Arabidopsis 2010: Analysis of Two-Component Signaling Elements from Arabidopsis

双组分系统是细菌感知和响应环境刺激的主要手段。这些系统由许多不同的元件组成，即组氨酸激酶、反应调节器和在磷酸转运体的情况下，组氨酸磷酸转移蛋白(HPTS)。在拟南芥中已经确定了编码与这些元素中的每一个相似的蛋白质的基因，而对于35个这样的基因中的大多数，还没有确定的功能。提出了一种综合的方法来阐明这些蛋白在拟南芥中的功能。将结合基因敲除和可诱导的过度表达来评估这些基因在植物生长和发育中的作用。突变的植物品系将根据它们对激素、光和渗透胁迫等生物和非生物因素的反应以及它们的基因表达模式来表征。这些基因在植物中的表达将通过GUS融合和原位RNA分析相结合来确定。同源蛋白在细胞内的位置也将被描绘出来。为了便于这种定位，将产生一系列10个单抗来标记蛋白质，每个蛋白质驻留在不同的膜上。来自拟南芥的蛋白质复合体将被提纯和分析，以确定这些元件之间的相互作用，并鉴定新的相互作用蛋白。总之，这些研究将阐明这些拟南芥两组分信号元件中的每一个起作用的信号通路，以及它们如何相互作用来控制植物的生长和发育。这些研究的数据将存放在一个公开可访问的网页上，该网页目前正在建设中，位于北卡罗来纳大学(http://www.bio.unc.edu/research/two-component/).我们将在TAIR数据库上建立到这个网站的链接，我们将与TAIR协调适当地存放数据。淘汰赛种子将通过存放在俄亥俄州立大学的ABRC股票中心公开获得。针对膜标记蛋白培育的单克隆体将用于通过UNH运输的费用，细胞系也将保存在美国模式培养库(ATCC)。为了与2010年项目的目标保持一致，对两组分信号元件的功能分析将有助于我们在生物、细胞和进化水平上理解植物。这项研究将为拟南芥双组分信号系统在植物生长发育中的作用提供功能信息。这项研究将有助于确定双组分信号转导通路的亚细胞位置、双组分信号元件之间的相互作用以及这些通路的下游靶点。这项研究应该阐明在细菌中产生的信号转导机制如何适应植物的信号转导。这些研究应该揭示拟南芥中几个基因家族的功能。这些基因家族编码的蛋白质被预测为相互作用，因此我们的研究应该有助于在大基因家族相互作用的成员之间发展信号特异性的范例。此外，还将开发普遍适用于确定拟南芥蛋白质亚细胞位置的工具。

Two-component systems are the primary means by which bacteria sense and respond to environmental stimuli. These systems are comprised of a number of distinct elements, namely histidine kinases, response regulators and in the case of phosphorelays, histidine phosphotransfer proteins (HPts). Genes encoding similar proteins to each of these elements have been identified in Arabidopsis, and for the majority of the 35 such genes no function has yet been definitively ascribed. An integrated approach to elucidate the function of these proteins in Arabidopsis is proposed. A combination of gene knockouts and inducible overexpression will be used to assess the roles of these genes in plant growth and development. The mutant plant lines will be characterized in terms of their response to biotic and abiotic factors such as hormones, light, and osmotic stress, and for their pattern of gene expression. Where in the plants these genes are expressed will be determined using a combination of GUS fusions and in situ RNA analysis. The location of the cognate proteins within the cell will also be delineated. To facilitate this localization, a series of 10 monoclonal antibodies will be generated to marker proteins, each of which resides on a distinct membrane. Protein complexes from Arabidopsis will be purified and analyzed to determine the interactions among these elements and to identify novel interacting proteins. Together, these studies will illuminate the signaling pathways in which each of these Arabidopsis two-component signaling elements function and how they interact to control plant growth and development.The data from these studies will be deposited on a publicly accessible web page that is currently under construction at UNC (http://www.bio.unc.edu/research/two-component/). A link to this web site will be established on the TAIR database, and we will coordinate with TAIR to deposit data as appropriate. The knockout seeds will be made publicly available through deposition in the ABRC Stock Center at Ohio State. The monoclonals raised against the membrane marker proteins will be available for the cost of shipping through UNH and the cell lines will also be deposited with the American Type Culture Collection (ATCC). In keeping with the goals of the 2010 project, functional analysis of the two-component signaling elements will aid in our understanding of plants at the organismal, cellular, and evolutionary levels. The research will provide functional information on the role of Arabidopsis two-component signaling systems in plant growth and development. The research will serve to define the subcellular location of the two-component signal transduction pathways, the interactions between the two-component signaling elements, and the downstream targets of the pathways. The research should clarify how a signal transduction mechanism that arose in bacteria has been adapted to plant signal transduction.These studies should uncover the functions of several gene families in Arabidopsis. The proteins encoded by these gene families are predicted to interact and thus our studies should aid in the development of a paradigm for signaling specificity among interacting members of large gene families. In addition, tools will be developed that will be generally applicable in defining the subcellular location of proteins in Arabidopsis.