Supporting Information Legends
Figures
Figure S1. Among mutants of the class II SDG, the efs mutant showed the highest
germination frequency. (A) Diagrams represent gene structures. Lines indicate genomic DNA;
Black boxes indicate exons of SDG26, EFS, SDG7, and SDG4 genes; Arrows indicate
transcription start sites; inverted triangles with vertical lines indicate T-DNA insertion sites;
and underlines indicate amplicons for quantification of mRNA levels. All mutants were
homozygous T-DNA insertion lines isolated from Salk collection: sdg26 (Salk_013895), efssk42 (Salk_026442), sdg7 (Salk_131218), and sdg4 (Salk_128444). efs-sk42 is also shown in
Figure 2 of the main text. (B) Expression levels of SDGs in T-DNA insertion lines. (C)
Germination frequencies of sdg mutants (SD, n=3 biological replicates).
Figure S2. The expression of PIF1, but not of PIF3, PIF4, and PIF5 was reduced in the efs
mutant. The expression levels of four PIF mRNAs were determined in imbibed phyBoff wild
type and efs mutant seeds and normalized with respect to that of PP2A (SD, n=3 biological
replicates).
Figure S3. An EFS transgene restores the expression of PIF1 target genes in the efs mutant.
(A) The expression of PIF1-activated direct target genes is restored in imbibed phyBoff
EFS/efs mutant seeds. The expression levels of PIF1 target gene mRNAs were normalized
with respect to that of PP2A, and the values relative to wild type are presented in the graph
(SD, n=3 biological replicates). (B) The expression of PIF1-repressed direct target genes is
restored in imbibed phyBoff EFS/efs mutant seeds (SD, n=3 biological replicates). (C) The
expression of PIF1 indirect target genes is restored in imbibed phyBoff EFS/efs mutant seeds
(SD, n=3 biological replicates).
Figure S4. Selective changes of gene expression in the efs mutant. (A) Reduced levels of
H3K36me3 and H3K36me2 on various loci in the efs mutant. ChIP assays were performed
using anti-H3K36me2, H3K36me3, and H3 antibodies in imbibed phyBoff wild type and efs
mutant seeds. The level of each modified H3 was normalized with respect to that of H3. The
ChIP value itself was calculated by normalization with respect to the level of input DNA.
Gene body fragments were amplified (SD, n=3 biological replicates). (B) Expression levels
of ACTIN2, four PIFs, and GA3ox2 mRNA (SD, n=3 biological replicates).
Figure S5. The efs mutant does not show increased cryptic transcription at the PIF1 locus. (A)
The genomic structure of the PIF1. The amplicons used to determine the levels of the 5’,
middle, and 3’ regions of the PIF1 mRNA are indicated as 5’ region, M region, and 3’ region,
respectively; the filled boxes indicate exons; the open boxes indicate UTRs; and the lines
indicate introns. (B) Expression levels of the 5’, M, and 3’ regions of the PIF1 mRNA (SD,
n=3 biological replicates).
Figure S6. Models. (A) EFS inhibits the cryptic transcription of gene X, which activates the
mRNA expression of PIF1. In the efs mutant, cryptic transcription suppresses gene X
expression, which decreases PIF1 mRNA expression in imbibed seeds. (B and C) EFS and
its binding complex (Y) recruit RNA polymerase II to the PIF1 promoter to activate PIF1
mRNA expression (B), or to the promoter of gene X to activate PIF1 mRNA expression (C).
In the efs mutant, RNA polymerase II shows less recruitment to the PIF1 gene or gene X
promoters, decreasing PIF1 mRNA expression in imbibed seeds.
Supporting Table
Table S1. Primers used in this study
Supporting Experimental procedures
Supporting Experimental Procedures