Hajnalka Laczkó-Dobos

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.

Lipid-assisted protein–protein interactions that support photosynthetic and other cellular activities

Glycoglycerolipids are dominant lipids of photosynthetic organisms, i.e. higher plants and cyanobacteria. X-ray crystallographic localization of glycerolipids revealed that they are present at functionally and structurally important sites of both the PS I and PS II reaction centres. Phosphatidylglycerol (PG) is an indispensible member of glycerolipids, including the formation of functionally active oligomers of the reaction centres both PS I and PS II. Lipids assist in the assembly of protein subunits of the photosynthetic machinery by pasting the individual protein components together. PG is needed to glue CP43 to the reaction centre core. PG and digalactosyldiacylglycerol (DGDG) interact in photosynthetic processes: PG alone controls electron transport at the acceptor site of PS II, and together with DGDG is involved in electron transport at the donor site of PS II. PG is crucial for the formation of division rings and is implicated in the fission of cyanobacteria.

Phosphatidylglycerol depletion affects photosystem II activity in Synechococcus sp. PCC 7942 cells

The role of phosphatidylglycerol (PG) in photosynthetic membranes of cyanobacteria was analyzed in a Synechococcus sp. PCC 7942 mutant produced by inactivating its cdsA gene presumably encoding cytidine 5′-diphosphate-diacylglycerol synthase, a key enzyme in PG synthesis. In a medium supplemented with PG the Synechococcus sp. PCC 7942/ΔcdsA cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a suppression of O2 evolving activity, and (c) in a modification of Chl fluorescence induction curves. Two-dimensional PAGE showed that in the absence of PG (a) the amount of the PSI monomers increased at the expense of the PSI trimers and (b) PSII dimers were decomposed into monomers. [35S]methionine labeling confirmed that PG depletion did not block the de novo synthesis of PSII proteins but slowed down the assembly of the newly synthesized D1 protein into PSII core complexes. Retailoring of PG was observed during PG depletion: the exogenously added artificial dioleoyl PG was transformed into photosynthetically more essential PG derivatives. Concomitantly with a decrease in PG content, SQDG content increased, but it could not restore photosynthetic activity.

Carotenoids Assist in Cyanobacterial Photosystem II Assembly and Function

Carotenoids (carotenes and xanthophylls) are ubiquitous constituents of living organisms. They are protective agents against oxidative stresses and serve as modulators of membrane microviscosity. As antioxidants they can protect photosynthetic organisms from free radicals like reactive oxygen species that originate from water splitting, the first step of photosynthesis. We summarize the structural and functional roles of carotenoids in connection with cyanobacterial Photosystem II. Although carotenoids are hydrophobic molecules, their complexes with proteins also allow cytoplasmic localization. In cyanobacterial cells such complexes are called orange carotenoid proteins, and they protect Photosystem II and Photosystem I by preventing their overexcitation through phycobilisomes (PBS). Recently it has been observed that carotenoids are not only required for the proper functioning, but also for the structural stability of PBSs.

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.

Phosphatidylglycerol Depletion Induces an Increase in Myxoxanthophyll Biosynthetic Activity in Synechocystis PCC6803 Cells

Phosphatidylglycerol (PG) depletion suppressed the oxygen-evolving activity of Synechocystis PCC6803 pgsA mutant cells. Shortage of PG led to decreased photosynthetic activity, which, similar to the effect of high light exposure, is likely to generate the production of reactive oxygen species (ROS) or free radicals. Protection of the PG-depleted cells against light-induced damage increased the echinenone and myxoxanthophyll content of the cells. The increased carotenoid content was localized in a soluble fraction of the cells as well as in isolated thylakoid and cytoplasmic membranes. The soluble carotenoid fraction contained carotene derivatives, which may bind to proteins. These carotene-protein complexes are similar to orange carotenoid protein that is involved in yielding protection against free radicals and ROS. An increase in the content of myxoxanthophyll and echinenone upon PG depletion suggests that PG depletion regulates the biosynthetic pathway of specific carotenoids.

Lipids, Proteins, and Their Interplay in the Dynamics of Temperature-Stressed Membranes of a Cyanobacterium, Synechocystis PCC 6803

Proper responses to low- and high-temperature stresses are essential for the survival of many organisms. It has been established that at low-temperature stress the sufficient microviscosity of the lipids is decisive in this respect. In many organisms, adapting the level of lipid unsaturation to the low growth temperature regulates this feature. At high-temperature stresses, however, there are no unequivocal results concerning the role of the lipids. In these temperature ranges, the lipids are all disordered and fluid and their physical parameters change slowly with increasing temperatures, while biological organisms give characteristic stress responses in rather narrow temperature ranges. Therefore, one may speculate that other membrane parameters/components, which change sharply in the range of the high-temperature stress, may give a signal to initiate the general response of the cells. For such a role, proteins are the trivial candidates. To reveal the role of the lipids and the proteins in these processes, we used a genetically engineered system, based on a cyanobacterium, Synechocystis PCC 6803. In the wild-type cells of this bacterium, by altering the growth temperature, the polyunsaturated lipid content of the cell membranes can be varied considerably (as required by the homeoviscous adaptation principle). In the case of desA(-)/desD(-) mutant cells, which can contain only monounsaturated fatty acyl chains in their lipids, homeoviscous adaptation of the lipids is not possible. Since desA(-)/desD(-) mutation affects only the lipids, additional perturbations (e.g., altered protein content) should minimally disturb the comparison of the lipid behaviors in wild-type and mutant cells. Infrared spectra of thylakoid and cytoplasmic membranes isolated from wild-type and mutant cells were recorded in 3 degrees C steps between 8 and 92 degrees C. By analyzing the rates of protein structural changes, hydrogen-deuterium exchange, in-membrane lipid disorder, and water-membrane interfacial order/hydration as functions of the temperature, we conclude that (i) the gel-to-liquid crystalline phase transition of the lipids correlates with the growth temperature in the wild-type cells but not in the desA(-)/desD(-) mutants, (ii) over the physiological temperature range, both protein and lipid dynamics are regulating/regulated, providing remarkably constant dynamics for both the thylakoid and cytoplasmic membrane, (iii) in the high-temperature stress region, protein structure and dynamics are changing sharply without any correlation with growth temperature and/or mutation, i.e., membrane protein stability does not seem to depend on the lipid composition of the membrane (this finding points to the possible primacy of proteins as initiators/targets of heat-shock alarms), and (iv) there are substantial differences between the dynamics of the proteins of the thylakoid and cytoplasmic membranes, reflecting their different protein complexes and lipid-to-protein ratios.

Elevated Growth Temperature Can Enhance Photosystem I Trimer Formation and Affects Xanthophyll Biosynthesis in Cyanobacterium Synechocystis sp. PCC6803 Cells

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.

Remodeling of phosphatidylglycerol in Synechocystis PCC6803

The phosphatidylglycerol deficient DeltapgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization-Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/DeltapgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A(1) and A(2) or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.

Effect of partial or complete elimination of light-harvesting complexes on the surface electric properties and the functions of cyanobacterial photosynthetic membranes

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.