G10-90

RB XVE

OlexA-46

Hygr PtaZFP2 LB

pNos

(a)

#30 #37 #39(b)

0

0,5

1

1,5

2

WT #30 #39

(c)

PtaZFP2

rela

tive

exp

ress

ion

(d)

PtaZFP2

rela

tive

exp

ress

ion

0

0,5

1

1,5

2

0 1 3 6 9 12 24 48 96 144

Time after 17β-estradiol addition (h)

Fig. S1 Details on transgene construct, southern-blot analysis and levels of PtaZFP2 expression without 17β-estradiol in PtaZFP2-OE (#30 and #39) and WT poplars

(a) Schematic diagram of the estrogen inducible LexA-46::PtaZFP2 construct subcloned into the PMDC7 vector (Zuo et al., 2000; Curtis & Grossniklaus, 2003). pNos; nopaline synthase promoter; HygR, Hyromycin phosphotransferase II coding sequence. XVE, DNA sequences encoding a chimeric transcription factor containing the DNA-binding domain of LexA, the transcription activation domain of VP16 and the regulatory region of the human estrogen receptor. This chimeric transcription factor is under the control of a synthetic promoter (G10-90) and its transactivating activity is strickly regulated by estrogens. The coding sequence of PtaZFP2 is under the control of a synthetic promoter containing eight copies of the LexA operator (OlexA-46). Arrows indicate the direction of transcription.(b) Southern-blot analysis of three PtaZFP2-OE poplar transgenic lines recovered from three independent transformation events. Two of theses lines contain one copie of the transgene ((#30 and #39) while the line #37 is a multi-copy transgenic line. In this experiment, the hybridation was performed on 10µg of genomic DNA with a 32P-labeled probe corresponding to the XVE DNA sequence.(c) Expression level of PtaZFP2 transgene in PtaZFP2-OE poplar transgenic lines (#30, #39) and wild-type poplars (WT) without 17β-estradiol (DMSO treated).(d) Time course of endogenous PtaZFP2 expression by 17β-estradiol (10 µM) in WT stem. Relative transcripts abundance was determined by comparing the PtaZFP2 expression level of treated to untreated wild-type plants.

Fig. S2

Fig. S2 Schematic representation of the stress versus strain curves during a 3 point bending test. Each experimental curve can be split in two parts. The first linear part of the loading curve was used in order to compute the Young modulus (E). The elastic limit el was defined as the breaking point of the experimental curve. The second part represents the plastic behaviour. We considered a plastic zone that ranged between el and lim; with lim =1.5 el. In this zone, the total strain is composed of an elastic strain e and a plastic strain p. The stress-strain relationship can be written as follows:

Where ET is the tangent modulus in the plastic zone. ET measures the apparent rigidity of the tissues when both elastic and plastic strains are involved. The hardening modulus (H) can be defined as the modulus that locally links the stress and the plastic strain and represents the contribution of the additional plastic strain p that does

not recover after unloading. The lower the hardening modulus is, the higher is the additional plastic strain p .

pp H

EEE

T

T

1

T

T

EE

EEH

lim el

el

2

3lim for with

Table S1 Sequences of primers used for qRT-PCR

Primer name Sequence (5'-3')Fragment length

(pb)Gene name Gene model

EF1α F1  5'- GCAGATGATTTGCTGTTGC -3'

EF1α R1  5'- TGTAACCAACCTCTTCAGG -3'

PE1S 5'- CGTGCGAGTCACAAGAAACC -3'

PE1AS 5'- CACAGAACTCTCTTGCTGCT -3'

ZFP1 F1 5'- CAGTGCTCTATTTGCGGG -3'

ZFP1 R1 5'- GCTCAAATCCAAGCACAGG -3'

TCH4 F2 5'- CAAGGCAGGAGAGGTTG -3'

TCH4 R2 5'- ACAGGTAATGCAAGGACG -3'

XET6-F1 5'- TGTATGGGGTCTAATGGAGTC -3'

XET6-R1 5'- GGAATCGCTTAGTGTCTGC -3'

ETR1 F1 5'- GGATTCTGGATGCGGTG -3'

ETR1 R1 5'- ACCATTACTTCGACTAGATCC -3'

ACO4 F1 5'- GCATCATCTTGCTATTCCAG -3'

ACO4 R1 5'- GCAATCACTCTGTGCTCC -3'

CLE1-like F1 5'- GAAACTCGTTCAATCGACCG -3'

CLE1-like R1 5'- ACTGAGCCTGAAAGACTCG -3'

PO2 F1 5'- GCTATGGAGCAGTGGTC -3'

PO2 R1 5'- GGCCAACAGAGTGACTT -3'

ABC F2 5'- GCTGTTAGATGAGGCAACC -3'

ABC R2 5'- AGGACTGCGATGAGATCAC -3'

AMY1 F1 5'- GGAAAGCCACCAGGATTG -3'

AMY1 R1 5'- AAAGATCAGCATCAGAGGC -3'

FER F1 5'- AGGGTATTGTCGGAGTAGAC -3'

FER R1 5'- CTTGGAGTTAAGCCATCAGAG -3'

CML42 F1 5'- AGATCAGGCTAGGATGAGAC -3'

CML42 R1 5'- CCCAACTTCCTCAACACG -3'

GDSL F1 5'- TTGAGAGCTTGTTGTGGG -3'

GDSL R1 5'- CCATCTTCAATGGAACTGGC -3'

NIMIN1 F2 5'- AGAAGTTTTCAGGAAGCACG -3'

NIMIN1 R2 5'- CTAGGGAAGATGAGAGGAGG -3'

PtaETR1

201

136

PtaZFP1

112 PtaEF1 α

PtaZFP2287

PtaTCH4

169

132

132

151

PtaACO4

PtaCLE1-like

PtaPO2

PtaXET6

91

Potri.006G110000PtaFERONIA

PtaCML42

147

283

178

120

PtaABC

PtaAMY1

Potri.002G201500

Potri.014G159000

Potri.017G074000

Potri.002G126300

PtaGDSL

PtaNIMIN1

229

177

Potri.006G112500

Potri.010G047900

Potri.002G190800

Potri.006G130900

Potri.001G235800

Potri.009G027700

Potri.018G095100

Potri.001G376100

Potri.006G129900

Potri.018G094900

Over-represented GO terms among up-regulated genes in poplar transgenic line #39 Biological Process

GO Term P-valueSample

frequencyBackground

frequencyGenes

GO:1901700 response to oxygen-containing compound 1.94e-04 17/51 (33.3%)

2456/30328 (8.1%)

WRKY53 RHL41 GolS2 AT5G57150 PDIL1-3 DFR EP3 PHO1 AT5G51160 WAKL2 SERK1 ERF-1 FER PLA2A WRKY40 EFE AMY1

GO:0010200 response to chitin 3.60e-04 8/51 (15.7%) 422/30328 (1.4%)

WRKY53 RHL41 EP3 WAKL2 ERF-1 PLA2A WRKY40 EFE

GO:0010243 response to organic nitrogen 4.77e-04 8/51 (15.7%) 438/30328 (1.4%)

WRKY53 RHL41 EP3 WAKL2 ERF-1 PLA2A WRKY40 EFE

GO:1901698 response to nitrogen compound 4.85e-04 10/51 (19.6%)

791/30328 (2.6%)

WRKY53 RHL41 EP3 PHO1 AT5G51160 WAKL2 ERF-1 PLA2A WRKY40 EFE

GO:0042221 response to chemical stimulus 9.08e-04 20/51 (39.2%)

3779/30328 (12.5%)

WRKY53 RHL41 GolS2 AT5G57150 PDIL1-3 DFR EP3 PHO1 CCD8 AT5G51160 WAKL2 AT5G10695 SERK1 HMA5 ERF-1 FER PLA2A WRKY40 EFE AMY1

GO:0044765 single-organism transport 4.07e-03 16/51 (31.4%)

2716/30328 (9.0%)

WRKY53 OPT3 EP3 PHO1 CCD8 AT5G51160 AT3G01350 AT5G10695 GLIP5 AT4G31980 FER PLA2A WRKY40 SPX2 EFE PDIL1-4

GO:0010033 response to organic substance 6.01e-03 16/51 (31.4%)

2800/30328 (9.2%)

WRKY53 RHL41 GolS2 AT5G57150 PDIL1-3 DFR EP3 CCD8 WAKL2 AT5G10695 SERK1 ERF-1 PLA2A WRKY40 EFE AMY1

GO:0009719 response to endogenous stimulus 6.97e-03 13/51 (25.5%)

1875/30328 (6.2%)

WRKY53 RHL41 GolS2 AT5G57150 EP3 CCD8 WAKL2 SERK1 ERF-1 PLA2A WRKY40 EFE AMY1

Over-represented GO terms among down-regulated genes in poplar transgenic line #39 Biological Process

GO Term P-valueSample

frequencyBackground

frequencyGenes

GO:0050896 response to stimulus 4.41e-03 46/112 (41.1%)

6606/30328 (21.8%)

DI21 AT1G53710 RVE1 TLP18.3 AT3G58610 AFB2 ARF6 LOS1 CRY1 ABCB15 AT1G62660 VHA-A SYNC1 PAB2 EMB1467 AT2G37130 NAC2 SDE3 NIP1;2 ARF1 PPC1 PEPC1 PGM2 SIP2 MAC3B ACX1 AT4G33070 AT1G33440 EIN3 AT5G63710 PPI1 ACBP4 ASN2 EBF1 GSTU8 ETR1 MUR4 BT1 AT3G09440 ERS1 NIK1 RCN1 NCED3 AVP1 FAB1 CLE1

GO:0006950 response to stress 7.08e-03 33/112 (29.5%)

4018/30328 (13.2%)

DI21 RVE1 AT3G58610 LOS1 CRY1 ABCB15 AT1G62660 VHA-A PAB2 EMB1467 AT2G37130 SDE3 ARF1 PPC1 PEPC1 SIP2 MAC3B ACX1 AT4G33070 EIN3 AT5G63710 PPI1 ASN2 GSTU8 ETR1 MUR4 BT1 AT3G09440 NIK1 RCN1 NCED3 AVP1 FAB1

GO:1901575 organic substance catabolic process 8.95e-03 19/112 (17.0%)

1611/30328 (5.3%)

AT3G58610 ABCB15 AT1G62660 VHA-A PAB2 EMB1467 AT1G21680 PPC1 AIM1 PGM2 SIP2 ACX1 AT4G33070 ABCF1 EBF1 GSTU8 AT3G09440 RCN1 FAB1

Cellular ComponentGO:0005829 cytosol 4.57e-03 20/112

(17.9%)1687/30328

(5.6%)

LOS1 TOC159 THY-1 AT3G08947 ACLB-2 ECT2 SYNC1 PAB2 AT5G52580 AT2G37130 PPC1 PGM2 AT2G25970 IMPA-2 AT4G33070 AT1G36730 ACBP4 ASN2 AT3G09440 STY17

Molecular FunctionGO:0003824 catalytic activity 7.75e-08 57/112

(50.9%)6861/30328

(22.6%)

AT4G24340 ALDH11A3 AT1G53710 STT3B TLP18.3 AT3G58610 AFB2 PEPKR1 AT1G49570 AT5G24010 TOC159 CRY1 THY-1 ABCB15 AT1G62660 ACLB-2 VHA-A CYP707A4 EMB1467 AT2G37130 AT2G40860 AT4G32250 SDE3 emb1138 AT1G28240 PPC1 PEPC1 AIM1 PGM2 AT2G38600 AT1G32930 SIP2 SLP3 ACX1 AT4G33070 CSR1 AT3G12620 XTH5 AT3G22560 AT5G63710 CYP78A9 ASN2 SBTI1.1 EBF1 AT3G45400 GSTU8 ETR1 LACS9 MUR4 FMO GS-OX4 ERS1 NIK1 NCED3 AVP1 EMB2813 STY17 FAB1

Table S3: List of over-represented Amigo GO terms in the Arabidopsis matches of the differentially expressed poplar genes in PtaZFP2 -OE plants. GO enrichment was tested by interrogating The Arabidopsis Information Resource (TAIR) Gene Ontology annotation search tool (http://www.arabidopsis.org/tools/bulk/go/).