ADe Novoregulation design shows an effectiveness in altering plant secondary metabolism

Abstract

Decrease of nicotine, other tobacco alkaloids (OTAs), and carcinogenic tobacco specific nitrosamines (TSNAs) is important for health. To reduce these compounds, we selected MRE and G-box elements of promoters of four Nicotiana tabacum Jasmonate ZIM-domain (NtJAZ) repressor genes as targets to screen transcription factors (TFs) and then identified Arabidopsis Production of Anthocyanin Pigment 1 (PAP1) and Transparent Testa 8 (TT8) for a novel regulation design. Electrophoretic mobility shift, cross-linked chromatin immunoprecipitation, and dual luciferase analyses demonstrated that PAP1, TT8, PAP1/TT8, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex bound to and activated NtJAZ promoters. Stacked PAP1 (P) and TT8 (T), which were introduced to commercial tobacco cultivars, Narrow Leaf Madole (NL) and KY171 (KY), upregulated four NtJAZs, downregulated seven/eight nicotine biosynthetic genes, and reduced nicotine and nornicotine in transgenic red P+T-NL and P+L-KY plants. Field trials of red P+T-NL, P+L-KY, and PAP1 tobacco genotypes and metabolic quantification showed significant reduction of nicotine and four OTAs in most or all cured leaves. Three TSNAs and the fourth one were diminished by 63-92% and 30-74% in contents in all leaves of P+T-NL and P+L-KY genotypes, respectively. The total content of each TSNA was significantly reduced by 25-60% in leaves of PAP1 plants. Taken together, this regulation designed crossing from Arabidopsis anthocyanin to tobacco alkaloid pathway is defined as a distant pathway-cross regulation (DPCR). The proof of concept of DPCR unearths new functions of two known TFs, new activators of NtJAZs, and a negative regulation pathway of nicotine biosynthesis in red tobacco.