NASA/NSF Collaborative Research: Molecular and Biochemical Analysis of CTR1, A Protein Involved in Ethylene Signal Transduction

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9416017 Kieber The proposal involves an analysis of ethylene signal transduction in the model plant Arabidopsis thaliana, with a focus on one particular element of this pathway, the CTR1 kinase. CTR1 was previously identified as a member of the Raf family of serine/threonine protein kinases that acts as a negative regulator of ethylene responses in Arabidopsis. The goals of the studies proposed here are to characterize the CTR1 biochemically, to define the function of the amino terminal domain of CTR1, to understand how the CTR1 protein and gene are regulated, and to identify additional elements involved in ethylene signal transduction. Specifically, it is hoped to demonstrate that CTR1 encodes a functional protein kinase and to characterize the activity in vitro by purifying this protein after expression in a heterologous system. The properties of the enzyme will be compared to other protein kinases, with particular attention given to other members of the Raf family. It will be determine whether the activity of the CTR1 kinase is regulated by differential phosphorylation using in vivo radiolabeling with P32 orthophosphate followed by immunoprecipitation with anti-CTR1 antibodies. Different domains of the CTR1 will be over-expressed in planta in an attempt to determine the function of the amino terminal domain and to examine the effect that inappropriate levels and spatial expression of CTR1 has on plant development. There is preliminary evidence that CTR1 translation is enhanced by ethylene, and this will be confirmed and investigated further. Finally, novel elements involved in ethylene signal transduction will be searched for using additional; genetic screens and the yeast two-hybrid system. The studies should lead to an increased understanding of ethylene signal transduction, and may provide insights into other environmental signal transduction pathways. Plant development is a complex interplay of various hormonal, environmental and endogeno us developmental signals. The elucidation of the molecular mechanism underlying the perception and transduction of these signals, and how these pathways are integrated with each other is crucial for an understanding of the mechanisms that regulate plant growth and development. One such signal is the gaseous plant hormone ethylene, which has been known to have significant effects on plants since the turn of the century. Ethylene affects seed germination, fruit ripening, and abscission and senescence of leaves and flowers. Ethylene is also involved in the response of plants to various environmental stresses such as wounding, pathogen attack, drought and flooding.. This proposal involves the analysis of the ethylene signal transduction pathway in the model plant Arabidopsis thaliana, with a focus of a particulate element of this pathway, the CTR1 kinase. The goals of the studies proposed here are to characterize the CTR1 kinase biochemically, to define the function of the various parts of the CRT protein, to understand how the CTR1 protein and gene are regulated, and to identify additional elements involved in ethylene signal transduction The results of these studies will shed light on mechanisms by which plants sense and respond to various environmental stimuli. They will also lay the basis for future biotechnological applications to interfere with flower senescence and fruit ripening.