The Role Of Ethylene, MAPK And Ca～(2+) In UV-B-induced Stomatal Closure And Their Relationships With H₂O₂ And NO In Vicia Faba

Previous study has indicated that NO and H₂O₂ can act as second messengersto mediate UV-B-induced stomatal closure. However, it is not clear what are theupstream and downstream signal molecules of NO and H₂O₂ in UV-B signaltransduction pathways in guard cells. In the signalling transduction network ofABA-induced stomatal closure, it has been shown that signal molecules ofmitogen-activated protein kinase (MAPK) , Ca²⁺, Calcium-dependent protein kinases(CDPK) and protein tyrosine phosphatase are all involved in the ABA signaltransduction pathway, and all function as the upstream elements of the ABA-inducedH₂O₂ and/or NO in guard cells. In addition, studies also show that Ca²⁺ and CDPK arealso the downstream elements of the ABA-induced H₂O₂ and NO in guard cells.Besides, other studies have shown that UV-B radiation can induce the generation ofethylene and that ethylene can regulate stomatal movement as well. However, there islittle information available about whether the signal molecules of ethylene, MAPK,CDPK and Ca²⁺ are also involved in the UV-B-induced stomatal closure. Furthermore,it is also not clear the relationships between these signal molecules and theUV-B-induced H₂O₂ and NO. In order to further understand the UV-B signaltransduction network in the guard cells, this study mainly investigated the role ofethylene, MAPK and Ca²⁺ in UV-B-induced stomatal closure and their relationshipswith H₂O₂ and NO in Vicia faba by epidermal strip bioassay and laser-scanningconfocal microscopy.The main results were followed:1. UV-B radiation could obviously induce ethylene release in Vicia faba strip, thepeak appeared at 1h after treatment, which was evidently prior to the marked increasein endogenous H₂O₂ and NO level in guard cells under UV-B radiation. ACC synthaseinhibitor AOA and AVG, ACC oxidase inhibitor CoCl₂ and NiCl₂. ethylene receptorinhibitor 1-MCP and AgNO₃ could all significantly inhibit UV-B-induced stomatalclosure and decrease UV-B-induced increases of endogenous H₂O₂ and NO level inguard cells. In addition, the effects of AOA and AVG on UV-B-induced stomatalclosure and generation of H₂O₂ and NO could be reversed significantly by exogenousACC treatment. The effect of AOA, AVG, 1-MCP and AgNO₃ on UV-B-induced stomatal closure could also be significantly reversed by exogenous H₂O₂ and NOtreatment. Exogenous ethylene donor ethephon could not only induce stomatal closurein visible light, but also increase endogenous H₂O₂ and NO level in guard cells. Inaddition, H₂O₂ scavenger catalase(CAT) and Vitamin C (Vc), NO scavenger c-PTIOcould all effectively inhibit ethephon-induced stomatal closure. The results indicatedthat ethylene was involved in the signal transduction pathway of UV-B-inducedstomatal closure and functioned in the upstream of UV-B-induced H₂O₂ and NO.Besides, the results also showed that exogenous ethylene induced stomatal closure bypromoting H₂O₂ and NO production in guard cells of Vicia faba.2. MAPK inhibitor PD98059, CDPK inhibitor TFP, nonspecific protein kinaseinhibitor STS and tyrosine protein phosphatase inhibitor PAO all had no obvious effecton both stomatal aperture and endogenetic H₂O₂ and NO level in guard cells undervisible light. But PD98059 could not only obviously reverse UV-B-induced stomatalclosure, but also decrease UV-B radiation-induced enhancement of endogenous H₂O₂and NO level in guard cells. The effect of STS was similar to PD98059 whereas thefunction was weaker. Yet TFP and PAO neither reversed UV-B-induced stomatalclosure, nor had effect on UV-B-induced endogenous H₂O₂ and NO level in guard cells.The results suggested that MAPK was involved in the signal transduction pathway ofUV-B-induced stomatal closure, and its function was to regulate the H₂O₂ and NOlevel in guard cells. However, CDPK and tyrosine protein phosphatase is not involvedin the signal transduction pathway of UV-B-induced stomatal closure in Vicia faba.3. When the closed stomata induced by UV-B radiation was moved to visible lightfor 2 h, there was no obvious effect on stomatal aperture and level of H₂O₂ and NO inguard cells. Whereas PD98059 could not only promote the closed stoma induced byUV-B to reopen, but also effectively decrease the UV-B-induced increases of H₂O₂and NO level in guard cells. Furthermore, PD98059 not only inhibited exogenousH₂O₂- and NO-induced stomatal closure, but also decreased H₂O₂ and NO level inguard cells when treated with exogenous H₂O₂ or NO respectively. These resultssuggested that in the signalling transduction pathway of UV-B-induced stomatalclosure, the UV-B-activated MAPK maybe inhibited the scavenging system of H₂O₂and NO, and resultantly caused the decreases in H₂O₂ and NO level in guard cells,which induced stomatal closure of Vicia faba. However, the results presented here didnot exclude the possibility of that the UV-B-activated MAPK maybe also induced the generation of H₂O₂ and NO in guard cells.4. Exogenous ethephon-induced stomatal closure could be obviously inhibited byMAPK inhibitor PD98059 in visible light. But the effect of PD98059 onUV-B-induced stomatal closure could not be effectively reversed by exogenousethephon treatment. This result suggested that MAPK maybe functioned in thedownstream of ethylene in the signal transduction pathway of UV-B-induced stomatalclosure of Vicia faba.5. Both Ca²⁺ chelator EDTA and Ca²⁺ channels inhibitor LaCl₃ inhibitedUV-B-induced stomatal closure, but had no effect on UV-B induced increases of H₂O₂and NO level in guard cells, indicating that Ca²⁺ signal was involved in the signaltransduction pathway of UV-B-induced stomatal closure and might function in thedownstream of H₂O₂ and NO.6. Under UV-B radiation, O_2·^- scavenger Tiron and cell wall peroxidase inhibitorSHAM could obviously inhibit not only the UV-B radiation-induced stomatal closure,but also the UV-B-increased H₂O₂ level in guard cells. However, NADPH oxidaseinhibitor DPI could neither inhibit UV-B-induced stomatal closure, nor decreaseUV-B-increased H₂O₂ level in guard cells. The results showed that guard cells of Viciafaba generated H₂O₂ in response to UV-B radiation via the cell wall peroxidase, butnot NADPH oxidase, which is obviously different from that ABA-induced H₂O₂generation in guard cells of Vicia faba via NADPH oxidase pathway.