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

Carrots possess a diverse of falcarin-type polyacetylenes (PAs), which emerge as not only important phytoalexins against pathogens but also potential anti-cancer agents. Despite abundantly found in carrot root tissues, the biosynthesis and evolutionary origins of falcarindiol, a C17-PA, remain unknown. Fatty acid desaturase 2 (FAD2) enzymes play crucial roles in diversifying PAs by introducing various double and/or triple carbon-carbon bonds on fatty acid chains. Here, we apply association analysis and 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 further uncovered unexpected synergistic and re-directive effects among FAD2 enzymes, complicating the biosynthetic pathway. CRISPR-Cas9-mediated mutagenesis and overexpression studies identified overlooked DcFAD2 hub genes as essential for falcarindiol production. Evolutionary analysis suggests that the expansion of DcFAD2 genes underpins the richness of falcarindiol in carrots, independently of the biosynthetic gene cluster previously identified in tomato. This work underscores the complexity of falcarin biosynthetic network and identifies hub genes essential for falcarindiol biosynthesis in carrot.