2.3.1. Metabolite extraction.

Mung bean samples were ground using a tissue grinder and passed through a 100-mesh sieve. The extraction procedure was adapted from previously reported methods [20,21]. Briefly, 20 mg of homogenized mung bean powder was mixed with 1000 μL of extraction solvent (methanol:water = 3:1, v/v). The mixture was vortexed and subjected to grinding at 35 Hz for 4 minutes, followed by sonication in an ice-water bath for 5 minutes. This process was repeated two to three times. The samples were then incubated at –40 °C for 1 hour and centrifuged at 12,000 rpm for 15 minutes at 4 °C. The supernatants were collected and transferred into autosampler vials for LC-MS/MS analysis. Quality control (QC) samples were prepared by pooling equal aliquots from each sample.

2.3.2. LC-MS/MS analysis.

Mobile phase A consisted of water containing 0.01% acetic acid, while mobile phase B was a mixture of isopropanol and acetonitrile (1:1, v/v). The autosampler tray temperature was set at 4 °C, and the injection volume was 2 μL. The mass spectrometry parameters were as follows: sheath gas flow rate, 50 Arb; auxiliary gas flow rate, 15 Arb; capillary temperature, 320 °C; full MS resolution, 60,000; MS/MS resolution, 15,000; collision energy (stepped), 20/30/40 eV; spray voltage, + 3.8 kV (positive mode) or –3.4 kV (negative mode). Raw data were collected using UPLC-Q-Orbitrap HRMS and processed with Compound Discoverer 3.3 for peak detection, retention time alignment, peak integration, and metabolite identification.

3.2.1. Metabolites and physiological and metabolic characteristics of BL13_1 mung bean samples.

As shown in Table 1, the BL13_1 sample showed significant advantages in metabolite composition, especially in its energy metabolism and environmental adaptability. It was found that the sample had a high L-lactic acid content, reflecting its high metabolic activity in the glycolytic pathway. This characteristic is closely related to the ability of plants to maintain energy supply in low oxygen or anaerobic environments [23]. The accumulation of L-lactic acid as the end product of glycolysis indicates that BL13_1 can effectively maintain energy metabolism under hypoxic conditions, thus supporting plant growth.In addition, the relatively high content of hydroxymeglutarate in the BL13_1 sample further reveals its high metabolic activity in the Mevalonate pathway. This pathway is a key pathway for the synthesis of membrane lipids and secondary metabolites, and is closely related to membrane stability and the generation of signal molecules [24]. This metabolic characteristic suggests that BL13_1 has significant advantages in maintaining cell membrane structure and promoting signal transduction.

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Table 1. Relative quantitative comparison of key differential metabolites between GLD07_03 and five mung bean varieties.

https://doi.org/10.1371/journal.pone.0327962.t001

In terms of plant hormone metabolism, the high levels of dinoprostol and deoxycortisone in BL13_1 samples suggest their potential advantages in growth and development and regulation of environmental responses. Plant hormones play an important role in regulating plant growth, differentiation and coping with environmental stress [25]. Increased levels of these hormones can promote growth and development of plants and improve their resilience in the face of biological and abiotic stresses. In addition, the up-regulation of L-asparagine content further indicates the activity of BL13_1 in the pathway of nitrogen metabolism and protein synthesis. Nitrogen metabolism plays a crucial role in plant growth, amino acid synthesis and overall protein construction [26]. This metabolic characteristic enables BL13_1 to maintain its growth advantage under the condition of nutrient scarcity, indicating that BL13_1 has an efficient metabolic regulation ability in a limited resource environment.

In summary, BL13_1 showed excellent metabolic characteristics in energy metabolism, lipid synthesis and environmental adaptability. The combination of its key metabolic components, such as L-lactic acid, hydroxymethylglutaric acid, dinoprost, deoxycortisone and L-asparagine, allows it to show good adaptability in a variable and challenging growing environment.

3.2.2. Metabolites and physiological metabolic characteristics of NL2_1 mung bean samples.

NL2_1 samples showed significant advantages in the accumulation of nucleosides, amino acids, and flavonoid metabolites (Table 1). Increased levels of nucleosides (such as nicotinamide adenine dinucleotide and uridine 5’ -diphosphate) indicate that NL2_1 is active in energy production and metabolic regulation. These nucleosides are important cofactors in energy metabolism in cells and are involved in a variety of metabolic pathways, such as REDOX reactions and nucleotide biosynthesis [27].At the same time, the upregulation of amino acids (such as L-aspartic acid, D-alanine, L-serine) indicates that NL2_1 has high metabolic activity in nitrogen metabolism and protein synthesis. Amino acids are not only the basic building blocks of proteins, but also play an important role in cell signaling and metabolic regulation [28]. This metabolic characteristic enables NL2_1 to support rapid growth and efficient use of nitrogen sources in resource-limited conditions, thereby improving its environmental adaptability. The up-regulation of flavonoids such as quercetin, genistein and astilbin further enhanced the antioxidant capacity of NL2_1. These compounds reduce cell damage by clearing reactive oxygen species (ROS), thereby improving their survival in stressed environments [29].Flavonoids are an important part of the plant antioxidant defense system, which can effectively protect cells from oxidative stress and maintain the stability of cell function. In addition, the high content of glycerophospholipids (such as glycerophosphoryl choline) suggests that NL2_1 has advantages in maintaining membrane stability and signaling. Glycerophospholipids are an important part of cell membranes, which participate in membrane fluidity and signal molecule synthesis, and help plants maintain physiological homeostasis in complex environments [30]. This glycerophospholipid rich property enables NL2_1 to maintain membrane integrity and functional stability under environmental changes, thereby enhancing its adaptability under stress conditions.

The metabolic characteristics of NL2_1 in nucleoside, amino acid and flavonoid metabolites, combined with its advantages in glycerophospholipid synthesis, make it show outstanding physiological characteristics in terms of rapid growth and environmental adaptation. This metabolic combination not only enhances its antioxidant capacity, but also supports its survival and development in variable environments by improving energy production and maintaining cell membrane homeostasis.

3.2.3. Metabolites and physiological and metabolic characteristics of NL4_1 mung bean samples.

As shown in Table 1, NL4_1 samples showed significant advantages in the accumulation of organic acids, amino acids and flavonoid metabolites. The abundance of organic acids (such as succinic acid and L-malic acid) indicates that NL4_1 samples have a high activity in the TCA cycle. TCA cycle is the core pathway of plant respiration and energy generation, and high levels of succinic acid and L-malic acid indicate that NL4_1 has significant energy metabolism ability in a growing environment with high energy demand [31]. Increased levels of amino acids (such as L-aspartate and D-alanine) further support their metabolic activity in nitrogen metabolism and protein synthesis [28]. By increasing the content of these amino acids, NL4_1 sample enhanced its adaptability and growth ability under high metabolic requirements. The accumulation of flavonoids, such as epicatechin and astilbin, provides NL4_1 with potent antioxidant capacity. Flavonoids act as natural antioxidants in plants, clearing ROS, reducing oxidative damage of cells, and protecting cell structure and function [29]. NL4_1 shows significant advantages in antioxidant defense mechanisms by increasing the concentration of these compounds, thereby improving its survival and stability in stressed environments.

NL4_1 samples demonstrated advantages in energy production and antioxidant defense by enhancing the accumulation of organic acids, amino acids and flavonoid metabolites. The high activity of the TCA cycle and glycolytic pathway provides it with sufficient energy to support rapid growth and cope with environmental stress. In addition, the upregulation of flavonoid metabolites enhances its antioxidant defenses, making it more adaptable to high metabolic demands in complex environments. These metabolic characteristics provide potential application value for NL4_1 in future breeding and functional component research.

3.2.4. Metabolites and physiological and metabolic characteristics of T1114111_1 mung bean samples.

In this study, the metabolic characteristics of mung bean sample T1114111_1 were analyzed in detail, and it was found that the contents of fatty acids, amino acids, organic acids and flavonoid metabolites in the sample were significantly increased (Table 1). This up-regulation of metabolite content is closely related to its advantages in energy production, cell stability, and antioxidant defense.

First, the high levels of L-malic acid, succinic acid and malonic acid detected in the samples indicate that they participate in the active metabolic process of the TCA cycle, which provides stable energy support for the rapid growth and adaptability of plants [31]. As the core pathway of cell metabolism, the tricarboxylic acid cycle meets the requirements of cell biosynthesis and energy requirements by providing ATP and intermediate metabolites [32]. Second, increased levels of flavonoids (such as epicatechin, geranin, and kaempferol) and aromatic compounds (such as gallic acid and 4-hydroxybenzoic acid) in the T1114111_1 sample helped to play a key role in antioxidant defense by clearing excess ROS, thereby reducing the damage caused by cellular oxidative stress. Maintain normal cell function [29].Some studies have pointed out that the antioxidant properties of flavonoids can not only enhance the physiological defense ability of plants, but also have a potential protective effect in response to environmental stress [33]. In addition, the high content of fatty acids and glycerophospholipid metabolites in the samples further enhanced the stability and signaling ability of the cell membrane. These metabolites play a particularly important role in biological and abiotic stress, maintaining the integrity of cell structure and regulating transmembrane signaling to promote adaptive responses [34]. Such enhanced stability and signal transduction provide plants with stronger tolerance and resilience in the face of abiotic stresses such as drought and high temperature [35]. Finally, the high content of alkaloid metabolites in the T1114111_1 sample, especially trigonelline, provides additional antimicrobial and anti-inflammatory defense functions. This alkaloid further improves the overall stress resistance of plants by inhibiting the growth of pathogenic microorganisms and reducing inflammatory responses [36]. The antibacterial properties of trigonelline in various plants have been extensively studied, and its potential in plant disease prevention and control cannot be ignored [37].

In summary, the comprehensive action of various metabolites in the sample of T1114111_1 mung bean makes it show significant advantages in energy metabolism, cell stability and antioxidant defense. This provides a solid metabolic basis for mung bean’s adaptability under different stress conditions, and provides a valuable research direction for improving plant stress resistance through metabolite regulation in the future.

3.2.5. Metabolites and physiological and metabolic characteristics of JL13_1 mung bean samples.

Analysis of JL13_1 mung bean samples revealed its metabolic characteristics, in particular the abundance of amino acids (such as D-proline, L-asparagine) and fatty acid metabolites (such as (7Z,10Z)-7, 10-hexadecadienoic acid) (Table 1). The high content of these metabolites indicates that the sample has significant advantages in nitrogen metabolism, osmotic regulation and membrane stability [28, 34].

As an osmoprotective agent, proline plays a key role in plant response to abiotic stresses such as drought and salt stress. It stabilizes protein structure, protects cells from dehydration damage, and helps maintain cellular water balance [38]. The content of D-proline in JL13_1 samples is prominent, which indicates that JL13_1 has strong adaptability in osmoregulation, which helps plants maintain growth activity in extreme environment. L-asparagine is another amino acid that is significantly present in JL13_1 samples and plays an important role in nitrogen storage and transport. Under the condition of limited nitrogen in plants, asparagine can promote nitrogen redistribution, thereby maintaining metabolic activities and growth [39]. This characteristic enables JL13_1 sample to maintain a stable physiological and metabolic state under the condition of nutrient limitation. The presence of fatty acids such as (7Z,10Z)-7, 10-hexadecadienoic acid in JL13_1 samples suggests that it has advantages in the construction and stability of cell membranes. Fatty acids play a key role in maintaining membrane structural integrity and fluidity, which is critical for responding to temperature fluctuations and changes in osmotic pressure [34]. The abundance of fatty acid metabolites helps to improve the adaptability of cell membranes and ensure that cells maintain homeostasis in complex environments.

Overall, the metabolic characteristics of JL13_1 samples reflect the physiological advantages of JL13_1 in coping with environmental stress. The high content of amino acids and fatty acids supports its advantages in osmoregulation, nitrogen metabolism and membrane stability, making it potentially useful in plant breeding.

3.2.6. Metabolites and physiological metabolic characteristics of GLD07_03 mung bean samples.

GLD07_03 Mung bean samples showed significant advantages in defense and stress resistance metabolites, and its metabolic characteristics were mainly reflected in the enrichment of defense-related metabolites such as lignans (such as 3-(3,4,5-trimethoxyphenyl)-2-allyl-1-ol, 4-hydroxycinnamic acid, terpenoid lactones (such as salicylate) and methyl salicylate (Table 1). These compounds are produced mainly through the phenylalanine pathway, giving GLD07_03 sample powerful antibacterial and antioxidant functions, contributing to its self-protection ability under pathogen invasion and environmental stress [40].

Lignans play an important role in plant defense, and their antibacterial and antioxidant properties have been confirmed in various studies [41]. GLD07_03 samples rich in 3-(3,4, 5-trimethoxyphenyl) −2-allyl-1-ol and 4-hydroxycinnamic acid can not only directly inhibit the growth of pathogens, but also reduce the accumulation of ROS under stress conditions by inducing antioxidant enzyme activity, and protect cells from oxidative damage. Terpenolactones such as salviolactone also play an important role in plant response to pests and abiotic stresses. They can improve the defense capability of plants by inhibiting the biosynthetic pathway of pathogens and promoting the strengthening of cell walls [42]. As a key signaling molecule, methyl salicylate plays a central role in activating systemic acquired resistance (SAR) [43]. SAR is a whole-plant immune mechanism that can enhance plant resistance to broad-spectrum pathogens through signal transduction pathways [44]. The high content of methyl salicylate in GLD07_03 samples further proves that GLD07_03 has strong signal transmission and defense response ability. The high content of carbohydrate and nucleoside metabolites in GLD07_03 samples, such as colocin, maltohexose and adenosine diphosphate, provided energy reserve and osmoregulation functions under stress. Carbohydrate metabolites not only play an important role in the regulation of cell osmosis, but also serve as carbon sources to support physiological activities of plants under stress conditions [45]. As a nucleoside metabolite, adenosine diphosphate is involved in energy metabolism and signal transmission, supporting plants to maintain metabolic activity under adverse conditions [46].

Overall, GLD07_03 mung bean samples showed significant physiological adaptability to pests and environmental stresses through accumulation of a range of defense and resistance metabolites. This not only revealed the ecological adaptation strategy of GLD07_03, but also provided a scientific basis and application prospect for the study of stress resistance and the development of functional components of mung bean.