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

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Biologia Plantarum

Abstract

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.

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Abbreviations

2,4-D:

2,4-dichlorophenoxyacetic acid

Con A:

concavalin A

DEC:

embryogenic line induced and maintained on MS2,4D medium

DIG:

digoxigenin

DSA:

lectin from Datura stramonium

GNA:

lectin from Galanthus nivalis

HEC:

habituated embryogenic line

MES:

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

MS:

Murashige and Skoog

MS2,4D:

MS medium supplemented with 2,4-D

MSNH4:

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

MSNH4MES:

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

NBT/BCIP:

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

PEDC:

embryogenic line induced and maintained on the MSNH4 medium

PNA:

lectin from Arachis hypogea

PVDF:

polyvinylidene difluoride membrane

SDS-PAGE:

sodium dodecyl sulphate polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Institute Ruđer Bošković, Bijenička cest 54, 10000, Zagreb, Croatia

    A. Crnković, R. Garić & S. Mihaljević

  2. Faculty of Science, University of Zagreb, Horvatovac 102a, 10000, Zagreb, Croatia

    D. Leljak-Levanić

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  1. A. Crnković
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  2. R. Garić
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  3. D. Leljak-Levanić
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  4. S. Mihaljević
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Correspondence to S. Mihaljević.

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Acknowledgements: We thank Prof. Sacco de Vries (the University of Wageningen, The Netherlands) for providing the antibody for EP3-1 chitinase. We also thank Drs. Biljana Balen and Lucia Neal for the critical reading of the manuscript. This research was supported by grants No. 098-0982913-2829 and 119-1191196-1225 funded by the Ministry of Science, Education and Sport of the Republic of Croatia. A. Crnković and R. Garić contributed equally to this paper.

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Crnković, A., Garić, R., Leljak-Levanić, D. et al. Reduced protein secretion and glycosylation induced by ammonium stress inhibits somatic embryo development in pumpkin. Biol Plant 58, 209–217 (2014). https://doi.org/10.1007/s10535-014-0400-2

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  • DOI: https://doi.org/10.1007/s10535-014-0400-2

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