Integrated multi-omics analyses reveal that p-coumaroyl-CoA 2’-hydroxylases act upstream of stilbene synthases to mediate oxyresveratrol biosynthesis in mulberry (Morus alba L.)

Oxyresveratrol is a bioactive stilbenoid with strong antioxidant, anti-inflammatory, and tyrosinase-inhibitory activities that accumulates in mulberry (Morus alba L.) tissues. Despite its relevance, the biosynthetic origin of oxyresveratrol has remained unclear, with competing hypotheses proposing either hydroxylation of resveratrol or synthesis from a distinct precursor. Moreover, resveratrol and oxyresveratrol naturally accumulate in non-renewable parts of mulberry trees, limiting their efficient extraction. To bypass these spatiotemporal constraints, we established cell suspension cultures from mulberry twigs and demonstrated that combined treatment with methyl jasmonate and methyl- or hydroxypropyl-β-cyclodextrins elicits high levels of both resveratrol and oxyresveratrol, accumulating intra- and extracellularly. Using this system, we addressed the biological question of how oxyresveratrol is synthesized at the molecular level in mulberry. We first improved the structural and functional annotation of the mulberry genome by integrating short- and long-read sequencing data derived from elicited cell suspension transcriptomes. By combining these resources with integrative transcriptomic, proteomic, and metabolomic analyses, we identified a coordinated induction of several stilbene synthases (STSs) and a group of p-coumaroyl-CoA 2'-hydroxylases (C2'Hs) that were strongly co-expressed with resveratrol and oxyresveratrol accumulation. Functional validation in Nicotiana benthamiana, grapevine cell cultures, and in vitro enzyme assays demonstrated that C2'Hs catalyze the hydroxylation of p-coumaroyl-CoA upstream of the STS reaction, generating 2',4'-dihydroxycinnamoyl-CoA as an alternative substrate for STSs. These findings reveal that oxyresveratrol is produced through a biosynthetic pathway parallel to resveratrol formation rather than via post-synthetic hydroxylation. In addition, we provide genomic and transcriptomic resources contextualized within jasmonate-mediated elicitation, enabling the discovery of novel phenylpropanoid structural and regulatory genes in the Morus genus. Together, our work establishes a new biosynthetic paradigm for stilbenoid diversification in plants and delivers molecular tools and resources for the biotechnological production of oxyresveratrol.