Piriformospora indica, an excellent system for heavy metal sequestration and amelioration of oxidative stress and DNA damage in Cassia angustifolia Vahl under copper stress

Ecotoxicology and Environmental Safety 156 (2018) 409–419

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Piriformospora indica, an excellent system for heavy metal sequestration and amelioration of oxidative stress and DNA damage in Cassia angustifolia Vahl under copper stress Rajeshwari Nanda, Veena Agrawal

T

⁎

Department of Botany, University of Delhi, Delhi 110007, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Cassia angustifolia Copper stress Antioxidant enzymes Piriformospora indica DNA damage Phytostabilization

Present investigation reveals copper induced phytotoxicity, oxidative stress and DNA damage in Cassia angustifolia Vahl and its amelioration by employing a symbiotic fungus, Piriformospora indica. Seeds were germinated on Knop's medium containing five Cu levels (0, 1, 10, 50, 100 and 200 mg L−1), with and without P. indica. Colonization with P. indica significantly (P < 0.05) ameliorated Cu induced oxidative stress. However, maximum amelioration was observed at 50 mg L−1 Cu with P. indica. Atomic absorption spectroscopy revealed that P. indica colonization significantly inhibited Cu accumulation in shoots. Maximum decline in Cu accumulation in shoots was observed at 50 mg L−1 (27.27%) with P. indica over Cu alone. Besides, P. indica colonized seedlings stored 16.86% higher Cu in roots as compared to Cu alone at 200 mg L−1. Similarly, maximum proline accumulation increased up to 19.32% over Cu alone at 50 mg L−1 Cu with P. indica. Significant elevation in antioxidant enzyme levels of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase and glutathione reductase was seen with P. indica. Contrary to increase in antioxidant level, toxic parameters such as lipid peroxidation and hydrogen peroxide decreased significantly with P. indica. Maximum decline in lipid peroxidation (13.76%) and hydrogen peroxide (18.58%) was observed at 50 mg L−1 with P. indica over Cu alone. P. indica significantly reduced DNA damage as well as changed the protein profile in C. angustifolia seedlings. Thus, P. indica proved to be an excellent system to alleviate Cu induced oxidative stress and might be useful as a phytostabilization tool.

1. Introduction

leading to lipid peroxidation, DNA and protein damage (Panda et al., 2016). Plants inherently have certain mechanisms to combat oxidative damage such as enzymatic antioxidants (SOD, CAT, POX and GR) and non-enzymatic antioxidants such as glutathione, thiols and carotenoids to counter damaging effects of ROS (Inzé and Van Montagu, 1995; Thounaojam et al., 2014; Munne-Bosch and Pinto-Marijuan, 2016). There are certain techniques by which amount of heavy metals can be removed or restricted to prevent harmful effect in plants and animals. These include physical, chemical and biological techniques. Phytoremediation technique is an effective strategy to remediate heavy metal contaminated land as it is simple and highly cost efficient as compared to other techniques (Sarwar et al., 2017). Symbiotic association of plants with arbuscular mycorrhizal fungi also play an important role in plant defence system as it can effectively restrict and alleviate heavy metals in the soil (Emamverdian et al., 2015; Latef et al., 2016). Various

Copper, an essential micronutrient is required for the normal plant growth as it acts as a cofactor in various enzymes such as superoxide dismutase, amino oxidase, plastocyanin and polyphenol oxidase. However, at higher concentrations, it causes growth inhibition, chlorosis and reduction in biomass due to increased production of reactive oxygen species and interfering with cellular molecules (Yruela, 2005; Burkheadd et al., 2009; Lange et al., 2017). High levels of Cu naturally occur in soils but mining, smelting and waste disposal contribute to increased level in soil. Typically, Cu concentrations in soil generally range from 2 to 250 ppm and plant tissues contain from 20 to 30 μg g−1 DW (Mittler et al., 2004; Khatun et al., 2008). Copper is a redox‒active metal which is directly involved in production of reactive oxygen species (ROS) which results in oxidative stress consequently

Abbreviations: GSH, Glutathione; GSSG, Glutathione Disulfide; NBT, Nitro Blue Tetrazolium; NADP, Nicotinamide Adenosine Dinucleotide Phosphate; SOD, Superoxide dismutase; CAT, Catalase; APX, Ascorbate peroxidase; GPX, Guaiacol peroxidase; GR, Glutathione reductase; H2O2, Hydrogen peroxide; MDA, Malondialdehyde; SDS, Sodium dodecyl sulphate; PAGE, Polyacrylamide Gel Electrophoresis ⁎ Corresponding author. E-mail address: [email protected] (V. Agrawal). https://doi.org/10.1016/j.ecoenv.2018.03.016 Received 4 November 2017; Received in revised form 3 March 2018; Accepted 6 March 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 156 (2018) 409–419

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2.1.2. Analysis of root colonization Root colonization analysis was performed according to the modified protocol of Dickson and Smith (1998) and Phillips and Hayman (1970). Root colonization in C. angustifolia co–cultivated with P. indica was analysed after 28 d of inoculation. Roots of C. angustifolia were washed thoroughly with ddH2O to remove attached medium and mycelium. Roots were then cut into 1.0 cm pieces, boiled for 10 min in 10% KOH solution and incubated in 1 M HCl for 10 min. Roots were washed with autoclaved ddH2O and stained with cotton blue overnight. Stained root segments were observed under light microscope at 40× magnification.

genes involved in heavy metal tolerance such as ZIP transporters, metallothioneins and glutathione-s-transferases were up regulated in mycorrhiza colonized plants. Additionally, heavy metal responsive genes up regulated in response to heavy metal exposure in colonized plants might be localized to fungal structures such as arbuscules (Hildebrandt et al., 2007). Therefore, these plant–fungi associations might be effectively utilized in enhancing heavy metal phytostabilization potential of plants. Piriformospora indica is a symbiotic mycorrhizal fungus as it has a beneficial role in plant growth and yield under normal and stressed condition (Varma et al., 1999). It enhances antioxidant defence system of plants which is crucial in stress tolerance. It also induces local and systemic disease resistance by modulating the level of antioxidants to combat oxidative stress (Vadassery et al., 2009). It has also shown a significant capacity to immobilise heavy metals in roots which can be very promising in phytoremediation (Shahabivand et al., 2017). Very few studies (Shahabivand et al., 2012; Sartipnia et al., 2013; Hui et al., 2015; Gill et al., 2016) have been reported in literature regarding heavy metal stress in plants where P. indica successfully alleviated phytotoxicity. Therefore, practical application of P. indica in clean up of heavy metal contaminated lands can prove to be a major tool in functioning of sustainable environment. C. angustifolia is drought tolerant fast growing shrub which is frequently used in wasteland development (Sharma et al., 1999). Metal accumulating plants are generally slow growing which greatly limits their full utilization in phytoremediation (Pilon-Smits, 2005). Therefore, the ability of C. angustifolia to grow in wastelands and enhanced heavy metal accumulation can provide a detoxification strategy to combat heavy metal pollution. We have previously reported that it can accumulate significant amount of heavy metals in its roots and has a great prospect in phytoremediation (Nanda and Agrawal, 2016). The present study was carried out to further explore the potential of mycorrhizal association of C. angustifolia with P. indica regarding heavy metal distribution in various plant parts and its beneficial effects. So far, there is no report of any study regarding alleviation of heavy metal stress in C. angustifolia, an important source of human drug. Therefore, following objectives were set to determine the potential of P. indica association with C. angustifolia: a) effect of this association on various physiological parameters, b) its effect on oxidative stress and defense system and c) its effect of heavy metal accumulation in various plant parts and its scope in phytoremediation.

2.2. Chlorophyll estimation Chlorophyll estimation was carried out according to Arnon (1949). Leaf samples (0.1 g) were homogenized in 5 mL chilled 80% acetone in dark conditions. Absorbance of supernatant was recorded at 645 and 663 nm after centrifugation at 5000 × g for 10 min at 4 °C. 2.3. Metal accumulation Treated seedlings were washed thoroughly with autoclaved ddH2O to remove residues of medium followed by 0.1 M HNO3 to remove metals adsorbed on root surface. Control and treated plant material was digested in HNO3/HCLO4 (3:1, v/v). After complete digestion, 10 mL of 0.1 N HNO3 was added and analysed in atomic absorption spectrophotometer (AAS) (Shimadzu, Japan). Limit of detection (LOD) was determined by analyzing the known concentration of samples at which it was reliably detected. Limit of quantification (LOQ) was determined by analyzing the known concentration of samples at which it was reliably quantified. 2.4. Antioxidant enzyme assays 2.4.1. Superoxide dismutase (SOD) assay SOD assay was performed according to a modified NBT method of Beyer and Fridovich (1987). Each reaction mixture contained phosphate buffer (pH 7.5), riboflavin (2 μM), methionine (13 mM), NBT (7.5 μM) and 50 µL protein extract. Detailed protocol has been described previously in Nanda and Agrawal (2016). 2.4.2. Catalase (CAT) assay Determination of CAT activity in the material was assayed according to Aebi (1974). Catalase activity was measured by monitoring the decrease in H2O2 concentration at 240 nm. Complete protocol has been described in Nanda and Agrawal (2016).

2. Materials and methods 2.1. Plant material and experimental conditions Seeds of C. angustifolia Vahl were surface sterilized and germinated on Knop's medium (Knop, 1865) according to the detailed protocol described in Nanda and Agrawal (2016). CuSO4 was used for heavy metal treatment in the present study. Aqueous solution of CuSO4 (Merck, Germany) was added to adjust Cu concentration in the medium. Treatment was given with five Cu levels (0, 1, 10, 50, 100 and 200 mg L−1), with and without P. indica. Range of Cu concentrations were selected on the basis of inhibition of growth of seedlings after exposure.

2.4.3. Ascorbate peroxidase (APX) assay APX activity was determined according to the modified protocol of Nakano and Asada (1981). APX was assayed using ascorbic acid and H2O2 as a substrate. Kinetic changes were measured at 290 nm for 500 s at 25 °C. Protocol for APX estimation has been described in detail in Nanda and Agrawal (2016). 2.4.4. Guaiacol peroxidase (GPX) assay Determination of GPX activity was performed according to the protocol of Thimmaiah (1999). GPX was measured using guaiacol as a substrate. Kinetic changes were recorded at 470 nm for 300 s at 25 °C. Chemical constituents and method has been previously illustrated in Nanda and Agrawal (2016).

2.1.1. Co-cultivation of C. angustifolia with P. indica The stock cultures of Piriformospora indica were obtained from Prof. Ajit Varma, AIMT (Amity University), Noida, India. Stock cultures of P. indica were sub–cultured in liquid modified Kaefer medium (pH 6.5) (Hill and Kafer, 2001). For co-cultivation of C. angustifolia seeds with P. indica, a 5 mm2 pit was prepared on the solid culture medium with help of a needle and same size of matured mycelium was placed in the pit (Sharma and Agrawal, 2013). Surface sterilized seeds were carefully placed in the mycelium filled pit for proper infection.

2.4.5. Glutathione reductase (GR) assay GR activity was determined according to the protocol of Schaedle and Bassham (1977). GR was assayed by monitoring a decrease in absorbance at 340 nm due to NADPH oxidation. Complete protocol has been given in detail in Nanda and Agrawal (2016). 410

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Table 1 Effect of P. indica (PI) colonization on average seed germination, average shoot and root length in C. angustifolia seedlings exposed to various concentrations of Cu. Different letters within each column indicate significantly different values within each treatment (two-way ANOVA, Duncan's multiple range test, P < 0.05). Copper concentration

Average germination (%)

(mg L−1)

Cu

0 1 10 50 100 200

Average shoot length (cm)

Cu + PI d

70.8 ± 4.2 72.22 ± 8.6d 73.60 ± 2.42d 51.32 ± 4.72c 45.56 ± 4.17b 26.33 ± 2.3a

(%) d

72.2 ± 2.4 75.0 ± 4.16d 76.36 ± 2.3d 59.53 ± 2.13c 48.6 ± 2.42b 27.77 ± 2.4a

2.0 3.84 3.75 15.9 6.98 5.46

Cu 5.67 5.82 5.39 2.73 2.24 1.47

Average root length (cm)

Cu + PI ± ± ± ± ± ±

e

0.13 0.12e 0.10d 0.06c 0.05b 0.08a

5.95 6.31 5.94 3.33 2.51 1.52

± ± ± ± ± ±

(%) e

0.15 0.16f 0.19d 0.15c 0.12b 0.02a

4.93 8.41 10.2 21.97 12.05 3.4

Cu 7.65 8.38 6.14 2.35 1.35 0.34

Cu + PI ± ± ± ± ± ±

e

0.4 0.2f 0.25d 0.15c 0.15b 0.07a

(%) e

8.0 ± 0.2 8.83 ± 0.25f 6.55 ± 0.25d 2.63 ± 0.21c 1.51 ± 0.1b 0.36 ± 0.05a

4.57 5.36 6.67 11.91 11.85 5.88

*Data represents the mean ± SD of three independent experiments. Different letters within each column indicate significantly different values at P < 0.05 (two-way ANOVA).

buffer (300 mM NaOH and 1 mM EDTA, ≥ pH 13) for 10 min followed by electrophoresis at 0.75 V cm−1 and 30 mA for 15 min in the same alkaline buffer at 4 °C. Slides were taken out from the tank and rinsed in distilled water and neutralized with chilled Tris buffer (pH 7.4) followed by staining with ethidium bromide solution. Extraction of nuclei, electrophoresis and microscopic analysis has been previously described in detail in Nanda and Agrawal (2016).

2.5. Estimation of lipid peroxidation Lipid peroxidation was estimated according to the protocol of Heath and Packer (1968). Shoot sample (0.5 g) was homogenized in 1% TCA solution (w/v) and centrifuged at 8500 × g for 10 min. Reaction mixture was prepared in a test tube with 1 mL of supernatant and 4 mL of 0.5% TBA/20% TCA (w/v) solution. Test tubes were kept into a boiling water bath for 30 min. Samples were then centrifuged at 10,000 × g and absorbance was recorded at 535 and 600 nm.

2.10. Statistical analysis

2.6. Estimation of hydrogen peroxide (H2O2)

Statistical analysis was performed using SPSS 22.0 (Software Package for the Social Sciences). Data were analysed by two–way ANOVA and tested for descriptive and homogeneity of variance. Duncan's multiple range test was used to determine the significant difference among means of different treatment group (P < 0.05). Data were also subjected to logarithmic transformation using compute variables to check the significance of interaction. Principal Component Analysis (PCA) was performed for all the enzymatic antioxidants. Pearson coefficient was calculated to determine the correlation between different variables.

H2O2 estimation was performed according to the modified protocol of Junglee et al. (2014). Shoot samples (0.5 g) were weighed and crushed in 0.1% TCA solution. Samples were then centrifuged at 12,879 × g for 15 min at 4 °C. Reaction mixture was prepared with 1.0 mL of 10 mM phosphate buffer (pH 7.0), 2.0 mL of 1 M potassium iodide and 1.0 mL of supernatant. Absorbance was measured at 390 nm and H2O2 content was determined with the help of standard curve. 2.7. Estimation of proline accumulation

3. Results

Proline content was estimated according to modified protocol of Bates et al. (1973). Shoot sample (0.2 g) was homogenized in 5 mL 3% sulphosalicylic acid and supernatant was collected. Reaction mixture was prepared with 2 mL of supernatant, 2 mL each of ninhydrin reagent and glacial acetic acid followed by boiling for 1 h. Toluene (4 mL) was added to the reaction mixture and vortexed vigorously. The upper phase containing brick red coloured chromophore was extracted and its absorbance was recorded at 520 nm.

3.1. Seed germination and seedling growth P. indica inoculation showed significant (P < 0.05) positive interaction with Cu regarding seed germination and seedling growth in C. angustifolia (two-way ANOVA) (Supplementary material). Log transformation of data also showed significant effect of the P. indica treatment on morphological parameters. Presence of Cu in growth medium significantly inhibited average seed germination in C. angustifolia above 10 mg L−1 Cu concentration (Table 1). P. indica alone exhibited 72.2% germination. Seed germination initially increased up to 72.20% and 73.60% at 1 and 10 mg L−1 as compared to control (70.80%) which increased further up to 75.0% and 76.36% with P. indica. Decline in seed germination started at 50 mg L−1 (51.32%) which increased to 59.53% with P. indica. At higher concentrations, it decreased to 45.56% and 26.33% at 100 and 200 mg L−1 Cu, respectively which increased to 48.60% and 27.77% at 100 and 200 mg L−1 with P. indica. Application of Cu did not have significant effect (P < 0.05) on shoot length up to 1 mg L−1 concentration. Average shoot length in control seedlings started with 5.67 cm which increased further with P. indica (5.95 cm). Lower concentration of Cu (1 mg L−1) promoted average shoot length up to 5.82 cm which increased to 6.31 cm with P. indica. Shoot length accounted for 5.39 cm at 10 mg L−1 but P. indica colonization increased it up to 5.94 cm. Higher concentrations of Cu proved toxic as shoot length declined sharply. It decreased up to 2.73, 2.24 and 1.47 cm at 50, 100 and 200 mg L−1 which increased up to 3.33, 2.51 and 1.52 cm with P. indica (Table 1). Copper exposure significantly (P < 0.05) affected root length in C. angustifolia seedlings at all concentrations. Inoculation with P. indica

2.8. SDS-PAGE analysis Seedlings (1.0 g) were homogenized in Zivy's buffer (30 mM TrisHCl, 1 mM EDTA) pH 8.5 at 4 °C (Zivy et al., 1983) and centrifuged at 12,879 × g for 10 min at 4 °C. Supernatant was centrifuged again and precipitate was discarded. Protein samples were loaded in to the wells and gels (10%) were run for 3–4 h using constant current of 25 mA followed by staining for 12–14 h in 0.2% CBB R-250 solution. Detailed method of SDS-PAGE analysis has been previously described in Nanda and Agrawal (2016). 2.9. Comet assay analysis Comet assay was performed using the protocol of Gichner and Plewa (1998). Leaves were sliced into fringes and nuclei were collected in to a micro-centrifuge tube. Equal volume of 0.75% agarose was added to the micro-centrifuge tube containing nuclei. This nuclear suspension was spread on microscope slides pre-coated with 1.0% agarose followed by a second layer of 80 µL 0.75% agarose. Slides were then immersed in a horizontal electrophoresis tank containing freshly prepared alkaline 411

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Fig. 1. Showing colonization of P. indica in C. angustifolia roots: chlamydospores visible in macerated root tissues (A), P. indica mycelium inside root epidermal cells (B) and P. indica chlamydospores and hyphae inside root epidermal cells (C) after 28 days of co-cultivation. Images were captures under light microscope at 40× magnification.

16 Chl a content (mg g−1 FW)

showed significant positive effect in alleviating Cu induced root growth reduction. Control seedlings exhibited 7.65 cm root length which enhanced up to 8.0 cm with P. indica. Root length initially increased up to 8.38 cm at 1 mg L−1 which further increased (8.83 cm) with P. indica. Root length slightly declined to 6.14 cm at 10 mg L−1 but increased up to 6.55 cm in P. indica colonized seedlings. A sharp decline in root length was seen at higher concentrations as it decreased to 2.35, 1.35 and 0.34 cm at 50, 100 and 200 mg L−1. Respective increase in root length was 2.63, 1.51 and 0.36 cm with P. indica (Table 1). 3.2. Root colonization analysis

14

f

f

e e

12

d

10

Cu

A

Cu+P. indica d c

c

8

b

b

a a

6 4 2 0 0

P. indica spores were visible in epidermal cells of all the root segments of co–cultivated seedlings. Fungal mycelium not only grew around the roots but also penetrated intracellularly. However, fungal hyphae did not penetrate vascular tissues. Spores were spherical and intracellular (Fig. 1A–C). Necrotization or injury of cellular structure was not visible in root cells of C. angustifolia.

1

10

50

100

200

Metal concentration (mg L−1)

Chl b content (mg g−1 FW)

12

3.3. Chlorophyll a and b estimation Chlorophyll a and b showed dose dependent decrease in Cu stressed seedlings at all concentrations (Fig. 2A and B). P. indica colonization significantly (P < 0.05) improved chlorophyll content in C. angustifolia seedlings over Cu alone. Analysis of results after log-transformation of data showed that there was significant effect of P. indica treatment on Chl a and b. Chl a content in control seedlings accounted for 13.59 mg g−1 FW which increased further up to 13.88 mg g−1 FW in P. indica colonized seedlings. Decline in Chl a content in Cu stressed seedlings were 13.02, 26.93, 43.78% at 1, 10 and 50 mg L−1 Cu. However, decrease in Chl a was less in P. indica colonized seedlings as only 9.71%, 21.19% and 35.39% decline was observed at 1, 10 and 50 mg L−1 Cu. More than 50% decline in Chl a content was observed at 100 and 200 mg L−1 Cu (50.02% and 61.73%, respectively) but after P. indica colonization, decline was 45.40% and 60.26%. Similar results were also observed regarding Chl b content in C. angustifolia. Control seedlings of both inoculated and non-inoculated seedlings exhibited no significant difference regarding Chl a content which was 9.92 and 10.1 mg g−1 FW, respectively. However, Chl b content declined by 22.68% and 32.86% at 1 and 10 mg L−1 Cu but P. indica colonization improved Chl b content where it was respectively 18.76% and 29.03%. Decrease in Chl b content at 50 mg L−1 Cu was 47.86% in non–colonized seedlings and 38.81% in P. indica colonized seedlings. However, a sharp decrease was observed at 100 and 200 mg L−1 Cu with 51.61% and 68.95% decline. However, at the same concentration with P. indica, this decline was 49.0% and 64.81%. Correlation analysis revealed that Chl a content was negatively correlated with Cu alone and Cu with P. indica (r = − 0.987, P < 0.01, r = − 0.998, P < 0.01 in

10

B

f

f

e e

8

Cu Cu+P. indica

d d c

6

c b b a a

4 2

0 0

1

10

50

100

200

Metal concentration (mg L−1) Fig. 2. Effect of Cu stress and its alleviation through P. indica on Chl a (A) and Chl b (B) in C. angustifolia seedlings after 28 d. Values represent the mean ± SD of three independent experiments. Different letters within each column indicate significantly different values within each treatment (two-way ANOVA, Duncan's multiple range test, P < 0.05).

Cu lone and Cu with P. indica, respectively). Similarly, Chl b content also showed strong negative correlation with Cu alone and with P. indica (r = − 0.968, P < 0.01, r = − 0.983, P < 0.01 in Cu alone and with P. indica, respectively). 3.4. Metal accumulation in various plant parts Atomic absorption spectroscopy analysis revealed that P. indica colonization significantly (P < 0.05) affected Cu accumulation in both shoot and root of C. angustifolia seedlings (Fig. 3A and B). Significance of this interaction in shoot was unchanged after logarithmic transformation of variables. However, in roots, logarithmic transformation of data showed that there was significant effect of P. indica on Cu accumulation but it was not dependent on Cu concentration. In shoots, control and P. indica alone seedlings accumulated similar 412

Ecotoxicology and Environmental Safety 156 (2018) 409–419

Copper content in shoots (mg Kg-1 DW )

R. Nanda, V. Agrawal

2000 1800 1600 1400 1200 1000 800 600 400 200 0

A

Cu Cu+P. indica

3.5. Estimation of antioxidant enzymes

e

d

e

f

100

200

Principal Component Analysis (PCA) was executed for all antioxidant enzymes to determine the interaction between Cu stress and P. indica treatment. PCA showed that the first principal component (F1) have the eigenvalue > 1. This component explains 95.25% of the variation in the data. In the scree plot, a straight line is formed after the first principal component (Fig. 6E and F). PCA Biplot shows the relation between treatment and parameters. In the biplot, observation 6, 11 and 12 are located far from other clusters which show its dissimilarity over other observations. Distinct clusters in biplot clearly show correlation between Cu stress, response of antioxidant enzymes in the presence and absence of P. indica treatment.

c d

a a

a b

0

1

b c

10

50

Copper content in roots (mg K g1 DW )

Metal concentration (mg L −1 ) 14000 12000

B

Cu Cu+ P. indica

e

10000

8000

d d

6000

c c

4000

2000 0

3.5.1. Superoxide dismutase activity Seedlings treated with Cu showed a dose dependent increase in SOD activity. P. indica inoculation too significantly (P < 0.05) enhanced SOD activity in C. angustifolia seedlings. Log-transformation of variables showed that P. indica exhibited significant effect on SOD activity. In P. indica alone, there was 12% increase in SOD activity as compared to control. Increase in SOD activity under Cu stressed seedlings was 44.0%, 80.0% and 120.0% at 1, 10 and 50 mg L−1 Cu over control which respectively increased up to 70.4%, 98.4% and 157.6% in P. indica colonized seedlings. At higher concentrations, increase in SOD activity was more than 150% which accounted for 162.4% and 195.2% at 100 and 200 mg L−1 Cu over control. Percent increase in SOD activity after P. indica colonization was 181.6% and 202.4% at 100 and 200 mg L−1 Cu. SOD activity was directly correlated with Cu alone and with P. indica (r = 0.998, P < 0.01, r = 0.971, P < 0.01 in Cu alone and with P. indica, respectively).

e

a a

a a

0

1

b b 10

50

Metal concentration (mg

100

200

L−1 )

Fig. 3. Effect of increasing Cu concentration on metal accumulation in shoots (A) and roots (B) in Cassia angustifolia seedlings after 28 d. Values represent the mean ± SD of three independent experiments. Different letters within each column indicate significantly different values within each treatment (two-way ANOVA, Duncan's multiple range test, P < 0.05).

3.5.2. Catalase activity Catalase activity in Cu treated seedlings showed a dose dependent response in C. angustifolia (Fig. 4B). However, there was no significant (P < 0.05) increase in CAT activity with P. indica inoculation. P. indica alone seedlings did not exhibit significant increase in CAT activity as compared to control. CAT activity increase in Cu stressed seedlings was 18.9, 31.71% at 1 and 10 mg L−1 which respectively increased up to 25.3% and 38.72% over control. At higher concentrations, more than 50% increase in CAT activity was observed which accounted for 58.54%, 74.39% and 88.41% at 50, 100 and 200 mg L−1 Cu. After P. indica colonization, CAT activity further increased up to 60.98%, 82.32% and 93.29% at 50, 100 and 200 mg L−1 Cu over control. Correlation analysis showed that CAT activity was directly correlated with Cu alone and with P. indica (r = 0.991, P < 0.01, r = 0.992, P < 0.01 in Cu alone and with P. indica, respectively).

amount of Cu (8.25 and 8.36 mg kg−1 DW). Limit of detection and quantification for Cu was respectively 0.4 and 5.1 μg L−1. At relatively lower concentrations, Cu accumulation accounted for 51.69 and 223.34 mg kg−1 DW, respectively at 1 and 10 mg L−1 in Cu alone. However, at the same concentrations with P. indica, Cu accumulation decreased to 8.36 and 49.85 mg kg−1 DW. Seedlings grown under higher concentrations of Cu alone exhibited 866.25, 1528.66 and 1654.83 mg kg−1 DW Cu content at 50, 100 and 200 mg L−1, respectively. At the same concentrations, it decreased to 630.0 and 1353.06 and 1409.0 mg kg−1 DW in P. indica colonized seedlings. In shoots, maximum decrease in Cu accumulation was observed at 50 mg L−1 Cu which was 27.27% less in P. indica colonized seedlings as compared to Cu alone. P. indica colonization significantly (P < 0.05) enhanced Cu accumulation in roots as compared to Cu alone. Cu accumulation in Cu alone seedlings was 9.11 and 82.34 mg kg−1 DW in control and 1 mg L−1 which respectively increased up to 9.9 and 87.17 mg kg−1 DW with P. indica. A significantly high amount of Cu was stored in roots at 10, 50 and 100 mg L−1 Cu alone where it was 1169.44, 3821.45 and 6117.0 mg kg−1 DW. At the same concentrations, P. indica colonization further enhanced it to 1263.66, 4108.0 and 6412.16 mg kg−1 DW. Maximum Cu accumulation (9624.33 mg kg−1 DW) was observed at 200 mg L−1 Cu alone seedlings which increased to 11,247.30 mg kg−1 DW with P. indica. However, maximum increase in Cu accumulation was achieved at 200 mg L−1 with P. indica which was 16.86% higher than Cu alone. Metal accumulation was directly correlated with Cu alone and with P. indica in shoots (r = 0.920, P < 0.01, r = 0.90, P < 0.01 in Cu and Cu + P. indica, respectively). Metal accumulation was directly correlated with Cu alone and with P. indica in roots (r = 0.917, P < 0.01, r = 0.897, P < 0.01 in Cu alone and with P. indica, respectively).

3.5.3. Ascorbate peroxidase activity Seedlings treated with Cu showed significant (P < 0.05) increase in APX activity in C. angustifolia (Fig. 4C). Significance of the interaction was same when data were transformed logarithmically. P. indica colonized seedlings without Cu did not exhibit significant increase regarding APX activity. APX activity in Cu stressed seedlings initially increased up to 3.43% and 7.72% at 1 and 10 mg L−1 Cu over control. However, after P. indica colonization it further increased up to 7.37% and 21.44% at 1 and 10 mg L−1 Cu over control. APX activity increased up to 21.97%, 28.41% and 33.44% at 50, 100 and 200 mg L−1 Cu over control. P. indica colonization increased it further up to 42.11%, 51.55% and 54.1% at 50, 100 and 200 mg L−1 Cu over control. Correlation analysis showed that APX activity was directly correlated with Cu alone and with P. indica (r = 0.962, P < 0.01, r = 0.952, P < 0.01 in Cu alone and with P. indica, respectively). 3.5.4. Guaiacol peroxidase activity GPX activity showed a significant (P < 0.05) increase under Cu stress as well as P. indica colonized seedlings (Fig. 4D). P. indica have a 413

Ecotoxicology and Environmental Safety 156 (2018) 409–419

7

A

Cu Cu+P. indica

b

b

d

c c

d

6

Cu+P. indica

5

4

B

Cu

a a

b b

d d

e e

f f

c c

3 2

1 0

1

10

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Fig. 4. Effect of Cu stress and its alleviation through P. indica on superoxide dismutase (A), catalase (B), ascorbate peroxidase (C) and guaiacol peroxidase (D) in C. angustifolia seedlings after 28 d. Values represent the mean ± SD of three independent experiments. Different letters within each column indicate significantly different values within each treatment (two-way ANOVA, Duncan's multiple range test, P < 0.05).

3.6. Lipid peroxidation

significant effect on GPX activity as shown by log transformation of data. Increase in GPX activity was 13.9% and 65.11% at 1 and 10 mg L−1 Cu over control which further increased up to 16.27% and 74.41% in P. indica colonized seedlings over control. GPX activity showed a significant increase at 50, 100 and 200 mg L−1 Cu which respectively accounted for 88.37%, 113.95% and 130.23% over control. In P. indica colonized seedlings, it further increased up to 102.32%, 137.2% and 160.46% at 50, 100 and 200 mg L−1 Cu over control. Correlation analysis showed that GPX activity was directly correlated with Cu alone and with P. indica (r = 0.972, P < 0.01, r = 0.980, P < 0.01 in Cu alone and with P. indica, respectively).

MDA content is used as an index for lipid peroxidation. MDA content significantly increased in Cu stress alone but decreased significantly (P < 0.05) in P. indica colonized seedlings (Fig. 5B). Effect of this interaction was significant as confirmed by log transformation of data. P. indica alone seedlings accumulated 13.09% less MDA content as compared to control. MDA content initially increased to 23.21% and 92.0% at 1 and 10 mg L−1 Cu over control but decreased to 14.54% and 79.29% with P. indica. Lipid peroxidation increased up to 127.27% at 50 mg L−1 Cu over control but with P. indica colonization, increase was only 96.0%. At higher concentrations, MDA content increased up to 167.54% and 235.63% at 100 and 200 mg L−1 Cu which further accounted for 141.45% and 228.18% with P. indica colonization. Correlation analysis showed that lipid peroxidation was directly correlated with Cu and Cu + P. indica (r = 0.990, P < 0.01, r = 0.962, P < 0.01 in Cu and Cu with P. indica, respectively).

3.5.5. Glutathione reductase activity P. indica inoculation exhibited significant (P < 0.05) effect on GR activity in stressed C. angustifolia seedlings (Fig. 5A). Log transformation of data also showed significant P. indica interaction with Cu stress regarding GR activity. P. indica alone seedlings exhibited 6.0% in GR activity as compared to control. Lower concentrations of Cu showed 6.81 and 21.96.0% increase in GR activity at 1 and 10 mg L−1 Cu over control which respectively increased up to 21.2% and 30.3% over control. There was 30.76% increase in GR activity at 50 mg L−1 Cu over control which further increased up to 53.84% when seedlings were colonized with P. indica along with Cu. At higher concentrations GR activity increased up to 46.15% and 92.3% at 100 and 200 mg L−1 Cu as compared to control. P. indica inoculation along with Cu significantly enhanced GR activity up to 61.53% and 100% at 100 and 200 mg L−1 Cu over control. Correlation analysis showed that GR activity was directly correlated with Cu alone and with P. indica (r = 0.880, P < 0.01, r = 0.950, P < 0.01 in Cu alone and with P. indica, respectively).

3.7. Estimation of hydrogen peroxide H2O2 content significantly (P < 0.05) increased in Cu treated C. angustifolia seedlings. However, with P. indica colonization, hydrogen peroxide decreased significantly (P < 0.05) under Cu stress (Fig. 5C). Log transformation of data confirmed the significant effect of P. indica inoculation. In P. indica alone seedlings, H2O2 content decreased up to 5.55% over control. In Cu stressed seedlings, extremely high amount of H2O2 was observed at 1 and 10 mg L−1 Cu which accounted for 38.88% and 80.15%, respectively over control. After P. indica colonization, amount of H2O2 content measured was 19.84% and 57.14% at 1 and 10 mg L−1 Cu, respectively over control. More than 100% increase was observed at higher concentrations of Cu. Increase in H2O2 content was 134.92%, 190.47% and 220.63% at 50, 100 and 200 mg L−1 Cu, 414

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Fig. 5. Effect of Cu stress and its alleviation through P. indica on glutathione reductase (A), MDA content (B), H2O2 content (C) and proline accumulation (D) in C. angustifolia seedlings after 28 d. Values represent the mean ± SD of three independent experiments. Different letters within each column indicate significantly different values within each treatment (two-way ANOVA, Duncan's multiple range test, P < 0.05).

3.8. Proline content

respectively over control but with P. indica colonization, respective increase was 91.26%, 156.34% and 201.58% over control. Correlation analysis showed that H2O2 content was positively correlated with Cu alone and with P. indica (r = 0.993, P < 0.01, r = 0.979, P < 0.01 in Cu alone and with P. indica, respectively).

Cu treated seedlings showed significantly (P < 0.05) increased proline content in C. angustifolia as compared to control (Fig. 5D). Inoculation with P. indica too significantly (P < 0.05) enhanced proline content in C. angustifolia seedlings as compared to Cu alone. It was also validated by log transformation of data. Seedlings inoculated with only

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Fig. 6. Principal component analysis of antioxidant enzymes, Cu stress and P. indica treatment. Determination of Principal component through Scree plot (A) and Principal Component Analysis Biplot of Cu stress and antioxidant enzymes response in the presence and absence of P. indica (B). Letters obs1, obs2, obs3, obs4, obs5, obs6, obs7, obs8, obs9, obs10, obs11 and obs12 represent control, 1 mg L−1 Cu, 10 mg L−1 Cu, 50 mg L−1 Cu, 100 mg L−1 Cu, 200 mg L−1 Cu, P. indica alone, 1 mg L−1 Cu + P. indica, 10 mg L−1 Cu + P. indica, 50 mg L−1 Cu + P. indica, 100 mg L−1 Cu + P. indica and 200 mg L−1 Cu + P. indica, respectively.

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Fig. 7. Showing DNA damage under Cu stress and its alleviation through P. indica in 28-day-old C. angustifolia seedlings. A dose dependent DNA damage was observed under Cu stress. Letters, A–F represent 0, 1, 10, 50, 100 and 200 mg L−1 Cu. Letters G–L represent 0, 1, 10, 50, 100 and 200 mg L−1 Cu + P. indica colonized seedlings. Arrows represent the extensive DNA damage at higher concentration of Cu. Decrease in extent of DNA damage is clearly visible in comet images. Images captured at 40× magnification under fluorescence microscope.

3.9. Comet assay

P. indica did not show any significant changes in proline content. There was 2.7 and 3.22 fold increase at 1 and 10 mg L−1 Cu over control which slightly increased up to 2.74 and 3.72 fold in P. indica colonized seedlings. However, proline content significantly increased up to 5.97 and 7.25 fold at 50 and 100 mg L−1 Cu over control. After P. indica colonization, it further increased up to 7.13 and 8.19 fold respectively at 50 and 100 mg L−1 Cu. Maximum increase in proline content was observed at 200 mg L−1 Cu which accounted for 10.52 fold. However, it further increased up to 11.53 fold when seedlings were inoculated with P. indica. Maximum increase in proline content was seen at 50 mg L−1 where it increased by 19.32% over Cu alone. Correlation analysis showed that proline accumulation was positively correlated with Cu alone and with P. indica (r = 0.963, P < 0.01, r = 0.971, P < 0.01 in Cu alone and with P. indica, respectively).

A dose dependent increase in tail length was observed in comet assay analysis. Concentration above 10 mg L−1 Cu induced high DNA damage in C. angustifolia seedlings. Maximum DNA damage was observed at 200 mg L−1 Cu (Fig. 7A–F). Co-cultivation of C. angustifolia with P. indica significantly improved DNA damage as compared to Cu stress alone as nuclear head was bigger and tail length was shorter at higher concentrations of Cu (Fig. 7G–L).

3.10. SDS-PAGE analysis Total protein was subjected to one dimensional SDS-PAGE analysis. Proteins were resolved in sharp separate bands between 14 and 43 kDa molecular weights. An increasing concentration of several low molecular weight bands were present in seedlings treated with Cu. However 416

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as roots stored maximum metal restricting their transport to shoots. High accumulation of Cu in P. indica colonized seedling roots might be explained by the symbiotic association of P. indica with C. angustifolia seedlings. Symbiotic association is an important plants defence system that can immobilize heavy metals in roots by binding it to hyphal cell wall and restricting their uptake to shoots (Emamverdian et al., 2015). So, it may be suggested that by restricting Cu in roots, P. indica may confers metal tolerance and provides an excellent strategy for the development of phytostabilization tool to restrict metals in contaminated areas. Superoxide dismutase rapidly converts superoxide into H2O2 in various cell compartments such as chloroplasts, mitochondria, nuclei, peroxisomes and apoplasts (Alscher et al., 2002; Gill and Tuteja, 2010). P. indica colonization in C. angustifolia seedlings resulted in further increase in SOD activity. Similar results have also been reported in P. indica colonized plants where SOD activity increased in colonized plants under Cd stress (Hui et al., 2015). Baltruschat et al. (2008) have suggested that fungus activate antioxidant enzyme system in plant leaves. So, it clearly indicates that P. indica might have used the same mechanism to activate antioxidant enzymes to prevent ROS induced oxidative damage. Catalase is found mainly in peroxisomes and neutralizes H2O2 without requiring any reductant. Excess accumulation of H2O2 can lead to severe disruption in photosynthesis as there are various thiol-regulated enzymes. Catalase neutralizes H2O2 produced throughout mitochondrial electron transport, β-oxidation of the fatty acids and photorespiration (Scandalios et al., 1997; Anjum et al., 2016). In the present study, there was a mild increase in catalase activity in P. indica colonized seedlings. Colonization with P. indica also increased catalase activity in Nicotiana tobacum under Cd stress (Hui et al., 2015). P. indica induced increase in catalase activity might be due to the fact that root endophytes facilitate stronger response against stress in symbiotic plants than non-symbiotic plants (Rodriguez et al., 2004). Ascorbate peroxidase utilizes ascorbate as specific electron donor to reduce H2O2 into H2O (Yamaguchi et al., 1995; Jim´enez et al., 1997). P. indica colonization up regulated the protein expression of APX in Hordeum vulgare seedlings exposed to drought stress (Ghabooli et al., 2013) which might be the reason for increased APX activity in C. angustifolia seedlings. Guaiacol peroxidases belong to class III peroxidases which are commonly found in apoplast, cell wall or vacuole. These are considered as biomarkers for metal stress (Jouili et al., 2011; Horemans et al., 2015). This enzyme uses guaiacol as a reductant to facilitate the decomposition of H2O2. In the present study, increase in GPX activity was observed in P. indica colonized C. angustifolia seedlings which might be involved in H2O2 neutralization. In the present study, P. indica colonization in C. angustifolia seedlings significantly increased GR activity. Glutathione reductase play an important role in maintaining high GSH/GSSG ratio which maintains the GSH pool necessary for active protein function under normal and adverse conditions (Thounaojam et al., 2012). Therefore, it is suggested that increase in GR activity in C. angustifolia after P. indica colonization might be responsible for increased redox balance, subsequently leading to decreased ROS production and hence reduced oxidative damage (Thounaojam et al., 2012). Malondialdehyde is produced by lipid peroxidation which is also considered as an indicator of oxidative stress induced membrane damage (Thounaojam et al., 2012). Polyunsaturated fatty acid (PUFA) is the main component of membrane lipid which is very susceptible to peroxidation. Hydroxyl radical and singlet oxygen produce lipid peroxy radicals and hydro peroxides by reacting with the methylene groups of PUFA (Smirnoff, 1998). Lipid peroxidation increased in Cu treated C. angustifolia seedlings as compared to control. However, P. indica colonized seedlings exhibited decreased levels of lipid peroxidation. Our results corroborated with Hui et al. (2015) where P. indica has been reported to decrease lipid peroxidation in N. tobacum under Cd stress. Decrease in lipid peroxidation in P. indica colonized C. angustifolia

Fig. 8. Showing effect of Cu stress on one dimensional SDS-PAGE analysis and its alleviation through P. indica in 28-day-old Cassia angustifolia seedlings. Arrows represent the increased synthesis of low molecular weight proteins at higher concentration of Cu stress (A) and Cu + P. indica (B) Letters 1–6 represent 0, 1, 10, 50, 100 and 200 mg L−1 Cu concentration. M represents PageRuler prestained protein ladder.

these bands were absent in control and 1 mg L−1 Cu stress (Fig. 8A). Protein profile of seedlings colonized with P. indica showed significant changes as compared to Cu stress alone. Low molecular weight bands of ~ 14 kDa were present only above 10 mg L−1 Cu (Fig. 8B).

4. Discussion Present study aims to evaluate the Cu induced phytotoxicity and its amelioration through P. indica colonization in C. angustifolia. P. indica inoculation in C. angustifolia proved highly beneficial in alleviating Cu toxicity. In the present study, P. indica significantly improved antioxidant defence system by enhancing enzymatic activities of SOD, APX, GPX and GR. Additionally, significant increase in proline level was also observed. In addition to enhancing protective parameters, it also lowered down damaging parameters such as hydrogen peroxide content, lipid peroxidation and DNA damage. Besides, it restricted the translocation of Cu from root to shoot by binding them in roots. In the present study, photosynthetic pigments such as Chl a and b in C. angustifolia seedlings were significantly reduced under Cu stress. Seedlings colonized with P. indica also showed significant improvement regarding chlorophyll content in C. angustifolia seedlings under Cu stress. Similar studies regarding P. indica induced increase in chlorophyll content have also been reported in Brassica rapa under drought stress (Sun et al., 2010) and Nicotiana tobacum (Hui et al., 2015). Decrease in chlorophyll content was slowed down by P. indica colonization in Brassica campestris under heat and osmotic stress suggesting its protective role against stress induced chlorophyll degradation (Kao et al., 2016). In P. indica colonized Oryza sativa seedlings, genes involved in chlorophyll synthesis were up regulated (Jogawat et al., 2016). Therefore, increase in chlorophyll in C. angustifolia might be due to overexpression of these genes. In some plants, heavy metal chelation and sequestration determines the basis of metal tolerance, translocation and storage. Low transportation of metals in shoots and its restriction in roots is the most significant characteristics (Dickinson and Lepp, 1997). In the present study, a significantly high amount of Cu was stored in roots of C. angustifolia seedlings in P. indica colonized seedlings. Additionally, root to shoot transport of Cu was significantly affected by P. indica colonization 417

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screened for Cu storage potential. Besides, more study is needed to understand the underlying mechanism of C. angustifolia and P. indica interaction.

seedlings might be due to increased accumulation of proline which plays a protective role in maintaining osmotic balance and protecting membrane degradation from oxidative damage. It might also be due to elimination of H2O2 content after P. indica colonization along with Cu in C. angustifolia seedlings. In the current study, Cu treatment alone significantly increased H2O2 content in C. angustifolia seedlings but colonization with P. indica significantly decreased H2O2 accumulation. Decrease in H2O2 content might be due to increased activities of CAT, APX and GPX. Similar results have also been reported in Arabidopsis thaliana seedlings (Camehl et al., 2011). Increased cell death is the result of oxidative stress induced membrane damage. So, stability of the membrane is necessary for normal physiological functions in the cell (Liu et al., 2014). In the present study, P. indica colonized C. angustifolia seedlings exhibited improved cell viability which might be due to decreased ROS accumulation and increased antioxidant activity. Reduced cell death in P. indica colonized seedlings might also be due to increased accumulation of proline and reduced lipid peroxidation which might have protected cell membrane under Cu stress in C. angustifolia seedlings. High proline accumulation is considered as one of the adaptive strategy of plants to survive under various stresses. Stress induced proline accumulation protects plants against heavy metal toxicity by protecting enzymatic components and it also removes H2O2 and maintains an high GSH/GSSG ratio (Anjum et al., 2014). Proline accumulation during osmotic stress is mainly due to increased synthesis and reduced degradation. Heavy metal induced plant water imbalance may also trigger the accumulation of proline (Chen et al., 2004). In the present study, proline content significantly increased in C. angustifolia seedlings after P. indica colonization. Mycorrhizal fungi have already been reported to improve accumulation of proline involved in protection of biomolecules against ROS damage (Talaat and Shawky, 2014). P. indica colonization also increased proline content in N. tobacum plants under Cd stress (Hui et al., 2015). Increased proline accumulation in C. angustifolia seedlings colonized with P. indica along with Cu clearly indicates their oxidative stress ameliorating potential. Heavy metals interfere with DNA and nuclear proteins which subsequently results in DNA damage and cell cycle alterations (Chang et al., 1996). In the present study, we observed that high concentration of Cu induced DNA damage might be due to DNA fragmentation (Gichner et al., 2005, 2006). High DNA damage under Cu stress can be explained by the increased ROS produced during oxidative stress or inactivation of proteins which are involved in DNA replication, transcription or repair processes (Kasprzak et al., 1999). In the present study, DNA damage significantly decreased in P. indica colonized seedlings as comet tails were shorter as compared to Cu alone which might be due to further increase in antioxidant enzymes involved in ROS detoxification. Copper directly affects the stability of protein leading to its turnover so control of protein homeostasis is essential for environmental acclimation of plants (Moore et al., 2016). Several proteins involved in antioxidant defence, superoxide dismutases, and glutathione-s-transferases were up regulated whereas proteins involved in protein synthesis and degradation were modulated in rice seedlings under Cu stress indicating oxidative stress (Chen et al., 2015). Mycorrhizal inoculation plays a very important part in preventing inhibition of protein synthesis by increasing nitrate assimilation in plants (Talaat and Shawky, 2014). P. indica is also reported to play a significant role in inducing antioxidant enzyme in colonized plants and conferring tolerance to heavy metal stress. Various proteins involved in oxidative stress defences were up regulated in barley plants colonized with P. indica which clearly indicates its involvement in inducing systemic response under drought stress (Ghabooli et al., 2013). Similar detoxification mechanism might be involved in C. angustifolia seedlings colonized with P. indica under Cu stress. Although, P. indica offer sustainable approach to combat heavy metal stress in C. angustifolia, other species of Cassia should also be

5. Conclusion Our findings concluded that colonization of plants with P. indica exerted a great ameliorating effect against Cu stress. Photosynthetic pigments were increased significantly after addition of P. indica. Increase in antioxidant activities of SOD, CAT, APX, GPX and GR suggest Cu induced oxidative stress in C. angustifolia seedlings. Decrease in lipid peroxidation and hydrogen peroxide content clearly indicates that colonization of P. indica resulted in successful reduction of ROS induced oxidative stress and providing stress tolerance against Cu stress. P. indica colonization in C. angustifolia seedlings significantly enhanced its Cu accumulation capacity which can be successfully utilized in phytostabilization purposes. Additionally, P. indica mediated amelioration of phytotoxicity and oxidative stress can be utilized in plants growing in Cu contaminated areas for increased stress tolerance and better performance. Symbiotic associations can offer better phytostabilization strategy for heavy metal contaminated areas and P. indica induced systemic response can be best utilized to combat heavy metal induced environmental degradation. Acknowledgements Authors are grateful to the University of Delhi, India for providing Research and Development Grant to VA. RN is indebted to University Grants Commission, New Delhi for the award of JRF and SRF. We are also grateful to Prof. Ajit Varma, Amity Institute of Microbial Technology, Noida, India for kindly providing the inoculum of Piriformospora indica. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2018.03.016. References Aebi, H., 1974. Catalase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, Academic Press Inc., New York, pp. 680. Alscher, R.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot. 5, 1331–1341. Anjum, N.A., Aref, I.M., Duarte, A.C., Pereira, E., Ahmad, I., Iqbal, M., 2014. Glutathione and proline can coordinately make plants withstand the joint attack of metal (loid) and salinity stresses. Front. Plant Sci. 5, 662. Anjum, N.A., Sharma, P., Gill, S.S., Hasanuzzaman, M., Khan, E.A., Kachhap, K., Mohamed, A.A., Thangavel, P., Devi, G.D., Vasudhevan, P., Sofo, A., Khan, N.A., Misra, A.N., Lukatkin, A.S., Singh, H.P., Pereira, E., Tuteja, N., 2016. Catalase and ascorbate peroxidase-representative H2O2-detoxifying heme enzymes in plants. Environ. Sci. Pollut. Res. 23, 19002–19029. Arnon, D., 1949. Copper enzymes isolated chloroplasts, polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Baltruschat, H., Fodor, J., Harrach, B.D., Niemczyk, E., Barna, B., Gullner, G., Janeczko, A., Kogel, K.H., Schäfer, P., Schwarczinger, I., Zuccaro, A., 2008. Salt tolerance of barley induced by the root endophyte Piriformospora indica is associated with a strong increase in antioxidants. New Phytol. 180, 501–510. Bates, L.E., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Beyer, W., Fridovich, I., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal. Biochem. 161, 559–566. Burkhead, J.L., Gogolin Reynolds, K.A., Abdel‐Ghany, S.E., Cohu, C.M., Pilon, M., 2009. Copper homeostasis. New Phytol. 182, 799–816. Camehl, I., Drzewiecki, C., Vadassery, J., Shahollari, B., Sherameti, I., Forzani, C., Munnik, T., Hirt, H., Oelmüller, R., 2011. The OXI1 kinase pathway mediates Piriformospora indica-induced growth promotion in Arabidopsis. PLoS Pathog. 7, e1002051. http://dx.doi.org/10.1371/journal.ppat.1002051. Chang, L.W., Magos, L., Suzuki, T. (Eds.), 1996. Toxicology of Metals. CRC Press, Boca Raton, FL, USA. Chen, C., Song, Y., Zhuang, K., Li, L., Xia, Y., Shen, Z., 2015. Proteomic analysis of copperbinding proteins in excess copper-stressed roots of two rice (Oryza sativa L.) varieties with different Cu tolerances. PLoS One 10, e0125367.

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