Transcript and metabolite profiling in cell cultures of 18 plant species that produce benzylisoquinoline alkaloids

Abstract

Benzylisoquinoline alkaloids (BIAs) are a large and diverse group of ∼2500 specialized metabolites found predominantly in plants of the order Ranunculales. Research focused on BIA metabolism in a restricted number of plant species has identified many enzymes and cognate genes involved in the biosynthesis of compounds such as morphine, sanguinarine and berberine. However, the formation of most BIAs remains uncharacterized at the molecular biochemical level. Herein a compendium of sequence- and metabolite-profiling resources from 18 species of BIA-accumulating cell cultures was established, representing four related plant families. Our integrated approach consisted of the construction of EST libraries each containing approximately 3500 unigenes per species for a total of 58,787 unigenes. The EST libraries were manually triaged using known BIA-biosynthetic genes as queries to identify putative homologs with similar or potentially different functions. Sequence resources were analyzed in the context of the targeted metabolite profiles obtained for each cell culture using electrospray-ionization and collision-induced dissociation mass spectrometry. Fragmentation analysis was used for the identification or structural characterization coupled with the relative quantification of 72 BIAs, which establishes a key resource for future work on alkaloid biosynthesis. The metabolite profile obtained for each species provides a rational basis for the prediction of enzyme function in BIA metabolism. The metabolic frameworks assembled through the integration of transcript and metabolite profiles allow a comparison of BIA metabolism across several plant species and families. Taken together, these data represent an important tool for the discovery of BIA biosynthetic genes.