Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
The extracellular proteins in three distinctly induced embryogenic lines of pumpkin (Cucurbita pepo L.) cultivated in four MS media modified regarding the nitrogen composition or auxin presence/absence have been analyzed. Extracellular glycoproteins containing α-D-mannose were specifically detected by the lectine concavalin A. During the cultivation of embryogenic tissue in the medium supplemented with reduced nitrogen, the embryos were mostly arrested at preglobular and globular developmental stages, which coincide with the absence of protein secretion. Secreted glycoproteins of 76, 68, 37 and 34 kDa were detected only if any of the three lines were cultivated in the medium that stimulates embryo development, irrespectively of the addition of 2,4-dichlorophenoxyacetic acid or tunicamycin. The glycoprotein of 64 kDa was detected in all lines cultivated in hormone-free MS medium with conventional nitrogen sources and it appears to be associated with embryo maturation. Tunicamycin treatment did not influence embryogenesis, although it specifically affected glycosylation of proteins in the investigated lines. Our results show that besides auxin, the source of nitrate is of great importance for proper protein glycosylation, excretion and developmental transition of pumpkin somatic embryos.Analizirani su izvanstanični proteini tri ustaljene embriogene linije bundeva (Cucurbita pepo L.) koje su uzgajane na četiri različite podloge MS s obzirom na udjel dušika i prisutnost/odsutnost auksina. Izvanstanični proteini što sadrže α-D-manozu specifično su dokazani lektinom konkavalinom A. Tijekom uzgoja embriogenoga tkiva na podlozi s dodatkom reduciranog dušika, razvitak većine embrija bio je zakočen u predglobularnom i globularnom razvojnom stadiju, što se podudaralo s nedostatkom izlučivanja proteina. Izlučivanje proteina od 76, 68, 37 i 34 kDa dokazano je u sve tri linije tijekom uzgoja na podlozi koja pospješuje razvoj embrija, neovisno o dodatku 2,4-diklorofenoksioctene kiseline ili tunikamicina. Glikoprotein od 64 kDa utvrđen je u sve tri linije uzgajane na podlozi MS bez dodatka hormona i s nepromijenjenim izvorima dušika, te se mogao povezati sa sazrijevanjem embrija na ovoj podlozi. Izlaganje tunikamicinu nije utjecalo na embriogenezu, iako je imalo specifično djelovanje na glikozilaciju proteina ispitanih linija. Rezultati pokazuju da osim auksina i izvori dušika imaju važnu ulogu u pravilnoj glikozilaciji proteina, njihovom izlučivanju i dinamici razvitka somatskih embrija bundeve
