Regulation of Arabidopsis HFR1 Function by Reversible Phosphorylation

植物利用一系列光感受器来感知周围的光环境，并启动一系列信号转导事件，导致基因表达改变和适应性生长发育。拟南芥HFR1编码bHLH转录因子，在远红光和蓝光条件下促进幼苗光形态发生。此外，在冠层遮荫条件下，HFR1在平衡避荫响应中起着关键作用。已有研究证实，HFR1蛋白在黑暗中被COP1-SPA1 E3泛素连接酶复合物通过泛素- 26s蛋白酶体途径靶向降解，并在光照条件下稳定，促进光信号传导。然而，光调节HFR1稳定性和功能的分子机制仍未完全阐明。本项目的目的是测试酪蛋白激酶II （CKII）和AtFyPP1 （pp6型丝氨酸/苏氨酸蛋白磷酸酶）可逆磷酸化HFR1可能作为控制HFR1稳定性和功能的分子开关的工作模型。该项目的第一个目标是利用诱变和转基因方法确定HFR1磷酸化的体内生理意义。HFR1磷酸化对其稳定性、亚细胞定位、核体形成、蛋白与COP1和SPA1的相互作用以及转录抑制活性的潜在影响将被研究。第二个目的是测试CKII和AtFyPP1分别作为HFR1磷酸化和去磷酸化的激酶和磷酸酶的模型。CKII和AtFyPP1在调节幼苗光形态发生、避荫反应和开花时间中的功能意义将通过生化和遗传方法进行研究。这些研究有望对调控HFR1稳定性和功能的分子机制提供重要的见解。这项工作的广泛影响有几个方面：首先，从拟议的研究中获得的知识将为作物植物的光信号调控提供有价值的见解，并增强在高种植密度下开发更好的转基因作物以提高产量的能力。其次，这项工作可能为研究可逆磷酸化在调节细胞信号传导中的作用提供一个新的范例。第三，这项工作将加深和拓宽我们对植物中激酶和磷酸酶的功能和调控的理解。此外，该项目将为从博士后到本科生的下一代植物科学家提供良好的培训机会。更广泛地说，PI将担任院校教育外展委员会成员，并将参加每年为高中教师举办的“教师培训和课程发展研讨会”。此外，PI还将在当地小学课堂上介绍由印第安纳大学教授罗杰·p·汉加特（Roger P. Hangarter）制作的“动态植物”电影，向学生们介绍植物如何对光和其他环境刺激作出反应。

Plants use an array of photoreceptors to sense the ambient light environment and initiate a cascade of signal transduction events, leading to altered gene expression and adaptive growth and development. Arabidopsis HFR1 encodes a bHLH transcription factor that acts to promote seedling photomorphogenesis under far-red and blue light conditions. In addition, HFR1 plays a critical role in balancing the shade avoidance response under canopy shade conditions. Previous studies have established that HFR1 protein is targeted for degradation in darkness by the COP1-SPA1 E3 ubiquitin ligase complex through the ubiquitin-26S proteasome pathway, and is stabilized under light conditions to promote light signaling. However, the molecular mechanisms by which light regulates HFR1 stability and functionality remain to be fully elucidated. The goal of this project is to test the working model that reversible phosphorylation of HFR1 by casein kinase II (CKII) and AtFyPP1 (a PP6-type serine/threonine protein phosphatase) may serve as the molecular switch controlling HFR1 stability and function. The first aim of the project will determine the in vivo physiological significance of HFR1 phosphorylation using mutagenesis and transgenic approaches. Potential effects of HFR1 phosphorylation on its stability, subcellular localization, nuclear body formation, protein-protein interaction with COP1 and SPA1, and transcriptional repression activity will be investigated. The second aim is designed to test the model that CKII and AtFyPP1 serve as the kinase and phosphatase responsible for phosphorylation and dephosphorylation of HFR1, respectively. The functional significance of CKII and AtFyPP1 in regulating seedling photomorphogenesis, shade avoidance response and flowering time will be investigated using biochemical and genetic approaches. These studies are expected to add significant insights into the molecular mechanism regulating HFR1 stability and function. The broader impacts of this work are several fold: First, knowledge gained from the proposed research will provide valuable insight into the regulation of light signaling in crop plants and enhance the ability to develop better transgenic crops for increased yield under high planting density. Second, this work may generate a new paradigm for studying the role of reversible phosphorylation in regulating cellular signaling in general. Third, this work will deepen and broaden our understanding of the function and regulation of kinases and phosphatases in plants. In addition, this project will provide excellent training opportunities for the next generation of plant scientists, ranging from postdoc fellows to undergraduate students. More broadly, the PI will serve on the Institutional Educational Outreach Committee, and will participate in the annual "Teacher Training and Curriculum Development Workshop" for high school teachers. Moreover, the PI will introduce the "Plants-In-Motion" movies, which were created by Professor Roger P. Hangarter at Indiana University, in local elementary school classes to teach school children about how plants respond to light and other environmental stimuli.