Molecular insights into ThMYB14-mediated flavonoid accumulation in Tetrastigma hemsleyanum Diels et Gilg in response to water stress

Tetrastigma hemsleyanum Diels et Gilg ( T. hemsleyanum ) is a prized Chinese medicinal plant renowned for its numerous health advantages. It has gained significant recognition in China for both its medicinal and economic importance. Our earlier research has demonstrated that flavonoids, the primary bioactive components, accumulated efficiently in T. hemsleyanum during water stress. Although R2R3 MYB transcription factors have been implicated in regulating flavonoid biosynthesis, the specific mechanisms remain unclear. Here, we discovered a novel R2R3 MYB transcription factor, ThMYB14 , whose transcript levels, along with those of several flavonoid biosynthesis-related genes, were consistently upregulated. To explore ThMYB14 ’s function in flavonoid biosynthesis, we generated ThMYB14 -overexpressing (OE) and CRISPR/Cas9-mediated knockdown transgenic hairy roots lines. The results revealed that overexpression and knockdown of ThMYB14 significantly promoted and inhibited flavonoid accumulation, respectively. Moreover, promoter sequence analysis identified numerous potential MYB binding sites, including CCAAT-box, MBS, and MBSI elements, upstream of flavonoid biosynthesis-related genes. Yeast one-hybrid assays proved that Th MYB14 could bind to these motifs and promote the expression of target genes involved in flavonoid biosynthesis. AlphaFold3-based predictions and molecular docking further confirmed that Th MYB14 boosts flavonoid accumulation by specifically recognizing AAC elements within these sites. Interestingly, key residues in Th MYB14, such as K51, N102, K105, and N106, were likely involved in this recognition. The results shed light on the molecular mechanisms through which Th MYB14 recognizes multiple cis-elements to regulate flavonoid accumulation in T. hemsleyanum , offering a theoretical foundation for improving medicinal quality through genetic regulation. • ThMYB14 was identified as a novel regulator of flavonoid biosynthesis . • Overexpression and knockout of ThMYB14 significantly impacts flavonoid accumulation. • Th MYB14 binds to AAC elements in CCAAT-box, MBS, and MBSI motifs of target genes. • Key residues K51, N102, K105, and N106 of Th MYB14 are likely crucial for DNA binding.