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A tandem array of UDP-glycosyltransferases from the UGT73C subfamily glycosylate sapogenins, forming a spectrum of mono- and bisdesmosidic saponins

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This study identifies six UGT73Cs all able to glucosylate sapogenins at positions 3 and/or 28 which demonstrates that B. vulgaris has a much richer arsenal of UGTs involved in saponin biosynthesis than initially anticipated.

Abstract

The wild cruciferous plant Barbarea vulgaris is resistant to some insects due to accumulation of two monodesmosidic triterpenoid saponins, oleanolic acid 3-O-β-cellobioside and hederagenin 3-O-β-cellobioside. Insect resistance depends on the structure of the sapogenin aglycone and the glycosylation pattern. The B. vulgaris saponin profile is complex with at least 49 saponin-like metabolites, derived from eight sapogenins and including up to five monosaccharide units. Two B. vulgaris UDP-glycosyltransferases, UGT73C11 and UGT73C13, O-glucosylate sapogenins at positions 3 and 28, forming mainly 3-O-β-d-glucosides. The aim of this study was to identify UGTs responsible for the diverse saponin oligoglycoside moieties observed in B. vulgaris. Twenty UGT genes from the insect resistant genotype were selected and heterologously expressed in Nicotiana benthamiana and/or Escherichia coli. The extracts were screened for their ability to glycosylate sapogenins (oleanolic acid, hederagenin), the hormone 24-epibrassinolide and sapogenin monoglucosides (hederagenin and oleanolic acid 3-O-β-d-glucosides). Six UGTs from the UGT73C subfamily were able to glucosylate both sapogenins and both monoglucosides at positions 3 and/or 28. Some UGTs formed bisdesmosidic saponins efficiently. At least four UGT73C genes were localized in a tandem array with UGT73C11 and possibly UGT73C13. This organization most likely reflects duplication events followed by sub- and neofunctionalization. Indeed, signs of positive selection on several amino acid sites were identified and modelled to be localized on the UGT protein surface. This tandem array is proposed to initiate higher order bisdesmosidic glycosylation of B. vulgaris saponins, leading to the recently discovered saponin structural diversity, however, not directly to known cellobiosidic saponins.

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Acknowledgements

We thank Dr. Francisco R. Badenes-Pérez for the generous gift of hederagenin 3-O-β-cellobioside, Dr. Carl Erik Olsen for NMR-spectroscopy of this saponin, Dr. Elizabeth Heather Jakobsen Neilson for assistance in LC-MS analysis, Dr. Mika Zagrobelny for assistance in phylogenetic analysis and analysis for positive selection, Dr. Qing Liu for discussions and sharing a standard, Dr. Bekzod Khakimov for discussions and sharing a protocol for alkaline hydrolysis, Mette Sørensen for thorough reading and constructive ideas during writing, Michael Court and the UGT nomenclature committee for systematic UGT names, and three anonymous reviewers for constructive comments and suggestions. This work was funded by the Danish Council for Independent Research, Technology and Production Sciences (Grant No. 1335-00151) and Department of Plant and Environmental Sciences, University of Copenhagen (PhD stipend to PØE).

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  1. Department of Plant and Environmental Sciences and Copenhagen Plant Science Center, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark

    Pernille Østerbye Erthmann, Niels Agerbirk & Søren Bak

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  1. Pernille Østerbye Erthmann
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Conceived research: PØE, SB. Planned experiments: All. Carried out research and analyzed data: PØE supervised by NA and SB. Wrote draft: PØE. Wrote paper: All.

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Correspondence to Søren Bak.

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Erthmann, P.Ø., Agerbirk, N. & Bak, S. A tandem array of UDP-glycosyltransferases from the UGT73C subfamily glycosylate sapogenins, forming a spectrum of mono- and bisdesmosidic saponins. Plant Mol Biol 97, 37–55 (2018). https://doi.org/10.1007/s11103-018-0723-z

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