Chemosphere xxx (2013) xxx–xxx

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Chemosphere

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Impact of nano-CuO stress on rice (Oryza sativa L.) seedlings

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.05.044

Abbreviations: DAB, 3030-diaminobenzidine; GSH, reduced glutathione; GSSG,oxidized glutathione; MDA, malondialdehyde; NBT, nitro blue tetrazolium; NPs,nanoparticles; ROS, reactive oxygen species.⇑ Corresponding author. Tel.: +91 33 25241975; fax: +91 33 2524 1977.

E-mail address: zahed_kly@yahoo.com (Z. Hossain).

Please cite this article in press as: Shaw, A.K., Hossain, Z. Impact of nano-CuO stress on rice (Oryza sativa L.) seedlings. Chemosphere (2013),dx.doi.org/10.1016/j.chemosphere.2013.05.044

Arun Kumar Shaw, Zahed Hossain ⇑Plant Stress Biology Lab, Department of Botany, West Bengal State University, Kolkata 700 126, West Bengal, India

h i g h l i g h t s

� The present study highlights the response of rice seedlings to nano-CuO stress.� Stress induced modulation of antioxidant enzymes and metabolites were studied.� Histochemical staining with NBT and DAB indicate severe oxidative burst.� Elevated APX and GR activity do not protect stressed cells from oxidative damage.� Decline in DHAR renders stressed cells in futile recycling of ascorbate pool.

a r t i c l e i n f o

Article history:Received 1 February 2013Received in revised form 16 May 2013Accepted 19 May 2013Available online xxxx

Keywords:AntioxidantNano-CuO stressNanoparticlesNanotoxicologyRiceROS

a b s t r a c t

Indiscriminate release of metal oxide nanoparticles (NPs) into the environment due to anthropogenicactivities has become a serious threat to the ecological system including plants. The present studyassesses the toxicity of nano-CuO on rice (Oryza sativa cv. Swarna) seedlings. Three different levels ofstress (0.5 mM, 1.0 mM and 1.5 mM suspensions of copper II oxide, <50 nm particle size) were imposedand seedling growth performance was studied along control at 7 and 14 d of experiment. Modulation ofascorbate–glutathione cycle, membrane damage, in vivo ROS detection, foliar H2O2 and proline accumu-lation under nano-CuO stress were investigated in detail to get an overview of nano-stress response ofrice. Seed germination percentage was significantly reduced under stress. Higher uptake of Evans blueby nano-CuO stressed roots over control indicates loss of root cells viability. Presence of dark blue anddeep brown spots on leaves evident after histochemical staining with NBT and DAB respectively indicatesevere oxidative burst under nano-copper stress. APX activity was found to be significantly increased in1.0 and 1.5 mM CuO treatments. Nevertheless, elevated APX activity might be insufficient to scavenge allH2O2 produced in excess under nano-CuO stress. That may be the reason why stressed leaves accumu-lated significantly higher H2O2 instead of having enhanced APX activity. In addition, increased GR activitycoupled with isolated increase in GSH/GSSG ratio does not seem to prevent cells from oxidative damages,as evident from higher MDA level in leaves of nano-CuO stressed seedlings over control. Enhanced prolineaccumulation also does not give much protection against nano-CuO stress. Decline in carotenoids levelmight be another determining factor of meager performance of rice seedlings in combating nano-CuOstress induced oxidative damages.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Indiscriminate release of metal oxide nanoparticles (NPs) intothe environment due to anthropogenic activities has become aserious threat to the ecological system including plants. Plantnanotoxicology is an emerging and less-explored area of researchfor the plant stress biologists. By definition, nanoparticles (NPs)

are ultrafine particles that typically have at least one dimensionless than 100 nm in size. Apart from its wide applications in drugand gene delivery, tissue engineering, bio-detection of pathogensand tumour therapy, NPs are also known to be toxic for biologicalsystems including plants. The negative impacts of NPs on plantdevelopment and metabolism depend on the size, concentrationand chemistry of NPs, as well as the chemical milieu of the subcel-lular sites to which the NPs are deposited (Dietz and Herth, 2011).

Uptake of NPs through primary roots is usually barred due topresence of suberinized exo- and endodermis. However, lateralroot junctions are the primary sites through which NPs could enterthe xylem via cortex and the central cylinder (Dietz and Herth,2011). NPs upon dissolution act as metal ions, able to interact with

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the sulfhydryl, carboxyl groups of proteins and thus alter activity.A toxic metal like copper is able to transfer electrons to molecularoxygen to form reactive superoxides and to H2O2 to form the mostdestructive ROS- hydroxyl radicals (Dietz and Herth, 2011). TheNP-dependent toxicity mediated excess ROS generation unbal-ances the cellular redox system in favor of oxidized forms, result-ing in oxidative damage to cellular components-lipids, proteinsand nucleic acid (Halliwell and Gutteridge, 1989). Plants areequipped with robust enzymatic and non-enzymatic antioxidantdefence systems to protect cells against the oxidative damages(Hossain et al., 2006, 2012).

Limited studies reported both negative and positive effects ofNPs on higher plants (Lin and Xing, 2007). Phytotoxicity of metaloxide nanoparticles on seed germination and root developmenthas recently been studied in lettuce, radish and cucumber (Wuet al., 2012). Lin and Xing (2007) performed similar kind of studywith five types of nanoparticles and six different plant species(radish, rape, ryegrass, lettuce, corn, and cucumber). Among thevarious metal oxide NPs, nano-TiO2 is by far the most well studiedNP whose toxicity has been tested in different crop systems. Casti-glione et al. (2011) investigated the effects of nano-TiO2 on seedgermination, seedling development and root mitosis of Vicia nar-bonensis and Zea mays. Their findings revealed concentration-dependent increase in the alterations in mitotic activity, chromo-somal aberrations, indicating genotoxic effects of NPs. Similarly,toxicity of ZnO and TiO2 nanoparticles on germinating rice seed-lings was explored in detail by Boonyanitipong et al. (2011).Authors claimed that nano-ZnO had more detrimental effects onroot growth and development as compared to TiO2. Interestingly,Lu et al. (2002) reported that application of nano-SiO2 and nano-TiO2 mixture on soybean (Glycine max) could increase nitratereductase, enhance water and fertilizer absorbing capacity, stimu-late antioxidant defence system and also hasten seed germinationand seedling growth. In addition, nano-TiO2 was reported to en-hance photosynthesis and nitrogen metabolism, and thus greatlyimprove spinach growth (Hong et al., 2005; Zheng et al., 2005;Yang et al., 2006). Larue et al. (2012) recently mapped nano-TiO2

distribution in wheat (Triticum aestivum) tissues by synchrotron-radiation micro-X-ray fluorescence. Whie investigating root-to-shoot translocation of NPs, authors have suggested a thresholddiameter of 140 nm, above which NPs are no longer accumulatedin roots, as well as a threshold diameter of 36 nm, over whichNPs are accumulated in root parenchyma but do not reach the steleand hence do not translocate to the shoot. Interestingly, this accu-mulation neither hampers seed germination, transpiration andphotosynthesis nor induces oxidative stress.

Copper (Cu), an essential micronutrient plays crucial role inplant growth and development. It actively participates in vital cel-lular processes like photosynthetic electron transport, mitochon-drial respiration, cell wall metabolism, hormone signaling,protein trafficking, and iron mobilization (Raven et al., 1999; Yru-ela, 2005, 2009). Nevertheless, at high concentration, Cu is extre-mely toxic to plant system causing chlorosis, necrosis, stunting,root growth inhibition (Xiong and Wang, 2005; Yruela, 2005).The excess copper ions interact with the sulfhydryl groups of pro-teins lead to protein dysfunctioning thus enzyme inactivation.Being a redox-active transition metal Cu could trigger the forma-tion of reactive oxygen species (ROS) through the Fenton or Ha-ber–Weiss reactions (Halliwell and Gutteridge, 1989). Plantresponse to Cu stress has been studied extensively over the pastdecade at physiological, molecular and proteomic levels(Drazkiewicz et al., 2004; Wang et al., 2004; Ahsan et al., 2007;Bona et al., 2007; Elisa et al., 2007; Sudo et al., 2008; Zhanget al., 2009; Ahmed et al., 2010; Ritter et al., 2010; Li et al., 2012;Thounaojam et al., 2012). Nevertheless, the impact of copper oxidenanoparticle (nano-CuO) on plants is not at all well explored. Much

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emphasis was given on the toxicity study of CuO nanoparticles onsoil bacteria (Rousk et al., 2012) and alga (Aruoja et al., 2009; Wanget al., 2011). Only few reports about nano-CuO toxicity on plantsare available. Dimkpa et al. (2012) recently demonstrated the im-pact of commercial CuO (<50 nm) and ZnO (<100 nm) NPs onwheat. So, there is an urgent need of understanding plants re-sponse to nano-CuO stress.

In the present experiment, an attempt was made to unravel thenano-CuO stress induced activation of defence mechanism ofmonocot model plant- rice (Oryza sativa L.). In addition of deter-mining antioxidant enzymes activities and contents of antioxidantmetabolites of ascorbate–glutathione cycle, membrane damage,in vivo ROS detection, foliar H2O2 and proline accumulation undernano-CuO stress were studied in detail to get an overview of nano-stress response of rice. To our best knowledge, this study is the firstendeavor in dissecting nano-CuO stress in rice seedlings.

2. Materials and methods

2.1. Plant material, treatments and sample collection

Rice seeds (Oryza sativa cv. Swarna) were used as plant materialfor the present investigation. Seeds were first disinfected in 3% So-dium hypochlorite solution for 5 min, followed by repeated wash-ing (at least three times) in water and overnight soaking in ddH2O.On the next day seeds were allowed to germinate on water soakedcotton pad placed on plastic tray (800 � 1000) and considered as con-trol. Three different concentrations (0.5 mM, 1.0 mM and 1.5 mM)of nano-CuO (copper II oxide, <50 nm particle size, Sigma–aldrich,USA) suspensions were used to impose stress. The overnight watersoaked seeds were allowed to germinate on the respective nano-CuO suspension saturated cotton pad just like control set. Seed-lings were maintained at 25 �C in growth chamber illuminatedwith white fluorescent light (600 lmol m–2 s–1, 16 h light periodd–1) and 70% relative humidity. Whenever needed, more nano-cop-per suspensions were added to the respective trays. Control seed-lings were maintained under the same condition just like stressedseedlings by adding more ddH2O whenever required. The day ger-mination first observed was considered as zero day of experiment.Thereafter, both control and stressed seedlings were maintainedfor consecutive 14 d. On 7th and 14th d (after germination), mor-phological parameters like root length, root weight, shoot lengthand shoot weight were recorded. Shoots were randomly harvestedon the same days (7 and 14) and subsequently stored at �80 �C forbiochemical analysis. In total three independent biological experi-ments were performed under the same growth conditions. Fordetection of ROS and cell death, freshly collected leaves and rootswere used respectively. In addition, germination percentage ofboth control and stressed seeds were determined on 2nd d ofexperiment.

2.2. In vivo detection of ROS

Foliar hydrogen peroxide (H2O2) accumulation was detectedusing 3030-diaminobenzidine (DAB) assay (Thordal-Christensenet al., 1997; Iriti et al., 2006). In vivo infiltration of leaves with5 mM DAB at pH 3.8 forms deep brown polymerization productsupon reaction with H2O2 in the presence of peroxidase (Thordal-Christensen et al., 1997). Superoxide was detected by infiltrationof leaves in 6 mM nitroblue tetrazolium (NBT) that produce darkblue insoluble formazan on reaction with superoxides (Fryeret al., 2002). Chlorophylls were removed from the leaves by infil-tration with lacto-glycerol-ethanol (1:1:4 v/v/v) solution followedby boiling in water bath for 10 min. Images were captured using

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Olympus CX41 microscope (Olympus, Tokyo, Japan) equipped withdigital camera (ProgRes CT3).

2.3. Root cell viability assay by Evans blue staining

Root cell viability was determined by Evans blue staining(Tamas et al., 2004). Freshly collected roots were washed thor-oughly in ddH2O followed by overnight staining in 0.25% (w/v)aqueous solution of Evans blue (Sigma, USA) at room temperature.On the next day, the stained roots were washed several times withddH2O until no further blue color eluted from the roots. Thestained roots were studied under Olympus CX41 microscope(Olympus, Tokyo, Japan) and images were captured using a digitalcamera (Canon, PowerShot, SX110 IS, Japan).

2.4. Hydrogen peroxide estimation

Foliar hydrogen peroxide content was estimated according tothe method of Brennan and Frenkel (1977). One hundred milligramof chilled leaf tissue was macerated in 4 mL cold acetone andhomogenate was filtered through Whatman No. 1 filter paper.Two mL of this filtrate was treated with 1 mL of titanium reagent(20% titanium tetrachloride in concentrated HCl, v/v) and 1 mL ofconcentrated ammonia solution to precipitate the titanium-hydro-peroxide complex. After centrifugation (at 5000g for 30 min) pre-cipitate was dissolved in 2 N H2SO4 and the absorbance was readat 415 nm. H2O2 content was calculated from a standard curve pre-pared in the similar way and expressed as lmol g�1 FW.

2.5. Measurement of Malondialdehyde concentration

Malondialdehyde (MDA) concentration was measured follow-ing the procedure of Hodges et al. (1999). Frozen leaf tissue washomogenized in 80% cold ethanol and centrifuged to pellet debris.Different aliquots of the supernatant were mixed either with 20%trichloroacetic acid or with a mixture of 20% trichloroacetic acidand 0.5% thiobarbituric acid. Both mixtures were allowed to reactin a water bath at 90 �C for 1 h. After that, samples were cooleddown in an ice bath and centrifuged. Absorbance of the superna-tant was read at 440, 534 and 600 nm against a blank. MDA con-centration was expressed in terms of nmol g�1 FW.

2.6. Estimation of free proline content

Proline content was measured spetrophotometrically using themethod of Bates et al. (1973). One hundred milligram of leaf tissuewas homogenized with 5 mL 3% sulphosalicylic acid and centri-fuged at 5000g for 10 min. Supernatant was treated with acid-nin-hydrin and acetic acid, boiled for 1 h at 100 �C. The reaction wasthen terminated in an ice bath. Reaction mixture was extractedwith 2 mL toluene. Absorbance of chromophore containing toluenewas determined at 520 nm. Proline content was expressed aslmol g�1 FW.

2.7. Estimation of carotenoids content

Carotenoids content was determined by the method of Lich-tenthaler (1987). Fifty milligram of leaf tissue was homogenizedin 5 mL chilled methanol (100%). Homogenate was centrifuged at4000g for 15 min. Absorbance of the supernatant was recorded at470 nm and the content was expressed as mg carotenoids g�1 FW.

2.8. Antioxidant enzymes assays

Fresh leaf tissue (0.5 g) was homogenized in 1.5 mL of 50 mMpotassium phosphate buffer (pH 7.8) containing 1 mM EDTA,

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1 mM dithiotreitol and 2% (w/v) polyvinyl pyrrolidone (PVP) usingchilled mortar and pestle kept in ice bath. The homogenate wascentrifuged at 15000g at 4 �C for 30 min. Clear supernatant wasused for all enzymes assays. For measuring APX activity, the tissuewas separately ground in homogenizing medium containing2.0 mM ascorbate in addition to the other ingredients. All assayswere done at 25 �C. Soluble protein content was determinedaccording to Bradford (1976) using BSA as a standard. All spectro-photometric analyses were conducted using an UV/visible Spectro-photometer-Genesis 10S UV–VIS (Thermo Scientific).

2.8.1. Ascorbate peroxidase (APX)APX (EC 1.11.1.11) activity was assayed according to the meth-

od of Nakano and Asada (1981). Three milliliter of the reactionmixture contained 50 mM potassium phosphate buffer (pH 7.0),0.1 mM EDTA, 0.5 mM ascorbate, 0.1 mM H2O2 and 0.1 mL enzymeextract. The hydrogen peroxide-dependent oxidation of ascorbatewas followed by a decrease in the absorbance at 290 nm(� ¼ 2:8 mM�1 cm�1). APX activity was expressed as lmol ascor-bate oxidized min�1 mg�1 protein.

2.8.2. Superoxide dismutase (SOD)SOD (EC 1.15.1.1) activity was determined by nitro blue tetrazo-

lium (NBT) photochemical assay according to Beyer and Fridovich(1987). In this method 1 mL of solution containing 50 mM potas-sium phosphate buffer (pH 7.8), 9.9 mM L-methionine, 57 lMNBT, 0.025% triton-X-100 was added into small glass tubes fol-lowed by 20 lL of sample. Reaction was started by adding 10 lLof riboflavin solution (4.4 mg 100 mL–1) followed by placing thetubes in an aluminum foil-lined box having two 20-W fluorescentlamps for 7 min. A parallel control was run where buffer was usedinstead of sample. After illumination absorbance of solution wasmeasured at 560 nm. A non-irradiated complete reaction mixturewas served as a blank. SOD activity was expressed as U mg�1 pro-tein. One unit of SOD was equal to that amount which causes a 50%decrease of SOD-inhibitable NBT reduction.

2.8.3. Glutathione reductase (GR)GR (EC 1.6.4.2) activity was determined by monitoring the glu-

tathione dependant oxidation of NADPH, as described by Carlbergand Mannervik (1985). In a cuvette, 0.75 mL 0.2 M potassiumphosphate buffer (pH 7) containing 2 mM EDTA, 75 lL NADPH(2 mM), 75 lL oxidized glutathione (20 mM) were mixed. Reactionwas initiated by adding 0.1 mL enzyme extract to the cuvette andthe decrease in absorbance at 340 nm was monitored for 2 min. GRactivity was calculated using the extinction coefficient for NADPHof 6.2 mM�1 cm�1 and expressed as lmol NADPH oxidized min�1 -mg�1 protein.

2.8.4. Dehydroascorbate reductase (DHAR)DHAR (EC 1.8.5.1) enzyme activity was measured according to

the method of Nakano and Asada (1981). The complete reactionmixture contained 50 mM potassium phosphate buffer (pH 7.0),2.5 mM GSH, 0.2 mM DHA and 0.1 mM EDTA in a final volume of1 mL. Reaction was initiated by adding suitable aliquot of enzymeextract and increase in absorbance was recorded at each 30 s inter-val for 3 min at 265 nm. Enzyme activity was expressed as lmolascorbate formed min�1 mg�1 protein.

2.8.5. Monodehydroascorbate reductase (MDAR)MDAR (EC 1.6.5.4) enzyme activity was measured as described

by Miyake and Asada (1992). Monodehydroascorbate was gener-ated by ascorbate oxidase using a reaction mixture (1 mL) contain-ing 50 mM HEPES-KOH buffer, pH 7.6, 0.1 mM NADPH, 2.5 mMascorbate, ascorbate oxidase (0.14U) and suitable aliquot of

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enzyme extract. MDAR activity was expressed as lmol NADPH oxi-dized min�1 mg�1 protein.

2.9. Estimation of foliar ascorbate and glutathione contents

Ascorbate content was determined according to Law et al.(1983). The assay is based on the reduction of Fe+3 to Fe+2 by ascor-bate in acidic solution. The Fe2+ forms a red chelate with bipyridylabsorbing at 525 nm. DHA was calculated by subtracting AsA fromtotal ascorbate. The DTNB � GSSG reductase recycling procedure ofAnderson (1985) was used for the determination of both total(GSH + GSSG) and GSSG levels. GSH content was calculated by sub-tracting GSSG content from the total glutathione content.

2.10. Statistical analysis

The results are presented as mean values ± standard errors(n = 9). Statistical significance between mean values was assessedusing analysis of variance and a conventional Duncan’s MultipleRange Test (DMRT), using SPSS-10 statistical software (SPSS Inc.,Chicago, IL, USA). A probability of p < 0.05 was consideredsignificant.

3. Results

3.1. Inhibition of seed germination and seedlings growth under nano-copper stress

The impact of metal stress is often assessed through seed ger-mination assay. Nano-copper stress treatment had clear effectson rice seed germination percentage. As compared to control(91.6%) significant gradual decrease in germination percentagewas observed with increase in CuO concentration- 0.5 mM(78.6%), 1.0 mM (75.6%) and 1.5 mM (71.6%). In addition, seedlinggrowth was significantly affected under nano-CuO stress(Figs. 1A, 2A, Table 1). As compared to control, significant reductionin shoot length as well as shoot weight was recorded in stressedseedlings both at 7 and 14 d of experiment. Root growth was se-verely affected under copper stress. At 7 d, gradual significant de-crease in root length and root weight was recorded withincreasing concentration of nano-CuO (Figs. 1A and B and Table 1).Maximum reduction in root growth was observed under 1.5 mMnano-copper treatment. After 7 d of stress treatment, no significantroot growth was further evident (Table 1).

3.2. Nano-CuO causes severe oxidative burst

Irrespective of CuO concentration and time period, NBT stainingof nano-copper stressed leaves revealed dark blue spots, indicativeof superoxides accumulation (Figs. 1C and E and 2C and E). Inter-estingly, no such clear spots were detected in control leavesthroughout the experiment. Similarly, after DAB staining stressedleaves exhibited deep brown spots pinpoint H2O2 deposits(Figs. 1D and F and 2D and F). Maximum spots were observed inleaves of 1.5 mM CuO treated seedlings. Presence of dark blueand deep brown spots indicate severe oxidative burst undernano-CuO stress.

Fig. 1. Effects of nano-CuO stress on growth performance of 7 d old rice (Oryzasativa cv. Swarna) seedlings (A). Roots stained with Evans blue indicates nonviablecells. Control, 0.5, 1.0 and 1.5 mM nano-CuO stressed roots are shown from left toright respectively (B). NBT stained leaves showing dark blue spots (formazan)indicate superoxides deposits (C). Enlarged microscopic view of dark blue spots(arrow marks) (E). Foliar H2O2 deposits analyzed by DAB staining (D). Enlargedmicroscopic view of a portion of stressed leaf showing dark brown spot (arrowmark) (F). (For interpretation of the references to colour in this figure legend, thereader is referred to the web version of this article.)

3.3. Nano-CuO induced loss of root cells viability

Differential uptake of Evans blue by the control and stressedroots was observed. Figs. 1B and 2B show the 7 d- and 14 d-old riceroots stained with Evans blue respectively. The uptake of Evansblue by nano-CuO stressed roots was much higher than the control

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both at 7 d and 14 d of treatment. Completely dark blue coloredroots indicate maximum cell death in 1.5 mM CuO exposed roots.

3.4. Higher hydrogen peroxide accumulation under nano-copper stress

Nano-CuO stressed rice seedlings showed an increasing trend infoliar hydrogen peroxide content with passage of time (Fig. 3A). Inaddition, with the increase in CuO concentration, gradual enhance-ment in H2O2 content was noticed. Even at 7 d, low copper concen-tration (0.5 mM) had prominent effect in elevating H2O2 content.

O stress on rice (Oryza sativa L.) seedlings. Chemosphere (2013), http://

Fig. 2. Impact of nano-CuO stress on growth performance of 14 d old rice (Oryzasativa cv. Swarna) seedlings (A). Loss of root cell viability as evident by Evans bluestaining. Control, 0.5, 1.0 and 1.5 mM nano-CuO stressed roots are shown from leftto right respectively (B). In vivo detection of superoxide accumulation by NBTstaining (C). Enlarged microscopic view of dark blue spots (arrow marks) (E). DABstained leaves showing dark brown spots indicative of H2O2 deposits (D). Enlargedmicroscopic view of a portion of stressed leaf showing dark brown spot (arrowmark) (F). (For interpretation of the references to colour in this figure legend, thereader is referred to the web version of this article.)

Table 1Effects of nano-CuO stress on growth of rice (Oryza sativa cv. Swarna) seedlings. Data werfollowed by a common letter in a column are not significantly different at the 5% level by

Treatment Shoot length (cm) Shoot weight (mg)

7 d 14 d 7 d 14 d

Control 4.07a ± 0.09 5.90a ± 0.12 15.10a ± 0.68 28.58a ± 10.5 mM 3.54b ± 0.09 4.26b ± 0.15 12.91b ± 0.54 22.53b ± 11.0 mM 3.37b ± 0.09 3.69c ± 0.16 12.99b ± 0.42 19.84b ± 11.5 mM 2.88c ± 0.16 3.25d ± 0.14 11.34c ± 0.51 13.41c ± 0

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At 14 d, a sharp increase (�1.7-fold over control) in foliar H2O2 le-vel was recorded in high CuO treatment (1.5 mM).

3.5. Nano-CuO treatment triggers oxidative damage to lipidmembranes

Tissue MDA level has been used as a general marker of oxidativedamage to lipid membranes. Throughout the experimental period,significantly high foliar MDA level was maintained in all CuO-stressed seedlings irrespective of CuO concentrations (Fig. 3B).Maximum increase (1.5-fold) was noticed in 1.5 mM CuO treat-ment at 7 d, while at 14 d, the 1.0 mM CuO exposed leaves showedhighest MDA level. The control seedlings maintain a steady level ofMDA throughout the experiment (Fig. 3B).

3.6. Nano-CuO stress induced high accumulation of proline

Nano-CuO stress had significant effect in enhancing endoge-nous free proline level. Throughout the experimental period,stressed seedlings accumulated significantly higher level of proline(Fig. 3C). Degree of accumulation was in close association with in-crease in CuO concentration. On both days (7 and 14), highestaccumulation of proline was noticed in 1.5 mM CuO exposedleaves.

3.7. Changes in carotenoids level under nano-copper stress

Leaf carotenoids content was decreased upon exposure to med-ium CuO stress (1.0 mM) at 7 d, while that was significantly in-creased in 1.5 mM CuO treated seedlings (Fig. 3D). However,consecutive 14 d of stress resulted significant decline (�1.2-fold)in carotenoids level (as compared to control) in all stressed seed-lings irrespective of concentrations.

3.8. Nano-CuO stress induced modulation of antioxidant enzymesactivity

Under nano-CuO stress, antioxidant enzymes activities of riceleaf were differentially modulated. Among the five studied antiox-idant enzymes of ascorbate–glutathione cycle, APX activity wassignificantly increased in 1.0 and 1.5 mM CuO treatments(Fig. 4A). However, at 7 d the magnitude of the increase was morein leaves of 1.5 mM CuO treated seedlings. In contrast, leaves ofboth 1.0 and 1.5 mM CuO treated seedlings exhibited almost equalincrease (�1.3-fold as compared to control leaf) in activity at 14 d.Interestingly, lowest concentration of CuO (0.5 mM) had mostly nosignificant effect on foliar antioxidant enzymes activity includingAPX (Fig. 4A).

SOD activity was found to be less affected under nano-CuOstress. At 7 d, increase in SOD activity (1.4-fold) was recorded onlyin 1.0 mM CuO treatment, while at 14 d, maximum significant in-crease was found in 1.5 mM CuO treated leaves (Fig. 4B). LikeAPX, nano-CuO had significant effect on GR activity. At 7 d, both1.0 mM and 1.5 mM nano-CuO exposed leaves exhibited almostequal significant increase (�1.2-fold) in GR activity (Fig. 4C). In

e recorded at 7 and 14 d of experiment and expressed as mean ± S.E. (n = 15). MeansDuncan’s Multiple Range Test (DMRT).

Root length (cm) Root weight (mg)

7 d 14 d 7 d 14 d

.44 3.15a ± 0.23 4.09a ± 0.26 4.77a ± 0.33 13.60a ± 0.52

.07 1.05b ± 0.04 1.06b ± 0.13 1.61b ± 0.09 1.81b ± 0.23

.52 0.73b ± 0.05 0.74bc ± 0.11 0.89c ± 0.08 0.90c ± 0.06

.81 0.33c ± 0.04 0.37c ± 0.03 0.50c ± 0.08 0.51c ± 0.08

O stress on rice (Oryza sativa L.) seedlings. Chemosphere (2013), http://

Fig. 3. Effects of nano-CuO stress on accumulations of H2O2 (A), MDA (B), proline (C) and carotenoids (D) in leaves of rice (Oryza sativa cv. Swarna) seedlings. Data areexpressed as the mean values ± standard errors (n = 9). Values in columns with different letters are significantly different at 5% level according to Duncan’s Multiple RangeTest.

Fig. 4. Nano-CuO stress induced changes in APX (A), SOD (B), GR (C), DHAR (D) and MDAR (E) activities in leaves of rice (Oryza sativa cv. Swarna) seedlings. Data are expressedas the mean values ± standard errors (n = 9). Values in columns with different letters are significantly different at 5% level according to Duncan’s Multiple Range Test.

6 A.K. Shaw, Z. Hossain / Chemosphere xxx (2013) xxx–xxx

contrast, at 14 d as compared to control, significant increase wasonly recorded in 1.0 mM CuO treatment.

Unlike other antioxidant enzymes, DHAR activity was not at allsignificantly affected under nano-CuO treatment at 7 d (Fig. 4D). In

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contrast, significantly reduced DHAR activity (�1.8-fold) was no-ticed at 14 d in all CuO exposed leaves irrespective of concentra-tion. Like APX, a gradual increase in MDAR activity was observedat 7 d in 1.0 mM and 1.5 mM nano-CuO treated rice seedlings

O stress on rice (Oryza sativa L.) seedlings. Chemosphere (2013), http://

Table 2Effects of nano-CuO stress on foliar ascorbate contents of rice (Oryza sativa cv. Swarna). Contents were measured at 7 and 14 d of experiment and expressed as mean ± S.E. (n = 9).Means followed by a common letter are not significantly different at the 5% level by Duncan’s Multiple Range Test (DMRT).

Treatment tAsA (lmol g�1 FW) AsA (lmol g�1 FW) DHA (lmol g�1 FW) AsA/DHA

Control 7 d 0.419a ± 0.062 0.052a ± 0.008 0.368a ± 0.061 0.179a ± 0.04614 d 2.810a ± 0.252 1.228a ± 0.036 1.592a ± 0.231 0.882a ± 0.100

0.5 mM 7 d 2.757b ± 0.214 0.132b ± 0.019 2.625b ± 0.232 0.059b ± 0.01214 d 8.093b ± 0.359 0.652c ± 0.049 7.441b ± 0.376 0.090b ± 0.010

1.0 mM 7 d 3.083b ± 0.222 0.233c ± 0.017 2.850b ± 0.207 0.082b ± 0.00314 d 13.219d ± 0.707 0.681c ± 0.011 12.538d ± 0.702 0.056b ± 0.003

1.5 mM 7 d 3.894c ± 0.114 0.342d ± 0.037 3.552c ± 0.130 0.099b ± 0.01214 d 10.789c ± 0.439 1.014b ± 0.021 9.775c ± 0.427 0.105b ± 0.004

A.K. Shaw, Z. Hossain / Chemosphere xxx (2013) xxx–xxx 7

(Fig. 4E). Interestingly, at 14 d, all the nano-CuO stressed leaves(irrespective of concentration) exhibited a sudden increase (�1.7-fold) in MDAR activity as compared to control.

3.9. Status of the ascorbate and glutathione pool under nano-CuOstress

Foliar reduced ascorbate content (AsA) significantly increasedin nano-CuO stressed seedlings at 7 d of experiment (Table 2).The degree of increase was in accordance with the increase inCuO treatment concentration. Highest accumulation (6.5-fold ascompared to control) was observed in leaves of 1.5 mM CuO ex-posed seedlings. Interestingly, at 14 d of experiment completelyopposite trend was observed. As compared to control, stressedleaves accumulated significantly low level of AsA. In contrast,dehydroascorbate (DHA) level was steadily remained high innano-CuO stressed leaves throughout the experimental period.However, the AsA/DHA ratio was found to be decreased (�3 to15-fold as compared to control) in all stressed leaves irrespectiveof concentration at both 7 and 14 d of experiment (Table 2).

Nano-CuO stress had significant effects on leaf glutathione pool(Table 3). Although no significant variation in total glutathione wasobserved, but level of GSH (reduced glutathione) was highly influ-enced under stress. The 1.0 and 1.5 mM CuO exposed leaves accu-mulated significantly higher (�1.2 to 1.5-fold) level of GSH ascompared to control. Interestingly, oxidized glutathione (GSSG) le-vel showed completely reverse trend. Application of nano-CuOstress resulted significant reduction in foliar GSSG level. But, over-all the GSH/GSSG ratio was significantly increased in all stressedseedlings irrespective of CuO concentration (except for 0.5 mMtreatment at 7 d). The 1.5 mM and 1.0 mM CuO treated rice seed-lings maintained highest GSH/GSSG ratio at 7 d and 14 d of exper-iment respectively (Table 3).

4. Discussion

To pinpoint the actual impact of nano-CuO stress on rice seed-lings growth, the present time and dose dependent experimentwas conducted. The impact of NPs depends on the size and/orthe shape of the particles, the applied concentrations, the specificconditions of experiments, and more importantly the plant speciesstudied (Brunner et al., 2006; Lin and Xing, 2007; Eichert et al.,2008). In has been documented in several physiological experi-ments that phytotoxic dose of NPs varies between crops underinvestigation. Fifty percent inhibitory concentrations (IC50) ofnano-Zn and nano-ZnO were estimated to be near 50 mg L�1 forradish, and about 20 mg L�1 for rape and ryegrass (Lin and Xing,2007). Furthermore, study on the phytotoxicity of nano-TiO2 on Vi-cia narbonensis L. and Zea mays L seed germination, root elongationand mitotic behavior revealed maximum root growth inhibition atvery high dose (40000 mg L�1), as indicated by decrease in mitotic

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index with concomitant increase in chromosomal aberration per-centage (Castiglione et al., 2011). However, for plant nanotoxicolo-gy study, comparatively low concentrations (100–1000 mg L�1) ofnano-TiO2 were used in rice (Boonyanitipong et al., 2011) andwheat (Larue et al., 2012). In separate study, EC50 (effective con-centration that exhibits half of its maximal effects) dose of nano-CuO for seed germination was found to be 13 mg L�1 for lettuce;398 mg L�1 for radish, and 228 mg L�1 for cucumber (Wu et al.,2012). In the present investigation, three different concentrationsof nano-CuO suspensions (low, 0.5 mM; medium, 1.0 mM and high,1.5 mM) were selected (Supplemental Fig. 1, pilot experiment re-sults) to impose the stress. Present findings clearly indicate thatnano-CuO concentration higher than 0.5 mM is highly toxic for riceseedling growth and development. However, seed germinationwas significantly affected even in 0.5 mM CuO treatment. Wuet al. (2012) recently tested biological effects of various metaloxide NPs and found germination was highly affected under CuONPs in lettuce, radish and cucumber seeds. Nano-TiO2 had similarnegative effects on Vicia narbonensis and Zea mays seed germina-tion (Castiglione et al., 2011). Ahsan et al. (2007) also reported de-creased seed germination rate in rice exposed to cupric sulfate(CuSO4, 5H2O). Under our experimental set up, clear effects ofnano-CuO toxicity become evident from day 7 of experiment bothin terms of shoot and root growth. Loss of root cells viability understress was established by Evans blue staining. This dye is unable tocross the intact membranes and thus used to assess the cells integ-rity (Gaff and Okongo-Ogola, 1971). Higher uptake of Evans blue byroots of nano-CuO stressed seedlings indicates higher cell deathover the control. Our finding is in accordance with the Cu inducedthe loss of plasma membrane integrity as evident in maize rootsstained with Evans blue (Wang et al., 2011). The high nano-CuOconcentration (1.5 mM suspension) was found to be detrimentalfor the rice seedlings, as after day 7 no significant root growthwas further evident (Table 1). Dimkpa et al. (2013) recently re-ported phytotoxic effects of Ag NPs on growth of wheat plants insand matrix. The study also revealed reduction of shoot and rootlengths under nano-Ag stress in a dose-dependent manner. Fur-thermore, treatment with high dose of Ag NPs (2.5 mg kg�1) in-creased root branching, thereby affecting overall plant biomass.

Enhanced activity of antioxidant enzymes of ascorbate glutathi-one cycle under nano-CuO stress is an indication of an increasedproduction of ROS as well as quick activation of plants defencemechanism to combat oxidative stress damage. In vivo detectionof superoxides and H2O2 as dark blue and deep brown spots inleaves further indicates severe oxidative burst under nano-copperstress. It is now well established that elevated antioxidant en-zymes levels could be well associated with plant stress tolerancemechanism (Gossett et al., 1994; Hernandez et al., 1995). In re-sponse to nano-CuO stress, seedlings exhibited a diverse antioxida-tive response. Consistent increases in APX activity of 1.0 and1.5 mM CuO exposed leaves ensure higher rate of hydrogenperoxide scavenging in stressed seedlings as compared to control.

O stress on rice (Oryza sativa L.) seedlings. Chemosphere (2013), http://

Table 3Effects of nano-CuO stress on foliar glutathione contents of rice (Oryza sativa cv. Swarna). Contents were measured at 7 and 14 d of experiment and expressed as mean ± S.E.(n = 9). Means followed by a common letter are not significantly different at the 5% level by Duncan’s Multiple Range Test (DMRT).

Treatment tGSH (nmol g�1 FW) GSH (nmol g�1 FW) GSSG (nmol g�1 FW) GSH/GSSG

Control 7 d 0.293ab ± 0.003 0.161a ± 0.007 0.132a ± 0.003 1.228a ± 0.08214 d 0.301a ± 0.003 0.154a ± 0.005 0.146a ± 0.002 1.058a ± 0.051

0.5 mM 7 d 0.299b ± 0.001 0.165a ± 0.003 0.134a ± 0.001 1.228a ± 0.03514 d 0.300a ± 0.022 0.189a,b ± 0.023 0.111c ± 0.002 1.720b ± 0.228

1.0 mM 7 d 0.289a ± 0.002 0.181b ± 0.003 0.108b ± 0.001 1.678b ± 0.04914 d 0.324a ± 0.003 0.238c ± 0.004 0.086d ± 0.001 2.775c ± 0.105

1.5 mM 7 d 0.288a ± 0.003 0.189b ± 0.005 0.100c ± 0.002 1.903c ± 0.08914 d 0.333a ± 0.017 0.201bc ± 0.017 0.132b ± 0.001 1.513b ± 0.129

8 A.K. Shaw, Z. Hossain / Chemosphere xxx (2013) xxx–xxx

Nevertheless, stressed leaves accumulated significantly high levelof H2O2 starting from day 7 and the degree of accumulation wasin consistency with the increase in the nano-CuO suspension con-centration (Fig. 3A). In vivo detection of H2O2 through DAB stainingfurther supports high accumulation of H2O2 in leaves of stressedseedlings. Instead of higher APX activity, presence of high endoge-nous H2O2 level implies very high rate of production of H2O2 undernano-CuO stress. The observed increase in the APX activity doesnot seem to give full protection to the nano-CuO stressed seedlingsin scavenging this deadly reactive oxygen species-H2O2. A similartrend in increased APX activity has been reported in maize rootssubjected to Cu stress (Wang et al., 2011).

Within plant cell, SOD acts as the first line of defense againstoxidative stress as its activity directly modulates the amount ofO��2 and H2O2, the two important Haber–Weiss reaction substrates(Bowler et al., 1992). The 1.0 and 1.5 mM nano-CuO treated leavesshowed significantly higher SOD activity over the control at 7 dand 14 d respectively. Similar trend of copper (CuSO4, 5H2O) in-duced activation of SOD was reported in Arabidopsis thaliana (Dra-zkiewicz et al., 2004). In our experiment, nano-CuO stressedseedlings exhibited significantly higher (�1.2-fold at 7 d) activityof GR, the rate-limiting enzyme of ascorbate–glutathione cycle thatmaintains the GSH/GSSG ratio favorable for AsA reduction (Gossettet al., 1996). The observed increases in GR activity might be thereason of significantly high GSH contents as well as high GSH/GSSGratio in leaves of 1.0 and 1.5 mM nano-CuO treated seedlings (Ta-ble 3). A similar pattern of gradual increase in GR activity over thecontrol has been reported previously in maize roots subjected toincreasing Cu stress (Wang et al., 2011). In our study, nano-CuOstress induced decrease in GSSG content might be attributed tothe significantly declined (�1.8-fold over control at 14 d) DHARactivity (Fig. 4D), the monomeric thiol enzyme that reduces DHAinto AsA at the expense of glutathione (GSH) as an electron donor,with subsequent formation of oxidized glutathione (GSSG) (Foyerand Noctor, 2005).

APX readily dismutes H2O2 using ascorbate as the electron do-nor but, at the same time, it generates another free radical namedmonodehydroascorbate (MDHA), which can either disproportion-ate spontaneously into dehydroascorbate (DHA) and reducedascorbate (AsA) or be enzymatically converted into DHA by the en-zyme MDAR (Hossain et al., 2009). In present experiment, at 14 dall the stressed seedlings exhibited significant increase (�1.7-fold)in MDAR activity irrespective of the nano-CuO treatment concen-tration. This may be the reason of significantly increased DHA lev-els (�8 to 9-fold over control) in stressed leaves (Table 2). On theother side, at 14 d sharp decline in DHAR activity (�1.8-fold) inall CuO treated seedlings lead to significant reduction in foliarAsA content as well as low AsA/DHA ratio.

Plants accumulate a number of osmoprotective substances asadditional physiological response to various abiotic stresses. Pro-line is one of them, reported to be accumulated under metal stress(Tripathi and Gaur, 2004; Choudhary et al., 2007; Nedjimi and

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Daoud, 2009). It acts as a reservoir of carbon and nitrogen sourcesfor post-stress growth (Fukutaku and Yamada, 1984), as cytoplas-mic osmoticum protects enzymes from high ionic concentration oftoxic metal ions within the cell. Additional roles for proline in ROSdetoxification and stabilization of cell membranes have also beenproposed (Hamilton and Heckathorn, 2001). Nevertheless, its rolein combating metal stress is a matter of debate. In the presentexperiment, nano-CuO stressed seedlings accumulated signifi-cantly high level of proline and showed a positive correlation be-tween stress pressure (increasing CuO concentration) and prolineaccumulation. However, this enhanced proline level does not seemto be very effective in protecting cells from nano-CuO induced oxi-dative stress damages, as evident from high MDA level in stressedseedlings (Fig. 3B). We suggest that enhanced accumulation ofendogenous free proline in leaves of nano-CuO exposed rice seed-lings is simply a stress effect, rather than stress protectant.

Carotenoids are known to be potent quenchers of ROS, particu-larly singlet oxygen (1O2) by intercepting the triplet-chlorophyll(Young, 1991). They also act as light harvesting pigments byabsorbing photons and transferring the excitation energy to chlo-rophyll, which eventually reaches the reaction center. In presentinvestigation, nano-CuO stressed seedlings exhibited significantdecreases (�1.2-fold) in leaf carotenoids contents (except for1.5 mM CuO treated seedlings at 7 d). In contrast, control leavesmaintained high carotenoids level (Fig. 3D). Decreased carotenoidslevels might render the nano-CuO exposed seedlings more vulner-able to stress.

5. Conclusions

In summary, from our experimental data following conclusionscould be drawn- (a) nano-CuO treatment exerts oxidative dam-ages to rice seedlings as evident from high ROS scavenging anti-oxidant enzymes activity and enhanced MDA level; (b) elevatedAPX activity might be insufficient to scavenge all H2O2 producedin excess under nano-CuO stress; (c) significant decline in DHARactivity renders the stressed cells in futile recycling of antioxidantmetabolites, such as DHA into AsA; (d) isolated increase in GSH/GSSG ratio does not seem to prevent cells from oxidative dam-ages; (e) enhanced proline accumulation does not give much pro-tection against nano-CuO stress; and (f) decline in carotenoidslevel might be another determining factor of meager performanceof rice seedlings in combating nano-CuO stress induced oxidativedamages.

Acknowledgement

The author A.K. Shaw thankfully acknowledges the financialsupport provided by the Department of Science & Technology,New Delhi as DST INSPIRE Fellow.

O stress on rice (Oryza sativa L.) seedlings. Chemosphere (2013), http://

A.K. Shaw, Z. Hossain / Chemosphere xxx (2013) xxx–xxx 9

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2013.05.044.

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