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

## TL;DR

This study identifies two genes in peanuts that control seed coat color by regulating pigment production, offering new targets for breeding more nutritious peanut varieties.

## Contribution

The discovery of AhWSC1a and AhWSC1b as master regulators of testa pigmentation in peanuts, linking their function to proanthocyanidin biosynthesis and gene regulation.

## Key findings

- Loss-of-function mutations in AhWSC1a/1b lead to white seed coats due to suppressed proanthocyanidin biosynthesis.
- AhWSC1 forms a complex with AhTc1 to activate late anthocyanin biosynthesis genes.
- AhWSC1 is a rate-limiting regulator of anthocyanin biosynthesis, as overexpression of AhTc1 alone does not induce pigmentation.

## Abstract

Seed coat color in peanut (Arachis hypogaea L.) is a critical agronomic trait that affects both nutritional quality and market appeal. In this study, we identified two bHLH transcription factor genes, AhWSC1a and AhWSC1b, homologues of Arabidopsis TRANSPARENT TESTA 8, 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 AhWSC1a/1b 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 (DFR, LDOX) 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 AhDFR and AhLDOX transcription. In this research, we also found that the black testa phenotype may arise from elevated AhTc1 expression associated with a structural variant (SV); however, in the SV background, the introduction of ahwsc1a/1b mutant leads to a significant suppression of AhTc1 expression. Notably, because AhWSC1 is transcriptionally silent in hairy-root systems, overexpression of AhTc1 alone failed to induce these late-stage anthocyanin biosynthesis genes, highlighting AhWSC1 as an indispensable, rate-limiting hub of anthocyanin biosynthesis pathway regulation. Collectively, our findings establish AhWSC1a and AhWSC1b 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.