关键词： CARBON-ISOTOPE DISCRIMINATION   ABSCISIC-ACID REGULATION   WATER-USE EFFICIENCY   G-BETA-DEFICIENT   STOMATAL DENSITY   GENETIC-VARIATION   PLASMA-MEMBRANE   G-GAMMA   CO2   THALIANA  

摘要： Land plants must balance CO2 assimilation with transpiration in order to minimize drought stress and maximize their reproductive success. The ratio of assimilation to transpiration is called transpiration efficiency (TE). TE is under genetic control, although only one specific gene, ERECTA, has been shown to regulate TE. We have found that the alpha-subunit of the heterotrimeric G protein in Arabidopsis (Arabidopsis thaliana), GPA1, is a regulator of TE. gpa1 mutants, despite having guard cells that are hyposensitive to abscisic acid-induced inhibition of stomatal opening, have increased TE under ample water and drought stress conditions and when treated with exogenous abscisic acid. Leaf-level gas-exchange analysis shows that gpa1 mutants have wild-type assimilation versus internal CO2 concentration responses but exhibit reduced stomatal conductance compared with ecotype Columbia at ambient and below-ambient internal CO2 concentrations. The increased TE and reduced whole leaf stomatal conductance of gpa1 can be primarily attributed to stomatal density, which is reduced in gpa1 mutants. GPA1 regulates stomatal density via the control of epidermal cell size and stomata formation. GPA1 promoter::beta-glucuronidase lines indicate that the GPA1 promoter is active in the stomatal cell lineage, further supporting a function for GPA1 in stomatal development in true leaves.

关键词： 水稻   拟南芥   核质间转运   小G蛋白Ran   非生物胁迫  

摘要： 植物作为固定生长的生物,必须对外界变化的生长环境条件作出迅速反应以适应环境的改变。盐害、干旱及极端温度等非生物胁迫严重影响植物生长发育过程,是造成全球农业减产的重要因素。研究植物响应非生物胁迫反应的信号传导分子机理,对农作物的基因改良,提高主要作物对非生物胁迫的耐受能力,从而保持全球粮食的稳定供应有着重要的意义。\n 小G蛋白Ran是importinβ受体家族介导的核质间转运过程中一个必需的调控蛋白因子。此外它在细胞有丝分裂过程及分裂后核组装过程中也有重要作用。迄今为止,愈来愈多的研究报道表明,调控蛋白在核质间的分隔在植物生长发育及响应外界环境变化的信号传导过程中起重要的调控作用。我们研究发现Ran在植物生长发育和响应非生物胁迫过程中发挥重要作用。实验主要包括以下两个部分:\n (1)水稻小G蛋白基因OsRAN2的功能研究。\n 关于植物中Ran的研究在拟南芥,西红柿,小麦中已有报道。而水稻中的这类蛋白目前为止还未有报道。我们实验结果发现:(ⅰ)水稻Ran家族(包括OsRAN1,OsRAN2和OsRAN3)与人类Ran/TC4以及拟南芥或者小麦中的同原物享有保守的蛋白序列和结构特征。OsRAN2主要定位在细胞核内,部分定位在细胞质中,这与动物细胞中Ran以RanGTP形式定位在细胞核内,以RanGDP形式定位在细胞质中的结果一致。(ⅱ)盐胁迫、渗透胁迫和外源ABA处理会下调OsRAN1和OsRAN2的转录表达。而且我们发现,ABA合成抑制剂氟啶酮会部分消除盐胁迫和渗透胁迫条件下OsRAN2的表达下降趋势,而外源添加ABA又会一定程度地恢复这种下调趋势。这就表明,OsRAN2在水稻响应非生物胁迫反应过程中的作用可能部分是通过ABA信号分子来介导的。另外我们还发现,外源水杨酸SA和茉莉酸JA处理也会下调OsRAN2的表达。(ⅲ)高表达OsRAN2的转基因水稻和拟南芥植株对这些非生物胁迫处理敏感。我们鉴定到在盐胁迫或者外源ABA处理条件下,胁迫响应基因AtNCED3、AtPLC1和AtMYB2在转基因拟南芥中有较高的诱导表达。玉米(Zea mays)转录因子Lc是一种入核转运的报告基因。而且高表达OsRAN2会破坏Lc在烟草(Nicotiana tabacum *** No.1)表皮细胞中的亚细胞定位,使得Lc不能完全定位到细胞核中。我们推测,高表达OsRAN2的转基因植株对胁迫反应的敏感,一方面是通过调节胁迫相应基因如AtNCED3、AtPLC1和AtMYB2的表达来实现的,另一方可能是由于一些重要调控蛋白在细胞核质间的转运被破坏而导致的。(ⅳ)OsRAN2高表达和RNAi(RNA interference)的转基因水稻植株还表现出多种生长发育表型。如高表达OsRAN2的转基因植株抽穗要比野生型植株晚；RNAi转基因植株为雄性不育,且分蘖不正常,花序发育不正常等等。我们推测,OsRAN2的过量表达或者OsRAN1-2下调表达很可能引起了转基因植株中不正常的RanGTPase循环,从而造成了不正常的核质间转运、细胞有丝分裂等过程,最终使得转基因植株表现出一系列的生长发育表型。\n (2)拟南芥Ran家族参与的非生物胁迫反应\n 有报道称,拟南芥中AtRAN1,AtRAN2和AtRAN3可能参与了光受体介导的植物光信号传导途径。我们实验发现,在盐胁迫和外源ABA处理条件下,AtRAN1,AtRAN2和AtRAN3表达被上调。而且双突变体ran1/ran3-1,ran-1/ran3-2,ran2/ran3-1和ran2/ran3-2表现出对盐胁迫和ABA处理的敏感。这些结果表明AtRAN1,AtRAN2和AtRAN3在拟南芥植株对非生物胁迫反应过程中起重要调控作用。我们推测,AtRAN1,AtRAN2和AtRAN3突变后,影响importinβ受体家族介导胁迫调控因子的核质间转运过程,最终引起了双突变体对非生物胁迫的敏感表型。

关键词： 拟南芥   突变体   再誉镰刀菌   抗性   植物激素   G蛋白  

摘要： 水杨酸(SA)、茉莉酸(JA)和乙烯(ET)被认为是传统的植物防卫激素，在调控植物对病原菌等生物性胁迫的抗性中发挥重要作用。它们通过诱导植物产生病程相关蛋白或植物防卫素等方式使植物产生系统获得性抗性和系统诱导性抗性并以网络状的信号传导形式共同抵御生物性胁迫。此外，脱落酸(ABA)和G蛋白也可以通过影响JA、ET和胼胝质的形成来抵抗病原菌的侵入。本论文以拟南芥JA、ET、SA和ABA生物合成和信号转导突变体以及G蛋白突变体为材料，研究了植物激素和G蛋白在拟南芥对再誉镰刀菌抗性中的作用及其机制。\n 1.建立了再誉镰刀菌侵染拟南芥的体系，发现再誉镰刀菌可侵染拟南芥Col-0生态型，症状首先从叶缘开始发生，逐渐形成褐色病斑，菌丝可以侵染开花期的顶端组织造成顶端组织坏死，进一步导致植株的黄化和萎蔫甚至死亡。\n 2.以SA信号转导突变体npr1和转基因植株NahG为材料结合外源SA处理研究了SA及其信号转导组分在拟南芥对再誉镰刀菌抗性中的作用，研究发现npr1和NahG对再誉镰刀菌的抗性降低，而外源SA处理后植株抗性增强，这表明SA及其信号转导组分在拟南芥对再誉镰刀菌抗性中起正向调节作用。npr1和NahG抗性的降低可能与病程相关基因表达受到抑制有关。\n 3.以JA信号转导突变体jar1和coi1为材料结合外源茉莉酸甲酯(MeJA)处理研究了JA及其信号转导组分在拟南芥对再誉镰刀菌抗性中的作用，研究发现jar1抗性降低，而coi1抗病性则显著增强。jar1抗性降低可能与JA与异亮氨酸衍生物合成受阻，导致JA诱导的应答反应被抑制有关。而coi1抗性的增强则有可能与其激活SA和ET途径有关，也有可能与COI1突变后再誉镰刀菌分泌的效应物挟持这一受体而侵染拟南芥的机制受阻断有关。\n 4.以ET信号转导突变体ein3为材料结合外源乙烯利和AgNO3处理研究了ET及其信号转导组分在拟南芥对再誉镰刀菌抗性中的作用，研究发现发现EIN3信号阻断和野生型植株外源AgNO3处理后其抗性降低，而外源乙烯利处理后植株抗性增强，这表明ET及其信号转导组分EIN3正向地参与拟南芥防御再誉镰刀菌的反应。基因表达分析表明ein3对再誉镰刀菌抗性的降低可能与其在发病的早期阶段PDF1.2诱导能力的减弱有关。\n 5.以ABA信号转导突变体abi5-1为材料结合外源ABA处理研究了ABA及其信号转导组分在拟南芥对再誉镰刀菌抗性中的作用，研究发现abi5-1对再誉镰刀菌的抗性降低，而外源ABA处理后植株抗性增强，这表明ABA正向地参与调控拟南芥对再誉镰刀菌的防卫反应。基因表达分析表明abi5-1对再誉镰刀菌抗性的降低也可能与JA、ET和SA等防卫途径途径相关，JA、ET和SA等途径有可能在再誉镰刀菌侵染早期被抑制从而导致抗性降低。\n 6.以G蛋白β亚基缺失突变体agb1-2为材料研究了对G蛋白信号途径在拟南芥对再誉镰刀菌抗性中的作用。结果表明agb1-2对再誉镰刀菌抗性增强，这可能与其能更快速地诱导JA和ET信号途径有关。

关键词： 细胞外ATP   ROS   异三聚体G蛋白   Ca2+   质子泵   气孔  

摘要： 气孔运动关系到植物与外界环境气体和水分的交换,气孔运动的调节机理是植物生理学研究的重要内容。细胞外ATP(extracellular ATP,eATP)作为一种信使分子,通过其特定的信号转导机制参与细胞代谢、生长和发育过程的调控。本文研究了eATP对气孔运动的影响,并对其信号转导过程中可能的信号传递组分进行了探索。 我实验室已经完成的研究结果显示,eATP能够作为信使分子促进拟南芥气孔开放,保卫细胞内活性氧(reactive oxygen species,ROS)在eATP信号转导过程中发挥介导作用,异三聚体G蛋白α亚基参与了eATP诱导ROS合成、促进气孔开放的过程。但是,对于eATP信号转导过程中活性氧下游的生理变化还不清楚,异三聚体G蛋白其他两个亚基(Gβ、Gγ)在eATP调节气孔运动的信号转导过程中是否发挥作用也不清楚。因此,本文以拟南芥野生型、异三聚体G蛋白基因缺失突变体和超表达株系、活性氧合成的关键酶—NADPH氧化酶的基因缺失突变体为材料,采用表皮条生物分析、激光共聚焦显微技术、非损伤微测技术对异三聚体G蛋白和ROS在eATP调节保卫细胞Ca2+流和H+流的过程中发挥的作用进行了探索。结果表明: 异三聚体G蛋白β、γ亚基基因缺失突变体在外加ATP处理之后,气孔开度明显增大,突变体及其野生型气孔对eATP刺激的反应没有明显差异;ATP处理后,野生型和突变体保卫细胞内ROS含量都有显著升高。这些结果显示,Gβ、Gγ可能不参与eATP诱导胞内ROS升高、促进气孔开放的过程。 为进一步探索eATP调节气孔运动的生理机制,我们利用非损伤微测技术测定了eATP处理后拟南芥保卫细胞Ca2+流和H+流的动态变化。结果显示,在eATP处理之后,保卫细胞跨膜Ca2+内流增加;在Gα基因缺失突变体中,外加ATP对Ca2+流没有产生明显的影响,而在Gα基因超表达株系中,Ca2+内流显著增强,Gβ、Gγ基因缺失突变体Ca2+流对eATP刺激的反应与野生型没有明显差异。在NADPH氧化酶的基因缺失突变体中,eATP处理几乎没有引起Ca2+流的变化。这些结果表明,eATP可能通过活性氧促进钙离子内流促进气孔开放,在此过程中,Gα起到了重要的介导作用,而Gβ、Gγ未参与其中。 H+流检测的结果显示,eATP处理引起保卫细胞跨膜H+外流增加。在NADPH氧化酶的基因缺失突变体中,eATP处理没有引起H+流的变化。在Gα基因缺失突变体中,外加ATP对H+流几乎没有影响,而在Gα基因超表达株系中,H+外流明显增强;Gβ、Gγ基因缺失突变体与野生型之间没有明显差异。这表明eATP可能通过ROS激活细胞质膜质子泵、促进细胞向外排放H+离子,并最终促进气孔开放程度加大。在此过程中,Gα可能参与了信号转导过程,而Gβ、Gγ未参与。

关键词： HETEROTRIMERIC G-PROTEIN   LATERAL ROOT INITIATION   ALPHA/BETA-HYDROLASE FOLD   BETA-GAMMA-SUBUNITS   DIFFERENTIATION-RELATED GENES   ABSCISIC-ACID REGULATION   CELL-PROLIFERATION   DEVELOPMENTAL PROCESSES   PLANT TRANSFORMATION   EXPRESSION ANALYSIS  

摘要： Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two G beta gamma dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single beta (AGB1) or the two gamma (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.

关键词： ABA   AGB1   Early seedling development   GPA1   RACK1   Seed germination   G-BETA-GAMMA   ABSCISIC-ACID REGULATION   SIGNAL-TRANSDUCTION   ALPHA-SUBUNIT   CELL-DIVISION   DEVELOPMENTAL PROCESSES   INTRACELLULAR RECEPTOR   TRANSCRIPTION FACTORS   MOLECULAR-CLONING   SEED-GERMINATION  

摘要： Mammalian receptor for activated C kinase 1 (RACK1) is a versatile scaffold protein, playing regulatory roles in multiple signal transduction pathways. Moreover, RACK1 interacts with the heterotrimeric G-proteins (G-proteins) and regulates some specific functions of G beta gamma. Although the protein sequences of both RACK1 and G-proteins are highly conserved in Arabidopsis, their relationship remains elusive. Here we provide genetic and biochemical evidence that Arabidopsis RACK1 and G-proteins may act through a mechanism that is distinct from their counterparts in mammals. Loss-of-function alleles of RACK1A (the most abundantly expressed RACK1 gene in Arabidopsis) do not appear to share morphological and developmental phenotypes with loss-of-function alleles of GPA1 (encoding the sole G alpha in Arabidopsis) or AGB1 (encoding the sole G beta in Arabidopsis). The analysis of gpa1 rack1a and agb1 rack1a double mutants suggested that the effect of RACK1A on morphological and developmental traits may occur independently of the presence or absence of the G-protein subunits. Although both RACK1A and G-protein subunits are negative regulators of ABA responses in the ABA inhibition of early seedling development, an additive ABA hypersensitivity was observed in gpa1 rack1a and agb1 rack1a double mutants. Biochemical analysis suggested that unlike their counterparts in mammals, RACK1 may not physically interact with AGB1. Taken together, these findings revealed some fundamental differences in the relationship of RACK1 and G-proteins between Arabidopsis and mammals.

关键词： AOS   Arabidopsis   heterotrimeric G-protein   G-protein alpha-subunit (GPA1)   jasmonic acid   PDF1   2   VSP   ABSCISIC-ACID   METHYL JASMONATE   BETA-SUBUNIT   TRANSCRIPTION FACTOR   DISEASE RESISTANCE   COUPLED RECEPTOR   GENE-EXPRESSION   GAMMA-SUBUNIT   CELL-DEATH   PHYTOCHROME  

摘要： Heterotrimeric G-proteins have been implicated in having a role in many plant signalling pathways. To understand further the role of G-proteins, a preliminary experiment was performed to assess the impact of the G alpha subunit loss-of-function mutation gpa1-1 on the Arabidopsis transcriptome. The analysis indicated that the G alpha subunit may play a role in response to jasmonic acid (JA). Consistent with this, G alpha mutants showed a reduced response to JA in inhibition of chlorophyll accumulation and root growth, whilst G alpha gain-of-function plants overexpressing G alpha showed the opposite phenotype. The levels of JA and related compounds were unaffected in the gpa1-1 mutant, as was autoregulation of the Allene Oxide Synthase (AOS) gene that encodes a key enzyme for JA biosynthesis. In contrast, further analyses using G alpha loss- and gain-of-function Arabidopsis lines indicated that G alpha positively modulates the expression of the Vegetative Storage Protein (VSP) gene. This indicates that the G alpha subunit regulates a subset of JA-regulated genes defining a branch point in this signalling pathway in Arabidopsis. Further analysis of the impact of G alpha loss of function upon the JA-regulated transcriptome using Arabidopsis full genome arrays indicated that up to 29% of genes that are > 2-fold regulated by JA in the wild type are misregulated in the G alpha mutant. This supports the observation that a significant proportion of, but not all, JA-regulated gene expression is mediated by G alpha.

关键词： SUSPENSION-CULTURED CELLS   ABSCISIC-ACID   ALPHA-SUBUNIT   HYPOCOTYL ELONGATION   POLLEN GERMINATION   CA2+   SYNTHASE   CALCIUM   ABA   CHANNELS  

摘要： Extracellular calmodulin (ExtCaM) regulates stomatal movement by eliciting a cascade of intracellular signaling events including heterotrimeric G protein, hydrogen peroxide (H2O2), and Ca2+. However, the ExtCaM-mediated guard cell signaling pathway remains poorly understood. In this report, we show that Arabidopsis (Arabidopsis thaliana) NITRIC OXIDE ASSOCIATED1 (AtNOA1)-dependent nitric oxide (NO) accumulation plays a crucial role in ExtCaM-induced stomatal closure. ExtCaM triggered a significant increase in NO levels associated with stomatal closure in the wild type, but both effects were abolished in the Atnoa1 mutant. Furthermore, we found that ExtCaM-mediated NO generation is regulated by GPA1, the G alpha-subunit of heterotrimeric G protein. The ExtCaM-dependent NO accumulation was nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cG alpha). In addition, cG alpha Atnoa1 and gpa1-2 Atnoa1 double mutants exhibited a similar response as did Atnoa1. The defect in gpa1 was rescued by overexpression of AtNOA1. Finally, we demonstrated that G protein activation of NO production depends on H2O2. Reduced H2O2 levels in guard cells blocked the stomatal response of cG alpha lines, whereas exogenously applied H2O2 rescued the defect in ExtCaM-mediated stomatal closure in gpa1 mutants. Moreover, the atrbohD/F mutant, which lacks the NADPH oxidase activity in guard cells, had impaired NO generation in response to ExtCaM, and H2O2-induced stomatal closure and NO accumulation were greatly impaired in Atnoa1. These findings have established a signaling pathway leading to ExtCaM-induced stomatal closure, which involves GPA1-dependent activation of H2O2 production and subsequent AtNOA1-dependent NO accumulation.

关键词： 拟南芥   低温胁迫   POD   SOD   MDA  

摘要： 采用分光光度法测定了低温胁迫下两种拟南芥G蛋白突变体的超氧化物歧化酶(SOD)、过氧化物酶(POD)的活性及丙二醛(MDA)含量的变化。结果表明:在低温12h,野生型(Ws)和突变体gpa1-1的SOD酶活性迅速升高,分别为15.501 U/mg protein和9.453 U/mg pro-tein,突变体gpa1-2则在低温24 h升至最高点,酶活性为6.325 U/mg protein。低温胁迫下,三种材料的POD酶活性都呈现降低趋势,而MDA含量呈上升趋势。初步证明外界低温刺激与植物细胞内G蛋白有一定的关联。

关键词： 拟南芥   AtRGS1蛋白   结构域   突变体   脱落酸   糖   转基因植株  

摘要： 本研究以拟南芥野生型Col-0、突变体rgs1-2、过表达35S:RGS1-N、35S:RGS1-C为材料,通过突变体分析、转基因植株构建等技术,用分子生物学、生物化学等方法研究了AtRGS1蛋白N-末端、C-末端结构域在拟南芥葡萄糖和ABA信号转导途径中的作用。取得了以下主要研究结果: 1、采用TOPO克隆的方法构建了35S:RGS1-N、35S:RGS1-C过表达转基因植株。 克隆了AtRGS1基因N-末端、C-末端cDNA,通过TOPO克隆构建了CaMV 35S为启动子,GFP为报告基因,含有融合基因35S:RGS1-N-GFP、35S:RGS1-C-GFP的植物表达载体。利用根癌农杆菌浸染转化法将35S:RGS1-N-GFP、35S:RGS1-C-GFP融合基因导入突变体rgs1-2植株,经过抗生素筛选、根部荧光观察和GFP基因PCR鉴定,获得了转化成功的阳性植株。 2、运用突变体分析技术研究AtRGS1蛋白N-末端、C-末端结构域在拟南芥葡萄糖和ABA信号转导途径中的作用及其调节机理。结果表明: (1)rgs1-2种子萌发对葡萄糖的抑制作用不敏感,而35S:RGS1-N、35S:RGS1-C种子表现为超敏感,其中35S:RGS1-N种子最敏感。四基因型种子萌发对甘露糖的抑制作用未表现出差异。表明这种抑制作用与糖的渗透胁迫无关,而是由葡萄糖介导的信号转导途径引起的。己糖激酶抑制剂NAG处理削弱了甘露糖对种子萌发的抑制效应,而对葡萄糖抑制萌发没有影响,表明葡萄糖抑制种子萌发不依赖于已糖激酶途径。 (2)rgs1-2种子萌发对ABA低敏,而35S:RGS1-N、35S:RGS1-C种子表现为超敏感,其中35S:RGS1-N种子最敏感。表明过表达AtRGS1-N、AtRGS1-C能互补rgs1-2的ABA不敏感性表型,增加了种子对ABA的敏感性。 (3)葡萄糖与ABA共处理结果显示葡萄糖与ABA对种子萌发的抑制作用存在叠加效应。Fluridone与葡萄糖共处理结果显示Fluridone能降低葡萄糖抑制种子萌发的效应。表明葡萄糖是通过增加内源ABA含量来抑制萌发的。 (4)rgs1-2幼苗主根显著长于Col,而35S:RGS1-N、35S:RGS1-C主根显著短于Col、rgs1-2。rgs1-2、35S:RGS1-N和35S:RGS1-C主根生长对葡萄糖和ABA的应答表现出相同趋势,即rgs1-2敏感性最低,其次是35S:RGS1-C,35S:RGS1-N敏感性最高。表明AtRGS1基因突变和过表达改变了代谢糖和ABA调节作用的敏感性。 (5)植株水平,rgs1-2离体叶片失水速率显著快于Col,而35S:RGS1-N、35S:RGS1-C显著低于Col。表明过表达AtRGS1-N、AtRGS1-C能降低植株叶片失水速率,增强耐旱性。 (6)干旱胁迫条件下,rgs1-2水势显著低于Col,而35S:RGS1-N、35S:RGS1-C水势显著高于Col、rgs1-2。表明rgs1-2保水能力较弱,而35S:RGS1-N、35S:RGS1-C具有较强的保水能力,在干旱胁迫下能够保持较高的叶片水势。相应地,Col、rgs1-2叶片ABA含量较低,而转基因株系35S:RGS1-N、35S:RGS1-C叶片ABA含量显著高于Col、rgs1-2。表明过表达AtRGS1 N-末端、C-末端结构域增强了拟南芥应对干旱胁迫的能力。