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Ultrasonic seed treatment improved cadmium (Cd) tolerance in Brassica napus L.
Ecotoxicology and Environmental Safety 185 (2019) 109659
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Ultrasonic seed treatment improved cadmium (Cd) tolerance in Brassica napus L.
T
Gangshun Raoa,b,c,1, Suihua Huangb,c,1, Umair Ashrafd,1, Zhaowen Mob,c, Meiyang Duanb,c, Shenggang Panb,c, Xiangru Tangb,c,∗ a
Department of Biotechnology, Faculty of Agricultural Science, Guangdong Ocean University, Zhanjiang, 524088, PR China Department of Crop Science and Technology, College of Agriculture, South China Agricultural University, Guangzhou, 510642, PR China c Scientific Observing and Experimental Station of Crop Cultivation in South China, Ministry of Agriculture, PR China, Guangzhou, 510642, China d Department of Botany, Division of Science and Technology, University of Education, Lahore, 54770, Punjab, Pakistan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antioxidants Cadmium stress Germination rate Rapeseed Ultrasonic waves Yield
Cadmium (Cd) affects crop growth and productivity by disrupting normal plant metabolism. To determinate whether ultrasonic (US) seed treatment can alleviate Cd stress in rape (Brassica napus L.), the seeds of two oilseed rape cultivars i.e., ‘Youyanzao18’ and ‘Zaoshu104’ were exposed to ultrasonic waves for 1 min at 20 KHz frequency. Seeds without US treatment were taken as control (CK). Results revealed that the germination rate of both cultivars was significantly (P < 0.05) higher in US treatment than CK only at 0 and 10 mg Cd L−1. The shoot and root length of both cultivars were significantly higher in US treatment than CK at all Cd treatments except the root length of Youyanzao18 at 50 mg Cd L−1. The fresh weight Youyanzao18 was significantly (P < 0.05) higher in US than CK except for Youyanzao18 at 25 mg Cd L−1. Moreover, the superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) activities and the proline, glutathione (GSH), and soluble protein contents in Youyanzao18 were relatively higher in the US treatment than CK. The malondialdehyde (MDA) contents were prominently reduced in US treatment than CK. The pods per plant, seeds per pod and rapeseed yield were increased by 15.9, 11.4, and 16.4% in Youyanzao18 and 10.3, 9.5, and 11.5% in Zaoshu104, respectively in US treatment, compared to CK. Moreover, the contents of Cd in root, stem, leaf, rape pod shell, and rapeseeds were comparatively less in US treatment than CK whereas the Cd concentrations in different plant parts of both rape cultivars were recorded as: leaf ˃ root ˃ stem ˃ rape pod shell ˃ rapeseed. In sum, the US treatment improved the morphological growth and rapeseed yield whereas reduced the Cd accumulation in different plant parts of rapeseed under Cd contaminated soil.
1. Introduction It is well known that heavy metals are polluting agricultural soil adversely (Angelova et al., 2017; Ashraf et al., 2018). Recently, paddy fields of China are contaminated with various potent heavy metals, especially cadmium (Cd) (Du et al., 2018; Kanu et al., 2019). The sources of these pollutants are mine industries, excessive use of fertilizer and pesticides, application of polluted irrigation water and industrial waste, etc (Herrero et al., 2003; Thakur et al., 2016; Al-Saleh and Abduljabbar, 2017). Generally, heavy metals are taken up by the roots, translocated via shoots and leaves, and then accumulated in grains and/or seeds (Li et al., 2017; Ashraf et al., 2017a,b). Physiologically, Cd could induce excessive production of reactive
oxygen species (ROS) that could result in oxidative damage to plants which may cause substantial reduction in morphological attributes and yield (Khan et al., 2015; He et al., 2017; Kanu et al., 2017). Various crop/soil management techniques such as soil cleansing, soil amendments, phyto-extraction, and physical, chemical and biological remediation of soil are purposed to reduce the Cd contents in edible parts of the plants (Yao et al., 2012; Li et al., 2016); however, such techniques require time, labor and heavy investment (Qiu et al., 2011). Therefore, the development of effective, safe, and simple techniques to decrease Cd content in plants is the need of today's agriculture. Ultrasonic seed treatment is considered safe, inexpensive, accessible and eco-friendly technique which had positive effects on seed
∗
Corresponding author.Department of Crop Science and Technology, College of Agriculture, South China Agricultural University, Guangzhou, 510642, PR China. E-mail address: [email protected] (X. Tang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ecoenv.2019.109659 Received 10 March 2019; Received in revised form 1 September 2019; Accepted 6 September 2019 Available online 18 September 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 185 (2019) 109659
G. Rao, et al.
compound fertilizer (N-P2O5-K2O, 15%-15%–15%) at 450 kg hm−1 and borax (boric acid sodium salt, 98%; boron (B), 15%) at 15 kg hm−2 were applied to the experimental units before sowing. Additionally, the compound fertilizer at 450 kg hm−1 was applied at five-leaf stage.
germination, plant metabolic activities and plant growth (Yang et al., 2015). It has been reported that the US seed treatment with suitable intensity could enhance cell growth by stimulating various enzymatic and physiological mechanisms (Liu et al., 2003; Pitt and Ross, 2003). Previously, US seed treatment was successfully employed in stimulating the germination and growth of different crops such as chickpea, wheat, pepper, watermelon, and rice (Goussous et al., 2010; Chen et al., 2013; Rao et al., 2018). Rapeseed (Brassica napus L.) is one of the major oilseeds and widely grown over the globe (Koubaa et al., 2016; Hatzig et al., 2018). In China, rapeseed is extensively grown in rotation with rice (Wollmer et al., 2018). Previously, various techniques such as bacterial mediated heavy metal alleviation, biochar amendments, phytoremediation, and irrigation management have been adopted to minimize the heavy metal accumulation in edible/consumable plant parts (Ali et al., 2013b; Etesami, 2018); however, the US seed treatment of rapeseeds to improve the morphological growth and to minimize the Cd concentrations in rapeseeds were rarely investigated (Koubaa et al., 2016). Therefore, the present study was conducted to assess the potential of the US seed treatment to enhance the morpho-physiological features coupled with reduced Cd contents in rapeseed under Cd contaminated soil.
2.2. Plant sampling Three plants from each replicate were uprooted at stem elongation, flowering, and maturity stages, thoroughly washed and leaves were separated and stored at −80 °C till biochemical analyses. At maturity stage, plants were divided into root, stem, leaves, rape pod shell and rapeseed, and oven-dried at 70 °C till constant weight to quantify the accumulated Cd content in respective plant parts.
2.3. Biochemical assays Fresh leaves samples (0.5 g) were grounded and homogenized with 9 ml of 50 mM sodium phosphate buffer (PBS) (pH 7.8) in an ice bath, followed by centrifuge at 10000×g at 4 °C for 15 min. The supernatant was used for determination of activities of antioxidant enzyme and contents of protein and malondialdehyde (MDA). The activities of SOD (EC 1.15.1.1), CAT (EC 1.11.1.6) and APX (EC 1.11.1.11) and contents of proline and malondialdehyde (MDA) were assayed as described by Rao et al. (2018). Peroxidase (POD, EC 1.11.1.7) activity was determined according to Ashraf and Tang (2017). Protein contents were determined according to Bradford (1976). The glutathione (GSH) content was determined using the method of Li et al. (2015). The reaction mixture contained 0.2 ml of the resulting supernatant, 4.4 ml of PBS, and 0.4 ml of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; 0.04%, w/v). The mixture was incubated in the dark at 30 °C for 5 min and the absorbance was read at 412 nm using a UV-VIS spectrophotometer (UV-2600). The GSH content was expressed as microgram per gram fresh leaves weight (μg g−1 FW).
2. Materials and methods 2.1. Experimental details Uniform rapeseeds (Brassica napus L. cv. Youyanzao18 and Zaoshu104), provided by Guizhou Hemufu Co., Ltd., and College of Agriculture, Hunan Agricultural University, China, respectively, were randomly divided into two groups. One group was placed in a Plant Seed Production Increase Processor (JD-1L, Guangzhou Golden Rice Agricultural Science & Technology Co., Ltd., Guangzhou, China) for 1 min at 20 KHz (US). The untreated seeds were taken as control (CK). The seeds were sterilized for 10 min in 0.5% NaClO solution, rinsed for 30 min in tap water and shade dried. To determine the US treatment on morphological traits under Cd stress, the seeds were randomly divided into six groups, respectively. Each group was comprised of three petri dishes (Ф 9 cm), and then thirty seeds were evenly placed on two sheets of filter papers in each Petri dish. Cd in the form of CdCl2·2.5H2O was applied at 10, 20, 50, 100, and 250 mg Cd l−1, whereas treatments without Cd application was taken as control (0 mg Cd l−1). Respective Cd concentrations were applied with 50 ml in each Petri dish. The dishes were placed in an illuminating incubator (PGX-280A-3H, Life Apparatus, Ningbo, China) maintained at 25 °C and 80% relative humidity with a photoperiod of 12:12 (L:D) h. The light intensity of the light period was 3000 lx. Distilled water (20 ml) was added to each group on daily basis. After 10 days of growth, seedlings were harvested and the germination rate, the root and shoot length, and the fresh weight of seedlings were recorded. In order to determine the effects of US treatment on the growth of rape under Cd contaminated soil, the 2nd part of the experiment was carried out in a rain-protected wire house at Experimental Area, College of Agriculture, South China Agricultural University, Guangzhou, P.R. China (23°09′ N, 113°21′ E) by making small plots during rape growing season (from December 2016 to April 2017). Before fertilizer application, the experimental soil was silty clay comprising 1.01 g kg−1 total N, 48.30 and 68.12 mg kg−1 available P and K, respectively, 22.31 g kg−1 organic matter, 33.014 mg kg−1 total Cd contents and 6.41 soil pH. (The soil in the experimental plots was previously used for pot experiment for Cd toxicity in rice from our research group (Kanu et al., 2017). After completion of the experiment, the soil from the entire pots were mixed thoroughly, placed and leveled in the experimental plots. The US treated and un-treated (CK) seeds of both cultivars i.e., Youyanzao18 and Zaoshu104 were uniformly sown at 7.5 kg hm−2 in the Cd contaminated soil on December 8, 2016. The treatments were arranged in a completely randomized design (CRD) in triplicate. A
2.4. Determination of yield and yield components Plant density was measured by counting the plant from 1 m2 in each plot and expressed as ten thousand per hectare ( × 104 hm−2). Five plants were randomly selected and uprooted from each plot at the maturity stage to measure the number of pods per plant, number of seeds per pod, and 1000-seed weight. At maturity, plants were harvested, threshed manually and then sun-dried to get the rapeseed yield from 1 m2, and then the yield was estimated in kilogram per hectare (kg hm−2).
2.5. Determination of Cd contents in different plant parts The oven-dried samples were ground into powder form and digested with nitric acid and perchloric acid in 4:1 ratio (v/v), respectively (Wu et al., 2016). The subsequent solutions were diluted with distilled water to 25 ml and the absorbance was read with Atomic Absorption Spectrophotometer (AA-6300C, Shimadzu, Japan). The working calibration solutions were made up from 1000 mg l−1 Cd standards and within the recommended linear ranges. The regression values (R2) of the calibration curve was > 0.999.
2.6. Statistical analysis The experimental data were analyzed by ANOVA using SPSS software (version 19.0; SPSS Inc., Chicago, USA). Differences amongst treatments were assessed using the least significant difference (LSD) test at 0.05 probability level. 2
Ecotoxicology and Environmental Safety 185 (2019) 109659
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Table 1 Effects of US seed treatment on germination rate, root length, shoot length, and fresh weight of rape seedlings under Cd stress. Cd concentration (mg L−1)
Cultivar
Treatment
Germination rate (%)
Root length (cm)
Shoot length (cm)
Fresh weight (mg plant−1)
0
Youyanzao18
CK US CK US CK US CK US CK US CK US CK US CK US CK US CK US CK US CK US
97.8 ± 1.1 ab 100.0 ± 0.0a 90.0 ± 1.9c 95.6 ± 1.1b 96.7 ± 0.0b 100.0 ± 0.0a 68.9 ± 1.1d 72.2 ± 1.1c 94.4 ± 1.1a 95.6 ± 1.1a 65.6 ± 1.1b 67.8 ± 1.1b 92.2 ± 1.1a 92.3 ± 1.1a 62.2 ± 1.1b 65.6 ± 1.1b 71.1 ± 1.1a 72.2 ± 1.1a 38.9 ± 1.1b 42.2 ± 1.1b 45.6 ± 1.1b 52.2 ± 1.1a 28.9 ± 1.1c 32.2 ± 1.1c
10.63 ± 0.18c 11.45 ± 0.04b 11.60 ± 0.15b 13.25 ± 0.08a 2.36 ± 0.06c 2.55 ± 0.04b 2.54 ± 0.03b 3.05 ± 0.07a 1.15 ± 0.02d 1.58 ± 0.02b 1.50 ± 0.03c 1.71 ± 0.01a 0.79 ± 0.01c 0.84 ± 0.02c 1.02 ± 0.01b 1.23 ± 0.03a 0.23 ± 0.01c 0.35 ± 0.02b 0.24 ± 0.02c 0.43 ± 0.01a 0.15 ± 0.01bc 0.26 ± 0.01a 0.13 ± 0.01c 0.16 ± 0.01b
4.85 5.23 5.53 5.97 2.75 3.11 3.93 4.57 2.66 3.05 3.45 4.10 1.94 2.48 3.16 3.35 1.36 1.56 1.27 1.64 1.03 1.35 1.09 1.33
66.69 ± 0.37b 73.44 ± 0.03a 59.69 ± 0.26d 64.69 ± 0.53c 57.52 ± 0.71b 60.32 ± 0.64a 46.72 ± 0.25c 48.06 ± 0.27c 50.82 ± 0.21a 51.63 ± 0.49a 30.92 ± 0.50c 32.41 ± 0.32b 34.33 ± 0.62b 39.93 ± 0.24a 26.85 ± 0.36c 28.17 ± 0.41c 24.52 ± 0.42b 26.58 ± 0.60a 12.47 ± 0.31c 13.16 ± 0.13c 15.46 ± 0.09b 21.43 ± 0.52a 9.71 ± 0.23c 10.59 ± 0.23c
Zaoshu104 10
Youyanzao18 Zaoshu104
25
Youyanzao18 Zaoshu104
50
Youyanzao18 Zaoshu104
100
Youyanzao18 Zaoshu104
250
Youyanzao18 Zaoshu104
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.04d 0.04c 0.09b 0.09a 0.04d 0.05c 0.09b 0.10a 0.06d 0.05c 0.08b 0.09a 0.04d 0.02c 0.09b 0.04a 0.01b 0.04a 0.03b 0.04a 0.01b 0.04a 0.02b 0.03a
Values are mean ± standard error (SE) of three replicates. Values with different lowercase alphabets differ significantly within Cd concentration according to LSD at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment. Table 2 Effects of US treatment on the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and, ascorbate peroxidase (APX) in the leaves of the rape at stem elongation, flowering and maturity stages. Sampling stage
Cultivar
Treatment
SOD activity (U g−1 FW)
POD activity (U min−1 g−1 FW)
CAT activity (U min−1 g−1 FW)
APX activity (U min−1 g−1 FW)
Stem elongation
Youyanzao18
CK US CK US CK US CK US CK US CK US
128.73 ± 0.45b 135.80 ± 0.23a 93.60 ± 0.13c 128.73 ± 0.23b 92.76 ± 0.21d 113.98 ± 0.35b 105.32 ± 0.23c 120.01 ± 0.57a 104.14 ± 0.23c 107.18 ± 0.57b 77.23 ± 0.35d 130.12 ± 0.18a
14.94 ± 0.41b 16.58 ± 0.16a 7.46 ± 0.09d 8.50 ± 0.19c 15.81 ± 0.14b 18.04 ± 0.14a 5.34 ± 0.07d 7.17 ± 0.14c 12.48 ± 0.21c 13.17 ± 0.27c 17.96 ± 0.31b 28.58 ± 0.87a
27.17 32.33 25.52 28.45 25.87 31.72 14.36 22.60 19.96 27.91 19.03 19.98
48.71 ± 0.99b 53.74 ± 0.73a 44.73 ± 1.19c 46.05 ± 0.91bc 44.55 ± 1.10c 70.90 ± 1.02a 44.44 ± 0.66c 53.57 ± 0.39b 80.99 ± 1.47c 133.66 ± 2.26a 97.83 ± 0.64b 133.14 ± 2.38a
Zaoshu104 Flowering
Youyanzao18 Zaoshu104
Maturity
Youyanzao18 Zaoshu104
± ± ± ± ± ± ± ± ± ± ± ±
0.66bc 0.82a 0.50c 0.49b 0.45b 0.45a 0.28d 0.49c 0.25b 0.34a 0.30b 0.32b
Values are mean ± standard error (SE) of three replicates. Values with different lowercase alphabets differ significantly within sampling stage according to LSD at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment.
3. Results
and 4.8% in Zaoshu104 at 0 and 25 mg Cd L−1, respectively (Table 1).
3.1. Morphological traits
3.2. Antioxidant enzymes
Cd stress decreased germination rate, root-shoot length, and fresh weight; however, US treatment modulated the morphological traits in both Youyanzao18 and Zaoshu104. For example, US treatment remained statistically similar (P˃0.05) with CK for germination rate at 0, 25, 50 and 100 Cd mg l−1 but a significant increment was observed in germination rate of Youyanzao18 with US treatment at 10 and 250 mg Cd l−1. For Zaoshu104, US treatment substantially increased the germination rate at 0 and 10 mg L−1. Compared to CK, the US treatment increased root length by 7.7, 8.1, 36.7, 51.4 and 71.1% in Youyanzao18 and by 14.2, 19.8, 14.2, 20.7, 79.2 and 20.5% in Zaoshu104 at 0, 10, 25, 50, 100 and 250 mg Cd l−1, respectively. US treatment enhanced shoot length by 8.0, 13.1, 14.8, 27.8, 14.7 and 31.4% in Youyanzao18 and 7.8, 16.1, 19.0, 6.0, 29.2, and 22.4% in Zaoshu104 at 0, 10, 25, 50, 100, 250 mg Cd l−1, respectively. Compared to CK, the US treatment increased the seedling fresh weight by 10.1, 4.9, 16.3, 8.4, 38.7% in Youyanzao18 at 0, 10, 50, 100, 250 mg Cd L−1, respectively, and by 8.4
US treatment substantially improved the antioxidant enzyme activities i.e., SOD, POD, CAT and APX in the leaves of rape under Cd contaminated soil. Compared with CK, US treatment enhanced the activity of SOD by 5.5, 22.9 and 2.9% for Youyanzao18 and 37.5, 13.9 and 68.5% for Zaoshu104 at stem elongation, flowering and maturity, respectively. Compared to CK, US treatment enhanced the activity of POD by 10.9 and 14.1% for Youyanzao18 at stem elongation and flowering stages, respectively, as well as by 14.0, 34.4, and 59.1% for Zaoshu104 at stem elongation, flowering and maturity, respectively. Compared to CK, US treatment significantly enhanced the activity of CAT by 19.0, 22.6, 39.8% for Youyanzao18 at stem elongation, flowering and maturity, respectively, and by 11.5 and 57.4% for Zaoshu104 at stem elongation and flowering, respectively. Compared to CK, US treatment significantly enhanced the activity of APX by 10.3, 59.1, 65.0% for Youyanzao18 at stem elongation, flowering and maturity, respectively, and by 20.5 and 36.1% for Zaoshu104 at flowering and 3
Ecotoxicology and Environmental Safety 185 (2019) 109659
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Table 3 Effects of ultrasonic treatment on malondialdehyde (MDA), proline, soluble protein and glutathione (GSH) contents in the leaves of rape at stem elongation, flowering and maturity stage. Sampling stage
Cultivar
Treatment
MDA content (μmol g−1 FW)
Soluble protein content (mg g−1 FW)
Proline content (μg g−1 FW)
GSH content (μg g−1 FW)
Stem elongation
Youyanzao18
CK US CK US CK US CK US CK US CK US
1.93 1.69 1.72 1.30 1.52 1.42 1.47 1.36 2.20 1.63 2.56 2.03
9.64 ± 0.10c 10.17 ± 0.09b 7.42 ± 0.14d 11.64 ± 0.13a 9.46 ± 0.21b 10.50 ± 0.09a 6.49 ± 0.09c 6.88 ± 0.08c 7.78 ± 0.14a 7.98 ± 0.10a 6.37 ± 0.05b 6.49 ± 0.12b
270.02 345.19 608.56 653.59 386.40 392.02 412.02 469.49 251.71 312.79 382.29 451.75
941.21 ± 7.06b 1496.65 ± 7.04a 802.47 ± 7.00c 831.16 ± 14.11c 963.27 ± 6.71b 1052.94 ± 7.00a 701.24 ± 6.87c 949.63 ± 7.16b 790.35 ± 7.03b 894.51 ± 7.32a 787.60 ± 7.02b 920.10 ± 13.98a
Zaoshu104 Flowering
Youyanzao18 Zaoshu104
Maturity
Youyanzao18 Zaoshu104
± ± ± ± ± ± ± ± ± ± ± ±
0.03a 0.03b 0.03b 0.02c 0.04a 0.03 ab 0.03a 0.03b 0.03b 0.04d 0.04a 0.02c
± ± ± ± ± ± ± ± ± ± ± ±
0.94d 0.94c 1.81b 3.07a 2.97c 2.11c 2.41b 1.84a 1.22d 1.85c 3.89b 12.00a
Values are mean ± standard error (SE) of three replicates. Values with different lowercase alphabets differ significantly within sampling stage according to LSD at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment.
rapeseed by 22.5, 6.7, 24.8, 47.1 and 43.1% (for Youyanzao18), and 13.3, 5.2, 5.6, 7.0, 27.1% (for Zaoshu104), respectively (Table 5).
maturity, respectively (Table 2). 3.3. MDA, proline, soluble protein and GSH contents
4. Discussion
US treatment substantially decreased the MDA contents in both rape cultivars. For example, the MDA contents 12.5, and 25.8% for Youyanzao18 at stem elongation and maturity stage, respectively, and by 24.3, 7.3 and 20.7% for Zaoshu104 at stem elongation, flowering and maturity stage, respectively, compared with CK. Compared to CK, US treatment increased the soluble protein contents by 5.5, and 11.0% for Youyanzao18 and by 56.9 and 6.0% for Zaoshu104 at stem elongation and flowering stage, respectively. Moreover, US treatment enhanced the proline contents in both rape cultivars i.e., 27.8, and 24.3% for Youyanzao18 at stem elongation and maturity stage, respectively, and by 7.4, 13.9 and 18.2% for Zaoshu104 at stem elongation, flowering and maturity stage, respectively, compared to CK. In addition, compared to CK, US treatment notably improved the GSH contents by 59.0, 9.3 and 13.2% for Youyanzao18 at stem elongation, flowering and maturity stage, respectively, and by 35.4 and 16.8% for Zaoshu104 at flowering and maturity stage, respectively (Table 3).
Cd, a toxic heavy metal, may cause adverse effects on plants, animals and human health (Zong et al., 2017a; Wang et al., 2017). Ultrasonic, acoustic waves at frequencies higher than 20 KHz, is a rapid, efficient, and safe technique that can be applied to improve and/or to stimulate the morphological growth and development of the crop plants (Tyagi et al., 2014). In the present study, it was found that Cd stress decreased germination rate, root-shoot length, and fresh weight of rape seedlings (Table 1). Reduction in morphological growth of rape under Cd stress is in agreement with prior reports of Meng et al. (2009), Ali et al. (2013a), and Zong et al. (2017b) who revealed the adverse effects of heavy metals on the seed germination and seedling growth of Brassica napus. Cd stress severely inhibits the cell division and growth, and dry biomass accumulation (Rady and Hemida, 2015). On the other hand, the US seed treatment improved the morphological traits and/or early growth of rape seedlings under Cd stress (Table 1). These results were in accordance with Chen et al. (2013) who found that US seed treatment could potentially reduce the toxic effects of Cd and Pb in the growing wheat seedlings. It is well known that plants are well-equipped with a sophisticated enzymatic and non-enzymatic antioxidative defense system to counter oxidative stress caused by over-production of various reactive oxygen species (ROS) (Ashraf et al., 2015). US treatment generally improved the activities of various antioxidants i.e., SOD, POD, CAT, and APX and modulated the proline, soluble protein, and GSH contents thereby ameliorated the Cd-induced oxidative stress as showed by lower MDA content in US treatment (Table 2; Table 3). The SOD, POD, CAT, and APX are important antioxidant enzymes in plants and play crucial roles in the defense system against various abiotic stresses (Pandey et al., 2017). Within plant cells, SOD acts as the front line of defense by converting O2− into H2O2, and then, its further detoxification and/or conversion to H2O and O2 with the involvement of CAT and APX (You
3.4. Yield and yield components US treatment improved the pods per plant, seeds per pod and seed yield of Brassica napus. For Youyanzao18, the US treatment increased the pods per plant, seeds per pod and rapeseed yield by 15.9, 11.4, and 16.4% compared to CK. For Zaoshu104, compared to CK, the pods per plant, seeds per pod and seed yield were increased by 10.3, 9.5, 11.5%, respectively at maturity (Table 4). 3.5. Cd accumulation different parts at maturity US treatment substantially reduced the Cd contents in rape at maturity and the trend of Cd contents in each part was recorded as: leaf ˃ root ˃ stem ˃ rape pod shell ˃ rapeseed. Compared to CK, the US treatment reduced Cd contents in root, stem, leaf, rape pod shell and
Table 4 Effects of US treatment on yield and yield components of rape under Cd contaminated soil. Cultivar
Treatment
Plant density ( × 104 hm−2)
1000-seed weight (g)
Pods per plant (No.)
Seeds per pod (No.)
Rapeseed yield (kg hm−2)
Youyanzao18 Youyanzao18 Zaoshu104 Zaoshu104
CK US CK US
33.00 34.00 34.00 36.00
3.85 3.89 2.96 2.97
142.7 165.4 126.6 139.6
16.3 18.2 19.9 21.7
1830.06 2131.01 1771.77 1975.03
± ± ± ±
0.58b 0.58b 0.58b 0.58a
± ± ± ±
0.03a 0.01a 0.04b 0.03b
± ± ± ±
2.0b 1.3a 2.6c 2.1b
± ± ± ±
0.3d 0.4c 0.4b 0.4a
± ± ± ±
43.91c 30.94a 30.71c 22.59b
Values are mean ± standard error (SE) of three replicates. Values sharing a letter in common do not differ significantly at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment. 4
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Table 5 Accumulation of Cd contents in different parts of rape under Cd contaminated soil at maturity (mg kg−1). Cultivar
Treatment
Root
Youyanzao18 Youyanzao18 Zaoshu104 Zaoshu104
CK US CK US
21.372 16.564 17.207 14.922
± ± ± ±
0.133a 0.277b 0.159b 0.218c
Stem
Leaf
11.462 ± 0.194a 10.698 ± 0.043b 9.342 ± 0.080c 8.854 ± 0.025d
40.011 30.074 24.522 23.157
± ± ± ±
0.139a 0.209b 0.233c 0.395d
Rape pod shell
Rapeseed
9.055 4.794 7.146 6.647
0.890 0.506 0.781 0.570
± ± ± ±
0.096a 0.094d 0.062b 0.110c
± ± ± ±
0.012a 0.009d 0.017b 0.016c
Values are mean ± standard error (SE) of three replicates. Values sharing a letter in common do not differ significantly at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment.
mechanisms lie behind this response need to be investigated. A suite of frequencies of ultrasonic waves according to the crop and also for different cultivars within the same crop also needs to be examined.
and Chan, 2015). Additionally, POD, which considered as another antioxidant enzyme that converts H2O2 into O2 and H2O (Sudhakar et al., 2001). The present study showed that US treatment substantially improved the activities of SOD, POD, CAT, and APX in rape leaves (Table 2). Similar to our findings, some previous reports of Chen et al. (2013) and Rao et al. (2018) stated that seed treatment with US improved the cellular antioxidant defense against cadmium and lead stress and abridged the ROS-induced oxidative stress in wheat and rice. In general, the MDA results from lipid peroxidation reflects the extent of membrane integrity (Jędrzejuk et al., 2018), therefore, an assay of MDA in plant tissues can be used as an indicator of oxidative damage. Our results demonstrated that US treatment could notably decrease the contents of MDA in rape leaves caused by Cd stress (Table 3). Furthermore, the biosynthesis of proline under stress conditions prevents membrane damage and acts as a hydroxyl radical scavenger (Kaur and Asthir, 2015). It is found that the US treatment significantly increased the proline contents in the leaves of rape under Cd stress (Table 3). Similarly, US treatment enhanced the soluble protein in rape leaves of both cultivars (Table 3). Proline generally helps to stabilize subcellular structures under stress conditions (Correa Molinari et al., 2007), whereas higher contents of soluble protein also related to the ability of plants to resist abiotic stresses (Zhang et al., 2015). Soluble proteins are commonly involved in the defense mechanism of plants which helps in osmoregulation and alleviating the oxidative damage in plants (Anjum et al., 2011; Anjum et al., 2017; Ashraf et al., 2017a,b), whereas, GSH plays crucial roles in oxidation/reduction metabolic cycles, protein synthesis and removal of ROS (Li et al., 2015). Moreover, GSH is also involved in Cd detoxification by forming phytochelatins, which may entangle with heavy metals and can be transported into the vacuoles in the form of complexes (Chen et al., 2012). In the current study, US treatment increased GSH content in rape leaves under Cd stress (Table 3). Similar results were also reported by Chen et al. (2013) in wheat. US treatment substantially improved the number of pods per plant, seeds per pod, and rapeseed yield of both rape cultivars under Cd toxicity (Table 4). Exposure of seeds to US waves may result in substantial changes in the physio-biochemical features of the seeds exposed (Teixeira Da Silva and Dobránszki, 2014). For example, seeds of some cereals treated with the US led to the improvement in the growth of coleoptile, mesocotyl, the seminal roots as well as the overall root volume and water and mineral uptake (Kratovalieva et al., 2012). The yield and yield components such as panicle numbers, grains per panicle, seed setting rate, grain weight and yield per plant were substantially increased in rice treated with US treatment (frequency 40 kHz) under Pb toxicity (Rao et al., 2018). Moreover, US treatment significantly decreased the Cd concentration in different plant parts of Brassica napus, especially in the seeds (Table 5). The less Cd contents in seeds might be related to the overall less uptake and accumulation of Cd contents in different plants parts in US treatment than CK. Previously, Rao et al. (2018) found US-treatment significantly reduced Pb uptake in rice. No doubt, present study on rape and our previous study on rice (Rao et al., 2018) denoted that US seed treatment causes possibly alterations in seed physiology that might results in the changes in plant morphological and yield response even under stressful environment; however, clear understandings about the changes caused and exact
5. Conclusion US seed treatment improved the morpho-physiological attributes of Brassica napus i.e., improved germination and seeding growth, increased the proline, GSH, and soluble protein contents and enhanced the activities of antioxidants e.g., SOD, POD, CAT, and APX, whilst reduced MDA content under Cd toxicity. Moreover, the US seed treatment decreased Cd contents in the root, stem, leaf, rape pod shell, and rapeseeds and improved the yield and its components under Cd contaminated soil. Although, US seed treatment improved the overall performance of Brassica napus under Cd toxicity; however, insights into the exact mechanism(s) and alterations induced by the US treatment in Brassica napus need further investigations. Acknowledgements This study was supported by the National Natural Science Foundation of China (31271646), the Natural Science Foundation of Guangdong Province (8151064201000017), the World Bank Loan Agricultural Pollution Control Project in Guangdong (07241510A08N3684), and the Agricultural Standardization Project of Guangdong Province (4100 F10003). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109659. References Al-Saleh, I., Abduljabbar, M., 2017. Heavy metals (lead, cadmium, methylmercury, arsenic) in commonly imported rice grains (Oryza sativa) sold in Saudi Arabia and their potential health risk. Int. J. Hyg Environ. Health 220, 1168–1178. 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, H., Khan, E., Sajad, M.A., 2013b. Phytoremediation of heavy metals-Concepts and applications. Chemosphere 91, 869–881. Angelova, V.R., Ivanova, R.I., Todorov, J.M., Ivanov, K.I., 2017. Potential of rapeseed (Brassica napus L.) for phytoremediation of soils contaminated with heavy metals. J. Environ. Prot. Ecol. 18, 468–478. Anjum, S.A., Wang, L.C., Farooq, M., Hussain, M., Xue, L.L., Zou, C.M., 2011. Brassinolide application improves the drought tolerance in maize through modulation of enzymatic antioxidants and leaf gas exchange. J. Agron. Crop Sci. 197, 177–185. Anjum, S.A., Ashraf, U., Khan, I., Tanveer, M., Shahid, M., Shakoor, A., Wang, L., 2017. Phyto-toxicity of chromium in maize: oxidative damage, osmolyte accumulation, anti-oxidative defense and chromium uptake. Pedosphere 27, 262–273. Ashraf, U., Tang, X., 2017. Yield and quality responses, plant metabolism and metal distribution pattern in aromatic rice under lead (Pb) toxicity. Chemosphere 176, 141–155. Ashraf, U., Kanu, A.S., Mo, Z., Hussain, S., Anjum, S.A., Khan, I., Abbas, R.N., Tang, X., 2015. Lead toxicity in rice: effects, mechanisms, and mitigation strategies—a mini review. Environ. Sci. Pollut. Res. 22, 18318–18332. Ashraf, U., Hussain, S., Anjum, S.A., Abbas, F., Tanveer, M., Noor, M.A., Tang, X., 2017a. Alterations in growth, oxidative damage, and metal uptake of five aromatic rice cultivars under lead toxicity. Plant Physiol. Biochem. 115, 461–471.
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Ultrasonic seed treatment improved cadmium (Cd) tolerance in Brassica napus L.
T
Gangshun Raoa,b,c,1, Suihua Huangb,c,1, Umair Ashrafd,1, Zhaowen Mob,c, Meiyang Duanb,c, Shenggang Panb,c, Xiangru Tangb,c,∗ a
Department of Biotechnology, Faculty of Agricultural Science, Guangdong Ocean University, Zhanjiang, 524088, PR China Department of Crop Science and Technology, College of Agriculture, South China Agricultural University, Guangzhou, 510642, PR China c Scientific Observing and Experimental Station of Crop Cultivation in South China, Ministry of Agriculture, PR China, Guangzhou, 510642, China d Department of Botany, Division of Science and Technology, University of Education, Lahore, 54770, Punjab, Pakistan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antioxidants Cadmium stress Germination rate Rapeseed Ultrasonic waves Yield
Cadmium (Cd) affects crop growth and productivity by disrupting normal plant metabolism. To determinate whether ultrasonic (US) seed treatment can alleviate Cd stress in rape (Brassica napus L.), the seeds of two oilseed rape cultivars i.e., ‘Youyanzao18’ and ‘Zaoshu104’ were exposed to ultrasonic waves for 1 min at 20 KHz frequency. Seeds without US treatment were taken as control (CK). Results revealed that the germination rate of both cultivars was significantly (P < 0.05) higher in US treatment than CK only at 0 and 10 mg Cd L−1. The shoot and root length of both cultivars were significantly higher in US treatment than CK at all Cd treatments except the root length of Youyanzao18 at 50 mg Cd L−1. The fresh weight Youyanzao18 was significantly (P < 0.05) higher in US than CK except for Youyanzao18 at 25 mg Cd L−1. Moreover, the superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) activities and the proline, glutathione (GSH), and soluble protein contents in Youyanzao18 were relatively higher in the US treatment than CK. The malondialdehyde (MDA) contents were prominently reduced in US treatment than CK. The pods per plant, seeds per pod and rapeseed yield were increased by 15.9, 11.4, and 16.4% in Youyanzao18 and 10.3, 9.5, and 11.5% in Zaoshu104, respectively in US treatment, compared to CK. Moreover, the contents of Cd in root, stem, leaf, rape pod shell, and rapeseeds were comparatively less in US treatment than CK whereas the Cd concentrations in different plant parts of both rape cultivars were recorded as: leaf ˃ root ˃ stem ˃ rape pod shell ˃ rapeseed. In sum, the US treatment improved the morphological growth and rapeseed yield whereas reduced the Cd accumulation in different plant parts of rapeseed under Cd contaminated soil.
1. Introduction It is well known that heavy metals are polluting agricultural soil adversely (Angelova et al., 2017; Ashraf et al., 2018). Recently, paddy fields of China are contaminated with various potent heavy metals, especially cadmium (Cd) (Du et al., 2018; Kanu et al., 2019). The sources of these pollutants are mine industries, excessive use of fertilizer and pesticides, application of polluted irrigation water and industrial waste, etc (Herrero et al., 2003; Thakur et al., 2016; Al-Saleh and Abduljabbar, 2017). Generally, heavy metals are taken up by the roots, translocated via shoots and leaves, and then accumulated in grains and/or seeds (Li et al., 2017; Ashraf et al., 2017a,b). Physiologically, Cd could induce excessive production of reactive
oxygen species (ROS) that could result in oxidative damage to plants which may cause substantial reduction in morphological attributes and yield (Khan et al., 2015; He et al., 2017; Kanu et al., 2017). Various crop/soil management techniques such as soil cleansing, soil amendments, phyto-extraction, and physical, chemical and biological remediation of soil are purposed to reduce the Cd contents in edible parts of the plants (Yao et al., 2012; Li et al., 2016); however, such techniques require time, labor and heavy investment (Qiu et al., 2011). Therefore, the development of effective, safe, and simple techniques to decrease Cd content in plants is the need of today's agriculture. Ultrasonic seed treatment is considered safe, inexpensive, accessible and eco-friendly technique which had positive effects on seed
∗
Corresponding author.Department of Crop Science and Technology, College of Agriculture, South China Agricultural University, Guangzhou, 510642, PR China. E-mail address: [email protected] (X. Tang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ecoenv.2019.109659 Received 10 March 2019; Received in revised form 1 September 2019; Accepted 6 September 2019 Available online 18 September 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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compound fertilizer (N-P2O5-K2O, 15%-15%–15%) at 450 kg hm−1 and borax (boric acid sodium salt, 98%; boron (B), 15%) at 15 kg hm−2 were applied to the experimental units before sowing. Additionally, the compound fertilizer at 450 kg hm−1 was applied at five-leaf stage.
germination, plant metabolic activities and plant growth (Yang et al., 2015). It has been reported that the US seed treatment with suitable intensity could enhance cell growth by stimulating various enzymatic and physiological mechanisms (Liu et al., 2003; Pitt and Ross, 2003). Previously, US seed treatment was successfully employed in stimulating the germination and growth of different crops such as chickpea, wheat, pepper, watermelon, and rice (Goussous et al., 2010; Chen et al., 2013; Rao et al., 2018). Rapeseed (Brassica napus L.) is one of the major oilseeds and widely grown over the globe (Koubaa et al., 2016; Hatzig et al., 2018). In China, rapeseed is extensively grown in rotation with rice (Wollmer et al., 2018). Previously, various techniques such as bacterial mediated heavy metal alleviation, biochar amendments, phytoremediation, and irrigation management have been adopted to minimize the heavy metal accumulation in edible/consumable plant parts (Ali et al., 2013b; Etesami, 2018); however, the US seed treatment of rapeseeds to improve the morphological growth and to minimize the Cd concentrations in rapeseeds were rarely investigated (Koubaa et al., 2016). Therefore, the present study was conducted to assess the potential of the US seed treatment to enhance the morpho-physiological features coupled with reduced Cd contents in rapeseed under Cd contaminated soil.
2.2. Plant sampling Three plants from each replicate were uprooted at stem elongation, flowering, and maturity stages, thoroughly washed and leaves were separated and stored at −80 °C till biochemical analyses. At maturity stage, plants were divided into root, stem, leaves, rape pod shell and rapeseed, and oven-dried at 70 °C till constant weight to quantify the accumulated Cd content in respective plant parts.
2.3. Biochemical assays Fresh leaves samples (0.5 g) were grounded and homogenized with 9 ml of 50 mM sodium phosphate buffer (PBS) (pH 7.8) in an ice bath, followed by centrifuge at 10000×g at 4 °C for 15 min. The supernatant was used for determination of activities of antioxidant enzyme and contents of protein and malondialdehyde (MDA). The activities of SOD (EC 1.15.1.1), CAT (EC 1.11.1.6) and APX (EC 1.11.1.11) and contents of proline and malondialdehyde (MDA) were assayed as described by Rao et al. (2018). Peroxidase (POD, EC 1.11.1.7) activity was determined according to Ashraf and Tang (2017). Protein contents were determined according to Bradford (1976). The glutathione (GSH) content was determined using the method of Li et al. (2015). The reaction mixture contained 0.2 ml of the resulting supernatant, 4.4 ml of PBS, and 0.4 ml of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; 0.04%, w/v). The mixture was incubated in the dark at 30 °C for 5 min and the absorbance was read at 412 nm using a UV-VIS spectrophotometer (UV-2600). The GSH content was expressed as microgram per gram fresh leaves weight (μg g−1 FW).
2. Materials and methods 2.1. Experimental details Uniform rapeseeds (Brassica napus L. cv. Youyanzao18 and Zaoshu104), provided by Guizhou Hemufu Co., Ltd., and College of Agriculture, Hunan Agricultural University, China, respectively, were randomly divided into two groups. One group was placed in a Plant Seed Production Increase Processor (JD-1L, Guangzhou Golden Rice Agricultural Science & Technology Co., Ltd., Guangzhou, China) for 1 min at 20 KHz (US). The untreated seeds were taken as control (CK). The seeds were sterilized for 10 min in 0.5% NaClO solution, rinsed for 30 min in tap water and shade dried. To determine the US treatment on morphological traits under Cd stress, the seeds were randomly divided into six groups, respectively. Each group was comprised of three petri dishes (Ф 9 cm), and then thirty seeds were evenly placed on two sheets of filter papers in each Petri dish. Cd in the form of CdCl2·2.5H2O was applied at 10, 20, 50, 100, and 250 mg Cd l−1, whereas treatments without Cd application was taken as control (0 mg Cd l−1). Respective Cd concentrations were applied with 50 ml in each Petri dish. The dishes were placed in an illuminating incubator (PGX-280A-3H, Life Apparatus, Ningbo, China) maintained at 25 °C and 80% relative humidity with a photoperiod of 12:12 (L:D) h. The light intensity of the light period was 3000 lx. Distilled water (20 ml) was added to each group on daily basis. After 10 days of growth, seedlings were harvested and the germination rate, the root and shoot length, and the fresh weight of seedlings were recorded. In order to determine the effects of US treatment on the growth of rape under Cd contaminated soil, the 2nd part of the experiment was carried out in a rain-protected wire house at Experimental Area, College of Agriculture, South China Agricultural University, Guangzhou, P.R. China (23°09′ N, 113°21′ E) by making small plots during rape growing season (from December 2016 to April 2017). Before fertilizer application, the experimental soil was silty clay comprising 1.01 g kg−1 total N, 48.30 and 68.12 mg kg−1 available P and K, respectively, 22.31 g kg−1 organic matter, 33.014 mg kg−1 total Cd contents and 6.41 soil pH. (The soil in the experimental plots was previously used for pot experiment for Cd toxicity in rice from our research group (Kanu et al., 2017). After completion of the experiment, the soil from the entire pots were mixed thoroughly, placed and leveled in the experimental plots. The US treated and un-treated (CK) seeds of both cultivars i.e., Youyanzao18 and Zaoshu104 were uniformly sown at 7.5 kg hm−2 in the Cd contaminated soil on December 8, 2016. The treatments were arranged in a completely randomized design (CRD) in triplicate. A
2.4. Determination of yield and yield components Plant density was measured by counting the plant from 1 m2 in each plot and expressed as ten thousand per hectare ( × 104 hm−2). Five plants were randomly selected and uprooted from each plot at the maturity stage to measure the number of pods per plant, number of seeds per pod, and 1000-seed weight. At maturity, plants were harvested, threshed manually and then sun-dried to get the rapeseed yield from 1 m2, and then the yield was estimated in kilogram per hectare (kg hm−2).
2.5. Determination of Cd contents in different plant parts The oven-dried samples were ground into powder form and digested with nitric acid and perchloric acid in 4:1 ratio (v/v), respectively (Wu et al., 2016). The subsequent solutions were diluted with distilled water to 25 ml and the absorbance was read with Atomic Absorption Spectrophotometer (AA-6300C, Shimadzu, Japan). The working calibration solutions were made up from 1000 mg l−1 Cd standards and within the recommended linear ranges. The regression values (R2) of the calibration curve was > 0.999.
2.6. Statistical analysis The experimental data were analyzed by ANOVA using SPSS software (version 19.0; SPSS Inc., Chicago, USA). Differences amongst treatments were assessed using the least significant difference (LSD) test at 0.05 probability level. 2
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Table 1 Effects of US seed treatment on germination rate, root length, shoot length, and fresh weight of rape seedlings under Cd stress. Cd concentration (mg L−1)
Cultivar
Treatment
Germination rate (%)
Root length (cm)
Shoot length (cm)
Fresh weight (mg plant−1)
0
Youyanzao18
CK US CK US CK US CK US CK US CK US CK US CK US CK US CK US CK US CK US
97.8 ± 1.1 ab 100.0 ± 0.0a 90.0 ± 1.9c 95.6 ± 1.1b 96.7 ± 0.0b 100.0 ± 0.0a 68.9 ± 1.1d 72.2 ± 1.1c 94.4 ± 1.1a 95.6 ± 1.1a 65.6 ± 1.1b 67.8 ± 1.1b 92.2 ± 1.1a 92.3 ± 1.1a 62.2 ± 1.1b 65.6 ± 1.1b 71.1 ± 1.1a 72.2 ± 1.1a 38.9 ± 1.1b 42.2 ± 1.1b 45.6 ± 1.1b 52.2 ± 1.1a 28.9 ± 1.1c 32.2 ± 1.1c
10.63 ± 0.18c 11.45 ± 0.04b 11.60 ± 0.15b 13.25 ± 0.08a 2.36 ± 0.06c 2.55 ± 0.04b 2.54 ± 0.03b 3.05 ± 0.07a 1.15 ± 0.02d 1.58 ± 0.02b 1.50 ± 0.03c 1.71 ± 0.01a 0.79 ± 0.01c 0.84 ± 0.02c 1.02 ± 0.01b 1.23 ± 0.03a 0.23 ± 0.01c 0.35 ± 0.02b 0.24 ± 0.02c 0.43 ± 0.01a 0.15 ± 0.01bc 0.26 ± 0.01a 0.13 ± 0.01c 0.16 ± 0.01b
4.85 5.23 5.53 5.97 2.75 3.11 3.93 4.57 2.66 3.05 3.45 4.10 1.94 2.48 3.16 3.35 1.36 1.56 1.27 1.64 1.03 1.35 1.09 1.33
66.69 ± 0.37b 73.44 ± 0.03a 59.69 ± 0.26d 64.69 ± 0.53c 57.52 ± 0.71b 60.32 ± 0.64a 46.72 ± 0.25c 48.06 ± 0.27c 50.82 ± 0.21a 51.63 ± 0.49a 30.92 ± 0.50c 32.41 ± 0.32b 34.33 ± 0.62b 39.93 ± 0.24a 26.85 ± 0.36c 28.17 ± 0.41c 24.52 ± 0.42b 26.58 ± 0.60a 12.47 ± 0.31c 13.16 ± 0.13c 15.46 ± 0.09b 21.43 ± 0.52a 9.71 ± 0.23c 10.59 ± 0.23c
Zaoshu104 10
Youyanzao18 Zaoshu104
25
Youyanzao18 Zaoshu104
50
Youyanzao18 Zaoshu104
100
Youyanzao18 Zaoshu104
250
Youyanzao18 Zaoshu104
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.04d 0.04c 0.09b 0.09a 0.04d 0.05c 0.09b 0.10a 0.06d 0.05c 0.08b 0.09a 0.04d 0.02c 0.09b 0.04a 0.01b 0.04a 0.03b 0.04a 0.01b 0.04a 0.02b 0.03a
Values are mean ± standard error (SE) of three replicates. Values with different lowercase alphabets differ significantly within Cd concentration according to LSD at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment. Table 2 Effects of US treatment on the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and, ascorbate peroxidase (APX) in the leaves of the rape at stem elongation, flowering and maturity stages. Sampling stage
Cultivar
Treatment
SOD activity (U g−1 FW)
POD activity (U min−1 g−1 FW)
CAT activity (U min−1 g−1 FW)
APX activity (U min−1 g−1 FW)
Stem elongation
Youyanzao18
CK US CK US CK US CK US CK US CK US
128.73 ± 0.45b 135.80 ± 0.23a 93.60 ± 0.13c 128.73 ± 0.23b 92.76 ± 0.21d 113.98 ± 0.35b 105.32 ± 0.23c 120.01 ± 0.57a 104.14 ± 0.23c 107.18 ± 0.57b 77.23 ± 0.35d 130.12 ± 0.18a
14.94 ± 0.41b 16.58 ± 0.16a 7.46 ± 0.09d 8.50 ± 0.19c 15.81 ± 0.14b 18.04 ± 0.14a 5.34 ± 0.07d 7.17 ± 0.14c 12.48 ± 0.21c 13.17 ± 0.27c 17.96 ± 0.31b 28.58 ± 0.87a
27.17 32.33 25.52 28.45 25.87 31.72 14.36 22.60 19.96 27.91 19.03 19.98
48.71 ± 0.99b 53.74 ± 0.73a 44.73 ± 1.19c 46.05 ± 0.91bc 44.55 ± 1.10c 70.90 ± 1.02a 44.44 ± 0.66c 53.57 ± 0.39b 80.99 ± 1.47c 133.66 ± 2.26a 97.83 ± 0.64b 133.14 ± 2.38a
Zaoshu104 Flowering
Youyanzao18 Zaoshu104
Maturity
Youyanzao18 Zaoshu104
± ± ± ± ± ± ± ± ± ± ± ±
0.66bc 0.82a 0.50c 0.49b 0.45b 0.45a 0.28d 0.49c 0.25b 0.34a 0.30b 0.32b
Values are mean ± standard error (SE) of three replicates. Values with different lowercase alphabets differ significantly within sampling stage according to LSD at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment.
3. Results
and 4.8% in Zaoshu104 at 0 and 25 mg Cd L−1, respectively (Table 1).
3.1. Morphological traits
3.2. Antioxidant enzymes
Cd stress decreased germination rate, root-shoot length, and fresh weight; however, US treatment modulated the morphological traits in both Youyanzao18 and Zaoshu104. For example, US treatment remained statistically similar (P˃0.05) with CK for germination rate at 0, 25, 50 and 100 Cd mg l−1 but a significant increment was observed in germination rate of Youyanzao18 with US treatment at 10 and 250 mg Cd l−1. For Zaoshu104, US treatment substantially increased the germination rate at 0 and 10 mg L−1. Compared to CK, the US treatment increased root length by 7.7, 8.1, 36.7, 51.4 and 71.1% in Youyanzao18 and by 14.2, 19.8, 14.2, 20.7, 79.2 and 20.5% in Zaoshu104 at 0, 10, 25, 50, 100 and 250 mg Cd l−1, respectively. US treatment enhanced shoot length by 8.0, 13.1, 14.8, 27.8, 14.7 and 31.4% in Youyanzao18 and 7.8, 16.1, 19.0, 6.0, 29.2, and 22.4% in Zaoshu104 at 0, 10, 25, 50, 100, 250 mg Cd l−1, respectively. Compared to CK, the US treatment increased the seedling fresh weight by 10.1, 4.9, 16.3, 8.4, 38.7% in Youyanzao18 at 0, 10, 50, 100, 250 mg Cd L−1, respectively, and by 8.4
US treatment substantially improved the antioxidant enzyme activities i.e., SOD, POD, CAT and APX in the leaves of rape under Cd contaminated soil. Compared with CK, US treatment enhanced the activity of SOD by 5.5, 22.9 and 2.9% for Youyanzao18 and 37.5, 13.9 and 68.5% for Zaoshu104 at stem elongation, flowering and maturity, respectively. Compared to CK, US treatment enhanced the activity of POD by 10.9 and 14.1% for Youyanzao18 at stem elongation and flowering stages, respectively, as well as by 14.0, 34.4, and 59.1% for Zaoshu104 at stem elongation, flowering and maturity, respectively. Compared to CK, US treatment significantly enhanced the activity of CAT by 19.0, 22.6, 39.8% for Youyanzao18 at stem elongation, flowering and maturity, respectively, and by 11.5 and 57.4% for Zaoshu104 at stem elongation and flowering, respectively. Compared to CK, US treatment significantly enhanced the activity of APX by 10.3, 59.1, 65.0% for Youyanzao18 at stem elongation, flowering and maturity, respectively, and by 20.5 and 36.1% for Zaoshu104 at flowering and 3
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Table 3 Effects of ultrasonic treatment on malondialdehyde (MDA), proline, soluble protein and glutathione (GSH) contents in the leaves of rape at stem elongation, flowering and maturity stage. Sampling stage
Cultivar
Treatment
MDA content (μmol g−1 FW)
Soluble protein content (mg g−1 FW)
Proline content (μg g−1 FW)
GSH content (μg g−1 FW)
Stem elongation
Youyanzao18
CK US CK US CK US CK US CK US CK US
1.93 1.69 1.72 1.30 1.52 1.42 1.47 1.36 2.20 1.63 2.56 2.03
9.64 ± 0.10c 10.17 ± 0.09b 7.42 ± 0.14d 11.64 ± 0.13a 9.46 ± 0.21b 10.50 ± 0.09a 6.49 ± 0.09c 6.88 ± 0.08c 7.78 ± 0.14a 7.98 ± 0.10a 6.37 ± 0.05b 6.49 ± 0.12b
270.02 345.19 608.56 653.59 386.40 392.02 412.02 469.49 251.71 312.79 382.29 451.75
941.21 ± 7.06b 1496.65 ± 7.04a 802.47 ± 7.00c 831.16 ± 14.11c 963.27 ± 6.71b 1052.94 ± 7.00a 701.24 ± 6.87c 949.63 ± 7.16b 790.35 ± 7.03b 894.51 ± 7.32a 787.60 ± 7.02b 920.10 ± 13.98a
Zaoshu104 Flowering
Youyanzao18 Zaoshu104
Maturity
Youyanzao18 Zaoshu104
± ± ± ± ± ± ± ± ± ± ± ±
0.03a 0.03b 0.03b 0.02c 0.04a 0.03 ab 0.03a 0.03b 0.03b 0.04d 0.04a 0.02c
± ± ± ± ± ± ± ± ± ± ± ±
0.94d 0.94c 1.81b 3.07a 2.97c 2.11c 2.41b 1.84a 1.22d 1.85c 3.89b 12.00a
Values are mean ± standard error (SE) of three replicates. Values with different lowercase alphabets differ significantly within sampling stage according to LSD at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment.
rapeseed by 22.5, 6.7, 24.8, 47.1 and 43.1% (for Youyanzao18), and 13.3, 5.2, 5.6, 7.0, 27.1% (for Zaoshu104), respectively (Table 5).
maturity, respectively (Table 2). 3.3. MDA, proline, soluble protein and GSH contents
4. Discussion
US treatment substantially decreased the MDA contents in both rape cultivars. For example, the MDA contents 12.5, and 25.8% for Youyanzao18 at stem elongation and maturity stage, respectively, and by 24.3, 7.3 and 20.7% for Zaoshu104 at stem elongation, flowering and maturity stage, respectively, compared with CK. Compared to CK, US treatment increased the soluble protein contents by 5.5, and 11.0% for Youyanzao18 and by 56.9 and 6.0% for Zaoshu104 at stem elongation and flowering stage, respectively. Moreover, US treatment enhanced the proline contents in both rape cultivars i.e., 27.8, and 24.3% for Youyanzao18 at stem elongation and maturity stage, respectively, and by 7.4, 13.9 and 18.2% for Zaoshu104 at stem elongation, flowering and maturity stage, respectively, compared to CK. In addition, compared to CK, US treatment notably improved the GSH contents by 59.0, 9.3 and 13.2% for Youyanzao18 at stem elongation, flowering and maturity stage, respectively, and by 35.4 and 16.8% for Zaoshu104 at flowering and maturity stage, respectively (Table 3).
Cd, a toxic heavy metal, may cause adverse effects on plants, animals and human health (Zong et al., 2017a; Wang et al., 2017). Ultrasonic, acoustic waves at frequencies higher than 20 KHz, is a rapid, efficient, and safe technique that can be applied to improve and/or to stimulate the morphological growth and development of the crop plants (Tyagi et al., 2014). In the present study, it was found that Cd stress decreased germination rate, root-shoot length, and fresh weight of rape seedlings (Table 1). Reduction in morphological growth of rape under Cd stress is in agreement with prior reports of Meng et al. (2009), Ali et al. (2013a), and Zong et al. (2017b) who revealed the adverse effects of heavy metals on the seed germination and seedling growth of Brassica napus. Cd stress severely inhibits the cell division and growth, and dry biomass accumulation (Rady and Hemida, 2015). On the other hand, the US seed treatment improved the morphological traits and/or early growth of rape seedlings under Cd stress (Table 1). These results were in accordance with Chen et al. (2013) who found that US seed treatment could potentially reduce the toxic effects of Cd and Pb in the growing wheat seedlings. It is well known that plants are well-equipped with a sophisticated enzymatic and non-enzymatic antioxidative defense system to counter oxidative stress caused by over-production of various reactive oxygen species (ROS) (Ashraf et al., 2015). US treatment generally improved the activities of various antioxidants i.e., SOD, POD, CAT, and APX and modulated the proline, soluble protein, and GSH contents thereby ameliorated the Cd-induced oxidative stress as showed by lower MDA content in US treatment (Table 2; Table 3). The SOD, POD, CAT, and APX are important antioxidant enzymes in plants and play crucial roles in the defense system against various abiotic stresses (Pandey et al., 2017). Within plant cells, SOD acts as the front line of defense by converting O2− into H2O2, and then, its further detoxification and/or conversion to H2O and O2 with the involvement of CAT and APX (You
3.4. Yield and yield components US treatment improved the pods per plant, seeds per pod and seed yield of Brassica napus. For Youyanzao18, the US treatment increased the pods per plant, seeds per pod and rapeseed yield by 15.9, 11.4, and 16.4% compared to CK. For Zaoshu104, compared to CK, the pods per plant, seeds per pod and seed yield were increased by 10.3, 9.5, 11.5%, respectively at maturity (Table 4). 3.5. Cd accumulation different parts at maturity US treatment substantially reduced the Cd contents in rape at maturity and the trend of Cd contents in each part was recorded as: leaf ˃ root ˃ stem ˃ rape pod shell ˃ rapeseed. Compared to CK, the US treatment reduced Cd contents in root, stem, leaf, rape pod shell and
Table 4 Effects of US treatment on yield and yield components of rape under Cd contaminated soil. Cultivar
Treatment
Plant density ( × 104 hm−2)
1000-seed weight (g)
Pods per plant (No.)
Seeds per pod (No.)
Rapeseed yield (kg hm−2)
Youyanzao18 Youyanzao18 Zaoshu104 Zaoshu104
CK US CK US
33.00 34.00 34.00 36.00
3.85 3.89 2.96 2.97
142.7 165.4 126.6 139.6
16.3 18.2 19.9 21.7
1830.06 2131.01 1771.77 1975.03
± ± ± ±
0.58b 0.58b 0.58b 0.58a
± ± ± ±
0.03a 0.01a 0.04b 0.03b
± ± ± ±
2.0b 1.3a 2.6c 2.1b
± ± ± ±
0.3d 0.4c 0.4b 0.4a
± ± ± ±
43.91c 30.94a 30.71c 22.59b
Values are mean ± standard error (SE) of three replicates. Values sharing a letter in common do not differ significantly at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment. 4
Ecotoxicology and Environmental Safety 185 (2019) 109659
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Table 5 Accumulation of Cd contents in different parts of rape under Cd contaminated soil at maturity (mg kg−1). Cultivar
Treatment
Root
Youyanzao18 Youyanzao18 Zaoshu104 Zaoshu104
CK US CK US
21.372 16.564 17.207 14.922
± ± ± ±
0.133a 0.277b 0.159b 0.218c
Stem
Leaf
11.462 ± 0.194a 10.698 ± 0.043b 9.342 ± 0.080c 8.854 ± 0.025d
40.011 30.074 24.522 23.157
± ± ± ±
0.139a 0.209b 0.233c 0.395d
Rape pod shell
Rapeseed
9.055 4.794 7.146 6.647
0.890 0.506 0.781 0.570
± ± ± ±
0.096a 0.094d 0.062b 0.110c
± ± ± ±
0.012a 0.009d 0.017b 0.016c
Values are mean ± standard error (SE) of three replicates. Values sharing a letter in common do not differ significantly at 0.05 probability level. US: ultrasonic seed treatment; CK: seeds without US treatment.
mechanisms lie behind this response need to be investigated. A suite of frequencies of ultrasonic waves according to the crop and also for different cultivars within the same crop also needs to be examined.
and Chan, 2015). Additionally, POD, which considered as another antioxidant enzyme that converts H2O2 into O2 and H2O (Sudhakar et al., 2001). The present study showed that US treatment substantially improved the activities of SOD, POD, CAT, and APX in rape leaves (Table 2). Similar to our findings, some previous reports of Chen et al. (2013) and Rao et al. (2018) stated that seed treatment with US improved the cellular antioxidant defense against cadmium and lead stress and abridged the ROS-induced oxidative stress in wheat and rice. In general, the MDA results from lipid peroxidation reflects the extent of membrane integrity (Jędrzejuk et al., 2018), therefore, an assay of MDA in plant tissues can be used as an indicator of oxidative damage. Our results demonstrated that US treatment could notably decrease the contents of MDA in rape leaves caused by Cd stress (Table 3). Furthermore, the biosynthesis of proline under stress conditions prevents membrane damage and acts as a hydroxyl radical scavenger (Kaur and Asthir, 2015). It is found that the US treatment significantly increased the proline contents in the leaves of rape under Cd stress (Table 3). Similarly, US treatment enhanced the soluble protein in rape leaves of both cultivars (Table 3). Proline generally helps to stabilize subcellular structures under stress conditions (Correa Molinari et al., 2007), whereas higher contents of soluble protein also related to the ability of plants to resist abiotic stresses (Zhang et al., 2015). Soluble proteins are commonly involved in the defense mechanism of plants which helps in osmoregulation and alleviating the oxidative damage in plants (Anjum et al., 2011; Anjum et al., 2017; Ashraf et al., 2017a,b), whereas, GSH plays crucial roles in oxidation/reduction metabolic cycles, protein synthesis and removal of ROS (Li et al., 2015). Moreover, GSH is also involved in Cd detoxification by forming phytochelatins, which may entangle with heavy metals and can be transported into the vacuoles in the form of complexes (Chen et al., 2012). In the current study, US treatment increased GSH content in rape leaves under Cd stress (Table 3). Similar results were also reported by Chen et al. (2013) in wheat. US treatment substantially improved the number of pods per plant, seeds per pod, and rapeseed yield of both rape cultivars under Cd toxicity (Table 4). Exposure of seeds to US waves may result in substantial changes in the physio-biochemical features of the seeds exposed (Teixeira Da Silva and Dobránszki, 2014). For example, seeds of some cereals treated with the US led to the improvement in the growth of coleoptile, mesocotyl, the seminal roots as well as the overall root volume and water and mineral uptake (Kratovalieva et al., 2012). The yield and yield components such as panicle numbers, grains per panicle, seed setting rate, grain weight and yield per plant were substantially increased in rice treated with US treatment (frequency 40 kHz) under Pb toxicity (Rao et al., 2018). Moreover, US treatment significantly decreased the Cd concentration in different plant parts of Brassica napus, especially in the seeds (Table 5). The less Cd contents in seeds might be related to the overall less uptake and accumulation of Cd contents in different plants parts in US treatment than CK. Previously, Rao et al. (2018) found US-treatment significantly reduced Pb uptake in rice. No doubt, present study on rape and our previous study on rice (Rao et al., 2018) denoted that US seed treatment causes possibly alterations in seed physiology that might results in the changes in plant morphological and yield response even under stressful environment; however, clear understandings about the changes caused and exact
5. Conclusion US seed treatment improved the morpho-physiological attributes of Brassica napus i.e., improved germination and seeding growth, increased the proline, GSH, and soluble protein contents and enhanced the activities of antioxidants e.g., SOD, POD, CAT, and APX, whilst reduced MDA content under Cd toxicity. Moreover, the US seed treatment decreased Cd contents in the root, stem, leaf, rape pod shell, and rapeseeds and improved the yield and its components under Cd contaminated soil. Although, US seed treatment improved the overall performance of Brassica napus under Cd toxicity; however, insights into the exact mechanism(s) and alterations induced by the US treatment in Brassica napus need further investigations. Acknowledgements This study was supported by the National Natural Science Foundation of China (31271646), the Natural Science Foundation of Guangdong Province (8151064201000017), the World Bank Loan Agricultural Pollution Control Project in Guangdong (07241510A08N3684), and the Agricultural Standardization Project of Guangdong Province (4100 F10003). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109659. References Al-Saleh, I., Abduljabbar, M., 2017. Heavy metals (lead, cadmium, methylmercury, arsenic) in commonly imported rice grains (Oryza sativa) sold in Saudi Arabia and their potential health risk. Int. J. Hyg Environ. Health 220, 1168–1178. 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, H., Khan, E., Sajad, M.A., 2013b. Phytoremediation of heavy metals-Concepts and applications. Chemosphere 91, 869–881. Angelova, V.R., Ivanova, R.I., Todorov, J.M., Ivanov, K.I., 2017. Potential of rapeseed (Brassica napus L.) for phytoremediation of soils contaminated with heavy metals. J. Environ. Prot. Ecol. 18, 468–478. Anjum, S.A., Wang, L.C., Farooq, M., Hussain, M., Xue, L.L., Zou, C.M., 2011. Brassinolide application improves the drought tolerance in maize through modulation of enzymatic antioxidants and leaf gas exchange. J. Agron. Crop Sci. 197, 177–185. Anjum, S.A., Ashraf, U., Khan, I., Tanveer, M., Shahid, M., Shakoor, A., Wang, L., 2017. Phyto-toxicity of chromium in maize: oxidative damage, osmolyte accumulation, anti-oxidative defense and chromium uptake. Pedosphere 27, 262–273. Ashraf, U., Tang, X., 2017. Yield and quality responses, plant metabolism and metal distribution pattern in aromatic rice under lead (Pb) toxicity. Chemosphere 176, 141–155. Ashraf, U., Kanu, A.S., Mo, Z., Hussain, S., Anjum, S.A., Khan, I., Abbas, R.N., Tang, X., 2015. Lead toxicity in rice: effects, mechanisms, and mitigation strategies—a mini review. Environ. Sci. Pollut. Res. 22, 18318–18332. Ashraf, U., Hussain, S., Anjum, S.A., Abbas, F., Tanveer, M., Noor, M.A., Tang, X., 2017a. Alterations in growth, oxidative damage, and metal uptake of five aromatic rice cultivars under lead toxicity. Plant Physiol. Biochem. 115, 461–471.
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