Introduction

Aromatic amino acids (AAA), including tryptophan, tyrosine, and phenylalanine, serve as precursors for numerous natural products (Parthasarathy et al. 2018) and play crucial roles in plant growth and development (Less et al. 2010). They are involved in various physiological processes such as auxin biosynthesis, enhancing drought resistance, promoting lignin synthesis, participating in signal transduction, and responding to biotic and abiotic stresses (Lynch et al. 2020; Pandey et al. 2017).

Shikimic acid serves as a precursor in the synthesis of AAA, also known as the shikimate pathway. Shikimic acid is synthesized through a series of enzyme-catalyzed reactions involving phosphoenolpyruvate (PEP) and erythros-4-phosphate, including 3-deoxyarabinoheptulose-7-phosphate, 5-enolpyruvate shikimate-3-phosphate synthase (EPSPS), dehydroguanic acid synthase, dehydroguanic acid dehydrase and shikimate dehydrogenase (Maeda et al. 2012; Tzin and Galli 2010; Wu et al. 2022; Suh et al. 1993). Shikimic acid is then used in enzyme-catalyzed reactions involving shikimate kinase, EPSPS enzyme, cladonic acid synthase, AAA, and other enzymes to produce secondary metabolites.

EPSPS enzyme is a key enzyme in the shikimate pathway and the sole target of the herbicide glyphosate (Okumu et al. 2019). Glyphosate is the most widely used broad-spectrum herbicide worldwide, making research on glyphosate and EPSPS enzyme in plants highly significant (Duke 2011). Based on the inherent sensitivity and catalytic efficiency of EPSPS enzyme towards glyphosate, EPSPS enzymes can be classified into two major classes. Class I EPSPS enzymes are found in all plants and certain bacterial species such as Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium, which are relatively sensitive to glyphosate inhibition. Class II EPSPS enzymes are only present in bacteria such as Staphylococcus aureus and Agrobacterium tumefaciens. Currently, the most widely used EPSPS gene is derived from Bacillus subtilis and has been extensively studied and applied in plants (Funke et al. 2006; Herrmann and Weaver 1999; Dill et al. 2008; Chhapekar et al. 2015).

The mechanism of action of glyphosate involves mimicking the carbon state of PEP. Glyphosate competitively binds reversibly with EPSPS enzyme to form a stable but non-covalent ternary complex EPSPS-S3P-glyphosate, leading to the loss of EPSPS enzyme activity. This diversion causes a substantial flow of carbon sources towards S3P, resulting in rapid accumulation of shikimic acid within tissues. Consequently, the synthesis of essential aromatic amino acids (AAA) necessary for protein biosynthesis is severely hindered, ultimately inhibiting plant growth (Duke and Powles 2009; Wiersma et al. 2015).

Frequent use of glyphosate can lead to the development of glyphosate resistance in weeds, with amplification of the EPSPS gene being the primary mechanism conferring glyphosate resistance in Amaranthus populations (Mahajan et al. 2018). Chao Ouyang discovered a naturally evolved TIPS-EPSPS mutation that has been shown to confer high resistance to glyphosate in goosegrass, with resistance levels 180 times that of the wild type (WT) (Ouyang et al. 2021). Overexpression of the EPSPS gene and site-specific mutations can enhance plant resistance to glyphosate to varying degrees. When the EPSPS gene is overexpressed, EPSPS enzyme synthesis increases, and recommended field doses of glyphosate do not kill the plants, thereby enhancing glyphosate resistance (Vázquez-García et al. 2020; Chandi et al. 2012; Alcántara-de la Cruz et al. 2016).

Although EPSPS enzyme is the sole key enzyme in the shikimate pathway (Ge et al. 2012), so far, the Class II EPSPS genes sourced from bacteria have been most widely studied and utilized in plants, while research on Class I EPSPS genes from plants remains insufficiently explored. Further investigation is needed to determine whether overexpressing endogenous EPSPS genes or optimizing them via codon usage can enhance plant resistance to glyphosate. Glyphosate reduces photosynthetic efficiency, increases chlorophyll degradation, inhibits chlorophyll function, carotenoid synthesis, ferredoxin enzyme activity, auxin transduction, and increases auxin oxidation (Ozturk et al. 2008; Vivancos et al. 2011). This study measured changes in chlorophyll content, soluble sugars, malondialdehyde (MDA), relative conductivity, and ROS levels before and after glyphosate treatment in overexpressing and wild-type plants to assess glyphosate’s impact on photosynthesis and plant stress resistance. The study explores whether codon optimization of maize’s endogenous EPSPS gene can enhance glyphosate resistance, optimizing the gene’s impact on the shikimate pathway, and the effects of glyphosate treatment on plants. This research lays a theoretical foundation for breeding glyphosate-resistant maize.

Materials and methods

Optimization of EPSPS gene and construction of overexpression vector

Using the online software GeneScript (https://www.genscript.com.cn/codon-opt.html), the codons of the endogenous EPSPS gene in maize were optimized to construct an expression vector.

Plant materials and growth conditions

Materials included WT Arabidopsis thaliana seeds and T2 seeds overexpressing the Y-EPSPS gene. Seeds were sterilized in 75% ethanol for 1 min, followed by 1% sodium hypochlorite for 10 min, and then rinsed 5–6 times with sterile water. Seeds were placed on 1/2 MS solid medium without glyphosate or with glyphosate, and incubated at 4 °C in the dark for 3 days before being transferred to continuous light for germination. T2 Arabidopsis seeds were germinated in vermiculite under conditions of 220 mmolm-2s-1 light intensity, a 16 h photoperiod, 8 h dark period, temperature maintained at 22–24 °C, relative humidity at 65%, and nutrient solution sprayed every 15 days.

Detection of overexpression positive plants

T2 generation Arabidopsis thaliana overexpressing the Y-EPSPS gene was screened with a 1/1000 concentration of glyphosate, sprayed every 2 days. Surviving seedlings were transplanted into soil (nutrient soil:vermiculite = 8:3). Arabidopsis young leaves were ground with a pestle in 1.5 mL centrifuge tubes containing 200 μL buffer, followed by incubation of Bar test strips in the tubes for 5 min, and subsequent PCR detection. After covering the entire plastic tray with leaves from overexpressing plants, samples were collected for genomic extraction, and PCR detection of the screened marker gene Bar was conducted (primer sequences in Supplementary Material Table 1). Positive plants were utilized for subsequent physiological and biochemical indicator assays.

Glyphosate resistance of overexpressed positive seeds and plants

Wild-type Arabidopsis seeds and seeds overexpressing the Y-EPSPS gene were separately placed on 1/2 MS solid medium without glyphosate or supplemented with 0.5 and 0.8 mM/L glyphosate, and their germination rates were assessed. Arabidopsis plants overexpressing the Y-EPSPS gene were cultivated in nutrient soil. When these plants reached 4 weeks of age, they were subjected to 0.8 mM/L glyphosate stress. One week later, agronomic traits of the plants were statistically analyzed and photographed.

Expression analysis of Y-EPSPS gene under glyphosate stress

Four-week-old T2 generation Arabidopsis overexpressing the Y-EPSPS gene were subjected to 0.8 mM/L glyphosate stress. Leaf samples were collected 3 days post-treatment, rapidly frozen in liquid nitrogen, and RNA was extracted for fluorescence quantitative PCR. The top two positive plants with the highest expression levels of the Y-EPSPS gene were selected for subsequent experiments.

Detection of H2O2 and O2 − content

DAB staining buffer was prepared (0.02 g DAB dissolved in 50 mL water) and placed in 50 mL centrifuge tubes. The fifth leaf of 4-week-old Arabidopsis thaliana was immersed in DAB staining solution and vacuum-infiltrated for 30 min, followed by overnight incubation at room temperature. Chlorophyll was removed in 95% ethanol at 80 °C, and photographs of the staining results were taken (Wang et al. 2022).

NBT staining buffer was prepared (0.5 g NBT, 500 µL sodium azide solution, 500 µL 1 M sodium phosphate buffer, diluted to 50 mL with deionized water) and placed in 50 mL centrifuge tubes. The fifth leaf of 4-week-old Arabidopsis thaliana was soaked in NBT staining buffer for 60 min, washed in 80 °C water containing 95% ethanol until chlorophyll was completely removed, and then photographed for staining results (Geerts and Roels 1981).

Measurement of physiological and biochemical indicators

Hydrogen peroxide, superoxide anion, soluble sugars, malondialdehyde, chlorophyll content, and activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and shikimate dehydrogenase (SD) were measured. Test kits were purchased from boxbio (http://www.boxbiological.com/) and experiments were conducted according to the manufacturer’s instructions.

Fluorescence quantitative PCR

Total RNA was extracted from maize leaves using the Trizol method. cDNA was synthesized from total RNA using M-MLV reverse transcriptase (Takara). qRT-PCR was performed using SYBR PreMix Ex Taq (Takara) with amplification conditions of: 95 °C for 10 min pre-denaturation, followed by 40 cycles of 95 °C denaturation for 10 s, 58 °C annealing for 20 s, and 72 °C extension for 15 s, with a melt curve analysis. Data were analyzed using the 2−ΔΔt method with AtActin1 gene as an internal reference, and each sample was biologically replicated three times. Primers used in this study are listed in Supplementary Material Table 1.

Statistical analysis

All results in this study were repeated at least three times. Experimental quantitative data were statistically analyzed using SPSS 19.0 software (SPSS Inc.Chicago, IL, USA), and differences in treatment outcomes were confirmed using t tests. Significance levels were denoted as *p < 0.05, **p < 0.01 and ***p < 0.001.

Results

Codon optimization results and expression cassette frame in overexpression vectors

We optimized the codons of the EPSPS gene using online software and observed changes in codon preferences favoring maize compared to the original sequence (Fig. 1). The expression cassette is illustrated in Fig. 2.

Fig. 1
figure 1

Genetic transposition of the expression cassette

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Fig. 2
figure 2

Y-EPSPS Amino acid sequence

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Results of overexpression in transgenic plants

Following PCR validation and strip tests (Fig. 3A, C), we successfully obtained 15 positive overexpression transgenic plants. Fluorescence quantitative PCR indicated that plants OE4 and OE13 exhibited the highest gene expression levels (Fig. 3B). Consequently, we selected these strains for propagation and subsequent experiments.

Fig. 3
figure 3

Results of overexpressed positive plants. M: DNA Marker DL 2000; P: positive control; N: water; 1–15: Overexpressed positive plants. A PCR test results. B qPCR test results. C Test result of bar gene test strip. P: Positive control; N: Negative control; 1–15: Overexpressed positive plants. The student’s test was used, with asterisks indicating statistically significant differences (*p < 0.05; **p < 0.01). The data is shown as the mean ± SD from three independent experiments

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Glyphosate resistance in Y-EPSPS overexpressing plants

EPSPS enzyme is the sole target of glyphosate action (Ge et al. 2014), reacting to glyphosate. Y-EPSPS overexpressing and wild-type (WT) plants were separately inoculated on 1/2MS solid media containing different concentrations of glyphosate, and seed germination was observed after 8 days. We found that under glyphosate-free conditions, germination in overexpressing plants was similar to WT plants and remained unaffected. However, in media containing 0.5 mM glyphosate, both overexpressing and WT plants showed reduced germination, with higher germination rates observed in overexpressing plants: WT 54.02%, E-OE4 74.44%, E-OE13 80% (Fig. 4A, C). In media with 0.8 mM glyphosate, significant inhibition of germination was observed in all strains: WT 34.48%, E-OE4 52.22%, E-OE13 43.48% (Fig. 4A, C). After spraying 0.8 mM glyphosate solution on overexpressing and WT plants, overexpressing plants remained viable while WT plants perished (Fig. 4B). These results demonstrate that the Y-EPSPS gene can reduce sensitivity of Arabidopsis to glyphosate, enhancing its resistance.

Fig. 4
figure 4

Results of positive seed germination experiment and glyphosate resistance. A Germination results of Arabidopsis overexpressed positive seeds with different concentrations of glyphosate. B Resistance results of overexpressed Arabidopsis positive plants to 0.8 mM glyphosate spray. C Seed germination of Arabidopsis thaliana under different concentrations of glyphosate. The student’s test was used, with asterisks indicating statistically significant differences (*p < 0.05; **p < 0.01)

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Glyphosate treatment increases H2O2 and O2 − levels

Reactive oxygen species (ROS) are crucial signaling molecules in response to various stress processes. Changes in hydrogen peroxide and superoxide anion levels play a vital role in plant stress (Sedigheh et al. 2011). Therefore, assessing ROS levels in Y-EPSPS overexpressing and WT plants is crucial. The sixth leaf of control and glyphosate-treated Arabidopsis plants was isolated and immersed in NBT and DAB staining solutions to reveal hydrogen peroxide and superoxide anion levels. Results showed no significant difference between overexpressing plants and WT under normal conditions. However, under glyphosate treatment, overexpressing plants exhibited lighter leaf color compared to WT (Fig. 5A, B). Quantitative results of H2O2 and O2− were consistent with chemical staining results (Fig. 5C, D). These findings indicate that Y-EPSPS overexpression reduces ROS accumulation in Arabidopsis leaves, thereby conferring glyphosate resistance.

Fig. 5
figure 5

H2O2 and O2− content detection results. A, B NBT, DAB staining. Overexpressed and positive plants at 4 weeks of age were stained. C, D The amount of H2O2 and O2−accumulated in plants. The student’s test was used, with asterisks indicating statistically significant differences (*p < 0.05; **p < 0.01). The data is shown as the mean ± SD from three independent experiments

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Impact of Y-EPSPS gene on chlorophyll content and Fv/Fm

To further understand the role of the Y-EPSPS gene in glyphosate resistance, two Y-EPSPS overexpressing Arabidopsis plants were genetically transformed, and their chlorophyll content and Fv/Fm were measured. Before glyphosate treatment, chlorophyll content and Fv/Fm levels were similar between overexpressing and WT plants. However, after glyphosate treatment, overexpressing plants showed significantly higher chlorophyll content and Fv/Fm compared to WT plants (Fig. 6). This suggests that overexpressing the Y-EPSPS gene reduces sensitivity of plants to glyphosate, mitigating its toxic effects.

Fig. 6
figure 6

Chlorophyll content and Fv/Fm detection results. The student’s test was used, with asterisks indicating statistically significant differences (*p < 0.05; **p < 0.01). The data is shown as the mean ± SD from three independent experiments

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Y-EPSPS gene enhances soluble sugar and MDA content

We measured the levels of soluble sugars and MDA in both Y-EPSPS overexpressing plants and wild-type plants before and after glyphosate treatment. Initially, there was nearly no difference in soluble sugar content between overexpressing and wild-type plants prior to glyphosate treatment. However, post-treatment, the soluble sugar content in overexpressing plants significantly exceeded that of WT plants. Similarly, before glyphosate treatment, there was minimal difference in MDA content between overexpressing and WT plants. Yet, after glyphosate treatment, MDA levels in overexpressing plants notably decreased compared to WT plants (Fig. 7), indicating enhanced stress resistance and antioxidant capability, thereby reducing glyphosate-induced damage and conferring resistance to glyphosate.

Fig. 7
figure 7

The content of soluble sugar and MDA was detected. The student’s test was used, with asterisks indicating statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001). The data is shown as the mean ± SD from three independent experiments

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Y-EPSPS gene alters ROS-related enzyme activities

The activity of reactive oxygen species ROS-related enzymes is an important indicator of the plant’s ability to scavenge ROS. Therefore, we assessed the activity of ROS-related enzymes. The results showed that before glyphosate treatment, the levels of POD, SOD, and CAT activities were similar between overexpressing plants and wild-type (WT) plants. After glyphosate treatment, however, the enzyme activities of POD, SOD, and CAT in overexpressing plants were significantly higher than those in WT plants (Fig. 8). This enables the breakdown of higher and more oxidative molecules produced in plants, reducing glyphosate’s toxic effects on plants.

Fig. 8
figure 8

Results of POD, SOD and CAT enzyme activity. The student’s t test was used, with asterisks indicating statistically significant differences (*p < 0.05; **p < 0.01: ***p < 0.001). The data is shown as the mean ± SD from three independent experiments

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Y-EPSPS gene reduces shikimate dehydrogenase activity

We measured the activity of shikimate dehydrogenase (SD), a bifunctional enzyme essential for shikimate acid synthesis. Before glyphosate treatment, SD activity showed little difference between overexpressing and wild-type plants. However, post-treatment, SD activity decreased in both overexpressing and wild-type plants. Yet, SD activity in overexpressing plants was notably higher than in WT plants, reaching twice the level observed in WT plants (Fig. 9). This suggests that glyphosate reduces EPSPS enzyme activity, leading to excessive accumulation of shikimate acid in plants, thereby inhibiting SD enzyme activity.

Fig. 9
figure 9

Results of SD enzyme activity detection. The student’s test was used, with asterisks indicating statistically significant differences (*p < 0.05; **p < 0.01: ***p < 0.001). The data is shown as the mean ± SD from three independent experiments

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Impact of Y-EPSPS gene on ROS detoxification genes, stress response genes, and shikimate synthesis enzyme gene expression under glyphosate stress

Under glyphosate stress, overexpression of the Y-EPSPS gene partially reduces H2O2 and O2− levels in plants. These findings indicate that Y-EPSPS is involved in ROS regulation and participates in plant growth and development. In this study, we analyzed the expression of several ROS detoxification genes (AtCAT3: Gene ID: 838651 and AtSOD1: Gene ID: 837405), stress defense-related genes (AtLTP3: Gene ID: 836051 and AtSOS1: Gene ID: 814729), and the DQSD gene (Gene ID: 819809) (primer sequences in Supplementary Table 1) to determine the role of Y-EPSPS in glyphosate resistance. Results showed that gene expression levels increased post glyphosate treatment (Fig. 10), in response to glyphosate stress, thereby reducing glyphosate toxicity.

Fig. 10
figure 10

Effects of Y-EPSPS gene on the expression of active oxygen-related genes, stress response genes and shikimic acid pathway genes. Expression analysis of ROS (A, B), stress response (C, D) and shikimic acid pathway related genes (E) in Col-0, transgenic lines OE4 and OE13 under control and glyphosate stress conditions. The student’s test was used, with asterisks indicating statistically significant differences (*p < 0.05; **p < 0.01: ***p < 0.001). The data is shown as the mean ± SD from three independent experiments

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Discussion

To produce high-quality glyphosate-resistant maize, we utilized the floral dip method to transfer the optimized Y-EPSPS gene into Arabidopsis thaliana (Ali et al. 2022; Zhang et al. 2006), resulting in 15 overexpressing plants. We assessed changes in chlorophyll, soluble sugars, MDA content, relative conductivity, and ROS levels in both overexpressing and wild-type plants before and after glyphosate treatment. Additionally, changes in enzyme activities such as POD, SOD, CAT, and SD were analyzed. Expression patterns of genes related to chlorophyll synthesis, ROS scavenging, and shikimate pathway were also examined.

Codon optimization involves altering gene sequences to enhance the expression of recombinant proteins. The degeneracy of the genetic code allows multiple codon sequences to encode the same protein. Codon usage bias prioritizes the use of specific synonymous codons (Gillen et al. 2021; Fu et al. 2020; López et al. 2020). Therefore, codon optimization can increase gene expression by altering the gene sequence without changing the amino acid sequence, thereby boosting protein content within cells. Traditionally, optimal codons are thought to enhance translation efficiency due to faster ribosomal movement and release along mRNA (Andersson and Kurland 1990; Ehrenberg and Kurland 1984; Sørensen and Pedersen 1991), potentially leading to faster growth rates through more efficient ribosome utilization. Another view suggests that optimal codon usage improves translation accuracy (Akashi 1994).

This study employed codon optimization to make the EPSPS gene sequence more favorable for maize-specific codon usage, enhancing EPSPS expression in plants. This increased the content of EPSP synthase expressed in Arabidopsis plants and verified its activity through indoor glyphosate tolerance experiments, aiming to acquire a novel EPSPS gene that enhances glyphosate tolerance.

SD is a key enzyme catalyzing the fourth step of the shikimate biosynthetic pathway (Yuan et al. 2022). In plants, SDH forms a DQD/SDH complex through its N-terminal to enhance metabolic cycling efficiency, and its activity analysis is crucial for shikimate pathway analysis (Akhlaghi et al. 2018). SD enzyme catalyzes shikimate to 3-dehydrogenated shikimic acid, while EPSPS catalyzes shikimate to 5-enol-shikimic acid, eventually synthesizing the aromatic amino acid precursor chorismate (Choi Si-Sun et al. 2019). Glyphosate competitively binds to EPSPS reversibly, forming a stable but non-covalent ternary complex EPSPS-S3P-glyphosate, leading to loss of EPSPS enzyme activity. This diverts substantial carbon sources towards shikimic acid, resulting in its rapid accumulation within tissues and severely hindering the synthesis of aromatic amino acids essential for protein biosynthesis, ultimately inhibiting plant growth (Duke and Powles 2009; Wiersma et al. 2015). Glyphosate spraying inhibits SD activity in plants. It is speculated that glyphosate forms a ternary complex with EPSPS, blocking the shikimate pathway and causing accumulation of shikimic acid, which in turn downregulates SD activity due to feedback regulation by shikimic acid, leading to decreased enzyme activity.

Previous studies have found that glyphosate can promote the formation of plant ROS (Jalal et al. 2021). In this study, following glyphosate stress, accumulations of H2O2 and O2− were observed in the leaves of both overexpressing plants and wild-type (WT) plants, though the accumulation in overexpressing plants was lower than in WT plants. Although the accumulation of O2− increased, it was not as significant as the accumulation of the ROS H2O2, which may be due to biochemical differences between these two ROS. Superoxide is a transient molecule, whereas hydrogen peroxide is the most stable ROS, making it easier to accumulate (Shaner et al. 2005). POD, CAT, and SOD constitute the most important protective enzyme systems in plants and are crucial for scavenging reactive oxygen species (ROS) (Shafiq et al. 2021). The activities of POD, CAT, and SOD indirectly reflect the vigor of plants. SOD is responsible for converting O2− to H2O2, while CAT and POD degrade H2O2 to H2O and O2, thereby clearing ROS generated by glyphosate stress and maintaining dynamic balance of ROS in plants (Hu et al. 2018; Jiang et al. 2023; Zhao et al. 2005). This study found that the enzyme activities of POD, SOD, and CAT decreased in plants subjected to glyphosate stress, with overexpressing plants showing higher enzyme activities compared to WT plants, suggesting that the Y-EPSPS gene can mitigate ROS accumulation and oxidative damage caused by glyphosate stress to some extent. After glyphosate stress, both malondialdehyde (MDA) and soluble sugar content increased in both overexpressing and wild-type plants. In this study, the soluble sugar content in overexpressing plants was higher than in wild-type plants, whereas the MDA content was lower (You et al. 2012). MDA, an active metabolic byproduct, is often used as an indicator of oxidative stress. When plant cells undergo oxidative damage, MDA accumulates inside the cells and may react with molecules such as proteins, nucleic acids, and lipids, leading to abnormal cellular functions or even cell death (Mao et al. 2023; Gill and Tuteja 2010; Noctor 2006). Numerous studies have indicated that adverse environments enhance lipid peroxidation in leaf tissues of plants, resulting in increased MDA content and relative conductivity. Lipid peroxidation is a secondary effect of oxidative stress, which has been observed following glyphosate stress in various plants, including soybean, rice (Oryza sativa L.), tobacco (Nicotiana tabacum L.), and peanut (Arachis hypogaea L.) (Li et al. 2019; Vennapusa et al. 2022; Daramola et al. 2023). The content of soluble sugars in plants typically reflects their resistance to stress because sugars serve as both an energy source and antioxidants, aiding plants in coping with adverse conditions (Barco-Antoñanzas et al. 2022). As signaling molecules for nutrition and metabolism, soluble sugars are crucial for maintaining plant structure, metabolism, growth, and cellular osmotic balance (Barco-Antoñanzas et al. 2022; Fernándes-Escalada et al. 2016). Therefore, the content of soluble sugars in plants serves as an important indicator for evaluating their physiological status and stress responses. The increase in soluble sugar content indicates that overexpressing plants exhibit greater tolerance to glyphosate (Li et al. 2020).

After spraying glyphosate, the chlorophyll content of both overexpressing and wild-type plants significantly decreased, while soluble sugars, MDA, relative conductivity, and ROS content markedly increased. The changes in overexpressing plants and wild-type plants were consistent with plant stress responses. Previous research has also indicated that glyphosate spraying reduces leaf area, subsequently lowering photosynthetic rates and aboveground biomass (Hoagland et al. 1980; Ding et al. 2008). Following glyphosate treatment, chlorophyll content in overexpressing plants initially declined but reached levels similar to untreated WT plants by day 30. This suggests glyphosate impacts plants, even those tolerant to it. Additionally, soluble sugars, MDA, relative conductivity, and ROS content in overexpressing plant leaves showed significant changes post-glyphosate treatment, albeit lower than WT plants. Therefore, these overexpressing plants exhibit significant resistance to glyphosate.

In this study, glyphosate treatment affected MDA, soluble sugars, and ROS production. As demonstrated, chlorophyll content decreased in both overexpressing and wild-type Arabidopsis. Glyphosate likely induces oxidative stress, leading to several minor or indirect effects on plant physiology (Gomes et al. 2014). As a result of oxidative stress, glyphosate damages plants, prompting increased production of soluble sugars in response to the damage. Glyphosate disrupts chlorophyll, impacting photosynthetic rates and metabolism. It affects the shikimate pathway, hindering plant growth and development. Ultimately, glyphosate can kill plants. Glyphosate increases ROS activity within plant tissues, and the plant’s ability to scavenge ROS is crucial for enhancing glyphosate tolerance. Overexpressing the Y-EPSPS gene promotes synthesis of aromatic amino acids, enhancing the plant’s ability to clear active substances. Previous studies utilized exogenous EPSPS genes sourced from other plants. This study seeks to identify endogenous EPSPS genes in plants and optimize their codons to enhance glyphosate tolerance. Optimizing endogenous genes can stabilize genetic traits and reduce responses to the biotic environment. To further explore the biological function of the Y-EPSPS gene, future work will involve yeast hybridization, dual luciferase assays, and HPLC detection. A series of experiments will also be conducted on maize to observe phenotypic changes in transgenic maize and measure related physiological and biochemical indicators. This research lays a solid foundation for understanding the herbicide resistance mechanisms in maize, thereby benefiting maize breeding efforts.