Primary Cell Wall Antigens The walls of angiosperms and gymnosperms are composed of cellulose, hemicelluloses, and pectic polysaccharides albeit in different amounts. There are two general types of wall based on the relative amounts of pectic polysaccharides and the structure and amounts of hemicellulosic polysaccharides (Carpita and Gibeaut, [1993] Plant Journal 3, 1-30). Type I walls which typically contain xyloglucan and/or glucomannan and 20-35% pectin. Type I walls are found in all dicotyledons, the non-graminaceous monocotyledons, and gymnosperms. Type II walls, that are rich in arabinoxylan but contain <10% pectin are present in the Poaceae (grasses). To maximize the likelyhood of obtaining diverse antibodies, we will utilize an array of plant cell wall-derived carbohydrates and present them to the animals' immune system in a variety of contexts through the use of different immunogens. A monoclonal approach using complex immunogens such as the structural complex carbohydrates of the plant cell wall is likely to yield specific antibodies against diverse carbohydrate structures. We will use intact cell walls isolated from dicots (Arabidopsis thaliana, Medicago truncatula, and Lycopersicon esculentum) and monocots (Oryza sativa and Zea mays) as one form of immunogen. These species are currently under intensive study in various genome projects. We will also use purified cell wall polysaccharides and plant cell wall extracts that contain mixtures of polysaccharides as immunogens (see Table below). These polysaccharides cover a range of known structural features of wall polysaccharides and will maximize the diversity of the antibodies that are likely to be produced. We will also isolate polysacchardies from the culture medium of suspension-cultured plant cells.

Polysaccharides to be used for immunogen preparation (Brief descriptions of the structural features of the polysaccharides can be obtained by following the links)

Polysaccharide

Plant Source

Structural Motifs

Pectins Rhamnogalacturonan I Homogalacturonan

B Sycamore Tomato Arabidopsis seed mucilage Various

B Diversity of side chains containing 3 and 3,5-linked α-L-Ara and 4 and 3,4-linked β-D-Gal (Type I arabinogalactan) Ripening-dependent changes in side chains RG-I backbone - no side chains Different degrees and patterns of O-methyl esterification

Hemicellulose Xyloglucan Galactoglucomannan Glucuronoarabinoxylan Mixed-linkage β-glucans

B Sycamore, Arabidopsis Tamarind seed Jojoba seed Tomato Tomato Zea mays Zea mays

B α-L-Fuc containing side chains No fucose on side chains α-L-Gal-containing side chains α-L-Ara-containg side chains typical of solanaceous plants 4-linked β-Man and β-Glc backbone substituted 1,4-linked β-xylans 3 and 4-linked β-Glc residues

Proteoglycans/glycoproteins Type II arabinogalactan Extensins

B commercial mucilages Tomato

B substituted 3 and 3,6-linked galactans arabinosylated hydroxyproline

We will also use oligosaccharide fragments generated from cell wall polysaccharides as immunogens. This will allow us to target particular structural features of a polysaccharide and provide us with additional structural variants to present to the immune system. Oligosaccharide fragments will be generated by selective enzymatic cleavage of the polysaccharide. For example a set of well-defined oligosaccharides are generated by treating xyloglucans with endo-1,4-β-glucanses. Oligosaccharide are also generated by treating RG-I with endo-arabinanase and endo-galactanase together with rhamnogalacturonan hydrolase or lyase. Back to Top

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A National Science Foundation-funded (Grant No DBI-0421683) research project at The Complex Carbohydrate Research Center of The University of Georgia