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 β-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 β-glucogallin accumulation,
respectively. Taken together, we have provided an overview of walnut
fruit development and uncovered genes involved in HT biosynthesis.

核桃（Juglans regia L.）是一类坚果营养丰富的重要果树。本研究对核桃果实发育过程进行了监测，收集了5个发育阶段的9份生物学样本，并通过转录组学与代谢组学分析手段开展检测。成熟果实果皮中富集大量酚类代谢物，而脂质、碳水化合物、氨基酸及其衍生物则主要积累于种仁中。脂肪酸生物合成起始于授粉后13周，核桃外果皮或可进行光合作用。通过转录组与代谢组的共表达分析，本研究预测了部分代谢通路的功能基因。针对可水解单宁（hydrolysable tannins, HTs）的生物合成途径，筛选出3个编码莽草酸脱氢酶（shikimate dehydrogenase, SDHs）的基因，其可将3-脱氢莽草酸转化为没食子酸（gallic acid, GA）；以及4个编码UDP-糖基转移酶（UDP-glycosyltransferase, UGT）的基因，其可在该途径中将GA转化为β-没食子酰葡萄糖，用于功能验证。随后将这3个SDH基因在大肠杆菌（Escherichia coli）中进行异源表达，其重组蛋白均表现出GA生成活性。此外，在杨树毛状根中异源表达这3个SDH基因与4个UGT基因后，GA与β-没食子酰葡萄糖的积累量分别出现显著提升。综上，本研究系统梳理了核桃果实的发育进程，并揭示了参与可水解单宁生物合成的功能基因。