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

这项建议概述了实验，以阐明吲哚-3-丁酸(IBA)在模式植物拟南芥中的功能。虽然IBA是植物生长激素生长素的一种自然存在形式，并在商业上用于促进许多物种的生根，但其作用的分子机制才刚刚开始被理解。已分离到一组IBA反应突变体，包括具有b？氧化和过氧化物酶体生物发生缺陷的突变体。对这些突变体的分析表明，IBA被转化为吲哚-3-乙酸(IAA)的反应类似于脂肪酸分解代谢(b？氧化)，这是植物中的一个主要过氧体过程。其他IBA反应突变体具有明显正常的过氧化体功能，可能在IBA的感知或运输方面存在缺陷。拟议的实验将使用这些IBA反应突变体来从化学、分子和生理上阐明IBA在植物发育中的作用。具有正常b型氧化表型的突变体将被用来揭示IBA功能的一些方面，这些功能不依赖于IBA向IAA的转化，例如运输、代谢和基因表达的调节。该提案有五个具体目标。1)对IBA反应突变体进行鉴定，以确定IBA产生的IAA在幼苗生长和侧根发育过程中的作用。2)比较野生型和IBA反应突变体的IBA水平和IBA代谢。3)利用阻止IBA向IAA转化的突变体进行全球基因表达分析，以寻找IBA直接调控的基因。4)突变筛选将继续使IBA反应途径饱和，包括筛选新的突变，现有突变的增强剂，以及IBR基因和IBA调节基因的零等位基因的反向遗传筛选。5)对突变体中存在缺陷的基因进行鉴定，并对编码的蛋白质进行生化鉴定。要了解生长素的作用，必须确定内源生长素的功能意义。识别IBA转化为IAA所涉及的基因是理解这种转化的调节和重要性的先决条件。这一知识对于确定IBA相对于其他输入对活性生长素库的贡献是必不可少的，包括从头合成和共轭水解。此外，阐明IBA在遗传易驯化植物中作用的分子机制可能会为IBA在农业上的应用提供见解。例如，识别和鉴定将IBA转化为IAA的特定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改。除了增加对生长素代谢和植物发育中过氧化体功能的知识外，这项研究还将为学生提供广泛的跨学科培训，将基因组学、分子遗传学和生物化学联系起来，这对下一代科学家来说是必不可少的。

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.