Hydrogen peroxide production is an early event during bicarbonate induced stomatal closure in abaxial epidermis of Arabidopsis

The presence of 2 mM bicarbonate in the incubation medium induced stomatal closure in abaxial epidermis of Arabidopsis. Exposure to 2 mM bicarbonate elevated the levels of H2O2 in guard cells within 5 min, as indicated by the fluorescent probe, dichlorofluorescein diacetate (H2DCF-DA). Bicarbonate-induced stomatal closure as well as H2O2 production were restricted by exogenous catalase or diphenylene iodonium (DPI, an inhibitor of NAD(P)H oxidase). The reduced sensitivity of stomata to bicarbonate and H2O2 production in homozygous atrbohD/F double mutant of Arabidopsis confirmed that NADP(H) oxidase is involved during bicarbonate induced ROS production in guard cells. The production of H2O2 was quicker and greater with ABA than that with bicarbonate. Such pattern of H2O2 production may be one of the reasons for ABA being more effective than bicarbonate, in promoting stomatal closure. Our results demonstrate that H2O2 is an essential secondary messenger during bicarbonate induced stomatal closure in Arabidopsis.