Metabolomic and Transcriptomic Analyses of Flavonoid Biosynthesis in Different Colors of Soybean Seed Coats

Abstract

Soybean has outstanding nutritional and medicinal value because of its abundant protein, oil, and flavonoid contents. This crop has rich seed coat colors, such as yellow, green, black, brown, and red, as well as bicolor variants. However, there are limited reports on the synthesis of flavonoids in the soybean seed coats of different colors. Thus, the seed coat metabolomes and transcriptomes of five soybean germplasms with yellow (S141), red (S26), brown (S62), green (S100), and black (S124) seed coats were measured. In this study, 1645 metabolites were detected in the soybean seed coat, including 426 flavonoid compounds. The flavonoids differed among the different-colored seed coats of soybean germplasms, and flavonoids were distributed in all varieties. Procyanidins A1, B1, B6, C1, and B2, cyanidin 3-O-(6″-malonyl-arabinoside), petunidin 3-(6″-p-coumaryl-glucoside) 5-glucoside, and malvidin 3-laminaribioside were significantly upregulated in S26_vs._S141, S62_vs._S141, S100_vs._S141, and S124_vs._S141 groups, with a variation of 1.43-2.97 × 10¹³ in terms of fold. The differences in the contents of cyanidin 3-O-(6″-malonyl-arabinoside) and proanthocyanidin A1 relate to the seed coat color differences of red soybean. Malvidin 3-laminaribioside, petunidin 3-(6″-p-coumaryl-glucoside) 5-glucoside, cyanidin 3-O-(6″-malonyl-arabinoside), and proanthocyanidin A1 affect the color of black soybean. The difference in the contents of procyanidin B1 and malvidin 3-glucoside-4-vinylphenol might be related to the seed coat color differences of brown soybeans. Cyanidin 3-gentiobioside affects the color of green soybean. The metabolomic-transcriptomic combined analysis showed that flavonoid biosynthesis is the key synthesis pathway for soybean seed color formation. Transcriptome analysis revealed that the upregulation of most flavonoid biosynthesis genes was observed in all groups, except for S62_vs._S141, and promoted flavonoid accumulation. Furthermore, CHS, CHI, DFR, FG3, ANR, FLS, LAR, and UGT88F4 exhibited differential expression in all groups. This study broadens our understanding of the metabolic and transcriptomic changes in soybean seed coats of different colors and provides new insights into developing bioactive substances from soybean seed coats.

Keywords: flavonoid; metabolome; seed coat color; soybean; transcriptome.

Figure 1

Phenotypes of soybean germplasms. S141: yellow seed coat; S26: red seed coat; S62: brown seed coat; S100: green seed coat; S124: black seed coat.

Figure 2

Differentially accumulated metabolites of the metabolome in soybeans with different seed coat colors. (a). Principal component analysis of the metabolites detected in the soybean seed coat using three biological replicates. (b). Numbers of differentially expressed metabolites among S26, S62, S100, and S124. (c–f). Volcano plots of differentially expressed metabolites among S26_vs._S141, S62_vs._S141, S100_vs._S141, and S124_vs._S141. Red dots indicate the upregulation of metabolites, blue dots indicate the downregulation of metabolites, and grey dots indicate that there is no significant difference in the metabolites.

Figure 3

The classification of all the identified metabolites in the soybean seed coat. (a) classification of all the identified metabolites; (b) number of the DAMs.

Figure 4

Analysis of transcriptomics data of soybeans with different seed coat colors. (a) Principal component analysis of the genes detected in the soybean seed coat with three biological replicates. (b) Number of differentially expressed genes among S26, S62, S100, and S124. (c–f). Volcano plots of differentially expressed genes among S26_vs._S141, S62_vs._S141, S100_vs._S141, and S124_vs._S141. Red dots indicate the upregulation of gene expression, green dots indicate the downregulation of gene expression, and grey dots indicate an absence of a significant difference in gene expression.

Figure 5

KEGG enrichment analysis of differentially accumulated metabolites and differentially expressed genes in soybeans with different seed coat colors. (a–d). KEGG enrichment analysis of differentially accumulated metabolites. The x- and y-axes represent the enrichment factor and pathway term, respectively. The colors and sizes of the dots represent the significance and number of metabolites, respectively. (e). KEGG enrichment analysis of differentially expressed genes.

Figure 6

Flavonoid pathway in soybean with different seed coat colors. CHS: chalcone synthase; CHS6: chalcone synthase 6; CHS1: chalcone synthase 1; UGT88F4: UDP-glycosyltransferase 88F4; FTH: Naringenin,2-oxoglutarate 3-dioxygenase (fragment); DFR: bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase; ANR: anthocyanidin reductase ((2S)-flavan-3-ol-forming), LAR: leucoanthocyanidin reductase. The colored rectangle represents the up- or downregulation of S141, S26, S62, S100, and S124, respectively, and the red rectangle represents a high FPKM value compared with the blue rectangle.

Figure 7

qRT-PCR detection of 6 key differentially expressed genes. The x-axis represents the relative expression of samples in qRT-PCR, and the y-axis represents the FPKM value in transcriptomics. The error bars indicate the SDs of three biological replicates. SoyZH13 14G066200 (DFR); SoyZH13 02G147700 (DFR); SoyZH13 01G196800 (ANT17); SoyZH13 08G058800 (ANR); SoyZH13 02G046100 (FHT); SoyZH13 20G171400 (LAR).