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Polyunsaturated lipids are important components of photosynthetic membranes. Xanthophylls are the main photoprotective agents, can assist in protection against light stress, and are crucial in the recovery from photoinhibition. We generated the xanthophyll- and polyunsaturated lipid-deficient ROAD mutant of Synechocystis sp. PCC6803 (Synechocystis) in order to study the little-known cooperative effects of lipids and carotenoids (Cars). Electron microscopic investigations confirmed that in the absence of xanthophylls the S-layer of the cellular envelope is missing. In wild-type (WT) cells, as well as the xanthophyll-less (RO), polyunsaturated lipid-less (AD), and the newly constructed ROAD mutants the lipid and Car compositions were determined by MS and HPLC, respectively. We found that, relative to the WT, the lipid composition of the mutants was remodeled and the Car content changed accordingly. In the mutants the ratio of non-bilayer-forming (NBL) to bilayer-forming (BL) lipids were found considerably lower. Xanthophyll to β-carotene ratio increased in the AD mutant. In vitro and in vivo methods demonstrated that saturated, monounsaturated lipids and xanthophylls may stabilize the trimerization of Photosystem I (PSI). Fluorescence induction and oxygen-evolving activity measurements revealed increased light sensitivity of RO cells compared to those of the WT. ROAD showed a robust increase in light susceptibility and reduced recovery capability, especially at moderate low (ML) and moderate high (MH) temperatures, indicating a cooperative effect of xanthophylls and polyunsaturated lipids. We suggest that both lipid unsaturation and xanthophylls are required for providing the proper structure and functioning of the membrane environment that protects against light and temperature stress.

We investigated the relation between the carotenoid composition and the structure of phycobilisome (PBS) antenna of cyanobacterium Synechocystis sp. PCC 6803. PBS is a large soluble protein complex enhances the light harvesting efficiency of the cells. It is composed of a central allophycocyanin core and radial phycocyanin rods, but it does not contain carotenoids. However, the absence or low level of carotenoids were previously shown to lead the co-existence of unconnected rod units and assembled PBS with shorter peripheral rods. Here we show that the lack of β-carotene, but not of xanthophylls or the distortion of photosystem structure, evoked unconnected rods. Thus, these essential β-carotene molecules are not bound by Photosystem I or Photosystem II. Our results do not show correlation between the reactive oxygen species (ROS) and PBS distortion despite the higher singlet oxygen producing capacity and light sensitivity of the mutant cells. Reduced cellular level of those linker proteins attaching the rod units together was also observed, but the direct damage of the linkers by ROS are not supported by our data. Enzymatic PBS proteolysis induced by nitrogen starvation in carotenoid mutant cells revealed a retarded degradation of the unconnected rod units.

In the thylakoid membranes of the mesophilic cyanobacterium Synechocystis PCC6803, PSI reaction centers (RCs) are organized as monomers and trimers. PsaL, a 16 kDa hydrophobic protein, a subunit of the PSI RC, was previously identified as crucial for the formation of PSI trimers. In this work, the physiological effects accompanied by PSI oligomerization were studied using a PsaL-deficient mutant (ΔpsaL), not able to form PSI trimers, grown at various temperatures. We demonstrate that in wild-type Synechocystis, the monomer to trimer ratio depends on the growth temperature. The inactivation of the psaL gene in Synechocystis grown phototropically at 30°C induces profound morphological changes, including the accumulation of glycogen granules localized in the cytoplasm, resulting in the separation of particular thylakoid layers. The carotenoid composition in ΔpsaL shows that PSI monomerization leads to an increased accumulation of myxoxantophyll, zeaxanthin and echinenone irrespective of the temperature conditions. These xanthophylls are formed at the expense of β-carotene. The measured H2O→CO2 oxygen evolution rates in the ΔpsaL mutant are higher than those observed in the wild type, irrespective of the growth temperature. Moreover, circular dichroism spectroscopy in the visible range reveals that a peak attributable to long-wavelength-absorbing carotenoids is apparently enhanced in the trimer-accumulating wild-type cells. These results suggest that specific carotenoids are accompanied by the accumulation of PSI oligomers and play a role in the formation of PSI oligomer structure.

The function of xanthophylls in the organisation and structure of the photosynthetic complexes is not completely clarified yet. Recently, we observed a reduced level of the photosystem oligomers upon xanthophyll deficiency, although xanthophylls are not considered to be part of the photosynthetic complexes of cyanobacteria. The present study aimed at further investigating the relationship between xanthophylls and photosytem I (PSI) complex in the cyanobacterium Synechocystis sp. PCC 6803. Interestingly, we recorded the presence of echinenone and zeaxanthin in the isolated PSI trimers. These two xanthophyll species are among the most abundant xanthophylls in this cyanobacterial species. Various xanthophyll biosynthesis mutants were used to investigate the specific role of these xanthophylls. Our spectroscopic results revealed specific structural changes manifested in altered pigment-pigment or pigment-protein interactions within PSI complex in the absence of zeaxanthin and echinenone. These structural modifications of the complexes seem to destabilize the PSI trimeric complexes and eventually result in an increased propensity for monomerization. Our results clearly demonstrate that xanthophylls are important for the fine-tuning of the PSI trimer structure. These xanthophylls could be part of the complex or be embedded in the membrane in the vicinity of PSI.

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 10 h 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, florescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683 nm with a long 1.2 ns 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.

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.

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.

A U3 snoRNA gene isolated from a Chlamydomonas reinhardtii (CRE:) genomic library contains putative pol III-specific transcription signals similar to those of RNA polymerase III-specific small nuclear (sn)RNA genes of higher plants. The 222 nt long CRE: U3 snoRNA was immunoprecipitated by anti-gamma-mpppN antisera, but not by anti-m(2,2,7)G antibodies, supporting the notion that it is a RNA polymerase III transcript. Tagged CRE: U3 snoRNA gene constructs were expressed in CRE: cells. Results of chemical and enzymatic structure probing of CRE: U3 snoRNA in solution and of DMS modification of CRE: U3 snoRNA under in vivo conditions revealed that the two-hairpin structure of the 5'-domain that is found in solution is no longer detected under in vivo conditions. The observed differences can be explained by the formation of several base pair interactions with the 18S and 5'-ETS parts of the pre-rRNA. A model that involves five intermolecular helices is proposed.

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 (CO(2)), mixotrophic (CO(2) 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.

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.

Influence of the modification of the cyanobacterial light-harvesting complex [i.e. phycobilisomes (PBS)] on the surface electric properties and the functions of photosynthetic membranes was investigated. We used four PBS mutant strains of Synechocystis sp. PCC6803 as follows: PAL (PBS-less), CK (phycocyanin-less), BE (PSII-PBS-less) and PSI-less/apcE(-) (PSI-less with detached PBS). Modifications of the PBS content lead to changes in the cell morphology and surface electric properties of the thylakoid membranes as well as in their functions, such as photosynthetic oxygen-evolving activity, P700 kinetics and energy transfer between the pigment-protein complexes. Data reveal that the complete elimination of PBS in the PAL mutant causes a slight decrease in the electric dipole moments of the thylakoid membranes, whereas significant perturbations of the surface charges were registered in the membranes without assembled PBS-PSII macrocomplex (BE mutant) or PSI complex (PSI-less mutant). These observations correlate with the detected alterations in the membrane structural organization. Using a polarographic oxygen rate electrode, we showed that the ratio of the fast to the slow oxygen-evolving PSII centers depends on the partial or complete elimination of light-harvesting complexes, as the slow operating PSII centers dominate in the PBS-less mutant and in the mutant with detached PBS.

We used differential scanning calorimetry (DSC) as a technique capable of identifying photosynthetic complexes on the basis of their calorimetric transitions. Annotation of thermal transitions was carried out with thylakoid membranes isolated from various photosynthetic mutants of Synechocystis sp. PCC6803. The thylakoid membranes exhibited seven major DSC bands between 40 and 85°C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherms and by a subsequent annealing procedure. The successive annealing procedure proved to be more reliable technique than mathematical deconvolution in assigning thermal transitions. The main DSC band, around 47°C, resulting from the high enthalpy change that corresponds to non-interacting complex of PSII, was assigned using the PSI-less/apcE(-) mutant cells. Another band around 68-70°C relates to the denaturation of PSII surrounded by other proteins of the photosynthetic complexes in wild type and PSI-less/apcE(-) cells. A further major transition found at 82-84°C corresponds to the PSI core complex of wild type and PSII-deficient BE cells. Other transition bands between 50-67 and 65-75°C are believed to relate to ATP synthase and cytochrome b(6)f, respectively. These thermal transitions were obtained with thylakoids isolated from PSI(-)/PSII(-) mutant cells. Some minor bands determined at 59 and 83-84°C correspond to an unknown complex and NADH dehydrogenase, respectively. These annotations were done by PSI-less/apcE(-) and PSI(-)/PSII(-) mutants.