SUPPLEMENTARY INFORMATION
Content:
1. Legends for supplementary figures S1 – S8
2. Supplementary tables S1 – S4
3. Descriptions for files Data S1 and S2
1. Legends for supplementary figures S1 – S8
Figure S1. Schematic representation of constructs used to express amiRNAs in an
inducible manner.
(a) Artificial miRNAs were placed under the control of the AlcA promoter (AlcApro),
which can be activated, after ethanol (EtOH) treatment, by the AlcR transcription
factor.
(b) Artificial miRNAs were placed under the control of the 6xOP promoter (6xOPpro),
which can be activated, after dexamethasone (DEX) treatment, by a fusion between
the hormone-binding domain of the rat glucocorticoid receptor (GR) and the chimeric
transcription factor LhG4.
(c) Artificial miRNAs were placed under the control of the LexA promoter (LexApro;
composed of eight LexA operator sequences), which can be activated, after 17estradiol treatment, by the chimeric XVE transcription factor.
35Spro: Cauliflower Mosaic Virus 35S promoter; 3’OCSter: terminator of octopine
synthase gene from Agrobacterium tumefaciens; rbcsE9ter: rbcS E9 poly(A) addition
sequence; rbcsS3Ater: rbcsS3A poly(A) addition sequence; 3’NOSter: terminator of
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nopaline synthase gene from Agrobacterium tumefaciens; NOSpro: promoter of
nopaline synthase gene from Agrobacterium tumefaciens; Hpt: hygromycin
phosphotransferase II coding sequence; G10-90pro: synthetic G10-90 promoter.
Figure S2. Kinetic of AP3 knockdown in 35Spro>>AP3-amiRNAGR-LhG4 plants.
35Spro>>AP3-amiRNAGR-LhG4 plants were treated once with a mock-solution or with
a solution containing 10 µM dexamethasone. At the indicated time-points, flowers up
to stage 10 were collected for analysis and AP3 mRNA levels in mock and
dexamethasone-treated samples (gray and black bars, respectively) were determined
by qRT-PCR. Error bars indicate standard errors calculated from data of three
independent experiments.
Figure S3. Genome-wide analysis of genes responsive to induction of an artifical
microRNA targeting AP3 using two different inducible promoter systems.
(a) The number of genes up- (black bars) and down-regulated (gray bars) in
microarray experiments with 35Spro>>AP3-amiRNAGR-LhG4 (left bar) and
35Spro>>AP3-amiRNAAlc (right bar) plants.
(b) Selected Gene Ontology terms identified as enriched (FDR<0.05) in the
microarray datasets stemming from the analysis of 35Spro>>AP3-amiRNAGR-LhG4
(gray bars) and 35Spro>>AP3-amiRNAAlc (black bars) plants. Stress-related GO
terms are shown on the left, and terms known to be associated with AP3 function on
the right.
Figure S4. Transcriptional response of selected genes after activation of the
OPpro/35Spro:GR-LhG4 promoter system.
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(a) Expression of selected genes (as indicated) in flowers of wild-type (white bars)
plants that were treated with a dexamethasone-containing solution relative to mocktreated control plants.
(b) Expression of selected genes (as indicated) in seedlings of wild-type (white bars)
and 35Spro>>AG-amiRNAGR-LhG4 (gray bars) plants that were treated with a
dexamethasone-containing solution relative to mock-treated control plants of the same
genotype.
Log2-transformed expression ratios derived from qRT-PCR assays of are shown.
Error bars indicate S.E.M. of three biological replicates.
Figure S5. Transcriptional response of selected genes in response to the expression of
a functional amiRNA.
(a) Relative levels of AlcR mRNA in seedlings of 35Spro>>AG-amiRNAAlc (white
bar) and AP1pro:AP1-GRFIS 35Spro>>CTRL-amiRNAAlc (black bar) plants.
(b) Levels of amiRNA precursors in dexamethasone-treated 35Spro>>AGamiRNAGR-LhG4 (white bar), ethanol-treated AP1pro:AP1-GRFIS 35Spro>>CTRLamiRNAAlc (light gray bar), ethanol-treated 35Spro>>AG-amiRNAAlc (dark gray bar),
relative to their mock-treated controls.
Expression ratios derived from qRT-PCR assays of are shown. Error bars represent
S.E.M. of three biological replicates.
Figure S6. Schematic representation of constructs used to generate the different floral
induction systems.
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(a) The AP1 coding region was translationally fused to the ligand-binding domain of
the rat glucocorticoid receptor (GR). This translational fusion was placed under the
control of the Cauliflower Mosaic Virus 35S promoter.
(b) The entire AP1 locus, apart from the 3’UTR, was PCR-amplified and the final
exon was translationally fused to the ligand-binding domain of the rat glucocorticoid
receptor.
(c) The AP1 coding region was placed downstream of the AlcA promoter while the
AlcR gene was constitutively expressed from the 35S promoter.
(d) The entire AP1 locus, apart from the 3’UTR, was PCR-amplified and the final
exon was translationally fused to the ligand-binding domain of the mouse androgen
receptor (AR).
(e) The AP1 coding region was placed downstream of the LexA promoter while the
XVE gene was constitutively expressed from the synthetic G10-90 promoter.
35Spro: Cauliflower Mosaic Virus 35S promoter; 3’OCSter: terminator of octopine
synthase gene from Agrobacterium tumefaciens; rbcsE9ter: rbcS E9 poly(A) addition
sequence; rbcsS3Ater: rbcsS3A poly(A) addition sequence; 3’NOSter: terminator of
nopaline synthase gene from Agrobacterium tumefaciens; NOSpro: promoter of
nopaline synthase gene from Agrobacterium tumefaciens; Hpt: hygromycin
phosphotransferase II coding sequence; G10-90pro: synthetic G10-90 promoter.
Figure S7. Characterization of glucorcorticoid-based floral induction systems.
(a) Inflorescence of a 35Spro:AP1-GRFIS ag-1 plant ~14 d after treatment with a
dexamethasone-containing solution. Petals are narrow and greenish.
(b) Inflorescence of a AP1pro:AP1-GRFIS ag-1 plant ~14 d after treatment with a
dexamethasone-containing solution. Petals are broader than in (b) and white.
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(c) Petals from wild-type plants (left) and from AP1pro:AP1-GRFIS (middle) and
35Spro:AP1-GRFIS (right) lines after treatment with a dexamethasone-containing
solution. Scale bar: 1 mm.
(d-f) A 35Spro:AP1-GRFIS floral induction system containing a cassette that confers
resistance to kanamycin.
(d) An untreated 35Spro:AP1-GRFIS inflorescence-like meristem.
(e) A 35Spro:AP1-GRFIS inflorescence-like meristem 5 d after treatment with a
dexamethasone-containing solution.
(f) A mature flower formed ~14 d after treatment with a dexamethasone-containing
solution.
(g-i) A glufosinate-resistant AP1pro:AP1-GRFIS floral induction system line with an
alternative genomic insertion site (see Table S4).
(g) An untreated AP1pro:AP1-GRFIS inflorescence-like meristem.
(h) An AP1pro:AP1-GRFIS inflorescence-like meristem 5 d after treatment with a
dexamethasone-containing solution.
(i) A mature flower formed ~14 d after dexamethasone treatment.
(j-l) An AP1pro:AP1-GRFIS floral induction system containing a cassette that confers
resistance to kanamycin.
(j) An untreated AP1pro:AP1-GRFIS inflorescence-like meristem.
(k) An AP1pro:AP1-GRFIS inflorescence-like meristem 5 d after treatment with a
dexamethasone-containing solution.
(l) A mature flower formed ~14 d after dexamethasone treatment.
Figure S8. Expression of AP1 in an estradiol-dependent manner using the
LexApro/XVE system.
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Two independent second generation Lexpro:AP1/G10-90pro:XVE lines were
germinated and grown on 0.5 x MS agar for seven days. Subsequently, seedlings were
tranferred to 0.5 x MS supplemented with (+ Est) or without (- Est) 5 M 17estradiol for 24 h. Samples were processed and RT-PCR was performed using AP1specific and control (REF1) primers (see Experimental Procedures). Complementary
DNA generated from mRNA of 8-day old wild-type (L-er) seedlings and no template
control (NTC) samples were used as controls, respectively. AP1-specific signals first
appeared after 25 cycles of PCR in the + Est samples only.
2. Supplementary tables S1 – S4
Table S1. Primer sequences used for generating constructs.
The oligonucleotide names and sequences used for PCR amplification of indicated
sequences and subsequent cloning. If a restriction site was incorporated, the
corresponding sequence is underlined.
Oligo ID
Sequence (5`->3`)
Application
DM-177
ATCTCGAGCGTGGTGGTTAG
AP1 genomic
DM-180
ACATTCTAGATGCGGCGAAGCAGC
AP1 genomic
DM-658
CAGAAGCTTCCCATCCCAGAAGATGACTGTAT
CAC
mouse AR ligand
binding domain
DM-659
GCCTCTAGATTCACTGTGTGTGGAAATAGATG
mouse AR ligand
binding domain
AR-33
AAAACTCGAGGATCAAAAATGGGAAGGGGTA
GG
AP1 CDS
AR-34
AAATACTAGTTCATGCGGCGAAGCAGC
AP1 CDS
SW481
gaGATACACGTCCGCAAGTGCAAtctctcttttgtattcc
CONTROL-amiRNA
SW482
gaTTGCACTTGCGGACGTGTATCtcaaagagaatcaatga
CONTROL-amiRNA
SW483
gaTTACACTTGCGGAGGTGTATCtcacaggtcgtgatatg
CONTROL-amiRNA
SW484
gaGATACACCTCCGCAAGTGTAAtctacatatatattcct
CONTROL-amiRNA
FW-100
GGAGCTCCCAGAAGCTCGAAAAACAAAGAAA
AAAATC
GR in situ probe
DM-183
CTTACTTGGCTTTGGCCGCC
GR in situ probe
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Table S2. Primer sequences used for TAIL-PCR.
The oligonucleotide names and sequences used for genotyping and TAIL-PCR are
indicated.
Oligo ID
Sequence (5`->3`)
Application
DM-400
TCCGACAAGATCCTTTGAGCAC
pAP1::AP1-5-GR
DM-401
ATAAATATGGATTGCACGACGAGTC
pAP1::AP1-5-GR
DM-456
GCAGCTTCTTCTCTGTCGC
pAP1::AP1-3-GR
DM-457
AGCTCGAACATGAGATGGAAGAG
pAP1::AP1-3-GR
DM-359
NTCGASTWTSGWGTT
TAIL AD1
DM-360
NGTCGASWGANAWGAA
TAIL AD2
DM-361
WGTGNAGWANCANAGA
TAIL AD3
DM-492
GCGACATCTATGATAGAGCGCCAC
T-DNA Left Border primer 1
DM-493
GGCAGAACCGGTCAAACCTAAAAG
T-DNA Left Border primer 2
DM-494
AGTACATTAAAAACGTCCGCAATGTG
T-DNA Left Border primer 3
DM-5
TAAGTGGAGAGTGACTTGGTA
OPpro:AP3-amiRNA/35Spro:GR-LhG4
DM-6
GTAGCATCTTCAACCAAGACA
OPpro:AP3-amiRNA/35Spro:GR-LhG4
Table S3. Primer sequences used for RT-PCR.
The oligonucleotide name and sequences used for RT-PCR are shown along with the
gene aliases and identifiers.
Oligo ID
Sequence (5`->3`)
Target
DM-250
CTGTTGAGGAAACTTGCGTGAGG
REF1 (At1g13320)
DM-251
CTGAAAGTCGCTTAGCCAGAGGAG
REF1 (At1g13320)
FW-107
CTCTAGAGATCAAAAATGGGAAGGGGTAGGGTTC
AP1 (At1g69120)
FW-108
CGGATCCTGCGGCGAAGCAGCCAAG
AP1 (At1g69120)
Table S4. Primer sequences used for qRT-PCR.
The oligonucleotide name and sequences used for qRT-PCR are shown along with the
gene aliases and identifiers.
Oligo ID
Sequence (5`->3`)
Target
DM-64
CTACAGATGGGCGTAGAGTGTGTGA
At4g22212
DM-65
GCTTTCCATTTCAGCACTGCTCTCT
At4g22212
DM-66
TGGAGCTTCCGCGCAACA
DM-67
AAACAACAACAAGTACGATGAGTACGA
KIN2 (At2g02800)
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KIN2 (At2g02800)
HB1 (At2g16060)
DM-70
GGAGCCAGCCATTCTAAATACGGT
DM-71
GTGACCACATCTCCGGCACTG
DM-80
GCTCTCCAAAGGAAGACTGGGT
DM-81
AGAAGAGGGTTCTTGGTCAAAGTTGAT
NAC1 (At1g56010)
SW-679
TTTTTCTTGGAACCACCATCGG
WAK2 (At1g21270)
SW-680
AGCTCTGTGTTCTTCCGGTGC
WAK2 (At1g21270)
SW-681
GTAAGCAGAGTGTTGTGAAGC
ERF15 (At2g31230)
SW-682
caattacaagaTCAACATGAGCTC
ERF15 (At2g31230)
SW-683
gctttcagcgtgtagtatgttg
BG2 (At3g57260)
SW-684
tccttatttatgcttgcagctc
BG2 (At3g57260)
SW-685
GGTGACCAGTTACAGGCATTAAG
DMR6 (At5g24530)
SW-686
ACAGAAACGATGCGACCGATAG
DMR6 (At5g24530)
DM-534
ACCAGATTCTTCGTGCAAAGATAGCTG
AG (At4g18960)
DM-535
AAGCTGCTCGTAGTTAGATCCTCCTG
AG (At4g18960)
HB1 (At2g16060)
NAC1 (At1g56010)
AP1 (At1g69120)
BT-33
CATGCTCTCTCATCAGCCATCTCC
BT-34
GGTTCAAGAGTCAGTTCGAGATCATTCC
DM-439
ACTAGTCCATAGTGAGGATCTTCAGCTC
DM-440
TCTCTTGGGAATCAGATCGAGACCAC
AP3 (At3g54340)
DM-442
AAATCAAAGGGATTCAGCAAGCCACTG
GR
DM-443
TCCAGCAGTGACACCAAGGTAGG
GR
DM-454
GTGGACCGTCGTTCTTATTCACTCG
AlcR
DM-455
ACCTCTCTCTAAATCCTTCGCAACCAG
AlcR
DM-544
AAACACACGCTCGGACGCATATTAC
amiRNA precursor
DM-545
GGCGACTCGGTATTTGGATGAATGAG
amiRNA precursor
DM-242
AAGCGGTTGTGGAGAACATGATACG
REF1 (At1g13320)
DM-243
TGGAGAGCTTGATTTGCGAAATACCG
REF1 (At1g13320)
AP1 (At1g69120)
AP3 (At3g54340)
3. Descriptions for files Data S1 and S2
Data S1. Differential expression of genes in the microarray experiments. The
expression of genes in response to activation of the AP3-amiRNA or AG-amiRNA
from both the AlcApro/AlcR and OPpro/GR-LhG4 systems are shown. Log2transformed fold-change values, p-values and the average of the expression values of
the probes from the untreated samples are shown.
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Data S2. GO terms identified as significantly enriched among differentially expressed
genes from the microarray experiments. The number of genes included in a GO
category is represented for both the genes annotated on the microarray (noted as
“Genomic”) and the genes identified as differentially expressed in the microarray
experiments. P-value and false discovery rate (up to FDR < 0.05) are indicated.
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