Citric acid improves lead (pb) phytoextraction in brassica napus L. by mitigating pb-induced morphological and biochemical damages

Ecotoxicology and Environmental Safety 109 (2014) 38–47

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Citric acid improves lead (pb) phytoextraction in brassica napus L. by mitigating pb-induced morphological and biochemical damages Muhammad Bilal Shakoor a, Shafaqat Ali a,n, Amjad Hameed b, Mujahid Farid a, Sabir Hussain a, Tahira Yasmeen a, Ullah Najeeb c, Saima Aslam Bharwana a, Ghulam Hasan Abbasi d a

Department of Environmental Sciences Government College University Allama Iqbal Road 38000 Faisalabad, Pakistan Nuclear Institute for Agriculture and Biology (NIAB), P.O. Box 128, Jhang road Faisalabad, Pakistan c Department of Plant and Food Sciences, Faculty of Agriculture and Environment, The University of Sydney, Eveleigh, NSW 2015, Australia d Department of Soil Science, University College of Agriculture and Environmental Sciences, The Islamia University of Bahawalpur, Bahawalpur, Pakistan b

art ic l e i nf o

a b s t r a c t

Article history: Received 17 February 2014 Received in revised form 24 July 2014 Accepted 24 July 2014

Phytoextraction is an environmentally friendly and a cost-effective strategy for remediation of heavy metal contaminated soils. However, lower bioavailability of some of the metals in polluted environments e.g. lead (Pb) is a major constraint of phytoextraction process that could be overcome by applying organic chelators. We conducted a glasshouse experiment to evaluate the role of citric acid (CA) in enhancing Pb phytoextraction. Brassica napus L. seedlings were grown in hydroponic media and exposed to various treatments of Pb (50 and 100 μM) as alone or in combination with CA (2.5 mM) for six weeks. Pbinduced damage in B. napus toxicity was evident from elevated levels of malondialdehyde (MDA) and H2O2 that significantly inhibited plant growth, biomass accumulation, leaf chlorophyll contents and gas exchange parameters. Alternatively, CA application to Pb-stressed B. napus plants arrested lipid membrane damage by limiting MDA and H2O2 production and by improving antioxidant enzyme activities. In addition, CA significantly increased the Pb accumulation in B. napus plants. The study concludes that CA has a potential to improve Pb phytoextraction without damaging plant growth. & 2014 Elsevier Inc. All rights reserved.

Keywords: Anitoxidant enzymes Brassica napus L Citric acid Lead Malondialdehyde

1. Introduction The introduction of toxic heavy metals/metalloids into water bodies from domestic, agricultural and industrial practices is a global environmental problem due to their well-recognized toxic effects on the mankind and ecosystem (Mataka et al., 2006). A wide range of research has been conducted to understand the toxicity mechanism of Pb to human health and to ecosystem (Schreck et al., 2012). Lead (Pb) toxicity in plants is associated with negative effects on nutrient uptake, photosynthesis and antioxidant enzymes (Bharwana et al., 2014a; Pourrut et al., 2011a). Antioxidant enzymes are one mechanism plants have evolved as a response to metal-induced toxicity (Piotrowska-Niczyporuk et al., 2012). For example, guaiacol peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT) signifi cantly contribute to regulate the cellular redox homeostasis to a safe level (Yin et al., 2008). However, under Pb toxicity, overproduction of reactive oxygen species (ROS) disturbs this redox equilibrium (Pourrut et al., 2011a; Shahid et al., 2013). ROS-induced lipid membrane n

Corresponding author. Fax: þ92 41 9200671. E-mail address: [email protected] (S. Ali).

http://dx.doi.org/10.1016/j.ecoenv.2014.07.033 0147-6513/& 2014 Elsevier Inc. All rights reserved.

peroxidation and oxidative stress interrupt normal metabolic activities and damage biological molecules such as proteins, lipids and nucleic acids; ultimately leading to cellular destruction (Pourrut et al., 2011b). Clearly, a viable and cost-effective method to remove Pb from the environment is needed. Phytoremediation is a method by which some plants uptake, sequester and detoxify contaminants (Jing et al., 2007; Shakoor et al., 2013). The first step of phytoremediation, phytoextraction, involves plants translocating heavy metals to aboveground parts such as shoots and ultimately harvesting these plants from contaminated area (Nascimento and Xing, 2006). Two phytoextraction approaches are commonly implemented (1) use of hyper-accumulator plants, and (2) addition of chemicals (synthetic/organic) to enhance the growth of high-biomass producing plants to accelerate metal uptake in their aerial parts (Evangelou et al., 2007; Murakami et al., 2007). In this context, Brassica species are generally considered heavy metal tolerant due to their high above-ground biomass production and have capacity to extract considerable amounts of heavy metals from contaminated media (Meng et al., 2008; Vangronsveld et al., 2009). Whilst a number of plants have a high phytoextraction potential, the low solubility and diffusion of some metals can limit plant

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phytoextraction (Turan and Esringü, 2007). Numerous studies suggested that the addition of chelating agents such as ethylenediaminetetraaceticacid (EDTA), citric acid (CA) and N-(2-hydroxyethyl)-ethylenediaminetriaceticacid (HEDTA) may enhance metal mobility and bioavailability, thereby increasing phytoextraction process (Cheng, 2003; Elless and Blaylock, 2000; Farid et al., 2013b; Huang et al., 1997; Liphadzi and Kirkham, 2006; Safari et al., 2008; Turgut et al., 2004). For enhancing phytoextraction, EDTA is the most commonly used synthetic chelator, however; its slow degradation rate and long persistence in soil makes it unsuitable for beneficial purpose. On the other hand, highly biodegradable (Ding et al., 2005; Farid et al., 2013a) low molecular weight organic acids (LMWOA) such as citric acid, can provide an alternate to EDTA in improving metal uptake from contaminated soil (Luo et al., 2005). The present study investigates the (1) physiological and biochemical effects of Pb-poisoning on Brassica napus L., (2) effect of CA on the growth, and activity of antioxidant enzymes on plants exposed to toxic levels of Pb and (3) effect of CA on Pb phytoextraction by B. napus L.. We hypothesized that CA has the potential to enhance Pb bioavailability and uptake without negatively affecting plant growth.

2. Materials and Methods 2.1. Experimental Site The experiment was performed in glasshouses at Ayub Agricultural Research Institute, Faisalabad, Pakistan.

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2.7. Determination of Chlorophyll contents Chlorophyll a, b, total chlorophyll and total carotenoids were spectrophotometrically determined (Metzner et al., 1965). The topmost fully expanded leaves were taken to extract the pigments. The photosynthetic pigments were extracted from a 0.5 g of leaves in an aqueous acetone 85% (v/v) solution. The extract was centrifuged at 4000 rpm for 10 min; the supernatants were then diluted with 85% aqueous acetone solution to the appropriate concentration for spectrophotometric analysis. The extinction was evaluated against a blank of a pure 85% aqueous acetone at wavelengths of 452.5, 644 and 663 nm. Total and chlorophyll, a, b, and total carotenoids were calculated by following equations: Chlorophyll aðmg=mLÞ ¼ 10:3n–E663 –0:98nE644 Chlorophyll bðmg=mLÞ ¼ 19:7nE644 –3 : 87nE663 Total chlorophyll ¼ chlorophyll a þ chlorophyll b Total carotenoidsðmg=mLÞ ¼ 4:2nE452:5 –fð0:0264nchlaÞ þ ð0 : 426nchl bÞg At the end, these pigment fractions were calculated as mg g  1 fresh weight.

2.8. Assessment of Electrolyte leakage At the end of experiment (after six weeks), the topmost fully expanded leaves were cut into 5 mm long fragments, and positioned in test tubes filled with 8 mL distilled water. The tubes were incubated in a water bath at 32 1C for 2 hours and initial electrical conductivity (EC) of the medium EC1 was noted. The samples were autoclaved at 121 1C for 20 min to discharge all electrolytes, and then cooled at 25 1C, and final EC2 was measured (Dionisio-Sese and Tobita, 1998). Electrolyte leakage (EL) was calculated by the following formula EL ¼ ðEC 1 =EC 2 Þ  100:

2.2. Growth Conditions Seeds of Brassica napus L. (variety Faisal canola) were obtained from Ayub Agricultural Research Institute, Faisalabad, Pakistan. Seeds were thoroughly rinsed with distilled water before sowing into plastic trays containing 6 cm layers of sterilized quartz sand and incubating at 20–22 1C in growth chambers. After four weeks, 24 seedlings were shifted to one of three 40 L iron tubs lined with polythene sheets (n¼3). The tubs were filled with modified Hoagland’s solution (in mML  1: K(NO3)2 3000; KH2(PO4) 100; Ca(NO3)2 2000; MgSO4 1000; H3BO3 50; ZnSO4. 7H2O 0.8; MnCl2. 4H2O 0.05; CuSO4. H2Mo4. H2O 0.10; 5H2O 0.3; and FeNa-EDTA 12.5). The growth media was constantly aerated with an air pump and changed every week. After two weeks, Pb(NO3)2 and citric acid (CA) treatments were applied as Pb (50 mM), Pb (100 mM ), CA (2.5 mM), Pb (50 mM ) þ CA (2.5 mM) and Pb (100 mM) þ CA (2.5 mM) with three replications, whereas no Pb(NO3) or CA was added to the control (Ck) treatment. The pH was maintained at 6.0 70.1 during the experiment by 1 M H2SO4 or NaOH. 2.3. Measurements of plant biomass and growth parameters Six weeks after the Pb and CA treatments were applied, the plants were harvested and partitioned into leaves, root and stem. Plant fresh weight and stem length were measured and samples were allowed to dry at 70 1C for at least 72 hours for dry biomass measurements. Fresh plant samples were used for biochemical analysis.

2.4. Leaf Area Leaf area of each individual plant was measured using a leaf area meter (L12000, L1-COR, USA).

2.5. Gas exchange Attributes Infrared gas analyzer (IRGA) was used for measuring leaf transpiration rate (E), stomatal conductance (gs), photosynthetic rate (A) and water use efficiency (A/E).

2.6. Soil plant analysis development (SPAD) value For the analysis of leaf greenness / SPAD (Soil-Plant Analysis Development) value, SPAD-502 (name of company) meter was used.

2.9. Determination of Antioxidant enzymes 2.9.1. Sample extraction In order to determine enzymatic activities, 0.5 g of fresh sample was ground in potassium phosphate buffer solution under pre-chilled condition. Different buffer solutions were used for each enzyme. Samples were centrifuged at 15,000 rpm at 4 1C for 20 min. The liquid was extracted and stored in micro centrifuge tubes. Supernatant was then used to analyze the activities of different antioxidant enzymes. The activities of SOD and POD were determined according to the method of Zhang (1992).

2.9.2. Catalase (CAT) Catalase (CAT, EC 1.11.1.6) activity was determined according to Aebi (1984). The analyte mixture (3.0 mL) contained 100 μL enzyme extract, 100 μL H2O2 (300 mM) and 2.8 mL 50 mM phosphate buffer with 2 mM EDTA (pH 7.0). The CAT activity was determined by monitoring reduction in absorbance of analyte mixture at 240 nm as a consequence of H2O2 disappearance (ε ¼ 39.4 mM  1 cm  1).

2.9.3. Ascorbate peroxidase (APX) Ascorbate peroxidase (APX, EC 1.11.1.11) activity was measured according to Nakano and Asada (1981).The assay mixture consisting of 100 μL enzyme extract, 100 μL ascorbate (7.5 mM), 100 μL H2O2 (300 mM) and 2.7 mL 25 mM potassium phosphate buffer was used for measuring APX activity. The oxidation of ascorbate was analyzed by the variation in absorbance at 290 nm (ε ¼ 2.8 mM  1 cm  1).

2.10. Malondialdehyde (MDA) content The lipid peroxidation level in the leaf and root tissues was assessed in terms of malondialdehyde (MDA, a product of lipid peroxidation) contents. 0.25 g of leaf / root sample was homogenized in 5 mL of 0.1% trichloro acetic acid TCA. The homogenate was centrifuged at 10,000g for 5 min. To 1 mL aliquot of the supernatant, 4 mL of 20% TCA containing 0.5% TBA was added. The mixture was then heated at 95 1C for 30 min, and then rapidly cooled down in an ice bath. After centrifugation at 10,000g for 10 min, the absorbance of supernatant was recorded at 532 nm, and the readings for nonspecific absorption at 600 nm were subtracted. The MDA contents were calculated by applying an extinction coefficient of 155 mM  1 cm  1.

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2.11. Hydrogen peroxide contents The H2O2 contents were assayed colorimetrically as documented by Jana and Choudhuri, (Jana and Choudhuri, 1981). Hydrogen peroxide was extracted by homogenizing 50 mg leaf or root tissues with 3 mL of phosphate buffer (50 mM, pH 6.5). The homogenate was centrifuged at 6,000 g for 25 min. To measure H2O2 contents, 3 mL of the extracted solution was mixed with 1 mL of 0.1% titanium sulphate in 20% (v/v) H2SO4, and the mixture was then centrifuged at 6,000 g for 15 min. The intensity of yellow color supernatant was analyzed at 410 nm. H2O2 contents were calculated by implying the extinction coefficient of 0.28 mmol  1 cm  1. 2.12. Soluble protein content Assessment of soluble protein contents was carried out according to Bradford, (Bradford, 1976) method, using Coomassie Brilliant Blue G-250 as a dye and albumin as a standard. 2.13. Determination of Lead (Pb) Concentration After six weeks of treatment, plants were harvested, cleansed with tap water, distilled water and deionized water comprehensively. Immediately after that, the plant samples were differentiated into leaves, stem, and roots, dehydrated at 80 1C in an oven for 48 hours, and then mashed into powder. 0.5 g of each sample was dry-ashed, extracted with HCl and then centrifuged at 3600 rpm for 15 min. Concentrations of Pb in leaves stem and roots were examined by flame atomic absorption spectrometry. The Pb concentration and accumulation in the root, stem and leaf tissues were calculated by the following formula: Pb concentrationðmg kg  1 Þ ¼ reading  dilution factor=dry wt: of plant part The accumulation of Pb in plant shoot and root was estimated by the following formula: Pb accumulationðmg plant  1 Þ ¼ conc:of Pb  dry wt: of plant organ

2.14. Statistical Analysis All readings documented in this investigation are the average of three replicates. Analysis of variance (ANOVA) was performed by putting the values in a statistical package, SPSS version 16.0 (SPSS, Chicago, IL) followed by Tukey test to identify significant differences among the treatment means.

3. Results 3.1. Plant Growth attributes Exposure of B. napus plants to increasing Pb levels (50, 100 μM) significantly inhibited various plant growth attributes such as plant height, root length, numbers of leaf per plant and leaf area (Fig. 1A-D). Although Pb-stressed plants had significantly lower growth attributes compared with non-stressed plants, CA application significantly increased the height, root length, and leaf growth of Pb-stressed plants. 3.2. Gas exchange Attributes Brassica napus L. leaves showed suppressed gas exchange characteristics such as net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (Gs) and water use efficiency (Pn/E) due to the toxicity caused by Pb at both levels. The inhibitory effect of higher Pb concentration (100 μM) on leaf gas exchange characteristics was significantly greater compared with control as well as plants treated with 50 μM Pb (Fig. 1 E-H). Citric acid addition to growth media significantly improved all the gas exchange parameters of the Pb-stressed (50 and 100 μM) plants. 3.3. Plant biomass Both levels of Pb significantly reduced plant fresh and dry biomass and the inhibitory effect was more severe under 100 μM

Pb (Fig. 2). Citric acid treatment significantly enhanced the fresh and dry weights of leaves, shoots and roots of Pb-stressed B. napus plants. 3.4. Chlorophyll contents and SPAD value Chlorophyll and carotenoids contents as well as SPAD value were significantly reduced by Pb stress alone (Fig. 3). The addition of CA (2.5 mM), dramatically improved the chlorophyll contents in leaves of Brassica plants. Citric acid supplementation to growth media significantly improved chlorophyll a, chlorophyll b, total chlorophyll, carotenoids and SPAD value of Pb-stressed or nonstressed plants compared with control. 3.5. Activities of antioxidant enzymes Antioxidant enzyme (SOD, POD, CAT, and APX) activities of B. napus leaves and roots variably responded to various treatments of Pb alone or with CA in the growth media (Fig. 4). Activities of SOD, POD, CAT and APX of B. napus roots and leaves significantly increased and then decreased by increasing Pb concentration from 50 to 100 μM, however, except POD. Activities of these enzymes remained significantly higher than the control plants under any level of Pb. Addition of CA (2.5 mM) significantly increased antioxidant enzyme activities of Pb-stressed (50 and 100 μM) B. napus plants. Total soluble protein contents of B. napus leaves and roots significantly inhibited by increasing concentrations of Pb in the growth media (Fig. 5 A, B). Citric acid (2.5 mM) application significantly increased the leaf protein contents of both Pbstressed or unstressed plants, while effect of CA on root protein contents was significant only in Pb-stressed plants. 3.6. Oxidative stress and electric conductivity Pb-induced oxidative stress in B. napus leaves and root tissues was estimated in term of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) contents. Significant increase in leaf and root MDA contents was recorded under any levels of Pb (50 and 100 μ M). Exogenous application of CA (2.5 mM) significantly reduced leaf and roots MDA content of plants experiencing Pb stress (Fig. 5 C & D). A similar increasing trend in H2 O 2 contents was observed in plants under various Pb concentrations (50 and 100 μ M), however, CA addition dramatically lowered H 2 O 2 content in both leaves and roots of the Pb-stressed plants (Fig. 5E & F). Pb-induced membrane injury and solute leakage in B. napus leaves is presented in Fig. 5 (G). A significant increase in the electrolyte leakage was recorded under Pb stress (50 and 100 μM). Citric acid application to Pb-stressed or unstressed B. napus significantly lowered the solute leakages in leaf tissues. 3.7. Lead contents Lead concentration in different parts of B. napus plants is expressed in Table 1. Increasing Pb concentration in growth media significantly increased the Pb uptake in all three plant parts viz. root, stem and leaves. Irrespective of Pb levels, B. napus roots accumulated maximum Pb contents followed by stem and leaves. Citric acid addition significantly increased the Pb uptake in all three plant organs at both levels of Pb (50, 100 μM) applied. Moreover, CA facilitated Pb translocation from plant roots to aboveground parts. Lead content in shoot and root of Brassica plants with respect to two levels of Pb alone and in combination with CA is expressed in

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Fig. 1. Effect of exogenous CA (2.5 mM) on plant height A, root length B, number of leaves plant  1 C and leaf area D of canola (Brassica napus L) seedlings under lead (Pb) (50, 100 mM) toxicity. Values are means of at least 3 replicates. Error bars are standard error of means. Bars bearing different letters differ significantly from each other at p o 0.05).

Table 1. A significant rise in per plant Pb uptake in shoot and root tissues was noted with increasing concentration of Pb in the nutrient culture. Addition of CA in conjunction with two levels

of Pb further enhanced per plant Pb uptake in shoot and root of Brassica napus L. plants causing the highest Pb accumulation at 100 μM as compared with control.

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Fig. 2. Effect of exogenous CA (2.5 mM) on leaf, stem and root fresh weight A,B,C and dry Weight D,E,F of canola (Brassica napus L) seedlings under lead (Pb) (50, 100 mM) toxicity. Values are means of at least 3 replicates. Error bars are standard error of means. Bars bearing different letters differ significantly from each other at p o 0.05).

4. Discussion Lead is an extremely phytotoxic heavy metal which induces several physiological and ultrastructural disorders in plants (Sharma and Dubey, 2005). Generally, sensitivity to metal toxicity in plants relies on metal concentration, exposure time, and plant species, age and tissue type (Gao et al., 2010). The decrease in plant biomass (Fig. 2) likely indicates heavy metal stress caused by the interference of lead with the plant physiology and metabolism (Alia and Saradhi, 1991; Baker and Walker, 1990). In particular, a reduction in protein synthesis (Stiborova et al., 1987) and photosynthesis, and damage to cell and sub-cellular organelles (Hauck et al., 2003). The addition of CA to Pb-stressed plants significantly enhanced plant growth and biomass (Figure??). This could be due to the ameliorating role of CA in facilitating various metabolic processes

of plants, e.g. through mobilizing weakly soluble essential nutrients (Strom et al., 2001). Gao et al. (2010) observed that plant biomass was increased in Solanum nigrum L. with the application of CA (how much?). This fact was also confirmed by Najeeb et al. (2011) and Ehsan et al. (2014), who observed similar promotive role of CA for plant growth under Mn and Cd stress in J. effusus and B. napus L. respectively. Additionally, Pb stress induced a negative impact on leaf chlorophyll contents and gas exchange attributes. This might be the consequence of disruption of chloroplast, protein complex, and photosynthetic apparatus when plants are exposed to heavy metal stress (Ali et al., 2013b, 2013a; Vassilev et al., 1995). Moreover, breakdown of chlorophyll could be due to a rise in chlorophyllase activity under heavy metal stress (Hegedus et al., 2001). Heavy metals can influence chlorophyll contents, gas exchange and stomatal conductance; leading to a reduction photosynthetic rate

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Fig. 3. Effect of exogenous CA (2.5 mM) on chlorophyll a A, Chlorophyll b B, total chlorophyll C, total carotenoids D and SPAD value E of canola (Brassica napus L) seedlings under lead (Pb) (50, 100 mM) toxicity. Values are means of at least 3 replicates. Error bars are standard error of means. Bars bearing different letters differ significantly from each other at po 0.05).

(Balakhnina et al., 2005). However, in present situation when CA was applied to plants under Pb stress, a remarkable increase in chlorophyll content, gas exchange characteristics and SPAD value was observed. The role of chelator in enhancing photosynthetic attributes has been found due to the substantial increase in chlorophyll content (Wang et al., 2004) and subsequently elevating light harvesting potential of the treated plants. Antioxidant enzymes and some specific metabolites play a key role in adaptation and survival of plants under metal toxicity. Oxidative stress alters the antioxidant activities, which are essential to mitigate this stress (Shamsi et al., 2008). Increase in hydrogen peroxide (H2O2) activity coupled with growth inhibition

might be the reason for reduction in antioxidants enzyme activity (Schützendübel et al., 2001). Decreased activities of SOD and CAT under Pb stressed plants were noticed in present study and this decrease was attributed to Pb uptake in the plant cells. The SOD is a vital constituent of plants antioxidative defense mechanism as it dismutase O2 to H2O2 and then O2. Likewise, POD and APX are major enzymes and disintegrate H2O2 (Scandalios, 1994). In the present experiment, the reduction of enzyme activity under higher Pb levels might be due to a delay in the elimination of H2O2 and toxic peroxides intervened by POD and APX. An increase in the activities of these antioxidative enzymes under the combined treatment of Pb and CA, suggest that CA acid might improve

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Fig. 4. Effect of exogenous CA (2.5 mM) on net photosynthetic rate A, transpiration rate B, stomatal conductance C and water use efficiency D of canola (Brassica napus L) seedlings under lead (Pb) (50, 100 mM) toxicity. Values are means of at least 3 replicates. Error bars are standard error of means. Bars bearing different letters differ significantly from each other at po 0.05).

antioxidant defense mechanisms by enhancing SOD, POD, CAT and APX activities in Brassica plants. Najeeb et al. (2011) also documented a positive role of CA in enhancing the activities of antioxidant enzymes and thus, mediated metal induced oxidative stress in J. effusus plants. To our knowledge, this is the first study to evaluate the role of CA in improving the antioxidant enzyme activities under Pb stress in B. napus L. plants. Protein contents (in leaf and root tissues) were significantly reduced at both stress levels (Pb 50, 100 mM). Damage to the soluble proteins present in leaves and roots of B. napas plants may be due to oxidative damage that caused the reduction in protein contents (Gupta et al., 2009). However, CA addition significantly increased the protein contents and decreased the effect of Pb stress as compared with Pb-stressed plants only. Similar positive effects of CA have been documented and suggested that plants would modify their metabolisms to mitigate harmful effect induced by metal toxicity (Bharwana et al., 2014b; Ganesh et al., 2008; Vernay et al., 2007).

Lipid peroxidation is the oxidative damage of lipids consisting of any number of carbon–carbon double bond, producing MDA as the ultimate final product. An increase in MDA level is generally regarded as an indicator of oxidative stress in metal treated stressed plants (Sandalio et al., 2001). Lead stress leads to the overproduction of ROS, which cause oxidative destruction in plant cells (Apel and Hirt, 2004). Increase in MDA and H2O2 contents under Pb stress accounted for lipid peroxidation or plasma membrane damage, which in turn inhibited plant growth (Zhang et al., 2009). In the current experiment, concentration-dependent rise in lipid peroxidation and H2O2 contents in Brassica plant shoot tissues was noticed, confirming Pb- induced oxidative damage in B. napus L. The reduction in EC, MDA and H2O2 with the addition of CA demonstrated the protective role of CA against the Pb toxicity that accelrated oxidative deterioration in the Brassica plants. Similar findings about protective role of CA were recorded in J. effusus by Najeeb et al. (2011), where CA safeguarded plants from metal-induced chloroplast disintegration.

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Fig. 5. Effect of exogenous CA (2.5 mM) on protein content in A leaves and B roots, melondialdehyde (MDA) in C leaves and D roots, hydrogen peroxide(H2O2) in E leaves and F roots and electrolyte leakage in G leaves of canola (Brassica napus L) seedlings respectively under lead (Pb) (50, 100 mM) toxicity. Values are means of at least 3 replicates. Error bars are standard error of means. Bars bearing different letters differ significantly from each other at p o 0.05).

Table 1 Effect of exogenous CA on Pb uptake and in root, stem and leaf of canola (Brassica napus L) seedlings under lead (Pb) toxicity. Treatment

Ck CA Pb 50 Pb 50þCA Pb 100 Pb 100þ CA

Pb accumulation (mg plant  1)

Pb concentration (mg g  1)

Shoot

Root

Root

Stem

0.016 e 0.035 e 2.18 d 8.55 c 10.14 b 13.36 a

0.0032 e 0.0047 e 1.81 d 4.55 c 5.76 b 7.41 a

2.13 e 3.07 e 2233.37 d 2850 c 4160.66 b 4785 a

0.97 1.91 217.58 272.20 407.95 485.45

Leaf e e d c b a

0.83 e 1.58 e 164.08 d 194.12 c 311.44 b 365.78 a

Values are means of at least 3 replicates. Error bars are standard error of means. Bars bearing different letters differ significantly from each other at p o 0.05).

Metal accumulation in plants is clearly related to the concentration of metal in the growing environment (Labra et al., 2006). Lead contents in all three parts of Brassica plants in present

experiment were enhanced, as we increased the Pb concentration in solution media. Our outcomes about more uptake of Pb contents in roots than shoots and leaves indicate the potential of plants to get rid of metal-induced damages (Najeeb et al., 2011). In general, the plants with greater resistance, take up a lower percentage of the total solution metal and encompass the lowest above ground metal in leaves (Liu and Kottke, 2004). ¼In the case of B. napus L., application of CA significantly enhanced Pb uptake and concentration in all three organs of plants such as roots, stem and leaves as compare to the Pb alone treated plants. Our results are in accordance with the findings of Hocking et al. (1997), who documented enhanced bioavailability of Mn in rhizosphere in the presence of low molecular weight organic acid (LMWOA). Citric acid and other LMWOA offer protons and electrons for removal of metals in the rhizosphere together with oxidation of CAv(Jones and Brassington, 1998). Increased uptake of Pb with CA addition is also in conformity with the results of Williams et al. (2006) who suggested, CA was efficient in increasing the concentration of Cd in shoots of Indian mustard.

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5. Conclusion Citric acid plays a key role in improving the growth of B. napus L. under Pb stress. Lead toxicity can reduce growth, biomass, pigments, photosynthetic attributes, antioxidant capacity and protein content of plants by inducing lipid membrane damage and electrolyte leakage. However, CA addition significantly improves the morphology, photosynthetic characteristics by limiting the cellular oxidative damage. Our results also elaborate that B. napus L might extract a considerable amount of pollutant like Pb and serves as a hyperaccumulator plant. Furthermore, this study is based on hydroponic conditions, where CA effectively improves Pb phytoextraction; however, field-based research is required to further verify the ameliorative effects of CA plants grown on Pb contaminated soils.

Acknowledgment Thanks to Higher Education Commission (HEC) of Pakistan for financial support. The results presented in this paper are a part of M. Phil studies of Mr. Muhammad Bilal Shakoor.

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