Supermolecular structure of photosystem II and location of the PsbS protein

This paper addresses the question of whether the PsbS protein of photosystem two (PS II) is located within the LHC II PS II supercomplex for which a three-dimensional structure has been obtained by cryoelectron microscopy and single particle analysis. The PsbS protein has recently been implicated as the site for non-photochemical quenching. Based both on immunoblotting analyses and structural considerations of an improved model of the spinach LHC II PS II supercomplex, we conclude that the PsbS protein is not located within the supercomplex. Analyses of other fractions resulting from the solubilization of PS Il-enriched membranes derived from spinach suggest that the PsbS protein is located in the LHC II-rich regions that interconnect the supercomplex within the membrane.     

Molecular analysis of the Chlamydomonas nuclear gene encoding PsbW and demonstration that PsbW is a subunit of photosystem II,but not photosystem I

PsbW is a nuclear-encoded protein located in the thylakoid membrane of the chloroplast. Studies in higher plants have provided substantial evidence that PsbW is a core component of photosystem II. However, recent data have been presented to suggest that PsbW is also a subunit of photosystem I. Such a sharing of subunits between the two photosystems would represent a novel phenomenon. To investigate this, we have cloned and characterized the psbW gene from the green alga Chlamydomonas reinhardtii. The gene is split by five introns and encodes a polypeptide of 115 residues comprising the 6.1 kDa mature PsbW protein preceded by a 59 amino acid bipartite transit sequence. Using antibodies raised to PsbW we have examined: (1) C. reinhardtii mutants lacking either photosystem and (2) purified photosystem preparations. We find that PsbW is a subunit of photosystem II, but not photosystem I.     

Isolation of monomeric photosystem II that retains the subunit PsbS

Photosystem II has been purified from a transplastomic strain of Nicotiana tabacum according to two different protocols. Using the procedure described in Piano et al. (Photosynth Res 106:221–226, 2010) it was possible to isolate highly active PSII composed of monomers and dimers but depleted in their PsbS protein content. A “milder” procedure than the protocol reported by Fey et al. (Biochim Biophys Acta 1777:1501–1509, 2008) led to almost exclusively monomeric PSII complexes which in part still bind the PsbS protein. This finding might support a role for PSII monomers in higher plants.     

Auxiliary proteins involved in the assembly and sustenance of photosystem II

Chloroplast proteins that regulate the biogenesis, performance and acclimation of the photosynthetic protein complexes are currently under intense research. Dozens, possibly even hundreds, of such proteins in the stroma, thylakoid membrane and the lumen assist the biogenesis and constant repair of the water splitting photosystem (PS) II complex. During the repair cycle, assistance is required at several levels including the degradation of photodamaged D1 protein, de novo synthesis, membrane insertion, folding of the nascent protein chains and the reassembly of released protein subunits and different co-factors into PSII in order to guarantee the maintenance of the PSII function. Here we review the present knowledge of the auxiliary proteins, which have been reported to be involved in the biogenesis and maintenance of PSII.     

The PsbW protein stabilizes the supramolecular organization of photosystem II in higher plants

PsbW, a 6.1-kDa low-molecular-weight protein, is exclusive to photosynthetic eukaryotes, and associates with the photosystem II (PSII) protein complex. In vivo and in vitro comparison of Arabidopsis thaliana wild-type plants with T-DNA insertion knock-out mutants completely lacking the PsbW protein, or with antisense inhibition plants exhibiting decreased levels of PsbW, demonstrated that the loss of PsbW destabilizes the supramolecular organization of PSII. No PSII-LHCII supercomplexes could be detected or isolated in the absence of the PsbW protein. These changes in macro-organization were accompanied by a minor decrease in the chlorophyll fluorescence parameter F(V) /F(M) , a strongly decreased PSII core protein phosphorylation and a modification of the redox state of the plastoquinone (PQ) pool in dark-adapted leaves. In addition, the absence of PsbW protein led to faster redox changes in the PQ pool, i.e. transitions from state 1 to state 2, as measured by changes in stationary fluorescence (F(S) ) kinetics, compared with the wild type. Despite these dramatic effects on macromolecular structure, the transgenic plants exhibited no significant phenotype under normal growth conditions. We suggest that the PsbW protein is located close to the minor antenna of the PSII complex, and is important for the contact and stability between several PSII-LHCII supercomplexes.     

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

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

In vitro synthesis and assembly of photosystem II proteins of spinach chloroplasts

The synthesis and assembly of photosystem II (PS II) proteins of spinach chloroplasts were investigated in three different in vitro systems, i.e., protein synthesis in isolated chloroplasts (in organello translation), read-out translation of thylakoid-bound ribosomes, and transport of translation products from spinach leaf polyadenylated RNA into isolated chloroplasts. Polyacrylamide gel electrophoresis of labeled thylakoid polypeptides in the presence of sodium dodecyl sulfate revealed that the first two systems were capable of synthesizing the reaction center proteins of PS II (47 and 43 kDa), the herbicide-binding protein, and cytochrome b559. The reaction center proteins synthesized in organello were shown to bind chlorophyll and to assemble properly into the PS II core complex. One of the reaction center proteins translated by the thylakoid-bound ribosomes (47 kDa) was also found to be integrated in situ into the complex but was lacking bound chlorophyll. Incorporation of radioactivity into the three extrinsic proteins of the oxygen-evolution system (33, 24, and 18 kDa) was detected only when intact chloroplasts were incubated with the translation products from polyadenylated RNA, showing that these proteins are coded for by nuclear DNA. The occurrence of a precursor polypeptide 6 kDa larger than the 33-kDa protein was immunochemically detected in the translation products.     

Interactions between the photosystem II subunit PsbS and xanthophylls studied in vivo and in vitro

The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type.     

Expression, assembly and auxiliary functions of photosystem II oxygen-evolving proteins in higher plants

The oxygen-evolving complex (OEC) of higher plant photosystem II (PSII) consists of an inorganic Mn₄Ca cluster and three nuclear-encoded proteins, PsbO, PsbP and PsbQ. In this review, we focus on the assembly of these OEC proteins, and especially on the role of the small intrinsic PSII proteins and recently found “novel” PSII proteins in the assembly process. The numerous auxiliary functions suggested during the past few years for the OEC proteins will likewise be discussed. For example, besides being a manganese-stabilizing protein, PsbO has been found to bind calcium and GTP and possess a carbonic anhydrase activity. In addition, specific roles have been suggested for the two isoforms of the PsbO protein in Arabidopsis thaliana. PsbP and PsbQ seem to play an additional role in the formation of PSII supercomplexes and in grana stacking, besides their originally recognized role in providing a proper calcium and chloride ion concentration for water splitting.     

The PsbS protein controls the organization of the photosystem II antenna in higher plant thylakoid membranes

The PsbS subunit of photosystem II (PSII) plays a key role in nonphotochemical quenching (NPQ), the major photoprotective regulatory mechanism in higher plant thylakoid membranes, but its mechanism of action is unknown. Here we describe direct evidence that PsbS controls the organization of PSII and its light harvesting system (LHCII). The changes in chlorophyll fluorescence amplitude associated with the Mg(2+)-dependent restacking of thylakoid membranes were measured in thylakoids prepared from wild-type plants, a PsbS-deficient mutant and a PsbS overexpresser. The Mg(2+) requirement and sigmoidicity of the titration curves for the fluorescence rise were negatively correlated with the level of PsbS. Using a range of PsbS mutants, this effect of PsbS was shown not to depend upon its efficacy in controlling NPQ, but to be related only to protein concentration. Electron microscopy and fluorescence spectroscopy showed that this effect was because of enhancement of the Mg(2+)-dependent re-association of PSII and LHCII by PsbS, rather than an effect on stacking per se. In the presence of PsbS the LHCII.PSII complex was also more readily removed from thylakoid membranes by detergent, and the level of PsbS protein correlated with the amplitude of the psi-type CD signal originating from features of LHCII.PSII organization. It is proposed that PsbS regulates the interaction between LHCII and PSII in the grana membranes, explaining how it acts as a pH-dependent trigger of the conformational changes within the PSII light harvesting system that result in NPQ.     

Subunit proteins of photosystem II

Recipient of the Society Award for Young Scientists 1991.     

Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant

The biochemical properties of PsbS protein, a nuclear-encoded Photosystem II subunit involved in the high energy quenching of chlorophyll fluorescence, have been studied using preparations purified from chloroplasts or obtained by overexpression in bacteria. Despite the homology with chlorophyll a/b/xanthophyll-binding proteins of the Lhc family, native PsbS protein does not show any detectable ability to bind chlorophylls or carotenoids in conditions in which Lhc proteins maintain full pigment binding. The recombinant protein, when refolded in vitro in the presence of purified pigments, neither binds chlorophylls nor xanthophylls, differently from the homologous proteins LHCII, CP26, and CP29 that refold into stable pigment-binding complexes. Thus, it is concluded that if PsbS is a pigment-binding protein in vivo, the binding mechanism must be different from that present in other Lhc proteins. Primary sequence analysis provides evidence for homology of PsbS helices I and III with the central 2-fold symmetric core of chlorophyll a/b-binding proteins. Moreover, a structural homology owed to the presence of acidic residues in each of the two lumen-exposed loops is found with the dicyclohexylcarbodiimide/Ca(2+)-binding domain of CP29. Consistently, both native and recombinant PsbS proteins showed [(14)C]dicyclohexylcarbodiimide binding, thus supporting a functional basis for its homology with CP29 on the lumen-exposed loops. This domain is suggested to be involved in sensing low luminal pH.     

Biogenesis,assembly and turnover of photosystem II units

Assembly of photosystem II, a multiprotein complex embedded in the thylakoid membrane, requires stoichiometric production of over 20 protein subunits. Since part of the protein subunits are encoded in the chloroplast genome and part in the nucleus, a signalling network operates between the two genetic compartments in order to prevent wasteful production of proteins. Coordinated synthesis of proteins also takes place among the chloroplast-encoded subunits, thus establishing a hierarchy in the protein components that allows a stepwise building of the complex. In addition to this dependence on assembly partners, other factors such as the developmental stage of the plastid and various photosynthesis-related parameters exert a strict control on the accumulation, membrane targeting and assembly of the PSII subunits. Here, we briefly review recent results on this field obtained with three major approaches: biogenesis of photosystem II during the development of chloroplasts from etioplasts, use of photosystem II-specific mutants and photosystem II turnover during its repair cycle.     

The PsbS protein controls the macro-organisation of photosystem II complexes in the grana membranes of higher plant chloroplasts

The PsbS protein is a critical component in the regulation of non-photochemical quenching (NPQ) in higher plant photosynthesis. Electron microscopy and image analysis of grana membrane fragments from wild type and mutant Arabidopsis plants showed that the semi-crystalline domains of photosystem II supercomplexes were identical in the presence and absence of PsbS. However, the frequency of the domains containing crystalline arrays was increased in the absence of PsbS. Conversely, there was a complete absence of such arrays in the membranes of plants containing elevated amounts of this protein. It is proposed that PsbS controls the macro-organisation of the grana membrane, providing an explanation of its role in NPQ.     

The low molecular mass PsbW protein is involved in the stabilization of the dimeric photosystem II complex in Arabidopsis thaliana

Arabidopsis thaliana plants have been transformed with an antisense gene to the psbW of photosystem II (PSII). Eight transgenic lines containing low levels of psbW mRNA have been obtained. Transgenic seedlings with low contents of PsbW protein (more than 96% reduced) were selected by Western blotting and used for photosynthetic functional studies. There were no distinct differences in phenotype between the antisense and wild type plants during vegetative period under normal growth light intensities. However, a sucrose gradient separation of briefly solubilized thylakoid membranes revealed that no dimeric PSII supracomplex could be detected in the transgenic plants lacking the PsbW protein. Furthermore, analysis of isolated thylakoids demonstrated that the oxygen-evolving rate in antisense plants decreased by 50% compared with the wild type. This was found to be due to up to 40% of D1 and D2 reaction center proteins of PSII disappearing in the transgenic plants. The absence of the PsbW protein also altered the contents of other PSII proteins to differing extents. These results show that in the absence of the PsbW protein, the stability of the dimeric PSII is diminished and consequently the total number of PSII complexes is greatly reduced. Thus the nuclear encoded PsbW protein may play a crucial role in the biogenesis and regulation of the photosynthetic apparatus.     

Recent advances in understanding the assembly and repair of photosystem II

Background

Photosystem II (PSII) is the light-driven water:plastoquinone oxidoreductase of oxygenic photosynthesis and is found in the thylakoid membrane of chloroplasts and cyanobacteria. Considerable attention is focused on how PSII is assembled in vivo and how it is repaired following irreversible damage by visible light (so-called photoinhibition). Understanding these processes might lead to the development of plants with improved growth characteristics especially under conditions of abiotic stress.

Scope

Here we summarize recent results on the assembly and repair of PSII in cyanobacteria, which are excellent model organisms to study higher plant photosynthesis.

Conclusions

Assembly of PSII is highly co-ordinated and proceeds through a number of distinct assembly intermediates. Associated with these assembly complexes are proteins that are not found in the final functional PSII complex. Structural information and possible functions are beginning to emerge for several of these ‘assembly’ factors, notably Ycf48/Hcf136, Psb27 and Psb28. A number of other auxiliary proteins have been identified that appear to have evolved since the divergence of chloroplasts and cyanobacteria. The repair of PSII involves partial disassembly of the damaged complex, the selective replacement of the damaged sub-unit (predominantly the D1 sub-unit) by a newly synthesized copy, and reassembly. It is likely that chlorophyll released during the repair process is temporarily stored by small CAB-like proteins (SCPs). A model is proposed in which damaged D1 is removed in Synechocystis sp. PCC 6803 by a hetero-oligomeric complex composed of two different types of FtsH sub-unit (FtsH2 and FtsH3), with degradation proceeding from the N-terminus of D1 in a highly processive reaction. It is postulated that a similar mechanism of D1 degradation also operates in chloroplasts. Deg proteases are not required for D1 degradation in Synechocystis 6803 but members of this protease family might play a supplementary role in D1 degradation in chloroplasts under extreme conditions.     

Subunit positioning and transmembrane helix organisation in the core dimer of photosystem II

Recently 3D structural models of the photosystem II (PSII) core dimer complexes of higher plants (spinach) and cyanobacteria (Synechococcus elongatus) have been derived by electron [Rhee et al. (1998) Nature 396, 283-286; Hankamer et al. (2001) J. Struct. Biol., in press] and X-ray [Zouni et al. (2001) Nature 409, 739-743] crystallography respectively. The intermediate resolutions of these structures do not allow direct identification of side chains and therefore many of the individual subunits within the structure are unassigned. Here we review the structure of the higher plant PSII core dimer and provide evidence for the tentative assignment of the low molecular weight subunits. In so doing we highlight the similarities and differences between the higher plant and cyanobacterial structures.