Allium ramosum was transplanted in Yu’er Mountain Pasture, Chengde City, Hebei Province, and the experiment was conducted from May to August 2023. The study field is located 41°44’ N and 140°16’ E at an altitude of 1460 m. The pasture is situated in a semi-arid continental monsoon climate zone with an accumulated temperature of 1513.1°C when the temperature is ≥10°C and a frost-free period of 85 days. The experiment was conducted using a completely randomized block design with a plot size of 4 x 9 = 36 m² and a 0.5 m interval between blocks. Two types of base fertilizers were used: conventional chemical fertilizer (The nitrogen fertilizer is urea containing 47% N, the phosphorus fertilizer is superphosphate containing 12% P₂O₅, and the potassium fertilizer is potassium chloride containing 50% K₂O.) and 225.00 kg/hm² compound microbial fertilizer (The compound microbial fertilizer is “Kunyijian Huolin Potassium Compound Microbial Fertilizer” manufactured by Tianjin Kunhe Biotechnology Group Co., Ltd. The nutrient content is as follows: N 3.76%, P₂O₅ 9.41%, K₂O 2.83%, and the number of effective living bacteria ≥ 2.0 billion/ml.). The foliar fertilizers included 0.2% amino acid solution and 0.2% algal extract (After applying the base fertilizer, spray every ten days, for a total of three times.). The following experimental treatments were used: CK (control: No treatment was applied), C+A (conventional chemical fertilizer combined with 0.2% amino acid solution), C+S (conventional chemical fertilizer combined with 0.2% algal extract), B+A (225.00 kg/hm² compound microbial fertilizer combined with 0.2% amino acid solution), and B+S (225.00 kg/hm² compound microbial fertilizer combined with 0.2% algal extract).

For statistical analysis, one square meter was randomly selected from each block and some samples were analyzed for physiological indicators at −80°C, while the rest were blanched at 105°C for 10 min and then dried to constant weight at 75°C for further experimentation.

To measure growth parameters, In each experimental plot, a one-square-meter area located within the interior of the plot is randomly selected as the sampling point and inflorescence stem height was measured using a ruler and corolla diameter using a Vernier caliper (victor vc5150s made in China). The fresh weight (FW) and dry weight (DW) of the A. ramosum flowers were measured using an electronic scale (made in China).

The scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) against free radicals was assessed following the protocol outlined by Xue et al. (2021) with the findings presented as the percentage of the dry weight (% DW). The iron-reducing antioxidant capacity of the A. ramosum flowers was measured using a ferric reducing antioxidant power (FRAP) kit according to with the manufacturer’s instructions (Beijing Solabao, Beijing, China). Briefly, flower tissue (0.1 g) was ground in 1 ml of pre-cooled extract, the homogenate centrifuged at 4°C for 10 min at 10,000×g, and the supernatant collected for detection.

The respective solutions were then combined according to the instructions provided with the FRAP kit and allowed to react in the dark at room temperature for 20 min. The absorbance at 593 nm was quantified using a spectrophotometer (UV-1100 spectrophotometer, Shanghai Mepu Instruments Co., Ltd, China), and the antioxidant potential of A. ramosum flowers was quantified by monitoring the change in absorbance, using Trolox as a standard control. The efficacy of 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in neutralizing free radicals was assessed using the method outlined by Schaich et al. (2015), and the results reported as percentage of dry weight (% DW). The hydroxyl radical scavenging capacity (HRSC) was measured using the method described by Schaich et al. (2015), and the results were reported as a percentage. The superoxide anion radical scavenging rate was quantified using the same method, and the results were expressed as percentages.

The SOD activity was quantified using the method described by Hosseinpour et al. (2020), and the results were reported as units per gram of FW per minute (U g^–1 min^–1 FW). The POD activity was measured following the protocol outlined by Khan et al. (2022) and the results were expressed as units per gram of FW per minute (U g^–1 min^–1 FW). Polyphenol oxidase (PPO) activity was assessed using the method described by Rasmussen et al. (2021) and the results were reported as units per gram FW per hour (U g^–1 h^–1 FW).

Methodology by Kamran et al. (2021) was employed to ascertain the rate of superoxide anion (O2• −) production, and concentration of malondialdehyde MDA (The product has a maximum absorption peak at 532nm and a minimum absorption peak at 600nm.) in A. ramosum flowers, with the results documented in moles per gram of FW [mol g⁻¹ (FW)], millimoles per gram FW per minute [mmolg⁻¹min⁻¹ (FW)], and nanomoles per gram of FW (nmol g⁻¹ (FW), respectively.

The GSH content was quantified using the method described by Zhong et al. (2023), with results reported as milligrams per gram of FW (mg g^–1 (FW)). Free amino acid content was measured using the ninhydrin colorimetric method outlined by Xu et al. (2022), and the results expressed as milligrams per gram of DW (mg g^–1 (DW)). The soluble sugars (SS) content was determined using the method reported by Kumar and Kumar (2023). The soluble protein (SP) content was measured using the method described by Xu et al. (2022). The total ascorbic acid (TAA) content was quantified according to the method described by Bida-Badi et al. (2023). Proline content was determined using the method described by Ghafoor et al. (2020), the flavonoid content was measured using the method described by Guo et al. (2022), and polyphenols were determined using the method described by Yang et al. (2018).

Precisely weight 3g of Allium ramosum flower (freshly harvested samples) samples and place them in a 20mL headspace vial, which is then immediately sealed for metabolomics analysis using headspace-coupled gas chromatography-mass spectrometry (HC-GC-MS). The samples are injected into the GC-MS system using split mode, with an injection volume of 1 µL and a split ratio of 10:1. After separation through a VF-WAXms capillary column (25m×0.25mm×0.2µm, Agilent CP9204), the samples are subjected to mass spectrometry detection. The inlet temperature is set at 180°C, with high-purity helium gas as the carrier gas at a flow rate of 2 mL/min, and the septum purge flow rate is 3 mL/min. The temperature program starts at 40°C, holds for 2 minutes, then increases to 100°C at a rate of 5°C/min, followed by a further increase to 230°C at a rate of 15°C/min, and holds for 5 minutes, with a post-run at 230°C for 2 minutes. The electron impact ion source (EI) is used, with a transfer line temperature of 310°C, an ion source temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scanning mode is full scan (SCAN), with a mass scan range of m/z 30-1000, and a scan frequency of 3.2 scans/s.

To evaluate the stability of the analysis system during the testing process, a quality control sample (QC: Mix all the samples to be tested according to the same mass.) is prepared. The QC sample is a mixture of all the samples to be tested and is treated in the same manner as the formal samples. During the instrument analysis, a QC sample is inserted every 5-15 samples. The repeatability of the QC samples can reflect the stability of the instrument throughout the entire analysis process during data analysis. It also serves to identify variables with high variability in the analysis system, ensuring the reliability of the results.

Experimental data were analyzed using SPSS 22.0 (IBM, Chicago, IL, USA)., with one-way analysis of variance and Duncan’s test employed to determine any significant differences among the groups (p ≤ 0.05). Principal component analysis scores and loadings were used to evaluate the effects of the five treatments on the A. ramosum flowers.

As seen in Supplementary Figures S1A–D , all fertilizer treatments enhanced the fresh weight, dry weight, inflorescence stem height, and corolla diameter of A. ramosum flowers as compared to the control (CK). Specifically, the B+S treatment was associated with notable 112, 223, and 29% increases in the fresh weight, dry weight, and corolla diameter, respectively, while B+A treatment significantly boosted the inflorescence stem height, with a 0.24-fold increase observed.

Compared with the control (CK), all treatments, C+A, C+S, B+A, and B+S, were found to notably augment the superoxide anion radical scavenging rate (SASR), with 102, 103, 112, and 104% increases observed, respectively. ( Figure 1A ). C+A and B+S led to significant improvements in the ABTS scavenging rate as compared to CK, with increases of 57.1% and 63.8%, respectively ( Figure 1B ), while all treatments, C+A, C+S, B+A, and B+S, led to marked enhancements of the DPPH scavenging rates (5%, 9%, 10%, and 11.7%, respectively; Figure 1C ); B+A and B+S increased the FRAP content 1.15- and 1.72-fold, respectively ( Figure 1D ); and C+A, C+S, B+A, and B+S significantly increased the HRSC by 53, 51.1, 43.9, and 48.7%, respectively ( Figure 1E ).

In contrast to the control (CK), C + A, C + S, B + A, and B + S notably augmented the POD content, with increases of 9, 18.1, 28.2, and 55.8% observed, respectively ( Supplementary Figure S2A ); while C+A, C+S, B+A, and B+S significantly increased the PPO content by 44.8, 71.8, 48.8, and 50.4%, respectively ( Supplementary Figure S2B ); and C+A, C+S, B+A, and B+S significantly increased the SOD content, with increases of 32.5, 34.4, 55.8, and 36.2%, respectively ( Supplementary Figure S2C ).

Compared to the control (CK), the C+A, C+S, B+A, and B+S significantly reduced the MDA content by 22.4, 34.4, 29.6, and 47.2%, respectively ( Supplementary Figure S3A ), while C+A, C+S, B+A, and B+S significantly reduced the superoxide anion (O2• −) content by 9, 12, 15.5, and 29.2%, respectively ( Supplementary Figure S3B ).

The application of various fertilizers substantially influenced the levels of GSH, SS, SA, and TAA within the A. ramosum flowers. Compared to the control (CK), C+A, C+S, B+A, and B+S notably elevated the GSH content by 28.9, 28.2, 29.6, and 58.1%, respectively ( Figure 2A ); B+A and B+S increased the SP content 80 and 85%, respectively ( Figure 2B ); and C+A, C+S, B+A, and B+S led to a significant increase in the SS content, with enhancements of 24.8, 9, 13.3, and 34.6%, respectively ( Figure 2C ).

The different fertilizers had significant impacts on the flavone, flavonoid, polyphenol, and proline contents in the A. ramosum flowers. Compared with the control (CK), C+A, C+S, B+A, and B+S significantly increased the flavone content by 28.1, 18.8, 33.9, and 56.1%, respectively ( Figure 3A ); C+A and B+S, significantly increased the flavonoid content by 31.3 and 32.6%, respectively ( Figure 3B ); B+S and B+A, significantly increased the polyphenol content by 44.2 and 27%, respectively ( Figure 3C ); and C+A, C+S, B+A, and B+S led to notable increases in the proline content, with enhancements of 38.1, 36.6, 49.7, and 57.1%, respectively ( Figure 3D ).

The different fertilizers also had a significant impact on the proline and free amino acid contents of the A. ramosum flowers, with C+A, C+S, B+A, and B+S treatments associated with significant increases of 74.7, 67.4, 31, and 71.9% in the proline content, respectively ( Supplementary Figure S4A ); and C+A, C+S, B+A, and B+S significantly increasing the Free Amino Acid content by 46.7, 49.6, 41.1, and 29.6%, respectively ( Supplementary Figure S4B ), as compared to CK.

Pearson’s correlation analysis was used to test the correlations between different variables ( Figure 4 ). The results indicated positive correlation between B+S and the SS content, flavonoid content, ABTS scavenging rate, proline content, polyphenol content, flavone content, POD, Corolla diameter, DW, FW, FRAP, and DPPH scavenging rate for A. ramosum flowers; with negative correlation observed for B+S and O2• − positive correlation observed between B+A and the TAA content, alkaloids, SOD, and Inflorescence stem height; negative correlation between B+A and MDA levels; significant positive correlation observed between C+S and both polyphenol oxidase activity and free amino acids content; significant positive correlation between C+A and proline content; and negatively correlation for C+A and FRAP.

The Spearman’s rank correlation method was employed to examine the relationships between the various fertilizer applications and the growth metrics, antioxidant proficiency, and nutritional attributes of the A. ramosum flowers, with results indicating positive correlation between the DPPH scavenging rate and inflorescence stem height, corolla diameter, polyphenol content, proline content, and SP content and negative correlation with MDA and superoxide anions (O2• −). FRAP was positively correlated with corolla diameter, dry weight, polyphenol content, and POD, and negatively correlated with O2• − and the ABTS scavenging rate was positively correlated with proline, alkaloids, flavones, flavonoids, soluble sugars, and HRSC ( Figure 5 ).

Before and after the biostimulant treatments, a total of 504 volatile compounds were identified in the Allium ramosum flowers, including 23 Terpenoids, 10 Amines, 8 organic nitrogen compounds, 178 Esters, 29 Acids, 134 Ketones, 18 Amides, 85 Alcohols, 70 Aldehydes, 88 Others, 22 Ethers, 87 Heterocyclic compounds, 27 Organosulfur compounds, 90 Hydrocarbons, and 7 Phenols ( Figure 6A ).

Compared to the CK, all treatments significantly increased the relative content of organic sulfur compounds. The B+S, B+A, C+S, and C+A treatments increased the content by 12.5%, 9.4%, 4.9%, and 3.1%, respectively, compared to CK ( Figure 6B ). The results indicate that the effects of B+S and B+A on the content of organic sulfur compounds are greater than those of C+S and C+A ( Figure 7 ). To visually present the trend of metabolite changes under biostimulant treatments, a clustering analysis was performed on the standardized differential metabolite content. When Allium ramosum flowers were treated with B+A, the relative expression levels of Disulfide, methyl 1-(methylthio)propyl, Disulfide, dimethyl, 2,3,5,7-Tetrathiaoctane 3,3-dioxide, Diallyl disulphide, Sulfide, allyl methyl, Disulfide, methyl 2-propenyl, Disulfide, methyl propyl, 1-(methylthio)-Pentane, Dimethyl sulfide, Disulfide, methyl 1-propenyl, Tetrasulfide, dimethyl, Diallyl sulfide, 2,3,5-trithiahexane 5-oxide, 1-(ethylthio)-1,3-Butadiene, 1,1’-thiobis-1-Propene, Trisulfide, methyl propyl, and 1-Propene-1-thiol were high. When Allium ramosum flowers were treated with B+S, the relative expression levels of Disulfide, dimethyl, 2,3,5,7-Tetrathiaoctane 3,3-dioxide, Sulfide, allyl methyl, Disulfide, methyl 2-propenyl, Disulfide, methyl propyl, 1-(methylthio)-Pentane, Disulfide, methyl (methylthio)methyl, (methylsulfinyl)(methylthio)-Methane, 3-Methylthio-6-methyl-5-thioxo-2,5-dihydro-1,2,4-triazine, 4-Methylmercaptoaniline, Tetrasulfide, dimethyl, Dimethyl trisulfide, Trisulfide, methyl 2-propenyl, and 1-(ethylthio)-1,3-Butadiene were high ( Figure 7 ). These 27 different organosulfur compounds could be classified into 6 subcategories based on their relative content. An overall upward trend was observed in the third subcategory, where the content of these 8 metabolites in the B+S and B+A samples was higher than that in the C+S, C+A, and CK samples, indicating that 7 of these metabolites (Diallyl disulphide, Disulfide, dimethyl, Sulfide, allyl methyl, Disulfide, methyl propyl, Disulfide, methyl 2-propenyl, 1-(methylthio)-Pentane, and 2,3,5,7-Tetrathiaoctane 3,3-dioxide) are associated with the B+S and B+A samples ( Figure 8 ).

Numerous studies have demonstrated that the application of external amino acid-based fertilizers or substances derived from algal extracts markedly enhances plant growth and development.

Within the context of this investigation, a notable enhancement in the corolla diameter was observed under all four treatments, along with dry mass, fresh mass, and the inflorescence stem length of the A. ramosum blossoms as compared to CK. Notably, B+A and B+S yielded significantly better outcomes than C+A and C+S ( Supplementary Figures S1A-D ), suggesting that using a compound microbial fertilizer as a base with amino acid or algal extract as a foliar fertilizer can effectively promote the growth of A. ramosum flowers. Echoing the conclusions of Khan et al. (2019), the current study confirmed that using amino acid-based fertilizers promotes plant growth and increases biomass (Khan et al., 2019). In parallel, a liquid amino acid fertilizer derived from pig bristles has also been found to augment crop yield (Wang et al., 2019). These results suggest that amino acids stimulate plant growth and augments agricultural yield. In the present study, the application of amino acid fertilizers resulted in an increase in the fresh and dry biomass of A. ramosum, which is consistent with earlier findings by Khan et al. (2019) and Wang et al. (2019) suggesting that amino acid fertilizers may enhance the growth of A. ramosum flowers by stimulating the endogenous synthesis of hormones, facilitating improved nutrient uptake. Amino acids have also been found able to activate specific transporters, facilitating the ability of plants to more effectively absorb and utilize nutrients (Sharma and Dietz, 2006). The promotion of inflorescence stem height observed for B+A was superior to that by C+A, possibly because of the interaction of microorganisms in the compound microbial fertilizer with the minerals and organic substances in the soil promoting the absorption of nutritional elements by the plants.

Previous research has indicated that both algal extracts and their isolated constituents induce pronounced physiological reactions in plants, resulting in enhanced stem elongation and increased root biomass (Yakhin et al., 2017; Hernández-Herrera et al., 2014). Microalgal extracts, particularly those containing polysaccharides, have been shown to foster the growth of agricultural crops (Ronga et al., 2019; Garcia-Gonzalez and Sommerfeld, 2016). Sugars function as both potent signaling agents and direct precursors for intermediary metabolism, thereby facilitating plant growth and development (Zheng et al., 2016). The presence of macro- and micronutrients, vitamins, and phytohormones in algal extracts may also contribute to this process (Garcia-Gonzalez and Sommerfeld, 2016; Ghaderiardakani et al., 2019). For example, studies have indicated that the potassium in seaweed extracts can positively modulate the water relations of the treated plants, enhance the photosynthetic activity, and promote meristematic growth (Hernández-Herrera et al., 2014). The superior promotion of corolla diameter, DW, and FW for A. ramosum flowers as a result of B+S application as compared to C+S is possibly because the combination of a variety of microorganisms in the compound microbial fertilizer with the active components in the algal extract enhances the biological activities of both. The growth hormones and trace elements in algal extracts can promote the growth and metabolism of microorganisms, which can further decompose and transform the nutrients in the algal extract, rendering them more easily absorbed by plants.

Allium plants are consumed globally as food, and their impact on human health is significant because of their rich content of bioactive compounds, with antioxidant, anti-inflammatory, and anticancer activities (Yang et al., 2023). In a study conducted by Pourzand et al. (2016), the authors reported the consumption of certain Allium plants in association with a reduced risk and incidence of breast cancer. The abundance of phytochemicals in Allium vegetables has meant that their health benefits have been fully explored, holding promise for the development of new foods and nutritional supplements.

Our findings indicate significantly higher FRAP, 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt

scavenging rate, DPPH scavenging rate, superoxide anion radical scavenging rate, and HRSC for A. ramosum flowers treated with C+A, C+S, B+A, and B+S as compared to CK ( Figures 1A–E ). Notably, the SASR was significantly higher for the B+A treatment than the other treatments, whereas the FRAP, ABTS scavenging rate, DPPH scavenging rate, and HRSC were significantly higher for the B+S treatment ( Figures 1A–E ).

The antioxidant properties of garlic are closely associated with its phenolic composition. Kumar and Kumar (2023) found that elevated concentrations of polyphenols led to enhanced antioxidant efficacy in various plant tissues in the Allium species, including the leaves, roots, bulbs, and pericarps. Concurrently, Mollica et al. (2018) revealed that the phenolic constituents of Allium scoroprasum L. floral extracts are abundant, conferring potent antioxidant capabilities and functioning as effective metal chelators and free radical scavengers (as evidenced by the DPPH and ABTS scavenging rates). Dai et al. (2023) highlighted the pivotal role of phenolic compounds and ascorbic acid in the antioxidant mechanism of leeks. Flavonoids, which are prevalent across a wide array of plant species, are integral to the antioxidant, anticancer, antimicrobial, and antimutagenic defenses of a plant and constitute a significant class of antioxidants synthesized by plants for their survival (Treutter, 2006; Ghasemzadeh et al., 2010; Sharma et al., 2014).

Correlation analysis indicated that proline content is significantly positively correlated with the DPPH, ABTS, and the superoxide anion radical scavenging rates, whereas polyphenol content is significantly positively correlated with the DPPH and ABTS scavenging rates ( Figure 5 ). These results suggest that an increase in the proline and flavonoid content, among other bioactive substances, may be the reason for the enhanced scavenging and reduction capacity of A. ramosum flowers ( Figures 3A–D ). The antioxidant activity (DPPH scavenging rate, ABTS scavenging rate, FRAP, superoxide anion radical scavenging rate, and HRSC) was significantly correlated with proline, polyphenol, flavonoid, flavone, and TAA contents ( Figures 4 , 7 ). B+A treatment yielded analogous outcomes. This finding provides additional evidence indicating that the enhanced antioxidant capacity of A. ramosum blossoms is likely associated with the augmentation of the bioactive compound content. Interestingly, the DPPH and ABTS scavenging rates, FRAP, superoxide anion radical scavenging rate, and HRSC treatments were significantly higher following B+A and B+S treatment, indicating the possibility of a synergistic effect between compound microbial fertilizer and tri-amino acid fertilizer or algal extracts.

MDA serves as an indicator for the extent of impairment in plant cell membranes (Du et al., 2024). The progressive generation of toxic oxygen species culminates in lipid peroxidation and subsequent deterioration of plant cell membrane integrity, hastening cellular apoptosis (Raju et al., 2020). ROS are noxious by-products of metabolism, in addition to an association with oxidative and nitrosative stressors, all of which exert detrimental effects on plant physiology (Samanta et al., 2024). In the present study, all treatments reduced the levels of MDA and O2• − ( Supplementary Figures S3A, B ) while significantly increasing the POD and SOD contents ( Supplementary Figures S2A, B ). These results align with the conclusions of Al-Karaki and Othman (2023), wherein the utilization of amino acid fertilizers was observed to enhance antioxidant activity in lettuce. However, the results are different from those reported by Zhang et al. (2022), who suggested a non-significant effect for amino acid fertilizers on the SOD content in Trollius chinensis, suggesting a need for additional investigation. However, Ali et al. (2023) observed an increase in POD activity, polyphenol content, and proline content following the application of algal extracts via foliar spray in tomato and sweet pepper plants, and Orhan et al. (2004) highlighted the fact that SOD and POD are principal cellular enzymes that safeguard plants against ROS-induced damage, a notion that concurs with our findings as indicated by correlation analysis ( Figures 4 , 5 ). Treatment with B + A and B + S had a significant effect on the POD and SOD contents in A. ramosum flowers, with MDA negatively correlated with POD and O2• − production negatively correlated with SOD.

The nutritional attributes of vegetables are instrumental in safeguarding their quality and guaranteeing their nutritional interity (Kathi et al., 2024; Wang et al., 2021). The data obtained in our investigation revealed that all studied treatments led to a marked enhancement in the concentrations of GSH, TAA, soluble sugars, SP, proline, polyphenols, flavones, and flavonoids in leeks as compared with CK ( Figures 2A–D , 3A–D ). GSH, an important non-enzymatic antioxidant, plays a crucial role in plants by clearing ROS and regulating the cellular redox balance. Studies have shown that GSH can directly react with ROS (O2• −) to form complexes, thereby reducing the oxidative damage (Hasanuzzaman et al., 2017). In addition, GSH can maintain the reduced state of other antioxidants (such as vitamins C and E) and indirectly protect cell membranes (Foyer and Noctor, 2005). These studies indicate that GSH plays a crucial role in plant growth and development (E et al., 2024). Numerous investigations have revealed that the incorporation of amino acid-derived biostimulants can exert a favorable influence on the proline content, flavone content, and antioxidant potential (Parađiković et al., 2011; Pourzand et al., 2016; Shafie et al., 2021). For instance, the administration of diverse biostimulants, such as algal extracts and amino acids, and protein hydrolysates, has been documented to augment the proline content and total flavonoid content and antioxidant capacity in hydroponically cultivated sweet pepper fruits (Parađiković et al., 2011), Achillea leaves (Shafie et al., 2021), tomato fruits (Caruso et al., 2019), and lettuce leaves (Zhou et al., 2022).

The results of these studies suggest that the application of biostimulants is a good strategy for increasing the yield of nutritious vegetable crops with lower environmental impact (Parađiković et al., 2011; Shafie et al., 2021). It is noteworthy that GSH, TAA, soluble sugar, SP, proline, polyphenol, flavone, and flavonoid contents were significantly increased under B+A and B+S-treatment ( Figures 3A–D , 4A–D , 4 , 5 ). According to previous studies, the accumulation of proline in plants aids in maintaining cellular osmotic equilibrium, which in turn enables plants to withstand various environmental challenges, such as drought, salt-induced stress, and cold temperatures (Wang et al., 2020). Proline has been found to alleviate oxidative stress in plants by modulating the activity and expression of antioxidant enzymes and acting as a scavenger for ROS (Sehar et al., 2021). For example, proline accumulation can enhance the antioxidant defense systems in wheat seeds (Sehar et al., 2021) and stabilize protein structures (Szabados and Savouré, 2010). These studies indicate that proline plays a multifaceted role in plant growth. However, proline accumulation is not isolated, and is closely related to the metabolism of free amino acids content. Free amino acids content are important nitrogen sources in plants and participate in various physiological processes, such as protein and hormone synthesis.

The types and content of free amino acids content differ in different plants under various conditions (Sun et al., 2017; Moore et al., 2022). Studies have shown the types and contents of free amino acids in plants can undergo significant changes to adapt to new physiological requirements when a plant is placed under adverse stress (Hanif et al., 2021). These changes can affect the growth, development, and antioxidant capacity of plants. Furthermore, free amino acids serve as substrates for antioxidants and participate in ROS scavenging to alleviate oxidative damage (Choińska et al., 2022).

The results of our study demonstrate that the proline and free amino acid contents were markedly elevated in leek plants subjected to all treatments as compared to the control ( Supplementary Figures S4A, B ). In particular, a substantial increase was observed in the proline and free amino acid content for the B + A and B + S treatment groups as compared to the C+A or C+S treatment groups ( Supplementary Figures S4A, B ; Figures 4 , 5 ).

Among the seven metabolites in the third subclass, Diallyl Disulphide, Disulfide, methyl 2-propenyl, and Sulfide, allyl methyl are metabolites associated with oxidation during the aging process. Studies have shown that Diallyl Disulphide can induce the expression of antioxidant enzymes by regulating the Nrf2-ARE signaling pathway, thereby protecting cells from oxidative stress. This suggests that Diallyl Disulphide may exert an indirect antioxidant effect by regulating the antioxidant defense mechanism in plants (Shang et al., 2019). Disulfide, methyl 2-propenyl is a natural sulfur-containing compound in garlic with antioxidant activity. Like other sulfur-containing compounds, Disulfide, methyl 2-propenyl can scavenge ROS, protecting cells from oxidative damage (Vega-Hissi et al., 2019). Therefore, we speculate that Diallyl Disulphide, Disulfide, methyl 2-propenyl, and Sulfide, allyl methyl are related to the increased 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) scavenging rate, DPPH scavenging rate, and FRAP content, as well as the decreased MDA content and O2• − content in the B+S and B+A samples.