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The sensitive to freezing6 (sfr6) mutant of Arabidopsis (Arabidopsis thaliana) is late flowering in long days due to reduced expression of components in the photoperiodic flowering pathway in long-day photoperiods. Microarray analysis of gene expression showed that a circadian clock-associated motif, the evening element, was overrepresented in promoters of genes down-regulated in sfr6 plants. Analysis of leaf movement rhythms found sfr6 plants showed a sucrose (Suc)-dependent long period phenotype; unlike wild-type Arabidopsis, the clock in sfr6 plants did not have a shorter rhythm in the presence of Suc. Other developmental responses to Suc were unaltered in sfr6 plants, suggesting insensitivity to Suc is restricted to the clock. We investigated the effect of sfr6 and Suc upon clock gene expression over 24 h. The sfr6 mutation resulted in reduced expression of the clock components CIRCADIAN CLOCK ASSOCIATED1, GIGANTEA, and TIMING OF CAB1. These changes occurred independently of Suc supplementation. Wild-type plants showed small increases in clock gene expression in the presence of Suc; this response to Suc was reduced in sfr6 plants. This study shows that large changes in level and timing of clock gene expression may have little effect upon clock outputs. Moreover, although Suc influences the period and accuracy of the Arabidopsis clock, it results in relatively minor changes in clock gene expression.

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.

The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex series of interacting feedback loops whereby proteins regulate their own expression across day and night. early bird (ebi) is a circadian mutation that causes the clock to speed up: ebi plants have short circadian periods, early phase of clock gene expression, and are early flowering. We show that EBI associates with ZEITLUPE (ZTL), known to act in the plant clock as a posttranslational mediator of protein degradation. However, EBI is not degraded by its interaction with ZTL. Instead, ZTL counteracts the effect of EBI during the day and increases it at night, modulating the expression of key circadian components. The partnership of EBI with ZTL reveals a novel mechanism involved in controlling the complex transcription-translation feedback loops of the clock. This work highlights the importance of cross talk between the ubiquitination pathway and transcriptional control for regulation of the plant clock.

Circadian clocks are required to coordinate metabolism and physiology with daily changes in the environment. Such clocks have several distinctive features, including a free-running rhythm of approximately 24 h and the ability to entrain to both light or temperature cycles (zeitgebers). We have previously characterized the EARLY FLOWERING4 (ELF4) locus of Arabidopsis (Arabidopsis thaliana) as being important for robust rhythms. Here, it is shown that ELF4 is necessary for at least two core clock functions: entrainment to an environmental cycle and rhythm sustainability under constant conditions. We show that elf4 demonstrates clock input defects in light responsiveness and in circadian gating. Rhythmicity in elf4 could be driven by an environmental cycle, but an increased sensitivity to light means the circadian system of elf4 plants does not entrain normally. Expression of putative core clock genes and outputs were characterized in various ELF4 backgrounds to establish the molecular network of action. ELF4 was found to be intimately associated with the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LONG ELONGATED HYPOCOTYL (LHY)-TIMING OF CAB EXPRESSION1 (TOC1) feedback loop because, under free run, ELF4 is required to regulate the expression of CCA1 and TOC1 and, further, elf4 is locked in the evening phase of this feedback loop. ELF4, therefore, can be considered a component of the central CCA1/LHY-TOC1 feedback loop in the plant circadian clock.

In response to exogenous rhythms of light and temperature, most organisms exhibit endogenous circadian rhythms (i.e. cycles of behavior and gene expression with a periodicity of approximately 24 h). One of the defining characteristics of the circadian clock is its ability to synchronize (entrain) to an environmental rhythm. Entrainment is arguably the most salient feature of the clock in evolutionary terms. Previous quantitative trait studies of circadian characteristics in Arabidopsis (Arabidopsis thaliana) considered leaf movement under constant (free-running) conditions. This study, however, addressed the important circadian parameter of phase, which reflects the entrained relationship between the clock and the external cycle. Here it is shown that, when exposed to the same photoperiod, Arabidopsis accessions differ dramatically in phase. Variation in the timing of circadian LUCIFERASE expression was used to map loci affecting the entrained phase of the clock in a recombinant population derived from two geographically distant accessions, Landsberg erecta and Cape Verde Islands. Four quantitative trait loci (QTL) were found with major effects on circadian phase. A QTL on chromosome 5 contained SIGNALING IN RED LIGHT REDUCED 1 and PSEUDORESPONSE REGULATOR 3, both genes known to affect the circadian clock. Previously unknown polymorphisms were found in both genes, making them candidates for the effect on phase. Fine mapping of two other QTL highlighted genomic regions not previously identified in any circadian screens, indicating their effects are likely due to genes not hitherto considered part of the circadian system.

Plants synchronize developmental and metabolic processes with the earth's 24-h rotation through the integration of circadian rhythms and responses to light. We characterize the time for coffee (tic) mutant that disrupts circadian gating, photoperiodism, and multiple circadian rhythms, with differential effects among rhythms. TIC is distinct in physiological functions and genetic map position from other rhythm mutants and their homologous loci. Detailed rhythm analysis shows that the chlorophyll a/b-binding protein gene expression rhythm requires TIC function in the mid to late subjective night, when human activity may require coffee, in contrast to the function of EARLY-FLOWERING3 (ELF3) in the late day to early night. tic mutants misexpress genes that are thought to be critical for circadian timing, consistent with our functional analysis. Thus, we identify TIC as a regulator of the clock gene circuit. In contrast to tic and elf3 single mutants, tic elf3 double mutants are completely arrhythmic. Even the robust circadian clock of plants cannot function with defects at two different phases.

Circadian clocks are important regulators of behavior and physiology. The circadian clock of Drosophila depends on an autoinhibitory feedback loop involving dCLOCK, CYCLE (also called dBMAL, for Drosophila brain and muscle ARNT-like protein), dPERIOD, and dTIMELESS. Recent studies suggest that the clock mechanism in other insect species may differ strikingly from that of Drosophila. We cloned Clock, Bmal, and Timeless homologs (apClock, apBmal, and apTimeless) from the silkmoth Antheraea pernyi, from which a Period homolog (apPeriod) has already been cloned. In Schneider 2 (S2) cell culture assays, apCLOCK:apBMAL activates transcription through an E-box enhancer element found in the 5′ region of the apPeriod gene. Furthermore, apPERIOD can robustly inhibit apCLOCK: apBMAL-mediated transactivation, and apTIMELESS can augment this inhibition. Thus, a complete feedback loop, resembling that found in Drosophila, can be constructed from silkmoth CLOCK, BMAL, PERIOD, and TIMELESS. Our results suggest that the circadian autoinhibitory feedback loop discovered in Drosophila is likely to be widespread among insects. However, whereas the transactivation domain in Drosophila lies in the C terminus of dCLOCK, in A. pernyi, it lies in the C terminus of apBMAL, which is highly conserved with the C termini of BMALs in other insects (except Drosophila) and in vertebrates. Our analysis sheds light on the molecular function and evolution of clock genes in the animal kingdom.

The circadian system of Neurospora crassa includes a molecular feedback loop that is entrainable by light. A recent study has shown that a second, elusive oscillator interacts with the feedback loop to drive output rhythms.

The circadian clock regulates many aspects of plant physiology, growth and development. It produces daily rhythms of growth and metabolism, and interacts with signalling pathways controlling environmental responses over the course of a day or a year. Over the last decade, a combination of empirical research in molecular genetics and mathematical modelling, mostly utilising Arabidopsis thaliana, has led to the identification of many plant clock components and an understanding of their interlocking roles within the biochemical mechanism. The plant clock shares many characteristics of circadian clocks in other taxa, being temperature-compensated, capable of generating endogenous rhythms, of entraining to environmental cycles and regulated by means of transcription–translation feedback loops; however, few, if any, components of the plant clock appear to be shared with other organisms, indicating an independent evolutionary origin. In this review, we describe our current understanding of the central clockwork and how it receives input and regulates outputs. We also discuss the interaction between the clock and the environment, identifying areas, such as the integration of non-photic stimuli, where future work will lead to a fuller understanding of how the circadian system is embedded in plant physiology.

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 arrhythmias. 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.