Involvement Of Hydrogen Peroxide In Stomatal Closure Induced By Salicylic Acid In Vicia FabaL.

A growing body of evidence suggests that salicylic acid is an endogenous signal for itsgeneral functions in plants, especially it plays a crucial role in disease-resistance responses,and much attention has been focused on this. Previous studies have suggested that SA caninhibit catalase, resulting in elevated levels of H₂O₂, which activate defense response in plants.At present, it was accepted that H₂O₂ may be a new singal in plants and it performs generalfunctions in plant physiological process. It was reported that H₂O₂ may mediate ABA topromote stomatal closure, but it was not known whether H₂O₂ was involved in stomatalclosuring induced by SA, which was advantageous for plant defense. Here we provide theevidence that H₂O₂ may function as an intermediate in SA signal in Vicia guard cells byepidermal strips bioassay, laser scanning confocal microscopy, microinjection and patchclamp.SA can induce stomatal closure with a concentration-dependeni manner, the changes instomata apertures were reversible when the concentration of SA between 1-1000μmol/L,butwhen the concentration of SA at 10^-2mol/L, the changes in stomatal apertures wereirreversible (Figure 2), The tests of cell viability by FDA indicated that the reversibility ofstomata challenged with SA correlated with whether the cells were viable or not. Similarly,the maximum promotion of stomatal closure was observed when concentration of H₂O₂ at 10^-3mol/L, and at this concentration the changes in stomatal aperture were irreversible andaccompanied by the loss of cell viability, but when the concentration of H₂O₂ at 10^-7 mol/L or10^-5mol/L, the changes in stomatal aperture were reversible and the challenged cell kept goodviability (Figure 3). CAT at 20U/ml reversed the SA-induced stomatal closuring by 83-90%(Figure 4); Similarly, DPI at 10μmol/L reversed the effect of SA on stomatal apertures by45-60% (Figure 5), but CAT and DPI treatment alone slightly increased the stomatal apertures,which indicated that the generation of H₂O₂ might be involved in the stomatal closuringinduced by SA.We directly examined the production of H₂O₂ by laser scanning confocal microscopybased on H₂DCFDA. Similarly to the result of H₂O₂ at 10^-5mol/L, SA at 100μmol/L byapplied externally or microinjection could induce rapid increasing in nuorescence of DCF inguard cells. Moreovef, time-course experiments showed the increase in fiuorescence intensityof the chloroplast occurred significantly earlier than that in the other regions of guard celIs(Figure 7). Microinjection into one guard cell with CAT 20U/ml comp1etely inhibited thegeneration of H2O2 in the microinjected guard cell (FigUre 9). In the presence of CAT or DPI,the guard cell treated with SA l00llmolth showed increase in DCF fiuorescence only in theregions of chloroplasts located, but the production of H2O2 was inhibitCd in the regions ofplasma membrane (Figure l2,Figure l3). These results suggests that the chloroplasts in guardcells might be the main source of H2O2 production when the stomata treated with SA,simultaneously, plasma membrane NADPH oxidase may also contribute to the H2O2generation in the guard cells.The protoplasts of Ncia guard ce1ls were isolated by two-stCp enZyme digest. Thevoltage-dependent K+-selective channels in the plasma membrane were recorded bywhole-cell configureuration. WC considered that the current we recorded should be carried byK+ based on the two results below: one was the determination of reversal pOtential and theother was the whole-cell current can be raPid blocked by eXternal lmmol1 of Ba2+. Thewhole-cell inward K+ currents were inhibited by 70% after 20min treated by SA l00pmola(Figure l8), similarly, H2O2 at l0-5mol/L inhibited inward K+ currents by 47% at the sametime (Figure l9). But intracellular application of CAT or DPI partly abolished SA-inhibitedinward K+ current by 52% or 34% (Figure 20;Figure 2l), respectively This ind...