Ecotoxicology and Environmental Safety 112 (2015) 29–38

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

http://d0147-65

n CorrE-m

madhoo

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

Application of ethylene diurea (EDU) in assessing the response of atropical soybean cultivar to ambient O3: Nitrogen metabolism,antioxidants, reproductive development and yield

Richa Rai a, Madhoolika Agrawal a,n, Krishna Kumar Choudhary a, S.B. Agrawal a,Lisa Emberson b, Patrick Büker b

a Department of Botany, Banaras Hindu University, Varanasi, Indiab Stockholm Environment Institute, University of York, United Kingdom

a r t i c l e i n f o

Article history:Received 10 July 2014Received in revised form20 October 2014Accepted 21 October 2014

Keywords:O3

SoybeanGrowthNitrogen assimilationEDUYield

x.doi.org/10.1016/j.ecoenv.2014.10.03113/& Elsevier Inc. All rights reserved.

esponding author.ail addresses: madhoo.agrawal@gmail.com,58@yahoo.com (M. Agrawal).

a b s t r a c t

The present study deals with assessment of response of a tropical soybean cultivar to O3 in relation tophotosynthetic pigments, chlorophyll fluorescence kinetics, antioxidative capacity, N assimilation en-zymes, metabolites, growth and yield using ethylene diurea (EDU) given as a soil drench (400) ppm at aninterval of 10 days after germination up to maturity. Mean O3 concentration was 42 ppb and accumulatedthreshold above 40 ppb (AOT 40) was 9.07 ppm h. Lipid peroxidation and total phenolics reduced, whileincreases in activities of antioxidative and nitrogen assimilation enzymes, ascorbic acid, protein, pho-tosynthetic pigments, Fv/Fm ratio, number of leaves, flowers, pods, branches and yield attributes werefound in EDU treated plants. EDU alleviated the negative effects of O3 by enhancing the first line ofdefense against ROS and protecting N assimilation enzymes at flowering and maintaining adequatesupply of photosynthates to developing pods during pod filling stage. EDU provided maximum protec-tion between flowering to pod filling stage.

& Elsevier Inc. All rights reserved.

1. Introduction

Tropospheric O3 is identified as the air pollutant most likely tothreaten food production across the globe due to its phytotoxicityand prevalence over important agricultural regions of NorthAmerica, Europe and Asia (Emberson et al., 2009; Van Dingenenet al., 2009). Van Dingenen et al. (2009) reported annual produc-tion losses of U.S $ 14 to 26 billion for major agriculture com-modities resulting from emissions under present day air qualitylegislation. Surface O3 concentrations have risen from a pre-in-dustrial value of 10 ppb to an average summer concentration be-tween 30 and 50 ppb in the mid latitudes of the northern hemi-sphere (Royal Society, 2008). Meehl et al. (2007) projected an in-crease of 20–25% in mean global O3 concentration between 2015and 2050 and 40–60% by 2100. Global photochemical modelspredict increase in O3 concentration by 2030 in several parts ofAsia under a current legislation emission scenario (Dentener et al.,2005).

Important crops such as wheat and soybean are highly sensi-tive to O3. To feed the growing population, new mitigation andmanagement techniques should be employed to protect cropsfrom O3 damage. Areas with elevated concentrations of O3 need tobe identified by using screening methods that may be either se-lecting sensitive species or the use of chemical protectants toidentify geographical locations where ambient O3 levels can conferdamage to crops. In the 1970s, an antioxidant ethylene diurea(EDU/N-[2-(2-oxo-1- imidazolidinyl) ethyl- N′-phenylurea] wasfound to protect agricultural plants from O3 injury (Carnahan et al.,1978). Since then a large number of experiments on various cropspecies have demonstrated the effectiveness of EDU as a researchtool for identifying susceptibility of crops to ambient O3 (Fenget al., 2010). EDU is a very cheap and effective method to assess theimpact of ambient O3 on crops in developing countries (Feng et al.,2010; Tiwari et al., 2005), where electricity is a major constraintand O3 monitoring equipments are not available. Hence biomo-nitoring programmes using EDU may provide a viable and suc-cessful tool to map O3 injury on plants.

The mode of action of EDU is still not completely understood. Itis not known whether EDU affects stomatal conductance or whe-ther it confers tolerance through induction of enzyme systemsinvolved in scavenging of O3 induced reactive oxygen species(ROS).

R. Rai et al. / Ecotoxicology and Environmental Safety 112 (2015) 29–3830

It is a well known fact that soybean is a very sensitive crop toO3 and wide variations in yield losses are reported from Europe,but such information under ambient levels of O3 from the Asianregion are scarce (Emberson et al., 2009; Van Dingenen et al.,2009). Emberson et al. (2009) reported that Asian cultivars of riceand wheat are more sensitive to O3 compared to North Americancultivars. Studies related to response of soybean cultivars underambient and elevated levels of O3 mainly concentrated on growthand yield parameters in the Asian region (Wahid et al., 2001, Singhet al., 2010; Singh and Agrawal, 2011). No study has attempted toevaluate the response of soybean cultivars growing at ambient O3

concentration using EDU as a tool with respect to changes in an-tioxidant defense and nitrogen metabolism enzymes affecting theplant performance at different developmental stages and re-productive output. Soybean, being a leguminous crop may show alarge magnitude of protection to N metabolism under EDU treat-ment at ambient concentrations of O3. It was hypothesized thatunder ambient O3 concentration, EDU will provide protection to N

Fig. 1. (A) Weekly variations in O3 concentrations, (B) AOT 40 during the experimentaperature and, (D) monthly variations in total rainfall, minimum and maximum relative

metabolism, growth and yield of soybean plants via strengtheningthe antioxidant system. The protection afforded against O3 by EDUwill vary with growth stage.

The present investigation was undertaken with the objective toevaluate the impacts of ambient ozone exposure on the develop-mental stages of soybean affecting its yield and to assess themechanism of action of EDU in a tropical leguminous crop in re-lation to its defense action and nitrogen metabolism

2. Materials and methods

2.1. Experimental site

The experiment was conducted under natural field conditionsduring rainy season from July to October, 2012 at a suburban areaof Varanasi city situated in the eastern Gangetic plains of Indiansubcontinent at 25°14′ N latitude, 82°03′E longitude and 76.2 m

l period, (C) monthly variations in sunshine hours, minimum and maximum tem-humidity during experimental period.

R. Rai et al. / Ecotoxicology and Environmental Safety 112 (2015) 29–38 31

above sea level. Mean monthly minimum and maximum tem-peratures were 24 and 33.5 °C. The mean monthly minimum andmaximum relative humidity were 75.6% and 87.9%. Total sunshinehours during the experimental period was 581.2 h and total rain-fall was 714.9 mm (Fig. 1).

2.2. Experimental plant

Seeds of test cultivar of soybean (Glycine max L. cv. JS 335) wereobtained from Directorate of Soybean Research, Indore. Soybean isa nutritious leguminous crop that provides oil seed and vegetableoil. It is also a very good source of high quality protein. JS 335(Jawahar Soybean 335) is a semi- determinate cultivar of soybean.Its maturity time is 95–100 days and was released in 1994. It iscommonly grown in central zone (M.P., Maharashtra, Rajasthan,Gujrat, Bundelkhand regions of Uttar Pradesh). It is resistant tobacterial pustule of bacterial blight and tolerant to green mosaicand susceptible to yellow mosaic virus. Its yield potential is2500–3000 kg ha�1.

2.3. Raising of plants

Plants were raised in earthen pots of 12.5 cm radius and 30 cmdepth. The soil for pot filling was prepared at one place aftermixing soil and farmyard manure in the ratio of 3:1. Seeds wereinoculated with Rhizobium culture of strain Bradyrhizobium japo-nicum L. Seeds of soybean were sown in 40 separate pots. Aftergermination, plants were thinned to one plant per pot. After aweek of germination, 20 pots were treated with 100 ml of EDU(400 ppm) solution as soil drench at 10 days interval upto 35 daysafter germination. Thereafter, 200 ml EDU plant�1 was applied tillthe maturity (R8 stage) of pods. Fresh solution of EDU was pre-pared each time using deionized water. Twenty pots were kept asnon EDU treated and deionized water was given equivalent to EDUsolution at same interval. Plants were subjected to identical waterregime both for EDU and non EDU treated plants. Pots were irri-gated using wicks to avoid water stress as given in experimentalprotocol by ICP vegetation (Mills et al., 1997). There were twentyreplicates each of EDU and non EDU treated plants and wereplaced randomly in the ambient air in an open area receiving fullsunlight.

2.4. O3 monitoring

Twelve hour (07:00–19:00 h) monitoring of O3 concentrationswas conducted by using an automatic O3 analyzer (Model 400 A,API, Inc., USA). Exceedance hours above 40 ppb were calculated.

Exposure index for ozone i.e. AOT 40 (Accumulated ozone overa concentration threshold of 40 ppb) was calculated by followingformula given by Mills et al. (2007).

∑= − ≥AOT40 [CO 40] for CO 40 ppb, [AOT 40 units: ppmh]i

n3 3

where, CO3 is the hourly O3 concentration in parts per billion(ppb), i is the index, n is the number of hours with CO3440 ppbover the 3-month growing period that has been set as the eva-luation period for respective crops.

2.5. Chlorophyll fluorescence kinetics

Ten plants each of EDU and non EDU treatments were selectedrandomly for measurement of chlorophyll fluorescence kinetics atflowering stage. Chlorophyll fluorescence was measured between09:00 and 11:00 h using a portable plant efficiency analyzer(Model MK 29414, Hansatech Instrument Ltd.) on second fullyexpanded trifoliate. Leaf clips for dark adaptation were placed on

the adaxial side of the leaves 30 min before measurement andthen exposed to red light of 650 nm through LED at excitation ir-radiance of 3000 mmol m�2 s�1. Minimum fluorescence (Fo) andmaximum fluorescence (Fm) were measured from which variablefluorescence (Fv) and ratio of variable and maximal fluorescence(Fv/Fm) were calculated.

2.6. Biochemical analyzes

After estimation of chlorophyll fluorescence kinetics, the sameten replicate plants were selected for analyzes of photosyntheticpigments, lipid peroxidation, antioxidative and N-assimilationenzymes and metabolites. Second fully expanded trifoliate was cutand placed in liquid N2 and stored in a deep freezer (�80 °C) foranalyzes.

2.6.1. Photosynthetic pigments and lipid peroxidationChlorophyll and carotenoids were extracted from the leaf discs

with 80% acetone and quantified according to the methods givenby Maclachlan and Zalik (1963) and Duxbury and Yentsch (1956),respectively. The extent of lipid peroxidation was estimated asmalondialdehyde (MDA) content following the protocol of Heathand Packer (1968).

2.6.2. Antioxidative enzymesSuperoxide dismutase (SOD) was isolated from homogenised

leaves in 100 mM EDTA phosphate buffer (pH 7.8), filtered andcentrifuged at 10,000 g for 15 min. SOD activity was determined inthe supernatant by inhibition of the photochemical reduction ofnitro-blue tetrazolium (NBT) at 560 nm (Fridovich, 1974). The re-action mixture consisted of 50 mM sodium-potassium phosphatebuffer (pH 7.8), 13 mM methionine, 2 μM riboflavin, 75 μM NBT,100 mM EDTA and 20 μl enzyme extract. Ascorbate peroxidase(APX) activity was determined at 290 nm, as ascorbate oxidized in50 mM phosphate buffer (pH 7.6), 0.1 mM EDTA, 0.5 mM ascorbateand 0.1 mM H2O2, according to the method of Nakano and Asada(1987). Glutathione reductase (GR) was assayed as a decrease inabsorbance of NADPH at 340 nm according to Schaedle and Bas-sham (1977) in 50 mM Tris–HCl (pH 7.6), 0.15 mM NADPH, 1 mMGSSG, 3 mM MgCl2 and enzyme extract.

2.6.3. Nitrogen metabolism enzymesNitrate reductase (NR) and nitrite reductase (NiR) activities

were estimated according to the methods of Nicholas and Nason(1957). Enzyme was extracted in 0.1 M phosphate buffer (pH 7.5)containing cysteine and EDTA. Extract was incubated with KNO3

for NR and KNO2 for NiR with NADH for 15 min at room tem-perature and the reaction was stopped by adding sulfanilamide.The mixture was then incubated for 5 min with 1-(napthyl)ethylenediamine (NNED) and absorbance of pink color was mea-sured at 540 nm. Appearance of pink color was measured for NRwhile the degradation in colour was measured for NiR and po-tassium nitrite (KNO2) was used as standard.

2.6.4. Primary and secondary metabolitesAscorbic acid content was estimated by using 2, 6 di-

chlorophenol-indophenol (DCPIP) dye reduction method given byKeller and Schwager (1977). Total phenolics were determined inacetone extracts followed by the addition of folin ciocalteau re-agent by following the protocol given by Bray and Thorpe (1954).Protein was determined by homogenizing the leaf tissue in Trisbuffer and then extracted in trichoroacetic acid (TCA) followed bycentrifugation. After drying, pellets were dissolved in 0.1 N NaOHand protein content in solution was measured after adding alka-line solution and folin ciocalteau reagent by following the protocolof Lowry et al. (1951).

R. Rai et al. / Ecotoxicology and Environmental Safety 112 (2015) 29–3832

2.7. Growth parameters

Growth parameters such as numbers of leaves, senesced leaves,branches, nodes and plant height were recorded at two days in-tervals during vegetative (V4, 15 days after germination ), flow-ering (R1, 30 DAG), pod setting (R5, 55 DAG) and seed setting (R8,85 DAG) stages for all the replicates of EDU and non EDU treatedplants. Data presented in the present study is mean of readingsrecorded at two day interval. Floral parts such as number of

Fig. 2. Variations in lipid peroxidation, photosynthetic pigments and chlorophyll fluoresrepresent Mean71SE, n¼10. Level of significance *po0.05; **po0.01; ***po0.001; N

flowers, abortive flowers, pods, abortive pods and pod/flower ratiowere counted at R1, R5 and R8 growth stages of soybean for all thereplicates of EDU and non-EDU plants.

2.8. Yield attributes

Plants were harvested at maturity and number of pods plant�1,weight of pods plant�1, number of seeds plant�1 and weight of

cence kinetics at flowering stage in EDU and non EDU treated soybean plants. Bars.S not significant. FW: fresh weight, DW: dry weight.

R. Rai et al. / Ecotoxicology and Environmental Safety 112 (2015) 29–38 33

seeds plant�1 were recorded. Harvest index (HI) was calculated asthe rate of economic yield (weight of seed plant�1) to aboveground biomass of the plant.

2.9. Statistical analysis

Data of vegetative and reproductive growth, floral parts, bio-chemical parameters, chlorophyll fluorescence kinetics, photo-synthetic pigments and yield attributes were subjected to Student′s t-test for assessing the significance of quantitative changes indifferent parameters due to EDU using the statistical package SPSS

Fig. 3. Variations in antioxidative and nitrogen assimilation enzymes, total protein andBars represent Mean71SE, n¼10. Level of significance *po0.05; **po0.01; ***po0.00

software (SPSS Inc., version 10.0). Two way ANOVA was used todetermine statistically significant differences in growth para-meters due to stage and treatment.

3. Results

3.1. O3 monitoring

Twelve hour daily mean O3 concentrations varied from 37.2 to46.9 ppb during the experimental period (Fig. 1). AOT 40 value

phenolics content at flowering stage in EDU and non EDU treated soybean plants.1; NS not significant. FW: fresh weight, DW: dry weight.

R. Rai et al. / Ecotoxicology and Environmental Safety 112 (2015) 29–3834

recorded was 9.04 ppm h (Fig. 1). Exceedance hours above 40, 50,60 and 70 ppb O3 concentration were 958, 397, 106 and 20 hduring the experimental period. Mean O3 concentrations

Fig. 4. Variations in growth parameters, number of flowers, pods abortive flowers and usoybean plants (Mean71SE, n¼20). Level of significance*po0.05; **po0.01; ***po0.0

during vegetative (V4), flowering (R1), pod setting (R5) and seedsetting (R8) stages were 37.2, 42.52, 43.36 and 46.9 ppb,respectively.

nfilled pods plant-1at different developmental stages in EDU and non EDU treated01; N.S not significant.

R. Rai et al. / Ecotoxicology and Environmental Safety 112 (2015) 29–38 35

3.2. Photosynthetic pigments and efficiency, enzymes andmetabolites

Lipid peroxidation measured as MDA content reduced sig-nificantly by 20% under EDU treatment compared to non-EDUtreatment (Fig. 2). Significant increases of 11.1%, 18.5% and 40% inactivities of SOD, APX and GR were recorded in EDU treated thannon EDU treated plants (Fig. 3). Under EDU treatment, enzymaticactivities of nitrate reductase, nitrite reductase and protein con-tent increased by 28.5%, 26.3% and 31.3%, respectively. Significantincrease in ascorbic acid content was recorded in EDU treatedplant. Total phenolics decreased by 20.8% in EDU treated comparedto non- EDU plants (Fig. 3).

Significant increases were recorded in Fm and Fv values in EDUtreated plants compared to non EDU treated ones (Fig. 2). Fo and Fv/Fm ratio did not vary significantly between the two treatments.Total chlorophyll and carotenoid contents of EDU treated plantsincreased significantly by 13.6% and 12.1% than non EDU treatedplants (Fig. 2).

3.3. Growth parameters

Number of leaves were significantly higher in EDU treatment atall ages of sampling i.e vegetative (V4), flowering (R1), pod setting(R5) and seed setting (R8) compared to non EDU treatment(Fig. 4 ). Number of senesced leaves decreased in EDU treatedplants than non EDU treated ones. Significant variations wereobserved in number of leaves and senesced leaves due to age andtreatment (Table 1).

Number of nodes and branches increased by 29.6% and 15.9% atflowering, 27.7% and 8.2% at pod setting and 19.7% and 6.8% at seedsetting stages of soybean, respectively in EDU treatment comparedto non EDU treatment (Fig. 4). Plant height was significantlyhigher at all stages of sampling in EDU treatment compared to nonEDU treatment (Fig. 4). Results of two way ANOVA showed thatnumber of branches, nodes and plant height varied significantlydue to age and treatment. Percent increases recorded in number offlowers were 57.1%, 18.8% and 12.4% at flowering (R1), pod (R5)and seed setting (R8) stages, respectively in plants under EDUtreatment. Significant increase in number of abortive flowers wasrecorded at R5 and R8 with maximum increase at seed settingstage in non EDU treated plants. Variations observed in number offlowers and abortive flowers were significant due to age, treat-ment and age� treatment (Table 1).

Number of pods was higher in EDU treated plant by 147.7% and33.6% at pod setting and seed setting stages, respectively (Fig. 4).Pod/flower ratio increased by 107.1% and 22.3% at pod setting andseed setting stages. Number of unfilled pods reduced at pod

Table 1F ratio and level of significance for different growth parameters in soybean cultivar.

Parameters Age Treatment Age� treatment

Number of leaves plant�1 62.1** 36.7*** 0.57N.S

Number of senesced leaves plant�1 12.9*** 2.1N.S 1.27N.S

Number branches plant�1 39.1*** 31.8*** 0.57N.S

Number nodes plant�1 49.6*** 13.4*** 0.89N.S

Plant height 125.43*** 18.3*** 0.22N.S

Number flowers plant�1 525.3*** 32.9*** 28.3***

Abortive flower plant�1 208.6*** 15.1*** 3.6**

Number of pods plant�1 562.9*** 89.4*** 30.12***

Number of unfilled pods plant�1 75.7*** 2.8N.S 2.51*

Number of pod/Number of flower 77.8*** 13.5*** 13.5***

N.S not significant.n po0.05.nn po0.01.nnn po0.001.

setting stage significantly under EDU treatment (Fig. 4). Results oftwo way ANOVA showed that number of pods, pod/flower ratioand unfilled/filled pod ratio varied significantly due to age, treat-ment and age� treatment (Table 1).

3.4. Yield attributes

Significant increase in number and weight of seeds plant(po0.01) were recorded in EDU treatment. Number and weight ofpods plant�1 increased significantly by 27.5% and 26.7%, respec-tively under EDU treatment compared to non EDU treatment(Fig. 5). Harvest index did not vary significantly in EDU treatmentcompared to non EDU treatment (Fig. 5).

4. Discussion

Visual evaluations of plants at two day intervals showed apositive influence of EDU applications on plant growth from ve-getative stage to maturation stage. No visible injury symptomswere, however recorded on soybean foliage under both thetreatments. The present O3 monitoring results clearly show theincreasing trend of O3 concentrations in rural areas of Varanasi,situated in the middle of Indo-Gangetic plains of India. Higher O3

concentrations observed during the reproductive stage could beattributed to higher number of sunshine hours and less rainfallduring this period, which favoured more O3 formation. AOT 40values associated with 5% yield reduction of soybean was reportedto be 4.4 ppm h (critical level) (Mills et al., 2007). However, in thepresent study, AOT 40 value was recorded as 9.04 ppm h, which isalmost double of the critical level. This trend could be attributed tohigher excedance hours above 40 ppb during the growth period ofsoybean.

Reduction in lipid peroxidation in EDU treated plants in thepresent study confirms the anti-senescence property of EDU(Whitaker et al., 1990). Tropospheric O3 induces ROS in plants,causing lipid peroxidation (Kangasjärvi and Kangasjärvi, 2014).Thetotal antioxidant capacity of cells is collectively constituted by therange of enzymatic and non-enzymatic antioxidant mechanisms(Ghisseli et al., 2000). Apoplastic antioxidant capacity is a primaryprotectant against O3 damage (Kangasjärvi et al., 2005). In thepresent study, enzymatic activities of SOD, APX and GR increasedin EDU treated plants more than non EDU treated plants. Ranieriand Soldatini (1995) reported 40–50% increases in the activities ofSOD, APX and GR in potted bean plants treated with 150 ppm EDUcompared to non EDU treatment, and suggested that EDU pro-tection is caused by the stimulation of detoxifying system involvedin the elimination of both H2O2 and superoxide radicals.

SOD is a primary antioxidative enzyme, which scavenges su-peroxide radicals generated from oxidative stress due to O3. Potatocultivars (Alexendrai and Abbis) exposed to 78.5 and 95.5 ppbmean O3 for 10 h showed significant increase in SOD activity inboth the cultivars treated with 300 ppm EDU (Hassan, 2006).

APX an important antioxidative enzyme of the Halliwell Asadapathway helps in removing H2O2 formed during oxidative stress(Wu and Tiedmann, 2002). Paoletti et al. (2008) reported increasein APX activity and ascorbic acid content in 500 ppm EDU infusedplants of Fraxinus excelsior as compared to water infused tree dueto decrease in apoplastic content of H2O2. During the presentstudy, increase in ascorbic acid content suggests higher re-generation of ascorbate to act as an e- donor to APX in Halliwell–Asada pathway. Ascorbic acid is a well known antioxidant andcellular reductant. EDU stimulated ascorbate content and APXactivities in apoplast (Manning et al., 2011). GR, maintaining thecellular ascorbate and glutathione pools also increased during the

Fig. 5. Variations in yield and yield attributes in EDU and non EDU treated soybean plants. Bars represent Mean7 1SE, n¼20. Level of significance *po0.05; **po0.01;***po0.001; N.S not significant.

R. Rai et al. / Ecotoxicology and Environmental Safety 112 (2015) 29–3836

present study suggesting the protection provided by EDU againstO3.

Increase in chlorophyll content in EDU compared to non EDUtreated plants suggests that EDU treatment helped to retainchlorophyll content by reducing oxidative stress and delaying se-nescence. O3 induced ROS can damage chloroplasts by alteration ofmembrane permeability causing damage to chlorophyll and thusinducing senescence (Heath, 2008). E. Singh et al. (2009a) reportedreductions in total chlorophyll by 26.6% and 34% in a sensitivesoybean cultivar PK 472 exposed to 70 and 100 ppb O3 for 4 hday�1. Carotenoids, which help in maintaining chlorophyll contentby providing protection against photooxidative damage increasedin EDU compared to non EDU treated plants. Increase in car-otenoid content in wheat (S. Singh et al., 2009b) and spinach(Tiwari and Agrawal, 2009) under EDU treatment compared to noEDU was reported, suggesting that EDU increased non enzymaticantioxidant content also.

Nitrate reductase is a rate-limiting enzyme in nitrogen assim-ilation in higher plants and the effect of O3 on nitrate reductaseactivity in plants has rarely been reported. Agrawal and Agrawal

(1990) reported decline in NR and NiR activities by 13.8% and24.5% in Vicia faba L. exposed to 100 ppb O3 for 2 h for 30 days. Inthe present study, NR and NiR activity increased under EDUtreatment, which could be correlated with a protective mechanismof EDU. Protein content was higher in EDU treated plants, whichmay be correlated with increase in N-assimilation enzymes in-volved in incorporating N into amino acids. Lee and Chen (1982)found that EDU preserved protein content longer by regulatingprotein catabolism in leaves. In EDU treatment, total phenolicscontent reduced and protein content increased suggesting thatrelative abundance of carbon in EDU treated plants influenced therate at which the substrate i.e. phenylalanine is diverted to proteinsynthesis (Jones and Hartley, 1999).

Studies have shown that reduction in the net photosyntheticrate under O3 exposure could be attributed to the alteration in theelectron transport rate by inducing reduction in Fv/Fm ratio, sug-gesting changes in PSII and its ability to reduce the primary ac-ceptor QA (Guidi et al., 2009; Feng et al., 2011; Rai and Agrawal,2014). Fv/Fm ratio, which denotes the photochemical efficiency ofplants slightly increased in EDU treated plants. No change in Fo

R. Rai et al. / Ecotoxicology and Environmental Safety 112 (2015) 29–38 37

and increase in Fm and Fv under EDU treatment compared to nonEDU suggest that O3 did not induce modification at the antennaepigment level, and did not damage the electron transport system.Similarly, Contran et al. (2009) reported that Fo, Fm and Fv/Fm didnot vary significantly in Ash trees (Fraxinus excelsior L.) exposed to450 ppm EDU given as gravitational infusion, but significant effectof EDU was recorded at stepwise flow of energy through PSII toreaction centers level (RC). This trend suggests that EDU preventedthe inactivation of reaction centers and first impact of O3 on thephotosynthetic apparatus is not on the photochemical reactions,but on the sites at which electrons are utilized in dark reactions.Tiwari and Agrawal (2009) also found increases in Fm and Fv andno change in Fo in Palak at 62.7 ppb ambient O3 for 8 h grown at300 ppm EDU as a soil drench.

During the present study, no significant increase in the numberof leaves recorded at vegetative stage, which can be correlatedwith low O3 concentrations due to higher rainfall. Lower numberof senescenced leaves at flowering and pod stages can be attrib-uted to higher protection provided by EDU at higher concentra-tions of O3 at these stages. Increases in number of nodes representadditional sites for pod initiation and more leaves to support poddevelopment. Tropospheric O3 influences plant reproductivestructures directly by affecting flowering and subsequent podformation (Black et al., 2000). Number of flower and abortiveflower varied significantly due to age, treatment and age� treat-ment suggesting higher occurrence of O3 at latter age influencingplant reproductive structures. The relationship between assimilatesupply and pod survival is much more complex and can be eval-uated at the level of the individual flower and pod. O3 adverselyaffects assimilate production and distribution to sink organs suchas root and reproductive organs and thus inhibits their growth anddevelopment (Black et al., 2007). Higher pod/flower ratio underEDU treatment also suggests that higher levels of O3 at the re-productive stage did not alter the assimilate supply betweenleaves and developing pods compared to non EDU treatment. Thiscould also be correlated with increase in content of photosyntheticpigments and less damage to PS II suggesting adequate supply ofassimilate in flowers and developing pods. Soybean yield is de-termined by the numbers of pods and seeds that survive up tomaturity. In the present study, yield response in non EDU treatedplants suggest that the increase in O3 concentration during podfilling stage led to greater yield losses in soybean. The results arein agreement with previous studies showing beneficial effects ofEDU on yield components of soybean plants (Singh and Agrawal,2011; Feng et al., 2010; Paoletti et al., 2009). Increases in numberof pods and weight of mature pods in EDU treated plants suggesthigher seed carrying capacity compared to non EDU treatment.Elagoz and Manning (2005) have reported decrease in number ofpods and seeds by 54% and 71% in bush bean cultivar S156 exposedto 480 ppb O3 for 14 h compared to filtered chambers.

In non EDU treated plants, greater reductions in yield and yieldcomponents could be related to higher numbers of abortiveflowers and low pod/ flower ratio, suggesting that higher levels ofO3 during the reproductive stage directly affected floral initiationand development of flowers. Ozone has been shown to reduceweight of seeds plant-1 of soybean cultivars PK 472 and Braggexposed to 70 and 100 ppb O3 for 4 h daily (Singh et al., 2010). Ahigher occurrence of O3 between flowering and seed maturityreduced the crop yield (Lee et al., 1988). Secondly, test cultivar JS335 has a semi determinate reproductive habit, hence is moreseverely affected by exposure during sensitive stages of re-productive development due to their limited capacity to com-pensate any losses of reproductive sites or impairment of polli-nation and fertilization. Increase in weight of seeds plant�1 in EDUtreated plants, can be related to increase in number of seeds andalso to increase in supply of assimilates to seeds. Singh and

Agrawal (2011) reported increases of 29.8% and 28.2% in weight ofseeds of soybean cultivars Pusa 9712 and Pusa 9814 treated at400 ppm EDU treatment at 50.2 ppb O3 concentration. The re-duction in yield during the present study was 26.6% at a mean O3

concentration of 42 ppb with 300 ppm EDU suggesting more orless similar magnitude of sensitivity with other tropical soybeancultivars that have been assessed previously.

5. Conclusions

The present study confirms that EDU is a powerful tool forassessing effect of O3 on crops. The present study further con-cludes that mode of action of EDU treatment is mainly by en-hancing first line of defense against ROS generated by O3, whichfurther helped in maintaining plant metabolic machinery andensuring an adequate supply towards developing pods even underhigh O3 concentrations at flowering stage. EDU provided moreprotection when O3 concentrations were high. The hypothesis thatEDU by inducing antioxidant defense will protect photosyntheticefficiency and pigments, affecting N metabolism, plant perfor-mance and yield positively has been proved through this experi-ment. Reproductive sites were maintained under EDU treatment,which helped in sustaining yield of soybean even at higher O3

concentration during reproductive stage. The study further con-cludes that magnitude of protection provided by EDU to plantdepends upon ambient O3 concentrations, which suggests a needof application of different doses of EDU during the growth stage ofplants depending upon O3 concentration.

Acknowledgements

The authors are thankful to the Head of the Department ofBotany for all the laboratory and field facilities and to the Uni-versity Grant Commission, New Delhi and Department of Scienceand Technology, New Delhi for providing fellowships to R R andKKC, respectively. We owe a special thanks to reviewers of thismanuscript for their valuable suggestions and corrections.

References

Agrawal, M., Agrawal, S.B., 1990. Effects of ozone exposure on enzymes and me-tabolites of nitrogen metabolism. Sci.. Hortic. 43, 169–177.

Black, V.J., Black, C.R., Roerts, J.A., Stewart, C.A., 2000. Impact of ozone on the re-productive development of plants. New Phytol. 147, 421–447.

Black, V.J., Stewart, C.A., Roberts, J.A., Black, C.R., 2007. Ozone affects gas exchange,growth and reproductive development in Brassica campestris (Wisconsin FastPlants). New Phytol. 176, 150–163.

Bray, H.G., Thorpe, W.Y., 1954. Analysis of phenolic compounds of interest in me-tabolism. Methods Biochem. Anal. 1, 27–52.

Carnahan, J.E., Jenner, E.L., Wat, E.K.W., 1978. Prevention of ozone injury to plantsby a new protectant chemical. Phytopathology 68, 1225–1229.

Contran, N., Paoletti, E., Manning, W.J., Tagliaferro, F., 2009. Ozone sensitivity andethylene diurea in ash trees assessed by JIP chlorophyll a fluorescence transientanalysis. Photosynthetica 47, 68–78.

Dentener, F., Stevenson, D., Cofala, J., Mechler, R., Amann, M., Bergamaschi, P., Raes,F., Derwent, R., 2005. The impact of air pollutant and methane emission con-trols on tropospheric ozone and radiative forcing: CTM calculations for theperiod 1990- 2030. Atmos. Chem. Phys. 5, 1731–1755.

Duxbury, A.C., Yentsch, C.S., 1956. Plankton pigment monographs. J. Mar. Res. 15,19–101.

Elagoz, V., Manning, W.J., 2005. Factors affecting the effects of EDU on growth andyield of field-grown bush beans (Phaseolus vulgaris L.), with varying degrees ofsensitivity to ozone. Environ. Pollut. 136, 385–395.

Emberson, L.D., Buker, P., Ashmore, M.R., Mills, G., Jackson, L.S., Agrawal, M., Ati-kuzzaman, M.D., Cinderby, S., Engardt, M., Jamir, C., Kobayashi, K., Oanh, N.T.K.,Quadir, Q.F., Wahid, A., 2009. A comparison of North- America and Asian ex-posure- response data for ozone effects on crop yields. Atmos. Environ. 43,1945–1953.

R. Rai et al. / Ecotoxicology and Environmental Safety 112 (2015) 29–3838

Feng, Z., Pang, J., Kobayashi, K., Zhu, J., Ort, D.R., 2011. Differential responses in twovarieties of winter wheat to elevated O3 concentration under fully open- airfield conditions. Glob. Ch. Biol. 17, 580–591.

Feng, Z., Wang, S., Szantoi, Z., Chen, S., Wang, X., 2010. Protection of plants fromambient ozone by applications of ethylenediurea (EDU): a meta-analytic re-view. Environ. Pollut. 158, 3236–3242.

Fridovich, I., 1974. Superoxide dismutase. Adv. Enzymol 41, 35–97.Ghisseli, A., Serafini, M., Natella, F., Scaccini, C., 2000. Total antioxidant capacity as a

tool to assess redox status: critical view and experimental data. Free Radic. Biol.Med 29, 1106–1114.

Guidi, L., Degl'Innocenti, E., Martinelli, F., Piras, M., 2009. Ozone effects on carbonmetabolism in sensitive and insensitive Phaseolus cultivars. Environ. Exp. Bot.66, 117–125.

Hassan, I.A., 2006. Physiological and biochemical responses of potato (Solanumtuberosum L. cv. Kara) to ozone and antioxidant enzymes. Ann. Appl. Biol. 148,375–386.

Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplast. I. Kineticsand stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125,189–198.

Heath, R.L., 2008. Modification of the biochemical pathways of plants induced byozone: What are the varied route to changes? Environ. Pollut. 155, 453–463.

Jones, C.G., Hartley, S.E., 1999. A protein competition model of phenolic allocation.Oikos 86, 27–44.

Kangasjärvi, S., Kangasjärvi, J., 2014. Towards understanding extracellular ROSsensory and signalling systems in plants. Adv. Bot. 2014, http://dx.doi.org/10.1155/2014/53894.

Kangasjärvi, J., Jaspers, P., Kollist, H., 2005. Signalling and cell death in ozone-ex-posed plants. Plant Cell Environ. 28, 1021–1036.

Keller, T., Schwager, H., 1977. Air pollution and ascorbic acid. Eur. J. For. Pathol. 7,338–350.

Lee, E.H., Chen, C.M., 1982. Studies on the mechanisms of ozone tolerance. Cyto-kinin like activity of N [2-(2-oxo-1-imidazolidinyl) ethyl]-N-phenylurea, acompound protecting against O3 injury. Physiol. Plant. 56, 486–491.

Lee, E.H., Tingey, D.T., Hogsett, W.E., 1988. Evaluation of ozone exposure indices inexposure- response modelling. Environ. Pollut. 53, 43–62.

Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurementwith folin phenol reagent. J. Biol. Chem. 193, 265–275.

Maclachlan, S., Zalik, S., 1963. Plastid structure, chlorophyll concentration and freeamino acid composition of a chlorophyll mutant of barley. Can. J. Bot. 41,1053–1062.

Manning, W.J., Paoletti, E., Sandermann Jr, H., Ernst, D., 2011. Ethylenediurea (EDU):a research tool for assessment and verification of the effects of ground levelozone on plants under natural conditions. Environ. Pollut. 159, 3283–3293.

Meehl, G.A., Stocker, T.F., Collins, W.D., Friedlingstein, P., Gaye, A.T., Gregory, J.M.,Kitoh, A., Knutti, R., Murphy, J.M., Noda, A., Raper, S.C.B., Watterson, I.G.,Weaver, A.J., Zhao, Z.C., 2007. Global climate projections. In: Solomon, S., Qin,D., Manning, M., Chen, Z., Marquis, M., Averyt, K.B., Tignor, M., Miller, H.L. (Eds.),Climate Change 2007: The Physical Science Basis. Contribution of WorkingGroup I to the Fourth Assessment Report of the Intergovernmental Panel onClimate Change. Cambridge University Press, Cambridge, United Kingdom andNew York, NY, USA, pp. 749–844.

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 ofozone for agricultural and horticultural crops. Atmos. Environ. 41, 2630–2643.

Mills, G., Benton, J., Skärby, L., Fuhrer, J., Gimeno, B., 1997. ICP Vegetation experi-mental protocol for the ICP-CROPS. ICP-CROPS coordination centre. The Not-tingham Trent University, U.K. p. 47.

Nakano, Y., Asada, K., 1987. Purification of ascorbate peroxidase in spinach chlor-oplast, its inactivation in ascorbate depleted medium and reactivation bymonodehydroascorbate radical. Plant Cell Physiol. 28, 131–140.

Nicholas, J.D., Nason, A., 1957. Determination of nitrate and nitrite. Methods En-zymol. 3, 981–984.

Paoletti, E., Contran, N., Manning, W.J., Ferrara, A.M., 2009. Use of the antiozonantethylenediurea (EDU) in Italy: Verification of the effects of ambient ozone oncrop plants and trees and investigation of EDU's mode of action. Environ. Pollut.157, 1453–1460.

Paoletti, E., Contran, N., Manning, W.J., Castagna, A., Ranieri, A., Tagliaferro, F., 2008.Protection of ash (Fraxinus excelsior) trees from ozone injury by ethylene-diurea. Environ. Pollut. 157, 1453–1460.

Rai, R., Agrawal, M., 2014. Assessment of competitive ability of two Indian wheatcultivars under ambient O3 at different developmental stages. Environ. Sci.Pollut. Res. 21, 1039–1053.

Ranieri, A., Soldatini, G.F., 1995. Detoxification system in bean plants grown inpolluted air: effects of the antioxidant EDU. Agric. Mediterr. 125, 375–386.

Royal Society 2008. Ground-level ozone in the 21st century: future trends, impactsand policy implications. Science Policy report 15/08. The Royal Society, London.

Schaedle, M., Bassham, J.A., 1977. Chloroplast glutathione reductase. Plant Physiol.59, 1011–1012.

Singh, S., Agrawal, S.B., 2011. Cultivar-specific response of soybean (Glycine max L.)to ambient and elevated concentrations of ozone under open top chambers.Water Air Soil Pollut. 217, 283–302.

Singh, E., Tiwari, S., Agrawal, M., 2010. Variability in antioxidant and metabolitelevels, growth and yield of two soybean varieties: an assessment of anticipatedyield losses under projected elevation of ozone. Agric. Ecosyst. Environ. 135,168–177.

Singh, E., Tiwari, S., Agrawal, M., 2009a. Effects of elevated ozone on photosynthesisand stomatal conductance of two soybean varieties: a case study to assessimpacts of one component of predicted global climate change. Plant Biol. 11,101–108.

Singh, S., Agrawal, S.B., Agrawal, M., 2009b. Differential protection of ethylene-diurea (EDU) against ambient ozone for five cultivars of tropical wheat. En-viron. Pollut. 157, 2359–2367.

Tiwari, S., Agrawal, M., 2009. Protection of palak (Beta vulgaris L. Var. All green)plants from ozone injury by ethylenediurea (EDU): Roles of biochemical andphysiological variations in all eviating the adverse impacts. Chemosphere 75,1492–1499.

Tiwari, S., Agrawal, M., Manning, J.W., 2005. Assessing the impact of ambient ozoneon growth and productivity of two cultivars of wheat in India using three ratesof application of ethylenediurea (EDU). Environ. Pollut. 138, 153–163.

Van Dingenen, R., Dentener, F.J., Raes, F., Krol, M.C., Emberson, L., Cofala, J., 2009.The global impact of ozone on agricultural crop yields under current and futureair quality legislation. Atmos. Environ. 43, 604–618.

Wahid, A., Milne, E., Shamsi, S.R.A., Ashmore, M.R., Marshall, F.M., 2001. Effects ofoxidants on soybean growth and yield in the Pakistan, Punjab. Environ. Pollut.113, 271–280.

Whitaker, B.D., Lee, E.H., Rowland, R.A., 1990. Ethylenediurea and ozone protection:foliar glycerolipids and steryl lipids in snapbean exposed to ozone. Physiol.Plant 80, 286–293.

Wu, Y., Tiedmann, von A., 2002. Evidence for oxidative stress involved in physio-logical leaf spot formation in winter and spring barley. Phytopathology 92,145–155.