Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1997 Apr;113(4):1293–1301. doi: 10.1104/pp.113.4.1293

Purification and immunological identification of metallothioneins 1 and 2 from Arabidopsis thaliana.

A Murphy 1, J Zhou 1, P B Goldsbrough 1, L Taiz 1
PMCID: PMC158252  PMID: 9112777

Abstract

Gene families encoding two types of metallothioneins (MTs) MT1 and MT2, have been identified in Arabidopsis thaliana, and their respective mRNAs have been shown to be regulated by copper in a tissue-specific manner (J. Zhou and P.B. Goldsbrough [1994] Plant Cell 6: 875-884; J. Zhou and P.B. Goldsbrough [1995] Mol Gen Genet 248: 318-328; A.S. Murphy and L. Taiz [1995] Plant Physiol 109: 1-10). However, to date the protein products have not been identified. To purify MT proteins from Arabidopsis, we isolated low-molecular-mass, copper-binding, thiol-rich proteins using selective precipitation followed by size-exclusion, copper-affinity, and thiol-affinity chromatographies. Polyclonal antibodies raised against Arabidopsis MT-glutathione-S-transferase fusion proteins cross-reacted with the 4.5- and 8-kD bands in immunoblots of low-molecular-mass, copper-binding proteins purified from seedling, mature leaf, and mature root tissues. The identity of the proteins was subsequently confirmed by amino acid sequencing. MT1 expression was constitutive in roots and inducible by copper in mature leaves; the reverse pattern was observed for MT2. MT2 expression was also concentrated in the growing tip of the root. The accumulation of the MT1- and MT2-encoded proteins thus parallels the regulation of their respective mRNAs with regard to tissue specificity and induction by copper. In addition, a new type of MT, designated MT3, was derived from the database, detected by reverse transcription-polymerase chain reaction, and tentatively identified at the protein level by amino acid sequencing of a 7-kD cysteine-rich polypeptide.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Buchanan-Wollaston V. Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Identification of a gene encoding a senescence-specific metallothionein-like protein. Plant Physiol. 1994 Jul;105(3):839–846. doi: 10.1104/pp.105.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bühler R. H., Kägi J. H. Human hepatic metallothioneins. FEBS Lett. 1974 Feb 15;39(2):229–234. doi: 10.1016/0014-5793(74)80057-8. [DOI] [PubMed] [Google Scholar]
  3. Choi D., Kim H. M., Yun H. K., Park J. A., Kim W. T., Bok S. H. Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection. Plant Physiol. 1996 Sep;112(1):353–359. doi: 10.1104/pp.112.1.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chubatsu L. S., Meneghini R. Metallothionein protects DNA from oxidative damage. Biochem J. 1993 Apr 1;291(Pt 1):193–198. doi: 10.1042/bj2910193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Evans I. M., Gatehouse L. N., Gatehouse J. A., Robinson N. J., Croy R. R. A gene from pea (Pisum sativum L.) with homology to metallothionein genes. FEBS Lett. 1990 Mar 12;262(1):29–32. doi: 10.1016/0014-5793(90)80145-9. [DOI] [PubMed] [Google Scholar]
  6. Evans K. M., Gatehouse J. A., Lindsay W. P., Shi J., Tommey A. M., Robinson N. J. 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. Plant Mol Biol. 1992 Dec;20(6):1019–1028. doi: 10.1007/BF00028889. [DOI] [PubMed] [Google Scholar]
  7. Ferreira A. M., Ciriolo M. R., Marcocci L., Rotilio G. Copper(I) transfer into metallothionein mediated by glutathione. Biochem J. 1993 Jun 15;292(Pt 3):673–676. doi: 10.1042/bj2920673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  9. Hamer D. H. Metallothionein. Annu Rev Biochem. 1986;55:913–951. doi: 10.1146/annurev.bi.55.070186.004405. [DOI] [PubMed] [Google Scholar]
  10. Hellman U., Wernstedt C., Góez J., Heldin C. H. Improvement of an "In-Gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal Biochem. 1995 Jan 1;224(1):451–455. doi: 10.1006/abio.1995.1070. [DOI] [PubMed] [Google Scholar]
  11. Howden R., Goldsbrough P. B., Andersen C. R., Cobbett C. S. Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol. 1995 Apr;107(4):1059–1066. doi: 10.1104/pp.107.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lane B. G. Cellular desiccation and hydration: developmentally regulated proteins, and the maturation and germination of seed embryos. FASEB J. 1991 Nov;5(14):2893–2901. doi: 10.1096/fasebj.5.14.1752357. [DOI] [PubMed] [Google Scholar]
  13. Loraine A. E., Yalovsky S., Fabry S., Gruissem W. Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells. Plant Physiol. 1996 Apr;110(4):1337–1347. doi: 10.1104/pp.110.4.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McCully M. How Do Real Roots Work? (Some New Views of Root Structure). Plant Physiol. 1995 Sep;109(1):1–6. doi: 10.1104/pp.109.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Murphy A., Taiz L. A New Vertical Mesh Transfer Technique for Metal-Tolerance Studies in Arabidopsis (Ecotypic Variation and Copper-Sensitive Mutants). Plant Physiol. 1995 May;108(1):29–38. doi: 10.1104/pp.108.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Omer C. A., Gibbs J. B. Protein prenylation in eukaryotic microorganisms: genetics, biology and biochemistry. Mol Microbiol. 1994 Jan;11(2):219–225. doi: 10.1111/j.1365-2958.1994.tb00302.x. [DOI] [PubMed] [Google Scholar]
  17. Rauser W. E. Phytochelatins and related peptides. Structure, biosynthesis, and function. Plant Physiol. 1995 Dec;109(4):1141–1149. doi: 10.1104/pp.109.4.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rauser W. E. Phytochelatins. Annu Rev Biochem. 1990;59:61–86. doi: 10.1146/annurev.bi.59.070190.000425. [DOI] [PubMed] [Google Scholar]
  19. Reese R. N., Mehra R. K., Tarbet E. B., Winge D. R. Studies on the gamma-glutamyl Cu-binding peptide from Schizosaccharomyces pombe. J Biol Chem. 1988 Mar 25;263(9):4186–4192. [PubMed] [Google Scholar]
  20. Robinson N. J., Tommey A. M., Kuske C., Jackson P. J. Plant metallothioneins. Biochem J. 1993 Oct 1;295(Pt 1):1–10. doi: 10.1042/bj2950001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  22. Tohoyama H., Inouhe M., Joho M., Murayama T. Production of metallothionein in copper- and cadmium-resistant strains of Saccharomyces cerevisiae. J Ind Microbiol. 1995 Feb;14(2):126–131. doi: 10.1007/BF01569894. [DOI] [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Zhou J., Goldsbrough P. B. Structure, organization and expression of the metallothionein gene family in Arabidopsis. Mol Gen Genet. 1995 Aug 21;248(3):318–328. doi: 10.1007/BF02191599. [DOI] [PubMed] [Google Scholar]
  25. de Miranda J. R., Thomas M. A., Thurman D. A., Tomsett A. B. Metallothionein genes from the flowering plant Mimulus guttatus. FEBS Lett. 1990 Jan 29;260(2):277–280. doi: 10.1016/0014-5793(90)80122-y. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES