Functional Analysis of Plant Mitogen-activated Protein Kinase Cascades in Stress and Hormonal Signaling

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The major goal of this project is to elucidate the molecular mechanisms by which plants respond to environmental stress and hormonal signals. Numerous studies have shown that so-called mitogen-activated protein kinase (MAPK) cascades are activated by abiotic stresses (e.g., cold, drought, salt, wounding and touch), pathogens and pathogen-derived elicitors and plant hormones. MAPK cascades are evolutionarily conserved protein modules that function in molecular signaling pathways in a variety of eukaryotes, including yeasts, worms, flies, frogs, mammals, and plants. MAPKs are protein kinases that activate regulatory proteins such as transcription factors by phosphorylating them. MAPKs are in turn phosphorylated and activated by MAPKKs, and MAPKKs are phosphorylated and activated by MAPKKKs. The Arabidopsis genome project has revealed large gene families encoding MAPKs, MAPKKs and MAPKKKs (referred to as MAPK cascade genes). However, little is known about the constitution of plant MAPK cascades and the specific roles that particular MAPK cascade genes play in particular plant signal transduction pathways. A comprehensive approach based on genomic information will be employed to generate a freely available set of MAPK-related resources including engineered MAPK cascade genes and transgenic plants overexpressing these genes. Engineered MAPK cascade genes will be transiently expressed in Arabidopsis protoplasts (individual cells stripped of their cell walls to facilitate DNA uptake) to determine the function of Arabidopsis MAPK cascade genes involved in essential plant signaling pathways. Since the functions of MAPK cascades in plant signal transduction pathways are likely conserved, studies using the Arabidopsis genome resources will have broad implications and applications in other plant species including important crop plants.The four MAPK cascades that will be the focus of this project include those involved in osmotic stress protection, bacterial peptide recognition, hormone signaling, and oxidative stress responses. The overall strategy will involve the identification and cloning of the complete set of Arabidopsis genes encoding the MAPKs, MAPKKs, MAPKKKs and relevant protein phosphatases. Transient expression of immunologically-tagged protein kinases will be used to match particular MAPK cascades with particular abiotic or biotic stress or hormone signals in Arabidopsis and maize protoplasts. Engineered MAPKKKs and MAPKKs, designed to be constitutively active or to act as inhibitors, will be used in conjunction with DNA microarray technology to identify Arabidopsis genes that are activated in response to particular signaling pathways. Identification of Arabidopsis knockout mutants corresponding to key MAPK-related regulatory genes will be initiated. Transgenic Arabidopsis, maize and soybean plants that over-express particular engineered MAPKKK genes will be generated. The transgenic plants will be tested for the exhibition of agronomically useful traits. The experimental design is based on Arabidopsis and maize protoplast technology. The transient nature of the protoplast systems allows direct functional analysis of plant genes at an unprecedented high throughput rate and at a relatively low cost. This approach takes full advantage of the Arabidopsis genome infrastructure that is being created as a part of the NSF Plant Genome Research Program, including DNA sequencing, gene expression technology, and knockout mutant libraries. The experimental approaches are especially powerful in unraveling the functions of genes that are difficult to tackle by traditional genetic and biochemical approaches due to redundancy, lethality or low levels of expression. The elucidation and manipulation of MAPK cascades in plants will reveal fundamentally important regulatory processes and provide new tools for crop improvement related to stress tolerance, disease resistance, and yield enhancement. This project integrates three laboratories that provide complementary expertise in stress and hormonal signaling, pathogen responses, and transgenic crop plant generation and analysis. The project will also provide excellent training opportunities in a multidisciplinary academic environment. Information and materials generated from this project, including the full set of cloned and engineered MAPK genes, will be made freely available to the plant community via a web-accessible database.