engleski

Comparative proteomic analysis of the secreted proteins of sugar beet cell lines

In the field of plant proteomics, the cell wall is an emerging area of study. Proteins secreted in the extracellular space, secretome, are crucial for plant development, morphogenesis, cell division, formation and elaboration of cell wall, extracellular signalization and defense. Three sugar beet cell lines grown for years on the PG0 medium serve as a model for studying plant cell differentiation and habituation: N – differentiated, growth hormones dependent, normal cell wall ; HNO – dedifferentiated, habituated, poor cell wall elaboration, and Tz – tumor line, dedifferentiated and habituated, obtained after transformation of leaf fragments with a wild type Agrobacterium tumefaciens B6S3 (Crèvecoeur, et al., 1992 ; Krsnik-Rasol, et al., 2001). The aim of this work was to compare the secretome of the cell lines, and to identify secreted proteins. Extracellular proteins were collected after cultivation of cell lines for 4 days in the liquid medium. Proteins were concentrated and separated by SDS-PAG electrophoresis in 12% T gels and stained with Coomassie Brilliant Blue. Selected protein candidates, 20 of them, were cut from the gel, digested by trypsin and analyzed by MALDI-TOF/TOF mass spectrometer. Extracellular protein patterns were cell line specific. Several of the identified proteins were classified as peroxidase and chitinase family of enzymes. Pectin esterase, xyloglucan endotransglucosylase, glucan endo-1, 3-β-D-glucosidase were also identified, few cytoplasmic proteins as well as few unidentified proteins. There were significant differences in the expression of identified proteins between the cell lines. Cytoplasmic proteins were probably a result of cellular budding and ruptures during subculture. Protein pattern of the secretome was clearly a function of cell morphology and differentiation state, but at the same time secreted proteins may induce extracellular signals influencing cell growth and differentiation. Next steps should include identification and elucidation of functions of unidentified proteins.

Proteomics; secretome; sugar beet; cell culture; extracellular proteins

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