Nicosulfuron stress on the glyoxalase system and endogenous hormone content in sweet maize seedlings

To reduce the harmful effects of nicosulfuron on sweet corn, the physiological regulation mechanism of sweet corn detoxification was studied. This study analyzed the effects of nicosulfuron stress on the glyoxalase system, hormone content, and key gene expression of nicosulfuron-tolerant “HK301” and nicosulfuron-sensitive “HK320” sweet corn seedling sister lines. After spraying nicosulfuron, the methylglyoxal (MG) content in HK301 increased first and then decreased. Glyoxalase I (GlyI) and glyoxalase II (GlyII) activities, non-enzymatic glutathione (GSH), and the glutathione redox state glutathione/(glutathione + glutathione disulfide) (GSH/(GSH + GSSG)) showed a similar trend as the MG content. Abscisic acid (ABA), gibberellin (GA), and zeatin nucleoside (ZR) also increased first and then decreased, whereas the auxin (IAA) increased continuously. In HK301, all indices after spraying nicosulfuron were significantly greater than those of the control. In HK320, MG accumulation continued to increase after nicosulfuron spraying and GlyI and GlyII activities, and GSH first increased and then decreased after 1 day of stress. The indicators above were significantly greater than the control. The GSH/(GSH + GSSG) ratio showed a decreasing trend and was significantly smaller than the control. Furthermore, ABA and IAA continued to increase, and the GA and ZR first increased and then decreased. Compared with HK320, HK301 significantly upregulated the transcription levels of GlyI and GlyII genes in roots, stems, and leaves. Comprehensive analysis showed that sweet maize seedlings improved their herbicide resistance by changing the glyoxalase system and regulating endogenous hormones. The results provide a theoretical basis for further understanding the response mechanism of the glyoxalase system and the regulation characteristics of endogenous hormones in maize under nicosulfuron stress.


Introduction
China is the world's second-largest sweet corn producer and main importer and exporter. Therefore, the sweet corn market has a good development prospect (Li and Huang 2021). Recently, the breeding of sweet corn varieties has developed with a focus on high sugar content to meet human needs. However, the increase of sugar content in sweet corn grains will inevitably reduce seed vigor, leading to the slow growth of the population canopy and the inability to cope with the competition with weeds and, subsequently, yield reduction. Using herbicides is a simple and effective method for sweet corn weed control in the field (Mccourt et al. 2006). In 1999, the use of nicosulfuron as a sweet corn post-seedling herbicide was approved in some areas of the USA, providing a new approach to controlling sweet corn postseedling weeds. As an inhibitor of acetyllactate synthase (ALS), nicosulfuron can effectively control various annual and perennial weeds and some broad-leaved weeds. Inhibiting ALS activity in plants blocks the synthesis of branched amino acids (valine, leucine, and isoleucine), thus inhibiting cell division (Mccourt et al. 2006). The harmful effects of nicosulfuron on different maize varieties are similar. Under early stress, sensitive varieties present leaf etiolation and wilting, accompanied by the purple leaf phenomenon. With the growth of plants, the leaves grow horsetail curls, cause plant dwarfness, and may produce secondary stems. With the further aggravation of nicosulfuron damage, sensitive species plants stop growing, resulting in purple stems and leaves turning yellow, and in serious cases, death occurs (Wang et al.2016).
Several studies have shown that under various abiotic stress conditions, plants produce high concentrations of reactive oxygen species (ROS) as an oxidative stress response, reduce the activity of photosynthetic electron transport, inactivate the PSI and PSII systems of the photoreaction centers, inhibit the biosynthesis of plant chlorophyll, and thus inhibit various metabolic activities in plants (Bigot et al. 2007;Sirhindi et al. 2016). In addition to ROS, another highly active toxic substance, methylglyoxal (MG), accumulates in large quantities in plant cells under various abiotic stress conditions. High concentrations of MG produce irreversible advanced glycation end products by combining with amino acid residues in proteins such as arginine, lysine, and cysteine. MG can also react with membrane lipids to produce irreversible advanced lipid peroxidation end products. Moreover, MG can induce ROS production, which can produce oxidative stress, destroy proteins, DNA, RNA, lipids, and biofilms, or inactivate the antioxidant defense system, leading to cell death (Mostofa et al. 2018). Plant cells have an MG detoxification glyoxalase system which consists of two main enzymes, glyoxalase I (GlyI) and glyoxalase II (GlyII). Firstly, S-D-lactate glutathione (SLG) is formed by the reaction of MG, and reduced glutathione GSH is catalyzed by Gly I. The hydrolysis of SLG catalyzed by GlyII regenerates GSH and lactate to maintain cellular redox balance (Ye et al. 2019). Ghosh et al. showed that the overexpression of the GlyII gene improved the tolerance of rice to salt stress (Ghosh et al.2014). Simultaneously, overexpression of GlyI and GlyII genes in transgenic tobacco plants also increased the activity of GSH metabolic enzymes, indicating a close interaction between the antioxidant defense system and the glyoxalase system (Singla-Pareek et al. 2006). Therefore, the glyoxalase system plays an important role in plant sensing, response, and adaptation to environmental stress. Although the exact mechanism related to abiotic stress tolerance remains to be elaborated, recent studies have found that plant responses to multiple abiotic stresses are regulated by complex signal networks (Devanathan et al.2014). ABA, IAA, GA, and JA can reduce ROS accumulation, improve the activities of antioxidant enzymes and detoxification enzymes, and regulate stomatal opening (Hossain et al.2015). The involvement of MG and plant hormones in signal transduction in removing oxidative pressure and restoring oxidative balance in plants is still unclear. MG controls stomatal opening and closure by regulating ROS products and calcium ion concentration in arabidopsis mesophyll cells, and ABA and JA are not involved in this signal transduction process (Hoque et al. 2012a, b). However, Hoque et al. (2012a, b) revealed that MG controls stomatal opening and closure by inhibiting potassium ion entry into guard cells, and ABA participates in this signal transduction process.
In response to various environmental stresses, changes in MG content, regulation of the glyoxal system and key genes, and changes in endogenous hormones in plants have been widely reported. However, the accumulation of MG, expression of the glyoxalase system and key genes, and response mechanism of endogenous hormones in sweet corn under nicosulfuron stress have been rarely reported. Previous studies have shown that compared with the resistant inbred line HK301, the photosynthetic capacity and electron transport activity of HK320 were reduced under nicosulfuron-pyrisulfuron stress, and an increased amount of ROS accumulated in the plant, leading to a significant decrease in the activity of its antioxidant enzyme system (Wu et al. 2022). Therefore, in this study, we continued using a pair of sister lines, nicosulfuron-tolerant HK301 and nicosulfuron-sensitive HK320, differing in nicosulfuron tolerance to study the changes of the glyoxalase system and regulation rules of endogenous hormones in sweet corn under nicosulfuron stress and elucidate the physiological mechanism of interaction between multiple enzyme systems to reduce herbicide damage. The results will provide a theoretical basis for enhancing the resistance of sweet corn and realizing stable and high yields by cultivation regulation.

Plant materials
A pair of sister lines (nicosulfuron-tolerant HK301 and nicosulfuron-sensitive HK320) was used as the plant material.

Field experiment
Our experiment was conducted at the Dongyang experimental station of the Zhejiang Academy of Agricultural Sciences (29°16′ N, 120°59′ E), Dongyang, Zhejiang Province, China, during 2021-2022. The Dongyang experimental station is located in a subtropical monsoon climate zone with sufficient light and rainfall. The temperature was 25 ± 3 ℃ (day)/15 ± 3 ℃ (night). The experiment was designed as a randomized complete block design with three replicates (6 m in length and width). Approximately 150 plants were planted in each block. Each inbred line was sown in four rows measuring 6 m in length, with 65 cm between rows and 27 cm between plants. Three seeds were planted in each hole to enhance the seedling rate. When maize seedlings reached the four-leaf stage, an electric backpack sprayer with a nozzle was used to spray nicosulfuron, with water used as the control. Nicosulfuron was soluble in water, and the effective concentration was 80 mg·kg −1 (Wu et al. 2022;Yang et al.2021;Wang et al.2021). Furthermore, HK301 plants could attain normal growth under this condition, whereas HK320 plants either wilted or completely died. Field investigation and sampling were conducted 1 day, 3 days, 5 days, and 7 days after treatment (DAT).

MG content
About 0.3 g of leaves was joined in 3 ml 5% perchloric acid extract, placed it on ice for 15 min, and centrifuged at 11,000 g at 4℃ for 10 min. The extract was decolorized with charcoal (10 mg/ml) and centrifuged at 11,000 g for 10 min after 15 min at room temperature. The supernatant was neutralized with saturated potassium carbonate for 15 min and then centrifuged at 11,000 g for 10 min to obtain the supernatant for MG determination. MG content was calculated according to the Wild et al. (2012) method.

Measurement of enzyme activities
The GlyI enzyme activity determination method used 1.7 mM GSH and 3.5 mM MG as a reactant. The increase of 240 nm absorbance value was recorded within 1 min after the reaction started, and the activity was calculated using the extinction coefficient of 3.37 mM −1 cm −1 (Hasanuzzaman and Fujita 2011). The GlyII enzyme activity was measured according to Hossain's method (Hossain et al. 2010). The reactants used were 0.2 mM 5,5′-dithiobis (2-nitro-benzoic acid) (DTNB, Ellman's reagent) and 1 mM S-D-lactoylglutathione (SLG), and the activity was calculated using 13.6 mM −1 cm −1 extinction coefficient.

Non-enzymatic substances assay
The determination method of GSH and GSSG contents was based on Queval and Noctor (2007). The absorbance value was determined at 412 nm using a GR-dependent reduction method dependent on DTNB. The content of GSH and GSSG was determined without pretreatment of the extract. Specific determination of the GSSG content was obtained after VPD pretreatment of the extract.

Hormone content
IAA, ZR, GA, and ABA in roots and leaves were determined by Agilent 1100-series high-performance liquid chromatography using the method of Hou et al. (2008). The Finnigan LC-MS/MS system (Thermo Electron, San Jose, CA, USA), HiQ Sil C 18 chromatographic column (250 mm × 4.6 mm i.d., 5 µm), and methanol/water containing 0.2% formic acid (50:50, v/v) were used to determine the hormone content. The elution flow rate was 1.0 mL/min, and benzoic acid was used as an internal standard (ISTD). The determination results were calculated according to the standard curve.

Gene expression assay
GlyI and GlyII gene sequences were obtained by GeneBank and maize gene database (www. maize gdb. org). Primer Premier 5 software was used to design gene primers according to the gene sequences in the database, and NCBI primer-BLAST (https:// www. ncbi. nlm. nih. gov/ tools/ primer-blast/ index. cgi? LINK_ LOC= Blast Home) was used to compare the primers on NCBI to ensure specificity. The internal reference gene was GAPDH (Table 1). Sample RNA was extracted using total RNA extraction reagents (TaKaRa, Japan). Data were analyzed using the 2 −ΔΔct method.

Statistical analysis
Data processing and mapping were performed using Microsoft Excel and SigmaPlot 12.5. Analysis of variance (ANOVA) and mean values were compared using the least significant difference (LSD) test in SPSS (V. 12.0; SPSS Inc., Chicago, IL, USA). Significant differences were identified at the P < 0.05 threshold.

MG content
In HK301, MG content first increased and then decreased after nicosulfuron treatment and was significantly greater than that of the control (Fig. 1A). The maximum MG content was reached at 3 DAT, which was significantly increased by 32.6% compared with the control. In contrast, after 1 DAT, the MG content in HK320 continued to increase and increased significantly by 80.1% compared with the control at 7 DAT (Fig. 1B).

GlyII activity
After nicosulfuron stress, GlyII activity in HK301 seedling leaves continued to increase and reached the maximum value at 5 days, then decreased with the extension of stress, although it was still significantly higher than that for the control. GlyII activity in HK301 significantly increased at 1, 3, 5, and 7 DAT by 86.1%, 179.9%, 200.1%, and 90.0%, respectively, compared with HK301-CK (Fig. 2C). Significant increases in GlyII activity in HK320 were detected 1 DAT owing to nicosulfuron, and with stress time extension, GlyII activity was significantly lower than HK320-CK. It decreased by 24.1%, 53.7%, and 45.6% at 3, 5, and 7 DAT, respectively (Fig. 2D).

GSH, GSSG content, and (GSH/GSH + GSSG) ratio
After nicosulfuron stress, the GSH content of HK301 reached the maximum value at 3 DAT, which was significantly increased by 115.8%, higher than HK301-CK. Subsequently, it showed a downward trend, but it was still significantly higher than that of the control. The GSH content of HK301 at 1, 3, 5, and 7 DAT was significantly increased by 42.6%, 115.8%, 110.8%, and 86.1%, respectively (Fig. 3A). However, the GSH content of HK320 reached the maximum at 1 DAT and then continued to decrease. At 5 and 7 DAT, the GSH content of HK320 decreased by 54.0% and 47.2%, Fig. 1 Effects of nicosulfuron on the MG content in leaves of maize seedlings. HK301-CK: water treatment in HK301; HK301: nicosulfuron 80 mg·kg -1 treatment in HK301; HK320-CK: water treatment in HK320; HK320: nicosulfuron 80 mg·kg -1 treatment in HK320; vertical bars represent the SE (n = 3). Small letters (a, b) indicate differences between values obtained on different days after nicosulfuron treatment (P < 0.05) according to a least significant difference (LSD) test respectively, compared with the control, and the difference reached a significant level (Fig. 3B).
With the prolonged duration of the nicosulfuron stress time, the GSSG content of HK301 continued to increase significantly and reached the maximum value at 7 DAT, which was 107.5% higher than HK301-CK (Fig. 3C). Except at 1 and 3 DAT, the GSSG content of HK320 increased by 17.4% and 25.6% compared with HK320-CK, and there was no significant change at other time points (Fig. 3D).
After the addition of nicosulfuron, the GSH/GSSG ratio of HK301 was significantly increased by 42.21% compared with HK301-CK. Subsequently, it showed a downward trend and was significantly lower at 7 DAT compared to HK301-CK (Fig. 3E). Compared to HK320-CK, the GSH/GSSG ratio of HK320 was significantly higher only at 1 day of stress, then showed a downward trend. Compared with the control, the GSH/GSSG ratio of HK320 was significantly decreased by 56.8% and 48.4% on days 5 and 7, respectively (Fig. 3F).
After treatment with nicosulfuron, the GA of the two sweet corn inbred lines significantly increased. Compared to HK301-CK, the proline content in HK301 increased by 68.0%, 323.3%, 396.3%, and 343.2% at 1, 3, 5, and 7 DAT, respectively (Fig. 4E). The GA of HK320 reached the maximum value at 3 DAT after nicosulfuron stress, which increased by 138% compared with the control and then decreased with the extension of treatment time. When the stress lasted for 5 days and 7 days, the GA increased by 24.5% and 55.0% compared with the control, which was still significantly higher than the control (Fig. 4F).

Nicosulfuron-induced alterations in glyoxalase-related genes
The differential expression patterns of the glyoxalase gene in roots, stems, and leaves of maize seedlings treated with nicosulfuron were determined. Our results demonstrated that the GlyI gene expression of HK301 in roots, stems, and leaves was significantly upregulated compared to the control. There was no significant difference in GlyI transcript levels in the roots and stems of HK320 after nicosulfuron treatment. However, GlyI in leaves of HK320 was significantly downregulated compared to the control. The increment of GlyI expression in roots, stems, and leaves in HK301 was significantly greater than that in HK320 after nicosulfuron stress (Fig. 5A).
The addition of nicosulfuron significantly affected the transcript level of GlyII. GlyII in HK301 was strongly induced to be transcribed by nicosulfuron treatment, which was significantly upregulated in roots, stems, and leaves. In contrast, GlyII in roots, stems, and leaves of HK320 was significantly downregulated compared to the control. Compared with HK320, HK301 significantly upregulated the relative expression of GlyII in roots, stems, and leaves (Fig. 5B).

Discussion
The preliminary results of this study showed that compared with the drug-resistant inbred line HK301, the photosynthetic capacity and electron transport activity of the sensitive inbred line HK320 were decreased, and a large amount of ROS accumulated in the plant, leading to a significant decrease in the activity of its antioxidant enzyme system. With ROS production, plants also produce a large amount of the highly active cytotoxic substance MG (Wu et al. 2022). Several studies have shown that MG is a byproduct of fatty acid, protein, and glucose metabolism in plants and an internal mediator of ROS production, which can intensify oxidative damage in plants (Hossain et al. 2009). This study showed that MG accumulation of the sensitive inbred HK320 was significantly increased after spraying with nicosulfuron compared with the resistant inbred HK301, and MG accumulation of HK320 was significantly higher than that of the control by 80.1% and 62.7% after 7 DAT of spraying. Several studies have shown that plants regulated the content of MG in vivo through the glyoxalase system, and the overexpression of GlyI and GlyII can over-improve the scavenging ability of MG in plants under various abiotic stresses. GlyI and GlyII activities of HK320 were significantly lower than those of HK301 after nicosulfuron stress, and GlyI and GlyII activities of HK320 were 15.5% and 74.3% lower than those of HK301 after 7 DAT, respectively, with a significant difference. In addition, the results showed that compared with HK301, GlyI of HK320 was significantly downregulated in roots, stems, and leaves, and the relative expression level of GlyI in leaves was the lowest. GlyII of HK320 was significantly downregulated in roots, stems, and leaves, and the relative expression level was the lowest in leaves. Chao et al. (2005) showed that under salt stress, the transcriptional expression level of Gly I in salttolerant rice varieties was higher than that in salt-sensitive rice varieties. Further studies showed that salt-tolerant rice varieties had higher Gly I and Gly II activities and lowered endogenous MG content. Yadav et al. (2005a) showed that drought stress, sorbitol, and ABA treatment can upregulate the gene expression of Gly I and Gly II in rice seedlings and reduce endogenous MG content, thus improving the drought tolerance of seedlings. This study was consistent with previous studies and proved that, compared with Effects of nicosulfuron treatment on the expression level of GlyoxalaseI (GlyI) and GlyoxalaseII (GlyII) related genes in roots, stems, and leaves of sweet corn seedlings. HK301-CK: water treatment in HK301; HK301: nicosulfuron 80 mg·kg -1 treatment in HK301; HK320-CK: water treatment in HK320; HK320: nicosulfuron 80 mg·kg -1 treatment in HK320. Small letters (a, b) indicate differences between values obtained on different days after nicosulfuron treatment (P < 0.05) according to a least significant difference (LSD) test HK320, Gly I and Gly II genes in HK301 can effectively respond to herbicide stress, improve Gly I and Gly II activities in vivo, and regulate MG content in plants.
GSH, as an electron donor, is an important link between the antioxidant and glyoxalase systems. GSH plays an important role in the detoxification of ROS and MG (Pei et al. 2019). This study showed that under nicosulfuron stress, the GSH content of HK301 was significantly higher than that in control, and the GSH content of HK320 was significantly lower than that of the control 3 to 7 days of stress. Meanwhile, compared with HK301, the ratio of GSH/ GSH + GSSG in HK320 decreased, and the redox-steady state was broken. Hasanuzzaman and Fujita (2011) think that by maintaining GSH homeostasis and antioxidant enzyme levels, the increase of ROS and MG levels under stress conditions could be limited by the overexpression of glyoxalase in plants. Yadav et al. (2005b) found that GSH may be the limiting factor of MG content in transgenic tobacco, and the increase of GSH plays a very important role in eliminating MG or Mg-induced oxidative stress response. Nahar et al. (2015) observed that exogenous GSH could reduce MG content in plants and reduce oxidative damage. Alla and Hassan (2007) found that the detoxification ability of ROS decreased with a GSH decrease through the effect of isopropanone on maize. Elkelish et al. (2019) observed that increasing GSH content can improve the oxidative defense system, and the up-regulation of the oxidative defense system can avoid the negative effects of oxidative damage caused by ROS (Ahanger et al. 2018). In our experiment, the MG content in HK320 increased significantly, possibly due to the decreased activity of glyoxalase, resulting in the inability to generate more GSH to mobilize the detoxification enzyme of the antioxidant system to clear it.
Endogenous hormones play an important role in regulating plant growth and development (Peleg and Blumwald 2011). Although endogenous hormones can induce the expression of plant defense genes under abiotic stress and improve plant stress resistance, multiple endogenous hormones coordinate to regulate multiple physiological processes in plants (Yuan et al. 2012). Yuan et al. (2012) showed that low sulfursulfuron chloride and sulfursulfuron aminophenyl concentrations increased GA and ZR in rice and soybean leaves and decreased ABA. In contrast, high concentrations of herbicide treatment reduced GA and ZR and increased ABA. Guo et al. showed that a low concentration of MTC increased IAA, GA, and ZR in millet seedlings, which showed a promoting effect on millet seedlings. In contrast, a high concentration of MTC resulted in a large accumulation of IAA and ABA in millet seedlings and reduced GA and ZR, leading to the final death of millet seedlings (Guo et al. 2020). This study showed that compared with HK301, the IAA and ABA of HK320 were significantly increased, and ZR and GA were significantly decreased. During 7 days of nicosulfuron stress, ABA and IAA in HK320 increased by 19.4% and 27.7% compared with that in HK301, whereas ZR and GA decreased by 23.8% and 54.1% on average. The high sensitivity of HK320 to nicosulfuron may lead to the accumulation of IAA in HK320, which induces the accumulation of ABA. Simultaneously, the decrease of ZR and GA in HK320 weakened its antagonistic effect on ABA accumulation, resulting in leaf senescence and death. Wang and Xiong (2016) showed that in ryegrass leaves, ABA, GA, IAA, and ZR were closely related to their hightemperature resistance. Bashandy et al. (2010) found that glutathione homeostasis plays an important role in IAA transport in arabidopsis. Han et al. (2013) suggested that blocking the regulation of GSH in arabidopsis can reduce SA accumulation and SA-dependent gene expression and proved that glutathione status is a determinant of JA synthesis, signaling, and transport in plants. The increase in ABA contributes to the increase of the total activity of glutathione reductase, whereas glutathione reductase reduces GSH (Zhang et al. 2006). In this study, under nicosulfuron stress, the ABA of HK320 was significantly higher than that of HK301, whereas IAA was significantly lower than that of HK301, and the GSH of HK301 was significantly higher than that of HK320. This study was consistent with previous studies and proved the coordinated response mechanism of various plant endogenous hormones to the stress of nicosulfuron.

Conclusion
Under nicosulfuron stress, compared with the resistant variety HK301, the sensitive variety HK320 could not degrade the absorbed nicosulfuron rapidly in the plant. Furthermore, the non-enzymatic substances GSH and GSH/GSH + GSSH were continuously decreased owing to the significant decrease in the activity of the glyoxalase system (GlyI and GlyII), and the redox-homeostasis was broken. As a result, the antioxidant defense system was inactivated, the toxic substance MG could not be cleared, and the accumulation increased significantly, leading to cell death. Moreover, owing to the high sensitivity of HK320 to nicosulfuron, the endogenous hormone content changed significantly, IAA and ABA increased, and the coordination mechanism was disturbed, eventually leading to leaf senescence and death. This study proved that the glyoxalase system, the expression of key genes, and the coordination mechanism of endogenous hormones play an important role in response to herbicide stress and MG clearance in different drug-resistant sweet maize varieties.