New trends in photobiology

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Journal of Photochemistry and Photobiology B: Biology 26 (1994) 3-27

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New Trends in Photobiology (Invited Review)

Short-term adaptation of plants to changing light intensities and its relation to Photosystem II photochemistry and fluorescence emission Holger Dau Fachbereich Biologie/Botanik, Phih'pps-Universitii~ Lahnberge, D-35032 Marbur~ Germany

Received 20 April 1994; accepted 15 July 1994

Abstract

The rapid response of the photosynthetic system to changes in light intensity (response within less than 30 min) is condidered. Variations in light intensity result in concentration changes in photosynthetic intermediates or protein states. These changes in turn affect Photosystem H (PS n) by a 'backpressure effect', resulting in the accumulation of PS II products (reduced plastoquinone, lumen protons). The product backpressure produces PS II states especially susceptible to photoinactivation and photodamage. By activation of special adaptation mechanisms, the efficiency of the photosynthetic system is optimized and photodamage is minimized. The following aspects are discussed: (1) long-term vs. short-term adaptation; (2) analysis of short-term adaptation by measurement of chlorophyll a fluorescence and photosynthetic oxygen evolution; (3) kinetics of the response of the photosynthetic system to changes in light intensity (induction curves, assignment of phases, time constants); (4) the 'product backpressure' on PS II (accumulation of reduced Q^, lumen pH effect on PS n donor side reactions, thylakoid voltage effect on PS II photochemistry); (5) the molecular mechanisms of short-term adaptation (pH-dependent energy quenching, reversible inactivation of the manganese complex, light-harvesting complex (LHC) phosphorylation); (6) induction of photoinactivation and photodamage; (7) relation between product backpressure, adaptation and photodamage. Keywords: Adaptation; C~lorophyll;Fluorescence; Photoinhibition;Photosynthesis;PhotosystemII

1. Introduction

Although outside the laboratory the sudden exposure of fully dark-adapted plants to bright light is a rare event, rapid changes in light intensity are likely to occur frequently in the natural habitat of the plant. Sources for short-term change in light intensity include water circulation and waves (for algae), the movement of clouds, changes in the mutual shading of leaves by wind or even the moving shade of a grazing cow. All these phenomena can result in changes in the light intensity by one to two orders of magnitude within a few seconds. Taking into consideration that 10 rain of exposure to bright sunlight can be sufficient for severe Photosystem II (PS II) damage (i.e. a decrease in PS II activity which is irreversible within several hours), 1011-1344/94/$07.00 © 1994 Elsevier Science S.A. All rights reserved SSDI 1011-1344(94)07047-4

the importance of protective short-term adaptation is obvious. Variations in light intensity result in concentration changes in photosynthetic intermediates and state changes of proteins; these changes affect the state of PS II. For example, the sudden exposure of a shaded leaf to bright sunlight results in a mismatch between the light-driven electron transport and the capability of the Calvin cycle to accept electrons; an over-reduction of the photosynthetic electron transport chain occurs. The reduction of the electron transport chain takes a few seconds [1,2]. As a result of the reduction of the electron transport chain, an accumulation of PS II with reduced primary quinone acceptor ( Q ^ - ) occurs. PS II in the QA- state is especially susceptible to photoinhibitory damage [3,4]; in this state, a few minutes of

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H. Dau ] J. Photochem. Photobiol. B: Biol. 26 (1994) 3-27

exposure to bright light can result in damage to many PS II entities. However, an increase in light intensity also results in the activation of protection mechanisms. One of these mechanisms is coupled to the formation of the thylakoid pH gradient, which occurs with a time constant of about 10 s [5]. This mechanism is sufficiently fast to provide some protection against detrimental effects resulting from the sudden increase in light intensity. A few minutes later other adaptation mechanisms will be activated (light-harvesting complex (LHC) phosphorylation [6], zeaxanthin formation [7]). This example demonstrates some aspects of importance for short-term adaptation. (1) A variation in light intensity results in concentration changes in photosynthetic intermediates and state changes of proteins. These changes do not occur instantaneously; changes occur with distinct time constants ranging from seconds to minutes. (2) PS II 'responds' to concentration changes and state changes of the photosynthetic system. A 'backpressure' effect (QA reduction due to reduction of the electron transport chain) results in a PS II state susceptible to damage; adaptation mechanisms provide protection. It is necessary to discuss short-term adaptation as a dynamic process; short response times are decisive for effective protection. The physiological role of shortterm adaptation is determined by the kinetics of the response (see Section 3) and the molecular mechanisms of the effect on PS II (see Section 4). To monitor the effect on PS II, measurements of the oxygen evolution of PS II and the fluorescence yield of chlorophyll a (Chl a) are the most prominent experimental tools (Section 2). The molecular mechanisms of photoinactivation and photodamage are only briefly discussed. Because of various recent survey articles [8--12], the xanthophyll cycle itself is not described; however, the role of zeaxanthin in the mechanisms of short-term adaptation of PS II is reported. Only the light response of higher plants and green algae is considered. 1.1. Long-term vs. short-term adaptation

Long-term adaptation involves a change in the balance between the synthesis and degradation of proteins and pigments. A typical example is the decrease in the number of LHC polypeptides per PS II in response to high light intensities [13-15]. This adaptation response is controlled by the antagonistic activity of two photoreceptors [16]. Long-term adaptation by protein and pigment synthesis takes more than 30 min, typically several hours or even several days. Long-term adaptation is usually controlled on the level of gene transcription or translation.

The function of photosynthetic short-term adaptation is twofold: (1) the coordination of the activities of the different components of the photosynthetic system (PS II, PS I, ATPase, Calvin cycle) and (2) protection against photodamage. Both aspects are interrelated. For example, overexcitation of PS II with respect to PS I excitation, i.e. a coordination problem, can result in the accumulation of reduced QA which in turn favours PS II photodamage. Thus optimal coordination of PS II and PSI activity could help to minimize photodamage. Short-term adaptation of the photosynthetic system has similar goals to long-term light adaptation. However, the adaptation mechanisms are distinctly different. Presumably, specialized photoreceptors are not involved in short-term light adaptation of the photosynthetic system. Instead, the concentration of intermediates (e.g. lumen proton concentration, the concentration of reduced plastoquinone) is sensed by proteins. The signal transduction may involve protein phosphorylation (e.g. LHC phosphorylation), conformational changes of proteins, the translocation of proteins and changes in the membrane topology. At the end of the signal transduction chain we find a redirection of fluxes of energy or matter. Because this redirection affects the sensed concentration, a (usually negative) feedback control is achieved. The synthesis of pigments or proteins is not involved in short-term adaptation. Usually shortterm adaptation proceeds within less than 30 min. 1.2. The green plant PS II

Of the known photosystems, PS II is unique with respect to its ability to use the solvent, i.e. water, as a substrate [17]. This astonishing evolutionary achievement has its price: the vulnerability of PS II [3]. The electron donors P680 (a special Chl) and Tyr-Z (Tyr161 of the D1 protein) (see Fig. 1) are characterized by an extraordinary positive redox potential (above + 1 V) which is needed to drive water oxidation. Therefore the pigments and amino acid residues which are direct neighbours of these redox factors are in permanent danger of oxidation, i.e. damage. Also, due to the oxygen evolution process, molecular oxygen is present. Chlorophyll triplets, which are by themselves not harmful, readily react with molecular oxygen to produce harmful singlet oxygen [18]. Due to its extreme vulnerability, PS II seems to be the main victim of photoinactivation and photodamage. Presumably for this reason PS II is the main target of light intensity adaptation of photosynthetic light reactions. PS II is a complex of pigments and proteins (approximately 25 distinct polypeptides) with a total molecular mass of greater than 400 kDa (including associated LHC pigment-protein complexes, see Fig. 1). The functional PS II holocomplex contains 200-300 Chls (about 30% are Chl b), different carotenes and

H. Dau / J. Photochem. Photobiol. B: Biol. 26 (1994) 3-27

t

~k

--

PS II

oz

LHCs

--

PS n Core Complex - -

Fig. 1. T h e green plant PS II. CP24, CP26 and CP29 (and LHCIIa, not shown) constitute t h e minor fraction o f the LHCs. T h e major fraction of the LHCs, which is of the LHCII type, is presumably organized as trimers. T h r e e LI-ICII trimers are shown. T h e indicated a r r a n g e m e n t of CP43, CP47 and LHCs is based on the work of Bassi and Dainese [19] a n d T h o r n b e r et al. [20]. T h e extrinsic 17 kDa, 24 k D a and 33 k D a proteins do not contain pigments; some smaller pigment-free proteins are not shown. T h e top is the o u t e r thylakoid side (stroma side, acceptor side); the bottom is the inner thylakoid side (lumen side, donor side).

xanthophylls. The number of peripheral LHC trimers and the LHC xanthophyll content are determined by short-term as well as long-term light adaptation [8,13-15,21-24]. It is unclear whether (and to what extent) the Chl and carotenoid stoichiometries of the individual pigment-protein complexes are variable. Kfihlbrandt et al. [25] recently determined the structure of the peripheral LHC trimers with 3.4 A resolution. The peripheral LHCs (LHCII, the major LHC type, about 75% of all LHC polypeptides) contain, per monomerle polypeptide, 7-9 Chl a, 6-7 Chl b and two luteins, which are embedded between membrane-spanning helices in the centre of the polypeptide [25]. Unfortunately, the LHC nomenclature is characterized by rapid changes and consequently considerable confusion [26]. In this paper, the nomenclature of Bassi and coworkers is used [19,23]. The following correspondences between proteins and genes (of higher plants) are valid [26]: CP24 = Lhcb6, CP26 ---Lhcb5, CP29-- Lhcb4, LHCIIa = Lhcb3, LHCII = Lhcb2 and Lhcbl. The minor LHCs (CP24, CP26, CP29, LHCIIa) are assumed to have a polypeptide structure similar to the peripheral LHCs; however, they usually contain less Chl b [23]. Presumably, the minor LHCs are located between the PS II core complex and the peripheral LHCs. It has been found that the xanthophylls zeaxanthin and violaxanthin are preferentially associated with the minor LHCs [23]. CP43 and CP47 contain about 20 Chl a each [27-30]; the pigment arrangement is unknown. The structure of the D1-D2 reaction centre heterodimer has been concluded from molecular modelling based on sequence

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homologies with the bacterial reaction centre [31,32]. The precise location of the Mn complex is unknown; however, it has been concluded from spectroscopic investigations that the Mn complex is located in the vicinity of Tyr-Z (for a review, see Debus [17]). Based on X-ray absorption studies, a model for the structure of the Mn complex and its orientation within the thylakoid membrane has been proposed [33-35]. The chemical mechanisms and intermediates involved in the process of water oxidation are still essentially unknown [17,34,36]. It has been proposed that the association of two multimeric core complexes, i.e. two times the core complex constituents shown in Fig. 1, results in a core complex dimer [37]. Possibly, in the stacked grana, the Chl a/b proteins and core complexes are part of a dynamic protein network [38-41] where the simultaneous association of an individual Chl a/b-containing protein with more than one core complex is not excluded. The aggregation dynamics of the proteins embedded in the thylakoid membrane are insufficiently understood (for related reviews, see Allen [21] and Melis [38]).

2. Technical aspects 2.1. In vivo vs. in vitro investigations

The response of PS II to a change in light intensity is determined by the response of the photosynthetic system located in the chloroplast, which interacts with the cytoplasm by membrane transport processes. Therefore the inescapable starting point for studies on the light adaptation of photosynthetic organisms is the exposure of the intact plant to changes in light intensity. (In the following, light which is applied to stimulate an adaptation response of the plant is denoted as actinic light). For the investigation of long-term adaptation, it is often possible to 'harvest' plants in different adaptation states and to analyse the light-induced changes by biochemical means. However, this approach is usually not feasible for analyses of short-term adaptation, because concentrations of intermediates or enzyme states are often not maintained during the biochemical preparation procedure. Therefore the most suitable starting point for the investigation of short-term adaptation is measurements on the intact organism using spectroscopic techniques or electrodes. In a later state, various aspects of the adaptation process can be studied on isolated subunits of the plant (e.g. the thylakoid membrane with its protein complexes) or purified protein complexes provided with an artificial environment which simulates the in vivo situation (pH buffer, salts, electron acceptors, etc.). These in vitro experiments are indispensable for elucidating the molecular details of an adaptation process. However, some care seems to be

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H. Dau / Z Photochem. Photobiol. B: Biol. 26 (1994) 3 - 2 7

advisable; there are numerous examples of misleading conclusions drawn from pure in vitro experiments. 2.Z Experimental techniques The recent progress in our understanding of shortterm light adaptation of photosynthetic organisms is, at least partially, due to technical progress which has resulted in a more widespread use of spectroscopic techniques for studies on intact plants. (For a collection of articles on the use of fluorescence measurements and related spectroscopic techniques in plant physiology and photosynthesis research, see Van Kooten and Snel [421.) In the following, a brief introduction is given into the terms and techniques which are of particular importance for fluorescence studies on short-term light adaptation. This introduction involves some simplifications; a more thorough discussion of the molecular mechanisms of variable PS II fluorescence and its relation to PS II photochemistry is given by Dau [43]. 2.2.1. Chl a fluorescence of intact plants The room-temperature fluorescence emission of plants is predominantly due to the Chl a fluorescence of the PS II antenna Chls; the contribution of PS I fluorescence emission is relatively small, but not fully negligible (typically 1%-5% at 685 nm, 5%-30% above 700 nm [43-46]). At room temperature, the PS II fluorescence yield is not dependent on the excitation wavelength used; the emission spectrum is almost independent of the excitation wavelength [43,47]. Although the excitation and emission spectra of PS II fluorescence usually remain unchanged, the PS II fluorescence yield is variable. 2.2.2. Fluorescence yield measurements The total Chl a fluorescence emission (F,o,) is obtained

by F,ot=CAF~L where CA is a usually constant absorption coefficient, F4, is the fluorescence yield and IL is the intensity of the illuminating light source. Ftot does not only depend on the PS II fluorescence yield (F~,), but also on the light intensity (IL). For example, an increase in light intensity by a factor of 1000 (saturation pulse technique, see Section 2.2.4) can result in a fourfold increase in the fluorescence yield. This corresponds to an increase in F,o, by a factor of 4000. Obviously, a special fluorometer is needed which delivers a signal proportional to the fluorescence yield but not to the total fluorescence emission. A standard instrument for monitoring changes in fluorescence yield is the PAM fluorometer (Walz, Effeltrich) developed by Schreiber and coworkers [48,49].

This instrument employs an intensity-modulated lightemitting diode (modulation with 1.6 kI-Iz or 100 kHz) for excitation at about 650 nm. The amplitude of the modulated fluorescence emission (detected above 700 nm) which results from the modulated excitation is determined by a special correlation technique. The fluorescence emission originating from non-modulated light itself does not contribute to the output signal of the PAM fluorometer. The use of this or other techniques employing a modulated light source (e.g. modulation by a rotating chopper disc) is essential for the so-called saturation pulse technique described below (Section 2.2.4). In the following, the fluorescence yield determined using modulated fluorometry is abbreviated by the letter F, the fluorescence yield in special states of PS II is denoted by the letter F plus subscript or superscript.

2.2.3. Photochemical and non-photochemical quenching Three decay paths determine the fate of excited PS II antenna states: (1) decays by fluorescence emission, (2) non-radiative decays resulting in heat release, and (3) photochemical decays due to initiation of primary charge separation. These decay paths compete with each other. Therefore an increase in the rate of photochemistry results in decreased heat release and decreased fluorescence emission. This type of fluorescence decrease is usually denoted as 'photochemical quenching'. Photochemical quenching is characterized by an increase in the photochemical yield (of an ensemble of PS II entities) occurring concomitantlywith a decrease in the fluorescence yield. The photochemical yield can be monitored by measurement of the PS II oxygen evolution (see Section 2.2.8). Fluorescence quenching which does not result from the use of light energy for driving PS II photochemistry is denoted as 'non-photochemical quenching'. A possible origin of non-photochemical quenching is an increase in the thermal deactivation probability (see Section 4.2.1). Increased thermal deactivation results in a decrease in photochemistry and fluorescence emission, because both decay paths compete with the thermal decay path for excited state energy. Thus a (more or less) parallel decrease in the photochemical yield and fluorescence yield is indicative of non-photochemical fluorescence quenching. 2.2.4. FM', the fluorescence yield in the presence of reduced QA In the presence of reduced QA, any further PS II photochemistry resulting in QA reduction is inhibited ('closed reaction centre' or 'closed trap') and the fluorescence yield is maximal. The fluorescence yield in the presence of fully reduced QA (reduced QA present in all PS II) and in the absence of any oxidized P680 is denoted as FM or FM'. According to the nomenclature

H. Dau I Z Photochera. Photobiol. B: Biol. 26 (1994) 3 - 2 7

of Van Kooten and Snel [50], FM is exclusively used in plants that are fully dark adapted, whereas FM' denotes the fluorescence level in the reduced QA state of plants which were exposed to light prior to the determination of FM'. (Because of its widespread use in plant physiology, the nomenclature of Van Kooten and Snel [50] is applied throughout this article. Often a distinction between FM and FM' is not necessary and the prime is dropped as in the related review of Dau [43].) By application of 300-1200 ms light pulses of saturating intensity (500-5000 W m-2), a complete reduction of QA can be achieved, whereas no significant accumulation of P680 + seems to occur [49,51,52]. Using this saturation pulse technique, FM' can be determined repeatedly (but not continuously) during the adaptation period of the plant to a change in fight intensity. The FM' level is affected by non-photochemical quenching but not by photochemical quenching. Thus the quenching of FM' is characteristic of non-photochemical fluorescence quenching. 2.2.5. Fo', the fluorescence yield in the presence o f oxidized

QA For fully oxidized QA (oxidized QA in all PS II), the photochemical fluorescence quenching is maximal and a minimal level of the fluorescence yield, called Fo (fully dark-adapted plants) or Fo' (after light exposure), is approached. The photochemical yield (see Section 2.2.8) in the presence of fully oxidized Qa is denoted as t/~p°. An estimation of Fo' for plants exposed to actinic light can be obtained by removal of the actinic light and the addition of far-red light (PS I light) for about 3 s (see, for example, Dau and Hansen [53]). The far-red P S I light results in quinone oxidation and the resulting fluorescence level is close to Fo'. In comparison with the determination of FM' by the saturation pulse technique, the determination of Fo' is clearly more critical. It is not always possible to achieve full QA oxidation before relaxation of non-photochemical quenching occurs. Moreover, a stray fight artefact of the PAM fluorometer (Walz, Effeltrich) can lead to erroneous Fo' determinations. Non-photochemical quenching which results from an increase in the rate of non-radiative decay of excited antenna states is characterized by the quenching of Fo' [43]. 2.2. 6. Quenching coefficients

The photochemical quenching coefficient qr (sometimes also denoted as qo or qQ) is calculated according to qv = ( F M ' - F ) / ( F M ' - F o ' )

(la)

where F is the fluorescence yield in the presence of usually subsaturating actinic light, FM' is the fluores-

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cence yield as determined by the saturation pulse method and Fo' is the fluorescence yield as determined by removal of the actinic light and application of far-red light as described above. The quenching coefficient qv is related to the degree of PS II trap closure due to reduced QA. For qp= 1, all QA are assumed to be in an oxidized state; for qp =0, all QA are assumed to be in a reduced state. Due to excitation energy transfer between PS II units (PS II connectivity), the fraction of PS II with oxidized QA is not necessarily linearly related to qp as discussed elsewhere [43]. Other quenching coefficients are in use as a measure of the extent of non-photochemical fluorescence quenching. The non-photochemical quenching coefficient qN is defined as follows [50] qN = 1 - (FM' - Fo')/(Fu - Fo)

(lb)

The term 'non-photochemical quenching' denotes the combined effect of different types of non-photochemical quenching: energy quenching (q,~), state transition quenching (q-r), irreversible quenching or photoinhibition quenching (qi) and others. Experimental procedures have been proposed to determine the relative contributions of various qr~ constituents [54-57]. Energy quenching and state transition quenching are discussed in detail in Sections 3.4, 3.6 and 4.2. 2.2.Z Relation between PS I I photochemistry and fluorescence parameters

The relation between the fluorescence yield and photochemical yield depends on the mechanisms of excitation energy transfer between PS II pigments and on PS II charge separation reactions. In order to obtain quantitative relations, the use of a quantitative model is inevitable. In plant physiology, the bipartite model of Butler and coworkers has been particularly popular [58,59]. However, this model is not in agreement with recent experimental results of picosecond measurements [43,60-62]. The reversible radical pair model (RRP model) of the Holzwarth group [62] seems to be more appropriate. The central assumptions of the RRP model (significant reversibility of primary charge separation, electric-field-dependent charge separation rate constants, rapid exciton equilibration) have been experimentally verified [47,63--66]; the significance of PS II heterogeneities, however, is still unclear [43,44,67]. The photochemical yield of PS II is defined as the quantum yield of QA reduction. The photochemical yield of an individual PS II entity with oxidized QA is denoted as q~,o (or ~o°). The effective photochemical yield for an ensemble of PS II entities with a fraction of PS II in the closed state (QA reduced) is denoted as q~p (or ~o)The photochemical yield of QA reduction is not identical with the photochemical yield of primary charge

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H. Dau I J. Photochem. Photobiol. B: Biol. 26 (1994) 3 - 2 7

separation. In PS II with reduced QA the primary charge separation, i.e. P680÷/Pheo - (pheo, a pheophytin molecule bound to the D1 protein) formation, can still proceed [62--64], but the yield of QA- formation is zero. In the presence of reduced QA, the yield of P680+/Pheo- formation is diminished due to the electric field which results from the negative charged Q ^ [43,63',65]. It has recently been shown [43] that the relations between the fluorescence parameters (F, FM', Fo') and the photochemical yield of PS II are essentially the same for the bipartite model of Butler and coworkers [58,59,68,69] and for the more recent RRP model of Holzwarth and coworkers [62]. However, the interpretation of non-photochemical FM' quenching in the absence of Fo' quenching (occurring on illumination with photoinhibitory light) is not model independent (for a more detailed discussion, see Dau [43]). For both models ~p=qp~p°

(2)

holds (qp according to Eq. 1). This relation allows a hypothetical qr~,° value to be calculated by dividing the detected quantum yield by qp. An estimate for ~p can be obtained, e.g. by measurement of the oxygen evolution of PS II. • When non-photochemical fluorescence quenching results from an increased rate of excited (antenna) state decay by thermal deactivation, for both models the following set of relations holds (for derivations see Dau [43])

~pot(Fu'-F)/FM'

(3)

~p° ctFo'

(4)

• F°

(5)

(FM'-Fo')/FM'

and

1/Fo' = 1/FM' + Co

inactive PS II centres which are not connected to the pool of active centres by excitation energy transfer).

2. 2.8. Measurement of the photochemical yield There are several approaches for the detection of changes in the photochemical yield of QA reduction. The well-studied DCMU (3(3,4-dichlorophenyl)-l,l-dimethylurea) induction curves should reflect changes in Or° [43]. Single turnover flash saturation curves (detection of fluorescence or electrochromic absorption changes) can results in an estimate of Op [71-74]. However, using these techniques, the time course of the photochemical yield after a change in light intensity is not accessible. To study time courses of the photochemical yield, oxygen evolution measurements have been employed [1,6,75-79]. The conventional oxygen electrode detects the oxygen concentration in an aqueous medium. To obtain a measure of the photochemical yield, the derivative (slope) of the oxygen concentration needs to be calculated. Usually the accuracy and temporal resolution of this approach are not satisfactory. For algae, a more direct measure of the yield of photosynthetic oxygen evolution is obtained using a rate electrode system with modulated measuring light [80]. Presently, for leaves, the only approach which allows the determination of the oxygen evolution yield with sufficient temporal resolution is the use of photoacoustic measurements [81,82] (for a recent review, see Fork and Herbert [83]). Both rate electrodes and photoacoustics do not detect respiratory oxygen uptake; however, both techniques are possibly affected by oxygen uptake resulting from the Mehler reaction [78,84,85]. Recently, Reising and Schreiber [86] proposed that, under some circumstances, the pH-dependent CO2 solubilization in chloroplast stroma can also result in a gas uptake contribution to the photoacoustic signal.

(6)

where Co is a constant which is, in contrast with F0' and FM', independent of the rate constant of nonradiative decay of excited antenna states [43]. The validity of these four relations (Eqs. (3)-(6)) is in agreement with the assumption that non-photochemical quenching results from increased thermal deactivation of excited antenna states. In the case of efficient excitation energy transfer between PS II units, nonphotochemical quenching by other mechanisms may result in a linear relation between ~ , and ( F M ' - F ) / FM' as predicted by Eq. (3) [70]. Deviations from Eqs. (3)-(6) can be explained by non-photochemical quenching which has its roots in processes involving the PS II reaction centre complex, e.g. an effect on primary or secondary charge separation. Another possible explanation for deviations from Eqs. (3)-(6) may be significant permanent heterogeneities (e.g. a/fl heterogeneity) or transient heterogeneities (formation of

2.3. Quantitative analysis of time courses A change in light intensity results in a multiphase response of the fluorescence yield and PS II photochemical yield (see Fig. 2, Section 3). Transients are readily observed; however, a meaningful quantitative description is not a trivial task. Occasionally the magnitude of the peaks in the transient are evaluated (e.g. Refs. [87-89]). The peak height is determined by a first process responsible for the increase and a second process responsible for the subsequent decrease. A decrease in peak height is not necessarily an indication that these two processes are less active or efficient. A decrease may result from the acceleration of the second process responsible for the peak. Therefore the peak height is usually not a particularly meaningful parameter.

H. Dau I J. Photochem. Photobiol. B: Biol. 26 (1994) 3 - 2 7

changes of QAredox state

Often time courses of an 'output signal' (e.g. the fluorescence yield) which result from a stepwise change of an 'input signal' (e.g. light intensity) are described by a sum of exponential functions [90-92]

y(t) =Yo + ~ 1 , ( 1 - e-'/r') i

9

I

[

changesof lumen pH

p

I

[ LHC dephosphorylation [

(7)

where t is the time after the stepwise change in light intensity, y(t) is the output signal as a function of time, Yo is the stationary level of the output signal before the stepwise change in the input signal is initiated, A~ is the amplitude factor, T~ is the time constant and i is the summation index. A description according to Eq. (7) is reasonable only if the response is indeed given by a sum of exponential functions. This is the ease for 'linear systems', which can be described by a set of linear different equations. The responses of the PS II fluorescence and photochemical yields, however, are often 'non-linear', i.e. not exponential. Therefore Hansen and coworkers [1,93-95] introduced the use of 'linearizing experimental conditions' for kinetic studies in photosynthesis research. Examples of linearizing experimental conditions are frequency response measurements (sinusoidal modulation of the light intensity with a sequence of different frequencies), which allow the determination of the amplitude factors Ai and the time constants T~ for different output signals (fluorescence yield, yield of oxygen evolution, absorbance changes, etc.) [1,2,93,96]. The kinetic parameters Ai and T~ determined under linearizing experimental conditions represent the linear part or linear kernel of the real step response.

3. Phases of the adaptation kinetics

The response of a plant to a change in light intensity is multiphase. Figure 2 shows a schematic representation of the response of the fluorescence yield and oxygen yield to a stepwise increase in light intensity from I^ (e.g. 10 W m -2) to IB (e.g. 30 W m-2). At least seven kinetic phases can be distinguished by analysis of fluorescence transients, transients in the yield of PS II oxygen evolution or absorption changes [1,94,95,97]. Using the techniques of linear kinetic analysis, these seven phases can be described in terms of amplitude factors and time constants as discussed above (Section 2.3). The values of the amplitude factors and time constants depend on the light intensities investigated; the time constants increase at higher light intensities [94,95,97]. Often not all of the seven phases are detectable. In Table 1 some characteristics of the individual kinetic phases are summarized. In the following, the adaptation response is discussed with the assumption that each time constant labels a distinct reaction or process, usually the formation or

$ ~ y F l u o r e s c e n c e

0

~ ......~..........1..... I

ls

I

lOs

I

lOOs

I

lO00s

IB IA I

Light Intensity

Fig. 2. Response of the PS II fluorescence yield and photochemical yield to an increase in light intensity (schematic representation). The logarithmic time scale gives the time after the increase in light intensity from I^ to lB. The numbers in squares denote the kinetic phases discussed in Section 3. The letters O, I, D and P are used to denote the original, initial, dip and peak level respectively of the fluorescence transient. The bars at the top indicate the time ranges for detectable changes in the concentrations of PS II with reduced Q^, lumen protons and dephosphorylated LHC proteins respectively.

decay of an intermediate product or state (for an overview, see Table 1 and Fig. 3). The time constants are numbered as proposed by Hansen and coworkers [1,94]; the response to a stepwise increase in light intensity (as shown in Fig. 2) is discussed.

3.1. T1 - QA reduction and increase in thylakoid membrane voltage The 7"1 phase is characterized by an increase in the fluorescence yield and a decrease in the oxygen yield [1], a behaviour which is typical of decreasing photochemical quenching of the PS II fluorescence. A more detailed analysis of the fluorescence response has revealed that this component is composed of several kinetic components with time constants in the millisecond time range [2,96]. Dau et al. [96] resolved four components in the fluorescence yield response with time constants of about 1, 5, 20 and 100 ms. The 1 ms component was assigned to an increase in the concentration of reduced QA; the 5 ms component has been demonstrated to result from a fluorescence increase due to an increase in the thylakoid membrane

10

H. Dau / Z Photochem. PhotobioL B: Biol. 26 (1994) 3-27

~q

"6

¢J

r~ O o . ~

"~ o

O

TSb - 180s

ATe

T 6 ~4005

[]

[] ~

C°2

.o ~.~ ~

Fig. 3. Assignment of time constants to changes in the concentration of intermediates or states of proteins. Thick black arrows indicate electron transport; open arrows indicate uptake or release of protons. Qa, primary quinone acceptor of PS II; Qb, secondary quinone acceptor of PS II; PQ, mobile plastoquinone; Cyt b/f, cytochrome b/f complex; PC, plastocyanin; Fd, ferredoxin; Phos, label for LHC (de)phosphorylation; FNR, ferredoxin-NADPH reductase (NADPH, reduced nicotinamide adenine dinucleotide phosphate).

g'.÷

m

Vl~

m

e~ m

~ ~

~.~

N o

m x..,

¢q O

v~

O e~

c

o "O

.~.~ 7.~ No

o

8

o~!9

3.2. T2 -- changes in the plastoquinone pool redox state

B

.8

i

voltage [46,65,96]; the 20 ms component is presumably related to reactions at the QB binding site. The origin of the 100 ms component is still unknown. The 7"1 phase corresponds to the O-I phase of the fluorescence induction curve of dark-adapted plants. It has been proposed that the O-I fluorescence increase mainly results from trap closure (by Q^ reduction) of PS II units of the 'QB-non-reducing' type (also called 'non-B type PS II') [98]. However, the assumption that the Q--I phase of dark-adapted chloroplast is only determined by QB-non-reducing PS II has been questioned [99-101]. As far as the light increase response of light-adapted plants is concerned, a major contribution stemming from QA reduction of QB-non-reducing units seems to be unlikely, because before the increase in light intensity the Q^ of most QB-non-reducing PS II should be in light-reduced state. Also, the observed 7"1 decrease in the oxygen yield cannot be reconciled with a major contribution of QB-non-reducing PS II units to the TI phase. In conclusion, the T1 phase results mainly from an increase in the concentration of reduced Q^ and, to a minor extent, from a fluorescence increase due to the higher thylakoid voltage.

After an increase in light intensity, the 7"2 phase is usually characterized by a decrease in the fluorescence yield and a simultaneous increase in the photochemical yield [1,93]. However, sometimes the T2 phase is absent

H. Dau / Z Photochem. Photobiol. B: Biol. 26 (1994) 3-27

or a fluorescence increase (oxygen yield decrease) is observed [2]. The direction of the T2 phase (increase or decrease) depends on the extent of stimulation of the P S I electron flux relative to the stimulation of the PS II electron flux by the actinic light applied to increase the light intensity from IA to IB [2]. Mainly because of this differential effect of PS II light vs. P S I light on the direction of the Tz fluorescence changes, Hansen and coworkers [1,2,93] suggested that the T2 phase results from redox changes of the plastoquinone (PQ) pool. For example, overexcitation of P S I with respect to PS II results in an oxidation of the PQ pool; the presence of more oxidized PQ pool molecules results in more PS II with oxidized QA, i.e. less PS II trap closure and lower fluorescence. The assignment of the T2 phase to changes in the PQ pool redox state is in agreement with results obtained for the I-D phase of the fluorescence induction curve of dark-adapted plants [102,103]. 3.3. T3 - reduction of ferredoxin and other PS I electron acceptors

The T3 phase reflects the reduction of electron acceptors located in the electron transport chain behind P S I [1,2,104]. The resulting 'electron backpressure' leads to the reduction of the plastocyanin (PC) pool, the PQ pool, the secondary PS II quinone acceptor QB and, eventually, of QA. Thus PS II trap closure (decrease in photochemical quenching, fluorescence yield increase, decrease in PS II photochemical yield) is the result. At present, it is not clear whether T3 labels the redox change of a 'ferredoxin pool' or the redox changes of an electron acceptor pool consisting of ferredoxin (Fd) plus nicotinamide adenine dinucleotide phosphate (NADP). The experiments of Satoh and Katoh [88] and Pschorn et al. [105] suggest that the D-P fluorescence increase of dark-adapted plants results from a reduction of electron acceptors located in the electron transport chain prior to the Fd-NADP oxido-reductase. 3.4. T4 -

increase in thylakoid p H gradient

The T4 phase is characterized by a parallel decrease in the photochemical yield and fluorescence yield [1,5]; also, an increase in the percentage of absorbed light energy which is dissipated in the form of heat is observed [5]. Moreover, this phase is characterized by a quenching of the F~' fluorescence and Fo' fluorescence [5]. All these characteristics are indicative of the dominating influence of a non-photochemical quenching mechanism. Furthermore, a light-induced alkalization of the cytoplasm as sensed by the plasmalemma voltage, occurs with/'4 [85,93-95,97] and the T4 phase is inhibited by the protonophore nigericin [6,106] indicating a relation

11

between T4 and proton fluxes. Apparent absorbance changes at 535 nm [1,94], which are assumed to be related to the formation of the thylakoid pH gradient [107-109], and an increase in the PS II luminescence [85] have been observed to occur concomitantly with T4 quenching of the PS II fluorescence. In conclusion, the characteristic features of the T4 phase indicate that non-photochemical fluorescence quenching of this phase is identical with the 'pH-dependent quenching' or 'energy quenching' of the chlorophyll fluorescence (see Section 4.2.1); T4 labels the increase in the thylakoid pH gradient. An increase in photochemical quenching (decrease in the concentration of reduced QA) also occurs with T4 [1,5]. This decrease in the fraction of PS II with reduced QA results, at least partially, from the lowered rate of Q^ reduction due to the decrease in the photochemical yield of PS II resulting from the increased pH gradient. However, at very low light intensities, a slight increase in the thylakoid pH gradient does not result in non-photochemical fluorescence quenching, but some photochemical quenching is still observed [94]. A pH-dependent activation of ferredoxin-NADPH reductase (FNR) [88,105] and/or Calvin cycle enzymes [110] may be the origin of this phenomenon. 3.5. Ts,, Tsb -- unknown origin

The Ts, component is often [94,95,97,111], but not always [1], present in the fluorescence response to a change in light intensity and is only rarely detectable in the response of the oxygen yield signal. Hansen et al. [85] presented evidence indicating that the Tsa fluorescence increase is coupled with a decrease in the oxygen evolution yield of PS II. Vanselow and Hansen [111] found that a light effect on the K ÷ channel in the plasmalemma occurs simultaneously with the Tsa increase in the PS II fluorescence yield; this light effect on the plasmalemma is presumably mediated by the light-induced uptake of Ca 2+ into the chloroplast [112-114]. It has been proposed that Tsa is the time constant of FNR activation or of changes in the chloroplast NADP ÷ pool [85,104]. The origin of Tsb is unknown. A Tsb fluorescence decrease and a Tsb oxygen yield increase are observed in response to an increase in light intensity [6]. Also, some quenching of the FM' fluorescence occurs during the T5b phase. Possibly, this kinetic component is related to the stimulation of Calvin cycle activity either by enzyme activation or by the formation of an increased ATP/ADP ratio; a more active Calvin cycle would lead to a decreased electron backpressure and therefore to a decrease in PS II trap closure. It is still unclear whether and how the Tsa and Tsb phases are related to the oscillations discussed in Section 3.7.

12

1-1. Dau / Z Photochem. Photobiol. B" Biol. 26 (1994) 3-27

3.6. T6 -- LHC (de)phosphorylation In the case of plants adapted to relatively low light intensities, an increase in light intensity leads to a considerable T~ increase in the oxygen yield and a relatively small increase in the fluorescence yield which can be described with a time constant T6 of 4-20 min [1,6,75,115]. Also, a T6 increase in FM' and Fo' occurs [6,106]. This phase is absent in Chl b-less mutants and inhibited by the phosphatase inhibitor NaF [6,10@ which inhibits LHC dephosphorylation. The T6 phase is not inhibited by nigericin, an uncoupler which inhibits the establishment of a transthylakoid proton gradient and energy quenching [6,106]. The relation between the oxygen yield and fluorescence parameters (F, FM' and Fo') is different from the relation observed for pH-dependent quenching [6,106]. Moreover, electrophoretic analysis of 32p-orthophosphate-labelled leaf discs adapted to low and high light intensities suggests that the T6 phase involves dephosphorylation of an LHC protein [6]. In conclusion the time constant T6 labels the phosphorylation/dephosphorylation of LHCII. The T6 oxygen yield increase is usually not observed on exposure of dark-adapted plants to light or lightadapted plants to saturating or photoinhibitory light intensities. This indicates that LHC dephosphorylation, induced by an increase in light intensity, only occurs within a limited range of light intensity. For lightadaptation of dark-adapted plants and for exposure to photoinhibitory light intensities, phosphorylation of LHCII is usually observed. Waiters and Horton [55] used 77 K fluorescence emission as a measure of the excitation energy redistribution resulting from LHC phosphorylation. Their data suggest that maximal LHC phosphorylation is observed at very low light intensities and LHC dephosphorylation occurs for any further increase in light intensity.

However, a direct and non-critical use of the results obtained for one phenomenon to explain certain features of the other phenomenon may be problematical. Presumably close analogies are not given for the slower phases. As already mentioned above, an increase in light intensity often results in LHC dephosphorylation, whereas the light adaptation of dark-adapted plants is often assumed to result in LHC phosphorylation. Furthermore, the induction curves of fully dark-adapted plants are affected by the dark/light activation of Calvin cycle enzymes and metabolite build-up [110,121]. The light activation of Calvin cycle enzymes will not affect the response to an increase in light intensity if the enzymes are already maximally activated at very low light intensities. The induction kinetics of dark-adapted plants are possibly more complex than the response kinetics to an increase in light intensity. 3.9. Response to photoinhibitory light intensities The response to oversaturating light intensities (IB > 100 W m-2) differs from the response to an increase in light intensity up to a subsaturating level with respect to the slower phases of the kinetics. As already mentioned above, an increase t o oversaturating light intensities may result in LHC phosphorylation rather than LHC dephosphorylation. Most important, however, the response to oversaturating light levels is determined by various phenomena which are often summarized under the term photoinhibition [3]. A conversion of violaxanthin to zeaxanthin results in additional fluorescence quenching [8-12,122] and intact PS II is usually irreversibly converted into different types of inactive or damaged PS II [3]. For extremely high light intensities (IB> 1000 W m-2), these processes can proceed within a few minutes [123]. 3.10. The meaning of 'time constants' and 'kinetic

3. Z Oscillations

phases'

In intact leaves an increase in light intensity can cause sinusoidal [77,97,116-119] or even chaotic [120] oscillations of the fluorescence yield and oxygen yield and diverse metabolite concentrations. These oscillations are favoured by extremely high CO2 concentrations. It is unclear what growth or experimental conditions are needed to make oscillations a fully reproducible phenomenon at physiological CO2 levels.

In principle, all time constant values depend on all processes involved. Thus the assignment of individual time constants to individual processes constitutes an approximation. This approximation might be unjustified for time constants of similar value which result from tightly coupled processes. Such a 'mixing' of time constants might occur for T2 and T3 [2,3]. During the T6 phase, which originates from LHC (de)phosphorylation changes in the thylakoid pH gradient or QA redox state can also occur (see the bars at the top of Fig. 2). In general, during the T~ phase, concentrations of intermediates or enzyme activation states can change if they have a time constant faster than T~. For example, by analysis of the T4 phase, the non-photochemical energy quenching is separated from other non-photochemical quenching mechanisms which

3.8. Induction curves of dark-adapted plants The fluorescence induction curves of dark-adapted plants and the response to an increase in light intensity from IA to IB subsequent to adaptation to IA show similarities. In particular, the first four phases (T1 to T4) of the fluorescence yield seem to be comparable.

H. D a u / J. Photochem. PhotobioL B" Biol. 26 (1994) 3-27

act on a slower time scale; however, changes in the extent of photochemical quenching (qe) occur during the T4 phase.

4. Molecular mechanism of changes in ~p and F

As discussed above (Section 3), the increase in the rate of PS II and P S I electron flux in response to an increase in light intensity results in changes in the concentration of diverse products or states, e.g. concentration of PQH2 or Fd, lumen pH value, membrane voltage, phosphorylation state of LHCII and activation state of Calvin cycle enzymes. These changes in turn affect the photochemical yield and fluorescence yield of PS II resulting in complex multiphase kinetics as shown in Fig. 2. In the following, the molecular mechanism of the PS II response to concentration changes of (intermediate) products or state changes of components of the photosynthetic system is discussed in more detail. Three types of response are distinguished: (1) passive response by product backpressure; (2) active response by special regulatory mechanisms; (3) essentially irreversible photoinactivation and photodamage to the pigment-protein complex. Possibly, reversible photoinactivation constitutes a regulatory mechanism (see Section 4.2.1). The discussion of subjects such as 'PS II downregulation', 'photosynthetic control' or 'photoinhibition' may gain clarity and focus by a clear distinction between these three types of PS II response.

4.1. Passive response by product backpressure Plant photosynthesis can be viewed as a complex sequence of enzymatic reactions, including two lightdriven reactions, which are the primary charge separation reactions of PS II and PS I. The primary light reactions are ultrafast; typical times for 'trapping' of the excited antenna state by formation of the primary radical pair are 100-300 ps [44,47,62,64,65]. Therefore, for a photon absorption rate below 109 s -a, which corresponds to about 106 times the light intensity o f bright sunlight, the primary photochemistry itself cannot limit the rate of photosynthesis. Also, at neutral pH and in the presence of sufficient amounts of extrinsic electron donors and acceptors, none of the PS II electron transfer and proton transfer reactions is rate limiting for plant photosynthesis (see Fig. 2 in Ref. [43]). Instead, saturation phenomena somewhere in the complex sequence of enzymatic reactions occurring outside the two photosystems can result in an accumulation of PS II products and/or depletion of PS II substrates. We can view this eventual effect on the photosystem photochemistry as a product backpressure. (The term 'feedback control' for the response by product backpressure

13

is avoided, because in control theory this term is usually used for an active response as discussed in Section 5.2). The PS II substrates are water, oxidized PQ and stroma protons (see Fig. 1). The products of PS II activity are double-reduced and protonated quinones (PQH2), molecular oxygen, lumen protons and a transthylakoid membrane voltage (negative at the stroma side). Under physiological conditions and normal laboratory conditions, neither 02 backpressure nor HzO depletion are of importance. The occurrence of backpressure by inhibition of QB protonation at high stroma pH values cannot be excluded. However, there are no indications that, in vivo, stroma pH backpressure is of any significance, presumably because no extreme changes in the stroma pH occur (less than 1 pH unit [124]). Thus, in vivo, product backpressure can lead to a decrease in the PS II photochemical yield by three paths: (1) redox (or electron) backpressure involving QA reduction (see Section 4.1.1); (2) lumen pH backpressure eventually resulting in P680 + accumulation (see Section 4.1.2); (3) thylakoid voltage backpressure by electric fields opposing electrogenic charge separation reactions (see Section 4.1.3).

4.1.1. QA-mediated redox backpressure A rate-limiting step somewhere in the photosynthetic electron transport chain behind the PS II quinone acceptors (Fig. 3) results in accumulation of (mobile) PQ in its reduced and protonated form (PQH2). There are two possible consequences for the QB binding site: (1) due to accumulation of PQH2, the QB binding site is occupied by fully reduced and protonated quinone, and/or (2) after PQH2 has left the QB binding site, rebinding of non-reduced PQ does not occur due to the absence of PQ in its non-reduced state. In both cases, further QA to QB electron transfer is impossible and an accumulation of reduced QA occurs. In the presence of reduced QA (i.e. QA-), the rate of primary charge separation (formation of P680÷/Pheo - ) is diminished [44,62,64] and the regular secondary charge separation (formation of QA-) is impossible, a phenomenon usually denoted as PS II trap closure. Because excited state decay competes with fluorescence decay, the restricted primary charge separation leads to an increase in the PS II fluorescence yield (for a review on the molecular details of PS II trap closure, see Dau [43]). In consequence, for an ensemble of PS II entities, with some containing reduced QA-, the fluorescence yield is increased and the photochemical yield of the PS II ensemble (Re) is decreased. (However, the photochemical yield of the individual PS II entities with oxidized QA, denoted as tPp°, remains unaffected.) The backpressure mediated by QA reduction seems to be the most significant of the three backpressure paths. The OA-mediated redox backpressure is a major

14

H. Dau / J. Photochem. PhotobioL B: Biol. 26

determinant of the shape of the oxygen evolution saturation curves and the light response kinetics of the PS II photochemical yield and fluorescence yield (see Fig. 2 and Table 1). The fluorescence parameter (1qp) provides a measure of the extent of the decrease in the PS II photochemical yield resulting from QA reduction (see Eq. (2)). The 1"2, T3 and T5 phases of the response to a change in light intensity are mainly determined by (transient) changes in the QA-mediated backpressure. It is generally accepted that the maximal steady state rate of photosynthetic electron flux (photosynthetic capacity) is determined by the limitations of the Calvin cycle CO2 supply. However, there are two distinct hypotheses of how CO2 limitation affects PS II by redox backpressure: (i) when the ATP usage of the Calvin cycle is limited due to CO2 limitation, the light-driven proton movement into the lumen exceeds the ATPase proton effiux, and the resulting low lumenal pH decreases the rate of PQH2 re-oxidation by the cytochrome (Cyt) bff complex [125-129]; (ii) the CO2 limitation of Calvin cycle usage of NADPH results in a redox backpressure mediated by Fd and the whole electron transport chain. The first hypothesis, which is often referred to as 'photosynthetic control', is widely accepted. Nonetheless, there are some experimental observations which are not in agreement with the first hypothesis. The lumen pH increase subsequent to an increase in light intensity is not related to an increase, but to a considerable decrease, in the amount of reduced QA (as indicated by the qp increase during the T4 phase, see Table 1). Also, based on investigations employing 820 um absorption changes as an indicator of P700 +, Laisk and Oja [130] concluded that photosynthetic control is not of importance under normal atmospheric conditions. Presumably, both phenomena, photosynthetic control and direct redox backpressure, can contribute to the QA-mediated product backpressure. Further investigations are needed in order to elucidate to what extent and under what circumstances photosynthetic control by pH inhibition of PQH2 oxidation is of importance.

4.1.2. Lumen pH backpressure In undamaged PS II, even at high intensities of continuous light, a sizeable accumulation of P680 + or Tyr-Z + has not been observed. However, after absorption of a photon, transient formation of donor side cation radicals occurs (P680 +, Tyr-Z +) [131-137]. Low lumen pH values might slow down the donor side electron transfer reactions resulting in an increased lifetime of the donor side radicals [135]. It seems possible that oxidation of PS II components by donor side radicals triggers photoinhibition (see Section 4.3). In conclusion, the presumed slowing down of PS II donor side reactions by lumen pH backpressure is not

(1994) 3 - 2 7

sufficient to result in a sizeable accumulation of P680 ÷ or Tyr-Z +. Thus the donor side radicals themselves do not result in a decrease in the photochemical yield; trap closure by P680 oxidation is insignificant under continuous illumination. However, an increase in the lifetime of these highly oxidizing radicals could be of significance for the induction of photoinhibition.

4.1.3. Thylakoid voltage backpressure PS II and PSI electron transfer results in a thylakoid voltage which is negative at the PS II aeceptor side and positive at the PS II donor side. Thus the electric field which stems from the light-induced thylakoid voltage opposes further PS II charge separation reactions. In the open reaction centre state, the rate constant for the excited state decay by charge separation (kp) is diminished about 10% per 100 mV of thylakoid voltage [46,65] as reviewed elsewhere [43]. Due to the decreased rate constant for excited state decay by charge separation, the excited state lifetime is lengthened and consequently the Fo' fluorescence yield increases by about 10% per 100 mV of thylakoid voltage. The in vivo light intensity dependence of the thylakoid voltage is not known; presumably the maximal in vivo thylakoid voltage is less than 200 mV. It is important to note that a decrease in the rate constant for excited state decay (kp) by 20% does not result in a decrease in the photochemical yield q~poby 20% as discussed below. The photochemical yield in the open reaction centre state is approximately given

by ~po= 1/(1 +kA/kp)

(8)

where /CA is the rate constant for excited state decay by fluorescence and non-radiative decay [43]. For example, for kA/kp = 8, the photochemical yield changes from only 0.89 to 0.865 (relative change of 2.7%) for a 20% decrease in kp. However, for kAIkp= 1, the photochemical yield changes from 0.50 to 0.44 (relative change of 11.2%) for a 20% decrease in kp. In conclusion, under most conditions, the effect of thylakoid voltage backpressure on the PS II photochemical yield should be negligibly small. Only when the PS II photochemical yield is already small, due to a high probability of thermal deactivation of excited antenna states, can the thylakoid voltage backpressure lead to a decrease in the photochemical yield which is not insignificant. It seems probable that the light-induced thylakoid voltage increases the lifetime of donor side cations (P680 ÷, Tyr-Z ÷) by decreasing the rate constant for electron transfer reactions which are electrogenic [43]. However, at present, it is not known whether this effect can contribute significantly to the induction of photoinhibition.

H. Dau I Z Photochem. PhotobioL B: Biol. 26 (1994) 3 - 2 7

It seems that the membrane voltage provides only a minor contribution to the proton-motive force, which is the driving force for the chloroplast ATPases (in contrast with the situation in mitochondria). High thylakoid membrane voltages are not present because the light-induced proton ettlux is compensated by counterion fluxes across the thylakoid membrane [138]. Presumably, the photosynthetic system avoids high thylakoid voltages in order to minimize voltage backpressure on the charge separation reactions of PS II, P S I and the Cyt b/f complex. Voltage-controlled ion channels may act as a 'relief valve' which provides protection against high thylakoid voltages [139-141].

4.2. Active response by regulation mechanisms Passive response by product backpressure is an inevitable consequence of the reversibility of chemical reactions and the exhaustion of substrate pools. The absence of any product backpressure would violate thermodynamic principles (reversibility of chemical reactions) or rules of logic (reaction without substrate). Passive product backpressure involves the same intermediates or intermediate states as the regular forward reaction. This is not the case for an active response which involves a special regulation mechanism. The distinction between 'passive response by product backpressure' and 'active response by regulation mechanism' is made with respect to PS II activity. Thus regulatory feedback loops which do not include PS II, e.g. the thioredoxin-dependent activation of Calvin cycle enzymes, are not considered an active PS II response.

4.2.1. Molecular mechanism of "energy quenching' The light-induced formation of a thylakoid pH gradient results in pronounced quenching of the PS II fluorescence [107,142-144]. Because of its relation to the 'energization' of the thylakoid membrane by the thylakoid pH gradient, this phenomenon is called 'energy quenching', 'high energy state quenching' or 'pH-dependent quenching'. In this article, the non-photochemical quenching of PS II fluorescence, which increases simultaneously with the light-induced formation of the thylakoid pH gradient, is called 'energy quenching'. This energy quenching is responsible for the nonphotochemical quenching occurring with T4 (see Section 3.4). In the following, pH-dependent effects which are delayed with respect to the light-induced formation of the pH gradient (e.g. the violaxanthin to zeaxanthin conversion [8-12]) are not considered. After an increase in light intensity, energy quenching is characterized by a decrease in the F u ' fluorescence and Fo' fluorescence and a simultaneous decrease in the photochemical yield of PS II in the open reaction centre state [5]. Because energy quenching is the dominant non-photochemical quenching effect at subphoto-

15

inhibitory light intensities, the light saturation curves of oxygen evolution or CO2 fixation and the fluorescence parameters (Fo', FM', qp) have been used for investigation [55,145-152]. Several studies indicate that energy quenching of the Fo' fluorescence occurs [7,45,151,153-155]. It has been proposed by Krause et al. [143] that energy quenching results from an increase in the probability of the non-radiative decay of excited antenna states (increased rate of thermal dissipation). Because non-radiative decay of excited antenna states competes with excited state decay by fluorescence emission and primary charge separation, the activation of a non-radiative decay path can explain the observed decrease in the fluorescence yield and photochemical yield. The relation between FM' quenching, Fo' quenching and the decrease in the photochemical yield of open reaction centres has been found to be in good agreement with the assumption of an increased rate of non-radiative decay of excited antenna states [154]. However, studies on the quantitative relation between Fo', FM' and qbp° [146,154] were based on the bipartite model of Butler and coworkers or similar models [156,157] which are not in agreement with the recent results of time-resolved spectroscopy (for a more extended discussion, see Holzwarth and Roelofs [61] and Dau [43]). Based on the 'outdated' bipartite model, it was concluded that energy quenching results from the increased thermal deactivation of excited antenna states (antenna quenching), but not from an increased rate of deactivation of P680", the excited state of the reaction centre Chl. As shown elsewhere [43], it is possible to 'translate' this conclusion. Assuming the validity of the RRP model, the above-mentioned experimental results on the relation between FM', Fo' and ~ o suggest that energy quenching results from the increased thermal deactivation of excited states of the PS II pigment system, including the reaction centre pigment P680, but not from an effect on the PS II charge separation reactions; increased thermal deactivation of P680" is spectroscopically indistinguishable from the increased thermal deactivation of the excited states of other PS II antenna pigments. Dau and Hansen [5] demonstrated by photoacoustic experiments that energy quenching of the PS II fluorescence is indeed coupled with an increase in the amount of energy dissipated in the form of heat. (Other photoacoustic studies on the same subject did not provide fully conclusive results as reviewed by Fork and Herbert [83].) However, the observed increase in heat release does not provide fully definitive proof that energy quenching results from the activation of a nonradiative decay path for excited antenna states. Fast PS II cyclic electron transport acting as a futile cycle would not be in conflict with the photoacoustic results. However, if such a futile cycle exists, it will need to have a turnover time faster than 50 gs [5].

16

H. Dau /.I. Photochem. PhotobioL B: Biol. 26 (1994) 3-27

Presumably, energy quenching does not originate from the pH gradient itself but from the acidification of the thylakoid lumen [142]. There are some indications that, in the intact thylakoid system, it is not the bulk lumen pH that is sensed, but the pH of proton domains [158--162]. PS II membrane particles seem to exhibit a pH-dependent quenching of the fluorescence emission and photochemical yield which is dependent on the pH of the medium [163--166]. Light-induced energy quenching is inhibited by uncouplers (nigericin, NH4C1 and others) which short circuit the thylakoid pH gradient. Energy quenching is also inhibited by antimycin A [167,168] and dicyclohexylcarbodiimide (DCCD) [169] which, at low concentrations, do not short circuit the pH gradient. The maximal extent (or capacity) of energy quenching and/ or the pH level for half-maximum quenching are correlated with the zeaxanthin content of the thylakoid [9-12,168,170]. However, despite the impressive correlations, full proof of the zeaxanthin effect on the energy quenching mechanism is still lacking. There are various different hypotheses and models for the molecular mechanism of energy quenching [171-174]. The two most elaborate models are discussed in detail. 4.2.1.1. The Ca 2÷ release/QA re-oxidation model

The model as proposed in 1992 by Krieger and Weis [174] is described below. (1) At lumen pH values lower than 5.5 (pK value of 4.7), one calcium per PS II is released at the lumen side of PS II by H+/Ca 2+ exchange. Presumably, this calcium stems from the oxygen-evolving complex (OEC) where it is a cofactor essential for oxygen evolution activity [17]. Calcium is released preferentially in higher S states of the OEC. Therefore the Ca 2÷ release requires light. In the presence of sufficient Ca 2÷ in the medium, the low-pH-induced Ca 2÷ release is reversible, i.e. a pH increase results in Ca 2÷ rebinding. (2) Due to the Ca 2+ release, the Mn cluster becomes inactivated; electron donation to Tyr-Z is no longer possible. As a consequence, the Tyr-Z ÷ and P680 ÷ states may become more stable, i.e. their lifetime is increased. (3) Another consequence of the lumen side Ca 2+ release seems to be a shift of the QA redox potential to more positive values, resulting either from electrostatic events (positive charge of the OEC) or from structural changes. (4) QA- is rapidly oxidized by electron donation to P680 +. This QA- re-oxidation by recombination with P680 ÷ becomes possible due to the increased lifetime of P680 ÷ and is somehow stimulated by the more positive redox potential of Q^. Krieger and Weis [174] did not state on what route this recombination occurs, the graphical representation presented in Ref. [174]

suggests direct radiationless P680 ÷/QA- recombination. In a more recent paper, Krieger and Weis [165] mention that the recombination reaction is possibly mediated by Cyt b559 as proposed by Schreiber and Neubauer [175]. By various elegant experiments, Krieger and Weis [165,166,174] provided convincing evidence supporting the statements (1)-(3). In particular, the occurrence of a low-pH-induced Ca z÷ release and the resulting inactivation of the OEC is, at least in vitro, well established [17]. The fourth statement, however, is mainly speculative. Picosecond fluorescence relaxation experiments were interpreted as supporting pH-dependent fluorescence quenching by rapid QA- recombination [164]. However, these results are ambiguous, because it is extraordinarily difficult, if not impossible, to distinguish on the basis of picosecond fluorescence decay between fluorescence quenching due to increased thermal deactivation of excited states and quenching due to less trap closure. Furthermore, the validity of the rationale used for the interpretation of the fluorescence decays is not well established and contradicts many recent results as discussed elsewhere [43]. There is considerable evidence that internal cyclic electron transport involving Cyt b559 is possible [176-180]. We may speculate that this cyclic electron transport is the mechanism of the fast QA-/P680 + recombination postulated by Krieger and Weis [174]. However, to explain the decrease in FM' fluorescence by light pulses of high intensity, this cyclic electron transport must be surprisingly efficient. Horton and coworkers [163,181] observed a presumably pH-dependent effect on the PS II oxygen field which was possibly related to cyclic electron transport involving Cyt b559. However, this effect does not seem to be related to the quenching of FM' fluorescence. It is not clear whether the pH/Ca 2÷ effect investigated by Krieger and Weis [165,166,174] is identical with the energy quenching observed under in vivo conditions. In particular, the well-established energy quenching of Fo' fluorescence [5,7,45,151,153-155] is difficult to reconcile with the model of Krieger and Weis [174]. (Unfortunately, Krieger and Weis did not present a pH dependence of Fo' fluorescence for the PS II particles used.) The finding that the extent and/or pH dependence of energy quenching correlates with the zeaxanthin content of the plant [9-12,168,170] is difficult to reconcile with the model described above. It is commonly assumed that energy quenching constitutes a protection mechanism against inactivation or damage by excessive light (photoinactivation and photodamage, see Section 4.3) [182]. Apparently, several aspects of the pH/Ca 2÷ phenomenon investigated by Krieger and Weis do not suggest a protection mechanism, but photoinactivation resulting from lumen pH backpressure. In particular, the proposed increase in

H. Dau / J. Photochem. Photobiol. B: Biol. 26 (1994) 3-27

the P680 ÷ lifetime (see point (2) of the model) should trigger rather than prevent damage to PS II. Only the so far mainly speculative assumption of rapid QArecombination by a triplet-free route could be considered as a protection mechanism. In conclusion, the interpretation of the pH-induced Ca 2+ release as a regulatory or protection mechanism needs experimental verification. Also, it is unclear to what extent the model proposed by Krieger and Weis [174] is of relevance for the phenomenon usually denoted as energy quenching.

4.2.1.2. The zeaxanthin/LHC quenching model Whereas Krieger and Weis [174] suggested that the molecular events resulting in energy quenching are located in the PS I1 core complex, Horton and coworkers (and others) assume that LHCII plays a central role [109,169,173,183,184]. Because the model of Horton and coworkers [57,173] is still in the process of rapid development, details are added or modified frequently. The description of the model given below is partially based on a presentation of Peter Horton at the Fifth Congress of the European Society for Photobiology in Marburg, Germany, September 1993. (0) Changes in the lumen pH result in various effects on PS II as reviewed in Horton and Ruban [57]. Under physiological conditions, the dominating mechanism is energy quenching, as described below, at least when sufficient zeaxanthin is present. (1) At low lumen pH values, specific LHC residues, probably special glutamates and/or aspartates, become protonated [57]. The conversion of violaxanthin to zeaxanthin results in a structural change of the LHC. As a consequence, the apparent pK value for the protonation reaction is shifted from about 4.5 in the absence of zeaxanthin to about 5.7 in the presence of sufficient zeaxanthin [57]. Presumably, the protonated residues, which are of importance in energy quenching, are located mainly at the minor LHCs often denoted as CP26 and CP29. The protonated residues are closer to the lumen side of the LHC. Possibly, they are located in a 'proton domain' [169]. Therefore energy quenching can be promoted by a localized pH. This proton domain might also function as a proton channel [185,186]. (2) The protonation of LHC residues results in a conformational change of the LHC polypeptide. As a consequence, the LHC Chls, densely packed within the polypeptide matrix [25], are affected. Another consequence of the conformational change of (some) LHC polypeptides is a long-range effect resulting in a special interaction between LHC trimers which is simulated by LHC aggregation in vitro [57]. (3) Eventually the changes in the protein structure result in the formation of Chl-carotenoid aggregates. Also, 'head-to-tail aggregation' of xanthophylls might be involved [187]. It has not been clearly stated whether

17

the formation of Chl--carotenoid aggregates results from an interaction between LHC trimers or occurs directly in or at the protonated LHC polypeptides. (4) The pigment aggregates are characterized by a high rate of excited state deactivation by non-radiative decay. Thus these pigment aggregates serve as quenching centres. Excited state deactivation by these quenching centres competes with fluorescence emission and the regular PS II photochemistry. As a consequence, the fluorescence yield F and photochemical yield ~po decrease. Concerning the physical nature of the quenching centres, recent results on the excited state phenomena of carotenoids could be of relevance [188,189]. It seems possible that the energy of the lowest lying carotenoid singlet state (Sa energy) is below the Sa energy of Chl a [187,189]. Thus a close association of C h l a and carotenoid could result in efficient Chl-to-carotenoid excitation energy transfer. Due to the short lifetime of the carotenoid $1 state, this excitation energy transfer would result in the quenching of excited states. The model of Horton and coworkers predicts a quenching of FM', Fo' and q~o as observed for energy quenching in vivo. Also, the mechanism is in agreement with the proposal that energy quenching serves as a protection mechanism against excessive light. The enhancement of energy quenching by zeaxanthin does not contradict the proposed model and the effect of several inhibitors can be rationalized. Nonetheless, many details of the model are still speculative and some aspects are not fully conclusive. Two structural changes in the LHCs are assumed to occur. First, the conversion of zeaxanthin to violaxanthin results in a structural change which affects the apparent pK value of residues, and is decisive for energy quenching. This effect on the pK value is assumed to be related to the aggregation of LHC trimers which is inhibited by antimycin A. Taking into consideration that the relevant residues, as well as the major fraction of zeaxanthin, seem to be located at the minor LHCs (in particular, CP26 and CP29 [23]), which are assumed not to form trimers, this proposal is not fully convincing. Also, Lokstein et al. [151] found for a Chl b-free barley mutant, which is commonly assumed to lack functional LHCs of the trimer-forming LHCII type [190], that the extent of energy quenching was decreased, but the phenomenon was not absent. Jahns and Krause [191] observed substantial energy quenching in intermittentlight-grown pea leaves lacking LHCII. Recently, it was found that, in a Chl b-free mutant of the green alga Scenedesmus obliquus, the extent of energy quenching was not significantly diminished in comparison with the wild-type organism [192]. These findings indicate that a fully functional LHC system is not indispensable for energy quenching. (However, Lokstein et al. [151] draw the opposite conclusion.) Thus a specific interaction

18

H. Dau / J. Photochem. Photobiol. B: Biol. 26 (1994) 3-27

between LHC trimers does not seem to be decisive for energy quenching. A second structural change in the LHC polypeptide is assumed to be the cause of special pigment-pigment interaction (see above, point (2)). Again, the proposed involvement of interactions between LHC trimers seems doubtful. Experimental evidence is needed which explains whether and how the LHC structure changes in response to protonation. Other scenarios seem possible. For example, the protonation of residues at the minor LHCs could result in changes in the electrostatic potential without affecting the polypeptide conformation itself. The changed electrostatic potential could either favour the binding association of a zeaxanthin close to Chl a, or could result in an electrochromic shift of singlet state energies which increases the likelihood of singlet energy transfer from Chl a to a neighbouring zeaxanthin. In both cases, the modified electrostatics could result in a Chl-zeaxanthin quenching centre.

4.2.1.3. The mechanisms of energy quenching - summary It is important to distinguish between various pHdependent effects of PS II. There is considerable evidence that, under in vivo conditions, the dominating phenomenon is the quenching of excited Chl states resulting in the thermal dissipation of excited state energy. This dominating part of energy quenching results in a considerable decrease in FM' and in a smaller decrease in Fo'. The decrease in the photochemical yield of PS II in the open reaction centres, ~ , seems to follow the Fo' decrease [5], whereas for partial trap closure the effective ~x, is reasonably well approximated by (FM'-F)/FM' [146]. Thus this energy quenching meets all the criteria for so-called 'antenna quenching' [43]. This does not necessarily mean that, for each individual PS II entity, the rate of excited state decay by thermal dissipation increases with increasing energy quenching continuously. The switching of some PS II into a quenched state and a continuous change in the properties of each PS II entity can result in the same syndrome (effect on F~', Fo', F, ~e ° and ti~ as described above), if PS II connectivity is given [43,70]. Most probably antenna quenching results from the formation of quenching centres. Candidates for in vivo fluorescence quenchers are C h i C h i dimers with special properties, carotenoids with low $1 state energies, extrinsic quenchers (e.g. mobile PQs) and Chl radicals. It is important to note that, with the assumption of rapid exciton equilibration [43,47,62], fluorescence quenching by P680 ÷ would also result in 'antenna quenching'. The model proposed by Horton and coworkers (see Section 4.2.1.2) can account for most experimental observations. However, as already pointed out above, several details are still speculative and some aspects of the model may need to be modified.

In particular, in plants with a low zeaxanthin content, other pH effects could be of relevance. It is conceivable that, after a pH-triggered reversible photoinactivation of the OEC by Ca 2÷ release, cyclic electron flow, possibly involving Cyt b559, provides some protection against irreversible damage. It remains to be clarified whether there are two types of relevant internal electron cycling: (1) a very fast cycle (turnover faster than about 500 /~s) which affects the saturation pulse FM' level as proposed by Krieger and Weis [174]; (2) a slower cycle (turnover slower than about 500 ~s) which does not affect the saturation pulse F~t' level [181,193]. 4.2.2. Mechanisms of state transitions by LHC phosphorylation Plants can (partially) remove an imbalance between the PS II and P S I electron transport rate by 'state transitions' [75]. Relative overexcitation of PS II results in a state 1 to state 2 transition which involves the phosphorylation of LHC polypeptides [193-196]. The imbalanced PS II and P S I electron flow may be the consequence of a rapid change in the spectral composition of actinic light. However, as discussed in Section 3.6, state transitions by LHC phosphorylation are also initiated by changes in light intensity. The subject of LHC phosphorylation has been reviewed recently by Allen [21]. Therefore only a brief summary of certain aspects is given and some unresolved questions involving the physiological role of LHC phosphorylation are discussed. State 1 to state 2 transitions involve the phosphorylation of the peripheral PS II LHCs of the LHCII type. The extent of LHC phosphorylation is determined by the relative rates of phosphorylation by a regulated LHC kinase and dephosphorylation by a LHC phosphatase. Presumably, the reduction of a component in the electron transport chain between PS II and P S I results in an increased kinase activity. Phosphorylated LHC polypeptides can disconnect from PS II and withdraw from the grana regions of the thylakoid. Also, a decrease in thylakoid stacking results from LHC phosphorylation. In the stroma region some, but usually not all and possibly none, of the phosphorylated LHC polypeptides connect functionally to P S I . Possibly the fraction of phosphorylated LHC connecting to PS I varies between different plant species; it might also depend on the adaptation state of the plant, i.e. on the state obtained by longterm and short-term adaptation (details and references are given in the excellent review by Allen [21]). Presumably, the kinase activity is controlled by the redox state of a component of the Cyt b/f complex [6,21,106,197-199]. The redox state of this component serves as a sensor for a mismatch between the PS IIdriven electron flow and the PS I-driven electron flow. It has often been proposed that state transitions serve to regulate the extent of cyclic P S I electron flow [195].

H. Dau / J. Photochem. Photobiol. B: Biol. 26 (1994) 3-27

However, it is not clear how the cyclic P S I electron flow involving the Cyt b/f complex affects the redox state of the component controlling the LHC kinase activity. The fate of the phosphorylated LHCs after disconnection from PS II is still fairly obscure. Because LHC phosphorylation results in a decrease in the number of LHCs functionally connected to PS II, the yield of PS II oxygen evolution in the open reaction centre state (molecules of O2/number of photons absorbed by PS II, P S I and IMCs) is decreased [18,200,201]. Moreover, on LHC phosphorylation, the fluorescence yield of the thylakoid system is decreased in the FM' state as well as in the Fo' state. In vitro, Fra' and Fo' decrease by the same percentage when sufficient Mg2+ is present [202-206]. In vivo, the FM' decrease is found to be more pronounced than the Fo' decrease for state transitions initiated by the removal of far-red light after previous adaptation of the plant to blue and far-red light of low intensity (e.g. blue light of 2 W m-2); a decrease in FM' and Fo' by the same percentage is observed after adaptation to light of higher intensities (e.g. blue light of 20 W m -2 [53]). Apparently, the molecular mechanism of the state transitions depends on the adaptation state of the plant. We could imagine that the uncoupling of the LHC from the PS II core complex results in an increase in Fo' fluorescence because the excited states of the uncoupled LHC are no longer quenched by the PS II charge separation reaction. Surprisingly, the opposite is observed as already mentioned above. The decrease in FM' and Fo' on LHC uncoupling from PS II has been explained by the coupling of these units to PS I, resulting in efficient fluorescence quenching by PS I charge separation. This explanation, however, cannot be valid when only a minority of phosphorylated LHC couples to PS I. Thus the uncoupled LHC must be in a low fluorescent state. "There may be a relation to the pronounced fluorescence quenching on LHC aggregation in vitro [183]. In conclusion, the functional implication of LHC phosphorylation are insufficiently understood; more work on this subject is needed. As discussed in Section 3.6, an increase in light intensity can initiate LHC dephosphorylation. This phenomenon seems to be of considerable physiological importance as demonstrated by the observation that intensity-induced LHC dephosphorylation results in an increase in the yield of PS II oxygen evolution by sometimes more than 50% [6,106]. However, it is essentially unknown how these light-intensity-induced state transitions are controlled and why such an enormous increase in the photochemical yield is observed. In the following, a working hypothesis is presented which needs experimental verification. With an increase in light intensity, the Calvin cycle activity comes closer to saturation. As a consequence,

19

the branching of the electron flow at the PS I acceptor side between linear electron flow from PS I to the Calvin cycle on the one hand and cyclic electron flow from P S I to the Cyt b/f complex on the other hand is changed in favour of cyclic electron flow. The increased cyclic electron flow results in an overenergization of the thylakoid membrane, i.e. a transthylakoid pH gradient which is unnecessarily high. The increased cyclic electron flow or imbalance in photosystem activities due to the decrease in 4~ of PS II by energy quenching and product backpressure affects the kinase redox sensor located in (or at) the Cyt b/f complex and LHC dephosphorylation is initiated. The LHC dephosphorylation results in a decrease in PS I cyclic electron flow and an increase in linear electron flow.

4.3. Photoinactivation and photodamage In the following, a brief summary of some aspects of photoinhibition is given. The arguments described below are extracted from the excellent review of Aro et al. [3] (see also Krause [207,208]). Aspects which are of importance for an understanding of the physiological role of short-term light adaptation are examined. The term 'photoinhibition' is avoided, because its precise meaning seems to be unclear. 'Photoinactivation' is used to denote the reversible inhibition of the PS II capability to evolve oxygen and to reduce PQ. 'Photodamage' is used to denote any inactivation or inhibition of PS II which is not readily reversible. Photodamage to PS II is assumed to result from an acceptor side effect and from a donor side effect [3].

4.3.1. Acceptor-side-induced photodamage (1) High light intensity results in product backpressure on PS II. The acceptor side is affected by depletion of reducible PQ; the QB binding site remains empty. (2) QA- accumulates. (3) In the absence of QB, the protonation and/or double reduction of QA- can occur, which results in a reversibly photoinactivated state of PS II. Also, QAH2 may leave PS II. QAH2 release is essentially irreversible. All these processes require high light intensities, because they proceed only with an extremely low quantum yield. (4) All the phenomena mentioned in (3) result in a triplet-formation state of PS II. This means that the reaction sequence 'light + P680~ 1P680"--* P680+Pheo - --*3p680", occurs with a significant quantum yield. (5) The 3p680 triplet allows the formation of singlet oxygen according to: 3P680" + 302 ~ 1P680 + 10 2. (6) The singlet oxygen causes damage to the PS II reaction centre complex. (7) This damage triggers proteolysis of the D1 protein. On illumination under aerobic conditions, the quantum yield of formation of the triplet-forming state is

20

H. Dau / Z Photochem. PhotobioL B: Biol. 26 (1994) 3 - 2 7

presumably smaller than the yield of the subsequent triplet formation (4), singlet oxygen formation (5) and reaction centre damage (6). Therefore it seems unlikely that the 'irregular' QA states (QAH, QAH2, empty QA binding site) accumulate under in vivo conditions. It is important to note that the first three steps all require high fight intensities. Therefore photodamage resulting from acceptor side effects is presumably irrelevant at subsaturating light intensities.

4.3.2. Donor-side-induced photodamage (1) Under continuous high intensity illumination, the average number of PS II entities containing donor side radicals (Tyr-Z + and/or P680 ÷) is increased. It is not clear what factor is the decisive cause of the increase in the concentration of these cation radicals. Possibilities are listed below. (la) Tyr-Z + and P680 + are transiently formed after primary charge separation has taken place. (This, however, may not be valid for PS II with reduced QA.) At high light intensities, the transient formation of these cation radicals occurs more often. (lb) High light intensities result in lumen acidification. The increased lumen proton concentration results in a deceleration of the donor side reactions, in particular Tyr-Z + re-reduction by the OEC. As a consequence, electron donation by the OEC cannot keep up with the light-drive removal of electrons by the primary charge separation reaction. Under these circumstances the lifetimes of P680 + and Tyr-Z + are significantly increased. (lc) High light intensities result in lumen acidification. The low lumen pH causes the inactivation of the Mn complex, e.g. by inducing the release of Ca or Mn. T h e inactivated Mn complex results in increased fifetimes, or even accumulation, of Tyr-Z + and P680 +. (2) Tyr-Z + and/or P680 + oxidize carotenoids, Chls or amino acid residues. (3) The damage to the PS II reaction centre complex triggers the proteolysis of the D1 protein. The molecular mechanisms and in vivo role of donorside-induced photodamage are still unclear (see Krause [208]). Possibly, all three routes to the formation of P680 ÷ and Tyr-Z + ((la)-(lc)) are effective. Mechanism (la) provides an explanation for the D1 turnover event at low light intensities. However, according to mechanism (la), the quantum yield for D1 degradation should be low in the case of PS II trap closure by QA reduction. Thus mechanism (la) is unlikely to be of relevance for photodamage occurring after exposure of the plant to high light intensities. The investigations of Krieger and Weis (see Section 4.2.1.1) suggest that the low lumen pH is decisive for reversible photoinaetivation of the OEC. The proposed protection against long-lived cation radicals by (cyclic) QA-/P680 + recombination could reduce the cation radical lifetime considerably, but this

mechanism may be insufficient to provide full protection against oxidative damage by P680 ÷ and/or Tyr-Z +.

5. The role of short-term adaptation in summary

5.1. Product backpressure/photodamage /adaptation Saturation of the Calvin cycle activity results in inevitable product backpressure. PS II is affected by (1) redox backpressure mediated by Q^ reduction and (2) backpressure mediated by a low lumen pH. Product backpressure by thylakoid voltages seem to be of less importance. Both events, QA reduction and a low lumen pH, could be important 'cofactors' of photoinactivation and photodamage to PS II. Presumably, the accumulation of reduced QA or a low lumen pH potentiates the quantum yield of photoinactivation and photodamage (see Section 4.3). In conclusion, product backpressure results in photodamage (see Fig. 4). Both phenomena, product backpressure and photodamage, are physical necessities; they are not an adaptation response of the plant. The pH-dependent PS II downregulation by the energy quenching mechanism constitutes an adaptation response which should provide significant protection against photodamage by decreasing the product backpressure. Light-intensity-induced LHC dephosphorylation seems to optimize the coordination of the PS II and PSI activities; one consequence seems to be a decreased lumen pH backpressure.

I

..... trotion chonge-~s [ ] of photosynthetic L____.Jadaptation intermediates~ " ~ mechanism Tt" T6

~ l

~3. product [ .backprt*sureI

OA

P" I

~¢ 4.1.

I photoinactivation and photodamag¢ see4.3.

s¢0 4.2,

Z]

W

PS II

fluorescence ~:~ and photochemistry see2

Fig. 4. Schematic representation of the relation between product hack'pressure, adaptation mechanism and photodamage. A change in light intensity causes changes in the concentration of intermediates which occur with distinct time constants (T~ to 7"6). The resulting product backpressure (accumulation of reduced Q^ and formation of the thylakoid pH gradient) favours PS II photoinactivation and photodamage. Product backpressure is decreased by adaptation mechanisms resulting in a negative feedback loop. Product backpressure, adaptation mechanisms and photodamage affect the fluorescence yield and photochemical yield of PS II.

H. Dau / Z Photochem. Photobiol. B: BioL 26 (1994) 3-27

Energy quenching decreases the quantum yield of primary charge separation. Often this decrease will not exceed 50%. However, because several steps in the sequence of events leading to photodamage depend on the primary charge separation (see Section 4.3), the protective effect should be more pronounced than just a decrease in the rate of photodamage formation by 50%. For example, the lowered quantum yield of PS II in the open reaction centre state decreases the rate of QA2- formation in two ways: (1) the decreased rate of PS II electron transport results in less redox backpressure, i.e. less PS II in the QA- state, the 'substrate' for QA2- formation; (2) the quantum yield of secondary reduction of QA- to QA2- is diminished. Thus, for a 50% reduction in PS II quantum yield, the rate of QA2- state formation, a state which is especially susceptible to photodamage (see Section 4.3), should be reduced by more than 50%. In conclusion, short-term adaptation by activation of the energy quenching mechanism could be of considerable importance as a protein mechanism against PS II photodamage. 5.2. Kinetics of the response to changes in light intensity After an increase in fight intensity a reduction in the electron transport chain occurs within a few seconds (7"1, T2 and T3 phases of the PS II response, see Sections 3.1-3.3). The resulting redox backpressure is potentially dangerous for PS II (see Section 5.1). The light-induced acidification of the lumen is characterized by a time constant of 3-30 s (see Section 3.4); the resulting pH backpressure could cause photoinactivation and photodamage (see Section 4.3.2). The acidification of the lumen results in an increased thermal dissipation of excitation energy by the energy quenching mechanism (see Section 4.2.1), at least in plants containing significant amounts of zeaxanthin. Consequently, the rate of PS II electron flow is decreased; redox backpressure and pH backpressure are released. Thus energy quenching provides a very fast adaptation with respect to light intensity changes. In addition, the lumen acidification may result in reversible inactivation of the OEC by Ca ~+ release (see Section 4.2.1.1). Irreversible damage to inactivated PS II may be minimized by rapid QA-/P680 + recombination mediated by a cyclic electron flow involving Cyt b559. An increase in the capability of the Calvin cycle to accept reducing equivalents (time constant Tsb of 50-250 s; see Section 3.5) results in a further decrease in the redox backpressure. At subsaturating light intensities, LHCII dephosphorylation occurs (time constant of 200-1000 s). Possibly the LHC dephosphorylation results in a downregulation of the cyclic P S I electron flow; as a

21

consequence, pH backpressure is released and fight usage is optimized (increase in the PS II antenna). However, the proposed control of the cyefic PSI electron flow by LHCII phosphorylation is speculative and needs experimental support. At subsaturating light intensities the PS II response to an increase in light intensity is characterized by an increase in energy quenching (T4 phase) which is partially reversed at later times (T~ phase). Thus, with respect to the adaptation to relatively low light intensities, the energy quenching mechanism seems to provide protection which is particularly effective during the transition phase. 6. Open questions Two adaptation mechanisms have been discussed in this article: protection against excessive fight by pHdependent energy quenching and coordination of PS II and P S I electron fluxes by LHC phosphorylation. In both cases, further work is needed to achieve an understanding of the molecular mechanisms involved in the observed redirection of energy fluxes. All models proposed so far contain various highly speculative aspects. Moreover, each single model proposed for energy quenching is not capable of explaining all aspects of the available experimental results. The dominating physiological role of LHC phosphorylation is still obscure. Energy quenching is commonly assumed to be a protection mechanism against excessive light. However, so far there is no unambiguous proof that (and to what extent) energy quenching does indeed protect against photodamage. The assumed relations between energy quenching and xanthophyll cycle pigments are based on correlations only; unambiguous proof is still lacking. Plants grown at different light intensities seem to exhibit different 'capacities' for adaptation by energy quenching and LHC phosphorylation [209]. Thus, not only the composition of the photosynthetic apparatus (stoichiometry of pigments, pigment-protein complexes, photosystems, etc.), but also its capacity for short-term adaptation, is influenced by long-term adaptation. Studies on the relation between long-term and short-term adaptation could help to elucidate the physiological and ecological role of light adaptation. Acknowledgements I would like to thank Professors U.-P. Hansen (Kiel, Germany), IC Sauer (Berkeley, USA) and H. Senger (Marburg, Germany) for their support and encouragement. I am grateful to H. Schiller and I. Heinze (Marburg, Germany) for their help in preparing the

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H. Dau / Z Photochem. Photobiol. B: Biol. 26 (1994) 3-27

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