Overexpression of MtTdp2α (tyrosyl-DNA phosphodiesterase 2) gene confers salt tolerance in transgenic Medicago truncatula

  • Massimo ConfalonieriEmail author
  • Maria Carelli
  • Aldo Tava
  • Lamberto Borrelli
Original Article


Soil salinity is one of the main abiotic stresses affecting yield in major crop plants, including legumes. Research carried out on model legumes such as barrel medic (Medicago truncatula Gaertn.) showed that the Tyrosyl-DNA phosphodiesterase 2α (MtTdp2α) DNA repair gene, involved in the removal of topoisomerase-DNA covalent complexes, play a key role in the plant response to osmotic and copper stresses. However, no informations are currently available about the involvement of MtTdp2α in response to salt stress and salt shock. In the present study we investigated the role of MtTdp2α under salinity (0, 50, 100, 150 and 200 mM NaCl) stress conditions in transgenic M. truncatula overexpressing the MtTdp2α gene. The level of salt tolerance of Tdp2-28 selected transgenic line was significantly higher than control as measured by the increase in shoot fresh weight, shoot dry weight and salt sensitivity index, in response to 100 mM NaCl. After salt stress, Tdp2-28 transgenic line showed significantly higher chlorophyll and carotenoid total contents, 2,2-Diphenyl-1-Picrylhydrazyl radical scavenging activity, and significantly lower levels of oxidative DNA damage than the control line. Interestingly, the expression levels of several genes, including genes linked to genome maintenance and regulation of DNA topology (MtTdp1α, MtTop2), base excision repair pathway (MtOGG1) and double strand break sensing/repair (MtMRE11) were enhanced in Tdp2-28 transgenic shoots under salt stress conditions as compared to their controls. These findings suggest that MtTdp2α play an important role in plant tolerance to salt stress.

Key message

Overexpression of MtTdp2α gene in Medicago truncatula confers salt stress tolerance through better plant growth performances, higher chlorophyll and carotenoid contents, increased antioxidant capacity, reduced oxidative DNA damage, and by up-regulation of several genes involved in DNA metabolism.


Medicago truncatula Salt tolerance Transgenic Tyrosyl-DNA phosphodiesterase 





Base excision repair


Double strand break


Salt sensitivity index




Quantitative real-time PCR


Polyethylene glycol


Murashige and Skoog (1962)


Reactive oxigen species


5-Tyrosyl DNA phosphodiesterase II


Author contributions

MC conceived and designed the research, conducted the salt stress, the pigment and antioxidant determination experiments and wrote the manuscript. LB, MC and AT performed molecular and ion determination analyses, and analyzed the data. All authors discussed the results and agreed to the final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2019_1560_MOESM1_ESM.doc (47 kb)
Supplementary material 1 (DOC 47 KB)


  1. Amin M, Elias SM, Hossain A, Ferdousi A, Rahman S, Tuteja N, Seraj ZI (2012) Over-expression of a DEAD-box helicase, PDH45, confers both seedling and reproductive stage salinity tolerance to rice (Oryza sativa L.). Mol Breed 30(1):345–354Google Scholar
  2. Araújo SS, Balestrazzi A, Faè M, Morano M, Carbonera D, Macovei A (2016) 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 Tissue Organ Cult 127:675–680Google Scholar
  3. Aydi S, Sassi S, Debouba M, Hessini K, Larrainzar E, Gouia H, Abdelly C (2010) Resistance of Medicago truncatula to salt stress is related to glutamine synthetase activity and sodium sequestration. J Plant Nutr Soil Sci 173:892–899Google Scholar
  4. Balestrazzi A, Confalonieri M, Macovei A, Carbonera D (2011) Seed imbibition in Medicago truncatula Gaertn.: expression profiles of DNA repair genes in relation to PEG-mediated stress. J Plant Physiol 168:706–713Google Scholar
  5. Balestrazzi A, Confalonieri M, Macovei A, Donà M, Carbonera D (2012) Genotoxic stress, DNA repair and crop productivity. In: Tuteja N, Gill SS (eds) Crop improvement under adverse conditions. Springer, Berlin, pp 153–170Google Scholar
  6. Bianco C, Defez R (2009) Medicago truncatula improves salt tolerance when nodulated by an indole-3-acetic acid-overproducing Sinorhizobium meliloti strain. J Exp Bot 60(11):3097–3107Google Scholar
  7. Bonatto D (2007) A systems biology analysis of protein-protein interaction between yeast superoxide dismutases and DNA repair pathways. Free Rad Biol Med 43:557–567Google Scholar
  8. Campanelli A, Ruta C, Morone-Fortunato I, De Mastro G (2013) Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection. Cent Eur J Biol 8(8):765–776Google Scholar
  9. Cerda A, Pardines J, Botella MA, Martinez V (1995) Effect of potassium on growth, water relations, and the inorganic and organic solute contents for two maize cultivars grown under saline conditions. J Plant Nutr 18:839–851Google Scholar
  10. Chavan JJ, Gaikwad NB, Umdale SD, Kshirsagar PR, Bhat KV, Yadav SR (2014) Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui. Plant Growth Regul 72:1–15Google Scholar
  11. Chen H, Chu P, Zhou Y, Li Y, Liu J, Ding Y, Tsang EW, Jiang L, Wu K, Huang S (2012) Overexpression of AtOGG1, a DNA glycosylase/AP lyase, enhances seed longevity and abiotic stress tolerance in Arabidopsis. J Exp Bot 63:4107–4121Google Scholar
  12. Chen SH, Chan NL, Hsieh TS (2013) New mechanistic and functional insights into DNA topoisomerases. Annu Rev Biochem 82:139–170Google Scholar
  13. Confalonieri M, Faè M, Balestrazzi A, Donà M, Macovei A, Valassi A, Giraffa G, Carbonera D (2014a) Enhanced osmotic stress tolerance in Medicago truncatula plants overexpressing the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2). Plant Cell Tissue Organ Cult 116(2):187–203Google Scholar
  14. Confalonieri M, Carelli M, Galimberti V, Macovei A, Panara F, Biggiogera M, Scotti C, Calderini O (2014b) Seed-specific expression of AINTEGUMENTA in Medicago truncatula led to the production of larger seeds and improved seed germination. Plant Mol Biol Rep 32:957–970Google Scholar
  15. de Lorenzo L, Merchan F, Laporte P, Thompson R, Clarke J, Sousa C, Crespi M (2009) A novel plant leucine-rich repeat receptor kinase regulates the response of Medicago truncatula roots to salt stress. Plant Cell 21:668–680Google Scholar
  16. 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 32:129–141Google Scholar
  17. 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) in Medicago truncatula plants. Plant Cell Rep 33:1071–1080Google Scholar
  18. Foyer CH, Noctor G (2003) Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiol Plant 119:335–364Google Scholar
  19. Foyer CH, Noctor G (2005) Oxidant and antioxidant signaling in plants: a reevaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28:1056–1071Google Scholar
  20. Garcia-Ortiz MV, Ariza R, Roldan-Arjona T (2001) An OGG1 orthologue encoding a functional 8-oxoguanine DNA glycosylase/lyase in Arabidopsis thaliana. Plant Mol Biol 47:795–804Google Scholar
  21. Gill SS, Tajrishi M, Madan M, Tuteja N (2013) A DESD-box helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. PB1). Plant Mol Biol 82:1–22Google Scholar
  22. Gruber V, Blanchet S, Diet A, Zahaf O, Boualem A, Kakar K, Alunni B, Udvardi M, Frugier F, Crespi M (2009) Identification of transcription factors involved in root apex responses to salt stress in Medicago truncatula. Mol Genet Genom 281:55–66Google Scholar
  23. Hossain Z, Mandal AKA, Datta KS, Biswas AK (2006) Development of NaCl-tolerant strain in Chrysanthemum morifolium Ramat. through in vitro mutagenesis. Plant Biol 8:450–461Google Scholar
  24. Hussain TM, Chandrasekhar T, Hazara M, Sultan Z, Saleh BK, Gopal GR (2008) Recent advances in salt stress biology: a review. Biotech Mol Biol Rev 3(1):8–13Google Scholar
  25. Jain M, Tyagi AK, Khurana JP (2006) Overexpression of putative topoisomerase 6 genes from rice confers stress tolerance in transgenic Arabidopsis plants. FEBS J 273:5245–5260Google Scholar
  26. John R, Ganeshan U, Singh BN, Kaul T, Reddy MK, Sopory SK, Rajam MV (2016) Over-expression of topoisomerase II enhances salt stress tolerance in tobacco. Front Plant Sci 7:1280Google Scholar
  27. Kim SH, Ahn YO, Ahn MJ, Lee HS, Kwak SS (2012) Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweet potato. Phytochemistry 74:69–78Google Scholar
  28. Läuchli A (1984) Salt exclusion: an adaptation of legumes for crops and pastures under saline conditions. In: Staples RC, Toenniessen GH (eds) Salinity tolerance in plants: strategies for crop improvement. Wiley, New York, pp 171–187Google Scholar
  29. 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 461:674–678Google Scholar
  30. Li D, Z Su Z, Dong J, Wang J T (2009) An expression database for roots of the model legume Medicago truncatula under salt stress. BMC Genom 10:517Google Scholar
  31. Li R, Shi F, Fukuda K, Yang Y (2010) Effects of salt and alkali stresses on germination, growth, photosynthesis and ion accumulation in alfalfa (Medicago sativa L.). Soil Sci Plant Nutr 56:725–733Google Scholar
  32. Luo Y, Liu YB, Dong YX, Gao XQ, Zhang XS (2009) Expression of a putative alfalfa helicase increases tolerance to abiotic stress in arabidopsis by enhancing the capacities for ROS scavenging and osmotic adjustment. J Plant Physiol 166(4):385–394Google Scholar
  33. Maathuis FJM (2013) Sodium in plants: perception, signalling, and regulation of sodium fluxes. J Exp Bot 65(3):849–858Google Scholar
  34. Macovei A, Tuteja N (2012) MicroRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.). BMC Plant Biol 12:183–195Google Scholar
  35. Macovei A, Balestrazzi A, Confalonieri M, Carbonera D (2010) Tyrosyl-DNA phosphodiesterase) gene family in barrel medic (Medicago truncatula Gaertn.): bioinformatic investigation and expression profiles in response to copper- and PEG-mediated stress. Planta 232:393–407 Tdp1Google Scholar
  36. Macovei A, Balestrazzi A, Confalonieri M, Faè M, Carbonera D (2011) New insights on the barrel medic MtOGG1 and MtFPG functions in relation to oxidative stress response in planta and during seed imbibition. Plant Physiol Biochem 49:1040–1050Google Scholar
  37. Meloni DA, Oliva MA, Martinez CA, Cambraia J (2003) Photosynthesis and activity of peroxide dismutase, peroxidase and glutathione reductase in cotton under salt stress. Environ Exp Bot 49:69–76Google Scholar
  38. Mhadhbi H, Fotopoulos V, Mylona PV, Jebara M, Elarbi Aouani M, Polidoros AN (2011) Antioxidant gene-enzyme responses in Medicago truncatula genotypes with different degree of sensitivity to salinity. Physiol Plant 141:201–214Google Scholar
  39. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681Google Scholar
  40. Murai J, Huang SY, Das BB, Dexheimer TS, Takeda S, Pommier Y (2012) Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerase I and II and base alkylation in vertebrate cells. J Biol Chem 287:12848–12857Google Scholar
  41. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:73–79Google Scholar
  42. Murphy TM, George A (2005) A comparison of two DNA base excision repair glycosylases from Arabidopsis thaliana. Biochem Biophys Res Commun 329:869–872Google Scholar
  43. Owttrim GW (2006) RNA helicases and abiotic stress. Nucleic Acids Res 34(11):3220–3230Google Scholar
  44. Paul D, Lade H (2014) Plant-growth-promoting rhizobacteria to improve crop growth in saline soils: a review. Agron Sustain Dev 34:737–752Google Scholar
  45. Paull TT, Deshpande RA (2014) The Mre11/Rad50/Nbs1 complex: recent insights into catalytic activities and ATP-driven conformational changes. Exp Cell Res 329:139–147Google Scholar
  46. Porra RJ, Thomson WA, Kriedermann PE (1989) Determination of accurate extintion coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophylls standards by absorption spetroscopy. Biochim Biophys Acta 975:348–394Google Scholar
  47. Sabatini ME, Pagano A, Araùjo S, Balestrazzi A, Macovei A (2017) The Tyrosyl-DNA phosphodiesterase 1β (Tdp1β) gene discloses an early response to abiotic stresses. Genes 8:305Google Scholar
  48. Saed-Moucheshi A, Shekoofa A, Pessarakli M (2014) Reactive oxygen species (ROS) generation and detoxifying in plants. J Plant Nutr 37(10):1573–1585Google Scholar
  49. Saha P, Mukherjee A, Biswas AK (2015) Modulation of NaCl induced DNA damage and oxidative stress in mungbean by pretreatment with sublethal dose. Biol Plant 59(1):139–146Google Scholar
  50. Sanan-Mishra N, Pham XH, Sopory SK, Tuteja N (2005) Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc Natl Acad Sci USA 102:509–514Google Scholar
  51. Scaramelli L, Balestrazzi A, Bonadei M, Piano E, Carbonera D, Confalonieri M (2009) Production of transgenic barrel medic (Medicago truncatula Gaertn.) using the ipt-type MAT vector system and impairment of Recombinase-mediated excision events. Plant Cell Rep 2:197–211Google Scholar
  52. Shah SH, Houborg R, McCabe RF (2017) Response of chlorophyll, carotenoid and SPAD-502 measurement to salinity and nutrient stress in wheat (Triticum aestivum L.). Agronomy 7(3):61. Google Scholar
  53. Sharma P, Jha AB, Dubey RS, Pessarakl M (2012) Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot 2012:217037. Google Scholar
  54. Shaul O (2002) Magnesium transport and function in plants: the tip of the iceberg. Biometals 15(3):309–323Google Scholar
  55. Šimková K, Moreau F, Pawlak P, Vriet C, Baruah A, Alexandre C et al (2012) Integration of stress-related and reactive oxygen species-mediated signals by Topoisomerase VI in Arabidopsis thaliana. Proc Natl Acad Sci USA 109:16360–16365Google Scholar
  56. Stepien P, Johnson GN (2009) Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte Thellungiella: role of the plastid terminal oxidase as an alternative electron sink. Plant Physiol 149:1154–1165Google Scholar
  57. Tang L, Cai H, Zhai H, Luo X, Wang Z, Cui L, Bai X (2014) Overexpression of Glycine soja WRKY20 enhances both drought and salt tolerance in transgenic alfalfa (Medicago sativa L.). Plant Cell Tissue Organ Cult 118(1):77–86Google Scholar
  58. Tuteja N, Tuteja R (2006) DNA helicases as molecular motors: an insight. Phys A 372:70–83Google Scholar
  59. Valavanidis A, Vlachogianni T, Fiotakis C (2009) 8-hydroxy-2′-deoxyguanosine (8-OHdG): a critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health C 27:120–139Google Scholar
  60. Vashisht AA, Tuteja N (2006) Stress responsive DEAD-box helicases: a new pathway to engineer plant stress tolerance. J Photochem Photobiol Biol 84:156–160Google Scholar
  61. Vos SM, Tretter EM, Schmidt BH, Berger JM (2011) All tangled up: how cells direct, manage and exploit topoisomerase function. Nat Rev Mol Cell Biol 12:827–841Google Scholar
  62. Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids using various solvents with spectrophotometers of different resolution. Plant Phisiol 144:307–313Google Scholar
  63. Xie S, Lam E (1994) Abundance of nuclear DNA topoisomerase 1 is correlated with proliferation in Arabidopsis thaliana. Nucl Acid Res 22:5729–5736Google Scholar
  64. Zahaf O, Blanchet S, De Zelicourt A, Alunni B, Plet J, Laffont C, de Lorenzo L, Imbeaud S, Ichanté JL, Diet A, Badri M, Zabalza A, González EM, Delacroix H, Gruber V, Frugier F, Crespi M (2012) Comparative transcriptomic analysis of salt adaptation in roots of contrasting Medicago truncatula genotypes. Mol Plant 5(5):1068–1081Google Scholar
  65. 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 286:403–409Google Scholar
  66. Zhou G-A, Chang R-Z, Qiu L-J (2010) Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis. Plant Mol Biol 72:357–367Google Scholar
  67. Zhu J-K (2003) Regulation of ion homeostasis under salt stress. Curr Opin Plant Biol 6:441–445Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.CREA Research Centre for Animal Production and Aquaculture (CREA-ZA)LodiItaly

Personalised recommendations