ABSTRACT
The cryptochrome photoreceptors of higher plants are dimeric proteins. Their N-terminal photosensory domain mediates dimerization, and the unique C-terminal extension (CCT) mediates signaling. We made use of the human FK506-binding protein (FKBP) that binds with high affinity to rapamycin or rapamycin analogs (rapalogs). The FKBP-rapamycin complex is recognized by another protein, FRB, thus allowing rapamycin-induced dimerization of two target proteins. Here we demonstrate by bioluminescence resonance energy transfer (BRET) assays the applicability of this regulated dimerization system to plants. Furthermore, we show that fusion proteins consisting of the C-terminal domain of Arabidopsis cryptochrome 2 fused to FKBP and FRB and coexpressed in Arabidopsis cells specifically induce the expression of cryptochrome-controlled reporter and endogenous genes in darkness upon incubation with the rapalog. These results demonstrate that the activation of cryptochrome signal transduction can be chemically induced in a dose-dependent fashion and uncoupled from the light signal, and provide the groundwork for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families.
Subject(s)
Cryptochromes/metabolism , Cryptochromes/radiation effects , Photoreceptors, Plant/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/radiation effects , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/radiation effects , Cryptochromes/drug effects , Dimerization , Energy Transfer , Genes, Reporter , Light , Luciferases/metabolism , Luminescence , Photoreceptors, Plant/drug effects , Photoreceptors, Plant/radiation effects , Plants/drug effects , Plants/metabolism , Plants, Genetically Modified , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/radiation effects , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Tacrolimus Binding Proteins/metabolism , Tacrolimus Binding Proteins/radiation effectsABSTRACT
Cryptochrome (Cry) photoreceptors share high sequence and structural similarity with DNA repair enzyme DNA-photolyase and carry the same flavin cofactor. Accordingly, DNA-photolyase was considered a model system for the light activation process of cryptochromes. In line with this view were recent spectroscopic studies on cryptochromes of the CryDASH subfamily that showed photoreduction of the flavin adenine dinucleotide (FAD) cofactor to its fully reduced form. However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA, which is absent for other members of the cryptochrome/photolyase family. Thus, CryDASH may have functions different from cryptochromes. The photocycle of other members of the cryptochrome family, such as Arabidopsis Cry1 and Cry2, which lack DNA repair activity but control photomorphogenesis and flowering time, remained elusive. Here we have shown that Arabidopsis Cry2 undergoes a photocycle in which semireduced flavin (FADH(.)) accumulates upon blue light irradiation. Green light irradiation of Cry2 causes a change in the equilibrium of flavin oxidation states and attenuates Cry2-controlled responses such as flowering. These results demonstrate that the active form of Cry2 contains FADH(.) (whereas catalytically active photolyase requires fully reduced flavin (FADH(-))) and suggest that cryptochromes could represent photoreceptors using flavin redox states for signaling differently from DNA-photolyase for photorepair.
