Arabidopsis 2010: MAPK Cascade Signaling Networks

本项目的目标是建立以6个MAPKKs （MKK1, 2, 4, 5, 7, 9）为中心的详细分子调控网络，这些MAPKKs在连接10个上游MAPKKs (MTKs)， 6个下游MAPKs (MPKs)， 12个转录因子（tf）和数千个靶基因响应控制拟南芥生长发育的12个信号中发挥重要作用。大量研究表明，植物的MAPK级联在控制各种胁迫和防御反应以及各种发育过程中起重要作用。该项目将确定特定的mpk及其直接上游调节因子mkk和mtk如何整合到拟南芥中连接上游信号和下游转录因子（TFs）和靶基因的植物信号网络中。在拟南芥MAPK级联信号功能基因组分析的第一阶段，我们在拟南芥叶肉原生质体中克隆并分析了20个MPK、10个MKK和60个推测的MTK基因，建立了MAPK级联在H2O2、flg22和乙烯信号通路中的功能。该信息已经建立了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。34个基因和12个信号在MAPK级联项目网站http://genetics.mgh.harvard.edu/sheenweb/mapk_cascades_nsf.html上列出。该项目结合全球基因表达谱和生物信息学工具，对单个mkk和tf进行功能获得和功能丧失突变分析，构建与关键tf和大量靶基因直接相关的重叠MAPK信号级联，以响应应激、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。原生质体系统的瞬态特性使我们能够以前所未有的高通量和相对较低的成本对MAPK级联和tf进行直接和动态的功能分析。提出的实验方法在揭示大量基因的调控方面尤其强大，这些基因由于冗余、致死率或低表达水平而难以通过传统的遗传和生化方法解决。我们的建议旨在通过结合基于细胞的分析、基因组和遗传工具以及整个植物分析来阐明MAPK信号级联在逆境、防御和发育中的功能作用。综合方法的独特优势是，在简化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，并扩大到基因组水平。阐明和操纵植物中的MAPK级联将揭示根本重要的信号传导过程。该项目整合了胁迫、防御、植物激素信号和基因调控方面的广泛经验、资源和信息，以促进对拟南芥和其他植物物种复杂但进化保守的信号网络的全面和分子理解和整合。该项目将在多学科的学术环境中，特别是在前沿功能基因组学和生物信息学的应用方面，为研究生和本科生、博士后和女性/少数民族提供优秀和独特的培训机会。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一个全面的网络可访问的MAPK级联和植物信号转导数据库（MAPKDB）以及TAIR、PlantsP和ABRC免费提供给植物界。

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.