Impaired photosystem I oxidation induces STN7-dependent phosphorylation of the light-harvesting complex I protein Lhca4 in Arabidopsis thaliana

Reduction of the plastoquinone (PQ) pool is known to activate phosphorylation of thylakoid proteins. In the Arabidopsis thaliana mutants psad1-1 and psae1-3, oxidation of photosystem I (PSI) is impaired, and the PQ pool is correspondingly over-reduced. We show here that, under these conditions, the antenna protein Lhca4 of PSI becomes a target for phosphorylation. Phosphorylation of the mature Lhca4 protein at Thr16 is suppressed in stn7 psad1 and stn7 psae1 double mutants. Thus, under extreme redox conditions, hyperactivation of thylakoid protein kinases and/or reorganization of thylakoid protein complex distribution increase the susceptibility of PSI to phosphorylation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Anna Ihnatowicz and Paolo Pesaresi contributed equally to the article.     

STN8 protein kinase in Arabidopsis thaliana is specific in phosphorylation of photosystem II core proteins

Combination of reversed genetics with analyses of in vivo protein phosphorylation in Arabidopsis thaliana revealed that STN8 protein kinase is specific in phosphorylation of N-terminal threonine residues in D1, D2, and CP43 proteins, and Thr-4 in the PsbH protein of photosystem II. Phosphorylation of D1, D2, and CP43 in the light-exposed leaves of two Arabidopsis lines with T-DNA insertions in the stn8 gene was found significantly reduced in the assays with anti-phosphothreonine antibodies. Protein phosphorylation in each of the mutants was quantified comparatively to the wild type by mass spectrometric analyses of phosphopeptides released from the photosynthetic membranes and differentially labeled with stable isotopes. The lack of STN8 caused 50-60% reduction in D1 and D2 phosphorylation, but did not change the phosphorylation level of two peptides that could correspond to light-harvesting proteins encoded by seven different genes in Arabidopsis. Phosphorylation of the PsbH protein at Thr-4 was completely abolished in the plants lacking STN8. Phosphorylation of Thr-4 in the wild type required both light and prior phosphorylation at Thr-2, indicating that STN8 is a light-activated kinase that phosphorylates Thr-4 only after another kinase phosphorylates Thr-2. Analysis of the STN8 catalytic domain suggests that selectivity of STN8 in phosphorylation of the very N-terminal residues in D1, D2, and CP43, and Thr-4 in PsbH pre-phosphorylated at Thr-2 may be explained by the long loops obstructing entrance into the kinase active site and seven additional basic residues in the vicinity of the catalytic site, as compared with the homologous STN7 kinase responsible for phosphorylation of light-harvesting proteins.     

The structure of photosystem II in Arabidopsis: localization of the CP26 and CP29 antenna complexes

A genetic approach has been adopted to investigate the organization of the light-harvesting proteins in the photosystem II (PSII) complex in plants. PSII membrane fragments were prepared from wild-type Arabidopis thaliana and plants expressing antisense constructs to Lhcb4 and Lhcb5 genes, lacking CP29 and CP26, respectively (Andersson et al. (2001) Plant Cell 13, 1193-1204). Ordered PS II arrays and PS II supercomplexes were isolated from the membranes of plants lacking CP26 but could not be prepared from those lacking CP29. Membranes and supercomplexes lacking CP26 were less stable than those prepared from the wild type. Transmission electron microscopy aided by single-particle image analysis was applied to the ordered arrays and the isolated PSII complexes. The difference between the images obtained from wild type and antisense plants showed the location of CP26 to be near CP43 and one of the light-harvesting complex trimers. Therefore, the location of the CP26 within PSII was directly established for the first time, and the location of the CP29 complex was determined by elimination. Alterations in the packing of the PSII complexes in the thylakoid membrane also resulted from the absence of CP26. The minor light-harvesting complexes each have a unique location and important roles in the stabilization of the oligomeric PSII structure.     

High Light-Dependent Phosphorylation of Photosystem II Inner Antenna CP29 in Monocots Is STN7 Independent and Enhances Nonphotochemical Quenching

Phosphorylation of the photosystem II antenna protein CP29 has been reported to be induced by excess light and further enhanced by low temperature, increasing resistance to these stressing factors. Moreover, high light-induced CP29 phosphorylation was specifically found in monocots, both C3 and C4, which include the large majority of food crops. Recently, knockout collections have become available in rice (Oryza sativa), a model organism for monocots. In this work, we have used reverse genetics coupled to biochemical and physiological analysis to elucidate the molecular basis of high light-induced phosphorylation of CP29 and the mechanisms by which it exerts a photoprotective effect. We found that kinases and phosphatases involved in CP29 phosphorylation are distinct from those reported to act in State 1-State 2 transitions. In addition, we elucidated the photoprotective role of CP29 phosphorylation in reducing singlet oxygen production and enhancing excess energy dissipation. We thus established, in monocots, a mechanistic connection between phosphorylation of CP29 and nonphotochemical quenching, two processes so far considered independent from one another.In eukaryotic photosynthesis, light-dependent reactions are performed by two supramolecular complexes, PSII and PSI, which catalyze light harvesting and electron transport from water to NADP⁺. To this aim, water is oxidized by PSII, which, in turn, is oxidized by PSI, which becomes a reductant for ferredoxin-NADP(+) oxidoreductase and NADP⁺ (Nelson and Ben-Shem, 2004). The two photosystems are functionally connected by the plastoquinone (PQ) and cytochrome (cyt) b₆/f, which catalyze the building of the transthylakoid proton gradient, which is dissipated by ATP synthase (ATPase) activity for ATP synthesis from ADP and inorganic phosphate (P_i). PSII and PSI have clearly distinct absorption spectra, with PSI-light-harvesting complex I (LHCI) complexes being enriched in red-shifted absorption forms (Gobets and van Grondelle, 2001). Within canopies, this leads to differential excitation depending on available light quality. This effect needs to be compensated to avoid imbalance of electron transport rates, yielding into either photoinhibition or decrease of photon use efficiency. Two major regulatory mechanisms counteract these effects. (1) State 1-State 2 transitions are active in limiting light conditions (Rintamäki et al., 2000) and inhibited by reduction of a disulfide bridge in high light (HL; Lemeille et al., 2009). This mechanism is activated by overreduction of PQ to plastoquinol (PQH₂) through activation of a thylakoid bound kinase, STN7, acting on LHCII (Depège et al., 2003; Bellafiore et al., 2005). This causes a fraction of PSII antenna system, mainly Lhcb2 (Leoni et al., 2013), to be transferred to PSI in stroma-exposed membranes. The consequent increase in PSI antenna size (Galka et al., 2012) bursts the electron transfer rate and reequilibrates PQ/PQH₂ redox poise, thus causing feedback inactivation of kinase activity. A phosphatase, PPH1-TAP38 (Pribil et al., 2010; Shapiguzov et al., 2010), dephosphorylates LHCII, allowing for its return to PSII in grana partitions. (2) The process of nonphotochemical quenching (NPQ) is rather aimed to photoprotection from excess light (Horton and Ruban, 2005; de Bianchi et al., 2010). In this condition, saturation of downstream metabolic reactions causes depletion of ADP and P_i and inhibition of ATPase activity, which normally brings back protons from lumen to the stromal compartment. This causes accumulation of protons in the lumen, triggering dissipation into heat of the energy absorbed in excess by PSII (de Bianchi et al., 2010; Niyogi and Truong, 2013). Thus, the synergic, although independent, activities of State-1-State 2 transitions and NPQ cover the needs for regulation of photosynthesis over the wide dynamic range of light intensity experienced by plants when shaded or by daily light changes. However, a number of experimental results do not fit within this scheme: phosphorylation of PSII antenna proteins, namely CP29 (LHCB4) has been reported to be induced in very HL and further enhanced by low temperature (Bergantino et al., 1995), implying inhibition of LHC protein phosphorylation by HL is not a general feature of plant photosynthesis. Moreover, CP29 phosphorylation has been shown to be protective in condition of HL combined with low temperature (Mauro et al., 1997), a major stressing factor limiting crop productivity. HL-induced CP29 phosphorylation has been specifically found in monocots, either C3 or C4 (Bergantino et al., 1998), which include the large majority of food crops, thus making the study of this process of interest for both basic and applied research. In dicots, CP29 phosphorylation has been detected at very low level (Fristedt and Vener, 2011) and targeted to different sites within the N-terminal domain with respect to monocots (Testi et al., 1996). Although early research focused on biochemical and physiological characterization of CP29 phosphorylation (Croce et al., 1996; Mauro et al., 1997; Hwang et al., 2003), genetic dissection of this regulation process has been hampered by lack of genetic resources. More recently, rice (Oryza sativa) has become a model organism for monocots, and knockout collections have become available. In this study, we have used reverse genetics coupled to biochemical and physiological analysis to elucidate the molecular basis of HL-induced phosphorylation of CP29 and the mechanisms by which it exerts a photoprotective effect. We found that a different set of kinases and phosphatases is involved in CP29 reversible phosphorylation with respect to that reported to act in State 1-State 2 transitions and that the photoprotection effect is mediated by an enhancement of excess energy dissipation. These results establish, for the first time, a mechanistic connection between thylakoid protein phosphorylation and NPQ, so far believed to be independent processes.     

Role of serine/threonine protein kinase STN7 in the formation of two distinct photosystem I supercomplexes in Physcomitrium patens

Reversible thylakoid protein phosphorylation provides most flowering plants with dynamic acclimation to short-term changes in environmental light conditions. Here, through generating Serine/Threonine protein kinase 7 (STN7)-depleted mutants in the moss Physcomitrella (Physcomitrium patens), we identified phosphorylation targets of STN7 kinase and their roles in short- and long-term acclimation of the moss to changing light conditions. Biochemical and mass spectrometry analyses revealed STN7-dependent phosphorylation of N-terminal Thr in specific Light-Harvesting Complex II (LHCII) trimer subunits (LHCBM2 and LHCBM4/8) and provided evidence that phospho-LHCBM accumulation is responsible for the assembly of two distinct Photosystem I (PSI) supercomplexes (SCs), both of which are largely absent in STN7-depleted mutants. Besides the canonical state transition complex (PSI-LHCI-LHCII), we isolated the larger moss-specific PSI-Large (PSI-LHCI-LHCB9-LHCII) from stroma-exposed thylakoids. Unlike PSI-LHCI-LHCII, PSI-Large did not demonstrate short-term dynamics for balancing the distribution of excitation energy between PSII and PSI. Instead, PSI-Large contributed to a more stable increase in PSI antenna size in Physcomitrella, except under prolonged high irradiance. Additionally, the STN7-depleted mutants revealed altered light-dependent phosphorylation of a monomeric antenna protein, LHCB6, whose phosphorylation displayed a complex regulation by multiple kinases. Collectively, the unique phosphorylation plasticity and dynamics of Physcomitrella monomeric LHCB6 and trimeric LHCBM isoforms, together with the presence of PSI SCs with different antenna sizes and responsiveness to light changes, reflect the evolutionary position of mosses between green algae and vascular plants, yet with clear moss-specific features emphasizing their adaptation to terrestrial low-light environments.

Phosphorylation-dependent formation of photosystem I supercomplexes provides both short- and long-term acclimation of moss photosynthetic apparatus to changing environmental cues.     

Evidence for direct interaction between the chlorophyll-proteins CP29 and CP47 in photosystem II.

Fractionation by anionic-exchange chromatography of an oxygen-evolving photosystem II complex solubilized with 10 mM dodecyl maltoside shows the existence of a sovra-molecular complex between the internal chlorophyll a antenna CP47 and the chlorophyll a/b minor antenna CP29. The chromatographic result is confirmed by a cross-linking experiment which brings about a binary conjugate formed by CP47 and CP29. The sovra-molecular complex between the two chlorophyll protein-complexes has a low temperature fluorescence emission red shifted with respect to the two isolated antenna components. A possible two arms antenna topology for photosystem II is suggested.     

Dephosphorylation of photosystem II proteins and phosphorylation of CP29 in barley photosynthetic membranes as a response to water stress

Kinetic studies of protein dephosphorylation in barley thylakoid membranes revealed accelerated dephosphorylation of photosystem II (PSII) proteins, and meanwhile rapidly induced phosphorylation of a light-harvesting complex (LHCII) b4, CP29 under water stress. Inhibition of dephosphorylation aggravates stress damages and hampers photosystem recovery after rewatering. This increased dephosphorylation is catalyzed by both intrinsic and extrinsic membrane protein phosphatase. Water stress did not cause any thylakoid destacking, and the lateral migration from granum membranes to stroma-exposed lamellae was only found to CP29, but not other PSII proteins. Activation of plastid proteases and release of TLP40, an inhibitor of the membrane phosphatases, were also enhanced during water stress. Phosphorylation of CP29 may facilitate disassociation of LHCII from PSII complex, disassembly of the LHCII trimer and its subsequent degradation, while general dephosphorylation of PSII proteins may be involved in repair cycle of PSII proteins and stress-response-signaling.     

氯霉素处理对拟南芥STN7和STN8基因表达的影响

本实验运用多肽抗体、磷酸化抗体和半定量RT-PCR技术,研究了叶绿体蛋白合成抑制剂--氯霉素(CAP)处理对拟南芥叶片在生长光强下LHCⅡ蛋白与PSⅡ核心蛋白的磷酸化、STN7和STN8基因在mRNA水平和蛋白水平的变化.结果显示:与对照相比,CAP处理叶片在生长光强下STN7基因表达的mRNA水平减少,类囊体膜上酶蛋白含量较低,LHCⅡ蛋白磷酸化水平也较低;而STN8基因表达的mRNA水平增加,类囊体膜上酶蛋白含量增加了1倍,与PSⅡ核心蛋白中D1、D2和CP43的磷酸化水平较高相吻合.研究表明,氯霉素抑制叶绿体蛋白合成后并影响核基因STN7和STN8的表达.     

Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29. Effects of zeaxanthin, pH and phosphorylation.

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.     

Conformational changes in photosystem II supercomplexes upon removal of extrinsic subunits

Photosystem II is a multisubunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts. It consists of a large number of intrinsic membrane proteins involved in light-harvesting and electron-transfer processes and of a number of extrinsic proteins required to stabilize photosynthetic oxygen evolution. We studied the structure of dimeric supercomplexes of photosystem II and its associated light-harvesting antenna by electron microscopy and single-particle image analysis. Comparison of averaged projections from native complexes and complexes without extrinsic polypeptides indicates that the removal of 17 and 23 kDa extrinsic subunits induces a shift of about 1.2 nm in the position of the monomeric peripheral antenna protein CP29 toward the central part of the supercomplex. Removal of the 33 kDa extrinsic protein induces an inward shift of the strongly bound trimeric light-harvesting complex II (S-LHCII) of about 0.9 nm, and in addition destabilizes the monomer-monomer interactions in the central core dimer, leading to structural rearrangements of the core monomers. It is concluded that the extrinsic subunits keep the S-LHCII and CP29 subunits in proper positions at some distance from the central part of the photosystem II core dimer to ensure a directed transfer of excitation energy through the monomeric peripheral antenna proteins CP26 and CP29 and/or to maintain sequestered domains of inorganic cofactors required for oxygen evolution.     

The phosphorylation status of the chloroplast protein kinase STN7 of Arabidopsis affects its turnover

The chloroplast serine-threonine protein kinase STN7 of Arabidopsis (Arabidopsis thaliana) is required for the phosphorylation of the light-harvesting system of photosystem II and for state transitions, a process that allows the photosynthetic machinery to balance the light excitation energy between photosystem II and photosystem I and thereby to optimize the photosynthetic yield. Because the STN7 protein kinase of Arabidopsis is known to be phosphorylated at four serine-threonine residues, we have changed these residues by site-directed mutagenesis to alanine (STN7-4A) or aspartic acid (STN7-4D) to assess the role of these phosphorylation events. The corresponding mutants were still able to phosphorylate the light-harvesting system of photosystem II and to perform state transitions. Moreover, we noticed a marked decrease in the level of the STN7 kinase in the wild-type strain under prolonged state 1 conditions that no longer occurs in the STN7-4D mutant. The results suggest a possible role of phosphorylation of the STN7 kinase in regulating its turnover.     

Unique organization of photosystem II supercomplexes and megacomplexes in Norway spruce

Photosystem II (PSII) complexes are organized into large supercomplexes with variable amounts of light‐harvesting proteins (Lhcb). A typical PSII supercomplex in plants is formed by four trimers of Lhcb proteins (LHCII trimers), which are bound to the PSII core dimer via monomeric antenna proteins. However, the architecture of PSII supercomplexes in Norway spruce[Picea abies (L.) Karst.] is different, most likely due to a lack of two Lhcb proteins, Lhcb6 and Lhcb3. Interestingly, the spruce PSII supercomplex shares similar structural features with its counterpart in the green alga Chlamydomonas reinhardtii [Kou?il et al. (2016) New Phytol. 210 , 808–814]. Here we present a single‐particle electron microscopy study of isolated PSII supercomplexes from Norway spruce that revealed binding of a variable amount of LHCII trimers to the PSII core dimer at positions that have never been observed in any other plant species so far. The largest spruce PSII supercomplex, which was found to bind eight LHCII trimers, is even larger than the current largest known PSII supercomplex from C. reinhardtii. We have also shown that the spruce PSII supercomplexes can form various types of PSII megacomplexes, which were also identified in intact grana membranes. Some of these large PSII supercomplexes and megacomplexes were identified also in Pinus sylvestris, another representative of the Pinaceae family. The structural variability and complexity of LHCII organization in Pinaceae seems to be related to the absence of Lhcb6 and Lhcb3 in this family, and may be beneficial for the optimization of light‐harvesting under varying environmental conditions.     

Optimizing photosynthesis under fluctuating light: The role of the Arabidopsis STN7 kinase

Optimal photosynthetic performance requires that equal amounts of light are absorbed by photosystem ii (PSii) and photosystem i (PSi), which are functionally linked through the photosynthetic electron transport chain. However, photosynthetic organisms must cope with light conditions that lead to the preferential stimulation of one or the other of the photosystems. Plants react to such imbalances by mounting acclimation responses that redistribute excitation energy between photosystems and restore the photosynthetic redox poise. in the short term, this involves the so-called state transition process, which, over periods of minutes, alters the antennal crosssections of the photosystems through the reversible association of a mobile fraction of light-harvesting complex ii (LHCii) with PSi or PSii. Longer-lasting changes in light quality initiate a long-term response (LTr), occurring on a timescale of hours to days, that redresses imbalances in excitation energy by changing the relative amounts of the two photosystems. Despite the differences in their timescales of action, state transitions and LTr are both triggered by the redox state of the plastoquinone (PQ) pool, via the activation of the thylakoid kinase STN7, which appears to act as a common redox sensor and/or signal transducer for both responses. This review highlights recent findings concerning the role of STN7 in coordinating short- and long-term photosynthetic acclimation responses.Key words: state transitions, long-term acclimation, photosynthesis, STN7, Arabidopsis     

Identification of a photosystem II phosphatase involved in light acclimation in Arabidopsis

Reversible protein phosphorylation plays a major role in the acclimation of the photosynthetic apparatus to changes in light. Two paralogous kinases phosphorylate subsets of thylakoid membrane proteins. STATE TRANSITION7 (STN7) phosphorylates LHCII, the light-harvesting antenna of photosystem II (PSII), to balance the activity of the two photosystems through state transitions. STN8, which is mainly involved in phosphorylation of PSII core subunits, influences folding of the thylakoid membranes and repair of PSII after photodamage. The rapid reversibility of these acclimatory responses requires the action of protein phosphatases. In a reverse genetic screen, we identified the chloroplast PP2C phosphatase, PHOTOSYSTEM II CORE PHOSPHATASE (PBCP), which is required for efficient dephosphorylation of PSII proteins. Its targets, identified by immunoblotting and mass spectrometry, largely coincide with those of the kinase STN8. The recombinant phosphatase is active in vitro on a synthetic substrate or on isolated thylakoids. Thylakoid folding is affected in the absence of PBCP, while its overexpression alters the kinetics of state transitions. PBCP and STN8 form an antagonistic kinase and phosphatase pair whose substrate specificity and physiological functions are distinct from those of STN7 and the counteracting phosphatase PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, but their activities may overlap to some degree.     

Phosphoproteomics of Arabidopsis chloroplasts reveals involvement of the STN7 kinase in phosphorylation of nucleoid protein pTAC16

Light-regulated protein kinases STN7 and STN8 phosphorylate thylakoid membrane proteins and also affect expression of several chloroplast proteins via yet unknown mechanisms. Comparative phosphoproteomics of acetic acid protein extracts of chloroplasts from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutants yielded two previously unknown findings: (i) neither STN7 nor STN8 kinase was required for phosphorylation of Ser-48 in Lhcb1.1-1.3 proteins; and (ii) phosphorylation of Thr-451 in pTAC16 protein was STN7-dependent. pTAC16 was found distributed between thylakoids and nucleoid. Its knockout did not affect the nucleoid protein composition and the Thr-451 phosphorylated protein was excluded from the nucleoid. Thr-451 of pTAC16 is conserved in all studied plants and its phosphorylation may regulate membrane-anchoring functions of the nucleoid.     

High light stimulates Deg1-dependent cleavage of the minor LHCII antenna proteins CP26 and CP29 and the PsbS protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The chloroplast Deg1 protein performs proteolytic cleavage of the photodamaged D1 protein of the photosystem II (PSII) reaction center, PSII extrinsic subunit PsbO and the soluble electron carrier plastocyanin. Using biochemical, immunological and mass spectrometry approaches we showed that the heterogeneously expressed Deg1 protease from Arabidopsis thaliana can be responsible for the degradation of the monomeric light-harvesting complex antenna subunits of PSII (LHCII), CP26 and CP29, as well as PSII-associated PsbS (CP22/NPQ4) protein. The results may indicate that cytochrome b ₆ protein and two previously unknown thylakoid proteins, Ptac16 and an 18.3-kDa protein, may be the substrates for Deg1. The interaction of Deg1 with the PsbS protein and the minor LHCII subunits implies its involvement in the regulation of both excess energy dissipation and state transition adaptation processes.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions

The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI-PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.     

The STN8 kinase‐PBCP phosphatase system is responsible for high‐light‐induced reversible phosphorylation of the PSII inner antenna subunit CP29 in rice

Reversible phosphorylation of thylakoid light‐harvesting proteins is a mechanism to compensate for unbalanced excitation of photosystem I (PSI) versus photosystem II (PSII) under limiting light. In monocots, an additional phosphorylation event on the PSII antenna CP29 occurs upon exposure to excess light, enhancing resistance to light stress. Different from the case of the major LHCII antenna complex, the STN7 kinase and its related PPH1 phosphatase were proven not to be involved in CP29 phosphorylation, indicating that a different set of enzymes act in the high‐light (HL) response. Here, we analyze a rice stn8 mutant in which both PSII core proteins and CP29 phosphorylation are suppressed in HL, implying that STN8 is the kinase catalyzing this reaction. In order to identify the phosphatase involved, we produced a recombinant enzyme encoded by the rice ortholog of AtPBCP, antagonist of AtSTN8, which catalyzes the dephosphorylation of PSII core proteins. The recombinant protein was active in dephosphorylating P‐CP29. Based on these data, we propose that the activities of the OsSTN8 kinase and the antagonistic OsPBCP phosphatase, in addition to being involved in the repair of photo‐damaged PSII, are also responsible for the HL‐dependent reversible phosphorylation of the inner antenna CP29.     

Restoration of light induced photosystem II inhibition without de novo protein synthesis

Illumination of isolated spinach thylakoid membranes under anaerobic conditions gave rise to severe inhibition of photosystem II electron transport but did not result in D1-protein degradation. When these photoinhibited thylakoids were incubated in total darkness the photosystem II activity could be fully restored in vitro in a process that required 1-2 h for completion.