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Plant Biol (Stuttg) 2002; 4(5): 612-618
DOI: 10.1055/s-2002-35432
Original Paper
Georg Thieme Verlag Stuttgart ·New York

A Putative Role for the Vacuolar Calcium/Manganese Proton Antiporter AtCAX2 in Heavy Metal Detoxification

G. Schaaf 1,4 , E. Catoni 2,4 , M. Fitz 2 , R. Schwacke 3 , A. Schneider 3 , N. von Wirén 1 , W. B. Frommer 2
  • 1 Plant Nutrition, University of Hohenheim, Stuttgart, Germany
  • 2 ZMBP, Plant Physiology, University of Tübingen, Tübingen, Germany
  • 3 Institut für Botanik, University of Cologne, Cologne, Germany
  • 4 both authors contributed equally to this manuscript
Further Information

Publication History

Received: January 28, 2002

Accepted: August 22, 2002

Publication Date:
15 November 2002 (online)

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Abstract

Regulation of uptake and compartmentation of metal ions is important for the maintenance of metal ion homeostasis. To identify mechanisms involved in the protection of plants from Mn toxicity, wild-type yeast was transformed with an Arabidopsis cDNA library and transformants were screened on toxic Mn concentrations. Wild-type yeast could not grow in the presence of 30 mM MnSO4, while two transformants carrying variants of the same gene were able to grow. Database searches revealed that the isolated cDNAs correspond to AtCAX2, previously described as a vacuolar calcium-proton antiporter. Since no other genes could be identified, AtCAX2 might represent a major function permitting Mn detoxification in this suppressor screen. Furthermore, yeast transformed with the two AtCAX2 cDNAs showed increased sensitivity towards hydrogen peroxide, pointing to a limited availability of cytoplasmic Mn in the presence of AtCAX2 activity. The open reading frames of the cDNA encoded polypeptides that have a 42 and a 92 amino acids shorter N-terminal region relative to the predicted full-length coding region of AtCAX2. In contrast to both truncated cDNAs, the full-length clone was unable to confer Mn resistance to yeast, indicating that, similar to AtCAX1, AtCAX2 also carries an autoinhibitory N-terminal domain regulating the activity of AtCAX2.

Abbreviations

aa: amino acid

AtCAX1, AtCAX2: Arabidopsis cDNAs (genes) encoding Ca2+ exchangers 1 and 2, respectively

EST: expressed sequence tag

5-FOA: 5-fluoroorotic acid

PGK: phosphoglycerol kinase

TMD: transmembrane spanning domain

Key words

Manganese toxicity - vacuolar transporter - Arabidopsis thaliana - autoinhibitory domain - reactive oxygen species (ROS)

References

  • 01 Ansorge,  W.,, Sproat,  B. S.,, Stegemann,  J.,, and Schwager,  C.. (1986);  A non-radioactive automated method for DNA sequence determination.  J. Biochem. Biophys. Methods. 13 315-323
    Search in Google Scholar
  • 02 Antz,  C.,, Bauer,  T.,, Kalbacher,  H.,, Frank,  R.,, Covarrubias,  M.,, Kalbitzer,  H. R.,, Ruppersberg,  J. P.,, Baukrowitz,  T.,, and Fakler,  B.. (1999);  Control of K+ channel gating by protein phosphorylation: structural switches of the inactivation gate.  Nat. Struct. Biol.. 6 146-150
    Search in Google Scholar
  • 03 Apse,  M. P.,, Aharon,  G. S.,, Snedden,  W. A.,, and Blumwald,  E.. (1999);  Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. .  Science. 285 1256-1258
    Search in Google Scholar
  • 04 Barceloux,  D. G.. (1999);  Manganese.  J. Toxicol. Clin. Toxicol.. 37 (2) 293-307
    Search in Google Scholar
  • 05 Blamey,  F. P. C.,, Joyce,  D. C.,, Edwards,  D. D.,, and Asher,  C. J.. (1986);  Role of trichomes in sunflower tolerance to manganese toxicity.  Plant Soil. 91 171-180
    Search in Google Scholar
  • 06 Burnell,  J. N.. (1988) The biochemistry of manganese in plants. Manganese in Soils and Plants. Graham, R. D., Hannam, R. J., and Uren, N. C., eds. Dordrecht; Kluwer Academic pp. 125-137
    Search in Google Scholar
  • 07 Clarkson,  D. T.. (1988) The uptake and translocation of manganese by plant roots. Manganese in Soils and Plants. Graham, R. D., Hannam, R. J., and Uren, N. C., eds. Dordrecht; Kluwer Academic pp. 101-111
    Search in Google Scholar
  • 08 Davis,  J. G.. (1996);  Soil pH and magnesium effects on manganese toxicity in peanuts.  J. Plant Nutr.. 19 535-550
    Search in Google Scholar
  • 09 Dohmen,  R. J.,, Strasser,  A. W.,, Honer,  C. B.,, and Hollenberg,  C. P.. (1991);  An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera.  Yeast. 7 691-692
    Search in Google Scholar
  • 10 Dürr,  G.,, Strayle,  J.,, Plemper,  R.,, Elbs,  S.,, Klee,  S. K.,, Catty,  P.,, Wolf,  D. H.,, and Rudolph,  H. K.. (1998);  The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation.  Mol. Biol. Cell.. 9 1149-1162
    PubMedSearch in Google Scholar
  • 11 El-Jaoual,  T., and Cox,  D. A.. (1998);  Manganese toxicity in plants.  J. Plant Nutr.. 21 353-386
    PubMedSearch in Google Scholar
  • 12 Finley,  J. W.. (1999);  Manganese absorption and retention by young women is associated with serum ferritin concentration.  Am. J. Clin. Nutr.. 70 37-43
    PubMedSearch in Google Scholar
  • 13 Fridovich,  I.. (1995);  Superoxide radical and superoxide dismutases.  Annu. Rev. Biochem.. 64 97-112
    CrossrefPubMedSearch in Google Scholar
  • 14 Frommer,  W. B.,, Ludewig,  U.,, and Rentsch,  D.. (1999);  Taking transgenic plants with a pinch of salt.  Science. 285 1222-1223
    CrossrefPubMedSearch in Google Scholar
  • 15 González,  A.,, Koren'kov,  V.,, and Wagner,  G. J.. (1999);  A comparison of Zn, Mn, Cd, and Ca transport mechanisms in oat root tonoplast vesicles.  Physiol. Plant.. 106 203-209
    CrossrefPubMedSearch in Google Scholar
  • 16 González,  A., and Lynch,  J.. (1999);  Tolerance of tropical common bean genotypes to manganese toxicity: performance under different growing conditions.  J. Plant Nutr.. 22 511-525
    PubMedSearch in Google Scholar
  • 17 González,  A.,, Steffen,  K. L.,, and Lynch,  J. P.. (1998);  Light and excess manganese. Implications for oxidative stress in common bean.  Plant Physiol.. 118 493-504
    CrossrefPubMedSearch in Google Scholar
  • 18 Handreck,  K. A.. (1997);  Low iron supply aggravates manganese toxicity in zonal geraniums growing in soilless potting media.  J. Plant Nutr.. 20 1593-1605
    PubMedSearch in Google Scholar
  • 19 Heenan,  D. P., and Campbell,  L. C.. (1981);  Influence of potassium and manganese on growth and uptake of magnesium by soybeans (Glycine max [L.] Merr. cv Bragg).  Plant Soil. 61 447-456
    PubMedSearch in Google Scholar
  • 20 Hirschi,  K. D.,, Zhen,  R. G.,, Cunningham,  K. W.,, Rea,  P. A.,, and Fink,  G. R.. (1996);  CAX1, an H+/Ca2+ antiporter from Arabidopsis. .  Proc. Natl. Acad. Sci. USA. 93 8782-8786
    CrossrefPubMedSearch in Google Scholar
  • 21 Hirschi,  K. D.,, Korenkov,  V. D.,, Wilganowski,  N. L.,, and Wagner,  G. J.. (2000);  Expression of Arabidopsis CAX2 in tobacco. Altered metal accumulation and increased manganese tolerance.  Plant Physiol.. 124 125-133
    CrossrefPubMedSearch in Google Scholar
  • 22 Horst,  W. J.. (1988) The physiology of manganese toxicity. Manganese in Soils and Plants. Graham, R. D., Hannam, R. J., and Uren, N. C., eds. Dordrecht; Kluwer Academic pp. 175-188
    Search in Google Scholar
  • 23 Hughes,  N. P., and Williams,  R. J. P.. (1988) An Introduction to manganese biological chemistry. Manganese in Soils and Plants. Graham, R. D., Hannam, R. J., and Uren, N. C., eds. Dordrecht; Kluwer Academic pp. 7-19
    Search in Google Scholar
  • 24 Kamprath,  E. J., and Foy,  C. D.. (1971) Lime-fertilizer-plant interactions in acid soils. Fertilizer Technology and Use. Olson, R. A., Army, T. J., Hanway, J. J., and Kilmer, V. J., eds. Madison (WI); Soil Science Society of America pp. 105-141
    Search in Google Scholar
  • 25 Korshunova,  Y. O.,, Eide,  D.,, Clark,  W. G.,, Guerinot,  M. L.,, and Pakrasi,  H. B.. (1999);  The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range.  Plant Mol. Biol.. 40 37-44
    CrossrefPubMedSearch in Google Scholar
  • 26 Lapinskas,  P. J.,, Lin,  S. J.,, and Culotta,  V. C.. (1996);  The role of the Saccharomyces cerevisiae CCC1 gene in the homeostasis of manganese ions.  Mol. Microbiol.. 21 519-528
    PubMedSearch in Google Scholar
  • 27 Liu,  X. F., and Culotta,  V. C.. (1999);  Post-translation control of Nramp metal transport in yeast. Role of metal ions and the BSD2 gene.  J. Biol. Chem.. 274 4863-4868
    CrossrefPubMedSearch in Google Scholar
  • 28 Malecki,  E. A.,, Huttner,  D. L.,, and Greger,  J. L.. (1994);  Manganese status, gut endogenous losses of manganese, and antioxidant enzyme activity in rats fed varying levels of manganese and fat.  Biol. Trace Elem. Res.. 42 17-29
    PubMedSearch in Google Scholar
  • 29 Malecki,  E. A., and Greger,  J. L.. (1996);  Manganese protects against heart mitochondrial lipid peroxidation in rats fed high levels of polyunsaturated fatty acids.  J. Nutr.. 126 27-33
    PubMedSearch in Google Scholar
  • 30 Maniatis,  T.,, Fritsch,  E. F.,, and Sambrook,  J.. (1984) Molecular Cloning: A Laboratory Manual. Plainview, NY; Cold Spring Harbor Lab. Press
    Search in Google Scholar
  • 31 Marschner,  H.. (1995) Mineral Nutrition of Higher Plants. London; Academic Press pp. 326-328
    Search in Google Scholar
  • 32 Minet,  M.,, Dufour,  M. E.,, and Lacroute,  F.. (1992);  Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs.  Plant J.. 2 417-422
    PubMedSearch in Google Scholar
  • 33 Morgan,  P. W.,, Taylor,  D. M.,, and Joham,  H. E.. (1976);  Manipulation of IAA-oxidase activity and auxine-deficiency symptoms in intact cotton plants with manganese nutrition.  Plant Physiol.. 37 149-156
    PubMedSearch in Google Scholar
  • 34 Paidhungat,  M., and Garret,  S.. (1998);  Cdc1 is required for growth and Mn2+ regulation in Saccharomyces cerevisiae. .  Genetics. 148 1777-1786
    PubMedSearch in Google Scholar
  • 35 Pfeffer,  P. E.,, Tu,  S. I.,, Gerasimovicz,  W. V.,, and Cavanaughk,  J. R.. (1986);  In vivo 31PNMR studies of corn root tissue and its uptake of toxic metals.  Plant Physiol.. 80 77-84
    PubMedSearch in Google Scholar
  • 36 Pittman,  J. K., and Hirschi,  K.. (2001);  Regulation of CAX1, an Arabidopsis Ca2+/H+ antiporter. Identification of an N-terminal autoinhibitory domain.  Plant Physiol.. 127 1020-1029
    CrossrefPubMedSearch in Google Scholar
  • 37 Pittman,  J. K.,, Shigaki,  T.,, Cheng,  N. H.,, and Hirschi,  K. D.. (2002);  Mechanism of N-terminal autoinhibition in the Arabidopsis Ca2+/H+ antiporter CAX1.  J. Biol. Chem.. 10 (e. pub. ahead of print)
    PubMedSearch in Google Scholar
  • 38 Portnoy,  M. E.,, Liu,  X. F.,, and Culotta,  V. C.. (2000);  Saccharomyces cerevisiae expresses three functionally distinct homologues of the Nramp family of metal transporters.  Mol. Cell. Biol.. 20 7893-7902
    CrossrefPubMedSearch in Google Scholar
  • 39 Reinders,  A.,, Schulze,  W.,, Kühn,  C.,, Barker,  L.,, Schulz,  A.,, Ward,  J. M.,, and Frommer,  W. B.. (2002);  Protein-protein interactions between sucrose transporters of different affinities co-localized in the same enucleate sieve element.  Plant Cell. 14 1567-1577
    CrossrefPubMedSearch in Google Scholar
  • 40 Rogers,  E. E.,, Eide,  D. J.,, and Guerinot,  M. L.. (2000);  Altered selectivity in an Arabidopsis metal transporter.  Proc. Natl. Acad. Sci. USA. 97 12356-12360
    CrossrefPubMedSearch in Google Scholar
  • 41 Schier,  G. A., and McQuattie,  C. J.. (2000);  Effect of manganese on endomycorrhizal sugar maple seedlings.  J. Plant Nutr.. 23 1533-1545
    PubMedSearch in Google Scholar
  • 42 Shigaki,  T.,, Cheng,  N.,, Pittman,  J. K.,, and Hirschi,  K.. (2001);  Structural determinants of Ca2+ transport in the Arabidopsis H+/Ca2+ antiporter CAX1.  J. Biol. Chem.. 276 43152-43159
    CrossrefPubMedSearch in Google Scholar
  • 43 Thomine,  S.,, Wang,  R.,, Ward,  J. M.,, Crawford,  N. M.,, and Schroeder,  J. I.. (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 4991-4996
    CrossrefPubMedSearch in Google Scholar
  • 44 Ueoka-Nakanishi,  H.,, Tsuchiya,  T.,, Sasaki,  M.,, Nakanishi,  Y.,, Cunningham,  K. W.,, and Maeshima,  M.. (2000);  Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells.  Eur. J. Biochem.. 267 3090-3098
    PubMedSearch in Google Scholar
  • 45 Ullmann,  P.,, Gondet,  L.,, Potier,  S.,, and Bach,  T. J.. (1996);  Cloning of Arabidopsis thaliana glutathione synthetase (GSH2) by functional complementation of a yeast gsh2 mutant.  Eur. J. Biochem.. 236 662-669
    CrossrefPubMedSearch in Google Scholar
  • 46 van Loon,  A. P.,, Pesold-Hurt,  B.,, and Schatz,  G.. (1986);  A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen.  Proc. Natl. Acad. Sci. USA. 83 3820-3824
    PubMedSearch in Google Scholar
  • 47 Viarengo,  A.,, Burlando,  B.,, Ceratto,  N.,, and Panfoli,  I.. (2000);  Antioxidant role of metallothioneins: a comparative overview.  Cell. Mol. Biol.. 46 407-417
    PubMedSearch in Google Scholar
  • 48 Zakour,  R. A., and Glickman,  B. W.. (1984);  Metal-induced mutagenesis in the lacI gene of Escherichia coli. .  Mutat. Res.. 126 9-18
    PubMedSearch in Google Scholar

W. B. Frommer

ZMBP
Eberhard-Karls-Universität Tübingen

72076 Tübingen
Germany

Email: wbf@zmbp.uni-tuebingen.de

Section Editor: H. Rennenberg