Chromium-induced physio-chemical and ultrastructural changes in four cultivars of Brassica napus L.

Chemosphere 120 (2015) 154–164

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Chromium-induced physio-chemical and ultrastructural changes in four cultivars of Brassica napus L. Rafaqat A. Gill a, Lili Zang a, Basharat Ali a, Muhammad A. Farooq a, Peng Cui a, Su Yang a, Shafaqat Ali b, Weijun Zhou a,⇑ a b

Institute of Crop Science and Zhejiang Key Laboratory of Crop Germplasm, Zhejiang University, Hangzhou 310058, China Department of Environmental Sciences, Government College University, Faisalabad 38000, Pakistan

h i g h l i g h t s  Cr-toxicity decreased the plant growth in four cultivars of oilseed rape.  Accumulation of reactive oxygen species and malondialdehyde was induced under Cr stress.  Anti-oxidant enzyme activities were enhanced in the tolerant cultivars under Cr stress.  Cr damaged the leaf and root ultra-structures in Cr sensitive cultivar.  Zheda 622 was found to be the most sensitive cultivar.

a r t i c l e

i n f o

Article history: Received 26 December 2013 Received in revised form 4 June 2014 Accepted 10 June 2014 Available online 11 July 2014 Handling Editor: A. Gies Keywords: Antioxidant enzyme activities Chromium stress Photosynthesis gas exchange capacity Microscopic study Oilseed rape Reactive oxygen species

a b s t r a c t In nature, plants are continuously exposed to several biotic and abiotic stresses. Among these stresses, chromium (Cr) stress is one of the most adverse factors that affects the plant growth, and productivity, and imposes a severe threat for sustainable crop production. In the present study, toxic effects of Cr were studied in hydroponically grown seedlings of four different cultivars of Brassica napus L. viz. ZS 758, Zheda 619, ZY 50 and Zheda 622. The study revealed that elevated Cr concentrations reduced the plant growth rate and biomass as compared to respective controls in all the cultivars and this decline was more obvious in Zheda 622. It was observed that reduction of photosynthetic attributes was more pronounced in Zheda 622 as compared to other cultivars; while, cultivar ZS 758 performed better under Cr-toxicity. Results showed that Cr contents in different parts of seedlings were higher in Zheda 622 as compared to other cultivars and Cr contents were higher in roots than shoots in all the cultivars. Accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) were induced under different Cr concentrations. Results showed that some of anti-oxidant enzyme activities in leaves and roots were increased under the Cr-toxicity. The electron microscopic study showed that ultrastructural damages in leaf mesophyll and root tip cells were more prominent in Zheda 622 as compared to other cultivars under 400 lM Cr stress. Under 400 lM Cr concentration, changes like broken cell wall, immature nucleus, a number of mitochondria, ruptured thylakoid membranes and large size of vacuole and starch grains were observed in leaf ultrastructures. The damages in root cells were observed in the form of disruption of golgibodies and diffused cell wall under the higher concentration of Cr (400 lM). On the basis of these observations, it was concluded that Zheda 622 was found to be more sensitive as followed by ZY 50, Zheda 619 and ZS 758 under Cr-toxicity. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Heavy metals are naturally occurring elements of soil that are present in traces and their enhancement in soil and water ⇑ Corresponding author. Tel.: +86 571 88982770; fax: +86 571 88981152. E-mail address: [email protected] (W. Zhou). http://dx.doi.org/10.1016/j.chemosphere.2014.06.029 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

resources is a potential hazard in several parts of the world. Their presence in the soil is as free metal ions, soluble metal complexes, organically bound metals, exchangeable metal ions, precipitated or insoluble compounds such as carbonates and oxides (Shanker et al., 2005). Chromium (Cr) among heavy metals is considered as a serious environmental contaminant for the biota and released into the environment mainly from the leather tanning, mining,

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

drilling mud, electroplating and in wood preservation (KabadaPendias and Pendias, 1992). It has been found that Cr caused stunting plant growth, chlorosis in newly leaves, wilting of tops, impaired photosynthesis, damaging of root and finally plant death (Sharma et al., 2003; Scoccianti et al., 2006). Furthermore, plants growing in abiotic-stressed environment also stimulate the formation of reactive oxygen species (ROS), which can harm the production of biomolecules such as lipids, proteins and nucleic acids, thereby, interrupting the both mitochondrial respiration and carbohydrate metabolism (Gill and Tuteja, 2010). In plants, metal-induced lipid peroxidation has been reported (Ali et al., 2013a), which profoundly alters the membrane structure and therefore, changes their enzymatic and transport activities. However, plants have their own protection mechanisms such as cellular antioxidants, which scavenge the ROS and protect from oxidative damages. The antioxidant hyperactivity and cellular accumulation of antioxidants in various plant parts under cadmium, aluminium, zinc and copper stress have been reported (Ali et al., 2013b; Van Assche and Clijsters, 1983). Moreover, it was investigated that Cr-toxicity caused the ultrastructural changes in the form of poorly developed lamellar system with widely spaced thylakoid and fewer grana (Ali et al., 2013c). These abnormalities in thylakoid membrane might have some negative impact on photosynthesis and excitation energy transfer imbalance may also occur (Ali et al., 2013c). Nevertheless, it is not clear at what concentration Cr induces the ultrastructural changes in chloroplast and causes the inhibition of photosynthesis (Vazques et al., 1987). Besides, it was found that Cr and other heavy metals caused ultrastructural abnormalities in plant leaf mesophyll, root cells, and increased metal deposition in different plant parts (Ali et al., 2013c, 2013d). Oilseed rape (Brassica napus L.) is well recognized as a major source of edible oil from around the world (Momoh et al., 2002). Due to rapid growth, greater biomass, and ability to absorb heavy metals, Brassica species are considered to be a potential candidate against heavy metal stress (Meng et al., 2009). These plants use unique mechanisms to defeat or tolerate metal toxicity in polluted soils (Papazoglou et al., 2005). Thus, it is imperative to evaluate the response and mechanism of Brassica species against Cr stress. Hence, present study was carried out to analyze the potential of B. napus resistance against Cr stress and its effects on plant growth, photosynthetic pigments, oxidative stress and the role of cellular antioxidant activities in protecting the plants from Cr-toxicity.

2. Materials and methods 2.1. Plant material and growth conditions Seeds of four cultivars of winter B. napus L. viz. ZS 758, Zheda 619, ZY 50 and Zheda 622 were obtained from the College of Agriculture and Biotechnology, Zhejiang University. The seeds were grown in plastic pots (170  220 mm) filled with peat moss. At the five-leaf stage, morphologically uniform seedlings were selected and plugged into plate holes in plastic pots (five plants per pot) containing a half-strength Hoagland nutrient solution (Hoagland and Arnon, 1941), aerated continuously with an air pump, and kept in a greenhouse. The pH of the solution was maintained at 6.0. The light intensity was in the range of 250– 350 lmol m2 s1, temperature was 16–20 °C and the relative humidity was approximately 55–60%. Each treatment contained four pots (replicates) and each pot had five plants. The nutrient solution was renewed after every 5 d. After two weeks of acclimatization, solutions were adjusted to the desired Cr concentration (0, 100, and 400 lM). The treatment concentrations were based on findings from pre-experimental studies, in which several concen-

155

trations of Cr were used i.e., 0, 100, 200, 400, 600 and 1000 lM. Cr at 100 lM concentration showed a little damage to plant growth and 400 lM Cr imposed a significant damage to plant growth, whereas concentrations higher than 400 lM were too toxic for plant growth. 2.2. Morphological parameters Fifteen days after treatment, plants were harvested and separated into leaves, stem, and roots. The length of the plant, stem, root and the leaf area of randomly selected six plants per treatment were measured manually. Dry and fresh biomass of the rapeseed plants was determined separately. Fresh leaves, stems, and roots of six plants per treatment were weighed immediately after harvesting and then placed into an oven 80 °C. The dried samples were weighed immediately after removal from the oven until biomass became stable (Momoh and Zhou, 2001). Photosynthetic gas exchange parameters were analyzed after 15 d of treatment by LiCor-6400 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA). Top most fully expanded leaf after 2 h of acclimation in a growth cabinet, at a temperature of 18 °C under a light intensity of 1000 lmol m2 s1, relative humidity of 60%, photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate were assessed. The total eight readings per treatment were taken from randomly selected plants (Zhou and Leul, 1998). 2.3. Determination of Cr contents For determination of Cr contents, plants were differentiated into root and shoot, and washed thoroughly with distilled water thrice. Then, the samples were dried at 70 °C for 48 h and were ground to fine powder, and wet digested in a 5 mL mixture of strong HNO3:HClO4 (2:1, v/v). After heating the mixture at 80 °C on water bath for about 2 h, Cr contents were measured using atomic absorption spectrometry (PE-100, PerkinElmer). 2.4. Analysis of lipid peroxidation and reactive oxygen species (ROS) For the analysis of lipid peroxidation and reactive oxygen species (ROS) in fresh leaves and roots, total four samples were taken per treatment. Lipid peroxidation was measured in terms of malondialdehyde (MDA) which was analyzed according to Zhou and Leul (1999). For determination of hydrogen peroxide (H2O2) contents, leaf and root samples (0.5 g) were extracted with 5.0 mL of trichloroacetic acid (TCA, 0.1%) in an ice bath, and the homogenate was centrifuged at 12 000g for 15 min (Velikova et al., 2000). The 0.5 mL supernatant was mixed with 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI was added. The absorbance was read at 390 nm and the H2O2 contents were calculated by using a standard curve. For estimation of extra-cellular hydroxyl radicals (OH), leaf and root samples (0.5 g) were incubated in 1 mL of 10 mM Na-phosphate buffer (pH 7.4) consisting 15 mM 2-deoxy-D-ribose (SRL, Mumbai) at 37 °C for 2 h (Halliwell et al., 1987). Following incubation an aliquot of 0.7 mL from the above mixture was added to reaction mixture containing 3 mL of 0.5% (w/v) thiobarbuteric acid (TBA, 1% stock solution made in 5 mM NaOH) and 1 mL glacial acetic acid, heated at 100 °C in a water bath for 30 min and cooled down to 41 °C for 10 min before measurement. Superoxide radical ðO 2 Þ was determined according to Jiang and Zhang (2001) with some modifications. The leaf and root samples (0.5 g) were homogenized in 3 mL of 65 mM potassium phosphate buffer (pH 7.8) and then homogenate was centrifuged at 5000g for 10 min at 4 °C. After that the supernatant (1 mL) was mixed with 0.9 mL of 65 mM potassium phosphate buffer (pH 7.8) and 0.1 mL of 10 mM

156

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

hydroxylamine hydrochloride, and then incubated at 25 °C for 24 h. After incubation 1 mL of 17 mM sulphanilamide and 1 mL of 7 mM a-naphthylamine were mixed in 1 mL solution for further 20 min at 25 °C. After incubation, n-butanol in the same volume was added and centrifuged at 1500g for 5 min. The absorbance in the supernatant was read at 530 nm. A standard curve was used to calculate the generation rate of O 2 .

2.5. Biochemical analysis To observe the toxic effects of Cr on B. napus cultivars, the effect of Cr on enzyme activities (SOD, CAT, APX, POD) and total soluble protein (TSP) contents in the leaves and roots were studied. For this purpose, four samples (0.5–0.6 g) of fresh leaves and roots were taken per treatment and homogenized in 8 mL of 50 mM potassium phosphate buffer (pH 7.8) under ice cold conditions. Homogenate was centrifuged at 10 000g for 20 min at 4 °C and the supernatant was used for the determination of the following enzyme activities. Total soluble protein contents were determined using the method of Bradford (1976) and bovine serum albumin was used as a standard. The assay for ascorbate peroxidase (APX, EC 1.11.1.11) activity was measured in a reaction mixture of 3 mL containing 100 mM phosphate (pH 7), 0.1 mM ethylenediaminetetraacetic acid disodium salt (EDTA-Na2), 0.3 mM ascorbic acid, 0.06 mM H2O2 and 100 lL enzyme extract. The change in absorption was taken at 290 nm 30 s after addition of H2O2 (Nakano and Asada, 1981). Catalase (CAT, EC 1.11.1.6) activity was measured according to Aebi (1984) with the use of H2O2 (extinction co-efficient 39.4 mM cm1) for 1 min at A240 in 3 mL reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 2 mM EDTA-Na2, 10 mM H2O2 and 100 lL enzyme extract. Glutathione reductase (GR, EC 1.6.4.2) activity was assayed by Jiang and Zhang (2002) with the oxidation of NADPH at 340 nm (extinction coefficient 6.2 mM cm1) for 1 min. The reaction mixture was comprised of 50 mM potassium phosphate buffer (pH 7.0), 2 mM EDTA-Na2, 0.15 mM NADPH, 0.5 mM oxidized glutathione (GSSG) and 100 lL enzyme extract in a 1 mL volume. The reaction was started by using NADPH. Total superoxide dismutase (SOD, EC 1.15.1.1) activity was determined with the method of Zhang et al. (2008) following the inhibition of photochemical reduction due to nitro blue tetrazolium (NBT). The reaction mixture was comprised of 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 lM NBT, 2 lM riboflavin, 0.1 mM EDTA and 100 lL of enzyme extract in a 3-mL volume. One unit of SOD activity was measured as the amount of enzyme required to cause 50% inhibition of the NBT reduction measured at 560 nm. Peroxidase (POD, EC1.11.1.7) activity was assayed by Zhou and Leul (1999) with some modifications. The reactant mixture contained 50 mM potassium phosphate buffer (pH 7.0), 1% guaiacol, 0.4% H2O2 and 100 lL enzyme extract. Variation due to guaiacol in absorbance was measured at 470 nm. Reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined according to Law et al. (1983) with some modifications. Samples (0.3 g) were homogenized with 5 mL of 10% (w/v) TCA and homogenate was centrifuged at 15 000g for 15 min. To assay total glutathione, 150 lL supernatant was added to 100 lL of 6 mM dithionitrobenzoate (DTNB), 50 lL of glutathione reductase (10 units mL1), and 700 lL 0.3 mM NADPH. The total glutathione contents were calculated from the standard curve. All the reagents were prepared in 125 mM NaH2PO4 buffer, containing 6.3 mM EDTA, at pH 7.5. To measure GSSG, 120 lL of supernatant was added to 10 lL of 2-vinylpyridine followed by 20 lL of 50% (v/ v) triethanolamine. The solution was vortex-mixed for 30 s and incubated at 25 °C for 25 min. The mixture was assayed as mentioned above. Calibration curve was developed by using GSSG sam-

ples treated exactly as above and GSH was determined by subtracting GSSG from the total glutathione contents. 2.6. Transmission electron microscopy After 15 d of treatment, topmost leaf fragments without veins and root tips (8–10 each per treatment) were collected from randomly selected plants and then fixed overnight in 2.5% glutaraldehyde (v/v) in 0.1 M PBS (sodium phosphate buffer, pH 7.4) and washed three times with the same PBS. Then the samples were post fixed in 1% OsO4 [osmium (VIII) oxide] for 1 h and washed three times in 0.1 M PBS (pH 7.4), with 10-min intervals between each washing. Then, with 15–20-min intervals, the samples were dehydrated in a graded series of ethanol (50%, 60%, 70%, 80%, 90%, 95% and 100%) and at the end washed by absolute acetone for 20 min. The samples were then infiltrated and embedded in Spurr’s resin overnight. After heating at 70 °C for 9 h, ultrathin sections (80 nm) of specimens were prepared and mounted on copper grids for viewing by a transmission electron microscope (JEOLTEM1230EX) at an accelerating voltage of 60.0 kV. 2.7. Statistical analysis All values described in results section are mean of at least three replicates ± standard deviation (SD). The data were analyzed using SPSS v16.0 (SPSS, Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was carried out, followed by Duncan’s multiple range test between the means of treatments to determine the significant difference (at the p < 0.05 and 0.01 level) between mean values. 3. Results 3.1. Cr-toxicity inhibits the plant growth The plant morphology in terms of plant length, stem length, root length, number of leaves and leaf area per plant under Cr stress is presented in Table 1. Results depicted that the maximum plant length, stem length and root length were observed under the control conditions in each cultivar. The increase in Cr concentrations induced significant reduction in plant length and minimum plant length was measured under the higher concentration of Cr (400 lM). The mean values of plant length showed decreasing trend with an increase in Cr concentration and this decrease was more pronounced in Zheda 622 followed by Zheda 619, ZY 50, and ZS 758 as compared to their respective controls. The stem and root length of cultivar ZS 758 under Cr stress was higher than other three cultivars in which Zheda 622 had the lowest stem and root length. Moreover, higher concentration of Cr (400 lM) did not show any significant difference among three cultivars except Zheda 622. It was noticed that higher concentration of Cr significantly reduced the number of leaves and leaf area per plant in all the cultivars as compared to their respective control (Table 1). The data regarding fresh and dry biomass of different parts of plant under different concentrations of Cr are presented in Table 2. The data expressed that fresh biomass of plant parts was significantly reduced at 400 lM Cr in all the cultivars over their respective controls. Results mentioned that there was no significant difference among all cultivars in leaf and stem fresh biomass, except for Zheda 622 which produced minimum fresh biomass of different plant parts under the higher concentration of Cr. Likewise, dry biomass of different plant parts was also statistically reduced under higher Cr concentration (400 lM) as compared to control plants in all cultivars. However, the reduction in dry biomass was more obvious in Zheda 622 as compared to other cultivars. Hence, it could be concluded that fresh and dry biomass

157

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

Table 1 Effects of different treatments of chromium (Cr) (lM) on length (cm) of plant, stem and root, number of leaves per plant, and leaf area (cm2 plant1) of four cultivars of Brassica napus. Cultivar

Cr conc.

Plant length

ZS 758

0 100 400 0 100 400 0 100 400 0 100 400

32.24 ± 1.95 27.1 ± 1.88 21.56 ± 2.07 31.44 ± 1.92 26.32 ± 1.97 20.57 ± 1.93 32.41 ± 2.32 27.35 ± 1.90 20.53 ± 2.21 31.14 ± 1.91 25.22 ± 2.04 16.57 ± 1.88

Zheda 619

ZY 50

Zheda 622

a b c a b c a b c a b d



Variety (V) Treatments (T) VT

Stem length

Root length

9.8 ± 1.06 7.13 ± 0.98 5.42 ± 1.02 9.73 ± 1.01 7.14 ± 0.94 5.22 ± 1.07 9.62 ± 1.05 6.87 ± 1.05 5.13 ± 0.98 9.66 ± 1.02 6.66 ± 0.72 4.2 ± 0.87 NS

13.59 ± 0.84 13.25 ± 0.90 10.47 ± 0.94 13.25 ± 0.97 12.94 ± 1.08 9.68 ± 0.87 13.84 ± 1.12 12.52 ± 1.06 9.42 ± 1.13 13.46 ± 1.16 12.57 ± 1.08 7.34 ± 1.06 NS

a b bc a b c a bc c a bc d

a a b a a b a a b a a c

Number of leaves/plant

Leaf area

8.62 ± 0.94 6.55 ± 0.96 4.21 ± 1.05 8.57 ± 1.06 6.48 ± 1.09 4.17 ± 1.24 8.51 ± 1.05 6.43 ± 0.78 4.05 ± 1.56 8.6 ± 0.98 6.38 ± 1.04 3.3 ± 0.87 NS

188.43 ± 10.32 a 144.3 ± 9.70 cd 89.18 ± 4.92 e 185.74 ± 10.46 ab 152.29 ± 9.72 c 82.97 ± 5.44 e 171.43 ± 9.12 b 135.83 ± 10.31 d 75.54 ± 5.90 e 173.61 ± 9.41 ab 142.81 ± 10.36 cd 60.3 ± 5.33 f

a b c a b c a b c a b d























The same letters within a column indicate there was no significant difference at a 95% probability level; respectively; NS, non-significant.



and



indicate significance at the p < 0.05 and 0.01 level,

Table 2 Effects of different treatments of chromium (Cr) (lM) on fresh and dry weights (g) of leaves, stem and root per plant of four different cultivars of Brassica napus. Cultivar

Cr conc.

Leaf fresh weight

Leaf dry weight

Stem fresh weight

Stem dry weight

Root fresh weight

Root dry weight

ZS 758

0 100 400 0 100 400 0 100 400 0 100 400

125.69 ± 7.94 a 105.37 ± 6.95 d 52.33 ± 4.96 e 117.9 ± 7.69 a–c 110.43 ± 6.79 b–d 46.32 ± 5.01 e 121.9 ± 8.26 ab 102.65 ± 7.41 d 45.77 ± 6.15 e 122.94 ± 6.82 a 107.52 ± 5.96 cd 30.35 ± 5.37 f NS

7.60 ± 0.10 a 5.70 ± 0.10 de 2.40 ± 0.10 g 7.20 ± 0.10 c 5.80 ± 0.10 d 2.30 ± 0.10 gh 7.40 ± 0.10 b 5.60 ± 0.10 e 2.20 ± 0.10 h 7.10 ± 0.10 c 5.10 ± 0.10 f 1.60 ± 0.10 i

21.53 ± 1.15 a 18.73 ± 1.01 cd 11.26 ± 0.99 f 20.32 ± 0.94 a-c 17.26 ± 0.96 de 10.79 ± 0.92 f 21.10 ± 1.03 ab 17.46 ± 0.81 de 9.82 ± 0.99 f 19.52 ± 0.94 bc 15.75 ± 1.06 e 6.82 ± 1.06 g

4.27 ± 0.09 3.67 ± 0.10 2.92 ± 0.12 4.13 ± 0.09 3.58 ± 0.10 2.61 ± 0.11 4.15 ± 0.09 3.63 ± 0.10 2.70 ± 0.10 3.97 ± 0.11 3.63 ± 0.10 1.47 ± 0.10

15.37 ± 1.02 12.50 ± 1.07 8.56 ± 1.03 15.51 ± 1.00 12.43 ± 0.98 7.15 ± 0.93 14.91 ± 0.99 11.83 ± 1.03 6.33 ± 0.95 15.15 ± 1.04 10.42 ± 0.94 4.39 ± 1.03

3.44 ± 0.10 3.11 ± 0.10 2.29 ± 0.10 3.42 ± 0.10 3.06 ± 0.10 2.22 ± 0.10 3.40 ± 0.10 3.03 ± 0.10 2.15 ± 0.11 3.41 ± 0.11 2.94 ± 0.10 1.11 ± 0.11



































Zheda 619

ZY 50

Zheda 622

Variety (V) Treatments (T) VT

The same letters within a column indicate there was no significant difference at a 95% probability level; respectively; NS, non-significant.

greatly reduced in Zheda 622, followed by ZY 50, Zheda 619 and ZS 758 under Cr stress conditions. 3.2. Cr reduces the photosynthetic parameters The exposure of B. napus cultivars to Cr stress resulted in reduction of net photosynthetic rate, stomatal conductance, intercellular CO2 concentration and transpiration rate in plant leaves (Table 3) and this effect of toxicity became more pronounced as the concentration of Cr was increased. When plants exposed to 400 lM Cr concentration, net photosynthetic rate decreased by 14.2%, 23.06%, 22.18% and 43.78%; stomatal conductance decreased by 69.44%, 76.32%, 78.38% and 88.58%; intercellular CO2 concentration decreased by 62.74%, 65.02%, 63.27% and 69.57% and transpiration rate decreased by 73.35%, 72.17%, 71.62% and 81.15% in ZS 758, Zheda 619, ZY 50 and Zheda 622, respectively as compared to control plants. It was also observed that there was no significant difference in transpiration rate at 400 lM Cr concentration among all cultivars, however, cultivar Zheda 622 showed significantly lower transpiration rate. Two cultivars Zheda 619 and ZS 758 showed better performance of net photosynthesis rate as compared to other cultivars, and cultivar Zheda 622 was proved to be most sensitive under Cr-toxicity.



and

a c d ab c e ab c e b c f



a b d a b de a bc e a c f

a b c a b c a b c b d

indicate significance at the p < 0.05 and 0.01 level,

3.3. Cr-toxicity enhances the Cr contents in plants Data regarding Cr contents in shoots and roots of four cultivars of B. napus under different Cr concentrations are presented in Table 6. It was observed that Cr contents were increased in all cultivars as Cr level was increased in the solution. Cr contents were found maximum in Zheda 622 among all cultivars, and contents of Cr were higher in roots than shoots in all cultivars under different concentrations of Cr (Table 6). 3.4. Cr stress enhances the contents of MDA and ROS The contents of reactive oxygen species (ROS) and malondialdehyde (MDA) were linearly increased in the leaves and roots of all cultivars, as Cr concentration was increased in the solution (Table 4). Under the higher concentration of Cr treatment (400 lM), contents of MDA were significantly higher in the leaves and roots of all cultivars and MDA contents were obviously higher in cultivar Zheda 622 as compared to other cultivars. Moreover, lower concentration of Cr (100 lM) did not show any significant change on the contents of MDA in all the cultivars. Data showed that there was not any significant in hydrogen peroxide (H2O2) contents of the leaves and roots in two cultivars ZS 758 and Zheda 619 under

158

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

Table 3 Effects of different treatments of chromium (Cr) (lM) on net photosynthetic rate (lmol CO2 m2 s1), stomatal conductance (mol H2O m2 s1), intercellular CO2 concentration (lmol CO2 mol1), and transpiration rate (mmol H2O m2 s1) of the youngest fully expanded leaf of four cultivars of Brassica napus. Cultivar

Cr conc.

Net photosynthetic rate

Stomatal conductance

Intercellular CO2 conc.

Transpiration rate

ZS 758

0 100 400 0 100 400 0 100 400 0 100 400

17.38 ± 2.09 16.68 ± 2.02 14.31 ± 2.04 17.6 ± 2.05 16.36 ± 2.02 13.54 ± 2.17 17.49 ± 2.06 16.55 ± 1.83 13.61 ± 2.13 17.52 ± 1.87 16.38 ± 1.95 9.85 ± 1.11 NS

0.36 ± 0.01 bc 0.19 ± 0.01 d 0.11 ± 0.01 g 0.38 ± 0.01 a 0.17 ± 0.01 e 0.09 ± 0.01 h 0.37 ± 0.01 ab 0.17 ± 0.01 e 0.08 ± 0.01 h 0.35 ± 0.01 c 0.14 ± 0.01 f 0.04 ± 0.01 I

200.50 ± 9.93 a 137.16 ± 8.29 b 74.71 ± 4.74 c 203.7 ± 9.68 a 138.38 ± 8.91 b 71.25 ± 4.90 cd 200.3 ± 8.95 a 140.47 ± 6.78 b 73.57 ± 4.63 cd 198.36 ± 10.01 a 132.82 ± 7.36 b 60.37 ± 5.01 d NS

4.39 ± 0.10 2.71 ± 0.10 1.17 ± 0.09 4.42 ± 0.09 1.68 ± 0.10 1.23 ± 0.10 4.51 ± 0.11 2.61 ± 0.11 1.28 ± 0.10 4.35 ± 0.09 2.40 ± 0.13 0.82 ± 0.11

Zheda 619

ZY 50

Zheda 622

Variety (V) Treatments (T) VT

a ab ab a ab b a ab b a ab c





















The same letters within a column indicate there was no significant difference at a 95% probability level; respectively; NS, non-significant.



and



a b e a d e a b e a c f

indicate significance at the p < 0.05 and 0.01 level,

Table 4 Effects of different treatments of chromium (Cr) (lM) on malondialdehyde (MDA) (nmol mg1 protein), hydrogen peroxide (H2O2) (mmol g1 FW), superoxide radical ðO 2 Þ (nmol min1 g1 FW) and hydroxyl ion (OH) (mmol g1 FW) contents in the leaves and roots of four cultivars of Brassica napus. Cultivar

Cr conc. MDA

ZS 758

0 100 400 0 100 400 0 100 400 0 100 400

Leaf

Zheda 619

ZY 50

Zheda 622

Variety (V) Treatments (T) VT

107.64 ± 7.23 113.93 ± 5.52 128.71 ± 5.47 106.35 ± 4.99 112.65 ± 7.01 132.42 ± 4.93 108.17 ± 5.01 116.32 ± 5.52 136.23 ± 7.97 106.52 ± 4.97 117.90 ± 7.35 148.21 ± 6.53 NS

O 2

H2O2

c c b c c b c c b c c a

OH

Root

Leaf

Root

Leaf

115.60 ± 7.66 e 120.54 ± 7.26 e 132.70 ± 5 bcd 116.52 ± 3.03 e 123.29 ± 7.83 de 134.62 ± 5.08 bc 114.70 ± 8.99 e 122.29 ± 6.19 de 138.71 ± 5.02 ab 114.88 ± 5.07 e 124.79 ± 5.62 cde 146.08 ± 4.12 a NS

25.68 ± 1.52 g 31.37 ± 2.08 ef 42.12 ± 1.98 d 26.68 ± 2.64 g 29.60 ± 2.05 fg 46.23 ± 3 c 26.22 ± 3.14 g 35.19 ± 2.52 e 56.76 ± 2.08 b 26.14 ± 1.98 g 39.28 ± 2.05 d 64.26 ± 1.90 a

7.97 ± 1.5 g 8.11 ± 1.1 g 17.54 ± 1.03 d 8.04 ± 1.07 g 8.87 ± 1.03 g 19.63 ± 1.30 c 7.86 ± 1.25 g 11.84 ± 1.15 f 25.31 ± 0.98 b 8.35 ± 1.43 g 14.6 ± 0.88 e 37.82 ± 1 a

53.44 ± 4.96 69.55 ± 5.50 138.87 ± 5.03 52.31 ± 6.01 73.33 ± 5.03 142.11 ± 5.56 53.66 ± 6.26 77.82 ± 6.06 154.30 ± 5.50 54.37 ± 6.24 84.63 ± 5.56 166.46 ± 5.03

f e c f e c f de b f d a

Root

Leaf

Root

41.32 ± 5.56 f 48.66 ± 4.98 def 62.50 ± 5.52 c 42.92 ± 4.50 ef 51.70 ± 5.03 de 65.32 ± 6.01 c 42.44 ± 5.27 ef 56.11 ± 4.14 cd 76.66 ± 4.32 b 43.49 ± 6.46 ef 61.79 ± 4.85 c 93.25 ± 6.04 a

0.13 ± 0.015 d 0.14 ± 0.009 cd 0.15 ± 0.011 bcd 0.13 ± 0.010 cd 0.14 ± 0.008 cd 0.15 ± 0.008 bc 0.13 ± 0.012 cd 0.14 ± 0.007 bcd 0.15 ± 0.009 b 0.13 ± 0.013 cd 0.14 ± 0.010 bcd 0.17 ± 0.012 a

0.02 ± 0.001 g 0.03 ± 0.002 g 0.04 ± 0.002 d 0.02 ± 0.002 g 0.03 ± 0.003 f 0.04 ± 0.001 c 0.02 ± 0.002 g 0.03 ± 0.001 ef 0.05 ± 0.002 b 0.02 ± 0.001 g 0.03 ± 0.001 ef 0.07 ± 0.002 a













































The same letters within a column indicate there was no significant difference at a 95% probability level; respectively; NS, non-significant.

lower concentration of Cr as compared to control (Table 4). However, application of 100 lM Cr showed a significant difference on H2O2 contents of leaves and roots in two cultivars ZY 50 and Zheda 622 as compared to control. The minimum H2O2 contents were observed in control plants in all the cultivars; however, the highest H2O2 contents were observed in Zheda 622 under 400 lM Cr. A significant difference in superoxide radical ðO 2 Þ contents was observed in all cultivars when subjected to higher Cr concentration (Table 4). Data also suggested that O 2 contents were lower under control level in all cultivars. The O 2 contents were found higher at 400 lM Cr in all the cultivars and Zheda 622 produced most O 2 contents at this level among all cultivars. It was observed that under higher concentration of Cr, there was no change in OH contents of leaves in two cultivars ZS 758 and Zheda 619; however, a significant increase was observed under the higher concentration of Cr in the cultivars ZY 50 and Zheda 622 as compared to their respective controls (Table 4). Moreover, lower concentration of Cr did not show any significant change on OH contents of leaves in all cultivars. 3.5. Cr induces the inhibition of enzyme activities The data regarding antioxidants enzyme activities (SOD, POD, APX) in the leaves and roots of four cultivars under different Cr



and



indicate significance at the p < 0.05 and 0.01 level,

concentrations have been shown in Table 5. The present study showed that lower concentration of Cr (100 lM) increased the SOD activity significantly in the leaves and roots in all cultivars as compared to their respective controls. SOD activity was found maximum under the higher concentration of Cr (400 lM) in all cultivars and at this Cr level, ZS 758 showed higher SOD activity as compared to other cultivars. The present study showed that lower concentration of Cr did not show any significant difference on POD activity of the leaves and roots in all cultivars as compared to their respective controls. POD activity was significantly enhanced under the higher concentration of Cr in all cultivars; except for Zheda 622, in which POD activity did not show any significant difference as compared to its control. Results indicated that POD activity was higher in leaves than roots in all cultivars. It was found that APX activity in the leaves and roots in all cultivars decreased linearly, as Cr concentration was increased in the solution. The minimum APX activity was found in both leaves and roots of Zheda 622 under the higher concentration of Cr as compared to other cultivars. Results showed that CAT activity was significantly higher in the leaves of all cultivars under the higher concentration of Cr as compared to their respective controls; except for Zheda 622, in which CAT activity was lower at this concentration (Table 6). However, maximum CAT activity was observed in the leaves and roots of cultivar ZS 758 under higher concentration of Cr, as compared to other

159

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

Table 5 Effects of different treatments of chromium (Cr) (lM) on superoxide dismutase (SOD) (U g1 FW), guaiacol peroxidase (POD), and ascorbate peroxidase (APX) activities (lmol min1 mg1 protein) in the leaves and roots of four cultivars of Brassica napus. Cultivar

ZS 758

Zheda 619

ZY 50

Zheda 622

Cr conc.

SOD

0 100 400 0 100 400 0 100 400 0 100 400

Variety (V) Treatments (T) VT

POD

Leaf

Root

Leaf

994.12 ± 10 f 1133.09 ± 14.52 de 1398.18 ± 5.13 a 1009.23 ± 11.53 f 1145.94 ± 12.05 d 1354.98 ± 10 b 998.01 ± 12.50 f 1119.81 ± 12.50 e 1307.33 ± 10.03 c 939.11 ± 14.91 f 1124.22 ± 12 e 1274.94 ± 10 d

974.26 ± 12.05 g 1074.77 ± 8.54 e 1223.99 ± 5.70 a 967.08 ± 11.01 g 1066.32 ± 12.01 e 1181.76 ± 12.50 b 978.34 ± 10 g 1059.74 ± 10.01 ef 1153.02 ± 12.52 c 971.35 ± 13.15 g 1044.21 ± 11.50 f 1116.52 ± 10 d





1.12 ± 0.08 1.22 ± 0.12 1.50 ± 0.13 1.13 ± 0.09 1.23 ± 0.12 1.50 ± 0.30 1.13 ± 0.10 1.20 ± 0.10 1.46 ± 0.12 1.12 ± 0.06 1.13 ± 0.12 1.35 ± 0.10 NS









APX Root

Leaf

Root

14.64 ± 3.01 cd 16.17 ± 0.50 bcd 21.23 ± 2.51 a 14.23 ± 2.99 d 16.23 ± 3.01 bcd 19.09 ± 1.99 abc 14.06 ± 2.03 d 17.13 ± 1.97 abcd 20.66 ± 2.05 ab 14.28 ± 1.85 d 15.78 ± 2.03 cd 18.11 ± 2 abcd NS

2.81 ± 0.03 a 2.31 ± 0.03 b 1.41 ± 0.02 c 2.58 ± 0.11 a 2.21 ± 0.03 b 1.23 ± 0.12 cd 2.59 ± 0.09 a 2.10 ± 0.03 b 1.24 ± 0.11 cd 2.57 ± 0.08 a 2.21 ± 0.13 b 1.12 ± 0.10 d

2.92 ± 0.03 2.62 ± 0.02 1.76 ± 0.12 2.78 ± 0.04 2.60 ± 0.11 1.73 ± 0.10 2.82 ± 0.08 2.66 ± 0.12 1.73 ± 0.09 2.81 ± 0.07 2.56 ± 0.10 1.47 ± 0.11





















c bc a c bc a c ab a c c abc

The same letters within a column indicate there was no significant difference at a 95% probability level; respectively; NS, non-significant.



and



a b c ab b c ab bc c ab b d

indicate significance at the p < 0.05 and 0.01 level,

Table 6 Effects of different treatments of chromium (Cr) (lM) on catalase (CAT) activity (lmol min1 mg1 protein), total soluble protein (TSP) (mg g1 FW), and Cr contents in the leaves and roots of four cultivars of Brassica napus. Cultivar

ZS 758

Zheda 619

ZY 50

Zheda 622

Variety (V) Treatments (T) VT

Cr conc.

0 100 400 0 100 400 0 100 400 0 100 400

CAT

TSP

Cr content

Leaf

Root

Leaf

Root

Shoot

Root

33.85 ± 2.04 de 36.04 ± 3 bcd 41.53 ± 2 a 34.35 ± 1.94 cd 35.28 ± 2.08 bcd 39.28 ± 3 ab 33.54 ± 2 de 36.59 ± 2.64 bcd 38.97 ± 2.05 abc 33.75 ± 2 de 34.31 ± 3.06 cd 29.55 ± 3.16 e

14.44 ± 3 cd 15.21 ± 2 cd 21.08 ± 2.12 a 13.66 ± 2.01 d 14.83 ± 4 cd 17.37 ± 2.01 bc 14.52 ± 4.01 cd 16.11 ± 3 bc 18.22 ± 1.93 bc 13.55 ± 1.97 e 15.26 ± 3.02 cd 17.95 ± 2.02 bc NS

457.82 ± 12.29 fg 519.61 ± 3.43 d 735.62 ± 4.44 a 461.53 ± 3.44 fg 507.49 ± 7.67 d 725.29 ± 4.72 ab 447 ± 13.72 g 487.93 ± 5.96 e 711.69 ± 8.39 b 464.03 ± 7.97 f 485.38 ± 7.15 e 662.09 ± 18.86 c

183.78 ± 12.57 c 203.79 ± 6.69 bc 258.97 ± 10.38 a 184.51 ± 5.94 c 177.91 ± 10 bc 243.45 ± 2.89 a 174.07 ± 3.63 c 184.90 ± 11.54 bc 242.72 ± 6.36 a 171.38 ± 9.99 c 185.96 ± 5.65 c 221.84 ± 9.97 ab

0.34 ± 0.02 g 31.35 ± 3.14 f 88.41 ± 5.30 c 0.35 ± 0.02 g 38.65 ± 3.29 e 105.99 ± 4.91 b 0.33 ± 0.02 g 41.36 ± 3.26 e 107.65 ± 5.53 b 0.37 ± 0.02 g 49.6 ± 3.61 d 141.8 ± 7.94 a

1.52 ± 0.14 f 1157.66 ± 115.50 e 4404.66 ± 105.51 c 1.42 ± 0.11 f 1422 ± 106.92 d 4726.66 ± 195.21 b 1.17 ± 0.11 f 1471 ± 86.50 d 4846.66 ± 184.13 b 1.72 ± 0.08 f 1566.83 ± 134.38 d 5417.70 ± 145.47 a



































The same letters within a column indicate there was no significant difference at a 95% probability level; respectively; NS, non-significant.

cultivars. Moreover, total soluble protein (TSP) contents were increased in the leaves and roots under higher concentration of Cr in all cultivars as compared to their respective controls (Table 6). Data also depicted that there was no significant difference in TSP contents among all cultivars under different Cr concentrations, except, cultivar Zheda 622 showed significantly lower TSP contents in leaves under higher concentration of Cr, as compared to other cultivars. The changes induced by different concentration of Cr on glutathione reduced (GSH), glutathione oxidized (GSSG), total glutathione contents (GSH + GSSG), and glutathione reduced/glutathione oxidized (GSH/GSSG) ratio in the leaves and roots of rapeseed plants have been shown in Table 7. It was observed that GSH contents were increased gradually in the leaves in all cultivars as we increased the Cr concentration, and maximum GSH contents were found under the higher concentration of Cr in all cultivars. However, lower concentration of Cr did not show any significant change in root GSH contents in all the cultivars as compared to their respective controls. The higher concentration of Cr significantly increased the GSH contents of the roots in all cultivars; except for Zheda 622, in which GSH contents did not show any significant difference as compared to control. Moreover, no significant



and



indicate significance at the p < 0.05 and 0.01 level,

difference was found in glutathione oxidized (GSSH) contents of leaves in all cultivars under different concentrations of Cr. The higher concentration of Cr significantly increased the GSSG contents in all cultivars as compared to their respective controls. A linear increase was observed in root GSSG contents in all cultivars under different Cr concentrations; however, maximum GSSG contents were found under the higher concentration of Cr in all cultivars. Results demonstrated that total glutathione (GSH + GSSG) contents in the leaves were increased significantly under the lower concentration of Cr but no any significant change was found at this concentration in total glutathione contents of roots in all the cultivars as compared to their respective controls. The higher concentration of Cr significantly enhanced the total glutathione contents of the leaves and roots in all cultivars as compared to control and at this level, maximum total glutathione contents were observed in the leaves and roots of ZS 758 as compared to other cultivars. The glutathione reduced/glutathione oxidized (GSH/ GSSG) ratio of roots in all cultivars decreased significantly under the higher concentration of Cr as compared to their respective controls. However, no any significant difference was observed in GSH/ GSSG ratio of leaves in all the cultivars under different Cr concentrations as compared to their respective controls (Table 7).

160

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

Table 7 Effects of different treatments of chromium (Cr) (lM) on the contents (lmol min1 mg1 protein) of reduced glutathione (GSH), oxidized glutathione (GSSG), total glutathione (GSH + GSSG) and glutathione reduced/glutathione oxidized (GSH/GSSG) ratio in the leaves and roots of four cultivars of Brassica napus. Cultivar

ZS 758

Zheda 619

ZY 50

Zheda 622

Variety (V) Treatments (T) VT

Cr conc.

GSH Leaf

Root

GSSG Leaf

GSH + GSSG

0 100 400 0 100 400 0 100 400 0 100 400

9.05 ± 0.05 fg 10.84 ± 0.37 cd 13.25 ± 0.61 a 9.03 ± 0.06 fg 10.52 ± 0.69 de 12.63 ± 0.74 ab 8.88 ± 1.01 fg 9.82 ± 0.20 def 11.66 ± 0.40 bc 8.06 ± 0.07 g 9.73 ± 0.20 ef 10.37 ± 1.19 de

8.48 ± 0.18 cd 8.65 ± 0.05 cd 8.98 ± 0.02 ab 8.49 ± 0.20 cd 8.58 ± 0.01 cd 9.04 ± 0.07 a 8.46 ± 0.22 cd 8.34 ± 0.10 d 8.74 ± 0.05 bc 8.45 ± 0.24 cd 8.35 ± 0.14 d 8.58 ± 0.05 cd





0.09 ± 0.002 0.11 ± 0.020 0.15 ± 0.003 0.11 ± 0.015 0.11 ± 0.010 0.13 ± 0.002 0.10 ± 0.007 0.11 ± 0.007 0.14 ± 0.004 0.10 ± 0.008 0.11 ± 0.003 0.13 ± 0.008 NS

















c bc a c c a c c a c c ab

GSH/GSSG

Root

Leaf

Root

Leaf

0.04 ± 0.003 g 0.03 ± 0.001 g 0.09 ± 0.002 b 0.04 ± 0.002 g 0.04 ± 0.001 f 0.09 ± 0.002 a 0.03 ± 0.003 fg 0.04 ± 0.003 e 0.09 ± 0.001 b 0.04 ± 0.001 g 0.05 ± 0.002 d 0.08 ± 0.001 c NS

9.16 ± 0.13 fg 10.96 ± 0.38 cd 13.40 ± 0.61 a 9.14 ± 0.12 fg 10.63 ± 0.70 d 12.76 ± 0.74 ab 8.99 ± 1.01 fg 9.93 ± 0.19 def 11.80 ± 0.39 bc 8.17 ± 0.07 g 9.84 ± 0.19 ef 10.50 ± 1.19 de

8.51 ± 0.18 de 8.70 ± 0.09 cd 9.07 ± 0.11 ab 8.52 ± 0.20 de 8.62 ± 0.09 cde 9.14 ± 0.07 a 8.49 ± 0.22 de 8.38 ± 0.10 e 8.83 ± 0.14 bc 8.48 ± 0.24 de 8.40 ± 0.14 de 8.69 ± 0.10 cd

87.40 ± 2.62 93.1 ± 8.34 87.41 ± 3.96 80.68 ± 7.97 94.79 ± 5.87 90.67 ± 5.80 83.42 ± 7.48 85.67 ± 6.96 82.61 ± 4.53 74.90 ± 4.96 86.17 ± 4.05 78.51 ± 9.18

























The same letters within a column indicate there was no significant difference at a 95% probability level; respectively; NS, non-significant.

3.6. Cr-toxicity induces ultrastructural changes The ultrastructural changes in leaf mesophyll and root tip cells under control and higher Cr concentration (400 lM) have been illustrated in Figs. 1 and 2. The TEM micrographs of leaf mesophyll cells of ZS 758, Zheda 619, ZY 50 and Zheda 622 at control with low and high magnifications are shown in Fig. 1A–H. The micrographs showed clean and thin cell walls. A well-developed chloroplast with thylakoid membranes containing the visible structures of grana, stroma and plastoglobuli were observed in the micrographs. The nucleus was present with the nuclear membrane and nucleolus. The TEM micrographs of leaf mesophyll cells of ZS 758, Zheda 619, ZY 50 and Zheda 622 at 400 lM Cr alone with low and high magnifications are displayed in Fig. 1I–P. It was found that at this concentration, ZS 758 and Zheda 619 showed no obvious changes in the leaf mesophyll cell as compared to their respective controls (Fig. 1I–L). However, the cultivar ZY 50 showed some changes like, more number of plastoglobuli as well as disappearance of starch grains in the chloroplast as compared to respective control (Fig. 1M–N). It was observed that the toxic effects of Cr were obvious in the mesophyll cell of Zheda 622. The changes like increase in the numbers of starch grain and plastoglobuli structures were observed under 400 lM Cr concentration (Fig. 1O–P). The TEM micrographs of root tip cells of ZS 758, Zheda 619, ZY 50 and Zheda 622 at control and 400 lM Cr with low and high magnifications are demonstrated in Fig. 2A–P. At control concentration, micrographs showed that root tip cells of all the cultivars presented clear cell walls, typical oval shaped mitochondria as well as a large size and well developed nucleus was also found in the micrographs (Fig. 2A–H). However, it was found that higher concentration of Cr did not show significant alterations in the root tip cells of all the cultivars; except, Zheda 622 which showed maximum damages under Cr-toxicity (Fig. 2I–P). Results showed that in cultivar Zheda 622, all the organelles were poorly developed and disappeared in cell under the Cr toxicity (Fig. 2O–P).

4. Discussion The present study was conducted to highlight the phyto-toxic effects of Cr on the normal function of the B. napus plants as well as the ultrastructure changes occurred in cell structure. In the



and



Root ab a ab bc a ab abc abc abc c abc bc

229.92 ± 13.63 bc 247.44 ± 5.65 a 103.29 ± 2.33 g 236.11 ± 7.57 abc 209.34 ± 4.86 d 96.25 ± 11.30 g 221.17 ± 11.98 cd 183.16 ± 10.38 e 96.77 ± 5.92 g 241.52 ± 10.05 ab 153.82 ± 5.02 f 108.22 ± 7.46 g

indicate significance at the p < 0.05 and 0.01 level,

present investigation, plant growth in terms of plant, stem and root lengths, number of leaves and leaf area per plant was decreased as we increased the Cr concentration in the solution (Table 1). Results showed that lower level of Cr (100 lM) did not show any significant change in root length of all the cultivars. However, a significant decrease was found under the higher Cr stress (400 lM) in all plant growth parameters which might be due to adverse effect of Cr on the roots of the plant and therefore, plants were not able to uptake nutrients and continue their normal activity (Ali et al., 2013c). Our present findings are in line with Sharma and Dubey (2005) who also concluded that Pb stress decreased the plant growth in different plants. It has been documented that the reduction in root growth may be due to metal-induced inhibition of cell division of cell wall (Eun et al., 2000). Moreover, our results confirm the findings of Dey et al. (2009) that root length was deteriorated in wheat plants under Cr stress. In another study, it was found that higher concentration of Cr (300 lg L1) can cause stunted shoot growth and overall considered inhibitory effects on plant growth (Faisal and Hasnain, 2005; Gbaruko and Friday, 2007). Fresh and dry biomass of leaf, stem and root has been considered as a prerequisite measurement for various abiotic stresses. Present study depicted that fresh and dry biomass of different plant parts was reduced significantly under both Cr treatments (100 and 400 lM) as compared to the control (Table 2); however, lower concentration of Cr did not show any significant change in leaf fresh weight of cultivar Zheda 619. The data further depicted a decreasing trend in fresh and dry biomasses of plant parts, which was ZS 758 > Zheda 622 > ZY 50 > Zheda 622. This decrease in plant biomass may be the result that heavy metals stress might also inhibit the photosynthetic electron transport chain (Mohanty et al., 1989), and ultimately resulting in biomass reduction. Moreover, inhibition of plant biomass might be due to inhibition of photosynthesis, inactivity of both photosystem II and the enzymes of carbon reduction cycles (Skorzynska and Baszynski, 1997). Moreover, Zurayk et al. (2001) found that Cr and salinity interaction decreased dry weight in a significant manner in Portulaca oleracea. It is now obvious that responsible features for controlling CO2 assimilation in plants are net photosynthetic rate, stomatal conductance, transpiration rate, carbon uptake or a combination of all of these parameters. Results showed that both concentrations of Cr (100 and 400 lM) significantly reduced photosynthetic parameters in all the cultivars except net photosynthetic rate

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

161

Fig. 1. Electron micrographs of leaf mesophyll of 15-d hydroponically grown seedlings of four cultivars of Brassica napus (cvs. ZS 758, Zheda 619, ZY 50 and Zheda 622) under control and 400 lM Cr concentration. (A and B) TEM micrographs of leaf mesophyll cells of ZS 758 under control with low and high magnification respectively show well developed chloroplast (Chl), nucleus (N) with nucleoli (Nue), mitochondria (M), and starch grain (SG). (C and D) TEM micrographs of leaf mesophyll cells of Zheda 619 under control with low and high magnifications respectively show distinct nucleus (N), a clear cell wall (CW), nuclear membrane (NM), thylakoid membranes (Thy), chloroplast (Chl) and starch grains (SG). (E and F) TEM micrographs of leaf mesophyll cells of ZY 50 under control with low and high magnification respectively show plastoglobuli structures (PG), starch grain (SG), a distinct cell wall (CW), visible nucleus (N) with nucleoli (Nue) and thylakoid membrane (Thy). (G and H) TEM micrographs of leaf mesophyll cells of Zheda 622 under control with low and high magnifications respectively show cell wall (CW), starch grain (SG), a well developed nucleus (N) with nucleoli (Nue) and thylakoid structures (Thy). (I and J) TEM micrographs of leaf mesophyll cells of ZS 758 under 400 lM Cr with low and high magnification respectively show cell wall (CW), a well developed chloroplast (Chl), starch grain (SG), nucleus (N) with nucleoli (Nue), a visible nuclear membrane (NM) and thylakoid membrane (Thy). (K and L) TEM micrographs of leaf mesophyll cells of Zheda 619 under 400 lM Cr with low and high magnification respectively show clear a well developed nucleus and thylakoid membranes as well as plastoglobuli (PG). (M and N) TEM micrographs of leaf mesophyll cells of ZY 50 under 400 lM Cr with low and high magnification respectively show thylakoid membranes (Thy), chloroplast (Chl), cell wall (CW), nucleus (N) with nucleolus (Nue) and plastoglobuli (PG). (O and P) TEM micrographs of leaf mesophyll cells of Zheda 622 under 400 lM Cr with low and high magnification respectively show ruptured chloroplast (Chl), increase size and number of starch grains (SG) in thylakoid membranes as compared to control.

which did not decrease significantly under both concentrations of Cr in ZS 758. Moreover, lower concentration of Cr also did not show any significant reduction in net photosynthetic rate in all other cultivars. The reduction in photosynthetic parameters under Cr stress might be due to that heavy metal can affect stomatal conductance, gas exchange and chlorophyll contents, and ultimately photosynthetic parameters get affected in metal-treated conditions (Ali et al., 2013c). The decline in rate of photosynthesis under Cr-toxicity might be due to interference of metal in nutrient uptake, respiration rate and permeability of the cell membrane (Sharma and Dubey, 2005) and moreover, may be due to decomposition of chlorophyll by rise in chlorophyllase activity under heavy metal stress (Hegedus et al., 2001). Furthermore, our findings are in line with Liu et al. (2008) and Ali et al. (2011), who stated that Cr at higher concentrations decreased net photosynthetic rate, stomatal conductance, intercellular CO2 concentration and transpiration rate in Amaranthus viridis and Hordeum vulgare, respectively. In present investigation, Cr contents in different plant parts were found dose dependent (Table 6). Cr contents in roots were higher than upper parts (shoots) of plants in all B. napus cultivars. The tendency of Cr to accumulate in higher amounts in roots than shoots might be the possible reason of longer plant length (upper portion) than roots in present study under different metal concentrations (Wong et al., 1984, Chugh et al., 1992). It is important to mention that high retention of heavy metals in root is desirable

in a crop like Brassica, which is mainly utilized as an oil crop. Moreover, more Cr contents in cultivar Zheda 622 showed its sensitiveness against the metal stress as compared to other cultivars. MDA contents are commonly used as an indicator of lipid peroxidation as well as stress level (Chaoui et al., 1997). Data showed that MDA and ROS contents were significantly enhanced in the leaves and roots of plants in all the cultivars after exposure to different Cr concentrations (100 and 400 lM); especially the cultivar Zheda 622 was much affected as compared to other cultivars which showed its sensitiveness against the Cr stress (Table 4). Halliwell and Gutteridge (1990) described that excess ROS reacts with lipids, proteins and nucleic acids resulting in rise of MDA, membrane leakage and DNA breakdown which cause severe damage to plant cell. Although, ROS may act as secondary messengers to regulate gene expression and protein biosynthesis involved in the stress defenses, however, they are cyto-toxic at higher concentrations (Jiang and Zhang, 2001). They may cause damages in the form of pigment loss, reduction in gas exchange capacity, and decreased protein and RNA concentrations (Masood et al., 2012). It has been reported that overproduction of MDA and ROS could be due to responses of heavy metal in biological system through various mechanisms involving electron transfer (Dietz et al., 1999). In our research, under higher concentrations of Cr, the effect of MDA and ROS increased which might be the result of cell damage in the plant tissues. These results may suggest that the harmful

162

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

Fig. 2. Electron micrographs of root tip cells of 15-d hydroponically grown seedlings of four cultivars of Brassica napus (cvs. ZS 758, Zheda 619, ZY 50 and Zheda 622) under control and 400 lM Cr concentration. (A and B) TEM micrographs of root tip cells of ZS 758 under control with low and high magnifications respectively show a clear cell wall (CW), oval shape mitochondria (M), large size elongated nucleus (N) with roundish nucleoli (Nue) and nuclear membrane (NM). (C and D) TEM micrographs of root tip cells of Zheda 619 under control with low and high magnifications respectively show large nucleus (N) with number of nucleoli (Nue), nuclear membrane (NM) and a clear cell wall (CW). (E and F) TEM micrographs of root tip cells of ZY 50 under control with low and high magnifications respectively show well develop mitochondria (M), nucleus (N) with number of nucleoli (Nue), nuclear membrane (NM) and cell wall (CW). (G and H) TEM micrographs of root tip cells of Zheda 622 control with low and high magnifications respectively show clear nucleus (N) with nucleoli (Nue), cell wall (CW), well developed mitochondria (M) and nuclear membrane (NM). (I and J) TEM micrographs of root tip cells of ZS 758 under 400 lM Cr with low and high magnifications respectively show giant size nucleus (N) with scattered nucleoli (Nue), well developed mitochondria (M). (K and L) TEM micrographs of root tip cells of Zheda 619 under 400 lM Cr with low and high magnifications respectively show clear cell wall (CW), large size nucleus (N) with nucleoli (Nue), visible golgibodies structures (GB), and a number of mitochondria (M). (M and N) TEM micrographs of root tip cells of ZY 50 under 400 lM Cr with low and high magnifications respectively show a visible cell wall (CW), nucleus (N) with nucleoli (Nue) and nuclear membrane (NM) but mitochondria (M) was undeveloped as compared to control. (O and P) TEM micrographs of root tip cells of Zheda 622 under 400 lM Cr with low and high magnifications respectively show a ruptured cell with undeveloped nucleus (N) and broken nuclear membrane as well as no mitochondria were found as compared to control.

impact of Cr on plants is probably exerted through the production of ROS. Higher production of H2O2 and O2 contents were observed in many plant species exposed to Cr and metal has been implicated in the generation of oxidative stress (Panda et al., 2003). Previous studies have been reported that tolerant plants showed a lower accumulation of lipid peroxidation and H2O2 as compared to sensitive plants when treated with heavy metals (Cho and Seo, 2005). Like other stresses, under Cr stress, plant cells have evolved antioxidant systems to cope with ROS or alleviate their damaging effect. The antioxidant enzymes systems comprise of SOD, POD, CAT, APX and GR which control the cellular concentration of O2. and H2O2, thereby preventing the formation of OH (RucinskaSobkowiak and Pukacki, 2006). In the present study, the activities of SOD, POD and CAT were enhanced under different Cr concentrations (100 and 400 lM) except CAT in leaves of Zheda 622 where CAT activity was reduced under higher concentration of Cr. While, APX activity was decreased in relation to different Cr concentrations. Moreover, no significant change was found in POD activity in all the cultivars under lower concentration of Cr (100 lM). Previously, Meng et al. (2009) also reported that SOD activity was increased under heavy metal stress, which showed a positive impact of antioxidant enzyme activity on physiological mechanisms of plants under stressed conditions by minimizing the production of ROS (Mittler, 2002). The increase in CAT activity in the leaves and roots under Cr stress suggested that its effective scavenging mechanism to remove H2O2 resulted from metal stress-

caused oxidative damage (Reddy et al., 2005). Moreover, Shamsi et al. (2008) also found that activities of SOD and POD were increased under heavy metal stress in soybean plants. It was also observed that POD activities were higher in the roots as compared to leaves in all the cultivars (Table 5), which can be explained by the probability that the glutathione/ascorbate cycle was operating at a high rate in order to detoxify the ROS formed in the roots (Liu et al., 2003). However, reduction in antioxidant enzyme activities might be due to the oxidative stress, inhibition of enzyme synthesis and change in the assemblage of enzyme subunits (Garnier et al., 2006). The increase in antioxidant activities in the leaves and roots was higher in the cultivars (ZS 758 and Zheda 619) under Cr stress which proved their tolerance against Cr stress. The significant decrease in APX activity under Cr stress in Zheda 622 as compared to other cultivars showed its sensitivity against the Crtoxicity (Ali et al., 2013d). It is well documented in the literature that glutathione (GSH) and some emerging antioxidants like proline and carbon monoxide are considered as non-enzymatic antioxidant systems and they play an important role in plants to response under abiotic stress (Sharma and Dietz, 2009). It has been considered that GSH and phytochelatins play a key role in heavy metal detoxification and subsequent stress tolerance (Masood et al., 2012). In the present study, different Cr concentrations (100 and 400 lM) increased non-enzymatic antioxidants activities i.e., GSH, GSSG and total glutathione in the leaves and roots of B. napus cultivars (Fig. 2). How-

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

ever, lower concentration of Cr (100 lM) did not show any significant change in GSH activity of root and GGSG activity of leaf in all the cultivars. Previously, Sun et al. (2011) found that GSH contents were increased under arsenic stress in Pteris vitatta. It can be suggested that plant might depend on considerable constitutive GSH to counteract the potentially harmful effects of heavy metal, and a relatively high contents were assumed to be more protective against the formation of ROS by Cr stress. In the present study, the ultrastructural changes occurred in different parts of plant cells were found to be dose-dependent. The mesophyll cells were significantly damaged at 400 lM Cr in Zheda 622 as compared to other cultivars (Fig. 1). The number of plastoglobuli and starch grains was increased in Zheda 622 under Cr stress which indicated that plants might be undergo in stress under metal stress (Ali et al., 2013c; Daud et al., 2013). The root tip cells of Zheda 622 were damaged significantly under Cr stress than other cultivars (Fig. 2). The cell structure was ruptured and disappearance of different organelles was observed in Zheda 622 under Cr stress (Fig. 2O–P). Previously, it was shown that heavy metal toxicity damaged the root tip cells of B. napus (Ali et al., 2013b; Panda, 2007). Moreover, Ali et al. (2013d) also reported that higher concentrations of Cr stress ruptured the root tip cell in barley.

5. Conclusions The difference observed among four oilseed rape cultivars in the present study, suggests that cultivars have different capability to face the Cr-toxicity. The study indicated that Cr-toxicity reduced the plant growth in all the cultivars and Zheda 622 proved to be more sensitive as followed by ZY 50, Zheda 619 and ZS 758 under Cr-toxicity. It was found that activities of SOD and POD were increased under the higher concentration of Cr (400 lM) in all the cultivars, except for Zheda 622, in which POD activity did not show any significant difference. The electron microscopic study revealed that ultrastructural damages in leaf mesophyll and root tip cells were more prominent in Zheda 622 as compared to other cultivars under 400 lM Cr stress. Moreover, our present study intends to investigate the toxic effects of Cr by using hydroponic conditions, and these findings would be of great interest to the scientists working on the phytoremediation and related area. In order to study its toxic effects on these Brassica cultivars in the real soil environment, further investigation is needed. Acknowledgements This study was supported by the National High Technology Research and Development Program of China (2013AA103007), Special Fund for Agro-scientific Research in the Public Interest (201303022), the National Key Science and Technology Supporting Program of China (2010BAD01B04), the National Natural Science Foundation of China (31170405), the Science and Technology Department of Zhejiang Province (2012C12902-1), and the Agriculture Department of Zhejiang Province (N20120624). References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Ali, B., Huang, C.R., Qi, Z.Y., Ali, S., Daud, M.K., Geng, X.X., Liu, H.B., Zhou, W.J., 2013a. 5-Aminolevulinic acid ameliorates cadmium-induced morphological, biochemical, and ultrastructural changes in seedlings of oilseed rape. Environ. Sci. Pollut. Res. 20, 7256–7267. Ali, B., Tao, Q., Zhou, Y., Gill, R.A., Ali, S., Rafiq, M.T., Xu, L., Zhou, W.J., 2013b. 5-Aminolevolinic acid mitigates the cadmium-induced changes in Brassica napus as revealed by the biochemical and ultra-structural evaluation of roots. Ecotoxicol. Environ. Saf. 92, 271–280.

163

Ali, B., Wang, B., Ali, S., Ghani, M.A., Hayat, M.T., Yang, C., Xu, L., Zhou, W.J., 2013c. 5-Aminolevulinic acid ameliorates the growth, photosynthetic gas exchange capacity, and ultrastructural changes under cadmium stress in Brassica napus L.. J. Plant Growth Regul. 32, 604–614. Ali, S., Farooq, M.H., Hussain, S., Yasmeen, T., Abbasi, G.H., Zhang, G., 2013d. Alleviation of chromium toxicity by hydrogen sulfide in barley. Environ. Toxicol. Chem. 32, 2234–2239. Ali, S., Zeng, F., Qiu, L., Zhang, G., 2011. The effect of chromium and aluminum on growth, root morphology, photosynthetic parameters and transpiration of the two barley cultivars. Biol. Plant. 55, 291–296. Bradford, N.M., 1976. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 72, 248–254. Chugh, L.K., Gupta, V.K., Sawhney, S.K., 1992. Effect of cadmium on enzymes of nitrogen metabolism in pea seedlings. Phytochemistry 3, 395–400. Chaoui, A., Mazhoudi, S., Ghorbal, M.H., Ferjani, E., 1997. Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean (Phaseolus vulgaris L.). Plant Sci. 127, 139–147. Cho, U.H., Seo, N.H., 2005. Oxidative stress in Arabidopsis thaliana exposed to cadmium is due to hydrogen peroxide accumulation. Plant Sci. 168, 113–120. Daud, M.K., Ali, S., Variath, M.T., Zhu, S.J., 2013. Differential physiological, ultramorphological and metabolic responses of cotton cultivars under cadmium stress. Chemosphere 93, 2593–2602. Dey, S.K., Jena, P.P., Kundu, S., 2009. Antioxidative efficiency of Triticum aestivum L. exposed to chromium stress. J. Environ. Biol. 30, 539–544. Dietz, K.J., Baier, M., Kramer, U., 1999. Free radicals and reactive oxygen species as mediators of heavy metal toxicity in plants. In: Prasad, M.N.V., Hagemeyer, J. (Eds.), Heavy Metal Stress in Plants: From Molecules to Ecosystems. SpringerVerlag, Berlin, pp. 73–97. Eun, S.O., Youn, H.S., Lee, Y., 2000. Lead disturbs microtubule organization in the root meristem of Zea mays. Physiol. Plant. 110, 357–365. Faisal, M., Hasnain, S., 2005. Chromate resistant Bacillus cereus augments sunflower growth by reducing toxicity Cr (VI). J. Plant Biol. 48, 187–194. Garnier, L., Simon-Plas, F., Thuleau, P., Agnel, J.P., Blein, J.P., Ranjeva, R., 2006. Cadmium affects tobacco cells by a series of three waves of reactive oxygen species that contribute to cytotoxicity. Plant Cell Environ. 29, 1956–1969. Gbaruko, B.C., Friday, O.U., 2007. Bio-accumulation of heavy metals in some fauna and flora. Int. J. Environ. Sci. Tech. 4, 197–202. Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909– 930. Halliwell, B., Gutteridge, J.M.C., 1990. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 186, 1–85. Halliwell, B., Gutteridge, J.M.C., Aruoma, O., 1987. The deoxyribose method: a simple ‘test tube’ assay for determination of rate constants for reactions of hydroxyl radicals. Anal. Biochem. 165, 215–219. Hegedus, A., Erdel, S., Horvath, G., 2001. Comparative studies of H2O2 detoxifying enzymes in green and greening barely seedlings under Cd stress. Plant Sci. 160, 1085–1093. Hoagland, D., Arnon, D., 1941. Physiological aspects of availability of nutrients for plant growth. Soil Sci. 51, 431–444. Jiang, M., Zhang, J., 2001. Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings. Plant Cell Physiol. 42, 1265–1273. Jiang, M., Zhang, J., 2002. Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves. J. Exp. Bot. 53, 2401– 2410. Kabada-Pendias, A., Pendias, H., 1992. Trace Elements in Soils and Plants, fourth ed. Boca Raton, London. Law, M.Y., Charles, S.A., Halliwell, B., 1983. Glutathione and ascorbic-acid in spinach (Spinacia oleracea) chloroplasts, the effect of hydrogen-peroxide and of paraquat. Biochem. J. 210, 899–903. Liu, D., Jiang, W., Gao, X., 2003. Effect of cadmium on root growth, cell division and nucleoli in root tip cells of garlic. Biol. Plant. 47, 79–83. Liu, D., Zou, J., Wang, M., Jiang, W., 2008. Hexavalent chromium uptake and its effects on mineral uptake, antioxidant defence system and photosynthesis in Amaranthus viridis L.. Bioresour. Technol. 99, 2628–2636. Masood, A., Iqbal, N., Khan, N.A., 2012. Role of ethylene in alleviation of cadmiuminduced photosynthetic capacity inhibition by sulphur in mustard. Plant Cell Environ. 35, 524–533. Meng, H.B., Hua, S.J., Shamsi, I.H., Jilani, G., Li, Y.L., Jiang, L.X., 2009. Cadmium induced stress on the seed germination and seedling growth of Brassica napus L., and its alleviation through exogenous plant growth regulators. Plant Growth Regul. 58, 47–59. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410. Mohanty, N., Vass, I., Demeter, S., 1989. Impairment of photosystem II activity at the level of secondary quinine acceptor in chloroplasts treated with cobalt, nickel and zinc ions. Physiol. Plant. 76, 386–390. Momoh, E., Zhou, W., 2001. Growth and yield responses to plant density and stage of transplanting in winter oilseed rape (Brassica napus L.). J. Agron. Crop Sci. 186, 253–259. Momoh, E.J.J., Zhou, W.J., Kristiansson, B., 2002. Variation in the development of secondary dormancy in oilseed rape genotypes under conditions of stress. Weed Res. 42, 446–455.

164

R.A. Gill et al. / Chemosphere 120 (2015) 154–164

Nakano, Y., Asada, K., 1981. Hydrogen-peroxide is scavenged by ascorbate-specific peroxidase in spinach-chloroplasts. Plant Cell Physiol. 22, 867–880. Panda, S.K., 2007. Chromium-mediated oxidative stress and ultrastructural changes in root cells of developing rice seedlings. J. Plant Physiol. 164, 1419–1428. Panda, S.K., Chaudhury, I., Khan, M.H., 2003. Heavy metals induce lipid peroxidation and affects antioxidants in wheat leaves. Biol. Plant. 46, 289– 294. Papazoglou, E.G., Karantounias, G.A., Vemmos, S.N., Bouranis, D.L., 2005. Photosynthesis and growth responses of giant reed (Arundo donax L.) to the heavy metals Cd and Ni. Environ. Int. 31, 243–249. Reddy, A.M., Kumar, S.G., Jyothsnakumari, J., Thimmanaik, S., Sudhakar, C., 2005. Lead induced changes in antioxidant metabolism of horsegram (Macrotyloma uniflorum (Lam.) Verdc.) and bengalgram (Cicer arietinum L.). Chemosphere 60, 97–104. Rucinska-Sobkowiak, R., Pukacki, P.M., 2006. Antioxidative defense system in lupin roots exposed to increasing concentrations of lead. Acta Physiol. Plant. 28, 357– 364. Scoccianti, V., Crinelli, R., Tirillini, B., Mancinelli, V., Speranza, A., 2006. Uptake and toxicity of Cr (III) in celery seedlings. Chemosphere 64, 1695–1703. Shamsi, I.H., Jilani, G., Zhang, G.P., Kang, W., 2008. Cadmium stress tolerance through potassium nutrition in soybean. Asian J. Chem. 20, 1099–1108. Shanker, A.K., Cervantes, C., Loza-Tavera, H., Avudainayagam, S., 2005. Chromium toxicity in plants. Environ. Int. 31, 739–753. Sharma, D.C., Sharma, C.P., Tripathi, R.D., 2003. Phytotoxic lesions of chromium in maize. Chemosphere 51, 63–68. Sharma, P.R., Dubey, S., 2005. Lead toxicity in plants. Braz. J. Plant Physiol. 17, 35– 52. Sharma, S.S., Dietz, K.J., 2009. The relationship between metal toxicity and cellular redox imbalance. Trends Plant Sci. 14, 43–50.

Skorzynska, E., Baszynski, T., 1997. Differences in sensitivity of the photosynthetic apparatus in Cd-stressed runner bean plants in relation to their age. Plant Sci. 128, 11–21. Sun, L., Yan, X., Liao, X., Wen, Y., Chong, Z., Liang, T., 2011. Interactions of arsenic and phenanthrene on their uptake and antioxidative response in Pteris vittata L.. Environ. Pollut. 159, 3398–3405. van Assche, F., Clijsters, H., 1983. Multiple effects of heavy metals on photosynthesis. In: Marcelle, R., Clijsters, H., Van Poucke, M. (Eds.), Effects of Stress on Photosynthesis. Martinus Nijhoff/Dr W. Junk Publishers, London, pp. 371–382. Vazques, M.D., Poschenrieder, C., Barcelo, J., 1987. Chromium VI induced structural and ultrastructural changes in bush bean plants (Phaseolus vulgaris L.). Annal. Bot. 59, 427–438. Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Plant Sci. 151, 59–66. Wong, M.K., Chuah, G.K., Koh, L.L., Ang, K.P., Hew, C.S., 1984. The uptake of cadmium by Brassica chinensis and its effect on plant zinc and iron distribution. Environ. Exp. Bot. 24, 189–195. Zhang, W.F., Zhang, F., Raziuddin, R., Gong, H.J., Yang, Z.M., Lu, L., Ye, Q.F., Zhou, W.J., 2008. Effects of 5-aminolevulinic acid on oilseed rape seedling growth under herbicide toxicity stress. J. Plant Growth Regul. 27, 159–169. Zhou, W., Leul, M., 1998. Uniconazole-induced alleviation of freezing injury in relation to changes in hormonal balance, enzyme activities and lipid peroxidation in winter rape. Plant Growth Regul. 26, 41–47. Zhou, W.J., Leul, M., 1999. Uniconazole-induced tolerance of rape plants to heat stress in relation to changes in hormonal levels, enzyme activities and lipid peroxidation. Plant Growth Regul. 27, 99–104. Zurayk, R., Sukkariyah, B., Baalbaki, R., 2001. Common hydrophytes as bioindicators of nickel, chromium and cadmium pollution. Water Air Soil Pollut. 127, 373–388.