Journal of Advanced Research

Journal of Advanced Research

Volume 37, March 2022, Pages 43-60
Journal of Advanced Research

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

https://doi.org/10.1016/j.jare.2021.06.017Get rights and content
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open access

Research Highlights

  • Arabidopsis PAP1 and TT8 transcription factors and cis-regulatory elements of tobacco JAZs are for the first time selected as molecular tools for a De Novo regulation design.

  • This design creates a distant pathway-cross regulation (DPCR) that effectively downregulates the alkaloid biosynthesis in tobacco.

  • The DPCR significantly reduces carcinogenic compounds.

  • This design unearths two novel regulatory functions of PAP1 and TT8 and their complex and a new regulation mechanism of four NtJAZs.

Abstract

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.

Keywords

Arabidopsis
Alkaloid
Anthocyanin
Molecular tools
Tobacco
Transcription factor

Cited by (0)

Peer review under responsibility of Cairo University.

1

Contributed equally (M.L., X.H, and C.L.H.).