Different Doses Of UV-B Radiation-induced Stomatal Closure In Arabidopsis Ethylene, Different G Proteins And The Role Of Hydrogen Peroxide Trimers And Their Relationships

As an important environmental signal, Ultraviolet B (UV-B) radiation has obvious effect on many aspects of plant the growth and development, thus, it is of important theoretical and practical significance to study the signal transduction mechanism of plant cells response to UV-B radiation. Previous research has shown that different doses of UV-B radiation can induce stomatal closure; Ethylene mediates0.8W·m-2UV-B-induced stomatal closure via peroxidases(POD)-dependent hydrogen peroxide (H2O2) generation in broad bean guard cells; Heterotrimeric G proteins a subunit(Ga) participates in the signal transduction pathways of0.5W·m-2UV-B-induced stomatal closure through activating NADPH oxidase-dependent H2O2production in arabidopsis (Arabidopsis thaliana). Although previous evidences have shown that ethylene、Ga and H2O2are involved in the signal transduction pathways of UV-B-induced stomatal closure, it is still unclear the similarities or differences between different doses of UV-B-induced guard cell signal transduction pathways as well as the relationship between these signal molecules due to the use of different plant species and UV-B radiation doses in the previous different experiments. By using leaves of wild Arabidopsis thaliana and various mutants combined with pharmacological, genetic and laser-scanning cofocal microscopy methods, this study mainly investigates (1) the effect of different doses of UV-B on Arabidopsis stomatal movement;(2) whether ethylene, Ga and H2O2are involved in different doses of UV-B induced stomatal closure;(3) whether the enzymatic sources of H2O2mediating different doses of UV-B-induced stomatal closure are the same or not; and (4) the relationship among ethylene, Ga and H2O2in low doses of UV-B-triggered guard cell signaling pathways. The following main results and conclusions are obtained:(1) UV-B radiation induced stomatal closure of wild-type Arabidopsis leaves in a dose-dependent manner, that were substantial at0.5W·m-2and maximal at0.65W·m-2. Based on these results,0.5and0.65W·m-2UV-B radiation for3h were choosed respectively as the low dose and high dose of UV-B for subsequent researches.(2) Stomatal closure in the mutants of ethylene receptors ETR1and EIN4, the copper transporter RANl as well as the ethylene signal componets—EIN2, EIN3and ARR2could not be induced by the low dose of UV-B and exogenous ethylene, but could be induced by high dose of UV-B, suggesting that ethylene mediates the low dose of UV-B-induced stomatal closure, but is not involved in the guard cell signal pathway triggered by high dose of UV-B. In addition, ethylene percepting and signaling elements including ETR1, EIN4, RAN1, EIN2, EIN3and ARR2are all involved in the low dose of UV-B-triggered guard cell signaling pathways.(3) G protein a subunit inhibitor pertussis toxin (PTX) could completely inhibit the low dose of UV-B-induced stamatal closure, but could not block high dose of UV-B-induced stamatal closure; G protein a subunit activator cholera toxin (CTX) induced stomatal closure in the absence of UV-B radiation; Stomata in loss of function mutants gpa1-1and gpa1-2did not response to the low dose of UV-B, but closed under the high dose of UV-B; Constructs of wGa (GPA1gene overexpressing line) and cGa (an overexpression line of a constitutively active form of GPA1) showed faster stomatal closure induced by the low dose of UV-B than the wild type. These evidences indicate that G protein a subunit is involved in the signal transduction pathway of low dose but not high dose of UV-B-induced stomatal closure in Arabidopsis.(4) The two different doses of UV-B-induced H2O2and stomatal closure were significantly scavenged or inhibited by H2O2scavengers catalase (CAT) and ascorbic acid (ASA), suggesting that H2O2is involved in the signal transduction pathways of different doses of UV-B-induced stomatal closure in Arabidopsis leaves. The low dose of UV-B-induced H2O2generation in guard cells and subseguent stomatal closure could be inhibited by NADPH oxidase inhibitor-diphenylene iodonium chloride (DPI), but not by cell wall peroxidase inhibitor-salicylhydroxamic acid (SHAM), while the high dose of UV-B-induced H2O2production and stomatal closure were inhibited by SHAM, but not by DPI; the stomta of single and double mutants of NADPH oxidase AtrbohD and AtrbohF failed to close and produce H2O2under the low dose of UV-B radiation, but did close and produce H2O2under the high dose of UV-B. However, the effect of high dose of UV-B could be reversed by SHAM. These results indicate that H2O2mediating low dose of UV-B-induced stomatal closure is mainly generated through NADPH oxidases—AtrbohD and AtrbohF, while H2O2mediating high dose of UV-B-induced stomatal closure is mainly sourced from cell wall peroxidases.(5) Both the low dose of UV-B and exogenous ethylene could induce H2O2generation and stomatal closure in wild-type Arabidopsis, but could not in the mutants of AtrbohD/F, ETR1, EIN4and RAN1, while they also induced H2O2production but not stomatal closure in the mutants of EIN2, EIN3and ARR2; Exogenous H2O2rescued the defect in the low dose of UV-B-and exogenous ethylene-induced stomatal closure in the mutants of AtrbohD/F, ETR1, EIN4and RANI, but could not rescue this defect in the mutants of EIN2, EIN3and ARR2. Taken together, our results strongly support the guard cell signaling pathway triggered by the low dose of UV-B as followings: UV-B firstly triggers ethylene production, which then induce NADPH oxidase-dependent H2O2generation via ethylene receptor ETRl and EIN4and copper ion transporter RAN1, the produced H2O2finally induces stomatal closure in an ethylene signal components—EIN2, EIN3, and ARR2dependent manner.(6) The low dose of UV-B and exogenous ethylene either alone or together could not induce H2O2production and stomatal closure in gpa1-1and gpa1-2mutants, while could induce those in wGa and cGa constructs; Under light without or with low dose of UV-B or exogenous ethylene, CTX could induce H2O2production and stamatal closure in the mutants of ETRl, EIN4and RANI, and induced H2O2production but did not stamatal closure in the mutants of EIN2, EIN3and ARR2. Together, the results show a linear intracellular signaling pathway induced by low dose of UV-B in guard cells of Arabidopsis, that is:UV-B-induced ethylene activates Ga through ETR1, EIN4and RAN1, then the activated Ga induces the generation of H2O2via NADPH oxidases, finally the generated H2O2induces stomatal closure by depending on ethylene signal components EIN2, EIN3, and ARR2.In conclusion, the data presented here indicate the following facts:different doses of UV-B induce stomatal closure in Arabidopsis thaliana via different signaling pathways; Ethylene and Ga participate in the guard cell signaling pathways triggered by the low dose of UV-B but not by the high dose of UV-B; H2O2is involved in the different doses of UV-B-triggered guard cell signaling, while H2O2mainly comes from NADPH oxidases AtrbohD and AtrbohF in the low dose of UV-B radiation, but mainly from cell wall peroxidases in the high dose of UV-B; The linear signal transductioin pathway mediated the low dose UV-B-induced stomatal closure is:UV-B induces ethylene generation, then ethylene activates Ga through its receptor ETR1and EIN4and copper transporter RANI, the activated Ga induces H2O2generation via NADPH oxidases, finally the generated H2O2induces stomatal closure by depending on ethylene signal components EIN2, EIN3, and ARR2.