Functional analysis of GT61 glycosyltransferases from grass species in xylan substitutions

Zhong, Ruiqin; Cui, Dongtao; Phillips, Dennis; Sims, Nathanael T.; Ye, Zheng‐Hua

doi:10.1007/s00425-021-03794-y

Cited by 12 publications

(8 citation statements)

References 49 publications

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“…Previous studies of rice GT61 members showed that eight of them, including OsXAT2–OsXAT7, OsXYXT1, and OsXAX1, mediate 3‐ O ‐Ara f , 2‐O‐ Xyl, and hydroxycinnamate‐esterified 3‐ O ‐Ara f substitutions of xylan, respectively (Anders et al., 2012; Chiniquy et al., 2012; Zhong, Cui, Phillips, & Ye, 2018; Zhong et al., 2021; Feijao et al., 2022). Whether any of the remaining rice GT61 members are involved in xylan substitutions remains elusive.…”

Section: Resultsmentioning

confidence: 99%

“…Clade A rice GT61 members phylogenetically fall into five noticeable subgroups (Figure 1a) and at least one member from each of the subgroups I, II III, and V has been implicated in xylan substitutions. None of the rice subgroup IV members was able to substitute xylan when heterologously expressed in the Arabidopsis gux1/2/3 mutant (Zhong et al., 2021). To find out whether any rice subgroup IV GT61 members are involved in the addition of disaccharide side chains (Xyl‐Ara f or Ara f ‐Ara f ) onto xylan, we co‐expressed them with the XAT, OsXAT2, in the Arabidopsis gux1/2/3 mutant, which is ideal for gain‐of‐function studies of glycosyltransferases involved in xylan substitutions owing to its xylan being devoid of any sugar side chains (Lee et al., 2012; Mortimer et al., 2010).…”

Section: Resultsmentioning

confidence: 99%

“…Although Os02g22190 is phylogenetically closely related to OsXAXT1 (Figure 1a), its co-expression with OsXAT2 in the gux1/2/3 mutant did not result in 2-Osubstitutions of the Araf residues of xylan and its recombinant protein expressed in HEK293 cells was unable to xylosylate the (Araf)Xyl 4 acceptor (Figure S6), implying that its biochemical function might differ from that of OsXAXT1. It is possible that similar to the scenario of OsXATs, which belong to different subgroups (Zhong et al, 2021;Figure 1a), some GT61 members residing in other subgroups may share redundant functions with OsXAXT1. It remains to be investigated which GT61 members might possess the same enzymatic activity as OsXAXT1.…”

Section: Discussionmentioning

confidence: 99%

“…Genetic and biochemical studies have identified a group of family GT61 glycosyltransferases as xylan 3‐ O ‐arabinosyltransferases (XATs) involved in 3‐ O ‐arabinosylation of grass xylan. Gain‐of‐function analyses showed that TaXAT2 from wheat and OsXAT2 to OsXAT7 from rice could mediate the addition of 3‐ O ‐Ara f side chains onto xylan when heterologously expressed in the Arabidopsis glucuronic acid substitution of xylan 1/2/3 ( gux1/2/3 ) triple mutant (Anders et al., 2012; Zhong et al., 2021). RNAi downregulation of TaXAT1 in wheat and simultaneous mutations of OsXAT2 and OsXAT3 in rice led to reduced arabinosylation of xylan (Anders et al., 2012; Chen et al., 2021).…”

Section: Introductionmentioning

confidence: 99%

“…RNAi downregulation of TaXAT1 in wheat and simultaneous mutations of OsXAT2 and OsXAT3 in rice led to reduced arabinosylation of xylan (Anders et al., 2012; Chen et al., 2021). Activity assays of recombinant proteins of XATs from rice as well as several other grass species, including maize, sorghum, Brachypodium , and switchgrass, have revealed that they possess arabinosyltransferase activities catalyzing Ara f transfer from UDP‐Ara f onto xylooligomer acceptors (Zhong et al., 2021; Zhong, Cui, Phillips, & Ye, 2018).…”

Section: Introductionmentioning

confidence: 99%

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Identification of xylan arabinosyl 2‐O‐xylosyltransferases catalyzing the addition of 2‐O‐xylosyl residue onto arabinosyl side chains of xylan in grass species

et al. 2022

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Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with a-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Consistent with its function as a xylan arabinosyl 2-Oxylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of xylan arabinosyl 2‐O‐xylosyltransferases catalyzing the addition of 2‐O‐xylosyl residue onto arabinosyl side chains of xylan in grass species

et al. 2022

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Chemo‐Enzymatic Synthesis of Long‐Chain Oligosaccharides for Studying Xylan‐Modifying Enzymes

Martinez

Ruprecht

Senf

et al. 2023

Chemistry A European J

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Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemoenzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α-glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthasemediated coupling reactions as key steps. The xylo-oligosaccharide donors were protected at the non-reducing end with a 4-O-tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3-O-methylether substitu-ent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyltransferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI-MS/ MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long-chain acceptors for assaying biosynthetic glycosyltransferases.

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2021–2022

Harvey

2024

Mass Spectrometry Reviews

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The use of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry for the analysis of carbohydrates and glycoconjugates is a well‐established technique and this review is the 12th update of the original article published in 1999 and brings coverage of the literature to the end of 2022. As with previous review, this review also includes a few papers that describe methods appropriate to analysis by MALDI, such as sample preparation, even though the ionization method is not MALDI. The review follows the same format as previous reviews. It is divided into three sections: (1) general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, quantification and the use of computer software for structural identification. (2) Applications to various structural types such as oligo‐ and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals, and (3) other general areas such as medicine, industrial processes, natural products and glycan synthesis where MALDI is extensively used. Much of the material relating to applications is presented in tabular form. MALDI is still an ideal technique for carbohydrate analysis, particularly in its ability to produce single ions from each analyte and advancements in the technique and range of applications show little sign of diminishing.

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Functional analysis of GT61 glycosyltransferases from grass species in xylan substitutions

Cited by 12 publications

References 49 publications

Identification of xylan arabinosyl 2‐O‐xylosyltransferases catalyzing the addition of 2‐O‐xylosyl residue onto arabinosyl side chains of xylan in grass species

Identification of xylan arabinosyl 2‐O‐xylosyltransferases catalyzing the addition of 2‐O‐xylosyl residue onto arabinosyl side chains of xylan in grass species

Chemo‐Enzymatic Synthesis of Long‐Chain Oligosaccharides for Studying Xylan‐Modifying Enzymes

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2021–2022

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