Bettina Ughy

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.

A second isoform of the ferredoxin:NADP oxidoreductase generated by an in-frame initiation of translation

Ferredoxin:NADP oxidoreductases (FNRs) constitute a family of flavoenzymes that catalyze the exchange of reducing equivalents between one-electron carriers and the two-electron-carrying NADP(H). The main role of FNRs in cyanobacteria and leaf plastids is to provide the NADPH for photoautotrophic metabolism. In root plastids, a distinct FNR isoform is found that has been postulated to function in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by heterotrophic metabolism. A multiple gene family encodes FNR isoenzymes in plants, whereas there is only one FNR gene (petH) in cyanobacteria. Nevertheless, we detected two FNR isoforms in the cyanobacterium Synechocystis sp. strain PCC6803. One of them (FNRS ≈34 kDa) is similar in size to the plastid FNR and specifically accumulates under heterotrophic conditions, whereas the other one (FNRL ≈46 kDa) contains an extra N-terminal domain that allows its association with the phycobilisome. Site-directed mutants allowed us to conclude that the smaller isoform, FNRS, is produced from an internal ribosome entry site within the petH ORF. Thus we have uncovered a mechanism by which two isoforms are produced from a single gene, which is, to our knowledge, novel in photosynthetic bacteria. Our results strongly suggest that FNRL is an NADP+ reductase, whereas FNRS is an NADPH oxidase.

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.

Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosynthetic membranes of the cyanobacterium Cylindrospermopsis raciborskii: An FTIR spectroscopic study

The effects of the growth temperature on the lipids and carotenoids of a filamentous cyanobacterium, Cylindrospermopsis raciborskii, were studied., The relative amounts of polyunsaturated glycerolipids and myxoxanthophylls in the thylakoid membranes increased markedly when this cyanobacterium was grown at 25°C instead of 35°C. Fourier transform infrared spectroscopy was used to analyze the low-temperature-induced structural alterations in the thylakoid membranes. Despite the higher amount of unsaturated lipids there, conventional analysis of the νsymCH2 band (characteristic of the lipid disorder) revealed more tightly arranged fatty-acyl chains for the thylakoids in the cells grown at 25°C as compared with those grown at 35°C. This apparent controversy was resolved by a two-component analysis of the νsymCH2 band, which demonstrated very rigid, myxoxanthophyll-related lipids in the thylakoid membranes. When this rigid component was excluded from the analysis of the thermotropic responses of the νsymCH2 bands, the expected higher fatty-acyl disorder was observed for the thylakoids prepared from cells grown at 25°C as compared with those grown at 35°C. Both the carotenoid composition and this rigid component in the thylakoid membranes were only growth temperature-dependent; the intensity of the illuminating light during cultivation had no apparent effect on these parameters. We propose that, besides their well-known protective functions, the polar carotenoids in particular may have structural effects on the thylakoid membranes. These effects should be exerted locally—by forming protective patches, in-membrane barriers of low dynamics—to prevent the access of reactive radicals generated in either enzymatic or photosynthetic processes to sensitive spots of the membranes.

Reversible membrane reorganizations during photosynthesis in vivo: Revealed by small-angle neutron scattering

In the present study, we determined characteristic repeat distances of the photosynthetic membranes in living cyanobacterial and eukaryotic algal cells, and in intact thylakoid membranes isolated from higher plants with time-resolved small-angle neutron scattering. This non-invasive technique reveals light-induced reversible reorganizations in the seconds-to-minutes time scale, which appear to be associated with functional changes in vivo.

Monitoring Photosynthesis in Individual Cells of Synechocystis sp. PCC 6803 on a Picosecond Timescale

Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I (PSI) in vivo for the first time, and the observed 6-ps equilibration process and 25-ps trapping process are the same as found previously for isolated PSI. No major differences are detected between different cells. The PAL mutant cells, devoid of phycobilisomes, show four lifetimes: ∼20 ps (PSI and PSII), ∼80 ps, ∼440 ps, and 2.8 ns (all due to PSII), but not all cells are identical and variations in the kinetics are traced back to differences in the PSI/PSII ratio. Finally, FLIM measurements on WT cells reveal that in some cells or parts of cells, phycobilisomes are disconnected from PSI/PSII. It is argued that the FLIM setup used can become instrumental in unraveling photosynthetic regulation mechanisms in the future.

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.

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.

Excitation energy transfer between Light-harvesting complex II and Photosystem i in reconstituted membranes

Light-harvesting complex II (LHCII), the major peripheral antenna of Photosystem II in plants, participates in several concerted mechanisms for regulation of the excitation energy and electron fluxes in thylakoid membranes. In part, these include interaction of LHCII with Photosystem I (PSI) enhancing the latters absorption cross-section - for example in the well-known state 1 - state 2 transitions or as a long-term acclimation to high light. In this work we examined the capability of LHCII to deliver excitations to PSI in reconstituted membranes in vitro. Proteoliposomes with native plant thylakoid membrane lipids and different stoichiometric ratios of LHCII:PSI were reconstituted and studied by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission from LHCII was strongly decreased in PSI-LHCII membranes due to trapping of excitations by PSI. Kinetic modelling of the time-resolved fluorescence data revealed the existence of separate pools of LHCII distinguished by the time scale of energy transfer. A strongly coupled pool, equivalent to one LHCII trimer per PSI, transferred excitations to PSI with near-unity efficiency on a time scale of less than 10ps but extra LHCIIs also contributed significantly to the effective antenna size of PSI, which could be increased by up to 47% in membranes containing 3 LHCII trimers per PSI. The results demonstrate a remarkable competence of LHCII to increase the absorption cross-section of PSI, given the opportunity that the two types of complexes interact in the membrane.