Circadian rhythms are determined by cell-autonomous transcription-translation feedback loops that entrain to environmental stimuli. In the model circadian clock of Drosophila melanogaster, the clock is set by the light-induced degradation of the core oscillator protein timeless (TIM) by the principal light-sensor cryptochrome (CRY). The cryo-EM structure of CRY bound to TIM revealed that within the extensive CRY:TIM interface, the TIM N-terminus binds into the CRY FAD pocket, in which FAD and the associated phosphate-binding loop (PBL) undergo substantial rearrangement. The TIM N-terminus involved in CRY binding varies in isoforms that facilitate the adaptation of flies to different light environments. Herein, we demonstrate, through peptide binding assays and pulsed-dipolar electron spin resonance (ESR) spectroscopy, that the TIM N-terminal peptide alone exhibits light-dependent binding to CRY and that the affinity of the interaction depends on the initiating methionine residue. Extensions to the TIM N-terminus that mimic less light-sensitive variants have substantially reduced interactions with CRY. Substitutions of CRY residues that couple to the flavin rearrangement in the CRY:TIM complex have dramatic effects on CRY light activation. CRY residues Arg237 on α8, Asn253, and Gln254 on the PBL are critical for the release of the CRY autoinhibitory C-terminal tail (CTT) and subsequent TIM binding. These key light-responsive elements of CRY are well conserved throughout Type I cryptochromes of invertebrates but not by cryptochromes of chordates and plants, which likely utilize a distinct light-activation mechanism.
more »
« less
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
- Award ID(s):
- 1658640
- NSF-PAR ID:
- 10450855
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Physiologia Plantarum
- Volume:
- 172
- Issue:
- 3
- ISSN:
- 0031-9317
- Page Range / eLocation ID:
- p. 1653-1661
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Cryptochromes are highly conserved blue light-absorbing flavoproteins which function as photoreceptors during plant development and in the entrainment of the circadian clock in animals. They have been linked to perception of electromagnetic fields in many organisms including plants, flies, and humans. The mechanism of magnetic field perception by cryptochromes is suggested to occur by the so-called radical pair mechanism, whereby the electron spins of radical pairs formed in the course of cryptochrome activation can be manipulated by external magnetic fields. However, the identity of the magnetosensitive step and of the magnetically sensitive radical pairs remains a matter of debate. Here we investigate the effect of a static magnetic field of 500 μT (10× earth's magnetic field) which was applied in the course of a series of iterated 5 min blue light/10 min dark pulses. Under the identical pulsed light conditions, cryptochrome responses were enhanced by a magnetic field even when exposure was provided exclusively in the 10 min dark intervals. However, when the magnetic stimulus was given exclusively during the 5 min light interval, no magnetic sensitivity could be detected. This result eliminates the possibility that magnetic field sensitivity could occur during forward electron transfer to the flavin in the course of the cryptochrome photocycle. By contrast, radical pair formation during cryptochrome flavin reoxidation would occur independently of light, and continue for minutes after the cessation of illumination. Our results therefore provide evidence that a magnetically sensitive reaction is entwined with dark-state processes following the cryptochrome photoreduction step.more » « less
-
more » « less
Abstract Many animals use the Earth’s geomagnetic field for orientation and navigation. Yet, the molecular and cellular underpinnings of the magnetic sense remain largely unknown. A biophysical model proposed that magnetoreception can be achieved through quantum effects of magnetically-sensitive radical pairs formed by the photoexcitation of cryptochrome (CRY) proteins. Studies in
Drosophila are the only ones to date to have provided compelling evidence for the ultraviolet (UV)-A/blue light-sensitive type 1 CRY (CRY1) involvement in animal magnetoreception, and surprisingly extended this discovery to the light-insensitive mammalian-like type 2 CRYs (CRY2s) of both monarchs and humans. Here, we show that monarchs respond to a reversal of the inclination of the Earth’s magnetic field in an UV-A/blue light and CRY1, but not CRY2, dependent manner. We further demonstrate that both antennae and eyes, which express CRY1, are magnetosensory organs. Our work argues that only light-sensitive CRYs function in animal light-dependent inclination-based magnetic sensing. -
Bollenbach, Tobias (Ed.)more » « less
The circadian clock is an evolutionarily-conserved molecular oscillator that enables species to anticipate rhythmic changes in their environment. At a molecular level, the core clock genes induce circadian oscillations in thousands of genes in a tissue–specific manner, orchestrating myriad biological processes. While previous studies have investigated how the core clock circuit responds to environmental perturbations such as temperature, the downstream effects of such perturbations on circadian regulation remain poorly understood. By analyzing bulk-RNA sequencing of
Drosophila fat bodies harvested from flies subjected to different environmental conditions, we demonstrate a highly condition-specific circadian transcriptome: genes are cycling in a temperature-specific manner, and the distributions of their phases also differ between the two conditions. Further employing a reference-based gene regulatory network (Reactome), we find evidence of increased gene-gene coordination at low temperatures and synchronization of rhythmic genes that are network neighbors. We report that the phase differences between cycling genes increase as a function of geodesic distance in the low temperature condition, suggesting increased coordination of cycling on the gene regulatory network. Our results suggest a potential mechanism whereby the circadian clock mediates the fly’s response to seasonal changes in temperature. -
more » « less
The members of the phytochrome (phy) family of bilin-containing photoreceptors are major regulators of plant photomorphogenesis through their unique ability to photointerconvert between a biologically inactive red light-absorbing Pr state and an active far-red light-absorbing Pfr state. While the initial steps in Pfr signaling are unclear, an early event for the phyB isoform after photoconversion is its redistribution from the cytoplasm into subnuclear foci known as photobodies (PBs), which dissipate after Pfr reverts back to Pr by far-red irradiation or by temperature-dependent nonphotochemical reversion. Here we present evidence that PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) functions both as an essential structural component of phyB-containing PBs and as a direct regulator of thermal reversion that is sufficient to stabilize phyB as Pfr in vitro. By examining the genetic interaction between a constitutively active phyBY276H-YFP allele (
YHB-YFP ) and PCH1, we show that the loss of PCH1 prevents YHB from coalescing into PBs without affecting its nuclear localization, whereas overexpression of PCH1 dramatically increases PB levels. Loss of PCH1, presumably by impacting phyB-PB assembly, compromises a number of events elicited inYHB-YFP plants, including their constitutive photomorphogenic phenotype, red light-regulated thermomorphogenesis, and input of phyB into the circadian clock. Conversely, elevated levels of both phyB and PCH1 generate stable, yet far-red light–reversible PBs that persisted for days. Collectively, our data demonstrate that the assembly of PCH1-containing PBs is critical for phyB signaling to multiple outputs and suggest that altering PB dynamics could be exploited to modulate plant responses to light and temperature.
