The Role Of NO In Extracellular CaM Regulated Stomatal Movement In Arabidopsis Thaliana And Its Possible Mechanism

Nitric oxide (NO) is a key signaling molecular in plants involved in various biological pathways. NO has been reported as a new member of signal transduction pathway in guard cells recently. Extracellular CaM (ExtCaM) regulates stomatal movement in Vicia faba and Arabidopsis thaliana as a peptide primary messenger and this process is mediated by G-protein and H₂O₂.In this study, by using Atnoslmutant, heterotrimeric G protein alpha subunit null mutants gpa1-1 and gpa1-2, overexpression lines wGa and cGa, and atrbohD/F double mutant, together with epidermal strip bioassay and fluorescence microscopy, we demonstrated that NO is involved in ExtCaM induced stomatal closure. In the same time we investigated the relationship among G protein, H₂O₂ and NO. The main results were as follows:Firstly, exogenous SNP (NO donor) induced stomatal closure in Arabidopsis wild type and endogenous NO mediated dark-induced stomatal closure. The intact leaf water loss in Atnosl was faster than that in wild type during drought stress, which indicated the Atnosl stomatal couldn't close in time.PTIO—a specific NO scavenger could reverse ExtCaM induced stomatal closure and L-NAME, an inhibitor of NOS also impaired the effect of ExtCaM. ExtCaM failed to induce Atnosl stomatal closure. The above data provide pharmacology and genetic evidence for the involvement of NO in the ExtCaM induced stomatal closure. ExtCaM increased the NO level in Arabidopsis of wild type, but failed in Atnosl mutant. It is presumed that ExtCaM induced stomatal closure via activating the NOS activity and increase NO content in guard cells.SNP induced stomatal closure both in gpal-1 and gpal-2, and in wGa and cGa, which indicated that NO acted downstream of Ga in ExtCaM signaling pathway. ExtCaM induced NO generation in Arabidopsis of wild type, wGa and cGa, but failed in gpal-1 and gpal-2, which suggested Ga was involved in ExtCaM induced NO generation in guard cells.H2O2 induced stomatal closure in Arabidopsis wild type, but after coincubated with PTIO or L-NAME, the stomatal closure was reversed or inhibited. Furthermore, H2O2 only induced slightly stomatal closure in Atnosl mutant. These results supported that endogenous NO was necessary for H2O2 induced stomal closure. Further study showed that H2O2 accelerated NO generation in guard cells of wild type, which indicating the requirement for NO synthesis and action during H2O2 induced stomatal closure.ExtCaM enhanced NO synthesis in guard cells, while after coincubated with CAT, CaM couldn't induce NO increase. ExtCaM also failed to induce NO increase in AtrbohD/F double mutant. These results suggest that H2O2 involved in the acceleration of NO promoted by ExtCaM.Our results first showed the involvement of NO in ExtCaM regulating stomatal closure in Arabidipsis thaliana. Combining with our former results (Ph. D. Dissertation of Chen YL, 2002;Chen et al, 2004) and the property of AtNOSl (Guo et al, 2003) and the regulation of NO on ion channels (Garcla-Mata et al, 2003), we put forward the possible way that ExtCaM induced stomatal closure.