Plant genomics: Identification and analysis of MAP kinase insertional knockout mutants in Arabidopsis thaliana

Functional genomics is regarded as one of the most important areas of future research in biology that will bring major benefits in medicine and agriculture. The principle aim of the present proposal is the identification of MAP kinase knockout mutants in Arabidopsis thaliana. MAP kinases are a large family of signalling molecules that are present in all eukaryotes, with functions that are fundamental for development and responses to changes in physiological conditions. The proposed strategy will use PCR-based screening of transposon insertion libraries of populations of mutagenized lines of Arabidopsis, an approach that has been shown to work efficiently. Knocking out a gene permits a link to the phenotype, but this reverse genetics approach must be supplemented by analyses at the structural level and at the biochemical and molecular levels. Reverse genetics will be complemented by a global expression analysis (expression profiling) of the MAP kinase genes. A DNA or protein chip enables one to identify those MAP kinases that are active in a particular tissue, and then these MAP kinase insertion mutants can be crossed which results in a complete knockout in this tissue. This approach obviates many of the problems inherent in other approaches, such as the use of antisense technology, is relatively rapid, and provides a means of overcoming the problem of genetic redundancy through crosses of different mutants. In the longer term, it also enables the identification of other components of the signalling pathways by suitable crosses to other mutants of MAP kinase signal transduction pathways, resulting in double mutants which can be analysed for an additive or epistatic effect. This proposal also provides a testing ground for the knockout of a gene family in a plant species, identified as one of the main aims of the Multinational Coordinated Arabidopsis thaliana Genome Research Project.