Hydrogen peroxide is a common signal for darkness- and ABA-induced stomatal closure in Pisum sativum

The requirement for hydrogen peroxide (H₂O₂) generation and action during stomatal closure induced by darkness and abscisic acid (ABA) was investigated in pea (Pisum sativum L.). Stomatal closure induced by darkness or ABA was inhibited by the H₂O₂-scavenging enzyme catalase or the antioxidant N-acetyl cysteine (NAC), or by diphenylene iodonium (DPI), an inhibitor of the H₂O₂-generating enzyme NADPH oxidase. Exogenous H₂O₂ induced stomatal closure in a dose- and time-dependent manner, and H₂O₂ was also required for ABA-inhibition of stomatal opening in the light. H₂O₂ accumulation in guard cells was increased by darkness or ABA, as assessed with the fluorescent dye dichlorodihydrofluorescein diacetate (H₂-DCFDA) and confocal microscopy. Such increases were inhibited by catalase, NAC or DPI, consistent with the effects of these compounds on stomatal apertures. Employing polymerase chain reaction (PCR) with degenerate oligonucleotide primers, several NADPH oxidase homologues were identified from pea genomic DNA that had substantial identity to the Arabidopsis thaliana (L.) Heynh. rboh (respiratory burst oxidase homologue) genes. Furthermore, an antibody raised against the tomato rboh identified immunoreactive proteins in epidermal, mesophyll and guard cells.