Life on Earth tends to follow a daily rhythm: some animals are awake during the day and asleep at night, whilst others are more active at night, or during the twilight around dawn and dusk. For many living things, these cycles of activity are driven by an internal body clock that helps the organism to adapt to the daily cycle of light and dark—and similar internal clocks also exist in plants.

These internal clocks define daily—or circadian—cycles whereby multiple genes are switched ‘on’ or ‘off’ at different time points in every 24-hr period. And, because light and ambient temperatures also vary with time of the day, many organisms use these external signals as cues to reset their own internal clocks. Moreover, the hours of daylight and temperature vary around the world, and also with the seasons, so plants and animals must be able to change how these external signals influence their internal clocks so that they stay in tune with the day/night cycle. However, it is not clear how they do this.

To explore this question, Anwer et al. grew plants that were from a cross between two types of the model plant Arabidopsis thaliana from different environments: one from Germany, and the other from Tajikistan in Central Asia. These offspring were also genetically engineered so that an enzyme that could give off light was produced under the control of the internal clock. Anwer et al. found that the plants continued to glow and fade with an almost daily rhythm even after external cues, such as changes in temperature or light, had been removed.

Different offspring plants consistently glowed and faded with different rhythms such that some had, for example, a 21-hr day and others a 28-hr day. These differences were caused by many genes that differed from the original German and Tajikistan parent plants, and Anwer et al. ‘mapped’ one of these genetic differences to a single gene. Offspring that inherited a version of a gene called ELF3 from the Tajikistan parent had internal clocks that ran faster when the plant was under the light. These plants also gradually stopped glowing as brightly as the German parent when they were kept in the dark, suggesting that their internal clocks were ‘ticking more softly’. It was already known that the ELF3 gene affected the circadian clock in plants, and Anwer et al. thus concluded that the plants with Tajikistan version of this gene, called ELF3-Sha, were also less able to reset their internal clocks to synchronize in response to external cues.

Anwer et al. also showed that the normal ELF3 protein is more likely to be found in the nucleus of a plant cell than the ELF3-Sha version, which might suggest that this protein is involved in switching genes off. Further research is now needed to uncover exactly how the ELF3 protein does this to keep the plant's internal clock ‘ticking’ correctly.

The rotation of the earth leads to environmental changes in ambient light and temperature that define a 24-hr cycle. During these diurnal cycles, light is correlated with warmth, while darkness is correlated with coolness. Thus, to ensure fitness in response to such regular environmental oscillations, many organisms have evolved an internal timing mechanism to predict these cyclic environmental cues, and this is termed the circadian clock. This internal clock synchronizes major developmental and physiological processes. For example, a considerable proportion of metabolism is under clock control (Graf et al., 2010; Haydon et al., 2011; Sanchez et al., 2011; Sanchez-Villarreal et al., 2013), and seasonal developmental programs require a circadian oscillator (Thines and Harmon, 2011). The coordination of molecular and physiological processes with the external environment thus provides an adaptive advantage under diverse climatic conditions (Dodd et al., 2005).

Circadian rhythms are produced by molecular oscillators, which are comprised of interlocking feedback loops having several components (Bujdoso and Davis, 2013). In Arabidopsis, the central loop consists of two closely related single-myb transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), as well as the pseudo response regulator (PRR) TIMING OF CAB EXPRESSION 1 (TOC1). In the morning, CCA1 and LHY repress TOC1 by directly binding to its promoter, resulting in the evening accumulation of TOC1, which, in turn, represses CCA1 and LHY expression (Gendron et al., 2012; Huang et al., 2012). The core oscillator is further tuned by an evening- and morning-phased loop. The evening loop is proposed to include GIGANTEA (GI) and TOC1, where GI activates the expression of TOC1, and TOC1 represses GI (Locke et al., 2006; Zeilinger et al., 2006; Huang et al., 2012). Two members of the pseudo response regulator gene family, PRR7 and PRR9, repress CCA1 and LHY during the day, and this establishes the morning loop (Locke et al., 2006; Zeilinger et al., 2006). Recently, an evening complex comprising EARLY FLOWERING 3 (ELF3), EARLY FLOWERING 4 (ELF4), and LUX ARRYTHMO (LUX) has been found to repress the morning loop by specifically binding to the PRR9 promoter to mediate transcription repression (Nozue et al., 2007; Kolmos et al., 2009; Dixon et al., 2011; Helfer et al., 2011; Nusinow et al., 2011; Chow et al., 2012; Herrero and Davis, 2012; Herrero et al., 2012).

Ambient light and temperature are two important environmental factors, termed zeitgebers, which reset the clock in a process referred to as entrainment (McClung and Davis, 2010; McClung, 2011). PRR7 and PRR9 have been reported to play a role in temperature compensation, which is the resistance of period change under differing mean ambient temperatures (Salomé and McClung, 2005; Salome et al., 2010). However, diurnal temperature regulation of the circadian clock is still poorly understood (McClung and Davis, 2010). In contrast, light has been established as a key factor for the entrainment of the clock where continuous irradiation shortens free-running period length in a fluence-rate-dependent manner as a process termed parametric entrainment (Aschoff, 1979; Covington et al., 2001; Johnson et al., 2003). Most genetic components of the clock were originally discovered from a light-entrained oscillator. Several studies have shown that many observable rhythms in plants dampen in constant darkness (DD), where dampening is defined as a reduction in circadian amplitude. For example, many genes have been associated with circadian rhythms of mRNA abundance that are expressed robustly under constant light (LL), but dampen in DD (Watillon et al., 1993; Zhong et al., 1997). Furthermore, the cyclic protein accumulation of several clock-regulated components, such as GI and ELF3 (Liu et al., 2001; David et al., 2006), was found to be reduced when shifted to darkness. Thus, the effects of various light-input signals on the Arabidopsis core oscillator remain to be elucidated.

Molecular and genetic studies support the involvement of ELF3 in light gating to the circadian clock, and ELF3 is also a required component of the core oscillator (Covington et al., 2001; Thines and Harmon, 2010; Kolmos et al., 2011; Herrero et al., 2012). The elf3 mutant was originally isolated in a screen for lines displaying photoperiod-independent early flowering (Zagotta et al., 1992, 1996). Further characterization of elf3 revealed other severe phenotypes, including defects in clock-regulated leaf movement rhythms, rhythmic hypocotyl elongation, and arrhythmic expression of gene expression under the free running condition of LL and in DD (Hicks et al., 1996; Reed et al., 2000; Thines and Harmon, 2010; Kolmos et al., 2011; Herrero et al., 2012). Natural variation at ELF3 has been detected that could be associated to an alteration in shade-avoidance responses, which included increased elongation of the hypocotyl, delay of cotyledon opening in seedlings, increased elongation of stems and petioles, and reduced developmental timing in adults, and to changes in circadian function (Tajima et al., 2007; Jimenez-Gomez et al., 2010; Coluccio et al., 2011; Undurraga et al., 2012). ELF3 was cloned and reported to encode a nuclear protein of unknown function, and it was proposed to work as a transcription factor (Hicks et al., 2001; Liu et al., 2001). Based on the accumulation of ELF3 protein upon shifting plants to LL, and its decrease when plants were shifted to DD, it was concluded that its abundance is dependent on ambient light (Liu et al., 2001). To extend this hypothesis based on the reported light-dependent phenotypes of elf3, McWatters et al. (2000) proposed that ELF3 fulfills the so-called zeitnehmer concept, in that this factor acts to bridge light perception to the clock. This idea was further strengthened when ELF3 was found to physically interact with the phytochromeB photoreceptor (Liu et al., 2001). The previously noted role of ELF3 in light input to the circadian clock (Kolmos et al., 2011) is not altered by new findings that ELF3 is core to the oscillator.

Many elements in the clock were isolated in screens for rhythm mutants using bioluminescence readout of the molecular oscillator. Worldwide, the species distribution of Arabidopsis ranges from the equator to extreme northern latitudes near the Arctic Circle (Koornneef et al., 2004). Therefore, as an alternative to induced mutants, another source of genetic variation can be found among naturally occurring populations of Arabidopsis. More specifically, environmental variations across and within local populations of Arabidopsis act as a discriminatory force on the gene pool from which only a few genetic variants of adaptive phenotypes will be selected and pass through reproduction. Arabidopsis populations based on Recombinant Inbred Lines (RILs) have been derived from parental accessions. Such populations are advantageous in mapping novel gene interactions. For example, quantitative trait locus (QTL) mapping was used to explain circadian parameters associated with natural variation in the circadian rhythmicity of leaf movement (Swarup et al., 1999; Michael et al., 2003; Edwards et al., 2005; Anwer and Davis, 2013). More recently, the rhythm of bioluminescence from modified firefly LUCIFERASE (LUC) genes coupled to the clock-controlled promoter was successfully used to map QTLs for circadian parameters (Darrah et al., 2006; Boikoglou et al., 2011). Thus, the luciferase-based system can be employed to accurately measure variation in circadian-rhythm parameters within RIL populations to detect phenotypically expressed genetic variation in circadian clock genes (Anwer and Davis, 2013).

In this study, we characterized a natural allele of ELF3 to determine its effect on rhythmicity in the Arabidopsis circadian clock. Classical QTL mapping in a modified Bay-0 x Shakdara mapping population, followed by positional cloning, revealed an allele of ELF3 (ELF3-Sha) that accelerates circadian oscillations in a light-dependent manner and a dampened oscillator in darkness. We determined that the periodicity phenotype of ELF3-Sha results from a single encoded amino-acid change A362V, which is an allelic variant that is largely confined to a latitudinal stripe in Central Asia. Furthermore, we identified that the circadian abnormalities in ELF3-Sha are associated to cellular localization defects of ELF3 protein that results in an inability to properly function under light and in darkness. Thus, by characterizing ELF3-Sha, we clarified the molecular mode-of-action of ELF3 in the circadian clock.

ELF3 has been established as a required component for the generation of circadian rhythms and the perception of light and temperature inputs (Covington et al., 2001; Thines and Harmon, 2010; Kolmos et al., 2011). However, most of the elf3 alleles used in these studies were identified in mutagenesis screens, and many of them displayed arrhythmicity under free-running conditions of LL and DD. Here, we report the cloning and characterization of ELF3-Sha as a natural allele of ELF3, which displayed a light-dependent short-period phenotype. Unlike elf3 loss-of-function mutants, this allelic state displayed robust circadian rhythms under continuous light. However, in prolonged darkness, the expression profile of many clock genes was dramatically damped (Figure 10). The association between the clock phenotypes of ELF3-Sha and their cellular basis led us to conclude that proper cellular distribution of ELF3 is required to perform normal repressive function in the circadian clock (Figures 12 and 13) which in turn, depends on its cyclic accumulation. Importantly, we could track the origin of ELF3-Sha to Central Asia, providing more insight about the molecular evolution of this allele.

The identification and validation of quantitative trait loci (QTLs) by genetic mapping is a well-established procedure. However, to understand the molecular basis of a locus by characterizing QTL to a quantitative trait nucleotide (QTN) level is still an arduous task (Koornneef et al., 2004). Here, we reported the characterization of a natural allele of ELF3 (ELF3-Sha) to a QTN level of understanding. In the quantitative analysis of circadian periodicity of a modified BxS population, we identified a QTL and further validated it in HIFs and a NIL (Table 1; Figure 2A,B). Our periodicity results were consistent with previous studies where the ELF3 in Sha was found to modulate the shade-avoidance response, including the action of shade on the oscillator (Jimenez-Gomez et al., 2010; Coluccio et al., 2011). In those studies, HIFs were the main genetic resource used. The authors of those papers proposed ELF3 to be a candidate locus for the detected shade-avoidance QTL, and transgenic efforts were used to complement that notion (Jimenez-Gomez et al., 2010). Here, we generated the appropriate NIL and showed that the introgression of ELF3-Sha to Bay-0 resulted in severe circadian alterations, including a curtailed period length (Figure 2B).

The light-dependent acceleration of circadian oscillations of ELF3-Sha could be associated with reduced ELF3 functionality (McWatters et al., 2000; Covington et al., 2001; Kolmos et al., 2011). The intermediate expression profile of several clock genes in ELF3-Sha, when compared to wild-type and null mutants, confirmed that it is a hypomorphic allele (Figures 10 and 11). This characteristic of ELF3-Sha is distinctly different from elf3 null mutants, which displayed complete arrhythmia under LL (Covington et al., 2001; Hicks et al., 2001; Thines and Harmon, 2010; Kolmos et al., 2011). However, the functionality of the oscillator in these mutants in darkness remained disputable. Based on the observation that the circadian rhythms persist in the elf3-1 mutant, it was proposed that ELF3 solely acts as a light zeitnehmer, or timekeeper, and that it is not a required component of the oscillator. In contrast, a recent study reported that the circadian oscillator was abolished in constant darkness (DD) in temperature-entrained elf3-1 seedlings, emphasizing that ELF3 is an integral part of the oscillator as well as acting as a zeitnehmer (Thines and Harmon, 2010). As these studies were mainly based on null mutants, a conclusive statement about ELF3 function in sustaining the oscillator in DD is hard to draw. Consequently, in this paper, we thus analyzed the profile of several clock genes in the functionally stable ELF3-Sha mutant in DD, comparing it with those in elf3-1 and elf3-4 null mutants. Consistent with a previous report (Thines and Harmon, 2010), we could not detect any sign of rhythmicity in null elf3 mutants. Interestingly, in the hypomorphic ELF3-Sha, a rapid dampening in the expression of several clock markers in DD was observed (Figures 3A, 5D, 6A, and right panels of Figure 10G–L). Thus, our results conclusively demonstrate that ELF3 is required for robust oscillation in darkness and further support the idea that ELF3 is an integral part of the oscillator (Thines and Harmon, 2010). Although ELF3-Sha lost robustness and precision in prolonged darkness, it is important to note that it quickly recovered when transferred back to the light (Figures 6A,B, 7A–C). Consequently, we further propose that the function of ELF3 is collectively defined by both the light–dark boundary and by circadian clock regulation.

elf3 loss-of-function mutants under free-running conditions were previously shown to display major defects in the expression of the core oscillator genes PRR7, PRR9, CCA1, LHY, TOC1, and GI (Kikis et al., 2005; Dixon et al., 2011; Kolmos et al., 2011). To monitor the expression of these clock genes in ELF3-Sha, we used the robust luciferase reporter-expression system and measured these expression profiles in Bay-0, NIL-S, and the nulls elf3-1 and elf3-4. Our results were consistent with previous reports showing the arrhythmic low levels of CCA1 and LHY and high levels of TOC1 and GI in elf3 loss-of-function mutants. In contrast, under LL, ELF3-Sha was rhythmic for all genes studied, and it displayed an intermediate level of expression compared to wild type and loss-of-function elf3 (Figure 10A–F, left panels). Specifically, NIL-S displayed higher expression of TOC1 and GI compared to Bay-0, but lower expression than elf3-1 and elf3-4. In contrast, expression of CCA1 and LHY was lower in NIL-S, compared to Bay-0, but higher than elf3-4. Consistent with the previous finding that CCA1 and LHY regulate PRR7 and PRR9 (Farre et al., 2005; Nakamichi et al., 2010), a higher transcript abundance of PRR7 and PRR9 was observed in NIL-S (Figures 10E,F and 11C,D). These data, along with recent findings showing the evening complex (ELF3, ELF4, and LUX) directly binds to the PRR9 promoter to repress transcription, support the repressive action of ELF3 in the core oscillator (Helfer et al., 2011; Nusinow et al., 2011; Chow et al., 2012; Herrero et al., 2012). Higher expression levels of the evening genes TOC1 and GI, as well as dramatically dampened oscillations of ELF3-Sha in darkness, cannot be simply explained by only considering ELF3 at PRR9. Considering the double loss-of-function mutant cca1-11 lhy-21 is rhythmic, an additional repressive role of ELF3 by targeted degradation of GI is conceptually plausible (Ding et al., 2007; Yu et al., 2008). Taken together, our data support a hypothesis which holds that the EC-containing ELF3 has more than one entry point in the circadian clock (Kolmos et al., 2009, 2011; Herrero and Davis, 2012).

ELF3 is both a cytosolic and nuclear localized protein. It appears to be multifunctional in that it has several binding partners. These include phyB, COP1, ELF4, and GI (Yu et al., 2008; Nusinow et al., 2011; Herrero et al., 2012). Different domains of ELF3 specifically interact with these different proteins. Both phyB and COP1 interact with the N-terminal domain (Liu et al., 2001; Yu et al., 2008; Kolmos et al., 2011), whereas ELF4 and GI interact with the middle domain (Yu et al., 2008; Herrero et al., 2012). Further, all these proteins co-localize in the nucleus where they form distinct nuclear bodies (Mas et al., 2000; Yu et al., 2008; Chen et al., 2010; Herrero et al., 2012). Such nuclear foci could be suspected as interaction points of binding proteins (Yu et al., 2008; Herrero and Davis, 2012; Herrero et al., 2012; Kim et al., 2013). Thus, formation of fewer ELF3-nuclear foci from encoded ELF3-Sha might be the result of a defect in the binding of one of its interacting proteins. Since the A362V variant is located in the middle domain of ELF3, such a hypothesis could be particularly attributed to either GI and/or ELF4. The rhythmic accumulation of both ELF3 and GI depends upon the activity of COP1 that mediates ubiquitination and targeted degradation of these proteins in dark conditions (Yu et al., 2008). It is noteworthy here that ELF3 is essential for this process. In the absence of ELF3, COP1 cannot interact with GI and thus cannot initiate its decay. However, GI is not required for COP1-mediated ELF3 degradation (Yu et al., 2008). When these data are taken together, the formation of a COP1-ELF3-GI complex could be considered a plausible active mechanism controlling the rhythmic accumulation of these proteins. Under these conditions, A362V mutation in ELF3-Sha would result in attenuated binding affinity with GI, disturbing the balance of the COP1-ELF3-GI complex and, in turn, resulting in the rapid decay of ELF3. Both the lower accumulation of ELF3-Sha protein, despite the generation of higher transcript (Figure 13E,F), and aberrant oscillator behavior of ELF3-Sha in prolonged darkness support this hypothesis. The short-period phenotype of ELF3-Sha under LL could also be explained by an early decay of ELF3-Sha during light/dark entrainment. As ELF3 directly represses PRR9 (Dixon et al., 2011; Herrero et al., 2012), the early depletion of ELF3-Sha results in an early expression of PRR9, an event that sets the pace of the oscillator during the preceding entrainment, which is maintained through LL. Based on these results and our observation that ELF3-Sha is defective in proper clock resetting (Figure 6D), we propose that ELF3 plays a pivotal role in defining entrainment properties of the circadian clock, which, in turn, depend on cyclic accumulation of ELF3 protein, but not on its absolute levels or, indirectly, its transcript abundance. This idea could be further supported by the observation that ELF3 protein rhythmically oscillates and drives a long period in plants that constitutively overexpress ELF3 (Covington et al., 2001; Dixon et al., 2011; Herrero et al., 2012). Discounting the important role that ELF3 plays in clock entrainment, it can be provisionally excluded that ELF3 is involved in parametric entrainment because we could not detect any differential effect of fluence rate on ELF3-Sha periodicity compared to ELF3-Bay (Figure 9A–C). Fluence rate curves based on ELF3-ox also support this notion (Covington et al., 2001).

Studies on ELF3-Sha as a natural allele disrupted in normal circadian behavior provide a perspective on its repressive action on clock periodicity. Notably, in the context of the Ws-2 and Bay-0 genomes, we could show that ELF3-Sha is a hypomorphic allele defective in proper localization of encoded ELF3 protein. This defect resulted in two distinct phenotypes: one displaying a light-dependent short period and the other exhibiting loss of rhythm robustness in darkness. These phenotypes were clearly observable in both the Bay-0 and Ws-2 genetic background. It is notable that Sha parental line did not itself display such obvious circadian defects (Figures 2B and 3A). Related to this, the transcript profile of clock genes in Sha followed the same pattern as observed in Bay-0, and such pattern was distinct from that seen in NIL-S (Figure 11). Moreover, ELF3-Sha did not affect hypocotyl length in NIL-S and Sha in the same way. Under all light qualities tested, the hypocotyl length of NIL-S was similar to that of Bay-0, but was significantly longer when compared to Sha (Figure 4B), leading to the possibility that other segregating QTLs within Sha wild type genetically interact with ELF3-Sha. Such similar background-dependent effects of natural alleles have been reported in Arabidopsis for seed longevity, axillary bud formation, and flowering time (Sugliani et al., 2009; Huang et al., 2012; Undurraga et al., 2012; Méndez-Vigo et al., 2013).

Our efforts to track the history of the ELF3-Sha revealed that this allele is present in a group of genetically related accessions that are predominately distributed in a geographical area of Central Asia (Figure 14A,B). This confined location of ELF3-Sha leads to the possibility that this allele provides local adaptive advantage to these accessions under their respective environmental conditions, and thus, has been positively selected during species migration. Such a hypothesis can only be confirmed in further studies using realistic environmental conditions in which these accessions are derived.

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Author details

1. Muhammad Usman Anwer

   1. Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany
   2. Department of Biology, University of York, York, United Kingdom

   Contribution

   MUA, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

2. Eleni Boikoglou

   1. Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany
   2. Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary

   Present address

   Cold Spring Harbour Laboratory, Laurel Hollow, Germany

   Contribution

   EB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

3. Eva Herrero

   Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany

   Present address

   MRC National Institute for Medical Research, London, United Kingdom

   Contribution

   EH, Conception and design, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

4. Marc Hallstein

   Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany

   Contribution

   MH, Acquisition of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

5. Amanda Melaragno Davis

   1. Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany
   2. Department of Biology, University of York, York, United Kingdom

   Contribution

   AMD, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Geo Velikkakam James

   Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany

   Contribution

   GVJ, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

7. Ferenc Nagy

   Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary

   Contribution

   FN, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Seth Jon Davis

   1. Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany
   2. Department of Biology, University of York, York, United Kingdom

   Contribution

   SJD, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   seth.davis@york.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

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