ELF4 is required for oscillatory properties of the circadian clock

Abstract

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.

Figure 1.

ELF4 is involved in red-light response and acts at night. A, Hypocotyl length of 1-week-old seedlings grown under continuous red light. elf4-1 has a long hypocotyl under a range of red-light fluences where ELF4-ox has no phenotype. B, ELF4 gates light input to the clock during the night (ZT13 to ZT23), here shown as the difference in elf4-1 CAB2:LUC luminescence in light-induced versus noninduced seedlings. Seedlings were entrained in 12/12 LD cycles and transferred to DD at dusk (ZT12). Time in hours since the start of transfer (hence time = 0) was ZT12. The experiment was repeated twice.

Figure 2.

The elf4 clock runs for 1 d and stops at subjective dusk (approximately time 32 h). A, Time to peak of CAB2:LUC activity in dark-adapted elf4-1 seedlings after red-light pulse treatment. Seedlings were entrained in 8/16 LD cycles and then transferred to darkness at dusk (ZT8). Time of pulse is shown as hours since last dawn; 5 min of red light were given at 3-h intervals from time 9. Error bars represent sem. The experiment was repeated twice. B to D, Normalized CCR2:LUC, CCA1:LUC, and CCR2:LUC profiles of elf4-1 and ELF4-ox seedlings, compared to the wild type, before and after exposure to jet lag (an extended night of 24 h long) under LD cycles. White bars indicate light intervals and gray bars indicate darkness.

Figure 3.

Dose-dependent effect of ELF4. A and B, ELF4-ox plants flower late under long days (white bars), but not short days (gray bars). C to F, ELF4-ox has long period and early phase under LL. CAB2:LUC (C and E) and CCR2:LUC (D and F). C and D, Gray bars indicate subjective night. Insets, RAE plots of luminescence rhythms (RAE versus period length). Each period estimate corresponds to one seedling. E and F, Peak time of ELF4-ox CAB2:LUC (E) and CCR2:LUC (F) in DD. Error bars represent sem. All seedlings were entrained in 16/8 LD cycles. Time is ZT.

Figure 4.

Morning gene expression (CCA1:LUC, CAB2:LUC) is less affected than expression of an evening-specific gene (CCR2:LUC). Luminescence profiles of elf4-1 and ELF4-ox kept under entraining conditions, 8/16 LD short day (left) and 16/8 LD long day (right). A and B, CCA1:LUC. C and D, CAB2:LUC. E and F, CCR2:LUC. Gray blocks indicate night time. Error bars represent sem.

Figure 5.

Temperature entrainment defects in elf4-1. A, C, and E, Seedlings were entrained to LD cycles and then transferred to LL. B, D, and F, One set of LD-entrained plants was subsequently given temperature cycles for 3 d (12:12 warm/cool [WC]) and then released into LL and constant temperature for 3 d. A and B, CAB2:LUC. C and D, CCA1:LUC. E and F, CCR2:LUC. White bars indicate free run under LL. A, C, and E, Gray bars indicate subjective night. B, D, and F, Red blocks indicate 24°C (daytime). Blue blocks indicate 18°C (nighttime). Red hatched bars indicate subjective warm day. Blue hatched bars indicate subjective cold night. Time is light ZT. Insets, RAE plots (RAE versus period). Each period estimate is an RAE-weighted mean of a group of seedlings. Hours 12 to 84 and 84 to 152 analyzed for free run under LL after LD and WC entrainment, respectively. Error bars represent sem.

Figure 6.

ELF4 is expressed in the night and influences the expression level of CCA1 and TOC1. A, ELF4:LUC luminescence activity compared to CCR2:LUC in wild type. B, Luminescence of ELF4:ELF4LUC and ELF4:LUC in the elf4-1 mutant. C, Long period and high amplitude of CCA1:LUC in ELF4-ox under LL. D, Long period of LHY:LUC in ELF4-ox. Inset, LHY:LUC expression in elf4-1 mutant; note that in elf4-1 the LUC levels are arrhythmic and more than 10-fold lower than the wild type. E, TOC1:LUC expression is high and arrhythmic in elf4-1. F, ELF4-ox displays low TOC1:LUC expression, which is robustly rhythmic. Gray bars indicate subjective day. Time is ZT. Error bars represent sem. All LUC seedlings were entrained in 16/8 LD cycles. G, ELF4 expression is rhythmic in the toc1-1 mutant. Seedlings were entrained in 12/12 LD cycles. ELF4 level is normalized to β-TUBULIN4 at each time point. Mean values are plotted; error bars represent sds. The experiment was replicated twice.