Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium
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Ohio University / OhioLINK
2013
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1368012677 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-ohiou13680126772021-08-03T05:23:05Z Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium Wiemels, Richard E. Plant Biology Molecular Biology Biochemistry fucosyltransferase xyloglucan wheat Brachypodium glycome profiling cell wall Plant cell walls are constructed from a complex network of polysaccharide polymers. Xyloglucan (XyG) is one polymer whose biosynthesis and structure in dicots has been well-studied <i>in vitro</i>. Xyloglucan in dicots has complex branching in its side chains which include xylose, galactose and fucose (Fuc) residues (XXXG type), while XyG in grasses is known only to include xylose, galactose and possibly arabinose residues (XXGG type) with less side-chain branching. Previous work has shown that detergent extracts from Golgi-enriched microsomal membranes from wheat roots were able to fucosylate tamarind XyG (TXyG, a Fuc-free XyG). Digestion with endoglucanase of the [<sup>14</sup>C]Fuc-containing product of the fucosyl transfer reaction co-eluted with authentic fucosylated XyG oligosaccharides, and mass spectrometry has shown the presence of Fuc in native XyG from the wheat root cell wall. In this study, glycome profiling of cell wall extracts from etiolated wheat and Brachypodium seedlings was used to further confirm the presence of fucosylated XyG in these species (collaboration with Dr. Michael Hahn, University of Georgia). Bioinformatics analysis was used to identify homologs to Arabidopsis XyG-specific fucosyltransferase (AtFUT1) in grasses including wheat, Brachypodium, and barley. Phylogenetic analysis and expression profiling of putative wheat <i>FUT</i>s were used to identify three promising wheat candidate genes: <i>TaFUT4</i>, <i>TaFUT6</i> and <i>TaFUT15</i>. Of these, the TaFUT15 protein sequence was found to have the highest sequence identity/similarity with AtFUT1. Two variants of TaFUT4 were chosen for expression, named TaFUT<sub>L126</sub> and TaFUT4<sub>P121</sub>. The full-length cDNAs encoding TaFUT<sub>L126</sub>, TaFUT4<sub>P121</sub>, TaFUT6, and TaFUT15 were expressed in <i>Pichia pastoris</i> for functional analysis. Two genes from Brachypodium provided by Dr. Samuel Hazen (University of Massachusetts, Amherst), <i>Bradi1g46020</i> and <i>Bradi3g58040</i>, were also tested. None of the proteins showed fucosyltransferase activity for TXyG. Defucosylated cell wall extracts were also tested as acceptors, but no activity was detected. 2013-09-26 English text Ohio University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1368012677 http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1368012677 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
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NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Plant Biology Molecular Biology Biochemistry fucosyltransferase xyloglucan wheat Brachypodium glycome profiling cell wall |
| spellingShingle |
Plant Biology Molecular Biology Biochemistry fucosyltransferase xyloglucan wheat Brachypodium glycome profiling cell wall Wiemels, Richard E. Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium |
| author |
Wiemels, Richard E. |
| author_facet |
Wiemels, Richard E. |
| author_sort |
Wiemels, Richard E. |
| title |
Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium |
| title_short |
Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium |
| title_full |
Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium |
| title_fullStr |
Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium |
| title_full_unstemmed |
Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium |
| title_sort |
cloning, expression, and biochemical assay of putative xyloglucan-specific fucosyltransferases from wheat and brachypodium |
| publisher |
Ohio University / OhioLINK |
| publishDate |
2013 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1368012677 |
| work_keys_str_mv |
AT wiemelsricharde cloningexpressionandbiochemicalassayofputativexyloglucanspecificfucosyltransferasesfromwheatandbrachypodium |
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