Recruitment of a duplicated primary metabolism gene into the nicotine biosynthesis regulon in tobacco

Gene duplication is a powerful source of phenotypic diversity in plants, but the molecular mechanisms that generate new functions in duplicated genes are not fully documented. Here, we analyzed how duplicated genes encoding quinolinate phosphoribosyltransferase (QPT), an enzyme involved in the synthesis of nicotinamide adenine dinucleotide (NAD) and the pyridine moiety of nicotine, are regulated by the jasmonate-responsive transcriptional factor ERF189 that functions critically for nicotine biosynthesis in tobacco (Nicotiana tabacum). The tobacco genome contains duplicated QPT genes; QPT1 is expressed at a constitutive basal level, whereas QPT2 is regulated coordinately with other structural genes involved in nicotine biosynthesis, in terms of tissue specificity, jasmonate induction, and regulation by ERF189. The binding-site specificity of ERF189 was defined as 5'-(A/C) GC(A/C)(A/C) NCC-3' by using a characterized tobacco putrescine N-methyltransferase promoter, and was then used to search for potential binding sites in the QPT promoters. Assays involving in vitro DNA binding, transient transactivation, and transgenic hairy roots revealed that the QPT2 promoter contains three functional ERF189-binding sites, which individually confer incremental ERF189-mediated activation to the promoter. The QPT1 promoter is not bound and regulated by ERF189. These results indicate that one copy of the duplicated QPT genes was recruited to a tobacco alkaloid regulon by evolving multiple target cis-regulatory elements of ERF189 in its promoter, to cope with an increased metabolic demand for pyridine precursors during active alkaloid biosynthesis.