Class of photoreceptors in plants and animals
Cryptochromes (from the Greek κρυπτός χρώμα, "hidden colour") are a class of flavoproteins found in plants and animals that are sensitive to blue light. They are involved in the circadian rhythms and the sensing of magnetic fields in a number of species. The name "cryptochrome" was proposed as a "portmanteau" combining the "chromatic" nature of the photoreceptor, and the "cryptogamic" organisms on which many blue-light studies were carried out.
The genes "CRY1" and "CRY2" encode the proteins CRY1 and CRY2, respectively. Cryptochromes are classified into plant Cry and animal Cry. Animal Cry can be further categorized into insect type (Type I) and mammal-like (Type II). CRY1 is a circadian photoreceptor whereas CRY2 is a clock repressor which represses Clock/Cycle (Bmal1) complex in insects and vertebrates. In plants, blue-light photoreception can be used to cue developmental signals. Besides chlorophylls, cryptochromes are the only proteins known to form photoinduced radical-pairs "in vivo". These appear to enable some animals to detect magnetic fields.
Cryptochromes have been the focus of several current efforts in optogenetics. Employing transfection, initial studies on yeast have capitalized on the potential of CRY2 heterodimerization to control cellular processes, including gene expression, by light.
Discovery.
Although Charles Darwin first documented plant responses to blue light in the 1880s, it was not until the 1980s that research began to identify the pigment responsible. In 1980, researchers discovered that the HY4 gene of the plant "Arabidopsis thaliana" was necessary for the plant's blue light sensitivity, and, when the gene was sequenced in 1993, it showed high sequence homology with photolyase, a DNA repair protein activated by blue light. Reference sequence analysis of cryptochrome-1 isoform d shows two conserved domains with photolyase proteins. Isoform d nucleotide positions 6 through 491 show a conserved domain with deoxyribodipyrimidine photolyase, and positions 288 through 486 show a conserved domain with the FAD binding domain of DNA photolyase. Comparative genomic analysis supports photolyase proteins as the ancestors of cryptochromes. However, by 1995 it became clear that the products of the HY4 gene and its two human homologs did not exhibit photolyase activity and were instead a new class of blue light photoreceptor hypothesized to be circadian photopigments. In 1996 and 1998, "Cry" homologs were identified in "Drosophila" and mice, respectively.
Evolutionary history.
Cryptochromes (CRY1, CRY2) are evolutionarily old and highly conserved proteins that belong to the flavoproteins superfamily that exists in all kingdoms of life. Cryptochromes are derived from and closely related to photolyases, which are bacterial enzymes that are activated by light and involved in the repair of UV-induced DNA damage.
In eukaryotes, cryptochromes no longer retain this original enzymatic activity. By using a T-DNA labeled allele of the "cry1" gene in the "Arabidopsis" plant, researchers determined that the "cry1" gene encoded a flavoprotein without photolyase activity and with a unique C-terminal tail. The protein encoded by this gene was named cryptochrome 1 to distinguish it from its ancestral photolyase proteins and was found to be involved in the photoreception of blue light. Studies of "Drosophila cry-"knockout mutants led to the later discovery that cryptochrome proteins are also involved in regulating the mammalian circadian clock. The "Drosophila cry" gene similarly encodes a flavoprotein without photolyase activity that also binds pterin chromophores. "Cry" mutants ("cryb)" were found to express arrhythmic levels of luciferase as well as PER and TIM proteins in photoreceptor cells. Despite the arrhythmicity of these protein levels, "cryb" mutants still showed rhythmicity in overall behavior but could not entrain to short pulses of light, leading researchers to conclude that the dorsal and ventral lateral neurons (the primary pacemaker cells of "Drosophila)" were still functioning effectively. When "cryb" mutants also had visually unresponsive compound eyes, though, they failed to behaviorally entrain to environmental cues. These findings led researchers to conclude that the cryptochrome protein encoded by "cry" is necessary for "Drosophila" photoentrainment. In mammals, a protein analog of the "Drosophila" cryptochrome protein was discovered with the characteristic property of lacking photolyase activity, prompting researchers to consider it in the same class of cryptochrome proteins. In mice, the greatest "cry1" expression is observed in the suprachiasmatic nucleus (SCN) where levels rhythmically fluctuate. Due to the role of the SCN as the primary mammalian pacemaker as well as the rhythmic fluctuations in "cry1" expression, researchers concluded "cry1" was also necessary for the entrainment of mammalian circadian rhythms.
A common misconception in the evolutionary history of cryptochrome proteins is that mammalian and plant proteins are orthologs of each other that evolved directly from a shared photolyase gene. However, genomic analysis indicates that mammalian and fly cryptochrome proteins show greater sequence similarity to the (6-4) photolyase proteins than to plant cryptochrome proteins. It is therefore likely that plant and animal cryptochrome proteins show a unique case of convergent evolution by repeatedly evolving new functions independently of each other from a single common ancestral "cry" gene.
Research by Worthington et al. (2003) indicates that cryptochromes first evolved in bacteria and were identified in "Vibrio cholerae". Genome sequencing of this bacteria identified three genes in the photolyase/cryptochrome family, all of which have the folate and flavin cofactors characteristic of these proteins. Of these genes, one encodes a photolyase, while the other two encode cryptochrome proteins designated VcCry1 and VcCry2. Cashmore AR et al. (1999) hypothesize that mammalian cryptochromes developed later in evolutionary history shortly after plants and animals diverged based on conserved genomic domains between animal cryptochromes and the "Arabidopsis" (6-4) photolyase protein. Based on the role of cryptochromes in the entrainment of mammalian circadian rhythms, current researchers hypothesize that they developed simultaneously with the coevolution of PER, TIM, CLOCK, and CYCLE proteins, but there is currently insufficient evidence to determine the exact evolution timing and mechanism of evolution.
Structure.
All members of the flavoprotein superfamily have the characteristics of an N-terminal photolyase homology (PHR) domain. The PHR domain can bind to the flavin adenine dinucleotide (FAD) cofactor and a light-harvesting chromophore. The structure of cryptochrome involves a fold very similar to that of photolyase, arranged as an orthogonal bundle with a single molecule of FAD noncovalently bound to the protein. These proteins have variable lengths and surfaces on the C-terminal end, due to the changes in genome and appearance that result from the lack of DNA repair enzymes. The Ramachandran plot shows that the secondary structure of the CRY1 protein is primarily a right-handed alpha helix with little to no steric overlap. The structure of CRY1 is almost entirely made up of alpha helices, with several loops and few beta sheets.
Function.
Phototropism.
In plants, cryptochromes mediate phototropism, or directional growth toward a light source, in response to blue light. This response is now known to have its own set of photoreceptors, the phototropins.
Unlike phytochromes and phototropins, cryptochromes are not kinases. Their flavin chromophore is reduced by light and transported into the cell nucleus, where it affects the turgor pressure and causes subsequent stem elongation. To be specific, "Cry2" is responsible for blue-light-mediated cotyledon and leaf expansion. "Cry2" overexpression in transgenic plants increases blue-light-stimulated cotyledon expansion, which results in many broad leaves and no flowers rather than a few primary leaves with a flower. A double loss-of-function mutation in Arabidopsis thaliana Early Flowering 3 (elf3) and Cry2 genes delays flowering under continuous light and was shown to accelerate it during long and short days, which suggests that Arabidopsis CRY2 may play a role in accelerating flowering time during continuous light.
Photomorphogenesis.
Cryptochromes receptors cause plants to respond to blue light via photomorphogenesis. They help control seed and seedling development, as well as the switch from the vegetative to the flowering stage of development.
In "Arabidopsis", CRY1 is the primary inhibitor of hypocotyl elongation but CRY2 inhibits hypocotyl elongation under low blue light intensity. CRY2 promotes flowering under long-day conditions.
CRY gene mediates photomorphogenesis in several ways. CRY C-terminal interacts with CONTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a E3 ubiquitin ligase that represses photomorphogenesis and flowering time. The interaction inhibits COP1 activity and allows transcription factors such as ELONGATED HYPOCOTYL 5 (HY5) to accumulate. HY5 is a basic leucine zipper (bZIP) factor that promotes photomorphogenesis by binding to light-responsive genes. CRY interacts with G protein β-subunit AGB1, where HY5 dissociates from AGB1 and becomes activated. CRY interacts with PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PIF5, repressors of photomorphogenesis and promoter of hypocotyl elongation, to repress PIF4 and PIF5 transcription activity. Lastly, CRY can inhibit auxin and brassinosterioid (BR) signaling to promote photomorphogenesis.
Light capture.
Despite much research on the topic, cryptochrome photoreception and phototransduction in "Drosophila" and "Arabidopsis thaliana" is still poorly understood. Cryptochromes are known to possess two chromophores: pterin (in the form of 5,10-methenyltetrahydrofolic acid (MTHF)) and flavin (in the form of FAD). Both may absorb a photon, and in "Arabidopsis", pterin appears to absorb at a wavelength of 380 nm and flavin at 450 nm. Past studies have supported a model by which energy captured by pterin is transferred to flavin. Under this model of phototransduction, FAD would then be reduced to FADH, which probably mediates the phosphorylation of a certain domain in cryptochrome. This could then trigger a signal transduction chain, possibly affecting gene regulation in the cell nucleus.
A new hypothesis proposes that partner molecules sense the transduction of a light signal into a chemical signal in plant cryptochromes, which could be triggered by a photo-induced negative charge on the FAD cofactor or on the neighboring aspartic acid within the protein. This negative charge would electrostatically repel the protein-bound ATP molecule and thereby also the protein C-terminal domain, which covers the ATP binding pocket prior to photon absorption. The resulting change in protein conformation could lead to phosphorylation of previously inaccessible phosphorylation sites on the C-terminus and the given phosphorylated segment could then liberate the transcription factor HY5 by competing for the same binding site at the negative regulator of photomorphogenesis COP1.
A different mechanism may function in "Drosophila". The true ground state of the flavin cofactor in "Drosophila" CRY is still debated, with some models indicating that the FAD is in an oxidized form, while others support a model in which the flavin cofactor exists in anion radical form, FAD-•. Recently, researchers have observed that oxidized FAD is readily reduced to FAD-• by light. Furthermore, mutations that blocked photoreduction had no effect on light-induced degradation of CRY, while mutations that altered the stability of FAD-• destroyed CRY photoreceptor function. These observations provide support for a ground state of FAD-•. Researchers have also recently proposed a model in which FAD- is excited to its doublet or quartet state by absorption of a photon, which then leads to a conformational change in the CRY protein.
Also the ring eyes of the demosponge larva of "Amphimedon queenslandica" express a blue-light-sensitive cryptochrome (Aq-Cry2), which might mediate phototaxis. In contrast, the eyes of most animals use photo-sensitive opsins expressed in photoreceptor cells, which communicate information about light from the environment to the nervous system. However, "A. queenslandica" lacks a nervous system, like other sponges. And it does not have an opsin gene in its fully sequenced genome either, despite having many other G-protein-coupled receptors (GPCRs). Therefore, the sponge's unique eyes must have evolved a different mechanism to detect light and mediate phototaxis, possibly with cryptochromes or other proteins.
Iris function.
Isolated irises constrict in response to light via a photomechanical transduction response (PMTR) in a variety of species and require either melanopsin or cryptochrome to do so. The iris of chicken embryos senses short-wavelength light via a cryptochrome, rather than opsins. Research by Margiotta and Howard (2020) shows that the PMTR of the chicken iris striated muscle occurs with "CRY" gene activation by 430 nm blue light. The PMTR was inhibited in "CRY" gene knockouts and decreased when flavin reductase was inhibited, but remained intact with the addition of melanopsin antagonists. Similarly, cytosolic "CRY1" and "CRY2" proteins were found in iris myotubes, and decreasing transcription of these genes inhibited PMTRs. The greatest iris PMTRs therefore correspond with the development of striated, rather than smooth, muscle fibers through "CRY"-mediated PMTRs.
Circadian rhythm.
Studies in animals and plants suggest that cryptochromes play a pivotal role in the generation and maintenance of circadian rhythms. Similarly, cryptochromes play an important role in the entrainment of circadian rhythms in plants. In "Drosophila", cryptochrome (dCRY) acts as a blue-light photoreceptor that directly modulates light input into the circadian clock, while in mammals, cryptochromes (CRY1 and CRY2) act as transcription repressors within the circadian clockwork. Some insects, including the monarch butterfly, have