Abstract：
Flavonol glycosides, contributing to the health benefits and distinctive flavors of tea (Camellia sinensis), accumulate predominantly as diglycosides and triglycosides in tea leaves. However, the UDP-glycosyltransferases (UGTs) mediating flavonol multiglycosylation remain largely uncharacterized. In this study, we employed an integrated proteomic and metabolomic strategy to identify and characterize key UGTs involved in flavonol triglycoside biosynthesis. The recombinant rCsUGT75AJ1 exhibited flavonoid 4\'-O-glucosyltransferase activity, while rCsUGT75L72 preferentially catalyzed 3-OH glucosylation. Notably, rCsUGT73AC15 displayed substrate promiscuity and regioselectivity, enabling glucosylation of rutin at multiple sites and kaempferol 3-O-rutinoside (K3R) at the 7-OH position. Kinetic analysis revealed rCsUGT73AC15\'s high affinity for rutin (Km = 9.64 μM). Across cultivars, CsUGT73AC15 expression inversely correlated with rutin levels. Moreover, transient CsUGT73AC15 silencing increased rutin and K3R accumulation while decreasing their respective triglycosides in tea plants. This study offers new mechanistic insights into the key roles of UGTs in regulating flavonol triglycosylation in tea plants.
摘要：
黄酮醇苷，有助于茶（山茶）的健康益处和独特的风味，在茶叶中主要以二糖苷和三糖苷的形式积累。然而,介导黄酮醇多糖基化的UDP-糖基转移酶（UGT）在很大程度上仍未表征。在这项研究中,我们采用整合的蛋白质组学和代谢组学策略来鉴定和表征黄酮醇三糖苷生物合成中涉及的关键UGT。重组rCsUGT75AJ1表现出类黄酮4'-O-葡萄糖基转移酶活性，而rCsUGT75L72优先催化3-OH糖基化。值得注意的是,rCsUGT73AC15显示底物混杂性和区域选择性，在多个位点实现芦丁的糖基化，在7-OH位置实现山奈酚3-O-rutinoside（K3R）的糖基化。动力学分析显示rCsUGT73AC15对芦丁的高亲和力(Km=9.64μM)。跨品种，CsUGT73AC15表达与芦丁水平呈负相关。此外，瞬时CsUGT73AC15沉默增加了茶树中的芦丁和K3R积累，同时减少了它们各自的三糖苷。这项研究为UGTs在调节茶树中黄酮醇三糖基化中的关键作用提供了新的机制见解。
