Integrated Metabolome and Transcriptome Analysis Reveals New Insights into the Walnut Seed Coat Coloration

The color of the walnut seed coat is a critical determinant of its market value; however, research into the mechanisms responsible for seed coat color formation is yet to be determined. Using two walnut clones with contrasting pale-yellow and light purple seed coats, we characterized pigmentation, particularly anthocyanin content, using spectrophotometry. We then conducted integrated transcriptomic and metabolomic analyses to identify the molecular mechanisms and pathways underlying their formation. The anthocyanin content in the light purple seed coat clone was significantly greater than that in the clone with a white seed coat. The results of comparative metabolomics indicated that four anthocyanins (delphinidin, cyanidin-3-(caffeoylglucoside), pelargonidin-3-(6″-caffeoylglucoside), and delphinidin-3-O-sophoroside) were significantly more abundant in the light purple seed coat clone. These anthocyanins were the key pigments responsible for the light purple coloration of the walnut seed coat. Furthermore, comparative transcriptomics revealed that structural genes in the anthocyanin biosynthesis pathway (e.g., phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, chalcone isomerase, and bronze-1) were significantly upregulated in the purple seed coat clone. Coexpression network analysis revealed that several transcription factors (e.g., ARF, bHLH, and MYB-related) were significantly correlated with the upregulation of these structural genes and the accumulation of four key anthocyanins. These transcription factors may serve as critical regulators influencing seed coat color formation. In conclusion, these findings establish a strong theoretical foundation for walnut breeding aimed at developing diverse seed coat colors.