Introduction

Nicosulfuron [2-[(4,6-dimethoxypyrimidin-2-ylcarbamoyl)sulfamoyl]-N,N-dimethylnicotinamide] is an effective, broad-spectrum, inner-absorption conducting sulfonylurea herbicide, which is mainly used for the control of annual weeds and broadleaf weed species during maize production (Stall and Bewick 1992; Williams and Harvey 2000; Wang et al. 2016). Nicosulfuron residue in both soil and rivers is degraded via hydrolysis and microbial activity; therefore, the toxicity of nicosulfuron has a minor effect on the next crop (Wang et al. 2011). The effectiveness of nicosulfuron compensates for the paucity of post-emergence herbicides in corn, and it has also been important for the development of the herbicide industry in China.

The different nicosulfuron susceptibilities of weeds and crops explain its ability to effectively control the growth of weeds. Nicosulfuron kills weeds selectively, while causing minimal harm to crops (Dobbels and Kapusta 1993; Beckie and Tardif 2012; Yu and Powles 2014). Nicosulfuron is a sulfonylurea herbicide that inhibits the activity of acetolactate synthase (ALS), reduces the synthesis of branched chain amino acids, and kills weeds (Rey-Caballero et al. 2016). In contrast, nicosulfuron can be degraded into inactive substances and thus loses its activity when it is combined with glucose in maize (Pataky et al. 2008; Meyer et al. 2010).

The resulting effects of corn exposure to nicosulfuron have been widely reported. A large number of studies indicated severe injury of maize seedlings after nicosulfuron treatment between 7 and 14 days. The severity of the resulting injury decreased in the following manner: common corn < waxy corn < sweet corn (O’Sullivan et al. 2002). Both sweet corn inbred and hybrid lines exhibit different resistance to nicosulfuron, and while some are extremely resistant, others are completely susceptible. Several studies have demonstrated that nicosulfuron induced significant injuries in some sweet corn hybrids, including Delmonte 2038, Merit, and Silver Extra Sweet, following nicosulfuron application at 25 g hm−2 or 50 g hm−2 (O’Sullivan et al. 1995; O’Sullivan and Bouw 1998). Different corn varieties basically suffer the same injury after nicosulfuron treatment: during the early stage of development, the leaves show local chlorosis and etiolation, which is accompanied by the purple leaf phenomenon. With further plant growth, new leaves show cauda equina curl. Consequently, plant growth is inhibited, which results in dwarfing plants, and secondary stems may be produced. With further aggravating phytotoxicity, sensitive maize lines cease to grow, stalks become purple, the leaves become yellowish, and plants eventually die. Several studies indicated that nicosulfuron can reduce the chlorophyll content and photosynthesis, which consequently inhibits plant growth (Hussain et al. 2010; Li et al. 2018; Tan et al. 2012; Yang et al. 2017). Herbicide application damages the photosynthetic apparatuses within both photosystem I (PSI) and photosystem II (PSII). This in turn reduces the maximum photochemical efficiency of photosystem II (Fv/Fm), the photochemical quenching coefficient (qP), and the quantum yield of PSII electron transport (nPSII) (Yuan et al. 2014; Hu et al. 2014). Therefore, it is vitally important to improve the nicosulfuron resistance of maize and to understand the resistance and detoxification mechanisms of maize.

Oxygenic photosynthetic organisms such as maize, photosystem I (PSI), photosystem II (PSII), and mitochondrial-mediated electron transport are major sites for the production of reactive oxygen species (ROS, e.g., O2·-, H2O2, and 1O2) under both natural and stress conditions (He and Häder 2010; Prasad et al. 2016). As a consequence, the presence of toxic factors, such as nicosulfuron, can enhance the production of ROS, using the same electron transport systems and thus accelerating the chance of physiological and biochemical damage (Sheeba et al. 2011). The antioxidant system plays an important role for scavenging ROS in plants and for preventing herbicide stress-related injuries in plant cells (Sun et al. 2017). Under herbicide stress, the redox balance in the plant is broken, which leads to a significant increase in ROS. Excessive O2.– in plants induces the generation of superoxide dismutase (SOD), which specifically removes superoxide anion radicals (Foyer et al. 1994; Baek and Skinner 2003; Gill and Tuteja 2010). SOD scavenges O2.– by catalyzing its dismutation, where one O2.– molecule is decreased to H2O2 and another is oxidized to O2 (Table 1). Part of H2O2 can be catalyzed to form H2O and O2 via catalase (CAT). However, overly abundant H2O2 can be removed via the ascorbate-glutathione cycle (AA-GSH). The ascorbate-glutathione metabolic pathway, also named the Foyer-Halliwell-Asada pathway, is an important antioxidant pathway for higher plants during the process of resisting biotic and abiotic stresses, and it is also an important pathway for the metabolism of toxic substances in higher plants (Li et al. 2015). In this pathway, ascorbate peroxidase (APX) catalyzes hydrogen peroxide and produces a monodehydroascorbate radical (MDHA) and water. Then, MDHA is converted to ascorbate (AA) in a reaction that is catalyzed via monodehydroascorbate reductase (MDHAR). Furthermore, MDHA can be used to generate dehydroascorbate (DHA) by disproportionation, and subsequently, it can be reduced via dehydroascorbate reductase (DHAR) to the AA. Then, the glutathione disulfide (GSSG) is converted to glutathione (GSH) in a reaction catalyzed via glutathione reductase (GR; Fig. 1, Table 1) (Zhang and Kirkham 1996; Kaur et al. 2015; Sytykiewicz 2016). At the physiological and biochemical levels, a variety of abiotic and biotic stresses (chilling, drought, heavy metals, salt, and aphid infection) have been reported to lead to changes in the ascorbate-glutathione pathway enzyme activity (Anjum et al. 2014; Gill and Tuteja 2010). Until now, no data has been published on the effects of nicosulfuron on the expression of genes that encode isozymes in higher plants that are involved in the AsA-GSH cycle.

Table 1 Major ROS scavenging antioxidant enzymes
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Fig. 1
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ROS and antioxidant defense mechanism

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Apparently, studying the gene expression involved in antioxidant enzyme activities can further enhance our understanding of the molecular adaptation and detoxification mechanisms of plants to various biotic and abiotic stresses. In fact, ROS that are overproduced in plant cells can regulate the expression of various genes related to antioxidant and other stresses. For example, overexpression of Cu/Zn SOD in transgenic tobacco plants caused an increased resistance to drought, chilling, and oxidative stress (Zhang et al. 2017). Upregulation in the transcript abundance of antioxidative enzyme genes was also observed in response to drought, salt, and stress (Li et al. 2017; He et al. 2017; Diaz-Vivancos et al. 2016). Sytykiewicz (2016) showed that GR1 and GR2 genes were significantly upregulated by aphid infestation of maize leaves. In response to a variety of abiotic stresses, the generation of ROS, increased antioxidant enzyme activity, and elevated transcript level of antioxidant enzymes has been extensively reported. In contrast, the regulatory mechanism of the family of antioxidant system genes in maize after spraying with nicosulfuron has not been reported. Therefore, we selected the pair of nicosulfuron-resistant and nicosulfuron-sensitive nearly isogenic lines (NILs) of maize SN509-R and SN509-S, to minimize effects caused by differences in the genetic background. Here, we aimed to (1) investigate the accumulation of both O2·- and H2O2 and the antioxidant enzyme activity of maize seedlings in the presence of nicosulfuron and (2) investigate changes in the expressions of gene for antioxidant enzymes in maize seedlings in response to nicosulfuron exposure.

Materials and methods

Experimental design

The experiment was designed at the southern farm of the Shenyang Agricultural University (41° 49′ N, 123° 34′ E), which is an area within the north temperate zone, with a monsoon-affected semi-humid continental climate. The fundamental nutrient content of the tested soil was 26.79 g kg−1 of organic matter, 2.45 g kg−1 of total nitrogen, 110.75 mg kg−1 of alkaline hydrolyzed nitrogen, 11.09 mg kg−1 of available phosphorous, and 105.74 mg kg−1 of available potassium. The pair of NILs SN509-R and SN509-S was developed by the Institute of Specialty Corn at the Shenyang Agricultural University. The nicosulfuron-resistant inbred line SN509-R developed normally after nicosulfuron exposure. In contrast, nicosulfuron can inhibit the growth of the nicosulfuron-susceptible inbred line SN509-S, which ultimately causes plant death (Fig. S1). A split-plot experimental design was used with nicosulfuron treatment defining main plots and inbred lines within subplots. The row length of one plot was 6 m, and the row width was 0.6 m. We set up eight rows per plot and each plot had an area of 28.8 m2.

Pesticide treatments

From 2015 to 2016, an experiment was conducted in an incubator maintained at 27 °C with a relative humidity of 85%. Uniformly sized seeds of two inbred lines were sown after rinsing with a small volume of distilled water in culture dishes. After 7 days, uniformly sized buds (0.01 m) were transferred to new culture dishes via filter paper. Nicosulfuron concentrations of 0 (control), 8, 40, 80, 120, 160, 240, 320, and 400 mg kg−1 were applied to each of the cultures to screen for the nicosulfuron resistances of SN509-R and SN509-S under a wide range of herbicide concentrations. Fifty buds of each line were evenly spread into the culture dishes, with three replicates. After 2 days, buds were transferred to the field, and the survival rate was investigated at the five-leaf stage. This initial screening test revealed that for a nicosulfuron concentration of 80 mg kg−1, SN509-R plants were able to attain normal growth via self-defense mechanisms, while SN509-S plants either wilted or died (Table 2). In 2016, an experiment was conducted in the field. Maize seedlings were treated with nicosulfuron at the five-leaf stage using a laboratory pot sprayer, equipped with a nozzle. The nicosulfuron concentration was maintained at 80 mg kg−1 to investigate both physiological parameters and antioxidant enzyme activities of maize seedlings. Due to the fact of seedlings of SN509-S either died or wilted after 15 days after nicosulfuron treatment (DAT), data were determined every 2 days after 0 DAT.

Table 2 Effect of nicosulfuron on germination rate of waxy maize seed of SN509-R and SN509-S. Asterisk indicates differences under different days after herbicide treatment at P < 0.05, according to the least significant difference (LSD) test. CK control
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H2O2 assay, O2 ·- production rate, and lipid peroxidation

A method described by Jena and Choudhuri (1981) was used to determine the content of H2O2. A reaction mixture containing 1 ml of 0.1% titanium sulfate in 20% H2SO4 (v/v) and 1 ml of supernatant was centrifuged at 8000×g for 15 min at 25 °C. The absorbance of the yellow color of the reaction solution was recorded at 415 nm. The content of H2O2 was calculated via a standard curve with known concentrations, and the extinction coefficient (Ɛ = 0.28 μmol−1 cm−1) was used to calculate the content of H2O2, which was expressed in micromoles per gram (FW). The O2·- production rate was assayed according to the method described by Jiang and Zhang (2002) with some modifications. 0.1-g seedling samples of waxy maize seedling leaves were homogenized with 1 ml of 50 mM Tris-HCl (pH 7.5). The homogenate was then centrifuged at 10,000×g at 4 °C for 10 min. The decrease in absorbance caused by the reduction of 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT sodium salt) was measured at 470 nm, and the extinction coefficient (Ɛ = 2.16 × 104 M−1 cm−1) was used to calculate the O2·- production rate. The O2·- production rate was expressed in nanomoles per minute per gram (FW).

After 0 DAT, the seedling leaves were sampled, placed in liquid nitrogen, and then stored at − 80 °C until further analyses. The thiobarbituric acid method was used to determine the malondialdehyde (MDA) content of samples (Heath and Packer 1968). MDA was extracted by grinding 0.5 g of leaf samples in 5 ml of phosphate buffer (0.05 mM, pH 7.8). The homogenate was centrifuged at 4500×g for 10 min and the supernatant was collected to measure MDA. The 2 ml of extract liquid and 3 ml of 0.5% thiobarbituric acid (TBA) containing 5% trichloroacetic acid (TCA) were boiled for 10 min, and the solution was then cooled to room temperature. This mixture was centrifuged at 4500×g for 10 min. The absorbance of the supernatant was recorded at 450, 532, and 600 nm with a UNICO™ UV-2000 spectrophotometer (UV-2000, UNICO, USA). MDA was expressed as nanomoles per gram (FW), and the extinction coefficient (155 mM−1 cm−1) was used to calculate lipid peroxidation.

Enzyme assay

After nicosulfuron treatment, at 0 DAT, the seedling leaves were sampled, frozen in liquid nitrogen, and then stored at − 80 °C until subsequent analysis. Enzyme activities were measured spectrophotometrically, and absorbances were recorded with a UNICO™ UV-2000 spectrophotometer (UV-2000, UNICO, USA). Enzymes were extracted by grinding 0.5 g of leaf samples in 5 ml of phosphate buffer (pH 7.5) containing 1 mM EDTA, 1% PVP, 1 mM DTT, and 1 mM PMSF. The homogenate was centrifuged at 15,000×g at 4 °C for 30 min, and the supernatant was collected to measure the enzyme activity.

Superoxide dismutase (SOD, EC 1.15.1.1) activity

The method described by Giannopolitis and Ries (1977) was used to measure the SOD activity. Three-milliliter reaction mixture contained 100 mM phosphate buffer (pH 7.8), 3.0 mM EDTA, 200 mM methionine, 2.25 mM nitroblue tetrazolium chloride (NBT), 60 μM riboflavin, and 1.5 M sodium carbonate. The photoreduction of NBT was measured by recording the absorbance at 560 nm. The enzyme activity was calculated as 50% inhibition expressed in units per gram (FW). One unit of SOD activity was defined as the enzyme activity required to inhibit the photoreduction of NBT to purple formazan by 50%.

Catalase (CAT, EC 1.11.1.6) activity

Catalase activity levels were assayed according to a method described by Aebi (1984). A decrease in the absorbance due to the disappearance of H2O2 was measured at 240 nm (Ɛ = 0.036 mM−1 cm−1). The catalyzed reaction system consisted of 100 mM phosphate buffer (pH 7.0), 10 mM H2O2, and enzyme extract. The enzyme activity was expressed in micromoles (H2O2 oxidized) per minute per gram (FW).

Ascorbate peroxidase (APX, EC 1.11.1.11) activity

The reaction mixture contained 50 mM phosphate buffer (pH 7.0), 0.3 mM ascorbate, 0.1 mM EDTA, 0.06 mM H2O2, and enzyme extract. The decrease in the absorbance caused by the reduction of ascorbate concentration was measured at 290 nm, and the extinction coefficient (2.8 mM−1 cm−1) was used to calculate enzyme activity. The enzyme activity of APX was expressed in micromoles (product) per minute per gram (FW) according to Nakano and Asada (1981).

Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activity

Monodehydroascorbate reductase activity was measured according to the method described by Hossain et al. (1984). A decrease in the absorbance was measured at 340 nm, and the extinction coefficient (6.2 mM−1 cm−1) was used to calculate the enzyme activity. The enzyme activity of MDHAR was expressed in nanomoles per minute per gram (FW).

Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity

A method initially described by Nakano and Asada (1981) was used to measure the DHAR activity. The catalyzed reaction system consisted of 2.5 mM GSH, 0.1 mM EDTA, 0.2 mM DAsA, 50 mM phosphate buffer (pH 7.0), and enzyme extract. An increase in the absorbance caused by the formation of AA was recorded at 265 nm (Ɛ = 14 mM−1 cm−1) up to 2 min, and it was expressed in nanomoles per minute per gram (FW).

Glutathione reductase (GR, EC 1.6.4.2) activity

A method initially described by Cakmak et al. (1993) was used to measure GR activity. The reaction mixture contained 1 mM EDTA, 0.2 mM NADPH, 100 mM phosphate buffer (pH 7.8), 1 mM GSSG, and enzyme extract. Changes in the absorbance of the reaction solution described below at 340 nm (Ɛ = 6.2 mM−1 cm−1) caused by NADPH oxidation were recorded for 3 min. The enzyme activity of GR was expressed in nanomoles per minute per gram (FW).

Glutathione-S-transferase (GST, EC 2.5.1.18) activity

GST activity levels were assayed according to a method initially described by Habig et al. (1974). Changes in the absorbance of the reaction solution described above at 340 nm (Ɛ = 9.6 mM−1 cm−1) caused by the formation of conjugate production were recorded for 5 min. The enzyme activity was calculated and expressed in nanomoles per minute per gram (FW).

GSH, GSSG, AA, and DHA contents

GSH and GSSG contents were measured according to a method described by Queval and Noctor (2007) with some modifications. 0.1 g of leaf sample was homogenized in 5% sulfosalicylic acid and centrifuged at 10,000×g for 10 min. The supernatant was collected to measure GSH and GSSG contents. The reaction mixture contained 120 mM phosphate buffer (pH 8.0), 0.6 mM 5,5′-dithiobis (2-nitro-benzoic acid) DTNB, 6 mM EDTA, and 0.1 mM NADPH. The reaction was initiated by adding 5 μl GR (0.12 unit μl−1). Absorbance was recorded at 412 nm, and GSH and GSSG content was expressed in micromoles per gram (FW). AA and DHA contents were measured according to a method initially described by Gossett et al. (1994). The increase in the absorbance caused by the formation of red chelate was determined at 265 nm. AA and DHA content was expressed in nanomoles per gram (FW).

RNA isolation and real-time RT-PCR

Total RNA of maize leaves (control and treatment of NILs) was isolated via the RNA pure Plant Kit (Kangwei Company, China). One microgram of total RNA was used to synthesize the cDNA using the HiScript II Q RT Super Mix reagent (Vazyme, China). Primers were designed using the Primer Premier 3 software (bioinfo.ut.ee/primer3-0.4.0/primer3/) according to gene sequences in the GenBank Database. The PCR efficiency of all primers for gene expression analysis was examined (Table 3). Only primers with higher amplification efficiency (> 90%) were used for this experiment. Maize cDNA sequences of SOD, APX, MDHAR, DHAR, GR, and GST were searched against GenBank, www.maizegdb.org and www.ncbi.nlm.nih.gov/ (Table 4). PCR amplification was performed with the HiScript II Q RT Super Mix reagent (Vazyme, China) on a real-time PCR detection system according to the manufacturer’s instructions (CFX Connect Optics Module, Singapore). The 2–ΔΔct method was used to calculate the relative transcript levels, and the gapdh gene was used as the house-keeping gene. Three biological repeats were performed for each analysis. The expression of each transcript was normalized against the amount of gapdh control transcript in each sample. Values are means ± standard error (SE) of three biological repeats.

Table 3 List of primers used for the real-time RT-PCR
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Table 4 Superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and glutathione-S-transferase genes of Zea mays
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Data analysis

Microsoft Excel and SigmaPlot 12.5 were used for data processing and mapping, and each reported data point represents the mean ± standard error (SE) of three replicates combined in the three experimental repeats. SPSS version 12.0 (SPSS Inc., Chicago, IL, USA) was used to conduct an analysis of variance (ANOVA), and mean values were compared via least significant difference (LSD) test at a P < 0.05 significance threshold indicated significant difference.

Results

O2 ·- production rate, H2O2 content, and lipid peroxidation

In SN509-R, the O2·- production rate first increased and then decreased with extended exposure time, while the O2·- production rate increased throughout in SN409-S (Fig. 2a). The average O2·- production rate of SN509-R was 8.93% higher than that of the control (NR). Compared to the control (NS), the O2·- production rate of SN509-S increased by 0.26, 18.43, 12.94, and 119.73% at 1, 3, 5, and 7 DAT, respectively. After nicosulfuron treatment, the average O2·- production rate of SN509-S was 14.18% higher than that of SN509-R. Our study showed that the accumulation of ROS in SN509-S was increased significantly, and the redox balance in the plant was severely damaged.

Fig. 2
figure 2

a–c Effects of nicosulfuron on the O2·- production rate, H2O2 content, and lipid peroxidation in leaves of maize seedlings. NR, water treatment in SN509-R; RT, nicosulfuron 80 mg kg−1 treatment in SN509-R; NS, water treatment in SN509-S; ST, nicosulfuron 80 mg kg−1 treatment in SN509-S. Vertical bars represent the SE (n = 3). Small letters indicate differences between values obtained on different days after nicosulfuron treatment (P < 0.05) according to a least significant difference (LSD) test

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After 1 DAT, nicosulfuron increased the H2O2 content by different amounts in the two inbred lines (Fig. 2c). Compared to the control (NR), nicosulfuron increased the H2O2 content of SN509-R at 1, 3, 5, and 7 DAT by up to 17.61, 22.01, 21.65, and 15.39%, respectively. Compared to the control (NS), nicosulfuron significantly enhanced the H2O2 content of SN509-S at 1, 3, 5, and 7 DAT by up to 28.10, 27.30, 44.98, and 65.07%, respectively. This suggested that H2O2 accumulation in SN509-S was increased throughout with the exposure time.

A large amount of ROS production will result in cell membrane lipid peroxidation. After 5 DAT, the MDA content of SN509-R reached a maximal value, followed by a decrease, while the MDA content of SN509-S increased with exposure time (Fig. 2b). Compared to control, MDA content of SN509-S significantly increased by 49.42, 119.38, 209.47, and 220.94% at 1, 3, 5, and 7 DAT, respectively; the MDA content of SN509-R increased by an average of 79.33% from 1 DAT to 7 DAT. Additionally, the MDA content of SN509-S was significantly higher than that of SN509-R at 3, 5, and 7 DAT, with increases of 53.33, 40.64, and 191.17%, respectively. The accumulation of MDA indicated that the resistant inbred line SN509-R has a greater capacity to adapt to herbicide stress.

Nicosulfuron induced changes in antioxidant enzymes activities

The accumulation of ROS induces the production of SOD (Fig. 3a). After herbicide treatment, the SOD enzyme activity of SN509-R was first increased and then decreased each day following treatments. However, the SOD enzyme activity of SN509-S was much lower than that of control. After 1 DAT, nicosulfuron decreased CAT activity by different amounts in the two inbred lines, while the CAT enzyme activity of SN509-S was much lower than that of SN509-R. The average CAT enzyme activity of SN509-R was 32.3% higher than that of SN509-S (Fig. 3b).

Fig. 3
figure 3

a–c Effects of nicosulfuron on superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in leaves of SN509-R and SN509-S. NR, water treatment in SN509-R; RT, nicosulfuron 80 mg kg−1 treatment in SN509-R; NS, water treatment in SN509-S; ST, nicosulfuron 80 mg kg−1 treatment in SN509-S. Vertical bars represent the SE (n = 3). Small letters indicate differences in values obtained on different days after nicosulfuron treatment (P < 0.05) according to a least significant difference (LSD) test

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In the AA-GSH cycle, The APX enzyme activity of the inbred lines showed different responses to nicosulfuron over time (Fig. 3c). The APX enzyme activity of SN509-S first increased and then reduced each day following treatments, while the APX enzyme activity of SN509-R increased significantly. The APX activity was compared between both inbred lines: compared to SN509-S, the nicosulfuron treatment significantly increased the APX enzyme activity of SN509-R at 1, 3, 5, and 7 DAT by up to 59.49, 14.26, 57.31, and 52.67%, respectively. After herbicide treatment, the MDHAR enzyme activity of SN509-R increased with increasing exposure time, while the MDHAR enzyme activity of SN509-S decreased with increasing exposure time. Compared to the control, the average MDHAR enzyme activity of SN509-R increased by 71.46% and the average MDHAR enzyme activity of SN509-S decreased by 23.33%. In addition, nicosulfuron treatment induced significant increases in DHAR enzyme activity in both inbred lines at each sampling time. The DHAR enzyme activity of SN509-R was much higher than that of SN509-S. After the spraying, the GR activity of SN509-R increased significantly, while the GR activity of SN509-S did not significantly changed (Fig. 4).

Fig. 4
figure 4

a–d Effects of nicosulfuron on monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) in leaves of SN509-R and SN509-S. NR, water treatment in SN509-R; RT, nicosulfuron 80 mg kg−1 treatment in SN509-R; NS, water treatment in SN509-S; ST, nicosulfuron 80 mg kg−1 treatment in SN509-S. Vertical bars represent the SE (n = 3). Small letters indicate differences in values obtained on different days after nicosulfuron treatment (P < 0.05) according to a least significant difference (LSD) test

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GST plays an important role in maintaining the plant redox balance. The most important biological function of GST is to regulate the binding of electron affinity substances to GSH, thus preventing continuous bursts of oxidation (Dixon et al. 2010). Following herbicide treatment, nicosulfuron caused an increase of GST activity in SN509-R, while GST activity of SN509-S increased only slightly. The average GST enzyme activity of SN509-R was 54.89% higher than that of SN509-S (Fig. 4d).

GSH, GSSG, AA, and DHA contents

After nicosulfuron treatment, GSH increased in leaves of SN509-R at 1 and 5 DAT. In contrast, GSSG of SN509-R significantly increased at each sampling time point. The GSH/GSSG value of SN509-R decreased with increased exposure time. In contrast, nicosulfuron had little effect on the GSH/GSSG of SN509-S (Fig. 5). Compared to SN509-S, nicosulfuron significantly increased both AA and DHA contents in SN509-R. AA/DHA of SN509-R remained at a high level at 7 DAT (Fig. 6).

Fig. 5
figure 5

a–c Effects of nicosulfuron on ascorbate (AA), dehydroascorbate content (DHA), and AA/DHA ratios in leaves of SN509-R and SN509-S. NR, water treatment in SN509-R; RT, nicosulfuron 80 mg kg−1 treatment in SN509-R; NS, water treatment in SN509-S; ST, nicosulfuron 80 mg kg−1 treatment in SN509-S. Vertical bars represent the SE (n = 3). Small letters indicate differences in values obtained on different days after nicosulfuron treatment (P < 0.05) according to a least significant difference (LSD) test

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Fig. 6
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a–c Effects of nicosulfuron on reduced glutathione (GSH), and oxidized glutathione content (GSSG), and GSH/GSSG ratios in leaves of SN509-R and SN509-S. SN509-R-CK, water treatment as control; SN509-R, nicosulfuron 80 mg kg−1 treatment; SN509-S-CK, water treatment as control; SN509-S, nicosulfuron 80 mg kg−1 treatment. Vertical bars represent the SE (n = 3). Small letters indicate differences in values obtained on different days after nicosulfuron treatment (P < 0.05) according to a least significant difference (LSD) test

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Nicosulfuron induced changes in gene expression

The expression levels of antioxidant enzymes in leaves of maize and their control group were measured 12 h after exposure. The obtained results showed that, compared to the control, the Cu/Zn sod9 gene of SN509-R was significantly upregulated, while the Cu/Zn sod9 gene of the sensitive inbred line SN509-S was remarkably downregulated (Fig. 7a).

Fig. 7
figure 7

a–d Transcriptional levels of different antioxidant enzymes, expressed relative to the control, in leaves of maize seedlings exposed to nicosulfuron (A. Cu/Zn SOD, B. APX1, C. APX2, D. APX3). NR, water treatment in SN509-R; RT, nicosulfuron 80 mg kg−1 treatment in SN509-R; NS, water treatment in SN509-S; ST, nicosulfuron 80 mg kg−1 treatment in SN509-S. Values are mean of three independent biological replicates. Vertical lines represent standard error. Asterisk refers to significant levels (P < 0.05)

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Nicosulfuron significantly affected the expression level of enzyme genes in the AA-GSH cycle. After herbicide treatment, compared to control, APX2 and APX3 transcripts of SN509-R were significantly increased. While the APX2 transcript of SN509-S was not altered by nicosulfuron stress, the APX3 transcript was significantly reduced in SN509-S (Fig. 7b–d). Nicosulfuron significantly affected the expression of MDHAR genes (Fig. 8a–d). After nicosulfuron spraying, MDHAR1, MDHAR2, MDHAR3, and MDHAR4 transcripts of SN509-R were significantly higher than those of control at 12 h treatment, showing increases of 156.05, 181.23, 91.37, and 193.56%, respectively. Nicosulfuron treatment significantly reduced the transcript levels of MDHAR1 and MDHAR3 in SN509-S, whereas nicosulfuron stress did not alter MDHAR2 and MDHAR4 transcripts of SN509-S. In SN509-R, the transcript levels of DHARs also increased significantly after nicosulfuron treatment (Fig. 8e, f; Fig. 9a, b). In contrast, the expressions of DHAR1, DHAR2, DHAR3, and DHAR4 in SN509-S were lower than those of the control at 12 h, showing reductions of 18.12, 36.51, 31.19, and 57.59%, respectively. GR1 and GR2 of SN509-R showed similar responses to nicosulfuron with strong transcript inductions (Fig. 9c, d). Changes in GRs expression levels were not significant in SN509-S, although slight decreases were found in GR1and GR2. Twelve hours after spraying and compared to the control, the Gst1 gene in SN509-R was not significantly changed; however, the Gst1 gene in SN509-S was significantly upregulated (Fig. 9e).

Fig. 8
figure 8

a–f Transcriptional levels of different antioxidant enzymes, expressed relative to the control, in leaves of maize seedlings exposed to nicosulfuron (A. MDHAR1, B. MDHAR2, C. MDHAR3, D. MDHAR4, E. DHAR1, F. DHAR2). NR, water treatment in SN509-R; RT, nicosulfuron 80 mg kg−1 treatment in SN509-R; NS, water treatment in SN509-S; ST, nicosulfuron 80 mg kg−1 treatment in SN509-S. Values are mean of three independent biological replicates. Vertical lines represent standard error. Asterisk refers to significant levels (P < 0.05)

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Fig. 9
figure 9

a–e Transcriptional levels of different antioxidant enzymes, expressed relative to the control, in leaves of maize seedlings exposed to nicosulfuron (A. DHAR3, B. DHAR4, C. GR1, D. GR2, E. Gst1). NR, water treatment in SN509-R; RT, nicosulfuron 80 mg kg−1 treatment in SN509-R; NS, water treatment in SN509-S; ST, nicosulfuron 80 mg kg−1 treatment in SN509-S.Values are mean of three independent biological replicates. Vertical lines represent standard error. Asterisk refers to significant levels (P < 0.05)

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Discussion

The damaging effect of nicosulfuron in plants caused an accumulation of O2·- and H2O2 that has been suggested to be associated with a reduction of electron transport activity as reported in earlier studies (Wang et al. Photosynthetica Wang et al. Accepted). Due to inhibited electron transport activity, an excess of electrons is delivered to O2, which results in the generation of O2·-. This O2·- induces the production of SOD enzymes, which results in a dismutation of O2·- to H2O2 (Prasad et al. 2016; Hassan and Alla 2005). In this study, a significant increase in the accumulation of O2·- in SN509-S was only observed at 7 DAT. Due to the low activities of SOD and CAT, the O2·- in SN509-S cannot be removed and eventually converted into a large number of H2O2 molecules. It has been suggested that nicosulfuron, as a toxic factor, creates oxidative stress by increasing ROS levels in plants, eventually inactivating SOD and CAT in plants. This is supported by previous results reported by Alla and Hassan (2007) showing that isoproturon significantly accelerated the production of H2O2 in maize seedlings. Compared to the unstable O2·-, H2O2 is more stable and easier to transport between cells. Under abiotic stress, high concentrations of H2O2 will produce hydroxyl radicals, thus exacerbating the toxicity of ROS (Basu et al. 2017; Gill and Tuteja 2010). Furthermore, excessive accumulation of H2O2 will lead to membrane lipid peroxidation. Numerous studies have demonstrated that membrane lipid peroxidation leads to the accumulation of MDA and herbicide treatment also causes the accumulation of MDA (He et al. 2017; Yu et al. 2015). Here, the MDA content of SN509-S increased with increasing exposure time, while the content of SN509-R was significantly lower than that of SN509-S. These findings suggest that oxidative stress in the leaves of SN509-S via nicosulfuron increased with exposure time. An effective defense mechanism in the leaves of SN509-R could protect plants from nicosulfuron damage.

H2O2 can be effectively detoxified in chloroplasts and other organelles; here, APX is a key enzyme of the AA-GSH pathway, which has been reported to be involved in the transformation of H2O2 into H2O that utilizing AA as electron donor (Gill and Tuteja 2010). In the present study, after nicosulfuron treatment, APX activity of SN509-R was higher than that of SN509-S. Under nicosulfuron stress, the low level of APX enzyme activity compared to the control might be associated with a low level of AA content in SN509-S, since the APX enzyme stability is closely related to the AA content (Prasad et al. 2016). Furthermore, MDHA and DHA regenerate AA via MDHAR and DHAR enzymes. The results of this study showed that the average MDHAR and DHAR activity of SN509-R was higher than that of SN509-S. Altered MDHAR and DHAR activities have been reported in the presence of patulin in maize (Ismaiel and Papenbrock 2017). The GR activity was significantly increased in SN509-R and GSH content continued to decline. However, compared to the control, the GSH content of SN509-R remained at a high level. In contrast, compared to control, the GR activity of SN509-S was not significantly different. In summary, these results suggest that nicosulfuron significantly suppresses MDHAR, DHAR, and GR activities in SN509-S, and this reduction in their activities is related to the depletion of GSH. Bashir et al. (2015) furthermore suggested that increased enzyme activity is closely related to changes in both GSH content and AA-GSH cycle activity in Indian mustard. In addition, the low level of MDHAR and DHAR activities in SN509-S may lead to a change in AA homeostasis, which is indicated by the severe decrease of the ratio of AA/DHA. Many studies have demonstrated that the regeneration of AA requires NADPH as the electron donor for enzyme activities (Foyer and Noctor 2011). Under nicosulfuron treatment, damage to the electron transport chain may be an important reason for the reduction of the ratio of AA/DHA. Bartoli et al. (2006) reported a direct interaction between the photosynthetic electron transport chain and AA. Therefore, disruption of photosynthetic electron transport activity by nicosulfuron may be a further reason for the decrease in AA content.

Many studies have demonstrated that SOD was strongly related to the quenching of superoxide into H2O2 (Baek and Skinner 2003; Sun et al. 2018; Xu et al. 2017). There are three types of SOD enzymes, including MnSOD, FeSOD, and Cu/ZnSOD. However, Cu/ZnSOD is more stable than MnSOD and FeSOD (Bueno et al. 1995). In this study, the expression of genes encoding antioxidative enzyme variations was assayed after nicosulfuron treatment in 12 h. After herbicide treatment, compared to control, the expression of sod9 in SN509-R was strongly upregulated, while the expression of sod9 was strongly downregulated in SN509-S. A similar result has been reported for Arabidopsis thaliana, maize, and rice (Yu et al. 2014; Xu et al. 2017). In contrast, the expression of Cu/ZnSOD in Kentucky bluegrass was downregulated under drought stress (Bian and Jiang 2009). This difference may be due to the different plant species used and different stress conditions.

Under nicosulfuron stress, the expression of different APX genes showed different responses, indicating that separate genes may respond to specific conditions. Compared to control, APX2 and APX3 of SN509-R showed the strongest responses to the nicosulfuron treatment, with 1.5–2-fold transcript increases at 12 h after treatments, while the expression of APX3 in SN509-S was strongly downregulated; however, the expression of APX1 remained unaltered in both inbred lines. MDHAR is the second enzyme in the AA-GSH cycle, which exhibited upregulation under a series of oxidative stresses and thus plays a critical role in plant defense against ROS (Begara-Morales et al. 2015; Johnston et al. 2015). Nicosulfuron also significantly increased the transcript levels of MDHAR1, MDHAR2, and MDHAR3 in SN509-R following 12 h of nicosulfuron treatment. In contrast, the nicosulfuron treatment led to significantly reduced transcript levels of MDHAR1, MDHAR2, and MDHAR3 in SN509-S. However, the transcript levels of MDHAR4 slightly decreased in both inbred lines. This suggested that MDHAR1, MDHAR2, and MDHAR3 may play more central roles to protect plants against nicosulfuron-induced oxidative stress compared to MDHAR4.

It has been demonstrated that DHAR overexpression also increases plant resistance to various oxidative stresses (Shin et al. 2013; Zhang et al. 2015). In this study, nicosulfuron treatment significantly increased the transcript levels of DHAR1, DHAR2, DHAR3, and DHAR4 in SN509-R, in contrast to SN509-S. These findings suggested that DHAR responded stronger to nicosulfuron stress caused after 12 h of nicosulfuron treatment. It was also shown that GR plays an important role in regulating a variety of oxidative stresses, and the overexpression of GR enhanced plant tolerance to heavy metal, salt, and drought stress (Seo et al. 2006; Torres-Franklin et al. 2008; Wu et al. 2015). We also observed that nicosulfuron increased the transcription of GR genes in SN509-R significantly, while the expression of GR genes in SN509-S remained unaltered, indicating the vital function of GR in the nicosulfuron stress tolerance of maize.

For the evaluation of detoxification capacity in a pair of nearly isogenic lines (NILs) of waxy maize, both activity and the transcript level of GST of maize treated with nicosulfuron were studied. Our results indicate that the average GST activity of SN509-R was significantly higher than that of SN509-S. Furthermore, the transcriptions of Gst1 were increased in both inbred lines. The abiotic stress caused an increase of GST activity and transcript, which is in agreement with previous studies. The overexpression of the Gst1 gene in transgenic potatoes can significantly increase the activity of GST and GPX enzymes and enhanced the resistance of transgenic plants to oxidative stress (Gill and Tuteja 2010).