, Volume 223, Issue 2, pp 180–190 | Cite as

Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2+ tolerance and accumulation but not translocation to the shoot

  • Mirella Pomponi
  • Vincenzo Censi
  • Valentina Di Girolamo
  • Angelo De Paolis
  • Luigi Sanità di Toppi
  • Rita Aromolo
  • Paolo Costantino
  • Maura Cardarelli
Original Article


Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd2+ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd2+ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd2+ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd2+ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd2+ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd2+ transport.


Cd accumulation Cd tolerance Glutathione PCS1 overexpression Tobacco 



Days after germination







Special thanks are given to Dr. Annette Pickford for helpful comments during manuscript revision. We thank Dr. Adele Figliolia (INP Rome, Italy) for helpful discussions, Prof. Rita Biasi and Dr. Patricia Gutierrez (University of Viterbo, Italy) for their help in ANOVA analysis. This work was partially supported by grants from Istituto Pasteur Fondazione Cenci-Bolognetti, and MIUR (FIRB, PRIN, Centro di Eccellenza in Biologia e Medicina Molecolare).


  1. Bellincampi D, Cardarelli M, Zaghi D, Serino G, Salvi G, Gatz C, Cervone F, Altamura MM, Costantino P, De Lorenzo G (1996) Oligogalacturonides prevent rhizogenesis in rolB-transformed tobacco explants by inhibiting auxin-induced expression of the rolB gene. Plant Cell 8:477–487PubMedCrossRefGoogle Scholar
  2. Broeks A, Gerrard B, Allikmets R, dean M, Plasterk RH (1996) Homologues of the human multidrug resistance genes MRP and MDR contribute to heavy metal resistance in the soil nematode Caenorhabditis elegans. EMBO J 15:6132–6143PubMedGoogle Scholar
  3. Capone I, Spanó L, Cardarelli M, Bellincampi D, Petit A, Costantino P (1989) Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA. Plant Mol Biol 13:43–52PubMedCrossRefGoogle Scholar
  4. Cardarelli M, Mariotti D, Pomponi M, Spanó L, Capone I, Costantino P (1987) Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype. Mol Gen Genet 209:475–480PubMedCrossRefGoogle Scholar
  5. Cecchetti V, Pomponi M, Altamura MM, Pezzotti M, Marsilio S, D’Angeli S, Tornielli GB, Costantino P, Cardarelli M (2004) Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation. Plant J 38:512–525PubMedCrossRefGoogle Scholar
  6. Clemens S, Kim EJ, Neumann D, Schroeder JL (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J 18:3325–3333PubMedCrossRefGoogle Scholar
  7. Clemens S, Schroeder JI, Degenkolb T (2001) Caenorhabditis elegans expresses a functional phytochelatin synthase. Eur J Biochem 268:3640–3643PubMedCrossRefGoogle Scholar
  8. Cobbett CS (1999) A family of phytochelatin synthase genes from plant, fungal and animal species. Trends Plant Sci 4:335–337PubMedCrossRefGoogle Scholar
  9. Cobbett CS (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physiol 123:825–833PubMedCrossRefGoogle Scholar
  10. Connolly EL, Fett JP, Guerinot ML (2002) Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. Plant Cell 14:1347–1357PubMedCrossRefGoogle Scholar
  11. Cunningham SD, Berti WR, Huang JWW (1995) Phytoremediation of contaminated soils. Trends Biotechnol 13:393–397CrossRefGoogle Scholar
  12. Curie C, Alonso JM, Le Jean M, Ecker JR, Briat JF (2000) Involvement of NRAMP1 from Arabidopsis thaliana in iron transport. Biochem J 3:749–755CrossRefGoogle Scholar
  13. De Knecht JA, Van Dillen M, Koevoets PLM, Schat H, Verkleji JAC, Ernst WHO (1994) Phytochelatins in cadmium-sensitive and cadmium-tolerant Silene vulgaris: chain length distribution and sulphide incorporation. Plant Physiol 104:255–261PubMedGoogle Scholar
  14. Gisbert C, Ros R, De Haro A, Walker DJ, Bernal MP, Serrano R, Navarro-Aviñó J (2003) A plant genetically modified that accumulates Pb is especially promising for phytoremediation. Biochem Biophys Res Commun 303:440–445PubMedCrossRefGoogle Scholar
  15. Glaeser H, Coblenz A, Kruczek R, Ruttke I, Ebert-Jung A, Wolf K (1991) Glutathione metabolism and heavy metal detoxification in Schizosaccharomyces pombe. Isolation and characterisation of glutathione-deficent cadmium-sensitive mutants. Curr Genet 19:207–213CrossRefGoogle Scholar
  16. Gong J-M, Lee DA, Schroeder JI (2003) Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis. Proc Natl Acad Sci USA 100:10118–10123PubMedCrossRefGoogle Scholar
  17. Grill E, Winnacker E-L, Zenk MH (1985) Phytochelatins: the principal heavy-metal complexing peptides of higher plants. Science 230:674–676PubMedCrossRefGoogle Scholar
  18. Grill E, Loffler S, Winnacker E-L, Zenk MH (1989) Phytochelatins, the heavy metal-binding peptides of plants, are synthesised from glutathione by a specific γ-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc Natl Acad Sci USA 86:6838–6842PubMedCrossRefGoogle Scholar
  19. Guerinot ML (2000) The ZIP family of metal transporters. Biochim Biophys Acta 1465:190–198PubMedCrossRefGoogle Scholar
  20. Guo DS, Xi YY, Wang AY, Zhang J, Yuan XY (1999) Contribution of an auxin to the uptake of nickel and cadmium in maize seedlings. Biomed Environ Sci 12:170–176PubMedGoogle Scholar
  21. Howden R, Goldsbrough PB, Anderson CR, Cobbett CS (1995) Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 107:1059–1066PubMedCrossRefGoogle Scholar
  22. Kubota H, Sato K, Yamada T, Maitani T (2000) Phytochelatin homologs induced in hairy roots of horseradish. Phytochemistry 53:239–245PubMedCrossRefGoogle Scholar
  23. Lee S, Moon JS, Ko T, Petros D, Golsbrough PB, Korban SS (2003) Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress. Plant Physiol 131:656–663PubMedCrossRefGoogle Scholar
  24. Li Y, Dhankher OM, Carreira L, Lee D, Chen A, Schroeder JI, Balish RS, Meagher RB (2004) Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity. Plant Cell Physiol 45:1787–1797PubMedCrossRefGoogle Scholar
  25. Maliga PA, Sz-Breznovits A, Marton L (1973) Streptomycin-resistant plants from callus culture of haploid tobacco. Nat New Biol 244:29–30PubMedGoogle Scholar
  26. Maurel C, Leblanc N, Barbier-Brigoo H, Perrot-Rochemann C, Bouvier-Durand M, Guern J (1994) Alteration of auxin perception in rolB-transformed tobacco protoplasts. Plant Physiol 105:1209–1215PubMedCrossRefGoogle Scholar
  27. Nedelkoska TV, Doran PM (2000) Hyperaccumulation of cadmium by hairy roots of Thlaspi caerulescens. Biotechnol Bioeng 67:607–615PubMedCrossRefGoogle Scholar
  28. Ortiz DF, Kreppel L, Speiser DM, Scheel G, McDonald G, Ow DW (1992) Heavy-metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter. EMBO J 11:3491–3499PubMedGoogle Scholar
  29. Papi M, Sabatini S, Bouchez D, Camilleri C, Costantino P, Vittorioso P (2000) Identification and disruption of an Arabidopsis zinc finger gene controlling seed germination. Genes Dev 1:28–33Google Scholar
  30. Rauser WE (1990) Phytochelatins. Annu Rev Biochem 59:61–86PubMedCrossRefGoogle Scholar
  31. Salt DE, Rauser WE (1995) MgATP-dependent transport of phytochelatins across the tonoplast of oat roots. Plant Physiol 107:1293–1301PubMedGoogle Scholar
  32. Salt DE, Prince RC, Pickering IJ, Raskin I (1995) Mechanisms of cadmium mobility and accumulation in Indian mustard. Plant Physiol 109:1427–1433PubMedGoogle Scholar
  33. Salt DE, Smith RD, Raskin I (1998) Phytoremediation. Annu Rev Plant Physiol Plant Mol Biol 49:643–668PubMedCrossRefGoogle Scholar
  34. Sanitá di Toppi L, Lambardi M, Pecchioni N, Pazzagli L, Durante M, Gabrielli R (1999) Effects of cadmium stress on hairy roots of Daucus carota. J Plant Physiol 154:385–391Google Scholar
  35. Sanitá di Toppi L, Prasad MNV, Ottonello S (2002) Metal chelating peptides and proteins in plants. In: Prasad MNV, Strzaka K (eds) Physiology and biochemistry of heavy metal detoxification and tolerance in plants. Kluwer, Dordrecht, pp 59–93Google Scholar
  36. Sauge-Merle S, Cuiné S, Carrier P, Lecomte-Pradines C, Luu DT, Peltier G (2003) Enhanced toxic metal accumulation in engineered bacterial cells expressing Arabidopsis thaliana phytochelatin synthase. Appl Environ Microbiol 69:490–494PubMedCrossRefGoogle Scholar
  37. Spanó L, Mariotti D, Cardarelli M, Branca C, Costantino P (1988) Morphogenesis and auxin sensitivity of transgenic tobacco with different complements of Ri T-DNA. Plant Physiol 87:479–483PubMedCrossRefGoogle Scholar
  38. Spena A, Schmulling T, Koncz C, Schell J (1987) Independent and synergistic activity of rolA, B, and C loci in stimulating abnormal growth in plants. EMBO J 6:3891–3899PubMedGoogle Scholar
  39. Thomine S, Wang R, Ward JM, Crawford NM, Schoereder JI (2000) Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes. Proc Natl Acad Sci USA 97:4991–4996PubMedCrossRefGoogle Scholar
  40. Vatamaniuk OK, Mari S, Lu Y, Rea PA (2000) Mechanism of heavy metal ion activation of phytochelatin (PC) synthase. Biol Chem 275:31451–31459CrossRefGoogle Scholar
  41. Vernoux T, Wilson RC, Seeley KA, Reichheld J, Muroy S, Brown S, Maughan SC, Cobbett CS, Van Montagu M, Inzè D, May MJ, Sung ZR (2000) The root meristemless1/cadmium sensitive2 gene defines a glutathione-dependent pathway involved in initiation and maintenance of cell division during postembryonic root development. Plant Cell 12:97–109PubMedCrossRefGoogle Scholar
  42. Vögeli-Lange R, Wagner GJ (1996) Relationship between cadmium, glutathione and cadmium-binding peptides (phytochelatins) in leaves of intact tobacco seedlings. Plant Sci 114:11–18CrossRefGoogle Scholar
  43. Williams LE, Pittman JK, Hall JL (2000) Emerging mechanism for heavy metal transport in plants. Biochim Biophys Acta 1465:104–126PubMedCrossRefGoogle Scholar
  44. Yamaguchi H, Nishizawa NK, Nakanishi H, Mori S (2002) IDI7, a new iron-regulated ABC transporter from barley roots, localizes to the tonoplast. J Exp Bot 53:727–735PubMedCrossRefGoogle Scholar
  45. Zhu YL, Pilon-Smits EAH, Tarun AS, Weber SU, Jouanin L, Terry N (1999) Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing γ-glutamylcysteine synthetase. Plant Physiol 121:1169–1177PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Mirella Pomponi
    • 1
  • Vincenzo Censi
    • 2
    • 6
  • Valentina Di Girolamo
    • 2
  • Angelo De Paolis
    • 3
  • Luigi Sanità di Toppi
    • 4
  • Rita Aromolo
    • 5
  • Paolo Costantino
    • 1
  • Maura Cardarelli
    • 2
  1. 1.Dipartimento di Genetica e Biologia MolecolareUniversitá La SapienzaRomeItaly
  2. 2.Istituto Biologia e Patologia Molecolari, CNRUniversitá La SapienzaRomeItaly
  3. 3.Istituto di Scienze delle Produzioni Alimentari CNR (ISPA)LecceItaly
  4. 4.Dipartimento di Biologia Evolutiva e FunzionaleUniversitá di ParmaParmaItaly
  5. 5.Istituto Sperimentale per la Nutrizione delle PianteRomeItaly
  6. 6.Istituto di Ricerche di Biologia Molecolare P. Angeletti, IRBMPomezia (Rome)Italy

Personalised recommendations