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

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.

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Figure 1: Characterization of A. halleri HMA4 RNAi lines.
Figure 2: Genomic organization and expression of HMA4 genes in A. halleri.
Figure 3: Levels and cell specificity of HMA4 promoter activity in A. halleri and A. thaliana.
Figure 4: Characterization of A. thaliana expressing AhHMA4.

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Primary accessions

GenBank/EMBL/DDBJ

Data deposits

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

References

  1. 1

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

    Article  Google Scholar 

  2. 2

    Koornneef, M., Alonso-Blanco, C. & Vreugdenhil, D. Naturally occurring genetic variation in Arabidopsis thaliana. Annu. Rev. Plant Biol. 55, 141–172 (2004)

    CAS  Article  Google Scholar 

  3. 3

    Baker, A. J. M., McGrath, S. P., Reeves, R. D. & Smith, J. A. C. in Phytoremediation of Contaminated Soil and Water (eds Terry, N. & Bañuelos, G. S.) 85–107 (CRC Press LLC, Boca Raton, Florida, 1999)

    Google Scholar 

  4. 4

    Talke, I. N., Hanikenne, M. & Krämer, U. 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)

    CAS  Article  Google Scholar 

  5. 5

    Becher, M., Talke, I. N., Krall, L. & Krämer, U. 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)

    CAS  Article  Google Scholar 

  6. 6

    Weber, M. 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)

    CAS  Article  Google Scholar 

  7. 7

    Dräger, D. B. 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)

    Article  Google Scholar 

  8. 8

    Courbot, M. 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)

    CAS  Article  Google Scholar 

  9. 9

    Willems, G. 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)

    CAS  Article  Google Scholar 

  10. 10

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

    Article  Google Scholar 

  11. 11

    Axelsen, K. B. & Palmgren, M. G. Inventory of the superfamily of P-type ion pumps in Arabidopsis. Plant Physiol. 126, 696–706 (2001)

    CAS  Article  Google Scholar 

  12. 12

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

    ADS  CAS  Article  Google Scholar 

  13. 13

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

    ADS  Article  Google Scholar 

  14. 14

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

    CAS  Article  Google Scholar 

  15. 15

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

    CAS  Article  Google Scholar 

  16. 16

    Sinclair, S. A. 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)

    CAS  Article  Google Scholar 

  17. 17

    Clemens, S., Palmgren, M. G. & Krämer, U. A long way ahead: understanding and engineering plant metal accumulation. Trends Plant Sci. 7, 309–315 (2002)

    CAS  Article  Google Scholar 

  18. 18

    Grotz, N. 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)

    ADS  CAS  Article  Google Scholar 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

    Küpper, H., Lombi, E., Zhao, F. J. & McGrath, S. P. Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta 212, 75–84 (2000)

    Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

    Clark, R. M., Wagler, T. N., Quijada, P. & Doebley, J. A distant upstream enhancer at the maize domestication gene tb1 has pleiotropic effects on plant and inflorescent architecture. Nature Genet. 38, 594–597 (2006)

    CAS  Article  Google Scholar 

  25. 25

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

    CAS  Article  Google Scholar 

  26. 26

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

    CAS  Article  Google Scholar 

  27. 27

    Zhong, S., Khodursky, A., Dykhuizen, D. E. & Dean, A. M. Evolutionary genomics of ecological specialization. Proc. Natl Acad. Sci. USA 101, 11719–11724 (2004)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Arrivault, S., Senger, T. & Krämer, U. 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)

    CAS  Article  Google Scholar 

  29. 29

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

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  Google Scholar 

  31. 31

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

    CAS  Article  Google Scholar 

  32. 32

    Vancanneyt, G. 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)

    CAS  Article  Google Scholar 

  33. 33

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

    ADS  CAS  Article  Google Scholar 

  34. 34

    Eppinger, M. 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)

    Article  Google Scholar 

  35. 35

    Walther, D., Bartha, G. & Morris, M. Basecalling with LifeTrace. Genome Res. 11, 875–888 (2001)

    CAS  Article  Google Scholar 

  36. 36

    Gordon, D., Abajian, C. & Green, P. Consed: a graphical tool for sequence finishing. Genome Res. 8, 195–202 (1998)

    CAS  Article  Google Scholar 

  37. 37

    Bray, N., Dubchak, I. & Pachter, L. AVID: A global alignment program. Genome Res. 13, 97–102 (2003)

    CAS  Article  Google Scholar 

  38. 38

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

    CAS  Article  Google Scholar 

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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.

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Correspondence to Ute Krämer.

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The file contains Supplementary Figures 1 -11 with Legends, Supplementary Table 1, Supplementary Methods and additional references. (PDF 3465 kb)

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Hanikenne, M., Talke, I., Haydon, M. et al. Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4. Nature 453, 391–395 (2008). https://doi.org/10.1038/nature06877

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