Fc Reduce The Guard Cells The Level Of No Inhibition Of Dark-induced Stomatal Closure,

Guard cells regulate water and gass exchange in leaves by closing and opening stomatal pores, and stomatal movements are regulated by multiple exteral and internal factors. Previous studies proved that fusicoccin(FC) can activate the plasma membrane H+-ATPase, and obviously cause stomatal opening. Nitric oxide(NO), as an important signaling molecule, participate in many plant physiological processes such as growth and development, pathogen defense reaction, programmed cell death(PCD)and stress tolerance. Rencently, various of studies suggested that NO mediate stomatal movement regulated by several phytohormones and environment factors. However,it is unclear whether FC inhibition of dark-induced stomatal closure is related to the change of NO levels in guard cells. In the present study, by means of epidermal strip bioassay and the laser scanning confocal microscopy(LSCM), we studied the relationship between inhibition of dark-induced stomatal closure by FC and change of NO levels in guard cells of broad bean, and the mechanism of FC-induced change of NO levels in guard cells was also explored.The results are as follows.1.Different concentration of FC(0～1.0μM),c-PTIO(0～250μM) and L-NAME(0-45μM) all evidently inhibited dark-induced stomatal closure, and the effects are in a dose-dependent manner. The optimal concentrations of FC, c-PTIO and L-NAME are 0.1μM,200μM and 25μM, respectively. Additionally, FC, c-PTIO and L-NAME decreased the level of NO in guard cells in darkness. The results show that inhibition of dark-induced stomatal closure by FC is associated with the decrease of NO levels in guard cells.2.Exogenous application of 100μM SNP remarkably promoted stomatal closure in light. FC and c-PTIO did not cause any changes of stomatal apertures in light. However, similar to c-PTIO, a NO scavenger, FC significantly prevented stomatal closure induced by SNP. Additionally, both FC and c-PTIO largely abolish SNP-induced DAF-2DA fluorescence in guard cells.The results provide evidence that FC induces NO removal,hence suppress stomatal closure caused by SNP in light.3.After the pre-treatment for 3h in the dark, NO had generated in guard cells and the stomata had been closed.Like c-PTIO, FC not only reopen the closed stomata induced by dark, but also lessen the level of NO generated in darkness.The results indicate that FC induces the removal of NO having been generated by dark, thus reopen the stomata had been closed by darkness.4.Different concentration of butyric acid did not cause any change in stomatal pores in light, but significantly suppressed SNP-induced stomatal closure and DAF-2DA fluorescence in guard cells. Considering that the effects of butyric acid on SNP-induced stomatal closure and DAF-2DA fluorescence are accordant with those of FC and FC can induce cytosolic acidification, we speculate that cytosolic acidification is involved in the removal of NO induced by FC.5.The pre-treatment for 3h in darkness caused stomatal closure and NO incrase in guard cells. Butyric acid evidently reopen the stomata have been closed by dark, and markedly reduced the level of NO generated by darkness. Considering that the effects of butyric acid on the stomata had been closed by dark and the level of NO had been generated by darkness are consistent with those of FC and FC can cause cytosolic acidification, we speculate that the removal of NO induced by FC is relative to guard cells cytosolic acidification.Taken together all the results, we conclude that FC causes the removal of NO via inducing guard cells cytosolic acidification, finally prevents dark-induced stomatal closure.