title: Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation.
abstract: Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine, forming methionine, which is then used for the synthesis of S-adenosyl-methionine, a universal methyl donor for numerous methylation reactions, to produce primary and secondary metabolites. Here, we demonstrate that manipulating tobacco (Nicotiana tabacum) MTHFR gene (NtMTHFR1) expression dramatically alters the alkaloid profile in transgenic tobacco plants by negatively regulating the expression of a secondary metabolic pathway nicotine N-demethylase gene, CYP82E4. Quantitative real-time polymerase chain reaction and alkaloid analyses revealed that reducing NtMTHFR expression by RNA interference dramatically induced CYP82E4 expression, resulting in higher nicotine-to-nornicotine conversion rates. Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower nicotine-to-nornicotine conversion rates. However, the reduced expression of NtMTHFR did not affect the methionine and S-adenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine N-demethylation and suggests that the negative regulation of CYP82E4 expression may serve to recruit methyl groups from nicotine into the C1 pool under C1-deficient conditions.
authors.affiliation: Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, North Carolina 27707, USA.
authors.forename: Chiu-Yueh
authors.initials: CY
authors.lastname: Hung
authors.forename: Longjiang
authors.initials: L
authors.lastname: Fan
authors.forename: Farooqahmed S
authors.initials: FS
authors.lastname: Kittur
authors.forename: Kehan
authors.initials: K
authors.lastname: Sun
authors.forename: Jie
authors.initials: J
authors.lastname: Qiu
authors.forename: She
authors.initials: S
authors.lastname: Tang
authors.forename: Bronwyn M
authors.initials: BM
authors.lastname: Holliday
authors.forename: Bingguang
authors.initials: B
authors.lastname: Xiao
authors.forename: Kent O
authors.initials: KO
authors.lastname: Burkey
authors.forename: Lowell P
authors.initials: LP
authors.lastname: Bush
authors.forename: Mark A
authors.initials: MA
authors.lastname: Conkling
authors.forename: Sanja
authors.initials: S
authors.lastname: Roje
authors.forename: Jiahua
authors.initials: J
authors.lastname: Xie
mesh_terms: D000470:Alkaloids / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D015151:Immunoblotting; D008745:Methylation; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009538:Nicotine / Q000031:analogs & derivatives / Q000378:metabolism*; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D018076:DNA, Complementary; D010940:Plant Proteins; D009538:Nicotine; C005311:nornicotine; D003577:Cytochrome P-450 Enzyme System; D005492:Folic Acid; C516919:nicotine demethylase, tobacco; D042965:Methylenetetrahydrofolate Reductase (NADPH2)