all wild - type , mutant and transgenic lines were in the arabidopsis thaliana ecotype columbia-0 ( col-0 ) .
the procedures for arabidopsis husbandry ; yeast one - hybrid , two - hybrid , and three - hybrid analyses ; biolumenscent imaging ; immunoprecipitations ; chromatin immunoprecipitations ; and hypocotyl measurements were as described previously , with modifications detailed in methods . in all growth chambers , light was supplied at 80 mol m-2 sec-1 by cool - white fluorescent bulbs at 22 c . for yeast two - hybrid analyses ,
sd - wl medium selects for the presence of both bait and prey vectors , and sd - wlha medium selects for an interaction between bait and prey proteins .
ipp2 , apx3 and at1g11910 were used to normalize real - time pcr expression analyses , and all primers for quantitative pcr are listed in supplemental table 1 .
elf4elf4-ha construct includes 580 bp of promoter sequence cloned from col-0 dna amplified using primers listed in supplemental table 1 .
antibodies were generated in rabbits ( sigma genosys ) against either an elf3 specific peptide ( csiqeerkrydsskp ) , or a full - length lux protein fused to glutathione - s - transferase ( gst ) .
antibodies were affinity purified against the same elf3 peptide using a sulfolink immobilization kit ( thermo scientific ) or using a gst - lux affinity column .
blots for elf4 represent 20% of the total ip sample , as elf4 must be run on a separate 15% gel , identified by ( * ) , due to its low molecular weight .
the dot (  ) denotes a background signal arising from the cross - linked ha beads ( data not shown ) .
lux runs as high and low molecular weight isoforms , denoted by (  ) .
hypocotyl measurements were performed on evenly spaced seedlings grown under 12l:12d , measured on day 10 .
 the circadian clock is required for adaptive responses to daily and seasonal changes in environmental conditions1 - 3 . 
 light and the circadian clock interact to consolidate the phase of hypocotyl cell elongation to dawn under diurnal cycles in arabidopsis thaliana4 - 7 . here 
 we identify a protein complex ( evening complex ) composed of early flowering 3 ( elf3 ) , early flowering 4 ( elf4 ) and the transcription factor lux arrhythmo ( lux ) that directly regulates plant growth8 - 12 . 
 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 phytochrome - interacting factor 4 ( pif4 ) and pif5 under diurnal conditions4,6,13 . 
 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 critical function of the complex . 
 therefore , the evening complex underlies the molecular basis for circadian gating of hypocotyl growth in the early evening . 
Methods Summary
Supplementary Material