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Supporting Information Legends
Supplemental Figure 1. Protein composition in dry wild type and val1 seeds. Seed storage
proteins 2S albumins and subunits of 12S globulins are shown on an SDS-PAGE gel. Lanes:
1. Protein ladder; 2. Wild type; 3. val1 mutant.
Supplemental Figure 2. Levels and composition of fatty acid in wild type and val1 dry seeds.
Total levels and composition of fatty acids were similar in the wild type and val1 mutant.
Total fatty acid levels were 356 ± 32 and 348 ± 38 μg mg-1 DW in wild type and val1
embryos, respectively. The GC-FID analysis was performed on 1.000 mg (± 5%) of dry seeds
harvested from individual plants (n = 26 for wild type, n = 19 for val1).
Supplemental Figure 3. SALK_088606C does not contain a T-DNA insertion in another
locus. (A) PCR amplification of the region (At5g63350 gene) potentially containing the
insertion. The top portion of the gel shows results from amplification of the gene using only
primers flanking the T-DNA insertion site (F +R). The bottom part of the gel shows results
when the T-DNA-specific primer (I) was included (F+I+R). In both cases, only the expected
950-bp band was produced and the expected 820-bp band for F+I+R primer combination was
absent. WT indicates control wild type DNA sample and 1 – 23 are individual homozygous
val1 plants. (B) Expression of At5g63350 is not affected in the val1. Collectively, these
results suggest that the T-DNA is no longer present in the At5g63350 locus.
Supplemental Figure 4. Validation of VAL1 expression profiles by Taqman qPCR.
Expression profiles of full-length (At2g30470.1) and novel truncated (At2g30470.N2) VAL1
SVs are shown. The Taqman probes and primers targeted unique regions (5’-region upstream
of the T-DNA insertion) in these two SVs such that they could be distinguished. Both SVs
showed similar levels and expression patterns in developing wild type and val1 mutant
embryos, which is consistent with similar read densities at the 5’-region of the VAL1 gene in
both wild type and val1 mutant.
Supplemental Figure 5. Selected transcripts de-repressed in developing val1embryos. All
transcripts shown are known, not novel, transcripts.
Supplemental Figure 6. Expression profiles of the core LAFL TF transcripts in wild type
and val1 embryos. RNA-Seq and qPCR results are shown in parallel and were consistent
between these two methods. In cases where multiple SVs were detected (e.g., FUS3),
expression of only the known SV are shown. No statistically significant changes were
observed between the wild type and val1 mutant.
Supplemental Figure 7. Expression profiles of five selected transcripts in wild type and val1
embryos. RNA-Seq and qPCR results are shown in parallel and were comparable between
these two approaches. Some transcripts were completely de-repressed in the absence of
VAL1.
Supplemental Figure 8. Venn diagram depicting the overlap of transcripts associated with
the H3K27me3 mark and the individual members of the core LAFL TF network. (A)
ABI3/LEC2; (B) FUS3/LEC2; (C) FUS3/LEC1; (D) FUS3/ABI3. The majority of the
differentially expressed transcripts were de-repressed in the val1 mutant epigenetically
through the PHD-L domain.
Supplemental Figure 9. Principal component analysis (PCA) on the correlations of
metabolite, fatty acid, and hydrophobic protein levels in developing wild type and val1
embryos. Metabolites from embryos (8 – 17 DAP) harvested from 3 independent experiments
(A - C; 3 biological replicates each) were analyzed and the resulting differences among
samples visualized by PCA score plots. No major differences were observed between wild
type and val1 metabolomes in developing embryos. Changes in individual metabolomes were
only observed for the actual embryo development in general, as a developmental trend from
day 8 to 17 was apparent based on component 1. Based on component 2, embryo
metabolomes at days 8 and 17 were similar, but different from the rest of the time points.
Solid-colored and white-filled colored circles represent individual replicates for the wild type
and val1 mutant, respectively. The numbers represent days after pollination from the time
course and are color coded with their respective samples (circles).
Supplemental Figure 10. Changes in the levels of phytohormones in developing wild type
and val1 mutant embryos. Phytohormone levels were analyzed by UPLCMS/MS as described
in Methods. Overall, phytohormone levels were similar in developing wild type and mutant
embryos. OPDA, JA, and JA-Ile are biosynthetically related and showed similar profiles,
with the highest accumulation in 8-day-old embryos. Both ABA and IAA peaked at day 10.
Statistically significant differences between wild type and mutant are shown by an asterisk (p
< 0.05).
Supplemental Figure 11. SDS-PAGE analysis of seed storage proteins. Seed storage
proteins were extracted by biphasic extractions and solubilized either from the interphase
containing insoluble material or from the combined interphase and aqueous phase.
Solubilized proteins were separated by SDS-PAGE. Results for three biological replicates for
12-day-old embryos are shown. No differences were observed in terms of seed storage
protein abundances between these two treatments, suggesting that these proteins partitioned
in the interphase. This enabled us to perform all analyses (polar metabolites in aqueous phase,
fatty acids in organic phase, and seed storage proteins in interphase) on a single embryo
sample.
Supplemental Figure 12. RNA-Seq analysis workflow. Green boxes are outputs and blue
boxes are tools.
Supplemental Table 1. All detected differentially expressed transcripts in val1 developing
embryos.
Supplemental Table 2. Transcripts affected in both seedlings and embryos in the val1
mutant.
Supplemental Table 3. Direct targets of each member of the core LAFL TFs that were also
de-repressed among the set of 3,293 transcripts in the val1 mutant.
Supplemental Table 4. Transcripts that were de-repressed through epigenetic regulation in
val1.
Supplemental Table 5. Selected genes up-regulated at all time points in val1 embryos.
Supplemental Table 6. Transcripts containing the Sph/RY motif that were de-repressed in
developing val1 embryos.
Supplemental Table 7. Primers, probes, and TaqMan kits used for qPCR or genotyping.
Supplemental File 1. Transcripts.fa.gz - Sequences of all transcripts detected during embryo
development.