GWAS and functional validation identify NtJMJ as a positive regulator of nicotine accumulation in Nicotiana tabacum

Abstract

Nicotine is an important metabolite in tobacco. However, the genetic and epigenetic bases underlying its natural variation remain poorly characterized. In this study, a multi-parent advanced generation intercross (MAGIC) population was used to dissect the genetic architecture of nicotine content across two environments. A total of 61,421 single nucleotide polymorphisms (SNPs) were used in genome wide association study, which identifyied 52 significant loci associated with nicotine accumulation. Among these, a stable locus on chromosome 2 (qNIC2) was consistently detected across environments and overlapped with previously reported nicotine-related quantitative trait loci (QTL). To identify the candidate gene within the qNIC2 interval, expression profile data from multiple tobacco tissues were integrated and analyzed using co-expression network analysis. A root enriched co-expression module containing all known nicotine biosynthesis and regulatory genes was identified. Within this module, NtJMJ, encoding a putative JmjC domain-containing histone demethylase, was selected as the candidate gene due to its strong root-specific and topping-inducible expression pattern. Genotype groups based on marker AX-117702345 showed significant differences in nicotine content, with TT accessions showing higher NtJMJ expression and consequently higher nicotine content. Subcellular localization analysis revealed the nuclear localization of NtJMJ, supporting its potential role as a transcriptional regulator. Functional validation further indicated that NtJMJ positively regulates nicotine accumulation, as edited lines showed reduced nicotine accumulation and overexpression lines increased it. Consistently, PMT expression was upregulated in overexpression lines and downregulated in mutant lines. Notably, phylogenetic and protein structural analyses indicated that NtJMJ belongs to the KDM4 subfamily and possesses a highly conserved JmjC catalytic core and trimethyl-lysine binding pocket, supporting its annotation as a putative histone demethylase. Collectively, our findings demonstrate that NtJMJ contributes to nicotine accumulation in tobacco, providing new insights into nicotine metabolism and offering a promising molecular target for breeding.