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

该提案涉及对模式植物拟南芥中乙烯信号转导的分析，重点关注该途径的一个特定元素，即CTR1激酶。CTR1先前被确定为丝氨酸/苏氨酸蛋白激酶Raf家族的成员，在拟南芥中作为乙烯反应的负调节因子。本文提出的研究目标是表征CTR1的生物化学特征，定义CTR1氨基末端结构域的功能，了解CTR1蛋白和基因是如何被调控的，并确定参与乙烯信号转导的其他元件。具体来说，我们希望证明CTR1编码一个功能性的蛋白激酶，并通过在异种系统中表达后纯化该蛋白来表征其体外活性。该酶的特性将与其他蛋白激酶进行比较，并特别关注Raf家族的其他成员。将确定CTR1激酶的活性是否通过使用P32正磷酸盐进行体内放射性标记，然后使用抗CTR1抗体进行免疫沉淀的差异磷酸化来调节。在植物中，CTR1的不同结构域被过表达，以确定氨基末端结构域的功能，并研究CTR1不适当的表达水平和空间表达对植物发育的影响。有初步证据表明，CTR1的翻译被乙烯增强，这将被进一步证实和研究。最后，利用附加的方法寻找参与乙烯信号转导的新元素；遗传筛选和酵母双杂交系统。这些研究将增加对乙烯信号转导的理解，并可能为其他环境信号转导途径提供见解。植物发育是多种激素、环境和内源性信号相互作用的复杂过程。阐明这些信号感知和转导的分子机制，以及这些途径如何相互整合，对于理解调节植物生长和发育的机制至关重要。其中一种信号是植物的气态激素乙烯，自世纪之交以来，人们就知道乙烯对植物有重大影响。乙烯影响种子发芽、果实成熟、叶和花的脱落和衰老。乙烯还参与植物对各种环境胁迫的反应，如伤害、病原体攻击、干旱和洪水。本研究涉及模式植物拟南芥（Arabidopsis thaliana）中乙烯信号转导通路的分析，重点关注该通路的一个微粒元件，CTR1激酶。本文提出的研究目标是对CTR1激酶进行生化表征，确定CRT蛋白各部分的功能，了解CTR1蛋白和基因是如何被调控的，并确定参与乙烯信号转导的其他元素。这些研究的结果将揭示植物感知和响应各种环境刺激的机制。它们还将为未来生物技术应用于干预花朵衰老和果实成熟奠定基础。

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.