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CORESTA Meeting, Agronomy/Phytopathology, 2021, Online, AP 49

Consequences of molecular genetic alteration of leaf nitrate levels on TSNAs, alkaloids, flowering time, and biomass in Burley tobaccos

DEWEY R.E.(1); BOVET L.(2); GOEPFERT S.(2); CAMPANONI P.(2); LEWIS R.S.(1)
(1) North Carolina State University, Dept. of Crop and Soil Sciences, Raleigh, NC, U.S.A.; (2) PMI R&D, Philip Morris Products S.A., Neuchatel, Switzerland

We have previously demonstrated that tobacco-specific nitrosamine (TSNA) levels in Burley tobaccos can be substantially lowered using two distinct molecular genetic techniques designed to impede the plant’s ability to store high levels of free nitrate in the cured leaf: (1) overexpression of a mutant nitrate reductase (NR) enzyme that is in a continually activated state and (2) downregulation of two closely related nitrate transporters, designated CLCNt2-S and CLCNt2-T. We have conducted multiple-year field trials to assess the effects of these genetic modifications on both the chemical (nitrate, alkaloids, and TSNAs) and agronomic (flowering time and plant biomass at harvest) properties of the plant. Plants expressing a constitutively activated NR enzyme flower prematurely and accumulate less biomass than wildtype control Burley tobaccos. However, by making F1 hybrids with Burley genotypes known to flower atypically late, the biomass and/or flowering time were largely restored to normal without compromising the ability of the NR transgene to lower nitrate and TSNA levels. Reducing leaf nitrate levels via the genome editing-mediated knockout of CLCNt2-S and CLCNt2-T can also influence flowering time and aerial biomass. Disruption of CLCNt2-S, but not CLCNt2-T, confers a very late-flowering phenotype, suggesting that the two closely related gene isoforms are not functionally redundant. The non-equivalence of CLCNt2-S and CLCNt2-T is also supported by the results of transcript profiling assays of the two genes across different tissue types and developmental stages. Nevertheless, in order to significantly lower cured leaf nitrate and TSNA levels, both CLCNt2-S and CLCNt2-T must be rendered non-functional. The results of these studies provide key insights into how promising TSNA-reduction techniques can best be deployed in a manner that minimally impacts key agronomic properties.