Ecotoxicology and Environmental Safety 101 (2014) 146–156

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Growth, yield and quality attributes of a tropical potato variety (Solanum tuberosum L. cv Kufri chandramukhi) under ambient and elevated carbon dioxide and ozone and their interactions Sumita Kumari, Madhoolika Agrawal n Laboratory of Air Pollution and Global Climate Change, 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 31 July 2013 Received in revised form 16 December 2013 Accepted 20 December 2013 Available online 15 January 2014

The present study was designed to study the growth and yield responses of a tropical potato variety (Solanum tuberosum L. cv. Kufri chandramukhi) to different levels of carbon dioxide (382 and 570 ppm) and ozone (50 and 70 ppb) in combinations using open top chambers (OTCs). Plants were exposed to three ozone levels in combination with ambient CO2 and two ozone levels at elevated CO2. Significant increments in leaf area and total biomass were observed under elevated CO2 in combination with ambient O3 (ECO2 þAO3) and elevated O3 (ECO2 þEO3), compared to the plants grown under ambient concentrations (ACO2 þAO3). Yield measured as fresh weight of potato also increased significantly under ECO2 þ AO3 and ECO2 þEO3. Yield, however, reduced under ambient (ACO2 þAO3) and elevated ozone (ACO2 þEO3) compared to ACO2 (filtered chamber). Number, fresh and dry weights of tubers of size 35–50 mm and4 50 mm used for direct consumption and industrial purposes, respectively increased maximally under ECO2 þAO3. Ambient as well as elevated levels of O3 negatively affected the growth parameters and yield mainly due to reductions in number and weight of tubers of sizes 435 mm. The quality of potato tubers was also modified under different treatments. Starch content increased and K, Zn and Fe concentrations decreased under ECO2 þAO3 and ECO2 þEO3 compared to ACO2 þ AO3. Starch content reduced under ACO2 þ AO3 and ACO2 þEO3 treatments compared to ACO2. These results clearly suggest that elevated CO2 has provided complete protection to ambient O3 as the potato yield was higher under ECO2 þAO3 compared to ACO2. However, ambient CO2 is not enough to protect the plants under ambient O3 levels. Elevated CO2 also provided protection against elevated O3 by improving the yield. Quality of tubers is modified by both CO2 and O3, which have serious implications on human health at present and in future. & 2013 Elsevier Inc. All rights reserved.

Keywords: Elevated CO2 Elevated O3 Growth Nutrients Yield Potato

1. Introduction Air pollution and climate change are considered as significant threats to global food production. Anthropogenic activities like deforestation and fossil fuel burning are responsible for the considerable increase in atmospheric carbon dioxide (CO2) and are expected to continue in future. Atmospheric CO2 concentration has increased from 280 ppm at the beginning of the industrial revolution to the current level of 395.68 ppm (NOAA, 2013). Global CO2 emissions represent 77% of the total anthropogenic green house gases (IPPC, 2007). Tropospheric ozone (O3) is a secondary air pollutant and an oxidant, which is produced through reactions between primary pollutants (NOx and HCs) from vehicular emissions, fossil fuel burning and industrial processes, on bright sunny

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days through the photochemical cycle (Atkinson, 2000). Ozone and its precursor gases have capacity of long range transport, thus affecting peri-urban and rural agricultural production sites (Rai et al., 2010; Wang et al., 2011). East and south Asia are amongst those parts of the world, where higher increases in average O3 concentrations have been projected for the future (The Royal Society, 2008). Rising tropospheric O3 concentrations have been reported from many Asian countries, including China and India (Wang et al., 2011; Roy et al., 2009; Rai and Agrawal, 2012). The simulated AOT40 values are reported to be substantially higher throughout the year over the most fertile Indo-Gangetic plains than the other regions of India, having potential adverse impact on agriculture and natural vegetation (Roy et al., 2009). A clear trend of increase in the tropospheric O3 values after 1990 was suggested to be influenced by the increased anthropogenic activities in and around the Indian cities. The IPCC report (2007) predicted that ambient levels of O3 may increase by 20–25% between 2015 and 2050 and by a further 40–60% by 2100.

S. Kumari, M. Agrawal / Ecotoxicology and Environmental Safety 101 (2014) 146–156

Direct O3 effects have been estimated to reduce crop productivity under two scenarios (IPCC SRES A2 and B1) of projected O3 precursor emissions (Avnery et al., 2011). The total crop production losses worth $17–35 billion under A2 scenario and $12–21 billion annually under B1 scenario are predicted (Avnery et al., 2011). Plants act as a sink for O3 through stomatal and nonstomatal processes. After entering the stomata, O3 reacts with the liquid components of the apoplast to create reactive oxygen species (ROS) (Kangasjarvi et al., 2005), that can oxidize the cell wall constituents to start a chain of reactions, which may lead to cellular death at the final stage. Ozone has been shown to negatively affect growth and productivity of crops (Wang et al., 2011; Kumari et al., 2013) along with quality deterioration (Mishra et al., 2013; Rai et al., 2010). Potato (Solanum tuberosum L.), a species having large sinks as tubers with greater stored starch content has 85% of edible part compared to 50% of cereals, hence is projected to be an important food for human nutrition in coming future (FAO, 2008). Miglietta et al. (1998) have reported that a doubling of CO2 concentration will increase tuber yield by 40%, whereas 30% increase was reported under controlled environment experiments (Wheeler et al., 1994). Conn and Cochran (2006) showed decrease in biomass allocation to leaves and stems and increase in tuber under elevated CO2. However, Persson et al. (2003) found that elevated CO2 reduced tuber yield by 2%, but there was significant increase in number of smaller sized tubers. In contrast, significant stimulation of tuber yield at elevated CO2 has been found by Hogy and Fangmeier (2009a). Ozone exposure generally induces premature leaf senescence, thus reducing the green leaf area available for assimilate production and consequent crop yield (Persson et al., 2003). Yield responses of potato under elevated CO2 and O3 have been estimated using open top chambers and Free Air CO2 Enrichment (FACE) approaches in temperate and subtropical regions (Craigon et al., 2002; Vorne et al., 2002) by the Commission of the European Union, under a network of experiments named Changing climate and potential impacts on potato yield and quality (CHIP). Fangmeier et al. (2002) reported reductions in potato tuber biomass by 6.5% under elevated O3 (47–65 ppb), while 15% increase under elevated CO2 at 680 ppm. Craigon et al. (2002) have found 17% increase in dry weight of potato under 680 ppm, while plant biomass and tuber dry weight reduced at 60 ppb O3. No CO2 and O3 interaction was, however, observed for growth and yield. High O3 concentrations can alter nutrients and product quality significantly in a number of crops (Wang and Frei, 2011). Crop quality modifications are also reported under elevated CO2

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(Loladze, 2002). Pleijel and Danielsson (2009) have reported alteration in important nutrients in wheat plants under elevated CO2. Among the major staple crops, only wheat has been studied with respect to interactive effects of O3 and CO2 in tropical region (Mishra et al., 2013). Therefore, the present study was undertaken to determine the impact of season long exposure of CO2 and O3 on growth, yield and nutritional quality of tubers (macro and micronutrients, carbohydrate pool and protein contents) under ambient and elevated levels. Interactive effects of CO2 and O3 under existing levels and future projections were also studied. Following hypotheses have been proposed for the present study: 1. O3 induced visible damage may directly correlate with reductions in tuber number and yield. 2. Elevated CO2 concentration may ameliorate the negative effects of ambient O3 more than elevated CO2 at elevated O3. 3. Ambient and elevated CO2 may modify metabolites and mineral nutrient concentrations in potato tubers under ambient and elevated O3. Information concerning the combined effects on the growth and yield of any of the potato variety has not been worked out in relation to O3 and CO2 in combination in tropical region under field conditions. 2. Material and methods 2.1. Study area and experimental set up The study was conducted in Botanical garden of Banaras Hindu University, Varanasi situated at 25o140 N latitude, 821030 E longitude and 76.19 m above mean sea level in the eastern Gangetic plains of India during the winter season from November 2010 to February 2011. Mean monthly maximum temperatures were 30.09, 25.5, 21.4, 27.15 1C, and mean monthly minimum temperatures were 17.16, 9.20, 7, 11.4 1C in November and December, 2010, and January and February 2011, respectively (Fig. 1). The relative humidity varied from 38–92%, whereas total rainfall recorded during the experimental period was 6.5 mm (Fig. 1). Open top chambers (OTCs) were used for the experiment and the detailed description is given in Tiwari et al. (2006). There were 3 air exchanges per min in OTCs. Ten OTCs were installed after preliminary preparation of the field at the experimental site. The treatments were: ambient CO2 with nearly no O3 (ACO2), Ambient CO2 þ Ambient O3 (ACO2 þ AO3), Ambient CO2 þElevated O3 (ACO2 þEO3), Elevated CO2 þ Ambient O3 (ECO2 þ AO3) and Elevated CO2 þ Elevated O3 (ECO2 þ EO3). ACO2 chamber were equipped with charcoal filters to reduce ambient O3 concentrations by 84–90%. All the chambers were provided with prefilters to remove the dust. The elevated concentration of O3 (20 7 0.5 ppb) was kept above the ambient as per the predictions made by Roy et al. (2009) for India. Elevated CO2 (570 7 25 ppm) was chosen to match with the predicted concentration in the middle of the century (IPPC, 2007). Ambient and elevated concentrations of CO2

Fig. 1. Mean eight hourly ozone concentrations, temperature and relative humidity during the experimental period from November 2010 to February 2011.

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were administered 24 h daily, whereas elevated O3 was given 8 h daily from 9:30 am to 4:30 pm during the day time. Both CO2 and O3 treatments were started after emergence and continued till the maturity of potato crop. The experiment was performed using randomized block design. There were two replication of each treatment randomly distributed within the whole plot. CO2 was supplied through CO2 cylinder having connection in air delivery system of OTCs, attached to the high speed blowers. The CO2 concentration was regulated by dilution with air stream generated by air blower. The concentration of CO2 in the chamber was monitored just above the crop canopy using an Infra- red CO2 analyzer (Model Li-6200 COR. Inc., Lincoln, NE, USA). Ozone gas was generated by passing oxygen gas across a UV lamp in an ozone generator (Systrocom, India). The O3 generator was connected to air delivery system of OTCs and additional O3 over the ambient was blown into the chamber for elevated O3 concentration through controlled system connected with solenoid valve. Ozone monitoring was done above the crop canopy for 8 h from 9:30 to 4:30 h through an inert Teflon tube from sowing to maturity using UV absorption photometric ambient O3 analyzer (Model APOA 370, HORIBA Ltd., Japan). The

100

ACO2+AO3

ACO2

variations in O3 and CO2 concentrations along with AOT 40 for O3 mean temperatures and mean relative humidity inside the chamber of different treatments are given in Supplementary Table 1. Exposure index for O3, i.e. AOT40 (accumulated O3 over a threshold concentration of 40 ppb) was calculated as per the formula given by Mills et al. (2007)

2.2. Plant material The test plant material was potato (Solanum tuberosum L.) cv kufri chandramukhi, a widely grown variety in north India. This variety is developed from parents having indigenous germplasm (S4485 X kufri kuber) at Central Potato Research Institute (CPRI), Shimla, and was released in year 1968. It is an early maturing (80–90 days) variety having yield potential of 25 t ha  1. A whole plot was prepared by plowing up to 30 cm deep using recommended agronomic practices and then OTCs were installed. Recommended doses of fertilizers (150, 100 and 30 kg ha  1 of N as urea, P as super phosphate and K as murate of potash) were

ACO2+EO3

ECO2+AO3

0.06 a b

c

d

b

c

0.04

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60

e 0.03 40 0.02 20

0.01

0

0.00 a b

Number of leaves

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b

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0.0006

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200

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0.0004

100

0.0002

0.0000

0 a 3000

a

300

b c

c

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200

d e 100

1000

0

Specific Leaf Area (SLA) (cm2 g-1)

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2000

Net Assimilation Rate (NAR) (g cm-2 day-1)

a

300

Leaf area (cm-2)

0.05

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Relative Growth Rate (RGR) (g g-1 day-1)

a 80

Plant height (cm)

ECO2+EO3

0 a a d

bc c d

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80

60

40

40

b 20

Leaf Area Ratio (LAR) (cm2 g-1)

Foliar injury Percent (FIP)

60

20

0 60 DAE

0 30 - 60 DAE

Fig. 2. Foliar injury percentage and growth parameters of potato plants at 60 DAE and RGR, NAR, SLA and LAR between 30 and 60 DAE under different treatments of O3 and CO2 in combination. Values are mean 7 SE (n ¼6). Bars showing different values indicate significant differences among treatments at p o0.05 according to Duncan0 s test. DAE: Days after emergence.

28.7 7 2.68b 18.3 7 4.5c 52.16 71.9a 4.6 7 2.8d 50.727 1.3a Values not followed by same letters within column at a particular age are significantly different at po 0.05. FW: Fresh weight; DW: Dry weight; DAE: Days after emergence.

55.82 71.9b 45.56 76.87bc 69 74.18a 24 71.9d 43.2 71.18c 12.54 7 2.29b 21.767 4.79a 2.54 7 0.61c 17.7 7 3.02ab 15.86 7 1.95ab 135.6 714.9c 67.56 7 19.81d 230 7 19a 20.28 7 12.5e 184.7 72.34b 227.4 7 17.73b 151.5 7 24.3c 292 7 19.81a 73.6 7 10.42d 220.34 7 4.53b 63.83 7 12.8a 64.647 9a 247 6.91b 54.8 7 6.22a 757 4.09a 1.6 70.24b 0.8 7 0.2c 2.6 7 0.24a 0.4 7 0.24c 2.4 7 0.24a 5.2 7 0.37b 3.2 7 0.37c 7.8 7 0.48a 2.2 7 0.2c 4.4 7 0.24b 3.6 7 0.24bc 47 0.31abc 4.2 7 0.2ab 3.2 7 0.2c 4.6 7 0.4a 426.8 7 2.5b 283.7 7 4.2c 545.8 7 41.8a 148.7 7 1.45d 479.9 7 9.01b 10.4 70.24b 8 70.54c 14.6 70.50a 5.8 70.2d 11.4 70.24b ACO2 ACO2 þ AO3 ECO2 þAO3 ACO2 þ EO3 ECO2 þEO3 90

97.86 7 0.99c 85.62 7 6.12d 123.7 7 2.48a 46.36 7 2.44e 109.7 7 1.83b

0.86 7 0.01b 0 5.26 7 0.03a 0 0 12.3 70.2b 6.3 70.17c 12.23 70.4b 2.76 70.6d 17.26 71.6a 6.9 7 0.58c 7.96 7 0.4b 15.6 7 0.24a 4.36 7 0.8c 5.66 7 1.4bc

Tuber DW 450 mm Tuber DW 35–50 mm Tuber DWo 35 mm Tuber FW 450 mm

14.767 1.4b 0 88.9 7 12.3b 0 0 152.6 7 5.8b 71.96 7 1.6c 1207 19.06b 53.7 7 10.4c 226.17 10.4a 92.3 7 8.03b 108.9 7 4.54b 150.8 7 5.1a 93.8 7 11.7b 75.5 7 18.3b 0.337 0.33b 0 1.337 0.3a 0 0 47 0.01b 27 0.01c 2.66 7 0.33c 1.337 0.33d 5.337 0.33a 20.06 7 0.37c 57 0.01b 14.337 0.46d 5.66 7 0.33b 33.737 0.56a 8.337 0.33a 7.137 0.17e 47 0.01d 22.92 7 0.24b 4.66 7 0.33c

2.3.4. Tuber quality parameters Starch and sugar contents in tubers were extracted in dried samples after boiling with 5 ml of 80% ethanol. After centrifugation pellets were washed with 80% ethanol for four times following centrifugation. Final washing was done with distilled water, and the supernatant collected after each washing was used for estimation of reducing sugar, and soluble sugar, whereas pellets were used for extracting starch. 2 ml of aliquot out of 20 ml of maintained supernatant was used for reducing sugar estimation by the method of Somogyi and Nelson (Herbart et al., 1971) and 0.5 ml for total soluble sugar by the method of Dubois et al. (1956). Pellets were washed with 52% perchloric acid (v/v) with successive centrifugation and centrifuged after dissolved in H2SO4. The supernatant was used for starch estimation by the method of Dubois et al. (1956). Total protein, was extracted by homogenizing leaf sample in tris buffer (pH 6.8). After centrifugation, 5 ml of 10% TCA (Trichloroacetic acid) was added and then centrifuged. After drying, pellets were dissolved in 0.1 N NaOH and supernatant was used to measure protein content following the method of Lowry et al. (1951). Total amino acids were estimated by the methodology given by Mishra et al. (2013) using dry powdered samples of potato. The method consists of extraction of amino acids in 80% ethanol twice from 0.2 g of sample then its colorimetric estimation was done using 1 ml Ninhydrin reagent and determination of OD (Optical density) at 570 nm wavelength. For nutrient analysis, oven-dried samples of tubers were ground in a stainless steel grinder and passed through a 2-mm sieve. For determination of K, Na, Mg, Fe, Zn, Mn, Ca and Cu, 0.1 g of powdered sample was digested in a mixture of HClO4, and HNO3 (9:4) following the method of Jackson (1973). The digested samples were

259.6 7 1.57c 180.6 7 5.43d 359.7 7 3.8a 150.8 7 2.08e 301.6 7 8.8b

2.3.3. Yield parameters Yield of the plant was calculated as fresh weight of tubers. Number of tubers plant  1 and biomass of tubers plant  1 were also estimated. Tubers were separated intoo 35, 35–50 and 450 mm size. Yield parameters were estimated on 6 replicate plants at 60 DAE (intermediate harvest) and at 90 DAE (final harvest) following the complete harvesting of plants as described earlier.

9.33 70.33b ACO2 ACO2 þ AO3 7.66 70.33c ECO2 þAO3 12.33 70.33a ACO2 þ EO3 5.33 70.33d ECO2 þEO3 9.66 70.33b

2.3.2. Measurements of foliar injury Three plants from each replicate chamber were assessed for foliar injury in terms of percentage at 60 DAE. Percentage of injury was calculated as compared to the total leaf area. All leaves were collected (excluding totally senesced leaves) and total surface area was measured through portable leaf area meter (Model Li-3100 Li-COR, Inc. USA) and then injured leaf area was cut and rest of the area was again measured with the help of leaf area meter. Foliar injury percentage (FIP) was calculated according to the Mishra et al. (2013).
Foliar Injury Percentage ðFIPÞ ¼ Total injured area=Total leaf area  100

149

60

where, W1 and W2 is total plant dry weight, LW is total leaf dry weight, LA is leaf area, LA1 ¼leaf area at time t1, LA2 ¼leaf area at time t2, t1 is initial time, t2 is final time and ln is natural log.

Tuber FW35– 50 mm

Leaf Area Ratio ðcm  2 g  1 Þ ¼ ½ðLA2  LA1 Þ ðln W 2  ln W 1 Þ=ðW 2  W 1 Þðln LA2  ln LA1 Þ

Tuber FW o 35 mm

½ðln LA2  ln LA1 Þ=ðLA2  LA1 Þ Specific Leaf Area ðcm2 g  1 Þ ¼ LA =LW

Tuber no 450 mm

Þ ¼ ½ðW 2  W 1 Þ=ðt 2  t 1 Þ

Tuber no 35–50 mm

1

Tuber noo 35 mm

Þ ¼ ðln W 2  ln W 1 Þ=ðt 2  t 1 Þ

Net Assimilation Rate ðNARÞ ðg cm  2 d

Total tuber DW

1

Total tuber FW

Relative Growth Rate ðg g  1 d

Total no. tuber

2.3.1. Growth parameters Three plants were randomly selected from each replicate chamber of different treatments for growth and biomass measurements at 60 (intermediate harvest) and 90 (final harvest) days after emergence (DAE) by carefully digging monoliths (20  20  20 cm3) containing intact below ground parts. Plants were thoroughly washed under running tap water to remove soil particles and then root and shoot lengths, leaf area and numbers of leaves were quantified. Leaf area was measured using a portable leaf area meter (Model Li-3100 Li-COR, Inc. USA). Plant parts were separated and oven dried at 80 1C till constant weight and then dry weight was taken for biomass determination. Biomass partitioning was calculated for individual treatment of potato by dividing the biomass of an organ by total biomass and multiplied by 100. Growth indices such as Relative Growth rate (RGR), Net Assimilation Rate (NAR), Specific Leaf Area (SLA) and Leaf Area Ratio (LAR), were calculated on dry weight data using modified formulae of Hunt (1982).

Treatments Parameter

2.3. Plant sampling and analysis

Age, DAE

added during the preparation of the field. Half dose of nitrogen and full dose of phosphorus and potassium were added as basal dressing before sowing tubers and another half of nitrogen as top dressing after 15 days of germination. Potato tubers were hand sown in rows on ridges with a distance of 30 cm between ridges in each chamber. After emergence plants were thinned to have 20 cm distance between each of them. Similar irrigation regime was followed through drip irrigation in order to maintain similar moisture content in each chamber.

Table 1 Fresh (g plant  1) and dry weights (g plant  1) and numbers (plant  1) of total and different sized tubers of potato plants under different treatments of O3 and CO2 in combinations at 60 and 90 DAE. Values are mean 7 SE (n¼ 6).

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S. Kumari, M. Agrawal / Ecotoxicology and Environmental Safety 101 (2014) 146–156

filtered through Whatman No. 42 filter paper and the volume was made up to 25 ml with distilled water. The concentrations of K, Na, Mg, Fe, Zn, Mn, Ca and Cu in the filtrate were determined with an atomic absorption spectrophotometer (Model 2380; Perkin Elmer, New Jersey, USA). Total N was quantified using the microKjeldahl technique in a Gerhardt Automatic N Analyzer (Model KB8S, Germany 4). The organic carbon content in tuber was measured by wet digestion following the modified method of Walkley and Black (1934) using 0.1 g powdered sample of tuber.

3.3. Growth and yield parameters Plant height and number of leaves reduced significantly under ACO2 þ EO3 (36.7 and 42.6%) and ACO2 þ AO3 (30.2 and 31.4%) compared to ACO2 (Fig. 2), whereas significant increments were observed under ECO2 þ AO3 (29.3 and 21.4%) and ECO2 þEO3 (15.5 and 13.3%) compared to ACO2 þAO3 (Fig. 2). Leaf area significantly increased under ECO2 þAO3 and ECO2 þEO3 by 71.3 and 58.3%, respectively compared to ACO2 þ AO3, whereas decreased under ACO2 þEO3 and ACO2 þ AO3 by 43.6 and 18%, respectively compared to ACO2 (Fig. 2). Number and fresh weight of total tubers increased significantly under ECO2 þ AO3 by 82.5 and 92.4%, and under ECO2 þ EO3 by 42.5 and 69.16% respectively at 90 DAG compared to ACO2 þAO3 however, the same decreased under ACO2 þ EO3 and ACO2 þ AO3 at both the ages of samplings compared to ACO2 (Table 1). Tuber dry weight was significantly increased under ECO2 þ AO3 (135%) and ECO2 þ EO3 (59%) to ACO2 þ AO3, but decreased under ACO2 þ EO3 (64.4%) and ACO2 þAO3 (28.5%) compared to ACO2 at 60 DAG (Table 1). Potato tubers were categorized into three sizes according to their diameter, as small of diametero35 mm, intermediate of diameter between 35 and 50 mm, used directly for the human consumption and the larger size 450 used for the industrial processes. At intermediate harvest, number of potato tubers ofo35 mm size increased significantly under ECO2 þAO3 (47%) and decreased under ECO2 þEO3 (17.6%) compared to ACO2 þAO3, while decreased significantly under ACO2 þ EO3 (20%) compared to ACO2 (Table 1). Potato tubers of size between 35 and 50 mm increased under ECO2 þAO3 (33.3%) and ECO2 þEO3 (166.6%), where as4 50 mm size was only recorded under ECO2 þ AO3 treatments at 60 DAE (Table 1). Numbers of all sized potato were significantly lower under ACO2 þEO3 and ACO2 þAO3 treatments compared to ACO2 (Table 1). Fresh and dry weights of tubers also followed a similar trend with respect to their size category. At final

2.3.5. Statistical analysis The statistical significance of data for yield parameters were analyzed through one way Analysis of Variance (ANOVA) to examine the treatment effects on all the measured parameters. Duncan0 s multiple range tests were performed as post hoc on all the parameters subjected to ANOVA test. The entire statistical tests were performed using SPSS software (Spss Inc, version 16.0).

3. Results 3.1. Ozone monitoring During the experiment, recorded mean monthly O3 concentrations (ppb) were 52.8 in November and 46.9 in December, 2010, 45.8 in January and 52.6 in February 2011, with overall mean concentration of 49.6 ppb (Fig. 1). The values of AOT 40 in different treatment chambers are given in Supplementary Table 1. 3.2. Foliar injury Foliar injuries as chlorosis and necrosis were observed on adaxial surface of the leaf after 30 days of exposure in ACO2 þAO3 and ACO2 þEO3 treatments, which later became more severe. Plants grown under ECO2 þAO3 and ECO2 þEO3 did not show any visible leaf injury at any stage of the growth. Foliar injury percentages were 55.1 and 21.8% in ACO2 þ EO3 and ACO2 þAO3 treatments, respectively (Fig. 2).

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ACO2 ACO2+AO3

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90 DAE

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Root biomass Shoot biomass

Leaf biomass Tuber biomass 120 90 DAE

a b

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Biomass allocation (%)

Total biomass (g plant-1)

ACO2+EO3 ECO2+EO3

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Biomass allocation (%)

Total biomass ( g plant-1)

100 60

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0 ACO2 ACO2+AO3 ECO2+AO3 EO3+ACO2 ECO2+EO3

Fig. 3. Total biomass and percent allocation of biomass in different plant parts of potato under different treatments of O3 and CO2 in combination at 60 and 90 DAE. Values are mean 7 SE (n¼ 6). Bars showing different letters indicate significant differences among treatments at p o 0.05 according to Duncan0 s test. DAE: Days after emergence.

S. Kumari, M. Agrawal / Ecotoxicology and Environmental Safety 101 (2014) 146–156

harvest (90 DAE), smaller size tuber did not vary in terms of number and fresh weight among all the treatments except under ECO2 þEO3, where tuber number increased by 15% compared to ACO2 þAO3. Fresh weight and number of tubers of size 35–50 mm (92.6 and 143.7%) as well as4 50 mm (225 and 240%) increased significantly under ECO2 þAO3 compared to ACO2 þAO3, whereas the same decreased under ACO2 þEO3 compared to ACO2. Potato grown under ECO2 þEO3 treatment showed significant increases in number and fresh weight of tubers of size 35–50 mm and 450 mm compared to ACO2 þAO3 treatment. At final harvest, dry weight of tubers of o35 mm was significantly higher under ACO2 þ AO3 (73.5%) and ACO2 þEO3 (41%) compared to ACO2 (Table 1). Tuber dry weight of size 35–50 mm

ACO2+AO3

ACO2

increased significantly only under ECO2 þAO3 (51.4) compared to ACO2 þ AO3. However, significant decrease was observed under ACO2 þ EO3 (57%) treatment compared to ACO2. Tubers of 50 mm size showed maximum increase in dry weight under ECO2 þAO3 (185%) followed by ECO2 þEO3 (177%) compared to ACO2 þAO3, while reduced under ACO2 þEO3 (83.7%) and ACO2 þAO3 (36.2%) treatments compared to ACO2. Total biomass of potato showed significant enhancement under ECO2 þAO3 and ECO2 þEO3 by 107.4 and 56.2% at 60 DAE and 17.4 and 10.9% at 90 DAE, respectively compared to ACO2 þAO3 (Fig. 3). However, significant reduction in total biomass was observed under ACO2 þEO3 60.1 and 40.8%, respectively at 60 and 90 (DAE) compared to ACO2. Total biomass did not vary between

ECO2 +AO3

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Soluble sugar [mg g-1(DM)]

C/N ratio

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Starch [mg g-1DM]

0.25

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Reducing sugar [mg g-1 DM]

ECO2+EO3

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Amino acid [mg g-1DM]

Protein content [mg g-1DM]

20

151

e 200

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0 90 DAE

90 DAE

Fig. 4. Protein, amino acid, sugar, total N, organic C contents and C/N ratio in potato tubers under different treatments of O3 and CO2 in combination at 90 DAE. Values are mean 7 SE (n¼ 6). Bars showing different letters indicate significant differences among treatments at p o 0.05 according to Duncan0 s test. DAE: Days after emergence.

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ACO2 þAO3 and ACO2 at 90 DAE (Fig. 3). Percent allocations of biomass at 60 DAE were lower in tubers compared to other parts in all the treatments. In contrast maximum allocation to biomass was into tubers at final harvest and the percentages were 77.2, 72.5, 55.6, 69.5, 62.8% under ECO2 þ AO3, ECO2 þEO3, ACO2 þEO3, ACO2, ACO2 þAO3, respectively (Fig. 3). Plants showed significant increase in RGR (9.3%) and NAR (12.5%) and decrease in SLA (28.78%) under ECO2 þ AO3 between 30 and 60 DAG compared to ACO2 þ AO3, whereas LAR did not vary significantly (Fig. 2). Under ACO2 þEO3 and ACO2 þAO3, RGR and NAR declined significantly compared to ACO2. SLA showed significant increase under ACO2 þEO3 (53.6%) and ACO2 þ AO3 (23.6%), whereas LAR increased only under ACO2 þ EO3 (25.7%) compared to ACO2 between 30 and 60 DAG (Fig. 2). 3.4. Tuber quality parameters Starch content significantly increased by 130.6 and 60.9% under ECO2 þAO3 and ECO2 þEO3, respectively compared to ACO2 þAO3, whereas decreased under ACO2 þEO3 (53.9%) and ACO2 þ AO3 (24.7%) compared to ACO2 (Fig. 4). Reducing sugar showed significant enhancement with maximum increase under ACO2 þEO3 (21.3%) followed by ACO2 þAO3 (7.9%) compared to ACO2. Under ECO2 þAO3 (32%) decrease in reducing sugar was recorded compared to ACO2 þAO3 (Fig. 4). Soluble sugar content was also increased under ECO2 þAO3 (63.8%) and ECO2 þ EO3 (26.6%) treatments compared to ACO2 þAO3 and decreased under ACO2 þ EO3 (53.4%) and ACO2 þAO3 (37.5%) compared to ACO2 (Fig. 4). Total nitrogen content in tubers was highest under ACO2 and declined maximally in ACO2 þEO3 (26.1%) followed by ACO2 þAO3 (17.3%), ECO2 þAO3 (15.2%) and ECO2 þEO3 (2.4%) and (Fig. 4). Organic carbon content in tuber increased significantly by 20 and 14.4%, respectively under ECO2 þAO3 and ECO2 þEO3 compared to ACO2 þAO3, but declined under EO3 þACO2 (16%) and ACO2 þAO3 (7.9%) compared to ACO2 (Fig. 4). C/N ratio in tubers increased significantly, under ECO2 þAO3 (41.6%), ECO2 þEO3 (17.2%) compared to ACO2 þAO3, and decreased under ACO2 þ EO3 (13.7%) and ACO2 þAO3 (11.4%) compared to ACO2. Protein and amino acid contents in tubers decreased significantly under all the treatments compared to ACO2 (Fig. 4). Potassium, Mg, Zn and Fe reduced significantly under all the treatments compared to ACO2 (Table 2). Recorded percent reductions were 25.7 for K, 24.5 for Mg, 20.7 for Zn under ACO2 þEO3, and 12.6, 46, 2.6 for K, Mg, Zn under ACO2 þAO3, respectively, compared to ACO2 (Table 2). Similarly reductions were 19.8 for K, 2.4 for Zn under ECO2 þ AO3, 9.2 for K, 13.7 for Zn under ECO2 þ EO3 compared to ACO2 þ AO3. However Mg significantly increased by 13.8 and 63.5% under ECO2 þAO3 and ECO2 þEO3, respectively compared to ACO2 þ AO3. Ca concentration was lowest under ECO2 þAO3 (Table 2). Na concentration showed significant increasing trend under all treatments compared to ACO2, with maximum in ACO2 þEO3 (24%) and minimum in ECO2 þ AO3 (6.6%). For Mn reductions were recorded under ACO2 þEO3 and ACO2 þ AO3 treatments compared to ACO2 whereas, significant increments under

ECO2 þAO3 and ECO2 þEO3 were observed compared to ACO2 þ AO3. Cu showed significant increase (80%) under ECO2 þAO3 and ECO2 þEO3 (18.9%) compared to ACO2 þ AO3, while reductions under ACO2 þAO3 (40.6%) and ACO2 þEO3 (47.5%) compared to ACO2 respectively.

4. Discussion 4.1. Impact on growth and yield The most noticeable effect of ambient (ACO2 þAO3) and elevated O3 (ACO2 þEO3) in the present study was the visible leaf damage after 30 days of exposure. However, no visible symptoms were recorded under ECO2 þEO3 treatment. De Temmerman et al. (2002), however, recorded foliar injury in all O3 exposed potato plants irrespective of additional CO2. Visible O3 injury as distinct reddish violet or brown necrotic spots under ambient (23 ppb) and elevated O3 (43 ppb) was reported 48 days after emergence in potato cv Bintje (Persson et al., 2003). In the present study the results of leaf damage clearly denote that present ambient ozone concentration at ambient CO2 level is more lethal than the elevated O3 at elevated CO2 for potato plant. Visible leaf injury under ambient and elevated O3 at ambient CO2 led to reductions in leaf area, number of leaves and plant height significantly compared to ambient CO2, and the magnitude of reduction was higher under ACO2 þEO3 compared to ACO2 þ AO3 treatment. Sarkar and Agrawal (2012) have also reported leaf injuries and consequent significant reductions in plant height, number of leaves and leaf area of two rice cultivars under ambient (45.3 ppb) and elevated O3 (ambient þ 20 ppb) at Varanasi. Tropospheric O3 is known to affect physiological and metabolic functions at higher concentrations, leading to severe growth reductions (Rai and Agrawal, 2014). The present study also showed that fresh and dry weights of tuber reduced significantly under ACO2 þ AO3 and ACO2 þEO3, with higher percentage of reductions under later treatment. Ozone effects on growth and development may change the partitioning of plant assimilates, leading to reduced biomass accumulation in tubers. At final harvest, reductions in yield under EO3 þACO2 and ACO2 þAO3 were due to reductions in tubers of intermediate (35–50 mm) and largest size (450 mm), which are mainly used for the human consumption and industrial processes. This finding is in accord with Vandermeiren et al. (2005), who reported similar trend of reductions in tuber at a range of 42–65 ppb of O3 in a FACE experiment. In both ACO2 þ EO3 and ACO2 þ AO3 treatments, considerable decrease in fresh weight of intermediate and largest size potato tubers indicates that sufficient proportion of assimilates was not distributed towards the sink, thus affecting the economic importance of the crop. Observed leaf injury in these treatments might have played major role in affecting photosynthesis as well as transport of assimilates towards the below ground sinks. Mosley et al. (1978) suggested that early maturing varieties of potato may

Table 2 Micro and macro nutrients in potato tubers under different treatments of O3 and CO2 in combinations at 90 DAE. Values are mean 7SE (n¼ 6). Treatments

ACO2 ACO2 þ AO3 ECO2 þAO3 ACO2 þ EO3 ECO2 þEO3

Parameters Ca (mg g  1)

Mg (mg g  1)

K (mg g  1)

Na (mg g  1)

Fe (mg g  1)

Mn (mg g  1)

Cu (mg g  1)

Zn (mg g  1)

0.73 70.002d 0.80 70.004c 0.65 70.002e 0.83 70.005b 1.01 70.005a

0.577 0.002a 0.30 7 0.003e 0.357 0.003d 0.43 7 0.003c 0.50 7 0.003b

33.04 7 0.33a 28.87 7 1.01b 23.157 0.02d 24.547 0.08d 26.20 7 0.22c

10.577 0.05d 11.59 7 0.02b 11.277 0.02c 13.117 0.03a 11.55 7 0.02b

2.52 7 0.002a 2.277 0.004b 1.707 0.002e 1.87 7 0.002d 2.02 7 0.002c

0.95 70.08a 0.83 70.02c 1.00 70.02a 0.90 70.01b 1.14 70.008a

13.337 0.22b 7.917 0.22d 14.25 7 0.28a 7.007 0.14e 9.147 0.22c

57.50 70.52a 56.00 70.28b 54.66 70.30c 45.58 70.36e 48.33 70.36d

Values not followed by same letters within column are significantly different at po 0.05. DAE: Day after emergence.

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be more sensitive to O3 induced injury than late maturing varieties. Potato variety used in this experiment is also an early maturing that may be a reason for higher negative effects of O3 on yield. In the present study, plants grown under ACO2 þ AO3 treatment also showed significant reductions in plant height, number of leaves, leaf area, number of tubers, and fresh and dry weights of tubers at mean O3 concentration of 49.6 ppb, suggesting that present ambient concentrations of ozone are high enough to affect physiological and metabolic functions of plants, resulting into substantial yield reduction. This result indicates that the plants mechanism of resistance towards O3 was not enough and gradually weakened as the number of tubers was not affected much by O3 but later on tuber dry weight declined drastically. Also the ambient concentration of CO2 was not able to provide much support in avoiding ozone damage to plant. Asensi-Fabado et al. (2010) reported 24% yield reduction in potato under ambient O3 concentration. Exposure at ACO2 þAO3, however, had no significant effect on above ground total biomass despite leaf damage in the present study. These results further proved the hypothesis that O3 at ambient and elevated levels may cause visible injuries on leaves leading to reductions in number and biomass accumulation in tubers; the reductions being higher at elevated compared to ambient O3. Number of tubers and their biomass increased under ECO2 þ AO3 and ECO2 þEO3 compared to ACO2 þ AO3 at both the harvest suggesting that ameliorative effects of elevated CO2 on tuber development continued till latter stages of growth. Donnelly et al. (2001) also found significant increments in above and below ground biomass at intermediate and final harvests under elevated CO2 with ambient O3. The enhancement in tuber yield may be ascribed to increase in photosynthesis under elevated CO2 (Conn and Cochran, 2006), and a higher transport efficiency of photosynthates to tubers, leading to high biomass accumulation and yield. The results of biomass allocation clearly show that maximum proportion of biomass to the tubers was under ECO2 þAO3 at both the ages of sampling. Finnan et al. (2002) reported that tuber yield was unaffected by elevated O3 (50–70 ppb) at ambient concentrations of CO2, while yield was decreased at elevated O3 concentration in both years of that study. The yield increase under elevated CO2 was due to larger tuber sizes rather than an increase in the number of tubers (Finnan et al. 2002). In the present study, however, enhancement of yield under ECO2 þ AO3 was found due to increase in number of tubers as well as sizes of tubers at both harvests. Tubers of intermediate and larger sizes increased under ECO2 þAO3 in terms of number, fresh and dry weights, indicating that excess assimilates as a result of increased photosynthesis were well utilized by the growing sinks. In the present study, biomass allocated to tubers increased with concomitant reductions in allocation to stem and leaf under ECO2 þ AO3. Lawson et al. (2001), however, reported that at final harvest under ECO2 and ambient O3, fresh weight of only tuber o 35 and 35–50 mm sizes increased significantly suggesting that tuber number increased, but the additional tubers remained small. The results of the present study further suggest the enhancement in the economical value of potato crop under elevated CO2, as intermediate and largest size tubers are mainly responsible for the increase in the yield under ambient and elevated CO2. Dry weight of tubers of 35–50 and 450 mm increased under ECO2 þAO3 treatment, while dry weight of smaller tuber did not vary compared to ACO2 þ AO3. The enhancement of marketable potato yield under elevated CO2 were more under current ambient ozone concentration than with the predicted elevated ozone concentration, suggesting that elevated ozone has capacity to counteract the positive effect of elevated CO2 in long term exposure conditions. However, the negative effect was less than the ACO2 þ AO3.

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In the present study, leaf area at the intermediate harvest increased under ECO2 þAO3 and ECO2 þEO3 compared to ACO2 þ AO3 with maximum increase in ECO2 þAO3. The increase in leaf area might have helped in enhancing light capture and photo assimilation, which was maximum under ECO2 with ambient O3 and hence leaf area was higher under ECO2 þ AO3 compared to ECO2 þEO3. Greater carbohydrate supply and improved wateruse efficiency have been shown to support larger leaves and more rapid leaf development under ECO2 (Ferris et al., 2001). Larger leaves may also provide a larger area susceptible to damage by O3. Under ECO2 þAO3 treatment, number of potato tubers and fresh weight increased maximally followed by ECO2 þEO3 compared to ACO2 þ AO3 at both harvests. This suggests that the elevated concentration of CO2 has lessen the negative effects of ambient O3 more than elevated O3 on tuber initiation, as the excess photosynthate might be utilized for repair process and avoiding leaf damage. This also indicates that O3 has caused more negative effects on growth of potato plants at ambient CO2 with ambient O3 compared to elevated CO2 with elevated O3. However no significant effects of CO2 (550 ppm) and O3 (50–60 ppb) on any of the growth and yield variables of potato were also reported (Lawson et al., 2001; Craigon et al., 2002). Plessel et al. (2007) reported that there was no elevated CO2 induced increase in tuber mass at double ambient O3, suggesting that O3 at elevated concentrations counteracted elevated CO2 by inhibiting the allocation of assimilates from leaves to tubers. In the present study, however, CO2 at elevated concentration enhanced above ground biomass and tuber biomass to a greater extent than the reduction under ambient and elevated O3, resulting in net increase under combined treatment of ECO2 þAO3 and ECO2 þEO3. Elevated CO2 concentration has been shown to alleviate the negative effects of ambient ozone concentration resulting into beneficial impact as considerable increase in yield was recorded (Craigon et al., 2002; Donnelly et al., 2001). The hypothesis that maximum ameliorating effects of elevated CO2 will be at ambient level of O3 was also found to be correct. However, ECO2 þEO3 was found to cause less damaging effects than ACO2 þAO3. 4.2. Growth indices Relative growth rate was maximum in ECO2 þAO3 due to increase in NAR. Increased photosynthetic activity may be the reason for an increase in NAR under ECO2 þAO3. Also increased leaf area under ECO2 þAO3 might have provided more area for assimilation of photosynthates. This suggests that assimilation rate is an important determining factor for maintenance of RGR. SLA declined under ECO2 þAO3, suggesting accumulation of more biomass per unit area. A decrease in SLA of potato leaves exposed at elevated CO2 but no change at ambient CO2 with elevated O3 was reported (Donnelly et al., 2001). Decrease in SLA is most likely a consequence of accumulation of starch. Larger LAR suggests major contribution of photosynthates in leaf expansion. In the present study, higher LAR under elevated O3 suggests major contribution of photosynthate in leaf expansion as a compensatory mechanism. Again increase in SLA justifies this response as it indicates that the leaf area expansion was relatively higher than the biomass of the leaves. Donnelly et al. (2001) and Hacour et al. (2002) reported no significant effect of elevated O3 on SLA of potato leaves. In the present study, RGR and NAR reduced under both elevated and ambient O3 concentrations at ambient CO2 significantly. Physiological alteration mainly in photosynthesis due to O3 might lead to reduction in RGR (Biswas et al., 2013). Reduction in NAR indicates that the efficiency of dry matter production in leaves was reduced by O3 even at ambient concentration. Reduction in net photosynthetic rate is suggested to be the most important factor leading to reduction in NAR of O3 exposed

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plants (Rai et al., 2010). Under combination of ECO2 þ EO3, RGR increased due to increase in LAR, indicating that the leaves utilized the excess CO2 and produced more photosynthates, which enhanced leaf expansion. This shows that RGR was affected more by the photosynthetic efficiency than the increased leaf expansion. Reductions in RGR and NAR of ACO2 þAO3 treated plants compared to ACO2 confirmed the adverse effects of ambient ozone on assimilation capacity. Increase in SLA of ACO2 þAO3 and ACO2 þEO3 suggests the production of thinner leaves and utilization of photosynthate for repair processes rather than biomass in leaves. Also lower NAR at ACO2 þ AO3 suggests higher O3 influx in presence of ACO2 causing reduction in assimilation capacity during the present study. Rai et al. (2010), however, found significant reductions in RGR and LAR, but no significant effect on SLA of rice crop at ambient O3 concentrations.

4.3. Impact on quality Potato quality was determined on the basis of starch and sugar contents and nutrient elements. Glucose and fructose are the primary reducing sugars in potato tubers. Potato starch is a polysaccharide. Starch and soluble sugar contents were highest and reducing sugar content was the lowest under ECO2 þAO3 in the present study. The greater starch content and dry matter of tubers under ECO2 þAO3 may be attributed to increases in leaf area and photosynthesis, which may increase the availability of photosynthate for storage in the tubers (Donnelly et al., 2001; Vorne et al., 2002). High dry matter content improves texture and crispness of the fried products, prevents excessive fat absorption in frying, and reduces the susceptibility to black spot as well as the risk of sogginess (Storey and Davies, 1992). The direct relationship between CO2 enrichment and dry matter content in potato tubers under ambient and enhanced O3 during the present study is consistent with earlier reports (Vorne et al., 2002; Heagle et al., 2003). The decline in reducing sugars found in this study resulting from ECO2 is beneficial to potato growers as high reducing sugar content is responsible for producing overly dark colors when tubers are fried in oil at high temperatures. Food industries prefer low concentration of reducing sugars in tubers to avoid the darkening of chips due to the Maillard browning reaction, which generates accrylamide, a known carcinogen (Mottarm et al., 2002). In the present study, exposure to ACO2 þ EO3 significantly increased the reducing sugar content of the tubers along with the reduction in total dry matter and starch content at final harvest. Assimilates are transported from the leaves to tubers in form of sucrose and are enzymatically converted to starch in the tubers (Oparka et al., 1992). As leaves were injured under ACO2 þAO3 and ACO2 þEO3 treatments, lower translocation of sucrose may have reduced starch accumulation in tubers. Donnelly et al. (2001) found no variation in starch content of tubers under ACO2 þEO3, while Kollner and Krause (2000) reported a decrease. As reducing sugar increased and starch content decreased under ambient and elevated ozone with ACO2 in the present study, the quality of the potato tubers deteriorated for industrial uses after O3 exposure. Reducing sugar was more or less same in ACO2 þAO3 and ECO2 þ EO3. Observed reductions in starch and soluble sugar contents under ACO2 þEO3 resulted due to induced disturbance/hindrance of assimilate allocation in potato resulted in reduced fresh weight and quality of tubers. This result accords with Vorne et al. (2002) who found reduced assimilate production and disturbance of carbohydrate allocation, resulting in reduced yield in potato fumigated with O3 in OTCs. Similarly, plants grown under ACO2 þAO3 showed increase in reducing sugar and decrease in starch content of tuber and thus affecting quality even at ambient O3. Carbohydrate content under ambient O3 was also negatively affected in wheat (Rai et al., 2010).

Mineral elements in staple food crops are important sources of mineral nutrients for human bodies. Studies focusing Meta analyses on crop species showed that elevated CO2 generally reduces plant element concentrations (Hogy and Fangmeier, 2009a,2009b). In the present study, ECO2 þAO3 treatment stimulated the biomass maximally, but the protein and amino acids and concentrations of Ca, K, Zn and Fe in tubers were lower than ACO2 þAO3. Decline in mineral nutrients under elevated CO2 is most likely a dilution effect due to enhancement of dry matter (Heagle et al., 2003; Kumari et al., 2013). In case of potato, tubers grow mainly by cell division and by expansion of the established cells (Bodin and Svensson, 1996). The cell expansion to a large extent is due to increased volume of vacuoles and incorporation of starch in the amyloplasts (Bodin and Svensson, 1996). Larger amount of stored starch in the amyloplasts results into decreased nutrient: starch ratio. Because starch is the major solid of potato tubers, the nutrient concentration in the tubers decreases. This phenomenon is referred as growth dilution. Loladze (2002) suspected that CO2 enrichment across the globe would exacerbate micronutrient (Cu, Zn and Fe) deficiencies in grains of main crops, and thus may intensify the problem of micro nutrient malnutrition. In spite of increased root length and amendment of recommended NPK, the dilution of elements was not overcome in the present study. Lower N and K concentrations reduce the susceptibility for sogginess, but increase the potential for non enzymatic browning of potato chips. In the present study nutrient elements Mg, K, Fe, Cu and Zn in potato tubers decreased significantly under ambient (ACO2 þAO3) and elevated ozone (ACO2 þEO3) treatments compared to ACO2. Likewise reduction in the above elements was found under combination of ECO2 þEO3 treatment compared to ACO2 þAO3. Ozone exposure probably caused a reduced assimilate supply to the growing tuber and thus reduced the nutrient levels in tuber as also observed by Singh et al. (2013) in mustard under ACO2 þ AO3 treatment. Since water plays a significant role in mineral mobilization, reduced stomatal conductance under ambient and elevated O3 (Rai et al., 2011) might be the reason to reduce the transpiration, thus affecting mineral uptake in the present study. Ozone induced reductions in root biomass may also impact the ability of the plants to take up the nutrients and water required to sustain growth and yield (Rai et al., 2011). Another study on mustard by Singh et al. (2009) have also reported that protein and nutrient (Ca, Mg, K, P, Zn) contents significantly decreased in plants grown in ACO2 þAO3 compared to ACO2 at mean ozone concentrations ranging from 41.65 to 54.2 ppb in a similar tropical area at Varanasi. Heagle et al. (2003), however, reported that in potato tuber, the concentrations of N, P, K and Zn increased under elevated ozone. Likewise, Fangmeier et al. (2002) found that elevated ozone increased nitrogen and manganese concentrations in tubers. Piikki et al. (2007) found that O3 exposure of potato increased the concentrations of K and Mg in the tubers, while Ca was unaffected. However, in the present only Ca and Na contents increased significantly under ACO2 þAO3 and ACO2 þEO3 treatments compared to ACO2. Under combination of ECO2 þ EO3, Ca content also increased significantly, while no significant variation was observed for Na. The increase in Ca concentration during the present study might be the strategy of plant to avoid the harmful effects of O3 on its metabolic activity to sustain growth and yield. Potato protein is of a fairly high quality because of the physiologically valuable amino acid composition. In the present study, potato grown under ECO2 þAO3 showed higher C/N ratio, which was associated with increase in C and decrease in total N, thus resulting in lower protein. CO2 enrichment has been reported to decrease total protein concentration (Hogy and Fangmeier, 2009b; Fangmeier et al., 2002), indicating deterioration of quality for consumers. In contrast, no impact on the protein concentration of potato under ECO2 þAO3 was observed by Piikki et al. (2007).

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Pleijel and Uddling (2012) analyzed 57 experiments with CO2 and O3 exposures and found that elevated CO2 has a direct negative effect on grain protein accumulation due to CO2 induced impairment of nitrate assimilation. In the present study, reduction in tuber N content under ECO2 þAO3 was mainly due to increase in the starch content The decreases in leaf protein may also contribute to the decrease in tuber protein concentration in potato (Fangmeier et al., 2002), as tubers receive approximately 50% of the N translocated from the leaves and stems. Amino acid concentrations in the present study decreased under CO2 enrichment due to lower N availability both under ambient and elevated levels of O3. Dilution effect is mainly responsible (Fangmeier et al., 2002) for decrease in amino acid contents as dry matter in tubers enhanced both under ECO2 þAO3 and ECO2 þEO3 treatments. The present study has also confirmed the hypothesis that both CO2 and O3 under ambient and elevated levels will modify nutritional qualities of tubers.

5. Conclusion Potato plants grown at elevated CO2 þAO3 showed most favorable growth response and highest total biomass accumulation and tuber yield among all the treatments of CO2 and O3 at ambient and elevated levels. Biomass allocated to stems and leaves declined, while that allocated to tubers increased under ECO2 þAO3. CO2 enrichment both at ambient and elevated levels of O3 improved the potato quality for industrial processing and marketing by increasing tuber size of4 50 mm; but reduced protein and mineral nutrients such as Ca, K, Fe and Zn. Ambient as well as predicted future increases in O3 concentrations were found to have detrimental effects on marketable tuber yield and tuber quality by causing visible damage to potato leaves. Negative effects of ambient and elevated O3 include reductions in starch, protein, amino acid and Mg, K, Fe, Mn, Cu, Zn contents. Increased reducing sugar under ACO2 þEO3 deteriorated the quality of potato affecting human consumption and industrial uses. ECO2 protected the leaves from O3 injury. Elevated CO2 protected yield losses also under elevated O3, but ACO2 was not found to protect the plants under present ambient O3 concentrations. In future, when both O3 and CO2 may increase to the concentrations opted in this experiment; the positive effects of CO2 on yield can be counteracted by the negative effects of O3. As food security is a most important issue due to population increase, qualitative and nutritional modifications in potato under futuristic concentrations of CO2 and O3 have serious implications on human health. Acknowledgment Sumita Kumari is grateful to University Grant Commission, India for research fellowship. Authors thank Head, Department of Botany, Banaras Hindu University, Varanasi for providing necessary laboratory facility. Our special thanks go to the reviewers of this paper for their valuable suggestions and corrections.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2013.12.021. References Asensi-Fabado, A., Garcia-Breijo, F.J., Reig-Arminana, J., 2010. Ozone induced reductions in below-ground biomass: an anatomical approach in potato. Plant Cell Environ. 33, 1070–1083.

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Atkinson, R., 2000. Atmospheric chemistry of VOCs and NOx. Atmos. Environ. 34, 2063–2101. Avnery, S., Mauzerall, D.L., Liu, J., Horowitz, L.W., 2011. Global crop yield reductions due to surface ozone exposure: 2. Year 2030 potential crop production losses and economic damage under two scenarios of O3 pollution. Atmos. Environ. 45, 2297–2309. Biswas, D.K., Xu, H., Li, Y.G., Ma, B.L., Jiang, G.M., 2013. Modification of photosynthesis and growth responses to elevated CO2 by ozone in two cultivars of winter wheat with different years of release. J. Exp. Bot. 64, 1485–1496. Bodin, B., Svensson, B., 1996. Potatis och Potatisproduktion. Institutionen for vaxtodlingslara, Sveriges Lantbruksuniversitet SLU, Uppsala; Sweden (in Swedish). Conn, J.S., Cochran, V.L., 2006. Responses of potato to elevated atmospheric CO2 in the North American Subarctic. Agric. Ecosyst. Environ. 112, 49–57. Craigon, J., Fangmeier, A., Jones, M., Donnelly, A., Bindi, M., De Temmerman, L., Persson, K., Ojanpera, K., 2002. Growth and marketable-yield responses of potato to increasing CO2 and ozone. Eur. J. Agron. 17, 273–290. De Temmerman, L., Pihl-Karlsson, G., Donnelly, A., Ojanpera, K., Jager, H.-J., Finnan, J., 2002. Factors influencing visible ozone injury on potato and the interaction with increasing CO2. Eur. J. Agron. 17, 291–302. Donnelly, A., Craigon, J., Black, R.C., Colls, J.J., Landon, G., 2001. Elevated CO2 increases biomass and tuber yield in potato even at high ozone concentrations. New Phytol. 149, 265–274. Dubois, M., Gilles, K.A., Hamilton, J.K., Roberts, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Fangmeier, A., De Temmerman, L., Black, C., Persson, K., Vorn, V., 2002. Effects of elevated CO2 and/or ozone on nutrient concentrations and nutrient uptake of potatoes. Eur. J. Agron. 17, 353–368. FAO, 2008 [email protected] 〈www.potato2008.org〉. Ferris, R., Sabatti, M., Miglietta, F., Mills, R, Taylor, G., F., 2001. Leaf area is stimulated in Populus by free air CO2 enrichment (POPFACE), through increased cell expansion and production. Plant Cell Environ. 24, 305–315. Finnan, J.M., Donnelly, A., Burke, J.I., Jones, M.B., 2002. The effects of elevated concentrations of carbon dioxideand ozone on potato (Solanum tuberosum L.) yield. Agric. Ecosyst. Environ. 88, 11–22. Hacour, A., Craigon, J., Vandermeiren, K., Ojanpera, K., Pleijel, H., Danielsson, H., Hogy, P., Finnan, J., Bindi, M., 2002. CO2 and ozone effects on canopy development of potato crops across Europe. Eur. J. Agron. 17, 257–272. Heagle, A.S., Miller., J.E., Pursley., W.A., 2003. Growth and yield responses of potato to mixtures of carbon dioxide and ozone. J. Environ. Qual. 32, 1603–1610. Herbart, D., Philipps, P.J., Strange, R.E., 1971. Estimation of reducing sugars. In: Noories, J.R., Robbins, D.W. (Eds.), Method Microbial. Cap III, vol. 10. Academic Press, London New York, pp. 209–344. Hogy, P., Fangmeier, A., 2009a. Atmospheric CO2 enrichment affects potatoes: 1. Above ground biomass production and tuber yield. Eur. J. Agron. 30, 78–84. Hogy, P., Fangmeier, A., 2009b. Atmospheric CO2 enrichment affects potatoes: 2. Tuber quality traits. Eur. J. Agron. 30, 85–94. Hunt, R., 1982. Growth Curves. Edward Arnold Publishers Ltd., London. IPPC, Climate Change 2007. The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press. Jackson, M.L., 1973. Soil Chemical Analysis. Prentice Hall of India pvt. Ltd., New Delhi, India p. 187. Kangasjarvi, J., Jaspers, P., Kollist, H., 2005. Signalling and cell death in ozoneexposed plants. Plant Cell Environ. 28, 1021–1036. Kollner, B., Krause, G.H.M., 2000. Changes in carbohydrates, leaf pigments and yield in potatoes induced by different ozone exposure regimes. Agric. Ecosyst. Environ. 78, 149–158. Kumari, S., Agrawal, M., Tiwari, S., 2013. Impact of elevated CO2 and elevated O3 on Beta vulgaris L.: pigments,metabolites, antioxidants, growth and yield. Environ. Pollut. 174, 279–288. Lawson, T., Craigon, J., Black, C.R., Colls, J.J., Tulloch, A.-M., Landon, G., 2001. Effects of elevated carbon dioxide and ozone on the growth and yield of potatoes (Solanum tuberosum) grown in open-top chambers. Environ. Pollut. 111, 479–491. Loladze, I., 2002. Rising atmospheric CO2 and human nutrition: towards globally imbalanced plant stoichiometry. Trends Ecol. Evol. 17, 457–461. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the foliar phenol reagent. J. Biol. Chem. 193, 265–275. Miglietta, F., Magliulo, V., Bindi, M., Cerio, L., Vaccari, F.P., Loduca, V., Peressotti, A., 1998. Free air CO2 enrichment of potato (Solanum tuberosum L.): development, growth and yield. Global Change Biol. 4, 163–172. Mills, G., Buse, A., Gimeno, B., Bermejo, V., Holland, M., Emberson, L., Pleijel, H., 2007. A synthesis of AOT40-based response functions and critical levels of ozone for agricultural and horticultural crops. Atmos. Environ. 41, 2630–2643. Mishra, A.K., Rai, R., Agrawal, S.B., 2013. Differential response of dwarf and tall tropical wheat cultivars to elevated ozone with and without carbon dioxide enrichment: growth, yield and grain quality. Field Crops Res. 145, 21–32. Mosley, A.R., Rowe, R.C., Weidensaul, T.C., 1978. Relationship of foliar ozone injury to maturity classification and yield of potatoes. AM Potato J. 55, 147–153. Mottram, D.S., Wedzicha, B.L., Dodson, A.T., 2002. Food chemistry: acrylamide is formed in the Maillard reaction. Nature 419, 448–449. NOAA/ESRL 2013 〈http://www.esrl.noaa.gov/gmd/ccgg/trends/〉. Oparka, K.J., Viola, R., Wright, K.M., Prior, DAM., 1992. Sugar The Benjamin Cummings Publishing Company transport in the potato tuber. In: Farrar, JF, Gordon, AJ, Trethewey, RN, Geigenberger, P, Henning, A, Notter-Fleischer, H,

156

S. Kumari, M. Agrawal / Ecotoxicology and Environmental Safety 101 (2014) 146–156

Pollock, M. (Eds.), Carbon Partitioning Within and Between Organisms. Bios Scientific Publishers, Oxford, pp. 91–114. Persson, K., Danielsson, H., Sellden, G., Pleijel, H, 2003. The effects of tropospheric ozone and elevated carbon dioxide on potato (Solanum tuberosum L.cv. Bintje) growth and yield. Sci. Total Environ. 310, 191–201. Piikki, K., Vorne, V., Ojanpera, K., Pleijel, H., 2007. Impact of elevated O3 and CO2 exposure on potato (Solanum tuberosum L. cv. Bintje) tuber macronutrients (N, P, K, Mg, Ca). Agric. Ecosyst. Environ. 118, 55–64. Pleijel, H., Uddling, J., 2012. Yield vs. quality trade-offs for wheat in response to carbon dioxide and ozone. Global Change Biol. 18, 596–605. Pleijel, H., Danielsson, H., 2009. Yield dilution of grain Zn in wheat grown in opentop chamber experiments with elevated CO2 and O3 exposure. J. Cereal Sci. 50, 278–282. Plessl, M., Elstner, E.F., Rennenberg, H., Habermeyer, J., Heiser, I., 2007. Influence of elevated CO2 and ozone concentrations on late blight resistance and growth of potato plants. Environ. Exp. Bot. 60, 447–457. Rai, R., Agrawal, M., 2012. Impact of tropospheric ozone on crop plants. Proc. Natl. Acad. Sci. India Sect. B: Biol. Sci. 82 (2), 241–257. Rai, R., Agrawal, M., Agrawal, S.B., 2010. Threat to food security under current levels of ground level ozone: a case study for Indian cultivars of rice. Atmos. Environ. 44, 4272–4282. Rai, R., Agrawal, M., Agrawal, S.B., 2011. Effects of ambient O3 on wheat during reproductive development: gas exchange, photosynthetic pigments, chlorophyll fluorescence, and carbohydrates. Photosynthetica 49, 285–294. Rai, R., Agrawal, M., 2014. Assessment of competitive ability of two Indian wheat cultivars under ambient O3 at different developmental stages. Environ. Sci. Pollut. Res. 21, 1039–1053. Roy, S., Beig, G., Ghude, S., 2009. Exposure plant response of ambient ozone over the tropical Indian region. Atmos. Chem. Phys. 9, 4141–4157. Sarkar, A., Agrawal, S.B., 2012. Evaluating the response of two high yielding Indian rice cultivars against ambient and elevated levels of ozone by using open top chambers. J. Environ. Manage. 95, S19–S24.

Singh, S., Bhatia, A., Tomer, R., Kumar, V., Singh, B., Singh, S.D., 2013. Synergistic action of tropospheric ozone and carbon dioxide on yield and nutritional quality of Indian mustard (Brassica juncea (L.) Czern.). Environ. Monit. Assessment 185, 6517–6529. Singh, P., Agrawal, M., Agrawal, S.B., 2009. Evaluation of physiological, growth and yield responses of a tropical oil crop (Brassica campestris L. var. Kranti) under ambient ozone pollution at varying NPK levels. Environ. Pollut. 157, 871–880. Storey, R.M.J., Davies, H.V., 1992. Tuber quality. In: Harris, P.M. (Ed.), The Potato Crop: The Scientific Basis for Improvement. Chapman & Hall, London, pp. 507–552. The Royal Society, 2008. Ground-level Ozone in the 21st Century: Future Trends, Impacts and Policy Implications. Science Policy Report 15/08. Tiwari, S., Agrawal, M., Marshall, F.M., 2006. Evaluation of ambient air pollution impact on carrot plants at a suburban site using open top chamber. Environ. Monit. Assessment 119, 15–30. Vandermeiren, K., Black, C., Pleijel, H., De Temmerman, L., 2005. Impact of rising tropospheric ozone on potato: effects on photosynthesis, growth, productivity and yield quality. Plant Cell Environ. 28, 982–996. Vorne, V., Ojanpera, K., De Temmerman, L., Bindi, M., Hogy, P., Jones, M.B., Lawson, T., Persson, K., 2002. Effects of elevated carbon dioxide and ozone on potato tuber quality in the European multiple-site experiment ‘CHIP project. Eur. J. Agron. 17, 369–381. Walkley, A., Black, C.A., 1934. An examination of de Gjareff methods for determining soil organic matter and proposed modification of the chromic acid titration method. Soil Sci. 37, 29–38. Wang, Y., Frei, M., 2011. Stressed food – the impact of abiotic environmental stresses on crop quality. Agric. Ecosyst. Environ. 141, 271–286. Wang, Y., Zhang, Y., Hao, J., Luo, M., 2011. Seasonal and spatial variability of surface ozone over China: contributions from background and domestic pollution. Atmos. Chem. Phys. 11, 3511–3525. Wheeler, R.M., Mackowiak, C.L., Sager, J.C., Knott, W.M., 1994. Growth of soybean and potato at high CO2 partial pressure. Adv. Space Res. 14, 251–255.