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First published online March 23, 2007; 10.1104/pp.107.096206 Plant Physiology 144:391-401 (2007) © 2007 American Society of Plant Biologists
ELF4 Is Required for Oscillatory Properties of the Circadian Clock1,[W]Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (H.G.M.); Max Planck Institute for Plant Breeding Research, D–50829 Cologne, Germany (E.K., S.J.D.); School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom (A.H.); Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 (M.R.D., R.M.A.); Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, H–6726 Szeged, Hungary (P.G., F.N.); and Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JH, United Kingdom (A.J.M.)
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.
Many organisms have evolved circadian clocks to facilitate optimal timing of rhythmic behaviors. Plants use an endogenous oscillator and predictable signals from the environment to anticipate changes in circadian time. Key outputs controlled by the clock include the timing of germination, optimization of photosynthetic processes relative to the time of day, and floral transition. Each of these has been shown to be crucial for plant fitness (Green et al., 2002  ; Dodd et al., 2005). In recent years, several molecular components associated with the plant clock have been identified. Most of these components are themselves circadian regulated, with peak expression of each phased to occur at a specific time of day. For example, the MYB-related transcription factors, CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and LONG ELONGATED HYPOCOTYL (LHY; Schaffer et al., 1998; Wang and Tobin, 1998), are morning-specific genes, both acting in a feedback loop on the pseudoresponse regulator TIMING OF CAB EXPRESSION1 (TOC1), which peaks in the evening (Strayer et al., 2000; Alabadi et al., 2001; Mas et al., 2003). This transcription-translation feedback loop has been placed at the core of the Arabidopsis (Arabidopsis thaliana) clock (Alabadi et al., 2001). The original single-loop model was recently extended to incorporate additional loops (Farre et al., 2005; Locke et al., 2005, 2006; Salome and McClung, 2005a; Zeilinger et al., 2006). Beyond this core, the wider plant circadian system constitutes a complex network of multiple and interconnected pathways, many of which feed back on each other, controlling responses to light, temperature, and day length. These features are poorly understood.
Previously, we identified early flowering4 (elf4) from Arabidopsis and showed that ELF4 is important for circadian precision and normal clock function (Doyle et al., 2002
Light signals perceived by photoreceptors, including phytochromes and cryptochromes (Lin, 2002
We have shown previously that ELF4 is expressed in the evening and that the elf4 loss-of-function mutant has low CCA1 expression leading to arrest of the elf4 oscillator after one cycle under free run (Doyle et al., 2002
Hypo- and Hypermorphic Red-Light Signaling in elf4 Plants
Under natural 24-h days, the LD rhythm defines the diurnal environment. However, signaling through light input pathways in plants is itself a clock-controlled process, being gated by so-called zeitnehmer functions, one of which requires ELF3 (McWatters et al., 2000
A gating assay was conducted to test whether the red-light defects in elf4-1 were in part due to alterations in circadian processing of light information. For this, wild-type and elf4-1 plants harboring the CAB2:luciferase (LUC) marker were entrained to 12/12 LD cycles and replicate samples were placed into continuous darkness (DD). From subjective dark (zeitgeber time [ZT] = 12, noted here as at the start of the Fig. 1B graph; transfer time = 0) at 2-h intervals, a set of replicate samples was given a 5-min pulse of red light and the acute response of light activation of CAB2:LUC induction was assayed. As reported previously for white-light pulses (McWatters et al., 2000 ), we could confirm that wild-type Arabidopsis has a gated response of CAB2 induction (Fig. 1B). The response to light was at a maximum during the early part of the subjective day, around the time when the plants anticipated the transition of dark to light (subjective dawn; time 12 h in Fig. 1B), indicating the gate is open during the (subjective) day but closed during the (subjective) night. The elf4-1 mutant showed defects in its response to red-light treatments over the course of the entire experiment, but most especially during subjective night (times 0–12 h and 24–36 h in Fig. 1B). In elf4-1 mutants, the gate was open during subjective night (Fig. 1B) when elf4-1 displayed high activation of CAB2 in response to the light pulse. These plants have increased sensitivity to light at night relative to wild type and thus elf4 is a partial gating mutant. Red-light perception in the elf4-1 mutant is thus altered, at least in part, because of an underlying clock defect that affects the gating of this red-light response pathway.
It was noted that, after transfer to constant conditions following exposure to LD cycles, elf4-1 mutant plants displayed weak rhythmicity on the first day (Doyle et al., 2002 Until 32 h after the last dawn (i.e. subjective dusk for these plants previously entrained to 8/16 LD), the timing of the peak in elf4 seedlings was indistinguishable from that of wild-type plants (Fig. 2A ). However, the two sets of seedlings responded differently to pulses given at or 36 h after the last dawn (t test; P < 0.05): Wild-type seedlings continued to show circadian control, but the peak of CAB2:LUC in elf4-1 occurred about 30 h after the pulse, regardless of when the pulse was given (Fig. 2A). Thus, the circadian clock in elf4 runs down at the end of the first subjective day in DD to a point where it is strongly reset by even a brief light pulse. We interpret this as although rhythmicity can be driven by a light zeitgeber in elf4, ELF4 is needed to sustain clock activity beyond the end of the first subjective day in DD.
Characterization of ELF4-ox Plants
We previously concluded based on loss-of-function studies that ELF4 is both a repressor of the floral transition and required to sustain normal clock function (Doyle et al., 2002
Because elf4-1 is a severe clock mutant under light or in darkness, it was reasoned that ELF4-ox lines should also show circadian alterations. Three independent transgenic lines were tested for alterations in circadian leaf movement rhythms. All lines showed an increased free-running period under LL (Table I ; Supplemental Fig. S1). These results were confirmed for molecular rhythms of ELF4-ox plants harboring the morning CAB2:LUC and the evening CCR2:LUC reporters (Table I; Fig. 3, C–F). These lines also had rhythms with longer periods under LL after entrainment to LD cycles (Fig. 3, C and D). In darkness, ELF4-ox peaked later than wild type most significantly for the evening marker CCR2:LUC (Fig. 3, E and F). Thus, ELF4 modulates rhythmicity of multiple clock outputs. Here, we define based on these misexpression studies that ELF4 is a strong genetic repressor of clock periodicity.
Entrainment to LD Cycles Is Altered in elf4-1 Mutants
The gating assay (Fig. 1B) showed us that elf4-1 plants display greater sensitivity to light than wild type. CCA1 and CAB2 are both under clock control and normally rise during the late night with peak at or shortly after dawn, respectively. They are also regulated directly by light. CCR2 expression is also clock controlled, but is less directly affected by light (Suarez-Lopez et al., 2001 In elf4-1 under long or short days, there was a strong reduction in the rising of gene expression during darkness and, instead, there was an abrupt increase in CCA1:LUC and CAB2:LUC expression immediately following lights on (Fig. 4 ), again implying an increase in light sensitivity in these plants relative to wild type. This suggests that the ability of the elf4-1 mutant to anticipate dawn was attenuated, extending the possibility that entrainment of the oscillator is altered in elf4-1. In contrast, ELF4-ox correctly anticipated the coming lights on before photic signals were present (Fig. 4). We interpret this as a strong suggestion that, whereas ELF4 is essential for normal entrainment to light, rhythmic accumulation of ELF4 transcript is not.
The transcription of CCR2 cycles, with a trough in the day and a peak in the night, is similar to the phase angle of ELF4 (Fig. 6A). Under short days, only a marginal rhythm is seen for CCR2:LUC in elf4-1; however, a weak rhythm that apparently is able to anticipate dusk is seen in long photoperiod conditions (Fig. 4, E and F), suggesting that the slave oscillator of CCR2 (Heintzen et al., 1997 ) still runs under these conditions even in the elf4-1 mutant. Again, the same phase of the CCR2 peak was seen in ELF4-ox plants compared to wild type (Fig. 4, E and F), reinforcing our earlier proposition that, whereas ELF4 is necessary for correct entrainment of plants, rhythmic ELF4 expression is not.
To further refine our understanding of clock resetting and ELF4's role in this entrainment process, we measured the time taken by wild type, elf4-1, and ELF4-ox seedlings harboring CCR2:LUC to reentrain to a 12/12 LD cycle following the inversion of day and night (equivalent to jumping across 12 time zones instantaneously). The rapid change in light regime induces jet lag because the circadian clock is no longer in its correct orientation with respect to the environmental cycle. This protocol is similar to that used to define entrainment defects in cca1 and lhy mutants (Kim et al., 2003 ; Fig. 2). Under this regime, the timing of peak CCR2:LUC activity, relative to the lights-out signal, was nearly restored in elf4-1 within the first day (Fig. 2B). In contrast, the wild-type line did not display near-normal timing of the peak in CCR2 expression until the second day. Thus, elf4 resets faster than the wild type. To understand the preliminary events that led to rapid clock resetting in elf4-1 relative to wild type, we repeated the assay with the three genotypes expressing LUC under the control of CCA1 or LHY promoters, respectively. This showed that the morning peak of CCA1 and LHY in wild type occurred when plants expected dawn (e.g. after time 48 for CCA1 [Fig. 2C] or time 72 for LHY [Fig. 2D]), although this point was now in darkness because of the extended night. Wild-type plants exhibited little response to the lights on that occurred at subjective dusk (listed as time 60 for CCA1 [Fig. 2C] and time 84 for LHY [Fig. 2D]), relative to the original entraining cycle (Fig. 2, C and D). These results can be explained by gated repression of light activation of these genes during the subjective night, similar to that shown for CAB2 in wild-type seedlings (Fig. 1B). In contrast, the peak of LUC activity in elf4-1 was much reduced after time 48, but the relative increase in gene induction in response to lights on at time 60 was much greater. This is consistent with the defective gating found in this mutant in which the gate for light responsiveness is open during subjective night. The light induction of CCA1 and LHY in elf4-1 is the likely cause of its rapid clock resetting. ELF4-ox plants also exhibited accelerated clock resetting of CCR2:LUC relative to wild type. However, expression of CCA1:LUC and LHY:LUC in ELF4-ox matched that of wild type between time 36 and 72 for CCA1 (Fig. 2C) and time 48 and 96 for LHY (Fig. 2D), implying that the resetting behavior here is not likely to be due to changes in the gating of light responsiveness. Instead, we suggest it may be due to the longer endogenous period allowing easier resetting via a single-phase delay.
Temperature cycles can rescue rhythmicity of the elf3 mutant, which acts to gate light input, in a subsequent interval of constant temperature (McWatters et al., 2000
Timing of ELF4 Action
ELF4 is required for robust rhythmicity and for a normal response to LD cycles. To aid the understanding of ELF4's role in the circadian signaling network, molecular expression phenotypes of core clock genes were measured in various ELF4 genotypic backgrounds. Luminescence rhythms were measured in wild-type plants expressing ELF4:LUC under LL after entrainment under LD cycles. Compared to the evening marker CCR2, ELF4:LUC generated a rhythm with peak expression in the middle of the night (Fig. 6A
). We compare this to our analysis on the ELF4 transcript under 12/12 LD photoperiods. There, we found peak expression at dusk (ZT12; Fig. 6G); we have previously shown that ELF4 transcript levels are clock controlled and peak in the evening and that ELF4 expression is affected by photoperiod (Doyle et al., 2002 As expected, ELF4:LUC activity in elf4 was arrhythmic (Fig. 6B), as was that of CCR2:LUC expression, in agreement with our previous reports. Rhythmicity in the elf4-1 mutant could be rescued by restoring ELF4 expression with the ELF4:ELF4-LUC construct (Fig. 6B). Like plants constitutively overexpressing ELF4, these plants had a long-period phenotype. Thus, ELF4 regulation appears to be primarily transcriptional and ELF4 activity is potentially dose dependent even under the control of its own promoter.
The elf4 mutant phenotype includes low transcription of the morning clock gene CCA1 (we confirmed our previous LUC data regarding CCA1 expression [Doyle et al., 2002
The current model of the CCA1/LHY-TOC1 loop (Alabadi et al., 2001
Our data illustrate that elf4-1 plants have a range of deficiencies in their circadian responses to light, photoperiod, and temperature. Importantly, such plants do not display sustained rhythmicity in the absence of environmental signals. Misexpression studies of ELF4 further confirm an important clock function for this gene. Analysis of gene expression of key components of the plant clock (CCA1, LHY, and TOC1) and targeted assays to define the abrogated rhythm in elf4-1 revealed that the central circadian feedback loop in elf4 was locked into the evening phase. However, constitutive overexpression of ELF4 does not produce arrhythmia, but acts to delay the clock, causing a long-period phenotype seen across a range of assays. Plants overexpressing ELF4 exhibited robust rhythms of clock gene expression and these lines were able to respond to photoperiods, for example, flowering earlier in long days than short days. These results showed that ELF4 is essential for free-running circadian rhythms. Here, we have presented evidence that expression of the various clock outputs is strongly affected by the LD zeitgeber in the elf4-1 mutant (Figs. 2 and 4). elf4-1 plants were found to show more rapid reentrainment following a change in the zeitgeber phase (Fig. 2, B–D), which indicates that the clock is reset more rapidly in these mutants than in the wild type. This is probably due to ELF4's role of gating light input to the clock. The gate in elf4-1 plants never fully closes (Fig. 1B); hence, these plants are more sensitive to photic cues due to increased activity of the light-signaling pathway. Increased light sensitivity is also seen in the pattern of CAB2:LUC and CCA1:LUC expression in elf4 mutants (Fig. 4). In the absence of a zeitgeber, elf4-1 does not show robust free-running rhythms in any of the various hands of the clock (Figs. 5 and 6). Regardless of how the clock is assayed, the elf4-1 mutant is weakly rhythmic or arrhythmic under LL and DD, regardless of the previous entrainment protocol. We thus conclude that ELF4 is required for entrainment.
Most importantly, the putative clock components CCA1, LHY, and TOC1 are virtually arrhythmic after the first 24 h in LL in an elf4-1 background, implying that this feedback loop cannot continue to cycle in the absence of ELF4. We have shown that CCA1 and LHY levels are both low in the elf4-1 mutant, whereas TOC1 is high—strong circumstantial evidence that ELF4 acts to promote the former while repressing the latter (Fig. 6; Supplemental Fig. S2). This evidence leads to the conclusion that ELF4 is essential for correct clock function in Arabidopsis and that, in the absence of ELF4, the clock will stop after a single cycle. ELF4 transcription is rhythmic, with a peak during the early night, coinciding with the point at which the clock arrests in elf4-1, implying that ELF4 acts at this point of the 24-h cycle (Fig. 2A). A recent study by Quail and colleagues reported that TOC1 expression was unchanged in another elf4 mutant allele (elf4-101, a T-DNA insertion in the Columbia background; Kikis et al., 2005
The near loss of circadian function in elf4 differs from all previously described recessive circadian mutants of Arabidopsis because elf4 mutants become quickly arrhythmic when transferred to all types of constant conditions. Other mutants, for example, elf3 and lux, are capable of maintaining rhythmicity in certain unchanging constant conditions, such as DD (Hicks et al., 1996
We have shown that, although the CCA1/LHY-TOC1 feedback loop is stalled in the evening phase in elf4-1, the clock has full oscillatory function in ELF4-ox, which shows robust rhythmicity of gene expression (Table I; Figs. 5 and 6), and this line is able to distinguish between long and short days for the purpose of controlling flowering time. However, the long-period phenotype and later flowering under long days of ELF4-ox plants highlights the notion that the level of ELF4 expression calibrates circadian period. We have previously observed that ELF4 levels are extremely low and lose rhythmicity in wild-type plants grown in extended darkness (Doyle et al., 2002
A previous report on an elf4 mutant allele that demonstrated arrhythmicity of the CCA1/LHY-TOC1 feedback loop in dark-grown seedlings indicated that ELF4 was required for light activation of this loop (Kikis et al., 2005
We suggest that ELF4 functions to convert an hourglass into a clock. Without ELF4, the CCA1/LHY-TOC1 feedback loop can be turned over by an environmental cycle of light and dark, but stops depending on the discontinuation of the environmental rhythm. The closest functional analog to ELF4 may be the FREQUENCY (FRQ) locus of Neurospora crassa. In the absence of FRQ, N. crassa rhythms are of low amplitude, variable length, and not temperature compensated (Merrow et al., 1999
Plant Material and Transgenics
Arabidopsis (Arabidopsis thaliana) ecotype Wassilewskija (Ws-2), elf4-1, and the LUC lines CAB2:LUC+ (6B insertion), CCA1:LUC, and CCR2:LUC are described (McWatters et al., 2000 ELF4:LUC was constructed by subcloning approximately a 1.5-kb fragment from the bacterial artificial chromosome clone T28M21 as a XbaI-NcoI fragment into pZIP221B. This fragment is between 1.7 kb and 260 bp upstream of the translational start site. The LUC+ gene from pLUC+ (Promega) was subcloned into this resultant vector as a NcoI-XbaI fragment (pELF4-incomplete:LUC+). A fragment was PCR amplified using an arbitrary upstream primer and the primer 5'-AACCATGGTCTCGCCGTTCCTCTTCATAA-3'. This PCR product was digested with NcoI and the fragment intersected into the NcoI site of pELF4-incomplete:LUC+, resulting in the completed transcriptional fusion pELF4P:LUC+. Similarly, an upstream primer and 5'-AAACCATGGCTCTAGTTCCGGCAGCACCAC-3' was used to generate a PCR that was subcloned as an NcoI fragment into pELF4-incomplete:LUC to generate the translational fusion construct pELF4P:ELF4-LUC+.
To generate a vector for TOC1:LUC, PCR against Ws genomic DNA using 5'-TCGCTCTAGACTTCTCTGAGGAATTTCATCAAAC-3' and 5'-ACTAGGATCCGATCAGATTAACAACTAAACCCACA-3' generated a 2,068-bp fragment that was subcloned into a LUC vector as a XbaI-BamHI insert and, for LHY:LUC, a similar PCR with 5'-TGCGGTCGACTGTTTCAAATAACTGTTATGTCCTA-3' and 5'-GGAAGGATCCAACAGGACCGGTGCAGCAT-3' generated a 1,812-bp fragment that was subcloned as a SalI-BamHI insert. These constructs were used to transform wild-type Ws or elf4-1, as described in the text, by the floral-dip method (Clough and Bent, 1998
For hypocotyl length measurements, seeds were surface sterilized and plated on 2.2 g/L Murashige and Skoog medium without Suc or vitamins (0.5x Murashige and Skoog) with 2.5 mM 2-(N-mopholino)ethanesulfonic acid (pH 5.7) and 8 g/L agar. Plates were stored in the dark for 3 to 4 d at 4°C, placed at 22°C in the darkness for 1 d, and irradiated with light for 6 d, as described (Davis et al., 2001
Luminescence levels were quantified on either a low-light imaging system or an adapted microtiter plate-reading scintillation counter and analyzed essentially as described (McWatters et al., 2000
Replicated samples of elf4-1, toc1-1, and the wild-type controls Ws-2 and C24 seedlings were collected and immediately frozen in liquid nitrogen, starting at dawn on day 8. toc1-1 and C24 seedlings were collected during a 12/12 LD cycle; elf4-1 and Ws-2 seedlings under LL following the discontinuation of such a cycle. RNA was extracted (Qiagen RNeasy kit) using an additional DNAse treatment step (Qiagen) as per the manufacturer's instructions. cDNA was synthesized (ABI TaqMan) and real-time PCR carried out in triplicate in an ABI Prism 3700 using SYBR Green master mix (ABI) and gene-specific primers (ELF4-F, 5'-CGACAATCACCAATCGAGAATG-3', ELF4-R, 5'-AATGTTTCCGTTGAGTTCTTGAATC-3'; TOC1-F, 5'-ATCTTCGCAGAATCCCTGTGATA-3', TOC1-R, 5'-GCACCTAGCTTCAAGCACTTTACA-3'; CCA1-F, 5'-TCTGTGTCTGACGAGGGTCGAATT-3', CCA1-R, ACTTTGCGGCAATACCTCTCTGG-3'; LHY-F, 5'-CAACAGCAACAACAATGCAACTAC-3', LHY-R, 5'-AGAGAGCCTGAAACGCTATACGA-3';
The following materials are available in the online version of this article.
We are particularly grateful to Dr. Arp Schnittger, Dr. Csaba Koncz, and Dr. Réka Tóth (Max-Planck-Institut für Züchtungsforschung) for critical reading and comments. Received January 19, 2007; accepted March 16, 2007; published March 23, 2007.
1 This work was supported by the Max Planck Society and the Life Sciences Research Foundation (S.J.D. group) and by the Biotechnology and Biological Sciences Research Council (A.J.M. group; award no. G10325) and the Human Frontier Science Program Organization (award no. RG0299/1999–M). E.K. was supported by a Max-Planck-Gesellschaft fellowship within the International Max Planck Research School program. Work in R.M.A.'s laboratory was supported by the College of Agricultural and Life Sciences and the Graduate School of the University of Wisconsin and by the National Science Foundation (grant no. 0209786). H.M.W. is a Royal Society University Research Fellow. 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Seth J. Davis (davis{at}mpiz-koeln.mpg.de).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.096206 * Corresponding author; e-mail davis{at}mpiz-koeln.mpg.de; fax 49–(0)221–5062267.
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ABSTRACT
RESULTS
-TUBULIN4 at each time point. Mean values are plotted; error bars represent SDs. The experiment was replicated twice.
