The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth

Abstract

The circadian clock is required for adaptive responses to daily and seasonal changes in environmental conditions. Light and the circadian clock interact to consolidate the phase of hypocotyl cell elongation to peak at dawn under diurnal cycles in Arabidopsis thaliana. Here we identify a protein complex (called the evening complex)--composed of the proteins encoded by EARLY FLOWERING 3 (ELF3), ELF4 and the transcription-factor-encoding gene LUX ARRHYTHMO (LUX; also known as PHYTOCLOCK 1)--that directly regulates plant growth. ELF3 is both necessary and sufficient to form a complex between ELF4 and LUX, and the complex is diurnally regulated, peaking at dusk. ELF3, ELF4 and LUX are required for the proper expression of the growth-promoting transcription factors encoded by PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 (also known as PHYTOCHROME INTERACTING FACTOR 3-LIKE 6) under diurnal conditions. LUX targets the complex to the promoters of PIF4 and PIF5 in vivo. Mutations in PIF4 and/or PIF5 are epistatic to the loss of the ELF4-ELF3-LUX complex, suggesting that regulation of PIF4 and PIF5 is a crucial function of the complex. Therefore, the evening complex underlies the molecular basis for circadian gating of hypocotyl growth in the early evening.

Figure 1. ELF3, ELF4 and LUX are co-expressed and ELF3 directly interacts with both ELF4 and LUX in yeast

a) Expression of ELF3, ELF4 and LUX under diurnal or circadian conditions. Normalization is relative to maximum. Bars above the graphs represent light conditions during harvesting; black=lights off, white=lights on, grey=lights on during subjective night. Error bars represent the S.E.M., n=3. b) Yeast two-hybrid assay between ELF4 and ELF3, ELF4, LUX, LUX-N or LUX-C. C) Yeast two-hybrid assay between LUX and ELF3, ELF4 or LUX. These experiments were repeated twice (b and c). d) Yeast three-hyrbid containing combinations of ELF4-GAL4-DBD, LUX-GAL4-AD and ELF3. Error bars are S.E.M., n=4, presented as fold of induction over control vectors.

Figure 2. ELF3 bridges a diurnally regulated complex containing ELF4 and LUX in vivo

a) ELF3 is necessary for ELF4 and LUX to co-precipitate in vivo. Immunoprecipitations (IP) were performed on day 12 at ZT12, 12L:12D. b) ELF3, ELF4 and LUX oscillate and form a complex. (*): 15% gel, (·): bead background band, (–): LUX isoforms. c and d) Evening Complex formation in short and long days. Seedlings were grown under short– or long–day photoperiods (8L:16D or 16L:8D, respectively) and harvested beginning at ZT0 on day 12. Experiments were performed three times with similar results. Bars represent light conditions during harvesting as denoted in Figure 1a.

Figure 3. The Evening Complex regulates PIF5 and PIF4 expression through recruitment by LUX

a) PIF5 and PIF4 expression in elf3, elf4, lux and wild type (Col-0). Bars indicate light conditions as denoted in Figure 1a. Error bars represent the S.E.M., n=3. b) PIF5 (left) and PIF4 (right) promoters denoting degenerate (GATWCK or GATWYG) or consensus (GATWCG) LBS, unfilled or filled arrowheads, respectively. Numbers are relative to transcriptional start (+1). Bars are ChIP amplicons (above) and fragments for yeast one-hybrid (below), red × denotes mutated LBS. c)Yeast one-hybrid (Y 1-H) with LUX-AD and PIF5 and PIF4 promoter fragments. Fold enrichment is relative to controls. d) LUX, e) and g) ELF3 and f) ELF4 ChIP on PIF5 and PIF4 at ZT14, extended light. CS=coding sequence (d-g). Error bars represents the S.E.M. n=3 (d and e). g) Endogenous ELF3 ChIP from elf3-1, LUX/NOX ami and wild type. Error bars represent the S.D. of the average of two technical replicates measured twice. Experiments were repeated with similar results (f and g).

Figure 4. Hypocotyl growth defects are rescued by loss of PIF5 and PIF4 in EC member mutant backgrounds

a) Growth defects in elf3-2 background require PIF4/5. Scale bar = 5mm. b) Scatter plot of hypocotyl measurements from wild type, elf3-2, pif4-101 and pif5-1 single and compound mutants. This experiment was repeated with similar results. c) Model represents EC action on PIF4/5 expression during early evening to gate hypocotyl growth in Arabidopsis seedlings. The circadian-regulated EC represses PIF4/5 expression in the evening. Throughout the day, post-transcriptional light-mediated degradation of PIF4/5 proteins inhibit growth. Near dawn, concomitant rise of PIF4/5 RNA and PIF4/5 protein levels promote growth (white arrow).