Characterisation of cuticular mutants in Arabidopsis thaliana

Plants are protected by the extracellular cuticle, which is made up of cutin, cutan and waxes. The cutin composition of a variety of plants has been known and models of the biosynthesis of cutin monomers exist but not many enzymes have been identified. It is generally accepted that a defect in the cuticle leads to an organ fusion phenotype. In the model plant A. thaliana many fusion mutants have been identified but the identification of genes involved have not lead to a complete picture of the biosynthetic pathway as the fragile Arabidopsis cutin could not be analysed so far. In this thesis a method to analyse cutin in Arabidopsis was developed and different procedures for the analysis were tested. The analysis of totally extracted leaves instead of isolated cuticles was found to be applicable to analyse Arabidopsis cutin. The main lipids in the Arabidopsis cutin were found to be w-OH-fatty acids, a-OH-fatty acids, a-w-diacids and VLCFAs. The optimal depolymerisation step was carried out using methanolic hydrochloric acid and the extraction of lipophilic cutin monomers was performed using hexane. After the subsequent derivatisation with BSTFA the cutin monomers were analysed with GC-MS and GC-FID. The application of the method to the fusion mutant ace/hth revealed together with other background information to the knowledge that it might have a defect in the w-oxidation reaction of w-hydroxy-fatty acids to w-oxo-fatty acids that are putative precursors for a-w-diacids that occur in the Arabidopsis cutin. Another organ fusion mutant that was analysed was bdg. BDG shows homology to a/b hydrolases and the cutin analysis revealed an increased amount of all cutin components by two to three times. This was reconfirmed by previous results from other experiments and the putative function of BDG was suggested to be the transesterification of the cutin components in the cell wall. The upregulation of the cutin biosynthetic pathway, which might be the cause for the increased amount of cutin, could be a compensatory effect of the plant trying to complement the improperly built cuticle. Other putative cuticular mutants were analysed and the cutin composition of pel3, cer13 and FDH:FAE was changed in an unspecific manner. Additional experiments will be necessary to verify the function of the genes as the results of the cutin analyses were rather due to secondary or compensatory effects than being caused directly by the knock-out (pel3 and cer13) or differential expression (FDH::FAE) of genes. The cer10/ecr mutant was characterised because CER10/ECR is a candidate for the last reduction step in the FAE complex on the cytosolic site of the ER. Defects of cer10/ecr were a change in the overall size of the plant, fusions in flowers, a reduced amount of wax on all aerial organs, difference in the sepal surface in comparison to the wild type and occasional organ duplication in flowers. It could be shown, that CER10 is allelic to ECR. So all defects should be caused by a mutation in ECR. Cutinanalysis of cer10 exhibited a reduction in a-hydroxy fatty acids and a slight accumulation in VLCFAs. Analysis of the expression of a-hydroxylases did not reveal the enzyme that is responsible for the a-hydroxylation in the cutin monomers. The analysis of lipids of the seed coat of cer10/ecr showed a primary effect of the lack of ECR as lipids with longer chain lengths are reduced when compared to the wild type but the elongation of VLCFAs in cer10 is not completely abolished. In another project the organ fusion mutant cer13 was characterised. In an approach to map the cer13 gene the map based cloning strategy was chosen and the analysis points to a specific location on chromosome 3 but further fine mapping strategies will be needed to identify CER13. The method of cutin analysis was applied to O.sativa japonica and the cutin composition of rice has been established. In the future it will be possible to analyse putative cutin mutants in rice to identify components of the cutin biosynthesis in O.sativa. The experiments in this thesis investigated the composition of cutin in Arabidopsis. The application of this method will lead to an understanding of the cutin biosynthesis and its involvement in signalling and plant protection. Still further studies on other plant species will be necessary as the cutin composition of Arabidopsis is different than in other plants and the biosynthesis of cutin components in Arabidopsis might not be true for all land plants.