Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant Nicotiana attenuata. Despite their ecological importance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Here, we show that three cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, and NaCYP716E107) from N. attenuata oxidize the pentacyclic triterpene skeleton, as evidenced by heterologous expression in Nicotiana benthamiana. NaCYP716A419 catalyzed a consecutive three-step oxidation reaction at the C28 position of β-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2α position of β-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6β position of β-amyrin/oleanolic acid. The genes encoding these three CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata, suggesting the ecological significance of these specialized metabolite biosynthetic steps.

中文翻译：

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细胞色素 P450 单加氧酶在三萜生物合成中的作用及其对生长发育的潜在影响


五环三萜类化合物以其天然生物活性而闻名，在生态模型植物烟草中表现出复杂的时空积累模式。尽管它们具有生态重要性，但减毒猪笼草中三萜类化合物合成的潜在生物合成酶和功能属性仍未被探索。在这里，我们发现来自减毒烟草的三种细胞色素 P450 单加氧酶（NaCYP716A419、NaCYP716C87 和 NaCYP716E107）氧化五环三萜骨架，如本塞姆氏烟草中的异源表达所证明。 NaCYP716A419在β-香树脂醇/羽扇豆醇/羽扇豆二醇的C28位上催化连续的三步氧化反应，产生相应的醇、醛和羧酸。 NaCYP716C87 羟基化 β-香树脂醇/羽扇豆醇/羽扇豆二醇/高根二醇/齐墩果酸/桦木酸的 C2α 位点，而 NaCYP716E107 羟基化 β-香树醇/齐墩果酸的 C6β 位点。编码这三种 CYP716 酶的基因在花中高度表达，并对 ABA、MeJA、SA、GA3 和非生物胁迫处理的诱导作出反应。利用 VIGS 技术，我们发现 NaCYP716A419 的沉默会影响 N. attenuata 的生长和繁殖，表明这些特殊代谢物生物合成步骤的生态意义。