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Ammonium nutrition mitigates cadmium toxicity in rice (Oryza sativa L.) through improving antioxidase system and the glutathione-ascorbate cycle efficiency
Ecotoxicology and Environmental Safety xxx (xxxx) xxxx
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Ammonium nutrition mitigates cadmium toxicity in rice (Oryza sativa L.) through improving antioxidase system and the glutathione-ascorbate cycle efficiency Zhichao Wua,b,c, Qi Jianga,b,c, Tao Yana,b,c, Xin Zhanga,b,c, Shoujun Xua,b,c, Hanzhi Shia,b,c, Teng-hao-bo Denga,b, Furong Lia,b, Yingqiong Dua,b, Ruiying Dua,b, Chengxiao Huc, Xu Wanga,b,∗, Fuhua Wanga,b,c,∗∗ a b c
Public Monitoring Center for Agro-Product of Guangdong Academy of Agricultural Sciences, China Key Laboratory of Testing and Evaluation for Agro-product Safety and Quality (Guangzhou), Ministry of Agriculture and Rural Affairs, China Research Center of Trace Elements/College of Resources and Environment for Huazhong Agricultural University, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitrogen form Cadmium Plant resistance Antioxidant enzyme Antioxidant Rice (Oryza sativa L.)
Nitrogen (N) forms not only affect cadmium (Cd) accumulation in plants, but also affect plant resistance to Cd toxicity. However, few researches have been reported underlying the mechanism of the relationship between nitrogen forms and plant resistance under Cd exposure. Here, we explored the mechanism on how different NO3−/NH4+ ratios affect antioxidase system and the glutathione-ascorbate cycle under five different ratios of NO3−/NH4+ (1:0, 2:1, 1:1, 1:2, 0:1) and three dosages of Cd exposure (0, 1, 5 μmol L−1 Cd) in rice (Oryza sativa L.). The results showed that high NO3− and high Cd exposure both significantly inhibited tissue growth of rice plants, and this inhibiting trend was mitigated with increasing NH4+ ratios as proved by the increased biomass and the decreased concentrations of malonaldehyde (MDA) and hydrogen peroxide (H2O2), as well as the levels of Cd contents in rice tissues. Additionally, high NH4+ ratios elevated the SOD activities in rice tissues, especially at high Cd treatment. However, other two antioxidases (CAT and APX) were insensitive to changes of NO3−/ NH4+ ratios (except the full NO3−). Furthermore, high NH4+ ratios induced increasing of the efficiency of glutathione-ascorbate cycle (GSH-AsA) under two levels of Cd exposure, as evidenced by increasing concentrations of GSH and AsA and the activities of GR and DHAR in rice tissues. Overall, these results revealed that ammonium nutrition caused an enhancement resistance to Cd stress in rice plants was responsible for increasing of partial antioxidase system and the efficiencies of GSH-AsA cycle.
1. Introduction
severe phyto-toxicity, including generating excess reactive oxygen species (ROS), decline in photosynthesis and respiration, deficiency in nutrient and water uptake, disturbation of various metabolic pathways, and even plant death (Sandalio et al., 2009; Wu et al., 2015). One of the negative effects caused by certain accumulation of Cd in plants is an enhanced generation of reactive oxygen species (ROS) (Nahakpam and Shah, 2011), which plays important roles in regulating plant metabolism in the forms of signal molecules when at a relatively low level. However, its excess generation causes various oxidative damages, including membrane lipid peroxidation, enzyme and protein inactivation, DNA molecule destruction, and growth and development inhibition (Sharma et al., 2012). To scavenge and minimize these oxidative damages from excess ROS, plants have evolved an efficient
Cadmium (Cd) is one of the most serious pollutants, which generally exists in agricultural environment (Wu et al., 2014). Due to anthropogenic and industrial activities, application of phosphate-containing fertilizers, sewage sludge, pesticides, and so on, a large amount of Cd has been imported into farmland areas (Sarwar et al., 2010), leading to Cd enrichment in edible parts of crops. In China, rice is one of the most important staple food crop with the largest planting fields, and quite a large proportion of planting soils has been reported to be suffered from high degree of Cd contamination (Lin et al., 2012a, 2012b), resulting in great concern of health risks to people (Jalloh et al., 2009). Plants when grown in Cd-contaminated environment frequently emerge a series of
∗
Corresponding author. Public Monitoring Center for Agro-Product of Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China. Corresponding author. Public Monitoring Center for Agro-Product of Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China. E-mail addresses: [email protected] (X. Wang), [email protected] (F. Wang).
∗∗
https://doi.org/10.1016/j.ecoenv.2019.110010 Received 27 August 2019; Received in revised form 22 October 2019; Accepted 26 November 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Zhichao Wu, et al., Ecotoxicology and Environmental Safety, https://doi.org/10.1016/j.ecoenv.2019.110010
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all experiments was in terms of Wu et al. (2018a). After another 4-day cultivation with 1/2-strength nutrient solution, the full-strength nutrient solution containing each treatment was used. The experimental treatments of five different ratios of NO3−/NH4+ were 1:0 (NO32.86 mmol L−1), 2:1 (NO3-1.91 mmol L−1 and NH4+-0.95 mmol L−1), 1:1 (NO3-1.43 mmol L−1 and NH4+-1.43 mmol L−1, considered as the normal NO3−/NH4+ ratio), 1:2 (NO3-0.95 mmol L−1 and NH4+1.91 mmol L−1), 0:1 (NH4+-2.86 mmol L−1), and the Cd-treated concentrations was 0, 1, 5 μmol L−1 by using of analytical reagent CdCl2. The five ratios of NO3−/NH4+ were obtained through adjusting the concentrations of analytical reagents of NH4Cl, NH4NO3, and NaNO3. Each treatment repeated four times. The nutrient solution was adjusted to pH 5.5 by addition of diluted NaOH or HCl solution, was renewed every 3 days. The experimental containers were placed in a controlled greenhouse with a light/dark (16 h/8 h) cycle and 30 °C during the day and 25 °C during the night with 180 μmol m−2 s−1 irradiance. After 25day growth with each treatment, plant seedlings were harvested. Some plant samples were stored at −80 °C in an ultra-low temperature freezer (Thermo Forma 900, USA).
antioxidative system, which involves a number of antioxidases, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and so on, as well as non-enzyme antioxidants such as ascorbic acid (AsA) and reduced glutathione (GSH) (Shah et al., 2001; Zornoza et al., 2010; Wu et al., 2018b). Nitrogen (N) is an essential nutrient for plant growth and development, which always exists high deficiency in most planting soils (Herandez et al., 1997). As important components of various structural, functional and genetic molecules in plant cells, nitrogen plays important roles in defensing various adverse stresses (Durner and Klessig, 1999; Wu et al., 2018a). Ammonium (NH4+) and nitrate (NO3−) are two important and the most forms of inorganic nitrogen taken up by plants. It has long been observed that supply of different nitrogen forms caused variant effects on plant resistance or tolerance to adverse stresses, especially for heavy-metal stress. One of these is that influences Cd uptake, accumulation, and even its chemical speciation in plant tissues. It has been reported that plant supplied with NH4+ enhanced Cd accumulation in potato (Larsson Jönsson and Asp, 2011), sunflower and tobacco (Cheng et al., 2018), Populus clones (Bi et al., 2019) and hyperaccumulation plants (Cheng et al., 2016). However, the inconsistent observations have also been pointed out that supply of NO3− improved Cd accumulation in plants, these including in wheat (Wångstrand et al., 2007), and rice (Wu et al., 2018a), and hyperaccumulator such as Rorippa globosa (Turcz.) Thell. (Wei et al., 2015). The other possible strategies against Cd toxicity is depended on changing in antioxidant system. Lin et al. (2011) reported that nitrogen deficiency decreased the activities of CAT, APX and GR, but increased SOD in leaves of rice seedlings under Cd stress. Lin et al. (2012a, 2012b) found that high nitrogen application showed greater effects on increasing alleviation of Cd toxicity than that of lower nitrogen level in Populus yunnanensis by improving the activities of some antioxidant enzymes. Nogueirol et al. (2018) suggested that tomato plants treated with NO3− increased the activities of APX and guaiacol peroxidase (GPOX) in both shoots and roots under Cd stress, but decreased the CAT activities. Bi et al. (2019) indicated that NH4+ supply did not affect the activities of POD and GR in leaves in Nanlin 895 and decreased the CAT activities in leaves in Nanlin 1388, while no differences were observed for NO3− treatment in two contrasting Populus clones. However, few literatures could be found on how different ratios of NH4+/NO3− influence plant resistance to Cd toxicity related to the antioxidase system and the GSH-AsA cycle in rice plants. The objectives of this study were to investigate application of different ratios of NH4+/NO3− affected the resistance mechanisms related to antioxidase system and the GSH-AsA cycle in rice plants under lowand-high dosages of Cd exposure. It was hypothesized that rice plants treated with NH4+, rather than NO3−, could promote plant resistance to Cd stress, which might be responsible for increasing the efficiencies of antioxidase system and the GSH-AsA cycle. These findings will provide a high-efficiency strategy for alleviating Cd stress and increasing the crop yield grown in Cd-contaminated areas.
2.2. Growth and Cd concentration measurement in plant tissues The harvested plant seedlings were washed clearly by using deionized water, and were divided into two parts of shoots and roots. Then using absorbing paper to suck off the surface water of plant tissues, and were measured as fresh weight (FW). Cadmium measurement in plant tissues was according to the method from Wu et al. (2018a). Generally, fresh tissues (1.0 g) were ground into powder, and were digested in a 10-ml mixture of HNO3–HClO4 (4:1). The Cd concentrations of the diluted digestive solution were measured by inductively coupled plasma mass spectrometry (Agilent 7900, ICP-MS, Japan). 2.3. Measurement of MDA and H2O2 in plant tissues Measurement of MDA and H2O2 concentrations in plant tissues of shoots and roots was used the methods from Ben Amor et al. (2005) and Alexieva et al. (2001). Briefly, fresh separated tissues (0.5 g) were homogenized with 5 mL of 0.1% cold trichloroacetic acid (TCA) at 4 °C. After centrifuging at 12,000 g for 15 min at 4 °C, the supernatant was used to measure the concentrations of H2O2 and MDA in plant tissues. For MDA measurement, the reaction system solution containing of 1 mL of the supernatant and 4 mL of 0.5% thiobarbituric acid (TBA in 20% TCA) was placed at 95 °C for 30 min, and was quickly cooled. After centrifuging at 12,000 g for 20 min, MDA concentrations in supernatant were measured from difference values at 532 and 600 nm by using a extinction coefficient of 155 mmol cm−1 through a spectrophotometer (TU-1901, China). For H2O2 measurement, the reaction mixture was consisted of 1 mL of the supernatant, 1 mL of 100 mmol L−1 K3PO4 buffer (pH 6.5) and 4 mL of 1 mol L−1 KI. After reaction in the dark condition for 1 h, the mixture was used for determination of H2O2 concentration at 390 nm by a spectrophotometer.
2. Materials and methods 2.4. Measurement of GSH and AsA in plant tissues 2.1. Plant material, growth condition and treatment Measurement of GSH and AsA in plant tissues of shoots and roots was used the methods described in Smith (1985) and Law et al. (1983). Simply, Fresh plant tissues (0.5 g) were homogenized in 5 mL of acidic buffer containing 5% K3PO4 and 1 mmol L−1 ethylenediaminetetraacetic acid at 4 °C. After centrifuging at 12,000 g at 4 °C for 20 min, the supernatant was used to measure the concentrations of GSH and AsA in plant tissues. Reduced glutathione assay was based on an enzyme-recycling at 412 nm, and AsA measurement was in terms of the decline of Fe3+ to Fe2+, and was measured the form complexes at 525 nm. Both GSH and AsA concentrations were calculated according to the respective standard curve by a spectrophotometer.
Plant material of ‘Nongzhan Gui’ (Oryza sativa L.) rice with high Cd accumulation in its edible parts was used for all experiments of this research. Seed surface was sterilized by immersing in 5% sodium hypochlorite solution for 15 min, washed with deionized water, and then soaking in deionized water at 28 °C for 24 h incubation. Subsequently, seeds were placed on wetted filter paper for germination in an incubator with a controlled dark environment at 28 °C. After one week, the morphological uniform seedlings were transferred to the plastic containers (37.8 × 27.8 × 9.0 cm) containing 6-L 1/4-strength nutrient solution for 4-day pre-cultivation. The nutrient solution used in 2
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3.2. Effects of different NO3−/NH4+ ratios on MDA and H2O2 concentrations in shoots and roots under Cd stress
2.5. Measurement of antioxidase in plant tissues Frozen plant tissues of shoots and roots (0.5 g) stored at −80 °C were homogenized under liquid nitrogen condition. After addition 5 mL of 50 mmol L−1 K3PO4 buffer (pH 7.0) containing 0.4 mmol L−1 EDTA, 5 mmol L−1 ascorbate, and 2% polyvinyl polypyrrolidone, and was centrifuged at 12,000 g at 4 °C for 30 min. The supernatant was used for measurement of protein concentrations and antioxidase activities in plant tissues. Protein measurement in the supernatant was according to the procedure from Bradford (1976). Superoxide dismutase was measured in terms of Stewart and Bewley (1980) by calculating the values of inhibiting 50% initial decline of nitro blue tetrazolium chloride under light at 560 nm. Catalase measurement was in terms of the method described by Aebi (1984) through calculating the decomposition values of H2O2 at 240 nm, and one unit of CAT activity was mean that CAT decomposed 1 μmol H2O2 in 1 min. Ascorbate peroxidase measurement was used the method from Nakano and Asada (1981) by calculating the decline values of AsA concentrations at 290 nm with a extinction coefficient (2.8 mmol−1 cm−1), and one unit of APX activity was mean that APX decomposed 1 μmol AsA in 1 min. Glutathione reductase measurement was in accordance with Foyer and Halliwell (1976) through calculating the decline values of oxidized glutathione at 340 nm, and one unit of GR activity was mean that GR decomposed 1 μmol oxidized glutathione in 1 min. Dehydroascorbate reductase measurement was used the method of Nakano and Asada (1981) through calculating the generation of reduced ascorbate at 265 nm, and one unit of DHAR was mean that DHAR decomposed 1 μmol dehydroascorbate in 1 min.
Malonaldehyde and hydrogen peroxide were another two important indices to evaluate the resistance degree to Cd stress. As shown in Fig. 1, high NO3− ratios (NO3−/NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) markedly promoted the MDA concentrations in shoots and roots. For non and low dosage of Cd exposure, the MDA concentrations in shoots and roots emerged little changes among high NH4+ ratio treatments, and were greatly lower than those of the full NO3− treatment being of 13–30% for shoots and 17–27% for roots under non Cd (Fig. 1A and D), and of 11–25% for shoots and 13–26% for roots under low dosage of Cd exposure (Fig. 1B and E). For high dosage of Cd exposure, the MDA concentrations in shoots and roots were gradually decreased with increasing of NH4+ ratios with values of 12–38% lower for shoots and 14–38% lower for roots than those the full NO3− treatment respectively (Fig. 1C and F). The changes of H2O2 concentrations in shoots and roots showed familiar trends with MDA. For non and low dosage of Cd exposure, high NH4+ ratios significantly reduced the H2O2 concentrations than those the full NO3− treatment, being of 15–23% lower for shoots and 15–35% lower for roots under non Cd (Fig. 1G and J), and of 16–28% for shoots and 12–30% for roots under low dosage of Cd exposure (Fig. 1H and K). For high dosage of Cd exposure, high NH4+ ratios markedly decreased the H2O2 concentrations in shoots and roots with values of 14–39% and 15–41% than those the full NO3− treatment respectively (Fig. 1I and L). Based on the findings of MDA and H2O2, these suggest that NH4+-N treated rice plants lead a higher resistance than NO3−-N, especially at high Cd exposure.
2.6. Statistical analysis
3.3. Effects of different NO3−/NH4+ ratios on GSH and AsA concentrations in shoots and roots under Cd stress
All data in this research was statistically analyzed through using one-way analysis of variance (ANOVA) followed by LSD test at significant level of P ≤ 0.05 by using SPSS 20.0 software. The diagrams in the figure part were drawn in terms of Sigmaplot 12.5 software.
Reduced glutathione and ascorbic acid, as two key antioxidants in GSH-AsA cycle, show important roles in maintaining its high efficiency to scavenge ROS. As shown in Fig. 2, both high NO3− ratios (NO3−/ NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) greatly reduced the GSH concentrations in shoots and roots, while high NH4+ ratios treatments significantly increased them. For non, low and high dosages of Cd exposure, the GSH concentrations in shoots and roots showed little changes among high NH4+ ratios treatments, and were markedly higher compared to the full NO3− with values of 29–45% for shoots and 30–38% for roots under non Cd (Fig. 2A and D), of 31–39% for shoots and 35–40% for roots under low dosage of Cd exposure (Fig. 2B and E), and of 35–70% for shoots and 24–56% for roots under high dosage of Cd exposure (Fig. 2C and F). Similar trends were also found for AsA in shoots and roots. The AsA concentrations in high NH4+ ratios treatments were significantly higher than those the full NO3− treatment being of 30–32% for shoots and 15–18% for roots under non Cd treatment (Fig. 2G and J), of 38–40% for shoots and 27–30% for roots under low dosage of Cd exposure (Fig. 2H and K), and of 20–38% for shoots and 15–29% for roots under high dosage of Cd exposure (Fig. 2I and L). These findings mean that NH4+-N treated rice plants have higher levels of GSH and AsA in plant tissues than NO3−-N, leading greater resistance to high Cd stress.
3. Results 3.1. Effects of different NO3−/NH4+ ratios on fresh weight and Cd distribution in shoots and roots Plant tissue weight is considered as one of the most important indices to evaluate the resistance degree to Cd stress. As shown in Table 1, both high NO3− ratios (NO3−/NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) significantly inhibited plant growth. Without Cd addition, plant growth was significantly inhibited under the treatments of high NO3−ratios, and these inhibition effects were gradually alleviated with increasing of NH4+ ratios being values of 26–51% higher for shoots and 13–37% higher for roots than those of the full NO3− treatment respectively. Similar trends were also found under low and high Cd treatments, with values of 31–52% higher for shoots and 13–42% higher for roots under low dosage of Cd exposure, and of 17–79% higher for shoots and 10–61% higher for roots under high dosage of Cd exposure. These findings mean that NH4+-N treated rice plants show a better growth than NO3−-N, especially at high Cd stress. Increasing NH4+ratios significantly reduced the Cd concentrations of shoots and roots with values of 14–42% and 16–40% lower for shoots, and of 16–39% and 12–34% lower for roots than NO3−/NH4+ (2:1) under low and high dosages of Cd exposure respectively, while the full NO3− was 11% and 15% lower for shoots and of 18% and 12% lower for roots than NO3−/NH4+ (2:1). For low dosage of Cd exposure, the Cd accumulation in each tissue, as well as in the whole plant showed declining tendencies with increasing of NH4+ ratios, while no significant differences were found for these indexes under high dosage of Cd exposure.
3.4. Effects of different NO3−/NH4+ ratios on antioxidase activities in shoots and roots under Cd stress The key antioxidases, such as SOD, CAT, APX, GR and DHAR, play important effects in minimizing the oxidative damage and maintaining the cellular redox status. Superoxide dismutase catalyzes superoxide radicals to less toxic substance of H2O2. As shown in Fig. 3, high treatment NO3− (NO3−/ NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) greatly reduced the SOD activities in shoots and roots, while high NH4+ ratios treatments greatly 3
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Table 1 Effects of five NO3−/NH4+ ratios on fresh weight and Cd distribution in shoots and roots under three dosages of Cd exposure. Treatments
Shoot FW g plant
−1
Root FW
Shoot Cd Conc.
−1
μg g
g plant
−1
Root Cd Conc. μg g
FW
−1
Shoot Cd Accum. −1
FW
Root Cd Accum.
μg plant
μg plant
ND ND ND ND ND
ND ND ND ND ND
−1
Total Cd Accum. μg plant−1
0 μmol L−1 Cd NO3−:NH4+(1:0) NO3−:NH4+(2:1) NO3−:NH4+(1:1) NO3−:NH4+(1:2) NO3−:NH4+(0:1)
8.81 ± 0.97 c 11.09 ± 1.59 b 13.17 ± 1.43 a 13.32 ± 1.17 a 12.92 ± 1.65 ab 1 μmol L−1 Cd
1.52 1.71 2.02 2.06 2.08
± ± ± ± ±
0.14 0.16 0.21 0.15 0.27
b b a a a
ND ND ND ND ND
NO3−:NH4+(1:0) NO3−:NH4+(2:1) NO3−:NH4+(1:1) NO3−:NH4+(1:2) NO3−:NH4+(0:1)
9.11 ± 0.68 c 11.94 ± 0.81 b 13.57 ± 1.48 a 13.29 ± 0.87 ab 13.88 ± 1.20 a 5 μmol L−1 Cd
1.49 1.68 2.12 2.09 2.08
± ± ± ± ±
0.11 0.13 0.16 0.12 0.17
b b a a a
2.47 2.79 2.40 2.02 1.63
± ± ± ± ±
0.22 0.25 0.19 0.17 0.13
b a b c d
37.7 45.9 38.4 33.5 28.2
b a b c d
22.5 33.3 32.4 27.1 22.5
± ± ± ± ±
1.59 2.47 2.63 2.24 1.95
c a a b c
56.3 77.4 81.4 70.1 58.2
± ± ± ± ±
4.18 5.67 6.81 6.04 4.11
c b a b c
78.8 ± 6.14 c 110 ± 9.89 a 113 ± 10.2 a 95.8 ± 7.36 b 81.3 ± 9.01 c
NO3−:NH4+(1:0) NO3−:NH4+(2:1) NO3−:NH4+(1:1) NO3−:NH4+(1:2) NO3−:NH4+(0:1)
6.51 ± 0.54 e 7.61 ± 0.36 d 8.85 ± 0.40 c 10.19 ± 0.61 b 11.63 ± 0.79 a
1.09 1.19 1.32 1.53 1.75
± ± ± ± ±
0.08 0.11 0.13 0.13 0.14
d cd c b a
5.08 5.97 5.02 4.25 3.58
± ± ± ± ±
0.42 0.60 0.45 0.35 0.30
b a b c d
99.2 ± 9.82 b 113 ± 10.2 a 100 ± 8.23 b 87.4 ± 7.48 c 74.8 ± 6.65 d
33.1 45.4 44.4 44.6 42.3
± ± ± ± ±
2.80 3.17 2.23 3.54 3.48
b a a a a
110 134 132 135 129
± ± ± ± ±
9.25 10.4 10.8 10.4 11.6
b a a a a
143 180 175 176 171
ND ND ND ND ND
± ± ± ± ±
3.21 3.78 2.85 2.47 1.96
ND ND ND ND ND
± ± ± ± ±
11.4 12.8 13.0 12.8 10.3
b a a a a
Values of data indicate as means ± standard deviation, and different lowercase letters mean significantly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘ND’, ‘Conc.’ and ‘Accum.’ mean ‘not detected’, ‘concentration’ and ‘accumulation’ respectively. The Cd accumulation of shoots or root is obtained by multiplying the fresh weight of shoots or roots with their Cd concentration respectively. The total Cd accumulation is through the Cd accumulation of shoots plus the Cd accumulation of roots.
higher resistance to Cd stress.
promoted them. With three levels of Cd exposure, the SOD activities in shoots and roots exhibited little differences among high NH4+ ratios treatments, and were markedly higher than those of the full NO3− treatment with values of 15–42% for shoots and 22–59% for roots under non Cd (Fig. 3A and D), of 18–59% for shoots and 22–60% for roots under low dosage of Cd exposure (Fig. 3B and F), and of 14–42% for shoots and 27–77% for roots under high dosage of Cd exposure (Fig. 3C and F). Catalase and ascorbate peroxidase convert excess of H2O2 into water and divalent oxygen. As shown in Fig. 4, full NO3− and high Cd remarkably reduced the CAT activities in shoots and roots under three levels of Cd exposure, and no great differences were observed among other four NO3−/NH4+ ratios. Similar trends were also found for APX under three levels of Cd exposure. Glutathione reductase and dehydroascorbate reductase are two key antioxidases in GSH-AsA cycle, show vital roles in maintaining high levels of GSH and AsA. As shown in Fig. 5, high NO3− (NO3−/NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) noteworthy reduced the GR activities in shoots and roots. For non and low dosages of Cd exposure, the GR activities in shoots and roots showed little differences among high NH4+ ratio treatments, and were greatly higher than those of the full NO3− treatment being of 14–41% for shoots and 13–45% for roots under non Cd (Fig. 5A and D), and of 20–49% for shoots and 14–47% for roots under low dosage of Cd exposure (Fig. 5B and E). For high dosage of Cd exposure, the GR activities in shoots and roots were gradually increased with increasing of NH4+ ratios being values of 14–62% higher for shoots and 13–78% higher for roots than those of the full NO3− treatment respectively (Fig. 5C and F). For DHAR, high NH4+ ratios significantly increased the DHAR activities in shoots and roots than those of the full NO3− treatment with values of 21–31% higher for shoots and 39–50% higher for roots under non Cd (Fig. 5G and J), of 31–39% higher for shoots and 55–69% higher for roots under low dosage of Cd exposure (Fig. 5H and K), and of 25–65% higher for shoots and 24–70% higher for roots under high dosage of Cd exposure (Fig. 5I and L). From this data of antioxidases, it means that NH4+-N treated rice plants have greater activities of antioxidase than NO3−-N, leading
4. Discussion It is generally known that plant growth biomass and the degree of oxidative damage were the most important indicators to evaluate plant resistance to various adverse stresses (Wu et al., 2017). In the present study, high Cd treatments and nitrogen forms both showed important effects on plant tissue biomass, with high NO3−-N ratios and high Cd reducing tissue biomass and increasing the accumulation of MDA and H2O2 in rice tissues, and these negative effects were gradually mitigated with increasing NH4+ ratios, especially at high Cd stress. Similar evidences were also observed in Oryza sativa (L.), Solanum lycopersicon and tanzania guinea, which found that NH4+-fed plants had higher growth and less levels of MDA accumulation than those grown with NO3−-fed plants under Cd exposure (Hassan et al., 2005; Nasraoui et al., 2012; de Sousa Leite and Monteiro, 2019). The reasons to this finding may be assumed that NO3−-N induced an enhancement of generation of organic acids, and thus promoted Cd migration to shoots within xylem by forming complexes with organic acid, leading more Cd accumulation in plant shoots and subsequently caused oxidative stress (Hassan et al., 2005; Wu et al., 2018a). Here, we also found that NH4+-fed rice plants showed lower Cd concentrations both in shoots and roots than NO3−. These results have been confirmed by several studies (Hassan et al., 2008; Jalloh et al., 2009; Nogueirol et al., 2018). In our previous research, we observed that inhibition of Cd uptake by NH4+ might be responsible for competing for binding sites with Cd2+ and changing the internal physiological characteristics of the root system (Wu et al., 2018a). From the results of this study, it indicates that NH4+-N nutrition has a greater function in alleviating Cd toxicity than NO3−-N nutrition, as proved by strengthening plant growth and reducing accumulation of H2O2 and MDA in plant tissues. Cadmium accumulation to a certain extent in plant cells causes serious damages on physiological/biochemical processes due to excess generation of ROS (Gallego et al., 2012). To decrease or avoid stress damages, plants have developed a series of defensive mechanisms (Rizwan et al., 2016). One of these defensive mechanisms is involved in 4
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Fig. 1. Effects of five NO3−/NH4+ ratios on MDA and H2O2 concentrations in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Conc.’ indicates ‘concentration’. ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
were mitigated with increasing of NH4+ ratios. This suggested that NH4+ treated rice plants caused higher SOD activities than those treated with NO3−-N, thus had a higher ability to eliminate ROS. Similar results have also been reported that SOD activities in plant tissues showed variation of difference derived from the two nitrogen regimes under Cd stress, were more pronouncedly enhanced in NH4+-N than
antioxidase system, which removes ROS through the sequential and simultaneous processes of enzymatic catalysis reaction. Superoxide dismutase, as the first enzyme in the detoxifying process, catalyzes the two molecules of superoxide into the less toxic H2O2 and oxygen at a very fast rate. Here, high Cd and high NO3− treatments remarkably reduced the activities of SOD in rice tissues, and these inhibition trends 5
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Fig. 2. Effects of five NO3−/NH4+ ratios on GSH and AsA concentrations in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Conc.’ indicates ‘concentration’. ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
NH4+-N in Oryza sativa (Jalloh et al., 2009) and Solanum (Nasraoui et al., 2012). It is participated that the enhanced SOD activity by NH4+N might be owing to maintaining of ionic microenvironment balance and enhancing expression of resistance genes in leaves, protecting plant
biochemical structures and synthetic machinery (Jalloh After ROS catalysis into oxidases of CAT and APX 6
minimizing the sensitivity of the photoet al., 2009; Nasraoui et al., 2010, 2012). H2O2 by SOD enzyme, another two antiare required to continue to catalyze its
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Fig. 3. Effects of five NO3−/NH4+ ratios on SOD activities in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
induced by NH4+-N under Cd stress (Finkemeier et al., 2003). In other hand, NH4+-N might induce generation of signal molecules which enhanced biosynthesis of DHAR and GR (Konotop et al., 2012). However, no strong evidences on the effects of different nitrogen forms affected the activities of these antioxidases in plant tissues were found, thus the mechanism involved in increasing of these enzymes by NH4+-N treatment still needs further researches.
reaction into molecular oxygen and water. Here, high Cd and full NO3−-N treatments both caused noteworthy decrease in activities of CAT and APX in plant tissues. However, no great changes were observed among other four NO3−/NH4+ ratios. These mean that the increased activities of CAT and APX under Cd stress were independent on different nitrogen forms, and only suggesting a compensatory and adaptive mechanism of plants by increasing H2O2 scavenging. Nasraoui et al. (2012) found the enhancement degrees of CAT and APX were higher in NH4+-treated tomato seedlings than those grown with NO3−, and pointed outed that antioxidative ability was higher induced by Cd when NH4+ used as nitrogen source. It was reported that the APX activities in plant tissues of tomato plants showed increasing trends, but the opposite results were also found for the CAT activities exposed to Cd (Nogueirol et al., 2018). Beside different nitrogen forms, the alteration of CAT and APX was dependent on nitrogen-treated levels, with less activities in nitrogen deficiency and higher activities in nitrogen sufficiency (Lin et al., 2011; Konotop et al., 2012). However, the reasons to these discrepant responses of CAT and APX to different nitrogen forms under Cd-stressed conditions in different plant species are needed further researches. Another important antioxidant system against Cd stress is the GSHAsA cycle, which is consisted of the main associated enzymes (GR and DHAR) that catalyzes and maintains high efficiency of generation of GSH and AsA to remove excess H2O2 (Hasanuzzaman et al., 2012). Several researches have been reported that the efficiencies of GSH-AsA cycle could be enhanced under adverse stresses (Gill and Tuteja, 2010; Kanwar et al., 2015), while severe stresses showed an opposite trend (Talukdar, 2012; Srivastava et al., 2014). Here, the efficiencies of GSHAsA cycle were significantly reduced under high Cd and the full NO3−N treatments, and these inhibition effects were alleviated with increasing of NH4+-N ratios. This increase in the efficiencies of GSH-AsA cycle could be caused by an increased rate of phytochelatin synthesis
5. Conclusions This research was investigated the relationship between the nitrogen forms and the plant resistance to Cd stress in rice seedlings. There was a strong evidence that NH4+-fed rice plants had greater effects on enhancement of plant resistance to Cd stress compared with NO3−-N, as proved by increasing of partial antioxidase system and the efficiency of GSH-AsA cycle. These findings promote our knowledge on the alleviating mechanism of NH4+-mediated resistance to Cd stress in crop plants, and imply that the application of reduced N fertilizers is an effective method for alleviating Cd stress and increasing the crop yield grown in Cd-contaminated areas. Further work is required to research the molecular and metabolic mechanisms of crop plants treated with different nitrogen forms under Cd stress, and needs confirm these findings in the field conditions.
Declaration of interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
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Fig. 4. Effects of five NO3−/NH4+ ratios on CAT and APX activities in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
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Fig. 5. Effects of five NO3−/NH4+ ratios on GR and DHAR activities in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
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Acknowledgements
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Ammonium nutrition mitigates cadmium toxicity in rice (Oryza sativa L.) through improving antioxidase system and the glutathione-ascorbate cycle efficiency Zhichao Wua,b,c, Qi Jianga,b,c, Tao Yana,b,c, Xin Zhanga,b,c, Shoujun Xua,b,c, Hanzhi Shia,b,c, Teng-hao-bo Denga,b, Furong Lia,b, Yingqiong Dua,b, Ruiying Dua,b, Chengxiao Huc, Xu Wanga,b,∗, Fuhua Wanga,b,c,∗∗ a b c
Public Monitoring Center for Agro-Product of Guangdong Academy of Agricultural Sciences, China Key Laboratory of Testing and Evaluation for Agro-product Safety and Quality (Guangzhou), Ministry of Agriculture and Rural Affairs, China Research Center of Trace Elements/College of Resources and Environment for Huazhong Agricultural University, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitrogen form Cadmium Plant resistance Antioxidant enzyme Antioxidant Rice (Oryza sativa L.)
Nitrogen (N) forms not only affect cadmium (Cd) accumulation in plants, but also affect plant resistance to Cd toxicity. However, few researches have been reported underlying the mechanism of the relationship between nitrogen forms and plant resistance under Cd exposure. Here, we explored the mechanism on how different NO3−/NH4+ ratios affect antioxidase system and the glutathione-ascorbate cycle under five different ratios of NO3−/NH4+ (1:0, 2:1, 1:1, 1:2, 0:1) and three dosages of Cd exposure (0, 1, 5 μmol L−1 Cd) in rice (Oryza sativa L.). The results showed that high NO3− and high Cd exposure both significantly inhibited tissue growth of rice plants, and this inhibiting trend was mitigated with increasing NH4+ ratios as proved by the increased biomass and the decreased concentrations of malonaldehyde (MDA) and hydrogen peroxide (H2O2), as well as the levels of Cd contents in rice tissues. Additionally, high NH4+ ratios elevated the SOD activities in rice tissues, especially at high Cd treatment. However, other two antioxidases (CAT and APX) were insensitive to changes of NO3−/ NH4+ ratios (except the full NO3−). Furthermore, high NH4+ ratios induced increasing of the efficiency of glutathione-ascorbate cycle (GSH-AsA) under two levels of Cd exposure, as evidenced by increasing concentrations of GSH and AsA and the activities of GR and DHAR in rice tissues. Overall, these results revealed that ammonium nutrition caused an enhancement resistance to Cd stress in rice plants was responsible for increasing of partial antioxidase system and the efficiencies of GSH-AsA cycle.
1. Introduction
severe phyto-toxicity, including generating excess reactive oxygen species (ROS), decline in photosynthesis and respiration, deficiency in nutrient and water uptake, disturbation of various metabolic pathways, and even plant death (Sandalio et al., 2009; Wu et al., 2015). One of the negative effects caused by certain accumulation of Cd in plants is an enhanced generation of reactive oxygen species (ROS) (Nahakpam and Shah, 2011), which plays important roles in regulating plant metabolism in the forms of signal molecules when at a relatively low level. However, its excess generation causes various oxidative damages, including membrane lipid peroxidation, enzyme and protein inactivation, DNA molecule destruction, and growth and development inhibition (Sharma et al., 2012). To scavenge and minimize these oxidative damages from excess ROS, plants have evolved an efficient
Cadmium (Cd) is one of the most serious pollutants, which generally exists in agricultural environment (Wu et al., 2014). Due to anthropogenic and industrial activities, application of phosphate-containing fertilizers, sewage sludge, pesticides, and so on, a large amount of Cd has been imported into farmland areas (Sarwar et al., 2010), leading to Cd enrichment in edible parts of crops. In China, rice is one of the most important staple food crop with the largest planting fields, and quite a large proportion of planting soils has been reported to be suffered from high degree of Cd contamination (Lin et al., 2012a, 2012b), resulting in great concern of health risks to people (Jalloh et al., 2009). Plants when grown in Cd-contaminated environment frequently emerge a series of
∗
Corresponding author. Public Monitoring Center for Agro-Product of Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China. Corresponding author. Public Monitoring Center for Agro-Product of Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China. E-mail addresses: [email protected] (X. Wang), [email protected] (F. Wang).
∗∗
https://doi.org/10.1016/j.ecoenv.2019.110010 Received 27 August 2019; Received in revised form 22 October 2019; Accepted 26 November 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Zhichao Wu, et al., Ecotoxicology and Environmental Safety, https://doi.org/10.1016/j.ecoenv.2019.110010
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all experiments was in terms of Wu et al. (2018a). After another 4-day cultivation with 1/2-strength nutrient solution, the full-strength nutrient solution containing each treatment was used. The experimental treatments of five different ratios of NO3−/NH4+ were 1:0 (NO32.86 mmol L−1), 2:1 (NO3-1.91 mmol L−1 and NH4+-0.95 mmol L−1), 1:1 (NO3-1.43 mmol L−1 and NH4+-1.43 mmol L−1, considered as the normal NO3−/NH4+ ratio), 1:2 (NO3-0.95 mmol L−1 and NH4+1.91 mmol L−1), 0:1 (NH4+-2.86 mmol L−1), and the Cd-treated concentrations was 0, 1, 5 μmol L−1 by using of analytical reagent CdCl2. The five ratios of NO3−/NH4+ were obtained through adjusting the concentrations of analytical reagents of NH4Cl, NH4NO3, and NaNO3. Each treatment repeated four times. The nutrient solution was adjusted to pH 5.5 by addition of diluted NaOH or HCl solution, was renewed every 3 days. The experimental containers were placed in a controlled greenhouse with a light/dark (16 h/8 h) cycle and 30 °C during the day and 25 °C during the night with 180 μmol m−2 s−1 irradiance. After 25day growth with each treatment, plant seedlings were harvested. Some plant samples were stored at −80 °C in an ultra-low temperature freezer (Thermo Forma 900, USA).
antioxidative system, which involves a number of antioxidases, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and so on, as well as non-enzyme antioxidants such as ascorbic acid (AsA) and reduced glutathione (GSH) (Shah et al., 2001; Zornoza et al., 2010; Wu et al., 2018b). Nitrogen (N) is an essential nutrient for plant growth and development, which always exists high deficiency in most planting soils (Herandez et al., 1997). As important components of various structural, functional and genetic molecules in plant cells, nitrogen plays important roles in defensing various adverse stresses (Durner and Klessig, 1999; Wu et al., 2018a). Ammonium (NH4+) and nitrate (NO3−) are two important and the most forms of inorganic nitrogen taken up by plants. It has long been observed that supply of different nitrogen forms caused variant effects on plant resistance or tolerance to adverse stresses, especially for heavy-metal stress. One of these is that influences Cd uptake, accumulation, and even its chemical speciation in plant tissues. It has been reported that plant supplied with NH4+ enhanced Cd accumulation in potato (Larsson Jönsson and Asp, 2011), sunflower and tobacco (Cheng et al., 2018), Populus clones (Bi et al., 2019) and hyperaccumulation plants (Cheng et al., 2016). However, the inconsistent observations have also been pointed out that supply of NO3− improved Cd accumulation in plants, these including in wheat (Wångstrand et al., 2007), and rice (Wu et al., 2018a), and hyperaccumulator such as Rorippa globosa (Turcz.) Thell. (Wei et al., 2015). The other possible strategies against Cd toxicity is depended on changing in antioxidant system. Lin et al. (2011) reported that nitrogen deficiency decreased the activities of CAT, APX and GR, but increased SOD in leaves of rice seedlings under Cd stress. Lin et al. (2012a, 2012b) found that high nitrogen application showed greater effects on increasing alleviation of Cd toxicity than that of lower nitrogen level in Populus yunnanensis by improving the activities of some antioxidant enzymes. Nogueirol et al. (2018) suggested that tomato plants treated with NO3− increased the activities of APX and guaiacol peroxidase (GPOX) in both shoots and roots under Cd stress, but decreased the CAT activities. Bi et al. (2019) indicated that NH4+ supply did not affect the activities of POD and GR in leaves in Nanlin 895 and decreased the CAT activities in leaves in Nanlin 1388, while no differences were observed for NO3− treatment in two contrasting Populus clones. However, few literatures could be found on how different ratios of NH4+/NO3− influence plant resistance to Cd toxicity related to the antioxidase system and the GSH-AsA cycle in rice plants. The objectives of this study were to investigate application of different ratios of NH4+/NO3− affected the resistance mechanisms related to antioxidase system and the GSH-AsA cycle in rice plants under lowand-high dosages of Cd exposure. It was hypothesized that rice plants treated with NH4+, rather than NO3−, could promote plant resistance to Cd stress, which might be responsible for increasing the efficiencies of antioxidase system and the GSH-AsA cycle. These findings will provide a high-efficiency strategy for alleviating Cd stress and increasing the crop yield grown in Cd-contaminated areas.
2.2. Growth and Cd concentration measurement in plant tissues The harvested plant seedlings were washed clearly by using deionized water, and were divided into two parts of shoots and roots. Then using absorbing paper to suck off the surface water of plant tissues, and were measured as fresh weight (FW). Cadmium measurement in plant tissues was according to the method from Wu et al. (2018a). Generally, fresh tissues (1.0 g) were ground into powder, and were digested in a 10-ml mixture of HNO3–HClO4 (4:1). The Cd concentrations of the diluted digestive solution were measured by inductively coupled plasma mass spectrometry (Agilent 7900, ICP-MS, Japan). 2.3. Measurement of MDA and H2O2 in plant tissues Measurement of MDA and H2O2 concentrations in plant tissues of shoots and roots was used the methods from Ben Amor et al. (2005) and Alexieva et al. (2001). Briefly, fresh separated tissues (0.5 g) were homogenized with 5 mL of 0.1% cold trichloroacetic acid (TCA) at 4 °C. After centrifuging at 12,000 g for 15 min at 4 °C, the supernatant was used to measure the concentrations of H2O2 and MDA in plant tissues. For MDA measurement, the reaction system solution containing of 1 mL of the supernatant and 4 mL of 0.5% thiobarbituric acid (TBA in 20% TCA) was placed at 95 °C for 30 min, and was quickly cooled. After centrifuging at 12,000 g for 20 min, MDA concentrations in supernatant were measured from difference values at 532 and 600 nm by using a extinction coefficient of 155 mmol cm−1 through a spectrophotometer (TU-1901, China). For H2O2 measurement, the reaction mixture was consisted of 1 mL of the supernatant, 1 mL of 100 mmol L−1 K3PO4 buffer (pH 6.5) and 4 mL of 1 mol L−1 KI. After reaction in the dark condition for 1 h, the mixture was used for determination of H2O2 concentration at 390 nm by a spectrophotometer.
2. Materials and methods 2.4. Measurement of GSH and AsA in plant tissues 2.1. Plant material, growth condition and treatment Measurement of GSH and AsA in plant tissues of shoots and roots was used the methods described in Smith (1985) and Law et al. (1983). Simply, Fresh plant tissues (0.5 g) were homogenized in 5 mL of acidic buffer containing 5% K3PO4 and 1 mmol L−1 ethylenediaminetetraacetic acid at 4 °C. After centrifuging at 12,000 g at 4 °C for 20 min, the supernatant was used to measure the concentrations of GSH and AsA in plant tissues. Reduced glutathione assay was based on an enzyme-recycling at 412 nm, and AsA measurement was in terms of the decline of Fe3+ to Fe2+, and was measured the form complexes at 525 nm. Both GSH and AsA concentrations were calculated according to the respective standard curve by a spectrophotometer.
Plant material of ‘Nongzhan Gui’ (Oryza sativa L.) rice with high Cd accumulation in its edible parts was used for all experiments of this research. Seed surface was sterilized by immersing in 5% sodium hypochlorite solution for 15 min, washed with deionized water, and then soaking in deionized water at 28 °C for 24 h incubation. Subsequently, seeds were placed on wetted filter paper for germination in an incubator with a controlled dark environment at 28 °C. After one week, the morphological uniform seedlings were transferred to the plastic containers (37.8 × 27.8 × 9.0 cm) containing 6-L 1/4-strength nutrient solution for 4-day pre-cultivation. The nutrient solution used in 2
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3.2. Effects of different NO3−/NH4+ ratios on MDA and H2O2 concentrations in shoots and roots under Cd stress
2.5. Measurement of antioxidase in plant tissues Frozen plant tissues of shoots and roots (0.5 g) stored at −80 °C were homogenized under liquid nitrogen condition. After addition 5 mL of 50 mmol L−1 K3PO4 buffer (pH 7.0) containing 0.4 mmol L−1 EDTA, 5 mmol L−1 ascorbate, and 2% polyvinyl polypyrrolidone, and was centrifuged at 12,000 g at 4 °C for 30 min. The supernatant was used for measurement of protein concentrations and antioxidase activities in plant tissues. Protein measurement in the supernatant was according to the procedure from Bradford (1976). Superoxide dismutase was measured in terms of Stewart and Bewley (1980) by calculating the values of inhibiting 50% initial decline of nitro blue tetrazolium chloride under light at 560 nm. Catalase measurement was in terms of the method described by Aebi (1984) through calculating the decomposition values of H2O2 at 240 nm, and one unit of CAT activity was mean that CAT decomposed 1 μmol H2O2 in 1 min. Ascorbate peroxidase measurement was used the method from Nakano and Asada (1981) by calculating the decline values of AsA concentrations at 290 nm with a extinction coefficient (2.8 mmol−1 cm−1), and one unit of APX activity was mean that APX decomposed 1 μmol AsA in 1 min. Glutathione reductase measurement was in accordance with Foyer and Halliwell (1976) through calculating the decline values of oxidized glutathione at 340 nm, and one unit of GR activity was mean that GR decomposed 1 μmol oxidized glutathione in 1 min. Dehydroascorbate reductase measurement was used the method of Nakano and Asada (1981) through calculating the generation of reduced ascorbate at 265 nm, and one unit of DHAR was mean that DHAR decomposed 1 μmol dehydroascorbate in 1 min.
Malonaldehyde and hydrogen peroxide were another two important indices to evaluate the resistance degree to Cd stress. As shown in Fig. 1, high NO3− ratios (NO3−/NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) markedly promoted the MDA concentrations in shoots and roots. For non and low dosage of Cd exposure, the MDA concentrations in shoots and roots emerged little changes among high NH4+ ratio treatments, and were greatly lower than those of the full NO3− treatment being of 13–30% for shoots and 17–27% for roots under non Cd (Fig. 1A and D), and of 11–25% for shoots and 13–26% for roots under low dosage of Cd exposure (Fig. 1B and E). For high dosage of Cd exposure, the MDA concentrations in shoots and roots were gradually decreased with increasing of NH4+ ratios with values of 12–38% lower for shoots and 14–38% lower for roots than those the full NO3− treatment respectively (Fig. 1C and F). The changes of H2O2 concentrations in shoots and roots showed familiar trends with MDA. For non and low dosage of Cd exposure, high NH4+ ratios significantly reduced the H2O2 concentrations than those the full NO3− treatment, being of 15–23% lower for shoots and 15–35% lower for roots under non Cd (Fig. 1G and J), and of 16–28% for shoots and 12–30% for roots under low dosage of Cd exposure (Fig. 1H and K). For high dosage of Cd exposure, high NH4+ ratios markedly decreased the H2O2 concentrations in shoots and roots with values of 14–39% and 15–41% than those the full NO3− treatment respectively (Fig. 1I and L). Based on the findings of MDA and H2O2, these suggest that NH4+-N treated rice plants lead a higher resistance than NO3−-N, especially at high Cd exposure.
2.6. Statistical analysis
3.3. Effects of different NO3−/NH4+ ratios on GSH and AsA concentrations in shoots and roots under Cd stress
All data in this research was statistically analyzed through using one-way analysis of variance (ANOVA) followed by LSD test at significant level of P ≤ 0.05 by using SPSS 20.0 software. The diagrams in the figure part were drawn in terms of Sigmaplot 12.5 software.
Reduced glutathione and ascorbic acid, as two key antioxidants in GSH-AsA cycle, show important roles in maintaining its high efficiency to scavenge ROS. As shown in Fig. 2, both high NO3− ratios (NO3−/ NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) greatly reduced the GSH concentrations in shoots and roots, while high NH4+ ratios treatments significantly increased them. For non, low and high dosages of Cd exposure, the GSH concentrations in shoots and roots showed little changes among high NH4+ ratios treatments, and were markedly higher compared to the full NO3− with values of 29–45% for shoots and 30–38% for roots under non Cd (Fig. 2A and D), of 31–39% for shoots and 35–40% for roots under low dosage of Cd exposure (Fig. 2B and E), and of 35–70% for shoots and 24–56% for roots under high dosage of Cd exposure (Fig. 2C and F). Similar trends were also found for AsA in shoots and roots. The AsA concentrations in high NH4+ ratios treatments were significantly higher than those the full NO3− treatment being of 30–32% for shoots and 15–18% for roots under non Cd treatment (Fig. 2G and J), of 38–40% for shoots and 27–30% for roots under low dosage of Cd exposure (Fig. 2H and K), and of 20–38% for shoots and 15–29% for roots under high dosage of Cd exposure (Fig. 2I and L). These findings mean that NH4+-N treated rice plants have higher levels of GSH and AsA in plant tissues than NO3−-N, leading greater resistance to high Cd stress.
3. Results 3.1. Effects of different NO3−/NH4+ ratios on fresh weight and Cd distribution in shoots and roots Plant tissue weight is considered as one of the most important indices to evaluate the resistance degree to Cd stress. As shown in Table 1, both high NO3− ratios (NO3−/NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) significantly inhibited plant growth. Without Cd addition, plant growth was significantly inhibited under the treatments of high NO3−ratios, and these inhibition effects were gradually alleviated with increasing of NH4+ ratios being values of 26–51% higher for shoots and 13–37% higher for roots than those of the full NO3− treatment respectively. Similar trends were also found under low and high Cd treatments, with values of 31–52% higher for shoots and 13–42% higher for roots under low dosage of Cd exposure, and of 17–79% higher for shoots and 10–61% higher for roots under high dosage of Cd exposure. These findings mean that NH4+-N treated rice plants show a better growth than NO3−-N, especially at high Cd stress. Increasing NH4+ratios significantly reduced the Cd concentrations of shoots and roots with values of 14–42% and 16–40% lower for shoots, and of 16–39% and 12–34% lower for roots than NO3−/NH4+ (2:1) under low and high dosages of Cd exposure respectively, while the full NO3− was 11% and 15% lower for shoots and of 18% and 12% lower for roots than NO3−/NH4+ (2:1). For low dosage of Cd exposure, the Cd accumulation in each tissue, as well as in the whole plant showed declining tendencies with increasing of NH4+ ratios, while no significant differences were found for these indexes under high dosage of Cd exposure.
3.4. Effects of different NO3−/NH4+ ratios on antioxidase activities in shoots and roots under Cd stress The key antioxidases, such as SOD, CAT, APX, GR and DHAR, play important effects in minimizing the oxidative damage and maintaining the cellular redox status. Superoxide dismutase catalyzes superoxide radicals to less toxic substance of H2O2. As shown in Fig. 3, high treatment NO3− (NO3−/ NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) greatly reduced the SOD activities in shoots and roots, while high NH4+ ratios treatments greatly 3
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Table 1 Effects of five NO3−/NH4+ ratios on fresh weight and Cd distribution in shoots and roots under three dosages of Cd exposure. Treatments
Shoot FW g plant
−1
Root FW
Shoot Cd Conc.
−1
μg g
g plant
−1
Root Cd Conc. μg g
FW
−1
Shoot Cd Accum. −1
FW
Root Cd Accum.
μg plant
μg plant
ND ND ND ND ND
ND ND ND ND ND
−1
Total Cd Accum. μg plant−1
0 μmol L−1 Cd NO3−:NH4+(1:0) NO3−:NH4+(2:1) NO3−:NH4+(1:1) NO3−:NH4+(1:2) NO3−:NH4+(0:1)
8.81 ± 0.97 c 11.09 ± 1.59 b 13.17 ± 1.43 a 13.32 ± 1.17 a 12.92 ± 1.65 ab 1 μmol L−1 Cd
1.52 1.71 2.02 2.06 2.08
± ± ± ± ±
0.14 0.16 0.21 0.15 0.27
b b a a a
ND ND ND ND ND
NO3−:NH4+(1:0) NO3−:NH4+(2:1) NO3−:NH4+(1:1) NO3−:NH4+(1:2) NO3−:NH4+(0:1)
9.11 ± 0.68 c 11.94 ± 0.81 b 13.57 ± 1.48 a 13.29 ± 0.87 ab 13.88 ± 1.20 a 5 μmol L−1 Cd
1.49 1.68 2.12 2.09 2.08
± ± ± ± ±
0.11 0.13 0.16 0.12 0.17
b b a a a
2.47 2.79 2.40 2.02 1.63
± ± ± ± ±
0.22 0.25 0.19 0.17 0.13
b a b c d
37.7 45.9 38.4 33.5 28.2
b a b c d
22.5 33.3 32.4 27.1 22.5
± ± ± ± ±
1.59 2.47 2.63 2.24 1.95
c a a b c
56.3 77.4 81.4 70.1 58.2
± ± ± ± ±
4.18 5.67 6.81 6.04 4.11
c b a b c
78.8 ± 6.14 c 110 ± 9.89 a 113 ± 10.2 a 95.8 ± 7.36 b 81.3 ± 9.01 c
NO3−:NH4+(1:0) NO3−:NH4+(2:1) NO3−:NH4+(1:1) NO3−:NH4+(1:2) NO3−:NH4+(0:1)
6.51 ± 0.54 e 7.61 ± 0.36 d 8.85 ± 0.40 c 10.19 ± 0.61 b 11.63 ± 0.79 a
1.09 1.19 1.32 1.53 1.75
± ± ± ± ±
0.08 0.11 0.13 0.13 0.14
d cd c b a
5.08 5.97 5.02 4.25 3.58
± ± ± ± ±
0.42 0.60 0.45 0.35 0.30
b a b c d
99.2 ± 9.82 b 113 ± 10.2 a 100 ± 8.23 b 87.4 ± 7.48 c 74.8 ± 6.65 d
33.1 45.4 44.4 44.6 42.3
± ± ± ± ±
2.80 3.17 2.23 3.54 3.48
b a a a a
110 134 132 135 129
± ± ± ± ±
9.25 10.4 10.8 10.4 11.6
b a a a a
143 180 175 176 171
ND ND ND ND ND
± ± ± ± ±
3.21 3.78 2.85 2.47 1.96
ND ND ND ND ND
± ± ± ± ±
11.4 12.8 13.0 12.8 10.3
b a a a a
Values of data indicate as means ± standard deviation, and different lowercase letters mean significantly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘ND’, ‘Conc.’ and ‘Accum.’ mean ‘not detected’, ‘concentration’ and ‘accumulation’ respectively. The Cd accumulation of shoots or root is obtained by multiplying the fresh weight of shoots or roots with their Cd concentration respectively. The total Cd accumulation is through the Cd accumulation of shoots plus the Cd accumulation of roots.
higher resistance to Cd stress.
promoted them. With three levels of Cd exposure, the SOD activities in shoots and roots exhibited little differences among high NH4+ ratios treatments, and were markedly higher than those of the full NO3− treatment with values of 15–42% for shoots and 22–59% for roots under non Cd (Fig. 3A and D), of 18–59% for shoots and 22–60% for roots under low dosage of Cd exposure (Fig. 3B and F), and of 14–42% for shoots and 27–77% for roots under high dosage of Cd exposure (Fig. 3C and F). Catalase and ascorbate peroxidase convert excess of H2O2 into water and divalent oxygen. As shown in Fig. 4, full NO3− and high Cd remarkably reduced the CAT activities in shoots and roots under three levels of Cd exposure, and no great differences were observed among other four NO3−/NH4+ ratios. Similar trends were also found for APX under three levels of Cd exposure. Glutathione reductase and dehydroascorbate reductase are two key antioxidases in GSH-AsA cycle, show vital roles in maintaining high levels of GSH and AsA. As shown in Fig. 5, high NO3− (NO3−/NH4+, 2:1 and 1:0) and high Cd (5 μmol L−1) noteworthy reduced the GR activities in shoots and roots. For non and low dosages of Cd exposure, the GR activities in shoots and roots showed little differences among high NH4+ ratio treatments, and were greatly higher than those of the full NO3− treatment being of 14–41% for shoots and 13–45% for roots under non Cd (Fig. 5A and D), and of 20–49% for shoots and 14–47% for roots under low dosage of Cd exposure (Fig. 5B and E). For high dosage of Cd exposure, the GR activities in shoots and roots were gradually increased with increasing of NH4+ ratios being values of 14–62% higher for shoots and 13–78% higher for roots than those of the full NO3− treatment respectively (Fig. 5C and F). For DHAR, high NH4+ ratios significantly increased the DHAR activities in shoots and roots than those of the full NO3− treatment with values of 21–31% higher for shoots and 39–50% higher for roots under non Cd (Fig. 5G and J), of 31–39% higher for shoots and 55–69% higher for roots under low dosage of Cd exposure (Fig. 5H and K), and of 25–65% higher for shoots and 24–70% higher for roots under high dosage of Cd exposure (Fig. 5I and L). From this data of antioxidases, it means that NH4+-N treated rice plants have greater activities of antioxidase than NO3−-N, leading
4. Discussion It is generally known that plant growth biomass and the degree of oxidative damage were the most important indicators to evaluate plant resistance to various adverse stresses (Wu et al., 2017). In the present study, high Cd treatments and nitrogen forms both showed important effects on plant tissue biomass, with high NO3−-N ratios and high Cd reducing tissue biomass and increasing the accumulation of MDA and H2O2 in rice tissues, and these negative effects were gradually mitigated with increasing NH4+ ratios, especially at high Cd stress. Similar evidences were also observed in Oryza sativa (L.), Solanum lycopersicon and tanzania guinea, which found that NH4+-fed plants had higher growth and less levels of MDA accumulation than those grown with NO3−-fed plants under Cd exposure (Hassan et al., 2005; Nasraoui et al., 2012; de Sousa Leite and Monteiro, 2019). The reasons to this finding may be assumed that NO3−-N induced an enhancement of generation of organic acids, and thus promoted Cd migration to shoots within xylem by forming complexes with organic acid, leading more Cd accumulation in plant shoots and subsequently caused oxidative stress (Hassan et al., 2005; Wu et al., 2018a). Here, we also found that NH4+-fed rice plants showed lower Cd concentrations both in shoots and roots than NO3−. These results have been confirmed by several studies (Hassan et al., 2008; Jalloh et al., 2009; Nogueirol et al., 2018). In our previous research, we observed that inhibition of Cd uptake by NH4+ might be responsible for competing for binding sites with Cd2+ and changing the internal physiological characteristics of the root system (Wu et al., 2018a). From the results of this study, it indicates that NH4+-N nutrition has a greater function in alleviating Cd toxicity than NO3−-N nutrition, as proved by strengthening plant growth and reducing accumulation of H2O2 and MDA in plant tissues. Cadmium accumulation to a certain extent in plant cells causes serious damages on physiological/biochemical processes due to excess generation of ROS (Gallego et al., 2012). To decrease or avoid stress damages, plants have developed a series of defensive mechanisms (Rizwan et al., 2016). One of these defensive mechanisms is involved in 4
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Fig. 1. Effects of five NO3−/NH4+ ratios on MDA and H2O2 concentrations in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Conc.’ indicates ‘concentration’. ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
were mitigated with increasing of NH4+ ratios. This suggested that NH4+ treated rice plants caused higher SOD activities than those treated with NO3−-N, thus had a higher ability to eliminate ROS. Similar results have also been reported that SOD activities in plant tissues showed variation of difference derived from the two nitrogen regimes under Cd stress, were more pronouncedly enhanced in NH4+-N than
antioxidase system, which removes ROS through the sequential and simultaneous processes of enzymatic catalysis reaction. Superoxide dismutase, as the first enzyme in the detoxifying process, catalyzes the two molecules of superoxide into the less toxic H2O2 and oxygen at a very fast rate. Here, high Cd and high NO3− treatments remarkably reduced the activities of SOD in rice tissues, and these inhibition trends 5
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Fig. 2. Effects of five NO3−/NH4+ ratios on GSH and AsA concentrations in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Conc.’ indicates ‘concentration’. ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
NH4+-N in Oryza sativa (Jalloh et al., 2009) and Solanum (Nasraoui et al., 2012). It is participated that the enhanced SOD activity by NH4+N might be owing to maintaining of ionic microenvironment balance and enhancing expression of resistance genes in leaves, protecting plant
biochemical structures and synthetic machinery (Jalloh After ROS catalysis into oxidases of CAT and APX 6
minimizing the sensitivity of the photoet al., 2009; Nasraoui et al., 2010, 2012). H2O2 by SOD enzyme, another two antiare required to continue to catalyze its
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Fig. 3. Effects of five NO3−/NH4+ ratios on SOD activities in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
induced by NH4+-N under Cd stress (Finkemeier et al., 2003). In other hand, NH4+-N might induce generation of signal molecules which enhanced biosynthesis of DHAR and GR (Konotop et al., 2012). However, no strong evidences on the effects of different nitrogen forms affected the activities of these antioxidases in plant tissues were found, thus the mechanism involved in increasing of these enzymes by NH4+-N treatment still needs further researches.
reaction into molecular oxygen and water. Here, high Cd and full NO3−-N treatments both caused noteworthy decrease in activities of CAT and APX in plant tissues. However, no great changes were observed among other four NO3−/NH4+ ratios. These mean that the increased activities of CAT and APX under Cd stress were independent on different nitrogen forms, and only suggesting a compensatory and adaptive mechanism of plants by increasing H2O2 scavenging. Nasraoui et al. (2012) found the enhancement degrees of CAT and APX were higher in NH4+-treated tomato seedlings than those grown with NO3−, and pointed outed that antioxidative ability was higher induced by Cd when NH4+ used as nitrogen source. It was reported that the APX activities in plant tissues of tomato plants showed increasing trends, but the opposite results were also found for the CAT activities exposed to Cd (Nogueirol et al., 2018). Beside different nitrogen forms, the alteration of CAT and APX was dependent on nitrogen-treated levels, with less activities in nitrogen deficiency and higher activities in nitrogen sufficiency (Lin et al., 2011; Konotop et al., 2012). However, the reasons to these discrepant responses of CAT and APX to different nitrogen forms under Cd-stressed conditions in different plant species are needed further researches. Another important antioxidant system against Cd stress is the GSHAsA cycle, which is consisted of the main associated enzymes (GR and DHAR) that catalyzes and maintains high efficiency of generation of GSH and AsA to remove excess H2O2 (Hasanuzzaman et al., 2012). Several researches have been reported that the efficiencies of GSH-AsA cycle could be enhanced under adverse stresses (Gill and Tuteja, 2010; Kanwar et al., 2015), while severe stresses showed an opposite trend (Talukdar, 2012; Srivastava et al., 2014). Here, the efficiencies of GSHAsA cycle were significantly reduced under high Cd and the full NO3−N treatments, and these inhibition effects were alleviated with increasing of NH4+-N ratios. This increase in the efficiencies of GSH-AsA cycle could be caused by an increased rate of phytochelatin synthesis
5. Conclusions This research was investigated the relationship between the nitrogen forms and the plant resistance to Cd stress in rice seedlings. There was a strong evidence that NH4+-fed rice plants had greater effects on enhancement of plant resistance to Cd stress compared with NO3−-N, as proved by increasing of partial antioxidase system and the efficiency of GSH-AsA cycle. These findings promote our knowledge on the alleviating mechanism of NH4+-mediated resistance to Cd stress in crop plants, and imply that the application of reduced N fertilizers is an effective method for alleviating Cd stress and increasing the crop yield grown in Cd-contaminated areas. Further work is required to research the molecular and metabolic mechanisms of crop plants treated with different nitrogen forms under Cd stress, and needs confirm these findings in the field conditions.
Declaration of interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
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Fig. 4. Effects of five NO3−/NH4+ ratios on CAT and APX activities in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
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Fig. 5. Effects of five NO3−/NH4+ ratios on GR and DHAR activities in leaves and roots under three dosages of Cd exposure. Different lowercase letters indicate markedly statistical differences under the same dosage of Cd exposure by one-way ANOVA analysis and LSD test (P ≤ 0.05, n = 4). ‘Cd0, Cd1 and Cd5′ means ‘0, 1 and 5 μmol L−1 Cd’ respectively.
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Acknowledgements
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