REGULATION OF THE PLANT GROWTH HORMONE INDOLEACETIC ACID

在高等植物中，大约95%的激素吲哚-3-醋酸(IAA， 生长素)以不活跃的结合形式存在。释放的IAA从 这些偶联物很可能对控制细胞 IAA水平以及它的行动。在博士后工作中，PI设计了一个 拟南芥对特定基因不敏感突变体的新筛选 IAA-氨基酸结合物对游离IAA仍然敏感。其中一个 在此屏幕中鉴定的基因被克隆并显示为编码一种水解酶 专用于特定的IAA-氨基酸结合物。克隆的基因具有 拟南芥中可能编码其他IAA-氨基酸的几个同源物 水解酶。 国际和平倡议提出了三个主要目标。第一个是确定新的IAA- 共轭水解酶。这将通过分离被破坏的基因来完成 在其他IAA结合不敏感突变体中，通过分离新的IAA- 通过同源结合水解酶基因。 第二个目标是确定细胞和发育 IAA结合水解酶基因的表达及细胞鉴定 相应蛋白质的定位。基因表达将是 通过RNA印迹分析，分析启动子-报告基因 构建，以及原位杂交。蛋白质定位将是 通过引入功能性表位标记版本的 将水解酶结合到相应的突变植物中。蜂窝 将使用市场上可获得的抗体分析定位 与表位相反。 第三个目标是通过失活来操纵植物中的IAA调节 和-过表达IAA结合水解酶基因。的表型分析 将进行新的和现有的突变体和双突变体。基因 将在植物中过度表达，反义实验将 对那些无法获得常规突变体的基因进行研究。

In higher plants, about 95% of the hormone indole-3-acetic acid (IAA, auxin) is found in inactive conjugated forms. The release of IAA from these conjugates is likely to be of importance to control the cellular levels of IAA and thus its action. In postdoctoral work, the PI designed a novel screen for Arabidopsis mutants that were insensitive to specific IAA-amino acid conjugates that remained sensitive to free IAA. One of the genes identified in this screen was cloned and shown to encode a hydrolase specific for particular IAA-amino acid conjugates. The cloned gene has several homologs in Arabidopsis that may encode other IAA-amino acid hydrolases. The PI proposed three main objectives. The first is identify new IAA- conjugate hydrolases. This will be done by isolating the genes disrupted in other IAA-conjugate insensitive mutants and by isolating new IAA- conjugate hydrolase genes by homology. The second objective is to determine the cellular and developmental expression of the IAA-conjugate hydrolase genes and determine the cellular localization of the corresponding proteins. Gene expression will be monitored by RNA blot analysis, analysis of promoter-reporter gene constructs, and in situ hybridization. Protein localization will be determined by introducing functional epitope-tagged versions of the conjugate hydrolases into the corresponding mutant plants. Cellular localization will be analyzed using commercially available antibodies against the epitope. The third objective is manipulate IAA regulation in plants by inactivating and -overexpressing IAA-conjugate hydrolase genes. Phenotypic analysis of new and existing mutants and double mutants will be carried out. The genes will be overexpressed in plants, and antisense experiments will be conducted on those genes for which no conventional mutants are available.