LsMYB3, an R2R3-MYB repressor without canonical repression motifs, negatively regulates anthocyanin biosynthesis in...

Anthocyanins contribute to pigmentation, nutritional quality, and stress responses in plants. Although red spines are a characteristic trait of wild lettuce (Lactuca serriola), the genetic basis underlying spine pigmentation remains unclear. Here, we identified LsMYB3, a gene controlling spine color variation in wild lettuce. LsMYB3 encodes an R2R3-MYB transcription factor lacking a canonical repression motif but functioning as a negative regulator of anthocyanin biosynthesis. CRISPR/Cas9-mediated knockout of LsMYB3 enhanced anthocyanin accumulation, whereas overexpression suppressed pigmentation and converted red spines to green. Sequence analysis revealed that the natural Lsmyb3 allele carries a conserved Cys-to-Ser substitution within the R2 domain and a 1-bp deletion causing premature protein truncation, indicating loss of function. Yeast one-hybrid and dual-luciferase assays demonstrated that LsMYB3 directly represses LsDFR, a key anthocyanin biosynthetic gene. Notably, the effects of LsMYB3 disruption were tissue-specific and varied among genetic backgrounds, resulting in increased anthocyanin accumulation in wild lettuce spines and cultivated lettuce leaves. In addition, LsMYB3 physically interacted with the bHLH regulator RLL1, suggesting its involvement in the lettuce MBW regulatory network. Together, our findings identify LsMYB3 as a key negative regulator of anthocyanin biosynthesis and provide new insights into the evolution and diversification of pigmentation traits in lettuce.