ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H2O2) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H2O2 have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H2O2 in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H2O2 production. Stomatal closure induced by H2O2 is inhibited by the removal of NO with NO scavenger, and both ABA and H2O2 stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H2O2 synthesis nor does NO treatment induce H2O2 production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H2O2 and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H2O2 synthesis. In the NR double mutant nia1, nia2 both ABA and H2O2 fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H2O2 are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H2O2 production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H2O2 and NO-induced stomatal closure.