In higher plants, about 95% of the hormone indole-3-acetic acid (IAA, auxin) is found in inactive conjugated forms. The release of IAA from these conjugates is likely to be of importance to control the cellular levels of IAA and thus its action. In postdoctoral work, the PI designed a novel screen for Arabidopsis mutants that were insensitive to specific IAA-amino acid conjugates that remained sensitive to free IAA. One of the genes identified in this screen was cloned and shown to encode a hydrolase specific for particular IAA-amino acid conjugates. The cloned gene has several homologs in Arabidopsis that may encode other IAA-amino acid hydrolases. The PI proposed three main objectives. The first is identify new IAA- conjugate hydrolases. This will be done by isolating the genes disrupted in other IAA-conjugate insensitive mutants and by isolating new IAA- conjugate hydrolase genes by homology. The second objective is to determine the cellular and developmental expression of the IAA-conjugate hydrolase genes and determine the cellular localization of the corresponding proteins. Gene expression will be monitored by RNA blot analysis, analysis of promoter-reporter gene constructs, and in situ hybridization. Protein localization will be determined by introducing functional epitope-tagged versions of the conjugate hydrolases into the corresponding mutant plants. Cellular localization will be analyzed using commercially available antibodies against the epitope. The third objective is manipulate IAA regulation in plants by inactivating and -overexpressing IAA-conjugate hydrolase genes. Phenotypic analysis of new and existing mutants and double mutants will be carried out. The genes will be overexpressed in plants, and antisense experiments will be conducted on those genes for which no conventional mutants are available.
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