Genetic Analysis of Indole-3-Butyric Acid Function in Plants

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This proposal outlines experiments to elucidate the function of indole-3-butyric acid (IBA) in the model plant Arabidopsis thaliana. Although IBA, a naturally occurring form of the plant growth hormone auxin, is used commercially to promote rooting in many species, the molecular mechanisms by which it acts are largely unknown. It has been suggested that IBA is converted into indole-3-acetic acid (IAA) using reactions analogous to those of fatty acid degradation (b-oxidation), a peroxisomal process in plants. A set of IBA-response mutants has been isolated and provisionally grouped into two classes: 1) mutants defective in peroxisome biogenesis or general boxidation, which may inefficiently convert IBA to IAA, and 2) mutants with apparently normal peroxisome function, which may be defective in IBA perception or signaling. The hypothesis that IBA functions in auxin signaling through its conversion to IAA in peroxisomes will be tested by characterizing these mutants. The following experiments will be conducted: 1) Phenotypes related to auxin and peroxisomal functions will be examined in the IBA-response mutants. 2) Specific defects of two IBA-response mutants for which the responsible genes have been identified will be investigated. One mutant is defective in a peroxisomal targeting receptor. Peroxisome morphology will be visualized and the subcellular distribution of peroxisomal proteins will be examined. The sequence of the gene defective in the second mutant suggests that the encoded protein hydrolyzes IAA-CoA to release free IAA following IBA chain shortening. The substrate specificity of this enzyme will be tested. 3) The genes defective in several other IBA-response mutants will be isolated, with emphasis on those having apparently normal peroxisomal function. These genes and mutants will be further characterized to elucidate their role in IBA action. 4) Additional IBA-response mutants will be isolated to identify a more complete set of genes involved in IBA function.To understand auxin action, the functional significance of the various auxins found in plants must be determined. Identifying genes involved in IBA to IAA conversion is a first step to understanding the regulation of this conversion. This knowledge is essential in determining the contributions of the various facets of IAA metabolism to the active IAA pool. Elucidating of the molecular mechanisms of IBA action in a genetically tractable plant may provide insights for agricultural and horticultural IBA uses. Identifying and characterizing the specific isozymes that convert IBA to IAA may facilitate their modification in difficult-to-root cultivars where IBA application is normally ineffective. This research will also reveal genes critical for the function and biogenesis of plant peroxisomes, which carry out functions not found in mammalian or yeast peroxisomes. Finally, the proposed experiments will expand understanding of plant boxidation, which is essential both for the utilization of seed storage lipids and for chain shortening of other substrates, including jasmonic acid, a plant hormone involved in defense responses.