MAPKs Signaling In Vicia Guard Cells

The stress messengers abscisic acid (ABA) and salicylic acid (SA) induce stomatal closure by mediating the generation of reactive oxygen species (ROS) in higher-plant leaves. H2O2 acts as signal molecular and intermediate in the downstream of ABA or SA signal transduction pathway, which has recently emerged as a secondary messenger of stress signaling. Mitogen-activated protein (MAP) kinase signal transduction cascades are routes through which eukaryotic cells deliver extracellular messages to the cytosol and nucleus, and the increasing evidences showed that MAPKs are involved in ABA-, SA- or H2O2-signaling respectively. In addition, plant guard cells have been a well-developed model system for understanding how components interact within a signaling network in a single cell. Based on epidermal strip bioassay, microinjection, patch-clamp and laser scanning confocal microscopy in the experiments, we provided the first evidence that MAP kinases, including MEK1/2 or P38/HOG1, plays an important role in ABA- or SA-induced H2O2 signal initial, amplification and specific targeting in response to stimuli in guard cells.ABA- or H2O2-induced Vicia faba stomatal closure.was inhibited or reversed by the specific inhibitor PD98059 of MEK1/2; the guard cells were pre-incubated or -microinjected by 10 umol L-1 PD98059, ABA could not enhance the fluorescence intensity of H2O2 probe dichlorofluorescein (DCF). Meanwhile, after ABA induced the H2O2 accumulation in guard cells, the exogenous or intracellular PD98059 could reduce the DCF fluorescence intensity. Most interestingly, on the contrary to ABA, the DCF fluorescence intensity of guard cells treated by 100 umol L-1 salicylic acid (SA) was not down-regulated by PD98059, yet PD98059 did not regulate the stomatal movement being induced by light, dark or salicylic acid.The voltage-dependent K+-selective channels in the plasma membrane were recorded by whole-cell configuration. The whole-cell patch-clamp analysis showed that the ABA or H2O2 could not induce the decreasing of inward K+ currents were abolished by the presence of PD98059 in the bothsolution; after ABA- or H2O2-induced the decreasing of inward K+ currents, PD98059 restored the decreasing of inward K+ currents. Moreover, the results, the combating Ca2+ antagonist BAPTA and PD98059 treatment, showed the same as the aforesaid, that is to say, PD98059 reversibly abolished ABA- or H2O2-induced the decreasing of inward K+ currents in independent manner. Together, these results suggest that MEK1/2 could mediate stomatal closure by abolishing the ABA-induced H2O2 generation/accumulation and ABA- or H2O2-induced the decreasing of inward K+ currents in the specific manner.P38 MAPK is first and well characterized in yeast, which allows yeast to adapt to conditions of high osmolarity by the production of glycerol in responsive to high osmotic stresses. ABA is generated to response to osmotic stress in plant cells. H2O2 is recognized as the second messenger in ABA signaling cascade. ABA and H2O2 reduce stomatal aperture by silencing the inward-rectifying K+ channels and activating the outward-rectifying K+ channels. Here using an epidermal strip bioassay and whole-cell patch clamp analysis, we provide evidence that p38 MAPK regulate stomatal closure by regulating the inward K+ current in guard cell of V. faba. Our research show that SB203580, a specific inhibitor of p38a and p38B, at the concentration of 1umol L-1, potentially could disrupt or reverse H2O2-induced stomatal closing or inward K+ current decreasing, besides the inhibitor partially down-regulates ABA-induced stomatal closing and inward K+ current lowering, but it does not reverse ABA-induced inward K+ current lowering. SB202474, a structurally related with SB203580 but inactive compound, does not; and SB203580 is specifically involved in the downstream H2O2 signaling cascades in guard cells.To insight into the p38 MAPK founctions in ABA- and SA-induced H2O2 accumulation in guard cells, we went on resaearching the DCF fluorescence intensity by the A...