DOI: 10.25442/hku.28159268
title: <b><i>Supporting data for </i></b>"Functional characterizations of stilbene biosynthesis and <i>O</i>-methylation enzymes in flowering plants"

description: Stilbenes are unique plant-specialized metabolites that accumulate in various plant species and organs to combat environmental stresses such as UV radiation and fungal infections. Stilbene synthase (STS) is the primary enzyme responsible for catalyzing the production of stilbenes. In the phylogenetic tree analysis of STSs, several sequences closely related to the known <i>STSs</i> in Poaceae and Fabaceae were identified as <i>STS</i>-like sequences, and their catalytic abilities were characterized through in vitro and in vivo studies. STSs and chalcone synthases (CHSs) both belong to the type III polyketide synthase family and utilize the same elongation mechanism to produce a tetraketide intermediate. However, distinct cyclization mechanisms between CHS and STS result in different reaction products. Specific amino acid residue changes in A1, A2, and A3 regions enable CHS enzymes to acquire STS catalytic activity, presenting opportunities for protein engineering. Furthermore, stilbenes were newly discovered in UV-treated mesocotyls of <i>Setaria italica</i> and roots of <i>Medicago truncatula</i> in this study.<br><i>O</i>-methylation enhances the stability and bioavailability of stilbenes, showing potential benefits for human health. However, <i>O</i>-methylated enzymes of stilbenes have not been extensively studied. To obtain more information about stilbene <i>O</i>-methylation, the role of a SAM-dependent stilbene <i>O</i>-methyltransferase (SOMT) in <i>Sorghum bicolor </i>(SbSOMT)<i> </i>was investigated and elucidated. <i>SbSOMT</i> gene expression is pathogen-inducible and is detected in sorghum seedlings following the infection of <i>Colletotrichum sublineola</i>. Concurrently, <i>O</i>-methylated<i> </i>resveratrol (pinostilbene and pterostilbene) was detectable in the sorghum tissues in response to the pathogen attack. In contrast to another pathogen-inducible enzyme, SbOMT4, SbSOMT demonstrates efficient <i>O</i>-methylation of resveratrol in both enzyme assays and <i>in planta</i> experiments. Sorghum <i>SbSOMT</i> CRISPR/Cas9 mutants failed to accumulate pterostilbene and pinostilbene, confirming the indispensable role of SbSOMT in resveratrol 3,5-bis-<i>O</i>-methylation.<br>The canonical caffeic acid <i>O</i>-methyltransferase (COMT) is constitutively expressed in sorghum with a limited ability to catalyze the <i>O</i>-methylation of the stilbenes. The phylogenetic analysis in this study suggested that genus-specific SOMTs were recruited from canonical caffeic acid <i>O</i>-methyltransferases (COMTs) after the divergence of <i>Sorghum</i> from other genera. In recombinant enzyme assays, SbSOMT and COMTs exhibit regioselective catalysis, with SbSOMT targeting <i>O</i>-methylation of the stilbene A-ring and COMTs focusing on the B-ring. Subsequently, crystal structures of SOMT-stilbene complexes are presented. While SbSOMT shares a global structural resemblance with SbCOMT, molecular characterizations highlight two hydrophobic residues (Ile144/Phe337) crucial for substrate binding orientation, leading to 3,5-bis-<i>O</i>-methylations in the A-ring. In contrast, the equivalent residues (Asn128/Asn323) in SbCOMT promote an opposite orientation favoring 3ʹ-<i>O</i>-methylation in the B-ring. Consistently, a highly conserved COMT likely participates in isorhapontigenin (a 3ʹ-<i>O</i>-methylated stilbene) formation in wounded wild sugarcane (<i>Saccharum spontaneum</i>). Finally, this study uncovers the potential of Saccharinae grasses as sources of <i>O</i>-methylated stilbenes and rationalizes the regioselectivity of SOMT activities for bioengineering <i>O</i>-methylated stilbenes.The experimental data file dataset includes enzyme assay, gene expression, and metabolite data for investigating the phylogeny and catalytic ability of newly identified stilbene synthases and <i>O</i>-methyltransferases. The enzyme assay data file includes statistics for evaluating specific enzyme catalytic ability. The plant gene expression data file includes statistical data and images illustrating the expression of candidate genes in treated and untreated plant tissues. The plant metabolite data file includes statistical data and images illustrating the metabolites analyzation in treated and untreated plant tissues.<br>