1. Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam, Germany
2. Max Planck Institute for Developmental Biology, D-72076 Tübingen, Germany
3. Plant Cell Biology, Department of Life Sciences,
4. Center for Assistance in Technology of Microscopy, Department of Chemistry, University of Liège, B-4000 Liège, Belgium
5. Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany
6. 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.).
7. These authors contributed equally to this work.

Correspondence to: Ute Krämer^1,6 Correspondence and requests for materials should be addressed to U.K. (Email: ute.kraemer@bioquant.uni-heidelberg.de).

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 adaptation^{1, 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. thaliana^{4, 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 hypertolerance^{7, 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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