Ecotoxicology and Environmental Safety 115 (2015) 101–111

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Cultivar specific variations in antioxidative defense system, genome and proteome of two tropical rice cultivars against ambient and elevated ozone$ Abhijit Sarkar a, Aditya Abha Singh a, Shashi Bhushan Agrawal a,n, Altaf Ahmad b, Shashi Pandey Rai c a b c

Laboratory of Air Pollution and Global Climate Change, Department of Botany, Banaras Hindu University, Varanasi 221005, India Department of Botany, Aligarh Muslim University, Aligarh 202001, India Laboratory of Morphogenesis, Department of Botany, Banaras Hindu University, Varanasi 221005, India

art ic l e i nf o

a b s t r a c t

Article history: Received 18 October 2014 Received in revised form 3 February 2015 Accepted 4 February 2015

For the past few decades continuous increase in the levels of tropospheric ozone (O3) concentrations is posing to be a threat for agricultural productivity. Two high yielding tropical rice cultivars (Malviya dhan 36 and Shivani) were evaluated against different concentrations of O3 under field conditions. Experimental design included filtered chambers, non-filtered chambers having ambient O3 and 10 and 20 ppb elevated O3 above the ambient. Study was conducted to assess differential response if any in induction of antioxidative defense system, genome stability, leaf proteome, yield and quality of the product in both the test cultivars. Superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) were induced under ambient and elevated levels of O3. Native polyacrylamide gel electrophoresis (PAGE) of SOD, CAT and POD also displayed increased enzymatic activity along with associated alterations in specific isoforms. Ascorbic acid, thiols and phenolics were also stimulated at ambient and elevated O3. Structural alterations in DNA of rice plants due to O3 affecting its genome template stability (GTS) was examined using RAPD technique. 2-D PAGE revealed 25 differential spots in Malviya dhan 36 and 36 spots in Shivani after O3 treatment with reductions in RuBisCO subunits. Reductions in yield and change in the quality of grains were also noticed. & 2015 Elsevier Inc. All rights reserved.

Keywords: Tropospheric ozone Antioxidative enzymes Native PAGE DNA Yield Grain quality

1. Introduction Tropospheric ozone (O3) has long been documented as a foremost threat to agriculture globally (Booker et al., 2009; Cho et al., 2011; Singh et al., 2014a). Rapid urbanization, industrialization, increased vehicle use coupled with uncontrolled fossil fuel burning and unwise management of natural resources have led to an increase in the concentration of ozone precursors and thus to tropospheric O3. Presently, background O3 concentrations have doubled since the last century (Meehl et al., 2007) and there are evidences of increase in its annual mean values ranging from 0.1 to 1 ppb per year (Coyle et al., 2003). According to the IPCC (2007)

☆ Present work forms a part of Council of Scientific and Industrial Research (CSIR), New Delhi funded research Project no. 24/292/06/EMR-II. n Corresponding author. Fax: þ 91 542 2368174. E-mail addresses: [email protected] (A. Sarkar), [email protected] (A.A. Singh), [email protected] (S.B. Agrawal), [email protected] (A. Ahmad), [email protected] (S.P. Rai).

http://dx.doi.org/10.1016/j.ecoenv.2015.02.010 0147-6513/& 2015 Elsevier Inc. All rights reserved.

report, mean daily O3 concentration is estimated to have increased from around 10 ppb, prior to the industrial revolution, to a current level of approximately 60 ppb during hot summer months. There are numerous model based reports which predicted an increase in future O3 concentrations associated with O3-induced damage to agricultural crops at global level including India (Ainsworth, 2008; Emberson et al., 2009). Ozone predominantly penetrates in internal environment of leaf tissues through stomatal openings where it generates a cascade of reactive oxygen species (ROS) in surrounding aqueous medium that causes membrane damage, alteration of gene expression, impairment of photosynthetic proteins, degradation of chlorophyll and alterations in metabolic activities (Booker et al., 2009; Fuhrer, 2009; Singh et al., 2014b). In response to this, plants adopt various defense mechanisms which includes an array of antioxidants which may be enzymatic/non-enzymatic biomolecules or low molecular weight compounds (Chen and Gallie, 2005). Ozone, being a potent oxidant, affects the genome structure of a living organism. It is documented that increased ROS

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production is the primary cause of mutaginicity of DNA which eventually affects DNA stability (Bray and West, 2005; Labuschagne, 2007). Apart from DNA damage, elevated ROS production leads to impairment of photosynthesis as a consequence of progressive loss in the amount and activity of enzymes, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (Agrawal et al., 2002; Cho et al., 2008; Singh et al., 2014b). These damaging effects of O3 are conclusively translated into yield reduction. Several researchers have documented reduction in rice yield due to O3 exposure (Ariyaphanphitak et al., 2005; Shi et al., 2009; Sawada and Kohno, 2009). Further, nutritional quality of product is also recognized as a major functional trait for the well being of society. Few reports have registered that this parameter was also adversely affected in rice due to O3 stress (Rai et al., 2010; Wang et al., 2012; Zheng et al., 2013). Rice is cultivated in around 95 countries globally as the main staple crop, and provides major source of nutrition for more than half the world's population (IRRI, 2002). It is the most important food source throughout the world with Asia, Africa and Latin America as the major rice consuming countries (Maclean et al., 2002; Ainsworth, 2008). Rice is the staple food crop of South East Asian countries, providing 21% of the calorific needs of world's population (Fitzgerald et al., 2009). Therefore, considering the importance of rice as a major food source, the present study was designed to evaluate the response of two high yielding cultivars of rice (Oryza sativa L. Cv Malviya dhan 36 and Shivani) against O3 stress by using open top chambers (OTCs) under near natural conditions. Study was done to evaluate cultivar specific response under ambient and elevated levels by using integrated approaches. We focused our investigation on assessment of changes in antioxidant defense system, stability of genome, proteomic responses, yield losses and alterations in the nutritional quality of the product. Till date, limited studies have addressed the response of rice plants against futuristic concentrations of O3 under field conditions which is also supported by documentation of Rai et al. (2010) that field experiments conducted with rice are still limited as compared to other crops. So far, effect of O3 on plant genome has not been studied in detail, thus findings of the present work could provide some new information related to genotoxic effect of O3. Results of the present study may be of helpful to researchers in understanding the mechanism of O3 action and related responses in rice plants vis-a-vis in screening and raising of resistant cultivars against O3.

The experiment was carried out in field conditions using Open top chambers (OTCs) constructed according to the design of Bell and Ashmore (1986). OTCs were ventilated with ambient air that passed through activated charcoal filters (filtered chambers: FCs), ambient non-filtered air (non-filtered chambers: NFCs), elevated levels of O3 exposure (non-filtered chambers with 10 ppb (19,600 ng m  3) O3 elevation: NFCLOs and non-filtered chambers with 20 ppb (39,200 ng m  3) elevated O3: NFCHOs). Exposure of elevated O3 was done with the help of O3 generators (Model Systrocom, India) daily at the peak hours of O3 concentration (10:00 h to 15:00 h) at the experimental site. The ozone generators were fed with pure oxygen (up to 95% purity) to generate O3 through oxygen cylinders to provide oxygen rich environment around the air inlet to minimize the formation of NOx or other by products. There were three replicate chambers for each treatment for both the cultivars. At the age of 21 days, rice seedlings were transplanted in rows maintaining a distance of 15 cm. In each chamber, there were 36 plants. Recommended dose of fertilizers (120, 80 and 60 kg ha  1 N, P and K as urea, single super phosphate and muriate of potash, respectively) were used in present study. Harvesting of crop was done in the second week of October. 2.3. Meteorological parameters Data regarding the meteorological parameters viz., maximum and minimum temperature, relative humidity, total rainfall and sunshine hours at experimental site were obtained from the Indian Meteorological Division (IMD), Banaras Hindu University. 2.4. Ozone monitoring During experiment, O3 monitoring was carried out for 12 h d  1 (06:00–18:00 h) at the experimental site in different OTCs by an O3 analyzer (Model APOA 370, HORIBA Ltd., Kyoto, Japan) at regular intervals. Air samples were collected with the help of teflon tube (0.35 cm diameter) placed above canopy of the plants. AOT40 (accumulated ozone over a threshold concentration of 40 ppb) value was calculated according to the formula of Mauzerall and Wang (2001). 2.5. Foliar injury Macroscopic symptoms due to O3 treatment was recorded in terms of foliar injury which was observed on the leaf surface as (non-parasitic) interveinal yellowing or chlorotic stippling.

2. Material and methods 2.1. Plant material Rice (O. sativa L.) was selected as the plant material. The rice cultivars tested in the present experiment are widely cultivated in Indo-Gangetic plain of India. Malviya dhan 36 is a semi-tall, mutant of Mahsuri with yield potential of 40–45 q ha  1 while Shivani is a dwarf, high yielding hybrid cultivar with yield potential of 55–60 q ha  1. 2.2. Experimental design The experiment was performed at the Agricultural Research Farm of Banaras Hindu University sited at the eastern Gangetic plains of India. It is located 76.19 m above mean sea level at 82°03′ E longitude and 25°14′N latitude. The soil of the experimental site is fertile with a sandy loam texture (sand 45%, silt 28% and clay 27%) and soil pH ranging between 7.270.2. Total N content varied from 0.02% to 0.04%, organic carbon from 0.15% to 0.34% and available P between 0.05 and 0.09 mg g  1.

2.6. Sampling of plants, antioxidants, lipid peroxidation (LPO), total protein and native PAGE Enzymatic and non-enzymatic antioxidants and lipid peroxidation were measured at three stages of development spectrophotometrically. First sampling was done at 25 days after transplantation (DAT); second at 50 DAT and third at 75 DAT. Three replicates of both cultivars were selected randomly from each chamber at specific sampling periods, hence making a set of nine replication for each treatment (n ¼9). Third fully expanded leaves from top of the plant canopy were selected for analyses. Protein for native PAGE assay and spectrophotometric analyses was isolated according to the procedure suggested by Singh et al. (2014b). Total protein was quantified by the method given by Lowry et al. (1951). For estimation of lipid peroxidation, ascorbic acid, phenolics, thiols and spectrophotometric detection of antioxidative enzymes, superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) activity was assayed by the method provided by Singh et al. (2014b). Native PAGE analyses for SOD, CAT and POD at 50 DAT was done for

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103

Table 1 Details of primer code, its sequence, number of polymorphic bands in NFCLOs and NFCHOs as compared to FCs and genome template stability at different O3 doses. Primer

Sequence (5′-3′)

Malviya dhan 36 FCs

OPD1 OPD4 OPD5 OPD6 OPB5 OPA7 OPA6 OPA9 OPA3 OPA10 OPA4 OPA12

ACC GCG AAG G TCT GGT GAG G TGA GCG GAC A ACC TGA ACG G TGC GCC CTT C GAA ACG GGT G GGT CCC TGA C GGG TAA CGC C AGT CAG CCA C GTG ATC GCA G AAT CGG GCT G TCG GCG ATA G

Total number of bands in control (FCs) Total number of polymorphic bands in treatments (A þ U) GTS (%)

9 6 8 2 3 5 2 4 3 6 3 8

Shivani

NFCLOs

NFCHOs

FCs

A

U

A

U

3 2 1 0 1 0 0 1 0 1 0 1

3 6 0 0 0 0 0 0 1 0 0 0

4 5 2 1 1 1 1 1 0 0 1 1

3 4 0 0 1 0 0 0 1 1 1 0

59 100

treatments FCs, NFCLOs and NFCHOs of both the cultivars.

8 5 11 2 4 4 2 4 3 5 4 8

NFCLOs

NFCHOs

A

U

A

U

4 2 0 1 1 0 0 0 0 1 1 1

2 1 0 0 3 1 0 1 2 0 0 0

3 4 0 1 2 0 0 3 0 1 1 1

2 2 0 0 4 0 0 0 3 0 0 0

60 20 66.1

29 50.8

100

18 70

27 55

calculated and were searched in the available bioinformatics public database (Uniprot) for their identification.

2.7. DNA isolation and amplification with RAPD markers 2.9. Yield parameters and grain/seed quality Genomic DNA was isolated from tissue of the third leaf from top at 50 DAT, according to the method of Khanuja et al. (1999). The isolated genomic DNA of rice plants from different experimental treatments were used as template for PCR amplification. Twelve different 10-mer oligonucleotide RAPD primers with an arbitrary sequence (Operon Technologies Inc., Alameda, CA, USA and Genie) were used in PCR amplifications. Primer codes are as follows; OPA3, OPA4, OPA6, OPA7, OPA9, OPA10, OPA12, OPB5, OPD1, OPD4, OPD5, OPD6. Sequences of individual primers have been provided in Table 1. Procedure for the PCR amplification was followed as elucidated by Singh et al. (2014). Amplification products were separated on 1.2% agarose gels, stained with ethidium bromide, visualized under UV light and photographed using Gel Doc (BioRad). Polymorphism was observed in RAPD profiles including the disappearance of a band present in control (A) or appearance of a band (U) in NFCLOs and NFCHOs treatments as compared to control (FCs). A band present was scored as 1 and an absent band was denoted by 0. Genome template stability (GTS) was calculated using the formula given by Cenkci et al. (2010).

Yield parameters were assessed at the time of harvest. Ten plants were sampled from each OTC. Number of grains/plant and weight of grains/plant were assessed for yield parameters. After estimation of yield, grains sampled from different treatments were crushed using a domestic electric grinder to obtain fine powder. For extracting total soluble sugar, reducing sugar, starch and protein, illustrations as mentioned in Mishra et al. (2013) were followed. Analysis of total N was conducted through Gerhardt Digestion and Distillation system-2000 (Model KB8S, Germany). For determination of nutrients, digestion of powdered seed samples was done by following the method given by Allen et al. (1986). P was estimated by the method of Jackson (1958). Concentration of Mg and Fe were determined using atomic absorption spectrophotometer (Model 2380, PerkinElmer, New Jersey, USA). Na, K and Ca contents of each extracted sample were determined using a flame photometer (Systronics Flame photometer 128, Ahemdabad, India). 2.10. Statistical analysis

2.8. Two dimensional gel electrophoresis (2-DGE) For 2-DGE analysis protein samples were isolated at 50 DAT from FCs, NFCLOs and NFCHOs. Protein isolation was carried out as per the method described in Sarkar et al. (2010). 2-DGE was carried out using 180 μg protein samples. IPG strip gels (11 cm, pH 4– 7) were used for carrying out first dimension on an IPGphor unit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) followed by the second dimension separation. After equilibration with equilibration buffers, IPG strip was placed onto the resolving gel and electrophoresis was carried out at 20 and 40 mA current. To visualize the protein spots, gels were silver stained. Post-staining gels were documented by a Multi-Imager Gel documentation system (Bio-Rad, Hercules, CA, USA) and analyzed with PDQuest software (Bio-Rad). During image analysis all the 2-D profiles of NFCLOs and NFCHOs were compared with their respective controls. After comparing 2-D images only those protein spots that were visually identifiable as changed/differentially expressed with respect to their controls were recognized as changed protein spots. Molecular weights of differentially expressed spots were

Statistical significance of antioxidants, LPO and total protein was analyzed through one way ANOVA. Duncan's multiple range test was performed as post-hoc to one way ANOVA test. Data on biochemical parameters of different treatments measured repeatedly at three ages were tested using an ANOVA with repeated measures (ANOVAR) to assess individual and combined effects of O3 treatment and age. A two way ANOVA test was performed for yield and seed quality parameters taking treatment and cultivar as two independent factors. All the statistical tests were performed using SPSS (SPSS Inc., Version 16.0).

3. Results 3.1. Meteorological parameters and ozone concentration Maximum mean temperature varied from 31.4 °C in July to 33.8 °C in September. Minimum and maximum temperatures (mean) were highest during the month of September (Fig. 1).

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Minimum Temp

Relative humidity

120

80

80

60

60

40

40

20 0 30

Monitoring days vs Col 6

Monitoring days vs Col 7

Rainfall

B

Area curve represents sunshine hours

Temprature (o C)

100

20 0 30

20

20

10

10

0

0

NFCs

FCs

NFCLOs

NFCHOs

15 j 17 j 20 j 21 j 22 j 23 j 25 j 27 j 28 j 29 j 30 j 1 jul 2 jul 3 jul 4 jul 5 jul 6 jul 8 jul 9 jul 10 jul 12 jul 13 jul 14 jul 16 jul 18 jul 19 jul 20 jul 22 jul 23 jul 24 jul 25 jul 26 jul 27 jul 29 jul 30 jul 31 jul 1 aug 2 aug 3 aug 4 aug 5 aug 7 aug 8 aug 9 aug 11 aug 12 aug 13 aug 15 aug 16 aug 18 aug 19 aug 20 aug 21 aug 22 aug 23 aug 24 aug 25 aug 26 aug 28 aug 29 aug 30 aug 31 aug 1 sep 2 sep 3 sep 4 sep 5 sep 7 sep 8 sep 10 sep 11 sep 12 sep 14 sep 15 sep 16 sep 17 sep 19 sept 20 sept 21 sept 23 sept 24 sept 25 sept 26 sept 29 sept 30 sept

120

Sunshine hours

100

Maximum Temp

A

C

100 80 60 40 20 0 15 Jun 17 Jun 20 Jun 21 Jun 22 Jun 23 Jun 25 Jun 27 Jun 28 Jun 29 Jun 30 Jun 1 Jul 2 Jul 3 Jul 4 Jul 5 Jul 6 Jul 8 Jul 9 Jul 10 Jul 12 Jul 13 Jul 14 Jul 16 Jul 18 Jul 19 Jul 20 Jul 22 Jul 23 Jul 24 Jul 25 Jul 26 Jul 27 Jul 29 Jul 30 Jul 31 Jul 1 Aug 2 Aug 3 Aug 4 Aug 5 Aug 7 Aug 8 Aug 9 Aug 11 Aug 12 Aug 13 Aug 15 Aug 16 Aug 18 Aug 19 Aug 20 Aug 21 Aug 22 Aug 23 Aug 24 Aug 25 Aug 26 Aug 28 Aug 29 Aug 30 Aug 31 Aug 1 Sept 2 Sept 3 Sept 4 Sept 5 Sept 7 Sept 8 Sept 10 Sept 11 Sept 12 Sept 14 Sept 15 Sept 16 Sept 17 Sept 19 Sept 20 Sept 21 Sept 23 Sept 24 Sept 25 Sept 26 Sept 29 Sept 30 Sept

Ozone concentration (ppb)

Rainfall (mm)

Relative humidity (%)

120

June

July

August

September

Fig. 1. Variation between meteorological variables and ozone formation. (A) Daily mean values of minimum temperature, maximum temperature and relative humidity, (B) Daily mean values of rainfall and sunshine hours. (C) Daily mean ozone concentration in different treatments; FCs, NFCs, NFCLOs and NFCHOs.

Relative humidity (mean) was highest (87.9%) during the month of June followed by 82.5% in July, 78.6% in August with minimum (72.4%) in the month of September. Maximum rainfall was recorded in the month of July whereas minimum precipitation was observed in September (Fig. 1). Mean daily sun shine hours were highest in September (7.1 h) while minimum during June (1.2 h). Recorded meteorological variables and O3 concentrations indicate that prevailing weather conditions (temperature, humidity, rainfall and sunshine hours) considerably affected O3 formation resulting least concentration of mean monthly ambient O3 in June and highest in September. Monthly mean O3 concentration was highest in the month of September, while daily mean O3 was maximum in the last week of July and first week of August. Ambient mean O3 concentration was 52.2 ppb for the entire growth period of rice ranging between a minimum concentration of 28.4 ppb (daily mean) and maximum concentration of 103 ppb (Fig. 1). Minimum O3 concentration was recorded in the month of June (40.8 ppb) whereas maximum was recorded in the month of September (59.4 ppb). Monitoring days on which there was precipitation coupled with higher relative humidity and low sunshine hours displayed low O3 concentrations. Mean day time O3 concentrations for the rice growth period were 5.6 ppb in FCs, 52.2 ppb in NFCs, 62.2 ppb in NFCLOs and 72.6 ppb in NFCHOs and the corresponding AOT40 values were 0, 11.2, 12.4 and 16.8 ppm hour in FCs, NFCs, NFCLOs and NFCHOs, respectively.

3.2. Foliar injury Foliar injury was observed in the form of chlorotic spots, interveinal yellowing/chlorosis and stippling on leaves. Injury was firstly observed with both the treatments of O3 at 30 DAT in Shivani while injury appeared one week later in Malviya dhan 36. Initially, there were minor injury symptoms in the form of chlorotic spots only but later these became more prominent and covered a larger surface area turning into interveinal chlorosis. Plants grown under NFCs showed only a few chlorotic spots while no foliar injury was detected in FCs throughout the experimental period in both the cultivars. 3.3. Antioxidants, LPO, total protein and native PAGE Lipid peroxidation (LPO) increased significantly at all the three stages of sampling in both the cultivars (Fig. 2), with significant variations due to treatment, age and treatment  age in ANOVAR test (Supplementary Table 1). At 75 DAT, LPO was significantly increased by 8.0%, 38.7%, and 53.4% in Malviya dhan 36, and 41.5%, 58.8%, and 76.8% in Shivani, at NFCs, NFCLOs, and NFCHOs, as compared to FCs, respectively. At all the sampling ages, ascorbic acid (AA) was increased significantly in both the rice cultivars. It increased by 24.5%, 35.3%, and 62.1% in Malviya dhan 36, and 8.6%, 19.6%, and 31.3% in Shivani, at NFCs, NFCLOs, and NFCHOs, respectively as compared to FCs at 75 DAT (Fig. 2). ANOVAR showed that AA varied significantly due to individual factors and

NFCLOs

NFCHOs a

c

6 a 4 d c bab

ba

d

a

a

b

a

d

c

b

d

0.8

dc

c

dc

b

1.0

c

b

c ba d

ba d d

a d

a dc b

c ab

0.6

c

0.4

a

0.2 0.0

20

a

c

b

ba

d bca dc

cba

c ba

b dc

c

c

a

10

50

a

a c b

d

b

a d

a

b cd

d

cc

a ba

0.5

a

0.4

b

c

d

c

a

d

a c

b

a

b

a

a

b

b

d a dc b

a

b

c

d

0.3 0.2

a

b

-1

Thiols (mg g fresh leaf)

30 c

20 10

a cb

d

d

25 20

c d

c

15

d

d 10

dc

c

5

0.0

0

a

b

a

c

c

b

b

a c

d

d

0.3

a

c

d

d

d

a

b

c

c

ba

a

b

a 30

c

d

20 c

b

a

a b d

d

cb

b d

c

ba

a

dc dc

0 0.00014

0.1

b

aa

0.00010 c

0.00008 d

b

c

a

b

b

a cb d

d

dc b

a

dc

d

0.0

a

c

aa

b

a

b

c d

c

0.2

b

d

10

0.00012

0 30

c d

b

c

b

d

0.1 POD activity -1 -1 (mM pur. formed min mg fresh leaf)

b

5 0

GR activity -1 -1 (µM NADPH oxidized min g fresh leaf)

40

c b

d

d

d

a

a

ba b

d

c d

c

40

b

a d

c

b dc

0.00006

a 30 20

0.00004 10 0.00002 0.00000

-1

a bc

Total protein (mg g fresh leaf)

-1

Phenolics (mg g fresh leaf)

0

15

1.2

b

SOD activity -1 (unit g fresh leaf)

2

c

b

1.4 -1

NFCs a

Lipid peroxidation (nM ml )

FCs

8

105

25 DAT 50 DAT 75 DAT

Malviya dhan 36

25 DAT50 DAT75 DAT

Shivani

25 DAT 50 DAT 75 DAT

Malviya dhan 36

25 DAT50 DAT75 DAT

0

CAT APX activity -1 -1 -1 -1 (mM acorbic acid oxidized min g fresh leaf) (mM H2O2 oxidized min g )

-1 Ascorbic acid (mg g fresh leaf)

A. Sarkar et al. / Ecotoxicology and Environmental Safety 115 (2015) 101–111

Shivani

Fig. 2. Lipid peroxidation, total protein, phenolics, ascorbic acid, thiols and antioxidative enzyme activities (SOD, POD, CAT, GR and APX) in rice cultivars, Malviya dhan 36 and Shivani under different O3 treatment regimes (NFCs, NFCLOs and NFCHOs) with respect to FCs at 25, 50 and 75 DAT. Values are mean 7S.E. Bars showing different letters indicate difference according to Duncan's test at P o0.05.

interaction for Malviya dhan 36; however, the interaction between treatment and age was not significant for Shivani (Supplementary Table S1). At 75 DAT phenolics were increased by 16.7%, 38.9%, and 51.8% in Malviya dhan 36 at NFCs, NFCLOs, and NFCHOs, as compared to FCs, respectively (Fig. 2), with significant results in ANOVAR for both the cultivars (Supplementary Table S1). Thiols were

increased by 15%, 60%, and 90% in Malviya dhan 36 and 23.3%, 54.2%, and 104% in Shivani at 75 DAT at NFCs, NFCLOs and NFCHOs, respectively as compared to FCs (Fig. 2). Total soluble protein decreased by 18.5%, 39.6%, and 40.8% at 75 DAT in Malviya dhan 36, and by 37.3%, 43.5%, and 49.5% at 75 DAT, in Shivani at NFCs, NFCLOs, and NFCHOs, respectively. Thiols and total soluble

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A. Sarkar et al. / Ecotoxicology and Environmental Safety 115 (2015) 101–111

proteins showed significant variations with treatment and interaction of age  treatment (Supplementary Table S1). All the antioxidative enzymes depicted an induction upon O3 exposure. Superoxide dismutase (SOD) was induced at all the three sampling stages in both the cultivars with more increase in Shivani while Catalse (CAT) activity showed more increase in Malviya dhan 36 (Fig. 2). Ascorbate peroxidase (APX) activity also increased by 19.4%, 43.1% and 55.3% in Malviya dhan 36, and 13.7%, 59.2% and 72.4% in Shivani, at NFCs, NFCLOs and NFCHOs, respectively as compared to FCs (Fig. 2). At 75 DAT, POD activity increased by 11%, 17.3% and 27.4% in Malviya dhan 36 and 8.8%, 24% and 28.5% in Shivani at NFCs, NFCLOs and NFCHOs, as compared to FCs, respectively. Similarly, activity of glutathione reductase (GR) also increased by 15%, 60% and 90% in Malviya dhan 36, and 23.3%, 54.2% and 104% in Shivani, at 75 DAT at NFCs, NFCLOs and NFCHOs, respectively as compared to FCs (Fig. 2). Variations due to treatment, age and treatment  age for SOD, CAT, APX, POD and GR activities have been provided in Supplementary Table S1. Native PAGE analysis of selected antioxidative enzymes in both the cultivars indicated a clear modulation in their respective activities upon O3 exposure. All the three isoforms of SOD showed differential response in both cultivars at FCs, NFCLOs and NFCHOs (Fig. 3). In Malviya dhan 36 SOD II showed increased activity only at NFCHOs whereas, SODI and SOD III showed increased band intensity at both the elevated dose of O3. For cultivar Shivani, SOD III displayed increased activity at NFCLOs and NFCHOs as compared to FCs (Fig. 3). In gel activity of CAT varied with O3 concentrations and showed increased activities at NFCLOs and NFCHOs. Enzymatic activity for CAT increased more in Shivani as

compared to Malviya dhan 36 (Fig. 3). There were four isoforms of POD in all the three treatments. Intensities of POD I, POD II and POD III bands were increased under NFCHOs. POD III showed increased activity in both the cultivars with higher intensity detected in Malviya dhan 36. Band intensity of POD IV was decreased in NFCLOs of both the cultivars (Fig. 3). For all three antioxidative enzymes, there were variation in activities of different isoforms under different treatments of O3 but in general, activities of these enzymes were more at elevated levels of O3. Results of Native PAGE were confirmed by the data of spectrophotometric analyses, where they showed significant increase in their activities upon O3 exposure. 3.4. DNA polymorphism and genome template stability (GTS) Results of present investigation confirmed the mutagenic potential of O3 by affecting template stability of DNA and demonstrated that DNA alterations were induced in rice genome due to O3. In Malviya dhan 36, 12 RAPD primers cumulatively generated a total of 266 DNA bands; out of which 59 in FCs, 59 in NFCLOs, and 52 in NFCHOs (Fig. 4). Out of the total 59 bands in NFCLOs, 20 bands were polymorphic and this number increased to 29 in NFCHOs. In Shivani, the 12 RAPD primers generated a total of 174 bands, out of which 60 in FCs, 59 in NFCLOs and 55 bands in NFCHOs. Total number of polymorphic bands in NFCLOs was 18 while polymorphic bands recorded in NFCHOs were 27. For Malviya dhan 36, primer OPD1 and OPD4 displayed maximum polymorphism by showing specific amplification for elevated O3 treatments. Shivani showed maximum polymorphism for primers OPD1, OPD4 and OPD5. Both the rice cultivars responded almost

Fig. 3. Native PAGE of antioxidative enzymes SOD, CAT and POD in rice cultivars, Malviya dhan 36 and Shivani at NFCLOs and NFCHOs as compared to FCs.

A. Sarkar et al. / Ecotoxicology and Environmental Safety 115 (2015) 101–111

107

Fig. 4. RAPD profile of genomic DNA isolated from FCs, NFCLOs and NFCHOs of both the test cultivars showing DNA polymorphism in NFCLOs and NFCHOs with respect to filtered chambers. M: marker lane, 1: DNA isolated from rice plants grown in FCs amplified with different RAPD primers, 2: DNA isolated from rice plants grown in NFCLOs amplified with different RAPD primers, and 3: DNA isolated from rice plants grown in NFCHOs amplified with different RAPD primers.

similarly as number of polymorphic bands increased with increasing dose of O3. As the FCs experienced almost zero O3; so, genome of FCs plants were unaffected and hence taken as the control. Any difference among treatments was considered due to the modification of genome by O3 exposure, so genome template stability (GTS) was calculated for both the elevated O3 dose. Genome template stability varied from 100% in FCs, to 66.1%, 50.8% in Malviya dhan 36 and 70% and 55% in cultivar Shivani under NFCLOs and NFCHOs, respectively (Table 1).

the larger subunit of RuBisCO (LSU) while spots 20, 21 and 22 could be correlated with smaller subunit of RuBisCO (SSU). Larger subunit of RuBisCO showed decreased abundance in Shivani as compared to Malviya dhan 36 whereas RuBisCO smaller subunit showed decreased abundance in Malviya dhan 36. Parallel to our spectrophotometic analysis, more loss of total protein in cultivar Shivani is also evident from 2-D PAGE analysis revealing more reduction in protein abundance qualitatively as well as quantitatively.

3.5. Two dimensional gel electrophoresis and detection of differentially expressed proteins under elevated O3 treatments

3.6. Yield parameters and seed quality

Two dimensional gel electrophoresis of both the rice cultivar revealed negative effects of O3-exposure on leaf proteins. Reduction in the protein abundance is highlighted with red arrow; proteins that showed their increased abundance are shown by pink arrow whereas newly appeared protein spots are marked by green arrow (Supplementary Fig. S2). Spot numbers are assigned to each differential spot numerically which are common in both the cultivars. While the protein spots that are differentially expressed in cultivar Shivani but not in Malviya dhan 36 are marked with alphabetical numbers. Newly expressed protein spots are highlighted using roman numerals. A total of 25 differential spots were observed for the cultivar Malviya dhan 36 out of which seven spots showed increased abundance, 17 spots revealed decrease in their abundance and there was appearance of one new protein spot in NFCLOs and NFCHOs treatment with respect to FCs (Supplementary Fig. S2). A total of 36 differential spots were observed in cultivar Shivani out of which five spots showed increment in their abundance, 29 spots showed decreased abundance, while two new spots appeared only at elevated O3 doses (Supplementary Fig. S2). Out of these, 17 spots were commonly increased or decreased in abundance in both the cultivars while, rest of the spots showed specific differential response for cultivar Shivani (Supplementary Fig. S2). Protein spots of relatively higher molecular weight (varying from 96 to 43 kDa) experienced decline in their abundance under increasing O3 treatments in both the rice cultivars. Proteins of molecular weights 66.7, 48.5, 38.5, 19.9 kDa experienced more damaging effects under higher levels of O3 (Supplementary Fig. S2). Spots 13, 14, 15 and 16 could be correlated to

Number of grains/plant decreased by 12.1%, 19.8% and 28.8% in Malviya dhan 36 and 6.7%, 17.3% and 27.2% in Shivani at NFCs, NFCLOs and NFCHOs, respectively as compared to FCs. The values of weight of grains/plant decreased by 13.1%, 28.6% and 43% in Shivani which were higher than the reduction detected for Malviya dhan 36 (Table 2). Two way ANOVA depicted that above two parameters varied significantly due to treatment, cultivar and their interaction (Table 2). Seed quality analysis of rice cultivars showed that O3 had negative effect on nutritional parameters of rice grains resulting in a decreased content of starch and an increased pool of total soluble sugars and reducing sugars (Table 2). Total soluble sugar showed a significant increment of 41.9% and 20.6% in plants growing under NFCHOs treatment of Malviya dhan 36 and Shivani, as compared to FCs, respectively. Two way ANOVA test showed significant variations in reducing sugar due to treatment, cultivar and their interaction. Total sugar showed significant variation with treatment and cultivar, while starch was significant only due to treatment (Table 2). Protein content declined significantly by 10.9%, 16.8%, 7.1% and 9.4% at NFCLOs and NFCHOs of Malviya dhan 36 and Shivani respectively as compared to controls. Nitrogen content showed maximum reduction in NFCHOs of cultivar Malviya dhan 36 (Table 2); and it varied significantly due to treatment, cultivar, and their interaction in two way ANOVA test. Ozone exposure also resulted in decreased amount of P, Na and Fe. P content decreased significantly by 24.1%, 35.8%, 43.7% and 19.0%, 41.2%, 48.4% at NFCs, NFCLOs and NFCHOs of Malviya dhan 36 and Shivani, respectively compared to FCs. However, other mineral nutrients (K, Ca, and Mg) resulted in their increased contents in rice grain upon O3 exposure

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Cultivar

Malviya dhan 36

Parameter Treatment

FCs

NFCs

NFCLOs

NFCHOs

FCs

NFCs

NFCLOs

NFCHOs

Treatment

Cultivar

Treatment  cultivar

Number of grains/plant Weight of grains/plant Reducing sugar (mg g  1) Total soluble sugar (mg g  1) Starch (mg g  1) Nitrogen (mg g  1) Protein (mg g  1) Phosphorus (mg g  1) Sodium (mg g  1) Potassium (mg g  1) Calcium (mg g  1) Magnesium (mg  1) Iron (mg g  1)

7707 7.83a 87.0 7 1.63a 2.107 0.11c 55.2 7 0.58d 1137 3.18a 3.90 7 0.03a 9.88 7 0.10a 2.50 7 0.09a 8.94 7 0.10a 2.06 7 0.03c 8.64 7 0.49b 6.93 7 0.18c 4.317 0.09a

6767 4.93b 77.3 7 2.15b 3.217 0.09b 63.7 7 2.03c 1077 1.71a 2.40 7 0.61b 8.98 7 0.21b 1.92 7 0.03b 8.707 0.08b 2.54 7 0.04b 13.9 7 0.56a 7.79 7 0.38b 3.337 0.05b

618 74.69c 67.3 71.91c 3.42 70.08ab 71.8 72.10b 97.0 72.60b 2.20 70.06c 8.81 70.08bc 1.61 70.05c 8.40 70.06c 3.65 70.03b 14.6 70.54a 9.21 70.31a 3.20 70.07b

548 7 4.51 d 55.17 1.77d 3.60 7 1.00a 78.3 7 1.52a 92.3 7 2.82b 1.60 7 0.05d 8.22 7 0.34c 1.40 7 0.06d 8.20 7 0.04c 3.88 7 0.05a 15.2 7 0.61a 9.90 7 0.60a 2.40 7 0.07c

9787 3.98a 1317 1.57a 1.40 7 0.04b 104 7 2.00c 1217 2.54a 4.43 7 0.06a 12.2 7 0.43a 2.727 0.08a 8.90 7 0.07ab 8.62 7 0.26c 6.34 7 0.37c 4.107 0.08b 6.90 7 0.23a

9127 4.14b 1147 1.91b 1.617 0.06a 1117 2.19b 1087 2.06b 3.83 7 0.07b 11.9 7 0.16ab 2.20 7 0.01b 8.29 7 0.16bc 9.617 0.44bc 7.60 7 0.38b 4.52 7 0.09b 5.50 7 0.02b

809 7 3.44c 93.5 7 0.89c 1.64 7 0.05a 1177 2.43b 98.1.7 1.48c 2.90 7 0.05c 11.3 7 0.27bc 1.60 7 0.04c 9.167 0.29a 10.2 7 0.39b 9.30 7 0.15a 4.78 7 0.04a 5.117 0.02c

7127 4.65d 74.7 7 1.09d 1.677 0.06a 1267 1.92a 91.9 7 1.85d 2.43 7 0.06d 11.0 7 0.38c 1.43 7 0.06d 7.87 7 0.25c 10.3 7 0.28a 8.26 7 0.10b 4.88 7 0.10a 4.80 7 0.01d

921nnn 269nnn 48.2nnn 48.9nnn 44.2nnn 481nnn 9.13nnn 151nnn 11.8nnn 9.39nnn 43.9nnn 16.6nnn 141nnn

333nnn 797nnn 697nnn 120nnn 1.94NS 430nnn 177nnn 8.37n 0.001NS 160nnn 280nnn 355nnn 102nnn

19.4nnn 22.9nnn 23.5nnn 0.38NS 1.10NS 21.7nnn 0.48NS 3.22n 5.44nn 1.81NS 10.9nnn 6.04nn 4.30nn

NS: not significant. nnn

Po 0.001. Po 0.01. Po 0.05.

nn n

Shivani

Two way ANOVA test

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Table 2 Yield parameters, seed quality characteristics and results of two way ANOVA test for the two rice cultivars grown in open top chambers under different O3 exposure regimes. Values are mean 7 SE. Different letters within a group of rows indicate significant differences according to Duncan's test. Two way ANOVA (F values) and p Value demonstrate the significance level due to treatment, cultivar and their interaction.

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(Table 2). A greater increase was observed for cultivar Malviya dhan 36 with respect to Shivani. Amount of calcium showed an increase of 68.9% and 76.0%, 46.5% and 30.2% at NFCLOs and NFCHOs of Malviya dhan 36 and Shivani, respectively. Two way ANOVA showed significant variation for all the nutrient elements at treatment, cultivar and their interaction with a non-significant variation only at cultivar for Na and treatment  cultivar for K (Table 2).

4. Discussion Tropospheric O3 is a secondary pollutant and a short-lived compound whose formation largely depends on several meteorological factors including temperature, rainfall, relative humidity and solar radiation. Meteorological conditions during the experiment were favorable for O3 formation. The lowest mean monthly O3 concentration was observed in June due to rainy days and the concomitant increased relative humidity and the fewer number of sunshine hours. A wash out of primary pollutants may also be a reason for low O3 levels in June as according to Tiwari et al. (2008) minimum O3 levels during monsoon has been attributed to lesser availability of solar radiation and wash out of precursors. The observed mean monthly maximum O3 concentration in September can be linked to increased temperature, lower humidity, almost lack of precipitation and increased number of sunshine hours, which favor photochemical production of O3. Earlier researchers have also reported high O3 concentration at agriculture farm site of Banaras Hindu University, Varanasi (Sarkar et al., 2010, Singh et al., 2014a, 2014b). Lipid components of cell membranes are important targets for attack by O3 generated ROS. Ranieri et al. (1996) demonstrated that lipid peroxidation occurs after O3 exposure evidenced by increase in malondialdehyde (MDA) concentrations in plant cells. A significant increment in LPO was observed in both cultivars, with higher peroxidation of lipids in cultivar Shivani indicating greater membrane damage. This could probably be due to increased production of ROS as evident by higher induction in the antioxidative enzymes in cultivar Shivani. First line of defense against O3 derived ROS lies in the apoplast, where ascorbate is believed to provide protection from the oxidative injury. AA can react directly with many types of free radicals such as superoxide, hydrogen peroxide and singlet oxygen (Hong-bo et al., 2008). Investigations in the past have documented that O3 tolerant genotypes have higher contents of AA than the sensitive ones (Robinson and Britz, 2000; Burkey et al., 2003). We also found more increment in AA content in Malviya dhan 36 (tolerant) than cultivar Shivani (sensitive). Phenols are efficient scavengers of ROS and their augmentation upon O3 exposure indicates plant's adaptation under oxidative stress (Sarkar et al., 2010). Decreased amount of phenolics was observed at 75 DAT compared to 50 DAT which might possibly be due to ageing of rice plants. Ageing of plants might have caused a reduction in photosynthesis leading to less availability of photosynthates for production of secondary metabolites. Rai and Agrawal (2008) observed an increment in total phenol content under ambient O3 pollution in different cultivars of rice. In our study similar increase in phenolics was also noticed under ambient and elevated O3 concentrations. A significant increment in SOD activity was observed in both test cultivars under O3 stress with lesser induction in Malviya dhan 36. Lower induction of SOD in this cultivar suggests less production of superoxide radical vis a vis CAT and POD required for detoxification of H2O2. Agrawal et al. (2002) also found an induced expression of Mn-SOD by elevated O3 in rice seedling. Sarkar et al. (2010) too reported induced accumulation of antioxidative enzymes like SOD and APX in wheat cultivars. Induction in SOD

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activity was followed simultaneously by increased CAT and POD activity to detoxify H2O2 in both the cultivars with more induction in Shivani. Cho et al. (2008) reported increased amounts of CAT and POD transcripts in rice seedling under elevated O3 exposure. APX is an efficient H2O2 scavenger that is particularly induced under O3 stress. A similar increment in APX activity under higher O3 treatment was noticed in the present experiment. As APX and GR are integral parts of the ascorbate glutathione cycle, for utilization and regeneration of AA, their increased activity might indicate strategic management of O3 induced oxidative stress in both cultivars. Among non-enzymatic antioxidants, thiols play an important role in scavenging ROS. Higher induction in GR activity is supported by increased production of thiols in Shivani than Malviya dhan 36. Increased thiols might be involved directly in the reduction of active oxygen radicals due to O3 stress as it acts as a potent antioxidant (Hong-bo et al., 2008). Quantitative decline in amount of thiols at 75 DAT as compared to 50 DAT might be due to ageing of rice plants. Today, RAPD technique is used for analysis of DNA damage; this technique may prove to be a promising tool for the development of novel biomarkers for DNA damage (Vardar et al., 2014) due to stress. RAPD method used in present study provides the speculation of DNA damage resulting of O3 exposure. Based on the results primer OPD1 and OPD4 can be used as biomarker for both the cultivars; while primer OPB5 can be used specifically for cultivar Shivani. In literature there are many reports on O3 induced DNA damage using different animal as models (Cheng et al., 2003; Labuschagne, 2007) but only a few studies have addressed the mutagenic effect of O3 on plants (Aras et al., 2010; Tripathi et al., 2011; Vardar et al., 2014). Previous studies have reported that ROS are the major cause of oxidative damage to DNA (Bray and West, 2005; Labuschagne, 2007). Oxidative attack on DNA generates altered bases as well as damaged sugar residues that undergo fragmentation and lead to strand breaks. In NFCLOs and NFCHOs different polymorphic bands were detected for each primer due to the loss and gain of PCR fragments. According to Liu et al. (2005) a probable cause for appearance or disappearance of bands may be genomic rearrangement leading to an altered priming site, strand breaks and DNA photoproducts which obstruct normal polymerization of DNA in PCR reaction. In our study cultivar Shivani which displayed more ROS production showed more GTS as compared to the one which had lesser production (Malviya dhan 36). The probable reason could be that although ROS might have been produced DNA strand breaks in Shivani, but the DNA repair mechanism in this cultivar might be more efficient leading to faster recovery and hence more GTS than Malviya dhan 36. The observed decline in total protein content could be due to the enhanced production of ROS due to O3 stress. These ROS structurally modify cellular proteins by altering their secondary and tertiary structures resulting in misfolded and unassembled proteins, thus enhancing their susceptibility to proteolysis (Stadtmann and Oliver, 1991). Rai and Agrawal (2008) also reported a significant loss in total protein under ambient O3 in two rice cultivars. Previous researchers have documented that O3, being a potent oxidant, affects the proteome of plants (Feng et al., 2008; Sarkar et al., 2010). In the present study, two dimensional separation of total proteins displayed reduced amount of RuBisCO in both the test cultivars that could be correlated with reduced photosynthetic ability under higher O3 exposure. Ozone affects the synthesis and causes degradation of RuBisCO subunits due to its oxidation (Agrawal et al., 2002). Agrawal et al. (2002) reported 52 differentially expressed proteins; including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and various defense/ stress and pathogenesis related (PR) proteins. PR proteins are rapidly generated after O3 exposure and provide protection against oxidative stress. Increased abundance in some of the protein spots

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in both rice cultivars could be linked to these PR or some other proteins involved in defense pathways. More number of upregulated protein spots in Malviya dhan 36 also suggests its lesser sensitivity towards O3 than Shivani. Earlier findings also suggested detrimental effect of O3 on photosynthetic proteins (including RuBisCO) and proteins involved in energy and metabolism (Sarkar et al., 2010; Feng et al., 2008). A change in the protein abundance pattern upon O3 treatment has also been reported by Ahsan et al. (2010) showing an increased abundance of some new proteins involved in defense metabolism. Most of the available reports have shown that O3 is a potential threat for yield in rice (Ariyaphanphitak et al., 2005; Shi et al., 2009). Sawada and Kohno (2009) reported significant yield loss under ambient and elevated levels of O3 exposure in 12 rice cultivars. Not only reductions in yield, O3 also resulted in variation of the grain quality. Some prior published reports have also documented that O3 exposure lead to alterations in seed quality (Rai et al., 2010; Wang et al., 2012; Zheng et al., 2013). Reduction in starch and associated increase in total soluble sugar as obtained in our investigation is supported by the findings of Rai et al. (2010). More accumulation of starch in Malviya dhan 36 than Shivani at NFCs and elevated O3 exposure can be correlated with more accumulation of Mg at increased O3 exposure, as Mg facilitates export of carbohydrates from source (leaves) to sink (seed) (Rai et al., 2010). More increase in K contents at increased O3 concentrations in Malviya dhan 36 led to greater increase in total soluble sugars as compared to Shivani. Reduced protein content in seed as obtained in present study showed an opposite trend to those reported by Wang et al. (2012) in rice and Pleijel et al. (2006) in wheat. Increase in K, Mg and Ca has also been reported by Wang et al. (2012) and was similar to the findings of present study, whereas, decrease in N and P was in agreement with the investigation of Rai et al. (2010). One of the obvious reasons of yield depression in rice could be the reduction in abundance of RuBisCO protein which might have resulted in the loss of its assimilation capacity leading to reduced carboxylation efficiency. Further, cultivar Shivani utilized more of its assimilates for defense and repair mechanisms leading to lower translocation of assimilates to reproductive parts resulting in higher yield reduction and accumulation of starch with respect to Malviya dhan 36.

5. Conclusion Present study concludes that O3 caused cultivar specific modulation of antioxidant defense system in rice to counteract the oxidative stress. The amounts of total cellular proteins were decreased under O3 stress in both the tested cultivars. Native PAGE confirmed induced accumulation and activities of several antioxidative enzymes. Two dimension gel electrophoresis revealed differentially expressed proteins in the tested cultivars under O3 exposure with more number of differential spots in cultivar Shivani. Elevated O3 led to mutational changes in DNA affecting genome template stability. Shivani showed a higher range of reduction in cellular proteins, greater induction of antioxidative defense system and maintained more genome template stability as compared to Malviya dhan 36. However, considering yield parameters, Shivani proved to be more sensitive than Malviya dhan 36 against O3. This reveals that Shivani utilized more of the photosynthates for neutralizing adverse effect of O3 rather than the translocation of photosynthates to reproductive parts resulting in more loss of yield. We believe that our results would add up in understanding differential sensitivity of cultivars against O3 and help in developing O3-resistant cultivars by using conventional breeding and molecular techniques.

Conflict of interest Authors declare no conflict of interest.

Acknowledgment Authors would like to express their sincere thanks to Council of Scientific and Industrial Research, India for providing financial support in the form of research project (Scheme number: 24/292/ 06/EMR-II). Authors are also thankful to Head, Department of Botany and Co-ordinator, CAS in Botany, Banaras Hindu University for laboratory facilities.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.02. 010.

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