floral organ identity determination in nigella damascena … · 2016-01-06 · floral organ...

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188

NATURE PLANTS | www.nature.com/natureplants 1

Floral organ identity determination in Nigella damascena

(Ranunculaceae), a species with spiral flowers

Peipei Wang1,2,*, Hong Liao1,2,*, Wengen Zhang1,2,*, Xianxian Yu1,2, Rui Zhang1,

Hongyan Shan1, Xiaoshan Duan1,2, Xu Yao1,2, Hongzhi Kong1

1State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany,

Chinese Academy of Sciences, Beijing 100093, China 2University of Chinese Academy of Sciences, Beijing 100049, China *These authors contributed equally to this work

Author for correspondence:

Hongzhi Kong

Tel: +86 010 62836489 Email: [email protected]

This PDF file includes:

Supplementary Methods Supplementary Tables 1 to 10 Supplementary Figures 1 to 12

Flexibility in the structure of spiral flowers and its underlying mechanisms

http://dx.doi.org/10.1038/nplants.2015.188

2 NATURE PLANTS | www.nature.com/natureplants

SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188

Supplementary Methods

Scanning electronic microscopy. Floral buds were fixed in fresh FAA. Images were

captured using a Hitachi S-4800 scanning electron microscope. Yeast two-hybrid (Y2H). The GAL4-based MATCHMAKER Two-Hybrid System

(Clontech) was employed to determine the interactions among proteins encoded by

the floral MADS-box genes. For each gene, a full-length coding sequence was

amplified from the floral cDNA using gene-specific primers in which the appropriate

restriction recognition sites was introduced (Supplementary Table 10). The PCR

product was digested with the corresponding restriction enzymes and ligated into

the GAL4 activation domain (AD) expression vector pGADT7 and the GAL4-binding

domain (BD) expression vector pGBKT7 using the T4 DNA ligase (Promega). The

capability of auto-activation was tested by co-transforming the gene-specific

recombinant pGBKT7 vector and the pGADT7 empty vector into yeast

(Saccharomyces cerevisiae strain AH109) competent cells through the Small-Scale

LiAc Yeast Transformation Procedure, following the manufacturer’s instructions. For

genes capable of auto-activation, truncated sequences lacking the C-terminal regions

were introduced into the pGBKT7 vector. For testing protein-protein interactions, the

corresponding pGADT7 and pGBKT7 plasmids were introduced into the yeast

competent cells. Transformants carrying both AD and BD recombinant plasmids were

confirmed by PCR and their growth on the selective synthetic dropout (SD) medium

lacking leucine and tryptophan (SD/–Leu/–Trp) at 22°C for 7 days. Positive

transformants were diluted serially and incubated on selective SD medium lacking

leucine, tryptophan, histidine and adenine but containing 5 mM

3-amino-1,2,4-trizole (SD/–Leu/–Trp/–His/–Ade/ +5 mM 3-AT) at 22°C for 7 days for

His and Ade reporter gene expression test, and LacZ reporter gene expression

induced by X-Gal was assessed through the colony-lift filter method. Growth was

confirmed up to 14 days at 22°C. For each combination, at least three technical

replicates were performed. Transformants carrying the empty pGADT7 and/or

pGBKT7 vectors were used as negative controls.




Supplementary Table 1. Genes and accession number information

Gene lineage Gene number Gene name Accession number

AP1 2 NdFRUITFULL1 (NdFL1)

NdFRUITFULL2 (NdFL2)

AP3 3

NdAPETALA3-1 (NdAP3-1)



PI 2 NdPISTILLATA1 (NdPI1)

NdPISTILLATA2 (NdPI2)

AG 2 NdAGAMOUS1 (NdAG1)

NdAGAMOUS2 (NdAG2)

SEP 3

NdSEPALATA1 (NdSEP1)



AGL6 1 NdAGAMOUS-LIKE6 (NdAGL6)

Total 13




Supplementary Table 2. Primers used for amplification of cDNA

Primer name Primer (5’-3’) cDNA synthesis

polyT CCGGATCCTCTAGAGCGGCCGC(T)17

Nested primers

FL_F1 GCTCATGARATNTCNRTKCTTTG

FL_F2 CTKTGYGAYGCTGAWGTTGCT

B_F1 AACAGGCAGGTSACCTAYTC

B_F2 TYACTGTTCTMTGTGATGCT

AG1_F1 CARGTSACCTTYTGCAARCG

AG1_F2 TCYGTKCTYTGTGATGCWG

AG2_F1 TKCBAARAGAAGAAATGG

AG2_F2 THTGYGATGCTGARGTTGC

SEP_F1 CTTTGTGATGCTGAAGTTG

SEP_F2 GAAAACAAGATAAATCGWCAAG

AGL6_F1 GAGAACAAGATYAAYMGDCAAG

AGL6_F2 TGYGATGCYGARGTTGSTCT

Universal reverse primer

AP CCGGATCCTCTACAGCGGCCGC

ANS-specific primers

ANS_F CTTGTSAACCATGGMATCC

ANS_R CCAATGTGCAYGATKATWGAG




Supplementary Table 3. Information on genes used in this study

Taxon and species Gene name Accession number Source

Brassicaceae

Arabidopsis thaliana AP1 At1g69120 TAIRa

CAL At1g26310 TAIR

FUL At5g60910 TAIR

AGL79 At3g30260 TAIR

AP3 At3g54340 TAIR

PI At5g20240 TAIR

AG At4g18960 TAIR

SHP1 At3g58780 TAIR

SHP2 At2g42830 TAIR

STK At4g09960 TAIR

SEP1 At5g15800 TAIR

SEP2 At3g02310 TAIR

SEP3 At1g24260 TAIR

SEP4 At2g03710 TAIR

AGL6 AT2G45650 TAIR

AGL13 At3g61120 TAIR

Ranunculaceae

Aquilegia coerulea AqcoFL1A JX680252.1 ref. 1b

AqcoFL1B JX680253.1 ref. 1

AqcoFL2 Aquca_006_00408.1 Phytozome v10.2.1c

AqcoAP3-1 Aquca_006_00074.1 Phytozome v10.2.1



AqcoAP3-3b HQ694798 ref. 2

AqcoPI Aquca_014_00308.1 Phytozome v10.2.1

AqcoAG1 Aquca_136_00009.1 Phytozome v10.2.1

AqcoAG1-2 Aquca_136_00010.1 Phytozome v10.2.1

AqcoAG2 Aquca_022_00039.1 Phytozome v10.2.1

AqcoSEP1 Aquca_006_00411.2 Phytozome v10.2.1

AqcoSEP2 Aquca_002_00916.1 Phytozome v10.2.1

AqcoAGL6 Aquca_013_00477.1 Phytozome v10.2.1

Aquilegia vulgaris AqvuAP3-1 EF489478 ref. 3

AqvuAP3-2 EF489477 ref. 3

AqvuAP3-3 EF489476 ref. 3

Nigella sativa NisaFL1 KF500137.1 ref. 4

NisaFL2 KF500169.1 ref. 4

NisaAP3-2 HQ694795.1 ref. 2

NisaAP3-3 HQ694794.1 ref. 2

NisaPI1 HQ694797.1 ref. 2

NisaPI2 HQ694796.1 ref. 2





Helleborus x hybridus HehyFL1 KF500135.1 ref. 4

HehyFL2 KF500171.1 ref. 4

Helleborus orientalis HeorAG1a AY464109.1 ref. 5

HeorAG1b AY464108.1 ref. 5

Ranunculus bulbosus RabuFL1 AY306179.1 ref. 6

RabuFL2 AY306180.1 ref. 6



Ranunculus sceleratus RascFL1 AB473875.1 —b

RascFL2 AB473876.1 —

RascFL3 AB473877.1 —

Ranunculus ficaria RafiAG1 AY464114.1 ref. 5

RafiAG2 AY464115.1 ref. 5

Hydrastis canadensis HycaFL1 KF500136.1 ref. 4

HycaFL2 KF500172.1 ref. 4

Eranthis hyemalis ErhyFL1 KF500138.1 ref. 4

ErhyFL2 KF500140.1 ref. 4

Clematis marmoraria x Clematis

paniculata (cultivar Joe) CljoFL1 KF500139.1 ref. 4

Clematis integrifolia ClinAG1 AY464113.1 ref. 5

ClinAG2 AY464112.1 ref. 5

Delphinium exaltatum DeexAP3-1 EU481804.1 ref. 7

DeexAP3-2 EU481803.1 ref. 7

DeexAP3-3 EU481802.1 ref. 7

DeexPI EU481805.1 ref. 7

Delphinium ajacis DeajPI1 AF052862.1 ref. 8

Cimicifuga racemosa CiraAP3-1 AY162862.1 ref. 9

CiraAP3-2 AY162863.1 ref. 9

CiraAP3-3 AY162864.1 ref. 9

CiraPI1 AY162865.1 ref. 9



Anemone sylvestris AnsyFL1 KF500141.1 ref. 4

Anemone nemorosa AnneAP3-1 AY162841.1 ref. 9

AnneAP3-2 AY162842.1 ref. 9

AnneAP3-3_1 AY162843.1 ref. 9

Aconitum sinomontanum AcsiPI1 EU481819.1 ref. 7

AcsiPI2 EU481820.1 ref. 7

Thalictrum thalictroides ThthAG1 JN887118.1 ref. 10

ThthAG2 AY867879.1 ref. 11

Berberidaceae

Berberis thunbergii BethunFL1 KF500149.1 ref. 4

Berberis gilgiana BegiFL1 KF500145.1 ref. 4

BegiFL2 KF500142.1 ref. 4







BegiAP3-1 AY162857.1 ref. 9

BegiAP3-2_1 AY162858.1 ref. 9

BegiAG AY464106.1 ref. 5

Berberis bealei BebeFL JN593334.1 —

Epimedium grandiflorum EpgrAP3-1 EU481793.1 ref. 7

EpgrAP3-3 EU481794.1 ref. 7

Epimedium sagittatum EpsaAGL2-2 JN590219.1 —

EpsaAGL2-1 JN590218.1 —

Menispermaceae

Menispermum dauricum MedaAP3-2 EU481784.1 ref. 7

MedaAP3-3 EU481783.1 ref. 7

MedaAP3-3_1 EU481786.1 ref. 7

Cocculus trilobus CotrAP3-1 HQ694788.1 ref. 2

CotrAP3-2 HQ694789.1 ref. 2

CotrAP3-3 HQ694790.1 ref. 2

Lardizabalaceae

Sinofranchetia chinensis SichFL1 DQ 656565.1 ref. 12

SichFL2 DQ656566.1 ref. 12

SichAP3-1 JQ806394.1 ref. 13

SichAP3-2 JQ806395.1 ref. 13

SichAP3-3 JQ806396.1 ref. 13

Akebia trifoliata AktrFL1 AY627632.1 ref. 14

AktrFL2 GU357459.1 ref. 15

AktrAP3-1 AY627630.1 ref. 14

AktrAP3-2 AY627631.1 —

AktrAP3-3 DQ303124.1 ref. 14

AktrAG1 AY627635.1 —

AktrSEP1_1 GU357447.1 ref. 15

AktrSEP1_2 GU357448.1 ref. 15

Akebia quinata AkquAP3-1_1 AY162835.1 ref. 9

AkquAP3-2_1 AY162839.1 ref. 9

Papaveraceae

Dicentra eximia DiexAP3 AF052875.1 ref. 8

Eschscholzia californica EscaFL3 KF500168.1 ref. 4

EscaFL1 HM592297.1 —

EscaFL2 HM592298.1 —

EscaDEF1 EF378697.1 ref. 16

EscaDEF2 EF378698.1 ref. 16

EscaAG1 DQ088996.1 ref. 17

EscaAG2 DQ088997.1 ref. 17

Papaver somniferum PasoFL1 AY306177.1 ref. 6

PasoFL2 AY306178.1 ref. 6

Lamprocapnos spectabilis LaspFL1 KF500114.1 ref. 4

LaspFL2 KF500152.1 ref. 4





Sanguinaria canadensis SacaFL2 KF500157.1 ref. 4

SacaAP3 AF130868 ref. 18

Eupteleaceae

Euptelea pleiosperma EuplFL1 DQ656558.1 ref. 12

EuplFL2 DQ656559.1 ref. 12

EuplAG1 GU357452.1 ref. 15

EuplAG2 GU357453.1 ref. 15

EuplSEP1 GU357461.1 ref. 15

Euptelea polyandra EupoAP3 EU481781.1 ref. 7

Trochodendraceae

Trochodendron aralioides TrarAP3 DQ453774.1 ref. 19

Sabiaceae

Meliosma dilleniifolia MediAP3-1 AY436709.1 ref. 20

Buxaceae

Pachysandra procumbens PaprAP3-1 DQ479360.1 ref. 19

Magnoliaceae

Magnolia grandiflora MagrAP1 AY821777.1 ref. 21

MagrAGL2 AY821781.1 ref. 21

Magnolia praecocissima MaprMADS7 AB050649.1 —

MaprMADS2 AB050644.1 —

Michelia figo MifiFL AY306159.1 ref. 6

Chloranthaceae

Chloranthus spicatus ChspAP1 AY316311.1 ref. 22

ChspAP3 AF230701.1 ref. 23

ChspAG AY464100.1 ref. 5

Nymphaeaceae

Nuphar advena NuadAP1 AY936223.1 —

NuadSEP2 GU048648.1 —

Amborellaceae

Amborella trichopoda AmtrAP1 XM_006856294.1 —

AmtrAG AY936231.1 —

AmtrAGL2 AY936232.1 — aTAIR: The Arabidopsis Information Resource, https://www.arabidopsis.org/ bNCBI: National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/ cPhytozome v10.2.1: http://phytozome.jgi.doe.gov/pz/portal.html

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Supplementary Table 4. Primers used for qRT-PCR

Primer name Forward primer (5’-3’) Reverse primer (5’-3’) FL1_RT CAAATCAAAGTCAGAGCTCAATG ATAACTTCCAAATCTCTTACCTGT

AP3-1_RT GAGGATCTGAATATAGCCGAG GGTGGTGTTGGGTTGGTTG

AP3-2_RT AAGCTTTCGATAGAGTTCGC TCCTGTTCAACCAAAGCATAG

AP3-3_RT GTGTTGATGACTTGACTTTTG CCCAGTCGTAAATTGTAATGT

PI1_RT GCCTTAACTATATCTTGCTCAAG GCTTAATAGCAGTAATACTTTGTAG

PI2_RT ACTACATCTTGCTTAAAACTCAAC AGGAGTTTCCAGCAATGAAC

AG1_RT TAAAGGGTCTCGAGAAAAAGAT ATTCGTTTGTTGGCATCAGAC

AG2_RT TGAAGCAGCAAATTGAAATC CATGGCCTCGTATTCATTC

SEP1_RT GCTTCAGAGCAAGGAAATTACGT GAAGGATCAGAACCTGGACG

SEP2_RT CATCACCCTGTTCGAACTG AGTTTCAAGGATAATCACATGT

SEP3_RT CCTTGAGAGGCAATTAGACT TGTTGCCTACCATATCCCAC

AGL6_RT AACTTCGTAGAAAGGAACGTC CAATCTGAAGGGTGGGCTC

ANS_RT CAACCAGACTGAGATCCCTAGTG CGATGTGCACGATGATAGAGTC




Supplementary Table 5. Primers used for in situ hybridization

Primer name Forward primer (5’-3’) Reverse primer (5’-3’) Probe length T7_adapter TAATACGACTCACTATAGGG

FL1_IS GAGCAACCAAATCAAAGTC GCCACTACCTCCAATATACAG 340bp

FL2_IS CTAAGCGATACTTGTCAATCC CTGACATATGCACACGCTC 360bp

AP3-1_IS AGAATTGAGGAAGCTTATGC GCCTTGAGAAGGTTGATGAG 309bp

AP3-2_IS GATCTCTTTCGTGAAATTGAAG GGTGAAATATTAATAGTGCAGG 296bp

PI1_IS GAGACTATCCTTCACAGATGC GTCGCAGGGTAAGAGAAAC 284bp

PI2_IS GGGAATGCTAGAGACTTGG GATGGCACAAATATGGTATC 324bp

AG1_IS GCTGAGAATGAAAGAACC CCTGGAGCTTTACTAAACAG 347bp

AG2_IS GGAGATTGACTTGCATAATG GGACGTCAAATATACCTAATC 367bp

SEP1_IS CTGCAATTAGGATACAACCG GTTGACAGTTATGTAGAGCC 291bp

SEP2_IS CAGGATGAGAGGAGTATGG TCAACCCATATGACATACAG 381bp

SEP3_IS CTTCCACCCTCTTGATTGTG GAGACCAACTTCAGCATCAC 334bp

AGL6_IS TGGAAACAACAATTTCTCTG GATTCATTTCCATGCTTGG 313bp




Supplementary Table 6. Primers used for VIGS construction

Primer name Forward primer (5’-3’) Reverse primer (5’-3’) FL_EcoRIF GCGAATTCAATCAAGGGAGATCAAATGGAG

FL_XbaIR GCTCTAGAAACCACTACCTCCAATATACAG

FL2_BamHIF ATGGATCCATCTAAGCGATACTTGTCAATC

FL2_KpnIR ACGGTACCAGCTGACATATGCACACGCTC

AP3-1_KpnIF ATGGTACCATGAAGAAAGAATTGAGGAAGC

AP3-1_XhoIR AACTCGAGATGAAGAATCCAGTAGATAAGA

AP3-2_BamHIF TTGGATCCTTCGATTGTCACTATGCTTTGG

AP3-2_KpnIR GTGGTACCTTACTAGACAGATATTTAGGTG

AP3-3_XbaIF GGTCTAGAGTTCGTGATCGGAAGTAT

AP3-3_BamHI AAGGATCCAACCCAGTCGTAAATTGTAATG

PI1_KpnIF AAGGTACCTTCCAGAACACTAGAGACTATC

PI1_XhoIR2 AACTCGAGATAGAAACCAATAACATCAGTG

PI2_EcoRIF ACGAATTCAGACACAATAGAAGGGATTATC

PI2_XbaIR GGTCTAGATTGATGGCACAAATATGGTATC

AG1_BamHIF AAGGATCCAATTGCTGAGAATGAAAGAACC

AG1_KpnIR ATGGTACCTTCCTGGAGCTTTACTAAACAG

AG2_KpnIF AAGGTACCAATGAGAACGAGCGAGCCCAAC

AG2_XhoIR AACTCGAGCTGACGTCAAATATACCTAATC

SEP1_KpnIF ATGGTACCGACTGCAATTAGGATACAACCG

SEP1_XhoIR GACTCGAGACGTTGACAGTTATGTAGAGCC

SEP2_EcoRIF ATGAATTCATATGCAGTAATACAGTTCCAG

SEP2_XbalIR GATCTAGATGAACTGTCTTTATTCTCGTTG

SEP3_BamHIF TTGGATCCTTACCCTCTTGATTGTGAACC

SEP3_KpnIR-2 TAGGTACCTTCAAGTCACCATTGTTCAAAC

AGL6_BamHIF AAGGATCCTTGTACCACCAATATGTTTCGG

AGL6_KpnIR TCGGTACCTCTCTATTATTGATTCATTTCC

AGL6_KpnIF TAGGTACCATGAGCCCACCCTTCAGATTG

AGL6_XhoIR CGCTCGAGATTCTATTATTGATTCATTTCC

ANS_XbaIF AATCTAGAGTGAGGAGTTGAAGAAGTGC

ANS_BamHIR ATGGATCCCTGGTTGTATACTCGCTTG

Bold font indicates introduced restriction enzyme recognition site.




Supplementary Table 7. Computational estimation of off-target silencing (sliding window size=21bp, step length=1bp)

NdFL1 NdFL2 NdAP3-1 NdAP3-2 NdAP3-3 NdPI1 NdPI2 NdAG1 NdAG2 NdSEP1 NdSEP2 NdSEP3 NdAGL6

NdFL1 81.0%# 71.4% 71.4% 71.4% 76.2% 76.2% 81.0% 71.4% 71.4% 76.2% 71.4% 76.2%

NdFL2 81.0% 71.4% 76.2% 76.2% 71.4% 71.4% 71.4% 71.4% 71.4% 76.2% 71.4% 71.4%

NdAP3-1 76.2% 76.2% 85.7% 85.7% 71.4% 71.4% 76.2% 76.2% 76.2% 76.2% 81.0% 76.2%

NdAP3-2 71.4% 71.4% 85.7% 90.5% 85.7% 76.2% 71.4% 76.2% 66.7% 71.4% 76.2% 71.4%

NdAP3-3 71.4% 76.1% 85.7% 90.5% 76.2% 76.2% 71.4% 66.7% 66.7% 71.4% 66.7% 71.4%

NdPI1 76.2% 71.4% 71.4% 85.7% 76.2% 81.0% 71.4% 71.4% 71.4% 71.4% 71.4% 71.4%

NdPI2 76.2% 71.4% 81.0% 76.2% 81.0% 81.0% 71.4% 76.2% 71.4% 71.4% 71.4% 76.2%

NdAG1 81.0% 71.4% 76.2% 71.4% 66.7% 71.4% 71.4% 85.7% 76.2% 71.4% 71.4% 76.2%

NdAG2 71.4% 71.4% 76.2% 76.2% 71.4% 81.0% 81.0% 85.7% 71.4% 71.4% 85.7% 66.7%

NdSEP1 71.4% 71.4% 71.4% 71.4% 81.0% 71.4% 66.7% 71.4% 71.4% 85.7% 76.2% 76.2%

NdSEP2 76.2% 71.4% 76.2% 71.4% 71.4% 71.4% 71.4% 66.7% 71.4% 85.7% 71.4% 71.4%

NdSEP3 71.4% 71.4% 71.4% 76.2% 76.2% 81.0% 81.0% 71.4% 71.4% 81.0% 71.4% 81.0%

NdAGL6 76.2% 71.4% 76.2% 71.4% 76.2% 76.2% 76.2% 76.2% 71.4% 76.2% 76.2% 71.4%

# indicates the highest percentage identity between sequences in the sliding windows of each pair of VIGS region and cDNA

sequences.

cDNA VIGS region




Supplementary Table 8. Information on VIGS experiments

VIGS treated plants BatchPlants Flowers

Treated Survived Recorded Strong Moderate Weak

TRV2–NdAP3-3

1st 155 95 32 67 2 4

2nd 110 99 66 68 34 3

total 265 194 98 135 36 7

TRV2–NdAP3-1–NdANS 1st 60 24 5

TRV2–NdAP3-2–NdANS 1st 65 20 10

TRV2–NdAP3-1–NdAP3-2

1st 91 33 5 0 5 0

2nd 65 32 30 6 49 10

3rd 60 41 22 17 25 14

total 216 106 57 23 79 24

TRV2–NdAP3-1–NdAP3-2–NdAP3-3

1st 101 63 27 35 15 14

2nd 82 75 50 75 12 15

total 183 138 77 110 27 29

TRV2–NdPI1–NdPI2

1st 13 5 4 6 0 3

2nd 66 34 34 124 9 17

total 79 39 38 130 9 20

TRV2–NdAG1–NdANS 1st 80 56 13

TRV2–NdAG2–NdANS 1st 80 46 25

TRV2–NdAG1–NdAG2

1st 56 45 17 9 5 9

2nd 99 60 47 129 93 76

total 155 105 64 138 98 85

TRV2–NdFL1–NdFL2–NdANS

1st 77 30 23 28 26 28

2nd 74 53 50 57 67 56

total 151 83 73 85 93 84

TRV2–NdAGL6

1st 45 18 9 7 8 18

2nd 150 36 25 14 18 9

3rd 107 58 58 9 38 30

total 302 112 92 30 64 57

TRV2–NdFL1–NdFL2–NdAGL6 1st 79 26 24 11 43 16

TRV2–NdSEP1–NdSEP2–NdSEP3

1st 92 55 42 10 39 69

2nd 96 53 14 5 12 26

total 188 108 56 15 51 95

ndap3-3 TRV2–NdAP3-1–NdAP3-2 1st 51 33 9 21 0 0

ndap3-3 TRV2–NdPI1–NdPI2

1st 18 18 11 30 4 6

2nd 1 1 1 3 0 0

total 19 19 12 33 4 6

ndap3-3 TRV2–NdAG1–NdAG2 1st 31 30 23

ndap3-3 TRV2–NdAGL6

1st 1 1 1 0 3 0

2nd 4 4 4 1 4 2

total 5 5 5 1 7 2




Supplementary Table 9. Numbers of flowers used for statistical analysis

Plants Number of flowers

1st 2nd 3rd

Wild-type 129 129 129

ndap3-3 82 82 82

Mock 105 76 46

TRV2–NdAP3-3 82 45 19

TRV2–NdAP3-1–NdAP3-2 9 6 5

TRV2–NdAP3-1–NdAP3-2–NdAP3-3 38 31 26

TRV2–NdPI1–NdPI2 27 24 25

TRV2–NdAG1–NdAG2 10 25 23

TRV2–NdFL1–NdFL2–NdANS 25 42 33

TRV2–NdAGL6 54 19 10

TRV2–NdFL1–NdFL2–NdAGL6 7 14 13

TRV2–NdSEP1–NdSEP2–NdSEP3 14 11 8

ndap3-3 TRV2–NdAP3-1–NdAP3-2 9 4 4

ndap3-3 TRV2–NdPI1–NdPI2 8

ndap3-3 TRV2–NdAGL6 2




Supplementary Table 10. Primers used for Y2H

Primer name Forward primer (5’-3’) Reverse primer (5’-3’) NdFL1_NdeI TCGCCCATATGGGTAGAGGTAG

NdFL1_EcoRI CATCAGAATTCTTACTACTCGTTAAC

NdFL2_NcoI GCATCCATGGGTAGAGGTAGAG

NdFL2_EcoRI GTAGGAATTCTTCATTACAACTCATTTATG

NdAP3-1_NdeI CAGACATATGGGAAGAGGAAAG

NdAP3-1_PstI TTCTGCAGATTAACCTAAACGGAG

NdAP3-1ΔC_NdeI CAGACATATGGGAAGAGGAAA

NdAP3-1ΔC_BamHI AATGGGATCCCTTACTAAATTCTTTCTTCATATT

NdAP3-2_NdeI CAGACATATGGGAAGAGGAAAG

NdAP3-2_BamHI ATCAGGATCCATTATTAACCTAAACGGAG

NdAP3-3_NcoI GGAGTACCATGGGTAGAGGG

NdAP3-3_EcoRI ATCTAAGAATTCCTATTACTTAACCC

NdPI1_NdeI AGGAACATATGGGGAGAGGAAAG

NdPI1_EcoRI CTTTGAATTCTTACTATTTATTCTCCTGC

NdPI2_NdeI GAAACATATGGGGAGAGGTAAGATTG

NdPI2_EcoRI TGAAGAATTCCTATTAATGCTCTTG

NdAG1_NcoI CAAAGAACCATGGGAAGAGGAAAG

NdAG1_EcoRI ATAGAATTCTCACCCAAGTTGAAGTGCCG

NdAG2_NcoI CCACACAAACCATGGGAAGAG

NdAG2_EcoRI GCTGAATTCTCAACTAATTAATTGGAG

NdSEP1_NdeI GAACATATGGGAAGAGGGAAAG

NdSEP1_EcoRI TGCTGAATTCTCAGAGCATCCACCCAGGA

NdSEP2_NdeI AGAACATATGGGAAGAGGGAAAG

NdSEP2_BamHI AATAGGATCCCTCAGAGCATCCATCTAGG

NdSEP2ΔC_NdeI AGAACATATGGGAAGAGGGAAAG

NdSEP2ΔC_BamHI TCTGGGATCCCCTAATCCTGGCTTTTTAATAATC

NdSEP3_NcoI TCAATCGCCATGGGTAGAGGAAG

NdSEP3_EcoRI TGAGGAATTCCTATCAAGCTAACCATC

NdAGL6_NdeI TTAGCATATGGGTAGAGGAAG

NdAGL6_EcoRI CATAGAATTCTCAGAGAACCCATCG

AD_r AGATGGTGCACGATGCACAG

BD_r TAAGAGTCACTTTAAAATTTGTAT

T7 TAATACGACTCACTATAGGGC

Bold font indicates introduced restriction enzyme recognition site.




S1

S2S3

S4

S5

S1

S2S3

S4

S5

d e1st

2nd

3rd

1st/2nd

a b c

# of

tota

l flo

ral o

rgan

s

0 2 4 640

50

60

70

80 ρ = 0.8291

P1

S1

S2

S3

S4

S5

P2

P4

P5

P6

P7

P8

St1

St2St3

St4

St5

St6

P3

h

S4

P2P1

S1

S2S3

S4

S5f g

3

4th

5th

6th

) 3

Height of receptacle (mm)

# of

tota

l flo

ral o

rgan

s

0 1 2 340

50

60

70

80

Width of receptacle (mm)0 2 4 6

0 9018

ρ = 0.7499

Supplementary Figure 1 Correlation between the number of floral organs per flower

C1-5

C2

C4C3

C5

-13-21

-8 -3

-16 -24

-11-19

-2-10

-18

-7-15-23

-4-12

-20

-1

-6

-9-17

-25

-14-22S4

S5

S3S1

P1P6

St6 P4St1St3

h i

Heig

ht o

f rec

epta

cle

(mm

Width of receptacle (mm)0 2 4 6

0

1

2

3 ρ = 0.9018

Supplementary Figure 1. Correlation between the number of floral organs per flower and the size of floral meristem in Nigella damascena. a, An individual plant at anthesis. b, A diagram showing the structure of the plant, in which the 1st, 2nd and 3rd bloomed flowers are indicated. c, Correlation between the total number of floral organs and the height and width of the floral receptacle in mature flowers (stage 16). A total of 20 flowers were investigated. The Spearman correlation coefficient (ρ) is shown for each chart. The regression equation for the bottom chart is height = -0.0254 + 0.4852*width. d-e Scanning electron microscopy (SEM) images of flower buds at stage 4 showing thee, Scanning electron microscopy (SEM) images of flower buds at stage 4, showing the difference in the size of floral meristem. f-i, SEM images of flower buds at stages 4, 5, 6 and 8, respectively. Br, bract; S, sepal; P, petal; St, stamen; C, carpel. The numbers indicate the forward (in f-h) or reverse (in i) order of the organ initiated. Scale bar = 100 µm.




a b Aquilegia AqvuAP3-1Aquilegia AqcoAP3-1

Delphinium DeexAP3-1Cimicifuga CiraAP3-1

Anemone AnneAP3-1Nigella NdAP3-1

Menispermum MedaAP3-3_1Cocculus CotrAP3-1

Epimedium EpgrAP3-1

100

100

100

AP3-

1

Arabido

p pgSinofranchetia SichAP3-1

Akebia AktrAP3-3Akebia AktrAP3-2Akebia AkquAP3-1_1

Eschscholzia EscaDEF1Sanguinaria SacaAP3

Dicentra DiexAP3

100100

9595

Aquilegia AqcoAP3-2Aquilegia AqvuAP3-2Anemone AnneAP3-2

Delphinium DeexAP3-2Nigella NisaAP3-2

Nigella NdAP3-2Cimicifuga CiraAP3-2

Berberis BegiAP3 2 1

100

100

51100

AAP

3-2

0.1

opsis SEP3

B2 (PI)

Aquilegia AqcoAP3-3bAquilegia AqvuAP3-3Aquilegia AqcoAP3-3

Anemone AnneAP3-3_1Delphinium DeexAP3-3

Nigella NdAP3-3Nigella NisaAP3-3

Cimicifuga CiraAP3-3E i di E AP3 3

10098

1005591

98

Berberis BegiAP3-2_1Cocculus CotrAP3-2

Menispermum MedaAP3-2Sinofranchetia SichAP3-2

Akebia AktrAP3-1Akebia AkquAP3-2_1

100100

97

AAP

3-3

Epimedium EpgrAP3-3Berberis BegiAP3-1

Menispermum MedaAP3-1Cocculus CotrAP3-3

Sinofranchetia SichAP3-3Eschscholzia EscaDEF2

Euptelea EuplAP3Trochodendron TrarAP3

Pachysandra PaprAP3-1Meliosma MediAP3-1

Magnolia MaprMADS7Chloranthus ChspAP3

100

87

96

100

64

93

0.1

A

RanunculaceaeBerberidaceaeMenispermaceaeLardizabalaceaePapaveraceaeEupteleaceaeOthers

Supplementary Figure 2. Classification and phylogenetic analysis of floral MADS-box genes. a, An unrooted tree showing the classification of the N. damascena genes into lineages. b-f, Phylogenetic trees of each gene lineage. Bootstrap values higher than 50% are indicated for each node. Yellow stars indicate inferred gene duplication events.




Delphinium DeajPI1Delphinium DeexPI

Aconitum AcsiPI1Aconitum AcsiPI2

Nigella NisaPI2Nigella NdPI2

Nigella NdPI1Nigella NisaPI1

Cimicifuga CiraPI2Cimicifuga CiraPI3

90

100

97

100

100100

100

100

dc Nigella NdFL2Nigella NisaFL2

Helleborus HehyFL2Ranunculus RabuFL1

Ranunculus RabuFL2Ranunculus RascFL3

Aquilegia AqcoFL2Hydrastis HycaFL2Sanguinaria SacaFL2

Eschscholzia EscaFL3Papaver PasoFL1

Lamprocapnos LaspFL2E ptelea E plFL2

5962

95

10088

100100

78100

FL2

0.1 Cimicifuga CiraPI1Euptelea EuplFL2Eranthis ErhyFL2Eranthis ErhyFL1

Clematis CljoFL1Helleborus HehyFL1

Ranunculus RascFL2Ranunculus RascFL1Ranunculus RabuFL3

Ranunculus RabuFL4Anemone AnsyFL1

Nigella NisaFL1Nigella NdFL1

Aquilegia AqcoFL1BAquilegia AqcoFL1A

Hydrastis HycaFL1Berberis BethFL174

100

100

97

5796

100

100

96100

10055

FL1

Clematis ClinAG1Ranunculus RafiAG1Aquilegia AqcoAG1Aquilegia AqcoAG1-2

Thalictrum ThthAG1Helleborus HeorAG1b

Helleborus HeorAG1a100 100

67

100100

AG1

e

Berberis BethFL1Berberis BegiFL1Berberis BebeFL

Berberis BegiFL2Berberis BegiFL3Berberis BegiFL4

Lamprocapnos LaspFL1Papaver PasoFL2

Eschscholzia EscaFL1Eschscholzia EscaFL2

Sinofranchetia SichFL1Akebia AktrFL1

Akebia AktrFL2Sinofranchetia SichFL2

Euptelea EuplFL1Chloranthus ChspAP1

M li M AP1

91

7492

99

100

80

54

10099

74100

52

F Helleborus HeorAG1aNigella NdAG1

Akebia AktrAG1Euptelea EuplAG2

Clematis ClinAG2Ranunculus RafiAG2

Aquilegia AqcoAG2Thalictrum ThthAG2

Nigella NdAG2Berberis BegiAG

Euptelea EuplAG1Eschscholzia EscaAGL2

Eschscholzia EscaAGL1Chloranthus ChspAG

100

9571

100

100

94

6360

53

AG2

Aquilegia AqcoSEP2Nigella NdSEP2

Epimedium EpsaAGL2-2Akebia AktrSEP1-2

Aquilegia AqcoSEP1Nigella NdSEP1

SEP2

191

5962

9889

f

0.1

Magnolia MagrAP1Michelia MifiFL

Amborella AmtrAP1Nuphar NuadAP1

100

10054

0.1Magnolia MaprMADS2

Amborella AmtrAG52

RanunculaceaeBerberidaceaeM i

Supplementary Figure 2 (Continued)

0.1

Nigella NdSEP1Epimedium EpsaAGL2-1

Akebia AktrSEP1-1Euptelea EuplSEP1Magnolia MagrAGL2

Amborella AmtrAGL2Nuphar NuadSEP2

SEP1

5290

MenispermaceaeLardizabalaceaePapaveraceaeEupteleaceaeOthers




0

0.5

1

1.5

2

Rela

tive

expr

essio

n

0

0.5

1

1.5

2NdAP3-1 NdAP3-2 NdAP3-3

B F S P St C B F S P St C0

0.5

1

1.5

2

B F S P St C

0

0.5

1

1.5

2

Rela

tive

expr

essio

n NdPI1 NdPI2

B F S P St C0

0.5

1

1.5

2

B F S P St C

2

ion 2

Rela

tive

expr

essio

n

0.5

1

1.5

2

0

NdAG1 NdAG2

NdSEP1 NdSEP2 NdSEP3

B F S P St C0

0.5

1

1.5

2

B F S P St C

2

0

1

B F S P St C

0.5

1.5

Rela

tive

expr

essi

0

0.5

1

1.5

1.5

2

essio

n NdAGL6 NdFL11.5

2

B F S P St C0

0.5

1

1.5

B F S P St C

0

0.5

1

Rela

tive

expr

0.5

1

0B F S P St CB F S P St C

Supplementary Figure 3. Expression patterns of 12 Nigella damascena genes as revealed by qRT-PCR analyses. Flowers at stage 12 were assayed, and the relative

i l l f th li d t th t f NdAP3 3 i t l E bexpression levels of other genes were normalized to that of NdAP3-3 in petals. Error bars indicate the collective standard deviations of three biological replicates and three technical replicates. B, bract; F, flower bud; S, sepal; P, petal; St, stamen; C, carpel.




a1 a2 a3 a4 a5S3 S5 S6 S9 S11

sepe

br se pepe

st

pe

sest

ca

stca

se

sepest

brse

br

stpe

cast

pe

se

cast

pese

br

br

b1 b2 b3 b4 b5S5 S6 S9 S10

se

S3

se

br

st

NdFL1

NdFL2

sepe

st

sepe

st

br br

sepest

ca

se

st

pe

ca

c1 c2 c3

d1 d2 d3

c4

d4

c5

d5

S4 S6

S4 S6

S6 S8

S9S6

S10

S10

sepe

NdFL2

NdAP3-1

stpe

se sepe

st

pese

st

ca

sepe

stca

pese

ca st

sepe

stca

se pe

ca

st

sepe

br

br

e1 e2 e3 e4 e5S2 S5 S7 S9 S10

sepe

st

NdAP3-2

NdPI1

pe

se sepe

st

pest

ca

se se

stpe

ca

br

f1 f2 f3 f4 f5S3 S4 S9S6 S10

NdPI1

NdPI2

ca

g1 g2 g3 g4 g5S5S3 S6 S8 S10

st

sepe

sepe

st

sepe

st castpesebr

br

sest

NdAG1

Supplementary Figure 4. Expression patterns of 12 Nigella damascena genes as revealed by RNA in situ hybridization. a, NdFL1. b, NdFL2. c, NdAP3-1. d, NdAP3-2. e,NdPI1. f, NdPI2. g, NdAG1. h, NdAG2. i, NdSEP1. j, NdSEP2. k, NdSEP3. l, NdAGL6. The stages of the flowers are shown in the top left corners of the images br bract; se sepal;stages of the flowers are shown in the top left corners of the images. br, bract; se, sepal; pe, petal; st, stamen; ca, carpel. Scale bar = 100 µm.




S3 S4

st ca

h1 h2 h3 h4 h5S6 S8 S10

sebr

se

st

se

ca

st

sepe

st cape

pe

br

S2 S6 S9 S10

pe

i1 i2 i3 i4 i5S4

br

se se sepe

st

pese

stca

stpese

pe

br

NdAG2

NdSEP1

br

se

se pest

ca

sepe

cast

sepe

st

S2 S3 S8S6 S10

br

j1 j2 j3 j4 j5

cast

k1 k2 k3 k4 k5S5S3 S6 S9 S10

st

NdSEP2

cast

pesesepe

stpe

se

st

sepe

sepe

sepe pe

sebr

stst

ca

brse

stca

pe

l1 l2 l3 l4 l5

se

br

br

S2 S4 S6 S9 S10

NdSEP3

NdAGL6





1.5 NdAP3-1

on 1.5 NdAP3-2 1.5 NdAP3-3

0

0.5

1

Rela

tive

expr

essi

S6~7 S9 S12 S16S P St C S P St C S P St C S P St C 0

0.5

1

S6~7 S9 S12 S16S P St C S P St C S P St C S P St C

1.5NdPI1

1.5

NdPI2

ssio

n

0

0.5

1


0

0.5

1

0

0.5

1

Rela

tive

expr

e



1

1.5 NdAG1

1

1.5 NdAG2

ress

ion

0

0.5

1

0

0.5

1

Rela

tive

exp



1

1.5

1

1.5NdSEP1 NdSEP2

1

1.5NdSEP3

xpre

ssio

n

0 5

1

1.5 NdFL1

0 5

1

1.5 NdFL2NdAGL6

0 5

1

1.5

e ex

pres

sion

0

0.5


0.5


0.5

Rela

tive

ex


0

0.5


0.5

S6~7 S9 S12 S16S P St C S P St C S P St C S P St C0

0.5

Rela

tive


Supplementary Figure 5. Expression patterns of 13 Nigella damascena genes as revealed by digital gene expression profiling (DGE). Flowers at stages 6-7, 9, 12 and 16 were used, and the relative expression levels of floral MADS-box genes were normalized to those of NdAP3-3 in petals at stage 12. Error bars indicate the collective standard deviations of three biological replicates. S, sepal; P, petal; St, stamen; C, carpel.




NdFL1 M I K C

NdFL2 M I K C

100bp EcoRI XbaI BamHIKpnIXhoI

NdFL1 NdANS

EcoRI XbaI BamHI KpnI XhoI

NdFL2NdANS

TRV2–NdFL1–NdANS

TRV2–NdFL2–NdANS

ca

NdAP3-1 M I K C

NdAP3-2 M I K C

NdAP3-3 M I K C

NdPI1 M I K C

EcoRI XbaI BamHIKpnI XhoI

EcoRI XbaI BamHI KpnIXhoI

NdAP3-3


NdAP3-1NdANS


NdAP3-2NdANS

EcoRI XbaI BamHIKpnI

XhoI

NdPI1NdANS

TRV2–NdAP3-3

TRV2–NdAP3-1–NdANS

TRV2–NdAP3-2–NdANS

TRV2–NdPI1–NdANS

NdPI2 M I K C

NdAG1 M I K C

NdAG2 M I K C

NdSEP2 M I K C

NdSEP1 M I K C


NdANS NdAG1


NdANS NdSEP1

EcoRI XbaI BamHIKpnI XhoI

NdANSNdSEP2

EcoRI XbaIBamHI

KpnI XhoI

NdANS NdAG2

NdANSNdPI2

TRV2–NdAG1–NdANS

TRV2–NdSEP1–NdANS


TRV2–NdAG2–NdANS

TRV2–NdPI2–NdANS

NdSEP3 M I K C

NdAGL6 M I K C

LB2×35S

CPEcoRIXbaI

b EcoRI XbaI BamHI KpnI XhoI

NdAP3-2 NdAP3-1NdAP3-3

EcoRIXbaI

BamHIKpnI

XhoI

EcoRI XbaIBamHI

KpnI XhoI

NdAP3-2 NdAP3-1

EcoRI XbaIBamHI KpnI XhoI

NdAGL6


NdSEP3NdANS



TRV2–NdAGL6


Kan BamHIKpnI

XhoIRzNOSt

RBpYL1569663bp

XbaI KpnINdPI1NdPI2

EcoRI XbaIBamHI KpnI XhoI

NdAG1 NdAG2


NdFL2NdFL1 NdANS


NdFL2NdFL1 NdAGL6-2


NdSEP3NdSEP2 NdSEP1


TRV2–NdAG1–NdAG2


TRV2–NdFL1–NdFL2–NdAGL6

TRV2–NdSEP1–NdSEP2 NdSEP3NdSEP2–NdSEP3

Supplementary Figure 6. VIGS construction. a, Schematics showing the positions of the primers used to amplify fragments for VIGS construction. Rectangles and lines indicate the coding regions and untranslated regions (UTRs), respectively. M, I, K and C stand for the sequences that encode the MADS, I, K and C regions of the proteins, respectively. Wavy lines indicate the regions that were used for the VIGS assays. The relative positions of the

i d f RT PCR d i it h b idi ti l i di t d b fill d dprimers used for qRT-PCR and in situ hybridization are also indicated by filled and open triangles, respectively. b, A simplified diagram of the tobacco rattle virus PYL156 vector. Red fonts indicate the restriction enzyme recognition sites used in this study. c, Schematics of VIGS constructs.




ba1.5

1

0.5

0

NdAP3-3 NdAP3-1 NdAP3-2

0

1

0.75

0.5

0.25

Rela

tive

expr

essio

n

NdPI1 NdPI21

0.75

0.5

0.25

dcNdAP3-1 NdAP3-2 NdAP3-3

0.5

1

0.75

0.25ativ

e ex

pres

sion

0

NdAG1 NdAG2

1.5

2fe

0

1

0.75

NdFL1 NdANS

Rela

xpre

ssio

n

Supplementary Figure 7. Relative expression levels of genes in wild-type, mock-treated,

0.5

1

0

0.5

0.25

0

Rela

tive

ex

and gene-specific construct-treated flowers with strong phenotypic changes. a, TRV2–NdAP3-3. b, TRV2–NdAP3-1–NdAP3-2. c, TRV2–NdAP3-1–NdAP3-2–NdAP3-3. d, TRV2–NdPI1–NdPI2. e, TRV2–NdAG1–NdAG2. f, TRV2–NdFL1–NdFL2–NdANS. g, TRV2–NdFL1–NdFL2–NdAGL6. h, TRV2–NdAGL6. i, TRV2–NdSEP1–NdSEP2–NdSEP3. Light grey, wild-type flowers; black, mock-treated flowers; colors, gene-specific construct-treated flowers with strong phenotypic changes. Error bars indicate the collective standard deviations of three technical replicates.




NdAGL61

0.75

0.5

0.25

0

hgNdFL1 NdAGL6

0

0.25

0.5

0.75

1

Rela

tive

expr

essio

n

0

i

0

1

0.75

0.5

NdSEP1 NdSEP2 NdSEP3

e ex

pres

sion

0.25

0

Rela

tive





a b c d

ndap3-3 TRV2–NdAP3-3, strong

e f

TRV2–NdAP3-3, moderate

g

h i j k

TRV2–NdAP3-2–NdANS, strongTRV2–NdAP3-1–NdANS, strong

h i j k

l m n o

TRV2–NdAP3-1–NdAP3-2, moderateTRV2–NdAP3-1–NdAP3-2, strong

p r s tq

ndap3-3 TRV2–NdAP3-1–NdAP3-2, strong

TRV2–NdAP3-1–NdAP3-2–NdAP3-3, strong

TRV2–NdPI1–NdPI2, strong

u v w x

Supplementary Figure 8. Phenotypes of the flowers in which AP3- and PI-lineage members were knocked down. a-x, Flowers and floral organs. For each flower, both side and top views are shown, and “strong” and “moderate” indicate the flowers with strong and moderate phenotypic changes, respectively. Note that: 1) five organs, which display different degree of petal-to-sepal transformation in TRV2–NdAP3-3–treated flowers with moderate phenotypic changes, are shown in (g); 2) a petal of TRV2–NdAP3-1–NdANS–treated flower with strong phenotypic changes is shown in (p); 3) a petal of TRV2–NdAP3-treated flower with strong phenotypic changes is shown in (p); 3) a petal of TRV2 NdAP32–NdANS–treated flower with strong phenotypic changes is shown in (q); and 4) two petals of TRV2–NdAP3-1–NdAP3-2–treated flowers with moderate phenotypic changes are shown in (r). y, Numbers of floral organs in the aforementioned flowers. 1st, 2nd and 3rd represent the first, second and third flowers, respectively.




40

60

Sepal

10

Petaly

75

Stamen 60 Carpel

0

20

1st 2nd 3rd

0

5

1st 2nd 3rd

0

25

50

75

0

20

40

20

40

60Sepal+Petal

50

75

100 Stamen+Carpel

1st 2nd 3rd 1st 2nd 3rd

Petal+Stamen100 90

Total

0

20

1st 2nd 3rd

0

25

1st 2nd 3rd

0

25

50

75

1st 2nd 3rd

0

30

60

1st 2nd 3rd

ndap3 3 TRV2 NdAP3 3MockWt ndap3-3


TRV2–NdAP3-3


MockWt


ndap3-3 TRV2–NdAP3-1–NdAP3-2





a b c d

TRV2–NdANS, strong

e fTRV2–NdAG1–NdANS, strong

TRV2 NdAG1 NdAG2 strongTRV2–NdAG2–NdANS, strong TRV2–NdAG1–NdAG2, strong

g h

TRV2–NdAG1–NdAG2, strongTRV2 NdAG2 NdANS, strong

TRV2–NdAG1–NdAG2, moderate TRV2–NdAG1–NdAG2, moderate

TRV2 NdAG1 NdAG2, strong

i j k l

m n o p

ndap3-3 TRV2–NdAG1–NdAG2, strong

q Sepal Petal Stamen Carpel Total

7.5

10

100

150

40

60

6

8

100

***

******100

150Petal+Stamen

TRV2–NdAG1–NdAG2Mock

0

2.5

5

1st 2nd 3rd0

50

1st 2nd 3rd0

20

1st 2nd 3rd0

2

4

1st 2nd 3rd0

50

1st 2nd 3rd0

50

100

1st 2nd 3rd

Supplementary Figure 9. Phenotypes of the flowers in which AG-lineage members were knocked down. a-p, Flowers and floral organs. For each flower, both side and top views are shown, and “strong” and “moderate” indicate the flowers with strong and moderate phenotypic changes, respectively. Note that: 1) three organs, which display different degree of stamen-to-petal transformation in TRV2–NdAG1–NdAG2–treated flowers with moderate phenotypic changes, are shown in (m); and 2) two organs showing different degree of carpel-to-sepal transformation in TRV2–NdAG1–NdAG2–treated flowers with strong and moderate phenotypes are shown in (n). q, Numbers of floral organs in the aforementioned flowers. 1st, 2nd and 3rd represent the first, second and third flowers, respectively, and stars (*) indicate the P values obtained with a Mann-Whitney U test. *, P<0.05; **, P< 0.01; ***, P< 0.001.




a b c d

e f g hTRV2–NdFL1–NdFL2–NdANS, strong TRV2–NdAGL6, strong

TRV2–NdFL1–NdFL2–NdAGL6, strong TRV2–NdAGL6, moderate

i j k l

TRV2–NdSEP1–NdSEP2–NdSEP3, moderate

q r s t

ndap3-3 TRV2–NdAGL6, strong

TRV2–NdSEP1–NdSEP2–NdSEP3, strong

m n o p

TRV2–NdSEP1–NdSEP2–NdSEP3, moderate TRV2–NdSEP1–NdSEP2–NdSEP3, moderate

Supplementary Figure 10. Phenotypes of the flowers in which AP1-, AGL6- and SEP-lineage members were knocked down a t Flowers and floral organs For each flowerlineage members were knocked down. a-t, Flowers and floral organs. For each flower, both side and top views are shown, and “strong” and “moderate” indicate the flowers with strong and moderate phenotypic changes, respectively. Two sepals of TRV2–NdAGL6–treated flowers with moderate (left) and weak (right) phenotypic changes are shown in (i). Three petals of TRV2–NdAGL6–treated flowers with nearly strong (left), moderate (middle) and weak (right) phenotypic changes are shown in (j). u, Numbers of floral organs in the aforementioned flowers. 1st, 2nd and 3rd represent the first, second and third flowers respectively and stars (*) indicate the P values obtained with a Mann-third flowers, respectively, and stars ( ) indicate the P values obtained with a MannWhitney U test. *, P<0.05; **, P< 0.01; ***, P< 0.001.




u10

Sepal20

Petal50

Stamen

2.5

5

7.5

10

20

10

20

30

40

0 0 0

5

7.5Carpel

20

Sepal+Petal

40

60 Stamen+Carpel

1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd

0

2.5

0

10

0

20

Petal+Stamen Total


Total

25

50

75

20

40

60eta Sta e

20

40

60

ota

****

ota

TRV2–NdAGL6


TRV2–NdFL1–NdFL2–NdAGL6

Mock

00 0


TRV2–NdSEP1–NdSEP2–NdSEP3





Supplementary Figure 11. Interactions among proteins of floral MADS-box genes as revealed by using Y2H assays a Test for auto activation b Test for interaction Left panelrevealed by using Y2H assays. a, Test for auto-activation. b, Test for interaction. Left panel, colonies grown on the SD-LW medium. Middle panel, colonies grown on the selective medium (SD-LWHA + 5 mM 3-AT). Right panel, lacZ test results of the positive colonies on the selective medium. Positive colonies in left panel indicate the co-existence of two vectors in the cells; positive colonies in middle panel and blue coloration in right panel indicate the capability of auto-activation in (a) and the interaction of the paired proteins in (b). ΔC indicates the truncation of the C-terminal regions in NdAP3-1 and NdSEP2, and the C-terminal truncated versions of NdAP3-1 and NdSEP2 were inserted into the pGBKT7the C terminal truncated versions of NdAP3 1 and NdSEP2 were inserted into the pGBKT7 vectors to avoid auto-activation in (b). L, leucine; W, tryptophan; H, histidine; A, adenine; 3-AT, 3-amino-1,2,4-triazole.




NdAG1 expression in wild-type andndap3 3 mutant floral organs

a dNdAG2 expression in mock and

NdAP3 1/2 silenced floral organsndap3-3 mutant floral organs

0

0.2

0.4

0.6

0.8

1

1.2

NdAP3-1/2-silenced floral organs

0

50

100

150

200Re

lativ

e ex

pres

sion

1

1.5

2

NdAP3-1 expression in wild-type and ndap3-3 mutant floral organs

NdAP3-3 expression in mock and NdAG1/2-silenced floral organs

200

300

b e

stamenstamen-derived

carpel

petalpetal-derived

sepal

outer stamenstamen-derived

sepal

inner stamenstamen

e ex

pres

sion

0

0.5

NdAP3-2 expression in wild-type and ndap3-3 mutant floral organs

NdAGL6 expression in mock and NdAG1/2-silenced floral organs


petal

0

100

c f

petalpetal-derived

sepal


sepal

inner stamenstamen

Rela

tive

ndap3-3 mutant floral organs NdAG1/2-silenced floral organs

0

2

4

6

8

0

0.5

1

1.5

2

2.5

Rela

tive

expr

essio

n


petal

petalpetal-derived

sepal


sepal

inner stamenstamen

Supplementary Figure 12. qRT-PCR assessment of gene expression levels in floral organs of wild-type, “Double Sepals” mutant (ndap3-3), mock and VIGS-treated flowers. a-c, “Double Sepals”. d-e, TRV2–NdAG1–NdAG2. f, TRV2–NdAP3-1–NdAP3-2. Light grey, wild-type floral organs; black, mock-treated floral organs; colors, “Double Sepals” or gene-specific construct treated floral organs with strong phenotypic changes Error barsspecific construct-treated floral organs with strong phenotypic changes. Error bars indicate the collective standard deviations of three technical replicates.


floral organ identity determination in nigella damascena … · 2016-01-06 · floral organ...

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