The R2R3-MYB transcription factor LcMYBC2-L1 represses anthocyanin biosynthesis in Litchi chinensis Sonn.

Abstract MYB transcription factors are key regulators of anthocyanin biosynthesis, acting as both activators and repressors. However, the functional characterization of R2R3-MYB repressors in litchi ( Litchi chinensis ) remains limited. In this study, we identified and characterized LcMYBC2-L1 as an R2R3-MYB transcriptional repressor that negatively regulates anthocyanin accumulation. LcMYBC2-L1 contained conserved basic helix-loop-helix (bHLH)-binding, C1 (LIsrGIDPxT/SHRxI/L), and C2 (pdLNLD/ELxiG/S) motifs, localizes to the nucleus, and exhibits transcriptional repression activity. Overexpression of LcMYBC2-L1 in tobacco suppressed anthocyanin biosynthesis, resulting in white petals. Heterologous expression of LcMYBC2-L1 in tobacco ( Nicotiana tabacum ) suppressed anthocyanin biosynthesis and resulted in the production of white petals. Importantly, overexpression of LcMYBC2-L1 and its mutation variants in red litchi calli overexpressing LcMYB1 led to significantly reduced anthocyanin accumulation and downregulation of biosynthetic genes, confirming the repressive role of LcMYBC2-L1 in anthocyanin biosynthesis. The C2 motif was shown to be essential for the inhibitory function of LcMYBC2-L1. Protein interaction assays revealed that LcMYBC2-L1 could interact with both LcMYB1 and LcbHLH1/3, thereby interfering with the formation of the activated LcMYB1–LcbHLH3 complex and suppressing the expression of downstream anthocyanin biosynthetic genes. Taken together, our findings establish that LcMYBC2-L1 represses anthocyanin biosynthesis by disrupting the LcMYB1–LcbHLH3 complex via its C2 repression motif, providing new insights into the R2R3-MYB-mediated regulatory mechanisms of anthocyanin production in plants.