Mutation in FvPAL2 leads to light red strawberry fruits and yellow-green petioles

In recent years, light red or white strawberries have attracted much attention because of their unusual color, however, the mechanism of strawberry color formation, especially light red strawberry color, is not well understood. By EMS mutagenesis of woodland strawberry (Fragaria vesca), we identified two mutants, rg40 and rg120, with light red fruit and yellow-green petiole, and allelic hybridization and BSA mixed-pool sequencing revealed that the phenotype was caused by mutation in the FvPAL2 protein in the anthocyanin synthesis pathway. Enzyme activity experiments showed that the mutant FvPAL2 protein barely catalyzed the substrate conversion normally, thus blocking anthocyanin synthesis, which in turn led to a decrease in anthocyanin accumulation in fruits and petioles. Analysis of the active pockets of the wild-type and mutant FvPAL2 proteins revealed that the mutant FvPAL2 could not bind to the substrate properly. The specific transcription factors FvMYB10 and FvMYB10L were further found to bind and activate the expression of FvPAL1 and FvPAL2 in both fruit and petiole. The discovery of the key site of FvPAL2 protein activity provides a clear modification target for the breeding of light red strawberry varieties, which has important application value.