Research Paper

Abstract: Suppression subtractive hybridization (SSH) was per-formed to isolate cDNAs representing genes that are differen-tially expressed in leaves of Fagus sylvatica upon ozone expo-sure. 1248 expressed sequence tags (ESTs) were obtained from2 subtractive libraries containing early and late ozone-respon-sive genes. Sequences of 1139 clones (91%) matched the EBI/NCBI database entries. For 578 clones, no putative functioncould be assigned. Most abundant transcripts were O-methyl-transferases, representing 7% of all sequenced clones. ESTs wereorganized into 12 functional categories according to the MIPSdatabase. Among them, 12% (early)/15% (late) were associatedwith disease and defence, 19/11% with cell structure, 4/10% withsignal transduction, and 9/6% with transcription. The expressionpattern of selected ESTs (ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit [rbcS], WRKY-type transcription factor, ul-traviolet-B-repressible protein, aquaporine, glutathione S-transfer-ase, catalase, caffeic acid O-methyltransferase, and pathogenesis-related protein 1 [PR1]) was analysed by quantitative real-timeRT-PCR (qRT-PCR) which confirmed changed transcript levelsupon ozone treatment of European beech saplings. The ESTscharacterized will contribute to a better understanding of foresttree genomics and also to a comparison of ozone-responsivegenes in woody and herbaceous plants.

Key words: Fagus sylvatica, cDNA, ozone, suppression subtrac-tive hybridization.

Abbreviations:EST: expressed sequence tagqRT-PCR: quantitative real-time RT-PCRRT: reverse transcriptionSSH: suppression subtractive hybridization

Introduction

As a result of anthropogenic activities, concentrations of tropo-spheric ozone have increased during recent decades. A criticallevel for ozone injury in plants has been established (AOT40)

and is given in an accumulated ozone dose over a threshold of40 nl/l (Kärenlampi and Skärby, 1996). However, these AOT40values are often exceeded in Central Europe and North Ameri-ca (Kley et al., 1999). Ozone enters the leaf through stomataand is converted into reactive oxygen species, thus triggeringan oxidative burst (Schraudner et al., 1997; Pellinen et al.,1999; Rao and Davis, 1999). This provokes plant reactions thatare known to occur during the hypersensitive response in anincompatible plant-pathogen interaction. More generally,ozone has been recognized as an abiotic elicitor, resulting inthe activation of defence-related processes at the transcrip-tional level (Kangasjärvi et al., 1994; Pell et al., 1997; Sharmaand Davis, 1997; Sandermann et al., 1998).

The impact of ozone on trees was studied in detail at the mor-phological and physiological level (Sandermann et al., 1997,and articles therein; review Matyssek and Sandermann,2003). However, at the transcriptional level, much less isknown in long-lived woody plants compared to herbaceousplants (Langebartels et al., 2002). In trees, 14 ozone-inducedgenes from Norway spruce, Scots pine, hybrid poplar, birch,and European beech have been described (Langebartels et al.,2002). Ozone exposure of 3-year-old European beech saplingsover a period of 7 weeks (0.06 to 0.150 μl/l, 10 h/d) resulted inan accumulation of extensin transcripts in 5-month-old leaves(Schneiderbauer et al., 1995). Recently it was shown that expo-sure of 4-year-old European beech saplings to 0.2 μl/l ozoneover a period of 46 d resulted in an increased transcript levelfor ACC synthase and ACC oxidase, whereas ribulose bisphos-phate carboxylase small subunit was not altered (Nunn et al.,2005). Knowledge about the molecular mechanisms at the lev-el of gene expression is important to understand an environ-mental impact, e.g., ozone, on forest trees (Matyssek et al.,2004; Sandermann and Matyssek, 2004). To obtain a compre-hensive view of an interaction between ozone and gene ex-pression in deciduous trees, it is important to identify genesthat are responsive towards this air pollutant. In addition, thiswill also contribute to a better comparison of environmentalimpacts on woody and herbaceous plants. There are manyways to identify differentially expressed genes. Suppressionsubtractive hybridization (SSH; Diatchenko et al., 1996) hasbeen successfully applied in plants to isolate ozone-, UV-B-,and metal ion-induced expressed sequence tags (ESTs) in her-baceous (Sävenstrand et al., 2000; Heidenreich et al., 2001;Sävenstrand et al., 2002; Mahalingam et al., 2003; Sahr et al.,2005) and pathogen- and safener-inducible cDNAs in trees

Transcriptome Analysis of Ozone-Responsive Genes in Leavesof European Beech (Fagus sylvatica L.)

M. Olbrich1,*, G. Betz1,*, E. Gerstner1, C. Langebartels1, H. Sandermann1, and D. Ernst1

1 GSF – National Research Center for Environment and Health, Institute of Biochemical Plant Pathology, Ingolstädter Landstraße 1,85764 Neuherberg, Germany

* Both authors contributed equally to this work

Received: April 28, 2005; Accepted: October 25, 2005

Plant Biol. 7 (2005): 670– 676© Georg Thieme Verlag KG Stuttgart · New YorkDOI 10.1055/s-2005-873001ISSN 1435-8603

670

(Asiegbu et al., 2004; Rishi et al., 2004). In the present study,we report the use of SSH to isolate two subtractive cDNA li-braries from ozone-treated leaves of 3-year-old saplings ofFagus sylvatica, the most important deciduous tree in CentralEurope (Schütt et al., 1992). The isolated ESTs were grouped in-to functional categories and an ozone-induced accumulationof selected transcripts was shown by qRT-PCR.

Materials and Methods

Accession numbers

The sequences reported here have been deposited in theEMBL nucleotide database (http://www.ebi.ac.uk/embl/index.html) under the accession numbers AJ972495 –AJ972503, AM051087 – AM051089, AM062766 –AM063038,and AM072430– AM072444.

Plants and growth conditions

Material for SSH

Three-year-old European beech saplings (Schlegel Baumschu-len, Riedlingen, Germany) were planted in 14-l pots that hadbeen filled with natural forest soil (site Höglwald, Bavaria, Ger-many; Grams et al., 2002). Saplings were kept in a climate-controlled greenhouse, from formation of buds until fullydeveloped leaves, under natural daylight, a relative humidityof 70 ± 10% and a temperature of 25 ± 2 �C during daytime(6 a.m. – 8 p.m.) and 20 ± 2 �C at night (8 p.m. – 6 a.m.) (experi-ment 1). For a period of 30 d, nine saplings were treated withozone (0.3 μl/l; 8 h/d) and another nine saplings were kept inpollutant-free air. A single leaf from controls and ozone-treat-ed saplings was harvested at 4 h, 8 h, 24 h, 32 h, 2 d, 3 d, 4 d, 5 d,7 d, 9 d, 12 d, 15 d, 22 d, 26 d, and 30 d after onset of exposure,frozen in liquid nitrogen, and stored at – 80 �C until used forRNA isolation.

Material for qRT-PCR

Four-year-old European beech saplings were planted as de-scribed above and cultivated for 69 d at 22 �C (6 a.m. –8 p.m.)and at 17 �C (8 p.m. – 6 a.m.) under natural daylight and a rela-tive humidity of 80% (experiment 2). Nine saplings were keptin clean air and another 9 were exposed to ozone for 8 h/d(0.15 μl/l). After 13 d, the ozone concentration was increasedto 0.19 μl/l. After 16 d, a single leaf from each sapling was har-vested and the material from 3 saplings was combined, frozenin liquid nitrogen, and stored at – 80 �C. A second harvest wascarried out after 69 d. This finally resulted in 6 control and 6ozone-exposed samples.

RNA isolation

Total RNA was isolated from leaves according to Kiefer et al.(2000). Poly(A)+ was isolated from total RNA using the Oligo-tex® mRNA kit (Quiagen, Hilden, Germany). The RNA yield andquality was determined by spectral photometry at 260 and280 nm. For the cDNA libraries, the identical amount of groundtissue from time points 4 h to 5 d and from 7 d to 30 d werepooled.

Suppression subtractive hybridisation (SSH)

Two μg of poly(A)+ was reverse-transcribed using the super-script™ II reverse transcriptase according to the manufacturer’sinstructions (Invitrogen, Karlsruhe, Germany). SSH was car-ried out with the PCR Select cDNA subtraction kit (Clontech,Heidelberg, Germany) (Heidenreich et al., 2001; Sahr et al.,2005). PCR fragments obtained by the SSH method werecloned into a pGEM-T vector (Promega, Mannheim, Germany)and transferred into E. coli JM109 (Promega) to create a sub-tractive library. Bacterial clones were stored at – 80 �C as stocksfor PCR amplification of the cDNA inserts. Two different sub-tractive cDNA libraries were constructed. Control and ozone-treated saplings – 4 h to 5 d (SSH1) and 7 d to 30 d (SSH2) –were used alternately as driver and tester, in order to obtainozone up- and down-regulated clones.

DNA sequence analysis

The cDNA library was kept in microtitre plates at – 80 �C.Clones were amplified using M13 primers. After the first am-plification, PCR products were labelled with BigDye® termi-nator mix (Applera Corporation, Darmstadt, Germany) in asecond PCR and sequencing was carried out with a 3730 DNAanalyzer (Applied Biosystems, Darmstadt, Germany). For data-base analysis, vector cDNA sequences were compared withGenBank database sequences using BlastN (http://www.ncbi.nlm.nih.gov/).

Microarray production, hybridization, and data analysis

Isolated cDNAs clones were amplified using flanking vectorDNA sequences. PCR products were purified and finally sus-pended in 20 μl spotting buffer (3 × SSC, 1.5 M betaine). Thesesolutions were arrayed from 96-well microtiter plates ontoaldehyde-coated microscope slides (Genetix, Hampshire, UK)using a Microgrid II spotter (Biorobotics, Cambridge, UK) andeach cDNA clone was spotted twice. After an incubation timeof 2 d at room temperature, unbound material was removedand residual free aldehyde groups were blocked (Huang et al.,2002).

RNA was isolated from control and ozone-treated plants thatwere also used for the construction of the SSH2 library. Thirtymicrograms of total RNA (of 2 controls and 2 treated samples)were separately reverse-transcribed with Superscript II (Invi-trogen) at 42 �C overnight and then incubated for 3 h in thepresence of Cy-3 and Cy-5 (Amersham Biosciences, Freiburg,Germany). cDNAs were purified using a Qiaquick PCR purifi-cation kit (Qiagen, Hilden, Germany). For each experiment,a dye-swap of Cy-3 and Cy-5 was performed (Huang et al.,2002; Plessl et al., 2005).

Pre-hybridization of slides and hybridization with labelledcDNA probes of slides were carried out as described by Huanget al. (2002). Labelled probes were dissolved in 60 μl hybridi-zation buffer and hybridized overnight under a 24 × 50 mm2

glass cover slip in hybridization chambers (VersArray™, Biorad,München, Germany). The subsequent washing steps at 42 �Cwere as follows: three times with 1 × SSC, 0.1% SDS for 2 mineach, twice with 0.1 × SSC, 0.1% SDS for 1 min each, and finallytwice with ddH2O for 2 min each. The slides were then driedunder a stream of N2 and stored in the dark.

Ozone-Induced Genes in Beech Plant Biology 7 (2005) 671

The arrays were scanned using a GenePix4000A scanner (AxonInstruments, Union City, USA). Primary data were processed,normalized, and analyzed using the GenePixPro6.0 software(GenePix).

Quantitative real-time RT-PCR (qRT-PCR)

For qRT-PCR, 10 μg of total RNA and random hexaprimer (50 –250 ng; Amersham Biosciences, Freiburg, Germany) were usedfor first strand cDNA synthesis. Reverse transcription was at42 �C using superscript III reverse transcriptase according tothe manufacturer’s instructions (Invitrogen). RT-PCR was per-formed with a 7500 real-time PCR system (Applied Biosys-tems). Each reaction consisted of 12.5 μl Absolute™ SYBR®

Green Rox Mix (ABgene, Surrey, UK), 0.5 μl of specific primers,and 10.5 μl of diluted cDNA. The PCR conditions were as fol-lows: 1 cycle at 50 �C for 2 min, 1 cycle at 95 �C for 15 min, 40cycles at 95 �C for 15 s, 60 �C for 1 min. The gene-specific prim-er sets are given in Table 1. As an internal standard, 18S RNAwas used. Three biological repetitions, consisting of combinedleaf material from 3 saplings have been carried out and eachtranscript has been quantified three times. That resulted in 9independent values for the calculation of the relative induc-tion according to Pfaffl et al. (2002) by group-wise comparisonof induced samples versus the control samples (REST©).

Results and Discussion

The SSH strategy was used to isolate ozone-induced genesfrom European beech. This method has been shown to be apowerful method for the isolation of differentially or specifi-cally expressed genes upon abiotic as well as biotic stress fac-tors (Sävenstrand et al., 2000; Heidenreich et al., 2001; Asieg-

bu et al., 2004; Rishi et al., 2004; Sahr et al., 2005). The SSH ap-proach, additionally, can contribute to the isolation of novelgenes that may be involved in the response of deciduous treesto the air pollutant ozone. The ozone concentration used inthis study (0.3 μl/l) was relatively high, by far exceeding am-bient concentrations (Matyssek et al., 2004). However, for amechanistic approach, the concentration used should resultin the isolation of ozone-responsive genes. During the earlystages of ozone exposure (up to 5 d), no leaf injury was ob-served. However, at the end of the ozone exposure, necroticleaf areas were visible and the bronzing for ozone-damagedbeech leaves appeared (Simons, 1993; Langebartels et al.,1997).

Two different subtractive libraries were constructed: (i) start-ing with the onset of exposure up to 5 d of exposure (SSH1, ear-ly stages) and (ii) during ozone exposure from day 5 up to day30 (SSH2, late stages; experiment 1). The sequence analysis of1139 putative ozone-responsive clones using BlastN revealedthat 583 (SSH1, 311 and SSH2, 272) did not have any match

Table 1 Primers used for qRT-PCR. An oligonucleotide primer pair was designed for selected genes using Primer Express 2,0 (Applied Biosys-tems). The primers are given in 5′→ 3′ direction

Gene name Primer set Classification

Aquaporin CGG TGC TGT GAA AGC TCT TG transporterTTC CCA GCA AAA AAC CAT GAA G

WRKY-type transcription factor TTT CTC ACT GGA CAC GCT GG transcriptionGAT GGC TAC CGT TGG AGG AA

Caffeic acid O-methyl transferase group 1 AAC AAT GGC ATG GCT GGT CT secondary metabolismCCA CCA CCA ACA TCA ATC AAT G

Catalase GAC GGT GCC TTT GGG TAT CA disease/defenceGTG CTC CAG GGT CGG ATC T

Glutathione S-transferase GTG GAC CTT GCT CTT GTC ACC disease/defenceGCC CAT GCA ATA ATC TTG GG

Ultraviolet-B-repressible protein CTG TCA CGG CCT TTT CCT TAA G transcriptionCCA GAG GCA GGC TTC AAG TG

Ribulose-bisphosphate carboxylase small subunit CTG GTA CAT GGA CAA CTG TG energyCTT TCT TCT CCA GCA ACT GG

Pathogenesis-related protein PR1 CAC TGT GAT TGA GGG TGA TG disease/defenceGCT CTT CAA CAC AGA TCC TC

18S RNA AAA CGG CTA CCA CAT CCA AGCCT CCA ATG GAT CCT CGT TA

Table 2 Summary of BlastN homology search of ozone-responsivebeech leaf ESTs against the GenBank database

Similarity E-value BlastN

High 0 8 (2.6%)High E–165 to E–100 26 (8.6%)Moderate E–99 to E–10 220 (72.4%)Low E–9 to E–1 50 (16.4%)

Total 304

Plant Biology 7 (2005) M. Olbrich et al.672

Table 3 A selected sub-set of ozone-responsive ESTs and corresponding EMBL accession numbers matching homologues in the Genbank data-base. Confirmation of differential expression detected by microarray analysis. EF, expression factor; SEM, mean standard error

Clone Accession number Greatest homology EF SEM

SSH2P4E03 AM072431 Caffeic acid O-methyltransferase (COMT) 23.68 0.89SSH2P6A03 AM072434 Stress and pathogenesis-related protein 9.39 0.67SSH2P4E05 AM072430 Beta-glucosidase 7.97 0.91SSH2P6G01 AM072435 ypr10 gene for allergen Cas s 1 6.67 0.10SSH2P5B12 AM072436 Catalase 4.24 0.42SSH1P2C04 AM062802 Peroxidase 4.18 0.28SSH2P3B04 AM062987 Plasma membrane H+-ATPase (srha1) 3.35 0.22SSH2P3G01 AM062996 Glutathione S-transferase GST 16 3.17 0.31SSH2P5G06 AM063014 Peptidylprolyl isomerase (cyclophilin) 3.12 0.25SSH2P6H07 AM063034 Heat-shock protein (Gmhsp26-A) 2.97 0.20SSH2P4F05 AM072432 Catalase (cat2 gene) 2.91 0.29SSH2P5G09 AJ972501 Glutathione S-transferase (GSTa) 2.86 0.15SSH2P1F04 AM062969 Cystathionine-gamma-synthase precursor 2.61 0.24SSH2P6E05 AM063027 Actin (ACT11) mRNA 2.59 0.09SSH1P2A06 AM062835 Flavanone 3-hydroxylase (F3H) 2.39 0.11SSH1P6D08 AM062921 Cysteine protease 2.33 0.15SSH1P1G02 AM062766 Aldo/keto reductase (AKR) 2.30 0.24SSH2P2A07 AM062976 Acetohydroxy acid isomeroreductase (aair) 2.29 0.13SSH1P2G07 AM062816 Histone H2B 2.23 0.05SSH1P1D08 AM072437 Resistance protein-like (NBS-LRR) 2.17 0.08SSH2P2E07 AM062977 Aldehyde dehydrogenase 2.14 0.09SSH2P2G12 AM062981 ACC oxidase 2 2.08 0.27SSH1P2E05 AM072438 Isoflavone reductase homolog 2.00 0.06SSH2P4C05 AM063003 Cysteine proteinase, putative 1.93 0.12SSH2P4D09 AM063005 ADP-ribosylation factor (ARF1) 1.90 0.18SSH2P1G02 AM072440 O-Methyltransferase (OMT1-BN-8) 1.74 0.05SSH2P1G05 AM062962 Inorganic phosphate transporter (PT1) 1.64 0.11SSH2P4A04 AM063000 Histone 3 1.63 0.13SSH1P2H02 AM072439 Chalcone isomerase 4, putative 1.54 0.08SSH1P3A01 AM062835 DnaJ protein 1.53 0.02SSH1P7H08 AM072441 Thiazole biosynthetic enzyme – 1.53 0.06SSH1P7F07 AM062947 CAB-8 gene chlorophyll a/b binding protein – 1.53 0.07SSH1P5H10 AM072442 6.1 kDa polypeptide of photosystem II – 1.55 0.04SSH1P4C02 AM062888 Ferredoxin-NADP+ oxidoreductase precursor – 1.59 0.05SSH1P3H07 AM062849 Chlorophyll a/b-binding protein CP29 – 1.59 0.07SSH2P4G08 AM063008 Beta-cyanoalanine synthase – 1.62 0.05SSH1P5C11 AM062903 Nucleoside diphosphate kinase – 1.64 0.07SSH1P2A12 AM062828 Glutaredoxin I – 1.65 0.03SSH1P4F09 AM062875 PS I reaction center psaN precursor – 1.65 0.04SSH1P6B07 AM062917 Protein phosphatase-2C (PP2C) – 1.72 0.03SSH2P5A01 AM063021 Acetyl Co-A acetyltransferase – 1.73 0.05SSH1P2D12 AM062831 PS I reaction center subunit X psaK – 1.74 0.04SSH2P4G07 AM072433 RUBISCO small subunit (rbcS) – 1.74 0.05SSH1P3E10 AM072443 PS I reaction center subunit II (psaD) – 1.77 0.05SSH1P2F12 AM062832 Aminotransferase 2 – 1.79 0.04SSH1P6B04 AM062909 Glyoxysomal malate dehydrogenase (mdhG) – 1.80 0.03SSH1P4G01 AM062889 Protein tyrosine phosphatase (ptpkis1) – 1.84 0.07SSH1P4D06 AM062869 Rubisco activase precursor – 1.94 0.04SSH1P7H03 AM062943 Putative protein kinase – 1.95 0.06SSH2P2H01 AM062983 Ribosomal protein L24 – 2.08 0.04SSH1P6G02 AM062908 Chlorophyll a/b-binding protein (lhcb1*7) – 2.18 0.04SSH1P1D03 AM062768 Carbonic anhydrase – 2.19 0.03SSH1P6F08 AJ972497 Aquaporin (PIP2-1) – 2.27 0.03SSH1P4E03 AM072444 Chlorophyll a/b-binding protein 4 – 2.51 0.03

Ozone-Induced Genes in Beech Plant Biology 7 (2005) 673

within the entire GenBank database. Thus the identification ofthese genes might provide additional important informationon the response of deciduous trees towards ozone. 556 ESTsshowed homologies to known genes in the database (SSH1,307 and SSH2, 249). After removing redundant clones thatwere present in both cDNA libraries, 304 single clones (SSH1,211 and SSH2, 93) turned out to be ozone-responsive, and asummary of BlastN similarity alignments against the GenBankdatabase is given in Table 2. The higher number of singletonsin library SSH1 (211) compared to SSH2 (93) is caused by agreater redundancy of clones present in library SSH2. Finally,an overlap of 28 clones between the early- and late-responsivelibraries was found. Therefore, in total, 276 ESTs turned out tobe ozone-responsive. Microarray analysis confirmed the ozoneresponsiveness and selected ESTs showing up- or down-regu-lation with a factor greater than 1.5 are given in Table 3. Therelatively high induction factor for a caffeic acid O-methyl-transferase is in agreement with the high number of clonesencoding O-methyltransferases that were found in the twolibraries.

The relative proportion of ESTs (304) in different functionalclassifications was estimated according to the MIPS classifica-tion (http://mips.gsf.de) and we compared these classes ofgenes that were expressed in the two libraries (Table 4). Alarge group of genes was related to disease/defence in both li-braries, with 25 clones in library SSH1 and 14 clones in librarySSH2 (Table 4), that indicates a rapid, direct defence responseof beech leaves towards ozone exposure, as has been shown forherbaceous plants (Sandermann et al., 1998; Ernst et al., 1999).The total percentage, about 13%, is similar to a stress cDNA col-lection, including a subtractive cDNA library of Arabidopsis,10% of which belonged to the disease/defence class (Mahalin-gam et al., 2003). Functional classification of randomly pickedozone-responsive ESTs resulted in 12 – 13% cell rescue/defencegenes (Tamaoki et al., 2003), and in a pathogen-induciblecDNA library of Scots pine roots 14% of genes were present inthis class (Asiegbu et al., 2004). Another class of prevalentgenes were grouped into cell structure (16%), with a greaterabundance for library SSH1 (40 versus 10 clones) (Table 4).The class of genes involved in protein destination increasedfrom 8 to 11% in the late ozone-responsive cDNA library. Thismay indicate senescence phenomena, as this class represented3% in young poplar leaves, whereas in autumn leaves 7% be-

long to this class (Bhalerao et al., 2003). A great differencewas observed between the libraries for category signal trans-duction. The early ozone-responsive library contained 4% ofthis class, whereas the late ozone-responsive library contained10% (Table 4). It is important to note that these predictions arebased on transcriptional changes that may not necessarily re-flect protein abundance or enzyme activities.

We analyzed the transcript abundance for 8 selected genes,representing different functional classes, by qRT-PCR in an ad-ditional experiment (Table 5, experiment 2). Aquaporin mRNA(highest homology to PIP2-1 from Vitis vinifera), belonging tothe transporter group was down-regulated after 16 d of ozonetreatment by a factor of 1.5 and after 69 d by a factor of 25.However, this down-regulation at day 69 was not significantaccording to the p value of 0.44. A WRKY-like transcription fac-tor was up-regulated after 16 d (2.2) and down-regulated after69 d (0.2). WRKY transcription factors might be involved inozone-induced gene regulation and, in a subtracted ozone-

Table 4 Functional grouping of ESTs according to the MIPS classifica-tion (http://www.mips.gsf.de) obtained from the 2 SSH libraries ofozone-treated Fagus sylvatica leaves

Functional category SSH1 SSH2

Metabolism 11 4Energy 29 10Cell growth/division 8 4Transcription 19 6Protein synthesis 21 6Protein destination 18 10Transporter 7 4Intracellular traffic 3 3Cell structure 40 10Signal transduction 9 9Disease/defence 25 14Secondary metabolism 8 5Unclear classification 3 3Unclassified 10 5

Total number of clones 211 93

Table 5 Ozone-responsive ESTs isolated by SSH and verified by qRT-PCR. Four-year-old European beech saplings were exposed to 0.15 μl/l ozonefor 13 d and then further 56 d with 0.19 μl/l (8 h/d; experiment 2). After 16 d, a single leaf of each sapling was harvested, the material from 3 sap-lings was combined for qRT-PCR, and each transcript was quantified three times (n = 9). A second harvest was carried out after 69 d

Accession number Homology Relative gene expression (16 d) Relative gene expression (69 d)

AJ972497 Aquaporin 0.7 (0.001) 0.04 (0.44)AJ972498 WRKY-type transcription factor 2.2 (0.56) 0.2 (0.001)AJ972499 Caffeic acid O-methyltransferase 1.5 (0.001) 1.2 (0.001)AJ972500 Catalase 0.7 (0.001) 2.2 (0.001)AJ972501 Glutathione S-transferase 0.7 (0.001) 1.4 (0.001)AJ972495 Ultraviolet-B-repressible protein 0.08 (0.001) 0.08 (0.001)AJ972502 rbcS 0.2 (0.001) 0.8 (0.001)AJ972503 PR1 2.8 (0.001) 6.4 (0.001)

Calculation of gene regulation and statistical analysis was as described by Pfaffl et al. (2002) and corresponding p-values are indicated in brackets.

Relative expression ratio = (Efficiencytarget)CTtarget (mean control – mean ozone-treated) / (Efficiency18S rRNA)CT18S rRNA (mean control – mean ozone-treated)

Plant Biology 7 (2005) M. Olbrich et al.674

induced cDNA library, genes with WRKY motifs were over-represented (Ernst and Aarts, 2004; Mahalingam et al., 2003).Caffeic acid O-methyltransferase involved in secondary metab-olism was weakly up-regulated (1.5/1.2). This is consistentwith the well-known inducibility by ozone (Koch et al., 1998;Chiron et al., 2000; Ludwikow et al., 2004). Catalase (highesthomology to cat2 gene from Prunus persica), glutathione S-transferase, and pathogenesis-related protein (PR1) belong tothe main class disease/defence (Table 4). All three transcriptswere up-regulated after 69 d of ozone exposure, whereas, after16 d of ozone treatment, only PR1 was induced (2.8). An induc-tion of these genes has been described many times in the liter-ature (reviewed by Langebartels et al., 2002). The down-regu-lation of the small subunit of ribulose-1.5-bisphosphate carbox-ylase/oxygenase (rbcS) (0.8/0.2) is in accordance with a declinein rbcS transcripts in herbaceous plants upon ozone exposure(Glick et al., 1995). An ultraviolet-B-repressible protein wasclearly down-regulated at both time points (0.08/0.08). The in-terference of UV-B radiation and ozone is given by the produc-tion of reactive oxygen species, thus resulting in the up- ordown-regulation of specific genes (Langebartels et al., 2002).

Conclusions

The development of ozone-responsive cDNA libraries in Euro-pean beech resulted in a functional classification of responsivegenes. However, about 50% of all EST could not be assigned to aputative function and are yet to be characterized. Main percen-tile differences between the early and late ozone-induced li-brary were observed in four functional classes: (i) disease/de-fence, (ii) cell structure, (iii) signal transduction, and (iv) pro-tein destination. A transcriptional analysis by microarray anal-ysis and by qRT-PCR of selected genes confirmed the ozone re-sponsiveness of the libraries, and indicates that herbaceousplants and deciduous long-lived trees use similar defencestrategies and mechanisms against oxidative stress. Theknown, as well yet unknown, ESTs will be a valuable resourcefor further research on growth and parasite defence in foresttrees.

Acknowledgements

We are grateful to the staff of the GSF Department of Environ-mental Engineering for their excellent technical support dur-ing the greenhouse experiments. This research was supportedby the Deutsche Forschungsgemeinschaft (SFB 607).

References

Asiegbu, F. O., Nahalkova, J., and Li, G. (2004) Pathogen-induciblecDNAs from the interaction of the root rot fungus Heterobasidionannosum with Scots pine (Pinus sylvestris L.). Plant Science 168,365 – 372.

Bhalerao, R., Keskitalo, J., Sterky, F., Erlandsson R., Björkbacka, H.,Birve, S. J., Karlsson, J., Gardeström, P., Gustafsson, P., Lundeberg,J., and Jansson, S. (2003). Gene expression in autumn leaves. PlantPhysiology 131, 430 – 442.

Chiron, H., Drouet, A., Claudot, A.-C., Eckerskorn, C., Trost, M., Heller,W., Ernst, D., and Sandermann, H. (2000) Molecular cloning andfunctional expression of a stress-induced multifunctional O-meth-yltransferase with pinosylvin methyltransferase activity from Scotspine (Pinus sylvestris L.). Plant Molecular Biology 44, 733– 745.

Diatchenko, L., Lau, Y. F. C., Campell, A. P., Chenchik, A., Moqadam, F.,Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D.,and Siebert, P. D. (1996) Suppression subtractive hybridization: amethod for generating differentially regulated or tissue-specificcDNA probes and libraries. Proceedings of the National Academyof Sciences of the USA 93, 6025 – 6030.

Ernst, D., Grimmig, B., Heidenreich, B., Schubert, R., and Sander-mann, H. (1999) Ozone-induced genes: mechanisms and biotech-nological applications. In Plant Responses to Environmental Stress(Smallwood, M. F., Calvert, C. M., and Bowles, D., eds.), Oxford:BIOS Scientific Publishers, pp. 33 – 41.

Ernst, D. and Aarts, M. (2004) Cis elements and transcription factorsregulating gene promoters in response to environmental stress. InMolecular Ecotoxicology of Plants (Sandermann, H., ed.), Berlin,Heidelberg: Springer-Verlag, pp.151 – 176.

Glick, R. E., Schlagnhaufer, C. D., Arteca, R. N., and Pell, E. J. (1995)Ozone-induced ethylene emission accelerates the loss of ribu-lose-1, 5-bisphosphate carboxylase/oxygenase and nuclear-encod-ed mRNAs in senescing potato leaves. Plant Physiology 109, 891 –898.

Grams, T. E. E., Kozovits, A. R., Reiter, M., Winkler, J. B., Sommerkorn,M., Blaschke, H., Häberle, K.-H., and Matyssek, R. (2002) Quantify-ing competitiveness in woody plants. Plant Biology 4, 153 – 158.

Heidenreich, B., Mayer, K., Sandermann, H., and Ernst, D. (2001) Mer-cury-induced genes in Arabidopsis thaliana: identification of in-duced genes upon long-term mercuric ion exposure. Plant, Celland Environment 24, 1227 – 1234.

Huang, X., von Rad, U., and Durner, J. (2002) Nitric oxide inducestranscriptional activation of the nitric oxide-tolerant alternativeoxidase in Arabidopsis suspension cultures. Planta 215, 914 – 923.

Kangasjärvi, J., Talvinen, J., Utriainen, M., and Karjalainen, R. (1994)Plant defence systems induced by ozone. Plant, Cell and Environ-ment 17, 783 – 794.

Kärenlampi, L. and Skärby, L. (1996) Critical levels for ozone in Eu-rope: testing and finalizing the concepts. UN-ECE workshop re-port, University of Kuopio, Kuopio, Finland.

Kiefer, E., Heller, W., and Ernst, D. (2000) A simple and efficient pro-tocol for isolation of functional RNA from plant tissue rich in sec-ondary metabolites. Plant Molecular Biology Reporter 18, 33 – 39.

Kley, D., Kleinman, M., Sandermann, H., and Krupa, S. (1999) Photo-chemical oxidants: state of the science. Environmental Pollution100, 19 – 42.

Koch, J. R., Scherzer, A. J., Eshita, S. M., and Davis, K. R. (1998) Ozonesensitivity in hybrid poplar is correlated with a lack of defense-gene activation. Plant Physiology 118, 1243 – 1252.

Langebartels, C., Heller, W., Ernst, D., Lütz, C., Payer, H.-D., Sander-mann, H. (1997) Ozone responses of trees: results from controlledchamber exposures at the GSF phytotron. In Forest Decline andOzone: A Comparison of Controlled Chamber and Field Experi-ments (Sandermann, H., Wellburn, A. R., and Heath, R. L., eds.),Ecol. Studies No.127, Berlin: Springer, pp.163 – 200.

Langebartels, C., Schraudner, M., Heller W., Ernst, D., and Sander-mann, H. (2002) Oxidative stress and defense reactions in plantsexposed to air pollutants and UV-B radiation. In Oxidative Stressin Plants (Inzé, D. and Van Montagu, M., eds.), London, New York:Taylor & Francis, pp.105– 135.

Ludwikow, A., Gallois, P., and Sadowski, J. (2004) Ozone-induced ox-idative stress response in Arabidopsis: transcription profiling bymicroarray approach. Cellular and Molecular Biology Letters 9,829 – 842.

Mahalingam, R., Gomez-Buitrago, A., Eckardt, N., Shah, N., Guevara-Garcia, A., Day, P., Raina, R., and Fedoroff, N. V. (2003) Characteriz-ing the stress/defense transcriptome of Arabidopsis. Genome Biol-ogy 4, R20.

Matyssek, R. and Sandermann, H. (2003) Impact of ozone on trees:an ecophysiological perspective. Progress in Botany 64, 349 – 404.

Ozone-Induced Genes in Beech Plant Biology 7 (2005) 675

Matyssek, R., Wieser, G., Nunn, A. J., Kozovits, A. R., Reiter, I. M.,Heerdt, C., Winkler, J. B., Baumgarten, M., Häberle, K.-H., Grams, T.E. E., Werner, H., Fabian, P., and Havranek, W. M. (2004) Compari-son between AOT40 and ozone uptake in forest trees of differentspecies, age and site conditions. Atmospheric Environment 38,2271 – 2281.

Nunn, A. J., Anegg, S., Betz, G., Simons, S., Kalisch, G., Seidlitz, H.,Grams, T. E. E., Häberle, K.-H., Matyssek, R., Bahnweg, G., Sander-mann, H., and Langebartels, C. (2005) Role of ethylene in the regu-lation of cell death and leaf loss in ozone-exposed European beech.Plant, Cell and Environment 28, 886 – 897.

Pell, E. J., Schlagnhaufer, C. D., and Arteca, R. N. (1997) Ozone-in-duced oxidative stress: mechanisms of action and reaction. Physi-ologia Plantarum 100, 264 – 273.

Pellinen, R., Palva, T., and Kangasjärvi, J. (1999) Subcellular localiza-tion of ozone-induced hydrogen peroxide production in birch (Be-tula pendula) leaf cells. The Plant Journal 20, 349 – 356.

Pfaffl, M. W., Horgan, G. W., and Dempfle, L. (2002) Relative expres-sion software tool (REST©) for group-wise comparison and statis-tical analysis of relative expression results in real-time PCR. Nucle-ic Acids Research 30, e36.

Plessl, M., Rigola, D., Hassinen, V., Aarts, M. G. M., Schat, H., and Ernst,D. (2005) Transcription profiling of the metal-hpyeraccumulatorThlaspi caerulescens (J. & C. PRESL). Biosciences 60, 216 – 223.

Rao, M. V. and Davis, K. R. (1999) Ozone-induced cell death occurs viatwo distinct mechanisms in Arabidopsis: the role of salicylic acid.The Plant Journal 17, 603 – 614.

Rishi, A. S., Munir, S., Kapur, V., Nelson, N. D., and Goyal, A. (2004) Iden-tification and analysis of safener-inducible expressed sequencetags in Populus using a cDNA microarray. Planta 220, 296 – 306.

Sahr, T., Voigt, G., Paretzke, H. G., Schramel, P., and Ernst, D. (2005)Caesium-affected gene expression in Arabidopsis thaliana. NewPhytologist 165, 747 – 754

Sandermann, H., Wellburn, A. R., and Heath, R. L. (1997) Forest De-cline and Ozone. Berlin, Heidelberg, New York: Springer-Verlag.

Sandermann, H., Ernst, D., Heller, W., and Langebartels, C. (1998)Ozone: an abiotic elicitor of plant defence reactions. Trends inPlant Science 3, 47 – 50.

Sandermann, H. and Matyssek, R. (2004) Scaling up from molecularto ecological processes. In Molecular Ecotoxicology of Plants (San-dermann, H., ed.), Berlin, Heidelberg: Springer-Verlag, pp. 207–226.

Sävenstrand, H., Brosché, M., Ängehagen, M., and Strid, A. (2000) Mo-lecular markers for ozone stress isolated by suppression subtrac-tive hybridization: specificity of gene expression and identifica-tion of a novel stress-regulated gene. Plant, Cell and Environment23, 689 – 700.

Sävenstrand, H., Brosché, M., and Strid A. (2002) Regulation of geneexpression by low levels of ultraviolet-B radiation in Pisum sati-vum: Isolation of novel genes by suppression subtractive hybridi-sation. Plant and Cell Physiology 43, 402 – 410.

Schneiderbauer, A., Back, E., Sandermann, H., and Ernst, D. (1995)Ozone induction of extensin mRNA in Scots pine, Norway spruceand European beech. New Phytolologist 130, 225 – 230.

Schraudner, M., Langebartels, C., and Sandermann, H. (1997) Changesin the biochemical status of plant cells induced by the environ-mental pollutant ozone. Physiologia Plantarum 100, 274 – 280.

Schütt, P., Schuck, H. J., and Stimm, B. (1992) Lexikon der Forstbota-nik, Landsberg: Ecomed.

Sharma, Y. K. and Davis, K. R. (1997) The effects of ozone on antioxi-dant responses in plants. Free Radical Biology and Medicine 23,480 – 488.

Simons, S. (1993) Biochemische Effekte und Symptomentwicklungbei Buchen (Fagus sylvatica L.) und Nadelgehölzen unter realenund proportional erhöhten Ozonkonzentrationen. PhD thesis, Lud-wig-Maximilians-Universität München.

Tamaoki, M., Nakajima, N., Kubo, A., Aono, M., Matsuyama, T., andSaji, H. (2003) Transcriptome analysis of O3-exposed Arabidopsisreveals that multiple signal pathways act mutually antagonistical-ly to induce gene expression. Plant Molecular Biology 53, 443 –456.

D. Ernst

GSF – National Research Center for Environment and HealthInstitute of Biochemical Plant PathologyIngolstädter Landstraße 185764 NeuherbergGermany

E-mail: ernst@gsf.de

Guest Editor: R. Matyssek

Plant Biology 7 (2005) M. Olbrich et al.676