Arsenic accumulation in Canna: Effect on antioxidative defense system

Arsenic accumulation in Canna: Effect on antioxidative defense system

Applied Geochemistry 108 (2019) 104360 Contents lists available at ScienceDirect Applied Geochemistry journal homepage: www.elsevier.com/locate/apge...

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Applied Geochemistry 108 (2019) 104360

Contents lists available at ScienceDirect

Applied Geochemistry journal homepage: www.elsevier.com/locate/apgeochem

Arsenic accumulation in Canna: Effect on antioxidative defense system a,b

Ashish Praveen

c,∗

, Vimal Chandra Pandey , Sonali Mehrotra

a,d

, Nandita Singh

a

T

a

Plant Ecology and Environmental Science Division, National Botanical Research Institute, Lucknow, 226001, Uttar Pradesh, India Academy of Scientific and Innovative Research (AcSIR), New Delhi, India Department of Environmental Science, Babasaheb Bhimrao Ambedkar (Central)University, Lucknow, 226025, Uttar Pradesh, India d Department of Botany, Dolphin (PG) Institute, Manduwala, Uttarakhand Technical University, Dehradun, Uttarakhand, India b c

ARTICLE INFO

ABSTRACT

Keywords: Canna Arsenic Antioxidant Phytoremediation

Three Canna cultivars namely Canna red dazzler (CR), Canna flaccida (CF) and Canna cala lillies (CL) were selected to examine the uptake of arsenic and antioxidative response at four different arsenic concentrations (10, 20, 30 and 50 mg kg¯1). The study consisted of three harvestings at an interval of 45 days each, and at each harvest arsenic uptake and antioxidant activity were examined. The arsenic uptake and antioxidant activity were more in roots as compared to shoots. The percentage uptake of arsenate by CL, CF and CR at 10, 20, 30 and 50 mg kg¯1 were 52, 43, 36, 35 for CL, 39, 40, 35, 34 for CF and 45, 41, 37, 33 for CR respectively. The antioxidant enzymatic activity was higher in CL and CR than CF cultivar. The overall enzymatic antioxidants had their activities increased from first to second harvesting and were significantly higher in the third harvesting in all the three varieties as compared to control. Results suggested that Canna cultivars can be a better option for rhizofiltration or phytostabilization rather than phytoextraction and as it has an effective antioxidative mechanism in reducing the arsenic stress.

1. Introduction Heavy metal contamination is increasing due to various anthropogenic activities which results in increased heavy metals concentration to levels that are unsafe for human health. It delivers its effect not merely on human health, but also on soil and plant productivity. Not all the metals are all important, but they are toxic at higher concentrations as they cause oxidative stress by forming free radicals. Arsenic is a recognized carcinogen and mutagen, its elevated concentration in the environment is of outstanding concern for public health (Gress et al., 2015; Tóth et al., 2016; Yang et al., 2018; Rasheed et al., 2018). Arsenic contamination of soil can come as a consequence of both natural sources and anthropogenic activities, which admits the use of arsenical pesticides and herbicides, atmospheric deposition, mining activity, waste disposal, and other sources (Mandal and Suzuki, 2002). Coal fly ash disposal is also a potential source of arsenic contamination across the world (Pandey et al., 2011). Irrigation of agricultural land with arsenic contaminated soil or waste water, particularly in Bangladesh, India and other regions of Southeast Asia, causes accumulation of arsenic in both soil and plant, posing risk to soil ecosystem and human health (Meharg and Rahman, 2003, Tóth et al., 2016). There are various physicochemical technologies for remediation of arsenic-contaminated sites, but a holistic approach is needed in today's scenario. ∗

Pandey and Singh (2015) suggested a holistic approach for the utilization of arsenic-contaminated soils by the cultivation of aromatic plants along with economic return. Additionally, Pandey et al. (2015) proposed sustainable phytoremediation based on naturally colonizing, economically valuable and unpalatable plant species are indeed a safe and sustainable approach. Numerous plant species have been reported to hyperaccumulate metals i.e. Ni, Cu, Cd, As, Cr, Se etc. (Baker et al., 2000; Luongo and Ma, 2005). Pteris vittata L., is an arsenic hyperaccumulator (Ma et al., 2001); besides this other arsenic hyperaccumulating plant, have also been reported (Francesconi et al. 2002; Zhao et al., 2003) like Pityrogramma calomelanos in Hemionitidaceae (Francesconi et al. 2002) and other ferns of Pteridaceae. However, P. calomelanos is also incorporated in Pteridaceae as well. Not all but most of the reported As-accumulators belong to family Pteridaceae. Our hypothesis was to analyze whether Canna of Cannaceae has the hyperaccumulating efficiency for arsenic? In order to find this, Canna was selected for the study as it has good biomass and does not require much attention to flourish. Canna is known to accumulate arsenic in hydroponic study and wetlands but its uptake capacity in soil is a subject of study. Canna indica L. of family Cannaceae is known to accumulate dioxins, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) on its leaf surfaces and can remove Cu, Zn, insecticides, Ni, Mn, Pb (Bose et al., 2008; Sun et al., 2009; Yadav et al., 2010),

Corresponding author. E-mail address: [email protected] (V.C. Pandey).

https://doi.org/10.1016/j.apgeochem.2019.06.001 Received 27 December 2018; Received in revised form 9 March 2019; Accepted 1 June 2019 Available online 08 June 2019 0883-2927/ © 2019 Elsevier Ltd. All rights reserved.

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triazophos which is a pesticide (Cheng et al., 2007). Hydroponic study for arsenic phytoremediation by Canna showed more accumulation of arsenic in root as compared to shoots (Visoottiviseth and Aksorn, 2004), but remediation of arsenic from soil by Canna is a subject to study. The accumulator plants have evolved defence mechanisms to cope up with the oxidative stress caused due to accumulation of different contaminants. A possible mechanism provides a strong antioxidant defence system which can make a plant species more tolerant to metal stress (Srivastava et al., 2005; Tripathi et al., 2007; Dwivedi et al., 2010). The objective of this study was 1) to evaluate the arsenic uptake by Canna cultivars from soil 2) The antioxidant defence response in the cultivars 3) Whether Canna of Cannaceae can be an option for phytoremediation of arsenic from soil in severely affected areas?

sample (leaf and roots) were homogenized in 5% TCA and centrifuged at 10,000×g for 10 min at 4 °C. After this supernatant was collected, taken in Eppendorf tubes and to it TBA-TCA mixture (0.5% TBA added to 20% TCA) was added and then the mixture was heated at 95 °C for 30 min and quickly cooled in ice. MDA was calculated from the difference in absorbance measured at 532 nm and 600 nm with extinction coefficient of 155 mM−1 cm−1 (Unit: nmol¯1 cm3). 2.6. Superoxide dismutase (SOD) EC 1.15.1.1 Assay for superoxide dismutase was performed in terms of SOD's ability to inhibit reduction of nitroblue tetrazolium (NBT) to form formazan by superoxide (Beyer and Fridivich, 1987). Plant tissue was homogenized in phosphate buffer (pH 7.5), 1 mM ethylenediaminetetraacetic acid and 2% (w/v) polyvinylpyrrolidone (PVP) in a chilled motor and pestle. The homogenate was centrifuged at 14,000×g for 10 min at 4 °C and supernatant separated and applied for the assay. The assay mixture consisted of phosphate buffer (pH 7.8), L-methionine, NBT, Triton X-100 and riboflavin. The photoreduction of NBT (formation of purple formazan) was measured at 560 nm (Unit: mg¯1 protein).

2. Material and methods 2.1. Plant material Rhizomes with plantlets of three cultivars of Canna sp. (Canna red dazzeler, Canna flaccida, Canna cala lillies) were collected from CSIRNBRI Lucknow.

2.7. Catalase (CAT) EC 1.11.1.6

2.2. Soil treatment and plantation

For the estimation of CAT plant sample (0.5 g) were homogenized in 5 ml containing 50 mM phosphate buffer (pH 7.0) and 1 mM dithiothreitol (DTT). CAT activity was assayed by a decrease in absorbance at 240 nm as H2O2 was consumed. The reaction mixture consisted of 50 mM phosphate buffer (pH 7.0) and enzyme extract. The reaction was initiated by adding H2O2 and rate of decomposition of H2O2 was measured at 240 nm (Del Rio et al., 1977) (Unit: nmol min¯1 mg¯1 protein).

For the experiment pots of about 8 inches in size filled with 6 kg of garden soil were taken. Arsenic as sodium arsenate was applied at concentrations of 10, 20, 30 and 50 mg kg−1 and garden soil as such was taken as control. For the three cultivars, 150 pots with ten pots each for 10, 20, 30 and 50 mg kg−1, the rest ten without any treatment (i.e. garden soil as such) in triplicate was maintained. The pots were kept on a tray so that after watering the arsenic leached with water in the tray is again filled in pots. The setup was left in the net house (temperature and relative humidity during experiment ranged from 30 °C to 38 °C and 55–75%, respectively) and water was given in the pots daily in the evening. Watering was done in such a manner that it can only wet the soil. Before plantation, soil was collected from the pots for arsenic estimation. Rhizomes of the plant survive very long and new plantlets come out from it. The older plant dies off before the rainy season and has stunted plant growth in winter season. The experiment was started during the rainy season in the month of July and was harvested in the month of November before the winter. Harvesting was done after an interval of 45 days each so that the final harvesting is finished before winter.

2.8. Glutathione reductase (GR) EC 1.6.4.2 To estimate GR plant tissue was homogenized in 0.1 M phosphate buffer pH 7.5 that contained 0.5 mM EDTA, then centrifuged at 20,000×g for 15 min at 4 °C.The reaction mixture consisted of 0.2M phosphate buffer pH 7.5 containing 1 mM EDTA, 3 mM DTNB, 2 mM NADPH and 20 mM of GSSG. The Reaction was initiated by NADPH and extinction coefficient used was 6.2 mM−1cm−1. The activity of GR was monitored by observing the increase in absorbance at 412 nm when DTNB was reduced by glutathione (GSH) to form the 5-Thio-2-nitrobenzoic acid (TNB) (Smith et al., 1988) (Unit: mg¯1 protein).

2.3. Chlorophyll determination

2.9. Glutathione determination

For total chlorophyll estimation SPAD-502 (Konica- Minolta, Osaka, Japan) was used before harvesting on three randomly picked out plants from each treatment and control (Unit: SPAD units).

The total glutathione level was done using the method of (Gosset et al., 1994), where two solutions were prepared, solution A consisted of 110 mM Na2PO4.7H2O, 40 mM NaH2PO4.H2O, 15 mM EDTA, 0.3 mM 5, 5-dithiobis-(2-nitrobenzoic acid) (DTNB) and 0.4 ml l.1 BSA and solution B consisted of 1 mM EDTA, 50 mM imidazole, 0.2 ml l.1 BSA and an equivalent of 1.5 units of glutathione reductase (GR). Total glutathione was measured in a reaction mixture consisting of 400 ml of solution A, 320 ml of solution B, 400 ml of a 1:50 dilution of the extract in 5% (w/v) Na2HPO4 (pH 7.5) and 80 ml of 3 mM NADPH. The reaction rate was measured spectrophotometrically by following the change in absorbance at 412 nm for 4 min (Unit: micromolgm¯1fresh weight).

2.4. Extraction and enzyme assay A part of the harvested plant samples was frozen in liquid nitrogen and stored at −80 °C for enzymatic analysis. The remainder of the plant samples were oven dried at −65 °C in hot air oven for digestion and arsenic analysis. Extraction of the enzyme of roots and shoots was done in 4 °C in triplicate by crushing the tissues in pre-chilled mortar and pestle from the samples that were stored in −80 °C. All biochemical estimations were performed using UV–Vis Spectrophotometer (Lambda 6 UV-vi's; Perkin-Elmer, Beaconsfield, UK).

2.10. Ascorbate concentration Total ascorbate was determined by the method of Hodges et al. (1996). Plant tissue was homogenized in 10% TCA and 50 mM phosphate buffer (pH 7.4) which contained 3 mM EDTA and 1 mM DTT. For assay of total ascorbate, DTT was used and the extract was centrifuged at 14,000×g for 15 transactions. Now the aliquot was incubated at 25 °C for 10 min. To the aliquot was added N-ethylmaleimide (0.5% w/

2.5. Lipid peroxidation determination Lipid peroxidation in plant tissues was assayed by the TBA method (Heath and Packer, 1968), where the final decay product of lipid peroxidation malondialdehyde (MDA) was utilized as an indicator. Plant 2

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Table 1 Showing arsenic concentration in soil, arsenic uptake in leaf and root grown in 0 (control), 10, 20, 30 and 50 ppm of arsenic amended soil after different harvests (0, 45 and 90) where H denotes harvesting. All values are mean of three replicates ± S.D. CF = Canna flaccida, CL = Canna catellia, CR = Canna red dazzler. Means followed by the same letter were not significantly different at p < 0.05 within cultivars at harvestings according to Tukey's test, numeral 1 and 2 being used with letters to differentiate the class between harvestings. Arsenic in mg/kg

CL

CF

CR

0 10 20 30 50 0 10 20 30 50 0 10 20 30 50

Arsenic Soil (mg/kg)

Arsenic Leaf (mg/kg)

Arsenic root (mg/kg) 1st H

0 day

1st H

2nd H

1st H

0.77 ± 0.10 e3 8.86 ± 0.61 d3 18.76 ± 1.57 c3 28.19 ± 3.43 b3 45.76 ± 5.10 a3 0.95 ± 0.26 e3 8.61 ± 0.19 d3 17.63 ± 0.54 c3 28.27 ± 3.36 b3 46.48 ± 3.87 a3 0.84 ± 0.17 e3 8.81 ± 0.30 d3 18.63 ± 1.04 c3 27.80 ± 2.56 b3 47.68 ± 3.53 a3

0.73 ± 0.24e2 6.80 ± 1.16d2 14.41 ± 1.60c2 22.27 ± 1.88b2 34.30 ± 3.26a2 0.87 ± 0.07 e2 7.13 ± 1.34 d2 14.69 ± 2.82 c2 23.05 ± 1.38 b2 36.08 ± 2.92 a2 0.73 ± 0.10 e2 6.52 ± 1.25 d2 14.42 ± 1.86 c2 21.70 ± 2.47 b2 37.56 ± 2.26 a2

0.72 ± 0.19e1 4.31 ± 0.88d1e1 10.11 ± 1.63b1c1 14.77 ± 2.69b1 22.13 ± 2.89a1 0.84 ± 0.10 e1 5.11 ± 0.90 d1e1 10.28 ± 1.69b1c1 15.45 ± 2.96b1 25.19 ± 2.63a1 0.65 ± 0.13 e1 4.07 ± 1.18 d1e1 9.71 ± 1.17b1c1d1 13.89 ± 2.55b1 24.08 ± 2.73a1

0.04 0.61 1.28 1.96 3.13 0.05 0.56 1.24 1.72 3.04 0.04 0.58 1.23 1.93 3.15

v), 0.61M TCA, 0.8M H3PO4, 65 mM α-α-bipyridil and 110 mM ferric chloride. The mixture was then incubated in a water bath at 55 °C for 10 min and absorbance was recorded at 525 nm (Unit: micromolmg¯1protein).

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2nd H 0.01d 0.08cd 0.14bc 0.37b 0.42a 0.01d 0.13cd 0.23bc 0.56b 0.85a 0.01d 0.03cd 0.13bc 0.43b 0.54a

0.07 0.86 1.62 2.96 4.22 0.09 0.81 1.57 2.63 4.17 0.08 0.83 1.52 2.90 4.30

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02d1 0.07c1d1 0.43b1c1 0.82a1b1 0.60a1 0.01d1 0.09 c1d1 0.32b1c1d1 0.48b1 0.90a1 0.01d1 0.12 c1d1 0.13b1c1d1 0.84a1b1 1.11a1

0.05 1.18 3.11 3.98 6.01 0.05 1.10 2.23 3.45 5.64 0.04 1.27 3.38 4.09 6.17

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2nd H 0.01e 0.26de 0.92cd 0.52bc 0.73 ab 0.01ef 0.23de 0.60cd 0.77c 0.74 ab 0.008e 0.33de 0.71c 1.15bc 1.27a

0.08 1.80 3.52 4.99 7.47 0.11 1.69 3.03 4.89 6.70 0.08 1.71 3.55 5.12 7.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02g1 0.46e1 0.43c1d1 0.75c1 0.77a1 0.02g1 0.13e1f1 0.42d1e1 0.27c1 0.68a1b1 0.01g1 0.11e1f1 0.78c1d1 1.11b1c1 0.56a1

(Supporting information Tables SI–S1). 3.2. Arsenic in soil The water used had 0.00085 μg l¯1 of arsenic, thus negating the external arsenic addition. The concentration of arsenic in the soil was found to decrease after each harvest (Table 1 and Fig. 1A). The soil arsenic concentration after 3rd harvest in pots at 10, 20, 30 and 50 mg kg¯1 for Cl was 3.77, 9.81, 17.19 and 28.41, for CF was 4.15, 10.74, 17.52 and 30.26, for CR was 3.65, 10.04, 16.87 and 29.19 mg kg¯1 respectively. So the percent removal of arsenic at 10, 20, 30 and 50 mg kg¯1 for Cl was 63, 51, 43 and 43, for CF was 59, 46, 42 and 40, for CR was 63, 51, 42 and 42 respectively (Fig. 2).

2.11. Arsenic determination and arsenic concentration ratio (ACR) Oven dried plant and soil samples 0.1 g were taken and mixed with 5:1 nitric acid HNO3 (60%) and hydrofluoric acid (HF, 40%) and digested in the hot block digester (Kjeldahl digestion Unit) for 6 h. Arsenic was determined in plant sample and soil on ICP-MS (Agilent technologies 7500cx). Soil samples were taken from a depth of 5 cms in all the three harvests, but after final harvest soil from pot was mixed and then sampling was performed to know the arsenic remaining in the soil. The arsenic concentration ratio was calculated as the ratio of the shoot to root arsenic concentration.

3.3. Percentage accumulation of arsenic by Canna cultivars Canna is a fast growing plant with a good shoot and root biomass. It sustains an extended root system. Percentage total arsenic accumulation by the cultivars after 135 days (3rd H i.e. final harvesting) of harvesting at 10, 20, 30 and 50 mg kg¯1 were 52, 43, 36 and 35 for CL, for CF it was 39, 40, 35 and 34, and in CR it was 45, 41, 37 and 33 respectively. (Fig. 1D, E, F).

2.12. Statistical analysis The data represent means from three replicate pots and was analyzed employing SPSS 17 for analysis of variance (ANOVA) using Tukey's test and was compared for changes at P ≤ 0.05 between cultivars in each harvest.

3.4. Arsenic accumulation and arsenic accumulation ratio

3. Results

The arsenic accumulation in root and shoot of Canna was found to show variation, with roots showing more accumulation compared to shoots (Table 1). In roots at 50, 30, 20 and 10 mg kg¯1 the arsenic concentration in the final harvesting of CL was 9.89, 6.12, 5.24 and 2.82 mg kg¯1, CF was 9.83, 6.00, 4.33 and 2.25 mg kg¯1, CR was 10.04, 6.16, 5.01 and 2.74 mg kg¯1 whereas in shoot it was 5.46, 3.86, 2.71 for CL and 1.27 mgkg¯1 for CF, 5.39, 3.82, 2.56 and 1.10 mg kg¯1 for CR, 5.45, 3.83, 2.68 and 1.22 mgkg¯1 respectively (Fig. 1B). The arsenic accumulated by the three cultivars was relatively higher in 3rd harvesting in the order 3rd > 2nd > 1st and also the plant biomass was of the same order (Table 2). The percentage of total arsenic uptake by the three cultivars decreased with increasing arsenic concentration. The percentage uptake by CL at 10, 20, 30 and 50 mgkg¯1 were 52, 43, 36 and 35, for CF it was 39, 40, 35 and 34, for CR it was 45, 41, 37 and 33 respectively (Fig. 1D, E, F). The percentage uptake for both shoot and root were calculated from the initial and final values and dry weight of the plant parts and soil respectively. For obtaining total arsenic uptake by plant the percentage accumulation of both shoot and root were

The study showed varied antioxidant response and arsenic uptake in the three cultivars of Canna that were selected for the study. The overall antioxidative enzymatic response and arsenic uptake were found enhanced in all the three cultivars with arsenic exposure in different harvestings while it was comparatively higher in 3rd harvesting, discussed in sections below. 3.1. Morphological study During the test period in all the three cultivars of Canna, the leaf and root did not show any morphological symptoms of toxicity as compared to the control. There were no injury symptoms in plants and were healthy like control. All the three cultivars of Canna (CL, CF and CR) which were chosen for the study did not indicate any substantial differences (P < 0.05) in their root and shoot length as well as total chlorophyll content at different concentrations of arsenic (10, 20, 30 and 50 mg kgˉ1) in each harvest as compared to their control 3

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Fig. 1. The arsenic concentration in soil at 0 days and after third harvesting (A), arsenic accumulation in leaf and root (B) after third harvesting, arsenic concentration ratio (C) and percentage removal (D, E, F) of arsenic in Canna cultivars after three harvestings.

added. The arsenic concentration ratio (ACR) was calculated as the ratio of the shoot to root arsenic concentration. In our study, the values of ACR obtained were low for all the three cultivars (Fig. 1C).

with highest activity in final harvesting (Table 3 and Table SI-S2, S3, S4). In all the cultivars enhanced antioxidant activity was observed in respective harvestings and at all treatments, but was higher at 50 mg kg¯1 arsenic. Lipid peroxidation was significantly increased in CR, CF and CL in all four concentrations in root and shoot in the three harvestings compared to their controls (Table 3 and Table SI-S2, S3) but an enhanced response was seen in CR and CL. MDA, a product of lipid peroxidation is used as an index for stress and it was found to be enhanced in the three cultivars at respective harvestings. Substantial

3.5. Biochemical activity The biochemical study included the antioxidative responses in all the cultivars. The responses in the biochemical parameters due to oxidative stress and antioxidative defence showed variation in cultivars 4

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(Table 3 and Table SI-S2, S3, S4). It was observed that the ascorbate concentration increased with arsenic concentration (10, 20, 30 and 50 mg kg¯1) and is also associated with the activities of APX and GR, which were also heightened. The concentration of total glutathione was enhanced in the three cultivars at all the harvests of both root and shoots, but more into the roots. Total glutathione was found to increase in successive harvestings with enhanced activity in 3rd harvesting (Tables SI–S4). The concentration of GR also increased in the three successive harvestings at all concentrations in both root and shoot (Table 3 and Table SI-S2, S3). In our experiment of Canna cultivars, we also detected AR activity in roots and its activity increased with arsenic concentration and duration (Table .3 and Tables SI–S2). 4. Discussion The present situation of contamination in soil and water all over the world requires immediate action as it has its impact on the health of human beings. The situation becomes grim when the contamination reaches unaffected parts of the world through crops grown in contaminated areas with its impact on human health (Rasheed et al., 2018). The present work was framed to reduce the contamination through environment friendly method of phytoremediation with Canna plant and to an extent we were successful in achieving it. The three cultivars of Canna were healthy and did not demonstrate any substantial differences in plant height, root length, and total chlorophyll content compared to their controls. However, the roots became brownish in arsenic treated plants while in control it was pale yellow in colour, this may be due to accumulation of arsenic in the roots and plasmolysis of roots (Marin et al., 1992). Likewise, in case of chlorophyll no visual symptoms of arsenic toxicity were found, the leaves were green, which indicated that no arsenic induced stress was observed (Mysliwa-Krdziel et al., 2004). So from the morphological study, it can be concluded that the given concentrations of arsenic did not cause any toxic effect on Canna. Arsenic uptake in roots and its translocation to shoots varied in the three Canna cultivars where uptake was more in roots compared to shoots. This indicated that there was a limited mobility in the plant which was also observed by Outridge and Noller (1991) who concluded that the concentrations of heavy metals in roots of freshwater macrophytes were higher. Jomjun et al. (2011) also found that Canna glauca had higher quantity of arsenic in its lower part (rhizomes and roots). They also noted a high arsenic removal rate as well as high arsenic accumulation in Canna glauca from submerged soil.

Fig. 2. Reduction in soil arsenic by the three cultivars of Canna at different concentrations (10, 20, 30 and 50 mg kg−1) after final harvestings.

differences in antioxidative enzymatic response to arsenic were seen in the three cultivars. In this experiment, it was observed that by application of varied concentration of arsenic on Canna cultivars the activity of superoxide dismutase was enhanced compared to control and with increased duration (45, 90 and 135 days) showing maximum activity after 135 days (final harvesting) which indicated oxidative stress caused by arsenic (Table 3 and Table SI-S2, S3). Catalase (CAT) was also observed to have increased activity in root and shoot in the three cultivars respectively, with CF showing decreased activity in comparison to CL and CR (Table .3 and Table SI-S2, S3). In addition to these other enzymes were also activated. Ascorbate peroxidase (APX) an important antioxidant for oxidative defence which showed differences in activity in three harvestings, a prominent increase was observed from first to second harvesting in both root and shoot (Table SI-S2 and S3). The present work suggests a twofold enhancement in APX activity in Canna cultivars in arsenic treated pots in second harvesting as compared to first harvesting. Total ascorbate concentration in all the cultivars showed an increment after each harvest and was more in the third harvest and at 50 mg kg¯1 concentration (Tables SI–S4). Our results demonstrated an enhanced APX and ascorbate in all the three cultivars, both in roots and shoots with a sudden increase after first harvesting and a sharp increase from second to third harvesting

Table 2 Root and Leaf dry weight (in grams) of the three Canna cultivars grown in 0(control), 10,20,30 and 50 ppm of arsenic amended soil after different harvests (45, 90 and 135 days) where H denotes harvesting. All values are mean of three replicates ± S.D. CF = Canna flaccida, CL = Canna catellia, CR = Canna red dazzler. Means followed by the same letter were not significantly different at P < 0.05 within cultivars at harvestings according to Tukey's test, numeral 1 and 2 being used to differentiate the class between harvestings. Arsenic in mg/kg

Root (Dry weight in gm) 1st H

CL

CF

CR

0 10 20 30 50 0 10 20 30 50 0 10 20 30 50

1.90 1.67 1.51 1.55 1.84 1.83 1.92 1.81 1.67 1.66 1.85 1.70 1.71 1.65 1.65

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Leaf (Dry weight in gm)

2nd H 0.32a 0.84a 0.24a 0.20a 0.92a 0.47a 0.90a 0.61a 1.78a 1.64a 0.37a 0.86a 0.54a 0.64a 0.72a

3.01 2.53 2.15 2.35 2.64 2.76 2.65 2.37 2.53 2.66 2.99 2.50 2.55 2.64 2.87

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3rd H 0.82a1 0.26a1 0.51a1 0.40a1 0.43a1 0.74a1 0.34a1 0.65a1 0.43a1 0.56a1 0.71a1 0.40a1 0.57a1 0.45a1 0.53a1

6.51 5.92 5.81 5.72 5.80 6.07 5.88 5.92 5.84 5.88 6.61 5.85 5.77 5.90 5.81

5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1st H 0.40a2 0.83a2 0.88a2 0.74a2 0.84a2 0.45a2 0.81a2 0.70a2 0.72a2 0.81a2 0.50a2 0.80a2 0.91a2 0.70a2 0.89a2

1.76 1.69 1.73 1.67 1.63 1.94 1.60 1.74 1.85 1.73 1.68 1.84 1.80 1.83 1.84

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2nd H 0.40a 0.28a 0.31a 0.60a 0.45a 0.70a 0.52a .054 a 0.63a 0.74a 0.70a 0.86a 0.51a 0.68a 0.71a

4.62 4.25 4.50 4.42 4.50 4.28 4.23 3.98 4.12 4.25 4.52 4.35 4.40 4.38 4.52

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3rd H 0.34 a1 0.55a1 0.50a1 0.73a1 0.68a1 0.82a1 0.77a1 0.62a1 0.56a1 0.65a1 0.44a1 0.55a1 0.60a1 0.72a1 0.70a1

6.88 6.38 6.45 6.52 6.73 6.92 6.37 6.44 6.50 6.70 6.87 6.37 6.44 6.53 6.73

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2a2 0.3a2 0.1a2 0.7a2 0.5a2 0.2a2 0.2a2 0.1a2 0.3a2 0.6a2 0.1a2 0.5a2 0.7a2 0.3a2 0.6a2

6

CR

CF

CL

Arsenic in mg kg−1

CR

CF

CL

Arsenic in mg kg−1

25.32 ± 4.34 d2 69.75 ± 1.55c2 77.50 ± 3.10c2 90.42 ± 4.73b2 127.10 ± 2.68a2 24.28 ± 3.22 d2 69.23 ± 3.57c2 76.98 ± 3.22c2 90.42 ± 2.36b2 125.55 ± 4.65a2 28.42 ± 5.44 d2 69.75 ± 1.55c2 77.50 ± 3.10c2 89.38 ± 3.22b2 127.62 ± 3.90a2

3 H 0.08 0.38 0.45 0.52 0.73 0.09 0.37 0.44 0.50 0.70 0.07 0.37 0.44 0.53 0.73

3 H 8.88 ± 0.17e2 28.45 ± 0.82d2 35.46 ± 1.28c2 42.50 ± 1.23b2 62.01 ± 3.03a2 8.85 ± 0.32e2 27.93 ± 2.90d2 35.00 ± 1.24c2 41.73 ± 1.97b2 58.51 ± 3.51a2 9.04 ± 0.31e2 28.88 ± 0.86d2 36.72 ± 1.06b2 c2 41.46 ± 1.76b2 61.50 ± 3.84a2

3 H

rd

rd

SOD

AR

rd

0.002d2 0.03c2 0.01b2c2 0.07b2 0.05a2 0.002d2 0.02c2 0.01b2c2 0.03b2c2 0.06a2 0.001d2 0.05c2 0.07b2c2 0.03b2 0.06a2

1.32g2 2.84e2f2 3.24c2d2e2 5.20b2c2 5.92a2 0.47g2 3.32f2 3.31d2e2f2 3.78c2d2 4.64a2b2 0.37g2 3.30e2f2 2.54d2e2 4.24b2c2 5.32a2

MDA

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Leaf

12.10 41.19 50.51 60.55 74.84 10.13 37.92 48.81 56.47 70.66 12.05 41.10 49.11 60.45 75.65

Root

0 10 20 30 50 0 10 20 30 50 0 10 20 30 50

1.80 ± 0.35e2 13.16 ± 0.67d2 21.04 ± 0.87c2 31.40 ± 0.71b2 52.87 ± 3.23a2 1.92 ± 0.26e2 12.00 ± 2.07d2 19.84 ± 0.36c2 29.87 ± 2.50b2 49.11 ± 3.36a2 1.40 ± 0.15e2 13.37 ± 0.34d2 20.75 ± 1.76c2 31.15 ± 0.70b2 52.68 ± 1.62a2

3 H

rd

CAT

3.01 ± 0.82f2 21.53 ± 0.26e2 32.15 ± 20.01d2 42.35 ± 2.40b2 66.64 ± 1.43a2 3.46 ± 0.34f2 18.65 ± 1.34e2 31.07 ± 0.65d2 39.33 ± 1.43b2c2 60.26 ± 2.76a2 2.99 ± 0.71f2 21.10 ± 2.65e2 35.55 ± 2.57c2d2 42.04 ± 3.75b2 66.87 ± 4.53a2

3 H

rd

rd

3 H

CAT

SOD

Root

34.52 ± 2.98h2 104.35 ± 5.57f2 148.41 ± 9.85d2 173.16 ± 4.95c2 212.78 ± 8.01a2 33.90 ± 3.37h2 89.70 ± 9.37g2 123.44 ± 9.45e2 140.78 ± 4.75d2 171.30 ± 8.82c2 27.54 ± 2.01h2 103.91 ± 10.81f2 150.04 ± 8.27d2 177.30 ± 8.56c2 199.75 ± 10.12b2

3 H

rd

APX

14.29 41.50 49.87 61.75 94.55 14.90 38.22 47.95 59.41 94.29 14.57 40.18 49.79 61.38 93.36

3 H

rd

GR

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

35.51 ± 2.40g2 138.92 ± 7.83f2 180.01 ± 7.48d2e2 197.61 ± 6.54c2d2 242.20 ± 9.74a2 31.37 ± 3.45g2 115.38 ± 11.31f2 167.52 ± 9.70e2 178.14 ± 1.72d2e2 216.42 ± 16.61b2c2 34.61 ± 4.10g2 127.85 ± 5.80f2 177.17 ± 2.91d2e2 200.90 ± 5.10c2d2 239.31 ± 11.89a2b2

3 H

rd

APX

0.13f2 1.33e2 1.70c2d2e2 3.90b2 8.72a2 0.20f2 1.38e2 1.58 d2e2 3.31b2c2d2 6.09a2 0.22f2 1.95e2 2.75c2d2e2 44.58 b2c2 6.78a2

23.76 50.63 58.90 65.62 86.80 23.77 49.60 57.35 64.58 85.25 24.80 51.66 59.42 65.10 84.22

3rd H

MDA

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.22e2 4.22c2d2 2.68b2c2 1.79 b2 3.10 a2 1.78e2 4.10d2 3.10b2c2d2 3.23 b2 2.68 a2 2.68e2 1.89c2d2 4.98b2c2 4.65 b2 2.36 a2

17.76 ± 0.40e2 47.09 ± 2.28d2 56.73 ± 3.31b2c2d2 67.13 ± 3.60b2 98.63 ± 7.45a2 17.94 ± 3.10e2 47.60 ± 2.32d2 54.94 ± 3.14 c2d2 66.85 ± 3.13b2 98.93 ± 4.34a2 15.68 ± 2.70e2 47.84 ± 1.96d2 57.40 ± 4.51b2c2d2 66.43 ± 4.28b2c2 100.84 ± 7.01a2

3rdH

GR

Table 3 Showing antioxidative enzyme activity in Canna cultivars, grown in 0(control), 10, 20, 30 and 50 ppm of arsenic amended soil after 3rd harvesting (where H denotes harvesting). All values are mean of three replicates ± S.D. CF = Canna flaccida, CL = Canna catellia, CR = Canna red dazzler. Means followed by the same letter were not significantly different at p < 0.05 within cultivars at harvestings according to Tukey's test, numeral 1 and 2 being used with letters to differentiate the class between harvestings. Superoxide dismutase (SOD), Catalase (CAT), Ascorbate peroxidise (APX), Glutathione reductase (GR), Malondialdehyde (MDA), Ascorbate reductase (AR).

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Among the three cultivars there were substantial differences in arsenic uptake in roots, Canna calla lilies (CL) and Canna red dazzler (CR) had more uptake of arsenic in roots as compared to Canna flaccida (CF) in all the three harvestings. As well, more uptakes by root in Canna indica L. have been observed in the hydroponic study by Visoottiviseth and Aksorn (2004). The arsenic accumulated by the three cultivars was relatively higher in 3rd harvestings compared to 1st and 2nd harvestings. This higher accumulation can be a result of the longer duration for which the plant was left exposed (135 days) and also due to the higher biomass of both root and shoot. The arsenic accumulation was also higher in 2nd harvesting compared to 1st harvesting and we found that the plant biomass was enhanced in 2nd harvesting from 1st harvesting. Thus, an increase in biomass could be the reason for higher metal accumulation in successive harvestings which has been noted in the results. The arsenic concentration ratio (ACR) indicates the transport and mobility of arsenic from root to shoot in the plant. The movement of arsenic in the upper parts (shoot) was less and it can be an avoidance method by the plant to limit the upward movement and therefore accumulated in the root. The removal of arsenic from soil also varied with concentration, results showed that at a lower concentration of arsenic the removal percentage was more in all the three cultivars. Similar outcomes were likewise obtained in P. vittata, A. capillus veneris and P. karka by Raj and Singh (2015), which showed more accumulation at a lesser concentration in soil. Therefore in all the three cultivars the percentage arsenic removal from soil at given four concentrations can be generalized in order 10 > 20 > 30 > 50 mg kg¯1. Plants have defence mechanisms to combat oxidative damage due to metal toxicity (Gratao et al., 2005). It may tolerate the metal toxicity but to some extent and if the metal is in excess then an important defence mechanism operates which constitutes the release of the antioxidants to combat the increased ROS production caused due to metal toxicity. ROS are not only produced during stress, rather it is also formed at a basal level, but its activity increases during stress. When there is metal toxicity then enhancement in the generation of reactive oxygen species (ROS) occur which damages membrane lipid by peroxidation. It has been described in the case of Co, Cu, Cd, As and various others (Li et al., 2005, Mazhaudi et al., 1997, Lombardi and Sebastiani, 2005; Dey et al., 2007, Singh and Ma, 2006). Normally the ROS are removed by the plants, but when formed in excess then enzymatic or non-enzymatic antioxidants are produced to quench them. In Canna cultivars lipid peroxidation increased with arsenic concentration (10, 20, 30 and 50 mgkg¯1) and duration, with more peroxidation in the case of root and in the final harvesting (i.e. 3rd harvesting). This may be due to differing metal accumulation in plant parts with root accumulating more arsenic than shoot and in this experiment root accumulated more arsenic than shoot in all the three harvestings. SODs constitute the first line of defence against ROS (Alscher et al., 2002). The activity of superoxide dismutase (SOD) in the cultivars increased in first and second harvestings (Supporting information Table SI-S2 and S3) but prominent enhancement was seen in final harvesting in both roots and shoot with roots responding more which may be due to long exposure to arsenic and more accumulation of arsenic by the root at the end of final harvesting (Table 3). It was noted that CR and CL had enhanced activity as compared to CF, the activity increased with increasing concentration of arsenic in all the three cultivars. Enhanced SOD was also found in red clover plant with arsenic concentration (Mascher et al., 2002). In addition to SOD, catalase activity was also found to be enhanced in all the three cultivars. CAT is the enzyme which works without any reducing agent and breaks down H2O2 one of the products of SOD (Asada, 1992) into H2O and O2 (Lin and Kao, 2000). The enhanced activity of SOD and CAT in all the studied cultivars infers that the plant responded to stress in a positive way by controlling the level of ROS and thus decreased oxidative damage (Miller et al., 2008). APX plays one of the important functions in the metabolism of H2O2 in plants. APX activity increased in all the three cultivars with arsenic concentration and duration for which it was

exposed to arsenic while the activity was more in the roots which may be due to more accumulation of arsenic in the roots. CL and CR showed increased activity as compared to CF. The AsA-GSH cycle prevents active accumulation of oxidants in cells where APX utilizes the AsA in the cycle and prevents H2O2 toxicity (Asada, 1992, 1997). It has been observed that generally APX activity increases along with other enzymes like SOD, CAT, and GSH (Shigeoka et al., 2002). Ascorbate acts as an antioxidant by removing hydrogen peroxide in PSI also known as Mehler reaction and APX plays a key part in this by scavenging the hydrogen peroxide as soon as it is formed (Asada, 2006). AsA plays two roles, first, it is a substrate for APX and second it can scavenge singlet oxygen, hydroxyl radicals and superoxides directly (Noctor, 2006). Therefore as is found in other abiotic stresses heavy metal induced oxidative stress also triggers the AsA-GSH cycle for H2O2 detoxification (Foyer and Nocter, 2005). Glutathione contains non-protein reduced sulphur and plays an important role in cellular defence by detoxifying the H2O2 in AsA-GSH cycle. The total glutathione was found enhanced in the cultivars. As discussed that there were no morphological symptoms of toxicity in any part of the plant, which may be ascribable to the active functioning of the antioxidants. One of the important reductase activities which reduce arsenate to arsenite in P. vittata, bacteria, and fungi (Shi et al., 1999; Duan et al., 2005) is done by arsenate reductase (AR). The activity of AR has found to be confined in roots of P. vittata although there has been some controversy on the site of arsenate reduction in P. vittata (Enxie et al., 2009) but it was found to function in roots of P. vittata showing that the reduction occurs only in roots (Duan et al., 2005). The activity of antioxidants was enhanced in CL and CR as compared to CF in all the harvestings and at all concentrations, but no significant difference in the morphological parameters was observed in the cultivars, this can be due to relatively less arsenic accumulation by CF. 5. Conclusion From the study, we can conclude that Canna accumulates arsenic and has an effective antioxidative system. The activity of SOD, CAT, APX, GR, MDA, AR, total glutathione, and total ascorbate were found to be enhanced in all the three cultivars in successive harvestings as compared to control plants which enables plant to cope up with the arsenic stress. The accumulation of arsenic is more in roots compared to shoots. This varied accumulation in root and shoot can be attributed to the extensive root system and also less mobility of the arsenic from root to shoot. On the basis of arsenic uptake in roots, shoots and percentage removal of arsenic from the soil by the three selected Canna cultivars, the cultivars can be inferred in the order CL > CR > CF. Results suggested that Canna cultivars can be suitable for rhizofiltration or phytostabilization rather than for phytoextraction. According to Dushenkov and Kapulnik (2000), plants to be selected for rhizofiltration should fulfil two important measures; first it should tolerate and accumulate the target metal significantly. Berti and Cunningham (2000) have contributed a comprehensive explanation of phytostabilization. Phytostabilizers should have a poor translocation of contaminants to the aboveground plant part as it possesses its own benefit i.e. if the shoot is consumed it does not present a danger. One significant detail to note is that this technology eliminates the need to harvest the shoot as a hazardous waste. The second point is that the plant should be easy to grow and possess an intense root system. Canna delivers a very intensive root system and also good canopy. All the three Canna cultivars have shown accumulation of arsenic both in roots and shoots although with reduced quantity in shoots compared to roots and with no toxicity symptoms at four concentrations. This meant it was capable to tolerate the given concentration of arsenic and can be a good alternative for long term arsenic removal from soil. It does not need any addition of nutrients as well as flourishes well without any extra care. 7

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AP is thankful to Council of Scientific and Industrial Research, Government of India for providing fellowship and CSIR-National Botanical Research Institute where the study was performed. Authors also thank director of CSIR-National Botanical Research Institute for providing facilities and Council of Scientific and Industrial Research for providing the funds (P-81-101). Financial assistance given to Dr. V.C. Pandey under the Scientist's Pool Scheme (Pool No. 13 (8931-A)/2017) by the Council of Scientific and Industrial Research, Government of India is gratefully acknowledged. The authors declare no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apgeochem.2019.06.001. References Sun, L., Liu, Y., Jin, H., 2009. Nitrogen removal from polluted river by enhanced floating bed grown Canna. Ecol. Eng. 35, 135–140. https://doi.org/10.1016/j.ecoleng.2008. 09.016. 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