NO May Function Downstream Of H₂O₂ In ABA-induced Stomatal Closure In Vicia Faba L.

Abscisic acid (ABA) can regulate a variety of physiological and molecular responses for plant against stresses, including drought, chilling, salinity, and so forth. More often stomatal closure occurred in these responses accompanying with ABA production. Previous research reported that ABA may bring about reduction in stomatal aperture by promoting the efflux of potassium salt from guard cells, and hydrogen oxide (H2O2) involves into this process as an intermediate. The guard cell has also become a model system for investigation of ABA signaling pathways.Nitric oxide (NO) is a very active molecule involvs in many and diverse biological pathways. NO touches upon multiple aspects of intracellular signaling pathways such as gene activation, protein expression and activity regulation of enzymes. Recent studies have demonstrated that NO is another signal that activates defense responses after pathogen attack.Signal interaction between NO and reactive oxygen intermediates (ROIs) in the plant have already been reported and recently nitric oxide was reported to be a novel component of abscisic acid signaling in stomatal guard cells. Delledonne and colleagues demonstrated that, in soybean cell suspension, the efficient induction of hypersensitive cell death requires a balance between ROIs and NO production. Lum and co-workers identified H2O2 as an upstream signal that leads to NO production. They also found that the H2O2 induced NO production was mediated via calcium ion flux, as it was blocked in the presence of a calcium ion channel blocker, verapamil.Recent documents suggested that either H2O2 or NO function as signal molecules to mediate ABA-induced stomatal closure in guard cells, but there was no report to study on the relationship between H2O2 and NO in ABA signal transduction pathway. Here, using stomatal analysis and laser scanning cofocal microscope techniques, we show firstly that NO as the downstream intermediate of H2O2 signaling mediates ABA-induced stomatal closure in Viciafaba L. Sodium nitroprusside (SNP, a NO donor) and H2O2 could mimic the effects of ABA on stomatal closure. Carboxy-PTIO (c-PTIO, a specific scavenger of NO) partly reversed the stomatal closure induced by ABA or H2O2. While catalase (CAT), a H2O2 scavenger, failed to reverse NO-induced aperturereduction in Vicia faba guard cells. Monitoring the changes of both NO and H2O2 generation in guard cells by using fluorescent probe of NO or H2O2, DAF-2DA or H2DCF-DA, respectively, we found that the generating rate of H2O2 in the guard cells was faster than that of NO after treated with 10 mol/L ABA. CAT almost completely inhibited the increase of DAF fluorescence induced by ABA. Similar to ABA, exogenous H2O2 provoked the production of NO. C-PTIO could slightly enhance the fluorescent intensity of DCF stimulated by ABA, while exogenous SNP could not increase DCF fluorescence in guard cells. Taken together, these results suggest that H2O2 could probably act as upstream component of NO signaling and NO negatively regulate H2O2 generation in ABA-induced stomatal closure in guard cells.