Supporting Information Methods S1, Figs S1–S5 and Tables S1–S4
Methods S1
Real-time RT-PCR
RNA extraction, cDNA synthesis and real-time RT-PCR experiment were
performed as described (Pan et al., 2011). RNA samples were extracted from various
tissues and organs from different developmental stages of P. aphrodite subsp.
formosana. Total RNA was treated with RNase-free DNase (NEB, Hertfordshire, UK)
to remove genomic DNA contamination. First-strand cDNA was synthesized by use
of SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) from total RNA.
Gene-specific primers for PeSEP1-4 were designed by use of Primer Express
(Applied Biosystems, Foster City, CA, USA) and are in Table S1. PeActin4 was used
as internal control. Data were analyzed by use of the Sequencing Detection System
v1.2.2 (Applied Biosystems). cDNAs for real-time RT-PCR analysis were from
vegetative and reproductive tissue, including two-leaf seedlings, leaf, root, floral stalk,
floral bud, and pedicel. Floral buds were divided into five developmental substages:
B1, 0.5-1.0 cm; B2, 1.0-1.5 cm; B3, 1.5-2.0 cm; B4, 2.0-2.5 cm; B5, 2.5-3.0 cm long.
Floral organs (sepals, lateral petals, lip, pollinaria and column) were collected.
Samples were examined after pollination: ovules [(56 days after pollination (DAP)],
embryos [(embryo 1, 80 (DAP) and embryo 2 (100 DAP)] and mature dry seeds (120
DAP).
Yeast two-hybrid and Spotting assay
Yeast two-hybrid analysis involved use of the MATCHMAKER II system
(Clontech, Palo Alto, CA). Because of autoactivation of the GAL4 reporter genes by
intrinsic transcription activation domains in PeSEP, we used truncated PeSEPΔC
proteins containing the MADS, I, and K regions (primers are in Table S1).
Transformed AH109 yeast cells were selected on medium lacking leucine and
tryptophane (SD-Leu/-Trp, Sigma-Aldrich, St. Louis, MO, USA). The yeast strains
were constructed by co-transformation of three constructs, including two pGADT7
constructs containing PeSEPΔC and PeMADS6 and one pGBKT7 construct
containing one AP3-like gene (PeMADS2-5). The protein–protein interactions of the
selected yeast strains were evaluated by spotting assay with serial dilutions as
mentioned above. All spotting assay sets involved at least three independent
experiments. The protein interaction strength was classified into five degrees on the
basis of the growth status and number of yeast colonies. Empty pGADT7 and
pGBKT7 vectors were negative controls. More than 5 individual transformed colonies
were chosen and streaked to another SD-Leu/-Trp plate. In all, 10 ml of the liquid cell
cultures were grown and harvested in mid-log phase at OD600=1.0 corresponding to
2×107 cells/ml. Ten-fold serial dilutions from 10-1 to 10-4 of each culture were spotted
on the control SD-Leu/-Trp growth plate and on the selected SD-Leu/-Trp/-His and
SD-Leu/-Trp/-His/-Ade plates.
Anthocyanin and chlorophyll extraction
Three sepals, two petals and one lip from each single flower at full blooming stage
were used for pigment extraction. Total anthocyanin content was quantified as
described (Hsieh et al., 2013). For the chlorophyll extraction, three sepals and two
petals of PeSEP-silenced Phalaenopsis flowers were ground with liquid N2. The
chlorophyll was extracted with 80％ acetone, filtered with use of Whatman filter
paper (No. 5, Whatman, UK) and collected in test tubes. Total chlorophyll content
was quantified by measuring absorbance at OD652 with a spectrophotometer and
calculated as described (Yoshida et al., 1971).
Cryo scanning electron microscopy (Cryo-SEM)
Sample preparation for Cryo-SEM was as described (Kumar et al., 2013). Fresh
flowers were dissected and loaded on a stub. Samples were frozen with liquid
nitrogen slush then transferred to a sample preparation chamber at -160℃. After 5
min, the temperature was raised to -85℃ and sublimed for 15 min. After being
coated with platinum at -130℃, samples were transferred to a cryo stage in an SEM
chamber and observed at < -160 ℃ by use of Cryo-SEM (FEI Quanta 200
SEM/Quorum Cryo System PP2000TR FEI, FEI Co., Hillsboro, OR) at 20 KV.
Ectopic expression of PeSEP1 and PeSEP3 in Arabidopsis
cDNA fragments containing the coding regions of PeSEP1 and PeSEP3 were
cloned into the pBI121 vector (primers are in Table S1). The constructs were then
introduced into Agrobacterium tumefaciens (strain GV3101). Wild-type Arabidopsis
thaliana ecotype Columbia plants were transformed by the floral dip method (Clough
& Bent, 1998). Transformation plants carrying each transgene expressed under the
control of the constitutive CaMV 35S promoter were selected on MS medium
supplemented with kanamycin and placed at 23℃ in a growth chamber under
long-day conditions (16-h light/8-h dark).
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Supporting Information figure legends
Fig. S1 Multiple alignment of regions in PeSEP genes used for VIGS experiment.
Red box indicates the regions for primer design to amplify cDNA fragments for
PeSEP2- and PeSEP3-silenced constructs.
Fig. S2 Multiple alignments of SEP-like MADS-box proteins. The DOMADS1 lacks
the 2 conserved motifs and DOMADS3, ZMM3 and OsM5 lack only the SEPⅡ
motif (arrows). PeSEP1-4 proteins identified are labeled with stars. The SEPⅠ and
SEPⅡ motifs are indicated with red boxes. Identical or similar residues are shaded
yellow or blue/green, respectively. Dashes indicate gaps.
Fig. S3 Overrepresented motifs discovered on the 2-kb promoters of PeSEP1-4 genes
using the MEME program. Motif locations are in the upper panel. Information about
the 10 predicted motifs is summarized in the lower table.
Fig. S4 Phenotypes of the PeSEP-silenced Phalaenopsis I-Hsin Sunrise Cinderella.
(a-i) complete and dissected mock-treated and PeSEP3- and PeSEP2-silenced flowers
from front and back views. (j-k) Flowers bearing severe and mild morphological
phenotypes and unopened floral buds in PeSEP3-silenced plants. (l) Chlorophyll
content in mock-treated and PeSEP3- and PeSEP2-silenced flowers. Scale bar=1 cm.
Fig. S5 Cell types of the PeSEP-silenced flowers in Phalaenopsis I-Hsin Sunrise
Cinderella. Fresh cross-sections of epidermal cells of mock-treated and PeSEP3- and
PeSEP2-silenced sepal (a-f), petal (g-l), lip (m-r), column (s-u), and leaf (v-w) in
adaxila and abaxial surfaces. Scale bar=50 μm.
Table S1 Primers used in this study.
Primer Name
Sequence
Used for cloning (3’RACE)
SEP_F1
5’-ATGGGRAGRGGRARRGTRGAGHTGAAGMGRAT-3’
SEP_F2
5’-SMAARGAKCTHGAKCARYTNGAGMGDCARYTDGA-3’
PeSEP_MADS
5’-ATGGGGAGAGGAAGGGTGGAGATGA-3’
Used for GenomeWalker
GenomeWalker_PeSEP1_GSP1_F 5’-GAGGTAACTTCCGGCTGGGAGTGGGAAGT-3’
GenomeWalker_PeSEP1_GSP2_F 5’-AAGACTGAGGGGGCCCTCCCTCCTCATT-3’
GenomeWalker_PeSEP1_GSP3_F 5’-GGGCCGCTACACAGCTCAAGT-3’
GenomeWalker_PeSEP2_GSP1_F 5’-CCTCCTCTTCGCAAACGTCACCTGCCTG-3’
GenomeWalker_PeSEP2_GSP2_F 5’-CTCCCTCACTCTTCTCTTTATCTCTCCCACGAA-3’
GenomeWalker_PeSEP3_GSP1_F 5’-GCATCGCAGAGGACGGAGAGCTCATAGG-3’
GenomeWalker_PeSEP3_GSP2_F 5’-GCATCGCAGAGGACGGAGAGCTCATAGG-3’
GenomeWalker_PeSEP4_GSP1_F 5’-GCATCACAGAGCACTGAAAGCTCATACGC-3’
GenomeWalker_PeSEP4_GSP2_F 5’-ATCGGAGGTGGGTTGATTGCGGGATTAG-3’
Used for RT-PCR analysis
PeSEP1_RT_F1
5’-GGGATATGATCAACGGCAGCC-3’
PeSEP1_RT_R1
5’-GCAACATGTATCGAAGTCTCGTATGATTA-3’
PeSEP2_RT_F1
5’-GATGCTCGATCAGCTCTGTGATCTC-3’
PeSEP2_RT_R1
5’-CATCCAACCCGAAATGAAGCC-3’
PeSEP3_RT_F1
5’-TACAAATTTCTGGCTTTCCCCC-3’
PeSEP3_RT_R1
5’-CGGACTAACGTATCTAACAACTG-3’
Actin_F
5’-GGCTAACAGAGAGAAGATGACC-3’
Actin_R
5’-AATAGACCCTCCAATCCAGAC-3’
Used for real-time RT-PCR analysis
PeSEP1_real time_F
5’-ACCTGGTTGGCTTGCATGAG-3’
PeSEP1_real time_R
5’-GAACAGACATGTCAACAATAAACACAA-3’
PeSEP2_real time_F
5’-TCATTTCGGGTTGGATGTGA-3’
PeSEP2_real time_R
5’-TTACGCAAAAAAGGGAAGTCTACA-3’
PeSEP3_real time_F
5’-TCTATTTGATTTCCTCAGGGTCATAA-3’
PeSEP3_real time_R
5’-TCTGAAAACAAAACCCGTCTTCTA-3’
PeSEP4_real time_F
5’-AAGGTTGACTATTGCATTTGTATTTTGTAG-3’
PeSEP4_real time_R
5’-TTCAGTGCCTGCTTATTTGTGTTC-3’
Actin4_F
5’-TTGTGAGCAACTGGGATGACAT-3’
Actin4_R
5’-GCCACGCGAAGTTCATTGT-3’
PeMADS2_real time_F
5’-AGGGAAACTTACCGCGCTCTA -3’
PeMADS2_real time_R
5’-GAGATGGGGGATCTATTCAGAGAGA -3’
PeMADS3_real time_F
5’-TCGCCTGATCTTTTATTATCTGCAT-3’
PeMADS3_real time_R
5’-TTGCAGTGCTAGACCCTACTTGTAA-3’
PeMADS4_real time_F
5’-TCACATATGCAGTCTTCATTTTATTGTT-3’
PeMADS4_real time_R
5’-GCAAGATAAGAGCAAGTTGCAGTAGTTA-3’
PeMADS5_real time_F
5’-AGCTCAATCTCAATGGCAAATCG -3’
PeMADS5_real time_R
5’-TTGGCTTTGATTGACCTGCAA -3’
PeMADS6_real time_F
5’-GGCTCACATACCTATTGCACC -3’
PeMADS6_real time_R
5’-TTCATATTGTAACTCTCCAAACTCATTAGT -3’
Used for Y2H
Y2H-EcoRI-PeSEP1ΔC_F
5’-GCGAATTCATGGGAAGAGGGAGAGTG-3’
Y2H-BamHI-PeSEP1ΔC_R
5’-GGATCCTCACTCCTCCAACCTTCTTTT-3’
Y2H-EcoRI-PeSEP2ΔC_F
5’-GCGAATTCATGGGGAGAGGAAGG-3’
Y2H-BamHI-PeSEP2ΔC_R
5’-GGATCCTCATTCTTGCAACTTCCTTCT-3’
Y2H-EcoRI-PeSEP3ΔC_F
5’-GAATTCATGGGAAGAGGGAGAGTGGAGC-3’
Y2H-BamHI-PeSEP4ΔC_F
5’-GGATCCGCATGGGAAGGGGAAG-3’
Y2H-BamHI-PeSEP4ΔC_R
5’-GGATCCTCATTCTTGCAACTTCATTCTCA-3’
Used for VIGS in Phalaenopsis
VIGS_PeSEP2_F
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGAACAAGATAAACAGGCAGG-3’
VIGS_PeSEP2_R
5’-GGGGACCACTTTGTACAAGAAAGCTAGCACCGATAGCTCATAAGCC-3’
VIGS_PeSEP3_F
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGAACAAGATTAACCGTCAAG-3’
VIGS_PeSEP3_R
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAGCTCATAGGCCTTCTTGAGAA-3’
Used for ectopic overexpression in Arabidopsis
PeSEP1_F
5’-CCCGGGATGGGAAGAGGGAGAGTG-3’
PeSEP1_R
5’-CCCGGGTACTCATGCAAGCCAACC-3’
PeSEP3_F
5’-TCTAGAGGAAGCAACCTCAGTTTTGGC-3’
PeSEP3_R
5’-TCTAGACGGACTAACGTATCTAACAACTG-3’
Table S2 Consensus CArG-box sequences used for patterning search by the fuzznuc
software.
Motif types
Consensus CArG-box sequences
M1
M2
M3
M4
M5
M6
M7
SRF-type
MEF-type
MEF-type
SEP3
Os-M1
Os-M2 (MADS58)
Os-M3 (MADS58)
CC(A/T)6GG
C(A/T)8G
CTA(A/T)4TAG
C(A/T)7GG
CC(A/T)6G
CC(A/T)8G
CC(A/T)7G
M8
M9
M10
Os-M4 (MADS3)
RIN-1
RIN-2
CC(A/T)4N2G
C(C/T)(A/T)6(A/G)G
C(C/T)(A/T)G(A/T)4(A/G)G
M11
M12
RIN-3
RIN-4
CC(A/T)6AG
CC(A/T)6A/GG
Table S3 Predicted CArG boxes on the promoters of PeSEP genes.
Promoter
pPeSEP1
pPeSEP3
pPeSEP2
pPeSEP4
CarG-box sequences (5’-3’)
CTATAAATAG, CTATTTATAG
CTAATAATTG, CAATTATTAG
CCATATTAG
CCATAATAG
CCTGTATTAG
CCATAATATG
(C)CTATAAATAG, CTATTTATAG
CTATTTTAGG, CCTAAAATAG
CCAATATGG, CCATATTGG
CCATATATG(G), (C)CATATATGG
CATTATTTGG, CCAAATAATG
CCAAATATG
CTAGATTAAG
CTATAAATAG, (C)TATTTATAG
CCAAAACTG
CTTTATATTG, CAATATAAAG
CCAAAAAAG
CCAAATCCG
(C)CATAAATTTG, CAAATTTATG
CAAATATATG, (C)CATATATTTG
CTTTTAATAG, CTATTAAAAG
CATATAAAAG, CTTTTATATG
CCTAATGTG
CCATAAGAG
CCATTTAAG
CCTTATTCG
CCAATAGAG
CTTGTTATGG
CCATTTTTG
CCTTATTGG, CCAATAAGG
CCTTTTGGG
CTTGAATTGG
CTTGATATGG
CAATTAAATG
CATTTAATTG
CCATTTTCG
CATATTTTTG, CAAAAATATG
CATAAATTAG, CTAATTTATG
CCATTTCTG
CATTTAAAGG, CCTTTAAATG
CATTTTAAGG, CCTTAAAATG
Motif types
M3
M2
M5
M5
M10
M7
M2, M3, M6, M9
M4, M7, M9, M12
M8
M1, M9, M12, M5, M8
M4, M7
M5, M8
M10
M2, M3, M9, M6
M8
M2
M5, M8
M8
M2, M6
M2, M6
M2, M9
M2
M8
M8
M5, M8
M8
M8
M10
M5, M8
M8
M8
M10
M10
M2
M2
M8
M2
M2
M8
M4, M7
M4, M7
Strands
(+) (-)
(+) (-)
(-)
(+)
(+)
(+)
(+) (-)
(+) (-)
(+) (-)
(+) (-)
(+) (-)
(+)
(+)
(+) (-)
(-)
(+) (-)
(-)
(-)
(+) (-)
(+) (-)
(+) (-)
(+) (-)
(-)
(+)
(+)
(+)
(+)
(-)
(-)
(+) (-)
(-)
(-)
(-)
(+)
(-)
(+)
(+) (-)
(+) (-)
(+)
(+) (-)
(+) (-)
Table S4 SEP-like MADS-box genes in Orchidaceae.
Species
Gene name
Dendrobium crumenatum
DcSEP1
Dendrobium grex Madame Thong-In DOMADS1
DOMADS3
Aranda deborah
OM1
Oncidium Gower Ramsey
OMADS6
OMADS11
a
PeSEP1
Phalaenopsis equestris
PeSEP2 a
a
PeSEP3
a
PeSEP4
a
Genes identified in this study.
Accession No. Length (nt)
DQ119842
732
AF198174
525
AF198176
663
X69107
753
HM140844
732
HM140847
747
KF673857
735
KF673858
728
KF6738589
753
KF6738560
741
Clade
SEP3
SEP3
SEP1/2
SEP3
SEP3
SEP1/2
SEP3
SEP1/2
SEP3
SEP1/2
Pan et al., Figure S1
Pan et al., Figure S2
Motif
width (bp) sites
E-vaule
conserved consensus sequence
C(Schranz & Mitchell-Olds; Zahn et al.)C[GA]AC(McGonigle et al.)A(Schranz & Mitchell-Olds)C[AGT]C[CT]G
Motif 1
15
13
3.4E-14
Motif 2
37
8
7.9E-05
G[CAG](Ohmori et al.)[CT]CC(Schranz & Mitchell-Olds)CTC(Schranz & Mitchell-Olds)[CGT]T[CT]T[TG]C(Jaillon et
al.)[TG][ACGT]TTTC[AC]C[CGT]TT[CT]T(McGonigle et al.; Schranz & Mitchell-Olds)CCT
Motif 3
14
18
5.0E-03
C[AG]A[GA]CC[CGA]AA(McGonigle et al.)A(Schranz & Mitchell-Olds)GA
Motif 4
21
6
1.4E+02
ACATTAC[TA]CTAC[AT]GCT[AG](Schranz & Mitchell-Olds)G[TA]A
Motif 5
41
6
1.6E+02
GCAAA(Schranz & Mitchell-Olds)TT[AG][TG]A[GC][AT]CA[AT][AT]T(Schranz & Mitchell-Olds)GA(Zahn et
al.)[CA][GA][TA]CT(Schranz & Mitchell-Olds)GAT[ACG]AT[AG][CT][GA]A[AC](McGonigle et al.; Schranz & Mitchell-Olds)
Motif 6
15
9
1.6E+02
[TA]TTT[TA]TT[CT]T[CT]AAA[AT]A
Motif 7
11
9
1.7E+04
T[GC][GC]GGC[ATG][AGT][TG]GA
Motif 8
21
12
3.1E+04
(Schranz & Mitchell-Olds)A[TA]G[TA](Zahn et al.)C(Schranz & Mitchell-Olds; Jaillon et
al.)[TAG][TA]T[TG]G[TA]C[TA][GTA][CA]C[TG]
Motif 9
7
2
1.1E+05
GGCGGAG
Motif 10
13
2
1.2E+05
GGAT[AG]GG(Zahn et al.)CCCAC
Pan et al., Figure S3
Pan et al., Figure S4
Pan et al., Figure S5