Nicotine is synthesized in the tobacco root and translocated to the leaf where it may be demethylated to nornicotine. Three functional nicotine demethylases (CYP82E4, CYP82E5 and CYP82E10) have been identified in tobacco to date. CYP82E4 (E4) is the major demethylase and mainly functions in senescent leaf. Expression of CYP82E10 (E10) is reported to be in the roots and CYP82E5 (E5) functions in both root and leaf.

In tobacco leaf, nicotine is comprised of 0.2 % of (R)-nicotine, but the nicotine metabolite, nornicotine, consists of 4-75 % of (R)-nornicotine. The reasons for this discrepancy between nicotine and nornicotine enantiomeric composition is not known. Previously, we have confirmed the preference of E4 for (R)-nicotine in vitro which can explain why a high (R)-nornicotine content could result from a low (R)-nicotine percentage. In this study, our goal was to evaluate the role of each demethylase in nicotine and nornicotine composition in root and leaf tissue.

Two sets of grafts were generated from nicotine demethylase tobacco mutants and tomato. First set of grafts were generated from nicotine demethylase tobacco mutants and tomato. These grafts were with a tomato (scion) on a tobacco mutant root stock. The second set was grafts with nicotine demethylase tobacco mutants (scion) on the triple mutant root stock. After recovery, the grafts were topped to stimulate nicotine production and then analyzed for nicotine and nornicotine composition. The results indicate that (1) in the root, originally synthesized nicotine may consist of 3% (R)-nicotine; (2) E5 and E10 selectively demethylate (R)-nicotine to (R)-nornicotine in root, before translocation of nicotine to leaf and (3) in leaf, E4 demethylates (S)-nicotine into (S)-nornicotine during senescence.