ABA And H₂O₂ Signaling In Nacl-induced Stomatal Closure In Arabidopsis Thaliana

The phytohomone abscisic acid (ABA) plays many significant roles in plant growth and developmental progresses and in plant responses to environmental stresses. ABA production is increased in plant tissues under stress conditions to regulate water loss by closing stomata. Despite the recognitions of the central role played by ABA in regulation stomatal functions, the signal transduction events leading to alterations of the stomatal aperture remain incompletely understood. The availability of a newly selected ABA-deficient Arabidopsis mutant Ios5 makes it convenient to study ABA signaling. Here, ABA-induced H2O2 generation and their roles in inducing stomatal closing were investigated by epidermal strips bioassay, laser scanning confocal microscopy and patch clamp with Ios5 and its wild type plant as materials.There were no clear differences between wild type and Ios5 in the density, shapes and aperture of their stomata. 1 OOmM NaCl induced the open stomata of wild type close effectively, while the effects of NaCl on stomatal aperture of Ios5 were not significant; 1 OOmM NaCI and lOuM ABA effectively induced stomatal closure of both wild type and Ios5. This further defines the central roles of ABA in inducing stmatal closure. H2O2 also promoted open stomata of both wild type and Ios5 closing, which could be reversed at concentration lower than 10"5M. Cell viability tested by FDA indicated that guard cells had high viability when treated with H2O2 at concentration lower than 10"5M. 10"3M H2O2 induced-stomatal closing was irreversibly and was accompanied by the loss of cell viability. CAT (20U/ml) and Vc (lOmM) partly abolished not only NaCl-induced stomatal closing of wild type, but also NaCl+ABA-induced stomatal closing of both wild type and Ios5. But the effects of CAT and Vc on the stomatal aperture of Ios5 under salinity stress were not obvious, and CAT or Vc alone only slightly increased the stomatal aperture. Our results indicated that ABA-induced stomatal closing under salinity stress and H2O2 may be an intermediate in ABA signaling.H2O2 generation in guard cells was examined by laser scanning confocal microscopy basedon fluorescence probe H2DCFDA.The fluorescence intensity in guard cells of wild type and Ios5 was essentially the same before treatment. NaCl (lOOmM) markedly induced the increasing in fluorescence of DCF in guard cells of wild type, but not in Ios5, and the fluorescence intensity in guard cells of wild type was nearly 230% as much as in Ios5 after 15 minutes treatment; NaCl (lOOmM) and ABA (lOuM) together induced rapid increasing in fluorescence in both wild type and Ios5. Exogenous Vc completely abolished the fluorescence increase induced by NaCl or NaCl+ABA, but exogenous CAT (20U/ml) could only abolish H2O2 generation in the region of plasma membrane, and had no effects on the H2O2 generation inside the guard cells, such as the regions of chloroplasts. Our results suggested that ABA induces H2O2 generation in guard cells under salinity stress and both the plasma membrane and the chloroplasts may be the regions where H2O2 generates.The protoplasts of Arabidopsis were isolated and the voltage-dependent K+-selective channels in the plasma membrane were recorded by whole-cell configuration. The reversal potential and the property that the current was rapidly blocked by external 1 mM BaCl2 made sure that the current we recorded was carried by K+. The current of Ios5 and wild type was essentially the same before treatment. NaCl (lOOmM) in the bath solution inhibited the inward current of wild type and Ios5 by 52.4% and 26.2% respectively after 30 minutes treatment. While NaCl (lOOmM) and ABA (lOuM) together inhibited the inward current of wild type and Ios5 at similar significant level-by 56.1% and 75.6% respectively, both of which could be abolished by intracelluar application of CAT (20U/ml). This indicated that H2O2 mediates ABA-induced stomatal closing under salinity stress by targeting inward K+ channels on plasma membrane.