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.