Evolution of Proteins of the DNA Photolyase/Cryptochrome Family.

Proteins of the cryptochrome/DNA photolyase family (CPF) are phylogenetically related and structurally conserved flavoproteins that perform various functions. DNA photolyases repair DNA damage caused by UV-B radiation by exposure to UV-A/blue light simultaneously or subsequently. Cryptochromes are photoreceptor proteins regulating circadian clock, morphogenesis, phototaxis, and other responses to UV and blue light in various organisms. The review describes the structure and functions of CPF proteins, their evolutionary relationship, and possible functions of the CPF ancestor protein.

Rapid allopolyploid radiation of moonwort ferns (Botrychium; Ophioglossaceae) revealed by PacBio sequencing of homologous and homeologous nuclear regions.

Polyploidy is a major speciation process in vascular plants, and is postulated to be particularly important in shaping the diversity of extant ferns. However, limitations in the availability of bi-parental markers for ferns have greatly limited phylogenetic investigation of polyploidy in this group. With a large number of allopolyploid species, the genus Botrychium is a classic example in ferns where recurrent polyploidy is postulated to have driven frequent speciation events. Here, we use PacBio sequencing and the PURC bioinformatics pipeline to capture all homeologous or allelic copies of four long (∼1 kb) low-copy nuclear regions from a sample of 45 specimens (25 diploids and 20 polyploids) representing 37 Botrychium taxa, and three outgroups. This sample includes most currently recognized Botrychium species in Europe and North America, and the majority of our specimens were genotyped with co-dominant nuclear allozymes to ensure species identification. We analyzed the sequence data using maximum likelihood (ML) and Bayesian inference (BI) concatenated-data ("gene tree") approaches to explore the relationships among Botrychium species. Finally, we estimated divergence times among Botrychium lineages and inferred the multi-labeled polyploid species tree showing the origins of the polyploid taxa, and their relationships to each other and to their diploid progenitors. We found strong support for the monophyly of the major lineages within Botrychium and identified most of the parental donors of the polyploids; these results largely corroborate earlier morphological and allozyme-based investigations. Each polyploid had at least two distinct homeologs, indicating that all sampled polyploids are likely allopolyploids (rather than autopolyploids). Our divergence-time analyses revealed that these allopolyploid lineages originated recently-within the last two million years-and thus that the genus has undergone a recent radiation, correlated with multiple independent allopolyploidizations across the phylogeny. Also, we found strong parental biases in the formation of allopolyploids, with individual diploid species participating multiple times as either the maternal or paternal donor (but not both). Finally, we discuss the role of polyploidy in the evolutionary history of Botrychium and the interspecific reproductive barriers possibly involved in these parental biases.

DASH-type cryptochromes regulate fruiting body development and secondary metabolism differently than CmWC-1 in the fungus Cordyceps militaris.

Cryptochromes (CRYs) belong to the photolyase/cryptochrome flavoprotein family, which is widely distributed in all kingdoms. A phylogenetic analysis indicated that three Cordyceps militaris proteins [i.e., cryptochrome DASH (CmCRY-DASH), (6-4) photolyase, and cyclobutane pyrimidine dimer (CPD) class I photolyase] belong to separate fungal photolyase/cryptochrome subfamilies. CmCRY-DASH consists of DNA photolyase and flavin adenine dinucleotide-binding domains, with RGG repeats in a C-terminal extension. Considerably, more carotenoids and cordycepin accumulated in the ΔCmcry-DASH strain than in the wild-type or ΔCmwc-1 strains, indicating an inhibitory role for CmCRY-DASH in these biosynthetic pathways. Fruiting body primordia could form in the ΔCmcry-DASH strain, but the fruiting bodies were unable to elongate normally, differently from the Cmwc-1 disruption strain, where primordium differentiation did not occur. Cmcry-DASH expression is induced by light in the wild-type strain, but not in the ΔCmwc-1 strain. CmCRY-DASH is also necessary for the expression of Cmwc-1, implying that Cmcry-DASH and Cmwc-1 exhibit interdependent expression. The Cmvvd expression levels in the wild-type and ΔCmcry-DASH strains increased considerably following irradiation, while Cmvvd expression in the ΔCmwc-1 strain was not induced by light. It is speculated that the photo adaptation may be faster in the Cmcry-DASH mutant based on Cmvvd transcript dynamics. These results provide new insights into the biological functions of fungal DASH CRYs. Furthermore, the DASH CRYs may regulate fruiting body development and secondary metabolism differently than WC-1.

Phylogenetic and Functional Classification of the Photolyase/Cryptochrome Family.

The photolyase/cryptochrome (PHR/CRY) family is a large group of proteins with similar structure but very diverge functions such as DNA repair, circadian clock resetting and regulation of transcription. As a result of advances in the biochemistry of the CRY/PHR family and identification of new members, several adjustments have been made to the classification of this protein family. For example, a new class of PHRs, Class III, has been proposed. Furthermore, CRYs have been suggested to function as photosensory proteins in the primordial eye of sponge larvae. Additionally, a magnetosensory function has been attributed to certain CRYs. Recent advances in the field enabled us to propose a comprehensive classification scheme and nomenclatural system for this family. This review focuses on the computational and biochemical classifications of the PHR/CRY family. Several examples show that computational analysis can give a hinge about the function of newly discovered members before performing any biochemical study.

Identification of zebrafish magnetoreceptor and cryptochrome homologs.

Magnetoreception is essential for magnetic orientation in animal migration. The molecular basis for magnetoreception has recently been elucidated in fruitfly as complexes between the magnetic receptor magnetoreceptor (MagR) and its ligand cryptochrome (Cry). MagR and Cry are present in the animal kingdom. However, it is unknown whether they perform a conserved role in diverse animals. Here we report the identification and expression of zebrafish MagR and Cry homologs towards understanding their roles in lower vertebrates. A single magr gene and 7 cry genes are present in the zebrafish genome. Zebrafish has four cry1 genes (cry1aa, cry1ab, cry1ba and cry1bb) homologous to human CRY1 and a single ortholog of human CRY2 as well as 2 cry-like genes (cry4 and cry5). By RT-PCR, magr exhibited a high level of ubiquitous RNA expression in embryos and adult organs, whereas cry genes displayed differential embryonic and adult expression. Importantly, magr depletion did not produce apparent abnormalities in organogenesis. Taken together, magr and cry2 exist as a single copy gene, whereas cry1 exists as multiple gene duplicates in zebrafish. Our result suggests that magr may play a dispensable role in organogenesis and predicts a possibility to generate magr mutants for analyzing its role in zebrafish.

Historical biogeography of the fern genus Deparia (Athyriaceae) and its relation with polyploidy.

The wide geographical distribution of many fern species is related to their high dispersal ability. However, very limited studies surveyed biological traits that could contribute to colonization success after dispersal. In this study, we applied phylogenetic approaches to infer historical biogeography of the fern genus Deparia (Athyriaceae, Eupolypods II). Because polyploids are suggested to have better colonization abilities and are abundant in Deparia, we also examined whether polyploidy could be correlated to long-distance dispersal events and whether polyploidy could play a role in these dispersals/establishment and range expansion. Maximum likelihood and Bayesian phylogenetic reconstructions were based on a four-region combined cpDNA dataset (rps16-matK IGS, trnL-L-F, matK and rbcL; a total of 4252 characters) generated from 50 ingroup (ca. 80% of the species diversity) and 13 outgroup taxa. Using the same sequence alignment and maximum likelihood trees, we carried out molecular dating analyses. The resulting chronogram was used to reconstruct ancestral distribution using the DEC model and ancestral ploidy level using ChromEvol. We found that Deparia originated around 27.7Ma in continental Asia/East Asia. A vicariant speciation might account for the disjunctive distribution of East Asia-northeast North America. There were multiple independent long-distance dispersals to Africa/Madagascar (at least once), Southeast Asia (at least once), south Pacific islands (at least twice), Australia/New Guinea/New Zealand (at least once), and the Hawaiian Islands (at least once). In particular, the long-distance dispersal to the Hawaiian Islands was associated with polyploidization, and the dispersal rate was slightly higher in the polyploids than in diploids. Moreover, we found five species showing recent infraspecific range expansions, all of which took place concurrently with polyploidization. In conclusion, our study provides the first investigation using phylogenetic and biogeographic analyses trying to explore the link between historical biogeography and ploidy evolution in a fern genus and our results imply that polyploids might be better colonizers than diploids.

Evolutionary History of the Photolyase/Cryptochrome Superfamily in Eukaryotes.

BACKGROUND: 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. RESULTS: 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.

dbCRY: a Web-based comparative and evolutionary genomics platform for blue-light receptors.

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. Database URL: http://www.dbcryptochrome.org/

A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of light-harvesting protein levels.

Diatoms possess several genes for proteins of the cryptochrome/photolyase family. A typical sequence for a plant cryptochrome was not found in our analysis of the Phaeodactylum tricornutum genome, but one protein grouped with higher plant and green algal cryptochromes. This protein, CryP, binds FAD and 5,10-methenyltetrahydrofolate, according to our spectroscopic studies on heterologously expressed protein. 5,10-Methenyltetrahydrofolate binding is a feature common to both cyclobutane pyrimidine dimer photolyases and DASH cryptochromes. In recombinant CryP, however, the FAD chromophore was present in its neutral radical state and had a red-shifted absorption maximum at 637 nm, which is more characteristic for a DASH cryptochrome than a cyclobutane pyrimidine dimer photolyase. Upon illumination with blue light, the fully reduced state of FAD was formed in the presence of reductant. Expression of CryP was silenced by antisense approaches, and the resulting cell lines showed increased levels of proteins of light-harvesting complexes, the Lhcf proteins, in vivo. In contrast, the levels of proteins active in light protection, the Lhcx proteins, were reduced. Thus, CryP cannot be directly grouped with known members of the cryptochrome/photolyase family. Of all P. tricornutum proteins, it is the most similar in sequence to a plant cryptochrome, and is involved in the regulation of light-harvesting protein expression, but shows spectroscopic features and a chromophore composition that are most typical of a DASH cryptochrome.

CryB from Rhodobacter sphaeroides: a unique class of cryptochromes with new cofactors.

Cryptochromes and photolyases are structurally related but have different biological functions in signalling and DNA repair. Proteobacteria and cyanobacteria harbour a new class of cryptochromes, called CryPro. We have solved the 2.7 Å structure of one of its members, cryptochrome B from Rhodobacter sphaeroides, which is a regulator of photosynthesis gene expression. The structure reveals that, in addition to the photolyase-like fold, CryB contains two cofactors only conserved in the CryPro subfamily: 6,7-dimethyl-8-ribityl-lumazine in the antenna-binding domain and a [4Fe-4S] cluster within the catalytic domain. The latter closely resembles the iron-sulphur cluster harbouring the large primase subunit PriL, indicating that PriL is evolutionarily related to the CryPro class of cryptochromes.

The cryptochromes: blue light photoreceptors in plants and animals.

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

Cryptochrome genes are highly expressed in the ovary of the African clawed frog, Xenopus tropicalis.

Cryptochromes (CRYs) are flavoproteins sharing high homology with photolyases. Some of them have function(s) including transcription regulation in the circadian clock oscillation, blue-light photoreception for resetting the clock phase, and light-dependent magnetoreception. Vertebrates retain multiple sets of CRY or CRY-related genes, but their functions are yet unclear especially in the lower vertebrates. Although CRYs and the other circadian clock components have been extensively studied in the higher vertebrates such as mice, only a few model species have been studied in the lower vertebrates. In this study, we identified two CRYs, XtCRY1 and XtCRY2 in Xenopus tropicalis, an excellent experimental model species. Examination of tissue specificity of their mRNA expression by real-time PCR analysis revealed that both the XtCRYs showed extremely high mRNA expression levels in the ovary. The mRNA levels in the ovary were about 28-fold (XtCry1) and 48-fold (XtCry2) higher than levels in the next abundant tissues, the retina and kidney, respectively. For the functional analysis of the XtCRYs, we cloned circadian positive regulator XtCLOCK and XtBMAL1, and found circadian enhancer E-box in the upstream of XtPer1 gene. XtCLOCK and XtBMAL1 exhibited strong transactivation from the XtPer1 E-box element, and both the XtCRYs inhibited the XtCLOCK:XtBMAL1-mediated transactivation, thereby suggesting this element to drive the circadian transcription. These results revealed a conserved main feedback loop in the X. tropicalis circadian clockwork and imply a possible physiological importance of CRYs in the ovarian functions such as synthesis of steroid hormones and/or control of estrus cycles via the transcription regulation.