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Reduced protein secretion and glycosylation induced by ammonium stress inhibits somatic embryo development and maintains the proembryogenic state of pumpkin embryogenic cultures

Extracellular proteins and glycoproteins secreted by ammonium- or auxin-induced somatic embryogenic cultures of pumpkin were analyzed. Despite an overall similarity in developmental characteristics between these embryogenic cultures, distinct expression patterns of extracellular proteins and glycoproteins were observed. When supplied as the sole source of nitrogen, ammonium caused acidification of the culture medium and significantly reduced protein secretion. Buffering of the ammonium-containing medium restored extracellular protein secretion and glycosylation and enhanced cell aggregation but not the development of later embryo stages. As revealed by Concavalin A immunodetection, extracellular glycoproteins containing alpha-D- mannose and alpha-D-glucose were most abundant in proembryogenic cultures grown in a medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D) and in a buffered ammonium-containing medium. We assume that extracellular proteins (28, 31, 44 kDa) and Con A-binding glycoproteins (26, 30, 40, 53, and 100 kDa) found in both proembryogenic cultures may have a role during somatic embryogenesis induction. The glycan components of proteins were further characterized by affinity blotting with different lectins (GNA, DSA, and PNA). Binding patterns of mannose- specific GNA lectin partially correlated with those detected with Concavalin A while no signal was observed with DSA and PNA, regardless of the treatment applied. Results indicate that complex N- or O-glycans are not typical for early phases of pumpkin embryo development. Accumulation of extracellular glycoproteins with high-mannose-type glycans from 30 to 34 kDa, observed after the transfer from 2, 4-D- or ammonium-containing medium into maturation medium, appears to be associated with development of later embryo stages. This study also revealed the presence of EP-3-like endochitinases in pumpkin embryogenic cultures, particularly in cultures grown in a buffered ammonium-containing medium, however, these proteins should be examined further.

Ammonium; cell aggregation; Cucurbita pepo; endochitinase; extracellular proteins; glycosylation; somatic embryogenesis

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