CLCNt2 Mediates Nitrate Content in Tobacco Leaf, Impacting the Production of Tobacco-Specific Nitrosamines in Cured Leaves.

Lucien Bovet,Samuel Kleinhans,Ralph E Dewey,Simon Goepfert,Prisca Campanoni,Joanne Schwaar,Jian Lu,Ramsey S Lewis,Hélène Laparra,Yuki Matsuba,Hong Ma,Aurore Hilfiker

doi:10.3389/fpls.2022.741078

Abstract

Nitrate accumulation in tobacco (Nicotiana tabacum L.) leaf, particularly in the burley (BU) type, is a reservoir for the generation of nitrosating agents responsible for the formation of tobacco-specific nitrosamines (TSNAs). TSNAs are mainly produced via the nitrosation of alkaloids occurring during the curing of tobacco leaves. Additional formation of TSNAs may also occur during tobacco storage, leaf processing and in some circumstances via pyrosynthesis during combustion. Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are found in the tobacco products and have been documented to be animal carcinogens. A previous study showed that decreasing the accumulation of nitrate in tobacco leaf via the overexpression of a deregulated form of nitrate reductase is efficient to reduce the production of TSNAs. We pursue in finding another molecular genetic target to lower nitrate in BU tobacco. Suppressing expression or knocking-out CLCNt2 has a direct impact on leaf nitrate and TSNA reduction in cured leaves without altering biomass. This study provides now a straight path toward the development of new commercial tobacco varieties with reduced TSNA levels by breeding of variants deficient in active CLCNt2 copies.

Highlights

The International Agency for Research on Cancer has identified eight tobacco-specific nitrosamines (TSNAs) in tobacco and tobacco smoke (IARC, 2004 and the references therein)
For such KO mutations, we used a TN90 as background, instead of TN90e4e5, to measure the impact of the lack CLCNt2 copies in a tobacco type used in agronomy, the TN90e4e5 plants being not implemented in the field to date
We have effectively demonstrated that decreasing the expression of CLCNt2-S and CLCNt2-T in tobacco limits the storage of nitrate in the leaf, reducing the propensity for TSNA formation in the cured lamina and in the smoke of cigarettes made from these tobaccos

Summary

Introduction

The International Agency for Research on Cancer has identified eight tobacco-specific nitrosamines (TSNAs) in tobacco and tobacco smoke (IARC, 2004 and the references therein). They are unique to tobacco and are present in smokeless tobacco, snuff, cigarettes, and electronic cigarette liquids. Two TSNAs have been classified as group 1 carcinogens: N-nitrosonornicotine (NNN) and 4-methylN-nitrosamino-1-(3-pyridyl)-1-butanone (NNK). For this reason, strategies for reducing TSNA levels in tobacco are being developed. No genetic solution has been found to significantly reduce the levels of NNK. A technique known as the ZyvertTM technology (Lusso et al, 2017) can reduce the levels of NNN and, its precursor nornicotine by knocking out three

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