Fruit color is a critical agronomic trait that determines consumer acceptance and market value in strawberry (Fragaria spp.). Fragaria nilgerrensis, a diploid strawberry endemic to Southwest China, exhibits a stable white fruit phenotype, but its genetic basis has not been experimentally validated. Here, we demonstrate that a single nucleotide mutation causing an R82S amino acid substitution in the core transcription factor FngMYB10 is the direct molecular cause of anthocyanin deficiency in F. nilgerrensis. Through site-directed mutagenesis, transient expression, and stable transformation experiments, we show that replacing serine with arginine at position 82 effectively activates the expression of key genes in the entire flavonoid biosynthesis pathway and recovers anthocyanin synthesis and fruit pigmentation. Yeast two-hybrid and bimolecular fluorescence complementation assays reveal that the R82S mutation specifically disrupts the critical protein-protein interaction between FngMYB10 and its transcriptional cofactor FngbHLH33, but does not alter its interaction with FngMYC1. These results indicate that FngbHLH33 is a key cofactor for anthocyanin synthesis in F. nilgerrensis, while FngMYC1 likely acts in other tissues or metabolic branches. Structural modeling revealed that this mutation eliminates essential hydrogen bonds within the conserved bHLH-interaction motif, preventing assembly of the functional MYB-bHLH-WD40 transcriptional complex required for anthocyanin gene activation. The results confirm that the R82S missense mutation in FngMYB10 of F. nilgerrensis specifically disrupts its interaction with the key cofactor FngbHLH33, blocking the assembly of the functional MBW complex and ultimately leading to the complete loss of fruit anthocyanin biosynthesis. These findings advance our understanding of the transcriptional regulatory network of fruit color formation and offer direct targets for molecular breeding of strawberry fruit color.
Cao et al. (Mon,) studied this question.