Molecular and Genetic Analysis of Cytokinin Signaling in Abrabidopsis

细胞分裂素是一组植物激素，几乎涉及生长和发育的各个方面。 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，将采用两种不同的战略。首先，将使用简单的幼苗反应筛选细胞分裂素信号传导受损的突变体。第二种方法将集中在IBC 6及其同系物在细胞分裂素信号传导中的作用。IBC 6与细菌反应调节因子相似，是一个拟南芥细胞分裂素初级反应基因，已通过差异显示鉴定。 一种新的遗传筛选已被开发，是基于乙烯介导的三重响应形态，黄化拟南芥幼苗通过细胞分裂素的存在下。先前的工作表明，在细胞分裂素的存在下，拟南芥幼苗中乙烯生物合成的升高是由于ACSS的转录后修饰，ACSS是ACC合成酶基因家族的一个成员，其编码乙烯生物合成的限速酶。初步研究表明，这种筛选分离细胞分裂素不敏感突变体的可行性。 将分离出在细胞分裂素存在下不能采用三重反应的其他突变体，并鉴定出在细胞分裂素诱导乙烯方面有缺陷的突变体。 将使用经典的细胞分裂素反应测定法（体外芽起始、叶衰老的延迟、花青素苷积累的诱导）测定突变体的细胞分裂素反应性，并将测试细胞分裂素调节的基因表达，以将在细胞分裂素信号传导至ACS 5的早期步骤中被破坏的突变体与更普遍地影响ACS 5调节的突变体区分开。 这些突变体的表型将进行评估方面的整体形态学变化，并对应于那些在细胞分裂素信号传导的早期步骤中明显受到影响的基因将被克隆。 这些突变体应确定细胞分裂素信号所需的元素，并可能有助于揭示内源性细胞分裂素的作用。 第二，密切相关的项目，集中在一些细胞分裂素调控的基因，确定使用差异显示。 这些基因将是非常有用的细胞分裂素不敏感的突变分析上述，也可能提供深入了解细胞分裂素的作用机制。 特别是，这些基因之一，IBC 6，是快速和特异性诱导的细胞分裂素和细菌反应调节器显示相似性。这一点，加上以前的工作牵连CKI 1组氨酸激酶在细胞分裂素信号转导，表明IBC 6可能参与细胞分裂素信号转导。 IBC 6是拟南芥中小基因家族的一部分;将评估这些其他基因家族成员对细胞分裂素的反应。 植物中IBC 6的表达将以多种方式被破坏，以确定该基因在细胞分裂素信号传导中的作用。 这些研究应该有助于开始解开细胞分裂素是如何被感知的，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.