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

eBook ISBN: Print ISBN:

0-306-48135-9 1-7923-4316-6

©2004 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print ©1996 Kluwer Academic Publishers Dordrecht All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's eBookstore at:

http://kluweronline.com http://ebooks.kluweronline.com

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 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 (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.

442

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

443

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.

References Adamse P and Britz SJ (1992) Amelioration of UV-B damage under high irradiance. I: Role of photosynthesis. Photochem Photobiol 56:645–650 Ahmad M and Cashmere AR (1993) HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366: 162–166 Allen LH Jr, Vu CV, Berg RH III and Garrard LA (1978) Impact of solar UV-B radiation on crops and crop canopies. In: UV-B Biological and Climatic Effects Research, Final Report, EPAIAG-D6-168, pp 1–134. USDA-EPA, Washington DC Ambler JE, Krizek DT and Seminiuk P (1975) Influence of UVB radiation on early seedling growth and translocation of 65Zn from cotyledons in cotton. Physiol Plant 34: 177–181 Aro E-M, Virgin I and Anderson B (1993) Photoinhibition of

Chapter 18 Ultraviolet-B Radiation Photosystem II: Inactivation, protein damage and turnover. Biochim Biophys Acta 1143: 113–134 Barber J (1987) Rethinking the structure of the photosystem two reaction centre. TIBS 12: 123–124 Barbato R, Frizzo A, Friso G, Rigoni F and Giacometti GM (1995) Degradation of the D1 protein of the Photosystem II reaction center by ultraviolet-B radiation requires the presence of functional manganese on the donor side. Eur J Biochem 227: 723–729 Barnes PW, Flint SD and Caldwell MM (1987) Photosynthesis damage and protective pigments in plants from a latitudinal arctic/alpine gradient exposed to supplemental UV-B radiation in the field. Arctic Alpine Res 19: 21–27 Barnes PW, Flint SD and Caldwell MM (1990) Morphological responses of crop and weed species of different growth forms to ultraviolet-B radiation. Amer J Bot 77: 1354–1360 Beggs CJ and Wellmann E (1994) Photocontrol of flavonoid biosynthesis. In: Kendrick RE and Kronenberg GHM (eds) Photomorphogenesis in Plants, pp 733–751. Kluwer Academic Publishers, Dordrecht Beggs CJ, Schneider-Ziebert U and Wellmann E (1986) UV-B radiation and adaptive mechanisms in plants. In: Worrest RC and Caldwell MM (eds) Stratospheric Ozone Reductions, Solar Ultraviolet Radiation and Plant Life, NATO ASI Series G, pp 235–250. Springer-Verlag, Berlin Bennett JH (1981) Photosynthesis and gas diffusion in leaves of selected crop plants exposed to ultraviolet-B radiation. J Environ Qual 10: 271–275 Biggs RH and Kossuth SV( 1978) Effects of ultraviolet-B radiation enhancements under field conditions. In: UV-B Biological and Climatic Effects Research (BACER), Final Report. US Environmental Protection Agency, Washington DC Biggs RH, Kossuth SV and Teramura AH (1981) Response of 19 cultivars of soybean to ultraviolet B irradiance. Physiol Plant 53: 19–26 Bogenrieder A and Klein R (1982) Does solar UV influence the competitive relationship of higher plants? In: Calkins J (ed) The Role of Solar Ultraviolet Radiation in Marine Ecosystems, pp 641–649. Plenum Press, New York Bornman JF and Teramura AH (1993) Effects of Ultraviolet-B Radiation on Terrestrial Plants. In: Young AR (ed) Environmental UV Photobiology, pp 427–471. Plenum Press, New York Bornman JF and Vogelmann TC (1988) Penetration of blue and UV radiation measured by fiber optics in spruce and fir needles. Physiol Plant 72: 699–705 Bornman JF and Vogelmann T (1991) The effect of UV-B radiation on leaf optical properties measured with fibre optics. J Exp Bot 42: 547–554 Bornman JF, Evert RF and Mierzwa RJ (1983) The effect of UVB and UV-C radiation on sugar beet leaves. Protoplasma 117: 7–16 Brandle JR, Campbell WF, Sisson WB and Caldwell MM (1977) Net photosynthesis, electron transport capacity and ultrastructure of Pisum sativum L. exposed to ultraviolet-B radiation. Plant Physiol. 60: 165–169 Caldwell MM, Robberecht R and Flint SD (1983) Internal filters: Prospects for UV acclimation in higher plants. Physiol Plant 58: 445–150 Caldwell MM, Teramura AH and Tevini M (1989) The changing solar ultraviolet climate and the ecological consequences for

447 higher plants. Trends Ecol Evol 4: 363–367 Cameron RJ (1970) Light intensity and growth of Eucalyptus seedlings. II. The effect of cuticular waxes on light absorption in leaves of Eucalyptus species. Aust J Bot 18: 275–284 Cen Y-P and Bornman JF (1990) The response of bean plants to UV-B radiation under different irradiances of background visible light. J Exp Bot 41: 1489–1495 Cen Y-P and Bornman JF (1993) The effect of exposure to enhanced UV-B radiation on the penetration of monochromatic and polychromatic UV-B radiation in leaves of Brassica rapus. Physiol Plant 87: 249–255 Chappell J and Hahlbrock K (1984) Transcription of plant defense genes in response to UV light or fungal elicitor. Nature 311: 76–78 Christopher DA and Mullet JE (1994) Separate photosensory pathways co-regular blue light/ultraviolet-A-activated psbDpsbC Transcription and light-induced D2 and CP43 degradation in barley (Hordeum vulgare) chloroplasts. Plant Physiology 104:1119–1129 Day TA, Vogelmann TC and DeLucia EH (1992) Are some plant life forms more effective than others in screening out ultravioletB adiation? Oecologia 83: 513–519 Deckmyn G and Impens CM (1994) The importance of the ratio UV-B/photosynthetic active radiation (PAR) during leaf development as determining factor of plant sensitivity to increased UV-B irradiance: effects on growth, gas exchange and pigmentation of bean plants (Phaseolis vulgaris cv. Label) Plant Cell Environ 17: 295–301 DeLucia EH, Day TA and Vogelmann (1992) Ultraviolet-B and visible light penetration into needles of two subalpine conifers during foliar development. Plant Cell Environ 15: 921–929 Douglas C, Hoffmann H, Schulz W and Hahlbrock K (1987) Structure and elicitor or UV-light stimulated expression of two 4 coumarate coenzyme ligase genes in parsley. EMBO J 6: 1189–1196 Ensminger PA and Schafer E (1992) Blue and ultraviolet-B light photoreceptors in parsley cells. Photochem Photobiol 55:437– 447 Fernbach E and Mohr H (1992) Photoreactivation of the UV light effects on growth of Scots pine (Pinus sylvestris L.) seedlings. Trees 6: 232–235 Garrard LA, Van TK, and West SH (1976) Plant response to middle ultraviolet (UV-B) radiation: Carbohydrate level and chloroplast reactions Soil Crop Sci Soc Fla Proc 36: 184–188 Gausman HW, Rodriguez RR and Escobar DE (1975) Ultraviolet radiation reflectance, transmittance and absorptance by plant leaf epidermises. Agron J 67: 720–724 Gehrke C, Johanson Ulf, Callaghan TV, Chadwick D and Robinson CH (1995) The impact of enhanced ultraviolet-B radiation on litter quality and decomposition processes in Vaccinium leaves from the subarctic. Oikos 72: 213–222 Greenberg BM, Gaba V, Canaani O, Malkin S, Mattoo AK and Edelman M (1989a) Separate photosensitizers mediate degradation of the 32-kDa Photosystem II reaction center protein in the visible and UV spectral regions. Proc Natl Acad Sci USA 86: 6617–6620 Greenberg BM, Gaba V, Mattoo AK and Edelman M (I989b) Degradation of the 32 kDa Photosystem II reaction center protein in W, visible and far-red light occurs through a common 23.5 kDa intermediate. Z Naturforsch 44: 450–452 Howland GP (1975) Dark-repair of ultraviolet-induced pyrimidine

448 dimers in the DNA of wild carrot protoplasts. Nature 254: 160–161 Ikenaga M, Kondo S and Fujii T (1974) Action spectrum for enzymatic photoreactivation in maize. Photochem Photobiol 19: 109–113 Iwanzik W, Tevini M, Dohnt G, Voss M, Weiss W, Graber P and Renger G (1983) Action of UV-B radiation on photosynthetic primary reactions in spinach chloroplasts. Physiol Plant 58: 401–407 Jackson JF (1987) DNA repair in pollen: A review. Mutation Res. 181: 17–29 Jagger J (1967) Introduction to Research in Ultraviolet Photobiology. Prentice Hall, New Jersey Jansen MAK, Depka B, Trebst A and Edelman M (1993) Engagement of specific sites in the plastoquinone niche regulates degradation of the Dl protein in Photosystem II. J Biol Chem 268: 21246–21252 Johanson U, Gehrke C, Björn LO, Callaghan TV, and Sonesson M (1995) The effects of enhanced UV-B radiation on a subarctic heath ecosystem. Ambio 24, 106–111 Jordan BR, Chow WS, Strid A and Anderson JM (1991) Reduction in cab and psb A RNA transcripts in response to supplementary ultraviolet-B radiation. FEBS Lett 284: 5–8 Jordan BR, He J, Chow WS and Anderson JM (1992) Changes in mRNA levels and polypeptide subunits of ribulose 1,5bisphosphate carboxylase in response to supplementary ultraviolet-B radiation. Plant Cell Environ 15: 91–98 Kramer GF, Norman HA, Krizek DT and Mirecki RM (1991) Influence of UV-B radiation on polyamines, lipid peroxidation and membrane lipids in cucumber. Phytochem 30: 2101–2108 Kramer GF, Krizek DT and Mirecki RM (1992) Influence of photosynthetically active radiation on spectral quality on UV-B induced polyamine accumulation in soybean. Phytochem 31: 1119–1125 Krupa SV and Kickert RN( 1989) The greenhouse effect: Impacts of ultraviolet-B (UV-B) radiation, carbon dioxide and ozone on vegetation. Environ Poll 61:263–393 Kubasek WL, Shirley BW, McKillop A, Goodman HM, Briggs W and Ausubel FM (1992) Regulation of flavonoid biosynthetic genes in germinating Arabidopsis seedings The Plant Cell 4L: 1229–1236 Larson RA, Garrison WJ and Carlson RW (1990) Differential responses of alpine and non-alpine Aquilegia species to increased ultraviolet-B radiation. Plant Cell Environ 13: 983– 987 Lee DW and Lowry JB (1980) Young-leaf Anthocyanin and solar ultraviolet. Biotropica 12: 75–76 Li J, Ou-Lee TM, Raba R, Amundson RG and Last RL (1993) Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell 5: 171–179 McLennan AG (1987) DNA damage, repair, and mutagenesis. In Bryant JB and Dunham VL (eds) DNA replication in Plants, pp 135–186. CRC Press, Boca Raton Melis A, Nemson JA and Harrison MA (1992) Damage to functional components and partial degradation of Photosystem II reaction center proteins upon chloroplast exposure to ultraviolet-B radiation. Biochim Biophys Acta 1100: 312–320 Middleton EM and Teramura AH (1994) The role of flavonol glycosides and carotenoids in protecting soybean from uitraviolet-B damage. Plant Physiol 103: 741–752 Mirecki RM and Teramura AH (1984) Effects of ultraviolet-B

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

Chapter 18 Ultraviolet-B Radiation UV-B conditions on photosynthesis in mixed wheat and wild oat canopies. Oecologia 82: 304–310 Sancar WB and Sancar GB (1988) DNA repair enzymes. Annu Rev Biochem. 57: 29–67 Saito N and Werbin H (1969) Evidence for a DNA photoreactivating enzyme in higher plants. Photochem Photobiol 19:389–393 Schulze-Lefert P, Dangl JL, Becker-Andre M, Hahlbrock K and Schulz W (1989) Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene, EMBO J 8: 651–656 Siggel U, Renger G, Stiehl H and Rumberg B (1972) Evidence for electronic and ionic interaction between electron transport chains in chloroplasts. Biochim Biophys Acta 256: 328–335 Sisson WB (1981) Photosynthesis, growth and ultraviolet irradiance absorbance of Cucurbia pepo L leaves exposed to ultraviolet-B radiation (280–315 nm). Plant Physiol 67:120– 124 Sisson WB and Caldwell MM (1976) Photosynthesis, dark respiration and growth of Rtinwx patientia L. exposed to ultraviolet irradiance (288 to 315 nm) simulating a reduced atmospheric ozone column. Plant Physiol 58: 563–568 Steinmuller D and Tevini M (1985) Action of ultraviolet radiation (UV-B) upon cuticular waxes in some crop plants. Planta 164: 557–564 Stewart JD and Hoddinott J (1993) Photosynthetic acclimation to elevated carbon dioxide and U V irradiation in Pinus banksiana. Physiol Plant 88: 493–500 Strid A and Porra RJ (1992) Alterations in pigment content in leaves of Pisum sativum after exposure to supplementary UVB. Plant Cell Physiol 33: 1015–1023 Strid A, Chow WS and Anderson J (1990) Effects of supplementary ultraviolet-B radiation on photosynthesis in Pisum sativum, Biochim Biophys Acta 1020: 260–268 Sullivan JH (1994) Temporal and fluence responses of tree foliage to UV-B radiation. In: RH Biggs and MEB Joyner (eds) Stratospheric Ozone Depletion/UV-B Radiation in the Biosphere, pp 67–76. Spring-Verlag, Berlin Sullivan JH and Teramura AH (1989) The effects of ultravioletB radiation on loblolly pines. I. Growth, photosynthesis and pigment production in greenhouse grown saplings. Physiol Plant 77: 202–207 Sullivan JH and Teramura AH (1990) Field study of the interaction between supplemental UV-B radiation and drought in soybean. Plant Physiol 92: 141–146 Sullivan JH and Teramura AH (1992) The effects of ultravioletB radiation on loblolly pines. 2. Growth of field grown seedlings. Trees 6: 115–120 Sutherland BM (1981) Photoreactivation. Bio Sci 31: 439–444 Takahama U (1983) Redox reactions between kaempferol and illuminated chloroplasts. Plant Physiol 71: 598–601 Teramura AH (1980) Effects of ultraviolet-B irradiance on soybean. I. Importance of photosynthetically active radiation in evaluating ultraviolet-B irradiance effects on soybean and wheat growth, Physiol Plant 48: 333–339 Teramura AH (1983) Effects of ultraviolet-B radiation on the growth and yield of crop plants. Physiol Plant 58: 415–427 Teramura AH (1990) Implication of stratospheric ozone depletion upon plant production. Hort Sci 25: 1557–1560 Teramura AH and Caldwell MM (1981) Effects of ultraviolet-B irradiation on soybean. IV. Leaf ontogeny as a factor in

449 evaluating ultraviolet-B irradiance effects on net photosynthesis. Physiol Plant 57: 175–180 Teramura AH and Murali NS (1987) Intraspecific differences in growth and yield of soybean exposed to ultraviolet-B radiation under greenhouse and field conditions. Environ Exp Bot 26: 89–95 Teramura AH and Sullivan JH (1994) Effects of UV-B radiation on photosynthesis and growth of terrestrial plants. Photosynth Res 39: 463–473 Teramura AH, Biggs RH and Kossuth S (1980) Effects of ultraviolet-B irradiances on soybean. II. Interaction between ultraviolet-B and photosynthetically active radiation on net photosynthesis, dark respiration and transpiration. Plant Physiol 65: 483–488 Teramura AH, Tevini M and Iwanzik W (1983) Effects of ultraviolet-B irradiation on plants during mild water stress. I. Effects on diurnal resistance. Physiol Plant 57: 175–180 Teramura AH, Sullivan JH and Ziska LH (1990) Interaction of elevated ultraviolet-B radiation and on productivity and photosynthetic characteristics in wheat, rice and soybean. Plant Physiol 94: 470–475 Teramura AH, Ziska LH and Sztein AE (1991) Changes in growth and photosynthetic capacity of rice with increased UVB radiation. Physiol Plant 83: 373–380 Tevini M and Steinmuller D (1987) Influence of light, UV-B radiation, and herbicides on wax biosynthesis of cucumber seedlings. J Plant Physiol 131: 111–121 Tevini M and Teramura AH (1989) UV-B effects on terrestrial plants. Photochem Photobiol 50: 479–487 Tevini M, Iwanzik MW and Thoma U (1981) Some effects of enhanced UV-B irradiation on the growth and composition of plants. Planta 153: 388–394 Tevini M, Iwanzik W and Thoma U (1982) The effects of UV-B irradiation on higher plants. In: Calkins J (ed) The Role of Solar Ultraviolet Radiation in Marine Ecosystems, pp 581– 615. Plenum Pub Corp, New York Tevini M, Steinmuller D and Iwanzik W (1986) Über die Wirkung erhohter U V - B - S t r a h l u n g in kombination mit anderen Stressfaktoren auf Wachstum and Funktion von Nutzpflanzen, BPT-Bericht, Gesellschaft für Strahlen und Umweltforschung, München 6: 1–172 Tevini M, Mark U, Fieser G, Saile M, Ros J and Braun J (1989) Action and action mechanisms of enhanced solar and artificial UV-B radiation on growth, composition and function of crop plants. Final Report 1989. Gesellsachaft für Strahlen-und Umweltforschung, München Tevini M, Braun J and Fieser G (1991) The protective function of the epidermal layer of rye seedlings against ultraviolet-B radiation. Photochem Photobiol 53: 329–333 Trosko JE and Mansour VH (1969) Photoreactivation of ultraviolet light-induced pyrimidine dimers in ginko cells grown in vitro. Mutat Res 7: 120–121 Van TK, Garrard LA and West SH (1977) Effects of 298-nm radiation on photosynthetic reactions of leaf discs and chloroplast preparations of some crop species. Environ Exp Bot 17: 107–112 Vierstra RD, John TR and Poff K.L (1982) Kaempferol 3-ogalactoside, 7-o-rhamnoside is the major green fluorescing compound in the epidermis of Vicia faba. Plant Physiol 69: 522–525 Vu CV, Allen LH Jr and Garrard LA (1981) Effects of

450 supplemental UV-B radiation on growth and leaf photosynthetic reactions of soybean (Glycine max). Physiol Plant 52: 353– 362 Vu CV, Allen LH Jr and Garrard LA (1982a) Effects of supplemental UV–B radiation on primary photosynthetic carboxylating enzymes and soluble proteins in leaves of and crop plants. Physiol Plant 55: 11–16 Vu CV, Allen LH Jr. and Garrard LA (1982b) Effects of UV–B radiation (280–320) on photosynthetic constituents and processes in expanding leaves of soybean (Glycine max L. Merr.). Environ Exp Bot 22: 465–473 Vu CV, Allen LH Jr. and Garrard LA (1984) Effects of UV–B radiation (280–320 nm) on ribulosel,5-bisphosphate carboxylase in pea and soybean. Env Exp Bot 24: 131–143 Wellmann E (1974) Regulation der Flavonoidsynthese durch UV-Licht und Phytochrom in Zellkulturen und Keimlingen der Petersilie. Ber Dtsch Bot Ges 87: 267–273 Wilson Ml and Greenberg BM (1993) Protection of the Dl Photosystem II reaction center protein from degradation in ultraviolet radiation following adaptation of Brassica napus L. to growth in ultraviolet-B. Photochem Photobiol 57: 556–563

Alan H. Teramura and Lewis H. Ziska Wingate VPM, Lawton MA and Lamb CJ (1988) Glutathione causes a massive and selective induction of plant defense genes. Plant Physiol 87: 206–210 Wiztum A, Keven O and Even-Chem Z (1978) The effect of UVradiation and sucrose on IAA levels in Spirodela oligorhiza. Ann Bot 42: 595–598 Wright LA and Murphy TM (1982) Short-wave ultraviolet light closes leaf stomata. Amer J Bot 69: 1196–1199 Ziska LH and Teramura AH (1992) enhancement of growth and photosynthesis in rice (Oryza saliva): Modification by increased ultraviolet-B radiation. Plant Physiol 99: 473–481 Ziska LH, Teramura AH and Sullivan JH (1992) Physiological sensitivity of plants along an elevational gradient to UV-B radiation. Amer J Bot 79: 863–871 Ziska LH, Teramura AH, Sullivan JH and McCoy A (1993) Influence of ultraviolet-B (UV-B) radiation on photosynthetic and growth characteristics in field-grown cassava (Manihot esculentum Crantz). Plant Cell Environ. 16:73–79

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

451 452 453 453 453 455 456 457 457 458 458 458 459 459 460 460 462 464 464 464 465 465

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.

452

H. Griffiths

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

453

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)

Chapter 19 Integration Using Isotope Discrimination

455

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

457

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

458

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

Chapter 19 Integration Using Isotope Discrimination

459

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

461

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

462

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

464

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.

References Badger MR, Pfanz H, Budel B, Heber U and Lange O (1993) Evidence for the functioning of a photosynthetic concentrating mechanism in lichens containing green algal and cyanobacterial photobionts. Planta 191: 59–72 Beardall J, Griffiths H and Raven JA (1982) Carbon isotope discrimination and the accumulating mechanism in Chlorella emersonii. J Exp Bot 33: 729–737 Beerling DJ and Woodward FI (1993) Ecophysiological responses of plants to global environmental change since the last glacial maximum. New Phytol 125: 641–648 Beerling DJ, Chaloner WJ, Huntley B, Pearson JA and Tooley MJ (1993) Stomatal density responds to the glacial cycle of environmental change. Proc Roy Soc B 251: 133–138 Borland AM and Griffiths H (1996) Variations in the phases of CAM and regulation of carboxylation patterns determined by carbon-isotope-discrimination techniques. In: Winter K, Smith AP and Smith JAC (eds) Crassulacean Acid Metabolism. Biochemistry, Ecophysiology and Evolution, pp 230–249. Springer Verlag, Berlin Borland AM, Griffiths H, Broadmeadow MSJ, Fordham MC and Maxwell C (1993) Short-term changes in carbon isotope discrimination in the -CAM intermediate Clusia minor L. growing in Trinidad. Oecologia, 95: 444–453 Borland AM, Griffiths H, Broadmeadow MSJ, Fordham MC and Maxwell C (1994) Carbon-isotope composition of biochemical fractions and the regulation of carbon balance in leaves of the CAM intermediate Clusia minor L. growing in Trinidad. Plant Physiol 106:493–501 Broadmeadow MSJ and Griffiths H (1993) Carbon isotope discrimination and the coupling of fluxes within forest

465

canopies. In: Ehleringer J, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp. 109– 129. Academic Press, San Diego Broadmeadow MSJ, Griffiths H, Maxwell C and Borland AM (1992) The carbon isotope ratio of plant organic material reflects temporal and spatial variations in within tropical forest formations in Trinidad. Oecologia, 89: 435–441 Brugnoli E, Hubick KT, von Caemmerer S, Wong SC and Farquhar GD (1988) Correlation between the carbon isotope discrimination in leaf starch and sugars of C4 plants and the ratio of intercellular and atmospheric partial pressures of carbon dioxide. Plant Physiol 88: 1418–1424 Caldwell MM and Richards JH (1989) Hydraulic lift: water efflux from upper roots improves water uptake by deep roots. Oecologia 79: 1–5 Condon AG Richards RA and Farquhar GD (1987) Carbon isotope discrimination is positively correlated with grain yield and dry matter production in field-grown wheat. Crop Sci 27: 996–1001 Cowan 1R, Lange O and Green TGA (1992) Carbon-dioxide exchange in lichens: D e t e r m i n a t i o n of transport and carboxylation characteristics. Planta 187: 282–294 Craig H and Gordon LI (1965) Deuterium and oxygen–18 variations in the ocean and the marine atmosphere. In: Tongiori E (ed) Proceedings of a Conference on Stable Isotopes in Oceanographic Studies and Palaeotemperatures, pp 9–130. Lischi and Figli, Pisa Dawson TE (1993a) Hydraulic lift and water use by plants: Implications for performance, water balance and plant-plant interactions. Oecologia 95: 565–574 Dawson TE (1993b) Water sources of plants as determined from xylem-water isotopic composition: perspectives on plant competition, distribution, and water relations. In: Ehleringer J, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp 465–496. Academic Press, San Diego Dawson TE and Ehleringer JR (1991) Streamside trees that do not use stream water. Nature 350: 335–337 Deleens E and Garnier-Dardart J (1977) Carbon isotope composition of biochemical fractions isolated from leaves of Bryophyllum daigremontianum Berger, a plant with crassulacean acid metabolism: Some physiological aspects related to dark fixation. Planta 135: 241–248 Deleens E, Cliquet J-B and Prioul J-L (1994) Use of plant label near natural abundance for monitoring carbon and nitrogen partitioning. Aust J Plant Physiol 21: 133–146 Eamus D (1991) The interaction of rising concentration and temperature with water use efficiency. Plant Cell Environ 14: 843–852 Ehleringer JR (1993a) Carbon isotope variation in Encelia farinosa: Implications for growth, competition and drought survival. Oecologia 95: 340–346 Ehleringer JR (1993b) Gas exchange implications of isotopic variation in arid-land plants. In: Smith JAC and Griffiths H (eds) Water Deficits. Plant Responses from Cell to Community pp 265–284. Bios Scientific Publishers, Oxford Ehleringer JR and Dawson TE (1992) Water uptake by plants: perspectives from stable isotopes. Plant Cell Environ 15: 1073–1082 Ehleringer JR and Osmond CB (1989) Stable Isotopes. In: Pearcy, RW, Ehleringer JR, Mooney HA and Rundel PW (eds) Plant

466 Physiological Ecology, pp 281–299. Chapman and Hall, London Ehleringer JR and Rundel PW (1989) Stable isotopes: History, units and instrumentation. In: Rundel PW, Ehleringer JR and Nagy KA (eds) Stable Isotopes in Ecological Research, pp. 1– 15. Springer-Verlag, New York Ehleringer JR and Vogel JC (1993) Historical aspects of stable isotopes in plant carbon and water relations. In: Ehleringer J, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp. 9–18. Academic Press, San Diego Ehleringer JR, Hall AE and Farquhar GD (1993) Stable Isotopes and Plant Carbon-Water Relations. Academic Press, San Diego Epstein S, Yapp CJ and Hall J (1976) The determination of the D/H ratio of non-exchangeable hydrogen in cellulose extracted from aquatic and land plants. Earth Planet Sci Lett 30: 241– 251 Evans JR, Sharkey TD, Berry JA and Farquhar GD (1986) Isotope discrimination measured concurrently with gas exchange to investigate diffusion of leaves in higher plants. Aust J Plant Physiol 9: 121–137 Farquhar GD and Lloyd J (1993) Carbon and oxygen isotope effects in the exchange of carbon dioxide between terrestrial plants and the atmosphere. In: Ehleringer J, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp. 47–70. Academic Press, San Diego Farquhar GD and Richards RA (1984) Isotopic composition of plant carbon correlates with water use efficiency of wheat genotypes. Aust J Plant Physiol 11: 539–552 Farquhar GD, O’Leary MH and Berry JA (1982) On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves. Aust J Plant Physiol 9: 121–137 Farquhar GD, Ehleringer JR and Hubick KT (1989a) Carbon isotope discrimination and photosynthesis. Ann Rev Plant Physiol Mol Biol 40: 503–537 Farquhar GD, Hubick KT, Condon AG and Richards RA (1989b) Carbon isotope fractionation and plant water-use efficiency. In: Rundel PW, Ehleringer JR and Nagy KA (eds) Stable Isotopes and Ecological Research, 21–40. Springer-Verlag, New York Farquhar GD, Lloyd J, Taylor JA, Flanagan LB, Syvertsen JP, Hubick KT, Wong SC and Ehleringer JR (1993) Vegetation effects on the isotope composition of oxygen in atmospheric Nature 363: 439–442 Flanagan LB (1993) Environmental and biological influences on the stable oxygen and hydrogen isotope composition of leaf water. In: Ehleringer JR, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp 71–90. Academic Press, San Diego Flanagan LB, and Varney GT (1995) Influence of vegetation and soil exchange on the concentration and stable isotope ratio of atmospheric within a Pinus resinosa canopy. Oecologia 101: 374–44 Flanagan LB, Comstock JP and Ehleringer JR (1991) Comparison of modeled and observed environmental influences on the stable oxygen and hydrogen isotope composition of leaf water in Phaseolus vulgaris L. Plant Physiol 96: 588–596 Flanagan LB, Phillips SL, Ehleringer JR, Lloyd J and Farquhar GD (1994) Effect of changes in leaf water oxygen isotopic composition on discrimination against during

H. Griffiths photosynthetic gas exchange. Aust J Plant Physiol 21: 221– 234 Grace J, Lloyd, J, McIntyre J, Miranda A, Meir P, Miranda H, Moncrieff J, Massheder J, Wright I, and Gash J (1995) Fluxes of carbon dioxide and water vapor over an undisturbed tropical forest in south-west Amazonia. Global Change Biology 1: 1– 12 Griffiths H (1991) Applications of stable isotope technology in physiological ecology. Funct Ecol 5: 254–269 Griffiths H (1992) Carbon isotope discrimination and the integration of carbon assimilation pathways in terrestrial CAM plants. Plant Cell Environ 15: 1051–1062 Griffiths H Broadmeadow MSJ Borland AM and Hetherington CS (1990) Short-term changes in carbon-isotope discrimination identify transitions between and carboxylation during Crassulacean acid metabolism. Planta 181: 604–610 Gutiérrez MV and Meinzer FC (1994) Carbon isotope discrimination and photosynthetic gas exchange in coffee hedgerows during canopy development. Aust J Plant Physiol 21:207–219 Guy RD, Berry JA, Fogel, ML and Hoering TC (1989) Differential fractionation of oxygen isotopes by cyanide-resistant and cyanide-sensitive respiration in plants. Planta 177: 483–491 Guy RD, Fogel ML and Berry JA (1993) Photosynthetic fractionation of the stable isotopes of oxygen and carbon. Plant Physiol 101:37–47 Hall AE, Ismail AM and Menendez CM (1993) Implications for plant breeding of genotypic and drought-induced differences in water-use efficiency, carbon isotope discrimination, and gas exchange. In: Ehleringer JR, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp 349– 370. Academic Press, San Diego Handley LL, Nevo E, Raven JA, Martinez-Carrasco R, Scrimgeour CM, Pakniyat H and Forster BP (1994a) Chromosome 4 controls potential water use efficiency ( C) in barley. J Exp Bot 45: 1661–1663 Handley LL, Odee D and Scrimgeour C (1994b) and patterns in savanna vegetation: Dependence on water availability and disturbance. Funct Ecol 8: 306–314 Henderson SA, von Caemerer S and Farquhar GD (1992) Shortterm measurements of carbon isotope discrimination in several C4 species. Aust J Plant Physiol 19: 263–285 Jones HG (1993) Drought tolerance and water-use efficiency. In: Smith JAC and Griffiths H (eds) Water Deficits. Plant Responses from Cell to Community pp 193–204. Bios Scientific Publishers, Oxford Keeling CD, Whorf TP, Wahlen M and van der Plicht J (1995) Interannual extremes in the rate of rise of atmospheric carbon dioxide since 1980. Nature 375: 666–670. Lange O, Büdel B, Heber U, Meyer A, Zellner H and Green TGA (1993) Temperate rainforest lichens in New Zealand: High thallus water content can severely limit photosynthetic exchange. Oecologia 95, 303–313 Lauteri M, Brugnoli E and Spacciano L (1993) Carbon isotope discrimination in leaf soluble sugars and in whole-plant dry matter in Helianthus annuus L. grown under different water conditions. In: Ehleringer JR, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp 93–108. Academic Press, San Diego Leaney FW, Osmond CB, Allison GB and Ziegler H (1985)

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,

467

the development of leaf area and crop productivity. In: Smith JAC and Griffiths H (eds) Water deficits. Plant Responses from Cell to Community, pp 253–264. Bios Scientific Publishers, Oxford Polley HW, Johnson HB, Marino BD and Mayeux HS (1993) Increase in water use efficiency and biomass over glacial to present concentrations. Nature 361: 61–64 Press MC, Shah N, Tuohy JM and Stewart GR (1987) Carbon isotope ratios demonstrate carbon flux from host to parasite. Plant Physiol 85: 1143–1145 Quick WP, Fichtner K, Schulze E-D, Wendler R, Leegood RC, Mooney H, Rodermal SR, Bogorad L and Stitt M (1992) Decreased ribulose-l,5-bisphosphate carboxylase-oxygenase in transgenic tobacco plants transformed with ‘antisense’ rbcS. IV. Impact on photosynthesis in conditions of altered nitrogen supply. Planta 188: 522–531 Raven JA (1987) The applications of mass spectrometry to biochemical and physiological studies. In: Davies DD (ed) The Biochemistry of Plants, Vol 13, pp. 127–180. Academic Press, New York Richards RA and Condon AG (1993) Challenges ahead in using carbon isotope discrimination in plant-breeding programs. In: Ehleringer J, Hall AE and Farquhar GD (eds) Stable Isotopes and Plant Carbon-Water Relations, pp. 451–462. Academic Press, San Diego Robinson SA, Yakir, D, Ribas-Carbo M, Giles L, Osmond CB, Siedow JN and Berry JA (1992) Measurements of the engagement of cyanide-resistant respiration in the crassulacean acid metabolism plant Kalanchoe daigremontiana with the use of on-line oxygen isotope discrimination. Plant Physiol 100:1087–1091 Rundel PW, Ehleringer JR and Nagy KA (1989) Stable Isotopes in Ecological Research. Springer-Verlag, New York Schulze E-D, Lange OL, Ziegler H and Gebauer G (1991) Carbon and nitrogen isotope ratios of mistletoes growing on nitrogen and non-nitrogen fixing hosts and on CAM plants in the Namid Desert confirm partial heterotrophy. Oecologia 88: 457–462 Sharkey TD and Berry JA (1985) Carbon isotope fractionation of algae as influenced by an inducible concentrating mechanism. In Lucas WJ and Berry JA (eds) Inorganic Carbon Uptake by Aquatic Organisms, pp 389–401. American Society of Plant Physiologists, Rockville, Maryland Smith BN and Ziegler H (1990) Isotopic fractionation of hydrogen in plants. Botanica Acta 103: 335–342 Smith EC and Griffiths H (1996) The occurrence of a pyrenoid is correlated with a concentrating mechanism and carbon isotope discrimination characteristics in lichens and bryophytes. Planta 198: 6–16 Sternberg LO (1989) A model to estimate carbon dioxide recycling in forests using ratios and concentrations of forest air. Agric For Meterol 48: 163–193 Sternberg LO, DeNiro MJ and Ting IP (1984) Carbon, hydrogen and oxygen isotope ratios of cellulose from plants having intermediate photosynthetic modes. Plant Physiol 74: 557– 561 Stewart GR and Press MC (1990) The physiology and biochemistry of parasitic angiosperms. Annu Rev Plant Physiol Mol Biol 41: 127–151 Syvertsen, JP, Lloyd J, McConchie C, Kriedemann PE, and

468 Farquhar GD (1995) On the relationship between leaf anatomy and diffusion through the mesophyll of hypostomatous leaves. Plant Ceil Environ 18: 149–157 Tardieu F and Davies WJ (1993) Root-shoot communication and whole-plant regulation of water flux. In: Smith JAC and Griffiths H (eds) Water deficits. Plant Responses from Cell to Community pp 147–162. Bios Scientific Publishers, Oxford Tissue DT, Yakir D and Nobel PS (1991) Diel water movement between parenchyma and chlorenchyma of two desert CAM plants under wet and dry conditions. Plant Cell Environ 14: 407–413 Treseder KK, Davison DW and Ehleringer J (1995) Absorption of ant-provided carbon dioxide and nitrogen by a tropical epiphyte. Nature 375: 137–139. Vogel JC (1980) Fractionation of the carbon isotopes during photosynthesis. In: Sitzungsberichte der Heidleberger Akadamie der Wissenschaften, mathematisch-naturwissenschaftliche Klasse Jahrgang 1980. 3. Abhandlung, pp. 111– 135. Springer-Verlag, Berlin von Caemmerer S (1989) A model of photosynthetic assimilation and carbon-isotope discrimination in leaves of certain intermediates. Planta 178: 463–474 von Caemmerer S and Evans JR (1991) Determination of the

H. Griffiths average partial pressure of in chloroplast from leaves of several plants. Planta 153: 376–387 von Caemmerer S and Hubick KT (1989) Variation in short-term carbon isotope discrimination among and intermediate species. Planta 178:475–481 White JWC, Cook ER, Lawrence JR and Broecker WS (1985) The D/H ratios of sap in trees: Implications for water sources and tree ring D/H ratios. Geochim Cosmochim Acta 49: 237– 246 Yakir D (1992) Variations in the natural abundance of oxygen18 and deuterium in plant carbohydrates. Plant Cell Environ 15:1005–1020 Yakir D, Osmond CB and Giles L (1991) Autotrophy in maize husk leaves. Evaluation using natural abundance of stable isotopes. Plant Physiol 97: 1196–1198 Yakir D, Berry, JA, Giles L and Osmond CB (1994) Isotopic heterogeneity of water in transpiring leaves: Identification of the component that controls the of atmospheric and Plant Cell Environ 17: 73–80 Zhang JW and Marshall JD (1995) Variation in carbon isotope discrimination and photosynthetic gas exchange among populations of Psudotsuga menziesii and Pinus ponderosa in different environments. Funct Ecol 9 402–412

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.

474

Neil R. Baker

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