Pulsing Cl⁻ channels in coat cells of developing bean seeds linked to hypo-osmotic turgor regulation

Abstract

Seed coat cells in the developing seeds of grain legumes release nutrients to the developing embryo. This occurs into an apoplastic space that separates the maternal (seed coat) and filial (embryo) generations. Protoplasts of seed coat cells from coats of Phaseolus vulgaris L. seeds were isolated and whole-cell current across their plasma membranes was characterized using the patch-clamp technique. A pulsing inward current that displayed a spontaneous activation and voltage-dependent inactivation was observed. The frequency and magnitude of the current pulses were positively dependent on cytoplasmic Cl⁻ concentrations and independent of external cations. The pulse current was inhibited by DIDS and La³⁺, but not by Gd³⁺. Single channel events (conductance=18 pS) could be identified with the inactivating phase of the pulses. Together, these findings are consistent with the current being carried by a burst of Cl⁻ efflux through Cl⁻-permeable channels that activate almost simultaneously. Neomycin caused a reversible inhibition of the pulsed current, suggesting that its activation is likely to be modulated by an IP₃-dependent intracellular Ca²⁺ release. The pharmacological profiles of Cl⁻ efflux from excised seed coats were comparable with those of the Cl⁻ channels in the whole cell configuration, suggesting that the Cl⁻ channels may underpin Cl⁻ efflux from the seed coats. Efflux of Cl⁻ from the seed coats was also stimulated by hypo-osmotic treatment as was the frequency and magnitude of Cl⁻ channel in whole-cell patch clamp experiments. This implies that the Cl⁻ channels responsible for the pulsed Cl⁻ currents are likely to be a component of the turgor-regulatory mechanism in developing bean seeds.