We used a series of in vitro-generated deletion and amino acid substitution derivatives of phytochrome B (phyB) expressed in transgenic Arabidopsis to identify regions of the molecule important for biological activity. Expression of the chromophore-bearing N-terminal domain of phyB alone resulted in a fully photoactive, monomeric molecule lacking normal regulatory activity. Expression of the C-terminal domain alone resulted in a photoinactive, dimeric molecule, also lacking normal activity. Thus, both domains are necessary, but neither is sufficient for phyB activity. Deletion of a small region on each major domain (residues 6 to 57 and 652 to 712, respectively) was shown to compromise phyB activity differentially without interfering with spectral activity or dimerization. Deletion of residues 6 to 57 caused a large increase in the fluence rate of continuous red light (Rc) required for maximal seedling responsiveness, indicating a marked decrease in efficiency of light signal perception or processing per mole of mutant phyB. In contrast,deletion of residues 652 to 712 resulted in a photoreceptor that retained saturation of seediing responsiveness to Rc at low fluence rates but at a response leve1 much below the maximal response elicited by the parent molecule. This deletion apparently reduces the maximal biological activity per mole of phyB without a major decrease in efficiency of signal perception, thus suggesting disruption of a process downstream of signal perception. In addition, certain phyB constructs caused dominant negative interference with endogenous phyA activity in continuous far-red light, suggesting that the two photoreceptors may share reaction partners. INTRODUCTIONPhytochromes are the best characterized of the photoreceptors that control many aspects of early seedling development. These molecules are dimers with a monomeric molecular mass of 4 2 5 kD. Each monomer consists of two major structural domains: a chromophore-bearing, globular N-terminal domain (Vierstra and Quail, 1986) and an extended C-terminal domain, which carries the dimerization sites (Jones et al., 1986;Vierstra et al., 1987;Edgerton and Jones, 1992;Cherry et al., 1993).In Arabidopsis, the phytochromes are encoded by five different genes-PHYA through PHYE (Sharrock and Quail, 1989;Clack et al., 1994). Contrasting photosensory functions of the phyA and phyB holoproteins have been defined through analysis of responses to constitutive overexpression of Quail, 1989, 1991;Kay et al., 1989;Keller et al., 1989;Cherry et al., 1991;Wagner et al., 1991;McCormac et al., 1993) and mutations in (Nagatani et al., 1991(Nagatani et al., , 1993Somers et al., 1991;Dehesh et al., 1993;Parks and Quail, 1993;Reed et al., 1993;Whitelam et al., 1993;Wagner and Quail, 1995;Xu et al., 1995) hypocotyl elongation, hook opening, and cotyledon expansion) in continuous far-red light (FRc), whereas phyB is primarily responsible for deetiolation in continuous red light (Rc). phyA is most abundant in dark-grown seedlings (m50-fold phyB levels), whereas, due ...

We used a series of in vitro-generated deletion and amino acid substitution derivatives of phytochrome B (phyB) expressed in transgenic Arabidopsis to identify regions of the molecule important for biological activity. Expression of the chromophore-bearing N-terminal domain of phyB alone resulted in a fully photoactive, monomeric molecule lacking normal regulatory activity. Expression of the C-terminal domain alone resulted in a photoinactive, dimeric molecule, also lacking normal activity. Thus, both domains are necessary, but neither is sufficient for phyB activity. Deletion of a small region on each major domain (residues 6 to 57 and 652 to 712, respectively) was shown to compromise phyB activity differentially without interfering with spectral activity or dimerization. Deletion of residues 6 to 57 caused a large increase in the fluence rate of continuous red light (Rc) required for maximal seedling responsiveness, indicating a marked decrease in efficiency of light signal perception or processing per mole of mutant phyB. In contrast, deletion of residues 652 to 712 resulted in a photoreceptor that retained saturation of seedling responsiveness to Rc at low fluence rates but at a response level much below the maximal response elicited by the parent molecule. This deletion apparently reduces the maximal biological activity per mole of phyB without a major decrease in efficiency of signal perception, thus suggesting disruption of a process downstream of signal perception. In addition, certain phyB constructs caused dominant negative interference with endogenous phyA activity in continuous far-red light, suggesting that the two photoreceptors may share reaction partners.