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Cytokinin/auxin、MEK/CDPK和CO在蚕豆气孔运动中的作用及其与H2O2和NO的关系研究

2020-11-28阅读(817)

Cytokinin/auxin、MEK/CDPK和CO在蚕豆气孔运动中的作用及其与H2O2和NO的关系研究

论文摘要

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。

论文目录

  • 摘要
  • Abstract
  • Chapter One.Summarization of Literature
  • Ⅰ-1.The stomata and the guard cell
  • Ⅰ-2.Light-induced stomatal opening
  • Ⅰ-3.ABA regulates stomatal movement
  • Ⅰ-4.Cytokinins and auxins regulate stomatal movement
  • Ⅰ-4-1.Cytokinins and stomatal movement
  • Ⅰ-4-2.Auxins and stomatal movement
  • Ⅰ-5.Hydrogen peroxide and its signalling role in guard cells
  • 2O2 in plant'>Ⅰ-5-1.The resource of H2O2 in plant
  • 2O2 in plant'>Ⅰ-5-2.The role of H2O2 in plant
  • 2O2 as a signal in guard cells'>Ⅰ-5-3.H2O2 as a signal in guard cells
  • Ⅰ-6.Nitric oxide and its signalling role in guard cells
  • Ⅰ-6-1.The resource of NO in plant
  • Ⅰ-6-1-1.Nitric oxide synthase（NOS）
  • Ⅰ-6-1-2.Nitrate reductase（NR）
  • Ⅰ-6-1-3.Other enzymatic sources of NO
  • Ⅰ-6-1-4.Nonenzymatic sources of NO
  • Ⅰ-6-2.The function of NO in plant
  • Ⅰ-6-3.NO as a signal in guard cells
  • Ⅰ-7.ROS,NO and SA in plant defense responses
  • 2O2 and NO signalling and cross-talk in plant cells'>Ⅰ-8.H2O2 and NO signalling and cross-talk in plant cells
  • 2O2 and NO signalling in guard cells'>Ⅰ-9.ABA,H2O2 and NO signalling in guard cells
  • Ⅰ-10.MAPKs and its signalling networks in guard cells
  • Ⅰ-10-1.Biochemical assay of MAPKs
  • Ⅰ-10-2.Plant MAPKs cascades
  • Ⅰ-10-3.The roles of MAPKs in plant responses
  • Ⅰ-10-4.MAPKs and ROS in pathogen signalling
  • Ⅰ-10-5.MAPKs signal pathway in guard cells
  • Ⅰ-11.CDPKs and its signalling in guard cells
  • Ⅰ-12.Carbon monoxide and its signalling in plant
  • Ⅰ-12-1.Biogenic sources of CO
  • Ⅰ-12-2.CO production by heme oxygenase enzyme（HO）
  • Ⅰ-12-3.Role of CO in animals
  • Ⅰ-12-4.HO-1 and CO in plants
  • Ⅰ-13.The intention and significance of this research
  • Chapter Two.The decrease of the hydrogen peroxide levels in guard cells during cytokinin- and auxin-induced stomatal opening
  • Ⅱ-1.Introduction
  • Ⅱ-2.Materials and methods
  • Ⅱ-2-1.Chemicals
  • Ⅱ-2-2.Plant materials
  • Ⅱ-2-3.Stomatal bioassay
  • 2DCF-DA'>Ⅱ-2-4.Dye loading of H2DCF-DA
  • Ⅱ-2-5.Laser-scanning confocal microscopy
  • Ⅱ-2-6.Statistical analysis
  • Ⅱ-3.Results
  • Ⅱ-3-1.Both cytokinins and auxins induce stomatal opening in darkness
  • 2O2 levels of guard cells in darkness'>Ⅱ-3-2.Both cytokinins and auxins decrease H2O2 levels of guard cells in darkness
  • 2O2-induced stomatal closure can be prevented by cytokinins,but not by auxins'>Ⅱ-3-3.Exogenous H2O2-induced stomatal closure can be prevented by cytokinins,but not by auxins
  • 2O2-induced DCF fluorescence in guard cells is reduced by cytokinins,but not by auxins'>Ⅱ-3-4.Exogenous H2O2-induced DCF fluorescence in guard cells is reduced by cytokinins,but not by auxins
  • Ⅱ-3-5.Closed stomata caused by dark can be reopened by cytokinins,but not by auxins
  • 2O2 generated by darkness,but auxins do not'>Ⅱ-3-6.Cytokinins reduce levels of H2O2 generated by darkness,but auxins do not
  • Ⅱ-4.Discussion
  • Chapter Three.Cytokinin- and auxin-induced stomatal opening involves the change of nitric oxide levels in guard cells
  • Ⅲ-1.Introduction
  • Ⅲ-2.Materials and methods
  • Ⅲ-2-1.Chemicals
  • Ⅲ-2-2.Plant materials
  • Ⅲ-2-3.Stomatal bioassay
  • Ⅲ-2-4.Dye loading of DAF-2 DA
  • Ⅲ-2-5.Laser-scanning confocal microscopy
  • Ⅲ-2-6.Statistical analysis
  • Ⅲ-3.Results
  • Ⅲ-3-1.Cytokinins and auxins can induce stomatal opening
  • Ⅲ-3-2.Effects of cytokinins and auxins on the darkness-induced NO levels in guard cells
  • Ⅲ-3-3.Cytokinins can,but auxins not,reverse SNP-induced stomatal closure
  • Ⅲ-3-4.Effects of cytokinins and auxins on the SNP-induced DAF-2 DA fluorescence levels in guard cells
  • Ⅲ-3-5.Cytokinins can,but auxins not,reopen the closed stomata caused by dark
  • Ⅲ-3-6.Effects of cytokinins and auxins on NO levels having been generated by darkness
  • Ⅲ-4.Discussion
  • Chapter Four.MAPK kinase and CDP kinase modulate hydrogen peroxide levels during darkness-induced stomatal closure
  • Ⅳ-1.Introduction
  • Ⅳ-2.Materials and methods
  • Ⅳ-2-1.Chemicals
  • Ⅳ-2-2.Plant materials
  • Ⅳ-2-3.Stomatal bioassay
  • 2DCF-DA'>Ⅳ-2-4.Dye loading of H2DCF-DA
  • Ⅳ2-5.Laser-scanning confocal microscopy
  • Ⅳ-2-6.Statistical analysis
  • Ⅳ-3.Results
  • Ⅳ-3-1.Effects of PD98059 and TFP on dark-induced stomatal closure
  • 2O2 levels of guard cells'>Ⅳ-3-2.Both PD98059 and TFP affect the dark-induced H2O2 levels of guard cells
  • 2O2'>Ⅳ-3-3.Effects of PD98059/TFP on stomatal closure and DCF fluorescence in guard cells induced by exogenous H2O2
  • Ⅳ-3-4.The closed stomata caused by dark can be reopened by PD98059 and TFP
  • 2O2 generated by darkness'>Ⅳ-3-5.PD98059 and TFP reduce levels of H2O2 generated by darkness
  • Ⅳ-4.Discussion
  • Chapter Five.The nitric oxide levels are modulated by MAPK kinase and CDP kinase during darkness-induced stomatal closure
  • Ⅴ-1.Introduction
  • Ⅴ-2.Materials and methods
  • Ⅴ-2-1.Chemicals
  • Ⅴ-2-2.Plant materials
  • Ⅴ-2-3.Stomatal bioassay
  • Ⅴ-2-4.Dye loading of DAF-2 DA
  • Ⅴ-2-5.Laser-scanning confocal microscopy
  • Ⅴ-2-6.Statistical analysis
  • Ⅴ-3.Results
  • Ⅴ-3-1.Both PD98059 and TFP reverse darkness-induced stomatal closure
  • Ⅴ-3-2.Both PD98059 and TFP affect the dark-induced NO levels of guard cells
  • Ⅴ-3-3.Effects of PD98059/TFP on stomatal closure and NO levels in guard cells by SNP
  • Ⅴ-3-4.PD98059 and TFP reopen the closed stomata caused by darkness
  • Ⅴ-3-5.PD98059 and TFP reduce levels of NO generated by darkness
  • Ⅴ-4.Discussion
  • Chapter Six.Carbon monoxide-induced stomatal closure involves generation of hydrogen peroxide in guard cells
  • Ⅵ-1.Introduction
  • Ⅵ-2.Materials and methods
  • Ⅵ-2-1.Chemicals
  • Ⅵ-2-2.Plant materials
  • Ⅵ-2-3.Preparation of the gaseous CO aqueous solution
  • Ⅵ-2-4.Stomatal bioassay
  • 2DCF-DA'>Ⅵ-2-5.Dye loading of H2DCF-DA
  • Ⅵ-2-6.Laser-scanning confocal microscopy
  • Ⅵ-2-7.Statistical analysis
  • Ⅵ-3.Results
  • Ⅵ-3-1.Exogenous CO induces stomatal closure in a dose- and time-dependent manner
  • 2O2 is involved in CO-regulated stomatal closure'>Ⅵ-3-2.H2O2 is involved in CO-regulated stomatal closure
  • 2O2 generation in Vicia faba guard cells'>Ⅵ-3-3.CO induces H2O2 generation in Vicia faba guard cells
  • 2O2 in Vicia faba'>Ⅵ-3-4.Darkness-induced stomatal closure involves CO and H2O2 in Vicia faba
  • 2O2 synthesis'>Ⅵ-3-5.CO is involved in darkness-induced H2O2synthesis
  • Ⅵ-4.Discussion
  • Chapter Seven.Carbon monoxide-induced stomatal closure is dependent on nitric oxide synthesis in guard cells
  • Ⅶ-1.Introduction
  • Ⅶ-2.Materials and methods
  • Ⅶ-2-1.Chemicals
  • Ⅶ-2-2.Plant materials
  • Ⅶ-2-3.Preparation of the gaseous CO aqueous solution
  • Ⅶ-2-4.Stomatal bioassay
  • Ⅶ-2-5.Dye loading of DAF-2 DA
  • Ⅶ-2-6.Laser-scanning confocal microscopy
  • Ⅶ-2-7.Statistical analysis
  • Ⅶ-3.Results
  • Ⅶ-3-1.Effect of exogenous CO on stomatal aperture of Vicia faba
  • Ⅶ-3-2.Effects of the scavenger/inhibitor of CO/NO on stomatal closure by CO
  • Ⅶ-3-3.CO induces NO generation in Vicia faba guard cells
  • Ⅶ-3-4.Darkness-induced stomatal closure involves CO and NO in Vicia faba
  • Ⅶ-3-5.CO is involved in darkness-induced NO synthesis
  • Ⅶ-4.Discussion
  • Chapter Eight.Conclusions and future perspectives
  • Ⅷ-1.Conclusions
  • Ⅷ-2.Future perspectives
  • References
  • Acknowledgments
  • The publications during studing for doctor's degree

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