Dominant Negative Suppression of Arabidopsis Photoresponses by Mutant Phytochrome A Sequences Identifies Spatially Discrete Regulatory Domains in the Photoreceptor

Abstract

We used the exaggerated short hypocotyl phenotype induced by oat phytochrome A overexpression in transgenic Arabidopsis to monitor the biological activity of mutant phytochrome A derivatives. Three different mutations, which were generated by removing 52 amino acids from the N terminus (delta N52), the entire C-terminal domain (delta C617), or amino acids 617-686 (delta 617-686) of the oat molecule, each caused striking dominant negative interference with the ability of endogenous Arabidopsis phytochrome A to inhibit hypocotyl growth in continuous far-red light ("far-red high irradiance response" conditions). By contrast, in continuous white or red light, delta N52 was as active as the unmutagenized oat phytochrome A protein in suppressing hypocotyl elongation, while delta C617 and delta 617-686 continued to exhibit dominant negative behavior under these conditions. These data suggest that at least three spatially discrete molecular domains coordinate the photoregulatory activities of phytochrome A in Arabidopsis seedlings. The first is the chromophore-bearing N-terminal domain between residues 53 and 616 that is apparently sufficient for the light-induced initiation but not the completion of productive interactions with transduction chain components. The second is the C-terminal domain between residues 617 and 1129 that is apparently necessary for completion of productive interactions under all irradiation conditions. The third is the N-terminal 52 amino acids that are apparently necessary for completion of productive interactions only under far-red high irradiance conditions and are completely dispensable under white and red light regimes.

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

“…The phyBlphyD group of phytochromes carries a 10-to 37-amino acid N-terminal extension, which is not found in the other phytochromes (Clack et al, 1994). Four N-terminal deletions were constructed to test (1) the ímportance of this N-terminal extension (AN57, lacking amino acids 6 to 57); (2) the function of additional N-terminal regions, which were shown to be important for phyA activity (Cherry et al, 1992;Boylan et al, 1994) (AN90 and AN103, lacking amino acids 6 to 90 and amino acids 6 to 103, respectively); and (3) the biological activity of the C-terminal domain when overexpressed by itself (AN633, which lacks amino acids 6 to 633). All four constructs can be detected immunochemically with a monoclonal antibody (MAb B2), which recognizes an epitope between amino acids 652 and 712 of phyB.…”

“…In attempts to identify functionally important structural domains of oat phyA, severa1 researchers have analyzed the effect of in vitro-generated deletion and amino acid substitution rnutants on the ability of the photoreceptor to cause increased inhibition of hypocotyl or stem elongation when overexpressed in transgenic Arabidopsis or tobacco, respectively (Cherryet al, 1992(Cherryet al, , 1993Stockhaus et al, 1992;Boylan et al, 1994). Taken together, these data indicate that the N-terminal domain is sufficient for spectral integrity but not for normal biological activity of the protein and that regions at the N and C terrnini of phyA are necessary for normal biological activity.…”

“…Taken together, these data indicate that the N-terminal domain is sufficient for spectral integrity but not for normal biological activity of the protein and that regions at the N and C terrnini of phyA are necessary for normal biological activity. Some differences were observed in the biological activity of similar constructs between tobacco and Arabidopsis, but the reasons for this observation are not understood (Cherry et al, 1992;Boylan et al, 1994). …”

“…The C357-S phyB derivative carries a single amino acid change, which converts the chromophore attachment site at cysteine 357 to serine. The resulting molecule is predicted to be spectrally inactive, as is phyA carrying this mutation (Boylan et al, 1994), because the thiol group involved in covalent chromophore attachment is changed to an inactive hydroxyl group.Two additional constructs were designed to test the functional importance in phyB of putative dimerization sites reported for phyA Jones, 1992,1993). A652-712 (lacking amino acids 652 to 712) removes the phyB equivalent of the putative central dimerization site and AC1087 (lacking amino acids 1087 to 1172) of the putative C-terminal dimerization site reported for phyA.…”

In Vivo Characterization of Chimeric Phytochromes in Yeast

“…The 4 -6-kDa N terminus of phyA is important for photoreceptor function. Deletion of this region results in a dominant-negative phenotype in overexpressing lines (17,18). Wagner et al (19) constructed PHYAB and PHYBA chimeras similar to ours, but used oat PHYA and rice PHYB and overexpressed the chimeric genes in wild-type Arabidopsis.…”