Glycosylation of extracellular proteins during somatic embryogenesis in pumpkin (Cucurbita pepo L.)

Abstract

Biljne stanice u suspenziji rastu i izlučuju proteine u hranidbenu podlogu i time utječu na daljnji rast i diferencijaciju stanica. Glikoproteini čine većinu izvanstaničnih proteina od kojih mnogi imaju strukturnu i/ili enzimatsku ulogu. Ranija istraţivanja pokazala su da se profil izlučenih proteina mijenja ovisno o promjenama vezanim uz indukciju somatske embriogeneze, a nepravilna glikozilacija može izazvati zastoj u razvitku embrija. Jedan od najbolje opisanih glikoproteina vezanih uz somatsku embriogenezu biljaka je endohitinaza EP3, karakteristična za rani stadij embriogeneze u mrkve i uročnjaka. Cilj istraživanja bio je usporediti status glikozilacije proteina izlučenih u staničnoj suspenziji dviju embriogenih linija bundeve induciranih pomoću 2,4-D ili na modificiranoj podlozi MS bez hormona koja sadrži 1 mM NH4Cl kao jedini izvor dušika. Uočeno je da je tijekom kultivacije embriogenog tkiva u podlozi s NH4Cl kao jedinim izvorom dušika dolazilo do značajnog zakiseljavanja podloge. Dodatkom 25 mM MES, vrijednost pH podloge je stabilizirana što je potaklo izlučivanje izvanstaničnih proteina. Prisustvo i promjene u aktivnosti specifičnih izvanstaničnih endohitinaza ovisile su o hranidbenoj podlozi i razvojnim promjenama tijekom somatske embriogeneze. Izvanstanični proteini razdvojeni su elekroforezom SDS-PAGE, preneseni na membranu i analizirani obradom membrane različitim lektinima (ConA, GNA, DSA, PNA). Rezultati rada pokazali su da postoji razlika u razini glikozilacije uzrokovane različitim sastavom i vrijednosti pH kultivacijske podloge.Plant cells cultivated in a suspension secrete proteins in the cultivation medium, thereby affecting the further growth and cell differentiation. Glycoproteins make up the majority of extracellular proteins, many of which have structural and/or enzymatic role. Previous research has shown that the profile of extracted protein is changed in relation to changes during somatic embryogenesis induction. Moreover, an abnormal glycosylation may cause delay in plant embryo development. One of the best described glycoprotein associated with somatic embryogenesis of plants is endochitinase EP3, characteristic for the early stage of embryogenesis in carrot and Arabidopsis. The objective of this work was to compare the glycosylation status of proteins extracted in cell suspension of two pumpkin embriogenic lines induced by 2,4-D or on a modified MS medium without hormones containing 1 mM NH4Cl as the sole source of nitrogen. It was noted that the cultivation medium with NH4Cl as the sole source of nitrogen undergo acidification. The addition of 25 mM MES stabilized pH of the culture medium, what enhanced the secretion of extracellular proteins. Presence of specific endochitinases and changes in their activity depended on the medium composition and developmental changes during somatic embryogenesis. Extracellular proteins were separated by SDS-PAGE electrophoresis, transferred to membrane and analyzed by processing membrane with different lectins (ConA, GNA, DSA, PNA). The results showed that there was a difference in the level of glycosylation caused by medium composition and pH