Global proteome response of Synechocystis 6803 to extreme copper environments applied to control the activity of the inducible petJ promoter.

AIMS: Cyanobacteria are prokaryotes performing oxygenic photosynthesis, and they can be engineered to harness solar energy for production of commodity and high-value chemicals by means of synthetic biology. The Cu2+ -regulated petJ promoter (PpetJ ), which controls the expression of the endogenous cytochrome c553, can be used for expression of foreign products in Synechocystis 6803. We aimed to disclose potential bottlenecks in application of the PpetJ in synthetic biology approaches. METHODS AND RESULTS: Quantitative label-free mass spectrometry revealed global proteome changes which occurred during nutrient conditions which repress or activate of PpetJ in Synechocystis 6803. CONCLUSIONS: Some irreversible proteome alterations were discovered due to the copper stress, including downregulation of the ribosomal proteins, significant changes in protein amounts of the cell surface layer and the outer and inner membranes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed limitations in the use of PpetJ for biotechnological applications.

The bacterial-type [4Fe-4S] ferredoxin 7 has a regulatory function under photooxidative stress conditions in the cyanobacterium Synechocystis sp. PCC 6803.

Ferredoxins function as electron carrier in a wide range of metabolic and regulatory reactions. It is not clear yet, whether the multiplicity of ferredoxin proteins is also reflected in functional multiplicity in photosynthetic organisms. We addressed the biological function of the bacterial-type ferredoxin, Fed7 in the cyanobacterium Synechocystis sp. PCC 6803. The expression of fed7 is induced under low CO2 conditions and further enhanced by additional high light treatment. These conditions are considered as promoting photooxidative stress, and prompted us to investigate the biological function of Fed7 under these conditions. Loss of Fed7 did not inhibit growth of the mutant strain Δfed7 but significantly modulated photosynthesis parameters when the mutant was grown under low CO2 and high light conditions. Characteristics of the Δfed7 mutant included elevated chlorophyll and photosystem I levels as well as reduced abundance and activity of photosystem II. Transcriptional profiling of the mutant under low CO2 conditions demonstrated changes in gene regulation of the carbon concentrating mechanism and photoprotective mechanisms such as the Flv2/4 electron valve, the PSII dimer stabilizing protein Sll0218, and chlorophyll biosynthesis. We conclude that the function of Fed7 is connected to coping with photooxidative stress, possibly by constituting a redox-responsive regulatory element in photoprotection. In photosynthetic eukaryotes domains homologous to Fed7 are exclusively found in chloroplast DnaJ-like proteins that are likely involved in remodeling of regulator protein complexes. It is conceivable that the regulatory function of Fed7 evolved in cyanobacteria and was recruited by Viridiplantae as the controller for the chloroplast DnaJ-like proteins.

Identification of new protein complexes of Escherichia coli inorganic pyrophosphatase using pull-down assay.

Inorganic pyrophosphatase (PPase) is a conserved and essential enzyme catalyzing the hydrolysis of pyrophosphate PP(i). Its activity is required to promote a lot of thermodynamically unfavorable reactions including biosynthesis of activated precursors of sugars and amino acids. Several protein partners of PPase were found so far in Escherichia coli by large-scale approaches. Functional role of these interactions was not studied. In this paper we report the identification of three protein partners of E. coli PPase not found earlier. Pull-down assay on the Ni(2+)-chelating column using 6His-tagged PPase as bait was used to isolate PPase complexes from stationary-phase cells. Of several isolated protein components, five were identified by MALDI-TOF mass-spectrometry: two chaperones (DnaK and GroEL) and three enzymes of carbohydrate and amino acid metabolism (FbaB, fructose-1,6-bisphosphate aldolase, class I; GadA, l-glutamate decarboxylase; and KduI, 5-keto-4-deoxyuronate isomerase). These three proteins were cloned, expressed and purified in 6His-tagged and/or tag-free forms. Their binary interactions with PPase were verified by independent approaches. Initial characterization of the complexes indicates that PPase may stabilize its protein partners against unfolding or degradation. Comparative analysis of the PPase protein partners allowed an insight into its possible involvement in the cell metabolic regulation.

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes.

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.

Chloroplast transcription at different light intensities. Glutathione-mediated phosphorylation of the major RNA polymerase involved in redox-regulated organellar gene expression.

Previous studies using purified RNA polymerase from mustard (Sinapis alba) chloroplasts showed control of transcription by an associated protein kinase. This kinase was found to respond to reversible thiol/disulfide formation mediated by glutathione (GSH), although at concentrations exceeding those thought to exist in vivo. In the present study, several lines of evidence are presented to substantiate the functioning of this regulation mechanism, also in vivo: (a) Studies on the polymerase-associated transcription kinase revealed that at appropriate ATP levels, GSH concentrations similar to those in vivo are sufficient to modulate the kinase activity; (b) GSH measurements from isolated mustard chloroplasts showed considerable differences in response to light intensity; (c) this was reflected by run-on transcription rates in isolated chloroplasts that were generally higher if organelles were prepared from seedlings incubated under high-light as compared with growth-light conditions; (d) the notion of a general transcriptional switch was strengthened by in vitro experiments showing that the kinase not only affects the transcription of a photosynthetic gene (psbA) but also that of a non-photosynthetic gene (trnQ); and (e) the polymerase-kinase complex revealed specific differences in the phosphorylation state of polypeptides depending on the light intensity to which the seedlings had been exposed prior to chloroplast isolation. Taken together, these data are consistent with GSH and phosphorylation-dependent regulation of chloroplast transcription in vivo.

A SecY homologue is involved in chloroplast-encoded D1 protein biogenesis.

We have used the photosystem II reaction center D1 protein as a model to study the mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins. The unusually high turnover rate and distinct pausing intermediates during translation make the D1 protein biogenesis particularly suitable for these purposes. Here we show that cpSecY, a chloroplast homologue of bacterial essential translocon component SecY, interacts tightly with thylakoid membrane-bound ribosomes, suggesting its involvement in protein translocation and insertion. Co-immunoprecipitation and cross-linking experiments indicated that cpSecY resides in the vicinity of D1 elongation intermediates and provided evidence for a transient interaction of cpSecY with D1 elongation intermediates during the biogenesis of D1. After termination of translation, such interactions no longer existed. Our results indicate that, in addition to a well characterized role of cpSecY in posttranslational translocation of nuclear-encoded proteins, it seems to be also involved in cotranslational membrane protein translocation and insertion in chloroplasts.

Coregulation of light-harvesting complex II phosphorylation and lhcb mRNA accumulation in winter rye.

Winter rye plants grown under contrasting environmental conditions or just transiently shifted to varying light and temperature conditions, were studied to elucidate the chloroplast signal involved in regulation of photosynthesis genes in the nucleus. Photosystem II excitation pressure, reflecting the plastoquinone redox state, and the phosphorylation level of thylakoid light-harvesting proteins (LHCII and CP29) were monitored together with changes occurring in the accumulation of lhcb, rbcS and psbA mRNAs. Short-term shifts of plants to changed conditions, from 1 h to 2 d, were postulated to reveal signals crucial for the initiation of the acclimation process. Comparison of these results with those obtained from plants acclimated during several weeks' growth at contrasting temperature and in different light regimes, allow us to make the following conclusions: (1) LHCII protein phosphoylation is a sensitive tool to monitor redox changes in chloroplasts; (2) LHCII protein phosphorylation and lhcb mRNA accumulation occur under similar conditions and are possibly coregulated via an activation state of the same kinase (the LHCII kinase); (3) Maximal accumulation of lhcb mRNA during the diurnal light phase seems to require an active LHCII kinase whereas inactivation of the kinase is accompanied by dampening of the circadian oscillation in the amount of lhcb mRNA; (4) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead (5) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for the regulation of lhcb gene expression in the nucleus.

Regulation of translation elongation in cyanobacteria: membrane targeting of the ribosome nascent-chain complexes controls the synthesis of D1 protein.

The photosystem II reaction centre protein D1 is encoded by the psbA gene. The D1 protein is stable in darkness but undergoes rapid turnover in the light. Here, we show that, in cyanobacterium Synechocystis sp. PCC6803, the synthesis of the D1 protein is regulated at the level of translation elongation in addition to the previously known transcriptional regulation. When Synechocystis sp. PCC6803 cells were transferred from light to darkness, the psbA mRNA remained abundant for hours. Cytosolic ribosomes were attached to psbA transcripts in the dark, and translation continued up to a distinct pausing site. However, ribosome nascent D1 chain complexes were not targeted to the thylakoid membrane, and no full-length D1 protein was produced in darkness. The arrest in translation elongation was released in the light, concomitantly with targeting of ribosome D1 nascent-chain complexes to the thylakoid membrane, allowing the synthesis of the full-length D1 protein. Downregulation of membrane targeting of ribosome complexes was also observed in the light if damage to the D1 protein was slow. This novel type of regulation of prokaryotic translation functions to balance the synthesis and degradation of the rapidly turning over photosystem II D1 protein in Synechocystis sp. PCC6803.

Abnormal regulation of photosynthetic electron transport in a chloroplast ycf9 inactivation mutant.

The ycf9 (orf62) gene of the plastid genome encodes a 6.6-kDa protein (ORF62) of thylakoid membranes. To elucidate the role of the ORF62 protein, the coding region of the gene was disrupted with an aadA cassette, yielding mutant plants that were nearly (more than 95%) homoplasmic for ycf9 inactivation. The ycf9 mutant had no altered phenotype under standard growth conditions, but its growth rate was severely reduced under suboptimal irradiances. On the other hand, it was less susceptible to photodamage than the wild type. ycf9 inactivation resulted in a clear reduction in protein amounts of CP26, the NAD(P)H dehydrogenase complex, and the plastid terminal oxidase. Furthermore, depletion of ORF62 led to a faster flow of electrons to photosystem I without a change in the maximum electron transfer capacity of photosystem II. Despite the reduction of CP26 in the mutant thylakoids, no differences in PSII oxygen evolution rates were evident even at low light intensities. On the other hand, the ycf9 mutant presented deficiencies in the capacity for PSII-independent electron transport (ferredoxin-dependent cyclic electron transport and NAD(P)H dehydrogenase-mediated plastoquinone reduction). Altogether, it is shown that depletion of ORF62 leads to anomalies in the photosynthetic electron transfer chain and in the regulation of electron partitioning among the different routes of electron transport.

Rubisco activase: an enzyme with a temperature-dependent dual function?

Heat treatment of intact spinach leaves was found to induce a unique thylakoid membrane association of an approximately 40 kDa stromal protein. This protein was identified as rubisco activase. Most of the rubisco activase was sequestered to the thylakoid membrane, particularly to the stroma-exposed regions, during the first 10 min of heat treatment at 42 degrees C. At lower temperatures (38-40 degrees C) the association of rubisco activase with the thylakoid membrane occurred more slowly. The temperature-dependent association of rubisco activase with the thylakoid membrane was due to a conformational change in the rubisco activase itself, not to heat-induced alterations in the thylakoid membrane. Association of the 41 kDa isoform of rubisco activase occurred first, followed by the binding of the 45 kDa isoform to the thylakoid membrane. Fractionation of thylakoid membranes revealed a specific association of rubisco activase with thylakoid-bound polysomes. Our results suggest a temperature-dependent dual function for rubisco activase. At optimal temperatures it functions in releasing inhibitory sugar phosphates from the active site of Rubisco. During a sudden and unexpected exposure of plants to heat stress, rubisco activase is likely to manifest a second role as a chaperone in association with thylakoid-bound ribosomes, possibly protecting, as a first aid, the thylakoid associated protein synthesis machinery against heat inactivation.

The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation.

A cytochrome b (6) f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a 'high light' acclimation state. The cytochrome b (6) f complex may be involved indirectly in the regulation of photoacclimation via 1) regulation of the plastoquinone redox state; 2) regulation of the redox-controlled thylakoid protein kinase allowing exposure of the dephosphorylated LHC II to acclimative proteolysis.

Cooperative regulation of light-harvesting complex II phosphorylation via the plastoquinol and ferredoxin-thioredoxin system in chloroplasts.

Light induces phosphorylation of photosystem II (PSII) proteins in chloroplasts by activating the protein kinase(s) via reduction of plastoquinone and the cytochrome b(6)f 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.

Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II.

Regulation of translation elongation, membrane insertion, and assembly of the chloroplast-encoded D1 protein of photosystem II (PSII) was studied using a chloroplast translation system in organello. Translation elongation of D1 protein was found to be regulated by (1) a redox component that can be activated not only by photosynthetic electron transfer but also by reduction with DTT; (2) the trans-thylakoid proton gradient, which is absolutely required for elongation of D1 nascent chains on the thylakoid membrane; and (3) the thiol reactants N-ethylmaleimide (NEM) and iodosobenzoic acid (IBZ), which inhibit translation elongation with concomitant accumulation of distinct D1 pausing intermediates. These results demonstrate that D1 translation elongation and membrane insertion are tightly coupled and highly regulated processes in that proper insertion is a prerequisite for translation elongation of D1. Cotranslational and post-translational assembly steps of D1 into PSII reaction center and core complexes occurred independently of photosynthetic electron transfer or trans-thylakoid proton gradient but were strongly affected by the thiol reactants DTT, NEM, and IBZ. These compounds reduced the stability of the early PSII assembly intermediates, hampered the C-terminal processing of the precursor of D1, and prevented the post-translational reassociation of CP43, indicating a strong dependence of the D1 assembly steps on proper redox conditions and the formation of disulfide bonds.

Dephosphorylation of photosystem II reaction center proteins in plant photosynthetic membranes as an immediate response to abrupt elevation of temperature.

Kinetic studies of protein dephosphorylation in photosynthetic thylakoid membranes revealed specifically accelerated dephosphorylation of photosystem II (PSII) core proteins at elevated temperatures. Raising the temperature from 22 degrees C to 42 degrees C resulted in a more than 10-fold increase in the dephosphorylation rates of the PSII reaction center proteins D1 and D2 and of the chlorophyll a binding protein CP43 in isolated spinach (Spinacia oleracea) thylakoids. In contrast the dephosphorylation rates of the light harvesting protein complex and the 9-kD protein of the PSII (PsbH) were accelerated only 2- to 3-fold. The use of a phospho-threonine antibody to measure in vivo phosphorylation levels in spinach leaves revealed a more than 20-fold acceleration in D1, D2, and CP43 dephosphorylation induced by abrupt elevation of temperature, but no increase in light harvesting protein complex dephosphorylation. This rapid dephosphorylation is catalyzed by a PSII-specific, intrinsic membrane protein phosphatase. Phosphatase assays, using intact thylakoids, solubilized membranes, and the isolated enzyme, revealed that the temperature-induced lateral migration of PSII to the stroma-exposed thylakoids only partially contributed to the rapid increase in the dephosphorylation rate. Significant activation of the phosphatase coincided with the temperature-induced release of TLP40 from the membrane into thylakoid lumen. TLP40 is a peptidyl-prolyl cis-trans isomerase, which acts as a regulatory subunit of the membrane phosphatase. Thus dissociation of TLP40 caused by an abrupt elevation in temperature and activation of the membrane protein phosphatase are suggested to trigger accelerated repair of photodamaged PSII and to operate as possible early signals initiating other heat shock responses in chloroplasts.

The ycf 9 (orf 62) gene in the plant chloroplast genome encodes a hydrophobic protein of stromal thylakoid membranes.

There are still some open reading frames, orfs, with unknown function in the higher plant chloroplast genome. Of these conserved orfs, designated as ycfs (hypothetical chloroplast open reading frames), one is ycf 9 (orf 62) in the transcription unit with the psbC and psbD genes. The aim of this work was to investigate the function of ycf 9 by insertional inactivation of the gene with a selectable marker cassette, consisting of the aadA coding region connected to the trc promoter and rrnB terminator. This cassette was inserted 19 bp downstream from the start of the coding region of the tobacco ycf 9 gene. Two DNA constructs with the aadA cassette in opposite orientations were precipitated on 1 micron gold particles and delivered into leaves of Nicotiana tabacum, cultivar Samsun, by the biolistic method. Spectinomycin-resistant plants regenerated following bombardment with only the construct containing the aadA gene in the opposite orientation as ycf 9. In spite of several subsequent regeneration cycles on spectinomycin, the transplastomic plants did not reach homoplasmicity. This suggests that the ycf 9 gene product is essential for chloroplast function. Using a polyclonal antibody raised against the inner part of the gene product, the polypeptide was localized in the stromal thylakoid membranes of chloroplasts.

Expression of psbA genes is regulated at multiple levels in the cyanobacterium Synechococcus sp. PCC 7942.

In cyanobacterium Synechococcus sp. PCC 7942 the photosystem II reaction-center protein D1 is encoded by three psbA genes. The psbAI gene encodes D1:1 protein, the form used for acclimated growth, and psbAII and psbAIII genes encode the stress-induced form, D1:2 protein. Strong light and low temperature have been shown to induce the expression of psbAII/III genes and down-regulate the expression of psbAI gene. Recently, we reported the involvement of reduced thiols in the up-regulation of psbAII/III genes. In this study, we have analyzed the regulation of psbA gene expression in Synechococcus further, at both the transcriptional and post-transcriptional levels. We show that the inhibitors of the photosynthetic electron-transfer chain, which have different effects on the redox state of the plastoquinone (PQ) pool, have similar effect on the transcription of psbA genes. The inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) do not cause any changes in psbA gene expression when added under low-light conditions, but dramatically reduce the high-light induction of psbAII/III genes when added upon a high-light shift. Moreover, when the thiol reductant, dithiothreitol, is added to Synechococcus cells together with DCMU concomitant with the high-light shift, no inhibition of psbAII/III gene up-regulation takes place, indicating that the thiol redox state rather than the redox state of the PQ pool regulates psbA gene transcription. We also provide evidence for post-transcriptional regulation of psbA gene expression, particularly, inhibition of translation of psbAI transcripts at high light, and demonstrate that the D1 protein synthesis and degradation processes are coregulated in Synechococcus.

Thiol redox state regulates expression of psbA genes in Synechococcus sp. PCC 7942.

Three psbA genes encode two different forms of the photosystem II reaction centre protein D1 in Synechococcus sp. PCC 7942. The psbAI gene encoding D1 protein form I (D1:1) is mainly expressed under low growth light conditions while the psbAII and psbAIII genes, encoding D1 protein form II (D1:2), are induced under stress conditions (e.g. high light or low temperature). In this paper we show that psbAII/III genes can be rapidly induced even under low growth light conditions by adding the thiol reductant (DTTred) to Synechococcus cell culture, at a concentration that does not affect cell growth or photosynthetic activity. Similar induction of psbAII/III genes was obtained by illuminating the cells with photosystem I light. In both instances psbAI gene down-regulation coincided with the up-regulation of psbAII/III genes. DTTred-induced exchange in transcript pools was subsequently followed by an exchange of D1:1 for D1:2 at the protein level. Thiol oxidants, iodosobenzoic acid or diamide, reverted the effects of DTTred on psbA gene expression. Thiol oxidants and the thiol-modifying agent N-ethylmaleimide also totally prevented high-light induction of psbAII/III genes. These data strongly suggest that the up-regulation of psbAII/III genes that occurs under stress conditions is mediated by production of thiol reductants, whereas the expression of the psbAI gene is sustained by the more oxidizing conditions that prevail during the steady-state growth of cells.

Co-translational assembly of the D1 protein into photosystem II.

Assembly of multi-subunit membrane protein complexes is poorly understood. In this study, we present direct evidence that the D1 protein, a multiple membrane spanning protein, assembles co-translationally into the large membrane-bound complex, photosystem II. During pulse-chase studies in intact chloroplasts, incorporation of the D1 protein occurred without transient accumulation of free labeled protein in the thylakoid membrane, and photosystem II subcomplexes contained nascent D1 intermediates of 17, 22, and 25 kDa. These N-terminal D1 intermediates could be co-immunoprecipitated with antiserum directed against the D2 protein, suggesting co-translational assembly of the D1 protein into PS II complexes. Further evidence for a co-translational assembly of the D1 protein into photosystem II was obtained by analyzing ribosome nascent chain complexes liberated from the thylakoid membrane after a short pulse labeling. Radiolabeled D1 intermediates could be immunoprecipitated under nondenaturing conditions with antisera raised against the D1 and D2 protein as well as CP47. However, when the ribosome pellets were solubilized with SDS, the interaction of these intermediates with CP47 was completely lost, but strong interaction of a 25-kDa D1 intermediate with the D2 protein still remained. Taken together, our results indicate that during the repair of photosystem II, the assembly of the newly synthesized D1 protein into photosystem II occurs co-translationally involving direct interaction of the nascent D1 chains with the D2 protein.

Thylakoid protein phosphorylation and the thiol redox state.

Illumination of thylakoid membranes leads to the phosphorylation of a number of photosystem II-related proteins, including the reaction center proteins D1 and D2 as well as the light-harvesting complex (LHCII). Regulation of light-activated thylakoid protein phosphorylation has mainly been ascribed to the redox state of the electron carrier plastoquinone. In this work, we show that this phosphorylation in vitro is also strongly influenced by the thiol disulfide redox state. Phosphorylation of the light-harvesting complex of photosystem II was found to be favored by thiol-oxidizing conditions and strongly downregulated at moderately thiol-reducing conditions. In contrast, phosphorylation of the photosystem II reaction center proteins D1 and D2 as well as that of other photosystem II subunits was found to be stimulated up to 2-fold by moderately thiol-reducing conditions and kept at a high level also at highly reducing conditions. These responses of the level of thylakoid protein phosphorylation to changes in the thiol disulfide redox state are reminiscent of those observed in vivo in response to changes in the light intensity and point to the possibility of a second loop of redox regulation of thylakoid protein phosphorylation via the ferredoxin-thioredoxin system.

Substitution of Ala-251 of the D1 reaction centre polypeptide with a charged residue results in impaired function of photosystem II.

Ala-251 in the membrane-parallel helix in the D-E loop of the D1 polypeptide close to the Q(B) pocket of photosystem II (PS II), was mutated to aspartate (D), lysine (K), leucine (L) or serine (S) in Synechocystis 6803. O2 evolution rates (H2O-->DCBQ; 2,6-dichloro-p-benzoquinone) of A251D, A251L and A251S were lower, being 38, 16, 62 and 70%, respectively, of that of the control, and there was an even more drastic impairment of O2 evolution when measured from H2O to DMBQ (2,5-dimethyl-p-benzoquinone), demonstrating modifications in the Q(B) pocket. However, in all other mutants but A251K, the Q(B) function could sustain O2 evolution at a level high enough to support photosynthetic growth. The mutant A251S, carrying a substitution of alanine for a chemically quite similar residue serine, was less severely affected. Substitution by a positively charged residue drastically delayed chlorophyll a fluorescence relaxation in the non-photosynthetic strain A251K, implying strong impairment of Q(A)-to-Q(B) electron transfer. Delay of fluorescence relaxation was clear in A251D as well, carrying a substitution of alanine for a negatively charged residue. The effects of the substitutions of A251 demonstrate the importance of this residue of the D1 polypeptide in the conformation of the acceptor side of PS II and, accordingly, the effect on the acceptor-side function of PS II was very clear. Nevertheless, the tolerance of PS II activity to high-light-induced photoinhibition in vivo and the subsequent D1 degradation were not much impaired in any of the photosynthetic mutant strains as compared to the control.