Mihály Kis

Phosphatidylglycerol Is Essential for Oligomerization of Photosystem I Reaction Center

Our earlier studies with the pgsA mutant of Synechocystis PCC6803 demonstrated the important role of phosphatidylglycerol (PG) in PSII dimer formation and in electron transport between the primary and secondary electron-accepting plastoquinones of PSII. Using a long-term depletion of PG from pgsA mutant cells, we could induce a decrease not only in PSII but also in PSI activity. Simultaneously with the decrease in PSI activity, dramatic structural changes of the PSI complex were detected. A 21-d PG depletion resulted in the degradation of PSI trimers and concomitant accumulation of monomer PSI. The analyses of PSI particles isolated by MonoQ chromatography showed that, following the 21-d depletion, PSI trimers were no longer detectable in the thylakoid membranes. Immunoblot analyses revealed that the PSI monomers accumulating in the PG-depleted mutant cells do not contain PsaL, the protein subunit thought to be responsible for the trimer formation. Nevertheless, the trimeric structure of PSI reaction center could be restored by readdition of PG, even in the presence of the protein synthesis inhibitor lincomycin, indicating that free PsaL was present in thylakoid membranes following the 21-d PG depletion. Our data suggest an indispensable role for PG in the PsaL-mediated assembly of the PSI reaction center.

Carotenoids, versatile components of oxygenic photosynthesis.

Carotenoids (CARs) are a group of pigments that perform several important physiological functions in all kingdoms of living organisms. CARs serve as protective agents, which are essential structural components of photosynthetic complexes and membranes, and they play an important role in the light harvesting mechanism of photosynthesizing plants and cyanobacteria. The protection against reactive oxygen species, realized by quenching of singlet oxygen and the excited states of photosensitizing molecules, as well as by the scavenging of free radicals, is one of the main biological functions of CARs. X-ray crystallographic localization of CARs revealed that they are present at functionally and structurally important sites of both the PSI and PSII reaction centers. Characterization of a CAR-less cyanobacterial mutant revealed that while the absence of CARs prevents the formation of PSII complexes, it does not abolish the assembly and function of PSI. CAR molecules assist in the formation of protein subunits of the photosynthetic complexes by gluing together their protein components. In addition to their aforementioned indispensable functions, CARs have a substantial role in the formation and maintenance of proper cellular architecture, and potentially also in the protection of the translational machinery under stress conditions.

Involvement of Carotenoids in the Synthesis and Assembly of Protein Subunits of Photosynthetic Reaction Centers of Synechocystis sp. PCC 6803

The crtB gene of Synechocystis sp. PCC 6803, encoding phytoene synthase, was inactivated in the Delta crtH mutant to generate a carotenoidless Delta crtH/B double mutant. Delta crtH mutant cells were used because they had better transformability than wild-type cells, most probably due to their adaptation to partial carotenoid deficiency. Cells of the Delta crtH/B mutant were light sensitive and could grow only under light-activated heterotrophic growth conditions in the presence of glucose. Carotenoid deficiency did not significantly affect the cellular content of phycobiliproteins while the chlorophyll content of the mutant cells decreased. The mutant cells exhibited no oxygen-evolving activity, suggesting the absence of photochemically active PSII complexes. This was confirmed by 2D electrophoresis of photosynthetic membrane complexes. Analyses identified only a small amount of a non-functional PSII core complex lacking CP43, while the monomeric and dimeric PSII core complexes were absent. On the other hand, carotenoid deficiency did not prevent formation of the cytochrome b(6)f complex and PSI, which predominantly accumulated in the monomeric form. Radioactive labeling revealed very limited synthesis of inner PSII antennae, CP47 and especially CP43. Thus, carotenoids are indispensable constituents of the photosynthetic apparatus, being essential not only for antioxidative protection but also for the efficient synthesis and accumulation of photosynthetic proteins and especially that of PSII antenna subunits.

Short- and long-term redox regulation of photosynthetic light energy distribution and photosystem stoichiometry by acetate metabolism in the green alga, Chlamydobotrys stellata

The effect of acetate metabolism on the light energy distribution between the two photosystems, on the PS II/PS I stoichiometry and on the expression of psbA and psbB and psaA genes was investigated in the green alga, Chlamydobotrys stellata during autotrophic (CO2), mixotrophic (CO2 plus acetate) and photoheterotrophic (only acetate) cultivation. It was observed that acetate assimilation in the glyoxylate cycle resulted in a large drop in the ATP content and a concomitant increase in the NADPH content of the cells. The combined effect of high NADPH concentration and linear electron transport brought about an over-reduction of the inter-photosystem electron transport components. The reduced state of the inter-photosystem components initiated a state 1/state 2 transition of LHC II and a decrease in the PS II/PS I ratio. The PS II/ PS I ratio was reduced because the synthesis of PS II reaction centers was repressed and that of the PS I reaction centers was slightly enhanced by acetate cultivation. The amount of PsbA and PsbB proteins of PS II and the abundance of psbA mRNA decreased. The abundance of PS I PsaA protein and psaAmRNA were only slightly increased. All of the acetate-induced effects were reversible when the cells were transferred back to an acetate-free medium. Our observations demonstrate that the expression of the PS II psbA and psbB and PS I psaA genes is regulated by the redox state of the inter-photosystem components at the transcriptional level. Experiments carried out in the presence of DBMIB which facilitates the reduction of plastoquinone pool indicate that the expression of genes encoding the components of PS II and PS I are controlled by the redox state of a component (cytochrome b/f complex) located behind the plastoquinone pool.

Cadmium exerts its toxic effects on photosynthesis via a cascade mechanism in the cyanobacterium, Synechocystis PCC 6803

Despite intense research, the mechanism of Cd(2+) toxicity on photosynthesis is still elusive because of the multiplicity of the inhibitory effects and different barriers in plants. The quick Cd(2+) uptake in Synechocystis PCC 6803 permits the direct interaction of cadmium with the photosynthetic machinery and allows the distinction between primary and secondary effects. We show that the CO(2) -dependent electron transport is rapidly inhibited upon exposing the cells to 40 µm Cd(2+) (50% inhibition in ∼15 min). However, during this time we observe only symptoms of photosystem I acceptor side limitation and a build of an excitation pressure on the reaction centres, as indicated by light-induced P700 redox transients, O(2) polarography and changes in chlorophyll a fluorescence parameters. Inhibitory effects on photosystem II electron transport and the degradation of the reaction centre protein D1 can only be observed after several hours, and only in the light, as revealed by chlorophyll a fluorescence transients, thermoluminescence and immunoblotting. Despite the marked differences in the manifestations of these short- and long-term effects, they exhibit virtually the same Cd(2+) concentration dependence. These data strongly suggest a cascade mechanism of the toxic effect, with a primary effect in the dark reactions.

Metagenome changes in the mesophilic biogas-producing community during fermentation of the green alga Scenedesmus obliquus

A microalgal biomass offers a potential alternative to the maize silage commonly used in biogas technology. In this study, photoautotrophically grown Scenedesmus obliquus was used as biogas substrate. This microalga has a low C/N ratio of 8.5 relative to the optimum 20-30. A significant increase in the ammonium ion content was not observed. The methane content of the biogas generated from Sc. obliquus proved to be higher than that from maize silage, but the specific biogas yield was lower. Semi-continuous steady biogas production lasted for 2 months. Because of the thick cell wall of Sc. obliquus, the biomass-degrading microorganisms require additional time to digest its biomass. The methane concentration in the biogas was also high, in co-digestion (i.e., 52-56%) as in alga-fed anaerobic digestion (i.e., 55-62%). These results may be related to the relative predominance of the order Clostridiales in co-digestion and to the more balanced C/N ratio of the mixed algal-maize biomass. Predominance of the order Methanosarcinales was observed in the domain Archaea, which supported the diversity of metabolic pathways in the process.

Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone QB in photosystem II

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of Q(B) to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PG(L)) enhances the affinity for BQ that inactivates Q(B). 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of Q(B) during 10 days of PG depletion, and was recovered rapidly within 10h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PG(H)) displaces Q(B) directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14 days decreased the chlorophyll/PBS ratio to about 1/4, flourescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683nm with a long 1.2ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PG(H), PG(L) and other PG molecules on PSII structure are discussed.

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

The lack of lipid phosphatidylglycerol inhibits chlorophyll biosynthesis and induces accumulation of an aberrant protein complex containing monomeric PSI and CP43 antenna of PSII. 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.

Carotenoids are essential for the assembly of cyanobacterial photosynthetic complexes

In photosynthetic organisms, carotenoids (carotenes and xanthophylls) are important for light harvesting, photoprotection and structural stability of a variety of pigment-protein complexes. Here, we investigated the consequences of altered carotenoid composition for the functional organization of photosynthetic complexes in wild-type and various mutant strains of the cyanobacterium Synechocystis sp. PCC 6803. Although it is generally accepted that xanthophylls do not play a role in cyanobacterial photosynthesis in low-light conditions, we have found that the absence of xanthophylls leads to reduced oligomerization of photosystems I and II. This is remarkable because these complexes do not bind xanthophylls. Oligomerization is even more disturbed in crtH mutant cells, which show limited carotenoid synthesis; in these cells also the phycobilisomes are distorted despite the fact that these extramembranous light-harvesting complexes do not contain carotenoids. The number of phycocyanin rods connected to the phycobilisome core is strongly reduced leading to high amounts of unattached phycocyanin units. In the absence of carotenoids the overall organization of the thylakoid membranes is disturbed: Photosystem II is not formed, photosystem I hardly oligomerizes and the assembly of phycobilisomes remains incomplete. These data underline the importance of carotenoids in the structural and functional organization of the cyanobacterial photosynthetic machinery.

Proteins, glycerolipids and carotenoids in the functional photosystem II architecture

Photosystem II (PSII), the first supercomplex of the electron transport chain, governs the energy transfer using harvested light energy, which is transformed into biochemical energy. Phosphatidylglycerol and sulfoquinovosyl diacylglycerol, the anionic lipids of photosynthetic organisms, together with a neutral lipid, digalactosyldiacylglycerol, assist in the assembly of photosynthetic complexes. These lipids and carotenoids serve as mortar for the proteins which act as bricks in the construction of the active photosynthetic machinery, and they have determinative roles in the oligomerization of protein subunits. X-ray crystallographic localization of glycerolipids and carotenoids revealed that they are present at functionally and structurally important sites of both the PSI and PSII reaction centers. Phosphatidylglycerol is involved in the formation of the reaction-center oligomers and controls electron transport at the acceptor site of PSII. Digalactosyldiacylglycerol, together with phosphatidylglycerol, is involved in the electron transport at the donor site. Phosphatidylglycerol and carotenoids are needed to glue CP43 to the reaction center core. Carotenoids are protective agents, which prevent photosynthetic complexes from degradation caused by reactive oxygen species.