The past 25 years have seen remarkable progress in plant circadian biology. This perspective highlights major advances in our understanding of the oscillator network, the processes it regulates, and circadian variation across tissues and cell types. Genomic and systems approaches uncovered pervasive circadian control of physiology, development, and fitness, including photoperiodic and domestication traits. Emerging tools now position the field to engineer clocks for agricultural resilience in a changing climate.

Circadian clocks provide plants with an adaptive advantage by enabling them to anticipate daily environmental changes. The periodicity of circadian clocks is regulated at multiple levels of gene expression, including transcription, mRNA processing, translation, and protein modification. Numerous mRNA splicing factors have been implicated in maintaining circadian period length. However, these factors often play additional roles in transcription, making it difficult to determine whether they affect the clock through splicing-dependent or splicing-independent mechanisms. We and others have shown that XAP5 CIRCADIAN TIMEKEEPER (XCT) and components of the PRE-MRNA-PROCESSING FACTOR 19 (PRP19) complex, including the functionally redundant PRP19A and PRP19B, physically associate and regulate both splicing and circadian rhythms. Here, our transcriptome analyses reveal that the antagonistic regulation of circadian period length by XCT and PRP19 likely occurs through splicing-independent mechanisms. Interestingly, both factors co-regulate expression of a substantial set of shared target genes involved in RNA metabolism, photosynthesis, and stress responses despite having largely distinct targets for splicing. Gene co-expression analysis followed by functional characterization identified anthocyanin biosynthesis as another process antagonistically regulated by XCT and PRP19. Nonetheless, we found genetic perturbation of anthocyanin production does not affect circadian period, suggesting that the observed correlation between anthocyanin levels and circadian period may instead reflect disruption of a shared upstream regulatory pathway. Together, our findings suggest involvement of XCT and PRP19 in the transcriptional coordination of anthocyanin biosynthesis and biological timing, expanding their known roles beyond mRNA splicing.

Daily rhythms in physiology are obvious and widespread. While for millennia it was thought that these cycles represent passive responses to environmental cycles, we now recognize that many of them are governed by circadian oscillators. In plants, these cell-autonomous oscillators regulate daily processes such as photosynthesis, organ growth, and hormone production, as well as seasonal transitions like flowering. Furthermore, the circadian system gates plant responses to biotic and abiotic stresses, modulating susceptibility to pathogens and environmental extremes in a time-of-day-dependent manner. Variants of circadian clock genes have been repeatedly selected during crop domestication and improvement, highlighting the importance of the circadian system to plants and its relevance for agriculture. Here, I review the history of circadian studies in plants and summarize our current understanding of the molecular nature of the circadian oscillator. I also discuss how this complex network both responds to and is buffered against changes in the environment. Next, I examine how circadian oscillators differ between various tissues and how their activities are coordinated throughout the plant body. Finally, I discuss emerging directions, such as ways in which this understanding can be applied to crop improvement in the face of climate change.

Several closely related Myb-like activator proteins are known to have partially redundant functions within the plant circadian clock, but their specific roles are not well understood. To clarify the function of the REVEILLE 4, REVEILLE 6, and REVEILLE 8 transcriptional activators, we characterized the growth and clock phenotypes of CRISPR-Cas9-generated single, double, and triple rve mutants. We found that these genes act synergistically to regulate flowering time, redundantly to regulate leaf growth, and antagonistically to regulate hypocotyl elongation. We previously reported that increasing intensities of monochromatic blue and red light have opposite effects on the period of triple rve468 mutants. Here, we further examined light quality-specific phenotypes of rve mutants and report that rve468 mutants lack the blue light-specific increase in expression of some circadian clock genes observed in wild type. To investigate the basis of these blue light-specific circadian phenotypes, we examined RVE protein abundances and degradation rates in blue and red light and found no significant differences between these conditions. We next examined genetic interactions between RVE genes and ZEITLUPE and ELONGATED HYPOCOTYL5, two factors with blue light-specific functions in the clock. We found that the RVEs interact additively with both ZEITLUPE and ELONGATED HYPOCOTYL5 to regulate circadian period, which suggests that neither of these factors are required for the blue light-specific differences that we observed. Overall, our results suggest that the RVEs have separable functions in plant growth and circadian regulation and that they are involved in blue light-specific circadian signaling via a novel mechanism.

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.

The functions of closely related Myb-like repressor and Myb-like activator proteins within the plant circadian oscillator have been well-studied as separate groups, but the genetic interactions between them are less clear. We hypothesized that these repressors and activators would interact additively to regulate both circadian and growth phenotypes. We used CRISPR-Cas9 to generate new mutant alleles and performed physiological and molecular characterization of plant mutants for five of these core Myb-like clock factors compared with a repressor mutant and an activator mutant. We first examined circadian clock function in plants likely null for both the repressor proteins, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and the activator proteins, REVEILLE 4 (RVE4), REVEILLE (RVE6), and REVEILLE (RVE8). The rve468 triple mutant has a long period and flowers late, while cca1 lhy rve468 quintuple mutants, similarly to cca1 lhy mutants, have poor circadian rhythms and flower early. This suggests that CCA1 and LHY are epistatic to RVE4, RVE6, and RVE8 for circadian clock and flowering time function. We next examined hypocotyl elongation and rosette leaf size in these mutants. The cca1 lhy rve468 mutants have growth phenotypes intermediate between cca1 lhy and rve468 mutants, suggesting that CCA1, LHY, RVE4, RVE6, and RVE8 interact additively to regulate growth. Together, our data suggest that these five Myb-like factors interact differently in regulation of the circadian clock versus growth. More generally, the near-norm al seedling phenotypes observed in the largely arrhythmic quintuple mutant demonstrate that circadian-regulated output processes, like control of hypocotyl elongation, do not always depend upon rhythmic oscillator function.

Biological rhythms are ubiquitous. They can be generated by circadian oscillators, which produce daily rhythms in physiology and behavior, as well as by developmental oscillators such as the segmentation clock, which periodically produces modular developmental units. Here, we show that the circadian clock controls the timing of late-stage floret development, or anthesis, in domesticated sunflowers. In these plants, up to thousands of individual florets are tightly packed onto a capitulum disk. While early floret development occurs continuously across capitula to generate iconic spiral phyllotaxy, during anthesis floret development occurs in discrete ring-like pseudowhorls with up to hundreds of florets undergoing simultaneous maturation. We demonstrate circadian regulation of floral organ growth and show that the effects of light on this process are time-of-day dependent. Delays in the phase of floral anthesis delay morning visits by pollinators, while disruption of circadian rhythms in floral organ development causes loss of pseudowhorl formation and large reductions in pollinator visits. We therefore show that the sunflower circadian clock acts in concert with environmental response pathways to tightly synchronize the anthesis of hundreds of florets each day, generating spatial patterns on the developing capitulum disk. This coordinated mass release of floral rewards at predictable times of day likely promotes pollinator visits and plant reproductive success.

Sunflowers are famous for their ability to track the sun throughout the day and then reorient at night to face east the following morning. This occurs by differential growth patterns, with the east sides of stems growing more during the day and the west sides of stems growing more at night. This process, termed heliotropism, is generally believed to be a specialized form of phototropism; however, the underlying mechanism is unknown. To better understand heliotropism, we compared gene expression patterns in plants undergoing phototropism in a controlled environment and in plants initiating and maintaining heliotropic growth in the field. We found the expected transcriptome signatures of phototropin-mediated phototropism in sunflower stems bending towards monochromatic blue light. Surprisingly, the expression patterns of these phototropism-regulated genes are quite different in heliotropic plants. Most genes rapidly induced during phototropism display only minor differences in expression across solar tracking stems. However, some genes that are both rapidly induced during phototropism and are implicated in growth responses to foliar shade are rapidly induced on the west sides of stems at the onset of heliotropism, suggesting a possible role for red light photoreceptors in solar tracking. To test the involvement of different photoreceptor signaling pathways in heliotropism, we modulated the light environment of plants initiating solar tracking. We found that depletion of either red and far-red light or blue light did not hinder the initiation or maintenance of heliotropism in the field. Together, our results suggest that the transcriptional regulation of heliotropism is distinct from phototropin-mediated phototropism and likely involves inputs from multiple light signaling pathways.

XCT plays genetically separable roles in controlling the fidelity of 3' splice site selection during pre-mRNA splicing and the pace of circadian clock in Arabidopsis. The circadian oscillator allows organisms to synchronize their cellular and physiological activities with diurnal environmental changes. In plants, the circadian clock is primarily composed of multiple transcriptional-translational feedback loops. Regulators of post-transcriptional events, such as precursor messenger RNAs (pre-mRNA) splicing factors, are also involved in controlling the pace of the clock. However, in most cases the underlying mechanisms remain unclear. We have previously identified XAP5 CIRCADIAN TIMEKEEPER (XCT) as an Arabidopsis thaliana circadian clock regulator with uncharacterized molecular functions. Here, we report that XCT physically interacts with components of the spliceosome, including members of the Nineteen Complex (NTC). PacBio Iso-Seq data show that xct mutants have transcriptome-wide pre-mRNA splicing defects, predominantly aberrant 3 ' splice site selection. Expression of a genomic copy of XCT fully rescues those splicing defects, demonstrating that functional XCT is important for splicing. Dawn-expressed genes are significantly enriched among those aberrantly spliced in xct mutants, suggesting that the splicing activity of XCT may be circadian regulated. Furthermore, we show that loss-of-function mutations in PRP19A or PRP19B, 2 homologous core NTC components, suppress the short circadian period phenotype of xct-2. However, we do not see rescue of the splicing defects of core clock genes in prp19 xct mutants. Therefore, our results suggest that XCT may regulate splicing and the clock function through genetically separable pathways.

In the 24 years since the first plant circadian oscillator genes were cloned (Schaffer et al., 1998; Wang and Tobin, 1998), there has been a concerted effort to identify the mechanistic basis of circadian rhythms and to understand how the circadian system impacts the biology of plants. Progress has been so great that there is now a good understanding of the circadian biology of Arabidopsis and insight into the circadian biology of the major crops is developing. For this Focus Issue on Circadian Rhythms, we encouraged submission of articles that emphasize less understood aspects, such as interactions between the circadian system and the natural environment, cell-specific function, setting of the circadian clock by environmental signals, and evolution of plant circadian oscillators. The Arabidopsis circadian oscillator was first modeled as a simple negative feedback loop, with two Myb-like transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), directly inhibiting expression of TIMING OF CAB EXPRESSION 1 (TOC1). TOC1, a member of the PSEUDO-RESPONSE REGULATOR family, was first proposed to promote CCA1 and LHY expression (Alabadi et al., 2001). However, later studies revealed that TOC1 directly represses CCA1 and LHY expression (Gendron et al., 2012; Huang et al., 2012; Pokhilko et al., 2012), with these three genes forming a double negative feedback loop. Subsequent work by many laboratories has revealed that the circadian oscillator mechanism in Arabidopsis is instead actually composed of dozens of genes that regulate each other’s expression, forming a very complex interconnected network (Nakamichi, 2020). Posttranslational regulation of circadian proteins is also pervasive, enhancing the robustness of the circadian system and fine-tuning circadian period (Yan et al., 2021). In addition to controlling each other’s expression, this slew of circadian oscillator transcription factors regulate a large fraction of the plant transcriptome, with up to 40% of Arabidopsis genes reported to be under circadian regulation (Romanowski et al., 2020). Given the widespread influence of circadian oscillators on gene expression, it is not surprising that many aspects of physiology, including photosynthesis, abiotic and biotic stress responses, and the transition from vegetative to reproductive growth are modulated by the circadian system (Sanchez and Kay, 2016). Indeed, breeding during domestication has selected for allelic variants in circadian clock genes in a large number of crop species (Steed et al., 2021; Maeda and Nakamichi, 2022). Future improvements in crop germplasm and agronomic practices will depend upon a better understanding of the circadian system and its interactions with the environment. While circadian clocks are present in most living organisms, they have independently arisen in different lineages (Jabbur and Johnson, 2022). In this issue, Peterson et al. provide an Update on recent advances in understanding the evolution of circadian components in the green lineage. Genomic surveys indicate deep conservation of core elements of circadian oscillators, not only in land plants but also in Chlorophyte and Charophyte algae. Moreover, Peterson and colleagues summarize our current understanding of the function of circadian components in several land plants and algae (Petersen et al., 2022). Maeda and Nakamichi (2022) provide an excellent Update covering human selection of varieties suited for local conditions (including photoperiod and other climatic features) which allowed the cultivation of plants outside the ecological range of their wild ancestors. These authors present examples from longday plants, such as wheat and barley, and short-day plants, such as rice and sorghum. How this process led to the selection of alleles that either flower early, or late, to avoid particularly dry, or wet times of the year, is well illustrated (Maeda and Nakamichi, 2022). Greenham et al. (2022) Ed it o ri al

Numerous links have been reported between immune response and DNA damage repair pathways in both plants and animals but the precise nature of the relationship between these fundamental processes is not entirely clear. Here, we report that XAP5 CIRCADIAN TIMEKEEPER (XCT), a protein highly conserved across eukaryotes, acts as a negative regulator of immunity in Arabidopsis thaliana and plays a positive role in responses to DNA damaging radiation. We find xct mutants have enhanced resistance to infection by a virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, and are hyper-responsive to the defense-activating hormone salicylic acid (SA) when compared to wild-type. Unlike most mutants with constitutive effector-triggered immunity (ETI), xct plants do not have increased levels of SA and retain enhanced immunity at elevated temperatures. Genetic analysis indicates XCT acts independently of NONEXPRESSOR OF PATHOGENESIS RELATED GENES1 (NPR1), which encodes a known SA receptor. Since DNA damage has been reported to potentiate immune responses, we next investigated the DNA damage response in our mutants. We found xct seedlings to be hypersensitive to UV-C and γ radiation and deficient in phosphorylation of the histone variant H2A.X, one of the earliest known responses to DNA damage. These data demonstrate that loss of XCT causes a defect in an early step of the DNA damage response pathway. Together, our data suggest that alterations in DNA damage response pathways may underlie the enhanced immunity seen in xct mutants.

The Targeting Induced Local Lesions in Genomes (TILLING) technology is a reverse genetic strategy broadly applicable to every kind of genome and represents an attractive tool for functional genomic and agronomic applications. It consists of chemical random mutagenesis followed by high-throughput screening of point mutations in targeted genomic regions. Although multiple methods for mutation discovery in amplicons have been described, next-generation sequencing (NGS) is the tool of choice for mutation detection because it quickly allows for the analysis of a large number of amplicons. The aim of the present work was to screen a previously generated sunflower TILLING population and identify alterations in genes involved in several important and complex physiological processes. Twenty-one candidate sunflower genes were chosen as targets for the screening. The TILLING by sequencing strategy allowed us to identify multiple mutations in selected genes and we subsequently validated 16 mutations in 11 different genes through Sanger sequencing. In addition to addressing challenges posed by outcrossing, our detection and validation of mutations in multiple regulatory loci highlights the importance of this sunflower population as a genetic resource.

Effective insect pollination requires appropriate responses to internal and external environmental cues in both the plant and the pollinator. Helianthus annuus, a highly outcrossing species, is marked for its uniform eastward orientation of mature pseudanthia, or capitula. Here we investigate how this orientation affects floral microclimate and the consequent effects on plant and pollinator interactions and reproductive fitness. We artificially manipulated sunflower capitulum orientation and temperature in both field and controlled conditions and assessed flower physiology, pollinator visits, seed traits and siring success. East-facing capitula were found to have earlier style elongation, pollen presentation and pollinator visits compared with capitula manipulated to face west. East-facing capitula also sired more offspring than west-facing capitula and under some conditions produced heavier and better-filled seeds. Local ambient temperature change on the capitulum was found to be a key factor regulating the timing of style elongation, pollen emergence and pollinator visits. These results indicate that eastward capitulum orientation helps to control daily rhythms in floral temperature, with direct consequences on the timing of style elongation and pollen emergence, pollinator visitation, and plant fitness.

The circadian system ensures that plants respond appropriately to environmental change by predicting regular transitions that occur during diel cycles. In order to be most useful, the circadian system needs to be compensated against daily and seasonal changes in temperature that would otherwise alter the pace of this biological oscillator. We demonstrate that an evening-phased protein, the putative histone demethylase JMJD5, contributes to temperature compensation. JMJD5 is co-expressed with components of the Evening Complex, an agglomeration of proteins including EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHYMO (LUX), which also integrates temperature changes into the molecular clockwork. One role of the Evening Complex is to regulate expression of PSEUDORESPONSE REGULATOR9 (PRR9) and PRR7, important components of the temperature compensation mechanism. Surprisingly we find that LUX, but not other Evening Complex components, is dispensable for clock function at low temperatures. Further genetic analysis suggests JMJD5 acts in a parallel pathway to LUX within the circadian system. Although an intact JMJD5 catalytic domain is required for its function within the clock, our findings suggest JMJD5 does not directly regulate H3K36 methylation at circadian loci. Such data refine our understanding of how JMDJ5 acts within the Arabidopsis circadian system.

The circadian oscillator is a complex network of interconnected feedback loops that regulates a wide range of physiological processes. Indeed, variation in clock genes has been implicated in an array of plant environmental adaptations, including growth regulation, photoperiodic control of flowering, and responses to abiotic and biotic stress. Although the clock is buffered against the environment, maintaining roughly 24-h rhythms across a wide range of conditions, it can also be reset by environmental cues such as acute changes in light or temperature. These competing demands may help explain the complexity of the links between the circadian clock network and environmental response pathways. Here, we discuss our current understanding of the clock and its interactions with light and temperature-signaling pathways. We also describe different clock gene alleles that have been implicated in the domestication of important staple crops.

While fast plant movements are spectacular but rare, almost all plants exhibit relatively slow, growth-mediated tropic movements that are key to their survival in the natural world. In this brief review, we discuss recent insights into the molecular mechanisms underlying phototropism, gravitropism, hydrotropism, and autostraightening. Careful molecular genetic and physiological studies have helped confirm the importance of lateral auxin gradients in gravitropic and phototropic responses. However, auxin signaling does not explain all tropisms: recent work has shown that abscisic acid signaling mediates root hydrotropism and has implicated mechanosensing in autostraightening, the organ straightening process recently modeled as a proprioceptive response. The interactions between distinct tropic signaling pathways and other internal and external sensory processes are also now being untangled.

Although circadian oscillators in diverse eukaryotes all depend on interlinked transcriptional feedback loops, specific components are not conserved across higher taxa. Moreover, the circadian network in the model plant Arabidopsis thaliana is notably more complex than those found in animals and fungi. Here, we combine mathematical modeling and experimental approaches to investigate the functions of two classes of Myb-like transcription factors that antagonistically regulate common target genes. Both CCA1/LHY- and RVE8-clade factors bind directly to the same cis-element, but the former proteins act primarily as repressors, while the latter act primarily as activators of gene expression. We find that simulation of either type of loss-of-function mutant recapitulates clock phenotypes previously reported in mutant plants, while simulated simultaneous loss of both type of factors largely rescues circadian phase at the expense of rhythmic amplitude. In accord with this prediction, we find that plants mutant for both activator- and repressor-type Mybs have near-normal circadian phase and period but reduced rhythmic amplitude. Although these mutants exhibit robust rhythms when grown at mild temperatures, they are largely arrhythmic at physiologically relevant but nonoptimal temperatures. LHY- and RVE8-type Mybs are found in separate clades across the land plant lineage and even in some unicellular green algae, suggesting that they both may have functioned in even the earliest arising plant circadian oscillators. Our data suggest that the complexity of the plant circadian network may have arisen to provide rhythmic robustness across the range of environmental extremes to which plants, as sessile organisms, are regularly subjected.

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding big data that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

The circadian clock is a complex regulatory network that enhances plant growth and fitness in a constantly changing environment. In Arabidopsis (Arabidopsis thaliana), the clock is composed of numerous regulatory feedback loops in which REVEILLE8 (RVE8) and its homologs RVE4 and RVE6 act in a partially redundant manner to promote clock pace. Here, we report that the remaining members of the RVE8 clade, RVE3 and RVE5, play only minor roles in the regulation of clock function. However, we find that RVE8 clade proteins have unexpected functions in the modulation of light input to the clock and the control of plant growth at multiple stages of development. In seedlings, these proteins repress hypocotyl elongation in a daylength- and sucrose-dependent manner. Strikingly, adult rve4 6 8 and rve3 4 5 6 8 mutants are much larger than wild-type plants, with both increased leaf area and biomass. This size phenotype is associated with a faster growth rate and larger cell size and is not simply due to a delay in the transition to flowering. Gene expression and epistasis analysis reveal that the growth phenotypes of rve mutants are due to the misregulation of PHYTOCHROME INTERACTING FACTOR4 (PIF4) and PIF5 expression. Our results show that even small changes in PIF gene expression caused by the perturbation of clock gene function can have large effects on the growth of adult plants.