Complementary immunolocalization patterns of cell wall hydroxyproline-rich glycoproteins studied with the use of antibodies directed against different carbohydrate epitopes.
Antisera raised against the major hydroxyproline-rich glycoprotein (HRGP) in carrot (Daucus carota L.) taproot, extensin-1, and a minor HRGP, extensin-2, were characterized by western blot analysis, enzyme-linked immunosorbent assay, and periodate oxidation and found to be directed against carbohydrate epitopes shared by both glycoproteins. The anti-extensin-1 antibodies (gE1) target periodate-sensitive epitopes and may recognize the terminal alpha-1,3-arabinoside of extensin-1. The anti-extensin-2 antibodies (gE2) recognize periodate-insensitive epitopes, possibly binding the reducing, internal beta-1,2-arabinosides on the carbohydrate side chains. Despite the cross-reactivity of these antibodies, immunolocalization studies of carrot taproot and green bean (Phaseolus vulgaris L.) leaf tissues reveal a spatial segregation of gE1- and gE2-labeling patterns. The gE1 antibodies bind only to the cellulose-rich region of the cell wall (J.P. Staehelin and L.A. Stafstrom [1988] Planta 174: 321-332), whereas gE2 labeling is restricted to the expanded middle lamella at three cell junctions. Periodate oxidation of nonosmicated, thin-sectioned tissue abolishes gE1 labeling but leads to labeling of the entire cell wall by gE2, presumably as a result of unmasking cryptic epitopes on extensin-1 in the cellulose layer. Purified extensin-2 protein is more efficient than extensin-1 protein at agglutinating avirulent Pseudomonas strains lacking extracellular polysaccharide. Our results indicate that extensin-2 does not form a heterologous HRGP network with extensin-1 and that, in contrast to extensin-1, which appears to serve a structural role, extensin-2 could participate in passive defense responses against phytopathogenic bacteria.
Swords
K M
KM
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309-0347.
Staehelin
L A
LA
D000372:Agglutination Tests; D000906:Antibodies; D001089:Arabinose / Q000032:analysis; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002473:Cell Wall / Q000648:ultrastructure; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D007887:Fabaceae / Q000166:cytology* / Q000276:immunology / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D007365:Intercellular Junctions / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis*; D010946:Plants, Medicinal; D011549:Pseudomonas; D014675:Vegetables / Q000166:cytology* / Q000276:immunology / Q000648:ultrastructure
D000906:Antibodies; D000939:Epitopes; D006023:Glycoproteins; D009844:Oligosaccharides; D010940:Plant Proteins; C005488:extensin protein, plant; D001089:Arabinose