Circadian Network Research Articles

Comparative physiological and transcriptomic analysis of pear leaves under distinct training systems

Canopy architecture is critical in determining the light interception and distribution, and subsequently the photosynthetic efficiency and productivity. However, the physiological responses and molecular mechanisms by which pear canopy architectural traits impact on photosynthesis remain poorly understood. Here, physiological investigations coupled with comparative transcriptomic analyses were performed in pear leaves under distinct training systems. Compared with traditional freestanding system, flat-type trellis system (DP) showed higher net photosynthetic rate (PN) levels at the most time points throughout the entire monitored period, especially for the interior of the canopy in sunny side. Gene ontology analysis revealed that photosynthesis, carbohydrate derivative catabolic process and fatty acid metabolic process were over-represented in leaves of DP system with open-canopy characteristics. Weighted gene co-expression network analysis uncovered a significant network module positive correlated with PN value. The hub genes (PpFKF1 and PpPRR5) of the module were enriched in circadian rhythm pathway, suggesting a functional role for circadian clock genes in mediating photosynthetic performance under distinct training systems. These results draw a link between pear photosynthetic response and specific canopy architectural traits, and highlight light harvesting and circadian clock network as potential targets for the input signals from the fluctuating light availability under distinct training systems.

Rapid Jetlag Resetting of Behavioral, Physiological, and Molecular Rhythms in Proestrous Female Mice.

A gradual adaptation to a shifted light-dark (LD) cycle is a key element of the circadian clock system and believed to be controlled by the central circadian pacemaker, the suprachiasmatic nucleus (SCN). Endocrine factors have a strong influence on the regulation of the circadian clock network and alter acute photic responses of the SCN clock. In females, endocrine function depends on the stage of the ovarian cycle. So far, however, little is known about the effect of the estrous cycle on behavioral and molecular responses to shifts in the LD rhythm. Based on this, we investigated whether estrous state affects the kinetics of phase shift during jetlag in behavior, physiology, and molecular clock rhythms in the SCN and in peripheral tissues. Female mice exposed to an advanced LD phase at proestrous or metestrous showed different phase-shift kinetics, with proestrous females displaying accelerated adaptation in behavior and physiology. Constant darkness release experiments suggest that these fast phase shifts do not reflect resetting of the SCN pacemaker. Explant experiments on SCN, adrenal gland, and uterus confirmed this finding with proestrous females showing significantly faster clock phase shifts in peripheral tissues compared with the SCN. Together, these findings provide strong evidence for an accelerated adaptation of proestrous compared with metestrous females to new LD conditions that is accompanied by rapid behavioral, physiological, and molecular rhythm resetting. Not only do these findings open up a new avenue to understand the effect of estrous cycle on the clock network under changing environmental conditions but also imply a greater susceptibility in proestrous females.

Circadian Structural Plasticity Drives Remodeling of E Cell Output

Behavioral outputs arise as a result of highly regulated yet flexible communication among neurons. The Drosophila circadian network includes 150 neurons that dictate the temporal organization of locomotor activity; under light-dark (LD) conditions, flies display a robust bimodal pattern. The pigment-dispersing factor (PDF)-positive small ventral lateral neurons (sLNv) have been linked to the generation of the morning activity peak (the "M cells"), whereas the Cryptochrome (CRY)-positive dorsal lateral neurons (LNds) and the PDF-negative sLNv are necessary for the evening activity peak (the "E cells") [1, 2]. While each group directly controls locomotor output pathways [3], an interplay between them along with a third dorsal cluster (the DN1ps) is necessary for the correct timing of each peak and for adjusting behavior to changes in the environment [4-7]. M cells set the phase of roughly half of the circadian neurons (including the E cells) through PDF [5, 8-10]. Here, we show the existence of synaptic input provided by the evening oscillator onto the M cells. Both structural and functional approaches revealed that E-to-M cell connectivity changes across the day, with higher excitatory input taking place before the day-to-night transition. We identified two different neurotransmitters, acetylcholine and glutamate, released by E cells that are relevant for robust circadian output. Indeed, we show that acetylcholine is responsible for the excitatory input from E cells to M cells, which show preferential responsiveness to acetylcholine during the evening. Our findings provide evidence of an excitatory feedback between circadian clusters and unveil an important plastic remodeling of the E cells' synaptic connections.

Expansion of the circadian transcriptome in Brassica rapa and genome-wide diversification of paralog expression patterns.

An important challenge of crop improvement strategies is assigning function to paralogs in polyploid crops. Here we describe the circadian transcriptome in the polyploid crop Brassica rapa. Strikingly, almost three-quarters of the expressed genes exhibited circadian rhythmicity. Genetic redundancy resulting from whole genome duplication is thought to facilitate evolutionary change through sub- and neo-functionalization among paralogous gene pairs. We observed genome-wide expansion of the circadian expression phase among retained paralogous pairs. Using gene regulatory network models, we compared transcription factor targets between B. rapa and Arabidopsis circadian networks to reveal evidence for divergence between B. rapa paralogs that may be driven in part by variation in conserved non-coding sequences (CNS). Additionally, differential drought response among retained paralogous pairs suggests further functional diversification. These findings support the rapid expansion and divergence of the transcriptional network in a polyploid crop and offer a new approach for assessing paralog activity at the transcript level.

Temperature robustness in Arabidopsis circadian clock models is facilitated by repressive interactions, autoregulation, and three-node feedbacks

The biological interactions underpinning the Arabidopsis circadian clock have been systematically uncovered and explored by biological experiments and mathematical models. This is captured by a series of published ordinary differential equation (ODE) models, which describe plant clock dynamics in response to light/dark conditions. However, understanding the role of temperature in resetting the clock (entrainment) and the mechanisms by which circadian rhythms maintain a near-24 h period over a range of temperatures (temperature compensation) is still unclear. Understanding entrainment and temperature compensation may elucidate the principles governing the structure of the circadian clock network. Here we explore the design principles of the Arabidopsis clock and its responses to changes in temperature. We analyse published clock models of Arabidopsis, spanning a range of complexity, and incorporate temperature-dependent dynamics into the parameters of translation rates in these models, to discern which regulatory patterns may best explain clock function and temperature compensation. We additionally construct three minimal clock models and explore what key features govern their rhythmicity and temperature robustness via a series of random parameterisations. Results show that the highly repressive interactions between the components of the plant clock, together with autoregulation patterns and three-node feedback loops, are associated with circadian function of the clock in general, and enhance its robustness to temperature variation in particular. However, because the networks governing clock function vary with time due to light and temperature conditions, we emphasise the importance of studying plant clock functionality in its entirety rather than as a set of discrete regulation patterns.

Brassinosteroids Regulate Circadian Oscillation via the BES1/TPL-CCA1/LHY Module in Arabidopsisthaliana.

SummaryBrassinosteroids (BRs) regulate a variety of physiological processes in plants via extensive crosstalk with diverse biological signaling networks. Although BRs are known to reciprocally regulate circadian oscillation, the molecular mechanism underlying BR-mediated regulation of circadian clock remains unknown. Here, we demonstrate that the BR-activated transcription factor bri1-EMS-SUPPRESSOR 1 (BES1) integrates BR signaling into the circadian network in Arabidopsis. BES1 repressed expression of CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) at night by binding to their promoters, together with TOPLESS (TPL). The repression of CCA1 and LHY by BR treatment, which occurred during the night, was compromised in bes1-ko and tpl-8 mutants. Consistently, long-term treatment with BR shortened the circadian period, and BR-induced rhythmic shortening was impaired in bes1-ko and tpl-8 single mutants and in the cca1-1lhy-21 double mutant. Overall, BR signaling is conveyed to the circadian oscillator via the BES1/TPL-CCA1/LHY module, contributing to gating diurnal BR responses in plants.

Ammonia exposure causes lung injuries and disturbs pulmonary circadian clock gene network in a pig study

Ammonia toxicity to respiratory system in pig faming is of particular concern, but the molecular mechanism remains still unclear. The present study was devoted to assess the impacts of the ammonia exposure on the lung tissues based on a pig study using 80 ppm ammonia exposing to piglets for different days. The histology analysis revealed ammonia exposure induced lung injury and inflammatory response, as indicated by epithelial-mesenchymal transition (EMT), significant thickening of alveolar septa, infiltration of inflammatory cells and excessive mucus production. The transcriptome analysis revealed many more up-regulated genes in exposure groups when compared with the control group, and these genes were significantly enriched in the GO term of extracellular exosome, proteolysis, and regulation of circadian rhythm. The study discovered the induction of seven genes (CRY2, CIART, CREM, NR1D1, NR1D2, PER1 and PER3) that encode repressors of circadian clock. One gene (ARNTL) that encodes activator of circadian clock was down-regulated after ammonia exposure. The results of this study suggest that ammonia exposure disturbed the pulmonary circadian clock gene expression, which may establish new evidence for further understanding the toxicity of ammonia to lungs.

Heritable gene expression variability and stochasticity govern clonal heterogeneity in circadian period.

A ubiquitous feature of the circadian clock across life forms is its organization as a network of cellular oscillators, with individual cellular oscillators within the network often exhibiting considerable heterogeneity in their intrinsic periods. The interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock's entrainability, but our knowledge of mechanisms governing period heterogeneity within circadian clock networks remains largely elusive. In this study, we aimed to explore the principles that underlie intercellular period variation in circadian clock networks (clonal period heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 to 28 h. We report that a single parent clone can produce progeny clones with a wide distribution of circadian periods, and this heterogeneity, in addition to being stochastically driven, has a heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our clone panel, we found that inheritance of expression patterns in at least three clock genes might govern clonal period heterogeneity in circadian clock networks. Furthermore, we provide evidence suggesting that heritable epigenetic variation in gene expression regulation might underlie period heterogeneity.

Gap Junction Protein INNEXIN2 Modulates the Period of Activity-Rest Rhythms in <i>Drosophila Melanogaster</i>

The circadian neuronal circuit of Drosophila melanogaster is made up of about 150 neurons, distributed bilaterally and distinguished into 7 clusters. Multiple lines of evidence suggest that coherent rhythms in behaviour are brought about when these clusters function as a network. Although chemical modes of communication amongst circadian neurons have been well-studied, there has been no report of communication via electrical synapses made up of gap junctions. Here, we report for the first time that gap junction proteins - Innexins play crucial roles in determining the period of free-running activity rhythms in flies. Our experiments reveal the presence of gap junction protein INNEXIN2 in the ventral lateral neurons. RNA-interference based knockdown of its expression in circadian pacemakers slows down the speed of locomotor activity rhythm. Concomitantly, we find alterations in the oscillation of a core-clock protein PERIOD and in the output molecule Pigment Dispersing Factor in the circadian pacemaker neuron network.

Global Hopf bifurcation in networks with fast feedback cycles

<p style='text-indent:20px;'>Autonomous sustained oscillations are ubiquitous in living and nonliving systems. As open systems, far from thermodynamic equilibrium, they defy entropic laws which mandate convergence to stationarity. We present structural conditions on network cycles which support global Hopf bifurcation, i.e. global bifurcation of non-stationary time-periodic solutions from stationary solutions. Specifically, we show how monotone feedback cycles of the linearization at stationary solutions give rise to global Hopf bifurcation, for sufficiently dominant coefficients along the cycle. <p style='text-indent:20px;'>We include four example networks which feature such strong feedback cycles of length three and larger: Oregonator chemical reaction networks, Lotka-Volterra ecological population dynamics, citric acid cycles, and a circadian gene regulatory network in mammals. Reaction kinetics in our approach are not limited to mass action or Michaelis-Menten type.

Sites of Circadian Clock Neuron Plasticity Mediate Sensory Integration and Entrainment

Networks of circadian timekeeping in the brain display marked daily changes in neuronal morphology. In Drosophila melanogaster, the striking daily structural remodeling of the dorsal medial termini of the small ventral lateral neurons has long been hypothesized to mediate endogenous circadian timekeeping. To test this model, we have specifically abrogated these sites of daily neuronal remodeling through the reprogramming of neural development and assessed the effects on circadian timekeeping and clock outputs. Remarkably, the loss of these sites has no measurable effects on endogenous circadian timekeeping or on any of the major output functions of the small ventral lateral neurons. Rather, their loss reduces sites of glutamatergic sensory neurotransmission that normally encodes naturalistic time cues from the environment. These results support an alternative model: structural plasticity in critical clock neurons is the basis for proper integration of light and temperature and gates sensory inputs into circadian clock neuron networks.

Segregation of an MSH1 RNAi transgene produces heritable non-genetic memory in association with methylome reprogramming

MSH1 is a plant-specific protein. RNAi suppression of MSH1 results in phenotype variability for developmental and stress response pathways. Segregation of the RNAi transgene produces non-genetic msh1 ‘memory’ with multi-generational inheritance. First-generation memory versus non-memory comparison, and six-generation inheritance studies, identifies gene-associated, heritable methylation repatterning. Genome-wide methylome analysis integrated with RNAseq and network-based enrichment studies identifies altered circadian clock networks, and phytohormone and stress response pathways that intersect with circadian control. A total of 373 differentially methylated loci comprising these networks are sufficient to discriminate memory from nonmemory full sibs. Methylation inhibitor 5-azacytidine diminishes the differences between memory and wild type for growth, gene expression and methylation patterning. The msh1 reprogramming is dependent on functional HISTONE DEACETYLASE 6 and methyltransferase MET1, and transition to memory requires the RNA-directed DNA methylation pathway. This system of phenotypic plasticity may serve as a potent model for defining accelerated plant adaptation during environmental change.

Clock-in, clock-out: circadian timekeeping between tissues.

Life evolved in the presence of alternating periods of light and dark that accompany the daily rotation of the Earth on its axis. This offered an advantage for organisms able to regulate their physiology to anticipate these daily cycles. In each light-sensitive organism studied, spanning single-celled bacteria to complex mammals, there exist timekeeping mechanisms able to control physiology over the course of 24 hours. Endowed with internal timekeeping, organisms can put their previously stored energy to the most efficient use, selectively ramping up biological processes at specific times of day or night according to when they'll be needed. Humans have evolved to be more active during the day (diurnal), likely due to the increased opportunities for foraging or hunting in our evolutionary past, and this daily activity is accompanied by an upregulation of genes involved in metabolism to increase the energy available for such behaviours. Remarkably, this happens without conscious thought, due to a complex organism-wide signalling apparatus known as the circadian clock network, that conveys time information between cells and tissues.

Circadian neuronal networks

Whatever you were doing just before reading this, walking to work, drinking a coffee after lunch or winding down in the evening in your favourite armchair, it is likely you will do it again tomorrow, more or less at the same time. This daily cycle is a reflection of the fact that we live in a 24-hour environment that, through rhythmic changes in light, temperature and social interactions, imposes temporal constraints on our lives. However, we do not simply react to those daily changes; rather, we anticipate them and behave accordingly.

MYC-Associated Factor MAX is a Regulator of the Circadian Clock.

The circadian transcriptional network is based on a competition between transcriptional activator and repressor complexes regulating the rhythmic expression of clock-controlled genes. We show here that the MYC-associated factor X, MAX, plays a repressive role in this network and operates through a MYC-independent binding to E-box-containing regulatory regions within the promoters of circadian BMAL1 targets. We further show that this “clock” function of MAX is required for maintaining a proper circadian rhythm and that MAX and BMAL1 contribute to two temporally alternating transcriptional complexes on clock-regulated promoters. We also identified MAX network transcriptional repressor, MNT, as a fundamental partner of MAX-mediated circadian regulation. Collectively, our data indicate that MAX regulates clock gene expression and contributes to keeping the balance between positive and negative elements of the molecular clock machinery.

The Circadian Clock Gene, TaPRR1, Is Associated With Yield-Related Traits in Wheat (Triticum aestivum L.).

Timing of flowering is crucial for the transformation from vegetative to reproductive growth in the important food crop, wheat (Triticum aestivum L.). The circadian clock is a central part of photoperiod regulation, with Pseudo-Response Regulators (PRRs) representing key components of circadian networks. However, little is known about the effects of PRR family members on yield-related traits in crop plants. In this study, we identified polymorphisms and haplotypes of TaPRR1, demonstrating that natural variations in TaPRR1 are associated with significant differences in yield-related traits including heading date, plant height and thousand grain weight. TaPRR1-6A-Hapla showed an earlier heading date, advanced by 0.9 to 1.7%. TaPRR1-6B-Hapla and TaPRR1-6D-Hapla displayed reduced plant height and increased thousand grain weight of up to 13.3 to 26.4% and 6.3 to 17.3%, respectively. Subcellular localization and transcriptional activity analysis showed that TaPRR1 is a nuclear localization protein with transcriptional activity controlled by an IR domain. The expression profiles of TaPRR1 genes over a 48-h period were characterized by circadian rhythms, which had two peaks under both short- and long- day conditions. In addition, geographical distribution analysis indicated higher distribution frequencies of TaPRR1-6A-Hapla, TaPRR1-6B-Haplb, and TaPRR1-6D-Haplb in different agro-ecological production zones. Furthermore, analysis of molecular variance of the distribution frequency of TaPRR1 haplotypes suggested significant differences in haplotype distribution frequency between landraces and modern cultivars. Our study provides a basis for in-depth understanding of TaPRR1 function on yield-related traits in wheat, as well as establishing theoretical guidance for wheat molecular marker-assisted breeding.

Regulation and function of extra-SCN circadian oscillators in the brain.

Most organisms evolved endogenous, so called circadian clocks as internal timekeeping mechanisms allowing them to adapt to recurring changes in environmental demands brought about by 24-hour rhythms such as the light-dark cycle, temperature variations or changes in humidity. The mammalian circadian clock system is based on cellular oscillators found in all tissues of the body that are organized in a hierarchical fashion. A master pacemaker located in the suprachiasmatic nucleus (SCN) synchronizes peripheral tissue clocks and extra-SCN oscillators in the brain with each other and with external time. Different time cues (so called Zeitgebers) such as light, food intake, activity and hormonal signals reset the clock system through the SCN or by direct action at the tissue clock level. While most studies on non-SCN clocks so far have focused on peripheral tissues, several extra-SCN central oscillators were characterized in terms of circadian rhythm regulation and output. Some of them are directly innervated by the SCN pacemaker, while others receive indirect input from the SCN via other neural circuits or extra-brain structures. The specific physiological function of these non-SCN brain oscillators as well as their role in the regulation of the circadian clock network remains understudied. In this review we summarize our current knowledge about the regulation and function of extra-SCN circadian oscillators in different brain regions and devise experimental approaches enabling us to unravel the organization of the circadian clock network in the central nervous system.

Waveform Plasticity under Entrainment to 12-h T-cycles in Drosophila melanogaster: Behavior, Neuronal Network, and Evolution.

A crucial property of circadian clocks is the ability to regulate the shape of an oscillation over its cycle length (waveform) appropriately, thus enhancing Darwinian fitness. Many studies over the past decade have revealed interesting ways in which the waveform of rodent behavior could be manipulated, one of which is that the activity bout bifurcates under environments that have 2 light/dark cycles within one 24-h day (LDLD). It has been observed that such unique, although unnatural, environments reveal acute changes in the circadian clock network. However, although adaptation of waveforms to different photoperiods is well studied, modulation of waveforms under LDLD has received relatively less attention in research on insect rhythms. Therefore, we undertook this study to ask the following questions: what is the extent of waveform plasticity that Drosophila melanogaster exhibits, and what are the neuronal underpinnings of such plasticity under LDLD? We found that the activity/rest rhythms of wild-type flies do not bifurcate under LDLD. Instead, they show similar but significantly different behavior from that under a long-day LD cycle. This behavior is accompanied by differences in the organization of the circadian neuronal network, which include changes in waveforms of a core clock component and an output molecule. In addition, to understand the functional significance of such variations in the waveform, we examined laboratory selected populations that exhibit divergent eclosion chronotypes (and therefore, waveforms). We found that populations selected for predominant eclosion in an evening window (late chronotypes) showed reduced amplitude plasticity and increased phase plasticity of activity/rest rhythms. This, we argue, is reflective of divergent evolution of circadian neuronal network organization in our laboratory selected flies.

Role of Tau Protein in Remodeling of Circadian Neuronal Circuits and Sleep.

Multiple neurological, physiological, and behavioral functions are synchronized by circadian clocks into daily rhythms. Neurodegenerative diseases such as Alzheimer’s disease and related tauopathies are associated with a decay of circadian rhythms, disruption of sleep patterns, and impaired cognitive function but the mechanisms underlying these alterations are still unclear. Traditional approaches in neurodegeneration research have focused on understanding how pathology impinges on circadian function. Since in Alzheimer’s disease and related tauopathies tau proteostasis is compromised, here we sought to understand the role of tau protein in neuronal circadian biology and related behavior. Considering molecular mechanisms underlying circadian rhythms are conserved from Drosophila to humans, here we took advantage of a recently developed tau-deficient Drosophila line to show that loss of tau promotes dysregulation of daily circadian rhythms and sleep patterns. Strikingly, tau deficiency dysregulates the structural plasticity of the small ventral lateral circadian pacemaker neurons by disrupting the temporal cytoskeletal remodeling of its dorsal axonal projections and by inducing a slight increase in the cytoplasmic accumulation of core clock proteins. Taken together, these results suggest that loss of tau function participates in the regulation of circadian rhythms by modulating the correct operation and connectivity of core circadian networks and related behavior.

Seasonal plasticity in GABAA signaling is necessary for restoring phase synchrony in the master circadian clock network.

Annual changes in the environment threaten survival, and numerous biological processes in mammals adjust to this challenge via seasonal encoding by the suprachiasmatic nucleus (SCN). To tune behavior according to day length, SCN neurons display unified rhythms with synchronous phasing when days are short, but will divide into two sub-clusters when days are long. The transition between SCN states is critical for maintaining behavioral responses to seasonal change, but the mechanisms regulating this form of neuroplasticity remain unclear. Here we identify that a switch in chloride transport and GABAA signaling is critical for maintaining state plasticity in the SCN network. Further, we reveal that blocking excitatory GABAA signaling locks the SCN into its long day state. Collectively, these data demonstrate that plasticity in GABAA signaling dictates how clock neurons interact to maintain environmental encoding. Further, this work highlights factors that may influence susceptibility to seasonal disorders in humans.