Identification of a Ca2+/H+ antiport in the plant chloroplast thylakoid membrane

Abstract

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+ mg-1 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.

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.

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.

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.

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.

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.

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.

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).