Mutant screen distinguishes between residues necessary for light-signal perception and signal transfer by phytochrome B.
The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651-amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, beta-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.
Oka
Yoshito
Y
Department of Biology, Graduate School of Science, Kyoto University, Kyoto, Japan.
Matsushita
Tomonao
T
Mochizuki
Nobuyoshi
N
Quail
Peter H
PH
Nagatani
Akira
A
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008027:Light; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051939:Phytochrome B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011092:Polyethylene Glycols / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016415:Sequence Alignment; D015398:Signal Transduction
D029681:Arabidopsis Proteins; C497952:PHYB protein, Arabidopsis; D045322:Photosynthetic Reaction Center Complex Proteins; D051939:Phytochrome B; D011092:Polyethylene Glycols; C018557:glycofurol