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

Abstract

Burley 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.

Keywords: NNK; NNN; burley; nitrate reductase; nitrogen-assimilation pathway; tobacco; tobacco-specific nitrosamines.

Figure 1

Nitrogen‐assimilation pathway in higher plants. Inorganic nitrogen in the form of nitrate or ammonia becomes incorporated into amino acids and other organic molecules as depicted. The specific steps shown include: nitrate transporters (NRT), nitrate reductase (NR), nitrite reductase (NiR), ammonium transporters (AMT), glutamine synthetase (GS), glutamate synthase (GOGAT), asparagine synthetase (AS), glutamate dehydrogenase (GDH), and isocitrate dehydrogenase (ICDH).

Figure 2

Fresh weights of WT plants and 35S:tr‐NR, 35S:S523D‐NR, 35S:GS1, 35S:GOGAT and 35S:ICDH transgenic lines grown under three levels of N‐fertilization. Values shown represent the nontransformed means ± standard errors of 4–6 plants for each genotype. Statistical tests were performed on transformed data (natural logarithmic transformation). Within each nitrate treatment level, means sharing the same letter are not significantly different from each other (P < 0.05); the letter ‘a’ only applies to plants treated with 19 mm nitrate, letters ‘b’ and ‘c’ apply only to plants watered with the 8 mm nitrate media, and ‘d’ and ‘e’ only apply to plants treated with 0.2 mm nitrate.

Figure 3

Total nitrate content in leaves of WT plants and 35S:tr‐NR, 35S:S523D‐NR, 35S:GS1, 35S:GOGAT and 35S:ICDH transgenic lines grown under three levels of N‐fertilization. Data from plants grown using the 19 mm ${\mathrm{NO}}_3^{-}$ and 8 mm ${\mathrm{NO}}_3^{-}$ treatments are shown on the left; results from the 0.2 mm ${\mathrm{NO}}_3^{-}$ treatment are shown on the right. Values shown represent the nontransformed means ± standard errors of 4–6 plants for each genotype. Statistical tests were performed on transformed data (natural logarithmic transformation). Within each nitrate treatment level, means sharing the same letter are not significantly different (P < 0.05) as explained in Figure 2.

Figure 4

Average nitrate and nicotine content in cut tobacco filler derived from the lamina of WT and 35S:S523D‐NR genotypes. All plant materials of each genotype were pooled according to location (about 30 plants each from Clayton and Rocky Mount for 35S:S523D‐NR; about 40 plants from each location for WT). Data shown represent the average and standard error across both locations.

Figure 5

Average content of NAB (a), NAT (b), NNK (c), NNN (d) and total TSNAs (e) in the cut filler and smoke of cigarettes made from the lamina of WT and 35S:S523D‐NR leaves. Leaf materials of each genotype were pooled according to each of two locations as described in Fig. 4. Data shown represent the combined means and standard error of two replications per location for the TSNA content of the cut filler, and three replications per location for TSNA content in mainstream smoke.