The long term goal of the proposed research is to understand hormonal regulation of plant cell growth with emphasis on auxin. The specific goal of this proposal is to determine the biochemical and cellular function of the AXR1 family of proteins in Arabidopsis and Saccharomyces cerevisiae. This family of proteins is related to the ubiquitin-activating enzyme (E1) and genetic studies have shown that, in Arabidopsis, the AXR1 gene has an important role in auxin response suggesting that regulated protein degradation may be important for auxin action. Genetic studies also indicate that one of the suppressors of the axr1 phenotype (SAR1) acts downstream of AXR1.
The specific aims of this proposal are to define: 1) Role of AXR1 and SAR1 in Cell Growth: Since auxin is known to regulate both cell division and cell elongation the PI will determine whether the axr1 mutants are deficient in cell division, cell elongation or both by characterizing the cell division and elongation zones of axr1 roots in detail. Cell structure will be determined by examination of thin sections with a phase contrast microscope. The pattern and rate of cell division will be determined using BrdU. The effects of the sar1 (suppressor of axr1) on cell structure will also be determined. The axr1 mutant's deficiency in root hair elongation will be investigated using microinjection techniques. 2) Molecular Characterization of SAR1: The major proposed function for AXR1 is to relieve SAR1-mediated inhibition of auxin response. This proposed function will be investigated by cloning SAR1 using a map-based approach. A project initiated to recover this gene has already identified a large number of informative recombinants in this region and these will be used to orient a chromosome walk. Once isolated, the gene it will be characterized using standard techniques. 3) Characterization of Additional SAR Genes: At least two additional SAR loci have been identified and these (SAR2 and SAR3) will be characterized in a fashion similar to that described for SAR1. Genetic tests will be conducted to determine if the three SAR loci function in a single pathway. If appropriate, the PI will proceed with a molecular characterization of these genes. 4) Cellular Localization of AXR1: The sequence of the AXR1 protein suggests that it is localized to the cytoplasm and/or nucleus of the cell and several putative nuclear localization sequences have been identified within the protein. To determine if AXR1 is localized to the nucleus the PI will introduce N-and C-terminal GUS fusions into plant cells by particle bombardment and/or protoplast transformation. 5) Role of the Ubiquitin Pathway in Auxin Action: The results of this PI's previous studies suggest that the ubiquitin-proteosome pathway may be involved in auxin response. To investigate this possibility, the effects of specific proteosome inhibitors on auxin regulated-gene expression will be determined. 6) Genetic Studies of ENR1 and ENR2 in Yeast: Two genes related to AXR1 have been identified in the yeast Saccharomyces cerevisiae. The PI has demonstrated that the ENR1 gene is essential for viability while deletion of ENR2 confers no phenotype. However, the demonstration that a synthetic lethal interaction between ern2D (deletion of ENR2) and the cdc34-2 mutation indicates that the ENR2 and CDC34 interact, either directly or indirectly. Since the CDC34 gene encodes a ubiquitin-conjugating enzyme, this result provides further evidence for a role for the AXR1-like proteins in the ubiquitin pathway. A number of genetic and molecular experiment are planned in yeast to investigate the biochemical and cellular function of ENR1 and ENR2 and these will hopefully provide important clues to the function of AXR1 and the AXR1-like gene family. 7) Interaction between AXR1 and Arabidopsis E1 and E2 proteins: In parallel with the studies described in Specific Aim 6, the PI will test for interactions between AXR1 and various E1 and E2 proteins from Arabidopsis. 8) Biochemical Function of the AXR1-like Proteins: The results of the experiments described above as to the biochemical function of the AXR1-like proteins will be extended by characterizing the functions of these protein in vitro.
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