The ACC Synthase Genes and Polypeptides

该提案的具体目的是：1)分离和结构表征番茄ACC合酶基因的其余部分。描述番茄生长发育过程中每个基因的表达特征，并确定诱导这些基因的诱导剂类型。研究拟南芥ACS4和ACS5基因在吲哚乙酸（IAA）和Li+存在下的表达特性。确定拟南芥ACS4基因IAA诱导的顺作用元件和ACS5基因的Li+响应元件。这些元件将通过启动子区域的ExoIII缺失分析来定义。用CAT报告基因构建的启动子将在拟南芥原生质体中进行瞬时测定。这些元素的功能将通过稳定变换实验来验证。利用反义OS-ACs1 RNA抑制水稻在缺氧条件下的生长，确定低氧胁迫下表达水稻OS-ACs1和3两个基因的细胞类型。通过体外诱变和大肠杆菌表达实验确定ACC合酶活性所需的关键残基。在大肠杆菌中进行分子间互补实验，以确定ACC合酶是否为具有两个活性位点的二聚体。通过特异同工酶显性负突变的过表达抑制乙烯依赖的发育过程。对拟南芥ACC合酶同工酶进行生物化学表征，以确定其生物化学多样性。乙烯是一种独特的植物信号分子。碳氢化合物气体，被称为果实成熟激素，控制着植物生长发育的许多方面，其合成受到高度调节。最近的实验证据表明，在不同的单子叶和双子叶植物中，乙烯生物合成途径的关键酶-1-氨基环丙烷-1-羧酸（ACC）合成酶是由一个高度分化的多基因家族编码的，每个家族的成员受不同的诱导剂和发育阶段的差异调控。本文拟对番茄（Lycopersicon esculentum）、水稻（Oryza sativa）和拟南芥（Arabidopsis thaliana）中ACC合酶基因的结构和调控进行研究，以深入了解其在各种激素和胁迫条件下表达调控的生化机制及其在植物生长中的作用。此外，还提出了体外诱变研究，以获得有关ACC合酶蛋白结构和功能的基本知识。＊＊＊

9316475 Theologis The specific aims of the proposal are : 1) To isolate and structurally characterize the rest of the tomato ACC synthase genes. To characterize the expression characteristics of each tomato gene during growth and development and to define the type of inducer(s) responsible for their induction. To characterize the expression characteristics of the Arabidopsis ACS4 and ACS5 genes in the presence of indoleacetic acid (IAA) and Li+, respectively. To define the cisacting elements for IAA inducibility of the Arabidopsis ACS4 and Li+ responsive elements of the ACS5 gene. The elements will be defined by ExoIII deletion analysis of the promoter regions. Promoter constructs with the CAT reporter gene will be transiently assayed in Arabidopsis protoplasts. The functionally of the elements will be verified by stable transformation experiments. To inhibit growth of rice under hypoxia using antisense OS-ACs1 RNA and to define the cell types that express two rice genes OS-ACS1 and 3 under low O2 tension. To define the critical residues required for enzymatic activity of ACC synthase by in vitro mutagenesis and expression experiments in E. coli. To perform intermolecular complementation experiments in E. coli for determining whether ACC synthase is a dimer with two active sites. To inhibit ethylene dependent developmental processes by overexpression of dominant negative mutations of specific isoenzymes. To biochemically characterize the Arabidopsis ACC synthase isoenzymesfor defining their biochemical diversity. %%% Ethylene is an unique plant signaling molecule. The hydrocarbon gas, known as the fruit ripening hormone, controls many aspects of plant growth and development, and its synthesis is highly regulated. Recent experimental evidence indicates that the key enzyme in the ethylene biosynthetic pathway, 1-aminocyclopropane-1- carboxylic acid (ACC) synthase is encoded by a highly divergent multigene family in various mono-and dicotyledonous plant s. The members of each family are differentially regulated by various inducers and developmental stages. Studies on the structure and regulation of the ACC synthase genes in tomato (Lycopersicon esculentum), rice (Oryza sativa) and Arabidopsis thaliana are proposed here to gain insight into the details of the biochemical mechanisms that regulate their expression by various hormones and stress conditions as well as their role in regulating plant growth. Furthermore, in vitro mutagenesis studies are proposed to obtain fundamental knowledge about the structure and function of the ACC synthase protein. ***