Cryptochrome signaling in plants

Cryptochromes are blue light receptors that mediate various light-induced responses in plants and animals. They share sequence similarity to photolyases, flavoproteins that catalyze the repair of UV light-damaged DNA, but do not have photolyase activity. Arabidopsis cryptochromes work together with the red/far-red light receptor phytochromes to regulate various light responses, including the regulation of cell elongation and photoperiodic flowering, and are also found to act together with the blue light receptor phototropins to mediate blue light regulation of stomatal opening. The signaling mechanism of Arabidopsis cryptochromes is mediated through negative regulation of COP1 by direct CRY-COP1 interaction through CRY C-terminal domain. Arabidopsis CRY dimerized through its N-terminal domain and dimerization of CRY is required for light activation of the photoreceptor activity. Recently, significant progresses have been made in our understanding of cryptochrome functions in other dicots such as pea and tomato and lower plants including moss and fern. This review will focus on recent advances in functional and mechanism characterization of cryptochromes in plants. It is not intended to cover every aspect of the field; readers are referred to other review articles for historical perspectives and a more comprehensive understanding of this photoreceptor.     

Observation of Magnetic Field Effects on Transient Fluorescence Spectra of Cryptochrome 1 From Homing Pigeons

Cryptochromes are suggested to be involved in the bird magnetoreception based on the radical pair mechanism (RPM), a well established theory of weak magnetic field effects on chemical reactions. Two members of cryptochrome/photolyase family were found to respond to magnetic field, however, no direct responses of bird cryptochrome to magnetic field as weak as the Earth's magnetic field have been obtained so far. In this study, we used transient fluorescence spectroscopy to characterize the weak magnetic field effects of bird cryptochromes. To do this, we cloned the cryptochrome 1 gene (clCRY1) from the retina of homing pigeons (Columba livia), expressed it in insect Sf9 cells and analyzed the transient fluorescence of purified clCRY1 by application of 45–300 μT magnetic fields. The flavin adenine dinucleotide (FAD_ox) and glucose oxidase (GOD) in PBS buffer were set as controls which could be excited by light to generate radicals, but would not be sensitive to magnetic field. We observed that the transient fluorescence spectra of clCRY1 were sensitive to the applied magnetic field at room temperature. Our result provides a new proof of the cryptochrome‐based model of avian magnetoreception in vitro.     

Light-induced conformational change and product release in DNA repair by (6-4) photolyase

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 h 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.     

Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light‐dependent repair of ultraviolet (UV)‐induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6‐4 photolyase, which repairs 6‐4 pyrimidine–pyrimidone photoproducts (6‐4PPs). Although the Cry‐DASH protein is classified as a cryptochrome, it also has light‐dependent DNA repair activity. To determine the significance of the three light‐dependent repair enzymes in recovering from solar UV‐induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light‐dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light‐dependent repair of CPD was lost in the CPD photolyase‐deficient mutant, whereas weak repair activity against 6‐4PPs persisted in the 6‐4 photolyase‐deficient mutant. These results suggest the existence of a heretofore unknown 6‐4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates.     

Ultrafast dynamics of resonance energy transfer in cryptochrome

In this communication, we report the ultrafast dynamics of resonance energy transfer in a blue-light photoreceptor, Vibrio cholerae cryptochrome. The transfer was observed to occur in 60 ps. We also studied the local rigidity and solvation around the binding site of the photoantenna molecule. The results for the first time show energy transfer in cryptochrome suggesting some mechanistic similarities between photolyase that repairs damaged DNA and cryptochrome that mediates blue-light signaling.     

Ultrafast dynamics and anionic active states of the flavin cofactor in cryptochrome and photolyase

We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state flavin adenine dinucleotide (FAD) in subpicosecond and of neutral radical semiquinone (FADH(*)) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD(*-)) and hydroquinone (FADH(-)) have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In photolyases, the excited active state (FADH(-)*) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD(*-)*) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family.     

Electron hopping through the 15 A triple tryptophan molecular wire in DNA photolyase occurs within 30 ps

DNA photolyase is a photoactive flavoprotein that contains three tryptophan residues between the FAD cofactor and the protein surface, the solvent-exposed Trp being located 14.8 A from the flavin. Photoreduction of the neutral radical FADH. form to the catalytically active FADH- form occurs via electron transfer through this chain. The first step in this chain takes 30 ps, the second less than 4 ps. Using a combination of site-directed mutagenesis and femtosecond polarization spectroscopy to discriminate the spectroscopically indistinguishable Trp residues, we show that the third step occurs in less than 30 ps. This implies that the first photoreduction step is rate limiting and that the Trp chain effectively acts as molecular "wire" ensuring rapid and directed long-range charge translocation across the protein. This finding is important for the functioning of the large class of cryptochrome blue-light receptors, where the Trp chain is conserved. In DNA photolyase we make use of the natural photoactivation of the process, but more generally chains of aromatic amino acids may allow very fast long-range electron transfer also in nonphotoactive proteins.     

The road not taken: a theoretical view of an unexpected cryptochrome charge transfer path

Motivated by recent progress in electron paramagnetic resonance spectroscopy, we describe hole transfer along a chain of tryptophan amino acids within the cryptochrome protein of Synechocystis sp.: surprisingly, despite a close sequential and structural similarity to E. coli DNA photolyase, the charge transfer paths and the final sites of charge localization are different for these two enzymes. We study this phenomenon using atomistic simulations and electronic structure computations as a theoretical basis, and we take a new look at the concepts of charge transfer and introduce a modification of Marcus' theory that incorporates dynamic polarization effects. Only this variant of theory describes the population of the correct branch on the subnanosecond time scale. Based on our numerical analysis, we further suggest that the Asp372-Arg374 salt bridge acts as a novel stepping stone in the charge transfer reaction.     

Flavin Adenine Dinucleotide and N5,N10‐Methenyltetrahydrofolate are the in planta Cofactors of Arabidopsis thaliana Cryptochrome 3

Members of the cryptochrome/photolyase family (CPF) of proteins utilize noncovalently bound light‐absorbing cofactors for their biological function. Usually, the identity of these cofactors is determined after expression in heterologous systems leaving the question unanswered whether these cofactors are identical to the indigenous ones. Here, cryptochrome 3 from Arabidopsis thaliana was expressed as a fusion with the green fluorescent protein in Arabidopsis plants. Besides the confirmation of the earlier report of its localization in chloroplasts, our data indicate that fractions of the fusion protein are present in the stroma and associated with thylakoids, respectively. Furthermore, it is shown that the fusion protein expressed in planta contains the same cofactors as the His₆‐tagged protein expressed in Escherichia coli, that is, flavin adenine dinucleotide and N⁵,N¹⁰‐methenyltetrahydrofolate. This demonstrates that the heterologously expressed cryptochrome 3, characterized in a number of previous studies, is a valid surrogate of the corresponding protein expressed in plants. To our knowledge, this is also a first conclusive analysis of cofactors bound to an Arabidopsis protein belonging to the CPF and purified from plant tissue.     

Single Amino Acid Substitution Reveals Latent Photolyase Activity in Arabidopsis cry1

A quick switch: A single amino acid substitution at a conserved residue (D396N) of Arabidopsis cryptochrome-1 (Atcry1) confers single-stranded DNA repair activity in?vitro, conferring photolyase activity onto the cryptochrome. The mutant protein undergoes photoreduction of flavin to the fully reduced anionic form, similar to photolyases and unlike wild-type cryptochromes.     

In‐Planta Expression: Searching for the Genuine Chromophores of Cryptochrome‐3 from Arabidopsis thaliana

Göbel et al. present in this issue an exemplary study of identification of chromophores from Arabidopsis thaliana cryptochrome‐3. Usually taken for granted, proteins and cofactors, respective chromophores, from heterologous expression are considered identical to material isolated from their genuine host. Cryptochromes carry two chromophores, an antenna cofactor and a functional flavin chromophore, both noncovalently embedded into the protein. In particular the antenna chromophore is loosely bound and often lost during protein purification. The authors identify from plant‐extracted Cry3 unambiguously N⁵,N¹⁰‐methenyltetrahydrofolate as antenna chromophore and flavin adenine dinucleotide as the functional chromophore.     

Nicotinamide Adenine Dinucleotides Arrest Photoreduction of Class II DNA Photolyases in FADH˙ State

All light‐sensitive members of the photolyase/cryptochrome family rely on FAD as catalytic cofactor. Its activity is regulated by photoreduction, a light‐triggered electron transfer process from a conserved tryptophan triad to the flavin. The stability of the reduced flavin depends on available external electron donors and oxygen. In this study, we show for the class II photolyase of Methanosarcina mazei , Mm CPDII , that it utilizes physiologically relevant redox cofactors NADH and NADPH for the formation of the semiquinoid FAD in a light‐dependent reaction. Using redox‐inert variants Mm CPDII /W388F and Mm CPDII /W360F, we demonstrate that photoreduction by NADH and NADPH requires the class II ‐specific tryptophan cascade of Mm CPDII . Finally, we confirmed that mutations in the tryptophan cascade can be introduced without any substantial structural disturbances by analyzing crystal structures of Mm CPDII /W388F, Mm CPDII /W360F and Mm CPDII /Y345F.     

THE SUBUNIT STRUCTURE OF YEAST DNA PHOTOLYASE AND THE PURIFICATION OF A FLUORESCENT ACTIVATOR OF THE ENZYME

Abstract— Yeast DNA photolyase purified twice by affinity chromatography was analyzed by electrophoresis on polyacrylamide gradient gels or by sedimentation velocity through 5–200/, sucrose gradients containing 0.4MKC1. Its molecular weight estimated by both these methods was 130 ,000 and 136 ,000, respectively. However, the enzyme dissociated into two bands having molecular weights of 60 ,000 and 85 ,000 when it was examined by electrophoresis on SDS polyacrylamide gradient gels. The subunit structure of the enzyme was confirmed when two absorption maxima corresponding to polypeptides of 54 ,000 and 82 ,500 daltons were observed in sucrose gradients run in 1.0 M KCI. Upon mixing these two fractions, a time-dependent increase in activity occurred, demonstrating that active enzyme could be reconstituted from these subunits.
The activity of photolyase purified by affinity chromatography is enhanced by a compound (activator III) obtained from yeast by acidification, neutralization, ion exchange chromatography and gel filtration. Activator III emits at 350 and 440 nm when excited at 290 nm, and emits at 440 nm when excited at 358 nm. After acid hydrolysis, emission at 440 nm is produced only by excitation at 358 nm, indicating that it contains two separate chromophoric moieties. The chromophore excited by 358 nm light has a pK of 9–11, while the other has a pK of 4–5. Enhancement of photolyase activity by activator III at a concentration equimolar with that of the enzyme and the similarity of the fluorescent spectra of the activator and heat-denatured photolyase suggest that the activator may be the chromophore associated with the enzyme.     

Flavin-based Blue-Light photosensors: a photobiophysics update

This review deals with the biophysical aspects of flavin-based photosensors, comprising cryptochromes, LOV (Light, Oxygen and Voltage) and BLUF (Blue Light sensing Using FAD) proteins. Special emphasis is given to structural issues, photocycle quantum yields and energetics, mechanism of the light-triggered reactions, early stages in signal transduction and oligomeric states of the light sensing protein modules. For BLUF and LOV domains important parallels are emerging, despite their different alpha/beta fold arrangement, whereas there is increasing evidence for a mechanicistic and functional splitting of the cryptochrome family.     

DNA PHOTOLYASE FROM THE FUNGUS NEUROSPORA CRASSA. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES

Abstract A phr-gene from the filamentous fungus Neurosporu crassa was overexpressed in Escherichia coli cells, yielding a biologically active photolyase. After purification till apparent homogeneity, the 66 kDa protein was found to contain equimolar amounts of 5,1O-methenyltetrahydrofolic acid (MTHF) and FAD, classifying it as an MTHF-type photolyase. Compared to other MTHF photolyases the absorption maximum of Neurosporu photolyase is shifted from ca 380 nm to 391 nm (t = 34 800), while an additional shoulder is present at 465 nm. In dark-adapted enzyme the FAD chromophore is predominantly present in the oxidized form, in contrast with E. coli and Saccharomyces cerevisiue photolyase, which contain mainly semiquinone or fully reduced FAD, respectively. Preillumination or dithionite treatment converted oxidized FAD in Neurospora photolyase into the fully reduced form, with a concomitant shift of the absorption maximum from 391 to 396 nm and disappearance of the 465 nm shoulder. The action spectrum of photoreactivation coincides with the absorption spectrum of preilluminated (reduced) photolyase, extending the spectral region of MTHF-type photolyases from 380 till 396 nm. A quantum yield of 0.57 was obtained for the overall repair reaction. Comparison of spectral properties of FAD in Neurospora photolyase and the model compound lumiflavin points to an apolar microenvironment of photolyase-bound FAD. Neurosporu photolyase has distinct advantages over E. coli photolyase as it is more stable and contains a full complement of chromophores.