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First published online April 2, 2004; 10.1104/pp.103.037754 Plant Physiology 134:1471-1478 (2004) © 2004 American Society of Plant Biologists Phosphatidylglycerol Is Essential for Oligomerization of Photosystem I Reaction Center1Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, H–6701 Szeged, Hungary (I.D., A.S., L.K., B.U., B.B., M.K., Z.G.); Department of Plant Physiology and Biochemistry, Faculty of Biotechnology, Jagiellonian University, PL–30–387 Krakow, Poland (P.M., K.S.); Division of Material Science (Physics), Graduate School of Science, Nagoya University, Furocho, Chikusa, Nagoya 464–8601, Japan (K.I., S.I.); Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802 (G.S.); Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komamba, Tokyo 153–8902, Japan (I.S., H.W.); and Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, H–6701 Szeged, Hungary (T.F.)
Our earlier studies with the pgsA mutant of Synechocystis PCC6803 demonstrated the important role of phosphatidylglycerol (PG) in PSII dimer formation and in electron transport between the primary and secondary electron-accepting plastoquinones of PSII. Using a long-term depletion of PG from pgsA mutant cells, we could induce a decrease not only in PSII but also in PSI activity. Simultaneously with the decrease in PSI activity, dramatic structural changes of the PSI complex were detected. A 21-d PG depletion resulted in the degradation of PSI trimers and concomitant accumulation of monomer PSI. The analyses of PSI particles isolated by MonoQ chromatography showed that, following the 21-d depletion, PSI trimers were no longer detectable in the thylakoid membranes. Immunoblot analyses revealed that the PSI monomers accumulating in the PG-depleted mutant cells do not contain PsaL, the protein subunit thought to be responsible for the trimer formation. Nevertheless, the trimeric structure of PSI reaction center could be restored by readdition of PG, even in the presence of the protein synthesis inhibitor lincomycin, indicating that free PsaL was present in thylakoid membranes following the 21-d PG depletion. Our data suggest an indispensable role for PG in the PsaL-mediated assembly of the PSI reaction center.
Phosphatidylglycerol (PG) is an integral component of photosynthetic membranes. PG molecules are important for both the formation and functioning of photosynthetic apparatus. In cyanobacterial cells, PG is the only representative of the phospholipid family (Wada and Murata, 1998  ), and the majority of PG molecules are localized in the thylakoid membranes that are the site of oxygenic electron transport. Photosynthesis is the basic energy source of cyanobacteria and generally for photosynthetic organisms. The availability of the complete genomic sequence of Synechocystis PCC6803 (Kaneko et al., 1996) opened the way for studying the structural and functional roles of PG via molecular genetic approaches. The pgsA gene encoding PG phosphate synthase was inactivated in Synechocystis PCC6803 cells by inserting a kanamycin resistance gene cassette (Hagio et al., 2000). Maintenance of the recently generated pgsA mutant strain requires exogenously supplied PG. A 40% decrease in photosynthetic oxygen-evolving activity could be detected following a 3-d depletion of PG, which resulted in an approximately 50% decrease in the amount of PG molecules in the cellular membranes. The photosynthetic processes, as measured by fluorescence induction in the PG-depleted cells, slowed down, and a perturbation of the surroundings of the secondary quinone acceptor (QB) was observed (Gombos et al., 2002). This perturbation was similar to the effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which can block electron transport between primary quinone acceptor (QA) and QB. Additionally, Fragata et al. (1991) demonstrated that in isolated barley (Hordeum vulgare) PSII particles, oxygen-evolving activity strongly depends on the presence of PG and Mg2+ in the system (Fragata et al., 1991). These results indicate that PG molecules are essential constituents of photosynthetic apparatus in both cyanobacteria and in higher plants. However, PSI activity was not affected by short-term PG deprivation. This observation seemed to contradict x-ray crystallographic data, which showed the presence of PG in PSI and the absence in PSII reaction centers (RCs; Jordan et al., 2001; Zouni et al., 2001). Nevertheless, the 3.8 Å resolution of these measurements did not allow the detection of lipid molecules in PSII (Zouni et al., 2001). Although x-ray crystallographic measurements indicated the presence of three PG molecules in PSI RCs (Jordan et al., 2001), PSI activity was not significantly affected, even after 90% depletion of the cellular PG content. This finding suggests that the strength of PG binding by PSII might be much weaker than those of the PSI RCs. Accordingly, following a 90% decrease in PG content, the cells still may contain enough PG molecules to maintain PSI activity, but looser binding of PG to PSII RC could lead to loss of PSII activity at an earlier stage. It was also demonstrated by using isolated PSII particles treated with phospholipase that PG molecules assist in PSII dimer formation (Kruse et al., 2000). Recent reports confirmed the deleterious effect of PG deprivation not only on the photosynthetic function but also the photosynthetic structures in cyanobacteria. Using a mutant defective in PG synthesis, it was evidenced that PG molecules in photosynthetic membranes are required for the efficient dimer formation of the PSII complex (Sakurai et al., 2003). Earlier studies indicated that in higher plants PG contribute to the development of chloroplasts (Hagio et al., 2002; Xu et al., 2002).
In contrast to higher plants, in which only the monomer form of PSI RC is present (Golbeck, 1992
PSI RCs contain several protein subunits (Sun et al., 1997 Using long-term PG depletion, we studied the effect of PG on the PSI oligomer formation in the PG-deficient pgsA. Following 21 d of depletion, there was no detectable amount of PG in the cells. Presumably, despite of the strong binding of PG molecules to the PSI RCs, by this stage (most of) the trimeric complexes lost their PG content. Using anion-exchange chromatography of solubilized thylakoid fractions, we observed a dramatic decrease in the level of PSI trimers. A concomitant increase of monomeric PSI content suggested that monomers were formed at the expense of trimers. To our knowledge, this is the first in vivo evidence of the essential role of PG in PSI trimer formation.
Lipid Analyses Using thin-layer chromatography and gas chromatography, there was no detectable amount of PG in isolated thylakoid membranes of the pgsA mutant cells following a 21-d PG depletion (data not shown).
The absorption spectra of intact cells (Fig. 1A ) show that the Chl content of the pgsA mutant decreases dramatically following 5 d of PG deprivation; however, the phycocyanin content remained constant. The phycocyanin content, as measured by the optical density at 630 nm (OD630; Fig. 1B), did not change appreciably, even after 21 d of PG depletion. By contrast, the relative Chl content monitored by OD680 showed a rapid decrease between days 2 and 7 of PG depletion, decreasing to about 30% of the initial value. Following day 7 of depletion, the decrease slowed down, and at the end of day 21 the Chl content still remained about 20% of the initial value. For the first 3 d following the transfer of mutant cells to PG-free medium, the growth rate was similar to that of the mutant cultures grown in PG-supplemented medium. Later on, the growth rate of PG-deprived cultures decreased significantly compared to those cultivated in the presence of PG. Following about 14 d of PG depletion, division of the cells was stopped, but it could be completely recovered by readdition of PG to the culture. This reversible stage of depletion lasted to about day 21 of PG deprivation (data not shown).
Effect of Long-Term PG Depletion on the PSI Activity PSI activity was monitored by measuring changes in the OD705 values in intact cells. These measurements can detect change in the redox state of PSI that is proportional to PSI activity. The results are summarized in Table I. All these data were calculated on the basis of Chl concentration. PSI activity did not show any significant change in the first week of PG depletion, but during the second week it decreased to about 80% of the initial activity. At the end of the 21-d PG depletion, only 40% of initial PSI activity was observed. Interestingly, if 20 µM of PG was added to the cells on day 21 of PG depletion, the PSI activity returned to initial level within 3 d of cultivation. Low temperature fluorescence emission dramatically decreased at longer wavelength characteristic for PSI emission following 14 d of PG depletion (data not shown). This observation, together with the decrease in the absorption change at 705 nm, clearly indicates the effect of long-term PG depletion on PSI activity.
PG Depletion Effect on the Oligomerization of PSI Complexes
To investigate the oligomerization of RCs, pigment-protein complexes were isolated from thylakoid membranes. The separation of monomeric and trimeric forms of PSI was performed by anion-exchange chromatography combined with fluorescence spectroscopy. Fractions eluted with MgSO4 gradient were analyzed by fluorescence emission at 77 K. Separation of the thylakoid fraction isolated from pgsA mutant grown in the presence of PG resulted in four peaks of pigment-protein complexes (Fig. 2A
). According to the fluorescence emission bands, peaks 1 and 4 contained monomeric and trimeric forms of PSI, respectively. The 77 K fluorescence emission spectra of these fractions were identical to those of purified PSI monomers and trimers described by other authors (Van der Lee et al., 1993
By contrast, the same separation of thylakoids isolated from cells after 21 d of PG depletion revealed only three peaks of pigment-protein complexes, with an additional shoulder at peak 2 (Fig. 2D). No trimeric form of PSI could be detected by 77 K fluorescence analysis and/or gel filtration chromatography. Additionally, the relative ratio of free pigments to pigment-protein complexes was much higher in comparison to PG supplied cells. Interestingly, in thylakoid fractions isolated from 7-d PG-depleted cultures (Fig. 2B), the ratio of PSI trimer (peak 4) to monomer (peak 1) was smaller than that in cells cultured in the presence of PG (Fig. 2B). The relative amount of PSI RC trimer further decreased in 14-d PG-depleted cells (Fig. 2C); however, there still was a detectable level of PSI RC trimer content. Following a 21-d PG depletion, no PSI RC trimer could be detected (Fig. 2D). In the thylakoid fraction isolated from PG-depleted cells 3 d after the readdition of PG to the culture medium, the trimeric form of PSI (peak 4) was restored (Fig. 2E). Following a 20-h recovery period, trimerization of PSI RCs was observed even in the presence of lincomycin, a protein synthesis inhibitor, at the concentration of 0.4 mg mL–1 (Fig. 2F).
The protein composition of fractions from anion-exchange chromatography containing PSI monomers (peak 1) and trimers (peak 4) was analyzed by SDS-PAGE (Fig. 5A ) and immunoblotting (Fig. 5B). In all PSI monomer- and trimer-containing fractions isolated from the pgsA mutant cells grown in the presence or absence of PG and following 3 d of PG readdition, the proteins reacting specifically with antibodies raised against individual subunits of cyanobacterial PSI (PsaA, PsaC, PsaD, and PsaL) were identified. Immunoblot analyses revealed a striking difference between the amounts of PsaL in peaks 1 and 4. The trimer fractions both in the PG-supplemented cells and in the 21-d PG-depleted cells following 3 d of PG readdition contained high amounts of PsaL (Fig. 5B, lanes 5 and 6). Compared to the trimer fractions, all the monomer fractions contained much lower amounts of PsaL. The PsaL content in the monomer fraction of 21-d PG-depleted cells was under the detectable level (Fig. 5B, lane 2). Despite of the remarkable decrease of PsaL in the 21-d PG-depleted PSI RC monomers, we could detect a high level of this protein in the isolated thylakoid fraction of the same cell sample (Fig. 5B, lane 8). This observation can explain the PG-induced recovery of the PSI trimers in the PG-depleted cells even in the presence of lincomycin.
In this article, we describe the effects of prolonged PG deprivation on the photosynthetic RCs of the cyanobacterium Synechocystis PCC6803. During our work, we took advantage of pgsA mutant, which, due to a lesion in the PG phosphate synthase gene, is incapable of PG synthesis. Cultivation for 4 weeks in PG-deficient medium was found to be lethal for this mutant. Figure 6 shows a sequence of physiological and biochemical changes of the photosynthetic apparatus taking place upon long-term PG depletion. The first 3 to 5 d of PG deprivation caused mainly a disturbance in the photosynthetic electron transport. A 40% decrease in PG content could modify the functioning PSII RC by perturbing the membrane environment. This moderate PG depletion could affect the environment of QB site, which may result in a slowdown of electron transport between QA and QB (Gombos et al., 2002 ). This could indicate that PG molecules may be loosely bound to PSII RC and easily detached from the quinone site. Simultaneously, with the change in its function, the structure of PSII RC was also affected. In the second week of PG deprivation, the light-induced monomerization of PSII dimers was significantly enhanced (Sakurai et al., 2003). A severe PG depletion occurred in the third week of culturing. Following day 21, no PG could be detected by thin-layer chromatography in the lipid extract. This loss of PG was not compensated by an increase in the level of sulfoquinovosyldiacylglycerol, another anionic lipid, as has been observed in other photosynthetic organisms (Essigmann et al., 1998). A considerable inactivation of PSI activity was observed between days 14 and 21 of PG deprivation, as measured by OD705 changes. The PSI RC catalyzes the oxidation of reduced plastocyanin or cytochrome c6 and the reduction of soluble ferredoxin or flavodoxin. It plays an important role as a central part of electron transport, while also regulating the maintenance of physiological homeostasis of photosynthetic organisms. In cyanobacteria, a unique trimeric organization of PSI complexes has been observed (Fromme et al., 2001). However, the regulation of the transition and balance between PSI trimers and monomers has not yet been elucidated. Under our experimental conditions, the decrease of PSI activity was correlated with monomerization of the trimeric form. Earlier in vitro studies indicated that trimer formation was enhanced by the lipid environment (Kruip et al., 1999). The depletion of PG, a specific anionic lipid and the only phospholipid of cyanobacterial photosynthetic membranes, resulted in an unbalanced oligomer formation. This anionic lipid contains one negatively charged phosphate group bound to glycerol backbone, which can accommodate Ca2+ that has been suggested to be required for trimer formation (Fromme et al., 2001). In vitro it was demonstrated that Ca2+ can induce the aggregation of negatively charged lipids (Hincha, 2003). Under PG depletion, monomer PSI complexes became dominant at the expense of the trimers. Disappearance of PG molecules and concomitant complete monomerization of PSI RCs strongly suggest a requirement for PG in maintenance of the trimeric form. Although, except in PsaL, all of the protein subunits of the PSI RC are present in the monomer separated from the cells after 21 d of PG depletion, trimer formation could not be observed. This observation clearly demonstrates the importance of PsaL in trimer formation, while also suggesting that PG is required for the binding of PsaL to the PSI RC. PG-induced reconstitution of PSI trimers in the presence of lincomycin reveals that de novo protein synthesis is not required for the restoration of PSI oligomers. The high level of PsaL observed in the thylakoids of 21-d PG-depleted cells can provide the source of this protein for the restoration of PSI RC trimers, while readded PG molecules can stabilize the reformed PSI RC trimers. Our data confirm the indispensable role of PG molecules in the formation of the structures required for the functioning of the photosynthetic RCs.
On the basis of our current knowledge, it can be asserted that (1) PG is needed for the formation of both PSII (Sakurai et al., 2003 ) and PSI oligomers, (2) PG acts by loosely binding to PSII RC for the maintenance of the quinone environment and supporting its function (Gombos et al., 2002), (3) PG molecules might regulate the optimal light capturing by balancing the PSI monomer to trimer ratio, and (4) PG could be important in protection against the excess of light exposure by supporting the formation of PSI trimers that might facilitate radiationless energy dissipation. Our pgsA mutant can be instrumental in the localization of PG-specific binding sites in the RCs by exploring the specificity of PG binding and interactions with RC protein components. Our studies can also lead to understanding the physiological relevance of PSI trimers.
Organism and Growth Conditions
The pgsA mutant cells of Synechocystis PCC6803 were grown photoautotrophically in BG 11 medium (Allen, 1968
Absorption spectra of cell suspensions were recorded on Shimadzu UV-3000 spectrophotometer (Columbia, MD). Chl and phycocyanin contents were estimated on the basis of the OD680 and OD630 values, respectively. Both pigment values were normalized on the basis of cell number. The Chl concentration was determined by the method of Arnon et al. (1974)
Lipids were extracted from intact cells according to Bligh and Dyer (1959)
Transient absorption changes at selected wavelengths in the microsecond to millisecond range were measured with a split-beam spectrophotometer, with the probing light obtained by a combination of two monochromators, a mechanical shutter and a 100-W tungsten-iodine lamp, according to Iwaki et al. (1999)
Thylakoid fraction was isolated from Synechocystis PCC6803 according to Komenda and Barber (1995)
Monomer and trimer PSI pigment-protein complexes were separated according to Rögner et al. (1990b)
HPLC separations were performed at 10°C with Varian Prostar (Varian, Palo Alto, CA) liquid chromatograph equipped with UV/Vis absorption and conductivity gradient detectors. Extracted pigment-protein complexes (100–300 µL) were loaded on MonoQ HR 5/5 column (Amersham-Pharmacia Biotech, Uppsala), equilibrated with buffer D containing 20 mM MES (pH 6.5), 10 mM CaCl2, 10 mM MgCl2, 0.5 M mannitol, 5 mM MgSO4, and 0.03% (w/v) DM, and eluted with a nonlinear gradient of 5 to 200 mM MgSO4, with a flow rate of 0.4 mL min–1, as described by Rögner et al. (1990b) Gel filtration chromatography was performed on a TSK 3000 SW (7.5- x 600-mm) column (Tosoh Bioscience, Tokyo) equilibrated with buffer D at a flow rate of 0.5 mL min–1. The column was calibrated using protein Mr standards (Bio-Rad, Hercules, CA).
SDS-PAGE was performed according to the standard procedure, as described by Schagger and von Jagow (1987)
For 77 K measurements, the fractions obtained from MonoQ separation were cooled in liquid nitrogen. Steady-state fluorescence emission spectra (600–780 nm) were recorded, using 437-nm excitation wavelength, with Perkin-Elmer LS 50 spectrofluorimeter (Foster City, CA) equipped with liquid nitrogen attachment. The emission spectra were corrected for the wavelength dependence of the detection system and normalized at maximum.
We thank Dr. Jochen Kruip and Mrs. Tatjana Schwabe (Plant Biochemistry, Faculty of Biology, Ruhr-University Bochum, Bochum, Germany) for the gift of PsaL antibody. We also thank Erika Zukic for her excellent technical assistance. We thank Dr. Miklós Szekeres (Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary) for reading and correcting the manuscript. Received December 15, 2003; returned for revision January 27, 2004; accepted January 27, 2004.
1 This work was supported by grants from the Hungarian Science Foundation (OTKA; grant nos. T 34174 and T 38408), by the Dr. Rollin D. Hotchkiss Foundation (I.D.), and cofinanced by the European Union (Center of Excellence grant; contract no. BIER ICA1–CT–2000–70012) and the Polish Committee for Scientific Research (grant no. 158/E–338–/SPUB–M/5 PR UE/DZ 9/2001–2003).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037754. * Corresponding author; e-mail domonkos{at}nucleus.szbk.u-szeged.hu; fax 36–62–433–434.
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ABSTRACT
RESULTS
-dodecylmaltoside-solubilized thylakoid fractions from Synechocystis pgsA mutant cells on an anion-exchange column (MonoQ HR 5/5). Absorbance was recorded at 437 nm. Peak 1, PSI monomer; peak 2, a mixture of PSI and PSII monomers; peak 3, PSII oligomer; and peak 4, PSI trimer. A, Thylakoids from cells grown in the presence of PG (20 µM). B, Thylakoids from cells following 7 d of PG depletion. C, Thylakoids from cells following 14 d of PG depletion. D, Thylakoids from cells following 21 d of PG depletion. E, Thylakoids from cells following 21 d of PG depletion and 3 d of PG readdition. F, Thylakoids from cells following 21 d of PG depletion and 20 h of PG readdition in the presence of lincomycin (0.4 mg mL–1). For details, see the text.
-amino-caproic acid, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM benzamidine. The suspension was mixed with an equal volume of 0.1-mm glass beads and placed into a Bead Beater homogenizer (BioSpec Products, Bartlesville, OK). Cell disruption was performed 15 times for 1 min. Intact cells were removed by centrifugation at 2,000g for 5 min, and the supernatant was centrifuged again at 20,000g for 30 min. Pelleted thylakoids were resuspended either in buffer B (50 mM Tris, pH 7.5, 1 M Suc, 1 mM PMSF, and 1 mM benzamidine) or in buffer C (20 mM MES, pH 6.5, 10 mM CaCl2, 10 mM MgCl2, 0.5 M mannitol, 20% (v/v) glycerol, 1 mM PMSF, and 1 mM benzamidine) and stored at –80°C. 
