Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors

The hyperaccumulation of zinc (Zn) and cadmium (Cd) is a constitutive property of the metallophyte Arabidopsis halleri. We therefore used Arabidopsis GeneChips to identify genes more active in roots of A. halleri as compared to A. thaliana under control conditions. The two genes showing highest expression in A. halleri roots relative to A. thaliana roots out of more than 8000 genes present on the chip encode a nicotianamine (NA) synthase and a putative Zn2+ uptake system. The significantly higher activity of these and other genes involved in metal homeostasis under various growth conditions was confirmed by Northern and RT-PCR analyses. A. halleri roots also show higher NA synthase protein levels. Furthermore, we developed a capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (CapLC-ESI-QTOF-MS)-based NA analysis procedure and consistently found higher NA levels in roots of A. halleri. Expression of a NA synthase in Zn2+-hypersensitive Schizosaccharomyces pombe cells demonstrated that formation of NA can confer Zn2+ tolerance. Taken together, these observations implicate NA in plant Zn homeostasis and NA synthase in the hyperaccumulation of Zn by A. halleri. Furthermore, the results show that comparative microarray analysis of closely related species can be a valuable tool for the elucidation of phenotypic differences between such species.     

A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance

The metal tolerance of metal hyper-accumulating plants is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the Zn hyper-accumulating plant Arabidopsis halleri ssp. halleri, we carried out a functional screening of an A. halleri cDNA library in the yeast Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. The screening revealed four A. halleri defensin genes (AhPDFs), which induced Zn but not cadmium (Cd) tolerance in yeast. The expression of AhPDF1.1 under the control of the 35S promoter in A. thaliana made the transgenic plants more tolerant to Zn than wild-type plants, but did not change the tolerance to Cd, copper (Cu), cobalt (Co), iron (Fe) or sodium (Na). Thus, AhPDF1.1 is able to confer Zn tolerance both to yeast and plants. In A. halleri, defensins are constitutively accumulated at a higher level in shoots than in A. thaliana. A. halleri defensin pools are Zn-responsive, both at the mRNA and protein levels. In A. thaliana, some but not all defensin genes are induced by ZnCl2 treatment, and these genes are not induced by NaCl treatment. Defensins, found in a very large number of organisms, are known to be involved in the innate immune system but have never been found to play any role in metal physiology. Our results support the proposition that defensins could be involved in Zn tolerance in A. halleri, and that a role for plant defensins in metal physiology should be considered.     

Forms of zinc accumulated in the hyperaccumulator Arabidopsis halleri

The chemical forms of zinc (Zn) in the Zn-tolerant and hyperaccumulator Arabidopsis halleri and in the non-tolerant and nonaccumulator Arabidopsis lyrata subsp. petraea were determined at the molecular level by combining chemical analyses, extended x-ray absorption spectroscopy (EXAFS), synchrotron-based x-ray microfluorescence, and muEXAFS. Plants were grown in hydroponics with various Zn concentrations, and A. halleri specimens growing naturally in a contaminated site were also collected. Zn speciation in A. halleri was independent of the origin of the plants (contaminated or non-contaminated) and Zn exposure. In aerial parts, Zn was predominantly octahedrally coordinated and complexed to malate. A secondary organic species was identified in the bases of the trichomes, which contained elevated Zn concentrations, and in which Zn was tetrahedrally coordinated and complexed to carboxyl and/or hydroxyl functional groups. This species was detected thanks to the good resolution and sensitivity of synchrotron-based x-ray microfluorescence and muEXAFS. In the roots of A. halleri grown in hydroponics, Zn phosphate was the only species detected, and is believed to result from chemical precipitation on the root surface. In the roots of A. halleri grown on the contaminated soil, Zn was distributed in Zn malate, Zn citrate, and Zn phosphate. Zn phosphate was present in both the roots and aerial part of A. lyrata subsp. petraea. This study illustrates the complementarity of bulk and spatially resolved techniques, allowing the identification of: (a) the predominant chemical forms of the metal, and (b) the minor forms present in particular cells, both types of information being essential for a better understanding of the bioaccumulation processes.     

Elevated nicotianamine levels in Arabidopsis halleri roots play a key role in zinc hyperaccumulation

Zn deficiency is among the leading health risk factors in developing countries. Breeding of Zn-enriched crops is expected to be facilitated by molecular dissection of plant Zn hyperaccumulation (i.e., the ability of certain plants to accumulate Zn to levels >100-fold higher than normal plants). The model hyperaccumulators Arabidopsis halleri and Noccaea caerulescens share elevated nicotianamine synthase (NAS) expression relative to nonaccumulators among a core of alterations in metal homeostasis. Suppression of Ah-NAS2 by RNA interference (RNAi) resulted in strongly reduced root nicotianamine (NA) accumulation and a concomitant decrease in root-to-shoot translocation of Zn. Speciation analysis by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry showed that the dominating Zn ligands in roots were NA and thiols. In NAS2-RNAi plants, a marked increase in Zn-thiol species was observed. Wild-type A. halleri plants cultivated on their native soil showed elemental profiles very similar to those found in field samples. Leaf Zn concentrations in NAS2-RNAi lines, however, did not reach the Zn hyperaccumulation threshold. Leaf Cd accumulation was also significantly reduced. These results demonstrate a role for NAS2 in Zn hyperaccumulation also under near-natural conditions. We propose that NA forms complexes with Zn(II) in root cells and facilitates symplastic passage of Zn(II) toward the xylem.     

A broad-scale analysis of population differentiation for Zn tolerance in an emerging model species for tolerance study: Arabidopsis halleri (Brassicaceae)

Although current knowledge about the overall distribution of zinc (Zn) tolerance in Arabidopsis halleri populations is scarce, the species is an emerging model for the study of heavy metal tolerance in plants. We attempted to improve this knowledge by testing the Zn tolerance of scattered European metallicolous (M) and nonmetallicolous (NM) populations of A. h. subsp. halleri and A. h. subsp. ovirensis in hydroponic culture. The occurrence of constitutive tolerance was unconditionally established in A. h. halleri and tolerance was extended to the subspecies ovirensis. M populations were the most tolerant but there was a continuous range of variation in tolerance from NM to M populations. Finally, relatively high levels of tolerance were detected in some NM populations, suggesting that enhanced tolerance could be present at high frequency in populations that have not experienced metal exposure. We used our results to argue the evolutionary dynamics and origin of Zn tolerance in A. halleri.     

Zinc tolerance and accumulation in metallicolous and nonmetallicolous populations of Arabidopsis halleri (Brassicaceae)

Zinc tolerance was investigated in five populations of Arabidopsis halleri (syn.: Cardaminopsis halleri ) raised from seeds collected from contaminated and uncontaminated sites. Tolerance was measured by determining the concentration which inhibited root growth (EC₁₀₀). A. halleri populations from contaminated and uncontaminated sites were found to be Zn-tolerant compared with the Zn-nontolerant species Arabidopsis thaliana and A. lyrata subsp. petraea . At very high Zn concentrations, populations of A. halleri from uncontaminated sites were slightly less Zn-tolerant than those from contaminated sites. These observations support the hypothesis that in A. halleri , Zn tolerance is largely a constitutive property. One population from an uncontaminated site and one population from a contaminated site were studied for Zn uptake. Zinc content was measured in shoots and roots using a colorimetric test under laboratory conditions. The results showed that whatever their origin, individuals from both populations are Zn accumulators compared with the nonaccumulator species A. thaliana . Moreover, the population from the uncontaminated area accumulated Zn in its shoots and roots more quickly than the population from the contaminated site. These results suggest that, in A. halleri , Zn accumulation to very high concentration is a constitutive property.     

The genetic basis of zinc tolerance in the metallophyte Arabidopsis halleri ssp. halleri (Brassicaceae): an analysis of quantitative trait loci

The species Arabidopsis halleri, an emerging model for the study of heavy metal tolerance and accumulation in plants, has evolved a high level of constitutive zinc tolerance. Mapping of quantitative trait loci (QTL) was used to investigate the genetic architecture of zinc tolerance in this species. A first-generation backcross progeny of A. halleri ssp. halleri from a highly contaminated industrial site and its nontolerant relative A. lyrata ssp. petraea was produced and used for QTL mapping of zinc tolerance. A genetic map covering most of the A. halleri genome was constructed using 85 markers. Among these markers, 65 were anchored in A. thaliana and revealed high synteny with other Arabidopsis genomes. Three QTL of comparable magnitude on three different linkage groups were identified. At all QTL positions zinc tolerance was enhanced by A. halleri alleles, indicating directional selection for higher zinc tolerance in this species. The two-LOD support intervals associated with these QTL cover 24, 4, and 13 cM. The importance of each of these three regions is emphasized by their colocalization with HMA4, MTP1-A, and MTP1-B, respectively, three genes well known to be involved in metal homeostasis and tolerance in plants.     

Traces of ancient range shifts in a mountain plant group (Androsace halleri complex, Primulaceae)

Phylogeographical studies frequently detect range shifts, both expansions (including long-distance dispersal) and contractions (including vicariance), in the studied taxa. These processes are usually inferred from the patterns and distribution of genetic variation, with the potential pitfall that different historical processes may result in similar genetic patterns. Using a combination of DNA sequence data from the plastid genome, AFLP fingerprinting, and rigorous phylogenetic and coalescence-based hypothesis testing, we show that Androsace halleri (currently distributed disjunctly in the northwestern Iberian Cordillera Cantábrica, the eastern Pyrenees, and the French Massif Central and Vosges), or its ancestor, was once more widely distributed in the Pyrenees. While there, it hybridized with Androsace laggeri and Androsace pyrenaica, both of which are currently allopatric with A. halleri. The common ancestor of A. halleri and the north Iberian local endemic Androsace rioxana probably existed in the north Iberian mountain ranges with subsequent range expansion (to the French mountain ranges of the Massif Central and the Vosges) and allopatric speciation (A. rioxana, A. halleri in the eastern Pyrenees, A. halleri elsewhere). We have thus been able to use the reticulate evolution in this species group to help elucidate its phylogeographical history, including evidence of range contraction.     

Nuclear and chloroplast DNA phylogeography reveals vicariance among European populations of the model species for the study of metal tolerance, Arabidopsis halleri (Brassicaceae)

Arabidopsis halleri is a pseudometallophyte involved in numerous molecular studies of the adaptation to anthropogenic metal stress. In order to test the representativeness of genetic accessions commonly used in these studies, we investigated the A. halleri population genetic structure in Europe. Microsatellite and nucleotide polymorphisms from the nuclear and chloroplast genomes, respectively, were used to genotype 65 populations scattered over Europe. The large-scale population structure was characterized by a significant phylogeographic signal between two major genetic units. The localization of the phylogeographic break was assumed to result from vicariance between large populations isolated in southern and central Europe, on either side of ice sheets covering the Alps during the Quaternary ice ages. Genetic isolation was shown to be maintained in western Europe by the high summits of the Alps, whereas admixture was detected in the Carpathians. Considering the phylogeographic literature, our results suggest a distinct phylogeographic pattern for European species occurring in both mountain and lowland habitats. Considering the evolution of metal adaptation in A. halleri, it appears that recent adaptations to anthropogenic metal stress that have occurred within either phylogeographic unit should be regarded as independent events that potentially have involved the evolution of a variety of genetic mechanisms.     

Does Speciation between Arabidopsis halleri and Arabidopsis lyrata Coincide with Major Changes in a Molecular Target of Adaptation?

Ever since Darwin proposed natural selection as the driving force for the origin of species, the role of adaptive processes in speciation has remained controversial. In particular, a largely unsolved issue is whether key divergent ecological adaptations are associated with speciation events or evolve secondarily within sister species after the split. The plant Arabidopsis halleri is one of the few species able to colonize soils highly enriched in zinc and cadmium. Recent advances in the molecular genetics of adaptation show that the physiology of this derived ecological trait involves copy number expansions of the AhHMA4 gene, for which orthologs are found in single copy in the closely related A. lyrata and the outgroup A. thaliana. To gain insight into the speciation process, we ask whether adaptive molecular changes at this candidate gene were contemporary with important stages of the speciation process. We first inferred the scenario and timescale of speciation by comparing patterns of variation across the genomic backgrounds of A. halleri and A. lyrata. Then, we estimated the timing of the first duplication of AhHMA4 in A. halleri. Our analysis suggests that the historical split between the two species closely coincides with major changes in this molecular target of adaptation in the A. halleri lineage. These results clearly indicate that these changes evolved in A. halleri well before industrial activities fostered the spread of Zn- and Cd-polluted areas, and suggest that adaptive processes related to heavy-metal homeostasis played a major role in the speciation process.     

Merging methods in molecular and ecological genetics to study the adaptation of plants to anthropogenic metal-polluted sites: implications for phytoremediation

Metallophyte species that occur naturally on metal-enriched soils represent major biological resources for the improvement of phytoremediation, a benign and cost-effective technology that uses plants to clean up anthropogenic metal-polluted soils. Within the last decade, molecular genetic studies carried out on several model organisms (including Arabidopsis halleri) have considerably enhanced our understanding of metal tolerance and hyperaccumulation in plants, but the identification of the genes of interest for phytoremediation purposes remains a challenge. To meet this challenge, we propose to combine '-omics' with molecular ecology methods. Using A. halleri, we confronted molecular genetic results with: (i) within-species polymorphism and large-scale population differentiation for zinc tolerance; (ii) the demographical context (e.g. migration pattern) of the species for zinc tolerance evolution; (iii) the Quantitative Trait Loci (QTL) analysis of the genetic architecture for zinc tolerance; and (iv) the fine-scale dissection of identified QTL regions, to discuss more precisely the nature of the genes potentially involved in the adaptation to zinc-polluted soils.     

Cadmium uptake, translocation and tolerance in the hyperaccumulator Arabidopsis halleri

Arabidopsis halleri is a well-known zinc (Zn) hyperaccumulator, but its status as a cadmium (Cd) hyperaccumulator is less certain. Here, we investigated whether A. halleri can hyperaccumulate Cd and whether Cd is transported via the Zn pathway. Growth and Cd and Zn uptake were determined in hydroponic experiments with different Cd and Zn concentrations. Short-term uptake and root-to-shoot transport were measured with radioactive 109Cd and 65Zn labelling. A. halleri accumulated > 1000 mg Cd kg(-1) in shoot dry weight at external Cd concentrations >or= 5 microm, but the short-term uptake rate of 109Cd was much lower than that of 65Zn. Zinc inhibited short-term 109Cd uptake kinetics and root-to-shoot translocation, as well as long-term Cd accumulation in shoots. Uptake of 109Cd and 65Zn were up-regulated, respectively, by low iron (Fe) or Zn status. A. halleri was much less tolerant to Cd than to Zn. We conclude that A. halleri is able to hyperaccumulate Cd partly, at least, through the Zn pathway, but the mechanisms responsible for cellular Zn tolerance cannot detoxify Cd effectively.