Revisiting the Supramolecular Organization of Photosystem II in Chlamydomonas reinhardtii

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-d-maltoside (β-DM) or n-dodecyl-α-d-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.     

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P(700) recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.     

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.     

Activation of a Reserve Pool of Photosystem II in Chlamydomonas reinhardtii Counteracts Photoinhibition

The effect of strong irradiance (2000 micromole photons per square meter per second) on PSII heterogeneity in intact cells of Chlamydomonas reinhardtii was investigated. Low light (LL, 15 micromole photons per square meter per second) grown C. reinhardtii are photoinhibited upon exposure to strong irradiance, and the loss of photosynthetic functioning is due to damage to PSII. Under physiological growth conditions, PSII is distributed into two pools. The large antenna size (PSII_α) centers account for about 70% of all PSII in the thylakoid membrane and are responsible for plastoquinone reduction (Q_b-reducing centers). The smaller antenna (PSII_β) account for the remainder of PSII and exist in a state not yet able to photoreduce plastoquinone (Q_b-nonreducing centers). The exposure of C. reinhardtii cells to 60 minutes of strong irradiance disabled about half of the primary charge separation between P680 and pheophytin. The PSII_β content remained the same or slightly increased during strong-irradiance treatment, whereas the photochemical activity of PSII_α decreased by 80%. Analysis of fluorescence induction transients displayed by intact cells indicated that strong irradiance led to a conversion of PSII_β from a Q_b-nonreducing to a Q_b-reducing state. Parallel measurements of the rate of oxygen evolution revealed that photosynthetic electron transport was maintained at high rates, despite the loss of activity by a majority of PSII_α. The results suggest that PSII_β in C. reinhardtii may serve as a reserve pool of PSII that augments photosynthetic electron-transport rates during exposure to strong irradiance and partially compensates for the adverse effect of photoinhibition on PSII_α.     

Suppression of Quantum Yield of Photosystem II by Hyperosmotic Stress in Chlamydomonas reinhardtii

Addition of ethylene glycol (EG) or NaCl to cells of Chlamydomonasreinhardtii induced transient non-photochemical quenching ofChl fluorescence correlated with the inhibition of photosyntheticoxygen evolution. The induction of the quenching and subsequentrecovery proceeded not only in the light but also in the dark.The quenching was almost unaffected by the protonophore nigericin,suggesting the involvement of a type of non-photochemical quenchingattributable to a state 2 transition. Higher concentrationsof EG or NaCl caused a delay of the recovery of the maximumfluorescence yield (Fm'). Dark reduction rate of P700⁺ afterthe application of a flash light in the presence of DCMU wasenhanced by the hyperosmotic shock, suggesting a stimulatedreduction of the intersystem electron carriers. It is proposedthat the osmotic stress stimulates electron donation from stromalcomponents via the NAD(P)H dehydrogenase, which results in thereduction of the intersystem chain and triggering of a state2 transition leading to stimulated cyclic PSI activity. (Received May 16, 1995; Accepted July 26, 1995)     

Protein Disulfide Isomerase 2 of Chlamydomonas reinhardtii Is Involved in Circadian Rhythm Regulation

Protein disulfide isomerases （PDIs） are known to play important roles in the folding of nascent proteins and in the formation of disulfide bonds. Recently, we identified a PDI from Chlamydomonas reinhardtii （CrPDI2） by a mass spectrometry approach that is specifically enriched by heparin affinity chromatography in samples taken during the night phase. Here, we show that the recombinant CrPDI2 is a redox-active protein. It is reduced by thioredoxin reductase and catalyzes itself the reduction of insulin chains and the oxidative refolding of scrambled RNase A. By immunoblots, we confirm a high-amplitude change in abundance of the heparin-bound CrPDI2 during subjective night. Interestingly, we find that CrPDI2 is present in protein complexes of different sizes at both day and night. Among three identified interac- tion partners, one （a 2-cys peroxiredoxin） is present only during the night phase. To study a potential function of CrPDI2 within the circadian system, we have overexpressed its gene. Two transgenic lines were used to measure the rhythm of phototaxis~ In the transgenic strains, a change in the acrophase was observed. This indicates that CrPDI2 is involved in the circadian signaling pathway and, together with the night phase-specific interaction of CrPDI2 and a peroxiredoxin, these findings suggest a close coupling of redox processes and the circadian clock in C. reinhardtii.     

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-reconstitution of the extrinsic proteins and Photosystem II complexes from Chlamydomonas reinhardtii and Spinacia oleracea

Cross-reconstitution of the extrinsic proteins and Photosystem II (PS II) from a green alga, Chlamydomonas reinhardtii, and a higher plant,Spinacia oleracea, was performed to clarify the differences of binding properties of the extrinsic proteins between these two species of organisms. (1) Chlamydomonas PsbP and PsbQ directly bound to Chlamydomonas PS II independent of the other extrinsic proteins but not to spinach PS II. (2) Chlamydomonas PsbP and PsbQ directly bound to the functional sites of Chlamydomonas PS II independent of the origins of PsbO, while spinach PsbP and PsbQ only bound to non-functional sites on Chlamydomonas PS II. (3) Both Chlamydomonas PsbP and spinach PsbP functionally bound to spinach PS II in the presence of spinach PsbO. (4) While Chlamydomonas PsbP functionally bound to spinach PS II in the presence of Chlamydomonas PsbO, spinach PsbP bound loosely to spinach PS II in the presence of Chlamydomonas PsbO with no concomitant restoration of oxygen evolution. (5) Chlamydomonas PsbQ bound to spinach PS II in the presence of Chlamydomonas PsbP and PsbO or spinach PsbO but not to spinach PS II in the presence of spinach PsbP and Chlamydomonas PsbO or spinach PsbO. (6) Spinach PsbQ did not bind to spinach PS II in the presence of Chlamydomonas PsbO and PsbP. On the basis of these results, we showed a simplified scheme for binding patterns of the green algal and higher plant extrinsic proteins with respective PS II.     

A knowledge-based three dimensional model of the Photosystem II reaction center of Chlamydomonas reinhardtii

In this Minireview, a comparison of the binding niches of the PS II cofactors from several existing models of the PS II reaction center is provided. In particular, it discusses a three dimensional model of the Photosystem II (PS II) reaction center including D1, D2 and cytochrome b559 proteins from the green alga Chlamydomonas reinhardtii that was specifically generated for this Minireview. This model is the most complete to date and includes accessory chlorophyll_zs, a manganese cluster, two molecules of -carotene and cytochrome b559, all of which are essential components of the PS II reaction center. The modeling of the D1 and D2 proteins was primarily based on homology with the L and M subunits of the anoxygenic purple bacterial photosynthetic reaction centers. The non-homologous loop regions were built using a sequence specific approach by searching for the best-matched protein segments in the Protein Data Bank, and by imposing the matching conformations on the corresponding D1 and D2 regions. Cytochrome b559 which is in close proximity to D1 and D2 was tentatively modeled in / conformation and docked on the Q_B side of the PS II reaction center according to experimental suggestions. An alternate docking on the Q_A side is also shown for comparison. The cofactors in the PS II reaction center were modeled either by adopting the structures from the bacterial counterparts, when available, with modifications based on existing experimental data or by de novo modeling and docking in the most probable positions in the reaction center complex. The specific features of this model are the inclusion of the tetramanganese cluster (with calcium and chloride ions) in a open, C-shaped structure modeled within the D1/D2/cytochrome b559 complex with D1-D170, D1-E189, D1-D342 and D1-A344 as putative ligands; and the modeling of two cis -carotenes and two accessory chlorophyll_zs liganded by D1-H118 and D2-H117. We also analyzed residues in the model which may be involved in the D1 and D2 inter-protein interactions, as well as residues which may be involved in putative bicarbonate and water binding and transport.     

A highly active histidine-tagged Chlamydomonas reinhardtii Photosystem II preparation for structural and biophysical analysis.

We describe a genetically engineered strain of Chlamydomonas reinhardtii where the PsbH subunit of Photosystem II (PSII) has been modified to include a C-terminal polyhistidine tag. The strain was generated by the rescue to photoautotrophic growth of a psbH insertional mutant following chloroplast transformation with the modified gene. This selection strategy confirms that the addition of the tag to PsbH does not prevent the assembly of functional PSII, and results in an engineered strain with tagged PSII but no antibiotic-resistance markers in the chloroplast genome. Consequently, the strain is suitable for subsequent genetic manipulation of chloroplast PSII genes. We also describe a rapid PSII isolation procedure that gives a preparation capable of high rates of oxygen evolution. This preparation is suitable for spectroscopic analysis as shown by EPR analysis of the S2 state of the water oxidation cycle. Furthermore, electron microscopy, coupled to single particle analysis, has revealed the isolated PSII to be structurally homogeneous core dimers that are ideally suited for higher resolution structural studies.     

Photosystem II peripheral accessory chlorophyll mutants in Chlamydomonas reinhardtii. Biochemical characterization and sensitivity to photo-inhibition

In addition to the four chlorophylls (Chls) involved in primary charge separation, the photosystem II (PSII) reaction center polypeptides, D1 and D2, coordinate a pair of symmetry-related, peripheral accessory Chls. These Chls are axially coordinated by the D1-H118 and D2-H117 residues and are in close association with the proximal Chl antennae proteins, CP43 and CP47. To gain insight into the function(s) of each of the peripheral Chls, we generated site-specific mutations of the amino acid residues that coordinate these Chls and characterized their energy and electron transfer properties. Our results demonstrate that D1-H118 and D2-H117 mutants differ with respect to: (a) their relative numbers of functional PSII complexes, (b) their relative ability to stabilize charge-separated states, (c) light-harvesting efficiency, and (d) their sensitivity to photo-inhibition. The D2-H117N and D2-H117Q mutants had reduced levels of functional PSII complexes and oxygen evolution capacity as well as reduced light-harvesting efficiencies relative to wild-type cells. In contrast, the D1-H118Q mutant was capable of near wild-type rates of oxygen evolution at saturating light intensities. The D1-H118Q mutant also was substantially more resistant to photo-inhibition than wild type. This reduced sensitivity to photo-inhibition is presumably associated with a reduced light-harvesting efficiency in this mutant. Finally, it is noted that the PSII peripheral accessory Chls have similarities to a to a pair of Chls also present in the PSI reaction center complex.     

PHOTOSYSTEM II SUBUNIT R Is Required for Efficient Binding of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 to Photosystem II-Light-Harvesting Supercomplexes in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, the LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) protein is crucial for efficient energy-dependent thermal dissipation of excess absorbed light energy and functionally associates with photosystem II-light-harvesting complex II (PSII-LHCII) supercomplexes. Currently, it is unknown how LHCSR3 binds to the PSII-LHCII supercomplex. In this study, we investigated the role of PHOTOSYSTEM II SUBUNIT R (PSBR) an intrinsic membrane-spanning PSII subunit, in the binding of LHCSR3 to PSII-LHCII supercomplexes. Down-regulation of PSBR expression diminished the efficiency of oxygen evolution and the extent of nonphotochemical quenching and had an impact on the stability of the oxygen-evolving complex as well as on PSII-LHCII-LHCSR3 supercomplex formation. Its down-regulation destabilized the PSII-LHCII supercomplex and strongly reduced the binding of LHCSR3 to PSII-LHCII supercomplexes, as revealed by quantitative proteomics. PHOTOSYSTEM II SUBUNIT P deletion, on the contrary, destabilized PHOTOSYSTEM II SUBUNIT Q binding but did not affect PSBR and LHCSR3 association with PSII-LHCII. In summary, these data provide clear evidence that PSBR is required for the stable binding of LHCSR3 to PSII-LHCII supercomplexes and is essential for efficient energy-dependent quenching and the integrity of the PSII-LHCII-LHCSR3 supercomplex under continuous high light.Plant photosynthetic electron transfer is conducted by a series of reactions at the chloroplast thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). Two separate photosystems (PSI and PSII) and an ATP synthase catalyze these reactions. PSI and PSII are multiprotein complexes that are mainly embedded in unstacked and stacked regions of the thylakoid membrane, respectively. PSI consists of more than 10 subunits and a number of cofactors such as chlorophyll a, β-carotene, phylloquinone, and three iron-sulfur (4Fe-4S) clusters (Busch and Hippler, 2011). PSI catalyzes light-driven electron transfer from luminal plastocyanin to stromal ferredoxin. The latter reduces the ferredoxin-NADP reductase that, in turn, leads to the formation of NADPH. PSII catalyzes light-induced electron transfer from water to the plastoquinone pool by using chlorophyll a, carotenoids, as well as redox-active cofactors, causing the release of oxygen and protons (Pagliano et al., 2013). The core complex is organized as a dimer. Monomers are composed of the reaction center subunits PSBA (D1) and PSBD (D2), the inner antenna proteins PSBB (CP47) and PSBC (CP43), the α- and β-subunits (PSBE and PSBF) of cytochrome b₅₅₉, as well as a number of intrinsic low-molecular-mass subunits. The core monomer is further associated with an inorganic Mn₄O₅Ca cluster and a few chloride ions (Rivalta et al., 2011; Umena et al., 2011) required for photosynthetic water oxidation. To optimize this process, the oxygen-evolving complex is formed at the luminal side by the extrinsic polypeptides PSBO, PSBP, PSBQ, and PSBR (for review, see Pagliano et al., 2013).To enhance the light-harvesting capacity of PSII, various light-harvesting proteins bind to dimeric PSII core complexes (Dekker and Boekema, 2005). A common structure found for vascular plants and green algae is the C₂S₂ supercomplex, where two copies of monomeric Lhcb4 and Lhcb5 and two LHCII trimers (S-trimer; Boekema et al., 1995) bind to the dimeric PSII core. In vascular plants, larger but less stable PSII supercomplexes, known as C₂S₂M₂, are composed of two extra copies of the monomeric Lhcb6 with two additional LHCII trimers (M-trimer) bound through Lhcb4 and Lhcb5 (Dekker and Boekema, 2005; Caffarri et al., 2009). Even larger complexes containing two additional LHCII trimers (L-trimer), bound via Lhcb6, are found and are known as C₂S₂M₂L_1–2 (Boekema et al., 1999). A recent study in Chlamydomonas reinhardtii identified PSII-LHCII supercomplexes with three LHCII trimers attached to each side of the core (C₂S₂M₂L₂; Tokutsu et al., 2012). Interestingly, such PSII-LHCII supercomplexes associate with LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3; Tokutsu and Minagawa, 2013), an ancient light-harvesting protein required for efficient energy-dependent (qE) quenching in the alga (Peers et al., 2009). The qE component of nonphotochemical quenching (NPQ) is an energy-dependent constituent of NPQ and regulates the thermal dissipation of excess absorbed light energy (Li et al., 2000; Peers et al., 2009). The qE capacity in C. reinhardtii increases proportionally to the light-dependent accumulation of the LHCSR3 protein (Peers et al., 2009). In contrast, in vascular plants, qE is constitutively active and dependent on PSBS, a PSII polypeptide (Li et al., 2000). Mass spectrometric analyses of isolated C₂S₂M₂ PSII supercomplexes revealed the presence of extrinsic subunits PSBP, PSBQ, and PSBR, while PSBS was not identified, suggesting that PSBS does not influence the association of the PSII core with the outer light-harvesting complex system (Pagliano et al., 2014). In line with the proteomic findings, recent data suggest that subunits PSBP, PSBQ, and PSBR contribute to the stability of PSII-LHCII supercomplexes in vascular plants (Caffarri et al., 2009; Ifuku et al., 2011; Allahverdiyeva et al., 2013). A recent quantitative proteomic study performed with C. reinhardtii identified PSBR as the only PSII subunit to be induced upon the shift from photoheterotrophic to photoautotrophic growth conditions similar to LHCSR3 (Höhner et al., 2013).In vascular plants and green algae, PSBR is nucleus encoded and has a mass of about 10 kD. The mature protein has a predicted 70-amino acid luminal N-terminal part and a C-terminal transmembrane span (Ljungberg et al., 1986; Lautner et al., 1988; Webber et al., 1989). An association of PSBR with the oxygen-evolving complex has been suggested, as its presence is required for the stable assembly of PSBP with the PSII core and its absence also impacts the binding of PSBQ to the core (Suorsa et al., 2006; Liu et al., 2009). For stable association with the PSII core complex, PSBR needs the presence of PSBJ (Suorsa et al., 2006). Functionally, the depletion of PSBR protein expression decreased rates of oxygen evolution (Allahverdiyeva et al., 2007, 2013) and quinone reoxidation (Allahverdiyeva et al., 2007). PSBR phosphorylation is known for Arabidopsis (Arabidopsis thaliana; Reiland et al., 2009, 2011; Nakagami et al., 2010) and in the green alga C. reinhardtii (Turkina et al., 2006), although phosphorylation sites are not conserved between the alga and the vascular plant.In this work, we addressed the question of whether down-regulation of PSBR expression would affect LHCSR3 binding to the PSII-LHCII supercomplex in C. reinhardtii. To this end, we took advantage of artificial microRNA (amiRNA) technology to down-regulate PSBR expression and investigated the impact of PSBR down-regulation on photosynthetic performance as well as on PSII-LHCII-LHCSR3 supercomplex formation. Our data provide evidence that PSBR is required for the stable binding of LHCSR3 to PSII-LHCII supercomplexes.     

The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II

Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of light-harvesting complexes (LHC) II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of Chlamydomonas reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains seven Lhcbs, in addition to the PSI core and the nine Lhcas that compose PSI-LHCI, together binding ∼320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ∼65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ∼78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ∼60 ps. This relatively slow transfer, as compared with that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.     

Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.     

Partial characterization of the biosynthesis and integration of the Photosystem II reaction centers in the thylakoid membrane of Chlamydomonas reinhardtii

Pulse-labeling of wild-type and a Photosystem II mutant strain of Chlamydomonas reinhardtii was carried out in the presence or absence of inhibitors of either cytoplasmic or chloroplast ribosomes, and their thylakoid membrane polypeptides were analyzed by polyacrylamide gel electrophoresis. A pulse-chase study was also done on the wild-type strain in the presence of anisomycin, an inhibitor of protein synthesis on cytoplasmic ribosomes. The following results were obtained: the Photosystem II reaction center is mainly composed of integral membrane proteins synthesized within the chloroplast. Several of the proteins of the Photosystem II reaction center are post-translationally modified, after they have been inserted in the thylakoid membrane.     

Photosystem II antenna complexes CP26 and CP29 are essential for nonphotochemical quenching in Chlamydomonas reinhardtii

Photosystems must balance between light harvesting to fuel the photosynthetic process for CO₂ fixation and mitigating the risk of photodamage due to absorption of light energy in excess. Eukaryotic photosynthetic organisms evolved an array of pigment-binding proteins called light harvesting complexes constituting the external antenna system in the photosystems, where both light harvesting and activation of photoprotective mechanisms occur. In this work, the balancing role of CP29 and CP26 photosystem II antenna subunits was investigated in Chlamydomonas reinhardtii using CRISPR-Cas9 technology to obtain single and double mutants depleted of monomeric antennas. Absence of CP26 and CP29 impaired both photosynthetic efficiency and photoprotection: Excitation energy transfer from external antenna to reaction centre was reduced, and state transitions were completely impaired. Moreover, differently from higher plants, photosystem II monomeric antenna proteins resulted to be essential for photoprotective thermal dissipation of excitation energy by nonphotochemical quenching.     

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)–tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2⁻ mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.     

Genetic Engineering of the Processing Site of D1 Precursor Protein of Photosystem II Reaction Center in Chlamydomonas reinhardtii

The D1 protein (D1) of photosystem II (PSII) reaction centeris synthesized as a precursor (pD1) and then processed at itscarboxyl terminus to establish the function of water cleavage.The amino acid sequence of the carboxyl terminal extension excisedby this process is poorly conserved except for a residue afterthe cleavage site at position of 345. We have constructed avector for site-directed mutagenesis of the chloroplast psbAgene encoding D1 of the green alga, Chlamydomonas reinhardtii.The vector enables one to transform the chloroplasts of a psbAdeletion mutant (Fud7) and directly select transformants forresistance to spectinomycin. Using this transforming vector,we have substituted Ser345 to Gly, Cys, Val and Phe in orderto investigate effects of the amino acid side chain at thisposition on the processing rate. All of the resulting transformantsexhibited the PSII activity as wild type and grew normally underphotoautotrophic conditions even under strong light where rapidturnover of Dl protein is expected to occur. Western blottinganalysis demonstrated that mature D1 accumulates in these transformantsat wild type level. Pulse and chase labeling of chloroplast-encodedproteins using [³⁵S]sulfate revealed that the processing ofD1 precursor protein occurs in all four transformants as efficientlyas in wild type, at least under the experimental conditionsexamined. The results suggest that either the amino acid sidechain at position of 345 (+1 position) is not crucial to theenzymatic cleavage of pD1 in vivo or the apparent rate of processingin vivo is not limited by the enzymatic cleavage. (Received September 22, 1995; Accepted December 25, 1995)     

Kinetics of excitation trapping in intact Photosystem I of Chlamydomonas reinhardtii and Arabidopsis thaliana

We measured picosecond time-resolved fluorescence of intact Photosystem I complexes from Chlamydomonas reinhardtii and Arabidopsis thaliana. The antenna system of C. reinhardtii contains about 30-60 chlorophylls more than that of A. thaliana, but lacks the so-called red chlorophylls, chlorophylls that absorb at longer wavelength than the primary electron donor. In C. reinhardtii, the main lifetimes of excitation trapping are about 27 and 68 ps. The overall lifetime of C. reinhardtii is considerably shorter than in A. thaliana. We conclude that the amount and energies of the red chlorophylls have a larger effect on excitation trapping time in Photosystem I than the antenna size.     

Dissection of the Blue-Light-Dependent Signal-Transduction Pathway Involved in Gametic Differentiation of Chlamydomonas reinhardtii

Gametogenesis of the green alga Chlamydomonas reinhardtii may be viewed as a two-step process that is controlled by the environmental cues of nitrogen deprivation and blue light. Initiation of gametogenesis is induced by nitrogen deprivation, resulting in mating-incompetent pregametes, when cells are kept in the dark. For the completion of gametic differentiation light is required. Pregametes were treated with pharmacological compounds to influence the light-dependent conversion to mature gametes. Dibutyryl-cyclic 3[prime]5[prime] adenosinemonophosphate, papaverine, and genistein were found to inhibit the progression of gametogenesis in the light. Treatment of pregametes in the dark with either staurosporine or papaverine resulted in their conversion to mature gametes. Apparently, papaverine has different effects in the dark and in the light; the effect of staurosporine suggested that a protein kinase C-like component inhibits the conversion of pregametes to gametes, a block that normally is relieved by illumination. This hypothesis was corroborated by the observation that activators of protein kinase C, N-heptyl-5-chloro-1-naphthalenesulfonamide, N- (6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, and the phorbolester phorbol-12-myristate 13-acetate inhibited gametogenesis in the light. Genistein and dibutyryl-cyclic 3[prime]5[prime] adenosinemonophosphate were able to inhibit the dark activation caused by staurosporine treatment, suggesting that their targets work downstream from the "protein kinase C-like" kinase. Surprisingly, staurosporine and papaverine worked synergystically on the activation of pregametes in the dark.