Gene expression profiles of O3-treated ... - Wiley Online Library

Arabidopsis gene expression profiles N. Tosti et al.

Plant, Cell and Environment (2006) 29, 1686–1702

doi: 10.1111/j.1365-3040.2006.01542.x

Gene expression profiles of O3-treated Arabidopsis plants NICOLA TOSTI2*, STEFANIA PASQUALINI1, ANDREA BORGOGNI1, LUISA EDERLI1, EGIZIA FALISTOCCO1, STEFANIA CRISPI3 & FRANCESCO PAOLOCCI2 1 Università degli Studi di Perugia, Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali e Zootecniche, Borgo XX Giugno, 74, I-06121 Perugia, 2CNR, Istituto di Genetica Vegetale, Via della Madonna Alta, 130, I-06128 Perugia and 3CNR, Istituto di Genetica e Biofisica ‘ABT’ Via P. Castellino, 111, I-80128, Napoli, Italia

ABSTRACT To analyse cellular response to O3, the tolerant Arabidopsis thaliana genotype Col-0 was exposed to O3 fumigation (300 ppb) for 6 h and the modulation of gene expression during the treatment (3 h after the beginning of the treatment, T3 h) and the recovery phase (6 h from the end of the treatment, T12 h) assessed by gene chip microarray and real-time reverse transcriptase (RT)-PCR analyses. The Arabidopsis transcriptional profile is complex, as new genes (i.e. reticuline oxidase) and pathways, other than those already reported as O3-responsive, appear to be involved in the O3 response. The steady-state transcript levels of several WRKY genes were increased in O3-treated plants and the W-box was the cis-element over-represented in the promoter region of T3 h up-regulated genes. The fact that the W-box element was also over-represented in almost all T3 h-induced receptor-like kinases (RLKs) suggests a WRKY-mediated control of RLKs under O3 stress and a mechanicistic similarity with the pathogen-induced transcriptional responses. We investigated the molecular and physiological implications of our findings in relation to O3induced plant stress response. Key-words: microarray analysis; ozone; real-time RT-PCR; RLKs; transcription factors; WRKY proteins.

INTRODUCTION The concentration of tropospheric O3 has increased during the past few decades because of human activities and it is estimated that by the year 2100, 50% of global forest will be exposed to potentially phytotoxic concentrations (Fowler et al. 1999). However, the risk assessment for vegetation based on relationships between external O3 concentration and plant response has been shown to be inadequate and, recently, new models assessing the future global impact of O3 on vegetation have been elaborated (Ashmore 2005; Karnosky et al. 2005).

Correspondence: Stefania Pasqualini. Fax: +39 0755856404; e-mail: [email protected] *Present address: &LAB Srl, Via Strozzacapponi, 89/a, 06071 Perugia, Italia.

1686

Plants respond to high O3 levels by displaying either sensitivity, manifested by rapid lesion formation, or tolerance. O3 phytotoxicity is the result of an accumulation of reactive oxygen species (ROS), such as superoxide radicals O2•−) and hydrogen peroxide (H2O2) (Wohlgemuth et al. 2002). Phenotypically, acute O3 exposure (a high dose for a short time) causes necrotic lesions similar to those provoked by avirulent pathogen infections, while chronic exposure (a low dose for an extended period) accelerates foliar senescence and produces symptoms not unlike those caused by virulent infections (Schlagnhaufer et al. 1995; Ruzsa, Mylona & Scandalios 1999). Furthermore, rapid generation of ROS and changes in transcript accumulation of defence-related genes overlap the pathogen defence response (Sandermann 1996; Sharma et al. 1996; Schraudner et al. 1998). It has been postulated, based on these similarities, that several signal molecules, including ethylene (ET) (Mehlhorn & Wellburn 1987; Pell, Schlagnhaufer & Arteca 1997; Tuomainen et al. 1997), jasmonic acid (JA) (Orvar, McPherson & Ellis 1997) and salicylic acid (SA) (Sharma et al. 1996; Rao & Davis 1999) act as second or third messengers for O3-induced gene expression. Studies on mutants defective in SA signalling have demonstrated that SA is pivotal in initiating lesions in Arabidopsis plants exposed to O3 (Rao & Davis 1999; Rao et al. 2000), and studies with rcd1 and ein2 mutants show that the specific role of ET is to propagate lesions by promoting superoxide radical production (Overmyer et al. 2000; Overmyer, Brosche & Ellis 2003). Rao, Lee & Davis’s (2002) demonstration that SA plays a role not only in initiating, but also in propagating lesions by controlling ET production adds a layer of complexity to this cell-death pathway. As lesions develop rapidly when jar1, an Arabidopsis JA-signalling mutant, is exposed to acute O3 (Rao et al. 2000), the JAsignalling pathway appears to be involved in the lesioncontainment processes. JA can antagonize lesion spread in several ways, one of which is by suppressing SA biosynthesis and signalling (Overmyer et al. 2003). Attenuation of ET sensitivity by JA contributes to halting the spread of cell death (Schenk et al. 2000). O3-induced cell death spread is stimulated by early, rapid accumulation of ET, which can suppress the protective function of JA and so allow cell death to proceed. By inhibiting the ET pathway, JA halts propagation of cell death (Tuominen et al. 2004). These various signal molecules often appear to interact and result © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd

Ozone effects on Arabidopsis transcriptome 1687 in a dynamic response to biotic and abiotic stimuli (for recent reviews see Baier et al. 2005; Foyer & Noctor 2005; Kangasjarvi, Jaspers & Kollist 2005). Despite the large number of mutants available and studies carried out to dissect the molecular and physiological events underpinning the O3-triggered plant responses, studies that monitor the O3-induced expression profile over the entire genome are few. With the aim to study the oxidative signalling mediated by acute O3 fumigation in the tolerant Arabidopsis thaliana Col-0 ecotype, we took advantage of the GeneChip Arabidopsis ATH1 Genome array developed by Affymetrix (Santa Clara, CA, USA) to investigate the O3-induced transcript alterations. As this method monitors the gene-expression profile throughout the entire genome, it allowed us both to investigate the complexity of the O3 stress response and to discover new genes and pathways likely to be modulated and implicated in the O3 response. We here report the molecular and physiological implications of these findings.

MATERIALS AND METHODS Plant growth conditions and treatments A. thaliana L. ecotype Columbia (Col-0) plants were employed for experimentation. Plants were raised in a growth chamber under a photosynthetic photon flux density (PPFD) of 150 µmol m−2 s−1, 14 h light/10 h dark. Growth temperature and relative humidity was kept 25 °C and 60%, respectively. Four-week-old plants were exposed to a single 300-ppb-O3 dose for 6 h (0800–1400 h). The O3 produced by UV irradiation (OEG50L lamp; Helios Italquartz s.r.l., Milan, Italy) was monitored continuously by a UV-Photometric O3 analyser (Thermo Electron Corporation, Franklin, MA, USA). Entire plant rosettes were harvested before (0 h), during (3 h) and after (6, 12 and 24 h) exposure to O3. The complete experimental set was repeated twice and for each time point, four plants were used.

RNA isolation and sample preparation For each experiment, a leaf tissue was pooled from four plants for each time point and immediately frozen in liquid nitrogen. Total RNA was prepared from the frozen tissue using the Nucleo Spin RNA Plant kit (Macherey-Nagel Inc. Easton, PA, USA) according to the supplier’s instructions, to which a further DNase I treatment was added (Ambion, Austin, TX, USA). RNA integrity was checked on 1.5% (w/ v) agarose gel prior to, and after the DNase I digestion. Absence of genomic DNA contamination was subsequently confirmed by the null PCR amplification of RNA samples in the presence of elongation factor 1-α primers (Robbins et al. 2003). Within each experimental set, RNA from control and O3-treated plants was isolated in triplicate and then pooled. Labelled cRNAs were synthesized following the Affymetrix protocol. Briefly, first strand cDNA was synthesized by

retrotranscription of 20 µg of total RNA with SuperScript II Rnase H– Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) using an oligo dT(24) that contains a T7 promoter on 5′ end (Genset, Evry, France), then the second strand cDNA was generated with RNase-H, DNA polymerase I and DNA ligase (Invitrogen). An in vitro transcription was performed using the Enzo BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Farmingdale, NY, USA) to synthesize and biotinylate the cRNA. After cleanup and quantification of the biotin-labelled cRNA, about 40 µg of cRNA was fragmented by heating at 94 °C for 35 min in a buffer containing 40 mM Tris-acetate (pH 8.1), 125 mM KOAc and 30 mM MgOAc. Before use for microarray analysis, cRNA probes were preliminarily tested on a chip test (Test3 Array; Affymetrix) to check for their quality and integrity. Fifteen micrograms of fragmented cRNAs (including control cRNA and grid alignment oligonucleotides) were hybridized overnight (∼16 h, 45 °C) to an ATH1 Arabidopsis GeneChip microarray (Affymetrix) containing 22 810 probe sets. The hybridization mix was prepared as recommended by the manufacturer (Affymetrix) and a set of spiked biotin-labelled bacterial RNAs including control cRNA and grid alignment oligonucleotides were added to each sample at the time of hybridization. GeneChip Fluidics station 400 (Affymetrix) was used for washing and staining (with antistreptavidin antibody) the arrays. Fluorescent signals were measured on the arrays using a specific scanner (Agilent GeneArray scanner, Affymetrix) to generate digitized image data (DAT files). The microarray experiments were performed twice for each selected time point as biological replicates.

Data analysis DAT files were analysed by MAS 5.0 to generate background-normalized image data (CEL files). The ATH1 22 810 probe sets were filtered to provide probe-set intensities by means of robust multiarray analysis method (Irizarry et al. 2003). The full data set was normalized according to the invariant set method (Li & Wong 2001). The funnel-shaped procedure described by Saviozzi et al. (2003) was then applied. This method permitted to obtain a subset of robust probe sets that were expressed in at least one sample analysed on the basis of their presence calls and signal distributions. This filtering yielded a data set of 6872 probe sets. Subsequently, significant analysis of microarray (SAM-software, Stanford University, CA, USA) (Tusher, Tibshirani & Chu 2001) was used to identify probe sets differentially expressed between control, T3 h and T12 h samples. Once the data set was generated, robustness of differential expression was evaluated by combining fold change with statistical validation. The data set was analysed with the two-class unpaired method, implemented in the SAM-software, to highlight probe sets transactivated only in the control or O3-treated samples. This test requires two user-set parameters: a minimal fold change value and a threshold value that can be adjusted to maximize the

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd, Plant, Cell and Environment, 29, 1686–1702

1688 N. Tosti et al. number of significant genes while minimizing the predicted false discovery rate. We conducted a blocked, two-class unpaired test using a 1.5-fold-change cutoff and a threshold that allows a false significant number of about one. This filtering yielded probe sets differentially expressed between control and T3 h and T12 h samples (Supplementary Table S1). Gene-expression changes are depicted in MapMan format version 1.8.0 (Thimm et al. 2004). Cluster analysis of the probe sets validated by SAM at both T3 h and T12 h that showed a greater than twofold difference in at least one pairwise comparison (0 h versus T3 h and 0 h versus T12 h, for a total of 569 genes; Supplementary material Table S2) was performed using the SOTA (selforganizing tree algorithm) at DNA array data clustering server (http://gepas. bioinfo.cnio.es/cgi-bin/sotarray) (Dopazo & Carazo 1997; Herrero, Valencia & Dopazo 2001). Gene ontology classification (Ashburner et al. 2001) was performed with the Gene Ontology at TAIR annotation tool (http://www.arabidopsis.org/tools/bulk/go/index.jsp).

Supplementary Table S3. Real-time RT-PCR was performed using the Taqman Universal PCR Master Mix (Applied Biosystems) according to the manufacturer’s instruction and using 2 µL of 1:100 diluted RT reaction product in a total of 20 µL per reaction. For each cDNA sample, four replicates were run and analysed on ABI Prism 5700 (Applied Biosystems) according to the manufacturer’s instructions. For each reaction, the threshold cycle value (Ct) was determined by setting the threshold within the logarithmic amplification phase. After the validation tests, normalization to elongation factor (At5g19510) gene levels was performed using the delta delta-Ct method (User Bulletin 2, ABI Prism 7700 sequence detection systems). For each given target gene, the mRNA quantity for the control sample at 0 h was arbitrarily set to 0, and all other samples were expressed in relation to this control sample. The reactions were repeated twice with two different cDNA preparations, and one of the experiments was plotted.

Promoter analysis

RESULTS

The Arabidopsis genomic sequence was obtained from ftp://ftp.arabidopsis.org/Sequences and 1 Kb long sequence upstream of a known or predicted translational start codon was retrieved for each gene whose relative probe sets on chips displayed statistically significant signals. The search for probable cis-elements on promoter sequences was performed using the oligo-analysis program by regulatory sequence analysis tools (RSA-tools) (http:// www.rsat.ulb.ac.be.rsat) (van Helden, Andre & ColladoVides 1998) and the motif analysis at the TAIR site (http://www.arabidopsis.org/tools/bulk/motiffinder), which calculated and detected the over-represented oligonucleotides in a set of sequences. The detected motifs were then compared with known transcriptional-factor binding motifs available in the PLACE database (http://www.dna.affrc.go. jp/htdocs/PLACE) (Higo et al. 1999). Microsoft Excel and a database created with FileMaker Pro (Filemaker, Inc., Santa Clara, CA, USA) were used to manage such data as well as to retrieve gene annotation, gene pathway and gene family classification at TAIR databases (http://www.tair.org).

To analyse the temporal evolution of the oxidative stress response, we exposed Col-0 plants to 300 ppb O3 for 6 h. This treatment is sufficient to evoke a physiological and transcriptional response, but does not induce tissue damage, as judged by either the development of visible lesions or enhanced ion leakage, which is a measure of cell membrane damage (not shown). GeneChip hybridization assays were performed at T3 h and T12 h to monitor the gene expression profiles during O3 treatment and in the recovery phase. The complete experimental set, from plant treatment to RNA isolation, cRNA labelling and chip hybridization was replicated twice to compute the correlation within each treatment. As the Pearson correlation values were highly significant (Table 1), they provided evidence that the experimental design (O3 treatment and leaf sampling) and technical procedures (RNA preparation and

Real-time reverse transcriptase (RT)-PCR analysis Five micrograms of de novo-isolated DNA-free RNA from the leaf pools of unfumigated plants sampled before the start of O3 fumigation and after 3, 6, 12 and 24 h from the onset of O3 fumigation were reverse transcribed in the presence of 200 units of SuperScript II H- Reverse Transcriptase (Invitrogen) and 100 pmoles of random hexamers (Pharmacia Biotech, Piscataway, NJ, USA). Primers and Taqman probes for amplification reactions for nine selected genes were synthesized by the Custom TaqMan Gene Expression Assay Service (Applied Biosystems, Foster City, CA, USA). The relative sequences are shown in

Table 1. Number and percentage of the Arabidopsis thaliana genes (probe sets) over 22 746 present in the GeneChip array, that hybridized with cRNAs from control and O3-treated (T3 h and T12 h) plants. ‘Present’ and ‘Absent’ designate hybridizing/nonhybridizing probes Present

0h 0 h 2° exp. 3h 3 h 2° exp. 12 h 12 h 2° exp.

Absent

No.

%

No.

%

14 003 12 792 14 381 14 387 14 374 14 230

61.56 56.24 63.22 63.25 63.19 62.56

8743 9954 8365 8359 8372 8516

38.44 43.76 36.78 36.75 36.81 37.44

Correlation coefficient

0.952* 0.974* 0.973*

The between replicate values were calculated by the Pearson correlation. *Statistical significance at P ≤ 0.01.


Ozone effects on Arabidopsis transcriptome 1689 analyses) were highly reliable and reproducible. The number and percentage of probes retrieved as ‘hybridizing’ or ‘non-hybridizing’ from control (0 h) and O3-treated plants at T3 h and T12 h are given in Table 1.

Analysis of up- and down-regulated genes in O3-treated plants Using the resources available at the TAIR site (http:// www.arabidopsis.org) and the NetAffx analysis tool at the Affymetrix site (http://www.affymetrix.com) the description of the relative gene and, when possible, its location in the biochemical pathways were searched for each probe set isolated by SAM analysis. Because the study of genes and pathways regarded as modulated under O3 stress is a test that evaluates the biological and technical relevance of the experiment, these pathways were investigated first. In addition, series of genes not belonging to a specific pathway but relevant in the O3 stress response were also considered. Supplementary Table S4 shows the biochemical pathways in which at least one gene was up- or down-regulated more than twofold after 3 h of O3 fumigation. Although some pathways displayed both up- and down-regulated genes (flavonoid biosynthesis, sucrose biosynthesis, anaerobic glycolisis, UDP-glucose conversion), there were generally only up- or down-regulated genes within most of the biosynthetic routes. Figure 1 displays the O3-induced changes in gene expression for core metabolism and general regulatory processes, during and after the treatment. Table 2 instead reports several O3-modulated genes along with the relative expression changes at T3 h and T12 h with respect to the control.

O3-modulated genes of the ET and JA pathways Activation of ET synthesis by induction of the genes encoding for 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is one of the earliest responses to O3 (Tuomainen et al. 1997). ACS is encoded by a multigene family with nine ACS genes characterized in Arabidopsis (Tsuchisaka & Theologis 2004). We found a 2.2 increase in ACS6 and a 3.9 increase in ACS2 expression after 3 h of O3 fumigation, but recorded no modification in other ACS genes (Table 2, Fig. 1c & d). After being synthesized, ET is perceived by membrane receptors and the signal transduced to trigger specific biological responses. In Arabidopsis at least five families of ET receptor members are involved (ETR1, ETR2, ERS1, ERS2 and EIN4) (Chang et al. 1993; Hua et al. 1995; Sakai et al. 1998), downstream from these, a CTR1 gene acts as a negative regulator of the signalling events (Kieber et al. 1993). We found that ERS1 and CTR1 were both increased at T3 h and T12 h, while EIN4 was induced only after 3 h of O3 fumigation. Five genes connected to the JA pathway were up-regulated early (T3 h): two genes encoding for lipoxygenases (At3g22400 and At1g72520) increased 3.2- and 3.0-fold, respectively, and three genes encoding for 12-oxo-phytodienoate reductase (OPR2, OPR3 and At1g17990) were induced 2.7-, 2.5- and

1.9-fold, respectively. Antimicrobial JA-induced PDF1.2 was also strongly increased at both T3 h and T12 h (Table 2, Fig. 1c & d).

The shikimate pathway and its derived secondary metabolites SA, phenylpropanoids and camalexin are three of the most important compounds involved in the plant O3 response that derive from the shikimate pathway. In Arabidopsis, SA is produced by isochorismate synthase (ICS), though it has been suggested that it is also synthesized via the phenylalanine ammonia lyase (PAL) pathway (Durner, Shah & Klessig 1997). We recorded a 6.7-fold increase in ICS1 after 3 h of O3 treatment (Table 2). Three of the genes that control the SA-signalling pathway (PAD4, EDS1 and EDS5) (Nawrath & Metraux 1999; Nawrath et al. 2002) were upregulated 2.6-, 1.8- and 7.7-fold, respectively, after 3 h of O3 stress (Table 2). Three phenylalanine biosynthetic pathway genes (At1g11790, At4g28420, TAT3) were only up-regulated at T3 h (Table 2). In subsequent steps leading to the formation of phenylpropanoid metabolites, some genes were upwhile others were down-regulated. At3g19010, a gene that encodes for a flavonol synthase, was only up-regulated at T3 h (Table 2). In contrast, a reduction was recorded in the expression of the TT6 gene that encodes for flavanone-3hydroxylase and lies a step upstream from flavonol synthase, as well as in the At1g01420 gene which encodes a flavonol 3-O-glycosyltransferase and is situated downstream (Table 2). There was a steep early rise in three other genes (At1g21100, At1g21120, At1g21130 of 12.2, 16.3 and 9.6, respectively) that code for O-methyltransferase, a phenylpropanoid pathway enzyme involved in lignin formation. Cytochrome P450, which is the key enzyme in camalexin synthesis and is encoded by the PAD3 gene (Zhou, Tootle & Glazebrook 1999) was increased 15.5-fold at T3 h and remained high at 12 h (Table 2). Supplementary Fig. S1 displays the expression changes of genes involved in the secondary metabolism.

Genes related to oxidative stress, photosynthesis, senescence and polyamines Antioxidant enzyme activities would be expected to rise during a situation that leads to increased oxidative stress. We found two glutahione S-transferase (GST) genes (GST1 and GST6) and two monodehydroascorbate reductase genes (At3g09940 and At5g03630) peaked 3 h after the onset of O3 exposure. We also found three early up-regulated genes (3 h) which maintained their high expression levels after 12 h (CAT1, At1g63940 and At1g32350). In contrast, CAT2, GST1, GPX1 and GPX7 were found to be down-regulated only after 12 h from the onset of O3 fumigation (Table 2, Supplementary Fig. S2). The oxidation reactions reduce net photosynthesis and possibly accelerate cell senescence (Reich & Amundson 1985; Dann & Pell 1989; Pell, Eckardt & Glick 1994). O3 causes a marked down-regulation of At2g28000 coding


1690 N. Tosti et al.

Figure 1. Effect of acute O3 treatment on gene expression in Arabidopsis. Gene-expression changes are depicted in MapMan format version 1.8.0 (Mapping_overview_AgiCode) where each square represents a gene. Blue, red and white meant up-regulation, down-regulation and no change in expression, respectively. The complete data set used for MapMan analysis is given in Supplementary Table S1. The figures are best viewed, and all data point annotations are provided at http://gabi.rzpd.de/projects/MapMan. (a) and (b) Panels A and B: gene expression of core metabolism (metabolism_overview, BINs from 1 to 5, 7 to 14 and 16, 19, 21, 23, 25) during (T3 h, A) and after (T12 h, B) O3 treatment. (c) and (d) Panels C and D: gene expression of general regulatory processes (regulation_overview, BINs 17, 21, 27, 29 and 30) during (T3 h, C) and after (T12 h, D) O3 treatment. MAPK, mitogen-activated protein kinase.


Ozone effects on Arabidopsis transcriptome 1691

Figure 1. Continued

for Rubisco subunit binding protein and RBCL, that encodes for large subunit of Rubisco. Miller, Arteca & Pell (1999) reported a direct relationship between induction of some senescence-associated genes and O3 exposure in Arabidopsis. We recorded raised expression of five senescence-related genes (Table 2). There was marked induction of ERD1 (early responsive to dehydration),

SAG21 and ATBCB (blue copper binding protein) at both T3 h and T12 h, while SAG101 and SAG20 were induced only at T3 h (Table 2). In regard to polyamine metabolism, we found a drastic down-regulation after 3 and 12 h of spermidine synthase and adenosylmethionine decarboxylase, two genes that lead to spermidine biosynthesis (Table 2).


1692 N. Tosti et al. Table 2. List of selected genes up- or down-regulated in response to O3 treatment (nc, no change) Fold change ID AGI Ethylene signalling At1g01480 At4g11280 At2g40940 At3g04580 At5g03730 Jasmonic acid signalling At1g76690 At1g17990 At2g06050 At5g44420 At3g22400 At1g72520 Salicylic acid signalling At3g52430 At4g39030 At3g48090 At1g74710 Tryptophane biosynthesis At1g25220 At5g05730 At5g17990 At2g04400 Phenylalanine biosynthesis At1g11790 At4g28420 At2g24850 Phenylpropanoid pathway At3g51240 At3g19010 At1g01420 At1g21100 At1g21120 At1g21130 Camalexin biosynthesis At3g26830 Polyamine biosynthesis At5g15950 At5g53120 Oxidative stress response At1g02930 At2g47730 At2g29450 At2g25080 At4g31870 At3g09940 At5g03630 At1g63940 At1g20630 At4g35090 At1g32350 Senescence At5g51070 At4g02380 At5g20230 At5g14930 At3g10980 Photosynthesis At2g28000 AtCg00490

Gene model

Full name or description

T3 h

T12 h

ACS2 ACS6 ERS1 EIN4 CTR1

ACC synthase ACC synthase ETHYLENE RESPONSE SENSOR 1 ETHYLENE INSENSITIVE 4 CONSTITUTIVE TRIPLE RESPONSE 1

2.2 3.9 2.2 1.9 1.9

nc nc 4.1 nc 3.0

OPR2 OPR3 PDF1.2

12-oxophytodienoate reductase 12-oxophytodienoate reductase putative 12-oxophytodienoate reductase PLANT DEFENSIN 1.2 Lipoxygenase Lipoxygenase

2.7 1.9 2.5 3.3 3.2 3.0

nc nc nc

PAD4 EDS5 EDS1 ICS1

PHYTOALEXIN DEFICIENT 4 ENHANCED DISEASE SUSCEPTIBILITY 5 ENHANCED DISEASE SUSCEPTIBILITY 1 Isochorisnmate synthase (SID2)

2.6 7.7 1.8 6.7

nc 2.5 nc nc

ASB1

Anthranilate synthase Anthranilate synthase Anthranilate phosphoribosyltransferase (PAT1) Indole-1-glycerol phosphate synthase

5.0 4.1 2.6 3.6

2.3 2.6 2.0 1.6

Prephenate dehydratase Aminotransferase, putative Tyrosine aminotransferase, jasmonic acid-responsive gene

1.9 4.1 1.8

TRP1

TAT3

3.2 nc nc

nc nc nc

TT6

TRANSPARENT TESTA 6 Flavonol synthase family Glycosyltransferase family O-methyltransferase, putative O-methyltransferase, putative O-methyltransferase, putative

−7.0 3.1 −2.3 12.2 16.3 9.6

−5.5 nc nc 3.4 2.0 2.0

PAD3

PHYTOALEXIN DEFICIENT 3

15.5

5.3

Adenosylmethionine decarboxylase Spermidine synthase

−3.6 −1.7

−9.3 −3.1

Glutathione S-transferase 1 (GST1) Glutathione S-transferase 16 (GST16) Glutathione S-transferase 1 (GST1) Glutathione peroxidase 1 Glutathione peroxidase 7 Monodehydroascorbate reductase putative Monodehydroascorbate reductase putative Monodehydroascorbate reductase putative Catalase 1 Catalase 2 Alternative oxidase putative

2.6 1.9 nc nc nc 2.5 2.4 1.8 2.0 nc 5.9

nc nc −2.7 −2.3 −14.7 nc nc 2.2 2.0 −3.9 4.2

2.5 3.5 3.1 1.8 1.7

2.2 4.8 2.4

ATGSTF6 ATGSTF8 ATGSTU5 ATGPX1 ATGPX7

MDHR CAT1 CAT2

ERD1 SAG21 ATBCB (At5g20230) SAG101 SAG20

EARLY RESPONSIVE TO DEHYDRATION SENESCENCE-ASSOCIATED GENE 21 ARABIDOPSIS BLUE-COPPER-BINDING PROTEIN (BC) SENESCENCE-ASSOCIATED GENE 101 SENESCENCE-ASSOCIATED GENE 20

RBCL

RuBisCo subunit binding protein alpha subunit Large subunit of RuBisCo

nc −2.8

nc nc −2.4 −6.8



SOTA analysis To gain further information on the gene-expression profile mediated by O3, the data relative to the T12 h sample hybridizations were combined with those of the control and T3 h samples and SOTA analysis performed. The result of the SOTA is a hierarchical clustering with the accuracy and the robustness of a neural network (Dopazo et al. 2001) and this analysis allowed us to cluster genes showing similar trends of expression across the treatment. Figure 2 depicts the dendrogram and the 22 clusters obtained. These clusters could be classified into two principal groups that approximately reflected the gene expression profile (reported as histograms). Group 1 includes mainly down-regulated genes, group 2 mainly up-regulated genes. Genes whose maximum expression occurred at T3 h are seen in the upper part (2a), those whose maximum 6 15 23 34 29

1

32 21 16 2 4 9

27 76 54 48 55 43

2b

Promoter motif analyses A 1 Kb sequence relative to the regulatory region of 0 h/ T3 h differentially regulated genes with a more than twofold variation was analysed and used to search for probable cis-elements. The most represented oligonucleotides in the promoter region of the 766 up-regulated genes (Supplementary Table S5) are reported in Table 3. A similar search failed to identify any over-represented cis-elements on the corresponding region of the down-regulated genes. To identify motifs identical or similar to known cis-elements, the retrieved sequences were compared to motifs available in the PLACE database. TGAC was the most represented motif on the target promoters (Table 3). TGAC is part of the sequence of the elicitor-responsive promoter element (ERE) [(T)(T)TGAC(Y)], known as the W-box. The W-box motif (TTGACY) was present on the promoter of 613 genes of the 766 total genes up-regulated more then twofold at T3 h. Then, the W-box motif frequency was studied on the promoter regions of the genes selected for SOTA analysis to evaluate the presence of such element on gene clusters showing different expression profiles. The W-box element resulted to be mainly concentrated in group 2a (1.914 W-box per promoter), which clusters genes characterized by an increment in their expression at T3 h and a decline at T12 h. The value of group 2b, which clusters genes with the highest steady-state mRNA levels at T12 h was 1.669. In contrast, the W-box motif was 0.928 in the group 1 down-regulated genes.

WRKY genes, receptor-like kinase (RLK) and mitogen-activated protein kinase (MAPK) cascades

6

2a

expression occurred at T12 h and were mainly up-regulated in the lower part (2b).

47 18 2 2 0 h 3 h 12 h

Figure 2. Self-organizing tree algorithm applied on twofold down- and up-regulated genes. Circles are proportional to the number of genes (given within or next to the circle) belonging to each cluster. Histograms represent (from left to right) the expression values after log transformation of the probes at 0 h (control), T3 h and T12 h. The number on the left of the branch points indicates the two main cluster groups.

The W-box element contains the invariant TGAC core essential for the binding of a class of transcription factors containing the WRKY domain, which appears to be unique to plants (Eulgem et al. 2000). We found 14 up-regulated WRKY genes at T3 h (Table 4). Nine WRKY genes maintained their over-expression at T12 h, though at a lower level. There were, however, two exceptions (AtWRKY26 and AtWRKY45) which registered a further increment. One WRKY gene (AtWRKY22) was not modulated at T3 h, but was up-regulated at T12 h and one (AtWRKY60) was down-regulated at T3 h only (Supplementary Fig. S3). Thirty-seven of the 303 RLKs present on the Arabidopsis genome (Ward 2001) were up-regulated after 3 h of O3 fumigation. The increase was above twofold in 30. Nine RLKs maintained their over-expression at T12 h, while four increased at T12 h and only one increased at T12 h only. Of the six RLKs that were down-regulated, one was downregulated at T3 h only, whereas five were decreased at T12 h (Supplementary Table S6). We found 99 TTGACY elements in the promoter regions of the 38 up-regulated RLKs, with an average of 2.605 motifs per promoter,


1694 N. Tosti et al. Table 3. List of the 4–8mer oligos over-represented in the 1.0 Kb promoter sequence of up-regulated genes after 3 h of O 3 treatment Sequence 4mer gtca|tgac agtc|gact aata|tatt 5mer gtcaa|ttgac aagtc|gactt agtca|tgact 6mer agtcaa|ttgact aaagtc|gacttt aagtca|tgactt 7mer aaaagtc|gactttt aagtcaa|ttgactt acttttc|gaaaagt 8mer gaaaagtc|gacttttc aaagtcaa|ttgacttt gactttga|tcaaagtc a

Occa

Exp occb

Occ Pc

Occ Ed

Occ sige

5 168 4 412 15 511

4 247.30 3 679.76 14 233.10

5.4e-43 4.7e-32 7.9e-27

7.4e-41 6.4e-30 1.1e-24

40.13 29.20 23.97

2 312 1 855 1 799

1 676.39 1 370.75 1 347.46

4.1e-49 1.2e-35 6.5e-32

2.1e-46 6.0e-33 3.3e-29

45.67 32.23 28.48

Wboxatnpr1 (TTGAC)

888 855 773

559.13 554.62 521.25

8.9e-38 2e-32 4.7e-25

1.9e-34 4.1e-29 9.7e-22

33.73 28.38 21.01

Wboxatnpr1 (TTGAC) Dofcorezm (AAAG)

402 408 367

225.97 233.52 226.88

3.3e-26 3.6e-25 8.4e-18

2.7e-22 2.9e-21 6.9e-14

21.56 20.53 13.16

Dofcorezm (AAAG) Wboxatnpr1 (TTGAC) Dofcorezm (AAAG)

127 204 105

41.61 105.88 43.51

1.8e-26 1.8e-17 2.2e-15

6.0e-22 6.1e-13 7.3e-11

21.22 12.22 10.14

Dofcorezm (AAAG) Wboxatnpr1 (TTGAC) Dofcorezm (AAAG)

Known cis-acting element

Number of occurrence; bexpected occurrence; coccurrence P-value; doccurrence E-value; eoccurrence significativity.

significantly higher than the average (1.986) of this ciselement on all the Arabidopsis RLK promoters (P = 0.01). MAPK-signalling pathways are reported to be actively involved in transducting oxidative signalling (Tournier et al. 1997; Klotz et al. 1999; Kovtun et al. 2000; Wrzaczek & Hirt 2001; Moon et al. 2003). We found that 11 MAPK of the four gene families classified in Arabidopsis (Kovtun et al. 2000; Tena et al. 2001; Asai et al. 2002; Ichimura et al. 2002; Jonak et al. 2002) were up-regulated after 3 h of fumigation and five MAPK at T12 h, while only two were downregulated at T12 h (Supplementary Table S7).

treatment, so confirming the microarray data. The RLK At4g23190 was up-regulated with respect to the control. Whereas WRKY55 reached peak transcription at T3 h, the WRKY26 peak, as assessed by real-time PCR analysis, was not achieved until 24 h. However, the WRKY26 level was significantly higher than the control value at T3 h and T12 h, as the microarray data confirmed. WRKY22

Table 4. Fold variation of WRKY transcriptional factors modulated at T3 h and T12 h Fold variation

Real-time RT-PCR analyses on selected genes A real-time RT-PCR approach on independent RNA sources was performed to validate the differential geneexpression values obtained by microarray analysis. However, other than for validation, we carried out real-time RTPCR analysis to test each gene over the entire time course, because they were monitored not only at the time points assessed by microarray, but also at T6 h and T24 h. Hence, a subset of seven target genes (belonging to either MAPK, RLK, WRKY or hormone signalling gene families) predicted by microarray analysis to be differentially expressed along with the negative regulator EDR1 (see Discussion) were deliberately chosen for real-time RT-PCR analysis based on the putative gene’s biological function rather than on the magnitude of differential transcript accumulation. Figure 3 gives the transcriptional profile of the selected genes. O3 triggered early transcriptional activation of AtMPK3, but the level dropped to near that of controls 24 h after starting O3 fumigation. The two tested RLKs were significantly up-regulated within the first 3 h of

ID AGI Up-regulated At5g24110 At5g13080 At1g80840 At1g62300 At2g40740 At2g38470 At4g23810 At5g07100 At2g23320 At2g46400 At4g31800 At3g01970 At4g01250 At2g30590 At2g30250 Down-regulated At2g25000

Name

T3 h

T12 h

AtWRKY30 AtWRKY75 AtWRKY40 AtWRKY6 AtWRKY55 AtWRKY33 AtWRKY53 AtWRKY26 AtWRKY15 AtWRKY46 AtWRKY18 AtWRKY45 AtWRKY22 AtWRKY39 AtWRKY25

18.5 8.6 7.0 5.8 4.4 4.2 3.7 2.7 2.0 2.2 2.1 2.5 nc 2.0 2.0

nc 5.2 2.5 3.4 nc nc 1.6 5.6 2.1 nc nc 5.5 2.1 nc 2.0

AtWRKY60

−2.4

nc

nc, no change with respect to the control (0 h).



Figure 3. Quantitative analysis at different time points of the transcript levels of eight target genes selected on the basis of microarray data. Data are expressed as a mean value (± SEM) of the transcript levels of each target gene relative to the elongation factor cDNA, as described in Materials and Methods.

up-regulation was also confirmed by real-time RT-PCR analysis, although, unlike in the microarray, up-regulation was recorded not only at T12 h, but also at T3 h. The kinetic PCR showed that CTR1 was up-regulated relatively late and that its expression started to increase at T3 h and continued to increase until 6 h from the end of the O3 treatment (T12 h).

Other genes Clearly, a large number of genes of different known functions were modulated by O3 treatment. Within gene families of transcription factors other than WRKY, such as MYB, bZIP and ERF as well as among genes that encode transport proteins, cytochrome, etc., both up- and downregulated genes were recorded (Supplementary Fig. S3). Thus, the picture is complex and clear description and gene/ family classification in relation to O3 far from easy. A summary of the modulation of the expression LEVELS of MYB and bZIP transcription factors is reported in Supplementary Table S8. Among the modulated genes reported to have putative function, the steepest up-regulation was registered in those related to reticuline oxidase. Expression was enhanced from 2.8- to 23.4-fold in six genes at T3 h and in all these genes remained high from 2.4- to 5.8-fold at T12 h. One gene was up-regulated (5.1-fold) only at T12 h (Supplementary Table S9).

DISCUSSION In plants, genome-wide gene-expression analyses using microarray has been applied to the characterization and identification of novel target genes and pathways associated

with specific biological processes and treatments (for a review see Zhu 2003). Recently, Ludwikow, Gallois & Sadowski (2004) performed genome-wide microarray analyses on Arabidopsis plants during O3 treatment to specifically monitor the fluctuation of genes that respond to oxidative stress, whereas Mahalingam et al. (2005) analysed the temporal evolution of a subset of about 1390 stress-related genes in fumigated Arabidopsis plants. Here, we monitored alterations in gene expression during and after O3 treatment using GeneChip arrays to shed light on the genetic networks that regulate the responses to O3 fumigation in the tolerant Columbia genotype. Our transcript profiling revealed that there is a differentiated and time-coordinated response among members of the same multigene family. More interestingly, microarray and real-time RT-PCR indicated that the WRKY family of transcription factors is involved in both the perception of O3 (and/or its by-products) and its signal-transduction pathway. To the best of our knowledge, this is one of the first reports, in plants, where microarray validation has been achieved by real-time RT-PCR using gene-specific fluorescent probes. This technique provides greater sensitivity, specificity, accuracy and reproducibility than SYBR Green does (Tian et al. 2004).

How do signalling molecules work in O3-treated Arabidopsis plants Tamaoki et al. (2003), who carried out transcriptome analysis on about 12 000 ESTs of O3-exposed Col-0 Arabidopsis, reported that a large number of defence genes were activated after 12 h of exposure, and that many of them were induced by ET and JA but suppressed by SA signalling, which suggests that the SA pathway is strongly antagonistic to gene expression induced by ET and JA signalling. They reported that a low level of ET seems to induce defence genes and proposed that ET and JA signalling might act for the acquisition of O3 tolerance in Col-0, whereas SA signalling might not be strongly activated by O3 in Col-0. Support for this comes from the observation of Rao & Davis (1999) that the O3-sensitive ecotype Cvi-0 accumulated higher levels of SA than Col-0 did. Pasqualini et al. (2002) reported that, in tobacco, high LEVEL of SA correlated with the high O3-sensitivity of the cv. BelW3, whereas SA content was low in the tolerant cv. BelB. Furthermore, there was a greater reduction of O3-induced SA accumulation and cell death in the transgenic Cvi-0 that expresses a bacterial salicylate hydroxylase gene than in the wild type (Rao et al. 2000). It therefore seems that SA signalling may propagate leaf injury by O3. Together these results indicate that in Col-0, O3 exposure activates the expression of many defence genes, mostly through ET and JA signalling. However, some SA signallings do not result in cell death. We demonstrated that ET, JA and SA are activated not only by enhanced expression of several genes belonging to their biosynthetic routes (e.g. genes encoding for the key enzymes ACS, OPDA reductase and ICS for ET, JA and SA synthesis, respectively, Fig. 1) but also by an increment


1696 N. Tosti et al. in the steady-state levels of genes involved in the signal transduction of these molecules. CTR1, a negative regulator of ET, was up-regulated at T12 h. Real-time RT-PCR analyses over the entire time-course confirmed that CTR1 increased early at T3 h and reached its maximum after 12 h. CTR1 is a Raf-like protein kinase and there is genetic and biochemical evidence that it interacts with receptor ETR1 as an ET negative regulator. EDR1 encodes a putative MAPK kinase kinase similar to CTR1 and regulates SAinducible defence responses negatively (Tang, Christiansen & Innes 2005). EDR1 was not differentially regulated at T3 and T12 h in the microarray analysis, but it was upregulated at T6 h in real-time RT-PCR analysis (Fig. 3). The activation of negative regulators of ET- and SA-signalling pathways probably explains why O3 did not trigger leaf injury in Col-0 plants.

Stress responses and induction of plant defences Genes involved in antioxidant systems were both up- and down-regulated by O3 treatment. Enhanced lipid peroxidation upon oxidative stress affects membrane properties and activates detoxifying enzymes such as GST. As predicted, O3 induces GST1 (At1g02930) and GST6 after 3 h of fumigation and these genes decline after 12 h (Conklin & Last 1995; Sharma et al. 1996; Mahalingam et al. 2005). As regards the glutathione peroxidase (GPX) genes, in accordance with Ludwikow et al. (2004), we did not find an activation but only a down-regulation of GPX1. We also documented a steep down-regulation of GPX7. H2O2 detoxification is also mediated by catalase. We identified two transcription patterns for CAT2 and CAT1 genes in plants exposed to a 300 ppb O3 dose. CAT1 exhibited twofold up-regulation after 3 and 12 h from the start of O3 fumigation, whereas there was a fourfold decline in CAT2 after 12 h, a picture also noted by Ludwikow et al. (2004). The functional role of CAT1 is uncertain, but the lack of a consensus sequence to target this protein to peroxisomes or glioxysomes suggests that it is either cytosolic or localized to a different subcellular compartment, for instance the mitochondrion (Frugoli et al. 1996). According to Zhong & McClung (1996), CAT2 is localized predominantly in the peroxisomes where it may scavenge the H2O2 produced during photorespiration. This divergence in function is reflected in expression profile variations seen during stress conditions. CAT2 is repressed, possibly as the result of a general decrease in photorespiratory activity, during stress conditions. The dramatic drop in mRNA levels of a large subunit of Rubisco we detected could, indeed compromise both photosynthetic and photorespiratory processes. We also found that a member of an alternative oxidase gene family (AOX), which aids in reducing ROS production in mitochondria (Robson & Vanlerberghe 2002), was strongly induced by O3. In addition, the acute O3 treatment resulted in a decreased accumulation of photosynthetic proteins, whereas induced an accumulation of senescence genes, and

several other genes located in O3-responsive pathways (shikimate, tyrosine, triptophane and phenylalanine). By contrast to acute treatment, chronic exposure to O3 caused smaller proportion of Arabidopsis trancriptome to be altered, with nearly 10 times more genes down-regulated than up-regulated (Miyazaki et al. 2004). The substantial down-regulation of polyamine synthesis genes in ozonated Col-0 plants is of interest. Although the contribution of polyamine molecules to plant protection against O3 damage has been well documented (Langebartels et al. 1991; Tuomainen et al. 1996; Navakoudis et al. 2003), their exact mode of action remains a matter of debate (Bors et al. 1989; Langebartels et al. 1991). Adenosylmethionine decarboxylase and spermidine synthase genes, which encode the enzymes that lead to the spermidine formation, were both severely down-regulated by O3 in our experiment. A similar O3-induced drop in mRNA levels has been reported for the arginine decarboxylase gene in tobacco plants, despite an increase in putrescine levels (Van Buuren et al. 2002). Putrescine is the most important polyamine involved in protecting the photosynthetic apparatus and, therefore the entire plant against O3, whereas spermidine and spermine seem to play a lesser role in the O3 protection (Langebartels et al. 1991; Navakoudis et al. 2003). O3 caused both up- and down-regulation of a number of genes within the phenylpropanoid pathway (Fig. 1). Genes that lead to phenylpropanoid derivatives known to be induced by O3, including phytoalexin (Zinser, Ernst & Sandermann 1998; Chiron et al. 2000) and lignin (Koch et al. 1998; Cabane et al. 2004) were strongly up-regulated. The early and strong up-regulation of OMT (O-methyltransferase), an enzyme operative in lignin biosynthesis, suggests that its biosynthesis is a response to O3, as Ludwikow et al. (2004) have also proposed. However, O3 severely downregulated both TT6, which encodes for flavol synthase, that catalyses the conversion of dihydroflavonols to flavonols (Shirley et al. 1995) and the glycosyltransferase At1g01420. Production of the indol phytoalexin, camalexin is known to be pathogen-induced in Arabidopsis (Tsuji et al. 1992). Camalexin is derived via the triptophane biosynthetic pathway by an unknown mechanism and only one putative biosynthetic enzyme, PAD3, which is a cytocrome P450, is known (Glazebrook et al. 1997). In our stress conditions, many tryptophane biosynthetic genes were induced and the PAD3 gene was dramatically up-regulated.

Other genes As previously mentioned, the response to O3 often differs among members within the same gene families, some being up-regulated and others down-regulated. An exception to this picture was the strong activation of many genes that encode putative reticuline oxidase, an enzyme essential for the formation of benzophenanthridine alkaloids in response to plant pathogen attack (Dittrich & Kutchan 1991). Our results reinforce Richards et al.’s finding (1998) that aluminium and O3 stress induce a reticuline oxidase in Arabidopsis plants. Such genes are therefore among the


Ozone effects on Arabidopsis transcriptome 1697 worthiest of study when dissecting the O3 response in Arabidopsis.

Signal perception and transduction There is mounting evidence that ROS plays a signalling role in the defence response to abiotic stress. However, although ROS and the cellular redox state are known to control expression of plant genes, reports on signalling pathways, transcription factors and promoter elements specific for redox regulation are very recent (for a review see Laloi, Apel & Danon 2004). Several candidates described as promoter elements and DNA-binding factors could act as redox-responsive sequences. The ERE [(T)(T)TGAC(Y)], known as the W-box, have been reported to be also present in O3-responsive promoters (Schubert et al. 1997). Here we report that the W-box is the most significatively over-represented motif in the promoter region of up-regulated genes after 3 h O3 stress. Intriguingly, Mahalingam et al. (2003) reported an over-representation of WRKY motifs in the promoter region of genes up-regulated by O3 as per suppressive subtractive hybridization (SSH). Genes containing the W-box promoter elements are likely targets of WRKY, including the WRKY genes themselves (Eulgem et al. 1999). The W-box mediates transcriptional responses to pathogen-derived elicitors and occurs in promoters of different defence-related genes, among them the PR-like genes (Rushton et al. 1996; Eulgem et al. 1999; Du & Chen 2000). The activity of many WRKY genes is increased rapidly by pathogen infection (Asai et al. 2002; Navarro et al. 2004), SA (Dong, Chen & Chen 2003), wounding (Hara et al. 2000; Laloi et al. 2004) and nitric oxide (NO; Parani et al. 2004). Apart from the WRKY genes described to be O3-modulated by Mahalingam et al. (2003, 2005) we found other WRKY genes over-expressed at T3 h, making it probable that they are involved in the response of Arabidopsis to O3 stress during the early phase of exposition. WRKY 33, 40 and 46 were also up-regulated in NO-treated Arabidopsis plants (Parani et al. 2004) and in KO-Apx1 mutants during light stress (Davletova et al. 2005), suggesting that O3, NO and light might, at least partially, trigger the same signalling pathway. The modulation of genes that reach maximum expression at T12 h, or are down-regulated at this time, does not appear to depend on WRKY because the number of W-box elements in their promoters is not significant. However, the transcript levels for some WRKY genes are still high at T12 h, while other WRKY genes reach their expression peak at this time point. Thus, it is conceivable that at least some genes differentially regulated at T12 h are controlled by as yet uncharacterized regulatory factors, which may be primary targets of WRKYs. RLKs serve as receivers and transducers of external and internal stimuli. Based on the presence of W-box within the promoter region of many RLKs, it has been proposed that RLKs are potential targets for WRKY proteins (Du & Chen 2000; Ohtake, Takahashi & Komeda 2000). RLK expression that depends on WRKY has been proved for

two of them: SIRK with AtWRKY6 (Robatzek & Somssich 2002) and RLK4 with AtWRKY18 (Du & Chen 2000). As many W-box elements were identified in promoter region of most RLKs that were up-regulated at T3 h, WRKY may activate RLKs. Nevertheless, a direct action of ROS on RLKs that leads to their initial activation cannot be excluded (Kovtun et al. 2000). WRKY may therefore be involved in the initial activation and/or the maintenance of RLK expression during O3 treatment, so keeping the response constant during stress. The progressive induction of RLK transcripts upon pathogen attack seems to be a sensitization process of cells to further pathogen recognition (Navarro et al. 2004). Many studies have shown that the MAPK cascades are the major downstream component of receptors or sensors that transduce extracellular stimuli in plant intracellular responses (Tena et al. 2001; Zhang & Klessig 2001). Moreover, direct activation of MAPK by O3 has been demonstrated in tobacco (Samuel, Miles & Ellis 2000; Samuel & Ellis 2002), rice (Kim et al. 2003) and Arabidopsis (Ahlfors et al. 2004).There is evidence to suggest that a subset of plant responses to biotic and abiotic stresses, for instance generation of ROS and activation of early defence genes, is shared (Somssich & Hahlbrock 1998). MAPKs are likely to be one of the convergence points in the defence-signalling network. Using northern analysis, Ahlfors et al. (2004) revealed that in Col-0, AtMPK3 responded to O3 transcriptionally and that both AtMPK3 and AtMPK6 were translocated to the nucleus during the early stages of O3 treatment. The role of MAPK3 and MAPK6 in O3-stressed Arabidopsis plants has recently been elucidated by Miles et al. (2005). These researchers demonstrated that both RNA-i silenced MAPK3 and MAPK6 plants were hypersensitive to O3. Ichimura et al. (2000) reported that environmental stresses induced rapid and transient activation of two MPKs (AtMPK4 and AtMPK6) in Arabidopsis plants. Their activation was associated with tyrosine phosphorylation, but unrelated to the amount of mRNA or protein. Our microarray results show that several MAPKs were induced (Supplementary Table S7), and that AtMPK3 was transcriptionally upregulated, as confirmed by real-time RT-PCR analysis (Fig. 3). Asai et al. (2002) identified a complete MAPK cascade that functions downstream of flagellin receptor FLS2 and induces WRKY22/WRKY29 transcription. In addition, Andreasson et al. (2005) showed that WRKY25 and 33 are in vitro substrates of AtMPK4 that functions as a regulator of pathogen-defence responses. In our experiments WRKY 22, 25 and 33 are among the WRKY genes that are upregulated by O3, suggesting that they might be targets of an upstream activation of a MAPK cascade even under O3 stress. However, as yet uncharacterized mechanisms that directly activate WRKY proteins at the early stage of O3 treatment can also be envisaged. ROS has been hypothesized to interact selectively with a target molecule that perceives the increased ROS concentration and then transforms this information into a change in gene expression. Such a change in transcriptional activity could be achieved either through oxidation of components of


1698 N. Tosti et al. signalling pathways that subsequently activate transcription factors or by direct modification of a redox-sensitive transcription factor (Laloi, Apel & Danon 2004). There is evidence that ROS and redox changes directly modify the redox state of transcription factor TGA1 (Despres et al. 2003). The fact that WRKY possess a redox-sensitive zincfinger DNA binding domain makes them excellent candidates for redox regulation (Arrigo 1999). In conclusion, we here show that O3 affects transcript levels of numerous genes and provide strong evidence that WRKY transcription factors are key components of stress perception/signal transduction pathways in O3-treated plants. Our data also indicate that the ET- and SAsignalling pathways are down-regulated in Col-0 during the recovery phase. The up-regulation of entire classes of gene families (i.e. reticuline oxidase) suggests that they are candidate genes that should be monitored in both O3-tolerant and susceptible genotypes. Further studies with WRKY transgenic plants and pathway-specific mutants should lead to a more profound understanding of the network of pathways that mediate the plant response to O3 stress.

ACKNOWLEDGMENTS This research was supported by a grant from MIUR (National Project, PRIN 2003) from Italy, and by a grant from the University of Perugia. The authors are grateful to Dr Remo Sanges (Tigem, Napoli, Italia) for assistance with microarray data acquisition.

REFERENCES Ahlfors R., Macioszek V., Rudd J., Brosché M., Schlichting R., Scheel D. & Kangasjärvi J. (2004) Stress hormone-independent activation and nuclear translocation of mitogen-activated protein kinases in Arabidopsis thaliana during ozone exposure. Plant Journal 40, 512–522. Andreasson E., Jenkins T., Brodersen P., et al. (2005) The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO Journal 24, 2579–2589. Arrigo A.P. (1999) Gene expression and the thiol redox state. Free Radical Biology and Medicine 27, 936–944. Asai T., Tena G., Plotnikova J., Willmann M.R., Chiu W.L., Gomez-Gomez L., Boller T., Ausubel F.M. & Sheen J. (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415, 977–983. Ashburner M., Ball C.A., Blake J.A., et al. (2001) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nature Genetics 25, 25–29. Ashmore M.R. (2005) Assessing the future global impacts of ozone on vegetation. Plant, Cell & Environment 28, 949–964. Baier M., Kandlbinder A., Golldack D. & Dietz K.-J. (2005) Oxidative stress and ozone: perception, signalling and response. Plant, Cell & Environment 28, 1012–1020. Bors W., Langebartels C., Michel C. & Sandermann H. (1989) Polyamunes as radicals scavengers and protectants against ozone damage. Phytochemistry 28, 1589–1595. Cabane M., Pireaux J.C., Leger E., Weber E., Dizengremel P., Pollet B. & Lapierre C. (2004) Condensed lignins are synthesized in poplar leaves exposed to ozone. Plant Physiology 134, 586–594.

Chang C., Kwok S.F., Bleecker A.B. & Meyerowitz E.M. (1993) Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science 262, 539–544. Chiron H., Drouet A., Lieutier F., Payer H.D., Ernst D. & Sandemann H. (2000) Gene induction of stilbene biosynthesis in scots pine in response to ozone treatment, wounding and fungal infection. Plant Physiology 4, 865–872. Conklin P.L. & Last R.L. (1995) Differential accumulation of antioxidant mRNA in Arabidopsis thaliana exposed to ozone. Plant Physiology 109, 203–212. Dann M.S. & Pell E.J. (1989) Decline of activity and quantity of ribulose bisphosphate carboxylase/oxygenase and net photosynthesis in ozone-treated potato foliage. Plant Physiology 91, 427– 432. Davletova S., Rizhsky L., Liang H.J., Zhong S.Q., Oliver D.J., Coutu J., Shulaev V., Schlauch K. & Mittler R. (2005) Cytosolic ascorbate peroxidase1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17, 268–281. Despres C., Chubak C., Rochon A., Clark R., Bethune T., Desveaux D. & Fobert P.R. (2003) The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1. Plant Cell 15, 2181–2191. Dittrich H. & Kutchan T.M. (1991) Molecular-cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack. Proceedings of the National Academy of Sciences of the USA 88, 9969–9973. Dong J., Chen C. & Chen Z. (2003) Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Molecular Biology 51, 21–37. Dopazo J. & Carazo J.M. (1997) Phylogenetic reconstruction using a growing neural network that adopts the topology of a phylogenetic tree. Journal of Molecular Evolution 44, 226–233. Dopazo. J., Zanders E., Dragoni I., Amphlett C. & Falciani F. (2001) Methods and approaches in the analysis of gene expression data. Journal of Immunological Methods 250, 93–112. Du L. & Chen Z. (2000) Identification of genes encoding receptorlike protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis. Plant Journal 24, 837–847. Durner J., Shah J. & Klessig D.F. (1997) Salicylic acid and disease resistance in plants. Trends in Plant Science 2, 266–274. Eulgem T., Rushton P.J., Elmon Schmelzer E., Hahlbrock K. & Somssich I.E. (1999) Early nuclear event in plant defence: rapid gene activation by WRKY transcription factors. EMBO Journal 18, 4689–4699. Eulgem T., Rushton P.J., Robatzek S. & Somssich I.E. (2000) The WRKY superfamily of plant transcription factors. Trends in Plant Science 5, 199–206. Fowler D., Cape J.N., Coyle M., Flechard C., Kuylenstierna J., Hicks K., Derwent D., Johnson C. & Stevenson D. (1999) The global exposure of forests to air pollution. Water, Air, and Soil Pollution 116, 5–32. Foyer C.H. & Noctor G. (2005) Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant, Cell & Environment 28, 1056–1071. Frugoli J.A., Zhong H.H., Nuccio M.L., McCourt P., McPeek M.A., Thomas T.L. & McClung C.R. (1996) Catalase is encoded by a multigene family in Arabidopsis thaliana (L) Heynh. Plant Physiology 112, 327–336. Glazebrook J., Zook M., Mert F., Kagan I., Rogers E.E., Crute I.R., Holub E.B., Hammerschmidt R. & Ausubel F.M. (1997) Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance. Genetics 146, 381–392.


Ozone effects on Arabidopsis transcriptome 1699 Hara K., Yagi M., Kusano T. & Sano H. (2000) Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding. Molecular and General Genetics 263, 30–37. van Helden J., Andre B. & Collado-Vides J. (1998) Extracting regulatory sites from the upstream region of yeast genes by computational analysis of oligonucleotide frequencies. Journal of Molecular Biology 281, 827–842. Herrero J., Valencia A. & Dopazo J. (2001) A hierarchical unsupervised growing neural network for clustering gene expression patterns. Bioinformatics 17, 126–136. Higo K., Ugawa M., Iwamoto M. & Korenaga T. (1999) Plant cisacting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Research 27, 297–300. Hua J., Chang C., Sun Q. & Meyerowitz E. (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene. Science 269, 1712– 1714. Ichimura K., Mizoguchi T., Yoshida R., Yuasa T. & Shinozaki K. (2000) Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. Plant Journal 24, 655– 665. Ichimura K., Shinozaki K., Tena G., et al. (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends in Plant Science 7, 301–308. Irizarry R.A., Bolstad B.M., Collin F., Cope L.M., Hobbs B. & Speed T.P. (2003) Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Research 31, e15. DOI: 10.1093/nar/ gng015. Jonak C., Ökrész L., Bögre L. & Hirt H. (2002) Complexity, cross talk and integration of plant MAP kinase signalling. Current Opinion in Plant Biology 5, 415–424. Kangasjarvi J., Jaspers P. & Kollist H. (2005) Signalling and cell death in ozone-exposed plants. Plant, Cell & Environment 28, 1021–1036. Karnosky D.F., Pregitzer K.S., Zak D.R., Kubiske M.E., Hendrey G.R., Weinstein D., Nosal M. & Percy K.E. (2005) Scaling ozone responses of forest trees to the ecosystem level in a changing climate. Plant, Cell & Environment 28, 965–981. Kieber J.J., Rothenberg M., Roman G., Feildmann K.A. & Ecker J.R. (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of Raf family of protein kinase. Cell 72, 427–551. Kim J.A., Agrawal G.K., Rakwal R., Han K.S., Kim K.N., Yun C.H., Heu S., Park S.Y., Lee Y.H. & Jwa N.S. (2003) Molecular cloning and mRNA expression analysis of a novel rice (Oryza sativa L.) MAPK kinase kinase, OsEDR1, an ortholog of Arabidopsis AtEDR1, reveal its role in defense/stress signalling pathways and development. Biochemical and Biophysical Research Communications 300, 868–876. Klotz L.O., Pellieux C., Briviba K., Pierlot C., Aubry J.M. & Sies H. (1999) Mitogen-activated protein kinase (p38-, JNK-, ERK-) activation pattern induced by extracellular and intracellular singlet oxygen and UVA. European Journal of Biochemistry 260, 917–922. Koch J.R., Scherzer A.J., Eshita S.M. & Davis K.R. (1998) Ozone sensitivity in hybrid polar is correlated with a lack of defencegene activation. Plant Physiology 118, 1243–1252. Kovtun Y., Chiu W.L., Tene G. & Sheen J. (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proceedings of the National Academy of Sciences of the USA 97, 2940–2945. Laloi C., Apel K. & Danon A. (2004) Reactive oxygen signalling: the latest news. Current Opinion in Plant Biology 7, 323– 328. Laloi C., Mestres-Ortega D., Marco Y., Meyer Y. & Reichheld J.P. (2004) The Arabidopsis cytosolic thioredoxin h5 gene induction

by oxidative stress and its W-box-mediated response to pathogen elicitor. Plant Physiology 134, 1006–1016 Langebartels C., Kerner K., Leonardi S., Schraudner M., Trost M., Heller W. & Sandermann H. (1991) Biochemical-plant responses to ozone. 1. Differential induction of polyamine and ethylene biosynthesis in tobacco. Plant Physiology 95, 882–889. Li C. & Wong W.H. (2001) Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proceedings of the National Academy of Sciences of the USA 98, 31–36. Ludwikow A., Gallois P. & Sadowski J. (2004) Ozone-induced oxidative stress response in Arabidopsis: transcription profiling by microarray approach. Cellular and Molecular Biology Letters 9, 829–842. Mahalingam R., Gomez-Buitrago A.M., Eckardt N., Shah N., Guevara-Garcia A., Day P., Raina R. & Fedoroff N. (2003) Characterizing the stress-defense transcriptome of Arabidopsis. Genome Biology 4, R20. Mahalingam R., Shah N., Scrymgeour A. & Fedoroff N. (2005) Temporal evolution of the Arabidopsis oxidative stress response. Plant Molecular Biology 57, 709–730. Mehlhorn H. & Wellburn A.R. (1987) Stress ethylene formation determines plant sensitivity to ozone. Nature 327, 417–418. Miles G.P., Samuel M.A., Zhang Y.C. & Ellis B.E. (2005) RNA interference-based (RNAi) suppression of AtMPK6, an Arabidopsis mitogen-activated protein kinase, results in hypersensitivity to ozone and misregulation of AtMPK3. Environmental Pollution 136, 230–237. Miller J.D., Arteca R.N. & Pell E.J. (1999) Senescence-associated gene expression during ozone-induced leaf senescence in Arabidopsis. Plant Physiology 120, 1015–1023. Miyazaki S., Fredricksen M., Hollis K.C., Poroyko V., Shepley D., Galbraith D.W., Long S.P. & Bohnert H.J. (2004) Trancript expression profiles of Arabidopsis thaliana grown under controlled conditions and open-air elevated concentrations of CO2 and O3. Field Crops Research 90, 47–59. Moon H., Lee B., Choi G., et al. (2003) NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants. Proceedings of the National Academy of Sciences of the USA 100, 358–363. Navakoudis E., Lutz C., Langebartels C., Lutz-Meindl U. & Kotzabasis K. (2003) Ozone impact on the photosynthetic apparatus and the protective role of polyamines. Biochimica et Biophysica Acta – General Subjecta 1621, 160–169. Navarro L., Zipfel C., Rowland O., Keller I., Robatzek S., Boller T. & Jones J.D.G. (2004) The transcriptional innate immune response to flg22. Interplay and overlap with Avr gene-dependent defense responses of bacterial pathogenesis. Plant Physiology 135, 1113–1128. Nawrath C. & Metraux J.P. (1999) Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high level of camalexin. Plant Cell 11, 1393–1404. Nawrath C., Heck S., Parinthawong N. & Metraux J.P. (2002) EDS5, an essential component of salicylic acid-dependent signaling for disease resistance in Arabidopsis, is a member of the MATE transporter family. Plant Cell 14, 275–286. Ohtake Y., Takahashi T. & Komeda Y. (2000) Salicylic acid induces expression of a number of receptor-like kinase genes in Arabidopsis thaliana. Plant and Cell Physiology 41, 1038–1044. Orvar B.L., McPherson J. & Ellis B.E. (1997) Pre-activating wounding response in tobacco prior to high-level ozone exposure prevents necrotic injury. Plant Journal 11, 203–212. Overmyer K., Tuominen H., Kettunen R., Betz C., Langebartels C., Sandermann H. & Kangasjarvi J. (2000) Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for ethylene and


1700 N. Tosti et al. jasmonate signaling pathways in regulating superoxide-dependent cell death. Plant Cell 12, 1849–1862. Overmyer K., Brosche M. & Kangasjarvi J. (2003) Reactive oxygen species and hormonal control of cell death. Trends in Plant Science 8, 335–342. Parani M., Rudrabhatla S., Myers R., Weirich H., Smith B., Leaman D.W. & Goldman S.L. (2004) Microarray analysis of nitric oxide responsive transcripts in Arabidopsis. Plant Biotechnology Journal 2, 359–366. Pasqualini S., Della Torre G., Ferranti F., Ederli L., Piccioni C., Reale L. & Antonielli M. (2002) Salicylic acid modulates ozoneinduced hypersensitive cell death in tobacco plants. Physiologia Plantarum 115, 204–212. Pell E.J., Eckardt N.A. & Glick R.E. (1994) Biochemical and molecular basis for impairment of photosynthetic potential. Photosynthesis Research 39, 453–462. Pell E.J., Schlagnhaufer C.D. & Arteca R.N. (1997) Ozoneinduced oxidative stress: mechanisms of action and reaction. Physiologia Plantarum 100, 264–273. Rao M.V. & Davis K.R. (1999) Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid. Plant Journal 17, 603–614. Rao M.V., Lee H.I., Creelman R.A., Mullet J.E. & Davis K.R. (2000) Jasmonic acid signaling modulates ozone-induced hypersensitive cell death. Plant Cell 12, 1633–1646. Rao M.V., Lee H. & Davis K.R. (2002) Ozone-induced ethylene production is dependent on salicylic acid, and both salicylic acid and ethylene act in concert to regulate ozone induced cell death. Plant Journal 32, 447–456. Reich P.B. & Amundson R.G. (1985) Ambient levels of ozone reduce net photosynthesis in tree and crop species. Science 230, 566–570. Richards K.D., Schott E.J., Sharma Y.K., Davis K.R. & Gardner R.C. (1998) Aluminium induces oxidative stress genes in Arabidopsis thaliana. Plant Physiology 116, 409–418. Robatzek S. & Somssich I.E. (2002) Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes and Development 16, 1139–1149. Robbins M.P., Paolocci F., Hughes J.W., Turchetti V., Allison G., Arcioni S., Morris. P. & Damiani F. (2003) Sn, a maize bHLH gene, modulates anthocyanin and condensed tannin pathways in Lotus corniculatus. Journal of Experimental Botany 54, 239–248. Robson C.A. & Vanlerberghe G.C. (2002) Transgenic plant cells lacking mitochondrial alternative oxidase have increased susceptibility to mitochondria-dependent and -independent pathways of programmed cell death. Plant Physiology 129, 1908– 1920. Rushton P.J., Torres J.T., Parniske M., Wernert P., Hahlbrock K. & Somssich I.E. (1996) Interaction of elicitor-induced DNAbinding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO Journal 15, 5690–5700. Ruzsa S.M., Mylona P. & Scandalios J.G. (1999) Differential response of antioxidant genes in maize leaves exposed to ozone. Redox Report 4, 95–103. Sakai H., Hua J., Chen Q.G., Chang C., Medrano L.I., Bleecker A.B. & Meyerowitz E.M. (1998) ETR2 is an ETR1-like gene involved in ethylene signalling in Arabidopsis. Proceedings of the National Academy of Sciences of the USA 95, 5812–5817. Samuel M.A. & Ellis B.E. (2002) Double jeopardy: both overexpression and suppression of a redox-activated plant mitogenactivated protein kinase render tobacco plants ozone sensitive. Plant Cell 14, 2059–2069. Samuel M.A., Miles G.P. & Ellis B.E. (2000) Ozone treatment rapidly activates MAP kinase signalling in plants. Plant Journal 22, 367–376.

Sandermann H. (1996) Ozone and plant health. Annual Review of Phytopathology 34, 347–366. Saviozzi S., Iazzetti G., Caserta E., Guffanti A. & Calogero R.A. (2003) Microarray data analysis and mining. In Methods in Molecular Medicine: Molecular Diagnosis of Infectious Diseases (eds J. Decker & U. Reischl), pp. 67–90. Humana Press Inc., Totowa, NJ, USA. Schenk P.M., Kazan K., Wilson I., Anderson J.P., Richmond T., Somerville S.C. & Manners J.M. (2000) Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proceedings of the National Academy of Sciences of the USA 97, 11655–11660. Schlagnhaufer C.D., Glick R.E., Arteca R.N. & Pell E.J. (1995) Molecular cloning of an ozone-induced 1-aminocyclopropane-1carboxylate synthase cDNA and its relationship with a loss of rbcS in potato (Solanum tuberosum L.) plants. Plant Molecular Biology 28, 93–103. Schraudner M., Moeder W., Wiese C., Van Camp W., Inze D., Langebartels C. & Sandermann H. (1998) Ozone-induced oxidative burst in the ozone biomonitor plant, tobacco Bel W3. Plant Journal 16, 265–245. Schubert R., Fischer R., Hain R., Schreier P.H., Bahnweg G., Ernst D. & Sandermann H. Jr. (1997) An ozone-responsive region of the grapevine resveratrol synthase promoter differs from the basal pathogen-responsive sequence. Plant Molecular Biology 34, 417–426. Sharma Y.K., Leon J., Raskin I. & Davis K.R. (1996) Ozoneinduced responses in Arabidopsis thaliana: the role of salicylic acid in the accumulation of defense-related transcripts and induced resistance. Proceedings of the National Academy of Sciences of the USA 93, 5099–5104. Shirley B.W., Kubasek W.L., Storz G., Brugemann E., Koornneef M., Ausubel F.M. & Godman H. (1995) Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis. Plant Journal 8, 659– 671. Somssich I.E. & Hahlbrock K. (1998) Pathogen defence in plants – a paradigm of biological complexity. Trends in Plant Science 3, 86–90. Tamaoki M., Nakajima N., Kubo A., Aono M., Matsuyama T. & Saji H. (2003) Transcriptome analysis of O3-exposed Arabidopsis reveals that multiple signal pathways act mutually antagonistically to induce gene expression. Plant Molecular Biology 53, 443–456. Tang D.Z., Christiansen K.M. & Innes R.W. (2005) Regulation of plant disease resistance, stress responses, cell death, and ethylene signaling in Arabidopsis by EDR1 protein kinase. Plant Physiology 138, 1018–1026. Tena G., Asai T., Chiu W.L. & Sheen J. (2001) Plant mitogenactivated protein kinase signaling cascades. Current Opinion in Plant Biology 4, 392–400. Thimm G., Blasing O., Gibon Y., Nagel A., Meyer S., Kruger P., Selbig J., Muler L.A., Rhee S.Y. & Stitt M. (2004) MAPMAM: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant Journal 37, 914–939. Tian B., Rentz S.S., Gorman G.S., Rogers T. & Page J.G. (2004) Comparison of real time PCR assay methods in detection and quantification of β-actin in mouse tissues. Preclinica 2, 214–220. Tournier C., Thomas G., Pierre J., Jacquemin C., Pierre M. & Saunier B. (1997) Mediation by arachidonic acid metabolites of the H2O2-induced stimulation of mitogen-activated protein kinases (extracellular-signal-regulated kinase and c-Jun NH2terminal kinase). European Journal of Biochemistry 244, 587– 595. Tsuchisaka A. & Theologis A. (2004) Unique and overlaping expression patterns among the Arabidopsis


Ozone effects on Arabidopsis transcriptome 1701 1-amino-cyclopropane-1-carboxylate synthase gene family members. Plant Physiology 136, 2982–3000. Tsuji J., Jackson E.P., Gage D.A., Hammerschmidt R. & Somerville S.C. (1992) Phytoalexin accumulation in Arabidopsis thaliana during the hypersensitive reaction to Pseudomonas syringae pv syringae. Plant Physiology 98, 1304–1309. Tuomainen J., Pellinen R., Roy S., Kiiskinen M., Eloranta T., Karjalainen R. & Kangasjárvi J. (1996) Ozone affect birch (Betula pendula Roth) phenylpropanoid, polyamine and active oxygen detoxifying pathways at biochemical and gene expression levels. Journal of Plant Physiology 148, 179–188. Tuomainen J., Betz C., Kangasjarvi J., Ernst D., Yin Z.H., Langebartels C. & Sandermann H. Jr. (1997) Ozone induction of ethylene emission in tomato plants: regulation by differential transcript accumulation for the biosynthetic enzymes. Plant Journal 12, 1151–1162. Tuominen H., Overmyer K., Keinanen M., Kollist H. & Kangasjarvi J. (2004) Mutual antagonism of ethylene and jasmonic acid regulates ozone-induced spreading cell death in Arabidopsis. Plant Journal 39, 59–69. Tusher V.G., Tibshirani R. & Chu G. (2001) Significance analysis of microarray applied to the ionizing radiation response. Proceedings of the National Academy of Sciences of the USA 98, 5116–5121. Van Buuren M.L., Guidi L., Fornalè S., Ghetti F., Franceschetti M., Soldatini G. & Bagni N. (2002) Ozone-response mechanisms in tobacco: implications of polyamine metabolism. New Phytologist 156, 389–398. Ward J. (2001) Identification of novel families of membrane proteins from the model plant Arabidopsis thaliana. Bioinformatics 17, 560–563. Wohlgemuth H., Mittelstrass K., Kschieschan S., Bender J., Weigel H.J., Overmyer K., Kangasjarvi J., Sandermann H. & Langebartels C. (2002) Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone. Plant, Cell & Environment 25, 717–726. Wrzaczek M. & Hirt H. (2001) Plant MAP kinase pathways: how many and what for? Biology of the Cell 93, 81–87. Zhang S.Q. & Klessig D.F. (2001) MAPK cascade in plant defense signaling. Trends in Plant Science 6, 520–527. Zhong H.H. & McClung C.R. (1996) The circadian clock gates expression of two Arabidopsis catalase genes to distinct and opposite circadian phases. Molecular Genetics and Genomics 251, 196–203. Zhou N., Tootle T.L. & Glazebrook J. (1999) Arabidopsis PAD3, a gene required for camalexin biosynthesis, encodes a putative cytochrome P450 monoxygenase. Plant Cell 11, 2419– 2428. Zhu T. (2003) Global analysis of gene expression using GeneChip microarrays. Current Opinion in Plant Biology 6, 418– 425. Zinser C., Ernst D. & Sandermann H. (1998) Induction of stilbene synthase and cinnamyl alcohol dehydrogenase mRNAs in Scots pine (Pinus sylvestris L.) seedlings. Planta 204, 169–176. Received 8 December 2005; received in revised form 31 March 2006; accepted for publication 19 April 2006

SUPPLEMENTARY MATERIAL The following supplementary material is available for this article online: Figure S1. Effect of acute O3 treatment on gene expression in Arabidopsis. Gene-expression changes are

depicted in MapMan format version 1.8.0 (Mapping_overview_AgiCode) where each square represents a gene. Blue, red and white meant up-regulation, down-regulation and no change in expression, respectively. The complete data set used for MapMan analysis is given in Supplementary Table S1. The figures are best viewed, and all data point annotations are provided at http://gabi.rzpd.de/projects/MapMan. Panels A and B: expression of genes involved in the secondary metabolism (BIN 16) during (T3 h, A) and after (T12 h, B) O3 treatment. Figure S2. Effect of acute O3 treatment on gene expression in Arabidopsis. Gene-expression changes are depicted in MapMan format version 1.8.0 (Mapping_overview_AgiCode) where each square represents a gene. Blue, red and white meant up-regulation, down-regulation and no change in expression, respectively. The complete data set used for MapMan analysis is given in Supplementary Table S1. The figures are best viewed, and all data point annotations are provided at http:// gabi.rzpd.de/projects/MapMan. Panels A and B: expression of genes involved in cellular response (BINs 20, 21, 31 and 33) during (T3 h, A) and after (T12 h, B) O3 treatment. Figure S3. Effect of acute O3 treatment on gene expression in Arabidopsis. Gene-expression changes are depicted in MapMan format version 1.8.0 (Mapping_overview_AgiCode) where each square represents a gene. Blue, red and white meant up-regulation, down-regulation and no change in expression, respectively. The complete data set used for MapMan analysis is given in Supplementary Table S1. The figures are best viewed, and all data point annotations are provided at http:// gabi.rzpd.de/projects/MapMan. Panels A and B: modulation of transcriptional factors (BIN 27) during (T3 h, A) and after (T12 h, B) O3 treatment. Table S1. List of genes used for MapMan analysis, in which all genes showing more than 1.5-fold variation in the comparisons T0–T3 h and T0–T12 h were considered. nc, no change. Table S2. List of selected genes for SOTA analysis. Table S3. Information relative to the primers and probes used in real-time RT-PCR analysis. Table S4. List of pathways in which at least one gene was found to be up- or down-regulated more than twofold in plants after 3 h O3 treatment compared with control plants (0 h). Table S5. Lists of genes found to be up- or down-regulated more than twofold in plants after 3 h O3 treatment compared with control plants (0 h).


1702 N. Tosti et al. Table S6. List of differentially regulated RLK genes during (T3 h) and after (T12 h) the O3 treatment [with respect to control (T0 h) plants]. nc, no change.

Table S9. List of genes coding for reticuline oxidative-like proteins with relative expression levels at T3 and T12 h. nc, no change.

Table S7. List of differentially regulated MPK genes during (T3 h) and after (T12 h) O3 treatment [with respect to control (T0 h) plants]. nc, no change.

This material is available as part of the online article from http://www.blackwell-synergy.com

Table S8. List of bZIP and MYB genes differentially regulated during (T3 h) and after (T12 h) O3 treatment [with respect to control (T0 h) plants]. nc, no change.
