Advertisement

Plant Molecular Biology

, Volume 20, Issue 6, pp 1019–1028 | Cite as

Expression of the pea metallothionein-like gene PsMTA in Escherichia coli and Arabidopsis thaliana and analysis of trace metal ion accumulation: Implications for PsMTA function

  • Katherine M. Evans
  • John A. Gatehouse
  • William P. Lindsay
  • Jianguo Shi
  • Andrew M. Tommey
  • Nigel J. Robinson
Research Articles

Abstract

The PsMTA gene from pea (Pisum sativum) shares similarity with metallothionein (MT) genes and related sequences have also been isolated from a number of other higher-plant species. The proteins encoded by these genes have not yet been purified from plants and their functions remain unclear although, by analogy to MT, roles in the metabolism and detoxification of metal ions have been proposed. By contrast, correlation between transcript abundance and Fe availability has led to an alternative proposal that these genes are involved in mechanisms of Fe efficiency.

Phenotypic effects of constitutive PsMTA expression were examined in Escherichia coli and Arabidopsis thaliana. Copper accumulation by E. coli cells expressing recombinant PsMTA protein was approximately 8-fold greater than in control cells. No significant effects on the accumulation of Zn or Cd were detected. In segregating A. thaliana progeny, derived from a transgenic F1 parent containing the PsMTA gene under the control of a CaMV 35S promoter, 75% of individuals accumulated more Cu (several-fold in some plants) than untransformed, control plants. These data suggest that PsMTA protein binds Cu in planta and that uncoupled (constitutive) expression of the PsMTA gene causes enhanced Cu accumulation.

Roots of P. sativum plants grown under conditions of low Fe availability showed elevated activity of root surface Fe(III) reductase and accumulated more Cu than roots of plants grown in an Fe-supplemented solution. Changes in the expression of MT-like genes, coincident with changes in Fe availability, are consistent with a role in Cu homoeostasis.

Key words

copper detoxification metallothionein metal tolerance pea Pisum sativum PsMTA 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Karin M, Richards RI: Human metallothionein genes: molecular cloning and sequence analysis of mRNA. Nucl Acids Res 10: 3165–3173 (1982).PubMedGoogle Scholar
  2. 2.
    Hamer DH: Metallothionein. Annu Rev Biochem 55: 913–955 (1986).PubMedGoogle Scholar
  3. 3.
    Kägi JHR, Schäffer A: Biochemistry of metallothioneins. Biochemistry 27: 8509–8515 (1988).PubMedGoogle Scholar
  4. 4.
    Olafson RW, McCubbin WD, Kay CM: Primary- and secondary-structural analysis of a unique prokaryotic metallothioncin from Synechococcus sp. cyanobacterium: Biochem J 251: 691–699 (1988).PubMedGoogle Scholar
  5. 5.
    Munger K, Lerch K: Copper metallothionein from the fungus Agaricus bisporus: chemical and spectroscopic properties. Biochemistry 24: 6751–6756 (1983).Google Scholar
  6. 6.
    Lerch K, Beltramini M: Neurospora copper metallothionein: molecular structure and biological significance. Chem Scripta 21: 109–115 (1983).Google Scholar
  7. 7.
    Munger K, Germann UA, Lerch K: The Neurospora crassa metallothionein gene. J Biol Chem 262: 7363–7367 (1987).PubMedGoogle Scholar
  8. 8.
    Palmiter RD: Molecular biology of metallothionein gene expression. In: Kägi JHR, Kojima Y (eds) Metallothionein II, pp. 63–80. Birkhauser Verlag, Basel (1987).Google Scholar
  9. 9.
    Karin M, Najarian R, Haslinger A, Valenzuela P, Welch J, Fogel S: Primary structure and transcription of an amplified genetic locus: The CUP1 locus of yeast. Proc Natl Acad Sci USA 81: 337–341 (1984).PubMedGoogle Scholar
  10. 10.
    Robinson NJ, Ratliff RI, Anderson PJ, Delhaize E, Berger JM, Jackson PJ: Biosynthesis of poly(γ-glutamylcysteinyl)glycines in cadmium-tolerant Datura innoxia (Mill.) cells. Plant Sci 56: 197–204 (1988).Google Scholar
  11. 11.
    Robinson NJ, Jackson PJ: Metallothionein-like metal complexes in angiosperms; their structure and function. Physiol Plant 67: 499–506 (1986).Google Scholar
  12. 12.
    Jackson PJ, Unkefer CJ, Doolen JA, Watt K, Robinson NJ: Poly(gamma-glutamylcysteinyl)glycine: its role in cadmium resistance in plant cells. Proc Natl Acad Sci USA 84: 6619–6623 (1987).PubMedGoogle Scholar
  13. 13.
    Grill E, Winnacker EL, Zenk MH: Synthesis of seven different homologous phytochelatins in metal-exposed Schizosaccharomyces pombe cells. FEBS Lett 197: 115–120 (1986).Google Scholar
  14. 14.
    Grill E, Winnacker EL, Zenk MH: Phytochelatins, a class of heavy metal-metal-binding peptides from plants, are functionally analogous to metallothioneins. Proc Natl Acad Sci USA 84: 439–443 (1987).Google Scholar
  15. 15.
    Kägi JHR, Kojima Y: Chemistry and biochemistry of metallothionein. In: Kägi JHR, Kojima Y (eds) Metallothionein II, pp. 25–62. Birkhauser Verlag, Basel (1987).Google Scholar
  16. 16.
    Grill E, Winnacker EL, Zenk MH: Phytochelatins, the principal heavy-metal complexing peptides of higher plants. Science 230: 674–676 (1985).Google Scholar
  17. 17.
    Murasugi A, Wada C, Hayashi Y: Occurrence of acidlabile sulphide in cadmium-binding peptide I from fission yeast. J Biochem 93: 661–664 (1983).PubMedGoogle Scholar
  18. 18.
    Kondo N, Isobe M, Imai K, Goto T: Synthesis of metallothionein-like peptides cadystin A and B occurring in fission yeast, and their isomers. Agric Biol Chem 49: 71–83 (1985).Google Scholar
  19. 19.
    Lane B, Kajioka R, Kennedy T: The wheat germ Ec protein is a zinc-containing metallothionein. Biochem Cell Biol 65: 1001–1005 (1987).Google Scholar
  20. 20.
    Evans IM, Gatehouse LN, Gatehouse JA, Robinson NJ, Croy RRD: A gene from pea (Pisum sativum L.) with homology to metallothionein genes. FEBS Lett 262: 29–32 (1990).CrossRefPubMedGoogle Scholar
  21. 21.
    DeMiranda JR, Thomas MA, Thurman DA, Tomsett AB: Metallothionein genes from the flowering plant Mimulus guttatus. FEBS Lett 260: 277–280 (1990).CrossRefPubMedGoogle Scholar
  22. 22.
    DeFramond AJ: A metallothionein-like gene from maize (Zea mays): Cloning and characterization. FEBS Lett 290: 103–106 (1991).CrossRefPubMedGoogle Scholar
  23. 23.
    Kawashima I, Inokuchi Y, Chino M, Kimura M, Shimizu N: Isolation of a gene for a metallothionein-like protein from soybean. Plant Cell Physiol 32: 913–916 (1991).Google Scholar
  24. 24.
    Okumura N, Nishizawa N-K, Umehara Y, Mori S: An iron defiency-specific cDNA from barley roots having two homologous cysteine-rich MT domains. Plant Mol Biol 17: 531–533 (1991).PubMedGoogle Scholar
  25. 25.
    Tommey AM, Shi J, Lindsay WP, Urwin PE, Robinson NJ: Expression of the pea gene PsMT A in E. coli: Metal-binding properties of the expressed protein. FEBS Lett 292: 48–52 (1991).CrossRefPubMedGoogle Scholar
  26. 26.
    Kille P, Winge DR, Harwood JL, Kay J: A plant metallothionein produced in E. coli. FEBS Lett 295: 171–175 (1991).CrossRefPubMedGoogle Scholar
  27. 27.
    Bragg A, Neilands JB: Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbial Rev 51: 509–518 (1987).Google Scholar
  28. 28.
    Robinson NJ, Gupta A, Fordham-Skelton AP, Croy RRD, Whitton BA, Huckle JW: Prokaryotic metallothionein gene characterization and expression: chromosome crawling by ligation-mediated PCR. Proc R Soc Lond B 242: 241–247 (1990).PubMedGoogle Scholar
  29. 29.
    Ditta G, Stanfield S, Corbin D, Helsinki D: Broad-hostrange DNA cloning system for gram negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347–7351 (1980).PubMedGoogle Scholar
  30. 30.
    Dhaese P, DeGreve H, Decraemer H, Schell J, VanMontagu M: Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. Nucl Acids Res 7: 1837–1849 (1979).PubMedGoogle Scholar
  31. 31.
    Feinberg AP, Vogelstein B: A technique for radiolabeling restriction endonuclease fragments to a high specific activity. Anal Biochem 132: 6–13 (1983).PubMedGoogle Scholar
  32. 32.
    Sheikholeslam SN, Weeks DP: Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol 8: 291–298 (1987).Google Scholar
  33. 33.
    Grusak MA, Welch RM, Kochian LV: Physiological characterization of a single-gene mutation of Pisum sativum exhibiting excess iron accumulation. Plant Physiol 93: 976–981 (1990).Google Scholar
  34. 34.
    Lang A, Thorpe MR: Xylem, phloem and transpiration flows in a grape: Application of a technique for measuring the volume of attached fruits to high resolution using Archimedes' principle. J Exp Bot 40: 1069–1078 (1989).Google Scholar
  35. 35.
    Lin C-M, Kosman DJ: Copper uptake in wildtype and copper metallothionein-deficient Schizosaccharomyces pombe. J Biol Chem 265: 9194–9200 (1990).PubMedGoogle Scholar
  36. 36.
    Silar P, Wegnez M: Expression of the Drosophila melanogaster metallothionein genes in yeast. FEBS Lett 269: 273–276 (1990).CrossRefPubMedGoogle Scholar
  37. 37.
    Kille P, Stephens P, Cryer A, Kay J: The expression of a synthetic rainbow trout metallothionein gene in E. coli. Biochim Biophys Acta 1048: 178–186 (1990).PubMedGoogle Scholar
  38. 38.
    Jacobs FA, Romeyer FM, Beauchemin M, Brousseau R: Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coli. Gene 83: 95–103 (1989).CrossRefPubMedGoogle Scholar
  39. 39.
    Robinson NJ, Evans IM, Mulcrone J, Bryden J, Tommey AM: Genes with similarity to metallothionein genes and Cu, Zn ligands of Pisum sativum L. Plant Soil, In press.Google Scholar

Copyright information

© Kluwer Academic Publishers 1992

Authors and Affiliations

  • Katherine M. Evans
    • 1
  • John A. Gatehouse
    • 1
  • William P. Lindsay
    • 1
  • Jianguo Shi
    • 1
  • Andrew M. Tommey
    • 1
  • Nigel J. Robinson
    • 1
  1. 1.Department of Biological SciencesUniversity of DurhamDurhamUK

Personalised recommendations