Light provides energy for photosynthesis and also signals for plants to grow and develop. Arabidopsis contains several photoreceptors that monitor 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 mutants carrying multiple mutations with lesions in various combinations of phyA signaling factors 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 negative regulator of phYA signaling, 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. Another COP1 target is the bZIP factor, HY5, which appears to mediate opposing effects on cell expansion in hypocotyls and cotyledons in response to FR light. We will investigate how HY5 differentially controls expression of the Membrane Steroid Binding Protein (MSBP) 1 in these two organs, which is known to be a negative regulator of cell expansion, Transgenic plants expressing the appropriate constructs will be used to determine whether regulation is at the transcriptional, post-transcriptional or post-translational level. 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 and also provide additional information on general mechanisms of eukaryotic cell signaling.
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