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.

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.

Site-directed mutagenesis of two amino acid residues in cytochrome b559 α subunit that interact with a phosphatidylglycerol molecule (PG772) induces quinone-dependent inhibition of photosystem II activity.

X-ray crystallographic analysis (1.9-Å resolution) of the cyanobacterial photosystem II (PSII) dimer showed the presence of five phosphatidylglycerol (PG) molecules per reaction center. One of the PG molecules, PG772, is located in the vicinity of the QB-binding site. To investigate the role of PG772 in PSII, we performed site-directed mutagenesis in the cytochrome (Cyt) b559 α subunit of Synechocystis sp. PCC 6803 to change two amino acids, Thr-5 and Ser-11, which interact with PG772. The photosynthetic activity of intact cells was slightly lower in all mutants than that of cells in the control strain; however, the oxygen-evolving PSII activity was decreased markedly in cells of mutants, as measured using artificial quinones (such as p-benzoquinone). Furthermore, electron transport from QA to QB was inhibited in mutants incubated with quinones, particularly under high-intensity light conditions. Lipid analysis of purified PSII showed approximately one PG molecule per reaction center, presumably PG772, was lost in the PSII dimer from the T5A and S11A mutants compared with that in the PSII dimer from the control strain. In addition, protein analysis of monomer and dimer showed decreased levels of PsbV and PsbU extrinsic proteins in the PSII monomer purified from T5A and S11A mutants. These results suggest that site-directed mutagenesis of Thr-5 and Ser-11, which presumably causes the loss of PG772, induces quinone-dependent inhibition of PSII activity under high-intensity light conditions and destabilizes the binding of extrinsic proteins to PSII.

Thylakoid membrane lipid sulfoquinovosyl-diacylglycerol (SQDG) is required for full functioning of photosystem II in Thermosynechococcus elongatus.

Sulfoquinovosyl-diacylglycerol (SQDG) is one of the four lipids present in the thylakoid membranes. Depletion of SQDG causes different degrees of effects on photosynthetic growth and activities in different organisms. Four SQDG molecules bind to each monomer of photosystem II (PSII), but their role in PSII function has not been characterized in detail, and no PSII structure without SQDG has been reported. We analyzed the activities of PSII from an SQDG-deficient mutant of the cyanobacterium Thermosynechococcus elongatus by various spectroscopic methods, which showed that depletion of SQDG partially impaired the PSII activity by impairing secondary quinone (QB) exchange at the acceptor site. We further solved the crystal structure of the PSII dimer from the SQDG deletion mutant at 2.1 Å resolution and found that all of the four SQDG-binding sites were occupied by other lipids, most likely PG molecules. Replacement of SQDG at a site near the head of QB provides a possible explanation for the QB impairment. The replacement of two SQDGs located at the monomer-monomer interface by other lipids decreased the stability of the PSII dimer, resulting in an increase in the amount of PSII monomer in the mutant. The present results thus suggest that although SQDG binding in all of the PSII-binding sites is necessary to fully maintain the activity and stability of PSII, replacement of SQDG by other lipids can partially compensate for their functions.

High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase.

Analysis of fatty acids from the cyanobacterium Cyanothece sp. PCC 8801 revealed that this species contained high levels of myristic acid (14:0) and linoleic acid in its glycerolipids, with minor contributions from palmitic acid (16:0), stearic acid, and oleic acid. The level of 14:0 relative to total fatty acids reached nearly 50%. This 14:0 fatty acid was esterified primarily to the sn-2 position of the glycerol moiety of glycerolipids. This characteristic is unique because, in most of the cyanobacterial strains, the sn-2 position is esterified exclusively with C16 fatty acids, generally 16:0. Transformation of Synechocystis sp. PCC 6803 with the PCC8801_1274 gene for lysophosphatidic acid acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) from Cyanothece sp. PCC 8801 increased the level of 14:0 from 2% to 17% in total lipids and the increase in the 14:0 content was observed in all lipid classes. These findings suggest that the high content of 14:0 in Cyanothece sp. PCC 8801 might be a result of the high specificity of this acyltransferase toward the 14:0-acyl-carrier protein.

Specific Distribution of Phosphatidylglycerol to Photosystem Complexes in the Thylakoid Membrane.

The thylakoid membrane is the site of photochemical and electron transport reactions of oxygenic photosynthesis. The lipid composition of the thylakoid membrane, with two galactolipids, one sulfolipid, and one phospholipid, is highly conserved among oxygenic photosynthetic organisms. Besides providing a lipid bilayer matrix, thylakoid lipids are integrated in photosynthetic complexes particularly in photosystems I and II and play important roles in electron transport processes. Thylakoid lipids are differentially allocated to photosynthetic complexes and the lipid bilayer fraction, but distribution of each lipid in the thylakoid membrane is unclear. In this study, based on published crystallographic and biochemical data, we estimated the proportions of photosynthetic complex-associated and bilayer-associated lipids in thylakoid membranes of cyanobacteria and plants. The data suggest that ∼30 mol% of phosphatidylglycerol (PG), the only major phospholipid in thylakoid membranes, is allocated to photosystem complexes, whereas glycolipids are mostly distributed to the lipid bilayer fraction and constitute the membrane lipid matrix. Because PG is essential for the structure and function of both photosystems, PG buried in these complexes might have been selectively conserved among oxygenic phototrophs. The specific and substantial allocation of PG to the deep sites of photosystems may need a unique mechanism to incorporate PG into the complexes possibly in coordination with the synthesis of photosynthetic proteins and pigments.

Sulfoquinovosyldiacylglycerol has an Essential Role in Thermosynechococcus elongatus BP-1 Under Phosphate-Deficient Conditions.

Anionic lipids, sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), are major classes of the thylakoid membrane lipids in cyanobacteria and plant chloroplasts. PG is essential for growth and photosynthesis of cyanobacteria, algae and plants, but the requirement for SQDG differs even among cyanobacterial species. Although SQDG and PG can compensate each other in part presumably to maintain proper balance of anionic charge in lipid bilayers, the functional relationship of these lipids is largely unknown. In this study, we inactivated the sqdB gene, encoding a UDP-sulfoquinovose synthase and involved in SQDG biosynthesis, in Thermosynechococcus elongatus BP-1. In wild-type cells, PG accounted for only approximately 3.5 mol% of total membrane lipids, but its content was substantially increased along with complete loss of SQDG in the sqdB mutant. Under phosphate (Pi)-sufficient conditions, the growth rate and PSII activity were slightly lower in sqdB than in wild-type cells. In addition, the formation of PSI trimers and PSII dimers and energy transfer in phycobilisomes were perturbed in the mutant. Under Pi-deficient conditions, the growth of sqdB cells was severely impaired, with a decrease in PSII activity. PG supplementation could partially rescue the defective growth and PSII activity of Pi-deficient sqdB cells but fully recovered the impaired growth of the pgsA mutant of T. elongatus, which is deficient in PG biosynthesis. These data suggest that SQDG has a specific role in the growth and photosynthesis of T. elongatus, which cannot be complemented by PG, particularly under Pi-deficient conditions.

Multiple Impacts of Loss of Plastidic Phosphatidylglycerol Biosynthesis on Photosynthesis during Seedling Growth of Arabidopsis.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane in cyanobacteria and plant chloroplasts. Although PG accounts only for ~10% of total thylakoid lipids, it plays indispensable roles in oxygenic photosynthesis. In contrast to the comprehensive analyses of PG-deprived mutants in cyanobacteria, in vivo roles of PG in photosynthesis during plant growth remain elusive. In this study, we characterized the photosynthesis of an Arabidopsis thaliana T-DNA insertional mutant (pgp1-2), which lacks plastidic PG biosynthesis. In the pgp1-2 mutant, energy transfer from antenna pigments to the photosystem II (PSII) reaction center was severely impaired, which resulted in low photochemical efficiency of PSII. Unlike in the wild type, in pgp1-2, the PSII complexes were susceptible to photodamage by red light irradiation. Manganese ions were mostly dissociated from protein systems in pgp1-2, with oxygen-evolving activity of PSII absent in the mutant thylakoids. The oxygen-evolving complex may be disrupted in pgp1-2, which may accelerate the photodamage to PSII by red light. On the acceptor side of the mutant PSII, decreased electron-accepting capacity was observed along with impaired electron transfer. Although the reaction center of PSI was relatively active in pgp1-2 compared to the severe impairment in PSII, the cyclic electron transport was dysfunctional. Chlorophyll fluorescence analysis at 77K revealed that PG may not be needed for the self-organization of the macromolecular protein network in grana thylakoids but is essential for the assembly of antenna-reaction center complexes. Our data clearly show that thylakoid glycolipids cannot substitute for the role of PG in photosynthesis during plant growth.

Roles of Lipids in Photosynthesis.

Thylakoid membranes in cyanobacterial cells and chloroplasts of algae and higher plants are the sites of oxygenic photosynthesis. The lipid composition of the thylakoid membrane is unique and highly conserved among oxygenic photosynthetic organisms. Major lipids in thylakoid membranes are glycolipids, monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol, and the phospholipid, phosphatidylglycerol. The identification of almost all genes involved in the biosynthesis of each lipid class over the past decade has allowed the generation and isolation of mutants of various photosynthetic organisms incapable of synthesizing specific lipids. Numerous studies using such mutants have revealed that these lipids play important roles not only in the formation of the lipid bilayers of thylakoid membranes but also in the folding and assembly of the protein subunits in photosynthetic complexes. In addition to the studies with the mutants, recent X-ray crystallography studies of photosynthetic complexes in thylakoid membranes have also provided critical information on the association of lipids with photosynthetic complexes and their activities. In this chapter, we summarize our current understanding about the structural and functional involvement of thylakoid lipids in oxygenic photosynthesis.

Characterization of Chlamydomonas reinhardtii phosphatidylglycerophosphate synthase in Synechocystis sp. PCC 6803.

Phosphatidylglycerol (PG) is an indispensable phospholipid class with photosynthetic function in plants and cyanobacteria. However, its biosynthesis in eukaryotic green microalgae is poorly studied. Here, we report the isolation and characterization of two homologs (CrPGP1 and CrPGP2) of phosphatidylglycerophosphate synthase (PGPS), the rate-limiting enzyme in PG biosynthesis, in Chlamydomonas reinhardtii. Heterologous complementation of Synechocystis sp. PCC 6803 pgsA mutant by CrPGP1 and CrPGP2 rescued the PG-dependent growth phenotype, but the PG level and its fatty acid composition were not fully rescued in the complemented strains. As well, oxygen evolution activity was not fully recovered, although electron transport activity of photosystem II was restored to the wild-type level. Gene expression study of CrPGP1 and CrPGP2 in nutrient-starved C. reinhardtii showed differential response to phosphorus and nitrogen deficiency. Taken together, these results highlight the distinct and overlapping function of PGPS in cyanobacteria and eukaryotic algae.

Site-directed mutagenesis of amino acid residues of D1 protein interacting with phosphatidylglycerol affects the function of plastoquinone QB in photosystem II.

Recent X-ray crystallographic analysis of photosystem (PS) II at 1.9-Å resolution identified 20 lipid molecules in the complex, five of which are phosphatidylglycerol (PG). In this study, we mutagenized amino acid residues S232 and N234 of D1, which interact with two of the PG molecules (PG664 and PG694), by site-directed mutagenesis in Synechocystis sp. PCC 6803 to investigate the role of the interaction in PSII. The serine and asparagine residues at positions 232 and 234 from the N-terminus were mutagenized to alanine and aspartic acid, respectively, and a mutant carrying both amino acid substitutions was also produced. Although the obtained mutants, S232A, N234D, and S232AN234D, exhibited normal growth, they showed decreased photosynthetic activities and slower electron transport from QA to QB than the control strain. Thermoluminescence analysis suggested that this slower electron transfer in the mutants was caused by more negative redox potential of QB, but not in those of QA and S2. In addition, the levels of extrinsic proteins, PsbV and PsbU, were decreased in PSII monomer purified from the S232AN234D mutant, while that of Psb28 was increased. In the S232AN234D mutant, the content of PG in PSII was slightly decreased, whereas that of monogalactosyldiacylglycerol was increased compared with the control strain. These results suggest that the interactions of S232 and N234 with PG664 and PG694 are important to maintain the function of QB and to stabilize the binding of extrinsic proteins to PSII.

Modified molecular interactions of the pheophytin and plastoquinone electron acceptors in photosystem II of chlorophyll D-containing Acaryochloris marina as revealed by FTIR spectroscopy.

Acaryochloris marina is a unique cyanobacterium that contains chlorophyll (Chl) d as a major pigment. Because Chl d has smaller excitation energy than Chl a used in ordinary photosynthetic organisms, the energetics of the photosystems of A. marina have been the subject of interest. It was previously shown that the redox potentials (E m's) of the redox-active pheophytin a (Pheo) and the primary plastoquinone electron acceptor (QA) in photosystem II (PSII) of A. marina are higher than those in Chl a-containing PSII, to compensate for the smaller excitation energy of Chl d (Allakhverdiev et al., Proc Natl Acad Sci USA 107: 3924-3929, 2010; ibid. 108: 8054-8058, 2011). To clarify the mechanisms of these E m increases, in this study, we have investigated the molecular interactions of Pheo and QA in PSII core complexes from A. marina using Fourier transform infrared (FTIR) spectroscopy. Light-induced FTIR difference spectra upon single reduction of Pheo and QA showed that spectral features in the regions of the keto and ester C=O stretches and the chlorin ring vibrations of Pheo and in the CO/CC stretching region of the Q A (-) semiquinone anion in A. marina are significantly different from those of the corresponding spectra in Chl a-containing cyanobacteria. These observations indicate that the molecular interactions, including the hydrogen bond interactions at the C=O groups, of these cofactors are modified in their binding sites of PSII proteins. From these results, along with the sequence information of the D1 and D2 proteins, it is suggested that A. marina tunes the E m's of Pheo and QA by altering nearby hydrogen bond networks to modify the structures of the binding pockets of these cofactors.