Poster: Secondary metabolism

Abs # 252: The Functional Characterization of Medicago truncatula Glycosyltransferases Involved in Natural Product Biosynthesis

Presenter:	Blount, Jack W., jwblount@noble.org
Authors	Blount, Jack W. (A)   Achnine, Lahoucine  (A)   Dixon, Richard A. (A)
Affiliations:	(A): The Samuel Roberts Noble Foundation

Glycosyltransferases (GTs) aid in the stabilization, compartmentation, and activation of secondary metabolites by transferring a nucleotide-diphosphate-activated sugar onto these low molecular weight compounds. Medicago truncatula, a model legume, contains many glycosylated secondary metabolites such as isoflavonoids, flavonoids, anthocyanins, and triterpene saponins. Using our extensive M. truncatula EST database, we have identified 63 full length putative GTs which may be involved in glycosylation of secondary metabolites. Of the 63 clones, 53 fit into 13 of the 14 Arabidopsis thaliana GT families, while the 10 remaining clones fit into two GT families not found in Arabidopsis. An efficient HPLC method was developed to analyze these 63 GT candidates against 32 potential substrates by dividing the substrates equally into four groups. The compounds in each group are readily separated by the HPLC method, and each compound within a particular group has a distinct UV spectrum. The HPLC method was tested using one of the M. truncatula GT clones and a human hepatic glucoronosyltransferase, UGT1A6 supersomes (GenTest), which glucoronidates a wide range of small planar phenolic compounds. The liver enzyme used UDP-glucuronic acid while the M. truncatula clone used UDP-glucose as the active sugar donor. The liver enzyme glycosylated the anthocyanin pelargonidin as well as vanillin, while the M. truncatula clone glycosylated quercetin. These results were reconfirmed for both enzymes by assaying them with individual substrates. This poster will present functional analysis of the soluble M. truncatula GTs assayed with the 32 compounds in the four groups and several different UDP-sugar donors.