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

Oxyresveratrol is a bioactive stilbenoid with strong antioxidant, anti-inflammatory, and tyrosinase-inhibitory activities that accumulates in mulberry (Morus alba) tissues. Despite its importance, 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 predominantly accumulate in non-renewable parts of mulberry trees, limiting their efficient extraction. To overcome 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, which accumulate both intra- and extracellularly. Using this system, we investigated the molecular basis of oxyresveratrol biosynthesis 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 coordinated induction of multiple 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 demonstrate that oxyresveratrol is produced via a biosynthetic pathway parallel to resveratrol formation rather than through post-synthetic hydroxylation. In addition, we provide genomic and transcriptomic resources within the context of jasmonate-mediated elicitation, facilitating 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.