Target theory and the photoinactivation of Photosystem II

Application of target theory to the photoinactivation of Photosystem II in pea leaf discs (Park et al. 1995, 1996a,b) reveals that there is a critical light dosage below which there is complete photoprotection and above which there is photoinactivation (i.e a light-induced loss of oxygen flash yield). The critical dosage is about 3 mol photons m^–2 for medium and high light-grown leaves and 0.36 mol photons m^–2 for low light-grown leaves. Photoinactivation is a one-hit process with an effective cross-section of 0.045 m² mol^–1 photons which does not vary with growth irradiance, unlike the cross-section for oxygen evolution which increases with decreasing growth irradiance. The cross-section for oxygen evolution increased by about 20% following exposure to 6.8 mol photons m^–2 which may be due to energy transfer from photoinactivated units to functional Photosystem II units. We propose that the photoinactivation of PS II begins when a small group of PS II pigment molecules whose structure is uninfluenced by growth irradiance, becomes uncoupled energetically from the rest of the photosynthetic unit and thus no longer transfers excitions to P680. De-excitation of this group of pigment molecules provides the energy which leads to the damage of Photosystem II. Treatment of pea leaves with dithiothreitol, an inhibitor of the xanthophyll cycle, decreases the critical dosage i.e. decreases photoprotection but has no effect on the PS II photoinactivation cross-section. Treatment with 1 M nigericin increased the photoinactivation cross-section of PS II as did exposure to lincomycin which inhibits D1 protein synthesis and thus the repair of PS II reaction centres.Abbreviations DTT- dithiothreitol - PS II- Photosystem II - Fm- maximum fluorescence - Fv- variable fluorescence - LHCIIb- main light harvesting pigment-protein complex of PS II - D1 protein- psbA gene product - P680- reaction centre chlorophyll of Photosystem II - Qa- first quinone electron acceptor of Photosystem II - (o₂)- cross-section for oxygen evolution - (pi)- cross-section for photoinactivation     

The effects of elevated light on Photosystem II function: A thermoluminescence study

We have used the technique of thermoluminescence (TL) to investigate high-light-induced chlorophyll fluorescence quenching phenomena in barley leaves, and have shown it to be a powerful tool in such investigations. TL measurements were taken from wild-type and chlorina f2 barley leaves which had been dark-adapted or exposed to 20 min illumination of varying irradiance or given varying periods of recovery following strong irradiance. We have found strong evidence that there is a sustained trans-thylakoid pH in leaves following illumination, and that this pH gives rise to quenching of chlorophyll fluorescence which has previously been identified as a slowly-relaxing component of antenna-related protective energy dissipation; we have identified a state of the PS II reaction centre resulting from high light treatments which is apparently able to perform normal charge separation and electron transport but which is non-photochemically quenched, in that the application of a light pulse of high irradiance cannot cause the formation of a high fluorescent state; and we have provided evidence that a transient state of the PS II reaction centre is formed during recovery from such high light treatments, in which electron transport from Q_Ato Q_Bis apparently impaired.     

The effect of outer antenna complexes on the photochemical trapping rate in barley thylakoid Photosystem II

We have investigated the previous suggestions in the literature that the outer antenna of Photosystem II of barley does not influence the effective photosystem primary photochemical trapping rate. It is shown by steady state fluorescence measurements at the F₀ fluorescence level of wild type and the chlorina f2 mutant, using the chlorophyll b fluorescence as a marker, that the outer antenna is thermally equilibrated with the core pigments, at room temperature, under conditions of photochemical trapping. This is in contrast with the conclusions of the earlier studies in which it was suggested that energy was transferred rapidly and irreversibly from the outer antenna to the Photosystem II core. Furthermore, the effective trapping time, determined by single photon counting, time-resolved measurements, was shown to increase from 0.17±0.017 ns in the chlorina Photosystem II core to a value within the range 0.42±0.036-0.47±0.044 ns for the wild-type Photosystem II with the outer antenna system. This 2.5-2.8-fold increase in the effective trapping time is, however, significantly less than that expected for a thermalised system. The data can be explained in terms of the outer antenna increasing the primary charge separation rate by about 50%.     

Electron transport to oxygen mitigates against the photoinactivation of Photosystem II in vivo

The role of electron transport to O₂ in mitigating against photoinactivation of Photosystem (PS) II was investigated in leaves of pea (Pisum sativum L.) grown in moderate light (250 mol m^–2 s^–1). During short-term illumination, the electron flux at PS II and non-radiative dissipation of absorbed quanta, calculated from chlorophyll fluorescence quenching, increased with increasing O₂ concentration at each light regime tested. The photoinactivation of PS II in pea leaves was monitored by the oxygen yield per repetitive flash as a function of photon exposure (mol photons m^–2). The number of functional PS II complexes decreased nonlinearly with increasing photon exposure, with greater photoinactivation of PS II at a lower O₂ concentration. The results suggest that electron transport to O₂, via the twin processes of oxygenase photorespiration and the Mehler reaction, mitigates against the photoinactivation of PS II in vivo, through both utilization of photons in electron transport and increased nonradiative dissipation of excitation. Photoprotection via electron transport to O₂ in vivo is a useful addition to the large extent of photoprotection mediated by carbon-assimilatory electron transport in 1.1% CO₂ alone.Abbreviations Fm, Fo, Fv- maximal, initial (corresponding to open PS II traps) and variable chlorophyll fluorescence yield, respectively - NPQ- non-photochemical quenching - PS- photosystem - Q_A- primary quinone acceptor - qP- photochemical quenching coefficient     

Unifying model for the photoinactivation of Photosystem II in vivo under steady-state photosynthesis

We present a unifying mechanism for photoinhibition based on current obsevations from in vivo studies rather than from in vitro studies with isolated thylakoids or PS II membranes. In vitro studies have limited relevance for in vivo photoinhibition because very high light is used with photon exposures rarely encountered in nature, and most of the multiple, interacting, protective strategies of PS II regulation in living cells are not functional. It is now established that the photoinactivation of Photosystem II in vivo is a probability and light-dosage event which depends on the photons absorbed and not the irradiance per se. As the reciprocity law is obeyed and target theory analysis strongly suggests that only one photon is required, we propose that a single dominant molecular mechanism occurs in vivo with one photon inactivating PS II under limiting, saturating or sustained high light. Two mechanisms have been proposed for photoinhibition under high light, acceptor-side and donor-side photoinhibition [see Aro et al. (1994) Biochim Biophys Acta 1143: 113–134], and another mechanism for very low light, the low-light syndrome [Keren et al. (1995) J Biol Chem 270: 806–814]. Based on the exciton-radical pair equilibrium model of exciton dynamics, we propose a unifying mechanism for the photoinactivation of PS II in vivo under steady-state photosynthesis that depends on the generation and maintenance of increased concentrations of the primary radical pair, P680⁺Pheo_–, and the different ways charge recombination is regulated under varying environmental conditions [Anderson et al. (1997) Physiol Plant 100: 214–223]. We suggest that the primary cause of damage to D1 protein is P680⁺, rather than singlet O₂ formed from triplet P680, or other reactive oxygen species.     

Dissecting the Photoprotective Mechanism Encoded by the flv4‐2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization

In Synechocystis sp. PCC 6803, the flv4‐2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4‐2 operon mutants. In the ?sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with ?sll0218‐flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the ?sll0218 and ?sll0218‐flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218‐lacking mutants. The YFP‐tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA‐defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the ?flv4‐2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting.     

A simple model relating photoinhibitory fluorescence quenching in chloroplasts to a population of altered Photosystem II reaction centers

A model is presented describing the relationship between chlorophyll fluorescence quenching and photoinhibition of Photosystem (PS) II-dependent electron transport in chloroplasts. The model is based on the hypothesis that excess light creates a population of inhibited PS II units in the thylakoids. Those units are supposed to posses photochemically inactive reaction centers which convert excitation energy to heat and thereby quench variable fluorescence. If predominant photoinhibition of PS II and cooperativity in energy transfer between inhibited and active units are presumed, a quasi-linear correlation between PS II activity and the ratio of variable to maximum fluorescence, F_VF_M, is obtained. However, the simulation does not result in an inherent linearity of the relationship between quantum yield of PS II and F_VF_M ratio. The model is used to fit experimental data on photoinhibited isolated chloroplasts. Results are discussed in view of current hypotheses of photoinhibition.Abbreviations F_M maximum total fluorescence - F₀ initial fluorescence - F_V maximum variable fluorescence - PS Photosystem - Q_A, Q_B primary and secondary electron acceptors of Photosystem II     

Photosystem II reaction centres stay intact during low temperature photoinhibition

Photoinhibition of photosynthesis was studied in intact barley leaves at 5 and 20°C, to reveal if Photosystem II becomes predisposed to photoinhibition at low temperature by 1) creation of excessive excitation of Photosystem II or, 2) inhibition of the repair process of Photosystem II. The light and temperature dependence of the reduction state of Q_A was measured by modulated fluorescence. Photon flux densities giving 60% of Q_A in a reduced state at steady-state photosynthesis (300 mol m^–2s^–1 at 5°C and 1200 mol m^–2s^–1 at 20°C) resulted in a depression of the photochemical efficiency of Photosystem II (F_v/F_m) at both 5 and 20°C. Inhibition of F_v/F_m occurred with initially similar kinetics at the two temperatures. After 6h, F_v/F_m was inhibited by 30% and had reached steady-state at 20°C. However, at 5°C, F_v/F_m continued to decrease and after 10h, F_v/F_m was depressed to 55% of control. The light response of the reduction state of Q_A did not change during photoinhibition at 20°C, whereas after photoinhibition at 5°C, the proportion of closed reaction centres at a given photon flux density was 10–20% lower than before photoinhibition.Changes in the D1-content were measured by immunoblotting and by the atrazine binding capacity during photoinhibition at high and low temperatures, with and without the addition of chloramphenicol to block chloroplast encoded protein synthesis. At 20°C, there was a close correlation between the amount of D1-protein and the photochemical efficiency of photosystem II, both in the presence or in the absence of an active repair cycle. At 5°C, an accumulation of inactive reaction centres occurred, since the photochemical efficiency of Photosystem II was much more depressed than the loss of D1-protein. Furthermore, at 5°C the repair cycle was largely inhibited as concluded from the finding that blockage of chloroplast encoded protein synthesis did not enhance the susceptibility to photoinhibition at 5°C.It is concluded that, the kinetics of the initial decrease of F_v/F_m was determined by the reduction state of the primary electron acceptor Q_A, at both temperatures. However, the further suppression of F_v/F_m at 5°C after several hours of photoinhibition implies that the inhibited repair cycle started to have an effect in determining the photochemical efficiency of Photosystem II.Abbreviations CAP D-threochloramphenicol - F₀ and F ₀ fluorescence when all Photosystem II reaction centres are open in dark- and light-acclimated leaves, respectively - F_m and F _m fluorescence when all Photosystem II reaction centres are closed in dark- and light-acclimated leaves, respectively - F_s fluorescence at steady state - Q_A the primary, stable quinone acceptor of Photosystem II - q_N non-photochemical quenching of fluorescence - q_P photochemical quenching of fluorescence     

Energy transfers from Photosystem II to Photosystem I in Cryptomonas rufescens (Cryptophyceae)

In Cryptomonas rufescens (Cryptophyceae), phycoerythrin located in the thylakoid lumen is the major accessory pigment. Oxygen action spectra prove phycoerythrin to be efficient in trapping light energy.The fluorescence excitation spectra at ?196°C obtained by the method of Butler and Kitajima (Butler, W.L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72–85) indicate that like in Rhodophycease, chlorophyll a is the exclusive light-harvesting pigment for Photosystem I.For Photosystem II we can observe two types of antennae: (1) a light-harvesting chlorophyll complex connected to Photosystem II reaction centers, which transfers excitation energy to Photosystem I reaction centers when all the Photosystem II traps are closed. (2) A light-harvesting phycoerythrin complex, which transfers excitation energy exclusively to the Photosystem II reaction complexes responsible for fluorescence at 690 nm.We conclude that in Cryptophyceae, phycoerythrin is an efficient light-harvesting pigment, organized as an antenna connected to Photosystem II centers, antenna situated in the lumen of the thylakoid. However, we cannot afford to exclude that a few parts of phycobilin pigments could be connected to inactive chlorophylls fluorescing at 690 nm.     

Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.     

Photosystem II electron transfer cycle and chlororespiration in planktonic diatoms

The dominance of diatoms in turbulent waters suggests special adaptations to the wide fluctuations in light intensity that phytoplankton must cope with in such an environment. Our recent demonstration of the unusually effective photoprotection by the xanthophyll cycle in diatoms [Lavaud et al. (2002) Plant Physiol 129 (3) (in press)] also revealed that failure of this protection led to inactivation of oxygen evolution, but not to the expected photoinhibition. Photo-oxidative damage might be prevented by an electron transfer cycle around Photosystem II (PS II). The induction of such a cycle at high light intensity was verified by measurements of the flash number dependence of oxygen production in a series of single-turnover flashes. After a few minutes of saturating illumination, the oxygen flash yields are temporarily decreased. The deficit in oxygen production amounts to at most 3 electrons per PS II, but continues to reappear with a half time of 2 min in the dark until the total pool of reducing equivalents accumulated during the illumination has been consumed by (chloro)respiration. This is attributed to an electron transfer pathway from the plastoquinone pool or the acceptor side of PS II to the donor side of PS II that is insignificant at limiting light intensity but is accelerated to milliseconds at excess light intensity. Partial filling of the 3-equivalents capacity of the cyclic electron transfer path in PS II may prevent both acceptor-side photoinhibition in oxygen-evolving PS II and donor-side photoinhibition when the oxygen-evolving complex is temporarily inactivated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electron donation in Photosystem II

The electron transfer resulting from illumination and dark storage of PS II has been studied using EPR signals from several electron carriers. The recombination of D⁺ (Signal II) and Q⁻_A formed by illumination occurred during dark storage at 77 K and was used to deplete reaction centres of D⁺. The donor D was then shown to be oxidized in the dark by the S₂ state of the oxygen-evolving complex. A slow change which occurred during dark storage of PS II samples was detected using the power saturation characteristics of D. We interpret this effect on D to be an indirect result of a rearrangement of the manganese complex during long-term dark adaptation. A role for D in the stability, protection and perhaps initial manganese binding of the oxygen-evolving complex is suggested.     

Mathematical modelling of the light response curve of photoinhibition of Photosystem II

The light response curves of the acceptor and donor side mechanisms of photoinhibition of Photosystem II were calculated, using Arabidopsis as a model organism. Acceptor-side photoinhibition was modelled as double reduction of Q_A, noting that non-photochemical quenching has the same effect on the quantum yield of Q_A double reduction in closed PSII centres as it has on the quantum yield of electron transport in open centres. The light response curve of acceptor-side photoinhibition in Arabidopsis shows very low efficiency under low intensity light and a relatively constant quantum yield above light saturation of photosynthesis. To calculate the light response curve of donor-side photoinhibition, we built a model describing the concentration of the oxidized primary donor P₆₈₀⁺ during steady-state photosynthesis. The model is based on literature values of rate constants of electron transfer reactions of PSII, and it was fitted with fluorescence parameters measured in the steady state. The modelling analysis showed that the quantum yield of donor-side photoinhibition peaks under moderate light. The deviation of the acceptor and donor side mechanisms from the direct proportionality between photoinhibition and photon flux density suggests that these mechanisms cannot solely account for photoinhibition in vivo, but contribution of a reaction whose quantum yield is independent of light intensity is needed. Furthermore, a simple kinetic calculation suggests that the acceptor-side mechanism may not explain singlet oxygen production by photoinhibited leaves. The theoretical framework described here can be used to estimate the yields of different photoinhibition mechanisms under different wavelengths or light intensities.     

D1 protein of photosystem II: The light sensor in chloroplasts

Light, controls the “blueprint” for chloroplast development, but at high intensities is toxic to the chloroplast. Excessive light intensities inhibit primarily photosystem II electron transport. This results in generation of toxic singlet oxygen due to impairment of electron transport on the acceptor side between pheophytin and Q_B -the secondary electron acceptor. High light stress also impairs electron transport on the donor side of photosystem II generating highly oxidizing species Z⁺ and P680⁺. A conformationsl change in the photosystem II reaction centre protein Dl affecting its Q_B-binding site is involved in turning the damaged protein into a substrate for proteolysis. The evidence indicates that the degradation of D1 is an enzymatic process and the protease that degrades D1 protein has been shown to be a serine protease Although there is evidence to indicate that the chlorophyll a-protein complex CP43 acts as a serine-type protease degrading Dl, the observed degradation of Dl protein in photosystem II reaction centre particlesin vitro argues against the involvement of CP43 in Dl degradation. Besides the degradation during high light stress of Dl, and to a lesser extent D2-the other reaction centre protein, CP43 and CP29 have also been shown to undergo degradation. In an oxygenic environment, Dl is cleaved from its N-and C-termini and the disassembly of the photosystem II complex involves simultaneous release of manganese and three extrinsic proteins involved in oxygen evolution. It is known that protein with PEST sequences are subject to degradation; D1 protein contains a PEST sequence adjacent to the site of cleavage on the outer side of thylakoid membrane between helices IV and V. The molecular processes of “triggering” of Dl for proteolytic degradation are not clearly understood. The changes in structural organization of photosystem II due to generation of oxy-radicals and other highly oxidizing species have also not been resolved. Whether CP43 or a component of the photosystem II reaction centre itself (Dl. D2 or cy1 b559 subunits), which may be responsible for degradation of Dl, is also subject to light modification to become an active protease, is also not known. The identity of proteases degrading Dl, LHCII and CP43 and C29 remains to be established     

The tetranuclear manganese complex of Photosystem II

A polynuclear manganese complex functions in Photosystem II both to accumulate oxidizing equivalents and to bind water and catalyze its four-electron oxidation. Recent electron paramagnetic resonance (EPR) spectroscopic studies of the manganese complex show that four manganese ions are required to account for its magnetic properties. The exchange couplings between manganese ions in the S₂ state are characteristic of a Mn₄O₄ cubane-like structure. Based on this structure for the manganese complex in the S₂ state, as well as a consideration of the known properties of the manganese complex in Photosystem II and the coordination chemistry of manganese, structures are proposed for the five intermediate oxidation states of the manganese complex. A molecular mechanism for the formation of an O-O bond and the displacement of O₂ from the S₄ state is suggested.     

Inhibition of Photosystem II activity by saturating single turnover flashes in calcium-depleted and active Photosystem II

Inhibition of Photosystem II (PS II) activity induced by continuous light or by saturating single turnover flashes was investigated in Ca²⁺-depleted, Mn-depleted and active PS II enriched membrane fragments. While Ca²⁺- and Mn-depleted PS II were more damaged under continuous illumination, active PS II was more susceptible to flash-induced photoinhibition. The extent of photoinactivation as a function of the duration of the dark interval between the saturating single turnover flashes was investigated. The active centres showed the most photodamage when the time interval between the flashes was long enough (32 s) to allow for charge recombination between the S₂ or S₃ and Q_B ⁻ to occur. Illumination with groups of consecutive flashes (spacing between the flashes 0.1 s followed by 32 s dark interval) resulted in a binary oscillation of the loss of PS II-activity in active samples as has been shown previously (Keren N, Gong H, Ohad I (1995), J Biol Chem 270: 806–814). Ca²⁺- and Mn-depleted PS II did not show this effect. The data are explained by assuming that charge recombination in active PS II results in a back reaction that generates P₆₈₀ triplet and thence singlet oxygen, while in Ca²⁺- and Mn-depleted PS II charge recombination occurs through a different pathway, that does not involve triplet generation. This correlates with an up-shift of the midpoint potential of Q_A in samples lacking Ca²⁺ or Mn that, in term, is predicted to result in the triplet generating pathway becoming thermodynamically less favourable (G.N. Johnson, A.W. Rutherford, A. Krieger, 1995, Biochim. Biophys. Acta 1229, 201–207). The diminished susceptibility to flash-induced photoinhibition in Ca²⁺- and Mn-depleted PS II is attributed at least in part to this mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interaction of 1,4-benzoquinones with Photosystem II in thylakoids and Photosystem II membrane fragments from spinach

The interaction of exogenous quinones with the Photosystem II (PS II) acceptor side has been analyzed by measurements of flash-induced 320 nm absorption changes, transient flash-induced variable fluorescence changes, thermoluminescence emission and oxygen yield in dark-adapted thylakoids and PS II membrane fragments. Two classes of 1,4-benzoquinones were shown to give rise to remarkably different reaction patterns. (A) Phenyl-p-benzoquinone (Ph-p-BQ) -type compounds give rise to a marked binary oscillation of the initial amplitudes of 320 nm absorption changes induced by a flash train in dark-adapted PS II membrane fragments and a retardation of the decay kinetics of the flash-induced variable fluorescence. The electron transfer reactions to these type of quinones are severely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (B) In the presence of tribromotoluquinone (TBTQ) a different oscillation pattern of the 320 nm absorption changes is observed characterized by a marked relaxation after the first flash in the 5 ms domain. This relaxation is insensitive to 10 μM DCMU. Likewise the decay of the flash-induced variable fluorescence in TBTQ-treated samples is much less sensitive to DCMU than in control. The thermoluminescence emission exhibits an oscillation in samples incubated for 5 min with TBTQ before addition of 30 μM DCMU. Under the same conditions a significant flash-induced oxygen evolution is observed only after the third and fourth flash, respectively, whereas in the presence of TBTQ alone a normal oscillation pattern is observed. The different functional patterns of PS II caused by the two types of classes of exogenous quinones are interpreted by their binding properties: a noncovalent association with the Q_B-site of Ph-p-BQ-type quinones versus a tight (covalent?) binding in the vicinity of Q_A (possibly also at the Q_B-site) in the case of halogenated 1,4-benzoquinones. The mechanistic implications of these findings are discussed.     

Grana stacking and protection of Photosystem II in thylakoid membranes of higher plant leaves under sustained high irradiance: An hypothesis

We propose yet another function for the unique appressed thylakoids of grana stacks of higher plants, namely that during prolonged high light, the non-functional, photoinhibited PS II centres accumulate as D1 protein degradation is prevented and may act as dissipative conduits to protect other functional PS II centres. The need for this photoprotective mechanism to prevent high D1 protein turnover under excess photons in higher plants, especially those grown in shade, is due to conflicting demands between efficient use of low irradiance and protection from periodic exposure to excessive irradiance.     

Evidence for slow turnover in a fraction of Photosystem II complexes in thylakoid membranes

We used two different techniques to measure the recovery time of Photosystem II following the transfer of a single electron from P-680 to Q_A in thylakoid membranes isolated from spinach. Electron transfer in Photosystem II reaction centers was probed first by spectroscopic measurements of the electrochromic shift at 518 nm due to charge separation within the reaction centers. Using two short actinic flashes separated by a variable time interval we determined the time required after the first flash for the electrochromic shift at 518 nm to recover to the full extent on the second flash. In the second technique the redox state of Q_A at variable times after a saturating flash was monitored by measurement of the fluorescence induction in the absence of an inhibitor and in the presence of ferricyanide. The objective was to determine the time required after the actinic flash for the fluorescence induction to recover to the value observed after a 60 s dark period. Measurements were done under conditions in which (1) the electron donor for Photosystem II was water and the acceptor was the endogenous plastoquinone pool, and (2) Q₄₀₀, the Fe²⁺ near Q_A, remained reduced and therefore was not a participant in the flash-induced electron-transfer reactions. The electrochromic shift at 518 nm and the fluorescence induction revealed a prominent biphasic recovery time for Photosystem II reaction centers. The majority of the Photosystem II reaction centers recovered in less than 50 ms. However, approx. one-third of the Photosystem II reaction centers required a half-time of 2–3 s to recover. Our interpretation of these data is that Photosystem II reaction centers consist of at least two distinct populations. One population, typically 68% of the total amount of Photosystem II as determined by the electrochromic shift, has a steady-state turnover rate for the electron-transfer reaction from water to the plastoquinone pool of approx. 250 e⁻ / s, sufficiently rapid to account for measured rates of steady-state electron transport. The other population, typically 32%, has a turnover rate of approx. 0.2 e⁻ / s. Since this turnover rate is over 1000-times slower than normally active Photosystem II complexes, we conclude that the slowly turning over Photosystem II complexes are inconsequential in contributing to energy transduction. The slowly turning over Photosystem II complexes are able to transfer an electron from P-680 to Q_A rapidly, but the reoxidation of Q⁻_A is slow (t1/2 = 2 s). The fluorescence induction measurements lead us to conclude that there is significant overlap between the slowly turning over fraction of Photosystem II complexes and PS II_β reaction centers. One corollary of this conclusion is that electron transfer from P-680 to Q_A in PS II_β reaction centers results in charge separation across the membrane and gives rise to an electrochromic shift.