Plant Physiol. (1999) 119: 1379-1386

Identification of a Ca²⁺/H⁺ Antiport in the Plant Chloroplast Thylakoid Membrane¹

William F. Ettinger^*, Anne M. Clear, Katheryn J. Fanning, and Mary Lou Peck

Department of Biology, Gonzaga University, E. 502 Boone Avenue, Spokane, Washington 99258

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

To assess the availability of Ca²⁺ in the lumen of the thylakoid membrane that is required to support the assembly of the oxygen-evolving complex of photosystem II, we have investigated the mechanism of ⁴⁵Ca²⁺ transport into the lumen of pea (Pisum sativum) thylakoid membranes using silicone-oil centrifugation. Trans-thylakoid Ca²⁺ transport is dependent on light or, in the dark, on exogenously added ATP. Both light and ATP hydrolysis are coupled to Ca²⁺ transport through the formation of a transthylakoid pH gradient. The H⁺-transporting ionophores nigericin/K⁺ and carbonyl cyanide 3-chlorophenylhydrazone inhibit the transport of Ca²⁺. Thylakoid membranes are capable of accumulating up to 30 nmol Ca²⁺ mg¹ chlorophyll from external concentrations of 15 µM over the course of a 15-min reaction. These results are consistent with the presence of an active Ca²⁺/H⁺ antiport in the thylakoid membrane. Ca²⁺ transport across the thylakoid membrane has significant implications for chloroplast and plant Ca²⁺ homeostasis. We propose a model of chloroplast Ca²⁺ regulation whereby the activity of the Ca²⁺/H⁺ antiporter facilitates the light-dependent uptake of Ca²⁺ by chloroplasts and reduces stromal Ca²⁺ levels.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

The physiology of Ca²⁺ in the plant cell and its distribution among various organelles, gradients, and transient fluxes continues to be the subject of research and the focus of several recent reviews (Evans et al., 1991; Bush, 1993, 1995; Gilroy et al., 1993). However, little attention has been given to the problem of Ca²⁺ transport across the thylakoid membrane. In fact, Ca²⁺ is required for several essential processes inside the chloroplast thylakoid lumen. In particular, Ca²⁺ ions are essential for the function of the OEC, a multimeric complex in the thylakoid lumen responsible for light-dependent oxygen evolution in plants. Functional assembly of PSII and the OEC requires that all essential polypeptides and cofactors are present in the stroma, thylakoid membrane, or thylakoid lumen.

The assembly of the OEC inside the thylakoid lumen requires the assembly of the OE33, OE23, and OE17 polypeptides and the essential inorganic ions Mn²⁺, Ca²⁺, and Cl to the OEC in a light-dependent process (Ghanotakis et al., 1984; Becker et al., 1985; Miller and Brudvig, 1989). Additionally, in saturating light the reaction center D1 protein of PSII rapidly becomes damaged in a process known as photoinhibition (Mattoo et al., 1989). Damage resulting from photoinhibition is repaired by the proteolytic degradation of the D1 protein, followed by the disassembly of the remaining PSII proteins and OEC polypeptides, the resynthesis of D1, and the reassembly of a new PSII core and OEC from existing polypeptides and ions (Broussac et al., 1990; Hundal et al., 1990a, 1990b; Virgin et al., 1990). Therefore, both the initial assembly of PSII and its subsequent reassembly after photoinhibition require Ca²⁺ availability in the chloroplast thylakoid lumen. Furthermore, Ca²⁺ in the thylakoid lumen has been implicated in the formation and maintenance of localized proton domains (Dilley and Chiang, 1989) and the stabilization of the high redox potential form of Cyt b₅₅₉ (McNamera and Gounaris, 1995). All of these processes require the availability of Ca²⁺ in the thylakoid lumen.

Ca²⁺ transported to the thylakoid lumen must originate from the cytosol and be transported through the chloroplast envelope membranes, the stroma, and the thylakoid membrane. It is well documented that Ca²⁺ is actively transported across the chloroplast envelope membranes in the light (Nobel and Packer, 1965; Nobel, 1967, 1969; Muto et al., 1982; Kreimer et al., 1985a, 1985b, 1988). Trans-envelope Ca²⁺ movement from the cytosol to the stroma is believed to be energetically coupled to a significant membrane potential across the inner envelope membrane through an electrogenic Ca²⁺ pump (Kreimer et al., 1985b).

Although it is conceivable that Ca²⁺ transport across the thylakoid membrane may be dependent on simple diffusion, the diffusion of a such a large divalent ion across the lipid bilayer of the thylakoid membrane could be a rate-limiting process of OEC biogenesis. Moreover, measurements of transthylakoid membrane potential in the light indicate that there is a 15- to 30-mV positive membrane potential across the thylakoid membrane (Bulychev et al., 1972; Hangarter and Good, 1982). According to the Nernst equation, neither simple nor facilitated diffusion of Ca²⁺ across the thylakoid membrane would be thermodynamically spontaneous unless the ratio of free Ca²⁺ were greater than about 3:1 (stromal:luminal). However, the low-affinity Ca²⁺-binding site for the photoactivation process of the OEC is reported to have a K_d of 0.3 mM (Miller and Brudvig, 1989; see also Debus, 1992). This suggests that significantly higher Ca²⁺ concentrations are required in the thylakoid lumen for PSII assembly than the 2 to 6 µM free Ca²⁺ in the stroma (Kreimer et al., 1988; Johnson et al., 1995).

In this study we investigated how the essential inorganic ion Ca²⁺ was translocated across the thylakoid membrane to the thylakoid lumen. Using isolated, intact thylakoid membranes we have demonstrated that a significant amount of ⁴⁵Ca²⁺ is translocated across the thylakoid membrane in an energy- and time-dependent process. Light (or ATP in the dark) is necessary to support ⁴⁵Ca²⁺ transport. Furthermore, the activity of the transport process was shown to be sensitive to proton-translocating uncouplers such as CCCP and nigericin/K⁺. On the basis of our results we propose that a Ca²⁺/H⁺ antiport actively moves Ca²⁺ from the stroma into the thylakoid lumen in the light. The antiporter provides the concentrations of luminal Ca²⁺ required for the assembly of the OEC and supports other Ca²⁺-requiring reactions in the thylakoid lumen.

	MATERIALS AND METHODS

Top Abstract Introduction Methods Results Discussion References

Materials

Chloroplast and Thylakoid Isolation

Measurement of ⁴⁵Ca Transport

1

1

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Energy-Dependent Transport of Ca²⁺ across the Thylakoid Membrane

1

1

View larger version (25K): [in this window] [in a new window]   Figure 1. Light-dependent accumulation of 45Ca2+ across intact, isolated thylakoid membranes. Duplicate transport reactions were initiated by adding 45Ca2+ (15 µM final concentration) to thylakoid membranes maintained in the light (open bars) or dark (hatched bars). After mixing and at the intervals indicated, 60-µL aliquots were removed from the reactions and the transport reactions were stopped by centrifugation of the membranes through silicone oil. Chl, Chlorophyll.

Light or ATP Hydrolysis Can Facilitate Ca²⁺ Transport

View larger version (27K): [in this window] [in a new window]   Figure 2. Light- or ATP-dependent accumulation of 45Ca2+. Transport reactions were initiated by adding 45Ca2+ (1.5 µM final concentration) to thylakoid membranes suspended in import buffer containing 5 mM MgCl2 and 3 mM ATP. Duplicate reactions were maintained in the dark or light. Reactions were terminated after 15 min. White bars, +ATP; hatched bars, ATP. Chl, Chlorophyll.

pH Is Necessary for Ca²⁺ Transport

View larger version (16K): [in this window] [in a new window]   Figure 3. The light-dependent 45Ca2+ transport reaction is sensitive to nigericin. Reactions were initiated by adding 45Ca2+ (1.5 µM final concentration) to thylakoid membranes suspended in import buffer in the light. Nigericin was added from an ethanolic stock to the concentration indicated. Potassium, approximately 25 mM, was present in the reaction as the counterion to the Tricine buffer. An equal volume of ethanol (1 µL per 60-µL reaction) was added to all reactions. Reactions were terminated after 15 min. Chl, Chlorophyll.

Light Energy Provides the pH Required for Ca²⁺ Import

View larger version (23K): [in this window] [in a new window]   Figure 4. ATP-dependent 45Ca2+ transport is sensitive to inhibitors of the H+-ATP synthase. Thylakoid membranes were preincubated with DCCD (100 µM) for 10 min and the membranes were repurified by centrifugation before use. Tentoxin (4 µM) was added to intact chloroplasts, which were then incubated on ice for 1 h before thylakoid membranes were isolated. AMP-PNP was added to a final concentration of 3 mM from an aqueous stock. Transport reactions were initiated by adding 45Ca2+ (1.5 µM final concentration) to thylakoid membranes suspended in import buffer containing 5 mM MgCl2 and 3 mM ATP in the light (white bars) or in the dark (hatched bars). Reactions were terminated after 15 min. Chl, Chlorophyll.

Characterization of the Ca²⁺/H⁺ Antiporter

View larger version (47K): [in this window] [in a new window]   Figure 5. Characterization of 45Ca2+ transport across the thylakoid membrane. Light-dependent 45Ca2+ transport was assessed in the presence of DCPIP (10 µM), K3FeCN6 (1 mM), Ca2+ ionophore A23187 (5 µM), CCCP (5 µM), LaCl3 (100 µM), and MnCl2 (100 µM). One microliter of a concentrated stock of each effector was added to the thylakoid membranes before the reaction was initiated. Transport reactions were initiated by adding 45Ca2+ (1.5 µM final concentration) to thylakoid membranes suspended in import buffer at 25°C in the light or dark. Reactions were terminated after 15 min. Chl, Chlorophyll.

We investigated the kinetics of Ca²⁺ uptake by chloroplast thylakoid membranes and intact chloroplasts by measuring the initial rate of Ca²⁺ uptake as a function of increasing Ca²⁺ concentration (Fig. 6). Analysis of these data demonstrated that the thylakoid transport reaction has a K_m for Ca²⁺ of 24 µM and a V_max of 7 nmol min¹ mg¹ chlorophyll. The K_m for Ca²⁺ of the thylakoid membrane Ca²⁺/H⁺ antiporter is comparable to reported K_m values (10-70 µM) for the Ca²⁺/H⁺ antiporters isolated from higher-plant tonoplast membranes (Schumaker and Sze, 1986; Blackford et al., 1990). The transport of Ca²⁺ across thylakoid membranes and intact chloroplasts showed similar kinetics. Intact chloroplasts have a K_m for Ca²⁺ of 22 µM and a V_max of 3 nmol min¹ mg¹ chlorophyll (Fig. 7). However, the value we obtained for the K_m for Ca²⁺ of the chloroplast Ca²⁺-transport reaction was significantly lower than the 180 and 188 µM values obtained by Muto et al. (1982) and Kreimer et al. (1985b), respectively. The difference in relative Ca²⁺ K_m values between our data and previously reported values may reflect our differing methods but may also indicate that, like mitochondria, chloroplasts have multiple Ca²⁺-transport mechanisms (Gunter et al., 1994).

View larger version (19K): [in this window] [in a new window]   Figure 6. Kinetic analysis of Ca2+ uptake by isolated thylakoids() and intact chloroplasts (). A series of reaction solutions were prepared by combining CaCl2 and 3 µM of 45Ca2+ to a final Ca2+ concentration of 3.0, 8.6, 25.5, 48.0, or 93.0 µM. Light-dependent reactions were run in triplicate and initiated by the addition of thylakoid membranes. Reactions were terminated after 1.0, 2.5, and 4.0 min. Reaction rates for each Ca2+ concentration were determined by linear-regression analysis of the average Ca2+ uptake at the three different time points. Reactions with intact chloroplasts were run in an identical manner, except the reactions were terminated by centrifugation of the membranes through a layer of 100% AR-200 silicone oil. Results shown are the averages of two independent experiments. Chl, Chlorophyll.

View larger version (21K): [in this window] [in a new window]   Figure 7. A model of Ca2+ flux in the intact chloroplast. Dark-adapted chloroplasts contain micromolar levels of free Ca2+ in the stroma, and Mg2+ ions are sequestered in the thylakoid lumen (A). The light-stimulated release of Mg2+ from the thylakoid lumen reduces the trans-thylakoid membrane potential (B). The Ca2+/H+ antiporter pumps Ca2+ into the thylakoid lumen and prevents Ca2+-mediated inhibition of CO2 fixation (C). Adaptation to the dark prompts the electroneutral exchange of Ca2+ and Mg2+ across the thylakoid membrane and results in Calvin-Benson-cycle inhibition by Ca2+ in the dark (D).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

We initiated this study to elucidate the mechanism of Ca²⁺ transport across the thylakoid membrane and to assess its availability for assembly into the OEC. We utilized thylakoid membrane fractions prepared by osmotic lysis from freshly isolated Percoll-gradient-purified chloroplasts to ensure membrane purity and stability. Thylakoid membranes prepared the same way have been shown to be competent in the pH- or ATP-dependent transport of the precursor forms of plastocyanin, OE33, OE23, and OE17 and in the integration of the precursor form of the light-harvesting chlorophyll a/b protein into the thylakoid membrane (Cline et al., 1992). Uptake of ⁴⁵Ca²⁺ by the thylakoid membranes was monitored by silicone oil centrifugation and liquid-scintillation counting of the recovered membranes (Heldt, 1980). This method has enabled us to demonstrate that the import of a significant amount Ca²⁺ into the thylakoids is time and energy dependent. Our results demonstrate that the transthylakoid pH is necessary and sufficient for Ca²⁺ import across the thylakoid membrane, and our results also strongly suggest that the mechanism for the import of Ca²⁺ is a Ca²⁺/H⁺ antiporter powered by a light- or ATP-induced proton gradient.

Early studies of Ca²⁺ transport across the membranes of isolated chloroplasts were initiated by Nobel and coworkers (Nobel and Packer, 1965; Nobel, 1967, 1969), who demonstrated that Ca²⁺ transport into chloroplast membranes is a light-dependent process that requires ATP and Mg²⁺ and is facilitated by the addition of thiol reagents and PMS. However, these studies did not suggest a mechanism of Ca²⁺ transport across the chloroplast membrane, nor did they separate out the role of the envelope or thylakoid membranes, light, pH, or ATP in the transport process. Studies with intact chloroplasts have been carried out by Muto et al. (1982) and Kreimer et al. (1985a, 1985b, 1988). These studies demonstrated that Ca²⁺ transport from the cytosol into the chloroplast is a light-dependent process. According to their model, the total Ca²⁺ concentration in the chloroplast is 4 to 23 mM. However, much of the stromal Ca²⁺ is bound or sequestered, and the free Ca²⁺ concentration in the stroma is only 2 to 6 µM. Therefore, stroma-free Ca²⁺ is about 10-fold more concentrated than in the cytosol (100-300 nM), and transport must be energetically facilitated by an electrogenic Ca²⁺ pump in the inner envelope membrane (Kreimer et al., 1985a, 1985b, 1988). Other information about transthylakoid ion transport comes from more recent patch-clamp studies demonstrating the existence of thylakoid membrane cation channels that have conductance for K⁺, Mg²⁺, and Ca²⁺ (in order of their decreasing conductance) (Enz et al., 1993; Pottosin and Schönknecht, 1996). However, patch-clamp studies do not address the direction, energetics, or mechanism of Ca²⁺ transport across the thylakoid membrane.

A H⁺/Ca²⁺ antiporter in the thylakoid membrane would certainly supply the Ca²⁺ required for the assembly and maintenance of PSII and other thylakoid functions noted above. Additionally, the transport of Ca²⁺ across the thylakoid membrane may be associated with several important processes in chloroplasts. The transition from dark to light is a key regulatory period for stromal enzymes involved in carbon reduction. However, the role of Ca²⁺ in the regulation of Calvin-Benson-cycle enzymes is more complex. It appears that low levels of Ca²⁺ in the stroma are necessary for the activation of FBPase in the presence of reduced thioredoxin. Ca²⁺ apparently inhibits the catalytic activity of the enzyme (Charles and Halliwell, 1980; Hertig and Wolosiuk, 1980). Thus, there is an apparent discrepancy between the light-dependent transport of Ca²⁺ from the cytosol to the stroma and the activation of Calvin-Benson-cycle enzymes, because Ca²⁺ is known to inhibit the activity of FBPase, and elevated Ca²⁺ levels have a pronounced inhibitory effect on CO₂ fixation. Ca²⁺ accumulated by chloroplasts in the light must be tightly bound in the stroma or sequestered in the thylakoid to prevent the inhibition of CO₂ fixation by Ca²⁺ (Wolosiuk et al., 1993). The thylakoid membrane Ca²⁺/H⁺ antiporter would provide the means to sequester chloroplast Ca²⁺ in the thylakoid lumen in the light and thus prevent the Ca²⁺-mediated inhibition of Calvin-Benson-cycle enzymes.

Stores of Ca²⁺ in the thylakoid lumen may play an important role in the regulation of Calvin-Benson-cycle enzymes. Recently, Johnson et al. (1995) noted a sharp rise in stromal Ca²⁺ concentrations after the transition of plants from light to dark. This rise in stromal Ca²⁺ peaked 20 to 25 min after the transition from light to dark and may play a role in signaling the light-to-dark transition through the inhibition of Calvin-Benson-cycle enzymes. The peak is believed to represent a change in stromal free Ca²⁺ levels from a basal level of 150 nM to approximately 5 to 10 µM. Such a pronounced increase is unlikely to originate from the cytosol without the expenditure of a great deal of cellular energy in the dark. However, the rapid release of Ca²⁺ from luminal stores could result in dramatic changes in stromal Ca²⁺ concentrations, even if the Ca²⁺ was free to move from the stroma to the cytosol. We propose that the dramatic rise in stromal Ca²⁺ after the light-to-dark transition that was observed by Johnson et al. (1995) is coupled to the release of stores of Ca²⁺ from the thylakoid lumen.

On the basis of our results and the observations by Johnson et al. (1995), we propose that there are dramatic fluxes of Ca²⁺ across the thylakoid membrane that are induced by the transitions from dark to light and light to dark (Fig. 7). When dark-adapted chloroplasts are transferred to the light, there is an initial increase in trans-thylakoid membrane potential to about 60 to 80 mV (lumen positive), which subsequently decays within a few seconds to a steady state of 15 to 30 mV (Bulychev et al., 1972; Hangarter and Good, 1982; Remis et al., 1986). The collapse of the transient membrane potential is caused by the efflux of Mg²⁺ from the thylakoid lumen to the stroma (Fig. 7B). The magnitude of light-induced Mg²⁺ efflux is approximately 26 to 120 nmol Mg²⁺ mg¹ chlorophyll (Barber et al., 1974; Hind et al., 1974; Portis and Heldt, 1976; Krause, 1977; Enz et al., 1993).

Several Calvin-Benson-cycle enzymes, including Rubisco, FBPase, and sedoheptulose-1,7-bisphosphatase, are activated by Mg²⁺ ions (Portis and Heldt, 1976), which is in apparent agreement with the light-induced efflux of Mg²⁺ ions from the thylakoid lumen. However, the mechanism by which Mg²⁺ is returned to the thylakoid lumen in the dark has not yet been elucidated. We propose that such a mechanism may be the coupling of the exchange of luminal Ca²⁺ accumulated in the light (Fig. 7C) for stromal Mg²⁺ (Fig. 7D). The counterexchange of these ions would be electrically neutral. Any mechanism for exchanging Ca²⁺ and Mg²⁺ across the thylakoid membrane in the dark would need to be tightly regulated to prevent a futile cycle from forming. This exchange may be regulated by the state of oxidation or by the reduction of thioredoxin, as are several enzymes of the Calvin-Benson cycle (for review, see Wolosiuk, 1993). Cycling of Ca²⁺ and Mg²⁺ across the thylakoid membrane is correlated with the activation of Calvin-Benson-cycle enzymes by Mg²⁺ in the light and their inhibition by Ca²⁺ in the dark.

In conclusion, we have demonstrated the existence of a Ca²⁺/H⁺ antiporter in the thylakoid membrane. This an-tiporter provides the mechanism and energetics necessary to transport Ca²⁺ into the thylakoid lumen in the light and thereby supply Ca²⁺ for Ca²⁺-dependent processes in the thylakoid lumen, such as the assembly of the OEC. Additionally, our model of chloroplast Ca²⁺ homeostasis proposes that the uptake of cytosolic Ca²⁺ by chloroplasts in the light does not lead to a pronounced increase in stromal Ca²⁺ but, rather, results in an increase of Ca²⁺ stores in the thylakoid lumen. The Ca²⁺/H⁺ antiporter is critical for transporting stromal Ca²⁺ into the thylakoid lumen in the light, where it would have no inhibitory effect on Calvin-Benson-cycle enzymes. We propose that pools of Ca²⁺ sequestered in the lumen during a period of light are released from the lumen after a period of dark adaptation and complement the activity of thioredoxin in the inhibition of Calvin-Benson-cycle enzymes in the dark.

	FOOTNOTES

   Received August 21, 1998; accepted January 7, 1999.
^*   Abbreviations: AMP-PNP, 5`-adenylyl imidodiphosphate; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; DCCD, N,N-dicyclohexylcarbodiimide; DCPIP, 2,6-dichlorophenolindophenol; FBPase, Fru-1,6-bisphosphatase; OEC, oxygen-evolving complex of PSII.

	ACKNOWLEDGMENTS

We would like to thank Dr. Steven Theg for his valuable suggestions and Amanda B. Lehman and Hilary I. Van Hole for their excellent technical assistance. Silicone oils AR-200 and AR-20 were generously donated by Wacker Silicones.

	LITERATURE  CITED

Top Abstract Introduction Methods Results Discussion References

Arnon DI (1949) Copper enzymes in isolated chloroplasts. Plant Physiol 24: 1-15 [Free Full Text]

Barber J, Mills JB, Nicolson J (1974) Studies with cation specific ionophores show that within the intact chloroplast Mg²⁺ acts as the main exchange cation for H⁺ pumping. FEBS Lett 49: 106-110 [Medline]

Becker DW, Callahan FE, Cheniae GM (1985) Photoactivation of NH₂OH-treated leaves: reassembly of released extrinsic PSII polypeptides and relegation of Mn into the polynuclear Mn catalyst of water oxidation. FEBS Lett 192: 209-214 [CrossRef]

Blackford S, Rea PA, Sanders D (1990) Voltage sensitivity of H⁺/Ca²⁺ antiport in higher plant tonoplast suggests a role in vacuolar calcium accumulation. J Biol Chem 265: 9617-9620 [Abstract/Free Full Text]

Broussac A, Zimmermann J-L, Rutherford AW, Lavergne J (1990) Histidine oxidation in the oxygen-evolving photosystem II enzyme. Nature 347: 303-306 [CrossRef]

Bulychev AA, Andrianov VK, Kurella GA, Litvin FF (1972) Micro-electrode measurements of the transmembrane potential of chloroplasts and its photoinduced changes. Nature 236: 175-177

Bush DS (1993) Regulation of cytosolic calcium in plants. Plant Physiol 103: 7-13 [ISI][Medline]

Bush DS (1995) Calcium regulation in plant cells and its role in signaling. Annu Rev Plant Physiol Plant Mol Biol 46: 95-122 [CrossRef][ISI]

Charles SA, Halliwell B (1980) Action of calcium ions on spinach (Spinacia oleracea) chloroplast fructose bisphosphatase and other enzymes of the Calvin cycle. Biochem J 188: 775-779 [Medline]

Cline K, Ettinger WF, Theg SM (1992) Protein-specific energy requirements for protein transport across or into thylakoid membranes: two lumenal proteins are translocated in the absence of ATP. J Biol Chem 267: 2688-2696 [Abstract/Free Full Text]

Cline K, Werner-Washburn M, Lubben TH, Keegstra K (1985) Precursors to two nuclear encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J Biol Chem 260: 3691-3696 [Abstract/Free Full Text]

Debus RJ (1992) The manganese and calcium ions of photosynthetic oxygen evolution. Biochim Biophys Acta 1102: 269-352 [CrossRef][Medline]

Dilley RA, Chiang GG (1989) Chloroplast thylakoid membrane-bound Ca²⁺ acts in a gating mechanism to regulate energy-coupled proton fluxes. Ann NY Acad Sci 574: 246-267 [Medline]

Enz C, Steinkanp T, Wagner R (1993) Ion channels in the thylakoid membrane (a patch-clamp study). Biochim Biophys Acta 1143: 67-76

Evans DE, Briars S-A, Williams LE (1991) Active calcium transport by plant cell membranes. J Exp Biol 42: 285-303

Ghanotakis DF, Babcock GT, Yocum CF (1984) Calcium reconstitutes high levels of oxygen evolution in polypeptide-depleted photosystem II preparations. FEBS Lett 167: 127-130 [CrossRef]

Gilroy S, Bethke PC, Jones RL (1993) Calcium homeostasis in plants. J Cell Sci 106: 453-462 [ISI][Medline]

Gunter TE, Gunter KK, Sheu S-S, Gavin CE (1994) Mitochondrial calcium transport: physiological and pathological relevance. Am J Physiol 267: 313-339

Hangarter RP, Good NE (1982) Energy thresholds for ATP synthesis in chloroplasts. Biochim Biophys Acta 681: 397-404

Heldt HW (1980) Measurement of metabolite movement across the envelope and of the pH in the stroma and the thylakoid space in intact chloroplasts. Methods Enzymol 69: 604-613

Hertig C, Wolosiuk RA (1980) A dual effect of Ca²⁺ on chloroplast fructose-1,6-bisphosphatase. Biochem Biophys Res Commun 97: 325-333 [Medline]

Hind G, Nakatani HY, Izawa S (1974) Light-dependent redistribution of ions in suspensions of chloroplast thylakoid membranes. Proc Natl Acad Sci USA 71: 1484-1488 [Abstract/Free Full Text]

Hundal T, Aro EM, Carlberg I, Andersson B (1990a) Restoration of light induced photosystem II inhibition without de novo protein synthesis. FEBS Lett 267: 203-206 [CrossRef][Medline]

Hundal T, Virgin I, Styring S, Andersson B (1990b) Changes in the organization of photosystem II following light-induced D1 protein degradation. Biochim Biophys Acta 1017: 235-241 [CrossRef]

Johnson CH, Knight MR, Kondo T, Masson P, Sedbrook J, Haley A, Trewavas A (1995) Circadian oscillations of cytosolic and chloroplastic free calcium in plants. Science 269: 1863-1865 [Abstract/Free Full Text]

Krause GH (1977) Light-induced movement of magnesium ions in intact chloroplasts: spectroscopic studies with eriochrome blue SE. Biochim Biophys Acta 460: 500-510 [Medline]

Kreimer G, Melkonian M, Holtum JAM, Latzko E (1985a) Characterization of calcium fluxes across the envelope of intact spinach chloroplasts. Planta 166: 515-523 [CrossRef]

Kreimer G, Melkonian M, Holtum JAM, Latzko E (1988) Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase. Plant Physiol 86: 423-428 [Abstract/Free Full Text]

Kreimer G, Melkonian M, Latzko E (1985b) An electrogenic uniport mediates light-dependent Ca²⁺-influx into intact spinach chloroplasts. FEBS Lett 180: 253-258 [CrossRef]

Mattoo AK, Marder JB, Edelman M (1989) Dynamics of the photosystem II reaction center. Cell 56: 241-246 [CrossRef][ISI][Medline]

McNamera VP, Gounaris K (1995) Granal photosystem II complexes contain only the high redox potential form of cytochrome b-559 which is stabilized by the ligation of calcium. Biochim Biophys Acta 1231: 289-296

Miller A-F, Brudvig GW (1989) Manganese and calcium requirements for reconstitution of oxygen-evolution in manganese-depleted photosystem II membranes. Biochemistry 28: 8181-8190 [CrossRef][Medline]

Muto S, Izawa S, Miyachi S (1982) Light-induced Ca²⁺ uptake by intact chloroplasts. FEBS Lett 139: 250-254 [CrossRef]

Nelson N, Eytan E, Notsani B-E, Sigrist H, Sigrist-Nelson K, Gitler C (1977) Isolation of a chloroplast N,N-dicyclohexylcarbodiimide-binding protein proteolipid, active in proton translocation. Proc Natl Acad Sci USA 74: 2375-2378 [Abstract/Free Full Text]

Nobel PS (1967) Calcium uptake, ATPase and photophosphorylation by chloroplasts in vitro. Nature 214: 875-877 [Medline]

Nobel P (1969) Light-induced changes in the ionic composition of chloroplasts in Pisum sativum. Biochim Biophys Acta 172: 134-143 [Medline]

Nobel PS, Packer L (1965) Light-dependent ion translocation in spinach chloroplasts. Plant Physiol 633-640

Portis AR, Heldt HW (1976) Light dependent changes of the Mg²⁺ concentration in the stroma in relation to the Mg²⁺ dependency of CO₂ fixation in intact chloroplasts. Biochim Biophys Acta 449: 434-446 [Medline]

Pottosin II, Schönknecht G (1996) Ion channel permeable for divalent and monovalent cations in native spinach thylakoid membranes. J Membr Biol 152: 223-233 [CrossRef][ISI][Medline]

Remis D, Bulychev AA, Kurella GA (1986) The electrical and chemical components of the proton motive force in chloroplasts as measured with capillary and pH-sensitive electrodes. Biochim Biophys Acta 852: 68-73

Robinson SP, Wiskich JT (1977) Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO₂ fixation and electron transport. Biochim Biophys Acta 461: 131-140 [Medline]

Schumaker KS, Sze H (1986) Calcium transport into the vacuole of oat roots: characterization of H⁺/Ca²⁺ exchange activity. J Biol Chem 261: 12172-12187 [Abstract/Free Full Text]

Shavit N, Dilley RA, San Peitro A (1968) Ion translocation in isolated chloroplasts uncoupling of photophosphorylation and translocation of K⁺ and H⁺ by nigericin. Biochemistry 7: 2356-2363 [Medline]

Steele JA, Uchytil TF, Durbin RD, Bhatnagar P, Rich DH (1976) Chloroplast coupling factor I: a species-specific receptor for tentoxin. Proc Natl Acad Sci USA 73: 2245-2248 [Abstract/Free Full Text]

Theg SM, Filar LJ, Dilley RA (1986) Photoinactivation of chloroplasts already inhibited on the oxidizing side of photosystem II. Biochim Biophys Acta 849: 104-111

Virgin I, Ghanotakis DF, Andersson B (1990) Light induced D1-protein degradation in isolated photosystem II core complexes. FEBS Lett 269: 45-48 [CrossRef][ISI][Medline]

Wolosiuk RA, Ballicora MA, Hagelin K (1993) The reductive pentose phosphate cycle for photosynthetic CO₂ assimilation: enzyme modulation. FASEB J 7: 622-737 [Abstract]

Copyright Clearance Center:   0032-0889/99/119//08
 
© 1999 American Society of Plant Physiologists