Plant Cryptochromes Research Articles

'Seeing' the electromagnetic spectrum: spotlight on the cryptochrome photocycle.

Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

A phytochrome/phototropin chimeric photoreceptor promotes growth of fern gametophytes under limited light conditions.

Many ferns thrive even in low-light niches such as under an angiosperm forest canopy. However, the shade adaptation strategy of ferns is not well understood. Phytochrome 3/neochrome (phy3/neo) is an unconventional photoreceptor, found in the fern Adiantum capillus-veneris, that controls both red and blue light-dependent phototropism and chloroplast photorelocation, which are considered to improve photosynthetic efficiency in ferns. Here we show that phy3/neo localizes not only at the plasma membrane but also in the nucleus. Since both phototropism and chloroplast photorelocation are mediated by membrane-associated phototropin photoreceptors, we speculated that nucleus-localized phy3/neo possesses a previously undescribed biological function. We reveal that phy3/neo directly interacts with Adiantum cryptochrome 3 (cry3) in the nucleus. Plant cryptochromes are blue light receptors that transcriptionally regulate photomorphogenesis; therefore, phy3/neo may function via cry3 to synchronize light-mediated development with phototropism and chloroplast photorelocation to promote fern growth under low-light conditions. Furthermore, we demonstrate that phy3/neo regulates the expression of the Cyclin-like gene AcCyc1 and promotes prothallium expansion growth. These findings provide insight into the shade adaptation strategy of ferns and suggest that phy3/neo plays a substantial role in the survival and growth of ferns during the tiny gametophytic stage under low-light conditions, such as those on the forest floor.

One More for Light-triggered Conformational Changes in Cryptochromes: CryP from Phaeodactylum tricornutum

Cryptochromes are a ubiquitously occurring class of photoreceptors. Together with photolyases, they form the Photolyase Cryptochrome Superfamily (PCSf) by sharing a common protein architecture and binding mode of the FAD chromophore. Despite these similarities, PCSf members exert different functions. Photolyases repair UV-induced DNA damage by photocatalytically driven electron transfer between FADH¯ and the DNA lesion, whereas cryptochromes are light-dependent signaling molecules and trigger various biological processes by photoconversion of their FAD redox and charge states. Given that most cryptochromes possess a C-terminal extension (CTE) of varying length, the functions of their CTE have not yet been fully elucidated and are hence highly debated. In this study, the role of the CTE was investigated for a novel subclass of the PCSf, the CryP-like cryptochromes, by hydrogen/deuterium exchange and mass-spectrometric analysis. Striking differences in the relative deuterium uptake were observed in different redox states of CryP from the diatom Phaeodactylum tricornutum. Based on these measurements we propose a model for light-triggered conformational changes in CryP-like cryptochromes that differs from other known cryptochrome families like the insect or plant cryptochromes.

BIC2, a Cryptochrome Function Inhibitor, Is Involved in the Regulation of ABA Responses in Arabidopsis.

The plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via regulating the expression of ABA response genes. BIC1 (Blue-light Inhibitor of Cryptochromes 1) and BIC2 have been identified as the inhibitors of plant cryptochrome functions, and are involved in the regulation of plant development and metabolism in Arabidopsis . In this study, we report the identification of BIC2 as a regulator of ABA responses in Arabidopsis . RT-PCR (Reverse Transcription-Polymerase Chain Reaction) results show that the expression level of BIC1 remained largely unchanged, but that of BIC2 increased significantly in response to ABA treatment. Transfection assays in Arabidopsis protoplasts show that both BIC1 and BIC2 were mainly localized in the nucleus, and were able to activate the expression of the co-transfected reporter gene. Results in seed germination and seedling greening assays show that ABA sensitivity was increased in the transgenic plants overexpressing BIC2, but increased slightly, if any, in the transgenic plants overexpressing BIC1. ABA sensitivity was also increased in the bic2 single mutants in seedling greening assays, but no further increase was observed in the bic1 bic2 double mutants. On the other hand, in root elongation assays, ABA sensitivity was decreased in the transgenic plants overexpressing BIC2, as well as the bic2 single mutants, but no further decrease was observed in the bic1 bic2 double mutants. By using qRT-PCR (quantitative RT-PCR), we further examined how BIC2 may regulate ABA responses in Arabidopsis , and found that inhibition of ABA on the expression of the ABA receptor genes PYL4 (PYR1-Like 4) and PYL5 were decreased, but promotion of ABA on the expression of the protein kinase gene SnRK2.6 (SNF1-Related Protein Kinases 2.6) was enhanced in both the bic1 bic2 double mutants and 35S:BIC2 overexpression transgenic plants. Taken together, our results suggest that BIC2 regulates ABA responses in Arabidopsis possibly by affecting the expression of ABA signaling key regulator genes.

Photochemistry and Signaling Activity of Plant Cryptochromes: A Review

Photochemistry and Signaling Activity of Plant Cryptochromes: A Review

Photochemistry and Signaling Activities of Plant Cryptochromes. Review

Cryptochromes compose the widespread class of blue-light sensory receptors that in plants regulate processes of development and circadian rhythm. These photoreceptors can also function as magnetoreceptors. Cryptochrome proteins bind flavin adenine dinucleotide (FAD) as a chromophore in the photolyase homology region (PHR) domain and contain the C-terminal extension (CCE) which is joined to PHR near the FAD-binding site. The cryptochrome activation is initiated by photochemical FAD conversions involving electron/proton transfer and the formation of redox forms. In plants, cryptochrome protein with photoreduced FAD undergoes conformational changes causing disengagement of the PHR domain and CCE that is accompanied by the formation of functionally active oligomers of cryptochrome molecules. Photooligomerization is considered as a key process necessary for cryptochrome signaling activity.

Effects of Dynamical Degrees of Freedom on Magnetic Compass Sensitivity: A Comparison of Plant and Avian Cryptochromes.

The magnetic compass of migratory birds is thought to rely on a radical pair reaction inside the blue-light photoreceptor protein cryptochrome. The sensitivity of such a sensor to weak external magnetic fields is determined by a variety of magnetic interactions, including electron-nuclear hyperfine interactions. Here, we investigate the implications of thermal motion, focusing on fluctuations in the dihedral and librational angles of flavin adenine dinucleotide (FAD) and tryptophan (Trp) radicals in cryptochrome 4a from European robin (Erithacus rubecula, ErCry4a) and pigeon (Columba livia, ClCry4a) and cryptochrome 1 from the plant Arabidopsis thaliana (AtCry1). Molecular dynamics simulations and density functional theory-derived hyperfine interactions are used to calculate the quantum yield of radical pair recombination dependent on the direction of the geomagnetic field. This quantity and various dynamical parameters are compared for [FAD•- Trp•+] in ErCry4a, ClCry4a, and AtCry1, with TrpC or TrpD being the third and fourth components of the tryptophan triad/tetrad in the respective proteins. We find that (i) differences in the average dihedral angles in the radical pairs are small, (ii) the librational motions of TrpC•+ in the avian cryptochromes are appreciably smaller than in AtCry1, (iii) the rapid vibrational motions of the radicals leading to strong fluctuations in the hyperfine couplings affect the spin dynamics depending on the usage of instantaneous or time-averaged interactions. Future investigations of radical pair compass sensitivity should therefore not be based on single snapshots of the protein structure but should include the ensemble properties of the hyperfine interactions.

Arabidopsis cryptochrome 2 forms photobodies with TCP22 under blue light and regulates the circadian clock

Cryptochromes are blue light receptors that regulate plant growth and development. They also act as the core components of the central clock oscillator in animals. Although plant cryptochromes have been reported to regulate the circadian clock in blue light, how they do so is unclear. Here we show that Arabidopsis cryptochrome 2 (CRY2) forms photobodies with the TCP22 transcription factor in response to blue light in plant cells. We provide evidence that PPK kinases influence the characteristics of these photobodies and that together these components, along with LWD transcriptional regulators, can positively regulate the expression of CCA1 encoding a central component of the circadian oscillator.

Stop CRYing! Inhibition of cryptochrome function by small proteins.

Plants can detect the presence of light using specialised photoreceptor proteins. These photoreceptors measure the intensity of light, but they can also respond to different spectra of light and thus ‘see' different colours. Cryptochromes, which are also present in animals, are flavin-based photoreceptors that enable plants to detect blue and ultraviolet-A (UV-A) light. In Arabidopsis, there are two cryptochromes, CRYPTOCHROME 1 (CRY1) and CRYPTOCHROME 2 (CRY2) with known sensory roles. They function in various processes such as blue-light mediated inhibition of hypocotyl elongation, photoperiodic promotion of floral initiation, cotyledon expansion, anthocyanin production, and magnetoreception, to name a few. In the dark, the cryptochromes are in an inactive monomeric state and undergo photochemical and conformational change in response to illumination. This results in flavin reduction, oligomerisation, and the formation of the ‘cryptochrome complexome'. Mechanisms of cryptochrome activation and signalling have been extensively studied and found to be conserved across phylogenetic lines. In this review, we will therefore focus on a far lesser-known mechanism of regulation that is unique to plant cryptochromes. This involves inhibition of cryptochrome activity by small proteins that prevent its dimerisation in response to light. The resulting inhibition of function cause profound alterations in economically important traits such as plant growth, flowering, and fruit production. This review will describe the known mechanisms of cryptochrome activation and signalling in the context of their modulation by these endogenous and artificial small inhibitor proteins. Promising new applications for biotechnological and agricultural applications will be discussed.

Plant Cryptochromes Illuminated: A Spectroscopic Perspective on the Mechanism.

Plant cryptochromes are central blue light receptors for the control of land plant and algal development including the circadian clock and the cell cycle. Cryptochromes share a photolyase homology region with about 500 amino acids and bind the chromophore flavin adenine dinucleotide. Characteristic for plant cryptochromes is a conserved aspartic acid close to flavin and an exceptionally long C-terminal extension. The mechanism of activation by excitation and reduction of the chromophore flavin adenine dinucleotide has been controversially discussed for many years. Various spectroscopic techniques have contributed to our understanding of plant cryptochromes by providing high time resolution, ambient conditions and even in-cell approaches. As a result, unifying and differing aspects of photoreaction and signal propagation have been revealed in comparison to members from other cryptochrome subfamilies. Here, we review the insight from spectroscopy on the flavin photoreaction in plant cryptochromes and present the current models on the signal propagation from flavin reduction to dissociation of the C-terminal extension.

C-Terminal Extension of a Plant Cryptochrome Dissociates from the β-Sheet of the Flavin-Binding Domain.

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting β-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the β-sheet signal, implying an association of the CCT close to the only β-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that β-sheet reorganization causes the CCT release and restructuring.

Cryptochromes and the Circadian Clock: The Story of a Very Complex Relationship in a Spinning World.

Cryptochromes are flavin-containing blue light photoreceptors, present in most kingdoms, including archaea, bacteria, plants, animals and fungi. They are structurally similar to photolyases, a class of flavoproteins involved in light-dependent repair of UV-damaged DNA. Cryptochromes were first discovered in Arabidopsis thaliana in which they control many light-regulated physiological processes like seed germination, de-etiolation, photoperiodic control of the flowering time, cotyledon opening and expansion, anthocyanin accumulation, chloroplast development and root growth. They also regulate the entrainment of plant circadian clock to the phase of light–dark daily cycles. Here, we review the molecular mechanisms by which plant cryptochromes control the synchronisation of the clock with the environmental light. Furthermore, we summarise the circadian clock-mediated changes in cell cycle regulation and chromatin organisation and, finally, we discuss a putative role for plant cryptochromes in the epigenetic regulation of genes.

Effect of temperature on the Arabidopsis cryptochrome photocycle.

Cryptochromes are blue light-absorbing photoreceptors found in plants and animals with many important signalling functions. These include control of plant growth, development, and the entrainment of the circadian clock. Plant cryptochromes have recently been implicated in adaptations to temperature variation, including temperature compensation of the circadian clock. However, the effect of temperature directly on the photochemical properties of the cryptochrome photoreceptor remains unknown. Here we show that the response to light of purified Arabidopsis Cry1 and Cry2 proteins was significantly altered by temperature. Spectral analysis at 15°C showed a pronounced decrease in flavin reoxidation rates from the biologically active, light-induced (FADH°) signalling state of cryptochrome to the inactive (FADox) resting redox state as compared to ambient (25°C) temperature. This result indicates that at low temperatures, the concentration of the biologically active FADH° redox form of Cry is increased, leading to the counterintuitive prediction that there should be an increased biological activity of Cry at lower temperatures. This was confirmed using Cry1 cryptochrome C-terminal phosphorylation as a direct biological assay for Cry activation in vivo. We conclude that enhanced cryptochrome function in vivo at low temperature is consistent with modulation by temperature of the cryptochrome photocycle.

Cryptochrome 1a depends on blue light fluence rate to mediate osmotic stress responses in tomato

The participation of plant cryptochromes in water deficit response mechanisms has been highlighted in several reports. However, the role of tomato (Solanum lycopersicum L.) cryptochrome 1a (cry1a) in the blue light fluence-dependent modulation of the water deficit response remains largely elusive. The tomato cry1a mutant and its wild-type counterpart were grown in water (no stress) or PEG6000 (osmotic stress) treatments under white light (60 μmol m−2 s-1) or from low to high blue light fluence (1, 5, 10, 15 and 25 μmol m−2 s-1). We first demonstrate that under nonstress conditions cry1a regulates seedling growth by mechanisms that involve pigmentation, lipid peroxidation and osmoprotectant accumulation in a blue light-dependent manner. In addition, we further highlighted under osmotic stress conditions that cry1a increased tomato growth by reduced malondialdehyde (MDA) and proline accumulation. Although blue light is an environmental signal that influences osmotic stress responses mediated by tomato cry1a, specific blue light fluence rates are required during these responses. Here, we show that CRY1a manipulation may be a potential biotechnological target to develop a drought-tolerant tomato variety. Nevertheless, the complete understanding of this phenomenon requires further investigation.

Structural insights into photoactivation of plant Cryptochrome-2

Cryptochromes (CRYs) are evolutionarily conserved photoreceptors that mediate various light-induced responses in bacteria, plants, and animals. Plant cryptochromes govern a variety of critical growth and developmental processes including seed germination, flowering time and entrainment of the circadian clock. CRY’s photocycle involves reduction of their flavin adenine dinucleotide (FAD)-bound chromophore, which is completely oxidized in the dark and semi to fully reduced in the light signaling-active state. Despite the progress in characterizing cryptochromes, important aspects of their photochemistry, regulation, and light-induced structural changes remain to be addressed. In this study, we determine the crystal structure of the photosensory domain of Arabidopsis CRY2 in a tetrameric active state. Systematic structure-based analyses of photo-activated and inactive plant CRYs elucidate distinct structural elements and critical residues that dynamically partake in photo-induced oligomerization. Our study offers an updated model of CRYs photoactivation mechanism as well as the mode of its regulation by interacting proteins.

The human CRY1 tail controls circadian timing by regulating its association with CLOCK:BMAL1

Circadian rhythms are generated by interlocked transcription-translation feedback loops that establish cell-autonomous biological timing of ∼24 h. Mutations in core clock genes that alter their stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR-tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR-tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity.

Coordinating light responses between the nucleus and the chloroplast, a role for plant cryptochromes and phytochromes.

To promote photomorphogenesis, including plastid development and metabolism, the phytochrome (phy) and the cryptochrome (cry) photoreceptors orchestrate genome-wide changes in gene expression in response to Red (R)- and Blue (B)-light cues. While phys and crys have a clear role in modulating photosynthesis, their role in the coordination of the nuclear genome and the plastome, essential for functional chloroplasts, remains underexplored. Using publicly available genome datasets for WT and phyABCDE or cry1cry2 Arabidopsis seedlings, grown, respectively, under R- or B-light, we bioinformatically analyzed the influence of light inputs and photoreceptors in the control of nuclear genes with a function in the chloroplast, and evaluated the role of phyB in the modulation of plastome-encoded genes. We show gene co-induction by R-phys and B-crys for genes with a chloroplastic function, and also apparent photoreceptor-driven preferential responses. Evidence from phyB in Arabidopsis together with published evidence from CRY2 in tomato also supports the participation of both photoreceptor families in the global modulation of the plastome genes. To begin addressing how these light-sensors orchestrate changes in an organellar genome, we evaluated their effect over genes with potential functions in plastid gene-expression regulation based on their TAIR annotation. Results indicate that both crys and phys modulate 'plastome-regulatory genes' with enrichment in the contribution of crys to all processes and of phys to post-transcription and transcription. Furthermore, we identified a new role for HY5 as a relevant light-signaling component in photoreceptor-based anterograde signaling leading to plastome gene regulation.

The oligomeric structures of plant cryptochromes

Cryptochromes (CRYs) are a group of evolutionarily conserved flavoproteins found in many organisms. In plants, the well-studied CRY photoreceptor, activated by blue light, plays essential roles in plant growth and development. However, the mechanism of activation remains largely unknown. Here, we determined the oligomeric structures of the blue-light-perceiving PHR domain of Zea mays CRY1 and an Arabidopsis CRY2 constitutively active mutant. The structures form dimers and tetramers whose functional importance is examined in vitro and in vivo with Arabidopsis CRY2. Structure-based analysis suggests that blue light may be perceived by CRY to cause conformational changes, whose precise nature remains to be determined, leading to oligomerization that is essential for downstream signaling. This photoactivation mechanism may be widely used by plant CRYs. Our study reveals a molecular mechanism of plant CRY activation and also paves the way for design of CRY as a more efficient optical switch.

Efficient Analytic Second Derivative of Electrostatic Embedding QM/MM Energy: Normal Mode Analysis of Plant Cryptochrome.

Analytic second derivatives of electrostatic embedding (EE) quantum mechanics/molecular mechanics (QM/MM) energy are important for performing vibrational analysis and simulating vibrational spectra of quantum systems interacting with an environment represented as a classical electrostatic potential. The main bottleneck of EE-QM/MM second derivatives is the solution of coupled perturbed equations for each MM atom perturbation. Here, we exploit the Q-vector method [J. Chem. Phys., 2019, 151, 041102] to workaround this bottleneck. We derive the full analytic second derivative of the EE-QM/MM energy, which allows us to compute QM, MM, and QM-MM Hessian blocks in an efficient and easy to implement manner. To show the capabilities of our method, we compute the normal modes for the full Arabidopsis thaliana plant cryptochrome. We show that the flavin adenine dinucleotide vibrations (QM subsystem) strongly mix with protein modes. We compute approximate vibronic couplings for the lowest bright transition, from which we extract spectral densities and the homogeneous broadening of FAD absorption spectrum in protein using vibrationally resolved electronic spectrum simulations.

A Tail of Night Owls: The Mammalian Cry1 C-Terminal Tail Controls Circadian Timing by Regulating its Association to CLOCK:BMAL1

Circadian rhythms are generated by a transcription-translation feedback loop that establishes cell-autonomous biological timing of ∼24-hours. A prevalent human variation in the core clock gene cryptochrome 1, Cry1Δ11, lengthens circadian period to cause Delayed Sleep Phase Disorder (DSPD). CRY1 has a 55 kDa photolyase homology region (PHR) followed by a ∼100 residue tail that is intrinsically disordered; the Δ11 variant lacks a short segment encoded by Exon 11 within its tail. Using fluorescence polarization binding assays and carbon detected NMR spectroscopy, we show here that the disordered tail of CRY1 interacts directly with its PHR, Furthermore we use fluorescence polarization and biolayer interferometry to show that Exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and CLOCK, a subunit of the primary circadian transcription factor. Competition between PER2 and the tail for the CRY1 PHR suggests a regulatory role for the tail in the early morning, when CRY1 binds to CLOCK:BMAL1 on DNA independently of PER2. Discovery of this autoregulatory role for mammalian CRY1 highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity.