Detalle de: Biosynthesis of oxyresveratrol in mulberry ( Morus alba L.) is mediated by a group of p-coumaroyl-CoA 2’-hydroxylases acting upstream of stilbene synthases

Mulberry ( Morus alba L.) is considered a millenary medicinal plant and a food source for silkworms. Different M. alba extracts offer a variety of biological and pharmacological properties that are in part attributed to stilbenoids, a small group of phenylpropanoids that include resveratrol and oxyresveratrol. These are naturally present in non-renewable parts of mulberry trees, impeding their efficient extraction. As a way to bypass this spatiotemporal restriction, we generated cell suspensions from mulberry twigs and demonstrated that the combined use of methyl jasmonate and methyl- or hydroxypropyl-β-cyclodextrins elicited a high production of resveratrol and oxyresveratrol, both intra and extracellularly. To identify oxyresveratrol-producing enzymes (unknown to date), we first improved the structural and functional annotation of the mulberry genome by integrating short and long-read sequencing data. We further combined this data with transcriptome, metabolite and proteome time-series evidence to identify a complete set of elicited phenylpropanoid- and stilbenoid-related genes. These included 22 stilbene synthase ( STS ) genes and a group of six p -coumaroyl-CoA 2’-hydroxylases (C2’Hs) that were highly co-expressed with resveratrol and oxyresveratrol accumulation. We transiently transformed Nicotiana benthamiana plants and grapevine ( Vitis vinifera L.) cell suspensions to functionally validate the role of C2’Hs as the first committed step of oxyresveratrol synthesis, providing an alternative substrate for STSs by hydroxylating p -coumaroyl-coA into 2’4’-dihydroxycinnamoyl-CoA. We offer tools for genomic and transcriptomic exploration in the context of jasmonate elicitation aiding in the characterization of novel stilbenoid-modifying and regulatory genes in the Morus genus.