Two Isatis Genomes Reveal the Biosynthesis and Evolutionary Origin of Indigo in Plants

Indigo, a plant-originated blue dye, has a long and well-documented history of extensive human use. The Isatis genus has long been a key source for indigo production, however, the biosynthetic pathway responsible for indigo within Isatis has remained elusive. Here, we conducted phylogenetic and metabolic analyses of various Isatis taxa, revealing that the capacity to produce indigo was apparently lost in some of these taxa. Following de novo genome sequencing, assembly, and comparative genomic analysis between Isatis indigotica and Isatis cappadocica, we delved into the origins and evolution of indigo biosynthesis. Homologous expression of candidate genes in Nicotiana benthamiana identified multiple oxidase families, including flavin-containing monooxygenase (FMO) and cytochrome P450 (CYP) protein that catalyze the oxidation steps leading to the indigo biosynthesis, indicating a metabolic innovation derived from the oxime pathway in plants. The evolutionary aspects concerning the neofunctionalization of CYPs-catalyzed biosynthesis of glucosides and FMOs-catalyzed oxime in Isatis taxa provide new insights into the evolution of these metabolic pathways in plants.