Light provides energy for phototsynthesis and also signals for plants to grow and develop. Arabidopsis contains several photoreceptors that measure photons at different wavelengths. Amongst these, phytochrome A (phyA), which is activated by far-red (FR) light, plays a major role in seedling deetiolation. Our long-term objective is to understand the inter-relationship between the various signaling intermediates and how their interactions lead to transduction of phyA signals for downstream developmental responses. To this end, we will analyze double and triple mutants with lesions in phyA signaling factor to establish the hierarchical relationship between the two homologous proteins, FHY1/FHL, and three transcription activators, LAF1, HFR1 and HY5. Interaction domains will be mapped by in vitro pull-down assays. Factor association in vivo in response to darkness and FR will be determined by co-immunoprecipitation and the physiological impact on phyA responses will be assayed during seedling deetiolation . Control of phy A signaling factor abundances has emerged to be an essential regulatory mechanism and COP1, a represser of photomorphogenesis, plays a major role. COP1 shows E3 ligase activity toward LAF1 and HFR1, which interact with one another in vitro. Using transgenic plants expressing both factors we will determine whether their association in vivo would lead to FR-hypersensitivity. In contrast to other signaling factors, COP1 is quite stable in darkness and FR probably due to rapid deubiquitination. We will use in vitro and in vivo assays to characterize dequiquitination enzyme that removes ubiquitin from COP1 and also Rad23 that appears to shuttle ubiquitinated COP1 to proteasomes for destruction. Finally, we will explore whether miRNA and siRNAs are involved in the regulation of phyA signaling. We expect results from these experiments to uncover previously unknown aspects of phyA signaling. Alterations in cell signaling pathways and misregulation in protein degradation are major causes of human diseases and the human homolog of Arabidopsis COP1 has been implicated in cancer. The work on phyA signaling network and the mechanism by which signaling factor abundances are controlled by COP1 will advance our understanding on how disease processes are regulated.
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