Control and Measurement of Photosynthetic Electron Transport in Vivo

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David M. Kramer and Antony R. Crofts high light, and aspects of instrumentation which relate to measurement of critical reactions of photosynthetic electron ...
Chapter 2 Control and Measurement of Photosynthetic Electron Transport in Vivo David Mark Kramer Institute of Biological Chemistry, Washington State University, 467 Clark Hall, Pullman WA 99164, USA

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

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

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

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David M. Kramer and Antony R. Crofts

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

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

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

I. Introduction

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

3. Thermoluminescence

III. What Reactions Can We Measure?

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

A. Optical Techniques

4. Absorbance

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

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

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

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

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

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

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

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