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Oxygenic photosynthesis produces various radicals and active oxygen species with harmful effects on photosystem II (PSII). Such photodamage occurs at all light intensities. Damaged PSII centres, however, do not usually accumulate in the thylakoid membrane due to a rapid and efficient repair mechanism. The excellent design of PSII gives protection to most of the protein components and the damage is most often targeted only to the reaction centre D1 protein. Repair of PSII via turnover of the damaged protein subunits is a complex process involving (i) highly regulated reversible phosphorylation of several PSII core subunits, (ii) monomerization and migration of the PSII core from the grana to the stroma lamellae, (iii) partial disassembly of the PSII core monomer, (iv) highly specific proteolysis of the damaged proteins, and finally (v) a multi-step replacement of the damaged proteins with de novo synthesized copies followed by (vi) the reassembly, dimerization, and photoactivation of the PSII complexes. These processes will shortly be reviewed paying particular attention to the damage, turnover, and assembly of the PSII complex in grana and stroma thylakoids during the photoinhibition–repair cycle of PSII. Moreover, a two-dimensional Blue-native gel map of thylakoid membrane protein complexes, and their modification in the grana and stroma lamellae during a high-light treatment, is presented.

Light induces phosphorylation of photosystem II (PSII) proteins in chloroplasts by activating the protein kinase(s) via reduction of plastoquinone and the cytochrome b6f complex. The recent finding of high-light-induced inactivation of the phosphorylation of chlorophyll a/b-binding proteins (LHCII) of the PSII antenna in floated leaf discs, but not in vitro, disclosed a second regulatory mechanism for LHCII phosphorylation. Here we show that this regulation of LHCII phosphorylation is likely to be mediated by the chloroplast ferredoxin-thioredoxin system. We present a cooperative model for the function of the two regulation mechanisms that determine the phosphorylation level of the LHCII proteins in vivo, based on the following results: (i) Chloroplast thioredoxins f and m efficiently inhibit LHCII phosphorylation. (ii) A disulfide bond in the LHCII kinase, rather than in its substrate, may be a target component regulated by thioredoxin. (iii) The target disulfide bond in inactive LHCII kinase from dark-adapted leaves is exposed and easily reduced by external thiol mediators, whereas in the activated LHCII kinase the regulatory disulfide bond is hidden. This finding suggests that the activation of the kinase induces a conformational change in the enzyme. The active state of LHCII kinase prevails in chloroplasts under low-light conditions, inducing maximal phosphorylation of LHCII proteins in vivo. (iv) Upon high-light illumination of leaves, the target disulfide bond becomes exposed and thus is made available for reduction by thioredoxin, resulting in a stable inactivation of LHCII kinase.

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 (kPI), 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 (kREC) 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 D1 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 μmol photons m-2s-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.

Phosphorylation of the light-harvesting chlorophyll a/b complex II (LHC II) proteins is induced in light via activation of the LHC II kinase by reduction of cytochrome b(6)f complex in thylakoid membranes. We have recently shown that, besides this activation, the LHC II kinase can be regulated in vitro by a thioredoxin-like component, and H2O2 that inserts an inhibitory loop in the regulation of LHC II protein phosphorylation in the chloroplast. In order to disclose the complex network for LHC II protein phosphorylation in vivo, we studied phosphorylation of LHC II proteins in the leaves of npq1-2 and npq4-1 mutants of Arabidopis thaliana. In comparison to wild-type, these mutants showed reduced non-photochemical quenching and increased excitation pressure of Photosystem II (PS II) under physiological light intensities. Peculiar regulation of LHC II protein phosphorylation was observed in mutant leaves under illumination. The npq4-1 mutant was able to maintain a high amount of phosphorylated LHC II proteins in thylakoid membranes at light intensities that induced inhibition of phosphorylation in wild-type leaves. Light intensity-dependent changes in the level of LHC II protein phosphorylation were smaller in the npq1-2 mutant compared to the wild-type. No significant differences in leaf thickness, dry weight, chlorophyll content, or the amount of LHC II proteins were observed between the two mutant and wild-type lines. We propose that the reduced capacity of the mutant lines to dissipate excess excitation energy induces changes in the production of reactive oxygen species in chloroplasts, which consequently affects the regulation of LHC II protein phosphorylation.

Light-induced phosphorylation of light-harvesting chlorophyll a/b complex II (LHCII) proteins in plant thylakoid membranes requires an activation of the LHCII kinase via binding of plastoquinol to cytochrome b6f complex. However, a gradual down-regulation of LHCII protein phosphorylation occurs in higher plant leaves in vivo with increasing light intensity. This inhibition is likely to be mediated by increasing concentration of thiol reductants in the chloroplast. Here, we have determined the components involved in thiol redox regulation of the LHCII kinase by studying the restoration of LHCII protein phosphorylation in thylakoid membranes isolated from high-light-illuminated leaves of pumpkin (Cucurbita pepo), spinach (Spinacia oleracea), and Arabidopsis. We demonstrate an experimental separation of two dynamic activities associated with isolated thylakoid membranes and involved in thiol regulation of the LHCII kinase. First, a thioredoxin-like compound, responsible for inhibition of the LHCII kinase, became tightly associated and/or activated within thylakoid membranes upon illumination of leaves at high light intensities. This reducing activity was completely missing from membranes isolated from leaves with active LHCII protein phosphorylation, such as dark-treated and low-light-illuminated leaves. Second, hydrogen peroxide was shown to serve as an oxidant that restored the catalytic activity of the LHCII kinase in thylakoids isolated from leaves with inhibited LHCII kinase. We propose a dynamic mechanism by which counteracting oxidizing and reducing activities exert a stimulatory and inhibitory effect, respectively, on the phosphorylation of LHCII proteins in vivo via a novel membrane-bound thiol component, which itself is controlled by the thiol redox potential in chloroplast stroma.

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 fluoresence (Fv/Fmax). 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 in Ceratodon 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 moss Ceratodon. The level of initial fluorescence (Fo) correlated with the production of inactive PSII centers depleted of D1 protein. The higher the Fo level, the more serve was the loss of D1 protein seen in the samples during photoinhibition. Restoration of Fv/Fmax 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.

The combined effects of partial defoliation and nutrient availability on net photosynthesis and related biochemical variables were studied in cloned Betula pendula Roth saplings. The saplings were randomly assigned to different nutrient levels (5, 1.5 and 0.5 mol N m-3) in aerated nutrient culture and to the following defoliation treatments: (1) control (no damage), (2) damage of the developing main stem leaves (half of the leaf lamina removed), and (3) removal of the developing main stem leaves (entire leaf lamina removed). The leaf immediately below the damaged area in the treated plants, and the corresponding leaf in the control plants, were selected for study. Net photosynthesis measurements and biochemical determinations were made 2, 8 and 14 d after assigning the treatments. At intermediate and low nutrient levels the final net photosynthetic capacity was significantly higher in the saplings with the topmost leaves removed than in the undamaged control saplings, indicating that the expression of compensatory photosynthesis after partial defoliation is not inhibited by nutrient deficiency. The photosynthetic enhancement was closely associated with the increased initial activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). However, the increased activity of Rubisco was not exclusively the result of a higher amount of Rubisco. The expression of compensatory photosynthesis after partial defoliation in our study cannot unequivocally be attributed to an increased flow of nitrogen to the remaining leaves.

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q(A) (-) to Q(B)(Q(B) (-)) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin(-)) and the EPR signal from the Q(A) (-) Fe(2+) complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S(2) state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S(2) multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S(2) multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b (559) was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed.

Reversible phosphorylation and turnover of the D1 protein in vivo were studied under low-temperature photoinhibition of pumpkin leaves and under subsequent recovery at low light at 4 °C or 23 °C. The inactivation of PS II and photodamage to D1 were not enhanced during low-temperature photoinhibition when compared to that at room temperature. The PS II repair cycle, however, was completely blocked at 4 °C at the level of D1 degradation. Both the recovery of the photochemical activity of PS II and the degradation of the damaged D1 protein at low light at 23 °C were delayed about 1 hour after low-temperature photoinhibition, suggesting that in addition to the decrease in catalytic turnover of the enzyme, the protease was specifically inactivated in vivo at low temperature. The effect of low temperature on the other regulatory enzymes of PS II repair, protein kinase and phosphatase [Rintamäki et al. (1996) J Biol Chem 271: 14870-14875] was variable. The D1 protein kinase was operational at low temperature while dephosphorylation of the D1 protein seemed to be completely inhibited during low temperature treatment. Under subsequent recovery conditions at low light and 23 °C, the high phosphorylation level of D1 was sustained in leaf discs photoinhibited at low temperature, despite the recovery of the phosphatase activity. This high phosphorylation level of D1 was due to the persistently active kinase. The D1 kinase, previously shown to get activated by reduction of plastoquinone, was, however, found to be maximally active already at relatively low redox state of the plastoquinone pool. We suggest that phosphorylation of PS II centers increases the stability of PS II complexes and concomitantly improves their survival under stress conditions.[PUBLICATION ABSTRACT]

The properties of the large and small subunit polypeptides of purified wheat ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco, E.C. 4.1.1.39) were studied. The protein was dissociated into subunits by extreme pH or detergent treatment. The separated subunits were unable to reassemble into functional holoprotein when the starting conditions were restored. Some of the separated small subunit polypeptides retained their ability to form functional heterologous Rubisco when mixed with large subunits from a cyanobacterial Rubisco. The separated large subunits of wheat Rubisco formed non-functional, high-molecular-weight aggregates. Treatment with both sodium dodecyl sulphate and thiol reductant was necessary to disrupt the aggregated structure, which indicates that the large subunits had been cross-linked by disulphide bridges. Since added thiol reductant did not prevent aggregation of the separated subunits during attempted reconstitution, oxidation of the sulphydryl groups apparently took place on contact faces sheltered by the secondary and tertiary structures of the polypeptides. High concentration of large subunits or freezing and thawing of the solution stimulated the formation of disulphide cross-links between the large subunits. The presence of small subunits did not prevent aggregation of large subunits. The results suggest that large subunits have a tendency to cross-link with disulphide bridges thus preventing proper assembly with small subunits.