Two bHLH Transcription Factor Genes AhWSC1a and AhWSC1b Act as Gatekeepers of Testa Pigmentation, Preventing White Seed Coats in Peanuts

Seed coat color in peanut (<i>Arachis hypogaea</i> L.) is a critical agronomic trait that affects both nutritional quality and market appeal. In this study, we identified two bHLH transcription factor genes, <i>AhWSC1a</i> and <i>AhWSC1b</i>, homologues of <i>Arabidopsis TRANSPARENT TESTA 8</i>, as indispensable gatekeepers of basal flavonoid pigmentation. QTL-seq analysis of a recombinant inbred line population derived from a black-testa parent (S3) and a white-testa parent (S2) revealed that recessive loss-of-function mutations in both <i>AhWSC1a/1b</i> abolish proanthocyanidin biosynthesis, resulting in a white testa. Integrated metabolomic and transcriptomic profiling confirmed the absence of proanthocyanidins and a strong repression of late anthocyanin-pathway genes (<i>DFR</i>, <i>LDOX</i>) in the mutants. Molecular assays further demonstrated that AhWSC1 physically interacts with the R2R3-MYB regulator AhTc1 to form a functional MBW complex that activates <i>AhDFR</i> and <i>AhLDOX</i> transcription. In this research, we also found that the black testa phenotype may arise from elevated <i>AhTc1</i> expression associated with a structural variant (SV); however, in the SV background, the introduction of <i>ahwsc1a</i>/<i>1b</i> mutant leads to a significant suppression of <i>AhTc1</i> expression. Notably, because <i>AhWSC1</i> is transcriptionally silent in hairy-root systems, overexpression of <i>AhTc1</i> alone failed to induce these late-stage anthocyanin biosynthesis genes, highlighting <i>AhWSC1</i> as an indispensable, rate-limiting hub of anthocyanin biosynthesis pathway regulation. Collectively, our findings establish <i>AhWSC1a</i> and <i>AhWSC1b</i> as master regulators of peanut testa pigmentation, elucidate the molecular basis of classical white testa inheritance, and provide genetic targets for precision-breeding of nutritionally enhanced cultivars.