Different Doses Of UV-B Radiation-induced Stomatal Closure In Arabidopsis Cytoplasmic Alkaline Hydrogen Peroxide And Nitric Oxide And Its Relationship With Each Other

It is well known that ultraviolet B (UV-B) radiation acting as an important environmental signal regulates stomatal movement in many plant species, but the underlying mechanism is poorly understood. Change in cytosolic pH (pHcyt) has long been proposed to participate in multiple signaling pathways in plants. During stomatal movement, evidence also show that cytosolic alkalization in guard cells mediates the stomatal closure induced by ABA, MJ, ethylene, darkness and CO2. However, at present, whether change in pHcyt is involved in UV-B-induced stomatal closure remains unknown. In addition, the relationship between cytosolic alkalization and hydrogen peroxide (H2O2) or nitric oxide (NO) in guard cell signaling is also not clear. For example, some evidences show that cytoplasmic alkalization mediates stomatal closure via inducing of production of H2O2or NO in guard cells, but other evidences also show that exogenous H2O2-and NO-induced stomatal closure are dependent on cytoplasmic alkalization. With regard to UV-B-induced stomatal closure, previous studies have shown that H2O2and NO acting as signaling molecules participate in the response of stomata to different doses of UV-B, but the sources of H2O2or NO induced by the different doses of UV-B are not clear. Furthermore, if cytoplasmic alkalization participates in UV-B guard cell signaling, the relationship among cytoplasmic alkalization, H2O2and NO in this signaling also need to be determined. In this study, by using Arabidopsis (Arabidopsis thaliana) wild-type and mutants combined with pharmacological and genetic analysis, the importance and interactions of cytosolic pH, H2O2and NO in different doses of UV-B-induced stomatal closure were explored. Our aim is to provide valuable evidence for further elucidating the mechanism of UV-B signaling transduction in plants and for making clear the relationship between cytoplasmic alkalization and H2O2or NO.In this study, the following main results and conclusions were obtained:1. The optimal dose for UV-B to induce stomatal closure in Arabidopsis leaves and epidermal strips, respectively, is0.5and0.7W·m-2.2. Different doses (0.5W·m-2and0.7W·m-2) of UV-B radiation could induce cytoplasmic alkalization in guard cells and stomatal closure, which could be inhibited by a weak acid butyrate. Similar to UV-B, a weak base methylamine could induce stomatal closure in Arabidopsis leaves and epidermal strips under light. These results indicate that cytoplasmic alkalization mediates different doses of UV-B-induced stomatal closure in Arabidopsis leaves and epidermal strip.3.0.7W·m-2UV-B-induced H2O2production in guard cells and subsequent stomatal closure in Arabidopsis wild type were significantly inhibited by. H2O2scavengers ascorbic acid and catalase and by cell wall peroxidase inhibitor salicylhydroxamic acid (SHAM), but could not by NADPH oxidase inhibitor diphenylene iodonium chloride; Meanwhile, this dose of UV-B could induce H2O2production and stomatal closure in single and double mutants of NADPH oxidase AtrbohD, AtrbohF and AtrbohD/F, which could be abolished by SHAM. Combined with our laboratory previous results about the NADPH oxidase source of H2O2induced by0.5W·m-2UV-B in Arabidopsis guard cells, Our results clearly show that H2O2is involved in different doses of UV-B-induced stomatal closure, while low dose of UV-B induces H2O2generation in guard cells via NADPH oxidase, but high dose of UV-B induces H2O2production in guard cells mainly via cell wall peroxidases.4.0.7W·m-2UV-B-induced NO production and stomatal closure in wild-type Arabidopsis could be significantly prevented by NO scavenger c-PTIO, nitrate reductase (NR) inhibitor sodium tungstate and NO synthase (NOS) inhibitor L-NAME, while the stomata of Atnoal, nial and nial/nia2failed to close and produce NO in response to this dose of UV-B, but this dose of UV-B induced NO generation and stomatal closure in NR single mutant nia2. These results were similar to the previous results about0.5W·m-2UV-B-induced Arabidopsis stomatal closure and NO production in guard cells. Together, it is concluded that NO originated from both NOS and Nial pathways mediates stomatal closure induced by different doses of UV-B in Arabidopsis.5. The time course results showed that under different doses of UV-B, the cytoplasmic alkalization was obviously prior to H2O2and NO production in guard cells. Furthermore, butyric acid inhibited two different doses of UV-B-induced H2O2and NO generation, methylamine also induced H2O2and NO production in guard cells under light. The results indicate that different doses of UV-B-induced H2O2and NO production in guard cells are dependent on cytoplasmic alkalization.6. When wild-type Arabidopsis leaves or epidermal strips under the two different doses of UV-B alone (control), cytoplasmic alkalization increased gradually with the time of UV-B radiation. However, when NR single mutant Nial under the two different doses of UV-B alone, NADPH oxidase double mutant AtrbohD/F under the low dose of UV-B alone, or wild-type under high dose of UV-B in the presence of SHAM, the cytoplasmic alkalization in guard cells reached a maximum at60min of UV-B and kept constant after60minof UV-B radiation, although the starting time and level of cytoplasmic alkalization in guard cells were not significantly different to that in control before60min of UV-B radiation. Combined with the results that exogenous H2O2or NO also induced cytoplasmic alkalization in guard cell, our results indicate that under different doses of UV-B, cytoplasmic alkalization-induced H2O2and NO production can feedback induce cytoplasmic alkalization and lead to its further rising. In summary, the data presented here show that the relationship between cytoplasmic alkalization and H2O2or NO in the guard cell signaling pathway initiated by different doses of UV-B is as the following:UV-B induces cytoplasmic alkalization in guard cells, then cytoplasmic alkalization induces H2O2and NO production, the generated H2O2and NO induce stomatal closure and feedback enhance cytoplasmic alkalization. These findings will not only help us to gain further insight into the signal transduction networks of UV-B-induced stomatal closure, but also benefit us to better understand the relationship between cytoplasmic alkalization and H2O2or NO under various stimulus.