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.     

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.     

HYPERSENSITIVE TO HIGH LIGHT1 Interacts with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 and Functions in Protection of Photosystem II from Photodamage in Arabidopsis

Under high-irradiance conditions, plants must efficiently protect photosystem II (PSII) from damage. In this study, we demonstrate that the chloroplast protein HYPERSENSITIVE TO HIGH LIGHT1 (HHL1) is expressed in response to high light and functions in protecting PSII against photodamage. Arabidopsis thaliana hhl1 mutants show hypersensitivity to high light, drastically decreased PSII photosynthetic activity, higher nonphotochemical quenching activity, a faster xanthophyll cycle, and increased accumulation of reactive oxygen species following high-light exposure. Moreover, HHL1 deficiency accelerated the degradation of PSII core subunits under high light, decreasing the accumulation of PSII core subunits and PSII–light-harvesting complex II supercomplex. HHL1 primarily localizes in the stroma-exposed thylakoid membranes and associates with the PSII core monomer complex through direct interaction with PSII core proteins CP43 and CP47. Interestingly, HHL1 also directly interacts, in vivo and in vitro, with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), which functions in the repair and reassembly of PSII. Furthermore, the hhl1 lqy1 double mutants show increased photosensitivity compared with single mutants. Taken together, these results suggest that HHL1 forms a complex with LQY1 and participates in photodamage repair of PSII under high light.     

PsbN Is Required for Assembly of the Photosystem II Reaction Center in Nicotiana tabacum

The chloroplast-encoded low molecular weight protein PsbN is annotated as a photosystem II (PSII) subunit. To elucidate the localization and function of PsbN, encoded on the opposite strand to the psbB gene cluster, we raised antibodies and inserted a resistance cassette into PsbN in both directions. Both homoplastomic tobacco (Nicotiana tabacum) mutants ∆psbN-F and ∆psbN-R show essentially the same PSII deficiencies. The mutants are extremely light sensitive and failed to recover from photoinhibition. Although synthesis of PSII proteins was not altered significantly, both mutants accumulated only ∼25% of PSII proteins compared with the wild type. Assembly of PSII precomplexes occurred at normal rates, but heterodimeric PSII reaction centers (RCs) and higher order PSII assemblies were not formed efficiently in the mutants. The ∆psbN-R mutant was complemented by allotopic expression of the PsbN gene fused to the sequence of a chloroplast transit peptide in the nuclear genome. PsbN represents a bitopic trans-membrane peptide localized in stroma lamellae with its highly conserved C terminus exposed to the stroma. Significant amounts of PsbN were already present in dark-grown seedling. Our data prove that PsbN is not a constituent subunit of PSII but is required for repair from photoinhibition and efficient assembly of the PSII RC.     

Nanodomains of Cytochrome b6f and Photosystem II Complexes in Spinach Grana Thylakoid Membranes

The cytochrome b₆f (cytb₆f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytb₆f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytb₆f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytb₆f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytb₆f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytb₆f complexes. We suggest that the close proximity between PSII and cytb₆f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency.     

Cytochrome P450 CYP89A9 Is Involved in the Formation of Major Chlorophyll Catabolites during Leaf Senescence in Arabidopsis

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.     

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.     

Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex

Photosynthetic organisms have the ability to adapt to changes in light quality by readjusting the cross sections of the light-harvesting systems of photosystem II (PSII) and photosystem I (PSI). This process, called state transitions, maintains the redox poise of the photosynthetic electron transfer chain and ensures a high photosynthetic yield when light is limiting. It is mediated by the Stt7/STN7 protein kinase, which is activated through the cytochrome b₆f complex upon reduction of the plastoquinone pool. Its probable major substrate, the light-harvesting complex of PSII, once phosphorylated, dissociates from PSII and docks to PSI, thereby restoring the balance of absorbed light excitation energy between the two photosystems. Although the kinase is known to be inactivated under high-light intensities, the molecular mechanisms governing its regulation remain unknown. In this study we monitored the redox state of a conserved and essential Cys pair of the Stt7/STN7 kinase and show that it forms a disulfide bridge. We could not detect any change in the redox state of these Cys during state transitions and high-light treatment. It is only after prolonged anaerobiosis that this disulfide bridge is reduced. It is likely to be mainly intramolecular, although kinase activation may involve a transient covalently linked kinase dimer with two intermolecular disulfide bonds. Using the yeast two-hybrid system, we have mapped one interaction site of the kinase on the Rieske protein of the cytochrome b₆f complex.Photosynthetic organisms are subjected to constant changes in light quality and quantity and need to adapt to these changes in order to optimize, on the one hand, their photosynthetic yield, and to minimize photo-oxidative damage on the other. The photosynthetic electron transfer chain consists of photosystem II (PSII), the plastoquinone (PQ) pool, the cytochrome b₆f complex (Cyt b₆f), plastocyanin, and photosystem I (PSI). All of these complexes and components are integrated or closely associated with the thylakoid membrane. The two antenna systems of PSII and PSI capture and direct the light excitation energy to the corresponding reaction centers in which a chlorophyll dimer is oxidized and charge separation occurs across the thylakoid membrane. These processes lead to the onset of electron flow from water on the donor side of PSII to ferredoxin on the acceptor side of PSI coupled with proton translocation across the thylakoid membrane. In order to sustain optimal electron flow along this electron transfer chain, the redox poise needs to be maintained under changing environmental conditions. Several mechanisms have evolved for the maintenance of this redox balance. In the case of over-reduction of the acceptor side of PSI, excess electrons can reduce molecular oxygen through the Mehler reaction to superoxide, which is then converted to hydrogen peroxide by a plastid superoxide dismutase and ultimately to water by a peroxidase (Asada, 2000). Over-reduction of the PQ pool can be alleviated by PTOX, the plastid terminal oxidase responsible for oxidizing PQH₂ to form hydrogen peroxide, which is subsequently converted to water (Carol et al., 1999; Cournac et al., 2000; Wu et al., 1999).In addition to these electron sinks that prevent the over-reduction of the electron transfer chain, the photosynthetic apparatus is able to maintain the redox poise of the PQ pool by readjusting the relative cross sections of the light harvesting systems of PSII and PSI upon unequal excitation of the two photosystems. This readjustment can occur both in the short term through state transitions and in the long term by changing the stoichiometry between PSII and PSI (Bonaventura and Myers, 1969; Murata, 1969; Pfannschmidt, 2003). State transitions occur because of perturbations of the redox state of the PQ pool due to unequal excitation of PSII and PSI, limitations in electron acceptors downstream of PSI, and/or in CO₂ availability. Excess excitation of PSII relative to PSI leads to reduction of the PQ pool and thus favors the docking of PQH₂ to the Qo site of the Cyt b₆f complex. This process activates the Stt7/STN7 protein kinase (Vener et al., 1997; Zito et al., 1999), which is closely associated with this complex and leads to the phosphorylation of some LHCII proteins and to their detachment from PSII and binding to PSI (Depège et al., 2003; Lemeille et al., 2009). Although both Lhcb1 and Lhcb2 are phosphorylated, only the phosphorylated form of Lhcb2 is associated with PSI whereas phosphorylated Lhcb1 is excluded from this complex (Longoni et al., 2015). This state corresponds to state 2. In this way the change in the relative antenna sizes of the two photosystems restores the redox poise of the PQ pool. The process is reversible as over-excitation of PSI relative to PSII leads to the oxidation of the PQ pool and to the inactivation of the kinase. Under these conditions, phosphorylated LHCII associated with PSI is dephosphorylated by the PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010) and returns to PSII (state 1). It should be noted, however, that a strict causal link between LHCII phosphorylation and its migration from PSII to PSI has been questioned recently by the finding that some phosphorylated LHCII remains associated with PSII supercomplexes and that LHCII serves as antenna for both photosystems under most natural light conditions (Drop et al., 2014; Wientjes et al., 2013).State transitions are important at low light but do not occur under high light because the LHCII kinase is inactivated under these conditions (Schuster et al., 1986). It was proposed that inactivation of the kinase is mediated by the ferredoxin-thioredoxin system and that a disulfide bond in the kinase rather than in the substrate may be the target site of thioredoxin (Rintamäki et al., 1997, 2000). Analysis of the Stt7/STN7 protein sequences indeed reveals the presence of two conserved Cys residues close to the N-terminal end of this kinase, which are conserved in all species examined and both are essential for kinase activity although they are located outside of the kinase catalytic domain (Fig. 1) (Depège et al., 2003; Lemeille et al., 2009). Based on protease protection studies, this model of the Stt7/STN7 kinase proposes that the N-terminal end of the kinase is on the lumen side of the thylakoid membrane separated from the catalytic domain on the stromal side by an unusual transmembrane domain containing several Pro residues (Lemeille et al., 2009). This configuration of the kinase allows its catalytic domain to act on the substrate sites of the LHCII proteins, which are exposed to the stroma. Although in this model the conserved Cys residues in the lumen are on the opposite side from the stromal thioredoxins, it is possible that thiol-reducing equivalents are transferred across the thylakoid membrane through the CcdA and Hcf164 proteins, which have been shown to operate in this way during heme and Cyt b₆f assembly (Lennartz et al., 2001; Page et al., 2004) or through the LTO1 protein (Du et al., 2015; Karamoko et al., 2011).Figure 1.Conserved Cys in the Stt7/STN7 kinase. Alignment of the sequences of the Stt7/STN protein kinase from Selaginella moelendorffii (Sm), Physcomitrella patens (Pp), Oryza sativa (Os), Populus trichocarpa (Pt), Arabidopsis thaliana (At), Chlamydomonas reinhardtii ...Here we have examined the redox state of the Stt7/STN7 kinase during state transitions and after illumination with high light to test the proposed model. We find that the Stt7/STN7 kinase contains a disulfide bridge that appears to be intramolecular and maintained not only during state transitions but also in high light when the kinase is inactive. Although these results suggest at first sight that the disulfide bridge of Stt7/STN7 is maintained during its activation and inactivation, we propose that a transient opening of this bridge occurs during the activation process followed by the formation of an intermolecular disulfide bridge and the appearance of a short-lived, covalently linked kinase dimer.     

The Requirement for Carotenoids in the Assembly and Function of the Photosynthetic Complexes in Chlamydomonas reinhardtii

We have investigated the importance of carotenoids on the accumulation and function of the photosynthetic apparatus using a mutant of the green alga Chlamydomonas reinhardtii lacking carotenoids. The FN68 mutant is deficient in phytoene synthase, the first enzyme of the carotenoid biosynthesis pathway, and therefore is unable to synthesize any carotenes and xanthophylls. We find that FN68 is unable to accumulate the light-harvesting complexes associated with both photosystems as well as the RC subunits of photosystem II. The accumulation of the cytochrome b₆f complex is also strongly reduced to a level approximately 10% that of the wild type. However, the residual fraction of assembled cytochrome b₆f complexes exhibits single-turnover electron transfer kinetics comparable to those observed in the wild-type strain. Surprisingly, photosystem I is assembled to significant levels in the absence of carotenoids in FN68 and possesses functional properties that are very similar to those of the wild-type complex.Carotenoids (Cars) are fundamental components of the photosynthetic apparatus (Young and Britton, 1993, and refs. therein). The vast majority of Cars are noncovalently bound to either the core or the antenna subunits of PSI or PSII (Siefermann-Harms, 1985; Bassi et al., 1993). The most abundant Car bound to the core subunits of both photosystems is β-carotene, which is found in the vast majority of oxygenic organisms (Siefermann-Harms, 1985; Bassi et al., 1993). The light-harvesting complexes (LHCs) that act as the outer antenna in plants and green algae bind a wider range of oxygenated Cars, known as xanthophylls, the most abundant of which is lutein (Siefermann-Harms, 1985; Bassi et al., 1993; Jennings et al., 1996). The stoichiometry of xanthophylls binding to LHC complexes depends on the particular complexes and often on the illumination conditions during the organism’s growth (Siefermann-Harms, 1985; Demmig-Adams, 1990; Horton et al., 1996). Intriguingly, a molecule of β-carotene (as well as a molecule of chlorophyll [Chl] a) is found also in the cytochrome (Cyt) b₆f complex (Kurisu et al., 2003; Stroebel et al., 2003).Cars have multiple functions in the photosynthetic process; they act as light-harvesting pigments (Frank and Cogdell, 1993), enlarging the optical cross section to radiation that is poorly absorbed by Chl. Moreover, Cars play a crucial role in processes such as nonphotochemical quenching that control the efficiency of light harvesting in response to the intensity of the incident radiation (for review, see Demmig-Adams, 1990; Horton et al., 1996; Niyogi, 1999). Probably the most important role of Cars in photosynthesis is the quenching of the excited triplet state of Chl (for review, see Frank and Cogdell, 1993; Giacometti et al., 2007), preventing the formation of highly reactive singlet oxygen, which represents the principal species active under high light stress (Hideg et al., 1994; Krieger-Liszkay, 2005). The importance of Cars is demonstrated by the observation that disruption of their biosynthesis through mutation, or by inhibition of a key enzyme in the pathway, leads to either lethal phenotypes or to rapid photobleaching of the photosynthetic tissue (Claes, 1957; Faludi-Dániel et al., 1968, 1970; Bolychevtseva et al., 1995; Trebst and Depka, 1997).Moreover, it has been shown that the presence of xanthophylls is absolutely necessary for refolding in vitro of LHC I and LHC II antenna complexes (Plumley and Schmidt, 1987; Paulsen et al., 1993; Sandonà et al., 1998). Such Cars, therefore, have a structural role, as well as their involvement in light harvesting, nonphotochemical quenching regulation, and the quenching of the Chl triplet state. Whether Cars also play a key structural role in the formation and stability of the core complexes of both PSI and PSII has not been systematically explored, since assembly of these complexes in vitro is not feasible. Studies in vivo using higher plants are complicated by the fact that Car deficiency is lethal and can be studied only during the early stages of greening and leaf development (Faludi-Dániel et al., 1968, 1970; Inwood et al., 2008). In these studies, it was shown that the accumulation of PSII complexes was greatly impaired in mutants of maize (Zea mays; Faludi-Dániel et al., 1968, 1970; Inwood et al., 2008), while the assembly of PSI appeared to be less sensitive to Car availability. In mutants of the cyanobacterium Synechocystis sp. PCC 6803 lacking the genes for phytoene desaturase or ζ-carotene desaturase, there was a complete loss of PSII assembly, while functional PSI complexes were assembled, albeit with slightly altered electron transfer kinetics with respect to the wild-type complex (Bautista et al., 2005). In agreement with the higher sensitivity of PSII assembly to Car availability, Trebst and Depka (1997) reported a specific effect on the synthesis of the D1 subunit of PSII RC upon treatment with phytoene desaturase inhibitors. On the other hand, it has recently been reported that in lycopene-β-cyclase mutants of Arabidopsis (Arabidopsis thaliana) that have a decreased amount of β-carotene (bound to the RC) with respect to most of the xanthophyll pool pigments (bound to the LHCs), the level of accumulation of PSI complexes, particularly that of the LHC I complement, was more affected that that of PSII, probably also because of an increased sensitivity to photodamage of mutated PSI RC (Cazzaniga et al., 2012; Fiore et al., 2012).In this investigation, we have studied the accumulation and functionality of the major chromophore-binding complexes of the photosynthetic apparatus, PSI, PSII, and Cyt b₆f, in a Car-less mutant of the green alga Chlamydomonas reinhardtii (FN68) that is blocked at the first committed step of Car biosynthesis, namely, phytoene synthesis (McCarthy et al., 2004). Although the mutant is incapable of growing under phototrophic or photomixotrophic conditions, it can grow in complete darkness on a medium supplemented with a carbon source. Here, we show that the PSII core and antenna complexes fail to accumulate in the mutant and that the Cyt b₆f complex accumulates to approximately one-tenth of the wild-type level. On the other hand, the PSI reaction center accumulates in FN68 and possesses electron transfer properties that are remarkably similar to those of wild-type PSI. Interestingly, we find that the level of PSI accumulation differs in other phytoene synthase null mutants, suggesting that additional mutations in one or other of these strains affect PSI stability. Nevertheless, our findings demonstrate that Cars are not required for either the assembly or the functionality of PSI in vivo.     

Structural Basis of Efficient Electron Transport between Photosynthetic Membrane Proteins and Plastocyanin in Spinach Revealed Using Nuclear Magnetic Resonance

In the photosynthetic light reactions of plants and cyanobacteria, plastocyanin (Pc) plays a crucial role as an electron carrier and shuttle protein between two membrane protein complexes: cytochrome b₆f (cyt b₆f) and photosystem I (PSI). The rapid turnover of Pc between cyt b₆f and PSI enables the efficient use of light energy. In the Pc-cyt b₆f and Pc-PSI electron transfer complexes, the electron transfer reactions are accomplished within <10⁻⁴ s. However, the mechanisms enabling the rapid association and dissociation of Pc are still unclear because of the lack of an appropriate method to study huge complexes with short lifetimes. Here, using the transferred cross-saturation method, we investigated the residues of spinach (Spinacia oleracea) Pc in close proximity to spinach PSI and cyt b₆f, in both the thylakoid vesicle–embedded and solubilized states. We demonstrated that the hydrophobic patch residues of Pc are in close proximity to PSI and cyt b₆f, whereas the acidic patch residues of Pc do not form stable salt bridges with either PSI or cyt b₆f, in the electron transfer complexes. The transient characteristics of the interactions on the acidic patch facilitate the rapid association and dissociation of Pc.     

CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component,Is Essential for Meiotic Recombination Initiation in Rice

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated CENTRAL REGION COMPONENT1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID RECEPTOR-INTERACTING PROTEIN13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.     

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.     

Lack of Phosphatidylglycerol Inhibits Chlorophyll Biosynthesis at Multiple Sites and Limits Chlorophyllide Reutilization in Synechocystis sp. Strain PCC 6803

The negatively charged lipid phosphatidylglycerol (PG) constitutes up to 10% of total lipids in photosynthetic membranes, and its deprivation in cyanobacteria is accompanied by chlorophyll (Chl) depletion. Indeed, radioactive labeling of the PG-depleted ΔpgsA mutant of Synechocystis sp. strain PCC 6803, which is not able to synthesize PG, proved the inhibition of Chl biosynthesis caused by restriction on the formation of 5-aminolevulinic acid and protochlorophyllide. Although the mutant accumulated chlorophyllide, the last Chl precursor, we showed that it originated from dephytylation of existing Chl and not from the block in the Chl biosynthesis. The lack of de novo-produced Chl under PG depletion was accompanied by a significantly weakened biosynthesis of both monomeric and trimeric photosystem I (PSI) complexes, although the decrease in cellular content was manifested only for the trimeric form. However, our analysis of ΔpgsA mutant, which lacked trimeric PSI because of the absence of the PsaL subunit, suggested that the virtual stability of monomeric PSI is a result of disintegration of PSI trimers. Interestingly, the loss of trimeric PSI was accompanied by accumulation of monomeric PSI associated with the newly synthesized CP43 subunit of photosystem II. We conclude that the absence of PG results in the inhibition of Chl biosynthetic pathway, which impairs synthesis of PSI, despite the accumulation of chlorophyllide released from the degraded Chl proteins. Based on the knowledge about the role of PG in prokaryotes, we hypothesize that the synthesis of Chl and PSI complexes are colocated in a membrane microdomain requiring PG for integrity.Photosynthetic membrane of oxygenic phototrophs has a unique lipid composition that has been conserved during billions of years of evolution from cyanobacteria and algae to modern higher plants. With no known exception, this membrane system always contains the uncharged glycolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG) as well as the negatively charged lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG; Murata and Siegenthaler, 1998). Interestingly, it seems that PG is the only lipid completely essential for the oxygenic photosynthesis. The loss of DGDG has only a mild impact on the cyanobacterial cell (Awai et al., 2007), and as shown recently in the cyanobacterium Synechocystis sp. strain PCC 6803, both galactolipids can be in fact replaced by glucolipids (Awai et al., 2014). SQDG and PG are only minor lipid components, each accounting for 5% to 12% of total lipids (Murata and Siegenthaler, 1998). SQDG is dispensable, although its lack results in various defects (Yu et al., 2002; Aoki et al., 2004), but PG plays an essential role in both cyanobacterial cells and plant chloroplasts (Hagio et al., 2000; Babiychuk et al., 2003).The critical role of PG has been mostly connected to the function of PSII. In both cyanobacteria and plants, lack of PG impairs the stability of PSII complexes and the electron transport between primary and secondary quinone acceptors inside the PSII reaction center. As shown in Synechocystis sp., PG molecules stabilize PSII dimers and facilitate the binding of inner antenna protein CP43 within the PSII core (Laczkó-Dobos et al., 2008). Indeed, according to the PSII crystal structure, two PG molecules are located at the interface between CP43 and the D1-D2 heterodimer (Guskov et al., 2009). As a consequence, the PG depletion inhibits and destabilizes PSII complexes and also, impairs assembly of new PSII complexes, although all PSII subunits are still synthesized in the cell (Laczkó-Dobos et al., 2008).Despite the fact that the vital link between PG and PSII is now well established, the phenotypic traits of PG-depleted cells signal that there are other sites in the photosynthetic membrane requiring strictly PG molecules. In Synechocystis sp., lack of PG triggers rapid loss of trimeric PSI complexes (Domonkos et al., 2004; Sato et al., 2004), and because PSI complexes bind more than 80% of chlorophyll (Chl) in the Synechocystis sp. cell, the PG depletion is accompanied by a characteristic Chl bleaching (Domonkos et al., 2004). However, the reasons for this symptom are still unclear. Chl metabolism is tightly coordinated with synthesis, assembly, and degradation of photosystem complexes (for review, see Komenda et al., 2012b; Sobotka, 2014), and we have shown recently that the PSI complexes are the main sink for de novo Chl produced in cyanobacteria (Kopečná et al., 2012). Given the drastic decrease in PSI content in the PG-depleted cells, Chl biosynthesis must be directly or indirectly affected after the PG concentration in membranes drops below a critical value. Although it was recently suggested that galactolipid and Chl biosyntheses are coregulated during chloroplast biogenesis (Kobayashi et al., 2014), a response of the Chl biosynthetic pathway to the altered lipid content has not been examined.To investigate Chl metabolism during PG starvation, we used the Synechocystis sp. ΔpgsA mutant, which is unable to synthesize PG (Hagio et al., 2000). The advantage of using the ΔpgsA strain is in its ability to utilize exogenous PG from growth medium, which allows monitoring of phenotypic changes from a wild type-like situation to completely PG-depleted cells. Chl biosynthesis shares the same metabolic pathway with heme and other tetrapyrroles. At the beginning of tetrapyrrole biosynthesis, the initial precursor, 5-aminolevulinic acid (ALA), is made from Glu through glutamyl-tRNA and subsequently converted in several steps to protoporphyrin IX. The pathway branches at the point where protoporphyrin IX is chelated by magnesium to produce Mg-protoporphyrin IX, the first intermediate on the Chl branch. This step is catalyzed by Mg-chelatase, a multisubunit enzyme that associates relatively weakly with the membrane; however, all following enzymes downward in the pathway are almost exclusively bound to membranes (Masuda and Fujita, 2008; Kopečná et al., 2012). The last enzyme of the Chl pathway, Chl synthase, is an integral membrane protein that attaches a phytyl chain to the last intermediate chlorophyllide (Chlide) to finalize Chl formation (Oster et al., 1997; Addlesee et al., 2000). According to current views, Chl synthase should also be involved in reutilization of Chl molecules from degraded Chl-binding proteins, which includes dephytylation and phytylation of Chl molecules with Chlide as an intermediate (Vavilin and Vermaas, 2007).In this study, we show a complex impact of PG deficiency on Chl metabolism. The lack of PG inhibited Chl biosynthesis at the two different steps: first, it drastically reduced formation of the initial precursor ALA, and second, it impaired the Mg-protoporphyrin methyl ester IX (MgPME) cyclase enzyme catalyzing synthesis of protochlorophyllide (Pchlide). The diminished rate of Chl formation was accompanied by impaired synthesis of both trimeric and monomeric PSI complexes and accumulation of a PSI monomer associated with the CP43 subunit of PSII. We also showed that the PG-depleted cells accumulated Chlide, originating from dephytylation of existing Chl, which suggests an inability to reutilize Chl for the PSI synthesis. We discuss a scenario that the Chl biosynthesis and synthesis of core PSI subunits are colocated in PG-enriched membrane microdomains.     

DAC Is Involved in the Accumulation of the Cytochrome b6/f Complex in Arabidopsis

The biogenesis and assembly of photosynthetic multisubunit protein complexes is assisted by a series of nucleus-encoded auxiliary protein factors. In this study, we characterize the dac mutant of Arabidopsis (Arabidopsis thaliana), which shows a severe defect in the accumulation of the cytochrome b₆/f complex, and provide evidence suggesting that the efficiency of cytochrome b₆/f complex assembly is affected in the mutant. DAC is a thylakoid membrane protein with two predicted transmembrane domains that is conserved from cyanobacteria to vascular plants. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation analyses revealed a specific interaction between DAC and PetD, a subunit of the cytochrome b₆/f complex. However, DAC was found not to be an intrinsic component of the cytochrome b₆/f complex. In vivo chloroplast protein labeling experiments showed that the labeling rates of the PetD and cytochrome f proteins were greatly reduced, whereas that of the cytochrome b₆ protein remained normal in the dac mutant. DAC appears to be a novel factor involved in the assembly/stabilization of the cytochrome b₆/f complex, possibly through interaction with the PetD protein.The cytochrome b₆/f (Cyt b6/f) complex is a multisubunit complex that resides in the thylakoid membrane and functions in linear and cyclic electron transport. In the linear process, the complex receives electrons from PSII and transfers them to PSI, a process that is accompanied by the generation of a proton gradient, which is essential for ATP synthesis (Mitchell, 1961; Saraste, 1999). The native form of this complex is present as a dimer with a mass of 310 kD that can be converted into a 140-kD monomer with increasing detergent concentrations (Huang et al., 1994; Breyton et al., 1997; Mosser et al., 1997; Baniulis et al., 2009). In higher plants, the Cyt b6/f monomer contains at least eight subunits: Cyt f, Cyt b₆, PetC, PetD, PetM, PetL, PetG, and PetN (Wollman, 2004). PetC and PetM are encoded by nuclear genes, whereas the others are encoded by plastid genes. It has been shown that PetG and PetN are necessary for complex stability in tobacco (Nicotiana tabacum; Schwenkert et al., 2007). By contrast, PetL is not required for the accumulation of other subunits of the Cyt b6/f complex, even though it is involved in the stability and formation of the functional dimer (Bendall et al., 1986; Schwenkert et al., 2007). Inactivation of PetC in Arabidopsis (Arabidopsis thaliana) resulted in significantly reduced amounts of Cyt b6/f subunits and completely blocked linear electron transport, indicating that PetC participates in the formation of the functionally assembled Cyt b6/f complex (Maiwald et al., 2003). In Synechocystis sp. PCC 6803, the PetM subunit has no essential role in Cyt b6/f complex electron transfer or accumulation; however, the absence of this subunit apparently affects the levels of other protein complexes involved in energy transduction (Schneider et al., 2001). In addition to the other proteins, FNR was identified as a subunit of the Cyt b6/f complex isolated from spinach (Spinacia oleracea) thylakoid membranes (Zhang et al., 2001).Previous research has revealed how the Cyt b6/f complex assembles into a functional dimer (Bendall et al., 1986; Lemaire et al., 1986; Kuras and Wollman, 1994). In the Cyt b6/f complex, Cyt b₆ and PetD form a mildly protease-resistant subcomplex that serves as a template for the assembly of Cyt f and PetG, producing a protease-resistant cytochrome moiety (Wollman, 2004). The PetC and PetL proteins then participate in the assembly of the functional dimer (Schwenkert et al., 2007). PetD becomes more unstable in the absence of Cyt b₆, and the synthesis of Cyt f is greatly reduced when either Cyt b₆ or PetD is inactivated, indicating that both Cyt b₆ and PetD are prerequisite for the synthesis of Cyt f (Kuras and Wollman, 1994). The reduced synthesis of Cyt f can be explained by the so-called CES (for controlled by epistasy of synthesis) mechanism. It is suggested that, in this mechanism, the synthesis rate of some chloroplast-encoded subunits of photosynthetic protein complexes is regulated by the availability of their assembly partners from the same complexes (Choquet et al., 2001). The mechanism of CES for Cyt f has been studied in detail in Chlamydomonas reinhardtii (Choquet et al., 1998; Choquet and Vallon, 2000). In it, the unassembled Cyt f inhibits its own translation through a negative feedback mechanism, and MCA1 and TCA1 have been demonstrated to be involved in the regulation of Cyt f synthesis (Boulouis et al., 2011).Many studies have focused on understanding the conversion of apocytochrome to holocytochrome via the covalent binding of heme in Cyt f and Cyt b₆ during the assembly of Cyt b6/f through the CCS and CCB pathways (Nakamoto et al., 2000; Wollman, 2004; de Vitry, 2011). The CCS pathway was originally discovered in the green alga C. reinhardtii through genetic studies of ccs mutants (for cytochrome c synthesis) that display a specific defect in membrane-bound Cyt f and soluble Cyt c₆, two thylakoid lumen-resident c-type cytochromes functioning in photosynthesis (Xie and Merchant, 1998). In the CCS pathway, six loci that include plastid ccsA and nuclear CCS1 to CCS5 have been found in C. reinhardtii (Xie and Merchant, 1998). In these mutants, the apocytochrome is normally synthesized, targeted, and processed, but heme attachment is perturbed. The CCB pathway is involved in the covalent attachment of heme c(i) to Cyt b₆ on the stromal side of the thylakoid membranes (Kuras et al., 2007). The ccb mutants show defects in the accumulation of subunits of the Cyt b6/f complex and covalent binding of heme to Cyt b₆ (Lyska et al., 2007; Lezhneva et al., 2008). However, heme binding is not a prerequisite for the assembly of Cyt b₆ into the Cyt b6/f complex, although the fully formed Cyt b6/f showed an increased sensitivity to protease (Saint-Marcoux et al., 2009).The assembly of the Cyt b6/f complex is a multistep process, and current studies have shown that the covalent binding of heme to Cyt f and Cyt b₆ is highly regulated. Thus, it is reasonable to speculate that, similar to the other photosynthetic protein complexes (Mulo et al., 2008; Nixon et al., 2010; Rochaix, 2011), the assembly of the Cyt b6/f complex is also assisted by many nucleus-encoded factors. In this study, we characterized an Arabidopsis protein, DAC (for defective accumulation of Cyt b6/f complex), that seems to be involved in the assembly of the Cyt b6/f complex. In addition, we provide evidence that DAC interacts directly with PetD before it assembles within the Cyt b6/f complex.     

Cell Growth Defect Factor1/CHAPERONE-LIKE PROTEIN OF POR1 Plays a Role in Stabilization of Light-Dependent Protochlorophyllide Oxidoreductase in Nicotiana benthamiana and Arabidopsis

Angiosperms require light for chlorophyll biosynthesis because one reaction in the pathway, the reduction of protochlorophyllide (Pchlide) to chlorophyllide, is catalyzed by the light-dependent protochlorophyllide oxidoreductase (POR). Here, we report that Cell growth defect factor1 (Cdf1), renamed here as CHAPERONE-LIKE PROTEIN OF POR1 (CPP1), an essential protein for chloroplast development, plays a role in the regulation of POR stability and function. Cdf1/CPP1 contains a J-like domain and three transmembrane domains, is localized in the thylakoid and envelope membranes, and interacts with POR isoforms in chloroplasts. CPP1 can stabilize POR proteins with its holdase chaperone activity. CPP1 deficiency results in diminished POR protein accumulation and defective chlorophyll synthesis, leading to photobleaching and growth inhibition of plants under light conditions. CPP1 depletion also causes reduced POR accumulation in etioplasts of dark-grown plants and as a result impairs the formation of prolamellar bodies, which subsequently affects chloroplast biogenesis upon illumination. Furthermore, in cyanobacteria, the CPP1 homolog critically regulates POR accumulation and chlorophyll synthesis under high-light conditions, in which the dark-operative Pchlide oxidoreductase is repressed by its oxygen sensitivity. These findings and the ubiquitous presence of CPP1 in oxygenic photosynthetic organisms suggest the conserved nature of CPP1 function in the regulation of POR.