@article{lewis_lopez_bowen_andres_steede_dewey_2015, title={Transgenic and Mutation-Based Suppression of a Berberine Bridge Enzyme-Like (BBL) Gene Family Reduces Alkaloid Content in Field-Grown Tobacco}, volume={10}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0117273}, abstractNote={Motivation exists to develop tobacco cultivars with reduced nicotine content for the purpose of facilitating compliance with expected tobacco product regulations that could mandate the lowering of nicotine levels per se, or the reduction of carcinogenic alkaloid-derived tobacco specific nitrosamines (TSNAs). A berberine bridge enzyme-like (BBL) gene family was recently characterized for N. tabacum and found to catalyze one of the final steps in pyridine alkaloid synthesis for this species. Because this gene family acts downstream in the nicotine biosynthetic pathway, it may represent an attractive target for genetic strategies with the objective of reducing alkaloid content in field-grown tobacco. In this research, we produced transgenic doubled haploid lines of tobacco cultivar K326 carrying an RNAi construct designed to reduce expression of the BBL gene family. Field-grown transgenic lines carrying functional RNAi constructs exhibited average cured leaf nicotine levels of 0.684%, in comparison to 2.454% for the untransformed control. Since numerous barriers would need to be overcome to commercialize transgenic tobacco cultivars, we subsequently pursued a mutation breeding approach to identify EMS-induced mutations in the three most highly expressed isoforms of the BBL gene family. Field evaluation of individuals possessing different homozygous combinations of truncation mutations in BBLa, BBLb, and BBLc indicated that a range of alkaloid phenotypes could be produced, with the triple homozygous knockout genotype exhibiting greater than a 13-fold reduction in percent total alkaloids. The novel source of genetic variability described here may be useful in future tobacco breeding for varied alkaloid levels.}, number={2}, journal={PLOS ONE}, author={Lewis, Ramsey S. and Lopez, Harry O. and Bowen, Steve W. and Andres, Karen R. and Steede, William T. and Dewey, Ralph E.}, year={2015}, month={Feb} }
 @article{li_lewis_jack_dewey_bowen_miller_2012, title={Development of CAPS and dCAPS markers for CYP82E4, CYP82E5v2 and CYP82E10 gene mutants reducing nicotine to nornicotine conversion in tobacco}, volume={29}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-011-9575-9}, number={3}, journal={MOLECULAR BREEDING}, author={Li, Dandan and Lewis, Ramsey S. and Jack, Anne M. and Dewey, Ralph E. and Bowen, Steve W. and Miller, Robert D.}, year={2012}, month={Mar}, pages={589–599} }
 @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{chakrabarti_bowen_coleman_meekins_dewey_siminszky_2008, title={CYP82E4-mediated nicotine to nornicotine conversion in tobacco is regulated by a senescence-specific signaling pathway}, volume={66}, ISSN={["1573-5028"]}, DOI={10.1007/s11103-007-9280-6}, number={4}, journal={PLANT MOLECULAR BIOLOGY}, author={Chakrabarti, Manohar and Bowen, Steven W. and Coleman, Nicholas P. and Meekins, Karen M. and Dewey, Ralph E. and Siminszky, Balazs}, year={2008}, month={Mar}, pages={415–427} }
 @article{gavilano_coleman_bowen_siminszky_2007, title={Functional analysis of nicotine demethylase genes reveals insights into the evolution of modern tobacco}, volume={282}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M609512200}, abstractNote={Tobacco (Nicotiana tabacum L.) is a natural allotetraploid derived from the interspecific hybridization between ancestral Nicotiana sylvestris and Nicotiana tomentosiformis. The majority of cultivated tobacco differs from both of its progenitor species in that tobacco typically contains nicotine as the primary alkaloid, in contrast to its two progenitors that accumulate nornicotine in the senescing leaves. However, most, if not all, tobacco cultivars possess an unstable mutation, commonly referred to as the conversion locus, that when activated mediates the conversion of a large percentage of nicotine to nornicotine in the senescing leaf. We have recently identified CYP82E4, a tobacco nicotine N-demethylase gene whose expression was highly induced during senescence in plants that have converted, and CYP82E3, a closely related homolog that exhibited no nicotine N-demethylase activity. In this study, domain swapping and site-directed mutagenesis studies identified a single amino acid change that fully restored nicotine N-demethylase activity to CYP82E3. An examination of the N. tomentosiformis orthologs of CYP82E3 and CYP82E4 revealed that both are functional nicotine N-demethylase genes in N. tomentosiformis. Collectively, our results suggest that a single base pair mutation in CYP82E3 and transcriptional suppression of CYP82E4 played important roles in the evolution of the alkaloid profile characteristic of modern tobacco.}, number={1}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Gavilano, Lily B. and Coleman, Nicholas P. and Bowen, Steven W. and Siminszky, Balazs}, year={2007}, month={Jan}, pages={249–256} }