Effects of Phenanthrene Soil Pollution on Cadmium Bioaccumulation and Metabolic Responses in Maize (Zea mays L.)

Abstract

Co-contamination of cadmium (Cd) and polycyclic aromatic hydrocarbons (PAHs) in agricultural soils poses a critical threat to crops and food safety, but how PAHs affect Cd uptake and plant metabolism is still unclear. Maize (Zea mays L.) of the variety Hanyu 702 (HY702) was previously identified by our group asaccumulating Cd at low levels when grown in soil containing Cd and phenanthrene (Phe). These contaminants were used here as model pollutions, alone and in combination, to assess the accumulation, growth, physiological, and metabolic responses of HY702 seedlings. Four treatments were compared, including a control without pollution, single Phe pollution, single Cd pollution, and Cd and Phe combined pollution. The experiments followed a completely randomized design with three replicates per treatment. The results revealed that Cd accumulation in the plants was significantly reduced when Phe was present as well (9% reduction in roots and 44% in stems and leaves compared to Cd single pollution). The combined Cd-Phe pollution had no significant impact on the height or chlorophyll content of the maize plants but markedly reduced their malondialdehyde (MDA) content. In addition, it increased the proline content by 56% and antioxidant enzyme activity by 15% (peroxidase, POD), 24% (superoxide dismutase, SOD), and 57% (catalase, CAT) compared to the control treatment. Metabolomics analysis revealed that the coexistence of Phe and Cd activated four key metabolic pathways: (a) alanine, aspartate, and glutamate metabolism; (b) valine, leucine, and isoleucine biosynthesis; (c) aminoacyl-tRNA biosynthesis; and (d) histidine metabolism. This activation resulted in increased levels of six differential metabolites: L-asparagine, L-methionine, L-glutamate, (S)-2-acetyl-2-hydroxybutanoic acid, urocanic acid, and 2-isopropylmalic acid. These metabolites induced detoxification pathways and reduced Cd accumulation. The findings reported here offer new insights into how plants metabolically adapt to the combined pollution of Cd and PAHs and provide an important scientific basis for pollution control strategies.

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are environmental organic pollutants with mutagenic, carcinogenic, and teratogenic effects [1].PAHs, which are among the most prevalent organic persistent pollutants in the environment, exhibit varying toxic effects, depending on their molecular weight. Low-molecular-weight PAHs, such as phenanthrene (Phe) and pyrene (Pyr), are generally considered acutely toxic [2], causing harm to human health and to the environment, whereas high-molecular-weight PAHs are typically regarded as genotoxic [3]. In agricultural soils, background concentrations of PAHs typically range from tens to hundreds of µg/kg. However, soil located near pollution sources or directly affected by sewage sludge application can contain PAH concentrations reaching hundreds to thousands of µg/kg, frequently surpassing regulatory soil quality standards. Soil contaminated by industrial pollution often contains PAHs together with heavy metals. Since the self-purifying capacity of soil is limited, such combined pollution frequently leads to levels of PAHs and heavy metals exceeding their permissible limits. Globally, over 10 million soil sites are considered to be contaminated, including locations in countries such as China, Australia, and the United States [4,5,6]. Among the many contamination species, the combined pollution of cadmium (Cd) and PAHs is particularly noteworthy [7]. For instance, a study in the United States found that a mixture of organic pollutants and heavy metals had contaminated 40% of the affected areas [8]. A soil sample from the port of Jaén, Spain, contained a total of 414.4 mg/kg of Σ16PAHs, together with 1.3 mg/kg of Cd [9]. Similarly, soil from Adelaide Hills in Australia contained 245.1 mg/kg of Σ16PAHs combined with 2.3 mg/kg of Cd [10]. In China, nationwide agricultural soil monitoring recorded maximum concentrations of 72.81 mg/kg for Cd and 6.28 mg/kg for PAHs [11], while regional surveys identified combined pollution ofup to 0.847 mg/kg Cd and 1.263 mg/kg of PAHs in the administrative area of Guangzhou Province [12]. Likewise, a mean Cd of 3.3 mg/kg with 2.231 mg/kg of PAHs peaked in Hezhang County, Guizhou Province [13], and a mean of 2.71 mg/kg of Cd with 1.09 mg/kg of PAHs was detected in the administrative area of Liaoning Province [11]. Soil analysis at co-contaminated sites in Dongying City measured Cd at 23.1 mg/kg and PAHs at 1238.6 mg/kg [14]. According to the Chinese National Soil Pollution Survey Bulletin, 7% of sites exceeded the standards for Cd contamination and 1.4% for those of PAHs [15]. In agricultural soil, the average concentration of Σ16PAHs was 0.993 mg/kg, with Phe accounting for 12.72% of the total [16]. Cd accounts for 33.54% of agricultural soil pollution, with individual concentrations reaching as high as 23.33 mg/kg [5,17]. Globally, the combined pollution of Cd and Phehas been recorded in Spain, where one site contained 0.27 mg/kg Cd together with ΣPAH concentrations of 414.1 mg/kg, while a particular contamination in South Korea reached 5.0 mg/kg of Cd and 288.7 mg/kg of ΣPAHs [18]. Levels of Cd and PAH combined pollution in agricultural soil areincreasing due to irrational human activities [14], and this has rightfully attracted research attention, as these pollutants can accumulate in crops [19].

Pollutant accumulation in crops is influenced by the complex interactions between soil heavy metals and PAH pollution. The effects of various pollutant interactions on plant absorption and accumulation vary significantly. For instance, when present, Cd promotes the accumulation of Σ16PAHs in PakChoi (Brassica chinensis L.), whereas lead (Pb) inhibits their accumulation [20]. In maize stems and leaves, combined pollution with Pyr promotes the accumulation of copper (Cu) but prevents the accumulation of Cd [21]. Additionally, inhibition or promotion of accumulation has been observed in many plants exposed to concurrent pollutants. For instance, combined pollution of Cd and Pyr promotesthe accumulation of both in the mangrove, Kandelia obovata [22]. In contrast, in Siberian Cocklebur (Xanthium sibiricum L.), Cd-Pyr combined pollution decreases the uptake and accumulation of both pollutants [23]. The interaction between Cd and Phe in maize plants, in which both pollutants are easily transported, absorbed, and accumulated, is not well understood and requires more extensive research in order to develop agronomic varieties that can besafely cultivated on lightly polluted farmland.

Plant growth, physiology, and metabolism all exhibit distinct response mechanisms when heavy metals and PAHs are present due to the interacting uptake and accumulation of these pollutants. Cd-PAH combined pollution significantly affects the growth of wheat and maize seedlings, reducing their biomass by 13.1~49.8% and 28.0~37.8% compared to single Cd and PAH pollution, respectively [24,25]. Similarly, a 33.5% decrease in the height of wheat seedlings was observedin [24], but there were positive effects on the biomass of the wetland plant Acorus calamus [26]. This suggests that different plant species may respond differently to Cd-PAH combined pollution. Thus, heavy metals and PAHs can have a considerable combined inhibitory effect on accumulation, up to a certain dose for one plant species, to be growth-promoting toward another. Under normal physiological conditions, plants maintain a balanced state of reactive oxygen species. However, as a result of external factors, an overproduction of reactive oxygen radicals can lead to increased lipid peroxidation of the cell membrane, which can result in membrane damage. The antioxidant system, consisting of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), is crucial for scavenging reactive oxygen radicals to maintain normal cellular metabolism, preserve the integrity of organ functions, and increase plant resistance. SOD can scavenge free radicals in plants and effectively reduce cellular damage, while CAT specifically inactivates hydrogen peroxide, preventing oxidative damage. POD catalyzes the breakdown of hydrogen peroxide and oxidized phenols into harmless aldehydes and oxygen, thereby also protecting cells from oxidative damage. These enzymes are involved in the plant’s response to the combined pollution of heavy metals and PAHs as well. For example, Cd-PAH combined pollution can increase POD enzyme activity in exposed wheat seedlings by 72.7% while inhibiting SOD and CAT activities by up to 45.2% [24]. In one study on maize seedlings, the activities of POD, CAT, and ascorbate peroxidase (APX) were all enhanced, whereas SOD was reduced by 14.9% [25]. In that study, the presence of malondialdehyde (MDA), an indicator of oxidative stress and redox signaling in plants, resulted in the greatest increase (57%) [25]. To determine the physiological status of plants, metabolomics can be used to identify metabolic markers and associated metabolic pathways, and these can be correlated with pollution exposure. For instance, it was shown that Cd pollution altered 33 metabolites in Sedum, affecting 16 Cd pollution-related metabolic pathways, including those involving organic acids, amino acids, lipids, and polyols [27]. Common metabolic pathways impacted in maize under PAH pollution include galactose metabolism; aminoacyl tRNA biosynthesis, alanine, aspartate, and glutamate metabolism; valine, leucine, and isoleucine biosynthesis; and tyrosine metabolism, with a particular focus on amino acid metabolism [28]. How these metabolic processes are affected by combined pollution remains unclear, as most research has primarily addressed metabolite differences and pathway modifications using single pollutants.

Maize represents a major food crop with a worldwide cultivation area of 1.97 × 10⁶ km². It plays a crucial role in meeting global food demands [29]. Maize is considered to have soil remediation potential, as it is capable of absorbing a variety of pollutants, including PAHs, and holds practical value in mitigating mildly to moderately polluted agricultural soils [30]. Our research team has evaluated 11 high-yield maize varieties commonly grown in Hebei Province for their capacity to absorb and accumulate Cd and Phe; the variety Hanyu 702 (HY702) emerged as a low accumulator of both contaminants [31]. However, its metabolic mechanisms in response to these pollutants remained unclear. We used the HY702 variety of maize to test the hypothesis that its low potential to accumulate Cd and Phe may be due to its antioxidant system (in particular, SOD and POD activity). Alternatively, or in combination, overexpression of metal-binding proteins (e.g., phytochelatins) and altered metabolism (e.g., phenolic compound accumulation) might contribute to this low accumulation potential. These mechanisms would ultimately lead to reduced biomass in combination with decreased Cd accumulation.In this study, the maize variety HY702 was cultured in pot experiments, and metabolomics technology was employed to investigate the interactive effects of Cd-Phe combined pollution on their uptake and accumulation; in addition, the growth, physiological, and biochemical response characteristics of the plants werestudied. We concentrated on the effect on maize HY702 seedlings, as the seedling stage of the plants provides a critical research window: the heightened physiological activity of the plants at this developmental stage facilitates the identification of effects on growth and physiological responses [32]. Moreover, seedlings are more sensitive to exogenous pollutants compared to mature plants [33], so that they can provide an optimal model system to uncover metabolic mechanisms under co-contamination stress.

2. Materials and Methods

2.1. Test Materials

Maize cultivar HY702 (F1 hybrid) was purchased from Hebei Jinong Seed Industry Co., Ltd. (Shijiazhuang, China). It is the main cultivated variety in the Huang–Huai–Hai plain of China [31]. The variety was previously found to have a low-accumulation potential in Cd-Phe co-contamination tests [31].

Chemical Reagents: Phe (>95% purity), CdCl₂·2.5H₂O, n-hexane (HPLC grade), dichloromethane (HPLC grade), and methanol (HPLC grade) were purchased from Beijing Bailing Wei Technology Co., Ltd., Beijing, China. AnSPE silica gel purification column was purchased from Shanghai Anpu Experimental Technology Co., Ltd., Shanghai, China. Acetone (HPLC grade), HNO₃ (UP-S grade), and H₂O₂ (UP-S grade) were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd., Tianjin, China. All other analytical grade chemical reagents were purchased from Baoding Wanke Reagent Co., Ltd., Baoding, China. Cd was administered in the form of CdCl₂·2.5H₂O, which was dissolved in deionized water to prepare a treatment solution with a concentration of 250 mg/L CdCl₂. Phe was dissolved in HPLC-grade acetone to generate stock solutions at a concentration of 100 mg/L.

The tested soil samples, collected at a depth of 0–20 cm from uncontaminated agricultural fields in Baoding, China, were identified as meadow cinnamon soil. The soil contained a Cd concentration of 0.19 mg/kg and a Phe concentration of 0.02 mg/kg. It had an organic matter content of 20.92 g/kg, a total nitrogen content of 0.95 g/kg, a total phosphorus content of 0.30 g/kg, an alkaline hydrolyzable nitrogen content of 72.80 mg/kg, an available phosphorus content of 62.03 mg/kg, and a pH of 7.8. The soil was air-dried at room temperature, cleared of plant and animal residues, and sieved through a 2 mm mesh before being used in pot experiments (Table S1).

For Cd pollution, 100 mL of a 250 mg/L CdCl₂ solution was added to 10 kg of soil and mixed well, yielding a final Cd concentration of 2.5 mg/kg. For Phe pollution, 100 mL of acetone containing 100 mg/L of Phe was added to 10 kg of soil and stirred intermittently until the acetone was completely evaporated and the contaminant was evenly distributed, providing a final Phe concentration of 1 mg/kg. For simulated Cd-Phe-co-contaminated soil, the same procedure was followed, but both contaminants were added together.

2.2. Pot Experiments

Based on documented contamination levels of Cd and Phe in China’s agricultural soils and mining-contaminated farmland soils, we used a commonly adopted soil content of 2.5 mg/kg of Cd and 1 mg/kg of Phe. The pot experiments included four different treatments, as outlined in

Table 1. Maize pot experiment protocol for Cd and Phe exposure (mg/kg).

Treatment Number	Treatment Description	Phenanthrene Concentration	Cadmium Concentration
CK	Control without pollution	0	0
P1	Single phenanthrene pollution	1	0
C1	Single cadmium pollution	0	2.5
P1C1	Cadmium and phenanthrene combined pollution	1	2.5

. In addition to the control without pollution (CK), single pollution was performed with soil containing 1 mg/kg Phe (P1) or 2.5 mg/kg Cd (C1), while combined pollution represented treatment with soil containing 1 mg/kg Phe and 2.5 mg/kg Cd (P1C1). The setup followed a completely randomized design (CRD), and each treatment was replicated three times. The used pots had a diameter of 16.5 cm, a height of 15 cm, and a bottom diameter of 12 cm. Each pot was filled with 1.2 kg of the designated soils, which were then equilibrated for two weeks at room temperature while maintaining a soil moisture level at 60% of the field water-holding capacity. Ten HY702 maize seeds of uniform size were sown in each pot. The indoor temperature was maintained at 25°C during the day and at 20°C at night throughout the entire incubation period. Additionally, each treatment received the same amount of fertilizer and was irrigated with deionized water. Five days after seedlings appeared, they were thinned to leave eight seedlings per pot. All plants were then harvested after 30 days and measured (length from root to longest leaf).

The harvested plants were divided into roots, stems, and leaves and then divided into six aliquots. Three aliquots of each treatment were heated at 105 °C for 30 min and dried at 40~50 °C until constant weight to provide their dry weight. The other three aliquots were rinsed with water, dried, and chopped. They were then wrapped in aluminum foil, quickly frozen in liquid nitrogen, and stored in a self-sealing bag at −80 °C to be shipped to Shanghai Meiji Biomedical Technology Co., Ltd., Shanghai, China, for metabolomics analysis.

2.3. Quantitative Analysis of Indicators

To determine the Cd content of maize tissue, 0.2 g of dried roots, stems, and leaves wasdigested at 120 °C in 5 mL of 68% (v/v) HNO₃ and 30% (v/v) H₂O₂ and diluted 10 times with deionized water. Digests were diluted 10-fold with deionized water and analyzed by ICP-MS (PerkinElmer NexION 350X, Shelton, CT, USA), employing ¹¹⁵In as the internal standard [34]. Quality assurance procedures included the following: (1) routine analysis of reagent blanks (every 10 samples), (2) duplicate measurements (10% of samples), and (3) validation with certified reference material (GBW10012 maize), demonstrating recoveries within the 70–130% range [34].

For the determination of the Phe content, 0.1 g of the dried plant parts was subjected to ultrasonication extraction using n-hexane/acetone (1:1, v/v) with the two internal standards, p-terphenyl-d14 and 4-bromo-2-fluorobiphenyl. These were added to each sample at a final concentration of 20 µg/L. Following the collection of the extraction solvent, the ultrasonication extraction was repeated for another hour with a new acetone–n-hexane solvent, and the extraction process was repeated three times in total for each sample. The resulting three extracts were then combined, evaporated, and dissolved in n-hexane, followed by filtration through a silica gel column, and eluted using 15.0 mL of 1:1 (v/v) n-hexane–dichloromethane. Following evaporation, the samples were reconstituted in 1.0 mL of n-hexane, passed through 0.22 μm PTFE membranes, and analyzed by High-Performance Liquid Chromatography (HPLC) (Agilent 1260, Santa Clara, CA, USA) with the following conditions: a 4.6 mm × 250 mm C18 reverse-phase column with a column temperature of 30 °C, a mobile phase of methanol/water (85/15, v/v), a flow rate of 1.0 mL/min, an injection volume of 10 µL, and a detection wavelength for Phe at 254 nm [35,36,37]. The recovery of the two internal standards was achieved at 70~130% to support the reliability and reproducibility of the analytical method.

Fresh stems and leaves (0.5 g) were homogenized in ice-cold 50 mM phosphate buffer (pH 7.8) containing 1% (w/v) polyvinylpyrrolidone. The homogenate was centrifuged at 12,000× g for 15 min at 4 °C, and the supernatant was immediately analyzed for SOD activity, CAT activity, POD activity, MDA content, and proline content as described in the literature [38,39,40,41,42]. Chlorophyll content was measured with a portable chlorophyll meter.

Non-targeted metabolomics analysis was performed as follows. From 50 mg of plant leaves, the metabolites were extracted using 400 µL of methanol–water (4:1, v/v). After settling at −20 °C, the mixture was homogenized with a high-throughput tissue crusher, Wonbio-96c (Shanghai Wanbo Biotechnology Co., Ltd., Shanghai, China), at 50 Hz for 6 min, vortexed for 30 s, and treated with ultrasound at 40 kHz for 30 min at 5 °C. Proteins were precipitated at −20 °C for 30 min and then removed by centrifugation at 13,000× g at 4 °C for 15 min, after which the collected supernatant was subjected to UPLC-MS/MS analysis. The metabolites were chromatographically separated on a Thermo (Waltham, MA, USA) UHPLC system equipped with an ACQUITY (Sydney, Australia) BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters, Milford, CT, USA) with mobile phases of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile–isopropanol (1:1, v/v) (solvent B). Mass spectrometry was performed with a Thermo UHPLC-Q Exactive Mass Spectrometer equipped with an electrospray ionization (ESI) source operating in either positive or negative ion mode. The quality control (QC) sample was prepared as described above, and this was included as every 8th sample.

2.4. Statistical Analysis Methods

Statistical analyses were performed using SPSS 23.0, Origin 2022, and Microsoft Excel 2016. Before performing parametric tests, each dataset underwent verification for (1) normality using the Shapiro–Wilk test (p > 0.05) and (2) homogeneity of variances through Levene’s test (p> 0.05). When these criteria were met, treatment effects were evaluated by one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) posthoc comparisons. Statistical differences between treatments were considered significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

The type of interaction between Cd and Phe was further calculated using the following formula:

C_exp = A + B − A × B/100
RI = OB/C_exp

where A and B represent the effects (percentage increase or decrease in measured indexes compared to the blank control group) of individual Cd and Phe pollution, respectively. RI is the ratio of the total impacts to C_exp. Synergistic interactions occur when RI > 1, while antagonistic interactions occur when RI < 1 [43].

The raw UPLC-MS/MS data from non-targeted metabolomics was pre-treated using the Progenesis QI 2.3 software (Waters Inc., Milford, MA, USA). The combined positive and negative data were analyzed on the Majorbio I-Sanger Cloud Platform (available online: www.i-sanger.com). After standard data management, including baseline filtering, peak identification, integration, retention time correction, and peak alignment, a data matrix containing information on retention time, mass-to-charge ratio, and peak intensities was produced. The metabolites were identified using secondary mass spectrometry matching scores, with the MS mass error set to be less than 10 ppm. The identified metabolites were searched using the following databases: Human Metabolome Database (HMDB) (available online: http://www.hmdb.ca/), Metlin database (Available online: http://metlin.scripps.edu/); LIPID MAPS (available online: https://www.lipidmaps.org/); and the Majorbio databases NIST (version 2017), Fiehn (version 2013), and MS-DIAL (version 2021). To further ensure the precision and scientific validity of the investigation outcomes, a pathway analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG).

Multivariate statistical analysis was applied, or ananalysis of metabolite differences was applied. For this, principal component analysis (PCA) was performed on the pre-processed matrix files by orthogonal least squares discriminant analysis (OPLS-DA) using the R package ropls (version 1.6.2). Statistical validation was performed using Student’s t-test and fold difference analysis. Differential metabolites were selected with VIP values reaching the threshold (VIP > 1) in OPLS-DA analysis and regarding fold changes, with a p-value of p < 0.05 in the univariate analysis, and volcano plots were produced. The identified differential metabolites among the two groups were mapped into their biochemical pathways through metabolic enrichment and pathway analysis by standard procedures with KEGG (available online: http://www.genome.jp/kegg/ (accessed on 20 December 2024)). Enrichment analysis enabled the identification of a group of metabolites in a function node, under the presumption that the annotation analysis of a single metabolite develops into an annotation analysis of a group of metabolites. Statistical significance of enriched pathways was established using Fisher’s exact test using Scipy.stats.fisherexact in the Python 3.8.5 package (available online: https://docs.scipy.org/doc/scipy/ (accessed on 20 December 2024)). In addition, information on the classification of metabolites was collected with reference to the Human Metabolome Database (HMDB) 4.0 database (available online: https://www.hmdb.ca/ (accessed on 30 December 2024)).

3. Results

3.1. Accumulation Characteristics of Cd and Phe in HY702

Figure 1 shows the accumulation of Cd and Phe in the roots of maize HY702 seedlings grown in soil containing Cd and Phe alone and in combination. The uptake and accumulation of Cd in the roots were significantly inhibited in the co-presence of these contaminants, reducing the Cd content in the roots by 9% compared to the single Cd treatment (p < 0.05) (Figure 1A). In contrast, compared to the single Phe pollution treatment, Cd-Phe combined pollution resulted in a higher uptake of Phe in the roots, but the difference was not significant (p > 0.05) (Figure 1B). Thus, the presence of Cd did not affect the uptake of Phe by the roots of HY702 at the tested concentrations, but the presence of Phe had an antagonistic effect on root uptake of Cd. Based on the observation that the coexistence of Phe had no significant effect on Cd accumulation in the roots, subsequent analyses focused on the comparison of Cd-Phe combined pollution and Cd single pollution to determine the effect of Phe coexistence.

The stems and leaves were also analyzed (in combination) (Figure 1C), which revealed that the presence of 1 mg/kg of Phe strongly inhibited the uptake and accumulation of Cd in these plant parts compared to the single Cd treatment. The Cd content was reduced by 44% (p < 0.001), which was a much stronger effect than observed for the roots. Thus, the coexistence of Phe significantly inhibited the accumulation of Cd in the stems, leaves, and (to some extent) the roots of the maize, thereby reducing the risk of Cd pollution inthe plants, while there was no significant effect on Phe uptake.

3.2. Effects on HY702 Growth, Physiological, and Biochemical Responses

3.2.1. Growth Responses

The presence of Cd alone resulted in a significant biomass increase of 35%compared to the CK (p < 0.05). The combined pollution of Cd-Phe resulted in a biomass increase of 16% compared to the CK, but the difference was not significant (p > 0.05) (Figure 2A). Likewise, the difference between single Cd and Cd-Phe combined pollution was not significant (p > 0.05) (Figure 2A). Plant height increased by 8% in the presence of Cd (p < 0.01) and by 6% in Cd-Phe combined pollution (p < 0.05) compared to the CK control. However, a difference between single Cd and Cd-Phe combined pollution treatment was again not significant (p > 0.05) (Figure 2B). Thus, at the concentration levels used here, Cd had a growth-promoting effect on the maize seedlings, which was not significantly reduced by the coexistence of Phe.

3.2.2. Physiological and Biochemical Responses

To some extent, the relative chlorophyll content can be used to characterize the senescence of plant tissues under pollution. The relative chlorophyll content in stems and leaves of the maize did not significantly differ among all of the treatments (Figure 3A). The unchanged chlorophyll content in the stems and leaves suggests that there was a minimal impact on the health of these tissues under Cd pollution and Cd-Phe combined pollution at the concentrations applied (p > 0.05).

The MDA levels were determined in stems and leaves (Figure 3B). Both single Cd and Cd-Phe combined pollution significantly reduced the MDA content compared to CK, by 62% (p < 0.001) and 55% (p < 0.01), respectively, but there was no significant difference between the two treatments (p > 0.05) (Figure 3B). This suggests that MDA content was not significantly aggravated by the coexistence of Phe. MDA is a direct product of membrane lipid peroxidation, and it is an indicator of the extent of plasma membrane peroxidation in plants under unfavorable conditions. Higher MDA concentrations indicate more severe damage to cell membranes. The observed decrease in MDA content suggests that pollution with either single Cd or combined Cd-Phe mitigated the damage to cell membranes in the stems and leaves, and the plant’s resistance was notably enhanced.

The proline content of stems and leaves was significantly higher in seedlings grown under Cd-Phe combined pollution than in the CK, with an increase of 57% (p < 0.01) (Figure 3C), and this was significantly higher (by 45%) than the proline content under single Cd pollution (p < 0.01). Cd alone increased proline content by 8%, but this was not significant (p > 0.05). Proline is an essential osmotic regulator, and its quantity can characterize plant pollution signals. The findings suggest that the coexistence of Phe and Cd can significantly alleviate the harm caused by Cd alone in the maize plants.

The activity of the three enzymes mainly participating in the oxidative stress response was also assessed (Figure 4). SOD activity (Figure 4A) was significantly reduced as a result of single Cd treatment, by 24% (p < 0.05) compared to the CK. Cd-Phe combined pollution increased SOD activity by 13% relative to the CK, but this change was not statistically significant (p > 0.05). However, it represented a significant 49% enhancement compared to the single Cd treatment (p < 0.01). SOD can scavenge free radicals in plants to effectively reduce the cellular damage caused by free radicals. The results indicate that Cd alone inhibits SOD enzyme activity and decreases the ability to scavenge free radicals in plants, thus increasing cellular damage; however, Cd in coexistence with Phe significantly enhances the scavenging capacity of free radicals and mitigates cell damage. Abbott’s formula analysis of the Cd-Phe interaction revealed an RI value of 2.25 (>1) under the P1C1 treatment, indicating a synergistic promotional effect of Phe and Cd coexistence on SOD activity.

CAT scavenges hydrogen peroxide to break it down into water and oxygen, thereby minimizing the oxidative exposure of the plant. It was found that neither the single Cd nor the combined Cd-Phe treatment had a significant effect on the CAT enzyme activity of the plants (p > 0.05) (Figure 4B). The findings suggest that the coexistence of Cd and Phe pollution did not affect the scavenging capacity toward hydrogen peroxide by CAT.

For POD activity (Figure 4C), the effect of single Cd application was not significant, but Cd-Phe combined pollution significantly increased POD activity by 15% (p < 0.05) compared to the CK (27% increase compared to C1, p < 0.01). POD can protect cells from oxidative damage by catalyzing the breakdown of oxidized phenolics and hydrogen peroxide-like compounds into harmless aldehydes and oxygen. Abbott’s formula analysis resulted in an RI value of 3.68 (>1) under the P1C1 treatment, suggesting a synergistic promotional effect of Phe and Cd coexistence on POD activity. The POD and SOD results are consistent, indicating that the coexistence of Phe and Cd significantly reduces the oxidative stress of the plants by activating the plant’s antioxidant enzyme system, strengthening the scavenging of reactive oxygen species and peroxide analogs, and increasing the plant’s resistance. In contrast, single Cd did not have this positive effect on the enzyme system.

To sum up, maize exhibited unrestricted growth and improved biochemical parameters under Cd-Phe co-pollution compared to single Cd exposure. This combined pollution enhanced the activities of antioxidant enzymes (SOD and POD) and proline content, collectively activating the antioxidant defense system and osmotic regulation. As summarized in Figure 5, these responses promoted cellular membrane repair, consequently decreasing Cd absorption and accumulation, and this ultimately enhanced the plant’s resistance to Cd.

3.3. Characterization of Metabolic Differences in HY702 in Response to Cd-Phe Co-Pollution

3.3.1. Differential Metabolites of HY702 Under Cd-Phe Co-Pollution

After characterizing the metabolomes of the plant tissues, the data were clustered by principal component analysis (PCA) and OPLS-DA. PCA (Figure 6A) identified significant differences between the C1 and P1C1 treatments, indicating that the coexistence of Cd-Phe affected the metabolism of HY702 in a different manner than Cd alone did. The difference was even stronger in the OPLA-DA analysis (Figure 6B and Figure S1). The relative metabolite expression levels of the two sample groups were also distributed differently, as illustrated in a volcano plot (Figure 6C). Comparing the two treatments, 126 differentially expressed metabolites were identified (Table S2). Of those, 62 were up-regulated, and 64 were down-regulated under Cd-Phe pollution compared to Cd single pollution. These differential metabolites were primarily classified as lipids and lipid-like molecules (44%), organic acids and their derivatives (20%), organic heterocyclic compounds (18%), and organic oxides (12%), with 6% belonging to other categories, according to the HDMB compound classification (Figure 6D). The most common lipid and lipid-like molecules were fatty acyls, isoprenoid lipids, and steroids. Most organic acids and their derivatives were amino acids; the organic oxides were primarily carbohydrates; and the organic heterocyclic compounds included a range of substances such as lactones, azoles, and thiol-alkanes.

The top 10 up-regulated and down-regulated differential metabolites in the combined pollution treatment were identified by analyzing the changes in metabolite expression (Figure 6E). These included eight organic acids and their derivatives, five organic oxygen compounds, five lipids or lipid-like molecules, and two organic heterocyclic compounds. These metabolites were annotated into the three main categories of steroids (lipids), carbohydrates (sugars), and amino acids. The top ten up-regulated metabolites were Leu-Ser-Thr-Thr, (E,E)-piperlonguminine, A-L-arabinofuranosyl-(1->2)-[a-D-mannopyranosyl-(1->6)]-D-mannose, ureidopropionic acid, 8-[(aminomethyl)sulfanyl]-6-sulfanyloctanoic acid, valyl-hydroxyproline, XI-dihydro-2-methyl-3(2H)-thiophenone, 5-aminopentanal, ophthalmic acid, and L-asparagine. The top ten down-regulated metabolites were galactaric acid, 2-(2,4-dihydroxyphenyl)-3-(3,7-dimethylocta-2,6-dien-1-yl)-5,7-dihydroxy-6-(3-methylbut-2-en-1-yl)-3,4-dihydro-2H-1-benzopyran-4-one, prednisolone, aldosterone, tryptophyl-arginine, (1beta,8beta)-1,8-dihydroxy-3,7(11)-eudesmadien-12,8-olide, withaperuvin G, linalool oxide D 3-[apiosyl-(1->6)-glucoside], 3-[3,4-dihydroxy-2-(4-hydroxy-3,7-dimethylocta-2,6-dien-1-yl)phenyl]propanoic acid, and thymidine 3′,5′-cyclic monophosphate.Their respective FC values are shown in Figure 6E. The strongest effects were demonstrated for the oligopeptide Leu-Ser-Thr-Thr, whose abundance was more than doubled, and galactaric acid, which was more than halved in abundance.

3.3.2. Key Metabolic Pathways of HY702 Under Cd-PheCo-Pollution

The KEGG database was used to identify which metabolic pathways were enriched, as predicted by the differential metabolites of HY702 under Cd-Phe coexistence pollution. Eleven KEGG metabolic pathways could be linked to twenty-one distinct differential metabolites (Figure 7A). The amino acid metabolism pathway was the most enriched in P1C1 treatment, with 14 types of differential metabolites, followed by the carbohydrate metabolism pathway, which was comparatively more enriched with six types of differential metabolites. Among these, (S)-2-acetyl-2-hydroxybutanoic acid, 2-isopropylmalic acid, and L-glutamate were involved in both the sugar and amino acid metabolism pathways. Betaine and L-glutamate were also implicated in membrane transport routes, in addition to their role in amino acid metabolism. Similarly, L-asparagine, L-methionine, and L-glutamate were involved in the translation machinery. The metabolic pathways significantly associated with HY702 under Cd-Phe coexistence pollution were further analyzed and validated by the two indicators of significance (p < 0.05) and their enrichment factor (Figure 7B). The metabolic pathways that were significantly enriched included four amino acid metabolic pathways, two sugar metabolic pathways, and the translation machinery, such as aminoacyl-tRNA biosynthesis, valine, leucine, and isoleucine biosynthesis; alanine, aspartate; metabolism of glutamate, pyruvate, glutathione, histidine, glycolate, and dicarboxylic acid; arginine and proline; and tryptophan (Table S3).

To highlight the metabolic pathways with significant sites in the entire metabolic network and target key metabolites, the KEGG analysis was filtered to identify the key metabolic pathways most strongly and significantly impacted by the Cd-Phe coexistence response. The results are visualized by the topological analysis in Figure 7C. This identified the following four major metabolic pathways with an impact value > 0.05, p < 0.05, in order of decreasing impact: (a) metabolism of alanine, aspartate, and glutamate; (b) biosynthesis of valine, leucine, and isoleucine; (c) biosynthesis of aminoacyl-tRNA; and (d) metabolism of histidine. Six distinct metabolites were engaged in these pathways, including three organic acids (2-isopropylmalic acid, (S)-2-acetyl-2-hydroxybutanoic acid, and urocanic acid) and three amino acids (L-asparagine, L-methionine, and L-glutamate), all of which showed markedly elevated expression levels. Additionally, four other metabolic pathways were significantly impacted: metabolism of pyruvate, glutathione, arginine, and proline; glyoxylate; and dicarboxylic acid (0 < impact value < 0.05, p < 0.05) (Table S4 and Figures S3–S9).

By integrating these metabolomics results, distribution maps of the eight metabolic pathways with significantly up-regulated differential metabolites were constructed (Figure 8). The metabolic pathways of amino acids and sugars, as well as translation, were significantly altered in HY702 by the presence of combined Cd and Phe, with amino acid metabolism being the primary impacted process. The coexistence of Phe and Cd significantly up-regulated the biosynthesis of several metabolites, including L-asparagine, L-methionine, L-glutamate, (S)-2-acetyl-2-hydroxybutanoic acid, urocanic acid, and 2-isopropylmalic acid. Among these, L-asparagine, L-methionine, and L-glutamate play a pivotal role in Cd detoxification by forming stable chelates with Cd, thereby reducing the bioavailability of free Cd²⁺ and its subsequent uptake by plants [44,45]. Mechanistically, L-glutamate triggers the activation of glutamate receptors (GLRs) on the plasma membrane, initiating Ca²⁺-mediated signaling cascades that up-regulate Cd²⁺ transport proteins such as HMA3 [46]. This facilitates the vacuolar sequestration of Cd²⁺, effectively restricting its translocation to aerial tissues. Concurrently, L-asparagine supports endoplasmic reticulum protein folding, ensuring the proper synthesis and membrane localization of Cd transporters (e.g., HMA3), further enhancing Cd compartmentalization [47]. Collectively, these Phe-induced metabolic adaptations not only diminished Cd absorption and accumulation in HY702 but also bolstered the plant’s antioxidant defense system. In addition, Cd-Phe coexistence also affected metabolites such as 4-hydroxy-2-oxoglutarate, 4-guanidinobutanoate, L-cysteinyl-glycine, and (R)-2-hydroxybutane-1,2,4-tricarboxylate, impacting the occurrence of their associated metabolic pathways. Due to the up-regulation of these differential metabolites under Cd-Phe coexistence, the metabolic pathways became enriched to produce more amino acids and other substances, ultimately inhibiting the uptake and accumulation of Cd. Furthermore, although the lipid metabolic pathway accounted for a greater number of differential metabolites in HDMB, it did not exhibit significant enrichment. This is likely due to the unique metabolic characteristics of HY702 or the complexity and variety of lipid metabolic pathways, which makes it challenging to represent them separately in metabolic pathway maps.

4. Discussion

The coexistence of 2.5 mg/kg Cd and 1 mg/kg Phe in the soil significantly inhibited the uptake and accumulation of Cd in the stems and leaves of HY702 maize seedlings compared to Cd being present alone: the presence of Phe reduced the Cd content in the plants by 44%. This antagonistic effect decreased the risk of maize absorbing and accumulating this toxic heavymetal pollutant. Similar observations have been described in the literature. For example, combined pollution of Cd and Pyr prevented Cd accumulation in maize [21] and in Xanthium sibiricum [23]. Various explanations have been given for this inhibition. Firstly, Phe may alter the enzyme activities and metabolic processes in maize plants, hindering the synthesis of secretions that promote Cd accumulation and thus reducing Cd absorption [21]. Our study confirmed that the coexistence of Phe and Cd in the soil significantly increased the proline content and the antioxidant enzyme activities of SOD and POD in the maize seedlings compared to single Cd contamination. The observed effect is most likely due to Phe being present. Others have shown that Phe can increase the accumulation of proline and the enzyme activities of SOD and POD in ryegrass and rice [48,49]. This suggests that the coexistence of Phe stimulates the activity of antioxidant enzymes, including SOD and POD. In addition to activating the antioxidant enzyme system, plants also engage in osmotic regulation for self-defense. Under the influence of Phe, the proline content in HY702, an important osmoregulatory molecule, significantly increased, as shown in Figure 3. Proline also plays a crucial role in scavenging ROS, reducing membrane lipid peroxidation, and maintaining the structure of proteins and membrane systems [50]. Thus, under combined Phe and Cd stress, the elevated SOD and POD activities synergize with increased proline levels to scavenge ROS, thereby preserving the structural integrity of cell membranes and impeding passive transmembrane transport of Cd. Concurrently, the carboxyl and imino groups of proline can form stable chelate complexes with Cd, reducing the availability of free Cd in plants and diminishing its translocation and accumulation in stems and leaves [51]. Secondly, Phe and Cd may also form complexes, which decrease the concentration of free Cd ions in the soil, thereby reducing their uptake by plants [52]. Thirdly, the impact of Phe on the diversity of soil microbial communities may also indirectly contribute to reduced Cd accumulation [53]. Some varieties, such as HY702, may possess certain genetic traits that result in a low uptake of Cd, but the genetic nature of this property requires further research. Nevertheless, even for this variety, the antagonistic effect of Phe on Cd was demonstrated, as described here.

The metabolic response of the maize to the coexistence of Cd and Phe induced four major metabolic pathways involving six key metabolites in HY702, including L-asparagine, L-methionine, L-glutamate, (S)-2-acetyl-2-hydroxybutanoic acid, urocanic acid, and 2-isopropylmalic acid. This indicates that the levels of the metabolic pathways in which these metabolites were involved were expressed significantly moreinPhe-Cd coexistence compared to Cd alone. Plant pollution tolerance relies on L-glutamate, which is reported to accumulate in plants exposed to pollution, where it functions as an antioxidant and chelator [54,55,56]. The current study found that the coexistence of Phe and Cd significantly increased the expression of L-glutamate, which is involved in glutathione metabolism and other metabolic pathways. Several studies have shown that glutathione is a vital component in the immobilization of Cd and functions as an antioxidant and precursor of plant-chelating peptides (PCs) in plants [57,58]. This suggests that significant production of L-glutamate, caused by the presence of Phe, can immobilize Cd in roots and inhibit its migration to the stems and leaves, thereby drastically reducing their Cd content, as shown in Figure 1. Asparagine can chelate Cd directly or indirectly, similar to glutamate, thereby reducing the heavy metal’s toxicity [45]. Asparagine was discovered to be a crucial molecule for the uptake, cycling, transport, and storage of nitrogen as well, which is necessary for plants to withstand Cd pollution [59]. The production of aminoacyl tRNAs is an essential step in protein biosynthesis. The action of aminoacyl-tRNA synthetases (ARSs), which bind specific amino acids to their corresponding tRNAs, is essential in the translation process [60]. It has been shown that Phe stress may increase ribosome biogenesis in wheat xylem sap [61]. A subset of the DEPs was associated with tran smembrane transporter activity. The change inDEPs stressed by Phe perhaps further affected Cd migration. Furthermore, methionine can form stable chelates by binding to heavy metal ions like Cd, Pb, and Zn, which reduces their toxicity [62] and, to some extent, hamperstheuptake and accumulation of Cd in plants. Urocanic acid, closely related to glutamate and methionine, is an intermediate product of histidine metabolism and may also be involved in the chelation of Cd [63]. However, the mechanism by which urocanic acid reduces Cd accumulation in plants is not fully understood. The metabolites 2-isopropylmalic acid and (S)-2-acetyl-2-hydroxybutanoic acid were both found to be increased as a result of Phe and Cd combined pollution. The former is a derivative of malic acid and can be catalyzed into pyruvate [64]. It is a component of the tricarboxylic acid (TCA) cycle and is required for the synthesis of leucine in plants. In addition, it plays a significant role in plant pollution response and energy consumption [65]. The presented data suggest that 2-isopropylmalic acid and (S)-2-acetyl-2-hydroxybutanoic acid are also important for plant pollution response and energy expenditure. Both metabolites are involved in pathways related to valine, leucine, and isoleucine biosynthesis, which help produce essential osmoregulators for plants to withstand osmotic imbalance under pollution exposure [44]. These pathways may have contributed to reducing the accumulation of Cd in HY702.

Overall, the metabolic pathways by which maize var. HY702 mitigates Cd accumulation in the presence of Phe can be divided into three primary mechanisms: (1) competitive chelation is enabled by up-regulated L-methionine metabolites and increased proline content, as these can form stable complexes with Cd, reducing the mobility of free Cd ions [44,45]. (2) Transport regulation is increased via glutamate. Under combined Cd-Phe stress, elevated proline levels undergo intensified catabolism, resulting in increased glutamate accumulation. This glutamate may significantly activate SnRK2 kinase, which up-regulates the expression of heavy metal transport proteins (e.g., HMA3) bybinding to ABRE (Abscisic Acid-Responsive Element). Consequently, Cd is sequestered into vacuoles, reducing Cd accumulation in shoot tissues [46]. (3) Enhanced detoxification is enabled by glutathione-mediated conjugation, which converts Cd into non-toxic chelates. This process helps safeguard the stability of the antioxidant enzyme system, maintains endoplasmic reticulum homeostasis, ensures proper folding and membrane localization of Cd-transport proteins, and enhances vacuolar sequestration efficiency, ultimately reducing Cd accumulation in shoot tissues [47].

5. Conclusions

This study utilized metabolomics technology to elucidate the effects of Cd-Phe coexistence pollution on the growth, physiological and biochemical characteristics, and metabolic response mechanisms of maize var. HY702. The coexistence of Phe and Cd significantly inhibited the uptake and accumulation of Cd in the roots, stems, and leaves of HY702, demonstrating an antagonistic effect compared to Cd alone. This antagonistic effect was specifically manifested by a significant increase in the activities of the antioxidant enzymes SOD and POD, as well as in proline content. Metabolomic results indicated that under Cd-Phe coexistence, amino acid, sugar, and lipid metabolic pathways were all significantly activated. Additionally, six differential metabolites, namely L-asparagine, L-methionine, L-glutamate, 2-isopropylmalic acid, urocanic acid, and (S)-2-acetyl-2-hydroxybutanoic acid, were significantly increased, thereby inhibiting the absorption and accumulation of Cd in maize HY702. A significant up-regulation of urocanic acid and (S)-2-acetyl-2-hydroxybutanoic acid under Cd-Phe coexistence is reported here for the first time. The precise mechanisms by which these metabolites contribute to the suppression of Cd absorption and accumulation in maize due to Phe coexistence are not yet clear. To elucidate the molecular mechanisms governing the uptake and accumulation of Cd in maize more fully, in the future, genomic and transcriptomic techniques need to be employed to investigate changes in gene expression in maize under Cd-Phe coexistence pollution. These findings provide an important scientific basis for the safe cultivation and production of maize on lightly polluted soil.