Hydrogen Peroxide Is Involved in Abscisic Acid-Induced Stomatal Closure in Vicia faba

Abstract

One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H2O2), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H2O2 may function as an intermediate in ABA signaling in Vicia faba guard cells. H2O2 inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10−5 m. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H2O2 scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H2O2 was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H2O2 production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H2O2 production involved, and H2O2 may be an intermediate in ABA signaling.