Circadian Gating Research Articles

Circadian and environmental signal integration in a natural population of Arabidopsis

Plants sense and respond to environmental cues during 24 h fluctuations in their environment. This requires the integration of internal cues such as circadian timing with environmental cues such as light and temperature to elicit cellular responses through signal transduction. However, the integration and transduction of circadian and environmental signals by plants growing in natural environments remains poorly understood. To gain insights into 24 h dynamics of environmental signaling in nature, we performed a field study of signal transduction from the nucleus to chloroplasts in a natural population of Arabidopsis halleri. Using several modeling approaches to interpret the data, we identified that the circadian clock and temperature are key regulators of this pathway under natural conditions. We identified potential time-delay steps between pathway components, and diel fluctuations in the response of the pathway to temperature cues that are reminiscent of the process of circadian gating. We found that our modeling framework can be extended to other signaling pathways that undergo diel oscillations and respond to environmental cues. This approach of combining studies of gene expression in the field with modeling allowed us to identify the dynamic integration and transduction of environmental cues, in plant cells, under naturally fluctuating diel cycles.

Circadian Gating of Thyroid Hormone Action in Hepatocytes.

Thyroid hormones, thyroxin (T4) and the biologically active triiodothyronine (T3), play important roles in liver metabolic regulation, including fatty acid biosynthesis, beta-oxidation, and cholesterol homeostasis. These functions position TH signaling as a potential target for the treatment of metabolic dysfunction-associated steatotic liver disease (MASLD). Elevated T3 levels in the circulation are associated with increased hepatic lipid turnover, which is also under the control of the circadian clock system. In this study, we developed a cell system to study the impact of hepatocyte circadian rhythms on the metabolic response to T3 treatment under control and steatotic conditions. Synchronized AML-12 circadian reporter hepatocytes were treated with T3 at different circadian phases and metabolic conditions. T3 treatment increased metabolic activity in a dose-independent fashion and had no significant effect on circadian rhythms in AML-12 cells. T3 had marked time-of-treatment-dependent effects on metabolic transcript expression. Steatosis induction altered metabolic transcript expression in AML-12 cells. In this condition, the circadian rhythm period was lengthened, and this effect was independent of T3. Under steatotic conditions, T3 had marked time-of-treatment dependent effects on metabolic transcript expression, which differed from those observed under control conditions. These findings reveal a time-of-day-dependent response of hepatocytes to T3, which is further modulated by the metabolic state. Our data suggest that time has a strong influence on liver TH action, which might be considered when treating MASLD.

Circadian control of tumor immunosuppression affects efficacy of immune checkpoint blockade.

The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation has an essential function in host defense and tumor immunosurveillance. Here we use a single-cell RNA sequencing approach and a genetic model of colorectal cancer to identify clock-dependent changes to the immune landscape that control the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8+ T cells. Of these immunosuppressive cell types, PD-L1-expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Disruption of the epithelial cell clock regulates the secretion of cytokines that promote heightened inflammation, recruitment of neutrophils and the subsequent development of MDSCs. We also show that time-of-day anti-PD-L1 delivery is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, these data indicate that circadian gating of tumor immunosuppression informs the timing and efficacy of immune checkpoint inhibitors.

Dynamic encoding of temperature in the central circadian circuit coordinates physiological activities

The circadian clock regulates animal physiological activities. How temperature reorganizes circadian-dependent physiological activities remains elusive. Here, using in-vivo two-photon imaging with the temperature control device, we investigated the response of the Drosophila central circadian circuit to temperature variation and identified that DN1as serves as the most sensitive temperature-sensing neurons. The circadian clock gate DN1a’s diurnal temperature response. Trans-synaptic tracing, connectome analysis, and functional imaging data reveal that DN1as bidirectionally targets two circadian neuronal subsets: activity-related E cells and sleep-promoting DN3s. Specifically, behavioral data demonstrate that the DN1a-E cell circuit modulates the evening locomotion peak in response to cold temperature, while the DN1a-DN3 circuit controls the warm temperature-induced nocturnal sleep reduction. Our findings systematically and comprehensively illustrate how the central circadian circuit dynamically integrates temperature and light signals to effectively coordinate wakefulness and sleep at different times of the day, shedding light on the conserved neural mechanisms underlying temperature-regulated circadian physiology in animals.

Timed receptor tyrosine kinase signaling couples the central and a peripheral circadian clock in Drosophila

Circadian clocks impose daily periodicities to behavior, physiology, and metabolism. This control is mediated by a central clock and by peripheral clocks, which are synchronized to provide the organism with a unified time through mechanisms that are not fully understood. Here, we characterized in Drosophila the cellular and molecular mechanisms involved in coupling the central clock and the peripheral clock located in the prothoracic gland (PG), which together control the circadian rhythm of emergence of adult flies. The time signal from central clock neurons is transmitted via small neuropeptide F (sNPF) to neurons that produce the neuropeptide Prothoracicotropic Hormone (PTTH), which is then translated into daily oscillations of Ca2+ concentration and PTTH levels. PTTH signaling is required at the end of metamorphosis and transmits time information to the PG through changes in the expression of the PTTH receptor tyrosine kinase (RTK), TORSO, and of ERK phosphorylation, a key component of PTTH transduction. In addition to PTTH, we demonstrate that signaling mediated by other RTKs contributes to the rhythmicity of emergence. Interestingly, the ligand to one of these receptors (Pvf2) plays an autocrine role in the PG, which may explain why both central brain and PG clocks are required for the circadian gating of emergence. Our findings show that the coupling between the central and the PG clock is unexpectedly complex and involves several RTKs that act in concert and could serve as a paradigm to understand how circadian clocks are coordinated.

Transcriptional dynamics in source-sink tissues identifies molecular factors regulating the corm development process in saffron (Crocus sativus L.).

Geophytic plants have evolved to develop underground storage organs (USO) in the active growing season to withstand harsh environments as well as to coordinate growth and reproduction when conditions are favourable. Saffron is an autumn flowering geophyte and an expensive spice crop restricted to certain geographical locations in the world. Saffron, being sterile, does not produce seeds and thus propagates only through corms, the quality of which determines its yield. Corm development in saffron is unexplored and the underlying molecular mechanism is still elusive. In this study, we performed an extensive characterisation of the transcriptional dynamics in the source (leaf) and sink (corm) tissues during corm development in saffron. Via morphological and transcriptome studies, we identified molecular factors regulating corm development process in saffron, which defined corm development into three stages: the initiation stage demonstrates enhanced vegetative growth aboveground and swelling of shoot base belowground due to active cell division & carbohydrate storage; the bulking stage comprises of increased source and sink strength, active photosynthesis, circadian gating and starch accumulation; the maturation stage represents reduced source and sink strength, lowered photosynthesis, sugar transport, starch synthesis and cell cycle arrest. The global view of transcriptional changes in source and sink identifies similar and new molecular factors involved in the saffron corm development process compared to USO formation in other geophytes and provides a valuable resource for dissecting the molecular network underlying the corm development. We propose a hypothetical model based on data analysis, of how molecular factors via environmental cues can regulate the corm development process in saffron.

Investigating Circadian Gating of Temperature Responsive Genes.

Understanding gene expression dynamics in the context of the time of day and temperature response is an important part of understanding plant thermotolerance in a changing climate. Performing "gating" experiments under constant conditions and light-dark cycles allows users to identify and dissect the contribution of the time of day and circadian clock to the dynamic nature of stress-responsive genes. Here, we describe the design of specific laboratory experiments in plants (Arabidopsis thaliana and bread wheat, Triticum aestivum) to investigate temporal responses to heat (1h at 37°C) or cold (3h at 4°C), and we include known marker genes that have circadian-gated responses to temperature changes.

Cytonuclear interactions modulate the plasticity of photosynthetic rhythmicity and growth in wild barley.

In plants, the contribution of the plasmotype (mitochondria and chloroplast) in controlling the circadian clock plasticity and possible consequences on cytonuclear genetic makeup have yet to be fully elucidated. A genome-wide association study in the wild barley (Hordeum vulgare ssp. spontaneum) B1K collection identified overlap with our previously mapped DRIVERS OF CLOCKS (DOCs) loci in wild-cultivated interspecific population. Moreover, we identified non-random segregation and epistatic interactions between nuclear DOCs loci and the chloroplastic RpoC1 gene, indicating an adaptive value for specific cytonuclear gene combinations. Furthermore, we show that DOC1.1, which harbours the candidate SIGMA FACTOR-B (SIG-B) gene, is linked with the differential expression of SIG-B and CCA1 genes and contributes to the circadian gating response to heat. High-resolution temporal growth and photosynthesis measurements of B1K also link the DOCs loci to differential growth, Chl content and quantum yield. To validate the involvement of the Plastid encoded polymerase (PEP) complex, we over-expressed the two barley chloroplastic RpoC1 alleles in Arabidopsis and identified significant differential plasticity under elevated temperatures. Finally, enhanced clock plasticity of de novo ENU (N-Ethyl-N-nitrosourea) -induced barley rpoB1 mutant further implicates the PEP complex as a key player in regulating the circadian clock output. Overall, this study highlights the contribution of specific cytonuclear interaction between rpoC1 (PEP gene) and SIG-B with distinct circadian timing regulation under heat, and their pleiotropic effects on growth implicate an adaptive value.

Time-dependent regulation of respiration is widespread across plant evolution.

Establishing the temperature dependence of respiration is critical for accurate predictions of the global carbon cycle under climate change. Diurnal temperature fluctuations, or changes in substrate availability, lead to variations in leaf respiration. Additionally, recent studies hint that the thermal sensitivity of respiration could be time-dependent. However, the role for endogenous processes, independent from substrate availability, as drivers of temporal changes in the sensitivity of respiration to temperature across phylogenies has not yet been addressed. Here, we examined the diurnal variation in the response of respiration to temperatures (R-T relationship) for different lycophyte, fern, gymnospermand angiosperm species. We tested whether time-dependent changes in the R-T relationship would impact leaf level respiration modelling. We hypothesized that interactions between endogenous processes, like the circadian clock, and leaf respiration would be independent from changes in substrate availability. Overall, we observed a time-dependent sensitivity in the R-T relationship across phylogenies, independent of temperature, that affected modelling parameters. These results are compatible with circadian gating of respiration, but further studies should analyse the possible involvement of the clock. Our results indicate time-dependent regulation of respiration might be widespread across phylogenies, and that endogenous regulation of respiration is likely affecting leaf-level respiration fluxes.

Genome-wide circadian gating of a cold temperature response in bread wheat.

Circadian rhythms coordinate the responses of organisms with their daily fluctuating environments, by establishing a temporal program of gene expression. This schedules aspects of metabolism, physiology, development and behaviour according to the time of day. Circadian regulation in plants is extremely pervasive, and is important because it underpins both productivity and seasonal reproduction. Circadian regulation extends to the control of environmental responses through a regulatory process known as circadian gating. Circadian gating is the process whereby the circadian clock regulates the response to an environmental cue, such that the magnitude of response to an identical cue varies according to the time of day of the cue. Here, we show that there is genome-wide circadian gating of responses to cold temperatures in plants. By using bread wheat as an experimental model, we establish that circadian gating is crucial to the programs of gene expression that underlie the environmental responses of a crop of major socioeconomic importance. Furthermore, we identify that circadian gating of cold temperature responses are distributed unevenly across the three wheat subgenomes, which might reflect the geographical origins of the ancestors of modern wheat.

LNKs-RVEs complex ticks in the circadian gating of plant temperature stress responses

Recently, Kidokoro et al. found that protein complex LNK3,4-RVE4,8 and LNK1,2-RVE4,8 of the circadian clock modulates plant cold- and high-temperature tolerance, respectively. Here, we reviewed the discovery of LNKs, the dynamically formed morning-phased clock complexes, and their critical role on endogenous circadian rhythms. In addition, we summarized the research work on LNKs with the interacting proteins RVEs, CCA1 in temperature responses and discussed how the circadian clock confer increased fitness via gating the rhythmic expression of their target genes.

The FLOWERING LOCUS T gene expression is controlled by high-irradiance response and external coincidence mechanism in long days in Arabidopsis.

In natural long days, the florigen gene FLOWERING LOCUS T (FT) shows a bimodal expression pattern with morning and dusk peaks in Arabidopsis. This pattern differs from the one observed in the laboratory, and little is known about underlying mechanisms. A red : far-red (R : FR) ratio difference between sunlight and fluorescent light causes this FT pattern mismatch. We showed that bimodal FT expression patterns were induced in a day longer than 14 h with sunlight R : FR (= c. 1) conditions. By circadian gating experiments, we found that cumulative exposure of R : FR-adjusted light (R : FR ratio was adjusted to 1 with FR supplement) spanning from the afternoon to the next morning required full induction of FT in the morning. Conversely, only 2 h of R : FR adjustment in the late afternoon was sufficient for FT induction at dusk. We identified that phytochrome A (phyA) is required for the morning FT expression in response to the R : FR adjustment on the previous day. As a part of this mechanism, we showed that PHYTOCHROME-INTERACTING FACTOR 7 contributes to FT regulation. Our results suggest that phyA-mediated high-irradiance response and the external coincidence mechanism contribute to morning FT induction under natural long-day conditions.

Circadian gating of light-induced arousal in Drosophila sleep

Circadian rhythms and sleep homeostasis constitute the two-process model for daily sleep regulation. However, evidence for circadian control of sleep-wake cycles has been relatively short since clock-less animals often show sleep behaviors quantitatively comparable to wild-type. Here we examine Drosophila sleep behaviors under different light-dark regimes and demonstrate that circadian clocks gate light-induced arousal. Genetic excitation of tyrosine decarboxylase 2 (TDC2)-expressing neurons suppressed sleep more evidently at night, causing nocturnal activity. The arousal effects were likely mediated in part by glutamate transmission from the octopaminergic neurons and substantially masked by light. Application of T12 cycles (6-h light: 6-h dark) further showed that the light-sensitive effects of TDC2 neurons depended on the time of the day. In particular, light-sensing via visual input pathway led to strong sleep suppression at subjective night, and such an effect disappeared in clock-less mutants. Transgenic mapping revealed that light-induced arousal and free-running behavioral rhythms require distinct groups of circadian pacemaker neurons. These results provide convincing evidence that circadian control of sleep is mediated by the dedicated clock neurons for light-induced arousal.

Cell size homeostasis under the circadian regulation of cell division in cyanobacteria

Bacterial cells maintain their characteristic cell size over many generations. Several rod-shaped bacteria, such as Escherichia coli and the cyanobacteria Synechococcus elongatus, divide after adding a constant length to their length at birth. Through this division control known as the adder mechanism, perturbation in cell length due to physiological fluctuation decays over generations at a rate of 2−1 per cell division. However, previous experiments have shown that the circadian clock in cyanobacteria reduces cell division frequency at a specific time of day under constant light. This circadian gating should modulate the division control by the adder mechanism, but its significance remains unknown. Here we address how the circadian gating affects cell length, doubling time, and cell length stability in cyanobacteria by using mathematical models. We show that a cell subject to circadian gating grows for a long time, and gives birth to elongated daughter cells. These elongated daughter cells grow faster than the previous generation, as elongation speed is proportional to cell length and divide in a short time before the next gating. Hence, the distributions of doubling time and cell length become bimodal, as observed in experimental data. Interestingly, the average doubling time over the population of cells is independent of gating because the extension of doubling time by gating is compensated by its reduction in the subsequent generation. On the other hand, average cell length is increased by gating, suggesting that the circadian clock controls cell length. We then show that the decay rate of perturbation in cell length depends on the ratio of delay in division by the gating τG to the average doubling time τ0 as 2τG/τ0−1. We estimated τG≈2.5, τ0≈13.6 hours, and τG/τ0≈0.18 from experimental data, indicating that a long doubling time in cyanobacteria maintains the decay rate similar to that of the adder mechanism. Thus, our analysis suggests that the acquisition of the circadian clock during evolution did not impose a constraint on cell size homeostasis in cyanobacteria.

OS038 - Targeting the liver circadian clock by REV-ERB-alpha activation improves liver fibrosis by circadian gating of TGF-beta signaling

OS038 - Targeting the liver circadian clock by REV-ERB-alpha activation improves liver fibrosis by circadian gating of TGF-beta signaling

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

External and Internal Reshaping of Plant Thermomorphogenesis.

Plants dynamically adapt to changing temperatures to ensure propagation and reproductive success, among which morphogenic responses to warm temperatures have been extensively studied in recent years. As readily inferred from the cyclic co-oscillations of environmental cues in nature, plant thermomorphogenesis is coordinately reshaped by various external conditions. Accumulating evidence supports that internal and developmental cues also contribute to harmonizing thermomorphogenic responses. The external and internal reshaping of thermomorphogenesis is facilitated by versatile temperature sensing and interorgan communication processes, circadian and photoperiodic gating of thermomorphogenic behaviors, and their metabolic coordination. Here, we discuss recent advances in plant thermal responses with focus on the diel and seasonal reshaping of thermomorphogenesis and briefly explore its application to developing climate-smart crops.

GmLCLs negatively regulate ABA perception and signalling genes in soybean leaf dehydration response.

The circadian clock allows plants to actively adapt to daily environmental changes through temporal regulation of physiological traits. In response to drought stress, circadian oscillators gate ABA signalling, but the molecular mechanisms remain unknown, especially in crops. Here, we investigated the role of soybean circadian oscillators GmLCLa1, GmLCLa2, GmLCLb1 and GmLCLb2 in leaf water stress response. Under dehydration stress, the GmLCL quadruple mutant had decreased leaf water loss. We found that the dehydration treatment delayed the peak expression of GmLCL genes by 4 hr. In addition, the circadian clock in hairy roots also responded to ABA, which led to a free-running rhythm with shortened period. Importantly, in the gmlclq quadruple mutant, diurnal expression phases of several circadian-regulated ABA receptor, ABA catabolism and ABA signalling-related genes were shifted significantly to daytime. Moreover, in the gmlclq mutant leaf, expression of GmPYL17, GmCYP707A, GmABI2 and GmSnRK2s was increased under water dehydration stress. In summary, our results show that GmLCLs act as negative regulators of ABA signalling in leaves during dehydration response.

Circadian gating of dark-induced increases in chloroplast- and cytosolic-free calcium in Arabidopsis.

Summary Changes in the spatiotemporal concentration of free Ca2+ ([Ca2+]) in different organelles of the cell contribute to responses of plants to physiological and environmental stimuli. One example are [Ca2+] increases in the stroma of chloroplasts during light‐to‐dark transitions; however, the function and mechanisms responsible are unknown, in part because there is a disagreement in the literature concerning whether corresponding dark‐induced changes in cytosolic [Ca2+] ([Ca2+]cyt) can be detected.We have measured changes in [Ca2+]cyt upon darkness in addition to the already known dark‐induced increases in [Ca2+]stroma in the aerial part of the Arabidopsis thaliana plant.These [Ca2+]cyt transients depend on the photoperiod and time of day, peaking at anticipated dusk, and are superimposed on daily 24 h oscillations in [Ca2+]cyt. We also find that the magnitude of the dark‐induced increases in Ca2+ in both the cytosol and chloroplasts are gated by the nuclear circadian oscillator.The modulation of the magnitude of dark‐induced increases in [Ca2+]stroma and [Ca2+]cyt by transcriptional regulators in the nucleus that are part of the circadian oscillator demonstrates a new role for the circadian system in subcellular Ca2+ signalling, in addition to its role in driving circadian oscillations of [Ca2+] in the cytosol and chloroplasts.

The ELF3-PIF7 Interaction Mediates the Circadian Gating of the Shade Response in Arabidopsis.

SummaryLight filtered through dense planting initiates the shade avoidance syndrome (SAS) in plants, which helps them compete against their neighbors. Quantitative trait loci (QTL)-based analysis identified the nighttime-expressed clock component ELF3 as a new player in the SAS, but its detailed mechanism is unclear. Here, we show that the circadian clock gates shade-induced gene expression and hypocotyl elongation at night. ELF3 is involved in nighttime suppression via interaction with and inactivation of PHYTOCHROME-INTERACTING FACTOR 7 (PIF7). Loss of function of ELF3 restores the shade induction, which is largely reduced in the absence of PIF7, indicating that ELF3 acts upstream of PIF7. Finally, we found that the repressive activity of ELF3 on the shade response is stronger under short days than under long days. Our results reveal that the interaction between ELF3 and PIF7 mediates the circadian gating of the SAS, which coordinates the daily control of physiological outputs.