An arginine-to-histidine mutation in flavanone-3-hydroxylase results in pink strawberry fruits

Fruit color is a very important external commodity factor for consumers. Compared to the most typical red octoploid strawberry (Fragaria × ananassa), the pink strawberry often sells for a more expensive price and has a higher economic benefit due to its outstanding color. However, few studies have examined the molecular basis of pink-colored strawberry fruit. Through an EMS mutagenesis of woodland strawberry (Fragaria vesca), we identified a mutant with pink fruits and green petioles. Bulked-segregant analysis sequencing analysis and gene function verification confirmed that the responsible mutation resides in a gene encoding flavanone-3-hydroxylase (F3H) in the anthocyanin synthesis pathway. This nonsynonymous mutation results in an arginine-to-histidine change at position 130 of F3H. Molecular docking experiments showed that the arginine-to-histidine mutation results in a reduction of intermolecular force-hydrogen bonding between the F3H protein and its substrates. Enzymatic experiments showed a greatly reduced ability of the mutated F3H protein to catalyze the conversion of the substrates and hence a blockage of the anthocyanin synthesis pathway. The discovery of a key residue in the F3H gene controlling anthocyanin synthesis provides a clear target of modification for the molecular breeding of strawberry varieties with pink-colored fruits, which may be of great commercial value.