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Alleviation of Cadmium Toxicity by 5-Aminolevulinic Acid is related to Improved Nutrients Uptake and Lowered Oxidative Stress in Brassica napus.

Byline: Ling Xu, Basharat Ali, Rafaqat A. Gill, Lan Li and Weijun Zhou

Abstract

The oilseed rape production (Brassica napus L.) has been shown to decrease under heavy-metal stress. This study tested the hypothesis that 5-aminolevulinic acid (ALA) has ameliorating role under cadmium (Cd)-toxicity in B. napus. In the present study, three levels (0, 100 and 500 uM) of Cd and three levels (0, 12.5 and 25 mg/L) of foliar ALA were applied hydroponically in greenhouse. Results demonstrated that ALA significantly enhanced the leaf chlorophyll contents under Cd stress as compared to control. Exogenously applied ALA enhanced the concentration of macro- and micronutrients in the leaves and roots of B. napus under Cd stress. Further, foliar application of ALA significantly decreased malondialdehyde and reactive oxygen species in the leaves of B. napus under Cd stress.

Moreover, the present study revealed that leaves treated with ALA at different concentrations increased the concentration of glutathione reduced (GSH), glutathione oxidized (GSSG), total glutathione and soluble protein contents as well as decreased the GSH/GSSG ratio under Cd-toxicity. The present study stated that ALA improved the concentration of nutrients and decreased oxidative stress due to its ameliorative capability under Cd stress conditions in B. napus.

Keywords: Cadmium; Non-antioxidant enzyme activities; Elements uptake; Oilseed rape; Reactive oxygen species

Introduction

In atmosphere, various anthropogenic activities such as mining, industrialization, and phosphate fertilizers are directly or indirectly responsible for the environmental pollution (Zhou and Qiu, 2005). Metal toxicity has received great attention worldwide because large amounts of these metals are released into the environment every year. Heavy metal toxicity, especially by cadmium (Cd), lead (Pb), arsenic (As) and mercury (Hg) constitute serious threats to human health (Wenneberg, 1994). A part of agricultural soils in the world is contaminated with Cd as slightly to moderately due to the usage of phosphate fertilizers, sewage water as well as sedimentation (Thawornchaisit and Polprasert, 2009). Cd is most toxic element to plants and at high concentration (5 mM), can reduce chlorophyll contents and photosynthesis in B. napus (Baryla et al., 2001; Zhou and Huang, 2001). Cd can disturb the uptake and distribution of macro- and micronutrients in the plants (Ramos et al., 2002).

Previously, Rivetta et al. (1997) reported the Cd competition with elements i.e. Fe, Zn, Mn, Cu, Ca, Mg and P in radish plants. Aerobic metabolism faces a constant risk of being disturbed by ROS like free radicals O2 Cd-toxicity causes the oxidative stress through accumulation of ROS in plant, which ultimately lead to excessive lipid peroxidation (Shah et al., 2001).

Application of plant growth regulators (PGRs) is a promising approach to increase the plant tolerance against abiotic stresses (Ai et al., 2008; Ali et al., 2013a). PGRs can improve the productivity of plants by regulating the physiological characters. In fact, many PGRs have been found to enhance the stress tolerance in plants (El-Tayeb, 2005; Zhang et al., 2008). ALA is an important PGR and it is a precursor for biosynthesis of chlorophyll (Von Wettstein et al., 1995). ALA exerts positive effects on the plants and increases the chlorophyll content and antioxidants under saline conditions (Watanabe et al., 2000; Naeem et al., 2012). Recently, it was found that ALA improved the concentration of different nutrients in B. napus under salinity stress conditions (Naeem et al., 2010). Similarly, an increase in antioxidant enzymes activities was reported by ALA under salinity in Indian mustard and sugar beet (Bor et al., 2003; Youssef and Awad, 2008).

ALA and ascorbic acid have been shown to ameliorate the effect of water stress in B. napus and Zea mays, respectively (Liu et al., 2011; Dolatabadian et al., 2009).

Nowadays, rapeseed Brassica crops are used in food and feed applications as well as in biofuel production. Phytoremediation with B. napus has greater potential to become a protable enterprise (Grispen et al., 2006). Rapeseed is the oldest cultivated oil-producing plant. A rapid increase in B. napus production has been observed during the last two decades all over the world (FAOSTAT, 2005). Therefore, it is necessary to resolve the Cd-toxicity problems in B. napus fields on urgent basis. However, in the previous study, we found that ALA improved the root growth in B. napus by improving biochemical and ultrastructural changes under Cd stress (Ali et al., 2013b). Keeping in view the alleviating ability of ALA, this study was planned to clarify the hypothesis that ALA has the ameliorating role to improve Cd-induced growth and nutrients uptake, and decrease the oxidative stress in B. napus.

Materials and Methods

Plant Material and Treatments

Seeds of oilseed rape (Brassica napus L. cv. ZS 758) were obtained from the College of Agriculture and Biotechnology, Zhejiang University. Seeds were washed with distilled water and then air-dried. Seeds were sown in plastic pots (170 mm x 220 mm) filled with peat moss. After thirty days of sowing, morphologically uniform seedlings were selected and plugged into plate holes on plastic pots (five plants per pot) containing a half strength nutrient solution (Arnon and Hoagland, 1940), aerated continuously with air pump in the greenhouse. The pH of solution was maintained at 6.0 and light intensity was in the range of 250-350 umol m-2 s-1. Temperature and relative humidity were adjusted at 16-20C and 55-60% respectively.

After acclimatization period, desired Cd concentrations (0, 100 and 500 uM Cd) were adjusted and plants were simultaneously sprayed with an aqueous solution of ALA (Cosmo Oil, Japan) at three concentrations (0, 12.5 and 25 mg/L ALA). The treatment concentrations were based on pre-experimental studies, in which Cd at 100 uM concentration showed a little damage on plant growth and 500 uM Cd imposed a significant damage to plant growth, while those higher than 500 uM were too toxic for plant growth (Ali et al., 2014). After every 5 days, a subsequent foliar spray of ALA was applied. The control plants were sprayed with distilled water. After fifteen days, all physiological data were measured and samples were collected for nutrient uptake and biochemical analysis.

Morphological and Elemental Measurements

Plant biomass (% of control) against each treatment was calculated according to Ali et al. (2011) by using this formula: (% of control) = (Mean value in treatment - Mean value in control/Mean value in control x 100). A chlorophyll meter (Minolta Co. Ltd., Japan) was used to take SPAD values of the fully expanded functional leaves (the 4th from the apex), which provided a rapid, accurate, and non-destructive estimate of leaf chlorophyll content. For this purpose, a total of 20 readings per treatment were taken from randomly selected plants to determine chlorophyll content according to Wu et al. (1998).

After 15 days of treatments, plants were differentiated to leaves and roots, washed with tap water and distilled water thrice, respectively and then blotted to remove the excessive water. Thereafter, plants were separated into roots and leaves (topmost fully expanded leaf) and then dried at 80C in an oven for 48 h, and ground into powder. Plant samples of roots and leaves (0.2 g) were digested with a mix of 5 mL of HNO3 (65%) + 1 mL of HClO4 (72%). After that the resultant solutions were diluted to 25 mL using 2% HNO3 and filtered. The concentrations of Na+, K+, Ca2+, Mg2+, Mn2+, Fe3+, Cu2+ and Zn2+ in the filtrate were determined using inductively coupled plasma-mass spectrometry (ICP-MS, Agilent, 7500a) following a standard procedure.

The nitrogen content was determined according to the method of Lindner (1944). Plant dried samples (leaves and roots) were digested with 3 mL of H2SO4-H2O2 (30%) to obtain a clear solution. To the digested material, 2.5 N NaOH, 1% sodium silicate and Nessler's reagent were added. The absorbance of solution was read at 525 nm, using the spectrophotometer. Phosphorus content was estimated according to the method of Murphey and Riley (1962) by using molybdenum blue method and absorbance was read at 660 nm in a spectrophotometer. Sulphur content was measured by Butters and Chenery (1959) method using barium sulfate and absorbance was taken at 440 nm with spectrophotometer.

Malondialdehyde and Reactive Oxygen Species

Lipid peroxidation was estimated in terms of malondialdehyde (MDA) contents and was determined as 2- thiobarbituric acid (TBA) reactive substances following the method of Zhou and Leul (1998). H2O2 contents were determined according to the method of Velikova et al. (2000) with some modifications. To 0.5 mL of the supernatant, 0.5 mL of phosphate buffer (pH 7.0) and 1.0 mL of potassium iodide (1 mM) were added. The absorbance of the mixture was taken at 390 nm. H2O2 content was determined using an extinction coefficient of 0.28 uM cm-1 and expressed as nmol g-1 FW. The O - contents were determined according to Jiang and Zhang (2001) method. For estimation of -OH contents in leaves, 50 mg of leaves were incubated in 1 mL of 10 mM Na- phosphate buffer (pH 7.4) consisting 15 mM 2-deoxy-D-ribose at 37C for 2 h (Halliwell et al., 1987).

Following incubation, an aliquot of 0.7 mL from the above mixture were added to reaction mixture containing 3 mL of 0.5% (w/v) TBA and 1 mL glacial acetic acid, heated at 100C in a water bath for 30 min and cooled down to 41C for 10 min before measurement. The absorbance was measured at 532 nm and concentration was calculated using an extinction coefficient (155 mM cm-1) and expressed in mmol g-1 FW.

Total Soluble Protein and Non-enzymatic Antioxidants Analysis

TSP contents were determined according to the method of Bradford (1976). GSH and GSSG contents were measured by following the method of Law et al. (1983) with some modications. Leaves (0.3 g) were homogenized with 5 mL 10% (w/v) TCA and homogenate was centrifuged at 15,000 g for 15 min. The supernatant was used for the determination of GSH and GSSG contents. To assay total glutathione, 150 uL supernatant was added to 100 uL 6 mM DTNB, 50 uL glutathione reductase (10 units mL-1), and 700 uL 0.3 mM NADPH. The total glutathione content was 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 content, 120 uL of supernatant was added to 10 uL 2-vinylpyridine followed by 20 uL of 50% (v/v) triethanolamine. The solution was vortex-mixed for 30 s and incubated at 25C for 25 min.

Calibration curve was developed using GSSG samples treated exactly as above and GSH content was determined by subtracting GSSG content from the total glutathione content. Ascorbic acid (AsA) contents were measured according to Singh et al. (2006).

Statistical Analysis

The experiment was carried out through a randomized design. The analysis of variance was computed for statistically significant differences determined based on the appropriate two-way variance analysis (ANOVA). The results were the mean SD of at least three independent replicates and were analyzed using data processing system SPSS version 16.0 (SPSS, Chicago, IL, USA), followed by the Duncan's Multiple Range Test (DMRT).

Results

ALA Regulates the Plant Growth under Cd Stress

The data regarding plant biomass (% of control) and chlorophyll contents (SPAD value) are shown in Fig. 1. Results showed that Cd alone inhibited the plant growth significantly, which led to the significant decrease in plant biomass. However, exogenously applied ALA improved the biomass of B. napus plants under Cd stress. The maximum plant biomass (fresh and dry weight) was observed with ALA application alone at 25 mg/L dosage. The contents of chlorophyll were decreased by 30% and 40% under 100 and 500 uM Cd stress alone, respectively. However, application of ALA at higher concentration (25 mg/L) increased chlorophyll contents (26% and 21%) under both Cd levels (100 and 500 uM) respectively, compared to their respective controls.

ALA Improves the Nutrient Concentration in Leaves and Roots

Results showed that concentration of Na+ increased as concentration of Cd increased alone in the solution and this uptake was more obvious in leaves than roots (Table 1). The maximum Na+ contents were found at 500 uM Cd alone; however, application of ALA decreased the Na+ concentration in different plant parts under Cd-toxicity.

Higher dosage of ALA (25 mg/L) reduced the concentration of Na+ in leaf (by 25% and 35%) and in root (by 14% and 28%) under 100 and 500 uM Cd stress respectively, than that of untreated plants. Exogenous ALA alone at 25 mg/L increased concentration of K+ in leaf and root and increased the K+ concentration in leaf (by 29% and 62%) under 100 and 500 uM Cd stress respectively, than that of untreated plants. However, K+ concentration in root did not increase significantly under different concentrations of ALA and Cd. Moreover, this study revealed that ALA significantly decreased the Na+/K+ ratio in leaves and roots. Lower concentration of Cd (100 uM) alone showed no effect on the concentrations of Ca2+ and Mg2+ in leaves and roots, but higher concentration of Cd (500 uM) significantly reduced the concentrations of both elements (Table 1).

ALA at 25 mg/L enhanced the contents of Ca2+ by 45% and 24% in leaf and root respectively; Mg2+ by 80% and 90% in leaf and root respectively, under 500 uM Cd stress level (Table 1).

The magnitude of macro- (N, P and S) and micro-nutrients (Mn, Zn, Fe and Cu) in leaves and roots under different levels of ALA and Cd are presented in Table 2.

Higher level of Cd (500 uM) alone significantly reduced the concentration of macro- and micro-nutrients except Fe. This decline was 29% in N, 48% in P, 37% in S, 41% in Mn, 18% in Zn and 44% in Cu as compared to control. However, when ALA at 25 mg/L was foliar applied to plants under 500 uM Cd, the concentrations of all the nutrients was enhanced. This increase was 35% in N, 65% in P, 12% in S, 22% in Mn, 67% in Zn, 25% in Fe and 29% in Cu as than that of their respective controls. Cd alone at different levels gradually decreased the concentrations of macro- and micro-nutrients in roots of B. napus plants except Fe which was increased. Moreover, ALA under different Cd levels did not increase the concentrations of macro-nutrients. Whereas, ALA at 25 mg/L increased the concentration of Mn by 28% and 43%, Zn by 26% and 20%, Fe by 62% and 32%, Cu by 41% and 71% in roots under 100 and 500 uM Cd stress respectively, as compared to their respective controls.

Table 1: Effects of different treatments of 5-aminolevulinic acid (ALA) (mg/L) and cadmium (Cd) (uM) on the concentrations of Na+, K+, Ca2+, Mg2+ and Na+/K+ and Ca2+/Mg2+ ratios in leaves and roots of B. napus

ALA###Cd###Leaf (mg g-1 DW)###Root (mg g-1 DW)

conc. conc.###Na+###K+###Ca2+###Mg2+###Na+ /K+ Ca2+/Mg2+###Na+###K+###Ca2+###Mg2+###Na+/K+###Ca2+/Mg2+

###ratio###ratio###ratio###ratio

0###0###1.03 g 55.07 b###29.63 ab###4.95cd###0.02 e 5.97 ab###1.05 f###38.63 b###6.66 cd###2.07 bc###0.03 de###3.21 b

###100###1.79 b 39.89 e###29.16 ab###4.56 d###0.04 c 6.37 ab###1.48 c###35.44 bc###6.99 cd###2.03 bc###0.04 b###3.45 b

###500###2.34 a 26.12 g###17.61 d###2.65 f###0.09 a 6.70 a###2.04 a###30.41 c###4.74 f###0.96 e###0.07 a###4.95 a

12.5 0###1.13 f 53.95 b###28.97 ab###4.76cd###0.02 e 6.07 ab###1.21 de 40.69 b###6.56 d###1.98 bc###0.02 de###3.34 b

###100###1.62 c 47.02cd###31.29 ab###5.10 c###0.03 d 6.10 ab###1.34 cd 35.79 bc###7.66 b###2.33 b###0.04 bc###3.30 b

###500###1.83 b 32.99 f###19.45 cd###3.53 e###0.06 b 5.54 ab###1.76 b###29.57 c###5.20 f###1.53 d###0.06 a###3.54 b

25###0###1.27 e 61.70 a###31.86 ab###6.35 a###0.02 e 5.01 ab###1.07 ef 47.66 a###7.13 c###2.20 bc###0.02 e###3.25 b

###100###1.35 e 51.46bc###34.82 a###5.88 b###0.03 e 5.96 b###1.27 d###40.22 b###8.80 a###2.75 a###0.03 cd###3.21 b

###500###1.52 d 42.28de###25.45 bc###4.77cd###0.04 d 5.33 ab###1.46 c###35.60 bc###5.88 e###1.83 cd###0.04 b###3.21 b

Table 2: Effects of different treatments of 5-aminolevulinic acid (ALA) (mg/L) and cadmium (Cd) (uM) on the concentrations of macronutrients (mg g-1 DW) and micronutrients (ug g-1 DW) contents in leaves and roots of B. napus

ALA conc. Cd conc.###Leaf###Root

###Na###Pa###Sa###Mnb###Znb###Feb###Cub###Na###Pa###Sa###Mnb###Znb###Feb###Cub

0###0###48.90 c###12.40 ab 4.46 a###0.06 cd###0.11 de 0.32 f 4.64 c###29.49 ab###4.25 b 3.38 a###0.36 c 0.24 bc 0.57 f 26.53 ab

###100###45.42 cd###9.38 bcd 3.51 ab###0.06 d###0.15 c 0.37 cd 3.65 e###21.46 cde 3.44 bc 2.65 ab 0.25 f 0.23 c###0.60 f 17.42 d

###500###34.58 e###6.39 d###2.82 b###0.04 f###0.09 f 0.32 f 2.62 h###16.66 e###2.67 c 2.23 b###0.16 h 0.15 f###0.99 c 11.55 e

12.5###0###52.63 bc 13.64 ab 4.45 a###0.06 d###0.10 ef 0.35 e 4.81 b###27.63 abc 4.43 b 3.33 a###0.37 b 0.20 d###0.87 d 24.67abc

###100###47.59 c###11.41abc 3.62 ab###0.06 c###0.17 b 0.39 b 4.18 d###23.72bcde 3.84 bc 2.69 ab 0.28 e 0.26 b###0.71 e 21.44bcd

###500###38.68 de###8.05 cd 2.96 b###0.04 f###0.12 d 0.35 de 3.11 g###19.57 de###3.28 bc 2.16 b###0.18 g 0.17 e###1.16 b 15.90 de

25###0###62.66 a###15.56 a###4.48 a###0.08 a###0.14 c 0.38 c 5.55 a###30.94 a###6.73 a 3.27 a###0.41 a 0.24 bc 0.87 d 27.77 a

###100###57.68 ab 13.48 ab 3.74 ab###0.07 b###0.21 a 0.42 a 4.58 c###26.64abcd 4.35 b 2.75 ab 0.32 d 0.29 a###0.97 c 24.63abc

###500###46.75 c###10.57bcd 3.17 b###0.04 e###0.15 c 0.40 b 3.37 f###21.72 cde 3.54 bc 2.30 b###0.23 f 0.18 de 1.31 a 19.72 cd

ALA Reduces Cd-induced MDA and Various ROS

The contents of MDA, H2O2, O2 and OH in the leaves of B. napus under different concentrations of Cd and ALA have been shown in Fig. 2. Figure illustrated that higher concentration of Cd (500 uM) alone significantly increased the MDA contents (78%) than that of control. Exogenous ALA at 25 mg/L decreased the MDA contents by (28% and 34%) under 100 and 500 uM Cd stress, respectively.

Moreover, the higher level of Cd (500 uM) alone elevated the concentration of ROS contents in the leaves. Lower level of ALA (12.5 mg/L) showed no significant effect on H2O2 contents; however, it greatly decreased the level of O - and -OH under Cd stress. Higher level of ALA (25 mg/L) significantly reduced the H2O2 content by 18 and 22%, O - content by 36% and 32% and -OH content by 77 and 55% under 100 and 500 uM Cd stress, respectively as compared to their respective controls.

ALA Alleviates Non-enzymatic Antioxidants and Soluble Protein Contents

Results showed that different levels of Cd (100 and 500 uM) alone did not show any significant changes in GSH (Fig. 3a) and GSSG (Fig. 3b) contents in B. napus leaves. However, ALA alone increased GSH and GSSG contents significantly as compared to control. This increase was (31% and 77%) in GSH contents and (by 81% and 53%) in GSSG contents under 12.5 and 25 mg/L ALA, respectively as compared to control. The GSH+GSSG contents were also increased significantly with different concentrations of ALA alone (Fig. 3c), and maximum total glutathione contents were observed at 25 mg/L ALA alone. This increase in GSH+GSSG contents was 76% more as compared to control. However, Cd alone with different concentrations showed no significant change in total glutathione contents. However, when exogenous ALA was applied to Cd-stressed plants, GSH+GSSG contents were increased significantly.

The present study showed that GSH/GSSG ratio exhibited no change under various concentrations of Cd (Fig. 3d). The higher level of ALA alone enhanced the ratio of GSH/GSSG and showed maximum GSH/GSSG ratio (1225.39) in the leaves of B. napus.

Results described that lower level of Cd (100 uM) alone decreased the AsA contents (Fig. 3e) significantly, but higher level of Cd (500 uM) increased AsA contents in the leaves. This increase was 12% more as compared to untreated plants. ALA alone also improved the AsA contents by 61% and 91%, under 12.5 and 25 mg/L ALA treatment respectively. The Cd-treated plants sprayed with ALA showed significant increase in AsA contents. The AsA contents were increased by 96% and 115% with application of ALA at 25 mg/L under 100 and 500 uM Cd stress, respectively. TSP contents showed no significant change under ALA alone or Cd alone conditions (Fig. 3f). However, TSP contents were increased under the combine treatment of ALA and Cd at different concentrations. Maximum TSP contents were observed with the application of 25 mg/L ALA under the higher level of Cd (500 uM), and this increase was 39% more as compared to 500 uM Cd treatment alone (Fig. 3f).

Discussion

Metal toxicity is a complex phenomenon which shows negative effects on physiology of plants through element uptake and oxidative stress. In our previous study, we evaluated that exogenous application of ALA under Cd- toxicity promoted the plant growth by improving root morphology, antioxidant enzyme activities and ultrastructural changes in B. napus (Ali et al., 2013b). To understand how ALA ameliorates the Cd-induced plant growth by regulating elements uptake and oxidative stress in the leaves of B. napus, the current study was conducted. In the present study, plant growth parameter like plant biomass (% of control) significantly increased, when ALA was applied to Cd-stressed plants (Fig. 1). This is possibly because of the potential role of ALA to improve tolerance by decreasing lipid peroxidation through activation of antioxidant systems (Nishihara et al., 2003; Youssef and Awad, 2008).

It has been shown that ALA improves the plant growth by decreasing the effect of herbicide and salinity stress in oilseed rape and cotton respectively (Watanabe et al. 2000; Zhang et al. 2008). It was found that chlorophyll contents were reduced under the Cd stress alone, which might be due to destruction of photosynthetic apparatus and protein complex (Vassilev et al., 1995; Ali et al., 2013a), and inhibition of photosynthetic electron transport chain (Mohanty et al., 1989). It was also observed that ALA alone at 25 mg/L significantly improved the chlorophyll pigments in the leaves of B. napus under Cd-toxicity (Fig. 1). ALA improves chlorophyll biosynthesis because it is a key precursor of biomolecules (Hotta et al., 1997a, b; Naeem et al., 2010).

Cd at different levels decreases the concentration of elements in plants (Larbi et al., 2002; Ramos et al., 2002). In the present investigation, effects of ALA on Na+/K+ and Ca2+/Mg2+ ratios under Cd stress were measured. Recently, it is stated that enhanced ratios of Na+/K+ and Ca2+/Mg2+ can affect the plant growth (Naeem et al., 2012). Results showed that these ratios were increased as Cd level increased in the solution; however, at the same time, application of ALA minimized these ratios under Cd- toxicity (Table 1). Previously, Watanabe et al. (2000) also concluded that ALA at different concentrations notably reduced the Na+/K+ ratio in roots and leaves of cotton seedlings under salinity stress. Moreover, the reduction in K+ uptake in leaves and roots under Cd stress might be due to antagonistic effect of Cd (Murphy et al., 1999). This study also revealed that nutrient concentrations in roots and leaves were reduced under Cd-toxicity.

Our observations about reduction of nutrients under Cd stress are in line with Ouariti et al. (1997) and Lagriffoul et al. (1998), who stated that toxicity of heavy metals, can reduce the uptake of nutrients. Moreover, in the previous study we found that Cd stress alone increased the Cd concentration in shoots and roots of B. napus plants, thus, might be Cd reduced the nutrients concentrations due to antagonistic effect in the present study (Ali et al., 2013a). It was found that exogenously applied ALA mitigated the Cd stress and enhanced the capacity of plants to store essential nutrients (Table 2). It may be due to the fact that ALA has a promotive role in regulating a number of metabolic processes, thereby, improving nutrient uptake under abiotic stress (Zhang et al., 2008). Previously, it was found that ALA increased the uptake of different elements in the leaves of B. napus under salinity stress (Naeem et al., 2010, 2012).

Previously, Ahmad et al. (2011) applied the salicylic acid under Cd stress and noted that salicylic acid can improve the nutrient uptake in mustard plants.

Cd stress alone increased the production of MDA in the leaves of B. napus, however exogenously applied ALA significantly decreased the MDA contents under Cd stress (Fig. 2). Previously, Naeem et al. (2011) also found that exogenous ALA decreased the MDA production in the leaves of B. napus under salinity stress. Similar to these findings, in previous study, we found that ALA suppressed the MDA contents under Cd stress in B. napus roots (Ali et al., 2013b). Moreover, ROS contents were increased under the Cd-toxicity alone conditions (Fig. 2). ROS production in plants at sub-cellular level under Cd stress was also demonstrated previously (Rodriguez-Serrano et al., 2006; Ortega-Villasante et al., 2007). Cd enhanced the level of ROS, whereas, exogenous ALA helped the plants to remove ROS by activating the activities of antioxidant enzymes as found in spinach (Nishihara et al., 2003).

GSH and some emerging antioxidants like carbon monoxide and proline are considered as non-enzymatic antioxidant systems (Zhang and Chen, 2011) and play a crucial role in response to heavy metal stress. The present results showed that GSH contents were increased with the higher concentration of ALA (25 mg/L) alone (Fig. 3a). Moreover, findings of Nishihara et al. (2003) are agreement of our results who stated that ALA increases the GSH/GSSG ratio in spinach leaves under NaCl stress. In the present research, contents of AsA were enhanced under the higher Cd concentration (500 uM). Previously, Zengin and Munzuroglu (2005) also found similar results in Phaseolus vulgaris under metal stress. However, ALA showed a synergetic effect and enhanced the AsA contents in the leaves of B. napus under Cd stress (Fig. 3). Similar to these findings, recently, we also found that exogenously applied ALA ameliorated the Cd stress and improved the AsA contents in the roots of B. napus (Ali et al., 2013b).

As conclusion, it is found that Cd-toxicity can reduce the concentration of nutrients in the leaves and roots of B. napus. Moreover, it was also observed that ROS contents were enhanced in the leaves of B. napus under Cd-stressed alone conditions. Meanwhile, exogenously applied ALA reduced the ROS production by promoting non-antioxidant activities in the leaves of B. napus under Cd stress. Thus, it can be concluded that ALA has ameliorative role on B. napus under Cd-toxicity.

Acknowledgements

Authors would like to appreciate the Jiangsu Collaborative Innovation Center for Modern Crop Production, Zhejiang Provincial Top Key Discipline of Biology and its Open Foundation (2014C03, 2015D24), the Science and Technology Department of Zhejiang Province (2012C12902-1, 2011R50026-25), Zhejiang Provincial Education Department (Y201533983), and the Science Foundation of Zhejiang Sci-Tech University (14042216-Y) to provide the financial support for this study.

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Author:Ling Xu; Ali, Basharat; Gill, Rafaqat A.; Lan Li; Weijun Zhou
Publication:International Journal of Agriculture and Biology
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Date:Jun 30, 2016
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