Transcriptome analysis of Leucojum aestivum and identification of genes involved in norbelladine biosynthesis

Transcriptome analysis of Leucojum aestivum led to the identification of 50 key genes associated with Amaryllidaceae alkaloid biosynthesis including norbelladine synthase which localized in the cytosol and catalyzed norbelladine formation. The Amaryllidaceae alkaloids (AAs) are a large group of plant specialized metabolites, which are known for their biological activities. Although the general chemical reactions in the AA biosynthetic pathway have been proposed, the genes and enzymes of the pathway remain largely unstudied. All AAs are synthesized from a common precursor, norbelladine, by the condensation of tyramine and 3,4-dihydroxybenzaldehyde. The enzyme norbelladine synthase (NBS) which catalyzes the condensation reaction has only been characterized at a molecular level from one species, and the subcellular localizations have not been explored. Hence, the intracellular compartments wherein the AAs are biosynthesized remain unknown. In this study, a first comprehensive transcriptomic analysis of summer snowflake (Leucojum aestivum) was done to identify key genes associated with AA biosynthesis. Fifty orthologous genes were identified and deposited into GenBank. In addition, we identified and further characterized NBS from the transcriptome of L. aestivum and previously reported Narcissus papyraceus. Phylogenetic analysis showed that LaNBS, NpNBS1 and NpNBS2 shared high amino acid identity. The heterologous expression of LaNBS produced a recombinant protein with NBS activity. Bioinformatic prediction and C-terminal GFP tagging in transiently transformed Nicotiana benthamiana showed that LaNBS, NpNBS1 and NpNBS2 were likely localized to the cytosol which suggests that the AA biosynthesis starts in the cytosol. This study provides an Amaryllidaceae transcriptome that will be very helpful to identify genes for characterization studies in AA metabolism in planta or using heterologous systems. In addition, our study will facilitate the bioengineering of AA biosynthetic pathway in plants or in microorganisms.