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.     

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.     

Zinc hyperaccumulation and cellular distribution in Arabidopsis halleri

Although Arabidopsis halleri ( = Cardaminopsis halleri) is known as a Zn hyperaccumulator, there have been no detailed studies on Zn accumulation, tolerance and cellular distribution in this species. In a hydroponic experiment, A. halleri grew healthily with Zn concentrations varying from 1 to 1000 μ M , without showing phytotoxicity or reduction in root or shoot dry weights. The concentration of Zn in the shoots increased from 300 μ g g ^{? 1} dry weight in the 1 μ M Zn treatment to 32 000 μ g g ^{? 1} in the 1000 μ M Zn treatment. Approximately 60% of the total Zn in the shoots were water‐soluble, and there was no evidence of Zn and P co‐precipitation. Both citric and malic acid concentrations in the shoots were not significantly affected by the Zn treatments, whereas in the roots there was a positive response in both organic acids to increasing Zn in solution. Cellular distribution of Zn, Ca and K in frozen hydrated leaf tissues was examined using energy‐dispersive X‐ray microanalysis. Zinc was sequestered in the base of trichomes, whereas the middle and upper parts of trichomes were highly enriched with Ca. Mesophyll cells appeared to have more Zn than the epidermis, probably because the latter were very small in size. Similarities and differences between A. halleri and the other well‐known Zn hyperaccumulator, Thlaspi caerulescens, are discussed.     

A quantitative trait loci analysis of zinc hyperaccumulation in Arabidopsis halleri

The mechanisms of metal hyperaccumulation are still not understood, so we conducted a quantitative trait locus (QTL) analysis of zinc (Zn) hyperaccumulation in Arabidopsis halleri, in a cross between this and its sister species, A. petraea, in order to determine the number and approximate location of the genomic regions significantly contributing to this adaptation. An F2 cross between the two species was made, and the leaf Zn concentration of 92 individuals was measured at both low (10 microm) and high (100 microm) Zn concentrations. Twenty-five markers were established that were distributed on all of the eight chromosomes. Mapping of the markers established that they were essentially collinear with previous studies. QTLs exceeding a logarithm to the base 10 of the odds (LOD) value of 3 were found on chromosomes 4 (low Zn), 6 (high Zn) and 7 (both high and low Zn). Evidence for a QTL on chromosome 3 (low Zn) was also found. This analysis validates a previously used method of QTL analysis, based on microarray analysis of segregating families. Genes that have altered during the evolution of this character should also be QTL: this analysis calls into question a number of candidate genes from consideration as such primary genes because they do not appear to be associated with QTLs.     

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.     

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.     

Proteomic analysis of Arabidopsis halleri shoots in response to the heavy metals cadmium and zinc and rhizosphere microorganisms

Arabidopsis halleri has the rare ability to colonize heavy metal‐polluted sites and is an emerging model for research on adaptation and metal hyperaccumulation. The aim of this study was to analyze the effect of plant–microbe interaction on the accumulation of cadmium (Cd) and zinc (Zn) in shoots of an ecotype of A. halleri grown in heavy metal‐contaminated soil and to compare the shoot proteome of plants grown solely in the presence of Cd and Zn or in the presence of these two metals and the autochthonous soil rhizosphere‐derived microorganisms. The results of this analysis emphasized the role of plant–microbe interaction in shoot metal accumulation. Differences in protein expression pattern, identified by a proteomic approach involving 2‐DE and MS, indicated a general upregulation of photosynthesis‐related proteins in plants exposed to metals and to metals plus microorganisms, suggesting that metal accumulation in shoots is an energy‐demanding process. The analysis also showed that proteins involved in plant defense mechanisms were downregulated indicating that heavy metals accumulation in leaves supplies a protection system and highlights a cross‐talk between heavy metal signaling and defense signaling.     

Genetic basis of Cd tolerance and hyperaccumulation in Arabidopsis halleri

The genetic basis of Cd tolerance and hyperaccumulation was investigated in Arabidopsis halleri. The study was conducted in hydroponic culture with a backcross progeny, derived from a cross between A. halleri and a non-tolerant and non-accumulating related species Arabidopsis lyrata ssp. petraea, as well as with the parents of the backcross. The backcross progeny segregates for both cadmium (Cd) tolerance and accumulation. The results support that (i) Cd tolerance may be governed by more than one major gene, (ii) Cd tolerance and Cd accumulation are independent characters, (iii) Cd and Zn tolerances co-segregate suggesting that they are under pleiotropic genetic control, at least to a certain degree, (iv) the same result was obtained for Cd and Zn accumulation.     

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.     

Different strategies of Cd tolerance and accumulation in Arabidopsis halleri and Arabidopsis arenosa

Pseudometallophytes are commonly used to study the evolution of metal tolerance and accumulation traits in plants. Within the Arabidopsis genus, the adaptation of Arabidopsis halleri to metalliferous soils has been widely studied, which is not the case for the closely related species Arabidopsis arenosa. We performed an in-depth physiological comparison between the A. halleri and A. arenosa populations from the same polluted site, together with the geographically close non-metallicolous (NM) populations of both species. The ionomes, growth, photosynthetic parameters and pigment content were characterized in the plants that were growing on their native site and in a hydroponic culture under Cd treatments. In situ, the metallicolous (M) populations of both species hyperaccumulated Cd and Zn. The NM population of A. halleri hyperaccumulated Cd and Zn while the NM A. arenosa did not. In the hydroponic experiments, the NM populations of both species accumulated more Cd in their shoots than the M populations. Our research suggests that the two Arabidopsis species evolved different strategies of adaptation to extreme metallic environments that involve fine regulation of metal homeostasis, adjustment of the photosynthetic apparatus and accumulation of flavonols and anthocyanins.     

The Arabidopsis defensin gene AtPDF2.5 mediates cadmium tolerance and accumulation

Although excess cadmium (Cd) accumulation is harmful to plants, the molecular mechanisms underlying Cd detoxification and accumulation in Arabidopsis thaliana remain largely undetermined. In this study, we demonstrated that the A. thaliana PLANT DEFENSIN 2 gene AtPDF2.5 is involved in Cd tolerance and accumulation. In vitro Cd‐binding assays revealed that AtPDF2.5 has Cd‐chelating activity. Site‐directed mutagenesis of AtPDF2.5 identified eight cysteine residues that were essential for mediating Cd tolerance and chelation. Histochemical analysis demonstrated that AtPDF2.5 was mainly expressed in root xylem vascular bundles, and that AtPDF2.5 was significantly induced by Cd. Subcellular localization analysis revealed that AtPDF2.5 was localized to the cell wall. The overexpression of AtPDF2.5 significantly enhanced Cd tolerance and accumulation in A. thaliana and its heterologous overexpression in rice increased Cd accumulation; however, the functional disruption of AtPDF2.5 decreased Cd tolerance and accumulation. Physiological analysis suggested that AtPDF2.5 promoted Cd efflux from the protoplast and its subsequent accumulation in the cell wall. These data suggest that AtPDF2.5 promotes cytoplasmic Cd efflux via chelation, thereby enhancing Cd detoxification and apoplastic accumulation.     

Protein lysine methylation contributes to modulating the response of sensitive and tolerant Arabidopsis species to cadmium stress

The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analysed the importance of protein lysine methylation for plants to cope with cadmium. We analysed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species, Arabidopsis thaliana and A. lyrata, and in three populations of A. halleri with contrasting cadmium accumulation and tolerance traits. We showed that some proteins are differentially methylated at lysine residues in response to Cd and that a few genes coding KMTs are regulated by cadmium. Also, we showed that 9 out of 23 A. thaliana mutants disrupted in KMT genes have a tolerance to cadmium that is significantly different from that of wild-type seedlings. We further characterized two of these mutants, one was knocked out in the calmodulin lysine methyltransferase gene and displayed increased tolerance to cadmium, and the other was interrupted in a KMT gene of unknown function and showed a decreased capacity to cope with cadmium. Together, our results showed that lysine methylation of non-histone proteins is impacted by cadmium and that several methylation events are important for modulating the response of Arabidopsis plants to cadmium stress.     

The effects of cadmium and the cadmium-zinc interaction on the axenic growth of ectomycorrhizal fungi

Eleven strains of ectomycorrhizal fungi belonging to seven species have been cultured on a cadmium-contaminated growth medium in order to determine their in vitro cadmium tolerance. Four strains were collected from a zinc and cadmium-polluted soil. Radial growth rate was a sensitive parameter to detect Cd toxicity. A wide differential response to Cd was obtained between the individual species. A clear relation between Cd tolerance and site origin of the isolates did not exist, although such a relationship was found when strains are compared within one species. Cd-sensitive and Cd-tolerant strains of Suillus bovinus were studied in more detail. Two isolates were grown on media with combinations of two non-toxic zinc concentrations and three cadmium levels. Adding a higher Zn concentration to the medium resulted in a reduction of the toxic effect of Cd. This antagonistic effect also resulted in a lowered Cd concentration in the mycelium.