Studies on the mechanism of photosystem II photoinhibition II. The involvement of toxic oxygen species

In a previous paper it was shown that photoinhibition of reaction centre II of spinach thylakoids was predominantly caused by the degradation of D1-protein. An initial inactivation step at the Q_B-site was distinguished from its breakdown. The present paper deals with the question as to whether this loss of Q_B-function is caused by oxygen radical attack. For this purpose the photoinhibition of thylakoids was induced at 20°C in the presence of either superoxide dismutase and catalase or the antioxidants glutathione and ascorbic acid. This resulted in comparable though not total protection of D1-protein, photochemistry and fluorescence from photoinhibition. The combined action of both the enzymatic and the non-enzymatic radical scavenging systems brought about an even more pronounced protective effect against photoinhibition than did either of the two systems singularly at saturating concentrations. The results signify a major contribution of activated oxygen species to the degradation process of D1-protein and the related phenomena of photoinhibition. Thylakoids treated with hydroxyl radicals generated through a Fenton reaction showed a loss of atrazine binding sites, electron transport capacity and variable fluorescence in a similar manner, though not to the same extent, as usually observed following photoinhibitory treatment.Abbreviations Asc ascorbate - Fecy ferricyanide - GSH reduced glutathione - PQ plastoquinone - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SOD superoxide dismutase     

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     

Regulation of D1-protein degradation during photoinhibition of photosystem II in vivo: Phosphorylation of the D1 protein in various plant groups

Photoinhibition of PSII and turnover of the D1 reaction-centre protein in vivo were studied in pumpkin leaves (Cucurbita pepo L.) acclimated to different growth irradiances and in low-light-grown moss, (Ceratodon purpureus) (Hedw.) Brid. The low-light-acclimated pumpkins were most susceptible to photoinhibition. The production rate of photoinhibited PSII centres (k_PI), determined in the presence of a chloroplast-encoded protein-synthesis inhibitor, showed no marked difference between the high- and low-light-grown pumpkin leaves. On the other hand, the rate constant for the repair cycle (k_REC) of PSII was nearly three times higher in the high-light-grown pumpkin when compared to low-light-grown pumpkin. The slower degradation rate of the damaged D1 protein in the low-light-acclimated leaves, determined by pulsechase experiments with [³⁵S]methionine suggested that the degradation of the Dl protein retards the repair cycle of PSII under photoinhibitory light. Slow degradation of the D1 protein in low-light-grown pumpkin was accompanied by accumulation of a phosphorylated form of the D1 protein, which we postulate as being involved in the regulation of D1-protein degradation and therefore the whole PSII repair cycle. In spite of low growth irradiance the repair cycle of PSII in the moss Ceratodon was rapid under high irradiance. When compared to the high- or low-light-acclimated pumpkin leaves, Ceratodon had the highest rate of D1-protein degradation at 1000 mol photons m^–2 s^–1. In contrast to the higher plants, the D1 protein of Ceratodon was not phosphorylated either under high irradiance in vivo or under in-vitro conditions, which readily phosphorylate the D1 protein of higher plants. This is consistent with the rapid degradation of the D1 protein in Ceratodon. Screening experiments indicated that D1 protein can be phosphorylated in the thylakoid membranes of angiosperms and conifers but not in lower plants. The postulated regulation mechanism of D1-protein degradation involving phosphorylation and the role of thylakoid organization in the function of PSII repair cycle are discussed.Abbreviations Chl Chlorophyll - D1^* phosphorylated form of D1 protein - F_max and F_v maximal and variable fluorescence respectively - k_PJ and k_REC rate constants of photoinhibition and concurrent recovery respectively - LHCII lightharvesting chlorophyll a/bprotein of PSII - PFD photon flux density Dr. R. Barbato (Dipartimento di Biologia, Universita di Padova, Padova, Italy), Prof. P. Böger (Lehrstuhl fur Physiologie und Biochemie der Pflanzen, Universität Konstanz, Konstanz, Germany), Prof. A. Melis (Department of Plant Biology, University of California, Berkeley, USA), Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) and Mr. A. Soitamo (Department of Biology, University of Turku, Turku, Finland) are gratefully acknowledged for the D1-protein-specific antibodies. The authors thank Ms. Virpi Paakkarinen for excellent technical assistance. This work was supported by the Academy of Finland and the Foundation of the University of Turku.     

Inhibition of CO2-fixation and its effect on the activity of Photosystem II,on D1-protein synthesis and phosphorylation

To study the effects of limitations in the Calvin-cycle on Photosystem (PS) II function and on its repair by D1-protein turnover, glycerinaldehyde (DLGA) was applied to 1 h dark-adapted pea leaves via the petiole. The application resulted in a 90% inhibition of photosynthetic oxygen evolution after 90 min illumination at either 120 or 500 µmol m^–2 s^–1. In the control leaves an increase of light-dependent oxygen production to 147 and 171% was observed after 90 min illumination. According to chlorophyll fluorescence quenching analysis the inhibition of photosynthetic electron transport by DLGA led to a substantial increase in the reduction state of the primary quinone acceptor of PS II, QA, and to a rise in membrane energetisation. However, PS II functionality was hardly affected by DLGA at the low light intensity as indicated by the constant high yield of variable fluorescence, Fv/Fm. Only at 500 µmol m^–2 s^–1 a 15% loss of Fv/Fm was observed in the presence of DLGA indicating that inactivated PS II centres had accumulated. The control leaves also showed a slight loss of Fv/Fm which did not affect photosynthetic electron transport due to a faster reoxidation of QA. The relative stability of PS II function in the presence of DLGA could not be ascribed to an increased repair by the rapid turnover of the D1-protein. Radioactive pulse-labelling studies with [14C] leucine in combination with immunological determination of the protein content revealed that both synthesis and degradation of the protein were inhibited in DLGA-treated leaves whereas in the control leaves a stimulation of D1-protein turnover was observed. The changes of D1-protein turnover could be explained by differences in the occupancy state of the QB-binding niche. A relation between the phosphorylation status of the PS II polypeptides and the turnover of the D1-protein could not be established. As shown by radioactive labelling with [32P]i, addition of DLGA led to an increase in the phosphorylation level of the PS II polypeptides D1 and D2 at the low light intensity when compared to the non-treated control. At the higher light intensity the phosphorylation level of the PS II polypeptides in control and DLGA-treated leaves were identical in spite of the substantial differences in D1-protein turnover.     

Two mechanisms of recovery from photoinhibition in vivo: Reactivation of photosystem II related and unrelated to D1-protein turnover

Recovery (at 20° C) of spinach (Spinacia oleracea L.) leaf sections from photoinhibition of photosynthesis was monitored by means of the fluorescence parameter F_V/F_M of intact leaf tissue and of PSII-driven electron-transport activity of isolated thylakoids. Different degrees of photoinactivation of PSII were obtained by preillumination in ambient air (at 4 or 20° C), CO₂-free air or at low and high O₂ levels (2 or 41 %) in N₂. The kinetics of recovery exhibited two distinct phases. The first phase usually was completed within about 20-60 min and was most pronounced after preillumination in low O₂. The slow phase proceeded for several hours leading to almost complete reactivation of PSII. Preincubation of the leaves with streptomycin (SM), which inhibits chloroplast-encoded protein synthesis, inhibited the slow recovery phase only, indicating the dependence of this phase on resynthesis of the reaction-centre protein, D1. The fast recovery phase remained largely unaffected by SM. Both phases were strongly but not totally dependent on irradiation of the leaf with low light. When SM was absent, net degradation of the D1 protein could neither be detected upon photoinhibitory irradiation nor during following incubation of the leaf sections in low light or darkness. In the presence of SM, net D1 degradation was seen and tended to increase with O₂ concentration during photoinhibition treatment. Based on these data, we suggest that photoinactivation of PSII in vivo occurs in at least two steps. From the first step, reactivation appears possible in low light without D1 turnover (fast recovery phase). Action of oxygen then may lead to a second step, in which the D1 protein is affected and reactivation requires its removal and replacement (slow phase).Abbreviations Chl chlorophyll - F₀, F_M and F_V initial, maximum total and maximum variable chlorophyll fluorescence yield, respectively - PFD photon flux density - SM streptomycin We thank Professor P. Böger (Department of Plant Physiology and Biochemistry, University of Konstanz, Germany) for a gift of D1-specific antibodies. The paper contains part of the thesis work of J.L. The study was supported by the Deutsche Forschungs-gemeinschaft (SFB 189).     

Rapid turnover of the D1 reaction-center protein of photosystem II as a protection mechanism against photoinhibition in a moss,Ceratodon purpureus (Hedw.) Brid.

Susceptibility of a moss,Ceratodon purpureus (Hedw.) Brid., to photoinhibition and subsequent recovery of the photochemical efficiency of PSII was studied in the presence and absence of the chloroplast-encoded protein-synthesis inhibitor lincomycin.Ceratodon had a good capacity for repairing the damage to PSII centers induced by strong light. Tolerance against photoinhibition was associated with rapid turnover of the D1 protein, since blocking of D1 protein synthesis more than doubled the photoinhibition rate measured as the decline in the ratio of variable fluorescence to maximal fluorescence (F_v/F_max). Under exposure to strong light in the absence of lincomycin a net loss of D1 protein occurred, indicating that the degradation of damaged D1 protein inCeratodon was rapid and independent of the resynthesis of the polypeptide. The result suggests that synthesis is the limiting factor in the turnover of D1 protein during photoinhibition of the mossCeratodon. The level of initial fluorescence (F_o) correlated with the production of inactive PSII centers depleted of D1 protein. The higher the F_o level, the more severe was the loss of D1 protein seen in the samples during photoinhibition. Restoration of F_v/F_max at recovery light consisted of a fast and slow phase. The recovery of fluorescence yield in the presence of lincomycin, which was added at different times in the recovery, indicated that the chloroplast-encoded protein-synthesis-dependent repair of damaged PSII centers took place during the fast phase of recovery. Pulse-labelling experiments with [³⁵S]methionine supported the conclusion drawn from fluorescence measurements, since the rate of D1 protein synthesis after photoinhibition exceeded that of the control plants during the first hours under recovery conditions.     

Changes in D1-protein turnover and recovery of photosystem II activity precede accumulation of chlorophyll in plants after release from mineral stress

After seven weeks of a combined magnesium and sulphur deficiency, spinach (Spinacea oleracea L.) plants showed a substantial accumulation of inactivated photosystem II (PSII) centres as indicated by a 40% decrease of the chlorophyll (Chl) fluorescence parameter F_v/F_m (F_v being the yield of variable fluorescence and F_m the yield of maximal fluorescence when all reaction centres are closed) together with a severe loss of leaf Chl content of 75%. The responses of the photosynthetic apparatus were examined when the deficient plants were transferred back to a rich nutrient medium. During the first 24 h of the recovery phase, thylakoid protein synthesis measured as incorporation of [¹⁴C]leucine per unit of Chl increased substantially. The synthesis rate of the D1 reaction-centre polypeptide of PSII, which in the deficient plants was reduced to 50% of the non-deficient control, was stimulated eight- to ninefold. D1-protein content, which in the deficient plants was reduced to 40% of the non-deficient control, started to increase 2 d later. Thus, D1-protein degradation was also enhanced. The increased D1-protein turnover led to a rapid repair of the existing PSII centres as indicated by the rise of F_v/F_m. It was completed at day 7 of the recovery phase. At day 2 of the recovery phase, the synthesis of other thylakoid proteins such as the D2 protein, cytochrome b ₅₅₉, CP 47 and the 33-kDa polypeptide of the water-splitting system, became stimulated. This process resulted in an accumulation of new PSII centres. During the first week, formation of new PSII centres was not associated with an increase in leaf Chl content. The Chl content of the recovering leaves only started to increase when the ratio of PSII polypeptides versus LHCII (light-harvesting complex of PSII), which was substantially diminished in the deficient plants, became comparable to that of the control. The recovery process was accompanied by substantial changes in thylakoid protein phosphorylation. Their relevance to thylakoid protein turnover and stability is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - F_o yield of intrinsic fluorescence when all PSII centres are open in the dark - F_m yield of maximal fluorescence when all reaction centres are closed - F_m fluorescence yield when all reaction centres are closed (after a saturating flash) under steady-state conditions - F_v yield of variable fluorescence, (difference between F_oand F_m) - F yield of variable fluorescence under steady state conditions - LHC light-harvesting complex - PQ plastoquinone - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII - q_P photochemical quenching - q_n non-photochemical quenching The authors like to thank Dipl. Biol. Britta Untereiser for determining the chlorophyll fluorescence quenching factors. This work was supported by grants from the Bundesminister für Forschung und Technologie, the Project Europäisches Forschungszentrum and the German Israeli Foundation in cooperation with Prof. I. Ohad, Hebrew University, Jerusalem, Israel.     

Mathematical modelling of photoinhibition and Photosystem II repair cycle. I. Photoinhibition and D1 protein degradation in vitro and in the absence of chloroplast protein synthesis in vivo

The kinetics of photoinhibition of Photosystem II and D1 protein degradation were studied by applying mathematical modelling to new and published data. The word photoinhibition refers here only to such inhibition of PS II activity that requires chloroplast protein synthesis for recovery. It is shown that acceptor-side photoinhibition in vitro as well as in vivo photoinhibition in higher plants and cyanobacteria in the presence of prokaryotic translation inhibitors follow first-order kinetics. Degradation of damaged D1 protein also fits in a first-order reaction equation with respect to the concentration of photoinhibited PS II centres. It is shown that photoprotective lowering of the ratio of variable to maximum fluorescence can be distinguished from the lowering of this ratio associated with photoinhibition.     

Effect of photosystem II reaction center closure on nanosecond fluorescence relaxation kinetics

The fluorescence decay of chlorophyll in spinach thylakoids was measured as a function of the degree of closure of Photosystem II reaction centers, which was set for the flowed sample by varying either the preillumination by actinic light or the exposure of the sample to the exciting pulsed laser light. Three exponential kinetic components originating in Photosystem II were fitted to the decays; a fourth component arising from Photosystem I was determined to be negligible at the emission wavelength of 685 nm at which the fluorescence decays were measured. Both the lifetimes and the amplitudes of the components vary with reaction center closure. A fast (170–330 ps) component reflects the trapping kinetics of open Photosystem II reaction centers capable of reducing the plastoquinone pool; its amplitude decreases gradually with trap closure, which is incompatible with the concept of photosynthetic unit connectivity where excitation energy which encounters a closed trap can find a different, possibly open one. For a connected system, the amplitude of the fast fluorescence component is expected to remain constant. The slow component (1.7–3.0 ns) is virtually absent when the reaction centers are open, and its growth is attributable to the appearance of closed centers. The middle component (0.4–1.7 ns) with approximately constant amplitude may originate from centers that are not functionally linked to the plastoquinone pool. To explain the continuous increase in the lifetimes of all three components upon reaction center closure, we propose that the transmembrane electric field generated by photosynthetic turnover modulates the trapping kinetics in Photosystem II and thereby affects the excited state lifetime in the antenna in the trap-limited case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PQ plastoquinone - PSI and PSII Photosystem I and II - Q_A and Q_B primary and secondary quinone acceptor of PSII     

Turnover of the D1 protein and of Photosystem II in a Synechocystis 6803 mutant lacking Tyrz

The Photosystem II reaction center is rapidly inactivated by light, particularly at higher light intensity. One of the possible factors causing this phenomenon is the oxidized primary donor, P680⁺, which may be harmful to Photosystem II because of its highly oxidizing nature. However, no direct evidence specificially implicating P680⁺ in photoinhibition has been obtained yet. To investigate whether P680⁺ is harmful to Photosystem II, turnover of the D1 protein and of the Photosystem II reaction center complex were measured in vivo in a mutant of the cyanobacterium Synechocystis sp. PCC 6803, in which the physiological donor to P680⁺, Tyr_z, was genetically deleted. In this mutant, D1 degradation in the light is an order of magnitude faster than in wild type. The most straightforward explanation of this phenomenon is that accumulation of P680⁺ leads to an increased rate of turnover of the Photosystem II reaction center complex, which is compatible with the hypothesis of destructive oxidation by P680⁺ that is damaging to the Photosystem II complex.     

Photoinactivation of functional photosystem II and D1-protein synthesis in vivo are independent of the modulation of the photosynthetic apparatus by growth irradiance

To investigate whether the in-vivo photoinhibition of photosystem II (PSII) function by excess light is an intrinsic property of PSII, the maximal photochemical efficiency of PSII (Fv/Fm) and the content of functional PSII (measured by repetitive flash yield of oxygen evolution) were determined in leaves of pea (Pisum sativum L.), grown in 50 (low light), 250 (medium light), and 650 (high light) mol photons·m^–2·s^–1. The modulation of PSII functionality in vivo was induced in 1.1% CO₂ by varying either (i) the duration (0–2 h) of light treatment (fixed at 1800 mol photons· m^–2·s^–1) or (ii) irradiance (0–3200 mol photons·m^–2·s^–1) at a fixed duration (1 h), after infiltration of leaves with water (control), lincomycin (an inhibitor of chloroplast-encoded protein synthesis), or a combination of lincomycin with nigericin (an uncoupler), through the cut petioles of leaves of 22-to 24-d-old plants. The reciprocity law of irradiance and duration of illumination for PSII function in vivo (Park et al. 1995, Planta 196: 401–411) holds in all differently light-grown peas, demonstrating that inactivation of functional PSII depends on photon exposure (mol photons·m^–2), not on the rate of photon absorption. In vivo, PSII acts as an intrinsic photon counter and at higher photon exposures is inactivated following absorption of about 3 × 10⁷ photons. There is a functional heterogeneity of PSII in vivo with 25% less-stable PSIIs that are inactivated at low photon exposure, compared to 75% more-stable PSIIs regardless of modulation of the photosynthetic apparatus. We suggest that the less-stable PSIIs represent monomers located in the nonappressed granal margins, while the more-stable PSIIs are dimers located in the appressed grana membrane cores. The capacity for D1-protein synthesis was the same in all the light-acclimated peas and saturated at low light, indicating that D1-protein repair is also an intrinsic property of PSII. This accounts for the low intensity required for recovery of photoinhibition in sun and shade plants which is independent of light-harvesting antennae size or PSII/PSI stoichiometries.Abbreviations D1-protein psbA gene product - D2 protein psbD gene product - Fo chlorophyll fluorescence corresponding to open PSII reaction centres - Fv, Fm variable and maximum fluorescence after dark incubation, respectively - PS photosystem - Q_B secondary quinone electron acceptor Financial support for this research by the Department of Employment, Education and Training/Australian Research Council International Research Fellowships Program (Korea) is gratefully acknowledged.     

The irreversible photoinhibition of the photosystem II complex in leaves of Vicia faba under strong light

Exposure of the photosynthetic machinery to strong light causes the photoinhibition of the photosystem II complex. The recovery from the photoinhibition in vivo was characterized by monitoring the ratio of variable to maximum fluorescence (F_v/F_m) in detached leaves of broad bean (Vicia faba). The changes in the ratio were explained in terms of three components, namely, two saturating exponential components with half rise-times of about 15 and 120 min, respectively, and a non-recovery component. The non-recovery component increased gradually as the exposure to strong light was prolonged. Our results suggest that this irreversible component of the photoinhibition of the photosystem II complex was caused by severe stress due to strong light under which repair of the photosystem II complex was insufficient to allow full recovery. The irreversible photoinhibition is discussed in terms of both the physiology and ecology of plants.     

Stress-induced chlorosis and increase in D1-protein turnover precede photoinhibition in spinach suffering under magnesium/sulphur deficiency

To test wether chlorosis is induced by photoinhibitory damage to photosystem II (PSII), onset of chlorosis and loss of PSII function were compared in young spinach (Spinaciae oleracea L.) plants suffering under a combined magnesium and sulphur deficiency. Loss of chlorophyll already occurred after the first week of deficiency and preceded any permanent functional inhibition of the photosynthetic apparatus. Permanent disturbancies of photosynthetic electron transport measured in isolated thylakoids and of PSII function, determined via the ratio of variable fluorescence to maximal fluorescence, F_v/F_m, could be detected only after the second week of deficiency. After the third week, the plants had lost about 60% of their chlorophyll; even so, fluorescence data indicated that 85% of the existing PSII was still capable of initiating photosynthetic electron transport. However, quenching analysis of steady-state fluorescence showed an early increase in non-photochemical quenching and in down-regulated PSII centres with low steady-state quantum efficiency. Together with the down-regulation of PSII centres, a 1.4-fold increase in D1-protein synthesis, measured as incorporation of [¹⁴C]leucine, could be observed at the end of the first week before any loss of D1 protein, chlorophyll or photosynthetic activity could be detected. Immunological determiation by Western-blotting did not show a change in D1-protein content; thus, at this time, D1 protein was not only faster synthesised but was also faster degraded than before the imposition of mineral deficiency. The increased turnover was high enough to prevent any loss or functional inhibition of PSII. After 3 weeks, D1-protein synthesis on a chlorophyll basis was further stimulated by a factor of 2. However, this was not enough to prevent a net loss of D1 protein of about 70%, showing that the D1-protein was now degraded faster than it was synthesised. Immunological determination and electron-transport measurements showed that together with the loss of D1 protein the other polypetides of PSII were also degraded, resulting in a specific loss of PSII centres. The degradation of PSII centres prevented a large accumulation of damaged PSII centres. We assume that the decrease in PSII centres initiates the breakdown of the other thylakoid proteins.Abbreviations Fo yield of intrinsic fluorescence when all PSII centres are open in the dark - F_m yield of maximal fluorescence when all reaction centres are closed - F_m fluorescence yield when all reaction centres are closed under steady-state conditions - F_v yield of variable fluorescence, (difference between F_o and F_m) - F yield of variable fluorescence under steady-state conditions, difference between F_m and F_t, the fluorescence yield under steady-state conditions - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII - q_p photochemical quenching - q_n non-photochemical quenching This work was supported by grants from the Bundesminister für Forschung und Technologie and the German Israeli Foundation. The authors thank Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) for fruitful discussions.     

Strong-light photoinhibition treatment accelerates the changes of protein secondary structures in triton-treated photosystem I and photosystem II complexes

Changes in the protein secondary structure and electron transport activity of the Triton X-100-treated photosystem I (PSI) and photosystem II (PSII) complexes after strong illumination treatment were studied using Fourier transform-infrared (FT-IR) spectroscopy and an oxygen electrode. Short periods of photoinhibitory treatment led to obvious decreases in the rates of PSI-mediated electron transport activity and PSII-mediated oxygen evolution in the native or Triton-treated PSI and PSII complexes. In the native PSI and PSII complexes, the protein secondary structures had little changes after the photoinhibitory treatment. However, in both Triton-treated PSI and PSII complexes, short photoinhibition times caused significant loss of -helical content and increase of -sheet structure, similar to the conformational changes in samples of Triton-treated PSI and PSII complexes after long periods of dark incubation. Our results demonstrate that strong-light treatment to the Triton-treated PSI and PSII complexes accelerates destruction of the transmembrane structure of proteins in the two photosynthetic membranes.     

Phosphorylation of photosystem II core proteins prevents undesirable cleavage of D1 and contributes to the fine‐tuned repair of photosystem II

Photosystem II (PSII) is a primary target for light‐induced damage in photosynthetic protein complexes. To avoid photoinhibition, chloroplasts have evolved a repair cycle with efficient degradation of the PSII reaction center protein, D1, by the proteases FtsH and Deg. Earlier reports have described that phosphorylated D1 is a poor substrate for proteolysis, suggesting a mechanistic role for protein phosphorylation in PSII quality control, but its precise role remains elusive. STN8, a protein kinase, plays a central role in this phosphorylation process. To elucidate the relationship between phosphorylation of D1 and the protease function we assessed in this study the involvement of STN8, using Arabidopsis thaliana mutants lacking FtsH2 [yellow variegated2 (var2)] and Deg5/Deg8 (deg5 deg8). In support of our presumption we found that phosphorylation of D1 increased more in var2. Furthermore, the coexistence of var2 and stn8 was shown to recover the delay in degradation of D1, resulting in mitigation of the high vulnerability to photoinhibition of var2. Partial D1 cleavage fragments that depended on Deg proteases tended to increase, with concomitant accumulation of reactive oxygen species in the mutants lacking STN8. We inferred that the accelerated degradation of D1 in var2 stn8 presents a tradeoff in that it improved the repair of PSII but simultaneously enhanced oxidative stress. Together, these results suggest that PSII core phosphorylation prevents undesirable cleavage of D1 by Deg proteases, which causes cytotoxicity, thereby balancing efficient linear electron flow and photo‐oxidative damage. We propose that PSII core phosphorylation contributes to fine‐tuned degradation of D1.     

D1 protein degradation during photoinhibition of intact leaves a modification of the D1 protein precedes degradation

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high-light modified form that was subsequently degraded.     

NaCl胁迫加重强光胁迫下超大甜椒叶片的光系统II和光系统I的光抑制

快速叶绿素荧光动力学可以在无损情况下探知叶片光合机构的损伤程度, 快速叶绿素荧光测定和分析技术(JIP-test)将测量值转化为多种具有生物学意义的参数, 因而被广泛应用于植物光合机构对环境的响应机制研究。该文研究了超大甜椒(Capsicum annuum)幼苗在强光及不同NaCl浓度胁迫下的荧光响应情况。与单纯强光胁迫相比, NaCl胁迫引起了叶绿素荧光诱导曲线的明显改变, 光系统II (PSII)光抑制加重, 同时PSII反应中心和受体侧受到明显影响, 而且高NaCl浓度胁迫下PSII供体侧受伤害明显, 同时PSI反应中心活性(P700⁺)在盐胁迫下明显降低。这些结果表明, NaCl胁迫会增强强光对超大甜椒光系统的光抑制, 并且浓度越高抑制越明显, 但对PSI的抑制作用低于PSII。高NaCl浓度胁迫易对PSII供体侧造成破坏, 且PSI光抑制严重。     

Photosystem II function and herbicide binding sites during photoinhibition of spinach chloroplasts in-vivo and in-vitro

The time courses of some Photosystem II (PS II) parameters have been monitored during in-vivo and in-vitro photoinhibition of spinach chloroplasts, at room temperature and at 10 °C or 0 °C. Exposing leaf discs of low-light grown spinach at 25 °C to high light led to photoinhibition of chloroplasts in-vivo as manifested by a parallel decrease in the number of functional PS II centres, the variable chlorophyll fluorescence at 77K (F _v /F _m ), and the number of atrazine-binding sites. When the photoinhibitory treatment was given at 10 °C, the former two parameters declined in parallel but the loss of atrazine-binding sites occurred more slowly and to a lesser extent. During in-vitro photoinhibition of chloroplast thylakoids at 25 °C, the loss of functional PS II centres proceeded slightly more rapidly than the loss of atrazine-binding sites, and this difference in rate was further increased when the thylakoids were photoinhibited at 0 °C. During the recovery phase of leaf discs (up to 9 h) the increases in F _v /F _m preceded that of the number of functional PS II centres, while only a further decline in the number of atrazine-binding sites was observed. The recovery of variable chlorophyll fluorescence and the concentration of functional PS II centres occurred more rapidly at 25 °C than at 10 °C. These results suggest that the photoinhibition of PS II function is a relatively temperature-independent early photochemical event, whereas the changes in the concentration of herbicide-binding sites appear to be a more complex biochemical process which can occur with a delayed time course.Abbreviations BSA bovine serum albumin - Chl chlorophyll - D1 32kDa herbicide-binding polypeptide in photosystem II and product of the psbA gene - D2 34kDa polypeptide in photosystem II which is the product of the psbD gene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolin-dophenol - F ₀, F _v , F _m chlorophyll fluorescence with reaction centres open, variable and maximum fluorescence, respectively - LDS lithium dodecyl sulfate - MES 2-(N-morpholino) ethanesulfonic acid - PSII photosystem II - Q_A, Q_B first and second quinone-type PS II acceptor, respectively     

Light-dependent phosphorylation of D1 reaction centre protein of photosystem II: hypothesis for the functional role in vivo

Reversible phosphorylation of the D1 reaction centre protein of photosystem II (PSII) occurs in thylakoid membranes of higher plants. The significance of D1 protein phosphorylation in the function of PSII is not yet clear. This paper summarizes the data implying that phosphorylation of D1 protein in higher plants is involved in the regulation of the repair cycle of photoinhibited PSII centres. Photoinhibition of PSII, D1 protein phosphorylation and degradation have been studied in vivo in higher plant leaves acclimated to different growth irradiances. It is shown that photoinhibitory illumination induces maximal phosphorylation of the D1 protein. Under these conditions D1 turnover is also saturated. We postulate that phosphorylation retards the degradation of damaged D1 protein under conditions where rapid replacement by a new D1 copy is not possible. This would protect PSII from total disassembly and degradation of all PSII subunits. We conclude that the phosphorylation of D1 protein and the regulation of D1 protein degradation may have evolved together. Furthermore, these characteristics seem to be related to the highly organized structure of higher-plant type thylakoid membranes, since the capability to phosphorylate D1 protein is restricted to seed plants.     

Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II

The degradation rate of the D1 polypeptide was measured in threeSynechocystis PCC 6803 mutantsin vivo. Mutations were introduced into a putative cleavage area of the D1 polypeptide (QEEET motif) and into the PEST-like area. PEST sequences are often found in proteins with a high turnover rate. The QEEET-motif mutants are CA1 [(E242-E244);Q241H] and E243K, and the third mutation, E229D, was directed to the PEST-like area. During high-light illumination (1500 mol photons m^-2s^-1) that induced photoinhibition of photosystem II (PSII), the half-life time of the D1 polypeptide in mutant E229D (t _1/2=35 min) was about twice as long as in AR (control strain) cells (t _1/2=19 min). In growth light (40 mol photons m^-2s^-1), the degradation rate of the D1 polypeptide in E229D and AR strains was the same (t _1/25 h). In growth light the D1 polypeptide was degraded faster in both QEEET-motif mutants than in the AR strain, but in photoinhibitory light the degradation rates were similar. According to these results, the highly conservative QEEET motif as such is not required for the proteolytic cut of the D1 polypeptide, but it does affect the rate of degradation. No simple correlation existed between the degradation rate of the D1 polypeptide and the susceptibility of PSII to photoinhibition in mutant and AR cells under our experimental conditions.