节点文献
G蛋白、PTKs和PLC/PLD通过调节保卫细胞H2O2、NO水平介导光/暗调控的气孔运动
【作者】 张媛华；
【导师】 佘小平；
【作者基本信息】 陕西师范大学 ， 植物学， 2010， 博士
【摘要】 气孔是控制植物与环境间气体和水分交换的主要通道,气孔运动受各种环境因素和植物激素调控。前人曾就保卫细胞脱落酸(ABA)信号转导机制进行了大量研究,但保卫细胞光／暗信号转导研究尚不充分。本文借助药理学方法和激光扫描共聚焦显微镜(LSCM)技术对异源三聚体G蛋白、蛋白酪氨酸激酶(PTKs)和磷脂酶C/磷脂酶D (PLC/PLD)在光／暗调控气孔运动中的作用及其与H2O2、NO的关系进行了初步研究。所得结果主要如下：1.光下CTX显著诱导蚕豆和野生型拟南芥气孔关闭但PTX无效应,暗中PTX显著促进蚕豆和野生型拟南芥气孔开放但CTX无效应；无论光下、暗中拟南芥突变体gpal-1、gpal-2气孔均开放,CTX、PTX均不影响其气孔开度。这些结果表明G蛋白参与光／暗调控气孔运动,光通过钝化G蛋白诱导气孔开放,暗通过激活G蛋白促进气孔关闭。光下H2O2显著诱导蚕豆和野生型拟南芥气孔关闭但Vc、CAT和DPI无效应,暗中Vc、CAT和DPI显著促进蚕豆和野生型拟南芥气孔开放但H202无效应；H202检测结果显示蚕豆和野生型拟南芥保卫细胞H202水平光下低而暗中高。这些结果证明H202参与光／暗调控气孔运动,光通过降低保卫细胞H202水平诱导气孔开放,暗通过提高H202水平促进气孔关闭。光下CTX提高蚕豆和野生型拟南芥保卫细胞H202水平,暗中PTX降低蚕豆和野生型拟南芥保卫细胞H202水平；无论光下、暗中拟南芥突变体gpal-1、gpal-2保卫细胞均无H202荧光。这些结果显示G蛋白钝化降低H202水平,G蛋白激活提高H202水平。2.光下SNP显著诱导蚕豆和野生型拟南芥气孔关闭但c-PTIO和L-NAME无效应,暗中c-PTIO和L-NAME显著促进蚕豆和野生型拟南芥气孔开放但SNP无效应；NO检测结果显示蚕豆和野生型拟南芥保卫细胞NO水平光下低而暗中高。这些结果证明NO参与光／暗调控气孔运动,光通过降低保卫细胞NO水平诱导气孔开放,暗通过提高NO水平促进气孔关闭。光下CTX提高蚕豆和野生型拟南芥保卫细胞NO水平,暗中PTX降低蚕豆和野生型拟南芥保卫细胞NO水平；无论光下、暗中拟南芥突变体gpal-1、gpal-2保卫细胞均无NO荧光。这些结果显示G蛋白钝化降低NO水平,G蛋白激活提高NO水平。3.酪氨酸蛋白激酶(PTKs)抑制剂genestein, tyrphostin A23均阻止暗诱导蚕豆气孔关闭并降低保卫细胞H202水平。进一步的研究表明,genestein和tyrphostin A23不仅抑制外源H202诱导的气孔关闭和保卫细胞DCF荧光,而且促进暗诱导已关闭气孔重新开放,并降低保卫细胞暗诱导下已产生的H2O2。这些结果表明,PTKs通过抑制H202清除进而提高保卫细胞H202水平介导暗诱导气孔关闭。4.蛋白酪氨酸激酶(PTKs)抑制剂genestein、tyrphostin A23均阻止暗诱导蚕豆气孔关闭并降低保卫细胞NO水平。进一步的研究表明,genestein和tyrphostin A23不仅抑制SNP诱导的气孔关闭和保卫细胞NO荧光,而且促进暗诱导已关闭气孔重新开放,并降低保卫细胞暗诱导下已产生的NO。这些结果表明,PTKs通过抑制NO清除进而提高保卫细胞NO水平介导暗诱导气孔关闭。5.PLC抑制剂U73122和PLD抑制剂1-丁醇(n-butanol)均显著阻止暗诱导蚕豆气孔关闭并降低保卫细胞H202水平,PLC和PLD的催化产物PA显著促进蚕豆气孔关闭并增加保卫细胞H202水平。这些结果表明,PLC和PLD经过其催化产物PA诱导H202产生参与暗诱导气孔关闭。6.U73122和1-丁醇(n-butanol)既显著阻止暗诱导蚕豆气孔关闭、降低保卫细胞NO水平,也显著阻止SNP诱导气孔关闭；PA虽诱导气孔关闭但不诱导NO产生。这些结果表明,PLC和PLD既介导NO产生,也介导NO诱导气孔关闭的作用,但PLC、PLD介导NO产生与其催化产物PA无关。综上所述,本文的结果证明G蛋白、PTKs和PLC/PLD均通过调节保卫细胞H2O2、NO水平参与光、暗调控气孔运动。
【Abstract】 Stoma is the main gate controlling transpiration and regulating gas exchange in leaves. Stomatal movement is regulated by multiple external and internal factors. Although guard cells abscisic acid (ABA) signaling pathway has been well studied, the mechanism of guard cells response to light/dark signal is still unclear. By means of pharmacological approach and laser scanning confocal microscope, the present work explored whether heterotrimeric GTP binding protein (G protein), protein tyrosine kinases (PTKs) and phospholipase C/phospholipase D (PLC/PLD) mediate light/dark-regulated stomatal movement, and whether the effects of G protein, PTKs and PLC/PLD on stomatal movement are related to the changes of hydrogen dioxide (H2O2), nitric oxide (NO) levels in guard cells. The main results are as follows:1. Cholera toxin (CTX), an activator of G protein, significantly induced stomatal closure in Vicia faba and Arabidopsis (WT) in light, but pertussis toxin (PTX), an inhibitor of G protein, did not. PTX obviously promoted stomatal opening in Vicia faba and Arabidopsis (WT) in the darkness, but CTX did not. Whether in light or in the darkness, stomata of Arabidopsis mutant gpal-1 and gpal-2 kept opening, and CTX and PTX did not influenced the stomatal apertures of gpal-1 and gpal-2. The results show that G protein involves in light/dark-regulated stomatal movement, light induces stomatal opening through inactivating G protein, and dark causes stomatal closure via activating G protein.H2O2 significantly induced stomatal closure in Vicia faba and Arabidopsis (WT) in light, but ascorbic acid (Vc), catalase (CAT) and diphenyleneiodonium chloride (DPI), an important reducing substrate for H2O2 removal, one.of H2O2 scavenging enzymes and an inhibitor of the H2O2-generating enzyme NADPH oxidase, did not. Vc, CAT and DPI obviously promoted stomatal opening in Vicia faba and Arabidopsis (WT) in the darkness, but H2O2 did not. The result indicates that H2O2 level within guard cells is higher in the dark than in light. The results show that H2O2 participates in light/dark-regulated stomatal movement, light induces stomatal opening through reducing H2O2, and dark causes stomatal closure via increasing H2O2.CTX increased H2O2 level in guard cells in Vicia faba and Arabidopsis (WT) in light. PTX decreased H2O2 in guard cells in Vicia faba and Arabidopsis (WT) in dark.. Whether in light or in the dark, H2O2 did not appear in guard cells of Arabidopsis mutant gpal-1 and gpal-2. The results show that inactivation of G protein decreases H2O2, and activation of G protein increases H2O2. 2. Sodium nitroprusside (SNP), a NO donor, significantly induced stomatal closure in Vicia faba and Arabidopsis (WT) in light, but c-PTIO and L-NAME, a NO scavenger and an inhibitor of nitric oxide synthase, did not. c-PTIO and L-NAME obviously promoted stomatal opening in Vicia faba and Arabidopsis (WT) in the darkness, but SNP did not. The results indicates that NO levels in guard cells are higher in the dark than in light. The results show that NO participates in light/dark-regulated stomatal movement, light induces stomatal opening through lessening NO, and dark causes stomatal closure via increasing NO.CTX increased NO in guard cells in Vicia faba and Arabidopsis (WT) in light, whereas PTX decreased NO in guard cells in Vicia faba and Arabidopsis (WT) in the dark. Whether in light or in the dark, NO did not appear in guard cells of Arabidopsis mutant gpal-1 and gpal-2. The results show that inactivation of G protein decreases NO, and activation of G protein increases NO.3. Genistein and tyrphostin A23, two specific PTKs inhibitor, significantly prevented dark-induced stomatal closure in Vicia faba and reduced H2O2 levels in guard cells in the darkness. Additionally, genistein and tyrphostin A23 not only suppressed exogenous H2O2-induced stomatal closure and reduced H2O2 levels induced by exogenous H2O2 in guard cells in light, but also reopened the closed stomata in the darkness and abolished H2O2 that had been generated by dark. The results indicate that PTKs mediates dark-induced stomatal closure via inhibiting. H2O2 scavenging, consequently increasing H2O2 levels in guard cells.4. Both genistein and tyrphostin A23 evidently inhibited dark-induced stomatal closure and reduced NO levels in guard cells in the darkness. Additionally, genistein and tyrphostin A23 not only suppressed SNP-induced stomatal closure and reduced NO levels in guard cells in light, but also reopened the closed stomata by darkness and abolished NO that had been generated by dark. The results indicate that PTKs mediates dark-induced stomatal closure via suppressing NO scavenging, consequently increasing the level of NO in guard cells.5. U73122 and n-butanol, one specific inhibitor of PLC and a selective inhibitor of phosphatidic acid production by PLD, remarkably prevented dark-induced stomatal closure in Vicia faba and reduced H2O2 levels in guard cells in the darkness. Phosphatidic acid (PA), a common product of PLC and PLD activity, evidently caused stomatal closure in Vicia faba and increased H2O2 levels in guard cells in light. The results indicate that PA generated by PLC/PLD mediated dark-induced stomatal closure via increasing H2O2 levels in guard cells of Vicia faba.6. U73122 and n-butanol not only markedly suppressed dark-induced stomatal closure in Vicia faba and reduced NO levels in guard cells of Vicia faba in the darkness, but also evidently prevented SNP-caused stomatal closure in Vicia faba in light. PA obviously induced stomatal closure, but did not increase NO levels in guard cells of Vicia faba in light. The results show that PLC/PLD not only involve in dark-induced NO production, but also mediate NO-induced stomatal closure. However, PA does not participate in PLC/PLD-mediated NO production during dark-induced stomatal closure.All together, the present results proved that G protein, PTKs and PLC/PLD mediated light/dark-regulated stomatal movement via adjusting H2O2, NO levels in guard cells.