Variation of terpene alkaloids in Daphniphyllum macropodum across plants and tissues.

Daphniphyllum macropodum produces alkaloids that are structurally complex with polycyclic, stereochemically rich carbon skeletons. Understanding how these compounds are formed by the plant may enable exploration of their biological function and bioactivities. We employed multiple metabolomics techniques, including a workflow to annotate compounds in the absence of standards, to compare alkaloid content across plants and tissues. Different alkaloid structural types were found to have distinct distributions between genotypes, between tissues and within tissues. Alkaloid structural types also showed different isotope labelling enrichments that matched their biosynthetic relationships. The work suggests that mevalonate derived 30-carbon alkaloids are formed in the phloem region before their conversion to 22-carbon alkaloids which accumulate in the epidermis. This sets the stage for further investigation into the biosynthetic pathway.

The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum).

Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold-forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via an inferred acylated intermediate. This work provides a genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into iridoid biosynthesis in insects.