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 largely attributed to stilbenoids, a small group of phenylpropanoids, including 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 leaf petioles and demonstrated that the combined use of methyl jasmonate and two different types of cyclodextrins were able to elicit a high production of resveratrol and oxyresveratrol. As oxyresveratrol-producing enzymes are still unknown, we improved the structural and functional annotation of the mulberry genome by integrating short and long-read sequencing data and combined it with time series transcriptome, metabolite and proteome data in response to cell elicitation to identify a complete set of phenylpropanoid and stilbenoid related genes. These included 23 stilbene synthase (STS) genes and a group of six p-coumaroyl-CoA 2'-hydroxylases (C2'Hs), all being highly co-expressed with resveratrol and oxyresveratrol production. We transiently transformed grapevine (Vitis vinifera L.) calli and Nicotiana benthamiana plants to functionally validate the role of C2'Hs in the first committed step of oxyresveratrol synthesis, which provides an alternative substrate for STSs by hydroxylating p-coumaroyl-coA into 2'4'-dihydroxycinnamoyl-CoA. Finally, we offer tools for genomic and transcriptomic exploration also in the context of jasmonate elicitation aiding in the characterization of novel stilbenoid-modifying and regulatory genes in the Morus genus. Overall design: To investigate the effect at different times of elicitation with methyl jasmonate and cyclodextrin in cell cultures obtained from Morus alba stems.