Photosystem II Supercomplexes Research Articles

Salicylic Acid Protects Photosystem II by Alleviating Photoinhibition in Arabidopsis thaliana under High Light.

Salicylic acid (SA) is considered to play an important role in plant responses to environmental stresses. However, the detailed protective mechanisms in photosynthesis are still unclear. We therefore explored the protective roles of SA in photosystem II (PSII) in Arabidopsis thaliana under high light. The results demonstrated that 3 h of high light exposure resulted in a decline in photochemical efficiency and the dissipation of excess excitation energy. However, SA application significantly improved the photosynthetic capacity and the dissipation of excitation energy under high light. Western blot analysis revealed that SA application alleviated the decrease in the levels of D1 and D2 protein and increased the amount of Lhcb5 and PsbS protein under high light. Results from photoinhibition highlighted that SA application could accelerate the repair of D1 protein. Furthermore, the phosphorylated levels of D1 and D2 proteins were significantly increased under high light in the presence of SA. In addition, we found that SA application significantly alleviated the disassembly of PSII-LHCII super complexes and LHCII under high light for 3 h. Overall, our findings demonstrated that SA may efficiently alleviate photoinhibition and improve photoprotection by dissipating excess excitation energy, enhancing the phosphorylation of PSII reaction center proteins, and preventing the disassembly of PSII super complexes.

PH-Responsive Binding Properties of Light-Harvesting Complexes in a Photosystem II Supercomplex Investigated by Thermodynamic Dissociation Kinetics Analysis.

Reorganization of photosynthetic proteins on the thylakoid membrane is an important regulatory process for photoacclimation in photosynthetic organisms. However, the underlying mechanism has been poorly understood due to the lack of methods to analyze the interactions between membrane proteins. To investigate the mechanism, we demonstrated the binding properties of light-harvesting complex proteins (LHCs) in a photosystem II (PSII) supercomplex regulated by pH conditions, which primarily responded to environmental light conditions, using a thermodynamic dissociation kinetics analysis. The results showed that the strongly bound LHCs (∼60%) were responsive to pH conditions, whereas the moderately and loosely bound LHCs (∼40%) were nonresponsive. This result implies that the pH condition alters the binding properties of LHCs in the PSII supercomplex, inducing the reorganization of protein complexes.

The P-class pentatricopeptide repeat protein PpPPR_21 is needed for accumulation of the psbI-ycf12 dicistronic mRNA in Physcomitrella chloroplasts.

Chloroplast gene expression is controlled by numerous nuclear-encoded RNA-binding proteins. Among these, pentatricopeptide repeat (PPR) proteins are known to be key players in post-transcriptional regulation in chloroplasts. However, the functions of many PPR proteins remain unknown. In this study, we characterized the function of a chloroplast-localized P-class PPR protein PpPPR_21 in Physcomitrella patens. Knockout (KO) mutants of PpPPR_21 exhibited reduced protonemata growth and lower photosynthetic activity. Immunoblot analysis and blue-native gel analysis showed a remarkable reduction of the photosystem II (PSII) reaction center protein and poor formation of the PSII supercomplexes in the KO mutants. To assess whether PpPPR_21 is involved in chloroplast gene expression, chloroplast genome-wide microarray analysis and Northern blot hybridization were performed. These analyses indicated that the psbI-ycf12 transcript encoding the low molecular weight subunits of PSII did not accumulate in the KO mutants while other psb transcripts accumulated at similar levels in wild-type and KO mutants. A complemented PpPPR_21KO moss transformed with the cognate full-length PpPPR_21cDNA rescued the level of accumulation of psbI-ycf12 transcript. RNA-binding experiments showed that the recombinant PpPPR_21 bound efficiently to the 5' untranslated and translated regions of psbImRNA. The present study suggests that PpPPR_21 may be essential for the accumulation of a stable psbI-ycf12mRNA.

Investigation of Thermodynamic Dissociation Kinetics to Determine the Binding Strengths within a Membrane Protein Complex: Photosystem II Supercomplex

Protein-protein interactions as well as the formations of protein complexes have been intensively studied by various methods in both in vivo and in vitro. However, although membrane proteins have important roles in cells, interactions within membrane protein complexes have been hardly studied than that within soluble proteins due to the lack of suitable methods, though one approach using mass spectrometry has been recently developed to elucidate membrane protein complexes. Here, we present an approach to determine binding strengths within a membrane protein complex by investigating the thermodynamic dissociation kinetics of an isolated membrane protein complex using time-resolved fluorescence spectroscopy. We demonstrated different bindings of light-harvesting complex II (LHCII) trimers in a photosystem II (PSII) supercomplex isolated from a green alga Chlamydomonas reinhardtii, where the different binding types of the LHCII trimers have been indirectly predicted by the presence or absence of LHCII trimers in negatively stained PSII supercomplex particles observed by electron microscopy but have not been determined. As a result of analysis on thermodynamic dissociation kinetics, we identified three different activation energies for the dissociations of LHCII trimers, which imply the presence of multiple hydrogen bonds and/or ion-dipole bonds between LHCII trimers and core complexes.

Investigation on the Thermodynamic Dissociation Kinetics of Photosystem II Supercomplexes To Determine the Binding Strengths of Light-Harvesting Complexes.

The photosystem II (PSII) supercomplex splits water utilizing light energy and is composed of a core dimer complex surrounded by light-harvesting complexes (LHCs). In green algae, the major LHCs which are LHCII trimers have thus far been categorized into strongly, moderately, or loosely binding LHCII trimers based on their predicted binding to core complexes. However, the binding energies have been indirectly predicted based on the presence or absence of LHCII trimers in the PSII supercomplex under electron microscopy and have not been determined experimentally. In this study, we investigated the binding of LHCII trimers by analyzing thermodynamic dissociation kinetics using isolated PSII supercomplexes. We identified two activation energies for dissociation of LHCII trimers: 54 ± 19 and 134 ± 8 kJ/mol. This result indicated the types of intermolecular interactions between LHCII trimers and core complexes.

A chloroplast thylakoid lumen protein is required for proper photosynthetic acclimation of plants under fluctuating light environments

Despite our increasingly sophisticated understanding of mechanisms ensuring efficient photosynthesis under laboratory-controlled light conditions, less is known about the regulation of photosynthesis under fluctuating light. This is important because-in nature-photosynthetic organisms experience rapid and extreme changes in sunlight, potentially causing deleterious effects on photosynthetic efficiency and productivity. Here we report that the chloroplast thylakoid lumenal protein MAINTENANCE OF PHOTOSYSTEM II UNDER HIGH LIGHT 2 (MPH2; encoded by At4g02530) is required for growth acclimation of Arabidopsis thaliana plants under controlled photoinhibitory light and fluctuating light environments. Evidence is presented that mph2 mutant light stress susceptibility results from a defect in photosystem II (PSII) repair, and our results are consistent with the hypothesis that MPH2 is involved in disassembling monomeric complexes during regeneration of dimeric functional PSII supercomplexes. Moreover, mph2-and previously characterized PSII repair-defective mutants-exhibited reduced growth under fluctuating light conditions, while PSII photoprotection-impaired mutants did not. These findings suggest that repair is not only required for PSII maintenance under static high-irradiance light conditions but is also a regulatory mechanism facilitating photosynthetic adaptation under fluctuating light environments. This work has implications for improvement of agricultural plant productivity through engineering PSII repair.

Evidence of the supercomplex organization of photosystem II and light-harvesting complexes in Nannochloropsis granulata.

Diverse light-harvesting complexes (LHCs) have been found in photosynthetic microalgae that originated from secondary endosymbiosis involving primary red algae. However, the associations between LHCs and photosystem I (PSI) and photosystem II (PSII) in these microalgae are not fully understood. Eustigmatophyta is a red algal lineage that appears to have a unique organization in its photosynthetic machinery, consisting of only chlorophyll a and carotenoids that are atypical compared with other closely related groups. In this study, the supramolecular organization of pigment-protein complexes in the eustigmatophyte alga, Nannochloropsis granulata was investigated using Clear Native (CN) PAGE coupled with two-dimensional (2D) SDS-PAGE. Our results showed two slowly migrating green bands that corresponded to PSII supercomplexes, which consisted of reaction centers and LHCs. These green bands were also characterized as PSII complexes by their low temperature fluorescence emission spectra. The protein subunits of the PSII-LHC resolved by 2D CN/SDS-PAGE were analyzed by mass spectrometry, and four different LHC proteins were identified. Phylogenetic analysis of the identified LHC protein sequences revealed that they belonged to four different Lhc groups; (1) stress-related Lhcx proteins, (2) fucoxanthin chlorophyll a/c-binding Lhcf proteins, (3) red-shifted Chromera light-harvesting proteins (Red-CLH), and (4) Lhcr proteins, which are commonly found in organisms possessing red algal plastids. This is the first report showing evidence of a pigment-protein supercomplex consisting of PSII and LHCs, and to identify PSII-associated LHC proteins in Nannochloropsis.

Biochemical and Spectroscopic Characterization of Highly Stable Photosystem II Supercomplexes from Arabidopsis

Photosystem II (PSII) is a large membrane supercomplex involved in the first step of oxygenic photosynthesis. It is organized as a dimer, with each monomer consisting of more than 20 subunits as well as several cofactors, including chlorophyll and carotenoid pigments, lipids, and ions. The isolation of stable and homogeneous PSII supercomplexes from plants has been a hindrance for their deep structural and functional characterization. In recent years, purification of complexes with different antenna sizes was achieved with mild detergent solubilization of photosynthetic membranes and fractionation on a sucrose gradient, but these preparations were only stable in the cold for a few hours. In this work, we present an improved protocol to obtain plant PSII supercomplexes that are stable for several hours/days at a wide range of temperatures and can be concentrated without degradation. Biochemical and spectroscopic properties of the purified PSII are presented, as well as a study of the complex solubility in the presence of salts. We also tested the impact of a large panel of detergents on PSII stability and found that very few are able to maintain the integrity of PSII. Such new PSII preparation opens the possibility of performing experiments that require room temperature conditions and/or high protein concentrations, and thus it will allow more detailed investigations into the structure and molecular mechanisms that underlie plant PSII function.

Testing the Role of the N-Terminal Tail of D1 in the Maintenance of Photosystem II in Tobacco Chloroplasts.

A key step in the repair of photoinactivated oxygen-evolving photosystem II (PSII) complexes is the selective recognition and degradation of the damaged PSII subunit, usually the D1 reaction center subunit. FtsH proteases play a major role in D1 degradation in both cyanobacteria and chloroplasts. In the case of the cyanobacterium Synechocystis sp. PCC 6803, analysis of an N-terminal truncation mutant of D1 lacking 20 amino-acid residues has provided evidence that FtsH complexes can remove damaged D1 in a processive reaction initiated at the exposed N-terminal tail. To test the importance of the N-terminal D1 tail in higher plants, we have constructed the equivalent truncation mutant in tobacco using chloroplast transformation techniques. The resulting mutant grew poorly and only accumulated about 25% of wild-type levels of PSII in young leaves which declined as the leaves grew so that there was little PSII activity in mature leaves. Truncating D1 led to the loss of PSII supercomplexes and dimeric complexes in the membrane. Extensive and rapid non-photochemical quenching (NPQ) was still induced in the mutant, supporting the conclusion that PSII complexes are not required for NPQ. Analysis of leaves exposed to high light indicated that PSII repair in the truncation mutant was impaired at the level of synthesis and/or assembly of PSII but that D1 could still be degraded. These data support the idea that tobacco plants possess a number of back-up and compensatory pathways for removal of damaged D1 upon severe light stress.

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.

Light-induced gradual activation of photosystem II in dark-grown Norway spruce seedlings

Gymnosperms, unlike angiosperms, are able to synthesize chlorophyll and form photosystems in complete darkness. Photosystem I (PSI) formed under such conditions is fully active, but photosystem II (PSII) is present in its latent form with inactive oxygen evolving complex (OEC). In this work we have studied light-induced gradual changes in PSII function in dark-grown cotyledons of Norway spruce (Picea abies) via the measurement of chlorophyll a fluorescence rise, absorption changes at 830nm, thermoluminescence glow curves (TL) and protein analysis. The results indicate that in dark-grown cotyledons, alternative reductants were able to act as electron donors to PSII with inactive OEC. Illumination of cotyledons for 5min led to partial activation of PSII, which was accompanied by detectable oxygen evolution, but still a substantial number of PSII centers remained in the so called PSII-QB-non-reducing form. Interestingly, even 24h long illumination was not sufficient for the full activation of PSII centers. This was evidenced by a weak attachment of PsbP protein and the absence of PsbQ protein in PSII particles, the absence of PSII supercomplexes, the suboptimal maximum yield of PSII photochemistry, the presence of C band in TL curve and also the presence of up-shifted Q band in TL in DCMU-treated cotyledons. This slow light-induced activation of PSII in dark-grown cotyledons could contribute to the prevention of PSII overexcitation before the light-induced increase in PSI/PSII ratio allows effective operation of linear electron flow.

Effect of Light Acclimation on the Organization of Photosystem II Super- and Sub-Complexes in Arabidopsis thaliana.

To survive under highly variable environmental conditions, higher plants have acquired a large variety of acclimation responses. Different strategies are used to cope with changes in light intensity with the common goal of modulating the functional antenna size of Photosystem II (PSII). Here we use a combination of biochemical and biophysical methods to study these changes in response to acclimation to high light (HL). After 2 h of exposure, a decrease in the amount of the large PSII supercomplexes is observed indicating that plants are already acclimating to HL at this stage. It is also shown that in HL the relative amount of antenna proteins decreases but this decrease is far less than the observed decrease of the functional antenna size, suggesting that part of the antenna present in the membranes in HL does not transfer energy efficiently to the reaction center. Finally, we observed LHCII monomers in all conditions. As the solubilization conditions used do not lead to monomerization of purified LHCII trimers, we should conclude that a population of LHCII monomers exists in the membrane. The relative amount of LHCII monomers strongly increases in plants acclimated to HL, while no changes in the trimer to monomer ratio are observed upon short exposure to stress.

Efficiency of energy funneling in the photosystem II supercomplex of higher plants.

The investigation of energy transfer properties in photosynthetic multi-protein networks gives insight into their underlying design principles. Here, we discuss the excitonic energy transfer mechanisms of the photosystem II (PS-II) C2S2M2 supercomplex, which is the largest isolated functional unit of the photosynthetic apparatus of higher plants. Despite the lack of a definite energy gradient in C2S2M2, we show that the energy transfer is directed by relaxation to low energy states. C2S2M2 is not organized to form pathways with strict energetically downhill transfer, which has direct consequences for the transfer efficiency, transfer pathways and transfer limiting steps. The exciton dynamics is sensitive to small changes in the energetic layout which, for instance, are induced by the reorganization of vibrational coordinates. In order to incorporate the reorganization process in our numerical simulations, we go beyond rate equations and use the hierarchically coupled equation of motion approach (HEOM). While transfer from the peripheral antenna to the proteins in proximity to the reaction center occurs on a faster time scale, the final step of the energy transfer to the RC core is rather slow, and thus the limiting step in the transfer chain. Our findings suggest that the structure of the PS-II supercomplex guarantees photoprotection rather than optimized efficiency.

TEF30 Interacts with Photosystem II Monomers and Is Involved in the Repair of Photodamaged Photosystem II in Chlamydomonas reinhardtii.

The remarkable capability of photosystem II (PSII) to oxidize water comes along with its vulnerability to oxidative damage. Accordingly, organisms harboring PSII have developed strategies to protect PSII from oxidative damage and to repair damaged PSII. Here, we report on the characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in Chlamydomonas reinhardtii, which is conserved in the green lineage and induced by high light. Fractionation studies revealed that TEF30 is associated with the stromal side of thylakoid membranes. By using blue native/Deriphat-polyacrylamide gel electrophoresis, sucrose density gradients, and isolated PSII particles, we found TEF30 to quantitatively interact with monomeric PSII complexes. Electron microscopy images revealed significantly reduced thylakoid membrane stacking in TEF30-underexpressing cells when compared with control cells. Biophysical and immunological data point to an impaired PSII repair cycle in TEF30-underexpressing cells and a reduced ability to form PSII supercomplexes after high-light exposure. Taken together, our data suggest potential roles for TEF30 in facilitating the incorporation of a new D1 protein and/or the reintegration of CP43 into repaired PSII monomers, protecting repaired PSII monomers from undergoing repeated repair cycles or facilitating the migration of repaired PSII monomers back to stacked regions for supercomplex reassembly.

Genotypic Variation under Fe Deficiency Results in Rapid Changes in Protein Expressions and Genes Involved in Fe Metabolism and Antioxidant Mechanisms in Tomato Seedlings (Solanum lycopersicum L.).

To investigate Fe deficiency tolerance in tomato cultivars, quantification of proteins and genes involved in Fe metabolism and antioxidant mechanisms were performed in “Roggusanmaru” and “Super Doterang”. Fe deficiency (Moderate, low and –Fe) significantly decreased the biomass, total, and apoplastic Fe concentration of “Roggusanmaru”, while a slight variation was observed in “Super Doterang” cultivar. The quantity of important photosynthetic pigments such as total chlorophyll and carotenoid contents significantly decreased in “Roggusanmaru” than “Super Doterang” cultivar. The total protein profile in leaves and roots determines that “Super Doterang” exhibited an optimal tolerance to Fe deficiency compared to “Roggusanmaru” cultivar. A reduction in expression of PSI (photosystem I), PSII (photosystem II) super-complexes and related thylakoid protein contents were detected in “Roggusanmaru” than “Super Doterang” cultivar. Moreover, the relative gene expression of SlPSI and SlPSII were well maintained in “Super Doterang” than “Roggusanmaru” cultivar. The relative expression of genes involved in Fe-transport (SlIRT1 and SlIRT2) and Fe(III) chelates reductase oxidase (SlFRO1) were relatively reduced in “Roggusanmaru”, while increased in “Super Doterang” cultivar under Fe deficient conditions. The H+-ATPase relative gene expression (SlAHA1) in roots were maintained in “Super Doterang” compared to “Roggusanmaru”. Furthermore, the gene expressions involved in antioxidant defense mechanisms (SlSOD, SlAPX and SlCAT) in leaves and roots showed that these genes were highly increased in “Super Doterang”, whereas decreased in “Roggusanmaru” cultivar under Fe deficiency. The present study suggested that “Super Doterang” is better tomato cultivar than “Roggusanmaru” for calcareous soils.

Metal Binding in Photosystem II Super- and Subcomplexes from Barley Thylakoids.

Metals exert important functions in the chloroplast of plants, where they act as cofactors and catalysts in the photosynthetic electron transport chain. In particular, manganese (Mn) has a key function because of its indispensable role in the water-splitting reaction of photosystem II (PSII). More and better knowledge is required on how the various complexes of PSII are affected in response to, for example, nutritional disorders and other environmental stress conditions. We here present, to our knowledge, a new method that allows the analysis of metal binding in intact photosynthetic complexes of barley (Hordeum vulgare) thylakoids. The method is based on size exclusion chromatography coupled to inductively coupled plasma triple-quadrupole mass spectrometry. Proper fractionation of PSII super- and subcomplexes was achieved by critical selection of elution buffers, detergents for protein solubilization, and stabilizers to maintain complex integrity. The applicability of the method was shown by quantification of Mn binding in PSII from thylakoids of two barley genotypes with contrasting Mn efficiency exposed to increasing levels of Mn deficiency. The amount of PSII supercomplexes was drastically reduced in response to Mn deficiency. The Mn efficient genotype bound significantly more Mn per unit of PSII under control and mild Mn deficiency conditions than the inefficient genotype, despite having lower or similar total leaf Mn concentrations. It is concluded that the new method facilitates studies of the internal use of Mn and other biometals in various PSII complexes as well as their relative dynamics according to changes in environmental conditions.

MET1 is a thylakoid-associated TPR protein involved in photosystem II supercomplex formation and repair in Arabidopsis.

Photosystem II (PSII) requires constant disassembly and reassembly to accommodate replacement of the D1 protein. Here, we characterize Arabidopsis thaliana MET1, a PSII assembly factor with PDZ and TPR domains. The maize (Zea mays) MET1 homolog is enriched in mesophyll chloroplasts compared with bundle sheath chloroplasts, and MET1 mRNA and protein levels increase during leaf development concomitant with the thylakoid machinery. MET1 is conserved in C3 and C4 plants and green algae but is not found in prokaryotes. Arabidopsis MET1 is a peripheral thylakoid protein enriched in stroma lamellae and is also present in grana. Split-ubiquitin assays and coimmunoprecipitations showed interaction of MET1 with stromal loops of PSII core components CP43 and CP47. From native gels, we inferred that MET1 associates with PSII subcomplexes formed during the PSII repair cycle. When grown under fluctuating light intensities, the Arabidopsis MET1 null mutant (met1) showed conditional reduced growth, near complete blockage in PSII supercomplex formation, and concomitant increase of unassembled CP43. Growth of met1 in high light resulted in loss of PSII supercomplexes and accelerated D1 degradation. We propose that MET1 functions as a CP43/CP47 chaperone on the stromal side of the membrane during PSII assembly and repair. This function is consistent with the observed differential MET1 accumulation across dimorphic maize chloroplasts.

Cross-linking Evidence for Multiple Interactions of the PsbP and PsbQ Proteins in a Higher Plant Photosystem II Supercomplex

The extrinsic subunits of membrane-bound photosystem II (PSII) maintain an essential role in optimizing the water-splitting reaction of the oxygen-evolving complex (OEC), even though they have undergone drastic change during the evolution of oxyphototrophs from symbiotic cyanobacteria to chloroplasts. Two specific extrinsic proteins, PsbP and PsbQ, bind to the lumenal surface of PSII in green plants and maintain OEC conformation and stabilize overall enzymatic function; however, their precise location has not been fully resolved. In this study, PSII-enriched membranes, isolated from spinach, were subjected to chemical cross-linking combined with release-reconstitution experiments. We observed direct interactions between PsbP and PsbE, as well as with PsbR. Intriguingly, PsbP and PsbQ were further linked to the CP26 and CP43 light-harvesting proteins. In addition, two cross-linked sites, between PsbP and PsbR, and that of PsbP and CP26, were identified by tandem mass spectrometry. These data were used to estimate the binding topology and location of PsbP, and the putative positioning of PsbQ and PsbR on the lumenal surface of the PSII. Our model gives new insights into the organization of PSII extrinsic subunits in higher plants and their function in stabilizing the OEC of the PSII supercomplex.

Investigating the photoprotective role of cytochrome b-559 in photosystem II in a mutant with altered ligation of the haem.

Despite many years of study, the physiological role of cytochrome b-559 (Cyt b-559) within the photosystem II (PSII) complex still remains unclear. Here we describe the analysis of a mutant of the green alga Chlamydomonas reinhardtii in which the His ligand to the haem, provided by the alpha subunit, has been replaced by a Cys residue. The mutant is unable to grow photoautotrophically but can assemble oxygen-evolving PSII supercomplexes to 15-20% of the levels found in the wild-type control. Haem is still detected in the isolated PSII supercomplexes but at sub-stoichiometric levels consistent with weaker binding to the mutated cytochrome. Analysis of PSII activity in cells indicates slowed electron transfer in the mutant between plastoquinones QA and QB. We show that PSII activity in the mutant is more sensitive to chronic photoinhibition than the WT control because of two effects: a faster rate of damage and an impaired PSII repair cycle at the level of synthesis and/or incorporation of D1 into PSII. We also demonstrate that Cyt b-559 plays a role during the critical stage of assembling the Mn4CaO5 cluster. Overall we conclude that Cyt b-559 optimises electron transfer on the acceptor side of PSII and plays physiologically important roles in the assembly, repair and maintenance of the complex.

Light-Harvesting Complex Protein LHCBM9 Is Critical for Photosystem II Activity and Hydrogen Production in Chlamydomonas reinhardtii

Photosynthetic organisms developed multiple strategies for balancing light-harvesting versus intracellular energy utilization to survive ever-changing environmental conditions. The light-harvesting complex (LHC) protein family is of paramount importance for this function and can form light-harvesting pigment protein complexes. In this work, we describe detailed analyses of the photosystem II (PSII) LHC protein LHCBM9 of the microalga Chlamydomonas reinhardtii in terms of expression kinetics, localization, and function. In contrast to most LHC members described before, LHCBM9 expression was determined to be very low during standard cell cultivation but strongly increased as a response to specific stress conditions, e.g., when nutrient availability was limited. LHCBM9 was localized as part of PSII supercomplexes but was not found in association with photosystem I complexes. Knockdown cell lines with 50 to 70% reduced amounts of LHCBM9 showed reduced photosynthetic activity upon illumination and severe perturbation of hydrogen production activity. Functional analysis, performed on isolated PSII supercomplexes and recombinant LHCBM9 proteins, demonstrated that presence of LHCBM9 resulted in faster chlorophyll fluorescence decay and reduced production of singlet oxygen, indicating upgraded photoprotection. We conclude that LHCBM9 has a special role within the family of LHCII proteins and serves an important protective function during stress conditions by promoting efficient light energy dissipation and stabilizing PSII supercomplexes.