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

Abstract

Summary: Nitric oxide (NO) and hydrogen peroxide (H 2 O 2) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H 2 O 2 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 H 2 O 2 in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA‐mediated NO generation is in fact dependent on ABA‐induced H 2 O 2 production. Stomatal closure induced by H 2 O 2 is inhibited by the removal of NO with NO scavenger, and both ABA and H 2 O 2 stimulate guard cell NO synthesis. Conversely, NO‐induced stomatal closure does not require H 2 O 2 synthesis nor does NO treatment induce H 2 O 2 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 H 2 O 2 and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA‐mediated H 2 O 2 synthesis. In the NR double mutant nia1, nia2 both ABA and H 2 O 2 fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H 2 O 2 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 H 2 O 2 production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter‐relationship between ABA, endogenous H 2 O 2 and NO‐induced stomatal closure.