The Arabidopsis Platform (provided by the Institute of Plant Genetics and Crop Research [IPK])

A major contribution of the group will be the establishment of large Arabidopsis population and the application of TILLING to the Arabidopsis model system.

According to the experience from previous mutagenesis programs several mutagen treatments of Col-0, C24 and Ler seed stocks (from homogenous stock) including 0.35%, 0,4 % and 0,45% EMS for 16 hrs were conducted and the cultivated M1 individuals were checked for fertility and the frequencies of aborted seeds in the developing pods. From one M1 one M2 individual was selected for DNA preparation and seed harvest. At the end 10,000 M2 plants will be selected from different mutant populations.

The TILLING screening procedure will be set up according to Colbert et al., 2001, Plant Physiol. 126: 480-484; McCallum et al., 2000, Nature Biotechnol. 18: 455-7, Plant Physiol. 123: 439-442. Using the reference system Arabidopsis thaliana, attempts were made to improve the throughput of the screening procedures at several levels: On the one hand, with a set of mutations positioned at various sites within the target region, the possibility and frequency of detection of cleavage products in much simpler gel systems such as agarose gels were evaluated. Furthermore, several procedures for pre-checking of pools for the presence of cleavage products will be tested. If successful, the optimised procedure will be automated and will be implemented to dramatically increase the throughput in routine screenings.

In collaboration with the partner at the University in Bielefeld the DNAs will be tested in Screens to enhance the throughput of analysis.

In different projects, the power of the TILLING technology to identify mutations will be demonstrated.

The aim of one proof of concept study is to generate a set of novel alleles for two genes which encode key regulatory enzymes of starch and sucrose synthesis, respectively. We are particularly interested in new forms of the enzymes with altered kinetic and regulatory properties - substrate affinity, sensitivity to allosteric regulators or altered post-translational regulation. Both enzymes are highly regulated in plants, and interact with allosteric metabolite effectors, and with other proteins. Conformational changes from binding of these ligands are likely to be transmitted throughout the protein, so even mutations outside the ligand binding sites could alter the enzymes properties. Therefore, we expect a high probability of finding change-of-function mutants. Alterations in the regulatory properties of these enzymes could lead to commercial application in crop plants. From the results with Arabidopsis thaliana, strategies will be considered to target one or both of these enzymes for TILLING in other species, e.g. to increase starch synthesis in potato tubers, or sucrose accumulation in sugar beet. This project is done in cooperation with a group at the MPI-MP in Golm.

Due to the small size of peptide or short protein (<= 100 amino acids) encoding genes, these are highly underepresented in T-DNA or transposon insertion mutant collections. These genes are to the largest extent of unknown function but their gene products can be expected to serve very important functions as signalling molecules. Mutants will be identified by TILLING in 10 small protein/peptide encoding genes and the mutants will be subjected to a morphological characterisation.

The currently available T-DNA and transposon insertion lines represent insertion mutations for ca. 60-70 % of the Arabidopsis genes. The remainder have either not been hit by an insertion due their unfavourable (small) size, or insertions causing k.o. mutations are (gametophytic) lethal. The UP group contributes to the completion of the mutant collections by performance of TILLING screens for 25 genes, which are neither listed as gene hits in any of the FST databases nor as characterized by any other mutation in 'The Arabidopsis Information Resource' (TAIR, Stanford). The selection was done with emphasis on genes studied in the GABI-KAT project. The mutants will be subjected to a morphological characterisation and the mutated gene segments will be amplified and sequenced.