ABSTRACT
Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized "on chip" by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.
Subject(s)
Glycosyltransferases/chemistry , Plants/enzymology , Polysaccharides/analysis , Cell Wall/chemistryABSTRACT
The plant cell wall provides the richest available resource of fermentable carbohydrates and biobased materials. The main component of plant cell walls is cellulose, which is the most abundant biomolecule on earth. Apart from cellulose, which is constructed from relatively simple ß-1,4-glucan chains, plant cell walls also contain structurally more complex heteropolysaccharides (hemicellulose and pectin), as well as lignin and cell-wall proteins. A detailed understanding of the molecular structures, functions, and biosyntheses of cell-wall components is required to further promote their industrial use. Plant cell-wall research is, to a large degree, hampered by a lsack of available well-defined oligosaccharide samples that represent the structural features of cell-wall glycans. One technique to access these oligosaccharides is automated glycan assembly; a technique in which monosaccharide building blocks are, similarly to automated peptide and oligonucleotide chemistry, successively added to a linker-functionalized resin in a fully automated manner. Herein, recent research into the automated glycan assembly of different classes of cell-wall glycans used as molecular tools for cell-wall biology is discussed. More than 60 synthetic oligosaccharides were prepared and printed as microarrays for screening monoclonal antibodies that recognize plant cell-wall polysaccharides. The synthesized oligosaccharides have also been used to investigate glycosyltransferases and glycoside hydrolases, which are involved in synthesis and degradation of plant cell walls, as well as for the analysis of cell-wall-remodeling enzymes.
Subject(s)
Cell Wall/metabolism , Plants/metabolism , Polysaccharides/metabolism , Antibodies, Monoclonal/immunology , Carbohydrate Sequence , Enzyme Assays , Glycoside Hydrolases/metabolism , Glycosyltransferases/chemistry , Glycosyltransferases/metabolism , Polysaccharides/chemical synthesis , Polysaccharides/immunology , Substrate SpecificityABSTRACT
In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision.
Subject(s)
Antibodies, Monoclonal/metabolism , Brachypodium/metabolism , Cell Wall/metabolism , Epitope Mapping , Microarray Analysis/methods , Polysaccharides/metabolism , Glycoside Hydrolases/metabolism , Staining and LabelingABSTRACT
Pectin is a structurally complex plant polysaccharide with many industrial applications in food products. The structural elucidation of pectin is aided by digestion assays with glycosyl hydrolases. We report the automated glycan assembly of oligosaccharides related to the arabinogalactan side chains of pectin as novel biochemical tools to determine the substrate specificities of endogalactanases. Analysis of the digestion products revealed different requirements for the lengths and arabinose substitution pattern of the oligosaccharides to be recognized and hydrolyzed by the galactanases.
Subject(s)
Automation , Galactans/chemistry , Glycoside Hydrolases/metabolism , Polysaccharides/biosynthesis , Molecular Structure , Polysaccharides/chemistry , Substrate SpecificityABSTRACT
Arabinogalactan proteins are heavily glycosylated proteoglycans in plants. Their glycan portion consists of type-II arabinogalactan polysaccharides whose heterogeneity hampers the assignment of the arabinogalactan protein function. Synthetic chemistry is key to the procurement of molecular probes for plant biologists. Described is the automated glycan assembly of 14 oligosaccharides from four monosaccharide building blocks. These linear and branched glycans represent key structural features of natural type-II arabinogalactans and will serve as tools for arabinogalactan biology.
