Two genes involved in trichome morphogenesis: Localisation and putative function of STI and At-CLASP in A. thaliana.

Abstract

Within the scope of this thesis two proteins were analysed whose mutant phenotype affects the branching of trichomes. at-clasp mutant plants posses unbranched trichomes and the strength of the phenotype depends on the allele used. Sterility is observed in the strongest alleles. Furthermore it was shown that the at-clasp mutant phenotype is temperature dependant. Growth conditions with temperatures up to 16°C are permissive for normal growth of all alleles. To determine the localisation of At-CLASP, fusions to fluorescent proteins were constructed. It was shown that At-CLASP is localised to microtubules. Contrary to the data from H. sapiens, At-CLASP localisation was not restricted to the +end of microtubules but marks filaments almost entirely. To understand the binding properties of At-CLASP to microtubules, it was dissected into two fragments. The C-terminal region of the protein was localised to the cell-cortex or the plasma membrane. In contrast, the N-terminal region was sufficient to mediate the binding to microtubules as this fragment was hardly distinguishable from the entire protein. Despite the altered localisation of At-CLASP compared to H. sapiens CLASP, several orthologs of H. sapiens CLASP interaction partners were identified in the A. thaliana genome. These putative A. thaliana binding partners were tested for interaction with At-CLASP in direct Yeast-Two-Hybrid experiments. However, no interactions were detected. One postulated function of CLASP from H. sapiens is the stabilisation of subsets of microtubules. To test a similar function of the A. thaliana protein, the rescue of the tfc-a mutant under an increased dosage of At-CLASP was analysed. Preliminary results suggest that a stabilizing function of At-CLASP might be evolutionary retained. Trichomes of strong stichel alleles are completely unbranched. To improve the understanding of STI function plants expressing GFP:STI fusions were analysed further. GFP:STI fusion proteins mark the tip of emerging trichomes. As soon as a trichomes becomes visible by means of morphological criteria, GFP:STI can be detected in the tip of trichomes. Trichomes that initiate the second branch showed GFP:STI signal even before the branch was visible. Plasmolysis experiments demonstrated that GFP:STI is likely localised to the plasma membrane. In order to get a closer insight into which part of the STI protein is responsible for its specific localization, fragments were analysed for showing a comparable localisation pattern. Fusions of parts of the STI protein with YFP could not reveal the likely present motif for the described localisation. Nevertheless one longer fragment S1/AS3 and one shorter fragment S3/AS3 showed a strong dominant negative effect when overexpressed in wild-type. To increase the understanding about the molecular function of STI, possible binding partners of GFP:STI were identified by an immunoprecipitation assay. Phospholipase Dα1 and one transcription factor of the MADS-box family were identified as candidates for interaction with STI. A. thaliana contains one gene (STI-HOM) which is very similar to STI. To test whether STI-HOM fulfils a comparable a role in trichome development T-DNA mutants were analysed. None of them showed a trichome phenotype. The analysis of a sti sti-hom double-mutant situation revealed no change in the sti mutant trichome phenotype. Promoter swapping experiments were performed to test whether both proteins are functionally exchangeable. However STI-HOM was not able to rescue the sti mutant trichome phenotype. Therefore STI and STI-HOM are likely functionally distinct