Ambient temperature response establishes ELF3 as a required component of the core

Bryan Thines and Frank G. Harmon1

Plant 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- 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 (11). In the Arabidopsis mutant, nor- grown, or etiolated, null elf3-1 , 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 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 | | 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 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 , 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 , 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. The arrhythmic behavior in etiolated seedlings may i photocycles. have arisen from two possible sources: ( ) ELF3 forms an integral The profound disruption of circadian rhythms in LD-entrained component of the core oscillator, and its absence impairs clock elf3-1 regardless of free-running condition led us to evaluate the function so that rhythms are never present, or (ii) ELF3 is a extent ofthe clock defect in this mutant. To eliminate light effects on temperature zeitnehmer, in which case the clock is entrained in the oscillator, rhythmic gene expression was examined in dark- thermocycles but arrests after exposure to continuous temper- grown, or etiolated, WT and elf3-1 seedlings, where light exposure ature. To explore these possibilities, the oscillator in etiolated was limited to a brief 3-h white-light pulse to promote seed germi- elf3-1 seedlings was examined for evidence of proper entrainment. nation. Following entrainment in thermocycles differing by 4 °C Frequency demultiplication is a hallmark of clock entrainment [hot/cold (HC)], etiolated WT seedlings exhibited sustained and function (21). In environmental cycles with time periods (T) + rhythmic TOC1::LUC expression for at least 5 days in continuous less than 24 h, circadian rhythms “frequency demultiply,” or skip 22 °C warm or 18 °C cool conditions (Fig. 1 C and D). The mean an environmental cycle(s), so that overall repeated patterns still estimated free-running period in WT was 26.70 ± 0.22 h and 25.97 ± occur approximately every 24 h. In contrast, organisms with 0.15 h in warm and cool conditions, respectively (Fig. S1B and Table nonfunctional oscillators display repeated patterns that typically S1). Rhythms from all WT spots were good enough to be assigned reflect the shorter environmental T cycle (22–24). To determine RAE values ≤0.6 (Fig. S1 C and D). Similar rhythmic TOC1 whether etiolated elf3-1 seedlings exhibit any signs of temperature expression was also observed in etiolated seedlings free running entrainment, seedlings were entrained in HC (T = 24 h) and then moved to 12-h thermocycles (T = 12 h; 6 h 22 °C/6 h 18 °C). Seedlings with properly entrained circadian clocks were expected to + 4 2.5 retain the T = 24-h entrainment. Accordingly, TOC1::LUC ex- pression was examined immediately on transfer of WT and elf3-1 3 A B 2.0 seedlings to T = 12 h. In WT, the original T = 24-h entrainment 2 period was evident after the transfer as a strong peak every 24 h at a 1.5 A 1 phase matching that in normal HC entrainment (Fig. 2 ). In

0 1.0 3.5 2.5 C D 1.5 ABWT elf3-1 TOC1::LUC+

(arbitrary units) 2.5 2.0

1.5 1.5 1.0 TOC1::LUC+ 0.5 1.0 (arbitrary units) 0 24487296120 0 24487296120 0.5 ZT, h ZT, h 0 1224364860720 122436486072 Time in T =12h Fig. 1. ELF3 is required for sustained rhythms in Arabidopsis seedlings. Mean bioluminescence from the TOC1::LUC+ reporter in WT (•) and elf3-1 Fig. 2. ELF3 is required for clock temperature entrainment in dark-grown (□) individuals (n = 15) entrained in LD for 6 days and then released at ZT0 seedlings. Mean TOC1::LUC+ expression from HC-entrained etiolated WT (A; •) on day 7 into LL (A)orDD(B). TOC1::LUC+ expression after HC entrainment and elf3-1 (B; □) etiolated seedlings transferred at ZT0 into T = 12 thermocycles. in DD for 6 days and then release on day 7 into continuous 22 °C (C)or18°C Representative data from two independent experimental replicates are shown, (D) while maintaining DD. Representative data are shown for three exper- where six groups of ∼50 etiolated seedlings for each genotype were imaged for imental replicates. 72 h in T = 12 thermocycles in complete darkness.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.0911006107 Thines and Harmon Downloaded by guest on September 24, 2021 addition, a prominent trough in expression was apparent on the first delay of just under 12 h achieved at CT18. A phase advance of day after transfer, and to a lesser degree 24 h later, which matched nearly 6 h occurred after CT18, and following this breakpoint, the nadir in TOC1 expression observed in free-running conditions advances were maintained for the remainder of the subjective following T = 24-h entrainment, and the transition between the night. The shape of this tPRC from etiolated seedlings was anal- trough and major peak showed anticipation typical of clock-driven ogous to that of light-grown Arabidopsis seedlings, where the clock expression. A driven rhythm was apparent as a weaker peak start- was reset by light pulses (13), and opposite to that of the resetting ing ∼16 h after the change in entrainment conditions. The persis- pattern elicited by cold pulses (16). The response of the oscillator tence of the 24-h timing in WT indicates correct clock entrainment in ELF3OX was nearly identical to that of WT (Fig. 3), indicating during the preceding thermocycles. The elf3-1 seedlings given the that ELF3 did not have a substantial effect on resetting of the clock same treatment lacked all these hallmarks of entrainment (Fig. 2B). by warm cues. Therefore, ELF3 on its own does not appear to + On transfer to the T = 12-h thermocycles, TOC1::LUC expression modulate temperature information feeding into the Arabidopsis in elf3-1 seedlings immediately adopted a pattern reflecting the oscillator directly. changed external temperature cycles and showed no indication of the prior exposure to the T = 24-h conditions. Notably, the wave- ELF3 Is Required for Appropriate Response to Ambient Temperature form in the mutant background was invariant, exhibiting a single Cues. The circadian clock and other physiological responses adjust square peak and trough consistent with expression driven by to environmental temperature changes through altered gene external temperature changes instead of an endogenous circadian expression (25–27). Expression of the integral clock component GI rhythm. The behavior of elf3-1 seedlings indicates that the oscillator is elevated by warm temperature (27 °C) (28). The activities of in this mutant could not generate strong circadian rhythms; there- PRR7 and PRR9 are required for temperature entrainment of the fore, the circadian clock itself appeared to be impaired without circadian clock (16), and their expression is induced following ELF3 activity instead of the oscillator arresting in the changed ambient temperature increases (see below). Outside of the core environmental conditions. clock, PIF4 is required for high-temperature signaling and growth responses, and a shift to 28 °C triggers its induction (29). These Clock Sensitivity to Warm Temperature Is Not Altered by Overex- temperature-responsive genes were examined to evaluate whether pression of ELF3. To examine the contribution of ELF3 to tem- ELF3 directly contributes to temperature responses or if defects in perature entrainment of the Arabidopsis clock, oscillator sensitivity elf3 are more consistent with aberrant clock function. to resetting by a step up in ambient temperature was determined in The acute response of PRR7, PRR9, PIF4, and GI to high ELF3 ELF3OX WT and an overexpression ( ) line by constructing a temperature was measured in dark-grown WT and elf3-1 seedlings. PLANT BIOLOGY tPRC. A tPRC represents the extent of phase change induced by a Each genotype was grown in typical HC conditions for 7 days; resetting temperature signal given at different times across a nor- while under these conditions, sets of seedlings received a 3-h 28 °C mal circadian day. The ELF3OX line, which contains ELF3 tran- pulse at either ZT4 (midday) or ZT16 (midnight). As observed in script at ≈50 times endogenous levels (11), was tested, because, light-grown WT seedlings (7, 17, 24, 30), abundance of PRR7, unlike elf3-1, this line exhibits robust circadian rhythms that are PRR9, PIF4, and GI transcripts in WT exhibited diurnal patterns of only of a moderately long period in light-grown seedlings (13). higher expression at ZT4 and limited levels at ZT16 (Fig. 4). A 3-h ELF3OX Etiolated seedlings also exhibited robust rhythms after warm pulse at ZT16 given to WT induced PRR7 transcript 20-fold temperature entrainment comparable to those observed in WT over the untreated control, which was an order of magnitude (Table S1). If ELF3 acts to repress temperature cues during normal greater than at ZT4 (Fig. 4A and Table 1). Stronger night-phased ELF3OX entrainment, the would be more resistant to resetting by a responses were also observed for PIF4 and GI: Inductions were temperature change. An analogous phenotype for light resetting of 7.8- and 17.2-fold, respectively, after a warm treatment at ZT16, ELF3OX the oscillator in has been described previously (13). but changes were <2-fold at ZT4 (Fig. 4 C and D and Table 1). TOC1::LUC+ The apparent phase change of expression after a Clearly, induction by warm temperature was set to a specific time step up from 18 °C to 22 °C was determined in etiolated WT and of day, and the response was strongest during the night in ELF3OX seedlings that experienced a day of free run at 18 °C after etiolated seedlings. 6 days in 10 °C thermocycles. Seedlings were transferred to 22 °C at 3-h intervals starting at subjective dawn [circadian time (CT) 0]. Phase differences relative to the CT0 sample were calculated so that phase advances and delays were positive and negative values, 12 PRR7 PRR9 respectively (Fig. 3). In WT, the 4 °C shift up in temperature 2 A B caused phase delays, with the magnitude of change increasing as 8 the subjective day progressed to subjective night, with a maximal 1 4

0 0 12 4 9 PIF4 GI 6 3 C 2 D

3 Change in Expression 0 2 (fold of untreated WT at ZT4) (fold of untreated WT (hr) -3 1 -6

Phase Change 1 -9 -12 0 0 0 6 12 18 24 30 36 42 48 Pulse - ++- - ++- Circadian Time of transfer, hr Fig. 4. High-temperature-induced gene expression is altered in elf3-1. Fig. 3. WT and ELF3OX seedlings are similarly responsive to resetting by warm PRR7 (A), PRR9 (B), PIF4 (C), and GI (D) transcript levels in etiolated seedlings temperature. Six-day-old WT (▲,solidline)andELF3OX (□, dashed line) etio- before and after a 3-h 28 °C pulse, as indicated at the bottom. In each panel, lated seedlings entrained in HC were transferred to 22 °C after free running for genotypes are depicted in sets of four, which were composed of WT (black 24 h at 18 °C every 3 h starting at CT0. Phase advances and delays are positive and bars), elf3-1 (open bars), ELF3OX (light grey bars), and CCA1OX (dark grey negative values, respectively. Each point and error bars represent the mean ± bars). Background indicates time of day: White is ZT4, and gray is ZT16. All SEM of three experimentalreplicates. Data from a single 24-h period are double- samples are normalized to the WT 22 °C sample at ZT4 in each panel. Values plotted to aid in identification of the features in the tPRC. and error bars are mean ± SEM of three experimental replicates.

Thines and Harmon PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 Table 1. Loss of ELF3 substantially alters induction of gene expression by a warm temperature treatment Time* ZT4/ZT16

Genotype

Gene WT elf3-1 ELF3OX CCA1OX

† Fold change ± SEM

PRR7 1.7 ± 0.1/20.4 ± 2.3 1.1 ± 0.1/0.9 ± 0.2 2.0 ± 0.3/8.9 ± 1.4 1.4 ± 0.1/1.5 ± 0.2 PRR9 2.3 ± 0.3/2.5 ± 0.9 0.7 ± 0.2/0.4 ± 0.1 4.3 ± 1.3/3.3 ± 0.4 2.2 ± 0.5/1.0 ± 0.1 PIF4 1.3 ± 0.3/7.8 ± 0.7 1.6 ± 0.5/1.3 ± 0.2 1.5 ± 0.2/7.4 ± 0.4 1.5 ± 0.3/2.2 ± 0.7 GI 1.5 ± 0.2/17.2 ± 1.7 0.6 ± 0.3/0.7 ± 0.1 1.8 ± 0.3/12.7 ± 3.6 1.0 ± 0.1/1.4 ± 0.5

*Etiolated seedlings grown in thermocycles were given a 3-h 28 °C pulse at ZT4 (midday) or ZT16 (midnight). † Fold change of indicated transcripts normalized to untreated control samples. Values are averages of three independent experimental replicates.

Gene expression in elf3-1 was substantially different from that in Discussion WT. PRR7 expression in this mutant showed no significant high- Temperature is an important environmental cue that sets circadian temperature induction regardless of the time of day (Fig. 4A and clocks to local time in ectothermic organisms. Rhythms dependent on Table 1). This was attributable to constitutively elevated PRR7 the Arabidopsis clock were not sustained in thermocycle entrained expression, because PRR7 transcript level in untreated elf3-1 etiolated elf3-1 seedlings, indicating that defects caused by loss of seedlings matched that of WT individuals receiving the ZT16 high- ELF3 extend to conditions wherein light is absent. Furthermore, the temperature pulse (Fig. 4A). Basal PRR9 expression in elf3 mutant circadian clock showed no evidence of temperature entrainment in seedlings was also substantially elevated, especially at ZT16, where elf3-1.Thesefindings demonstrate that the circadian system as a the expression in this mutant was ∼30-fold higher than levels whole is compromised in the absence of ELF3 activity and show reached in WT following the high-temperature pulse (Fig. 4B). that ELF3 acts as more than a zeitnehmer that represses light input to High-temperature treatment of elf3-1 led to a reduction in PRR9 the clock. Therefore, ELF3 appears to be an integral component of expression at both times, but these transcript levels remained the core oscillator that is needed for circadian rhythms. substantially higher than the maximal response achieved in WT In contrast to our findings, previous models placed ELF3 as a under any condition. Like PRR7 and PRR9, basal expression of zeitnehmer responsible for gating light input to the central oscillator both PIF4 and GI in elf3-1 was drastically higher than in WT at based on the perceived light-dependent arrhythmic phenotype of ZT16 (Fig. 4 C and D)andGI expression in elf3-1 was repressed the elf3 mutant. Hicks et al. (12) and Covington et al. (13) reported somewhat in response to the elevated temperature. Collectively, that light-entrained elf3 seedlings transferred to DD exhibit circa- these data indicate that normal repression of PRR7, PRR9, PIF4, dian rhythms for the CAB2::LUC and CCR2::LUC reporters, and GI during the night was not achieved in elf3-1 and, as a result, respectively. Hicks et al. (12) also described CAB2::LUC rhythms in response to warm temperature cues was dramatically altered in the etiolated elf3 seedlings. Expression from the CAB2 promoter rap- elf3 mutant. Thus, ELF3 appeared critical to normal gating of the idly damps in the dark (20), rendering CAB2::LUC problematic for expression for these warm temperature-responsive genes. analysis of circadian rhythms in the dark. Typically, WT seedlings Because ELF3 function appeared to be required for nighttime exhibit a single discernable CAB2 expression peak, appearing within repression of these warm-responsive genes, expression of PRR7, the first 24 h in DD, after which rhythmic expression becomes dif- PRR9, PIF4, and GI in response to warm treatment was evaluated ficult to detect (12, 20, 31). The elf3 mutants show a comparable first in ELF3OX and the arrhythmic CCA1 overexpression (CCA1OX) peak in DD (12, 14). The question is whether this single CAB2::LUC line (2). ELF3OX was included to determine what, if any, imme- peak in elf3 represents bona fide oscillator activity or, instead, diate effect ELF3 had on acute high-temperature gene induction. reflects the last driven cycle of a clock that is running down after The arrhythmic CCA1OX line served to test what effect an receiving a final zeitgeber before free run (32). We favor the latter arrhythmic clock had on temperature responses and, therefore, to interpretation and for this reason our characterization of elf3-1 + distinguish elf3-specific phenotypes from general effects induced focused mainly on the behavior of TOC1::LUC expression, by clock dysfunction. In ELF3OX, the expression profiles of PRR7, because this reporter in WT was strongly rhythmic in the dark fol- PRR9, PIF4, and GI largely mirrored that of WT, where these lowing light or temperature entrainment (Fig. 1). With this more genes showed high expression at ZT4 but not at ZT16 (Fig. 4). Also appropriate reporter construct, rhythms were absent from elf3-1 a 28 °C pulse given at ZT16 was more effective at inducing under all tested conditions (Fig. 1 and Figs. S1 and S2). Similar expression than one given in the morning (Table 1). The absence of arrhythmic behavior was observed from CCR2::LUC and FKF1:: + + a notable phenotype in the rhythmic ELF3OX suggests that ELF3 LUC in the mutant, thereby reinforcing TOC1::LUC as an does not directly influence response to temperature signals. The accurate readout of the elf3-1 phenotype. phenotype of CCA1OX mirrored that observed in elf3-1. In par- In addition, without close control of ambient temperature, the ticular, basal transcript levels for PRR9, PRR7, PIF4, and GI were influence of inadvertent temperature cues on LUC expression can- elevated compared with WT, and a warm treatment at either ZT4 not be ruled out. Indeed, CCR2 promoter is temperature-responsive or ZT16 had little positive effect on expression (Fig. 4 and Table (33, 34). Arabidopsis seedlings respond to 4 °C steps in temperature, 1). The similarity between CCA1OX and elf3-1 phenotypes points but the lower limit of responsiveness for temperature cues has not toward the possibility that without a functional circadian clock, been defined in this species. Strikingly, temperature steps of 1 °C Arabidopsis seedlings were unable to respond to temperature cues entrain the Kalanchoë circadian oscillator (35). Given the possibility properly. Therefore, ELF3 appeared vital for circadian clock of similar sensitivity for Arabidopsis, we designed our experimental function; without this protein, the circadian clock was unable to system to avoid zeitgeber information arising from unintended produce robust rhythms, which ultimately precipitated a defect in ambient temperature cycles or cues that could drive expression temperature response. rhythms and mask the underlying state of the oscillator in elf3.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.0911006107 Thines and Harmon Downloaded by guest on September 24, 2021 Whether this precaution was also included in the previous studies is growth. Recently, the arrhythmic CCA1OX line was used to show difficult to know. that loss of circadian-regulated expression of the growth-promoting Taking these factors into account, the absence of robust cir- transcription factors PIF4 and PIF5 is the molecular basis for the cadian expression from the distinct CCR2, TOC1, and FKF1 long hypocotyl phenotype in this clock mutant (24). We propose the promoters in etiolated elf3-1 seedlings (Fig. 1 and Figs. S1 and S2) same molecular explanation for the ELF3 hypocotyl growth phe- is compelling evidence of severe clock dysfunction in this back- notype. PIF4 and PIF5 together promote hypocotyl elongation, and ground. Furthermore, dark-grown elf3-1 seedlings exposed to their action is limited by phyB-mediated light-dependent degrada- temperature cycles exhibited obviously arrhythmic expression tion, effectively limiting growth to just before dawn. Because PIF4 of PIF4, PRR7, PRR9, and GI that was similar to the well- expression is constitutively high in elf3-1, PIF4 protein is also likely characterized arrhythmic CCA1OX line (Fig. 4). Nozue et al. (24) elevated and could promote the exaggerated hypocotyl growth have also demonstrated that elf3-1 seedlings no longer show time typical of elf3 mutants, as it does in seedlings in which PIF4 is of day control of hypocotyl elongation consistent with an overexpressed (40). Loss of clock control over PIF4 in elf3-1 is arrhythmic clock. The simplest interpretation of this collection of consistent with previous hypocotyl measurements for this mutant arrhythmic phenotypes is that ELF3 serves a fundamental role in (10): Hypocotyl elongation is greatest under short day conditions, Arabidopsis the core circadian oscillator under all conditions. where longer nights support elevated PIF4 abundance, and hypo- A formal possibility exists that ELF3 is both a temperature cotyls are shorter in long days, where the longer light periods serve zeitnehmer (36) and a light zeitnehmer (14). As a dual zeitnehmer, to repress accumulation of PIF4. Therefore, a fully arrhythmic clock ELF3 could blunt daytime cues (light and warm temperature) to readily explains the apparent light-associated hypocotyl growth the clock during the transition to night to avoid inappropriate phenotypes for elf3. resetting during this part of its cycle. If this is the case, altered The elf3 circadian phenotype suggests a role for ELF3 in the ELF3 activity should modify the clock’s sensitivity to temperature ELF3OX central oscillator as a contributor to the X and Y activities suggested cues; however, seedlings were no more or less responsive by Locke et al. (41). Loss of X activity is expected to diminish to resetting by a shift to warm temperature compared with WT expression of CCA1/LHY,andbothCCA1 and LHY are expressed (Fig. 3). Absolute gene expression levels caused by a warm tem- at very low levels in elf3-1 (42). Conversely, these genes are perature shift were also similar between WT and ELF3OX (Fig. 4), expressed more highly in ELF3OX. Y function is modeled to pro- rather than showing a drastically diminished response in ELF3OX mote TOC1 expression, and GI has been proposed as a component as would be predicted if a primary function of ELF3 is to inhibit TOC1::LUC+ elf3-1 elf3 of Y activity. is expressed at elevated levels in high-temperature cues (Fig. 3). Furthermore, mutant seed- GI CCA1/LHY PLANT BIOLOGY lings lacked obvious hypersensitivity to warm treatments; instead, (42), as is (43). The low levels of expression could, in part, explain the markedly high TOC1 and GI expression (5, 6), but these plants were essentially nonresponsive to acute warm pulses elf3 that trigger strong induction of gene expression in WT (Fig. 4). By another potential contributor may be the effect of the mutation these measures, the principal role of ELF3 in etiolated seedlings on GI accumulation. ELF3 promotes degradation of GI by acting as appears not to be that of a temperature zeitnehmer. a scaffold for the association of GI with the E3 ubiquitin ligase Several contradictions have been observed for light responses in COP1 (15); consequently, ELF3 is formally a repressor of Y activity. the elf3 mutant; for example, this mutant shows excessive hypocotyl Thus, experimental data suggest that ELF3 may contribute to both PRR7 PRR9 elongation under white-light and monochromatic light conditions X and Y activity. Interestingly, and expression is also elf3 (10, 37), which suggests that ELF3 promotes light signaling; it also elevated in , and what role ELF3 may have in repressing PRR7 has enhanced acute induction of CAB2 expression by light (14), and PRR9 is presently unclear, because these regulatory details are which indicates that ELF3 represses light signals. Similarly, ELF3 missing from the present mathematical model of the clock. Clearly, physically interacts with phyB in vitro (11), consistent with ELF3 further work is needed to determine whether ELF3, in fact, serves in acting downstream of phyB, but hypocotyl and flowering time this dual capacity and to define the biochemical means by which this phenotypes of elf3 mutants are additive with those of a phyB mutant protein acts. Nevertheless, ELF3 is most likely an essential part of (10, 37). Additive phenotypes are also found with elf3 combined the core oscillator, allowing passage into the nighttime phase of the individually with phyA or hy4,acryptochrome 1 mutant background circadian cycle under all conditions. (37). Clearly, the relationship between ELF3 and light signaling components is more complicated than a model in which ELF3 is Materials and Methods a direct repressor within a linear phototransduction pathway. Plant Material and Growth Conditions. A. thaliana (Columbia) was used unless A model that reconciles these contradictions in the elf3 pheno- otherwise noted. Seeds were surface-sterilized and plated on 1× Murashige and Skoog basal salt medium with 0.8% agar and 3% (wt/vol) sucrose (MS type is one in which ELF3 activity is required by the core oscillator fi to produce circadian rhythms. Reed et al. (37) suggested that plates) (44). After strati cation in the dark at 4 °C for 3 days, plates were transferred to a Percival (percival-scientific.com) incubator set to the indi- aberrant circadian rhythms may be the basis for the apparent light elf3 cated light or temperature conditions. Light entrainment was a 12 h light/12 phenotypes of . The gating role of the plant oscillator attenuates h dark photoperiod at a continuous temperature of 22 °C. Light was sup- fi − − or intensi es cellular activities to create permissive or non- plied by cool-white fluorescent bulbs at 50 μmol·m 2·sec 1. For etiolated permissive times of the day for responses, including light, hormone, seedlings, imbibed and stratified seeds were exposed to 3 h of white light at and, as shown here, temperature signaling (38, 39). In arrhythmic the same fluence rate, wrapped in two layers of foil, and transferred to a clock mutants, the oscillator is likely locked in one particular phase, dark incubator. Unless otherwise noted, etiolated seedlings were grown in such as morning, and the activities and responses observed in such a thermocycles of 12 h 22 °C/12 h 18 °C. The TOC1::LUC+ (45), CAB2::LUC (20), mutant reflect the phase state of the arrested oscillator. We propose and FKF1::LUC+ (46) reporters were in WT or the elf3-1 mutant (10). CCR2:: + + that the clock in elf3 exists in a constitutively day state, not unlike the LUC was in Wassilewskija (Ws) with elf3-3 (Ws) (47). The TOC1::LUC arrested oscillator predicted previously (14). In fact, the hyper- reporter was crossed into the ELF3OX line from Liu et al. (11) and into the responsiveness of the CAB promoter in an elf3 mutant appears to CCA1OX line from Wang and Tobin (2). stem from this clock-gated pathway being locked into a permissive PRR7 PRR9 PIF4 GI Bioluminescence Assays. Seedlings were sprayed with ∼1mLof5mMfirefly state for light signals (14). Because , , ,and are luciferin (Biosynth, biosynth.com; Gold Biotechnology, goldbio.com) pre- normally daytime-phased by the oscillator, their unchanging high elf3 pared in 0.01% (vol/vol) Triton X-100 (Sigma Aldrich) 24 h before imaging. expression in etiolated is in line with the notion of a dysfunc- While in continuous conditions, seedlings were imaged every hour (etio- tional clock stuck in a state more like day than night. lated) or 2.5 h (light-grown) with a Berthold NightOwl II imaging system The excessive hypocotyl elongation observed in elf3 is consistent (berthold.com). Bioluminescence data from the images was extracted with with the idea of an arrested clock that no longer properly gates Berthold WinLight software and curve-fitting analysis of bioluminescence

Thines and Harmon PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 traces with fast Fourier transform-nonlinear least squares (FFT-NLLS) using transferred at ZT4 or ZT16 in the dark via the filter paper to plates pre- the Biological Rhythms Analysis Software System 3.0 (48). equilibrated to the pulse temperature (28 °C) or the current growth chamber temperature (22 °C or 18 °C). Three hours after transfer, seedlings were

Temperature Phase Response Curve. For each experimental replicate, nine MS harvested under a green safe light and immediately placed in liquid N2. RNA plates were sown with six spots each of WT and ELF3OX seeds. Each spot was was extracted using a Qiagen RNeasy Plant Mini Kit (qiagen.com). First- composed of ≈50 seeds. Seeds were given a 3-h white-light pulse to induce strand cDNA was synthesized using 2 μg of total RNA with an Invitrogen uniform germination and were then grown in the dark under thermocycles SuperScript III First Strand Synthesis Kit (invitrogen.com). Quantitative RT- of 12 h 22 °C/12 h 12 °C. After 6 days, all plates were transferred to 18 °C at PCR was performed as described previously (45). Transcript levels of target − ZT0, and starting 24 h later, one plate was transferred to 22 °C every 3 h. genes were calculated using the equation 2[C (Control) C (Experimental)], where + t t Bioluminescence from TOC1::LUC was recorded for 7 days after transfer. C is the mean threshold cycle for each sample. Isopentenyl pyrophosphate/ fi t Period and phase were calculated with FFT-NLLS (48), and the rst 24 h were dimethylallyl pyrophosphate isomerase (At3g02780) was used as the nor- excluded from analysis to measure stable phase change more accurately. The malization control (49). Primers sequences are shown in Table S2. phase was divided by the period and multiplied by 24 to determine the circadian time (CT, in hours) phase. CT phase values were averaged for three ACKNOWLEDGMENTS. We thank Sadaf Khan, Scott Rowe, and Tim Richardson experimental replicates and compared with the average CT0 phase. Phase for helpful discussions. We thank Steve A. Kay (University of California, San delays were calculated as negative numbers, whereas phase advances were Diego), David Somers (Ohio State University), and Elaine Tobin (University of calculated as positive numbers. California, Los Angeles) for generously providing transgenic lines. This work is supported by US Department of Agriculture Grant CRIS 5335-21000-025-00D (to Expression Analysis by Quantitative RT-PCR. Seedlings grown for 7 days on F.G.H) and Ruth L. Kirschstein National Research Service Award F32GM083536- Whatman filter paper in the dark under the indicated thermocycles were 01 (to B.T.).

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