Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco-specific nitrosamine accumulation in cured leaves and cigarette smoke.

Jianli Lu,Huihua Ji,Anne M Jack,James D Crutchfield,Ralph E Dewey,Lucien Bovet,Ramsey S Lewis,Leichen Zhang,Simon Goepfert

doi:10.1111/pbi.12510

Abstract

SummaryBurley tobaccos (Nicotiana tabacum) display a nitrogen‐use‐deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco‐specific nitrosamines (TSNAs). Two TSNA species, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and N‐nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen‐assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field‐grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well‐documented animal carcinogens found in tobacco products.

Highlights

Cured leaves of different tobacco market types are typically blended together in the production of cigarettes
Genetic studies have determined that loci designated yb1 and yb2 account for the major differences observed between flue-cured and burley tobaccos (Legg et al, 1977)
tobacco-specific nitrosamines (TSNAs) are produced via nitrosation of naturally occurring tobacco alkaloids in processes that predominantly take place during the curing of tobacco leaves, additional formation can occur in the subsequent storage and processing of the leaf, and in some circumstances via pyrosynthesis during combustion

Summary

Introduction

Cured leaves of different tobacco market types (flue-cured, burley and Oriental) are typically blended together in the production of cigarettes. Of these market types, burley tobaccos are unique in that they display a chlorophyll-deficient phenotype and are greatly impaired in their nitrogen-use and nitrogen utilization efficiencies (N-USE and N-UTL, respectively) (Henika, 1932; Lewis et al, 2012; Stines and Mann, 1960). As a likely consequence of their deficient N-UTL phenotype, burley plants accumulate higher levels of free nitrate in their leaves than most other tobacco types (Burton et al, 1994), a trait that is attributable to the yb and yb loci (Lewis et al, 2012). Given that NNN and NNK are among the most potent animal carcinogens found in tobacco products (Hecht, 1998, 2008), there is great interest in reducing their levels in these products

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