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bicarbonate is also observed in thylakoid membranes from Synechococcus sp. PCC 7002 ... effects of glycinebetaine and bicarbonate are of a different nature; ...
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Functional Plant Biology, 2003, 30, 797–803

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Stabilization of the oxygen-evolving complex of photosystem II by bicarbonate and glycinebetaine in thylakoid and subthylakoid preparations Vyacheslav V. KlimovA,B,C, Suleyman I. AllakhverdievA,B, Yoshitaka NishiyamaA, Andrei A. KhorobrykhB and Norio MurataA ADepartment

of Regulation Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. BInstitute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia. CCorresponding author; email: [email protected]

Abstract. The protective effect of 1 M glycinebetaine on thermal inactivation of photosynthetic oxygen evolution in isolated photosystem II membrane fragments from spinach is observed in CO2-free medium in both the presence and absence of added 2 mM bicarbonate. Conversely, the protective effect of 2 mM bicarbonate against thermoinactivation is seen in the absence as well as in the presence of 1 M glycinebetaine. The stabilizing effect of bicarbonate is also observed in thylakoid membranes from Synechococcus sp. PCC 7002 treated with 0.1% Triton X-100, and in unbroken spinach thylakoids. It is shown for the first time that bicarbonate protects the wateroxidizing complex against inactivation induced by pre-incubation of photosystem II membrane fragments (25°C) and thylakoids (40°C) at low pH (5.0–5.5) in non-bicarbonate-depleted medium. We conclude that the protective effects of glycinebetaine and bicarbonate are of a different nature; glycinebetaine acts as a non-specific, compatible, zwitterionic osmolyte while bicarbonate is considered an essential constituent of the water-oxidizing complex of photosystem II, important for its functioning and stabilization. Keywords: bicarbonate, glycinebetaine, photosystem II, thermostability, water-oxidizing complex. Introduction Photosystem II (PS II) of green plants and cyanobacteria is responsible for photosynthetic oxygen evolution and consists of two main functional blocks: (1) the photochemical reaction center (RC), which converts the excitation energy of chlorophyll into the energy of separated charges and produces the strongest biological oxidant, P680+ [with redox-potential of 1.12 V (Klimov et al. 1979)], and (2) the water-oxidizing complex (WOC), which is repeatedly oxidized by P680+ via the secondary electron donor YZ and, in turn, oxidizes H2O to produce O2 (for review see Renger 2001). The accumulation of oxidative equivalents required for water oxidation occurs mainly in a manganese cluster of the WOC containing four Mn ions, which are evidently coordinated by ligands provided by the D1- and D2-intrinsic proteins (also carrying cofactors of the RC) and probably by the chlorophyll–protein complex CP47. In higher plants, the WOC contains three extrinsic proteins of 18, 23 and 33 kDa.

The latter (named the ‘Mn-stabilizing protein’) associates strongly with the intrinsic proteins and provides sites for the tight association of the 23- and 18-kDa proteins (Miyao and Murata 1984, 1989). In cyanobacteria, the 18- and 23-kDa proteins are not found; in contrast the WOC of cyanobacteria contains two other extrinsic proteins not found in higher plants, cytochrome c550 and the 13-kDa protein, which is essential for WOC stability (Nishiyama et al. 1997). The Mn-cluster is characterized by low stability, which is mainly responsible for the high sensitivity of PS II and the WOC to inhibitory effects of elevated temperatures and other stress factors. A ‘compatible osmolyte’, glycinebetaine (GB), at a concentration of 1 M is extremely efficient in protecting the WOC against inactivation, which occurs when isolated thylakoid membranes and PS II membrane fragments are incubated at room temperature, alkaline pH or at high concentrations of salts. Glycinebetaine prevents the release

Abbreviations used: BC, bicarbonate; Chl, chlorophyll; cyt, cytochrome; DCBQ, 2,6-dichloro-p-benzoquinone; ddDT-20, DT-20 depleted of the 18-, 23-, and 33-kDa extrinsic proteins; DT-20, PS II membrane fragments from spinach; GB, glycinebetaine; PBQ, phenyl-1,4-benzoquinone; P680 and Z, the primary and secondary electron donors of PS II, respectively; RC, reaction center of PS II; WOC, the water-oxidizing complex of PS II. © CSIRO 2003

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of the extrinsic polypeptides and Mn from the WOC under these conditions (Miyao and Murata 1989; Mamedov et al. 1991; Papageorgiou et al. 1991; Murata et al. 1992; Mohanty et al. 1993; Papageorgiou and Murata 1995; Allakhverdiev et al. 1996; Nishiyama et al. 1997). The protecting effect of GB was especially pronounced in PS II membrane fragments depleted of the 33-kDa protein (Mohanty et al. 1993). Recently, a protective effect of bicarbonate (BC) ions against both photoinactivation and thermoinactivation of the donor side of PS II in subchloroplast membrane fragments has been demonstrated (Klimov et al. 1997a). The effect is consistent with recent data (Klimov et al. 1995a, b; Wincencjusz et al. 1996; Allakhverdiev et al. 1997; Klimov et al. 1997b; Baranov et al. 2000) showing that BC is required for the functioning of the WOC, along with its known requirement for the acceptor side of PS II (for recent review see Van Rensen and Govindjee 1999; Klimov and Baranov 2001 and references therein). In the present work, we compared the stabilizing effects of bicarbonate and glycinebetaine on the WOC in PS II membrane fragments and in thylakoid membrane fragments from spinach, and Synechococcus sp. PCC 7002 and found that the effects are of a different nature; GB acts as nonspecific compatible solute while BC is considered an essential constituent of the WOC, important for its functioning and stabilization.

centrifuged at 35000 g for 15 min, and the pelleted PS II membranes were suspended at a concentration that corresponded to 2 mg Chl mL–1 in 300 mM sucrose, 200 mM NaCl, 10 mM CaCl2 and 50 mM MES–NaOH (pH 6.2) (here after designated as medium S) and stored at –82°C. The depleted PS II membrane fragments (designated as ddDT-20) contained extrinsic proteins at 10–15% of the original level found in initial DT-20 preparation, as determined by SDS–PAGE. Bicarbonate removal from thylakoid membranes and PS II membrane fragments was achieved as described previously (Klimov et al. 1995a, b; Wincencjusz et al. 1996; Allakhverdiev et al. 1997; Klimov et al. 1997a), by diluting concentrated preparations 150–200-fold into the corresponding medium (medium A, B, and S for Synechococcus thylakoids, spinach thylakoids and ddDT-20, respectively) depleted of endogenous BC by means of flushing with nitrogen gas for 60 min and/or 60 min boiling. Thermoinactivation was carried out at 25 or 40°C in the dark, in medium depleted of CO2 and to which 2 mM NaHCO3 was added where indicated. Before the measurements of PS II activities, 2 mM NaHCO3 was added to all samples. Photosynthetic oxygen evolution was measured by monitoring the concentration of oxygen with a Clark-type oxygen electrode for 30–60 s after the start of actinic illumination. The measurements were carried out at 25°C in the presence of 1.0 m M phenyl-1,4benzoquinone (PBQ) or 0.5 mM 2,6-dichloro-p-benzoquinone (DCBQ) plus 0.5 mM K3[Fe(CN)6] (as indicated in the figure legends) as the exogenous electron acceptors. Red actinic light at an intensity of 2000 µmol photons m–2 s–1 was provided by an incandescent lamp used in conjunction with a heat-absorbing optical filter (HA-50, Hoya Glass, Tokyo, Japan) and a red optical filter (R-60, Toshiba, Tokyo, Japan).

Materials and methods

Figure 1A shows that both glycinebetaine and bicarbonate considerably decreased the rate of thermal inactivation of oxygen evolution in PS II membrane fragments lacking all three extrinsic proteins of the WOC (ddDT-20). In the medium not containing GB and depleted of CO2, nearly 50% of the activity was already lost during the first 30 min of incubation at 25°C and the activity was completely eliminated after 3 h incubation (curve 1). In contrast, if both 1 M GB and 2 mM BC were added to the medium before the incubation nearly 100% and 85% of the activity remained after 1 and 4 h treatment, respectively (curve 4). Even a 6.5-h incubation in the presence of both the agents eliminated only 50% of the activity. Only a partial protection of the WOC activity was observed when either of the agents, GB or BC, was added alone at concentrations saturating for this effect (1 M and 2 mM, respectively). It is noteworthy that all the measurements of O2-evolving activity were made at 25°C in the medium containing 2 mM BC. Thus, an irreversible inhibitory effect of temporary incubation in the medium depleted of BC is reported here. An alternative approach to reduce BC concentration in the medium is to lower pH. Figure 1B shows that incubation of ddDT-20 for 20 min at 25°C in medium S not depleted of BC does not lead to a significant loss of the O2-evolving activity if the pH of the medium is shifted from 7.0 to 6.5, while the activity is decreased by factors of 2 and 4 after

Synechococcus sp. PCC 7002 was obtained from the culture collection of the Pasteur Institute (Paris, France). The cells were grown photoautotrophically at 40°C for 3 d, and thylakoid membranes were isolated from the cells as described previously (Nishiyama et al. 1993). The isolated membranes were then suspended in medium A [50 mM Hepes–NaOH (pH 7.5), 800 mM sorbitol, 30 mM CaCl2, 1 M glycinebetaine] and stored in liquid nitrogen at a chlorophyll (Chl) concentration of 2 mg mL–1. Thylakoid membranes from spinach leaves were isolated as described previously (Robinson et al. 1980) with some modifications (20 mM Hepes–NaOH, pH 7.5, and 30 mM KCl were used instead of 20 mM Tricine, pH 8.0, and 2 mM MgCl2, respectively) in a medium containing 400 mM sucrose, 15 mM NaCl, 30 mM KCl, 1 mM EDTA and 20 mM Hepes–NaOH (pH 7.5). After centrifugation at 5000 g for 15 min, the pellet was twice washed in medium B, containing 10 mM NaCl, 30 mM KCl, 2 mM MgCl2 and 10 mM Hepes–NaOH (pH 7.5) and diluted in the final suspension medium containing 800 mM sucrose, 10 mM NaCl, 30 mM KCl, 2 mM MgCl2 and 50 mM Hepes–NaOH (pH 7.5) to obtain a Chl concentration of 4 mg mL–1. Subchloroplast PS II membrane fragments, designated here as DT-20, were isolated from spinach chloroplasts with 0.4% (w /v) digitonin and 0.15% (w /v) Triton X-100 as described by Shutilova et al. (1975) with modifications described by Allakhverdiev et al. (1988), except that pH 6.5 was used instead of pH 7.8. To remove the extrinsic proteins of 33-, 23- and 18-kDa without removing the manganese center of the WOC, the PS II membranes were incubated in a medium containing 300 mM sucrose, 200 mM NaCl, 10 mM CaCl2, 25 mM MES–NaOH (pH 6.5) and 3.0 M urea (Miyao and Murata 1984; Mohanty et al. 1993) at 0°C for 10 min. The suspension was

Results Photosystem II membrane fragments

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incubation at pH 6.0 and pH 5.5, respectively. Bicarbonate (2 mM) added to the samples before the incubation considerably protected the WOC from thermal inactivation, so that at

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pH 6.0 and 5.5 the remaining O2-evolving activity was 2–3 times higher than samples incubated without BC; at pH 6.5–7.0 there was no protective effect of the BC additions (Fig. 1B). Note that 1 M GB was present in the medium of all experiments and that 2 mM BC was added to all the samples before the measurements of O2-evolving activity. Spinach thylakoids

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pH of incubation at 25°C for 20 min Fig. 1. Effects of bicarbonate (BC) and glycinebetaine (GB) on the inactivation of oxygen evolution during dark incubation of spinach PS II membrane fragments depleted of the 18-, 23- and 33-kDa proteins (ddDT-20) at 25°C. ddDT-20 membrane fragments were suspended at a concentration corresponding to 15 µg Chl mL–1 and incubated (A) in medium S (pH 6.2), depleted of CO 2 in the absence of bicarbonate and glycinebetaine (1) and in the presence of 2 mM NaHCO3 (2), 1 M glycinebetaine (3), 2 mM NaHCO3 and 1 M glycinebetaine (4), or (B) in medium S, not depleted of CO2, at the designated pH for 20 min in darkness in the absence (1) or presence (2) of 2 mM NaHCO3. Before measurements of the oxygen evolution (at 25°C), 2 mM NaHCO3, 0.5 mM DCBQ and 0.5 mM ferricyanide were added to all samples. The O2-evolving activity taken as 100% was 135 µmol O2 mg–1 Chl h–1.

Figure 2 shows that the rate of thermoinactivation of O2-evolution in spinach thylakoid membranes also depends on the presence of BC in the medium. When the thermoinactivation was performed in the BC-depleted medium, the oxygen-evolving activity (measured in the presence of DCBQ and ferricyanide as electron acceptors) was decreased by factors of 2 and 5 after incubation of thylakoid membranes at 40°C in the dark for 10 and 20 min, respectively, (Fig. 2A, curve 1). However, if 2 mM NaHCO3 was added to the medium before the incubation at 40°C, the rate of thermoinactivation was considerably decreased (curve 2) so that nearly 80% and 40% of the activity remained after incubation for 10 and 20 min, respectively. Comparison of curves 1 and 3 shows that after thermoinactivation in the absence of BC, the rate of O2-evolution depended slightly on the presence of BC during the measurements of the activity. Similar results were obtained when the measurements were made in the presence of 5 µM gramicidin (1 mM ferricyanide was used as an electron acceptor in this case). In medium not depleted of CO2, the rate of thermoinactivation of oxygen evolution in thylakoids depended considerably on pH of the medium during the heat treatment (Fig. 2B). After a 10-min incubation at 40°C and pH 5.0, nearly 85% of the activity was lost, while the heat treatment at pH 7.0 eliminated nearly 50% of the activity. In contrast, if the heat treatment was applied in the presence of added 2 mM NaHCO3, the remaining activity at pH 5.0 was more than twice (42%) that remaining in the absence of NaHCO3 (curve 2). The effect of BC on thermoinactivation was also observed at pH 5.5 and pH 6.0, while at pH 6.5–7.5 it was not seen. Note that all the measurements of the O2-evolving activity were made after the addition of 2 mM NaHCO3. Thylakoid membranes from Synechococcus sp. PCC 7002 Before the experiments, the thylakoid membranes were treated with 0.1% Triton X-100 in order to remove the two extrinsic proteins of the WOC in cyanobacteria, cyt c550 and the product of the PsbU gene (Nishiyama et al. 1997). As shown previously (Nishiyama et al. 1997) this treatment resulted in a remarkable decrease in the heat stability of the O2-evolving machinery. The experiments were performed in medium containing 1 M GB and depleted of CO2. Figure 3A (curve 1) shows that the O2-evolving activity of the preparations decreased by

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pH of incubation at 40°C for 10 min Fig. 2. Effect of bicarbonate on thermal inactivation of oxygen evolution during dark incubation of spinach thylakoid membranes. (A) Time course of inactivation during dark incubation at 40°C in medium B depleted of CO2. (B) Dependence of the O2-evolving activity on pH during dark incubation for 10 min in medium B, not depleted of CO2 (50 mM Hepes and 50 mM MES were used for pH 7.0 and pH 5.0–6.5, respectively). Thylakoid membranes were suspended at a concentration corresponding to 20 µg Chl mL–1 and incubated at 40°C for the designated period of time in the absence (1) and presence (2) of 2 mM NaHCO3. Before the oxygen evolution measurements (at 20°C), 2 mM NaHCO3, 0.5 mM DCBQ and 0.5 mM ferricyanide were added to all samples. Before the measurements of O 2-evolution in (B) suspension of thylakoids treated at given pH was centrifuged and the pellet was resuspended in medium B (pH 7.0). Curve (3) in (A) is same as curve (1) except that 2 mM NaHCO3 was not added before measurement of oxygen evolution. The oxygen evolution activity taken as 100% was 156 µmol O2 mg–1 Chl h–1.

Fig. 3. Effects of bicarbonate on inactivation of oxygen evolution during incubation of thylakoid membranes isolated from Synechococcus sp. PCC 7002 and pre-treated with 0.1% Triton X-100 to remove the two extrinsic proteins, cyt c550 and the product of the PsbU gene (Nishiyama et al. 1997). (A) Time course of inactivation at 25°C in medium A, containing 1 M glycinebetaine and depleted of CO2. Thylakoid membranes suspended at a concentration corresponding to 15 µg Chl mL–1 were incubated in darkness at 25°C for the designated period in the absence (1–3) and presence (4–5) of 2 m M NaHCO3. Curves 1 and 4, no other additions; curves 2, 3 and 5, 100 µM MnCl2 was added before measurements of oxygen evolution; 3, same as 2 except the sample was pre-illuminated with continuous red light with an intensity of 70–80 µmol photons m –2 s–1 at 25°C for 10 min. (B) Dependence of O2-evolving activity on pH during incubation for 20 min at 40°C in medium A, containing 1 M glycinebetaine and not depleted of CO2; 50 mM Hepes and 50 mM MES were used for pH 7.0–7.5 and pH 5.0–6.5, respectively. Before the measurements of oxygen evolution, suspension of thylakoids treated at given pH was centrifuged and the pellet was resuspended in medium A (pH 7.5). Before the measurements of oxygen evolution (at 25°C) 2 mM NaHCO3 and 1 mM PBQ were added to all samples. The O2-evolving activity taken as 100% was 182 µmol O2 mg–1 Chl h–1.

Stabilization of the oxygen-evolving complex by bicarbonate

factors of 2 and 10 after dark incubation at 25°C for 1 and 8 h, respectively. However, if the treatment was performed in the presence of 2 mM BC, a remarkable protection of the O2-evolving machinery was revealed; nearly 90% of the activity remained even after incubation for 8 h, and incubation for 3 h did not inhibit the activity (curve 4) (note again that the activity was measured in the presence of 2 mM NaHCO3). If 0.1 mM MnCl2 was added to the samples pre-incubated for a few hours in the absence of added BC, oxygen evolution was considerably re-activated (curve 2) and the effect of Mn2+ was especially strong if the measurements of oxygen-evolution were made after photoactivation — pre-illumination with continuous red light (curve 3). Pre-illumination in the absence of added Mn2+ did not produce any effect on reactivation. Some reactivation after addition of Mn2+ was also seen after thermoinactivation in the presence of 2 mM NaHCO3 so that nearly 100% of the activity remained, even after incubation for 8 h at 25°C (curve 5). In the medium not depleted of CO2, the rate of thermal inactivation of oxygen evolution in thylakoid membranes from Synechococcus sp. PCC 7002 depended on pH of the medium used during the heat treatment (Fig. 3B). Nearly 30% of the activity is lost after a 20-min incubation at 40°C and pH 6.5–7.5, while the treatment at pH 5.0 eliminated more than 90% of the activity (curve 1). Bicarbonate (2 mM) added to the sample before the incubation considerably protected the WOC against thermal inactivation so that at pH 5.0 the remaining activity was five times higher (curve 2). Similar protecting effects were seen at pH 5.5 and pH 6.0 while at pH 6.5–7.5 there was no effect of the BC addition. Discussion The results obtained in this work are consistent with previous publications demonstrating the stabilizing effect of both glycinebetaine (Mamedov et al. 1991; Papageorgiou et al. 1991; Murata et al. 1992; Mohanty et al. 1993; Papageorgiou and Murata 1995; Allakhverdiev et al. 1996) and bicarbonate (Klimov et al. 1997a) during inactivation of the WOC of PS II. Each of these substances added alone efficiently protects the WOC from thermal inactivation (Fig. 1A) [related to the release of manganese from the PS II complex in the PS II membrane fragments as shown earlier (Mohanty et al. 1993)]. However, neither of them, added at saturating concentration for this effect, produced the maximal protection, which was reached only upon joint addition of BC and GB. This synergistic effect shows that the stabilizing actions of these two agents on PS II are of different natures, which is confirmed by the following arguments. (1) The effect of GB is observed at very high (1.0 M) concentration (Mamedov et al. 1991; Papageorgiou et al. 1991; Murata et al. 1992; Mohanty et al. 1993; Papageorgiou and Murata 1995; Allakhverdiev et al. 1996, and this paper), which is

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characteristic of other compatible solutes, for instance, sucrose (Mamedov et al. 1991; Papageorgiou et al. 1991; Murata et al. 1992; Mohanty et al. 1993; Papageorgiou and Murata 1995; Allakhverdiev et al. 1996) although GB reveals an especially strong stabilizing effect (Papageorgiou and Murata 1995). The protecting effect of BC is observed at concentrations more than 1000 times lower. The effect is saturated at 1 mM of added NaHCO3, which corresponds to the acting equilibrium concentration of HCO3– at pH 6.2 equal to 400 µM. (2) An especially pronounced protection effect of GB is observed for the activity and stability of the Mn-containing WOC of PS II (Mamedov et al. 1991; Papageorgiou et al. 1991; Murata et al. 1992; Mohanty et al. 1993; Papageorgiou and Murata 1995; Allakhverdiev et al. 1996). At the same time, the effect is shown for other PS II photoreactions, for example photooxidation of P680, photoreduction of pheophytin (Allakhverdiev et al. 1996), related to electron transfer in the PS II reaction center, and the effect is clearly seen in PS II preparations lacking Mn (Allakhverdiev et al. 1996). GB also stabilizes binding of the 18-, 23- and 33-kDa extrinsic proteins of the WOC (Murata et al. 1992; Mohanty et al. 1993; Papageorgiou and Murata 1995). In contrast, the effects of BC on the activities of the donor side of PS II (Klimov et al. 1995a, b; Wincencjusz et al. 1996; Allakhverdiev et al. 1997; Klimov et al. 1997b; Baranov et al. 2000), as well as on its stability, (Klimov et al. 1997a) are revealed only in the presence of Mn in the samples. These facts are consistent with the idea that GB, as an amino-acid zwitterion belonging to the class of ‘compatible’ or ‘counteracting’ solutes, protects macromolecular structures and enzymatic activities (including those of PS II) from structure-randomizing and inactivating factors, owing to them assuming a more folded (more ‘native’) configuration of proteins in its presence. On the contrary, BC is considered as an essential constituent of the WOC (Klimov et al. 1995a, b; Wincencjusz et al. 1996; Allakhverdiev et al. 1997; Klimov et al. 1997a, b). Therefore, in the absence of BC the Mn-cluster of the WOC becomes unstable, confirmed by data on the requirement for Mn2+ addition and photoactivation of the WOC in the samples incubated in the absence of BC (Fig. 3). The earlier results on effects of both BC and Mn2+ on thermoinactivation of photoinduced changes in the yield of chlorophyll fluorescence in PS II preparations showed that the Mn-cluster of the WOC is protected by BC (Klimov et al. 1997a). Our data also show that the protecting effect of BC on thermal inactivation of the WOC is revealed in various O2-evolving preparations — untreated spinach thylakoids and isolated PS II membrane fragments depleted of the extrinsic proteins [along with the untreated PS II membrane fragments (Klimov et al. 1997a)], thylakoid membranes from Synechococcus sp. PCC 7002 treated with 0.1% Triton X-100 to remove cyt c550 and the 13-kDa protein. These results demonstrate that the effects of BC are not provoked

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by possible damage to the WOC during isolation of PS II preparations and removal of the extrinsic proteins. It is known that activity of the WOC is inhibited upon lowering pH from 7.0 to 5.0–5.5 (Krieger and Weis 1993; Allakhverdiev et al. 1997) and this activity can be considerably restored when BC is added to the medium (Allakhverdiev et al. 1997). The low-pH inhibition was attributed to decreasing the HCO3– concentration (owing to conversion of HCO3– to CO2) required for the WOC activity (Allakhverdiev et al. 1997). The present work demonstrates that even a temporary incubation at low pH in medium not depleted of CO2 /HCO3– results in the irreversible loss of WOC activity (the activity can not be restored by subsequent addition of BC), while the samples incubated in the presence of 2 mM NaHCO3 are much more stable. These results show that the HCO3– ion (rather than CO2, H2CO3, or CO32–) is the species essential for both the activity and stability of the WOC. It is important that the protecting effect of BC at low pH is seen in both PS II membranes lacking the extrinsic proteins, and unbroken thylakoids. It is known that during ‘natural’ photosynthesis the intrathylakoid pH can be as low as 4.5–5 owing to light-induced creation of a pH gradient used for ATP-synthesis (Krieger and Weis 1993 and references therein). The low pH causes inactivation of the WOC and Ca2+-depletion may play an essential role in the pH-induced inactivation of oxygen evolution (Krieger and Weis 1993). Our data demonstrate that a decrease of BC concentration induced by lowering the pH may be responsible for the inactivation of the WOC. The importance of optimal pH in both the stroma and lumen for photoprotection and recovery from photoinactivation of PSII has been shown recently (Lee et al. 2002). Acknowledgments This work was supported by a Grant-in-Aid for Specially Promoted Research (No. 08102011) from the Ministry of Education, Science and Culture, Japan, by the National Institute for Basic Biology Cooperative Research Program on the Stress Tolerance of Plants, by a Fellowship from Japan Society for the Promotion of Science to VVK, and by the Russian Foundation of Basic Research (Grant 02-0448776 and 02-04-39015 to VVK). References Allakhverdiev SI, Klimov VV, Proskuryakov II (1988) ESP signals of photosystem II after a complete removal of manganese from pea subchloroplast particles. Biofizika 33, 600–604. Allakhverdiev SI, Feyziev YM, Ahmed A, Hayashi H, Aliev JA, Klimov VV, Murata N, Carpentier R (1996) Stabilization of oxygen evolution and primary electron transport reactions. Journal of Photochemistry and Photobiology B: Biology 34, 149–157. Allakhverdiev SI, Yruela I. Picorel R, Klimov VV (1997) Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem II. Proceedings of the National Academy of Sciences USA 94, 5050–5054.

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Manuscript received 10 April 2003, accepted 26 June 2003

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