Skip to main content
SpringerLink
Log in

MtTdp2α-overexpression boosts the growth phase of Medicago truncatula cell suspension and increases the expression of key genes involved in the antioxidant response and genome stability

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

Abstract

Aside from the great importance of Leguminosae in food and agriculture industry, legume model systems like Medicago truncatula are also essential tools to dissect complex cellular pathways and retrieve valuable information to other crops. Here, we investigated the roles played by the tyrosyl-DNA phosphodiesterase 2α (MtTdp2α) gene in cell viability and proliferation using M. truncatula suspension cultures. Our research hypothesis is that the overexpression of MtTdp2α, implicated in the removal of transient topoisomerase/DNA covalent complexes, can impact on cell suspension viability. M. truncatula suspension cultures derived from leaf explants of MtTdp2α-overexpressing lines and a control line carrying the empty vector were used. Our results showed that the control line reached the stationary growth phase by the fourth day of culture while the transgenic lines presented an extended exponential growth, reaching the stationary phase at day six following culture. The MtTdp2α-overexpressing lines also showed increased viability as compared to the control line. The transcript levels of MtSOD, MtAPX, MtMT2, MtMRE11, MtNBS1, MtRad50, MtOGG1 and MtFPG were significantly enhanced in the transgenic lines as compared to control. Overall, our results show that the MtTdp2α overexpression impacts in a positive manner on cell viability and proliferation in suspension cultures. Additionally, our study provides an insight on the suitability of M. truncatula cell suspension cultures as a promising alternative to evaluate potential protective mechanisms, with results comparable to those obtained when using whole plants.

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

References

  • Balestrazzi A, Macovei A, Tava A, Avato A, Raimondi E, Carbonera D (2011) Unraveling the response of plant cells to cytotoxic saponins. Role of metallothionein and nitric oxide. Plant Signal Behav 6:516–519

    Article  CAS  PubMed Central  Google Scholar 

  • Balestrazzi A, Confalomiri M, Macovei A, Donà M, Carbonera D (2013) Genotoxic stress, DNA repair and crop productivity. In: Tuteja N, Gill SS (eds) Crop improvement under adverse conditions. Springer-Verlag, Heidelberg, pp 153–169

    Chapter  Google Scholar 

  • Bhat SA, Srinivasan S (2002) Molecular and genetic analyses of transgenic plants: considerations and approaches. Plant Sci 163:673–681

    Article  CAS  Google Scholar 

  • Bonatto D (2007) A systems biology analysis of protein–protein interaction between yeast superoxide dismutases and DNA repair pathways. Free Rad Biol Med 43:557–567

    Article  CAS  PubMed  Google Scholar 

  • Butaye KMJ, Cammue BPA, Delauré ST, De Bolle MFG (2005) Approaches to minimize variation of transgene expression in plants. Mol Breed 16:79–91

    Article  Google Scholar 

  • Carimi F, Zottini M, Formentin E, Terzi M, Lo Schiavo F (2003) Cytokinins: new apoptotic inducers in plants. Planta 216:413–421

    CAS  PubMed  Google Scholar 

  • Chen SH, Chan NL, Hsieh TS (2013) New mechanistic and functional insights into DNA topoisomerases. Annu Rev Biochem 82:139–170

    Article  CAS  PubMed  Google Scholar 

  • Confalonieri M, Faè M, Balestrazzi A, Donà M, Macovei A, Valassi A, Giraffa G, Carbonera D (2014) Enhanced osmotic stress tolerance in Medicago truncatula plants overexpressing the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2). Plant Cell Tissue Organ Cult 116:187–203

    Article  CAS  Google Scholar 

  • Cortes Ledesma FC, El Khamisy SF, Zuma MC, Osborn K, Caldecott KW (2009) A human 5′-tyrosyl-DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage. Nature 461:674–678

    Article  PubMed  Google Scholar 

  • Donà M, Confalonieri M, Minio A, Biggiogera M, Buttafava A, Raimondi E, Delledonne M, Ventura L, Sabatini ME, Macovei A, Giraffa G, Carbonera D, Balestrazzi A (2013) RNA-Seq analysis discloses early senescence and nucleolar dysfunction triggered by Tdp1α depletion in Medicago truncatula. J Exp Bot 64:1941–1951

    Article  PubMed  Google Scholar 

  • Donà M, Ventura L, Balestrazzi A, Buttafava A, Carbonera D, Confalonieri M, Giraffa G, Macovei A (2014) Dose-dependent reactive species accumulation and preferential double strand breaks repair are featured in the γ-ray response in Medicago truncatula cells. Plant Mol Biol Rep 32:129–141

    Article  Google Scholar 

  • Faè M, Balestrazzi A, Confalonieri M, Donà M, Macovei A, Valassi A, Giraffa G, Carbonera D (2014) Copper-mediated genotoxic stress is attenuated by the overexpression of the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2 alpha) in Medicago truncatula plants. Plant Cell Rep 33:1071–1080

    Article  PubMed  Google Scholar 

  • Gepts P, Beavis WD, Brummer EC, Shoemaker RC, Stalker HT, Weeedn NF, Young ND (2005) Legumes as a model plant family. Genomics for food and feed report of the cross-legume advances through genomics conference. Plant Physiol 137:1228–1235. doi:10.1104/pp.105.060871

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Godoy-Hernández G, Vázquez-Flota FA (2006) Growth measurements: estimation of cell division and cell expansion. In: Loyola-Vargas VM, Vazquez-Flota F (eds) Plant cell culture protocols, 2nd edn. Humana Press Inc., Totowa, pp 51–58

    Google Scholar 

  • Gómez-Herreros F, Romero-Granados R, Zeng Z, Alarez-Quilón A, Quitero C, Ju L, Umans L, Vermiere L, Huylebroeck D, Caldecott KW, Cortés-Ledesma F (2013) TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo. PLoS Genet 9:e1003226

    Article  PubMed  PubMed Central  Google Scholar 

  • Guo D, Dexheimer TS, Pommier Y, Nash HA (2014) Neuroprotection and repair of 3′-blocking DNA ends by glaikit (gkt) encoding Drosophila tyrosyl-DNA phosphodiesterase 1 (TDP1). Proc Natl Acad Sci USA 111:15816–15820

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hassinen VT, Tervahauta AI, Schat H, Kärenlampi SO (2011) Plant metallothioneins-metal chelators with ROS scavenging activity? Plant Biol 13:225–232. doi:10.1111/j.1438-8677.2010.00398.x

    Article  CAS  PubMed  Google Scholar 

  • Lamarche BJ, Orazio NI, Weitzman MD (2010) The MRN complex in double-strand break repair and telomere maintenance. FEBS Lett 584:3682–3695

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li C, Sun S-Y, Khuri FR, Li R (2011) Pleiotropic functions of EAPII/TTRAP/TDP2-cancer development, chemoresistance and beyond. Cell Cycle 10:1–10

    Article  Google Scholar 

  • Macovei A, Balestrazzi A, Confalonieri M, Carbonera D (2010) The tyrosyl-DNA phosphodiesterase gene family in Medicago truncatula Gaertn.: bioinformatic investigation and expression profiles in response to copper- and PEG-mediated stress. Planta 232:303–307

    Article  Google Scholar 

  • Macovei A, Donà M, Carbonera D, Balestrazzi A (2016) Plant response to genotoxic stress: a crucial role in the context of global climate change. In: Tuteja N, Gill SS (eds) Abiotic stress response in plants. Wiley-VCH Verlag GmbH & Co. KgaA, Weinheim, pp 13–26

    Chapter  Google Scholar 

  • Mhadhbi H, Fotopoulus V, Mylona PV, Jebara M, Aouani ME, Polidoros AN (2011) Antioxidant gene-enzyme responses in Medicago truncatula genotypes with different degree of sensitivity to salinity. Physiol Plant 141:201–204

    Article  CAS  PubMed  Google Scholar 

  • Millet A, Strauss F, Delagoutte E (2015) Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex. Sci Rep 24:13154

    Article  Google Scholar 

  • Nagano I, Murakami T, Manabe Y, Abe K (2002) Early decrease of survival factors and DNA repair enzyme in spinal motor neurons of presymptomatic transgenic mice that express a mutant SOD1 gene. Life Sci 72:541–548

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acid Res 9:336

    Google Scholar 

  • Pype S, Declercq W, Ibrahimi A, Michiels C, Van Rietschoten JG, Dewulf N, de Boer M, Vandenabeele P, Huylebroeck D, Remacle JE (2000) TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-κB activation. J Biol Chem 275:18586–18593

    Article  CAS  PubMed  Google Scholar 

  • Shin R, Schachtman DP (2004) Hydrogen peroxide mediates plant root cell response to nutrient depravation. PNAS 101:8827–8832. doi:10.1073/pnas.0401707101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Simon HU, Haj-Yehia A, Levi-Schaffer F (2000) Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5:415–418

    Article  CAS  PubMed  Google Scholar 

  • Takahashi S (2012) Molecular functions of metallothionein and its role in hematological malignancies. J Hematol Oncol 5:41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Varady G, Sarkadi B, Fatyol K (2011) TTRAP is a novel component of the non-canonical TRAF6-TAK1 TGF-β signaling pathway. PLoS One 6:e25548

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ventura L, Macovei A, Donà M, Paparella S, Buttafava A, Giovannini A, Carbonera D, Balestrazzi A (2013) Genotoxic effects due to in vitro culture and H2O2 treatments in Petunia x hybrida cells monitored through DNA diffusion assay, FPG-SCGE and gene expression profile analyses. Acta Physiol Plant 36:331–341

    Article  Google Scholar 

  • Zeng Z, Cortes-Ledesma F, El Khamisy SF, Caldecott KW (2011) TDP2/TTRAP is the major 5′-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular resistance to topoisomerase II-induced DNA damage. J Biol Chem 286:403–409

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This research was supported by grants from the University of Pavia. S.A. has been awarded by a Research Contract funded by CARIPLO Foundation (Action 3, Code 2013-1727)—Integrated Project ‘Advanced Priming Technologies for the Lombardy Agro-Seed Industry-PRIMTECH’. S.A. also acknowledges the financial support from Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through research unit GREEN-it “Bioresources for Sustainability” (UID/Multi/04551/2013) and post-doctoral grant (SFRH/BPD/108032/2015).

Author information

Authors and Affiliations

  1. Department of Biology and Biotechnology ‘L. Spallanzani’, University of Pavia, via Ferrata 9, 27100, Pavia, Italy

    Susana Araújo, Alma Balestrazzi, Matteo Faè, Martina Morano, Daniela Carbonera & Anca Macovei

  2. Plant Cell Biotechnology Laboratory, Instituto de Tecnologia Química e Biológica ‘António Xavier’, Universidade Nova de Lisboa (ITQB-NOVA), Avenida da Republica, Estação Agronómica Nacional, 2780-157, Oeiras, Portugal

    Susana Araújo

Authors
  1. Susana Araújo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alma Balestrazzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matteo Faè
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Martina Morano
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daniela Carbonera
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anca Macovei
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anca Macovei.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Araújo, S., Balestrazzi, A., Faè, M. et al. MtTdp2α-overexpression boosts the growth phase of Medicago truncatula cell suspension and increases the expression of key genes involved in the antioxidant response and genome stability. Plant Cell Tiss Organ Cult 127, 675–680 (2016). https://doi.org/10.1007/s11240-016-1075-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-016-1075-5

Keywords

Search

Navigation