Skip to main content
SpringerLink
Log in

Molecular changes in Pisum sativum L. roots during arbuscular mycorrhiza buffering of cadmium stress

  • Original Paper
  • Published:
Mycorrhiza Aims and scope Submit manuscript

Abstract

Molecular responses to cadmium (Cd) stress were studied in mycorrhizal and non-mycorrhizal Pisum sativum L. cv. Frisson inoculated with Glomus intraradices. Biomass decreases caused by the heavy metal were significantly less in mycorrhizal than in non-mycorrhizal plants. Real-time reverse transcriptase–polymerase chain reaction showed that genes implicated in pathways of Cd detoxification varied in response to mycorrhiza development or Cd application. Expression of a metallothionein-encoding gene increased strongly in roots of Cd-treated non-mycorrhizal plants. Genes encoding γ-glutamylcysteine synthetase and glutathione (GSH) synthetase, responsible for the synthesis of the phytochelatin (PC) precursor GSH, were activated by Cd in mycorrhizal and non-mycorrhizal plants. Cd stress decreased accumulation of GSH/homoglutathione (hGSH) and increased thiol groups in pea roots, whether mycorrhizal or not, suggesting synthesis of PCs and/or homophytochelatins. An hGSH synthetase gene, involved in hGSH synthesis, did not respond to Cd alone but was activated by mycorrhizal development in the presence of Cd. Transcript levels of a glutathione reductase gene were only increased in non-mycorrhizal roots treated with Cd. Studies of three stress-related genes showed that a heat-shock protein gene was activated in mycorrhizal roots or by Cd and chitinase gene transcripts increased under Cd stress to a greater extent in mycorrhizal roots, whilst a chalcone isomerase gene was only up-regulated by Cd. Results indicate that although heavy metal chelation pathways contribute to Cd stress responses in pea, they may not make a major contribution to Cd tolerance strategies operating in the arbuscular mycorrhizal symbiosis.

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

Similar content being viewed by others

References

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Arisi A-CM, Mocquot B, Lagriffould A, Mench M, Foyer CH, Jouanin L (2000) Responses to cadmium in leaves of transformed poplars overexpressing γ-glutamylcysteine synthetase. Physiol Plant 109:143–149

    Article  CAS  Google Scholar 

  • Brune A, Urbach W, Dietz K-J (1995) Differential toxicity of heavy metals is partly related to a loss of preferential extraplasmic compartmentation: a comparison of Cd-, Mo-, Ni- and Zn-stress. New Phytol 129:403–409

    Article  CAS  Google Scholar 

  • Cairney JWG, Meharg AA (1999) Influences of anthropogenic pollution on mycorrhizal fungal communities. Environ Pollut 106:169–182

    Article  CAS  PubMed  Google Scholar 

  • Cobbett CS, Goldsbrough PB (2000) Mechanisms of metal resistance: phytochelatins and metallothioneins. In: Raskin I, Ensley BD (eds) Phytoremediation of toxic metals: using plants to clean up the environment. Wiley, London, pp 247–269

    Google Scholar 

  • Cruz-Ortega R, Ownby JD (1993) A protein similar to PR (pathogenesis-related) proteins is elicited by metal toxicity in wheat roots. Physiol Plant 89:211–219

    Article  CAS  Google Scholar 

  • del Val C, Barea JM, Azcón-Aguilar C (1999) Diversity of arbuscular mycorrhizal fungus populations in heavy-metal-contaminated soils. Appl Environ Microbiol 65:718–723

    CAS  PubMed  PubMed Central  Google Scholar 

  • Devers M, Soulas G, Martin-Laurent F (2004) Real-time reverse transcription PCR analysis of expression of atrazine catabolism genes in two bacterial strains isolated from soil. J Microbiol Methods 56:3–15

    Article  CAS  PubMed  Google Scholar 

  • Dixit V, Pandey V, Shyam R (2001) Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad). J Exp Bot 52:1101–1109

    Article  CAS  PubMed  Google Scholar 

  • Dubois JP, Benitez N, Liebig T, Baudraz M, Okopnik F (2002) Le cadmium dans les sols du haut Jura suisses. In: Baize D, Tercé M (eds) Les éléments traces métalliques dans les sols. Approches fonctionnelles et spatiales. INRA Editions, Paris, pp 33–52

    Google Scholar 

  • Dumas-Gaudot E, Guillaume P, Tahiri-Alaoui A, Gianinazzi-Pearson V, Gianinazzi S (1994) Changes in polypeptide patterns in tobacco roots colonized by two Glomus species. Mycorrhiza 4:215–221

    Article  CAS  Google Scholar 

  • Evans IM, Gatehouse LN, Gatehouse JA, Robinson NJ, Croy RRD (1990) A gene from pea (Pisum sativum L.) with homology to metallothionein genes. FEBS Lett 262:29–32

    Article  CAS  PubMed  Google Scholar 

  • Fadzilla NM, Finch RP, Burdon RH (1997) Salinity, oxidative stress and antioxidant responses in shoot cultures of rice. J Exp Bot 48:325–331

    Article  CAS  Google Scholar 

  • Finkemeier I, Kluge C, Metwally A, Georgi M, Grotjohann N, Dietz KJ (2003) Alterations in Cd-induced gene expression under nitrogen deficiency in Hordeum vulgare. Plant Cell Environ 26:821–833

    Article  CAS  PubMed  Google Scholar 

  • Fodor E, Szabó-Nagy A, László E (1995) The effects of cadmium on the fluidity and H+-ATPase activity of plasma membrane from sunflower and wheat roots. J Plant Physiol 147:87–92

    Article  CAS  Google Scholar 

  • Franken P, Gnädinger F (1994) Analysis of parsley arbuscular endomycorrhiza: infection development and mRNA levels of defense-related genes. Mol Plant-Microb Interact 7:612–620

    Article  CAS  Google Scholar 

  • Frendo P, Gallesi D, Turnbull R, van de Sype G, Hérouart D, Puppo A (1999) Localisation of glutathione and homoglutathione in Medicago truncatula is correlated to a differential expression of genes involved in their synthesis. Plant J 17:215–219

    Article  CAS  Google Scholar 

  • Gianinazzi-Pearson V (1996) Plant cell responses to arbuscular mycorrhizal fungi: getting to the roots of the symbiosis. Plant Cell 8:1871–1883

    Article  PubMed  PubMed Central  Google Scholar 

  • González-Chávez C, D'Haen J, Vangronsveld J, Dodd JC (2002) Copper sorption and accumulation by the extraradical mycelium of different Glomus spp. (arbuscular mycorrhizal fungi) isolated from the same polluted soil. Plant Soil 240:287–297

    Article  Google Scholar 

  • Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106:207–212

    Article  CAS  PubMed  Google Scholar 

  • Grill E, Winnacker E-L, Zenk MH (1987) Phytochelatins, a class of heavy-metal-binding peptides from plants are functionally analogous to metallothioneins. Proc Natl Acad Sci U S A 84:439–443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grill E, Löffler S, Winnacker E-L, Zenk MH (1989) Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific γ-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc Natl Acad Sci U S A 86:6838–6842

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Güssov D, Clackson T (1989) Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res 17:4000

    Article  Google Scholar 

  • Heggo A, Angle JS, Chaney RL (1990) Effects of vesicular–arbuscular mycorrhizal fungi on heavy metal uptake by soybeans. Soil Biol Biochem 22:865–869

    Article  CAS  Google Scholar 

  • Howarth JR, Doménguez-Solés JR, Gutiérrez-Alcalá G, Wray JL, Romero LC, Gotor C (2003) The serine acetyltransferase gene family in Arabidopsis thaliana and the regulation of its expression by cadmium. Plant Mol Biol 51:589–598

    Article  CAS  PubMed  Google Scholar 

  • Joner EJ, Leyval C (1997) Uptake of 109Cd by roots and hyphae of a Glomus mosseae/Trifolium subterraneum mycorrhiza from soil amended with high and low concentrations of cadmium. New Phytol 135:353–360

    Article  CAS  Google Scholar 

  • Joner EJ, Briones R, Leyval C (2000) Metal-binding capacity of arbuscular mycorrhizal mycelium. Plant Soil 226:227–234

    Article  CAS  Google Scholar 

  • Journet E-P, van Tuinen D, Gouzy J, Crespeau H, Carreau V, Farmer M-J, Niebel A, Schiex T, Jaillon O, Chatagnier O, Godiard L, Micheli F, Kahn D, Gianinazzi-Pearson V, Gamas P (2002) Exploring root symbiotic programs in the model legume Medicago truncatula using EST analysis. Nucleic Acids Res 30:5579–5592

    Article  PubMed  PubMed Central  Google Scholar 

  • Kaldorf M, Kuhn AJ, Schröder WH, Hildebrandt U, Bothe H (1999) Selective element deposits in maize colonized by a heavy metal tolerance conferring arbuscular mycorrhizal fungus. J Plant Physiol 154:718–728

    Article  CAS  Google Scholar 

  • Klapheck S (1988) Homoglutathione: isolation, quantification and ocurrence in legumes. Physiol Plant 74:727–732

    Article  CAS  Google Scholar 

  • Küster H, Hohnjec N, Krajinski F, Yahyaoui FE, Manthey K, Gouzy J, Dondrup M, Meyer F, Kalinowski J, Brechenmacher L, van Tuinen D, Gianinazzi-Pearson V, Pühler A, Gamas P, Becker A (2004) Construction and validation of comprehensive cDNA-based macro- and microarrays to explore root endosymbioses in the model legume Medicago truncatula. J Biotechnol 108:95–113

    Article  PubMed  Google Scholar 

  • Lanfranco L, Bolchi A, Ros EC, Ottonello S, Bonfante P (2002) Differential expression of a metallothionein gene during the presymbiotic versus the symbiotic phase of an arbuscular mycorrhizal fungus. Plant Physiol 130:58–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Laplaze L, Gherbi H, Duhoux E, Pawlowski K, Auguy F, Guermache F, Franche C, Bogusz D (2002) Symbiotic and non-symbiotic expression of cgMT1, a metallothionein-like gene from the actinorhizal tree Casuarina glauca. Plant Mol Biol 49:91–92

    Article  Google Scholar 

  • Lebourg A, Sterckeman T, Ciesielski H, Proix N, Gomez A (1996) Estimation of soil trace metal bioavailability using unbuffered salt solutions: degree of saturation of polluted soil extracts. Environ Technol 19:243–252

    Article  Google Scholar 

  • Leyval C, Turnau K, Haselwandter K (1997) Effect of heavy metal pollution on mycorrhizal colonization and function: physiological, ecological and applied aspects. Mycorrhiza 7:139–153

    Article  CAS  Google Scholar 

  • Leyval C, Joner EJ, del Val C, Haselwandter K (2002) Potential of arbuscular mycorrhizal fungi for bioremediation. In: Gianinazzi S, Schüepp H, Barea JM, Haselwandter K (eds) Mycorrhizal technology in agriculture. From genes to bioproducts. Birkhäuser Verlag, Basel, pp 175–186

    Chapter  Google Scholar 

  • Marshall JS, Keegstra K (1992) Isolation and characterization of a cDNA clone encoding the major hsp70 of the pea chloroplastic stroma. Plant Physiol 100:1048–1054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Matamoros MA, Moran JF, Iturbe-Ormaetxe I, Rubio MC, Becana M (1999) Glutathione and homoglutathione synthesis in legume root nodules. Plant Physiol 121:879–888

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McGrath SP, Zhao FJ, Lombi E (2001) Plant and rhizosphere processes involved in phytoremediation of metal-contaminated soils. Plant Soil 232:207–214

    Article  CAS  Google Scholar 

  • Meharg AA, Cairney JWG (2000) Co-evolution of mycorrhizal symbionts and their hosts to metal-contaminated environments. Adv Ecol Res 30:69–112

    Article  CAS  Google Scholar 

  • Moran JF, Iturbe-Ormaetxe I, Matamoros MA, Rubio MC, Clemente MR, Brewin NJ, Becana M (2000) Glutathione and homoglutathione synthetases of legume nodules. Cloning, expression, and subcellular localization. Plant Physiol 124:1381–1392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Murphy A, Taiz L (1995) Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes. Correlation with copper tolerance. Plant Physiol 109:945–954

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Neumann D, Lichtenberger O, Günther D, Tschiersch K, Nover L (1994) Heat-shock proteins induce heavy-metal tolerance in higher plants. Planta 194:360–367

    Article  CAS  Google Scholar 

  • Noctor G, Foyer CH (1998) Simultaneous measurement of foliar glutathione, γ-glutamylcysteine, and amino acids by high-performance liquid chromatography: comparison with two other assay methods for glutathione. Anal Biochem 264:98–110

    Article  CAS  PubMed  Google Scholar 

  • 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 11:3491–3499

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pilon-Smits EAH, Zhu YL, Sears T, Terry N (2000) Overexpression of glutathione reductase in Brassica juncea: effects on cadmium accumulation and tolerance. Plant Physiol 110:455–460

    Article  CAS  Google Scholar 

  • Porcel R, Barea JM, Ruiz-Lozano JM (2003) Antioxidant activities in mycorrhizal soybean plants under drought stress and their possible relationship to the process of nodule senescence. New Phytol 157:135–143

    Article  CAS  Google Scholar 

  • Pueyo M, López-Sánchez JF, Rauret G (2004) Assessment of CaCl2, NaNO3 and NH4NO3 extraction procedures for the study of Cd, Cu, Pb and Zn extractability in contaminated soils. Anal Chim Acta 504:217–226

    Article  CAS  Google Scholar 

  • Repetto O, Bestel-Corre G, Dumas-Gaudot E, Berta G, Gianinazzi-Pearson V, Gianinazzi S (2003) Targeted proteomics to identify cadmium-induced protein modifications in Glomus mosseae-inoculated pea roots. New Phytol 157:555–567

    Article  CAS  Google Scholar 

  • Rivera-Becerril F (2003) Physiological and molecular responses to cadmium in mycorrhizal and non mycorrhizal pea. PhD Thesis, Université de Bourgogne, Dijon

  • Rivera-Becerril F, Calantzis C, Turnau K, Caussanel J-P, Belimov AA, Gianinazzi S, Strasser RJ, Gianinazzi-Pearson V (2002) Cadmium accumulation and buffering of cadmium-induced stress by arbuscular mycorrhiza in three Pisum sativum L. genotypes. J Exp Bot 53:1177–1185

    Article  CAS  PubMed  Google Scholar 

  • Rüegsegger A, Brunold C (1992) Effect of cadmium on γ-glutamylcysteine synthesis in maize seedlings. Plant Physiol 99:428–433

    Article  PubMed  PubMed Central  Google Scholar 

  • Rüegsegger A, Schmutz D, Brunold C (1990) Regulation of glutathione synthesis by cadmium in Pisum sativum L. Plant Physiol 93:1579–1584

    Article  PubMed  PubMed Central  Google Scholar 

  • Ruiz-Lozano JM, Roussel H, Gianinazzi S, Gianinazzi-Pearson V (1999) Defence genes are differentially induced by a mycorrhizal fungus and Rhizobium in wild type and symbiosis-defective pea genotypes. Mol Plant-Microb Interact 12:976–984

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • SAS Institute Inc (1986) Introductory guide for personal computers. SAS Institute Inc, Cary

    Google Scholar 

  • Schützendübel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. J Exp Bot 53:1351–1365

    PubMed  Google Scholar 

  • Stevens RG, Creissen GP, Mullineaux PM (1997) Cloning and characterisation of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress. Plant Mol Biol 35:641–654

    Article  CAS  PubMed  Google Scholar 

  • Trouvelot A, Kough JL, Gianinazzi-Pearson V (1986) Mesure du taux de mycorhization VA d'un système radiculaire ayant une signification fonctionnelle. In: Gianinazzi-Pearson V, Gianinazzi S (eds) Physiological and genetical aspects of mycorrhizae. INRA Presse, Paris, pp 217–221

    Google Scholar 

  • Turnau K, Ryszka P, Gianinazzi-Pearson V, van Tuinen D (2001) Identification of arbuscular mycorrhizal fungi in soils and roots of plants colonizing zinc wastes in southern Poland. Mycorrhiza 10:169–174

    Article  CAS  Google Scholar 

  • Wagner GJ (1993) Accumulation of cadmium in crop plants and its consequences to human health. Adv Agron 51:173–212

    Article  CAS  Google Scholar 

  • Wollgiehn R, Neumann D (1999) Metal stress response and tolerance of cultured cells from Silene vulgaris and Lycopersicon peruvianum: role of heat stress proteins. J Plant Physiol 154:547–553

    Article  CAS  Google Scholar 

  • Xiang C, Oliver DJ (1998) Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10:1539–1550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu YL, Pilon-Smits EAH, Jouanin L, Terry N (1999a) Overexpression of glutathione synthetase in Indian mustard enhances cadmium accumulation and tolerance. Plant Physiol 119:73–79

    Article  CAS  Google Scholar 

  • Zhu YL, Pilon-Smits 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 121:1169–1177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was performed within the framework of a European Union-RTD INCO-Copernicus project (IC15-CT 98-0116). We thank G. Duc (INRA, Dijon, France) for providing seeds of cv. Frisson, and P. Franken (Max-PIanck Institut, Marburg, Germany) for the ß-tub primers. O. Chatagnier and V. Monfort are acknowledged for technical assistance. F. Rivera-Becerril was supported by a fellowship from the CONACyT-SFERE (Mexico–France) programme.

Author information

Authors and Affiliations

  1. UMR 1088 INRA/5184 CNRS/U., Bourgogne Plante-Microbe-Environnement, INRA-CMSE, BP 86510, 21065, Dijon Cédex, France

    Facundo Rivera-Becerril, Diederik van Tuinen, Silvio Gianinazzi & Vivienne Gianinazzi-Pearson

  2. Depto. El Hombre y su Ambiente, CBS, Universidad Autónoma Metropolitana-Xochimilco, Calz. del Hueso 1100, Col. Villa Quietud, 04960, México DF, Mexico

    Facundo Rivera-Becerril

  3. Microbiologie et Géochimie des Sols, UMR A111 INRA/U. Bourgogne, BP 86510, 21065, Dijon Cédex, France

    Fabrice Martin-Laurent

  4. Department of Physiology and Biochemistry of Plants, University of Bielefeld, Universitätstraße 25, 33501, Bielefeld, Germany

    Ashraf Metwally & Karl-Josef Dietz

  5. Botany Department, Faculty of Science, Assiut University, 71516, Assiut, Egypt

    Ashraf Metwally

Authors
  1. Facundo Rivera-Becerril
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Diederik van Tuinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fabrice Martin-Laurent
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ashraf Metwally
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karl-Josef Dietz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Silvio Gianinazzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Vivienne Gianinazzi-Pearson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vivienne Gianinazzi-Pearson.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rivera-Becerril, F., van Tuinen, D., Martin-Laurent, F. et al. Molecular changes in Pisum sativum L. roots during arbuscular mycorrhiza buffering of cadmium stress. Mycorrhiza 16, 51–60 (2005). https://doi.org/10.1007/s00572-005-0016-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00572-005-0016-7

Keywords

Search

Navigation