Letter | Published:

Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4

Nature 453, 391–395 (15 May 2008) | Download Citation

Subjects

Abstract

Little is known about the types of mutations underlying the evolution of species-specific traits. The metal hyperaccumulator Arabidopsis halleri has the rare ability to colonize heavy-metal-polluted soils, and, as an extremophile sister species of Arabidopsis thaliana, it is a powerful model for research on adaptation1,2,3. A. halleri naturally accumulates and tolerates leaf concentrations as high as 2.2% zinc and 0.28% cadmium in dry biomass4. On the basis of transcriptomics studies, metal hyperaccumulation in A. halleri has been associated with more than 30 candidate genes that are expressed at higher levels in A. halleri than in A. thaliana4,5,6. Some of these genes have been genetically mapped to broad chromosomal segments of between 4 and 24 cM co-segregating with Zn and Cd hypertolerance7,8,9. However, the in planta loss-of-function approaches required to demonstrate the contribution of a given candidate gene to metal hyperaccumulation or hypertolerance have not been pursued to date. Using RNA interference to downregulate HMA4 (HEAVY METAL ATPASE 4) expression, we show here that Zn hyperaccumulation and full hypertolerance to Cd and Zn in A. halleri depend on the metal pump HMA4. Contrary to a postulated global trans regulatory factor governing high expression of numerous metal hyperaccumulation genes, we demonstrate that enhanced expression of HMA4 in A. halleri is attributable to a combination of modified cis-regulatory sequences and copy number expansion, in comparison to A. thaliana. Transfer of an A. halleri HMA4 gene to A. thaliana recapitulates Zn partitioning into xylem vessels and the constitutive transcriptional upregulation of Zn deficiency response genes characteristic of Zn hyperaccumulators. Our results demonstrate the importance of cis-regulatory mutations and gene copy number expansion in the evolution of a complex naturally selected extreme trait10. The elucidation of a natural strategy for metal hyperaccumulation enables the rational design of technologies for the clean-up of metal-contaminated soils and for bio-fortification.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

Rent or Buy

All prices are NET prices.

Accessions

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

BAC sequences are available online (Genbank accession numbers EU382072, EU382073).

References

  1. 1.

    Arabidopsis thaliana and its wild relatives. Trends Ecol. Evol. 16, 693–700 (2001)

  2. 2.

    , & Naturally occurring genetic variation in Arabidopsis thaliana. Annu. Rev. Plant Biol. 55, 141–172 (2004)

  3. 3.

    , , & in Phytoremediation of Contaminated Soil and Water (eds Terry, N. & Bañuelos, G. S.) 85–107 (CRC Press LLC, Boca Raton, Florida, 1999)

  4. 4.

    , & Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri. Plant Physiol. 142, 148–167 (2006)

  5. 5.

    , , & Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. Plant J. 37, 251–268 (2004)

  6. 6.

    et al. Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors. Plant J. 37, 269–281 (2004)

  7. 7.

    et al. Two genes encoding Arabidopsis halleri MTP1 metal transport proteins co-segregate with zinc tolerance and account for high MTP1 transcript levels. Plant J. 39, 425–439 (2004)

  8. 8.

    et al. A major QTL for Cd tolerance in Arabidopsis halleri co-localizes with HMA4, a gene encoding a heavy metal ATPase. Plant Physiol. 144, 1052–1065 (2007)

  9. 9.

    et al. The genetic basis of zinc tolerance in the metallophyte Arabidopsis halleri ssp. halleri (Brassicaceae): An analysis of quantitative trait loci. Genetics 176, 659–674 (2007)

  10. 10.

    & The locus of evolution: evo devo and the genetics of adaptation. Evolution Int. J. Org. Evolution 61, 995–1016 (2007)

  11. 11.

    & Inventory of the superfamily of P-type ion pumps in Arabidopsis. Plant Physiol. 126, 696–706 (2001)

  12. 12.

    et al. Total silencing by intron-spliced hairpin RNAs. Nature 407, 319–320 (2000)

  13. 13.

    et al. Free histidine as a metal chelator in plants that hyperaccumulate nickel. Nature 379, 635–638 (1996)

  14. 14.

    et al. Molecular physiology of zinc transport in the Zn hyperaccumulator Thlaspi caerulescens. J. Exp. Bot. 51, 71–79 (2000)

  15. 15.

    et al. P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis. Plant Cell 16, 1327–1339 (2004)

  16. 16.

    et al. The use of the zinc-fluorophore, Zinpyr-1, in the study of zinc homeostasis in Arabidopsis roots. New Phytol. 174, 39–45 (2007)

  17. 17.

    , & A long way ahead: understanding and engineering plant metal accumulation. Trends Plant Sci. 7, 309–315 (2002)

  18. 18.

    et al. Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency. Proc. Natl Acad. Sci. USA 95, 7220–7224 (1998)

  19. 19.

    et al. Genetic basis of Cd tolerance and hyperaccumulation in Arabidopsis halleri. Plant Soil 249, 9–18 (2003)

  20. 20.

    et al. Zinc tolerance and hyperaccumulation are genetically independent characters. Proc. R. Soc. Lond. B 266, 2175–2179 (1999)

  21. 21.

    et al. Partial shotgun sequencing of the Boechera stricta genome reveals extensive microsynteny and promoter conservation with Arabidopsis. Plant Physiol. 140, 1169–1182 (2006)

  22. 22.

    , , & Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta 212, 75–84 (2000)

  23. 23.

    & Natural variation in Arabidopsis. How do we find the causal genes? Plant Physiol. 138, 567–568 (2005)

  24. 24.

    , , & A distant upstream enhancer at the maize domestication gene tb1 has pleiotropic effects on plant and inflorescent architecture. Nature Genet. 38, 594–597 (2006)

  25. 25.

    , & Copy number variants and genetic traits: closer to the resolution of phenotypic to genotypic variability. Nature Rev. Genet. 8, 639–646 (2007)

  26. 26.

    & Selection for more of the same product as a force to enhance concerted evolution of duplicated genes. Trends Genet. 22, 642–644 (2006)

  27. 27.

    , , & Evolutionary genomics of ecological specialization. Proc. Natl Acad. Sci. USA 101, 11719–11724 (2004)

  28. 28.

    , & The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deficiency and Zn oversupply. Plant J. 46, 861–879 (2006)

  29. 29.

    & Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998)

  30. 30.

    , & Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones. J. Exp. Bot. 51, 1961–1968 (2000)

  31. 31.

    & A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469 (2003)

  32. 32.

    et al. Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. 220, 245–250 (1990)

  33. 33.

    et al. Positive selection driving diversification in plant secondary metabolism. Proc. Natl Acad. Sci. USA 103, 9118–9123 (2006)

  34. 34.

    et al. Who ate whom? Adaptive Helicobacter genomic changes that accompanied a host jump from early humans to large felines. PLoS Genet. 2, e120 (2006)

  35. 35.

    , & Basecalling with LifeTrace. Genome Res. 11, 875–888 (2001)

  36. 36.

    , & Consed: a graphical tool for sequence finishing. Genome Res. 8, 195–202 (1998)

  37. 37.

    , & AVID: A global alignment program. Genome Res. 13, 97–102 (2003)

  38. 38.

    et al. Strategies and tools for whole-genome alignments. Genome Res. 13, 73–80 (2003)

Download references

Acknowledgements

We thank D. Baurain, D. Walther, C. Galante, T. Werner and the gardeners of the Max Planck Institute of Molecular Plant Physiology for assistance, R. Schmidt for A. thaliana 35SP-GUS lines, I. Somssich for pJAWOHL8, and S. Thomine for comments on the manuscript. This work was funded by: German Research Foundation Kr1967/3-1, Heisenberg Fellowship Kr1967/4-1; German Federal Ministry of Education and Research Biofuture 0311877 and GABI-ADVANCIS 0315037A; European Union RTN “METALHOME” HPRN–CT–2002–00243, InP ‘‘PHIME’’ FOOD-CT-2006-016253 (U.K.). Further funding was from ‘Fonds spéciaux pour la Recherche, University of Liège, Belgium’ (M.H., P.M.), ‘Fonds de la Recherche Scientifique – FNRS’, Belgium (M.H.), and the Max Planck Society (D.W.).

Author Contributions I.N.T., M.H., M.J.H., A.N., U.K., P.M. and J.K. performed experiments, C.L. the BAC sequencing and assembly, M.H. assembly and BAC annotation; D.W. and J.K. provided the BAC library and filters; U.K., M.H. and I.N.T. jointly designed experiments; D.W. gave experimental advice and edited the manuscript; U.K. conceived of the study and directed the research; U.K., M.H. and I.N.T. wrote and edited the manuscript; all authors commented on the manuscript.

Author information

    • Marc Hanikenne
    • , Ina N. Talke
    • , Michael J. Haydon
    • , Andrea Nolte
    •  & Ute Krämer

    Present addresses: Plant Cell Biology, Department of Life Sciences, University of Liège, B-4000 Liège, Belgium (M.H.); Department of Thoracic, Cardiac and Vascular Surgery, University of Tübingen, D-72076 Tübingen, Germany (A.N.); BIOQUANT Center, Heidelberg Institute of Plant Sciences, University of Heidelberg, D-69120 Heidelberg, Germany (I.N.T., M.J.H., U.K.).

    • Marc Hanikenne
    •  & Ina N. Talke

    These authors contributed equally to this work.

Affiliations

  1. Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam, Germany

    • Marc Hanikenne
    • , Ina N. Talke
    • , Michael J. Haydon
    • , Andrea Nolte
    •  & Ute Krämer

  2. Max Planck Institute for Developmental Biology, D-72076 Tübingen, Germany

    • Christa Lanz
    •  & Detlef Weigel

  3. Plant Cell Biology, Department of Life Sciences,

    • Patrick Motte

  4. Center for Assistance in Technology of Microscopy, Department of Chemistry, University of Liège, B-4000 Liège, Belgium

    • Patrick Motte

  5. Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany

    • Juergen Kroymann

Authors

  1. Search for Marc Hanikenne in:

  2. Search for Ina N. Talke in:

  3. Search for Michael J. Haydon in:

  4. Search for Christa Lanz in:

  5. Search for Andrea Nolte in:

  6. Search for Patrick Motte in:

  7. Search for Juergen Kroymann in:

  8. Search for Detlef Weigel in:

  9. Search for Ute Krämer in:

Corresponding author

Correspondence to Ute Krämer.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    The file contains Supplementary Figures 1 -11 with Legends, Supplementary Table 1, Supplementary Methods and additional references.

To obtain permission to re-use content from this article visit RightsLink.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature06877

Further reading Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.