Unravel the Mystery ofNIC1-locus on Nicotine Biosynthesis Regulation in Tobacco
Authors: Sui Xueyi , Xie He , Tong Zhijun , Zhang Hongbo , Song Zhongbang , Gao Yulong , Zhao Lu , Li Wenzheng , Li Meiyun , Li Yong , Li Yongping , Wang Bingwu 
Published: July 2020

ABSTRACTBackgroundNicotine biosynthesis is mainly regulated by jasmonate (JA) signaling cascade inNicotiana tabacum. As an allotetraploid species, the regulation of nicotine biosynthesis has been genetically verified via two unlinkedNICloci (named asNIC1andNIC2) which are possibly originated from its two ancestral diploids. Previously, aN. tomentosiformisoriginated ethylene response factor (ERF) gene cluster was identified as theNIC2-locus which has been demonstrated positively regulates nicotine accumulation inN. tabacum.ResultsHere, we describe the genetic mapping ofNIC1-locus, the major nicotine regulatory locus, by using aNIC1-locus segregating population through bulked segregant analysis. We identified two linkage marker TM23004 and TM22038 were delimited theNIC1-locus within a ~34.3-Mb genomic region at pseudochromosome 07 of tobacco genome. Genomic scan within this region revealed aNIC2-likelocusERFgene cluster exist in. To verify thisERFgene cluster is the genetically called “NIC1-locus”, different functional experiments based on most of theERFsin regulating nicotine biosynthesis and their influences on alkaloid accumulations have been carried out. Collinearity analysis showed thatNIC1-locusERFgenes are originated fromN. sylvestrisand exclusively expressed in root tissues. In addition, transcriptomic results indicate thatNIC1-locusERFgenes are coexpressed with theNIC2-locusERFgenes and other nicotine biosynthetic genes and regulators after JA induction. Furthermore, the suppressed expression of fourERFsof theNIC1-locus genes corresponding with decreasedNtPMTandNtQPTexpression inNtMYC2-RNAi lines indicates the selectedNIC1-locusERFsfunction in downstream ofNtMYC2in the JA signaling cascades. In the meanwhile, the alkaloid levels are also determined by the amplitude of the fourERFgene expressions in both wild type and LA mutant. Additionally,in vitrobinding assays, transient activation assays, and ectopic expression in transgenic plants demonstrate that theseERFgenes are able to bind the GCC-box elements residing in the step-limiting gene promoters (such asNtPMT2,NtQPT2) and functional redundant but quantitatively transactivate nicotine biosynthetic gene expression. Fornic1-locus mutation, two different sizes of deletions (nic1-Sandnic1-B) were identified which occurred at the surrounding regions of theNIC1-locus gene cluster, which might disrupt, to some extent, chromosomal microenvironment and change gene expression around the deletion regions (includingNIC1-locusERFs), resulting in the decreased expression levels ofNIC1-locusERFs(such asNtERF199) and reduced alkaloid accumulation in thenic1-locus mutant.ConclusionsOur findings not only provide insight in to the mechanism of theNIC1-locusERFsin the regulatory network of nicotine biosynthesis, but also unraveled the theoretical basis of thenic1-locus mutation in low nicotine mutant. These functional verifiedNIC1-locusERFgenes can be further used as potential target(s) for ethyl methanesulfonate-based mutagenesis to manipulate nicotine level in tobacco variety in tobacco breeding program.