title: Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix-loop-helix factor PIF3 in a reaction requiring the PAS domain of PIF3.
abstract: The signaling pathways by which the phytochrome (phy) family of photoreceptors transmits sensory information to light-regulated genes remain to be fully defined. Evidence for a relatively direct pathway has been provided by the binding of one member of the family, phyB, to a promoter-element-bound, basic helix-loop-helix protein, PIF3, specifically upon light-induced conversion of the photoreceptor molecule to its biologically active conformer (Pfr). Here, we show that phyA also binds selectively and reversibly to PIF3 upon photoconversion to Pfr, but that the apparent affinity of PIF3 for phyA is 10-fold lower than for phyB. This result is consistent with previous in vivo data from PIF3-deficient Arabidopsis, indicating that PIF3 has a major role in phyB signaling, but a more minor role in phyA signaling. We also show that phyB binds stoichiometrically to PIF3 at an equimolar ratio, suggesting that the resultant complex is the unit active in transcriptional regulation at target promoters. Deletion mapping suggests that a 37-aa segment present at the N terminus of phyB, but absent from phyA, contributes strongly to the high binding affinity of phyB for PIF3. Conversely, deletion mapping and point mutation analysis of PIF3 for determinants involved in recognition of phyB indicates that the PAS domain of PIF3 is a major contributor to this interaction, but that a second determinant in the C-terminal domain is also necessary.
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhu
authors.forename: J M
authors.initials: JM
authors.lastname: Tepperman
authors.forename: C D
authors.initials: CD
authors.lastname: Fairchild
authors.forename: P H
authors.initials: PH
authors.lastname: Quail
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002467:Cell Nucleus / Q000502:physiology; D018257:Helix-Loop-Helix Motifs; D007700:Kinetics; D016296:Mutagenesis; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010786:Photoreceptor Cells; D010834:Phytochrome / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051942:Phytochrome A; D051939:Phytochrome B; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D014157:Transcription Factors
chemical_list: D029681:Arabidopsis Proteins; D051792:Basic Helix-Loop-Helix Proteins; D010455:Peptides; D010834:Phytochrome; D051942:Phytochrome A; D051939:Phytochrome B; D011994:Recombinant Proteins; D014157:Transcription Factors; C497973:PHYA protein, Arabidopsis; C497952:PHYB protein, Arabidopsis; C116162:PIF3 protein, Arabidopsis