Combined reduced expression of two gene families lowers nicotine content to ultra-low levels in cultivated tobacco.

KEY MESSAGE: Reduced expression of two gene families results in ultra-low nicotine accumulation in Nicotiana tabacum. The potential for mandated lowering of tobacco cigarette filler nicotine levels to below 0.4 mg g-1 is currently being discussed by regulatory and public health organizations. Commercial tobacco cultivars that would routinely meet this proposed standard do not currently exist. Inactivation or silencing of gene families corresponding to single enzymatic steps in the nicotine biosynthetic pathways have not resulted in tobacco genotypes that would meet this standard under conventional agronomic management. Here, we produced and evaluated under field conditions tobacco genotypes expressing an RNAi construct designed to reduce expression of the Methyl Putrescine Oxidase (MPO) gene family associated with nicotine biosynthesis. In a standard flue-cured genetic background, cured leaf nicotine levels were reduced to only 1.08 to 1.65 mg g-1. When MPO RNAi was combined with reduced Berberine Bridge Like (BBL) activity conferred by induced mutations, genotypes producing cured leaf nicotine levels slightly lower than 0.4 mg g-1 were generated. Past research has suggested that MPO activity may contribute to the biosynthesis of nornicotine in a route that does not involve nicotine. However, nornicotine was not reduced to zero in MPO-silenced plants that were also homozygous for induced mutations in known Nicotine Demethylase genes that are responsible for the vast majority of nornicotine accumulation.