| Over the past decade, stomatal guard cells of higher-plant leaves have been a well-developed model system for understanding how components interact within a signaling network in a single cell. Up to now, the more comprehensive signaling model of guard cell was developed by Assmann in 1993, which including various principal signaling components and their signaling cascades. Besides these known components, the question has been raised whether there are additional components and signaling cascade in above traditional signaling model of guard cell. Based on epidermal strip bioassay, ABA-induced H2O2 generation and its signaling were investigated by microinjection, patch-clamp technique and laser scanning confocal microscopy in the experiment.H2O2 inhibited stomatal opening and promoted stomatal closure, which could be reversed and abolished by ascorbic acid (Vc) at concentrations lower than 10-5 M. More importantly, ABA-induced stomatal closure was also abolished partly by external catalase (CAT) and diphenylene iodonium (DPI), which are a H2O2 scavenger and a NADPH oxidase inhibitor, respectively. Time-course experiments of single-cell assays based on the fluorescent probe 2,7-dichlorofluorescin diacetate (H2DCFDA) showed that the generation of H2O2 was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than it did in the other regions of guard cells. The ABA-induced changes in fluorescence intensity in guard cells were abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H2O2 production, which preceded stomatal closure. These effects could be abolished by CAT or DPI microinjection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H2O2 production involved, and H2O2 may be an intermediate in ABA signaling. Meanwhile, our results further indicate the internal and external acting sites for ABA in guard cells.The protoplasts of Vicia guard cells were isolated by two-step enzyme digest. 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 inward K+ currents were inhibited by 76% (at -190 mV) with 1.0 ?M ABA in the bath solutions and that intracellular application of CAT or DPI partly abolished ABA-inhibited inward K+ current across the plasma membrane of guard cells. H2O2 mimicked ABA to inhibit inward K+ current, which effect was reversed by the addition of ascorbic acid (Vc) in the patch clamping micropipettes. These results suggested that H2O2 mediated ABA-induced stomatal movement by targeting inward K+ channels on plasma membrane.Epidermal bioassay demonstrated that benzylamine, a membrane-permeable weak base, mimicked hydrogen peroxide (H2O2) to induce stomatal closure, and butyric acid, a membrane-permeable weak base, abolished partly the H2O2-induced stomatal closure. Confocal pH mapping with the probe 5-(and-6)-carboxy seminaphthorhodafluor-1-acetoxymethylester (SNARF-1-AM) revealed that H2O2 leads to rapid changes in cytoplasmic and vacuolar pH in guard cells of Vicia faba L, i.e. alkalinization of cytoplasmic areas occurred in parallel with a decrease of the vacuolar pH, and that butyric acid pretreatment abolished alkalinization of cytoplasmic areas and acidification of vacuolar areas of guard cells challenged with H2O2. These results implied that the alkalinization of cytoplasm via efflux of cytosol protons into the vacuole in guard cells challenged with H2O2 was important at an early stage in the signal cascade leading to stomatal closure.
|
PDF Full Text Request