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Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium

Wiemels, Richard E.
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1368012677

Abstract Details

2013, Master of Science (MS), Ohio University, Plant Biology (Arts and Sciences).
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 in vitro. 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 [14C]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 FUTs were used to identify three promising wheat candidate genes: TaFUT4, TaFUT6 and TaFUT15. 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 TaFUTL126 and TaFUT4P121. The full-length cDNAs encoding TaFUTL126, TaFUT4P121, TaFUT6, and TaFUT15 were expressed in Pichia pastoris for functional analysis. Two genes from Brachypodium provided by Dr. Samuel Hazen (University of Massachusetts, Amherst), Bradi1g46020 and Bradi3g58040, 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.
Ahmed Faik (Advisor)
Allan Showalter (Committee Member)
Marcia Kieliszewski (Committee Member)
Michael Held (Committee Member)
Biochemistry; Molecular Biology; Plant Biology
fucosyltransferase; xyloglucan; wheat; Brachypodium; glycome profiling; cell wall

Recommended Citations

Citations

  • Wiemels, R. E. (2013). Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium [Master's thesis, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1368012677

    APA Style (7th edition)

  • Wiemels, Richard. Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium. 2013. Ohio University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1368012677.

    MLA Style (8th edition)

  • Wiemels, Richard. "Cloning, Expression, and Biochemical Assay of Putative Xyloglucan-specific Fucosyltransferases from Wheat and Brachypodium." Master's thesis, Ohio University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1368012677

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Ohio University and OhioLINK.