Novel substrate affinity of FaCCR1 and FaCCR1/FaOCT4 expression control the content of medium-chain esters in strawberry fruit

Fruit flavor and aroma are influenced by the sugars-to-acid ratio and most importantly by the specific profile and abundance of volatile organic compounds (VOCs). While over 550 VOCs have been identified in strawberry fruit, fewer than 20 are believed to be key for aroma, being esters the major components. A major and stable quantitative trait locus (QTL) explaining a large percentage (40%) of phenotypic variation for several medium-chain esters (MCE) was mapped at about 19 Mb in chromosome 6A using both a biparental population and genome-wide association studies (GWAS). Using a combination of genetic mapping, global transcriptomic studies of bulked contrasting lines, and in vivo and in silico functional analyses, we identified the cinnamoyl Co-A reductase FaCCR1(6A) and the organic cation/carnitine transporter FaOCT4(6A) as underlying genes. Subcellular localization experiments indicated that FaCCR1(6A) and FaOCT4(6A) localize at the cytosol and tonoplast membrane respectively. Transient overexpression of FaCCR1(6A) and FaOCT4(6A) in fruits resulted in a significant increase in MCE content. Despite the well-described function of CCRs in the biosynthesis of monolignols, in vitro enzymatic assays and molecular docking revealed that FaCCR1(6A) also presents high affinity for MCE precursors, indicating a previously unidentified function of this protein in volatile biosynthesis. Based in our findings, we have developed a predictive KASP assay in FaOCT4(6A) for selecting superior cultivars with enhanced fruit aroma.