Transcriptomic and Metabolic Analysis of Fruit Development and Identification of Genes Involved in Raffinose and Hydrolysable Tannin Biosynthesis in Walnuts

Walnut (Juglans regia L.) is an important fruit tree with high nutrition in its nuts. Here, the development of walnut fruits was monitored, and nine biological samples at five developmental stages were collected and analyzed by transcriptomic and metabolic assays. Many phenolic metabolites accumulated in the peel of mature fruits, while lipids, carbohydrates, and amino acids and their derivatives mainly accumulated in the kernel. Fatty acid biosynthesis occurred at 13 weeks after pollination, and photosynthesis might occur in the exocarp of walnuts. By coexpression analysis of the transcriptome and metabolome, genes responsible for some metabolic pathways were predicted. Three genes encoding shikimate dehydrogenases (SDHs) that convert 3-dehydroshikimic acid to gallic acid (GA) and four genes encoding UDP-glycosyltransferase (UGT) that convert GA to beta-glucogallin in the biosynthesis of hydrolysable tannins (HTs) were selected for functional confirmation. These three SDH genes were then expressed in Escherichia coli, and their recombinant proteins showed GA formation activity. Moreover, heterologous expression of the three SDH and four UGT genes in poplar hairy roots also showed a significant increase in GA and beta-glucogallin accumulation, respectively. Taken together, we have provided an overview of walnut fruit development and uncovered genes involved in HT biosynthesis.