Different Trimer G Protein Involved In Regulating The Uv - Induced Arabidopsis Stomatal Closure And B H < Sub > 2 < / Sub > O < Sub > 2 < / Sub > And No Relationship

Ultraviolet B (UV-B) radiation acting as a key environmental signal regulates many processes of plant growth and development, but their underlining mechanisms are much less known. In mammalian cells, heterotrimeric G proteins have been shown to transmit UV-B signals, but whether they also transmit UV-B signals in plant cells is not clear. Meanwhile, many evidences have indicated that G proteins mediate many factors such as abscisic acid (ABA)-, extracellular calmodulin (ExtCaM)-, pathogens-and ozone-regulated stomatal movement via regulating the production of hydrogen peroxide (H2O2) and nitric oxide (NO). With regard to the UV-B-induced stamatal closure, our previous studies have indicated that H2O2and NO acting as signal molecules participate in this process, but a series of issues are still not clear, such as (1) what are the specific zymetology pathways of H2O2and NO generation induced by UV-B;(2) whether G proteins participate in the signal transduction pathways of UV-B-induced stomatal closure;(3) what are the interrelationship among heterotrimeric G proteins, H2O2and NO in the signal transduction pathways of UV-B-induced stomatal closure. In this report, the above problems were studied in Arabidopsis thaliana by pharmacological, genetic and cell biological methods and the following main results and conclusions were obtained:1. When UV-B dose was up to0.5W·m-2, it could induce stomatal closure significantly in Arabidopsis leaves; and when leaves were exposed to0.5W·m-2UV-B for1-4h, stomatal aperture decreased most at3h. These results indicate that0.5W·m-2UV-B radiating for3h are the optimal radiation dose and time to induce stomatal closure in Arabidopsis leaves. So0.5W·m-2UV-B radiating for3h was chosen for subsequent researches.2. G protein inhibitor pertussis toxin (PTX) could inhibit0.5W·m-2UV-B-induced stamatal closure, while similar to UV-B, G protein activator cholera toxin (CTX) significantly induced stomatal closure of leaves under light. UV-B could not induce stomatal closure in G protein a subunit gene GPA1knockout mutants gpal-1and gpal-2, while it could induce stamatal closure in GPA1gene overexpressing line wGa and an overexpression line cGa of a constitutively active form of GPA1. Furthermore, faster stomatal closure was induced by UV-B in wGa and cGa than that in the wild type. These evidences indicate that G protein a subunit is involved in the signal transduction pathway of UV-B-induced stomatal closure in Arabidopsis.3. Both H2O2scavengers ascorbic acid (ASA) and catalase (CAT) and a NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) could effectively inhibit UV-B-induced stomatal closure and H2O2generation in guard cell of wild type. But UV-B could not induce stomatal closure and H2O2production in guard cells of NADPH oxidase single and double mutants of AtrbohD and AtrbohF. These results not only confirm that H2O2is involved in the signal transduction pathway of UV-B-induced stomatal closure, but also further show that UV-B-induced H2O2generation in guard cells depends on both NADPH oxidase AtrbohD and AtrbohF in Arabidopsis.4. PTX could inhibit significantly UV-B-induced H2O2generation in guard cells, while exogenous H2O2also reversed the inhibtory effect of PTX on UV-B-induced stomatal closure; CTX could induce H2O2generation in guard cells under light, which could be significantly inhibited by ASA, CAT and DPI; UV-B could not induce H2O2generation in guard cells of mutants gpal-1and gpal-2, but it induced more H2O2generation in guard cells of wGa and cGa, than that of the wild type; Exogenous H2O2could induce stomatal closure in mutants gpal-1and gpal-2under light and UV-B radiation, while CTX could not induce stomatal closure and H2O2generation in guard cells of NADPH oxidase single and double mutants of AtrbohD and AtrbohF. These results indicate that G proteins function upstream of H2O2in the signal transduction pathway of UV-B-induced stomatal closure.5. NO scavenger c-PTIO and nitrate reductase (NR) inhibitor sodium tungstate (Na2WO4) could all effectively inhibit the UV-B-induced stomatal closure and NO generation in guard cell of wild type; UV-B could not induce stomatal closure and NO generation in guard cells of NR single mutant nial and double mutant nialnia2, but it could induce stomatal closure and NO generation in guard cells in NR single mutant nia2. The results not only confirm again that NO participates in the signal transduction pathway of UV-B-induced stomatal closure, but also indicate that UV-B-induced NO generation in guard cells depends on Nial, but not Nia2.6. PTX could inhibit significantly UV-B-induced NO generation in guard cells, and exogenous NO donor sodium nitroprusside (SNP) also reversed the inhibitory effect of PTX on UV-B-induced stomatal closure; CTX could induce NO generation in guard cells under light, which was remarkably inhibited by c-PTIO and Na2WO4: UV-B could not induce NO generation in guard cells of mutants gpal-1and gpal-2, but it inudced more NO generation in guard cells of wGa and cGa than that of the wild type; SNP could induce stomatal closure of mutants gpal-1and gpal-2under light and UV-B radiation, and CTX could not induce stomatal closure and NO generation of guard cells in mutants nial and nialnia2. These results indicate that UV-B-induced NO generation in guard cells depends on Gα.7. Under visible light or UV-B radiation, exogenous H2O2-induced stomatal closure was abolished in mutants nial and nialnia2, but SNP could stimulate stomatal closure in NADPH oxidase single and double mutants of AtrbohD and AtrbohF; UV-B could not induce NO generation in guard cells of the single and double mutants of AtrbohD and AtrbohF, but it could induce H2O2generation in guard cells of mutants nial and nia1nia2. The results show that H2O2functions upstream of NO in the signal cascade of UV-B-induced stomatal closure.In conclusion, the results of this report indicate that0.5W m-2UV-B induces stomatal closure in Arabidopsis thaliana by eliciting a cascade of intracellular signaling events including Gα protein, H2O2and NO, in which the Gα protein transmits UV-B signals to elicit H2O2via NADPH oxidase AtrbohD and AtrbohF, which then elicit NO via Nial in guard cells to regulate UV-B dependent stomatal closure. These findings not only extend the functions of plant G proteins, but also provide clues to further understanding the UV-B signal transduction mechanisms in plants.