Biologia Plantarum

, Volume 58, Issue 2, pp 209–217 | Cite as

Reduced protein secretion and glycosylation induced by ammonium stress inhibits somatic embryo development in pumpkin

  • A. Crnković
  • R. Garić
  • D. Leljak-Levanić
  • S. Mihaljević
Original Papers


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. Ammonium, when supplied as the sole source of nitrogen, caused acidification of the culture medium and significantly reduced protein secretion. Buffering pH in the ammonium-containing medium restored extracellular protein secretion and glycosylation and an enhanced cell aggregation but not the development of later embryo stages. As revealed by Concavalin A (Con A) immunodetection, extracellular glycoproteins containing α-D-mannose and α-D-glucose were most abundant in proembryogenic cultures grown in a buffered ammonium-containing medium and in a medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). We assume that extracellular proteins (Mr 28, 31, and 44 kDa) and Con Abinding glycoproteins (Mr 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. Binding patterns of mannose-specific lectin from Galanthus nivalis partially correlated with those detected with Con A, whereas no signal was observed with lectins from Datura stramonium and Arachis hypogea regardless of the treatment applied. Results indicate that complex N- or O-glycans are not typical for early phases of pumpkin embryo development. The accumulation of extracellular glycoproteins with high-mannose-type glycans from 30 to 34 kDa, observed after the transfer from the ammonium- or 2,4-D-containing media into a maturation medium, appeared 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 the buffered ammonium-containing medium, however, these proteins should be examined further.

Additional key words

auxin cell aggregation Concavalin A immunodetection Cucurbita pepo endochitinase extracellular proteins lectins 



2,4-dichlorophenoxyacetic acid

Con A

concavalin A


embryogenic line induced and maintained on MS2,4D medium




lectin from Datura stramonium


lectin from Galanthus nivalis


habituated embryogenic line


2-(N-morpholino)-ethane-sulfonic acid


Murashige and Skoog


MS medium supplemented with 2,4-D


hormone-free MS medium supplemented with NH4 + as the sole source of nitrogen


hormone-free MS medium supplemented with NH4 + as the sole source of nitrogen and buffered with MES


nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate


embryogenic line induced and maintained on the MSNH4 medium


lectin from Arachis hypogea


polyvinylidene difluoride membrane


sodium dodecyl sulphate polyacrylamide gel electrophoresis


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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • A. Crnković
    • 1
  • R. Garić
    • 1
  • D. Leljak-Levanić
    • 2
  • S. Mihaljević
    • 1
  1. 1.Institute Ruđer BoškovićZagrebCroatia
  2. 2.Faculty of ScienceUniversity of ZagrebZagrebCroatia

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