Gene expression and organization of thylakoid protein complexes in the PSII-less mutant of Synechocystis sp. PCC 6803.

Photosystems I and II (PSI and PSII) are the integral components of the photosynthetic electron transport chain that utilize light to provide chemical energy for CO2 fixation. In this study, we investigated how the deficiency of PSII affects the gene expression, accumulation, and organization of thylakoid protein complexes as well as physiological characteristics of Synechocystis sp. PCC 6803 by combining biochemical, biophysical, and transcriptomic approaches. RNA-seq analysis showed upregulated expression of genes encoding the PSII core proteins, and downregulation of genes associated with interaction between light-harvesting phycobilisomes and PSI. Two-dimensional separation of thylakoid protein complexes confirmed the lack of PSII complexes, yet unassembled PSII subunits were detected. The content of PsaB representing PSI was lower, while the content of cytochrome b6f complexes was higher in the PSII-less strain as compared with control (CS). Application of oxygraph measurements revealed higher rates of dark respiration and lower PSI activity in the mutant. The latter likely resulted from the detected decrease in the accumulation of PSI, PSI monomerization, increased proportion of energetically decoupled phycobilisomes in PSII-less cultures, and low abundance of phycocyanin. Merging the functional consequences of PSII depletion with differential protein and transcript accumulation in the mutant, in comparison to CS, identified signal transduction from the photosynthetic apparatus to the genome level.

Psb28 is involved in recovery of photosystem II at high temperature in Synechocystis sp. PCC 6803.

Psb28 is an extrinsic protein of photosystem II (PSII), which is conserved among photosynthetic organisms from cyanobacteria to higher plants. A unicellular cyanobacterium, Synechocystis sp. PCC 6803, has two homologs of Psb28, Psb28-1 and Psb28-2. However, the role of these proteins remains poorly understood. In this study, we disrupted the psb28-1 (sll1398) and psb28-2 (slr1739) genes in wild-type Synechocystis sp. PCC 6803 and examined their photosynthetic properties to elucidate the physiological role of Psb28 in photosynthesis. We also disrupted the psb28-1 gene in a dgdA mutant defective in the biosynthesis of digalactosyldiacylglycerol, in which Psb28-1 significantly accumulates in PSII. The disruption of the psb28-1 gene in the wild-type resulted in growth retardation under high-light conditions at high temperatures with a low rate of restoration of photodamaged photosynthetic machinery. Similar phenomena were observed at normal growth temperatures in the psb28-1/dgdA double mutant. In contrast, disruption of psb28-2 in the wild-type and dgdA mutant did not affect host strain phenotype, suggesting that Psb28-2 does not contribute to the recovery of PSII. In addition, protein analysis using strains expressing His-tagged Psb28-1 revealed that Psb28-1 is mainly associated with the CP43-less PSII monomer. In the dgdA mutant, the CP43-less PSII monomer accumulated to a greater extent than in the wild-type, and its accumulation caused greater accumulation of Psb28-1 in PSII. These results demonstrate that Psb28-1 plays an important role in PSII repair through association with the CP43-less monomer, particularly at high temperatures.

Positive regulation of psbA gene expression by cis-encoded antisense RNAs in Synechocystis sp. PCC 6803.

The D1 protein of photosystem II in the thylakoid membrane of photosynthetic organisms is encoded by psbA genes, which in cyanobacteria occur in the form of a small gene family. Light-dependent up-regulation of psbA gene expression is crucial to ensure the proper replacement of the D1 protein. To gain a high level of gene expression, psbA transcription can be enhanced by several orders of magnitude. Recent transcriptome analyses demonstrated a high number of cis-encoded antisense RNAs (asRNAs) in bacteria, but very little is known about their possible functions. Here, we show the presence of two cis-encoded asRNAs (PsbA2R and PsbA3R) of psbA2 and psbA3 from Synechocystis sp. PCC 6803. These asRNAs are located in the 5' untranslated region of psbA2 and psbA3 genes. Their expression becomes up-regulated by light and down-regulated by darkness, similar to their target mRNAs. In the PsbA2R-suppressing strain [PsbA2R(-)], the amount of psbA2 mRNA was only about 50% compared with the control strain. Likewise, we identified a 15% lowered activity of photosystem II and a reduced amount of the D1 protein in PsbA2R(-) compared with the control strain. The function of PsbA2R in the stabilization of psbA2 mRNA was shown from in vitro RNase E assay when the AU box and the ribosome-binding site in the 5' untranslated region of psbA2 mRNA were both covered by PsbA2R. These results add another layer of complexity to the mechanisms that contribute to psbA gene expression and show PsbA2R as a positively acting factor to achieve a maximum level of D1 synthesis.

The antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply.

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (C(i)), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the Q(B) site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by C(i) limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in C(i) conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon.

Strategies for psbA gene expression in cyanobacteria, green algae and higher plants: from transcription to PSII repair.

The Photosystem (PS) II of cyanobacteria, green algae and higher plants is prone to light-induced inactivation, the D1 protein being the primary target of such damage. As a consequence, the D1 protein, encoded by the psbA gene, is degraded and re-synthesized in a multistep process called PSII repair cycle. In cyanobacteria, a small gene family codes for the various, functionally distinct D1 isoforms. In these organisms, the regulation of the psbA gene expression occurs mainly at the level of transcription, but the expression is fine-tuned by regulation of translation elongation. In plants and green algae, the D1 protein is encoded by a single psbA gene located in the chloroplast genome. In chloroplasts of Chlamydomonas reinhardtii the psbA gene expression is strongly regulated by mRNA processing, and particularly at the level of translation initiation. In chloroplasts of higher plants, translation elongation is the prevalent mechanism for regulation of the psbA gene expression. The pre-existing pool of psbA transcripts forms translation initiation complexes in plant chloroplasts even in darkness, while the D1 synthesis can be completed only in the light. Replacement of damaged D1 protein requires also the assistance by a number of auxiliary proteins, which are encoded by the nuclear genome in green algae and higher plants. Nevertheless, many of these chaperones are conserved between prokaryotes and eukaryotes. Here, we describe the specific features and fundamental differences of the psbA gene expression and the regeneration of the PSII reaction center protein D1 in cyanobacteria, green algae and higher plants. This article is part of a Special Issue entitled Photosystem II.

Isolation and characterization of a barley yellow stripe-like gene, HvYSL5.

Yellow stripe-like (YSL) family transporters, belonging to a novel subfamily of oligopeptide transporter (OPT), has been proposed to be involved in metal uptake and long-distance transport, but only a few of them have been functionally characterized so far. In the present study, we isolated an uncharacterized member of the YSL family, HvYSL5, in barley based on expressed sequence tag (EST) information. HvYSL5 shared 50% identity with HvYS1, a transporter for the ferric-mugineic acid complex, at the amino acid level. Promoter analysis showed that the HvYSL5 upstream sequence contains both iron deficiency response element 1 and 2 (IDE1 and 2). HvYSL5 was expressed in the roots and the expression was greatly induced by Fe deficiency, but not by deficiency of other metals including Zn, Cu and Mn. Spatial investigation showed that much higher expression of HvYSL5 was found in the mature zones of the roots, but not in the root tips. Furthermore, the expression showed a diurnal rhythm, being the highest in the morning, but with no expression in the afternoon. HvYSL5 was localized in all root cells, and subcellular localization analysis showed that HvYSL5 is likely to be localized in the vesicles. Knockdown of HvYSL5 did not result in any detectable phenotype changes. Although the exact role of HvYSL5 remains to be examined, our results suggest that it is involved in the transient storage of Fe or phytosiderophores.

Increased expression of peroxisome proliferator-activated receptor (PPAR)-alpha and PPAR-gamma in human atherosclerosis.

To clarify the mechanism of atherosclerosis development in humans, we studied the mRNA and protein expression of PPAR subtypes in various types of atherosclerotic lesions and their correlation with cell proliferation and macrophage invasion. Human aortas were obtained from 35 autopsied cases, and each sample was divided into halves. One half was used for the analysis of mRNA or protein expression with RT-PCR or Western blotting, respectively. The other was microscopically classified into atheromatous plaque (AP), fatty streak (FS), and diffuse intimal thickening (DIT), and was analyzed immunohistochemically. The mRNA levels of both PPARs increased significantly in atherosclerosis and tended to increase in proportion to the severity of the lesion, and the expression of PPAR-alpha correlated with that of PPAR-gamma in FS and AP. The PPAR-gamma protein increased in AP. Monocytes/macrophages, as well as endothelial and smooth muscle cells, expressed the PPAR-gamma protein in plaques. This expression in the DIT was noted mainly in macrophages but was not correlated with the density of macrophages, suggesting that only certain macrophages express the PPARs in DIT. Cell proliferation did not correlate with PPARs expression in any lesion type. These findings suggest that PPARs may be associated with atheromatous plaque formation, and that PPAR-gamma may be involved in the early stages of human atherosclerosis.

The PsbK subunit is required for the stable assembly and stability of other small subunits in the PSII complex in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

PsbK is a small membrane protein of the PSII core complex and is highly conserved from cyanobacteria to plants. Here, we studied its role in the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, by focusing on a psbK disruptant with hexahistidine-tagged CP47. The psbK disruptant showed photoautotrophic growth comparable with that of the wild type under a wide range of light conditions. The mutant PSII complex retained the oxygen-evolving activity with a unique modification of the acceptor Q(B) site. N-terminal sequencing revealed that Ycf12 and PsbZ proteins were lost in the PSII complex prepared from the mutant. Immunoblotting detected reduced accumulation of PsbZ in the mutant thylakoid. These results suggest that PsbK is required not only for association of PsbZ and Ycf12 with the isolated PSII complex but also for the stabilization of PsbZ in the thylakoid membrane.

Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits.

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni2+-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b(6)f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.

Involvement of digalactosyldiacylglycerol in cellular thermotolerance in Synechocystis sp. PCC 6803.

The effects of digalactosyldiacylglycerol (DGDG) deficiency on photosynthesis at high temperatures were examined using a dgdA mutant of Synechocystis sp. PCC 6803 incapable of DGDG biosynthesis. The dgdA mutant cells showed significant growth retardation when the temperature was increased from 30 to 38 degrees C, although wild-type cells grew normally. The degree of growth retardation was enhanced by increasing light intensity. In addition, dgdA mutant cells showed increased sensitivity to the photoinhibition of photosynthesis when illuminated at 38 degrees C. Analysis of photosynthesis in intact cells suggested that the inhibition of repair processes and accelerated photodamage resulted in growth retardation in dgdA mutant cells at high temperatures.

Lack of digalactosyldiacylglycerol increases the sensitivity of Synechocystis sp. PCC 6803 to high light stress.

The physiological role of digalactosyldiacylglycerol (DGDG) in photosynthesis was examined using a dgdA mutant of Synechocystis sp. PCC 6803 that is defective in the biosynthesis of DGDG. The dgdA mutant cells showed normal growth under low light (LL) conditions. However, their growth was retarded under high light (HL) conditions and under Ca(2+)- and/or Cl(-)-limited conditions compared to wild-type cells. The retardation in growth of the mutant cells was recovered by exogenous supply of DGDG in the growth medium. The dgdA mutant showed increased sensitivity to photoinhibition. Although both photodamage and repair processes of photosynthesis were affected, the repair process was more severely affected than the photodamage process, suggesting that DGDG plays an important role in the photosynthetic repair cycle.

Effects of phospholipase and lipase treatments on photosystem II core dimer from a thermophilic cyanobacterium.

Lipids are important components of transmembrane protein complexes. In order to study the roles of lipids in photosystem II (PSII), we treated the PSII core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus with phospholipase A(2) (PLA(2)) and lipase, and examined their effects on PSII structure and function. PLA(2)-treatment decreased the content of phospholipid, phosphatidylglycerol (PG) by 59%, leading to a decrease of oxygen evolution by 40%. On the other hand, although treatment with lipase specifically decreased the content of monogalactosyldiacylglycerol (MGDG) by 52%, it decreased oxygen evolution only by 16%. This indicates that PG plays a more important role in PSII than MGDG. Both PLA(2)- and lipase-treatments induced neither the dissociation of PSII dimer, nor any loss of polypeptides. The degradation of PG resulted in a damage to the Q(B)-binding site as demonstrated from photoreduction activity of 2,6-dichlorophenolindophenol and chlorophyll fluorescence yields in the absence or presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and the dependencies of oxygen evolution on various electron acceptors before and after PLA(2)- or lipase-treatments. However, there were approximately three and five molecules of PG and MGDG per PSII reaction center left in the PSII dimeric complex after the PLA(2)- and lipase-treatments. These lipids are therefore bound to the interior of the protein matrix and resistant to the lipase treatments. The resistance of these lipids against PLA(2)- and lipase-treatments may be a specific feature of PSII from the thermophilic cyanobacterium, suggesting a possible correlation between binding of lipids and thermostability of PSII.

A method for analyzing lipid-modified proteins with mass spectrometry.

In protein analysis using mass spectrometry, proteins are usually separated by electrophoresis and digested within the gel with proteases such as trypsin. However, analysis of lipid-modified proteins is difficult due to the low recovery of lipid-modified peptide fragments from the gel as well as their low ionization efficiency during mass spectrometry. In this study, we developed a simple extraction method with n-dodecyl-beta-D-maltoside following chloroform/methanol extraction that efficiently elutes lipid-modified fragments from gels. This method allowed us to analyze the structure of lipid-modified fragments, suggesting the applicability of the method for analysis of lipid-modified fragments by mass spectrometry.

Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II.

The galactolipid digalactosyldiacylglycerol (DGDG) is present in the thylakoid membranes of oxygenic photosynthetic organisms such as higher plants and cyanobacteria. Recent x-ray crystallographic analysis of protein-cofactor supercomplexes in thylakoid membranes revealed that DGDG molecules are present in the photosystem II (PSII) complex (four molecules per monomer), suggesting that DGDG molecules play important roles in folding and assembly of subunits in the PSII complex. However, the specific role of DGDG in PSII has not been fully clarified. In this study, we identified the dgdA gene (slr1508, a ycf82 homolog) of Synechocystis sp. PCC6803 that presumably encodes a DGDG synthase involved in the biosynthesis of DGDG by comparison of genomic sequence data. Disruption of the dgdA gene resulted in a mutant defective in DGDG synthesis. Despite the lack of DGDG, the mutant cells grew as rapidly as the wild-type cells, indicating that DGDG is not essential for growth in Synechocystis. However, we found that oxygen-evolving activity of PSII was significantly decreased in the mutant. Analyses of the PSII complex purified from the mutant cells indicated that the extrinsic proteins PsbU, PsbV, and PsbO, which stabilize the oxygen-evolving complex, were substantially dissociated from the PSII complex. In addition, we found that heat susceptibility but not dark-induced inactivation of oxygen-evolving activity was notably increased in the mutant cells in comparison to the wild-type cells, suggesting that the PsbU subunit is dissociated from the PSII complex even in vivo. These results demonstrate that DGDG plays important roles in PSII through the binding of extrinsic proteins required for stabilization of the oxygen-evolving complex.

Effects of the lack of phosphatidylglycerol on the donor side of photosystem II.

Our previous studies with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 (hereafter termed pgsA mutant), which is defective for the biosynthesis of phosphatidylglycerol (PG), revealed an important role for PG in the electron acceptor side of photosystem II (PSII), especially in the electron transport between plastoquinones Q(A) and Q(B). This study now shows that PG also plays an important role in the electron donor side of PSII, namely, the oxygen-evolving system. Analyses of purified PSII complexes indicated that PSII from PG-depleted pgsA mutant cells sustained only approximately 50% of the oxygen-evolving activity compared to wild-type cells. Dissociation of the extrinsic proteins PsbO, PsbV, and PsbU, which are required for stabilization of the manganese (Mn) cluster, followed by the release of a Mn atom, was observed in PSII of the PG-depleted mutant cells. The released PsbO rebound to PSII when PG was added back to the PG-depleted mutant cells, even when de novo protein synthesis was inhibited. Changes in photosynthetic activity of the PG-depleted pgsA mutant cells induced by heat treatment or dark incubation resembled those of DeltapsbO, DeltapsbV, and DeltapsbU mutant cells. These results suggest that PG plays an important role in binding extrinsic proteins required for sustaining a functional Mn cluster on the donor side of PSII.

Lipids in oxygen-evolving photosystem II complexes of cyanobacteria and higher plants.

Lipids in dimeric photosystem II complexes prepared from two species of cyanobacteria, Thermosynechococcus vulcanus and Synechocystis sp. PCC6803, and two higher plants, spinach and rice, were analyzed to determine how many lipid molecules and what class of lipids are present in the photosystem II complexes. It was estimated that 27, 20, 8, and 7 lipid molecules per monomer are bound to the dimeric photosystem II complexes of T. vulcanus, Synechocystis, spinach, and rice, respectively. In each of the organisms, the lipid composition of the photosystem II complexes was quite different from that of the thylakoid membranes used for preparation of the complexes. The content of phosphatidylglycerol in the photosystem II complexes of each organism was much higher than that in the thylakoid membranes. Phospholipase A2 treatment of the photosystem II complexes of Synechocystis that degraded phosphatidylglycerol resulted in impairment of QB-mediated but not QA-mediated electron transport. These findings suggest that phosphatidylglycerol plays important roles in the electron transport at the QB-binding site in photosystem II complexes.

Identification of an Arabidopsis thaliana gene for cardiolipin synthase located in mitochondria.

Cardiolipin (CL) is an anionic phospholipid with a dimeric structure. In eukaryotes, it is primarily localized in the inner membranes of mitochondria. Although the biosynthetic pathway of CL is well known, the gene for CL synthase has not been identified in any higher organisms. In this study, the CLS gene for a CL synthase has been identified in a higher plant, Arabidopsis thaliana. We have shown that the CLS gene encodes a CL synthase by demonstrating its ability to catalyze the reaction of CL synthesis from CDP-diacylglycerol and phosphatidylglycerol, and that CLS is targeted into mitochondria. These findings demonstrate that CLS is a CL synthase located in mitochondria.

Requirement of carotene isomerization for the assembly of photosystem II in Synechocystis sp. PCC 6803.

Carotene isomerase mutant (crtH mutant) cells of Synechocystis sp. PCC 6803 can accumulate beta-carotene under light conditions. However, the mutant cells grown under a light-activated heterotrophic growth condition contained detectable levels of neither beta-carotene nor D1 protein of the photosystem (PS) II reaction center, and no oxygen-evolving activity of PSII was detected. beta-Carotene and D1 protein appeared and a high level of PSII activity was detected after the cells were transferred to a continuous light condition. The PSI activities of thylakoid membranes from mutant cells were almost the same as those of thylakoid membranes from wild-type cells, both before and after transfer to the continuous light condition. These results suggest that beta-carotene is required for the assembly of PSII but not for that of PSI.

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.

Requirement of phosphatidylglycerol for maintenance of photosynthetic machinery.

Phosphatidylglycerol (PG) is a ubiquitous component of thylakoid membranes. Experiments with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 defective in biosynthesis of PG have demonstrated an indispensable role of PG in photosynthesis. In the present study, we have investigated the light susceptibility of the pgsA mutant with regard to the maintenance of the photosynthetic machinery. Growth of the mutant cells without PG increased the light susceptibility of the cells and resulted in severe photoinhibition of photosynthesis upon a high-light treatment, whereas the growth in the presence of PG was protected against photoinhibition. Photoinhibition induced by PG deprivation was mainly caused by an impairment of the restoration process. The primary target of the light-induced damage in thylakoid membranes, the D1 protein of photosystem (PS) II was, however, synthesized and degraded with similar rates irrespective of whether the mutant cells were incubated with PG or not. Intriguingly, it was found that instead of the synthesis of the D1 protein, the dimerization of the PSII core monomers was impaired in the PG-deprived mutant cells. Addition of PG to photoinhibited cells restored the dimerization capacity of PSII core monomers. These results suggest that PG plays an important role in the maintenance of the photosynthetic machinery through the dimerization and reactivation of the PSII core complex.