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.

Inhibition of FvMYB10 transcriptional activity promotes color loss in strawberry fruit.

FvMYB10 protein has been proved to be a transcriptional switch for anthocyanin biosynthesis in strawberry. A single nucleotide mutation in R2 domain of FvMYB10, named as FvmMYB10, is found to be responsible for the white color in strawberry variety 'Yellow Wonder'. However, the mechanism of FvmMYB10 suppresses anthocyanin biosynthesis in strawberry is largely unknown. Here, we show that the transcriptional level of FvMYB10 and key enzyme genes involved in anthocyanin biosynthesis in 'Yellow Wonder' were lower than that in red color variety 'Ruegen', especially at turning to ripening stage. The low expression level of FvmMYB10 may due to his inability to bind to its promoter region and activate its own expression. We found FvMYB10-overexpressing, but not FvmMYB10-overexpressing, promote anthocyanin accumulation in Arabidopsis and strawberry fruit despite of their similar expression levels. In addition, subcellular localization assay indicated that FvMYB10-YFP, but not FvmMYB10-YFP, localized to sub-nucleus foci (speckles) in the nucleus, implying the mutation of FvMYB10 might inhibit its transcription factor activity and eventually interfere with its function. Subsequently, we confirmed that FvMYB10 bind to the promoter region of some specific key enzyme genes, including FvCHS2 and FvDFR1 and activated their expression. While FvmMYB10 failed to binding and transcriptional activating these genes. Our findings provide insights into molecular mechanism of anthocyanin biosynthesis regulated by MYB10 in strawberry fruits.