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 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.

The Brassica juncea BjCdR15, an ortholog of Arabidopsis TGA3, is a regulator of cadmium uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants

“…The P-type ion pump HMA4 has been studied extensively (Verret et al, 2004(Verret et al, , 2005Mills et al, 2005;Talke et al, 2006;Courbot et al, 2007;Hanikenne et al, 2008). It has also been proposed that AtNRAMP3 functions as a metal transporter and can modulate heavy metal toxicity in plants ( Thomine et al, 2003).…”

“…The P 1B -type ATPase HMA4 plays a role in cadmium detoxification, being involved in its transport from root to shoot (Mills et al, 2003(Mills et al, , 2005Verret et al, 2004Verret et al, , 2005. A major role of HMA4 in zinc hyperaccumulation and cadmium and zinc hypertolerance has recently been demonstrated in the hyperaccumulator species Arabidopsis halleri (Hanikenne et al, 2008). Phytochelatins have also been studied extensively for their role in heavy metal detoxification (Goldsbrough, 1998;Clemens et al, 1999;Cobbett, 2000), and phytochelatin synthases (PCSs) have been overexpressed in plants, although the results obtained are contradictory.…”

Arabidopsisand relatives as models for the study of genetic and genomic incompatibilities

“…An important component is the increased capacity of A. halleri roots to export metals such as cadmium or zinc to the leaves, which in A. halleri are more tolerant to high-level metal concentrations than in A. lyrata or A. thaliana. The more efficient xylem loading of metals in the root is to a large extent caused by increased expression of a heavy metal ATPase gene, HMA4, because of the combination of cis-regulatory differences and triplication of the gene (Hanikenne et al 2008). HMA4 may also have been involved in the independent evolution of heavy metal tolerance and hyperaccumulation in another Brassicaceae, Thlaspi caerulescens (Papoyan & Kochian 2004).…”

Evolutionary aspects of elemental hyperaccumulation

“…From studies to date on different hyperaccumulator species, there appear to be several different cases by which hyperaccumulators have modified transporter abundance or activity. Some hyperaccumulators have increased the gene copy number of transporters via gene duplication events; examples include the HMA (heavy metal binding) transporter in A. halleri (Hanikenne et al 2008) and N. caerulescens (Ueno et al 2001;Craciun et al 2012). In A. halleri, Hanikenne et al (2008) sequenced bacterial artificial chromosomes and found three tandem copies of metal ATPase 4 (HMA4) relative to A. thaliana.…”

“…Some hyperaccumulators have increased the gene copy number of transporters via gene duplication events; examples include the HMA (heavy metal binding) transporter in A. halleri (Hanikenne et al 2008) and N. caerulescens (Ueno et al 2001;Craciun et al 2012). In A. halleri, Hanikenne et al (2008) sequenced bacterial artificial chromosomes and found three tandem copies of metal ATPase 4 (HMA4) relative to A. thaliana. In N. caerulescens, HMA4 was again shown to have a variable copy number in populations that differ in Cd accumulation and tolerance.…”