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. A. halleri naturally accumulates and tolerates leaf concentrations as high as 2.2% zinc and 0.28% cadmium in dry biomass. 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. 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. 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 trait. 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.

During the history of life on Earth, tectonic and climatic change repeatedly generated large territories that were virtually devoid of life and exhibited harsh environmental conditions. The ability of a few specialist pioneer plants to colonize such hostile environments was thus of paramount ecological importance for the continuous maintenance of primary production over time. Yet, we know very little about how extreme traits evolve and function in plants. Recent breakthroughs have given first insights into the molecular basis underlying the complex extreme model trait of metal hyperaccumulation and associated metal hypertolerance. This review gives an introduction into the hyperaccumulator research field and its history; provides an overview of hyperaccumulator germplasm; describes the state of the art of our understanding of the physiological, molecular, and genetic basis underlying metal hyperaccumulation and its evolution; and highlights future research needs and opportunities.