La Trobe
A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family.pdf (3.99 MB)
Download file

A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family

Download (3.99 MB)
journal contribution
posted on 22.03.2021, 06:13 by L Xuan, J Zhang, W Lu, P Gluza, B Ebert, T Kotake, M Lu, Y Zhang, MH Clausen, Kim JohnsonKim Johnson, Monika DoblinMonika Doblin, JL Heazlewood, Tony BacicTony Bacic, Lili SongLili Song, W Zeng
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The Arabidopsis thaliana genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI Arabidopsis GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-com-patible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (Nicotiana benthamiana) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identify-ing effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic accep-tors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal4) and β-1,3-galacto-pentose (β-1,3-Gal5) to microsomes expressing individual GT14 enzymes, we verified the β-glucu-ronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In ad-dition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosyn-thesis for these GT14 members. Together, the updated Arabidopsis GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.

Funding

This research was funded by the National Natural Science Foundation of China (grant no. 31971619) and the Overseas Expertise Introduction Project for Discipline Innovation (111 Project D18008) and by grants from Australia Research Council to the ARC Centre of Excellence in Plant Cell Walls (CE1101007) to A.B., M.S.D., K.L.J. and W.Z. M.H.C. acknowledges funding from the Novo Nordisk Foundation (grant nos. NNF18OC0053048 and NNF20OC0065094) and the Carlsberg Foundation (grant no. CF19-0072).

History

Publication Date

01/02/2021

Journal

International Journal of Molecular Sciences

Volume

22

Issue

3

Article Number

ARTN 1360

Pagination

(p. 1-22)

Publisher

MDPI

ISSN

1661-6596

Rights Statement

The Author reserves all moral rights over the deposited text and must be credited if any re-use occurs. Documents deposited in OPAL are the Open Access versions of outputs published elsewhere. Changes resulting from the publishing process may therefore not be reflected in this document. The final published version may be obtained via the publisher’s DOI. Please note that additional copyright and access restrictions may apply to the published version.