Limited water stress modulates expression of circadian clock genes in Brachypodium distachyon roots.

Organisms have evolved a circadian clock for the precise timing of their biological processes. Studies primarily on model dicots have shown the complexity of the inner timekeeper responsible for maintaining circadian oscillation in plants and have highlighted that circadian regulation is more than relevant to a wide range of biological processes, especially organ development and timing of flowering. Contribution of the circadian clock to overall plant fitness and yield has also long been known. Nevertheless, the organ- and species-specific functions of the circadian clock and its relation to stress adaptation have only recently been identified. Here we report transcriptional changes of core clock genes of the model monocot Brachypodium distachyon under three different light regimes (18:6 light:dark, 24:0 light and 0:24 dark) in response to mild drought stress in roots and green plant parts. Comparative monitoring of core clock gene expression in roots and green plant parts has shown that both phase and amplitude of expression in the roots of Brachypodium plants differ markedly from those in the green plant parts, even under well-watered conditions. Moreover, circadian clock genes responded to water depletion differently in root and shoot. These results suggest an organ-specific form and functions of the circadian clock in Brachypodium roots.

SUMOylation of PHYTOCHROME INTERACTING FACTOR 3 promotes photomorphogenesis in Arabidopsis thaliana.

In Arabidopsis thaliana, phytochrome B (phyB) is the dominant receptor of photomorphogenic development under red light. Phytochrome B interacts with a set of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR 3 (PIF3). The interaction between PIF3 and photoactivated phyB leads to the rapid phosphorylation and degradation of PIF3 and also to the degradation of phyB, events which are required for proper photomorphogenesis. Here we report that PIF3 is SUMOylated at the Lys13 (K13) residue and that we could detect this posttranslational modification in a heterologous experimental system and also in planta. We also found that the SUMO acceptor site mutant PIF3(K13R) binds more strongly to the target promoters than its SUMOylated, wild-type counterpart. Seedlings expressing PIF3(K13R) show an elongated hypocotyl response, elevated photoprotection and higher transcriptional induction of red-light responsive genes compared with plantlets expressing wild-type PIF3. These observations are supported by the lower level of phyB in plants which possess only PIF3(K13R), indicating that SUMOylation of PIF3 also alters photomorphogenesis via the regulation of phyB levels. In conclusion, whereas SUMOylation is generally connected to different stress responses, it also fine-tunes light signalling by reducing the biological activity of PIF3, thus promoting photomorphogenesis.

Light Control of Salt-Induced Proline Accumulation Is Mediated by ELONGATED HYPOCOTYL 5 in Arabidopsis.

Plants have to adapt their metabolism to constantly changing environmental conditions, among which the availability of light and water is crucial in determining growth and development. Proline accumulation is one of the sensitive metabolic responses to extreme conditions; it is triggered by salinity or drought and is regulated by light. Here we show that red and blue but not far-red light is essential for salt-induced proline accumulation, upregulation of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) and downregulation of PROLINE DEHYDROGENASE 1 (PDH1) genes, which control proline biosynthetic and catabolic pathways, respectively. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the transcription factor ELONGATED HYPOCOTYL 5 (HY5) binds to G-box and C-box elements of P5CS1 and a C-box motif of PDH1. Salt-induced proline accumulation and P5CS1 expression were reduced in the hy5hyh double mutant, suggesting that HY5 promotes proline biosynthesis through connecting light and stress signals. Our results improve our understanding on interactions between stress and light signals, confirming HY5 as a key regulator in proline metabolism.

ELONGATED HYPOCOTYL 5 mediates blue light signalling to the Arabidopsis circadian clock.

Circadian clocks are gene networks producing 24-h oscillations at the level of clock gene expression that are synchronized to environmental cycles via light signals. The ELONGATED HYPOCOTYL 5 (HY5) transcription factor is a signalling hub acting downstream of several photoreceptors and is a key mediator of photomorphogenesis. Here we describe a mechanism by which light quality could modulate the pace of the circadian clock through governing abundance of HY5. We show that hy5 mutants display remarkably shorter period rhythms in blue but not in red light or darkness, and blue light is more efficient than red to induce accumulation of HY5 at transcriptional and post-transcriptional levels. We demonstrate that the pattern and level of HY5 accumulation modulates its binding to specific promoter elements of the majority of clock genes, but only a few of these show altered transcription in the hy5 mutant. Mathematical modelling suggests that the direct effect of HY5 on the apparently non-responsive clock genes could be masked by feedback from the clock gene network. We conclude that the information on the ratio of blue and red components of the white light spectrum is decoded and relayed to the circadian oscillator, at least partially, by HY5.

Coordination of robust single cell rhythms in the Arabidopsis circadian clock via spatial waves of gene expression.

The Arabidopsis circadian clock orchestrates gene regulation across the day/night cycle. Although a multiple feedback loop circuit has been shown to generate the 24-hr rhythm, it remains unclear how robust the clock is in individual cells, or how clock timing is coordinated across the plant. Here we examine clock activity at the single cell level across Arabidopsis seedlings over several days under constant environmental conditions. Our data reveal robust single cell oscillations, albeit desynchronised. In particular, we observe two waves of clock activity; one going down, and one up the root. We also find evidence of cell-to-cell coupling of the clock, especially in the root tip. A simple model shows that cell-to-cell coupling and our measured period differences between cells can generate the observed waves. Our results reveal the spatial structure of the plant clock and suggest that unlike the centralised mammalian clock, the Arabidopsis clock has multiple coordination points.

Circadian and Light Regulated Expression of CBFs and their Upstream Signalling Genes in Barley.

CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light-including its spectra-also has a crucial role in regulating CBF expression. Earlier, studies established that the expression patterns of some of these regulatory genes follow circadian rhythms. To understand more of this complex acclimation process, we studied the expression patterns of the signal transducing pathways, including signal perception, the circadian clock and phospholipid signalling pathways, upstream of the CBF gene regulatory hub. To exclude the confounding effect of cold, experiments were carried out at 22 °C. Our results show that the expression of genes implicated in the phospholipid signalling pathway follow a circadian rhythm. We demonstrated that, from among the tested CBF genes expressed in Hordeumvulgare (Hv) under our conditions, only the members of the HvCBF4-phylogenetic subgroup showed a circadian pattern. We found that the HvCBF4-subgroup genes were expressed late in the afternoon or early in the night. We also determined the expression changes under supplemental far-red illumination and established that the transcript accumulation had appeared four hours earlier and more intensely in several cases. Based on our results, we propose a model to illustrate the effect of the circadian clock and the quality of the light on the elements of signalling pathways upstream of the HvCBFs, thus integrating the complex regulation of the early cellular responses, which finally lead to an elevated abiotic stress tolerance.

Light and Temperature Signalling at the Level of CBF14 Gene Expression in Wheat and Barley.

The wheat and barley CBF14 genes have been newly defined as key components of the light quality-dependent regulation of the freezing tolerance by the integration of phytochrome-mediated light and temperature signals. To further investigate the wavelength dependence of light-induced CBF14 expression in cereals, we carried out a detailed study using monochromatic light treatments at an inductive and a non-inductive temperature. Transcript levels of CBF14 gene in winter wheat Cheyenne, winter einkorn G3116 and winter barley Nure genotypes were monitored. We demonstrated that (1) CBF14 is most effectively induced by blue light and (2) provide evidence that this induction does not arise from light-controlled CRY gene expression. (3) We demonstrate that temperature shifts induce CBF14 transcription independent of the light conditions and that (4) the effect of temperature and light treatments are additive. Based on these data, it can be assumed that temperature and light signals are relayed to the level of CBF14 expression via separate signalling routes.

Light-quality and temperature-dependent CBF14 gene expression modulates freezing tolerance in cereals.

UNLABELLED: C-repeat binding factor 14 (CBF14) is a plant transcription factor that regulates a set of cold-induced genes, contributing to enhanced frost tolerance during cold acclimation. Many CBF genes are induced by cool temperatures and regulated by day length and light quality, which affect the amount of accumulated freezing tolerance. Here we show that a low red to far-red ratio in white light enhances CBF14 expression and increases frost tolerance at 15°C in winter Triticum aesitivum and Hordeum vulgare genotypes, but not in T. monococcum (einkorn), which has a relatively low freezing tolerance. Low red to far-red ratio enhances the expression of PHYA in all three species, but induces PHYB expression only in einkorn. Based on our results, a model is proposed to illustrate the supposed positive effect of phytochrome A and the negative influence of phytochrome B on the enhancement of freezing tolerance in cereals in response to spectral changes of incident light. KEY WORDS: CBF-regulon, barley, cereals, cold acclimation, freezing tolerance, light regulation, low red/far-red ratio, phytochrome, wheat.

SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana.

The red/far red light absorbing photoreceptor phytochrome-B (phyB) cycles between the biologically inactive (Pr, λmax, 660 nm) and active (Pfr; λmax, 730 nm) forms and functions as a light quality and quantity controlled switch to regulate photomorphogenesis in Arabidopsis. At the molecular level, phyB interacts in a conformation-dependent fashion with a battery of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR transcription factors, and by modulating their activity/abundance, it alters expression patterns of genes underlying photomorphogenesis. Here we report that the small ubiquitin-like modifier (SUMO) is conjugated (SUMOylation) to the C terminus of phyB; the accumulation of SUMOylated phyB is enhanced by red light and displays a diurnal pattern in plants grown under light/dark cycles. Our data demonstrate that (i) transgenic plants expressing the mutant phyB(Lys996Arg)-YFP photoreceptor are hypersensitive to red light, (ii) light-induced SUMOylation of the mutant phyB is drastically decreased compared with phyB-YFP, and (iii) SUMOylation of phyB inhibits binding of PHYTOCHROME INTERACTING FACTOR 5 to phyB Pfr. In addition, we show that OVERLY TOLERANT TO SALT 1 (OTS1) de-SUMOylates phyB in vitro, it interacts with phyB in vivo, and the ots1/ots2 mutant is hyposensitive to red light. Taken together, we conclude that SUMOylation of phyB negatively regulates light signaling and it is mediated, at least partly, by the action of OTS SUMO proteases.

High-level expression and phosphorylation of phytochrome B modulates flowering time in Arabidopsis.

Optimal timing of flowering in higher plants is crucial for successful reproduction and is coordinated by external and internal factors, including light and the circadian clock. In Arabidopsis, light-dependent stabilization of the rhythmically expressed CONSTANS (CO) is required for the activation of FLOWERING LOCUS T (FT), resulting in the initiation of flowering. Phytochrome A and cryptochrome photoreceptors stabilize CO in the evening by attenuating the activity of the CONSTITUTIVE PHOTOMORPHOGENIC 1-SUPPRESSOR OF PHYA-105 1 (COP1-SPA1) ubiquitin ligase complex, which promotes turnover of CO. In contrast, phytochrome B (phyB) facilitates degradation of CO in the morning and delays flowering. Accordingly, flowering is accelerated in phyB mutants. Paradoxically, plants overexpressing phyB also show early flowering, which may arise from an early phase of rhythmic CO expression. Here we demonstrate that overexpression of phyB induces FT transcription at dusk and in the night without affecting the phase or level of CO transcription. This response depends on the light-activated Pfr form of phyB that inhibits the function of the COP1-SPA1 complex by direct interactions. Our data suggest that attenuation of COP1 activity results in the accumulation of CO protein and subsequent induction of FT. We show that phosphorylation of Ser-86 inhibits this function of phyB by accelerating dark reversion and thus depletion of Pfr forms in the night. Our results explain the early flowering phenotype of phyB overexpression and reveal additional features of the molecular machinery by which photoreceptors mediate photoperiodism.

UV-B-responsive association of the Arabidopsis bZIP transcription factor ELONGATED HYPOCOTYL5 with target genes, including its own promoter.

In plants subjected to UV-B radiation, responses are activated that minimize damage caused by UV-B. The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) acts downstream of the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) and promotes UV-B-induced photomorphogenesis and acclimation. Expression of HY5 is induced by UV-B; however, the transcription factor(s) that regulate HY5 transcription in response to UV-B and the impact of UV-B on the association of HY5 with its target promoters are currently unclear. Here, we show that HY5 binding to the promoters of UV-B-responsive genes is enhanced by UV-B in a UVR8-dependent manner in Arabidopsis thaliana. In agreement, overexpression of REPRESSOR OF UV-B PHOTOMORPHOGENESIS2, a negative regulator of UVR8 function, blocks UV-B-responsive HY5 enrichment at target promoters. Moreover, we have identified a T/G-box in the HY5 promoter that is required for its UV-B responsiveness. We show that HY5 and its homolog HYH bind to the T/G(HY5)-box cis-acting element and that they act redundantly in the induction of HY5 expression upon UV-B exposure. Therefore, HY5 is enriched at target promoters in response to UV-B in a UVR8 photoreceptor-dependent manner, and HY5 and HYH interact directly with a T/G-box cis-acting element of the HY5 promoter, mediating the transcriptional activation of HY5 in response to UV-B.

The Arabidopsis ZINC FINGER PROTEIN3 Interferes with Abscisic Acid and Light Signaling in Seed Germination and Plant Development.

Seed germination is controlled by environmental signals, including light and endogenous phytohormones. Abscisic acid (ABA) inhibits, whereas gibberellin promotes, germination and early seedling development, respectively. Here, we report that ZFP3, a nuclear C2H2 zinc finger protein, acts as a negative regulator of ABA suppression of seed germination in Arabidopsis (Arabidopsis thaliana). Accordingly, regulated overexpression of ZFP3 and the closely related ZFP1, ZFP4, ZFP6, and ZFP7 zinc finger factors confers ABA insensitivity to seed germination, while the zfp3 zfp4 double mutant displays enhanced ABA susceptibility. Reduced expression of several ABA-induced genes, such as RESPONSIVE TO ABSCISIC ACID18 and transcription factor ABSCISIC ACID-INSENSITIVE4 (ABI4), in ZFP3 overexpression seedlings suggests that ZFP3 negatively regulates ABA signaling. Analysis of ZFP3 overexpression plants revealed multiple phenotypic alterations, such as semidwarf growth habit, defects in fertility, and enhanced sensitivity of hypocotyl elongation to red but not to far-red or blue light. Analysis of genetic interactions with phytochrome and abi mutants indicates that ZFP3 enhances red light signaling by photoreceptors other than phytochrome A and additively increases ABA insensitivity conferred by the abi2, abi4, and abi5 mutations. These data support the conclusion that ZFP3 and the related ZFP subfamily of zinc finger factors regulate light and ABA responses during germination and early seedling development.

Differential expression of the brassinosteroid receptor-encoding BRI1 gene in Arabidopsis.

Brassinosteroid (BR)-regulated growth and development in Arabidopsis depends on BRASSINOSTEROID INSENSITIVE 1 (BRI1), the BR receptor that is responsible for initiating the events of BR signalling. We analysed the temporal and spatial regulation of BRI1 expression using stable transgenic lines that carried BRI1 promoter:reporter fusions. In both seedlings and mature plants the tissues undergoing elongation or differentiation showed elevated BRI1 gene activity, and it could be demonstrated that in the hypocotyl this was accompanied by accumulation of the BRI1 transcript and its receptor protein product. In seedlings the BRI1 promoter was also found to be under diurnal regulation, determined primarily by light repression and a superimposed circadian control. To determine the functional importance of transcriptional regulation we complemented the severely BR insensitive bri1-101 mutant with a BRI1-luciferase fusion construct that was driven by promoters with contrasting specificities. Whereas the BRI1 promoter-driven transgene fully restored the wild phenotype, expression from the photosynthesis-associated CAB3 and the vasculature-specific SUC2 and ATHB8 promoters resulted in plants with varying morphogenic defects. Our results reveal complex differential regulation of BRI1 expression, and suggest that by influencing the distribution and abundance of the receptor this regulation can enhance or attenuate BR signalling.

The circadian clock-associated small GTPase LIGHT INSENSITIVE PERIOD1 suppresses light-controlled endoreplication and affects tolerance to salt stress in Arabidopsis.

Circadian clocks are biochemical timers regulating many physiological and molecular processes according to the day/night cycle. The small GTPase LIGHT INSENSITIVE PERIOD1 (LIP1) is a circadian clock-associated protein that regulates light input to the clock. In the absence of LIP1, the effect of light on free-running period length is much reduced. Here, we show that in addition to suppressing red and blue light-mediated photomorphogenesis, LIP1 is also required for light-controlled inhibition of endoreplication and tolerance to salt stress in Arabidopsis (Arabidopsis thaliana). We demonstrate that in the processes of endoreplication and photomorphogenesis, LIP1 acts downstream of the red and blue light photoreceptors phytochrome B and cryptochromes. Manipulation of the subcellular distribution of LIP1 revealed that the circadian function of LIP1 requires nuclear localization of the protein. Our data collectively suggest that LIP1 influences several signaling cascades and that its role in the entrainment of the circadian clock is independent from the other pleiotropic effects. Since these functions of LIP1 are important for the early stages of development or under conditions normally experienced by germinating seedlings, we suggest that LIP1 is a regulator of seedling establishment.

Light-regulated gene expression in yeast.

An important basic requirement of synthetic genetic networks is the option of external control of gene expression. Although several chemically inducible systems are available, all of these suffer from the common problem: the chemical inducers are difficult to remove so that to terminate the response. We have described a regulatory expression system for yeast, which employs light as inducer. This light switch translates light-controlled protein-protein interactions into the transcription of selected genes in a dose-dependent and reversible manner.

Functional interaction of the circadian clock and UV RESISTANCE LOCUS 8-controlled UV-B signaling pathways in Arabidopsis thaliana.

Circadian clocks regulate many molecular and physiological processes in Arabidopsis (Arabidopsis thaliana), allowing the timing of these processes to occur at the most appropriate time of the day in a 24-h period. The accuracy of timing relies on the synchrony of the clock and the environmental day/night cycle. Visible light is the most potent signal for such synchronization, but light-induced responses are also rhythmically attenuated (gated) by the clock. Here, we report a similar mutual interaction of the circadian clock and non-damaging photomorphogenic UV-B light. We show that low-intensity UV-B radiation acts as entraining signal for the clock. UV RESISTANCE LOCUS 8 (UVR8) and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) are required, but ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG (HYH) are dispensable for this process. UV-B responsiveness of clock gene expression suggests that photomorphogenic UV-B entrains the plant clock through transcriptional activation. We also demonstrate that UV-B induction of gene expression under these conditions is gated by the clock in a HY5/HYH-independent manner. The arrhythmic early flowering 3-4 mutant showed non-gated, high-level gene induction by UV-B, yet displayed no increased tolerance to UV-B stress. Thus, the temporal restriction of UV-B responsiveness by the circadian clock can be considered as saving resources during acclimation without losing fitness.

Light inputs shape the Arabidopsis circadian system.

The circadian clock is a fundamental feature of eukaryotic gene regulation that is emerging as an exemplar genetic sub-network for systems biology. The circadian system in Arabidopsis plants is complex, in part due to its phototransduction pathways, which are themselves under circadian control. We therefore analysed two simpler experimental systems. Etiolated seedlings entrained by temperature cycles showed circadian rhythms in the expression of genes that are important for the clock mechanism, but only a restricted set of downstream target genes were rhythmic in microarray assays. Clock control of phototransduction pathways remained robust across a range of light inputs, despite the arrhythmic transcription of light-signalling genes. Circadian interactions with light signalling were then analysed using a single active photoreceptor. Phytochrome A (phyA) is expected to be the only active photoreceptor that can mediate far-red (FR) light input to the circadian clock. Surprisingly, rhythmic gene expression was profoundly altered under constant FR light, in a phyA-dependent manner, resulting in high expression of evening genes and low expression of morning genes. Dark intervals were required to allow high-amplitude rhythms across the transcriptome. Clock genes involved in this response were identified by mutant analysis, showing that the EARLY FLOWERING 4 gene is a likely target and mediator of the FR effects. Both experimental systems illustrate how profoundly the light input pathways affect the plant circadian clock, and provide strong experimental manipulations to understand critical steps in the plant clock mechanism.

Temporal repression of core circadian genes is mediated through EARLY FLOWERING 3 in Arabidopsis.

The circadian clock provides robust, ∼24 hr biological rhythms throughout the eukaryotes. The clock gene circuit in plants comprises interlocking transcriptional feedback loops, reviewed in [1], whereby the morning-expressed transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) repress the expression of evening genes, notably TIMING OF CAB EXPRESSION 1 (TOC1). EARLY FLOWERING 3 (ELF3) has been implicated as a repressor of light signaling to the clock [2, 3] and, paradoxically, as an activator of the light-induced genes CCA1 and LHY [4, 5]. We use cca1-11 lhy-21 elf3-4 plants to separate the repressive function of ELF3 from its downstream targets CCA1 and LHY. We further demonstrate that ELF3 associates physically with the promoter of PSEUDO-RESPONSE REGULATOR 9 (PRR9), a repressor of CCA1 and LHY expression, in a time-dependent fashion. The repressive function of ELF3 is thus consistent with indirect activation of LHY and CCA1, in a double-negative connection via a direct ELF3 target, PRR9. This mechanism reconciles the functions of ELF3 in the clock network during the night and points to further effects of ELF3 during the day.

Light-regulated nuclear import and degradation of Arabidopsis phytochrome-A N-terminal fragments.

The photoreceptor phytochrome-A (phyA) regulates germination and seedling establishment by mediating very low fluence (VLFR) and far-red high irradiance (FR-HIR) responses in Arabidopsis thaliana. In darkness, phyA homodimers exist in the biologically inactive Pr form and are localized in the cytoplasm. Light induces formation of the biologically active Pfr form and subsequent rapid nuclear import. PhyA Pfr, in contrast to the Pr form, is labile and has a half-life of ∼30 min. We produced transgenic plants in a phyA-201 null background that express the PHYA-yellow fluorescent protein (YFP) or the PHYA686-YFP-dimerization domain (DD) and PHYA686-YFP-DD-nuclear localization signal (NLS) or PHYA686-YFP-DD-nuclear exclusion signal (NES) fusion proteins. The PHYA686-YFP fusion proteins contained the N-terminal domain of phyA (686 amino acid residues), a short DD and the YFP. Here we report that (i) PHYA686-YFP-DD fusion protein is imported into the nucleus in a light-dependent fashion; (ii) neither of the PHYA686 fusion proteins is functional in FR-HIR and nuclear VLFR; and (iii) the phyA-dependent, blue light-induced inhibition of hypocotyl growth is mediated by the PHYA686-YFP-DD-NES but not by the PHYA686-YFP-DD-NLS and PHYA686-YFP-DD fusion proteins. We demonstrate that (i) light induces degradation of all PHYA N-terminal-containing fusion proteins and (ii) these N-terminal domain-containing fusion proteins including the constitutively nuclear PHYA686-YFP-DD-NLS and predominantly cytoplasmic PHYA686-YFP-DD-NES degrade at comparable rates but markedly more slowly than PHYA-YFP, whereas (iii) light-induced degradation of the native phyA is faster compared with PHYA-YFP.

Quantitative analysis of regulatory flexibility under changing environmental conditions.

The circadian clock controls 24-h rhythms in many biological processes, allowing appropriate timing of biological rhythms relative to dawn and dusk. Known clock circuits include multiple, interlocked feedback loops. Theory suggested that multiple loops contribute the flexibility for molecular rhythms to track multiple phases of the external cycle. Clear dawn- and dusk-tracking rhythms illustrate the flexibility of timing in Ipomoea nil. Molecular clock components in Arabidopsis thaliana showed complex, photoperiod-dependent regulation, which was analysed by comparison with three contrasting models. A simple, quantitative measure, Dusk Sensitivity, was introduced to compare the behaviour of clock models with varying loop complexity. Evening-expressed clock genes showed photoperiod-dependent dusk sensitivity, as predicted by the three-loop model, whereas the one- and two-loop models tracked dawn and dusk, respectively. Output genes for starch degradation achieved dusk-tracking expression through light regulation, rather than a dusk-tracking rhythm. Model analysis predicted which biochemical processes could be manipulated to extend dusk tracking. Our results reveal how an operating principle of biological regulators applies specifically to the plant circadian clock.