Anthocyanin biosynthesis in plants is spatiotemporally controlled by a suite of transcription factors, with MYB proteins playing a key regulatory role. However, the evolution of the distinct roles of MYB paralogs remains poorly understood. Our previous studies have established GmMYBA2 and GmMYBA3 as the regulators of seed coat and floral anthocyanin production in soybean (Glycine max), respectively. In this study, we reveal the functional divergence of their paralog GmMYBA1 in orchestrating light-responsive anthocyanin biosynthesis in juvenile tissues and stems. In brief, hypocotyl/stem- and young leaf-predominant expression of GmMYBA1 correlates with photoprotective anthocyanin accumulation. Ectopic overexpression of GmMYBA1 induces systemic pigmentation across leaves, stems, and reproductive organs, whereas RNAi-mediated silencing of GmMYBA1 significantly reduces anthocyanin accumulation in the hypocotyl. Light-dark shift assays confirmed that GmMYBA1 is required for hypocotyl pigmentation, while dual-luciferase assays revealed the specific regulation of the GmMYBA paralogs by GmSTF1/2 (soybean TGACG-motif binding factor 1/2). GmSTF1/2 both activate GmMYBA1, with only GmSTF2 weakly inducing GmMYBA2 and neither affecting GmMYBA3. Further investigation indicated that the differential transactivation of GmMYBA promoters largely resulted from their cis-element difference, suggesting regulatory divergence as a driver of MYB paralog diversification. Our findings position GmMYBA1 as the central MYB activator integrating light signaling with anthocyanin biosynthesis, with paralog specialization reflecting evolutionary subfunctionalization post-gene duplication.

Anthocyanins play crucial roles in pollen protection and pollinator attraction in flowering plants. However, the mechanisms underlying flower color determination and whether floral anthocyanin regulators participate in other processes remain largely unresolved in soybeans (Glycine max). In this study, we investigated the genetic components and mechanisms governing anthocyanin biosynthesis in soybean flowers. Molecular and genetic studies have characterized two antagonistic regulators, the positive activator GmMYBA3 and the negative repressor GmMYBR1, that modulate the gene expression of anthocyanin biosynthesis in soybean flowers. Further findings revealed a regulatory interplay between GmMYBA3 and GmMYBR1 bridged by GmTT8a, highlighting the complexity of anthocyanin regulation in different soybean organs. Exploration of additional soybean cultivars demonstrated the universality of GmMYBA3 and GmMYBR1 in regulating floral anthocyanin biosynthesis- related genes, with GmF3′5′H identified as a crucial determinant of white flower color. This study provides a molecular mechanism underlying soybean flower color determination, paving the way for the molecular modification of soybean flowers to probably enhance their resistance to abiotic stresses and attractiveness to pollinators.