Ambient temperature response establishes ELF3 as a required component of the core Arabidopsis circadian clock Bryan Thines and Frank G. Harmon1 Plant Gene Expression Center, US Department of Agriculture–Agricultural Research Service, Albany, CA 94710; and Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 Edited by Steve A. Kay, University of California, San Diego, La Jolla, CA, and approved December 30, 2009 (received for review September 28, 2009) Circadian clocks synchronize internal processes with environmental GI activates TOC1 expression, whereas GI itself is negatively cycles to ensure optimal timing of biological events on daily and regulated by CCA1/LHY and TOC1 (7, 8). seasonal time scales. External light and temperature cues set the Light and temperature environmental cues, or zeitgebers, set core molecular oscillator to local conditions. In Arabidopsis, EARLY clock feedback loops to the correct time of day (9). Among factors FLOWERING 3 (ELF3) is thought to act as an evening-specific believed to be important for entrainment is EARLY FLOWER- repressor of light signals to the clock, thus serving a zeitnehmer ING 3 (ELF3) (10), which encodes a highly conserved plant- function. Circadian rhythms were examined in completely dark- specific nuclear protein (11). In the Arabidopsis elf3 mutant, nor- grown, or etiolated, null elf3-1 seedlings, with the clock entrained mally rhythmic CCR2 and CAB2 expression is arrhythmic under by thermocycles, to evaluate whether the elf3 mutant phenotype continuous light (LL) (12), although rhythms have been reported was light-dependent. Circadian rhythms were absent from etiolated in continuous darkness (DD) (12, 13). ELF3 protein normally elf3-1 seedlings after exposure to temperature cycles, and this accumulates in the evening (11), and, in the context of the clock, mutant failed to exhibit classic indicators of entrainment by temper- ELF3 is thought to allow progression through this light-sensitive ature cues, consistent with global clock dysfunction or strong per- phase (14). This hypothesis is bolstered by the observations that turbation of temperature signaling in this background. Warm the elf3 clock seems to arrest following approximately 11 h in LL temperature pulses failed to elicit acute induction of temperature- (14) and that light resetting of the clock is reduced when ELF3 is PLANT BIOLOGY responsive genes in elf3-1. In fact, warm temperature-responsive present in excess (13). Recently, ELF3 has been described as a genes remained in a constitutively “ON” state because of clock dys- substrate adaptor that promotes interaction between the E3 function and, therefore, were insensitive to temperature signals in ubiquitin ligase COP1 and GI to influence Arabidopsis flowering the normal time of day-specific manner. These results show ELF3 is time (15). broadly required for circadian clock function regardless of light con- Genetic components governing clock entrainment by temper- ditions, where ELF3 activity is needed by the core oscillator to allow ature remain largely undefined in Arabidopsis. Exceptions are progression from day to night during either light or temperature PRR7 and PRR9, which are partially redundant and required for entrainment. Furthermore, robust circadian rhythms appear to be a clock temperature entrainment (16). Because both PRR7 and prerequisite for etiolated seedlings to respond correctly to PRR9 also participate in clock responses to light (17), temperature temperature signals. entrainment and light entrainment appear to share components. However, the relative contributions of light and temperature to temperature signaling | temperature entrainment | luciferase | circadian entrainment of the Arabidopsis clock are not well understood, rhythms | transcription except for the possibility of two oscillators capable of distin- guishing light and temperature cues (18). he rotation of Planet Earth creates predictable daily environ- Our investigation of the circadian clock in photocycle-entrained Tmental fluctuations of light and dark along with concomitant elf3-1 revealed arrhythmic TOC1 expression in this null mutant oscillations in temperature. The circadian clock is an endogenous over a range of free-running conditions, including DD, a condition timekeeper that anticipates these predictable changes in the where this mutant was previously found to exhibit rhythms (12, 13). environment, confers rhythmic behavior to biological processes, Therefore, circadian gene expression in elf3-1 was examined in and optimally phases biological activities to specific times of the dark-grown, or etiolated, seedlings exposed to thermocycles in day. Circadian clocks are widespread in nature, and processes place of photocycles to determine if light signals alone cause clock under their control range from sleep–wake cycles in humans to arrhythmia. Rhythmic gene expression was not sustained in ther- elf3-1 daily expression of photosynthetic genes in plants. Clocks also time mocycle-entrained etiolated seedlings released into con- seasonal responses, such as the flowering transition in many tinuous temperatures; furthermore, the clock in this mutant was unable to entrain to thermal cues. ELF3 was required to restrict plant species. PRR7 Core molecular oscillators in eukaryotes incorporate inter- normal expression of temperature-sensitive genes like and PRR9 to the day and to establish enhanced sensitivity of these genes locked transcription–translation feedback loops. In the model to warm temperature cues during the night. These results indicate plant Arabidopsis thaliana, three such loops are critical to gen- fi that ELF3 serves as part of the core molecular oscillator instead eration and maintenance of circadian rhythms. The loop rst of solely modulating clock sensitivity to environmental cues. discovered is composed of the pseudoresponse regulator TOC1 (1) and two partially redundant Myb-like transcription factors, CCA1 (2) and LHY (3). Morning expression of CCA1 and LHY Author contributions: B.T. and F.G.H. designed research; B.T. and F.G.H. performed re- represses TOC1 expression by binding to its promoter (4), and search; B.T. and F.G.H. analyzed data; and B.T. and F.G.H. wrote the paper. circadian accumulation of TOC1 in the evening helps to induce The authors declare no conflict of interest. CCA1 and LHY. A second morning-phased loop includes two This article is a PNAS Direct Submission. TOC1-related proteins, PRR7 and PRR9 (5, 6). CCA1 and LHY 1To whom correspondence should be addressed. E-mail: [email protected]. PRR7 PRR9 induce and expression, whereas the two PRRs sub- This article contains supporting information online at www.pnas.org/cgi/content/full/ sequently repress CCA1 and LHY. In a third evening-phased loop, 0911006107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0911006107 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Results under warm conditions following entrainment in thermocycles dif- Circadian Rhythms in Dark-Grown Arabidopsis Seedlings Require fering by 10 °C (Fig. S1 C and D and Table S1). Therefore, the 4 °C ELF3. Rhythmic gene expression was evaluated in the null elf3-1 change in ambient temperature was an effective zeitgeber com- + mutant with a transcriptional fusion of firefly LUC to the TOC1 parable to the larger temperature differentials described elsewhere + promoter (TOC1::LUC ). WT and elf3-1 seedlings were grown in (16). Importantly, the strong rhythmic expression of TOC1 indicates photocycles [light/dark (LD)] for 6 days and then released at ZT0 that the clock system in the etiolated seedlings is similar to that in (time of dark-to-light transition) on day 7 into free-running con- light-grown seedlings instead of the circadian clock in root tissue, TOC1::LUC+ where TOC1 does not appear to cycle (19). Expression from the ditions, either LL or DD. As expected, expression in + WT was robustly rhythmic on release into either LL or DD, and FKF1::LUC reporter was also strongly rhythmic in etiolated WT cycles persisted for at least 5 days (Fig. 1 A and B). These seedlings seedlings (Fig. S1C and Table S1). On the other hand, WT seedlings exhibited statistically significant rhythms under both free-running harboring the CAB2::LUC reporter did not consistently exhibit conditions with relative amplitude error (RAE) values ≤0.6 for all robust rhythms across several independent experiments, likely individuals (Fig. S1 A and B and Table S1). The WT period on because CAB2 expression is light-dependent (20). release into LL and DD was 24.47 ± 0.09 h (±SEM) and 26.52 ± Incontrast to WT, etiolated elf3-1 seedlings exposedto equivalent TOC1 elf3-1 HC entrainment conditions did not show obvious circadian TOC1:: 0.49 h, respectively. expression in LD-entrained + + seedlings released into LL was arrhythmic, as was TOC1 expres- LUC and FKF1::LUC expression on release into either con- sion in elf3-1 seedlings released into DD (Fig. 1 A and B). Esti- tinuous warm or cool conditions (Fig. 1 C and D). Cryptic rhythms mated period values were not returned for most elf3-1 seedlings, were not present in elf3-1, because curve fit analysis failed to detect and in the limited instances in which period values were returned coherent rhythms in the traces from the mutant background (Fig. S1 for elf3-1, these were accompanied by an RAE >0.6 or a period C and D and Table S1). Identical results were obtained with seed- outside a physiologically relevant range of 15–30 h in almost all lings entrained in thermocycles differing by 10 °C (Fig. S1 C and D cases (Fig. S1 A and B and Table S1). Poor rhythms for elf3-1 in and Table S1). Thus, ELF3 is required for circadian rhythms in DD were not confined to TOC1 expression, because comparable seedlings deprived of light exposure, indicating that the elf3 arrhythmic expression was observed from the clock-driven output arrhythmic phenotype is independent of light cues. + promoters CCR2::LUC and FKF1::LUC (Figs. S1B and S2 and Table S1). Thus, elf3 mutant seedlings do not exhibit circadian ELF3 Activity Is Needed for Temperature Entrainment of the Circadian elf3-1 rhythms under continuous conditions following entrainment with Clock.