Bioresource Technology 100 (2009) 179–185

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Bioresource Technology

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Antioxidant response in sesame plants grown on industrially contaminatedsoil: Effect on oil yield and tolerance to lipid peroxidation

Amit K. Gupta a,b, Sarita Sinha a,*

a Ecotoxicology and Bioremediation, Environmental Sciences Division, National Botanical Research Institute, Lucknow 226 001, Indiab Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan, ROC

a r t i c l e i n f o

Article history:Received 22 February 2008Received in revised form 7 May 2008Accepted 7 May 2008Available online 24 June 2008

Keywords:SesameMetalsAntioxidantsOil yieldLipid peroxidation

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.05.013

* Corresponding author. Tel.: +91 522 205831/35x2E-mail addresses: sinha_sarita@rediffmail.com,

(S. Sinha).

a b s t r a c t

The plants of sesame white (Sesamum indicum L. var. T55) grown on tannery sludge (TS) contaminatedsoil have shown that Cr level in the seeds was found below detection limits in 10% and 25% TS, however,the levels of Ni, Pb and Cd were found above the recommended limits. In roots, the level of antioxidantsincreased in the plants grown upto 35% TS at 30 d over their respective controls. Total chlorophyll contentincreased significantly (p < 0.5) in the plants (leaves) grown on lower sludge amendments (upto 35% TS at30 d and 25% TS at 60 d) over their respective controls. In addition, the oil content increased (35% increaseover control) in the plants grown on 35% TS. No significant change was observed in thiobarbituric acidreactive substances (TBARS), a lipid peroxidation index, in the plants (upto 50% TS). The number of tric-homes in the leaves of treated plants was found more than control. In lower and upper leaves surfaces,the anterior end of the trichomes was found acute tipped and bent downwards, whereas, the trichome tipwas straight and blunt in control. The stomata on upper and lower surfaces of the leaves were found par-tially or totally closed in the plants grown on 100% TS as compared to control. The toxicity was observedat higher amendments which are evident from the observed morphological changes and decrease in chlo-rophyll content. This study concludes that it is not advisable to grow the plants on contaminated area,besides its healthy growth.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Heavy metals enter an agro-ecosystem through both indus-trial and geochemical activities. Among industrial waste, tannerywaste contributes significantly towards the contamination of theagro-ecosystem due to use of untreated/treated wastewater ontoagricultural field which act as a sink and leading to building uphigh metal concentration in the agricultural field and plantsgrowing therein. The effects on health, agriculture and environ-mental quality in the tannery wastewater receiving area havebeen reported (Singh et al., 2004a). Recently, Sinha et al.(2006) analyzed the plants growing on tannery wastewater con-taminated soil, and have shown high accumulation of metals inthe leafy vegetables than fruit bearing vegetables/crops. Guptaand Sinha (2006a) emphasized that the consumption of vegeta-bles grown on tannery waste contaminated area is not safe forhuman health. There are various reports on edible plants grow-ing on metals contaminated soil which have shown high accu-mulation of metals (Barman et al., 2000; Fytianos et al., 2001;Armienta et al., 2001; Wong et al., 2001; Sinha et al., 2007a;

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21; fax: +91 522 205839/836.sinha_sarita52@yahoo.com

Gupta and Sinha, 2006b). To overcome these menace, previouslyauthors lab studied the translocation of metals in the seeds of oilbearing plants namely Helianthus annuus L. (Singh et al., 2004b),Brassica juncea L. Czern (cv. rohini) (Singh and Sinha, 2005)grown on tannery waste contaminated soil and found no accu-mulation of toxic metals (Cr) in the seeds at lower amendments.Thus, it has been earlier observed that the oil bearing plantshave shown higher tolerance towards metal contaminated sub-strates. However, its impact in the fully grown plants and the ef-fect on the agricultural produce, grown under stress condition israther scanty under natural simulated conditions which approx-imates to field conditions. Thus, there is a need to investigateother annual crop plants which are grown at large scale for agri-cultural produce and also to evaluate its safety in terms of toxicmetal accumulation.

Among oil crops, sesame (Sesamum indicum) has one of thehighest oil yielding crop (35–63%) (Ashri, 1998; Baydar et al.,1999), cultivated in India, Sudan, China and Myanmar, which arethe major sesame-producing countries, contributing to 60% of theglobal yield. Sesame serves as a nutritious food for humans andis used widely in bakery and confectionery products as the seedis composed of 55% lipid and 20% protein. The oil is very stabledue to the presence of a number of antioxidants such as sesamin,sesamolin and sesamol (Suja et al., 2004). Therefore, it has a long

180 A.K. Gupta, S. Sinha / Bioresource Technology 100 (2009) 179–185

shelf life and can be blended with less stable vegetable oils to im-prove their stability and longevity (Chung et al., 2004; Suja et al.,2004). Shahidi et al., 2006 also reported that defatted sesame ex-tracts and their hulls possess good antioxidant activity. Recently,Gupta and Sinha (2006b) reported single and sequential extractionof metals from tannery waste contaminated soil and eventuallymetal accumulation in the fully grown plants of S. indicum L. var.T55. These plants have shown healthy growth on lower amend-ments of tannery sludge. The present study reports the antioxidantresponse, malondialdehyde, oil yield and morphological changes inthe fully grown plant of sesame (S. indicum L.) and level of metals inthe seeds.

2. Methods

Dewatered anaerobically digested tannery sludge cakes werecollected from up flow anaerobic sludge blanket (UASB) wastewa-ter treatment plant of Jajmau, Kanpur (Uttar Pradesh, India) inlarge plastic bags. The sub soil was collected from National Botan-ical Research Institute, Lucknow (India). The tannery sludge (TS)and soil were air-dried, finely ground and sieved (2 mm mesh) be-fore use. The various amendments of tannery sludge (10%, 25%,35%, 50% and 100%) were prepared using soil, served as control.For the purpose of convenience, the amendments have been de-noted as 10%, 25%, 35%, 50%, 75% and 100% TS, respectively andcontrol soil has been denoted as CS.

2.1. Physico-chemical analysis

Rhizospheric soil was obtained by firstly gently shaking off theloosely bound soil, while the rhizosphere soil adhering to the rootsystem was collected by hand from different amendments of tan-nery sludge along with control soil before the growth of the plants.Physico-chemical parameters are estimated using the method ofKalra and Maynard (1991).

2.2. Total metals in soil

For the estimation of total metals content in the soil, 0.50 gsoil + 10 ml double distilled water + 5 ml HNO3 + 4 ml HF + 1 mlHCl for 120 min at 630 W and 120 PSI was using Microwave Diges-tion System (MDS 2000). The suspension was filtered throughWhatman filter paper (No. 42) and the metal contents in the fil-trate were measured using GBC Avanta, Atomic Absorption Spec-trophotometer (AAS). All the analyses were carried out intriplicates.

2.3. Experimental design

The seeds of sesame white (S. indicum L. var. T55) were steril-ized with mercuric chloride (0.1%) for 5 min, washed with doubledistilled water and soaked (overnight) with water. The soakedseeds were evenly sown in pots (23 cm in diameter), which werefilled (12 kg) with different amendments (10%, 25%, 35%, 50% and100%) of TS along with one set of control soil, each in three repli-cates. Ten seeds were sown in each pot to a depth of 0.5 cm. Thepots were watered daily till seed germination. When the seedlingshave developed five or six leaves, they were thinned out to retainsix uniform ones per pot and allowed to grow. Pots were placedin the field laboratory at an average diurnal temperature of25–45 �C. The water level was made up as and when requiredusing tap water. The plants were harvested after 60 d after sowing.All the plants were free from any disease in whole of the experi-ment duration. The plants were uprooted from the pots after 30and 60 d.

2.4. Metal accumulation

After dry weight determination, the oven-dried samples wereground and digested in HNO3 (70%) for 20 min at 630 W and 40PSI using Microwave Digestion System MDS 2000 and metal con-tents were estimated using Atomic Absorption Spectrophotometer(GBC Avanta).

2.5. Estimation of various parameters

Chlorophyll content was estimated by the method of Arnon(1949) and carotenoid content by Duxbury and Yentsch (1956).Protein content was estimated following the method of (Peterson,1977) using serum albumin as the standard protein. Determinationof TBARS as an index of lipid peroxidation, was measured in termsof malondialdehyde (MDA) content using thiobarbutaric acid (TBA)reaction (Heath and Packer, 1968). Cysteine content was measuredfollowing the method of Gaitonde (1967), ascorbic acid contentwas estimated by the method of Keller and Schwager (1977),non-protein thiol (NPSH) content was measured following themethod by Ellman (1959) and free proline content was estimatedfollowing the method by Bates et al. (1973).

2.6. Scanning electron microscopy

Middle part of the leaves from selected amendments was col-lected for studying the surface structure through Electron Micros-copy. The leaves were kept in 2.5% gluteraldehyde for overnight,for fixation followed by dehydration in ethanol series (30%, 50%,70%, 90% and 100%). Further dehydration was done in BAL - TECCPD-030 critical point drier using liquid CO2 as carrier gas. Theleaves were mounted on a stub and were coated with 15 lm con-ductive gold, in an ion sputter coater (TFC 1100). Coated specimenswere mounted on Philips XL-20 Scanning Electron Microscope.

2.7. Quality control and quality assurance

For sludge samples, analytical data quality of metals was en-sured through repeated analysis of sewage sludge samples of Re-source Technology Corporation (EPA Certified Reference material)(Catalog No. CRM029-050; Lot No. JC029a) and results were foundto be within Prediction Intervals. The blanks were run in triplicateto check the precision of the method with each set of samples.

2.8. Statistical analysis

The experiment was performed in completely randomizedblock design involving six amendments of tannery sludge with gar-den soil at two durations. To confirm the variability of the data andvalidity of the results, two-way analysis of variance (ANOVA) wasperformed and least significant difference (LSD) was calculated.

3. Results and discussion

3.1. Physico-chemical analysis, total metals content and metalaccumulation in seeds

The results of physico-chemical properties and total metalsanalysis in the CS and TS are presented in Table 1. Recently, Guptaand Sinha (2006b) reported the results of pH, EC, CEC, OC and OM.The results of total metal analysis in the TS showed that the level oftoxic metals (Cr, Ni, Cd) was significantly high as compared to CS.The level of Pb was non-significantly high in TS than in CS.

Recently, Gupta and Sinha (2006b) reported the accumulationof metals in the plants of S. indicum. They have reported that the

Table 1Physico-chemical analysis and total metals (lg g�1 dw) in tannery sludge (TS) andcontrol soil (CS) (w/w) before the growth of the plants of sesame

Parameters Amendments (%)

CS 100% TS

BD (Mg g�3) 0.4 0.8Clay (%) 10 29pH (1:2.5)a 6.69 ± 0.0 7.73 ± 0.01EC (S cm�1)a 461.0 ± 0.0 3830 ± 10***

CEC (cmol kg�1)a 32.20 ± 2.60 174.77 ± 11.91**

OC (%)a 0.42 ± 0.08 5.87 ± 0.03***

Na+ (mg kg�1) 36.2 ± 2.62 99.8 ± 8.0**

K+ (mg kg�1) 16.9 ± 1.65 17.9 ± 2.51OM (%)a 0.72 ± 0.02 10.13 ± 0.06***

N–NO3 (mg kg�1) 8.2 ± 0.12 455.0 ± 15.41***

N–NH4 (mg kg�1) 2.1 ± 0.71 156.0 ± 12.31***

P+ (g g�1) 11.99 ± 1.01 46.80 ± 2.30**

MetalsNa 10964 ± 871 25876 ± 1145***

K 52136 ± 8219 12305 ± 1662**

Fe 17579 ± 37.68 9401 ± 61****

Zn 83.38 ± 0.94 340.69 ± 11.10****

Mn 229.64 ± 25.34 174.19 ± 15.50*

Cu 11.14 ± 0.38 148.08 ± 0.81****

Cr 18.29 ± 15 74899.76 ± 203****

Pb 9.11 ± 0.25 13.43 ± 2.72Ni 138.23 ± 16.66 419.16 ± 21.73***

Cd 0.46 ± 0.09 51.1 ± 1.25****

All the values of mean of three replicate ± SD, Students t- test (one tailed as com-pared to CS).* p < 0.05.** p < 0.02.*** p < 0.01.**** p < 0.001.a Values reported (Gupta and Sinha, 2006a,b).

A.K. Gupta, S. Sinha / Bioresource Technology 100 (2009) 179–185 181

accumulation Ni, Cd, Pb was found more in upper part of the plantthan Cr. The results of metals accumulation in seeds (Table 2) ofthe plants showed that the essential metals (Fe and Mn) concentra-tion increased upto 50% TS, except Zn, where, it was increased upto100% TS. However, in case of toxic elements (Ni and Cd), the accu-mulation in seeds was found to increase with increase in sludgeamendments. Interestingly, the level of Cr was recorded high intannery sludge and found below detection limits in lower amend-ments (10% and 25% TS). Whereas, at 35% and 50% TS, the level ofCr was less than other toxic metals (Pb, Ni and Cd).

The increase in Ni accumulation in seeds may be due to highmobility of the metal through xylem tissues of shoot and its greatertransport from root to shoot (Krämer et al., 1996). The level of Ni,Pb and Cd contents in the seed was higher than its prescribed safelimit in food given by Prevention of food adulteration Act, 1954(Awashthi, 2000). Besides high level of Cr in sludge, Cr was foundbelow detection limit in the seeds at lower amendments. The lessaccumulation of Cr in the seeds may be due to less accumulation ofmetal in the upper part of the plant (Gupta and Sinha, 2006b).Many reports demonstrated that the metal accumulation in the

Table 2Accumulation of metalsa (lg g�1 dw) in the seeds of sesame plants grown in different am

Amendments Metal accumulation

Fe Zn Mn Cu Na

CS 446.84 ± 78.97 51.98 ± 1.43 36.99 ± 4.99 17.86 ± 2.95 887 ±10% TS 358.84 ± 10.71 73.84 ± 8.83 13.91 ± 0.58 17.80 ± 4.66 2651 ±25% TS 510.65 ± 35.65 74.65 ± 5.98 14.56 ± 3.32 20.36 ± 2.51 2516 ±35% TS 851.04 ± 34.43 74.10 ± 3.60 16.87 ± 1.87 23.46 ± 1.16 1455 ±50% TS 1374.73 ± 77.48 77.18 ± 5.37 36.00 ± 19.91 23.66 ± 1.76 1232 ±100% TS 352.36 ± 2.58 105.65 ± 6.54 12.65 ± 2.36 29.30 ± 3.25 635 ±

All the values are mean of three replicates ±SD. BDL = below detection limit.a Accumulation of metals in both the part of the S. indicum reported earlier (Gupta an

plants is depended on its mobility and availability in soils andplant species growing on these soils (Gupta and Sinha, 2006b,2007a,b). Recently, Gupta and Sinha (2006b) reported less accumu-lation of Cr in the upper part of the plant and consequently noaccumulation of Cr was observed in the seeds of the plants grownon lower amendments. There are many reports (Barman et al.,2000; Fytianos et al., 2001; Armienta et al., 2001) where the accu-mulation of Cr has been found high in the plants growing on tan-nery waste contaminated sites. These authors have not assessedthe level of other toxic metals which are injuries to health.

3.2. Morphological changes in the leaves

The effect on the trichomes (figure not shown) was studied inthe upper and lower part of leaves of the plants grown in tannerysludge amendments, respectively after 60 d of growth. The analysisrevealed that trichomes were non-glandular, uniseriate and unicel-lular. The number of trichomes in the leaves of treated plants wasfound more than CS. It was analyzed that the size of trichome wasrecorded more in the plants grown on sludge i.e., 210 � 37.5 m(25% TS), than the size in CS (195 � 42 m) and (151 � 44.7 m in50% TS; 140 � 42 m in 100% TS. In upper leaves, the reductionin the size of trichomes was found more in CS (126 � 28 m) thanin TS (109 � 28 m in 25% TS, 121 � 30.8 m in 50% TS and 96.23 �27 m in 100% TS). In lower and upper leaves surfaces, the anteriorend of the trichomes was found acute tipped and bent downwards,whereas, the trichome tip was straight and blunt in CS.

It is apparent that the stomata (figure not shown) on upper andlower surface of the leaves were found partially or totally closed inthe plants grown on 100% TS as compared to CS. The change in waxstructure at the leaf surfaces (upper and lower) were found to besame, however, the epidermal cells of the leaf showed someshrinkage in all the amendments as compared to CS. The size andnumber of stomata per unit area were increased with increase insludge amendment ratio as compared to CS in both the surfaces.The size of the stomata (both the surfaces) was found differentand the decrease in size was more prominent in upper surface ascompared to lower surface. Size of stomata (particularly stomatallength) in the lower surface of the leaves was more in CS (15.8 �5.86 m) than 25% TS (12.3 � 4.59 m), 50% TS (11.2 � 1.02 m) and100% TS (9.2 � 1.4 m). In case of upper surface of the leaf, size ofstomata (particularly stomatal length) was found different andmore in CS (15.8 � 5.86 m) than 25% TS (7.67 � 3.12 m), 50% TS(7.24 � 1.53 m) and 100% TS (4.91 � 1.02 m). The observationsshowed slight elevation of stomata from the normal surface thanCS.

The changes at structural levels are either due to the result of adirect interaction of the toxic metals with structural componentsor due to a more indirect consequence of changes in signaltransduction and/or metabolism. The plants grown in tannerysludge exhibited folding of the cuticle of the epidermal layer andclosing of stomata of the leaves, which might be due to thinning

endment of tannery sludge (TS) and control soil (CS)

K Cr Ni Pb Cd

35.99 19812. ± 205.28 BDL 1.29 ± 0.62 1.01 ± 0.47 BDL61.3 18921 ± 868.08 BDL 4.73 ± 0.38 21.90 ± 9.98 4.73 ± 0.3841.56 18745 ± 142.32 BDL 5.22 ± 1.05 18.65 ± 2.51 5.22 ± 1.0571.85 22215 ± 210.57 3.04 ± 0.97 7.35 ± 1.47 11.68 ± 3.49 7.35 ± 1.4794.47 22564 ± 825.70 7.49 ± 0.98 11.21 ± 2.96 12.46 ± 0.04 9.21 ± 2.9626.58 1105 ± 123.65 43.65 ± 2.55 13.55 ± 1.55 11.25 ± 1.52 15.55 ± 1.65

d Sinha, 2006b)

Table 4Effect on photosynthetic pigments and carotenoid (mg g�1 fw) contents of leaves ofsesame grown on different amendments of tannery sludge (TS) and control soil (CS)

Amendments Growth periods (d) Photosynthetic pigments

30 60

CS 1.22 ± 0.07c 1.22 ± 0.08b Chlorophyll a0.37 ± 0.02c 0.38 ± 0.02b Chlorophyll b1.59 ± 0.10c 1.59 ± 0.1b Total chlorophyll0.43 ± 0.01b 0.57 ± 0.2b Carotenoids

10% TS 1.38 ± 0.18abc 1.55 ± 0.02a Chlorophyll a0.44 ± 0.03abc 0.53 ± 0.03a Chlorophyll b1.82 ± 0.12abc 2.08 ± 0.06a Total chlorophyll0.50 ± 0.02a 0.71 ± 0.04ab Carotenoids

25% TS 1.23 ± 0.18bc 1.29 ± 0.26b Chlorophyll a0.41 ± 0.05bc 0.44 ± 0.01ab Chlorophyll b1.64 ± 0.23bc 1.72 ± 0.36b Total chlorophyll0.46 ± 0.06b 0.59 ± 0.12ab Carotenoids

35% TS 1.49 ± 0.1a 1.19 ± 0.01b Chlorophyll a0.51 ± 0.09a 0.43 ± 0.07ab Chlorophyll b2.03 ± 0.03a 1.62 ± 0.06b Total chlorophyll0.54 ± 0.01ab 0.62 ± 0.02a Carotenoids

50% TS 1.47 ± 0.18ab 1.19 ± 0.09b Chlorophyll a0.51 ± 0.09ab 0.37 ± 0.02b Chlorophyll b1.98 ± 0.26ab 1.56 ± 0.11b Total chlorophyll0.50 ± 0.08b 0.55 ± 0.04ab Carotenoids

100% TS 1.45 ± 0.16abc 1.18 ± 0.06b Chlorophyll a0.50 ± 0.09ab 0.39 ± 0.00b Chlorophyll b1.94 ± 0.25abc 1.56 ± 0.02b Total chlorophyll0.47 ± 0.06b 0.55 ± 0.01ab Carotenoids

All the values are mean of three replicates ± SD. Different letters indicate signifi-cantly different values at a particular duration (DMRT, p < 0.05).

182 A.K. Gupta, S. Sinha / Bioresource Technology 100 (2009) 179–185

of epidermal layer under stress conditions. Closed stomata of theleaf results in slower rate of diffusion which may be a strategy toprevent the water loss through transpiration, as the translocationof water and solutes get disturbed in the presence of metals. Theelevation of stomata and its distraction at higher concentrationof metal have also been reported which is due to the stress condi-tions induced by heavy metals (Singh and Sinha, 2004b). Studieshave also shown that aquaporins are present in guard cells (Mau-rel, 1997) and these toxins interfere with the polymerization anddepolymerization of actin filaments altering K+ channel activitiesin guard cells (Hwang et al., 1997). These may be the possiblemechanism of direct effects of toxic metal ions on stomatal closure.Singh and Sinha (2004a) reported an increase in the frequency ofstomata and trichomes and closer of stomata and degenerationof certain cells in H. annuus grown on tannery sludge amendments.In the present study, the small weathered particles were also ob-served on the leaves surface and the deposition may be due tothe weathering of epidermal layers. Weathering of wax layer wasalso visible on the trichome of the plant which might be due tothe toxicity of metals. The oil glands and epidermal layer showeddemarcation in their structures. Structural changes in the oilglands may be considered as a process of dissolution of the lipidcontents by these metals.

3.3. Effect on seed weight and oil content

The weight of the seeds (Table 3) per plants was increased withincrease in sludge amendments upto 25% followed by decrease athigher amendments as compared to CS. The effect on oil content(Table 3) in the seeds of the plants grown on sludge amendmentswas also recorded which showed increase with increase in sludgeamendment upto 35% followed by decrease in higher amendments.

In earlier studies in oil yielding plants grown on tannery sludgecontaminated soil, an increase in oil content was observed (Singhet al., 2004b; Singh and Sinha, 2005).

3.4. Effect on chlorophyll, malondialdehyde, protein, ascorbic acid,cysteine, non-protein thiol and proline contents

Total chlorophyll content (Table 4) was increased significantly(p < 0.05) in the leaves of the plant at 30 d of growth, whereas,non-significant increase was observed at 60 d except 10% TS ascompared to their respective CS. Carotenoid content was increasedin the leaves grown upto 50% TS at 30 d and 35% TS at 60 d as com-pared to their respective CS followed by decrease.

The level of lipid peroxidation was determined in terms of thethiobarbituric acid (TBA)-reactive substances (malondialdehyde)(Table 5). No significant change in malondialdehyde content wasobserved in the roots and leaves of the plants except in roots at50% and 100% TS after 60 d as compared to CS.

The root protein content (Fig. 1) was increased significantly(p < 0.05) upto 25% TS followed by decrease as compared to their

Table 3Effect on weight of seeds (g) and oil contents (%) in sesame grown on differentamendments of tannery sludge (TS) and control soil (CS)

Amendments (%) Weight of seeds (g) Oil contents (%)

CS 2.05 ± 0.3 46.8010% TS 2.41 ± 0.3 56.09 (+19.85)25% TS 2.60 ± 0.2 58.01 (+23.95)35% TS 1.91 ± 0.4 61.08 (+30.51)50% TS 1.70 ± 0.2 38.02 (�18.76)100% TS 1.52 ± 0.3 25.34 (�45.85)

All the values are mean of three replicates ±SD. Values in the parenthesis are theincrease (+)/decrease (�) in oil content (%) than CS.

respective CS at 30 d. At 60 d, the protein content was decreasedin the roots with increase in sludge amendment ratio. Leaf proteincontent (Fig. 1) was found to decrease significantly (p < 0.05) in allthe amendments except 100% TS after 60 d over CS values. Theascorbic acid content was increased significantly in the roots andleaves of the plants in all the amendments after 60 d of growth(Fig. 1). Maximum increase (25.9%) was observed in the roots at50% TS, whereas, 17.27% in leaves in 100% TS after 60 d of growthover with their respective CS. Cysteine, one of the amino acids ofglutathione, plays an important role in detoxification of the metals.The results showed that root cysteine content (Fig. 2) wasincreased significantly (p < 0.05) in the plants grown in 10% and25% TS and in leaves (Fig. 2) upto 50% TS as compared to theirrespective CS after 60 d. The results on non-protein thiol contentin the roots (Fig. 2) and leaves (Fig. 2) of the plants grown at 50%and 100% TS showed significant decrease (p < 0.05) as comparedto their respective CS after 60 d. The results showed that the freeproline content (Fig. 2) in the roots of the plant increased signifi-cantly (p < 0.05) in all the sludge amendments at 30 d, whereas,non-significant (p < 0.05) increase at 60 d, compared to theirrespective controls. In the leaves (Fig. 2), it was increased signifi-cantly (p < 0.05) with increase in sludge amendments ratio upto

Table 5Effect on TBARS (m mol g�1 fw) content in roots and leaves in sesame grown ondifferent amendments of tannery sludge (TS) and control soil (CS)

Amendments Roots Leaves

30 d 60 d 30 d 60 d

CS 44.73 ± 0.42a 35.9 ± 0.14a 8.26 ± 0.25b 4.35 ± 0.47ab

10% TS 46.19 ± 0.25a 35.99 ± 0.63a 8.18 ± 0.35a 5.4 ± 0.54a

25% TS 46.33 ± 0.74a 37.01 ± 0.97a 8.17 ± 0.06ab 5.37 ± 0.53a

35% TS 45.19 ± 0.65a 36.61 ± 0.57a 8.14 ± 0.06ab 3.14 ± 0.28b

50% TS 43.5 ± 0.83a 37.6 ± 0.62a 9.45 ± 0.55a 3.1 ± 0.28b

100% TS 45.48 ± 0.35a 38.18 ± 0.48a 7.13 ± 0.3b 6.06 ± 0.5a

All the values are mean of three replicates ± SD. TBARS Different letters indicatesignificantly different values at a particular duration (DMRT, p < 0.05).

Fig. 1. Effect on protein (mg g�1 fw), ascorbic acid (mg g�1 fw) in the plant grown on various amendments of tannery sludge (TS). All the values are mean of three replicates ±SD. Different letters indicate significantly different values at a particular duration (DMRT, p < 0.05).

A.K. Gupta, S. Sinha / Bioresource Technology 100 (2009) 179–185 183

25% and non-significant decrease at higher amendments after 30 das compared to CS. In leaves, free proline content increased non-significantly as compared to CS after 60 d.

Increased level of photosynthetic pigment in the plants may bedue to availability of essential nutrients required for physiologicalactivities. It has also been reported in the oil yielding plants of B.juncea (Singh and Sinha, 2004b) and H. annuus (Singh et al.,2004b) grown on tannery sludge. Carotenoids are supposed to actas free radical-scavengers by electron transfer to their double bondstructure and plays significant role in the protection of chlorophyllpigment under stress conditions by quenching the photodynamicreactions, replacing peroxidation and collapsing of membrane inchloroplasts (Kenneth et al., 2000). In agreement to our results,an increase in carotenoid content was also observed in the plantsof H. annuus (Singh et al., 2004b) and fenugreek (Sinha et al.,2007b) grown on tannery sludge. The plants grown on tannerysludge have shown healthy growth (Singh and Sinha, 2004a,b).The metals generate reactive oxygen species in the plants grownunder stress conditions which damage photosynthetic apparatusand may also catalyze degradation of proteins through oxidativemodification and increased proteolytic activity (Romero-Puertaset al., 2002).

Metals are reported to induce oxidative stress by generatingtoxic oxygen species, causes oxidative damage to the biomoleculessuch as lipids, proteins and nucleic acids (Halliwell and Gutteridge,2004), which results in the disruption of the cellular metabolism.The plant cells develop an antioxidant defense mechanism forthe protection against reactive oxygen species that vary at variouscellular and subcellular levels in different plants, however, thelevel of the response varies with plant species and intensity ofstress. Plants use a diverse array of enzymes as well as low molec-ular weight antioxidants like cysteine, non-protein thiol and ascor-bic acid to scavenge different types of reactive oxygen species(ROS), thereby protecting potential cell injury. In plants, changes

in membrane permeability lead to water-stress like conditions,which results in an increase in proline levels.

The TBARS as an index of lipid peroxidation (measured as mal-ondialdehyde), and an indicator of free radical production was notfound to increase at lower sludge amendments in S. indicum. Wereet al. (2006) studied seed oil content and fatty acid composition inEast African sesame ( S. indicum L.) accessions (30 nos.) and re-ported that linoleic acid was the major component comprising42.9–54.0% of the total fatty acids, followed by oleic (31.6–42%),palmitic (7.2–9.7%) and stearic (3.8–5.6%) acids. In total, these fourconstituted about 98% of the total fatty acids. Linolenic acids con-stitutes only 0.4–0.5% of the total fatty acids. The malondialdehydeis the peroxidation product of poly unsaturated fatty acids partic-ularly linolenic acid. This may be the reason for non-significantchange in malondialdehyde content in the plants of sesame grownon lower amendments. In contrast, previous studies (Singh et al.,2004b; Singh and Sinha, 2005; Sinha et al., 2007a,b) reported sig-nificant increase in malondialdehyde content in the plants grownunder metal stress. The antioxidant enzyme activity also increasessignificantly to combat stress preventing lipid peroxidation.

Carotenoids and other antioxidants interact with these reactiveoxygen species, preventing the initiation of lipid peroxidation. Aconstitutively high antioxidant capacity or increase in its levelcould prevent the oxidative damage and improve resistance to oxi-dative stress. It indicates that this plant have strong defense mech-anism to crub the lipid peroxidation. A –SH containing amino acid(cysteine) is a key constituent of phytochelatins and plays animportant role in metal detoxification. Non-protein thiols, whichcontain a high percentage of Cys sulfhydryl residues in plants, playa pivotal role in heavy metal detoxification by oxidation of sulfhy-dryl moieties to disulphides. The reduced form of glutathione(GSH) is one of the most important components of non-proteinthiols metabolism which play several roles in heavy metal toler-ance and sequestration (Zhu et al., 1999). The pattern of cysteine,

Fig. 2. Effect of on cysteine (l mol g�1 fw), non-protein thiol (l mol g�1 fw) and free-proline (l mol g�1 fw) contents in the plant grown on various amendments of tannerysludge (TS). All the values are mean of three replicates ± SD. Different letters indicate significantly different values at a particular duration (DMRT, p < 0.05).

184 A.K. Gupta, S. Sinha / Bioresource Technology 100 (2009) 179–185

non-protein thiol and ascorbic acid contents in these plants, are inconformity with the reports (Singh et al., 2004b; Singh and Sinha2005; Sinha et al., 2007a,b) from other crop plants as they have re-ported an increase in the plants exposed to low level of metal. Thisindicates that the plants grown at lower amendments had consid-erable detoxification mechanism which can counter the stress. Pro-line is another antioxidant imparting defense mechanism towardsstress. Synthesis of proline in response to toxic metals has been re-ported (Singh and Sinha, 2005; Sinha et al., 2007a). Binding withmetal ions due to the chelating ability of proline (an amino acid)can also be a defense mechanism for survival and the observed po-sitive correlation between proline content and MDA content inboth roots and leaves (Sinha and Saxena, 2006) is in conformitywith this explanation.

4. Conclusion

The early-activated antioxidative defence mechanisms controlestablished the redox balance initially in the plants of sesamegrown on lower amendments (upto 35% TS), but reduction in chlo-

rophyll content and morphological effects do occur after a longergrowth period at higher amendments. No marked change in TBARSwas observed in the plants grown on lower amendments. In addi-tion, the oil content increased (35% increase over control) in theplants grown on 35% TS. Besides high level of Cr in the sludge, Crwas not translocated up to the fruits part of sesame at loweramendment of tannery sludge, however, Ni, Cd and Pb were foundabove the recommended limits as reported by Prevention of foodadulteration Act 1954 in all the amendments. Although, the plantshave shown healthy growth when grown on such contaminatedsoil; however, it is not advisable for human consumption of theseeds.

Acknowledgements

Authors are thankful to Dr. Rakesh Tuli, Director, NationalBotanical Research Institute, Lucknow for the facilities provided.AKG is grateful to National River Conservation Directorate, Minis-try of Environment and forests, Government of India, New Delhi,India for the award of fellowship.

A.K. Gupta, S. Sinha / Bioresource Technology 100 (2009) 179–185 185

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