Supplemental Figure 1

Zhao et al. 2013

PEAM4 (Pisum sativum_AAL66379)

MTPIM (Medicago truncatula_AAZ67068)

GmAP1 (Glycine max_XM_003547744)

CAL (Arabidopsis thaliana_NP-564243)

AP1 (Arabidopsis thaliana_AAM65504)

SQUA (Antirrhinum majus_X63701)

StMC (Solanum tuberosum, in this study)

LeMADS-MC (Solanum lycopersicon _AK328446)

MSM3 (Solanum marcrocarpon, in this study)

MPF3 (Physalis floridana, in this study)

NtMADS5 (Nicotiana tobacum_AAD39035)

NsMADS1 (Nicotiana sylvestris_AAD39036)

LeFUL2 (Solanum lycopersicon _SGNU581535)

PhFUL (Petunia hybrida_AAP83394)

NtMADS11 (Nicotiana tobacum_AAO12211)

PFFUL2 (Physalis floridana, in this study)

LeFUL1 (Solanum lycopersicon_AAM33098)

PFFUL1 (Physalis floridana, in this study)

NTFUL (Nicotiana tobacum_ABF82231)

AMFUL (Antirrhinum majus_AAP83363)

FUL (Arabidopsis thaliana_At3g60910)

AMDEFH28 (Antirrhinum majus_AAK75467)

GSQUA2 (Gerbera hybrida_FN298387)

PhFL (Petunia hybrida_AAP83396)

SLMBP20 (Solanum lycopersicon _BT013126)

PFFL (Physalis floridana, in this study)

TDR4 (Vaccinium myrtillus_FJ418852)

AGL79 (Arabidopsis thaliana_AAE77628)

Eupteleaful2 (Euptelea pleiosperm_ABG49518)

Eupteleaful1 (Euptelea pleiosperm_ABG49519)0.1

0.99

931.00

99

1.00

93

0.93

0.99

880. 98

741.00

801.00

100

1.00

77

1.00

100

1.00

77

0.99

58

0.97

1.00

98

0.94

51

0.91

62

euAP1

euFUL

FL

Supplemental Figure 1 Bayesian interference of AP1/SQUA-like genes.The native host and accession number of the corresponding gene are given in parentheses. MPF3, PFFUL1/2 and PFFL from Physalis fall into the three subcladesof euAP1, euFUL and FL, respectively. The maximum likelihood (ML) tree has a topology similar to that of the Bayesian tree. The supporting value for the Bayesian method above 0.90 and for ML above 50 is given for each branch. The scale at the bottom is the number (in units) of amino acid substitutions per site. The sequence alignment is presented in Supplemental Dataset 1 online.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

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Supplemental Figure 2

Zhao et al. 2013

0

1

2

3

MP

F3-MP

F2

Rela

tive β

-ga

lacto

sid

ase

acti

vit

y

MP

F3-PFA

G

MP

F3-PFG

LO1

MP

F3-PFG

LO2

MP

F3-PFS

EP

3

Negative control

MP

F2-MP

F2

MP

F3-MP

F3

PFG

LO1-P

FGLO

1

PFG

LO1-P

FGLO

2

PFA

G-P

FAG

Negative control

A

B

Supplemental Figure 2 Protein-protein dimerization among MPF3-interacting partners within the yeast two-hybrid system.(A) Relative quantification of protein-protein interaction strength of MPF3-associated heterodimerization. The relative β-galactosidase activity was determined in yeast for the combination of the MPF3-associated heterodimersindicated. The experiments were performed three times. The mean and standard deviation are shown. (B) Homodimerization of proteins. Homodimerization of MPF2 was also reported by He et al. (2007). The co-transformed yeast cells were grown on SD/-Leu-His-Trp selective plates. The co-transformation of empty vectors of pGBKT7 and pGADT7 was used as negative control.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

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MPF2-RNAi-

MPF3-VIGS-2

MPF2-RNAi-

MPF3-VIGS-1

MPF3-VIGS

MPF2-MPF3-

VIGS-2

WT MPF2-RNAi MPF3-RNAi

WT

A

B

C

F

D

G

E

H

Supplemental Figure 3

Zhao et al. 2013

Supplemental Figure 3 Anther transverse section analyses.(A, B) Stamen from a wild-type (WT) flower. (C) Stamen from the 35S:MPF2-RNAi plant. (D) Stamen from the 35S:MPF3-RNAi plant. (E) Stamen from a MPF3-VIGS flower. (F-H) Double-gene down-regulated stamens as indicated by MPF2-RNAi-MPF3-VIGS-1 (F), MPF2-RNAi-MPF3-VIGS-2 (G) and MPF2-

MPF3-VIGS-2 (H). Bar=100 μm.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

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PFACTIN (25)

PFMST8 (28 )

PFSUT1 (28)

PFSUT2 (28 )

PFINV4 (28 )

PFCSA (28)

WT

WT

MP

F2

-RN

Ai

MP

F3

-RN

Ai

MP

F3

-VIG

S

MP

F2-R

NA

i-MP

F3-V

IGS-

1

MP

F2-

RN

Ai-M

PF

3-V

IGS-

2

MP

F2-

-MP

F3-

VIG

S-1

MP

F2-

-MP

F3-

VIG

S-2

Leaves Androecium

Unigene17675_P106

Unigene70960_P106

Unigene38938_P106

Unigene54711_P106

Unigene13767_P106L

an

e 1

La

ne 2

La

ne 3

La

ne 4

La

ne 5

La

ne 6

La

ne 7

La

ne 8

La

ne 9

Unigene950_P106

Supplemental Figure 4

Zhao et al. 2013

Supplemental Figure 4 Expression of the candidate genes involved in sugar partitioning pathways. Total RNA was isolated from wild-type leaves (lane 1), wild-type androecium(lane 2), androecium from MPF2-RNAi (lane 3), androecium from the MPF3-RNAi line (lane 4), androecium from MPF3-VIGS (lane 5), androecium from the MPF2-RNAi-MPF3-VIGS-1 line (lane 6), MPF2-RNAi-MPF3-VIGS-2 line (lane 7), MPF2-MPF3-VIGS-1 line (lane 8) and MPF2-MPF3-VIGS-2 line (lane 9). PFACTIN from Physalis was used as an internal control. RT-PCR for each gene was performed three times using independent biological samples, and a representative example is shown. Amplification cycle number is given in parentheses. The sequence information of these genes is presented in Supplemental Dataset 2 online.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

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Supplemental Figure 5

Zhao et al. 2013

A

D

B

E

C

F

Rela

tive g

en

e e

xp

ressio

n

0

1

2

3

4

WT V1 V2 V3

MPF3 PFFUL2 PFFUL1 PFFL

G

Rela

tiv

e P

FIN

V4

e

xp

res

sio

n

WT

Androecium

0.00520

0.3

0.9

1.2

0.6

MPF3-VIGSI

H calyx androecium0

Rela

tive M

PF

2exp

ressio

n

50

250

150

100

200

44.0

9.3

Supplemental Figure 5 VIGS-mediated MPF3 silencing phenocopies MPF3-RNAi.(A) An intact WT flower bud. (B) A WT flower. (C) A WT ICS. (D) An intact MPF3-VIGS flower bud. (E) A MPF3-VIGS flower. (F) A MPF3-VIGS ICS. Bar=5 mm in (A), (C), (D) and (F). Bar=1 mm in (B) and (E). (G) MPF3 was silenced via a VIGS approach. MPF3 expression was evaluated in three VIGS flowers and is indicated as V1-V3. The severe MPF3 mRNA residual in V3 was only 4.1% of wild-type (WT) flowers. (H) Expression of MPF2. (I) Expression of PFINV4. Total RNA from the indicated mutated flowers was subjected to real-time RT-PCR. Gene expression in the androecium of the WT was taken as 1, and actin was used as an internal control. The experiments were repeated thrice using three independent biological samples. Mean expression and standard deviation are shown.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

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Supplemental Figure 6

Zhao et al. 2013

Po

llen

matu

rati

on

(%

)

0

0.4

1.2

0.8

WT0

40

120

80

P1 P2

Rela

tive e

xp

ressio

n

0.46

0.13

A B C

G

D E F

B

Supplemental Figure 6 Normal pollen development in the 35S:PDS-VIGS flowers.(A) An intact WT flower. (B) A partially bleached PDS-VIGS flower (P1). (C) A nearly fully bleached flower (P2). Bar=5 mm. (D-F) I2-KI stained pollen from WT (D) and 35S:PDS-RNAi transgenic flowers P1 (E) and P2 (F). Active pollen is blue and sterile pollen is tawny. Bar=100 μm. (G) Statistics for genotype and pollen phenotype in 35S:PDS-VIGS flowers. Pollen maturation rate (empty column) was recorded. Expression of PDS (black column) and PFINV4 (grey column) was investigated in P1 and P2. Total RNA from the indicated flowers was subjected to real time RT-PCR. Gene expression in the WT androecium was taken as 1, and actin was used as an internal control. The experiments were repeated three times. Mean expression and standard deviation are shown.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

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A B

E

D

F

C

Supplemental Figure 7

Zhao et al. 2013

Supplemental Figure 7 Cell morphology in the various ICSs. Epidermal cells of ICS are from the WT (A), the MPF2-RNAi (B), the MPF3-VIGS (C), the MPF2-RNAi-MPF3-VIGS-1 (D), the MPF2-RNAi-MPF3-VIGS-2 (E) and the MPF2-MPF3-VIGS-2 (F). Bar=100 µm. Quantification is presented in Figure 7G.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

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wild

-typ

e

35S

:AP

1

35S

:St-M

C

35S

:MP

F3

wild

-typ

e

35S

:AP

1

35S

:St-M

C

35S

:MP

F3

Ro

ssete

leaves n

um

be

r

10

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70

60

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LD SD

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-typ

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:AP

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:St-M

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:MP

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-typ

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:St-M

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:MP

F3

Bo

ltin

g t

ime (

da

ys)

10

0

70

60

50

40

30

20

LD SD

A

Supplemental Figure 8

Zhao et al. 2013

Supplemental Figure 8 Functional analyses in transgenic Arabidopsis.

Analysis of transgenic AP1, StMC and MPF3 in wild-type Arabidopsis. Bolting time (A) and numbers of rosette leaves (B) of a single-copy, target-inserted transgenic line were recorded under long-day (LD) and short-day (SD) conditions. More than ten plants of each line were included for analyses, and error bar is standard deviation.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

8

Genotype LineFlowering timea Node numberb

M ± SD P value M ± SD P value

Wild type WT 30.3±0.7 / 8.8±0.4 /

35S:MPF3

OE1 28.0±1.1 P=4.2×10-5 ** 6.1±0.6 P=1.6×10-10 **OE2 28.1±0.8 P=1.5×10-5 ** 6.1±0.4 P=1.0×10-12 **OE3 27.9±1.0 P=1.3×10-5 ** 6.1±0.4 P=1.0×10-12 **

35S:MPF3-RNAi

R1 29.5±1.3 P=0.1 8.4±0.5 P=0.1R3 29.9±0.8 P=0.2 8.9±1.1 P=0.9R5 29.6±0.9 P=0.1 8.4±0.9 P=0.1R7 29.6±0.9 P=0.1 9.3±0.7 P=0.1R8 29.8±1.0 P=0.2 9.5±1.2 P=0.1

R14 30.3±0.7 P=0.9 9.0±0.1 P=0.3

Supplemental Table 1 Variation in flowering time in transgenic Physalis plants.

a The time (day) from the germination to the time that the first flower opens. b The node number on the main stem once the first flower bud appears. M ± SD stands for average ± standard deviation, n=6. P value is relative to WT. Star (*) indicates the significance determined using two-tailed Student's t-test.

Supplemental Table 1

Zhao et al. 2013

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

9

a On variation, the plus indicates an increase, and the minus indicates a decrease.b Cell division (cd) index=organ size variation/cell size variation. For floral calyx, both organ and cell sizes are increased, therefore, cd index﹥1 indicates that cell division is promoted, otherwise, cell division is inhibited. For ICS, the organ size is reduced in all cases, while the cell size is either reduced or increased, therefore, 0﹤cd index﹤1 indicates that cell division is promoted, otherwise, cell division is repressed. The minus or plus in parenthesis indicates that cell division is inhibited or promoted.

Supplemental Table 2 MPF3 and MPF2 modulate cell division and cell expansion during calyx development in Physalis.

Supplemental Table 2

Zhao et al. 2013

MPF2-RNAiMPF2-MPF3-

VIGS-1

MPF2-RNAi-

MPF3-VIGS-1WT

MPF2-RNAi-

MPF3-VIGS-2

MPF2-MPF3-

VIGS-2MPF3-RNAi MPF3-VIGS

MPF2/MPF3 ratio 0.16 0.26 0.60 1 1.53 8.69 145.01 233.94

Floral calyx

P value Organ 0.0187 0.0873 0.0979 — 3.38×10-11 1.85×10-6 1.63×10-14 1.01×10-21

Cell 8.53×10-17 9.94×10-10 1.29×10-10 — 3.80×10-27 2.16×10-35 4.26×10-10 2.77×10-16

Variationa Organ +0.1338 +0.0734 +0.0929 — +0.6638 +0.3594 +1.4957 +1.8006Cell +1.0611 +0.3225 +0.6613 — +7.7034 +2.2255 +0.8474 +1.1434

cd indexb 0.13(-) 0.23(-) 0.14(-) — 0.09(-) 0.16(-) 1.77(+) 1.57(+)

Numbers Organ 28 20 14 50 22 15 25 61Cell 20 20 20 20 20 20 18 20

ICS

P value Organ 7.60×10-17 — 0.0009 — 0.6523 0.0194 0. 0358 0.0492Cell 3.81×10-7 — 3.29×10-14 — 0.4410 0.0001 1.32×10-7 0.0016

Variationa Organ -0.7265 — -0.2825 — -0.0344 -0.1494 -0.1894 -0.1084Cell +0.6384 — +0.9870 — -0.0481 -0.2305 -0.3145 -0.1774

cd indexb -1.14(-) — -0.29(-) — 0.72(+) 0.65(+) 0.60(+) 0.61(+)

Numbers Organ 17 — 12 46 15 43 60 62Cell 14 — 14 20 15 22 20 20

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

10

Probe

name

Position in

the MPF2

promoter

Sequences

(CArG-box is highlighted in red)

M0 -714~-695 TTCAGCTCCGACACCAAACT

M1 -2095~-2076 TCTAACAATTTTATGCTATT

M2 -1787~-1768 AAAGACATATAAATGCATAT

M3 -1491~-1472 AATCTCCATTTAAGGGTCAT

M4 -595~-576 TTTTACCATAAAAAGTAAAG

M5 -355~-336 TATATCAAAATTAGGTCTGC

M6 -329~-310 ATGTTCAATTATTGGTCCTT

Supplemental Table 3

Zhao et al. 2013

Supplemental Table 3 Probe sequences for EMSA.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

11

Supplemental Table 4 Parameter estimates under branch and branch-site models.

model P InL Estimate of parameters Models compared 2∆L

Branch model

M0：ω0=ω1=ωx

(x=2~11)13 -3195.77 ω0=ω1=ωx=0.122

Mf：ω free 23 -3468.92 ωx (0.05~999) Mf vs M0 546.30**

M2:ω0, ω1, ωx 21 -3469.62 ω0=0.09, ω1=999,

ωx=(0.05~0.33)M2 vs M0 547.70**

Site-specific model

M 1:nearly neutral 16 -3429.40 P0=0.80, ω0=0.18P1=0.08, ω1=1.00

Branch –site model

Model A 16 -3425.30 p0=0.80,p1=0.18,p2=0.02, ω=15.36

MA vs M1 8.20**

Supplemental Table 4

Zhao et al. 2013

**Codon site positively selected having P>95%.

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

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Supplemental Table 5 List of primers used in the present work.

Supplemental Table 5

Zhao et al. 2013

UsageGene name

Sequence UsageGene name

Sequence

Real time RT-PCR

MPF3F: 5’-GGA GAT GAA GCA AGG AGA AAT G-3’

Yeast two-hybrid

analysis

MPF3F: 5’-CAT CCA TGG GAA GAG GGA AAG TAG AAT-3’

R: 5’-AAC TTC CCA AGC AAA GAT TA-3’ R: 5’-GAT GGA TCC AAG TTT GCA GAT GAT ATG GAG-3’

PFFUL1F: 5’-GAG GTT GGT CTG ATT GTT-3’

MPF2F: 5’-CAT CCA TGG CAA GAG AGA AGA TTA AGA-3’

R: 5’-ATC TTC TCC TGC GTA GTG-3’ R: 5’-GAT GGA TCC CTA GCT GAG TGG TAG CCC TAA-3’

PFFUL2F: 5’-ACG AAG AAG GTG AAG GAA A-3’

PFGLO1F: 5’-GCA CCA TGG GGA GAG GAA AGA TAG AG-3’

R: 5’-GTG TGG CAA AAC GAA TGA A-3’ R: 5’-GTG GAT CCT TAG AAC CTC TCC TGC AAA TTA GG-3’

PFFLF: 5’-ACA CTG CTG ATC CCA AGG AA-3’

PFGLO2F: 5’-GCT CAT ATG GGG AGA GGG AAA ATA GAG-3’

R: 5’-GAT GAA GGC ACA TGA AAG GT-3’ R: 5’-GAT GGA TCC GCC TTG CCA CCA AGT CTT AGC T-3’

MPF2F: 5’-GTG CTG AGC TGA TGG AAG AA-3’

PFAGF: 5’-CAT CCA TGG ACT TCC AAA GTG ATC TAA C-3’

R: 5’-CTT CAA CAA AAT CTT CCC AC-3’ R: 5’-CTA GGA TCC ACA TCA GAA AGA AGA CCC TCC C-3’

PFINV4F: 5’-GTT GAG ATA GTA GGG ATG GC-3’

PFSEP1F: 5’-CTA CCA TGG GAA GGG GAA GAG TAG AAC TG-3’

R: 5’-TGG TCT TAT GTA TTT TGT CG-3’ R: 5’-CAT GGA TCC AAA GAT GGG AGC AGC TGC TTC-3’

PDSF: 5’-CCA GTG GAT ATT CTC AAG C-3’

PFSEP3F: 5’-CAT CCA TGG GAA GGG GTA GGG TTG AGC-3’

R: 5’-GAC ATG TCA GCA TAC ACA C-3’ R: 5’-CAT GGA TCC AGA ATC ATA AGC TTT CAA GG-3’

PFACTINF: 5’-AAC CGG AAT GGT GAA GGC TGG-3’

BiFC

MPF3F: 5’-GCT CTA GAA TGG GAA GAG GGA AAG TAG AAT-3’

R: 5’-CCA TAT CAT CCC AAT TGC TGA C-3’ R: 5’-GCG GAT CCT AGA TGT TTA TTC ATG TTG TAA-3’

Pull-downand EMSA

MPF3F: 5’-GAG GAT CCA TGG GAA GAG GGA AAG TAG AAT-3’

MPF2F: 5’-GCT CTA GAA TGG CAA GAG AGA AGA TTA AGA-3’

R: 5’-GTC TCG AGT CAT AGA TGT TTA TTC ATG TTG-3’ R: 5’-GCG GAT CCG CTG AGT GGT AGC CCT AAC TTC-3’

PFGLO1F: 5’-GCA CCA TGG GGA GAG GAA AGA TAG AG-3’

PFGLO1F: 5’-GCT CTA GAA TGG GGA GAG GAA AGA TAG AGA-3’

R: 5’-GTG GAT CCT TAG AAC CTC TCC TGC AAA TTA GG-3’ R: 5’-GCG GAT CCG AAC CTC TCC TGC AAA TTA GGC-3’

PFGLO2F: 5’-GCT CAT ATG GGG AGA GGG AAA ATA GAG-3’

PFGLO2F: 5’-GCT CTA GAA TGG GGA GAG GGA AAA TAG AGA-3’

R: 5’-GAT GGA TCC GCC TTG CCA CCA AGT CTT AGC T-3’ R: 5’-GCG GAT CCC ATT CTT TCA TGT AGA TTT GGC-3’

MPF2F: 5’-GAG GAT CCA TGG CAA GAG AGA AGA TTA AGA-3’

PFAGF: 5’-GCT CTA GAA TGG ACT TCC AAA GTG ATC TAA C-3’

R: 5’-GTC TCG AGC TAG CTG AGT GGT AGC CCT AAC-3’ R: 5’-GCG GAT CCG ACT AGT TGA AGA GGA GGT TGG-3’

PFAGF: 5’-CAT CCA TGG ACT TCC AAA GTG ATC TAA C-3’

cDNAisolation

PFFUL1F: 5’-GGA ACC AAA AGC ACT ACG CAG-3’

R: 5’-CTA GGA TCC ACA TCA GAA AGA AGA CCC TCC C-3’ R: 5’-GTT GAG AAA ACA ATC AGA CCA-3’

Subcellularlocalization

MPF3F: 5’-GCT CTA GAT GGG AAG AGG GAA AGT AGA AT-3’

PFFUL2F: 5’-GAA ATG GGG AGA GGA AGA-3’

R: 5’-GCG GTA CCT AGA TGT TTA TTC ATG TTG TAA-3’ R: 5’-ATA ATC ACT TGA CTC AGG TT-3’

RT-PCR

PFINV4F: 5’-TGG GTA GTT GAG ATA GTA GGG-3’

PFFLF: 5’-ATG GGA AGA GGT AGG GTA GAG-3’

R: 5’-GTT CAA TAA TGG CAC AGA GAG-3’ R: 5’-TCA ACC TTG GTT GCT GAC ATG G-3’

PFMST8F: 5’-TCA CCA CCA CTA TTG CAT ACC-3’

RNAi MPF3F: 5’-TCA CTA GTG GCG CGC CTC AAG AAA GAA CCA ACT CAT-3’

R: 5’-TGA TTA TGT TCT ACG CAC CAG R: 5’-AAG GAT CCA TTT AAA TAG ATT TTA CGA GAA CAT ATT-3’

PFSUT1F: 5’-AGT CAT AGC CAA GCA AGT AGC-3’

VIGS

PDSF: 5’-CTA CCA TGG GGC ACT CAA CTT TAT AAA CC-3’

R: 5’-GCT TTA AGA GAT TTA CCA AGA C-3’ R: 5’-CTA GGA TCC CTT CAG TTT TCT GTC AAA CC-3’

PFSUT2F: 5’-AGT GAC CAA TGT GCA TAG TGT C-3’

MPF3F: 5’-CGC CAT GGT CAA GAA AGA ACC AAC TCA T-3’

R: 5’-TGT GCA GGG TCC AGC TCG AGC-3’ R: 5’-GCG GAT CCA GAT TTT ACG AGA ACA TAT T-3’

PFCSAF: 5’-TTG TGC TAG AGG TCA TTG GAG-3’

MPF2-MPF3

F: 5’-CGC CAT GGC CAG CAT GAA GGA TAT CCT T-3’

R: 5’-CCA TGC GAG CCC ATT TGT TGC-3’ R: 5’-CTT CAT GCT GGA GAT TTT ACG AGA ACA TAT T-3’

PFACTINF: 5’-AAC CGG AAT GGT GAA GGC TGG-3’ F: 5’-TCG TAA AAT CTC CAG CAT GAA GGA TAT CCT T-3’

R: 5’-CCA TAT CAT CCC AAT TGC TGA C-3’ R: 5’-GCG GAT CCA GAT TTT ACG AGA ACA TAT T-3’

Supplemental Data. Zhao et al. (2013). Plant Cell 10.1105/tpc.113.111757

13