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

The signaling pathways by which the phytochrome (phy) family of photorecept ors transmits sensory information to light-regulated genes remain to be ful ly defined. Evidence for a relatively direct pathway has been provided by t he binding of one member of the family, phyB, to a promoter-element-bound. basic helix-loop-helix protein, PIF3, specifically upon light-induced conve rsion of the photoreceptor molecule to its biologically active conformer (P fr). Here, we show that phyA also binds selectively and reversibly to PIF3 upon photoconversion to Pfr, but that the apparent affinity of PIF3 for phy A is 10-fold lower than for phyB, This result is consistent with previous i n vivo data from PIF3-deficient Arabidopsis, indicating that PIF3 has a maj or 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. sug gesting that the resultant complex is the unit active in transcriptional re gulation at target promoters. Deletion mapping suggests that a 37-aa segmen t present at the N terminus of phyB, but absent from phyA, contributes stro ngly to the high binding affinity of phyB for PIF3, Conversely, deletion ma pping and point mutation analysis of PIF3 for determinants involved in reco gnition of phyB indicates that the PAS domain of PIF3 is a major contributo r to this interaction, but that a second determinant in the C-terminal doma in is also necessary.