Bafna et al. Int. J. Res. Chem. Environ. Vol.

3 Issue 3 July 2013(20-25)

International Journal of Research in Chemistry and Environment

Vol. 3 Issue 3 July 2013(20-25) ISSN 2248-9649 Research Paper

Effect of Lead Stress on Chlorophyll Content, Malondialdehyde and peroxidase activity in Seedlings of mung bean (Vigna radiata)
Dugar Deshna, *Bafna A.
Department of Biochemistry, Govt. Holkar Science College, Indore- 452001, (M.P.), INDIA

Available online at: www.ijrce.org (Received 06th March 2013, Accepted 18th May 2013) Abstract: The presence of heavy metals in solid and liquid wastes is an important issue that is related to the pollution of the environment. These are one of the most important environmental pollutants and reaching dangerous amount requires more research. Among the heavy metals, lead particularly has become a cosmopolitan enviornmental pollutant. This study was conducted to investigate the effects of Pb stress on different varieties (Virat, SML-668, A1 Gold and K-851) of mung bean (Vigna radiata). V.radiata occupies a very important position in Indian agriculture. Seeds of four different mung bean varieties were grown under laboratory conditions in dark at 0- 1500 M concentration of lead nitrate [Pb(NO3)2]. Distilled water was used as control treatment. To access the effect of lead stress on peroxidase activity, Malondialdehyde (MDA) and chlorophyll content,7 day old seedlings were used. The results revealed that the chlorophyll content of of mung bean seedlings significantly (p<0.05) decreased with increasing concentrations of [Pb(NO3)2]. MDA and peroxidase activity were significantly (p<0.05) increased proportionately (r > 0.9) to the increasing concentration of [Pb(NO3)2]. Increased amount of MDA was indicative for the formation of free radicals in plants under heavy metal stress, while increased level of peroxidase activity pointed to the occurrence of a scavenging mechanism. The decrease in chlorophyll content is an indication of reduction in the growth of the plants leading to a decrease in the yield.
Keywords: Chlorophyll, Heavy metal stress, Lead nitrate [Pb(NO3)2], Malondialdehyde(MDA), Peroxidase, Seedling:Vigna radiata etc.

Introduction
The industrialization and urbanization during the past few years have given rise to serious problems of environmental pollution. General increases in the level of heavy metals pose a pervasive threat to the natural ecosystem [1]. The presence of heavy metals in the environment is a serious ecological problem, because these elements can enter food chains and the biological cycle [2]. The plants under stress conditions are most likely to be adversely affected by high concentration of heavy metals. The possible adverse effects of heavy metal pollution and their phytotoxic effects have been reported in Shorea robusta [3], Capsicum annum [4], Albizia procera [5], Sinapis arvensis [6], Triticum aestivum [7].

systems [9]. The process of photosynthesis is adversely affected by Pb toxicity. One of the phytotoxic effects of Pb appears to be induction of oxidative stress in growing plant parts due to enhanced production of reactive oxygen species (ROS) resulting in an unbalanced cellular redox status. A number of different ROS, including the superoxide anion (O2.-), singlet oxygen (1O2), hydrogen peroxide (H2O2) and the hydroxyl radical (.OH) are produced during normal oxidative metabolism in aerobic organisms, but these ROS can pose a severe threat when produced in larger amounts [8]. Although the ROS generating processes are slow under normal conditions, Pb accelerates them [10].
In the present study, an attempt is made to elucidate the effect of different concentrations of lead nitrate on chlorophyll content, lipid peroxidation and peroxidase activity in V. radiata. The knowledge of physiological and biochemical basis of phytotoxicity thus

Among the metals, lead particularly has become a cosmopolitan environmental pollutant [8]. Lead is a toxic environmental contaminant that induces many biochemical and structural changes in biological

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gained may help in understanding the possible constraints in the role of lead pollutants in the plants. Estimation of Malondialdehyde :The level of lipid peroxidation was measured by estimating Malondialdehyde (MDA), a decomposition product of polyunsaturated fatty acid component of membrane lipid, using thiobarbituric acid (TBA) as the reactive material and measuring absorbance spectrophotometrically at 532 nm using an extinction coefficient of 155 mM-1 cm-1 following the method of Heath and Packer, (1968) [13] Peroxidase assay: The enzyme activity was assayed using o-dianisidine as hydrogen donor and H2O2 as electron acceptor. The rate of formation of yellow orange colored dianisidine dehydrogenation product is a measure of peroxidase activity and can be assayed spectrophotometrically at 430nm in terms of units/min/g according to Summer and Gjessing, (1943)[14] Statistical Analysis: Results were expressed as mean standard deviation (SD). ANOVA (Analysis of Variance) was used to compare these data between lead nitrate treated seedlings and control seedlings. P values (denotes level of significance) less than 0.05 was considered to be significant. Correlation analysis was performed for the assessment of change due to lead treatment.

Material and Methods

The seeds of Mung bean (Vigna radiata) used in present study were obtained from Sachidanand Krishi Seva Kendra at Nandlalpura and Manish Traders, seed depot at Tejaji Nagar, Indore (M.P). The four varieties selected for study were: Virat, SML-668, A1 Gold and K-851. The present research work has been completed in the Department of Biochemistry, Holkar Science College, Indore (M.P.) from Dec- May 2012.Seeds of uniform size were selected and surface sterilized with 0.1% solution of mercuric chloride for 5min to avoid any fungal growth, followed by washing for 4-5 times with distilled water [11]. 50 seeds were then placed in10 cm diameter Petri dishes lined with Whatman No. 1 filter paper moistened with distilled water for 24 hours in dark for germination. Seed was considered to have germinated when the emerging radical elongated to 1 mm [11]. Fifteen germinated seeds were transferred in each Petri dish lined with Whatman No. 1 filter paper on top and bottom as well and seeds were arranged in such a way that each seed did not touch each other nor touch the side of the plate. Vigna radiata seeds of all four varieties were subjected to stress (lead nitrate) with a concentration gradient of 300 M, 600 M, 900 M, 1200 M and 1500 M prepared in distill water. The choice of lead treatments represents the moderate and high concentrations mimicking polluted soils. The experiment was completely randomized and consisted of five treatments replicated three times. Control treatments were supplied with distilled water. At the start of experiment 3 ml of respective treatment was added in order to moisten the filter paper in each Petri dish and every day, 2ml respective solution was added for consecutive six days. All the Petri dishes were kept at room temperature (253C) in dark. On the 7 days old seedlings, various parameters chlorophyll content, malondialdehyde content and peroxidase activity were estimated. Estimation of chlorophyll content: Fresh leaf samples were extracted with 80% acetone. For spectrophotometric determination of chlorophyll a, chlorophyll b and total chlorophyll contents, the absorbance of the extracts were measured at 645 and 663 nm, respectively following the method by Sadasivam et al., (1992)[12] and calculated using following formula Chlorophyll (a) in mg/g tissue = 12.7(A663) 2.69(A645) V/1000 W Chlorophyll (b) in mg/g tissue = 22.9(A645) 4.68(A663) V/1000 W Total chlorophyll (mg per g tissue) = 20.2(A645) + 8.02(A663) V/1000 W

Results and Discussion

After six days of lead nitrate exposure at various concentrations, 7 day old parameters and impact of different concentration of [Pb(NO3)2] on these parameters was investigated. A concentration dependent decrease in chlorophyll-a content in seedlings of studied varieties was observed at all concentrations of [Pb(NO3)2] as compared to control seedlings. However, insignificant (p>0.05) decrease was observed at 300 M treatment of Pb while significant (p<0.05) decrease became evident from 600 M upto the highest concentration of Pb taken (Table 1). The correlation coefficient (r) between [Pb(NO3)2] concentrations and chlorophyll-a content was -0.99, 0.97, -0.99 and -0.93 in Virat, SML-668, A1 gold and K851 seedlings respectively. At 1500 M treatment of Pb, the maximum percent reduction in chlorophyll-a content observed was 90.54% in K-851 seedlings. Leaves of all four varieties of V.radiata seedlings exhibited reduction in chlorophyll- b content at all concentrations over control seedlings. However, at 300 M treatment of [Pb(NO3)2] decrease in chlorophyll-b content observed was insignificant (p>0.05) while significant (p<0.05) decrease was observed consistently from 600 M upto the highest concentration of Pb taken (Table 2). The correlation coefficient (r) of [Pb(NO3)2] concentrations vs chlorophyll-b was -0.97, -0.95, -1 and -0.99 in Virat, SML-668, A1 gold and K-851 seedlings respectively. Maximum reduction in chlorophyll-b content was observed at 1500 M treatment of Pb i.e. 74.12% in K-851 seedlings as compared to the control values.

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Total chlorophyll contents of studied seedlings showed a gradual decrease in response to increasing concentrations of [Pb(NO3)2]. Decrease in total chlorophyll content was found to be insignificant (p>0.05) at 300 M treatment of Pb. Nevertheless, total chlorophyll content pertaining to 600 M and above treatment of Pb decreased significantly (p<0.05) compared to control seedlings (Table 3). The correlation coefficient (r) was -0.86, -0.96, -1 and -0.99 in Virat, SML-668, A1 gold and K-851 seedlings respectively. At 1500 M treatment of [Pb(NO3)2], the maximum percent reduction in total chlorophyll content observed was 74.12% in K-851 seedlings compared to the control values. The reduction in Chlorophyll a, chlorophyll b and total chlorophyll content due to heavy metal stress has also been reported by John et al, (2008)[15] in Lemna polyrrhiza L. Decreased chlorophyll content associated with heavy metal stress may be the result of inhibition of the enzymes responsible for chlorophyll biosynthesis. MDA content increased with the increasing concentration of [Pb(NO3)2] in all studied seedlings, indicating a concentration-dependent free radical generation (Table 4). The increase in MDA content was significant (p<0.05) from 600 M and above treatment of [Pb(NO3)2]. The correlation coefficient (r) between Pb concentrations vs MDA content was 0.99, 0.98, 0.99 and 0.99 in Virat, SML-668, A1 gold and K-851 seedlings respectively. 56.67% was the maximum percent increase in MDA content in Virat seedlings at 1500 M treatment of [Pb(NO3)2]. This heavy metal induced increase in lipidperoxidation, corroborate well with the data of De Britto et al., (2011)[4] in Capsicum annum. This result suggests that stress directly or indirectly leads to the production of oxygen radicals, which results in the increased lipid peroxidation and oxidative stress in the plant, thus results in increased MDA. Thus the increased MDA indicates the prevalence of oxidative stress and perhaps this may be one of the possible mechanisms by which toxicity due to Pb stress could be manifested in plant tissues. The level of lipid peroxidation measured in terms of MDA indicates the prevalence of oxidative stress and perhaps this may be one of the possible mechanisms by which toxicity due to Pb stress could be manifested in plant tissues [10]. The activity of peroxidase showed a concomitant increase in seedlings of all varieties of V. radiata with increase in treatment of [Pb(NO3)2] (Table 5). Increase in peroxidase activity was found to be significant (p<0.05) at all concentrations compared to control. The correlation coefficient (r) was 0.91, 0.90, 0.93 and 0.97 in Virat, SML668, A1 gold and K-851 seedlings respectively. At 1500 M treatment of [Pb(NO3)2], the maximum increase in peroxidase activity observed was 43.93% in K-851 seedlings as compared to their control values. This concentration dependent increase in peroxidase activity due to heavy metal stress is considered in parallel to the finding of Verma and Dubey, (2003)[10] in rice plants. Enhancement in the activity of peroxidase suggests that this enzyme serves as an intrinsic defense tool to resist Pbinduced oxidative damage in plants.

Table 1 Data showing the impact of various treatments of lead nitrate on Chlorophyll- a content (mg/gm) in different varieties of Vigna radiata Chlorophyll-a content (mg/gm) Virat SML-668 A1 gold 0 (control) 1.260.02 1.350.03 1.320.05 NS NS 1.040.07 1.210.04 1.080.07NS 300 (17.46%) (10.37%) (18.18%) 0.890.03* 1.170.04* 0.880.07* 600 (29.37%) (13.33%) (33.33%) 0.750.03** 1.080.08** 0.670.05** 900 (40.21%) (20%) (49.24%) 0.640.04** 0.880.07** 0.550.04** 1200 (49.21%) (34.81%) (58.33%) 0.520.04** 0.680.06** 0.430.05** 1500 (58.73%) (49.63%) (67.42%) Values expressed are means standard deviation of three separate experiments, *Significance at 0.05 ** Significance at 0.01, NS = not significant, Values given in parentheses are decreased percent compared to control. Conc. of lead(M)

K-851 1.480.05 1.440.06NS (2.7%) 1.310.06* (11.49%) 1.10.04** (25.68%) 0.800.05** (45.95%) 0.140.07** (90.54%)

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Table 2 Data showing the impact of various treatments of lead nitrate on Chlorophyll-b content (mg/gm) in different varieties of Vigna radiate Chlorophyll-b content (mg/gm) Virat SML-668 A1 gold 0 (control) 1.270.05 1.960.05 1.550.06 1.190.07NS 1.900.1NS 1.270.05NS 300 (6.3%) (3.06%) (18.06%) 0.930.04NS 1.710.07NS 1.080.04NS 600 (26.77%) (12.76%) (30.32%) 0.850.04* 1.560.02* 0.870.03* 900 (33.07%) (20.41%) (43.87%) 0.840.05* 1.510.06* 0.610.06* 1200 (33.86%) (22.96%) (60.65%) 0.610.06** 1.020.1** 0.470.07** 1500 (51.97%) (47.96%) (69.68%) Values expressed are means standard deviation of three separate experiments, *Significance at 0.05 ** Significance at 0.01, NS = not significant, Values given in parentheses are decreased percent compared to contro Conc. of lead(M)

K-851 1.720.06 1.330.05NS (22.67%) 1.130.04NS (34.3%) 0.830.05* (51.74%) 0.660.07* (61.63%) 0.480.04** (72.09%)

Table 3 Data showing the impact of various treatments of lead nitrate on total Chlorophyll content (mg/gm) in different varieties of Vigna radiata Conc. of lead(M) 0 (control) Total Chlorophyll content (mg/gm) SML-668 A1 gold

Virat

K-851

1.300.02 1.910.02 1.500.02 1.700.20 1.070.02NS 1.880.03NS 1.260.02NS 1.260.04NS 300 (17.69%) (1.57%) (16%) (25.88%) 0.870.01* 1.690.02* 1.060.02* 1.040.04* 600 (33.08%) (11.52%) (29.33%) (38.82%) 0.860.02* 1.470.01* 0.850.02* 0.840.04* 900 (33.85%) (23.04%) (43.33%) (50.59%) 0.870.02* 1.450.02* 0.640.02* 0.640.04* 1200 (33.08%) (24.08%) (57.33%) (62.35%) 0.830.02** 1.040.03** 0.440.01** 0.440.02** 1500 (36.015%) (45.55%) (70.67%) (74.12%) Values expressed are means standard deviation of three separate experiments, *Significance at 0.05 ** Significance at 0.01, NS = not significant, Values given in parentheses are decreased percent compared to control.

Conclusion
From the observed results, it is concluded that the activity of malondialdehyde, peroxidase increased in response to the increased metal ion concentration from 300 M to 1500 M in all the selected varieties of V.radiata (Virat, SML-668, A1 Gold and K-851). The increased MDA content indicates the production of free oxygen radical, whereas the increased peroxidase activity indicates the scavenging mechanism of the plants against the ROS produced in response to the metal stress. The chlorophyll content of the metal stressed leaves decreased with increased concentration of metal ion from 300 M to 1500 M. The decreased chlorophyll content is a visible symptom of reduced growth. The defense mechanism of

the plants against metal stress was thus observed with the increased production of peroxidase, and the reduced growth by the decrease in the chlorophyll content. From the present study it can be concluded that K851 variety of V. radiata was most sensitive among the selected varieties as maximum reduction in chlorophyll-a, chlorophyll-b, total chlorophyll and MDA content was observed in it alongwith maximum increase in peroxidase activity. Since no nutrient solution was supplemented for growing seedlings for seven days, the results of this study can be taken as reference for analyzing any antagonistic or synergistic effects of nutrients with Pb toxicity.

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Table 4 Data showing the impact of various treatments of lead nitrate on malondialdehyde in different varieties of Vigna radiate Conc. of lead(M) 0 (control) Amount of malondialdehyde (mM/mg) SML-668 A1 gold

Virat

K-851 0.00330.0002 0.0040.0003NS (17.5%) 0.00480.0002NS (37.5%) 0.00560.0001* (41.07%) 0.0060.0002** (45%) 0.00640.0003** (48.44%)

0.00260.0002 0.00390.0003 0.00230.0002 0.00310.0002NS 0.00480.0001NS 0.00260.0001NS 300 (16.13%) (18.75%) (11.54%) 0.00360.0001NS 0.00560.0002NS 0.00310.0003NS 600 (27.78%) (30.36%) (25.81%) 0.00480.0003* 0.00760.0001* 0.00380.0002* 900 (45.83%) (48.68%) (39.47%) 0.00560.0002** 0.0080.0002** 0.0040.0001** 1200 (53.57%) (51.25%) (42.5%) 0.0060.0001** 0.00840.0002** 0.00480.0002** 1500 (56.67%) (56.25%) (52.08%) Values expressed are means standard deviation of three separate experiments, *Significance at 0.05 ** Significance at 0.01, NS = not significant, Values given in parentheses are increased percent compared to control.

Table 5 Data showing the impact of various treatments of lead nitrate on Peroxidase activity in different varieties of Vigna radiate Conc. of lead(M) 0 (control) Peroxidase activity (units/min/g) SML-668 A1 gold

Virat

K-851 0.60.02 0.670.02* (10.45%) 0.730.03* (17.81%) 0.80.04* (25%) 0.870.02** (31.03%) 1.070.03** (49.93%)

0.630.03 0.690.02 0.530.04 0.750.02* 0.850.03* 0.670.02* 300 (16%) (18.82%) (20.9%) 0.690.04* 0.920.03* 0.730.03* 600 (8.7%) (25%) (27.4%) 0.750.03* 10.04* 0.670.02* 900 (16%) (31%) (20.9%) 0.810.03** 0.920.02** 0.870.02** 1200 (22.22%) (25%) (39.08%) 0.880.02** 1.150.02** 0.930.04** 1500 (28.41%) (40%) (43.01%) Values expressed are means standard deviation of three separate experiments, *Significance at 0.05 ** Significance at 0.01, NS = not significant, Values given in parentheses are increased percent compared to control.

Acknowledgement
We express our sincere thanks to Dr R.K. Tugnawat, Principal and Prof. R.S. Maheshwari, Head of Department of Biochemistry, Govt. Holkar Science College Indore (M.P.)for providing necessary laboratory facilities and encouragement.

Physiological changes and defense mechanisms induced by cadmium stress in maize, Journal of Plant Nutrition and Soil Science, 169, 239246 (2006) 3. Pant P.P., Tripathi A.K. and Dwivedi V., Effect of Heavy Metals on Some Biochemical Parameters of Sal (Shorea robusta) Seedling at Nursery Level, Doon Valley, India. J Agri Sci, 2(1), 45-51, (2011) 4. De Britto A.J., Sebastian S.R., Gracelin D.H.S., Effect of Lead on malondialdehyde, superoxide dismutase, proline activity and chlorophyll content in Capsicum annum. Bioresearch Bulletin 5, 357-362, (2011) 5. Pandey P., Tripathi.A.K., Effect of Heavy metals on Morphological and Biochemical characteristics of Albizia

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