Copper tolerance and response of antioxidative enzymes in axenically grown Brassica juncea (L.) plants

Ecotoxicology and Environmental Safety 73 (2010) 1975–1981

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Copper tolerance and response of antioxidative enzymes in axenically grown Brassica juncea (L.) plants Sudhir Singh, Shraddha Singh, V. Ramachandran, Susan Eapen n Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085, India

a r t i c l e in fo

abstract

Article history: Received 28 January 2010 Received in revised form 2 August 2010 Accepted 15 August 2010 Available online 9 September 2010

Copper is an essential element for proper functioning of all living organisms including plants, but it can cause toxicity at elevated concentrations. In the present study, two varieties of Brassica juncea L. i.e. Pusa JK and TM 4 grown axenically were compared for Cu tolerance and accumulation ability. For further detailed biochemical studies, var. TM 4 was used because of its fast growth and better Cu accumulation in shoots. Toxic effects of Cu were manifested by a reduction in photosynthetic pigments and an increase in the levels of thiobarbituric acid reactive substances. The activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase and catalase showed an increase in a concentration and exposure time dependent manner in roots of B. juncea exposed to copper, indicating that they play an important role in combating copper stress in this species. & 2010 Elsevier Inc. All rights reserved.

Keywords: Antioxidant enzymes Thiobarbituric acid reactive substances Copper-induced oxidative stress Indian mustard

1. Introduction Copper is an essential element for growth and development of every living organism, including plants, since it acts as a co-factor in numerous proteins, particularly in those which are involved in the photosynthetic and the respiratory electron transport chains (Linder and Goode, 1991; Burkhead et al., 2009). Despite being an essential micronutrient for plants, Cu can be toxic when in excess. Redox cycling between Cu + and Cu2 + can catalyze the production of highly toxic hydroxyl radicals, which subsequently damage the macromolecules (Halliwell and Gutteridge, 1984). Copper is also highly reactive to thiols and can possibly displace other essential metals in proteins (Lippard and Berg, 1994). Because of this, organisms have tightly controlled homeostatic mechanisms to take up and maintain adequate concentrations of Cu in their cellular compartments (Burkhead et al., 2009). Excess concentrations of Cu have a cytotoxic effect through the production of reactive oxygen species (ROS) including hydroxyl radicals and hydrogen peroxide by Haber–Weiss reaction (Hegedus et al., 2001; Mittler et al., 2004). Reactive oxygen species are generally highly reactive molecules possessing an unpaired electron and when in excess, cause oxidative damage to the cell. To control the level of ROS and protect the cells from oxidative damage, plants have developed a complex antioxidant system.

Abbreviations: Pusa JK, Pusa Jai Kisan; TM 4, Trombay Mustard 4; SOD, Superoxide dismutase; APX, Ascorbate peroxidase; GPX, Guaiacol peroxidase; CAT, Catalase; TBARS, Thiobarbituric acid reactive substances n Corresponding author. Fax: + 91 22 25505342. E-mail addresses: [email protected], [email protected] (S. Eapen). 0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2010.08.020

Superoxide dismutase (SOD, EC 1.15.1.1), the first major enzyme found in all aerobes, causes the dismutation of superoxide radical to hydrogen peroxide (H2O2) and dioxygen (O2). The intracellular level of H2O2 is then regulated by a wide range of enzymes, the most important ones being peroxidases and catalase (Rusina et al., 2004). Degradation of H2O2 to water and oxygen is carried out by catalase (CAT, EC 1.11.1.6) in peroxisomes, by guaiacol peroxidase (GPX, EC 1.11.1.7) in vacuoles, cell wall and cytosol and by ascorbate peroxidase (APX, EC 1.11.1.11) particularly in chloroplast and mitochondria (Dietz et al., 1999). In the last few decades, enhanced industrial and mining activities have increased the levels of heavy metals including Cu in the ecosystems. Copper is a widespread contaminant, which is released into the environment by anthropogenic activities such as the use of pesticides, fungicides and the release of industrial wastes (Yruela, 2005). Plants growing in Cu-contaminated environments may develop a variety of defense mechanisms to combat its toxicity. Antioxidant responses and degrees of tolerance to oxidative stress are known to vary among different plant species/genotypes. The use of plants for remediation of heavy metal contaminated environments is an esthetically pleasing and environmentally friendly approach (Eapen and D’Souza, 2005; Eapen et al., 2007). Indian mustard (Brassica juncea L.) is a fast growing high biomass plant, which has been shown to have the potential to accumulate several toxic elements (Zhu et al., 1999; Minglin et al., 2005). Purakayastha et al. (2008) used five different species of Brassica and showed that B. juncea phytoextracted the highest amount of Cu from soil. Earlier studies on the use of B. juncea plants for remediation of Cu and Cu-induced oxidative stress are limited to low Cu concentrations (Wang et al., 2004), short durations

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S. Singh et al. / Ecotoxicology and Environmental Safety 73 (2010) 1975–1981

(Devi and Prasad, 2005) or restricted to plants growing in soil (Purakayastha et al., 2008). The present study has been carried out to compare the potential of two varieties of B. juncea grown under in vitro conditions for their tolerance to Cu and to determine the response of various antioxidant enzymes to counteract the toxic effects of Cu-induced stress. Use of in vitro grown plants for studies on molecular mechanisms has the advantage that any contamination with microbes, which may lead to erroneous results can be avoided (Doran, 2009). In addition, in vitro grown plants are smaller in size and require small volumes of solutions for the experiments. This study will be helpful in understanding the biochemical detoxification mechanisms of B. juncea exposed to Cu and its potential use in phytoextraction strategies. 2. Materials and methods 2.1. Plant material Seeds of two varieties of Indian mustard (B. juncea L. Czern and Coss) namely, Pusa Jai Kisan (Pusa JK) and Trombay Mustard 4 (TM 4) were surface sterilized using mercuric chloride following the methodology reported elsewhere (Singh et al., 2006) and inoculated in Hoagland’s liquid medium (Hoagland and Arnon, 1938) in long test tubes plugged with cotton plugs under sterile conditions and kept on a gyratory shaker at 40–50 rpm. The normal Hoagland’s solution contains 2.4 mM Ca(NO3)2, 1.0 mM KH2PO4, 3.0 mM KNO3, 1.0 mM MgSO4, 46.1 mM H3BO3, 9.1 mM MnCl2
4H2O, 0.76 mM ZnSO4
7H2O, 0.32 mM CuSO4
5H2O, 0.12 mM H2MoO4 and 44.6 mM FeSO4. After germination, plants were incubated at 25 72 1C under white fluorescent light (12.2 mM photon m 2 s 1) for a photoperiod of 12 h for elongation. Every 7 days, fresh Hoagland’s medium was supplied to the plants. Six week old seedlings were used for the experiments. 2.2. Copper tolerance and accumulation from solution Six week old B. juncea var. TM 4 and Pusa JK plants grown in liquid Hoagland’s medium in long test tubes under in vitro conditions were used for the experiments. Plants were exposed to 10 ml of fresh Hoagland’s medium spiked with different concentrations of Cu (10, 50, 75, 100, 150, 200 and 300 mM) and plants grown for a period of 21 days and their survival and growth recorded. Plants without Cu2 + served as control. Copper doses used for the present study were environmentally relevant and chosen to expose the plants from low to high levels of Cu (Khatun et al., 2008). Since B. juncea plants did not survive when exposed to 300 mM Cu, for studies on copper uptake, they were treated with Hoagland’s medium supplemented with 10, 50, 100 and 200 mM Cu for 14 days and levels of Cu depletion from the solution as well as Cu content in the plants estimated. 2.3. Metal analysis On days 0, 1, 3, 7 and 14, aliquots (500 mL) were drawn out from each test tube (both having control and treated plants) under aseptic conditions in a laminar air

flow chamber and Cu concentration was determined using GBC Model 932 B + Atomic Absorption Spectrophotometer (GBC, Australia). Also, 500 mL aliquots of stock solution (without plants) of each concentration were analyzed initially for Cu to ensure that the concentrations selected were of the same value without much variation. The actual analyzed Cu concentrations are shown in Table 1. Plants were harvested at the end of 14 days and washed thoroughly with distilled water. The roots and shoots of the plants were separated and dried in an oven (65 1C) for one week. Dried plant tissues were ground and acid digested using HNO3:HClO4 acid mixture (5:1, v/v). Copper concentrations were determined as described above for the liquid medium.

2.4. Effect of Cu on biochemical parameters Due to its fast growth and better Cu accumulation in shoots, B. juncea var. TM 4 was used for studying the biochemical parameters. To estimate the extent of oxidative damage caused by Cu and to understand the tolerance mechanisms, B. juncea var. TM 4 plants were incubated in 10 ml of Hoagland’s solution supplemented with four different concentrations of Cu (viz. 10, 50, 100 and 200 mM) along with one set of non-spiked control under aseptic conditions. Plants were harvested at 3, 7 and 14 days and were analyzed for different biochemical parameters, such as photosynthetic pigments and thiobarbituric acid content. For estimation of chlorophyll content, leaves (100 mg fw) were extracted in 80% chilled acetone and chlorophyll estimation conducted according to the method of Arnon (1949). Levels of carotenoid in the same extract were calculated by the formula given by Duxbury and Yentsch (1956). The levels of lipid peroxidation, in terms of TBARS content in the plant roots and shoots were estimated by thiobarbituric acid (TBA) reaction (Heath and Packer, 1968).

2.5. Estimation of antioxidants 2.5.1. Extraction of enzymes Roots and shoots (200 mg fw each) of the control and treated plants harvested on day 3, 7 and 14 were homogenized in 1 ml of 0.1 M Na–phosphate buffer (pH 7) followed by centrifugation at 12,000g for 10 min at 4 1C. All steps in the preparation of enzyme extract were carried out at 0–4 1C. This supernatant was used to measure the following enzymatic activities at 25 1C using a spectrophotometer (JASCO V-530, Japan).

2.5.2. Superoxide dismutase (SOD) (EC 1.15.1.1) The activity of SOD in the plant extracts was measured by the method of Nishikimi et al. (1972). SOD activity was assayed by its ability to inhibit the photochemical reduction of Nitro Blue Tetrazolium (NBT). One unit (U) corresponds to the amount of enzyme that inhibits reduction of NBT by 50% at 257 2 1C. A system free of enzyme served as control.

2.5.3. Catalase (CAT) (EC 1.11.1.6) The CAT activity was estimated in plant parts following the method of Aebi (1984). One ml reaction mixture contained 0.1 M phosphate buffer (pH 7.0), 6.6 mM H2O2 and 50 mL plant extract. The activity was estimated by monitoring the decrease in absorbance due to H2O2 reduction (extinction coefficient: 39.4 M 1 cm 1) at 240 nm. The activity was expressed in terms of mmol of H2O2 reduced min 1 g 1 fw at 257 2 1C.

Table 1 Removal of Cu by B. juncea var. Pusa JK and B. juncea var. TM 4 and its relative accumulation in shoot in both varieties exposed to different concentrations of Cu. Initial Cu content in solution (lg)

Final Cu content in solution (lg)

Cu concentration (lM)

Actual Cu concentration (lM 7 SE)1

Control Pusa JK 10 50 100 200 Control TM 4 10 50 100 200

2

–

–

9.8 7 0.3 49 7 1.9 97 7 2.9 196 7 1.3

6.2 31.1 61.6 124.5 – 6.2 31.1 61.6 124.5

1.0 8.6 11.8 38.5 – 0.9 7.5 15.2 28.3

2

9.8 7 0.3 49 7 1.9 97 7 2.9 196 7 1.3

Dry weight (dw) of plant tissue (g)

Cu concentration in plant tissue (lg g 1 dw)

Shoot

Root

Shoot

Root

0.135 0.132 0.127 0.129 0.125* 0.142 0.136 0.133 0.135 0.132*

0.0322 0.0298 0.0287 0.0275 0.0263* 0.0323 0.0317 0.0291 0.0301 0.0271*

4.9 7.5* 22.3** 28.7*** 40.0*** 4.8 8.1* 27.3** 48.1*** 77.0***

78.6 111.3* 464.2** 1137.6*** 2037.6*** 81.3 118.6* 512.2** 1105.6*** 2051.3***

* ** , and *** indicates significant differences at p r 0.05, p r0.01 and p r 0.001, respectively, from their respective control. Values followed by same superscripts letters are not significantly different at p r 0.01 for relative Cu accumulation in shoot (%). 1 2

After estimating actual Cu content in stock solutions of each concentration. Basal Hoagland’s solution has 0.32 mM of Cu2 + and was below linear detection limit of instrument.

Relative Cu accumulation in shoot (%)

20.7ab 22.9a 17.5bc 10.6d 8.5d 20.5ab 22.6a 19.6abc 16.3bc 15.5c

2.5.5. Guaiacol peroxidase (GPX) (EC 1.11.1.7) GPX activity was determined following the formation of tetraguaiacol as described by Singh et al. (2006). One unit of peroxidase activity represents the amount of enzyme catalyzing oxidation of 1 mmol of guaiacol in 1 min at 25 72 1C. 2.6. Statistical analysis All experiments were conducted in completely randomized design with at least three replicates. The data were subjected to ANOVA using the software IRRISTAT. Correlation coefficients (r) between Cu treatment and different oxidative stress parameters were estimated using MS Excel 2003.

3. Results Seeds of both varieties of B. juncea cultured on Hoagland’s medium germinated within 2–3 days of inoculation. Growth of variety TM 4 was relatively faster and it took 5–6 weeks to obtain a plant with the desired biomass (approx. 3 g fw), while variety Pusa JK took 6–7 weeks to get to a similar size. 3.1. Comparison of two varieties of B. juncea for Cu tolerance and accumulation from solution When B. juncea vars. TM 4 and Pusa JK were exposed to different concentrations of Cu, both could tolerate Cu up to a concentration of 200 mM and survived for at least 21 days. Above 200 mM of Cu, plants of both varieties did not survive after 14 days of exposure. Growth of plants was not affected by the presence of Cu in solution within an exposure time of 14 days for concentrations up to 100 mM (Table 1). When the potential of plants (roots +shoots) for Cu phytoextraction from medium was compared, both varieties showed similar potential. At low concentrations (10 and 50 mM), both varieties could extract up to 85% of added Cu within 14 days (Fig. 1A, B). Even at higher concentrations of Cu (100 and 200 mM), both varieties could extract up to 70% of externally added Cu (Fig. 1A, B). Removal of Cu by B. juncea var. Pusa JK and B. juncea var. TM 4 and its relative accumulation in shoots of both varieties exposed to different Cu concentrations are shown in Table 1. Data indicated that as the Cu concentrations of solution increased from 10 to 200 mM, the Cu concentration in plant tissues of both varieties also increased significantly. In general, Cu concentration (mg g 1 dw) in the plant tissues and Cu accumulation in shoot (at high concentration of 100 and 200 mM) of TM 4 was more than that of Pusa JK. However, dry weight of plant tissues did not show any significant difference except at 200 mM Cu, where significant reduction was noticed as compared to control in both varieties. Brassica juncea var. TM 4 was selected for further studies because of better Cu accumulation in shoot and its fast growth. 3.2. Photosynthetic pigments In the present study using B. juncea var. TM 4, the level of total chlorophyll did not show any change for 3 days when exposed to low concentrations of Cu (up to 10 mM), while at higher concentrations ( 410 mM), it showed a significant decline (Table 2). After 7 and 14 days of Cu exposure, total chlorophyll concentration of Cu-treated B. juncea plants decreased and maximum reduction in total chlorophyll (61%) was recorded at 200 mM Cu as compared to control after 14 days. Carotenoid

Residual Cu in solution treated witih TM 4 (μM)

2.5.4. Ascorbate peroxidase (APX) (EC 1.11.1.11) The APX activity was determined in the roots and shoots of the B. juncea var. TM 4 by the method of Nakano and Asada (1981), estimating the rate of ascorbate oxidation at 290 nm (extinction coefficient: 2.8 mM 1 cm 1). The enzyme activity was expressed in terms of mmol of ascorbate oxidized min 1 g 1 fw at 25 72 1C.

Residual Cu in solution treated with Pusa JK (μM)

S. Singh et al. / Ecotoxicology and Environmental Safety 73 (2010) 1975–1981

1977

10 μM 50 μM 100μM 200μM

200 175 150 125 100 75 50 25 0 0

2

4

6

10

8 Days

12

14

10 μM 50 μM 100μM 200μM

200 175 150 125 100 75 50 25 0 0

2

4

6

8 Days

10

12

14

Fig. 1. Removal of Cu by B. juncea var. Pusa JK (A) and B. juncea var. TM 4 (B) exposed to different concentrations of Cu.

Table 2 Effect of Cu on photosynthetic pigments (mg g different concentrations and exposure times. Cu concentrations (lM)

Control 10 50 100 200 Control 10 50 100 200 * **

Photosynthetic pigments (mg g 1 fw)

Total chlorophyll

Carotenoids

1

fw) of B. juncea var. TM 4 at

Exposure time (days)

3

7

14

2.48 2.38 1.95*** 1.72*** 1.62*** 0.81 0.76 0.65** 0.60** 0.52***

2.42 2.02*** 1.72*** 1.49*** 1.38*** 0.79 0.65** 0.61** 0.48*** 0.42***

2.41 1.65*** 1.43*** 1.20*** 0.94*** 0.77 0.59*** 0.55*** 0.41*** 0.35***

, and *** indicates significant differences from control at p r 0.05, pr 0.01 and pr 0.001, respectively at a particular exposure time.

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S. Singh et al. / Ecotoxicology and Environmental Safety 73 (2010) 1975–1981

content in Cu-treated B. juncea plants declined with increase in Cu concentration and exposure time (Table 2).

Oxidative stress parameters

3.3. Effect of Cu on lipid peroxidation Effect of Cu on lipid peroxidation was evaluated by determining TBARS content in plant parts. The levels of TBARS was not much affected in shoots of the treated plants after 3 days (except at 200 mM Cu), but increased significantly at and above 10 mM of Cu after 7 and 14 days of treatment (Fig. 2A). In roots, TBARS concentration increased above 10 mM Cu after 3 days and at all the concentrations after 7 and 14 days (Fig. 2B). It was noted that 200 mM Cu (after 14 days) in nutrient medium resulted in the maximum build-up of TBARS content, approximately 3–4-fold higher in shoots and roots of the treated B. juncea plants in comparison to their controls (Fig. 2A, B). Correlation analysis further confirmed that an increase in Cu concentrations led to an

16

∗∗∗ ∗∗∗

12 ∗∗∗ ∗∗∗

8

∗

∗

4

∗∗∗

∗

**

14

TBARS SOD CAT GPX APX

0.700 0.070 0.850*** 0.173 0.126

0.926 0.903*** 0.973*** 0.670** 0.968***

0.923*** 0.744** 0.870*** 0.989*** 0.973***

Roots

TBARS SOD CAT GPX APX

0.971*** 0.427 0.973*** 0.886*** 0.959***

0.991*** 0.944*** 0.968*** 0.908*** 0.971***

0.995*** 0.973*** 0.961*** 0.893*** 0.977***

***

* ** , and *** indicates significant correlation coefficients at pr 0.05, pr 0.01 and pr 0.001, respectively.

0

80

Day 3 Day 7 Day 14

∗∗∗

∗∗∗

∗∗∗ ∗∗∗

∗∗∗

∗∗

60 ∗

40

20

0

24

10 100 50 Cu concentration (μM)

200

200

Day 3 Day 7 Day 14

20

180 ∗∗∗ ∗∗∗

16 ∗∗∗

12

∗∗∗ ∗∗∗ ∗∗∗ ∗∗

8

0

∗∗∗

∗∗∗ ∗∗∗

∗∗∗

4

SOD activity in roots (U g-1 fw)

0

TBARS level in roots (m mol g-1 fw)

7

Shoots

100

Day 3 Day 7 Day 14

Exposure time (days) 3

SOD activity in shoot (U g-1 fw)

TBARS level in shoot (m mol g-1 fw)

20

Table 3 Correlation coefficient between Cu treatments and different oxidative stress parameters in shoots and roots of B. juncea var. TM 4 at different exposure times.

160

10 50 100 Cu concentration (μM)

200

Day 3 Day 7 Day 14

∗∗∗

140

∗∗∗

120 ∗∗∗

100 ∗∗

80

∗∗∗

∗∗∗

∗

∗∗∗ ∗ ∗∗

60 40 20 0

0 0

10 50 100 Cu concentration (μM)

200

Fig. 2. The effect of Cu treatment on TBARS (m mol g 1 fw) concentration in the shoots (A) and roots (B) of B. juncea var. TM 4. Significant differences from control (i.e. 0 mM Cu) are marked with asterisks (*p r0.05, **p r 0.01 and ***p r0.001) at a particular exposure time.

0

10 50 100 Cu concentration (μM)

200

Fig. 3. The effect of Cu treatment on SOD (U g 1 fw) activity in the shoots (A) and roots (B) of B. juncea var. TM 4. Significant differences from control (i.e. 0 mM Cu) are marked with asterisks (*p r 0.05, **p r0.01 and ***p r 0.001) at a particular exposure time.

S. Singh et al. / Ecotoxicology and Environmental Safety 73 (2010) 1975–1981

increase in TBARS levels in both roots and shoots at different exposure times (Table 3).

3.4. Effect of Cu on antioxidant enzymes Protection against enhanced ROS generation in plants is achieved through stimulation of various antioxidant enzymes. Antioxidant enzymes examined in this study (SOD, CAT, APX and GPX) showed differing responses at various Cu concentrations. In the present study, SOD activity, which converts superoxide (O2 d ) into H2O2 did not show any change in shoots at all Cu concentrations at day 3 (except at 10 mM Cu) and up to 50 mM Cu at day 7, but increased significantly above this concentration at 7 and 14 days (Fig. 3A). In roots of treated plants, SOD activity increased significantly at all the concentrations of Cu and exposure time (except for day 7 for which increase in activity was observed above 50 mM) (Fig. 3B). At 200 mM Cu after 14 days,

Day 3 Day 7 Day 14

250

∗∗∗ ∗∗∗

∗∗∗

∗∗∗

∗∗∗

200

∗∗∗

∗∗∗

∗∗∗

∗∗∗

150

100

50

APX actvity in shoot (μmol min-1g-1 fw)

CAT activity in shoot (μmol min-1 g-1 fw)

114% and 33% increases were observed in SOD activity in roots and shoots, respectively (Fig. 3A, B). Strong positive correlation was also found between SOD levels and Cu concentrations at 7 and 14 days exposure in roots and shoots of B. juncea (Table 3). CAT activity in the shoots of B. juncea plants showed an increase above 50 mM Cu after 3 days, above 10 mM after 7 days and for all the concentrations after 14 days (Fig. 4A). Similarly, CAT activity for roots increased significantly for all Cu concentrations and exposure time (except for day 3 at 10 mM) with a maximum increase of 97.9% at 200 mM Cu on day 14 as compared to control plants (Fig. 4B). Such an association between CAT activity and Cu levels was also substantiated by positive correlation coefficients (Table 2). APX and GPX activities showed a similar response in Cutreated B. juncea plants. In plant shoots, the activity of APX and GPX remained unaffected at all the Cu concentrations up to 3 days of exposure, while it was found to be higher at 7 and 14 days (Figs. 5A and 6A). Activities of APX and GPX increased in a concentration and exposure time dependent manner in roots of

600

300

Day 3 Day 7 Day 14

500

∗∗∗ ∗∗∗

400

∗∗∗

∗

∗∗∗

∗∗

∗∗

300

200

100

0

0 10 50 100 Cu concentration (μM)

Day 3 Day 7 Day 14

600

∗∗∗

500 ∗∗∗

400 ∗∗

∗∗∗

∗∗∗ ∗∗∗ ∗

∗∗∗

∗∗∗

0

200

∗∗∗ ∗∗∗

300 200 100

APX activity in roots (μmol min-1 g-1 fw)

0

CAT activity in roots (μmol min-1 g-1 fw)

1979

600

10 50 100 Cu concentration (μM)

200

Day 3 Day 7 Day 14

500

∗∗∗

∗∗∗

400 ∗

∗

∗∗

∗∗∗ ∗∗∗

∗∗∗

∗∗∗ ∗∗∗

∗∗∗ ∗∗∗

300

200

100

0

0 0

10 50 100 Cu concentration (μM)

200

Fig. 4. The effect of Cu treatment on CAT (mmol min 1 g 1 fw) activity in the shoots (A) and roots (B) of B. juncea var. TM 4. Significant differences from control (i.e. 0 mM Cu) are marked with asterisks (*p r0.05, **p r 0.01 and ***p r 0.001) at a particular exposure time.

0

10 50 100 Cu concentration (μM)

200

Fig. 5. The effect of Cu treatment on APX (mmol min 1 g 1 fw) activity in the shoots (A) and roots (B) of B. juncea var. TM 4. Significant differences from control (i.e. 0 mM Cu) are marked with asterisks (*pr 0.05, **p r0.01 and ***p r0.001) at a particular exposure time.

GPX activity in shoot (U g-1 fw)

1980

S. Singh et al. / Ecotoxicology and Environmental Safety 73 (2010) 1975–1981

Day 3 Day 7 Day 14

4

∗∗∗

3 ∗∗∗ ∗

∗∗∗

2 ∗∗∗

1

0 100 10 50 Cu concentration (μM)

0

GPX activity in roots (U g-1 fw)

40

200

Day 3 Day 7 Δαψ 14

∗∗∗

30

∗∗∗

∗∗∗ ∗∗∗ ∗∗∗

∗∗∗ ∗∗∗

∗∗∗ ∗∗∗

∗∗∗

20 ∗ ∗∗∗

10

0 0

10 50 100 Cu concentration (μM)

200

Fig. 6. The effect of Cu treatment on GPX (U g 1 fw) activity in the shoots (A) and roots (B) of B. juncea var. TM 4. Significant differences from control (i.e. 0 mM Cu) are marked with asterisks (*pr 0.05, **p r 0.01 and ***p r 0.001) at a particular exposure time.

the plant (Figs. 5B and 6B). The activities of both these enzymes showed positive correlations with Cu concentrations in roots at all exposure time and in shoots at 7 and 14 days (Table 3).

4. Discussion Brassica juncea is recognized as a candidate plant for remediation of metal-polluted soils and water bodies because of its potential to tolerate and accumulate high levels of toxic trace elements (Zhu et al., 1999; Purakayastha et al., 2008). In the present study, both varieties of B. juncea were found to be similar in Cu tolerance and its potential for removal of Cu from solutions over a broad concentration range (10–200 mM). Such performance is important as concentration of pollutants in effluents is prone to variations. Copper accumulation in shoots was found to be higher in var. TM 4 compared to Pusa JK especially at higher concentrations,

although most of the Cu taken up by plants was retained in roots. Roots of higher plants directly come in contact with solutions containing metals and are known to act as a storage house for heavy metals and restrict their translocation to the shoot biomass (Mazhoudi et al., 1997). Toxicity of Cu is due to its existence in two readily interconvertible oxidation states (Cu + –Cu2 + ) and it can catalyze the formation of free radicals through the Haber–Weiss reaction. These free radicals damage the photosynthetic apparatus (Devi and Prasad, 1998; Vajpayee et al., 2005) and may also catalyze degradation of proteins through oxidative modification and increased proteolytic activity (Romero-Puertas et al., 2002). In the present study, levels of photosynthetic pigments in B. juncea plants treated with Cu were found to decrease in a concentration and exposure time dependent manner. Copper primarily disturbs the integrity of thylakoid membranes, changes their fatty acid composition (De Vos et al., 1991), inhibits chlorophyll and carotenoid biosynthesis and retards the incorporation of these pigments in photosystems (Caspi et al., 1999; Boswell et al., 2002). Apart from its highly reactive redox state, Cu has the ability to bind thiol groups and disrupt redox status of the cell, thereby enhancing the production of ROS such as hydroxyl, peroxyl and alkoxyl radicals, which induce lipid peroxidation (Dietz et al., 1999). Lipid peroxidation leads to membrane damage and changes in lipid peroxidation serve as an indicator of the extent of oxidative damage under stress (Halliwell and Gutteridge, 1993). The present observation of an increase in TBARS content in B. juncea on day 7 and 14 when exposed to high concentrations of Cu treatment is in conformity with those observed in Ceratophyllum demersum (Devi and Prasad, 1998) and Pistia stratiotes (Sinha et al., 2003). Protection against enhanced ROS generation is achieved through activation of antioxidant mechanism of plant, which includes both enzymatic and non-enzymatic antioxidants. In the case of oxidative stress, activity of one or more antioxidant enzymes like SOD, CAT, APX, GPX etc. generally increases in plants and this elevated activity is usually correlated with increased stress tolerance. Superoxide dismutase is the first line of enzymatic defense against oxidative stress. The results of the present experiment are in agreement with the findings of Luna et al. (1994), who observed an increase in SOD activity in Cu-treated Avena sativa plants. Srivastava et al. (2005) also reported an increase in SOD activity in arsenic-treated Pteris vittata plants, which was tolerant to arsenic. The intracellular level of H2O2 is regulated by a wide range of enzymes, the most important ones being catalase (CAT) and peroxidase (POD) (Rusina et al., 2004). Both are known to play significant roles in regulating oxidative stress. Increase in APX and GPX activities in Cu-treated B. juncea plants is possibly for removal of H2O2 generated by SOD. These enzymes serve as an intrinsic defense tools to resist Cu-induced oxidative damage. Increase in APX and GPX activities in Cu-treated Phaseolus vulgaris, C. demersum, Lemna minor, Hydrilla verticillata and Salix viminalis has also been reported (Weckx and Clijsters, 1996; Gupta et al., 1996; Devi and Prasad 1998; Teisseire and Guy, 2000; Landberg and Greger, 2002), which is in agreement with the results of the present study using B. juncea. In earlier studies, it has been shown that when Cu is present in excess, it can promote and stimulate generation of Fenton-type reactive oxygen species leading to an increase in antioxidant enzyme activities (Devi and Prasad, 1998; Lombardi and Sebastiani, 2005). Increase in activities of these enzymes in the present study using B. juncea plants exposed to Cu suggests that there was a quick breakdown of superoxide radicals by SOD to keep their levels in control at the place of their generation, followed by detoxification of H2O2 by CAT, APX and GPX.

S. Singh et al. / Ecotoxicology and Environmental Safety 73 (2010) 1975–1981

5. Conclusions The present study has shown that in vitro grown B. juncea plants could effectively phytoextract Cu from solutions, but most of it was sequestered in roots with low levels of translocation to shoot biomass. The results of the present study have also shown that B. juncea var. TM 4 is equipped with efficient antioxidant enzymes, such as SOD, CAT, GPX and APX, the levels of which are enhanced to combat Cu-induced ROS.

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