REGULATION OF THE PLANT GROWTH HORMONE INDOLEACETIC ACID

在高等植物中，约95%的激素吲哚-3-乙酸（IAA， 生长素）以无活性的共轭形式存在。IAA的释放 这些缀合物可能对控制细胞内的 IAA的水平，从而其行动。在博士后工作中，PI设计了一个 新的筛选拟南芥突变体， IAA-氨基酸缀合物对游离IAA保持敏感。之一 克隆了在该筛选中鉴定的基因，并显示其编码水解酶 对特定的IAA-氨基酸缀合物具有特异性。克隆的基因具有 拟南芥中可能编码其他IAA-氨基酸的几种同源物 水解酶 PI提出了三个主要目标。首先是确定新的IAA- 共轭水解酶。这将通过分离被破坏的基因 在其它IAA缀合物不敏感突变体中，并通过分离新的IAA- 通过同源性缀合水解酶基因。 第二个目标是确定细胞和发育 IAA-缀合物水解酶基因的表达，并确定细胞的 定位相应的蛋白质。基因表达将是 通过RNA印迹分析、启动子-报告基因分析 构建体和原位杂交。蛋白质定位将是 通过引入功能性表位标记形式的 将水解酶缀合到相应的突变体植物中。蜂窝 将使用市售抗体分析定位 针对表位。 第三个目标是通过灭活植物中的IAA来操纵植物中的IAA调节。 和-过表达IAA-缀合物水解酶基因。的表型分析 将进行新的和现有的突变体和双重突变体。的基因 将在植物中过度表达，反义实验将在 在没有常规突变体可用的那些基因上进行。

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.