(WT) PC V SC SC - The Plant Cell · 1 Supplemental Data. Huang et al. Plant Cell (2015)...

Supplemental Figure 1. The Phenotypes of Rice GA-Related Mutants.

(A) to (C) Appearance of the GA-related mutants and the corresponding wild-type

plants. Bar = 10 cm.

(D) to (F) Anatomical analysis of the cross-sections of internodes (D) and (E) and leaf

sheaths (F) from GA-related mutants and the corresponding wild-type plants, to show

the distribution patterns of sclerenchyma cells.

SC, sclerenchyma cells; PC, parenchyma cells; V, vascular bundles. Arrows indicate the

altered sclerenchyma cells. Bar = 0.5 cm.

Nanjing6 (WT)

sd1-8

gid1-20 slr1-6

Zhonghua11 (WT)

d18-AD

Akibare (WT)

D

Nanjing6 (WT) sd1-8 gid1-20A Akibare (WT) d18-ADC

E F

BZhonghua11

(WT)

slr1-6

SCVPC

SC

SC

SC SC

SCV

PC

V

PC

PC

PC

V

V

SC

SC

Supplemental Data. Huang et al. Plant Cell (2015) 10.1105/tpc.15.00015

Supplemental Figure 2. Phylogenetic Analysis of the Secondary Wall NACs in Rice and

Arabidopsis.

In Arabidopsis, SND1 and NST1are key regulators for secondary wall biosynthesis in fibers;

VNDs are key regulators for secondary wall biosynthesis in vessels; SND2/3 functions

downstream of SND1 (Demura and Ye, 2010; Zhao and Dixon, 2011; Zhong and Ye, 2007).

The numbers at each node represent the bootstrap support (percentage). The scale bar is an

indicator of genetic distance based on branch length.

VND like

SND1 like

SND2/3 like

Os-NAC29

Os-NAC7

NST2

NST1

SND1

VND6

Os-NAC90

VND7

Os-NAC31

SND3

Os-NAC3

SND2

Os-NAC62

Os-NAC7399

77

97

100

100

97

96

99

57

77

100

0.2


Supplemental Figure 3. NAC29 and NAC31 Are Functional TFs That Regulate Cellulose

Synthesis.

(A) The four month-old wild type and NAC29- and NAC31- overexpressing (Ox) plants. Bar

= 10 cm.

(B) qRT-PCR examination of the NAC29 and NAC31 transcripts in the transgenic plants.

Rice TP1 was used as an internal control.

(C) Diagrams of the constructs used for transactivation assay.

(D) Luciferase activities in the rice protoplasts cotransfected with the constructs shown in (C).

(E) and (F) Subcellular localization pattern of NACs by transfecting the rice protoplasts with

GFP tagged TFs. The nuclei were counterstained with DAPI. Merge, merged images of GFP

and DAPI. Bars = 5 μm.

Error bars in this figure indicate the SD of three biological repeats.

Reporter

NAC29-GFP DAPI Merge DIC

NAC31-GFP DAPI Merge DIC

E

F

Rel

ativ

e ex

pres

sion

leve

l

WT0123456789B

WT NAC29 Ox NAC31 Ox

A

Rel

ativ

e lu

cife

rase

act

ivity

Mock NAC29 NAC3101

2

3

4

5

6

7D

CEffector

NOS ter35S mini proGal4BD NAC29/31

CaMV 35S proLUC

NOS ter35S mini pro5X GAL4 UAS


Supplemental Figure 4. MYB61 Is Required for Cellulose Synthesis.

(A) qRT-PCR analysis of the expression levels of R2R3-type MYBs in the internodes from

the control (–GA) and 6-hrs10 μM GA3 treated (+GA) wild-type plants.

(B) The three month-old wild type and MYB61 overexpressing (MYB61 Ox) plants. The

images inserted at the right bottom show an upward curved leaf. Bar = 15 cm.

(C) qRT-PCR examination of the expression of MYB61 and MYB61-like (MYB61L) gene in

MYB61 Ox. TP1 was used as the internal control in (A) and (C). Error bars in (A) and (C)

indicate the SD of three biological repeats.

(D) Observation of sclerenchyma cell walls in the internodes from the three month-old wild-

type and MYB61 Ox plants via transmission electron microscope. Bar = 2 μm.

(E) and (F) Enlargement and quantification of thickness of sclerenchyma cell wall examined

in (D). Error bars, SE (n = 30). *p < 0.01 determined by Student's t test. Bar = 1μm.

(G) Western blot of three secondary wall CESAs in wild type and MYB61 Ox with their

specific antibodies. ARF1 probed with anti-ARF1 antibody was used as a loading control.

A

G

Rel

ativ

e ex

pres

sion

leve

l

02468

101214161820

WT L1 L2

MYB61MYB61L

MYB61 Ox

WT MYB61 Ox

MYB61 OxWT

Anti-CESA4

Anti-CESA7

Anti-CESA9

Anti-ARF1

L1 L2

Rel

ativ

e ex

pres

sion

leve

l

0

1

2

3

4

5

6

–GA +GA

MYB61

MYB61L

MYB58

MYB103

CB

E F

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

WT MYB61Ox

Thic

knes

s of

SC

wal

l (µm

)

**

MYB61 Ox

WT

D

MYB61 Ox

WT


Supplemental Figure 5. NAC29 and NAC31 Bind to the SNBE Motif in the MYB61 Promoter.

(A) Diagram of MYB61 showing two SNBE elements within the 2-kb MYB61 promoter region.

Blue boxes indicate the SNBE elements. The DNA sequences containing a core motif of

SNBEs (the underlined green bases) were subjected to the EMSA assay. Red letters indicate

the mutated bases.

(B) and (C) EMSA assay showing that the recombinant NAC29 and NAC31 proteins directly

bound to the biotin-labeled SNBE2; but did not bind to SNBE1 (B) and point-mutated SNBE2

(C).

SNBE1–– –

++ –

SNBE2–– –

++–

SNBE1GST

TF–– –

++ –

SNBE2–– –

++–

probe

GST-NAC29 GST-NAC31

Free probes

TF

GSTTF

probeTF

Free probes

B

A

5'-TCCTTTACTTTTAACTGGCTTGTGCAAAAAGTAACATACCTGATATGTA-3'SNBE2

MYB61

-1000-2000 ATG

SNBE 1SNBE 25'-TTACCTGTGTAGGTGTTGTGTGCTGTGGAAGTTTCTTGGGAGGGCAAGC-3'SNBE1

5’-TCCTTTACTTTTAACTGGCTCGTGCAAAAAGTAACATACCTGATATGTA-3’

5’-TCCTTTACTTTTAACTGGCTTGTGCAAAGAGTAACATACCTGATATGTA-3’

SNBE2m1

SNBE2m2

C

SNBE2

GST-NAC29

m1 m2 +

GST-NAC31

m1 m2++TF

+– – + + ++++

Free probes


5’-CCTCACCTCGCGCACGAACCGCCCGTTCGCTAGT-3’

CESA7p P1

MBPCompetitor

MBP-MYB61 + + +– –– – – –+– – – + ++

+ +– –– – – –– – – + ++

CESA9p P1+

+

P1

-1500 0-1000CESA7 promoter

P2

-1000-1500 0

P2 P1

CESA9 promoter

Effector

Reporter

F

D

0

10

CESA4 CESA7 CESA9

MockMYB61

Rel

ativ

e lu

cife

rase

act

ivity

5

15

E

G

MBP-MYB61P1 P2 P3 P4

5’-CCTCACCTCGCGCACCAACCGCCCGTTCGCTAGT-3’GAMYB

GAMYBm

– + – + – + – +

CESA4 promoter-1000

P1P2P3P4

0

A

B

Rel

ativ

e lu

cife

rase

ac

tivity

01

2

3

4

Mock MYB61MYB61-GFP DAPI Merge DIC

C

ReporterEffector

CESA4p P4

++

MBP-MYB61MBP

–+ –

–

–

–––

+ –+

+ –+

Free probes

Competitor

NOS ter35S mini proGal4BD MYB61

CaMV 35S proLUC

NOS ter35S mini pro5X GAL4 UAS

LUC

CESA4/7/9 pro NOS ter35S mini pro

NOS ter

MYB61

CaMV 35S pro 35S mini pro


Supplemental Figure 6. MYB61 Regulates Secondary Wall CESA Transcription.

(A) Diagrams of the constructs used for transactivation assay.

(B) Luciferase activities in the rice protoplasts cotransfected with the constructs in (A).

(C) Subcellular localization pattern of MYB61 by transfecting the rice protoplasts with

GFP tagged MYB61. The nuclei were counterstained with DAPI. Merge, merged images

of GFP and DAPI. Bars = 5 μm.

(D) Luciferase activities in the protoplasts cotransfected with constructs shown above.

The transactivation activity was monitored by assaying the luciferase activity in the

protoplasts, with those transfected with an empty effector construct defined as 1.

(E) The 1-kb CESA4 promoter was divided into four fragments and subjected to EMSA

showing that all the four fragments were bound by MBP-MYB61.

(F) EMSA assay showing the direct binding between MYB61 and the biotin-labelled P4

fragment of CESA4. Unlabeled P4 fragments ([10 (+) and 50 (++) fold]) was applied for

binding competition.

(G) The 1.5-kb CESA7 and CESA9 promoter fragments (P1 and P2) were subjected to

EMSA, showing that the increasing unlabeled DNA fragments ([10 (+) and 50 (++) fold])

competed for the binding to fragment P1of CESA7 and CESA9. Blue boxes in (E) and

(G) indicate the GAMYB elements (the underlined green bases). The mutated base in

GAMYB (GAMYBm) is indicated by a red letter.

Error bars in this figure indicate the SD of three biological repeats.


Supplemental Figure 7. Development of Rice Internodes Involves Secondary Wall Accumulation.

(A) A nine-centimetre internode was evenly divided into nine sections. Bar = 1 cm.

(B) Fresh hand cut cross-sections of the segments circled in (A). Bars = 0.1 cm.

1 2 3

4 5 6

7 8 9A

1

B

2

3

4

5

6

7

9

8


Supplemental Figure 8. Impacts of GA on Cellulose Biosynthesis in Arabidopsis and

Poplar and Saccharification Assay of Cell Wall Residues of Transgenic Rice Plants.

(A) and (B) qRT-PCR analyses of the expression levels of secondary wall hierarchical TFs

in the control (–GA) and 6-hrs GA-treated (+GA) Arabidopsis, including R2R3-type MYBs

and NACs (A), and the secondary wall CESAs in the 9-hrs GA-treated poplar (B). Error

bars, the SD of three biological replicates. At-UBQ10 and Pto-Actin were used as internal

controls, respectively.

(C) Saccharification of destarched AIR from the internodes of wild type and transgenic rice

plants using an enzyme cocktail of cellulase and β-glucosidase, revealing releasing more

sugars from the internodes of TF-overexpression plants; but releasing fewer sugars from

the internodes of plants overexpressing MYB61-SRDX. Error bars, the SD of four biological

replicates. *, Significant difference between the wild-type and transgenic plants (p < 0.01).

C

Free

sug

ar y

ield

(%A

IR)

0

10

20

30

40

50

60

70

0 3 12 24Enzyme treatment (h)

WT (Zhonghua-11)NAC29 Ox L1NAC29 Ox L2NAC31 Ox L1NAC31 Ox L2MYB61 Ox L1MYB61 Ox L2WT (Nipponbare)MYB61-SRDX L1MYB61-SRDX L2MYB61-SRDX L3

Enzyme treatment (h)

Free

sug

ar y

ield

(%A

IR)

* *

0102030405060708090

0 3 12 24

* **

80

A

Rel

ativ

e ex

pres

sion

leve

l

0

1

2

3

4

5

6

7

8

–GA +GA

SND1VND7At-MYB46

At-MYB83At-MYB61

B

Rel

ativ

e ex

pres

sion

leve

l

0

1

2

3

4

5

6

–GA +GA

Pto-CESA1Pto-CESA2Pto-CESA17


1


Supplemental Table 1. List of the TFs Coexpressed with Secondary Wall CESAs in Rice.

TF type Gene Gene ID OsCESA41 OsCESA71 OsCESA91

R2R3-

type

MYBs

MYB61 Os01g18240 0.7605 0.7778 0.7169

MYB103 Os08g05520 0.7327 0.7142 0.7633

MYB86L Os01g36460 0.7337 0.7192 0.7010

MYB61L Os05g04820 0.7023 0.7153 0.6624

MYB58 Os02g46780 0.6695 0.6947 0.6691

SW

NACs

NAC62 Os05g48850 0.8475 0.8292 0.8404

NAC73 Os01g48130 0.7583 0.7616 0.7773

NAC7 Os06g04090 0.7583 0.7368 0.7482

NAC29 Os08g02300 0.6208 0.6169 0.6054

NAC3 Os01g09550 0.6172 0.6319 0.6537

Co-expression analysis of secondary wall CESAs with TF candidates in cell wall synthesis was

performed using the expressing database at http://www.ricearray.org and http://genecat.mpg.de/.

1, The Pearson correlation coefficient (PCC). The PCC of coexpressed R2R3-type MYBs and

NACs was set above 0.65 and 0.60, respectively.

2


Supplemental Table 2. Composition Analysis of Sugar and Lignin Content of Wall Residues

of the Internodes from Wild Type and Transgenic Rice Plants.

Residues Rhamnose Fucose Arabinose Xylose Mannose Galactose Glucose Lignin

Wild type (Zhonghua11)

0.8±0.1 0.8±0.0 24.2±0.7 240.2±4.4 1.2±0.1 8.5±0.1 65.1±1.1 131.0±2.3

NAC29 Ox-L1 1.6±0.0 0.8±0.0 28.6±0.7 222.3±2.9 1.1±0.1 11.1±0.3* 71.3±1.0 122.3±1.1

NAC29 Ox-L2 1.4±0.1 0.8±0.0 27.6±0.7 228.1±1.2 1.2±0.1 10.7±0.2* 65.1±1.4 131.1±4.5

NAC31 Ox-L1 1.5±0.0 0.7±0.0 26.4±0.6 268.4±4.4 1.0±0.1 10.6±0.2* 55.2±1.3 130.6±3.8

NAC31 Ox-L2 1.6±0.0 0.8±0.0 27.1±0.8 244.4±5.1 1.1±0.1 11.9±0.5* 62.0±2.2 125.2±3.7

MYB61 Ox-L1 2.3±0.0 0.9±0.0 34.3±2.9* 226.0±2.9 1.4±0.1 17.0±0.9* 70.1±4.8 132.6±8.9

MYB61 Ox-L2 3.2±0.8 1.1±0.2 38.7±5.9* 224.7±7.8 1.8±0.3 22.5±0.4* 70.7±6.4 124.6±5.1

Wild type (Nipponbare)

2.4±0.0 1.0±0.0 42.9±1.6 319.9±10.8 1.4±0.0 15.5±0.6 71.8±5.7 122.7±1.9

MYB61-SRDX-L1 2.4±0.0 1.0±0.0 32.2±2.0* 293.3±20.2 2.5±0.1 9.9±0.4* 69.6±1.3 124.7±4.8

MYB61-SRDX-L2 2.2±0.0 1.0±0.0 34.9±2.9* 302.1±14.9 2.0±0.1 10.5±0.7* 66.2±3.4 125.6±3.7

The alcohol insoluble residues (AIRs) were obtained as described in Methods. The amounts of sugars were

determined by GC-MS analysis of alditol acetate derivatives and are shown as the mean value ± SD (μg mg-1 AIR).

*Significant difference (t test at P < 0.05) with respect to wild-type plants (n=4) ± SD.

3


Supplemental Table 3. Identifying GAMYB Motif in the Promoter Region of CESA Genes

in the Rice, Arabidopsis, and Poplar Genomes.

Gene Locus Type Number Sequence Position1 Os-CESA4 LOC_Os01g54620.1 SCW 7 CAACCAAT; CAACCGCC;

CAACCTTG; CAACCCCA; CAACCTCC; CAACCAAA; CAACCATA

-142; -214; -269; -484; -753; -1094; -1379

Os-CESA7 LOC_Os10g32980.1 SCW 6 CAACCATC; CAACCATA; CAACCCAC; CAACCAGC; CAACCATC; CAACCGTG

-69; -131; -377; -1350; -1478; -1663

Os-CESA9 LOC_Os09g25490.1 SCW 11 CAACCTAC, CAACCATG, CAACCCCT, CAACCAAC, CAACCCGC, CAACCACT, CAACCACA, CAACCGAA, CAACCACT, CAACCGAT, CAACCCGC

-239; -243; -285; -432; -452; -604; -854; -1018; -1046; -1245; -1875

Os-CESA1 LOC_Os05g08370.1 PCW 3 CAACCGCC, CAACCTTG, CAACCCGA

-234; -1006; -1726

Os-CESA2 LOC_Os03g59340.1 PCW 7 CAACCGCG, CAACCCAA, CAACCTCA, CAACCCAA, CAACCATC, CAACCGCT, CAACCAAG

-348; -412; -417; -633; -1135; -1371; -1827

Os-CESA3 LOC_Os07g24190.1 PCW 6 CAACCGAT, CAACCCTG, CAACCCTA, CAACCGTT, CAACCAGA, CAACCACG

-30; -96; -452; -704; -1878; -1923

Os-CESA5 LOC_Os03g62090.1 PCW 2 CAACCTTT, CAACCAAA -1045; -1202Os-CESA6 LOC_Os07g14850.1 PCW 5 CAACCCCT, CAACCTTT,

CAACCCCC, CAACCCCC, CAACCACC

-125; -286; -360; -448; -1359

Os-CESA8 LOC_Os07g10770.1 PCW 1 CAACCACT -1698 Os-CESA10 LOC_Os12g29300.1 PCW 5 CAACCTTG, CAACCTAA,

CAACCATG, CAACCACT, CAACCAGT

-381; -1249; -1349; -1385; -1536

At-CESA4 AT5G44030.1 SCW 6 CAACCATT, CAACCTCA, CAACCGTT, CAACCATA, CAACCAAC, CAACCAGA

-136; -723; -1065; -1124; -1857; -1880

At-CESA7 AT5G17420.1 SCW 2 CAACCATG, CAACCGCT -792; -1631 At-CESA8 AT4G18780.1 SCW 1 CAACCCTT -1035 At-CESA1 AT4G32410.1 PCW 1 CAACCGGC -1413 At-CESA2 AT4G39350.1 PCW 2 CAACCAAA, CAACCAAA -1122; -1320At-CESA3 AT5G05170.1 PCW 4 CAACCAAA, CAACCCTA,

CAACCACC, CAACCAAG -374; -385; -1466; -1937

At-CESA5 AT5G09870.1 PCW 4 CAACCGAG, CAACCTGT, CAACCCAA, CAACCGGG

-73; -87; -94; -1837

4

At-CESA6 AT5G64740.1 PCW 1 CAACCTTT -281 At-CESA9 AT2G21770.1 PCW 3 CAACCGTA, CAACCGTC,

CAACCTAA -112; -1310; -1714

At-CESA10 AT2G25540.1 PCW 1 CAACCCAT -902 Pto-CESA1 Potri.002G257900.1 SCW 5 CAACCCCT, CAACCACT,

CAACCACC, CAACCAAG, CAACCCCA

-92; -790; -991; -1829; -1966

Pto-CESA2 Potri.018G103900.1 SCW 7 CAACCAAC, CAACCGCC, CAACCTCT, CAACCGGT, CAACCCCT, CAACCCTT, CAACCACC

-34; -152; -200; -941; -1045; -1746; -1818

Pto-CESA3 Potri.011G069600.1 SCW 6 CAACCAGA, CAACCGCC, CAACCTCA, CAACCCTC, CAACCAAG, CAACCTGT

-134; -182; -188; -205; -573; -1208

Pto-CESA10 Potri.006G181900.1 SCW 3 CAACCAAC, CAACCCAG, CAACCGGT

-31; -147; -1654

Pto-CESA17 Potri.004G059600.1 SCW 7 CAACCCAC, CAACCAGA, CAACCGCC, CAACCCAC, CAACCTTC, CAACCTTT, CAACCTTA

-185; -199; -216; -590; -1303; -1899; -1975

Pto-CESA4 Potri.005G087500.1 PCW 2 CAACCTGA, CAACCTTT -1651; -1886Pto-CESA5 Potri.001G266400.1 PCW 3 CAACCATA,

CAACCCGA,CAACCTCT -136; -1222; -1951

Pto-CESA6 Potri.013G019800.1 PCW 4 CAACCTCT, CAACCACA, CAACCACT, CAACCAAA

-54; -936; -989; -1867

Pto-CESA7 Potri.006G251900.1 PCW 1 CAACCATA -1751 Pto-CESA8 Potri.009G060800.1 PCW 5 CAACCACA, CAACCGTG,

CAACCGTC, CAACCTCT, CAACCTCA

-122; -134; -1018; -1481; -1719

Pto-CESA9 Potri.018G029400.1 PCW 2 CAACCAAT, CAACCGGT -341; -684 Pto-CESA11 Potri.002G066600.1 PCW 3 CAACCAGA, CAACCAGC,

CAACCACT -1172; -1719; -1770

Pto-CESA12 Potri.005G194200.1 PCW 5 CAACCACA, CAACCGAC, CAACCAGT, CAACCAGC, CAACCCAT

-331; -382; -599; -1518; -1613

Pto-CESA13 Potri.007G076500.1 PCW 1 CAACCACA -1936 Pto-CESA14 Potri.005G027600.1 PCW 1 CAACCTAG -717 Pto-CESA15 Potri.006G052600.1 PCW 1 CAACCCTC -76 Pto-CESA16 Potri.016G054900.1 PCW 4 CAACCTTG, CAACCAAT,

CAACCATG, CAACCAAA -265; -448; -556; -972

Two-kb promoter of CESAs from rice, Arabidopsis, and polar were used for identifying GAMYB

motif (CAACCNNN). PCW, primary cell wall; SCW, secondary cell wall. 1 The position of GAMYB motifs, which is not one-to-one correspondence with the order of motifs

shown in the sequence column.

5


Supplemental Table 4. The Primers Used for Generation of Transgenic Rice Plants and

Relevant Analyses in This Study.

Construct Primer sequence (5’-3’)

NAC29 Ox Forward CGGGATCCGCCACCATGAGCATCTCGGTGAA

Reverse GACTAGTCTTACAGTGCCACGTGGCT

NAC31 Ox Forward CGGGATCCGCCACCATGGATCGGCATGAGGAGGA

Reverse GGGGTACCCTCAGTCTCGGTCATCGATGAA

MYB61 Ox Forward CGGGATCCGCCACCATGGGGAGACATTCCTGCTGCTACA

Reverse GGGGTACCCCTAGATATTCTCAAAAGACAAGGACA

MYB61-SRDX Forward TCGACGGGCCCCTCGATCTGGATCTAGAACTCCGTTTGGGTTTCGCTTAAGGTAC

Reverse CTTAAGCGAAACCCAAACGGAGTTCTAGATCCAGATCGAGGGGCCCG

GST-NAC29 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAGCATCTCGGTGAAC

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTACAGTGCCACGTGGCTGAGACGCTC

GST-NAC31 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGATCGGCATGAGGAG

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTAGTCTCGGTCATCGATGAA

MBP-MYB61 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGGGAGACATTCCTGC

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTAGATATTCTCAAAAGACAA

FLAG-SLR1 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAAGCGCGAGTACCAA

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTACGCCGCGGCGACGCGCCATG

NAC29-BD Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAGCATCTCGGTGAAC Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTAGAGGCGGTACTCGTGCAT

NAC31-BD Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGATCGGCATGAGGAG

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTAGAGGCGGTACTCGTGGAT

SLR1-N Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAAGCGCGAGTACCAA

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTACTCCTGCGTGTCAACCAC

SLR1-C Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGCTGGGATCCGGCTGGTG

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTACGCCGCGGCGACGCGCCATG

RGA-Luc Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAAGAGAGATCATCACC

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTAGTACGCCGCCGTCGAGAGTTTC

SND1-Luc Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCTGATAATAAGGTCAAT

Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTATACAGATAAATGAAGAAGTGG

CESA4-promoter Forward CCCAAGCTTAATACACAGCAAAATCGGCA

Reverse GGGGTACCGGCTCACCCCCCTCTCCTCTCC

CESA7-promoter Forward CCCAAGCTTGAGCTCTCTGTTCATCAAATTTGTCCA

Reverse GGGGTACCGGTGAGGTGCCGGGGAAGCGA

6

CESA9-promoter Forward CGAGCTCGACGCTTATACAATGCGCAT

Reverse GGGGTACCGGCCGCGCAACAACGGCCGG

MYB61-promoter Forward CGAGCTCCGCTCGAGTAAAGAAAAACA

Reverse GGGGTACCTGAATCAATGCAGAAACTGAA

7


Supplemental Table 5. qRT-PCR and ChIP-PCR Primers Used in This Study.

Genes Forward primer sequence (5’-3’) Reverse primer sequence (5’-3’)

Os-CESA4 CTCCGAGACCACCACCACCAAC ACCCATCGTCTTCGTCGCATTAG

Os-CESA7 AAGCCATGCGGGGTCTCGTG CATCCATCCGGTCATCCCTCTTG

Os-CESA9 AGCGATTTCTGAAGATTTGT AACATAGCACGAACTCAAC

Os-XTH8 CTCCTTCTACCTCTCCTC GGATTCCAGATGATCTTGTA

Os-PIL1 AAGGCAGAGAAGATTCAG GCAATGAGGTATGAGTTCT

Os-MYB61 ATATCTTGAGCCACTTGT ATCCTCTTGGTGATTCAG

Os-MYB61L AGACAGCACAATCATCAA GGTCAAGAAACAACTCCT

Os-MYB103 CAAAGTGGTGCTCATTCC CAAGATCAGATCAGAGATGG

Os-MYB58 GTAGTAGAGACACAACAC ATATAGCACTTCTCATTGG

Os-NAC29 GCTGCTCCACCACCACCAC GAGTAGTAGGCGAGGCCGT

Os-NAC31 CTGAGTTCAGAGTCCAAG TGACGAAGAAATGTTGTTG

SLR1 GACGTCAACGAACGCTCAATT CGGAGTCCAGTCGTCGATCT

Os-TP1 TAACTGGTGCGAACTGCAAG CGGAGTTGATGATGTCGATG

Os-HNR GGCAGGTTCTGCAGTGGTAT TAAGGTCGGTATCGCCAATC

Os-Actin1 CTTCATAGGAATGGAAGCTGCGGGTA CGACCACCTTGATCTTCATGCTGCTA

Os-eEF1a TTTCACTCTTGGTGTGAAGCAGAT GACTTCCTTCACGATTTCATCGTAA

At-CESA4 GAGTGATGATAAAACGATGAGCAG TCTCAAAATTCTTCTGCGACATTA

At-CESA7 ATGGGTAGACAGAACAGAACACCAA CTTCAGCAGTTGATGCCACACTT

At-CESA8 CACTTCTTTGCCTCTTGTTGCTTAC GAAGCTCGAGGACACTCGTTAAGAT

At-MYB46 GAATGTGAAGAAGGTGATTGGTACA CGAAGGAACCTCAGTGTTCATCA

At-MYB83 CGCTTCTTCAATGTCGAGTTCCA CCATTCTTGGTAACAGGTGGTCTC

At-MYB61 TGTGTTTGGGTCAATGCCTCTTT TCCTTGGAAACAGATGGTCACAG

VND7 GGACGAATAAAGATCAGAACGA ATGCGGATGTATGACTTGTGTC

SND1 TCCTCTTGAATATCCCAAGGCTCA CCCACCGAAGAAGAACTTCTCCAT

At-UBQ10 AACTTTGGTGGTTTGTGTTTTGG TCGACTTGTCATTAGAAAGAAAGAGATAA

Pto-Actin AAACTGTAATGGTCCTCCCTCCG GCATCATCACAATCACTCTCCGA

Pto-CESA1 GAGTTGGAGAAATCATCA GAGAGTTAGTTCCTTCAG

Pto-CESA2 AACCACTGCAACCATCTCA ATGTTCCATGACAGCTCAGG

Pto-CESA17 AAGGACCAAGCAAACATT AAGTGGATGTAACGGTAAG

Os-Actin1(intron) TGGCATCTCTCAGCACATTC GGCAAGCAACATTGTAAGCA

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Os-CESA4 ChIP CTTCCATTCTTTCCATCC TGGTGATATAGCCATAGC

Os-CESA7 ChIP TTGAGATTTGAGCATTACCT ATTTCTTGCCAATGTTTACC

Os-CESA9 ChIP ATCAACTGGAGAGGACTT TTGGTGAGTGGTAGTAGT

(WT) PC V SC SC - The Plant Cell · 1 Supplemental Data. Huang et al. Plant Cell (2015)...

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Transcript of (WT) PC V SC SC - The Plant Cell · 1 Supplemental Data. Huang et al. Plant Cell (2015)...