@article{lewis_bowen_keogh_dewey_2010, title={Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L Functional characterization of the CYP82E10 gene}, volume={71}, ISSN={["1873-3700"]}, DOI={10.1016/j.phytochem.2010.09.011}, abstractNote={In most tobacco (Nicotiana tabacum L.) plants, nornicotine is a relatively minor alkaloid, comprising about 2–5% of the total pyridine alkaloid pool in the mature leaf. Changes in gene expression at an unstable locus, however, can give rise to plants that produce high levels of nornicotine, specifically during leaf senescence and curing. Minimizing the nornicotine content in tobacco is highly desirable, because this compound serves as the direct precursor in the synthesis of N′-nitrosonornicotine, a potent carcinogen in laboratory animals. Nornicotine is likely produced almost entirely via the N-demethylation of nicotine, in a process called nicotine conversion that is catalyzed by the enzyme nicotine N-demethylase (NND). Previous studies have identified CYP82E4 as the specific NND gene responsible for the unstable conversion phenomenon, and CYP82E5v2 as a putative minor NND gene. Here, by discovery and characterization of CYP82E10, a tobacco NND gene, is reported. PCR amplification studies showed that CYP82E10 originated from the N. sylvestris ancestral parent of modern tobacco. Using a chemical mutagenesis strategy, knockout mutations were induced and identified in all three tobacco NND genes. By generating a series of mutant NND genotypes, the relative contribution of each NND gene toward the nornicotine content of the plant was assessed. Plants possessing knockout mutations in all three genes displayed nornicotine phenotypes that were much lower (∼0.5% of total alkaloid content) than that found in conventional tobacco cultivars. The introduction of these mutations into commercial breeding lines promises to be a viable strategy for reducing the levels of one of the best characterized animal carcinogens found in tobacco products.}, number={17-18}, journal={PHYTOCHEMISTRY}, author={Lewis, Ramsey S. and Bowen, Steven W. and Keogh, Matthew R. and Dewey, Ralph E.}, year={2010}, month={Dec}, pages={1988–1998} }
 @article{keogh_courtney_kinney_dewey_2009, title={Functional Characterization of Phospholipid N-Methyltransferases from Arabidopsis and Soybean}, volume={284}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M109.005991}, abstractNote={Phospholipid N-methyltransferase (PLMT) enzymes catalyze the S-adenosylmethionine-dependent methylation of ethanolamine-containing phospholipids to produce the abundant membrane lipid phosphatidylcholine (PtdCho). In mammals and yeast, PLMT activities are required for the de novo synthesis of the choline headgroup found in PtdCho. PLMT enzyme activities have also been reported in plants, yet their roles in PtdCho biosynthesis are less clear because most plants can produce the choline headgroup entirely via soluble substrates, initiated by the methylation of free ethanolamine-phosphate. To gain further insights into the function of PLMT enzymes in plants, we isolated PLMT cDNAs from Arabidopsis and soybean (Glycine max) based upon primary amino acid sequence homology to the rat PLMT, phosphatidylethanolamine N-methyltransferase. Using a heterologous yeast expression system, it was shown that plant PLMTs methylate phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine but cannot utilize phosphatidylethanolamine as a substrate. Identification of an Arabidopsis line containing a knock-out dissociator transposon insertion within the single copy AtPLMT gene allowed us to investigate the consequences of loss of PLMT function. Although the accumulation of the PLMT substrates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine was considerably elevated in the atplmt knock-out line, PtdCho levels remained normal, and no obvious differences were observed in plant morphology or development under standard growth conditions. However, because the metabolic routes through which PtdCho is synthesized in plants vary greatly among differing species, it is predicted that the degree with which PtdCho synthesis is dependent upon PLMT activities will also vary widely throughout the plant kingdom.}, number={23}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Keogh, Matthew R. and Courtney, Polly D. and Kinney, Anthony J. and Dewey, Ralph E.}, year={2009}, month={Jun}, pages={15439–15447} }