磷脂酰肌醇3-磷酸在紫外B辐射、乙烯或暗诱导蚕豆气孔关闭中的作用

【作者】 胡洁；

【导师】 贺军民；

【作者基本信息】 陕西师范大学 ， 植物学， 2008， 硕士

【摘要】 有文献表明保卫细胞对脱落酸（ABA）应答的过程中,通过NADPH氧化酶产生活性氧进而诱导气孔关闭。本实验室已经证明紫外中UV-B辐射能够诱导保卫细胞中过氧化氢（H₂O₂）（一种活性氧）的生成,从而引起气孔关闭,但是其产生H₂O₂是通过细胞壁过氧化物酶途径,乙烯也能通过细胞壁过氧化物酶途径生成H₂O₂中,这与ABA诱导H₂O₂产生的酶学途径不同。本实验室还证明暗诱导气孔关闭也是通过NADPH氧化酶途径产生H₂O₂从而引起气孔关闭的。另有研究表明,在ABA诱导气孔关闭的信号转导途径中,磷脂酰肌醇3-磷酸（PI3P）参与了该信号转导过程,且作用机理是PI3P通过与NADPH氧化酶结合,从而激活NADPH氧化酶活性进而生成H₂O₂。关于PI3P是否也参与UV-B、乙烯和暗诱导气孔关闭信号途径,以及在这些过程中与H₂O₂的关系如何,却尚未见报道。一氧化氮（NO）作为信号分子参与保卫细胞信号转导的研究已经很广泛。有研究表明保卫细胞中NO的产生主要来源于一氧化氮合酶（NOS）途径。本实验室已证明UV-B、乙烯和暗诱导气孔关闭途径中均有NO参与,但是NO和PI3P在调控气孔运动的过程中相互关系如何尚没有相关研究。本论文以蚕豆表皮条为实验材料,借助表皮条生物学分析法和激光扫描共聚焦显微镜检技术,主要探讨了PI3P在UV-B、乙烯、暗诱导气孔关闭信号途径中的作用及其与H₂O₂和NO的关系,旨在进一步完善UV-B、乙烯、暗诱导气孔关闭的信号转导途径,也为探明PI3P在植物细胞信号转导途径中的作用和信号网络积累资料和奠定基础。本文的主要结果如下:1.PI3P是磷脂酰肌醇3-激酶（PI3K）的催化产物,PI3K的两种抑制剂沃曼青霉素（WM）、LY294002（LY）能显著抑制UV-B辐射诱导的气孔关闭,说明PI3P参与了UV-B诱导气孔关闭途径。通过测定保卫细胞内源H₂O₂水平可知,PI3K的两种抑制剂也能显著降低UV-B辐射下保卫细胞内源H₂O₂水平的升高;同时,WM、LY抑制UV-B诱导的气孔关闭效应也能被外源H₂O₂处理有效逆转。由于前人工作表明UV-B诱导H₂O₂产生是通过细胞壁过氧化物酶途径,我们由此推断PI3P参与UV-B途径的作用机制可能是通过影响细胞壁过氧化物酶影响了保卫细胞内H₂O₂的生成。2.PI3K的两种抑制剂WM、LY能显著降低UV-B辐射下保卫细胞内源NO水平的升高;同时,WM、LY抑制UV-B诱导的气孔关闭效应也能被外源硝普钠（SNP,NO的一种供体）处理有效逆转,说明PI3P在NO上游起作用。因为NO合成是通过NOS途径,所以推测PI3P可能是通过影响NOS途径影响保卫细胞内NO的生成。3.PI3K的两种抑制剂WM、LY能显著抑制外源乙烯诱导的气孔关闭,且它们也能显著降低外源乙烯诱导的保卫细胞内源H₂O₂水平的升高;同时,WM、LY抑制外源乙烯诱导气孔关闭的效应也能被外源H₂O₂处理有效逆转。说明PI3P参与乙烯信号途径,并影响了保卫细胞内H₂O₂的生成。4.PI3K的两种抑制剂WM、LY能显著降低外源乙烯诱导的保卫细胞内源NO水平的升高;同时,WM、LY的抑制外源乙烯诱导气孔关闭的效应也能被外源SNP处理有效逆转。说明PI3P能够影响乙烯诱导的保卫细胞中NO的生成。5.PI3K的两种抑制剂WM、LY能显著抑制暗诱导的气孔关闭,且它们也能显著降低暗诱导的保卫细胞内源H₂O₂水平的升高;同时,WM、LY抑制暗诱导气孔关闭的效应也能被外源H₂O₂处理有效逆转。说明PI3P参与了暗诱导的气孔关闭,作用机理和ABA途径一样,是通过影响NADPH氧化酶途径影响保卫细胞内H₂O₂的生成。6.PI3K的两种抑制剂WM、LY能显著降低暗诱导的保卫细胞内源NO水平的升高;同时,WM、LY抑制暗诱导气孔关闭的效应也能被外源SNP处理有效逆转。说明PI3P参与了暗诱导的保卫细胞中NO的生成。更多还原

【Abstract】 Previous study has indicated that guard cell generate reactive oxygen species in response to abscisic acid by NADPH oxidase,which leads to stomatal closing.Our laboratory research had proved that cell-wall peroxidase is an enzyme responsible for H₂O₂ generation and stomatal closure in UV-B-treated guard cells,ethylene also could induced H₂O₂ by cell-wall peroxidase,it is defferent from ABA-induced H₂O₂ in guard cells.We still found that dark could induced H₂O₂ generation by NADPH oxidase。In addition,studies also show that PI3P play a role in ABA-induced H₂O₂ generation,the NADPH oxidase complex is responsible for H₂O₂ generation,and is activated by the binding of PI3P to one of the components.However,there is little information available about whether the signal molecule PI3P is also involved in the UV-B,ethylene,or dark-induced stomatal closure. Furthermore,it is also not clear the relationships between PI3P and H₂O₂ in these pathways.NO,which generated by NOS in guard cells,act as second messengers to mediate UV-B, ethylene,dark-induced stomatal closure.However,it is not clear whether PI3P involves in NO generation in guard cells.In order to further understand the signal transduction network in guard cells,we mainly investigated the role of PI3P in UV-B,ethylene or dark-induced stomatal closure and the relationships between PI3P and H₂O₂ or NO in Vicia faba by epidermal strip bioassay and laser-scanning confocal microscopy.The main results were followed:1.PI3P is a product of phosphatidylinositol 3-kinase（PI3K）,PI3K inhibitor wortmannin（WM） or LY294002（LY） inhibited UV-B-induced H₂O₂ generation and stomatal closure.In addition,H₂O₂ partially rescues the inhibitory effect of WM or LY on UV-B-induced stomatal closure.This result showed that PI3P involves in UV-B-induced stomatal closure.Cell-wall peroxidase has been suggested to be a H₂O₂-generating enzyme during UV-B signaling in guard cells,the inhibitory effect of WM or LY on UV-B-induced H₂O₂ generation indicates that PI3P involves UV-B-induced stomatal closure through effecting H₂O₂ generation by cell-wall peroxidase.2.PI3K inhibitor WM or LY inhibited UV-B-induced endogenous NO generation in guard cells. In addition,the inhibitory effects of WM and LY on UV-B-induced stomatal closure could be recovered significantly by exogenous sodium nitroprusside（SNP,exogenous NO donor） treatment.It showed that PI3P involves in UV-B-induced NO generation,NOS has been suggested to be a NO-generating enzyme during UV-B signaling in guard cells.It showed that PI3P involves UV-B-induced stomatal closure through effecting NO generation by NOS.3.PI3K inhibitor WM and LY inhibited ethlene-induced stomatal closure and H₂O₂ generation in guard cells.In addition,the inhibitory effects of WM and LY on ethlene-induced stomatal closure and H₂O₂ generation could be reversed significantly by exogenous H₂O₂ treatment.It showed that PI3P involves in ethlene-induced stomatal closure and H₂O₂ generation in guard cells.4.PI3K inhibitor WM or LY inhibited ethlene-induced endogenous NO generation in guard cells.In addition,the inhibitory effects of WM and LY on ethlene-induced stomatal closure could be recovered significantly by exogenous SNP treatment.It showed that PI3P involves in ethlene-induced NO generation.It showed that PI3P involves ethlene-induced stomatal closure through effecting NO generation in guard cells.5.PI3K inhibitor WM and LY inhibited dark-induced stomatal closure and H₂O₂ generation in guard cells.In addition,the inhibitory effects of WM and LY on dark-induced stomatal closure and H₂O₂ generation could be reversed significantly by exogenous H₂O₂ treatment.It showed that PI3P involves in dark-induced stomatal closure and H₂O₂ generation.NADPH oxidase has been suggested to be a H₂O₂-generating enzyme during dark signaling in guard cells,the inhibitory effect of WM or LY on dark-induced H₂O₂ generation indicates that PI3P involves dark-induced stomatal closure through effecting H₂O₂ generation by NADPH oxidase.6.PI3K inhibitor WM or LY inhibited dark-induced endogenous NO generation in guard cells. In addition,the inhibitory effects of WM and LY on dark-induced stomatal closure could be recovered significantly by exogenous SNP treatment.It showed that PI3P involves in dark-induced NO generation.It showed that PI3P involves dark-induced stomatal closure through effecting NO generation in guard cells.更多还原