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.

Arabidopsis thaliana EARLY FLOWERING3 (ELF3) is essential for the generation of circadian rhythms. ELF3 has been proposed to restrict light signals to the oscillator through phytochrome photoreceptors, but that has not been explicitly shown. Furthermore, the genetic action of ELF3 within the clock had remained elusive. Here, we report a functional characterization of ELF3 through the analysis of the elf3-12 allele, which encodes an amino acid replacement in a conserved domain. Circadian oscillations persisted, and unlike elf3 null alleles, elf3-12 resulted in a short circadian period only under ambient light. The period shortening effect of elf3-12 was enhanced by the overexpression of phytochromes phyA and phyB. We found that elf3-12 was only modestly perturbed in resetting of the oscillator and in gating light-regulated gene expression. Furthermore, elf3-12 essentially displayed wild-type development. We identified targets of ELF3 transcriptional repression in the oscillator, highlighting the action at the morning gene PSEUDO-RESPONSE REGULATOR9. Taken together, we identified two separable roles for ELF3, one affecting the circadian network and the other affecting light input to the oscillator. This is consistent with a dual function of ELF3 as both an integrator of phytochrome signals and a repressor component of the core oscillator.

The circadian clock perceives environmental signals to reset to local time, but the underlying molecular mechanisms are not well understood. Here we present data revealing that a member of the heat shock factor (Hsf) family is involved in the input pathway to the plant circadian clock. Using the yeast one-hybrid approach, we isolated several Hsfs, including HEAT SHOCK FACTOR B2b (HsfB2b), a transcriptional repressor that binds the promoter of PSEUDO RESPONSE REGULATOR 7 (PRR7) at a conserved binding site. The constitutive expression of HsfB2b leads to severely reduced levels of the PRR7 transcript and late flowering and elongated hypocotyls. HsfB2b function is important during heat and salt stress because HsfB2b overexpression sustains circadian rhythms, and the hsfB2b mutant has a short circadian period under these conditions. HsfB2b is also involved in the regulation of hypocotyl growth under warm, short days. Our findings highlight the role of the circadian clock as an integrator of ambient abiotic stress signals important for the growth and fitness of plants.circadian clock | Hsf | heat compensation | salt tolerance T he circadian clock is an endogenous timing mechanism that ensures that daily rhythmic processes are synchronized with the environment. The circadian oscillator runs with a period of ∼24 h. It entrains to environmental signals, such as light and temperature, and in this way is able to anticipate daily environmental changes (1). Molecular genetics and modeling have revealed that the circadian system comprises several interconnected feedback loops involving multiple phase-specific gene products (2).In the model plant Arabidopsis, the morning feedback loops are composed of the major oscillator proteins CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), which peak in abundance at dawn and activate the expression of early-late morning genes PSEUDO RESPONSE REGULATOR 9 (PRR9) and PRR7 (3). Pseudoresponse regulator (PRR) proteins are transcriptional repressors, and PRR9 and PRR7 close the morning loops by binding to the CCA1 and LHY promoters (4). The transcription of PRR9 is acutely activated by light, a feature that is not shared by PRR7 (3, 5). Disruption of either locus results in a mild clock phenotype, including an increased clock period of ∼1 h and an elongated hypocotyl (6-8). The double loss-of-function mutant exhibits a strong synergistic phenotype with free-running period lengths of up to 35 h and loss of temperature entrainment and compensation, indicating overlapping roles for PRR9 and PRR7 (3, 9, 10).The ambient environment influences the circadian clock in a number of different ways. Although the most well-studied input cue is light, temperature is a major zeitgeber as well (11). Brief pulses (from minutes to hours, depending on the intensity) result in acute responses from the clock. Importantly, the severity of such responses is time-dependent and gated by the oscillator (12-14). Longer durations of zeitgeber change can function as an entrainment signal and general...

Many plants in temperate regions have a requirement for vernalization in order to initiate the reproductive growth phase. In cereals, this requirement has been linked to the VRN1 locus, which encodes an APETALA1-like (AP1-like) MADS-box gene. In perennial ryegrass (Lolium perenne L.), we have isolated two MADS-box genes that are regulated by vernalization, LpMADS1, which co-localize to the VRN1 locus in ryegrass, and LpMADS10, which is an SVP-like MADS-box gene. In the shoot apex, LpMADS1 is increasingly induced by cold exposure, whereas LpMADS10 is increasingly repressed. Comparison of LpMADS1 promoter regions from several ryegrass varieties, with and without vernalization requirement, suggests that a putative MADS-box protein-binding site (CArG-box) might be important for the vernalization-regulated expression of LpMADS1. Although the LpMADS10 expression pattern suggests it to be involved in floral repression, ectopic expression of LpMADS10 did neither affect flowering time significantly in Arabidopsis thaliana nor in L. perenne. Interestingly, we found that LpMADS1 interacts with LpMADS10 in a yeast two-hybrid assay. This finding is discussed in regard to the regulation of vernalization response in perennial ryegrass.

The light-dark cycle of the environment serves as one of the major Zeitgebers in entrainment of the circadian clock. The circadian system consists of interconnected feedback loops in which the CCA1/LHY-TOC1 loop has a central position. Genetic analyses of the elf4 mutant suggested that it is a positive regulator of CCA1 and LHY expression. Recently, we refined the mode-of-action of ELF4 in entrainment of the clock, and here hypothesize that ELF4 expression is interlocked with the CCA1/LHY-TOC1 loop.

BackgroundThe plant circadian clock has at its core a feedback loop that includes TIMING OF CAB2 EXPRESSION 1 (TOC1). This protein has an as of yet unknown biochemical activity. It has been noted that the extreme amino-terminus of this protein is distantly related in sequence to response regulators (RR), and thus TOC1 is a member of the so-called pseudo response regulator (PRR) family. As well, the extreme carboxy-terminus has a small sequence stretch related to the other PRRs and CONSTANS (CO)-like proteins, and this peptide stretch has been termed the CCT (for CONSTANS, CONSTANS-LIKE, TOC1) domain.MethodsTo extend further our understanding of the TOC1 protein, we performed a ROSETTA structural prediction on TOC1 orthologues from four plant species. Phylogenetic interpretations assisted in model construction.ResultsFrom our models, we suggest that TOC1 is a three-domain protein: TOC1 has an amino-terminal signaling-domain related to response receivers, a carboxy-terminal domain that could participate both in metal binding and in transcriptional regulation, and a linker domain that connects the two.ConclusionThe models we present should prove useful in future hypothesis-driven biochemical analyses to test the predictions that TOC1 is a multi-domain signaling component of the plant circadian clock.