Molecular and Genetic Analysis of Cytokinin Signaling in Abrabidopsis

细胞分裂素是一组植物激素，几乎涉及生长发育的方方面面。这项研究的主要目标是开始解开细胞分裂素感知的分子基础。为此，将采取两种不同的策略。首先，细胞分裂素信号受损的突变体将被筛选为使用简单的幼苗反应。第二种方法将重点放在IBC6及其同系物在细胞分裂素信号中所起的作用。IBC6与细菌反应调节因子类似，是一个拟南芥细胞分裂素初级反应基因，已通过差异显示技术鉴定出来。一种新的基于乙烯介导的三重反应形态的遗传筛选已经被开发出来，该形态是在细胞分裂素存在的情况下黄化的拟南芥幼苗所采用的。以前的工作表明，在细胞分裂素存在下，拟南芥幼苗乙烯生物合成的增加是由于编码乙烯生物合成限速酶的ACC合成酶家族成员ACSS的转录后修饰。初步研究表明，这种筛选方法用于分离细胞分裂素不敏感突变株是可行的。将分离出在细胞分裂素存在下未能采取三重反应的其他突变体，以及那些在细胞分裂素诱导乙烯方面存在缺陷的突变体。这些突变体将使用经典的细胞分裂素反应分析(体外萌芽、延缓叶片衰老、诱导花青素积累)来检测细胞分裂素的反应性，并将测试细胞分裂素调控的基因表达，以区分在细胞分裂素信号向ACS5传递信号的早期步骤中被破坏的突变体与那些更广泛地影响ACS5调控的突变体。这些突变体的表型将根据整体形态变化进行评估，并将克隆与那些在细胞分裂素信号传递早期明显受影响的基因相对应的基因。这些突变体应该识别细胞分裂素信号所需的元件，并可能有助于揭示内源性细胞分裂素的作用。第二个密切相关的项目，重点是一些细胞分裂素调节的基因，这些基因是用差异显示技术鉴定出来的。这些基因在上述细胞分裂素不敏感突变的分析中将非常有用，也可能为细胞分裂素的作用机制提供洞察力。特别是，其中一个基因IBC6能被细胞分裂素快速而特异地诱导，并显示出与细菌反应调节因子的相似性。这一点，再加上先前关于CKI-1组氨酸蛋白激酶参与细胞分裂素信号转导的研究，提示IBC6可能参与了细胞分裂素信号转导。IBC6是拟南芥中一个小基因家族的一部分；这些基因家族的其他成员对细胞分裂素的反应将被评估。IBC6在植物中的表达将以多种方式被干扰，以确定该基因在细胞分裂素信号转导中的作用。这些研究应该有助于开始解开细胞分裂素是如何被理解的，以及它是如何影响植物发育的。

Cytokinins are a group of plant hormones that have been implicated in almost every aspect of growth and development. The major goal of this research is to begin to unravel the molecular basis for cytokinin perception. To this end, two different strategies will be employed. Firstly, mutants impaired in cytokinin signaling will be screened for using a simple seedling response. The second approach will focus on the role that IBC6 and its homologs play in cytokinin signaling. IBC6, which is similar to bacterial response regulators, is an Arabidopsis cytokinin primary response gene that has been identified using differential display. A novel genetic screen has been developed that is based on the ethylene-mediated triple response morphology that etiolated Arabidopsis seedlings adopt in the presence of cytokinin. Previous work revealed that the elevation of ethylene biosynthesis in Arabidopsis seedlings in the presence of cytokinin is due to a post-transcriptional modification of ACSS, one member of the ACC synthase gene family, which encodes the rate-limiting enzyme of ethylene biosynthesis. Preliminary studies have demonstrated the feasibility of this screen for isolating cytokinin-insensitive mutants. Additional mutants will be isolated that fail to adopt the triple response in the presence of cytokinin, and those that are defective in the induction of ethylene by cytokinin will be identified. The mutants will be assayed for cytokinin responsiveness using classic cytokinin response assays (shoot initiation in vitro, retardation of leaf senescence, induction of anthocyanin accumulation), and will be tested for cytokinin-regulated gene expression in order to distinguish mutants disrupted in early steps of cytokinin signaling to ACS5 from those affecting ACS5 regulation more generally. The phenotypes of these mutants will be assessed with regard to overall morphological changes, and the genes corresponding to those that are clearly affected in early steps in cytokinin signaling will be cloned. These mutants should identify elements required for cytokinin signaling and may help to reveal the role of endogenous cytokinins. A second, closely-related project, focuses on a number of cytokinin-regulated genes that were identified using differential display. These genes will be extremely useful in the analysis of the cytokinin-insensitive mutations described above, and may also provide insights into the mechanism of cytokinin action. In particular, one of these genes, IBC6, is rapidly and specifically induced by cytokinin and shows similarity to bacterial response regulators. This, coupled with previous work implicating the CKI 1 histidine kinase in cytokinin signaling, suggests that IBC6 may be involved in cytokinin signal transduction. IBC6 is part of a small gene family in Arabidopsis; the response of these other gene family members to cytokinin will be assessed. Expression of IBC6 in planta will be disrupted in a variety of ways to determine the role of this gene in cytokinin signaling. These studies should help to begin to unravel how cytokinin is perceived and how it influences plant development.