【作者】 安振锋；

【导师】 章文华；

【作者基本信息】 南京农业大学 ， 植物学， 2008， 博士

【摘要】 在逆境胁迫下,脱落酸（ABA）通过一个复杂的信号转导网络来调节气孔的开度、减少水分的散失是植物获得抗逆性的重要机理之一。H₂O₂作为一种重要的信号物质参与了ABA诱导的气孔运动。植物体内的H₂O₂可以通过NADPH氧化酶、胺氧化酶等途径产生。目前已证实NADPH氧化酶产生的H₂O₂参与了ABA诱导的气孔关闭。植物中的胺氧化酶可以通过降解二胺或多胺产生H₂O₂,但是这一来源的H₂O₂是否参与ABA诱导的气孔关闭尚未见报道。以前在拟南芥中的研究表明,磷脂酶D（PLD）水解磷脂产生磷脂酸（PA）,PA通过激活NADPH氧化酶产生H₂O₂从而诱导气孔关闭,但这一H₂O₂的产生途径在蚕豆中是否存在及其与胺氧化酶产生H₂O₂的途径之间的关系尚未见报道。本研究选用蚕豆保卫细胞为研究体系,采用药理学、生理学、细胞生物学及生物化学的方法系统的研究了蚕豆中二胺氧化酶（CuAO）、磷脂酶D、NADPH氧化酶在ABA诱导气孔运动过程中的作用及其相互关系。研究表明CuAO的抑制剂可以明显抑制ABA诱导的蚕豆气孔关闭,在CuAO的几种产物中只有H₂O₂可以诱导气孔关闭。蚕豆中的CuAO主要存在于质外体,ABA迅速提高其活性,并使保卫细胞中H₂O₂含量升高,CuAO的抑制剂明显抑制了这一过程。这说明蚕豆中CuAO来源的H₂O₂参与了ABA诱导的气孔关闭。蚕豆的CuAO通过降解腐胺产生H₂O₂,另两种常见多胺亚精胺、精胺不能作为CuAO的底物。作为CuAO的底物,腐胺也可以提高CuAO的活性和保卫细胞中H₂O₂含量,并诱导气孔关闭,CuAO的抑制剂明显抑制了这一过程。这进一步说明蚕豆保卫细胞中的CuAO通过降解腐胺产生H₂O₂来促进气孔关闭。ABA和腐胺明显提高了蚕豆保卫细胞内Ca²⁺含量,CuAO抑制剂明显抑制了这一过程。这进一步说明CuAO降解腐胺产生的H₂O₂通过诱导保卫细胞中Ca²⁺含量升高从而参与了ABA诱导的气孔关闭。ABA明显提高了蚕豆叶片质膜上另一种产生H₂O₂的酶-NADPH氧化酶的活性。NADPH氧化酶的抑制剂明显抑制了ABA诱导的保卫细胞内H₂O₂含量升高和气孔关闭,如同时加入CuAO的抑制剂又可以更大程度的抑制ABA诱导的保卫细胞内H₂O₂含量升高。这说明NADPH氧化酶和CuAO可能各自独立的参与了ABA诱导的保卫细胞内H₂O₂的产生。CuAO的抑制剂对ABA诱导的NADPH氧化酶活性的升高无明显影响,NADPH氧化酶抑制剂对ABA和腐胺诱导的CuAO活性的升高也没有明显影响。这进一步说明蚕豆中CuAO和NADPH氧化酶各自独立的参与了ABA诱导的保卫细胞内H₂O₂的产生和气孔关闭。ABA对气孔运动的调节还包括其对气孔打开的抑制。本研究表明作为ABA的下游信号,H₂O₂也明显抑制了蚕豆气孔的打开,并且H₂O₂的清除剂可以部分的解除H₂O₂和ABA对蚕豆气孔打开的抑制,说明H₂O₂参与了ABA对气孔打开的抑制。CuAO的抑制剂可以部分的解除ABA对蚕豆气孔打开的抑制,并且在CuAO的几种产物中只有H₂O₂可以抑制气孔打开,说明CuAO途径产生的H₂O₂参与了ABA对气孔打开的抑制。作为CuAO的底物,腐胺通过提高CuAO的活性产生H₂O₂来抑制气孔打开,CuAO的抑制剂可以部分的解除腐胺对气孔打开的抑制。这些结果说明CuAO通过降解put产生的H₂O₂参与了ABA对气孔打开的抑制。PLD的抑制剂部分抑制了ABA诱导的蚕豆保卫细胞内H₂O₂含量的升高和气孔关闭,如同时加入CuAO的抑制剂又有更大程度的抑制作用。这说明蚕豆保卫细胞中的PLD也参与了ABA诱导的H₂O₂含量升高和气孔关闭,并且在这一过程中PLD与CuAO之间可能是平行的关系。PLD的产物PA也明显提高了蚕豆保卫细胞内H₂O₂的含量并诱导气孔关闭,CuAO的抑制剂对PA的作用无明显影响,但是NADPH氧化酶的抑制剂部分的抑制了PA的效应。这说明PLD的产物PA通过激活NADPH氧化酶产生H₂O₂来促进气孔关闭,ABA诱导的蚕豆叶片CuAO活性的升高不受PLD的调控,这进一步说明ABA经PLD、NADPH氧化酶产生H₂O₂诱导气孔关闭和ABA经CuAO产生H₂O₂诱导气孔关闭这两条途径是彼此独立的。本研究表明在蚕豆的保卫细胞中存在一条尚未报道过的信号转导途径:ABA→二胺氧化酶→过氧化氢→钙离子→气孔关闭。本研究还表明在蚕豆保卫细胞中存在与拟南芥中类似的信号转导途径:ABA→PLD→NADPH氧化酶→过氧化氢→气孔关闭。这两条途径之间是彼此独立的。这些发现对于深入认识并完善保卫细胞中ABA的信号转导网络有着重要的意义。更多还原

【Abstract】 In stress conditions,abscisic acid（ABA） modulates stomatal apertures through an intricate signalling network to reduce water loss and this is crucial to improve plant tolerance.H₂O₂ is an important signalling molecule that involved in ABA-induced stomatal movement and it comes from NADPH oxidase,amine oxidase and other sources in plants. It has been reported that the H₂O₂ generated by NADPH oxidase is involved in ABA-induced stomatal closure.The amine oxidase in plants catalyzes the oxidation of diamine or polyamine and produce H₂O₂.But whether the H₂O₂ generated by amine oxidase is involved in ABA-induced stomatal closure has not been reported.It has been reported that the phospholipase D（PLD） in Arabidopsis hydrolyzes phospholipids, producing phosphatidic acid（PA）.PA activates NADPH oxidase producing H₂O₂ and then induces stomatal closure.But whether this route is exist in Vicia faba and its relationship with the route that the H₂O₂ generated by CuAO have not been reported.In this study,we investigated the role and the relationship of CuAO,PLD and NADPH oxidase in ABA-induced stomatal closure in Vicia faba using pharmacological, physiological,cytobiological and biochemical approaches.Our results showed that the CuAO inhibitor inhibited the ABA-induced stomatal closure significantly and in the metabolized produces of CuAO,only H₂O₂ could induce stomatal closure.The CuAO in Vicia faba mainly existed in apoplast.ABA elevated the CuAO activity and the H₂O₂ level in guard cells rapidly,and the CuAO inhibitor inhibited the process.CuAO in Vicia faba oxidized putrescine（put） but not spermidine（spd） or spermine（spm） to produce H₂O₂.As the substrate of CuAO,put also activated the CuAO activity,and induced the elevation of H₂O₂ content in guard cells and stomatal closure.The CuAO inhibitor inhibited the process significently.These results indicate that CuAO is involved in ABA-induced stomatal closure via its degradation of put to produce H₂O₂.ABA and put elevated the Ca²⁺ content in Vicia faba guard cells and the CuAO inhibitor inhibited the process.This indicates that the H₂O₂ generated by CuAO is involved in ABA-induced stomatal closure via its elevation the Ca²⁺ content in guard cells.ABA elevated the activity of NADPH oxidase in Vicia faba plasma membrane which is another source of H₂O₂.The inhibitor of NADPH oxidase inhibited the ABA-induced elevation of H₂O₂ content in guard cells and stomatal closure.The elevation of the H₂O₂ content was more inhibited when the CuAO inhibitor was used together.These results indicated that NADPH oxidase and CuAO are involved in ABA-induced H₂O₂ production in guard cells independently.The CuAO inhibitor had no effect on the elevation of NADPH oxidase activity induced by ABA.The inhibitor of NADPH oxidase had no effect on the elevation of CuAO activity induced by ABA and put.These results further prove that the two enzymes are involved in ABA-induced H₂O₂ production in guard cells and stomatal closure independently.Inhibition of stomatal opening is another role of ABA in modulation stomatal movement.As the downstream signal of ABA,H₂O₂ inhibited stomatal opening in vicia faba.The H₂O₂ scavengers partly relieved the H₂O₂-or ABA-suppressed stomatal opening. This indicates that H₂O₂ is involed in ABA-suppressed stomatal opening.CuAO inhibitor partly relieved the ABA-suppressed stomatal opening and in the metabolized produces of CuAO only H₂O₂ inhibited stomatal opening.This indicates that the H₂O₂ generated by CuAO is involved in ABA-inhibited stomatal opening.As the substrate of CuAO,put also inhibited stomatal opening via elevation CuAO activity to produce H₂O₂.CuAO inhibitor partly relieved the put-suppressed stomatal opening.These results indicate that CuAO is involved in ABA-suppressed stomatal opening via its degradation of put to produce H₂O₂.PLD inhibitor partly inhibited the elevation of H₂O₂ content in vicia faba guard cells and stomatal closure induced by ABA,and a more inhibition was found when CuAO inhibitor was used together.This indicates that the PLD in Vicia faba guard cells is involved in ABA-induced H₂O₂ production and stomatal closure and PLD and CuAO may involved in this process independently.As the product of PLD,PA also induced the elevation of H₂O₂ content in vicia faba guard cells and stomatal closure and the CuAO inhibitor had no effect on the process,but the inhibitor of NADPH oxidase partly inhibited the process.This indicates that PA induces stomatal closure via activation NADPH oxidase to produce H₂O₂. The elevation of CuAO activity in vicia faba leaves induced by ABA was not mediated by PLD.This further proves that ABA induces H₂O₂ production and stomatal closure via its activation of PLD,NADPH oxidase and CuAO activity,and the two routes are independent.Based on these results,we propose a new signal route in vicia faba guard cells: ABA→CuAO→H₂O₂→Ca²⁺→stomatal closure.The signal route:ABA→PLD→NADPH oxidase→H₂O₂→stomatal closure is also exist in Vicia faba guard cells which is similar to that in Arabidopsis and the two routes are independently.These discoveries have important significance for thorough understanding and consummation of the ABA signalling pathway network in guard cells.更多还原