CYP71P2 regulated by MYB44 modulates oxygenated sesquiterpenoids biosynthesis in Atractylodes lancea

Oxygenated sesquiterpenoids (OSTs) represent a major class of bioactive plant metabolites with significant medicinal value, but most of their biosynthetic pathways and regulatory networks remain unclear. To address this problem, we identified a cytochrome P450 gene, named AlCYP71P2, as a key biosynthetic gene by leveraging a unique high-sesquiterpenoid mutant Atractylodes lancea. Functional characterization in yeast demonstrated that CYP71P2 catalyzes the conversion of β-farnesene to an OST, trans-nerolidol, while the application of exogenous trans-nerolidol feeding increased the accumulation of β-eudesmol, thereby establishing its role as a pivotal biosynthetic precursor for the characteristic OST. We further identified MYB44 as a direct transcriptional repressor of AlCYP71P2 through promoter analysis, yeast one-hybrid screening, and functional validation. Transient overexpression assays also showed that MYB44 suppresses the expression of AlCYP71P2, thereby reducing trans-nerolidol and β-eudesmol levels, while the overexpression of AlCYP71P2 enhanced trans-nerolidol and β-eudesmol biosynthesis. Therefore, this work unveils a core MYB44-CYP71P2 transcriptional repressor module that controls the biosynthesis of OSTs in A. lancea. The current study has developed a rapid, visual transient transformation system based on the RUBY reporter, which enables efficient in planta functional gene characterization, thereby overcoming a major technical bottleneck in non-model medicinal plants. This study also provides a research paradigm for elucidating the synthesis and regulation of bioactive compounds in non-model medicinal plants.