Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

Abstract

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl⁻ uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations≥50 mM. In contrast, in the absence of Cl⁻, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid, 1 mM 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, 200 μM Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl⁻/vanadate ratios may result from competition between vanadate and Cl⁻ for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.