Inhibition of dark-induced stomatal closure by fusicoccin involves a removal of hydrogen peroxide in guard cells of Vicia faba

Abstract

Fusicoccin (FC) treatment prevents dark-induced stomatal closure, the mechanism of which is still obscure. By using pharmacological approaches and laser-scanning confocal microscopy, the relationship between FC inhibition of dark-induced stomatal closure and the hydrogen peroxide (H₂O₂) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H₂O₂ and diphenylene iodonium (DPI), an inhibitor of H₂O₂-generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H₂O₂ levels in guard cells in darkness, indicating that FC-caused inhibition of dark-induced stomatal closure is related to the reduction of H₂O₂ levels in guard cells. Furthermore, like ASA, FC not only suppressed H₂O₂-induced stomatal closure and H₂O₂ levels in guard cells treated with H₂O₂ in light, but also reopened the stomata which had been closed by darkness and reduced the level of H₂O₂ that had been generated by darkness, showing that FC causes H₂O₂ removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H₂O₂ and had been closed by dark, and on H₂O₂ levels in guard cells of stomata treated with H₂O₂ and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H₂O₂ and had been closed by dark, which demonstrates that cytosolic acidification mediates FC-induced H₂O₂ removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H₂O₂ removal, and finally prevents dark-induced stomatal closure.