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Summary: Flavonoids are a class of secondary metabolites widely distributed in plants. Regiospecific modification by methylation and glycosylation determines flavonoid diversity. A rare flavone glycoside, diosmin (luteolin-4′-methoxyl-7-O-glucosyl-rhamnoside), occurs in Chrysanthemum indicum. How Chrysanthemum plants evolve new biosynthetic capacities remains elusive. Here, we assemble a 3.11-Gb high-quality C. indicum genome with a contig N50 value of 4.39 Mb and annotate 50,606 protein-coding genes. One (CiCOMT10) of the tandemly repeated O-methyltransferase genes undergoes neofunctionalization, preferentially transferring the methyl group to the 4′-hydroxyl group of luteolin with ortho-substituents to form diosmetin. In addition, CiUGT11 (UGT88B3) specifically glucosylates 7-OH group of diosmetin. Next, we construct a one-pot cascade biocatalyst system by combining CiCOMT10, CiUGT11, and our previously identified rhamnosyltransferase, effectively producing diosmin with over 80% conversion from luteolin. This study clarifies the role of transferases in flavonoid diversity and provides important gene elements essential for producing rare flavone.

Introduction: Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives: We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods: G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results: PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion: G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.