Role of isotropic lipid phase in the fusion of photosystem II membranes.

It has been thoroughly documented, by using 31P-NMR spectroscopy, that plant thylakoid membranes (TMs), in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (HII) phase. However, our knowledge concerning the structural and functional roles of the non-bilayer phases is still rudimentary. The objective of the present study is to elucidate the origin of I phases which have been hypothesized to arise, in part, from the fusion of TMs (Garab et al. 2022 Progr Lipid Res 101,163). We take advantage of the selectivity of wheat germ lipase (WGL) in eliminating the I phases of TMs (Dlouhý et al. 2022 Cells 11: 2681), and the tendency of the so-called BBY particles, stacked photosystem II (PSII) enriched membrane pairs of 300-500 nm in diameter, to form large laterally fused sheets (Dunahay et al. 1984 BBA 764: 179). Our 31P-NMR spectroscopy data show that BBY membranes contain L and I phases. Similar to TMs, WGL selectively eliminated the I phases, which at the same time exerted no effect on the molecular organization and functional activity of PSII membranes. As revealed by sucrose-density centrifugation, magnetic linear dichroism spectroscopy and scanning electron microscopy, WGL disassembled the large laterally fused sheets. These data provide direct experimental evidence on the involvement of I phase(s) in the fusion of stacked PSII membrane pairs, and strongly suggest the role of non-bilayer lipids in the self-assembly of the TM system.

Trimeric photosystem I facilitates energy transfer from phycobilisomes in Synechocystis sp. PCC 6803.

In cyanobacteria, phycobilisomes (PBS) serve as peripheral light-harvesting complexes of the two photosystems, extending their antenna size and the wavelength range of photons available for photosynthesis. The abundance of PBS, the number of phycobiliproteins they contain, and their light-harvesting function are dynamically adjusted in response to the physiological conditions. PBS are also thought to be involved in state transitions that maintain the excitation balance between the two photosystems. Unlike its eukaryotic counterpart, PSI is trimeric in many cyanobacterial species and the physiological significance of this is not well understood. Here, we compared the composition and light-harvesting function of PBS in cells of Synechocystis sp. PCC 6803, which has primarily trimeric PSI, and the ΔpsaL mutant, which lacks the PsaL subunit of PSI and is unable to form trimers. We also investigated a mutant additionally lacking the PsaJ and PsaF subunits of PSI. Both strains with monomeric PSI accumulated significantly more allophycocyanin per chlorophyll, indicating higher abundance of PBS. On the other hand, a higher phycocyanin:allophycocyanin ratio in the wild type suggests larger PBS or the presence of APC-less PBS (CpcL-type) that are not assembled in cells with monomeric PSI. Steady-state and time-resolved fluorescence spectroscopy at room temperature and 77 K revealed that PSII receives more energy from the PBS at the expense of PSI in cells with monomeric PSI, regardless of the presence of PsaF. Taken together, these results show that the oligomeric state of PSI impacts the excitation energy flow in Synechocystis.

The Interplay of Apoes with Syndecans in Influencing Key Cellular Events of Amyloid Pathology.

Apolipoprotein E (ApoE) isoforms exert intricate effects on cellular physiology beyond lipid transport and metabolism. ApoEs influence the onset of Alzheimer's disease (AD) in an isoform-dependent manner: ApoE4 increases AD risk, while ApoE2 decreases it. Previously we demonstrated that syndecans, a transmembrane proteoglycan family with increased expression in AD, trigger the aggregation and modulate the cellular uptake of amyloid beta (Aß). Utilizing our previously established syndecan-overexpressing cellular assays, we now explore how the interplay of ApoEs with syndecans contributes to key events, namely uptake and aggregation, in Aß pathology. The interaction of ApoEs with syndecans indicates isoform-specific characteristics arising beyond the frequently studied ApoE-heparan sulfate interactions. Syndecans, and among them the neuronal syndecan-3, increased the cellular uptake of ApoEs, especially ApoE2 and ApoE3, while ApoEs exerted opposing effects on syndecan-3-mediated Aß uptake and aggregation. ApoE2 increased the cellular internalization of monomeric Aß, hence preventing its extracellular aggregation, while ApoE4 decreased it, thus helping the buildup of extracellular plaques. The contrary effects of ApoE2 and ApoE4 remained once Aß aggregated: while ApoE2 reduced the uptake of Aß aggregates, ApoE4 facilitated it. Fibrillation studies also revealed ApoE4's tendency to form fibrillar aggregates. Our results uncover yet unknown details of ApoE cellular biology and deepen our molecular understanding of the ApoE-dependent mechanism of Aß pathology.

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species.

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin.

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.

Lipid and carotenoid cooperation-driven adaptation to light and temperature stress in Synechocystis sp. PCC6803.

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 was found considerably lower. Xanthophyll to ß-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.

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.

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 ß-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.

Label-free LC-MS/MS identification of phosphatidylglycerol-regulated proteins in Synechocystis sp. PCC6803.

We present a proteomics dataset combining SDS-PAGE prefractionation and data-dependent LC-MS/MS that enables the identification of phosphatidylglycerol-regulated proteins in the pgsA(-) mutant of Synechocystis sp. PCC6803, a cyanobacterium strain that grows with this indispensable phospholipid added exogenously. We searched the acquired raw data against a composite protein sequence database of Synechocystis using MASCOT, and employed Progenesis LC-MS software for label-free quantification based on extracted peptide intensities to detect changes in protein abundances upon phospholipid withdrawal. Protein identifications were validated using rigorous criteria, and our analysis of the dataset revealed 80 phosphatidylglycerol-regulated proteins involved in various cellular processes including photosynthesis, respiration, metabolism, transport, transcription, and translation. The data have been deposited to the ProteomeXchange with identifier PXD000363 (http://proteomecentral.proteomexchange.org/dataset/PXD000363).

Effects of lipids on the rate-limiting steps in the dark-to-light transition of Photosystem II core complex of Thermostichus vulcanus.

In our earlier works, we have shown that the rate-limiting steps, associated with the dark-to-light transition of Photosystem II (PSII), reflecting the photochemical activity and structural dynamics of the reaction center complex, depend largely on the lipidic environment of the protein matrix. Using chlorophyll-a fluorescence transients (ChlF) elicited by single-turnover saturating flashes, it was shown that the half-waiting time (Δτ 1/2) between consecutive excitations, at which 50% of the fluorescence increment was reached, was considerably larger in isolated PSII complexes of Thermostichus (T.) vulcanus than in the native thylakoid membrane (TM). Further, it was shown that the addition of a TM lipid extract shortened Δτ 1/2 of isolated PSII, indicating that at least a fraction of the 'missing' lipid molecules, replaced by detergent molecules, caused the elongation of Δτ 1/2. Here, we performed systematic experiments to obtain information on the nature of TM lipids that are capable of decreasing Δτ 1/2. Our data show that while all lipid species shorten Δτ 1/2, the negatively charged lipid phosphatidylglycerol appears to be the most efficient species - suggesting its prominent role in determining the structural dynamics of PSII reaction center.

Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone Q(B) 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.

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.

The zinc finger protein 3 of Arabidopsis thaliana regulates vegetative growth and root hair development.

Introduction: Zinc finger protein 3 (ZFP3) and closely related C2H2 zinc finger proteins have been identified as regulators of abscisic acid signals and photomorphogenic responses during germination. Whether ZFP3 and related ZFP factors regulate plant development is, however, not known. Results: ZFP3 overexpression reduced plant growth, limited cell expansion in leaves, and compromised root hair development. The T-DNA insertion zfp3 mutant and transgenic lines with silenced ZFP1, ZFP3, ZFP4, and ZFP7 genes were similar to wild-type plants or had only minor differences in plant growth and morphology, probably due to functional redundancy. RNAseq transcript profiling identified ZFP3-controlled gene sets, including targets of ABA signaling with reduced transcript abundance. The largest gene set that was downregulated by ZFP3 encoded regulatory and structural proteins in cell wall biogenesis, cell differentiation, and root hair formation. Chromatin immunoprecipitation confirmed ZFP3 binding to several target promoters. Discussion: Our results suggest that ZFP3 and related ZnF proteins can modulate cellular differentiation and plant vegetative development by regulating the expression of genes implicated in cell wall biogenesis.

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.

Identification of thylakoid membrane thermal transitions in Synechocystis sp. PCC6803 photosynthetic mutants.

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.

Salt Stress Induces Paramylon Accumulation and Fine-Tuning of the Macro-Organization of Thylakoid Membranes in Euglena gracilis Cells.

The effects of salt stress condition on the growth, morphology, photosynthetic performance, and paramylon content were examined in the mixotrophic, unicellular, flagellate Euglena gracilis. We found that salt stress negatively influenced cell growth, accompanied by a decrease in chlorophyll (Chl) content. Circular dichroism (CD) spectroscopy revealed the changes in the macro-organization of pigment-protein complexes due to salt treatment, while the small-angle neutron scattering (SANS) investigations suggested a reduction in the thylakoid stacking, an effect confirmed by the transmission electron microscopy (TEM). At the same time, the analysis of the thylakoid membrane complexes using native-polyacrylamide gel electrophoresis (PAGE) revealed no significant change in the composition of supercomplexes of the photosynthetic apparatus. Salt stress did not substantially affect the photosynthetic activity, as reflected by the fact that Chl fluorescence yield, electron transport rate (ETR), and energy transfer between the photosystems did not change considerably in the salt-grown cells. We have observed notable increases in the carotenoid-to-Chl ratio and the accumulation of paramylon in the salt-treated cells. We propose that the accumulation of storage polysaccharides and changes in the pigment composition and thylakoid membrane organization help the adaptation of E. gracilis cells to salt stress and contribute to the maintenance of cellular processes under stress conditions.

Lipid Polymorphism of the Subchloroplast-Granum and Stroma Thylakoid Membrane-Particles. II. Structure and Functions.

In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.

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.

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/DeltacdsA cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a suppression of O(2) 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. [(35)S]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.

A simple method to produce Synechocystis PCC6803 biofilm under laboratory conditions for electron microscopic and functional studies.

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacterium Synechocystis PCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to form Synechocystis PCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, the Synechocystis PCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies.