(R)-nicotine biosynthesis, metabolism and translocation in tobacco as determined by nicotine demethylase mutants

The accumulation and translocation of (R)-nicotine were investigated in tobacco with different mutation combinations of three nicotine demethylases. •(R)-nicotine accumulation and translocation was investigated.•Four percent of nicotine was in the (R) form at synthesis in root.•Most (R)-nicotine was selectively demethylated before being translocated to leaf.•The rest was demethylated in the leaf, resulting in low (R)-nicotine content in leaf. Nicotine is a chiral compound and consequently exists as two enantiomers. Since (R)-nicotine consists of less than 0.5% of total nicotine pool in tobacco, few investigations relating to (R)-nicotine have been reported. However, previous studies of nicotine demethylases suggested there was substantial amount of (R)-nicotine at synthesis in the tobacco plant. In this study, the accumulation and translocation of (R)-nicotine in tobacco was analyzed. The accumulation of nicotine and its demethylation product the nornicotine enantiomers, were investigated in different tobacco plant parts and at different growth and post-harvest stages. Scion/rootstock grafts were used to separate the contributions of roots (source) from leaves (sink) to the final accumulation of nicotine and nornicotine in leaf tissue. The results indicate that 4% of nicotine is in the (R) form at synthesis in the root. After the majority of (R)-nicotine is selectively demethylated by CYP82E4, CYP82E5v2 and CYP82E10 in the root, nicotine and nornicotine are translocated to leaf, where more nicotine becomes demethylated. Depending on the CYP82E4 activity in senescing leaf, constant low (R)-nicotine remains in the tobacco leaf and variable nornicotine composition is produced. These results confirmed the enantioselectivity of three nicotine demethylases in planta, could be used to predict the changes of nicotine and nornicotine composition, and may facilitate demethylase discovery in the future.