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United States Department of Agriculture

Agricultural Research Service

Research Project: Strategies to Predict and Manipulate Responses of Crops and Crop Disease to Anticipated Changes of Carbon Dioxide, Ozone and Temperature

Location: Plant Science Research

Title: Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation

item Hung, Chiu-yueh
item Fan, Longjiang
item Kittur, Farooqahmed
item Sun, Kehan
item Qui, Jie
item Tang, She
item Holliday, Bronwyn
item Xiao, Bingguang
item Burkey, Kent
item Bush, Lowell
item Conkling, Mark
item Roje, Sanja
item Xie, Jiahua

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/29/2012
Publication Date: 2/1/2013
Citation: Hung, C., Fan, L., Kittur, F.S., Sun, K., Qui, J., Tang, S., Holliday, B.M., Xiao, B., Burkey, K.O., Bush, L.P., Conkling, M.A., Roje, S., Xie, J. 2013. Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation. Plant Physiology. 161:1049-1060.

Interpretive Summary: Engineering plants with altered chemical composition requires understanding the complex regulation underlying interactions between metabolic pathways. In this study, genetic manipulation of a key enzyme responsible for controlling availability of single carbon units dramatically altered the expression of genes that control the composition of plant compounds called alkaloids. The results suggest that alkaloids serve as a source of one-carbon units when metabolic demand is high. This is an example of how metabolism is fine-tuned through co-regulation of genes that appear to be unrelated based on our previous understanding.

Technical Abstract: Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine forming Met, which is then used for the synthesis of S-adenosylmethionine (AdoMet), a universal methyl donor for numerous methylation reactions to produce primary and secondary metabolites. Here, we demonstrate that manipulating the 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 PCR (qRT-PCR) and alkaloid analyses revealed that reducing NtMTHFR expression by RNAi dramatically induced CYP82E4 expression, resulting in higher nicotine to nornicotine conversion rates (NCRs). Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower NCRs. However, the reduced expression of NtMTHFR did not affect the Met and AdoMet 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.

Last Modified: 06/27/2017
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