Xuhong Yu

Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis.

Cryptochromes (CRY) are photolyase-like blue-light receptors that mediate light responses in plants and animals. How plant cryptochromes act in response to blue light is not well understood. We report here the identification and characterization of the Arabidopsis CIB1 (cryptochrome-interacting basic-helix-loop-helix) protein. CIB1 interacts with CRY2 (cryptochrome 2) in a blue light–specific manner in yeast and Arabidopsis cells, and it acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation. CIB1 binds to G box (CACGTG) in vitro with a higher affinity than its interaction with other E-box elements (CANNTG). However, CIB1 stimulates FT messenger RNA expression, and it interacts with chromatin DNA of the FT gene that possesses various E-box elements except G box. We propose that the blue light–dependent interaction of cryptochrome(s) with CIB1 and CIB1-related proteins represents an early photoreceptor signaling mechanism in plants.

Blue Light–Dependent in Vivo and in Vitro Phosphorylation of Arabidopsis Cryptochrome 1

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light–dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light–dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light–induced phosphorylation. These results demonstrate that blue light–dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.

The Cryptochrome Blue Light Receptors

Cryptochromes are photolyase-like blue light receptors originally discovered in Arabidopsis but later found in other plants, microbes, and animals. Arabidopsis has two cryptochromes, CRY1 and CRY2, which mediate primarily blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation, respectively. In addition, cryptochromes also regulate over a dozen other light responses, including circadian rhythms, tropic growth, stomata opening, guard cell development, root development, bacterial and viral pathogen responses, abiotic stress responses, cell cycles, programmed cell death, apical dominance, fruit and ovule development, seed dormancy, and magnetoreception. Cryptochromes have two domains, the N-terminal PHR (Photolyase-Homologous Region) domain that bind the chromophore FAD (flavin adenine dinucleotide), and the CCE (CRY C-terminal Extension) domain that appears intrinsically unstructured but critical to the function and regulation of cryptochromes. Most cryptochromes accumulate in the nucleus, and they undergo blue light-dependent phosphorylation or ubiquitination. It is hypothesized that photons excite electrons of the flavin molecule, resulting in redox reaction or circular electron shuttle and conformational changes of the photoreceptors. The photoexcited cryptochrome are phosphorylated to adopt an open conformation, which interacts with signaling partner proteins to alter gene expression at both transcriptional and posttranslational levels and consequently the metabolic and developmental programs of plants.

A Study of Gibberellin Homeostasis and Cryptochrome-Mediated Blue Light Inhibition of Hypocotyl Elongation

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA4 in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA4 content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs.

Arabidopsis Cryptochrome 2 Completes Its Posttranslational Life Cycle in the Nucleus

CRY2 is a blue light receptor regulating light inhibition of hypocotyl elongation and photoperiodic flowering in Arabidopsis thaliana. The CRY2 protein is found primarily in the nucleus, and it is known to undergo blue light–dependent phosphorylation and degradation. However, the subcellular location where CRY2 exerts its function or undergoes blue light–dependent phosphorylation and degradation remains unclear. In this study, we analyzed the function and regulation of conditionally nuclear-localized CRY2. Our results show that CRY2 mediates blue light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation in the nucleus. Consistent with this result and a hypothesis that blue light–dependent phosphorylation is associated with CRY2 function, we demonstrate that CRY2 undergoes blue light–dependent phosphorylation in the nucleus. CRY2 phosphorylation is required for blue light–dependent CRY2 degradation, but only a limited quantity of CRY2 is phosphorylated at any given moment in seedlings exposed to blue light, which explains why continuous blue light illumination is required for CRY2 degradation. Finally, we showed that CRY2 is ubiquitinated in response to blue light and that ubiquitinated CRY2 is degraded by the 26S proteasome in the nucleus. These findings demonstrate that a photoreceptor can complete its posttranslational life cycle (from protein modification, to function, to degradation) inside the nucleus.

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

Arabidopsis thaliana cryptochrome 2 (CRY2) mediates photoperiodic promotion of floral initiation and blue light inhibition of hypocotyl elongation. It has been hypothesized that photoexcitation derepresses CRY2 by disengaging its C-terminal domain from the N-terminal PHR domain. To test this hypothesis, we analyzed activities of CRY2 fused to green fluorescent protein (GFP) at either the N terminus (GFP-CRY2) or the C terminus (CRY2-GFP). While GFP-CRY2 exerts light-dependent biochemical and physiological activities similar to those of the endogenous CRY2, CRY2-GFP showed constitutive biochemical and physiological activities. CRY2-GFP is constitutively phosphorylated, it promotes deetiolation in both dark and light, and it activates floral initiation in both long-day and short-day photoperiods. These results are consistent with the hypothesis that photoexcited CRY2 disengages its C-terminal domain from the PHR domain to become active. Surprisingly, we found that CRY2-GFP, but not GFP-CRY2, formed distinct nuclear bodies in response to blue light. Compared with GFP-CRY2 or the endogenous CRY2, CRY2-GFP degradation was significantly retarded in response to blue light, suggesting that the nuclear bodies may result from accumulation of photoexcited CRY2-GFP waiting to be degraded. Consistent with this interpretation, we showed that both GFP-CRY2 and endogenous CRY2 formed nuclear bodies in the presence of the 26S-proteasome inhibitors that block blue light–dependent CRY2 degradation.

Derepression of the NC80 motif is critical for the photoactivation of Arabidopsis CRY2

Cryptochromes are blue light receptors that regulate photomorphogenesis in plants and the circadian clock in animals and plants. Arabidopsis cryptochrome 2 (CRY2) mediates blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation. CRY2 undergoes blue light-induced phosphorylation, which was hypothesized to be associated with CRY2 photoactivation. To further investigate how light activates CRY2, we analyzed the physiological activities and phosphorylation of various CRY2 fusion proteins in transgenic plants. Our results showed that an 80-residue motif, referred to as NC80, was sufficient to confer the physiological function of CRY2. The GUS-NC80 fusion protein expressed in transgenic plants is constitutively active but unphosphorylated, suggesting that the blue light-induced CRY2 phosphorylation causes a conformational change to derepress the NC80 motif. Consistent with this hypothesis, the CRY2 C-terminal tail was found to be required for the blue light-induced CRY2 phosphorylation but not for the CRY2 activity. We propose that the PHR domain and the C-terminal tail of the unphosphorylated CRY2 form a “closed” conformation to suppress the NC80 motif in the absence of light. In response to blue light, the C-terminal tail of CRY2 is phosphorylated and electrostatically repelled from the surface of the PHR domain to form an “open” conformation, resulting in derepression of the NC80 motif and signal transduction to trigger photomorphogenic responses.

Arabidopsis cryptochrome 2 (CRY2) functions by the photoactivation mechanism distinct from the tryptophan (trp) triad-dependent photoreduction

Cryptochromes are blue-light receptors mediating various light responses in plants and animals. The photochemical mechanism of cryptochromes is not well understood. It has been proposed that photoactivation of cryptochromes involves the blue-light–dependent photoreduction of flavin adenine dinucleotide via the electron transport chain composed of three evolutionarily conserved tryptophan residues known as the “trp triad.” We investigated this hypothesis by analyzing the photochemical and physiological activities of Arabidopsis cryptochrome 2 (CRY2) mutations altered in each of the three trp-triad residues. We found that all trp-triad mutations of CRY2 tested lost photoreduction activity in vitro but retained the physiological and biochemical activities in vivo. Some of the trp-triad mutations of CRY2 remained responsive to blue light; others, such as CRY2W374A, became constitutively active. In contrast to wild-type CRY2, which undergoes blue-light–dependent interaction with the CRY2-signaling proteins SUPPRESSOR OF PHYA 1 (SPA1) and cryptochrome-interaction basic helix–loop–helix 1 (CIB1), the constitutively active CRY2W374A interacts with SPA1 and CIB1 constitutively. These results support the hypothesis that cryptochromes mediate blue-light responses via a photochemistry distinct from trp-triad–dependent photoreduction and that the trp-triad residues are evolutionarily conserved in the photolyase/cryptochrome superfamily for reasons of structural integrity rather than for photochemistry per se.

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

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.

Blue-light-independent activity of Arabidopsis cryptochromes in the regulation of steady-state levels of protein and mRNA expression.

Cryptochromes are blue-light receptors that mediate blue-light inhibition of hypocotyl elongation and blue-light stimulation of floral initiation in Arabidopsis. In addition to their blue-light-dependent functions, cryptochromes are also involved in blue-light-independent regulation of the circadian clock, cotyledon unfolding, and hypocotyl inhibition. However, the molecular mechanism associated with the blue-light-independent function of cryptochromes remains unclear. We reported here a comparative proteomics study of the light regulation of protein expression. We showed that, as expected, the protein expression of many metabolic enzymes changed in response to both blue light and red light. Surprisingly, some light-regulated protein expression changes are impaired in the cry1cry2 mutant in both blue light and red light. This result suggests that, in addition to mediating blue-light-dependent regulation of protein expression, cryptochromes are also involved in the blue-light-independent regulation of gene expression. Consistent with this hypothesis, the cry1cry2 mutant exhibited reduced changes of mRNA expression in response to not only blue light, but also red light, although the cryptochrome effects on the red-light-dependent gene expression changes are generally less pronounced. These results support a hypothesis that, in addition to their blue-light-specific functions, cryptochromes also play roles in the control of gene expression mediated by the red/far-red-light receptor phytochromes.