H⁺ Fluxes at Plasmalemma Level: In Vivo Evidence for a Significant Contribution of the Ca²⁺-ATPase and for the Involvement of its Activity in the Abscisic Acid-Induced Changes in Egeria Densa Leaves

Abstract:

The features of Ca²⁺ fluxes, the importance of the Ca²⁺ pump-mediated H⁺/Ca²⁺ exchanges at plasmalemma level, and the possible involvement of Ca²⁺-ATPase activity in ABA-induced changes of H⁺ fluxes were studied in Egeria densa leaves. The results presented show that, while in basal conditions no net Ca²⁺ flux was evident, a conspicuous Ca²⁺ influx (about 1.1 ìmol g⁻¹ FW h⁻¹) occurred. The concomitant efflux of Ca²⁺ was markedly reduced by treatment with 5 ìM eosin Y (EY), a specific inhibitor of the Ca²⁺-ATPase, that completely blocked the transport of Ca²⁺ after the first 20 - 30 min. The decrease in Ca²⁺ efflux induced by EY was associated with a significant increase in net H⁺ extrusion (−ÄH⁺) and a small but significant cytoplasmic alkalinization. The shift of external [Ca²⁺] from 0.3 to 0.2 mM (reducing Ca²⁺ uptake by about 30 %) and the hindrance of Ca²⁺ influx by La³⁺ were accompanied by progressively higher −ÄH⁺ increases, in agreement with a gradual decrease in the activity of a mechanism counteracting the Ca²⁺ influx by an nH⁺/Ca²⁺ exchange. The ABA-induced decreases in −ÄH⁺ and pH_cyt were accompanied by a significant increase in Ca²⁺ efflux, all these effects being almost completely suppressed by EY, in line with the view that the ABA effects on H⁺ fluxes are due to activation of the plasmalemma Ca²⁺-ATPase. These results substantially stress the high sensitivity and efficacy of the plasmalemma Ca²⁺ pump in removing from the cytoplasm the Ca²⁺ taken up, and the importance of the contribution of Ca²⁺ pump-mediated H⁺/Ca²⁺ fluxes in bringing about global changes of H⁺ fluxes at plasmalemma level.

Abbreviations:

ABA: abscisic acid BTP: bis-tris propane(1,3-bis[tris(hydroxymethyl) methylamino]propane) [Ca²⁺]_cyt: free cytoplasmic Ca²⁺ concentration −ÄH⁺: net H⁺ extrusion DMO: 5,5-dimethyloxazolidine-2,4-dione EB: erythrosin B EY: eosin Y pH_cyt: cytoplasmic pH

Key words:

ABA - Ca²⁺-ATPase - cytoplasmic pH - Egeria densa - H⁺-ATPase - H⁺/Ca²⁺ fluxes

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