Identification and biosynthesis of plant papanridins, a group of novel oligomeric flavonoids.

The discovery of novel flavonoids and elucidation of their biosynthesis are fundamental to understanding their roles in plants and their benefits for human and animal health. Here, we report a new pathway for polymerization of a group of novel oligomeric flavonoids in plants. We engineered red cells for discovering genes of interest involved in the flavonoid pathway and identified a gene encoding a novel flavanol polymerase (FP) localized in the central vacuole. FP catalyzes the polymerization of flavanols, such as epicatechin and catechin, to produce yellowish dimers or oligomers. Structural elucidation shows that these compounds feature a novel oligomeric flaven-flavan (FF) skeleton linked by interflavan-flaven and interflaven bonds, distinguishing them from proanthocyanidins and dehydrodicatechins. Detailed chemical and physical characterizations further confirmed the novel FFs as flavonoids. Mechanistic investigations demonstrated that FP polymerizes flavan-3-ols and flav-2-en-3-ol carbocation, forming dimeric or oligomeric flaven-4→8-flavans, which we term "papanridins." Data from transgenic experiments, mutant analysis, metabolic profiling, and phylogenetic analyses show that the biosynthesis of papanridins is prevalent in cacao, grape, blueberry, corn, rice, Arabidopsis, and other species in the plant kingdom. In summary, our study discoveries a group of novel oligomeric flavonoids, namely papanridins, and reveals that a novel FP-mediated polymerization mechanism for the biosynthesis of papanridins in plants.

A de novo regulation design shows an effectiveness in altering plant secondary metabolism.

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.