Cell Reports, Volume 22

Supplemental Information

PHB3 Maintains Root Stem Cell

Niche Identity through ROS-Responsive

AP2/ERF Transcription Factors in Arabidopsis

Xiangpei Kong, Huiyu Tian, Qianqian Yu, Feng Zhang, Rong Wang, Shan Gao, WenhongXu, Jiajia Liu, Eilon Shani, Chunxiang Fu, Gongke Zhou, Liangran Zhang, XianshengZhang, and Zhaojun Ding

Figu

Figu

The

pCY

C) B

muta

repre

(p<0

ure S1. The

ure 1

in planta

YCB1;1::CYCB

Bars: 50 μm. A

ant plants. (D

esent SE from

0.01).

expression o

a expression

B1;1-GFP in f

Abundance of

D-F) At least

m triplicate exp

of RBR1, KN

n of (A)

five-day old r

f RBR1 (D), K

100 seedling

periments. As

NOLLE and C

pRBR1::GU

roots of WT an

KNOLLE (E),

gs were exam

sterisks indica

CYCB1;1 in

US, (B) pK

nd phb3 muta

and CYCB1;1

mined for eac

ate means diff

the phb3 mu

KNOLLE::KN

ant plants. (A)

1 (F) transcrip

ch biological

fering significa

utant, Relate

NOLLE-GFP,

) Bars: 100 μm

pt in WT and

repeat. Error

antly from the

ed to

(C)

m; (B,

phb3

r bars

e WT

Figu

diffe

(A)

respe

F1 h

bars

Col (

ure S2. The p

erentiation, R

The sites of

ectively, exon

hybrid of these

represent SE

(p<0.01).

phb3 mutan

Related to Fig

f T-DNA ins

ns and introns.

e two alleles.

from triplicat

t seedlings s

gure 1

sertion in the

. (B) QC cell d

At least 100

te experiment

showed a hig

e two phb3

division and r

seedlings wer

ts. Asterisks in

ghly increase

mutant allele

root DSC diffe

re examined f

ndicate means

ed QC cell d

es. Boxes an

erentiation in

for each biolo

s differing sign

division and

nd lines repre

two alleles an

ogical repeat.

nificantly from

DSC

esent,

nd the

Error

m the

Figu

(A, C

D) Q

PHB

Diffe

ure S3. PLT1,

C) pPLT1::erC

Quantification

B3 OE seedlin

ferent letters

, PTL2 , SCR

CFP and pPL

n of the pPLT

ngs. The data a

indicate s

and SHR tra

LT2:: erCFP tr

T1::erCFP an

are shown as m

significant d

anscription in

ranscription in

nd pPLT2::er

mean ±SD (n=

differences (

n the AtPHB3

n five-day old

rCFP fluoresc

=15) with one

(P<0.01). (E

3 OE, Related

d seedling root

cence intensit

e-way ANOVA

E, G) pSCR

d to Figure 2

ts. Bars: 50 μm

ty in the WT

A and Tukey’

R::SCR-GFP

m. (B,

T and

’s test.

and

pSHR::SHR-GFP transcription in five-day old seedling roots. Bars: 50 μm. (F, H) Quantification of the

pSCR::SCR-GFP and pSHR::SHR-GFP fluorescence intensity in the WT and PHB3 OE seedlings. The

data are shown as mean ±SD (n=15) with one-way ANOVA and Tukey’s test. Different letters indicate

significant differences (P<0.01). (I) Abundance of PLT1, PTL2, SCR, SHR transcript in WT and

AtPHB3 OE plants. At least 100 seedlings were examined for each biological repeat. Error bars

represent SE from triplicate experiments. Asterisks indicate means differing significantly from the WT

(p<0.01).

Figu

(A) T

and t

treat

diffe

ure S4. The ph

The fluoresce

the phb3 muta

tment. Error b

erences (P<0.0

hb3 mutant h

ence microgra

ant. Bars: 50

bars represen

01).

hyper-accumu

aph illustrates

μm. (B) H2O2

t SE from tri

ulates ROS in

the presence

2 contents of W

iplicate exper

n its mitocho

of mitochond

WT and the ph

riments. Diffe

ndria, Relate

drial cpYFP in

hb3 mutant w

erent letters in

ed to Figure 3

n the roots of

with or withou

ndicate signif

3

f WT,

t DPI

ficant

Figu

(A)

trans

facto

ure S5. The tr

Genes altered

scribed genes

or genes. (D) q

ranscriptome

d in their tra

in Col-0 and

qRT-PCR ana

e of the phb3 m

anscription in

phb3 mutant.

lysis of select

mutant, Rela

the mutant.

. (C) The perc

ted differentia

ated to Figure

(B) Enriched

cent of differe

ally transcribed

es 3 and 4

d GO categor

entially transc

d genes.

ry of differen

cribed transcri

ntially

iption

Figu

(A) T

The

mean

ure S6. ERF11

The phenotyp

primary root

ns ±SE (n=25

14 OE shows

pes of the Col-

length of fiv

). Asterisks in

short root ph

-0 and ERF11

ve-day old Co

ndicate means

henotype, Re

14 over-expres

ol-0 and ERF1

s differing sign

elated to Figu

ssor five-day

114 over-expr

nificantly from

ure 4

old seedlings.

ressor seedling

m Col-0 (p<0.0

. Bars: 0.5 cm

gs. Data show

01).

m. (B)

wn as

Figu

Figu

Prop

vary

Bars

ure S7. H2O2

ure 5

pidium iodide

ying concentra

s: 50 μm.

did not indu

(PI) staining

ations of H2O2

uced cell dea

g of the roots

2 for 2 h. At le

ath in root PM

s of five-day-

east 20 seedlin

M under low

-old Col-0 se

ngs were exam

wer concentra

edlings treate

mined for each

ations, Relat

ed without or

h biological re

ed to

with

epeat.

Supplemental Experimental Procedures

Generation of Binary Constructs and Transformation

The coding sequence of ERF114 was amplified from the cDNA by RT-PCR using primers cloned into

pENTR (Invitrogen) to generate pENTR-ERF114. pENTR-ERF114 was cloned into pK7WG2 (Ghent

University) to generate 35S::ERF114 using LR reactions. For the SRDX dominant-negative construct,

the ERF114 cDNA was fused upstream of the SRDX sequence via PCR adaptor ligation and

recombined into the pK7GW2 (Ghent University) expression vector under control of the CaMV 35S

promoter via Gateway recombination. For GUS reporter construct, the 2000-bp promoter region

upstream of the ERF114 start codon was recombined into the pKGWFS7 plasmid (Ghent University),

in front of the β-glucuronidase ORF. The constructs were transformed into Agrobacterium tumefaciens

GV3101 by electroporation method and transformed into Arabidopsis as described previously.

RNA isolation and qRT-PCR

For quantitative real time PCR (qRT-PCR) assays, RNA was isolated using a RNeasy PlantMini kit

(Qiagen, Hilden, Germany) following the manufacturer’s protocol, treated with DNase I to remove

contaminating DNA and reverse transcribed to cDNA using a Transcriptor First Strand cDNA Synthesis

kit (Roche, Basel, Switzerland) based on a 1 μg RNA template, following the manufacturer’s protocol.

Subsequent reactions were performed in a Bio-RAD MyiQTM Real-time PCR Detection System

(Bio-Rad, Hercules, CA, USA) using the FastStart Universal SYBR Green Master mix (Roche,

Germany), according to the manufacturer’s instructions. Each sample was represented by three

biological replicates, and each biological replicate by three technical replicates. The reference sequence

was AtACTIN2. Details of all primers are given in Dataset S2.

Dual-luciferase transient expression assay in Arabidopsis protoplasts

To generate luciferase reporter constructs, the promoter fragments of PSK2 and PSK5 were amplified

and cloned into the transient expression vector pGreenII 0800-Luc. To generate 35S::ERF109 and

35S::ERF114 effector construct, the ERF109 and ERF114 coding sequence were amplified by PCR and

inserted into pENTR. pENTR-ERF109 and pENTR-ERF114 were cloned into pBI221 to generate

35S::ERF109 and 35S::ERF114 using LR reactions, respectively. The empty vector was included as a

control.

Arabidopsis mesophyll cell protoplast was isolated and transformed as described. 35S::ERF109

and 35S::ERF114 effector construct together with pPSK2::LUC or pPSK5::LUC reporter constructs

were introduced into protoplasts by PEG-mediated transformation. Transformed protoplasts were

incubated overnight, and firefly and Renilla luciferase activity was quantified using Dual-Luciferase

Reporter Assay kit (Promega, USA) and detected using a Synergy 2 multi-mode microplate (Bio-Tek)

according to the manufacturer’s instructions.

Accession numbers

The sequence data used in this article are represented in the Arabidopsis Genome Initiative and the

GenBank/EMBL databases under the following accession numbers: PHB3: At5g40770; AOX1a:

At3g22370; AOX1c: At3g27620.1; NDA1: At1g07180; NDB2: At4g05020; NDB3: At4g21490; NDB4:

At2g20800; ERF109: At4g34410; ERF1114: At5g61890; ERF115: At5g07310; PSK2: At2g22860;

PSK5: At5g65870; ACTIN2: At3g18780.