Nano-CuO stress induced modulation of antioxidative defense and photosynthetic performance of Syrian barley (Hordeum vulgare L.)

Nano-CuO stress induced modulation of antioxidative defense and photosynthetic performance of Syrian barley (Hordeum vulgare L.)

Environmental and Experimental Botany 102 (2014) 37–47 Contents lists available at ScienceDirect Environmental and Experimental Botany journal homep...

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Environmental and Experimental Botany 102 (2014) 37–47

Contents lists available at ScienceDirect

Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Nano-CuO stress induced modulation of antioxidative defense and photosynthetic performance of Syrian barley (Hordeum vulgare L.) Arun Kumar Shaw a , Supriya Ghosh a , Hazem M. Kalaji b , Karolina Bosa c , Marian Brestic d , Marek Zivcak d , Zahed Hossain a,∗ a

Plant Stress Biology Lab, Department of Botany, West Bengal State University, Kolkata 700 126, West Bengal, India Department of Plant Physiology, Warsaw University of Life Sciences SGGW, Nowoursynowska 159, 02-776, Warsaw, Poland c Department of Pomology, Warsaw University of Life Sciences SGGW, Warsaw, Poland d Department of Plant Physiology, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia b

a r t i c l e

i n f o

Article history: Received 9 December 2013 Received in revised form 3 February 2014 Accepted 23 February 2014 Keywords: Antioxidant Barley Chlorophyll fluorescence Nanoparticle Nanotoxicology

a b s t r a c t Nanoparticles (NPs), a new class of pollutant has raised global environmental concern. The present study highlights the impact of nano-CuO stress on Syrian barley (Hordeum vulgare L., landrace Arabi Aswad). Seedling performances in terms of antioxidant defence and chlorophyll fluorescence were studied under three different levels of stress (0.5 mM, 1 mM and 1.5 mM suspensions of copper II oxide, <50 nm particle size, prepared in ½ MS medium) at 10 and 20 day of treatment along with control. Dose dependent reduction in shoot and root growth was recorded with passage of time. The maximal quantum yield of PS II photosynthetic apparatus (Fv/Fm) did not alter after stress application. However, performance index parameter was found to be significantly decreased irrespective of stress level and treatment period. Enhanced flavonol level with concomitant increase in APX activity found to be insufficient to enforce a light control over the H2 O2 level under nano-stress. Furthermore, an impairment of the collaborative action of DHAR and MDAR in stressed leaves results in a lower ability for efficient enzymatic recycling of DHA into AsA. Overall the nano-stressed leaves exhibited significant decline in GSH/GSSG ratio that might not contribute in maintaining high GSH pool essential for sustaining balanced redox status under stress condition. In addition, an isolated increase in GR activity in 1.0 and 1.5 mM nano-CuO treated leaves at 20 day does not give much protection to the nano-CuO stressed seedlings from oxidative damages. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The phytotoxicity study of ultrafine particles is an emerging issue to elucidate its potential impacts on plant system. Accidental or incidental release of commercial products like cosmetics and medicines which contain manufactured nanomaterials (MNMs) has become a real threat to the environment (Colvin, 2003; Service, 2008; Lee et al., 2010). Over the recent years, much emphasis has been given on the impact of nanoparticles (NPs) on animal system and only few studies have highlighted the phytotoxicity of NPs

Abbreviations: APX, ascorbate peroxidase; AsA, reduced ascorbate; tAsa, total ascorbate; DHA, dehydroascorbate; GR, glutathione reductase; GSH, reduced glutathione; tGSH, total glutathione; GSSG, oxidized glutathione; MDA, malondialdehyde; MS, Murashige and Skoog; NBI, nitrogen balance index; PIABS , performance index; ROS, reactive oxygen species; SOD, superoxide dismutase. ∗ Corresponding author. Tel.: +91 33 2524 1975; fax: +91 33 2524 1977. E-mail address: zahed [email protected] (Z. Hossain). http://dx.doi.org/10.1016/j.envexpbot.2014.02.016 0098-8472/© 2014 Elsevier B.V. All rights reserved.

(Lee et al., 2010; Castiglione et al., 2011; Wu et al., 2012; Shaw and Hossain, 2013). Nanoparticles (NPs) are typically ultrafine particles with lengths between 1 nm and 100 nm in at least two of their dimensions (Varela et al., 2012). The negative effects of NPs on plant development and metabolism depend on the size, concentration and chemistry of NPs, as well as the chemical milieu of the subcellular sites to which the NPs are deposited (Dietz and Herth, 2011). Lateral root junctions are the primary sites through which NPs could enter the xylem via cortex and the central cylinder (Dietz and Herth, 2011). NPs upon dissolution act as metal ions, able to interact with the sulfhydryl, carboxyl groups of proteins and thus alter activity. In addition, prolonged NP treatments trigger excess formation of reactive oxygen species (ROS) resulting severe oxidative burst (Shaw and Hossain, 2013; Thwala et al., 2013). The ROS unbalances the cellular redox system in favor of oxidized forms, resulting in oxidative damage to cellular components–lipids, proteins and nucleic acid (Halliwell and Gutteridge, 1999). In addition, ROS like singlet oxygen (1 O2 ) directly or by means of secondary

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radicals, attacks photosynthetic apparatus component proteins such as D1 protein of PSII and causes its degradation (Adir et al., 2003). However, recent in vivo photoinhibition studies have demonstrated that ROS act primarily by inactivating the repair of PSII and not by damaging PSII directly. In cyanobacterial cells, the de novo synthesis of the D1 protein was found to be markedly suppressed by the elevated intracellular level of ROS (Nishiyama et al., 2005). To scavenge and neutralize these toxic radicals, plants have evolved complex antioxidant defense mechanism comprised of both enzymatic and non-enzymatic networks (Hossain et al., 2012). Among the various metal oxide NPs, nano-TiO2 is by far the most well studied NP whose toxicity has been tested in different crop systems (Boonyanitipong et al., 2011; Castiglione et al., 2011). Recently, Thwala et al. (2013) reported the potential risks imposed by the engineered nanoparticles (ENPs) toward higher aquatic plants. Furthermore, changes in enzymes activities and ascorbate and free thiols levels resulting higher membrane damage and photosynthetic stress in the shoots of germinating rice seedlings on exposure to very high concentration of cerium oxide nanoparticles (nCeO2 ) have been documented (Rico et al., 2013). In contrast, limited number of published papers on phytotoxicity of nano-CuO is available (Dimkpa et al., 2012; Shaw and Hossain, 2013). Chlorophyll a fluorescence kinetics study using portable devices known as Fluorimeters or Plant Stress Meters is one of the finest methods to investigate the function of PSII and its reactions to changes in the environment and plant growth conditions (Kalaji et al., 2012). Changes in chlorophyll fluorescence patterns in response to drought (Burling et al., 2013), light (Kalaji et al., 2012) and temperature stress (Kalaji et al., 2011) have been monitored in different crops. Nevertheless, photosynthetic performances of barley by exploiting multiparametric fluorescence devices under nano-CuO stress have not yet been explored at all. To better understand the mechanistic details and the extent of nano-CuO induced changes in the photosynthetic activity, chlorophyll fluorescence kinetics were measured. Moreover, modulation of each enzymatic component of ascorbate–glutathione cycle and contribution of AsA and GSH pools are discussed to get the new insights into the plant cell response to cope with the NP-induced oxidative stress.

(after germination) of stress treatment, plants photosynthetic performances were evaluated noninvasively by analyzing chlorophyll fluorescence signals. Moreover, chlorophyll and epidermal flavonols contents were measured non-destructively using a handheld leaf-clip sensor. Morphological parameters like root length, root weight, shoot length and shoot weight were also recorded on the same day. Seedlings were randomly selected and shoots were harvested on the same days (10 and 20) and subsequently stored at −80 ◦ C for biochemical analysis. For detection of ROS and cell death, freshly collected leaves and roots were used respectively. In total three independent biological experiments were performed under the same growth conditions. To account for experimental variation, at least six biological repeats resulting from independent experiments were used for each sample. 2.2. In vivo detection of ROS Foliar hydrogen peroxide (H2 O2 ) accumulation was detected using 3 ,3 -diaminobenzidine (DAB) assay (Thordal-Christensen et al., 1997; Iriti et al., 2006). In vivo infiltration of leaves with 5 mM DAB forms deep brown polymerization products upon reaction with H2 O2 in the presence of peroxidase (Thordal-Christensen et al., 1997). Chlorophylls were removed from the leaves by infiltration with lacto–glycerol–ethanol (1:1:4, v/v/v) solution followed by boiling in water bath for 10 min. Images were captured using Olympus CX41 microscope (Olympus, Tokyo, Japan) equipped with digital 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 thoroughly in ddH2 O 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 with ddH2 O until no further blue color eluted from the roots. The stained roots were studied under Olympus CX41 microscope (Olympus, Tokyo, Japan) and images were captured using a digital camera (Canon, PowerShot, SX110 IS, Japan). 2.4. Chlorophyll fluorescence, chlorophyll and epidermal flavonols contents

2. Materials and methods 2.1. Plant material, nano-copper treatment and sample collection Seeds of Syrian barley (Hordeum vulgare L.) landrace Arabi Aswad were used as plant material for the present nanophytotoxicity study. Seeds were first disinfected in 3% sodium hypochlorite solution for 5 min, followed by three times washing and overnight soaking in distilled water. On the next day seeds were allowed to germinate on cotton pad soaked with ½ MS medium in plastic tray and considered as control. Three different concentrations (low, 0.5 mM; medium, 1.0 mM and high, 1.5 mM) of nano-CuO (copper II oxide, <50 nm particle size, Sigma–Aldrich, USA) suspensions prepared in ½ MS medium were used to impose nano-stress. For stress treatment, randomly selected water soaked seeds were allowed to germinate on similar cotton pads soaked with respective nano-CuO suspension. Seedlings (30 seedlings/treatment/replicate) were maintained at 25 ◦ C in plant growth chamber illuminated with white fluorescent light (600 ␮mol m−2 s−1 , 16 h light period day−1 ) and 65% RH. The day germination first observed was considered as zero day of experiment. Thereafter, both control and stressed seedlings were maintained for consecutive 20 days. On 10 and 20 day

Slow fluorescence kinetics measured by the pulse-modulated fluorometer system FMS-2 (Hansatech Instruments Ltd., Pentney, King’s Lynn, Norfolk, England) enabled calculation of PSII quantum efficiency of PS II (PSII), based on the values of the steady-state fluorescence (Fs) and the light-saturated chlorophyll fluorescence (Fm ), according to Maxwell and Johnson (2000). The fast chlorophyll fluorescence rise recorded using a handheld portable continuous excitation fluorescence analyzer Pocket PEA (Hansatech Instruments Ltd., Pentney, King’s Lynn, Norfolk, England) during the first second of illumination allows to record the fluorescence value in a sequence of phases (labeled as O, K, J, I, P) from the initial (Fo) to the maximal (Fm) fluorescence value. Fo and Fm values were used for calculation of maximum quantum yield of PSII photochemistry (Fv/Fm). The mathematical model of the polyphasic (OKJIP) transient named as JIP-test (Strasser and Strasser, 1995) was used for calculation of partial biophysical determinants used for calculation of a single integrative parameter denoted as performance index (PIABS ) according to Strasser et al. (2000). Measurements of fluorescence records were performed on the middle region of attached leaves after 45 min of dark adaptation at room temperature on 10 and 20 day of stress treatment. A total

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of nine measurements (replications) were recorded from randomly selected leaves in each treatment group. In addition, mesophyll chlorophyll and epidermal flavonols contents were non-destructively evaluated using a hand-held leafclip sensor (Dualex® Scientific+, ForceA) according to instrument manual. Determination of the leaf chlorophyll content is based on the measurement of the difference in the transmission of two distinct wavelengths, one close to red and one in near infrared (Cerovic et al., 2012; Burling et al., 2013). The measurement of the UV optical absorbance in the epidermis is based on the fluorescence emitted by the chlorophyll located in the mesophyll and is directly linked to the concentration of flavonols present in the epidermis. Both chlorophyll and epidermal flavonols contents were expressed in ␮g/cm2 . Additionally, nitrogen balance index (NBI) was calculated using chlorophyll/flavonols ratio (Burling et al., 2013). 2.5. Hydrogen peroxide measurement Foliar hydrogen peroxide content was estimated according to the method of Brennan and Frenkel (1977). One hundred milligram of chilled leaf tissue was macerated in 4 mL cold acetone and homogenate was filtered through Whatman No. 1 filter paper. Two milliliters of this filtrate were treated with 1 mL of titanium reagent (20% titanium tetrachloride in concentrated HCl, v/v) and 1 mL of concentrated ammonia solution to precipitate the titanium–hydroperoxide complex. After centrifugation (at 5000 × g for 30 min) precipitate was dissolved in 2 N H2 SO4 and the absorbance was read at 415 nm. H2 O2 content was calculated from a standard curve prepared in the similar way and expressed as ␮mol g−1 FW.

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ascorbate was followed by a decrease in the absorbance at 290 nm (ε = 2.8 mM−1 cm−1 ). APX activity was expressed as ␮mol ascorbate oxidized min−1 mg−1 protein. 2.7.2. Superoxide dismutase (SOD) SOD (EC 1.15.1.1) activity was determined by nitro blue tetrazolium (NBT) photochemical assay according to Beyer and Fridovich (1987). In this method 1 mL of solution containing 50 mM potassium phosphate buffer (pH 7.8), 9.9 mM l-methionine, 57 ␮M NBT, 0.025% triton-X-100 was added into small glass tubes followed by 20 ␮L of sample. Reaction was started by adding 10 ␮L of riboflavin solution (4.4 mg/100 mL) followed by placing the tubes in an aluminum foil-lined box having two 20-W fluorescent lamps for 7 min. A parallel control was run where buffer was used instead of sample. After illumination absorbance of solution was measured at 560 nm. A non-irradiated complete reaction mixture was served as a blank. SOD activity was expressed as U mg−1 protein. One unit of SOD was equal to that amount which causes a 50% decrease of SOD-inhibitable NBT reduction. 2.7.3. Glutathione reductase (GR) GR (EC 1.6.4.2) activity was determined by monitoring the glutathione dependant oxidation of NADPH, as described by Carlberg and Mannervik (1985). In a cuvette, 0.75 mL 0.2 M potassium phosphate buffer (pH 7) containing 2 mM EDTA, 75 ␮L NADPH (2 mM), 75 ␮L oxidized glutathione (20 mM) were mixed. Reaction was initiated by adding 0.1 mL enzyme extract to the cuvette and the decrease in absorbance at 340 nm was monitored for 2 min. GR activity was calculated using the extinction coefficient for NADPH of 6.2 mM−1 cm−1 and expressed as ␮mol NADPH oxidized min−1 mg−1 protein.

2.6. Measurement of malondialdehyde concentration Malondialdehyde (MDA) concentration was measured following the procedure of Hodges et al. (1999). Frozen leaf tissue was homogenized 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 acid and 0.5% thiobarbituric acid. Both mixtures were allowed to react in a water bath at 90 ◦ C for 1 h. After that, samples were cooled down in an ice bath and centrifuged. Absorbance of the supernatant was read at 440, 532 and 600 nm against a blank. MDA concentration was expressed in terms of nmol g−1 FW. 2.7. Antioxidant enzymes assays Frozen leaf tissue (0.5 g) was homogenized in 1.5 mL of 50 mM potassium phosphate buffer (PBS, pH 7.8) containing 1 mM EDTA, 1 mM dithiotreitol (DTT) and 2% (w/v) polyvinyl pyrrolidone (PVP) using chilled mortar and pestle kept in ice bath. The homogenate was centrifuged at 15,000 × g at 4 ◦ C for 30 min. Clear supernatant was used for SOD, GR, DHAR and MDAR enzymes assays. For measuring APX activity, the tissue was separately ground in 50 mM PBS (pH 7.8) supplemented with 2.0 mM ascorbate, 1 mM EDTA, 1 mM DTT and 2% (w/v) PVP. All assays were done at 25 ◦ C. Soluble protein content was determined according to Bradford (1976) using BSA as a standard. All spectrophotometric analyses were conducted using an UV/visible Spectrophotometer-Genesis 10S UV–vis (Thermo Scientific). 2.7.1. Ascorbate peroxidase (APX) APX (EC 1.11.1.11) activity was assayed according to the method of Nakano and Asada (1981). Three milliliter of the reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbate, 0.1 mM H2 O2 and 0.1 mL enzyme extract. The hydrogen peroxide-dependent oxidation of

2.7.4. Dehydroascorbate reductase (DHAR) DHAR (EC 1.8.5.1) enzyme activity was measured according to the method of Nakano and Asada (1981). The complete reaction mixture 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 of 1 mL. Reaction was initiated by adding suitable aliquot of enzyme extract and increase in absorbance was recorded at each 30 s interval for 3 min at 265 nm. Enzyme activity was expressed as ␮mol ascorbate formed min−1 mg−1 protein. 2.7.5. Monodehydroascorbate reductase (MDAR) MDAR (EC 1.6.5.4) enzyme activity was measured as described by Miyake and Asada (1992). Monodehydroascorbate was generated by ascorbate oxidase using a reaction mixture (1 mL) containing 50 mM HEPES–KOH buffer, pH 7.6, 0.1 mM NADPH, 2.5 mM ascorbate, ascorbate oxidase (0.14 U) and suitable aliquot of enzyme extract. MDAR activity was expressed as ␮mol NADPH oxidized min−1 mg−1 protein. 2.8. 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 Fe3+ to Fe2+ by ascorbate in acidic solution. The Fe2+ forms a red chelate with bipyridyl absorbing at 525 nm. DHA was calculated by subtracting AsA from total ascorbate. The DTNB–GSSG reductase recycling procedure of Anderson (1985) was used for the determination of both total (GSH + GSSG) and GSSG levels. GSH content was calculated by subtracting GSSG content from the total glutathione content. 2.9. Statistical analysis The results are presented as mean values ± standard errors. Statistical significance between mean values was assessed using

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Fig. 1. Comparative growth performances of 10-day old Syrian barley (Hordeum vulgare L., landrace Arabi Aswad) seedlings under nano-CuO stress (A). Evans blue stained roots of 10 day old seedlings showing nonviable cells. Control, 0.5, 1.0 and 1.5 mM nano-CuO stressed roots are presented from left to right respectively (B). DAB staining for in vivo detection of foliar H2 O2 . 10-day old DAB stained barley leaves of control, 0.5, 1.0 and 1.5 mM nano-CuO stressed seedlings are arranged from left to right respectively (C). Enlarged microscopic view of a portion of control leaf showing no indication of brown spots (D). Magnified view of a portion of nanocopper stressed leaf (C, circle) showing dark brown spots (arrow marks) indicative of H2 O2 deposits (E).

Fig. 2. Nano-CuO mediated stress impact on growth performance of 20-day old Syrian barley (Hordeum vulgare L., landrace Arabi Aswad) seedlings (A). Roots of 20day old seedlings stained with Evans blue indicate nonviable cells. Control, 0.5, 1.0 and 1.5 mM nano-CuO stressed roots are presented from left to right respectively (B). DAB staining for in vivo detection of foliar H2 O2 . 20 day old DAB stained barley leaves of control, 0.5, 1.0 and 1.5 mM nano-CuO stressed seedlings are arranged from left to right respectively (C). Enlarged microscopic view of a portion of control leaf showing no indication of brown spots (D). Magnified view of a portion of nanocopper stressed leaf (C, circle) showing dark brown spots (arrow marks) indicative of H2 O2 deposits (E).

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analysis of variance and a conventional Duncan’s Multiple Range Test (DMRT), using SPSS-10 statistical software (SPSS Inc., Chicago, IL, USA). A probability of p < 0.05 was considered significant. 3. Results 3.1. Impact of nano-copper stress on seedling growth Nano-CuO treatment had marked effects on barley seedlings growth. As compare to control significant gradual decreases in shoot length as well as shoot weight were observed with increasing nano-CuO concentration both at 10 and 20 days (Fig. 1 and Table 1). Maximum decline in shoot length was recorded under 1.5 mM nano-copper treatment. Moreover, at 20 day of stress, leaf tip yellowing was noticed in all stressed leaves irrespective of nano-CuO concentration (Fig. 2A). Root growth was also significantly affected on exposure to nano-CuO stress. With the increase in nano-CuO concentration, significant decline in root length and weight were observed (Table 1). Even at 10 day of stress, significant decrease in root length was recorded at lowest nano-CuO concentration (0.5 mM). Maximum reduction in root growth was evident under 1.5 mM nano-copper treatment. Notably, after 10 day of stress treatment, no significant root growth was further evident (Table 1). 3.2. In vivo detection of ROS Histochemical staining with DAB offers a very sensitive technique for in vivo detection of cellular H2 O2 . The presence of deep brown spots on the nano-copper stressed leaves clearly indicates the location of H2 O2 accumulation (Figs. 1C, E and 2C, E). Interestingly, at 10 day of stress, prominent brown spots were only observed in 1.5 mM nano-CuO treated leaves (Fig. 1C). In contrast, H2 O2 deposits were evident in all stressed leaves irrespective of CuO concentration (Fig. 2C). However, intensity and the occurrence of spots were found to be more in high concentrations (1.0 and 1.5 mM) as compared to 0.5 mM CuO. Presence of deep brown spots indicate severe oxidative burst under nano-CuO stress. 3.3. Root cell viability Staining with Evans blue has been the most widely used technique for determining cell viability. As this dye is unable to cross the intact membranes, Evans blue staining is a reliable method to assess the cells integrity (Gaff and Okongo-Ogola, 1971). Figs. 1B and 2B represent the 10- and 20-day-old barley roots stained with Evans blue respectively. As compared to control, the uptake of Evans blue by nano-copper stressed roots was much higher irrespective of CuO concentrations. Completely dark blue colored roots indicate maximum cell death in 1.0 and 1.5 mM nano-CuO exposed roots (Figs. 1B and 2B).

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at both 10 and 20 days (Fig. 3C). Moreover, stress had no significant effects on maximal quantum yield of PSII (Fv/Fm) irrespective of nano-copper concentration and treatment time (Fig. 3D). At 10 day, the Fv/Fm value for both control and the stressed leaves was around 0.77. However, with passage of time, the average Fv/Fm value was found to be lower (∼0.73) (Fig. 3D). Interestingly, nanocopper treatment had negative impact on the performance index calculated on the basis of energy absorption (PIABS ). At 10 day, a significant decline (∼1.3-fold) in PIABS was recorded even at 0.5 mM nano-CuO treatment (Fig. 4A). With increase in nano-copper concentration, no further change in PIABS was observed. Similar trend of nano-copper stress induced decreasing PIABS (as compared to control) was recorded at 20 day of treatment (Fig. 4A). As compared to control, the stressed leaves showed ∼1.4-fold decrease in PIABS irrespective of nano-CuO concentrations (Fig. 4A). 3.5. Changes in chlorophyll, flavonols contents and nitrogen balance index (NBI) No apparent changes in the chlorophyll, epidermal flavonols contents and NBI were observed under nano-CuO stress at 10 day (Fig. 4B–D). However, at 20 day, all stressed leaves irrespective of treatment concentration showed a sudden decline (∼1.8-fold) in chlorophyll content as compared to control (Fig. 4C). Similar trend was observed in case of nitrogen balance index (NBI) (Fig. 4B). Even 0.5 mM nano-copper stress treatment caused ∼2-fold decrease in NBI. No further significant change in NBI was observed at 1.0 and 1.5 mM nano-CuO treatments (Fig. 4B). Completely reverse trend was noticed in case of epidermal flavonols content. At 20 day of stress, all nano-stressed leaves exhibited significantly increased flavonols level (∼1.2-fold over the control) irrespective of treatment dose (Fig. 4D). 3.6. Hydrogen peroxide concentration Lowest concentration of nano-CuO (0.5 mM) had no apparent effect on foliar hydrogen peroxide content (Fig. 5A). However, with the further increase in CuO concentration, significant enhancement in H2 O2 content was noticed. Interestingly, at 20 day, both 1.0 and 1.5 mM nano-copper exposed leaves exhibited a sharp increase (∼2–8-fold over control) in foliar H2 O2 level (Fig. 5A). 3.7. MDA concentration Oxidative damage to lipid membranes is usually monitored with the tissue MDA level. With the increase in nano-CuO concentration, a gradual increase in foliar MDA level was recorded (Fig. 5B). Accumulation of high level of MDA indicates nano-copper stress induced membrane damages. Maximum increase (∼1.8-fold) in foliar MDA concentration was noticed in 1.5 mM CuO treatment irrespective of stress period. 3.8. Modulation of antioxidant enzymes

3.4. Fs, Fm , PSII, Fv/Fm and PIABS Fig. 3A–C shows the changes in the steady state fluorescence (Fs), light-saturated chlorophyll fluorescence (Fm ) and quantum efficiency of PS II (PSII) respectively in response to nano-copper stress. At 10 day, a marked increase (∼1.5-fold over control) was noticed in both Fs and Fm (Fig. 3A and B) with increase in treatment concentration. However, at highest concentration (1.5 mM nano-CuO), the values were decreased up to the control level. No further significant changes in these two parameters were observed at later stage (20 days) of nano-stress (Fig. 3A and B). In contrast, nano-copper induced increase in quantum efficiency of PS II (PSII) was only recorded in highest nano-CuO concentration (1.5 mM)

Under nano-copper stress, activities of five antioxidant enzymes of ascorbate–glutathione cycle were differentially modulated. Among them, APX activity was significantly increased in 1.0 and 1.5 mM nano-CuO treatments (Fig. 6A). However, magnitude of the increase was more (∼2.5-fold) evident at 10 day than at 20 day of treatment (∼1.5-fold). Notably, lowest concentration of nanoCuO (0.5 mM) had mostly no significant effect on foliar antioxidant enzymes activities except SOD (Fig. 6B). Interestingly, long exposure of nano-copper stress (20 days) had significant effect in enhancing SOD activity (∼1.4-fold). In contrast, both GR and DHAR activities were found to be less affected under nano-CuO stress at 10 day (Fig. 6C and D).

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Table 1 Effects of nano-CuO stress on growth of barley (Hordeum vulgare L. landrace Arabi Aswad) seedlings. Data were recorded at 10 and 20 day of experiment and expressed as mean ± S.E. (n = 9). Means followed by a common letter in a column are not significantly different at the 5% level by Duncan’s Multiple Range Test (DMRT). Treatment

Shoot length (cm) 10 day

Control 0.5 mM 1.0 mM 1.5 mM

14.61a 12.92b 10.83c 9.92c

Shoot weight (mg) 20 day

± ± ± ±

0.60 0.39 0.34 0.12

17.61a 15.70b 14.04b 11.61c

10 day ± ± ± ±

0.73 0.54 0.67 0.43

95.56a 72.93b 65.23b 62.27b

Root length (cm) 20 day

± ± ± ±

7.36 4.19 6.82 1.93

111.11a 87.91b 82.97bc 70.10c

Nevertheless, a drastic increase (∼8-fold) in GR activity was evident on exposure to high nano-CuO (1.0 and 1.5 mM) at 20 day of treatment (Fig. 6C). Completely opposite trend was observed in case of DHAR. Both 1.0 and 1.5 mM nano-copper exposed leaves exhibited ∼1.5-fold decline in DHAR activity (Fig. 6D). Unlike other antioxidant enzymes, a sudden decline in MDAR activity was recorded under 1.5 mM nano-copper treatment (Fig. 6E). Notably, the magnitude of decrease was more (∼2-fold) evident at 10 day as compared to 20 day of stress (∼1.3-fold).

3.9. Ascorbate and glutathione levels Nano-copper stress had significant impact on the foliar ascorbate pool. Marked changes in the reduced ascorbate (AsA) contents were observed at 20 day of treatment (Table 2). The gradual increase was in accordance with the increase in CuO concentration. Highest AsA accumulation (10-fold over the control) was observed in the leaves of 1.5 mM CuO exposed seedlings. Similar increasing trends were recorded in total as well as in oxidized (DHA) ascorbate contents (Table 2). Nevertheless, as compared to control, mostly no significant changes in the AsA/DHA ratio were found in stressed leaves, except for the 1.5 mM nano-copper treatment at 20 day (Table 2).

10 day ± ± ± ±

5.12 6.57 5.53 4.96

2.73a 1.09b 0.59b 0.52b

± ± ± ±

Root weight (mg) 20 day

0.42 0.11 0.04 0.07

8.62a 1.29b 0.90b 0.79b

± ± ± ±

10 day 1.01 0.14 0.07 0.09

21.97a 13.19b 10.04bc 8.33c

20 day ± ± ± ±

2.18 1.36 0.93 0.42

27.98a 10.81b 7.60bc 6.11c

± ± ± ±

1.85 1.09 0.90 0.63

Completely reverse trend was observed in case of foliar glutathione pool (Table 3). At 10 day, sudden declines in both total (tGSH) and reduced (GSH) glutathione levels were noted even in 0.5 mM nano-CuO treated leaves. Thereafter, no further significant change was recorded with increasing nano-copper concentration. In contrast, 1.5 mM nano-CuO treated leaves exhibited maximum decreases (∼2-fold) in both tGSH and GSH contents (Table 3). Interestingly, nano-copper stress had no significant effect on oxidized glutathione (GSSG) level irrespective of treatment time and nano-copper concentration. But, overall the GSH/GSSG ratio was significantly decreased in all nano-stressed leaves irrespective of CuO concentration (except for 0.5 mM treatment at 20 day). Lowest GSH/GSSG ratio (∼2.5-fold decline as compared to control) was observed in the 1.5 mM nano-CuO treated leaves at 20 day of stress treatment (Table 3). 4. Discussion The present time scale experiment shows the negative impacts of nano-CuO stress on physio-biochemical as well as photosynthetic performance of Syrian barley. Although a dose dependent inhibition in seedling growth was noticed, however nano-CuO treatment concentration higher than 0.5 mM had marked impact in disrupting plant defense system. Recent studies on biological

Fig. 3. Impact of nano-CuO stress on chlorophyll fluorescence parameters: (a) steady state fluorescence (Fs), (b) light-saturated chlorophyll fluorescence (Fm ), (c) quantum efficiency of PS II (PSII) and (d) maximal quantum yield of PSII (Fv/Fm) of Syrian barley (Hordeum vulgare L., landrace Arabi Aswad) leaves. Data are expressed 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 Range Test.

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Fig. 4. Non-invasive determination of (a) performance index (PIABS ), (b) nitrogen balance index (NBI), (c) chlorophyll and (d) epidermal flavonols contents of nano-CuO stressed leaves of Syrian barley (Hordeum vulgare L., landrace Arabi Aswad). Data are expressed 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 Range Test. Table 2 Effects of nano-CuO stress on foliar ascorbate contents of barley (Hordeum vulgare L. landrace Arabi Aswad) seedlings. Contents were measured at 10 and 20 day of experiment and expressed as mean ± S.E. (n = 6). Means followed by a common letter are not significantly different at the 5% level by Duncan’s Multiple Range Test (DMRT). Treatment Control 10 day 20 day 0.5 mM 10 day 20 day 1.0 mM 10 day 20 day 1.5 mM 10 day 20 day

tAsA (␮mol g−1 FW)

AsA (␮mol g−1 FW)

DHA (␮mol g−1 FW)

AsA/DHA

0.92a ± 0.045 0.67a ± 0.190

0.08ab ± 0.037 0.04a ± 0.014

0.84a ± 0.049 0.62a ± 0.195

0.10a ± 0.049 0.11a ± 0.045

1.31b ± 0.178 1.35b ± 0.233

0.04a ± 0.008 0.33b ± 0.055

1.27b ± 0.175 1.02ab ± 0.191

0.04a ± 0.007 0.35ab ± 0.044

1.23ab ± 0.098 1.38b ± 0.075

0.04a ± 0.017 0.35b ± 0.070

1.19b ± 0.097 1.03ab ± 0.120

0.04a ± 0.014 0.42ab ± 0.148

1.32b ± 0.079 2.22c ± 0.111

0.13b ± 0.031 0.83c ± 0.129

1.19b ± 0.056 1.39b ± 0.129

0.10a ± 0.024 0.66b ± 0.163

effects of different metal oxide NPs have shown their negative effects on seed germination and plant growth in various crops like lettuce, radish, cucumber, maize (Castiglione et al., 2011; Wu et al., 2012). Interestingly, in the present experiment nano-copper stress had no apparent effects on seed germination. Nevertheless,

in case of rice, we reported significant inhibition in seed germination even under 0.5 mM nano-CuO treatment (Shaw and Hossain, 2013). As compared to shoot, root growth was found to be more adversely affected on exposure to stress. Significant reduction in

Table 3 Effects of nano-CuO stress on foliar glutathione contents of barley (Hordeum vulgare L. landrace Arabi Aswad) seedlings. Contents were measured at 10 and 20 day of experiment and expressed as mean ± S.E. (n = 6). Means followed by a common letter are not significantly different at the 5% level by Duncan’s Multiple Range Test (DMRT). Treatment Control 10 day 20 day 0.5 mM 10 day 20 day 1.0 mM 10 day 20 day 1.5 mM 10 day 20 day

tGSH (nmol g−1 FW)

GSH (nmol g−1 FW)

GSSG (nmol g−1 FW)

GSH/GSSG

0.29a ± 0.023 0.29a ± 0.034

0.20a ± 0.023 0.20a ± 0.033

0.09a ± 0.001 0.08a ± 0.002

2.28a ± 0.254 2.41a ± 0.390

0.21b ± 0.009 0.27a ± 0.029

0.12b ± 0.009 0.18a ± 0.029

0.09a ± 0.001 0.08a ± 0.001

1.40b ± 0.105 2.16a ± 0.343

0.23b ± 0.019 0.22ab ± 0.039

0.14b ± 0.019 0.14ab ± 0.039

0.09a ± 0.002 0.09a ± 0.001

1.67b ± 0.222 1.63ab ± 0.455

0.21b ± 0.014 0.17b ± 0.021

0.12b ± 0.013 0.08b ± 0.021

0.09a ± 0.001 0.09a ± 0.001

1.41b ± 0.132 0.98b ± 0.247

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µ

44

Fig. 5. Nano-CuO stress induced accumulations of H2 O2 (A) and MDA (B) in leaves of Syrian barley (Hordeum vulgare L., landrace Arabi Aswad). Data are expressed as the mean values ± standard errors (n = 6). Values in columns with different letters are significantly different at 5% level according to Duncan’s Multiple Range Test.

both root length and biomass even at lowest concentration of nanoCuO (0.5 mM) was in accordance with our earlier observation in nano-stressed rice (Shaw and Hossain, 2013). Nano-CuO induced loss of root cell viability as indicated by Evans blue staining may be a reason of drastic inhibition in barley root growth. This particular dye could not cross the intact membranes and thus used to assess the cells integrity (Gaff and Okongo-Ogola, 1971). Higher uptake of Evans blue by roots of nano-CuO stressed seedlings indicates higher cell death over the control. Completely dark blue colored roots of 1.0 and 1.5 mM nano-copper stressed barley seedlings indicate maximum cell death in particular at 20 day. This observation is in agreement with the nano-CuO induced loss of cell viability in roots of rice seedlings (Shaw and Hossain, 2013). Chlorophyll fluorescence study of 1.5 mM nano-CuO challenged leaves revealed an increase in PSII indicating high photochemical efficiency of PSII under nano-stress (Fig. 3C). This might be a physiological adaptation of barley seedlings to cope with the nanoCuO induced oxidative stress. In contrast of our findings, decrease in PSII was documented in rice leaves exposed to chilling stress (Yang et al., 2013). Similarly, a positive correlation between the steady-state chlorophyll fluorescence (Fs) and net photosynthetic rate (A) has been demonstrated in C3 plants (Flexas et al., 2002). Thus, Fs is often used as an indirect measure of plants’ photosynthetic activity. In the present study, an initial increase in Fs value was noticed under 0.5 and 1.0 mM nano-CuO treatment at 10 day of stress followed by decline up to the control level (Fig. 3A). However, no such change over the control was recorded at 20 day of stress. Nano-stressed leaves exhibited similar trend for light saturated chlorophyll fluorescence (Fm ) (Fig. 3B). Such increases in both Fs and Fm at an early stage of stress (10 day) might be a strategy to adapt with the nano-copper stressed condition. Under

our experimental set up, non-significant change in the Fv/Fm ratio was observed under nano-copper stress (Fig. 3D). On the other hand, the second parameter derived from fast fluorescence induction, performance index, decreased significantly. This parameter was introduced by Strasser et al. (2000) as the product of three independent parameters combining structural and functional criteria: density of reaction centers, the quantum efficiency of primary photochemistry and conversion of excitation energy in electron transport. Unaltered values of PSII quantum efficiency (shown here as Fv/Fm values) associated with PIABS decrease suggest that the nano-copper stress causes decrease in number of reaction centers and capacity of electron transport carriers, as confirmed by detailed JIP-test analysis (not shown here). This decline in PIABS is in accordance with the previous findings on the impact of light stress on photosynthetic apparatus of barley seedlings (Kalaji et al., 2012). Our results also confirm that the performance index (PIABS ) reflects the changes in photochemistry more sensitively than the commonly used parameter Fv/Fm, as established earlier in Triticum aestivum under water stress (Zivcak et al., 2008). Antioxidant enzymes have long been considered as the first line of defense against ROS generation, however, their actions need to be complemented by that of other ROS scavenging systems during severe stress conditions (Apel and Hirt, 2004). Flavonols are a class of flavonoids that constitute a secondary ROS-scavenging system in plants exposed to prolonged stress conditions (Fini et al., 2011). These scavengers may either inhibit the generation or reducing the level of ROS once they are formed (Agati et al., 2007). The 10 day of nano-CuO exposure had no apparent effects on the chlorophyll and epidermal flavonol contents which had also been reflected in the non-significant changes in the nitrogen balance index (NBI), an indicator of nitrogen status of plants. However, Dualex® device recorded significant enhancement in the flavonol content in the epidermis of all nano-stressed leaves at 20 day (Fig. 4D). The parallel marked reduction (∼1.6-fold as compared to control) in the chlorophyll contents at 20 day irrespective of stress level (Fig. 4C) might contribute for the sudden reduction in the NBI parameter (Fig. 4B). Under stress condition, flavonoids play a key functional role in keeping the H2 O2 concentration at a sub-lethal level (Fini et al., 2011). Nevertheless, in our case, enhanced flavonol level with concomitant increase in APX activity found to be totally inefficient to enforce a light control over the H2 O2 level (Fig. 5A). Change in MDA concentration has been used as a parameter to assess oxidative stress damages to lipid membranes (Hossain et al., 2006, 2009; Arbona et al., 2008). Under our experimental conditions, MDA level significantly increased in nano-stressed leaves suggesting higher membrane damage. This observation is in agreement with a recent finding on the impact of cerium oxide nanoparticles (nCeO2 ) on the oxidative stress and antioxidant defense system in germinating rice seeds (Rico et al., 2013). Hydroxyl radical (OH• ) is the most reactive of all ROS capable of abstracting hydrogen atom from a methylene ( CH2 ) group present in polyunsaturated fatty acid side chains of membrane lipids and initiating lipid peroxidation (Barber and Thomas, 1978). This hydroxyl radical is in turn generated from H2 O2 as a result of one electron reduction through Haber–Weiss or Fenton reactions by using metal catalyst (Halliwell and Gutteridge, 1999). Thus, H2 O2 scavenging enzymes have significant roles in protecting lipid membranes. Leaves of 1.0 and 1.5 mM nano-CuO stressed barley seedlings exhibited significant increase (∼1.5–2.5-fold) in APX activity (Fig. 6A). Instead of having higher APX activity, higher accumulation of endogenous H2 O2 level was evident in nano-stressed leaves. However, as compared to 10 day of stress, the magnitude of increase was more evident in 1.0 and 1.5 mM treatments at 20 day (Fig. 5A). Results imply that increased APX activity was not sufficient to scavenge the excess H2 O2 generated in response to nano-copper stress. In vivo detection of H2 O2 through DAB staining

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µ

µ

µ

µ

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Fig. 6. Nano-CuO stress induced modulation of APX (A), SOD (B), GR (C), DHAR (D) and MDAR (E) activities in leaves of Syrian barley (Hordeum vulgare L., landrace Arabi Aswad). Control seedlings were grown without CuO stress. Data are expressed as the mean values ± standard errors (n = 6). Values in columns with different letters are significantly different at 5% level according to Duncan’s Multiple Range Test.

further supports high accumulation of H2 O2 in leaves of nano-CuO stressed seedlings in particular at 20 day of stress treatment. A similar trend in enhanced APX activity with concomitant increases in H2 O2 and MDA levels have been reported in leaves of rice seedlings subjected to nano-CuO stress (Shaw and Hossain, 2013). In summary, lack of complete scavenging of the highly reactive ROS (H2 O2 ) through enhanced APX activity, might be one of the crucial factors causing higher membrane damages in leaves of nano-copper stressed barley seedlings. The activities of other antioxidant enzymes of ascorbate– glutathione cycle were found to be less affected at 10 day of nanocopper stress, except for SOD and MDAR (Fig. 6B–E). While at later stage (20 day) stressed leaves exhibited differential modulation in ROS scavenging enzymes. Copper (CuSO4 ·5H2 O) had similar effect on the activation of SOD in Arabidopsis (Drazkiewicz et al., 2004). Significant enhancement in SOD activity is also in accordance with the recent report on cerium oxide nanoparticles (nCeO2 ) mediated ROS scavenging in rice seedling (Rico et al., 2013). Within plant • cell, SOD directly modulates the amount of O2 − and H2 O2 , the two

important Haber–Weiss reaction substrates (Bowler et al., 1992). In our case, enhanced SOD activity with insufficient increased APX might contribute in higher accumulation of foliar H2 O2 and subsequent membrane damages. Using reduced ascorbate as the electron donor, APX readily dismutes the toxic H2 O2 . While scavenging H2 O2 , APX generates another free radical named monodehydroascorbate (MDHA), which may either disproportionate spontaneously into dehydroascorbate (DHA) and reduced ascorbate (AsA) or be enzymatically converted into DHA by the enzyme MDAR (Hossain et al., 2009). Dehydroascorbate reductase (DHAR), another important antioxidant enzyme component of ascorbate glutathione cycle catalyses the chemical conversion of DHA into AsA using reduced glutathione (GSH) as electron donor. In our experimental condition, the DHAR and MDAR activities were found to be reduced in nanocopper stressed leaves in particular at 20 day (Fig. 6D and E). Instead of showing decreased DHAR activity, significantly high level of AsA was evident in nano-stressed leaves (Fig. 6D and Table 2). Similarly, DHA level was also found to be enhanced, despite of reduced MDAR

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activity (Fig. 6E and Table 2). This disagreement in enzymes activity and antioxidant metabolite level strongly indicates non-enzymatic disproportion of MDHA into DHA and AsA. Gradual enhancement in both total ascorbate (tAsA) and AsA concentrations with increasing nano-copper stress level ultimately leads to significant increase in AsA/DHA ratio. In our experiment, 10 day of nano-stress had no significant effect on the activity of glutathione reductase (GR), the ratelimiting enzyme of ascorbate–glutathione cycle that maintains the GSH/GSSG ratio favorable for AsA reduction (Gossett et al., 1996). However, at later stage (20 day) drastic increase in GR activity was evident in 1.0 and 1.5 mM nano-CuO treated leaves (Fig. 6E). GR is basically a flavoprotein catalyzing the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). Instead of having high GR activity, the nano-copper stressed leaves exhibited decreasing trend in the GSH content as well as GSH/GSSG ratio with increasing stress level (Table 3). This observation is in contrary with our earlier findings, where concomitant increases in GR as well as GSH/GSSG ratio were observed in leaves of 1.0 and 1.5 mM nano-CuO treated rice seedlings (Shaw and Hossain, 2013). Within plant cell, GSH plays several important roles in stress mitigation. In addition to its function as an antioxidant, intimately involved in cellular redox balance, it is instrumental for the detoxification of xenobiotics and heavy metals (Foyer et al., 1995; Hossain and Komatsu, 2013). Enhanced GSH metabolism through the enzyme GST (glutathione-S-transferase) might be one possible explanation of decreased GSH content in the nano-stressed leaves.

5. Conclusions From the obtained results and the above-discussion, the following conclusions could be drawn to explain the nano-CuOmediated toxicity in barley seedlings: (a) prolonged nano-CuO treatment triggers oxidative burst; (b) nano-stress induced changes in antioxidant enzymes activity strongly indicate disruption of ROS/antioxidant balance; (c) application of nano-copper stress does not cause significant alteration in the maximal quantum yield of PS II photosynthetic apparatus (Fv/Fm); (d) elevated MDA level is indicative of high membrane damages; (e) enhanced APX activity coupled with increased flavonol level might not be sufficient to decompose the excess H2 O2 produced under nano-CuO stress; (f) presence of dark brown spots on the nano-copper stressed leaves after DAB staining clearly indicate H2 O2 deposits and thus reflects inefficient H2 O2 scavenging; (g) an impairment of the collaborative action of DHAR and MDAR might contribute to a lower ability for efficient enzymatic recycling of DHA into AsA; (h) significant decline in GSH/GSSG ratio with increasing treatment concentration might not contribute in maintaining high GSH pool essential for sustaining balanced redox status; and (i) isolated increase in GR activity in 1.0 and 1.5 mM nano-CuO treated leaves at 20 day does not give much protection to the nano-CuO stressed seedlings from oxidative damages.

Acknowledgements Seeds of Syrian barley landrace Arabi Aswad were kindly gifted by Dr. Adonis Kourieh, International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syrian Arab Republic. We are also thankful to the NBPGR, New Delhi for facilitating the transfer of barley seeds from Syrian Arab Republic to WBSU, Kolkata. The author A.K.S. wish to acknowledge the support of the Department of Science & Technology, New Delhi as DST INSPIRE Fellow.

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