title: Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.).
abstract: Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.
authors.affiliation: Institute Ruđer Bošković, Bijenička Cesta 54, 10000 Zagreb, Croatia. mihaljev@irb.hr
authors.forename: Snježana
authors.initials: S
authors.lastname: Mihaljević
authors.forename: Sandra
authors.initials: S
authors.lastname: Radić
authors.forename: Nataša
authors.initials: N
authors.lastname: Bauer
authors.forename: Rade
authors.initials: R
authors.lastname: Garić
authors.forename: Branka
authors.initials: B
authors.lastname: Mihaljević
authors.forename: Gordana
authors.initials: G
authors.lastname: Horvat
authors.forename: Dunja
authors.initials: D
authors.lastname: Leljak-Levanić
authors.forename: Sibila
authors.initials: S
authors.lastname: Jelaska
mesh_terms: D028464:Cucurbita / Q000187:drug effects / Q000196:embryology / Q000201:enzymology / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism; D005936:Glucans / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007210:Indoleacetic Acids / Q000494:pharmacology; D009566:Nitrates / Q000378:metabolism / Q000494:pharmacology*; D009584:Nitrogen / Q000378:metabolism / Q000494:pharmacology; D018384:Oxidative Stress; D009195:Peroxidase / Q000378:metabolism; D010650:Phenylalanine Ammonia-Lyase / Q000187:drug effects / Q000378:metabolism; D058439:Plant Somatic Embryogenesis Techniques; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D013312:Stress, Physiological; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors
chemical_list: D005936:Glucans; D007210:Indoleacetic Acids; D009566:Nitrates; D000644:Quaternary Ammonium Compounds; C048306:callose; D009195:Peroxidase; D013482:Superoxide Dismutase; D004950:Esterases; D010650:Phenylalanine Ammonia-Lyase; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen