The plant circadian clock is proposed to be a network of several interconnected feedback loops, and loss of any component leads to changes in oscillator speed. We previously reported that Arabidopsis thaliana EARLY FLOWERING4 (ELF4) is required to sustain this oscillator and that the elf4 mutant is arrhythmic. This phenotype is shared with both elf3 and lux. Here, we show that overexpression of either ELF3 or LUX ARRHYTHMO (LUX) complements the elf4 mutant phenotype. Furthermore, ELF4 causes ELF3 to form foci in the nucleus. We used expression data to direct a mathematical position of ELF3 in the clock network. This revealed direct effects on the morning clock gene PRR9, and we determined association of ELF3 to a conserved region of the PRR9 promoter. A cis-element in this region was suggestive of ELF3 recruitment by the transcription factor LUX, consistent with both ELF3 and LUX acting genetically downstream of ELF4. Taken together, using integrated approaches, we identified ELF4/ELF3 together with LUX to be pivotal for sustenance of plant circadian rhythms.

The circadian clock is a timekeeping mechanism that enables anticipation of daily environmental changes. In the plant Arabidopsis thaliana, the circadian system is a multiloop series of interlocked transcription-translation feedbacks. Several genes have been arranged in these oscillation loops, but the position of the core-clock gene ELF4 in this network was previously undetermined. ELF4 lacks sequence similarity to known domains, and functional homologs have not yet been identified. Here we show that ELF4 is functionally conserved within a subclade of related sequences, and forms an alpha-helical homodimer with a likely electrostatic interface that could be structurally modeled. We support this hypothesis by expression analysis of new elf4 hypomorphic alleles. These weak mutants were found to have expression level phenotypes of both morning and evening clock genes, implicating multiple entry points of ELF4 within the multiloop network. This could be mathematically modeled. Furthermore, morning-expression defects were particular to some elf4 alleles, suggesting predominant ELF4 action just preceding dawn. We provide a new hypothesis about ELF4 in the oscillator-it acts as a homodimer to integrate two arms of the circadian 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.