Circadian clocks coordinate physiology, development and behaviour with cycles of day and night, which contributes to organismal fitness. For this to occur, it is crucial that the circadian clock is aligned with the natural fluctuations of environmental conditions. Here, we investigated under natural conditions the role of environmental cues in adjusting the circadian clock, and how this affects clock outputs. By combining novel field-based experimentation in a natural plant population with genome-wide analysis and machine-learning strategies for data interpretation, we find that temperature cues change circadian timing. We identify unbalanced effects of light and temperature in the entrainment of circadian clock components, and seasonal changes in the effects of temperature and light cues upon circadian entrainment. We also identified extensive circadian modulation of temperature responses under field conditions. Therefore, plasticity of circadian timing under natural conditions allows flexible responses of both the clock and its outputs to environmental stimuli. ### Competing Interest Statement The authors have declared no competing interest. BBSRC, BB/Y513945/1, BB/P013511/1, BB/X01102X/1 Leverhulme Trust, RPG-2018-216 JST CREST, JPMJCR15O1 Japan Society for the Promotion of Science, JP21H04977 European Research Council, 101166968

Agrochemicals play an important role in maximizing agricultural yields. One class of agrochemicals is herbicides, which are used for weed control and to kill the top growth of certain crops before harvest. We examine the influence of plant circadian regulation on herbicide effectiveness to exemplify how knowledge of the circadian clock could be mobilized to optimize agrochemical use. First, we briefly introduce the circadian clock, highlighting its role in plant fitness and regulating physiological and metabolic pathways. Second, we discuss principles of chronotoxicity and the range of herbicides across which agrochemical interventions might be optimized with knowledge of circadian rhythms. Using existing data, we find that a substantial number of pathways targeted by herbicides are subject to circadian clock regulation, opening the possibility that clock control of herbicide effectiveness could be widespread. Finally, we suggest potential practical applications, explaining how this could enhance resource use efficiency, reduce inputs, and mitigate environmental impacts.

Chronobiology is a multidisciplinary field that extends across the tree of life, transcends all scales of biological organization, and has huge translational potential. For the UK to harness the opportunities presented within applied chronobiology, we need to build our network outwards to reach stakeholders that can directly benefit from our discoveries. In this article, we discuss the importance of biological rhythms to our health, society, economy and environment, with a particular focus on circadian rhythms. We subsequently introduce the vision and objectives of BioClocks UK, a newly formed research network, whose mission is to stimulate researcher interactions and sustain discovery-impact cycles between chronobiologists, wider research communities and multiple industry sectors. This article is part of the Theo Murphy meeting issue ‘Circadian rhythms in infection and immunity’.

The circadian clock of plants contributes to their survival and fitness. However, understanding clock function at the transcriptome level and its response to the environment requires assaying across high resolution time-course experiments. Generating these datasets is labour-intensive, costly and, in most cases, performed under tightly controlled laboratory conditions. To overcome these barriers, we have developed ChronoGauge: an ensemble model that can reliably estimate the endogenous circadian time of Arabidopsis plants using the expression of a handful of time-indicating genes within a single time-pointed transcriptomic sample. ChronoGauge can predict a plant's circadian time with high accuracy across unseen Arabidopsis bulk RNA-seq and microarray samples, and can be further applied to make non-random predictions across samples in non-model species, including field samples. Finally, we demonstrate how ChronoGauge can be applied to generate hypotheses regarding the response of the circadian transcriptome to specific genotypes or environmental conditions.

Circadian clocks provide a biological measure of time that coordinates metabolism, physiology and behaviour with 24 h cycles in the environment. Circadian systems have a variety of characteristic properties, such as entrainment to environmental cues, a self-sustaining rhythm of about 24 h and temperature compensation of the circadian rhythm. In this perspective, we discuss the process of circadian gating, which refers to the restriction of a biological event to particular times of day by the circadian clock. We introduce principles and processes associated with circadian gating in a variety of organisms, including some associated mechanisms. We highlight socioeconomic opportunities presented by the investigation of circadian gating, using selected examples from circadian medicine and agricultural crop production to illustrate its importance. This article is part of the Theo Murphy meeting issue ‘Circadian rhythms in infection and immunity’.

Life on Earth evolved in environmental conditions that fluctuate with a daily cycle. Organisms from all kingdoms of life, including plants and their associated microorganisms, harbour circadian clocks as an adaptation to these environmental changes. We review the involvement of circadian clocks in associations between plants and microbes, focusing first on mechanisms of specific circadian clock-regulated plant-microbe interactions. We then discuss more general ecological and evolutionary consequences of clock regulation of plant-microbe interactions and argue that circadian clocks can choreograph complex plant-microbe community interactions across multiple levels of biological organisation. We also discuss the difficulties in determining the precise nature of interactions in such communities, for both individual rhythmic interactions and the emergent rhythmic properties of these communities. An understanding of the regulation of these interactions by circadian clocks is likely to have implications for crop performance, fertiliser and pesticide use, soil and ecosystem health, biogeochemical cycling, and the impacts of global warming.

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.

Summary Chromatin architecture in the cells of animals and fungi influences gene expression. The molecular factors that influence higher genome architecture in plants and their effects on gene expression remain unknown. Cohesin complexes, conserved in eukaryotes, are essential factors in genome structuring. Here, we investigated the relevance of the plant-specific somatic cohesin subunit SYN4 for chromatin organisation in Arabidopsis thaliana . Plants mutated in SYN4 were studied using HRM, Hi-C, RNA sequencing, untargeted and targeted metabolomics and physiological assays to understand the role of this plant-specific cohesin. We show that syn4 mutants exhibit altered intra- and interchromosomal interactions, expressed as sharply reduced contacts between telomeres and chromosome arms but not between centromeres, and differences in the placement and number of topologically associated domains (TADs)-like structures. By transcriptome sequencing, we also show that syn4 mutants have altered gene expression, including numerous genes that control abiotic stress responses. The response to drought stress in Arabidopsis is strongly influenced by the genome structure in syn4 mutants, potentially due to altered expression of CYP707A3 , an ABA 8’-hydroxylase. In brief The 3D architecture of the genome extensively influences gene expression in animals and fungi. We show that the plant-specific cohesin subunit SYN4 affects intra- and interchromosomal interactions including telomere clustering, with consequences for the expression of genes of transient and induced biological pathways and the biosynthesis of bioactive compounds. Highly condensed genome structures at the CYP707A3 locus positively affects the stress response to water deprivation regulated by abscisic acid.

The circadian clock of plants contributes to their survival and fitness. However, understanding clock function at the transcriptome level and its response to the environment requires assaying across high resolution time-course experiments. Generating these datasets is labour-intensive, costly and, in most cases, performed under tightly controlled laboratory conditions. To overcome this barrier, we have developed ChronoGauge: an ensemble model which can reliably estimate the endogenous circadian time of plants using the expression of a handful of time-indicating genes within a single time-pointed transcriptomic sample. ChronoGauge can predict the plant circadian time with high accuracy across unseen Arabidopsis bulk RNA-seq and microarray samples, and can be further applied across samples in non-model species, including field samples. Finally, we demonstrate how ChronoGauge can be applied to test hypotheses regarding the response of the circadian transcriptome to specific genotypes or environmental conditions. ### Competing Interest Statement The authors have declared no competing interest.

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.

A classical question in biology is how different processes are controlled in space and time, with research pointing to different mechanisms as timers. In this collection of Voices, we asked researchers to define their scientific questions related to time-keeping and the approaches they use to answer them.

Surely most chronobiologists believe circadian clocks are an adaptation of organisms that enhances fitness, but are we certain that this focus of our research effort really confers a fitness advantage? What is the evidence, and how do we evaluate it? What are the best criteria? These questions are the topic of this review. In addition, we will discuss selective pressures that might have led to the historical evolution of circadian systems while considering the intriguing question of whether the ongoing climate change is modulating these selective pressures so that the clock is still evolving.

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.

The circadian clock is an internal time-keeping system that generates c. 24 h circadian rhythms. These rhythms are thought to align the phase of biological processes with the time of day in the environment. Three key properties of circadian rhythms are: free running of the rhythm without external cues, entrainment to external cycles, and temperature compensation of the period. These properties are widely conserved across organisms. Circadian rhythms in plants are thought to be generated by a transcriptional/translational feedback loop (TTFL) formed from core clock genes and proteins (Nohales & Kay, 2016). These circadian oscillations, and the circadian clock, can function even in single plant cells. Temporal information derived at the single-cell level is thought to be shared among cells, tissues, and organs, resulting in coordinated rhythms at the organismal level. In other words, circadian oscillators can produce physiological rhythms that have hierarchical organizational architectures. The mechanisms of the circadian clock have been studied extensively using the model plant Arabidopsis thaliana (Arabidopsis). However, there have been exciting developments in the investigation of circadian regulation in divergent plants ranging from green algae to crops. The knowledge obtained from various plant species is advancing understanding of the similarities and diversities of circadian clock mechanisms in the green lineage (Fig. 1). Addressing some of these questions formed part of the 33rd International Conference on Arabidopsis Research (ICAR2023), held in Japan. The conference included a session entitled, ‘Understanding circadian regulation in unpredictable environments’, organized by Antony Dodd (John Innes Centre, UK) and Tokitaka Oyama (Kyoto University, Japan). Several speakers studying various plants shared their findings to provide new insights into the questions above. The three properties of circadian rhythms described above, free running, entrainment, and temperature compensation, are crucial for understanding circadian clock systems. Free running is the property of maintaining a circadian rhythm, with a period of c. 24 h, without rhythmic environmental cues (for example, under constant light and constant temperature conditions) (Fig. 2a). Entrainment allows organisms to align their intrinsic rhythm with day–night cycles. Entrainment causes a phase shift (i.e. phase advance or phase delay), depending on the circadian phase, in response to an environmental cue (Fig. 2b). The circadian oscillator also regulates the magnitude of responses to stimuli according to the phase of the circadian clock (circadian gating). Such responses include gene expression, metabolism, and physiological outputs. Temperature compensation is the ability of the circadian clock to maintain a period of c. 24 h over a range of physiological temperatures (Fig. 2c). These three properties have been studied extensively, across life, over the past 70–80 yr (McClung, 2006). Although many clock-related genes have been identified in Arabidopsis, the molecular mechanisms underlying these three properties remain unclear. In the session at ICAR2023, Shu-Hsing Wu (Academia Sinica, Taiwan) provided a new molecular mechanism underlying the oscillation of the circadian clock. With colleagues, she investigated upstream open reading frame (uORF)-mediated translational regulation to understand why a stable circadian rhythm can be maintained within an essentially noisy process (Wu et al., 2022). The autonomous oscillation of gene expression is driven by the TTFL of circadian clock genes, so precise regulation of transcription and translation is required. However, bursts of de novo transcription and translation can produce fluctuations in mRNA and protein levels (Eldar & Elowitz, 2010). The Wu group has demonstrated that the uORFs upstream of the core clock gene TIMING OF CAB EXPRESSION 1 (TOC1) are involved in reducing such intrinsic noise. In addition, they suggested that these uORFs may effectively buffer TOC1 protein production, and discussed the possibility that this uORF-mediated regulation is a shared mechanism for maintaining the period length in other organisms. Antony Dodd presented the genome-wide circadian gating of cold responses in bread wheat (Graham et al., 2023). Low temperature is known to reduce crop yield by reducing photosynthetic capacity and causing photoinhibition (Hurry & Huner, 1992; Li et al., 2015). Therefore, with colleagues, he considered the possibility that the circadian gating of low temperature responses might affect crop performance. They found genome-wide circadian gating of low temperature responses in hexaploid bread wheat, as previously demonstrated in Arabidopsis, suggesting conserved regulatory mechanisms for temporal environmental response across angiosperms. These findings aid our understanding of circadian clock systems in crops and also provide helpful information for producing more climate change resilient crops in the future. Akari Maeda (Nagoya University, Japan) focused on temperature compensation in Arabidopsis. Previous studies reported that some mutants impair temperature compensation (Salome et al., 2010). However, the molecular mechanisms of temperature compensation are still unclear. She and her colleagues found that the quantitative control of clock protein abundance is essential for temperature compensation in Arabidopsis. They also reported that ubiquitin-dependent degradation is associated with temperature-dependent quantitative regulation of the clock protein abundance, revealing a novel molecular mechanism underlying temperature compensation. Takuya Matsuo (Kitasato University, Japan) presented the similarities and differences between the clock genes in Arabidopsis and Chlamydomonas reinhardtii (Chlamydomonas), a eukaryotic unicellular alga. He and his colleagues have identified clock genes in Chlamydomonas (Matsuo et al., 2008). They found that one of the clock proteins, RHYTHM OF CHLOROPLAST 15 (ROC15), is degraded by light stimuli, and this light-inducible degradation of ROC15 is involved in resetting the circadian clock (Niwa et al., 2013). In this session, he explained how Chlamydomonas-ELF3-like (CETL) is involved in the degradation pathway of ROC15. Interestingly, CETL has a very weak similarity to EARLY FLOWERING 3 (ELF3) in Arabidopsis (Gururaj et al., 2022). These findings are very helpful for understanding the evolution of the circadian clock genes in the green lineage. Individual plant cells possess a functional circadian clock (Davis et al., 2022). At the same time, there are differences in the period and phase among different parts of a plant. There is accumulating evidence for the coupling of circadian rhythms between cells and tissues in plants (Davis et al., 2022). However, how these circadian oscillators interact with each other remains poorly understood. Tokitaka Oyama presented an investigation of a noncell-autonomous circadian rhythm and its characteristics. Using single-cell level imaging with a duckweed species (Lemna minor), his group succeeded in monitoring dual-color bioluminescence reporters (Arabidopsis CIRCADIAN CLOCK-ASSOCIATED1::luciferase + (AtCCA1::LUC+) under the circadian clock and 35S::modified click-beetle red-color luciferase (CaMV35S::PtRLUC)) (Muranaka & Oyama, 2016; Watanabe et al., 2023). Both AtCCA1::LUC+ and CaMV35S::PtRLUC had bioluminescence rhythms with periods of c. 24 h. The amplitude of the rhythm of AtCCA1::LUC+ was reduced by overexpressing an effector of the clock (e.g. LmZTL), whereas the rhythm of CaMV35S::PtRLUC was not altered. In comparison, plasmolysis disrupted the CaMV35S::PtRLUC rhythm but did not affect AtCCA1::LUC+ rhythm. Thus, the CaMV35S::PtRLUC rhythm is noncell-autonomous and likely to be driven by symplast/apoplast mediated cellular interactions. This process could be involved in the coupling between cellular oscillators. James Locke (University of Cambridge, UK) used a mathematical approach to explain complex spatial waves of clock gene expression observed by fluorescence imaging (Greenwood et al., 2022). It was found that the interaction among oscillators possessing different periods possibly generates spatial waves. The different sensitivity to environmental stimuli between different types of tissues could be a factor for the period variations within a plant (Greenwood et al., 2022). With colleagues, he demonstrated that different sensitivity to stimuli can explain the period differences observed within plants. In addition, they demonstrated that the local coupling of clock components is necessary for forming spatial waves through a clock network model with spatial information. Finally, they analyzed the model behavior under a noisy light–dark cycle and showed that the local coupling minimized the timing errors, while the clocks in each tissue maintained phase differences (Greenwood et al., 2022). This session provided a valuable opportunity to discuss the circadian clock system with researchers studying not only Arabidopsis but also other plant species. It is possible that most of the core clock components in Arabidopsis have been identified. This provides an excellent opportunity to move on to the next mysteries of the circadian clock, which were discussed deeply during this session. Furthermore, this session taught us how advanced molecular techniques and mathematical approaches help our understanding of cell-, tissue-, and organ-specific clock properties, and the integration of temporal information from cell levels to organ levels. The clock properties discussed in this session, such as translational regulation, posttranslational regulation, gating, resetting, and coupling are all important for keeping time and responding accurately to fluctuating and unpredictable environments. In addition to the speakers, many well-established participants made this session very active and fruitful. The insights provided by this session will advance our understanding of not only the circadian clock of Arabidopsis, but also the mysteries of circadian clocks across the green lineages. The authors thank all session presenters and participants, ICAR for hosting several circadian clock sessions, and sponsors including the New Phytologist Foundation that made it possible to organize this session.

Circadian clocks are pervasive throughout nature, yet only recently has this adaptive regulatory program been described in nonphotosynthetic bacteria. Here, we describe an inherent complexity in the Bacillus subtilis circadian clock. We find that B. subtilis entrains to blue and red light and that circadian entrainment is separable from masking through fluence titration and frequency demultiplication protocols. We identify circadian rhythmicity in constant light, consistent with the Aschoff's rule, and entrainment aftereffects, both of which are properties described for eukaryotic circadian clocks. We report that circadian rhythms occur in wild isolates of this prokaryote, thus establishing them as a general property of this species, and that its circadian system responds to the environment in a complex fashion that is consistent with multicellular eukaryotic circadian systems.

Chloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis , a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions.

Diel cycles of gene expression are thought to adapt plants to 24-h changes in environmental conditions. The circadian clock contributes to this process, but less is known about circadian programs in developing reproductive organs. While model plants and controlled conditions have contributed greatly to our knowledge of circadian clock function, there is a need to better understand its role in crop plants under field conditions with fluctuating light and temperature. In this study, we investigated changes in the circadian clock during the development of grape berries of Vitis vinifera L. We found that the transcripts of circadian clock homologs had high-amplitude oscillations prior to, but not during, ripening. As ripening progressed, the amplitude and rhythmicity of the diel oscillations decreased until most transcripts tested had no significant fluctuation over the 24-h cycle. Despite this loss of rhythmicity, the majority of circadian clock genes investigated were expressed at or near their abundance at the nadir of their pre-ripening oscillation although the berries remained transcriptionally active. From this, it can be concluded that cycling of the canonical circadian clock appears unnecessary for berry ripening. Our data suggest that changes in circadian clock dynamics during reproductive organ development may have important functional consequences.

The malaria parasite Plasmodium falciparum has a nonphotosynthetic plastid called the apicoplast, which contains its own genome. Regulatory mechanisms for apicoplast gene expression remain poorly understood, despite this organelle being crucial for the parasite life cycle. Here, we identify a nuclear-encoded apicoplast RNA polymerase σ subunit (sigma factor) which, along with the α subunit, appears to mediate apicoplast transcript accumulation. This has a periodicity reminiscent of parasite circadian or developmental control. Expression of the apicoplast subunit gene, apSig, together with apicoplast transcripts, increased in the presence of the blood circadian signaling hormone melatonin. Our data suggest that the host circadian rhythm is integrated with intrinsic parasite cues to coordinate apicoplast genome transcription. This evolutionarily conserved regulatory system might be a future target for malaria treatment.

SUMMARY Circadian regulation produces a biological measure of time within cells. The daily cycle in the availability of light for photosynthesis causes dramatic changes in biochemical processes in photosynthetic organisms, with the circadian clock having crucial roles in adaptation to these fluctuating conditions. Correct alignment between the circadian clock and environmental day–night cycles maximizes plant productivity through its regulation of metabolism. Therefore, the processes that integrate circadian regulation with metabolism are key to understanding how the circadian clock contributes to plant productivity. This forms an important part of exploiting knowledge of circadian regulation to enhance sustainable crop production. Here, we examine the roles of circadian regulation in metabolic processes in source and sink organ structures of Arabidopsis. We also evaluate possible roles for circadian regulation in root exudation processes that deposit carbon into the soil, and the nature of the rhythmic interactions between plants and their associated microbial communities. Finally, we examine shared and differing aspects of the circadian regulation of metabolism between Arabidopsis and other model photosynthetic organisms, and between circadian control of metabolism in photosynthetic and non‐photosynthetic organisms. This synthesis identifies a variety of future research topics, including a focus on metabolic processes that underlie biotic interactions within ecosystems.