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

Abstract

Circadian clocks synchronize internal processes with environmental cycles to ensure optimal timing of biological events on daily and seasonal time scales. External light and temperature cues set the core molecular oscillator to local conditions. In Arabidopsis, EARLY FLOWERING 3 (ELF3) is thought to act as an evening-specific repressor of light signals to the clock, thus serving a zeitnehmer function. Circadian rhythms were examined in completely dark-grown, or etiolated, null elf3-1 seedlings, with the clock entrained by thermocycles, to evaluate whether the elf3 mutant phenotype was light-dependent. Circadian rhythms were absent from etiolated elf3-1 seedlings after exposure to temperature cycles, and this mutant failed to exhibit classic indicators of entrainment by temperature cues, consistent with global clock dysfunction or strong perturbation of temperature signaling in this background. Warm temperature pulses failed to elicit acute induction of temperature-responsive genes in elf3-1. In fact, warm temperature-responsive genes remained in a constitutively "ON" state because of clock dysfunction and, therefore, were insensitive to temperature signals in the normal time of day-specific manner. These results show ELF3 is broadly required for circadian clock function regardless of light conditions, where ELF3 activity is needed by the core oscillator to allow progression from day to night during either light or temperature entrainment. Furthermore, robust circadian rhythms appear to be a prerequisite for etiolated seedlings to respond correctly to temperature signals.

Fig. 1.

ELF3 is required for sustained rhythms in Arabidopsis seedlings. Mean bioluminescence from the TOC1::LUC⁺ reporter in WT (•) and elf3-1 (□) individuals (n = 15) entrained in LD for 6 days and then released at ZT0 on day 7 into LL (A) or DD (B). TOC1::LUC⁺ expression after HC entrainment in DD for 6 days and then release on day 7 into continuous 22 °C (C) or 18 °C (D) while maintaining DD. Representative data are shown for three experimental replicates.

Fig. 2.

ELF3 is required for clock temperature entrainment in dark-grown seedlings. Mean TOC1::LUC⁺ expression from HC-entrained etiolated WT (A; •) and elf3-1 (B; □) etiolated seedlings transferred at ZT0 into T = 12 thermocycles. Representative data from two independent experimental replicates are shown, where six groups of ∼50 etiolated seedlings for each genotype were imaged for 72 h in T = 12 thermocycles in complete darkness.

Fig. 3.

WT and ELF3OX seedlings are similarly responsive to resetting by warm temperature. Six-day-old WT (▲, solid line) and ELF3OX (□, dashed line) etiolated seedlings entrained in HC were transferred to 22 °C after free running for 24 h at 18 °C every 3 h starting at CT0. Phase advances and delays are positive and negative values, respectively. Each point and error bars represent the mean ± SEM of three experimental replicates. Data from a single 24-h period are double-plotted to aid in identification of the features in the tPRC.

Fig. 4.

High-temperature-induced gene expression is altered in elf3-1. PRR7 (A), PRR9 (B), PIF4 (C), and GI (D) transcript levels in etiolated seedlings before and after a 3-h 28 °C pulse, as indicated at the bottom. In each panel, genotypes are depicted in sets of four, which were composed of WT (black bars), elf3-1 (open bars), ELF3OX (light grey bars), and CCA1OX (dark grey bars). Background indicates time of day: White is ZT4, and gray is ZT16. All samples are normalized to the WT 22 °C sample at ZT4 in each panel. Values and error bars are mean ± SEM of three experimental replicates.