Lithospermum erythrorhizon is a medicinal plant valued for its ability to produce shikonin and its derivatives, a compound with important pharmacological properties. While R2R3-MYB transcription factors are known regulators of plant metabolism, no genome-wide analysis had been conducted in L. erythrorhizon . In this study, we present the comprehensive identification and evolutionary study of R2R3-MYBs in L. erythrorhizon , characterizing 176 LeMYB genes. Structural and conserved motif analyses revealed that the R2 and R3 domains of LeMYBs are relatively conserved, while sequence diversity has progressively emerged during evolution. Phylogenetic analysis classified the LeMYBs into 31 subfamilies, expanded mainly through whole-genome duplication, transposed duplication, and dispersed duplication. Expression profiling identified that certain LeMYBs exhibit root/leaf/flower-specific expression, with LeMYB1 predominantly expressed in roots, showing a 63-fold increase in transcript levels compared to leaves and significant upregulation under shikonin-inducing conditions. Subcellular localization confirmed that LeMYB1 is a nucleus-localized protein. Finally, yeast one-hybrid (Y1H) and dual-luciferase reporter assays collectively demonstrate that LeMYB1 binds to the promoters of key shikonin biosynthetic enzyme genes ( LeHMGR2 , LeGPPS1 , LePGT1 , LeGHQ3’’H2 , and LeSAT1 ) and activates their transcription, thereby promoting shikonin biosynthesis. This study provides the first genome-wide evolutionary analysis of R2R3-MYBs in L. erythrorhizon and reveals that LeMYB1 coordinately regulates multiple shikonin biosynthetic genes, laying the foundation for elucidating the regulatory mechanisms of shikonin biosynthesis and for mining potential transcription factors for genetic improvement.
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