【作者】 吕东；

【导师】 宋纯鹏； 张骁；

【作者基本信息】 河南大学 ， 植物学， 2004， 硕士

【摘要】 脱落酸（ABA）可以调节植物抗逆（包括干旱、冷害、盐害等）的多种生理反应和分子生物学效应。逆境下，伴随着体内ABA水平的升高植物气孔发生关闭。以前的研究报道ABA能通过促进保卫细胞K⁺外流来诱导气孔关闭，并且过氧化氢（H₂O₂）也参与其中。保卫细胞也已成为研究ABA信号转导的模式材料。 一氧化氮（NO）是一种非常活泼的分子，它参与多种生理活动。NO参与了跨膜信号转导的多个层面，如基因活化、蛋白质表达和酶活性的调节。最近的研究表明NO是植物在病原菌侵害后激活防御反应的信号之一。在植物中，NO和活性氧（ROIs）的相互作用已有报道。Delledonne等揭示在蚕豆悬浮细胞中诱导细胞超敏感死亡需要NO和ROIs的产生有一个平衡。Lum和同事发现H₂O₂作为上游信号诱导NO的产生，他们还发现ABA诱导的NO产生是经由钙离子介导的，因为它可以被钙离子通道阻断剂异搏定（verapamil）所抑制。最近的研究结果显示H₂O₂和NO都能作为信号分子介导ABA诱导的气孔关闭过程，但有关H₂O₂和NO在ABA信号转导途径中的相互关系知之甚少。 本文通过药理学和细胞学方法分析了H₂O₂与NO之间可能存在的关系并证明NO可能作为H₂O₂下游信号参与ABA诱导的蚕豆气孔关闭过程。表皮条生物分析显示和ABA作用相同，NO的供体硝普钠（SNP）和H₂O₂均能诱导蚕豆气孔关闭。由ABA或H₂O₂所诱导的蚕豆气孔关闭过程可部分地被NO的专一清除剂c-PTIO所逆转，而H₂O₂的清除剂过氧化氢酶（CAT）则不能逆转NO诱导的气孔关闭过程。用NO或H₂O₂专一的荧光探针二氨基荧光素二乙酸酯（DAF-2DA）或二氯荧光素二乙酸酯（H₂DCF-DA）分别标记蚕豆保卫细胞来检测蚕豆保卫细胞内NO或H₂O₂的水平变化发现，10 μmol／L ABA处理后，胞内H₂O₂的产生速率明显高于NO的产生速率。H₂O₂的清除剂CAT几乎可完全抑制ABA所诱导的DAF的荧光增加；外源H₂O₂和ABA一样显著诱导胞内DAF的荧光增加。NO的专一清除剂c-PTIO对ABA诱导的DCF荧光略有促进作用，但外源SNP不能诱导胞内DCF荧光的增加。这些结果表明，在ABA通过诱导H₂O₂和NO产生从而促进气孔关闭的过程中，H₂O₂可能在NO的上游起作用并受NO的负反馈调节。更多还原

【Abstract】 Abscisic acid (ABA) can regulate a variety of physiological and molecular responses for plant against stresses, including drought, chilling, salinity, and so forth. More often stomatal closure occurred in these responses accompanying with ABA production. Previous research reported that ABA may bring about reduction in stomatal aperture by promoting the efflux of potassium salt from guard cells, and hydrogen oxide (H2O2) involves into this process as an intermediate. The guard cell has also become a model system for investigation of ABA signaling pathways.Nitric oxide (NO) is a very active molecule involvs in many and diverse biological pathways. NO touches upon multiple aspects of intracellular signaling pathways such as gene activation, protein expression and activity regulation of enzymes. Recent studies have demonstrated that NO is another signal that activates defense responses after pathogen attack.Signal interaction between NO and reactive oxygen intermediates (ROIs) in the plant have already been reported and recently nitric oxide was reported to be a novel component of abscisic acid signaling in stomatal guard cells. Delledonne and colleagues demonstrated that, in soybean cell suspension, the efficient induction of hypersensitive cell death requires a balance between ROIs and NO production. Lum and co-workers identified H2O2 as an upstream signal that leads to NO production. They also found that the H2O2 induced NO production was mediated via calcium ion flux, as it was blocked in the presence of a calcium ion channel blocker, verapamil.Recent documents suggested that either H2O2 or NO function as signal molecules to mediate ABA-induced stomatal closure in guard cells, but there was no report to study on the relationship between H2O2 and NO in ABA signal transduction pathway. Here, using stomatal analysis and laser scanning cofocal microscope techniques, we show firstly that NO as the downstream intermediate of H2O2 signaling mediates ABA-induced stomatal closure in Viciafaba L. Sodium nitroprusside (SNP, a NO donor) and H2O2 could mimic the effects of ABA on stomatal closure. Carboxy-PTIO (c-PTIO, a specific scavenger of NO) partly reversed the stomatal closure induced by ABA or H2O2. While catalase (CAT), a H2O2 scavenger, failed to reverse NO-induced aperturereduction in Vicia faba guard cells. Monitoring the changes of both NO and H2O2 generation in guard cells by using fluorescent probe of NO or H2O2, DAF-2DA or H2DCF-DA, respectively, we found that the generating rate of H2O2 in the guard cells was faster than that of NO after treated with 10 mol/L ABA. CAT almost completely inhibited the increase of DAF fluorescence induced by ABA. Similar to ABA, exogenous H2O2 provoked the production of NO. C-PTIO could slightly enhance the fluorescent intensity of DCF stimulated by ABA, while exogenous SNP could not increase DCF fluorescence in guard cells. Taken together, these results suggest that H2O2 could probably act as upstream component of NO signaling and NO negatively regulate H2O2 generation in ABA-induced stomatal closure in guard cells.更多还原