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.
