Abstract
Five glucosyltransferases were cloned by RT-PCR amplification using total RNA from Hieracium pilosella L. (Asteraceae) inflorescences as template. Expression was accomplished in Escherichia coli, and three of the HIS-tagged enzymes, UGT90A7, UGT95A1, and UGT72B11 were partially purified and functionally characterised as UDP-glucose:flavonoid O-glucosyltransferases. Both UGT90A7 and UGT95A1 preferred luteolin as substrate, but possessed different regiospecificity profiles. UGT95A1 established a new subgroup within the UGT family showing high regiospecificity towards the C-3′ hydroxyl group of luteolin, while UGT90A7 primarily yielded the 4′-O-glucoside, but concomitantly catalysed also the formation of the 7-O-glucoside, which could account for this flavones glucoside in H. pilosella flower heads. Semi quantitative expression profiles revealed that UGT95A1 was expressed at all stages of inflorescence development as well as in leaf and stem tissue, whereas UGT90A7 transcript abundance was nearly limited to flower tissue and started to develop with the pigmentation of closed buds. Other than these enzymes, UGT72B11 showed rather broad substrate acceptance, with highest activity towards flavones and flavonols which have not been reported from H. pilosella. As umbelliferone was also readily accepted, this enzyme could be involved in the glucosylation of coumarins and other metabolites.
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Abbreviations
- GT:
-
Glycosyltransferases
- UGT:
-
UDP-Glycosyltransferases
- PSPG:
-
Plant secondary product glycosyltransferases
- RACE:
-
Rapid amplification of cDNA ends
- HSCCC:
-
High-speed counter current chromatography
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Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft and Dr. Sofia Moco is financed by the EU project “META-PHOR” (FOOD-CT-2006-036220).
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425_2009_902_MOESM2_ESM.tif
Alignment of H. pilosella UGT polypeptides using the ClustalW program. The conserved PSPG motif is shown in bold letters. UGT72B11 (GenBank accession number EU561016), UGT88A8 (EU561017), UG88A9 (EU561018), UGT90A7 (EU561019), UGT95A1 (EU561020) (TIFF 65 kb)
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Witte, S., Moco, S., Vervoort, J. et al. Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.. Planta 229, 1135–1146 (2009). https://doi.org/10.1007/s00425-009-0902-x
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DOI: https://doi.org/10.1007/s00425-009-0902-x