Supplemental Figures(1-4).ppt [兼容模式] · R3 c Band K568 mR3 c Band E568 a b c a b c a b c a...

A DRUS1

DRUS2

OsActin1

R S L FL SB SA UI FS

D St 4 St 8 St 9 St 10 St 11 St 12

E

B

C

H

IG

F

DR

US

2

NU

AN

CA

L

NUI CA

M

NU CA

N

DR

US

1

CANU

K

NU

AN

CA

J

100

70

55

130

100

70

Panicle length

IB: anti-DRUS1CT

DRUS1DRUS2

Non-specific*

kD

O

130

Supplemental Data. Pu et al. (2017). Plant Cell 10.1105/tpc.16.00218

Supplemental Figure 1. Expression patterns of DRUS1 and DRUS2 in rice.

(A) The mRNA level of DRUS1 and 2 in different tissues as assessed by RT-PCR. R, root; S, sheath;L, leaf blade of two-week-old seedlings; FL, flag leaf; SB, spikelet before pollination; SA, spikelet afterpollination (within 3 days); UI, uppermost internode; FS, flag leaf sheath. OsActin1 was used to ensureequal RNA loading.(B) and (C) GUS staining of the stems of ProDRUS1:GUS (B) and ProDRUS2:GUS (C) reporter riceplants. Upper panel, transverse section of stem; bottom panel, the basal node.(D–I) GUS staining in the inflorescence at different stages of anther development (D and E), pistil andstamen (F and G), and callus (H and I) of ProDRUS1:GUS (D, F and H) and ProDRUS2:GUS (E, Gand I) reporter rice plants.(J–N) In situ analysis of DRUS1 (J and K) and DRUS2 (L-N) in wild-type (WT) pistils beforefertilization. (K) and (N) are the corresponding sense controls. AN, antipodal; CA, carpel; I, integument;NU, nucellus.(O) Immunoblot analysis of DRUS1 and DRUS2 in the indicated tissues. The asterisk marks thenonspecific band. Coomassie Bright Blue staining of the total protein was used as a loading control.Bar = 1 mm in (F) and (G), 20 μm in (J) to (N).


DRUS1△

K-GFP

MergedFluorescence

GFP

Supplemental Figure 2. Transient expression of DRUS1ΔK-GFP in onion epidermal cells.

DRUS1ΔK-GFP was localized to the cell plasma-membrane after plasmolysis resulting from 0.15 Msucrose and 0.45 M mannitol treatment. Pro35S:GFP was used as a control. Arrows the plasmamembrane. Bar = 100 μm.


A CrRLK

At2g39360 CURVY1

At3g46290 HERCULES1

At5g59700

Os01g06280

Os03g17300

Os05g06990

Os06g22810

At5g54380 THESEUS1

Os03g55210

At5g61350 CAP1/ERULUS

At2g21480

At4g39110

At5g24010

At2g23200

Os03g03280

Os10g39010

Os03g03290

At1g30570 HERCULES2

Os04g52860

Os07g05370

At5g38990

At5g39000

At5g39020

At5g39030

Os05g20150

Os01g14930

At3g04690 ANXUR1

At5g28680 ANXUR2

Os01g56330 DRUS2

Os03g21540 DRUS1

At3g51550 FERONIA

Os04g49690

Os05g25430

Os05g25350

Os05g25370

Os05g25450

Os06g03610 RUPO

100

100

100

100

100

100

100

100

72

85

100

100

99

100

99

100

68

100

99

98

95

92

100

98

56

76

88

98

94

73

59

61

32

27

38

0.05


Malectin-like domain Ⅰ

Malectin-like domain Ⅱ

B

TM

CT

m1

m2

CDRUS1-EXa (23-451 aa) versus DRUS2-EX (24-452aa) 74.71%

DRUS1-EX versus FER-EX (28-446 aa) 51.79%

DRUS2-EX versus FER-EX 53.46%

DRUS1-INb (475-893 aa) versus DRUS2-IN (476-896 aa) 94.99%

DRUS1-IN versus FER-IN (470-895 aa) 79.71%

DRUS1-IN versus FER-IN 79.57%

Identity


Supplemental Figure 3. Protein sequence analysis of the CrRLK1L subfamily.

(A) Phylogenetic tree of the CrRLK1Ls from Arabidopsis thaliana (At) and Oryza sativa (Os) wasconstructed with MEGA(Version 6.0)(http://megasoftware.net/index.html), and Neighbor-Joining(NJ)statistical methods were used with 1000 bootstrap replications, with a p-distance substitution model,and pairwise deletion. RUPO, RUPTURED POLLEN TUBE; CAP1, [Ca2+] cyt-associated proteinkinase 1; DRUS1, DWARF AND RUNTISH SPIKELET1 and DRUS2, DWARF AND RUNTISHSPIKELET2. Bootstrapping values are indicated as percentages along the branches. The scale barcorresponds to 0.05 estimated amino acid substitutions per site.(B) Amino acid sequence alignment of DRUS1, DRUS2, and Arabidopsis FER. Dark blue highlighting,the same amino acids in all three proteins; Light blue highlighting , the same amino acids in two ofthree proteins; white, different amino acids in three proteins. Red lines, the Malectin-like domain I andII; Black line, the transmembrane domain (TM) and C-terminal region (CT); Dashed line, the deletedregion in the construct Pro35S:DRUS1/2ΔK-GFP. Arrows m1 and m2, respectively, indicate the K569Eand K667E mutations in the kinase domain.(C) Percent identity of the extracellular and intracellular domains among DRUS1, DRUS2, and FER.EXa, extracellular domain; INb, intracellular domain.


R3

c B

and

K568

E568

mR

3 c

Ban

da b c a b c a b c a b c a b c

C D

F

T-DNAr

550-f 550-rA

ATG TGATM KDSP

fer-4

E

FER 3' Tnos p35S HYG LBDRUS1/m1DRUS1pFERB

RB

Supplemental Figure 4. Molecular characterization of Arabidopsis fer-4 complementation plants with DRUS1 and m1DRUS1.

(A) Schematic representation of the fer-4 T-DNA insertion site. The filled boxes indicate exons. The empty boxes indicate 5'-, and 3’-UTR. Arrows indicate the primers used for genotyping. SP, signal peptide; TM, transmembrane domain; KD, kinase domain. (B) Structural diagram of the chimeric constructs used to complement fer-4. DRUS1 and m1DRUS1were expressed under the control of the FER promoter (pFER) and FER 3'-UTR (FER3'), respectively. Tnos, NOS terminator; p35S, CaMV 35S promoter; HYG, hygromycin-resistance gene; RB, right border; LB, left border. (C) Genotyping of Col-0, homozygous and heterozygous fer-4, and complementation plants. PCR was performed using the primer pair 550-f/550-r for WT FER (a band), 550-f/T-DNAr for the fer-4 T-DNA insertion (b band), and DRUS1-ex-f/DRUS1-ex-r for DRUS1 or m1DRUS1 (c band). (D) and (E) DNA sequencing of the c band in R3 and mR3 plants to identify WT DRUS1 (D) and m1DRUS1 (E). The WT K568 and mutated site E568 are boxed. (F) fer-4 plants complemented with DRUS1 and m1DRUS1, showing the rescued stature at about 45 days after germination. FER is the relative WT plant segregated from the fer-4+/- progeny.


Supplemental Figure 5. DRUS1 rescued fer-4 fertility better than did m1DRUS1.

(A) Comparison of seed setting in Col-0, fer-4 heterozygous (fer-4+/-), homozygous (fer-4), and therecovery plants R3 (DRUS1) and mR3 (m1DRUS1). Arrows indicate aborted seeds.(B) Statistical analysis of the normal and aborted seeds set by the plants shown in (A). Error barsindicate the means±SD; n ≥ 10 siliques from three individual plants were investigated.(C) and (D) Analysis of the mRNA expression of DRUS1 (C) and m1DRUS1 (D) in a rescued fer-4homozygous plant by RT-PCR. AtActin7 was used as a loading control. Col-0 and fer-4+/- were used aspositive controls for FER expression and a negative control for DRUS1 and m1DRUS1 expression.(E–G) Immunoblot analysis of DRUS1 (E) and m1DUS1 (G) protein expression in the siliques of therescued plants shown in (A) using an antibody raised against the extracellular domain (ED) of DRUS1(anti-DRUS1ED). DRUS1 protein was detected in the microsomal fraction but not in the soluble fractionin line R4 (F). The asterisk indicates the nonspecific band used as a loading reference. T, total protein; S,soluble protein; M, microsomal protein.

*Col-0 mR3 mR7a

Non-specific

DRUS1

E

Non-specific

DRUS1

Col-0 R4 R3

130

95

72

56

kD Col-0 R4 Col-0 R4 Col-0 R4

DRUS1

Non-specific

T S M

A

F

FER

DRUS1

AtActin7

Col-0 fer-4+/- R3 R4

FER

m1DRUS1

AtActin7

fer-4+/- mR3 mR7a

C

D

G*

*

*

kD

130

130kD

See

d N

umbe

r pe

r S

iliqu

e

BNormal

Aborted

0

10

20

30

40

50

60

70

56

56

IB: anti-DRUS1ED

IB: anti-DRUS1ED

IB: anti-DRUS1ED


Supplemental Figure 6. Phenotypic analysis of drus1-2 mutants from a BC2F2 population.

(A) Diagram showing the generation of drus1-2 BC2F2 progeny by backcrossing with wild type HWplants.(B) Genotyping of drus1-2 using the primer pairs shown in Figure 3A. The a and b bands represent WTDRUS1 and drus1-2 T-DNA insertions respectively, as in Supplemental Figure 7B.(C) The mRNA levels of DRUS1 and 2 in drus1-2 plants as assessed by RT-PCR. OsActin1 was used asa loading control. Primer pairs F2/R2 and F5/R5 were used, respectively, to detect DRUS1 and DRUS2mRNA.(D) and (E) The stature of drus1-2 mutant plants at the seed-filling stage (D), and the measurement ofplant height (E). Error bars indicate the means±SD; n=30 tillers from 10 plants.(F) to (H) A comparison of inflorescences (F), panicle length and primary branch number (G), the spikeletnumber (H) between HW and drus1-2 mutant plants at the flowering stage. Error bars indicate themeans±SD; n=30 panicles from 10 plants.(I) and (J) A comparison of seed setting between HW and drus1-2 plants at the harvest stage. Error barsindicate the means ± SD; n=30 panicles from 10 plants.Bar=10 cm in (D) and 5 cm in (F) and (I).

E

Pla

nt H

eigh

t (cm

)

0

30

60

90

120

HW drus1-2

HW drus1-2

F

I

HW drus1-2

DA♀ drus1-2 × HW ♂

♀BC1F2 (drus1-2) × HW♂

BC1F1(drus1-2+/-)

BC2F1(drus1-2+/-)

BC2F2(drus1-2)

J

0

20

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60

100

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d S

et R

ate

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150

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HW drus1-2

DRUS2

CDRUS1

OsActin1

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30

20

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Prim

ary Branch

Num

ber per PanicleP

anic

le L

engt

h (c

m)

a b a b

B HW drus1-2


(1: DJ, 2: drus1-1, 3: drus2, 4: drus1-1-/- drus2+/-, 5: drus1-1+/- drus2-/-, 6: drus1-1 drus2)

♀ drus2 × drus1-1 ♂

BC1F1 (drus1-1+/- drus2+/- )

♀DJ × BC1F2 (drus1-1-/- drus2+/- ) ♂

BC2F1 (drus1-1+/- drus2+/- )

BC2F2

A

a b c da b c d a b c d a b c d a b c d a b c d

B 1 2 3 4 5 6

D

DRUS1-3' (F3 and R3)DRUS1 (F2 and R2)DRUS1-5' (F1 and R1)

OsActin1DRUS2-3' (F6 and R6)DRUS2 (F5 and R5)DRUS2-5' (F4 and R4)

C 41 2 3 5 6

ab

a a

d

E

c

1 2 3 4 5 60

20

40

60

80

100

120

Inte

rnod

e Le

ngth

(cm

)

ⅠⅡⅢⅣⅤ

P

532 4 61

Ⅰ

Ⅱ

Ⅲ

Ⅳ

Ⅴ

P,

See

d S

et R

ate

(%)

20

40

1ND

2 3 4 5 6

G >10 DAF

1-3 DAF4-7 DAF

WSBF

MS100

80

60

0

F

1 2 3 4 5 6

Truncated DRUS2

DRUS1

*

Truncated DRUS1

IB: anti-DRUS1ED

H

130

100

55

70

170

40

1 2 3 4 5 6kD

6

55IB: anti-α-tubulin

DRUS2


Supplemental Figure 7. Molecular characterization and phenotype comparison of six genotypes.

(A) Diagram showing the generation of drus1-1+/- drus2+/- BC2F2 progeny; six genotypes were identifiedand used in subsequent analysis.(B) Genotyping of the drus1-1 and drus2 insertions in the six genotypes using the primer pairs shown inFigure 3A. Among them, F2/R2 was used for wild-type (WT) DRUS1 (a band), Ngus-r/R2 was used fordrus1-2 T-DNA (b band), F5/R5 was used for WT DRUS2 (c band), and 2715-LB/R5 was used for drus2T-DNA (d band).(C) RT-PCR analysis of full-length or partial transcripts of DRUS1 and DRUS2 in the six genotypesshown in (A). The primer pairs (see Figure 3A) used to amplify each fragment are shown on the right.OsActin1 was used as an internal control.(D and E) Comparison of culms and the internode length among the six genotypes at the harvest stage.The diamonds in (D) indicate nodes. P, I, II, III, IV, and V in (D) and (E), respectively, denote the panicleand internodes from the apical to the basal end. Error bars indicate the means ± SD; n=30 panicles from10 plants. a, b, c, and d indicate a significant difference at p < 0.001 (Student’s t-test).(F) Seed setting in the panicles of six genotypes at the harvest stage. Red arrows mark aborted seeds.(G) Seed setting rate. Mature and aborted seeds at sequential developmental stages (indicated to theright) were calculated and normalized to the total spikelet number. The different green color boxesindicate the green spikelets, and the white box represents white spikelet. BF, before fertilization; DAF,days after fertilization; MS, mature seed; ND, not detected. WS, white spikelet. Error bars indicate themeans±SD; n=30 panicles from 10 plants.(H) Immunoblot analysis of DRUS1 protein expression in spikelets (at anther stage 10) of the sixgenotypes using anti-DRUS1ED antibodies, which recognized DRUS1 strongly (arrows) and DRUS2(arrowheads) weakly when DRUS1 was absent. The asterisk indicates a nonspecific band. Theexpression of α-tubulin was used as a loading control.Bar=10 cm in (D), 5 cm in (F), and 1 mm in (G).


J

See

d N

umbe

r pe

r P

anic

le 140

12010080

6040

2020100

WT 3 edRi

14 16 18 24 27

WT 3 edRi

E

Pla

nt H

eigh

t (cm

)

100

80

60

40

20

014 16 18 24 27

WT

D

edRi

14 18WT

edRi

*

** ***

14 18

C WkS MoS SS

AP1 P2

ATG TGATM KDSP

edRi

Os03g21540

3 14 18

edRiWT RG-1

B

KWT RG-1 3 14 18

DRUS1

DRUS2

Os04g49690

OsActin1

edRi

H

Ⅰ

Ⅱ

Ⅲ

Ⅳ

Ⅴ

P

I

P Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ

WTedRi-3

Inte

rnod

e Le

ngth

(cm

)

0

5

10

20

30

15

25

(+131) (+768)

F25

20

15

10

5

0

Prim

ary Branch

Num

ber per Panicle

WT 3 14 16 18 24 27 edRi

25

20

15

10

5

0Pan

icle

Len

gth

(cm

)

Spi

kele

t Num

ber

per

Pan

icle

140120

100

80

60

40

200

WT 3 edRi

14 16 18 24 27

SSMoSGS or WkS

G


Supplemental Figure 8. Phenotype of edRi plants at the reproductive stage.

(A) Schematic representation of the cDNA of Os03g21540. The lower black bar denotes the target site ofthe edRi constructs. P1 and P2 are the primers used for RT-PCR. The filled boxes indicate exons. Theempty boxes indicate 5'-, and 3’-UTR. SP, signal peptide; TM, transmembrane domain; KD, kinasedomain.(B) Wild-type (WT, Nipponbare), RG-1 (regenerated from a callus without the construct), and threeindependent T0 plants at the flowering stage.(C) Inflorescences shown in (B). Inset showed close-up view of three spikelet grades. The weak spikelets(WkS) were green, with a width similar to those of wild type; The moderate spikelets (MoS) were lightgreen and narrower than those of the wild type; The severe spikelets (SS) were pale or white and muchnarrower than those of the wild type. Bar=5 mm.(D) Seed setting at the harvest stage.(E) Height of the T1 plants. Error bars indicate the means±SD; n=20 plants per line.(F) Panicle length and primary branch number in T1 plants. Error bars represent the means±SD; n=30panicles from 10 plants.(G) and (J) The spikelet number in three spikelet grades (G) and seed number (J) in T1 plants. Error barsrepresent the means ± SD; n=30 panicles from 10 plants.(H) Comparison of culms from WT and one representive T1 plants. Arrows indicate the nodes.(I) Statistical analysis of the internode length shown in (H). Bars indicate the means±SD; n ≥ 26 plants.(K) RT-PCR analysis of the mRNA level of DRUS1, DRUS2 and a related gene in WT and threeindependent T0 plants. OsActin1 was used as an internal control.


♀BC1F2 (drus1-1-/- drus2+/-) × ProDRUS1:DRUS1-7Myc-6His(DRUS1+/+)♂

F1 (DRUS1+/-/drus1-1+/- drus2+/-)

F2 (DRUS1+/-/dk)

A

F3 (DRUS1+/+/dk, DRUS1+/-/dk, dk)

21 23a b c d a b c da b c d a b c d

11 16C

GS WS

F

WS

GSGDRUS1+/-/dk

11 16 21 23 NCB

DRUS1-MH

100

12 21 23 24 11 16 17 20 PC

D

P.S.

DRUS1+/+/dkDRUS1+/-/ dk

DRUS1-MH130170

55

kD

IB: anti-cMyc

ND

E

See

d S

et R

ate

(%)

100

80

60

40

20

0

Supplemental Figure 9. Molecular characterization and flower organ phenotypes of DRUS1complementation plants.

(A) Diagram showing the generation of the complementation plants by crossing drus1-1-/- drus2+/- withProDRUS1:DRUS1-7myc-6His/WT. F3 progeny were used for phenotype analysis.(B) and (C) Genotyping of the DRUS1 complementation plants. The DRUS1-7Myc-6His DNA band(arrow in B) was amplified using the primer pair F2/7Myc-6His-r; the b and d bands in (C), respectively,represent the drus1-1 and drus2 T-DNA insertions, as in Supplemental Figure 7B.(D) Immunoblot analysis of the DRUS1-7Myc-6His fusion protein in the complemented plants. Rubiscostained with Ponceau S (P.S.) was used as a loading reference.(E) Seed setting rate was compared among DRUS1+/+/dk, DRUS1+/-/dk and dk plants. Error bars indicatethe means±SD; n=30 panicles from 10 plants.(F) and (G) Anther (F) and pistil (G) from green spikelet (GS) and white spikelet (WS) in DRUS1+/-/dk.Bar= 1 mm in (F) and (G).


Ex

WT

E

MP

F

EX

IN

SG

G

MP

H

EX

IN

SG

edRi

WT WkS MoS

I 2-K

I Sta

inin

gT

TC

Sta

inin

g

C edRi D

WT WkS MoSedRi

% o

f Def

ectiv

e P

olle

nby

I 2-K

I Sta

inin

g

80

60

40

20

0

edRi WT

BWkS/MoS SS

AWT

edRi WkS MoS SS

Supplemental Figure 10. Defects in floral organs and pollen grains in edRi plants.

(A) Anthers from three spikelet grades.(B) Pistils from three spikelet grades.(C) I2-KI (upper panel) and (TTC) (lower panel) staining of pollen grains from weak spikelet (WkS) andmoderate spikelet (MoS). Arrows indicate sterile pollen grains that lack staining.(D) Statistical analysis of the inviable pollen grains in (C). Error bars indicate the means±SD; n ≥ 500pollen grains.(E)-(H) Transmission electron microscopy of wild-type (WT) (E and F) and edRi (G and H) maturepollen grains. (F) and (H) are high magnification view of the pollen grains in (E) and (G), respectively.EX, exine; IN, intine; SG, starch granules; MP, mature pollen.Bar=1 mm in (A) and (B), 100 µm in (C) , 10 μm in (E) and (G), and 500 nm in (F) and (H).


drus1-1 drus2H

F

(n=725 258)

% o

f I2-K

I Sta

inin

g 100

80

60

40

20

120

0

G

Best

Better

Good

No Staining

(n=1828 2592)

% o

f TT

C S

tain

ing

100

80

60

40

20

120

0

drus1-1 drus2

I 2-K

I Sta

inin

gT

TC

Sta

inin

g

E

DJ drus1-1 drus2

A

HW drus1-2

BHW drus1-2D

CDJ

drus1-1

drus2

Supplemental Figure 11. Floral organs and pollen viability in the drus1-1 and drus2 single mutants.

(A) and (B) Anthers.(C) and (D) Pistils.(E) I2-KI (upper panel) and (TTC) (lower panel) staining. Arrow indicates the unstained pollen.(F) and (G) Statistical analysis of pollen viability based on the I2-KI staining and TTC stainingintensities (E). The pollen staining grades are shown on the right. The total number of pollen grainscounted for each genotype is indicated in parentheses below the graphs. Error bars indicate themeans±SD.(H) DAPI staining.Bar=1 mm in (A) to (D), 100 μm in (E); 20 μm in (H).


drus1-1

E MP

J

St 12

MPE

K

St 12

drus2

MP

I

St 12

drus1-1-/- drus2+/-

E

St 10

EEnT

Msp

drus1-1+/- drus2-/- (WS)

G

E MP

drus1-1+/- drus2-/- (GS)

H

St 12

edR

i

A

St 1

BAr

St 2 St 4

Sp

D E EMLT

SpC

V

St 5

Ar

C

St 3

edRi

St 11

E

Msp

F

Supplemental Figure 12. Transverse sections of edRi, drus1-1, drus2, drus1-1+/-drus2-/-, and drus1-1-

/- drus2+/- anthers at the indicated stages.

(A–E) edRi anthers at stages 1–5.(F) edRi anthers at stages 11.(G) drus1-1+/- drus2-/- anthers in a white spikelet at stage 10.(H) drus1-1+/- drus2-/- anthers in a green spikelet at stage 12.(I) A drus1-1-/- drus2+/- anther at stage 12.(J) A drus1-1 anther at stage 12.(K) A drus2 anther at stage 12.Ar, Archesporial cell; C, connective tissue; E, epidermis; En, endothecium; ML, middle layer; Msp,microspores; MP, mature pollen; Sp, sporogenous cell; T, tapetum; V, vascular tissue. Bar=20 μm.


edR

iW

T

Dy

St 6

V

TMMC

St 6

T

MMC

antisense

CESt 8

antisense

T

DSt 10

antisense

Msp

T

E

T

MMC

V

antisense

G

antisense

T

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antisense

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I T

MMC

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sense

J

Rel

ativ

e E

xpre

ssio

n Le

vel

（D

RU

S1

plus

DR

US

2）

WT edRi

ADRUS1DRUS2

0

1

2

3

4

5 B

0

1

2

3

4

WT edRi

Rel

ativ

e E

xpre

ssio

n Le

vel

（O

sCP

1）

Supplemental Figure 13. OsCP1 was upregulated in edRi anthers.

(A) and (B) The relative mRNA level of DRUS1 plus DRUS2 (A) and OsCP1 (B) in wild-type (WT) andedRi anthers from stage 8 to stage 12. The mRNA level of DRUS1, DURS2 or OsCP1 in WT anthers atstage 8 was set to 1. OsUBQ was used as an internal control. Error bars indicate the means±SD ofthree biological replicates.(C–J) In situ analysis of OsCP1 in WT (C-F) and edRi (G-J) anthers at the indicated stages. (F) and (J)correspond to the sense control.Dy, dyad cell; ESt 8, early stage 8; MMC, microspore mother cell; Msp, microspores; T, tapetum; V,vacular tissue. Bar=20 μm in (C-J).

F

sense

Dy

V

VVV


0

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4

6TDR

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ativ

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xpre

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API5

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0.5

F

GGAMYB

0

6

2

4

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H

WT anther edRi anther

**

**

Supplemental Figure 14. The expression of tapetal PCD-related genes in edRi anthers, as determinedby RT-qPCR.

OsUBE2 was used as an internal control. The mRNA level in wild-type (WT) anthers at stage 8 was setto 1. Error bars indicate the means ± SD of three biological replicates. Asterisks indicate a significantdifference by Student’s t-test; **p < 0.01.


Rel

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CSA

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UDT1

0

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20

40

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vel OsMADS3

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CYP704B2

0

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1.2

0.8

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DPW1

0

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1.2

0.8

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2.0

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F

BA

D E

C

G

WT anther edRi anther

******

***

***

***

***

***

***

***

***

Supplemental Figure 15. The expression of anther developmental genes in edRi anthers, asdetermined by RT-qPCR.

OsUBE2 was used as an internal control. The mRNA level in WT anthers at stage 8 was set to 1. Errorbars indicate the means ± SD of three biological replicates. Asterisks indicate a significant difference byStudent’s t-test; ***p < 0.001.


DJ anther drus1-1+/- drus2-/- green spikelet anther

*

A CSA

Rel

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4

3

2

1

0

*** ***

***

B

15

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0Rel

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MST8

***

C

20

10

0

30

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INV4

***

D UGP2

0

3000

2000

1000

4000

5000

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6000

8000

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10105

1

4

******25

***

***

***

Supplemental Figure 16. The altered expression of sugar metabolism-associated genes in drus1-1+/-

drus2-/- anthers, as determined by RT-qPCR.

OsUBE2 was used as an internal control. The mRNA level in DJ anthers at stage 9 was set to 1. Errorbars indicate the means ± SD of three biological replicates. Asterisks indicate a significant difference byStudent’s t-test; *p < 0.05, ***p < 0.001.

25


DJ

CP

W/m

g F

W0

100

200

300

400

500DJ

CP

W/m

g F

W

0

500

1000

1500

2000A B

0

500

1000

1500

DJ

CP

W/m

g F

W

drus1-1+/- drus2-/-

drus1-1+/- drus2-/- drus1-1+/- drus2-/-

DJdrus1-1+/- drus2-/-

CP

W/m

g F

W

0

500

1000

1500

2000

C

*

Supplemental Figure 17. 14C-signal accumulation in the flowers and stem.

(A) and (B) 14C-signal accumulation in the palea/lemma (A) and anther (B) at stage 11 (St 11) and stage12 (St 12).(C) and (D) 14C-signal accumulation in the stem at stage 11 (C) and stage 12 (D). S1, S2 and S3represent the internode III, II and I (see Supplemental Figure 7D), respectively.Error bars indicate the means ± SD of three biological replicates. Asterisks indicate a significantdifference by Student’s t-test; *p < 0.05.

D


A0 min 15 min 30 min

GA3

DRUS1DRUS2

Non-specific*

130

100

70

kD

B

DJ

DJ +Uni

DJ +Uni +GA3

dk

dk +Uni

dk +Uni +GA3

16

12

8

4

0

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E

She

ath

grow

th (

cm)

15

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0

C

+ +-++-

GA3

Uni

-- +-- +

DJ

dk

D

Non-specific*

130

100

70

kD

DRUS1DRUS2

IB: anti-DRUS1CT IB: anti-DRUS1CT

DJ

DJ +Uni

DJ +Uni +GA3

dk

dk +Uni

dk +Uni +GA3


Supplemental Figure 18. The relationship between the DRUS1/2 and GA signaling pathway.

(A) Protein expression of DRUS1 and 2 in 11-day-old seedlings in response to 10-6 M GA3 treatment. Thenon-specific band marked by an asterisk was used as a loading control.(B) Protein expression of DRUS1 and 2 in 18-day-old GA-deficient mutants sd1-8 and d18-AD and theGA-response mutant gid1-20. Akibare is the wild-type control for d18-AD, and Nanjing6 is the wild-typecontrol for sd1-6 and gid1-20. The non-specific band marked by an asterisk was used as a loadingcontrol.(C) and (D) Seedling growth in response to Uniconazol (Uni) and GA3 treatment. The seedlings weregrown in medium with (+) or without (-) Uni for 17 d, then treated with GA3 for one more week (C). Thelength of the last leaf sheath before and after GA3 treatment was measured (D). Arrowheads in (C) markthe lamina joint of the leaf sheath used for measurement. The experiment was performed three timeswith different plants and one representative result is shown. See Methods for more details. Error barsindicate the means ± SD. n=11-19. Bar=1 cm in (C).(E) The GA-responsive genes in dk changed in a similar way as those in DJ in response to GA3

treatment. Error bars indicate the means ± SD.


MP

EEn

EEn

anti-DRUS1ED Control

St 8 St 9 ESt 10 St 8

LSt 10 St 11 St 12 LSt 10

Mer

ged

F

ITC

Mer

ged

F

ITC

T

ML

MspTds

T

ML

Tds

T

ML

Msp

T

ML

Msp

T

ML

ML

T

Msp

TdsT

ML

A1 B1 C1 G1

A2 B2 C2 G2

EnT

D1 E1 F1 H1

BP

EnT

Msp

T

Msp

D2 E2 F2 H2

Supplemental Figure 19. Immunofluorescence assay of DRUS1 protein expression in wild-typeanthers at the indicated stages.

(A1-F2) Immunofluorescence images using anti-DRUS1ED antibody and FITC-conjugated secondaryantibody.(G1-H2) Negative control using antibody-depleted solution and FITC-conjugated secondary antibody.BP, bicellular pollen; E, epidermis; En, endothecium; ESt10, early stage 10; ML, middle layer; MP,mature pollen; Msp, microspore; LSt10, late stage 10; T, tapetum; Tds, tetrads; V, vascular tissue.Bar= 20 μm.


Seed number per silique

Line Genotype Total Normal (%) Aborted (%) Unfertilized (%)

Col-0 FER 55.93 ± 4.1954.98 ± 4.41

(98.30%)0.30 ± 0.46

(0.54%)0.65 ± 0.89

(1.16%)

fer-4+/- fer-4+/- 49.48 ± 4.1837.23 ± 5.63

(75.24%)2.53 ± 2.51

(5.10%)9.33 ± 3.77

(18.85%)

fer-4 fer-4 40.05±5.7716.10 ± 5.55

(40.20%)3.25 ± 2.07

(8.11%)20.70 ± 4.40

(50.11%)

R3 DRUS1/fer-4 43.83 ± 3.9132.23 ± 6.41

(73.53%)2.30 ± 2.04

(5.25%)9.30 ± 4.51

(21.22%)

R4 DRUS1/fer-4 51.98 ± 4.9443.43 ± 7.87

(83.55%)3.13 ± 2.90

(6.01%)5.38 ± 3.89

(10.34%)

R5 DRUS1/fer-4 49.50 ± 4.2138.58 ± 4.62

(77.93%)3.13 ± 2.74

(6.31%)7.83 ± 3.42

(15.81%)

HM3aDRUS1/fer-4 53.10 ± 4.19

45.40 ± 5.91(85.50%)

2.30 ± 2.82(4.33%)

5.43 ± 3.37(10.22%)

HM4a DRUS1/fer-4 49.60 ± 3.8945.40 ± 5.23

(91.53%)1.38 ± 1.25

(2.77%)2.93 ± 2.86

(5.90%)

HM5a DRUS1/fer-4 49.68 ± 4.3644.43 ± 6.51

(89.43%)1.70 ± 1.40

(3.42%)3.55 ± 3.50

(7.15%)

HZ3b DRUS1/fer-4+/- 53.85 ± 4.8449.15 ± 5.97

(91.27%)1.28 ± 1.30

(2.37%)3.43 ± 3.22

(6.36%)

HZ4b DRUS1/fer-4+/- 56.73 ± 4.4351.85 ± 7.04

(91.41%)1.95 ± 1.78

(3.44%)2.95 ± 3.52

(5.20%)

HZ5b DRUS1/fer-4+/- 52.25 ± 3.4147.60 ± 4.25

(91.10%)1.80 ± 1.50

(3.44%)2.85 ± 2.09

(5.45%)

aSegregated T2 homozygote from a T1 fer-4+/-. bSegregated T2 heterozygote from a T1 fer-4+/- (n=20-40).

Supplemental Table 1. fer-4 fertility is rescued by ProFER:DRUS1-FER3'


Seed number per silique

Line Genotype Total Normal (%) Aborted (%) Unfertilized (%)

Col-0 FER 51.63 ± 3.2350.70 ± 3.47

(98.21%)0.40 ± 0.71

(0.77%)0.53 ± 0.85

(1.02%)

fer-4+/- fer-4+/- 46.83 ± 3.8635.55 ± 5.65

(75.92%)2.15 ± 1.82

(4.59%)9.13 ± 4.06

(19.49%)

fer-4 fer-4 39.7±5.4316.15 ± 5.78

(40.68%)3.00 ± 1.97

(7.56%)20.55 ± 3.25

(51.76%)

mR3 m1DRUS1/fer-4 43.50 ± 3.6730.58 ± 5.11

(70.29%)1.55 ± 1.38

(3.56%)11.38 ± 3.91

(26.15%)

mR5a m1DRUS1/fer-4 41.65 ± 4.2527.30 ± 6.37

(65.55%)2.23 ± 1.75

(5.34%)12.13 ± 4.67

(29.11%)

mR7a m1DRUS1/fer-4 38.53 ± 4.3621.18 ± 5.75

(54.96%)3.23 ± 2.90

(8.37%)14.13 ± 3.63

(36.67%)

mR22a m1DRUS1/fer-4 40.80 ± 4.8826.55 ± 5.86

(65.07%)1.83 ± 1.58

(4.47%)12.43 ± 2.76

(30.45%)

mR5b m1DRUS1/fer-4+/- 44.40 ± 3.8834.53 ± 5.44

(77.76%)1.40 ± 1.22

(3.15%)8.48 ± 3.40

(19.09%)

mR7b m1DRUS1/fer-4+/- 43.70 ± 4.3533.10 ± 6.83

(75.74%)1.48 ± 2.15

(3.38%)9.13 ± 4.33

(20.88%)

mR22b m1DRUS1/fer-4+/- 44.15 ± 4.6937.15 ± 6.20

(84.14%)0.93 ± 1.16

(2.10%)6.08 ± 2.80

(13.76%)

mR4b m1DRUS1/fer-4+/- 41.25 ± 4.5228.65 ± 7.03

(69.45%)1.25 ± 1.46

(3.03%)11.35 ± 4.89

(27.52%)

aSegregated T2 homozygote from a T1 fer-4+/-. bSegregated T2 heterozygote from a T1 fer-4+/- (n=20-40).

Supplemental Table 2. fer-4 fertility is rescued by ProFER:m1DRUS1-FER3'


Number of seedlings

Parent genotype

Paniclea

#

Germin-ated

seeds drus1-1

drus1-1-/-

drus2+/- drus2drus1-1+/-

drus2-/-drus1-1 drus2

Ratio χ2

drus1-1-/-

drus2+/-

1 99 25 52 - - 22

DRUS2: drus2+/- : drus2

202 : 339: 1081: 1.67: 0.53

28.5254

p < 0.005

2 100 32 43 - - 25

3 183 48 103 - - 32

4 129 41 73 - - 15

5 138 56 68 - - 14

drus1-1+/-

drus2-/-

1 66 - - 16 31 19

DRUS1: drus1-1+/-: drus1-1

136 : 199 : 871 : 1.46 : 0.64

12.7441

p < 0.005

2 96 - - 24 54 18

3 113 - - 43 49 21

4 84 - - 27 41 16

5 63 - - 26 24 13

aPanicles were randomly collected from different plants.

Supplemental Table 3. Segregation of drus1-1-/- drus2+/- and drus1-1+/- drus2-/- selfed progeny


Progeny

Parents (♀×♂) drus1-1+/- DRUS1 drus2+/- DRUS2 Total TEF TEM χ2

DJ × drus1-1+/- drus2-/- 88 89 - - 177 NA 97.7%0.0227

0.75 < p < 0.9

drus1-1+/- drus2-/- × DJ 46 54 - - 100 85.2% NA0.32

0.5 < p < 0.75

DJ × drus1-1-/- drus2+/- - - 55 54 109 NA 102%0.01

0.9 < p

drus1-1-/- drus2+/- × DJ - - 66 65 131 103% NA0.03

0.75 < p < 0.9

TEF, transmission efficiency of female gametes; TEM, transmission efficiency of male gametes; NA, not applicable.

Supplemental Table 4. Transmission efficiency of the drus1-1 drus2 male and female gametophyte


Supplemental Table 5. Complete and partial complementation of drus1-1 drus2 with ProDRUS1:DRUS1-7Myc-6His

Parentphenotypes

Paniclea

#Germinated

seeds

Number of seedlings

ParentgenotypeDRUS1/dk dk

Completecomplementation

1 39 39 0

DRUS1+/+/dk2 55 55 0

3 84 84 0

Total 178 178 0

Partialcomplementation

1 49 42 6

DRUS1+/-/dk

2 54 43 11

3 27 21 6

4 54 40 11

5 59 46 16

Total 242 192 50

Segregation ratio 3.84 1

aPanicles were randomly collected from different plants.


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