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Plant Physiology and Biochemistry 52 (2012) 169e178

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Plant Physiology and Biochemistry

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

Research article

Contrasting responses of salinity-stressed salt-tolerant and intolerant winterwheat (Triticum aestivum L.) cultivars to ozone pollution

Y.H. Zheng a,b,*, X. Li a,e, Y.G. Li a, B.H. Miao d, H. Xu a, M. Simmons f, X.H. Yang c,**

a State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, 100093 Beijing, Chinab State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Water and Soil Conservation, Chinese Academy of Sciences and Ministry of WaterResources, Yangling, 712100 Shaanxi, Chinac State Key Laboratory of Crop Biology, Shandong Agricultural University, 61 Daizong Street, 271018 Taian, Chinad Institute of Upland Ecology and Technology of Beijing, Beijing 102300, ChinaeGraduate University of The Chinese Academy of Sciences, Beijing 100049, ChinafAgriculture and Natural Resources Department, University of Minnesota-Crookston, Crookston, MN 56716, USA

a r t i c l e i n f o

Article history:Received 9 September 2011Accepted 4 January 2012Available online 11 January 2012

Keywords:Abscisic acidAntioxidant enzymeGas exchangeO3

SalinityTriticum aestivum L

Abbreviations: Asat, the light-saturated CO2 assimABA, abscisic acid; Chl, chlorophyll; EL, electrolyte ltance; MDA, malondialdehyde; POD, peroxidase (EC 1species.* Corresponding author. State Key Laboratory of V

Change, Institute of Botany, The Chinese AcademyXiangshan, 100093 Beijing, China. Tel.: þ86 10628365** Corresponding author.

E-mail addresses: zhengyanhai333@163.com (Y.H.(X.H. Yang).

0981-9428/$ e see front matter � 2012 Elsevier Masdoi:10.1016/j.plaphy.2012.01.007

a b s t r a c t

Contrasting winter wheat cultivars, salt-tolerant DK961 and intolerant JN17, which sown in no salinity(�S) and salinity (þS) boxes were exposed to charcoal filtered air (CF) and elevated O3 (þO3) in open topchambers (OTCs) for 30 days. In �S DK961 and JN17 plants, þO3 DK961 and JN17 plants had significantlylower light-saturated net photosynthetic rates (Asat, 26% and 24%), stomatal conductance (gs, 20% and32%) and chlorophyll contents (10% and 21%), while O3 considerably increased foliar electrolyte leakage(13% and 39%), malondialdehyde content (9% and 23%), POD activity and ABA content. However,responses of these parameters to O3 were significant in DK961 but not in JN17 in þS treatment. Corre-lation coefficient of DK961 reached significance level of 0.01, but it was not significant in JN17 underinteraction of O3 and salinity. O3-induced reductions were larger in shoot than in root in both cultivars.Results indicate that the salt-tolerant cultivar sustained less damage from salinity than did the intolerantcultivar but was severely injured by O3 under þS condition. Therefore, selecting for greater salt tolerancemay not lead to the expected gains in yield in areas of moderate (100 mM) salinity when O3 is present inhigh concentrations. In contrast, salinity-induced stomatal closure effectively reduced sensitivity to O3 inthe salt-intolerant cultivar. Hence we suggest salt-tolerant winter wheat cultivars might be well adaptedto areas of high (>100 mM) salinity and O3 stress, while intolerant cultivars might be adaptable to areasof mild/moderate salinity but high O3 pollution.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Ozone (O3) pollution has been increasing remarkably over thepast several decades [1,2]. Currently, O3 concentrations in manycountries have reached levels that have reduced crop productivity[3,4], and some predict that crop yield losses caused by O3 pollutionin East Asia will increase substantially by 2020 [5,6].

ilation rate (mmol m�2 s�1);eakage; gs, stomatal conduc-.11.1.7); ROS, reactive oxygen

egetation and Environmentalof Sciences, 20 Nanxincun,96; fax: þ86 1082596146.

Zheng), xhyang@sdau.edu.cn

son SAS. All rights reserved.

Salinity is a common stress that severely limits crop productivity[7e9]. For example, in China 10% of arable lands are exposed tosalinity stress, and these areas are expanding due to poor irrigationpractices [10]. Numerous studies have investigated how to alleviatesalinity damage inwinter wheat, but planting salt-tolerant cultivarsis considered the best method to obtain high grain yields in salinecroplands [11,12]. Welfare et al. [13,14] reported additive effects ofsalinity and O3 on gas exchange in rice and chickpea plants.However, little is known about the responses of salt-tolerant winterwheat cultivars to O3 under saline conditions, including whethersalt-tolerant cultivars are also tolerant of O3 and whether responsesto O3 differ between salt-tolerant and intolerant cultivars. Given thesteady increase in O3 pollution and large areas of arable landsaffected by salinity, studies on the physiological responses of varioussalinity-treated winter wheat cultivars to O3 are urgently needed.

Stomatal closure is one plant response that limitsdamage under stress conditions [15,16]. Regardless of the exact

Fig. 1. Photographs from a scanning electron microscope (operated at 12 kV) of leafstomata of salt-tolerant DK961 and intolerant JN17. A, B, C and D are stomata of DK961,and E, F, G and H are stomata of JN17 under CF�S, CFþS, þO3�S and þO3þS conditions,respectively. CF: charcoal filtered air; þO3: CFþO3 (80 � 5 ppb); �S: no salinity; þS:salinity-treated.

Y.H. Zheng et al. / Plant Physiology and Biochemistry 52 (2012) 169e178170

physiological mechanism and species in which O3 damage mayoccur, O3 likely enters into the apoplastic space, crosses cellularmembranes and reaches the cellular compartments that affect thephotosynthetic apparatus and ultimately the yield of photosyn-thesis [17]. Maggio et al. [18] reported that salinity reduced bothstomatal conductance and O3 uptake thus linearly reducing O3effects on yield. Therefore, salinity-induced stomatal closure mightreduce the O3 flux in intercellular spaces and protect wheat fromO3 damage.

Environmental stresses mainly induce oxidative stress in planttissues [19]. Oxidative stress reduces membrane permeability andcauses lipid peroxidation [20,21]. At the chloroplast level, super-oxide accumulation may become more sensitive to intense light,inducing photoinhibition and photooxidation [22,23]. To cope withoxidative stress, plants synthesize antioxidants to metabolizereactive oxygen species (ROS) before cellular damage occurs in thechloroplast [24]. Therefore, activities of antioxidant enzymes mayindicate tolerances to stress conditions. In addition, the abscisicacid (ABA) content of plant tissues is used as a key indicator of plantstress [25].

The objectives of this study were to investigate whether salinitystress can reduce ozone damage in winter wheat and to determinewhether different responses existed between salt-tolerant andintolerant cultivars. Thus, specific soil conditions and plant varietieswere considered in order to develop reliable prediction models ofO3 damage.

2. Materials and methods

2.1. Plant culture and salinity treatments

The experiment was carried out in four open top chambers(OTC, 1.2 m diameter, 1.6 m height), which were installed ina temperature-controlled double-glazed greenhouse. Two con-trasting winter wheat cultivars, salt-tolerant DK961 and intolerantJN17, were used in this experiment. Seeds of individual cultivarwere sown in 24 plastic boxes (26 � 16 � 10 cm), which were filledwith 3 kg sand that had been washed and sterilized. Thirty seedswere sown in each box, and six boxes of each cultivar were placedin an OTC, i.e., each OTC held twelve boxes. Plants irrigated with fullstrength Hoagland nutrient solution (�S, no NaCl) were used as thecontrol, and experimental plants were irrigated with NaCl modifiedHoagland solution (þS, 100 mM NaCl). Water lost by evapotrans-piration was replenished daily for the duration of the experiment.Plants were thinned to 20 individuals per box 10 d after sowing. Themaximum photosynthetic photon flux density (PPFD) in chamberswas 1600 mmol m�2 s�1 at canopy height during the 14-h photo-period. The temperature in the OTCs fluctuated from 17 �C (night)to 36 �C (day), and the relative humidity (RH) was 75e86% duringthe experiment.

2.2. O3 fumigation and treatments

The OTCs were ventilated continuously (24 h day�1) withcharcoal filtered air (CF, <5 ppb O3). The average air velocity in thechambers corresponded to approximately one complete air changeper minute. O3 was generated by electrically discharging ambientair using an O3 generator (JQ-6A, Telihuo Co., Beijing, China). The airstream was bubbled through distilled water before going to thechambers to remove harmful compounds other than O3 [26].Produced O3 was dispensed randomly into the CF air streamentering two of the four chambers for 8 h day�1 (09:00e17:00) for30 days from sowing to harvest. O3 concentrations in the OTCswerecontinuously monitored by an O3 analyzer (APOA-360, Horiba,Japan) to ensure a concentration of 80 � 5 ppb. To minimize effects

of chamber and environmental heterogeneity on plant responses,plants and O3 treatments were switched among chambers everyother day. Locations of plants were also randomized within eachchamber.

Y.H. Zheng et al. / Plant Physiology and Biochemistry 52 (2012) 169e178 171

2.3. Stomata scanning and gas exchange measurements

Sections of lamina (about 9 mm2) taken from the middle of themost recent fully-expanded leaves of DK961 and JN17 plants wereexcised after completion of the experiment. A total of 8lamina sections were selected from both cultivars under salinityand O3 treatments. Excised lamina sections were fixed in a solutionof buffered glutaraldehyde (2.5%) for 24 h. Thereafter, thelaminae were dehydrated in a series of ethanolewater solu-tions (30, 50, 60, 70, 80, 90 and 100% ethanol) and incubated inan ethanoleisoamyl acetate mixture for 1 h. The laminaewere further dried then coated with gold. The mountedspecimens were examined and photographed with a scanningelectron microscope operated at 12 kV (Hitachi S-570, Hitachi,Japan).

Gas exchange was measured on the third (the most recent fully-expanded) leaves using a portable Gas Exchange FluorescenceSystem (SGF-3000, Heinz Walz, Germany) 30 d after O3 fumigation.Relative humidity wasmaintained at 70%, and leaf temperaturewasset at 25 �C in the leaf chamber. The flow rate was set at600 mmol s�1, and CO2 concentration in the leaf chamber wasmaintained at 400 mmol mol�1. The leaf was illuminated with1500 mmol m�2 s�1 PPFD from an internal light source in the leafchamber. After conditions for gas exchange measurements werestable, light-saturation net photosynthetic rate (Asat), stomatalconductance (gs) and transpiration rate (E) were simultaneouslyrecorded.

Fig. 2. Responses of light-saturated net photosynthetic rate (Asat) and stomatal conductanconditions. �S: no salinity; þS: 100 mM NaCl; CF: charcoal filtered air and þO3: CFþO3 (8differences between CF and þO3 are marked by * while �S and þS treatments are marked

2.4. Shoot and root biomass measurements and leaf sampling

Plants were harvested after 30 d of treatments. Twenty plants ofeach cultivar were cleaned and separated into shoots and roots andoven-dried at 75 �C to constant weight. Biomass of shoots and rootswas then recorded.

For each treatment, the eighty third plants leaves exhibiting novisible symptoms of salinity/ozone damage were harvested,washed with distilled water, dried by paper tissues and immedi-ately frozen in liquid nitrogen and transferred to an ultra-freezerat �80 �C until the time of assay.

2.5. Chlorophyll content

Chlorophyll content was measured following the methoddescribed by Hiscox and Isrealstam [27]. Frozen leaf samples (0.2 g)were crushed into fine homogenate and extracted in 95% ethanol.The absorbance of the extract was recorded at 663 and 645 nm,and chlorophyll content was calculated using the followingformula:

Chlorophyll content�mg g�1 FW

�¼8:02� OD663

þ 20:20� OD645

Where OD663 and OD645 are absorbances at 663 and 645 nm,respectively.

ce (gs) of salt-tolerant DK961 and intolerant JN17 to O3 pollution under �S and þS0 � 5 ppb). Vertical bars are means � SE (n ¼ 6). Within each treatment, significantby letters at p � 0.05.

Y.H. Zheng et al. / Plant Physiology and Biochemistry 52 (2012) 169e178172

2.6. Membrane permeability and lipid peroxidation measurements

Leaf membrane permeability was measured by electrolyteleakage (EL) following the method described by Dionisio-Sese andTobita [28]. Ten 4-cm pieces from the middle section of leaveswere placed in a tube containing 10 mL deionized water and thenincubated in a water bath at 25 �C for 2 h. The initial electricalconductivity of the medium (EC1) was analyzed using an electricalconductivity analyzer (KL-220, Xingzhou Company Ltd, China).The samples were boiled at 100 �C for 30 min to release elec-trolytes and then cooled to 25 �C at which time final electricalconductivity (EC2) was measured. EL was calculated using theformula:

ELð%Þ ¼ EC1=EC2 � 100

Lipid peroxidation was determined by estimating the malon-dialdehyde (MDA) content according to Kramer et al. [29]. Frozensamples (0.5 g) of shoots mixed with 5 mL phosphate buffer(pH 7.8) were crushed into fine powder in a mortar and pestleunder liquid nitrogen. The homogenate was centrifuged at4000 rpm for 20 min at 4 �C with the supernatant being used forMDA determination. A mixture of 1 mL extracts (MDA) and 2 mL0.6% thiobarbituric acid (TBA) was produced, boiled for 15 min,cooled and centrifuged for 10 min (4000 rpm). Absorbances ofsupernatant were recorded at 600, 532 and 450 nm, and MDAcontent was calculated with the following formula:

MDA�mmol g�1 FW

�¼ð6:45� ðD532 � D600Þ� 0:56D450Þ � V=W

where D532, D600 and D450 are the absorbances at 600, 532 and450 nm, respectively; V is the volume of extraction, and W is thefresh weight of the sample.

2.7. Antioxidant enzyme activity and ABA measurement

Frozen leaf samples (about 0.5 g) were homogenized in a pre-chilled mortar and pestle placed in ice with 5 mL 0.05 M potas-sium phosphate buffer (pH 7.8) containing 8.5% (v/v) 0.2 M KH2PO4and 91.5% 0.2 M K2HPO4. The homogenate was centrifuged at4000 rpm for 20 min. The supernatant was further used tomeasure guaiacol peroxidase (POD) activity. The assay mixturecontained 50 mL 0.1 M sodium phosphate (pH 6.0), 28 mL guaiacoland 19 mL 30% H2O2. The absorbance was continuously recordedfive times at 470 nm at 30-s intervals. Variation of absorbance perminute per gram fresh weight (DA470 min�1 g�1 FW) representedPOD activity. All spectrophotometric analyses were performed at0e4 �C with a UV/visible light spectrophotometer (UV-365, Shi-madzu, Japan).

Frozen leaf samples (about 0.5 g) were pulverized in liquidnitrogen and extracted in 80% methanol containing 1 mmol L�1 ofbutylated hydroxyl toluene (BHT). After 4 h of extraction, thehomogenate was centrifuged at 4000 rpm for 15 min at 4 �C. Thesupernatant was used to determine ABA content with an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibodyfor ABA (AFRCMAC 252) according to Asch [30].

Fig. 3. Leaf chlorophyll content of salt-tolerant DK961 (A) and intolerant JN17 (B) Inresponse to O3 under �S and þS conditions. CF: charcoal filtered air; þO3: CFþO3

(80 � 5 ppb); �S: no salinity; þS: salinity-treated. Vertical bars are means � SE (n ¼ 6).Within each treatment, significant differences between CF and þO3 are marked by*while �S and þS treatments are marked by letters at p � 0.05.

2.8. Regression and correlation analysis

Statistical analyses of data were performed using analysis ofvariance (ANOVA) in the general linear model procedure SPSSpackage (Ver. 11.5, SPSS, Chicago, IL, USA). The effects of O3, salinityand their interactions were analyzed on the measured variables

in salt-tolerant and intolerant wheat cultivars, respectively.Regression equations and correlation coefficients were determinedby “Fit Curves” in SigmaPlot 10.0. Differences between treatmentswere considered significant if p � 0.05.

3. Results

3.1. Leaf stomata scanning observation

Without salinity (�S), the stomatal length of DK961 (Fig. 1A,B) was decreased by 3% by O3, while stomatal length of JN17(Fig. 1E, F) declined by 12%. Salinity-caused significant stomatalshrinkage by 19% in JN17 (Fig. 1C, D) but only by 3% in DK961(Fig. 1G, H). In the saline (þS) treatment, reductions of stomatallength caused by O3 were 18% in DK961 but no reductions werenoted in JN17.

3.2. Gas exchange, chlorophyll content and biomass yield

Ozone-induced reductions of light-saturated net photosyntheticrate (Asat) (Fig. 2A, B) and stomatal conductance (gs) (Fig. 2C, D)

Y.H. Zheng et al. / Plant Physiology and Biochemistry 52 (2012) 169e178 173

were significant in both cultivars in the �S treatment. Salinityinduced considerable decreases in both Asat and gs in JN17 but not inDK961. In the þS treatment, both gs and Asat declined significantlyin DK961 in response to O3, while no significant changes weremeasured in JN17. Transpiration rate (E) was similar to Asat and gsresults in both �S and þS treatments.

For chlorophyll (Chl) content, significant interactions alsooccurred between salinity and O3 exposure. Considerable reduc-tions of Chl content were noted in both cultivars in response to O3in the �S treatment, while Chl content decreased significantly inDK961 (Fig. 3A) but not in JN17 (Fig. 3B) in the þS treatment. Noconsiderable salinity-induced reduction of Chl contentwas noted in

Fig. 4. Responses of shoots, roots and shoot/root ratio of salt-tolerant DK961 and intolerant Jcharcoal filtered air and þO3: CFþO3 (80 � 5 ppb). Vertical bars are means � SE (n ¼ 6). Withand þS treatments are marked by letters at p � 0.05.

DK961 in CF treatment, while drastic reduction was measuredin þO3 treatment.

Shoot (Fig. 4A, B) and root (Fig. 4C, D) biomass yields weresignificantly reduced by O3 in both cultivars (14% and 21%, respec-tively, in DK961; 27% and 33%, respectively, in JN17) in the �Streatment. In theþS treatment, O3 induced considerable reductionsin shoot and root biomass yields of DK961 but not in JN17. Salinity-induced reductions were larger in roots than in shoots while O3-induced reductions were larger in shoots than in roots in bothcultivars. The shoot/root ratio was larger in CF plants than in þO3plants inboth�S andþS treatments (Fig. 4E, F). These adverseeffectswere more significant in DK961 than in JN17 in the þS treatment.

N17 to O3 pollution under �S and þS conditions. �S: no salinity; þS: 100 mM NaCl; CF:in each treatment, significant differences between CF and þO3 are marked by *while �S

Y.H. Zheng et al. / Plant Physiology and Biochemistry 52 (2012) 169e178174

3.3. Membrane permeability and lipid peroxidation

Compared with CF plants, O3 significantly increased electro-lyte leakage (EL) and malondialdehyde (MDA) contents of bothcultivars in the �S treatment (12% and 8%, respectively for DK961(Fig. 5A, C) and 48% and 27%, respectively for JN17 (Fig. 5B, D)).Salinity dampened increases of EL and MDA caused by O3 in JN17(only 8% and 4%, respectively), while both parameters increasedgreatly by O3 in DK961 (18% and 13%, respectively) in the þStreatment. Salinity-induced increases of EL and MDA weresmaller in DK961 than in JN17. However, O3-induced increases ofEL and MDA were larger in DK961 than in JN17 in the þStreatment.

3.4. Antioxidant enzyme activity and ABA content

Higher original POD activity and lower ABA content weremeasured in DK961 than in JN17 plants. In the �S plants of bothcultivars, O3 significantly elevated POD activities. Salinity increasedPOD activity in both cultivars, but the extent of increase wassmaller in DK961 (Fig. 6A) than in JN17 (Fig. 6B). In the þS plants,O3 induced a significant increase of POD activity in DK961 but notin JN17.

In the �S treatment, ABA content increased significantly inresponse to O3 in both cultivars, with the extent being smaller inDK961 (Fig. 6C) than in JN17 (Fig. 6D). No significant differenceswere noted in ABA content of DK961 in response to salinity but

Fig. 5. Electrolyte leakage (EL) and MDA contents of salt-tolerant DK961 (A, C) and intoleair; þO3: CFþO3 (80 � 5 ppb); �S: no salinity; þS: salinity-treated. Vertical bars are means �*while �S and þS treatments are marked by letters at p � 0.05.

considerably increased in JN17. In the þS treatment, ABA contentincreased greatly by O3 in DK961, but there was no significantchange in JN17.

3.5. Relationships between gs and Asat

Positive correlations between gs and Asat of DK961 (Fig. 7A, B)were noted in all treatments of salinity and O3, with the slopesbeing greater in O3 than in CF plants in both no salinity and salinitytreatments. The correlation coefficient r1, r2 and r5 reacheda significance level of 0.001, and r6 reached a level of 0.01. In JN17(Fig. 7C, D), positive correlations between gs and Asat also existed insalinity and O3 treatments, however, the slopes were smaller in O3than in CF plants in both no salinity and salinity treatments. r3 wassignificant at the 0.01 level, r4 and r7 reached significance level of0.05, and r8 was not significant.

3.6. Effects of O3, salinity and their interaction

For the salt-tolerant DK961, the effects of salinity on Asat, gs, Chl,shoot, EL, MDA, POD and ABA did not reach the significant level(p � 0.05), but significant effect was noted on root (Table 1). Theeffects of O3 on gs and EL reached the significant level, while nosignificances were noted in Asat, Chl, shoot, root, MDA, POD andABA. The effects of the O3 � salinity on those parameters were allsignificant except root. For the salt-intolerant JN17, the effects ofsalinity on those parameters all reached significant level.

rant JN17 (B, D) in response to O3 under �S and þS conditions. CF: charcoal filteredSE. Within each treatment, significant differences between CF and þO3 are marked by

Fig. 6. Antioxidant enzyme (POD) activity and abscisic acid (ABA) content in leaves of salt-tolerant DK961 (A, C) and intolerant JN17 (B, D) In response to O3 under �S and þSconditions. CF: charcoal filtered air; þO3: CFþO3 (80 � 5 ppb); �S: no salinity; þS: salinity-treated. Vertical bars are means � SE. Within each treatment, significant differencesbetween CF and þO3 are marked by *while �S and þS treatments are marked by letters at p � 0.05.

Y.H. Zheng et al. / Plant Physiology and Biochemistry 52 (2012) 169e178 175

Significant effects of O3 were noted on those parameters exceptroot and ABA. However, the effects of the O3 � salinity on thoseparameters were not significant except shoot and ABA.

4. Discussion

4.1. Salinity-induced stomatal closure reduced O3 flux

Ozone mainly enters plant tissues and induces damage throughstomata, and stomatal control of O3 uptake plays a key role indetermining O3 sensitivity of crops [31]. Salinity-induced stomatalclosure increases the resistance of crops to O3 pollution byreducing O3 uptake [32]. In this study, O3 or salinity alone did notsignificantly reduce the stomatal length of the salt-tolerantcultivar, while considerable reductions were measured in thesalt-intolerant cultivar (Fig. 1). However, under saline condition,significant reductions of stomatal length caused by O3 were notedin the salt-tolerant cultivar but not in the salt-intolerant cultivar.This might be because the salt-tolerant cultivar can maintain highstomatal conductance (gs) under mild to moderate salinity stresses,while the gs of salt-intolerant cultivars significantly declines[33,34]. Thus, more O3 might be able to enter plant tissues of thesalt-tolerant cultivar and induce significant injuries in the þStreatment. In contrast, larger salinity-induced reductions of gs inthe salt-intolerant cultivar might reduce O3 uptake more signifi-cantly in the salt-tolerant cultivar in the þS treatment. This is inagreement with the previous report on alfalfa that salinity

could reduce both stomatal conductance and O3 uptake Maggioet al. [18].

4.2. Gas exchange, chlorophyll content and biomass yield

Stress-induced (salinity, ozone etc.) reductions of gs may limitplant photosynthetic capacity [35]. In this study, greater reductionscaused by O3 in Asat and gs were noted in the salt-tolerant cultivarthan in the intolerant cultivar under saline condition (Fig. 2).Therefore, the salinity-treated, salt-tolerant cultivar might be moreseverely injured by O3 than the salinity-treated, salt-intolerantcultivar under þS condition, in consequence O3 might linearlyreduce the grain yield of salt-tolerant wheat under the presentsalinity treatment. Therefore, additive effect of salinity and O3existing in salt-tolerant cultivar was in agreement with Welfareet al. [13,14] who reported on rice and chickpea plants, but antag-onistic effects occurred in salt-intolerant cultivar.

Ozone-induced oxidative stress not only decreases Chl synthesisbut also decomposes the original Chl in plant tissues [36,37]. In thisstudy, O3-induced reduction of Chl content was greater in the salt-tolerant cultivar than in the intolerant cultivar in the þS treatment(Fig. 3). This is likely because the salt-tolerant cultivar maintainedlarger stomata in the þS treatment leading to more O3 uptake. Incontrast, considerable stomatal closure induced by salinity in thesalt-intolerant cultivar might significantly reduce O3 flux andweaken oxidative stress resulting in little relative loss of Chlcontent under saline condition.

Fig. 7. Correlations between light-saturated net photosynthetic rate (Asat) and stomatal conductance (gs) in salt-tolerant DK961 and salt-sensitive JN17 under different O3 andsalinity treatments. Results for DK961 in �S and þS treatments are shown in figures A and C, respectively, and those for JN17 are shown in figures B and D, respectively. CF: charcoalfiltered air; þO3: CFþO3 (80 � 5 ppb); �S: no salinity; þS: salinity-treated. Regression equations and coefficients were determined by “Fit Curves” in SigmaPlot 10.0 (SPSS Inc.Chicago, Illinois, USA). *, **, *** Express significance at p ¼ 0.05, 0.01 and 0.001, respectively.

Y.H. Zheng et al. / Plant Physiology and Biochemistry 52 (2012) 169e178176

Salinity induces injuries to plant growth by causing physiolog-ical water stress [38], so it mainly damages plant roots (Fig. 4). Incontrast, O3 mainly enters plant tissues through leaf stomaand induces peroxidation [36], so its major injury performs inshoots.

4.3. Cell membrane permeability and lipid peroxidation

Electrolyte leakage (EL) and malondialdehyde (MDA) contentscould externally indicate cell membrane permeability and lipidperoxidation [37e39]. Significant increases in EL andMDA contentsof both cultivars might show that the integrity of cell membraneswas seriously damaged by O3 in �S plants. However, negativeeffects of O3 were dampened in the þS treatment, especially in the

Table 1Effects (p-values) of ozone, salinity and O3 � salinity on plant physiological and biochemAsat, light-saturated photosynthetic rate; gs, stomatal conductance; Chl, chlorophyll; EL,

Cultivar Source of variation Asat gs Chl

DK961 O3 0.063 0.042 0.058Salinity 0.365 0.359 0.054O3 � Salinity 0.024 0.045 0.038

JN17 O3 0.032 0.027 <0.001Salinity <0.001 <0.001 0.033O3 � Salinity 0.365 0.359 0.054

salt-intolerant cultivar (Fig. 5). This might because higher O3uptake due to greater gs resulted in more serious O3-inducedoxidative damage to cell membranes in the salt-tolerant cultivarthan in the intolerant cultivar under saline condition. Salinity-caused significant stomatal closure in the salt-intolerant cultivarmight have effectively reduced O3 uptake, resulting in less O3-induced reductions in both EL and MDA content under salinitystress.

4.4. Role of salinity in stimulating synthesis of antioxidant enzymeand O3 detoxification

Adaptation of plants to stress conditions has been correlatedwith increased levels of antioxidant compounds and enzymes in

ical parameters of salt-tolerant and intolerant wheat cultivars analyzed by ANOVAs.electrolyte leakage. The bold numerals highlight the significance at p � 0.05.

Shoot Root EL MDA POD ABA

0.137 0.324 0.034 0.066 0.132 0.0510.829 0.047 0.059 0.306 0.778 0.0910.047 0.374 0.036 0.049 0.036 0.038

0.018 0.136 <0.001 <0.001 <0.001 0.0740.021 <0.001 <0.001 0.043 0.046 0.0420.020 0.829 0.049 0.806 0.778 0.039

Y.H. Zheng et al. / Plant Physiology and Biochemistry 52 (2012) 169e178 177

plant tissues involved in detoxification responses of reactiveoxygen species (ROS) [40,41]. Salinity-elevated antioxidantenzymes activities may enhance the ability of plants scavengingO3-induced ROS [42,43]. Ozone alone can also stimulate antioxi-dant enzymes activities in most crops [39]. In the present study,POD activity of the salt-tolerant cultivar was significantly enhancedby O3 while no considerable increase was measured in the salt-intolerant cultivar under saline condition (Fig. 6). This mightindicate that greater salinity-induced increase in POD activity ofthe salt-intolerant cultivar might have enhanced the ability ofscavenging O3-induced ROS in the þS treatment. The presence ofABA, as an indicator of environmental stress, also indicated that thesalt-tolerant cultivar was injured more severely than the salt-intolerant cultivar in the þS treatment under these experimentalconditions.

4.5. Correlations between gs and Asat

Positive correlations exist between gs and Asat in most crops[44,45]. In this study, greater slope of correlation line between gsand Asat was noted in the salt-tolerant cultivar (correlation coeffi-cient r6 ¼ 0.84 p < 0.01) than that in the intolerant cultivar(r8 ¼ 0.11 p > 0.05) in the þO3þS treatment (Fig. 7). This result mayalso have been caused by high O3 flux through greater gs in the salt-tolerant cultivar in the þS treatment, which induced significantreduction in Asat. In contrast, the size of stomatal openingsdecreased substantially, resulting in less O3 uptake, and less O3-induced reduction of Asat existed in the salt-intolerant cultivarunder saline condition.

4.6. Effects of O3, salinity and their interaction

No significant effects of O3 and salinity alone on the mostphysiological and biochemical parameters occurred in the salt-tolerant cultivar but significant effects were noted under theinteraction of O3 and salinity. In the contrast, drastic effects of O3and salinity alone on those parameters in the salt-intolerantcultivar but no significant effects occurred under the interactionof O3 and salinity. These results might ensure that salinity-inducedstomatal closure in salt-intolerant wheat effectively reduced O3

flux and alleviated O3 injuries.

4.7. Conclusions and future perspectives

Salinity-induced stomatal closure may significantly reduce O3uptake and alleviate O3 injuries to wheat plants. O3-inducedinjuries were more serious in the salt-tolerant cultivar than in theintolerant cultivar under this experiment saline condition. Asa consequence, we suggest that selecting for greater salt tolerancemay not lead to the expected gains in yield in areas of moderate(100 mM) salinity when O3 is present in high concentrations. Salt-tolerant winter wheat cultivars may be used in areas of high(>100 mM) salinity and O3 stress, while salt-intolerant wheatcultivars may be adaptable to areas of mild/moderate salinity buthigh O3 pollution in the future.

Acknowledgements

We thank Dr. Hua Su for her help on artworks. Financialsupports by the State Key Laboratory of Soil Erosion and DrylandFarming on the Loess Plateau (10501-263), the State Key Laboratoryof Crop Biology at Shandong Agricultural University of China(2010KF08) and the National Science Foundation of China(30900200 and 31170367) are gratefully acknowledged.

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