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.

Plants use different classes of photoreceptors to collect information about their light environment. Cryptochromes are blue light photoreceptors that control deetiolation, entrain the circadian clock, and are involved in flowering time control. Here, we describe the cry1-L407F allele of Arabidopsis (Arabidopsis thaliana), which encodes a hypersensitive cryptochrome1 (cry1) protein. Plants carrying the cry1-L407F point mutation have elevated expression of CONSTANS and FLOWERING LOCUS T under short-day conditions, leading to very early flowering. These results demonstrate that not only the well-studied cry2, with an unequivocal role in flowering promotion, but also cry1 can function as an activator of the floral transition. The cry1-L407F mutants are also hypersensitive toward blue, red, and far-red light in hypocotyl growth inhibition. In addition, cry1-L407F seeds are hypersensitive to germination-inducing red light pulses, but the far-red reversibility of this response is not compromised. This demonstrates that the cry1-L407F photoreceptor can increase the sensitivity of phytochrome signaling cascades. Molecular dynamics simulation of wild-type and mutant cry1 proteins indicated that the L407F mutation considerably reduces the structural flexibility of two solvent-exposed regions of the protein, suggesting that the hypersensitivity might result from a reduced entropic penalty of binding events during downstream signal transduction. Other nonmutually exclusive potential reasons for the cry1-L407F gain of function are the location of phenylalanine-407 close to three conserved tryptophans, which could change cry1's photochemical properties, and stabilization of ATP binding, which could extend the lifetime of the signaling state of cry1.Light determines the plant's life, because light is the essential energy source for plant metabolism. The spatial, temporal, and spectral variability of light provides cues about the time of day, the season, and the presence of competitors for light. Sensitive and precise light perception, therefore, is essential to properly adjust plant development for maximal photosynthetic efficiency, to correlate vegetative and reproductive growth with favorable seasons, and eventually to maximize fitness. To cope with this task, plants have evolved several types of photoreceptors, including the phytochromes and cryptochromes (for review, see Banerjee and Batschauer, 2005;Josse et al., 2008;Mü ller and Carell, 2009). Phytochromes are red and far-red light receptors and regulate different aspects of plant development, such as hypocotyl elongation in red and far-red light and shade avoidance responses (Franklin et al., 2005). In addition, the two major phytochromes in Arabidopsis (Arabidopsis thaliana), phytochrome A (phyA) and phyB, are involved in flowering time control: phyA promotes flowering under short-day (SD) and long-day (LD) photoperiods (Johnson et al., 1994), while phyB acts as a floral inhibitor (Reed et al., 1993;Mockler et al., 1999).Cryptochromes are flavoproteins with two chromoph...