Arsenic induced modulation of antioxidative defense system and brassinosteroids in Brassica juncea L.

Arsenic induced modulation of antioxidative defense system and brassinosteroids in Brassica juncea L.

Ecotoxicology and Environmental Safety 115 (2015) 119–125 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal h...

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Ecotoxicology and Environmental Safety 115 (2015) 119–125

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Arsenic induced modulation of antioxidative defense system and brassinosteroids in Brassica juncea L. Mukesh Kumar Kanwar a,b, Poonam b, Renu Bhardwaj b,n a b

Department of Environmental Sciences, Sri Guru Granth Sahib World University, Fatehgarh Sahib 140406, Punjab, India Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143005, Punjab, India

art ic l e i nf o

a b s t r a c t

Article history: Received 1 September 2014 Received in revised form 6 February 2015 Accepted 8 February 2015

Brassica juncea (Indian mustard) L. plants were exposed to different concentrations (0.0, 0.1, 0.2 and 0.3 mM) of arsenic (V) and harvested after 30 and 60 days of sowing for the analysis of growth parameters, metal uptake, brassinosteroids (BRs) synthesis and oxidative stress markers. As (V) significantly hampered the growth of B. juncea plants and triggered the modulations of various stress markers like proteins, antioxidative enzymes (SOD, CAT, POD, APX, GR, MDHAR and DHAR) and MDA content. Furthermore, As (V) induced the synthesis of 4 BRs, castasterone, teasterone, 24-epibrassinolide, and typhasterol, which were isolated and characterized by gas chromatography–mass spectrometry (GC–MS). The study further highlig5895hted the significant uptake of arsenic ions by mustard plants. & 2015 Elsevier Inc. All rights reserved.

Keywords: Brassinosteroid Brassica juncea Antioxidant enzymes Metal uptake Arsenic metal ions

1. Introduction During the last century, the biosphere has been contaminated with heavy metals due to smelting, mining and waste disposal practices (Diwan et al., 2010). Amendment of agricultural soils with municipal sewage sludge is another important activity contributing to the load of these metals. The excess amount of these hazardous metals in the environment is reported to be dangerous to human health (Olowoyo et al., 2012). Metals like mercury (Hg), cadmium (Cd), lead (Pb), arsenic (As), copper (Cu), zinc (Zn), tin (Sn), and chromium (Cr) are of prime concern owing to their toxicities (Wright, 2007; Ghosh, 2010). These metals and metalloids are fatal to plants and can cause adverse effects even in trace amounts (Kanwar et al., 2012). Contamination of the environment with the metalloid As has grasped a worldwide interest. It is released by geological activities, smelting operations, fossil fuel combustion and by the use of pesticides and herbicides (Gupta et al., 2011; Selvaraj et al., 2013). Arsenic exists in  3, 0, þ 3 and þ5 oxidation states in nature and its known forms are arsenious acids (H3AsO4, H3AsO4  , H3AsO42  ) and arsenic acids (H3AsO4, H2AsO4–, HAsO42  ), arsenates, arsenites, methylarsenic acid, dimethylarsinic acid and arsine etc. in environment (Mohan and Pittman, 2007). Arsenic toxicity in plants leads to oxidative stress, resulting in the formation of free n

Corresponding author. Fax: þ 91 183 2258820. E-mail address: [email protected] (R. Bhardwaj).

http://dx.doi.org/10.1016/j.ecoenv.2015.02.016 0147-6513/& 2015 Elsevier Inc. All rights reserved.

radicals (Flora, 2011). These free radicals damage cell constituents causing cell damage or death (Chardi et al., 2009). Plants cope up the deleterious effects of metals, through activation of the antioxidative defense system comprising of enzymatic and non-enzymatic components. In addition to these, phytohormones also provide stress tolerance to plants under various stresses (Peleg and Blumwald, 2011). Brassinosteroids (BRs), a group of polyhydroxylated steroidal hormones have been reported to provide stress protection to plants. Recent studies revealed stress protective properties of BRs in plants under various stresses like heavy metals, drought, salt, high and low temperature and pathogen attack (Bajguz and Hayat, 2009; Kanwar et al., 2013). BRs provide tolerance to plants by interacting with other hormones. Their crosstalks with auxins, cytokinins, jasmonic acid, salicylic acid etc. play a significant role in triggering defense mechanisms (Bajguz and Hayat, 2009). Reports have confirmed the potential of plant hormones to improve crop performance synergistically under abnormal environmental conditions (Bajguz and Hayat, 2009). Brassica juncea (B. juncea) is raised as an oilseed crop in India. It is also known as hyper-accumulator of heavy metals (Kanwar et al., 2013). Metal induced antioxidative defense system and BRs synthesis needed to be investigated in B. juncea plants. Keeping this in mind, the present study was framed to study modulation of antioxidative defense system and BRs in B. juncea plants under As (V) stress. The effect of increased concentrations of As on growth, antioxidative capacity (as SOD, POD, APX, CAT, GR, DHAR and MDHAR), lipid peroxidation

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and synthesis of BRs in 30 and 60 days old plants of Brassica was investigated to have a better understanding of mechanism of defense against As stress.

2. Material and methods 2.1. Standards and Chemicals used 24-epibrassinolide (24-EBL), sephadex LH-20 and methane boronic acid were procured from Sigma-Aldrich, India Pvt. Ltd., New Delhi. Castasterone (CS), typhasterol (TY) and teasterone (TE) were procured from Chemical Clones Pvt. Ltd. Canada. Silica gel (60–120 mesh size) was obtained from Qualigens fine chemicals, Glaxo India Ltd, Mumbai. For TLC analysis pre-coated ALUGRAM SIL G/UV 254 plates were used and bought from Macherey–Nagel, Germany. All the solvents used in the extraction process were of HPLC grade. 2.2. Plant material

2.5.1.3. Guaiacol peroxidase (POD, EC 1.11.1.7). The activity of peroxidase was measured according to the method proposed by Putter (1974). 2.5.1.4. Superoxide dismutase (SOD, EC 1.15.1.1). SOD activity was estimated according to Kono (1978) by noting its potential to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) dye by superoxide radicals, which are produced by the auto-oxidation of hydroxylamine hydrochloride. 2.5.1.5. Glutathione reductase (GR, EC 1.6.4.2). Glutathione reductase was measured by the method proposed by Carlberg and Mannervik (1975). 2.5.1.6. Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4). Monodehydroascorbate reductase activity was determined according to the method proposed by Hossain et al., (1984). 2.5.1.7. Dehydroascorbate reductase (DHAR, EC 1.8.5.1). Dehydroascorbate reductase activity was measured by the method given by Dalton et al., (1986).

Certified seeds of B. juncea L. variety PBR 91 were arranged from Department of plant breeding, Punjab Agriculture University (Punjab) India. Seeds were surface sterilized with 0.01% sodium hypochlorite and rinsed five times with double distilled water. Then rose in earthen pots containing different concentrations of Arsenic metal stress in the form of Na2HAsO4  7H2O (0, 0.1, 0.3, and 0.5 mM) in the Botanical garden of Guru Nanak Dev University, Amritsar, Punjab, India under natural conditions.

2.5.3. Malondialdehyde content The malondialdehyde (MDA) content was measured using the method described by Heath and Packer (1968).

2.3. Studies on morphological parameters

2.6. Analysis of BRs

After 30 and 60 days of sowing, plants were harvested and analyzed for morphological parameters like shoot length and number of leaves per plant. Fifteen plants from three replicates of same concentration were selected for analysis.

2.6.1. Extraction and purification of BRs 30 and 60 days old plants B. juncea plants exposed to As (V) stress (0.0, 0.1, 0.2 and 0.3 mM) were harvested and processed for extraction and purification of BRs as per the method described earlier by Kanwar et al., (2013). The biologically active fractions obtained after extraction and purification were subjected to TLC and GC–MS analysis for their characterization.

2.4. Heavy metal analysis The leaves and shoots of 30 and 60 days old B. juncea L. plants were harvested. The collected samples were oven dried at 80 °C for 24 h. The dried samples were digested in a mixture of concentrated nitric acid (HNO3) and perchloric acid (HClO4) (V/V 3:1). The solution was mixed well and kept on a hot water bath at 60– 70 °C for about 20 min until clear solutions were left. The solutions were supplemented with double distilled water after cooling, filtered through Whatman filter paper. The samples were then analyzed in triplicate for arsenic content by atomic absorption spectroscopy (AAS) through SHIMADZU AAS-6300 attached with HVG. 2.5. Analysis of biochemical parameters 2.5.1. Antioxidative enzymes For estimation of antioxidative enzyme activities and protein, content 0.5 g of 30 and 60 days old B. juncea L. plants were homogenized in 5.0 ml of 100 mM potassium phosphate buffer (pH-7.0). The homogenate was centrifuged at 4 °C for 20 min at 15,000g.

2.5.2. Protein estimation Protein content was determined following the method of Lowry et al., (1951).

2.6.2. Radish hypocotyl bioassay The bioactivity of isolated fractions was determined using intact seedlings of Raphanus sativus L. with minor modifications (Takatsuto et al., 1983). Three days old seedlings of radish were transferred to test solutions. After incubation at 25°C in the darkness for 24 h, the elongation percentage of the hypocotyls with respect to the control, determined the biological activity. 2.6.3. Characterization of brassinosteroids 2.6.3.1. Thin layer chromatography. The bioactive fractions and the standards were spotted on TLC plates coated with silica gel 60 F 254, and developed with CHCl3: MeOH (8:2) as mobile phase. The spots were detected by spraying Liebermann–Burchard. Rf values for the standard and samples were recorded.

2.5.1.1. Ascorbate peroxidase (APX, EC 1.11.1.11). Ascorbate peroxidase activity was determined following the method proposed by Nakano and Asada (1981).

2.6.3.2. Derivatization of purified fraction. Methaneboronic acid (100 μg) and dry pyridine (60 μL) were mixed and 20 μL of this mixture was added to the active fractions. These were heated to 80 °C for 25–30 min. Further trimethyl silylation of methaneboronates was conducted by reacting with N-methyl-N-trimethylsilyltriflouroacetamide (MSTFA). 3 μL of this solution was injected into GC–MS. The standard BRs were also derivatized and subjected to GC–MS analysis.

2.5.1.2. Catalase (CAT, EC 1.11.1.6). Catalase activity was calculated by the method suggested by Aebi (1984).

2.6.3.3. GC–MS analysis. The GC–MS (Shimadzu, GC–MS, QP 2010) analysis of BRs was carried out with the following conditions: EI

22.0 7 1.12a 17.0 7 0.60b 12.7 7 0.70c 9.0 7 0.60d 85.7 7 2.33a 69.0 7 2.08b 48.57 1.90c 39.5 7 1.80d 0.0 0.1 0.2 0.3 60 days

Data represents the Mean 7SE. Different letters (a, b, c & d) within various concentrations of As (0, 0.1, 0.2 and 0.3 mM) are significantly different (Fisher LSD post hoc test, p r 0.05) and signify the effect of As stress on morphological and biochemical parameters.

10.5 7 0.60a 18.2 7 0.99b 22.97 1.524c 17.2 7 0.62b 15.27 1.80a 13.3 7 1.54a 11.5 7 1.80a 9.3 7 0.90b 32.27 2.07a 23.8 7 2.22b 20.3 7 1.23bc 15.6 7 2.02c 28.2 7 1.33a 37.7 7 1.33b 46.5 7 1.75c 50.37 2.00c 26.5 7 1.24a 38.37 1.53b 24.3 7 0.70a 19.5 7 0.70c 24.3 7 1.40a 30.9 7 2.54b 36.87 2.61b 23.2 7 0.90a 26.0 7 1.44ab 29.8 7 1.70a 22.7 7 0.90b 11.17 0.50c 20.4 7 1.27a 23.8 7 0.70ab 28.3 7 1.20b 35.37 2.50c

6.7 7 0.70a 6.3 7 0.33a 4.7 7 0.42b 3.7 7 0.53b

14.9 7 1.11a 13.0 7 1.03ab 11.7 7 0.48bc 9.7 7 0.71c

10.3 7 1.13a 13.5 7 1.50a 16.17 2.08ab 20.07 2.52b 9.5 7 0.83a 13.9 7 1.70b 20.27 1.30c 6.9 7 1.20a 6.7 7 0.90a 13.4 7 0.70b 18.57 0.60c 17.7 7 2.36bc 9.9 7 0.30a 17.6 7 1.53b 21.7 7 0.51c 26.97 0.80d 10.5 7 1.73a 13.5 7 0.52ab 14.9 7 0.64b 19.57 0.44c

POD (UA mg  1 protein) CAT (UA mg  1 protein) SOD (UA mg  1 protein) Protein content (mg g  1 FW)

15.0 7 0.60a 11.8 7 1.09b 8.3 7 1.20c 6.6 7 0.44c

3.3.1. Protein content The protein content was found to increase with the higher

0.0 0.1 0.2 0.3

3.3. Analysis of biochemical parameter’s in B. juncea plants

30 days

3.2.2. In leaves As (V) accumulation was also studied in the leaves of B. juncea plants and it was noticed that the metal accumulation was lesser than those of shoots. 0.3 mM treatment of As (V) revealed maximum accumulation of As (V) in 30 days old plants (Fig. 1). In leaves of 60 days old plants, maximum accumulation was recorded in 0.1 mM treatment. A decreasing trend in accumulation was observed in 0.2 mM and 0.3 mM As (V) treated plants as the accumulation was decreased by 1.17 folds and 1.26 folds respectively. The leaves of control plants did not reveal any metal ions (Fig. 1).

Number of leaves

3.2.1. In shoots Significant accumulation of As (V) was noticed in 30 days old B. juncea plants. It was observed that the metal accumulation efficiency of B. juncea plants increased with the increasing concentration of As (V). Maximum accumulation of As (V) was noticed in 0.3 mM treated plants (Fig. 1). While, the trend of accumulation was reversed in 60 days old plants, as the efficiency of Brassica plants decreased with increasing concentrations of As ions. Maximum accumulation was noticed in lower concentration of As (V) (0.1 mM). It got decreased by 1.14 folds in 0.2 mM and 1.34 folds in 0.3 mM As (V) treatment. Control plants were also analyzed for metal uptake, but no metal content was detected in untreated plants (Fig. 1).

Shoot length (cm)

3.2. As accumulation in 30 and 60 days old B. juncea plants

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Days and Treatments (mM)

As (V) induced phytotoxicity was studied in terms of reduced shoot length and number of leaves. It was observed that the increasing concentrations of As (V) drastically affected the growth of 30 days old plants. The shoot length (15 cm) and number of leaves (6.7) were highest in control plants only. The shoot length was significantly decreased by 2.26 folds and number of leaves by 1.81 folds in 0.3 mM of As (V) treatment (Table 1). Whereas, in 60 days old plants, growth was further severely reduced under the effect of As (V). A dose dependent decrease was observed in all concentrations of As (V) treatment, as the minimum shoot length (2.16 folds) and number of leaves (2.44 folds) was recorded in the higher dose of As (0.3 mM) treatment and was statistically different from control plants (Table 1).

Table 1 Effect of As (V) stress on morphological parameters, biochemical parameters and MDA content in 30 and 60 days old B. juncea plants.

3.1. Morphological parameter

APX (UA mg  1 protein)

GR (UA mg  1 protein)

3. Results

5.2 7 0.63a 7.4 7 0.50ab 10.9 7 0.90bc 14.7 7 2.40c

All data were subjected to one-way analysis of variance (ANOVA) for scrutinizing the effect of As stress on various morphological and biochemical parameters and expressed as the mean 7standard error of five replicates. The Fisher LSD post hoc test (p r 0.05) was applied for the comparisons against control values using SigmaStat Version 3.5.

7.4 7 0.52a 9.8 7 0.36ab 11.5 7 1.15b 15.07 0.73c

2.7. Statistical analysis

9.9 7 0.13a 12.5 7 0.80b 15.2 7 0.30c 16.6 7 0.40c

DHAR (UA mg  1 MDHAR (UA protein) mg  1 protein)

MDA (μmol g  1 FW)

(70 eV), source temperature 250 °C, column Rxt-1 (Length 30 m, Diameter 0.25 mm and 0.1 mm thickness), injection temperature 280 °C, column temperature programmed 200 °C for 5 min, then raised to 280 °C at rate of 20 °C min–1 and held on this temperature for 35 min; interphase temperature 290 °C, carrier gas He, flow rate 1.0 mL min–1 with split injection.

5.17 0.35a 7.9 7 1.11a 11.4 7 0.63b 17.67 1.23c

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Fig. 1. Arsenic accumulation (μg g–1 DW) in shoots and leaves of 30 and 60 days old B. juncea plants grown under different concentrations (0.0, 0.1, 0.2 and 0.3 mM) of the metal.

concentrations of As (V) in 30 days old B. juncea plants. Maximum protein content was recorded in plants treated with 0.3 mM As (16.56 mg g–1 FW) (Table 1). On the other hand, the protein content was noticed to decrease with the increasing concentration of As (V) in 60 days old plants. Highest content of proteins was recorded in control plants (14.9 mg g–1 FW). Whereas the protein content of stressed plants was sharply decreased and maximum decline by 1.53 folds was noticed in 0.3 mM concentration of As (V) as compared to control Brassica plants (Table 1). 3.3.2. Activities of antioxidative enzymes As (V) toxicity significantly increased the activities of antioxidant enzymes in 30 days old B. juncea plants. Activities of all the enzymes SOD, CAT, POD, APX, GR, and MDHAR were increased with the concentrations of As (V) stress as compared to control plants except in DHAR activity where no particular trend was observed (Table 1). Maximum SOD activity was recorded in 0.3 mM of As (V) concentrations (15.0 UA mg–1 protein). Similarly, activities of CAT (14.7 UA mg–1 protein), POD (19.5 UA mg–1 protein), APX (26.9 UA mg–1 protein), and MDAHR (20.0 UA mg–1 protein) were also maximum in 0.3 mM concentration of As (V) than untreated B. juncea plants. DHAR activity was first increased in low concentration of As (V) and then a sharp decline in its activity was observed. A maximum drop of 1.38 folds in its activity was noticed in 0.3 mM of As (V) stressed plants (Table 1). In 60 days old B. juncea plants, activities of SOD and GR were increased with increasing concentration of As (V) when compared to control plants. SOD (35.3 UA mg–1 protein) and GR (50.3 UA mg–1 protein) activity was maximum in 0.3 mM concentration of As (V). Decreasing trend in the activity of CAT, DHAR and MDHAR was noticed with the increasing concentration of As

(V) (Table 1). The lowest activity of CAT (11.1 UA mg–1 protein), DHAR (15.6 UA mg–1 protein) and MDHAR (9.3 UA mg–1 protein) activities were recorded in 0.3 mM treatment of heavy metal (Table 1). In case of POD and APX, activity got increased by 1.51 folds and 1.44 folds respectively in 0.1 mM of As (V) treated plants. Further the activity of POD and APX was decreased in 0.3 mM of As (V) treated plants as compared to untreated ones (Table 1). 3.3.3. MDA content With the increasing concentration of As (V), the levels of MDA content were also increased in a dose dependent manner in 30 days old plants. Maximum MDA content (17.6 μmol g–1 FW) was noticed in 0.3 mM As (V) treatment as compared to control plants respectively (Table 1). In 60 days old plants, maximum value (22.9 μmol g–1 FW) for MDA content was recorded in 0.2 mM of As (V) treated plants and got decreased in higher dose As (V) treatment (Table 1). 3.4. Characterization of BRs in 30 and 60 days old B. juncea plants The 30 and 60 days old plants of B. juncea exposed to As (V) stress (0.0, 0.1, 0.2 and 0.3 mM) were analyzed for the presence of BRs. The biological active fractions showing positive response in TLC were derivatized with methaneboronic acids and further their TMS were formed and subjected to GC–MS for characterization. The mass spectra of the samples obtained by GC–MS, clearly demonstrated the presence of castasterone (CS), teasterone (TE), 24-Epibrassinolide (24-EBL) and typhasterol (TY) in 30 days old plants when compared with spectra of the standard compounds. The analysis of 60 days old plants confirmed the presence of CS and 24EBL. These bismethane-borates (BMBs) and trimethyl silylates

Fig. 2. GC–MS fraction pattern along with m/z ions of endogenous castasterone isolated from B. juncea plants grown under As (V) stress.

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Fig. 3. GC–MS fraction pattern along with m/z ions of endogenous teasterone isolated from B. juncea plants grown under As (V) stress.

(TMS) of the bioactive compounds were found to be identical with standards. Isolated CS showed a molecular peak at 512 (Fig. 2) and other fragmented m/z were 415, 356, 287 and 155 and TE showed its molecular ion peak at 544 (Fig. 3) and other m/z were 529, 515, 454, 229, 155 and 107 respectively. On the other hand, 24-EBL showed a molecular peak at 528 (Fig. 4) and other fragmented m/z were 457, 415, 374, 345, 332, 177, 155 and 95, whereas TY showed its molecular peak at 544 (Fig. 5) and other m/z were 529, 515, 454, 229, 155 and 85. Based on GC retention time, mass spectral data and TLC with known standards, the isolated active fractions were determined as CS {(22R,23R,24S)-2α,3α,22,23-tetrahydroxy-2- methyl-5α-cholestane-6-one}, teasterone {(22R, 23R,24S)-3,22,23-trihydroxy-24-methyl -5 -cholestan-6-one}, 24-EBL {(22R,23R,24S)2α,3α,22,23-tetrahydroxy-2- methyl-5α-cholestane-6-one} and typhasterol {(2-deoxycastasterone (22R,23R,24S)- 3,22,23-trihydroxy24-methylz-5 -cholestan-6-one}.

4. Discussion Metal toxicity results in the alterations in physiological and cellular levels, which is finally ascribed to distorted plant metabolism (Hossain et al., 2012). In the present study it is clearly evident that shoot length and number of leaves significantly decreased in 30 and 60 days old B. juncea under higher concentration of As (V). It was reported by Garg and Singla, 2011 that the excess of As (V) was harmful to plants as it affected transpiration rate, root activity uptake and transport of water and essential nutrients. Arsenic reacts with the sulphydryl groups of proteins which result in abscission in plant leaves, severe inhibition of plant growth, and

steep reduction of biomass (Shao et al., 2011). Reduction in plant growth parameters was observed in As-treated Indian mustard grown hydroponically (Khan et al., 2009). The treatment caused the overall reduction of 9.3% and 8.0% in the root and shoot dry weights respectively when compared to controls. Similar to the present study, Karimi et al., (2013) reported decline in overall growth of the plants under arsenic, cadmium and mercury stress. The root and shoot biomass of artichoke and savory were reduced significantly under the raised levels of these metals. The reduction in shoot length and number of leaves could be interrelated to the As (V) accumulation in plants which decreased the availability of nutrients required for normal growth. The observations made on metal uptake studies showed the better accumulation efficiency of B. juncea plants, as accumulation was dose dependently increased in 30 days old plants. This is because of the fact that metals usually accumulated during the vegetative stage, which is consistent with the earlier reports in the literature (Kashiwagi et al., 2009). Chaturvedi (2006) carried out a study using two genotypes of B. juncea (Varuna and DHR-9504) to check the As ions uptake. He observed that arsenic extraction by plants increased with escalating concentrations of As in soils. However, in case of 60 days old plants, maximum uptake was observed in lower concentration of As (V) in both the cases (Fig. 1). It is not surprising that plant biomass decreases with maturity. The decrease in overall growth in terms of shoot length and number of leaves with increasing concentration of As (V) stress was well established from the present study (Fig. 1). The above finding can be interrelated with the decrease in As (V) accumulation with its increasing concentration in 60 days old plants. A comparative study was performed to note the accumulation of arsenic in leaves

Fig. 4. GC–MS fraction pattern along with m/z ions of endogenous 24-Epibrassinolide isolated from B. juncea plants grown under As (V) stress.

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Fig. 5. GC–MS fraction pattern along with m/z ions of endogenous typhasterol isolated from B. juncea plants grown under As (V) stress.

and roots of two varieties of B. juncea viz. varuna and pusa bold. It was observed that the trend of metal accumulation was concentration and time dependent. More As ions were found in roots after two days whereas the concentration was higher in leaves after four days of experiment (Gupta et al., 2009). Heavy metal toxicity leads to the production of ROS causing oxidative damage. It results in lipid peroxidation, modulation of antioxidative enzymes, deterioration of biological macromolecule, membrane dismantling, and DNA damage (Quartacci et al., 2001). Malondialdehyde (MDA) is a cytotoxic byproduct of lipid peroxidation and serves as a marker of free radical generation and tissue damage in plants and animals. In the present study it was found that MDA content increased with the increasing concentration of As (V) during different stages of growth and development. The increase in MDA level with increasing As (V) metal concentrations initiated the production of superoxide radicals, leading to enhanced lipid peroxidation. The results obtained are in concordance with studies on B. juncea which are exposed to different concentrations of As and showed enhancement in stress marker parameters like MDA and proline (Khan et al., 2009; Ahmad and Gupta, 2013). Similarly higher MDA content was noticed in rice genotypes under As stress (Dave et al., 2013; Mallick et al., 2014). In order to counteract oxidative stress, plants switch on several defense mechanisms involving antioxidative defense system (Kanwar et al., 2012). It was observed by Seth et al., (2007,) that the plants produced stress protective proteins under metal stress. Treatment of B. juncea plants with As (V) in the present finding revealed the enhanced protein content and antioxidant enzymes which is a clear response of defense strategy adopted by the plants. It was found that protein content and the activity of antioxidative enzyme activities got increased in 30 days old plants. Whereas, in 60 days old plants, protein content and the enzyme activity got decreased (Table 1). At a later stage of development, decrease in protein content was because of overall decrease in plant growth parameters and photosynthetic activity (Palma et al., 2002). Enhancement in the activity of antioxidants viz. SOD, CAT and GPX was observed in the varieties of B. juncea because of As metal ions (Gupta et al., 2009). The enzymes showed higher activities at lower concentration of As exposure (50 mM and 150 mM) followed by decrease at higher concentration (300 mM) compared to controls for different durations of experiment. Similarly, exposure of 20 days old plants of B. juncea to 5 and 25 mM As for 96 h in hydroponic cultures led to reduced plant growth, higher MDA content, and increased activities of SOD, APX and GR (Khan et al., 2009). The up-regulation of antioxidative defense system in rice seedlings was observed under As stress by Shri et al., (2009). Similar results were obtained in rice plants where enhanced antioxidative potential was noticed with As stress (Dave et al., 2013, Mallick et al., 2014).

Synthesis of phytohormones is another response exhibited by plants under heavy metals stress (Peleg and Blumwald, 2011). Elevated amounts of abscisic acid or ethylene were reported by Zengin (2006) and Maksymiec (2011) under heavy metal stress. Similarly, Maksymiec (2011) reported the contribution of jasmonic acid in Phaseolus coccineus as an early response to cadmium metal stress. Findings of the present study clearly revealed the synthesis of BRs like CS, TY and TE and 24-EBL in 30 and 60 days old metal treated plants (Figs. 2–5), pointing out the involvement of BRs in conferring metal induced stress protection. In our previous study, different BRs were isolated and characterized from leaves B. juncea L. grown under nickel metal stress (Kanwar et al. 2012, 2013). It was clear from the present study that isolated BRs belonged to C28 group and were of 6-oxo and 7-oxolactone types. It is further postulated that 24-EBL, CS, TY and TE are synthesized during an early C6 oxidation pathway, suggesting the possible role of the pathway operative under As (V) stress in B. juncea L. From the results obtained we concluded that the exposure of As (V) stress induces the modulation of antioxidative enzymes and synthesis of BRs which could be one of the anti-stress defense strategies adopted by B. juncea plants. Further, present study revealed the significant translocation of As (V) and its differential distribution in different parts of brassica. These studies are in concordance with earlier studies where As hyper-accumulator plants have capacity to store more As in aerial tissue as compared to roots (Finnegan and Chen, 2012). However, a more in-depth analysis is needed for defining the appropriate mechanistic relationship of implicated antioxidative defense and synthesis of BRs under As (V) stress in B. juncea.

5. Conclusion It is well established from the study undertaken that the significant accumulation of As (V) by B. juncea L. plants resulted in the reduced growth and modulations in the pool of various biochemical stress markers. The toxic effects of As (V) on growth, and other stress markers (protein content, lipid peroxidation and antioxidative enzymes viz. SOD, CAT, POD, APX, GR, DHAR and MDHAR) in B. juncea L. was concentration and time dependent. Another novel observation made from the present study is the synthesis of brassinosteroids, which is an example of a direct effect of either heavy metals induced stress or it may be a stress protective response of B. juncea plant itself to alleviate the ill effects of the induced stress. Our findings lay a ground for future work aimed at studying molecular mechanism of synergistic interactions between the BRs synthesis and stress indicators in coping the stress produced by As (V) in B. juncea plants.

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Acknowledgements Authors are thankful to Department of Biotechnology and Council Scientific and Industrial Research, Government of India for providing financial assistance.

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