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

Abstract

AbstractMulberry (Morus albaL.) is considered a millenary medicinal plant and a food source for silkworms. DifferentM. albaextracts 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 sixp-coumaroyl-CoA 2’-hydroxylases (C2’Hs) that were highly co-expressed with resveratrol and oxyresveratrol accumulation. We transiently transformedNicotiana benthamianaplants and grapevine (Vitis viniferaL.) 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 hydroxylatingp-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 theMorusgenus.