth of reviews have recently provided a comprehensive summary of the use of carbon isotope discrimination for the selection and improvement of crop plants (Farquhar et al 1989a,b; Ehleringer et al., 1993; Jones, 1993). Despite the resources which have been directed towards elucidating the relationship between water use and it is perhaps pertinent to consider whether any commercial varieties have yet arisen from these studies. However, there is overwhelming evidence in favor of a negative relationship between and transpiration efficiency (g dry matter for plants in semi-arid or drought stressed conditions, whether for herbaceous or woody crops. The advantages are that integrates the plant life history without the need to monitor water use directly, analyses can be performed rapidly on a small amount of dry matter, with it being preferable to use vegetative material. This can speed the selection of varieties for hybridization, with the heritability of these traits found to be strong for a range of species including wheat, barley, peanut.

Chapter 19 Integration Using Isotope Discrimination (Farquhar and Richards, 1984; Martin et al 1989; Hall et al., 1993; Richards and Condon, 1993). The use of carbon isotope discrimination has also been used to evaluate woody species, such as coffee (Gutierrez and Meinzer, 1994), and pine (Zhang and Marshall, 1995), and there is also the potential for the selection and improvement of agroforestry and intercropping multi-purpose tree varieties. A comparison for the leguminous Sesbania sesban, with high potential productivity, shows that there is a spectrum of responses between two varieties, encompassing the drought tolerant (var. Nubica) and intolerant (var. Sesban). Instantaneous gas exchange measurements show the expected relationship between and which translates into a negative association with instantaneous water use efficiency (Fig. 3). Dry matter accumulation and transpiration efficiency were inversely related to following the imposition of drought stress (Table 2). In general terms, variations in could come about through alterations in either photosynthetic capacity or stomatal conductance (Richards and Condon, 1993), which may be related to stress through effects of nitrogen status, nutrient supply, leaf morphology and temperature and hydraulic efficiency. However, for a particular pair of closely related genotypes, provided that similar conditions prevail, then the variation in can be formulated as an Alg weighted comparison of water use (Hall et al., 1993; Jones, 1993). There have been difficulties in finding a unifying theory to define the interaction between genes and environment which will allow evaluation of plant responses to drought stress across contrasting latitudes, climatic conditions and agronomic practices (Ehleringer et al., 1993). Once a consensus can be reached as to the optimal timing and material for sampling, and the best basis for expressing plant water use, biomass production, grain yield, rootshoot partitioning for a particular locale, progress may then be made (Jones, 1993; Passioura et al., 1993). Molecular biological techniques will also provide a more quantitative basis to the heritability of those characteristics currently integrated by Antisense mutants are a useful probe for the physiological basis for A, with a significant reduction in Rubisco compensated by increased and in tobacco (Quick et al., 1992), Recently, responses under elevated suggest that when nitrogen is limiting there is a significant effect on in antisense Rubisco mutants which must reflect mesophyll conductance

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or some other related parameter (H. Griffiths and W. P. Quick, unpublished data). Polymorphisms associated with drought tolerance in tomato have been identified on three chromosomes (Martin et al., 1989), the potential for use of other molecular markers has also been reviewed (Masle et al., 1993). In wild barley populations, chromosome addition techniques and analysis have identified a specific chromosome associated with carbon isotope composition (Handley et al., 1994a). Finally, there have also been reports of a positive relationship between and water use efficiency, particularly for irrigated crops or well watered

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conditions, as opposed to pot experiments (Condon et al., 1987; Richards and Condon, 1993). The coupling of gas exchange between a uniform canopy and the atmosphere, and timing of growth and water utilization form part of the complex considerations which determine the interactions between and water use efficiency for a particular crop (Farquhar and Lloyd, 1993; Jones, 1993; Richards and Condon, 1993; Passioura et al., 1993).

2. Natural Vegetation The approaches adopted by crop physiologists and ecologists may not be so distant. It has been shown that ‘conservative’ and ‘water saving’ responses can equally be found in crop ecotypes (Passioura et al., 1993; Richards and Condon, 1993) and natural vegetation (Ehleringer, 1993a; Jones, 1993). Thus utilization and competition for available water is intimately related to crop phenology or seasonal growth under natural conditions, with both reflected in carbon isotope composition. Observations based predominantly on the responses of natural communities to drought stress in arid regions has led to one useful definition which unifies approaches: the concept of as a metabolic ‘set point,’ with integrating gas exchange, plant water and nutrient status (Ehleringer, 1993a). In an elegant series of studies, the long term lifehistory responses of desert shrub communities have been related both to utilization of water sources and carbon isotope discrimination (Ehleringer, 1993a). The ranking of remained constant over long periods, allowing to be correlated with survival and growth rate for both inter- and intra-specific variation. Selection for occurred under natural

H. Griffiths

conditions, as determined from mortality rates and competition for water in populations of Encelia farinosa. Low individuals reflected slow growth rates and persistence through adverse conditions, whereas high was associated with high growth rates when water was available (Ehleringer, 1993a,b). The occurrence of high and low individuals represents a compromise allowing the population to exploit the intermittent seasonal rainfall. Perhaps this message should be considered by agronomists seeking to develop varieties for arid regions where rains may fail in certain years. Carbon isotope discrimination can also be used in the laboratory to distinguish phenotypic and genotypic responses to elevated provided that source isotope composition is controlled and measured routinely. Having screened the variation in for a range of Plantago major populations, a detailed comparison was made for three ecotypes each from temperate or tropical/Mediterranean climates. Although the dry matter productivity was stimulated by elevated for both groups, there were systematic differences between morphology, root/shoot allocation and photosynthetic physiology (Table 3). Assimilation rates were similar, but a lower stomatal conductance led to high instantaneous water use efficiency and low instantaneous for tropical populations, which also showed a higher conversion efficiency with carbon allocated to shoots and starch reserves (Table 3). One notable characteristic was the lower stomatal density in the tropical populations, which was further reduced under elevated Stomatal density and instantaneous were directly correlated (Fig. 4), being lower in the tropical ecotypes irrespective of growth regime, and were also reflected in organic

Chapter 19 Integration Using Isotope Discrimination

material (Table 3). Instantaneous water use was also higher at low stomatal density, but conductance and instantaneous water use were consistently higher for plants grown under elevated Thus morphology and assimilation characteristics control suggesting that long term genotypic acclimation to temperate and tropical habitats has optimized water use, but may be modified by phenotypic responses to elevated (Table 3, Fig. 4). The relationship between and stomatal density has long term implications for assessing climatic variations in the context of rising atmospheric (Beerling and Woodward, 1993; Beerling et al., 1993; Poliey et al., 1993). Carbon isotopes have also made a major contribution to the understanding of host parasite interactions, both as a comparison of water use and for inferring the proportion of carbon acquired from the host (Marshall et al., 1994). These interactions are best served by a host plant with CAM (Schulze et al., 1991) or the pathway, (Press et al., 1987) where carbon sources are demonstrably different. High rates of respiration are found in parasites such as Striga, which are in some circumstances equal to net uptake. The extent that a high reflects refixation by Rubisco in the light, rather than net metabolism, remains to be determined (Press et al., 1987; Stewart and Press, 1990; Griffiths, 1991;section II. A). Most recently, stable isotopes of carbon and nitrogen have revealed the extent that respiratory and excretory N, derived from ants, contribute to a myremecophilous vine with CAM (Treseder et al., 1995).

463

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H. Griffiths

C. Interactions Between Water Sources and Efficiency of Utilization

D. Exchanges of Atmosphere

Despite the different concepts of water use efficiency defined by agronomist and physiologist (Eamus, 1991) and the need to monitor the addition of irrigation water and utilization of available soil water, the use of natural abundance stable isotope studies (as opposed to the use of enriched water sources) has lagged behind diverse ecological applications. As mentioned above, the studies undertaken by Ehleringer and collaborators represent a superb combination of technology being used to solve ecological problems (Ehleringer, 1993a). A range of papers have shown how seasonal water utilization of meteoric or groundwater is related to growth form and life history pattern, determined from analysis (Rundel et al., 1989; Ehleringer et al., 1993). Perhaps the most potent demonstration, illustrating the potential for natural abundance studies to take over from isotope enrichment, has been proof for the occurrence of ‘hydraulic lift.’ Plants at the surface can acquire water drawn up from ground water by deep rooted perennials, because of a reverse gradient of plant-to-soil water potential near the surface (Caldwell and Richards, 1989). Because of the different signal associated with groundwater and surface water, the expectations that this process could be quantified by analysis (Griffiths, 1991) has now been elegantly justified (Dawson, 1993a,b). This work has demonstrated that hydraulic lift can be found in mesic (for Acer saccharum) as well as arid environments, with the neighboring vegetation acquiring from 3 to 60% of plant water by ‘piracy’ up to 5 m away from the phreatophyte. Intriguingly, the carbon isotope discrimination of the herbaceous vegetation corresponded to the degree of water stress alleviated by hydraulic lift (Dawson, 1993a). Finally, while the effects of soil water deficit can be physically avoided because of soil shrinkage away from the root, effectively isolating many succulent plants, the demobilization of stored water from parenchyma to chlorenchyma has been demonstrated by analysis (Tissue et al., 1991; Nobel and Cui, 1992). Alternatively, in agronomic environments, while there has been much interest of late in the role of roots in transducing and signaling soil water deficits (Tardieu and Davies, 1993), the potential for stable isotopes to augment these studies has yet to be realized.

The problem of scaling crop vegetational responses from studies utilizing individual plants has already been alluded to above. Analysis of the of atmospheric can distinguish seasonal and annual increments in global carbon exchange (Keeling et al., 1995) and may also be used to estimate the extent of respiratory refixation within forest canopies (Sternberg, 1989; Broadmeadow et al., 1992; Broadmeadow and Griffiths, 1993). However, the internal partial pressure, or isotope composition within the leaf can only be determined indirectly (whether measured as or see Section II.A.1) from gradients in VPD or instantaneous discrimination. While we know the water vapor concentration within the leaf, knowledge of the isotopic composition of metabolic water would allow the exchange with the atmosphere to be quantified. This underlies the interest that has been engendered by variations in the estimate of chloroplast water (Farquhar et al., 1993; Flanagan et al., 1994; cf. Yakir et al., 1994). Should the assumption hold that the chloroplast water signal is represented by that at the evaporating sites, then the diffusing out of leaves could allow vegetation processes to dominate the global atmospheric budget, rather than exchange with seawater, as previously thought (Farquhar et al., 1993). The relatively depleted composition of northern temperate latitudes can be used to calculate the relative contributions of vegetation (Farquhar et al., 1993), including net photosynthesis and soil respiration, by mass balance (Yakir, 1992). When combined with direct measurements of gaseous fluxes (Flanagan and Varney, 1995; Grace et al., 1995) across contrasting biomes, such approaches will hopefully lead to an understanding of how regional trends in the carbon cycle could account for the missing sink for carbon (Keeling et al., 1995).

and Water with the

IV. Future Potential While a review such as this must by necessity be selective in the scope and application, it is evident that stable isotopes are the key to many new approaches for the integration and evaluation of plant responses to environmental stress. It must be emphasized that this chapter has not considered the potential interactions with other stable isotopes,

Chapter 19 Integration Using Isotope Discrimination particularly nitrogen, which can provide an additional dimension to ecological studies (e.g. Deleens et al., 1994; Handley et al., 1994b). Improvements in technology will lead to more accessible analytical services, which, together with a more rigorous understanding of fractionation processes, will lead to increasing numbers of applications. However, as has been sagely pointed out by O’ Leary (1981, 1993), differences of less than for carbon should be viewed with caution, and it is always important to check the experimental manipulations have not introduced fractionation by changes in source composition or contamination during sample collection or purification.

Acknowledgments Despite the patient tutoring of Susanne von Caemmerer, I still have a re-fixation; I am grateful to colleagues for discussions and access to data, and to NERC, UK for support.

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Chapter 19 Integration Using Isotope Discrimination Hydrogen-isotope composition of leaf water in and plants: Its relation to the hydrogen-isotope composition of dry matter. Planta 164: 215–220 Lin G and Sternberg L da SL (1993) Hydrogen isotopic fractionation by plant roots during water uptake in coastal wetland plants. In: Ehleringer JR, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp 497–510. Academic Press, San Diego Lloyd J, Syvertsen JP, Kreidemann PE and Farquhar GD (1992) Low conductance for diffusion from stomata to the sites of carboxylation in leaves of woody species. Plant Cell Environ 15:873–899 Loreto F, Harley PC, Di Marco G and Sharkey TD (1992) Estimation of mesophyll conductance to flux by three different methods. Plant Physiol 98: 1437–1443 Madhaven S, Treichel I and O’Leary MH (1991) Effects of relative humidity on carbon isotope fractionation in plants. Botanica Acta 104: 292–294 Máguas C, Griffiths H, Ehleringer JR and Serodio J (1993) Characterisation of photobiont associations in lichens using carbon isotope discrimination techniques. In: Ehleringer J, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp. 201–212 Academic Press, San Diego Máguas C, Griffiths H and Broadmeadow MSJ (1995) Gas exchange and carbon isotope discrimination in lichens: evidence for interactions between concentrating mechanisms and diffusion limitation. Planta 196: 95–102 Marshall JD, Ehleringer JR, Schulze E-D and Farquhar G (1994) Carbon isotope composition, gas exchange and heterotrophy in mistletoes. Fund Ecol 8: 237–241 Martin B, Nienhuis J, King G and Schaefer A (1989) Restriction length polymorphisms associated with water-use efficiency in tomato. Science 243: 1725–1728 Masle J, Shin JS and Farquhar GD (1993) Analysis of restriction length polymorphisms associated with variation of carbon isotope discrimination among ecotypes of Arabidopsis thaliana. In: Ehleringer J, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp. 371–386. Academic Press, San Diego Maxwell C, Griffiths H, Borland AM, Broadmeadow MSJ and McDavid CR (1992) Photoinhibitory responses of the epiphytic bromeliad Guzmania monostachia during the dry season in Trinidad maintain photochemical integrity under adverse conditions. Plant Cell Environ 15:37–47 Mook WG, Bommerson JC and Staverman WH (1974) Carbon isotope fractionations between dissolved bicarbonate and gaseous carbon dioxide. Earth Plan Sci Lett 22: 169–176 Nobel PS and Cui M (1992) hydraulic conductances of the soil, the root-air gap, and the root: Changes for desert succulents in drying soil. J Exp Bot 43: 319–326 O’Leary MH (1981) Carbon isotope fractionation in plants. Phytochem 20: 553–567 O’Leary MH (1993) Biochemical basis of carbon isotope fractionation. In: Ehleringer J, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp. 19–28. Academic Press, San Diego Parkhurst DF (1994) Diffusion of and other gases inside leaves. New Phytol 126: 449–479 Passioura JB, Condon AG and Richards RA (1993) Water deficits,

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Chapter 20 Environmental Constraints on Photosynthesis: An Overview of Some Future Prospects Neil R. Baker Department of Biological and Chemical Sciences, University of Essex, Colchester, CO4 3SQ, UK

Summary I. Introduction II. Light Energy Transduction by Thylakoids III. Carbon Metabolism IV. Leaf Gas Exchange V. Scaling from the Chloroplast and Leaf to the Canopy Acknowledgments References

469 469 470 472 472 475 475 475

Summary An overview of some of the major issues that emerge from the book is presented. Consideration is given to possible future developments in studies of the impact of environmental changes on photosynthesis and the role that these may play in predictive modeling of the effects of global climate change on plant productivity.

I. Introduction Photosynthesis has been the subject of extensive investigation at many levels of organization, as evidenced by the recent proceedings of the International Photosynthesis Congresses held triennially (Baltscheffsky, 1990; Murata, 1992; Mathis, 1995), Recently considerable advances in knowledge of the molecular structure and function of components of the photosynthetic apparatus have led to a detailed understanding of the systems and mechanisms involved in the processes of light capture, energy transduction and carbohydrate biosynthesis. Particularly impressive developments have been the elucidation of the 3-dimensional structures of the reaction center of the purple bacterium Rhodopseudomonas viridis (Michel and Deisenhofer, 1988),

Neil R. Baker (ed): Photosynthesis and the Environment, pp. 469–476. © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

the light-harvesting complexes of Rhodopseudomonas acidophila (McDermott et al., 1995) and higher plants (Kühlbrandt et al., 1994), and of the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of Synechococcus (Newman and Gutteridge, 1993). However, such detailed knowledge of the structure and reactions of the photosynthetic apparatus does not in itself provide an understanding of the determinants and regulation of photosynthesis in organisms. Many other intrinsic biological factors, as well as edaphic and climatic factors, can be involved, often interactively, in determining photosynthetic performance of plants. Consequently, the task of resolving the limitations to photosynthesis of plants in different environments can be daunting. In many cases investigations of the factors limiting photosynthesis in an environmentally stressed

470 organism are based on the assumption that the operating systems involved in the photosynthetic process are essentially the same under different environmental conditions and that environmental factors will affect the different component reactions in different ways with a consequential change in overall photosynthetic rate and possibly the rate limiting process or processes. However, this approach is likely to be too simplistic for many field situations where environmental changes not only produce changes in the rate of operation of the photosynthetic apparatus, but also can induce modifications to the composition, organization and functioning of the apparatus. It is clear that adaptation of plants to field conditions often involves developmental processes and extensive turnover of components of the photosynthetic apparatus (Good, 1992). Consequently, in order to achieve a sound understanding of the consequences of environmental change for photosynthesis an integrative approach is generally required which considers not only the biophysics, biochemistry and physiology of photosynthetic mechanisms and processes, but also the genetics and developmental biology of the photosynthetic system and the interactions of the system with other plant processes. The structure of this volume was developed with this in mind. Scientists from a range of disciplines, but all having active research interests in the responses of photosynthesis to environmental change, albeit at different levels of organization, have contributed the initial chapters in which a multitude of important issues are raised in respect to specific research areas of photosynthesis. The later chapters examine the effects of specific environmental variables on photosynthesis and clearly demonstrate the need for a multidisciplinary approach in resolving the limitations to photosynthesis in different environments. This brief overview of some of the issues emerging from the book attempts to identify important problems to be addressed in the future. Clearly it is impossible to be completely impartial in writing such an overview and inevitably the content reflects my background, current interests and limitations. Abbreviations: A – rate of assimilation; – intercellular concentration; D1 – protein component of PS II reaction center core and product of psbA gene; – primary quinone electron acceptor of PS II; Rubisco – ribulose 1,5-bisphosphate carboxylase-oxygenase; UV-B – ultraviolet-B radiation between 290 and 320 nm; – quantum yield of assimilation; – quantum yield of linear electron transport through PS II

Neil R. Baker II. Light Energy Transduction by Thylakoids It has only been in the past two decades that an understanding of the factors determining light energy transduction by thylakoids in vivo has developed from information obtained from non-invasive measurements made on intact leaves and cells. Previously predictions of the limitations to electron transport and ATP synthesis were based primarily on the extensive studies made with isolated thylakoids. A good example of how misleading the application of such information generated with in vitro systems can be is the prediction of the redox state of the primary quinone acceptor of PS II under steady state light conditions in leaves. Prior to the development of the saturation pulse method to estimate photochemical quenching of chlorophyll fluorescence in leaves, it was widely predicted from extensive in vitro measurements on isolated thylakoids that, at steady state electron transport in saturating light, the pool would be highly reduced. However, it is now well established that the quenching of fluorescence due to photochemical quenching by oxidized is considerable in non-stressed leaves operating at steady state photosynthesis in saturating light and plays a major role in limiting photodamage to the PS II reaction centers (see Chapters 2 and 3). Although the exact mechanisms involved with quenching of excess excitation energy in the PS II antennae have not yet been resolved (Chapters 1 and 2), it is clear that this quenching process is an essential feature of the higher plant photosynthetic apparatus. Under non-limiting light conditions linear electron transport in non-stressed leaves appears to be restricted by the rate of oxidation of plastoquinol by the cytochrome complex and modulated in response to sink capacity via changes in the intrathylakoid pH (Chapter 3). Consequently, the PS II antenna quenching would not appear to be a limiting factor in energy transduction by thylakoids and perhaps is better considered as a mechanism to minimize the possibility of overexcitation of the PS II reaction centers and thus limit the rate of photodamage to the D1 protein (Chapters 1–3). Even under moderate light levels there is photoinactivation and degradation of the D1 protein occurring in leaves, which necessitates constant repair (Chapter 4). In order for a leaf to maintain a fully competent photosynthetic apparatus, it is essential that the rate of damage of D1 is compensated for by an equal rate of repair. Under conditions where leaf photosynthesis

Chapter 20 Future Prospects becomes severely sink-limited the excitation energy within the PS II antennae may not be effectively dissipated by photochemistry or non-photochemical quenching processes, the rate of photodamage and loss of D1 will then increase and may become greater than the rate of D1 synthesis. At this point there will be a decline in the population of photochemically competent PS II complexes. Clearly, an increase in antennae quenching of excitation energy can alleviate this situation, but at high light levels with severe sink limitations on photosynthesis the capacity for such an increase may be small. In such circumstances damage to PS II, and eventually to other thylakoid membrane components, could be prevented by transfer of electrons to an alternative acceptor to An obvious candidate for such an electron acceptor would be oxygen (Chapter 5), although reduction of oxygen via a Mehler reaction would result in proton pumping at rates comparable to those found when is being reduced. Consequently, for oxygen reduction to act as an effective protective sink for excess excitation energy either a sink for the ATP produced or a mechanism by which the proton motive force could be uncoupled from ATP synthesis would also be required. The development of non-invasive spectroscopic techniques and portable, easy to use instruments has led to a rapid increase in an understanding of the factors determining the rate of light energy transduction by the thylakoids in vivo as described in Chapter 3 by Genty and Harbinson. More complex techniques and their potential future applications are reviewed by Kramer and Crofts in Chapter 2. Clearly technological advances are resulting in increasingly sophisticated instrumentation with the capability of monitoring a wide range of excitation and electron transfer processes in leaves. However, in the context of resolving the limitations to photosynthetic productivity of plants in the field, it is essential to couple such techniques with simultaneous measurements of and water exchange. It has been argued that for mature, non-stressed maize leaves assimilation can be accurately predicted from modulated fluorescence parameters that estimate the quantum efficiency of PS II (Edwards and Baker, 1993). This prediction is based on the assumption that the relationship between linear electron transport through PS II and assimilation is constant in the leaves. Recent studies of leaves of a maize crop during the early season growing season in the UK, when chilling conditions are experienced, have shown

471 that this relationship is not constant and, in fact, shows very large deviations from the predicted relationship of ca. 12 electrons transported through PS II for each molecule assimilated (Fig. 1; Baker et al., 1995). Similar deviations from the expected relationship between linear electron transport and assimilation in leaves of droughted mangroves are considered by Cheeseman in Chapter

472 8, who suggests that electron flow to oxygen via the Mehler-ascorbate peroxidase cycle (see also Chapter 5) may be an important factor in accounting for this phenomenon. Other possible contributing factors to such deviations in leaves include changes in photorespiration and mesophyll conductance to (see Chapter 8), although this is unlikely to be the case in leaves. To a lesser extent, and only at low light, errors in the estimation of the PS II quantum efficiency by the saturation pulse fluorescence technique may result from quenching by oxidized plastoquinone (see Chapter 2). Clearly such deviations from the expected relationship between linear electron flow and assimilation in stressed leaves requires further investigation, since they may be indicative of the operation of an alternate electron acceptor to This would have important implications for understanding the mechanisms associated with the prevention of photoinhibitory damage to PS II reaction centers in stressed leaves experiencing high light levels.

III. Carbon Metabolism Although the pathways and constituent enzymes involved in carbon metabolism have been known for a considerable time, the constraints on the rate of carbon metabolism under different environmental conditions are complex (Chapters 6 and 7) and not yet fully understood. Considerable advances have been made in understanding the regulatory properties of key enzymes in carbon metabolism, such as fructose 1,6-bisphosphatase, Rubisco and sucrose phosphate synthase (Chapter 6). However, it is still not clear whether such control of enzyme activities is important in determining the rates of sucrose synthesis and export from leaf cells. It has been argued that downregulation of the rate of sucrose synthesis by changes in the levels of fructose 2,6-bisphosphate and the phosphorylation of sucrose phosphate synthase may be essential mechanisms to maintain sufficiently high concentrations of metabolites in the chloroplast to enable the Calvin Cycle to operate and to allow a supply of carbon for other metabolic processes in the leaf, such as starch and amino acid synthesis (Stitt, 1996). Undoubtedly, future studies using genetically manipulated plants with modified levels of enzymes will continue to play a key role in providing an increasingly detailed understanding of the complexity of metabolic regulation and the

Neil R. Baker physiological importance of particular reactions and pathways (see Chapter 12; Furbank and Taylor, 1995). Metabolite regulation of gene expression is playing an increasingly prominent role in understanding the responses of plants to environmental stimuli. It has recently been established that carbon metabolites are involved in regulating the expression of genes coding for components of the photosynthetic apparatus (Sheen, 1994). Consequently it is not at all surprising that metabolites are likely to play a major role in mediating responses of plants to environmental change, and in particular in acclimatory response to increasing atmospheric concentration (Chapter 16). In Chapter 10 Pollock and Farrar speculate that sucrose, besides being the major product of photosynthesis in most plants, may also have a crucial regulatory role in determining rates of leaf photosynthesis and sink growth. It is proposed that sucrose regulates photosynthetic metabolism in leaves by inducing downregulation of genes encoding component proteins of the photosynthetic apparatus, and also controls metabolism in sink tissues by inducing upregulation of genes encoding enzymes involved with sucrose hydrolysis and growth. Clearly these claims warrant further investigation. Although there is an increasing body of information indicating that assimilate status can play an important role in acclimation of plants to environmental changes, it is evident that other factors, particularly nitrogen nutrition and light (see Chapters 11 and 13), can also be centrally involved. Reductionist studies of the mechanisms by which metabolites can induce or repress gene expression are clearly essential for providing the fundamental molecular basis on which to develop an understanding of mechanisms of acclimation to environmental change. However, sufficient evidence now exists to argue strongly that a more holistic approach that considers the interaction of many environmental and physiological factors will be needed to fully understand the role of metabolites in the regulation of plant responses to environmental stimuli.

IV. Leaf Gas Exchange For many years analytical studies of leaf gas exchange, primarily based on the models of Farquhar and von Caemmerer (see Chapter 8), have played a central role in resolving the physiological activities which are limiting for photosynthesis in a given environ-

Chapter 20

Future Prospects

ment. Analyses of plots of assimilation (A) against intercellular concentration commonly referred to as curves, allow resolution of effects associated with Rubisco activity (carboxylation efficiency), the rate of regeneration of ribulose 1,5-bisphosphate and stomatal conductance (Chapters 8 and 17), whereas plots of A against absorbed light enable changes in quantum efficiency to be identified. Such gas exchange studies on leaves, when conducted simultaneously with absorption and fluorescence spectroscopic measurements, provide a very powerful analytical tool for locating potential limiting sites to photosynthesis (see Chapters 2, 3 and 8), which will undoubtedly play a prominent role in future studies of responses of plants to environmental change. However, the value of this approach inevitably is dependent, not only on the precision of the measurements made, but also on the validity of the mechanistic models used to interpret the changes in the parameters being monitored. In this context it is important to be aware that the mechanisms operating under a given environmental condition may not always be the same as those which operate in a different environment, and consequently application of a mechanistically based model appropriate to one environment may be inappropriate for another. Resolution of such problems will often require an integration of studies across a range of disciplines from molecular biology, biochemistry and biophysics to environmental leaf physiology. Heterogeneity of photosynthetic performance is another important factor that warrants serious consideration in the future when interpreting gas exchange data from whole leaves, or sections of leaves. Most models of leaf gas exchange implicitly assume that the photosynthetic capacity of a mature leaf is spatially uniform. However, it is now becoming increasingly evident that the complexity of leaves and the environmental stresses imposed on them frequently result in a spatial and temporal heterogeneity. Perhaps the most widely recognized demonstration of heterogeneity in plants is the patchy stomatal closure in leaves produced by application of the stress hormone, abscisic acid (see Chapters 9 and 14). This heterogeneity of stomatal response results in a non-uniform distribution of assimilation across leaves, as shown by starch staining (Terashima etal, 1988), autoradiography (Downton et al., 1988) and chlorophyll fluorescence imaging (Daley et al., 1989; Mott et al., 1993; Meyer and Genty, 1995) and clearly has implications for photosynthetic

473 performance of leaves under stress conditions. The development of computer-controlled fluorescence imaging instruments which allow fluorescence yield images to be taken at steady state and during a saturating light pulse have enabled quantitative imaging of the quantum yield of electron transport through PS II across leaves (Genty and Meyer, 1994; see also Chapter 2). This rapid, powerful, non-invasive technique will doubtlessly become an increasingly important tool for analyzing environmentally-induced changes in photosynthesis within leaves. Fluorescence imaging studies on the effects of exposure of mature leaves of oil-seed rape to elevated levels of UV-B radiation have demonstrated surprisingly large differences in the quantum efficiency of linear electron transport in adjacent leaf cells which were not apparent from monitoring the leaves with conventional fiber optic probe fluorimeters (Fig. 2). From preliminary studies, this heterogeneity would appear to be associated with UV-B-induced changes in the content and activity of Rubisco (D. J. Allen and N. R. Baker, unpublished; also see Chapter 18). Using a cooled, high resolution charge-coupled device camera attached to an inverted fluorescence microscope it is possible to image successfully chlorophyll fluorescence, and thus the quantum efficiency of linear electron transport, from individual chloroplasts within leaves (Fig. 3). Consequently, this imaging technique will enable rapid resolution of spatial heterogeneity of photosynthetic activity in leaves at tissue, cellular and subcellular levels. Heterogeneity of photosynthetic performance in leaves may be due to a number of different factors. It is evident that leaf growth during exposure to environmental stresses can result in heterogeneity of photosynthetic performance that is considerably more complex than the stomatal patchiness produced by abscissic acid applications. Such heterogeneity in the development of the photosynthetic apparatus is technically difficult to characterize and consequently has not been widely studied, although it would be predicted to play an important role in determining physiological performance and biological success of plants. Recently immunocytological studies of chloroplast development in maize leaves grown at low temperatures demonstrated a surprising degree of heterogeneity in the development of thylakoid proteins and the ability of the leaf to accumulate these proteins when temperature was increased (Robertson et al., 1993; Nie et al., 1995; see also Chapter 15). This heterogeneity takes many forms.

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Chapter 20 Future Prospects Levels of some thylakoid proteins were depressed specifically in some mesophyll cells, but not others; this was particularly the case for chloroplast-encoded thylakoid proteins. The chloroplasts of some mesophyll cells were seemingly entirely devoid of the chloroplast-encoded thylakoid proteins, cytochrome D1 and the and of the ATP synthetase. The distribution of PSI proteins in the thylakoids of bundle sheath chloroplasts was particularly patchy, whereas these appeared to be uniformly distributed in bundle sheath cells of control leaves. It is almost certainly the case that this heterogeneity in the photosynthetic apparatus contributes to the well-documented depressions in the efficiency and capacity of photosynthesis of maize crops grown at low temperatures (Baker and Nie, 1994). Fluorescence imaging techniques capable of resolving differences in photosynthetic performance at the cellular level have the potential to identify cells in which the development of a fully functional photosynthetic apparatus has been prohibited by a given environmental pressure. Consequently, it should be possible using micromanipulative techniques to sample mRNA from such cells, which can then be amplified using a polymerase chain reaction (PCR) and allow the genes which are affected by the environmental condition to be identified by differential display (see Chapter 12).

475 performance and water use efficiency (Chapter 19). Integration of mechanistic leaf photosynthesis models (Chapter 8) with radiation interception models enables calculations of canopy photosynthesis to be made, which consequently allow critical testing of the validity of the more simplistic models generally used to predict canopy productivity over prolonged periods and the effects of specific changing environmental variables on this process. The ability to develop accurate mechanistic models of leaf photosynthesis based on sound molecular and physiological studies is consequently crucial to the development of satisfactory models of canopy productivity. It is hoped that this volume will aid such developments and, perhaps more importantly, that funding agencies will view favorably the potential global implications of the many recent exciting developments in understanding the interactions of the environment with photosynthesis.

Acknowledgments I am grateful to Dan Bush, Julie Lloyd, Steve Long, James Morison, Don Ort, Kevin Oxborough, Christine Raines and John Whitmarsh for many stimulating discussions during the editing of the book and preparation of the manuscript.

References V. Scaling from the Chloroplast and Leaf to the Canopy The contents of this volume have concentrated primarily on leaf photosynthesis and the underlying mechanisms that determine environmental effects on this process. However, in the context of global environmental change and future world food production it is now pressing to develop accurate predictive models for crop productivity and vegetation change. The construction of such models will require an integration of a wide range of information since stress and acclimatory responses to environmental change can occur at many levels of organization, e.g. from thylakoid function to canopy architecture. Particularly important contributions will certainly be made by studies of environmental stress using stable isotopes, which have the potential to integrate information relating metabolism, photosynthetic

Baker, NR and Nie GY (1994) Chilling sensitivity of photosynthesis in maize. In Bajaj YPS (ed) Biotechnology of Maize, pp 465–481. Springer-Verlag, Berlin Baker NR, Oxborough K and Andrews JR (1995) Operation of an alternative electron acceptor to in maize crops during periods of low temperatures. In: Mathis P (ed) Photosynthesis: From Light to Biosphere Vol IV, pp 771–776. Kluwer Academic Publishers, Dordrecht Baltscheffsky M (ed) (1990) Current Research in Photosynthesis. Kluwer Academic Publishers, Dordrecht Daley PF, Raschke K, Ball JT and Berry JA (1989) Topography of photosynthetic activity of leaves obtained from video images of chlorophyll fluorescence. Plant Physiol 90: 1233–1238 Downton WJS, Loveys BR and Grant WJR (1988) Stomatal closure fully accounts for the inhibition of photosynthesis by abscisic acid. New Phytol 108: 263–266 Edwards GE and Baker NR (1993) Can assimilation in maize leaves be predicted accurately from chlorophyll fluorescence analysis? Photosynth Res 37: 89–102 Genty B and Meyer S (1994) Quantitative mapping of leaf photosynthesis using chlorophyll fluorescence imaging. Aust J Plant Physiol 22: 277–284

476 Furbank RT and Taylor WC (1995) Regulation of photosynthesis in and plants: A molecular approach. Plant Cell 7: 797– 807 Good NE (1992) Foreword. In: Baker NR and Thomas H (eds) Crop Photosynthesis: Spatial and Temporal Determinants, pp ix–xi. Elsevier Science Publishers BV, Amsterdam Kühlbrandt W, Wang DN and Fujiyoshi Y (1994) Atomic model of plant light-harvesting complex. Nature 350: 130–134 Mathis, P (ed) (1995) Photosynthesis: From Light to Biosphere. Kluwer Academic Publishers, Dordrecht McDermott G, Prince SM, Freer AA, Hawthornthwaite- Lawless AM, Papiz MZ, Cogdell RJ and Isaacs NW (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374: 517–521 Meyer S and Genty B (1995) Mapping of intercellular molar fraction in Rosa leaf fed with ABA. Significance of estimated from leaf gas exchange. In: Mathis P (ed) Photosynthesis: From Light to Biosphere, Vol V, pp 603–606. Kluwer Academic Publishers, Dordrecht M i c h e l H and Deisenhofer J (1988) Relevance of the photosynthetic reaction center from purple bacteria to the structure of PS II. Biochemistry 27: 1–7 Mott K.A, Cardon ZG and Berry JA (1993) Asymmetric patchy stomatal closure for two surfaces of Xanthium strumarium L.

Neil R. Baker leaves at low humidity. Plant Cell Environ 16: 25–34 Murata N (ed) (1992) Research in Photosynthesis. Kluwer Academic Publishers, Dordrecht Newman J and Gutteridge S (1993) The X-ray structure of Synechococcus ribulose-bisphosphate carboxylase/oxygenase activated complex at 2.2 Å resolution. J Biol Chem 268: 25876–25886 Nie G-Y, Robertson EJ, Fryer MJ, Leech RM and Baker NR (1995) Response of the photosynthetic apparatus in maize leaves grown at low temperature on transfer to normal growth temperature. Plant Cell Environ 18: 1–12 Sheen J (1994) Feedback control of gene expression. Photosynth Res 39: 427–438 Stitt M (1996) Plasmodesmata play an essential role in sucrose export from leaves: A step towards an integration of metabolic biochemistry and cell biology. Plant Cell 8: 565–571. Robertson EJ, Baker NR and Leech RM (1993) Chloroplast thylakoid protein changes induced by low growth temperature in maize revealed by immunocytology. Plant Cell Environ 16: 809–818 Terashima I, Wong S-C, Osmond B and Farquhar GD (1988) Characterization of nonuniform photosynthesis induced by abscisic acid in leaves having different mesophyll anatomies. Plant Cell Physiol 29: 385–394

Index A curve 193, 226, 227, 231, 232, 326, 349, 395, 398, 420, 421, 423 83 83 abscisic acid (ABA) 45, 241–243, 245, 247–249, 349, 358, 359, 363, 473 absorbance 59, 72 cross sections 5, 72 cytochrome f 74 spectroscopy 27, 31, 48, 59 acceptor side-induced photoinhibition 106, 112, 128 acclimation 175, 2 1 1 , 214, 287, 288, 293, 299, 324, 368, 380, 393, 395, 396 growth irradiance 86–90 temperature 380 Acer saccharum 464 acid invertase 272 activase 332 Adenocaulon bicolor 326, 341 ADP 34, 80, 81 ADP/ATP 82 ADPglucose pyrophosphorylase 154, 165, 209, 210, 212, 268, 269 ADPglucose starch synthase 209 Agave americana 206 AGPase 172, 179 flux control coefficients 173 overexpression 179 Agrobacterium tumefaciens 312 air pollution 410, 411, 418 245 aldolase 178 algae 375, 455 alkoxy radical 126 Allium cepa 437 Alocasia 232, 234, 285, 286, 287, 288, 333 macrorrhiza 289, 297, 325, 326, 330, 332, 335, 340, 342 amino acid sequence ascorbate peroxidase 135 monodehydroascorbate reductase 138 aminoethoxyvinylglycine (AVG) 423 amphistomatous 292 anion channels 245–246 ankyrin proteins 313 Antarctic diatoms 374 antenna 5–9, 46 complexes core 4 peripheral 4 pigments 2-21 detrapping 10 light-harvesting function 5 PS II 52 quenching sites 18 size 89 antheraxanthin 28, 32, 36, 37, 39 Anthoceros 455

anthocyanins 444 antioxidant 234 antisense mutations 374,461 rbcS DNA 233 rbcS tobacco plants 174 RNA 312 apoplast 243, 252 guard cell 249 aquatic 391 angiosperms 392 environment 389,391,392 plants 398 Arabidopsis 45, 162, 179, 444, 445 thaliana 309, 315, 393 rca mutant 160 Arachis hypogea 439 Arbutus unedo 351 arctic plants 214 Argyrodendron 288 Arrhenius plots 194 ascorbate 138,412,445 ascorbate peroxidase 132, 135, 136, 138, 139, 141, 142, 144 inhibitors 137 aspen canopies 342 assimilatory charge 336, 340, 342 ATP 29, 53, 68, 71, 77, 80 generation 73 synthase 32, 350, 475 synthesis 54,73, 81, 158 [ATP]/[ADP][Pi] ratio 80 ATP-phosphofructokinase 250 ATPase 81,82,87, 170,438 241, 242 413 Atriplex 202 glabriuscula 212 lentiformis 212 nummularia 212 patula 294 rosea 200 sabulosa 205 autoproteolytic event 115 autotrophs submersed 398 auxin 244,443 AVG. See aminoethoxyvinylglycine avonoid 437 azide 107

B Bacillus subtilis 274 thuringiensis 306 barium 245 barley 263, 272, 311, 371 barnyard grass 398

478 bean 210, 212 French 354, 362 kidney 396 Benson-Calvin cycle 154–156, 162, 177, 197, 214, 224, 231, 316, 370, 371, 472 Beta vulgaris 295 betaïne 356 biotechnology 305 blue fluorescence 32, 34, 57 blue light effects 244 blue light receptor 330 protein 315 Boehmeria cylindrica 393 Boltzman equilibrium 47 boundary layer 170, 454 conductance 173 flux control coefficients 173 Bouteloua gracilis 211 branching enzyme 179 Brassica carinata 443 campestris 443 napus 373, 378 oleracea 439, 442 bronzing 410 brussels sprouts 442 Bryophyllum tubiflorum 205 bryophytes 455 Bryopsis 129 Bt toxin 306 bundle sheath 200, 267, 340, 378 Bunsen coefficient ozone 415

C C24 14

192, 453, 458, 459 intermediates 456 192, 202, 205, 458, 459 cycle 204 enzymes 205 pathways 458 photosynthesis 200–205, 340 chilling sensitivity 202 photosynthetic pathway 453 species 203, 397 tropical grasses 202 C550 34 166, 243, 245-8, 423, 425 balance 429 channel 243, 246 homeostasis 414 cab 378 cabbage 395 cacti 206 Callitriche cophocarpa 399 calmodulin 247, 315 Caltha intraloba 212 Calvin cycle 154–156, 162, 177, 197, 214, 224, 231, 316, 370, 371, 472 intermediates 154 CAM. See crassulacean acid metabolism canopies 323

Index canopy photosynthesis 294 CAP. See chloramphenicol carbamylation 160, 233, 329 carbodiimides 38 carbohydrate 213, 266 content 207 export 207 partitioning 356 production 396 soluble 422, 460 carbon partitioning 224 stable isotopes 452 carbon cycle 388 carbon dioxide. See carbon isotope ratio 341 carbon metabolism 249–253, 373, 472 carbon partitioning starch 207 sucrose 207 carbon reduction photosynthetic 352 carbon reduction cycle photosynthetic 350 carbonic anhydrase 297 carboxyarabatinol 1 -phosphate 161 carboxyarabinitol (CA) 232 carboxyarabmitol 1-phosphate 206, 232, 329 carboxyarabitinol 1,5-bisphosphate (CABP) 158 carboxylation 70, 195, 440 capacity 226, 340 conductance 454 efficiency 193, 198, 202, 227, 391, 394, 473 energetic requirements 71 rate 354, 355 carboxysome 455 carotene 57 4,110, 445 carotenoid 6, 37, 48, 103, 427, 439, 442 electrical field-induced shifts 53 excited states 15 triplet 109 carrot 400 cassava 443 Castanea 231 catalase 134, 2 1 2 , 412 cattails 398 CCM. See concentrating mechanism cdc2a 273 cDNA. See complementary DNA Cecropia 286 cell division 272 cell volume 350, 351 cercosporin 126 156, 328 synthase 329 chalcone synthase 444 chalcone-flavanone isomerase 444 Chara corallina 247 charge separation 10, 106 chemical stress 47 Chenopodium album 360 rubium 267 chilling 198, 316, 233

Index photosynthesis 202, 471 damage to PS I 41 injury 376 sensitivity 376 stress 233 chimeric gene constructs 313 Chlamydomonas 108, 445 reinhardtii 29,45, 373 chloramphenicol (CAP) 102, 103, 116 Chlorella 418,427 sorokiniana 417 vulgaris 372, 373, 374, 375, 379 chlorofluorocarbons (CFCs) 436 chlorophyll 48, 442 a 4, 5 a/b 289, 299 a/b ratio 88, 89, 290, 293, 298 b 6 content 87, 286, 290 electrical field-induced shifts 53 excited states 2 fluorescence 12, 18, 28, 32, 74, 83, 349, 417, 456 singlet state 80 triplet state 6, 32, 84, 107, 126 chlorophyll 670 110 chloroplast biogenesis 306, 307, 377 development 308, 313, 377, 473 genome 307 movement 82 ribosomes 200, 205 surface 291 chlorosis 410, 427 chromanoxy radical 126, 138 chromosome addition 461 circadian control 378 cis-acting elements 313 citric acid 459 citrus 231 Cl 245, 246 Clarkia 165 mutants 165 xanthiana 179 cloud cover 331 clover 272 Clusia minor 459, 460 compensation point 226, 388, 390, 392, 402, 454 concentrating mechanism 389–391, 396, 397, 399, 455 concentration 175, 244, 348, 354 chloroplast 355, 356 intercellular 287, 349, 355, 412, 419, 423 conductance 174 enrichment 399, 400 external partial pressures 453 fixation 68, 69, 70, 198 predicting rates 201 quantum yield 71, 72, 87, 198, 202 photorespiratory 456, 458 respiratory 456, 463 response curves 287, 288 sensitivity 209 transfer conductance 456 uptake 343, 372

479 cold 167 cold acclimation proteins 214 cold hardening 375 cold-responsive genes 309 collard 439 Colocasia 288 Commelina communis 252 compartmentation 265 compensation point 226, 388, 390, 392, 402, 454 complementary DNA (cDNA) 309 library 310 conifers 373, 375 connectivity theorem 170 continuously stirred tank reactors (CSTR) 418 control analysis connectivity theorem 169, 170 deviation index 172 elasticity coefficient 169, 170 flux control coefficient 168, 171 response coefficient 169 control coefficients 170 core antenna complexes 4 cotton 395, 396 4-coumerate-CoA-ligase 444 coupling factor 283, 284, 290, 297, 378 328 cowpea 400 CP24 27, 36–38, 40 CP26 14, 27, 36–38, 40 CP29 14, 27, 36–40 CP43 4, 37, 1 1 5 CP47 4, 37 Crassula argentea 206 crassulacean acid metabolism (CAM) 192, 205, 316, 456, 458, 459 pathways 458 plants 397 Cretaceous 390, 391 Criegee mechanism 414 crop canopies 324, 341 crop ecotypes 462 cryoprotectant 374 CSTR. See continuously stirred tank reactors (CSTR) cucumber 129, 138 Cucumis 286, 296 cuticular conductance 326 cuticular transpiration 349, 356 cyanobacteria 376, 377, 455 cyclic electron transport 52, 73, 82, 125, 362, 372 cyclic photophosphorylation 203 cyclobutane pyrimidine 445 Cyperus papyrus 398 cytochrome b 34, 48 36, 37, 106, 115 32 complex 76–79, 81, 85–87, 283, 284, 288, 290 bc complex 115 bf complex 29, 53, 54 c oxidase 272, 273 f 32, 34, 48, 74, 76, 77, 87, 88, 284, 293, 297, 298, 378, 439, 475 cytosol 243, 250

480 D Dl protein 35, 37, 85, 102, 105, 106, 111, 129, 427, 428, 445, 470, 475 degradation, 104, 112 folding model 105, 113 phosphorylation 1 12 turnover 90, 116, 291 D1/D2 heterodimer 106 D2 protein 37, 105, 106, 111, 445 degradation 114 phosphorylation 112 protein folding model 105 Dactylis glomerata 212 dark respiration 206 DBMIB. See 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone DCCD. See dicyclohexylcarbodiimide DCMU. See 3-(3,4-dichlorophenyl)-1,1-dimethyl urea DCPIP. See 2,6-dichlorophenolindophenol de-epoxidase 32 de-epoxidation 33, 38, 53 violaxanthin 27, 138 dehydroascorbate 140 dehydroascorbate reductase 132, 140, 141 delayed fluorescence 31, 36 delayed light emission 27, 428 delayed luminescence (DL) 46, 47 photometer 47 desA 376 deuterium 458 development 267 developmental response 368 deviation index 172 diacylglycerol 248 di-isopropylfluorophosphate 115 1,2 diacylglyerol (DAG) 247 diamine oxidase 412 diatoms 373 Antarctic 374 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) 114 dicarboxylate carrier 158 2,6-dichlorophenolindophenol (DCP IP) 439 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) 36, 106, 111, 114 dicyclohexylcarbodiimide (DCCD) 38, 40 DIDS. See 4,4' diisothiocyanatostilbene-2,2' disulfonic acid differential display 311 differential expression 312 differential screening 309, 310, 314 diffused beam spectrophotometer 54 diffusion 454 Digitaria decumbens 210 sanguinalis 204 dihydroxyacetone phosphate/phosphate translocator 226 4,4' diisothiocyanatostilbene-2,2' disulfonic acid (DIDS) 246 discrimination 453 instantaneous 459 dissipation of excitation energy PS I 82 PS II 83 dithiothreitol 137 DL. See delayed luminescence DNA damage 445

Index DNA dimerization 445 DNA-binding proteins 311 down regulation 33, 225, 234, 267 PS II 125, 144 drought 348–363 drought-induced stomatal closure 71 duckweeds 398 Dunaliella tertiolecta 212, 375 dyes pH-indicating 34 dynamic model 342

E Echinochloa crusgalli 204, 398 ecological 390 compensation point 390 ecotypes 453, 460 efficiency photochemical 11 Eichhornia crassipes 398 elasticity coefficients 153 Elatostema repens 351 electrical field-induced shifts carotenoids 53 chlorophylls 53 electrochromic change 32, 53, 55 electrochromic shift 34, 48, 50, 53, 55, 199 electron acceptor primary 104 electron donor primary 104 electron transfer chain 28–3, 48 electron transport 26, 68, 70, 78, 226, 227, 231, 333, 374 apparent rate constant 77 capacity 290 control 81 cyclic 52, 73, 362 in vivo 74 limitation 76 pseudo-cyclic 52, 73 site of limitation 75 temperature dependence 197 whole chain 349, 352, 354, 355 Eleusina indica 352 Elodea canadensis 390, 399 Emerson enhancement 56, 88 emission spectra 57 Encelia farinosa 462 end-product inhibition of photosynthesis 207 energetic regulation 14 energetic requirements carboxylation 71 photorespiration 71 energy storage photosynthetic 46 energy-dependent quenching 13, 28, 29, 32, 33, 35–38, 56, 371 enzyme regulation 332 epicuticular waxes 443 epidermis 267 Eragrostis orcuttiana 397 Eriophorum vaginatum 395

Index ethylene 414 ethylene production 422, 423 ethylene release 423 Eucalyptus 211, 212 grandis 230 pauciflora 214 Euphorbia forbesii 340 evaporation 457 evergreens 380 excitation energy non-photochemical quenching 8, 12, 26, 35, 41, 74, 81, 83, 84, 375 transfer 4, 9, 16 excited states carotenoid 15 chlorophyll 2 dynamics 9, 19 lifetime 9, 20 exciton density PS II 84 exciton life-time PS II 84

F 37 83 Fagus 231 sylvativca 340 Faris banding 205 fast vacuolar (FV) channels 245 fatty acids 376 FBPase. See fructose 1,6-bisphosphatase Fe-SOD 130 feedback 202 inhibition photosynthesis 371 ferredoxin 34, 50, 54, 80, 81, 83, 87, 91, 125, 132, 142 Festuca arundinacea 213 fiber optic microprobe 437 field photosynthesis 224 flash measuring-beam kinetic spectrophotometer 53 Flaveria linearis 209 pringlei 196 trinervia 196 flavins 57, 244 flavonoids 444, 445 flavoprotein 244 Flindersia 288, 291 flowering 274 fluctuating light 321–343 fluidity of the membrane 377 fluorescence 12, 26, 31, 33, 41, 43, 50, 51, 55, 57, 82, 224, 234, 471 blue 32 chlorophyll 12, 18, 28 decay kinetics 57 imaging 44, 473 induction 32 measurements 59 pulse-modulated chlorophyll 339 pulsed kinetic 34

481 video imaging 44 yield 33 fluorimeter modulated 41 multi-flash kinetic 44 PAM 44 pulsed kinetic 41, 42 flux control coefficients 153, 168, 171, 173, 181 footprinting 314 forest 323 understories 323 fractionation occurring against 452 free radicals 2, 126, 445 free-air enrichment systems 393 French bean 354, 362 fructan 264, 267, 268, 270, 374 synthesis 374 fructose 1,5-bisphosphate 328 fructose 1,6-bisphosphatase (FBPase) 80, 129, 154, 155, 173, 207, 212, 233, 250, 251, 269, 328, 329, 332, 370, 396, 425, 426, 472 cytosolic 154, 162, 164, 166, 180, 203, 209, 210, 212, 213 plastidic 155, 156, 170, 178 flux control coefficients 173 fructose 1,6-bisphosphate (FBP) 233 fructose 2,6-bisphosphate 162, 251, 269, 472 fructose 6-phosphate 328 fruit abortion 274 fumarase 272, 273 fungal infections 47 fusicoccin 244 83, 234 83

G G-proteins 248, 315 gas exchange 69 gel retardation assays 314 gene phosphoenolpyruvate carboxylase 316 cold-responsive 309 gene expression 166, 207, 262, 267, 270, 307, 472 light-regulated 314 gene regulation 274–275 genetic engineering 306 genomic clone 313 genotype 306 genotypic differences 460 geological time 388 glucan synthase 412 glucans 460 glucose 396 glucose 6-phosphate 250, 251 glutamate 40 glutamine synthetase 129, 143 glutathione 133, 412, 444, 445 glutathione reductase 140, 374 glyceraldehyde-phosphate dehydrogenase 206, 297 glycerate 3-phosphate kinase 205 glycerol 3-phosphate acyl transferase 312 glycine decarboxylase 143, 391 glycolate oxidase 134, 143

482 glycolytic 251 groundwater 458 growth irradiance 285, 287, 288, 292, 294 acclimation 86–90 photosynthetic acclimation 284 GTP248 GTP-binding proteins 245 guard cell 242–244, 422, 442 apoplast 249 plasmalemma 241 ATPase 250

H ATPase 244 electrochemical potential difference 80-82 extruding ATPase 241, 242 extrusion 244 pumps 246 247 73 247 heat stress 370 heat-shock protein hsp-90 115 Hedera helix 230, 275 Helianthus 295 annuus 363 heliotropism 359 Henry’s Law 412 herbicides 45, 306 heterogeneity 473 hexokinase 212, 274 hexose 266, 274 hexose phosphate 275 hexose-repression 274 high energy state quenching 371 h i g h salt stress 47 high temperature 210 stress 47, 205 Hill activity 283–288, 294, 296–298 Holocene 388 Hordeum vulgare 195 host-parasite interactions 463 hydraulic conductivity 230, 292 hydraulic lift 464 Hydrilla verticillata 390 hydrogen stable isotopes 452 hydrogen peroxide 132, 133, 412, 445 scavenging 134, 141 sensitive enzymes 129 production spontaneous disproportionation of superoxide 132 hydroxyl radical 108, 127, 412 hydroxylamine 137 hydroxyurea 137 Hymenoclea salsola 360

I IAA. See indole acetic acid

Index imaging fluorescence video 44, 473, 474 immunocytology 378 Impatiens valeriana 351 in vitro transcription assay 309 indole acetic acid (IAA) 443 induction 324, 326, 329 responses 340 state 232, 325 infrared gas analysis 30 spectroscopy 56 inositol l,4,5-trisphosphate(IP3) 247, 248 insertional mutagenesis 315 intercellular 287 concentration 70 pressure 326 intercellular transport 200 internal concentration 45, 348 intracellular compartmentation 264 intraleaf acclimation 293 intrathylakoid pH 78, 81, 84 invertase 213, 267, 396 invertase genes 274 hexose-repression 274 ion channels 245, 246 ionic control of photosynthesis 425 IP3. See inositol 1,4,5-trisphosphate 1RMS. See isotope ratio mass spectrometers iron-sulfur acceptor complex 41 iron-sulfur centers 372 irradiance 166 isolines 444 isotope effect 453 isotope ratio mass spectrometers ( I R M S ) 453, 457, 458 isotopic composition water 457 isotopic steady state 457, 458

K 242, 243, 244, 245, 246, 247, 252 channels 245, 248 exchange via ATPase reactions 413 salts 243 stimulated ATPase 416 kidney bean 395, 396 kinetic absorbance measurements 48 kinetic fluorimeter 34 kinetic properties 172 kinetic spectrophotometry 48 kinetics rapid fluorescence rise 34

L Laminaria saccharina 373 Larrea divaricata 213 leaf absorbance 87 anatomy 444 chlorophyll fluorescence 353 conductance 249

Index development 291, 442 disc oxygen electrode 349, 351 energy budgets 359 flutter 342 forms 174 gas exchange 472–475 models 224 longevity 295 morphology 283, 442 movement 359 nitrogen 283, 287, 291, 294, 296, 297, 299 protein accumulation 378 proteins 283 pubescence 443 temperature 230, 428 thickness 291, 443 wilting 82 leaf water deficit (LWD) 350, 351, 360 Lemna species 398 LHCII. See light harvesting complex II lichens 455, 456 Liebig’s law of the minimum 70 light absorption 8 excess 2 energy transduction thylakoids 470 fluctuating 322–343 modulation 206 scattering 49 changes 50 stress 379 utilization 359 PS II 360 regulation 349 light-harvesting apparatus 29 light-harvesting complex 27, 36, 37, 38, 289, 290, 361 light-harvesting complex I {LHC I) 5, 378 light-harvesting complex II (LHC II) 5, 13, 27, 36, 37-41, 370, 372, 378, 379 aggregation 18, 19 phosphorylation 13, 20 light-harvesting function antenna 5 light-induced absorbance change around 820 nm 74 light-induced scattering changes 55 light-regulated gene expression 314 light-scattering 32, 48, 55, 199 lightfleck 324, 334–340 lightfleck use efficiency (LUE) 336, 337, 340 utilization 229 lignin 57 lipid 127, 375 peroxidation 445 peroxides 140 synthesis 375, 424 loblolly pine 440, 443 Lolium 264 perenne 272, 402 temulentum 209, 210, 267, 270, 271 low night temperatures 198 low temperature 164, 209, 475 sensitivity 202 stress 47

483 LUE. See lightfleck use efficiency lumenal pH 26, 29 luminescence 34, 107 delayed 47 LWD. See leaf water deficit Lycopersicon esculentum 440

M Macadamia 231 integrifolia 230 macroalgae 399 Macroptilium purpureum 360 magnesium 155 maize 129, 164, 204, 205, 270, 271, 272, 340, 352–354, 360, 378, 379, 460, 471, 473 chloroplast development 473 malate 246, 249, 250, 251 malic acid 459 malondialdehyde 417 manganese cluster 105 mangroves 225 Rhizophora 234 Manihot esculentum 445 mannose 209 Marchantia polymorpha 307 marine phytoplankton 376 maximum apparent quantum yield 352 maximum capacity for photosynthesis 87 maximum quantum yield 362 MDA. See monodehydroascorbate MDA reductase. See monodehydroascorbate reductase Medicago sativa 443 Mehler reaction 68, 71, 73, 78, 234, 339, 359, 361, 362, 471 Mehler-ascorbate peroxidase cycle 234 membrane 413 fluidity 377 permeability 409, 413, 415, 417, 418 potential 245 structure 423 transport 417 viscosity 376 Mesembryanthum crystallinum 316 mesophyll 267 cells 378 conductance 202, 231, 298, 420 resistance 419 metabolic demand 80 meteoric water 458 methane 436 microcompartmentation scavenging systems 141 CuZn-superoxide dismutase 131, 132 microprobe fiber optic 437 mid-day depression 225 Miocene 390, 391 mirage effect photothermal beam deflection 57 mitochondrial 158 mitochondrion 250 Mn-cluster 35 Mn-superoxide dismutase 130 mobilization of starch 210

484 model of photosynthesis 71, 287 modulated fluorimeters 41 molecular genetics 308 molecular techniques 305 monocyclic sinapic acid ester phenolic compounds 444 monodehydroascorbate 126, 132, 138, 139, 144, 234 ferredoxin-dependent photoreduction 139 monodehydroascorbate reductase 132, 138, 139, 141, 142 morphogen 273 mRNA 440 accumulation 378 stability 308 multi-flash kinetic fluorimeter 44 multi-wavelength modulated spectrophotometer 54 mutagenesis 315 insertional 315 random 315 mutants transgenic plants 171 mycorrhiza 422

N NAD(P)H-mediation 125 NADH 57 NADP 30, 32, 34, 76, 80 NADP-glyceraldehyde-phosphate dehydrogenase 129, 205, 212, 328 NADP-malate dehydrogenase (NADP-MDH) 75, 156, 158, 204 NADP-malic enzyme 205, 206 NADP-MDH. See NADP-malate dehydrogenase NADP-ME. See NADP-malic enzyme N A D P H 34, 57, 58, 68, 71, 77, 80, 438 generation 73 pyridine nucleotide 58 [NADPH]/[NADP] 80 near infrared absorbance spectroscopy 31, 34 near infrared ( N I R ) 49, 52 absorbance changes 55 34, 91, 92 necrosis 410, 427 negative thermal modulation 194 Nerium oleander 199, 211, 212, 213, 363, 376 neutral invertase 272 36 Nicotiana tabacum 285, 292, 307 N I R . See near infrared nitrate 165 nitrate reductase 166, 206, 357, 396 nitrate reduction 158, 356 5-nitro-2,3-phenylpropylaminobenzoic acid (NPPB) 246 nitrogen 176, 283, 284, 294 deficiency 299 effect of limiting 175 leaf 291, 294, 296, 297, 299 plant 296 supply 167, 214 thylakoid 285, 286, 289 nitrous oxide 436 Nitzschia closterium 375 non-heme iron 105 non-photochemical quenching 8, 12, 26, 35, 41, 74, 81, 83, 84, 375

Index non-photochemical quenching coefficient 83 non-radiative relaxation (heat) 55 418

NPPB. See 5-nitro-2,3-phenylpropylaminobenzoic acid NPQ. See non-photochemical quenching nuclear run-off analysis 309 nucleolar activity 214 nucleotide sequence 311 numbra 324 nutrients 392, 393, 395, 399, 401, 402 nutrient deficiency 299 nutrition 281, 282–300

O See superoxide 209, 213 solubility ratio 357 composition 464 fractionation 452 oscillations 47 oat, wild 443 OEC. See oxygen evolving complex Oenothera stricta 445 128 Olea europaea 392 ontogeny 268 open-top chambers 393 optimum temperature 211 Opuntia inermis 206 oscillatory behavior 208 osmosis stomatal movements 242 osmotic adjustment 351, 359 osmotic water flow 242 osmotin 314 overwintering 375 oxaloacetate carrier 158 oxidation water 104 oxidative products 410 oxygen active species 127 singlet 126, 128 stable isotopes 452 oxygen electrode 352, 353 oxygen evolution 57, 69, 372 quantum efficiency 72 quantum yield 72, 87 S-state model 42 oxygen evolving complex (OEC) 36, 371, 372 oxygen polarography 34 oxygen production 30 oxygen radical 108, 445 oxygen-evolving complex 29, 41, 42 oxygen-evolving reactions 36 oxygenase 70 Rubisco 352, 354, 355 oxygenase/carboxylase activities 196 oxygenation 70, 195, 357 rate 354, 355 Oxyria 214 digyna 2 1 2

Index ozone 409, 410–429 Bunsen coefficient 415 depletion 436 solubility 414

P p-aminophenol 137 p-nitrodiphenyl ether 126 absorbance changes 34, 91, 92 P680 32, 49, 91, 108, 110 primary electron donor 104

32, 34, 35, 36, 37, 42, 51, 72 triplet 128 P700 21, 32, 48, 49, 52, 53, 74, 77, 78, 79, 80, 83, 91, 298, 372 triplet 83 29, 34, 41, 51, 52 reduction 81 Paleozoic 390, 391 palisade cells 443 palisade parenchyma 424 PAM-fluorimeter 44 Panicum maximum 203, 204, 294 miliaceum 203, 204 virgatum 210 Paphiopedilum tonsum 248 papyrus 398 paraheliotropic 298 paraheliotropism 359, 360 paraquat 126 radicals 127 parenchymatous bundle sheath 267 particle bombardment 312 partitioning 178, 182 between starch and soluble sugar 356 carbon 224 Paspalum conjugatum 340 patch-clamp 244 patchy distributions 45 patchy stomatal behavior 228, 326, 349, 356 PC. See plastocyanin PCO. See photorespiratory carbon oxidation PCR. See polymerase chain reaction PCR cycle. See photosynthetic carbon reduction cycle pea 139, 209, 439 peanut 205, 439 penumbra 324 PEP. See phosphoenolpyruvate PEPC. See phosphoenolpyruvate carboxylase Peperomia obtusifolia 437 Perilla 290 peroxide 412 peroxisomes 132, 133 peroxy radical 126 pH indicating dyes 34 lumenal 26, 29 regulation 245 Phaeodactylum tricornutum 375 phase transition 376 Phaseolus 232 vulgaris 193, 232, 395

485 phenotypic differences 460 responses 453 phenoxy radicals 126 phenylalanine ammonia-lyase 444 pheophytin 35, 36, 104, 439 phloem 265 phosphate 34, 80, 81, 82, 162, 209 limitation 336, 370, 371 optimum 208 sequestering agents 208 status 213 translocator 198, 207, 251, 370 phosphatidylglycerol (PG) 376 phosphoenoipyruvate 250 phosphoenolpyruvate carboxylase (PEPC) 203, 205, 206, 212, 250, 251,316,391,397 genes 316 phosphofructokinase 203, 250, 251 phosphoglucose isomerase 165, 180, 212 phosphoglucose mutase 179, 212 phosphoglycerate kinase 212, 297 phosphoglyceric acid 328, 338, 352 ratio with triose 370 phosphoglycolate phosphatase 209 phosphohexoseisomerase 212 phosphoinositide 247 phospholipase C 247 phospholipid hydroperoxide glutathione peroxidase 141 phosphoribulokinase 154-156, 170, 173, 178, 206, 297 phosphoroscope 48 Becquerel-type 47 phosphorylation D1 112 D2 112 LHCII 13, 20 phosphotidylinositol 4,5-bisphosphate 247 photoacoustic spectrometry 32 photoacoustic spectroscopy 18, 34, 56, 73, 74 photobaric signals 57 photobleaching 427 photochemical efficiency 1 1 , 74, 80, 193 open (oxidized) PS II 353 photochemical quenching (q p ) 8, 12, 41, 76, 83, 234, 353, 371, 470 photochemistry 26 efficiency 6 in vivo 74 PS II quantum yield 353 photodamage 2, 84, 470 acceptor side-induced 106 donor side-induced 109 photodamaged Photosystem II repair 116 photoinactivation donor side-induced 113 PS II 104, 106 photoinduced stress 103 photoinhibition 13, 33, 35, 68, 83–85, 101–117, 127, 198, 202, 225, 350, 361, 368, 375, 378, 427 acceptor-side 128 donor-side 128

486 photoinhibition (cont’d) protection 144 PS II 102–117 photoinhibitory damage 339 photoinhibitory quenching (q1) 13 photolyase 445 photomorphogenesis 314 photon flux density 323 photooxidation 425 photooxidative damage 37 photophosphorylation 199, 208, 337, 350, 439 photoprotection 2, 225, 233, 445 photorepair 437 photorespiration 68, 70, 73, 82, 125, 143, 158, 162, 195, 197, 209, 228, 336, 355–357, 361, 362, 389–391, 396, 454 energetic requirements 71 photorespiratory carbon oxidation (PCO) 389–391, 396, 456, 458 pholorespiratory release 335 photosynthesis feedback inhibition 371 maximum capacity 87 model 71, 287 temperature response 369, 372–375 photosynthetic acclimation 211, 283, 286, 289, 380 growth irradiance 284 photosynthetic apparatus stoichiometry 86 photosynthetic carbon reduction cycle 350, 352, 356, 357 photosynthetic control 77, 226 photosynthetic efficiency 83, 378 photosynthetic electron transport 26, 68, 70, 78, 226, 227, 231, 333, 374 apparent rate constant 77 capacity 290 control 81 cyclic 52, 73, 362 in vivo 74 limitation 72, 76 poising 158 pseudo-cyclic 52, 73 site of limitation 75 temperature dependence 197 whole chain 349, 352, 354, 355 photosynthetic energy storage 46 photosynthetic gene expression 308 photosynthetic oxygenation cycle 357 photosynthetic units size and composition 89 Photosystem I 4, 30, 32, 36, 41, 50, 52, 53, 54, 58, 68, 72, 74, 80, 86, 87, 129, 130, 438 cyclic electron flow 372 dissipation of excess energy 82 light-induced damage 83 photochemistry 82 effective cross-section 88 quantum efficiency 52, 74 reaction center stoichiometry 88 turnover time 90 regulation 20 Photosystem II 4, 31, 36, 37, 39, 41, 43, 45, 54, 56, 68, 74, 80, 86, 87, 102–117, 127, 128, 439 antenna 52

Index Photosystem II (cont'd) charge separation 72 cores 112 dissipation of excess energy 83 donor side mechanism 102 donor side reactions 32 donor side-induced photodamage 109 donor side-induced photoinactivation 113 donor-side photoinhibition 128 down regulation 125, 144 efficiency 199 exciton density 84 exciton life-time 84 light utilization 360 loss of efficiency 83 photochemical efficiency 353 short and long term regulation 85 photochemistry 427, 428 effective cross-section 88 quantum yield 353 photoinactivation 85, 104, 106 photoinhibition 102–117 polypeptides 372 PS II:PS I 289, 299 372 5, 20, 372 quantum efficiency 74, 339, 471 reaction center 445 stoichiometry 88 reaction center complex 104 regulation 85 repair of photodamage 116 thermal inactivation 199 thermal stability 372 turnover time 90 photosystems relative absorbance cross-sections 88 photothermal beam deflection 56, 57 mirage effect 57 photothermal radiometry 34, 56, 74 photothermal signal 57 Phragmites australis 398 phytochrome 174, 314, 315, 316 phytohormones 437 phytoluminography 48 phytoplankton 399 Pi. See phosphate pigment-protein 284, 289 complexes 4, 282, 284, 297 pigments antenna 2–21 pineapple 397 Pinus banksiana 446 sylvestris 445 taeda 440 Piper auritum 327, 330, 331 Pisum 284, 285, 286, 287, 288, 290 sativum 252, 289, 439 plant nitrogen 296 plant water content 358 Plantago major 462, 463 plasma membrane 413, 429 ATPase 416

Index plasmalemma 243, 244-245, 245-246 ATPase guard cell 241, 250 ATPase 250 pump 242 P-type ATPase 244 plastid fructose bisphosphatase 170, 178 plastid gene 307 expression 308 plastid phosphoglucose isomerase 179 plastocyanin (PC) 29, 32, 48, 50, 77, 83, 87, 91 plastoglobuli 424 plastoquinol 29, 43, 76, 78, 81, 85-87 plastoquinone (PQ) 32, 33, 43, 46, 104, 338, 439, 440 reduction state 76 plastosemiquinone anion 439 Poa pratensis 395 poising photosynthetic electron transport 158 polyacrylamide gel electrophoresis 311 polyamines 445 polymerase chain reaction (PCR) 311 polymorphisms 461 polyphenolic compounds 57 poplar 129 Populus deltoides 330 fremontii 323 tremuloides 323, 330, 340 Porphyra yezoensis 399 post-translational modification 378 potassium channels 244–245 potato 129, 138 tubers 210 PPDK. See pyruvate orthophosphate dikinase PQ. See plastoquinone See plastoquinol. precipitation 457 procaine 248 proline 356 protease inhibitors 111 protection from photoinhibition 142, 144 protein blue light receptor 315 DNA-binding 311 import 378 kinase 413 kinase C 247 phosphorylation 1 1 6 soluble 286, 288 synthesis 214 trans-acting regulatory 315 turnover 102 proteolytic cleavage 111 proton ATPase 244 domain 14 extruding ATPase 241, 242 extrusion 242, 244 pumps 246, 248 proton/electron stoichiometry 73 protoporphyrin IX 126 Prunus 231

487 PS I. See Photosystem I PS II. See Photosystem II psbA 440 psbI 106 psbO-less mutant Synechocystis 6803 110 pseudo-cyclic electron transport 52, 73, 82 psychrophilic 369 pteridines 57 pulse-chase labeling 116 pulse-modulated chlorophyll fluorescence 339 pulse-saturation fluorescence techniques 56 pulsed kinetic fluorescence 34 pulsed kinetic fluorimeter 41, 42 pulvini action 82 pumpkin 371 purple bacteria 104 pyrenoids 455 pyridine nucleotide 58 pyrophosphatase 180 pyrophosphate 425 pyrophosphate: fructose 6-phosphate phosphotransfer 180 pyruvate kinase 212 pyruvate orthophosphate dikinase (PPDK) 201, 203, 204, 316

Q Q cycle 30, 73 Q-enzyme 213 See energy-dependent quenching See photoinhibitory quenching See non-photochemical quenching See non-photochemical quenching See photochemical quenching 32, 33 194, 197, 199, 210 32, 33, 36, 42, 76, 83, 104, 108 41, 44, 72 42, 43, 44, 46, 104, 108 Site 106 quantum yield fixation 71, 72, 87, 193, 198, 202, 471 oxygen evolution 72, 87 PS I photochemistry 41, 52, 70,74, PS II photochemistry 52, 70, 74, 202, 339, 353, 402, 471, 473 quencher 82 quenching energy-dependent 371 high energy state 371 mechanisms 14 non-photochemical 8, 12, 26, 35, 41, 74, 81, 83, 84, 375 photochemical 8, 12, 41, 76, 83, 234, 353, 371, 470 quenching sites antenna 18 reaction center 18 Quercus macrocarpa 332 pubescens 393 quinone analogs 57 quinones 57

488 R R-type channel 246 radiation interception 82 radical pair 101 radical scavengers 109 radish 400 Ramonda mykoni 351 random mutagenesis 315 rapeseed 212 Raphanus sativus 251 rbcS-3A 313 rca mutant Arabidopsis thaliana 160 reaction center 4, 32 P680 32, 34, 35, 36, 37, 42, 51, 72, 49, 91, 104, 108, 110, 128, 445 P700 21, 29, 32, 34, 48, 49, 51, 52, 53, 74, 77, 78, 79, 80, 83, 91, 298, 372 reduction 81 quenching 18 quenching sites 18 stoichiometries 88 recombination 55 red light stimulation of stomatal openings 244 redox potential thioredoxin 81 ferredoxin 81 redwood forest understories 341 reed 398 regeneration 226 regulation short-term 262 sucrose 269 regulation of light utilization 349 regulation of PS II 85 regulatory capacity 153, 167 regulatory genes 306 relative humidity 229 relative water content 350 repair process 111 reproduction 400 reproductive growth 400 respiration 207, 272, 273, 275, 388, 389, 392, 400, 401, 417, 441, 454 respiratory 456, 463 reverse 209 Rheum rhaponiticum 442 Rhizophora mangroves 234 Rhodophyta 399 rhubarb 442 riboflavin 57 ribose 5-phosphate 328 ribosomes 205 ribulose 1,5-bisphosphate (RuBP) 69, 159, 269, 324, 352, 356 regeneration 70, 198, 199, 207, 233, 287, 325, 327, 332, 352, 356 capacity 227 limitation 326, 395 substrate 441 ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) 69, 154, 155, 158, 159, 162, 167, 168, 170, 174, 175, 177, 182,

Index ribulose 1,5-bisphosphate carboxylase-oxygenase (cont’d) 195, 196, 198–201, 204, 206, 207, 211, 212, 214, 231, 233, 252, 268, 282, 285, 287, 288, 291, 293–299, 325, 327, 332, 334, 342, 354, 355, 356, 357, 373, 374, 308–397, 389, 400, 402, 414, 420, 424, 428, 429, 436, 440, 452, 454, 472 activase 160, 161, 199, 206, 232, 330, 371, 378 activation 200, 231, 298, 371 carbamylation (activation) state 160 concentration 86 flux control coefficients 173, 176 oxygenase function 143, 199, 352–355 ribulose 1,5-bisphosphate saturation 159, 168 specificity factors 195, 355, 390, 391 ribulose 5-phosphate 328 ribulose 5-phosphate kinase 129, 328, 329 rice 212, 316, 396, 398, 442, 443 chilling sensitivity 316 RNA (Northern) blot 311 root 249, 264, 272 mycorrhiza 422 signal 358 Rubisco. See ribulose 1,5-bisphosphate carboxylase-oxygenase RuBP. See ribulose 1,5-bisphosphate Rumex patentia 441 rye 369, 373, 379 rye grass 402

S S-states 48 model for oxygen evolution 42 42 42 transitions 29 S-type channel 246 sac X 275 sac Y 275 salinity-stress 401 Salix herbacea 392 saturating pulse 33, 45 saturation pulse technique 27 SBPase. See seduheptulose 1,7-bisphosphatase scavenging systems 79, 134 microcompartmentation 141 Scenedesmus 1 1 0 Scirpus 398, 402 olneyi 395, 398 Scots pine 373 seagrasses 399 sedoheptulose 1,7-bisphosphatase (SBPase) 80, 129, 154-156, 162, 170, 206, 212, 328 sedoheptulose 1,7-bisphosphate (SBP) 233, 328 Selanastrum minutum 165 semiquinone 44 semiquinone radical 126, 127 Senecio douglasii 360 senescence 267, 4 1 1 serine-proteases 115 Sesbania sesban 461, 462 shade 215, 283 flecks 324 plants 286 -intolerant species 329

Index signal molecule 265 signal perception 314 signal transduction 315 silicomolybdate 110 singlet 46, 47, 80 oxygen 2, 6, 83, 84, 101, 107, 108 quencher 107 sink 264, 271–274, 398 activity 207, 210 alternative 71 capacity 394, 396 limitation 81, 262, 370 metabolism 272 regulation 167 Skeletonema costatum 214, 373, 374 slow vacuolar (SV) channels 245 snfI 274 SOD. See superoxide dismutase soil drying 249, 358 soil-plant-air research (SPAR) units 393 Solanum aviculare 286 dulcamara 286 sorghum 205 Sorghum bicolor 203, 204, 210 source-sink capacities 396 source:sink ratios 264 soybean 138, 205, 327, 328, 333, 395, 402 canopy 342 trypsin inhibitor 115 Spartina patens 397, 398 townsendii 202 specific leaf weight 442 specificity factor Rubisco 195, 355, 390, 391 spectrophotometer diffused beam 54 flash measuring-beam kinetic 53 multi-wavelength modulated 54 spectroscopy absorption 59 FTIR 111 kinetic 48 near infrared and visible absorption 34 photoacoustic 18, 56, 73, 74 UV absorption 34 spin-polarized chlorophyll triplet state 107 Spinacea 284–287, 290, 293, 296, 298 oleracea 292 spinach 129, 142, 212, 351, 371 cold tolerant 369 Spirodella oligorhiza 443 spongy mesophyll 443 SPS. See sucrose phosphate synthase stable isotope 452–459 starch 172, 209, 210, 249, 250, 251, 252, 270, 396, 398 carbon partitioning 207 degradation 166 mobilization 210 synthase 210 synthesis 165, 179, 181, 182, 357, 374 low temperature 213 State transitions 13, 20, 32, 89, 125

489 steady-state model 342 Stern-Volmer equation 361 stomata 45,71, 168, 170, 228, 241, 330, 348, 418, 422, 442, 473 flux control coefficients 173 stomatal aperture 412 stomatal closure 33, 85, 349, 350, 356, 361, 362 drought-induced 71 stomatal conductance 193, 227, 229, 230, 249, 299, 325, 326, 331, 342, 357, 389, 392, 394, 397, 398, 400, 401, 424, 428, 429, 453, 473 stomatal control 224 stomatal density 392, 393, 462, 463 stomatal efficiency 392 stomatal limitations 231 stomatal models 229 stomatal movements 241, 247 osmosis 242 stomatal opening 47, 329 oscillations 47 stomatal openings red light stimulation 244 stomatal optimization 230 stomatal resistance 45, 357, 369 storage 268 stress chemical 47 drought 348–363 environmental 47 high salt 47 high temperature 47 light 379 low temperature 47 water 45, 82 stromal pH 155 submersed vegetation 391, 398 subsidiary cells 422 subtractive hybridization 311 sucrose 209, 213, 250–252, 262–264, 266, 270, 272–275, 396, 426 carbon partitioning 207 cytosolic 267 sucrose phosphate synthase (SPS) 162, 165, 166, 167, 206, 210, 213, 268, 269, 357, 358, 396, 425, 472 activation state 213 activity increase in cold 167 flux control coefficients 173 phosphorylation 163 sucrose regulation 269 sucrose synthase 210, 213, 252, 272, 426 sucrose synthesis 162, 164, 165, 180, 181, 200, 357, 370, 374 low temperature 213 sucrose-phosphate synthase 154, 180, 210, 212, 252 sucrose-sucrose fructosyl transferase 210 sucrose/starch accumulation 371 sugar soluble 460 sugar pools 263 sugar synthesis 182 suicide inhibitors 137 sulfhydryls 412 sun leaves 174 sun plants 286

490 sun-shade transitions 86, 331 sunflecks 294, 322–343 sunflower 233, 351, 400, 442 superoxide 2, 71, 79, 83, 110, 130–133, 141, 234, 412 superoxide dismutase (SOD) 110, 130–133, 144, 142, 312, 412, 445 susl 272 sycamore 273 Synechococcus 390 lividus 380 psbO-less mutant 110 PCC6803 116, 376, 377, 380

T Taraxacum officinate 209 temperature 71 acclimation 380 compensation 193 dependence electron transport 197 response 194, 195 photosynthesis 369, 372–375 tetcyclis 358 tetraethylammonium 245 thermal breakpoints 205 thermal dissipation 360 thermal inactivation PS II 199 thermal radiometry 27, 32, 55, 56 thermal stability

PS II 372 thermoluminescence 31 thermophilic algae 369 thiol groups 54 thiols 137 thioredoxin 54, 68, 155, 156, 158, 206, 233 redox potential 81 thioredoxin f 328, 329 thioredoxin-regulated enzymes 54 thiyl 138 thylakoid energization 350 light energy transduction 470 lipid composition 378 membranes 438 membrane electrical potential difference 30, 31, 39, 46 nitrogen 285, 286, 289 protein 298 proton electrochemical potential difference 14, 16, 30, 31, 36, 46, 55 stacking 87 Tidestromia oblongifolia 205 time geological 388 tissue culture 273 tissue wounding 422 tobacco 212, 396 antisense rbcS 161, 174 transgenic 160 tocopherol 57, 144, 445 tomato 233, 371, 378, 233 cold sensitive 369

Index tonoplast 243, 246–247 transport processes 246–247 Toona 288 Tradescantia albiflora 252, 290 ohioensis 252 trans-acting factor 313, 314 trans-acting regulatory proteins 315 transcription 270, 308, 313 circadian control 378 in vitro assay 309 transfer conductance 231 456 transfer equilibrium 10 in quenching 18 transgenic plants chimeric gene constructs 313 mutants 171 technology 305, 312 tobacco 233 transition metal ions 126, 127 translocation 210, 370, 422 transpiration 358, 400 efficiency 460 transport processes 417 transthylakoid 80-83, 330, 361 transthylakoidal proton gradient 80-83, 330, 361 Trifolium repens 401 triose phosphate 269, 328, 352, 422, 426 triose phosphate translocator 154, 180 triplet state 36, 55, 101, 125, chlorophyll 6 spin-polarized chlorophyll 107 Triticum aestivum 292, 297 tropical forest 342 understories 341 trypsin inhibitors 140 turgor 249, 265 turnover time PS I 90 PS II 90 two-electron gate 41,43,48 Typha 231, 398 tyrosine 113, 439

U Udotea flabellum 391 UDPglucose pyrophosphorylase 213 ultraviolet-B radiation. See UV-B radiation Ulva lactuca 399 umbra 324 understory aspen 342 redwood forest 341 tropical forest 341 unprimed ADP glucose starch synthase 209 Urtica 291 UV absorption spectroscopy 34 irradiation 35 radiation 44, 47 UV-A (320–400 nm)/blue light 437

Index UV-B 114, 435–446, 473 UV-damage 45

V vacuole 243, 247, 250, 264, 267 valve reaction 109 vanadate 244 vapor pressure deficit 193, 230, 358 vascular tissue 273 veins 267 Vicia faba 245, 246, 251, 252, 272, 295, 420 video imaging fluorescence 44, 473, 474 Vigna luteola 195 violaxanthin 14, 32, 37, 38, 53, 138, 144, 234 deepoxidation 27, 33, 38, 85, 138 viral infection 45, 47 viral resistance 306 vitamin E 445 vitamin K 57

491 water-logging 410, 424 water use efficiency (WUE) 389, 392, 397, 401, 402, 462, 464 wheat 199, 212, 369, 373, 400, 443 endosperm 210 white clover 272 white oak 395 wild-oat 443 winter rye 213 wounding response 413, 416 WUE. See water use efficiency

X Xanthium strumarium 360 strumerium 45 xanthophyll 234, 361 xanthophyll cycle 14, 27, 32, 37, 103 xenon flashlamp 41 xylem ABA concentration 249

Y W water deficiency 249 deficit 348, 349, 350, 356 groundwater 458 isotopic composition 457 meteoric 458 oxidation 104 potential 230, 348 stress 45, 82, 166, 167, 174, 201, 372 uptake 359 water hyacinth 398

42 105 yellow poplar 395 Yucca treculeana 437 105

Z Z scheme 72 Zea mays 369, 441 zeaxanthin 14, 28, 32, 36–39, 53, 138, 227, 234, 361 Zebrina pendula 363