Benzoxazinoids: a model for the repeated evolution of specialized metabolite biosynthesis in plants

Specialized metabolites are critical for plant interactions with the environment. While many metabolites are lineage-specific, independent evolution has led to identical compounds arising in distant taxa. Benzoxazinoids (BXD) exemplify this, occurring sporadically across angiosperms. Initially characterized in Zea mays (Poaceae - monocots), BXD serve as defense compounds, allelochemicals, and phytosiderophores. However, BXD biosynthesis in eudicots remained largely unknown. Through metabolomic and transcriptomic analyses, we uncovered BXD biosynthesis in three eudicot families: Acanthaceae, Lamiaceae, and Ranunculaceae. Although the biosynthetic pathways involve analogous reactions, the enzymes involved belong to distinct classes, distinct cytochrome P450 and UDP-glucosyltransferase families, as well as independently evolved 2-oxoglutarate-dependent dioxygenases, and O-methyltransferases. Phylogenetic analysis showed that BXD biosynthetic pathway evolved independently in Poaceae, Acanthaceae, Lamiaceae and Ranunculaceae. We further identified, in core eudicots, a pseudoenzyme, TSB-like, that mediates indole production by allosterically activating the tryptophan synthase α-subunit. TSB-like is a paralog of the canonical tryptophan synthase β-subunit, but lacks tryptophan biosynthetic activity. This pseudoenzyme is involved in the production of indole for BXD biosynthesis but its wider expression profile suggests a broader role in indole biosynthesis in core eudicots also for defense and pollinator attraction. Additionally, Z. mays exudes high levels of BXD in the soil, which result in the accumulation of the antimicrobial benzoxazolinone MBOA. The root-associated microbiota of Z. mays is enriched with bacteria strains able of catabolizing and metabolizing MBOA to the allelopathic aminophenol AMPO. We identified a N-acyl homoserine lactonase that catalyzes the conversion of MBOA to AMPO, enabling the adaptation of Z. mays root-associated bacteria to BXD.