Identification of CYP82E21 as a functional nicotine N-demethylase in tobacco flowers

Abstract

In the tobacco plant, nicotine N-demethylase enzymes (NND) belonging to the cytochrome P450 family catalyse the conversion of nicotine to nornicotine, the precursor of the carcinogenic tobacco-specific N-nitrosamine, N-nitrosonornicotine. To date three demethylase genes, namely CYP82E4, CYP82E5 and CYP82E10, have been shown to be involved in this process, while the related CYP82E2 and CYP82E3 genes are not functional. We have identified a further gene named CYP82E21 encoding a putative nicotine N-demethylase closely related to the CYP82E genes. The CYP82E21 gene was found in all Nicotiana tabacum cultivars analysed and originates from the tobacco ancestor Nicotiana tomentosiformis. We show that, in contrast to all other previously characterized NND genes, CYP82E21 is not expressed in green or senescent leaves, but in flowers, more specifically in ovaries. The nicotine N-demethylase activity of CYP82E21 was confirmed by ectopic expression of the coding sequence in a tobacco line lacking functional CYP82E4, CYP82E5 and CYP82E10 genes, resulting in an eightfold increase of nicotine demethylation compared to the control plants. Furthermore, nornicotine formation can be reduced in ovaries by introducing a CYP82E21-specific RNAi construct. Together, our results demonstrate that the CYP82E21 gene encodes a functional ovary-specific nicotine N-demethylase.

Investigation of benzylisoquinoline alkaloid biosynthetic pathway and its transcriptional regulation in lotus

“…Likewise, an intramolecular C–C phenol-coupling reaction together with a methylenedioxy bridge-forming reaction catalyzed by CYP719A 40 are required for the biosynthesis of roemerine. Additional N -demethylation and O -demethylation reactions that are catalyzed by N -demethylase (NDM) or O -demethylase (ODM), respectively 41 , 42 , result in structurally diversified BIAs. Analysis of the expressed genes in the leaf revealed one, three, one, three, and four copies of CYP80G , 7OMT , CYP719A , NDM , and ODM genes, respectively.…”

Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes

“…To date, four NND genes mediating nicotine to nornicotine conversion have been cloned from burley tobacco: CYP82E4, CYP82E5, CYP82E10, and CYP82E21. [5][6][7][8][9][10] CYP82E21 encodes an active demethylase, but it is only expressed in the ovary and may contribute little to nornicotine formation. 7 The nicotine to nornicotine conversion rate (NCR) in a mutant of CYP82E4 (e4/e4/E5E5/E10E10) was about 2.2% and is much lower than that in control plants, indicating that CYP82E4 is the major NND for nicotine demethylation in burley tobacco.…”

“…[5][6][7][8][9][10] CYP82E21 encodes an active demethylase, but it is only expressed in the ovary and may contribute little to nornicotine formation. 7 The nicotine to nornicotine conversion rate (NCR) in a mutant of CYP82E4 (e4/e4/E5E5/E10E10) was about 2.2% and is much lower than that in control plants, indicating that CYP82E4 is the major NND for nicotine demethylation in burley tobacco. Consistent with this observation, no significant difference was found between single mutant of CYP82E10 (E4E4/E5E5/ e10e10), a double mutant of CYP82E5 and CYP82E10 (E4E4/ e5e5/e10e10), and control plants.…”

Metabolomics-guided discovery of cytochrome P450s involved in pseudotropine-dependent biosynthesis of modified tropane alkaloids

“…5 ). With the exception of tropinone synthase (CYP82M3) from Atropa belladonna and N -demethylases (CYP82E4v1, CYP82E4v2, CYP82E21, CYP82E5v2, CYP82E10) from Nicotiana tabacum involved in pyridine/pyrrolidine alkaloid metabolism, no other cytochrome P450 from the Solanaceae family with roles in alkaloid biosynthesis were previously reported in the CYP71 and CYP82 families 16 , 46 , 47 .…”