Botanical Studies (2006) 47: 409-416.

*

Corresponding author: E-mail: yananruan@hotmail.

com; ruanyanan@163.com; Tel: +86-24-83970349; Fax:

+86-24-83970300.

INTRODUCTION

The total amount of O

3

in the troposphere is estimated

to have increased by 36% since pre-industrial times,

due primarily to anthropogenic emissions of several O

3

-

forming gases (IPCC, 2001). Elevated concentrations

of ozone at ground-level are known to have negative

impacts on human health, ecosystems, and materials. The

biological response of plants to ozone stress is dependent

on a number of factors, including the species involved, its

development stage, and environmental conditions (Heck

and Miller, 1994).

Ozone is one of the most powerful oxidants known.

In plants, primary damage is largely confined to the leaf

mesophyll, where ozone dissolves into the wet surface

of the exposed cell walls (Kangasjarvi et al., 1994).

Reactions of ozone with water and solutes in the apoplasm

lead to the formation of other reactive oxygen species

(ROS) including hydrogen peroxide (H

2

O

2

), superoxide

anion (O

2

-

£»), hydroxyl radicals (OH

.

), and singlet oxygen

(Oksanen et al., 2003). To scavenge the reactive oxygen

species, a relevant defense system is represented by

enzymes such as superoxide dismutase (SOD), ascorbate

peroxidase (APX), dehydroascorbate reductase (DHAR),

monodehydroascorbate reductase (MDAR), and

glutathione reductase (GR) (van Montagu and Inze, 1992),

together with small molecular antioxidants like ascorbate

(ASA), carotenoids, polyamines and glutathione, SOD

dismutase O

2

-

£» to O

2

, and H

2

O

2

. The ascorbate-glutathione

cycle is responsible for the removal of H

2

O

2

. Strong

induction of these Halliwell-Asada pathway enzymes has

been reported in various stress situations (Bowler et al.,

1992; Kangasjarvi et al., 1994). SOD cooperates with this

cycle in scavenging reactive oxygen species.

PHYSIOLOGY

Responses of the anti-oxidative system in leaves of

Ginkgo biloba to elevated ozone concentration in an

urban area

Xingyuan HE, Yanan RUAN*, Wei CHEN, and Tao LU

Department of Urban Forest, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, 72 Wenhua Road,

Shenyang 110016, P.R. China

(Received January 20, 2006; Accepted March 24, 2006)

ABSTRACT.

To study the effects of elevated tropospheric ozone concentrations on the anti-oxidative

systems of trees, the O

2

-

£»

generating rate, H

2

O

2

content, activities of SOD and all enzymes in the Halliwell-

Asada pathway and ascorbic acid content were periodically analyzed in leaves of Ginkgo biloba grown in

open-top chambers at either ambient (.45 nmol mol

-1

) or elevated (80 nmol mol

-1

) ozone concentrations

in an urban area for a growing season. The results show that elevated ozone exposure induced a greater

superoxide anion (O

2

-

£») generating rate and higher hydrogen peroxide (H

2

O

2

) content. Malondialdehyde

(MDA) content as an index of lipid peroxidation also increased. The activities of superoxide dismutase (SOD),

ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR), and

dehydroascorbate reductase (DHAR) were enhanced by high ozone exposure in the first half of the season

before falling to levels lower than those of control. The ascorbic acid content was always lower in the high-

ozone exposed leaves. We conclude that the antioxidant system in Ginkgo biloba did respond by acclimating

in the early season. However, the constant higher level of reactive oxygen species and declining enzyme

activities late in the season indicate that the system could not withstand the long-term exposure although no

visible injury was observed.

Keywords: Anti-oxidantive system; Elevated ozone concentration; Lipid peroxidation; Reactive oxygen

species.

Abbreviations: ASA, ascorbate; SOD, superoxide dismutase; APX, ascorbate peroxidase; MDAR,

monodehydroascorbate reductase; GR, glutathione reductase; DHAR, dehydroascorbate reductase; DHA,

dehydroascorbate; ROS, reactive oxygen species; O

2

-

£», superoxide anion; H

2

O

2

, hydrogen peroxide; MDA,

malondialdehyde; DW, dry weight.

410

Botanical Studies, Vol. 47, 2006

Urban air contains high concentrations of many

gaseous, particulate, and photochemical pollutants (such

as NOx, O

3

). Trees in urban areas can sequestrate CO

2

(McPherson, 1998; Nowak, 1994b), and reduce ozone

(Nowak, 1994a; McPherson and Simpson, 1998). Until

now, the few studies done on urban forests, however,

have mainly dealt with ecological functions (Akbari

and Taha, 1992; Simpson, 1998) while biochemical and

physiological changes have received scant attention.

Therefore, in order to better understand how the main

species of urban forests respond to increased ozone

concentration, Ginkgo biloba grown in an urban area was

exposed to 80 nmol mol

-1

ozone for a growing season,

and changes in the activities of related enzymes and

antioxidant contents were followed.

MATERIALS AND METHODS

Plant material

Six-year-old Ginkgo biloba trees were placed in open

top chambers (OTCs) in April 2005. Each OTC contained

thirty trees. These young trees were exposed to ambient

air or elevated (80 nmol/mol) ozone concentration from 10

June to 30 September 2005. Healthy Ginkgo biloba leaves,

collected at 9 A.M. every ten days, were then analyzed

immediately. To calculate dry weight, parallel samples

were dried at 80¢XC for 8 h.

Experimental site and OTCs

The experiment site was established at the Shenyang

Arboretum of Chinese Academy of Sciences (41¢X46¡¦ N,

123¢X26¡¦ E) in an urban environment. The factorial design

comprising three replicates of elevated O

3

and three of

ambient O

3

was used in 6 OTCs. The OTCs imitated the

Heagle design (Heagle et al., 1973), were 4 m in diameter

and 3 m in height with a 45o sloping frustum, and were

placed on 4-m centers (north-south and east-west) to avoid

mutual shading. Ozone was produced from pure oxygen

with an ozone generator (GP-5J, China). Ozone-enriched

air was injected into OTCs through the vertical vent pipes

in center of each chamber. Ozone concentrations were

continuously monitored by an ozone monitor (S-900,

Aeroqual, New Zealand) within the canopy in each OTC.

The elevated ozone concentration (80 ¡Ó 8 nmol mol

-1

)

was approximately 1.7 times the ambient ozone level (45

nmol mol

-1

), based on predicted near-future concentrations

for the city of Shenyang. The elevated O

3

treatment was

maintained and monitored for 8 h (08:00-16:00) everyday.

Growth parameters

Te n Ginkgo biloba trees from each treatment were

sampled randomly at the 20th and 90th days. Lengths of

axial shoot and lateral shoot were measured.

Generating rate of O

2

-

£»

and content of H

2

O

2

The generating rate of superoxide anion was

determined following the method of Ke et al. (2002) with

a slight modification. Fresh leaves (0.5 g) were ground

with a mortar and pestle in 5 mL of 50 mmol/L (pH 7.8)

phosphate buffer. The homogenate was filtered through a

45-£gm nylon mesh and centrifuged at 13,000 g for 20 min.

One mL Hydroxylammonium chloride (1 mmol/L) was

added into 0.5 mL of the above supernatant and incubated

for 1 h at 25¢XC. The color was developed by the addition

of 1 mL 4-minobenzenesulfonic acid (17 mmol/L) and 1

mL £\-Naphthylamine (7 mmol/L) for 20 min at 25¢XC. The

specific absorption at 530 nm was determined. Sodium

nitrite was used as standard solution to calculate the

content of O

2

-

.

Hydrogen peroxide concentrations were estimated by

forming a titanium-hydro peroxide complex (Dagmar et

al., 2001). Leaves (0.5 g) were ground with 5 mL cooled

acetone in an ice bath. This mixture was then filtered

through nylon cloth followed by the addition of 4 mL

titanium reagent and 5 mL ammonia to precipitate the

titanium-hydro peroxide complex. The reaction mixture

was centrifuged at 10,000 g for 10 min. Precipitate was

dissolved in 5 mL 2 M H

2

SO

4

and then re-centrifuged.

Supernatant was read at 415 nm against a reagent blank

in a Shimadzu (UV-1601) spectrophotometer. H

2

O

2

concentration was determined using a standard curve

plotted with a known concentration of H

2

O

2

.

Lipid peroxidation

Lipid peroxidation was determined by estimating the

malondialdehyde (MDA) content according to the method

of Buege and Aust (1978). MDA is a product of lipid

peroxidation. The quantity is determined by thiobarbituric

acid reaction. One g leaf was homogenized in 5 mL of

0.6% (v/v) TBA solution in 10% (v/v) trichloroacetic

acid. The homogenate was centrifuged at 12,000 g for 15

min. and the supernatant was heated in a boiling water

bath for 15 min and then cooled quickly in an ice bath.

The resulting mixture was centrifuged at 12,000 g for 15

min, and the absorbance of the supernatant was measured

at 532 nm. Measurements were corrected for unspecific

turbidity by subtracting the absorbance at 600 nm. MDA

concentrations were calculated by means of an extinction

coefficient of 155 (mmol/L)

-1

cm

- 1

(Zhangyuan and

Bramlage, 1992).

Ascorbate content

Ascorbate was estimated according to Mukherjee and

Choudhuri (1983) with a slight modification. ASA was

extracted from 0.2 g fresh leaf tissue with 5 mL of 6%

trichloroacetic acid. Two mL of the extract was mixed

with 1 mL of 2% dinitrophenylhydrazine (in acidic

medium) followed by the addition of one drop 10%

thiourea (in 70% ethanol). The mixture was boiled for 20

min in a water bath. After cooling to room temperature, 5

mL of 80% (v/v) H

2

SO

4

was added to the mixture at 0¢XC

(in an ice bath). The absorbance was recorded at 530 nm.

The concentration of ASA was calculated from a standard

curve plotted with a known concentration of ASA.

HE et al. ¡X Elevated ozone and

Ginkgo biloba

411

Enzyme extraction and assays

All enzyme extractions and centrifugations were carried

out at 4¢XC, and the extracts were stored on ice. All assays

were made at room temperature and repeated thrice.

Extraction of enzymes: superoxide dismutase (SOD;

EC 1.15.1.1) was extracted from 0.5 g leaves (fresh

weight) ground with a mortar and pestle in 5 mL of 50

mmol/L phosphate buffer (pH 7.8). The homogenate was

filtered through a 45-£gm nylon mesh and centrifuged at

13,000 g for 20 min.

Ascorbate peroxidase (APX; EC 1.11.1.6),

monodehydroascorbate reductase (MDAR; EC 1.6.5.4),

dehydroascorbate reductase (DHAR; EC 1.8.5.1), and

glutathione reductase (GR; EC 1.6.4.2) were extracted

together from 0.5 g of leaves (fresh weight) that had been

ground in a mortar. Three and a half mL phosphate buffer

(pH 7.6) containing 4% polyvinylprolidone (PVP), EDTA

1 mmol/L and ascorbate (sodium salt, freshly prepared) 1

mmol/L were added. Then 1.5 mL of saturated amonium

sulfate in the same buffer was added and stirred well to

minimize the interference of resin that would otherwise

inhibit APX activity (Krivosheeva et al., 1996). After

filtering through nylon cloth, the filtrate was centrifuged,

transferred into vials, and then kept on ice. The enzyme

activities were assayed immediately.

Assay of enzyme activity: SOD activity was estimated

by the inhibition of nitroblueterazolium (NBT) reduction

(Beyer and Fridovich 1987). The reaction mixture

contained 0.3 ml each of 13 £gmol/L riboflavin, 130

mmol/L L-methionine, and 630 £gmol/L NBT and enzyme

extracts of 0.01, 0.02, 0.04 or 0.08 ml. The phosphate

buffer (pH 7.8) was added to give a final volume of 3 ml.

The reaction was started by adding riboflavin and carried

out for 20 min under the light of two 27-w fluorescent

lamps. The absorbance at 560 nm was determined

regularly, and the extract volume causing a 50% inhibition

of NBT reduction was taken as one unit of activity.

For the APX assay, a 1-mL reaction mixture containing

0.83 mL of 0.5 mmol/L ASA in phosphate buffer (pH

7.0), 0.13 mL of 2 mmol/L H

2

O

2

(both were made

fresh) and 0.04 mL of crude enzyme was used. The

ASA consumption was monitored by the reduction of

absorbance at 290 nm taking 2.8 (mmol/L)

-1

cm

-1

as the

absorption coefficient (Nakano and Asada, 1981). For the

MDAR assay, the reaction mixture containing 0.9 mL of

2 mmol/L ASA in phosphate buffer (pH 7.0), 0.04 mL

of ascorbate oxidase (2 units) in phosphate buffer (pH

5.6), 0.03 mL of 2 mmol/L NADPH in phosphate buffer

(pH 7.6), and 0.03 mL crude enzyme was used. The

consumption of NADPH was monitored by the reduction

of absorbance at 340 nm taking 6.2 (mmol/L)

-1

cm

-1

as

the absorbance coefficient (Krivosheeva et al., 1996). For

the DHAR assay, a reaction mixture containing phosphate

buffer (pH 7.0) 0.7 mL, reduced glutathione (GSH) 20

mmol/L 0.1 ml in the phosphate buffer (pH 7.0), 2 mmol/L

DHA 0.1 mL, and crude enzyme 0.1 mL was used. DHA

was freshly prepared and kept on ice until it was added

to the reaction mixture in the cuvette to prevent its fast

oxidation at room temperature. The reduction of DHA

to ASA was monitored by the increase in absorbance at

290 nm, taking 2.8 (mmol/L)

-1

cm

-1

as the absorbance

coefficient (Krivosheeva et al., 1996). For GR assay, the

reaction mixture containing 0.86 mL of 1 mmol/L oxidized

glutathione (GSSG), 0.1 mL of 2 mmol/L NADPH in

phosphate buffer (pH 7.6), and 0.04 mL crude enzyme was

used. The consumption of NADPH was monitored for GR

assay (Krivosheeva et al., 1996).

All the ascorbate-glutathione cycle enzyme reactions

were monitored for 1 min in the cuvette of a Shimazu

UV-1601 spectrophotometer. NADPH was bought from

Merk and DHA, GSSG, GSH, AsA (sodium salt), PVP and

ascorbate oxidase were bought from Sigma.

Statistical analysis

The results presented are the means (n=3-8) of all the

measurements. One-way analysis of variance (ANOVA)

was performed using the SPSS computer package (SPSS

Inc. 1999) for all sets of data, and the mean differences

were compared by t-test (P < 0.05). Sample variability is

given as the standard deviation (S. D.) for presentation

with line diagram.

RESULTS

Growth parameters

The increment of axial shoot and lateral shoot

decreased 66% and 47%, respectively, from 20 d to 90 d

of elevated O

3

exposure, compared to plants grown under

ambient O

3

(Table 1).

Generating rate of superoxide anion (O

2

-

) and

content of hydrogen peroxide (H

2

O

2

)

Elevated ozone induced O

2

-

£» and H

2

O

2

accumulation

(Figure 1). The generating rate of O

2

-.

and content of H

2

O

2

increased by 30.7% and 31.7%, respectively, during the

first 10 days (on 21 June), compared to those of plants

grown under ambient O

3

. However, prolonged exposure

to elevated O

3

resulted in a gradual decrease in the O

2

-

£»

generating rate, which bottomed out after 40 days of

exposure and then increased rapidly after 60 days (Figure

1A). H

2

O

2

content always increased in the growing season

in response to elevated ozone (Figure 1B).

Lipid peroxidation

MDA content increased under elevated O

3

, compared

to plants grown under ambient O

3

in the growing season

(Figure 2). The fluctuation of MDA content was parallel to

the O

2

-

£» generating rate.

Ascorbate content

Ascorbate is an integral weapon in the defense against

ROS generated by ozone (Conklin and Barth, 2004).

Under elevated O

3

, ascorbate content fell significantly

412

Botanical Studies, Vol. 47, 2006

after 20 days exposure (Figure 3) compared to plants

grown under ambient O

3

.

Activity of antioxidative enzymes

A single peak appeared after 50 days of exposure (on

31 July). Elevated O

3

exposure enhanced SOD activity

significantly in the first 40 days. However, prolonged O

3

exposure gradually decreased SOD activity compared to

plants grown under ambient O

3

(Figure 4).

The activities of APX, MDAR and GR increased by

21%, 21%, and 22%, respectively, in the first 10 days,

compared to plants grown under ambient O

3

(Figure 5).

With prolonged exposure under high O

3

, APX, and MDAR

activities decreased after 40 days of exposure while GR

activity underwent no significant difference from 30 day

to 80 day. An evident increase appeared in the last 10 day

of exposure. Under high O

3

exposure, DHAR activity was

20.8% lower than under ambient O

3

in the first 10 day.

Then, higher activities were shown in the high O

3

exposed

leaves. However, after prolonged exposure, DHAR

activity became again lower than that under ambient O

3

in the last 20 day. APX activity was consistently much

higher than the sum of DHAR and MDAR activities in

this growing season.

DISCUSSION

Tropospheric ozone is a major component of a

photochemical air pollutant responsible for significant

damage in both natural and cultivated plants. In this study,

elevated O

3

caused a significant decrease in the growth of

axial and lateral shoots in Ginkgo biloba trees. However,

no visible foliar injury symptom was observed on Ginkgo

biloba leaves exposed to high ozone concentration.

The phytotoxicity of O

3

is due to its ability to generate

other ROS such as superoxide anion (O

2

-

£») and hydrogen

peroxide (H

2

O

2

) (Mudd, 1997; Runeckles and Vaarthou,

1997). Growth under elevated O

3

significantly increased

O

2

-

£» generating rate and H

2

O

2

content compared to those

of plants grown under ambient O

3

(Figure 1). Superoxide

anion is relatively unstable and is readily dismuted to

Figure 1. Generating rate of O

2

-

£»

(A) and content of H

2

O

2

(B)

in Ginkgo biloba leaves related to time of elevated O

3

exposure.

Elevated O

3

: the trees were grown under elevated (80 nmol/mol)

O

3

; Ambient O

3

: the trees were grown under ambient O

3

; means

on n=3-6. The same for all the following figures.

Figure 2. MDA content in Ginkgo biloba leaves related to time

of elevated O

3

exposure.

Table 1. Effects of elevated O

3

exposure during 20 and 90 days on growth parameters (axial shoot and lateral shoot lengths) in

Ginkgo biloba.

Axial shoot (cm)

Lateral shoot (cm)

Ambient O

3

20 day

25.10 ¡Ó 5.39

19.59 ¡Ó 3.75

90 day

33.67 ¡Ó 5.69

25.63 ¡Ó 3.46

Increment (90 day-20 day)

8.57 ¡Ó 1.01

6.04 ¡Ó 2.23

Elevated O

3

20 day

31.07 ¡Ó 4.74

24.08 ¡Ó 3.29

90 day

34.00 ¡Ó 5.29

27.25 ¡Ó 3.77

Increment (90 day-20 day)

2.93 ¡Ó 0.67

3.18 ¡Ó 1.70

Means ¡Ó S.D. (n=8) for length of axial shoot and lateral shoot.

HE et al. ¡X Elevated ozone and

Ginkgo biloba

413

H

2

O

2

spontaneously and/or enzymatically by SOD (Rao

and Davis, 1999). H

2

O

2

is a toxic ROS with deleterious

effects in plant tissue. Ozone-induced H

2

O

2

accumulation

within leaf mesophyll cells led to structural injuries

(Oksanen et al., 2003). Much accumulation of H

2

O

2

and

O

2

-

caused oxidative stress in Ginkgo biloba leaves under

high O

3

exposure. Accumulation of H

2

O

2

in response to

high ozone concentration has been reported in tobacco

(Schraudner et al., 1998), birch (Pellinen et al., 1999),

and aspen (Oksanen et al., 2003) while the accumulation

of both O

2

-

£» and H

2

O

2

has been observed in Arabidopsis

(Rao and Davis, 1999). Injury caused by these ROS is

one of the major damaging factors in plants exposed to

environmental stresses.

ROS generated by O

3

might induce lipid peroxidation,

therefore affecting the structure of cell membranes

(Calatayud et al., 2003). It has been suggested that

decreases in cell membrane stability reflect the extent of

lipid peroxidation caused by ROS (Sairam and Srivastava,

2002). In this study, MDA value was higher in Ginkgo

blioba leaves exposed to elevated O

3

than in those

exposed only to ambient O

3

, confirming an oxidative

stress (Figure 2). MDA concentration, which represents

the state of membrane lipid peroxidation, has been shown

to be correlated with the degree of elevated O

3

exposure

to plants (Prince et al., 1990; Yoshida et al., 1994; Ranieri

et al., 1996). Data obtained by MDA analysis confirmed

the occurrence of lipid peroxidation in leaves exposed

to elevated O

3

. Elevated ozone-induced membrane lipid

peroxidation has been reported in pumpkin (Ranieri et al.,

1996), sunflower (Cagno et al., 2001), lettuce (Calatayud

et al., 2002), and spinach (Calatayud et al., 2003).

It has been established that plant tolerance to O

3

depends largely on the ability to detoxify ROS (Rao et al.,

Figure 3. Ascorbate content in Ginkgo biloba leaves related to

time of elevated O

3

exposure.

Figure 4. SOD activity in Ginkgo biloba leaves related to time

of elevated O

3

exposure.

Figure 5. Activity of APX (A); activity of MDAR (B); activity of DHAR(C) and activity of GR (D) in Ginkgo biloba leaves related

to time of elevated O

3

exposure.

414

Botanical Studies, Vol. 47, 2006

1995). ASA is an integral weapon in the defense against

ROS generated by ozone, able to donate electrons to the

reactive oxygen intermediates produced by O

3

(Tanaka

et al., 1985) and to react with O

3

directly (Chameides,

1989; Van Hove et al., 2001; D¡¦Haese et al., 2005). In

this experiment, ASA content in Ginkgo biloba leaves

decreased significantly under O

3

exposure, apparently

as a consequence of substantial oxidative stress. ASA

acting as an ozone scavenger was sensitive to ozone

stress. It was not able to resist the elevated O

3

exposure,

as documented by other authors in the course of pollutant

stress (Nouchi, 1993; Wellburn and Wellburen, 1996;

Calatayud et al., 2002). The minimum ASA concentration

of Ginkgo biloba required to protect against ozone stress

is not known. In this study, elevated O

3

exposure enhanced

the activity of APX in the first 20 days. The results show

that the ascorbate content is still sufficient to maintain

APX activity in the early stages of O

3

exposure. However,

under prolonged exposure, APX activity fell below that

under ambient O

3

. This may be associated with the even

lower

ASA content. APX activity could be used as a

sensitive indicator for stress tolerance in tree species, for

acclimation, and for the injury caused by environmental

stresses (Jin et al., 2003). APX activity in consuming

ASA is much higher than the sum of MDAR and DHAR

activity in reproducing ASA (Figure 5). APX was very

sensitive to the environmental changes whether the change

led to acclimation or injury, but MDAR and DHAR

always responded more slowly (Jin et al., 2003). MDAR

activity was enhanced, but DHAR activity declined or

showed no significant change after 40 day exposure to

elevated O

3

. This result suggests that, from this moment

on, the regeneration of ASA is mostly catalyzed by

MDAR, and not by DHAR. The increase of MDAR and

decrease of DHAR activity mentioned above agree with

the findings reported for other types of stress (Anderson

et al., 1991; Krivosheeva et al., 1996; Gupta et al., 1999).

With prolonged O

3

exposure MDAR activity decreased,

but dehydroascorbate (DHA) content increased, which

stimulated the activity of DHAR. The pathway through

which DHAR catalyzed the regeneration of ASA became

dominant, however, not sufficiently to prevent the

decrease of ASA content. On the other hand, GR plays

an important role in the detoxification of oxygen radicals

by converting the oxidized glutathione into its reduced

form in ascorbate-glutathione cycle (Tanaka et al., 1985;

1990). The GR activity does not usually increase to more

than double under stress conditions and sometimes fails

to increase at all (Pasqualini et al., 2001). In this study,

GR activity was enhanced in the first 20 days in response

to elevated O

3

exposure. After prolonged exposure, an

apparent decrease appeared in the last 10 days. Rao et al.

(1995) reported O

3

exposure for 2 weeks enhanced GR

activity in wheat leaves but prolonged exposure decreased

it.

The ability of plants to scavenge ROS depends largely

on the induction of the activity of SOD and ascorbate-

glutathione cycle enzymes. SOD is the only enzyme for

the reduction of O

2

-

£» to H

2

O

2

or the oxidation of O

2

-

£»

t o O

2

to have been found to date in either plants or

other organisms (Asada, 2000). In the present study,

a significant correlation (R=-0.902) between the O

2

-

£»

generating rate and SOD activity of Ginkgo biloba was

shown in the growing season. Although elevated O

3

exposure for 30 days enhanced the activity of SOD, with

prolonging exposure SOD activity began declining (Figure

4). This suggests SOD activity might be stimulated by

superoxide anion, but could not resist continuous O

3

exposure throughout growing season. The result is similar

to the report in wheat by Rao et al. (1995).

In present study, the activities of all antioxidation

enzymes showed an evident seasonal fluctuation. The

maximum activity of all enzymes appeared between 30

July and 30 August, when weather became warmer, and

the daily maximum temperature attained 37¢XC in the

OTCs. High temperature might stimulate anti-oxidative

enzyme activities as well, but it did not eliminate the O

3

effect (Figures 4, 5). In the Mediterranean, SOD activity

o f Pinus halepensis was enhanced under O

3

exposure

in summer (Elvira et al., 1998). Similar responses by

antioxidants to ozone were found by several authors in

conifers (Castillo et al., 1987; Tandy et al., 1989) as well.

In conclusion, short term O

3

exposure induced

acclimation of the antioxidation defence system in Ginkgo

biloba leaves. However, the acclimation effect was not

sufficient to prevent the accumulation of reactive oxygen

species in leaves and, after prolonged exposure, the system

itself became affected, and the lowered protection level

led to lipid peroxidation of the leave cells.

Acknowledgements. We are grateful to Professor Tao

Dali for critical reading of the manuscript. Illuminating

comments from the editor of BBAS and two anonymous

reviewers are also appreciated. This work was funded

by the National Natural Science Foundation of China

Important Project 90411019, the Foundation of

Knowledge Innovation Program of the Chinese Academy

of Sciences kzcx3-sw-43, and the Innovation Program

of Institute of Applied Ecology, Chinese Academy of

Sciences SLYQY0414.

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HE et al. ¡X Elevated ozone and

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