Divergent fatty acid desaturase 2 is essential for falcarindiol biosynthesis in carrot

Carrots produce diverse falcarin-type polyacetylenes (PAs), which function as important antipathogenic phytoalexins and have potential as anticancer agents. Despite their abundance in carrot root tissues, the biosynthesis and evolutionary origins of falcarindiol, a C17-PA, remain unknown. Fatty acid desaturase 2 (FAD2) enzymes diversify PAs by introducing various double and/or triple carbon-carbon bonds into fatty acid chains. Here, we use association analysis to identify candidate FAD2 genes involved in falcarindiol biosynthesis. Using a rapid tobacco transient expression system, we found that DcFAD2 enzymes are highly functionally redundant and promiscuous. Combinatorial assays also revealed unexpected synergistic and redirective effects among FAD2 enzymes, further complicating the biosynthetic pathway. CRISPR-Cas9-mediated mutagenesis and overexpression studies identified previously overlooked DcFAD2 hub genes as essential for falcarindiol production. Evolutionary analysis suggests that the expansion of DcFAD2 genes underpins the abundance of falcarindiol in carrot, independent of the biosynthetic gene cluster previously identified in tomato. This work underscores the complexity of the falcarin biosynthetic network and identifies hub genes essential for falcarindiol biosynthesis in carrot.