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-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 Sadenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine Ndemethylation 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. Tetrahydrofolate (THF)-mediated one-carbon (C1) metabolism generates and provides C1 units in different oxidation states for various anabolic pathways, including alkaloid biosynthesis in plants (Hanson et al., 2000). Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylene-THF into the most reduced C1 derivative, 5-methyl-THF (Guenther et al., 1999;Roje et al., 1999;Gelling et al., 2004). The latter then serves as the methyl group donor for Met synthesis from homocysteine (Guenther et al., 1999;Hanson et al., 2000). More than 80% of synthesized Met is further converted to S-adenosyl-methionine (AdoMet), which is the universal methyl group donor in reactions leading to modifications of various primary and secondary metabolites (Giovanelli et al., 1985;Hanson et al., 2000;Roje, 2006). In plants, MTHFRs have been cloned and characterized from Arabidopsis (Arabidopsis thaliana) and maize (Zea mays; Roje et al., 1999). In contrast to the mammalian enzymes, which are NADPH dependent and inhibited by AdoMet, plant MTHFRs are NADH dependent and AdoMet insensitive. In addition, the reaction that they catalyze is likely reversible in the cytosol (Roje et al., 1999). To 1 This work was supported by the Golden LEAF Foundation (a startup fund to the Biomanufacturing Research Institute & Technology Enterprise), the National Institute of General Medical Sciences (grant no. SC3GM088084 to J.X.), the National Science Foundation of China and the Yunnan Tobacco Company (grant nos. 31060046 and 2011YN04 to B.X.), and the National Science Foundation (grant nos. MCB-04...

Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

“…A binary vector designated as A56 ( Figure 1A ) containing a 2×35S promoter and a tobacco glyceraldehyde-3-phosphate dehydrogenase gene ( GapC ) promoter (1291 bp) [40] driving human EPO and GalT [41], respectively was constructed as follows. The Gap C promoter was shown to direct GUS gene expression at high levels both in leaves and roots [40].…”

Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

“…The homologous reductases from mammals differ mainly in that the homomeric enzyme is allosterically regulated by S-adenosylmethionine and is NADP specific (37). The plant reductases are NADH dependent and S-adenosylmethionine insensitive (38). The E. coli type of methylene-H 4 F reductase has been structurally well studied and has an NAD(P) and FAD binding site (39).…”

Effects of overexpression of jasmonic acid biosynthesis genes on nicotine accumulation in tobacco

“…The isoflavone reductase-like enzyme A622 and a berberine bridgelike (BBL) enzyme are proposed to be involved in the condensation step of the pyridine and pyrrolidine rings to form nicotine (DeBoer, Lye, Aitken, Su, & Hamill, 2009;Kajikawa, Hirai, & Hashimoto, 2009). The synthesis and accumulation of the major and minor alkaloids is closely related and dynamically regulated (Chintapakorn & Hamill, 2003;Hung et al, 2013;Kajikawa et al, 2009;Lewis et al, 2015).…”

“…The isoflavone reductase-like enzyme A622 and a berberine bridgelike (BBL) enzyme are proposed to be involved in the condensation step of the pyridine and pyrrolidine rings to form nicotine (DeBoer, Lye, Aitken, Su, & Hamill, 2009;Kajikawa, Hirai, & Hashimoto, 2009). The synthesis and accumulation of the major and minor alkaloids is closely related and dynamically regulated (Chintapakorn & Hamill, 2003;Hung et al, 2013;Kajikawa et al, 2009;Lewis et al, 2015).Previous research indicates that many factors affect nicotine biosynthesis, including mechanical wounding, topping (decapitation of the apical meristem), plant hormones, transcription factors, and negative feedback by pathway products (Baldwin, Schmelz, & Ohnmeiss, 1994;Elliot, 1966;Wasternack & Hause, 2013) In this study, we intended to gain more insights on JA regulated nicotine synthesis in tobacco by modifying expression of the JA biosynthetic genes. As we do not know which enzyme controls the rate-limiting step of JA biosynthesis, we investigated all five major genes in the pathway.…”