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

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 (H(2)O(2)) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H(2)O(2) and diphenylene iodonium (DPI), an inhibitor of H(2)O(2)-generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H(2)O(2) levels in guard cells in darkness, indicating that FC-caused inhibition of dark-induced stomatal closure is related to the reduction of H(2)O(2) levels in guard cells. Furthermore, like ASA, FC not only suppressed H(2)O(2)-induced stomatal closure and H(2)O(2) levels in guard cells treated with H(2)O(2) in light, but also reopened the stomata which had been closed by darkness and reduced the level of H(2)O(2) that had been generated by darkness, showing that FC causes H(2)O(2) removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H(2)O(2) and had been closed by dark, and on H(2)O(2) levels in guard cells of stomata treated with H(2)O(2) and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H(2)O(2) and had been closed by dark, which demonstrates that cytosolic acidification mediates FC-induced H(2)O(2) removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H(2)O(2) removal, and finally prevents dark-induced stomatal closure.