Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation

• Published in: Plant physiology (Bethesda) 2013-02, Vol.161 (2), p.1049-1060
• Main Authors: Hung, Chiu-Yueh, Fan, Longjiang, Kittur, Farooqahmed S., Sun, Kehan, Qiu, Jie, Tang, She, Holliday, Bronwyn M., Xiao, Bingguang, Burkey, Kent O., Bush, Lowell P., Conkling, Mark A., Roje, Sanja, Xie, Jiahua
• Format: Article
• Language: English
• Subjects: Alkaloids; Alkaloids - metabolism; Amino Acid Sequence; Amino acids; Base Sequence; Biological and medical sciences; Biosynthesis; Cloning, Molecular; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; DNA, Complementary - chemistry; DNA, Complementary - genetics; Folic Acid - metabolism; Fundamental and applied biological sciences. Psychology; Gene expression regulation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Immunoblotting; Leaves; Metabolism; Methylation; Methylenetetrahydrofolate Reductase (NADPH2) - genetics; Methylenetetrahydrofolate Reductase (NADPH2) - metabolism; Molecular Sequence Data; Nicotiana - genetics; Nicotiana - metabolism; Nicotine - analogs & derivatives; Nicotine - metabolism; Plant Leaves - genetics; Plant Leaves - metabolism; Plant physiology and development; Plant Proteins - genetics; Plant Proteins - metabolism; Plants; Plants, Genetically Modified; Polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; SYSTEMES AND SYNTHETIC BIOLOGY; Transgenic plants; Yeasts
• ISSN: 0032-0889; 1532-2548; 1532-2548
• DOI: 10.1104/pp.112.209247

Methylenetetrahydrofolate reducíase (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-tonornicotine 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.