Molecular and Genetic Analysis of Cytokinin Signaling in Abrabidopsis

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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.