Responses of Fraxinus excelsior L. seedlings to ambient ozone exposure in urban and mountain areas...

Environmental ScienceProcesses & Impacts

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aInstitute of Biodiversity and Ecosystem Re

Gagarin Street, Soa 1113, Bulgaria. E-ma

bg; Tel: +359 28717195/301bDepartment of Forestry, University of Fores

Bulgaria. E-mail: nikolina_tzvetkova@ma

291907-441cShumen University – Department of Biolog

Bulgaria. E-mail: [email protected]; Tel: +35dUniversity of Soa – Department of Mete

Boulvrd, Soa 1164, Bulgaria. E-mail: done

Cite this: Environ. Sci.: ProcessesImpacts, 2013, 15, 1452

Received 29th July 2012Accepted 13th May 2013

DOI: 10.1039/c3em30614c

rsc.li/process-impacts

1452 | Environ. Sci.: Processes Impacts

Responses of Fraxinus excelsior L. seedlings to ambientozone exposure in urban and mountain areas based onphysiological characteristics and antioxidant activity

Petya Parvanova,*a Nikolina Tzvetkova,b Svetla Bratanova-Doncheva,*a

Nesho Chipev,c Radka Fikovaa and Evgeni Donevd

Effects of ozone on the sensitive tree species Fraxinus excelsior L. exposed to ambient air were investigated.

The dynamics of photosynthesis, transpiration, stomatal conductance and the activity of the antioxidant

enzymes superoxide dismutase (SOD) and catalase (CAT) in three-year-old ash seedlings were studied

during a four-month period (June–September). Seedlings were exposed to ambient ozone in an urban

(the Central City Park of Sofia – Borisova Gradina) and a mountain (Plana Mountain) area in Bulgaria.

The sites were located near climate monitoring stations, providing data on ozone concentrations and

meteorological parameters. Ozone exposure at the mountain site (AOT40) was more than two times

higher compared to the urban site. Significantly higher values of sun radiation, transpiration, stomatal

conductance and enzyme activity at the mountain site were also observed. At the urban site higher

values of temperature and air humidity were registered. Effects of the measured variables on ash

seedlings were complex and interdependent. No direct effect of ozone concentration in ambient air on

the leaf physiology and biochemistry could be proved. However, intensified SOD and CAT activity in the

presence of elevated ozone suggested antioxidant reaction in response to ozone uptake.

Environmental impact

The ozone sensitivity of tree species is affected by genetic factors, making it difficult to establish a direct correlation between exposure and induced effects.Competitive interaction with environmental factors oen leads to the dominance of ozone-tolerant species and reduces biodiversity in natural habitats.Southern Europe is characterized by higher ozone concentrations in comparison with northern Europe. This research can contribute to the European researchon the ozone effects on widespread tree species in different regions of Europe. This study is relevant to a relatively new study method of tree seedlings as a modelsystem for the analysis of complex physiological and biochemical changes in the exposure to ambient ozone in urban and mountainous environments.

Introduction

Tropospheric ozone (O3) is being currently considered as themost important phytotoxic photochemical air pollutant in mostparts of the world, as it causes signicant injury and damage toboth cultivated and native tree species.1–5 Ozone precursors areoen produced in urban areas and subsequently they can betransported to long distances by prevailing winds. High O3

concentrations affecting the growth and productivity of forests

search – Bulgarian academy of science,

il: [email protected]; sbrat@abv.

try, 10 Kliment Ohridski av., Soa 1756,

il.bg; Fax: +359 28622830; Tel: +359

y, 115 Universitetska Str., Shumen 9712,

9 88831687

orology and Geophysics, James Bouchier

[email protected]; Tel: +359 28161253

, 2013, 15, 1452–1458

are oen reported in rural, remote and mountain regions.6

Some of the negative effects induced by ozone includedecreased chlorophyll content, decline in photosynthesis andstomatal conductance, altered antioxidant activity levels,accelerated leaf senescence, and reductions in total plantbiomass and nutritive yield.1,3 The harmful effects of ozone onplants are principally associated with the cumulative dose takenup through the stomata.7 This uptake can cause degradation ofcell membranes particularly in photosynthetic cells.7

Ozone is one of the most powerful oxidants.11 Antioxidantmetabolism is considered to be a critical component of plantresponse to O3 stress. Antioxidant's changes have been sug-gested to take part in cellular repair processes, which alleviatethe initial oxidative damage caused by O3 especially on theplasma membrane structure and function.40

Common ash (Fraxinus excelsior L.) is a deciduous broad-leaved tree species, very common in the temperate mountainareas of Europe. It can be found at the edge of forests, in openareas and city park-lands. This tree species was proved to be

This journal is ª The Royal Society of Chemistry 2013

http://dx.doi.org/10.1039/c3em30614c

http://pubs.rsc.org/en/journals/journal/EM

http://pubs.rsc.org/en/journals/journal/EM?issueid=EM015007

Paper Environmental Science: Processes & Impacts

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sensitive to ozone;19–21 this sensitivity refers mostly to foliarinjury.

Most of the information regarding ozone effects on trees wasprovided by controlled experiments with tree seedlings. Fewstudies have been conducted under eld conditions. It istherefore important to monitor the response of plants exposedto ambient ozone.

The aim of the present investigation was to examine theeffects of O3 levels in ambient air on the physiological processesand oxidative stress in ash seedlings experimentally exposed toambient air at a mountain and an urban site.

Materials and methodsSites, plants and experiments

The study was carried out using three-year-old ash seedlingsexposed to ambient ozone at a mountain and an urban site inthe summer of 2009. The experimental sites were chosen to besituated near the Air Quality Monitoring Stations in the CentralPark of Soa city (Station Borisova Gradina) and in the PlanaMountain (Station Plana) – near Soa. Continuously workingautomatic climate stations provided 1 h O3 averages along with1 h averages of air temperature, relative humidity, general solarradiation, wind speed and direction.

Station Borisova Gradinawas situated in the Central Park of thecity of Soa not far from the city center, at an altitude of 540 ma.s.l. (42�400390 0N, 23�2003100E). The area is characterized by slowcirculation of air masses due to low wind speed.41 The meanannual temperature was 10 �C and precipitation was 570 mm. Alot of windless days and strong retention of air pollutants for along timewere common in this area. The parklandwas inuencedby different types of pollution sources. Ozone precursors in theregion came mainly from heavy traffic and also from organiccompounds emitted by trees. The vegetation was representedmainly by broadleaved tree species (horse-chestnut, sycamore,ash-tree, plane-tree, acacia, lime-tree, ornamental shrubs) andalso by some coniferous (pine-trees, r, spruce) and grasses. Theparkland Borisova Gradina was considered as an urban site.

Station Plana was situated in the Plana Mountain near Soaat an altitude of 1234 m (42�28034.6500N, 23�2503900E). The sitehad moderate-continental climate, with mean annual temper-ature varying between 6 and 8 �C and average sum of precipi-tation 800–850 mm per year. Station Plana was considered as anon-urban (mountain) site.

One-year-old seedlings of Fraxinus excelsior were transplantedfrom the nursery and grown as monocultures in greenhouse 6-lplastic pots, lled with sand/potting classical soil mixture.Seedlings were irrigated regularly. Plants of a similar size (3 yearsold) were selected. In the beginning of May 2009 six pots con-taining one seedling each were exposed at each site. Air qualitywas continuously monitored at regular intervals using ozonemonitors. These monitors were calibrated periodically.

Gas exchange measurements

Gas exchange was measured with an infrared gas analyzer(IRGA) (LICOR-6400, Li-cor Inc., Lincoln, NE, USA) in 6 plants


per treatment. The block temperature of the cuvette was xed at25.0 �C, photon ux density (PPFD) was set at a saturatingintensity of 1100 mmol m�2 s�1, a relative humidity of 50% and350 mmol mol�1 CO2. All measurements were taken in themorning. Tracking of gas exchange in the same marked leaveswas carried out monthly (30 June, 27 July, 20 August and 20September). Three fully expanded and developed leaves fromeach experimental plant were analysed, taking 20 measure-ments per leaf. The leaves were acclimated to the cuvette forabout 60 s before every measurement.

Enzyme assay

Three leaves from each plant were sampled in the same timeperiod during which physiological measurements were carriedout. The leaves were collected so as to be adjacent to the leaves,which were analysed for physiological activity. Aer cuttingthem, they were frozen in liquid nitrogen.

A 100 mg aliquot of crushed leaves was homogenized in10 ml of 0.1 M phosphate buffer pH 7.8. The homogenate wascentrifuged at 15 000g for 20 min at 4 �C, and the supernatantwas immediately used for SOD assays. The total SOD [EC1.15.1.1] activity was determined according to the method ofBeyer and Fridovich (1987 (ref. 22)) based on the ability of SODto inhibit the reduction of nitroblue tetrazolium (NBT) by theO2

� generated by the xanthine/xanthine oxidase system. Oneunit of SOD was dened as the amount of the enzyme thatinhibited the rate of NBT reduction by 50%. SOD activity wasexpressed as units per mg protein.

For the analysis of CAT activity 100 mg of the plant materialwas homogenized in 3 ml of 0.1 M phosphate buffer and wascentrifuged at 15 000g for 20 min at 4 �C. The reaction wasstarted by adding 250 ml of 40 mM H2O2 and the decrease ofH2O2 at 240 nm was followed for 3 min.23 CAT activity wasexpressed as units per mg protein.

The total protein concentration was measured with theprotein-dye binding assay24 using a protein assay reagent(Coomassie blue, Pierce) with BSA as a standard.

Data analysis

Statistical analyses were carried out using common sowarepackages (STATISTICA, SYSTAT, SPSS). Means were comparedby the t-statistics and effects were studied using multipleregression techniques. When signicance is noted in this paper,it refers to the statistical signicance at the *p < 0.05 level.

Results and discussionOzone exposure and climatic data

The measured levels of ozone in ambient air are presented inTable 1. The AOT40 index assumes that the harmful O3

concentrations for vegetation are only those exceeding athreshold of 40 ppb. Stomata are opened and more preciselywhen the solar radiation was above 50 W m�2.8 AOT40 remainsthe basis for estimating the potential risk to forests due to O3

and for setting the environmental quality objectives within the

Environ. Sci.: Processes Impacts, 2013, 15, 1452–1458 | 1453


Table

1Clim

aticdata,

ozo

neco

ncentrationan

dAOT4

0fortheJune–

September

periodof20

09at

theurban

site

(Borisova

Gradina)

andthemountain

site

(Plana)

Station

BorisovaGradina

Plan

a

Ozoneconcentration

(ppb

)(m

in–m

ax)

Sunradiation

(Wm

�2)(m

in–m

ax)

Airtempe

rature

(�C)(m

in–m

ax)

Airhum

idity(%

)(m

in–m

ax)

Ozoneconcentration

(ppb

)(m

in–m

ax)

Sunradiation

(Wm

�2)(m

in–m

ax)

Airtempe

rature

(�C)(m

in–m

ax)

Airhum

idity

(%)(m

in–m

ax)

Mean

AOT40

(pbb

h�1)

Mean

Mean

Mean

Mean

AOT40

(pbb

h�1)

Mean

Mean

Mean

Mon

thJun.

23.83(1–81)

1571

249.8(0–100

9)18

.7(8.1–34.4)

70.3

(17.7–97

.1)

50.3*(25–76

)40

92*

255.1*

(0–1

040)

15.4*(7.1–27.4)

73.1*(23.7–

100)

Jul.

25.97(1–98)

2102

258.3(0–985

)21

.0(11.8–38

.5)

66.2

(18.4–96

.9)

47.5*(17–76

)29

99*

273.3*

(0–1

014)

19.0*(8.9–31.0)

57.5*(25.7–

90)

Aug.

18.12(1–87)

887

221.1(0–904

)20

.1(12.1–34

.1)

71.0

(24.2–97

.4)

44.8*(19–72

)20

93*

220.6(0–968

)16

.9*(10.4–27

.6)

70.0

(32.4–

95.5)

Sept.

12.68(1–71)

412

169.8(0–837

)15

.7(5.5–32.8)

76.8

(26.8–96

.8)

38.3*(13–65

)51

4*16

6.0(0–878

)13

.9*(6.6–27.0)

75.7

(29.9–

96.2)

1454 | Environ. Sci.: Processes Impacts, 2013, 15, 1452–1458

Environmental Science: Processes & Impacts Paper

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European Union (EU) and the United Nation EconomicCommission for Europe (UN/ECE).42,43

The overall ozone exposure was signicantly higher at thestation Plana than at the station Borisova Gradina ( p < 0.0001).A trend of the gradual decrease of ozone from June toSeptember at both sites was present. The monthly values ofAOT40 weremaximal in June (4092 ppb) at the station Plana andin July at the station Borisova Gradina (2102 ppb). For the entireexperimental period AOT40 at the mountain station was higherthan the 6-month critical value of 5000 ppb h�1 accepted fortree vegetation.8 Higher ozone concentrations at the stationPlana were probably due to the transfer of a higher amount ofscavenging pollutants from the city. Some authors have foundthat ozone and its precursors could be easily transported fromurban to mountain areas during the periods of high atmo-spheric pressure and low wind speed.26,27

Signicantly higher ozone exposure values in mountainareas have been reported by many authors.12,22,28,29 Furthermore,higher concentrations at night depended on the topographyand remoteness of urban sources.25

Sun radiation was different for the two sites. A signicantlyhigher value of mean sun radiation was found at Plana ( p <0.0001) (Table 1). The difference was mainly due to the higherradiation at Plana during June and July.

During the study period air temperature was constantlyhigher (by 2–3 �C) at the station Borisova Gradina ( p < 0.0001)(Table 1).

Air humidity at Plana was overall signicantly ( p ¼ 0.00073)lower than at Borisova Gradina (Table 1). We assume that this isdue to the urban smog and heat island. The lowest air humidityat both sites was found in July.

Gas exchange

The overall rate of photosynthesis did not differ signicantlyamong the studied sites (Fig. 1a).

Higher photosynthetic activity at the mountain site wasfound only in July. At the urban site the rate of photosynthesiswas lowest in July, when the photosynthetic rate was maximal atthe mountain site. Aer July at both sites the net-photosyn-thesis remained almost constant up to the end of the experi-mental period. A signicant negative partial correlation wasfound between photosynthesis and AOT40 at the urban station(R ¼ �0.700*). Regardless of the higher ozone exposure at themountain site no signicant correlation of photosynthesis andAOT40 was found.

The rate of photosynthesis is assumed to indicate thesensitivity of plants to ozone, because the intensity of thisprocess is related to the antioxidant protection.32 Novak et al.33

observed 18% reduction of the photosynthesis aer exposure ofash seedlings to high ozone concentration. It was reported thatchronic O3 exposure can cause reduction in the intensity ofphotosynthesis.4

Gravano et al.30 suggested that in the leaves of F. excelsior, atleast at the beginning of the exposure, the onset of injurysymptoms was combined with an enhanced photosyntheticefficiency (compensatory mechanism). The increased rate of



Fig. 1 Photosynthesis (a), transpiration (b) and stomatal conductance (c) ofFraxinus excelsior L. foliage exposed at the urban (Borisova Gradina) andmountain (Plana) sites. Mean � SE. * Significance of differences between the sites(p < 0.05).


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photosynthesis found in July at the station Plana, when ozoneexposure was maximal, may be viewed as the initial compen-satory activity. We assume that the more intensive photosyn-thesis results in more synthesized substances in plants,suggesting more efficient antioxidant systems. So the plants willmore easily cope with the stress caused by the maximumconcentrations of ozone. Gerosa et al.31 found that ash trees,being highly ozone-sensitive, begin to show foliar symptoms atAOT40 levels of around 5000 ppb h�1. However, we did notobserve visual injuries at the mountain station in our studydespite the higher values of AOT40.


One of the responses of plants to the effect of ozone is thechange of cellular and tissue turgidity associated with the lossof water. The general rate of transpiration was found to behigher at the mountain station ( p ¼ 0.010) (Fig. 1b). Themaximal rate of ash seedlings transpiration was found inSeptember at the mountain site. Transpiration at the urban siteshowed almost constant values during the entire experimentalperiod (Fig. 1b).

Stomatal regulation is accepted to be an important factor incontrolling ozone uptake. Ash seedlings exposed to the moun-tain air conditions showed an overall higher stomatal conduc-tance ( p < 0.0001) compared to the urban site where thestomata maintained permanently low conductance. A signi-cant increase was observed in September at the mountain site(Fig. 1c). In our experiments the seedlings of F. excelsiordemonstrated lower stomatal conductance during periods ofhigher AOT40 both at the urban and mountain sites. Stomataluptake is generally dependent on a number of environmentalvariables including relative air humidity (vapour pressuredecit), soil moisture, solar radiation, air temperature and mayalso vary among tree species and with their age.

Ozone exposure may reduce stomatal conductance inparallel with photosynthetic decline.9 Novak et al.33 established8% reduction in stomatal conductance at increased ozoneconcentrations. The ability for a rapid closure of stomata maybe one of the mechanisms for decreasing ozone uptake by ashleaves.

Enzyme activity

Ozone is one of the most powerful oxidants.10 Antioxidantmetabolism is considered to be a critical component of plantresponses to O3 stress. A signicantly higher SOD activity wasfound in ash leaves at the station Plana during most of theexperimental period ( p < 0.001) compared to the BorisovaGradina station. Only in June SOD was signicantly more activein seedlings, exposed to urban air.

The lowest SOD activity was found in August at both stations.No signicant correlations between the SOD activity, ozoneexposure and climatic parameters were found at both experi-mental sites.

Antioxidant's changes have been suggested to be part of thecellular repair processes, which alleviate the initial oxidativedamage caused by O3 especially on the plasma membranestructure and function. Borowiak et al.,11 Paoletti et al.,12 andHerbinger et al.13 investigated the potential role of the antioxi-dant enzyme superoxide dismutase as a biochemical marker ofthe impact of tropospheric ozone. Seedlings exposed to ambientair at the station Plana showed signicantly higher CAT activityin comparison with those located at the station Borisova Gra-dina ( p < 0.0001) (Fig. 2b). Strong activation of the enzyme wasestablished in July at the station Plana. The highest CAT activityat the urban station Borisova Gradina was found in June. Asignicant negative correlation between CAT activity and airhumidity (R ¼ �0.863*) was established at the mountain site. Asignicant but lower correlation between CAT activity and airtemperature (R ¼ 0.651*) was also found. In contrast, at the



Fig. 2 SOD activity (a) and CAT activity (b) of leaves of 3-year-old Fraxinusexcelsior L. seedlings exposed to ambient ozone concentrations in urbanand mountain areas. Mean � SE. * Significance of differences between the sites(p < 0.05). Table 2 Results from the multiple forward stepwise regressions for the effect of

ozone and climatic factors on the activity of SOD in the urban and mountain sites

Variables

R ¼ 0.973; F ¼ 465.21 (6, 154); p < 0.0000

Partial corr. R-square t (154) p-level

Air humidity �0.858285 0.884164 �20.6878 0.000000Air temperature �0.694434 0.868565 �11.9375 0.000000AOT40 �0.219508 0.896665 �2.7830 0.006060Transpiration 0.278372 0.666574 0.5850 0.000452Photosynthesis 0.610902 0.707943 9.5445 0.000000Leaf conductance 0.469747 0.733163 6.5818 0.000000Solar radiation 0.225778 0.943922 2.8667 0.004728

Table 3 Results from the multiple forward stepwise regressions for the effect ofozone and climatic factors on the activity of CAT in the urban and mountain sites

Variables

R ¼ 0.973; F ¼ 465.21 (6, 154); p < 0.0000

Partial corr. R-square t (155) p-level

Leaf conductance 0.636 0.510847 10.2535 0.000000Temperature �0.954 0.868278 �39.5422 0.000000Air humidity 0.490 0.882730 �6.9847 0.000000AOT40 �0.943 0.895187 �35.3110 0.000000Solar radiation 0.935 0.943919 32.7747 0.000000Photosynthesis �0.419 0.697053 �5.7295 0.000000


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urban site no signicant relationships between CAT activity andair humidity (R ¼ �0.322), and between CAT activity and airtemperature (R ¼ 0.325) were established. These ndings sug-gested that local synoptic situations could play a role in theinitiation of the antioxidant activity reaction to ozone by plants.

Catalase is one of the best known and most effective anti-oxidant enzymes.14 It is known to play an important role in thedefense against ozone.15 According to many authors, ozonestimulates expression of cell catalase,16,17 but high ozoneconcentrations may inactivate this enzyme.18

The activation of SOD34 and CAT35 can indicate the responseof plants to elevated concentrations of ozone. The low activity ofSOD at the experimental site Borisova Gradina was establishedin July, when a maximum concentration of ozone wasmeasured. Ozone exposure decreased in August and so did theenzyme activity. Ash is considered to have low resistance toozone and this may be due, at least partly, to the inability ofleaves to activate the enzyme in their tissues.36

Ozone concentration in ambient air was signicantly higherat the mountain site. SOD activity was higher during most of theexperimental period. The highest concentrations of ozone atthis site were found in June. However SOD activity was highest


in July when ozone exposure was twofold lower. The stronginduction of SOD in July could be interpreted as a response ofplants to high ozone concentration in the previous month,which provoked activation of the defense system in leaves. Thiscould have been a late response demonstrating the so-called“memory effect”.44

However, no consensus exists on the impact of ozone on theactivity of SOD in the scientic literature at present.

Some authors support the hypothesis that high concentra-tions of ozone stimulate enzyme activity, while others supportthe opposite. According to Tanaka and Sugahara37 increasedresistance of tree plants is correlated with increased SODactivity. Strong induction of SOD in the summer associated withlarger concentrations of ozone and its precursors has beenestablished for other tree species.38 Increased activity of SODwas found to limit the effects of the toxic active oxygen species38

on tissues. Keutgen and Pawelzik39 observed a signicantreduction of SOD activity at high ozone levels both in the ozone-sensitive and ozone-tolerant cultivars of strawberry (Fragariaananassa Duchesne).

Regression analysis

In order to study the complex relationships among themeasured variables the total data matrix for both sites wasanalyzed using the multiple forward stepwise regressionmethod with the two enzymes as dependent variables. Theresults are presented in Tables 2 and 3.

As can be seen from our results the antioxidant systemactivation in ash seedlings was completely dependent on thestudied variables. In general, leaves responded to O3-induced




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stress by antioxidant enzyme system activation and mecha-nisms of avoidance and defense such as restriction of O3 uptakeby stomatal closure or detoxication through biochemicalreactions in the tissues. Hence, the effects of ambient ozone onthe physiological and biochemical mechanisms in the leaves ofash seedlings were not straightforward depending on the otherstudied factors, as well.

Conclusions

No direct effect on the leaf physiology and biochemistry due tothe elevated ozone concentration in ambient air could beproved in our study. However, our results suggested that Frax-inus excelsior L. seedlings did respond to ozone uptake byincreased antioxidant reaction. Antioxidant enzyme activityseemed to be an integral indicator of stress in trees exposed toambient ozone.

Obviously, more investigations are needed to reveal trendsand mechanisms of tree response to ambient ozone. Thephysiological and biochemical indicators which were studied inthis paper may serve as a basis for this future research.

Acknowledgements

This study was funded by the following grants: DO 02-127/2008Projects of the Ministry of Education and Science (Bulgaria),Project 108/2008 of the University of Forestry, Soa and ProjectRD 08-266 of Shumen University.

Notes and references

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Responses of Fraxinus excelsior L. seedlings to ambient ozone exposure in urban and mountain areas...

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