Identification of late O3-responsive genes in Arabidopsisthaliana by cDNA microarray analysisDavid D’Haesea,b,*, Nele Horemansa, Wim De Coena and Yves Guiseza

aDepartment of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, BelgiumbSchool of Biology and Psychology, Division of Biology, University of Newcastle, Newcastle-Upon-Tyne NE1-7RU, UK

Correspondence

*Corresponding author,

e-mail: david.dhaese@ncl.ac.uk

Received 31 January 2006

doi: 10.1111/j.1399-3054.2006.00711.x

To better understand the response of a plant to O3 stress, an integratedmicroarray analysis was performed on Arabidopsis plants exposed during 2days to purified air or 150 nl l�1 O3, 8 h day�1. Agilent Arabidopsis 2 OligoMicroarrays were used of which the reliability was confirmed by quantitativereal-time PCR of nine randomly selected genes. We confirmed the O3

responsiveness of heat shock proteins (HSPs), glutathione-S-tranferases andgenes involved in cell wall stiffening and microbial defence. Whereas, aprevious study revealed that during an early stage of the O3 stress response,gene expression was strongly dependent on jasmonic acid and ethylene, wereport that at a later stage (48 h) synthesis of jasmonic acid and ethylene wasdownregulated. In addition, we observed the simultaneous induction ofsalicylic acid synthesis and genes involved in programmed cell death andsenescence. Also typically, the later stage of the response to O3 appeared tobe the induction of the complete pathway leading to the biosynthesis ofanthocyanin diglucosides and the induction of thioredoxin-based redox con-trol. Surprisingly absent in the list of induced genes were genes involved inASC-dependent antioxidation, few of which were found to be induced after12 h of O3 exposure in another study. We discuss these and other particularresults of the microarray analysis and provide a map depicting significantlyaffected genes and their pathways highlighting their interrelationships andsubcellular localization.

Introduction

For more than six decades now, ozone (O3) has beenrecognized as an air pollutant (reviewed in Sandermannet al. 1998). Yet, the exact sequence of events followingthe diffusion of gaseous O3 into the apoplastic fluid of aplant still calls for further investigation. Because it is astrong oxidant, much effort has been done to understandthe reactions that O3 undergoes upon dissolving into theapoplast as well as the importance of antioxidants thatcould detoxify O3. Indeed, several authors have argued

in the past that low molecular antioxidants – and morein particular ascorbate (ASC) – contribute significantlyto the protection of a plant against visible injuriescaused by O3 (Chen and Gallie 2005). But except forlow molecular antioxidants, O3 was also shown toinduce the activity of antioxidative enzymes or the tran-scription of the corresponding genes (Sharma and Davis1994, Willekens et al. 1994, Conklin and Last 1995).These include catalases, glutathione (GSH) peroxidases,GSH-S-transferases (GSTs), superoxide dismutases and

Abbreviations – ABA, abscisic acid; ACC, aminocyclopropane-1-carboxylate; ASC, ascorbate; GSH, glutathione; GST, GSH-

S-transferase; HSP, heat shock protein; XTH, xyloglucan xyloglucosyl transferases.

Physiologia Plantarum 128: 70–79. 2006 Copyright � Physiologia Plantarum 2006, ISSN 0031-9317

70 Physiol. Plant. 128, 2006

ASC peroxidases. Enzymic antioxidants may scavengethe reactive oxygen species (ROS) produced as directreaction products of O3 or produced during the plant’sown biphasic NADPH-oxidase-dependent oxidativeburst (Moeder et al., 2002). The induction of antioxida-tive enzymes at the level of the transcript has beenshown to be coordinated by salicylic acid (Overmyeret al. 2000, Wang et al. 2002). In fact, the appearance ofthe typical O3 damage reported as necrotic spots spreadacross the leaf surface was shown to depend on theharmonized action of salicylic acid and ethylene(Moeder et al. 2002). However, while some authorsreport that the O3-inducible jasmonic acid is believedto attenuate the ethylene- and salicylic acid-dependentcell death (Rao et al. 2000, Kanna et al. 2003) othersclaim that jasmonic acid and ethylene work in concertand antagonistically to salicylic acid (Tamaoki et al.2003b).

Recently, advances have been made on the wayArabidopsis responds to O3 by way of full-genome orquasi-full genome expression analysis (Tamaoki et al.2003b, Miyazaki et al. 2004). In one paper, Tamaokiet al. (2003b) explored the role of jasmonic acid, sal-icylic acid and ethylene on transcriptome changesinduced by 12 h of 200 nl l�1 O3. At that stage duringthe exposure to ozone, only one third of the affectedgenes were not dependent on any of the growth regula-tors mentioned. In the second paper, Miyazaki et al.(2004) exposed Arabidopsis during 8–12 days at1.2 · ambient concentrations (60–90 nl l�1). Theycompared the expression profiles between plants inambient field conditions, plants exposed in open air toelevated O3 or CO2 and plants fumigated with O3 ingrowth chambers. Unfortunately, because of a lack ofan adequate growth chamber control condition, theyfound their results clouded by differences in growthcondition rather than exposure regimes. We decided tofocus our attention on changes in transcripts at an inter-mediate stage (48 h) of O3 exposure. At this stage, thesignalling pathways of ethylene and jasmonic acid areexpected to have completed their transient induction,which is believed to reach its maximum approximately3–6 h after the onset of the treatment (Kramell et al.2000, Reymond et al. 2000, Wang et al. 2002,Tamaoki et al. 2003a). In doing so, we hope to identifywhich O3-affected genes do not follow a similar trend asthe expression of genes related with ethylene and jas-monic acid signalling. Moreover, instead of merely sum-ming up the genes found to be induced or repressed bythe action of O3 (Miyazaki et al. 2004), considerableeffort was taken to present the results of the microarrayanalysis in a more integrated manner. A functional ana-lysis of the differentially expressed genes resulted in a

map showing the genes in the biochemical pathwaythey belong to and in the subcellular organelle theirproducts act in (Fig. 1S).

Materials and methods

Plant material, O3 treatment and RNA extraction

Seeds of Arabidopsis thaliana cv. Columbia were ster-ilized for 2 min in 70% ethanol, 15 min in 7% bleach,rinsed five times with sterile water and plated on 3025type Petri dishes (Ø 150 mm · 25 mm) filled with2.15 g l�1 Murashige and Skoog-salts, B5-vitamins,15 g l�1 sucrose and 7 g l�1 agar (pH 5.8). Plates werekept at 24�C with a day/night regime of 16 h/8 h. Threeweeks after sowing, the plants were transferred to 250-mlpots containing peat : vermiculite (1:1) with 0.77 gOsmocote� fertilizer (NPK 15:10:12) and grown at20�C/18�C (RH 60/70%) during day/night. Plants werethen placed in control chambers receiving 150 nl l�1

O3, 8 h day�1 or charcoal and Purafill� filtered air.The O3 regime applied here did not cause any macro-scopic lesions. After the exposure period, leaves origi-nating from four separate healthy plants were pooled,divided into aliquots of �0.5 g fresh weight and snapfrozen in liquid N2. Leaf samples were kept at �80�Cuntil RNA extraction.

For RNA extraction, frozen leave samples wereground using a pre-cooled mortar and pestle.Powdered tissue was added to a microcentrifuge tubecontaining Concert� Plant RNA reagent (Invitrogen,Carlsbad, CA). The extraction procedure was done

Fold induction with microarray

Fol

d in

duct

ion

with

qR

T-P

CR

0

1

2

3

0 1 2 3 4 5

Fig. 1. Correlation between the fold induction of nine randomly selected

O3-responsive transcripts as measured by either microarray analysis or by

real time–polymerase chain reaction (qRT–PCR). One transcript was

found to be differentially induced in the two experiments (arrow).

Physiol. Plant. 128, 2006 71

according to the manufacturer’s instructions and yieldedbetween 80 and 100 mg of high quality total RNA.

cDNA microarray analysis

Microarray chips (Agilent Arabidopsis 2 OligoMicroarray Kit; Agilent Technologies, Palo Alto, CA)spotted with approximately 21 500 unique 60-mers ofthe Arabidopsis thaliana genome were hybridized withsample RNA from leaves treated with filtered air or withO3. Before hybridizing the microarrays, the quality ofthe total RNA was examined for integrity and quantifiedby capillary electrophoresis on a RNA 6000 NanoLabChip� kit (Agilent Technologies) using an Agilent2100 BioAnalyser. The quality check as well as themicroarray analysis was performed by the MicroArrayFacility of the Flanders Interuniversity Institute forBiotechnology (VIB), Leuven, Belgium. The hybridiza-tion was completed as described elsewhere (Puskaset al. 2002). For this study, four hybridizations (equiva-lent of two microarray chips) were performed. Aliquotsof total RNA from control leaves and from O3-treatedleaves were hybridized once with Cy3 and once withCy5 to account for fluorophore effects.After microarray analysis, fluorescence data were cor-

rected with the ‘lowess’ (locally weighted scatter plotsmoother) normalization procedure (Yoon et al. 2003).Subsequently, the fluorescence ratio of the O3-treatedvs. control sample was taken. If the ratio was smallerthan one, it was replaced by the negative of its recipro-cal value (fold change). Genes were selected for closerinvestigation only if both the hybridizations with Cy3and Cy5 resulted in a more than two-fold repression orin a more than two-fold induction.

Quantitative real time–PCR

The expression of 10 transcripts (nine genes þ onehousekeeping gene) was measured using quantitativereal time–polymerase chain reaction (qRT–PCR) with aLightcycler� 1.5 instrument (Roche Applied Science,Mannheim, Germany). From a separate set of plants asfor the microarray experiment, total RNA was extractedand reverse transcribed to first strand cDNA using theSuperscript III Reverse Transcriptase kit (Invitrogen).Primers for the selected genes were predicted using theLightCycler� Probe Design Software (Roche AppliedScience), setting melting temperature Tm and the GCpercentage to 60�C and 50%, respectively. The secondstep of the qRT–PCR was performed using theLightCycler� FastStart DNA MasterPLUS SYBR Green Ikit (Roche Applied Science), according to the manufac-turer’s instructions. Quantification was performed as

described elsewhere (Pfaffl 2001). Amplification ofevery gene resulted in a single product with correctmass and melting temperature.

Results and discussion

We confirmed the reliability of the microarray results byway of analysing the expression level of nine genes plusone housekeeping gene using qRT–PCR. Only one of thenine target genes varied between microarray andqRT–PCR analysis (Fig. 1). Therefore, especially if groupsof related genes are considered collectively, the micro-array analysis enabled us to draw valid conclusions.

Functional analysis

A total of 582 O3-responsive transcripts were individu-ally examined in silico for function, structure and cel-lular localization. Of the differentially regulated genes,276 (47%) had a predicted or experimentally testedfunction, of which 118 (43%) were induced and 158(57%) were repressed. Interestingly, not only the expo-sure time but also the ratio of repressed vs. total affectedgenes is intermediate between the ratios revealed byTamaoki et al. (2003b) and Miyazaki et al. (2004)where Arabidopsis was exposed to O3 for 12 h (23%repressed) and 12 days (91%), respectively. This indi-cates that genes induced by O3 in an early stage areactively repressed in a later stage.

We created amap showing the annotatedO3-responsivegenes and their intergene relationships. This map waspasted as supplementary information. The proteinsshown on the map were sorted by function and weresuperimposed on the subcellular compartments accord-ing to the localization of their corresponding pathway orsubstrate(s). Apart from pasting O3-responsive genes onthe map, they were also assigned to functional classes(Table 1S; supplementary information). It should benoted, however, that the classification is very arbitrarysince many genes can be assigned to multiple classes.The main function here is to allow easy reference to thecorrect entry of the table. From hereon, alphanumericalreferences will be added to genes, proteins or entirepathways. These references refer to coordinates of themap and to the functional class in Table 1S. Forinstance, (4.d – C4) means that the corresponding genewas assigned to the subclass 4.d in Table 1S and can befound at coordinates C4 on the map.

Secondary metabolism

Eleven transcripts coding for subsequent steps in flavo-noid synthesis (3.b – D4/D5) were induced by the effect

72 Physiol. Plant. 128, 2006

of the applied O3 exposure. Three of these transcripts wereinducedmore than five-fold: naringenin chalcone synthase(8.2-fold), dihydroflavonol reductase (16.1-fold) andleucoanthocyanidin dioxygenase (anthocyanidin synthase;9.4-fold). The end-products of the upregulated pathwayseem to be anthocyanin diglucosides. In addition, O3

induced MYB75 (3.b – E4), a transcription factor knownto promote the transcription of chalcone reductase anddihydroflavonol reductase (Borevitz et al. 2000, Hiratsuet al. 2003). A red shade seen on the treated Arabidopsisleaves (data not shown) supports the upregulation of theanthocyanin pathway by O3 in vivo and shows thatchanges on the transcript level represent changes in corre-sponding protein levels and enzyme activities.

In the pathway starting from chorismate, three reac-tions seem upregulated by O3 (3.a – B2-C3). Two ofthese reactions lead to an increased production of anti-microbial compounds and will be discussed below. Athird reaction, the conversion of chorismate to isochor-ismate by isochorismate synthase, initiates the alterna-tive pathway for salicylic acid synthesis (Wildermuthet al. 2001). It is tempting to see this alternative routefor salicylic acid synthesis as a ‘stress-dependent’ route.In any case, this result indicates that O3 stress causes thesynthesis of salicylic acid. Exactly oppositely, the largestcluster of O3-responsive genes observed by Tamaokiet al. (2003b) in their microarray analysis containedgenes induced by ethylene and jasmonic acid, butrepressed by salicylic acid, confirming that salicylicacid acts antagonistically to ethylene and jasmonicacid in defence gene induction, even after their initialtransient expression phase (Tamaoki et al. 2003b).

Cell wall lignification and antimicrobial genes

A considerable fraction of the differentially regulatedgenes was associated with the cell wall (7.6%). Thiswas in accordance to an earlier report where the sameproportion (7%) was found in a study conducted onArabidopsis treated with pathogens and plant growthregulators (Schenk et al. 2000). Among the O3-responsivetranscripts, four were involved in lignin biosynthesis.Three cinnamyl alcohol dehydrogenases (1 – C1) catalysingthe reduction of synapylaldehyde, p-coumaraldehyde,coniferaldehyde and the dirigent protein (1 – B1) – cata-lysing the final step in the lignification – were induced. Inaddition, O3 also affected genes involved in callosedeposition and hydrolysis (PR2; 2.a – C1), cellulosesynthesis (2.b – D1) and pectin esterification (2.c – D1).Former work showed that the so-called stress-lignins canbe induced by O3 and by fungal elicitation but was alsofound in compressionwood and during early plant devel-opment (Schenk et al. 2000, Cheong et al. 2002, Cabane

et al. 2004). This study reveals that simultaneously withthe induction of cell wall stiffening, O3 also causes therepression of four xyloglucan xyloglucosyl transferases(XTHs) and expansin (2.a – C1), all involved in cell elon-gation. To our knowledge, this is the first time that O3 isshown to actively suppress cell elongation.

Interestingly, O3 also seems to affect antimicrobialgenes. For instance, O3 induces a monooxygenase(5.a – D1) involved in the production of the phytoalexincamalexin. Other O3-responsive antimicrobial genescoding for anthranilate N-benzoyl-transferase (3.a – B2),tropinone reductase (5.a – F2), reticulin oxidases (ber-berine bridge enzymes; 5.a –D4) and alcoholO-cinnamoyltransferase (3.a – C3) are responsible for the productionof dianthramides, tropane alkaloids, berberine-likealkaloids and alkyl cinnamates, respectively. A similarinduction of tropinone reductase was observed byTamaoki et al. (2003b), although it concerns anothertranscript. The polyamine putrescine is the precursorfor tropane alkaloids and may also be increased throughthe action of the enzyme ornithine aminotransferase(5.b – E1/F1). In addition to the production of antimi-crobial compounds, four chitinase genes (5.a – E1) andthree b-1,3-glucanases (2.a – C1) were upregulated.One of the chitinase transcripts (AT2G43570) and oneof the glucanases (AT3G57260) was previously alsofound to be induced by a 12-h O3 exposure (Tamaokiet al. 2003b). In contrast, the polygalacturonase-inhibit-ing protein 2 (PGIP2; 5.a – D1/E1) was downregulated.

During a pathogen attack, chitinase- and glucanase-like genes were shown to be induced (Cheong et al.2002). This makes more sense than the induction ofthese genes by O3. Indeed, genes involved in cell walllignification and antimicrobial activity are more likelyintended for pathogen exclusion and poisoning. It can-not be ruled out that the activity of the correspondinggene products contribute to O3 tolerance. For instance,the deposition of lignin might promote the scavengingof O3 molecules or its secondary products in the cellwall. However, it is more probable that the induction ofantimicrobial genes is an undirected response to oxida-tive stress, because the same genes were also inducedby wounding (Cheong et al. 2002).

Five genes containing a Toll interleukin resistancedomain (TIR; 9.a – E2) were strongly repressed by O3.These are believed to be involved in the plant recogni-tion of fungi, bacteria, viruses, nematodes and insects,and subsequent signal transduction (Meyers et al. 2003).

Antioxidation and detoxification

Remarkably, only few O3-affected transcripts wereinvolved in the antioxidation. For instance, a 2-day

Physiol. Plant. 128, 2006 73

exposure to O3 did not affect the expression levels ofcatalases, ASC peroxidases, GSH reductases, superox-ide dismutases or dehydro ASC reductases known to beinvolved in pathogenesis (Schenk et al. 2000).Surprisingly, genes related with ASC antioxidationwere not significantly induced, even though manyhave been annotated in the Arabidopsis genome (fourASC oxidases, eight ASC peroxidases, six dehydro-ASCreductases, six monodehydro-ASC reductases, L-galac-tono-1,4-lactone dehydrogenase, ASC biosynthesis geneVTC2). This is remarkable since ASC has been put for-ward as an important determinant for O3 sensitivity(Chen and Gallie 2005). One gene, the monodehydro-ASC reductase (6.d – C2), was even repressed by afactor 2.4. Similarly, Tamaoki et al. (2003b) alsoobserved the downregulation of two Cu/Zn-superoxidedismutases in Arabidopsis after 12 h of 200 nl l�1 O3.However, these authors also observed the ethylene- andjasmonic acid-dependent upregulation of transcriptscoding for a monodehydro-ASC reductase, an ASC per-oxidase and a GDP-mannose pyrophosphorylase(involved in ASC biosynthesis), indicating that in anearlier stage of O3 stress, ASC-dependent antioxidationmight be more important.The second most abundant antioxidant is GSH (May

et al. 1998). The synthesis of the amino acids that makeup the tripeptide GSH (Glu, Cys and Gly) were inducedby O3 (Glu, Cys: 4 – B4-D4; Gly through glycolateoxidase: 6.d – F2). Transcripts of the GSH biosynthesisenzymes, g-glutamylcysteine synthase (gsh1) and GSHsynthase (gsh2), were not responsive to O3, but thesehave been proposed to be regulated post-transciptionally(Foyer and Rennenberg 2000). The GSH-dependentdetoxification pathways, on the other hand, wereclearly induced by the O3 exposure. More specifically,five out of six GSH-S-transferases (GSTs) (6.b – F2/F3)were upregulated by O3 (one of which more then10-fold), confirming previous results (Sharma and Davis1994, Tamaoki et al. 2003b). Accordingly, the degrada-tion of GSH as well as its polymerization into phytoche-latins was repressed as indicated by changes in theexpression of phytochelatin synthase and l-glutamyltranspeptidase (6.d – F2). The above findings thereforesuggest that, at this stage during the administration of O3,GSH rather than ASC may be involved in redox-control.The idea of O3-induced repair of disulphide bridges is

reinforced by two other observations. Firstly, someGSH-independent reductants were shown to be inducedby O3 as well. More in detail, two out of three tran-scripts coding for thioredoxins were upregulated.Thioredoxins also reduce protein disulphide bonds andhave been proposed to serve as back-up reduction sys-tem at times when GSH and/or glutaredoxin (6.b – F2)

are oxidized (Trotter and Grant 2003). Indeed, a tran-script coding for glutaredoxin was repressed more thanfive times, while in an earlier stage of O3 fumigation,glutaredoxin was oppositely found to be induced(Tamaoki et al. 2003b). Secondly, we found 11 O3-induced transcripts that were involved in correct proteinfolding or protein repair. Among these induced tran-scripts, four could be identified as heat shock proteins(HSPs; 6.e – F3/F4). This is in agreement with the resultsof Tamaoki et al. (2003b). Three of the induced genesinvolved in protein folding were also found to beresponsive to 12 h of O3 in the latter study(AT5G28540, AT5G42020 and AT4G24190).

Signal transduction

Ethylene emission was previously implicated in the for-mation of lesions during O3 attack (Overmyer et al.2000, Tamaoki et al. 2003a). Since the induction ofethylene synthesis is known to be only transient peakingafter 3 h of oxidative stress (Wang et al. 2002, Tamaokiet al. 2003a), we hypothesized that the relevant geneswould not be significantly induced after 2 days of O3

exposure. Indeed, a tyrosine transaminase with amino-cyclopropane-1-carboxylate (ACC) synthase activitywas repressed. In addition, four out of five transcriptionfactors annotated as ethylene-responsive element-binding proteins (ERFs; 12.c – E5) and AP2 domaincontaining transcription factors involved in ethyleneresponse (Riechmann and Meyerowitz 1998) weredownregulated.

Jasmonic acid and its derivatives – commonly knownas jasmonates – are lipid-derived cyclopentanoneswhich are signals in plant defence and in distinct devel-opmental stages (Creelman and Mullet 1997). Throughtranscriptional activation, jasmonates are able to inducethe formation of toxic aldehydes that serve to protectagainst invading microorganisms (Hamberg et al. 2003).In respect to O3 stress, jasmonic acid was previouslyshown to attenuate salicylic acid-dependent cell death(Overmyer et al. 2000, Tamaoki et al. 2003b). Here, fiveout of six transcripts directly involved in the jasmonicacid synthesis (9.c – D3/D4) were repressed by O3,three of which more than five-fold. Moreover, genesknown to be strongly dependent on jasmonic acid, likea strictosidine synthase (3.a – B3) and a flavonolsynthase (3.b – D5; Menke et al. 1999), were alsorepressed by O3. As for ethylene, many studies showthat in response to an environmental or developmentalstress, jasmonic acid metabolism is generally inducedrather than repressed but again only transiently (Kramellet al. 2000, Reymond et al. 2000). In agreement withprevious studies (Wang et al. 2002, Tamaoki et al.

74 Physiol. Plant. 128, 2006

2003b), we therefore found that ethylene and jasmonicacid pathways are affected by O3 in a concert mannerand not antagonistically as proposed by others (Rao et al.2000, Kanna et al. 2003). Moreover, an O3-dependentrepression of a NADPH oxidase (9.c – E2) indicates that,in general, genes involved in early oxidative burst aredownregulated 2 days after the onset of the fumigation.

Cell death and senescence

Cysteine proteases have been indicated as mediators ofpathogen-induced cell death (Solomon et al. 1999).Here, two cysteine proteases (9.f – D4) were induced,2.9 and 4.6-fold, respectively, whereas a cysteine pro-tease inhibitor was repressed. In accordance, a copine-like protein (9.f – D2/D1) known to repress cell-deathwas downregulated by O3. These findings therefore canbe indicative for O3-induced cell death, similar to thepathogen-induced response. Two transcripts coding forglutamyl-tRNA reductase (HEMA1-2; 10 – D2/D3) andone transcript coding for the ferrochelatase (10 – D2) arerepressed by O3, whereas ferritin (fer1; 10 – D2) isinduced. This indicates a controlled downregulationof chlorophyll synthesis. Moreover, other senescence-associated genes (SAG) were found to be induced:SAG13 coding for the tropinone reductase (5.a – F2) andSAG29 coding for an ER-targeted ion channel (9.d – E3/F3).

Transporters

Among the affected transporters (8.b – C2-D2), manywere involved in the translocation of sugars and aminoacids. The treatment with O3 induced a glucose-6-phosphate transporter, while it repressed a sucrose/Hþ

symporter and a mannitol transporter. Transporter oligo-peptides were differentially affected by O3. These findingsindicate that O3 alters the redistribution of sugars withincells or across organs. In addition, two metal transporters,ZIP4 and ZIP9 (8.c – B2/C2), were induced while a Ca2þ–Naþ-antiporter (9.d – E2) and syntaxin involved in vesicletrafficking (8.d – D1) were repressed by O3.

Lipid breakdown

Six enzymes seem to work in an orchestrated way tobreak down phospholipids and triglycerides (11.c – D2/E3): acylglycerolphosphate acyltransferase, phosphati-date phosphatase, lipase class 3, lysophospholipase,glycerophosphodiester phosphodiesterase and cholinekinase. We found that all enzymes were repressed bythe action of O3, except for a copy of the glyceropho-sphodiester phosphodiesterase. It is not clear, however,whether the activated copy of this latter enzyme does

have preferential specificity for glycero-3-phosphocho-line, because it has been shown to have specificity forother glycerophosphodiesters (van der Rest et al. 2002).In addition, an ER-predicted protein involved in mem-brane degradation (11.c – E4) and two transcripts oflipid transporters (6.e – E3) were induced. This indicatesthat O3 not only induces lipid breakdown but moregenerally lipid turnover. One explanation may be thatwhile the de novo synthesis of lipids is repressed in thechloroplasts, the production of fatty acids from ER-localized triglycerides and its transport to the chloroplastis promoted. A shift of chloroplastic to non-chloroplasticmembrane lipids has indeed been shown previously tooccur under O3 stress (Carlsson et al. 1996). In thechloroplast, newly imported fatty acids could serve assubstrates for the a-dioxygenase (9.c – F3/F4) dependentsignal transduction pathway (Hamberg et al. 2003), alsofound induced by O3 in this study.

Growth regulation

Auxin has previously been implicated in many cellulardevelopmental processes (Normanly and Bartel 1999)and was also shown to be actively repressed duringH2O2-dependent signal transduction (Kovtun et al.2000). While the NIT2 gene – a nitrilase which has beenimplicated in auxin synthesis (Park et al. 2003) – wasinduced by O3, three transcripts coding for auxin-aminoacid conjugate hydrolase were repressed (12.a – E3/E4).These results indicate that auxin recovery from theconjugated pool as observed after 12 h of O3 (Tamaokiet al. 2003b) in a later phase switches to its de novosynthesis. Because of the apparent repression of the Trp-dependent auxin synthesis pathway (3.a – D3), it maybe that the Trp-independent pathway is favoured(Normanly and Bartel 1999). We also report a positiveeffect of O3 on an amino acid/auxin transporter (12.a – F2)as well as a repression of two chloroplastic purine per-meases (PUPs; 8.g – D1/2), known to transportcytokinins.

The repression of transcripts of the gibberellin cata-bolizing enzyme gibberellin-2-oxidase and of two inhi-bitors of gibberelin-20-oxidase (12.b – A2) suggests thepromotion of gibberellin-synthesis. Gibberellins act asregulators of plant developmental processes such asstem elongation, fruit and seed development, seed ger-mination and dormancy. However, to our knowledge,this is the first time that gibberellin metabolism has beenshown to respond to O3 stress. Interestingly, theseresults indicate that O3 stress might stimulate flowering.

In accordance to the well-characterized antagonisticrelation between gibberellins and abscisic acid (ABA),genes involved in ABA biosynthesis were repressed by

Physiol. Plant. 128, 2006 75

O3 as were two basic helix-loop-helix (bHLH) proteins,one of which (Jin1) is known to activate ABA-responsivegenes (12.d – F4) (Abe et al. 2003). In addition, thehomeobox leucine zipper transcription factors, ATHB-7 and the paralogous ATHB-12 (14.e – E5), wereinduced by O3. Both genes bare 80% similarity andare known to be responsive to water deficit conditionsand to be under control of ABA (Olsson et al. 2004).They operate as ABA-dependent regulators of inflores-cence stem elongation and branching in conditions ofwater-deficit.

Other O3-responsive genes

Polyamines have previously also been shown to preventO3-dependent lesion formation in a concentration-dependent manner (Bors et al. 1989). The possibleantioxidative capacity of polyamines conjugated tohydroxycinnamic acids and their capacity to inhibitlipid peroxidation have originally been put forward asputative defence mechanisms against O3. Polyaminesmay also affect transcription and modulate proteasespost-transciptionally so as to counteract senescence(Kasukabe et al. 2004). Our results show that polyaminesynthesis may be promoted under O3 conditions, sinceornithine aminotransferase (5.b – E1/F1) was inducedand polyamine oxidase, a putrescine degradationenzyme, was inhibited (5.b – E1/F1). A similar upregula-tion of polyamine synthesis was found in several otherstudies (Kasukabe et al. 2004 and references therein).Two transcripts coding for a- and b-amylase (11.a –

E3/E2), respectively, were induced about three-fold byO3 exposure compared with control plants. Takentogether with the inhibition of chlorophyll synthesisgenes described earlier, this might be indicative forfavouring respiration processes over photosynthesis.However, it should be mentioned that the Atb-AMYgene is believed to be targeted to the cytosol(Chandler et al. 2001 and references therein). Sincestarch is located in the chloroplast, the physiologicalrole of this enzyme is still unclear. To our knowledge,this is the first time a- and b-amylase are identified asO3-responsive genes. Amylase genes were previouslyshown to be under transcriptional regulation of gibber-ellin (Willmott et al. 1998), which we found also to beinduced by O3. It would therefore be interesting toexamine the role of gibberellins in stress-dependentinduction of starch catabolism.

Conclusion

To discover genes responsive to O3 in a stage of expo-sure between that of two previously published papers

(12 h in Tamaoki et al. 2003b and 8–12 days inMiyazaki et al. 2004), we have conducted a genome-wide microarray analysis on Arabidopsis plants treatedwith near-ambient O3 concentrations during 48 h. Athorough examination of each individual O3-responsivegene produced a more complete picture of O3-dependenttranscriptome changes. Considerable effort was made toarrange all differentially expressed genes on a singlemap indicating the genes’ subcellular localizations aswell as their functional links with other differentiallyexpressed genes. Table 1 summarizes the processesthat were significantly influenced by the 2-day O3

treatment.Among the responsive genes found, many have been

previously reported to be affected by O3. For instance,genes related to polyamine synthesis were upregulatedby O3, as reported already many years ago (Bors et al.1989). Similarly, b-1,3-glucanases, endochitinases,GSTs and enzymes involved in antimicrobial defence(tropinone reductase, anthranylate-N-benzoyl transfer-ase) and in programmed cell death and senescence(salicylic acid synthesis, cyteine proteases, SAGs) werepreviously reported to respond to O3. In addition, webelieve to be the first to report the unambiguous induc-tion of the complete pathway for anthocyanin digluco-sides and the induction of a-dioxygenase-dependentsignalling transduction pathway, gibberellin synthesisand amylase activity as well as the repression of XTHsand expansin.

Comparing our results (after 48 h 150 nl l�1 O3) withthe work of Tamaoki et al. (2003b; 12 h of 200 nl l�1

O3), we revealed that with longer exposure periods theratio of repressed vs. significantly affected genes drama-tically increases, indicative for an active repressionmechanism following an initial induction phase. Inaddition, we confirmed the antagonistic relation

Table 1. Summary of processes in Arabidopsis significantly up or

downregulated by a 2-day O3-exposure

Upregulated Downregulated

Flavonoid (anthocyanin) synthesis Synthesis of alkyl cinnamates

Cell wall stiffening Ethylene synthesis and signalling

Antimicrobial activity Jasmonate synthesis

GSH-dependent detoxification Lipid breakdown

Synthesis of thioredoxin

Protein folding by heat shock

proteins

a-Dioxygenase dependent

signalling

Salicylic acid, auxin and

gibberellin synthesis

Starch hydrolysis

76 Physiol. Plant. 128, 2006

between salicylic acid and the growth regulators jasmo-nic acid and ethylene, but here, exactly oppositely, thesynthesis of jasmonic acid and ethylene was repressedwhile salicylic acid appeared to be induced by O3. Inaccordance with the role of salicylic acid, severalchanges in transcript expression could be related withprogrammed cell death including the induction ofcysteine proteases, SAGs and the repression of chloro-phyll synthesis. Synchronized with the apparent induc-tion of programmed cell death, we observed therepression of early response processes such as pathogenrecognition (TIR-domain containing genes) and oxida-tive burst (NADPH oxidase) as well as the induction ofthe recently identified a-dioxygenase-dependent signaltransduction pathway.

Among other genes reported by Tamaoki and collea-gues to be upregulated after 12 h of O3 but downregu-lated in this study are genes coding for ASC-dependentantioxidation. This is in contradiction with reports inwhich ASC has been identified as the detoxifier of O3

(Chen and Gallie 2005 and references therein) and withthe role of salicylic acid to induce antioxidativeenzymes (Tamaoki et al. 2003a). Possibly, in the laterstages of stress response, other molecules like the antho-cyanins take over the role of principle antioxidants. Amore in-depth investigation of the ASC-related genes inArabidopsis undergoing a similar O3 treatment couldresolve the apparent inconsistency.

But not all genes were differentially regulated after 12or 48 h of O3. The processes governing cell wall stiffen-ing, antimicrobial activity, protein folding and repairand GSH-dependent detoxification of xenobioticsseemed to be promoted in this study as well as in thestudy conducted by Tamaoki et al. (2003b). It is notclear, however, if cell wall stiffening (or the repressionof cell wall loosening by XTHs) and antimicrobialactively take part in the protection of plants againstO3, but it would make an interesting line of research.Uninterrupted induction of HSPs and GSTs, on the otherhand, can easily be explained as a response to theO3-dependent oxidation of disulphide bridges or lowmolecular cell constituents. However, whereas someof the induced transcripts coding for HSP were thesame in our study and the study of Tamaoki and collea-gues, the transcript coding for GSTs were all different. Itremains to be elucidated what the differential function isof the GSTs and whether they are associated with spe-cific stages during abiotic stress response.

Acknowledgements – The authors thank Prof Dr Paul van

Hummelen (MicroArray Facility of the Flanders

Interuniversity Institute for Biotechnology) for performing

the microarray hybridizations.

Supplementary material

The following materials are available from http://www.blackwell-synergy.com

Fig. 1S. Map of transcriptome changes in Arabidopsisthaliana induced by a 2-day O3 exposure.

Table 1S. List of genes affected by the action of a2-day O3 exposure. All genes are indicated with theirgene locus, RefSeq accession number and mean foldchange (fold) in a colour coded manner (red, induced;green, repressed). Primary functional categories and sub-categories were numbered for easier reference. Whenappropriate, EC numbers were added. Coordinates(coor.) refer to the figure ‘Map of transcriptome changesin Arabidopsis thaliana induced by a 2-day O3 exposure’.

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