Blue Light-dependent Phosphorylation Research Articles

Insights into cryptochrome modulation of ABA signaling to mediate dormancy regulation in Marchantia polymorpha.

The acquisition of dormancy capabilities has enabled plants to survive in adverse terrestrial environmental conditions. Dormancy accumulation and release is coupled with light signaling, which is well studied in Arabidopsis, but it is unclear in the distant nonvascular relative. We study the characteristics and function on dormancy regulation of a blue light receptor cryptochrome in Marchantia polymorpha (MpCRY). Here, we identified MpCRY via bioinformatics and mutant complement analysis. The biochemical characteristics were assessed by multiple protein-binding assays. The function of MpCRY in gemma dormancy was clarified by overexpression and mutation of MpCRY, and its mechanism was analyzed via RNA sequencing and quantitative PCR analyses associated with hormone treatment. We found that the unique MpCRY protein in M. polymorpha undergoes both blue light-promoted interaction with itself (self-interaction) and blue light-dependent phosphorylation. MpCRY has the specific characteristics of blue light-induced nuclear localization and degradation. We further demonstrated that MpCRY transcriptionally represses abscisic acid (ABA) signaling-related gene expression to suppress gemma dormancy, which is dependent on blue light signaling. Our findings indicate that MpCRY possesses specific biochemical and molecular characteristics, and modulates ABA signaling under blue light conditions to regulate gemma dormancy in M. polymorpha.

Blue light-induced phosphorylation of Arabidopsis cryptochrome 1 is essential for its photosensitivity.

Plants possess two cryptochrome photoreceptors, cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2), that mediate overlapping and distinct physiological responses. Both CRY1 and CRY2 undergo blue light-induced phosphorylation, but the molecular details of CRY1 phosphorylation remain unclear. Here we identify 19 in vivo phosphorylation sites in CRY1 using mass spectrometry and systematically analyze the physiological and photobiochemical activities of CRY1 variants with phosphosite substitutions. We demonstrate that nonphosphorylatable CRY1 variants have impaired phosphorylation, degradation, and physiological functions, whereas phosphomimetic variants mimic the physiological functions of phosphorylated CRY1 to constitutively inhibit hypocotyl elongation. We further demonstrate that phosphomimetic CRY1 variants exhibit enhanced interaction with the E3 ubiquitin ligase COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1). This finding is consistent with the hypothesis that phosphorylation of CRY1 is required for COP1-dependent signaling and regulation of CRY1. We also determine that PHOTOREGULATORY PROTEIN KINASEs (PPKs) phosphorylate CRY1 in a blue light-dependent manner and that this phosphorylation is critical for CRY1 signaling and regulation. These results indicate that, similar to CRY2, blue light-dependent phosphorylation of CRY1 determines its photosensitivity.

Differential photoregulation of the nuclear and cytoplasmic CRY1 in Arabidopsis.

Arabidopsis cryptochrome 1 (CRY1) is a blue light receptor distributed in the nucleus and cytoplasm. The nuclear CRY1, but not cytoplasmic CRY1, mediates blue light inhibition of hypocotyl elongation. However, the photobiochemical mechanisms distinguishing the CRY1 protein in the two subcellular compartments remains unclear. Here we show that the nuclear CRY1, but not the cytoplasmic CRY1, is regulated by phosphorylation, polyubiquitination and 26S proteasome-dependent proteolysis in response to blue light. The blue light-dependent CRY1 degradation is observed only under high fluences of blue light. The nuclear specificity and high fluence dependency of CRY1 explain why this photochemical regulatory mechanism of CRY1 was not observed previously and it further supports the hypothesis that CRY1 is a high light receptor regulating photomorphogenesis. We further show that the nuclear CRY1, but not cytoplasmic CRY1, undergoes blue light-dependent phosphorylation by photoregulatory protein kinase 1 (PPK1) followed by polyubiquitination by the E3 ubiquitin ligase Cul4COP1/SPAs , resulting in the blue light-dependent proteolysis. Both phosphorylation and ubiquitination of nuclear CRY1 are inhibited by blue-light inhibitor of cryptochromes 1 (BIC1), demonstrating the involvement of photo-oligomerization of the nuclear CRY1. These finding reveals a photochemical mechanism that differentially regulates the physiological activity of the CRY1 photoreceptor in distinct subcellular compartments.

Molecular basis for blue light-dependent phosphorylation of Arabidopsis cryptochrome 2

Plant cryptochromes undergo blue light-dependent phosphorylation to regulate their activity and abundance, but the protein kinases that phosphorylate plant cryptochromes have remained unclear. Here we show that photoexcited Arabidopsis cryptochrome 2 (CRY2) is phosphorylated in vivo on as many as 24 different residues, including 7 major phosphoserines. We demonstrate that four closely related Photoregulatory Protein Kinases (previously referred to as MUT9-like kinases) interact with and phosphorylate photoexcited CRY2. Analyses of the ppk123 and ppk124 triple mutants and amiR4k artificial microRNA-expressing lines demonstrate that PPKs catalyse blue light-dependent CRY2 phosphorylation to both activate and destabilize the photoreceptor. Phenotypic analyses of these mutant lines indicate that PPKs may have additional substrates, including those involved in the phytochrome signal transduction pathway. These results reveal a mechanism underlying the co-action of cryptochromes and phytochromes to coordinate plant growth and development in response to different wavelengths of solar radiation in nature.

The Blue Light-Dependent Phosphorylation of the CCE Domain Determines the Photosensitivity of Arabidopsis CRY2

Arabidopsis cryptochrome 2 (CRY2) is a blue light receptor that mediates light inhibition of hypocotyl elongation and long-day promotion of floral initiation. CRY2 is known to undergo blue light-dependent phosphorylation, which is believed to serve regulatory roles in the function of CRY2. We report here on a biochemical and genetics study of CRY2 phosphorylation. Using mass spectrometry analysis, we identified three serine residues in the CCE domain of CRY2 (S588, S599, and S605) that undergo blue light-dependent phosphorylation in Arabidopsis seedlings. A study of serine-substitution mutations in the CCE domain of CRY2 demonstrates that CRY2 contains two types of phosphorylation in the CCE domain, one in the serine cluster that causes electrophoretic mobility upshift and the other outside the serine cluster that does not seem to cause mobility upshift. We showed that mutations in the serine residues within and outside the serine cluster diminished blue light-dependent CRY2 phosphorylation, degradation, and physiological activities. These results support the hypothesis that blue light-dependent phosphorylation of the CCE domain determines the photosensitivity of Arabidopsis CRY2.

Blue light-dependent phosphorylations of cryptochromes are affected by magnetic fields in Arabidopsis

The blue light receptor cryptochrome that could form radical pairs after exposure to blue light was suggested to be a magnetoreceptor based on the proposition that radical pairs were involved in the magnetoreception. But the effects of magnetic fields on the function of cryptochrome are poorly understood. Phosphorylation of cryptochrome in Arabidopsis was closely associated with the function of this photoreceptor. Here, we grew Arabidopsis seedlings in a 500μT magnetic field and a near-null magnetic field and found that the 500μT magnetic field enhanced the blue light-dependent phosphorylations of CRY1 and CRY2, and the near-null magnetic field weakened the blue light-dependent phosphorylation of CRY2 but not CRY1. Dephosphorylations of CRY1 and CRY2 in the darkness were slowed down in the 500μT magnetic field, whereas dephosphorylations of CRY1 and CRY2 were accelerated in the near-null magnetic field. These results suggest that magnetic field with strength higher or weaker than the local geomagnetic field affects the activated states of cryptochromes, which thus modifies the functions of cryptochromes.

Arabidopsis Casein Kinase1 Proteins CK1.3 and CK1.4 Phosphorylate Cryptochrome2 to Regulate Blue Light Signaling

Casein kinase1 (CK1) plays crucial roles in regulating growth and development via phosphorylating various substrates throughout the eukaryote kingdom. Blue light is crucial for normal growth of both plants and animals, and blue light receptor cryptochrome2 (CRY2) undergoes blue light-dependent phosphorylation and degradation in planta. To study the function of plant CK1s, systematic genetic analysis showed that deficiency of two paralogous Arabidopsis thaliana CK1s, CK1.3 and CK1.4, caused shortened hypocotyls, especially under blue light, while overexpression of either CK1.3 or CK1.4 resulted in the insensitive response to blue light and delayed flowering under long-day conditions. CK1.3 or CK1.4 act dependently on CRY2, and overexpression of CK1.3 or CK1.4 significantly suppresses the hypersensitive response to blue light by CRY2 overexpression. Biochemical studies showed that CK1.3 and CK1.4 directly phosphorylate CRY2 at Ser-587 and Thr-603 in vitro and negatively regulate CRY2 stability in planta, which are stimulated by blue light, further confirming the crucial roles of CK1.3 and CK1.4 in blue light responses through phosphorylating CRY2. Interestingly, expression of CK1.3 and CK1.4 is stimulated by blue light and feedback regulated by CRY2-mediated signaling. These results provide direct evidence for CRY2 phosphorylation and informative clues on the mechanisms of CRY2-mediated light responses.

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.

ArabidopsisCryptochrome 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.

Protein phosphatase 1 positively regulates stomatal opening in response to blue light in Vicia faba

Phototropins, plant blue light receptors, mediate stomatal opening through the activation of the plasma membrane H(+)-ATPase by unknown mechanisms. Here we report that type 1 protein phosphatase (PP1) positively regulates the blue light signaling between phototropins and the H(+)-ATPase in guard cells of Vicia faba. We cloned the four catalytic subunits of PP1 (PP1c) from guard cells and determined the expression of the isoforms in various tissues. Transformation of Vicia guard cells with PP1c isoforms that had lost enzymatic activity by one amino acid mutation, or with human inhibitor-2, a specific inhibitor protein of PP1c, suppressed blue light-induced stomatal opening. Addition of fusicoccin, an activator of the plasma membrane H(+)-ATPase, to these transformed guard cells induced normal stomatal opening, suggesting that the transformations did not affect the basic mechanisms for stomatal opening. Tautomycin, an inhibitor of PP1, inhibited blue light-induced H(+) pumping, phosphorylation of the plasma membrane H(+)-ATPase in guard cell protoplasts, and stomatal opening. However, tautomycin did not inhibit the blue light-dependent phosphorylation of phototropins. We conclude that PP1 functions downstream of phototropins and upstream of the H(+)-ATPase in the blue light signaling pathway of guard cells.

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.

Cryptochrome structure and signal transduction.

Cryptochromes are photosensory receptors mediating light regulation of growth and development in plants. Since the isolation of the Arabidopsis CRY1 gene in 1993, cryptochromes have been found in every multicellular eukaryote examined. Most plant cryptochromes have a chromophore-binding domain that shares similar structure with DNA photolyase, and a carboxyl terminal extension that contains a DQXVP-acidic-STAES (DAS) domain conserved from moss, to fern, to angiosperm. In Arabidopsis, cryptochromes are nuclear proteins that mediate light control of stem elongation, leaf expansion, photoperiodic flowering, and the circadian clock. Cryptochromes may act by interacting with proteins such as phytochromes, COP1, and clock proteins, or/and chromatin and DNA. Recent studies suggest that cryptochromes undergo a blue light-dependent phosphorylation that affects the conformation, intermolecular interactions, physiological activities, and protein abundance of the photoreceptors.

PP7 is a positive regulator of blue light signaling in Arabidopsis.

The cryptochrome blue light photoreceptors mediate various photomorphogenic responses in plants, including hypocotyl elongation, cotyledon expansion, and control of flowering time. The molecular mechanism of cryptochrome function in Arabidopsis is becoming increasingly clear, with recent studies showing that both CRY1 and CRY2 are localized in the nucleus and that CRY2 is regulated by blue light-dependent phosphorylation. Despite these advances, no positive cryptochrome signaling component has been identified to date. Here, we demonstrate that a novel Ser/Thr protein phosphatase (AtPP7) with high sequence similarity to the Drosophila retinal degeneration C protein phosphatase acts as an intermediate in blue light signaling. Transgenic Arabidopsis seedlings with reduced AtPP7 expression levels exhibit loss of hypocotyl growth inhibition and display limited cotyledon expansion in response to blue light irradiation. These effects are as striking as those seen in hy4 mutant seedlings, which are deficient in CRY1. We further demonstrate that AtPP7 transcript levels are not rate limiting and that AtPP7 probably acts downstream of cryptochrome in the nucleus, ensuring signal flux through the pathway. Based on our findings and recent data regarding cryptochrome action, we propose that AtPP7 acts as a positive regulator of cryptochrome signaling in Arabidopsis.

Regulation of Arabidopsis cryptochrome 2 by blue-light-dependent phosphorylation.

Cryptochromes are blue/ultraviolet-A light receptors that mediate various light responses in plants and animals. But the initial photochemical reaction of cryptochrome is still unclear. For example, although most photoreceptors are known to undergo light-dependent protein modification such as phosphorylation, no blue-light dependent phosphorylation has been reported for a cryptochrome. Arabidopsis cryptochrome 2 (cry2) mediates light regulation of seedling development and photoperiodic flowering. The physiological activity and cellular level of cry2 protein are light-dependent, and protein protein interactions are important for cry2 function. Here we report that cry2 undergoes a blue-light-dependent phosphorylation, and that cry2 phosphorylation is associated with its function and regulation. Our results suggest that, in the absence of light, cry2 remains unphosphorylated, inactive and stable; absorption of blue light induces the phosphorylation of cry2, triggering photomorphogenic responses and eventually degradation of the photoreceptor.

Asymmetric, blue light-dependent phosphorylation of a 116-kilodalton plasma membrane protein can be correlated with the first- and second-positive phototropic curvature of oat coleoptiles.

The possible correlation between blue light-dependent phosphorylation of a 116-kD protein and phototropic responses of etiolated oat (Avena sativa L.) seedlings was tested by a micromethod for protein phosphorylation. Quantitation of the basipetal distribution of this protein showed that the in vitro 32p phosphorylation values declined exponentially from tip to node, with more than 50% of the total label being found in the uppermost 5 mm. Nonsaturating preirradiation of the coleoptiles in vivo resulted in partial phosphorylation with endogenous ATP. Subsequent in vitro phosphorylation under saturating irradiation allowed the determination of the degree of in vivo phosphorylation. Unilateral preirradiation resulted in higher in vivo phosphorylation on the irradiated than on the shaded side of the coleoptile. The fluence-response curve for the difference in phosphorylation between both sides of the coleoptile resembles the fluence-response curve for first-positive phototropic curvature, although it is shifted by two orders of magnitude to higher fluences. Possible reasons for this shift are discussed. In the coleoptile base the phosphorylation gradient across the coleoptile becomes larger with increasing time of irradiation at a constant fluence. Thus, phosphorylation of the 116-kD protein, in accordance with second-positive phototropic curvature, does not obey the Bunsen-Roscoe reciprocity law.