Arabidopsis 2010: MAPK Cascade Signaling Networks

本项目的目标是构建一个详细的分子调控网络，该网络以6个MAPKK（MKK 1，2，4，5，7，9）为中心，在控制拟南芥生长和发育的12个信号中，连接10个上游MAPKK（MTK）、6个下游MAPKs（MPK）、12个转录因子（TF）和数千个靶基因。大量研究表明，植物MAPK级联反应在控制各种胁迫和防御反应以及各种发育过程中起着重要作用。该项目将确定特定的MPKs及其直接上游调节因子，MKKs和MTKs如何整合到拟南芥中连接上游信号和下游转录因子（TF）和靶基因的植物信号网络中。在拟南芥MAPK级联信号的功能基因组学分析的第一阶段，20 MPK，10 MKK和60假定的MTK基因已被克隆和分析在拟南芥叶肉原生质体中建立MAPK级联功能的H2 O2，flg 22，和乙烯信号通路。这些信息已经建立了MAPK级联信号网络的核心，包括10个MTK，6个MKK和6个MPK，这将作为启动新的全基因组研究的基础，连接12个动态和重叠的信号转导通路。这34个基因和12个信号列在MAPK级联项目网站http://genetics.mgh.harvard.edu/sheenweb/mapk_cascades_nsf.html上。该项目结合了全球基因表达谱和生物信息学工具，用于个体MKK和TF的功能获得和功能丧失突变体分析，以构建直接与关键TF和大量靶基因相关的重叠MAPK信号级联反应，以响应压力，激发子和激素信号。 实验设计是基于新的拟南芥原生质体技术结合微阵列和生物信息学分析在WT和突变体。原生质体系统的瞬时性质使得能够以前所未有的高通量速率和相对低的成本对MAPK级联和TF进行直接和动态的功能分析。所提出的实验方法在解开大量基因的调控方面特别强大，这些基因由于冗余、致死性或低水平表达而难以通过传统的遗传和生化方法处理。我们的建议旨在阐明MAPK信号级联反应在胁迫，防御和发展中的功能作用，结合细胞为基础的检测与基因组和遗传工具和整个植物分析。综合方法的独特优势是，在使用简化的细胞和分子测定方法建立的假设的指导下，进行全植物研究，并将其扩大到基因组水平。阐明和操纵MAPK级联反应在植物中将揭示根本重要的信号转导过程。该项目整合了关于胁迫、防御和植物激素信号传导和基因调控的广泛经验、资源和信息，以促进对拟南芥和其他植物物种中复杂但进化上保守的信号传导网络的全面和分子理解和整合。 该项目将为研究生和本科生，博士后研究员和妇女/少数民族提供优秀和独特的培训机会，在多学科的学术环境中，特别是在尖端功能基因组学和生物信息学的应用。该项目产生的信息，工具，协议，材料，讲座，服务和出版物将继续通过全面的网络访问MAPK级联和植物信号转导数据库（MAPKDB）和TAIR，PlantsP和ABRC免费提供给植物社区。

The goal of this project is to build a detailed molecular regulatory network centered on six MAPKKs (MKK1, 2, 4, 5, 7, 9) that play essential roles in linking ten upstream MAPKKKs (MTKs), six downstream MAPKs (MPKs), 12 transcription factors (TFs), and thousands of target genes in response to 12 signals that control Arabidopsis growth and development. Numerous studies have indicated that plant MAPK cascades are important for controlling diverse stress and defense responses and various developmental processes. The project will determine how specific MPKs and their immediate upstream regulators, MKKs and MTKs are integrated into the plant-signaling network connecting upstream signals and downstream transcription factors (TFs) and target genes in Arabidopsis. In the first phase of the functional genomic analysis of Arabidopsis MAPK cascade signaling, 20 MPK, 10 MKK and 60 putative MTK genes have been cloned and analyzed in Arabidopsis mesophyll protoplasts to establish MAPK cascade functions in the H2O2, flg22, and ethylene signaling pathways. The information has established the core of a MAPK cascade-signaling network including 10 MTKs, 6 MKKs and 6 MPKs that will serve as the foundation to launch new genome-wide studies linking 12 dynamic and overlapping signal transduction pathways. The 34 genes and 12 signals are listed on the MAPK Cascade Project website http://genetics.mgh.harvard.edu/sheenweb/mapk_cascades_nsf.html. This project combines global gene expression profiling and bioinformatics tools for individual MKKs and TFs with gain-of-function and loss-of-function mutant analyses to construct overlapping MAPK signaling cascades linked directly to key TFs and a large number of target genes in response to stress, elicitors, and hormonal signals. The experimental design is based on the novel Arabidopsis protoplast technology combined with microarray and bioinformatics analysis in WT and mutants. The transient nature of the protoplast system enables direct and dynamic functional analyses of MAPK cascades and TFs at an unprecedented high throughput rate and at relatively low cost. The proposed experimental approaches are especially powerful in unraveling the regulation of a large number of genes that are difficult to tackle by traditional genetic and biochemical approaches due to redundancy, lethality or low levels of expression. Our proposal aims to elucidate the functional roles of MAPK signaling cascades in stress, defense and development by combining cell-based assays with genomic and genetic tools and whole plant analyses. The unique advantage of the integrated approach is to carry out whole plant studies guided by hypotheses established using simplified cell and molecular assays scaled up to the genome level. The elucidation and manipulation of MAPK cascades in plants will reveal fundamentally important signaling processes. This project integrates broad experience, resources and information on stress, defense, and plant hormone signaling and gene regulation to facilitate comprehensive and molecular understanding and integration of complex but evolutionarily conserved signaling networks in Arabidopsis and other plant species. The project will provide excellent and unique training opportunities for graduate and undergraduate students, postdoctoral fellows and women/minority in a multidisciplinary academic environment, especially in the application of cutting-edge functional genomics and bioinformatics. Information, tools, protocols, materials, lectures, services and publications generated from this project will continue to be freely available to the plant community via a comprehensive web-accessible MAPK cascade and plant signal transduction database (MAPKDB) and TAIR, PlantsP and ABRC.