Abstract
In Nicotinia tabacum, nicotine is a predominant alkaloid which functions as an effective defensive toxin against herbivores. Nicotine is formed by condensation of N-methylpyrrolinium cation and a nicotinic acid derivative, but the enzyme(s) involved in this final condensation step remains elusive. We here report that the previously reported gene A622, which encodes an isoflavone reductase-like protein, and NBB1, which encodes a berberine bridge enzyme-like protein, are both involved in the final condensation steps. Expressions of both genes were tightly regulated by NIC regulatory loci for nicotine biosynthesis. Both genes were specifically expressed in the root, and induced by methyl jasmonate. Suppression of either A622 or NBB1 by RNAi reduced levels of nicotine and related alkaloids in the hairy roots and BY-2 cultured cells. A622-GFP and NBB1-GFP fusion proteins were localized in the plastid and the vacuole, respectively, indicating that final steps of nicotine synthesis may occur in these organelles.
