PEG6000 Stress Signaling In Vicia Faba Guard Cell

Stomatal movement is important for plants to exchange gas and waterwith environment. A good example is stomatal closure under osmotic stress to avoidosmotic water deficit and alleviate the harm of plants. So, it's very important to studythe mechanism of stomatal movement under osmotic stress. The studies in the pastshowed that stomatal movement under drought condition is mediated through receptionand transduction hydraulic & chemical signals by guard cell. A large body of study hasfocused on the chemical signals (such as ABA), but there is little information availableabout hydraulic signaling of guard cell. In this study, the abaxial epidermis of Vicia fabawas used as materials, PEG6000 was used as the osmolyte to simulate the decreasing ofwater potential, and the role and source of NO and H₂O₂ in stomatal movement causingby hydraulic signal were investigated by epidermal strip bioassay and laser scanningconfocal microscopy (LSCM). Additionally, the dynamics of vacuoles and relationshipsamong NO, H₂O₂, the dynamic of vacuoles and that of actin cytoskeleton were studied.The results were as follows:1. The treatment of PEG6000 induced stomatal closure significantly, and theeffects were in a dose- and time-dependent manner. The PEG6000-induced stomatalclosure can be reversed by NO scavenger c-PTIO, NOS inhibitor L-NAME, H₂O₂scavenger ASA and CAT, NADPH oxidase inhibitor DPA and cell wall peroxidaseinhibitor SHAM, which have no significant effect on stomatal aperture used alone.These data indicated that PEG6000 stress promotes stomatal closure via a pathwayinvolving H₂O₂ and NO.2. To determine whether PEG6000 stress could induce the NO and H₂O₂generation in guard cells, we used the specific fluorophore2', 7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA) and 4, 5-diaminofluoresceindiacetate (DAF-2 DA) combining LSCM to measure changes in intracellular H₂O₂ andNO level directly. PEG6000 stress induced an intense H₂O₂ and NO fluorescence inguard cells, and the control not. ASA, CAT, DPI and SHAM can suppress PEG6000stress-induced H₂DCF-DA fluorescence. Similarly, both c-PTIO and L-NAME canprevent PEG6000 stress-induced DAF-2 DA fluorescence. These results support clearlythat PEG6000 stress can induce H₂O₂ and NO generation, and the stomatal closureinduced by PEG6000 stress is mediated by H₂O₂ and NO. Furthermore, SHAM (an inhibitor of cell wall peroxidase), DPI (an inhibitor of NADPH oxidase) and L-NAME(an inhibitor of NOS) all can readily reverse stomatal closure and H2O2 and NOgeneration induced by PEG6000 stress, the data imply that H2O2 and NO generation inguard cells of Vicia faba is likely related to cell wall peroxidase, NADPH oxidase andNOS, respectively.3. To determine whether guard cell microfilaments are involved in the regulation ofPEG6000 stress-, NO- and H2O2-induced stomatal closure, we treated the epidermalstrips with CB, an inhibitor of F-actin polymerization and examined the effect of thedrug by measuring the size of the stomatal aperture and endogenous fluorescence.Stomatal apertures did not change significantly by pretreatment of CB alone, butstomatal closuring induced by PEG6000 stress, NO and H₂O₂ were suppressed.Pretreatment with CB didn't suppress the NO and H₂O₂ fluorescence, but changed thedistribution fluorescence visibly in guard cells treated by PEG6000 stress. These resultssuggest that actin remodeling participate in PEG6000 stress-induced stomatal closureand acting in downstream of H₂O₂.4. To visualize the vacuoles in guard cells, we stained V. faba guard cells withvacuole-specific probes and imaged the vacuoles using LSCM. The guard cell in theopened stomata contained only a few big vacuoles, and the cytoplasm of the guard cellwas pressed to surrounding the nucleus mainly. After PEG6000 stress treatment, largevacuoles did split into a large number of small vacuoles. Cytoplasm slices were alsobeen observed cling to the surface of vacuoles and between two vacuoles. Folding andruga were always been observed in tonoplast, which maybe as a reposition of tonoplast.5. PEG6000 stress, exogenous H₂O₂ and NO promoted stomatal closuresignificantly, and also promoted large vacuoles splitting into small vacuoles. Theseeffects were suppressed by c-PTIO,L-NAME,ASC,CAT,DPI and SHAM.Analogously, exogenous H₂O₂ and NO also can promote large vacuoles splitting intosmall vacuoles. These results indicate that the division of big vacuoles into smallvacuoles is required for NO and H₂O₂ induced stomatal closure, and the dynamics of thevacuoles play key role in stomatal closure.6. CB inhibited the change which big vacuoles split into small vacuoles response toPEG6000 stress, NO and H₂O₂, the stomatal closuring was also been inhibited. Theseresults indicated that actin microfilaments participated in PEG6000 stress, NO andH₂O₂-induced changes of vacuoles volume of guard cells. Actin polymerization maybe facilitates the vacuolar division.All together, the rough clues of hydraulic signaling of guard cell were outlined:PEG6000 stress(?)microfilament→vacuoles→stomatal closure...