Generation of polyclonal and monoclonal antibodies against arabinoxylans and their use for immunocytochemical location of arabinoxylans in cell walls of endosperm of wheat

Abstract

Polyclonal and monoclonal antibodies (pAbs and mAbs) against arabinoxylans (AX) were produced and used in addition to a mAb against (1→3,1→4)-β-d-glucan to investigate cell-wall structure in endosperm of wheat by light and electron microscopy. pAbs were raised against a xylotetraose BSA conjugate (pAbs anti-X3) while mAbs were produced from a mixture of arabino-xylo-oligosaccharides conjugated to keyhole limpet hemocyanin (KLH) (mAbs anti-AX1). The pAbs anti-X3 and the mAb anti-AX1 recognised unsubstituted (1→4)-β-d-xylosyl sequences but differed in capacity to bind arabinosylated xylo-oligosaccharides. The pAb anti-X3 did not bind to xylotetrasaccharides with the third xylosyl unit bearing arabinose at C(0)3 or at C(0)3 and C(0)2 while the mAb anti-AX1 did. The pAbs anti-X3 reacted strongly with the aleurone and central starchy endosperm cell walls and less strongly with subaleurone cell walls. The mAb anti-AX1 bound strongly and equally to all the cell walls of the endosperm. The distribution pattern of the AX epitopes was distinct according to cell types. (1→3,1→4)-β-d-Glucans were abundant in the aleurone and subaleurone cell walls but were not detected in the junction region between aleurone cells at the nucellus epidermis interface. The epitopes occurred in discreet domains of the walls of central cells.