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

该建议概述了阐明吲哚-3-丁酸（IBA）在模式植物拟南芥中的功能的实验。 虽然IBA是植物生长激素生长素的一种天然形式，并在商业上用于促进许多物种的生根，但它的分子机制才刚刚开始被理解。 一组IBA反应突变体已被分离，包括突变体与B？氧化和过氧化物酶体生物合成缺陷。 这些突变体的分析表明，IBA转化为吲哚-3-乙酸（IAA）使用类似的反应，脂肪酸catenase（B？氧化），在植物中主要是过氧化物酶体过程。 其他IBA反应突变体具有明显正常的过氧化物酶体功能，并且可能在IBA感知或运输方面存在缺陷。 拟议的实验将使用这些IBA反应突变体的化学，分子和生理的IBA在植物发育中的作用阐明。 有正常B的突变体？氧化表型将用于揭示IBA功能的各个方面，而不依赖于其向IAA的转化，例如转运、代谢和基因表达的调节。 该提案有五个具体目标。 1)IBA反应突变体的特点，以确定从IBA产生的IAA在幼苗生长和侧根发育过程中的作用。 2)将在野生型和IBA应答突变体之间比较IBA水平和IBA代谢。 3)使用IBA向IAA转化中阻断的突变体的全局基因表达分析将用于发现由IBA直接调控的基因。 4)将继续进行突变体筛选以使IBA反应途径饱和，包括筛选新的突变体、现有突变体的增强子以及反向遗传筛选IBR基因和IBA调节基因的无效等位基因。 5)突变体中的缺陷基因将被鉴定，编码的蛋白质将被生化鉴定。为了理解生长素的作用，必须确定内源生长素的功能意义。 确定基因参与转换IBA到IAA是了解这种转换的调节和重要性的先决条件。 这方面的知识是必不可少的，以确定IBA相对于其他投入的活性生长素池，包括从头合成和共轭水解的贡献。 此外，阐明IBA在遗传上易处理的植物中作用的分子机制可能为IBA的农业用途提供见解。 例如，鉴定和表征将IBA转化为IAA的特异性同工酶可能有助于在IBA应用通常无效的难生根品种中对其进行修饰。除了增加植物发育中生长素代谢和过氧化物酶体功能的知识外，这项研究将为学生提供广泛的跨学科培训，和生物化学，这将是下一代科学家必不可少的。

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.