TY - JOUR
AU - Wada, Hajime
AB - Abstract 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. lipid, phosphatidylglycerol, photosynthesis, photosystem II complex, thylakoid membrane Chl, chlorophyll, DCBQ, 2,6-dichloro-p-benzoquinone, DCMU, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, DGDG, digalactosyldiacylglycerol, DM, n-dodecyl β-d-maltoside, DMBQ, 2,6-dimethyl-p-benzoquinone, Fecy, potassium ferricyanide, MGDG, monogalactosyldiacylglycerol, MALDI-TOF MS, matrix-assisted laser desorption ionization time of flight mass spectrometry, PG, phosphatidylglycerol, Pheo, pheophytin, PLA2, phospholipase A2, PSI, photosystem I, PSII, photosystem II, SQDG, sulfoquinovosyldiacylglycerol, X:Y(Z), fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxy terminus In thylakoid membranes, the sites of primary photosynthetic reactions, electrons are transferred through a series of photosynthetic machineries to convert light energy into biologically useful chemical energy (1). The lipids that constitute thylakoid membranes show unique compositions compared with other biological membranes (2, 3). Specifically, thylakoid membranes have high proportions of glycolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG). They also have low proportions of phospholipid phosphatidylglycerol (PG). Biochemical and molecular biological analyses have indicated the importance of these lipids in the photosynthesis and heterogeneous distribution of lipids in various protein complexes in thylakoid membranes. The depletion of PG from thylakoid membranes by treatment with a phospholipase caused suppression of the electron transport activity in photosystem II (PSII) (4, 5). The oxygen-evolving activity of a PSII preparation derived through solubilization of spinach thylakoid membranes with Triton X-100 was stimulated by exogenously added DGDG and phosphatidylcholine (6). The treatment of a PSII complex prepared from thylakoid membranes of spinach with phospholipase A2 (PLA2), which cuts the ester bond at the sn-2 position of phospholipids, resulted in dissociation of the complex from dimer to monomer, suggesting that PG is involved in dimerization of the PSII complex (7). The activity of the cytochrome b6/f complex was reconstituted with DGDG, PG, and phosphatidylcholine, but not with MGDG or SQDG (8). DGDG and PG are bound to the trimeric form of light-harvesting chlorophyll a/b binding protein complexes (LHCII) and play important roles in the construction of these protein complexes (9). The roles of lipids in photosynthesis were also investigated with mutants defective in the biosynthesis of thylakoid lipids. An mgd1 mutant of Arabidopsis thaliana that is deficient in the biosynthesis of MGDG in chloroplasts was isolated and used to demonstrate that MGDG is required for the development of chloroplast membranes (10). Involvement of the molecular species of PG which contains a Δ3-trans-hexadecenoic acid [16:1(3t)] in the trimerization of LHCII was suggested by a series of studies involving a mutant of Chlamydomonas reinhardtii defective in the biosynthesis of 16:1(3t) (11). The role of SQDG in PSII complexes was studied with the hf-2 mutant of C. reinhardtii, and the results demonstrated that it has an important role in the electron transfer from water to tyrosine Z on the donor side of PSII (12, 13). We recently constructed mutants of Synechocystis sp. PCC6803 defective in the biosynthesis of PG and investigated the function of PG in photosynthesis (14–16). In a series of reports, we demonstrated that PG deficiency induces conformational changes in the binding site for plastoquinone QB in the D1 protein and thereby leads to impairment of electron transport from QA to QB in the PSII reaction center. These findings suggested that PG has an indispensable role in maintenance of the structure of the QB-binding site of D1 protein (16). PG was found to be necessary also for the assembly and stabilization of the photosynthetic machineries, because the presence of PG enhanced the formation of dimeric PSII complexes and trimeric photosystem I (PSI) complexes (17–19). In addition to the findings in the biochemical and molecular biological studies mentioned above, from a structural viewpoint the importance of thylakoid lipids in the protein complexes involved in photosynthesis has become apparent. One molecule of MGDG and three molecules of PG were identified in the crystal structure of the PSI complex, suggesting that these lipids contribute to the structure of this protein complex (20). It has also been reported that phosphatidylcholine is needed to obtain high-quality crystals of the cytochrome b6/f complex because it stabilizes this protein complex, although it is not a lipid present in thylakoid membranes (21). The crystal structure of the PSII complex at different resolutions has been reported (22–24), and 14 lipids, namely, four DGDG, six MGDG, three SQDG, and one PG, were assigned in the most recently determined crystal structure of the PSII complex at 3.0 Å resolution (25). In the structure, some other lipid-like components, which were not assigned, were also found, especially in the cavity harboring the QB-binding site. These findings suggest that the lipids have structural and functional important roles in the PSII complex, but the actual numbers of lipid molecules bound to the PSII complex remain obscure. Further biochemical analyses are required to determine the numbers of lipids, and to understand their structural and functional roles in the PSII complex. In this study, we analyzed the lipid compositions in dimeric PSII complexes to determine how many lipid molecules and what classes of lipids are present in PSII complexes. The PSII complexes used for lipid analysis were purified from two species of cyanobacteria, Thermosynechococcus vulcanus and Synechocystis sp. PCC6803, and from two higher plants, spinach and rice. In addition to the lipid analysis, we also examined the effects of PLA2 treatment on the activity of the PSII complex prepared from Synechocystis to study functional roles of PG in the PSII complex. MATERIALS AND METHODS Organisms and Growth Conditions For the purification of PSII complexes from Synechocystis sp. PCC6803, a transformant with six histidine residues as a tag at the C-terminus of the CP47 subunit of a PSII complex was constructed as described by Bricker et al. (26). The obtained transformant of Synechocystis sp. PCC6803 carrying the six-histidine tag is referred to as Synechocystis. Wild-type cells of T. vulcanus and Synechocystis cells were grown photoautotrophically at 55°C and 30°C, respectively, as described previously (27). Spinach was purchased from a local market and rice was grown in a local field for 2 months. Preparation of Thylakoid Membranes and PSII Complexes Thylakoid membranes and PSII dimer complexes from T. vulcanus were purified as described by Shen and Inoue (28). Thylakoid membranes and PSII complexes of Synechocystis were prepared according to Kashino et al. (29) with minor modifications as follows. Protein complexes in thylakoid membranes were solubilized with 1% n-dodecyl β-d-maltoside (DM) (D4641; Sigma) and the solubilized protein complexes were subjected to Ni-affinity column chromatography (Ni-NTA column; Qiagen) to purify the PSII complexes. The resultant PSII complexes were further purified by ultracentrifugation through a linear glycerol density gradient containing 5–30% glycerol (w/v) in a medium comprising 10 mM MgCl2, 5 mM CaCl2, 1 M glycine betaine, 0.03% DM, and 50 mM MES-NaOH (pH 6.5), made with a Gradient Master (Model 107ip; Biocomp Inc., New Brunswick, Canada). The gradient was centrifuged at 180,000 × g for 16 h at 4°C (P40ST rotor; Hitachi). Fractions containing the dimeric form of PSII complexes were collected and concentrated by ultrafiltration (Amicon Ultra-4; Millipore). PSII dimer complexes of spinach and rice were prepared from BBY-type (30) PSII membranes (31). The PSII membranes were treated with 2.3% n-heptyl β-d-thioglucoside to remove major LHCII proteins (32). The resultant PSII particles were treated with 1.2% DM at a chlorophyll (Chl) concentration of 1 mg/ml at 4°C for 30 min and then purified on a MonoQ column (Amersham Pharmacia Biotech). The obtained PSII was concentrated by ultrafiltration through a membrane filter (Amicon YM30; Millipore) and then treated again with 1.0% DM at 4°C for 30 min, followed by size-exclusion chromatography on two columns of HiLoad 26/60 Superdex 200pg (Amersham Pharmacia Biotech) linked serially. The obtained PSII dimer complexes described above were used for lipid analysis. The concentration of Chl was determined by the method of Arnon et al. (33), and the molar ratio of Chl to pheophytin (Pheo) was determined by HPLC as described previously (34) SDS-PAGE The polypeptide compositions of PSII dimer complexes prepared from T. vulcanus and Synechocystis were analyzed by SDS-PAGE according to the method described by Kashino et al. (35), with a gradient gel of 18–24% polyacrylamide containing 6 M urea. While those of PSII dimer complexes prepared from spinach and rice were analyzed by SDS-PAGE as described by Ikeuchi and Inoue (36), with a gradient gel of 16–22% polyacrylamide containing 7.5 M urea. Polypeptides separated on the gels were visualized with Coomassie Brilliant Blue R250 (Fulka). Mass Spectrometry Subunits of PSII dimer complexes prepared from Synechocystis were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Individual polypeptides separated by SDS-PAGE were excised and subjected to in-gel digestion with trypsin (V5280; Promega). The digested peptide fragments were extracted from the gel and analyzed by MALDI-TOF MS after removal of salts on a reversed-phase micro-column (Zip-Tip; Millipore). MALDI-TOF MS was performed on AXIMA-CFR (Shimadzu) using α-cyano 4-hydoroxycinnamic acid (C2020; Sigma) as the matrix. Proteins were identified as the highest ranking result by searching in the National Center for Biotechnology Information database using Mascot (http://www.matrix-scinence.com). Measurement of Photosynthetic Activities Photosynthetic activities were measured by means of oxygen exchange with a Clark-type oxygen electrode as described by Gombos et al. (37). In the case of T. vulcanus, the activities were measured at 30°C in a medium comprising 30 mM Mes (pH 6.0), 20 mM NaCl, and 3 mM CaCl2. The concentrations of 2,6-dichloro-p-benzoquinone (DCBQ) and potassium ferricyanide (Fecy), which were used as electron acceptors, were 0.5 mM and 1 mM, respectively. The activities of the PSII dimer complex prepared from Synechocystis were measured at 30°C in a medium comprising 50 mM Mes (pH 6.0), 10 mM NaCl, 20 mM CaCl2, and 1 M sucrose. The concentrations of DCBQ and Fecy were 0.5 mM and 6 mM, respectively. In the case of spinach and rice, the activities were measured at 25°C in a medium comprising 30 mM Mes (pH 6.5), 20 mM NaCl, 5 mM CaCl2, and 0.5 mM each of electron acceptors. Lipid Analysis Lipids were extracted from thylakoid membranes and PSII dimer complexes by the method of Bligh and Dyer (38). Lipids from thylakoid membranes and PSII complexes, which were not treated with PLA2, were separated into lipid classes on TLC plates as described previously (39, 40). Lipids extracted from the PLA2-treated PSII dimer complex of Synechocystis were separated into lipid classes by two-dimensional TLC. For the first dimension development was performed with CHCl3/CH3OH/28% NH4OH (65:35:5, by volume) and for the second dimension with CH3COCH3/C6H6/H2O (91:30:8, by volume). Quantification of lipid classes by gas chromatography was performed as described previously (39). The mobility of the DM that was extracted together with lipids in the PSII complexes was similar to that of DGDG on the TLC plates. However, DM did not interfere with the quantification of DGDG, because fatty acid methylesters derived from DM were detected on gas chromatography at retention times different from those of fatty acid methylesters derived from DGDG. Phospholipase A2 Treatment The PSII dimer complex of Synechocystis was diluted in a medium comprising 10 mM MgCl2, 5 mM CaCl2, 25% glycerol (w/v), and 50 mM MES-NaOH (pH 6.0) to the concentration of 0.5 mg Chl/ml. The PSII complex was incubated with or without PLA2 (P9279; Sigma) at a concentration of 200 units/mg Chl in the dark at 20°C. To monitor the changes in oxygen-evolving activity on PLA2 treatment, 2, 6-dimethyl-p-benzoquinone (DMBQ) was used as an electron acceptor in addition to DCBQ and Fecy. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was used at 1 μM to inhibit the electron transport at the QB-binding site. RESULTS The photosynthetic oxygen-evolving activities of the dimeric PSII complexes are shown in Table 1. The activities of the PSII complex prepared from T. vulcanus were very high and similar to those of the PSII complex that was used for crystal structure analysis (41). In the case of Synechocystis, the activities of the PSII complex before separation into the monomer and dimer by ultracentrifugation through a glycerol density gradient were similar to those of the PSII complex purified previously by Kashino et al. (29) (data not shown). Although the activities slightly decreased after separation of the dimer on ultracentrifugation, they were still very high. The PSII complexes prepared from spinach and rice exhibited reasonably high levels of oxygen-evolving activities that were similar to those reported previously (31, 42). The polypeptide compositions of the dimeric PSII complexes are shown in Fig. 1. Major PSII subunits CP47, CP43, PsbO (manganese-stabilizing protein), D2, D1, and PsbE were found in all PSII complexes. In addition to these subunits, PsbU and PsbV, which are known as extrinsic polypeptides in cyanobacterial PSII complexes, were also found in the PSII complex of T. vulcanus. These results demonstrate that the PSII complexes prepared from each organism are active and highly purified, and thus are suitable for lipid analysis. Fig. 1. View largeDownload slide Polypeptide compositions of the dimeric forms of PSII complexes. PSII complexes of T. vulcanus (lane 1), Synechocystis (lane 2), spinach (lane 3), and rice (lane 4) corresponding to 5 μg Chl were loaded on each lane. Fig. 1. View largeDownload slide Polypeptide compositions of the dimeric forms of PSII complexes. PSII complexes of T. vulcanus (lane 1), Synechocystis (lane 2), spinach (lane 3), and rice (lane 4) corresponding to 5 μg Chl were loaded on each lane. Table 1. Photosynthetic oxygen-evolving activities of photosystem II complexes. Acceptor   T. vulcanus   Synechocystis   Spinach   Rice     (μmol O2/mg Chl/h)        2,6-DCBQ  2,920 ± 190  1,040 ± 110  460 ± 60  420 ± 50  Fecy  1,210 ± 100  2,080 ± 200  840 ± 100  710 ± 80  Acceptor   T. vulcanus   Synechocystis   Spinach   Rice     (μmol O2/mg Chl/h)        2,6-DCBQ  2,920 ± 190  1,040 ± 110  460 ± 60  420 ± 50  Fecy  1,210 ± 100  2,080 ± 200  840 ± 100  710 ± 80  The values are averages and SD of independent measurements. View Large The lipid compositions of thylakoid membranes and PSII dimer complexes are shown in Table 2. The lipid compositions of thylakoid membranes prepared from all organisms were similar to those reported previously (14, 43–45). MGDG accounted for about 40 to 50% of the total lipids in all organisms. DGDG and SQDG together occupied a large part of the remaining total lipids. PG was a minor lipid, comprising 6%, 14%, 13%, and 5% of the total lipids in the thylakoid membranes of T. vulcanus, Synechocystis, spinach, and rice, respectively. There was a remarkable difference between the lipid composition of the thylakoid membranes and that of the PSII complexes. The contents of MGDG, DGDG, and SQDG were lower in the PSII complexes than in the thylakoid membranes. By contrast, the contents of PG in the PSII complexes of T. vulcanus, Synechocystis, spinach, and rice were 5, 2, 2, and 5 times higher than those in the thylakoid membranes prepared from the respective organisms. These results demonstrate that PG is enriched in PSII complexes. The numbers of lipid molecules bound to the PSII dimer complexes were estimated based on the contents of Chl and Pheo in the PSII complexes. As shown in Table 3, it was estimated that 27, 20, 8, and 7 lipid molecules per monomer are bound to the PSII dimer complexes of T. vulcanus, Synechocystis, spinach and rice, respectively. Table 2. Lipid compositions of thylakoid membranes and photosystem II complexes. Organism   Sample   Total   Lipid class               MGDG   DGDG   SQDG   PG       (nmol/μg Chl)    (mol%)      T. vulcanus  TM  2.63 ± 0.03  43.5 ± 1.5  25.6 ± 0.2  24.8 ± 1.1  6.1 ± 0.6    PSII  0.79 ± 0.04  29.8 ± 1.0  21.1 ± 2.1  20.4 ± 2.3  28.7 ± 2.3  Synechocystis  TM  2.39 ± 0.08  37.4 ± 1.8  20.0 ± 1.6  28.9 ± 0.6  13.7 ± 0.8    PSII  0.60 ± 0.03  28.7 ± 2.1  16.4 ± 0.8  25.5 ± 0.8  29.4 ± 1.6  Spinach  TM  1.39 ± 0.06  40.1 ± 0.7  32.1 ± 0.8  15.2 ± 0.0  12.6 ± 0.1    PSII  0.25 ± 0.02  39.9 ± 1.9  25.1 ± 0.3  9.7 ± 0.6  25.3 ± 3.7  Rice  TM  2.65 ± 0.07  48.4 ± 0.1  32.9 ± 0.1  13.9 ± 0.3  4.8 ± 0.3    PSII  0.23 ± 0.02  40.2 ± 0.3  26.2 ± 1.3  11.3 ± 0.1  22.3 ± 1.0  Organism   Sample   Total   Lipid class               MGDG   DGDG   SQDG   PG       (nmol/μg Chl)    (mol%)      T. vulcanus  TM  2.63 ± 0.03  43.5 ± 1.5  25.6 ± 0.2  24.8 ± 1.1  6.1 ± 0.6    PSII  0.79 ± 0.04  29.8 ± 1.0  21.1 ± 2.1  20.4 ± 2.3  28.7 ± 2.3  Synechocystis  TM  2.39 ± 0.08  37.4 ± 1.8  20.0 ± 1.6  28.9 ± 0.6  13.7 ± 0.8    PSII  0.60 ± 0.03  28.7 ± 2.1  16.4 ± 0.8  25.5 ± 0.8  29.4 ± 1.6  Spinach  TM  1.39 ± 0.06  40.1 ± 0.7  32.1 ± 0.8  15.2 ± 0.0  12.6 ± 0.1    PSII  0.25 ± 0.02  39.9 ± 1.9  25.1 ± 0.3  9.7 ± 0.6  25.3 ± 3.7  Rice  TM  2.65 ± 0.07  48.4 ± 0.1  32.9 ± 0.1  13.9 ± 0.3  4.8 ± 0.3    PSII  0.23 ± 0.02  40.2 ± 0.3  26.2 ± 1.3  11.3 ± 0.1  22.3 ± 1.0  The values are averages and SD of independent measurements. TM, thylakoid membranes. View Large Table 3. Numbers of Chl and lipid molecules in photosystem II complexes. Organism   Chl   Lipid               Total   MGDG   DGDG   SQDG   PG     (numbers/2 Pheo)            T. vulcanus  38.8 ± 3.0  27.4 ± 0.9  8.2 ± 0.5  5.8 ± 0.6  5.6 ± 0.5  7.8 ± 0.9  Synechocystis  37.4 ± 2.8  20.1 ± 1.1  5.8 ± 0.1  3.3 ± 0.3  5.1 ± 0.3  5.9 ± 0.6  Spinach  36.2 ± 2.8  7.9 ± 0.2  3.1 ± 0.2  2.0 ± 0.1  0.8 ± 0.1  2.0 ± 0.3  Rice  32.0 ± 2.4  6.6 ± 0.5  2.7 ± 0.2  1.7 ± 0.2  0.7 ± 0.1  1.5 ± 0.2  Organism   Chl   Lipid               Total   MGDG   DGDG   SQDG   PG     (numbers/2 Pheo)            T. vulcanus  38.8 ± 3.0  27.4 ± 0.9  8.2 ± 0.5  5.8 ± 0.6  5.6 ± 0.5  7.8 ± 0.9  Synechocystis  37.4 ± 2.8  20.1 ± 1.1  5.8 ± 0.1  3.3 ± 0.3  5.1 ± 0.3  5.9 ± 0.6  Spinach  36.2 ± 2.8  7.9 ± 0.2  3.1 ± 0.2  2.0 ± 0.1  0.8 ± 0.1  2.0 ± 0.3  Rice  32.0 ± 2.4  6.6 ± 0.5  2.7 ± 0.2  1.7 ± 0.2  0.7 ± 0.1  1.5 ± 0.2  View Large Tables 4 and 5 show the fatty acid compositions of lipid classes in thylakoid membranes and PSII complexes prepared from the cyanobacteria, T. vulcanus and Synechocystis, as well as those from the higher plants, spinach and rice. The fatty acid compositions of lipid classes in the thylakoid membranes were similar to those reported previously (39, 43–45). In each organism, the fatty acid compositions of the lipid classes in the thylakoid membranes were slightly different from those of the lipid classes in the PSII complexes. In the PSII complexes of T. vulcanus, the contents of 16:0 in MGDG, DGDG, and PG, and of 18:0 in SQDG and PG were significantly higher than those of the lipid classes in the thylakoid membranes. By contrast, the contents of 16:0 in SQDG, 18:1(11) in MGDG and DGDG, and 18:1(9) in DGDG in the PSII complexes were lower than those of the lipid classes in the thylakoid membranes. In Synechocystis, the contents of 18:0 and 18:1(9) in PG in the PSII complexes were higher than those of PG in the thylakoid membranes, whereas the contents of 18:2(9,12) were lower than those of PG in the thylakoid membranes. The contents of 16:0 in SQDG and 18:3(6,9,12) in DGDG in the PSII complexes were lower than those of the lipid classes in the thylakoid membranes, whereas the contents of 18:0 in SQDG and 18:2(9,12) in DGDG in the PSII complexes were higher than those of the lipid classes in the thylakoid membranes. In the higher plants, significant differences between the PSII complexes and thylakoid membranes were observed mainly in the contents of 18:0 and 18:1(9) in SQDG and PG, and 16:0 and 18:3(9,12,15) in DGDG. Although many differences between the fatty acid compositions of lipid classes in the PSII complexes and thylakoid membranes were found in each of the organisms, as described above, it was clarified that the specific molecular species of each lipid class are not enriched in PSII complexes compared to those of each lipid class in thylakoid membranes. Table 4. Fatty acid compositions of thylakoid membranes and photosystem II complexes prepared from cyanobacteria T. vulcanus and Synechocystis. Organism   Fatty acid   Thylakoid membrane         PSII complex             MGDG   DGDG   SQDG   PG   MGDG   DGDG   SQDG   PG       (mol%)                T. vulcanus  16:0  45.1  43.6  62.0  47.9  51.1  57.0  58.3  52.2    16:1(9)  15.5  15.1  3.9  10.7  16.4  14.3  2.1  4.9    18:0  3.9  2.5  8.0  8.9  3.6  2.4  19.8  12.6    18:1(9)  27.9  28.1  20.9  26.3  25.1  22.8  14.6  26.9    18:1(11)  7.6  10.7  5.2  6.2  3.8  3.5  5.2  3.4  Synechocystis  16:0  53.1  51.4  69.1  51.3  51.7  50.6  62.9  51.6    16:1(9)  5.4  7.8  7.8  3.4  5.9  7.2  9.5  5.4    18:0  0.2  1.5  nd  3.9  1.9  2.6  2.5  6.9    18:1(9)  9.7  4.6  12.3  16.1  9.4  6.9  14.0  18.6    18:2(9,12)  11.7  10.6  10.2  21.2  12.3  13.8  9.3  15.4    18:3(6,9,12)  19.9  24.1  nd  1.9  18.8  18.9  nd  nd    18:3(9,12,15)  nd  nd  0.6  2.2  nd  nd  2.0  2.1  Organism   Fatty acid   Thylakoid membrane         PSII complex             MGDG   DGDG   SQDG   PG   MGDG   DGDG   SQDG   PG       (mol%)                T. vulcanus  16:0  45.1  43.6  62.0  47.9  51.1  57.0  58.3  52.2    16:1(9)  15.5  15.1  3.9  10.7  16.4  14.3  2.1  4.9    18:0  3.9  2.5  8.0  8.9  3.6  2.4  19.8  12.6    18:1(9)  27.9  28.1  20.9  26.3  25.1  22.8  14.6  26.9    18:1(11)  7.6  10.7  5.2  6.2  3.8  3.5  5.2  3.4  Synechocystis  16:0  53.1  51.4  69.1  51.3  51.7  50.6  62.9  51.6    16:1(9)  5.4  7.8  7.8  3.4  5.9  7.2  9.5  5.4    18:0  0.2  1.5  nd  3.9  1.9  2.6  2.5  6.9    18:1(9)  9.7  4.6  12.3  16.1  9.4  6.9  14.0  18.6    18:2(9,12)  11.7  10.6  10.2  21.2  12.3  13.8  9.3  15.4    18:3(6,9,12)  19.9  24.1  nd  1.9  18.8  18.9  nd  nd    18:3(9,12,15)  nd  nd  0.6  2.2  nd  nd  2.0  2.1  The values are averages of independent measurements. Deviation of values was within 3%. nd, not detected. View Large Table 5. Fatty acid compositions of thylakoid membranes and photosystem II complexes prepared from spinach and rice. Organism   Fatty acid   Thylakoid membrane         PSII complex             MGDG   DGDG   SQDG   PG   MGDG   DGDG   SQDG   PG       (mol%)                Spinach  16:0  3.1  6.7  49.2  15.6  1.4  10.1  46.6  13.5    16:1(7)  0.3  0.2  1.4  nd  nd  1.6  0.8  nd    16:1(3t)  nd  nd  nd  46.8  nd  nd  nd  44.6    16:3(7,10,13)  13.6  4.1  1.0  nd  18.5  3.5  nd  nd    18:0  0.6  0.6  2.3  0.4  1.2  2.5  5.9  2.4    18:1(9)  1.1  1.7  2.9  nd  2.1  4.3  6.9  3.5    18:3(9,12)  3.1  2.4  6.3  2.2  1.7  3.3  7.0  3.4    18:1(9,12,15)  78.2  84.3  36.9  35.0  75.1  74.7  32.8  32.6  Rice  16:0  2.1  14.0  50.9  28.2  2.9  10.9  47.4  30.4    16:1(3t)  nd  nd  nd  40.6  nd  nd  nd  43.1    18:0  0.6  3.2  3.5  4.7  0.9  3.6  7.6  2.6    18:1(9)  1.0  1.3  1.8  9.2  1.1  2.1  6.2  3.8    18:2(9,12)  4.8  5.6  4.5  8.5  4.2  4.2  3.5  7.1    18:3(9,12,15)  91.5  75.9  39.3  8.8  90.9  79.2  35.3  13.0  Organism   Fatty acid   Thylakoid membrane         PSII complex             MGDG   DGDG   SQDG   PG   MGDG   DGDG   SQDG   PG       (mol%)                Spinach  16:0  3.1  6.7  49.2  15.6  1.4  10.1  46.6  13.5    16:1(7)  0.3  0.2  1.4  nd  nd  1.6  0.8  nd    16:1(3t)  nd  nd  nd  46.8  nd  nd  nd  44.6    16:3(7,10,13)  13.6  4.1  1.0  nd  18.5  3.5  nd  nd    18:0  0.6  0.6  2.3  0.4  1.2  2.5  5.9  2.4    18:1(9)  1.1  1.7  2.9  nd  2.1  4.3  6.9  3.5    18:3(9,12)  3.1  2.4  6.3  2.2  1.7  3.3  7.0  3.4    18:1(9,12,15)  78.2  84.3  36.9  35.0  75.1  74.7  32.8  32.6  Rice  16:0  2.1  14.0  50.9  28.2  2.9  10.9  47.4  30.4    16:1(3t)  nd  nd  nd  40.6  nd  nd  nd  43.1    18:0  0.6  3.2  3.5  4.7  0.9  3.6  7.6  2.6    18:1(9)  1.0  1.3  1.8  9.2  1.1  2.1  6.2  3.8    18:2(9,12)  4.8  5.6  4.5  8.5  4.2  4.2  3.5  7.1    18:3(9,12,15)  91.5  75.9  39.3  8.8  90.9  79.2  35.3  13.0  The values are averages of independent measurements. Deviation of values was within 3%. nd, not detected. View Large As mentioned above, it was demonstrated that PG is enriched in the PSII complex. This finding suggests that PG may play important roles in the PSII complex. To examine the functional importance of PG in the PSII complex, the PSII dimer complex of Synechocystis was treated with PLA2, which degrades PG in the complex, and the change in oxygen-evolving activity was monitored. In addition to DCBQ and Fecy, DMBQ was used as an electron acceptor. As shown in Table 6, the oxygen-evolving activity of the PSII complex with DCBQ or Fecy was partially inhibited by DCMU to 40% and 49%, respectively. However, DCMU almost completely inhibited the oxygen-evolving activity of the PSII complex when DMBQ was used. These results indicate that DCBQ and Fecy can accept electrons not only from QB but also from QA, while DMBQ preferentially accepts electrons from QB, consistent with the previous findings reported by Satoh et al. (46). After PLA2 treatment for 120 min, the oxygen-evolving activity with DCBQ or Fecy decreased to 83% and 77%, respectively, of the initial level (Table 6 and Fig. 2, A and B). By contrast, the oxygen-evolving activity monitored with DMBQ decreased to about 30% of the initial level (Table 6 and Fig. 2C). It was also found that the PLA2 treatment slightly decreased the extent of inhibition of the activity by DCMU (Table 6), indicating that the decrease in the PG content in the PSII complex increased the affinity of the electron acceptors to the QA site. This is also consistent with the finding previously reported that the extent of inhibition of activity by DCMU decreased in the PSII complex, which does not sustain the native structure of the QB-binding site (47). In addition to these results, it was also found that PLA2 digested only PG, i.e., not other lipids, in the PSII complex (Table 7). The content of PG in the PSII complex decreased to more than half of the initial level after the PLA2 treatment. The subunit composition of the PSII complex was also analyzed before and after the treatment with PLA2 to confirm that inactivation of the PSII complex was not induced by degradation of some subunits. As shown in Fig. 3, no change in the subunit composition was observed with the exception of an extra band for the PLA2-treated PSII complex that corresponds to PLA2. These results suggest that some of PG molecules degraded on treatment with PLA2 play important roles in the electron transport at the QB-binding site in the PSII complex. Fig. 2. View largeDownload slide Changes in photosynthetic activity of the PSII dimer complex of Synechocystis on PLA2 treatment. The oxygen-evolving activity of the PSII complex, which was incubated without (square) or with (circle) PLA2, was monitored using DCBQ (A), Fecy (B), or DMBQ (C) as the electron acceptor. The initial activities are shown in Table 6. Bars represent the SD (n > 3). Fig. 2. View largeDownload slide Changes in photosynthetic activity of the PSII dimer complex of Synechocystis on PLA2 treatment. The oxygen-evolving activity of the PSII complex, which was incubated without (square) or with (circle) PLA2, was monitored using DCBQ (A), Fecy (B), or DMBQ (C) as the electron acceptor. The initial activities are shown in Table 6. Bars represent the SD (n > 3). Fig. 3. View largeDownload slide Effects of PLA2 treatment on polypeptide compositions of the PSII dimer complex prepared from Synechocystis. The polypeptide composition of the PSII complex before (lane 1) and after (lane 2) PLA2 treatment for 120 min. The star in lane 2 indicates the polypeptide band corresponding to PLA2. Fig. 3. View largeDownload slide Effects of PLA2 treatment on polypeptide compositions of the PSII dimer complex prepared from Synechocystis. The polypeptide composition of the PSII complex before (lane 1) and after (lane 2) PLA2 treatment for 120 min. The star in lane 2 indicates the polypeptide band corresponding to PLA2. Table 6. Effect of PLA2 treatment on photosynthetic oxygen-evolving activity of the PS II complex of Synechocystis. Treatment  Acceptor               DCBQ     Fecy     DMBQ       −DCMU   +DCMU   −DCMU   +DCMU   −DCMU   +DCMU     (μmolO2/mg Chl/h)            0 min  1,040 ± 110  420 ± 40  2,080 ± 200  1,020 ± 140  370 ± 50  40 ± 10  120 min –PLA2 (Control)  970 ± 90  480 ± 60  1,900 ± 170  1,080 ± 100  410 ± 70  40 ± 10  120 min +PLA2  860 ± 90  670 ± 90  1,600 ± 90  1,280 ± 80  120 ± 20  90 ± 20  Treatment  Acceptor               DCBQ     Fecy     DMBQ       −DCMU   +DCMU   −DCMU   +DCMU   −DCMU   +DCMU     (μmolO2/mg Chl/h)            0 min  1,040 ± 110  420 ± 40  2,080 ± 200  1,020 ± 140  370 ± 50  40 ± 10  120 min –PLA2 (Control)  970 ± 90  480 ± 60  1,900 ± 170  1,080 ± 100  410 ± 70  40 ± 10  120 min +PLA2  860 ± 90  670 ± 90  1,600 ± 90  1,280 ± 80  120 ± 20  90 ± 20  The values are averages and SD of independent measurements. View Large Table 7. Effect of PLA2 treatment on the lipid composition of the PSII complex. Treatment   Lipid class           MGDG   DGDG   SQDG   PG     (nmol/μg Chl)        0 min  0.18 ± 0.01  0.10 ± 0.01  0.15 ± 0.02  0.18 ± 0.03  120 min –PLA2(Control)  0.21 ± 0.02  0.13 ± 0.02  0.17 ± 0.03  0.19 ± 0.02  120 min +PLA2  0.22 ± 0.01  0.13 ± 0.02  0.17 ± 0.03  0.07 ± 0.01  Treatment   Lipid class           MGDG   DGDG   SQDG   PG     (nmol/μg Chl)        0 min  0.18 ± 0.01  0.10 ± 0.01  0.15 ± 0.02  0.18 ± 0.03  120 min –PLA2(Control)  0.21 ± 0.02  0.13 ± 0.02  0.17 ± 0.03  0.19 ± 0.02  120 min +PLA2  0.22 ± 0.01  0.13 ± 0.02  0.17 ± 0.03  0.07 ± 0.01  The values are averages and SD of independent measurements. View Large DISCUSSION Lipid molecules bound to PSII complexes were analyzed previously using PSII preparations, which were solubilized from thylakoid membranes with n-octyl β-d-glucopyranoside or Triton X-100 (44, 48). Compared with these PSII preparations, the PSII dimer complexes used in this study were highly purified and retained higher oxygen-evolving activities, mainly because of solubilization of the thylakoid membranes with DM (Table 1 and Fig. 1). This suggested that the DM-solubilized PSII complexes had kept the native structure of the PSII complex in the thylakoid membranes. With these purified PSII complexes, we were able to analyze lipid molecules bound to PSII complexes more precisely (Table 2). The PSII complexes of higher plants contain much lower amounts of SQDG and higher amounts of MGDG compared with the PSII complexes of cyanobacteria. Interestingly, the lipid compositions of the PSII complexes were quite different from those of the thylakoid membranes in each of the organisms. The numbers of lipid molecules per monomer were 27, 20, 8, and 7 in the dimeric PSII complexes of T. vulcanus, Synechocystis, spinach, and rice, respectively (Table 3). The lipid contents in the PSII complexes of the higher plants were lower than those in the PSII complexes of the cyanobacteria. This difference might be caused by the structural difference between the PSII complexes of cyanobacteria and those of higher plants. The PSII complexes of higher plants are surrounded by LHCII in their thylakoid membranes (1) and the binding of lipid molecules might be limited compared to that in PSII complexes of cyanobacteria, in which phycobillisomes are attached to the surface of PSII complexes in thylakoid membranes as antennae (49). On the other hand, a remarkable and common characteristic was found in the lipid composition of the PSII complexes prepared from each of the organisms. The PG content was 2 to 5 times higher in the PSII complexes than in the thylakoid membranes, occupying about 20–30% of the total lipids present in the PSII complexes (Table 2). The enrichment of PG in PSII membranes prepared from cucumber was also reported previously by Yang et al. (50), and it was observed even under phosphate-deficient conditions, under which the PG content in thylakoid membranes decreased. These findings are consistent with our previous findings that PG plays important roles in the electron transport and dimerization of PSII complexes in Synechocystis sp. PCC6803 (16, 17). It was estimated that 8 and 6 molecules of PG per monomer are bound to the PSII dimer complexes of T. vulcanus and Synechocystis, respectively (Table 3). These numbers of PG molecules in the PSII complexes are much higher than that found in the most recent crystal structure of the PSII complex of T. elongatus at 3.0 Å resolution, in which only one PG molecule was identified in the complex (25). This difference suggests that many PG molecules still remain to be assigned in the crystal structure. As described in that paper (25), two or three disordered lipophylic molecules were found in the cavity harboring the QB-binding site. In addition, our previous findings suggested that PG plays important roles in electron transport at the QB-binding site of the PSII complex (14, 16). This information indicates the possibility that unassigned lipid-like structures in the QB-binding site can be attributed to PG. Supporting our hypothesis, PLA2 treatment of the PSII complex purified from Synechocystis resulted in a significant decrease in the oxygen-evolving activity with DMBQ as the electron acceptor, but there was only a small effect when DCBQ or Fecy was used instead of DMBQ (Fig. 2). These results indicate that the decrease in the PG content in the PSII complex induces a severe defect in electron transport mediated by QB, suggesting that some PG molecules are required to support the QB-mediated electron transport in the PSII complex. After the PLA2 treatment, about 3 molecules of PG still remained in the PSII complex. These PG molecules would be embedded in the PSII complex, thus they were not degraded on the PLA2 treatment. One of these PG molecules can be attributed to the PG molecule that has been identified in the crystal structure (25). The other PG molecules might be located at the monomer–monomer interface in PSII complexes since it was previously found that PG is involved in the dimerization of the PSII complex (7, 17). Kruse et al. (7) reported that the monomeric and dimeric PSII complexes prepared from spinach contained about 110 and 74 lipid molecules per monomer, respectively. The content of lipids in the dimeric PSII complex is 9 times higher than that of lipids in the dimeric PSII complex of spinach analyzed in this work (Table 3). This contradictory result might be caused by the differences in the procedures used for purification of the PSII complex. In their case, PSII membranes isolated from spinach were solubilized with n-octyl β-d-glucopyranoside and DM, and then subjected to size-exclusion chromatography to purify the PSII complex. Although we used similar procedures for the solubilization of PSII membranes, PSII complexes were purified by ion-exchange chromatography on a MonoQ column and by size-exclusion chromatography on two columns linked serially. It might be possible that the purification of the PSII complexes by ion-exchange chromatography and size-exclusion chromatography caused the removal of lipid molecules. However, in spite of the much lower content of lipids in our PSII complex compared with that of Kruse et al. (7), the activities of both the PSII complexes were almost the same. Thus it is likely that lipids lost from our PSII complex during the purification steps are non-specifically and/or loosely bound lipids that are not required for the activities of the PSII complex. When lipid molecules bound to PSII complexes were identified, we expected that specific molecular species of lipids would be bound to PSII complexes. It was reported that 16:1(3t) bound to PG was enriched in PSII complexes prepared from spinach compared with thylakoid membranes, and that the dimeric PSII complex contains a higher level of PG containing 16:1(3t) than the monomeric PSII complex (7). This finding suggested that the PG molecular species containing 16:1(3t) is involved in dimerization of the PSII complex. However, concerning the fatty acid composition of lipid classes, we found no remarkable differences between the thylakoid membranes and PSII complexes in any of the organisms (Tables 4 and 5). As demonstrated by the previous studies involving mutants of cyanobacteria and higher plants defective in fatty acid desaturases, the fatty acid composition of lipids in thylakoid membranes affects the process of recovery of PSII activity from photoinhibition of photosynthesis (51, 52). It has also been demonstrated, with a pgsA mutant of Synechocystis sp. PCC6803 defective in the biosynthesis of PG, that the effect of PG on the recovery from photoinhibition of photosynthesis depends on the molecular species of PG (17). Although the fatty acid composition of lipids in PSII complexes was similar to that of lipids in thylakoid membranes, it should be noted that the acyl groups of lipid molecules also contribute to the function of PSII complexes. In summary, 27 and 20 lipid molecules per monomer were identified in the dimeric forms of PSII complexes of two cyanobacteria, T. vulcanus and Synechocystis, as were 8 and 7 lipid molecules in those of the higher plants, spinach and rice, respectively. The lipid composition of PSII complexes was remarkably different from that of thylakoid membranes. In particular, the content of PG was significantly higher in PSII complexes than in thylakoid membranes. Digestion of PG in the PSII complex of Synechocystis by PLA2 treatment resulted in a decrease in the oxygen-evolving activity with DMBQ as the electron acceptor, suggesting that PG plays important roles in the electron transport at the QB-binding site in the PSII complex. This work was supported by Grants-in-Aid for Scientific Research (No. 16570029 to HW, and No. 14340257 to JRS) from the Ministry of Education, Science, Sports, and Culture of Japan. REFERENCES 1. Malkin, R. and Niyogi, K. 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TI - Lipids in Oxygen-Evolving Photosystem II Complexes of Cyanobacteria and Higher Plants
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvj141
DA - 2006-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/lipids-in-oxygen-evolving-photosystem-ii-complexes-of-cyanobacteria-2ia0sIPn20
SP - 201
EP - 209
VL - 140
IS - 2
DP - DeepDyve
ER -