SGER: A Novel Protein-linked Route for the Biosynthesis of Indole-3-acetic Acid in Maize Endosperm

生长素型植物激素吲哚-3-乙酸(IAA)的生物合成在半个多世纪以来一直是一个活跃的研究领域，已经预测了许多不同的途径，但很少有人被证明是植物正常发育的关键。在玉米籽粒发育后期，IAA的生物合成对于积累相对大量的储存的结合IAA是重要的，并且已被证明起源于氨基酸色氨酸。在胚乳发育的短时间内，与生理相关的高水平IAA的产生使其成为分析IAA生物合成途径的独特系统。使用两种互补的方法来鉴定负责该途径中不同转化的酶以及中间化合物，结果表明以前报道的途径不能解释测量的IAA产生。对潜在中间体的代谢物筛选显示，大多数既不是色氨酸也不是IAA的放射性与较高的分子量分数有关。凝胶电泳法和大小排除高效液相色谱法表明，放射性与10-20 kDa的蛋白质有关，其中色氨酸显然是通过硫代酯键连接的。该项目的重点是利用生化分析来分离、鉴定与色氨酸结合的蛋白质并对其进行微序列测定，以便随后克隆相应的基因。大量和高纯度生产色氨酸蛋白的能力将有助于解决它们在IAA生物合成中的作用(S)。广泛影响：该项目将在研究和拨款撰写过程中培训博士后科学家。该项目所取得的成果将作为单独资助的努力的一部分，用于培训和指导少数族裔本科生。此外，该实验室将接待来自大学和四年制学院的来访学生、博士后和其他科学家，学习与项目目标相关的技术。

The biosynthesis of the primary auxin-type plant hormone, indole-3-acetic acid (IAA), has remained an active area of study for more than half a century and numerous different pathways have been predicted but few have been shown to be critical for normal plant development. The biosynthesis of IAA during late kernel development in maize is important for the accumulation of relatively large quantities of stored conjugated IAA and has been shown to originate from the amino acid tryptophan. The physiologically relevant production of high levels of IAA during a short time period in endosperm development makes this a unique system for analysis of IAA biosynthesis pathways. Using two complementary approaches to identify the enzymes responsible for the different conversions in the pathway as well as the identification of the intermediate compounds, it was shown that previously reported pathways could not account for the measured IAA production. A metabolite screen for potential intermediates revealed the majority of the radioactivity that was not either tryptophan or IAA was associated with a higher molecular weight fraction. Gel electrophoresis and size exclusion HPLC indicated radioactivity was associated with proteins in the size range of 10-20 kDa, where tryptophan was linked apparently via a thio-ester bond. The focus of this project is to utilize biochemical analyses to isolate, characterize and microsequence proteins, which become bound to tryptophan, for subsequent cloning of corresponding genes. The ability to produce the tryptophyl-proteins in larger amounts and at higher purities will help resolve their role(s) in IAA biosynthesis.Broader impacts: This project will train a postdoctoral scientist in both research and in the grant writing process. Results obtained in this project will be used as part of a separately funded effort to train and mentor minority undergraduate students. In addition, the laboratory will host visiting students, postdocs and other scientists from universities and 4-year colleges to learn techniques related to the project goals.