Elucidation and reconstitution of the poplar salicortin biosynthetic pathway.

Salicinoids are specialized benzenoid (C6-C1) metabolites characteristic of the Salicaceae family. Although salicinoids have long been recognized as effective defense compounds and pharmacologically active constituents in willows and poplars, the biosynthetic pathway underlying their formation remains largely unresolved. Through integrated metabolomic and transcriptomic analyses of black cottonwood (Populus trichocarpa) roots, we identified candidate genes involved in salicinoid biosynthesis belonging to the cytochrome P450 monooxygenase family 92 (CYP92) and the progesterone 5β-reductase/iridoid synthase-like enzyme (PRISE) family of short-chain dehydrogenases/reductases. Co-expression of all identified candidate genes, together with the previously characterized glycosyltransferase gene PtUGT71L1, acyltransferase gene PtACT47, and cinnamic acid-CoA ligase gene PtCNL5, in Nicotiana benthamiana resulted in the production of salicortin, the most abundant salicinoid in poplar. Further characterization of the P450 candidates revealed that two enzymes catalyze ortho-hydroxylation of both aromatic rings of the proposed salicortin precursor, benzyl benzoate, while two other P450s catalyze the subsequent oxidative dearomatization of the benzoic acid-derived ring of benzyl benzoate. The resulting dieneone intermediate is converted by PRISE enzymes via a 1,4-reduction to form the characteristic 1-hydroxy-6-oxocyclohex-2-en-1-carboxylic ester moiety of salicortin. Functional redundancy among the identified CYP92 and PRISE enzymes may reflect the central role of these compounds in plant defense.