Genetic Analysis of Indole-3-Butyric Acid Functions 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 is a naturally occurring form of the plant growth hormone auxin and is used commercially to promote rooting in many species, the molecular mechanisms by which it acts are only beginning to be understood. A set of IBA-response mutants has been isolated, including mutants with b?oxidation and peroxisome biogenesis defects. Analysis of these mutants suggests that IBA is converted into indole-3-acetic acid (IAA) using reactions analogous to those of fatty acid catabolism (b?oxidation), a largely peroxisomal process in plants. Other IBA-response mutants have apparently normal peroxisome function and may have defects in IBA perception or transport. The proposed experiments will use these IBA-response mutants for the chemical, molecular, and physiological elucidation of the roles of IBA in plant development. The mutants with normal b?oxidation phenotypes will be used to uncover aspects of IBA function independent of its conversion to IAA, such as transport, metabolism, and regulation of gene expression. The proposal has five specific objectives. 1) The IBA-response mutants will be characterized to determine the roles of the IAA generated from IBA during seedling growth and lateral root development. 2) IBA levels and IBA metabolism will be compared between wild type and the IBA-response mutants. 3) Global gene expression analysis using mutants blocked in IBA to IAA conversion will be used to find genes directly regulated by IBA. 4) Mutant screens will be continued to saturate the IBA-response pathway, including screens for new mutants, enhancers of existing mutants, and reverse genetic screens for null alleles of IBR genes and IBA-regulated genes. 5) The genes defective in the mutants will be identified and the encoded proteins characterized biochemically.To understand auxin action, the functional significance of the endogenous auxins must be determined. Identifying genes involved in converting IBA to IAA is a prerequisite to understanding the regulation and importance of this conversion. This knowledge is essential to determine the contributions of IBA relative to other inputs to the active auxin pool, including de novo synthesis and conjugate hydrolysis. In addition, elucidating the molecular mechanisms of IBA action in a genetically tractable plant may provide insights for agricultural IBA uses. For example, 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.In addition to increasing knowledge of auxin metabolism and peroxisomal function in plant development, this research will provide students with broad interdisciplinary training bridging genomics, molecular genetics, and biochemistry that will be essential to the next generation of scientists.