Introduction

Copper (Cu) is an essential trace element and affects plant growth and development by regulating multiple metabolic pathways (Shabbir et al.2020). However, higher level of Cu can be toxic to plants and can impair shoot growth and cause leaf chlorosis and necrosis in leaf tips and edges (Ameh and Sayes 2019; Chen et al. 2015). It also can inhibit photosynthesis and metabolic functions, interfere with nutrient absorption, trigger oxidative bursts, damage cell structure, and increase plant mortality (Bouazizi et al. 2010; Gong et al. 2019). The Cu phytotoxicity affects the efficiency of phytoremediation and phytostablization in Cu-contaminated soils, especially Cu mining and metal processing areas. Several strategies, such as breeding of resistant plant varieties, solidifying soil Cu, fertilizer management, have been developed to reduce metal toxicity in plants (Rizwan et al. 2016). Nitrogen management has been regarded as the most economical and time-saving agronomic strategy to control the accumulation of metal elements, including Cd, Mn, and Zn in plants (Gao et al. 2016; Zhao and Shen 2018; Yang et al. 2020).

Nitrogen (N) is the most abundant macro-nutrient element for plant growth and developmental processes (Makino 2011; Yang et al. 2020) and has been well-known to be related to metal accumulation in plant, as well as plant tolerance to metal toxicity (Mitchell et al. 2000; Wu et al. 2019). Nitrate (NO3−-N) and ammonium (NH4+-N) are the two major forms of inorganic nitrogen absorbed by the roots of higher plants, showing variant effects on uptake, translocation, and accumulation of metals in plants (Ata-UI-Karim et al. 2020; Cheng et al. 2020; Hassan et al. 2005; Jalloh et al. 2009; Yang et al. 2020). For instance, compared to NO3−-N, NH4+-N was reported to accelerate Cd accumulation in tobacco (Tsadilas et al. 2005), sunflower (Zaccheo et al. 2006), Populus (Qasim et al. 2015), and Carpobrotus rossii (Cheng et al. 2016). Wang et al. (2021) found that adding NH4+-N induced remobilization of the immobilized Cd in soil for reduced rhizosphere pH, which resulted in Cd accumulation in Amaranthus mangostanus. However, the other studies showed that NO3−-N enhanced Cd uptake and accumulation in rice (Hassan et al. 2005; Jalloh et al. 2009; Wu et al. 2019), potato (Larsson and Asp 2013), sweet sorghum (Bai et al. 2021), and Sedum plumbizincicola (Hu et al. 2013). Similarly, NH4+-N reduced the accumulation of Al and alleviated the metal’s toxicity to rice (Wang et al. 2015) and Lespedeza bicolor (Chen et al. 2010). Therefore, the practical use of nitrogen fertilizer should take into account plant species, nitrogen forms, metal types and levels, and soil characteristics (Yang et al. 2020). However, not enough information is available about the effects of nitrogen fertilizers on Cu toxicity and stabilization capacity by pioneer plants in Cu contaminated soils.

Nitrogen plays an important role in promoting plant tolerance to toxicity of metals by regulating antioxidant system to eliminate excessive metal-induced oxidative damage (Jalloh et al. 2009; Yang et al. 2020). Giansoldati et al. (2012) found that N application ameliorated boron-induced oxidative stress in Brassica juncea by progressively lessening the activity of SOD, APX, PPX, and GR to the control levels. Wu et al. (2020) presented NH4+-N enhanced Cd tolerance in rice by increased SOD enzyme and the efficiencies of GSH-AsA cycle with increasing concentration of AsA and GSH and GR and DHAR activities. As a redox active metal, excessive Cu induced the activities of CAT, SOD, APX, and GR in Tanzania guinea grass, and the combination of NO3− with NH4+-N favored the antioxidant system to promote the efficiency of copper phytoextraction (de Souza Junior et al. 2019). Thus, enzymatic and non-enzymatic antioxidants were distinctly regulated by nitrogen forms in different plant species under different metals stress. However, the effects of different NH4+/NO3− proportion on plant resistance to Cu toxicity related to the antioxidant enzyme system and GSH-AsA cycle are still poorly understood, especially in trees.

Liriodendron chinense (Hemsl.) Sarg. is a widely used tree species in landscaping and afforestation programs, due to its fast growth rate, beautiful tree shape, good ornamental, and biomass accumulation (Huang and Guo 2015). Liriodendron species are planted as a crucial hardwood species in old field and mine wasteland (Sena et al. 2015). Previously, we performed preliminary experiments to understand how Liriodendron chinense responded in Cu wasteland by field planting in Dexing copper mine and found that Liriodendron chinense was strongly affected by Cu more than by other metals, such as Cd and Pb. Given this fact, we decided to investigate the effects of different NH4+/NO3− ratios on Cu toxicity in Liriodendron chinense in oxidative stress, antioxidant enzyme system, GSH-AsA cycle, Cu uptake, translocation and accumulation, and selected elements in plant tissues. To the best of our knowledge, this study is the first report to research the effects and underlying mechanisms of different nitrogen forms in protecting plants from Cu, and the potential of developing N management strategies to promote the stabilization in Cu-polluted soils.

Material and methods

Plant material and experimental design

The plant materials were derived by stem cutting Liriodendron chinense, which collected from an adult tree in a provenance trail plantation located in Huangma, Nanchang country, Jiangxi province of China. The tree originated from Mount Lu Nature Reserve, Jiangxi Province. Annual seedlings were transferred to pots containing soil culture medium using inert silica pellets and perlite (3:2, v/v), avoiding other factors in soils during the experiment. Each pot had one seedling. The pots were kept in a greenhouse with natural light and temperature conditions (average day, 18–30 °C; night, 16–24 °C), watered every 2 days by filtered water and added 1/4 Hoagland nutrient solution every week.

Uniform seedlings (about 20–30 green leaves and 40–50 cm tall) were selected and used for Cu and N treatments after 30 days of transplantation. In a preliminary experiment, the seedlings had damage symptoms under Cu treatment (25 mg.kg−1) and slight damage above 10 mg.kg−1 of sole supply of NH4+-N. In this study, we treated with 25 mg/kg Cu added as CuCl2 in combination with three molar percentage ratios of NH4+ to NO3− (1:0, 1:1, 0:1). Therefore, the treatments were designed as follows: (1) CK: plants untreated with Cu and N; (2) Cu: plants treated with 25 mg kg−1 Cu; (3) Cu-NH4+: plants treated with 25 mg kg−1 Cu + 300 mg kg−1 N (NH4Cl); (4) Cu-NH4+  + NO3−: plants treated with 25 mg kg−1 Cu + 150 mg kg−1 N (NH4Cl) + 150 mg kg−1 N (KNO3); (5) Cu-NO3−: plants treated with 25 mg kg−1 Cu + 300 mg kg−1 N (KNO3). To maintain the balance of potassium (K) for the addition of NO3−-N, KCl was added in the Cu-NH4+-N and Cu-NH4+ + NO3−treatments, respectively. Each treatment was replicated with six pots using a randomized complete block design. Obvious toxicity symptoms had been observed after 5th day of the treatment in Cu-treated plants, so the experiment harvest took place at the seventh day of the treatment when we observed old leaf senescence. Leave samples were harvested with a scissor from the fifth or sixth expanded leaves (counting from the top of the plant) and stored immediately at − 80 °C for physiological and biochemical analyses. Then, different tissues (roots, stem, and leaves) were collected for measuring the concentration of N and Cu. The roots were immersed in 20 mM Na2-EDTA for 30 min and rinsed with de-ionized water thoroughly.

Measurements of chlorophyll concentration in leaves

Chlorophyll concentration was determined following the method of Sun et al. (2015). Briefly, 0.1 g fresh leaves were added to 10 mL dimethyl sulfoxide (DMSO) and shaken in the dark for 72 h. The absorbance of the extract was measured at 663 nm and 645 nm with a spectrophotometer (SPAD-502, Minolta, Camera Co. Ltd., Osaka, Japan).

Crude enzyme extraction, MDA, and H2O2 contents analysis

For crude enzyme extraction, 1.0 g fresh leaves were ground in liquid nitrogen and transferred to 10-mL pre-cooling sodium phosphate buffer (100 mM, pH 7.0) and centrifuged for 20 min at 13,000 × g at 4 °C. Then, the supernatant was used to determine the concentrations of malondialdehyde (MDA) and reactive oxygen species (H2O2) and activities of antioxidant enzymes.

To measure MDA, 1 mL of the supernatant was mixed with 4 mL of reaction solution (0.5% (w/v) thiobarbituric acid and 5% (w/v) trichloroacetic acid), incubated at 95 °C for 30 min, and cooled in ice bath. The mixture was centrifuged at 10,000 × g for 10 min and the absorbance of the supernatant was measured at 532 nm and 600 nm (Khator and Shekhawat 2020).

H2O2 concentration was measured according to Shi et al. (2015). In brief, 100-µL supernatant was mixed with 1-mL solution containing 0.1% titanium sulfate and 20% (v/v) H2SO4 and incubated at 25 °C for 10 min. Then, the mixture was centrifuged at 12,000 × g for 10 min at room temperature, and the absorbance was read at 410 nm.

Measurement of antioxidant enzymes activity

The activities of four antioxidant enzymes, including superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7), catalase (CAT; EC 1.11.1.6), and ascorbate peroxidase (APX; EC1.11.1.1), were determined using a Total SOD Assay Kit (A001-3), a Plant POD Assay Kit (A084-3), a Plant CAT Assay Kit (A007-1), and a Plant APX Test Kit (A123-1–1), respectively, Xanthine oxidase method was used to measure superoxide dismutase (SOD; EC 1.15.1.1) activity by a Total SOD Assay Kit (A001-3), which was calculated by the values of inhibiting 50% initial decline of nitro blue tetrazolium at 550 nm. Peroxidase (POD; EC 1.11.1.7) activity was determined in a 3-mL reaction mixture containing 100-µL enzyme extract, 50 mM phosphate buffer (pH 7.0), 28-µL guaiacol, and 19-µL H2O2 by a Plant POD Assay Kit (A084-3), which was read five times at 420 nm within 2 min at 30-s intervals and measured the absorbance change of 0.01. Catalase (CAT; EC 1.11.1.6) activity was detected by a Plant CAT Assay Kit (A007-1). One unit of activity was expressed as the amount of enzymes for 1-mg tissue proteins consuming 1 µM H2O2 at 405 nm. Ascorbate peroxidase (APX) activity (EC1.11.1.1) was measured by a Plant APX Test Kit (A123-1–1). The reaction solution (1 mL) was composed 50 mM phosphate buffer (pH 7.8), 0.1 mM H2O2, 0.1 mM EDTA, 0.2 mM ascorbate (AsA), and 100µL supernatant. The reaction was started by addition of H2O2, and the oxidation rate of ascorbic acid was estimated by following the decrease in absorbance at 290 nm. APX activity was calculated by using the molar extinction coefficient for AsA (Zeng et al.2017). The four enzyme assay kits were obtained from Nanjing Jiancheng Bioengineering Institute, China.

Activity determination of nitrate reductase, glutathione reductase, dehydroascorbate reductase, AsA, and GSH

Nitrate reductase (NR) (EC.1.7.1.1) activity was determined according to the method described by Li et al. (2007). The 1-mL crude enzyme extract was mixed with a solution containing 100 mM potassium phosphate buffer (pH 7.5), 50 mM KNO3, and 1% (v/v) iso-propanol, then incubated at 30 °C for 30 min. Subsequently, 1 mL 30% (w/v) TCA was added into the reaction solution to stop the enzyme activity. The nitrite was released to the medium by adding 1% (w/v) sulfanilamide in 2.4 M HCl, 0.02% (w/v) naphthyl-ethylenediamine (1:1) into the reaction solution. Then, the absorbance was read at 540 nm. Controls were measured before the incubation period.

Glutathione reductase (GR) (EC1.6.4.2) activity was assayed according to Shi et al. (2015) using a Plant GR Assay Kit (A062-1–1; Nanjing Jiancheng Bioengineering Institute, China). The reaction solution was composed of 20-µL crude extract, 100-µL 2.5 mM GSSH, 10-µL 2 mM NADPH, and 70-µL GR assay solution. The oxidation rate was corrected for the non-enzymatic oxidation of NADP by GSSG. Then, GR activity was determined by a decrease in absorbance at 340 nm.

Dehydroascorbate reductase (DHAR) was assayed according to the method of Chen and Gallie (2006). Briefly, 0.1-g fresh leaves were ground in 1-mL pre-cooling extraction buffer containing 50 mM Tris–HCl, 2 mM EDTA, and 1 mM MgCl2 (pH 7.5), and the homogenate was centrifuged at 13,000 × g for 10 min at 4 °C. Then, 100-µL supernatant was mixed with 700 µL K2HPO4/KH2PO4 (pH 6.5, 50 mM), 100 µL DHA (0.5 mM), and 100 µL reduced glutathione (1 mM). The DHAR activity was calculated based on the absorbance at 265 nm.

AsA and glutathione (GSH) contents were determined according to the methods of Chen and Gallie (2006) and Brehe and Burch (1976), respectively. Briefly, 0.5-g leaves were ground in liquid nitrogen and transferred into 4-mL pre-chilled 5% (w/v) TCA solution, containing 2 mM Na2-EDTA. The mixture was centrifuged at 16,500 × g for 20 min at 4 °C, and the supernatant was collected for AsA and GSH analysis. The reaction solution for measuring AsA content was prepared by mixing 1 mL of supernatant, 1 mL of ethanol, 1 mL of 0.5% (w/v) 4,7-diphenyl-1,10-phenanthroline (bathophenanthroline), 1 mL of 5% (w/v) TCA, 0.5 mL of 0.03% (w/v) FeCl3, and 0.5 mL of 0.4% (w/v) H3PO4. The reaction solution was incubated at 30 °C for 1 h until the color changed and used to read the absorbance at 525 nm. For GSH determination, the reaction mixture consisted of 1 mL of supernatant and 0.5 mL of 4 mM 5,5-dithio-bis-(2-nitrobenzoic acid), and 1 mL of phosphate buffer (100 mM, pH 7.7) was kept at 25 °C for 10 min until the color changed. The GSH content was determined based on the absorbance at 405 nm.

Contents of Cu, N, P, and K in plant tissue

For Cu content analysis, 0.5-g harvested dried samples (root, stem, leave) were crushed and digested in acid mixture of HNO3–HClO4 (4:1, v/v) at 220 °C for 4 h. The concentrations of Cu in the diluted digestive solution were determined by inductive coupled plasma optical emission spectroscopy (ICP-OES) (Optima 8000; Perkin-Elmer, Waltham, Massachusetts, USA). Cu translocation from roots to shoots has been calculated by shoot-to root concentration ratio.

Total nitrogen was determined using oven dry roots, stems, and leaves according to the Kjeldahl method (Helrich 1990) after digesting with H2SO4 and H2O2. Total potassium was measured using NaOH-melting-flame photometric method (FP6431, Shanghai, China), and total phosphorus was determined using H2SO4–HClO4 digestion-phosphomolybdate blue spectrophotometry (UV/VIS-4802, UNICO, Shanghai, China),

Statistical analysis

All treatments were conducted in triplicate independent experiments, and all data were analyzed using the SPSS software (Version 18.0; IBM Corp., Armonk, NY). A one-way analysis of variance (ANOVA) was used to compare means among treatments. The significant differences among treatments were separated based on the least significant difference test (multi-range comparison) at P < 0.05. The data presented in tables and figures, expressed means ± SD, are based on three biological independent experiments.

Results

Supply of NO3 −-N alleviates Cu toxicity in Liriodendron chinense

Liriodendron chinense exposed to Cu for 7 days showed obvious toxicity symptoms, including curled, yellowed, and wilted leave. These effects were consistent with decreased chlorophyll content (Fig. 1a). Meanwhile, effects of NO3−-N and NH4+-N were apparently different in Cu-treated plants. Supply of exclusive NO3−-N showed mild toxicity symptoms, as evidenced by less leaf chlorosis and greater chlorophyll content by 1.48-fold compared to the N-Null under Cu treatment (Fig. 1a). However, regardless of the presence or absence of NO3−-N, adding NH4+-N intensified the Cu-induced inhibitory effects on L. chinense growth in the first 3 days. Afterward, leaf necrosis and wilting occurred more quickly when ammonium was used as the nitrogen source, especially when supplied as NH4+-N + NO3−-N. The chlorophyll content in the supply of NH4+-N and NH4+-N + NO3−-N was reduced by 29.4% and 39.7%, respectively, compared to controls.

Fig. 1
figure 1

Chlorophyll content, MDA level, and H2O2 concentration in leaves of Liriodendron chinense. (a) Total chlorophyll (Chlt) content, (b) malonaldehyde (MDA) concentration, (c) H2O2 concentration. Data are means ± SD (n = 4). Different letters indicate significant differences between different treatments at P < 0.05 by least significant difference test

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Supply of NO3 −-N reduces oxidative damage

Cu significantly increased the contents of H2O2 and MDA in Liriodendron chinense leaves, compared to controls (Fig. 1b, c). Adding NH4 + -N, with or without NO3−-N, significantly promoted H2O2 concentration by 41.8% and 46.3%, respectively, and MDA concentration was also increased compared to the N-Null. However, sole NO3−-N significantly reduced the concentrations of H2O2 and MDA in leaves under treatment of Cu.

Effects of nitrogen forms on antioxidant enzymes in response to Cu

Cu significantly inhibited SOD activity, compared to controls (Fig. 3). Sole NO3−-N significantly increased the SOD activity by 25.4%, compared to the N-Null (Fig. 3a). Supply of NH4+-N, with or without NO3−-N, had no apparent effects. Cu significantly inhibited APX activity. Surprisingly, NO3−-N treatments significantly stimulated APX activity in L. chinense leaves by 23.2% and 31.0%, respectively, compared to control and N-Null treatments (Fig. 3b). Cu treatments and adding NH4+-N, with or without NO3−-N, markedly increased POD and CAT activities, but NO3−-N restored the Cu-induced activities of POD and CAT (Fig. 2c, d).

Fig. 2
figure 2

Activity of leaf (a) superoxide dismutase (SOD), (b) ascorbate peroxidase (APX), (c) peroxidase (POD) and (d) catalase (CAT) in L. chinense. Data are means ± SD (n = 4). Different letters indicate significant differences between different treatments at P < 0.05 by least significant difference test

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Effects of nitrogen forms on NR activity and GSH-AsA cycle under Cu stress

Sole NO3−-N significantly stimulated NR activity in Cu-treated plants; however, supply NH4+-N alone significantly inhibited NR activity, when compared to N-Null (Fig. 3a). Cu treatment significantly activated GR activity and increased concentration of GHS and AsA in leaves (Fig. 3b, c, d), but significantly inhibited the DHAR activity (p < 0.05, Fig. 3e), which was 2.2-fold compared to controls. NO3−-N alone dramatically increased the DHAR activity by 64.4%, whereas NH4+-N, with or without NO3−-N, did not affect DHAR activities, compared with N-Null. Interestingly, adding N in two forms reduced the GR activity in Liriodendron chinense leaves compared to N-Null (Fig. 3b). Moreover, NH4+-N, with or without NO3−-N, greatly increased the GSH concentration in Cu-treated plant leaves, by 3.2- and 2.2-fold, respectively, compared to the N-Null (Fig. 3c). In contrast, NO3−-N alone restored Cu-induced GSH content compared to the N-Null. Similarly, NH4+-N, with or without NO3−-N, showed no remarkable effect in AsA concentrations compared to the N-Null (Fig. 3d), although NO3−-N alone significantly reduced AsA content to levels lower than those of controls.

Fig. 3
figure 3

Activity of leaf (a) nitrate reductase (NR), (b) glutathione reductase (GR), (c) glutathione (GSH), (d) ascorbic acid (AsA) concentration, and (e) dehydroascorbate reductase (DHAR) in L. chinense. Data are means ± SD (n = 4). Different letters indicate significant differences between different treatment at P < 0.05 by least significant difference test

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Nitrogen forms regulate Cu uptake, accumulation, and translocation from roots to shoots

Different forms of N differently influence Cu concentrations in roots, stems, and leaves and changed Cu translocation from roots to shoots, compared to N-Null treatment (Fig. 4). The greatest Cu concentration in roots was occurred when N was added as NO3−-N, which was 1.39-fold higher than that of N-Null. However, Cu concentrations in leaves were 24.8% lower compared to N-Null, in agreement with a significant lower translocation factor from roots to shoots by 35.2 (Fig. 4d). NH4+-N, with or without NO3−-N, increased Cu accumulation in roots, stem, and leaves, with increasing by 17.0%, 61.1%, and 21.9% in NH4+-N alone and 15.1%, 66.6%, and 36.9% in NH4+-N + NO3−-N; respectively, when compared with N-Null. Thus, compared to the N-Null treatment, adding NH4+-N, with or without NO3−-N, promoted Cu translocation from roots to shoots by 17.4% and 29.4%, respectively.

Fig. 4
figure 4

Copper (Cu) accumulation and translocation in L. chinense. Cu concentration in root (a), stem (b), leaves (c), and Cu translocation from root to shoot (d). Data are means ± SD (n = 4). Different letters indicate significant differences between different treatments at P < 0.05 by least significant difference test

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NO3 −-N regulates nutrient uptake in Liriodendron Chinense

Cu treatment inhibited nitrogen (N) uptake, increased slightly phosphorus (P) uptake, and reduced potassium (K) concentration of root in Liriodendron chinense (Table 1). Compared to the Cu only treatment, NO3−-N obviously increased N content in leaves and increased K content in roots. However, NO3−-N had little effect on N content in roots and stems, or on the content of K in stems and leaves, or on P content in roots and leaves. On the other hand, NH4+-N, with or without NO3−-N, markedly decreased K and P concentrations in L. chinense leaves. Overall, the concentrations of N in the leaves of Cu-treated plants were consistently higher in NO3−-N treatment than those in NH4+-N treatments.

Table 1 Nitrogen (N), potassium (K), and phosphorus (P) accumulations in L. chinense
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Discussion

Copper is essential for plant growth, but only in small quantity (5–20 mg kg−1 DW in leaves). Cu levels above 20–30 mg.kg−1 DW in plant leaves can damage many plant species (Marschner 2011). In our study, the Cu concentration in L. Chinense leaves in the sole Cu treatment was 34.2 mg kg−1, and plants in this treatment showed toxicity symptoms and indeed reduced chlorophyll contents. Excessive Cu in leaves causes reactive oxygen species bursts, which can trigger lipid peroxidation, disturb integrity of thylakoid membranes, and destroy chlorophyll structure (Khator and Shekhawat 2020). Interestingly, NO3−-N exclusively alleviated the Cu-induced toxicity in L. chinense, in which the Cu concentration of leaves was 22.4 mg kg−1. Conversely, NH4+N, with or without NO3−-N, stimulated Cu accumulations in plant tissues, leading to greater toxic effects in leaves. Such different effects in NO3−-N and NH4+-N may provide a new vision to phytostabilize or phytoextract on Cu-contaminated soils.

Effects of NO3 −-N/NH4 +-N on oxidative stress indicators

Cu catalyzes the generation of reactive oxygen species (Li et al. 2007), and Cu treatment increased H2O2 and MDA production in the L. chinense seedlings (Fig. 2). These findings suggest that Cu-induced ROS accumulation could cause oxidative damage and disturb cellular homeostasis (Cui et al. 2010; Khator and Shekhawat 2020; Shabbir et al. 2020). Evidence suggests that nitrogen plays important roles in defensing oxidative stress, and additional nitrogen prevents Cd-induced oxidative stress in rice (Wu et al. 2020), boron-induced toxicity in Brassica juncea (Giansoldati et al. 2012), and reduced Al-toxicity in Lespedeza bicolor (Chen et al. 2010). Furthermore, the form of N could influence cellular homeostasis and plant tolerance to Cd, Mn, Cu, and Al (Cheng et al. 2020; de Souza Junior et al. 2018, 2019; Jalloh et al. 2009). In our study, we observed that adding NO3−-N ameliorated Cu-induced oxidative damage in L. chinense leaves by reducing the levels of H2O2 and MDA to those of the non-Cu treatment. These observations suggest that ROS bursts were not induced by adding NO3−-N under Cu treatments and indicated that nitrate had a positive role in mitigating Cu toxicity to L. chinense. Conversely, NH4+-N, alone or combined with NO3−-N (1:1), further aggravated Cu toxicity in L. chinense seedlings, evidenced by significantly higher MDA and H2O2 contents compared to those in the alone Cu treatment. Similar results were reported by Zhu et al. (2016) in submerged plants Vallisneria natans. Also, it is well known that if the molar ratio of NH4+ to NO3− in a solution exceeds 1:1, NH4+-N can cause toxicity by increasing oxidative stress and lipid peroxidation in Panicum maximum (Santos et al. 2013). de Souza Junior et al. (2019) also reported that greater oxidative stress and Cu toxicity was induced by NH4+ above 50% of applied N rate in Tanzania guinea grass. However, NH4+-N-preferring plant species such as rice and Lespedeza bicolor are native to acidic soils and have evolved mechanisms to use NH4+-N efficiently, so N added as NH4+-N might alleviate Cd and Al toxicity, respectively (Wu et al. 2020; Jalloh et al. 2009; Zhao et al. 2009). For these reasons, we speculate that the effects of different N forms on metal toxicity should take into consideration the specificity of plant species.

Effect of NO3 −-N/NH4 +-N on antioxidant systems

Many studies have reported the crucial roles of different antioxidant enzymes including SOD, POD, and CAT in scavenging Cu-induced ROS accumulation (Buapet et al. 2019). SOD is considered as the first line of defense against ROS bursts (Chen et al.2015), and in our study, SOD enzyme was greatly reduced in the sole-Cu treatment and in the NH4+-N treatment. However, the inhibitory effects of Cu were alleviated by NO3−-N by increasing the enzyme activity to the level of the control. Indeed, the positive effect of NO3−-N on SOD activity was also found in APX activity and suggested a higher capacity to eliminate ROS as well as a protective role of NO3−-N to Cu stress.

In general, after ROS catalyzes into H2O2 by SOD enzyme, CAT and POD have been converted H2O2 to molecular oxygen and water (Fidalgo et al. 2013; Bai et al. 2021). Here, Cu stress and adding NH4+-N caused increased CAT and POD activities in leaves, which was concomitant with higher H2O2 accumulation. Cu-induced activation of POD and CAT enzymes also occurred in Solanum lycopersicum (Nazir et al. 2019) and Lemna minor (Hu et al. 2018), suggesting oxidative damage in plant leaves. However, N added as NO3−-N restored the Cu-induced increases in POD and CAT activities, accompanying with the lower H2O2 concentration in leaves. The CAT and POD inhibition by NO3−-N may be contributed to the low oxidative stress.

GSH-AsA cycle is also important to ROS detoxification in plants, which included the main enzyme (GR and DHAR) sustaining high efficiency of generation of GSH and AsA to catalyze excess H2O2 (Wu et al. 2020). In this cycle, GR plays important role in maintaining a high GSSG/GSH ratio under metal-induced oxidative stress by catalyzing to synthesize GSH (Giansoldati et al. 2012). Also, DHAR regulates the cellular AsA redox state by regenerating AsA from an oxidized state, thus enhancing tolerance to ROS (Chen and Gallie 2006). We found that GSH-AsA was activated by Cu, and adding NH4+-N increased GR activity and AsA and GSH concentrations. But NO3−-N alone recovered Cu-induced GR activity with lower levels of AsA and GSH concentration and alleviated the inhibition of Cu-induced DHAR activity. These findings suggest that NO3−-N modulated the levels of antioxidants and activities of antioxidase to provide greater ROS-scavenging capacity (Song et al. 2019). Another possible mechanism of NH4+-N in promoting efficiency of GSH-AsA cycle may contribute to an urgent demand of phytochelatin synthesis induced by Cu stress (Finkemeier et al. 2003). GSH not only has crucial role in scavenging metal-induced ROS, but also chelates toxic metals in cytoplasm of plant cells by compartmentalizing metals in vacuoles (Xu et al. 2010). Cd2+ is transported from roots to aerial organs via xylem and phloem in the form of GSH-Cd and PC-Cd complexes (Mendoza-Cózatl et al. 2008). Thus, the concomitant increased GR activity in L. chinense leaves could be due to the increased demand for reduced GSH to binding excessive Cu (Giansoldati et al. 2012).

Nitrate reductase is sensitive to oxidative stress (Campbell 1999), which could be as a result of the Cu-SH formation or oxidative damage to NR enzymes (Burzyński 2001; Li et al. 2007). In the present study, adding NH4+-N aggravated the Cu-induced inhibition of NR activity; however, adding sole NO3−-N significantly promoted the activity of NR, suggesting adding NO3−-N alleviated the oxidative stress by exposure to Cu. However, few studies explain the effects of different nitrogen forms on enzymatic and non-enzymatic antioxidant responses to Cu stress in plants. Thus, further study about mechanism of metal detoxification of nitrogen forms in enzyme system is needed to secure more detail.

Different effects of NO3 −-N/NH4 +-N on Cu uptake, translocation and accumulation of L. chinense seedlings

In general, restrained Cu translocation from roots to shoots by sequestrating excessive Cu in the roots is a vital mechanism of plant defense against Cu stress (Brunner et al. 2008; Konno et al. 2010; Sahi et al. 2007). In this study, adding exclusive NO3−-N obviously inhibited Cu translocation from roots to shoots, which resulted in a remarkably reduced Cu concentration in leaves. Conversely, adding NH4+-N, with or without NO3−-N, enhanced Cu translocation and increased Cu concentration in leaves. These changes were concomitant with the greater Cu toxicity in L. chinense, compared with N-Null. Therefore, the stronger affinities for Cu2+ of roots and lower Cu translocation from roots to shoots in NO3−-N treatments may have helped protect L. chinense from Cu.

NO3− and NH4+ could change the rhizosphere pH in different ways by regulating release of OH− and H+ ions, which affects the availability of metals in soil and solution (Hinsinger et al. 2003; Jalloh et al. 2009). Many pot experiments found that NH4+-N can enhance Cd accumulation in plants by regulating ion exchange reactions, availability of metal elements, and rhizosphere pH (Qasim et al. 2015; Cheng et al. 2020). However, other studies showed that supply of NO3−-N encourages accumulation of metals such as Cd, Cu, Mn, Al, and Mg in roots (Chen et al. 2010; Hu et al. 2019; de Souza Junior et al. 2018, 2019; Yang et al. 2020). NO3−-N hyperpolarizes the plasma membrane potential that facilitates the membrane transport of Cd, thus promoting Cd uptake by roots of Sedum plumbizincicola (Hu et al. 2013). Similar result was noted in Triticum polonicum L. (Cheng et al. 2020). Furthermore, Zhao and Shen (2018) also found that NH4+-N promoted the solubilization of Al compared to NO3−-N, but inhibited Al accumulation in rice roots. This result was explained by that the NH4+-N-induced protons compete with Al3+ for adsorption sites on root surfaces (Cumming 1990). In our study, both nitrogen forms promoted Cu uptake and accumulation in roots and stem, compared with plants in the N-Null treatment, but had different effects on Cu translocation from roots to shoots. So, the discrepant effects of different N forms on Cu uptake of plant roots take into account plant species, metal types and levels, soil properties, and so on.

Effects of NO3−-N on Cu uptake and Cu translocation from roots to shoots were different, depending upon the presence or absence of NH4+-N, because adding NO3−-N inhibited Cu translocation from roots to shoots to reduce Cu accumulation in leaves in the absence of NH4+-N, but promoted those in the presence of NH4+-N. These results indicate that adding NO3−-N alone may favor Cu accumulation in roots, whereas NH4+-N, with or without NO3−-N, increased Cu translocation from to shoots considering aggravating symptoms of Cu toxicity. The similar observations were confirmed by de Sousa Leite and Monteiro (2019) in Tanzania guinea exposed to Cd; in the experiment, NO3−-N mitigated Cd phytotoxicity by favoring Cd accumulation in roots and inhibiting Cd translocation from roots to shoots. Meanwhile, the uptake, translocation, and accumulation of Cd in Carpobrotus rossii and S. nigrum were promoted by NH4+-N (Cheng et al. 2016). There are many possible reasons for the different effects. Cheng et al. (2020) suggested that NO3−-N regulated the accumulation of lactose in roots and expression of metal transport genes to promote more Cd binding in cell walls and higher Cd sequestration in vacuoles, which result in increasing Cd uptake and accumulation in root and limiting Cd translocation from roots to shoots. These results also suggest addition of NO3−-rich fertilizers might enhance the Cu phytoextraction efficiency by L. chinense in areas that are moderate copper contaminated.

Conclusion

We demonstrate the alleviating effects of NO3−-N on Cu toxicity to L. chinense, likely due to inhibiting Cu transportation from roots to shoots and reducing oxidative stress. In contrast, NH4+-N alone, or combined with NO3−-N, intensified oxidative stress caused by Cu, despite the increased activities of CAT and POD and greater efficiency of GSH-AsA cycle. These results suggest that NO3−-N could ensure L. chinense grown in Cu-contaminated soil, which can be used for efficient metal phytostabilization. Also, considering the increase of Cu accumulation in roots, stems, and leaves caused by NH4+-N alone or combined with NO3−-N, NH4+-N can contribute to metal phytoextraction by promoting Cu accumulation in L. chinense.