High myristic acid in glycerolipids enhances the repair of photodamaged photosystem II under strong light.

Cyanobacteria inhabit areas with a broad range of light, temperature, and nutrient conditions. The robustness of cyanobacterial cells, which can survive under different conditions, may depend on the resilience of photosynthetic activity. Cyanothece sp. PCC 8801 (Cyanothece), a freshwater cyanobacterium isolated from a Taiwanese rice field, had a higher repair activity of photodamaged photosystem II (PSII) under intense light than Synechocystis sp. PCC 6803 (Synechocystis), another freshwater cyanobacterium. Cyanothece contains myristic acid (14:0) as the major fatty acid at the sn-2 position of the glycerolipids. To investigate the role of 14:0 in the repair of photodamaged PSII, we used a Synechocystis transformant expressing a T-1274 encoding a lysophosphatidic acid acyltransferase (LPAAT) from Cyanothece. The wild-type and transformant cells contained 0.2 and 20.1 mol% of 14:0 in glycerolipids, respectively. The higher content of 14:0 in the transformants increased the fluidity of the thylakoid membrane. In the transformants, PSII repair was accelerated due to an enhancement in the de novo synthesis of D1 protein, and the production of singlet oxygen (1O2), which inhibited protein synthesis, was suppressed. The high content of 14:0 increased transfer of light energy received by phycobilisomes to PSI and CP47 in PSII and the content of carotenoids. These results indicated that an increase in 14:0 reduced 1O2 formation and enhanced PSII repair. The higher content of 14:0 in the glycerolipids may be required as a survival strategy for Cyanothece inhabiting a rice field under direct sunlight.

Biosynthesis of phosphatidylglycerol in photosynthetic organisms.

Phosphatidylglycerol (PG) is a unique phospholipid class with its indispensable role in photosynthesis and growth in land plants, algae, and cyanobacteria. PG is the only major phospholipid in the thylakoid membrane of cyanobacteria and plant chloroplasts and a main lipid component in photosynthetic protein-cofactor complexes such as photosystem I and photosystem II. In plants and algae, PG is also essential as a substrate for the biosynthesis of cardiolipin, which is a unique lipid present only in mitochondrial membranes and crucial for the functions of mitochondria. PG biosynthesis pathways in plants include three membranous organelles, plastids, mitochondria, and the endoplasmic reticulum in a complex manner. While the molecular biology underlying the role of PG in photosynthetic functions is well established, many enzymes responsible for the PG biosynthesis are only recently cloned and functionally characterized in the model plant species including Arabidopsis thaliana and Chlamydomonas reinhardtii and cyanobacteria such as Synechocystis sp. PCC 6803. The characterization of those enzymes helps understand not only the metabolic flow for PG production but also the crosstalk of biosynthesis pathways between PG and other lipids. This review aims to summarize recent advances in the understanding of the PG biosynthesis pathway and functions of involved enzymes.

Deacylation of galactolipids decomposes photosystem II dimers to enhance degradation of damaged D1 protein.

Photosystem II (PSII) contains many lipid molecules that are essential for the function and maintenance of PSII. Under strong light conditions, PSII complexes are dynamically modified during the repair process; however, the molecular mechanism of the dynamic changes in the PSII structure is still unclear. In the present study, we investigated the role of a lipase in the repair of PSII in Synechocystis sp. PCC 6803. We identified a protein encoded by the sll1969 gene, previously named lipase A (lipA), in the Synechocystis sp. PCC 6803 genome as a candidate for the lipase involved in PSII repair. Recombinant protein expressed in Escherichia coli cells hydrolyzed fatty acids at the sn-1 position of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol as well as triacylglycerol esterified with stearic acids. PSII repair in a disrupted mutant of the lipA gene was suppressed by the slow degradation of damaged D1 protein under strong light. The level of the PSII dimer remained higher in lipA mutant cells than wild-type (WT) cells under strong light. LipA protein was associated with the PSII dimer in vivo, and recombinant LipA protein decomposed PSII dimers purified from WT cells to monomers by reducing MGDG content in the PSII complex. These results indicate that LipA reacts with PSII dimers, dissociates them into monomers by digesting MGDG, and enhances D1 degradation during PSII repair.

Crucial importance of length of fatty-acyl chains bound to the sn-2 position of phosphatidylglycerol for growth and photosynthesis of Synechocystis sp. PCC 6803.

Phosphatidylglycerol (PG) in thylakoid membrane is essential for growth and photosynthesis of photosynthetic organisms. Although the sn-2 position of PG in thylakoid membrane is exclusively esterified with C16 fatty acids, the functional importance of the C16 fatty-acyl chains at the sn-2 position has not been clarified. In this study, we chemically synthesized non-metabolizable PG molecules: we introduced linoleic acid (18:2, fatty acid containing 18 carbons with 2 double bonds) and one of the saturated fatty acids with different chain length (12:0, 14:0, 16:0, 18:0 and 20:0) by ether linkage to the sn-1 and sn-2 positions, respectively. With the synthesized ether-linked PG molecules, we checked whether they could complement the growth and photosynthesis of pgsA mutant cells of Synechocystis sp. PCC 6803 to understand the importance of length of fatty chains at the sn-2 position of PG. The pgsA mutant is incapable of synthesizing PG, so it requires exogenous PG added to medium for growth. The growth rate and photosynthetic activity of mutant cells depended on the length of fatty chains: the PG molecular species binding 16:0 most effectively complemented the growth and photosynthesis of mutant cells, and other PG molecular species with fatty chains shorter or longer than 16:0 were less effective; especially, those binding 12:0 inhibited the growth and photosynthetic activity of the mutant cells. These data demonstrate that length of fatty chains bound to the sn-2 position of PG is critical for PG performance in growth and photosynthesis.

Specific Incorporation of Polyunsaturated Fatty Acids into the sn-2 Position of Phosphatidylglycerol Accelerates Photodamage to Photosystem II under Strong Light.

Free fatty acids (FFAs) are generated by the reaction of lipases with membrane lipids. Generated polyunsaturated fatty acids (PUFAs) containing more than two double bonds have toxic effects in photosynthetic organisms. In the present study, we examined the effect of exogenous FFAs in the growth medium on the activity of photosystem II (PSII) under strong light in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PUFAs but not monounsaturated fatty acids accelerated the rate of photodamage to PSII by inactivating electron transfer at the oxygen-evolving complex. Moreover, supplemented PUFAs were specifically incorporated into the sn-2 position of phosphatidylglycerol (PG), which usually contains C16 fatty acids at the sn-2 position in Synechocystis cells. The disruption of the gene for an acyl-ACP synthetase reduced the effect of PUFAs on the photoinhibition of PSII. Thus, the specific incorporation of PUFAs into PG molecules requires acyl-ACP synthetase and leads to an unstable PSII, thereby accelerating photodamage to PSII. Our results are a breakthrough into elucidating the molecular mechanism of the toxicity of PUFAs to photosynthetic organisms.

Dissection of the Mechanisms of Growth Inhibition Resulting from Loss of the PII Protein in the Cyanobacterium Synechococcus elongatus PCC 7942.

In cyanobacteria, the PII protein (the glnB gene product) regulates a number of proteins involved in nitrogen assimilation including PipX, the coactivator of the global nitrogen regulator protein NtcA. In Synechococcus elongatus PCC 7942, construction of a PII-less mutant retaining the wild-type pipX gene is difficult because of the toxicity of uncontrolled action of PipX and the other defect(s) resulting from the loss of PIIper se, but the nature of the PipX toxicity and the PipX-independent defect(s) remains unclear. Characterization of a PipX-less glnB mutant (PD4) in this study showed that the loss of PII increases the sensitivity of PSII to ammonium. Ammonium was shown to stimulate the formation of reactive oxygen species in the mutant cells. The ammonium-sensitive growth phenotype of PD4 was rescued by the addition of an antioxidant α-tocopherol, confirming that photo-oxidative damage was the major cause of the growth defect. A targeted PII mutant retaining wild-type pipX was successfully constructed from the wild-type S. elongatus strain (SPc) in the presence of α-tocopherol. The resulting mutant (PD1X) showed an unusual chlorophyll fluorescence profile, indicating extremely slow reduction and re-oxidation of QA, which was not observed in mutants defective in both glnB and pipX. These results showed that the aberrant action of uncontrolled PipX resulted in an impairment of the electron transport reactions in both the reducing and oxidizing sides of QA.

Membrane lipid remodeling is required for photosystem II function under low CO2.

Membrane lipid remodeling in plants and microalgae has a crucial role in their survival under nutrient-deficient conditions. Aquatic microalgae have low access to CO2 , an essential carbon source for photosynthetic assimilates; however, 70-90 mol% of their membrane lipids are sugar-derived lipids (glycolipids) such as monogalactosyldiacylglycerol (MGDG). In this study, we discovered a new system of membrane lipid remodeling responding to CO2 in Synechocystis sp. PCC 6803, a unicellular, freshwater cyanobacterium. As compared with higher CO2 (HC; 1% CO2 ), under ambient air (lower CO2 : LC), phosphatidylglycerol (PG) content was increased at the expense of MGDG content. To explore the biological significance of this alteration in content, we generated a transformant of Synechocystis sp. PCC 6803 overexpressing sll0545 gene encoding a putative phosphatidic acid phosphate (oxPAP), which produces diacylglycerol that is used for the synthesis of glycolipids, and examined the effect on membrane lipid remodeling and phototrophic growth responding to LC. Photosystem II (PSII) activity and growth rate were inhibited under LC in oxPAP cells. PG content was substantially reduced, and MGDG and sulfoquinovosyldiacylglycerol contents were increased in oxPAP cells as compared with control cells. These phenotypes in oxPAP cells were recovered under the HC condition or PG supplementation. Increased PG content may be required for proper functioning of PSII under LC conditions.

A START domain-containing protein is involved in the incorporation of ER-derived fatty acids into chloroplast glycolipids in Marchantia polymorpha.

The appropriate regulation of thylakoid lipid synthesis is essential for the function of chloroplasts. In plant cells, membrane lipids synthesized in the ER are utilized as a precursor for the synthesis of chloroplast glycolipids. This pathway is thought to be mediated by the transport of glycerolipids synthesized in the ER into chloroplasts. However, we have little knowledge about the proteins involved in the lipid transfer between these organelles in plant cells. Here we show a protein, STAR2, containing the START (Steroidogenic acute regulatory protein-related lipid transfer) domain known to function as a lipid transporter, is involved in the incorporation of ER-derived fatty acids into chloroplast glycolipids in Marchantia polymorpha. We found that STAR2 localizes on the chloroplast envelope membrane as a punctuate structure and is required for the increase of C20 fatty acids, which are synthesized in the ER, in chloroplast glycolipids in response to phosphate deprivation. Our results indicate that STAR2 of M. polymorpha is likely to be involved in the lipid transfer from ER to chloroplast, presumably as a lipid transporter.

Long-Chain Saturated Fatty Acids, Palmitic and Stearic Acids, Enhance the Repair of Photosystem II.

Free fatty acids (FFA) generated in cyanobacterial cells can be utilized for the biodiesel that is required for our sustainable future. The combination of FFA and strong light induces severe photoinhibition of photosystem II (PSII), which suppresses the production of FFA in cyanobacterial cells. In the present study, we examined the effects of exogenously added FFA on the photoinhibition of PSII in Synechocystis sp. PCC 6803. The addition of lauric acid (12:0) to cells accelerated the photoinhibition of PSII by inhibiting the repair of PSII and the de novo synthesis of D1. α-Linolenic acid (18:3) affected both the repair of and photodamage to PSII. Surprisingly, palmitic (16:0) and stearic acids (18:0) enhanced the repair of PSII by accelerating the de novo synthesis of D1 with the mitigation of the photoinhibition of PSII. Our results show chemical potential of FFA in the regulation of PSII without genetic manipulation.

Elevated Levels of Specific Carotenoids During Acclimation to Strong Light Protect the Repair of Photosystem II in Synechocystis sp. PCC 6803.

The tolerance of photosynthesis to strong light increases in photosynthetic organisms during acclimation to strong light. We investigated the role of carotenoids in the protection of photosystem II (PSII) from photoinhibition after acclimation to strong light in the cyanobacterium Synechocystis sp. PCC 6803. In cells that had been grown under strong light at 1,000 µmol photons m-2 s-1 (SL), specific carotenoids, namely, zeaxanthin, echinenone, and myxoxanthophyll, accumulated at high levels, and the photoinhibition of PSII was less marked than in cells that had been grown under standard growth light at 70 µmol photons m-2 s-1 (GL). The rate of photodamage to PSII, as monitored in the presence of lincomycin, did not differ between cells grown under SL and GL, suggesting that the mitigation of photoinhibition after acclimation to SL might be attributable to the enhanced ability to repair PSII. When cells grown under GL were transferred to SL, the mitigation of photoinhibition of PSII occurred in two distinct stages: a first stage that lasted 4 h and the second stage that occurred after 8 h. During the second stage, the accumulation of specific carotenoids was detected, together with enhanced synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, and suppression of the production of singlet oxygen (1O2). In the ΔcrtRΔcrtO mutant of Synechocystis, which lacks zeaxanthin, echinenone, and myxoxanthophyll, the mitigation of photoinhibition of PSII, the enhancement of protein synthesis, and the suppression of production of 1O2 were significantly impaired during the second stage of acclimation. Thus, elevated levels of the specific carotenoids during acclimation to strong light appeared to protect protein synthesis from 1O2, with the resultant mitigation of photoinhibition of PSII.

Light-inducible expression of translation factor EF-Tu during acclimation to strong light enhances the repair of photosystem II.

In photosynthetic organisms, the repair of photosystem II (PSII) is enhanced after acclimation to strong light, with the resultant mitigation of photoinhibition of PSII. We previously reported that oxidation of translation elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, depresses the repair of PSII in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we investigated the role of EF-Tu in the repair of PSII after acclimation of Synechocystis to strong light. In cells that had been grown under strong light, both the repair of PSII and the synthesis of proteins de novo were enhanced under strong light, with the resultant mitigation of photoinhibition of PSII. Moreover, levels of EF-Tu were elevated, whereas levels of other components of the translation machinery, such as translation factor EF-G and ribosomal proteins L2 and S12, did not change significantly. The expression of the gene for EF-Tu was induced by light, as monitored at the transcriptional level. Elevation of the level of EF-Tu was strongly correlated with the subsequent enhancement of PSII repair in cells that had been grown under light at various intensities. Furthermore, overexpression of EF-Tu in Synechocystis enhanced protein synthesis and PSII repair under strong light, even after cell culture under nonacclimating conditions. These observations suggest that elevation of the level of EF-Tu might be a critical factor in enhancing the capacity for repair of PSII that develops during acclimation to strong light.

Oxidation of Translation Factor EF-Tu Inhibits the Repair of Photosystem II.

The repair of photosystem II (PSII) is particularly sensitive to oxidative stress and the inhibition of repair is associated with oxidative damage to the translational elongation system in the cyanobacterium Synechocystis sp. PCC 6803. However, the molecular mechanisms underlying this inhibition are unknown. We previously demonstrated in vitro that EF-Tu, a translation factor that delivers aminoacyl-tRNA to the ribosome, is inactivated by reactive oxygen species via oxidation of the Cys residue Cys-82. In this study, we examined the physiological role of the oxidation of EF-Tu in Synechocystis Under strong light, EF-Tu was rapidly oxidized to yield oxidized monomers in vivo. We generated a Synechocystis transformant that expressed mutated EF-Tu in which Cys-82 had been replaced with a Ser residue. Under strong light, the de novo synthesis of proteins that are required for PSII repair, such as D1, was enhanced in the transformant and photoinhibition of PSII was alleviated. However, photodamage to PSII, measured in the presence of lincomycin, was similar between the transformant and wild-type cells, suggesting that expression of mutated EF-Tu might enhance the repair of PSII. Alleviating photoinhibition through mutation of EF-Tu did not alter cell growth under strong light, perhaps due to the enhanced production of reactive oxygen species. These observations suggest that the oxidation of EF-Tu under strong light inhibits PSII repair, resulting in the stimulation of photoinhibition.

Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803.

Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

Zeaxanthin and Echinenone Protect the Repair of Photosystem II from Inhibition by Singlet Oxygen in Synechocystis sp. PCC 6803.

Carotenoids are important components of antioxidative systems in photosynthetic organisms. We investigated the roles of zeaxanthin and echinenone in the protection of PSII from photoinhibition in Synechocystis sp. PCC 6803, using mutants of the cyanobacterium that lack these carotenoids. The activity of PSII in mutant cells deficient in either zeaxanthin or echinenone was more sensitive to strong light than the activity in wild-type cells, and the activity in mutant cells deficient in both carotenoids was hypersensitive to strong light, indicating that the absence of these carotenoids increased the extent of photoinhibition. Nonetheless, the rate of photodamage to PSII, as measured in the presence of chloramphenicol, which blocks the repair of PSII, was unaffected by the absence of either carotenoid, suggesting that these carotenoids might act by protecting the repair of PSII. Knockout of the gene for the so-called orange carotenoid protein (OCP), in which the 3'-hydroxyechinenone cofactor, a derivative of echinenone, is responsible for the thermal dissipation of excitation energy, increased the extent of photoinhibition but did not affect photodamage, suggesting that thermal dissipation also protects the repair of PSII. In mutant cells lacking OCP, as well as those lacking zeaxanthin and echinenone, the production of singlet oxygen was stimulated and the synthesis de novo of various proteins, including the D1 protein, was markedly suppressed under strong light. These observations suggest that the carotenoids and thermal dissipation might protect the repair of photodamaged PSII by depressing the levels of singlet oxygen that inhibits protein synthesis.

Expression of a highly active catalase VktA in the cyanobacterium Synechococcus elongatus PCC 7942 alleviates the photoinhibition of photosystem II.

The repair of photosystem II (PSII) after photodamage is particularly sensitive to reactive oxygen species-such as H2O2, which is abundantly produced during the photoinhibition of PSII. In the present study, we generated a transformant of the cyanobacterium Synechococcus elongatus PCC 7942 that expressed a highly active catalase, VktA, which is derived from a facultatively psychrophilic bacterium Vibrio rumoiensis, and examined the effect of expression of VktA on the photoinhibition of PSII. The activity of PSII in transformed cells declined much more slowly than in wild-type cells when cells were exposed to strong light in the presence of H2O2. However, the rate of photodamage to PSII, as monitored in the presence of chloramphenicol, was the same in the two lines of cells, suggesting that the repair of PSII was protected by the expression of VktA. The de novo synthesis of the D1 protein, which is required for the repair of PSII, was activated in transformed cells under the same stress conditions. Similar protection of the repair of PSII in transformed cells was also observed under strong light at a relatively low temperature. Thus, the expression of the highly active catalase mitigates photoinhibition of PSII by protecting protein synthesis against damage by H2O2 with subsequent enhancement of the repair of PSII.