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Environmental Pollution 158 (2010) 3580e3587

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Environmental Pollution

journal homepage: www.elsevier .com/locate/envpol

Contrasting ozone sensitivity in related evergreen and deciduous shrubs

Vicent Calatayud a,*, Francisco Marco a, Júlia Cerveró a, Gerardo Sánchez-Peña b, María José Sanz a

a Fundación CEAM, c/ Charles R. Darwin 14, Parque Tecnológico, 46980 Paterna, Valencia, Spainb SPCAN, Dir. Gral. de Medio Natural y Política Forestal, Ministerio de Medio Ambiente, y Medio Rural y Marino, Ríos Rosas 24, 28003 Madrid, Spain

Mediterranean evergreen shrubs have a constitutively higher capacity

to tolerate ozone stress than their deciduous relatives.

a r t i c l e i n f o

Article history:Received 13 February 2010Received in revised form18 August 2010Accepted 18 August 2010

Keywords:OzoneVisible injuryOxidative stressPhotosynthesisModulated fluorescenceRubisco

Abbreviations: AA, Ascorbate; Asat, light saturateaccumulated exposure over threshold 40 ppb; CF, Chcellular CO2 concentration; DHA, dehydroascorbate;oxidant Power assay; gmax, maximum stomatalconductance to water vapor; GPX, Peroxidase; HACapacity; LAC, Liposoluble Antioxidant Capacity; NFþozone; OTC, Open Top Chamber; PPFD, photosynthetSuperoxide dismutase; TAC, Total Antioxidant Capacminimal fluorescence; Fm, maximum fluorescence;efficiency of photosystem II (PSII) primary photocherescence value; NPQ, non-photochemical quenchingexcitation capture by oxidized reaction centers ofelectron transfer at PSII.* Corresponding author.

E-mail address: vicent@ceam.es (V. Calatayud).

0269-7491/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.envpol.2010.08.013

a b s t r a c t

Plant responses to enhanced ozone levels have been studied in two pairs of evergreen-deciduous species(Pistacia terebinthus vs. P. lentiscus; Viburnum lantana vs. V. tinus) in Open Top Chambers. Ozone inducedwidespread visible injury, significantly reduced CO2 assimilation and stomatal conductance (gs), impairedRubisco efficiency and regeneration capacity (Vc,max, Jmax) and altered fluorescence parameters only inthe deciduous species. Differences in stomatal conductance could not explain the observed differences insensitivity. In control plants, deciduous species showed higher superoxide dismutase (SOD) activity thantheir evergreen counterparts, suggesting metabolic differences that could make them more prone toredox imbalances. Ozone induced increases in SOD and/or peroxidase activities in all the species, butonly evergreens were able to cope with the oxidative stress. The relevancy of these results for theeffective ozone flux approach and for the current ozone Critical Levels is also discussed.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Tropospheric ozone is the most relevant pollutant both for cropsand native vegetation. It causes a series of deleterious physiologicaland biochemical effects, visible injury and growth reductions, andmay also interact with other biotic and abiotic stresses (Krupa andManning, 1988; Krupa et al., 2000; Manning and von Tiedemann,1995). Model predictions using IPCC scenarios suggest that back-ground levels of this pollutant will increaseworldwide in the future(Vingarzan, 2004). Furthermore, ozone is itself a greenhouse gascontributing to radiative forcing (IPCC, 2007).

d CO2 assimilation; AOT40,arcoal Filtered Air; Ci, inter-FRAP, Ferric Reducing Anti-conductance; gs, stomatal

C, Hydrosoluble Antioxidant, Non Filtered Air þ 30 ppbic photon flux density; SOD,ity; Tr, transpiration rate.; F0,Fv:Fm, maximum quantum

mistry; Fs, steady state fluo-; Fexc, quantum efficiency ofPSII; FPSII, quantum yield of

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In Europe, the Mediterranean area experiences relatively highozone levels (Sanz et al., 2007) due to favorable conditions forozone formation, and a combination of meso-scale re-circulatoryprocesses and long-range transport (Millán et al., 1992, 1997, 2000;Sanz and Millán, 1998; Lelieveld et al., 2002). On forest sites, theselevels regularly exceed the critical level of 5000 ppb h [AOT40,daytime hours, April to September] (e.g., Gerosa et al., 2007),established to protect sensitive species from growth losses (ICPModelling and Mapping, 2004). While in the Mediterranean areathere is field evidence of widespread injury in irrigated crops(Hayes et al., 2007), ozone-like symptoms in broadleaf evergreenspecies have only been reported sporadically (Skelly et al., 1999);for a number of species, they have been induced experimentally(Sanz et al., 2001). Both field and experimental evidence suggestthat Mediterranean broadleaf evergreen species are comparativelyozone-tolerant (e.g., Bussotti and Gerosa, 2002; Nali et al., 2004).However, the sensitivity of evergreen species to this pollutant islargely under-investigated (Paoletti, 2006): not only have fewspecies been studied, but also our knowledge on the mechanismsunderlying their capacity to tolerate ozone stress is very fragmen-tary.While for many crops and deciduous plants exposure- or dose-response functions describing the effects of ozone have beenestablished (ICPModelling andMapping, 2004), these functions arestill lacking for typical Mediterranean species.

It is well-known that ozone effects on plants depend not onlyon the pollutant dose (concentration� exposure duration) enteringthe plant, i.e., on the ozone flux (Matyssek et al., 2007), but also

V. Calatayud et al. / Environmental Pollution 158 (2010) 3580e3587 3581

on the balance between the flux and effectiveness of the defencemechanisms to cope with oxidative stress (Paoletti et al., 2008;Castagna and Ranieri, 2009). Therefore, in order to better under-stand how this pollutant affects plants, both physiological (espe-cially gas exchange) and biochemical (antioxidant systems) aspectshave to be taken into account. While there is extensive literature onthe effects of ozone on broadleaf deciduous species, as mentionedabove, information for broadleaf evergreen shrubs is still scarce(e.g., Bussotti et al., 2003; Reig-Armiñana et al., 2004; Nali et al.,2004; Paoletti et al., 2009). As far as we know, a direct compar-ison between evergreen and deciduous shrub responses under thesame ozone exposure conditions has not been carried out. In thepresent paper, we compare the responses of two pairs of decid-uous-evergreen species in terms of visible injury, gas exchange,chlorophyll fluorescence, SPAD measurements, and antioxidantcapacity. Comparability between both types of plants is assured byusing two pairs of congeneric species. This is an important aspect,as some of the characteristics (e.g., constitutive antioxidant levels)and types of effects (e.g., enhanced senescence or not, type ofvisible injury, physiological responses) are expected to be genus-related. The experiment has been carried out under well-wateredconditions, in order to achieve optimal conditions for ozone uptake.To the best of our knowledge, this is the first time in which anti-oxidant metabolites have been studied in any of the four speciesconsidered in relation to ozone stress.

Specifically, we test the following hypotheses: 1) Evergreenspecies are more tolerant to ozone that related deciduous species(Paoletti, 2006). 2) Stomatal conductance may explain differencesin ozone sensitivity between evergreen and deciduous species(Reich, 1987). 3) Sensitivity of the species is related to its consti-tutive or induced antioxidant levels (Nali et al., 2004).

2. Materials and methods

2.1. Plant material

Plant seedlings were obtained from several Spanish nurseries. Plants of a similarsize (1 or 2 years old) were selected. Ten-liter containers were filled with 70% peat,15% sand, and 15% vermiculite, soil pH being close to 7.0. A slow release fertilizer wasincorporated (Osmocote plus), with NPK 20:20:20 and additional micronutrients.Plants were irrigated twice a day using a droplet irrigation system.

2.2. Open top chambers and treatments

The experiment was conducted in an OTC experimental field (“La Peira”) locatedin a rural area, in Benifaió (39�160 14.800N, 00�26059.600W), at an altitude of 30 m. Airquality inside and outside the chambers was continuously monitored at regularintervals with an ozone monitor (Dasibi 1008-AH, Environmental Corp.); thesemonitors were calibrated periodically. Plants were placed in three Open TopChambers (OTC), with two ozone treatments: Charcoal Filtered air (CF), and Non-Filtered air plus 30 ppb ozone (NFþ). Plants were fumigated 8 h a day, from 10:00 to18:00 CET, during the whole week. Ozone was generated from oxygen using a high-voltage electrical discharge generator (Sir sa). The experiment started on 18 May2005, and ended on 16 September 2005. The critical level for ozone, accumulatedexposure over a threshold of 40 ppb, was calculated according to the methodsdescribed by the EU 2002/3/EC Directive (EU, 2002), using mean hourly values from8:00 CET to 20:00 CET. Ozone concentration data of from experimental site(ambient) and treatments are provided in Table 1. The AOT40 value of 35 755 ppb h

Table 1Mean ozone concentrations for different daily time windows, maximum hourlyvalue, and cumulative ozone exposures AOT00 and AOT40. The 8 h window, from 10to 18 h, covered the 8 h in which plants of the NFþ treatment were exposed toincreased ozone levels. CF ¼ Charcoal Filtered Air; NFþ¼Non Filtered Air þ 30 ppbozone. Ambient air is not a treatment but refers to the ozone levels measured at theexperimental site outside the Open Top Chambers.

24 h mean(ppb)

12 h mean(ppb)

8 h mean(ppb)

hourly max(ppb)

AOT00(ppb*h)

AOT40(ppb*h)

CF 11 9 12 31 31 403 0NFþ 40 63 76 110 11 5293 35 733Ambient 31 44 48 86 91865 10 664

of the NFþ treatment is slightly above the AOT40 values regularly measured in innermountain areas of eastern Spain (e.g., up to 30 000 ppb h in Morella station someyears, data of the regional Air Quality Network), although well above the valuesmeasured at the experimental site, which is placed in an area with particularly lowozone levels.

2.3. Specific leaf area

At the beginning of the experiment, mature leaves were collected for calculationof dry weight and Specific Leaf Area (SLA); the latter was calculated as SLA ¼ Leafsurface (mm2)/Dry weight (mg). Leaf dry weight was determined by oven-dryingleaves at 60 �C to stable weight, and leaf surface was assessed by image analysis.

2.4. Visible injury assessment

All the plants were examined once aweek for ozone injury symptoms, recordingthe first date of symptom onset in each plant, the percentage of affected leaves perplant (LA), and the percentage of area affected for the symptomatic leaves (AAr). Thelatter was scored using a 5% step scale. To evaluate whole plant injury, a Plant InjuryIndex (PII) was calculated combining these two parameters: PII ¼ (LA � AAr)/100.

2.5. Gas exchange measurements

Gas exchange was measured with an infrared gas analyzer (IRGA) (LICOR-6400,Li-cor Inc., Lincon, NE, USA) in 8 plants per species and treatment. Block temperatureof the cuvette was fixed at 25 �C, and photon flux density (PPFD) was set at a satu-rating intensity of 1200 mmol / m2 s. All measurements were taken during themorning. Tracking of gas exchange in the same marked leaves was carried outmonthly (20 May, 21 June, 20 July and 23 August) after the starting of fumigation.The gmax for water value has been chosen by taking the 95th percentile of all thevalid values of stomatal conductance.

2.6. A/Ci curves

The response of the assimilation rate to changing intercellular CO2 partialpressure (i.e., A/Ci response curves, not shown) was measured on 29e30 August2005 in 4 representative mature attached leaves per plant and ozone treatment.While these leaves showed no apparent visible injury, symptoms were sometimespresent in other leaves of similar age. An infrared gas analyzer (IRGA) (LICOR-6400,Li-Cor Inc., Lincon, NE, USA) was used for the measurements. Maximum rate ofRubisco carboxylation (Vc,max), maximum RuBP regeneration capacity mediated bylight harvesting and electron transport (Jmax) were estimated by fitting thebiochemical model of Farquhar et al., (1980), with modifications by Sharkey (1985),to A/Ci response curves, using non-linear least-square regression methods. Blocktemperature of the cuvette was fixed at 25 �C, and photon flux density (PPFD) at1200 mmol /m2s. Air relative humidity was kept at about 50%. Photosynthetic rateswere taken into account when the coefficient of variation was lower than 1%.

2.7. Chlorophyll content

Chlorophyll content was measured non-destructively with a portable chloro-phyll meter (SPAD-520, Minolta). This instrument uses measurements of trans-mitted radiation in the red and near infrared wavelengths to provide numericalvalues related to leaf chlorophyll content. The average of 3 measurements wascalculated for each leaf, 2 leaves were measured per plant and 8 plants pertreatment.

2.8. Chlorophyll a fluorescence measurements

In the tracked leaves, modulated chlorophyll fluorescence measurements weretaken at ambient temperature at the same time as gas exchange determinations(n ¼ 8 leaves per species and treatment). Measurements were carried out witha portable fluorometer (PAM-2000, Walz, Effeltrich, Germany). Leaves were dark-adapted for at least 30 min prior to the measurements. After dark adaptation, theminimal fluorescence (Fo) was determined using the measuring light; then,a subsequent application of a saturating flash of white light (0.8 s at 8000 mmol/m2 s)raised fluorescence to its maximum value (Fm). This made it possible to determinethe maximum quantum efficiency of photosystem II (PSII) primary photochemistry,given by Fv:Fm ¼ (Fm � Fo):Fm.

At the end of August, 4 leaves per species and treatment were also selected foranalysis of quenching components using the saturation pulse method (Schreiberet al., 1986). After Fv:Fm determination, as described above, intermittent pulses ofsaturating strong white light (0.8 s at 8000 mmol /m2s) were applied in the presenceof white actinic light (PPFD at 1200 mmol/m2s), so that both the steady-state fluo-rescence value (Fs) and the maximum fluorescence value (Fm0) in the light-adaptedstate were determined; the minimum fluorescence in the light-adapted state (Fo0)was also measured by applying a pulse of far red-light during a brief interruption inactinic illumination. At each saturating pulse, the quenching due to non-photo-chemical dissipation of absorbed light energy (NPQ) was determined according to

Table 2Area, dry weight and Specific Leaf Area (SLA) (mean � SE, n ¼ 22) of the leaves(Viburnum species) or leaflets (Pistacia species) of the four species considered. Foreach column, significant differences between the four species are indicated withdifferent letters (ANOVA, Tuckey HSD test, p < 0.05).

Area (cm2) Dry weight (mg) SLA (mm2/mg)

P. lentiscus 8.29 � 0.47 a 123.51 � 8.28 a 6.94 � 0.29 aP. terebinthus 29.08 � 2.02 b 381.26 � 21.96 b 7.57 � 0.20 aV. lantana 81.03 � 5.54 c 579.41 � 50.96 c 14.78 � 0.65 cV. tinus 9.88 � 0.43 a 112.44 � 5.07 a 8.83 � 0.15 b

V. Calatayud et al. / Environmental Pollution 158 (2010) 3580e35873582

the equation NPQ ¼ (Fm � Fm0) / Fm0 . The coefficient for photochemical quenching(qP), which represents the redox state of the primary electron acceptor of PSII, Qa,was calculated as (Fm0 � Fs) / (Fm0 � Fo). The quantumyield of electron transfer at PSII(FPSII) was estimated as FPSII ¼ (Fm0 � Fs)/Fm0 (Genty et al., 1989), and the quantumefficiency of excitation capture by oxidized reaction centers of PSII was calculatedfrom the equation Fexc ¼ (Fm0 � Fo0)/Fm0 . For analyses, only the data of the last pulsewere considered.

2.9. Antioxidants

Towards the end of the experiment, asymptomatic leaves of the four species werecollected for antioxidant assays. Plants sampleswere collectedbetween11:00 and12:00(GMT), frozen in liquid nitrogen, crushed in a mortar and stored at �80 �C until used.

2.9.1. Ascorbic acid (AA) analysisMeasurements of reduced, oxidized and total ascorbate content in plants were

carried out by an adaptation of the colorimetric method by Gillespie and Ainsworth(2007). About 100 mg of ground frozen tissue was extracted in 1 ml of 6% (w/v)trichloroacetic acid (TCA) and centrifuged at 13 000 g for 5 min at 4 �C. AA wasmeasured from 100 ml of the extract by adding 50 ml of Phosphate Buffer 75 mM pH7.0 and 750 ml of working reagent (TCA 10%, H3PO4 43%, aa0-bipyridl 4%, FeCl3 3%).Samples were incubated for 1 h at 37 �C, and diluted with 1 ml of 6% TCA. Theferrous-dipyridyl complex formed was monitored at 525 nm (Okamura, 1980). Totalascorbic content (AA þ DHA) was also measured by a previous incubation with DTTto reduce DHA to AA (Okamura, 1980). Two determinations were made for eachsample. AA was determined by the use of a standard curve of known quantities ofascorbate (0e1.5 mM), and expressed as nmol AA/g FW.

2.9.2. Total antioxidant capacity (TAC)TAC determinations were carried out by the ferric reducing antioxidant power

(FRAP) assay (Benzie and Strain, 1996), adapted by Kerchev and Ivanov (2008) toallow discrimination between water-soluble (Hydrosoluble Antioxidant Capacity,HAC) and water-insoluble lipophilic antioxidants (Liposoluble Antioxidant Capacity,LAC). 50 mg of frozen tissue were ground and extracted with 1 ml of 0.1% (w/v) TCA.The homogenates were centrifuged at 15 000 g for 30 min and the resultingsupernatants (water-soluble fraction) were analyzed for antioxidant capacity, asreported below. Pellets were incubated with 1 ml of acetone for 30 min, centrifugedat 15 000 g for 15 min and the supernatant collected for analysis (water-insolublefraction). 50 ml of diluted extract (1/50 for Viburnum species, 1/200 for Pistacia) wasused in the FRAP assay, by adding 1.5 ml of freshly prepared FRAP reagent (Benzieand Strain, 1996), and later incubating at 37 �C for 24 h. Finally, the absorbance at593 nm was measured. Two replicate assays were made for each sample. Resultswere calculated by standard curves prepared with known concentrations of Fe2þ

(0e50 mM), and were expressed as mmol Fe2þ/g FW.

2.9.3. Superoxide dismutase (SOD) activityA 50e100 mg aliquot of crushed leaves was homogenized in 1 ml of 50 mM

phosphate buffer (pH 7.0) containing 0.1% Triton X-100 and 1% polyvinylpyrrolidonePVP-40 (w/v) (Drazkiewicz et al., 2007). The homogenate was centrifuged at15 000 g for 15 min at 4 �C, and the supernatant was immediately used for SODassays. Total SOD [EC 1.15.1.1] activity was determined according to the method ofBeauchamp and Fridovich, 1971, based on the ability of SOD to inhibit the reductionof nitroblue tetrazolium (NBT) by the O�

2 generated by the xanthine/xanthineoxidase system. The reactionmixture contained 50� 10�3 M Na2CO3/NaHCO3 buffer(pH 10.2), 1 �10�4 M EDTA, 1 �10�4 M xanthine, 2.5 � 10�5 M NBT and 12.5 � 10�3

units / ml of xanthine oxidase. One unit of SOD was defined as the amount of theenzyme that inhibited the rate of NBT reduction by 50%. SOD activity was expressedas units /mg protein). Three determinations were made for each sample.

2.9.4. Peroxidase (GPX) activityAbout 50e100 mg of frozen, ground leaf tissue was extracted with 1 ml of

100 mM phosphate buffer (pH 7.0), and then centrifuged at 15 000 g for 15 minat 4 �C. The supernatant was diluted with extraction buffer as required and assayedfor cytosolic guaiacol peroxidase (GPX) [EC 1.11.1.7] activity. GPXwas measured in anassay containing 100 mM phosphate buffer pH 7.0, 8 mM guaiacol. Reaction wasstarted by addition of H2O2 2 mM, and the rate of tetraguaiacol production wasmonitored by absorbance measured at 470 nm, using an extinction coefficient of26.6 mM�1 cm�1 (Polle et al., 1990; Maehly, 1955). Activity was calculated as mmolstetraguaiacol /(minmg protein). Each sample was assayed by triplicate.

2.9.5. Total proteinProtein in leaf extracts was determined using Bradford’s reagent (Sigma) under

manufacturer-recommended conditions, which involves constructing a standardcurve with known quantities of bovine gamma-Globulin (Bio-Rad).

2.10. Statistical analyses

Depending on the type of data, for two level analyses, independent t-test orManneWithney U test were applied. For more than two cases, one-way Analysis of

Variance (ANOVA) followed by LSD or Tuckey HDS tests, or KruskaleWallis H testfollowed by pairwise multiple comparisons were carried out. A probabilitylevel < 0.05 was considered statistically significant. Data were analyzed using SPSS10.0 for Windows (SPSS Inc.).

3. Results

3.1. Morphological traits of the species

The two evergreen species (P. lentiscus, V. tinus) are character-ized by having the smallest leaves or leaflets, while the deciduousspecies showed much larger leaves (Table 2). By far the largest leafarea was that of V. lantana, with leaves or about 30 cm2. Under theexperimental growing conditions, with well-watered plants,Viburnum species developed leaves with significantly higher SLAthan Pistacia species. SLA of V. lantana was about two times higherthan that of the other species, i.e., its leaves presented the lowestweight per surface unit. Although SLA in P. terebinthus was higherthan in P. lentiscus, differences between both species were notsignificant.

3.2. Symptom onset and development

At the end of the experiment, all species except V. tinus showedvisible injury. The first symptoms were observed in V. lantana, afteronly 6 days of fumigation (AOT40 ¼ 2755 ppb h). In P. terebinthus,symptom onset began after 22 days (AOT40 ¼ 7621 ppb h) and inP. lentiscus began after 62 days (AOT40 ¼ 19 798 ppb h). At the endof the experiment, all (12 out of 12 plants in V. lantana) or almost allplants (11 out of 12 in P. terebinthus) of the deciduous species weresymptomatic, while very scarce symptoms were observed in 3P. lentiscus plants. In V. lantana, ozone induced leaf reddening, whilein P. terebinthus and in P. lentiscus it caused brown stippling,sometimes with associated chlorosis. It has to be noted that underthe experimental conditions, P. terebinthus developed relativelylarge and thin leaves in comparison with leaves of plants grownunder water-stress conditions, which seem to be more ozone-tolerant (García-Breijo et al., 2008). The evolution of visible injury(Fig. 1) was consistent with the results of ozone onset: symptoms inthe two deciduous species were by far higher than in evergreenspecies.

3.3. Stomatal conductance

In all species, stomatal conductance (gs) experienced a declinefrom the beginning (MayeJune) to the end (JulyeAugust) of theexperiment, when increasing temperatures and associated higherVPDvaluespromote stomatal closure.Mean gs values throughout theexperiment, separating ozone treatments and periods (MayeJuneand JuneeJuly) are provided in Table 3. Under the well-irrigatedconditions of thepresent experiment, the rankingof species from thehighest to the lowest gs was not related to the leaf habit strategy(evergreen vs. broadleaves) of the plant. Stomatal conductancevalues were higher in both Pistacia species than in Viburnum,

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Fig. 1. Onset and evolution of visible injury in the four species considered (mean � SE,n ¼ 12).

V. Calatayud et al. / Environmental Pollution 158 (2010) 3580e3587 3583

especially at the beginning of the experiment (May and June). Inconsonance with the above mentioned results, the maximumstomatal conductance (gmax), a parameter which greatly affects themagnitude of ozone fluxes (e.g., Emberson et al., 2007) was likewisenot related to the leaf habit strategy of the plant (Table 3).

3.4. Evolution of gas exchange, chlorophyll, and fluorescenceparameters

Although all species experienced a tendency towards a reduc-tion in Asat at the end of the experiment, a significant reduction inCO2 assimilation was only observed in the two deciduous species.This reduction, which had already started in July, was followed bya decline in gs in August. Internal CO2 concentration increasedsignificantly in P. terebinthus (Fig. 2).

Consistent with the visual observation of leaf yellowing inP. terebinthus as an ozone response, chlorophyll content and Fv:Fm(Fig. 2) significantly declined in this species. In V. lantana, Fv:Fmtended to decrease, although not significantly, but other fluores-cence parameters were also altered (see below).

3.5. Physiological changes in pre-injured leaves

The two deciduous species experienced a significant decline inthe maximum rate of Rubisco carboxylation (Vc,max), and in themaximumRuBP regeneration capacitymediated by light harvestingand electron transport (Jmax) (Table 4). By contrast, in the twoevergreen species only slight, non-significant reductions in thesetwo parameters were observed. In P. terebinthus, changes in Rubiscowere paralleled by a significant decline in Fv:Fm (as was alsoobserved in tracked leaves). In V. lantana, significant reductionsin FPSII and in the coefficient for photochemical quenching (qP)were observed. That is, there was a reduction in the proportion ofabsorbed energy being used in photochemistry (FPSII) and in the

Table 3Stomatal conductance (gs) values under light-saturating conditions for different species (mperiods are considered separately), and gmax (95th percentile, n¼ 50e63). For each columnTuckey HSD test, p < 0.05).

gs (mmol H2O/m2 s)

CF & NFþ CF NFþP. lentiscus 123 � 9 b 126 � 12 ab 120 � 13 aP. terebinthus 123 � 8 b 133 � 10 b 113 � 13 aV. lantana 89 � 5 a 93 � 7 a 84 � 7 aV. tinus 99 � 6 ab 109 � 9 ab 90 � 7 a

proportion of open PSII reaction centers (qP). Quenching due tonon-photochemical dissipation of absorbed light energy (NPQ) wasnot affected significantly.

3.6. Antioxidant capacity

If the antioxidant levels measured with the FRAP assay incontrol plants (CF) are considered as indicative of the constitutivelevels of the species, the total antioxidant capacity (separated intoHAC and LAC) of both Pistacia species was much higher than inViburnum species (Table 5). The same is true for total (AA þ DHA)and reduced ascorbate (AA). The ozone treatment affected the twoevergreens, but by inducing opposite changes: a significantincrease in HAC and AA in V. tinus and a decrease in HAC in P. len-tiscus. Differences in AA contribute to the observed differences inHAC only to a certain degree: about 8% of HAC in Pistacia and about3% in Viburnum can be explained by AA content.

Constitutive (those of control plants) levels of SOD and GPXwere genus-related: SOD activity was much higher in Pistacia thanin Viburnum species, but the contrary occurred for GPX. Comparingpairs of species of the same genus, SOD activity was higher in thedeciduous species than in their evergreen relatives, and the samewas true for GPX in Viburnum species (Fig. 3). Ozone exposuresignificantly increased SOD levels in all species except V. lantana,while in both Viburnum species GPX activity increased significantly.In contrast, GPX activity was practically undetectable in both Pis-tacia species (not represented).

4. Discussion

Visible injury responses confirmed a much higher ozone toler-ance in the two evergreen species than in their two deciduousrelatives [photos available at Sanz and Calatayud (accessed on 20January 2010)]. Anatomical responses to enhanced ozone levels inPistacia species have been studied elsewhere (Reig-Armiñana et al.,2004 and García-Breijo et al., 2008), showing similar effects butdiffering in intensity. V. lantana is considered a very sensitivespecies, frequently showing symptoms in the field in areas expe-riencing high ozone levels (e.g., Novak et al., 2003). V. tinus wasfumigated at concentrations up to 120 ppb for 6 weeks withoutdeveloping symptoms (Orendovici et al., 2003), which is consistentwith the low sensitivity shown in the present study.

Results from the physiological measurements are also consis-tent with previous conclusions. Significant reductions in Asat and gsand related changes in Rubisco carboxilation efficiency (Vc,max) andregeneration capacity (Jmax) were observed only in ozone-exposedplants of the two deciduous species. Rubisco-related biochemicallimitations are involved in the decline in CO2 assimilation in ozonefumigated plants (e.g., Farage and Long, 1999; Enyedi et al., 1992;Pell et al., 1994; Reddy et al., 1993), and in the increase of Cilevels (as observed in P. terebinthus), which in turn can promotestomatal closure (Mikkelsen, 1995; Calatayud et al., 2007).A decrease in Rubisco may also trigger a series of senescence events

ean � SE, n ¼ 50e63, all measurements; or n ¼ 30e32, when the two treatments or, significant differences between species are indicatedwith different letters (ANOVA,

gmax (mmol H2O/m2 s)

May & June June & July

156 � 12 b 94 � 10 a 229.2163 � 10 b 83 � 7 a 216.2102 � 6 a 74 � 7 a 149.0119 � 6 a 77 � 9 a 161.8

**

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P. lentiscus P. terebinthus V. lantana V. tinus

Fv:F

m)

FC

fo

%(

Fig. 2. Gas exchange (Asat, gs, Ci), chlorophyll content (SPAD) and fluorescenceparameters (Fv:Fm). Data represented are NFþ / CF ratios, expressed as percentages.Significant differences between the CF and the NF þ treatment at the differentmeasuring times are indicated as *p < 0.05, and **p < 0.01 (t-test, mean � SE, n ¼ 8).

Table 4Rubisco-related and fluorescence parameters. Data are NFþ/CF ratios, expressed aspercentages. For each column and species, significant differences between the CFand the NFþ treatment are indicated with *p < 0.05 (ManneWithney U test,mean � SE, n ¼ 4).

Rubisco-relatedparameters(% of CF)

Fluorescence parameters (% of CF)

Vc,max Jmax Fv:Fm Fo Fm FPSII qP NPQ Fexc

P. lentiscus 80 82 97.2 94.6 84.0 91.2 94.1 97.1 93.0P. terebinthus 52* 39* 90.7* 123.3 89.2 70.1 82.4 101.9 87.4V. lantana 58* 34* 90.9 111.5 87.4 51.8* 60.3* 93.6 86.5V. tinus 86 89 99.1 112.2 107.1 78.6 82.6 94.7 97.1

V. Calatayud et al. / Environmental Pollution 158 (2010) 3580e35873584

(Pell et al., 1994; Crafts-Brandner et al., 1990), as those observed inP. terebinthus: chlorophyll content was reduced and Fv:Fm declined,indicating photoinhibitory damage to PSII reaction centers(Maxwell and Johnson, 2000). Degradation of thylakoid proteinsand effects in the light harvesting system occurs during senescence(Humbeck et al., 1996). In the case of V. lantana, the observeddecline in the quantum yield of electron transfer at PSII (FPSII) andin the photochemical quenching coefficient (qP) may result froma down-regulatory process associated with an inhibition of theCalvin cycle (Calatayud et al., 2007).

It has been argued that the most appropriate way to explainplant ozone responses is the effective ozone flux (Paoletti et al.,2008). This approach considers the balance between flux anddefence, i.e., the capacity of the detoxification mechanisms tocounteract the deleterious effects of the ozone taken up by theplant. On this basis, both gas exchange and (enzymatic and non-enzymatic) antioxidant systems have been taken into account inthe present study so as to better understand ozone responses.

With regard to the stomatal conductance component, thepresent study shows that, under well-irrigated conditions, thehypothesis that the sensitivity of a species was related to gs (Reich,1987) or to gmax, which is the main driver of ozone flux (Embersonet al., 2007), is not supported.

The study of non-enzymatic antioxidative systems (TAC, sepa-rated in HAC and LAC, and AA and total ascorbate) shows importantdifferences between constitutive (i.e., those of control plants) levelsof HAC and AA between genera. The FRAP assay provides a generalpicture on the TAC of the plant, while AA levelsmay vary in responseto environmental stress, scavenging harmful radicals produced inendogenous metabolism (Smirnoff, 2000). The much higher HAC inboth Pistacia species is most likely related to the accumulation ofantioxidant molecules such as polyphenols (Topçu et al., 2007;Atmani et al., 2009), with AA (likewise a hydrosoluble antioxi-dant) also contributing moderately; in tea leaves, a high correlationbetweenpolyphenols and the antioxidant powermeasured by FRAPwas demonstrated (Liu et al., 2009). Therefore, constitutive non-enzymatic antioxidant levels were not related to the plant capacitytowithstand ozone stress. A correlation between total AA levels andozone tolerance could be demonstrated in several studies (e.g.,Ferreira-Severino et al., 2007; Scebba et al., 2003), especially whenonly the apoplastic component e the first line of defence againstozonee was considered (Lyons et al., 2001; Castagna and Ranieri,2009), but not in others (D’Haese et al., 2005). Ozone inducedsignificant changes in HAC and total AA in both evergreen species,but in opposite directions: while in V. tinusHAC and total AAmay berelated to an active defence response by this plant against ozone, thereduction of HAC in P. lentiscus suggests oxidation of antioxidantmolecules (e.g., altered tannins, Reig-Armiñana et al., 2004).

Complementarily, two members of the enzymatic antioxidativeresponse (SOD and GPX) were studied. Both enzymes play

Table 5Non-enzymatic antioxidant responses in Viburnum and Pistacia species: Hydrosoluble (HAC) and liposoluble (LAC) antioxidant capacity determined by the FRAP assay; total(AA þ DHA) and reduced (AA) ascorbate content, and AA redox state (AA / AAþDHA). For species of the same genus, statistical differences are indicated by different letters(mean � SE, n ¼ 4e8, Mann Whitney U test followed by pairwise comparisons, p < 0.05).

HAC(mmol Fe2þ/g FW)

LAC(mmol Fe2þ/g FW)

Total ascorbate (AA þ DHA)(nmol/g FW)

Reduced ascorbate (AA)(nmol/g FW)

Ascorbate redox state(AA/(AA þ DHA)) (%)

P. lentiscus CF 2011 � 100 b 636 � 35 a 332 � 27 a 166 � 16 a 50 � 1 aNFþ 1349 � 68 a 646 � 53 a 271 � 28 a 128 � 15 a 47 � 1 a

P. terebinthus CF 1793 � 69 b 737 � 39 a 288 � 23 a 128 � 15 a 44 � 4 aNFþ 1956 � 79 b 676 � 115 a 348 � 14 a 147 � 6 a 42 � 0 a

V. lantana CF 429 � 24 a 219 � 19 a 18 � 1 a 13 � 0 a 72 � 1 aNFþ 382 � 34 a 203 � 34 a 18 � 2 a 13 � 1 a 73 � 1 a

V. tinus CF 461 � 19 a 215 � 28 a 14 � 1 a 10 � 0 a 75 � 1 aNFþ 574 � 60 b 259 � 30 a 21 � 2 b 15 � 1 a 74 � 3 a

V. Calatayud et al. / Environmental Pollution 158 (2010) 3580e3587 3585

important roles in the cellular defence strategy against oxidativestress (Castagna and Ranieri, 2009). Results of the present studysuggest that, at least towards the end of the growing season,deciduous species could present a constitutively higher presence ofintracellular superoxide-generating and peroxide-generatingprocesses than their evergreen relatives. This could render theirleaf cells more prone to imbalances in their redox state whenexposed to enhanced ozone. The uptake of this pollutant wouldgenerate different ROS species, promoting SOD and/or GPX activity,and triggering several pathways, such as pathogen-like response,oxidative burst or ROS signaling, which in combination would giverise to the visible ozone response in leaves (Heath, 2008), and mayalso accelerate senescence processes (Bhattacharjee, 2005). Inbeech, Polle et al., (2001) consistently found higher SOD activitylevels in stress-sensitive than in stress-resistant mature leavesbefore being submitted to oxidative stress. A more favorable redoxstate in evergreen shrubs would allow them to better cope withROS increases caused by ozone entry, maintaining them below thethreshold that triggers visible responses. Such a state may berelated to a metabolically higher capacity to counteract oxidativestress (resulting from an adaptation to habitats with high UV and

S

b

a

b

c

0

2000

4000

6000

8000

10000

12000

P. lentiscus P. terebinthus

)ni

et

or

pg

m/s

tin

u(

yti

vit

ca

DO

S

ab

c

0

100

200

300

400

500

V. lantana

)ni

et

or

pg

m/s

tin

u(

yti

vit

ca

XP

G

Fig. 3. Enzymatic antioxidant responses in leaves Viburnum and Pistacia species: Superoundetectable in both Pistacia species (not represented). For species of the same genus, statisttest followed by pairwise comparisons, p < 0.05).

light radiation or with drought periods which are known to favorROS formation, e.g., Davey et al., 2002; Cruz de Carbalho, 2008)and/or to constitutional avoidance: the development of thick,coriaceous and sclerophyllous leaves with strongly lignified wallsand a compact mesophyll with few intercellular air spaces i.e., lessexposed cell surface area may complicate the entrance of ozoneinto the cells (Lyons et al., 2001; Paoletti, 2006). An overlapping ofozone tolerance with the development of coriaceous leaves and thelevel of drought tolerance has been observed both in deciduous andin evergreen species (Nali et al., 2004; Calatayud et al., 2007).Pathways triggered as ozone responses varied between species:three of them increased SOD activity in response to superoxide,while in the two Viburnum, a GPX induction suggest peroxidegeneration, which can be acting as a propagation signal and/ortaking part in cell wall changes. Previous studies have also shownthat generation and accumulation of superoxide and/or peroxidemay differ among species (Wohlgemuth et al., 2002). In summary,as shown here, the study the antioxidant systems can significantlycontribute towards a better understanding of the ozone responsesof plants. However, given the complexity of the processes involved,we are still far from having a unified and consistent framework

ba

bb

V. lantana V. tinus

ab

V. tinus

CF NF+

xide dismutase (SOD) and peroxidase (GPX) activities. GPX activity was practicallyical differences are indicated by different letters (mean � SE, n ¼ 4e8, Mann Whitney U

V. Calatayud et al. / Environmental Pollution 158 (2010) 3580e35873586

with regard to the effective ozone flux that could be used formodelling and assessment purposes (Paoletti et al., 2008).

In terms of the current standards for protection of vegetation, itis noteworthy that evergreen species were able to withstand anAOT40 which was more than sevenfold above the critical level(CLec) of 5000 ppb h (OAT40) without developing symptoms (orhaving very scarce symptoms), and with limited physiologicalresponses. Although this CLec is intended to protect sensitivespecies from growth losses (ICP Modelling and Mapping, 2004),such a low value is exceeded over a large part of the Mediterraneanarea, and might be unrealistic as a risk indicator (Ferretti et al.,2007), especially for evergreen vegetation types (cf. also Naliet al., 2004). The development of exposure- and dose-responsefunctions, ideally taking into account a drought period, wouldcertainly contribute towards the establishment of more realisticCLe. The case of evergreens, however, cannot be extrapolated forthe whole Mediterranean area, a diverse territory in which somerelatively sensitive deciduous species grow, e.g., P. terebinthus, asdocumented here, Acer opalus subsp. granatense (Calatayud et al.,2007), or Quercus pyrenaica (Calatayud et al., 2009).

Acknowledgements

We thank the Ministerio de Medio Ambiente y Medio Rural yMarino, Conselleria de Medi Ambient, Aigua i Habitatge and FEDERfunds (project VegetPollOzone, Interreg IIIb, Meddoc) for support-ing parts of this study. Fundación CEAM is partly supported byGeneralitat Valenciana, Fundación Bancaja, and benefits form theprojects CONSOLIDER-INGENIO 2010 (GRACCIE) and PrometeoProgram (Generalitat Valenciana). Carmen Martín is thanked fortaking care of the plants.

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