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 H 2 O 2 in guard cells within 5 min, as indicated by the fluorescent probe, dichlorofluorescein diacetate (H 2 DCF-DA). Bicarbonate-induced stomatal closure as well as H 2 O 2 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 H 2 O 2 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 H 2 O 2 was quicker and greater with ABA than that with bicarbonate. Such pattern of H 2 O 2 production may be one of the reasons for ABA being more effective than bicarbonate, in promoting stomatal closure. Our results demonstrate that H 2 O 2 is an essential secondary messenger during bicarbonate induced stomatal closure in Arabidopsis.