Novel roles of ascorbate in plants: Induction of cytosolic Ca 2+ signals and efflux from cells via anion channels

Abstract

Ascorbate is not often considered as a signalling molecule in plants. This study demonstrates that, in Arabidopsis roots, exogenous l-ascorbic acid triggers a transient increase of the cytosolic free calcium activity ([Ca 2+ ] cyt.) that is central to plant signalling. Exogenous copper and iron stimulate the ascorbate-induced [Ca 2+ ] cyt. elevation, while cation channel blockers, free radical scavengers, low extracellular [Ca 2+ ], transition metal chelators, and removal of the cell wall inhibit this reaction. These data show that apoplastic redox-active transition metals are involved in the ascorbate-induced [Ca 2+ ] cyt. elevation. Exogenous ascorbate also induces a moderate increase in programmed cell death symptoms in intact roots, but it does not activate Ca 2+ influx currents in patch-clamped root protoplasts. Intriguingly, the replacement of gluconate with ascorbate in the patch-clamp pipette reveals a large ascorbate efflux current, which shows sensitivity to the anion channel blocker, anthracene-9-carboxylic acid (A9C), indicative of the ascorbate release via anion channels. EPR spectroscopy measurements demonstrate that salinity (NaCl) triggers the accumulation of root apoplastic ascorbyl radicals in an A9C-dependent manner, confirming that l-ascorbate leaks through anion channels under depolarization. This mechanism may underlie ascorbate release, signalling phenomena, apoplastic redox reactions, iron acquisition, and control the ionic and electrical equilibrium (together with K + efflux via GORK channels).