【作者】 张艳艳；

【导师】 章文华；

【作者基本信息】 南京农业大学 ， 植物学， 2007， 博士

【摘要】 盐胁迫和干旱一直是威胁农业生产的主要因素。盐胁迫下，质膜和液泡膜上的Na⁺／H⁺交换运输将过量的Na⁺分别排出细胞外或驱隔于液泡中，降低胞质中Na⁺含量、减轻过量Na⁺对细胞质中代谢酶的毒害，是提高植物耐盐性的重要方式。以玉米为材料的研究表明盐胁迫下，NaCl诱导玉米叶片NO短暂上升，NO通过提高液泡膜H⁺-ATPase和H⁺-PPase活性，提高质子转运活性和Na⁺／H⁺交换活性，从而将Na⁺泵入液泡，提高玉米的耐盐性。磷脂酶D（phospholipase D，PLD）及其水解产物磷脂酸（phosphatidic acid，PA）参与NO诱导质子转运活性和Na⁺／H⁺交换活性增加过程。干旱环境下，植物体内激素脱落酸（abscisic acid，ABA）的含量升高和重新分布诱导叶片表面气孔关闭、减少水分散失，从而维持植物体内水分平衡是提高植物抗干旱能力的重要方式。ABA诱导气孔关闭的内部信号系统、各种信号物质之间的互作和对话非常复杂，了解各种信号中间分子之间的位置关系将有助于我们更好地调控、操纵ABA诱导气孔关闭过程，为增强农作物的抗旱能力、提高农业生产提供理论依据。PLD、过氧化氢（hydrogen peroxide，H₂O₂）、一氧化氮（nitric oxide，NO）是已经发现的在ABA诱导气孔关闭中起关键作用的信号物质，PLDctl功能缺失或抑制H₂O₂、NO产生，ABA诱导气孔关闭受到抑制，但PLD和H₂O₂、NO的位置关系和相互作用还不是很清楚。拟南芥中存在12种PLD基因，具有不同的结构、生化和调节特性，因而参与不同的细胞过程。我们以拟南芥保卫细胞为研究体系，利用药理学、遗传学、分子生物学、细胞生物学和植物生理学手段，研究了ABA诱导气孔关闭过程中PLDα1、PLDδ、H₂O₂、NO之间的位置关系。结果发现：PLDα1、PLDδ都参与ABA诱导气孔关闭，pldα1、pldδ突变体气孔关闭对ABA处理不敏感；H₂O₂诱导pldα1气孔关闭，但不能诱导pldδ气孔关闭；PLDα1参与ABA诱导H₂O₂产生过程，而PLDδ响应H₂O₂作用。质膜NADPH氧化酶是保卫细胞中H₂O₂产生的主要来源，研究发现PLDα1通过介导ABA激活保卫细胞原生质体NADPH氧化酶活性参与ABA诱导H₂O₂产生：pldα1中ABA诱导NADPH氧化酶活性升高受到抑制，外施PLDα1的水解产物PA重新激活NADPH氧化酶活性；PLDα1／PA和蛋白磷酸酶ABI1结合正调控ABA诱导气孔关闭，突变PLDα1／PA-ABI1结合的必需氨基酸(第73位精氨酸，ABIl_R73A)对ABA诱导H₂O₂产生没有影响，说明PLDa1／PA参与H₂O₂产生不依赖PLDα1／PA-ABI1结合，但H₂O₂不能充分诱导ABIl_R73A气孔关闭，PA和NADAPH氧化酶、ABI1同时结合发挥作用是响应ABA信号所必需的。同时我们研究了PLDαl和NO在ABA诱导气孔关闭过程中的位置关系：pldal突变体中，NO产生对ABA不敏感，用正丁醇抑制PA产生部分抑制了ABA诱导NO产生，而外施二软脂酰磷脂酸（dipalmitoyl PA，16：0 PA）、二硬脂酰磷脂酸（distearoyl PA，18：0 PA）和二油酰磷脂酸（Dioleoyl PA 18：1PA）重新诱导NO产生，暗示PLDα1／PA在ABA诱导NO产生过程中发挥重要作用；外源NO诱导pldα1气孔关闭，PLDα1不参与H₂O₂诱导NO产生，进一步证实ABA诱导气孔关闭过程中，H₂O₂、NO位于PLDα1／PA下游起作用。硝酸还原酶（nitrate reductase，NR）与拟南芥中和NO产生相关的酶（Arabidopsis NO associated 1，AtNOA1）是植物体中已经确定的NO产生的主要来源，保卫细胞中NO产生主要来源于NR（Desikan等2002；Bright等2006）。为了进一步研究PLDa1／PA如何参与NO产生过程，我们克隆了拟南芥NR的两个结构基因NLA1和NLA2，并将其在大肠杆菌和烟草中表达，研究结果表明16：0 PA和18：0 PA结合NIA2、并激活其活性是PLDα1／PA参与NO产生的重要机理。同时我们也检测了NO对PLD活性的影响，活体实验表明NO可以提高叶肉细胞PLD活性，从而使得PLD和NO的关系更加复杂。我们提出了保卫细胞中PLDα1、H₂O₂、NO、PLDδ的位置关系模式图为：更多还原

【Abstract】 Salinity and drought have been the major threats affecting agricultural productivity. Salt stress elevates cellular Na+ level. To remove excess Na+ from the cytoplasm by the compartmentation of Na+ into the vacuole or exclusion of Na+ to the apoplast by Na+/H+ antiporters associated with vacuolar membrane （tonoplast） or plasma membrane respectively is crucial to improve salt tolerance.We report that NaC1 induced a transient increase in NO accumulation in maize leaves. NO induced the increase of vacuolar H+-ATPase and H+-PPase activities, along with an increase of Na+/H+ exchange activity, thereby increasing salt tolerance in maize. Phospholipase D （PLD） and its product phosphatidic acid （PA） may contribute to NO-induced H+-pump activation.Under drought stress, elevated abscisic acid （ABA） content and redistribution in plants induces stomatal closure and consequently reduces transpiration water loss to increase plant tolerance against drought. The signaling process of ABA-induced stomatal closure is a very complicated network and a large number of ABA signaling intermediates have been found in guard cells. Understanding relationship, interaction and "cross-talk" of these intermediates will help us manipulate ABA signaling transduction, decrease water loss through stomatal pores and enhance drought tolerance.Phospholipase D （PLD）, hydrogen peroxide （H202） and nitric oxide （NO） are involved in ABA-induced stomatal closure. PLD-loss-function or blocked H202 and NO production impaired ABA-induced stomatal closure. Interaction and relationship of PLD, H202 and NO remain unclear. 12 PLD genes are in Arabidopsis. There is distinguishable biochemical and regulatory properties in different PLDs, which determines these PLDs to mediate different cell processes. The present work proves both PLDα1 and PLDδare involved in the signaling process of ABA-induced stomatal closure. Stomatal closure is insensitive to ABA treatment in pldαland pldδ. H₂O₂ promotes stomatal closure in pldα1, but fails to induce stomatal closure in pldδ. PLDal mediates H₂O₂ production in ABA signal and PLDδ responds to H₂O₂ function. Further studies suggest PLDal mediates H₂O₂ production via its involvement in ABA-activated NADPH oxidase activity. Enhanced NADPH oxidase response to ABA treatment was impaired in pldal. Exogenously added phosphatidic acid （PA）, one of PLDal hydrolysis products, rescues activation of NADPH oxidase. PA interacting with ABI1 phosphatase 2C positively regulates stomatal closure. Our data that H₂O₂ production in ABI_R73A （the essential amino acid mutation for PA-ABI1 binding） response to ABA treatment suggests PLDal/PA-mediated NADPH oxidase activation and H₂O₂ production is independent in PA-ABI1 binding. The data suggest that PLDal-derived PA has two targets at least, ABI1 and NADPH oxidase, and PA interaction with both of the targets are required for mediating ABA response. Without PA binding to ABI1, ABI1 is presumed to be localized in the nucleus to inhibit ABA response. So, in ABI1_R734 mutant, even though the mutant still makes H₂O₂, H₂O₂ itself is insufficient to mediate ABA-promoted stomatal closure.PLDal and NO are required in the process of ABA-induced stomatal closure. The relationship between PLDal and NO in ABA-induced stomatal closure has also been studied in this work. NO production is insensitive to ABA treatment in pldal mutant. Exogenously applied 16:0, 18:0 and 18:1 PA promote NO production in pldal, suggesting a crucial role of PLDal/PA in ABA-induced NO production. Meanwhile, exogenous NO promotes stomatal closure inpldal. All these proofs indicate PLDα1/PA is upstream of NO function in the signal process of ABA-induced stomatal closure. PLDα1 is not involved in H₂O₂-induced NO production, which further proves both H₂O₂ and NO is downstream of PLDα1.Nitrate reductase （NR） is the main source for NO production in guard cell mediating ABA-induced stomatal closure. To address how PA regulates NO production, we cloned two Arabidopsis NR structural genes NIA1, NIA2 and expressed them in E.coli and tobacco via transient expression system. The study indicates two PA, 16:0 and 18:0 PA binds to NIA2, which is responsible for 90% of NR activity and promotes NIA2 activity.The effect of NO on PLD activity in vivo has also been investigated. The data suggests NO activates PLD activity at least in some specific cells such as mesophyll cell protoplasts. Thus, a working model depicting the relationship of ABA, PLDal, H202, NO and PLDδhas been proposed.更多还原