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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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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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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