EAGER: Development of Fluorescence-based Nanosensors for Dynamic Measurement of Active Cytokinins in Plants

PI: G. Eric Schaller （Dartmouth学院）CoPIs: Joseph J. Kieber（北卡罗来纳大学教堂山分校）和Sakiko Okumoto（弗吉尼亚理工学院和州立大学）细胞分裂素是植物调节多种生长和发育过程的激素，如细胞分裂、代谢、叶绿体发育和维持、衰老，以及对生物和非生物因素的反应。尽管细胞分裂素在植物中起着至关重要的作用，但目前还没有一种方法可以准确地确定细胞分裂素水平的细胞和亚细胞动力学。这个EAGER项目寻求开发基于荧光的纳米传感器，能够测量单子叶植物（水稻）和双子叶植物（拟南芥）的细胞分裂素水平。将开发和测试基于FRET和单荧光蛋白的纳米传感器，使用拟南芥细胞分裂素受体AHK4的细胞分裂素结合CHASE结构域和水稻细胞分裂素受体OsHK4的相应结构域作为配体结合结构域。使用放射性细胞分裂素结合试验对CHASE结构域结合细胞分裂素的要求进行评估，以促进纳米传感器的设计。纳米传感器将首先在体外进行测试，利用细菌表达的融合蛋白，随后在植物中进行测试，最初使用瞬时原生质体转化系统，然后通过产生稳定的转基因品系。将测试对外源性细胞分裂素和内源性细胞分裂素水平改变的反应，以确认纳米传感器报告体内细胞分裂素水平的能力。最后，利用不同的靶向序列，纳米传感器将靶向细胞质溶胶和内质网，以探索细胞分裂素水平在细胞内的变化以及细胞分裂素响应各种输入的运动动力学。这对细胞分裂素特别重要，因为它的受体定位于内质网膜，而感知部位在内质网腔内。如果成功，该项目将允许在细胞和亚细胞水平上定量生物活性细胞分裂素，并在活细胞中进行动态测量。细胞分裂素在调节许多具有农业意义的植物特性中起着关键作用。例如，细胞分裂素在调节水稻籽粒产量方面起着关键作用。纳米传感器技术的发展将促进和推进改善这些细胞分裂素调节特性的研究。用于纳米传感器的载体和转基因拟南芥和水稻品系将根据要求和/或通过包括拟南芥生物资源中心在内的适当库存中心提供给研究界。该项目将通过为本科生、研究生以及博士后研究助理提供跨学科的实践培训，加强研究和教育的基础设施。

PI: G. Eric Schaller (Dartmouth College) CoPIs: Joseph J. Kieber (University of North Carolina-Chapel Hill) and Sakiko Okumoto (Virginia Polytechnic Institute and State University) Cytokinins are plant hormones that regulate diverse growth and development processes, such as cell division, metabolism, chloroplast development and maintenance, and senescence, as well as responses to biotic and abiotic factors. In spite of their critical role in plants, no method has yet been developed to accurately determine the cellular and subcellular dynamics of cytokinin levels. This EAGER project seeks to develop fluorescence-based nanosensors capable of measuring cytokinin levels in a monocot (rice) and a dicot (Arabidopsis). FRET- and single fluorescent protein-based nanosensors will be developed and tested using the cytokinin-binding CHASE domain from the Arabidopsis cytokinin receptor AHK4 and the corresponding domain of the rice OsHK4 receptor as ligand-binding domains. The requirements for cytokinin binding by the CHASE domain using a radioactive cytokinin-binding assay will be assessed to facilitate nanosensor design. Nanosensors will initially be tested in vitro, making use of bacterially expressed fusion proteins and subsequently be tested in planta, initially using a transient protoplast transformation system and then by generation of stable transgenic lines. The response to exogenous cytokinin and to altered levels of endogenous cytokinin will be tested to confirm the ability of the nanosensors to report cytokinin levels in vivo. Finally, using different targeting sequences, nanosensors will be targeted to the cytosol and the endoplasmic reticulum to explore how cytokinin levels vary within the cell and the dynamics of cytokinin movement in response to various inputs. This is of particularly importance for cytokinin because its receptors are localized to the ER membrane, and the site of perception is within the ER lumen. If successful, this project will allow for the quantification of bioactive cytokinins at the cellular and subcellular levels and for dynamic measurements in living cells. Cytokinins play key roles in regulating many plant properties of agricultural significance. For example, cytokinins play a critical role in regulating grain yield in rice. Development of nanosensor technology will facilitate and advance studies to improve these cytokinin-regulated traits. Vectors and transgenic Arabidopsis and rice lines for the nanosensors will be made available to the research community on request and/or through appropriate stock centers including the Arabidopsis Biological Resource Center. The project will enhance the infrastructure of research and education by providing hands-on interdisciplinary training for undergraduate and graduate students as well as postdoctoral research associates.