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Antioxidative responses in relation to growth of mustard (Brassica juncea cv. Pusa Jaikisan) plants exposed to hexavalent chromium
Chemosphere 61 (2005) 40–47 www.elsevier.com/locate/chemosphere
Antioxidative responses in relation to growth of mustard (Brassica juncea cv. Pusa Jaikisan) plants exposed to hexavalent chromium Vivek Pandey *, Vivek Dixit, Radhey Shyam Stress Physiology, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India Received 13 August 2004; received in revised form 1 March 2005; accepted 8 March 2005 Available online 22 April 2005
Abstract Effect of hexavalent chromium (Cr6+) was seen on Brassica juncea cv. Pusa Jaikisan grown for 15 days in hydroponic culture supplemented with 0.2, 2 and 20 lM Cr. The inhibitory response of Cr6+ on growth of B. juncea was concentration and time dependent. The stimulation of plant growth, observed in response to exposure to 0.2 lM Cr6+, during initial 5 days was reversed on prolonged treatment and at higher Cr6+ concentrations (2 and 20 lM Cr6+). Despite reduction in growth, chlorophyll content increased substantially on 15 days exposure to 20 lM Cr6+. Significant increases in lipid peroxidation and tissue concentration of H2O2 occurred in plants exposed to 2 and 20 lM Cr6+. Effect of Cr6+ on antioxidative enzymes in roots and leaves was differential. SOD and CAT activities at lower levels of Cr6+ supply remained higher all through the treatment. While APX was very susceptible to excess Cr6+, GR and GST increased at elevated levels of Cr6+. The results suggested Cr6+ induced depression in plant growth of B. juncea to be a function of increased cellular accumulation of Cr despite increase in the activities of some of the antioxidative enzymes. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Antioxidative enzymes; Brassica juncea; Hexavalent chromium; Oxidative stress
1. Introduction Chromium (Cr) is a toxic element that occurs in highly variable oxidation states. Trivalent chromic (Cr3+) and hexavalent chromate (Cr6+) compounds are widely used industrial chemicals and are major contaminants of both soil and water. Both Cr3+ and Cr6+ are biologically active oxidation states. The latter is more toxic
* Corresponding author. Tel.: +91 522 2205831; fax: +91 522 2205836. E-mail address: [email protected] (V. Pandey).
and produces severe oxidative stress (Von Burg and Liu, 1993). The reaction of Cr with biological reductants produces short- or long-lived Cr intermediates of different valency states, that in turn react with hydrogen peroxide to generate hydroxyl radical (Stohs and Bagchi, 1995). It has now been invariably demonstrated that oxidative mechanisms are involved in the toxicity of metal ions in plants. Heavy metals stimulate the formation of reactive oxygen species (ROS) either by direct electron transfer involving metal cations, or as a consequence of metal mediated inhibition of metabolic reactions (Halliwell and Gutteridge, 1984). Similar to other stresses, plantÕs response to heavy metals result in changes in the levels
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.03.026
V. Pandey et al. / Chemosphere 61 (2005) 40–47
of antioxidants and antioxidative enzymes to detoxify the ROS. In addition to superoxide dismutase (SOD), catalase (CAT), and glutathione transferase (GST), the enzymes of ascorbate-glutathione cycle, ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) are reported to play an important role in overcoming the metal induced oxidative stress at cellular and sub-cellular levels (Foyer et al., 1997). Ascorbate and glutathione, other components of the antioxidative system, are found to increase under metal stress (Prasad et al., 1999). Since Cr6+ is a strong oxidant, it may cause severe oxidative stress in plant tissue. The antioxidative responses of plants to excess chromium have not been studied critically. The present study was undertaken to assess growth and antioxidative responses of mustard (Brassica juncea cv. Pusa Jaikisan) on exposure to Cr6+ at three levels (0.2, 2 and 20 lM). B. juncea was taken as the model because it has been suggested as a potent candidate for phytoremediation of metal enriched soils (Salt et al., 1995).
2. Material and methods B. juncea cv. Pusa Jaikisan plants were grown hydroponically in thermostatically controlled culture room maintained at 25 ± 1 °C. The plants were grown with half strength Hoagland solution and were provided with photosynthetic photon flux density (PPFD) at 200 lmol m2 s1 by fluorescent lamps for 16 h daily. Detailed methodology is described elsewhere (Dixit et al., 2001). After 15 days growth, plants were exposed to different Cr6+ concentrations (0.0, 0.2, 2 and 20 lM) supplied as potassium dichromate (K2Cr2O7). Plants were harvested after day 1, 2, 3, 4, 5 and 15 of the commencement of the treatment and washed thoroughly with sterile distilled water to determine various biophysical parameters. 2.1. Plant growth and chromium content Leaf area was measured on a Delta-T Devices Area Measurement System. Plant biomass was evaluated by drying fresh tissues at 80 °C. Chlorophyll and carotenoid contents were estimated according to Porra et al. (1989) and Duxbury and Yentsch (1956), respectively. The chromium content in roots and leaves was determined by atomic absorption spectrophotometry (Perkin-Elmer AAnalyst 300) after wet digestion of the samples as described by Veillon (1988). 2.2. Determination of anti-oxidative enzymes Roots and fully expanded leaves were ground with a chilled mortar and pestle in ice-cold homogenization
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buffer. The extraction buffer for APX and CAT consisted of 100 mM phosphate buffer (pH 7.0), 1 mM ascorbate (AsA), 1 mM ethylene diamine tetraacetic acid (EDTA) and 1 mM DL-dithiothreitol (DTT). The extraction buffer for SOD, GR and GST contained 100 mM phosphate buffer (pH 7.5), 1 mM EDTA, 1 mM DTT and 1 mM phenylmethanesulfonyl fluoride (PMSF). The homogenate was filtered through four layers of muslin cloth and centrifuged at 12 000g for 10 min at 4 °C. The supernatant was desalted with Sephadex G-25 column equilibrated with buffer suitable for different enzymes. Protein estimation was done using bovine serum albumin (Sigma) as standard (Peterson, 1979). Activity of SOD (EC 1.15.1.1) was assayed by using the photochemical nitro blue tetrazolium (NBT) method. The assay was performed in terms of SODÕs ability to inhibit reduction of NBT to form formazan by superoxide radical as described by Beyer and Fridovich (1987). The assay mixture in 3 ml contained 50 mM phosphate buffer, pH 7.8, 9.9 mM L-methionine, 57 lM NBT, 0.025% (w/v) Triton X-100, 0.0044% (w/v) riboflavin. The photoreduction of NBT (formation of purple formazan) was measured at 560 nm and an inhibition curve was made against different volumes of extract. One unit of SOD was defined as that being present in the volume of extract that caused inhibition of the photo-reduction of NBT by 50%. Activity of APX (EC 1.11.1.11) was measured by following the rate of hydrogen peroxide-dependent oxidation of ascorbic acid in a reaction mixture that contained 50 mM phosphate buffer (pH 7.0), 0.6 mM ascorbic acid and the enzyme extract (Chen and Asada, 1989). The reaction was initiated by addition of 10 ll of 10% (v/v) H2O2 and the oxidation rate of ascorbic acid was estimated by following the decrease in absorbance at 290 nm for 3 min. Activity of CAT (EC 1.11.1.6) was measured in 50 mM phosphate buffer, pH 7.0 by monitoring the production of oxygen (O2) from H2O2 (33.5 mM), using a Clark-type oxygen electrode (Hansatech, U.K.) as in del Rı´o et al. (1977). Activity of GR (EC 1.6.4.2) was monitored by following the increase in absorbance at 412 nm when 5, 5 0 -dithiobis (2-nitrobenzoic acid) (DTNB) was reduced by glutathione (GSH) to form 2-nitro-5-thiobenzoic acid (TNB) (Smith et al., 1988). The reaction mixture contained 100 mM phosphate buffer (pH 7.5), 0.5 mM EDTA, 0.75 mM DTNB, 0.1 mM NADPH and 1 mM oxidized glutathione (GSSG). To express GR activity, the increase in absorbance was plotted against a known amount of glutathione reductase. Activity of GST (EC 2.5.1.18) was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The enzyme activity was assayed in a reaction mixture containing 50 mM phosphate buffer (pH 7.5), 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and enzyme extract
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V. Pandey et al. / Chemosphere 61 (2005) 40–47
equivalent to 100 lg of protein. The reaction was initiated by the addition of 1 mM GSH and formation of S-(2,4-dinitrophenyl)glutathione (DNP-GS) was monitored as increase in absorbance at 334 nm to calculate the GST activity (Li et al., 1995). 2.3. Lipid peroxidation The level of lipid peroxidation in plant tissues was determined as 2-thiobarbituric acid (TBA) reactive metabolites chiefly malondialdehyde (MDA) as described by Heath and Packer (1968). 2.4. H2O2 accumulation H2O2 was measured spectrofluorometrically by measuring fluorescent compound dichlorofluorescein (DCF) produced during horseradish peroxidase-catalyzed reaction with H2O2 as substrate (Black and Brandt, 1974). 2.5. Statistical analysis All values reported in this work are mean of at least three independent experiments. A two-way ANOVA test was used to confirm the significance of the data. Comparison with control and between means of treatments was done by Tukey test.
3. Results The first visible symptom of Cr6+ exposure to B. juncea appeared in the form of leaf area reduction. On day 5 reduction in leaf area was 8%, 17%, and 28% in plants exposed to 0.2, 2 and 20 lM Cr6+, respectively. As the Cr6+ treatment prolonged, leaf area was significantly reduced at all the three levels of Cr6+ supply (Table 1). Up till day 5, 0.2 and 2 lM Cr6+ supply made little effect on biomass accumulation. In fact, there was a 15% increase in plant biomass at 0.2 lM Cr6+ on day 5. The biomass was, however, reduced by 19% and 38% respectively in the plants exposed to 2 and 20 lM Cr6+ on day 15 (Table 1). Chlorophyll content in leaves of Cr6+ treated B. juncea was mostly higher than the control during 15 days of metal exposure (Table 1). During initial 5 days of treatment this increment was evident at 0.2 and 2 lM Cr6+ but not at 20 lM Cr6+. Chlorophyll concentration at 20 lM Cr6+, however, increased significantly after day 5 when expansion of leaves was restricted. The dark green, small sized leaves of plants exposed to 20 lM Cr6+ for 15 days appeared quite distinct from those of the plants growing at 0.2 and 2 lM Cr6+. Carotenoid content in leaves of B. juncea increased invariably at 0.2 and 2 lM Cr6+ during 15 days of treatment (Table 1). As the treatment continued, level of
carotenoids at 20 lM Cr6+ increased to 36% over the control. Chromium content of Cr6+ treated B. juncea plants showed that the metal was largely restricted to the roots and very little of it was transported to the aerial parts. The roots of plants exposed to 0.2, 2 and 20 lM Cr6+ accumulated 76, 410 and 897 lg Cr g1 DW, respectively by day 5. Maximum accumulation of Cr was found in roots of plants grown for 15 days (Table 1). The amount of Cr detected in leaves by day 5 was quite low. The maximum amount of Cr (56.5 lg Cr g1 DW) was accumulated in leaves of plants grown at 20 lM Cr6+ for 15 days (Table 1). Exposure of B. juncea to Cr6+ resulted in accumulation of lipid peroxidation products (MDA), more in roots than in leaves. Significant increase in lipid peroxidation occurred from day 3 in the roots exposed to 20 lM Cr6+ (Table 2). In leaves, significant accumulation of MDA was observed from day 4, both at 2 and 20 lM Cr6+. Plant response to 2 and 20 lM Cr6+ produced significant accumulation of H2O2 from day 1 of the excess Cr6+ treatment (Table 2). At 0.2 lM Cr6+, H2O2 accumulation in roots and leaves was evident on day 5 and 15 (Table 2). Superoxide dismutase activity increased in roots but not in leaves during initial 3 days of treatment. Significant increase in SOD activity observed in roots at 0.2 lM Cr6+ on day 3 was maintained till day 15. At 2 lM Cr6+, SOD activity increased on day 2, 3 and 15. However, at 20 lM Cr6+ significant reduction in SOD activity took place in roots (Table 3). In leaves, increase in SOD activity was observed throughout the treatment at all levels of Cr6+ supply but the effect was not large enough to be significant. Ascorbate peroxidase activity was significantly increased in roots of Cr6+ treated plants until day 5. At 20 lM Cr6+, the APX activity showed maximum increase from day 1 to 5 but decreased significantly by day 15 (Table 3). APX activity in leaves at 0.2 and 2 lM Cr6+ was significantly higher than the control from day 2 to 5. While the increase at 20 lM Cr6+ was marginal from day 1 to 5, a significant decrease in the APX activity was observed on day 15 (Table 3). Catalase activity in roots was invariably higher than the control both at 0.2 and 2 lM Cr6+ supply. The increase was significant from day 3 to 15 at 0.2 lM Cr6+ and from day 1 to 5 at 2 lM Cr6+ (Table 3). The increase or decrease in catalase activity at 20 lM Cr6+ in roots was, however not significant. In leaves, catalase activity was stimulated throughout the treatment at all the three levels of Cr6+ supply (Table 3). Glutathione reductase, responsible for reduction of oxidized glutathione, showed a concentration dependent enhanced activity more in leaves than in roots. GR activity was significantly higher than the control in roots
V. Pandey et al. / Chemosphere 61 (2005) 40–47
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Table 1 Growth, chlorophyll, carotenoid and chromium accumulation in B. juncea exposed to different concentrations of hexavalent chromiuma,b Cr6+ supply (lM)
Day
0 2
Leaf area (cm )
5 15 5 15 5 15 5 15 5 15 5 15
Biomass (g) Chlorophyll (lg cm2) Carotenoids (lg cm2) Cr (lg g1 DW) Root Leaf
0.2 bA
2 bA
0.99 ± 0.02 1.27 ± 0.09aA 0.07 ± 0.007bB 0.09 ± 0.01aA 88.7 ± 4.76aB 105 ± 14.20aB 38.5 ± 0.25aA 49.5 ± 3.62aA 3.3 ± 0.26bD 37.0 ± 1.73aD ND 3.25 ± 0.29aC
0.98 ± 0.02 1.19 ± 0.08aB 0.08 ± 0.004aA 0.09 ± 0.001aA 107 ± 8.62aB 105 ± 13.39aB 41.6 ± 3.60aA 51.3 ± 4.69aA 75.9 ± 7.10bC 236 ± 10.00aC 6.48 ± 0.01bC 11.0 ± 0.80aB
20 aA
0.82 ± 0.04 0.95 ± 0.08aC 0.07 ± 0.001aB 0.07 ± 0.005aB 114 ± 22.95aA 101 ± 9.32aB 42.3 ± 7.74aA 50.0 ± 4.76aA 410 ± 12.53bB 634 ± 7.54aB 6.94 ± 0.02bB 24.5 ± 3.50aA
0.71 ± 0.05aB 0.71 ± 0.05aD 0.05 ± 0.003aC 0.06 ± 0.004bC 86.7 ± 8.53bB 133 ± 7.10aA 34.8 ± 2.45bA 67.3 ± 4.51aB 897 ± 7.54bA 1116 ± 26.0aA 16.0 ± 0.51bA 56.5 ± 1.50aA
a
Values are expressed as mean ± SD, n = 4. Values followed by the same lowercase letter in each column or uppercase letter in each row for each variable are not significantly different based on Tukey test (P = 0.05). b
Table 2 Effect of hexavalent chromium on lipid peroxidation and H2O2 accumulation in B. junceaa,b Cr6+ (lM)
MDA (nmol g FW Root 0.0 0.2 2.0 20 Leaf
0.0 0.2 2.0 20
1
Day 1
2
3
4
5
15
32 ± 4.0bA 32 ± 5.8bA 35.2 ± 2.9cdA 38.1 ± 2.6dA
33 ± 2.9bA 31.6 ± 2.9bA 32.8 ± 2.0dA 39.6 ± 3.2dA
32.2 ± 3.0bB 33 ± 4.4bB 41.4 ± 4.2bcAB 47.4 ± 4.2cdA
43.6 ± 4.0 abB 45 ± 3.4aB 47.5 ± 4.1bcAB 56.6 ± 3.2bcA
45.6 ± 3.0aB 49 ± 3.4aB 51.2 ± 3.5abB 65.7 ± 6.2abA
48 ± 2.9aB 54.8 ± 3.2aB 58.8 ± 5.6aB 70.1 ± 4.1aA
108 ± 10.7cA 102 ± 6.8cA 122 ± 14.3cA 125 ± 6.3cA
127 ± 11.9bcAB 112 ± 23.2cB 120 ± 10.1cB 157 ± 9.6deA
148 ± 16.4abAB 131 ± 20. 3bcB 162 ± 9.9bAB 170 ± 11.4cdA
154 ± 6.1abB 148 ± 10.4bB 171 ± 4.6bA 171 ± 4.6bA
155 ± 22.7abB 153 ± 11.5bB 180 ± 9.5bAB 209 ± 12.1bA
166 ± 2.5aB 180 ± 16.7aB 223 ± 10aA 248 ± 12aA
39.3 ± 4.1bcC 50.6 ± 6.1bC 70.6 ± 6.1bB 95.3 ± 5.0bA
44.6 ± 6.4abC 62 ± 2.0bB 91.6 ± 3.0aA 100 ± 6.0bA
53.3 ± 1.5aD 76 ± 5.2aC 106 ± 4.0aB 124 ± 4.0aA
54 ± 0.0aC 76.6 ± 2.3aB 102 ± 2.0aA 113 ± 1.1aA
50 ± 4.5aD 82.6 ± 2.0aC 103 ± 2.8aB 121 ± 3.2aA
132 ± 6.4aC 143 ± 3.0aB 182 ± 4.6aA 192 ± 2.0aA
88.3 ± 1.1cdC 94.6 ± 2.5cdC 106 ± 1.1dB 116 ± 1.1dA
84.3 ± 0.5cdC 90.6 ± 7.6dBC 96.6 ± 1.5dAB 106 ± 2.6eA
80.0 ± 0.0dD 90.6 ± 1.1dC 108.0 ± 4.0cdB 122.6 ± 1.1dA
93.3 ± 1.1cD 103 ± 6.3cC 116 ± 2.3cB 134 ± 2.0cA
)
H2O2 accumulation (nmol g FW 1) Root 0.0 28.6 ± 4.1cC 0.2 38.0 ± 4.0cBC 2.0 41.6 ± 2.5cB 20 59 ± 7.0cA Leaf
0.0 0.2 2.0 20
115 ± 5.0bC 124 ± 2.0bC 140 ± 3.0bB 162 ± 3.0bA
a
Values are expressed as means ± SD, n = 4. Values followed by the same lowercase letter in each row or uppercase letter in each column for each variable are not significantly different based on Tukey test (P = 0.05). b
at 2 and 20 lM Cr6+ from day 3 to 15 (Table 3). Similarly, significant increase in GR activity was observed from day 3 to 15 in leaves. On day 15, however, the activity declined from day 5 at all the levels of Cr6+ supply but remained higher than the control.
Activity of GST in roots of plants exposed to Cr6+ was invariably higher than the control but the increase was significant only at 2 and 20 lM Cr6+ during 3–5 day (Table 3). In leaves significant change in GST activity was not observed except at 2 lM Cr6+ supply.
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V. Pandey et al. / Chemosphere 61 (2005) 40–47
Table 3 Effect of hexavalent chromium on antioxidative enzymes in B. junceaa,b Cr6+ (lM)
Day 1
2
3
4
5
15
22.4 ± 1.1bcB 25.8 ± 1.0cAB 31.4 ± 2.7bcA 25.0 ± 2.3aAB
25.0 ± 1.6bcB 35.5 ± 2.1bA 29.0 ± 2.8bA 28.0 ± 2.7aB
28.4 ± 1.4 bB 40.4 ± 1.1abA 36.0 ± 2.7bAB 24.0 ± 2.2aC
30.3 ± 2.0bB 47.0 ± 2.4aA 42.0 ± 2.3bB 23.0 ± 2.7aC
36.5 ± 2.3aB 42.8 ± 2.9abA 46.8 ± 3.6aA 22.0 ± 3.2aC
22.1 ± 0.6bA 27.0 ± 2.1bA 25.5 ± 1.0bcA 24.4 ± 4.1abA
26.2 ± 0.3abA 30.9 ± 2.1abA 30.8 ± 2.3bA 27.5 ± 1.9abA
26.9 ± 2.2abA 33.7 ± 2.3abA 36.7 ± 2.2abA 30.7 ± 3.1aA
30.9 ± 1.2aA 38.6 ± 3.5aA 37.8 ± 3.7abA 34.6 ± 4.3aA
34.0 ± 2.4aA 42.5 ± 2.9aA 41.0 ± 2.5aA 35.4 ± 3.7aA
1.40 ± 0.04bB 2.48 ± 0.17bcA 2.30 ± 0.32aA 1.88 ± 0.17abAB
1.66 ± 0.06bC 3.12 ± 0.12aA 2.80 ± 0.26aA 2.14 ± 0.11aBC
1.87 ± 0.14bB 2.88 ± 0.10abA 2.71 ± 0.18aA 2.28 ± 0.11aAB
2.02 ± 0.07aB 2.89 ± 0.18abA 2.65 ± 0.27aA 2.36 ± 0.22aAB
2.30 ± 0.14aA 2.35 ± 0.23bcA 2.20 ± 0.15abA 1.20 ± 0.19cB
1.10 ± 0.08aB 1.65 ± 0.06abA 1.45 ± 0.07abA 1.23 ± 0.02aB
1.12 ± 0.15aC 1.85 ± 0.21aA 1.55 ± 0.08abAB 1.42 ± 0.14aBC
1.16 ± 0.10aC 1.94 ± 0.14aA 1.65 ± 0.07aAB 1.40 ± 0.09aBC
1.22 ± 0.13aB 1.78 ± 0.13aA 1.56 ± 0.11abAB 1.44 ± 0.04aAB
1.40 ± 0.09aB 1.50 ± 0.10bAB 1.60 ± 0.06aAB 0.93 ± 0.01bC
Catalase (U mg protein1) Root 0.0 60.0 ± 3.4bB 0.2 75.0 ± 4.0cAB 2.0 90.0 ± 8.9cA 20 70.0 ± 4.3bAB
63.0 ± 7.2abB 80.0 ± 3.6cAB 87.0 ± 5.5cA 75.0 ± 5.0bAB
65.0 ± 5.0abC 92.0 ± 8.5bcAB 105 ± 8.9bcA 80.0 ± 7.0bBC
85.0 ± 7.0aB 148 ± 6.7aA 153 ± 9.7aA 108 ± 11.0aB
80.0 ± 6.9abB 115 ± 9.5bA 125 ± 8.0bA 75.0 ± 7.5bB
85.0 ± 6.0aBC 110 ± 9.5bA 108 ± 12.5bcAB 65.0 ± 6.5bC
90.0 ± 4.4aC 195 ± 11.0abA 190 ± 9.9abA 128 ± 6.0bB
103 ± 6.5aC 185 ± 9.9bcA 170 ± 8.1abAB 140 ± 4.5bB
105 ± 7.5aC 220 ± 15.8aA 195 ± 10.2aA 142 ± 9.0bB
108 ± 5.0aB 175 ± 5.5bcA 160 ± 8.3bcA 150 ± 10.8abA
108 ± 4.2aB 155 ± 10.3cdA 150 ± 8.3cA 153 ± 8.6abA
120 ± 8.0aC 140 ± 6.0dBC 155 ± 5.8cAB 177 ± 12.1aA
(U mg protein1) 0.05 ± 0.006eA 0.05 ± 0.004eA 0.05 ± 0.004eA 0.07 ± 0.005fA
0.09 ± 0.006dB 0.09 ± 0.009dB 0.10 ± 0.005dAB 0.12 ± 0.006eA
0.12 ± 0.005cB 0.13 ± 0.007cB 0.15 ± 0.006cA 0.17 ± 0.006dA
0.15 ± 0.007bC 0.15 ± 0.009bC 0.18 ± 0.006bB 0.22 ± 0.009cA
0.23 ± 0.006aC 0.24 ± 0.010aC 0.26 ± 0.007aB 0.28 ± 0.009bA
0.24 ± 0.005aC 0.26 ± 0.006aB 0.26 ± 0.005aB 0.33 ± 0.01aA
0.20 ± 0.005dA 0.20 ± 0.01eA 0.23 ± 0.01dA 0.22 ± 0.01eA
0.28 ± 0.003cC 0.29 ± 0.01dBC 0.32 ± 0.01cB 0.35 ± 0.01dA
0.30 ± 0.01bcC 0.33 ± 0.01cB 0.37 ± 0.01bA 0.39 ± 0.01cA
0.30 ± 0.01bcC 0.35 ± 0.01bcB 0.40 ± 0.02bA 0.43 ± 0.01bA
0.37 ± 0.01aC 0.47 ± 0.01aB 0.49 ± 0.02aB 0.53 ± 0.02aA
0.31 ± 0.01bC 0.38 ± 0.01bB 0.39 ± 0.01bB 0.52 ± 0.01aA
0.25 ± 0.01aB 0.30 ± 0.02abAB 0.31 ± 0.03aA 0.31 ± 0.01aA
0.22 ± 0.01aB 0.28 ± 0.01abAB 0.32 ± 0.01aA 0.33 ± 0.01aA
0.26 ± 0.01aB 0.33 ± 0.02aA 0.34 ± 0.02aA 0.31 ± 0.02aA
0.26 ± 0.01aA 0.30 ± 0.01abA 0.31 ± 0.01aA 0.29 ± 0.01abA
0.19 ± 0.01aA 0.20 ± 0.01abA 0.20 ± 0.01cA 0.19 ± 0.01bcA
0.19 ± 0.01aB 0.21 ± 0.01aAB 0.25 ± 0.01bcA 0.20 ± 0.01abAB
0.20 ± 0.02aB 0.22 ± 0.01aB 0.29 ± 0.00aA 0.22 ± 0.01abB
0.21 ± 0.01aA 0.22 ± 0.01aA 0.22 ± 0.01bcA 0.23 ± 0.01aA
Superoxide dismutase Root 0.0 0.2 2.0 20 Leaf
0.0 0.2 2.0 20
Ascorbate peroxidase Root 0.0 0.2 2.0 20 Leaf
Leaf
0.0 0.2 2.0 20
0.0 0.2 2.0 20
Glutathione reductase Root 0.0 0.2 2.0 20 Leaf
0.0 0.2 2.0 20
(U mg protein1) 19.6 ± 1.1cA 20.8 ± 0.8cA 25.4 ± 2.6cA 21.9 ± 2.3aA 18.6 ± 1.1bA 23.0 ± 1.7bA 20.9 ± 2.1cA 19.0 ± 3.1bA (U mg protein1) 1.43 ± 0.10bA 2.00 ± 0.13cA 1.90 ± 0.09bA 1.67 ± 0.09bA 1.07 ± 0.10aA 1.40 ± 0.15bA 1.24 ± 0.14bA 1.19 ± 0.06aA
Glutathione-S-transferase (U mg protein1) Root 0.0 0.20 ± 0.02aA 0.21 ± 0.01aB 0.2 0.22 ± 0.01cA 0.26 ± 0.01bcAB bA 2.0 0.25 ± 0.01 0.29 ± 0.01abA 20 0.25 ± 0.01bA 0.25 ± 0.01abAB Leaf
a
0.0 0.2 2.0 20
0.16 ± 0.01aA 0.16 ± 0.01bA 0.17 ± 0.01cA 0.17 ± 0.01cA
0.16 ± 0.01aA 0.17 ± 0.01bA 0.17 ± 0.01cA 0.17 ± 0.01cA
Values are expressed as means ± SD, n = 4. Values followed by the same lowercase letter in each row or uppercase letter in each column for each variable are not significantly different based on Tukey test (P = 0.05). b
V. Pandey et al. / Chemosphere 61 (2005) 40–47
4. Discussion Chromium stimulates growth in certain plant species (Bonet et al., 1991; Han et al., 2004). In the present study, marginal stimulation of growth in B. juncea in the beginning at 0.2 lM Cr6+ could not be sustained during 15 days of growth as apparent from minor change in biomass accumulation and significant reduction in leaf area as compared to control. Plant biomass was severely reduced particularly at 2 and 20 lM Cr6+. Decreased biomass production in Cr6+ exposed B. juncea is in conformity with earlier findings on different plant species (Barcelo´ et al., 1985; Sharma and Sharma, 1993, 1996; Dixit et al., 2002). Chromium treatment affected leaf expansion in the present study as evident from production of smaller leaves. The severity of reduction in leaf size increased with increasing Cr6+ concentration and duration of exposure. Similar observations with reference to Cr6+ were also made in Phaseolus vulgaris (Barcelo´ et al., 1985), Pisum sativum (Dixit et al., 2002) and Vigna radiata (Shanker et al., 2004). One possible mechanism to explain the reduction in leaf area under metal stress is the restricted availability of water for leaf expansion (Radin and Boyer, 1982). There is also a good possibility of Cr6+ interaction with endogenous phytohormones that control plant growth processes (Moya et al., 1995). Significant increase in chlorophyll and carotenoids was seen in plants exposed to 20 lM Cr6+ on day 15. These observations are interesting and contrary as heavy metals are known to interact with the sulphydryl site on enzymes, d-aminolevulinic acid dehydrogenase and protochlorophyllide reductase of chlorophyll biosynthesis (Ouzounidou, 1995). However, both decrease and increase in the level of chlorophyll has been reported in different plant species exposed to Cr6+ (Sharma et al., 1995; Sharma and Sharma, 1996; Dixit et al., 2002; Samantary, 2002). An instantaneous change in level of carotenoids along with chlorophylls in Cr6+ stressed B. juncea was an interesting phenomenon observed in the present study. Generation of reactive oxygen species particularly singlet oxygen and oxygen radical may enhance under influence of Cr6+ ions during growth light exposure. Carotenoids can interact with the reactive oxygen species, preventing the initiation of potentially lethal processes such as lipid peroxidation (Pallett and Young, 1993). Low transport of Cr6+ from root to aerial parts in B. juncea is in accord with observations on pea (Dixit et al., 2002), cauliflower and cabbage (Zayed et al., 1998), and Indian mustard (Han et al., 2004). Hexavalent chromium, being a strong oxidant, produces oxidative stress as evident from enhanced level of lipid peroxidation and increasing tissue concentration of H2O2 in plants exposed to 2 and 20 lM Cr6+. Most heavy metals cause oxidative stress via generation of
45
reactive oxygen species (Dietz et al., 1999). There is, however, no evidence as yet of Cr6+ participation in generation of ROS in plants although Cr6+ induced enhanced generation of superoxide radicals has been demonstrated in vivo in pea root mitochondria (Dixit et al., 2002). The first line of defense against ROS-mediated toxicity is achieved by SOD that catalyzes the dismutation of superoxide radicals to H2O2 and O2. Significant increase in the SOD activity in roots at 0.2 and 2 lM Cr6+ may be a part of this defense mechanism. Significant decrease in SOD activity at 20 lM Cr6+ indicated interaction of Cr6+ with the enzyme. The role of SOD in Cr6+ induced toxicity in leaves seems to be limited in the present study as there was no significant increase in the activity of this enzyme. Plants exposed to different metals respond differently with reference to activity of SOD. Toxic concentrations of Zn could not induce SOD activity in beans (Weckx and Clijsters, 1996) but caused multifold enhancement in the SOD activity in mustard (Prasad et al., 1999). SOD activity decreased or remained unchanged in certain plant species exposed to cadmium (Cd) while in others it increased (Somashekaraiah et al., 1992; Dixit et al., 2001). Hexavalent chromium increased H2O2 accumulation both in roots and leaves. Plants exposed to 20 lM Cr6+ accumulated high level of H2O2 but showed decreased or poor SOD activity. This shows that either H2O2 is being generated by other enzymatic or nonenzymatic processes other than SOD dismutation or it is not being efficiently scavenged in Cr6+ treated plants. APX and CAT are two potent scavengers of H2O2, which minimize its accumulation and diffusion across the cellular membranes, preventing peroxidative damage to cellular constituents. Enhancement in the activity of both APX and CAT was recorded at 20 lM Cr6+ in roots as well as in leaves and this possibly contributed to substantial accumulation of H2O2 in these plant parts. Foyer et al. (1994) have shown that GR is one of the key enzymes that helps in reduction of GSSG to GSH by oxidizing NAD(P)H to NAD(P)+ and suggested its crucial role in combating oxidative stress in leaves. We found that GR activity was elevated more in leaves than in root. Prasad et al. (1999) have also reported up to 158% increase in shoot GR activity in B. juncea seedlings raised under Zn toxicity. However Satyakala and Jamil (1992) reported decreased GR activity in leaves and increased activity in roots of two Cr6+ treated aquatic weeds. GST catalyses conjugation of electrophilic xenobiotic substrates with glutathione. In the present study, Cr6+ induced differential GST response. While there was an increase in GST activity in roots, no significant changes were observed in leaves. Increase in GST activity was also found in Cd treated pea roots (Dixit et al., 2001). This low activity of GST in the present study can be
46
V. Pandey et al. / Chemosphere 61 (2005) 40–47
attributed to low amount of Cr6+ present in treated leaves. The present study showed a differential response of Cr6+ to antioxidative enzymes in roots and leaves (Fig. 1). While certain enzymes are expressed in excess,
1200
A
A
200 175
1000
150 800
125
600
100 75
400
50 200 0 1200
25
B
B
0 200 175
1000
125
600
100
400
75 50
200
25
0 120
C
C
0 200 175
100
Relative Enzyme Activity
Cellular chromium (µg g-1 dry wt)
150 800
150 80
125
60
100
40
75 50
20 0 120
others are inhibited at higher levels of cellular Cr. APX was found to be the most susceptible enzyme to Cr6+ while GST and GR were optimally increased at elevated cellular Cr concentration. With the increasing Cr accumulation, SOD and CAT activities mostly decreased but remained always higher than the control both in roots and leaves. The activities of these two enzymes were, however, severely inhibited in roots at 20 lM Cr6+ wherein cellular Cr showed highest accumulation. Our observations indicate that up to certain level of cellular Cr, CAT compensates the Cr induced loss in APX activity. As the cellular concentration of Cr increased, plant growth was affected despite increase in the activities of some of the enzymes. These enzymes seem to overcome oxidative stress induced by Cr6+ as no evidence of oxidative symptoms was found even at 20 lM Cr6+ in leaves. Due to increased Cr accumulation, plants can no longer maintain metal homoeostasis. The role of antioxidative defense may also become limiting, as Cr6+ is also known to interact with certain micronutrients essential for plant growth and limit their availability either by decreased uptake or immobilization in roots (Barcelo´ et al., 1985). The possibility of Cr6+ interacting synergistically with other physiological and biochemical components to impair plant growth cannot be ruled out. Although enzymatic detoxification is an efficient mechanism for scavenging the damaging oxygen species, more efficient protective processes (such as metal exclusion, immobilization, compartmentalization and complexing) are needed to prevent the generation of these species and availability of free metal ions to sensitive sites in the first instance.
25
D
D
0 200 175
100
150 80
125
60
Acknowledgments This work was undertaken under Institutional Project No. OLP-230525. We are grateful to the Director, NBRI, Lucknow for necessary facilities and encouragement. One of us (VD) is grateful to Council of Scientific and Industrial Research for providing Senior Research fellowship. Thanks are also due to Prof. C.P. Sharma for critically going through the manuscript and helpful suggestions.
100 75
40
References
50 20 0
25
Cr6+
0
SOD CAT APX GR
GST
Fig. 1. Relative enzyme activity in relation to cellular chromium in B. juncea exposed to Cr6+. Day 5 root (A), Day 15 root (B), Day 5 leaf (C), Day 15 leaf (D); (j) 0.2 lM Cr, ( ) 2 lM Cr, ( ) 20 lM Cr.
Barcelo´, J., Poschenrieder, Ch., Gunse´, B., 1985. Effect of chromium VI on mineral element composition of bush beans. J. Plant Nutr. 8, 211–217. Beyer, W.F., Fridovich, Y., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal. Biochem. 161, 559–566. Black, M.J., Brandt, R.B., 1974. Spectrofluorometric analysis of hydrogen peroxide. Anal. Biochem. 58, 246–254.
V. Pandey et al. / Chemosphere 61 (2005) 40–47 Bonet, A., Poschenrieder, Ch., Barcelo´, J., 1991. Chromium III–iron interaction in Fe-deficient and Fe-sufficient bean plants. I. Growth and nutrient content. J. Plant Nutr. 14, 403–414. Chen, G.X., Asada, K., 1989. Ascorbate peroxidase in tea leaves: occurrence of two isozymes and the differences in their enzymatic and molecular properties. Plant Cell Physiol. 30, 987–998. Dietz, K.-J., Baier, M., Kra¨mer, U., 1999. Free radicals and reactive oxygen species as mediators of heavy metal toxicity in plants. In: Prasad, M.N.V., Hagemeyer, J. (Eds.), Heavy Metal Stress in Plants: from Molecules to Ecosystems. Springer-Verlag, Berlin, pp. 73–99. del Rı´o, L.A., Ortega, M.G., Lopez, A.L., Gorge, J.L., 1977. A more sensitive modification of the catalase assay with the Clark oxygen electrode: application to the kinetic study of the pea leaf enzyme. Anal. Biochem. 80, 409–415. Dixit, V., Pandey, V., Shyam, R., 2001. Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad). J. Exp. Bot. 52, 1101–1109. Dixit, V., Pandey, V., Shyam, R., 2002. Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria. Plant Cell Environ. 25, 687–693. Duxbury, A.C., Yentsch, C.S., 1956. Plankton pigment monograph. J. Marine Res. 15, 92–101. Foyer, C.H., Descourvieres, P., Kunert, K.J., 1994. Protection against oxygen radicals: an important defence mechanism studied in transgenic plants. Plant Cell Environ. 17, 507– 523. Foyer, C.H., Lopez-Delgado, H., Dat, J.F., Scott, I.M., 1997. Hydrogen peroxide-and glutathione-associated mechanisms of acclamatory stress tolerance and signaling. Physiol. Plant 100, 241–254. Halliwell, B., Gutteridge, M.C., 1984. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, 1–14. Han, F.X., Maruthi Sridhar, B.B., Monts, D.L., Su, Y., 2004. Phytoavailability and toxicity of trivalent and hexavalent chromium to Brassica juncea. New Phytol. 162, 489–499. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–190. Li, Z.-S., Zhen, R.-G., Rea, P.A., 1995. 1-Chloro-2, 4-dinitrobenzene-elicited increase in vacuolar glutathione-S-conjugate transport activity. Plant Physiol. 109, 177–185. Moya, J.L., Ros, R., Picazo, I., 1995. Heavy-metal hormone interactions in rice plants: effects on growth, net photosynthesis, and carbohydrate distribution. J. Plant Growth Regulation 14, 61–67. Ouzounidou, G., 1995. Cu-ions mediated changes in growth, chlorophyll and other ion contents in a Cu-tolerant Koeleria splendens. Biol. Plant 37, 71–79. Pallett, K.E., Young, A.J., 1993. Carotenoids. In: Alscher, R.G., Hess, J.L. (Eds.), Antioxidants in Higher Plants. CRC Press, Boca Raton, pp. 59–89. Peterson, G.L., 1979. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83, 346–356.
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Porra, R.J., Thompson, W.A., Kriedman, P.E., 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochem. Biophys. Acta 975, 384–394. Prasad, K.V.S.K., Paradha Saradhi, P., Sharmila, P., 1999. Concerted action of antioxidant and curtailed growth under zinc toxicity in Brasica juncea. Environ. Exp. Bot. 42, 1–10. Radin, J.W., Boyer, J.S., 1982. Control of leaf expansion by nitrogen nutrition in sunflower plants: role of hydraulic conductivity and turgor. Plant Physiol. 69, 771–775. Salt, D.E., Prince, R.C., Pickering, I.J., Raskin, I., 1995. Mechanisms of cadmium mobility and accumulation in Indian mustard. Plant Physiol. 109, 1427–1433. Samantary, S., 2002. Biochemical responses of Cr-tolerant and Cr-sensitive mung bean grown on varying levels of chromium. Chemosphere 47, 1065–1072. Satyakala, G., Jamil, K., 1992. Chromium induced biochemical changes in Eichhornia crassipes (Mort) Solms and Pistia stratiotes L. Bull. Environ. Contam. Toxicol. 48, 921–928. Shanker, A.K., Djanaguiraman, M., Sudhagar, R., Chandrashekhar, C.N., Pathmanabhan, G., 2004. Differential antioxidative response of ascorbate glutathione pathway enzymes and metabolites to chromium speciation stress in green gram (Vigna radiata (L.) R. Wilczek. Cv CO 4) roots. Plant Sci. 166, 1035–1043. Sharma, D.C., Sharma, C.P., 1993. Effect of chromium on growth and biological yield of maize (Zea mays L. cv. Ganga 5). Indian J. Plant Physiol. 36, 61–64. Sharma, D.C., Sharma, C.P., 1996. Chromium uptake and toxicity effects on growth and metabolic activities in wheat, Triticum aestivum L. cv. UP 2003. Indian J. Exp. Biol. 34, 689–691. Sharma, D.C., Chatterjee, C., Sharma, C.P., 1995. Chromium accumulation and its effects on wheat (Triticum aestivum L. cv. HD 2204) metabolism. Plant Sci. 111, 145–151. Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase in crude tissue homogenates using 5,5 0 -dithiobis(2-nitrobenzoic acid). Anal. Biochem. 175, 408–413. Somashekaraiah, B.V., Padmaja, K., Prasad, A.R.K., 1992. Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris): involvement of lipid peroxidation in chlorophyll degradation. Physiol. Plant 85, 85–89. Stohs, S.J., Bagchi, D., 1995. Oxidative mechanism in the toxicity of metal ions. Free Radical Biol. Med. 18, 321–336. Veillon, C., 1988. Chromium. Methods Enzymol. 158, 334–343. Von Burg, R., Liu, D., 1993. Chromium and hexavalent chromium. J. Appl. Toxicol. 13, 225–230. Weckx, J.E.J., Clijsters, H.M.M., 1996. Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper. Physiol. Plant 96, 506–512. Zayed, A.C., Lytle, M., Qian, J., Terry, N., 1998. Chromium accumulation, translocation and chemical speciation in vegetable crops. Planta 206, 293–299.
Antioxidative responses in relation to growth of mustard (Brassica juncea cv. Pusa Jaikisan) plants exposed to hexavalent chromium Vivek Pandey *, Vivek Dixit, Radhey Shyam Stress Physiology, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India Received 13 August 2004; received in revised form 1 March 2005; accepted 8 March 2005 Available online 22 April 2005
Abstract Effect of hexavalent chromium (Cr6+) was seen on Brassica juncea cv. Pusa Jaikisan grown for 15 days in hydroponic culture supplemented with 0.2, 2 and 20 lM Cr. The inhibitory response of Cr6+ on growth of B. juncea was concentration and time dependent. The stimulation of plant growth, observed in response to exposure to 0.2 lM Cr6+, during initial 5 days was reversed on prolonged treatment and at higher Cr6+ concentrations (2 and 20 lM Cr6+). Despite reduction in growth, chlorophyll content increased substantially on 15 days exposure to 20 lM Cr6+. Significant increases in lipid peroxidation and tissue concentration of H2O2 occurred in plants exposed to 2 and 20 lM Cr6+. Effect of Cr6+ on antioxidative enzymes in roots and leaves was differential. SOD and CAT activities at lower levels of Cr6+ supply remained higher all through the treatment. While APX was very susceptible to excess Cr6+, GR and GST increased at elevated levels of Cr6+. The results suggested Cr6+ induced depression in plant growth of B. juncea to be a function of increased cellular accumulation of Cr despite increase in the activities of some of the antioxidative enzymes. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Antioxidative enzymes; Brassica juncea; Hexavalent chromium; Oxidative stress
1. Introduction Chromium (Cr) is a toxic element that occurs in highly variable oxidation states. Trivalent chromic (Cr3+) and hexavalent chromate (Cr6+) compounds are widely used industrial chemicals and are major contaminants of both soil and water. Both Cr3+ and Cr6+ are biologically active oxidation states. The latter is more toxic
* Corresponding author. Tel.: +91 522 2205831; fax: +91 522 2205836. E-mail address: [email protected] (V. Pandey).
and produces severe oxidative stress (Von Burg and Liu, 1993). The reaction of Cr with biological reductants produces short- or long-lived Cr intermediates of different valency states, that in turn react with hydrogen peroxide to generate hydroxyl radical (Stohs and Bagchi, 1995). It has now been invariably demonstrated that oxidative mechanisms are involved in the toxicity of metal ions in plants. Heavy metals stimulate the formation of reactive oxygen species (ROS) either by direct electron transfer involving metal cations, or as a consequence of metal mediated inhibition of metabolic reactions (Halliwell and Gutteridge, 1984). Similar to other stresses, plantÕs response to heavy metals result in changes in the levels
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.03.026
V. Pandey et al. / Chemosphere 61 (2005) 40–47
of antioxidants and antioxidative enzymes to detoxify the ROS. In addition to superoxide dismutase (SOD), catalase (CAT), and glutathione transferase (GST), the enzymes of ascorbate-glutathione cycle, ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) are reported to play an important role in overcoming the metal induced oxidative stress at cellular and sub-cellular levels (Foyer et al., 1997). Ascorbate and glutathione, other components of the antioxidative system, are found to increase under metal stress (Prasad et al., 1999). Since Cr6+ is a strong oxidant, it may cause severe oxidative stress in plant tissue. The antioxidative responses of plants to excess chromium have not been studied critically. The present study was undertaken to assess growth and antioxidative responses of mustard (Brassica juncea cv. Pusa Jaikisan) on exposure to Cr6+ at three levels (0.2, 2 and 20 lM). B. juncea was taken as the model because it has been suggested as a potent candidate for phytoremediation of metal enriched soils (Salt et al., 1995).
2. Material and methods B. juncea cv. Pusa Jaikisan plants were grown hydroponically in thermostatically controlled culture room maintained at 25 ± 1 °C. The plants were grown with half strength Hoagland solution and were provided with photosynthetic photon flux density (PPFD) at 200 lmol m2 s1 by fluorescent lamps for 16 h daily. Detailed methodology is described elsewhere (Dixit et al., 2001). After 15 days growth, plants were exposed to different Cr6+ concentrations (0.0, 0.2, 2 and 20 lM) supplied as potassium dichromate (K2Cr2O7). Plants were harvested after day 1, 2, 3, 4, 5 and 15 of the commencement of the treatment and washed thoroughly with sterile distilled water to determine various biophysical parameters. 2.1. Plant growth and chromium content Leaf area was measured on a Delta-T Devices Area Measurement System. Plant biomass was evaluated by drying fresh tissues at 80 °C. Chlorophyll and carotenoid contents were estimated according to Porra et al. (1989) and Duxbury and Yentsch (1956), respectively. The chromium content in roots and leaves was determined by atomic absorption spectrophotometry (Perkin-Elmer AAnalyst 300) after wet digestion of the samples as described by Veillon (1988). 2.2. Determination of anti-oxidative enzymes Roots and fully expanded leaves were ground with a chilled mortar and pestle in ice-cold homogenization
41
buffer. The extraction buffer for APX and CAT consisted of 100 mM phosphate buffer (pH 7.0), 1 mM ascorbate (AsA), 1 mM ethylene diamine tetraacetic acid (EDTA) and 1 mM DL-dithiothreitol (DTT). The extraction buffer for SOD, GR and GST contained 100 mM phosphate buffer (pH 7.5), 1 mM EDTA, 1 mM DTT and 1 mM phenylmethanesulfonyl fluoride (PMSF). The homogenate was filtered through four layers of muslin cloth and centrifuged at 12 000g for 10 min at 4 °C. The supernatant was desalted with Sephadex G-25 column equilibrated with buffer suitable for different enzymes. Protein estimation was done using bovine serum albumin (Sigma) as standard (Peterson, 1979). Activity of SOD (EC 1.15.1.1) was assayed by using the photochemical nitro blue tetrazolium (NBT) method. The assay was performed in terms of SODÕs ability to inhibit reduction of NBT to form formazan by superoxide radical as described by Beyer and Fridovich (1987). The assay mixture in 3 ml contained 50 mM phosphate buffer, pH 7.8, 9.9 mM L-methionine, 57 lM NBT, 0.025% (w/v) Triton X-100, 0.0044% (w/v) riboflavin. The photoreduction of NBT (formation of purple formazan) was measured at 560 nm and an inhibition curve was made against different volumes of extract. One unit of SOD was defined as that being present in the volume of extract that caused inhibition of the photo-reduction of NBT by 50%. Activity of APX (EC 1.11.1.11) was measured by following the rate of hydrogen peroxide-dependent oxidation of ascorbic acid in a reaction mixture that contained 50 mM phosphate buffer (pH 7.0), 0.6 mM ascorbic acid and the enzyme extract (Chen and Asada, 1989). The reaction was initiated by addition of 10 ll of 10% (v/v) H2O2 and the oxidation rate of ascorbic acid was estimated by following the decrease in absorbance at 290 nm for 3 min. Activity of CAT (EC 1.11.1.6) was measured in 50 mM phosphate buffer, pH 7.0 by monitoring the production of oxygen (O2) from H2O2 (33.5 mM), using a Clark-type oxygen electrode (Hansatech, U.K.) as in del Rı´o et al. (1977). Activity of GR (EC 1.6.4.2) was monitored by following the increase in absorbance at 412 nm when 5, 5 0 -dithiobis (2-nitrobenzoic acid) (DTNB) was reduced by glutathione (GSH) to form 2-nitro-5-thiobenzoic acid (TNB) (Smith et al., 1988). The reaction mixture contained 100 mM phosphate buffer (pH 7.5), 0.5 mM EDTA, 0.75 mM DTNB, 0.1 mM NADPH and 1 mM oxidized glutathione (GSSG). To express GR activity, the increase in absorbance was plotted against a known amount of glutathione reductase. Activity of GST (EC 2.5.1.18) was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The enzyme activity was assayed in a reaction mixture containing 50 mM phosphate buffer (pH 7.5), 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and enzyme extract
42
V. Pandey et al. / Chemosphere 61 (2005) 40–47
equivalent to 100 lg of protein. The reaction was initiated by the addition of 1 mM GSH and formation of S-(2,4-dinitrophenyl)glutathione (DNP-GS) was monitored as increase in absorbance at 334 nm to calculate the GST activity (Li et al., 1995). 2.3. Lipid peroxidation The level of lipid peroxidation in plant tissues was determined as 2-thiobarbituric acid (TBA) reactive metabolites chiefly malondialdehyde (MDA) as described by Heath and Packer (1968). 2.4. H2O2 accumulation H2O2 was measured spectrofluorometrically by measuring fluorescent compound dichlorofluorescein (DCF) produced during horseradish peroxidase-catalyzed reaction with H2O2 as substrate (Black and Brandt, 1974). 2.5. Statistical analysis All values reported in this work are mean of at least three independent experiments. A two-way ANOVA test was used to confirm the significance of the data. Comparison with control and between means of treatments was done by Tukey test.
3. Results The first visible symptom of Cr6+ exposure to B. juncea appeared in the form of leaf area reduction. On day 5 reduction in leaf area was 8%, 17%, and 28% in plants exposed to 0.2, 2 and 20 lM Cr6+, respectively. As the Cr6+ treatment prolonged, leaf area was significantly reduced at all the three levels of Cr6+ supply (Table 1). Up till day 5, 0.2 and 2 lM Cr6+ supply made little effect on biomass accumulation. In fact, there was a 15% increase in plant biomass at 0.2 lM Cr6+ on day 5. The biomass was, however, reduced by 19% and 38% respectively in the plants exposed to 2 and 20 lM Cr6+ on day 15 (Table 1). Chlorophyll content in leaves of Cr6+ treated B. juncea was mostly higher than the control during 15 days of metal exposure (Table 1). During initial 5 days of treatment this increment was evident at 0.2 and 2 lM Cr6+ but not at 20 lM Cr6+. Chlorophyll concentration at 20 lM Cr6+, however, increased significantly after day 5 when expansion of leaves was restricted. The dark green, small sized leaves of plants exposed to 20 lM Cr6+ for 15 days appeared quite distinct from those of the plants growing at 0.2 and 2 lM Cr6+. Carotenoid content in leaves of B. juncea increased invariably at 0.2 and 2 lM Cr6+ during 15 days of treatment (Table 1). As the treatment continued, level of
carotenoids at 20 lM Cr6+ increased to 36% over the control. Chromium content of Cr6+ treated B. juncea plants showed that the metal was largely restricted to the roots and very little of it was transported to the aerial parts. The roots of plants exposed to 0.2, 2 and 20 lM Cr6+ accumulated 76, 410 and 897 lg Cr g1 DW, respectively by day 5. Maximum accumulation of Cr was found in roots of plants grown for 15 days (Table 1). The amount of Cr detected in leaves by day 5 was quite low. The maximum amount of Cr (56.5 lg Cr g1 DW) was accumulated in leaves of plants grown at 20 lM Cr6+ for 15 days (Table 1). Exposure of B. juncea to Cr6+ resulted in accumulation of lipid peroxidation products (MDA), more in roots than in leaves. Significant increase in lipid peroxidation occurred from day 3 in the roots exposed to 20 lM Cr6+ (Table 2). In leaves, significant accumulation of MDA was observed from day 4, both at 2 and 20 lM Cr6+. Plant response to 2 and 20 lM Cr6+ produced significant accumulation of H2O2 from day 1 of the excess Cr6+ treatment (Table 2). At 0.2 lM Cr6+, H2O2 accumulation in roots and leaves was evident on day 5 and 15 (Table 2). Superoxide dismutase activity increased in roots but not in leaves during initial 3 days of treatment. Significant increase in SOD activity observed in roots at 0.2 lM Cr6+ on day 3 was maintained till day 15. At 2 lM Cr6+, SOD activity increased on day 2, 3 and 15. However, at 20 lM Cr6+ significant reduction in SOD activity took place in roots (Table 3). In leaves, increase in SOD activity was observed throughout the treatment at all levels of Cr6+ supply but the effect was not large enough to be significant. Ascorbate peroxidase activity was significantly increased in roots of Cr6+ treated plants until day 5. At 20 lM Cr6+, the APX activity showed maximum increase from day 1 to 5 but decreased significantly by day 15 (Table 3). APX activity in leaves at 0.2 and 2 lM Cr6+ was significantly higher than the control from day 2 to 5. While the increase at 20 lM Cr6+ was marginal from day 1 to 5, a significant decrease in the APX activity was observed on day 15 (Table 3). Catalase activity in roots was invariably higher than the control both at 0.2 and 2 lM Cr6+ supply. The increase was significant from day 3 to 15 at 0.2 lM Cr6+ and from day 1 to 5 at 2 lM Cr6+ (Table 3). The increase or decrease in catalase activity at 20 lM Cr6+ in roots was, however not significant. In leaves, catalase activity was stimulated throughout the treatment at all the three levels of Cr6+ supply (Table 3). Glutathione reductase, responsible for reduction of oxidized glutathione, showed a concentration dependent enhanced activity more in leaves than in roots. GR activity was significantly higher than the control in roots
V. Pandey et al. / Chemosphere 61 (2005) 40–47
43
Table 1 Growth, chlorophyll, carotenoid and chromium accumulation in B. juncea exposed to different concentrations of hexavalent chromiuma,b Cr6+ supply (lM)
Day
0 2
Leaf area (cm )
5 15 5 15 5 15 5 15 5 15 5 15
Biomass (g) Chlorophyll (lg cm2) Carotenoids (lg cm2) Cr (lg g1 DW) Root Leaf
0.2 bA
2 bA
0.99 ± 0.02 1.27 ± 0.09aA 0.07 ± 0.007bB 0.09 ± 0.01aA 88.7 ± 4.76aB 105 ± 14.20aB 38.5 ± 0.25aA 49.5 ± 3.62aA 3.3 ± 0.26bD 37.0 ± 1.73aD ND 3.25 ± 0.29aC
0.98 ± 0.02 1.19 ± 0.08aB 0.08 ± 0.004aA 0.09 ± 0.001aA 107 ± 8.62aB 105 ± 13.39aB 41.6 ± 3.60aA 51.3 ± 4.69aA 75.9 ± 7.10bC 236 ± 10.00aC 6.48 ± 0.01bC 11.0 ± 0.80aB
20 aA
0.82 ± 0.04 0.95 ± 0.08aC 0.07 ± 0.001aB 0.07 ± 0.005aB 114 ± 22.95aA 101 ± 9.32aB 42.3 ± 7.74aA 50.0 ± 4.76aA 410 ± 12.53bB 634 ± 7.54aB 6.94 ± 0.02bB 24.5 ± 3.50aA
0.71 ± 0.05aB 0.71 ± 0.05aD 0.05 ± 0.003aC 0.06 ± 0.004bC 86.7 ± 8.53bB 133 ± 7.10aA 34.8 ± 2.45bA 67.3 ± 4.51aB 897 ± 7.54bA 1116 ± 26.0aA 16.0 ± 0.51bA 56.5 ± 1.50aA
a
Values are expressed as mean ± SD, n = 4. Values followed by the same lowercase letter in each column or uppercase letter in each row for each variable are not significantly different based on Tukey test (P = 0.05). b
Table 2 Effect of hexavalent chromium on lipid peroxidation and H2O2 accumulation in B. junceaa,b Cr6+ (lM)
MDA (nmol g FW Root 0.0 0.2 2.0 20 Leaf
0.0 0.2 2.0 20
1
Day 1
2
3
4
5
15
32 ± 4.0bA 32 ± 5.8bA 35.2 ± 2.9cdA 38.1 ± 2.6dA
33 ± 2.9bA 31.6 ± 2.9bA 32.8 ± 2.0dA 39.6 ± 3.2dA
32.2 ± 3.0bB 33 ± 4.4bB 41.4 ± 4.2bcAB 47.4 ± 4.2cdA
43.6 ± 4.0 abB 45 ± 3.4aB 47.5 ± 4.1bcAB 56.6 ± 3.2bcA
45.6 ± 3.0aB 49 ± 3.4aB 51.2 ± 3.5abB 65.7 ± 6.2abA
48 ± 2.9aB 54.8 ± 3.2aB 58.8 ± 5.6aB 70.1 ± 4.1aA
108 ± 10.7cA 102 ± 6.8cA 122 ± 14.3cA 125 ± 6.3cA
127 ± 11.9bcAB 112 ± 23.2cB 120 ± 10.1cB 157 ± 9.6deA
148 ± 16.4abAB 131 ± 20. 3bcB 162 ± 9.9bAB 170 ± 11.4cdA
154 ± 6.1abB 148 ± 10.4bB 171 ± 4.6bA 171 ± 4.6bA
155 ± 22.7abB 153 ± 11.5bB 180 ± 9.5bAB 209 ± 12.1bA
166 ± 2.5aB 180 ± 16.7aB 223 ± 10aA 248 ± 12aA
39.3 ± 4.1bcC 50.6 ± 6.1bC 70.6 ± 6.1bB 95.3 ± 5.0bA
44.6 ± 6.4abC 62 ± 2.0bB 91.6 ± 3.0aA 100 ± 6.0bA
53.3 ± 1.5aD 76 ± 5.2aC 106 ± 4.0aB 124 ± 4.0aA
54 ± 0.0aC 76.6 ± 2.3aB 102 ± 2.0aA 113 ± 1.1aA
50 ± 4.5aD 82.6 ± 2.0aC 103 ± 2.8aB 121 ± 3.2aA
132 ± 6.4aC 143 ± 3.0aB 182 ± 4.6aA 192 ± 2.0aA
88.3 ± 1.1cdC 94.6 ± 2.5cdC 106 ± 1.1dB 116 ± 1.1dA
84.3 ± 0.5cdC 90.6 ± 7.6dBC 96.6 ± 1.5dAB 106 ± 2.6eA
80.0 ± 0.0dD 90.6 ± 1.1dC 108.0 ± 4.0cdB 122.6 ± 1.1dA
93.3 ± 1.1cD 103 ± 6.3cC 116 ± 2.3cB 134 ± 2.0cA
)
H2O2 accumulation (nmol g FW 1) Root 0.0 28.6 ± 4.1cC 0.2 38.0 ± 4.0cBC 2.0 41.6 ± 2.5cB 20 59 ± 7.0cA Leaf
0.0 0.2 2.0 20
115 ± 5.0bC 124 ± 2.0bC 140 ± 3.0bB 162 ± 3.0bA
a
Values are expressed as means ± SD, n = 4. Values followed by the same lowercase letter in each row or uppercase letter in each column for each variable are not significantly different based on Tukey test (P = 0.05). b
at 2 and 20 lM Cr6+ from day 3 to 15 (Table 3). Similarly, significant increase in GR activity was observed from day 3 to 15 in leaves. On day 15, however, the activity declined from day 5 at all the levels of Cr6+ supply but remained higher than the control.
Activity of GST in roots of plants exposed to Cr6+ was invariably higher than the control but the increase was significant only at 2 and 20 lM Cr6+ during 3–5 day (Table 3). In leaves significant change in GST activity was not observed except at 2 lM Cr6+ supply.
44
V. Pandey et al. / Chemosphere 61 (2005) 40–47
Table 3 Effect of hexavalent chromium on antioxidative enzymes in B. junceaa,b Cr6+ (lM)
Day 1
2
3
4
5
15
22.4 ± 1.1bcB 25.8 ± 1.0cAB 31.4 ± 2.7bcA 25.0 ± 2.3aAB
25.0 ± 1.6bcB 35.5 ± 2.1bA 29.0 ± 2.8bA 28.0 ± 2.7aB
28.4 ± 1.4 bB 40.4 ± 1.1abA 36.0 ± 2.7bAB 24.0 ± 2.2aC
30.3 ± 2.0bB 47.0 ± 2.4aA 42.0 ± 2.3bB 23.0 ± 2.7aC
36.5 ± 2.3aB 42.8 ± 2.9abA 46.8 ± 3.6aA 22.0 ± 3.2aC
22.1 ± 0.6bA 27.0 ± 2.1bA 25.5 ± 1.0bcA 24.4 ± 4.1abA
26.2 ± 0.3abA 30.9 ± 2.1abA 30.8 ± 2.3bA 27.5 ± 1.9abA
26.9 ± 2.2abA 33.7 ± 2.3abA 36.7 ± 2.2abA 30.7 ± 3.1aA
30.9 ± 1.2aA 38.6 ± 3.5aA 37.8 ± 3.7abA 34.6 ± 4.3aA
34.0 ± 2.4aA 42.5 ± 2.9aA 41.0 ± 2.5aA 35.4 ± 3.7aA
1.40 ± 0.04bB 2.48 ± 0.17bcA 2.30 ± 0.32aA 1.88 ± 0.17abAB
1.66 ± 0.06bC 3.12 ± 0.12aA 2.80 ± 0.26aA 2.14 ± 0.11aBC
1.87 ± 0.14bB 2.88 ± 0.10abA 2.71 ± 0.18aA 2.28 ± 0.11aAB
2.02 ± 0.07aB 2.89 ± 0.18abA 2.65 ± 0.27aA 2.36 ± 0.22aAB
2.30 ± 0.14aA 2.35 ± 0.23bcA 2.20 ± 0.15abA 1.20 ± 0.19cB
1.10 ± 0.08aB 1.65 ± 0.06abA 1.45 ± 0.07abA 1.23 ± 0.02aB
1.12 ± 0.15aC 1.85 ± 0.21aA 1.55 ± 0.08abAB 1.42 ± 0.14aBC
1.16 ± 0.10aC 1.94 ± 0.14aA 1.65 ± 0.07aAB 1.40 ± 0.09aBC
1.22 ± 0.13aB 1.78 ± 0.13aA 1.56 ± 0.11abAB 1.44 ± 0.04aAB
1.40 ± 0.09aB 1.50 ± 0.10bAB 1.60 ± 0.06aAB 0.93 ± 0.01bC
Catalase (U mg protein1) Root 0.0 60.0 ± 3.4bB 0.2 75.0 ± 4.0cAB 2.0 90.0 ± 8.9cA 20 70.0 ± 4.3bAB
63.0 ± 7.2abB 80.0 ± 3.6cAB 87.0 ± 5.5cA 75.0 ± 5.0bAB
65.0 ± 5.0abC 92.0 ± 8.5bcAB 105 ± 8.9bcA 80.0 ± 7.0bBC
85.0 ± 7.0aB 148 ± 6.7aA 153 ± 9.7aA 108 ± 11.0aB
80.0 ± 6.9abB 115 ± 9.5bA 125 ± 8.0bA 75.0 ± 7.5bB
85.0 ± 6.0aBC 110 ± 9.5bA 108 ± 12.5bcAB 65.0 ± 6.5bC
90.0 ± 4.4aC 195 ± 11.0abA 190 ± 9.9abA 128 ± 6.0bB
103 ± 6.5aC 185 ± 9.9bcA 170 ± 8.1abAB 140 ± 4.5bB
105 ± 7.5aC 220 ± 15.8aA 195 ± 10.2aA 142 ± 9.0bB
108 ± 5.0aB 175 ± 5.5bcA 160 ± 8.3bcA 150 ± 10.8abA
108 ± 4.2aB 155 ± 10.3cdA 150 ± 8.3cA 153 ± 8.6abA
120 ± 8.0aC 140 ± 6.0dBC 155 ± 5.8cAB 177 ± 12.1aA
(U mg protein1) 0.05 ± 0.006eA 0.05 ± 0.004eA 0.05 ± 0.004eA 0.07 ± 0.005fA
0.09 ± 0.006dB 0.09 ± 0.009dB 0.10 ± 0.005dAB 0.12 ± 0.006eA
0.12 ± 0.005cB 0.13 ± 0.007cB 0.15 ± 0.006cA 0.17 ± 0.006dA
0.15 ± 0.007bC 0.15 ± 0.009bC 0.18 ± 0.006bB 0.22 ± 0.009cA
0.23 ± 0.006aC 0.24 ± 0.010aC 0.26 ± 0.007aB 0.28 ± 0.009bA
0.24 ± 0.005aC 0.26 ± 0.006aB 0.26 ± 0.005aB 0.33 ± 0.01aA
0.20 ± 0.005dA 0.20 ± 0.01eA 0.23 ± 0.01dA 0.22 ± 0.01eA
0.28 ± 0.003cC 0.29 ± 0.01dBC 0.32 ± 0.01cB 0.35 ± 0.01dA
0.30 ± 0.01bcC 0.33 ± 0.01cB 0.37 ± 0.01bA 0.39 ± 0.01cA
0.30 ± 0.01bcC 0.35 ± 0.01bcB 0.40 ± 0.02bA 0.43 ± 0.01bA
0.37 ± 0.01aC 0.47 ± 0.01aB 0.49 ± 0.02aB 0.53 ± 0.02aA
0.31 ± 0.01bC 0.38 ± 0.01bB 0.39 ± 0.01bB 0.52 ± 0.01aA
0.25 ± 0.01aB 0.30 ± 0.02abAB 0.31 ± 0.03aA 0.31 ± 0.01aA
0.22 ± 0.01aB 0.28 ± 0.01abAB 0.32 ± 0.01aA 0.33 ± 0.01aA
0.26 ± 0.01aB 0.33 ± 0.02aA 0.34 ± 0.02aA 0.31 ± 0.02aA
0.26 ± 0.01aA 0.30 ± 0.01abA 0.31 ± 0.01aA 0.29 ± 0.01abA
0.19 ± 0.01aA 0.20 ± 0.01abA 0.20 ± 0.01cA 0.19 ± 0.01bcA
0.19 ± 0.01aB 0.21 ± 0.01aAB 0.25 ± 0.01bcA 0.20 ± 0.01abAB
0.20 ± 0.02aB 0.22 ± 0.01aB 0.29 ± 0.00aA 0.22 ± 0.01abB
0.21 ± 0.01aA 0.22 ± 0.01aA 0.22 ± 0.01bcA 0.23 ± 0.01aA
Superoxide dismutase Root 0.0 0.2 2.0 20 Leaf
0.0 0.2 2.0 20
Ascorbate peroxidase Root 0.0 0.2 2.0 20 Leaf
Leaf
0.0 0.2 2.0 20
0.0 0.2 2.0 20
Glutathione reductase Root 0.0 0.2 2.0 20 Leaf
0.0 0.2 2.0 20
(U mg protein1) 19.6 ± 1.1cA 20.8 ± 0.8cA 25.4 ± 2.6cA 21.9 ± 2.3aA 18.6 ± 1.1bA 23.0 ± 1.7bA 20.9 ± 2.1cA 19.0 ± 3.1bA (U mg protein1) 1.43 ± 0.10bA 2.00 ± 0.13cA 1.90 ± 0.09bA 1.67 ± 0.09bA 1.07 ± 0.10aA 1.40 ± 0.15bA 1.24 ± 0.14bA 1.19 ± 0.06aA
Glutathione-S-transferase (U mg protein1) Root 0.0 0.20 ± 0.02aA 0.21 ± 0.01aB 0.2 0.22 ± 0.01cA 0.26 ± 0.01bcAB bA 2.0 0.25 ± 0.01 0.29 ± 0.01abA 20 0.25 ± 0.01bA 0.25 ± 0.01abAB Leaf
a
0.0 0.2 2.0 20
0.16 ± 0.01aA 0.16 ± 0.01bA 0.17 ± 0.01cA 0.17 ± 0.01cA
0.16 ± 0.01aA 0.17 ± 0.01bA 0.17 ± 0.01cA 0.17 ± 0.01cA
Values are expressed as means ± SD, n = 4. Values followed by the same lowercase letter in each row or uppercase letter in each column for each variable are not significantly different based on Tukey test (P = 0.05). b
V. Pandey et al. / Chemosphere 61 (2005) 40–47
4. Discussion Chromium stimulates growth in certain plant species (Bonet et al., 1991; Han et al., 2004). In the present study, marginal stimulation of growth in B. juncea in the beginning at 0.2 lM Cr6+ could not be sustained during 15 days of growth as apparent from minor change in biomass accumulation and significant reduction in leaf area as compared to control. Plant biomass was severely reduced particularly at 2 and 20 lM Cr6+. Decreased biomass production in Cr6+ exposed B. juncea is in conformity with earlier findings on different plant species (Barcelo´ et al., 1985; Sharma and Sharma, 1993, 1996; Dixit et al., 2002). Chromium treatment affected leaf expansion in the present study as evident from production of smaller leaves. The severity of reduction in leaf size increased with increasing Cr6+ concentration and duration of exposure. Similar observations with reference to Cr6+ were also made in Phaseolus vulgaris (Barcelo´ et al., 1985), Pisum sativum (Dixit et al., 2002) and Vigna radiata (Shanker et al., 2004). One possible mechanism to explain the reduction in leaf area under metal stress is the restricted availability of water for leaf expansion (Radin and Boyer, 1982). There is also a good possibility of Cr6+ interaction with endogenous phytohormones that control plant growth processes (Moya et al., 1995). Significant increase in chlorophyll and carotenoids was seen in plants exposed to 20 lM Cr6+ on day 15. These observations are interesting and contrary as heavy metals are known to interact with the sulphydryl site on enzymes, d-aminolevulinic acid dehydrogenase and protochlorophyllide reductase of chlorophyll biosynthesis (Ouzounidou, 1995). However, both decrease and increase in the level of chlorophyll has been reported in different plant species exposed to Cr6+ (Sharma et al., 1995; Sharma and Sharma, 1996; Dixit et al., 2002; Samantary, 2002). An instantaneous change in level of carotenoids along with chlorophylls in Cr6+ stressed B. juncea was an interesting phenomenon observed in the present study. Generation of reactive oxygen species particularly singlet oxygen and oxygen radical may enhance under influence of Cr6+ ions during growth light exposure. Carotenoids can interact with the reactive oxygen species, preventing the initiation of potentially lethal processes such as lipid peroxidation (Pallett and Young, 1993). Low transport of Cr6+ from root to aerial parts in B. juncea is in accord with observations on pea (Dixit et al., 2002), cauliflower and cabbage (Zayed et al., 1998), and Indian mustard (Han et al., 2004). Hexavalent chromium, being a strong oxidant, produces oxidative stress as evident from enhanced level of lipid peroxidation and increasing tissue concentration of H2O2 in plants exposed to 2 and 20 lM Cr6+. Most heavy metals cause oxidative stress via generation of
45
reactive oxygen species (Dietz et al., 1999). There is, however, no evidence as yet of Cr6+ participation in generation of ROS in plants although Cr6+ induced enhanced generation of superoxide radicals has been demonstrated in vivo in pea root mitochondria (Dixit et al., 2002). The first line of defense against ROS-mediated toxicity is achieved by SOD that catalyzes the dismutation of superoxide radicals to H2O2 and O2. Significant increase in the SOD activity in roots at 0.2 and 2 lM Cr6+ may be a part of this defense mechanism. Significant decrease in SOD activity at 20 lM Cr6+ indicated interaction of Cr6+ with the enzyme. The role of SOD in Cr6+ induced toxicity in leaves seems to be limited in the present study as there was no significant increase in the activity of this enzyme. Plants exposed to different metals respond differently with reference to activity of SOD. Toxic concentrations of Zn could not induce SOD activity in beans (Weckx and Clijsters, 1996) but caused multifold enhancement in the SOD activity in mustard (Prasad et al., 1999). SOD activity decreased or remained unchanged in certain plant species exposed to cadmium (Cd) while in others it increased (Somashekaraiah et al., 1992; Dixit et al., 2001). Hexavalent chromium increased H2O2 accumulation both in roots and leaves. Plants exposed to 20 lM Cr6+ accumulated high level of H2O2 but showed decreased or poor SOD activity. This shows that either H2O2 is being generated by other enzymatic or nonenzymatic processes other than SOD dismutation or it is not being efficiently scavenged in Cr6+ treated plants. APX and CAT are two potent scavengers of H2O2, which minimize its accumulation and diffusion across the cellular membranes, preventing peroxidative damage to cellular constituents. Enhancement in the activity of both APX and CAT was recorded at 20 lM Cr6+ in roots as well as in leaves and this possibly contributed to substantial accumulation of H2O2 in these plant parts. Foyer et al. (1994) have shown that GR is one of the key enzymes that helps in reduction of GSSG to GSH by oxidizing NAD(P)H to NAD(P)+ and suggested its crucial role in combating oxidative stress in leaves. We found that GR activity was elevated more in leaves than in root. Prasad et al. (1999) have also reported up to 158% increase in shoot GR activity in B. juncea seedlings raised under Zn toxicity. However Satyakala and Jamil (1992) reported decreased GR activity in leaves and increased activity in roots of two Cr6+ treated aquatic weeds. GST catalyses conjugation of electrophilic xenobiotic substrates with glutathione. In the present study, Cr6+ induced differential GST response. While there was an increase in GST activity in roots, no significant changes were observed in leaves. Increase in GST activity was also found in Cd treated pea roots (Dixit et al., 2001). This low activity of GST in the present study can be
46
V. Pandey et al. / Chemosphere 61 (2005) 40–47
attributed to low amount of Cr6+ present in treated leaves. The present study showed a differential response of Cr6+ to antioxidative enzymes in roots and leaves (Fig. 1). While certain enzymes are expressed in excess,
1200
A
A
200 175
1000
150 800
125
600
100 75
400
50 200 0 1200
25
B
B
0 200 175
1000
125
600
100
400
75 50
200
25
0 120
C
C
0 200 175
100
Relative Enzyme Activity
Cellular chromium (µg g-1 dry wt)
150 800
150 80
125
60
100
40
75 50
20 0 120
others are inhibited at higher levels of cellular Cr. APX was found to be the most susceptible enzyme to Cr6+ while GST and GR were optimally increased at elevated cellular Cr concentration. With the increasing Cr accumulation, SOD and CAT activities mostly decreased but remained always higher than the control both in roots and leaves. The activities of these two enzymes were, however, severely inhibited in roots at 20 lM Cr6+ wherein cellular Cr showed highest accumulation. Our observations indicate that up to certain level of cellular Cr, CAT compensates the Cr induced loss in APX activity. As the cellular concentration of Cr increased, plant growth was affected despite increase in the activities of some of the enzymes. These enzymes seem to overcome oxidative stress induced by Cr6+ as no evidence of oxidative symptoms was found even at 20 lM Cr6+ in leaves. Due to increased Cr accumulation, plants can no longer maintain metal homoeostasis. The role of antioxidative defense may also become limiting, as Cr6+ is also known to interact with certain micronutrients essential for plant growth and limit their availability either by decreased uptake or immobilization in roots (Barcelo´ et al., 1985). The possibility of Cr6+ interacting synergistically with other physiological and biochemical components to impair plant growth cannot be ruled out. Although enzymatic detoxification is an efficient mechanism for scavenging the damaging oxygen species, more efficient protective processes (such as metal exclusion, immobilization, compartmentalization and complexing) are needed to prevent the generation of these species and availability of free metal ions to sensitive sites in the first instance.
25
D
D
0 200 175
100
150 80
125
60
Acknowledgments This work was undertaken under Institutional Project No. OLP-230525. We are grateful to the Director, NBRI, Lucknow for necessary facilities and encouragement. One of us (VD) is grateful to Council of Scientific and Industrial Research for providing Senior Research fellowship. Thanks are also due to Prof. C.P. Sharma for critically going through the manuscript and helpful suggestions.
100 75
40
References
50 20 0
25
Cr6+
0
SOD CAT APX GR
GST
Fig. 1. Relative enzyme activity in relation to cellular chromium in B. juncea exposed to Cr6+. Day 5 root (A), Day 15 root (B), Day 5 leaf (C), Day 15 leaf (D); (j) 0.2 lM Cr, ( ) 2 lM Cr, ( ) 20 lM Cr.
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