Molecular mechanisms of signal transfer from phytochromes tobasic helix-loop-helix transcription factor PIF3

Definition of the molecular mechanism by which the phytochrome (phy) family of photoreceptors (phyA-E) perceive and transduce informational light signals to downstream cellular components has been sought for many years. This dissertation provides evidence that: (1) The phy interacting basic helix-loop-helix transcription factor PIF3 is rapidly degraded by photoactivated phys A and B, with a minor contribution from phyD, via the ubiquitin proteasome system (UPS). This degradation of PIF3 appears to be independent of the Skip-Cullin-F box (SCF) E3 ligase branch of the UPS. (2) phys A, B and D dynamically control PIF3 protein levels, making PIF3 available when active phy is removed in natural night or vegetative-shade conditions. This dynamic control of PIF3 protein levels provides a cellular mechanism for rapid phy-regulation of target genes by controlling the abundance of transcriptional regulators, such as PIF3, in light. (3) The biochemical mechanism of signal transfer from photoactivated phy to PIF3 comprises a rapid light-induced phosphorylation event in PIF3 which flags the transcription factor for subsequent degradation. (4) Phosphorylation, light-induced intranuclear PIF3 re-distribution (speckling) and degradation of PIF3 all require direct association in vivo with photoactivated phyA or phyB. Early light-induced speckles may the site of PIF3 processing by the UPS or a site where PIF3 phosphorylation, ubiquitination and degradation all occur. (5) Early light induction of a phy and PIF3 dependent maker gene, EARLY LIGHT INDUCED PROTEIN (ELIP), requires DNA-bound PIF3, but not modification of PIF3 by photoactivated phys, providing evidence that PIF3 acts in light signaling as a constitutive transcription factor and is not sufficient to mediate the light signal to target genes. These data provide indirect evidence for the existence of a second factor required to mediate light-induction of the ELIP gene. The kinetics of light-induced ELIP gene expression displays a transient profile, which is consistent with rapid phy-induced PIF3 degradation. The expression of this gene is sustained when PIF3 is artificially stabilized, providing evidence that PIF3 degradation by phys might serve as a timing gate for PIF3 activity. (6) Evidence from analysis of functional determinants, within the PIF3 molecule, necessary for rapid light-induced gene expression at the dark- to light transition, versus PIF3 action on seedling morphogenesis in long-term, continuous-irradiation conditions, shows that the determinants necessary for PIF3 early and late action function in a temporally separable fashion. In a broader sense, this provides evidence for a bifurcation in the phy-mediated light signaling to PIF3.