Step 1 Step 2 Phas Gus Phas ter ABA - The Plant Cell filePhas Gus Phas ter PvALF Tightly coiled...
Transcript of Step 1 Step 2 Phas Gus Phas ter ABA - The Plant Cell filePhas Gus Phas ter PvALF Tightly coiled...
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
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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
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RL
T2-
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AIL
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AIL
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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
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1.5(a) FUS3
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1.5(b) ABI4
amiRNA-lines
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Pv
-AL
F l
ine
RL
T2
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RL
T2
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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