Differential gene expression in senescing leaves of two silver birch genotypes in response to...

Differential gene expression in senescing leaves of twosilver birch genotypes in response to elevated CO2 andtropospheric ozonepce_2123 1016..1028

SARI KONTUNEN-SOPPELA1,2, JOHANNA RIIKONEN1,3, HANNA RUHANEN1, MIKAEL BROSCHÉ4,PANU SOMERVUO5, PETRI PELTONEN1, JAAKKO KANGASJÄRVI4, PETRI AUVINEN5, LARS PAULIN5,MARKKU KEINÄNEN2, ELINA OKSANEN2 & ELINA VAPAAVUORI1

1Suonenjoki Research Unit, Finnish Forest Research Institute, 77600 Suonenjoki, Finland, 2Department of Biology,University of Eastern Finland, 80101 Joensuu, Finland, 3Department of Environmental Science, University of EasternFinland, 70211 Kuopio, Finland and 4Department of Biosciences and 5Institute of Biotechnology, University of Helsinki,00014 Helsinki, Finland

ABSTRACT

Long-term effects of elevated CO2 and O3 concentrationson gene expression in silver birch (Betula pendula Roth)leaves were studied during the end of the growing season.Two birch genotypes, clones 4 and 80, with different ozonegrowth responses, were exposed to 2¥ ambient CO2 and/orO3 in open-top chambers (OTCs). Microarray analyseswere performed after 2 years of exposure, and the transcrip-tional profiles were compared to key physiological charac-teristics during leaf senescence. There were genotypicdifferences in the responses to CO2 and O3. Clone 80 exhib-ited greater transcriptional response and capacity to altermetabolism, resulting in better stress tolerance. The geneexpression patterns of birch leaves indicated contrastingresponses of senescence-related genes to elevated CO2 andO3. Elevated CO2 delayed leaf senescence and reducedassociated transcriptional changes, whereas elevated O3

advanced leaf senescence because of increased oxidativestress. The combined treatment demonstrated that elevatedCO2 only temporarily alleviated the negative effects of O3.Gene expression data alone were insufficient to explain theO3 response in birch, and additional physiological and bio-chemical data were required to understand the true O3

sensitivity of these clones.

Key-words: Betula pendula; gas exchange; leaf senescence;long-term experiment; O3; phenology.

Abbreviations: CC, chamber control; DHPPG, 3,4′-dihydroxypropiophenone 3-b-d-glucoside; EC, elevatedCO2; EC + EO, combined elevated CO2 + elevated O3; EO,elevated O3; EST, expressed sequence tag; FDR, false dis-covery rate; NPQ, non-photochemical quenching of chloro-phyll fluorescence; PSII, photosystem II; RbcS, Rubiscosmall subunit gene.

INTRODUCTION

Over the last few decades, human activity has acceleratedatmospheric changes. CO2 emissions have increased byabout 80% between 1970 and 2004 (IPCC 2007). In par-allel, increasing NOx and hydrocarbon emissions causehigher tropospheric ozone (O3) concentrations (Percy &Ferretti 2004). For plants, these changes have contrastingeffects. Rising CO2 concentration increases photosynthesis,which in C3 plants is limited by atmospheric [CO2] (e.g.Drake, Jacob & Gonzalez-Meler 2000; Woodward 2002).However, the CO2 responses in plants show great varia-tion because of differences between species and in otherenvironmental factors (Drake et al. 2000; Woodward 2002;Nowak, Ellsworth & Smith 2004). Forest trees are gener-ally the most responsive to elevated [CO2] (Ainsworth &Long 2005). O3 is considered the most harmful tropo-spheric pollutant to plants (Matyssek & Sandermann2003) because it affects nearly all photosynthetic processes(Long & Naidu 2002) and causes damage to cell structures(Oksanen et al. 2004). Therefore, O3 accelerates climatechange by reducing the ability of plants to accumulatecarbon via photosynthesis to biomass production. It hasrecently been suggested that the role of O3 in decreasingthe CO2 uptake is underestimated in the current carbonsequestration models (Sitch et al. 2007; Wittig, Ainsworth& Long 2007).

The impact of O3 on plants is determined by [O3], O3

stomatal flux, exposure length and the plant defence capa-bilities such as the antioxidative capacity of the plant (Baieret al. 2005). O3 sensitivity is also dependent on developmen-tal factors such as the age of plant (Nunn et al. 2005; Kar-nosky et al. 2007;Wittig et al. 2007), leaf ontogeny (Oksanenet al. 2005) and the timing of exposure and tree size(Oksanen 2003). Chronic O3 stress during several growingseasons with fluctuating [O3] and adaptation to the stressrequires the adjustment of metabolism. The adjustmentresults in changes in resource allocation, in favour of repair,defence and compensations mechanisms (Dizengremel2001).The metabolic modifications in chronic O3 are seen as

Correspondence: S. Kontunen-Soppela. Fax: +358 13 2513590;e-mail: [email protected]

Plant, Cell and Environment (2010) 33, 1016–1028 doi: 10.1111/j.1365-3040.2010.02123.x

© 2010 Blackwell Publishing Ltd1016

altered contents of phenolic compounds (Yamaji et al. 2003;Peltonen, Vapaavuori & Julkunen-Tiitto 2005; Kontunen-Soppela et al. 2007) and chemical changes in tree leaves(Oksanen et al. 2005; Valkama, Koricheva & Oksanen2007).

The simultaneous exposure of plants to elevated [CO2]and [O3] has resulted in both decreased growth andcompensation for the O3-induced growth losses by CO2

(Rebbeck & Scherzer 2002; Kubiske et al. 2006). In silverbirch, only small growth losses were found in elevated [O3],and the growth responses in a combined elevated [CO2] and[O3] treatment were similar to responses in elevated [CO2](Riikonen et al. 2004). These two greenhouse gases alsohave opposite effects on leaf senescence. While the onset ofleaf abscission (Riikonen et al. 2004) and leaf yellowing(Syrjälä et al. unpublished) are delayed in elevated [CO2],they are accelerated by O3 promoting early leaf abscission(Karnosky et al. 1996; Nowak et al. 2004; Riikonen et al.2004).

The effects of elevated [CO2] on the transcriptome havebeen studied, for example, in soy bean (Ainsworth et al.2006; Casteel et al. 2008), Arabidopsis (Li et al. 2006b) andin the tree Populus (Gupta et al. 2005; Taylor et al. 2005;Druart et al. 2006; Cseke et al. 2009). These studies showthat responses to elevated [CO2] vary depending on species,genotypes and leaf ages. Both decreased (Li et al. 2006b)and increased (Gupta et al. 2005) accumulation of photo-synthesis light-harvesting transcripts have been observed.Elevated [CO2] also alters the flux through various biosyn-thetic routes, and changes secondary metabolite composi-tion. In Populus, the flavonoid biosynthesis route wasactivated (Druart et al. 2006). Expression of phenylpro-panoid pathway genes decreased (Gupta et al. 2005), oralternatively decreased or increased depending on thePopulus genotype (Cseke et al. 2009). Elevated [CO2]enhances cell expansion-related genes (Gupta et al. 2005;Taylor et al. 2005; Druart et al. 2006) and mediates changesin metabolism by inducing genes related to signalling andregulation (Ainsworth et al. 2006; Li et al. 2006b). In addi-tion, candidate genes for determining plant adaptation toelevated [CO2] have been studied with Populus (Rae et al.2006, 2007).

Acute O3 exposure of tree leaves elicits defence geneexpression commonly induced by all kinds of oxidativestress (Olbrich et al. 2005; Rizzo et al. 2007), correspond-ingly to data on herbaceous plants (e.g. Tamaoki et al. 2003;Li et al. 2006a;Tosti et al. 2006).The changes in gene expres-sion observed for chronic O3 exposure in mature trees aresimilar to, but often of smaller magnitude than in acutestress, and are influenced by other stresses in field experi-ments (Gupta et al. 2005; Jehnes et al. 2007; Paolacci et al.2007). Furthermore, different O3-responsive mechanismsmay function, depending on the level of O3 and on acute orchronic stress (Vahala et al. 2003).

Changes in gene expression caused by the combinedtreatment with elevated [CO2] and [O3] have so far beenreported in only two studies. In soy bean, the combinedtreatment produced relatively few alterations in gene

expression (Casteel et al. 2008). The combined treatment inPopulus led to gene expression patterns different fromeither gas alone (Gupta et al. 2005).

In the current study, as a part of a larger climatechange experiment (see e.g. Riikonen et al. 2004;Vapaavuori et al. 2009), we report genotypic differences inthe gene expression patterns of birch (Betula pendulaRoth) leaves caused by long-term exposure to elevated[CO2] and [O3]. Two birch clones, showing different sensi-tivity to O3 in terms of growth (Riikonen et al. 2004), wereexposed to elevated [CO2] and [O3] alone and in combi-nation, in open-top chambers (OTCs). This study focuseson leaf senescence phenomenon, because our previousphenological data showed differences in leaf abscissionbetween genotypes, delayed abscission in elevated CO2

and a trend for accelerated senescence in elevated O3

(Riikonen et al. 2004). Leaf samples were harvestedtwice at the end of the second exposure summer, andgene expression patterns were analysed with DNAmicroarrays. The gene expression data are compared tokey physiological parameters in order to understandmechanistic links.

MATERIALS AND METHODS

Experimental design

An OTC system in Suonenjoki Research Station,Finland (62°05′N, 27°00′E) was run during 1999–2001in order to study the responses of silver birch (B.pendula Roth) trees to elevated [CO2] and [O3] aloneand in combination. The experiment is described indetail in Vapaavuori et al. (2002). Two birch genotypes,clone 4 and clone 80 with different responses to O3, wereincluded in the study. Clone 4 was more sensitive to O3

than clone 80 according to growth responses (Riikonen2004). In addition, clone 80 was characterized as a physi-ologically more active genotype, which appeared, forexample, as higher gas exchange and growth rates. Theclones have a different origin: clone 4 was from Valkeako-ski (61°08′N, 28°49′E), while clone 80 was from Eno(62°48′N, 30°05′E) (Mutikainen et al. 2000). Both cloneswere represented as four replicate trees in each treatment.The experiment included altogether 32 tree individuals,each growing in a separate OTC, assigned to the followingtreatments: CC, 2¥ background CO2 (EC), 2¥ backgroundO3 (EO) and combined 2¥ background CO2 + 2¥ back-ground O3 (EC + EO). The exposure and meteorologicaldata are presented in more detail in Riikonen et al.(2005).

Samplings for the microarray analyses were done on 1August and 6 September 2000 (day of the year 214 and 250).Ten short shoot leaves of each tree were collected in eachsampling, and leaf discs (diameter: 2 cm) were punched,pooled, frozen immediately in liquid N2 and stored at-80 °C. To obtain a common reference sample for eachclone (4 and 80) and time-point (August and September),the leaf discs of the four CC trees were pooled.

Leaf senescence at elevated CO2 and O3 1017

© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1016–1028

Photosynthesis, chlorophyll fluorescence,ribulose 1·5-bisphosphatecarboxylase/oxygenase (Rubisco), solublesugars and starch and leaf abscission

Gas exchange measurements were carried out in late Julyand late August on short shoot leaves from three differenttree heights, using a portable gas exchange apparatusLi-6400 (Li-Cor Inc., Lincoln, NE, USA). The measure-ments were done at 20 °C, the growth conditions of CO2,saturating light intensity and at ambient relative humidityof the air. Chlorophyll fluorescence measurements weremade with a portable pulse amplitude-modulated fluorom-eter (MINI-PAM, Walz, Effeltrich, Germany). More detailsfrom the measurements are described in Riikonen et al.(2004). Immediately after gas exchange measurements, theleaves were detached, and a 2 cm2 disc was frozen in liquidN2 for the determination of Rubisco, chlorophyll, solublesugars and starch (Hansen & Møller 1975). In data analysis,the samples from the different tree heights were pooledand analysed as described in Riikonen et al. (2004). Leafabscission was monitored by collecting the fallen leaveswithin the chambers weekly as described in Riikonen et al.(2004).

RNA extraction and amplification

RNA was isolated from birch leaves according to Chang,Puryear & Cairney (1993) with the following modifications:frozen leaf discs (200–300 mg) were homogenated first inliquid N2 with sand, and then in prewarmed (65 °C) extrac-tion buffer where 1% Tween 80 (Fluka, Buchs, Switzerland)was added. The homogenates were incubated at 65 °C for50 min to 2 h, shaken for 15 min and incubated at 65 °C for15 min before the extraction was continued with chloro-form:IAA and RNA precipitation with LiCl as in Changet al. (1993). The isolated RNA (1 mg) was amplified withAminoAllyl MessageAmp aRNA Kit (Ambion,Austin,TX,USA) according to the manufacturer’s instructions.

Hybridization on microarrays

A common reference design was used for the microarrayanalyses (Churchill 2002; Smyth 2005). All treatmentswithin each clone and time-point were hybridized againstthe common reference obtained from the respectiveambient control chambers. All hybridizations were donewith dye swaps. The hybridization design of the experimentis shown in Fig. 1.

The cDNA microarrays used in this study consisted of8143 Populus euphratica ESTs representing approximately6340 distinct genes, and an undetermined number of para-logues (Brosché et al. 2005). The ESTs were obtained fromP. euphratica leaf, shoot and root control library, and severalstress-related cDNA libraries of plants subjected to thefollowing treatments: elevated CO2, different irradiancelevels, drought stress, flooding stress, O3, cold and freezing,salt stress and cadmium stress. The array has previously

been successfully applied for birch (Ruonala et al. 2006).The cDNA was spotted in triplicate on epoxy silane-coatedNexterion E-borosilicate glass (SCHOTT AG, Mainz,Germany), with the size of 75.6 ¥ 25.0 mm, at the FinnishDNA Microarray Centre in Turku.The array design is avail-able in ArrayExpress with the accession number A-MEXP-1042 (http://www.ebi.ac.uk/microarray-as/aer/entry).

The aRNA was labelled with Cy3 or Cy5 (AmershamBiosciences, Buckingshire, England) before the hybridiza-tion according to the instructions in the AminoAllyl Mes-sageAmp aRNA Kit (Ambion). The slides were pre-hybridized in pre-hybridization buffer (2% BSA, 5¥ SSC,0.1% SDS) for 30 min to 3 h at 65 °C. The arrays werehybridized in 50% formamide, 5¥SSC, 0.1% SDS, 5¥Den-hardt’s solution (Sigma, St Louis, MO, USA) and 10%Herring sperm (1 mg mL-1) (Sigma) for 16–18 h at 42 °C.The slides were then washed and scanned immediately afterthe hybridization with GenePix 4200AL scanner (AxonInstruments, Union City, CA, USA) at 635 and 532 nm.

Microarray data analysis

Images were analysed in GenePixPro 5.0 (Axon Instru-ments). Visually bad spots or areas on the array, small spots(diameter <50 mm) and low-intensity spots were markedwith negative flags. Low-intensity spots were determined asspots where less than 55% of the pixels had intensity abovethe background +1SD in either channel.

The data from GenePixPro were analysed with thelimma-package (linear models for microarray data) (Smyth2004) in R (http://www.r-project.org/). The spot qualityweights were set at 0.1 for all negative flags, obtained fromthe GenePixPro program, and at 1 for the rest of the spots.The background correction for the data was done withnormexp-function (Ritchie et al. 2007), and the backgroundcorrected mean intensities were used for calculations.

EO

EO EC + EO

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EC

EC

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August

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September

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Figure 1. Hybridization design of the experiment. Commonreference for both time-points (August, September) was obtainedby pooling the leaves of four individual trees in ambient controlchambers. The common reference was hybridized against samplesfrom four replicate trees in each treatment at each time-point.The treatments were CC, EC, EO and EC + EO. The designshown in the figure was repeated for both clones 4 and 80. Allhybridizations were done with dye swaps.

1018 S. Kontunen-Soppela et al.


The average of triplicate spots on each slide was used foranalyses. The data were normalized with the loess method.Linear models with moderated t-statistic (set at P < 0.005)were used to find genes that were differentially expressed(Smyth 2004, 2005).

Hierarchical clustering of the gene expression changesbetween control and treatments was done in R. Euclideanclustering with complete linkage method was used tocluster the gene expression data by treatment/samplingtime/clone that was significantly changed in at least one ofthe clones or treatments or at a sampling time.

Gene annotation and gene ontology(GO) analysis

Annotations, as well as GO term annotation and thefunction-based analysis of genes that were differentiallyexpressed, were performed using the software Blast2GO(Conesa et al. 2005; Götz et al. 2008). GO terms for each ofthe three main categories (biological process, molecularfunction and cellular component) were obtained fromsequence similarity (E value 1e-5) of the EST sequences onthe array. The application default parameters were used forthe other analyses.

Statistical analysis of physiological data

The main effects and interactions of clone, CO2, O3 andsampling time on leaf physiological parameters, and con-centrations of phenolic compounds were analysed by meansof the linear mixed models analysis of variance (anova),using the replicate tree number as a random factor (SPSS14.0 for Windows, SPPS Inc. 2005, Chicago, IL, USA).Where necessary, the data were ln transformed to achievethe normal distribution of residuals. The P values <0.05 arereported as significant.

RESULTS

Natural leaf senescence

Leaf senescence, monitored as the time-course of leafabscission, started earlier in the elevated O3 treatment(EO), and was significantly delayed by the elevated [CO2](P < 0.001) (Fig. 2). The difference between clones in leafabscission was significant (P < 0.001). In September, therewas a significant decrease in chlorophyll fluorescenceparameters, the maximal photochemical efficiency of PSII(Fv/Fm), the actual photochemical efficiency of PSII (Fv′/Fm′)and the NPQ of leaves when compared with the Augustsamples (Table 1). The starch concentration of leavesincreased in September, but no changes were seen in theamount of chlorophyll, concentration of soluble sugars orRubisco amount or activity (Fig. 3; Table 1). Leaf age alsoaffected the concentrations of many phenolic compoundgroups (Table 2; Supporting Information Table S2).

Responses to treatments

Elevated [CO2]Gene expression changes caused by elevated CO2 (EC)were similar in both clones according to the hierarchicalclustering (Fig. 4), and the number of genes (18) showingstatistically significant changes in expression was the same

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Figure 2. Effects of treatments on the timing of leaf abscission.Leaf abscission is presented as the percentage of total leaf areain clones 4 (a) and 80 (b) during the autumn 2000. Thetreatments were as in Fig. 1. Data show the mean values � SEfor four replicate samples. The samples for the microarray andphenolic compound analyses were taken at days 214 and 250. Themeasurements for physiological parameters were made at days210 and 237.



in the September samples. In contrast, in August only, oneEST was similar for both clones (Table 3; Supporting Infor-mation Table S1). In September, genes with decreased geneexpression were mainly related to photosynthesis light reac-tions and protein synthesis (five ESTs of ribosomal genes).In clone 4 in September, decreased expression of enzymesrelated to secondary metabolism (flavonoid 3-hydroxylase,UDP-glucosyl-transferase, sinapyl alcohol dehydrogenase)was observed (Table 3; Supporting Information Table S1).

Photosynthetic activity (Pn, measured at growth [CO2])and the actual photochemical efficiency of PSII (Fv′/Fm′)increased in EC (Table 1), and the NPQ decreased (Fig. 3;Table 1). Total activity and amount of Rubisco proteindecreased in EC (Table 1; Fig. 3). The down-regulation ofRubisco in EC was greater in clone 80 than in clone 4.Therewere a few CO2 ¥ clone and CO2 ¥ sampling time interac-tions, indicating clonal and time-dependant variation inresponse to EC.

Elevated [CO2] had a significant impact on the concen-tration of phenolic acids that increased, and the concentra-tions of DHPPG and flavone aglycons that decreased(Table 2; Supporting Information Table S2). There weremany clone ¥ CO2 interactions in the concentrations ofphenolics, showing a differential response of the studiedclones to EC (Table 2).

Elevated [O3]According to hierarchical clustering, the gene expressionprofile in the EO treatment in August was different fromthe other treatments (Fig. 4). Elevated [O3] had a greatereffect on the gene expression of clone 80 than on clone 4especially in August, when the number of genes withdecreased expression was markedly higher in clone 80 (256ESTs in clone 80, 81 ESTs in clone 4) (Supporting Informa-tion Table S1). Many genes down-regulated in EO wereconnected to general metabolism, including translation-related (24 ESTs) genes associated with photosynthesis (30ESTs) and glycolysis (six ESTs). In August, increasedexpression in clone 80 included enzymes related to second-ary metabolism (cinnamyl alcohol dehydrogenase), cell wallbiosynthesis-related enzymes (e.g. rhamnose synthase) andmany unknown ESTs (Supporting Information Table S1).In clone 4, three ESTs increasing in expression coded forNADH plastoquinone oxidoreductase. In September, onlyone EST was up-regulated in clone 80 in EO, while in clone4 the ESTs with increased expression were related to stress.

EO also decreased the amounts of Rubisco, chlorophylland concentrations of soluble sugars, and increased NPQ(Fig. 3; Table 1). Furthermore, elevated O3 increased theconcentrations of DHPPG and flavone aglycons in theleaves (Table 2; Supporting Information Table S2).

Combined elevated [CO2] and [O3] treatmentIn contrast to single EO and EC treatments, combinedEC + EO exposure induced more gene changes in expres-sion in clone 4 than in clone 80 in August (SupportingTa

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Information Table S1). The expression pattern of EC + EOin August resembled that of EC treatment in Septemberespecially in clone 4 (Fig. 4; Table 3). Expression of ESTsrepresenting NADH plastoquinone oxidoreductase andribosomal proteins, as well as senescence-associated proteinand serine carboxypeptidase, was decreased in both clonesin EC + EO in August, whereas in EC these changes wereseen in September (Table 3). In September, the clusteringanalysis showed the similarity of EC + EO treatment to EOtreatment (Fig. 4), although significant changes in geneexpression were not similar in these treatments (Table 3).

Elevated [CO2] affected leaf abscission and the param-eters related to photosynthesis similarly at both O3

levels. However, a significant CO2 ¥ O3 ¥ clone interactionrevealed that the chlorophyll concentration was reducedunder elevated [O3] only in ambient [CO2] in clone 4,while inclone 80 it was reduced also in combined elevated [CO2] and[O3] (Fig. 3;Table 1). Interaction effects of [CO2] and [O3] onphenolic concentrations were observed in DHPPG,phenolicacids and flavone aglycons (Table 2;Supporting InformationTable S2).

DISCUSSION

Senescence phenomena differedbetween clones

Leaf senescence is an active process during which somemetabolic pathways are turned on, whereas others are deac-tivated. Previous studies with birch in greenhouse condi-tions have shown that leaf senescence is associated withdecreases in expression of photosynthesis and nutrientremobilization-related genes (Valjakka et al. 1999; Sillan-pää et al. 2005). Growth cessation, bud set and leaf senes-cence are under photoperiodic control in birch (Li et al.2003), and therefore the sampling in this study was timed tothe period at the end of the growing season when leafsenescence normally starts. Decreased chlorophyll fluores-cence indicated the beginning of leaf senescence at the timeof sampling (Fig. 3).

The two genotypes studied differed in phenology. Clone80, originating from a slightly northern location in Finland,is adapted to shorter growing period than clone 4 (Muti-kainen et al. 2000), and is expected to senesce earlier. Ourdata show that senescence started at around the same timein the two clones, but proceeded faster in clone 80 (Fig. 2and Riikonen et al. 2003). Riikonen et al. (2003) also sug-gested that clone 80 is more effective in the translocation ofleaf nutrients than clone 4.

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Figure 3. Physiological parameters of clones 4 and 80 in thedifferent treatments � SD. Ribulose 1·5-bisphosphatecarboxylase/oxygenase (Rubisco) amount, Rubisco activity, NPQ,chlorophyll amount and the soluble sugars and starch presentedfor clones 4 (left panel) and 80 (right panel) at days 210 and 237.Data are the means � SE from four replicate trees. Thetreatments were as in Fig. 1.



Elevated [CO2] decreases the expression of Cmetabolism and senescence-related genes

Samples for gene expression analysis were harvestedtowards the end of the growing season, and the leaves havethus been exposed to the treatments continuously since budburst in early May. Accordingly, the leaves had reached anew state of homeostasis, and only a few genes showedaltered expression in elevated [CO2]. In agreement withprevious studies on mature aspen leaves, most changeswere seen as decreased transcript levels (Gupta et al. 2005;Taylor et al. 2005; Cseke et al. 2009).

In the September samples, expression of aphosphatidylinositol/phosphatidylcholine transfer proteinwas strongly decreased in both clones.This protein is relatedto phospholipid-mediated signal transduction processes,andit may regulate lipid biosynthetic processes in plants (Li, Xie& Bankaitis 2000). Lower levels of transcripts related tosenescence of birch leaves, such as b-amylase, metallothion-eins (Bhalerao et al. 2003) and a senescence-related protein,indicate a slow-down in senescence processes. The delayedleaf senescence of trees in elevated [CO2], based on pheno-logical and physiological measurements, has been reportedearlier (Riikonen et al. 2004;Taylor et al. 2008),but this is thefirst study indicating alterations also in senescence-relatedgene expression.

The changes in gene expression under elevated [CO2]were supported by the physiological, chemical and struc-tural changes in leaves, and the growth responses of thetrees. Despite down-regulation of net photosynthesis whenmeasured at ambient [CO2], the net photosynthesisincreased at 720 ppm CO2 (Table 1). This caused accumula-tion of starch in the leaves of both clones (Table 1; see alsoOksanen et al. 2005). The concentration of hemicelluloseTa

ble

2.T

heP

valu

esfo

rth

em

ain

effe

cts

ofth

eti

me

ofsa

mpl

ing,

clon

e,el

evat

edO

3an

dC

O2

conc

entr

atio

ns,a

ndth

eir

inte

ract

ions

onco

ncen

trat

ions

ofph

enol

icco

mpo

unds

and

com

poun

dgr

oups

insi

lver

birc

hsh

ort

shoo

tle

aves

Tim

eC

lone

CO

2O

3T

ime

¥cl

one

CO

2¥

O3

Clo

ne¥

CO

2¥

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Clo

ne¥

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2C

lone

¥O

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ime¥

clon

e¥

CO

2¥

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DH

PP

G<0

.000

<0.0

01↓

0.00

7↑0.

017

0.06

7P

enta

gallo

ylgl

ucos

e<0

.001

↓0.

044

0.03

20.

049

0.05

7P

heno

licac

ids

<0.0

01↓

<0.0

01<0

.001

↑0.

070

0.03

6F

lavo

neag

lyco

ns0.

013↓

0.00

10.

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des

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ves

<0.0

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ins

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sure

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ics

0.00

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006

Dat

ash

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est

atis

tics

for

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atio

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olic

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here

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lam

ount

ofph

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wer

ete

sted

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WP

:low

-mol

ecul

ar-w

eigh

tph

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ics.

Val

ues

show

nin

bold

are

sign

ifica

ntat

P<

0.05

leve

l,P

valu

es>0

.05

and

<0.1

are

rega

rded

asa

tren

dan

dth

eno

n-si

gnifi

cant

chan

ges

are

left

open

.Arr

ows

indi

cate

incr

ease

(↑)

orde

crea

se(↓

)co

mpa

red

toam

bien

tco

ntro

lor

Aug

ust

sam

plin

g(t

ime)

.

4Aug

EC

+ E

O

4Sep

EC

80S

ep E

C

80A

ug E

O

4Aug

EO

4Aug

EC

80A

ug E

C

4Sep

EO

4Sep

EC

+ E

O

80S

ep E

C +

EO

80A

ug E

C +

EO

80S

ep E

O

45

67

89

1011

Hei

ght

Figure 4. Dendrogram showing dissimilarities in GE betweentreatments and the sampling times. Euclidean clusteringwith complete linkage method was used to cluster thetreatment/sampling time/clone with gene expression data thatwere significantly changed in at least one of the clones (4 and 80)in one of the treatments at a sampling time (August andSeptember). The treatments were as in Fig. 1. Height shows thedistance between clusters in the dendrogram.



Tab

le3.

Sele

cted

gene

ssh

owin

gsi

gnifi

cant

diff

eren

ces

inex

pres

sion

inle

aves

ofsi

lver

birc

hgr

own

unde

rel

evat

edC

O2

,(E

C)

and

O3

(EO

)co

ncen

trat

ions

alon

eor

inco

mbi

nati

on(E

C+E

O)

com

pare

dw

ith

cont

rola

mbi

ent

cond

itio

ns

Gen

Ban

kE

STac

cess

ion

no.

Gen

efu

ncti

on

EC

EO

EC

+EO

Aug

Sep

Aug

Sep

Aug

Sep

804

804

804

804

804

804

AJ7

7659

1X

ylos

idas

e-0

.40

-0.1

5-0

.46

-1.0

1-0

.44

-0.6

7-0

.74

-0.2

5-0

.07

-0.2

20.

00-0

.34

AJ7

7508

0C

ytoc

hrom

eb6

fco

mpl

exsu

buni

tIV

0.03

0.81

-0.4

0-0

.93

-0.5

50.

08-0

.72

-0.5

1-0

.07

-0.7

70.

080.

10A

J777

882

AT

P-c

itra

tesy

ntha

se-0

.11

-0.4

10.

020.

11-0

.66

0.07

0.11

0.22

0.08

0.11

-0.0

80.

01A

J780

854

Sina

pyla

lcoh

olde

hydr

ogen

ase

-0.2

8-0

.62

-0.1

1-0

.36

-0.2

0-0

.27

-0.0

2-0

.09

-0.2

80.

00-0

.28

-0.1

6A

J768

767

Pho

tosy

stem

IIpr

otei

nD

1-0

.10

0.10

1.03

-0.1

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.57

-0.0

1-0

.04

0.33

0.17

0.05

0.03

0.39

AJ7

6977

8B

eta-

amyl

ase

0.02

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.56

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1-0

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0.02

-0.1

7-0

.16

0.04

-0.3

9-0

.06

-0.1

9A

J770

490

Pho

tosy

stem

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sem

bly

prot

ein

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00.

06-0

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-0.5

60.

140.

33-0

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-0.2

6-0

.71

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3-0

.12

AJ7

6940

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-0.4

3-0

.59

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6-0

.28

-0.7

9A

J771

235

sene

scen

ce-a

ssoc

iate

dpr

otei

n-0

.03

0.25

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.94

0.11

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0.05

-0.5

7-1

.16

0.04

0.62

AJ7

6869

5ea

rly

light

-ind

ucib

lepr

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90.

05-0

.08

0.14

0.05

0.42

-0.1

60.

20A

J769

270

NA

DH

-pla

stoq

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neox

idor

educ

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1-0

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8-0

.80

-0.8

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160.

80-0

.57

-0.2

0-0

.64

-0.9

4-0

.07

-0.2

1A

J769

365

NA

DH

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6742

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AD

Hde

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ogen

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0.43

-0.8

2-0

.38

-1.2

6-1

.82

-0.1

7-0

.03

AJ7

6861

3C

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0.40

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4-0

.41

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0.04

0.26

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0A

J767

435

Seri

neca

rbox

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80.

01A

J772

359

Thi

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-lik

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40.

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7A

J769

394

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0.08

AJ7

7602

440

sri

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mal

prot

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360

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7894

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60.

01A

J768

301

Car

boni

can

hydr

ase

-0.4

2-0

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0.04

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40.

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AJ7

7088

7C

hlor

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0.09

-0.0

40.

02-0

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AJ7

7295

0P

hoto

syst

emI

prot

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-0.2

9-0

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.35

-0.1

0-0

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-0.0

20.

21A

J778

941

Pho

tosy

stem

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buni

tx

-0.6

9-0

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7083

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6746

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264

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Res

ults

are

show

nse

para

tely

for

the

two

geno

type

s(c

lone

s80

and

4)an

dsa

mpl

ing

tim

es(A

ugus

tan

dSe

ptem

ber)

.Log

fold

chan

ges

(log

2va

lue

offo

ldch

ange

toam

bien

t)sh

own

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ldar

esi

gnifi

cant

(P<

0.00

5).



was higher under EC (Oksanen et al. 2005), which may berelated to the decreased expression of xylosidase genepotentially involved in the hemicellulose metabolism of sec-ondary cell wall (Goujon et al. 2003). The observed surplusof carbohydrates in EC was shunted to non-structural com-pounds, seen as higher concentration of starch (Riikonenet al. 2005); larger size of chloroplasts and starch grains(Oksanen et al. 2005); and accumulation of compoundsof the phenylpropanoid pathway, such as phenolic acids(Oksanen et al. 2005; Peltonen et al. 2005).

There were some clonal differences in gene expressionin response to EC. Genes related to secondary metabolismwere decreasing in expression in clone 4, and accumula-tion of phenolic compounds was different betweenclones (Peltonen et al. 2005). Clonal variation in carbonpartitioning to growth and secondary metabolism underelevated [CO2] were also observed in Populus (Cseke et al.2009).

Ozone-induced changes in gene expression aredependent on clone

Many studies show that O3 sensitivity and gene expressionprofiles vary both between species and within ecotypes ofthe same species (Li et al. 2006a; Rizzo et al. 2007; Puckette,Tang & Mahalingam 2008). This study illustrates the samephenomenon with large differences in gene expressionbetween the clones 4 and 80.

In early August, the majority of genes with alteredexpression had decreased transcript levels. Similar resultshave been previously reported in Arabidopsis where fivetimes more genes were down- than up-regulated underchronic O3 exposure (Miyazaki et al. 2004). In clone 80, theEO treatment decreased the expression of many ribosomalgenes which may be related to decreased protein synthesisin leaves. Many photosynthesis-related (Chl a/b binding,and PSI and II proteins, GAPDH, CP12) and carbonassimilation-related transcripts (RbcS, carbonic anhydrase)had reduced expression as previously shown in Populus(Gupta et al. 2005). The observed changes in gene expres-sion were in line with the decrease in physiological param-eters. The decline in photosynthetic light reactions was alsoseen as an increase in the NPQ, indicating increased energydissipation as heat when the capacity to electron transferdecreases (Muller, Li & Niyogi 2001; Niyogi et al. 2005).Theincreased NPQ may be associated with the increasedNADH plastoquinone oxidoreductase expression in EO inAugust. NADH plastoquinone oxidoreductase (ndh) isinduced by natural senescence (Catal, Sabater & Guéra1997) and O3 (Guéra et al. 2005). NDH is related to theacclimation of photosynthesis in changing environmentalconditions, and suggested to protect PSI from photoinhibi-tion (Rumeau, Peltier & Cournac 2007). However, thedecreased expression of photosynthesis-related genes andbiochemistry of photosynthesis could not be recorded asdecreasing net photosynthesis (Riikonen et al. 2005), nor ingrowth or total biomass, which was very little affected(Riikonen et al. 2004).

In response to EO in September, clone 80 had few alter-ations in gene expression which included decreased expres-sion of NADH plastoquinone oxidoreductase that has beenassociated with oxidative stress (Rumeau et al. 2007). Incontrast, clone 4 had increased expression of genes associ-ated with leaf ageing and/or oxidative stress including: fer-ritin (Murgia et al. 2007), 12-oxophytodienoate reductase(He et al. 2002) and ubiquitin (Wegener et al. 1997). Theincreased expression of 12-oxophytodienoate reductasepoints to the synthesis of jasmonic acid (Schaller 2001), oneregulator of O3 responses in plants (see e.g. Baier et al.2005). These findings are in accordance with the long-termO3 exposures of trembling aspen (Gupta et al. 2005) orbeech (Jehnes et al. 2007), where the expression of oxida-tive stress-induced signalling and defence-related genesincreased in leaves. Although oxidative stress responses inSeptember were more marked in clone 4 in terms of geneexpression, the physiological parameters measured indi-cated stronger oxidative stress in clone 80 under EO. Thestomatal conductance and thus O3 uptake were greater inclone 80, but also the ascorbate concentration was higher,indicating superior scavenging of reactive oxygen species inclone 80 (Padu et al. 2005). The number of mitochondriaand peroxisomes increased, and there was accumulation ofH2O2 (Oksanen et al. 2005) and some phenolic compounds,such as chlorogenic acid, myricetin glycosides and flavoneaglycons in clone 80 (see also Peltonen et al. 2005).Recently, Kontunen-Soppela et al. (2007) reported that amore O3-tolerant birch genotype showed a stronger shift inthe leaf metabolome towards defence-related compounds,such as phenolics, than a sensitive one. The clonal differ-ences in gene expression and other factors in response to O3

suggest that a wide set of changes are needed for a better O3

tolerance, and that increased expression of oxidative stress-related genes does not automatically signify a better toler-ance of the stress. Accordingly, Calfapietra et al. (2008) andRyan et al. (2009) stated that good O3 tolerance in Populusis a combination of different factors, such as reduced O3

uptake through stomata, higher isoprene emissions andcarotenoid concentrations.

CO2 alleviates the harmful effects of ozone incombined CO2 and O3 treatment

Currently, [CO2] and [O3] in the atmosphere increase inparallel, and thus it is important to understand the com-bined effects of these gases on plant performance. Elevated[CO2] mitigates the harmful effects of O3 on plants(Riikonen et al. 2004, 2005; Oksanen et al. 2005; Peltonenet al. 2005), but more importantly O3 may offset the benefi-cial effects of CO2 on plant photosynthesis and growth(Wustman et al. 2001; Karnosky et al. 2003). These observa-tions are supported by the gene expression data from thecurrent study. In clone 4, the gene expression changes inEC + EO treatment in August were similar to the EC treat-ment in September. The results are in contrast to data fromexperiment with aspen, where the effects of combinedelevated [CO2] and [O3] to gene expression showed more



resemblance to O3 treatment (Gupta et al. 2005). Our phe-nological and physiological data support the similaritybetween EC + EO and EC treatments.The timetable of leafabscission was very similar in the EC and EC + EO treat-ments, and most of the physiological measurements showthat in the EC + EO treatment, CO2 was able to alleviatethe negative effect of O3 (see Oksanen et al. 2005).

In contrast to the August sampling, the gene expressionprofile in the EC + EO treatment in September was moresimilar to the EO than to the EC treatment. This suggeststhat the CO2 effect on alleviating EO effects on geneexpression was temporary.This was also seen in the concen-trations of most phenolic compounds and physiologicalparameters where the EC + EO treatment resembled theEC treatment in August, but not in September.Accordingly,Riikonen et al. (2008) reported the disappearance of ben-eficial CO2 effects on paper birch photosynthesis in com-bined elevated [CO2] and [O3] from mid-August onwards insenescing leaves.

CONCLUSIONS

In this birch O3 and CO2 enrichment study, we combineinformation on responses derived from gene expression,phenological and physiological data. By using two geno-types with differential O3 responses, we show that the betteradaptation capacity of one birch clone to increasing green-house gases is linked to a greater capability to alter itsmetabolism. Furthermore, increased expression of oxida-tive stress-related genes alone cannot predict the sensitivityto O3, but other data (e.g. growth and physiological param-eters) are needed to assess the O3 tolerance in birch. Themain CO2-induced changes were seen as a general down-regulation of carbohydrate metabolism and delayed senes-cence of leaves. In elevated O3, many senescence-associatedgenes were up-regulated, indicating earlier leaf senescencecaused by increased oxidative stress. Analysis of geneexpression does not necessarily reflect the activity of thecorresponding proteins or biochemical processes, becauseseveral other processes, including post-translational modi-fications and protein stability, provide additional regulatorysteps. This can be seen in our data where elevated O3

decreased expression of photosynthesis-related geneswithout affecting net photosynthesis. Thus, gene expressiondata need to be interpreted carefully and should, if possible,be complemented with physiological data. In an ecologicalperspective, the deleterious impact of increasing tropo-spheric [O3] in leaves may be diminished by elevated [CO2],but the beneficial effect decreases towards the end of thegrowing season. This study increases the understanding ofthe mechanisms behind the O3 tolerance, the responsive-ness of birch to elevated [CO2] and the interactive effects ofthese two greenhouse gases in nature.

ACKNOWLEDGMENTS

The research was supported by the Academy of Finland(projects 40924, 047074, 240016) and the European

Commission (project ERBIC15CT-980102). We thankMarja-Leena Jalkanen, Pia Lappalainen and ElinaVälimäki for their skillful technical assistance, and Dr KirkOvermyer for revising the English language. Turku Centrefor Biotechnology, University of Turku and Åbo AkademiUniversity are acknowledged for providing the arrays.

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Received 9 September 2009; received in revised form 13 January2010; accepted for publication 14 January 2010

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Table S1. Gene expression log2 FC between ambient andEC-, EO- and EC + EO-treated samples of clones 4 and 80

in August and September. Significant changes (P < 0.005)between treatment and ambient are shown in bold.Table S2. Concentrations of phenolic compounds in clones4 and 80 in August and September. The treatments wereCC, EC, EO and EC + EO. Data show the mean values andSD for four replicate samples. The samples were taken atdays 214 and 250.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials sup-plied by the authors. Any queries (other than missing mate-rial) should be directed to the corresponding author for thearticle.



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