Phas Gus Phaster

PvALF

Tightly coiled

repressed phas

chromatin

Relaxing of phas

chromatin during

potentiation step

Activation of phas

gene expression during

activation step

Step 1 Step 2

(A) (B) (C)

PvALF

ABA

Supplemental Figure 1. Dynamic two-step system used to study potentiation and activation of the

phas promoter in transgenic Arabidopsis leaves. (A) In all non-seed tissues, the phas promoter is

repressed due to the presence of tightly coiled inhibitory chromatin. (B) Following estradiol-induced Pv-

ALF expression in leaves (step 1), the inhibitory chromatin in relaxed, potentiating the phas promoter for

expression. (C) In step 2, after the addition of ABA, the phas promoter is activated and the phas-gus

reporter is expressed.

Pv-ALF Pv-ALF

Supplemental Figure 2. Assessment of reproducibility between the two RNA-Seq biological replicates. (A) Density plot

of distributions of RPKM scores across each treatment and two replicates is shown. Correlation plots of the RPKM reads for

each replicate for 4MS (B) and 4A (C) treatments are shown. Coverage across transcripts was calculated by counting the

number of reads on aligned transcripts generated by TopHat. All correlation analysis and plots were performed using the R

statistical package. X and Y axes show the number of counts generated with the program HTSeq-count. Values of Pearson’s

correlation coefficient (r) are shown on each plot.

(B)

de

nsi

ty

0.1-

0.2-

0.3-

0.4-

0.5-

Log 10 (RPKM)

-5 0 5

(C)

(A)

Supplemental Figure 3: Analysis of overlapping genes that are down-regulated during the Pv-

ALF-mediated phas activation. Venn diagram of down-regulated genes specific to the potentiation

[4E] and activation [4E4A] steps of phas-gus expression is shown. The data consist of genes that

have ≥ 2-fold change in expression (see Supplemental Dataset 3 A,B for the list of genes specifically

down-regulated in 4E and 4E4A conditions).

4A 4E

4E4A

Supplemental Figure 4. Comparison of the number of genes identified in this study with genes detected

in related studies. Venn diagram was plotted using the Pv-ALF- and ABA-activated genes and genes induced

upon heterologous expression of maize VP1 in Arabidopsis abi3 background (Suzuki et al., 2003), Chip-on-chip

analysis of the Arabidopsis ABI3 regulon (Monke et al., 2012) and seed-specific genes activated only in seeds

(Le et al., 2010). See Supplemental Dataset 10 for genes overlapped with other related studies.

Chromatin 3%

Membrane associated

5%

Metabolism 12% Protein secreted/

processing 3%

Seed lipid 8%

Seed storage 17%

Signaling 5%

Stress 6%

Stress: Dessication

13%

Transcription 1%

Transport 7%

Uncharacterized 20%

Supplemental Figure 5. Functional classification of seed-specific Arabidopsis genes

expressed during activation of the phas promoter in leaves. The data consist of genes that

have ≥ 2-fold change and p (Corr) value of ≤ 0.05 from two independent RNA-Seq experiments.

(A)

Supplemental Figure 6. Expression profiles of genes involved in

ABA signaling pathway and transcription factors preferentially

expressed during Pv-ALF-mediated phas activation. Hierarchical

clustering heat map was performed using Euclidean distance method. (A)

Heat map of ABA biosynthetic pathway genes differentially expressed in

Pv-ALF/phas-gus heterologous system. (B) Heat map of TFs differentially

expressed during the two-step activation process. Color code indicates

degree of fold change.

(B)

4E 4A4E4A

4E 4A4E4A

4A 4E4A 4E

AP

M

YB

N

AC

W

RK

Y

Supplemental Figure 7. Co-expression analysis of transcription factors regulated by Pv-ALF

expression. (A) Relationship among ABI3 and 87 identified transcription factors up-regulated in the 4E and

4E4A treatments are visualized by co-expression analysis using ATTED-II database. The gene pocket circled

in red represents interactions of ABI3 with AIL5 (At5g57390) and extensin-like gene (At2g27380). (B) Table

showing the extent of gene coexpression network view with ABI3.

MR* Cor

* Locus Function

ABI3 3.6 0.92 At2g27380 ATEPR1

ABI3 16.4 0.78 At5g57390 AIL5 (AINTEGUMENTA-LIKE 5)

(A)

(B)

0

0.4

0.8

1.2

1.6

0

0.4

0.8

1.2

1.6(a) LEA

(b) Cupin

(c) Oleosin

Re

lati

ve

Ex

pre

ss

ion

(R

.E)

Supplemental Figure 8. RLT2 and AIL5 knock-down in Pv-ALF background reduces expression of seed-

specific genes in 4E4A-treated leaves. RT-PCR analysis of expression levels of LEA At3g17520 (a), Cupin

At4g36700 (b) and Oleosin At5g40420 (c) was performed with RNA isolated from 4E4A-treated leaves. Results

for three independent super-transformed lines carrying either amiRNA-RLT2 or amiRNA-AIL5 constructs, as well

as for untransformed control Pv-ALF plants and plants super-transformed with vector only (VC) are shown. Error

bars represent standard deviations.

0

0.4

0.8

1.2

1.6P

v-A

LF

lin

e

RL

T2-1

3

RL

T2-1

1

RL

T2-

2

AIL

5-1

AIL

5-2

AIL

5-5

VC

lin

e

Supplemental Figure 9. Seeds of RLT2 and AIL5 amiRNA knock-down plants do not show reduction in the

expression of endogenous Arabidopsis ABI3-related genes. Expression of FUS3 (a), ABI4 (b) and ABI5 (c)

genes was analyzed by quantitative RT-PCR using RNA isolated from fully developed seeds of three independent

super-transformed lines carrying either amiRNA-RLT2 or amiRNA-AIL5 constructs, as well as from untransformed

control Pv-ALF plants. Seeds were not treated with estradiol or exogenous ABA prior to analysis. Three biological

replicates were used to determine standard deviation values.

0

0.5

1

1.5(a) FUS3

0

0.5

1

1.5(b) ABI4

amiRNA-lines

0

0.5

1

1.5

Pv

-AL

F l

ine

RL

T2

-13

RL

T2

-11

RL

T2

-2

AIL

5-1

AIL

5-2

AIL

5-5

(c) ABI5

Supplemental Table 1. RNA-Seq sequence summary. Alignment of Illumina GAII 72-mer

reads to the Arabidopsis reference genome. The leaves were subjected to the four treatments as

described in Figure 1 legend.

*Number of genes was calculated by applying 1X median coverage filter.

Replicate 1

Total reads Mapped reads Un mapped reads

% coverage

No of genes passed filter

4MS 18,955,522 17,174,138 1,781,384 91 16610

4E 22,949,045 20,835,245 2,113,800 91 16673

4A 16,848,316 14,203,392 2,644,924 84 16423

4E4A 22,296,947 18,894,601 3,402,346 85 16332

Replicate 2

Total reads Mapped reads Un mapped reads

% coverage

No of genes passed filter

4MS 32,806,974 31,490,884 1,316,090 96 16507

4E 33,263,290 31,669,906 1,593,384 95 18175

4A 33,415,433 28,487,329 4,928,104 85 16453

4E4A 25,700,327 23,375,527 3,893,666 91 16399

Supplemental Table 2. Occurrence of specific binding sites enriched in the promoters of ABA-

independent (up in 4E only) and ABA-dependent (up in 4E4A only) genes upregulated upon Pv-

ALF induction. p-value from binomial distribution is provided in parentheses.

Upregulated genes

Sph/RY motif

ABRE motif

G-Box motif

RAV1-A motif

RAV1-B motif

up in 4E only

41 (35%)

(5.4 E-02)

26 (23%)

(2.7 E-02)

16 (19%)

(1.0E-01)

32 (28%)

(6.24E-02)

17 (30%)

(1.03E-01)

up in 4E4A only

90 (33%)

(4.77 E-02)

122 (44%)

(2.03E-18)

110 (40%)

(5.53E-24)

128 (47%)

(3.93E-03)

45 (17%)

( 3.83E-02)

Supplemental Table 3: Validation of RNA-seq data by qRT-PCR. RNA-Seq data and qRT-

PCR data are indicated as fold change in gene expression compared to 4MS control. The

statistical significance of qRT-PCR changes in gene expression was determined using Student’s

t-test (p-value < 0.05) and the RNA-Seq data was corrected for the false discovery rate using

Benjamini and Hochberg method. Pearson correlation coefficients (r) of 4E (r = 0.90) and 4E4A

(r = 0.94) were obtained by plotting RNA-Seq data on y-axis and qRT-PCR data on Y-axis. High

correlation value indicates a positive correlation between the two datasets. Primers used for this

study are listed in Supplemental Table 5.

GENE ID QPCR-4E

RNA-Seq4E

QPCR-4A

RNA-Seq4A

QPCR-4E4A RNA-Seq4E4A Other names

At3g13682 1.27 2.08 0.99 -1.21 0.89 -1.12 LDL2

At3g42670 2.38 1.98 1.51 -1.28 1.01 -1.36 CHROMATIN REMODELING 38

At5g44180 5.41 3.56 1.82 1.25 3.45 1.58 RLT2

At4g24190 2.48 3.18 -1.45 0.62 0.54 -2.16 ATHSP90-7, SHEPHERD

At1g32960 13.45 22.38 1.31 2.82 1.21 2.81 SBT3.3

At2g22795 4.79 2.32 0.82 -3.66 0.00 -1.92 unknown protein

At1g21240 19.56 18.06 1.81 3.092 2.66 5.89 WAK3

At1g21700 2.92 1.08 1.13 -1.72 0.83 -1.05 Stswi 3C

At4g02390 1.58 1.47 0.75 -1.17 0.59 -1.09 Parp

At5g40340 33.59 10.21 5.82 -1.47 0.24 -1.03 Tudor/PWWP/MBT superfamily protein

At1g15520 60.82 145.16 2.75 27.42 59.14 88.84 ABCG40

At3g17520 1.24 -1.95 174.54 47.02 1260.69 967.61 Late embryogenesis abundant protein (LEA) family

protein

At5g62490 1.04 -1.03 9.51 3.82 28.44 99.04 ATHVA22B,

At5g57390 1.12 1.43 2.05 3.40 2.68 4.51 AIL5

At4g36600 1.35 -1.04 24.57 15.99 3843.14 1616.41 LEA protein

At3g15670 2.30 -1.35 16.29 7.21 1879.51 495.25 LEA protein

At4g36700 0.87 -1.03 4.63 1.615 596.00 109.98 RmlC-like cupins superfamily protein

At1g24580 1.12 1.80 55.33 12.11 995.00 197.71 RING/U-box superfamily protein

At2g36270 1.49 1.41 10.41 6.88 159.00 63.01 ABA INSENSITIVE 5,

At5g40420 6.06 1.03 78.79 1.65 496.67 257.32 OLEOSIN 2

Supplemental Table 4. List of primers used for amiRNA cloning.

Name Sequence ( 5'-3') Brief description

Primer A CTG CAA GGC GAT TAA GTT GGG TAA C for all amiRNA cloning

Primer B GCG GAT AAC AAT TTC ACA CAG GAA ACA G for all amiRNA cloning

HBTF-I miR-s gaTTGATATTCACGAATAGGCGTtctctcttttgtattcc for all amiRNA-RLT2 cloning

HBTF-II miR-a gaACGCCTATTCGTGAATATCAAtcaaagagaatcaatga for all amiRNA-RLT2 cloning

HBTF-III miR*s gaACACCTATTCGTGTATATCATtcacaggtcgtgatatg for all amiRNA-RLT2 cloning

HBTF-IV miR*a gaATGATATACACGAATAGGTGTtctacatatatattcct for all amiRNA-RLT2 cloning

AIL5-I miR-s gaTGGAATGATTGTTATACCCATtctctcttttgtattcc for all amiRNA-AIL5 cloning

AIL5-II miR-a gaATGGGTATAACAATCATTCCAtcaaagagaatcaatga for all amiRNA-AIL5 cloning

AIL5-III miR*s gaATAGGTATAACAAACATTCCTtcacaggtcgtgatatg for all amiRNA-AIL5 cloning

AIL5-IV miR*a gaAGGAATGTTTGTTATACCTATtctacatatatattcct for all amiRNA-AIL5 cloning

Supplemental Table 5. List of primers used for semi-quantitative RT-PCR analysis.

Gene name Primer name Sequence

PvALF 700PvALF-rev 5'-CTTCACACACTGGAACCTTGCGATC-3'

PvALF Apa-3xHA-for 5'-CGGGCCCATGTACCCATACGATGTTC-3'

EF1a EF1-for 5'-TGCTGTCCTTATCATTGACTCCACCAC-3'

EF1a EF1-rev 5'-TTGGAGTACTTGGGGGTAGTGGCATC-3'

GUS 1Gus-for 5'-ATGGTCCGTCCTGTAGAAACCC-3'

GUS 515gus-rev 5'-CGATGGATTCCGGCATAGTTAAAGA-3'

Supplemental Table 6. List of primers used for q-RTPCR.

Name Sequence ( 5'-3') Brief description

LDL2-F TGACAGGTTGGGGTTTGGCCT AT3G13682, LDL

LDL2-R ACCAAGGCAGGGCCACCAGA AT3G13682 LDL2

CHR38-F AGCCGAGCAAAAGGCGCCTC At3g42670 CHR38

CHR38-R TGCAAGCGCTCATCGCCCAC At3g42670 CHR38

HBTF-F2 ACGGACCGGTTCTTGGAATGGA At5g44180 HBTFL1/RLT2

HBTF-R2 TGCGATGGCTTGGCATCTCTGC At5g44180 HBTFL1/RLT2

SHD-F AGCCGACGAGGAAATCGAGGCA AT4G24190 SHD (SHEPHERD);

SHD-R TGTTGCTCAACGGGTTCGGCTT AT4G24190 SHD (SHEPHERD);

SBT3.3-F GCACGTCGCCTCCATAGCGG AT1G32960 SBT3.3 serine-type endopeptidase

SBT3.3-R GCGAGCACGAGGTGCACCAC AT1G32960 SBT3.3 serine-type endopeptidase

2795-F AGGGATAATGGCGGTGGTACGG AT2G22795 hypothetical protein

2795-R ACCTCACTTTCCTCTGTCCCACTCT AT2G22795 hypothetical protein

WAK3-F TATGAGCTGCACGAGGCCCGA AT1G21240 WAK3 (wall associated kinase 3);

WAK3-R CGCAGCAAGCAGGAGGACCAA AT1G21240 WAK3 (wall associated kinase 3);

Stswi 3C-F GCTTCAGCAAAACGGCGGCG At1g21700 ATSWI3C

Stswi 3C-R TTACAGACGCGTGTGGCCGG At1g21700 ATSWI3C

Parp-F AGCTCCTCCTGAAGCGCCTG At4g02390 APP

Parp-R CTCGCAGAGGAGCAGAACGCC At4g02390 APP

PWWP-F GGCGTGTTTCGGAGACGGGA AT5G40340 PWWP domain-containing protein

PWWP-R ACCGCCTCTTCCACTGCACCT AT5G40340 PWWP domain-containing protein

ABCG40-F CGAGTGGACGGCGGTGAAGT AT1G15520 PDR12

ABCG40-R CGACGGAGGCGATGTGGTGG AT1G15520 PDR12

LEA_F CGAGGACGCAAAGGAGGCGG AT3G17520 LEA

LEA-R GTCTCGTATGCCGAACCCGCT AT3G17520 LEA

AVA22B-F ACCGGGCATGAACGGTGCTG AT5G62490 ATHVA22B ATHVA22B

AVA22B-R TGCCAGCGGTTGCTCCCAAG AT5G62490 ATHVA22B ATHVA22B

AIL5-F2 TCCGGTGGACACTCGTCGGA At5g57390 AIL5

AIL5-R2 TGGTTATACGCACCTTGTGACTCCT At5g57390 AIL5

pp2C-F GACCGCCTGCTCTGTAGCGC AT2G29380 protein phosphatase 2C

pp2C-R GCCTTGTCCGAAATCGCCGGA AT2G29380 protein phosphatase 2C

LEA600-F GGGCGAGCGGTGGACTATGC AT4G36600 LEA protein

LEA600-R CTTCCGGCTGCTTCGCTCCC AT4G36600 LEA protein

LEA670-F AACTGCCCAAGCGGCTCAGC AT3G15670 LEA protein, putative

LEA670-R CCGGCCTTGTCCTTGGCCTC AT3G15670 LEA protein, putative

cupin-F CTTGGCAGCTCCAACCGCGA AT4G36700 cupin family protein

cupin-R ACGACCGTAGGGGCTCTCGAA AT4G36700 cupin family protein

zinc f-F GGTGTGGAGTGTTGTGTGTGCCT AT1G24580 zinc finger protein

zinc f-R GGACCTGCAAAGAGGGCATGTGG AT1G24580 zinc finger protein

ABI5-F CAGCAGGCGCCACCAGTTCA AT2G36270 ABI5 ABI5 (ABA INSENSITIVE 5)

ABI5-R GCTGTCCACCCGCTCCAAACC AT2G36270 ABI5 ABI5 (ABA INSENSITIVE 5)

OLEO2-F GTCGCCCTATGAAGGCGGCC AT5G40420 OLEO2 (OLEOSIN 2)

OLEO2_R AGCGAACCGACGACAGGGACT AT5G40420 OLEO2 (OLEOSIN 2)

Name Sequence ( 5'-3') Gene ID

HBTF-F2 ACGGACCGGTTCTTGGAATGGA At5g44180

HBTF-R2 TGCGATGGCTTGGCATCTCTGC At5g44180

AIL5-F2 TCCGGTGGACACTCGTCGGA At5g57390

AIL5-R2 TGGTTATACGCACCTTGTGACTCCT At5g57390

LEA_F CGAGGACGCAAAGGAGGCGG At3g17520

LEA-R GTCTCGTATGCCGAACCCGCT At3g17520

OLEO2-F GTCGCCCTATGAAGGCGGCC At5g40420

OLEO2_R AGCGAACCGACGACAGGGACT At5g40420

Cupin_F CTTGGCAGCTCCAACCGCGA At4g36700

Cupin_R ACGACCGTAGGGGCTCTCGAA At4g36700

1319EF1a-

for GACATGAGGCAGACTGTTGCA EF1a

1381EF1a-

rev CCGGTTGGGTCCTTCTTGT EF1a

3HA-for GTACCCATACGATGTTCCTGACTA' PvALF

76HA-rev CATATGGATAGGATCCTGCATAGTCC PvALF

1417GUS-

for CAAAGCGGCGATTTGGAA GUS

1474GUS-

rev GCCAGGCCAGAAGTTCTTTTT' GUS

ABI3 -F GTGGTCGCTTCACCAACTTCTC C At3g24650

ABI3 -R AGCTTTAATCATGACCCTC CA At3g24650

FUS3 -F GGATGCCTAGACAGAGACGA At3g26790

FUS3 -R TTCGGGAGTATCATACGTCGGAGAG At3g26790

ABI4-F GGGCAGGAACAAGGAGGAAGTG At2g40220

ABI4-R TCTCCTCCAAAAGGCCAAATGGT At2g40220

ABI5-F 5'-CAGCAGGCGCCACCAGTTCA-3' At2g36270

ABI5-R 5'-GCTGTCCACCCGCTCCAAACC-3' At2g36270

CRC-F 5'-TTGACGTTCAGTTGGCTCAGCAG-3' At4g28520

CRC-R 5'-TGCAGATAGTCTCCTCAAGGCCG-3' At4g28520