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

Abstract

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.