Downregulation of a putative nitrate transporter gene substantially reduces the accumulation of TSNAs in air-cured tobaccos
Air-cured Burley tobacco leaves typically exhibit high levels of nicotine-derived nitrosamine ketone (NNK) and N'-nitrosonornicotine (NNN). It has been previously demonstrated that leaf nitrate accumulation in air-cured tobaccos serves as the source of nitrosating agents that contributes toward the production of both NNK and NNN. Within this context, the reduction of leaf nitrate stores might represent a viable strategy for efficiently reducing NNK and NNN in tobacco products. In Arabidopsis, several genes encoding nitrate transporters have been identified, including AtCLCa, whose tonoplast-localized gene product mediates sequestration of nitrate into the vacuole. The best AtCLCa orthologue gene candidate in Nicotiana tabacum was named CLC-Nt2 and is present as two copies within the genome, with one originating from N. sylvestris (CLC-Nt2-S) and the other from N. tomentosiformis (CLC-Nt2-T). For proof of concept, we generated anti-CLC-Nt2 constructs to silence both gene copies in transgenic plants also lacking the functional nicotine demethylase genes, CYP82E4 and CYP82E5. CLC-Nt2-RNAi plants and control plants were grown in the field. Interestingly, downregulation of CLC-Nt2 reduced nitrate storage in cured Burley leaves by 60-70 % without impacting yield. Consequently, NNK was reduced by around 40 % and NNN by 40-50 % in the leaf lamina. Smoke analysis using prototype cigarettes manufactured using the low-nitrate leaves demonstrated a 35 % reduction in NNK and a 47 % reduction in NNN. Our data confirm that decreasing nitrate levels in air-cured tobacco leaves contributes to decreased NNK and NNN in both lamina and smoke by impacting the nitrosation process. Inducing, selecting, and pyramiding knockout mutations within CLC-Nt2 genes likely represents an efficacious means of generating non-transgenic, reduced-tobacco-specific nitrosamine Burley varieties. Finally, maximal reductions in NNN content would be predicted by combining knockout CLC-Nt2 technologies with the Zyvert trait.