COMPLEMENTARY IMMUNOLOCALIZATION PATTERNS OF CELL-WALL HYDROXYPROLINE-RICH GLYCOPROTEINS STUDIED WITH THE USE OF ANTIBODIES DIRECTED AGAINST DIFFERENT CARBOHYDRATE EPITOPES

Antisera raised against the maj or hydroxyproline-rich glycoprotein (H RGP) in carrot (Daucus carota L.) taproot, extensin-1, and a minor HRG P, extensin-2, were characterized by western blot analysis, enzyme-lin ked immunosorbent assay, and periodate oxidation and found to be direc ted against carbohydrate epitopes shared by both glycoproteins. The an ti-extensin-1 antibodies (gE1) target periodate-sensitive epitopes and may recognize the terminal alpha-1,3-arabinoside of extensin-1. The a nti-extensin-2 antibodies (gE2) recognize periodate-insensitive epitop es, possibly binding the reducing, internal beta-1,2-arabinosides on t he carbohydrate side chains. Despite the cross-reactivity of these ant ibodies, immunolocalization-studies of carrot taproot and green bean ( Phaseolus vulgaris L.) leaf tissues reveal a spatial segregation of gE 1- and gE2-labeling patterns. The gE1 antibodies bind only to the cell ulose-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 lead s to labeling of the entire cell wall by gE2, presumably as a result o f unmasking cryptic epitopes on extensin-1 in the cellulose layer. Pur ified extensin-2 protein is more efficient than extensin-I protein at agglutinating avirulent Pseudomonas strains lacking extracellular poly saccharide. Our results indicate that extensin-2 does not form a heter ologous HRGP network with extensin-1 and that, in contrast to extensin -1, which appears to serve a structural role, extensin-2 could partici pate in passive defense responses against phytopathogenic bacteria.