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

Introduction: Transcription factors (TFs) and -regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for regulation designs to engineer plants.

Objectives: We selected two Arabidopsis TF types and two tobacco CRE types to design a regulation and evaluated its effectiveness in plant engineering.

Methods: G-box and MYB recognition elements (MREs) were identified in four () 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 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 promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four 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 regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.