Functional and biochemical analysis of the N-terminal domain of phytochrome A.
Phytochrome A (phyA) is a versatile plant photoreceptor that mediates responses to brief light exposures (very low fluence responses, VLFR) as well as to prolonged irradiation (high irradiance responses, HIR). We identified the phyA-303 mutant allele of Arabidopsis thaliana bearing an R384K substitution in the GAF subdomain of the N-terminal half of phyA. phyA-303 showed reduced phyA spectral activity, almost normal VLFR, and severely impaired HIR. Recombinant N-terminal half oat of PHYA bearing the phyA-303 mutation showed poor incorporation of chromophore in vitro, despite the predicted relatively long distance (>13 A) between the mutation and the closest ring of the chromophore. Fusion proteins bearing the N-terminal domain of oat phyA, beta-glucuronidase, green fluorescent protein, and a nuclear localization signal showed physiological activity in darkness and mediated VLFR but not HIR. At equal protein levels, the phyA-303 mutation caused slightly less activity than the fusions containing the wild-type sequence. Taken together, these studies highlight the role of the N-terminal domain of phyA in signaling and of distant residues of the GAF subdomain in the regulation of phytochrome bilin-lyase activity.
Mateos
Julieta L
JL
Max-Planck-Institut für Bioanorganische Chemie, Postfach 101356, D-45413 Mülheim an der Ruhr, Germany.
Luppi
Juan Pablo
JP
Ogorodnikova
Ouliana B
OB
Sineshchekov
Vitaly A
VA
Yanovsky
Marcelo J
MJ
Braslavsky
Silvia E
SE
Gärtner
Wolfgang
W
Casal
Jorge J
JJ
D017360:Arabidopsis / Q000502:physiology / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003624:Darkness; D005966:Glucuronidase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D018546:Hypocotyl / Q000254:growth & development; D008027:Light; D009154:Mutation; D010786:Photoreceptor Cells / Q000378:metabolism; D051942:Phytochrome A / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000502:physiology / Q000528:radiation effects; D015398:Signal Transduction
D029681:Arabidopsis Proteins; C497973:PHYA protein, Arabidopsis; D051942:Phytochrome A; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005966:Glucuronidase