Immunohistochemical localization of a glycoprotein, GP80, in the outermost layer of the developing endosperm of immature seeds of carrot

Abstract

A glycoprotein (GP80) from the immature fruits of carrot (Daucus carota L.) plants was purified through the sequential application of salt extraction, (NH4)2SO4 precipitation, chromatography on columns of concanavalin A-Sepharose, hydroxylapatite, and boronate, and electroelution, with subsequent sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its apparent relative molecular mass (Mr) was estimated to be 80 k by both SDS-PAGE and gel filtration at high ionic strength. The Mr of the polypeptide portion was estimated to be 74 k after deglycosylation by trifluoromethanesulfonic acid (TFMS). Antibodies were produced by immunizing rabbits with purified and deglycosylated GP80. Using immunoblotting of total protein extracts from each organ and tissue GP80 was detected only in immature seeds. It appeared at 4 d after flowering (DAF), accumulated until 15 DAF, then its level suddenly decreased and it disappeared completely at 23 DAF. Another glycoprotein, with an Mr of 78 k (GP78), cross-reacted with GP80-specific antibodies. GP78 appeared at 17 DAF and disappeared at 37 DAF. Histochemical examination of fruits using Sudan Black B and periodic acid-Schiff reagent (PAS) showed that the appearance of GP78 coincided with the rapid filling of the central cavity with endosperm cells, which have cell walls and contain lipid storage granules. In immunohistochemical studies, both GP80 and GP78 were found at the outermost layer of the developing endosperm.