Identification of CYP82E21 as a functional nicotine N-demethylase in tobacco flowers.
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.
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Verena.Liedschulte@pmi.com.
Verena
V
Liedschulte
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Joanne.Schwaar@pmi.com.
Joanne Deborah
JD
Schwaar
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Helene.Laparra@pmi.com.
Hélène
H
Laparra
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Aline.Vuarnoz@pmi.com.
Aline
A
Vuarnoz
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Berangere.Philippon@pmi.com.
Bérangère
B
Philippon
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Nicolas.Bakaher@pmi.com.
Nicolas
N
Bakaher
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Nicolas.Sierro@pmi.com.
Nicolas
N
Sierro
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Lucien.Bovet@pmi.com.
Lucien
L
Bovet
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Gerhard.Lang@pmi.com.
Gerhard
G
Lang
PMI R&D, Philip Morris Products SA, (part of Philip Morris International Group of Companies), Neuchâtel, Switzerland. Electronic address: Simon.Goepfert@pmi.com.
Simon
S
Goepfert
D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D035264:Flowers / Q000378:metabolism; D009538:Nicotine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D009602:Nitrosamines / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D014026:Nicotiana / Q000201:enzymology*
D009602:Nitrosamines; D010940:Plant Proteins; D009538:Nicotine; C005311:nornicotine; D003577:Cytochrome P-450 Enzyme System; C516919:nicotine demethylase, tobacco; D010089:Oxidoreductases, N-Demethylating; C008655:N'-nitrosonornicotine
Expression study; Gene function; Nicotiana tabacum; Nicotine N-demethylase; Nicotine conversion; Nornicotine biosynthesis; Solanaceae; Tobacco