Cd Hypertolerance Research Articles

Functional characterization of a DNA-damage repair/tolerance 100 (DRT100) gene in Sedum alfredii Hance for genome stability maintenance and Cd hypertolerance

Cd contamination is a world-wild concern for its toxicity and accumulation in food chain. Sedum alfredii Hance (Crassulaceae) is a zinc (Zn) and cadmium (Cd) hyperaccumulator native to China and widely applied for the phytoremediation at Zn or Cd contaminated sites. Although many studies report the uptake, translocation and storage of Cd in S. alfredii Hance, limited information is known about the genes and underlying mechanisms of genome stability maintenance under Cd stress. In this study, a gene resembling DNA-damage repair/toleration 100 (DRT100) was Cd inducible and designated as SaDRT100. Heterologous expression of SaDRT100 gene in yeasts and Arabidopsis thaliana enhanced Cd tolerance capability. Under Cd stress, transgenic Arabidopsis with SaDRT100 gene exhibited lower levels of reactive oxygen species (ROS), fewer Cd uptake in roots and less Cd-induced DNA damage. Evidenced by the subcellular location in cellular nucleus and expression in aerial parts, we suggested the involvement of SaDRT100 in combating Cd-induced DNA damage. Our findings firstly uncovered the roles of SaDRT100 gene in Cd hypertolerance and genome stability maintenance in S. alfredii Hance. The potential functions of DNA protection make SaDRT100 gene a candidate in genetic engineering for phytoremediation at multi-component contaminated sites.

SaMT3 in Sedum alfredii drives Cd detoxification by chelation and ROS-scavenging via Cys residues

Metallothioneins (MTs), a group of cysteine-rich proteins, are effective chelators of cadmium (Cd) and play a key role in plant Cd detoxification. However, little is known about the role of single cysteine (Cys) residues in the MTs involved in the adaptation of plants to Cd stress, especially, in hyperaccumulators. In the present study, we functionally characterised SaMT3 in S. alfredii, a Cd/Zn hyperaccumulator native to China. Our results showed that the C- and N- terminal regions of SaMT3 had differential functional natures in S. alfredii and determined its Cd hypertolerance and detoxification. Two CXC motifs within the C-terminal region were revealed to play a crucial role in Cd sensing and binding, whereas the four Cys-residues within the N-terminal region were involved in scavenging reactive oxygen species (ROS). An S. alfredii transgenic system based on callus transformation was developed to further investigate the in-planta gene function. The SaMT3-overexpressing transgenic plant roots were more tolerant to Cd than those of wild-type plants. Knockout of SaMT3 resulted in significantly decreased Cd concentrations and increased ROS levels after exposure to Cd stress. We demonstrated the SaMT3-mediated adaptation strategy in S. alfredii, which uses metal chelation and ROS scavenging in response to Cd stress. Our results further reveal the molecular mechanisms underlying Cd detoxification in hyperaccumulating plants, as well as the relation between Cys-related motifs and the metal binding properties of MTs. This research provides valuable insights into the functions of SaMT3 in S. alfredii, and improves our understanding of Cd hyperaccumulation in plants.

Quantitative proteome analysis reveals changes of membrane transport proteins in Sedum plumbizincicola under cadmium stress

Sedum plumbizincicola is an herbaceous species tolerant of excessive cadmium accumulation in above-ground tissues. The implications of membrane proteins, especially integrative membrane proteins, in Cd detoxification of plants have received attention in recent years, but a comprehensive profiling of Cd-responsive membrane proteins from Cd hyperaccumulator plants is lacking. In this study, the membrane proteins of root, stem, and leaf tissues of S. plumbizincicola seedlings treated with Cd solution for 0, 1 or 4 days were analyzed by Tandem Mass Tag (TMT) labeling-based proteome quantification (Data are available via ProteomeXchange with identifier PXD025302). Total 3353 proteins with predicted transmembrane helices were identified and quantified in at least one tissue group. 1667 proteins were defined as DAPs (differentially abundant proteins) using fold change >1.5 with p-values <0.05. The number of DAPs involved in metabolism, transport protein, and signal transduction was significantly increased after exposure to Cd, suggesting that the synthesis and decomposition of organic compounds and the transport of ions were actively involved in the Cd tolerance process. The number of up-regulated transport proteins increased significantly from 1-day exposure to 4-day exposure, from 5 to 112, 16 to 42, 18 to 44, in root, stem, and leaf, respectively. Total 352 Cd-regulated transport proteins were identified, including ABC transporters, ion transport proteins, aquaporins, proton pumps, and organic transport proteins. Heterologous expression of SpABCB28, SpMTP5, SpNRAMP5, and SpHMA2 in yeast and subcellular localization showed the Cd-specific transport activity. The results will enhance our understanding of the molecular mechanism of Cd hypertolerance and hyperaccumulation in S. plumbizincicola and will be benefit for future genetic engineering in phytoremediation.

Intra-specific variation in zinc, cadmium and nickel hypertolerance and hyperaccumulation capacities in Noccaea caerulescens

The study aimed at characterizing the patterns of natural variation in the tolerance and accumulation capacities for zinc (Zn), cadmium (Cd), and nickel (Ni) between and within edaphic ecotypes of the Zn/Cd/Ni hyperaccumulator, Noccaea caerulescens. Tolerance was assessed in a hydroponic ‘sequential exposure’ test, using the lowest concentration that completely arrested root growth as an end point. Accumulation was measured as the foliar metal concentration after six weeks of growth at 5 µM Zn, 2 µM Cd, or 1 µM Ni. Zn and Cd tolerance were positively correlated, and highest in the calamine ecotype. Ni tolerance was without significant ecotypic variation. The ultramafic ecotype was as Zn-tolerant as the non-metallicolous one, but much more sensitive to Cd. The accumulation capacities for Zn, Cd and Ni were all positively correlated and without significant ecotypic variation. Zn hyperaccumulation capacity was species-wide, but Cd and Ni hyperaccumulation capacities were lacking in four populations (all calamine). There is considerable independent variation among populations regarding their Zn, Cd, and Ni accumulation capacities. This variation is most pronounced within the calamine ecotype, because some populations apparently had adopted an exclusion strategy for Zn or Cd hypertolerance, whereas others had not.

&lt;i&gt;De novo&lt;/i&gt; transcriptome analysis reveals an unperturbed transcriptome under high cadmium conditions in the Cd-hypertolerant fern &lt;i&gt;Athyrium yokoscense&lt;/i&gt;

Athyrium yokoscense shows strong tolerance to cadmium exposure, even at levels that are many times greater than the toxic levels in ordinary plants. To determine the mechanism of Cd tolerance in A. yokoscense, we grew these plants under high Cd conditions and observed the tissue-specific accumulation of Cd and generation of reactive oxygen species, which is one of the major physiological responses to Cd stress. Fuchsin staining indicated the existence of a casparian strip in A. yokoscense roots, which may participate in Cd hypertolerance in A. yokoscense. Moreover, we performed RNA-seq of RNA samples from A. yokoscense plants treated with or without Cd exposure and obtained comprehensive RNA sequences as well as the Cd-responsive expression patterns of individual genes. Through de novo transcriptome assembly and gene expression analysis, we found that A. yokoscense showed normal features with no significant change in the expression levels of any transporter genes, even under high Cd exposure conditions. Our results demonstrate that A. yokoscense has an unusual mechanism that allows the invading Cd to partition into the distal roots, thus avoiding translocation of Cd into the xylem.

CDNA Library for Mining Functional Genes in Sedum alfredii Hance Related to Cadmium Tolerance and Characterization of the Roles of a Novel SaCTP2 Gene in Enhancing Cadmium Hyperaccumulation.

Heavy metal contamination presents serious threats to living organisms. Functional genes related to cadmium (Cd) hypertolerance or hyperaccumulation must be explored to enhance phytoremediation. Sedum alfredii Hance is a Zn/Cd cohyperaccumulator exhibiting abundant genes associated with Cd hypertolerance. Here, we developed a method for screening genes related to Cd tolerance by expressing a cDNA-library for S. alfredii Hance. Yeast functional complementation validated 42 of 48 full-length genes involved in Cd tolerance, and the majority of them were strongly induced in roots and exhibited diverse expression profiles across tissues. Coexpression network analysis suggested that 15 hub genes were connected with genes involved in metabolic processes, response to stimuli, and metal transporter and antioxidant activity. The functions of a novel SaCTP2 gene were validated by heterologous expression in Arabidopsis, responsible for retarding chlorophyll content decrease, maintaining membrane integrity, promoting reactive oxygen species (ROS) scavenger activities, and reducing ROS levels. Our findings suggest a highly complex network of genes related to Cd hypertolerance in S. alfredii Hance, accomplished via the antioxidant system, defense genes induction, and the calcium signaling pathway. The proposed cDNA-library method is an effective approach for mining candidate genes associated with Cd hypertolerance to develop genetically engineered plants for use in phytoremediation.

SpHMA1 is a chloroplast cadmium exporter protecting photochemical reactions in the Cd hyperaccumulator Sedum plumbizincicola.

Sedum plumbizincicola is able to hyperaccumulate cadmium (Cd), a nonessential and highly toxic metal, in the above-ground tissues, but the mechanisms for its Cd hypertolerance are not fully understood. Here, we show that the heavy metal ATPase 1 (SpHMA1) of S.plumbizincicola plays an important role in chloroplast Cd detoxification. Compared with the HMA1 ortholog in the Cd nonhyperaccumulating ecotype of Sedum alfredii, the expression of SpHMA1 in the leaves of S.plumbizincicola was >200 times higher. Heterologous expression of SpHMA1 in Saccharomyces cerevisiae increased Cd sensitivity and Cd transport activity in the yeast cells. The SpHMA1 protein was localized to the chloroplast envelope. SpHMA1 RNA interference transgenic plants and CRISPR/Cas9-induced mutant lines showed significantly increased Cd accumulation in the chloroplasts compared with wild-type plants. Chlorophyll fluorescence imaging analysis revealed that the photosystem II of SpHMA1 knockdown and knockout lines suffered from a much higher degree of Cd toxicity than wild type. Taken together, these results suggest that SpHMA1 functions as a chloroplast Cd exporter and protects photosynthesis by preventing Cd accumulation in the chloroplast in S.plumbizincicola and hyperexpression of SpHMA1 is an important component contributing to Cd hypertolerance in S.plumbizincicola.

Young leaf protection from cadmium accumulation and regulation of nitrilotriacetic acid in tall fescue (Festuca arundinacea) and Kentucky bluegrass (Poa pratensis)

Phytoextraction efficiency of cadmium (Cd) contaminated soil mainly depended upon the mechanism of plants in absorption, translocation, distribution, and detoxification of Cd. A pot experiment was designed to investigate Cd distribution and accumulation among the different leaves of tall fescue (Festuca arundinacea) and Kentucky bluegrass (Poa pratensis) and its regulation by Nitrilotriacetic acid (NTA), a biodegradable chelating agent. The results showed that Cd concentrations in the senescent and dead leaves were 3.2 and 5.3 fold of that in the emerging leaves of tall fescue, and 19.3 and 25.1 fold of that in the emerging leaves of Kentucky bluegrass, respectively. The lower Cd concentrations were maintained in the emerging and mature leaves to avoid Cd toxicity. In the emerging and mature leaves, Cd was mainly accumulated in the vascular bundles and epidermis. No Cd dithizonate color was observed in the mesophyll tissues of Kentucky bluegrass and only minor Cd was observed in the mesophyll tissues of tall fescue. In the senescent leaves, Cd dithizonate complexes were located in the protoplasts and cell walls of all leaf tissues. NTA greatly promoted Cd translocation and distribution to the senescent and dead leaves of tall fescue, but no significant effect was observed in Kentucky bluegrass. Our results indicate that a young leaf protection mechanism might be involved in their Cd hypertolerance. The Cd preferential accumulation could lead a novel phytoextraction strategy by the continuously harvesting the senescent and dead leaves of tall fescue and Kentucky bluegrass.

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.

A Genetic Transformation Method for Cadmium Hyperaccumulator Sedum plumbizincicola and Non-hyperaccumulating Ecotype of Sedum alfredii

The present study demonstrates the development of an Agrobacterium-mediated genetic transformation method for species of the Sedum genus, which includes the Cd/Zn hyperaccumulator Sedum plumbizincicola and the non-hyperaccumulating ecotype of S. alfredii. Multiple shoots were induced from stem nodes of two Sedum plants using Murashige and Skoog (MS) medium containing 0.1 mg/L cytokinin 6-benzyladenine (6-BA) and 1.0 mg/L auxin 1-naphthaleneacetic acid (NAA). The shoot primordia were used as direct targets for Agrobacterium infection. Selection on hygromycin was highly effective in generating Agrobacterium-transformed explants. This callus-free procedure allowed us to obtain transgenic plantlets after rooting hygromycin-resistant shoots on phytohormone-free MS medium containing the antibiotic. The presence and expression of the reporter genes gusA and GFP in transgenic plants were confirmed by a real-time polymerase chain reaction, histochemical GUS assays, and confocal microscopy. This reliable method for genetic transformation of Sedum plants will help us to understand gene functions and the molecular mechanisms underlying Cd hypertolerance and hyperaccumulation in these species.

Physiological mechanism of hypertolerance of cadmium in Kentucky bluegrass and tall fescue: Chemical forms and tissue distribution

Kentucky bluegrass (Poa pratensis) and tall fescue (Festuca arundinacea) are hypertolerant grasses to soil cadmium contamination. Little information is available on their tolerance mechanism. A sand culture and a hydroponic culture experiment were designed to investigate the Cd chemical form changes and its translocation in different tissues. The results showed that Kentucky bluegrass and tall fescue can tolerate 50–200mgkg−1 of soil Cd stresses and accumulate as high as 4275 and 2559mgCdkg−1 DW, respectively, in their shoots without the loss of shoot biomass. Their Cd hypertolerance was correlated with an increase of the undissolved Cd phosphates in the leaves in both grass species, as determined by sequential solvent extraction procedures. The superior Cd tolerance of tall fescue to Kentucky bluegrass was associated with less Cd translocation into the stele of roots and less Cd transported to leaves. The pectate- and protein-integrated Cd forms may be involved in the symplastic translocation of Cd from cortex into stele, and this may lead the higher Cd concentrations in the stele of roots and then above ground leaves via long-distance transport in Kentucky bluegrass.

Lead, zinc and cadmium accumulation from two metalliferous soils with contrasting calcium contents in heavy metal-hyperaccumulating and non-hyperaccumulating metallophytes: a comparative study

Aims and background We previously compared metallicolous (M) and non-metallicolous (NM) populations of Noccaea (=Thlaspi) caerulescens, Silene vulgaris, and Matthiola flavida for their abilities to tolerate and (hyper)-accumulate lead (Pb) in hydroponics. In the present study we aimed 1) to check the hyperaccumulation and tolerance abilities of these populations in controlled experiments using metalliferous soils, 2) to test the M. flavida M population for Zn and Cd hypertolerance in hydroponics.

Cd-induced changes in leaf proteome of the hyperaccumulator plant Phytolacca americana

Cadmium (Cd) is highly toxic to all organisms. Soil contamination by Cd has become an increasing problem worldwide due to the intensive use of Cd-containing phosphate fertilizers and industrial zinc mining. Phytolacca americana L. is a Cd hyperaccumulator plant that can grow in Cd-polluted areas. However, the molecular basis for its remarkable Cd resistance is not known. In this study, the effects of Cd exposure on protein expression patterns in P.americana was investigated by 2-dimensional gel electrophoresis (2-DE). 2-DE profiles of leaf proteins from both control and Cd-treated (400μM, 48h) seedlings were compared quantitatively using ImageMaster software. In total, 32 differentially expressed protein spots were identified using MALDI-TOF/TOF mass spectrometry coupled to protein database search, corresponding to 25 unique gene products. Of those 14 were enhanced/induced while 11 reduced under Cd treatment. The alteration pattern of protein expression was verified for several key proteins involved in distinct metabolic pathways by immuno-blot analysis. Major changes were found for the proteins involved in photosynthetic pathways as well as in the sulfur- and GSH-related metabolisms. One-third of the up-regulated proteins were attributed to transcription, translation and molecular chaperones including a protein belonging to the calreticulin family. Other proteins include antioxidative enzymes such as 2-cys-peroxidase and oxidoreductases. The results of this proteomic analysis provide the first and primary information regarding the molecular basis of Cd hypertolerance in P. americana.

Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd‐hyperaccumulating ecotype of Thlaspi caerulescens

Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.

Metal accumulation in tobacco expressing Arabidopsis halleri metal hyperaccumulation gene depends on external supply

Engineering enhanced transport of zinc to the aerial parts of plants is a major goal in bio-fortification. In Arabidopsis halleri, high constitutive expression of the AhHMA4 gene encoding a metal pump of the P1B-ATPase family is necessary for both Zn hyperaccumulation and the full extent of Zn and Cd hypertolerance that are characteristic of this species. In this study, an AhHMA4 cDNA was introduced into N. tabacum var. Xanthi for expression under the control of its endogenous A. halleri promoter known to confer high and cell-type specific expression levels in both A. halleri and the non-hyperaccumulator A. thaliana. The transgene was expressed at similar levels in both roots and shoots upon long-term exposure to low Zn, control, and increased Zn concentrations. A down-regulation of AhHMA4 transcript levels was detected with 10 μM Zn resupply to tobacco plants cultivated in low Zn concentrations. In general, a transcriptional regulation of AhHMA4 in tobacco contrasted with the constitutively high expression previously observed in A. halleri. Differences in root/shoot partitioning of Zn and Cd between transgenic lines and the wild type were strongly dependent on metal concentrations in the hydroponic medium. Under low Zn conditions, an increased Zn accumulation in the upper leaves in the AhHMA4-expressing lines was detected. Moreover, transgenic plants exposed to cadmium accumulated less metal than the wild type. Both modifications of zinc and cadmium accumulation are noteworthy outcomes from the biofortification perspective and healthy food production. Expression of AhHMA4 may be useful in crops grown on soils poor in Zn.

Mechanisms to cope with arsenic or cadmium excess in plants

The metalloid arsenic and the heavy metal cadmium have no demonstrated biological function in plants. Both elements are highly toxic and of major concern with respect to their accumulation in soils, in the food-chain or in drinking water. Arsenate is taken up by phosphate transporters and rapidly reduced to arsenite, As(III). In reducing environments, As(III) is taken up by aquaporin nodulin 26-like intrinsic proteins. Cd(2+) enters the root via essential metal uptake systems. As(III) and Cd(2+) share some similarity between their toxicology and sequestration machineries. Recent progress in understanding the mechanisms of As and Cd uptake and detoxification is presented, including the elucidation of why rice takes up so much arsenic from soil and of mechanisms of As and Cd hypertolerance.

Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4

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.

High-level Zn and Cd tolerance in Silene paradoxa L. from a moderately Cd- and Zn-contaminated copper mine tailing

Cadmium and zinc tolerance were examined in populations of Silene paradoxa, one from uncontaminated calcareous soil (CVD) and one from a mine tailing (FC) (Cd<1-15 ppm, Zn 400-1300 ppm, pH 2-6). The mine population exhibited extremely high Zn and Cd tolerance levels, although the degrees of Cd and Zn enrichment relatively low at the population site. Cd and Zn hypertolerance in FC were associated with reduced rates of accumulation of these metals, both in roots and shoots (Cd), or exclusively in shoots (Zn). However, exclusion potentially explained only a minor part of the superior tolerance in FC. Cd hypertolerance in FC was associated with decreased, rather than enhanced phytochelatin accumulation. The remarkably high levels of Cd and Zn hypertolerance in FC might relate to the low soil pH, due to oxidation of sulphide minerals, and the absence of soil organic matter at the FC site.

Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri.

The metal hyperaccumulator Arabidopsis halleri exhibits naturally selected zinc (Zn) and cadmium (Cd) hypertolerance and accumulates extraordinarily high Zn concentrations in its leaves. With these extreme physiological traits, A. halleri phylogenetically belongs to the sister clade of Arabidopsis thaliana. Using a combination of genome-wide cross species microarray analysis and real-time reverse transcription-PCR, a set of candidate genes is identified for Zn hyperaccumulation, Zn and Cd hypertolerance, and the adjustment of micronutrient homeostasis in A. halleri. Eighteen putative metal homeostasis genes are newly identified to be more highly expressed in A. halleri than in A. thaliana, and 11 previously identified candidate genes are confirmed. The encoded proteins include HMA4, known to contribute to root-shoot transport of Zn in A. thaliana. Expression of either AtHMA4 or AhHMA4 confers cellular Zn and Cd tolerance to yeast (Saccharomyces cerevisiae). Among further newly implicated proteins are IRT3 and ZIP10, which have been proposed to contribute to cytoplasmic Zn influx, and FRD3 required for iron partitioning in A. thaliana. In A. halleri, the presence of more than a single genomic copy is a hallmark of several highly expressed candidate genes with possible roles in metal hyperaccumulation and metal hypertolerance. Both A. halleri and A. thaliana exert tight regulatory control over Zn homeostasis at the transcript level. Zn hyperaccumulation in A. halleri involves enhanced partitioning of Zn from roots into shoots. The transcriptional regulation of marker genes suggests that in the steady state, A. halleri roots, but not the shoots, act as physiologically Zn deficient under conditions of moderate Zn supply.

Synthesis of low molecular weight thiols in response to Cd exposure in Thlaspi caerulescens

In this study, we investigated the accumulation of phytochelatins (PCs) and other low molecular weight (LMW) thiols in response to Cd exposure in two contrasting ecotypes differing in Cd accumulation. Using a root elongation test, we found that the highly accumulating ecotype Ganges was more tolerant to Cd than the low Cd-accumulation ecotype Prayon. L-buthionine-(S,R)-sulphoximine (BSO), a potent inhibitor of the gamma-glutamylcysteine synthetase gamma-ECS) (an enzyme involved in the PC biosynthetic pathway), increased the Cd sensitivity of Prayon, but had no effect on Ganges. Although PC accumulation increased in response to Cd exposure, no significant differences were observed between the two ecotypes. Cd exposure induced a dose-dependent accumulation of both Cys and a still unidentified LMW thiol in roots of both ecotypes. Root accumulation of Cys and this thiol was higher in Ganges than in Prayon; the ecotypic differences were more pronounced when the plants were treated with BSO. These findings suggest that PCs do not contribute to the Cd hypertolerance displayed by the Ganges ecotype of Thlaspi caerulescens, whereas Cys and other LMW thiols might be involved.