Growth, tolerance efficiency and phytoremediation potential of Ricinus communis (L.) and Brassica juncea (L.) in salinity and drought affected cadmium contaminated soil

Ecotoxicology and Environmental Safety 85 (2012) 13–22

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Frontier Article

Growth, tolerance efficiency and phytoremediation potential of Ricinus communis (L.) and Brassica juncea (L.) in salinity and drought affected cadmium contaminated soil Kuldeep Bauddh, Rana P. Singh n Department of Environmental Science, B.B. Ambedkar University, Lucknow-226025, India

a r t i c l e i n f o

abstract

Article history: Received 30 May 2012 Received in revised form 17 August 2012 Accepted 18 August 2012 Available online 6 September 2012

We have previously reported that Ricinus communis (castor) is more tolerant to soil cadmium (Cd) and more efficient for Cd phytoremediation than Brassica juncea (Indian mustard) (Bauddh and Singh, 2012). In the present study, R. communis was found more tolerant to salinity and drought in presence of Cd and removed more Cd in a given time than Indian mustard. R. communis produced 23 and twelve folds higher biomass in terms of fresh weight and dry weight, respectively than that in B. juncea during three months when grown in Cd contaminated soil in presence of 100 mM NaCl salinity and ten day water withdrawal based drought at 90 day after sowing (DAS). Castor plants showed stronger selfprotection ability in form of proline bioaccumulation (r2 ¼ 0.949) than Indian mustard (r2 ¼ 0.932), whereas a lower r2 for malondialdehyde (MDA) and total soluble protein in R. communis (r2 ¼ 0.914 and r2 ¼ 0.915, respectively) than that of B. juncea (r2 ¼ 0.947 and r2 ¼ 0.927, respectively) indicated a greater damage to cell membrane in Indian mustard during the multiple stress conditions. Though, the amount of Cd accumulated in the roots and shoots of Indian mustard was higher as per unit biomass than that in castor, total removal of the metal from soil was much higher in castor on per plant basis in the same period in presence of the stresses. R. communis accumulated about seventeen and 1.5 fold higher Cd in their roots and shoots, respectively than that of B. juncea in 90 DAS under the multiple stresses. Salinity alone enhanced Cd uptake, whereas drought stress reduced its uptake in both the plants. & 2012 Elsevier Inc. All rights reserved.

Keywords: Bioaccumulation Castor Drought Indian mustard Salinity Translocation factor

1. Introduction Amongst the various strategies adopted for removal of toxic heavy metals from the contaminated sites, phytoremeditiation has emerged as an economical, eco-friendly and aesthetically acceptable technology in the recent years (Cecchi and Zanchi, 2005; Liu et al., 2010; Sylwia et al., 2010; Huang et al., 2011; Santana et al., 2012; Witters et al., 2012; Stingu et al., 2012). Brassica juncea is one of the most studied plant found tolerant to most of the toxic heavy metals and suggested to be a potential phytoremediator (Singh et al., 2007; Nouairi and Ammar, 2009; Bauddh and Singh, 2009, 2011, 2012; Sharma et al., 2010). Most of the contaminated lands bearing industrial wastes and waste water are waste lands possessing multiple stresses like salinity, drought and excessive heavy metals simultaneously. B. juncea have been found to be sensitive to most of the stresses and its green leaves are used as vegetable and fodder in many part of the Indian subcontinent, therefore other alternatives are required to

n

Corresponding author. E-mail address: [email protected] (R.P. Singh).

0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.08.019

be investigated for removal of the metals from the contaminated agricultural ecosystems. We have recently reported Ricinus communis (Bauddh and Singh, 2012) and Jatropha curcas (Ghavri et al., 2010; Ghavri and Singh, 2010, 2012; Pandey et al., 2012) as better alternative to B. juncea. A considerable portion of the agricultural ecosystem supporting cultivation of B. juncea in Indian subcontinent is affected by drought and salinity; therefore it will be very significant to investigate comparative tolerance and phytoremediation efficiency of B. juncea and R. communis from the metal contaminated soil under these stresses. The present study is planned with this perspective to study the tolerance capability and cadmium removal by these two plants in similar agroclimatic conditions from the Cd contaminated soil under the influence of the multiple stresses. 2. Materials and methods 2.1. Plant materials and experimental design The seeds of Indian mustard (B. juncea L.) cv Pusa Jai Kisan were obtained from the National Seed Disposal Centre of Indian Agricultural Research Institute (IARI), New Delhi and that of Castor (R. communis L.) cv Kalpi were procured from the

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K. Bauddh, R.P. Singh / Ecotoxicology and Environmental Safety 85 (2012) 13–22

National Seed Disposal Centre, Lucknow. Seeds were sown in 30-cm-diameter earthen pots filled with 8 kg soil. The soil salinity was created by adding NaCl (100 mM) solution one liter per ten day. Ten days regime (ten day water withdrawal) was applied to create drought stress. The control plants or plants not subjected to the drought stress were irrigated routinely with one liter ground water on each three day. Soil was mixed with appropriate amount of CdCl2 to maintain 100 mg Cd kg  1 soil. Nitrogen (N), phosphorus (P) and K (potassium) (120, 30 and 80 mg kg  1 soil, respectively) were applied at the time of sowing in the form of urea, single super phosphate and potash. The detail of treatments used for the experiments is given in Table 1. The pots with seeds of B. juncea were kept directly in naturally illuminated net house of research field station in the Department of Environmental Science BBA University, Lucknow, India (during October–December months) having the minimum temperature ranged between 5.4–20.8 1C, maximum temperature 16.9– 33.5 1C and averaged temperature 11.5–29.55 1C with relative humidity 87–99 percent at 7.18 am and 37.6–63 percent at 2.15 pm. The seeds were germinated in five day and one plant was maintained in each pot for further studies. R. communis needs a comparatively higher temperature for seed germination; hence the pots containing its seeds were kept in Green House with controlled conditions for ten day at 3070.5 1C with 8570.5 percent relative humidity. After germination, the pots of R. communis were shifted in the same net house for comparative study (with B. juncea). After seedling emergence, one plant per pot was maintained. The details of irrigation and stress conditions are given in Table 1. All the measurements were performed at 30, 60 and 90 day after sowing (DAS). Physicochemical characteristics of the experimental soil was analyzed before seed sowing and presented in Table 2.

2.2. Plant growth analysis The plants were removed from pots at 30, 60 and 90 DAS and dipped in a water filled bucket. The plant roots were washed carefully to remove the adhering soil particles. Fresh weight of roots and shoots was taken by digital single pan balance. The

Table 1 Experimental design for the pot experiment with application of Cd, Salinity and drought. Treatment

Details

T0

Control (irrigation on 3 day periodicity) Drought (irrigation on 10 d periodicity) Salinity (100 mM NaCl solution), irrigation with ground water on each 3 day Salinity þ Drought Cd (100 mg Cd kg  1 soil) Cdþ Drought Cdþ Salinity Cdþ Salinity þDrought 6

T1 T2

T3 T4 T5 T6 T7 Replication (n)

same plant parts were then placed in an oven at 70 1C till the weight became constant. The dried plant parts were weighed to record the dry mass of roots and shoots. The average fresh and dry biomass accumulation/day/plant was measured as follows Biomass ðmgÞ=plant=day of 30 days old plant ðB1Þ ¼ Biomass of 30 days old plant=30 Biomass ðmgÞ=plant=day of 60 days old plant ðB2Þ ¼ Biomass of 60 days old plant=60 Biomass ðmgÞ=plant=day of 90 days old plant ðB3Þ ¼ Biomass of 90 days old plant=90

Average Biomass accumulation mg=plant=day ¼

B1 þ B2þ B3 3

2.3. Estimation of protein, proline and malondialdehyde (MDA) Leaves of the freshly harvested mustard and castor plants from various treatments were separated manually. Protein, Proline and MDA were determined at 30, 60 and 90 DAS. 2.3.1. Protein Protein was estimated by method of Lowry et al. (1951). Fresh leaves (100 mg) of the control and treated plants were homogenized separately in 3 ml of 10 percent chilled trichloroacetic acid (TCA) in pestle and mortar and centrifuged at 10,000 rpm for 10 min. After decanting the supernatants, the pellets were washed and heated for 7 min with 3 ml of 1 N NaOH (sodium hydroxide), cooled and centrifuged again at 10,000 rpm for 10 min. The 0.5 ml of extracted sample was taken in 2.5 ml of 0.5 percent CuSO4 (copper sulfate in 1 percent potassium sodium tartarate), 48 ml of 5 percent Na2CO3 (sodium carbonate) was added. 0.5 ml (1 N) of folin-phenol reagent was added after 10 min. 30-min incubation developed a blue color complex in the mixture. Absorbance was taken at 700 nm against a blank without sample. Protein content was calculated by a standard curve made by bovine serum albumin (BSA). 2.3.2. Proline Proline was determined according to the procedure described by Bates et al. (1973). Fresh leaves and roots (500 mg) was extracted with 3 percent aqueous 5-sulphosalicylic acid, centrifuged at 5000 r min  1. The sample of the supernatant was used for the proline assay and measured at 520 nm. Proline content was expressed as mg g  1 fresh weight (FW). 2.3.3. Malondialdehyde (MDA) The level of lipid peroxidation products in leaf samples was expressed as MDA content and was determined by Heath and Packer (1968). About 200 mg fresh leaves were ground in 0.25 percent 2-thiobarbituric acid (TBA) in 10 percent trichloroacetic acid (TCA) using a mortar and pestle. After heating at 951C for 30 min, the mixture was quickly cooled in an ice bath and centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was read at 532 nm and corrected for unspecific turbidity by subtracting the absorbance of the same at 600 nm. The blank was 0.25 percent TBA in 10 percent TCA. The concentration of lipid peroxides together with oxidatively modified proteins of plants were thus quantified in terms of MDA level using an extinction coefficient of 155 mM  1 cm  1 and expressed as n mol g  1 fresh weight (FW). 2.4. Estimation of cadmium, sodium and potassium content

Table 2 Physicochemical characteristics of experimental soil. Parameters

Values

pH EC(dsm  1) C organic (%) N (g kg  1) P(g kg  1) K(g kg  1) Ca(g kg  1) Na(g kg  1) S (ppm) Zn (ppm) Fe (ppm) Mn (ppm) Ni (ppm) Cd (ppm) Cr (ppm) Pb (ppm) Cu (ppm)

7.03 0.44 1.21 1.43 0.837 2.24 3.02 4.29 19.56 3.22 118.13 7.92 0.021 0.014 0.002 1.41 5.062

Cadmium, sodium and potassium content were estimated after digesting the sample in perchloric acid–nitric acid mixture (1:3 v/v) by atomic absorption spectrophotometer (AA 240 FS, Varian). Per plant (by roots and shoots) Cd accumulation was calculated as follow (Ali et al., 2002; Monni et al., 2000) Total Cd extraction by roots=shoot ðmg=plantÞ ¼ Dry biomass of roots=shoot ðgÞ  metal accumulated ðmg=g dry wtÞ by roots=shoot 2.5. Calculation of translocation factor (TF) and concentration index (CI) of Cd Translocation factor (TF), which was computed from the treatments concentrations, will be used to discuss the results from this study (Mattina et al., 2003). Translocation factor ðTFÞ ¼

C aerial C root

where C¼concentration of metal in mg g  1, in aerial part i.e., shoot. The concentration index (CI) was calculated by dividing Cd levels in the treatment plants by the Cd level in control plants (Kiekens and Camerlynck, 1982). Concentration index ðCIÞ ¼

Concentration of metal in treated plant Concetration of metal in normal plant

K. Bauddh, R.P. Singh / Ecotoxicology and Environmental Safety 85 (2012) 13–22

2.6. Statistical analysis Data (n ¼6) were analyzed statistically by one way analysis of variance (SPSS, Statistical package and MS Excel) using Duncan’s Multiple Range Tests (DMRT) to determine the significance of differences among treatments at probability (p) 0.05 and 0.01.

3. Results 3.1. Soil properties The experimental soil was investigated for its physicochemical and nutritional properties before the seed sowing (Table 2). The soil had pH 7.03, EC 0.44 ds m  1 and organic carbon 1.22 percent. It was rich in Fe (118.13 ppm), Na (4.29 g kg  1), Ca (3.02 g kg  1), S (19.56 ppm), Mn (7.92 ppm) and Zn (3.22 ppm). The soil was having N 1.43 g kg  1, P 0.837 g kg  1 and K (2.24 g kg  1). Other trace metals i.e., Ni, Cu, Cr and Pb were available in small amount. 3.2. Root and shoot biomass production in response to Cd, salinity and drought Application of the stresses to the plants resulted in general reduction in the growth of B. juncea and R. communis in terms of fresh and dry biomass production (Tables 3 and 4). All three stresses alone or in combination caused very significant decrease in biomass production in B. juncea. Roots are generally more affected than the shoots. Cadmium alone (100 mg Cd kg  1 soil) caused more inhibition in biomass production in comparison to drought (ten day water withdrawn) and salinity (100 mM alone) in both the plants. However, combination of drought and salinity caused more pronounced decreased in growth of these plants than the cadmium alone. A decrease of 80.0 and 50.93 percent in the root fresh wt. and 80.57 and 60.59 percent in the shoots fresh wt. in 90 DAS was found in B. juncea and R. communis, respectively when all three stresses viz salinity, drought and Cd were present at a time over the no stress controls. Similarly the root dry wt. decreased by 50.77 and 42.92 percent and shoot dry wt. decreased by

15

80.58 and 55.11 percent in B. juncea and R. communis, respectively under the stresses. Both the oil seed plants studied affected more drastically by Cd followed by drought and salinity when single stress was simulated, however, the effects of stress was more deteriorating in B. juncea when compared with R. communis. Further, the rate of fresh and dry biomass production per plant basis was much higher in roots and shoots of R. communis than that in B. juncea (Fig. 1). The increase in rate of root fresh weight was twelve fold higher and that of shoot fresh weight was two fold higher in R. communis than B. juncea. Similarly the rate of root dry weight production was ten fold higher and shoot dry weight production was three fold higher in R. communis.

3.3. Levels of protein, proline and MDA in the plants in response to cadmium, salinity and drought The effect of Cd alone and that in combination to salinity and drought on leaf total soluble proteins of B. juncea and R. communis was shown in Fig. 2(A–B). The protein content of leaves on 30, 60 and 90 DAS significantly (po0.01) decreased in both the species with application of individual as well as combined stresses. The decrease in total soluble protein was higher in B. juncea (61.72 percent) on 90 DAS under the multiple stresses as compared to R. communis (48.43 percent). The magnitude of decrease in leaf protein was higher in B. juncea (r2 ¼0.927) than that of R. communis (r2 ¼0.915) under these stresses (Fig. 2IA–IB). Cadmium was more detrimental in decreasing leaf protein followed by drought and salinity alone. The combination of the two or three stresses was, however, more effective in decreasing leaf protein in either of the plants. Proline accumulation increased by 2.6 fold in B. juncea and 17.7 fold in R. communis on 90 DAS when all three stresses applied in combination i.e., Cd, salinity and drought (Fig. 2IIA–IIB). Cumulative capacity of free proline is a manifest action of selfprotection ability of plants grown in stressed environment. The proline bioaccumulation in the plants subjected to single or multiple stresses was well correlated with the decrease in protein content in leaves. Castor plants showed stronger self-protection

Table 3 Effect of cadmium chloride on the fresh and dry biomass of B. juncea in presence of NaCl-salinity and drought at 30, 60 and 90 day of sowing. Data was analyses by one way analysis of variance (DMRT) at p o0.05. Different alphabets show significant differences between the treatments. Where: T0 ¼ Control (Irrigation on 3 day periodicity), T1¼ Drought (irrigation on 10 d periodicity), T2¼ Salinity (100 mM NaCl), T3¼ Salinity þDrought, T4 ¼Cd (100 mg Cd kg  1 soil), T5¼ Cdþ Drought, T6¼ CdþSalinity, T7¼ Cdþ Salinity þDrought. Treatments

Fresh biomass (mg) T0 T1 T2 T3 T4 T5 T6 T7 F value at p o 0.5

Roots

Shoots

30 DAS

60 DAS

90 DAS

30 DAS

60 DAS

90 DAS

0.089 70.003a 0.056 70.002c 0.064 70.000b 0.031 70.001f 0.041 70.001d 0.0307 0.00f 0.037 70.002e 0.026 70.001g 416.31

0.521 7 0.009a 0.364 7 0.002c 0.421 7 0.002b 0.234 7 0.014e 0.276 7 0.005d 0.215 7 0.004f 0.241 7 0.010e 0.184 7 0.002g 710.318

1.270 7 0.056a 0.891 7 0.046c 0.913 7 0.028c 0.425 7 0.035e 0.991 7 0.054b 0.321 7 0.009f 0.723 7 0.019d 0.254 7 0.013g 283.212

4.956 7 0.182a 3.235 7 0.200c 3.537 7 0.100b 2.648 7 0.035e 3.056 7 0.084c 2.545 7 0.022e 2.862 7 0.010d 2.044 7 0.071f 187.242

10.268 7 0.234a 7.455 7 0.292bc 8.123 7 0.202b 5.764 7 0.359d 6.879 7 0.0385c 4.543 7 0.112ef 5.138 7 0.084de 4.214 7 1.230f 50.437

16.2417 0.131a 11.5497 0.219c 12.2557 0.196b 7.778 7 0.217d 11.1577 0.100c 6.546 7 0.415e 6.938 7 0.332e 3.154 7 0.116f 888.822

0.179 7 0.001a 0.142 7 0.002c 0.153 7 0.002b 0.124 7 0.002d 0.1267 7 0.001d 0.119 7 0.001e 0.094 7 0.001f 0.071 7 0.00g 1333.016

0.195 7 0.002a 0.165 7 0.003c 0.170 7 0.001b 0.142 7 0.001e 0.146 7 0.001d 0.127 7 0.001g 0.134 7 0.003f 0.096 7 0.00h 592.881

0.58 70.016a 0.413 7 0.007b 0.4276 7 0.005b 0.265 7 0.003de 0.298 7 0.013cd 0.365 7 0.012bc 0.550 70.026a 0.204 70.007e 33.561

1.757 7 0.030a 1.377 7 0.022c 1.424 7 0.018b 0.950 70.106d 1.001 70.041d 0.765 70.024e 0.809 70.025e 0.523 70.011f 247.012

1.946 7 0.074a 1.651 7 0.030b 1.742 7 0.036b 1.349 7 0.080c 1.411 7 0.079c 1.037 70.046d 1.094 70.062d 0.8607 0.060e 113.552

Dry biomass production (mg) T0 0.021 70.001a T1 0.017 70.002b T2 0.018 70.002b T3 0.014 70.001c T4 0.013 70.00cd T5 0.011 70.00f T6 0.012 70.00de T7 0.0097 0.00g F value at p o 0.5 66.146

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Table 4 Effect of cadmium with salinity and drought on fresh and dry biomass production in R. communis after 30, 60 and 90 day of sowing. Data was analyses by one way analysis of variance (DMRT) at p o0.05. Different alphabets show significant differences between the treatments. Where: T0 ¼ Control (Irrigation on 3 day periodicity), T1¼ Drought (irrigation on 10 d periodicity), T2¼ Salinity (100 mM NaCl), T3¼ Salinity þDrought, T4¼ Cd (100 mg Cd kg  1 soil), T5¼ Cdþ Drought, T6¼Cd þSalinity, T7¼ Cdþ Salinityþ Drought. Treatments

Roots

Shoots 60 DAS

90 DAS

30 DAS

60 DAS

90 DAS

Fresh biomass production (mg) T0 0.678 70.045a T1 0.593 70.030b T2 0.615 70.022b T3 0.498 70.015c T4 0.511 70.011c T5 0.425 70.011d T6 0.442 70.009d T7 0.389 70.015d F value at p o 0.5 416.31

8.517 7 0.433a 6.388 7 0.555c 7.439 7 0.105b 6.136 7 0.104cd 5.942 7 0.0629cd 5.205 7 0.170e 5.453 7 0.156de 4.179 7 0.178f 710.318

12.7977 1.558a 10.802 7 0.2444b 10.981 7 0.258b 9.161 7 0.160c 9.097 7 0.976c 7.933 7 0.182c 7.844 7 0.091c 6.279 7 0.169d 283.212

7.478 7 0.531a 6.819 7 0.206b 7.124 7 0.0914ab 5.843 7 0.429c 4.448 7 0.369d 4.062 7 0.187de 4.131 7 0.102de 3.712 7 0.156f 187.242

27.21 7 0.950a 24.82 7 0.604b 24.9223 70.402b 20.73 7 0.360c 18.621 70.503d 16.803 7 0.600e 16.970 7 0.138e 14.720 7 1.325f 50.437

39.8927 01.586a 33.2817 0.0.975b 32.2357 0.841b 26.130 70.710c 20.181 70.1.051d 17.2847 0.646e 17.8547 0.332e 15.7187 0.551e 888.822

Dry biomass production T0 T1 T2 T3 T4 T5 T6 T7 F value at p o 0.5

1.254 7 0.062a 1.0843 7 0.019b 0.998 7 0.023b 0.810 70.023e 0.978 7 0.014c 0.893 7 0.040d 0.724 7 0.002f 0.659 7 0.013g 1333.016

2.789 7 0.113a 2.463 7 0.047b 2.449 7 0.042b 1.929 7 0.025d 2.161 7 0.123c 1.793 7 0.008d 1.824 7 0.125d 1.592 7 0.032e 592.881

1.315 7 0.046a 1.126 7 0.009b 1.146 7 0.004b 0.917 7 0.004dc 0.627 7 0.012cd 0.551 7 0.005be 0.483 7 0.004f 0.420 7 0.002g 33.561

5.418 7 0.594a 5.219 7 0.098a 5.474 7 0.027a 4.950 7 0.020a 3.519 7 0.056c 4.071 7 0.024b 3.986 7 0.025b 4.083 7 0.011b 247.012

9.097 70.174a 7.818 70.290b 7.915 70.136b 6.808 70.240c 5.823 70.071d 5.251 70.026d 5.362 70.162d 4.084 70.160e 113.552

(mg) 0.148 70.148a 0.126 70.126b 0.129 70.129b 0.0987 0.098c 0.0787 0.078de 0.0727 0.07de 0.07207 0.072de 0.0677 0.067de 66.146

Fresh wt of roots mg/plant/day

12

B.juncea

45

R. communis

Fresh wt of shoots mg/plant/day

30 DAS

10 8 6 4 2 0

R. communis

35 30 25 20 15 10 5 0

T0

T1

T2

T3 T4 T5 Treatments

T6

T7

T0

2.5

9 R. communis Dry wt of shoots mg/plant/day

B.juncea Dry wt of roots mg/plant/day

B. juncea

40

2 1.5 1 0.5

T1

T2

T3 T4 T5 Treatments

B. juncea

8

T6

T7

R. communis

7 6 5 4 3 2 1 0

0 T0

T1

T2

T3 T4 T5 Treatments

T6

T7

T0

T1

T2

T3 T4 T5 Treatments

T6

T7

Fig. 1. Effect of cadmium with salinity and drought on average fresh and dry biomass production (mg/plant/day) in B. juncea after 30, 60 and 90 day of sowing. Where: T0 ¼Control (Irrigation on 3 day periodicity), T1 ¼Drought (irrigation on 10 d periodicity), T2¼Salinity (100 mM NaCl), T3¼ Salinity þDrought, T4¼ Cd (100 mg Cd kg  1 soil), T5 ¼Cdþ Drought, T6¼ CdþSalinity, T7¼ Cdþ Salinity þDrought.

ability in form of proline bioaccumulation (r2 ¼0.961) than Indian mustard (r2 ¼ 0.918) (Fig. 3IIA–IIB). The level of malondialdehyde (MDA), one of the major thiobarbituric acid (TBA) reactive metabolites, increased significantly

in both the plants with Cd as well as salinity and drought stresses in the soil. On 90 DAS there was increase of MDA by three fold in B. juncea and 3.36 fold in R. communis over the control (Fig. 2 IIIA– IIIB). A higher correlation values for MDA which indicates more

K. Bauddh, R.P. Singh / Ecotoxicology and Environmental Safety 85 (2012) 13–22

Protien content (mg/g Fresh leaves)

30

T0 T4

25

T1 T5

T2 T6

T3 T7

T1 T5

T2 T6

IB

T3 T7

40 30

15 a

10 5

a b b c

d

ee

a cb

c

bb de e

f

20 cc

f

dd

e

10

a

b bbcd

a bcbcd d ee

0

30

60

90

400 Proline content (mg/g Fresh leaves)

T0 T4

20

0

300

a bbc c cd ee

f

30

II A

dd

f

60

e

90

200

II B

150

200 b c bb

100

aa cb

a

dd

a c

dd

ed

e

e

ba a

a

100

50

d

0

0 30

MDA content (µ mol/g Fresh leaves

50

IA

17

60

90

a

f f f ed

ee

30

III A

40

bc

bc

a

d

c

bb

e

a

d

gf f

60

90

30

III B

25

30

20 15

20 10 0

f

e dd

c

aaa f ed

d

c

aa

bab

a e dd

a 10

c 0

30

60 90 Time interval (Days)

c

a

5

f ee d

cb b

30

f ee d

bb

a

c

bb

a

e de d

60

90

Time interval (Days)

Fig. 2. Effect of cadmium with salinity and drought on protein, proline and MDA content in fresh leaves of B. juncea [A] and R. communis [B] after 30, 60 and 90 day of sowing. Data was analyses by one way analysis of variance (DMRT) at p o0.05. Different alphabets on the bars show significant differences between the treatments. Where: T0 ¼Control (Irrigation on 3 day periodicity), T1¼ Drought (irrigation on 10 d periodicity), T2¼Salinity (100 mM NaCl), T3¼ Salinity þ Drought, T4¼ Cd (100 mg Cd kg  1 soil), T5 ¼Cdþ Drought, T6¼ CdþSalinity, T7¼ Cdþ Salinity þDrought.

lipid peroxidation in leaves was calculated for B. juncea (r2 ¼0.947) with multiple stresses as compared to R. communis (r2 ¼0.914) (Fig. 3 IIIA–IIIB). 3.4. Cadmium accumulation The bioaccumulation of Cd was 1.437 fold and 5.078 fold higher in the roots than that in the shoots in B. juncea and R. communis, respectively (Table 5). Cadmium bioaccumulation per unit biomass was higher in B. juncea; whereas, rate of Cd bioaccumulation was more in R. communis as Indian mustard produced less biomass in the same duration under the stresses. Salinity enhanced the translocation of Cd from the soil to the roots; however, drought reduced it. Salinity increased 17.53 and 15.57 percent Cd accumulation in the roots and 27.94 and 17.83 percent Cd accumulation in shoots of B. juncea and R. communis, respectively. In the salinity and drought stresses total Cd accumulation per plant was much higher in the roots of R. communis than that of B. juncea. R. communis accumulated seventeen and 1.5 fold higher Cd in their roots and shoots, respectively than that of B. juncea (Fig. 4). A total count of Cd phytoextraction per plant was significantly more in R. communis (3.588 fold higher than B. juncea) which was able to remove higher Cd in the same duration from the contaminated soil in the presence of salinity

and drought stresses. In shoots also, Ricinus accumulated 1.397 fold higher Cd even in the presence of salinity and drought stresses on the basis of per plant Cd accumulation. Concentration index (CI) for roots of B. juncea was 176.24 on 90 DAS under the multiple stresses and 338.211 for roots of R. communis. On the other hand CI for shoot was higher i.e., 182.10 in B. juncea than that in R. communis (111.344) (Table 6). However, Translocation factor (TF) was higher (32–48 percent) in B. juncea than that in R. communis which suggested a high translocation of the metal from roots to shoots in B. juncea (Table 6).

3.5. Sodium and potassium accumulation in the roots and shoots Sodium and potassium were accumulated in higher amount in the shoots of both the plants under salinity stress (Fig. 5). Drought and cadmium stresses reduced the sodium level in leaves of both the plants, whereas these stresses increased potassium levels under the similar conditions. R. communis accumulated 1.7 fold higher Na þ than that of B. juncea when plants were exposed to Cd with salinity stress, however, highest K þ accumulated in R. communis when the plants get exposed to Cd with drought stress. Sodium and potassium ratio was found more than two fold higher in R. communis than that of B. juncea at 90 DAS (Fig. 5IIIA–IIIB).

18

K. Bauddh, R.P. Singh / Ecotoxicology and Environmental Safety 85 (2012) 13–22

IA

Protein content (mg/g fresh leaves)

30

Proiten Linear (Proiten)

25 20

30 15 20

10 y = -2.0229x + 25.342 r² = 0.927

5

10

y = -3.413x + 45.526 r² = 0.9152

0 0

Proline content (mg/g fresh leaves)

Protein Linear (Protein)

40

0 1

2

400

3

4

5

6

7

8

II A

Proline Linear (Proline)

0

2

3

4

5

6

7

8

II B

Proline Linear (Proline)

150

200

100

y = 34.116x + 104.54 r² = 0.932

100

1

200

300

50

y = 24.853x -25.771 r² = 0.949

0

0 0

MDA content (uM/ g fresh leaves)

IB 50

1

2

40

3

4

5

6

7

8

0

III A

MDA Linear (MDA)

1

30

3

4

5

6

7

8

III B

MDA Linear (MDA)

25

30

2

20 20

15 10

10

y = 3.1161x + 9.2152 r² = 0.947

0

y = 2.7283x + 2.473 r² = 0.9147

5 0

0

1

2

3

4

5

6

7

8

0

1

2

Treatments

3

4

5

6

7

8

Treatments

Fig. 3. Correlation analysis between different treatments and protein, proline and MDA content in fresh leaves of B. juncea [A] and R. communis [B]after 90 day of sowing. Where: T0 ¼Control (Irrigation on 3 day periodicity), T1¼ Drought (irrigation on 10 d periodicity), T2¼Salinity (100 mM NaCl), T3¼ Salinity þDrought, T4¼ Cd (100 mg Cd kg  1 soil), T5¼ Cdþ Drought, T6¼Cd þSalinity, T7¼ Cdþ Salinity þDrought.

Table 5 Cadmium accumulation in the roots and shoots of B. juncea and R. communis 30, 60 and 90 day of sowing. Data was analyses by one way analysis of variance (DMRT) at p o0.05. Different alphabets show significant differences between the treatments. Where: T0 ¼ Control (Irrigation on 3 day periodicity), T1¼ Drought (irrigation on 10 d periodicity), T2¼ Salinity (100 mM NaCl), T3 ¼Salinity þ Drought, T4 ¼Cd (100 mg Cd kg  1 soil), T5¼ Cdþ Drought, T6¼Cd þSalinity, T7¼ Cdþ Salinity þDrought. Treatments

Cd level in roots (mg g  1 dry wt)

Cd level in shoots (mg g  1 dry wt)

30 DAS

60 DAS

90 DAS

30 DAS

60 DAS

90 DAS

B. juncea T0 T1 T2 T3 T4 T5 T6 T7 F value at p o 0.5

1.417 0.007e 1.067 0.023e 1.437 0.032e 0.967 0.035e 922.187 9.538b 722.827 5.316d 1025.65740.212a 782.07 77.099c 28078.983

4.867 0.268e 2.557 0.027e 2.847 0.102e 2.147 0.099e 1185.647 58.168b 810.867 24.627d 1384.257 9.804a 983.81726.942c 1818.654

5.90 7 0.486e 6.15 7 0.0366e 7.18 7 0.0806e 6.73 7 0.449e 1314.12 735.752b 944.29 712.687d 1544.87 739.376a 1034.247 18.015c 3225.503

0.307 0.035e 0.25 70.011e 0.53 70.029e 0.25 70.031e 283.827 14.502b 205.46 7 3.786d 324.357 6.802a 263.127 6.325c 1919.811

2.157 0.098d 1.337 0.015d 2.637 0.097d 1.437 0.186d 605.917 5.760b 537.63714.265c 793.18730.253a 597.99726.054b 1756.954

3.96 70.286d 3.52 70.102d 4.907 0.066d 3.707 0.072d 827.457 26.972b 685.967 13.408c 1058.657 69.672a 719.637 7.157c 841.205

R. communis T0 T1 T2 T3 T4 T5 T6 T7 F value P o 0.5

1.814 70.007e 1.422 70.023e 1.928 70.032e 1.263 70.035e 395.8697 9.538b 351.3487 5.316c 448.024740.212a 365.7377 7.099c 28078.983

2.329 70.268e 1.924 70.027e 2.425 70.102e 2.0867 0.099e 707.839758.168c 604.188724.627d 788.5837 9.804a 742.6487 26.942b 1818.654

3.181 7 0.486d 2.728 7 0.0366d 3.216 7 0.0806d 2.745 7 0.449d 1086.356 735.752b 837.618 712.687c 1255.529 7 39.376a 1075.572 718.015b 3225.503

0.676 7 0.035d 0.421 7 0.011d 0.597 7 0.029d 0.402 70.031d 80.912 7 14.502c 80.820 7 3.786c 90.641 7 6.802a 86.8437 6.325b 1919.811

1.625 70.098c 1.468 70.015c 1.799 70.097c 1.523 70.186c 160.8307 5.760b 156.5487 14.265b 195.5127 30.253a 187.9947 26.054ab 1756.954

1.912 7 0.286d 1.789 7 0.102d 1.949 7 0.066d 1.640 70.072d 200.134 726.972ab 195.9557 13.408c 235.8217 69.672a 211.7787 7.157b 841.205

Total Cd accumulation mg/plant on 90 DAS

Cd accumulation mg/plant 90 on DAS

Cd accumulation mg/plant on 90 DAS

K. Bauddh, R.P. Singh / Ecotoxicology and Environmental Safety 85 (2012) 13–22

2700 2400 2100 1800 1500 1200 900 600 300 0

B. juncea

19

R. communis

T0

T1

T2

T3

T4

T5

T6

T7

T0

T1

T2

T3

T4

T5

T6

T7

T0

T1

T2

T3 T4 Treatments

1400 1200

1000 800 600 400 200 0

4000 3500 3000 2500 2000 1500 1000 500 0 T5

T6

T7

Fig. 4. Total Cd accumulation [mg/plant: roots (A), shoots (B) and total Cd accumulation (C)] in B. juncea and R. communis [90 DAS]. Where: T0 ¼ Control (Irrigation on 3 day periodicity), T1¼ Drought (irrigation on 10 d periodicity), T2¼ Salinity (100 mM NaCl), T3 ¼Salinity þ Drought, T4¼Cd (100 mg Cd kg  1 soil), T5¼Cd þDrought, T6¼ Cdþ Salinity, T7 ¼Cdþ Salinity þ Drought.

4. Discussion The study revealed that Cd in the soil possessing salinity or drought or both the stresses simultaneously influenced not only growth, protein, proline and Malondialdehyde (MDA) content but also Cd bioaccumulation in roots and its translocation to the shoots in R. communis and B. juncea. R. communis appeared relatively less sensitive towards the deteriorating effects of Cd, salinity and drought alone or in various combinations as compared to B. juncea. The results are in accordance to our earlier report on superiority of R. communis in relation to growth, enhanced levels of stress metabolites and phyto-accumulation of the metal over B. juncea, when Cd stress was applied alone (Bauddh and Singh, 2012). A reduction in the root and shoot biomass was observed as a function of quantity of the metal, salinity and drought stresses as well as exposure time. A few reports are available which indicate that R. communis has good tolerance and phytoremediation potential for Cd and other heavy metals (Rockwood et al., 2004; Cecchi and Zanchi, 2005; Rajkumar and Freitas, 2008; Shi and Cai,

2009; Huang et al., 2011), however, no study is available to investigate its effectiveness when salinity and drought also exist with the metal contamination which is a common situation in the urban and peri-urban regions of north Indian states. Similarly the suitability of R. communis over the widely studied oil seed crop B. juncea has also been studied rarely (Bauddh and Singh, 2012). The effect of varying stresses on leaf soluble proteins of B. juncea and R. communis was shown in Fig. 2. Cadmium contamination has decreased soluble protein in leaves of both the plants. The decrease in protein contents in plant parts as a result of increasing salinity, drought or Cd as single stress have been reported in many plants (Mohammadkhani and Heidari, 2008; Best et al., 2011; Farouk et al., 2011; Mafakheri et al., 2011; Muneer et al., 2011; Bauddh and Singh, 2012). It appears that similar to growth and biomass production, leaf protein levels also decrease more effectively when multiple abiotic stress occur simultaneously. Abiotic stresses e.g., heavy metal, salinity and drought are known to cause oxidative damage to plants through the formation of reactive oxygen species, which cause damage to membrane

167.0857 14.2 153.0937 11.9 167.5837 18.1 169.6827 12.7 543.8887 19.4 428.3337 17.1 532.4677 14.3 508.030 710.9 143.3887 5.2 131.0587 4.9 135.8577 14.2 137.2407 7.83 440.3187 13.0 386.0937 9.22 404.8997 29.8 403.1747 21.5 269.6617 12.51 337.9467 19.63 322.8177 9.51 314.3837 3.423 489.3967 12.12 434.7517 9.032 494.2937 3.317 421.1667 24.57 0 0.944 70.15 1.031 70.24 0.866 70.19 105.3667 11.13 102.700 75.08 123.9057 12.71 111.3447 11.30 R. communis T0 T1 T2 T3 T4 T5 T6 T7

0 0.858 7 0.04 1.011 7 0.22 0.863 7 0.03 341.839 721.7 263.461 710.5 395.0237 26.8 338.211 76.38

0 0.626 7 0.05 0.886 7 0.06 0.596 7 0.043 120.1457 10.9 119.885 78.31 134.464 79.15 128.8097 8.74 0 0.830 7 0.05 1.051 7 0.13 0.904 7 0.1 306.388 731.9 261.742 7 31.5 341.038 721.4 321.354 7 26.1 0 0.788 7 0.08 1.0677 0.08 0.700 7 0.06 219.097717.3 194.354 7 13.3 248.0017 20.1 202.936726.4

0 0.909 70.08 1.108 7 0.55 0.950 70.15 99.9467 4.15 97.3517 2.48 121.5897 12.2 117.7147 16.1

67.2197 2.08 57.306 7 2.00 68.2727 0.57 55.1617 4.77 62.960 7 0.61 72.6387 0.45 68.480 7 2.79 69.601 7 1.83 44.1377 0.46 52.540 7 3.95 92.4617 0.66 67.409 7 4.26 51.200 72.97 66.3417 1.85 57.2927 1.80 60.767 7 1.16 21.3367 1.56 23.8897 3.99 37.5997 4.10 26.4117 1.25 30.768 7 0.32 28.504 7 0.62 28.6237 2.12 30.965 7 2.25 0 0.89 70.04 1.24 7 0.10 0.94 70.05 209.82 7 22.39 173.50 7 9.51 268.857 31.26 182.10 7 11.70 B. juncea T0 T1 T2 T3 T4 T5 T6 T7

0 1.05 7 0.09 1.22 7 0.12 1.15 7 0.16 224.04 7 25.26 160.64 7 11.95 263.32 729.01 176.24 718.42

0 0.85 7 0.01 1.78 7 0.15 0.83 7 0.04 954.127 16.73 686.897 73.15 1087.367 99.54 880.82 7 19.23 0 0.52 7 0.02 0.58 7 0.01 0.44 7 0.02 243.85 73.10 167.24 714.13 285.12 713.56 202.50 7 5.84 0 0.75 7 0.01 1.017 0.10 0.68 7 0.03 652.5279.97 511.42711.55 725.7277.68 553.38715.26

0 0.62 70.02 1.22 7 0.02 0.67 70.02 282.817 14.80 250.91 7 11.99 369.747 5.55 278.70 7 2.65

30 DAS 30 DAS 60 DAS 30 DAS

Treatments

90 DAS

Concentration index in shoots Concentration index in roots

60 DAS

90 DAS

Translocation factor

60 DAS

90 DAS

K. Bauddh, R.P. Singh / Ecotoxicology and Environmental Safety 85 (2012) 13–22 Table 6 Effect of cadmium with salinity and drought on Concentration Index (CI) and Translocation Factor (TF) in B. juncea and R. communis 30, 60 and 90 day of sowing. Data was analyses by one way analysis of variance (DMRT) at p o0.05. Different alphabets show significant differences between the treatments. Where: T0 ¼Control (Irrigation on 3 day periodicity), T1 ¼Drought (irrigation on 10 d periodicity), T2¼ Salinity (100 mM NaCl), T3¼ Salinity þDrought, T4¼ Cd (100 mg Cd kg  1 soil), T5¼Cd þDrought, T6¼ Cdþ Salinity, T7¼Cd þSalinity þ Drought

20

lipids and proteins etc., however, to resist this damage plants have an antioxidative defense system by producing antioxidant compounds as stress metabolites (Ayala-Astorga and AlcarazMelendez, 2010; Singh et al., 2010; Chugh et al., 2011; Bauddh and Singh, 2012; Chutipaijit et al., 2012). Proline is one of these metabolites, which has been shown to accumulate high under the exposure of drought (Fariduddin et al., 2009) salinity (Demiral and Turkan, 2005; Ali et al., 2007) and heavy metal including cadmium (Xu et al., 2009; Sharma et al., 2010; Anjum et al., 2011; Muneer et al., 2011; Zhao, 2011). It plays an important role in osmo-regulation and osmotolerance. R. communis appeared to have relatively stronger selfprotection ability in terms of proline bioaccumulation (r2 ¼0.949) than that of B. juncea (r2 ¼0.0.932) grown in Cd contaminated soil affected with salinity and drought (Fig. 3IIA–IIB). It has been suggested that protein degradation might contribute to proline accumulation in the metal treated plants (Chen et al., 2001). The level of lipid peroxidation that was determined by malondialdehyde content showed to be increased in a time and stress amount dependent manner for both the species, though the MDA content was slightly higher in B. juncea than that in R. communis (Fig. 2IIIA–IIIB). An increased MDA levels have been reported in many plants under Cd (Zhang et al., 2010; Muneer et al., 2011; Zhao, 2011), salinity (Ayala-Astorga and Alcaraz-Melendez, 2010) and drought stresses (Fu et al., 2010, 2011; Chugh et al., 2011; Chutipaijit et al., 2012). When the correlation was plotted between stresses applied (Cd, salinity and drought) and MDA content in the leaves, lower r2 value was found in R. communis (r2 ¼0.914) than that of B. juncea (r2 ¼0.947) which indicate the greater damage to cell membrane in the B. juncea than that in R. communis (Fig. 3IIIA–IIIB). The plants are known to differentially accumulate Cd in its various tissues especially diverted to cell vacuoles using membrane vacuolar transporters (see Paulose et al., 2008 and references there in; Kato et al., 2010). Though, the amount of the metal accumulated in the roots and shoots of Indian mustard was higher on the basis of per unit biomass than that in castor, the total removal of metal from soil on per plant basis was much higher in castor because it produces large amount of underground and aerial biomass during the same period. R. communis is a perennial plant and can maintain stabilization/removal of the metal from the contaminated soil throughout the year for longer time in the same sowing which will reduce the operational and maintenance cost. Being tolerant to the various stresses, R. communis was proved still better phytoremediator of Cd than B. juncea when the contaminated soil is suffering from the other stresses e.g., salinity and drought simultaneously. Drought reduced the translocation of Cd from the soil to plants in both the crops, however, salinity caused more accumulation of Cd in B. juncea and R. communis. Salt induced Cd mobilization may be due to higher bioavailability of the metal in the soil (WegglerBeaton et al., 2000). Weggler-Beaton et al. (2000) have reported that the accumulation of Cd in plants is due to the formation of a Cl–Cd complex; hence, the uptake of Cd increases during the salinity stress. Shao et al. (2008) have reported that pretreatment with salt enhanced the defensive ability of plants against oxidative stress by increasing the activities of antioxidative enzymes. No reports are available, however, on Cd bioaccumulation under the drought stress as per our data base.

5. Conclusions The data presented in this paper indicate that R. communis is more tolerant to salinity, drought or Cd contamination when the stress is applied singly or in combination of the two or three than

K. Bauddh, R.P. Singh / Ecotoxicology and Environmental Safety 85 (2012) 13–22

21

[I A]

Sodium content µg/g dry leaves

1800

T1 T5

T2 T6

T3 T7

1500 1200 900 600 300

h

ed

b

c

gf

ba

dc

a ge

hf

aba cc

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d

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ee

ba ed

f

aa aa a c b bc bb d cbc f e

2500 2000 1500 b c d abcbca bc

bd e c

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4000

Potasium content µg/g dry leaves

Potasium content µg/g dry leaves

T3 T7

2000

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3500 3000 2500 2000 1500 1000

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a d

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ba

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a e

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a f

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cb e

d

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0.7 Sodium/Potasium ratio

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T2 T6

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Sodium content µg/g dry leaves

T0 T4

2100

0.6 0.5 0.4 0.3 0.2 0.1 0

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90 DAS [III B]

1.2 1 0.8 0.6 0.4 0.2 0

30 DAS 60 DAS 90 DAS Time interval (Days)

30 DAS 60 DAS 90 DAS Time interval (Days)

Fig. 5. Sodium and potassium accumulation and its ratio in the shoots of B. juncea (A) and R. communis (B) at 30, 60 and 90 DAS. Data was analyses by one way analysis of variance (DMRT) at p o 0.05. Where: T0 ¼Control (Irrigation on 3 day periodicity), T1¼ Drought (irrigation on 10 d periodicity), T2 ¼Salinity (100 mM NaCl), T3¼ Salinity þDrought, T4¼ Cd (100 mg Cd kg  1 soil), T5¼ CdþDrought, T6¼ Cdþ Salinity, T7¼Cd þSalinity þ Drought.

B. juncea in terms of plant growth, bioaccumulation of proline and MDA, protein levels and Cd accumulation in plant parts. The amount of Cd removed from soil in three months under the single or multiple stresses is significantly higher in R. communis than Indian mustard. The salinity increased Cd uptake, whereas drought reduced it in both the plants. Co-cultivation of both the plants as mixed cropping can be seen as potential rapid phytoremediation system.

Acknowledgment Rajeev Gandhi National Fellowship (RGNF) by UGC, New Delhi to Kuldeep Bauddh has provided the financial support for this work. It is gratefully acknowledged. References Ali, B., Hayat, S., Ahmad, A., 2007. 28-homobrassinolide ameliorates the saline stress in chickpea (Cicer arietinum L). Environ. Exp. Bot. 59, 217–223. Ali, N.A., Bernal, M.P., Ater, M., 2002. Tolerance and bioaccumulation of copper in Phragmites australis and Zeamays. Plant Soil 239, 103–111. Anjum, N.A., Umar, S., Iqbal, M., Khan, N.A., 2011. Cadmium causes oxidative stress in mung bean by affecting the antioxidant enzyme system and ascorbate– glutathione cycle metabolism. Russ. J. Plant Physiol. 58 (1), 92–99.

Ayala-Astorga, G.I., Alcaraz-Melendez, L., 2010. Salinity effects on protein content, lipid peroxidation, pigments and proline in Paulownia imperialis (Siebold & Zuccarini) and Paulownia fortune (Seemann & Hemsley) grown in vitro. Elect. J. Biotechnol. 13 (5), 1–9. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of proline for water stress studies. Plant Soil 39, 205–207. Bauddh, K., Singh, R.P., 2009. Genotypic differences in nickel (Ni) toxicity in Indian mustard (Brassica juncea L.). Pollut. Res. 28 (4), 699–704. Bauddh, K., Singh, R.P., 2011. Differential toxicity of cadmium to mustard (Brassica juncea L.) genotypes is not maintained at higher metal levels. J. Environ. Biol. 32, 355–362. Bauddh, K., Singh, R.P., 2012. Cadmium tolerance and its phytoremediation by two oil yielding plants Ricinus communis (L.) and Brassica juncea (L.) from the contaminated soil. Int. J. Phytorem. 14, 772–785. Best, M., Koenig, K., McDonald, K., Schueller, M., Rogers, A., Ferrieri, R.A., 2011. Inhibition of trehalose breakdown increases new carbon partitioning in to cellulosic biomass in Nicotiana tabacum. Carbohydr. Res. 346, 595–601. Cecchi, C.G.S., Zanchi, C., 2005. Phytoremediation of soil polluted by nickel using agricultural crops. Environ. Manage. 36, 675–681. Chen, C.T., Chen, L.M., Lin, C.C., Kao, C.H., 2001. Regulation of proline accumulation in detached rice leaves exposed to copper stress. Plant Sci. 160, 283–290. Chugh, V., Kaur, N., Gupta, A.K., 2011. Evaluation of oxidative stress tolerance in maize (Zea mays L.) seedlings in response to drought. Indian J. Biochem. Biophys. 48, 47–53. Chutipaijit, S., Cha-um, S., Sompornpailin, K., 2012. An evaluation of water deficit tolerance screening in pigmented indica rice genotypes. Pak. J. Bot. 44 (1), 65–72.

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