Phytochelatins Bind Zn(II) with Micro- to Picomolar Affinities without the Formation of Binuclear Complexes, Exhibiting Zinc Buffering and Muffling Rather than Storing Functions.

Phytochelatins (PCs) are poly-Cys peptides containing a repeating γ-Glu-Cys motif synthesized in plants, algae, certain fungi, and worms by PC synthase from reduced glutathione. It has been shown that an excess of toxic metal ions induces their biosynthesis and that they are responsible for the detoxification process. Little is known about their participation in essential metal binding under nontoxic, basal conditions under which PC synthase is active. This study presents spectroscopic and thermodynamic interactions with the PC2-PC5 series, mainly focusing on the relations between Zn(II) complex stability and cellular Zn(II) availability. The investigations employed mass spectrometry, UV-vis spectroscopy, potentiometry, competition assays with zinc probes, and isothermal titration calorimetry (ITC). All peptides form ZnL complexes, while ZnL2 was found only for PC2, containing two to four sulfur donors in the coordination sphere. Binuclear species typical of Cd(II)-PC complexes are not formed in the case of Zn(II). Results demonstrate that the affinity for Zn(II) increases linearly from PC2 to PC4, ranging from micro- to low-picomolar. Further elongation does not significantly increase the stability. Stability elevation is driven mainly by entropic factors related to the chelate effect and conformational restriction rather than enthalpic factors related to the increasing number of sulfur donors. The affinity of the investigated PCs falls within the range of exchangeable Zn(II) concentrations (hundreds of pM) observed in plants, supporting for the first time a role of PCs both in buffering and in muffling cytosolic Zn(II) concentrations under normal conditions, not exposed to zinc excess, where short PCs have been identified in numerous studies. Furthermore, we found that Cd(II)-PC complexes demonstrate significantly higher metal capacities due to the formation of polynuclear species, which are lacking for Zn(II), supporting the role of PCs in Cd(II) storage (detoxification) and Zn(II) buffering and muffling. Our results on phytochelatins' coordination chemistry and thermodynamics are important for zinc biology and understanding the molecular basis of cadmium toxicity, leaving room for future studies.

In Planta Arsenic Thiolation in Rice and Arabidopsis thaliana.

Inorganic and methylated thioarsenates have recently been reported to form in paddy soil pore waters and accumulate in rice grains. Among them, dimethylmonothioarsenate (DMMTA) is particularly relevant because of its high cytotoxicity and potential misidentification as nonregulated dimethylarsenate (DMA). Studying DMMTA uptake and flag leaf, grain, and husk accumulation in rice plants during grain filling, substantial dethiolation to DMA was observed with only 8.0 ± 0.1, 9.1 ± 0.6, and 1.4 ± 0.2% DMMTA remaining, respectively. More surprisingly, similar shares of DMMTA were observed in control experiments with DMA, indicating in planta DMA thiolation. Exposure of different rice seedling varieties to not only DMA but also to arsenite and monomethylarsenate (MMA) revealed in planta thiolation as a common process in rice. Up to 35 ± 7% DMA thiolation was further observed in the shoots and roots of the model plant Arabidopsis thaliana. Parameters determining the ratio and kinetics of thiolation versus dethiolation are unknown, yet, but less DMA thiolation in glutathione-deficient mutants compared to wild-type plants suggested glutathione concentration as one potential parameter. Our results demonstrate that pore water is not the only source for thioarsenates in rice grains and that especially the currently nonregulated DMA needs to be monitored as a potential precursor of DMMTA formation inside rice plants.

Phytochelatin-mediated metal detoxification pathway is crucial for an organomercurial phenylmercury tolerance in Arabidopsis.

KEY MESSAGE: An organomercurial phenylmercury activates AtPCS1, an enzyme known for detoxification of inorganic metal(loid) ions in Arabidopsis and the induced metal-chelating peptides phytochelatins are essential for detoxification of phenylmercury. Small thiol-rich peptides phytochelatins (PCs) and their synthases (PCSs) are crucial for plants to mitigate the stress derived from various metal(loid) ions in their inorganic form including inorganic mercury [Hg(II)]. However, the possible roles of the PC/PCS system in organic mercury detoxification in plants remain elusive. We found that an organomercury phenylmercury (PheHg) induced PC synthesis in Arabidopsis thaliana plants as Hg(II), whereas methylmercury did not. The analyses of AtPCS1 mutant plants and in vitro assays using the AtPCS1-recombinant protein demonstrated that AtPCS1, the major PCS in A. thaliana, was responsible for the PheHg-responsive PC synthesis. AtPCS1 mutants cad1-3 and cad1-6, and the double mutant of PC-metal(loid) complex transporters AtABCC1 and AtABCC2 showed enhanced sensitivity to PheHg as well as to Hg(II). The hypersensitivity of cad1-3 to PheHg stress was complemented by the own-promoter-driven expression of AtPCS1-GFP. The confocal microscopy of the complementation lines showed that the AtPCS1-GFP was preferentially expressed in epidermal cells of the mature and elongation zones, and the outer-most layer of the lateral root cap cells in the meristematic zone. Moreover, in vitro PC-metal binding assay demonstrated that binding affinity between PC and PheHg was comparable to Hg(II). However, plant ionomic profiles, as well as root morphology under PheHg and Hg(II) stress, were divergent. These results suggest that PheHg phytotoxicity is different from Hg(II), but AtPCS1-mediated PC synthesis, complex formation, and vacuolar sequestration by AtABCC1 and AtABCC2 are similarly functional for both PheHg and Hg(II) detoxification in root surficial cell types.

The cell biology of zinc.

Nearly 10% of all plant proteins belong to the zinc (Zn) proteome. They require Zn either for catalysis or as a structural element. Most of the protein-bound Zn in eukaryotic cells is found in the cytosol. The fundamental differences between transition metal cations in the stability of their complexes with organic ligands, as described by the Irving-Williams series, necessitate buffering of cytosolic Zn (the 'free Zn' pool) in the picomolar range (i.e. ~6 orders of magnitude lower than the total cellular concentration). Various metabolites and peptides, including nicotianamine, glutathione, and phytochelatins, serve as Zn buffers. They are hypothesized to supply Zn to enzymes, transporters, or the recently identified sensor proteins. Zn2+ acquisition is mediated by ZRT/IRT-like proteins. Metal tolerance proteins transport Zn2+ into vacuoles and the endoplasmic reticulum, the major Zn storage sites. Heavy metal ATPase-dependent efflux of Zn2+ is another mechanism to control cytosolic Zn. Spatially controlled Zn2+ influx or release from intracellular stores would result in dynamic modulation of cellular Zn pools, which may directly influence protein-protein interactions or the activities of enzymes involved in signaling cascades. Possible regulatory roles of such changes, as recently elucidated in mammalian cells, are discussed.

Micronutrient homeostasis in plants for more sustainable agriculture and healthier human nutrition.

The provision of sustainable, sufficient, and nutritious food to the growing population is a major challenge for agriculture and the plant research community. In this respect, the mineral micronutrient content of food crops deserves particular attention. Micronutrient deficiencies in cultivated soils and plants are a global problem that adversely affects crop production and plant nutritional value, as well as human health and well-being. In this review, we call for awareness of the importance and relevance of micronutrients in crop production and quality. We stress the need for better micronutrient nutrition in human populations, not only in developing but also in developed nations, and describe strategies to identify and characterize new varieties with high micronutrient content. Furthermore, we explain how adequate nutrition of plants with micronutrients impacts metabolic functions and the capacity of plants to express tolerance mechanisms against abiotic and biotic constraints. Finally, we provide a brief overview and a critical discussion on current knowledge, future challenges, and specific technological needs for research on plant micronutrient homeostasis. Research in this area is expected to foster the sustainable development of nutritious and healthy food crops for human consumption.

Dimethylmonothioarsenate Is Highly Toxic for Plants and Readily Translocated to Shoots.

Arsenic is one of the most relevant environmental pollutants and human health threats. Several arsenic species occur in soil pore waters. Recently, it was discovered that these include inorganic and organic thioarsenates. Among the latter, dimethylmonothioarsenate (DMMTA) is of particular concern because in mammalian cells, its toxicity was found to exceed even that of arsenite. We investigated DMMTA toxicity for plants in experiments with Arabidopsis thaliana and indeed observed stronger growth inhibition than with arsenite. DMMTA caused a specific, localized deformation of root epidermal cells. Toxicity mechanisms apparently differ from those of arsenite since no accumulation of reactive oxygen species was observed in DMMTA-exposed root tips. Also, there was no contribution of the phytochelatin pathway to the DMMTA detoxification as indicated by exposure experiments with respective mutants and thiol profiling. RNA-seq analysis found strong transcriptome changes dominated by stress-responsive genes. DMMTA was taken up more efficiently than the methylated oxyarsenate dimethylarsenate and highly mobile within plants as revealed by speciation analysis. Shoots showed clear indications of DMMTA toxicity such as anthocyanin accumulation and a decrease in chlorophyll and carotenoid levels. The toxicity and efficient translocation of DMMTA within plants raise important food safety issues.

A mutation in the essential and widely conserved DAMAGED DNA BINDING1-Cullin4 ASSOCIATED FACTOR gene OZS3 causes hypersensitivity to zinc excess, cold and UV stress in Arabidopsis thaliana.

The overly zinc sensitive Arabidopsis thaliana mutant ozs3 shows reduced growth of the primary root, which is exacerbated by an excess specifically of Zn ions. In addition, ozs3 plants display various subtle developmental phenotypes, such as longer petioles and early flowering. Also, ozs3 seedlings are completely but reversibly growth-arrested when shifted to 4°C. The causal mutation was mapped to a gene encoding a putative substrate-recognition receptor of cullin4 E3 ligases. OZS3 orthologous genes can be found in almost all eukaryotic genomes. Most species from Schizosaccharomyces pombe to Homo sapiens, and including A. thaliana, possess one ortholog. No functional data are available for these genes in any of the multicellular model systems. CRISPR-Cas9-mediated knockout demonstrated that a complete loss of OZS3 function is embryo-lethal, indicating essentiality of OZS3 and its orthologs. The OZS3 protein interacts with the adaptor protein DAMAGED DNA BINDING1 (DDB1) in the nucleus. Thus, it is indeed a member of the large yet poorly characterized family of DDB1-cullin4 associated factors in plants. Mutant phenotypes of ozs3 plants are apparently caused by the weakened DDB1-OZS3 interaction as a result of the exchange of a conserved amino acid near the conserved WDxR motif.

Between the devil and the deep blue sea: herbivory induces foraging for and uptake of cadmium in a metal hyperaccumulating plant.

Plants have been shown to change their foraging behaviour in response to resource heterogeneity. However, an unexplored hypothesis is that foraging could be induced by environmental stressors, such as herbivory, which might increase the demand for particular resources, such as those required for herbivore defence. This study examined the way simulated herbivory affects both root foraging for and uptake of cadmium (Cd), in the metal-hyperaccumulating plant Arabidopsis halleri, which uses this heavy metal as herbivore defence. Simulated herbivory elicited enhanced relative allocation of roots to Cd-rich patches as well as enhanced Cd uptake, and these responses were exhibited particularly by plants from non-metalliferous origin, which have lower metal tolerance. By contrast, plants from a metalliferous origin, which are more tolerant to Cd, did not show any preference in root allocation, yet enhanced Cd sharing between ramets when exposed to herbivory. These results suggest that foraging for heavy metals, as well as their uptake and clonal-sharing, could be stimulated in A. halleri by herbivory impact. Our study provides first support for the idea that herbivory can induce not only defence responses in plants but also affect their foraging, resource uptake and clonal sharing responses.

Stealing sugar from the honey fungus.

This article comments on: GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae).

Competition for light induces metal accumulation in a metal hyperaccumulating plant.

Plants can respond to competition with a myriad of physiological or morphological changes. Competition has also been shown to affect the foraging decisions of plants belowground. However, a completely unexplored idea is that competition might also affect plants' foraging for specific elements required to inhibit the growth of their competitors. In this study, we examined the effect of simulated competition on root foraging and accumulation of heavy metals in the metal hyperaccumulating perennial plant Arabidopsis halleri, whose metal accumulation has been shown to provide allelopathic ability. A. halleri plants originating from both metalliferous and non-metalliferous soils were grown in a "split-root" setup with one root in a high-metal pot and the other in a low-metal one. The plants were then assigned to either simulated light competition or no-competition (control) treatments, using vertical green or clear plastic filters, respectively. While simulated light competition did not induce greater root allocation into the high-metal pots, it did result in enhanced metal accumulation by A. halleri, particularly in the less metal-tolerant plants, originating from non-metalliferous soils. Interestingly, this accumulation response was particularly enhanced for zinc rather than cadmium. These results provide support to the idea that the accumulation of metals by hyperaccumulating plants can be facultative and change according to their demand following competition.

Arabidopsis halleri shows hyperbioindicator behaviour for Pb and leaf Pb accumulation spatially separated from Zn.

Lead (Pb) ranks among the most problematic environmental pollutants. Background contamination of soils is nearly ubiquitous, yet plant Pb accumulation is barely understood. In a survey covering 165 European populations of the metallophyte Arabidopsis halleri, several field samples had indicated Pb hyperaccumulation, offering a chance to dissect plant Pb accumulation. Accumulation of Pb was analysed in A. halleri individuals from contrasting habitats under controlled conditions to rule out aerial deposition as a source of apparent Pb accumulation. Several elemental imaging techniques were employed to study the spatial distribution and ligand environment of Pb. Regardless of genetic background, A. halleri individuals showed higher shoot Pb accumulation than A. thaliana. However, dose-response curves revealed indicator rather than hyperaccumulator behaviour. Xylem sap data and elemental imaging unequivocally demonstrated the in planta mobility of Pb. Highest Pb concentrations were found in epidermal and vascular tissues. Distribution of Pb was distinct from that of the hyperaccumulated metal zinc. Most Pb was bound by oxygen ligands in bidentate coordination. A. halleri accumulates Pb whenever soil conditions render Pb phytoavailable. Considerable Pb accumulation under such circumstances, even in leaves of A. thaliana, strongly suggests that Pb can enter food webs and may pose a food safety risk.

The two copies of the zinc and cadmium ZIP6 transporter of Arabidopsis halleri have distinct effects on cadmium tolerance.

Plants have the ability to colonize highly diverse environments. The zinc and cadmium hyperaccumulator Arabidopsis halleri has adapted to establish populations on soils covering an extreme range of metal availabilities. The A. halleri ZIP6 gene presents several hallmarks of hyperaccumulation candidate genes: it is constitutively highly expressed in roots and shoots and is associated with a zinc accumulation quantitative trait locus. Here, we show that AhZIP6 is duplicated in the A. halleri genome. The two copies are expressed mainly in the vasculature in both A. halleri and Arabidopsis thaliana, indicative of conserved cis regulation, and acquired partial organ specialization. Yeast complementation assays determined that AhZIP6 is a zinc and cadmium transporter. AhZIP6 silencing in A. halleri or expression in A. thaliana alters cadmium tolerance, but has no impact on zinc and cadmium accumulation. AhZIP6-silenced plants display reduced cadmium uptake upon short-term exposure, adding AhZIP6 to the limited number of Cd transporters supported by in planta evidence. Altogether, our data suggest that AhZIP6 is key to fine-tune metal homeostasis in specific cell types. This study additionally highlights the distinct fates of duplicated genes in A. halleri.

The search for candidate genes associated with natural variation of grain Zn accumulation in barley.

Combating hidden hunger through molecular breeding of nutritionally enriched crops requires a better understanding of micronutrient accumulation. We studied natural variation in grain micronutrient accumulation in barley (Hordeum vulgare L.) and searched for candidate genes by assessing marker-trait associations (MTAs) and by analyzing transcriptional differences between low and high zinc (Zn) accumulating cultivars during grain filling. A collection of 180 barley lines was grown in three different environments. Our results show a pronounced variation in Zn accumulation, which was under strong genotype influence across different environments. Genome-wide association mapping revealed 13 shared MTAs. Across three environments, the most significantly associated marker was on chromosome 2H at 82.8 cM and in close vicinity to two yellow stripe like (YSL) genes. A subset of two pairs of lines with contrasting Zn accumulation was chosen for detailed analysis. Whole ears and flag leaves were analyzed 15 days after pollination to detect transcriptional differences associated with elevated Zn concentrations in the grain. A putative α-amylase/trypsin inhibitor CMb precursor was decidedly higher expressed in high Zn cultivars in whole ears in all comparisons. Additionally, a gene similar to barley metal tolerance protein 5 (MTP5) was found to be a potential candidate gene.

Metal ligands in micronutrient acquisition and homeostasis.

Acquisition and homeostasis of micronutrients such as iron (Fe) and zinc (Zn) pose specific challenges. Poor solubility and high reactivity require controlled synthesis and supply of ligands to complex these metals extracellularly and intracellularly. Cytosolic labile pools represent only a minute fraction of the total cellular content. Several low-molecular-weight ligands are known in plants, including sulfur ligands (cysteine and peptides), nitrogen/oxygen ligands (S-adenosyl-l-methionine-derived molecules and histidine), and oxygen ligands (phenolics and organic acids). Some ligands are secreted into the extracellular space and influence the phytoavailability of metal ions. A second principal function is the intracellular buffering of micronutrients as well as the facilitation of long-distance transport in xylem and phloem. Furthermore, low-molecular-weight ligands are involved in the storage of metals, predominantly in vacuoles. A detailed molecular understanding is hampered by technical limitations, in particular the difficulty to detect and quantify cellular metal-ligand complexes. More, but still too little, is known about ligand synthesis and the transport across membranes, either with or without a complexed metal. Metal ligands have an immediate impact on human well-being. Engineering metal ligand synthesis and distribution in crops has tremendous potential to improve the nutritional quality of food and to tackle major human health risks.

Elevated root nicotianamine concentrations are critical for Zn hyperaccumulation across diverse edaphic environments.

The metallophyte Arabidopsis halleri thrives across an extremely broad edaphic range. Zn hyperaccumulation is found on soils differing in available Zn by up to six orders of magnitude, raising the question as to whether a common set of mechanisms confers this species-wide ability. Elevated root concentrations of the metal chelator nicotianamine due to strong constitutive expression of AhNAS2 are important for hyperaccumulation. In order to analyse the relevance of AhNAS2 under more natural conditions representing a range of metalliferous and nonmetalliferous habitats, we collected soil at eight different A. halleri sites and cultivated wild-type and AhNAS2-RNAi lines in these soils. AhNAS2 transcript abundance and root nicotianamine concentrations in wild-type plants were barely influenced by soil metal concentrations. The RNAi effect was fully expressed in different soils. Zn hyperaccumulation in AhNAS2-silenced lines was significantly reduced in seven soils. Root-to-shoot translocation of Cd, Mn, Cu, Ni, and Co was also affected by AhNAS2 silencing, albeit to a lower extent and less consistently. Leaf Fe levels were unaffected by AhNAS2 knockdown. Results demonstrate that elevated nicotianamine production in roots of A. halleri is a Zn hyperaccumulation factor regardless of the edaphic environment, that is, contributes to Zn hyperaccumulation in soils with contrasting Zn availability.

Interference between arsenic-induced toxicity and hypoxia.

Plants often face combinatorial stresses in their natural environment. Here, arsenic (As) toxicity was combined with hypoxia (Hpx) in the roots of Arabidopsis thaliana as it often occurs in nature. Arsenic inhibited growth of both roots and leaves, whereas root growth almost entirely ceased in Hpx. Growth efficiently resumed, and Hpx marker transcripts decreased upon reaeration. Compromised recovery from HpxAs treatment following reaeration indicated some persistent effects of combined stresses despite lower As accumulation. Root glutathione redox potential turned more oxidized in Hpx and most strongly in HpxAs. The more oxidizing root cell redox potential and the lowered glutathione amounts may be conducive to the growth arrest of plants exposed to HpxAs. The stresses elicited changes in elemental and transcriptomic composition. Thus, calcium, magnesium, and phosphorous amounts decreased in rosettes, but the strongest decline was seen for potassium. The reorganized potassium-related transcriptome supports the conclusion that disturbed potassium homeostasis contributes to the growth phenotype. In a converse manner, photosynthesis-related parameters were hardly affected, whereas accumulated carbohydrates under all stresses and anthocyanins under Hpx exclude carbohydrate limitation. The study demonstrates the existence of both synergistic since mutually aggravating effects and antagonistic effects of single and combined stresses.

Safer food through plant science: reducing toxic element accumulation in crops.

Natural processes and human activities have caused widespread background contamination with non-essential toxic elements. The uptake and accumulation of cadmium (Cd), arsenic (As), and lead (Pb) by crop plants results in chronic dietary exposure and is associated with various health risks. Current human intake levels are close to what is provisionally regarded as safe. This has recently triggered legislative actions to introduce or lower limits for toxic elements in food. Arguably, the most effective way to reduce the risk of slow poisoning is the breeding of crops with much lower accumulation of contaminants. The past years have seen tremendous progress in elucidating molecular mechanisms of toxic element transport. This was achieved in the model systems Arabidopsis thaliana and, most importantly, rice, the major source of exposure to As and Cd for a large fraction of the global population. Many components of entry and sequestration pathways have been identified. This knowledge can now be applied to engineer crops with reduced toxic element accumulation especially in edible organs. Most obvious in the case of Cd, it appears likely that subtle genetic intervention has the potential to reduce human exposure to non-essential toxic elements almost immediately. This review outlines the risks and discusses our current state of knowledge with emphasis on transgenic and gene editing approaches.

Methylated Thioarsenates and Monothioarsenate Differ in Uptake, Transformation, and Contribution to Total Arsenic Translocation in Rice Plants.

Methylated and inorganic thioarsenates have recently been reported from paddy fields besides the better-known oxyarsenates. Methylated thioarsenates are highly toxic for humans, yet their uptake, transformation, and translocation in rice plants is unknown. Here, hydroponic experiments with 20 day old rice plants showed that monomethylmonothioarsenate (MMMTA), dimethylmonothioarsenate (DMMTA), and monothioarsenate (MTA) were taken up by rice roots and could be detected in the xylem. Total arsenic (As) translocation from roots to shoots was higher for plants exposed to DMMTA, MTA, and dimethylarsenate (DMAV) compared to MMMTA and monomethylarsenate (MMAV). All thioarsenates were partially transformed in the presence of rice roots, but processes and extents differed. MMMTA was subject to abiotic oxidation and largely dethiolated to MMAV already outside the plant, probably due to root oxygen loss. DMMTA and MTA were not oxidized abiotically. Crude protein extracts showed rapid enzymatic reduction for MTA but not for DMMTA. Our study implies that DMMTA has the highest potential to contribute to total As accumulation in grains either as DMAV or partially as DMMTA. DMMTA has once been detected in rice grains using enzymatic extraction. By routine acid extraction, DMMTA is determined as DMAV and thus escapes regulation despite its toxicity.

Identification of C-terminal Regions in Arabidopsis thaliana Phytochelatin Synthase 1 Specifically Involved in Activation by Arsenite.

Phytochelatins (PCs) are major chelators of toxic elements including inorganic arsenic (As) in plant cells. Their synthesis confers tolerance and influences within-plant mobility. Previous studies had shown that various metal/metalloid ions differentially activate PC synthesis. Here we identified C-terminal parts involved in arsenite- [As(III)] dependent activation of AtPCS1, the primary Arabidopsis PC synthase. The T-DNA insertion in the AtPCS1 mutant cad1-6 causes a truncation in the C-terminal regulatory domain that differentially affects activation by cadmium (Cd) and zinc (Zn). Comparisons of cad1-6 with the AtPCS1 null mutant cad1-3 and the double mutant of tonoplast PC transporters abcc1/2 revealed As(III) hypersensitivity of cad1-6 equal to that of cad1-3. Both cad1-6 and cad1-3 showed increased As distribution to shoots compared with Col-0, whereas Zn accumulation in shoots was equally lower in cad1-6 and cad1-3. Supporting these phenotypes of cad1-6, PC accumulation in the As(III)-exposed plants were at trace level in both cad1-6 and cad1-3, suggesting that the truncated AtPCS1 of cad1-6 is defective in PCS activity in response to As(III). Analysis of a C-terminal deletion series of AtPCS1 using the PCS-deficient mutant of fission yeast suggested important regions within the C-terminal domain for As(III)-dependent PC synthesis, which were different from the regions previously suggested for Cd- or Zn-dependent activation. Interestingly, we identified a truncated variant more strongly activated than the wild-type protein. This variant could potentially be used as a tool to better restrict As mobility in plants.

CAX1 suppresses Cd-induced generation of reactive oxygen species in Arabidopsis halleri.

The molecular analysis of metal hyperaccumulation in species such as Arabidopsis halleri offers the chance to gain insights into metal homeostasis and into the evolution of adaptation to extreme habitats. A prerequisite of metal hyperaccumulation is metal hypertolerance. Genetic analysis of a backcross population derived from Arabidopsis lyrata × A. halleri crosses revealed three quantitative trait loci for Cd hypertolerance. A candidate gene for Cdtol2 is AhCAX1, encoding a vacuolar Ca2+ /H+ antiporter. We developed a method for the transformation of vegetatively propagated A. halleri plants and generated AhCAX1-silenced lines. Upon Cd2+ exposure, several-fold higher accumulation of reactive oxygen species (ROS) was detectable in roots of AhCAX1-silenced plants. In accordance with the dependence of Cdtol2 on external Ca2+ concentration, this phenotype was exclusively observed in low Ca2+ conditions. The effects of external Ca2+ on Cd accumulation cannot explain the phenotype as they were not influenced by the genotype. Our data strongly support the hypothesis that higher expression of CAX1 in A. halleri relative to other Arabidopsis species represents a Cd hypertolerance factor. We propose a function of AhCAX1 in preventing a positive feedback loop of Cd-elicited ROS production triggering further Ca2+ -dependent ROS accumulation.