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.     

Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 mol photons·m^–2·s^–1 and became photoinhibited, as indicated by the decrease in F_v/F_m (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound Q_B, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q _a ^- , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound Q_B and in Q_B-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [¹⁴C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.Abbreviations amp_(f,m,s) amplitude of the fast, (medium and slow) exponential component of fluorescence decay - F_m yield of maximum fluorescenc when all reaction centers are closed - F_o yield of intrinsic fluorescence at open PSII reaction centers in the dark - F_v yield of variable fluorescence, (difference between F_m and F_o) - LHC light-harvesting complex - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII Dedicated to Professor Dr. Dres. hc. Achim Trebst on the occasion of his 65th birthdayThis work was supported by grants from the BMFT and the Ministerium für Umwelt, Raumordnung and Landwirtschaft, Nordrhein-Westfalen. The authors thank H. Wietoska and M. Bronzel for skilful technical assistance.     

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.     

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.     

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.     

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).     

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.     

CO2 assimilation, xanthophyll cycle pigments and PSII efficiency in pumpkin plants as affected by ozone fumigation

CO₂ assimilation, xanthophyll cycle pigments and PSII efficiency were analyzed in two different ages of pumpkin leaves (Cucurbita pepo L. cv. Ambassador) exposed to 150 nmol mol^-1 of ozone (5 days, 5 h day^-1). Gas-exchange measurements revealed a reduction in CO₂ assimilation and stomatal conductance, accompanied by an increase in the intercellular CO₂ concentration both in young and in mature leaves as compared to their respective controls. In both leaves, F₀ remained unchanged, while F_m and the F_v/F_m ratio decreased after O₃ fumigation, indicating that ozone may induce an alteration in the capability of photosystem II (PSII) to reduce the primary acceptor Q_A. In the mature leaves the photochemical quenching (q_p) was significantly lowered by the pollutant, but this was not the case in the young leaves where q_p did not change. In both mature and young ozonated pumpkin leaves, the development of non-photochemical quenching caused a decrease in the PSII photochemical rate, as shown by the correlation between F_v/F_m and the de-epoxidation state of dark-adapted leaves. Decreases in the F_v/F_m ratio are generally attributed to damage to the PSII reaction centre, apart from the down-regulation of the capacity of PSII electron transport. While in young ozonated leaves the decrease in the F_v/F_m ratio was not associated with damage to the D1 protein, in mature ozonated pumpkin leaves, the decrease in the F_v/F_m was accompanied by a significant decline in the D1 content. In conclusion, ozone exposure induces alterations in the light reactions of photosynthesis in both young and mature leaves. However, in young leaves the engagement of the xanthophyll cycle appears to counteract ozone effects against the photosynthetic apparatus as demonstrated by the absence of damage to the D1 protein. On the other hand, the loss of D1 protein in mature fumigated leaves suggests that the activation of the xanthophyll cycle is not sufficient to prevent photoinhibition, probably because a physiological state of senescence adds to the oxidative stress.     

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.     

Photoinhibition and repair in Dunaliella salina acclimated to different growth irradiances

The light-dependent rate of photosystem-II (PSII) damage and repair was measured in photoautotrophic cultures of Dunaliella salina Teod. grown at different irradiances in the range 50–3000 mol photons · m^–2· s^–1. Rates of cell growth increased in the range of 50–800 mol photons·m^–2·s^–1, remained constant at a maximum in the range of 800–1,500 mol photons·m^–2 ·s^–1, and declined due to photoinhibition in the range of 1500–3000 mol photons·m^–2·s^–1. Western blot analyses, upon addition of lincomycin to the cultures, revealed first-order kinetics for the loss of the PSII reaction-center protein (D1) from the 32-kDa position, occurring as a result of photodamage. The rate constant of this 32-kDa protein loss was a linear function of cell growth irradiance. In the presence of lincomycin, loss of the other PSII reaction-center protein (D2) from the 34-kDa position was also observed, occurring with kinetics similar to those of the 32-kDa form of D1. Increasing rates of photodamage as a function of irradiance were accompanied by an increase in the steady-state level of a higher-molecular-weight protein complex ( 160-kDa) that cross-reacted with D1 antibodies. The steady-state level of the 160-kDa complex in thylakoids was also a linear function of cell growth irradiance. These observations suggest that photodamage to D1 converts stoichiometric amounts of D1 and D2 (i.e., the D1/D2 heterodimer) into a 160-kDa complex. This complex may help to stabilize the reaction-center proteins until degradation and replacement of D1 can occur. The results indicated an intrinsic half-time of about 60 min for the repair of individual PSII units, supporting the idea that degradation of D1 after photodamage is the rate-limiting step in the PSII repair process.Abbreviations Chl chlorophyll - PSI photosystem I - PSII photosystem II - D1 the 32-kDa reaction-center protein of PSII, encoded by the chloroplast psbA gene - D2 the 34-kDa reactioncenter protein of PSII, encoded by the chloroplast psbD gene - Q_A primary electron-accepting plastoquinone of PSII The work was supported by grant 94-37100-7529 from the US Department of Agriculture, National Research Initiative Competitive Grants Program.     

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.     

Effects of salicylic acid on protein kinase activity and chloroplast D1 protein degradation in wheat leaves subjected to heat and high light stress

In the north of China, wheat plants are often stressed by heat and high light during grain-filling stage, which leads to injury in photosynthetic apparatus and decline in photosynthetic rate. In order to develop a method to protect photosynthetic apparatus in wheat leaves subjected to heat and high light stress, the effects of SA (salicylic acid) and FSBA (5′-p-fluorosulfonylbenzoyl adenosine) on PK (protein kinase) activity, D1 protein degradation and the performance of PSII were investigated in present work. Our results showed that PK activity enhanced under heat and high light stress and declined when stress was removed. FSBA pretreatment resulted in marked decreases in PK activity and D1 protein level, suggesting a correlationship between degradation of D1 protein and phosphorylation. After 2 h of stress, D1 protein level in water-pretreated leaves decreased to 79% of control and then recovered to 81% after 3 h of recovery. This clearly indicated that the damage of D1 protein induced by heat and high light stress was reversible. Compared to the control, SA pretreatment could not only increase PK activity, retard the degradation of D1 protein during heat and high light stress, but also accelerate the recovery of D1 protein level when the stress was removed. Correspondingly, F_v/F_m (maximum photochemical efficiency of PSII), Φ_PSII (actual photochemical efficiency of PSII), ETR (electron transfer rate) and Pn (net photosynthetic rate) in SA-treated leaves were higher than that in leaves of control under both stress and non-stress conditions. Taken together, our results revealed that SA pretreatment could significantly alleviate damages of heat and high light stress on D1 protein and PSII of wheat leaves, and accelerate restoration of photosynthetic function.     

Up-regulation of a photosystem II core protein phosphatase inhibitor and sustained D1 phosphorylation in zeaxanthin-retaining, photoinhibited needles of overwintering Douglas fir

Overwintering needles of the evergreen conifer Douglas fir exhibited an association between arrest of the xanthophyll cycle in the dissipating state (as zeaxanthin + antheraxanthin; Z + A) with a strongly elevated predawn phosphorylation state of the D1 protein of the photosystem II (PSII) core. Furthermore, the high predawn phosphorylation state of PSII core proteins was associated with strongly increased levels of TLP40, the cyclophilin-like inhibitor of PSII core protein phosphatase, in winter versus summer. In turn, decreases in predawn PSII efficiency, F_v/F_m, in winter were positively correlated with pronounced decreases in the non-phosphorylated form of D1. In contrast to PSII core proteins, the light-harvesting complex of photosystem II (LHCII) did not exhibit any nocturnally sustained phosphorylation. The total level of the D1 protein was found to be the same in summer and winter in Douglas fir when proteins were extracted in a single step from whole needles. In contrast, total D1 protein levels were lower in thylakoid preparations of overwintering needles versus needles collected in summer, indicating that D1 was lost during thylakoid preparation from overwintering Douglas fir needles. In contrast to total D1, the ratio of phosphorylated to non-phosphorylated D1 as well as the levels of the PsbS protein were similar in thylakoid versus whole needle preparations. The level of the PsbS protein, that is required for pH-dependent thermal dissipation, exhibited an increase in winter, whereas LHCII levels remained unchanged.     

Slow degradation of the d1 protein is related to the susceptibility of low-light-grown pumpkin plants to photoinhibition

Photoinhibition of photosystem II (PSII) electron transport and subsequent degradation of the D1 protein were studied in pumpkin (Cucurbita pepo L.) leaves developed under high (1000 μmol m⁻² s⁻¹) and low (80 μmol m⁻² s⁻¹) photon flux densities. The low-light leaves were more susceptible to high light. This difference was greatly diminished when illumination was performed in the presence of chloramphenicol, indicating that a poor capacity to repair photodamaged PSII centers is decisive in the susceptibility of low-light leaves to photoinhibition. In fact, the first phases of the repair cycle, degradation and removal of photodamaged D1 protein from the reaction center complex, occurred slowly in low-light leaves, whereas in high-light leaves the degradation of the D1 protein more readily followed photoinhibition of PSII electron transport. A modified form of the D1 protein, with slightly slower electrophoretic mobility than the original D1, accumulated in the appressed thylakoid membranes of low-light leaves during illumination and was subsequently degraded only slowly.     

Over-production of the D1:2 protein makes Synechococcus cells more tolerant to photoinhibition of photosystem II

Over-expression of the psbAIII gene encoding for the D1 protein (form II; D1:2) of the photosystem II reaction centre in the Synechococcus sp. PCC 7942 was studied using a tac promoter and the lacI ^Q system. Over-expression was induced with 40 g/ml IPTG in the growth medium for either 6 or 12 h at growth irradiance (50 mol photons m^-2 s^-1). This treatment doubled the amount of psbAII/III mRNA and the D1:2 protein in membranes but decreased the amount of psbAI messages and the D1:1 protein. The total amount of both heterodimeric reaction centre proteins, D1 and D2, remained constant under growth light conditions, indicating that the number of PSII centres in the membranes was not affected, only the form of the D1 protein was changed from D1:1 to D1:2 in most centres. When the cells were photoinhibited either at 500 or 1000 mol photons m^-2 s^-1, in the presence or absence of the protein synthesis inhibitor lincomycin, the D1:2 protein remained at a higher level in cells in which over-expression had been induced by IPTG. These cells were also less prone to photoinhibition of PSII. It is suggested that the tolerance of cells to photoinhibition increases when most PSII reaction centres contain the D1:2 protein at the beginning of high irradiance. This tolerance is further strengthened by maintaining psbAIII gene over-expression during the photoinhibitory treatment.     

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein.The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the D1 polypeptide, as shown with PS II membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.Abbreviations atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine - Chl chlorophyll, diuron - (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DPC diphenylcarbazide - ioxynil 4-cyano-2,6-diiodophenol - k_b binding constant - Mes 4-morpholinoethanesulfonic acid - P-680 reaction-center chlorophyll a of photosystem-II - PAGE polyacrylamide gel electrophoresis - PS II photosystem-II - Q_A and Q_B primary and secondary quinone electron acceptors - Z electron donor to the photosystem-II reaction center - SDS sodium dodecylsulfate - Tricine N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine     

EFFECTS OF UV‐B RADIATION ON THE D1 PROTEIN REPAIR CYCLE OF NATURAL PHYTOPLANKTON COMMUNITIES FROM THREE LATITUDES (CANADA,BRAZIL, AND ARGENTINA)1

Ultraviolet radiation effects were examined in natural phytoplankton communities from Rimouski (Canada), Ubatuba (Brazil), and Ushuaia (Argentina). Outdoor pump‐mixed mesocosms were submitted to ambient solar radiation (NUVB) and ambient with additional UV‐B radiation (UVBR) from lamps (HUVB), corresponding to a local 60% ozone depletion scenario. At all sites, neither algal biomass nor dark‐adapted F_v/F_m were significantly affected by additional UVBR, suggesting the presence of active UV protection or repair mechanisms. To examine the role of D1 protein turnover, essential for PSII repair, short‐term surface incubations were performed in the presence or absence of lincomycin, a chloroplast protein synthesis inhibitor. Effects on PSII were determined using chl a in vivo fluorescence, whereas the D1 protein was detected immunochemically. In the absence of D1 repair, D1 pools and F_v/F_m decreased to a similar extent under both light treatments. In the presence of D1 repair, D1 pools suffered faster net degradation under HUVB compared with NUVB, whereas F_v/F_m was maintained for both light treatments, suggesting that HUVB exposure in field populations had more effect on D1 synthesis and PSII repair than on D1 degradation. The fewer undamaged reaction centers remaining in phytoplankton under HUVB were able to maintain F_v/F_m or actually recovered during the dark acclimation before F_v/F_m measurements. The D1 pools suffered faster net degradation at the tropical site where high irradiance drove faster D1 degradation and high water temperature enabled fast enzymatic activities. This study shows the crucial role of dynamic changes in D1 turnover in the photobiology of natural planktonic communities across a range of latitudes.     

Studies on the mechanism of photosystem II photoinhibition I. A two-step degradation of D1-protein

The role of D1-protein in photoinhibition was examined. Photoinhibition of spinach thylakoids at 20°C caused considerable degradation of D1-protein and a parallel loss of variable fluorescence, Q_B-independent electron flow and Q_B-dependent electron flow. The breakdown of D1-protein as well as the loss of variable fluorescence and Q_B-independent electron flow were largely prevented when thylakoids were photoinhibited at 0°C. The Q_B-dependent electron flow markedly decreased under the same conditions. This inactivation may represent the primary event in photoinhibition and could be the result of some modification at the Q_B-site of D1-protein. Evidence for this comes from fluorescence relaxation kinetics following photoinhibition at 0°C which indicate a partial inactivation of Q_A ^--reoxidation. These results support the idea of D1-protein breakdown during photoinhibition as a two step process consisting of an initial inactivation at the Q_B-site of the protein followed by its degradation. The latter is accompanied by the loss of PS II-reaction centre function.Abbreviations Asc ascorbate - p-BQ 1, 4-benzoquinone - DAD diaminodurene - DPC diphenylcarbazide - DQH₂ duroquinole - Fecy ferricyanide - MV methylviologen - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SiMo silicomolybdate     

Nitric oxide donor-mediated inhibition of phosphorylation shows that light-mediated degradation of photosystem II D1 protein and phosphorylation are not tightly linked

An outcome of the photochemistry during oxygenic photosynthesis is the rapid turn over of the D1 protein in the light compared to the other proteins of the photosystem II (PS II) reaction center. D1 is a major factor of PS II instability and its replacement a primary event of the PS II repair cycle. D1 also undergoes redox-dependent phosphorylation prior to its degradation. Although it has been suggested that phosphorylation modulates D1 metabolism, reversible D1 phosphorylation was reported not to be essential for PS II repair in Arabidopsis. Thus, the involvement of phosphorylation in D1 degradation is controversial. We show here that nitric oxide donors inhibit in vivo phosphorylation of the D1 protein in Spirodela without inhibiting degradation of the protein. Thus, D1 phosphorylation is not tightly linked to D1 degradation in the intact plant.