Skip to main content
SpringerLink
Log in

Ammonium regulates embryogenic potential in Cucurbita pepo through pH-mediated changes in endogenous auxin and abscisic acid

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

When supplied as the sole nitrogen source, ammonium can induce somatic embryogenesis in some plant species, including pumpkin. To get an insight into the involvement of phytohormones in this adaptive developmental response to nitrogen availability, the status of endogenous abscisic acid (ABA), indole-3-acetic acid (IAA), and their conjugates was analyzed in an ammonium-induced embryogenic pumpkin culture. Results were compared with endogenous hormone status in an embryogenic culture induced by exogenously supplied 2,4-dichlorophenoxyacetic acid (2,4-D). In the NH4 +-induced somatic embryogenic culture, the maximum concentration of ABA was measured after 21 days of cultivation, while in the 2,4-D-induced culture ABA peaked after 7 days of cultivation, which suggested a possible activation of different ABA-regulated stress responses. The NH4 +-induced embryogenic culture was also characterized by increased accumulation of endogenous IAA to the level similar or higher than the one induced by 2,4-D. A considerable decrease (threefold) in endogenous IAA content was observed after buffering the NH4 +-medium with MES or after nitrate re-supply. In contrast, buffering the NH4 +-medium increased endogenous ABA by five times. Significant ammonium- and pH-related changes were also detected in ascorbate and glutathione redox status, and discussed regarding the corresponding changes in cell size and mitotic activity in the cultures. Furthermore, a negative correlation between free IAA content and the activity of ascorbate peroxidase (EC 1.11.1.11) and auxin-sensitive peroxidase CpAPRX (EC 1.11.1.7) was observed. The results strongly suggest a mediating role of external pH in the regulation of endogenous phytohormones and the ascorbate–glutathione cycle during ammonium-induced acquisition of embryogenic competence in pumpkin.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Bielawski W, Joy KW (1986) Reduced and oxidized glutathione and glutathione-reductase activity in tissues of Pisum sativum. Planta 169:267–272

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brewitz E, Larsson C-M, Larsson M (1995) Influence of nitrate supply on concentration and translocation of abscisic acid in barley (Hordeum vulgaris). Physiol Plant 95:499–506

    Article  CAS  Google Scholar 

  • Britto DT, Kronzucker HJ (2002) NH4 + toxicity in higher plants: a critical review. J Plant Physiol 159:567–584

    Article  CAS  Google Scholar 

  • Caba JM, Centeno ML, Fernández B, Gresshoff PM, Ligero F (2000) Inoculation and nitrate alter phytohormone levels in soybean roots: differences between a supernodulating mutant and the wild type. Planta 211:98–104

    Article  CAS  PubMed  Google Scholar 

  • Chapin FS, Walter CHS, Clarkson DT (1988) Growth response of barley and tomato to nitrogen stress and its control by abscisic acid, water relations and photosynthesis. Planta 173:352–366

    Article  CAS  PubMed  Google Scholar 

  • Correa-Aragunde N, Foresi N, Delledonne M, Lamattiina L (2013) Auxin induces redox regulation of ascorbate peroxidase 1 activity by S-nitrosylation/denitrosylation balance resulting in changes of root growth pattern in Arabidopsis. J Exp Bot 64:3339–3349

    Article  CAS  PubMed  Google Scholar 

  • Cosio C, Vuillemin L, De Mayer M, Kevers C, Penel C, Dunand C (2009) An anionic class II peroxidase from zucchini may regulate hypocotyl elongation through its auxin oxidase activity. Planta 229:823–836

    Article  CAS  PubMed  Google Scholar 

  • Davey MW, Dekempeneer E, Keulemans J (2003) Rocket-powered high-performance liquid chromatographic analysis of plant ascorbate and glutathione. Anal Biochem 316:74–81

    Article  CAS  PubMed  Google Scholar 

  • Davies WJ, Kudoyarova G, Hartung W (2005) Long-distance ABA signaling and its relation to other signaling pathways in the detection of soil drying and the mediation of the plant’s response to drought. J Plant Growth Regulat 24:285–295

    Article  CAS  Google Scholar 

  • Endres L, Souza BM, Mercier H (2002) In vitro nitrogen nutrition and hormonal pattern in bromeliads. In Vitro Cell Dev Biol Plant 38:481–486

    Article  CAS  Google Scholar 

  • Fei and Vessey (2003) Involvement of cytokinin in the stimulation of nodulation by low concentrations of ammonium in Pisum sativum. Physiol Plant 118:447–455

    Article  Google Scholar 

  • Foyer CH, Noctor G (2011) Ascorbate and glutathione: the heart of the redox hub. Plant Phsiol 155:2–18

    Article  CAS  Google Scholar 

  • Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jürgens G (2003) Efflux-dependent auxin gradients establish the apical–basal axis of Arabidopsis. Nature 426:147–152

    Article  CAS  PubMed  Google Scholar 

  • Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinypyridine. Anal Biochem 106:207–212

    Article  CAS  PubMed  Google Scholar 

  • Hachiya T, Noguchi K (2011) Mutation of NRT1.1 enhances ammonium/low pH-tolerance in Arabidopsis thaliana. Plant Signal Behav 6:706–708

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Higashi K, Kamada H, Harada H (1996) The effect of reduced nitrogenous compounds suggests that glutamine synthetase activity is involved in the development of somatic embryos in carrot. Plant Cell, Tissue Organ Cult 45:109–114

    Article  CAS  Google Scholar 

  • Hradecká V, Novák O, Havlíček L, Strnad M (2007) Immunoaffinity chromatography of abscisic acid combined with electrospray liquid chromatography–mass spectrometry. J Chromatogr B 847:162–173

    Article  Google Scholar 

  • Ikeda-Iwai M, Umehara M, Satoh S, Kamada H (2003) Stress-induced somatic embryogenesis in vegetative tissues of Arabidopsis thaliana. Plant J 34:107–114

    Article  CAS  PubMed  Google Scholar 

  • Ilić N, Magnus V, Ostin A, Sandberg G (1997) Stable-isotope labeled metabolites of the phytohormone, indole-3-acetic acid. J Label Compd Radiopharm 39:433–440

    Article  Google Scholar 

  • Karami O, Saidi A (2010) The molecular basis for stress-induced acquisition of somatic embryogenesis. Mol Biol Rep 37:2493–2507

    Article  CAS  PubMed  Google Scholar 

  • Kiba T, Kudo T, Kojima M, Sakakibara H (2011) Hormonal control of nitrogen acquisition: roles of auxin, abscisic acid, and cytokinin. J Exp Bot 62:1399–1409

    Article  CAS  PubMed  Google Scholar 

  • Kikuchi A, Sanuki N, Higashi K, Koshiba T, Kamada H (2006) Abscisic acid and stress treatment are essential for the acquisition of embryogenic competence by carrot somatic cells. Planta 223:637–645

    Article  CAS  PubMed  Google Scholar 

  • Kikuchi A, Asahin M, Tanaka M, Satoh S, Kamada H (2013) Acquisition of embryogenic competence does not require cell division in carrot somatic cells. J Plant Res 126:243–250

    Article  PubMed  Google Scholar 

  • Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, Hoyerova K, Tillard P, Leon S, Ljung K, Zazimalova E, Benkova E, Nacry P, Gojon A (2010) Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Dev Cell 18:927–937

    Article  CAS  PubMed  Google Scholar 

  • Krouk G, Ruffel S, Gutiérrez RA, Gojon A, Crawford NM, Coruzzi GM, Lacombe B (2011) A framework integrating plant growth with hormones and nutrients. Trends Plant Sci 16:178–182  

    Article  CAS  PubMed  Google Scholar 

  • Kudoyarova GR,  Farkhutdinov RG, Veselov SYu  (1997) Comparison of the effects of nitrate and ammonium forms of nitrogen on auxin content in roots and the growth of plants under different temperature conditions. Plant Growth Regul 23:207–208

  • Lager I, Andréasson O, Dunbar TL, Andréasson E, Escobar MA, Rasmusson AG (2010) Changes in external pH rapidly alter plant gene expression and modulate auxin and elicitor responses. Plant, Cell Environ 33:1513–1528

    CAS  Google Scholar 

  • Leljak D, Jelaska S (1995) Callus formation and somatic embryo production in pumpkin Cucurbita pepo L. explants on hormone-free medium. Period Biol 97:327–332

    Google Scholar 

  • Leljak-Levanić D, Bauer N, Mihaljević S, Jelaska S (2004a) Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds. J Plant Physiol 161:229–236

    Article  PubMed  Google Scholar 

  • Leljak-Levanić D, Leljak-Levanić D, Bauer N, Mihaljević S, Jelaska S (2004b) Changes in DNA methylation during somatic embryogenesis in Cucurbita pepo L. Plant Cell Rep 23:120–127

    Article  PubMed  Google Scholar 

  • Lima JE, Kojima S, Takahashi H, von Wiren N (2010) Ammonium triggers lateral root branching in Arabidopsis in an ammonium transporter1;3-dependent manner. Plant Cell 22:3621–3633

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Linkohr BI, Williamson LC, Fitter AH, Leyser HM (2002) Nitrate and phosphate availability and distribution have different effects on root system architecture of Arabidopsis. Plant J 29:751–760

    Article  CAS  PubMed  Google Scholar 

  • Logan BA, Demming-Adam B, Rosenstiel TN, Adams WA III (1999) Effect of nitrogen limitation on foliar antioxidants in relationship to other metabolic characteristics. Planta 209:213–220

    Article  CAS  PubMed  Google Scholar 

  • Mashayekhi K, Neumann KH (2006) Effect of boron on somatic embryogenesis of Daucus carrota. Plant Cell Tiss Organ Cult 84(3):279–283

    Article  CAS  Google Scholar 

  • Mattiello L, Kirst M, da Silva F, Jorge RA, Menossi M (2010) Transcriptional profiling of maize root under acid soil growth. BMC Plant Biol 10:196–210

    Article  PubMed Central  PubMed  Google Scholar 

  • Michalczuk L, Cooke TJ, Cohen JD (1992) Auxin levels at different stages of carrot somatic embryogenesis. Phytochemistry 31:1097–1103

    Article  CAS  Google Scholar 

  • Mihaljević S, Radić S, Bauer N, Garić R, Mihaljević B, Horvat G, Leljak-Levanić D, Jelaska S (2011) Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.). J Plant Physiol 168:1943–1951

    Article  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for the rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nakano Y, Asad K (1981) Hydrogen peroxide is scavenged by ascorbate peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880

    CAS  Google Scholar 

  • Palmer SJ, Berridge DM, McDonald AJS, Davies WJ (1996) Control of leaf expansion in sunflower (Helianthus annuus L.) by nitrogen nutrition. J Exp Bot 47:359–368

    Article  CAS  Google Scholar 

  • Pasternak TP, Prinsen E, Ayaydin F, Miskolczi P, Potters G, Asard H, Vanonckelen HA, Dudits D, Fehér A (2002) The role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa. Plant Physiol 129:1807–1819

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pasternak T, Potters G, Caubergs R, Jansen MAK (2005) Complementary interactions between oxidative stress and auxins control plant growth responses at plant, organ, and cellular level. J Exp Bot 56:1991–2001

    Article  CAS  PubMed  Google Scholar 

  • Patterson K, Cakman T, Cooper A, Lager I, Rasmusson AG, Escobar MA (2010) Distinct signaling pathways and transcriptome response signatures differentiate ammonium- and nitrate-supplied plants. Plant, Cell Environ 33:1486–1501

    CAS  Google Scholar 

  • Penčik A, Rolčik J, Novák O, Magnus V, Bartak P, Buchtik R, Salopek- Sondi B, Strnad M (2009) Isolation of novel indole-3-acetic acid conjugates by immunoaffinity extraction. Talanta 80:651–655

    Article  PubMed  Google Scholar 

  • Peuke AD, Jeschke WD, Hartung W (1994) The uptake and flow of C, N and ions between roots and shoots in Ricinus communis L. III. Long distance transport of abscisic acid depending on nitrogen nutrition and salt stress. J Exp Bot 45:741–747

    Article  CAS  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Piotrowska A, Bajguz A (2011) Conjugates of abscisic acid, brassinosteroids, ethylene, gibberellins, and jasmonates. Phytochemistry 72:2097–2112

    Article  CAS  PubMed  Google Scholar 

  • Potters G, De Gara L, Ansard H, Horemans N (2002) Ascorbate and glutathione: guardians of the cell cycle, partners in crime? Plant Physiol Biochem 40:207–216

    Article  Google Scholar 

  • Potters G, Pasternak TP, Guisez Y, Palme KJ, Jansen MAK (2007) Stress induced morphogenic responses: growing out of trouble? Trends Plant Sci 12:98–105

    Article  CAS  PubMed  Google Scholar 

  • Rahayu YS, Walch-Liu P, Neumann G, Römheld V, von Wirén N, Bangerth F (2005) Root-derived cytokinins as long-distance signals for NO3 −-induced stimulation of leaf growth. J Exp Bot 56:1143–1152

    Article  CAS  PubMed  Google Scholar 

  • Ramage CV, Williams RR (2002) Inorganic nitrogen requirements during shoot organogenesis in tobacco leaf discs. J Exp Bot 53:1437–1443

    Article  CAS  PubMed  Google Scholar 

  • Rasband WS (2012). ImageJ (version 1.45) software, U. S. National Institutes of Health, Bethesda, Maryland, USA. http://imagej.nih.gov/ij/. Accessed 5 June 2014

  • Sakakibara H, Takei K, Hirose N (2006) Interactions between nitrogen and cytokinin in the regulation of metabolism and development. Trends Plant Sci 11:440–448

    Article  CAS  PubMed  Google Scholar 

  • Sánchez-Fernández R, Fricker M, Corben LB, White NS, Sheard N, Leaver CJ, Van Montague M, Inzé D, May MJ (1997) Cell proliferation and hair tip growth in the Arabidopsis root are under mechanistically different forms or redox control. Proc Natl Acad Sci USA 94:2745–2750

    Article  PubMed Central  PubMed  Google Scholar 

  • Smith DL, Krikorian AD (1989) Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryogenic cultures in hormone-free medium. Am J Bot 76:1834–1845

    Google Scholar 

  • Smith DL, Krikorian AD (1991) Growth and maintenance of an embryogenic cell culture of daylily (Hemerocallis) on hormone-free medium. Ann Bot 67:443–445

    CAS  PubMed  Google Scholar 

  • Smith DL, Krikorian AD (1992) Low external pH prevents cell elongation but not multiplication of embryogenic carrot cells. Physiol Plant 84:495–501

    Article  CAS  Google Scholar 

  • Stasolla C (2010) Glutathione redox regulation of in vitro embryogenesis. Plant Physiol Biochem 48:319–327

    Article  CAS  PubMed  Google Scholar 

  • Tamaki and Mercier (2007) Cytokinins and auxin communicate nitrogen availability as long-distance signal molecules in pineapple (Ananas comosus). J Plant Physiol 164:1543–1547

    Article  Google Scholar 

  • Tognetti VB, Muhlenbock P, Van Breusegem F (2012) Stress homeostasis—the redox and auxin perspective. Plant, Cell Environ 35:321–333

    Article  CAS  Google Scholar 

  • Turečková V, Novák O, Strnad M (2009) Profiling ABA metabolites in Nicotiana tabacum L. leaves by ultra-performance liquid chromatography-electrospray tandem mass spectrometry. Talanta 80:390–399

    Article  PubMed  Google Scholar 

  • Walch-Liu P, Neumann G, Bangerth F, Engels C (2000) Rapid effects of nitrogen form on leaf morphogenesis in tobacco. J Exp Bot 51:227–237

    Article  CAS  PubMed  Google Scholar 

  • Zavattieri MA, Frederico AM, Lima M, Sabino R, Arnholdt-Schmitt B (2010) Induction of somatic embryogenesis as an example of stress-related plant reactions. Electron J Biotechnol 13:1–9

    Article  Google Scholar 

  • Zdunek E, Lips SH (2001) Transport and accumulation rates of abscisic acid and aldehyde oxidase activity in Pisum sativum L. in response to suboptimal growth conditions. J Exp Bot 52:1269–1276

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. sc. Branka Salopek-Sondi on helpful advice during HPLC analysis of ascorbate. This research was funded by the Ministry of Science, Education and Sport of the Republic of Croatia (098-0982913-2829), the Centre of the Region Haná for Biotechnological and Agricultural Research (ED0007/01/01) and the Internal Grant Agency of Palacký University (PrF_2013_023).

Author information

Authors and Affiliations

  1. Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic

    Aleš Pěnčík, Veronika Turečková, Jakub Rolčík & Miroslav Strnad

  2. Institute Ruđer Bošković, Bijenička cesta 54, 10000, Zagreb, Croatia

    Sandi Paulišić & Snježana Mihaljević

Authors
  1. Aleš Pěnčík
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Veronika Turečková
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sandi Paulišić
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jakub Rolčík
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Miroslav Strnad
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Snježana Mihaljević
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Snježana Mihaljević.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pěnčík, A., Turečková, V., Paulišić, S. et al. Ammonium regulates embryogenic potential in Cucurbita pepo through pH-mediated changes in endogenous auxin and abscisic acid. Plant Cell Tiss Organ Cult 122, 89–100 (2015). https://doi.org/10.1007/s11240-015-0752-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-015-0752-0

Keywords

Search

Navigation