A Study of the Blue-Light-Dependent Phosphorylation, Degradation, and Photobody Formation of Arabidopsis CRY2

Abstract

Arabidopsis cryptochrome 2 (CRY2) is a blue-light receptor mediating blue-light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation. CRY2 is a constitutive nuclear protein that undergoes blue-light-dependent phosphorylation, ubiquitination, photobody formation, and degradation in the nucleus, but the relationship between these blue-light-dependent events remains unclear. It has been proposed that CRY2 phosphorylation triggers a conformational change responsible for the subsequent ubiquitination and photobody formation, leading to CRY2 function and/or degradation. We tested this hypothesis by a structure-function study, using mutant CRY2-GFP fusion proteins expressed in transgenic Arabidopsis. We show that changes of lysine residues of the NLS (Nuclear Localization Signal) sequence of CRY2 to arginine residues partially impair the nuclear importation of the CRY2K541R and CRY2K554/5R mutant proteins, resulting in reduced phosphorylation, physiological activities, and degradation in response to blue light. In contrast to the wild-type CRY2 protein that forms photobodies exclusively in the nucleus, the CRY2K541R and CRY2K554/5R mutant proteins form protein bodies in both the nucleus and cytosol in response to blue light. These results suggest that photoexcited CRY2 molecules can aggregate to form photobody-like structure without the nucleus-dependent protein modifications or the association with the nuclear CRY2-interacting proteins. Taken together, the observation that CRY2 forms photobodies markedly faster than CRY2 phosphorylation in response to blue light, we hypothesize that the photoexcited cryptochromes form oligomers, preceding other biochemical changes of CRY2, to facilitate photobody formation, signal amplification, and propagation, as well as desensitization by degradation.

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

“…Thus, it is generally accepted that photoactivated cry1 and cry2 transduce light signals to downstream components, primarily in the nucleus. Different from cry1 but similar to phys, photoactivated cry2 forms photobodies in the nucleus [56,64,65]. Photobody formation is closely correlated with cry2 function and degradation.…”

Formation of Arabidopsis Cryptochrome 2 Photobodies in Mammalian Nuclei

“…Secondly, to demonstrate that the blue light-induced checkpoint activation was due to clustering of TopBP1 in photobodies, we used the mutated AtCRY2 (D387A) as a control. The D387A mutation in the FAD binding pocket completely eliminates FAD binding to AtCRY2 and photobody formation (8). We constructed AtCRY2(D387A)-GFP-TopBP1 (Fig.…”

“…Among plant photosensory proteins, the red light receptor phytochromes (PHYs) are the most extensively studied with respect to photobody formation. More recently, it has been found that the blue light photosensory receptor cryptochrome (CRY) forms photobodies as well (8,9).…”

“…A genetic screen has identified several genes that are required for PHY-PB formation, and one of these proteins, hemera, has been found to associate with PHY-PBs (10). CRY-PBs have been analyzed mostly by using AtCRY2 (8,9). Recent work suggests that AtCRY2 controls signaling pathways by two mechanisms (11).…”

Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways

“…A photon is absorbed by the N-terminal flavin binding domain and initiates a chemical reaction within the protein, which in turn triggers a conformational change. The resulting "lit" state renders the cryptochrome receptor accessible to interacting proteins that trigger photomorphogenesis and signaling, while at the same time exposing ubiquitination sites elsewhere on the protein that target cry2 for degradation (Zuo et al, , 2012. The current challenge in this field is to determine which of the known primary photoreactions occurring in isolated cryptochromes in vitro are required for the initiation of biological activity and photomorphogenesis in vivo.…”