RESUMO
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 CRY2(W374A), 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 CRY2(W374A) 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.
Assuntos
Arabidopsis/metabolismo , Criptocromos/química , Triptofano/química , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Criptocromos/genética , Criptocromos/fisiologia , Luz , Modelos Genéticos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Fotoquímica/métodos , Estrutura Terciária de ProteínaRESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flavoproteínas/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Luz , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efeitos da radiação , Criptocromos , Flavoproteínas/genética , Flavoproteínas/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/efeitos da radiação , Modelos Moleculares , Fosforilação , Fotorreceptores de Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA de Plantas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiaçãoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Flores/crescimento & desenvolvimento , Transcrição Gênica , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Criptocromos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Criptocromos/genética , Criptocromos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , RNA Mensageiro/genética , RNA de Plantas/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/efeitos da radiação , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Anidrases Carbônicas/efeitos da radiação , Criptocromos/efeitos da radiação , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Desoxirribodipirimidina Fotoliase/efeitos da radiação , Cinética , Mutagênese , Mutação , Plântula/genética , Plântula/metabolismo , Plântula/efeitos da radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Criptocromos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Hipocótilo/genética , Hipocótilo/metabolismo , Hipocótilo/efeitos da radiação , Immunoblotting , Luz , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinação/efeitos da radiaçãoRESUMO
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.