Plant Molecular Biology

, Volume 64, Issue 4, pp 361–369 | Cite as

Transgenic Indian mustard (Brassica juncea) plants expressing an Arabidopsis phytochelatin synthase (AtPCS1) exhibit enhanced As and Cd tolerance

Article

Abstract

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an ArabidopsisthalianaAtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.

Keywords

Arsenic Brassica juncea L. Cadmium Heavy metal accumulation Phytochelatin synthase Phytoremediation 

Abbreviations

AtPCS

Arabidopsis phytochelatin synthase

γ-EC

γ-glutamylcysteine

γ-ECS

γ- glutamylcysteine synthetase

GS

glutathione synthase

GSH

glutathione

PCs

phytochelatins

PCS

phytochelatin synthase

References

  1. Bennett LE, Burkhead JL, Hale KL, Terry N, Pilon M, Pilon-Smits EA (2003) Analysis of transgenic Indian mustard plants for phytoremediation of metal-contaminated mine tailings. J Environ Qual 31:431–440Google Scholar
  2. Blaylock MJ, Salt DE, Dushenkov S, Zakharova O, Gussman C, Kapulnik Y, Ensley BD, Raskin I (1997) Enhanced accumulation of Pb in Indian mustard by soil-applied chelating agent. Environ Sci Technol 31:860–865CrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Ann Biochem 72:248–254CrossRefGoogle Scholar
  4. Clemens S, Kim EJ, Neumann D, Schroeder JI (1999) Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. EMBO J 18:3315–3333CrossRefGoogle Scholar
  5. Cobbett CS, Goldsbrough PB (2002) Phytochelatins and metallothionenins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol 53:159–182PubMedCrossRefGoogle Scholar
  6. Cobbett CS, May MJ, Howden R, Rolls B (1998) The gluthatione-deficient, cadmium-sensitive mutant, cad2-, of Arabidopsis thaliana is deficient in γ-glutamylcysteine synthetase. Plant J 16:73–78PubMedCrossRefGoogle Scholar
  7. Creissen G, Firmin J, Fryer M, Kular B, Leyland N, Reynolds H, Pastori G, Wellburn F, Baker N, Wellburn A, Mullineaux P (1999) Elevated gluthatione biosynthetic capacity in the chloroplasts of the tobacco plants paradoxically causes increased oxidative stress. Plant Cell 11:1277–1291PubMedCrossRefGoogle Scholar
  8. De Vos RCH, Vonk MJ, Voojis R, Schat H (1992) Gluthatione depletion due to copper-induced phytochelatin synthesis causes oxidative stress in Silene cucubalus. Plant Physiol 98:853–858PubMedGoogle Scholar
  9. Dhankher OP, Li Y, Rosen BP, Shi J, Salt D, Senecoff JF, Sashti NA, Meagher RB (2002) Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and γ-glutamylcysteine synthetase expression. Nat Biotechnol 20:1140–1145PubMedCrossRefGoogle Scholar
  10. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70–77PubMedCrossRefGoogle Scholar
  11. Fassel VA (1978) Quantitative elemental analyses by plasma emission spectroscopy. Science 202:183–191CrossRefPubMedGoogle Scholar
  12. Gasic K, Korban SS (2005) Nonspecific binding of monoclonal anti-flag M2 antibody in Indian mustard (Brassica juncea). Plant Mol Biol Rep 23:9–16Google Scholar
  13. Gasic K, Korban SS (2006) Heavy metal stress. In: Madhava Rao KV, Raghavendra AS, Janardhan Reddy K (eds) Physiology and molecular biology of stress tolerance in plant. Springer, The Netherlands, pp 219–254CrossRefGoogle Scholar
  14. Gasic K, Korban SS (2007) Expression of Arabidopsis phytochelatin synthase in Indian mustard (Brassica juncea) plants enhances tolerance for Cd and Zn. Planta 225:1277–1285PubMedCrossRefGoogle Scholar
  15. 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
  16. Gupta SC, Goldsbrough PB (1991) Phytochelatin accumulation and cadmium tolerance in selected tomato cell lines. Plant Physiol 97:306–312PubMedGoogle Scholar
  17. Ha SB, Smith AP, Howden R, Dietrich WM, Bugg S, O’Connell MJ, Goldsbrough PB, Cobbett CS (1999) Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe. Plant Cell 11:1153–1163PubMedCrossRefGoogle Scholar
  18. Hartley-Whitaker J, Ainsworth G, Mehrang AA (2001) Copper- and arsenate-induced oxidative stress in Holcus lanatus L. clones with differential sensitivity. Plant Cell Environ 24:713–722CrossRefGoogle Scholar
  19. Heiss S, Wachter A, Bogs J, Cobbett C, Rausch T (2003) Phytochelatin synthase (PCS) protein is induced in Brassica juncea leaves after prolonged Cd exposure. J Exp Bot 54:1833–1839PubMedCrossRefGoogle Scholar
  20. Howden R, Goldsbrough PB, Andersen CR, Cobbett CS (1995) Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 107:1059–1066PubMedCrossRefGoogle Scholar
  21. Lee SM, Moon JS, Domier LL, Korban SS (2002) Molecular characterization of phytochelatin synthase expression in transgenic Arabidopsis. Plant Physiol Biochem 40:727–733CrossRefGoogle Scholar
  22. Lee S, Moon JS, Ko TS, Petros D, Goldsbrough PB, Korban SS (2003) Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress. Plant Physiol 131:656–663PubMedCrossRefGoogle Scholar
  23. Li Y, Dhankher OP, 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 sensitivity. Plant Cell Physiol 45:1787–1797PubMedCrossRefGoogle Scholar
  24. Mukhopadhyay R, Rosen BP (2002) Arsenate reductases in prokaryotes and eukaryotes. Environ Health Perspect 110:745–748PubMedGoogle Scholar
  25. Murashige T, Skoog T (1962) A revised medium for growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479CrossRefGoogle Scholar
  26. Nriagu JO, Pacyna JM (1998) Quantitative assessment of worldwide contamination of air, water and soils with trace metals. Nature 333:134–139CrossRefGoogle Scholar
  27. Pickering IJ, Prince RC, George MJ, Smith RD, George GN, Salt DE (2000) Reduction and coordination of arsenic in Indian mustard. Plant Physiol 122:1171–1177PubMedCrossRefGoogle Scholar
  28. Pilon-Smits E (2005) Phytoremediation. Annu Rev Plant Biol 56:15–39PubMedCrossRefGoogle Scholar
  29. Pilon-Smits EAH, Hwang S, Lytle CM, Zhu Y, Tai JC, Bravo RC, Chen Y, Leustek T, Terry N (1999) Overexpression of ATP sulfurylase in Indian mustard leads to increased selenate uptake, reduction, and tolerance. Plant Physiol 119:123–131PubMedCrossRefGoogle Scholar
  30. Quaghebeur M, Rengel Z (2004) Arsenic uptake, translocation and speciation in pho1 and pho2 mutants of Arabidopsis thaliana. Physiol Plant 120:280–286PubMedCrossRefGoogle Scholar
  31. Raab A, Schat H, Meharg AA, Feldmann J (2005) Uptake, translocation and transformation of arsenate and arsenite in sunflower (Helianthus annuus): formation of arsenic-phytochelatin complexes during exposure to high arsenic concentrations. New Phytol 168:551–558PubMedCrossRefGoogle Scholar
  32. Raskin I, Smith RD, Salt DE (1997) Phytoremediation of metals: using plants to remove pollutants from the environment. Curr Opin Biotechnol 8:221–226PubMedCrossRefGoogle Scholar
  33. Roche Diagnostics (2000) DIG Application manual for filter hybridization. In: Eisel D et al. (ed). Roche Diagnostics GmbH, Mannheim, GermanysGoogle Scholar
  34. Salt DE, Rauser WE (1995) MgATP-dependent transport of phytochelatins across the tonoplast of oat roots. Plant Physiol 107:1293–1301PubMedGoogle Scholar
  35. Salt DE, Prince RC, Pickering IJ, Raskin I (1995) Mechanisms of cadmium mobility and accumulation in Indian mustard. Plant Physiol 109:1427–1433PubMedGoogle 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. Schmöger MEV, Oven M, Grill E (2000) Detoxification of arsenic by phytochelatins in plants. Plant Physiol 122:793–801PubMedCrossRefGoogle Scholar
  38. Van Huysen T, Abdel-Ghany S, Hale KL, LeDuc D, Terry N, Pilon-Smits EA (2003) Overexpression of cystathionine-gamma-synthase enhances selenium volatilization in Brassica juncea. Planta 218:71–78PubMedCrossRefGoogle Scholar
  39. Vatamaniuk OK, Mari S, Lu YP, Rea PA (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proc Natl Acad Sci USA 96:7110–7115PubMedCrossRefGoogle Scholar
  40. Vatamaniuk OK, Mari S, Lu YP, Rea PA (2000) Mechanism of heavy metal ion activation of phytochelatin (PC) synthase. J Biol Chem 275:31451–31459PubMedCrossRefGoogle Scholar
  41. Vatamaniuk OK, Mari S, Lang A, Chalasani S, Demkiv LO, Rea PA (2004) Phytochelatin synthase, a dipeptidyltransferase that undergoes multisite acylation with γ-glutamylcysteine during catalysis. J Biol Chem 279:22449–22460PubMedCrossRefGoogle Scholar
  42. 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
  43. Zarcinas BA, Cartwright B, Spouncer LR (1987) Nitric acid digestion and multi-element analysis of plant material by inductively coupled plasma spectrometry. Commun Soil Sci Plant Anal 18:131–146CrossRefGoogle Scholar
  44. Zhu YL, Pilon-Smiths EAH, Jouanin L, Terry N (1999a) Overexpression of glutathione synthetase in Indian mustard enhances cadmium accumulation and tolerance. Plant Physiol 119:73–79CrossRefGoogle Scholar
  45. Zhu YL, Pilon-Smiths EAH, Tarun AS, Weber SU, Jouanin L, Terry N (1999b) Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing γ(-glutamylcysteine synthetase. Plant Physiol 121:1169–1177CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.Department of Natural Resources and Environmental SciencesUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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