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Plant Molecular Biology

, Volume 62, Issue 6, pp 845–857 | Cite as

Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli

  • Bala RathinasabapathiEmail author
  • Shan Wu
  • Sabarinath Sundaram
  • Jean Rivoal
  • Mrittunjai Srivastava
  • Lena Q. Ma
Article

Abstract

Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI’s role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells expressing E. coli TPI. Excised frond tissue infiltrated with arsenate reduced arsenate significantly more under light than dark. This research highlights a novel role for P. vittata cTPI in arsenate reduction.

Keywords

Arsenic tolerance Escherichia coli Expression cloning Glyceraldehyde-3-phosphate Glycolysis Pteris vittata Triosephosphate isomerase 

Abbreviations

cTPI

Cytosolic triosephosphate isomerase

DTT

Dithiothereitol

EDTA

Ethylene diamine tetraacetic acid

pTPI

Plastidic triosephosphate isomerase

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

3-PGA

3-Phosphoglycerate

TPI

Triosephosphate isomerase

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Notes

Acknowledgements

This research was partly funded by a mini-grant to B.R and L.M. by the School of Natural Resources and Environment, University of Florida. We thank Dr. Barry P. Rosen (Wayne State University) for providing E. coli strains AW3110 and WC3110 and for useful discussions and an anonymous reviewer for constructive comments.

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Bala Rathinasabapathi
    • 1
    Email author
  • Shan Wu
    • 1
  • Sabarinath Sundaram
    • 1
  • Jean Rivoal
    • 2
  • Mrittunjai Srivastava
    • 3
  • Lena Q. Ma
    • 3
  1. 1.Horticultural Sciences DepartmentUniversity of FloridaGainesvilleUSA
  2. 2.Institut de Recherche en Biologie Végétale, Départment de Sciences BiologiquesUniversité de MontréalMontréalCanada
  3. 3.Department of Soil and Water ScienceUniversity of FloridaGainesvilleUSA

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