Comprehensive analysis of R2R3-MYB transcription factors reveals OsMYB1 as a key regulator of anthocyanin biosynthesis in rice.

R2R3-MYB transcription factors (TFs) are key regulators of plant development, stress responses, and secondary metabolism, with a central role in anthocyanin biosynthesis. However, a comprehensive understanding of the R2R3-MYB TFs involved in anthocyanin accumulation in rice (Oryza sativa L.) remains limited. In this study, we identified 105 R2R3-MYB TFs in the rice genome and performed extensive analyses of their phylogenetic relationships, conserved motifs, gene structures, and syntenic conservation across species. Integrating phylogenetic and expression profiling data, OsMYB1-belonging to subfamily 4 (S4) and homologous to AtMYB4-emerged as a key negative regulator of anthocyanin biosynthesis. Subcellular localization analysis confirmed that OsMYB1 is localized predominantly in the nucleus. Functional assays demonstrated that OsMYB1 binds to the promoters of OsDFR and OsANS, repressing their transcription. Moreover, OsMYB1 interacts with core components of the MYB-bHLH-WD40 (MBW) activation complex, specifically OsB2 and OsPAC1, to modulate anthocyanin biosynthesis via a negative feedback mechanism. CRISPR-Cas9-mediated knockout of OsMYB1 in purple rice varieties led to a marked increase in anthocyanin accumulation in pericarps and leaves, accompanied by upregulation of key biosynthetic genes such as OsF3'H, OsDFR, and OsANS. These findings establish OsMYB1 as a pivotal transcriptional repressor orchestrating anthocyanin biosynthesis in rice, providing valuable insights into the regulatory networks governing flavonoid pathways. This work offers promising genetic targets for biofortification and crop improvement strategies aimed at enhancing nutritional quality and stress resilience in cereal crops.