Andrew J. Millar

Circadian clock mutants in Arabidopsis identified by luciferase imaging

The cycling bioluminescence of Arabidopsis plants carrying a firefly luciferase fusion construct was used to identify mutant individuals with aberrant cycling patterns. Both long- and short-period mutants were recovered. A semidominant short-period mutation, timing of CAB expression (toc1), was mapped to chromosome 5. The toc1 mutation shortens the period of two distinct circadian rhythms, the expression of chlorophyll a/b-binding protein (CAB) genes and the movements of primary leaves, although toc1 mutants do not show extensive pleiotropy for other phenotypes.

Peroxiredoxins are conserved markers of circadian rhythms

Cellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth’s rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation–reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription–translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago.

Experimental validation of a predicted feedback loop in the multi‐oscillator clock of Arabidopsis thaliana

Our computational model of the circadian clock comprised the feedback loop between LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1), and a predicted, interlocking feedback loop involving TOC1 and a hypothetical component Y. Experiments based on model predictions suggested GIGANTEA (GI) as a candidate for Y. We now extend the model to include a recently demonstrated feedback loop between the TOC1 homologues PSEUDO‐RESPONSE REGULATOR 7 (PRR7), PRR9 and LHY and CCA1. This three‐loop network explains the rhythmic phenotype of toc1 mutant alleles. Model predictions fit closely to new data on the gi;lhy;cca1 mutant, which confirm that GI is a major contributor to Y function. Analysis of the three‐loop network suggests that the plant clock consists of morning and evening oscillators, coupled intracellularly, which may be analogous to coupled, morning and evening clock cells in Drosophila and the mouse.

The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana

Many plants use day length as an environmental cue to ensure proper timing of the switch from vegetative to reproductive growth. Day-length sensing involves an interaction between the relative length of day and night, and endogenous rhythms that are controlled by the plant circadian clock. Thus, plants with defects in circadian regulation cannot properly regulate the timing of the floral transition. Here we describe the gene EARLY FLOWERING 4 (ELF4), which is involved in photoperiod perception and circadian regulation. ELF4 promotes clock accuracy and is required for sustained rhythms in the absence of daily light/dark cycles. elf4 mutants show attenuated expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a gene that is thought to function as a central oscillator component. In addition, elf4 plants transiently show output rhythms with highly variable period lengths before becoming arrhythmic. Mutations in elf4 result in early flowering in non-inductive photoperiods, which is probably caused by elevated amounts of CONSTANS (CO), a gene that promotes floral induction.

Circadian rhythms persist without transcription in a eukaryote

Circadian rhythms are ubiquitous in eukaryotes, and coordinate numerous aspects of behaviour, physiology and metabolism, from sleep/wake cycles in mammals to growth and photosynthesis in plants. This daily timekeeping is thought to be driven by transcriptional–translational feedback loops, whereby rhythmic expression of ‘clock’ gene products regulates the expression of associated genes in approximately 24-hour cycles. The specific transcriptional components differ between phylogenetic kingdoms. The unicellular pico-eukaryotic alga Ostreococcus tauri possesses a naturally minimized clock, which includes many features that are shared with plants, such as a central negative feedback loop that involves the morning-expressed CCA1 and evening-expressed TOC1 genes. Given that recent observations in animals and plants have revealed prominent post-translational contributions to timekeeping, a reappraisal of the transcriptional contribution to oscillator function is overdue. Here we show that non-transcriptional mechanisms are sufficient to sustain circadian timekeeping in the eukaryotic lineage, although they normally function in conjunction with transcriptional components. We identify oxidation of peroxiredoxin proteins as a transcription-independent rhythmic biomarker, which is also rhythmic in mammals. Moreover we show that pharmacological modulators of the mammalian clock mechanism have the same effects on rhythms in Ostreococcus. Post-translational mechanisms, and at least one rhythmic marker, seem to be better conserved than transcriptional clock regulators. It is plausible that the oldest oscillator components are non-transcriptional in nature, as in cyanobacteria, and are conserved across kingdoms.

A novel circadian phenotype based on firefly luciferase expression in transgenic plants.

A 320-bp fragment of the Arabidopsis cab2 promoter is sufficient to mediate transcriptional regulation by both phytochrome and the circadian clock. We fused this promoter fragment to the firefly luciferase (Luc) gene to create a real-time reporter for regulated gene expression in intact plants. Cab2::Luc transcript accumulated in the expected patterns and luciferase activity was closely correlated to cab2::Luc mRNA abundance in both etiolated and green seedlings. The concentration of the bulk of luciferase protein did not reflect these patterns but maintained a relatively constant level, implying that a post-translational mechanism(s) leads to the high-amplitude regulation of luciferase activity. We used a low-light video imaging system to establish that luciferase bioluminescence in vivo accurately reports the temporal and spatial regulation of cab2 transcription in single seedlings. The unique qualities of the firefly luciferase system allowed us to monitor regulated gene expression in real time in individual multicellular organisms. This noninvasive marker for temporal regulation at the molecular level constitutes a circadian phenotype, which may be used to isolate mutants in the circadian clock.

Extension of a genetic network model by iterative experimentation and mathematical analysis.

Circadian clocks involve feedback loops that generate rhythmic expression of key genes. Molecular genetic studies in the higher plant Arabidopsis thaliana have revealed a complex clock network. The first part of the network to be identified, a transcriptional feedback loop comprising TIMING OF CAB EXPRESSION 1 (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), fails to account for significant experimental data. We develop an extended model that is based upon a wider range of data and accurately predicts additional experimental results. The model comprises interlocking feedback loops comparable to those identified experimentally in other circadian systems. We propose that each loop receives input signals from light, and that each loop includes a hypothetical component that had not been explicitly identified. Analysis of the model predicted the properties of these components, including an acute light induction at dawn that is rapidly repressed by LHY and CCA1. We found this unexpected regulation in RNA levels of the evening‐expressed gene GIGANTEA (GI), supporting our proposed network and making GI a strong candidate for this component.

Conditional Circadian Dysfunction of the Arabidopsis early-flowering 3 Mutant

Photoperiodic responses, such as the daylength-dependent control of reproductive development, are associated with a circadian biological clock. The photoperiod-insensitive early-flowering 3 (elf3) mutant of Arabidopsis thaliana lacks rhythmicity in two distinct circadian-regulated processes. This defect was apparent only when plants were assayed under constant light conditions. elf3 mutants retain rhythmicity in constant dark and anticipate light/dark transitions under most light/dark regimes. The conditional arrhythmic phenotype suggests that the circadian pacemaker is intact in darkness in elf3 mutant plants, but the transduction of light signals to the circadian clock is impaired.

The ELF3 zeitnehmer regulates light signalling to the circadian clock

The circadian system regulates 24-hour biological rhythms and seasonal rhythms, such as flowering. Long-day flowering plants like Arabidopsis thaliana, measure day length with a rhythm that is not reset at lights-off, whereas short-day plants measure night length on the basis of circadian rhythm of light sensitivity that is set from dusk. early flowering 3 (elf3) mutants of Arabidopsis are aphotoperiodic and exhibit light-conditional arrhythmia. Here we show that the elf3-7 mutant retains oscillator function in the light but blunts circadian gating of CAB gene activation, indicating that deregulated phototransduction may mask rhythmicity. Furthermore, elf3 mutations confer the resetting pattern of short-day photoperiodism, indicating that gating of phototransduction may control resetting. Temperature entrainment can bypass the requirement for normal ELF3 function for the oscillator and partially restore rhythmic CAB expression. Therefore, ELF3 specifically affects light input to the oscillator, similar to its function in gating CAB activation, allowing oscillator progression past a light-sensitive phase in the subjective evening. ELF3 provides experimental demonstration of the zeitnehmer (‘time-taker’) concept.

The clock gene circuit in Arabidopsis includes a repressilator with additional feedback loops

Circadian clocks synchronise biological processes with the day/night cycle, using molecular mechanisms that include interlocked, transcriptional feedback loops. Recent experiments identified the evening complex (EC) as a repressor that can be essential for gene expression rhythms in plants. Integrating the EC components in this role significantly alters our mechanistic, mathematical model of the clock gene circuit. Negative autoregulation of the EC genes constitutes the clocks evening loop, replacing the hypothetical component Y. The EC explains our earlier conjecture that the morning gene PSEUDO‐RESPONSE REGULATOR 9 was repressed by an evening gene, previously identified with TIMING OF CAB EXPRESSION1 (TOC1). Our computational analysis suggests that TOC1 is a repressor of the morning genes LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK ASSOCIATED1 rather than an activator as first conceived. This removes the necessity for the unknown component X (or TOC1mod) from previous clock models. As well as matching timeseries and phase‐response data, the model provides a new conceptual framework for the plant clock that includes a three‐component repressilator circuit in its complex structure.