The ascorbic acid cycle mediates signal transduction leading to stress-induced stomatal closure

Using a combination of pharmacological approaches, mutation analysis and a gene silencing strategy, we present evidence that treatment of tomato (Lycopersicon esculentum Mill.) plants with exogenous ascorbate (AsA) subsequently increases the level of cellular AsA and causes stomatal closure. Using the ABA-deficient mutants flacca and sitiens, we show that the AsA-mediated induction of stomatal closure requires the participation of ABA. In addition, ABA acts independently of its role in mediating another stress response, proline accumulation. Because cellular AsA level was not elevated during stomatal closure, we hypothesized that stomatal closure relies on the activation of the AsA cycle and possible accumulation of intermediate components, such as monodehydroascorbate, that have been reported to be involved in mediating stress-induced responses. To establish a link between H2O2 production, the AsA cycle and stomatal closure, we also evaluated the effect of AsA treatment on catalase-deficient transgenic plants, which have a constitutively high level of H2O2. Interestingly, stomata of catalase-deficient plants were much more responsive to AsA treatment, compared with wild-type control plants. Because an increase in cellular H2O2 upon stress has been widely documented in many organisms and has been interpreted as a signal that initiates a cascade of stress-induced responses, we suggest that stress-induced stomatal closure is mediated by H2O2 and activation of the AsA cycle.