Comparative sequencing of alkaloid plant transcriptomes
Project Contact: Toni Kutchan, Danforth Plant Science Center
Plants from widely distributed families produce a variety of alkaloids that contain similar structural elements. The monoterpenoid-tetrahydroisoquinoline alkaloids (Rubiaceae) that contain a secologanin and two dopamine moieties are related to the tetrahydroisoquinoline alkaloids (Papaveraceae, Berberidaceae; two dopamine moieties). Although the structures of multiple alkaloids from these general classes have been known for decades, only a partial understanding of the enzymology and molecular genetics of their formation has been gained. Availability of these biosynthetic cDNAs will widen the alkaloid field to a variety of studies leading to new knowledge on regulation, localization, transcription factors and synthetic biology of known and new medicinal compounds.
Plant alkaloid formation is developmentally regulated. A comparison of the tissue-specific, time-dependent expression of genes in a sampling of alkaloid-producing plant species is expected to provide insight into candidate genes encoding novel alkaloid biosynthetic enzymes. Constructing the biosynthetic pathways that produce plant-derived pharmaceuticals will be aided by elucidation of the origin and diversification of novel pathways within larger metabolic and regulatory networks. Cross-species comparative analyses of the enzyme encoding and the regulatory genes involved in alkaloid biosynthesis is expected to elucidate the evolutionary origins of pathways producing specific classes of alkaloids.
To achieve the above, five tissues - leaf, stem, root, flower bud, developing fruit - will be sampled from 7 medicinal plant species with interesting alkaloid profiles. Each tissue samples will be divided in two for RNA isolation and extraction of organic compounds, respectively. Three biological replicates should be done per tissue/species for proper analyses of covariation between gene expression levels and alkaloid concentrations. RNAs will be sent to the 1KP labs for cDNA synthesis and alkaloids will be quantitatively characterized in the Kutchan lab.
Possible alkaloid biosynthesis genes will be identified through analyses of covariation among gene expression levels and alkaloid content among the seven tissues. Phylogenetically-based cross-species comparisons are expected to narrow our set of candidate alkaloid biosynthetic and regulatory genes. These analyses will also allow us to hypothesize the identity of genes contributing to the synthesis of novel alkaloids. All hypotheses drawn from analyses of covariation will need to be tested experimentally in order to make sound conclusions about the gene and biochemical processes involved in alkaloid biosynthesis.