Role of N-glycosylation of 66 and 69 kDa glycoproteins in wall formation during pollen tube growth in vitro.

Two abundant cell wall glycoproteins (66 and 69 kDa) accumulate during growth in pollen tubes of tobacco. Glycosylation of the proteins was experimentally modified by application of the specific inhibitors tunicamycin and castanospermine to in vitro cultured pollen. Newly synthesized proteins were labeled with a 14C-amino acid mixture supplied to the medium. Modified glycoproteins were extracted from pollen tubes and isolated cell walls, and separated by 1-D and 2-D electrophoresis. The size of the molecules was reduced by tunicamycin and increased by castanospermine, effects which were measurable from the beginning of cultivation. The modification of the glycan moiety did not affect deposition of the proteins in the wall. Cultivation in the continuous presence of either inhibitor led to reduced callose deposition in the secondary cell wall and to inhibition of pollen tube growth. The results suggest that the two proteins play a role in the formation of the callose wall, and that this function depends on proper glycosylation of the molecules. As a consequence, the glycoproteins are essential for growth of the pollen tube.