Hydrogen sulfide alleviates cadmium-induced morpho-physiological and ultrastructural changes in Brassica napus

Ecotoxicology and Environmental Safety 110 (2014) 197–207

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Hydrogen sulfide alleviates cadmium-induced morpho-physiological and ultrastructural changes in Brassica napus Basharat Ali a, Rafaqat A. Gill a, Su Yang a, Muhammad B. Gill a, Shafaqat Ali b, Muhammad T. Rafiq c, Weijun Zhou a,n a

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 c Department of Environmental Sciences, International Islamic University, Islamabad 44000, Pakistan b

art ic l e i nf o

a b s t r a c t

Article history: Received 11 May 2014 Received in revised form 20 August 2014 Accepted 21 August 2014

In the present study, role of hydrogen sulfide (H2S) in alleviating cadmium (Cd) induced stress in oilseed rape (Brassica napus L.) was studied under greenhouse conditions. Plants were grown hydroponically under three levels (0, 100, and 500 mM) of Cd and three levels (0, 100 and 200 mM) of H2S donor, sodium hydrosulfide (NaHS). Results showed that application of H2S significantly improved the plant growth, root morphology, chlorophyll contents, elements uptake and photosynthetic activity in B. napus plants under Cd stress. Moreover, addition of H2S reduced the Cd concentration in the leaves and roots of B. napus plants under Cd-toxicity. Exogenously applied H2S decreased the production of malondialdehyde and reactive oxygen species in the leaves and roots by improving the enzymatic antioxidant activities under Cd stress conditions. The microscopic examination indicated that application of exogenous H2S improved the cell structures and enabled a clean mesophyll cell having a well developed chloroplast with thylakoid membranes, and a number of mitochondria could be observed in the micrographs. A number of modifications could be found in root tip cell i.e. mature mitochondria, long endoplasmic reticulum and golgibodies under combined application of H2S and Cd. On the basis of these findings, it can be concluded that application of exogenous H2S has a protective role on plant growth, photosynthetic parameters, elements uptake, antioxidants enzyme activities and ultrastructural changes in B. napus under high Cd stress conditions. & 2014 Elsevier Inc. All rights reserved.

Keywords: Plant growth Nutrients uptake Cadmium Microscopic ultrastructures Hydrogen sulfide Oilseed rape

1. Introduction Oilseed rape (Brassica napus L.) is rapidly expanding as a rotation crop following rice worldwide. Nowadays, it is considered as one of major oilseed crops, cultivated all over the world for the production of edible oil (Zhou, 2001; Momoh et al., 2002). Commonly, plants of genus Brassica are recognized as metals tolerant due to their rapid growth, higher biomass production and capability to absorb metals (Momoh and Zhou, 2001). It is reported that cadmium (Cd), mercury (Hg) and lead (Pb) are one of the major environmental toxicants (Chen et al., 2006). Cd has become a serious environmental threat due to the wide range application of pesticides, herbicides, fertilizers and expansion of industrialization (Folgar et al., 2009). It interferes with many metabolic processes, and reduced the water and nutrients uptake in plants (Najeeb et al., 2011). Recently, it was found that higher Cd levels resulted in plant growth retardation, yellowing of leaves and

n

Corresponding author. Fax: þ 86 571 88981152. E-mail address: [email protected] (W. Zhou).

http://dx.doi.org/10.1016/j.ecoenv.2014.08.027 0147-6513/& 2014 Elsevier Inc. All rights reserved.

disruption of chloroplasts ultrastructures in B. napus (Ali et al., 2013a, 2013b). Cd-toxicity has been found to affect the photosynthetic pigments, and consequently reduction in use of carbon and respiration process occurs in plants (Ali et al., 2013a). The toxicity of heavy metals causes effect on root surface area that reduces water absorption by preventing the growth of root hairs (Gouia et al., 2000). Uptake and distribution of macro- and micronutrients in plants are also disturbed by Cd (Ramos et al., 2002). Cd can cause oxidative stress in the plant cells by production of different reactive oxygen species (ROS) i.e. superoxide radicals (O∙2ˉ, hydrogen peroxide (H2O2) and hydroxyl radicals (OH∙) (Hendry et al., 1992). Absence of effective mechanisms which scavenge or remove these ROS, can cause degradation of protein, DNA breakage and cell death by lipid peroxidation (Thian and Li, 2006). In the plants, there are a number of antioxidant systems which protect the plants from oxidative damage, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), as well as glutathione reductase (GR) and ascorbate peroxidase (APX) (Kanazawa et al., 2000). It was reported that activities of antioxidant enzymes such as APX, CAT, GR and SOD were increased to scavenge ROS in rice

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(Oryza sativa L.) under abiotic stress conditions (Farooq et al., 2008). Phytoremediation is an environmental friendly and economical remediation technique for the control of metal contamination (McGrath et al., 2002). The combination of high biomass producing plant species and chemically supported phytoremediation would be useful against this constraint (Evangelou et al., 2004). Role of hydrogen sulfide (H2S) in reducing Cd-toxicity through chemically has been established formerly in Alfalfa (Li et al., 2012). In plants, H2S has been implicated in a variety of developmental processes, such as adventitious root and lateral root formation (Zhang et al., 2009). It is suggested that H2S plays a crucial role in alleviating the negative effect of aluminum (Al) stress in wheat and barley respectively (Zhang et al., 2008a; Dawood et al., 2012). H2S is proposed to be utilized against the toxicity of Al and boron (Zhang et al., 2010; Wang et al., 2010). Even though, the effects of H2S on stress physiology have been acknowledged much attention and in recent times, there are a number of reports providing evidence that H2S enhances the plant tolerance against the heavy metals stress in different crops (Wang et al., 2010; Zhang et al., 2010; Ali et al., 2014a). Keeping in view the importance of rapeseed and alleviating role of H2S under anoxic conditions, the present study was conducted to test the hypothesis that H2S has the ameliorating role on plant growth, photosynthetic gas exchange capacity, elements uptake, antioxidants machinery and ultrastructural changes under Cd stress in B. napus plants.

2. Materials and methods 2.1. Plant material and growth conditions For the experiment, seeds of B. napus L. cv. ZS 758 were taken from the College of Agriculture and Biotechnology, Zhejiang University. The seeds were grown in plastic pots (170 mm  220 mm) filled with peat moss. At five leaf stage, seedlings with uniform size were selected and transferred into plate holes on plastic pots (six plants per pot) containing a half strength Hoagland nutrient solution (Arnon and Hoagland, 1940). The composition of Hoagland nutrient solution was as follows (in mmol l  1): 3000 KNO3, 2000 Ca(NO3)2  4H2O, 1000 MgSO4  7H2O, 10 KH2PO4, 12 FeC6H6O7, 500 H3BO3, 800 ZnSO4  7H2O, 50 Mncl2, 300 CuSO4  5H2O, and 100 Na2MoO4. The pH of solution was maintained at 5.5 with 1 M of NaOH or HCl. Aeration was given continuously through air pump in the nutrient medium. Nutrient solution was replaced after every four days. The light intensity was in the range of 250–350 mmol m  2 s  1, temperature was 16–20 1C and the relative humidity was approximately 55–60 percent. After acclimatization period of 7 days, Cd as CdCl2 and H2S as NaHS were added into full strength Hoagland solution making 9 treatments: (1) control (Ck), (2) 100 mM CdCl2 alone, (3) 500 mM CdCl2 alone, (4) 100 mM NaHS alone, (5) 200 mM NaHS alone, (6) 100 mM CdCl2 þ 100 mM NaHS, (7) 500 mM CdCl2 þ100 mM NaHS, (8) 100 mM CdCl2 þ200 mM NaHS, and (9) 500 mM CdCl2 þ200 mM NaHS. The treatment concentrations were based on preexperimental studies, in which a number of lower and higher levels of Cd were applied, i.e., 100, 200, 300, 400, 500, 600 and 1000 mM of Cd. The NaHS was purchased from Sigma (St Louis, MO, USA) and used as the exogenous H2S donor as described previously by Chen et al. (2011). They applied a series of sulphur- and sodium-containing chemicals including NaHS, Na2S, Na2SO4, Na2SO3, NaHSO4, NaHSO3, and NaAC on Spinacia oleracea seedlings and found that H2S rather than other sulphur-containing compounds or sodium, was responsible for the increase in plant growth in S. oleracea after NaHS treatment. Moreover, it was also found that H2S contents generated from NaHS were the highest among other chemicals and previously, NaHS was also used as H2S donor under Cd and B stress respectively (Chen et al., 2011; Li et al., 2012; Wang et al., 2010). After fifteen days of treatment, all morphological data and photosynthetic parameters were measured. Samples for elemental analysis, biochemical and microscopic studies of leaves and roots were collected as described below.

automatism scan apparatus (MIN MAC, STD1600 þ ), equipped with WinRHIZO software offered by Regent Instruments Co. Photosynthetic gas exchange parameters were analyzed by LiCor-6400 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA). Topmost fully expanded leaf after 2 h of acclimatization in a growth cabinet, at a temperature of 18 1C under a light intensity of 1000 mmol m  2 s  1 and relative humidity of 60 percent, was sampled, and photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate were measured. The total eight readings per treatment were taken from randomly selected plants (Zhou and Leul, 1998). Chlorophyll content was determined according to the method of Pei et al. (2010). 2.3. Elemental analysis After 15 days of treatment, plants were harvested from each treatment, washed with tap water and distilled water three times, respectively, and then blotted to remove the excessive water. Thereafter, plants were separated into leaves and roots (topmost fully expanded leaf), dried at 80 1C in an oven for 48 h, and ground into powder. Plant samples of leaves and roots (0.2 g) were digested with a mix of 5 ml of HNO3 þ 1 ml of HClO4. After that the resultant solutions were diluted to 25 ml using 2 percent HNO3 and filtered. The concentrations of Na, K, Ca, Mg, Mn, Fe, Cu and Zn in the filtrate were determined using inductively coupled plasma–mass spectrometry (ICP–MS, Agilent, 7500a) following a standard procedure. The nitrogen contents were determined according to the method of Lindner (1944). Plant dried samples (leaves and roots) were digested with H2SO4–H2O2 to obtain a clear solution. To the digested material, 2.5 N NaOH, 1 percent sodium silicate and Nessler’s reagent was added. The absorbance of solution was read at 525 nm, using the spectrophotometer. Phosphorus contents were estimated according to the method of Murphy and Riley (1962) by using molybdenum blue method and absorbance was read at 660 nm in a spectrophotometer. Sulphur contents were measured by Butters and Chenery (1959) method using barium sulphate and absorbance was taken at 440 nm with a spectrophotometer. For determination of Cd contents in shoots and roots, samples were dried at 65 1C for 24 h, and then ashed in Muffle furnace at 550 1C for 20 h. After that ash was incubated with 31 percent HNO3 and 17.5 percent H2O2 at 70 1C for about 2 h, and dissolved in distilled water. Cd concentration in the digest was investigated using an atomic absorption spectrophotometer (AA-6800, Shimadzu Co. Ltd., Japan). 2.4. Analysis of lipid peroxidation and reactive oxygen species The oxidative damage to lipids was determined according to Zhou and Leul (1998) as lipid peroxidation in terms of malondialdehyde (MDA) production. Fresh samples (0.2 g) were homogenized and extracted in 10 ml of 0.25 percent TBA made in 10 percent trichloroacetic acid (TCA) then extract was heated at 95 1C for 30 min and cooled on ice. The samples were centrifuged at 5000g for 10 min. The absorbance was measured at 532 nm. Correction of non-specific turbidity was made by subtracting the absorbance value taken at 600 nm. The level of MDA was expressed as μmol g  1 fresh weight by using extinction coefficient of 155 mM cm  1. For determination of hydrogen peroxide (H2O2) contents, samples (0.1–0.4 g) were extracted with 5.0 ml of TCA (0.1 percent, w/v) in an ice bath, and the homogenate was centrifuged at 12,000g for 15 min (Velikova et al., 2000). In 0.5 ml of the supernatant, 0.5 ml of phosphate buffer (pH 7.0) and 1.0 ml of potassium iodide (1 M) were added. The absorbance of the mixture was measured at 390 nm. H2O2 contents were determined using extinction coefficient of 0.28 μM cm  1 and expressed as μmol g  1 DW. Superoxide radical (O∙ 2 ) was determined according to Jiang and Zhang (2001) method with some modifications. The fresh samples (0.3–0.5 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 1C. After that the supernatant (1 ml) was added with 0.9 ml of 65 mM potassium phosphate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine hydrochloride, and then incubated it at 25 1C for 24 h. After incubation, 1 ml of 17 mM sulphanilamide and 1 ml of 7 mM a-naphthylamine was mixed in 1 ml solution for further 20 min at 25 1C. After that, 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. Standard curve was used to calculate the generation rate of O2 . For estimation of extra-cellular hydroxyl radicals (OH∙), 0.1 g fresh samples were incubated in 1 ml of 10 mM Na-phosphate buffer (pH 7.4) consisting 15 mM 2-deoxy-Dribose (SRL, Mumbai) at 37 1C 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 percent (w/v) thiobirbuteric acid (TBA, Hi Media, Mumbai, 1 percent stock solution made in 5 mM NaOH) and 1 ml glacial acetic acid, heated at 100 1C in a water bath for 30 min and cooled down to 41 1C for 10 min before measurement. The absorbance was measured at 532 nm and concentration was calculated using an extinction coefficient (ε ¼ 155 mM cm  1) and expressed in μmol g  1 FW.

2.2. Morphological parameters 2.5. Determination of antioxidant enzyme activities After 15 days of treatment, plants were harvested and separated into leaf, stem and root. The plant height, stem and root length of six randomly selected plants per treatment were measured. The root surface area, volume, diameter and number of root tips of six randomly selected plants per treatment were determined using root

For enzyme activities, samples (0.5–0.6 g) were 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 1C and the supernatant was used for the

B. Ali et al. / Ecotoxicology and Environmental Safety 110 (2014) 197–207 determination of the following enzyme activities. 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 EDTA-Na2, 0.3 mM ascorbic acid, 0.06 mM H2O2 and 100 μl 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 coefficient 39.4 mM cm  1) 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 μl 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 cm  1) 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 GSSG and 100 μl 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. (2008b) 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 μΜ NBT, 2 μΜ riboflavin, 0.1 mM EDTA and 100 μl of enzyme extract in a 3-ml volume. One unit of SOD activity was measured as the amount of enzyme required to cause 50 percent 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 percent guaiacol, 0.4 percent H2O2 and 100 μl enzyme extract. Variation due to guaiacol in absorbance was measured at 470 nm. 2.6. Transmission electron microscopy After 15 days 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 4 percent glutaraldehyde (v/v) in 0.1 M PBS (Sodium Phosphate Buffer, pH 7.4) and cleansed three times with the same PBS. Samples were postfixed in 1 percent OsO4 (osmium (VIII) oxide) for 1 h, washed three times in 0.1 M PBS (pH 7.4) with 10 min intermission between each washing. After interval for 15–20 min, the samples were dried in a graded series of ethanol (50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, and 100 percent) 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 1C for 9 h, ultra-thin sections (80 nm) of specimens were prepared and mounted on copper grids for viewing by a transmission electron microscope (JEOL TEM-1230EX, Japan) at an accelerating voltage of 60.0 kV. 2.7. 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 are the means7S.D of at least three independent replicates. The mean differences were compared utilizing the Duncan’s multiple range test by using a statistical package, SPSS version 16.0 (SPSS, Chicago, IL, USA).

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was increased in the solution culture. The higher concentration of Cd (500 μM) alone caused reduction in root diameter by 23 percent, in root surface area by 27 percent, in root volume by 37 percent and in number of root tips by 34 percent as compared to control plants. The application of NaHS at different concentrations (100 and 200 mM) significantly improved all the root morphological parameters under Cd stress conditions. However, different levels of NaHS alone exhibited no change in root morphology as compared to control plants (Table 2S).

3.2. H2S alleviates Cd-induced inhibition of photosynthetic parameters The data regarding chlorophyll a, chlorophyll b, total chlorophyll and carotenoid contents under different treatments of H2S donor NaHS and Cd have been shown in Table 3S. Data exhibited that after 15 days of treatments, higher concentration of Cd (500 mM) single-handedly caused reduction in chlorophyll a by 48 percent, in chlorophyll b by 51 percent, in total chlorophyll by 49 percent and in carotenoid contents by 30 percent as compared to their respective controls. Exogenous application of H2S significantly enhanced the chlorophyll and carotenoid contents under the different Cd concentrations. Moreover, application of H2S donor NaHS alone at different concentrations did not show any significant affect on chlorophyll contents, as compared to control plants. The data regarding the effect of different treatments of H2S donor NaHS and Cd on photosynthetic parameters i.e. net photosynthetic rate, stomatal conductance, internal CO2 concentration, and transpiration rate have been presented in Fig. 1S. Results showed that Cd stress alone at both concentrations (100 and 500 mM) dramatically suppressed the photosynthetic parameters as compared to control plants. Higher concentration of Cd (500 mM) alone displayed minimum values of net photosynthetic rate, stomatal conductance, internal CO2 concentration and transpiration rate respectively, as compared to control. However, application of NaHS at different concentrations improved the photosynthetic parameters under Cd stress conditions. Thus, these results suggested that application of H2S donor NaHS alleviated Cd-induced photosynthetic changes in B. napus plants (Fig. 1S).

3.3. H2S alleviates the ion uptake in B. napus under Cd stress 3. Results 3.1. Effects of H2S and Cd stress on plant morphology The data regarding plant growth in term of plant height, stem length, root length and number of leaves per plant under different concentrations of H2S donor NaHS and Cd are shown in Table 1S. Results showed that Cd stress at higher concentration (500 mM) caused reduction in plant height by 29 percent, in stem length by 36 percent, in root length by 32 percent and in number of leaves by 26 percent as compared to control. However, when plants were treated with different concentrations of H2S, it eliminated the negative effect of Cd, thus, the plant morphology was improved. The higher NaHS concentration (200 mM) improved the plant length by 23 percent, stem length by 39 percent, root length by 32 percent and number of leaves by 33 percent as compared to their respective controls. However, application of NaHS alone at 100 and 200 mM concentrations did not show any change in plant morphology with respect to control. The effects of different treatments of H2S donor NaHS and Cd on root morphology of B. napus have been displayed in Table 2S. The results demonstrated that root morphology significantly reduced, as Cd concentration

The uptake of Na þ , K þ , Ca2 þ , Mg2 þ and their ratios Na þ /K þ and Ca2 þ / Mg2 þ in the leaves and roots of B. napus plants under different treatments of H2S donor NaHS and Cd concentrations is demonstrated in Table 1. Results showed that uptake of Na þ increased linearly as Cd levels increased and this uptake was found higher in upper parts of plants compared to the lower parts. However, addition of NaHS at different concentrations markedly lowered Na þ uptake in different plant parts under Cd stress. Application of NaHS at 200 mM decreased the Na þ concentration in leaf by (31 percent and 26 percent) and in roots by (30 percent and 21 percent) under 100 and 500 μM Cd respectively, as compared to their respective controls. Moreover, application of NaHS at different concentrations (100 and 200 μM) remarkably enhanced the uptake of K þ , Ca2 þ and Mg2 þ in the leaves and roots of B. napus under Cd-toxicity. The application of NaHS at 200 μM improved the concentration of K þ by (29 percent and 44 percent), Ca2 þ by (52 percent and 57 percent) and Mg2 þ by (28 percent and 70 percent) in the leaves and roots respectively, under the higher concentration of Cd (500 μM). Data also depicted that exogenous application of H2S reduce the Na þ /K þ ratio and increased the Ca2 þ /Mg2 þ ratio in the leaves and roots of B. napus under Cd-toxicity.

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Table 1 Effects of different treatments of H2S-donor NaHS (μM) and cadmium (Cd) (μM) on Na þ , K þ , Ca2 þ , Mg2 þ contents and Na þ /K þ and Ca2 þ /Mg2 þ ratios in leaves and roots of Brassica napus cv. ZS 758. NAHS conc.

0

100

200

Root (mg g  1 DW) 0

100

200

Cd conc.

Leaf (mg g  1 DW) Na þ

Kþ

Ca2 þ

Mg2 þ

Na þ /K þ ratio

Ca2 þ /Mg2 þ ratio

0 100 500 0 100 500 0 100 500

4.517 0.14 ef 7.28 7 0.36 c 10.38 7 0.63 a 4.81 7 0.22 e 6.197 0.33 d 9.127 0.27 b 4.117 0.27 f 5.09 7 0.22 e 7.747 0.39 c

62.3 7 2.66 a 43.6 7 1.45 d 28.5 7 1.65 f 60.4 7 2.70 a 47.6 71.73 c 31.5 71.68 f 63.4 7 2.51 a 53.3 7 2.05 b 36.7 7 1.60 e

25.5 7 1.07 a 18.4 7 0.86 d 11.4 7 0.60 f 23.5 7 1.20 b 21.4 71.14 c 14.5 7 0.80 e 25.6 7 1.22 a 23.5 7 1.09 b 17.3 70.48 d

8.54 7 0.28 a 6.3570.26 d 4.017 0.30 g 8.80 7 0.22 a 7.247 0.20 c 4.737 0.19 f 8.117 0.16 b 7.53 7 0.23 c 5.157 0.27 e

0.077 0.004 f 0.16 70.013 d 0.36 7 0.041 a 0.077 0.007 f 0.13 70.010 e 0.29 7 0.024 b 0.06 70.006 f 0.09 7 0.002 f 0.21 70.018 c

2.98 7 0.03 bc 2.89 7 0.01c 2.85 7 0.02cd 2.6770.07d 2.95 7 0.08 bc 3.05 7 0.06 bc 3.157 0.09 b 3.117 0.05 b 3.36 7 0.03 a

0 100 500 0 100 500 0 100 500

1.767 0.15 ef 2.95 7 0.24 c 4.127 0.29 a 1.82 7 0.15 e 2.53 7 0.31 d 3.727 0.22 b 1.38 7 0.23 f 2.0770.18 e 3.28 7 0.23 c

33.67 1.06 a 22.5 7 0.86 e 15.7 7 1.01 f 34.6 7 1.05 a 24.67 1.08 d 16.4 7 0.85 f 31.5 71.01 b 27.5 7 1.05 c 22.5 7 1.02 c

8.417 0.33 b 5.45 7 0.28 e 3.077 0.16 h 8.96 7 0.34 a 6.30 7 0.41 d 3.99 7 0.37 g 8.737 0.19 ab 7.127 0.25 c 4.82 7 0.21 f

2.75 7 0.17 ab 1.95 7 0.16 d 0.917 0.10 f 2.38 7 0.24 c 2.45 7 0.21 bc 1.32 7 0.20 e 2.90 7 0.20 a 2.6770.19 abc 1.55 7 0.23 e

0.05 7 0.003 ef 0.13 70.008 c 0.26 7 0.033 a 0.05 7 0.003 ef 0.10 70.008 d 0.22 7 0.008 b 0.04 70.006 f 0.077 0.004 e 0.14 70.006 c

3.05 7 0.11 bcd 2.79 7 0.09 cde 3.377 0.35 b 3.7770.25 a 2.577 0.08 e 3.047 0.21 bcd 3.017 0.14 bcd 2.6770.13 de 3.147 0.37 bc

Values are mean7 S.D. (n¼3). Means followed by the same letter did not significantly differ at Pr 0.05 according to Duncan’s multiple range test.

3.4. H2S regulates the elements uptake in B. napus under Cd stress The effects of H2S donor NaHS on the uptake of macronutrients (N, P, S) and micronutrients (Mn, Zn, Fe, Cu) in the leaves and roots of B. napus under different concentrations of Cd are delineated in Table 2. Data displayed that higher concentration of Cd (500 μM) significantly reduced the uptake of macro- and micronutrients in the leaves and roots as compared to control plants. Meanwhile, when different levels of NaHS were applied to plants under Cd stress, the concentration of all the nutrients was improved. Results showed that higher level of NaHS (200 μM) improved the concentration of N by (21 percent and 20 percent), P by (51 percent and 39 percent), S by (65 percent and 41 percent), Mn by (10 percent and 9 percent), Zn by (44 percent and 33 percent), Fe by (17 percent and 10 percent) and Cu by (38 percent and 13 percent) in the leaves and roots respectively, under higher concentration of Cd (500 μM). Moreover, it was also found in the present study that Cd stress alone significantly elevated the Cd contents in the leaves and roots of B. napus plants (Table 2). However, application of H2S donor NaHS in Cd-treated plants significantly decreased the Cd contents in different plant parts (Table 2). 3.5. H2S alleviates Cd-induced MDA and ROS The data regarding malondialdehyde (MDA) contents, hydrogen peroxide (H2O2) contents, superoxide radical (O∙2 ) contents and hydroxyl ion (  OH) contents in the leaves and roots of B. napus under different treatments of NaHS and Cd have been presented in Table 4S. Application of H2S donor NaHS alone at different concentrations did not reduce the production of MDA and reactive oxygen species (ROS) in B. napus leaves and roots as compared to control. Data exhibited that higher concentration of Cd (500 μM) alone significantly increased MDA contents by (134 percent and 157 percent) in the leaves and roots respectively, as compared to control. Exogenous application of H2S significantly reduced the MDA contents in the leaves and roots of B. napus under different concentrations of Cd. The higher level of Cd alone significantly enhanced the levels of ROS (H2O2, O∙2  and  OH) contents in the leaves and roots of B. napus. However, application

of NaHS at 200 μM lowered the H2O2 content by (23 percent and 37 percent), O∙2  content by (18 percent and 34 percent) and –OH content by (25 percent and 30 percent) in the leaves and roots respectively, under 500 μM Cd concentration, than that of respective controls (Table 4S).

3.6. H2S regulates various antioxidants activities under Cd stress To observe the alleviating role of H2S on Cd stress tolerance in B. napus seedlings, the effect of H2S donor NaHS on enzyme activities (SOD, POD, APX, CAT, GR) in the leaves and roots under Cd stress were studied (Fig. 1). Results showed that activities of SOD and POD were increased in the leaves and roots under the lower of Cd (100 μM) alone as compared to control; however, their contents were declined under the higher concentration of Cd (500 μM). Exogenously applied H2S ameliorated the Cd stress and enhanced the activities of SOD (Fig. 1a and b) and POD (Fig. 1c and d) in the leaves and roots under different concentrations of Cd. Data showed that higher level of Cd alone significantly increased the activity of APX in the leaves and roots of plants (Fig. 1e and f), as compared to control. Exogenous application of H2S had a synergetic effect on APX activity and increased the APX activity in the leaves and roots under Cd stress. The activity of CAT decreased at lower level of Cd in the leaves and roots, however, it increased under higher level of Cd in leaves (Fig. 1g and h). Meanwhile, application of NaHS under different Cd concentrations showed an ameliorating effect and enhanced the CAT activity in the leaves and roots of B. napus under Cd stress. The activity of GR (Fig. 1i and j) increased significantly in the leaves and roots under higher concentration of Cd. However, addition of exogenous H2S decreased the toxic effect of Cd and improved the GR activity in the leaves and roots of B. napus. The application of NaHS at 200 μM increased the GR activity by (9 percent and 6 percent) in the leaves and roots respectively, under 500 μM Cd stress as compared to respective control. Moreover, results indicated that addition of NaHS alone did not affect significantly on antioxidants enzyme activities in the leaves and roots as compared to control plants (Fig. 1a–j).

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Table 2 Effects of different treatments of H2S-donor NaHS (μM) and cadmium (Cd) (μM) on macro- and micronutrients content and Cd contents in leaves and roots of Brassica napus cv. ZS 758. NAHS conc.

Cd conc.

Leaf (mg g  1 DW) Na

Pa

Sa

Mnb

Znb

Feb

Cub

Cda

0 100 500 0 100 500 0 100 500

54.5 7 1.77 a 42.5 7 1.87c 29.3 7 0.99 e 55.3 7 1.81 a 46.4 7 1.94 b 32.8 7 1.73 d 56.4 7 2.01 a 48.37 1.71 b 35.571.40 d

16.8 7 0.55a 11.4 7 0.94c 7.6 70.75d 18.4 7 1.68a 14.3 7 1.01b 9.7 7 1.04c 18.7 7 1.03a 17.4 70.96a 11.4 7 0.93c

6.25 70.29a 3.62 70.23e 1.96 7 0.21g 5.447 0.29b 4.09 70.20d 2.38 70.24f 5.59 70.25b 4.76 70.17c 3.23 70.19e

0.08 7 0.002c 0.09 7 0.003ab 0.077 0.002e 0.08 7 0.002c 0.09 7 0.003ab 0.077 0.002de 0.09 7 0.003b 0.107 0.004a 0.077 0.002d

0.167 0.01 bc 0.17 70.02abc 0.09 7 0.01 d 0.157 0.02 bc 0.197 0.02 ab 0.107 0.01 d 0.157 0.02 bc 0.217 0.03 a 0.137 0.01 cd

0.63 7 0.02 a 0.59 7 0.02 a 0.46 7 0.02 c 0.647 0.02 a 0.617 0.03 a 0.477 0.02 c 0.62 7 0.02 a 0.647 0.02 a 0.54 7 0.02 b

0.04 70.002 a 0.02 7 0.002 d 0.017 0.001 f 0.04 70.003 a 0.03 7 0.003 cd 0.017 0.001 ef 0.03 7 0.003 ab 0.03 7 0.002 bc 0.02 7 0.002 e

0.5 7 0.02 g 213.4 7 12.09 d 642.97 11.95 a 0.5 7 0.01 g 165.5 7 12.22 e 511.9 7 14.28 b 0.5 7 0.02 g 105.9 7 11.23 f 399.7 7 10.37 c

Root (mg g  1 DW) 0 0 100 500 100 0 100 500 200 0 100 500

45.3 7 1.79 a 39.4 7 1.42 c 24.571.01 e 45.4 7 1.68 a 41.3 7 1.19bc 25.4 7 1.03 e 47.4 7 1.73 a 43.9 7 2.29ab 29.6 7 2.51 d

5.58 7 0.38 b 4.22 7 0.27 d 2.777 0.17 f 6.10 70.15 a 4.777 0.17 c 3.32 7 0.28 e 6.29 7 0.32 a 5.317 0.25 b 3.86 7 0.18 d

4.52 70.22 a 3.22 70.19bc 2.08 70.20d 4.49 70.18 a 3.55 70.17 b 2.34 70.21 d 4.177 0.19 a 4.187 0.18 a 2.94 70.16 c

0.09 7 0.003de 0.11 70.004b 0.08 7 0.002f 0.09 7 0.003cd 0.11 70.004ab 0.08 7 0.003f 0.107 0.003c 0.127 0.005a 0.08 7 0.002ef

0.217 0.02 b 0.22 7 0.02 b 0.12 70.01 c 0.247 0.03 ab 0.25 7 0.02 ab 0.137 0.01 c 0.23 7 0.02 ab 0.27 70.03 a 0.167 0.01 c

2.167 0.05 f 2.53 7 0.03 d 3.137 0.04 b 2.40 7 0.04 e 2.58 7 0.03 d 3.187 0.04 b 2.3570.03 e 2.95 7 0.03 c 3.43 7 0.04 a

0.04 70.002 bc 0.04 70.003 ab 0.03 7 0.002 e 0.04 70.003 bcd 0.04 70.004 ab 0.03 7 0.002 de 0.04 7 0.003 bcd 0.05 7 0.004 a 0.03 7 0.003 cde

0.7 7 0.01 g 1302.9 7 21.70 d 3761.9 7 24.85 a 0.7 7 0.01 g 931.2 7 20.90 e 2335.5 727.99 b 0.7 7 0.01 g 631.5 7 11.44 f 1347.1 724.83 c

0

100

200

Values are mean7 S.D. (n¼3). Means followed by the same letter did not significantly differ at P r0.05 according to Duncan’s multiple range test. a b

mg g  1 DW. mg g  1 DW.

3.7. H2S alleviates Cd-induced ultrastructural changes in B. napus The ultrastructural transformations in leaf mesophyll and root tip cells under different treatments of H2S donor NaHS and Cd have been illustrated in Figs. 2 and 3. The TEM micrographs of leaf mesophyll cells of ZS 758 at control, 100 mM NaHS alone and 200 mM NaHS alone with low and high magnification are shown in Fig. 2A–F. At these three levels, all the organelles in the cells were noticeably differentiated and well developed. A well developed chloroplast having regular arrangements of thylakoid membranes could be observed. A number of mitochondria could be observed in the micrographs. The TEM micrographs of leaf mesophyll cells of ZS 758 at 500 mM Cd alone, 100 mM NaHS under 500 mM Cd and 200 mM NaHS under 500 mM Cd are displayed in Fig. 2G–L. At higher concentration of Cd (500 μM), a number of visible symptoms of Cd-toxicity were found in measopyll cells (Fig. 2G and H). There was a spongy chloroplast having dissolved thylakoid membranes. Starch grains were observed in the chloroplast under the Cd-toxicity. Meanwhile, changes in the fine structures of leaf mesophyll cells due to application of H2S donor NaHS under 500 μM Cd were improved (Fig. 2I–L). Chloroplast was clear having well developed and visible thylakoid membranes. Moreover, starch grains were vanished and a clear cell wall and cell membrane could be observed in the micrographs. The TEM micrographs of root tip cells of ZS 758 at control, 100 mM NaHS alone and 200 mM NaHS alone with low and high magnifications are demonstrated in Fig. 3A–F. At these three levels, there were clear cells having rich cytoplasm and organelles. A big nucleus could be noticed with well developed nuclear membrane and nucleolus. The cells also comprised of a number of mitochondria and golgibodies. The endoplasmic reticulum could also be found in the root tip micrographs under control and NaHS alone conditions (Fig. 3A–F. Ultrastructural studies of Cd-toxicity showed undesirable effects on the root tip cells of B. napus under 500 μM Cd alone conditions (Fig. 3G and H). The higher level of Cd (500 μM) totally damaged the root tip cell. Any organelles could not be found in the micrographs. Heavy metal deposition was located in the form of small granules in vacuole and around the

cell wall. The TEM micrographs of root tip cells at 100 and 200 mM NaHS under 500 μM Cd showed well developed organelles in the cell (Fig. 3I–L). There were well developed mitochondria and endoplasmic reticulum in the micrographs. Nucleus was detected with nucleolus and nuclear membrane. Thus, H2S donor NaHS improved the cell structure and presented the well developed organelles under Cd stress conditions.

4. Discussion Hydrogen sulfide (H2S) is an emerging signaling molecule that regulates a variety of physiological activities in plants (Garcia-Mata and Lamattina, 2010; Ali et al., 2014a). In the present study, an effort has made to find out, how the exogenous application of H2S alleviates Cd-induced effects in various parameters of oilseed rape i.e., plant growth, gas exchange capacity, nutrients uptake, antioxidants machinery and ultrastructural modifications. According to the findings, a significant reduction in plant growth was observed that might be due to the adverse effect of Cd on the plant roots and thus, plants could not absorb the nutrients or continue their normal activity. Previously, it was also found that Cd suppressed the plant growth by interfering in a range of metabolic processes, i.e., inhibition of proton pump, decline in root elongation, and damage to photosynthetic machinery (Najeeb et al., 2011). It is considered that slower growth and development are a consequence of osmotic stress (Shani and Ben-Gal, 2005), which can be formed due to heavy metal toxicity. Addition of H2S alleviated the plant growth under Cd stress conditions (Table 1S). These results are consistent with the findings of Zhang et al. (2009), who determined that H2S increased the plant growth by improving chlorophyll contents and embryo length in plants (Qian et al., 2014). Earlier, studies have been carried out on root morphology and focused on the relationship between root morphology and yield (Schwarz and Grosch, 2003). In the present investigation, Cdtoxicity significantly reduced the root morphology; however, application of H2S significantly improved the root morphology

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Fig. 1. Effects of different treatments of NaHS (μM) and cadmium (Cd) (μM) on activities of superoxide dismutase (SOD) in (a) leaves and (b) roots, guaiacol peroxidase (POD) in (c) leaves and (d) roots, ascorbate peroxidase (APX) in (e) leaves and (f) roots, catalase (CAT) activities in (g) leaves and (h) roots, and glutathione reductase (GR) activity in (i) leaves and (j) roots of Brassica napus cv. ZS 758. Values are mean7 S.D. (n¼ 3). Means followed by the same letter did not significantly differ at P r 0.05 according to Duncan’s multiple range test.

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(Ck)

203

(Ck)

(100 µM NaHS)

(100 µM NaHS)

(200 µM NaHS )

(200 µM NaHS)

(500 µM Cd )

(500 µM Cd)

(100 µM NaHS + 500 µM Cd)

(100 µM NaHS + 500 µM Cd )

(200 µM NaHS + 500 µM Cd )

(200 µM NaHS + 500 µM Cd )

Fig. 2. Electron micrographs of leaf mesophyll cells of 15-day-hydroponically grown seedlings of Brassica napus cv. ZS 758. (A) and (B) TEM micrographs of mesophyll cells of leaves of ZS 758 at control level at low and high magnifications respectively show a clear cell wall (CW), well developed chloroplast (Chl) with thylakoids and grana and mitochondria (MC) is also present. (C) and (D) TEM micrographs of mesophyll cells of ZS 758 at 100 mM NaHS alone at low and high magnifications respectively show an elongated nucleus (N) with developed nucleolus (Nue), well developed chloroplast (Chl) with thylakoids and grana, and mitochondria (M) also could be observed in the micrographs. (E) and (F) TEM micrographs of mesophyll cells of ZS 758 at 200 mM NaHS alone at low and high magnifications respectively show a nucleus (N) with developed nucleolus (Nue), chloroplast (Chl) with thylakoids and grana, and mature mitochondria (M) could be observed in the micrographs. (G) and (H) TEM micrographs of mesophyll cells of leaves of ZS 758 at 500 mM Cd alone at low and high magnifications respectively show a chloroplast (Chl) with swollen thylakoid membranes and a big starch grain (S). (I) and (J) TEM micrographs of mesophyll cells of ZS 758 at 100 mM NaHS under 500 mM Cd concentrations at low and high magnifications respectively show a well developed chloroplast (Chl) with thylakoids and grana, and mitochondria (M) is also present in the cell. Cell wall (CW) and cell membrane (CM) are also clear in the micrographs. (K) and (L) TEM micrographs of mesophyll cells of ZS 758 at 200 mM NaHS under 500 mM Cd concentrations at low and high magnifications respectively show a well developed nucleus (N) and chloroplast (Chl) with thylakoids and grana.

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(Ck)

(Ck)

(100 µM NaHS)

(100 µM NaHS)

(200 µM NaHS)

(500 µM Cd)

(100 µM NaHS + 500 µM Cd)

(200 µM NaHS + 500 µM Cd)

(200 µM NaHS)

(500 µM Cd)

(100 µM NaHS + 500 µM Cd)

(200 µM NaHS + 500 µM Cd)

Fig. 3. Electron micrographs of root tip cells of 15-day-hydroponically grown seedlings of Brassica napus cv. ZS 758. (A) and (B) TEM micrographs of root tip cells of ZS 758 at control level at low and high magnifications respectively show well-developed nucleus (N) with nucleolus (Nue), smooth cell wall (CW), clear endoplasmic reticulum (ER) and mitochondria (MC) and golgibodies (GB). (C) and (D) TEM micrographs of root tip cells of ZS 758 at 100 mM NaHS alone at low and high magnifications respectively show a big well-developed nucleus (N) with nucleoli (Nue) and a distinct nuclear membrane, mitochondria (MC) and golgi bodies (GB). (E) and (F) TEM micrographs of root tip cells of ZS 758 at 200 mM NaHS alone at low and high magnifications respectively show a clear cell with well-developed nucleus (N), cell wall (CW), mitochondria (MC) and glogibodies (GB). (G) and (H) TEM micrographs of root tip cells of ZS 758 at 500 μM Cd alone at low and high magnifications respectively show a totally damaged cell and no organelles are clear in the micrographs. (I) and (J) TEM micrographs of root tip cells of ZS 758 at 100 μM NaHS under 500 μM Cd concentration at low and high magnifications respectively show a clear nucleolus (N), developed mitochondria (M), and golgibodies (GB). (K) and (L) TEM micrographs of root tip cells of ZS 758 at 200 μM NaHS under 500 μM Cd concentration at low and high magnifications respectively show a clear cell with big nucleus (N), long endoplasmic reticulum (ER) and mitochondria (M).

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by decreasing the toxic effect of Cd (Table 2S). Similarly, Panda and Upadhyay (2004) found that Cd exhibited more injurious effects on roots than shoots, and application of salicylic acid ameliorated the Cd deleterious effects in roots. Lately, it was observed that Cd decreased the root morphological parameters in B. napus (Ali et al., 2013b). Retardation in the root morphology might be due to inhibition in cell division and elongation rate of cells due to Cd stress (Liu et al., 2003). The improvement in root morphology under the exogenous application of H2S might be due to increase in APX activity in the roots (Li et al., 2012). Moreover, Dawood et al. (2012) demonstrated that NaHS released H2S which mitigated the toxic effect of Al and improved the root morphology in barley. Moreover, it was observed that Cd-toxicity lowered the chlorophyll (Table 3S) and photosynthetic parameters (Fig. 1S). Photosynthetic activity is dependent on different factors like cell size, number of stomata, stomatal conductance and leaf area (Kosobrukhov et al., 2004). The reason in inhibition of photosynthetic activity may be due to that Cd can affect on stomatal conductance, gas exchange and chlorophyll contents, and ultimately photosynthesis activity gets affected in Cd-treated conditions (Ali et al., 2013a; Wahid et al., 2007). Moreover, decomposition of chlorophyll by increase in chlorophyllase activity might cause inhibition under heavy metal stress (Hegedus et al., 2001). Application of H2S alleviated the Cd stress and significantly improved the chlorophyll and photosynthetic parameters in B. napus plants. As an emerging signal molecule, H2S could improve the photosynthetic activity by diminishing the effect of heavy metals (Dawood et al., 2012; Ali et al., 2014a). The possible reason behind is that H2S induces stomatal closure and participates in the abscisic acid (ABA)-dependent signaling pathway by regulating ATP-binding cassette (ABC) transporters in guard cells (GarciaMata and Lamattina, 2010). Hence, addition of exogenous H2S can regulate the ABA-induced H2O2 mediated stomatal closure and improve the intercellular CO2. Further, it was also found that application of H2S elevated the activity of ribulose-1, 5bisphosphate carboxylase in S. oleracea plants, which plays a cardinal role in controlling net photosynthesis rate (Chen et al., 2011). The toxicity of Cd significantly reduced the Ca2 þ /Mg2 þ ratio and increased the Na þ /K þ ratio in the leaves and roots of B. napus plants (Table 1). The reduction in K þ uptake in leaves and roots under Cd stress might be due to an antagonistic effect of the Cd ion (Murphy et al., 1999). Results also stated that exposure of Cd stress caused considerable reduction in macro- and micro-nutrients in the leaves and roots of B. napus plants; except Fe concentration in roots which was increased under different Cd concentrations (Table 2). Therefore, excessive Cd accumulation could affect the uptake and distribution of certain nutrients in the plants, and hence, it would be responsible for mineral deficiencies or imbalance and depression of plant growth (Kabata-Pendias and Pendias, 1992). These results are in line with the findings of Ali et al. (2014b), who reported that toxicity of heavy metals can reduce the uptake of nutrients in plants. However, exogenous application of H2S mitigated the Cd stress and restored the capability of plants to accumulate essential nutrients. Thus, elevated level of elements maybe due to the mitigatory mechanisms of external H2S (Dawood et al., 2012). Our present study showed that microelements such as Zn and Cu are taken up more after addition of H2S. This might be due to competition effect: lower Cd uptake thus more elements uptake because these elements have similar uptake mechanisms via Ca2 þ channels. Another possible explanation for this result may be less uptake of Cd under the application of H2S, as observed in the same study. This study intended that Cd-accumulation was higher in the roots than shoots of B. napus plants which explains the capability of plants to avoid Cd-induced changes (Najeeb et al.,

205

2011; Ali et al., 2013a). However, application of exogenous H2S significantly reduced the Cd contents in B. napus plants (Table 2). A similar role was reported previously by Xu et al. (2010) and Li et al. (2012) under the combined treatment of NaHS and Cd in Medicago truncatula and Medicago sativa, respectively. However, the mechanism behind the declining effects of Cd-uptake induced by H2S is still unclear. Malondialdehyde (MDA) concentration has been considered as an indicator of lipid peroxidation as well as stress level (Chaoui et al., 1959). Excess reactive oxygen species (ROS) react with lipids, proteins and nucleic acids resulting in rise of MDA, membrane leakage and DNA breakdown, causing severe damage to plant cell (Halliwell and Gutteridge, 2000). Results showed that MDA and ROS increased in leaves and roots of B. napus plants under Cd stress conditions. However, application of H2S lowered the production of ROS and MDA in leaves and roots under Cd-toxicity (Table 4S). These results are in accordance with the findings of Hsu and Kao (2004), who showed that exogenously supplied H2S, could attenuate heavy metal toxicity probably due to its ability to act as an antioxidant scavenging ROS. Therefore, it can be suggested that an activated antioxidant system under the metal stress may be beneficial for plants to remove excess ROS and inhibit lipid peroxidation (Dawood et al., 2012; Qian et al., 2014). In the present study, antioxidant enzyme (SOD, POD, CAT) activities decreased as well as APX and GR increased under the Cd stress conditions (Fig. 1). At the same time, application of H2S significantly enhanced these activities in the leaves and roots of B. napus under Cd stress conditions. The increase in APX and GR activity suggested their effective scavenging mechanism to remove H2O2 which resulted from metal stress-caused oxidative damage (Reddy et al., 2005). The decrease in SOD, POD and CAT activities might indicate their inactivation by accumulation of H2O2 induced by metal stress (Qureshi et al., 2007). Moreover, POD activity was higher in roots than leaves, so, it 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). The antioxidant enzymes enhanced their activities under the combined application of H2S and Cd at different concentrations (Fig. 1). This may be due to the fact that H2S has an alleviating role in Cd-induced oxidative stress, scavenges ROS accumulation and then up-regulates the antioxidant enzyme activities (Dawood et al., 2012; Qian et al., 2014). This study showed that plant mesophyll cell and root tip cell were significantly damaged under the Cd-toxicity conditions (Figs. 2 and 3). The chloroplast had been considered as highly vulnerable to oxidative stress (Zhang et al., 2003), which was damaged under Cd stress. Cd-induced stress resulted in dissolved thylakoid membranes and presence of starch granules inside the chloroplast (Fig. 2G and H). The presence of starch grains under the heavy metal stress indicates that plants may encounter stress, which was also described previously (Daud et al., 2009). Under Cd stress, root tip cells exhibited modifications including deposition of metals in vacuoles and cell walls, and disruption of all organelles inside the cell (Fig. 3G and H). Similarly, several studies revealed that Cd caused ultrastructural disorder in plant root and leaf cells, and caused an increase in metal deposition (Daud et al., 2009; Ali et al., 2013a, 2013b). However, H2S application improved these parameters under Cd-stress in both measopyll cell (Fig. 2I–L) and root tip cell (Fig. 3I–L). These results proposed that H2S has ability to promote the biogenesis of chloroplasts by increasing the number of grana lamellae and chlorophyll biosynthesis, which lead to an enhancement of photosynthesis (Ali et al., 2014a). Some other studies illustrated that H2S reduced the oxidative damage plant cells and improved the cell structures under lead (Ali et al., 2014a), aluminum (Qian et al., 2014) and boron toxicity (Wang et al., 2010).

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5. Conclusions On the basis of present study, it can be concluded that addition of H2S showed significant effects on Cd-exposed B. napus plants and improved the plant growth under Cd stress. It effectively decreased the uptake of Cd contents and alleviated Cd-induced growth inhibition. This alleviation was related to a significant improvement in photosynthetic performance, nutrients uptake, antioxidants enzyme activities and ultrastructural changes in the leaves and roots. Thus, it is suggested that exogenous H2S regulates the Cd-induced morpho-physiological and ultrastructural changes in B. napus plants. However, it needs an intensive and molecular level research at various growth stages of plant to understand, how H2S interacts with different physiological processes and improves the B. napus growth under Cd stress conditions.

Acknowledgments This work was supported by the National High Technology Research and Development Program of China (2013AA103007), the Special Fund for Agro-scientific Research in the Public Interest (201303022), the National Key Science and Technology Supporting Program of China (2010BAD01B01), the National Natural Science Foundation of China (31170405), and the Science and Technology Department of Zhejiang Province (2012C12902-1).

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.08.027. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Ali, B., Song, W.J., Hu, W.Z., Luo, X.N., Gill, R.A., Wang, J., Zhou, W.J., 2014a. Hydrogen sulfide alleviates lead-induced photosynthetic and ultrastructural changes in oilseed rape. Ecotoxicol. Environ. Saf. 102, 25–33. Ali, B., Tao, Q.J., Zhou, Y.F., 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. Ali, B., Wang, B., Ali, S., Ghani, M.A., Hayat, M.T., Yang, C., Xu, L., Zhou, W.J., 2013a. 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, B., Xu, X., Gill, R.A., Yang, S., Ali, S., Tahir, M., Zhou, W., 2014b. Promotive role of 5-aminolevulinic acid on mineral nutrients and antioxidative defense system under lead toxicity in Brassica napus. Ind. Crops Prod. 52, 617–626. Arnon, D.I., Hoagland, D.R., 1940. Crop production in artificial solution with special reference to factors affecting yield and absorption of inorganic nutrients. Soil Sci. 50, 463–485. Butters, B., Chenery, E.M., 1959. A rapid method for the determination of total sulphur in soils and plants. Analyst 84, 239–245. Chaoui, A., Mazhoudi, S., Ghorbal, M.H., Ferjani, E., 1959. Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean (Phaseolus vulgaris L.). Plant Sci. 127, 139–147. Chen, A., Komives, E.A., Schroeder, J.I., 2006. An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis. Plant Physiol. 141, 108–120. Chen, J., Wu, F.H., Wang, W.H., Zheng, C.J., Lin, G.H., Dong, X.J., He, J.X., Pei, Z.M., Zheng, H.L., 2011. Hydrogen sulphide enhances photosynthesis through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings. J. Exp. Bot. 62, 4481–4493. Daud, M.K., Variath, M.T., Ali, S., Najeeb, U., Jamil, M., Hayat, Y., Dawood, M., Khan, M.I., Zaffar, M., Sardar, A., Tong, X.H., Zhu, S., 2009. Cadmium-induced ultramorphological and physiological changes in leaves of two transgenic cotton cultivars and their wild relative. J. Hazard. Mater. 168, 614–625. Dawood, M., Cao, F., Jahangir, M.M., Zhang, G.P., Wu, F., 2012. Alleviation of aluminum toxicity by hydrogen sulfide is related to elevated ATPase, and suppressed aluminum uptake and oxidative stress in barley. J. Hazard. Mater. 210, 121–128.

Evangelou, M.W.H., Daghan, H., Schaeffer, A., 2004. The influence of humic acids on the phytoextraction of cadmium from soil. Chemosphere 57, 207–213. Farooq, M., Basra, S.M.A., Wahid, A., Cheema, Z.A., Cheema, M.A., Khaliq, A., 2008. Physiological role of exogenously applied glycinebetaine to improve drought tolerance in fine grain aromatic rice (Oryza sativa L.). J. Agron. Crop Sci. 194, 325–333. Folgar, S., Torres, E., Perez-Rama, M., Cid, A., Herrero, C., Abalde, J., 2009. Dunaliella salina as marine microalga highly tolerant to but a poor remover of cadmium. J. Hazard. Mater. 165, 486–493. Garcia-Mata, C., Lamattina, L., 2010. Hydrogen sulphide, a novel gasotransmitter involved in guard cell signaling. New Phytol. 188, 97–984. Gouia, H., Ghorbal, M., Meyer, C., 2000. Effect of cadmium on activity of nitrate reductase and other enzymes of the nitrate assimilation pathway in bean. Plant Physiol. Biochem. 38, 629–638. Halliwell, B., Gutteridge, J.M.C., 2000. 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. Hendry, G.A.F., Baker, A.J.M., Ewart, C.F., 1992. Cadmium tolerance and toxicity, oxygen radical processes and molecular damage in cadmium-tolerant and cadmium sensitive clones of Holcus lanatus. Acta Bot. Neerl 41, 271–281. Hsu, Y.H., Kao, C.H., 2004. Cadmium toxicity is reduced by nitric oxide in rice leaves. Plant Growth Regul. 42, 227–238. 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. Kabata-Pendias, A., Pendias, H., 1992. Trace Elements in Soils and Plants, second ed. CRC Press, Boca Raton, London. Kanazawa, S., Sano, S., Koshiba, T., Ushimaru, T., 2000. Changes in anti-oxidative in cucumber cotyledons during natural senescence: comparison with those during dark-induced senescence. Physiol. Plant. 109, 211–216. Kosobrukhov, A., Kyyazera, I., Mudrik, V., 2004. Plantago major plants responses to increase content of lead in soil: growth and photosynthesis. Plant Growth Regul. 42, 145–151. Li, L., Wang, Y., Shen, W., 2012. Roles of hydrogen sulfide and nitric oxide in the alleviation of cadmium-induced oxidative damage in alfalfa seedling roots. Biometals 25, 617–631. Lindner, R.C., 1944. Rapid analytical method for some of the more common inorganic constituents of plant tissues. Plant Physiol. 19, 76–89. 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. McGrath, S.P., Zhao, F.J., Lombi, E., 2002. Phytoremediation of metals, metalloids, and radionuclides. Adv. Agron. 75, 1–56. Momoh, E.J.J., Zhou, W.J., 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. Murphy, A., Eisinger, W., Shaff, J., Kochien, L., Taiz, L., 1999. Early copper induced leakage of K þ from Arabidopsis seedlings is mediated by ion channels and coupled to citrate efflux. Plant Physiol. 121, 1375–1382. Murphy, J., Riley, J.P., 1962. A modified single solution method for determination of phosphate in natural waters. Anal Chim. Acta 27, 31–36. Najeeb, U., Jilani, G., Ali, S., Sarwar, M., Xu, L., Zhou, W.J., 2011. Insight into cadmium induced physiological and ultra-structural disorders in Juncus effusus L. and its remediation through exogenous citric acid. J. Hazard. Mater. 186, 565–574. 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., Upadhyay, R.K., 2004. Salt stress induces oxidative alterations and antioxidative defense in the roots of Lemna minor. Biol. Plant. 48, 249–253. Pei, Z.F., Ming, D.F., Liu, D., Wan, G.L., Geng, X.X., Gong, H.J., Zhou, W.J., 2010. Silicon improves the tolerance to water-deficit stress induced by polyethylene glycol in wheat (Triticum aestivum L.) seedlings. J. Plant Growth Regul. 29, 106–115. Qian, P., Sun, R., Ali, B., Gill, R.A., Xu, L., Zhou, W.J., 2014. Effects of hydrogen sulfide on growth, antioxidative capacity, and ultrastructural changes in oilseed rape seedlings under aluminum toxicity. J. Plant Growth Regul. 33, 526–538. Qureshi, M.I., Abdin, M.Z., Qadir, S., Iqbal, M., 2007. Lead-induced oxidative stress and metabolic alterations in Cassia angustifolia Vahl. Biol. Plant. 51, 121–128. Ramos, I., Esteban, E., Lucena, J.J., Garate, A., 2002. Cadmium uptake and subcellular distribution in plants of Lactuca sativa sp. Cd Mn interaction. Plant Sci. 162, 761–767. 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. Schwarz, D., Grosch, R., 2003. Influence of nutrient solution concentration and a root pathogen on tomato root growth and morphology. Sci. Hortic 97, 109–120.

B. Ali et al. / Ecotoxicology and Environmental Safety 110 (2014) 197–207

Shani, U., Ben-Gal, A., 2005. Long-term response of grapevines to salinity: osmotic effects and ion toxicity. Am. J. Enol. Vitic 56, 148–154. Thian, X., Li, Y., 2006. Nitric oxide treatment alleviates drought stress in wheat seedlings. Biol. Plant. 50, 775–778. Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Plant Sci. 151, 59–66. Wahid, A., Ghani, A., Ali, I., Ashraf, M.Y., 2007. Effects of cadmium on carbon and nitrogen assimilation in shoots of mungbean [Vigna radiata (L.) Wilczek] seedlings. J. Agron. Crop Sci. 193, 357–365. Wang, B.L., Shi, L., Li, Y.X., Zhang, W.H., 2010. Boron toxicity is alleviated by hydrogen sulfide in cucumber (Cucumis sativus L.) seedlings. Planta 231, 1301–1309. Xu, J., Wang, W.Y., Yin, H.X., Liu, X.J., Sun, H., Mi, Q., 2010. Exogenous nitric oxide improves antioxidative capacity and reduces auxin degradation in roots of Medicago truncatula seedlings under cadmium stress. Plant Soil 326, 321–330. Zhang, G.Q., Zhou, W.J., Gu, H.H., Song, W.J., Momoh, E.J.J., 2003. Plant regeneration from the hybridization of Brassica juncea and B. napus through embryo culture. J. Agron. Crop Sci. 189, 347–350.

207

Zhang, H., Hu, L.Y., Hu, K.D., He, Y.D., Wang, S.H., Luo, J.P., 2008a. Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper stress. J. Integr. Plant Biol. 50, 1518–1529. Zhang, H., Tan, Z.Q., Hu, L.Y., Wang, S.H., Luo, J.P., Jones, R.L., 2010. Hydrogen sulfide alleviates aluminum toxicity in germinating wheat seedlings. J. Integr. Plant Biol. 52, 556–567. Zhang, H., Tang, J., Liu, X.P., Wang, Y., Yu, W., Peng, W.Y., Fang, F., Ma, D.F., Wei, Z.J., Hu, L.Y., 2009. Hydrogen sulfide promotes root organogenesis in Ipomoea batatas, Salix matsudana and Glycine max. J. Integr. Plant Biol. 51, 1086–1094. Zhang, W.F., Zhang, F., Raziuddin, R., Gong, H.J., Yang, Z.M., Lu, L., Ye, Q.F., Zhou, W.J., 2008b. Effects of 5-aminolevulinic acid on oilseed rape seedling growth under herbicide toxicity stress. J. Plant Growth Regul. 27, 159–169. Zhou, W.J., 2001. Oilseed rape. In: Zhang, GP, Zhou, WJ (Eds.), Crop Production. Zhejiang University Press, Hangzhou China, pp. 153–178. Zhou, W.J., 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.