Schaftoside and isoschaftoside are bioactive natural products widely distributed in higher plants including cereal crops and medicinal herbs. Their biosynthesis may be related with plant defense. However, little is known on the glycosylation biosynthetic pathway of these flavonoid di-C-glycosides with different sugar residues. Herein, we report that the biosynthesis of (iso)schaftosides is sequentially catalyzed by two C-glycosyltransferases (CGTs), i.e., CGTa for C-glucosylation of the 2-hydroxyflavanone aglycone and CGTb for C-arabinosylation of the mono-C-glucoside. The two enzymes of the same plant exhibit high homology but remarkably different sugar acceptor and donor selectivities. A total of 14 CGTa and CGTb enzymes were cloned and characterized from seven dicot and monocot plants, including Scutellaria baicalensis, Glycyrrhiza uralensis, Oryza sativa ssp. japonica, and Zea mays, and the in vivo functions for three enzymes were verified by RNA interference and overexpression. Through transcriptome analysis, we found homologous genes in 119 other plants, indicating this pathway is general for the biosynthesis of (iso)schaftosides. Furthermore, we resolved the crystal structures of five CGTs and realized the functional switch of SbCGTb to SbCGTa by structural analysis and mutagenesis of key amino acids. The CGT enzymes discovered in this paper allow efficient synthesis of (iso)schaftosides, and the general glycosylation pathway presents a platform to study the chemical defense mechanisms of higher plants.

af草苷和异sch草苷是具有生物活性的天然产物，广泛分布于高等植物中，包括谷类作物和草药。它们的生物合成可能与植物防御有关。然而，很少对这些类黄酮二-糖基化生物合成途径已知Ç -glycosides具有不同糖残基。在本文中，我们报道的（异）schaftosides生物合成顺序由两个催化Ç -glycosyltransferases（CGTs），即，CGTA为c ^ 2-hydroxyflavanone苷元和CGTb为-glucosylation Ç的单- -arabinosylation Ç-葡萄糖苷。同一植物的两种酶显示出很高的同源性，但糖受体和供体的选择性却截然不同。从7种双子叶和单子叶植物中克隆并鉴定了14种CGTa和CGTb酶，其中包括黄，，甘草，水稻。粳稻和玉米，并通过RNA干扰和过表达验证了三种酶的体内功能。通过转录组分析，我们在119种其他植物中发现了同源基因，这表明该途径对于（iso）af草苷的生物合成是通用的。此外，我们解析了五个CGT的晶体结构，并通过关键氨基酸的结构分析和诱变实现了SbCGTb向SbCGTa的功能转换。本文中发现的CGT酶可以有效合成（异）沙糖苷，而一般的糖基化途径为研究高等植物的化学防御机制提供了一个平台。