Sequence Variations in MYB (v-myb Myeloblastosis Viral Oncogene Homolog) Genes Impair Anthocyanin Biosynthesis and Contribute to Yellow Flower Phenotype in Rehmannia glutinosa

The corolla of <i>Rehmannia glutinosa</i> typically exhibits a stable reddish-purple color, but a naturally occurring yellow-flowered variant has recently been identified. To clarify the molecular basis of flower color variant, metabolomics, transcriptomics, and variant analyses were integrated. Metabolomic profiling revealed that the yellow phenotype was associated with lower anthocyanin levels and higher carotenoid levels. Specifically, the decreased cyanidin-3-O-glucoside led to a loss of red, while increased lutein provided the basis for the yellow color. Transcriptomic analysis revealed a downregulation of anthocyanin biosynthetic genes, including <i>CHS</i>, <i>CHI</i>, <i>F3H</i>, <i>DFR</i>, and <i>ANS</i>, in the yellow-flowered variant, and three S6-subgroup R2R3-MYB genes, including the known anthocyanin activator <i>RgMYB41</i> (<i>gene-DH2020_015992</i>), were downregulated. Variant analysis showed that A12S and G255E in the gene-DH2020_015992 transcription factor were predicted to markedly alter protein conformation and potentially impair regulatory function. Subcellular localization and transcriptional activation assays further supported the functional characterization of <i>gene-DH2020_015992</i> as a transcription factor. Collectively, these findings suggest that flower color variation in <i>R. glutinosa</i> is driven by MYB-mediated repression of anthocyanin biosynthesis and by carotenoid accumulation. This study provides a comprehensive genetic explanation for flower color variation in <i>R. glutinosa</i> and offers a theoretical foundation for floral pigmentation in plants.