Growth and development are highly regulated by environmental light signals at all phases of a plant's life cycle. Plants have evolved several light receptors including the phytochrome (phy) family of photoreceptors to monitor the red and far-red light, the cryptochromes, the phototropins and the ZTL/FKF1 family of F-box proteins to monitor the UV-A and blue light, and an unidentified receptor to monitor the UV-B light. Although much has been learned on these photoreceptors, the primary biochemical mechanism by which they regulate plant growth and development is still unknown. In recent years, degradation of transcription factors both in the dark and light has been shown to play central roles in the light signaling pathways. In general, one class of light signaling factors are degraded in the dark. They include HY5, LAF1 and HFR1, most of which are positive regulators of photomorphogenesis. The second class factors are degraded in light. This class includes Phytochrome Interacting Factors(PIFs), most of which negatively regulate photomorphogenesis. Although, the mechanisms of dark-induced degradation are well investigated, the light-induced degradation of PIFs is less well understood. This project is focused on investigating the early events in the light-induced degradation of the bHLH transcription factor, PIF1, using biochemical, molecular and genetic approaches. PIF1 is rapidly degraded under both red and far-red light conditions. Light-induced proteolytic removal of PIF1 relieves the negative regulation by PIF1 and promote seed germination, chlorophyll biosynthesis and hypocotyl growth inhibition. Since PIF1 is a primary phytochrome-signaling partner, whose stability is regulated by phytochromes through direct physical interaction, the results from this project will provide fundamental information on the mechanism of phytochrome regulated developmental pathways. This study will have broad implications in several ways. First, understanding the biochemical mechanism of regulation of PIF1 in plants will provide fundamental knowledge and molecular tools applicable in agricultural biotechnology for enhanced crop and biomass production. Second, the results and the research tools will be distributed to the community through publications, conference presentations, and website postings directly from our laboratories. Third, this project will contribute to training the next generation scientists in modern functional genomics and molecular genetics, including undergraduate, graduate and postdoctoral students. This training will provide them with the unique opportunity to make significant contributions to the field of plant biology and biotechnology and will prepare them for future careers as independent researchers.
Intellectual Merit Light is a critical factor regulating plant growth and development. For example, red (650 nm) and far-red (730 nm) light regulate important processes like seed germination, greening, flowering time and overall plant growth. The phytochrome family of photoreceptors monitors the red (R)/far-red (FR) light and initiates these responses. This project helped to elucidate poorly understood biochemical mechanisms of phytochrome action. The discovery of Phytochrome Interacting Factors (PIFs) heralded a new era not only in phytochrome signaling, but also in many other signaling pathways in plants. There were many oral presentations showing research on PIFs at this years ASPB conference held in Austin, TX. Our results and those of others showed that PIFs and phytochromes have antagonistic activities. All PIFs act as repressors of photomorphogenesis in the dark and light-activated phytochromes induce rapid degradation of PIFs under all three (red/far-red/blue) light conditions to promote photomorphogenesis. In support of this view, we demonstrated that pifQ (pif1pif3pif4pif5) mutants display phenotypes in the dark that are similar to light grown plants. These data suggest that multiple PIFs are acting additively to repress photomorphogenesis in the dark. We have also identified CK2 as a kinase that phosphorylates PIF1, and this phosphorylation enhances the instability of PIF1 under light. However, PIF1 was still robustly phosphorylated under light, suggesting that there are additional kinases that phosphorylate PIF1 in response to light and promote PIF1 degradation. These data suggest that multiple kinases act in concert to phosphorylate PIF1; one functions in the dark and the other functions only in response to light. Both phosphorylations are necessary for robust light-induced degradation of PIF1. We have also isolated a number of altered PIF1 proteins that are defective in light-induced degradation from genetic screen. Cloning and characterization of these mutants are in progress. Successful demonstration of these data will answer two of the most important questions in our field, namely what is the kinase and the E3 ligase responsible for the light-induced phosphorylation and ubiquitylation of PIF1. This grant not only helped to contribute within our discipline, but also to other disciplines in science. We have generated and distributed research materials to other labs that will help understand other biological processes. Support from this grant helped to publish 8 research articles in peer reviewed journals, 2 review articles and one book chapter. Broader Impacts This grant helped in teaching and training of a number of undergraduate, graduate and postdoctoral fellows. Seven undergraduate students went to medical schools and three to graduate schools. Other undergrads are still at UT or graduated. Seven Undergraduate students independently wrote and received Undergraduate research grant at UT. They worked on their projects independently and hope to publish their results. One postdoc and five graduate students were supported by this grant in various ways. Three Ph.D. students received their Ph.D. during the tenure of this grant. Graduate students and postdocs gained writing skills, research skills and mentoring skills. Extensive training of these students and postdocs will prepare them for their future career in Plant Biology. In addition, I have incorporated primary results from our lab in my classes for teaching graduate and undergraduate students (BIO388E: Plant Growth and Development, BIO344: Molecular Biology and BIO205L: Laboratory Techniques in Cell and Molecular Biology). Overall, this grant contributed to create resources that supported both research and education at UT. Although, we did not commercialize technology yet, we believe training undergraduate, graduate students and postdocs go far beyond their academic preparation. These students are receiving first hand experience in producing transgenic plants. They will increase awareness of their parents, family members and friends about the transgenic technology and help reduce the confusion about this technology many people still have. Moreover, we helped high school and middle school students to do their science fair projects in our lab. We hosted 4 high school students and one middle school student for their science fair projects during the tenure of this grant. Two of them secured first and second places at their school fair and went to regional competition. We have also hosted middle school students for field trips. I gave lectures to undergraduate research organizations at UT to motivate them to participate in research. I also participated at the annual parents day at UT to motivate and encourage young students to come to UT. I have been serving as a judge for poster session for undergraduate students sponsored by the college of natural sciences at UT. This is a fun filled day complementing undergraduates for their accomplishments and also to motivate them to go into research. I have also served as a panelist for the ASPB Women in Plant Biology workshop entitled: "Getting the most of the graduate school". ASPB Conference 2010, Montreal, Canada. All these activities supported by this grant are expected to have broad impacts on our society.
