Two Small Spatially Distinct Regions of Phytochrome B Are Required for Efficient Signaling Rates.

Abstract

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 ...

Cotyledon-Generated Auxin Is Required for Shade-Induced Hypocotyl Growth in Brassica rapa

“…The predicted B. rapa FPsc phyB protein (Bra022192) is more than 90% identical to that encoded by Arabidopsis locus AT2G18790, with most divergence in the N-terminal region (approximately 60% identity over the first 55 amino acids). Previous work has shown that deleting amino acids 1 to 57 has little effect on the function of Arabidopsis phyB, suggesting that this less-conserved region is nonessential (Wagner et al, 1996). We find that ein194 seedlings have reduced induction of shade-regulated genes in low R:FR light (Fig.…”

“…In addition, we find that overexpression of the highly conserved Arabidopsis PHYB sequence (AtPHYB) in transgenic B. rapa FPsc plants resulted in a stereotypical PHYB overexpression phenotype (Wagner et al, 1996), with short hypocotyls in W light (Supplemental Fig. S5; a single line was generated, likely with two insertions of the transgene).…”

Photoresponses of Transgenic Arabidopsis Overexpressing the Fern Adiantum capillus-veneris PHY1

“…It is possible that phyl forms inactive heterodimers with the endogenous phyA, which would reduce the leve1 of functional phyA. However, in previous studies of the expression of heterologous phytochromes in Arabidopsis, no heterodimers have ever been detected (Boylan et al, 1994;Sakamoto and Nagatani, 1996;Wagner et al, 1996b), and we favor the hypothesis that phyl is affecting the signal transduction of phyA by competing with endogenous phyA for binding to its reaction partner.…”

“…This might be caused by either the loss of functional endogenous phyA molecules or through an effect on the phyA signal transduction pathway. A dominant negative response under FR has also been observed in overexpressors of phyA lacking an N terminus (Boylan et al, 1994;Emmler et al, 1995), full-length phyB, and a truncated phyB (Sakamoto and Nagatani, 1996;Wagner et al, 1996b). Experiments in which the N-and C-terminal domains of phyA and phyB were exchanged (Wagner et al, 1996a) suggest that these two photoreceptors may share the same reaction partners (Wagner et al, 1996a(Wagner et al, , 1996b.…”

“…Overexpression of mutated phyA lacking the N-terminal 52 amino acids resulted in a dominant negative phenotype under FR (Boylan et al, 1994). It has also been shown that overexpression of the C-terminal (Sakamoto and Nagatani, 1996;Wagner et al, 199613) and chromophore-bearing N-terminal domains of phyB (Wagner et al, 1996b) cause a dominant negative phenotype under FR but not R. Thus, it was proposed that phyA and phyB may share reaction partners (Wagner et al, 1996a(Wagner et al, , 1996b.…”