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Figure S1. Sequence conservation within the ELF4 family. Multiple alignment of partial and fulllength ELF4 sequences (with GenBank numbering). Identity of sequences as in Figure 1, see also
Methods. The position of the α-helical folds is indicated by α1 and α2. Lower case letters refer to highly
conserved residues (consensus). Predicted proteins obtained by BLAST in genome databases (with EST
clone number): Cr, 280056 (Mittag et al., 2005); Nb, CN741587; Nt, TC11698; Pg, CK43569; Pp,
997049901; Ptr, VTK45573.b2 and YFS280019.b1. Species codes as follows. Am, Antirrhinum majus;
Bv, Beta vulgaris; Cr, Chlamydomonas rheinhardtii; Cs, Citrus sinensis; Ga, Gossypium arboreum; Gm,
Glycine max; Ha, Helianthus annuus; Ht, Hedyotis terminalis; Hv, Hordeum vulgare; In, Ipomoea nil; Le,
Solanum lycopersicon; Ls, Lactuca serriola; Mc, Mesembryanthemum crystallinum; Mt, Medicago
truncatula; Nb, Nicotiana benthamiana; Nt, N. tabacum; Os, Oryza sativa; Pg, Picea glauca; Pp,
Physcomitrella patens; Pt, Pinus taeda; Ptr, Populus trichocarpa; Sb, Sorghum bicolor; So, Saccharum
officinarum; St, Solanum tuberosum; Zm, Zea mays.
Figure S2. ELF4 and EFL structure. Structural models of ELF4 and EFL sequences. (A) ELF4 and EFL
models have similar α-helical folds predicted at their central regions. The colors from blue to red represent
directionality from the amino- to the carboxy-terminus. Identity of sequences as in Figure 1. (B) The
structural model of ELF4. Side view (left) and top view (right) of the refined ELF4 monomer. (C)
Structural map of elf4 encoded residue changes. See also Table 1. The encoded amino-acid residues
affected in the elf4 TILLING alleles are labeled along one monomer of the dimer. This ELF4 model
corresponds to the structural prediction of ELF4 in Figure S2B. (D) Movie of the ELF4 dimer. Color of
residues as in Figure 8C. (E) PDB file of the ELF4 dimer.
Figure S3. EFL expression. (A) DIURNAL EFL and ELF4 data. Circadian EFL and ELF4 expression in
Col-0 after entrainment for 9 days under diurnal conditions (light-dark and 22-12˚C) and release into
constant light and temperature (22˚C) (http://diurnal.cgrb.oregonstate.edu). EFL2 (Affy Id 259863_at),
EFL3 (Affy Id 263772_at), EFL4 (Affy Id 261039_at) and ELF4 (Affy Id 267364_at). The expression of
EFL3 is not circadian. (B) EFL1 expression. Real-time qPCR of EFL1 mRNA expression in Col-0 under
LL after 12L:12D entrainment.
Figure S4. An allelic elf4 series derived from TILLING. Sequence map of elf4 TILLING mutations.
Schematic positions of the TILLING promoter, missense and nonsense (*) mutations are indicated along
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the ELF4 gene. The nature of each missense mutation is shown below the allele name. ELF4 has a single
ORF of 333 bp that is depicted as a box. The gray band indicates the putative nuclear localization signal
within the encoded polypeptide. The position of the most downstream evening element (EE) in the ELF4
promoter is shown. The remaining two EEs in the promoter are located at positions -600 and -315. The
approximate sites of the T-DNA insertions, in elf4-101 and elf4-102 (Khanna et al., 2003), and the
deletion in elf4-1 (Doyle et al., 2002) are shown for comparison.
Figure S5. Allelic strength of elf4 alleles. The elf4 TILLING mutants (Col-0) were crossed to elf4-1
(Ws) harboring either CCA1:LUC or CCR2:LUC. The seedlings were monitored for bioluminescence
under LL, after 12L:12D entrainment, and period estimates were calculated for the rhythmic plants. The
R.A.E.-weighted mean of the period length was scored using the student’s t-test, as indicated with *, **,
and *** for P<0.05, P<0.01, and P<0.001, respectively. (A) Attenuated and short period of CCA1:LUC
expression in elf4-207  elf4-1 seedlings. Average luminescence of CCA1:LUC in elf4-207  elf4-1
(26.8±0.4h***; n=16) compared to Col-0  elf4-1 (29.0±1.6h; n=15). (B) Short period of CCA1:LUC
expression in elf4-203  elf4-1 seedlings. Average luminescence of CCA1:LUC in elf4-203  elf4-1
(27.9±1.6h*; n=21) compared to Col-0  elf4-1 (29.3±1.7h; n=13). (C) Short period of CCA1:LUC
expression in elf4-212  elf4-1 seedlings. Average luminescence of CCA1:LUC in elf4-212  elf4-1
(27.2±1.3h*; n=19) compared to Col-0  elf4-1 (29.4±1.7h; n=11). (D) Short period of CCR2:LUC
expression in elf4-207  elf4-1 seedlings. Normalized luminescence of CCR2:LUC in elf4-207  elf4-1
(27.5±2.9h***†; n=21) compared to Col-0  elf4-1 (27.5±1.2h; n=21). †: The R.A.E. values of the period
estimates were significantly more variable for the elf4-207  elf4-1 population than for Col-0  elf4-1
(precision phenotype). (E) Short period of CCR2:LUC expression in elf4-203  elf4-1 seedlings.
Normalized luminescence of CCR2:LUC in elf4-203  elf4-1 (26.6±1.5h*; n=19) compared to Col-0 
elf4-1 (27.9±1.0h; n=23). (F) Short period of CCR2:LUC expression in elf4-212  elf4-1 seedlings.
Normalized luminescence of CCR2:LUC in elf4-212  elf4-1 (26.4±1.1h*; n=24) compared to Col-0 
elf4-1 (27.7±1.0h; n=23).
Figure S6. elf4-204, elf4-201 and elf4-202. Clock transcript accumulation in elf4-204 (green), elf4-201
(violet), elf4-202 (brown), and Col-0 (black) under the third day under LL (lower panel), or the third day
in DD (upper panel). Normalized data and Col-0 data as in Figure 4. (A) GI. elf4-204 average: LL, 0.46,
DD, 0.64, P=0.31. (B) TOC1. elf4-204: LL, 0.54, DD, 0.71, P=0.15. elf4-201 DD average: elf4-201, 0.95,
Col-0, 0.50, P=9.7E-06. elf4-202 DD average: elf4-202, 0.83, Col-0, 0.50, P=0.03. (C) LUX. elf4-204
average: LL, 1.17, DD, 1.09, P=0.66.
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Figure S7. PRR9 and PRR7 expression in medium and weak elf4 alleles. PRR9 and PRR7 transcript
accumulation in elf4-204 (dark green), elf4-201 (violet), elf4-202 (brown), elf4-208 (blue), elf4-205 (light
green), elf4-211 (lavender), elf4-213 (red), and Col-0 (black) under the third day under LL (left panel), or
the third day in DD (right panel). Normalized data and Col-0 data as in Figure 4. (A) The mean PRR9
expression level is similar in DD and under LL for the elf4 alleles (de-repressed in DD). Mean expression
values as follows. elf4-204: LL, 0.71; DD, 0.44; P=0.30. elf4-201: LL, 0.60; DD, 0.73; P=0.44. elf4-202:
LL, 0.29; DD, 0.55; P=0.10. elf4-208: LL, 0.53; DD, 0.27; P=0.10. elf4-205: LL, 0.51; DD, 0.38; P=0.41.
elf4-211: LL, 0.36; DD, 0.26; P=0.35. elf4-213: LL, 0.78; DD, 0.34; P=0.06. Col-0: LL, 0.60; DD, 0.18;
P=0.03. (B) The mean level of PRR7 expression is elevated in DD compared to under LL in weak
hypomorphic elf4 alleles (compare position of arrowheads between LL and DD). Mean expression values
as follows. elf4-204: LL, 0.40; DD, 1.48; P=0.0002. elf4-201: LL, 0.62; DD, 1.56; P=0.0007. elf4-202:
LL, 0.58; DD, 1.22; P=0.02. elf4-208: LL, 0.60; DD, 1.10; P=0.03. elf4-205: LL, 0.31; DD, 1.30;
P=0.00003. elf4-211: LL, 0.30; DD, 0.61; P=0.03. elf4-213: LL, 0.57; DD, 0.97; P=0.04. Col-0: LL, 0.82;
DD, 0.95; P=0.60.
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