Identification of CYP82E21 as a functional nicotine N-demethylase in tobacco flowers. (November 2016)

• Record Type: Journal Article
• Authors: Liedschulte, Verena; Schwaar, Joanne Deborah; Laparra, Hélène; Vuarnoz, Aline; Philippon, Bérangère; Bakaher, Nicolas; Sierro, Nicolas; Bovet, Lucien; Lang, Gerhard; Goepfert, Simon
• Is Part Of: Phytochemistry. Volume 131(2016:Nov.)
• Page Start: 9
• Page End: 16
• Publication Date: 2016-11
• Subjects: Nicotiana tabacum -- Solanaceae -- Tobacco -- Gene function -- Expression study -- Nicotine N-demethylase -- Nicotine conversion -- Nornicotine biosynthesis; Botanical chemistry -- Periodicals; Biochemistry -- Periodicals; Botany -- Periodicals; Chimie végétale -- Périodiques; 572.2
• DOI: 10.1016/j.phytochem.2016.08.004
• Languages: English
• ISSNs: 0031-9422

Abstract

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. Graphical abstract: A nicotine N -demethylase (CYP82E21) was identified and its expression was shown in flowers, more specifically in ovaries. Nicotine demethylase function was confirmed using expression and RNAi silencing experiments. Highlights: A nicotine N -demethylase gene ( CYP82E21 ) was identified. Unlike the other known nicotine N -demethylase genes, CYP82E21 is not expressed in leaves. CYP82E21 is expressed in flowers, more specifically in ovaries. CYP82E21 function was confirmed using expression and RNAi silencing experiments.