【作者】 李婧；

【导师】 章文华；

【作者基本信息】 南京农业大学 ， 植物学， 2009， 硕士

【摘要】 磷脂酶D及其水解产物磷脂酸在植物信号转导途径中具有重要作用,包括参与调控植物生长发育,如根毛的发生及生长、花粉管的萌发及伸长；介导植物对外界环境的响应,如盐胁迫、渗透胁迫、病原微生物感染等；参与多种植物激素的信号转导,如脱落酸、生长素等。目前已有研究证实PLDal是ABA诱导拟南芥气孔关闭过程中一个不可缺少的因素,PLDα1的缺失导致气孔关闭对ABA处理不敏感。但是PLDα1在植物根系响应ABA信号转导链中的作用尚未见报道。此外,外施PLD抑制剂抑制了根毛的发生和生长,但未见在突变体中验证PLDα对于高等植物根毛发育的确切机制。本文以拟南芥PLDα1突变体(pldα1)和野生型(WT, Columbia生态型)为材料初步研究了PLDα1在植物根系响应ABA、盐胁迫信号转导中的作用以及PLDα1调控拟南芥根毛发育的机制。我们首先探讨了PLDα1在拟南芥根毛尖端生长中的作用。结果显示：pldα1突变体根毛长度小于WT,根毛尖端ROS和Ca2+浓度低于WT。pldα1突变体和野生型之间根毛密度没有显著差异。据此推测,PLDα1很可能是通过调控根毛尖端ROS的生成,影响Ca2+浓度梯度的形成和维持来调控根毛的尖端生长的,而与根毛的发生没有直接关系。我们研究了PLDα1在ABA抑制拟南芥主根伸长过程中的作用,发现在ABA处理下,pldα1突变体主根伸长受抑制程度小于野生型。我们采用3H标记的PC测定了根系PLDα1活性,实验结果显示,根系PLDα1活性在ABA处理下大幅度升高；用H2DCF-DA作为胞内ROS的荧光探针载入拟南芥根细胞原生质体,用激光共聚焦显微镜检测根细胞原生质体中ROS含量,实验结果显示,根细胞原生质体中ROS含量在ABA处理下升高,pldα1突变体升高程度小于WT。同样以根细胞原生质体为材料,用HO2·/O2-特异性染料XTT测定NADPH氧化酶产生的HO2·/O2-来表示NADPH氧化酶的活性,结果发现根系NADPH氧化酶活性在ABA处理下升高,pldal突变体升高程度小于WT；外源加入10μmol/L PA后pldα1突变体NADPH氧化酶活性显著升高,升高程度大于WT。外源加入H2O2可诱导WT和pldα1主根伸长都受到抑制,且二者差异不明显。这些结果表明,PLDα1产生的PA通过激活NADPH氧化酶产生ROS介导ABA调控的拟南芥主根伸长过程。我们还探讨了PLDα1在NaCl处理下拟南芥主根伸长中的作用。发现在NaCl处理下,pldal突变体主根伸长受抑制程度大于野生型；外源施加PA缓解pldal突变体主根伸长所受的抑制。我们采用3H标记的PC测定了根系PLDα1活性,实验结果显示,根系PLDα1活性在NaCl处理下大幅度升高。提取根细胞原生质体,用H2DCF-DA作为胞内ROS的荧光探针标记拟南芥根细胞原生质体,用荧光显微镜检测根细胞原生质体中ROS含量,实验结果显示,根细胞原生质体中ROS含量在NaCl处理下升高，pldal突变体升高程度小于WT；外源施加DPI处理后WT和pldal突变体根细胞原生质体中ROS含量在NaCl处理下均无明显升高。以上实验结果表明,PLDα1介导ABA和NaCl处理下的拟南芥主根伸长并参与根毛生长。更多还原

【Abstract】 PLD and it’s product PA functioned in various signaling pathways. They modulated growth and development of plants like the formation and growth of root hair; responsed to many stresses:salt stress, water deficit stress, and biotic stress. They also functioned in signaling transductions of ABA. Previous studies have indicated that PLDal was an essential factor in ABA-induced stomal close, knockout of PLDα1 showed insensitive to ABA in stomal closure. But the role of PLDal in ABA signal transduction of root was not reported. Besides, the formation and growth of root hair was suppressed when treated with PLDa inhibitor, but there was no report about the certain mechanism verified in mutant. In this study, the mechanism of PLDal in root ABA and salt stress signaling and root hair formation, growth was investigated using pldal mutant.Firstly, function of PLDα1 in root hair formantion and growth was studied using pldal and WT as research material. Root hair of pldal was shorter than the one of WT; at the tip of root hair, pldal had lower concentration of ROS and Ca2+ than WT. The root hair density of pldal and WT showed no significant difference.The functions of PLDal in ABA-modulated main root elongation were studied. When treated with ABA, main root elongation of Arabidopsis was inhibited, but the inhibition on pldal mutant was less than on WT. We determinated PLDal activity using 3H marked PC as substrate and founded that PLDα1 activity increased greatly under ABA treatment. Then we used H2DCF-DA as intracellular ROS fluorescent probe in root cell protoplast of Arabidopsis, and detected ROS concentration with LSCM(laser scan confocal microscope). The result indicated that ROS concentration in root hair protoplast increased under ABA treatment, the one of pldal mutant increased less than WT. NADPH oxidase oxidized NADPH and produced HO2·/O2-. We next used XTT as HO2·/O2- indicator to determinate NADPH oxidase activity. The results was as follows:the NADPH oxidase activity of both WT and pldα1 mutant increased; but the pldα1 mutant NADPH oxidase activity increased less than WT; when added with 10μmol/L PA, the NADPH oxidase activity of both WT and pldα1 mutant increased, but increased more in pldα1 mutant than in WT. When added with H2O2 in the culture medium, main root elongation of both pldα1 and WT were inhibited with no significant differences. All these results suggested that, PA produced by PLDα1 functioned in ABA-modulated root elongation of Arabidopsis by activating NADPH oxidase.The functions of PLDα1 in main root elongation under NaCl treatment were also studied. When seedlings were treated with NaCl, main root elongation was inhibited obviously in pldα1 than that in WT. We determinated PLDa activity using 3H marked PC as substrate and founded that PLDαactivity increased greatly under NaCl treatment. We then used fluorescent probe H2DCF-DA to detect intracellular ROS in root protoplasts with LSCM (laser scan confocal microscope). The result indicated that ROS concentration in root hair protoplast increased less in pldα1 than WT under NaCl treatment. Whereas, when seedlings were pre-treated with DPI, ROS concentration didn’t increased significantly in WT or pldα1.All these results suggested that, PLDα1 modulated main root elongation under NaCl and ABA treatment and regulated root hair growth.更多还原