In vivo quality control of photosystem II in cyanobacteria Synechocystis sp. PCC 6803: D1 protein degradation and repair under the influence of light, heat and darkness

The cells of Synechocystis sp. PCC 6803 were subjected under photoinhibitory irradiation (600 micromolm(-2)s(-1)) at various temperatures (20-40 degrees C) to study in vivo quality control of photosystem II (PSII). The protease biogenesis and its consequences on photosynthetic efficiency (chlorophyll fluorescence ratio Fv/Fm) of the PSII, D1 degradation and repair were monitored during illumination and darkness. The loss in Fv/Fm value and degradation of D1 protein occurred not only under high light exposure, but also continued when the cells were subjected under dark restoration process after high light exposure. No loss in Fv/Fm value or D1 degradation occurred during recovery under growth/low light (30 micromol m(-2) s(-1)). Further, it helped the resynthesis of new D1 protein, essential to sustain quality control of PSII. In vivo triggering of D1 protein required high light exposure to switch-on the protease biosynthesis to maintain protease pool which induced temperature-dependent enzymatic proteolysis of photodamaged D1 protein during photoinhition and dark incubation. Our findings suggested the involvement and overexpression of a membrane-bound FtsH protease during high light exposure which caused degradation of D1 protein, strictly regulated by high temperature (30-40 degrees C). However, lower temperature (20 degrees C) prevented further loss of photoinhibited PSII efficiency in vivo and also retarded temperature-dependent proteolytic process of D1 degradation.     

The acyl lipid composition of wheat leaves and moss protonemata using a new, non-carcinogenic extraction solvent system

As chloroform has proved to be carcinogenic we were looking for an alternative solvent system for chloroform:methanol widely used in plant lipid investigations. The lipids from leaves of wheat ( Triticum aestivum L. cv. Vakka) and from protonemata of the moss Ceratodon purpureus (Hedw.) Brid. were extracted with two petroleum ether:methanol solvent systems. The polar lipids were separated by two-dimensional thin-layer chromatography and the amounts of each lipid class were compared with those obtained from chloroform:methanol (2:1, v/v) extractions. The significantly higher amounts of phosphatidylinositol observed in petroleum ether:methanol (1:1, v/v) extraction suggest that the small amounts reported earlier in plants may be an artefact relating to the solvent system used. As petroleum ether:methanol (1:1, v/v) proved to be at least as good a solvent system as chloroform:methanol (2:1, v/v) we propose it as an alternative extractant for plant polar lipids.     

Structural characterization of NDH-1 complexes of Thermosynechococcus elongatus by single particle electron microscopy

The structure of the multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes from cyanobacteria was investigated by growing the wild type and specific ndh His-tag mutants of Thermosynechococcus elongatus BP-1 under different CO(2) conditions, followed by an electron microscopy (EM) analysis of their purified membrane protein complexes. Single particle averaging showed that the complete NDH-1 complex (NDH-1L) is L-shaped, with a relatively short hydrophilic arm. Two smaller complexes were observed, differing only at the tip of the membrane-embedded arm. The smallest one is considered to be similar to NDH-1M, lacking the NdhD1 and NdhF1 subunits. The other fragment, named NDH-1I, is intermediate between NDH-1L and NDH-1M and only lacks a mass compatible with the size of the NdhF1 subunit. Both smaller complexes were observed under low- and high-CO(2) growth conditions, but were much more abundant under the latter conditions. EM characterization of cyanobacterial NDH-1 further showed small numbers of NDH-1 complexes with additional masses. One type of particle has a much longer peripheral arm, similar to the one of NADH: ubiquinone oxidoreductase (complex I) in E. coli and other organisms. This indicates that Thermosynechococcus elongatus must have protein(s) which are structurally homologous to the E. coli NuoE, -F, and -G subunits. Another low-abundance type of particle (NDH-1U) has a second labile hydrophilic arm at the tip of the membrane-embedded arm. This U-shaped particle has not been observed before by EM in a NDH-I preparation.     

Single particle analysis of thylakoid proteins from Thermosynechococcus elongatus and Synechocystis 6803: localization of the CupA subunit of NDH-1

The larger protein complexes of the cyanobacterial photosynthetic membrane of Thermosynechoccus elongatus and Synechocystis 6803 were studied by single particle electron microscopy after detergent solubilization, without any purification steps. Besides the "standard" L-shaped NDH-1L complex, related to complex I, large numbers of a U-shaped NDH-1MS complex were found in both cyanobacteria. In membranes from Synechocystis DeltacupA and DeltacupA/cupB mutants the U-shaped complexes were absent, indicating that CupA is responsible for the U-shape by binding at the tip of the membrane-bound arm of NDH-1MS. Comparison of membranes grown under air levels of CO(2) or 3% CO(2) indicates that the number of NDH-1MS particles is 30-fold higher under low-CO(2).     

Structure of the chloroplast NADH dehydrogenase-like complex: nomenclature for nuclear-encoded subunits

The chloroplast NADH dehydrogenase-like complex (NDH) was first discovered based on its similarity to complex I in respiratory electron transport, and is involved in electron transport from photoproduced stromal reductants such as NADPH and ferredoxin to the intersystem plastoqunone pool. However, a recent study suggested that it is a ferredoxin-dependent plastoquinone reductase rather than an NAD(P)H dehydrogenase. Furthermore, recent advances in subunit analysis of NDH have revealed the presence of a novel hydrophilic subcomplex on the stromal side of the thylakoid membrane, as well as an unexpected lumenal subcomplex. This review discusses these new studies on the structure of NDH, and proposes a unified nomenclature for newly discovered NDH subunits.     

Towards a critical understanding of the photosystem II repair mechanism and its regulation during stress conditions

Photosystem II (PSII) is vulnerable to high light (HL) illumination resulting in photoinhibition. In addition to photoprotection mechanisms, plants have developed an efficient PSII repair mechanism to save themselves from irreversible damage to PSII under abiotic stresses including HL illumination. The phosphorylation/dephosphorylation cycle along with subsequent degradation of photodamaged D1 protein to be replaced by the insertion of a newly synthesized copy of D1 into the PSII complex, is the core function of the PSII repair cycle. The exact mechanism of this process is still under discussion. We describe the recent progress in identifying the kinases, phosphatases and proteases, and in understanding their involvement in the maintenance of thylakoid structure and the quality control of proteins by PSII repair cycle during photoinhibition.     

Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes

Gel-based analysis of thylakoid membrane protein complexes represents a valuable tool to monitor the dynamics of the photosynthetic machinery. Native-PAGE preserves the components and often also the conformation of the protein complexes, thus enabling the analysis of their subunit composition. Nevertheless, the literature and practical experimentation in the field sometimes raise confusion owing to a great variety of native-PAGE and thylakoid-solubilization systems. In the present paper, we describe optimized methods for separation of higher plant thylakoid membrane protein complexes by native-PAGE addressing particularly: (i) the use of detergent; (ii) the use of solubilization buffer; and (iii) the gel electrophoresis method. Special attention is paid to separation of high-molecular-mass thylakoid membrane super- and mega-complexes from Arabidopsis thaliana leaves. Several novel super- and mega-complexes including PS (photosystem) I, PSII and LHCs (light-harvesting complexes) in various combinations are reported.     

Membrane attachment of Slr0006 in Synechocystis sp. PCC 6803 is determined by divalent ions

Slr0006 is one of the Synechocystis sp. PCC 6803 proteins strongly induced under carbon limiting conditions. Slr0006 has no predicted transmembrane helices or signal peptide sequence, yet it was exclusively recovered in the membrane fraction of Synechocystis, when the cells were broken in isolation buffers which contain divalent cations and are generally used for photosynthesis studies. Even subsequent washing of the membranes with high salt or various detergents did not release Slr0006, indicating strong binding of the Slr0006 protein to the membranes. Further, DNAse or RNAse treatment did not disturb the tight binding of Slr0006 protein to the membranes. Nevertheless, when the cells were broken in the absence of divalent cations, Slr0006 remained completely soluble. Binding of the Slr0006 to the membrane could not be properly reconstituted if the cations were added after breaking the cells in the absence of divalent ions. This unusual phenomenon has to be considered in identification and localization of other yet uncharacterized cyanobacterial proteins.