Genomics‐based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula

Abstract

SummaryThe biosynthesis of triterpene saponins is poorly characterized in spite of the importance of these glycosylated secondary metabolites for plant defense and animal health. The model legume Medicago truncatula synthesizes more than 30 different saponins based on at least five triterpene aglycones; soyasapogenols B and E, medicagenic acid, hederagenin and bayogenin. We have employed an inducible cell culture system, DNA array-based and in silico transcript profiling, and targeted metabolite profiling, to identify triterpene glycosyltransferases (GTs) from among the more than 300 GTs expressed in M. truncatula. Two uridine diphosphate glucosyltransferases were functionally characterized; UGT73K1 with specificity for hederagenin and soyasapogenols B and E, and UGT71G1 with specificity for medicagenic acid. The latter enzyme also glycosylated certain isoflavones and the flavonol quercetin with higher efficiency than triterpenes; however, integrated transcript and metabolite profiling supported a function for UGT71G1 in terpenoid but not (iso)flavonoid biosynthesis in the elicited cell cultures.

Phenylpropanoid glycosyltransferases from osage orange (Maclura pomifera) fruit

“…The possession of low affinity and turnover in vitro does not preclude an enzyme of secondary metabolism from functioning with a ''poor'' substrate in vivo. Examples include the potential involvement of a catalytically inefficient O-methyltransferase in flavor production in strawberry [27] and of a glycosyltransferase with preference for quercetin and genistein in the glycosylation of triterpenes in Medicago truncatula [18]. Interpretation of in vivo substrate specificity from in vitro measurements is dangerous in the absence of knowledge of the actual metabolites existing in vivo.…”

“…The reaction mix was incubated at 30°C for 3 h and stopped by adding 2 ll of trichloroacetic acid (TCA; 240 mg/ ml). Ten microliter of methanol was added to each reaction mix, and 50 ll of the reaction was injected on a reverse phase HPLC column and separation performed as described previously [18]. Identification of glycosylated product was based on both retention time and UV spectrum, by comparison either to authentic standards or to aglycones, where the conjugates showed similar UV spectra but altered retention time.…”

“…The fine powder was extracted with ethyl acetate and dried under a stream of nitrogen. Dried residue was resuspended in methanol and injected on a reverse phase high performance liquid chromatography (HPLC) column as previously described, with a slight modification of the HPLC gradient [18]. Elution from 50% B (acetonitrile) to 100% B was for 24 min, instead of 4 min as in the original program.…”

Metabolomics Reveals Novel Pathways and Differential Mechanistic and Elicitor-Specific Responses in Phenylpropanoid and Isoflavonoid Biosynthesis in Medicago truncatula Cell Cultures

“…Previous studies used targeted metabolite profiling to demonstrate that MeJA elicits the accumulation of triterpene saponins (Suzuki et al, 2002(Suzuki et al, , 2005Achnine et al, 2005), whereas YE, a pathogen mimic, induces the accumulation of both free and glycosylated isoflavonoids (Kessmann et al, 1990;Suzuki et al, 2005). The changes in primary metabolism in these elicited cell cultures have been described earlier (Broeckling et al, 2005), and a detailed analysis of transcript profiles will appear in a parallel article (Naoumkina et al, 2007).…”

Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells

“…Combined transcript and metabolite profiling approaches to tackle the MeJA-mediated regulation of secondary metabolism resulted in the establishment of gene-to-metabolite networks involved in the biosynthesis of the pharmaceutically valuable terpenoid indole alkaloids in periwinkle (Catharanthus roseus) cells (7), in the characterization of enzymatic steps in the isoflavone and triterpene biosynthetic pathways in barrel medic (Medicago truncatula) cells (8,9), and in the isolation of novel regulators of nicotine and phenylpropanoid conjugate biosynthesis in tobacco (Nicotiana tabacum) cells (10)(11)(12)(13) or of aliphatic glucosinolate biosynthesis in Arabidopsis thaliana cells (14).…”