【作者】 宋喜贵；

【导师】 佘小平；

【作者基本信息】 陕西师范大学 ， 植物学， 2008， 博士

【摘要】 保卫细胞通过调节气孔运动控制植物与环境间的水分和气体交换。内源因素和外界因子均调控气孔运动。目前植物激素脱落酸对气孔运动的调控研究较为详尽,但细胞分裂素和生长素调控气孔运动的研究却欠深入。已有资料表明促细胞分裂原蛋白激酶（MAPKs）和钙依赖型蛋白激酶（CDPKs）也参与气孔运动,但详细机制仍未明确。最近10多年以来,过氧化氢（H₂O₂）和一氧化氮（NO）作为植物“第二信使”的研究已经成为逆境生物学的一个重要领域,尤其在植物-病原体和动物-病原体相互作用过程中H₂O₂和NO作为信号分子的研究已取得了一些进展。许多实验表明,H₂O₂和NO作为重要的信号分子参与气孔运动调节。我们此前工作证明光/暗调控气孔运动也与H₂O₂和NO有关,并且二者之间相互对话。迄今为止,仍然未见光/暗调控气孔运动中MAPKs/CDPKs与H₂O₂/NO、细胞分裂素/生长素与H₂O₂/NO以及一氧化碳（CO）与H₂O₂/NO关系的报道。本论文以蚕豆为研究材料,借助药理学方法和激光共聚焦扫描显微镜（LSCM）技术对上述问题进行了探索,主要结果如下:1.暗中一定浓度范围内的细胞分裂素（6-BA,KT 0-0.6μM）和生长素（IAA,NAA 0-10μM）明显诱导气孔开放,且表现浓度依赖效应,其最适浓度分别为0.2和10μM。细胞分裂素和生长素诱导气孔开放与他们能够降低H₂O₂水平有关。另外,和H₂O₂的清除剂ASA相类似,细胞分裂素不仅能够逆转外源H₂O₂引起的气孔关闭和降低胞内H₂O₂水平,而且能够减少黑暗已经诱导产生的内源H₂O₂并促进已经关闭的气孔重新开放。然而和H₂O₂的合成酶NADPH氧化酶的专一性抑制剂二苯基碘（DPI）作用方式类似,生长素既不能降低外源H₂O₂和黑暗引起的胞内H₂O₂水平,且不能促进气孔开放。这些结果说明细胞分裂素可能主要通过清除保卫细胞中暗诱导产生的H₂O₂进而引起气孔开放,而生长素可能通过抑制暗中H₂O₂生成降低胞内H₂O₂水平进而促进气孔开放。2.暗中细胞分裂素和生长素也能降低NO水平从而诱导气孔开放。与NO的专一性清除剂cPTIO相似,细胞分裂素不仅能够逆转SNP引起的气孔关闭和降低SNP引起的胞内NO水平,而且能够减少暗诱导已产生的内源NO水平进而促进已经关闭的气孔再开放。和一氧化氮合酶（NOS）的专一性抑制剂L-NAME作用类似,生长素既不能降低SNP和暗引起的胞内H₂O₂水平的增加,也不能促进SNP和暗诱导关闭的气孔开放。说明细胞分裂素降低暗诱导胞内NO水平可能主要通过清除方式进行,而生长素可能通过抑制NO的生成进而引起气孔开放。3.促细胞分裂原蛋白激酶激酶（MEK）抑制剂PD98059和钙依赖型蛋白激酶（CDPK）专一性抑制剂三氟拉嗪（TFP）都明显降低暗引起的H₂O₂水平逆转暗诱导气孔关闭,暗示MEK和CDPK参与暗诱导气孔关闭和H₂O₂产生。进一步的试验证明,与ASA类似而和DPI不一样,PD98059和TFP不仅能够减少光下外源H₂O₂引起的胞内H₂O₂水平进而促进气孔开放,而且能够降低暗诱导已产生的H₂O₂,促进暗诱导已关闭气孔重新开放。这些结果表明MEK和CDPK可能主要通过抑制H₂O₂的清除酶提高胞内H₂O₂水平参与暗诱导气孔关闭。当然也不排除MEK和CDPK在H₂O₂下游起作用的可能性。4.暗中PD98059和TFP明显促进气孔开放,且能降低暗诱导产生的NO。说明MEK和CDPK参与暗诱导NO增加,从而导致气孔关闭。另外,与L-NAME不同,与cPTIO类似,PD98059和TFP能降低光下SNP引起的胞内NO并逆转SNP引起的气孔关闭,也能减少暗诱导已产生的NO,进而引起已关闭气孔重新开放。暗示MEK和CDPK可能主要通过抑制NO的清除系统提高胞内NO水平参与暗诱导气孔关闭,MEK和CDPK在NO下游起作用的可能性也不能排除。5.最近,在动物中研究发现一氧化碳是另一种生理信使或生物活性分子。已有资料表明亚铁血红素加氧酶-1（HO-1,EC 1.14.99.3）能够促进亚铁血红素转变成一氧化碳和胆绿素,并同时有铁的释放。然而对植物中一氧化碳生理作用的了解还很有限。本实验首先探讨了CO在蚕豆气孔运动中的作用。结果表明,与H₂O₂作用效果相类似,CO的供体高铁血红素（Hematin）能以时间和剂量依赖的方式诱导气孔关闭,CO的气体饱和溶液亦如此,首次证明了CO和H₂O₂表现出相似的调控气孔运动的效应。我们还发现H₂O₂清除剂ASA和H₂O₂合成酶NADPH氧化酶专一性抑制剂二苯基碘（DPI）不仅能够逆转CO引起的气孔关闭还能抑制CO诱导的H₂O₂荧光,证实了CO引起的气孔关闭确实与保卫细胞中H₂O₂水平有关。另外,CO/NO清除剂血红蛋白（hemoglobin,Hb）,HO-1的抑制剂ZnPPIX,ASA和DPI不但都能够逆转黑暗引起的气孔关闭,而且都能抑制黑暗诱导的保卫细胞H₂O₂产生。这些结果表明保卫细胞CO水平也许光下低而暗中高;血红素加氧酶-1（HO-1）和NADPH氧化酶分别是保卫细胞CO和H₂O₂的合成酶源;来源于HO-1的CO介导了黑暗诱导保卫细胞H₂O₂的积累。6.SNP,Hematin和CO气体饱和溶液均能以时间和剂量依赖方式诱导气孔关闭,说明CO和NO也表现相似效应。我们的结果还显示cPTIO和L-NAME不仅能逆转CO引起的气孔关闭,还能清除CO诱导的NO产生,暗示CO引起的气孔关闭与NO/NOS信使系统有关。另外,CO/NO清除剂Hb和HO-1抑制剂ZnPPIX,cPTIO和L-NAME均逆转暗诱导气孔关闭和NO产生。这些结果表明,保卫细胞CO水平也许像NO一样光下低而暗中高;血红素加氧酶（HO-1）和一氧化氮合酶（NOS）分别是保卫细胞CO和NO的合成酶源;来源于HO-1的CO介导保卫细胞暗诱导NO的积累。综上所述,在结论与展望部分（P.152-FigureⅧ-1）,我们还勾勒出了保卫细胞中关于光/暗,cytokinins/auxins,MAPKs/CDPKs,一氧化碳和H₂O₂/NO的信号转导途径的大致线索。更多还原

【Abstract】 Guard cells control transpiration in plants and regulate gas exchange in leaves by opening and closing stomatal pores.Stomatal movements are regulated by both internal and external factors. Previous studies have shown that cytokinins and auxins can affect stomatal behaviour,and MAPKs/CDPKs are involved in the stomatal movement.However,little was still known about these exact mechanisms.From being molecules of somewhat novelty interest,in the last few years,H₂O₂ and NO have emerged to be central players in the world of plant guard cells signalling,articularly under various stressful situations.Our previous studies provided evidence that light/dark regulate stomatal movement via influencing hydrogen peroxide（H₂O₂） and nitric oxide（NO） production in guard cells of Vicia faba,and H₂O₂ and NO cross talk in the process.Up to now,there is little report to insight into the relation between mitogen-activated protein kinase kinase（MEK）/ calcium-dependent protein kinase（CDPK） and H₂O₂/NO,no report between cytokinins,auxins, carbon monoxide（CO） and H₂O₂/NO during light/darkness-regulated stomatal movement.Here,we select Vicia faba as plant material,through pharmacological and surgical approaches combined with laser scanning confocal microscope（LSCM） method to seek for these above problems.The main results are as follows.1.Cytokinins at concentrations of≥0.05μM significantly induced stomatal opening in darkness, as did auxins at concentrations of≥0.1μM.The optimum concentrations of cytokinins and auxins were 0.2 and 10μM,respectively.Both cytokinins and auxins reduced the levels of H₂O₂ in guard cells and induced stomatal opening in darkness.Additionally,cytokinins not only reduced exogenous H₂O₂ levels in guard cells caused by exposure to light,but also abolished H₂O₂ that had been generated during a dark period,and promoted stomatal opening,as did ascorbic acid（ASA,an important reducing substrate for H₂O₂ removal）.However,unlike cytokinins,auxins did not reduce exogenous H₂O₂,did not abolish H₂O₂ that had been generated in the dark,and therefore did not promote reopening of stoma induced to close in the dark.The above-mentioned effects of auxins were similar to that of diphenylene iodonium（DPI,an inhibitor of the H₂O₂-generating enzyme NADPH oxidase）.Taken together our results indicate that cytokinins probably reduce the levels of H₂O₂ in guard cells by scavenging,whereas auxins limit H₂O₂ levels through restraining H₂O₂ generation,inducing stomatal opening in darkness.2.Both cytokinins and auxins reduced the levels of NO in guard cells and induced stomatal opening in darkness.Additionally,cytokinins not only reduced NO levels in guard cells caused by sodium nitroprusside（SNP） in light but also abolished NO that had been generated by dark,and then promoted the closed stomata reopening,as did NO scavenger 2-（4-carboxyphenyl）-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide（cPTIO）.However,unlike cytokinins,auxins not only had incapability to reduce NO levels by SNP but also could not abolish NO having been generated by dark,so auxins could not promote the closed stomata to reopen.The above-mentioned effects of auxins were similar to that of nitric oxide synthase（NOS,enzyme commission 1.14.13.39） inhibitor N^G-nitro-L-Arg-methyl ester（L-NAME）.Hence,it is concluded that cytokinins reduced probably the levels of NO in guard cells via scavenging,and auxins reduced NO levels through restraining NO generation in all probability,and then induced stomatal opening in darkness.3.Both 2`-amino-3`-methoxyflavone（PD98059）（an inhibitor of mitogen-activated protein kinase kinase,MEK） and Trifluoperazine（TFP）（a specific inhibitor of calcium-dependent protein kinase,CDPK） reduced the levels of H₂O₂ in guard cells and promoted stomatal opening significantly in dark,implying that MEK/CDPK mediate dark-induced stomatal closure by influencing H₂O₂ levels of guard cells.In addition,like ASA,but unlike DPI,PD98059 and TFP not only reduced exogenous H₂O₂ levels in guard cells in light,but also abolished H₂O₂ that had been generated during a dark period,and promoted stomatal opening,the results suggest MEK and CDPK are probably relational to restraining the H₂O₂ scavenging enzyme to elevate H₂O₂ levels in guard cells during dark-induced stomatal closure,of course,the probability of MEK and CDPK acting as the target downstream of H₂O₂ in the signaling transduction chain is not excluded.4.Both PD98059 and TFP reduced the levels of NO in guard cells and promoted stomatal opening significantly in dark,implying that MEK/CDPK mediate dark-induced stomatal closure by affecting NO levels in guard cells.In addition,like NO scavenger cPTIO,but unlikeL-NAME, PD98059 and TFP not only reduced 4,5-diaminofluorescein diacetate（DAF-2 DA） fluorescence in guard cells by sodium nitroprusside（SNP） in light,but also abolished NO that had been generated during a dark period,and promoted stomatal opening,suggesting MEK and CDPK are probably relational to restraining the NO scavenging to elevate NO levels in guard cells during dark-induced stomatal closure.5.Recently,carbon monoxide（CO） was implicated as another important physiological messenger or bioactive molecule in animals.Previous researches indicate that heine oxygenase-1 （HO-1,EC 1.14.99.3） catalyzes the oxidative conversion of heine to CO and biliverdinⅨa with the concomitant release of iron.However,little is known about the physiological roles of CO in plant.In the portion,the regulatory role of CO during stomatal movement in Vicia faba was surveyed.Results indicated that,like hydrogen peroxide（H₂O₂）,CO donor Hematin induced stomatal closure in dose-and time-dependent manners.These responses were also proven by the addition of gaseous CO aqueous solution with different concentrations,showing the first times that CO and H₂O₂ exhibit the similar regulation role in the stomatal movement.Moreover,our data showed that ascorbic acid （ASA,an important reducing substrate for H₂O₂ removal） and diphenylene iodonium（DPI,an inhibitor of the H₂O₂-generating enzyme NADPH oxidase） not only reversed stomatal closure by CO, but also suppressed the H₂O₂ fluorescence induced by CO,implying that CO induced-stomatal closure probably involves H₂O₂ signal.Additionally,the CO/NO scavenger hemoglobin（Hb） and CO specific synthetic inhibitor ZnPPIX,ASA and DPI reversed the darkness-induced stomatal closure and H₂O₂ fluorescence.These results show that,maybe like H₂O₂,the levels of CO in guard cells of Vicia faba is higher in dark than that in light,HO-1 and NADPH oxidase are the enzyme systems responsible for generating endogenous CO and H₂O₂ in darkness respectively,and that CO being from HO-1 mediated darkness-induced H₂O₂ synthesis in guard cells stomatal closure of Vicia faba.6.Like SNP,CO donor Hematin induced stomatal closure in dose- and time-dependent manners.These responses were also proven by the addition of gaseous CO aqueous solution with different concentrations,showing the first times that CO and NO exhibite the same regulation role in the stomatal movement.Moreover,our data showed that cPTIO/L-NAME not only reversed stomatal closure by CO,but also suppressed the NO fluorescence induced by CO,implying that CO induced-stomatal closure probably involves NO/NOS signal system.Additionally,the CO/NO scavenger hemoglobin（Hb） and CO specific synthetic inhibitor ZnPPIX,NO scavenger cPTIO and nitric oxide synthase（NOS） inhibitor L-NAME reversed the darkness-induced stomatal closure and NO fluorescence.These results show that,maybe like NO,the levels of CO in guard cells of Vicia faba is higher in dark than that in light,HO-1 and NOS are the enzyme systems responsible for generating endogenous CO and NO in darkness respectively,and that CO being from HO-1 mediated darkness-induced NO synthesis in guard cells stomatal closure of Vicia faba.All together,the rough outlines of light/darkness,cytokinins/auxins,MAPKs/CDPKs,CO and H₂O₂/NO signalling of guard cell were drawn in the conclusions and future perspectives section （P.152-FigureⅧ-1）.更多还原