GENETICS AND GENOMICS

Transcriptional profiling in response to inhibition of cellulosesynthesis by thaxtomin A and isoxaben in Arabidopsis thalianasuspension cells

Isabelle Duval Æ Nathalie Beaudoin

Received: 13 October 2008 / Revised: 16 December 2008 / Accepted: 7 January 2009 / Published online: 7 February 2009

� Springer-Verlag 2009

Abstract The plant cell wall determines cell shape and is

the main barrier against environmental challenges. Pertur-

bations in the cellulose content of the wall lead to global

modifications in cellular homeostasis, as seen in cellulose

synthase mutants or after inhibiting cellulose synthesis. In

particular, application of inhibitors of cellulose synthesis

such as thaxtomin A (TA) and isoxaben (IXB) initiates a

programmed cell death (PCD) in Arabidopsis thaliana

suspension cells that is dependent on de novo gene tran-

scription. To further understand how TA and IXB activate

PCD, a whole genome microarray analysis was performed

on mRNA isolated from Arabidopsis suspension cells

exposed to TA and IXB. More than 75% of the genes

upregulated by TA were also upregulated by IXB, includ-

ing genes encoding cell wall-related and calcium-binding

proteins, defence/stress-related transcription factors, sig-

nalling components and cell death-related proteins.

Comparisons with published transcriptional analyses

revealed that half of these genes were also induced by

ozone, wounding, bacterial elicitor, Yariv reagent, chitin

and H2O2. These data indicate that both IXB and TA

activate a similar gene expression profile, which includes

an important subset of genes generally induced in response

to various biotic and abiotic stress. However, genes typi-

cally activated during the defence response mediated by

classical salicylic acid, jasmonate or ethylene signalling

pathways were not upregulated in response to TA and IXB.

These results suggest that inhibition of cellulose synthesis

induces PCD by the activation of common stress-related

pathways that would somehow bypass the classical hor-

mone-dependent defence pathways.

Keywords Cellulose synthesis � Isoxaben � Microarray �Programmed cell death � Thaxtomin A

Introduction

Unlike animal cells, plant cells are surrounded by a cell

wall that allows them to maintain their shape and stability

by restraining the high turgor pressure applied by the

vacuole. This extracellular network is composed of cellu-

lose microfibrils embedded in a matrix of polysaccharides

constituted by pectins and hemicelluloses. The strength of

the cell wall is driven by cellulose microfibrils that are

synthesised at the membrane surface by enzymatic com-

plexes formed by cellulose synthases (CesAs). On the other

hand, hemicelluloses and pectins are produced in the Golgi

apparatus and are further transported to the cell wall by

exocytosis vesicles. Hemicelluloses bind to cellulose

microfibrils, driving some resilience to this rigid but

flexible cell wall (reviewed in Fry 2004 and Cosgrove

2005). Proteins associated with this complex network of

polysaccharides are classified according to their amino

acid composition. These include hydroxyproline-rich

glycoproteins (HRGPs), arabinogalactan-proteins (AGPs),

Communicated by R. Rose.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-009-0670-x) contains supplementarymaterial, which is available to authorized users.

I. Duval � N. Beaudoin (&)

Departement de biologie, Universite de Sherbrooke,

Sherbrooke, QC J1K 2R1, Canada

e-mail: Nathalie.Beaudoin@usherbrooke.ca

Present Address:I. Duval

Natural Resources Canada, Canadian Forest Service,

Laurentian Forestry Centre, Quebec, QC G1V 4C7, Canada

123

Plant Cell Rep (2009) 28:811–830

DOI 10.1007/s00299-009-0670-x

glycine-rich proteins (GRPs) and proline-rich proteins

(PRPs). All of these proteins are glycosylated and contain

hydroxyproline, except for GRPs (reviewed in Cassab

1998). AGPs, also classified as proteoglycans, are the most

glycosylated proteins of the cell wall, with a carbohydrate

component contributing to more than 90% of the total

weight (Rumyantseva 2005).

The plant cell wall is the first barrier against pathogen

attack and environmental challenges. Consequently, any

important perturbations in the organisation or composition

of the cell wall must be perceived by the cell in order to

activate the appropriate signalling cascades required for

plant defence. For example, reduced cellulose synthesis in

the cellulose synthase CesA3 Arabidopsis mutants or after

treatment with the cellulose synthesis inhibitor isoxaben

(IXB) triggered ectopic lignin deposition and defence

responses through the ethylene and jasmonate (JA) path-

ways (Ellis et al. 2002; Cano-Delgado et al. 2003).

Similarly, a decrease in cellulose synthesis resulting from a

mutation that affects the activity of a vacuolar H?-ATPase

subunit was associated with a reduction in cell expansion

and an extensive deposition of lignin in cells that normally

do not lignify. These plants also displayed an increased

expression of defence-related genes, suggesting that

reduced cellulose synthesis in the cell wall leads to defence

response (Schumacher et al. 1999; Cano-Delgado et al.

2003). Activation of defence mechanisms in plants is often

associated with the induction of the hypersensitive

response (HR), a type of programmed cell death (PCD) that

would restrain pathogen growth and progression (Heath

2000; Greenberg and Yao 2004). We have previously

shown that application of inhibitors of cellulose synthesis

such as thaxtomin A (TA) and IXB on Arabidopsis sus-

pension cells also triggered PCD (Duval et al. 2005).

However, this PCD was not accompanied by classic

defence responses normally associated with the HR, such

as production of extracellular hydrogen peroxide and

expression of typical defence-related genes. Nonetheless,

our results indicated that the TA- and IXB-induced PCD

was an active process dependent on de novo gene expres-

sion and protein synthesis (Duval et al. 2005).

Experimental evidence and mutant analyses have shown

that IXB inhibits cellulose synthesis by perturbing cellu-

lose synthase (CesA) complexes through the recognition of

an epitope associated with complexes containing CesA3

and CesA6 (Scheible et al. 2001; Desprez et al. 2002;

Desprez et al. 2007). In particular, application of IXB leads

to depletion of CesA6-containing complexes from the

plasma membrane (Paredez et al. 2006). While there is

good evidence that TA is a cellulose biosynthesis inhibitor

(Scheible et al. 2003; Robert et al. 2004), its specific mode

of action is still unknown. TA is the main phytotoxin

produced by Streptomyces scabiei, one of the causative

agents of potato common scab disease. This toxin is

essential for the apparition of disease symptoms (Goyer

et al. 1998). Application of small concentrations of TA led

to shoot and root stunting and tissue thickening due to cell

hypertrophy, while application of higher levels reduced

total length of radish seedlings and caused necrosis (Leiner

et al. 1996). While some of these effects are related to TA

ability to inhibit cellulose synthesis, there are some indi-

cations that TA may also have additional effects (King

et al. 2001). However, the fact that both TA and IXB can

induce a similar cell death program suggests that it is their

capacity to inhibit cellulose synthesis that activates PCD.

To further understand how perturbations of the cell wall

induced by inhibitors of cellulose synthesis can lead to

PCD, we performed a whole genome microarray analysis

on mRNA from Arabidopsis suspension cells exposed to

TA or IXB. Genes with modified expression were classified

into functional categories. The transcriptional profiles

induced by each of these inhibitors of cellulose biosyn-

thesis were very similar. Results were also compared with

transcriptional changes induced by various abiotic and

biotic stress, including PCD-inducing treatments. More

than half of TA and IXB-upregulated genes were also

upregulated by each of ozone, wounding, bacterial elicitor,

Yariv reagent, chitin and H2O2, indicating the activation of

genes in common stress and defence signalling pathways.

These results suggest that TA and IXB induced comparable

cell wall perturbations which are sensed by the cell to

activate the expression of a common set of stress-related

genes that ultimately lead the cell to PCD.

Materials and methods

Plant material and treatments

Arabidopsis thaliana ecotype Landsberg erecta suspen-

sion-cultured cells were grown in Murashige and Skoog

(MS) medium (pH 5.7, 0.6 g l-1 MES) supplemented with

B5 vitamins and 1 mg l-1 2,4-D. Cells were maintained in

the dark under continuous shaking (gyratory shaker) at

120 rpm at 21�C. Suspensions of 100 ml were sub-cultured

weekly using 3:7 dilution. TA was purified as previously

described (Duval et al. 2005). TA diluted in methanol

(10 mM) was added to Arabidopsis cell suspensions at a

final concentration of 2 lM, and IXB (1 mM in methanol;

Crescent Chemicals Co., Inc., Islandia, NY, USA) was

added at a final concentration of 100 nM. The same volume

of methanol (20 ll) was added to all cells. Treatments were

carried out the third day after sub-culture, at the beginning

of the exponential phase. Samples consisted of four repli-

cates for each condition, and each replicate was the

combination of two 250 ml flasks containing 100 ml of

812 Plant Cell Rep (2009) 28:811–830

123

cells pooled together before freezing in liquid N2 after 6 h

of contact with the inhibitors. A 20 ml aliquot of suspen-

sion cells was conserved for each condition to check cell

death after 6 h and up to 72 h, using trypan blue staining as

previously described (Duval et al. 2005). Cells were stored

at -80�C until RNA extraction was performed.

Sample preparation and microarray data collection

and analysis

Total RNA was extracted as previously described (Duval

et al. 2005). Synthesis of cRNA and hybridizations to

Arabidopsis ATH1 GeneChip (Arabidopsis Genome ATH1

Array) were carried out by Genome Quebec, Innovation

Center (McGill University, Montreal, Canada) following

Affymetrix recommended protocols. Twelve arrays were

hybridized, four per condition. Data and statistical analyses

were performed using FlexArray software (Blazejczyk et al.

2007). FlexArray implements Bioconductor (Gentleman

et al. 2004) and R (http://www.r-project.org/), two open

source software and language respectively that provide

statistical tools for the analysis of genomic data. Raw signal

intensities were normalized with the Robust Multi-array

Average methodology (RMA; Irizarry et al. 2003). RMA

was chosen because of its better reproducibility and its less

number of false positive (Millenaar et al. 2006). Signifi-

cance analysis of microarrays (SAM; Tusher et al. 2001)

was carried out to determine the differentially expressed

genes. Genes with at least twofold (TA treatment) and

1.5-fold (IXB treatment) changes when compared to control

levels and a P value less than 0.05 were selected.

Functional classification of upregulated genes

Each gene upregulated by TA (twofold or more) or by IXB

(1.5-fold or more) was assigned a functional category

based on the known or putative function of its protein using

TAIR GO annotations and functional categorization

tools (Berardini et al. 2004). Normalization of functional

classification was performed using the Classification

SuperViewer tool (Provart and Zhu 2003).

RT-PCR confirmation of transcription profile

An RT-PCR was performed to confirm results obtained

with GeneChips. Nine genes with low basal expression in

control were selected. First strand cDNA synthesis was

carried out on 2.0 lg of total RNA, using polydT oligo-

nucleotide (Promega, http://www.promega.com) and AMV

reverse transcriptase (Promega, http://www.promega.com)

following the manufacturer’s instructions. One ll of cDNA

was amplified with primers presented in Table 1.

Comparison of expression data

Comparison of expression of genes upregulated in com-

mon by TA and IXB was performed using Genevestigator

Table 1 Primer sequences used

for RT-PCRGene Primer sequences Accession number

AGP18 50-ACATTGATCTGCATTGTCGTCGCCGGTGTC-30 At4g37450

50-CTACATTCCTCAACACCCTTGTGCTCGCTG-30

Aspartyl protease 50-GTCTCCCATTCCAAAACCACTCTTCTTC-30 At1g66180

50-CTCACACTACTCTGCTACAATCAGCTTTAG-30

AtATG8h 50-AATGGGGATTGTTGTCAAGTCTTTCAAGGA-30 At3g06420

50-TGCTGTAGCACATGTACAAGAACCCGTC-30

CYP83B1 50-GTAGCACGACCAAGAAATCTCTCCGGTTAC-30 At4g31500

50-CATCAGATGTGTTTCGTTGGTGCAAGAAC-30

AtEXP12 50-GTTAGTACGTTAAGTGTGGGGATGTGTTC-30 At3g15370

50-GTCCGTCAGAGTAACTTTGAAGGAGAGTC-30

GCN5-related 50-GTCAATGGAGATGGACTCAACTACTACGA-30 At2g32030

50-CAGTGCAACGAATCAGAAGGCAAGAAAC-30

AtMYB15 50-GAAGAGAGGACCATGGACACCTGAAGAAG-30 At3g23250

50-CTAGAGCCCGGCTAAGAGATCTTGTTC-30

Pectin esterase 50-CATGCTATCTCTCAAACTCTTCCTCGTAAC-30 At4g02330

50-CTAATATAAGGTACACCACTCTGAACCATC-30

WRKY75 50-ATATGGAGGGATATGATAATGGGTCGTTG-30 At5g13080

50-GAAGTTTTCGGTGGATTTCTCGATGGGATG-30

Actin11 50-ATGGCAGATGGTGAAGACATTCA-30 At3g12110

50-GCCTTTGCTATCCACATCTGTTG-30

Plant Cell Rep (2009) 28:811–830 813

123

Table 2 Upregulated genes by both TA (at least 2-FC after 6 h, P value \0.05) and IXB (at least 1.5-FC after 6 h, P value \0.05)

Experimentsa

Fold-changeProbe set

number AGI locus Description TA IXB Ozo

ne

wou

ndin

g

EF-

Tu

Yar

iv r

eage

nt

Chi

tin

H2O

2

P. s

yrin

gae

Am

inot

riaz

ole

AA

L-t

oxin

Sene

scen

ce

IXB

-hab

ituat

ed

Cell wall 264005_at At2g22470 Arabinogalactan-protein 2 (AGP2) 6.13 3.57 + + + + 250437_at At5g10430 Arabinogalactan-protein 4 (AGP4) 2.36 1.80 - - 253050_at At4g37450 Arabinogalactan-protein 18 (AGP18) 7.67 3.43 - + - - - - 251281_at At3g61640 Arabinogalactan-protein 20 (AGP20) 2.28 1.62 + + + - +248252_at At5g53250 Arabinogalactan-protein 22 (AGP22) 5.36 4.49 - + - - - 264315_at At1g70370

BURP domain-containing protein / polygalacturonase, putative

2.14 1.62 - - - - +23.399.4)21APXEtA(21nisnapxE07351g3tAta_883852 + - -

252563_at At3g45970 Expansin-like A1 (AtEXLA1) 2.64 2.28 + + + 252997_at At4g38400 Expansin-like A2 (AtEXLA2) 3.42 2.70 - + + - 252557_at At3g45960 Expansin-like A3 (AtEXLA3) 4.10 2.65 - + + + - 265066_at At1g03870 Fasciclin-like arabinogalactan-protein (FLA9) 3.48 2.30 - + - - - 263942_at At2g35860

Fascilin-like arabinogalactan protein 16 precursor (FLA16)

2.65 1.98 - - - 256633_at At3g28340 Galacturonosyltransferase-like 10 (GATL10) 2.51 1.81 + + + + + + - 255756_at At1g19940 Glucosyl hydrolase 9B5 (AtGH9B5) 4.73 3.06 - + - - 265648_at At2g27500 Glycosyl hydrolase family 17 protein 3.39 2.32 + + + + + + + 264280_at At1g61820

Hydrolase, hydrolyzing O-glycosyl compounds (BGLU46)

3.86 2.13 + + + + + + + - 248968_at At5g45280 Pectinacetylesterase, putative 2.88 1.77 + + - - 255524_at At4g02330 Pectinesterase (AtPMEPCRB) 5.90 3.63 + + + + + + - 245151_at At2g47550 Pectinesterase family protein 10.05 4.19 + + + - - 252255_at At3g49220 Pectinesterase family protein 3.08 2.26 + - - 252989_at At4g38420

SKU5 Similar 9 (SKS9) / pectinesterase / copper ion binding / oxidoreductase

12.26 6.15 + - - -

253628_at At4g30280 Putative xyloglucan endotransglucosylase / hydrolase (AtXTH18)

11.26 5.54 + + + + + + + + -

253608_at At4g30290 Putative xyloglucan endotransglucosylase / hydrolase (AtXTH19)

3.30 2.38 + + - -

253666_at At4g30270 Similar to endo-xyloglucan transferase / MERISTEM-5 (MERI5B) (SEN4)

3.66 2.24 + + +

247925_at At5g57560 Xyloglucan:xyloglucosyl transferase activity (XTH22) / touch 4 (TCH4)

4.75 3.27 + + + + + + - + - -

245794_at At1g32170 Xyloglucan endotransglycosylase 4 (XTH30) (XTR4)

2.53 1.86 + + + - 254042_at At4g25810 Xyloglucan endotransglycosylase 6 (XTR6) 8.44 4.37 + + + + + + + + - - 245325_at At4g14130

Xyloglucan endotransglycosylase-related protein (XTR7)

2.31 1.92 - - +

251336_at At3g61190 BON1-associa

Calcium-related

ted protein 1 (BAP1) 4.36 2.66 + + + + + + + + + - 259879_at At1g76650 Calcium ion binding (CML38) 6.24 1.94 + + + + + + + + - - 249417_at At5g39670 Calcium-binding EF hand family protein 2.85 1.76 + + + + + + + + - - 253915_at At4g27280

Calcium-binding EF hand family protein similar to PBP1

6.75 4.65 + + + + + + - -

265460_at At2g46600 Calcium-binding protein, putative; similar to PBP1 (PINOID-BINDING PROTEIN 1)

2.90 1.85 +08.130.2evitatup,niludomlaC00652g3tAta_557652 + + + + +

247493_at At5g61900 Copine (CPN1) / bonzai1 (BON1) 2.53 1.52 + + + + + + - 248164_at At5g54490 Pinoid-binding protein 1 (PBP1) 3.49 2.47 + + + + + + - -

98.344.6)3HCT(evisnopser-hcuoT00114g2tAta_380762 + + + + + + + + - -

814 Plant Cell Rep (2009) 28:811–830

123

Table 2 continued

Phosphorylation

249730_at At5g24430 Calcium dependent kinase (CDPK), putative 2.12 1.63 + + + + - 261506_at At1g71697 Choline kinase (AtCK1) (CK) 3.18 1.74 + + + + + + + 266124_at At2g45080 Cyclin-dependent protein kinase (CYCP3;1) 2.66 1.86 + - 267518_at At2g30500 Kinase interacting family protein 2.64 1.98 + + + - 246858_at At5g25930 Leucine-rich repeat family protein/ RLK5-like 2.43 2.04 + + + + + + + + - 245731_at At1g73500 MAP kinase kinase 9 (AtMKK9) 3.53 1.86 + + + + + 253937_at At4g26890 MAP kinase kinase kinase 16 (MKKK16) 2.54 1.75 + 257536_at At3g02800

Phosphoprotein phosphatase, similar to tyrosine specific protein phosphatase family protein

3.36 1.56 + + + + + +

258682_at At3g08720 Protein kinase 19 (AtPK19) / serine/threonine protein kinase 2 (AtPK2) (AtS6K2)

2.61 1.96 + + + + + + - - 256177_at At1g51620 Protein kinase family protein 2.30 1.62 + + + +

79.175.2evitatup,esaniknietorP04950g2tAta_730662 + + + + + + - - 34.267.4evitatup,esaniknietorP01193g2tAta_691662 + + - 66.151.3evitatup,esaniknietorP07074g5tAta_128842 + + + + + + +

245528_at At4g15530 Pyruvate orthophosphate dikinase (PPDK) 2.59 1.87 + + + + +251494_at At3g59350 Serine/threonine protein kinase, putative 2.38 1.78 + + + + + - Transcription factors

260203_at At1g52890 Arabidopsis NAC domain containing protein 19 (ANAC019)

4.37 1.79 + + - + + + -

265260_at At2g43000 Arabidopsis NAC domain containing protein 42 (ANAC042)/transcription factor

2.88 1.88 + + + + + + + + 248606_at At5g49450 Basic leucine-zipper 1 (ATBZIP1) 3.66 2.10 + + + 251861_at At3g54810

Blue micropylar end 3 (BME3) (BME3-ZF) /zinc finger (GATA type) family protein

2.07 1.98 + + + - 252679_at At3g44260 CCR4-NOT transcription complex protein, putative 2.25 1.86 + + + + + + - - 250910_at At5g03720 Heat stress transcription factor A3 (AtHSFA3) 2.79 1.61 + + - - 248794_at At5g47220

Member of subfamily B-3 of ERF / AP2 transcription factor family (AtERF-2)

5.89 1.87 + + + + - -

261984_at At1g33760 Member of the DREB subfamily A-4 of ERF/ AP2 transcription factor family

8.10 4.67 + + + + + - -

259729_at At1g77640 Member of the DREB subfamily A-5 of ERF / AP2 transcription factor family

4.18 2.33 + + -

260856_at At1g21910 Member of the DREB subfamily A-5 of ERF / AP2 transcription factor family.

3.22 2.76 + + + + -

248448_at At5g51190 Member of the subfamily B-3 of ERF/AP2 transcription factor family

2.38 1.52 + + + + - +

248799_at At5g47230 Member of the subfamily B-3 of ERF/AP2 transcription factor family (AtERF-5)

2.56 1.61 + + + + + +

247543_at At5g61600 Member of the subfamily B-3 of ERF/AP2 transcription factor family

2.66 1.59 + + + + + - 257919_at At3g23250 Myb domain protein 15 (MYB15) 4.50 2.27 + + + + + + + - 247535_at At5g61620 Myb family transcription factor 2.04 1.66 - + + 256332_at At1g76890 Plant trihelix DNA-binding protein (AtGT2) 2.91 1.86 - - 260037_at At1g68840

Regulator of the ATPase of the vacuolar membrane (RAV2) (RAP2.8)

2.56 2.15 + + + + + - + - -

247655_at At5g59820 Responsive to high light 41 (RHL41) / zinc finger family protein (ZAT12)

4.43 2.47 + + + + + + + + + - 261648_at At1g27730 Salt tolerance zinc finger (STZ) (ZAT10) 5.13 2.11 + + + + + + + 267028_at At2g38470 WRKY DNA-binding protein 33 (WRKY33) 9.07 4.51 + + + + + + -

Experimentsa

Fold-changeProbe set

number AGI locus Description TA IXB Ozo

ne

wou

ndin

g

EF-

Tu

Yar

iv r

eage

nt

Chi

tin

H2O

2

P. s

yrin

gae

Am

inot

riaz

ole

AA

L-t

oxin

Sene

scen

ce

IXB

-hab

ituat

ed

263783_at At2g46400 WRKY DNA-binding protein 46 (WRKY46) 5.63 2.05 + + + + + + - 245976_at At5g13080 WRKY DNA-binding protein 75 (WRKY75) 6.22 2.10 + + + + + + + + + - 251745_at At3g55980 Zinc finger (CCCH-type) family protein 3.49 2.48 + + + + + + - - -

245711_at At5g04340 Zinc finger of Arabidopsis thaliana 6 (C2H2) (CZF2) (ZAT6)

3.22 1.50 + + + + + + +

Plant Cell Rep (2009) 28:811–830 815

123

Table 2 continued

Stress, defence and cell death

256525_at At1g66180 Aspartyl protease family protein 3.28 2.31 + + + - - 246099_at At5g20230 Blue copper-binding protein (AtBCB) 3.27 2.10 + + + + + + + + + 253046_at At4g37370

Cytochrome P450, family 81, subfamily D, polypeptide 8 (CYP81D8)

2.07 1.58 + + + + + + + - 267411_at At2g34930 Disease resistance family protein 7.18 1.81 + + + + + + + + - 252126_at At3g50950

Disease resistance protein (CC-NBS-LRR class), putative

2.45 1.83 + + + + + -

265597_at At2g20142 Similar to disease resistance protein (TIR-NBS-LRR class), putative

3.23 2.03 + + + + + - - 266746_s_at At2g02930 Glutathione S-transferase 16 (AtGSTF3) (GST16) 2.54 1.73 + + + 260405_at At1g69930

Glutathione S-transferase (class tau) 11 (AtGSTU11)

2.22 1.66 + + + + + + + - - 250351_at At5g12030 17.6 kDa heat shock protein (AtHSP17.6A) 2.96 1.89 + + + + + - +250676_at At5g06320 NDR1/HIN1 97.126.2)3LHN(ekil- + + + + + + + - 245738_at At1g44130 Nucellin pr 02.287.3evitatup,nieto + + + + - - 254784_at At4g12720 Nudix hydrolase homolog 7 (AtNUDT7) (GFG1) 2.90 1.83 + + + + + + + - - 253104_at At4g36010 Pathogenesis-related thaumatin family protein 2.17 1.50 + + + + + +

87.112.2evitatup,esadixoreP07780g4tAta_011552 + + + - 21.299.2evitatup,esadixoreP02760g5tAta_646052 + - 22.229.2evitatup,esadixoreP03760g5tAta_207052 + + + -

249459_at At5g39580 Peroxidase, putative/identical to Peroxidase 62 precursor (PER62)

6.48 4.00 + + + + + + + + 251984_at At3g53260 Phenylalanine ammonia-lyase 2 (PAL2) 2.92 1.65 + + + -

55.115.2detaler-rotibihniesaetorP07883g2tAta_861662 + + + + + + + +248719_at At5g47910 Respiratory burst oxidase protein D (RbohD) 2.52 1.63 + + + + + + + 255479_at At4g02380

Senescence-associated gene 21 (SAG21)/late embryogenesis abundant like 5 (AtLEA5)

8.88 3.54 + + + + + - - 59.161.4)8TET(8NINAPSARTET01832g2tAta_392762 + + + + + + + 66.234.4)9TET(9NINAPSARTET03403g4tAta_236352 + + + + + - -

263935_at At2g35930 U-box domain-containing protein 2.59 1.81 + + + + + + - +249234_at At5g42200

Zinc finger (C3HC4-type RING finger) family protein

2.90 1.58 - Metabolism

255177_at At4g08040 1-amino-cyclopropane-1-carboxylate synthase 11 (ACS11)

2.66 1.84 + + -

259439_at At1g01480 1-aminocyclopropane-1-carboxylate synthase 2/ACC synthase 2 (ACS2)

2.24 1.64 + + + + + + + -

254926_at At4g11280 1-aminocyclopropane-1-carboxylate synthase 6 (ACS6)

2.78 1.96 + + + + + + + -

253999_at At4g26200 1-amino-cyclopropane-1-carboxylate synthase 7 (ACS7)

2.81 1.68 + + + - 257644_at At3g25780 Allene oxide cyclase 3 (AOC3) 5.83 3.02 + + + + + + - - 252652_at At3g44720 Arogenate dehydratase 4 (ADT4) 3.76 1.85 + + + + + - - 264313_at At1g70410

Carbonic anhydrase, putative/carbonate dehydratase, putative

2.66 1.76 - 256787_at At3g13790 Cell wall invertase 1 (AtBFRUCT1) (AtCWINV1) 3.24 1.85 + + + + - 248964_at At5g45340

Cytochrome P450, family 707, subfamily A, polypetide 3 (CYP707A3)

3.49 2.70 + + + + + - - -

Experimentsa

Fold-changeProbe set

number AGI locus Description TA IXB Ozo

ne

wou

ndin

g

EF-

Tu

Yar

iv r

eage

nt

Chi

tin

H2O

2

P. s

yrin

gae

Am

inot

riaz

ole

AA

L-t

oxin

Sene

scen

ce

IXB

-hab

ituat

ed

260023_at At1g30040 Gibberellin 2-oxidase (AtGA2OX2) 3.03 1.90 + + + + + + + - - 258075_at At3g25900 Homocysteine S-methyltransferase 1 (HMT-1) 2.68 1.94 + - +261459_at At1g21100 Putative O-methyltransferase 1 3.69 2.07 + + + + + + - - 256319_at At1g35910 Trehalose-6-phosphate phosphatase, putative 2.38 1.76 + + + + + - -

816 Plant Cell Rep (2009) 28:811–830

123

Table 2 continued

Secondary metabolism

261899_at At1g80820 Cinnamoyl CoA reductase isoform 2 (CCR2) 6.16 1.95 + + + + + + + + - - 256186_at At1g51680 4-coumarate:CoA ligase 1 (4CL1) (At4CL1) 5.85 2.27 - - 266693_at At2g19800 Myo-inositol oxygenase 2 (AtMIOX2) 3.36 2.56 + + + 245141_at At2g45400

Oxidoreductase, regulation of brassinosteroid metabolic pathway (BEN1)

2.37 1.86 - + +258277_at At3g26830 Phytoalexin deficient 3 (PAD3) (CYP71B15) 2.19 1.89 + + + + -

08.125.2esanegyxopilevitatuP02527g1tAta_993062 + + + + + + + - - Cellular transport

46.132.2)6HTAtA(6golomohCBA08774g3tAta_383252 + + + + - 249283_at At5g41800 Amino acid transporter family protein 2.26 1.79 + + 264624_at At1g08930

Early-respone to dehydration (ERD6)/sugar transporter family protein

2.74 1.83 + + + + + - 246238_at At4g36670 Mannitol transporter, putative 2.74 1.64 + 264289_at At1g61890 MATE efflux family prot 87.138.2nie + + + + + + 264000_at At2g22500 Mitochondrial substrate carrier family protein 2.09 1.57 + + + + + + - - 254120_at At4g24570 Mitochondrial substrate carrier family protein 2.57 2.21 + + + + + + 245866_s_at At1g57990 Purine permease 18 (AtPUP18) 2.40 1.66 + + + + + + + + - Other functions

252131_at At3g50930 AAA-type ATPase family protein 5.61 3.24 + + + + + + + + - - 263800_at At2g24600 Ankyrin repeat family protein 2.85 1.87 + + + + + + - 260427_at At1g72430 Auxin-responsive protein-related 4.39 2.46 + - -

31.269.5nietorpylimaf7dnaB09210g3tAta_272952 + + + + + + + + - 09.172.2nietorpylimaf7dnaB04896g1tAta_104062 + + + + + + + -

253284_at At4g34150 C2 domain-containing protein 2.38 1.57 + + + + + + + + - 256763_at At3g16860 Cobra-like protein precursor 8 (COBL8) 2.65 1.65 + + + + + + 254331_s_at At4g22710

Cytochrome P450, family 706, subfamily A, polypeptide 2 (CYP706A2)

2.29 1.81 + + + - - -

247949_at At5g57220 Cytochrome P450, family 81, subfamily F, polypeptide 2 (CYP81F2)

2.37 1.54 + + + + + + - + - - 249097_at At5g43520 DC1 domain-containing protein 2.78 1.60 - - - 259516_at At1g20450 Early responsive to dehydration 10 (ERD10) 3.61 1.92 + + - +254447_at At4g20860 FAD-binding domain-containing protein 3.69 2.09 + + + + + + + 264758_at At1g61340 F-box fa 48.191.4nietorpylim + + + + + + + 265725_at At2g32030

GCN5-related N-acetyltransferase (GNAT) family protein

5.13 2.61 + + + + + + - - 245226_at At3g29970 Germinatio 99.106.3detaler-nietorpn - - 256017_at At1g19180 Jasmonate-ZIM-domain protein (JAZ1) 3.57 1.83 + + + + + + + + + 265938_at At2g19620 Ndr fami 46.183.2nietorpyl + + - - 262549_at At1g31290

PAZ domain-containing protein / piwi domain-containing protein

2.36 1.67 + - - 245757_at At1g35140 Phosphate induced-1 (PHI-1) 4.18 2.92 + + + + + + + - 252624_at At3g44735 Phytosulfokine 3 precursor (ATPSK3) (PSK1) 2.54 1.65 + - - - 261975_at At1g64640 Plastocyanin-like domain-containing protein 2.50 1.55 - - - 267055_at At2g38360 Prenylated rab acceptor (PRA1) family protein 2.27 1.78 + + + - -

Experimentsa

Fold-changeProbe set

number AGI locus Description TA IXB Ozo

ne

wou

ndin

g

EF-

Tu

Yar

iv r

eage

nt

Chi

tin

H2O

2

P. s

yrin

gae

Am

inot

riaz

ole

AA

L-t

oxin

Sene

scen

ce

IXB

-hab

ituat

ed

254314_at At4g22470 Protease inhibitor/seed storage/lipid transfer protein (LTP) family protein

3.03 2.22 + + + + + +253140_at At4g35480 Putative RING-H2 finger protein (RHA3b) 2.04 1.56 + + + + -

04.224.2)82LFLAR(82ekil-FLAR01511g4tAta_009452 + + - 261476_at At1g14480 Similar to ankyrin repeat family protein 2.14 1.56 + + + -

Plant Cell Rep (2009) 28:811–830 817

123

MetaProfile Analysis tool (Hruz et al. 2008) with the

following experiments: ozone (AT-025), H2O2 (AT-185),

wounding (AT-120), Pseudomonas syringae (AT-106),

chitin (AT-169), EF-Tu (AT-128), PCD senescence

(AT-026), IXB-habituated cells (AT-010). Additional data

were taken from published work for treatment with Yariv

Table 2 continued

0 0

17.173.321TEBtAotralimiS07132g3tAta_529752 + + + - - 266800_at At2g22880 VQ motif containing prot 98.159.3nie + + + + + + + + 250435_at At5g10380

Zinc finger (C3HC4-type RING finger) family protein

4.86 2.61 + + - -

260327_at At1g63840 Zinc finger (C3HC4-type RING finger) family protein

2.27 1.65 + + Unknown functions

261056_at At1g01360 Expres 98.174.2nietorpdes - 264580_at At1g05340 Expres 33.213.4nietorpdes + + + + + + + + + - 259410_at At1g13340 Expres 35.157.2nietorpdes + + + + + + + + + - 262832_s_at At1g14870 Expressed protei 75.245.5n + + + + + + - 260686_at At1g17620 Expres 12.219.2nietorpdes + + + + - - 261247_at At1g20070 Expres 18.178.6nietorpdes + - 259566_at At1g20520 Expres 19.110.2nietorpdes + + - 261193_at At1g32920 Expres 98.161.3nietorpdes + + + + + + + - 245840_at At1g58420 Expres 95.151.2nietorpdes + + + + - - 265732_at At2g01300 Expres 61.215.4nietorpdes + - 267209_at At2g30930 Expres 64.290.3nietorpdes + - 265674_at At2g32190 Expres 81.356.01nietorpdes + + + + + + + + + -

35.211.01nietorpdesserpxE01223g2tAta_s_076562 + + + + + + + + + - 266545_at At2g35290 Expres 24.281.3nietorpdes + + + + + - 267623_at At2g39650 Expres 85.150.2nietorpdes + + + + + + 267357_at At2g40000 Expres 75.193.2nietorpdes + + + + + + + - + - 245119_at At2g41640 Expres 28.102.2nietorpdes + + + + + + + - 267393_at At2g44500 Expres 67.143.2nietorpdes + + + + + - - 258402_at At3g15450 Expres 06.153.2nietorpdes + + + 251774_at At3g55840 Expres 57.125.2nietorpdes + + + + + + + 251640_at At3g57450 Expres 00.255.2nietorpdes + + + + + + + - 251400_at At3g60420 Expres 08.100.4nietorpdes + + + + + + + - - 255602_at At4g01026 Expres 75.112.2nietorpdes + - 255532_at At4g02170 Expres 30.354.5nietorpdes + -

66.193.2nietorpdesserpxE01142g4tAta_002452 + + + + + + + + - 253830_at At4g27652 Expres 05.242.3nietorpdes + + + + 253643_at At4g29780 Expres 40.247.3nietorpdes + + + + + + +

49.181.3nietorpdesserpxE01153g4tAta_371352 + + + + + - 246270_at At4g36500 Expres 02.223.3nietorpdes + + + + + + + - 252882_at At4g39675 Expres 19.189.3nietorpdes + - - - +252866_at At4g39840 Expres 17.180.2nietorpdes + - +246018_at At5g10695 Expres 45.138.2nietorpdes + + + + + + + + - 250289_at At5g13190 Expres 30.242.3nietorpdes + + + + + + + + + 249522_at At5g38700 Expres 13.246.5nietorpdes + - + -

Total number of upregulated genes 123 120 115 113 109 96 88 53 31 33 14192192

Total number of downregulated genes 8 2 3 0 1 1 21 12 1 91 102

Experimentsa

Fold-changeProbe set

number AGI locus Description TA IXB Ozo

ne

wou

ndin

g

EF-

Tu

Yar

iv r

eage

nt

Chi

tin

H2O

2

P. s

yrin

gae

Am

inot

riaz

ole

AA

L-t

oxin

Sene

scen

ce

IXB

-hab

ituat

ed

a Data were compared with published results from other microarray experiments, with ‘‘?’’ indicating upregulation (at least 2-FC) and ‘‘-’’

indicating downregulation (at least 2-FC). Experiments are described in ‘‘Materials and methods’’ and ‘‘Supplementary Table S1’’

818 Plant Cell Rep (2009) 28:811–830

123

reagent, 1 h (Guan and Nothnagel 2004); AAL-toxin, 7 h

(Gechev et al. 2004) and aminotriazole which stimulates

H2O2 production by inhibiting catalase activity (Gechev

et al. 2005). Additional details on these experiments are

provided in ‘‘Supplementary Table S1’’. In Table 2, we

used relative expression levels compared to control levels

for comparison and genes with a fold-change (FC) of 2

and more were considered as upregulated (?) or down-

regulated (-), accordingly. Empty spaces indicated no

changes in expression. However, since these data origi-

nated from several different laboratories using various

experimental setups, the risk of obtaining false positive/

negative data must be kept in mind in this comparative

analysis.

Real-time quantitative PCR (RTqPCR)

For RTqPCR, cells were treated with 2.0 lM TA and cells

were harvested after 6, 12 and 24 h. Control cells were

treated with the same volume of methanol or without any

treatment to be harvested at the time of induction (t = 0).

Three biological replicates represented by three indepen-

dent flasks were collected. First strand cDNA was

synthesized as described for RT-PCR, except that 1.0 lg of

total RNA was used. For quantification, QuantiTect

SYBRGreen Mix (Qiagen, http://www1.quiagen.com) was

used and each sample was amplified in two replicates.

Primers are presented in Table 3.

We applied the mathematical model of Pfaffl (Pfaffl

2001). Efficiencies (E) of amplifications were determined

for each pair of primers by calculating the slope of a

standard curve and according to: E = 10-1/slope. This

standard curve was obtained by determining the amplifi-

cation signal of successive dilutions of the cDNA template.

Relative expression was determined with the following

equation: relative expression = EGOIDCt /Eref

DCt, where EGOI is

the efficiency of the amplification of the gene of interest

and Eref is the efficiency of the amplification of the refer-

ence gene. DCt is the variation in the amplification signal

between treated and control samples.

Protein extraction and western blot analyses

Protein extraction was performed as previously described

(Hamel et al. 2005). Thirty micrograms of proteins were

fractionated on a 10% SDS-PAGE and transferred to PVDF

membranes (Amersham, http://www.amersham.com).

Blocking, probing and washing conditions were previously

described (Hamel et al. 2005). Detection was performed

with ECL reagent (Amersham, http://www.amersham.com)

following manufacturer’s instructions.

Ethylene synthesis assay

Cell death was determined on Arabidopsis suspension cells

treated with 2.0 lM TA and pre-treated for 15 min either

with 10 lM ethylene synthesis inhibitor AVG (aminoeth-

oxyvinylglycine; Sigma, http://www.sigmaaldrich.com)

or with 10 lM ethylene synthesis precursor, ACC (1-

aminocyclopropane-1-carboxylic acid; Sigma, http://www.

sigmaaldrich.com). Cell death was determined as previ-

ously described in Duval et al. (2005).

Results and discussion

Thaxtomin A (TA) and isoxaben (IXB) activate

a similar reprogramming of gene expression

We reported previously that both TA and IXB activate a

particular form of PCD in Arabidopsis cell suspensions that

was dependant on de novo gene transcription and protein

synthesis (Duval et al. 2005). To gain a better under-

standing on how TA and IXB activate PCD, we studied the

early transcriptional changes that occur in Arabidopsis cell

Table 3 Primer sequences used

for RTqPCRGene Primer sequences Accession number

AtMKK9 50-CCGCTTCTCCACCTCTTCC-30 At1g73500

50-GTAAACAATCCCGCCGTTTC-30

AtMPK3 50-TGGCTACTTAGTATCTTTGCCTGTT-30 At3g45640

50-CATTGGAGCTACACTTAATCACTAGC-30

WRKY33 50-GCCACCAAAGGATTTTACTACTTAC-30 At2g38470

50-CCGTGTTCTAGTTCTATGGTACAAA-30

WRKY46 50-TACCAGCGAGGTTTTATCTGCAC-30 At2g46400

50-ATTATTCAAAGCTACGACCACAACC-30

AtATG8 h 50-CCAAAGCTCTCTTTGTTTTCG-30 At3g06420

50-AAGAACCCGTCTTCTTCCTTG-30

Actin2 50-ATCGGTGGTTCCATTCTTG-30 At3g18780

50-CTTTGATCTTGAGAGCTTAGAAAC-30

Plant Cell Rep (2009) 28:811–830 819

123

suspensions treated with TA or IXB independently using

Affymetrix ATH1 GeneChips.

It was found that DNA laddering, a typical hallmark of

PCD, occurred from 12 h after TA and IXB application,

with a significant increase in the number of dead cells

detected after 24 h of treatment (Duval et al. 2005). It is

also known from earlier work that inhibition of cellulose

synthesis by IXB occurs rapidly in plant cells. Incubation

of soybean cells for 2 h with 40 nM IXB caused half

inhibition of incorporation of radiolabeled glucose into the

acid-insoluble cell wall material representing the cellulose

fraction (Corio-Costet et al. 1991b). Change in expression

of the defence-related phenylalanine ammonia-lyase (PAL)

gene was also detected as early as 2 h after TA or IXB

addition (Duval et al. 2005). To target early transcriptional

modifications in response to inhibition of cellulose syn-

thesis that occur before the onset of PCD, gene expression

was analysed in cells after 6 h of treatment. At this stage, it

is expected that most of the cells have sensed the cell wall

perturbations caused by inhibition of cellulose synthesis

and that this has in turn activated the expression of genes

required to respond to these perturbations, including genes

involved in maintaining cell wall integrity and cell survival

as well as genes that are preparing the cells for PCD.

Microarray analyses were performed using four chips per

treatment, and data were processed using FlexArray

(Blazejczyk et al. 2007) selecting values with a P value less

than 0.05 for significant changes in expression.

As shown in Fig. 1, induction of cell death by 2.0 lM

TA occurred faster than with 100 nM IXB after 24 h and

48 h of treatment, but mortality was equivalent after 72 h.

TA application upregulated 255 genes by 2-fold and more

within 6 h and no genes were downregulated

(‘‘Supplementary Table S2’’). The delayed cell death pro-

cess in IXB-treated samples correlated with a lower level

of induction of gene expression. Hence, only 83 genes were

upregulated with more than 2-fold change (FC) after IXB

application (‘‘Supplementary Table S3’’). However, when

the limit was set at 1.5-FC for IXB-treated samples, a

similar number of genes displayed a modified expression

after TA and IXB treatments, with 273 genes upregulated

and five genes downregulated by IXB. A total of 192 genes

were upregulated in common by both inhibitors, which

represent more than 75% of TA-induced genes (Table 2).

Reproducibility of these results was confirmed by RT-PCR

for nine upregulated genes (Fig. 2). Functional classifica-

tion using TAIR GO annotations and functional

categorization tools (Berardini et al. 2004) showed con-

servation of the proportion of functional annotations in

genes upregulated either by TA or IXB (Fig. 3a, b). In

particular, stress-related annotations (‘‘response to stress’’

and ‘‘response to abitotic or biotic stimuli’’) were highly

represented for both TA and IXB-upregulated genes, with a

total of 30 and 28% of annotations respectively. To have a

better representation of upregulated genes in a given cat-

egory, we normalized the data by calculating the number of

upregulated genes in relation to the total number of genes

in that category using the Functional Classification Su-

perViewer (Provart and Zhu 2003; Fig. 3c, d). The

category with the most important proportion of genes

upregulated by TA and IXB (7 and 8% respectively) was

that of cell wall-related genes followed again by stress-

related annotations (4 and 5%) and the ‘‘extracellular’’

category (3 and 4%). Since a gene can belong to more than

AtEXPA12

AGP18

Pectin esterase

ATG8h

WRKY75

MYB15

Aspartyl protease

GCN5-related

CYP83B1

ACTIN11

C TA IXB

At3g15370

At4g37450

At4g02330

At3g06420

At5g13080

At3g23250

At1g66180

At2g32030

At4g31500

At3g12110

Fig. 2 Confirmation of transcription profile by RT-PCR. To validate

the ATH1 microarray data, expression of nine genes upregulated by

both treatments was confirmed by RT-PCR using the RNA samples

analysed in the microarray. C control, TA thaxtomin A-treated

samples, IXB isoxaben-treated samples

Time (h)

Dea

d ce

lls (

%)

0

10

20

30

40

50

60

70

80

24 48 72

Control

TA

IXB

Fig. 1 Cell death in Arabidopsis suspension cells treated with

thaxtomin A (TA) and isoxaben (IXB). Percentage of dead cells

after 24, 48 and 72 h detected by trypan blue staining in Arabidopsissuspension cultures treated with methanol (Control), TA (2.0 lM)

and IXB (100 nM). Data represent means (±SE) of four independent

experiments including at least 500 cells each

820 Plant Cell Rep (2009) 28:811–830

123

one functional class, each gene upregulated in common by

TA and IXB was put in a single functional category

according to its role in cell wall, calcium, phosphorylation,

etc. as shown in Table 2. There were 63 genes specifically

upregulated by TA, 81 genes upregulated by IXB and 5

genes downregulated by IXB only. These genes were also

classified into the various functional categories mentioned

above (‘‘Supplementary Table S4 and S5’’). Despite some

differences between the transcriptional profiles induced by

TA and IXB with regards to the genes upregulated and

their level of expression, the important overlap between the

two genes sets that are upregulated by both TA and IXB

suggests that they cause similar cell wall perturbations in

the first 6 h of treatment and that these perturbations are

sensed in the same way by the cells. These results also add

support to the hypothesis that PCD is activated as a result

of inhibition of cellulose synthesis and not because of an

additional effect that could be attributed to each molecule

independently.

Comparison of gene expression in other transcriptional

analyses

To identify genes or gene families whose expression would

correlate with the sensing of cell wall perturbations and the

activation of signalling cascades leading to PCD, TA and

IXB-induced transcriptional profiles were compared with

results obtained in published experiments and from data

available in Genevestigator (Hruz et al. 2008) (see

‘‘Materials and methods’’ and ‘‘Supplementary Table S1’’

for details). These experiments were selected based on their

effect on cell wall [e.g. wounding; treatment with Yariv

reagent, which perturbs cell wall through binding and

aggregation of AGP proteins (Guan and Nothnagel 2004)],

or among abiotic and biotic stress, including some that can

activate cell death (ozone, H2O2, AAL-toxin, Yariv

reagent, senescence, EF-Tu, chitin, Pseudomonas syringae,

aminotriazole). We also included a transcriptional analysis

performed on isoxaben-habituated cells (Manfield et al.

Fig. 3 Functional categorization of genes upregulated by TA and

IXB. Proportion of biological process annotations (Berardini et al.

2004) for genes upregulated by: a TA (C2.0 FC); b IXB (C1.5 FC).

Proportion of upregulated genes in a functional category as calculated

with the Classification SuperViewer (Provart and Zhu 2003); c TA

treatment (C2.0 FC); d IXB treatment (C1.5 FC). Only proportions

above 0.9% are shown. Error bars indicate bootstrap standard

deviation

Plant Cell Rep (2009) 28:811–830 821

123

2004). Genes were considered upregulated (?) or down-

regulated (-) when expression levels compared with that

of control levels gave more than a 2-fold ratio (Table 2).

However, because of the variety of experimental setups,

timing of experiment, number and type of chips analysed

and the difficulty in performing complex statistical analy-

ses on these data, the chance of having false positive/

negative data must be kept in mind. Hence, these com-

parisons must be interpreted with caution. This was

particularly well illustrated when we compared our data

with two different experiments where the effect of H2O2

was studied (Table 2). The first experiment analysed data

obtained after a 1 h-treatment with H2O2 (NASC-338,

Mittler) and the second one concerned data obtained after a

7 h-treatment with aminotriazole, which stimulates the

accumulation of H2O2 in tissues by inhibiting catalase

activity (Gechev et al. 2005). In the first case, 96 (50%) of

the TA and IXB-upregulated genes were also upregulated

by H2O2 whereas only 53 (28%) were found in the

experiment performed by Gechev et al. (2005), with 37

genes upregulated in common in both experiments. Several

reasons may explain these differences, including the way

the effect of H2O2 was investigated (treatment with H2O2

vs treatment with aminotriazole), the type and time of

sampling and the type and number of chips analysed.

Consequently, we have selected a large number of exper-

iments using related stress to have a more general and

representative picture of changes in gene expression that

may correlate with our results.

We found that more than half of the genes upregulated

by both TA and IXB were also upregulated by H2O2

(50.0%), chitin (56.8%) Yariv reagent (58.9%), EF-Tu

treatment (59.9%), wounding (62.5%) and ozone (64.0%)

(Table 2). Hence, inhibition of cellulose synthesis by TA

and IXB induced the expression of a common set of genes

also upregulated in response to abiotic and biotic stress.

These results also corroborate with several previous reports

(e.g. Durrant et al. 2000; Holley et al. 2003; Narusaka et al.

2004; Truman et al. 2007) that demonstrated an important

overlap in the gene sets expressed during stress, defence

response and wounding. However, very few genes (less

than 18%) were upregulated in common with our results in

the PCD-related experiments using the AAL-toxin (Gechev

et al. 2004) and in senescing cell cultures (Buchanan-

Wollaston et al. 2005). Different reasons can explain these

differences. For example, cell death induced by the AAL-

toxin was only detected after 3 days of treatment, while the

expression data used for comparison were from samples

taken after 7 h of treatment. This time point may represent

a very early stage before PCD is induced. In comparison,

transcriptional changes in response to TA and IXB were

evaluated after 6 h of treatment, while cell death was

detected at 24 h. In contrast, senescing cell cultures with

35% of dead cells were analysed several days after cell

proliferation had ceased, suggesting that gene expression at

this stage was associated with changes occurring during the

actual process of dying by PCD. Consequently, it is pos-

sible that some of the 33 genes also upregulated in

senescing cells may have a critical function in the cell

death process induced by TA and IXB.

Expression of cell wall-related genes

The proportion of commonly upregulated genes in the

different categories was fairly similar in the first six

experiments (Table 2), except for the cell wall category

where the proportion of genes upregulated was below 33%

for ozone, EF-Tu, H2O2 and chitin, but over 57% after

wounding and 64% in the Yariv reagent experiment. Yariv

reagent binds specifically to AGPs and triggers their

aggregation (Guan and Nothnagel 2004). AGPs are mem-

brane anchored hydroxyproline-rich family proteins that

are thought to play a role in plasticizing cell wall during

plant growth and particularly during cell elongation (Ding

and Zhu 1997; Lu et al. 2001; Rumyantseva 2005) and

which may play a role in cell adhesion (Johnson et al.

2003). Interestingly, application of Yariv phenylglycoside

(b-D-galactosyl)3 led to the activation of PCD in Arabid-

opsis cell suspensions (Gao and Showalter 1999).

Moreover, it was found that treatment of Arabidopsis

suspensions with Yariv reagent (b-D-glucosyl)3 perturbed

cell surface and caused various cell wall alterations that

were associated with changes in gene expression similar to

those reported in response to wounding (Guan and Noth-

nagel 2004). Alterations in cell wall induced by wounding

first activate the transcription of specific genes aimed at

healing the damaged cells in order to maintain cell wall

integrity and insure a protective barrier against potential

invading pathogens (Leon et al. 2001). Accordingly, our

findings indicate that TA and IXB activated a similar set of

cell wall-related genes. These include several genes

encoding proteins involved in cell wall loosening, such

as expansin (AtEXPA12 [At3g15370]), expansin-like

genes (AtEXLA1, AtEXLA2 and AtEXLA3 [At3g45970,

At4g38400 and At3g45960 respectively]), arabinogalactan-

proteins (AGP2, 4, 18, 20 and 22) (At2g22470, At5g10430,

At4g37450, At3g61640 and At5g53250 respectively), two

members of a specific kind of AGPs, the fasciclin-like

AGPs FLA9 (At1g03870) and the related protein, FLA16

(At2g35860). Additional genes potentially implicated in

strengthening the cell wall were also upregulated, such as

genes encoding proteins of the pectin esterase family

(At2g47550, At3g49220 and At4g02330) and one pectin

acetylesterase (At5g45280). Pectin esterases, and particu-

larly pectin methylesterases, are involved in the de-

esterification of pectins in muro leading to the formation of

822 Plant Cell Rep (2009) 28:811–830

123

ionisable free acid groups (-COO-). These groups can

easily crosslink to Ca2? and make bridges between pectin

chains, thus restricting cell expansion (Fry 2004). Other

genes encoding enzymes belonging to xyloglucan endo-

transglycosylases/hydrolases (XTHs) can also be required

to reinforce a damaged or altered cell wall. XTHs are

involved in cell wall assembly where they would be

required for the addition of newly secreted xyloglucans to

the forming cellulose microfibrils. Upregulated genes

encoding XTHs include XTR4, 6 and 7 (At1g32170,

At4g25810 and At4g14130, respectively), XTH18 and 19

(At4g30280 and At4g30290, respectively), TCH4

(At5g57560), which is rapidly induced after mechanical

stimuli at elongation sites (Antosiewicz et al. 1997) and

MERI5B (At4g30270), which is preferentially expressed in

shoot apical meristems and whose overexpression leads to

aberrant cell expansion (Verica and Medford 1997). Inhi-

bition of cellulose synthesis by TA or IXB did not activate

the expression of genes directly implicated in cellulose

synthesis such as cellulose synthase genes, but two cellu-

lose synthase-like (CSL) genes (At3g28180 and

At1g23480) were specifically upregulated after addition of

IXB (‘‘Supplementary Table S5’’). The CSLs would be

implicated in the synthesis of non-cellulosic polysaccha-

rides in the cell wall (Richmond and Somerville 2000).

Supporting this hypothesis, enzymes from the AtCSLA

family have been shown to exhibit different activities

according to the substrate provided (Liepman et al. 2005).

Consequently, it is possible that early inhibition of cellu-

lose synthesis by IXB could be compensated by the

activation of genes implicated in the accumulation or

modification of other cell wall components.

Expression of genes involved in calcium-binding

and phosphorylation

One of the first event activated by perception of wound-

stress and pathogen-related signals is an important change

in calcium fluxes which further translates in the activation

of various signalling cascades, including activation of

calmodulin and protein kinases (reviewed in Ma and

Berkowitz 2007). Some of these downstream signalling

events can in turn amplify the calcium signal by further

activating Ca2? transporters. In 2005, Tegg et al. found that

application of TA on Arabidopsis roots and pollen tubes

resulted in a rapid and short-lived Ca2? influx (within the

first minute). While it is not known whether Ca2? fluxes

are activated by IXB, we found several genes encoding

calcium-binding proteins that were upregulated after both

TA and IXB treatments. An important proportion of these

were also upregulated in the first six experiments presented

in Table 2. Of particular interest are the PINOID-BIND-

ING protein (PBP1) gene (At5g54490), which encodes a

calmodulin-related protein rapidly induced by mechanical

stimuli and wind (Braam and Davis 1990), and the

TOUCH3 gene (TCH3, [At2g41100]), which would be

implicated in increasing strength and flexibility in cells or

tissues exposed to mechanical stress (Sistrunk et al. 1994).

Both PBP1 and TCH3 bind to PINOID (PID), a Ser/Thr

protein kinase that acts as a positive regulator of auxin

efflux (Lee and Cho 2006). It was proposed that PBP1 and

TCH3 would be involved in a fine-tuning mechanism that

negatively regulates PID activity and overall polar auxin

transport. Moreover, binding of PBP1 and TCH3 to PID

was shown to be calcium-dependent, suggesting that

mechanical stress and calcium responses would be linked

to auxin transport (Benjamins et al. 2003). Knowing that

auxin can act as a potent stimulator of root hair growth, it is

interesting to note that TA treatment, in addition to activate

Ca2? influx, also enhanced root hair density (Tegg et al.

2005). It can be speculated that upregulation of PBP1 and

TCH3 in response to TA together with a Ca2? influx could

negatively regulate PID and maintain higher levels of auxin

in the cell to increase root hair formation. Tegg et al.

(2005) have also reported that the auxin-sensitive mutant

ucu2-2/gi2 was more sensitive to TA. In their account, TA

treatment stimulated root hair formation more importantly

in the ucu2/gi2 mutant than in wild-type plants. They

suggested that the increased sensitivity was due to a

structural similarity between TA and the auxin indole

acetic acid (IAA) and that TA would interact with an

auxin-related receptor, thus mimicking the action of auxin

and stimulating root hair formation. However, our results

do not support such a mechanism since very few auxin-

responsive genes are upregulated by TA. Most importantly,

treatment with IXB, whose structure is very different from

that of TA and IAA, also enhanced root hair formation in

seedlings (data not shown) and increased expression of

TCH3 and PBP1 in cell suspensions. So, instead of a

structural similarity between TA and IAA, it may be the

induction of cell wall perturbations by TA that somehow

modifies auxin transport, thus increasing the sensitivity to

TA reported in the ucu2/gi2 mutant. However, the effect of

inhibition of cellulose synthesis on auxin transport remains

to be shown. Other genes of interest encode two negative

regulators of defence responses, the membrane-associated

protein BON1-associated protein1 (BAP1) (At3g61190)

which contains a calcium-dependent phospholipid-binding

C2 domain, and its functional and interacting partner

BONZAI1/COPINE1 (BON1/CPN1) (At5g61900), a co-

pine protein which also harbours Ca2?-dependent

phospholipids-binding C2 domains (Hua et al. 2001; Yang

et al. 2007). Overexpression of BAP1 with its partner

BON1 inhibits PCD induced by pathogens, by the mam-

malian proapoptotic Bax and by oxidative stress,

suggesting that BAP1 acts as a negative regulator and

Plant Cell Rep (2009) 28:811–830 823

123

general inhibitor of PCD (Yang et al. 2007). It was pro-

posed that BAP and BON serve as signalling molecules or

maintain calcium or lipid homeostasis in stress responses

(Yang et al. 2007). Absence of BAP or BON activities

would then result in cell death. While several factors may

be involved in regulating BAP or BON activities, upregu-

lation of these genes in our system may indicate that

perception of alterations in the cell wall first activates a cell

survival mechanism that could involve BAP1 and BON1.

PCD would then be activated only when cell death signals

overcome the survival signals.

Calcium signalling activates various downstream cas-

cades that are highly regulated by changes in the

phosphorylation status of various regulatory or effector

proteins. Accordingly, TA and IXB also enhanced

expression of 12 kinase genes and one phosphatase gene.

Among these genes were the mitogen-activated protein

kinase kinase gene AtMKK9 (At1g73500), the putative

AtMKKK16 gene (At4g26890), and the stress-related At-

MPK3 (At3g45640) which was upregulated by TA only

(‘‘Supplementary Table S4’’). AtMKK9 was recently

shown to activate MPK3/MPK6 cascade in response to

ethylene, leading to activation of the EIN3 transcription

factor (Yoo et al. 2008), and synthesis of ethylene and

camalexin (Xu et al. 2008). In the present study, genes

encoding 1-aminocyclopropane-1-carboxylate synthase 2

and 6 (ACS2 and 6) (At1g01480 and At4g11280 respec-

tively), which are rate-limiting enzymes implicated in the

ethylene biosynthesis pathway and that have been identi-

fied as substrates of AtMPK6 (Liu and Zhang 2004) were

upregulated by TA and IXB treatment. Since AtMPK3 and

6 belong to the same subfamily and often act in the same

way, it is possible that at least one of the ACS genes

induced here encodes a substrate for AtMPK3. A time

course analysis of the expression of AtMPK3 and AtMKK9

was performed to evaluate their possible implication in

TA-induced cell death. Real-time quantitative PCR

(RTqPCR) analysis was carried out on cells treated with

2 lM TA for 6, 12 and 24 h (Fig. 4). Expression level of

AtMKK9 at 6 h was similar to that obtained with the

GeneChip analysis. Transcripts level increased at 12 h and

decreased after 24 h before going back to the 6 h level.

However, AtMPK3 was not significantly upregulated in this

experiment, with a less than 2-fold induction when ana-

lysed by RTqPCR. This may be explained by the fact that

these samples are different from those analysed on the

GeneChip. Although transcriptional regulation may be

important for sustained activation of MAPKs, phosphory-

lation by an upstream MAPKK is a prerequisite for MAPK

activity. To determine whether TA treatment induced At-

MPK3 activity, proteins extracted from cells treated with

TA (2, 6, 12, 24 and 48 h) were analysed by western blot

using the p-ERK antibody which specifically binds the

phosphorylated motif of MAPK. We could not detect any

phosphorylated proteins, suggesting that AtMPK3 was not

activated in these conditions (data not shown). Hence, this

finding suggests that the program of cell death activated in

TA-treated cells is most probably not mediated by a MAPK

signal transduction cascade. This result is intriguing since

activation of plant MAPK signalling pathways have been

Fig. 4 Relative expression of WRKY33, WRKY46, MKK9, MPK3 and

ATG8h genes. Equal amounts of cDNA were used for each RTqPCR

analysis. Relative gene expression levels were obtained as described

in ‘‘Materials and methods’’

824 Plant Cell Rep (2009) 28:811–830

123

widely implicated in response to wounding, stress and

defence responses and in the process of PCD (reviewed in

Nakagami et al. 2005). However, it was found that Raf-1,

one of the key regulators of MEK-MAPK/ERK cascade in

mammals, can regulate processes such as apoptosis inde-

pendently of MAPK (reviewed in Hindley and Kolch

2002). Results obtained by Asai et al. (2002) in Arabid-

opsis also hint the importance of MAPK-independent

pathways in flg22 signalling. Further characterization of

this putative MAPK-independent pathway will be required

to unravel the complex signalling network that seems to be

activated by TA and IXB.

Expression of defence and stress-related genes

Transcriptional reprogramming is an important feature

activated in response to upstream stress signals. Twenty-

four transcription factors (TF) genes were upregulated in

response to TA and IXB, including members from the

WRKY family, ERF/AP2 family, myb family and zinc

finger family. A high proportion of these transcription

factors (58–75%) was also upregulated in the first six

experiments presented in Table 2. In particular, three

members of the WRKY genes were upregulated by both TA

and IXB: WRKY33, 46 and 75 (At2g38470, At2g46400 and

At5g13080 respectively). In addition, WRKY40

(At1g80840) was upregulated by TA only (‘‘Supplemen-

tary Table S4’’). The WRKYs are plant zinc finger TFs that

would be implicated in the transcription of defence-related

genes and may be involved in regulating various devel-

opmental processes, such as trichome formation, hormone

signalling or fruit maturation (Ulker and Somssich 2004).

WRKY genes can be induced by biotic as well as abiotic

stresses, such as drought, wounding or cold adaptation. It

was reported that WRKY33, 40, 46 and 48 are transcrip-

tionally induced during defence responses and particularly

after inoculation with the avirulent strain of P. syringae,

with an increase in transcripts accumulation that reached a

maximum at 4 h (2 h for WRKY48) before returning to

control levels within 24 h (Dong et al. 2003). To determine

whether the expression of these WRKYs was sustained or

temporary after TA treatment, the expression patterns of

WRKY33 and 46 was followed in suspension cells treated

with 2 lM TA for 6, 12 and 24 h and compared with

control (methanol-treated) cells using RTqPCR. As shown

in Fig. 4, expression levels after 6 h of treatment were

slightly lower but representative of what we found with the

GeneChip hybridization. Expression decreased at 12 and

24 h, as shown after inoculation with avirulent bacteria

(Dong et al. 2003). Zheng et al. (2006) have also demon-

strated that plants constitutively expressing WRKY33 were

more resistant to the necrotrophic fungal pathogen Botrytis

cinerea but more susceptible to virulent biotrophic

pathogen Pseudomonas syringae, exhibiting enhanced

pathogen growth and disease symptoms. This was associ-

ated with a reduction in expression of the defence-related

and salicylic acid (SA)-regulated gene PR1, suggesting that

WRKY33 would act as a negative regulator of the SA

pathway (Zheng et al. 2006). More recent work has also

demonstrated the importance of WRKY33 in regulating the

expression of PAD3 (phytoalexin deficient3), which

encodes an enzyme involved in the synthesis of the phy-

toalexin camalexin (Qiu et al. 2008). Experimental

evidence suggests that the WRKY33-enhanced resistance

to Botrytis cinerea is mediated by a PAD3 dependent

pathway and functions independently of JA, SA and eth-

ylene-mediated signalling (Ferrari et al. 2007).

Interestingly, TA and IXB both enhanced expression of

WRKY33 and PAD3 and there was no expression of the SA-

responsive PR1 (Duval et al. 2005) Moreover, the fact that

there was no upregulation of SA-responsive genes such as

PR1, PR2 or PR5 by TA and IXB suggests that cellular

responses to inhibition of cellulose synthesis are not

mediated through an SA-dependent pathway. However,

additional information will be required to determine the

possible function of WRKYs, PAD3 and SA in perception

of cell wall stress and/or in the process of cell death.

While no production of extracellular H2O2 was detected

during the cell death induced by TA (Duval et al. 2005),

the TA and IXB-induced transcriptional profiles presented

some evidence that oxidative stress could be involved in

the response to inhibition of cellulose synthesis. For

example, expression of the NADPH oxidase AtRBOHD

gene (At5g47910) was upregulated after treatment by TA

and IXB. In mammals, NADPH oxidases catalyze the

production of superoxides, a type of reactive oxygen

species (ROS) implicated during the respiratory burst in

phagocytes. Plant homologs of NADPH oxidases, called

RBOH, have been identified in several plant species where

they are involved in biotic and abiotic stress and during

development (Sagi and Fluhr 2006). In Arabidopsis, At-

RBOHD was shown to be required for the generation of

extracellular ROS in plant defence response during an

incompatible reaction (Torres et al. 2002). In contrast, a

mutation in the AtRBOHD gene eliminated ROS accu-

mulation during a disease resistance reaction in

Arabidopsis plants inoculated with avirulent P. syringae

pv. tomato DC3000 and Peronospora parasitica. This was

associated with reduced cell death and electrolyte leakage,

indicating a decrease in HR. It was suggested that At-

RBOHD would be necessary to prevent the spread of cell

death from the HR site to the surrounding cells. ROS

generated by this NADPH oxidase could antagonize the

SA dependent pro-death signal, which is associated with

the HR to trigger the systemic defence response (Torres

et al. 2005). Alternatively, it was also shown that ROS

Plant Cell Rep (2009) 28:811–830 825

123

could interact directly with the cell wall either to

strengthen it by oxidative cross-linking of its components

(Apel and Hirt 2004; Kerr and Fry 2004) or in a general

loosening mechanism that may be required for cell

expansion (Schopfer 2001; Rodriguez et al. 2002).

Therefore, it can also be speculated that AtRBOHD

expression increases in response to TA and IXB to adjust

the cell wall structure that was perturbed by inhibitors of

cellulose synthesis. Finally, ROS are also thought to play

an important role in signal transduction. While RBOH

activity can be stimulated by calcium (Potocky et al.

2007), ROS production catalyzed by RBOH can also

trigger a large Ca2? influx, as seen during acid abscisic-

induced stomatal closure (Kwak et al. 2003). Whether TA

could modulate AtRBOHD gene transcription in response

to the Ca2? influx or whether AtRBOHD is required for

the influx of calcium remains to be specified.

Expression of genes related to senescence

and cell death

Genes that have been more specifically associated with cell

death were also found to be upregulated in response to TA

and IXB. In particular, three senescence-associated genes

(At4g02380, At4g30430, At2g23810) were upregulated by

both inhibitors, and three additional genes by TA treatment

only (At3g10980, At4g35770 and At2g17850) (‘‘Supple-

mentary Table S4’’). One of these genes (SEN1,

At4g35770, also called DARK-INDUCIBLE1) was strongly

induced by sucrose starvation (Fujiki et al. 2000) and in

senescing leaves (Oh et al. 1996). Another one, SAG20

(Senescence associated gene 20, At3g10980, also called

WOUND-INDUCED PROTEIN 12) was upregulated after

application of the necrotrophic fungus nep-1 protein, a

fungal protein causing cell death and ethylene production

in dicotyledons (Keates et al. 2003).

Since ethylene is known to be involved in leaf senes-

cence (Grbic and Bleecker 1995; Weaver et al. 1998) and

is required for the control of some PCDs (Asai et al.

2000; Greenberg et al. 2000; Moeder et al. 2002), we

tested whether production of ethylene was required for the

cell death induced by TA. Arabidopsis cells were pre-

treated with either a specific ACS inhibitor, AVG, or with

the ethylene precursor, ACC, before adding TA. The

percentage of cell death was estimated after 24, 48 and

72 h of treatment. While we could detect a slight reduc-

tion in cell death with the AVG pre-treatment and a slight

increase in cell death with the ACC pre-treatment, these

changes were not significant to propose an implication of

the ethylene pathway on TA-induced cell death (data not

shown).

We also found upregulated expression in response to TA

only of AtATG8h (At3g06420), a gene related to autophagy

(‘‘Supplementary Table S4’’). Autophagy is a kind of PCD

found in mammals, yeast and plants. In plants, it is char-

acterized by a catabolic degradation of cellular content in

the vacuolar apparatus with the objective of recycling

nutrients for other plant cells during period of stress.

AtATG8h is represented by nine homologs in Arabidopsis

(AtATG8a–AtATG8i), and all genes are preferentially

expressed in roots, in growing tissues, with nearly no

expression found in shoots (Slavikova et al. 2005). In

Arabidopsis suspension cells, transcripts level of AtATG8a,

c, g, h and i increased transiently during sucrose starvation

(Rose et al. 2006). Analysis of AtATG8h expression in

response to TA using RTqPCR confirmed its upregulation

at 6 h with a level maintained at least for 24 h after TA

application. However, since no other AtATG8 genes or

other autophagy-related genes (e.g. AtATG3, AtATG4,

AtATG7) were upregulated by TA or IXB in our experi-

ments, it is unlikely that autophagy is implicated in the cell

death process activated in response to inhibition of cellu-

lose synthesis.

An increase in the expression of the NDR1 (Nonrace-

specific disease resistance) gene NHL3 (for NDR1/HIN1-

like3, At5g06320) was also detected in response to both

TA and IXB. NHL3 is a glycosylated membrane protein

supposed to be involved in disease resistance against

pathogenic bacteria (Varet et al. 2003). The NHL3 pro-

tein is required for the proper execution of the HR cell

death, but in some particular cases only (Century et al.

1997). Members of the NDR1 family share some simi-

larities with the tobacco HIN1 (harpin-induced) protein,

whose gene is activated by the bacterial elicitor harpin

and the functional hrp gene cluster. NHL3 transcripts

accumulation was stimulated after inoculation with

avirulent P. syringae pv. tomato strains, but not by the

virulent strains (Varet et al. 2002). Also, overexpression

of NHL3 increased resistance to the avirulent strain P.

syringae pv. tomato DC3000 (Varet et al. 2003). Inter-

estingly, these plants did not display enhanced

expression of the defence genes PR1, PR2 and PDF1.2

but it was not determined how overexpression of NHL3

would lead to enhanced resistance in the absence of

constitutive PR gene expression (Varet et al. 2003). We

have found that PCD induced in response to inhibition of

cellulose synthesis also occurs in the absence of

expression of SA-responsive PR1 and PR2 genes and of

JA and ethylene-dependent PDF1.2 genes. Since classi-

cal SA, JA or ethylene-defence signalling pathways do

not seem to be activated by TA and IXB in Arabidopsis

cell suspensions, we suggest that an atypical stress or

defence-related pathway, such as that activated and/or

mediated by NHL3, could be implicated in regulating the

process of cell death induced by inhibition of cellulose

synthesis.

826 Plant Cell Rep (2009) 28:811–830

123

A majority of genes upregulated by TA and IXB

are downregulated in IXB-habituated cells

It is possible to habituate cells to proliferate in the presence

of inhibitors of cellulose synthesis such as dichlobenil

(Shedletzky et al. 1992; Sabba et al. 1999; Encina et al.

2002; Alonso-Simon et al. 2004), isoxaben (Corio-Costet

et al. 1991a; Corio-Costet et al. 1991b; Dıaz-Cacho et al.

1999; Manfield et al. 2004) and TA (unpublished results)

by adding increasing amounts of the inhibitors at each

subculture over a period of several months. Habituation to

isoxaben has been performed in Arabidopsis suspensions

(Manfield et al. 2004). Characterization of IXB-habituated

cells has revealed changes in the cell wall composition and

organisation, with increased levels in uronic acids and

mannose and changes in the distribution of xyloglucan

(XG). These results suggested that disruption in cellulose

synthesis was compensated by the accumulation of other

cell wall components. Transcriptional analysis in these

cells (NASCArrays-27; Manfield et al. 2004) indicated

important changes in the expression of cell wall-related

genes, but the only cellulose synthase gene upregulated in

these cells was AtCslD5. When comparing these results

with gene expression in TA and IXB-treated cells, we

found that 103 genes of the 192 genes upregulated by TA

and IXB treatment were downregulated ([2-FC) in habit-

uated cells, while only 14 were upregulated ([2-FC). It is

possible that repression of most of the genes upregulated in

the early response to IXB may be required to ensure cell

survival in IXB-habituated cells. Alternatively, it can be

postulated that the synthesis of a modified cell wall in

habituted cells requires an elevated energy expenditure that

could divert energy from other cellular processes that are

not essential in habituated cells, such as defence and stress-

related mechanisms. In any case, our findings indicate that

the stress-related mechanisms activated in response to IXB

are turned off in habituated cells.

Conclusion

Our results suggest that cell wall perturbations caused by

inhibitors of cellulose synthesis such as TA and IXB are

initially sensed as a general wound-like stress that acti-

vates the expression of genes required for cell wall repair

as well as several others genes involved in common stress

signalling pathways. Similarly, Arabidopsis cells treated

with the cell wall perturbing Yariv reagent also activated

gene expression profiles that resembled those induced in

response to wounding (Guan and Nothnagel 2004).

Wounding can activate several complex signalling path-

ways that can be either dependent or independent on JA.

JA-dependent pathway would be more important for long-

distance activation of wound-responses, while JA-inde-

pendent pathways maybe more important in the vicinity

of the wound site (Leon et al. 2001). In this work and as

reported after Yariv reagent treatment, key genes involved

in JA synthesis (except for allene oxide cyclase or AOC)

and JA-responsive genes, such as PDF1.2, were not

upregulated by TA and IXB, suggesting that the wound-

like response was perceived as a local signal that would

not be mediated by JA-dependent pathways. Combined

with the previous findings that TA and IXB do not acti-

vate the expression of defence-related genes that are

typically associated with the SA and ethylene-defence

signalling pathways, our results suggest that inhibition of

cellulose synthesis activate an atypical stress-related

pathway that can induce PCD independently of the clas-

sical hormone-dependent defence pathways. The

upcoming challenge will be to identify the key regulators

involved in this process.

Acknowledgments Financial support was provided by NSERC,

FQRNT and Universite de Sherbrooke as individual grants to N.B.

We also wish to thank L.-P. Hamel and A. Seguin for providing help

and antibodies for the western blot analysis, and Genome Quebec,

Innovation Center (McGill University, Montreal, Canada) for

microarray data collection and preliminary analysis.

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