Skip to main content
SpringerLink
Log in

Improved procedure for detection of superoxide dismutase isoforms in potato, Solanum tuberosum L.

  • Original Paper
  • Published:
Acta Physiologiae Plantarum Aims and scope Submit manuscript

Abstract

Superoxide dismutase (SOD) in-gel activity assay with selective inhibitors (KCN and H2O2) is one of the most commonly used methods for identification of SOD isoform types, i.e., FeSOD, MnSOD or Cu/ZnSOD, and evaluation of oxidative stress response in plants. However, there are potential pitfalls that surround this assay, such as problem to detect isoforms with low activity, comigration of SOD isoforms or application of inappropriate inhibitor concentration. We propose an improved method based on the combination of in-gel analysis of SOD activity and native-PAGE immunoblotting for identification of isoforms and determination of SOD isoenzyme activity pattern in potato. Depending on cultivar and growing conditions, one MnSOD, 3 FeSOD and 5–6 Cu/ZnSOD isoforms were identified in potato leaves. The most important qualitative difference between ex vitro- and in vitro-grown plants was the presence of additional FeSOD and Cu/ZnSOD isoforms in plantlets grown in vitro. Compared with results of in-gel activity assay with selective inhibitors, new method allowed accurate identification of comigrating FeSOD and Cu/ZnSOD isoforms and two protein bands of ambiguous identities. Potato SODs were also characterized by SDS-PAGE immunoblotting and single MnSOD (23.6 kDa), three Cu/ZnSOD polypeptides (17.9, 17 and 16.3 kDa) and single FeSOD (25.1 kDa) polypeptide were detected in leaves of four examined cultivars. The difference in the number of FeSOD and Cu/ZnSOD isoforms/polypeptides between native-PAGE and SDS-PAGE immunoblots suggests that SOD proteins may have undergone post-translational modifications affecting protein mobility or existence of isoforms that differ from each other in total protein charge, but not in molecular weight.

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

Similar content being viewed by others

References

  • Abdel-Ghany SE, Müller-Moulé P, Niyogi KK, Pilon M, Shikanai T (2005) Two P-type ATPases are required for copper delivery in Arabidopsis thaliana chloroplasts. Plant Cell 17:1233–1251

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Alscher RG, Erturk N, Heath LS (2002) Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot 53:1331–1341

    Article  CAS  PubMed  Google Scholar 

  • Anderson MD, Prasad TK, Stewart CR (1995) Changes in isozyme profiles of catalase, peroxidase, and glutathione reductase during acclimation to chilling in mesocotyls of maize seedlings. Plant Physiol 109:1247–1257

    CAS  PubMed Central  PubMed  Google Scholar 

  • Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287

    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 

  • Drotar A, Phelps P, Fall R (1985) Evidence for glutathione peroxidase activities in cultured plant cells. Plant Sci 42:35–40

    Article  CAS  Google Scholar 

  • Dugas DV, Bartel B (2008) Sucrose induction of Arabidopsis miR398 represses two Cu/Zn superoxide dismutases. Plant Mol Biol 67:403–417

    Article  CAS  PubMed  Google Scholar 

  • Fidalgo F, Santos A, Santos I, Salema R (2004) Effects of long-term salt stress on antioxidant defence systems, leaf water relations and chloroplast ultrastructure of potato plants. Ann Appl Biol 145:185–192

    Article  CAS  Google Scholar 

  • Fridovich I (1986) Superoxide dismutases. Adv Enzymol Relat Areas Mol Biol 58(6):61–97

    CAS  PubMed  Google Scholar 

  • Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930

    Article  CAS  PubMed  Google Scholar 

  • Kukavica B, Mojović M, Vučinić Ž, Maksimović V, Takahama U, Veljović-Jovanović S (2009) Generation of hydroxyl radical in isolated pea root cell wall, and the role of cell wall-bound peroxidase, Mn-SOD and phenolics in their production. Plant Cell Physiol 50:304–317

    Article  CAS  PubMed  Google Scholar 

  • Martinez CA, Loureiro ME, Oliva MA, Maestri M (2001) Differential responses of superoxide dismutase in freezing resistant Solanum curtilobum and freezing sensitive Solanum tuberosum subjected to oxidative and water stress. Plant Sci 160:505–515

    Article  CAS  PubMed  Google Scholar 

  • Meloni DA, Oliva MA, Martinez CA, Cambraia J (2003) Photosynthesis and activity of superoxide dismutase, peroxidase and glutathione reductase in cotton under salt stress. Environ Exp Bot 49:69–76

    Article  CAS  Google Scholar 

  • Miller A-F (2012) Superoxide dismutases: ancient enzymes and new insights. FEBS Lett 586:585–595

    Article  CAS  PubMed  Google Scholar 

  • Miszalski Z, Ślesak I, Niewiadomska E, Baczek-Kwinta R, Lüttge U, Ratajczak R (1998) Subcellular localization and stress responses of superoxide dismutase isoforms from leaves in the C3-CAM intermediate halophyte Mesembryanthemum crystallinum L. Plant, Cell Environ 21:169–179

    Article  CAS  Google Scholar 

  • Momcilovic I, Ristic Z (2007) Expression of chloroplast protein synthesis elongation factor, EF-Tu, in two lines of maize with contrasting tolerance to heat stress during early stages of plant development. J Plant Physiol 164:90–99

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Pan S-M, Yau Y–Y (1992) Characterization of superoxide dismutase in Arabidopsis. Taiwania 37:58–66

    Google Scholar 

  • Pilon M, Ravet K, Tapken W (2011) The biogenesis and physiological function of chloroplast superoxide dismutases. Biochim Biophys Acta 1807:989–998

    Article  CAS  PubMed  Google Scholar 

  • Queirós F, Rodrigues JA, Almeida JM, Almeida DPF, Fidalgo F (2011) Differential responses of the antioxidant defence system and ultrastructure in a salt-adapted potato cell line. Plant Physiol Biochem 49:1410–1419

    Article  PubMed  Google Scholar 

  • Queval G, Noctor G (2007) A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: application to redox profiling during Arabidopsis rosette development. Anal Biochem 363:58–69

    Article  CAS  PubMed  Google Scholar 

  • Rahnama H, Ebrahimzadeh H (2006) Antioxidant isozymes activities in potato plants (Solanum tuberosum L.) under salt stress. J Sci Islam Repub Iran 17:225–230

    CAS  Google Scholar 

  • Rojas-Beltran JA, Dejaeghere F, Abd Alla Kotb M, Du Jardin P (2000) Expression and activity of antioxidant enzymes during potato tuber dormancy. Potato Res 43:383–393

    Article  CAS  Google Scholar 

  • Santos I, Almeida J, Salema R (1999) The influence of UV-B radiation on the superoxide dismutase of maize, potato, sorghum, and wheat leaves. Can J Bot 77:70–76

    CAS  Google Scholar 

  • Weisiger RA, Fridovich I (1973) Superoxide dismutase organelle specificity. J Biol Chem 248:3582–3592

    CAS  PubMed  Google Scholar 

  • Yamakura F, Kawasaki H (2010) Post-translational modifications of superoxide dismutase. Biochim Biophys Acta 1804:318–325

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Ministry of Education, Science and Technological Development of the Republic of Serbia, Project Grant No. TR31049.

Author information

Authors and Affiliations

  1. Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar Despota Stefana 142, 11060, Belgrade, Serbia

    Ivana Momčilović, Danijel Pantelić, Jelena Savić & Dragan Vinterhalter

  2. Faculty of Biology, University of Belgrade, Studentski Trg 3, 11000, Belgrade, Serbia

    Masoud Hfidan

Authors
  1. Ivana Momčilović
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Danijel Pantelić
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Masoud Hfidan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jelena Savić
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dragan Vinterhalter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ivana Momčilović.

Additional information

Communicated by L. A. Kleczkowski.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Momčilović, I., Pantelić, D., Hfidan, M. et al. Improved procedure for detection of superoxide dismutase isoforms in potato, Solanum tuberosum L.. Acta Physiol Plant 36, 2059–2066 (2014). https://doi.org/10.1007/s11738-014-1583-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11738-014-1583-z

Keywords

Search

Navigation