Developmental Cell

Supplemental Information

Organizer-Derived WOX5 Signal Maintains

Root Columella Stem Cells through

Chromatin-Mediated Repression of CDF4 Expression

Limin Pi, Ernst Aichinger, Eric van der Graaff, Cristina I. Llavata-Peris, Dolf Weijers,

Lars Hennig, Edwin Groot, and Thomas Laux

Figure S1

A DCB

QC

QC

QCQC

Q1630 Q1630::WOX5 Q0680::WOX5Q068010/10 10/10 10/10 10/10

Figure S1. Expression patterns of Q1630 and Q0680. Related to Figure 1. (A-D) The expression of Q1630 and Q0680 is strongly reduced in the induced columella stem cell-like cells of Q1630::WOX5 (B compared to A) and Q0680::WOX5 (D Q ( p ) Q (compared to C) plants, respectively. Scale bars, 50 μm.

Figure S2

QC

Figure S2. CDF4-GFP protein localizes to nucleus. Related to Figure 2. gExpression pattern of pCDF4:CDF4-GFP.Scale bar, 50 μm.

Figure S3

A BIP_α‐WOX5

MW (kDa)

GST

GST‐WOX5

200x unlabeled probe

labeled probe + + + + + + + + +

+

+ + + + + +

+ +

+ + +

‐

‐ ‐

‐ ‐

‐

‐

‐ ‐ ‐ ‐

‐

‐ ‐ ‐

BS1 BS2 BS3+4

A B

WOX5‐GR

72

55

43

p

[

35

Figure S3. WOX5 protein binds to three conserved motifs of the CDF4 promoterRelated to Figure 3.A) Western blot of root protein extracts, probed with anti-WOX5 antibodies. Arrow indicates expected size for WOX5-GR of 55 kDa.B) EMSA of the oligonucleotide probes (BS1-BS4) with GST tagged WOX5 protein. The labeled probes are shifted by GST-WOX5 protein, but the shifts are (white arrowhead) greatly reduced when incubated with 200x excess unlabelled BS ) g ycompetitor. Square bracket indicates free probe.

Figure S4

A B C D E

(100%, n = 74) (90.1%, n = 81) (100%, n = 101) (70.7%, n = 75) (100%, n = 48)

F Gp35S:VP16‐ΔNCDF4

wox5‐1 QC184

QC

10.9 

13.8 12.9 

18.1 

15.0 

20.0 

25.0 

CS

C p

ostio

n

16/160.0 

5.1 

0.3  0.0 

0.0 

5.0 

10.0 

Em

pty

vect

or

J234

1::C

DF

4

35S

:CD

F4

DF

4-G

R -

DE

X

F4-

GR

+D

EX

35S

:∆N

CD

F4

RD

X-∆

NC

DF

4

wox

5-1st

arch

gra

ins

at C

Figure S4. Overexpression of CDF4 variants. Related to Figure 5.(A-E) Seedling phenotypes of 10-days-old plants transformed with empty vector (A), p35S:CDF4 (B), p35S:ΔNCDF4 (C), p35S:SRDX-ΔNCDF4 (D), and p35S:VP16-ΔNCDF4 (E).

35S

:CD

35S

:CD

3

35S

:SR

and p35S:VP16 ΔNCDF4 (E).(F) Quantification of starch grain accumulation in columella stem cells shown in Figures 5J-5O. Error bars represent SD (n = 8 for each genotype).(G) p35S::VP16-ΔNCDF4 does not complement wox5-1 defects (compare to Figure 5E).Scale bars, 0.5 cm (A-E); 50 μm (G).

AYFPc‐TPL YFPc‐TPR1 YFPc‐TPR2 YFPc‐TPR3 YFPc‐TPR4 YFPc‐WOX9

WOX5‐YFPn

Figure S5

2.2%, n = 89 5.6%, n = 72 3.2%, n = 93 3.6%, n = 83 3.1%, n = 64 0%, n > 1000

B

WBmWB

WOX5

WOX5ΔEAR

TLQLFPVN

TAQAFPVN

Homeodomain (HD)

Acidic domain (AD)

WUS box (WB)

D

C

WOX5mWBEAR motif (EAR)

WOX5mWB‐YFPn

WOX5△EAR‐YFPn

SRDX‐WOX5mWB‐YFPn

YFPc‐TPR1

REV‐YFPn

α- WOX5

47 kDa-

32 kDa-

3.9%, n = 103 2.1%, n = 960%, n > 1000 0%, n > 1000

CBB

GE FQC

QC

02468

C-li

ke l

ayer

s

***

35S:WOX5-GR

200300400500600700800

of e

xpre

ssio

n QC

CRC

H

Figure S5. WOX5 protein forms a complex with TPL/TPR co-repressors in Arabidopsisprotoplasts and TSA treatment suppressed WOX5-induced stem-cell-like cells. Related to Figure 6.(A) BiFC assays in transiently transfected Arabidopsis leaf protoplasts Nuclear YFP signals

DEX+TSAp35S:WOX5‐GR

DEXp35S:WOX5‐GR

15/1515/15

0

CS

C

0100

Leve

ls

(A) BiFC assays in transiently transfected Arabidopsis leaf protoplasts. Nuclear YFP signals show that WOX5 interacts with all TPL family members, but not with WOX9 as a negative control.(B) Schematic representation of the WOX5 protein variants. Deletion of the EAR domain is designated as ΔEAR, the mutated WUS box as mWB. (C) BiFC assays in transiently transfected Arabidopsis leaf protoplasts. WOX5 without the EAR motif interacts with TPR1. Mutations of the conserved amino acids in the WB abolish h i i i hil h f i f SRDX i REV (REVOLUTA)the protein interaction, while the fusion of SRDX restores it. REV (REVOLUTA) serves as a

negative control for interaction with TPR1.The frequency of cells with BiFC signal is given in percentage (A and C). YFP, green; chloroplast, red.(D) Western blot of leaf protoplasts transfected with the indicated constructs, probed with anti-WOX5 antibody. (E and F) phenotypes of the root tips of 35S:WOX5-GR plants treated as indicated for 24h. 10 µM TSA treatment (B) largely suppresses the formation of stem-cell-like cells (waved brackets). Scale bars, 50 μm.(G) Quantification of columella stem cell-like layers in (A and B). Error bars represent SD (n = 10, *** P < 0.001, Student’s t-test)(H) Expression levels of TPL, TPR2, TPR3 and HDA19 in QC and CRC. Values are extracted from the public microarray database (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi)

Supplemental Tables

Table S1. WOX5 direct responsive genes, related to Figure 2.

No. AGI number Description D+C/C

(LFC)

D/M

(LFC)

1 AT2G21560 similar to unknown protein (TAIR: AT4G39190) -3.1 -1.2

2 AT5G25160 ZFP3 (ZINC FINGER PROTEIN 3) -2.6 -1.9

3 AT4G10390 protein kinase family protein -1.9 -1.0

4 AT2G32660 disease resistance family protein / LRR family protein -1.9 -1.4

5 AT1G70470 similar to unknown protein (TAIR: AT1G23530) -1.9 -1.7

6 AT2G34140 Identical to Dof zinc finger protein AtDOF2.3/CDF4 -1.8 -1.4

7 AT4G21870 26.5 kDa class P-related heat shock protein (HSP26.5-P) -1.6 -1.5

8 AT4G31730 GDU1 (GLUTAMINE DUMPER 1) -1.5 -2.1

9 AT1G13740 similar to unknown protein (TAIR:AT1G69260) -1.4 -1.7

10 AT5G66440 protein of unknown function DUF1675 -1.3 -1.3

11 AT5G48800 phototropic-responsive NPH3 family protein -1.2 -1.5

12 AT5G01740 similar to SAG20 (WOUND-INDUCED PROTEIN 12) -1.2 -1.6

13 AT5G43380 TOPP6 (Type one serine/threonine protein phosphatase

6)

-1.1 -1.2

14 AT2G38320 similar to unknown protein (TAIR:AT5G01620) -1.1 -1.1

15 AT1G43160 RAP2.6 (related to AP2 6) ; DNA binding / transcription

factor

-1.1 -2.0

16 AT1G69850 ATNRT1:2 (NITRATE TRANSPORTER 1:2) -1.0 -1.4

17 AT1G13670 unknown protein -1.0 -1.3

18 AT5G67430 GCN5-related N-acetyltransferase (GNAT) family

protein

1 1.2

D+C/C and D/M, the mean normalized signal obtained by application of dexamethasone plus

cycloheximide (D+C), divided by the signal obtained by application of cycloheximide (C), or signals

from dexamethasone treatment (D) divided by those from mock treatment (D/M), respectively. LFC,

log2 fold change. Shaded lines indicate the candidate genes chosen for subsequent reporter analysis.

Table S2. Expression of VP16-CDF4 increases the frequency of roots with two

columella stem cell-like layers, related to Figure 5.

Lines 2 columella

stem cells

1 columella

stem cell

n

p35S:VP16-ΔN CDF4 _#1 19 (29.2%) 46 65

p35S:VP16-ΔN CDF4 _#2 20 (28.6%) 50 70

p35S:VP16-ΔN CDF4 _#3 27 (35.5%) 49 76

p35S:VP16-ΔN CDF4 _#4 19 (25.3%) 56 75

p35S:VP16-ΔN CDF4 _#5

Mean

31 (32.0%) 68 97

23.2 (30.3%) 53.8 76.6

Empty vector_#1 10 (12.8%) 68 78

Empty vector_#2

Mean

11 (16.2%) 57 68

10.5 (14.4%) 62.5 73

Frequencies of 5-day-old roots with one and two columella stem cell layers in T3

homozygous independent T3 lines are shown. n, number of roots counted.

Table S3. Overview of the IP-MS results. Related to Figure 6.

Number Ratio P-value Protein IDs Unique

Peptides

Description

1 5506.3 0.075 C0SVA6 8 WOX5

2 1507.8 0.005 A8MRL0 1 At4g40030

3 630.2 0.071 Q0V7R6 4 At4g40040

4 579.8 0.127 CON__P42212 5 YFP

5 370.2 0.002 Q93VY4 6 GUS

6 299.6 0.001 F4I460 1 At1g04160

7 289.2 0.002 Q9LFX8 5 At1g27090

8 242.4 0.004 Q6ICZ8 1 At5g13850

9 229.6 0.055 P49689 1 At2g19750

10 193.1 0.045 Q94AI7 3 TPL

11 150.2 0.000 C0SVQ6 1 At5g26170

12 101.1 0.010 F4IVN6 1 AT5G37780

13 82.8 0.027 Q27GK7 7 TPR4

14 80.3 0.134 Q9LNU5 2 At1g20225

15 63.6 0.000 P22954 3 At1g20220

16 47.1 0.089 Q8RWN5 2 At5g04280

17 44.5 0.155 Q9SCU2 3 At5g04280

18 39.4 0.139 Q9SJU0 1 At2g21380

19 34.9 0.055 F4K2T3 4 TPR3

20 31.8 0.150 F4KDR2 1 At5g02610

21 31.3 0.015 Q38896 1 At2g21060

22 29.7 0.049 Q9M2F9 1 At3g58570

23 27.2 0.040 Q93VA8 3 At1g76010

24 24.2 0.297 Q9LUT2 1 At3g17390

25 23.0 0.037 Q94KD0 1 At5g58470

26 18.3 0.009 F4JXE3 1 At5g59950

27 17.7 0.038 Q9LRZ0 2 TPR2

28 17.6 0.114 Q0WV90 3 TPR1

29 15.7 0.029 Q9ZSI6 1 At4g01700

30 15.5 0.327 O65719 4 At3g09440

The table shows the first 30 interactors in the list after MaxQuant and Perseus satistical analysis.The

ratio was obtained from p35S::WOX5-YFP vs. WT, and the p-value obtained from two biological

replicates. Shaded lines indicate the TPL/TPR proteins.

Table S4. Complementation of the wox5-1 mutant by WOX5 protein variants,

related to Figure 6.

Constructs complementation (%) n

Full Partial No

pWOX5:WOX5 34.4 31.3 34.4 64

pWOX5:WOX5ΔEAR 2.9 25.7 71.4 70

pWOX5:WOX5mWB 0 0 100 74

pWOX5:SRDX-WOX5mWB 13.3 30 56.7 60

pWOX5:WOX5mWB-TPR1 0 23.5 76.5 34

The frequencies of root phenotypes of primary transformants are shown. Full complementation: strong

QC184 expression and no starch accumulation in columella stem cells. Partial complementation: weak

QC184 expression in QC and accumulation of starch granules at the position of columella stem cells.

No complementation class is indistinguishable from wox5-1. n, total number of independent primary

transformants of each construct analyzed.

Supplemental Experimental Procedures

Plant growth conditions

Seeds were surface sterilized and germinated on 0.5x Murashige and Skoog (MS)

agar medium supplemented with 1% sucrose in a growth chamber with 16 h light / 8 h

dark at 22°C. For DEX induction, seedlings at 5 dpg were transferred onto agar plate

supplemented with 10 µM DEX (Sigma-Aldrich) for an appropriate time.

Transgenic work

For construction of p35S:WOX5-GR, the WOX5 cDNA was amplified by PCR from

AKS16 (Sarkar et al., 2007) and fused in frame to the ligand-binding domain of a rat

glucocorticoid receptor (GR) (Aoyama and Chua, 1997). The resulting WOX5-GR

was placed under the control of the CaMV p35S promoter (Benfey and Chua, 1990)

and then cloned into the binary vector pBarMAP (Ueda et al., 2011).

To generate UAS:CDF4, CDF4 cDNA amplified from a Col cDNA library was placed

under the transcriptional control of the GAL4 binding sites (UAS) amplified from

AKS50 (Sarkar et al., 2007). The resulting UAS:CDF4 was cloned into the binary

vector pGII0125 (a gift from Ben Scheres) and transformed into AKS50 and J2341

(designated as pWOX5::CDF4and J2341::CDF4 respectively). Two independent

pWOX5::CDF4 transformants were crossed to QC184 and F1 plants were analyzed.

To generate pCDF4:3xnlsGFP, about 3 Kb of promoter sequence upstream of the

predicted start codon were amplified from Col genomic DNA and cloned into the

binary vector pGIIK containing SV40-3xGFP (De Rybel et al., 2011).

pCDF4:3xnlsGFP was transformed into wox5-1 harboring pWOX5:erCFP, and two

representative transgenic lines were crossed to Col and wox5-1 respectively. F1 plants

were imaged under a confocal laser scanning microscope.

To generate pWOX5:WOX5-GFP, GFP was fused in frame to the C-terminus of

WOX5 and the resulting WOX5-GFP was placed under the control of the WOX5

promoter amplified by PCR from AKS50. pWOX5:WOX5-GFP was cloned into the

binary vector pGII0125 and then transformed into wild-type and wox5-1 QC184

plants, respectively. gWOX5-3xYFP was cloned by fusing a genomic WOX5 construct,

starting 4.5 kb upstream of the WOX5 locus, and including the 2 exons and the intron

of WOX5 but without the stop codon, in frame to a triple YFP. The 3’UTR was cloned

behind the stop codon of the 3rd YFP. gWOX5 -3xYFP was cloned into the binary

vector pGII0125 and then transformed into wox5-1 QC184.

For p35S:LhGR; pOp:WOX5-YFP (p35S::WOX5-YFP), YFP was fused in frame to

the C-terminus of WOX5, and the resulting WOX5-YFP was cloned into the Gateway

binary vector pOpOn2.1, containing the p35S promoter (Samalova et al., 2005).

The coding sequences of WOX5, WOX5ΔEAR, WOX5mWB, and SRDX-WOX5mWB

were cloned into pUC-SPYNE (Walter et al., 2004), to yield WOX5-YFPn,

WOX5ΔEAR-YFPn, WOX5mWB-YFPn and SRDX-WOX5mWB-YFPn, respectively.

TPL/TPRs cDNAs were cloned into pRT-SPYCE (a gift from Wolfgang Werr) to yield

YFPC-TPL/TPRs.

The full length of CDF4 and truncated ΔNCDF4 cDNAs were placed under the

control of p35S promoter and then cloned into pGII0125, yielding p35S:CDF4 and

p35S:ΔNCDF4, respectively. PCR-amplified SRDX-ΔNCDF4 replaced ΔNCDF4 to

yield p35S:SRDX-ΔN CDF4. For construction of p35S:VP16-ΔNCDF4, VP16 was

amplified from AKS50 and then fused to the N-terminus of ΔNCDF4. The resulting

VP16-ΔNCDF4 replaced ΔNCDF4 to yield p35S:VP16-ΔNCDF4.

To generate p35S:HDA19-GFP, HDA19 cDNA was amplified from a Col cDNA

library and fused in frame to the N-terminus of GFP. The resulting HDA19-GFP was

placed under the control of the p35S promoter and then cloned into pGII0125.

Detailed cloning information is available upon request. Plants were transformed by

the floral dip method (Clough and Bent, 1998) and T1 plants were selected on agar

plates containing appropriate antibiotics.

Western blotting

Total proteins from 5-day-old roots of p35S:WOX5-GR seedlings after 24 h of DEX

induction were extracted with cold extraction buffer (50 mM Tris-HCl, pH 7.5, 150

mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.2% NP-40, 1 mM PMSF) containing a

proteinase inhibitor cocktail (Roche). WOX5-GR was immunoprecipitated with

anti-WOX5 antibody coupled to Dynabeads (Invitrogen). The immunoprecipitated

protein was blotted onto a PVDF membrane and WOX5-GR was detected with

anti-WOX5 antibody.

LUC assay

The effector, reporter, and internal control plasmids were transformed into

Arabidopsis mesophyll protoplasts according to the procedure published previously

(Yoo et al., 2007). Luciferase activity was measured by the Dual-Luciferase assay

system (Promega) according to the manufacturer's instructions after incubation at

room temperature for 16 h. Renilla luciferase activity driven by the p35S promoter

was used as internal control for normalization.

EMSA

GST-WOX5 protein was expressed in E. coli BL21 (DE3) and purified with

Glutathione Sepharose 4B (GE Healthcare, 17-0756-01) according to the

manufacturer’s instructions. Complementary single-stranded oligonucleotides

(Supplemental Table for primers) were synthesized and labeled with biotin using

Biotin 3’ End DNA Labeling Kit (Thermo Scientific, 89818). Biotin-labeled

oligonucleotides were annealed as probes for EMSA. 2 µg GST-WOX5 and 20 fmol

biotin-labeled probes were incubated at room temperature for 20 min in 10 mM Tris

(pH 7.5), 50 mM KCl, 1 mM DTT, 50 ng/µL poly (dI-dC), 2.5 % glycerol, 0.05 %

Nonidet P-40, and 5 mM MgCl2. For supershift experiments, 2 µL polyclonal

anti-WOX5 antibodies were added to the binding reaction. EMSA was performed

using the LightShift Chemiluminescent EMSA kit (Thermo Scientific, 20148)

according to the manufacturer’s instructions.

IP-MS

IP experiments were performed as described (De Rybel et al., 2013) using 1.4 g of

root tips from DEX-treated p35S::WOX5-YFP transgenic seedlings in the Col-0

background for each sample. Interacting proteins were isolated by applying total

protein extracts to anti-GFP-coupled magnetic beads (Milteny Biotech). Two

biological replicates of each sample were compared with two DEX-treated Col

wild-type samples. MS and statistical analysis using MaxQuant and Perseus software

were performed as described previously (Hubner and Mann, 2011; Lu et al., 2011)

with minor modifications.

BiFC

About 10 µg DNA of each construct for BiFC was transformed into Arabidopsis

mesophyll protoplasts according to the protocol published (Yoo et al., 2007). YFP

signal was examined under confocal laser scanning microscopy after incubating

protoplasts at room temperature for 16 h.

RT-PCR

Total RNA of 5-days-old roots was extracted using the RNeasy Plant Mini Kit

(Qiagen, Cat. No. 74904) and an on-column DNAse (Qiagen, Cat. No. 79254)

treatment. RNA was reverse transcribed using the Superscript III system (Invitrogen,

Cat. No. 11752) according to the manufacturer’s instructions. qPCR was performed

with SYBR® Green (Roche, Cat. No. 04707516001) on a LightCycler 480 (Roche).

Three biological replicates of each sample were performed in all experiments and data

were analyzed with LightCycler 480 software release 1.5.0., version 1.5.0.39 (Roche).

Reference genes At4g34270 and AT2G28390 (Czechowski et al., 2005) were used for

data normalization. Primer sequences are listed in Supplemental Table.

Oligonucleotides used in this study. Related to Figures 1-7.

Name Sequence (5‘ - 3‘) Purpose

oLP135-F TGGTCGTGTCGTGGTTGGTATG

oLP135-R ATAAGCTCGACTTGGCGAACACC qPCR for CDF4

oLP150-F TTGTAGCTTGATCCGCATTG

oLP150-R GCGCATTCATCGCTTTACTT ChIP-qPCR_P1

oLP151-F TTTCGACAACCCTGAAATTCT

oLP151-R GATGCTTGAACGTGATCCAA ChIP-qPCR_P2

oLP152-F ACGCACAAATCCATCCAAAT

oLP152-R CAGTGCACGAGTCACGAGTT ChIP-qPCR_P3

oLP153-F CTGACGAGCCGGATCATACT

oLP153-R GGTCGGACGAATGAGCTAAG ChIP-qPCR_P4

oLP154-F TTCGTTAAAGAACGGTCATGG

oLP154-R ACCGATAATGCTTTTCTTTTCAA ChIP-qPCR_P5

oCP021 CACGCCCTGGAGTTCCAACAAC

oCP022 GCAAATTGAGAAGGTCATGAGG ChIP-qPCR_eIF4a

oLP236-F CATGTACTCGTTTCGCTTTCC

oLP236-R AGCAGCAAAATCAAGCGAAC ChIP-qPCR_ACTIN2/7

oLP351-F GCACTTTTCAAATAATTAGCTTTTTAAA

oLP351-R TTTAAAAAGCTAATTATTTGAAAAGTGC EMSA_pCDF4(BS1)

oLP352-F ATGAATGCGCCTATTACTACATGCTTT

oLP352-R TAAAGCATGTAGTAATAGGCGCATTCA EMSA_pCDF4(BS2)

oLP353-F AATATTTTAATCTCCACTAATTCAGTCA

oLP353-R TGACTGAATTAGTGGAGATTAAAATATT EMSA_pCDF4(BS3+4)

oLP364-F AATATTTTGGTCTCCACTAATTCAGTCA

oLP364-R TGACTGAATTAGTGGAGACCAAAATATT EMSA_pCDF4(mBS1)

oLP352-F AATATTTTAATCTCCACTGGTTCAGTCA

oLP352-R TGACTGAACCAGTGGAGATTAAAATATT EMSA_pCDF4(mBS2)

oLP353-F AATATTTTGGTCTCCACTGGTTCAGTCA

oLP353-R TGACTGAACCAGTGGAGACCAAAATATT EMSA_pCDF4(mBS3+4)

oLP368-F GGTACCTGTACACTGGATTTGTACAC

oLP368-R GGATCCGAAAACGTGCAGAGAATGG CDF4 promoter reporter

oLP384-F GGATCCATGGCGACTCAAGATTCT

oLP383-R GTCGACTCAGCACGATTGACCGTC CDF4 CDS

oLP383-F GGATCCATGCTGACGGCCGAGAAG

oLP383-R GTCGACTCAGCACGATTGACCGTC ΔN CDF4 CDS

oLP384-F GGATCCATGGCGACTCAAGATTCT

oLP384-R GTCGACTCAGCGAACACCATTTCC CDF4ΔC CDS

oLP229-F GGATCCACCGCCCCCCCGACCGAC

oLP229-R TCTAGACTACCCACCGTACTCGTCAATTC VP16 CDS

oLP201-F TTAATTAAGCAGAAAGACTTTTATCTACC

oLP301-R CCATGGGTTCAGATGTAAAGTCCTCA WOX5 promoter

oEA34 CCCGGGTGGTAGCGAACTAGGAATTGTATGTGC

oEA88 GGCGCGCCTCCGACGGAACTTGAGTTTGCTTC gWOX5-WOX5

oEA38 GGCGCGCCTCCGACGGAACTTGAGTTTGC

oEA40 GGTACCGTCATTGACCACAATAACAAAAG 3’UTR WOX5

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