Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Circadian disturbances by altering the light-dark cycle negatively affects hematopoietic function of bone marrow in mice.

Circadian rhythms in metabolically active tissues are crucial for maintaining physical health. Circadian disturbance (CD) can cause various health issues, such as metabolic abnormalities and immune and cognitive dysfunctions. However, studies on the role of CD in immune cell development and differentiation, as well as the rhythmic expression of the core clock genes and their altered expression under CD, remain unclear. Therefore, we exposed C57bl/6j mice to repeated reversed light-dark cycles for 90 days to research the effects of CD on bone marrow (BM) hematopoietic function. We also researched the effects of CD on endogenous circadian rhythms, temporally dependent expression in peripheral blood and myeloid leukocytes, environmental homeostasis within BM, and circadian oscillations of hematopoietic-extrinsic cues. Our results confirmed that when the light and dark cycles around mice were frequently reversed, the circadian rhythmic expression of the two main circadian rhythm markers, the hypothalamic clock gene, and serum melatonin, was disturbed, indicating that the body was in a state of endogenous CD. Furthermore, CD altered the temporally dependent expression of peripheral blood and BM leukocytes and destroyed environmental homeostasis within the BM as well as circadian oscillations of hematopoietic-extrinsic cues, which may negatively affect BM hematopoiesis in mice. Collectively, these results demonstrate that circadian rhythms are vital for maintaining health and suggest that the association between CD and hematopoietic dysfunction warrants further investigation.

Molecular circadian rhythms are robust in marine annelids lacking rhythmic behavior.

The circadian clock controls behavior and metabolism in various organisms. However, the exact timing and strength of rhythmic phenotypes can vary significantly between individuals of the same species. This is highly relevant for rhythmically complex marine environments where organismal rhythmic diversity likely permits the occupation of different microenvironments. When investigating circadian locomotor behavior of Platynereis dumerilii, a model system for marine molecular chronobiology, we found strain-specific, high variability between individual worms. The individual patterns were maintained for several weeks. A diel head transcriptome comparison of behaviorally rhythmic versus arrhythmic wild-type worms showed that 24-h cycling of core circadian clock transcripts is identical between both behavioral phenotypes. While behaviorally arrhythmic worms showed a similar total number of cycling transcripts compared to their behaviorally rhythmic counterparts, the annotation categories of their transcripts, however, differed substantially. Consistent with their locomotor phenotype, behaviorally rhythmic worms exhibit an enrichment of cycling transcripts related to neuronal/behavioral processes. In contrast, behaviorally arrhythmic worms showed significantly increased diel cycling for metabolism- and physiology-related transcripts. The prominent role of the neuropeptide pigment-dispersing factor (PDF) in Drosophila circadian behavior prompted us to test for a possible functional involvement of Platynereis pdf. Differing from its role in Drosophila, loss of pdf impacts overall activity levels but shows only indirect effects on rhythmicity. Our results show that individuals arrhythmic in a given process can show increased rhythmicity in others. Across the Platynereis population, rhythmic phenotypes exist as a continuum, with no distinct "boundaries" between rhythmicity and arrhythmicity. We suggest that such diel rhythm breadth is an important biodiversity resource enabling the species to quickly adapt to heterogeneous or changing marine environments. In times of massive sequencing, our work also emphasizes the importance of time series and functional tests.

A Luciferase Imaging-Based Assay for Studying Temperature Compensation of the Circadian Clock.

The pace of circadian rhythms remains relatively unchanged across a physiologically relevant range of temperatures, a phenomenon known as temperature compensation. Temperature compensation is a defining characteristic of circadian rhythms, ensuring that clock-regulated processes occur at approximately the same time of day across a wide range of conditions. Despite the identification of several genes involved in the regulation of temperature compensation, the molecular mechanisms underlying this process are still not well understood. High-throughput assays of circadian period are essential for the investigation of temperature compensation. In this chapter, we present a luciferase imaging-based method that enables robust and accurate examination of temperature compensation in the plant circadian clock.

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 (1 h at 37 °C) or cold (3 h at 4 °C), and we include known marker genes that have circadian-gated responses to temperature changes.

Circadian Effects on Vascular Immunopathologies.

Circadian rhythms exert a profound impact on most aspects of mammalian physiology, including the immune and cardiovascular systems. Leukocytes engage in time-of-day-dependent interactions with the vasculature, facilitating the emigration to and the immune surveillance of tissues. This review provides an overview of circadian control of immune-vascular interactions in both the steady state and cardiovascular diseases such as atherosclerosis and infarction. Circadian rhythms impact both the immune and vascular facets of these interactions, primarily through the regulation of chemoattractant and adhesion molecules on immune and endothelial cells. Misaligned light conditions disrupt this rhythm, generally exacerbating atherosclerosis and infarction. In cardiovascular diseases, distinct circadian clock genes, while functioning as part of an integrated circadian system, can have proinflammatory or anti-inflammatory effects on these immune-vascular interactions. Here, we discuss the mechanisms and relevance of circadian rhythms in vascular immunopathologies.

Revisiting the role and mechanism of ELF3 in circadian clock modulation.

The gene encoding EARLY FLOWERING3 (ELF3) is necessary for photoperiodic flowering and the normal regulation of circadian rhythms. It provides important information at the cellular level to uncover the biological mechanisms that improve plant growth and development. ELF3 interactions with transcription factors such as BROTHER OF LUX ARRHYTHMO (BOA), LIGHT-REGULATED WD1 (LWD1), PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), PHYTOCHROME-INTERACTING FACTOR 7 (PIF7), and LUX ARRHYTHMO (LUX) suggest a role in evening complex (EC) independent pathways, demanding further investigation to elucidate the EC-dependent versus EC-independent mechanisms. The ELF3 regulation of flowering time about photoperiod and temperature variations can also optimize crop cultivation across diverse latitudes. In this review paper, we summarize how ELF3's role in the circadian clock and light-responsive flowering control in crops offers substantial potential for scientific advancement and practical applications in biotechnology and agriculture. Despite its essential role in crop adaptation, very little is known in many important crops. Consequently, comprehensive and targeted research is essential for extrapolating ELF3-related insights from Arabidopsis to other crops, utilizing both computational and experimental methodologies. This research should prioritize investigations into ELF3's protein-protein interactions, post-translational modifications, and genomic targets to elucidate its contribution to accurate circadian clock regulation.

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.

Ginseng extracts improve circadian clock gene expression and reduce inflammation directly and indirectly through gut microbiota and PI3K signaling pathway.

Despite the potential benefits of herbal medicines for therapeutic application in preventing and treating various metabolic disorders, the mechanisms of action were understood incompletely. Ginseng (Panax ginseng), a commonly employed plant as a dietary supplement, has been reported to play its hot property in increasing body temperature and improving gut health. However, a comprehensive understanding of the mechanisms by which ginseng regulates body temperature and gut health is still incomplete. This paper illustrates that intermittent supplementation with ginseng extracts improved body temperature rhythm and suppressed inflammatory responses in peripheral metabolic organs of propylthiouracil (PTU)-induced hypothermic rats. These effects were associated with changes in gut hormone secretion and the microbiota profile. The in-vitro studies in ICE-6 cells indicate that ginseng extracts can not only act directly on the cell to regulate the genes related to circadian clock and inflammation, but also may function through the gut microbiota and their byproducts such as lipopolysaccharide. Furthermore, administration of PI3K inhibitor blocked ginseng or microbiota-induced gene expression related with circadian clock and inflammation in vitro. These findings demonstrate that the hot property of ginseng may be mediated by improving circadian clock and suppressing inflammation directly or indirectly through the gut microbiota and PI3K-AKT signaling pathways.

The circadian molecular clock in the suprachiasmatic nucleus is necessary but not sufficient for fear entrainment.

We show that nocturnal aversive stimuli presented to mice while they are eating and drinking outside of their safe nest can entrain circadian behaviors, leading to a shift toward daytime activity. We also show that the canonical molecular circadian clock is necessary for fear entrainment and that an intact molecular clockwork in the suprachiasmatic nucleus, the site of the central circadian pacemaker, is necessary but not sufficient to sustain fear entrainment of circadian rhythms. Our results demonstrate that entrainment of a circadian clock by cyclic fearful stimuli can lead to severely mistimed circadian behavior that persists even after the aversive stimulus is removed. Together, our findings support the interpretation that circadian and sleep symptoms associated with fear and anxiety disorders are, in part, the output of a fear-entrained clock, and provide a mechanistic insight into this clock.

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock.

Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH), and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks.

Natural oscillations known as circadian rhythms influence many processes in humans and other animals including sleep, eating, brain activity and body temperature. These rhythms allow us to anticipate and prepare for regular changes in our environment including day-night cycles and the temperature of our surroundings. Circadian clocks in animals, fungi and other 'eukaryotic' organisms rely on networks of components that repress their own production to generate oscillations in their levels in cells over the course of a 24-hour period. The components in animal and fungus circadian clocks are different but there are strong similarities in their properties and how the networks operate. As a result, a type of fungus known as Neurospora crassa is often used as a model to study how circadian rhythms work in animals. A central component in the N. crassa circadian clock is a protein known as Frequency (FRQ). It is a large protein that, unlike most proteins, lacks a well-defined, three-dimensional structure. Despite this, it is able to bind to and regulate other proteins to repress its own production. One of its protein partners known as CK1 attaches small tags known as phosphate groups to FRQ to set the length of the circadian rhythm. However, it remains unclear how FRQ interacts with its protein partners or what effect the phosphate groups have on its activity. To address this question, Tariq, Maurici et al. used biochemical approaches to study the structure of FRQ. The experiments revealed that it contains a compact core that is able to bind to CK1 and other protein partners. The way FRQ regulates its protein partners is unusual: it undergoes a chemical process known as liquid-liquid phase separation to sequester other circadian clock proteins and modulate their enzymatic activities. In this process, a solution containing molecules of FRQ separates into two distinct components (known as phases), one of which contains FRQ and its partners in a concentrated liquid-like mixture. Evidence for such mixtures has also been found in living fungal cells. Further experiments suggest that liquid-liquid phase separation of FRQ may allow the clock to compensate for changes in temperature to maintain a regular rhythm. The circadian clocks of animals and other organisms all have proteins that perform similar roles as FRQ and maintain sequence properties that promote liquid-liquid phase separation. Therefore, it is possible that liquid-liquid phase separation may be a common feature of circadian rhythms in nature.

The emergence of circadian timekeeping in the intestine.

The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-h period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, we track how and when the circadian clock emerges in specific cell types. We find that the circadian clock begins abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in Ecdysone and Hnf4 signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. Our data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages.

Ror homolog nhr-23 is essential for both developmental clock and circadian clock in C. elegans.

Animals have internal clocks that generate biological rhythms. In mammals, clock genes such as Period form the circadian clock to generate approximately 24-h biological rhythms. In C. elegans, the clock gene homologs constitute the "developmental clock", which has an 8-h period during larval development to determine the timing of molting. Thus, the ancestral circadian clock has been believed to evolve into the oscillator with a shorter period in C. elegans. However, circadian rhythms have also been observed in adult C. elegans, albeit relatively weak. This prompts the question: if the clock gene homologs drive the developmental rhythm with 8-h period, which genes generate the circadian rhythms in C. elegans? In this study, we discovered that nhr-23, a homolog of the mammalian circadian clock gene Ror, is essential for circadian transcriptional rhythms in adult C. elegans. Interestingly, nhr-23 was also known to be essential for the molting clock. The bilaterian ancestral circadian clock genes might have evolved to function over multiple periods depending on developmental contexts rather than a single 8-h period in C. elegans.

TimeTeller: A tool to probe the circadian clock as a multigene dynamical system.

Recent studies have established that the circadian clock influences onset, progression and therapeutic outcomes in a number of diseases including cancer and heart diseases. Therefore, there is a need for tools to measure the functional state of the molecular circadian clock and its downstream targets in patients. Moreover, the clock is a multi-dimensional stochastic oscillator and there are few tools for analysing it as a noisy multigene dynamical system. In this paper we consider the methodology behind TimeTeller, a machine learning tool that analyses the clock as a noisy multigene dynamical system and aims to estimate circadian clock function from a single transcriptome by modelling the multi-dimensional state of the clock. We demonstrate its potential for clock systems assessment by applying it to mouse, baboon and human microarray and RNA-seq data and show how to visualise and quantify the global structure of the clock, quantitatively stratify individual transcriptomic samples by clock dysfunction and globally compare clocks across individuals, conditions and tissues thus highlighting its potential relevance for advancing circadian medicine.

Circadian clock-related genome-wide mendelian randomization identifies putatively genes for ulcerative colitis and its comorbidity.

BACKGROUND: Circadian rhythm is crucial to the function of the immune system. Disorders of the circadian rhythm can contribute to inflammatory diseases such as Ulcerative colitis (UC). This Mendelian Randomization (MR) analysis applies genetic tools to represent the aggregated statistical results of exposure to circadian rhythm disorders and UC and its comorbidities, allowing for causal inferences. METHODS: Summary statistics of protein, DNA methylation and gene expression quantitative trait loci in individuals of European ancestry (pQTL, mQTL, and eQTL, respectively) were used. Genetic variants located within or near 152 circadian clock-related genes and closely related to circadian rhythm disorders were selected as instrumental variables. Causal relationships with UC and its comorbidities were then estimated through employed Summary data-based Mendelian Randomization (SMR) and Inverse-Variance-Weighted MR (IVW-MR). RESULTS: Through preliminary SMR analysis, we identified a potential causal relationship between circadian clock-related genes and UC along with its comorbidities, which was further confirmed by IVW-MR analysis. Our study identified strong evidence of positive correlation involving seven overlapping genes (CSNK1E, OPRL1, PIWIL2, RORC, MAX, PPP5C, and AANAT) through MWAS and TWAS in UC, four overlapping genes (OPRL1, CHRNB2, FBXL17, and SIRT1) in UC with PSC, and three overlapping genes (ARNTL, USP7, and KRAS) in UC with arthropathy. CONCLUSIONS: This SMR study demonstrates the causal effect of circadian rhythm disorders in UC and its comorbidities. Furthermore, our investigation pinpointed candidate genes that could potentially serve as drug targets.

Anti-obesity effect of butyrate links to modulation of gut microbiome and epigenetic regulation of muscular circadian clock.

The role of the muscle circadian clock in regulating oxidative metabolism exerts a significant influence on whole-body energy metabolism; however, research on the connection between the muscle circadian clock and obesity is limited. Moreover, there is a lack of studies demonstrating the regulatory effects of dietary butyrate on muscle circadian clock and the resulting antiobesity effects. This study aimed to investigate the impacts of dietary butyrate on metabolic and microbiome alterations and muscle circadian clock in a diet-induced obesity model. Male Sprague-Dawley rats were fed a high-fat diet with or without butyrate. Gut microbiota and serum metabolome were analyzed, and molecular changes were examined using tissues and a cell line. Further correlation analysis was performed on butyrate-induced results. Butyrate supplementation reduced weight gain, even with increased food intake. Gut microbiome analysis revealed an increased abundance of Firmicutes in butyrate group. Serum metabolite profile in butyrate group exhibited reduced amino acid and increased fatty acid content. Muscle circadian clock genes were upregulated, resulting in increased transcription of fatty acid oxidation-related genes. In myoblast cells, butyrate also enhanced pan-histone acetylation via histone deacetylase inhibition, particularly modulating acetylation at the promoter of circadian clock genes. Correlation analysis revealed potential links between Firmicutes phylum, including certain genera within it, and butyrate-induced molecular changes in muscle as well as phenotypic alterations. The butyrate-driven effects on diet-induced obesity were associated with alterations in gut microbiota and a muscle-specific increase in histone acetylation, leading to the transcriptional activation of circadian clock genes and their controlled genes.

Interorgan rhythmicity as a feature of healthful metabolism.

The finding that animals with circadian gene mutations exhibit diet-induced obesity and metabolic syndrome with hypoinsulinemia revealed a distinct role for the clock in the brain and peripheral tissues. Obesogenic diets disrupt rhythmic sleep/wake patterns, feeding behavior, and transcriptional networks, showing that metabolic signals reciprocally control the clock. Providing access to high-fat diet only during the sleep phase (light period) in mice accelerates weight gain, whereas isocaloric time-restricted feeding during the active period enhances energy expenditure due to circadian induction of adipose thermogenesis. This perspective focuses on advances and unanswered questions in understanding the interorgan circadian control of healthful metabolism.

Effects of circadian clock disruption on gene expression and biological processes in Aedes aegypti.

BACKGROUND: This study explores the impact of disrupting the circadian clock through a Cycle gene knockout (KO) on the transcriptome of Aedes aegypti mosquitoes. The investigation aims to uncover the resulting alterations in gene expression patterns and physiological processes. RESULTS: Transcriptome analysis was conducted on Cyc knockout (AeCyc-/-) and wild-type mosquitoes at four time points in a light-dark cycle. The study identified system-driven genes that exhibit rhythmic expression independently of the core clock machinery. Cyc disruption led to altered expression of essential clock genes, affecting metabolic processes, signaling pathways, stimulus responses and immune responses. Notably, gene ontology enrichment of odorant binding proteins, indicating the clock's role in sensory perception. The absence of Cyc also impacted various regulation of metabolic and cell cycle processes was observed in all time points. CONCLUSIONS: The intricate circadian regulation in Ae. aegypti encompasses both core clock-driven and system-driven genes. The KO of Cyc gene instigated extensive gene expression changes, impacting various processes, thereby potentially affecting cellular and metabolic functions, immune responses, and sensory perception. The circadian clock's multifaceted involvement in diverse biological processes, along with its role in the mosquito's daily rhythms, forms a nexus that influences the vector's capacity to transmit diseases. These insights shed light on the circadian clock's role in shaping mosquito biology and behavior, opening new avenues for innovative disease control strategies.

Antidepressant mechanism of traditional Chinese medicine: Involving regulation of circadian clock genes.

Numerous studies have demonstrated an intimate relationship between circadian rhythm disorders and the development and prevention of depression. The biological clock genes, which constitute the molecular basis of endogenous circadian rhythms, hold promising prospects for depression treatment. Based on an extensive review of recent domestic and international research, this article presents a comprehensive analysis of how traditional Chinese medicine (TCM) intervenes in depression by regulating circadian rhythms. The findings indicate that TCM exerts its antidepressant effects by targeting specific biological clock genes such as Bmal1, clock, Arntl, Per1, Per2, Per3, Nr1d1, Cry2, and Dbp, as well as regulating circadian rhythms of hormone secretion. However, most current research is still confined to basic experimental studies, lacking clinical double-blind control trials to further validate these viewpoints. Furthermore, there is insufficient research on the signal transduction pathway between biological clock genes and pathological changes in depression. Additionally, further clarification is needed regarding the specific targets of TCM on the biological clock genes.

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.