| The identification of phytochrome signaling intermediates, and the elucidation of molecular events involving the phyB photoreceptor in response to light are critical to our understanding of the mechanism of phytochrome action in Arabidopsis. To this end, I took a two-pronged approach in this study. In the first part of this dissertation, I describe the role of a novel gene, EARLY FLOWERING 4 (ELF4) in phytochrome signal transduction, and in light input to the circadian clock. In the second part of this dissertation I describe the identification of phyB missense mutations affecting binding to the bHLH transcription factor, PHYTOCHROME INTERACTING FACTOR 3 (PIF3). The data show that ELF4 is one of a five-member gene family in Arabidopsis and is required for phytochrome B-mediated de-etiolation of Arabidopsis seedlings. To elucidate the role of ELF4 in circadian clock function, I developed methods to probe the initiation of circadian oscillations in response to light. I show that the central circadian oscillator genes, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB (TOC1), are expressed at constant, basal levels in dark grown seedlings. Transfer to constant red light rapidly induces a biphasic pattern of CCA1 and LHY expression, and a reciprocal TOC1 expression pattern, consistent with initial induction of this synchronous oscillation of clock components by the light signal. I show that ELF4 is necessary for light-induced oscillations of both CCA1 and LHY, and conversely, CCA1 and LHY act negatively on light-induced ELF4 expression. Together with the observation that the temporal light-induced expression profile of ELF4 is counter-phased to that of CCA1 and LHY and parallels that of TOC1, these data are consistent with a previously unrecognized negative feedback loop in the clock formed by CCA1/LHY and ELF4, functioning in a manner analogous to the proposed CCA1/LHY/TOC1 oscillator. With respect to phyB, I developed a yeast reverse-hybrid screen that allowed the identification of twelve phyB missense mutations that are disrupted in binding to PIF3 and other phyB-binding bHLHs, but are unaffected in their ability to undergo Pr-Pfr photoconversion. Ultimately, using a bioinformatics comparison of Arabidopsis phyB to that of D. radiodurans for which a crystal structure has been solved, five of these mutated phyB amino acid residues were predicted to be solvent exposed in a cleft between the PAS and GAF domains. These residues could be directly required for the physical interaction of phyB with bHLH transcription factors. |
