Functional genomics of the Arabidopsis MAPK gene family

Research project P 14751 Functional genomics of the Arabidopsis MAPK gene family Erwin HEBERLE-BORS 27.11.2000 Mitogen-activated protein kinases (MAPKs) are present in all eukaryotes, with functions that are central to growth, development, and stress. Numerous reports describe the activation of MAPKs by a variety of different stimuli in plants. However, these largely descriptive studies do not adequately address the functional significance of such responses. We wish to use the model plant Arabidopsis thaliana to analyze its complete gene family of MAPKs in a functional genomics approach. To date we have identified 18 NLAPK genes in the nearly fully sequenced Arabidopsis genome. The principle aims of the present proposal are the identification and analysis of MAPK knockout mutants in Arabidopsis thaliana and the expression profiling of NIAPK genes, on a genome-wide basis. The proposed strategy will use PCR-based screening of transposon and T-DNA insertion libraries of populations of mutagenized lines of Arabidopsis: a putative knockout has already been isolated using this approach. This reverse genetics approach will be supplemented by analyses at the phenotypic level and at the biochemical and molecular levels. Expression profiling of all the MAPK genes using microar-ray technology will provide, a rapid screen for identifying those MAPKs that are expressed at a particular time, in a particular tissue, or following a particular stimulus. These results will also direct crossings of individual MAPK knockouts to produce multiple knockouts. An Arabidopsis MAPK microarray has already been developed for this purpose. The Rinctional genomics approach obviates many of the problems inherent in other approaches of analysing genes in gene families, such as the use of antisense technology. In the longer term, it also enables the identification of other components of the signaling pathways by suitable crosses to other mutants of MAPK signal transduction pathways. Together with global expression analysis it will facilitate the identification of MAPK interacting/dependent molecules.

Our most interesting results comes from the third part of the project in which we continued our work on tobacco MAPK genes. MAP kinases are highly conserved protein kinases that play central roles in the transduction of signals that are recognised by a cell and have to be transmitted to various parts of the cell, including the nucleus and the cytoskeleton. When dry pollen - in a state of suspended life - lands on a stigma, it takes up water and is re- animated to life. Then, formation of the actin cytoskeleton is the decisive event in pollen tube formation, leading eventually to fertilization. We identified a MAP kinase kinase which was activated by rehydration which in turn activated a particular MAP kinase, and this MAP kinase activated profilin, an actin-binding protein. Activation leads to profilactin complex dissociation and actin polymerisation. Thus, the hitherto unknown link between pollen hydration and pollen tube formation has been identified. Incidentally, profilin is an allergen. Apart from this, the P14751-B12 project was an attempt to develop innovative approaches to analyse one particular large gene family, the MAP kinase gene family, of the model plant species Arabidopsis thaliana, the first plant species whose genome has been fully sequenced. In recent years a number of genomes have been sequenced, and bioinformatics has lead to the identification of genes with predictable or unknown functions (genomics). The task now is to really identify the function of the many genes (functional genomics). A recurrent theme of functional genomics has been gene redundancy, i.e. the presence of gene families with sequence and functional similarity. We identified 20 members (paralogs) in the Arabidopsis MAPK gene family and tried to put them as oligonucleotides on a microarray (DNA-chip) for simultaneous gene expression analysis. We however failed so far. We nevertheless succeeded in doing a comprehensive gene expression analysis by other methods. In the second part of the project we knocked out MAPK genes, expecting to find changes in phenotye. We, however, did not find phenotypes in any of the single gene knock-outs tested. But we saw changes in the expression of a second MAPK gene when one particular MAPK gene was knocked out. Double knock-outs were then done in such a way that we crossed knock-out mutants in two MAPK genes that were expressed in the same tissue. Again no phenotype was found but it is likely that in some cases the double knock-out plants died during embryogenesis.