【作者】 穆娟；

【导师】 佘小平；

【作者基本信息】 陕西师范大学 ， 植物学， 2009， 硕士

【摘要】 目前,植物体内重要信号分子过氧化氢（hydrogen peroxide,H₂O₂）的作用及其生成途径已成为植物细胞信号转导研究的重要问题。已有研究发现NADPH氧化酶催化产生的H₂O₂参与ABA及光/暗调控气孔运动,还有不少报道表明二胺/多胺氧化酶催化产生的H₂O₂在植物抗性反应、维管组织发育、细胞程序性死亡、侧根发生中发挥重要作用,最近又有文献报道铜胺氧化酶（copper amine oxidase,CuAO）催化产生的H₂O₂参与了ABA诱导的气孔关闭过程,但时至今日尚未见CuAO及其催化产物H₂O₂参与光/暗调控气孔运动的报道。本实验以蚕豆（Vicia faba L.）为材料,借助表皮条实验、药理学分析及激光共聚焦扫描显微镜技术,研究了CuAO及其催化产物H₂O₂在光/暗调控气孔运动中的作用,并对光/暗调控气孔运动中CuAO及其催化产物H₂O₂与另一重要气体信号分子NO的关系进行了初步探索。本研究对于进一步了解保卫细胞光/暗信号转导途径和机制有重要理论意义。主要实验结果如下:1.光下所试浓度CuAO抑制剂氨基胍（Aminoguanidine,AG）和2-溴乙胺（2-Bromoethylamine,BEA）对气孔开度无显著影响,但暗中显著促进气孔开放。此结果表明CuAO参与光/暗调控气孔运动过程,且暗示CuAO活性光下低而暗中高。2.现已明了CuAO催化腐胺（putrescine,Put）氧化降解产生H₂O₂,NH₃和4-氨基丁醛,后者再经几步反应生成γ-氨基丁酸（GABA）和琥珀酸（Succ）。我们发现光下CuAO催化产物H₂O₂显著促进气孔关闭,暗中则明显逆转AG、BEA促进气孔开放的作用,但CuAO其余催化产物NH₃,GABA和Succ无上述效应。这些结果表明,CuAO催化产物中仅H₂O₂参与光/暗调控气孔运动。3.由于Put为CuAO催化反应底物,因此暗中CuAO抑制剂促进气孔开放的效应也可能与Put增加有关。我们的结果证实,外源Put并无促进气孔开放的效应,可见CuAO抑制剂促进气孔开放与Put无关。4.借助H₂DCF-DA荧光探针和激光共聚焦显微镜检测内源H₂O₂水平的结果表明,暗确实诱导H₂O₂产生,而且暗诱导H₂O₂产生的效应被CuAO抑制剂AG、BEA阻止。此结果表明,暗确实通过CuAO途径诱导H₂O₂产生。5.借助DCF-2DA荧光探针和激光共聚焦显微镜检测内源NO水平的结果表明,暗和H₂O₂处理均诱导NO产生,而且暗诱导NO产生的效应可被CuAO抑制剂AG、BEA阻止。此结果表明,暗通过CuAO催化产生的H₂O₂诱导NO生成。综上所述,本研究的结果表明,光/暗通过调控CuAO活性影响保卫细胞H₂O₂水平进而调节NO合成,最终实现对气孔运动的调控。更多还原

【Abstract】 Now,people show increasingly interest in the sources and the physiological roles of hydrogen peroxide（H₂O₂）,which is an important plant signalling molecule.Previous studies proved that H₂O₂ generated by the plasma membrane NADPH oxidase was involved in abscisic acid（ABA）- and light/dark-regulated stomatal movement,and a lot of evidence indicated that H₂O₂,a catalysate of copper amine oxidase（CuAO） and Polyamine Oxidase（FAD-PAO）,participated in many key plant physiological processes,such as plant defense reaction,development of vascular tissues, programmed cell death（PCD）,occurrence of lateral root.Recent research reported that H₂O₂ generated by CuAO was involved in ABA-induced stomatal closure.However,the physiological roles of CuAO and its catalysate H₂O₂ in light/dark - regulated stomatal movement was still unclear. In the present study,using the abaxial epidermis of Vicia faba as materials,by means of the epidermal strip bioassay and the laser scanning confocal microscopy（LSCM）,the roles of CuAO and its catalysate H₂O₂ in light/dark-regulated stomatal movement were investigated,and the relationship between H₂O₂ generated by CuAO and nitric oxide（NO）,another important plant signalling molecule,was explored.The present results had an important theoretical significance for the further understanding of the guard cells signal transduction pathways and the mechanisms of responding to light/dark stimulus.The results were as follows:1.Aminoguanidine（AG） and 2-Bromoethylamine（BEA）,which are the inhibitors of the CuAO, had no significant impacts on the stomatal aperture in light,but both could significantly prevent dark-induced stomata closure.The results indicated that CuAO involved in light/dark-regulated stomatal movement and the activity of CuAO seemed higher in dark than in light.2.The products from putrescine（Put） oxidation by CuAO include H₂O₂,ammonia（NH₃） and 4-aminobutanal.4-aminobutanal can be easily catalyzed toγ-aminobutyric acid（GABA）,which is subsequently transaminated and oxidized to succinic acid（Succ）.The present results suggested that only H₂O₂ had significant effects on stomatal aperture in light,and could reverse AG- and BEA-promoted stomatal opening in darkness.These results indicated that H₂O₂ generated by CuAO was involved in light/dark-regulated stomatal movement. 3.Put is a substrate of CuAO,so CuAO inhibitors-induced stomatal opening might relate to the accumulation of Put.The present results showed that exogenous Put did not induce stomatal opening in darkness,showing that the effect of AG and BEA on stomatal opening is not related to the accumulation of Put.4.By means of LSCM based on H₂DCF-DA,a specific molecular probe of H₂O₂,we provided evidence that dark indeed induced the generation of endogenous H₂O₂ and the fluorescence derived by darkness was very striking compared with that in light condition.The striking fluorescence was prevented by AG and BEA,the inhibitors of CuAO,indicating that dark induced H₂O₂ generation could through CuAO pathway.5.By means of LSCM based on DCF-2DA,a specific molecular probe of NO,we provided evidence that both darkness and exogenous H₂O₂ induced NO increasing,and the effect of darkness were also suppressed by the inhibitors of CuAO,AG and BEA,suggesting that NO generated by darkness was dependent on H₂O₂ synthesis through CuAO pathway.In summary,the present results shown that light/dark adjusted the production of H₂O₂ by governing the activity of CuAO,and then regulated NO synthesis,finally regulating stomatal movement.更多还原