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、反应调节剂和在磷酸化中继的情况下的组氨酸磷酸转移蛋白（HPts）。在拟南芥中已经鉴定出编码与这些元件中的每一个相似的蛋白质的基因，并且对于35个这样的基因中的大多数，还没有明确的功能。提出了一个完整的方法来阐明这些蛋白在拟南芥中的功能。基因敲除和诱导过表达的组合将用于评估这些基因在植物生长和发育中的作用。突变体植物系将根据它们对生物和非生物因素如激素、光和渗透胁迫的响应以及它们的基因表达模式来表征。将使用GUS融合和原位RNA分析的组合来确定这些基因在植物中的何处表达。同源蛋白在细胞内的位置也将被描绘。为了促进这种定位，将产生一系列的10个单克隆抗体的标记蛋白，其中每一个驻留在不同的膜上。从拟南芥蛋白质复合物将被纯化和分析，以确定这些元素之间的相互作用，并确定新的相互作用的蛋白质。总之，这些研究将阐明这些拟南芥双组分信号元件中的每一个的信号通路，以及它们如何相互作用以控制植物生长和发育。这些研究的数据将被存放在一个公开访问的网页上，该网页目前正在建设中，在拟南芥（http：//www.bio.unc.edu/research/two-component/）。将在TAIR数据库中建立与该网站的链接，我们将与TAIR协调，酌情存放存款数据。淘汰种子将公开提供，通过沉积在ABRC股票中心在俄亥俄州。针对膜标志物蛋白产生的单克隆抗体将可通过UNH以运输成本获得，并且细胞系也将保藏于美国典型培养物保藏中心（ATCC）。为了与2010年项目的目标保持一致，双组分信号元件的功能分析将有助于我们在生物体，细胞和进化水平上理解植物。该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。 该研究将有助于确定双组分信号转导通路的亚细胞定位，双组分信号元件之间的相互作用，以及通路的下游靶点。 这些研究将有助于阐明起源于细菌的信号转导机制如何适用于植物的信号转导，并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预计相互作用，因此我们的研究应该有助于发展一个范例之间的相互作用的大基因家族成员的信号特异性。 此外，将开发的工具，将普遍适用于定义蛋白质在拟南芥的亚细胞位置。

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.