Secondary metabolites in plants, including phenolic compounds and flavonoids, are produced through defense mechanisms against biotic and abiotic stresses (Hartmann, 2007; Zandalinas et al., 2017). These are known to have diverse physical and chemical traits, such as color, flavor, and medicinal effects, leading to high economic value (Pagare et al., 2015). Legume crops, such as soybean, mungbean (Vigna radiata L.), and pigeonpea (Cajanus cajan), were reported to contain large amounts of functional substances, including catechin, neo/chlorogenic acid, and iso/vitexin (Fu et al., 2008; Cao et al., 2011). Catechin and chlorogenic acid have been reported to have protective effects against colorectal cancers, reducing the incidence by 77–83% in humans (Lu et al., 2022). Vitexin also provides many health benefits including antioxidant, anti-inflammatory, antinociceptive, cardioprotective, and neuroprotective effects (He et al., 2016).

Mungbeans, one of the major legume crops in Asia, are cultivated on over six million hectares worldwide, with an annual grain production of three million tons (Gayacharan et al., 2020). Because of their high carbohydrate content (50–60%) and protein content (20–24%), mungbeans are an important nutritional source in many developing countries and are mostly consumed as starch grains or germinated sprouts (Peng et al., 2008; Tang et al., 2014). Ethanol extracts of mungbean seeds contain high levels of phenolic compounds, such as catechin, chlorogenic acid, and vitexin (An et al., 2020). Germination increases the nutritional value of the seeds (Hung et al., 2012). Germination can improve the functional and nutritional quality of legumes by increasing protein digestibility and reducing anti-nutritional factors, and germinated legumes can be consumed as protein supplements with functional agents (Kartikeyan et al., 2022). In mungbeans, the contents of functional substances, including carotenoids, vitamin E, and various phenolic compounds, increase after germination (Li et al., 2021). Mungbean sprouts grow rapidly indoors without being affected by the weather. Ethanol extracts of mungbean sprouts were reported to contain higher amounts of phenolic compounds than the seeds, even when compared with other legume crops (Peng et al., 2008). Therefore, mungbean sprouts are a healthy food that is easy to produce and can efficiently supply functional substances to the human diet.

The secondary metabolite contents in soybeans vary depending on the cultivar, cultivation period, and tissue (Yun et al., 2020). In cauliflowers (Brassica aleracea L. ssp. botrytis), secondary metabolites, such as anthocyanins, carotenoids, and phenolic compounds, were reported to vary according to genotypes with different floret colors (Park et al., 2013). Although the contents of secondary metabolites in mungbeans can vary depending on the cultivation period, genotype, and environmental conditions, these studies only examined a few major cultivars (Guo et al., 2012; An et al., 2020). Therefore, we hypothesized that the secondary metabolites would vary significantly among the 50 different mungbean genotypes and they would be regulated in the level of transcription. In this study, the metabolic profiling of 50 mungbean genotypes of diverse origins and phenotypes, including wild species, was performed using ultra-high-performance liquid chromatography (UPLC). Key genetic factors underlying the biosynthesis of secondary metabolites were identified. This study provides new insights into the value of mungbean sprouts as a nutritional resource with high functional substance contents.

Legume crops have high nutritional and health benefits and can produce high yields over relatively short cultivation periods, which are important traits for food crops (Iqbal et al., 2006; Rebello et al., 2014). Legume crops contain high amounts of secondary metabolites, including flavonoids and isoflavones, which can positively and negatively affect food quality. On the positive side, secondary metabolites have antioxidant, anti-inflammatory, and anti-cancer effects in humans (Ali Reza et al., 2021). However, some secondary metabolites can lower the food quality, taste bitter, decrease legume storage capacity, and discolor food (Oz et al., 2017). In this study, 20 secondary metabolites were profiled in 50 mungbean genotypes using UPLC to provide useful information for improving the food quality of mungbean sprouts.

Neochlorogenic acid was the most abundant component in most genotypes, followed by chlorogenic acid and catechin identified in the HCA ( Figure 1A ), whereas chlorogenic acid, catechin, and neochlorogenic acid were the major components in the PCA ( Figure 1B ). Although neochlorogenic acid was the most abundant secondary metabolite, it had less of an effect on clustering in the PCA because catechin and chlorogenic acid had higher variances among the genotypes ( Figure 2 ). Among the 20 functional substances, the chlorogenic acid and catechin contents showed the greatest variation, suggesting that these two compounds could be the first targets of genetic engineering or molecular breeding to improve the nutritional value of mungbean sprouts.

The mungbean reference genotype VC1973A (genotype 45) mungbean contained the highest amount of gallic acid ( Figure 2 ). Gallic acid is known to have effects of antioxidant, anti-inflammatory and neuroprotective capacity, that are beneficial for human health (Kroes et al., 1992; Lu et al., 2006). However, VC1973A had relatively lower contents of most compounds than the other genotypes. Commercial strawberry (Fragaria × ananassa) cultivars have lower anthocyanin contents and diversity than non-commercial cultivars and wild types, which is one of the problems to be addressed in breeding programs (Dzhanfezova et al., 2020). Wild mungbeans (genotype no. 36–42) had relatively higher isoflavone contents, including daidzein, genistin, and glycitin, than other genotypes, including VC1973A ( Figure 2 ). Isoflavones have physiological functions, such as phytoestrogens that have beneficial effects on bone health and reduce menopausal symptoms and the risk of breast cancer (Chen and Chen, 2021). Thus, the results of this study suggest that wild mungbeans can be a useful resource for producing functional substances that promote the nutritional value of mungbean sprouts.

Among the secondary metabolites identified in the mungbean sprouts, isoflavones, including daidzein, formononetin, genistin, glycitein, and glycitin, showed relatively high positive correlations ( Figure 3 ). The expression levels of transcripts encoding key enzymes in the biosynthetic pathways of isoflavones were measured using qRT-PCR ( Figure 4 ). Positive correlations were identified between secondary metabolite contents and the expression of genes that catalyze the biosynthetic pathways of secondary metabolites, such as IFS and daidzein, and IF7GT and daidzin. Soybean IFS has been shown to catalyze the biosynthesis of daidzein and genistein, which are the precursors of daidzin and genistin in rapeseed (Li et al., 2011). In contrast, negative correlations were detected between the formononetin content and IF7GT expression levels, where formononetin is catalyzed by formononetin 7-O glucoside by IF7GT. In soybeans, the aglycosylation of isoflavones decreased as the expression of IF7GT decreased (Gupta et al., 2017). A negative correlation was reported between formononetin contents and expression level of IF7GT because IF7GT is associated with the aglycosylation of formononetin (De Vega et al., 2015). The catechin contents and expression levels of F3’5’H, which catalyzes the other side of the biosynthetic pathway of dihydroquercetin, a precursor of catechin, also had a negative correlation ( Figure 4 ). F3’5’H had been reported to catalyze the biosynthesis of dihydromyricetin from dihydroquercetin in grape (Vitis vinifera) and tea plant (Camellia sinensis) (Jeong et al., 2006; Zhou et al., 2016). Within the pathway, the biosynthesis of delphinidin-based anthocyanins and cyanidin-based anthocyanins increased and decreased, respectively, as the expression level of F3’5’H increased. When dihydroquercetin, a precursor of leucocyanidin, was synthesized into dihydromyricetin by F3’5’H, the contents of leucocyanidin decreased. Thus, in the current study, when the expression level of F3’5’H increased, the leucocyanidin contents decreased, which is a precursor of catechin, resulting in a negative correlation between the catechin contents and expression of F3’5’H. In summary, significant correlations were identified between the contents of secondary metabolites and the expression levels of the genes catalyzing the biosynthetic pathways of these metabolites, indicating that the secondary metabolite contents are regulated at the level of the transcription of key genes in their pathways in mungbeans. These results suggest that functional substance content can be regulated by manipulating target genes for molecular breeding or genetic engineering.

In this study, metabolic profiling and transcriptome analysis were conducted using UPLC and qRT-PCR, respectively, on 50 mungbean genotypes of different origins. The key genes involved in the biosynthesis of secondary metabolites with beneficial effects on human health were identified in the mungbean sprouts. We found that wild mungbeans contained significantly higher levels of isoflavones, such as daidzin, genistin, and glycitin, than cultivated mungbeans. The chlorogenic acid and catechin contents showed the highest variance among the genotypes. A strong positive correlation was detected among the isoflavone groups in which the mainstream biosynthetic pathways were regulated by the same key enzymes ( Figure 4 ; Supplementary Figure 2 ). The results of this study will help enable improving the nutritional value of mungbean sprouts containing functional substances for desired purposes while reducing secondary metabolites that negatively impact food quality.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

BK designed the experiments and wrote the manuscript. BK and IL collected the material data and conducted experiments. BK performed the qualifying and quantifying of the metabolites and analyzed the transcriptomic results. IL contributed the transcriptomic results analyse. JH supervised and revised the manuscript. All authors contributed to the article and approved the submitted version.