Integrated proteomics and histochemical analysis of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) in embryogenic suspension culture

Abstract

t Cell suspension culture is a useful in vitro model-system for both scaling up and conserving the Brazilian conifer Araucaria angustifolia. In the present work, cell suspension of Araucaria was subjected to proteomics, biochemical and histochemical analyses. The results revealed new insights underlying the molecular mechanism of proembryogenic masses transition in cell suspension. Embryogenic cell cultures were cultivated in a basal liquid medium modified in a Steward apparatus (orbital agitator). Cell growth dynamics was evaluated using cell volume after sedimentation, fresh weight, mitotic index, conductivity, pH, and the number of proembryogenic masses (PEMs: I, II, III). Histochemical parameters, cell viability, and cell death analyses were performed to pinpoint growth rates. Proteomics analysis was performed using two-dimensional electrophoresis, and protein identification was carried out by MALDI-TOF-TOF tandem mass spectrometry. Cell growth dynamics showed a predominance of PEM III. Maximum slope of the exponential phase growth in fresh weight occurred at exponential phase after 15 days (optimal cultivation time), after which cell viability and pH decreased, thereby allowing the identification of stressrelated proteins. Several metabolism and growth proteins were abundant, such as: cytoskeletal, WOX1, cytokinin-related, and auxin-related proteins acting on cell wall modification, suspensor cell formation, and PEM I to PEM III transition.