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First published online May 18, 2007; 10.1104/pp.107.098731 Plant Physiology 144:1336-1346 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Effects of the Lack of Phosphatidylglycerol on the Donor Side of Photosystem II1,[OA]Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153–8902, Japan (I.S., N.M., H.W.); and Institute of Materials Science, University of Tsukuba, Ibaraki 305–8573, Japan (S.O., M.K.)
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 QA and QB. 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  psbO, psbV, and psbU 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.
Phosphatidylglycerol (PG) is one of the ubiquitous lipid components in thylakoid membranes from chloroplasts and cyanobacteria. The major lipids found in thylakoid membranes are glycolipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and the phospholipid PG (Block et al., 1983  ; Dorne et al., 1990; Somerville et al., 2000). Because PG is the only phospholipid in thylakoid membranes and is a negatively charged molecule at neutral pH (Wada and Murata, 1998; Frentzen, 2004), it is likely that PG mediates indispensable interactions with the components of photosynthetic complexes in thylakoid membranes and that PG plays a specific role in photosynthesis for which glycolipids cannot substitute.
The photosynthetic electron transport system involved in the primary processes of photosynthesis is composed of several protein-cofactor supercomplexes (Melis, 1991
Biochemical studies have indicated that PG plays important roles in photosynthesis. Decomposition of approximately 70% of PG from thylakoid membranes by treatment with phospholipase A2 was found to strongly inhibit photosynthetic electron transport in PSII without any significant effect on photosynthetic electron transport in PSI (Jordan et al., 1983
In addition to these in vitro experiments, our previous molecular biological analyses using a pgsA mutant of Synechocystis sp. PCC6803 have advanced our understanding of the function of PG in vivo (Hagio et al., 2000 In our previous studies, we clarified the important role that PG plays in the QB site of PSII, specifically in the acceptor side of PSII. In this study, however, we found that PG also plays an important role in the donor side of PSII. The results obtained by biochemical analyses of the PSII complex purified from the PG-depleted pgsA mutant cells suggest that PG plays a crucial role in the donor side of PSII for the binding of extrinsic proteins required for sustaining functional manganese (Mn) cluster.
PG Content and Fluorescence Parameters in pgsA Mutant Cells
Figure 1
shows changes in PG content in thylakoid membranes after the pgsA mutant cells grown in the presence of PG were transferred to medium with or without PG. As reported previously (Hagio et al., 2000
Wild-type and mutant parameters of room temperature chlorophyll (Chl) fluorescence are shown in Table I . pgsA mutant cells grown in the absence of PG for 8 d displayed much higher dark levels of fluorescence (F0) and a much lower level of Fv/Fm, which indicates the photochemical efficiency of PSII, compared with wild-type cells. Mutant cells grown in the presence of PG showed intermediate levels of F0 and Fv/Fm between wild-type and PG-depleted mutant cells. As we reported previously (Gombos et al., 2002 ), absorption spectra of intact cells indicated that the content of phycobilisomes in pgsA mutant cells is not significantly changed compared to wild-type cells. Thus, the contribution of phycobilisomes to fluorescence parameters is similar to that in wild-type cells and altered F0 level is not due to changes in phycobilisomes. However, it is possible that F0 level was altered due to the altered excitation energy transfer from phycobilisomes to Chl. These results indicate that deprivation of PG decreased the photochemical efficiency of PSII in mutant cells. In search of further alteration in the antenna system of mutant cells, low-temperature (77 K) fluorescence emission spectra were measured (Fig. 2
). In the case of wild-type and PG-supplemented mutant cells, excitation of Chl at 440 nm resulted in a large emission peak at 722 nm arising from PSI and two small peaks at 685 and 695 nm arising from PSII core antenna proteins, CP43 (Nakatani et al., 1984) and CP47 (Vermaas et al., 1986), respectively. By contrast, PG-depleted mutant cells showed a significantly increased emission peak from PSII at 685 nm and an emission peak from PSI that was shifted to a slightly shorter wavelength (720 nm; Fig. 2A). Moreover, excitation of phycobilisomes at 590 nm resulted in drastic increase of emission peaks from PSII (685 nm) and phycobilisomes (660 nm) in PG-depleted mutant cells (Fig. 2B). Low-temperature fluorescence emission spectra of intact cells were also measured with different Chl concentrations and similar results were obtained even with lower concentration of Chl (data not shown). It is likely that emission from phycobilisomes contributes to the emission at 685 nm from PSII because the emission from phycobilisomes is strongly increased in mutant spectra.
Photosynthetic Electron Transport Activity of PSII
Functional changes in PSII concomitant with the decrease in PG content were also analyzed by monitoring the photoreduction of 2,6-dichloro-phenol-indophenol (DCPIP), which accepts electrons from QB and/or the plastoquinone pool. As shown in Figure 3
, the photosynthetic electron transport activity from diphenylcarbazide (DPC), which donates electrons to Tyr Z, to DCPIP decreased gradually after mutant cells were transferred to the PG-free medium. By contrast, activity decreased only slightly when mutant cells were maintained in medium with PG. It was also found that, after transfer to PG-free medium, the photosynthetic electron transport activity from water, which donates electrons to the Mn cluster to DCPIP, decreased more than that from DPC to DCPIP. In addition, changes in the activity from DPC to DCPIP and from water to DCPIP of thylakoid membranes of wild-type cells were similar to those of thylakoid membranes of PG-supplemented mutant cells (data not shown). These results indicate that depletion of PG impairs photosynthetic electron transport within PSII, not only in the acceptor side, but also in the donor side at the oxygen-evolving system, including the Mn cluster. Although the impairment in the acceptor side of PSII probably occurs at the QB site, as reported previously (Gombos et al., 2002
Separation of Monomers and Dimers of PSII
As shown in Figure 4
, monomers and dimers were well separated by ultracentrifugation on a glycerol density gradient. In wild-type cells, the dimer was more abundant than the monomer and the ratio of monomer to dimer based on Chl was approximately 1:4. By contrast, in PG-depleted mutant cells, the monomer was more abundant than the dimer and the ratio of monomer to dimer was approximately 3:2. These results are consistent with our previous finding that PG is important for dimerization of PSII (Sakurai et al., 2003
Photosynthetic Oxygen-Evolving Activity of PSII Table II presents the oxygen-evolving activity of the purified PSII. Before the monomer and dimer were separated by ultracentrifugation on a glycerol density gradient, the activity of PSII of wild-type cells was approximately 3,000 and 850 µmol O2 mg Chl–1 h–1 when potassium ferricyanide and 2,6-dichloro-p-benzoquinone were used as electron acceptors, respectively (data not shown). Although the separation of the monomer and dimer by ultracentrifugation resulted in loss of activity, to some extent, the dimer of wild-type cells still sustained high levels of oxygen-evolving activity. However, the activity of the monomer was much lower than that of the dimer. These results suggest that the dimer is a functional form of PSII. In the case of the PG-depleted mutant cells, the ratio of dimer-to-monomer activity was similar to wild-type cells. However, the relative activity of the monomer and dimer of PG-depleted mutant cells was much lower overall than that of the monomer and dimer of wild-type cells.
Lipid Composition of PSII Although PG content in thylakoid membranes decreased in mutant cells, it remained unclear whether PG content in PSII was also decreased. Thus, the lipids in PSII were analyzed (Table III ). The monomer and dimer purified from the PG-depleted pgsA mutant cells still contained 12% and 15% PG, respectively, whereas thylakoid membranes contained only 3% PG. These results suggest that PG molecules remaining in the thylakoid membranes of PG-depleted mutant cells were preferentially retained by PSII. However, the total PG content in PSII of mutant cells was much lower than that in PSII of wild-type cells. Therefore, it is likely that the overall decrease in PG content is responsible for the observed PSII dysfunction in PG-depleted mutant cells.
Dissociation of Extrinsic Proteins from PSII
The composition of the PSII complex has been well characterized and it contains many protein subunits and cofactors, such as pigments, metals, and lipids, which optimize performance (Murata et al., 1984
To investigate whether the extrinsic proteins that dissociated from the PSII complex were synthesized in PG-depleted mutant cells, immunochemical analyses were performed using thylakoid membranes and PSII complexes (Fig. 6 ). Again, PsbV and PsbU were not detected and the amount of PsbO was significantly decreased in the mutant PSII. However, these extrinsic proteins were present at near wild-type amounts in the thylakoid membranes of the PG-depleted mutant cells. These results indicate that synthesis of extrinsic proteins proceeds normally in the PG-depleted mutant, but that the localization profile is altered. It is possible that the extrinsic proteins from the PG-depleted mutant dissociated from the complex and localized in the lumen. However, we cannot rule out that PSII dissociated during purification due to a lower core complex binding affinity caused by PG depletion.
Release of Mn from PSII Chl and Mn contents in the PSII complexes were also analyzed. Table IV shows that 36 to 37 Chl molecules per monomer were present in all purified PSII from wild-type or PG-depleted mutant cells. The monomer/dimer profile for Mn, however, was significantly different. In both wild-type and mutant cells, PSII dimers contained much higher amounts of Mn than PSII monomers, thus explaining the result that the dimer sustained higher oxygen-evolving activity than the monomer. It seems that the monomer is a premature complex in which a functional Mn cluster is not yet constituted or in which an unstable cluster is formed that readily dissociates Mn. Mn content in the dimer of the mutant cells was approximately 25% lower compared with wild-type cells, suggesting that the ability to sustain the functional Mn cluster was impaired in mutant cells, likely due to the dissociation of extrinsic proteins.
Rebinding of the PsbO Subunit to PSII
To investigate whether PG promotes rebinding of dissociated extrinsic proteins to the PSII complex, PG-depleted mutant cells were reincubated with PG and binding of PsbO to PSII was analyzed. After reincubation with PG, thylakoid membranes were isolated and solubilized with n-dodecyl-
Dissociation of Extrinsic Proteins in Vivo
It was unclear whether extrinsic protein dissociation from PSII occurred in vivo or whether this phenomenon was a result of the purification process. To address this issue, heat susceptibility of PG-depleted mutant cells was analyzed in comparison with wild-type cells. Mutants
It has been reported that, in mutant cells lacking PsbO or PsbV, Mn atoms in the Mn cluster are reduced under dark conditions and released from PSII (Burnap et al., 1996 ; Shen et al., 1998). Release of Mn inactivates oxygen-evolving activity, but decreased activity is restored under light conditions due to reincorporation of Mn atoms. Thus, we analyzed dark-induced inactivation of oxygen-evolving activity of mutant cells. In agreement with previous findings, oxygen-evolving activity of psbO and psbV mutant cells decreased during dark incubation (Fig. 9
). Activity of PG-depleted pgsA mutant cells also decreased under dark conditions. By contrast, activity of wild-type and psbU mutant cells did not decrease. Decreased activity of PG-depleted pgsA, psbO, and psbV mutant cells was restored after mutant cells were transferred to light (data not shown). These results suggest that the extrinsic proteins are dissociated from or weakly bound to PSII in PG-depleted mutant cells in vivo.
Low-temperature fluorescence spectra of intact cells, which are adjusted based on Chl, indicated that fluorescence from PSII following Chl excitation at 440 nm was higher in PG-depleted mutant cells compared to wild-type and PG-supplemented mutant cells (Fig. 2A). This increase could be attributed to the increased number of PSII complexes and/or the decreased energy transfer from antenna Chl to the PSII reaction center. We previously reported that Chl content in psgA mutant cells decreased after deprivation of PG (Gombos et al., 2002 ). This decrease in Chl content occurs mainly in PSI because the Chl content in PSII was not significantly changed in PG-depleted mutant cells (Table IV). Thus, it is likely that increased fluorescence from PSII is due to an increase in the ratio of antenna Chl in PSII to that of Chl in PSI. Fluorescence from phycobilisomes following excitation at 590 nm was also increased in PG-depleted mutant cells (Fig. 2B). This increase in fluorescence from phycobilisomes could be caused by an increase in the ratio of phycobilin to Chl in PG-depleted mutant cells. However, we cannot exclude the possibility that part of the increased fluorescence from PSII and phycobilisomes is caused by a decrease in energy transfer from antenna Chl to the reaction center and from phycobilisomes to the reaction center (or within phycobilisomes). It was previously reported that low-temperature fluorescence from PSII is higher in psbU mutant cells (Veerman et al., 2005). We also found that low-temperature fluorescence from PSII in mutants psbO and psbV increased similar to the PG-depleted pgsA mutant cells (data not shown). These findings suggest that the observed increase in fluorescence from PSII may also be induced to some extent by the dissociation of extrinsic proteins from PSII. Under stress conditions, the Chl a-binding protein (IsiA) accumulates in cyanobacteria. This protein may also contribute in part to the 77 K fluorescence as reported by Shpilyov et al. (2005).
In this work, we demonstrate that PG depletion induces dysfunction not only in the electron acceptor side of PSII, but also in the donor side (Fig. 3). Impairment in the acceptor side likely occurs at the QB site, as reported previously (Gombos et al., 2002
Siegenthaler and coworkers have shown asymmetric distribution of glycerolipids in the inner and outer leaflets of the lipid bilayer in thylakoid membranes (Rawyler and Siegenthaler, 1985 In parallel with the dissociation of extrinsic proteins, accumulation of Psb28 (Sll1398) was also observed in PSII from the PG-depleted mutant cells (Fig. 5). Although the reason for Psb28 accumulation was not determined, we suggest that this subunit may be involved in PSII assembly, which was interrupted by the depletion of PG. Design of a Synechocystis sp. PCC6803 mutant in which the Psb28 gene is disrupted is forthcoming and will be required to discern the role of Psb28 in PSII assembly. Subunit analysis of PSII also showed aberrant distribution of extrinsic proteins between monomer and dimer in pgsA mutant cells. Specifically, PsbV, PsbU, and Psb27 were exclusively found in the monomer, whereas PsbO and PsbQ were preferentially found in the dimer (Fig. 5). The dimer contained significantly higher amounts of Mn than the monomer (Table IV). These results imply distinct roles for these extrinsic proteins in dimerization of PSII and construction of the Mn cluster. However, further studies are required to clarify their individual functions.
In PG-depleted pgsA mutant cells, we observed accumulation of the PSII monomer (Fig. 4), which is consistent with our previous finding that PG is involved in the dimerization of PSII (Sakurai et al., 2003
Based on the results shown in Table III, we estimated that approximately six PG molecules per monomer are bound to PSII of wild-type cells, whereas three per monomer are bound to PSII of PG-depleted mutant cells. The three PG molecules lost from PSII of mutant cells may be located near the QB binding site, as predicted from our previous studies (Hagio et al., 2000
Thylakoid membranes contain SQDG as an additional anionic lipid. Because SQDG and PG molecules are negatively charged at neutral pH, it is possible that they interact with components in protein-cofactor supercomplexes in thylakoid membranes and would thus have important structural roles in those complexes. Güler et al. (1996)
In summary, we show that PG depletion from thylakoid membranes causes PSII dysfunction in the donor side and that these events correlate with the dissociation of extrinsic proteins from PSII. The dissociation of extrinsic proteins corresponded with the release of Mn from PSII and loss of oxygen-evolving activity. However, reincubation of PG-depleted mutant cells with PG induced rebinding of PsbO and dimerization of the PSII complex, even when de novo protein synthesis was inhibited. These findings suggest that PG plays an important role in binding the extrinsic proteins required for sustaining a functional Mn cluster on the electron donor side of PSII.
Organisms and Growth Conditions
CP47-His transformants made from the wild-type and the pgsA mutant of Synechocystis sp. PCC6803 were used in this study. CP47-His transformants expressing the CP47 subunit of the PSII complex with six His residues as a tag at the C terminus were constructed as described (Bricker et al., 1998
In vivo photosynthetic activity was monitored by a Clark-type oxygen electrode as described (Gombos et al., 1991
Thylakoid membranes were prepared as described below. Cells containing 200 µg Chl were harvested and washed with 1 mL of buffer A (50 mM MES-NaOH, pH 6.0, 10 mM MgCl2, 5 mM CaCl2, and 25% glycerol [w/v]), then resuspended in 200 µL of buffer A containing proteinase inhibitors (1 µM benzamidine, 1 µM phenylmethylsulfonyl fluoride, and 1 µM hexanoic acid). Glass beads (300 µL) suspended in buffer A were added to the cell suspension and shaken by FastPrep (FP120; Qbiogene). Cell debris and unbroken cells were removed by centrifugation at 2,000g for 5 min and the supernatant was further centrifuged at 20,000g for 20 min at 4°C. The pelleted thylakoid membranes were suspended in buffer A to a final concentration of 1 mg Chl mL–1.
PSII was purified as reported by Kashino et al. (2002)
DCPIP photoreduction activity of thylakoid membranes was measured in reaction buffer (50 mM MES-NaOH, pH 6.0, 10 mM NaCl, 20 mM CaCl2, 1 M Suc) containing 50 µM DCPIP. Where indicated, 1 mM DPC was added to the reaction. Samples were illuminated with white light filtered through thermocutting and red filters, and photoreduction of DCPIP was measured by the change in absorption at 600 nm (UV-3000; Shimazdu) at room temperature.
Lipids were extracted from thylakoid membranes and PSII by the method of Bligh and Dyer (1959)
SDS-PAGE analysis using an 18% to 24% polyacrylamide gradient gel and identification of separated polypeptides by MALDI-TOF MS were conducted as described by Sakurai et al. (2006)
Chl concentrations were determined by the method of Arnon et al. (1974)
Mutant cells grown in PG-free medium for 8 d were incubated with PG for 10 h in the absence or presence of 100 µM lincomycin and thylakoid membranes were prepared as described above. Protein complexes solubilized with DM from thylakoid membranes were separated by ultracentrifugation through a 5% to 30% linear density gradient of glycerol. The glycerol gradient was fractionated into 20 fractions of equal volume and distributions of D1 and PsbO in the gradient were analyzed by western blotting as described (Sakurai et al., 2003
We are grateful to Dr. Masahiko Ikeuchi (University of Tokyo) and Dr. Yoshitaka Nishiyama (Ehime University, Japan) for providing antibodies against PsbO and PsbV, respectively. We also thank Dr. Mitsue Miyao-Tokutomi (National Institute of Agrobiological Science, Japan) for supporting the measurement of DCPIP photoreduction activity. Received March 1, 2007; accepted May 14, 2007; published May 18, 2007.
1 This work was supported by the Grant-in-Aid for Scientific Research (grant nos. 16570029 [to H.W.] and 1870029 [to N.M.]) and the Research Fellowship for Young Scientists (grant no. 11578 to I.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hajime Wada (hwada{at}bio.c.u-tokyo.ac.jp).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.098731 * Corresponding author; e-mail hwada{at}bio.c.u-tokyo.ac.jp; fax 81–3–5454–6656.
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ABSTRACT
RESULTS
-subunit of cyt b559), comprising the reaction center of PSII, were similarly observed in the monomer and dimer of wild-type and PG-depleted mutant cells. However, the extrinsic proteins PsbV and PsbU, which were primarily present in the wild-type monomer, were not detected in the mutant PSII. The extrinsic proteins Psb27 (Slr1645) and PsbQ (Sll1638), which were preferentially found in the wild-type monomer and dimer, respectively, were also less abundant in mutant cells, as was the PsbO subunit. These extrinsic proteins bind to the lumenal side of the PSII complex and have an important function in sustaining the Mn cluster (Ono and Inoue, 1984
-D-maltoside (DM). Solubilized complexes, including PSII, were separated by ultracentrifugation in a 5% to 30% glycerol density gradient. The distribution of D1 and PsbO in the separated fractions was determined by western blotting. D1 was detected in two fraction areas, numbers 8 to 10 and 11 to 14, corresponding to the PSII monomer and dimer, respectively (
