Chemically Induced and Light-Independent Cryptochrome Photoreceptor Activation

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.

Coordination of Cryptochrome and Phytochrome Signals in the Regulation of Plant Light Responses

“…Both crys undergo B light-dependent phosphorylation, and the phosphorylation of the crys is closely correlated with their functions [47][48][49]. Homodimerization is required for the phosphorylation and physiological activity of the crys [50][51][52]. Recently, it was found that cry2 forms a homodimer in response to B light [53].…”

Formation of Nuclear Bodies of Arabidopsis CRY2 in Response to Blue Light Is Associated with Its Blue Light–Dependent Degradation

“…It is not clear as to how exactly CRY2-GFP could affect phosphorylation of the endogenous CRY2. However, given that cryptochromes form homodimers via the PHR domain (Sang et al, 2005;Rosenfeldt et al, 2007;Yu et al, 2007a), it is conceivable that phosphorylated CRY2-GFP might interact with the endogenous CRY2 to enable phosphorylation of the latter in the dark. Moreover, it is unclear as to whether or not CRY2-GFP itself might act as a kinase.…”

Increased DNA repair in Arabidopsis plants overexpressing CPD photolyase

“…The coding sequence of At-PHR1 was introduced into the NcoI site of pAVA393 (von Arnim et al 1998) 5Ј and in frame with the coding region of the GFP4/ GFP5 hybrid and 3Ј of the tandem CaMV 35S promoter. Protoplasts of an Arabidopsis Landsberg erecta mesophyll cell culture were transformed with the resulting construct pAVA393 At-PHR1::GFP as described by (Rosenfeldt et al 2008). The cellular localisation of the fusion protein was studied 16 h after transfection with a confocal microscope (Leica, Wetzlar, Germany) consisting of a DMRE stand equipped with an PL APO 63x/1.32-0.60 oil objective, a 65 nW Ar Laser and a TCS SP2 confocal scanner.…”