Downregulation of a putative nitrate transporter (CLC-Nt2) substantially reduces the accumulation of TSNAs in air-cured tobacco

Nicotine-derived nitrosamine ketone (NNK) and N’-nitrosonornicotine (NNN) are tobacco-specific nitrosamines naturally synthesized in tobacco leaves during the curing process and combustion of tobacco. They are classified as carcinogenic. Air-cured Burley tobacco leaves typically exhibit high levels of NNK and NNN. It has been previously demonstrated that leaf nitrate accumulation in air-cured tobaccos serves as the source of nitrosating agents that contributes to the production of both NNK and NNN. In this context, the reduction of leaf nitrate stores might represent an efficient strategy for 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, one originating from N. sylvestris (CLC-Nt2-S) and the other one from N. tomentosiformis (CLC-Nt2-T). For proof of concept, we generated anti-CLC-Nt2 constructs to silence both gene copies in transgenic plants that 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.