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.
Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 (AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This interdomain modulation would be critical for CRYs’ ability to transduce a bluelight signal into altered protein–protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.
blue-light receptor; flavin; photoreduction; diffusion; conformational dynamics
Cryptochromes are photolyase-like blue/UV-A light receptors that evolved from photolyases. In plants, cryptochromes regulate various aspects of plant growth and development. Despite of their involvement in the control of important plant traits, however, most studies on cryptochromes have focused on lower plants and herbaceous crops, and no data on cryptochrome function are available for forest trees. In this study, we isolated a cryptochrome gene, PeCRY1, from Euphrates poplar (Populus euphratica), and analyzed its structure and function in detail. The deduced PeCRY1 amino acid sequence contained a conserved N-terminal photolyase-homologous region (PHR) domain as well as a C-terminal DQXVP-acidic-STAES (DAS) domain. Secondary and tertiary structure analysis showed that PeCRY1 shares high similarity with AtCRY1 from Arabidopsis thaliana. PeCRY1 expression was upregulated at the mRNA level by light. Using heterologous expression in Arabidopsis, we showed that PeCRY1 overexpression rescued the cry1 mutant phenotype. In addition, PeCRY1 overexpression inhibited hypocotyl elongation, promoted root growth, and enhanced anthocyanin accumulation in wild-type background seedlings grown under blue light. Furthermore, we examined the interaction between PeCRY1 and AtCOP1 using a bimolecular fluorescence complementation (BiFc) assay. Our data provide evidence for the involvement of PeCRY1 in the control of photomorphogenesis in poplar.
Cryptochromes are photoreceptors that regulate entrainment of the circadian clock by light in plants and animals. They are related to DNA photolyases and have similar three-dimensional structures, characterized by a α/β domain and a helical domain and including a chromophore, flavin adenine dinucleotide.
Cryptochromes are photoreceptors that regulate entrainment by light of the circadian clock in plants and animals. They also act as integral parts of the central circadian oscillator in animal brains and as receptors controlling photomorphogenesis in response to blue or ultraviolet (UV-A) light in plants. Cryptochromes are probably the evolutionary descendents of DNA photolyases, which are light-activated DNA-repair enzymes, and are classified into three groups - plant cryptochromes, animal cryptochromes, and CRY-DASH proteins. Cryptochromes and photolyases have similar three-dimensional structures, characterized by an α/β domain and a helical domain. The structure also includes a chromophore, flavin adenine dinucleotide (FAD). The FAD-access cavity of the helical domain is the catalytic site of photolyases, and it is predicted also to be important in the mechanism of cryptochromes.
Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non–light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.
Vision in animals is generally associated with light-sensitive rhodopsin pigments located in the eyes. However, animals ranging from flies to humans also possess ancient visual receptors known as cryptochromes in multiple cell types. In this work, we study the mechanism of light sensing in two representative animal cryptochromes: a light-sensitive Drosophila cryptochrome (Dmcry) and a presumed light-insensitive mammalian cryptochrome from humans (Hscry1). We expressed recombinant cryptochromes to high levels in living cells, irradiated the cells with blue light, and analyzed the proteins' response to irradiation with electron paramagnetic resonance and fluorescence spectroscopic techniques. Photoreduction of protein-bound oxidized FAD cofactor to its radical form emerged as the primary cryptochrome photoreaction in living cells, and was correlated with a light-sensitive biological response in whole organisms. These results indicate that both Dmcry and Hscry1 are capable of undergoing similar light-driven reactions and suggest the possibility of an as-yet unknown photo-perception role for human cryptochromes in tissues exposed to light.
Cryptochromes are blue-light-absorbing receptors found in plants, animals, and humans. In mammals, they are not thought to respond to light, but this study demonstrates contrary evidence that indeed, human cryptochromes undergo a photochemical transformation in response to light.
Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster.
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.
protein phosphorylation; signal transduction; fluorescence imaging; protein degradation; photobody
Cryptochromes are blue-light absorbing flavoproteins with multiple signaling roles. In plants, cryptochrome (cry1, cry2) biological activity has been linked to flavin photoreduction via an electron transport chain to the protein surface comprising 3 evolutionarily conserved tryptophan residues known as the ‘Trp triad.’ Mutation of any of the Trp triad residues abolishes photoreduction in isolated cryptochrome protein in vitro and therefore had been suggested as essential for electron transfer to the flavin. However, photoreduction of the flavin in Arabidopsis cry2 proteins occurs in vivo even with mutations in the Trp triad, indicating the existence of alternative electron transfer pathways to the flavin. These pathways are potentiated by metabolites in the intracellular environment including ATP, ADP, AMP, and NADH. In the present work we extend these observations to Arabidopsis cryptochrome 1 and demonstrate that Trp triad substitution mutants at W400F and W324F positions which are not photoreduced in vitro can be photoreduced in whole cell extracts, albeit with reduced efficiency. We further show that the flavin signaling state (FADH°) is stabilized in an in vivo context. These data illustrate that in vivo modulation by metabolites in the cellular environment may play an important role in cryptochrome signaling, and are discussed with respect to possible effects on the conformation of the C-terminal domain to generate the biologically active conformational state.
cryptochrome; electron transfer; light signaling; photoreceptor; photoreduction; Trp triad
The blue light receptors cryptochromes mediate various light responses in plants. The photoexcited cryptochrome molecules undergo a number of biophysical and biochemical changes, including electron transfer, phosphorylation, and ubiquitination, resulting in conformational changes to propagate light signals. Two modes of cryptochrome signal transduction have been recently discovered, the CIB (cryptochrome-interacting basic-helix-loop-helix 1)-dependent CRY2 regulation of transcription and the SPA1/COP1 (SUPPRESSOR OF PHYA /CONSTITUTIVELY PHOTOMORPHOGENIC1)-dependent cryptochrome regulation of proteolysis. Both cryptochrome signaling pathways rely on blue light-dependent interactions between the cryptochrome photoreceptor and its signaling proteins to modulate gene expression changes in response to blue light, leading to altered developmental programs of plants.
Cryptochromes are FAD-based blue-light photoreceptors that regulate growth and development in plants and the circadian clock in animals. Arabidopsis thaliana and humans possess two cryptochromes. Recently, it was found that Arabidopsis cryptochrome 1 (AtCry1) binds ATP and exhibits autokinase activity that is simulated by blue light. Similarly, it was reported that human cryptochrome 1 (HsCry1) exhibited autophosphorylation activity under blue light. To test the generality of light stimulated kinase function of cryptochromes, we purified AtCry1, AtCry2, HsCry1 and HsCry2 and probed them for kinase activity under a variety of conditions. We find that AtCry1, which contains near stoichiometric amount of FAD and human HsCry1 and HsCry2, which contain only trace amounts of FAD have autokinase activity but AtCry2, which also contains stoichiometric amounts of FAD does not. Finally, we find that the kinase activity of AtCry1 is not significantly affected by light or the redox status of the flavin cofactor.
The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.
Low frequency electromagnetic fields (EMFs) are associated with electrical power lines and have been implicated in the development of childhood leukemias. However, the Earth also has a natural EMF that animals can detect and which they use in order to navigate and orient themselves, particularly during migrations. One way they might do this is by using specialised photoreceptors called cryptochromes, which when activated by light, generate changes within the molecule that are susceptible to EMFs. Cryptochromes are important components of animal circadian clocks, the 24 hour timers that determine daily behavioral and physiological cycles. We have studied the circadian behavior of the fruitfly and have observed some novel and robust effects of EMFs on the fly's sleep-wake cycle that are mediated by cryptochrome. By using cryptochrome mutants we find that our results do not support the classic model for how this molecule might respond to EMFs. We also show that mammalian cryptochromes can respond to EMF when placed into transgenic Drosophila, whereas in mammalian clock neurons, they cannot. Consequently, the EMF responsiveness of cryptochrome is determined by its intracellular environment, suggesting that other, unknown molecules that interact with cryptochrome are also very important.
Photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases; and cryptochromes that regulate blue-light dependent growth and development in plants, and light-dependent and light-independent circadian clock-setting in animals. Phylogenetic analysis has revealed a new branch of the family which co-segregates with plant cryptochromes. Here we describe the isolation and characterization of a member of this family named Type III photolyase, from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl-tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either or both chromophores we were able to determine the efficiency and rate of energy transfer from MTHF to FAD. Photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases; and cryptochromes that regulate blue-light dependent growth and development in plants, and light-dependent and light-independent circadian clock-setting in animals. Phylogenetic analysis has revealed a new branch of the family which co-segregates with plant cryptochromes. Here we describe the isolation and characterization of a member of this family named Type III photolyase, from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl-tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either or both chromophores we were able to determine the efficiency and rate of energy transfer from MTHF to FAD.
The photolyase/cryptochrome family of proteins are FAD-containing flavoproteins which carry out blue-light dependent functions including DNA repair, plant growth and development, and regulation of the circadian clock. In addition to FAD, many members of the family contain a second chromophore which functions as a photoantenna, harvesting light and transferring the excitation energy to FAD and thus increasing the efficiency of the system. The second chromophore is methenyltetrahydrofolate (MTHF) in most photolyases characterized to date and FAD, FMN, or 5-deazariboflavin in others. To date no second chromophore has been identified in cryptochromes. Drosophila contains 3 members of the cryptochrome/photolyase family: cyclobutane pyrimidine dimer (CPD) photolyase, (6-4) photoproduct photolyase, and cryptochrome. We developed an expression system capable of incorporating all known second chromophores into the cognate cryptochrome/photolyase family members. Using this system we demonstrate that Drosophila CPD photolyase and (6-4) photolyase employ 5-deazariboflavin as their second chromophore but Drosophila cryptochrome, which is evolutionarily closer to (6-4) photolyase than the CPD photolyase, lacks a second chromophore.
Recombinant cryptochrome 3 from A. thaliana with FAD and MTHF cofactors has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P212121 and X-ray diffraction data were collected to 1.9 Å resolution.
Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome–antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 Å. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.
cryptochrome 3; light receptors
Cryptochromes (Crys) and photolyases (Phrs) are flavoproteins that contain an identical cofactor (flavin adenine dinucleotide, FAD) within the same protein architecture, but whose physiological functions are entirely different. In this study, we investigated light-induced conformational changes of a cyanobacterium Cry/Phr-like protein (SCry-DASH) with UV–visible and Fourier transform infrared (FTIR) spectroscopy. We developed a system to measure light-induced difference spectra under the concentrated conditions. In the presence of reducing agent, SCry-DASH showed photoreduction to the reduced form, and we identified a signal unique for an anionic form in the process. Difference FTIR spectra enabled us to assign characteristic FTIR bands to the respective redox forms of FAD. An asparagine residue, which anchors the FAD embedded within the protein is conserved in not only the cyanobaterial protein, but also in Phrs and other Crys including the mammalian clock-related Crys. By characterizing an asparagine-to-cysteine (N392C) mutant of SCry-DASH, which mimics an insect specific Cry, we identified structural changes of the carbonyl group of this conserved asparagine upon light-irradiation. We also found that the N392C mutant is stabilized in the anionic form. We did not observe a signal from protonated carboxylic acid residues during the reduction process, suggesting that the carboxylic acid moiety would not be directly involved as a proton donor to FAD in the system. These results are in contrast to plant specific Crys represented by Arabidopsis thaliana Cry1 which carry Asp at the position. We discuss potential roles for this conserved asparagine position and functional diversity in the Cry/Phr frame.
Cryptochromes are flavoproteins that play a central role in the circadian oscillations of all living organisms except archaea. Cryptochromes are clustered into three subfamilies: plant-type cryptochromes, animal-type cryptochromes and cryptochrome-DASH proteins. These subfamilies are composed of photolyase/cryptochrome superfamily with 6–4 photolyase and cyclobutane pyrimidine dimer photolyase. Cryptochromes have conserved domain architectures with two distinct domains, an N-terminal photolyase-related domain and a C-terminal domain. Although the molecular function and domain architecture of cryptochromes are conserved, their molecular mechanisms differ between plants and animals. Thus, cryptochromes are one of the best candidates for comparative and evolutionary studies. Here, we have developed a Web-based platform for comparative and evolutionary studies of cryptochromes, dbCRY (http://www.dbcryptochrome.org/). A pipeline built upon the consensus domain profile was applied to 1438 genomes and identified 1309 genes. To support comparative and evolutionary genomics studies, the Web interface provides diverse functions such as (i) browsing by species, (ii) protein domain analysis, (iii) multiple sequence alignment, (iv) homology search and (v) extended analysis opportunities through the implementation of ‘Favorite Browser’ powered by the Comparative Fungal Genomics Platform 2.0 (CFGP 2.0; http://cfgp.snu.ac.kr/). dbCRY would serve as a standardized and systematic solution for cryptochrome genomics studies.
The stomatal pores are located on the plant leaf epidermis and regulate CO2 uptake for photosynthesis and the loss of water by transpiration. Their stomatal aperture therefore affects photosynthesis, water use efficiency, and agricultural crop yields. Blue light, one of the environmental signals that regulates the plant stomatal aperture, is perceived by the blue/UV-A light-absorbing cryptochromes and phototropins. The signal transduction cascades that link the perception of light to the stomatal opening response are still largely unknown. Here, we report two new players, Hypersensitive to Red and Blue 1 (HRB1) and Protein Phosphatase 7 (PP7), and their genetic and biochemical interactions in the control of stomatal aperture. Mutations in either HRB1 or PP7 lead to the misregulation of the stomatal aperture and reduce water loss under blue light. Both HRB1 and PP7 are expressed in the guard cells in response to a light-to-dark or dark-to-light transition. HRB1 interacts with PP7 through its N-terminal ZZ-type zinc finger motif and requires a functional PP7 for its stomatal opening response. HRB1 is phosphorylated in vivo, and PP7 can dephosphorylate HRB1. HRB1 is mostly dephosphorylated in a protein complex of 193 kDa in the dark, and blue light increases complex size to 285 kDa. In the pp7 mutant, this size shift is impaired, and HRB1 is predominately phosphorylated. We propose that a modification of HRB1 by PP7 under blue light is essential to acquire a proper conformation or to bring in new components for the assembly of a functional HRB1 protein complex. Guard cells control stomatal opening in response to multiple environmental or biotic stimuli. This study may furnish strategies that allow plants to enjoy the advantages of both constitutive and ABA-induced protection under water-limiting conditions.
Stomatal aperture is regulated by many environmental and biotic cues such as blue light, drought, elevated CO2 concentrations, high humidity, and pathogenic elicitors. Stomatal apertures vary over diurnal cycles, and stomata tend to be open during the day in response to blue light and tend to be closed at night. The blue/UV-A light-absorbing cryptochromes and phototropins are the receptors for the blue light response. We report the action of HRB1, a nuclear ZZ-type zinc finger protein, and PP7, a positive regulator of blue light signaling in the nucleus, in the signal transduction cascades downstream of blue light perception. Both hrb1 and pp7 mutants are more resistant to dehydration and show reductions in both water loss and blue light-regulated stomatal aperture. Our studies on their genetic and biochemical interactions offer novel insights on the network structure of the light signaling machinery and plant interactions with the environment. Periodic drought is one of the major environmental factors that limits biomass production and crop yield in a changing global climate. Our studies may open new possibilities to engineer plants to survive desiccation.
•The flavin chromophore of the photoreceptor cryptochrome is reduced by light.•The neutral flavin semiquinone is accumulated under anaerobic conditions.•Fully-reduced flavin forms in presence of external reductant (anaerobic conditions).•Stopped-flow experiments reveal light-independent recovery of the ground state.•The dark recovery process requires molecular oxygen.
Cryptochromes (crys) are flavoprotein photoreceptors present throughout the biological kingdom that play important roles in plant development and entrainment of the circadian clock in several organisms. Crys non-covalently bind flavin adenine dinucleotide (FAD) which undergoes photoreduction from the oxidised state to a radical form suggested to be active in signalling in vivo. Although the photoreduction reactions have been well characterised by a number of approaches, little is known of the oxidation reactions of crys and their mechanisms. In this work, a stopped-flow kinetics approach is used to investigate the mechanism of cry oxidation in the presence and absence of an external electron donor. This in vitro study extends earlier investigations of the oxidation of Arabidopsis cryptochrome1 by molecular oxygen and demonstrates that, under some conditions, a more complex model for oxidation of the flavin than was previously proposed is required to accommodate the spectral evidence (see P. Müller and M. Ahmad (2011) J. Biol. Chem. 286, 21033–21040 ). In the absence of an electron donor, photoreduction leads predominantly to the formation of the radical FADH•. Dark recovery most likely forms flavin hydroperoxide (FADHOOH) requiring superoxide. In the presence of reductant (DTT), illumination yields the fully reduced flavin species (FADH−). Reaction of this with dioxygen leads to transient radical (FADH•) and simultaneous accumulation of oxidised species (FAD), possibly governed by interplay between different cryptochrome molecules or cooperativity effects within the cry homodimer.
crys, cryptochromes; DTT, dithiothreitol; FAD, flavin adenine dinucleotide; FADHOOH, flavin hydroperoxide; MTHF, methenyltetrahydrofolate; NOS, nitric oxide synthase; Stopped-flow; Flavin adenine dinucleotide; Photoreduction; Spectroscopy; Global analysis; Flavin hydroperoxide
Proteins of the cryptochrome/photolyase family share high sequence
similarities, common folds and the flavin adenine dinucleotide (FAD) cofactor,
but exhibit diverse physiological functions. Mammalian cryptochromes are
essential regulatory components of the 24-hour circadian clock, whereas (6-4)
photolyases recognize and repair UV-induced DNA damage by using light energy
absorbed by FAD. Despite increasing knowledge about physiological functions from
genetic analyses, the molecular mechanisms and conformational dynamics involved
in clock signaling and DNA repair remain poorly understood. The (6-4)
photolyase, which has strikingly high similarity to human clock cryptochromes,
is a prototypic biological system to study conformational dynamics of
cryptochrome/photolyase family proteins. The entire light-dependent DNA repair
process for (6-4) photolyase can be reproduced in a simple in
vitro system. To decipher pivotal reactions of the common FAD
cofactor, we accomplished time resolved measurements of radical formation,
diffusion, and protein conformational changes during light-dependent repair by
full-length (6-4) photolyase on DNA carrying a single UV-induced damage. The
(6-4) photolyase by itself showed significant volume changes after blue light
activation, indicating protein conformational changes distant from the flavin
cofactor. A drastic diffusion change was observed only in the presence of both
(6-4) photolyase and damaged DNA, and not for (6-4) photolyase alone or with
undamaged DNA. Thus, we propose that this diffusion change reflects the rapid
(50 μs time constant) dissociation of the protein from the repaired DNA
product. Conformational changes with such fast turnover would likely enable DNA
repair photolyases to access the entire genome in cells.
blue light receptor; DNA damage and repair; flavoprotein; flavin adenine dinucleotide; transient grating
The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions.
Photolyases and cryptochromes are evolutionarily related flavoproteins, which however perform distinct physiological functions. Photolyases (PHR) are evolutionarily ancient enzymes. They are activated by light and repair DNA damage caused by UV radiation. Although cryptochromes share structural similarity with DNA photolyases, they lack DNA repair activity. Cryptochrome (CRY) is one of the key elements of the circadian system in animals. In plants, CRY acts as a blue light receptor to entrain circadian rhythms, and mediates a variety of light responses, such as the regulation of flowering and seedling growth.
We performed a comprehensive evolutionary analysis of the CRY/PHR superfamily. The superfamily consists of 7 major subfamilies: CPD class I and CPD class II photolyases, (6–4) photolyases, CRY-DASH, plant PHR2, plant CRY and animal CRY. Although the whole superfamily evolved primarily under strong purifying selection (average ω = 0.0168), some subfamilies did experience strong episodic positive selection during their evolution. Photolyases were lost in higher animals that suggests natural selection apparently became weaker in the late stage of evolutionary history. The evolutionary time estimates suggested that plant and animal CRYs evolved in the Neoproterozoic Era (~1000–541 Mya), which might be a result of adaptation to the major climate and global light regime changes occurred in that period of the Earth’s geological history.
The Arabidopsis COP1/SPA E3 ubiquitin ligase is a key negative regulator that represses light signaling in darkness by targeting transcription factors involved in the light response for degradation. The COP1/SPA complex consists of COP1 and members of the four-member SPA protein family (SPA1-SPA4). Genetic analysis indicated that COP1/SPA2 function is particularly strongly repressed by light when compared to complexes carrying the other three SPAs, thereby promoting a light response after exposure of plants to extremely low light. Here, we show that the SPA2 protein is degraded within 5–15 min after exposure of dark-grown seedlings to a pulse of light. Phytochrome photoreceptors are required for the rapid degradation of SPA2 in red, far-red and also in blue light, whereas cryptochromes are not involved in the rapid, blue light-induced reduction in SPA2 protein levels. These results uncover a photoreceptor-specific mechanism of light-induced inhibition of COP1/SPA2 function. Phytochrome A (phyA) is required for the severe blue light responsiveness of spa triple mutants expressing only SPA2, thus confirming the important role of phyA in downregulating SPA2 function in blue light. In blue light, SPA2 forms a complex with cryptochrome 1 (cry1), but not with cryptochrome 2 (cry2) in vivo, indicating that the lack of a rapid blue light response of the SPA2 protein is only in part caused by a failure to interact with cryptochromes. Since SPA1 interacts with both cry1 and cry2, these results provide first molecular evidence that the light-regulation of different SPA proteins diverged during evolution. SPA2 degradation in the light requires COP1 and the COP1-interacting coiled-coil domain of SPA2, supporting that SPA2 is ubiquitinated by COP1. We propose that light perceived by phytochromes causes a switch in the ubiquitination activity of COP1/SPA2 from ubiquitinating downstream substrates to ubiquitinating SPA2, which subsequently causes a repression of COP1/SPA2 function.
Plants have evolved photoreceptors that initiate a signaling cascade to adjust growth and development to the ambient light environment. The CUL4-dependent COP1/SPA E3 ubiquitin ligase is a key negative regulator of light signaling whose function is repressed by light. Recent research has identified mechanisms that are common to both phytochrome and cryptochrome photoreceptors. Here, we have identified a mechanism of light-induced COP1/SPA repression that is specific to phytochrome photoreceptors. We show that the SPA2 protein is very rapidly degraded in red, far-red and blue light in a phytochrome-dependent fashion. We further show that SPA2 degradation in the light depends on COP1 and on the interaction of SPA2 with COP1. Hence, our results suggest a light-induced degradation of SPA2, but not of COP1, by the COP1/SPA2 ubiquitin ligase. The human ortholog of COP1, which functions without the plant-specific SPA proteins, is known to be regulated by autodegradation following DNA damage. Hence, autodegradation of components of this E3 ligase is a regulatory mechanism used in both humans and plants.
The flavoprotein cryptochromes (CRYs) act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCFFbxl3 ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitylation and degradation. Yet, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound, and Fbxl3-Skp1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Strikingly, the F-box protein Fbxl3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved C-terminal tail and burying its PER-binding interface. This novel F-box protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitylation.
Despite the sequence and structural conservation between cryptochromes and photolyases, members of the cryptochrome/photolyase (flavo)protein family, their functions are divergent. Whereas photolyases are DNA repair enzymes that use visible light to lesion-specifically remove UV-induced DNA damage, cryptochromes act as photoreceptors and circadian clock proteins. To address the functional diversity of cryptochromes and photolyases, we investigated the effect of ectopically expressed Arabidopsis thaliana (6-4)PP photolyase and Potorous tridactylus CPD-photolyase (close and distant relatives of mammalian cryptochromes, respectively), on the performance of the mammalian cryptochromes in the mammalian circadian clock. Using photolyase transgenic mice, we show that Potorous CPD-photolyase affects the clock by shortening the period of behavioral rhythms. Furthermore, constitutively expressed CPD-photolyase is shown to reduce the amplitude of circadian oscillations in cultured cells and to inhibit CLOCK/BMAL1 driven transcription by interacting with CLOCK. Importantly, we show that Potorous CPD-photolyase can restore the molecular oscillator in the liver of (clock-deficient) Cry1/Cry2 double knockout mice. These data demonstrate that a photolyase can act as a true cryptochrome. These findings shed new light on the importance of the core structure of mammalian cryptochromes in relation to its function in the circadian clock and contribute to our further understanding of the evolution of the cryptochrome/photolyase protein family.
The cyclobutane pyrimidine dimer (CPD) and 6–4 lesion formations along with the specific breaks on strands are the most common type of DNA damage caused by Ultraviolet light (UV) irradiation. CPD photolyase I and II construct two subfamilies of flavoproteins, which have recognition and repair capabilities of CPD sites on both single stranded (ssDNA) and double stranded DNA (dsDNA) with the aid of blue light energy. The other types of flavoprotein family consist of cryptochromes (CRY) that act as photoreceptors in plants, or circadian rhythm regulators in animals. Recent findings showed that a specific type of Cryptochrome-Drosophila, Arabidopsis, Synechocystis, Human (CRY-DASH) has photorepair activity on ssDNA. In this work, real-time interactions between CRY-DASH and ss/dsDNA as well as the interactions between Vibrio cholerae photolyase (VcPHR) and ss/dsDNA were investigated using Surface Plasmon Resonance (SPR). The interactions were then characterized and compared in order to investigate the effect of different types of flavoprotein on UV damaged ss/dsDNA. SPR results confirm the specific binding of VcPHR and CRY-DASH with UV treated DNA. This study is the first instance to quantify the interactions of UV treated and untreated DNA with flavoproteins.