miR5298b regulated taxol biosynthesis by acting on TcNPR3, resulting in an alleviation of the strong inhibition of the TcNPR3-TcTGA6 complex in Taxus chinensis

Taxol, a valuable but rare secondary metabolite of the genus Taxus , is an effective anticancer drug. Understanding the regulation of taxol biosynthesis may provide a means to increase taxol content. The microRNA miR5298b was found to promote the accumulation of taxol and upregulate several taxol biosynthesis genes, including DBAT , TASY , and T5H , as demonstrated by experiments using the overexpression and mimicry of transient leaves. Moreover, miR5298b cleaves the mRNA sequence of TcNPR3 , a homolog of the salicylic acid receptor AtNPR3/4. Overexpression and knockdown by RNA interference of TcNPR3 confirmed that it repressed taxol biosynthesis. These results indicate that miR5298b enhances taxol biosynthesis via the cleavage of TcNPR3 . Yeast two-hybrid bimolecular fluorescence complementation and pull-down assays revealed that TcTGA6, a TGA transcription factor, physically interacted with TcNPR3. Functional experiments showed that TcTGA6 negatively regulates taxol biosynthesis by directly combining with the TGACG motif in the promoters of TASY , T5H , and T10H . TcNPR3 enhances TcTGA6 inhibition Luciferase assays showed that miR5298b alleviated the repression of the TcNPR3–TcTGA6 complex. In summary, miR5298b can cleave TcNPR3 , thereby alleviating the inhibition of the TcNPR3–TcTGA6 complex to upregulate taxol biosynthesis genes.