Screening and functional identification of CtMYB2 as a positive regulator of flavonoid biosynthesis in safflower (Carthamus tinctorius L.).

BACKGROUND: Safflower (Carthamus tinctorius L.), an annual herb of the Asteraceae family, possesses dried tubular florets that are commonly used in traditional Chinese medicine to promote blood circulation and remove blood stasis. Flavonoids are the main active constituents of safflower, yet their regulatory mechanisms remain largely unclear. While MYB transcription factors have been widely reported to participate in regulating flavonoid biosynthesis, few transcription factors that regulate this pathway have been functionally characterized in safflower.

RESULTS: Based on safflower transcriptome data, this study screened six MYB transcription factors potentially associated with flavonoid biosynthesis through co-expression analysis. CtMYB2 was selected for further cloning and characterization. The full length of the CtMYB2 gene is 1394 bp, with a 942 bp coding sequence (CDS). Bioinformatic analysis indicated that CtMYB2 encodes a hydrophilic nuclear protein, and subcellular localization confirmed its nuclear localization. A transient VIGS silencing and overexpression system was established in safflower petals. The optimal infiltration conditions were determined as treating petals at the full blooming stage (2 days post-anthesis) under a vacuum pressure of -0.10 to །0.09 MPa for 6 min, with the best activity duration being 5-7 days post-infiltration. Functional validation results demonstrated that silencing CtMYB2 suppressed the expression of CtC3H1, CtCHS3, and CtCHS1, leading to reduced total flavonoid content as well as decreased levels of the flavonol kaempferol and hydroxysafflor yellow A (HSYA), accompanied by lighter petal color. Conversely, overexpression of CtMYB2 upregulated the expression of CtCHI3 and CtCHS3, elevated total flavonoid accumulation together with enhanced contents of HSYA and kaempferol, and deepened petal color.

CONCLUSIONS: These findings indicate that CtMYB2 acts as a positive regulator of flavonoid biosynthesis in safflower, providing a foundation for elucidating the regulatory network of safflower flavonoids and breeding superior medicinal safflower varieties.