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 γ -glutamylcysteine synthetase ( γ -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.     

Characteristics of cadmium uptake in two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens

Uptake of Cd and Zn by intact seedlings of two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens was characterized using radioactive tracers. Uptake of Cd and Zn at 2 degrees C was assumed to represent mainly apoplastic binding in the roots, whereas the difference in uptake between 22 degrees C and 2 degrees C represented metabolically dependent influx. There was no significant difference between the two ecotypes in the apoplastic binding of Cd or Zn. Metabolically dependent uptake of Cd was 4.5-fold higher in the high Cd-accumulating ecotype, Ganges, than in the low Cd-accumulating ecotype, Prayon. By contrast, there was only a 1.5-fold difference in the Zn uptake between the two ecotypes. For the Ganges ecotype, Cd uptake could be described by Michaelis-Menten kinetics with a V(max) of 143 nmol g(-1) root FW h(-1) and a K(m) of 0.45 microM. Uptake of Cd by the Ganges ecotype was not inhibited by La, Zn, Cu, Co, Mn, Ni or Fe(II), and neither by increasing the Ca concentration. By contrast, addition of La, Zn or Mn, or increasing the Ca concentration in the uptake solution decreased Cd uptake by Prayon. Uptake of Ca was larger in Prayon than in Ganges. The results suggest that Cd uptake by the low Cd-accumulating ecotype (Prayon) may be mediated partly via Ca channels or transporters for Zn and Mn. By contrast, there may exist a highly selective Cd transport system in the root cell membranes of the high Cd-accumulating ecotype (Ganges) of T. caerulescens.     

Influence of iron status on cadmium and zinc uptake by different ecotypes of the hyperaccumulator Thlaspi caerulescens

We have previously identified an ecotype of the hyperaccumulator Thlaspi caerulescens (Ganges), which is far superior to other ecotypes (including Prayon) in Cd uptake. In this study, we investigated the effect of Fe status on the uptake of Cd and Zn in the Ganges and Prayon ecotypes, and the kinetics of Cd and Zn influx using radioisotopes. Furthermore, the T. caerulescens ZIP (Zn-regulated transporter/Fe-regulated transporter-like protein) genes TcZNT1-G and TcIRT1-G were cloned from the Ganges ecotype and their expression under Fe-sufficient and -deficient conditions was analyzed. Both short- and long-term studies revealed that Cd uptake was significantly enhanced by Fe deficiency only in the Ganges ecotype. The concentration-dependent kinetics of Cd influx showed that the V(max) of Cd was 3 times greater in Fe-deficient Ganges plants compared with Fe-sufficient plants. In Prayon, Fe deficiency did not induce a significant increase in V(max) for Cd. Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes. These results are in agreement with the gene expression study. The abundance of ZNT1-G mRNA was similar between the Fe treatments and between the two ecotypes. In contrast, abundance of the TcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions. The present results indicate that the stimulatory effect of Fe deficiency on Cd uptake in Ganges may be related to an up-regulation in the expression of genes encoding for Fe(2+) uptake, possibly TcIRT1-G.     

A novel CPx-ATPase from the cadmium hyperaccumulator Thlaspi caerulescens

Thlaspi caerulescens exhibits a unique capacity for cadmium tolerance and accumulation. We investigated the molecular basis of this exceptional Cd(2+) tolerance by screening for T. caerulescens genes, which alleviate Cd(2+) toxicity upon expression in Saccharomyces cerevisiae. This allowed for the isolation of a cDNA encoding a peptide with homology to the C-terminal part of a heavy metal ATPase. The corresponding TcHMA4 full-length sequence was isolated from T. caerulescens and compared to its homolog from Arabidopsis thaliana (AtHMA4). Expression of TcHMA4 and AtHMA4 cDNAs conferred Cd sensitivity in yeast, while expression of TcHMA4-C and AtHMA4-C cDNAs encoding the C-termini of, respectively, TcHMA4 and AtHMA4 conferred Cd tolerance. Moreover, heterologous expression in yeast suggested a higher Cd binding capacity of TcHMA4-C compared to AtHMA4-C. In planta, both HMA4 genes were expressed at a higher level in roots than in shoots. However, TcHMA4 shows a much higher constitutive expression than AtHMA4. Our data indicate that HMA4 could be involved in Cd(2+) transport and possibly in the Cd hyperaccumulation character.     

Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase

Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 microg g(-1) Zn and 10,000 microg g(-1) Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils. In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T. caerulescens. A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P1B subfamily of purported heavy metal-translocating ATPases. A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead [Pb], and copper [Cu]) out of the cell. However, in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance. Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens. Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding. When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation. The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.     

Cadmium hyperaccumulation protects Thlaspi caerulescens from leaf feeding damage by thrips (Frankliniella occidentalis)

Metal hyperaccumulation has been proposed as a plant defensive strategy. Here, we investigated whether cadmium (Cd) hyperaccumulation protected Thlaspi caerulescens from leaf feeding damage by thrips (Frankliniella occidentalis). Two ecotypes differing in Cd accumulation, Ganges (high) and Prayon (low), were grown in compost amended with 0-1000 mg Cd kg(-1) in two experiments under glasshouse conditions. F2 and F3 plants from the Prayon x Ganges crosses were grown with 5 mg Cd kg(-1). Plants were naturally colonized by thrips and the leaf feeding damage index (LFDI) was assessed. The LFDI decreased significantly with increasing Cd in both ecotypes, and correlated with shoot Cd concentration in a log-linear fashion. Prayon was more attractive to thrips than Ganges, but the ecotypic difference in the LFDI was largely accounted for by the shoot Cd concentration. In the F2 and F3 plants, the LFDI correlated significantly and negatively with shoot Cd, but not with shoot zinc (Zn) or sulphur (S) concentrations. We conclude that Cd hyperaccumulation deters thrips from feeding on T. caerulescens leaves, which may offer an adaptive benefit to the plant.     

Variation in root-to-shoot translocation of cadmium and zinc among different accessions of the hyperaccumulators Thlaspi caerulescens and Thlaspi praecox

Efficient root-to-shoot translocation is a key trait of the zinc/cadmium hyperaccumulators Thlaspi caerulescens and Thlaspi praecox, but the extent of variation among different accessions and the underlying mechanisms remain unclear. Root-to-shoot translocation of Cd and Zn and apoplastic bypass flow were determined in 10 accessions of T. caerulescens and one of T. praecox, using radiolabels (109)Cd and (65)Zn. Two contrasting accessions (Pr and Ga) of T. caerulescens were further characterized for TcHMA4 expression and metal compartmentation in roots. Root-to-shoot translocation of (109)Cd and (65)Zn after 1 d exposure varied 4.4 to 5-fold among the 11 accessions, with a significant correlation between the two metals, but no significant correlation with uptake or the apoplastic bypass flow. The F(2) progeny from a cross between accessions from Prayon, Belgium (Pr) and Ganges, France (Ga) showed a continuous phenotype pattern and transgression. There was no significant difference in the TcHMA4 expression in roots between Pr and Ga. Compartmentation analysis showed a higher percentage of (109)Cd sequestered in the root vacuoles of Ga than Pr, the former being less efficient in translocation than the latter. Substantial natural variation exists in the root-to-shoot translocation of Cd and Zn, and root vacuolar sequestration may be an important factor related to this variation.     

Distribution of cadmium in leaves of Thlaspi caerulescens

Knowledge of the intracellular distribution of Cd in leaves is necessary in order to understand the mechanisms of hyperaccumulation in Thlaspi caerulescens. Ganges and Prayon, two ecotypes accumulating Cd to different levels, were grown in nutrient medium containing varying concentrations (0, 5, 10, 50, and 100 microM) of Cd. Several different approaches were combined in this study to (i) validate the results obtained by a specific method and (ii) establish the link between observations and measurements performed at different scales. In both ecotypes, Cd, localized by autoradiography, was found mainly at the edges of the leaves, but also in points of higher concentration spread over the whole limb surface. This localization was clearly correlated with the necrotic spots observed on Prayon leaves. Scanning electron microscopy coupled with energy dispersive X-ray microanalysis (cryo-SEM-EDXMA) and tissue fractionation (apoplasm, cell walls, mesophyll protoplasts, and lower epidermis) showed that Cd had similar patterns of distribution in leaf cells of both ecotypes. Cadmium was found both inside the cells and in the cell walls, mainly in the large epidermal cells but also in small epidermal cells. All the methods used agreed well and the results indicated that metal storage in the plants studied involves more than one compartment and that Cd is stored principally in the less metabolically active parts of leaf cells.     

Hyperaccumulation of cadmium and zinc in Thlaspi caerulescens and Arabidopsis halleri at the leaf cellular level

Vacuolar compartmentalization or cell wall binding in leaves could play a major role in hyperaccumulation of heavy metals. However, little is known about the physiology of intracellular cadmium (Cd) sequestration in plants. We investigated the role of the leaf cells in allocating metal in hyperaccumulating plants by measuring short-term (109)Cd and (65)Zn uptake in mesophyll protoplasts of Thlaspi caerulescens "Ganges" and Arabidopsis halleri, both hyperaccumulators of zinc (Zn) and Cd, and T. caerulescens "Prayon," accumulating Cd at a lower degree. The effects of low temperature, several divalent cations, and pre-exposure of the plants to metals were investigated. There was no significant difference between the Michaelis-Menten kinetic constants of the three plants. It indicates that differences in metal uptake cannot be explained by different constitutive transport capacities at the leaf protoplast level and that plasma and vacuole membranes of mesophyll cells are not responsible for the differences observed in heavy metal allocation. This suggests the existence of regulation mechanisms before the plasma membrane of leaf mesophyll protoplasts. However, pre-exposure of the plants to Cd induced an increase in Cd accumulation in protoplasts of "Ganges," whereas it decreased Cd accumulation in A. halleri protoplasts, indicating that Cd-permeable transport proteins are differentially regulated. The experiment with competitors has shown that probably more than one single transport system is carrying Cd in parallel into the cell and that in T. caerulescens "Prayon," Cd could be transported by a Zn and Ca pathway, whereas in "Ganges," Cd could be transported mainly by other pathways.     

Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescens (J. & C. Presl)

Thlaspi caerulescens (J. & C. Presl, "Prayon") is a heavy-metal hyperaccumulator that accumulates Zn and Cd to high concentrations (40,000 and 4,000 mg kg DW-1 respectively) without phytotoxicity. The mechanism of Cd tolerance has not been characterized but reportedly involves vacuolar sequestration. The role of phytochelatins (PCs) in metal tolerance in T. caerulescens and the related non-accumulator T. arvense was examined. Although PCs were produced by both species in response to Cd, these peptides do not appear to be involved in metal tolerance in the hyperaccumulator. Leaf and root PC levels for both species showed a similar positive correlation with tissue Cd, but total PC levels in the hyperaccumulator were generally lower, despite correspondingly higher metal concentrations. The lack of a role for PCs in the hyperaccumulator's response to metal stress suggests that other mechanisms are responsible Cd tolerance. The lower level of leaf PCs in T. caerulescens also implies that Cd in the shoot is sequestered in a compartment or form that does not elicit a PC response.     

Investigation of heavy metal hyperaccumulation at the cellular level: development and characterization of Thlaspi caerulescens suspension cell lines

The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens.     

Biochemical and biophysical characterisation yields insights into the mechanism of a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens

TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P(1B)-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10μM), and all three metal ions activated the ATPase at a concentration of 0.3μM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (E(A)=38kJ?mol(-1)) over the whole concentration range while it increased from 17 to 42kJ?mol(-1) with rising copper concentration and decreased from 39 to 23kJ?mol(-1) with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine.     

Enhanced arsenate reduction by a CDC25-like tyrosine phosphatase explains increased phytochelatin accumulation in arsenate-tolerant Holcus lanatus

Decreased arsenate [As(V)] uptake is the major mechanism of naturally selected As(V) hypertolerance in plants. However, As(V)-hypertolerant ecotypes also show enhanced rates of phytochelatin (PC) accumulation, suggesting that improved sequestration might additionally contribute to the hypertolerance phenotype. Here, we show that enhanced PC-based sequestration in As(V)-hypertolerant Holcus lanatus is not due to an enhanced capacity for PC synthesis as such, but to increased As(V) reductase activity. Vacuolar transport of arsenite-thiol complexes was equal in both ecotypes. Based on homology with the yeast As(V) reductase, Acr2p, we identified a Cdc25-like plant candidate, HlAsr, and confirmed the As(V) reductase activity of both HlAsr and the homologous protein from Arabidopsis thaliana. The gene appeared to be As(V)-inducible and its expression was enhanced in the As(V)-hypertolerant H. lanatus ecotype, compared with the non-tolerant ecotype. Homologous ectopic overexpression of the AtASR cDNA in A. thaliana produced a dual phenotype. It improved tolerance to mildly toxic levels of As(V) exposure, but caused hypersensitivity to more toxic levels. Arabidopsis asr T-DNA mutants showed increased As(V) sensitivity at low exposure levels and enhanced arsenic retention in the root. It is argued that, next to decreased uptake, enhanced expression of HlASR might act as an additional determinant of As(V) hypertolerance and As transport in H. lanatus.     

The role of phytochelatins in constitutive and adaptive heavy metal tolerances in hyperaccumulator and non-hyperaccumulator metallophytes

Using the gamma-glutamylcysteine synthetase inhibitor, L-buthionine-[S,R]-sulphoximine (BSO), the role for phytochelatins (PCs) was evaluated in Cu, Cd, Zn, As, Ni, and Co tolerance in non-metallicolous and metallicolous, hypertolerant populations of Silene vulgaris (Moench) Garcke, Thlaspi caerulescens J.&C. Presl., Holcus lanatus L., and Agrostis castellana Boiss. et Reuter. Based on plant-internal PC-thiol to metal molar ratios, the metals' tendency to induce PC accumulation decreased in the order As/Cd/Cu > Zn > Ni/Co, and was consistently higher in non-metallicolous plants than in hypertolerant ones, except for the case of As. The sensitivities to Cu, Zn, Ni, and Co were consistently unaffected by BSO treatment, both in non-metallicolous and hypertolerant plants, suggesting that PC-based sequestration is not essential for constitutive tolerance or hypertolerance to these metals. Cd sensitivity was considerably increased by BSO, though exclusively in plants lacking Cd hypertolerance, suggesting that adaptive cadmium hypertolerance is not dependent on PC-mediated sequestration. BSO dramatically increased As sensitivity, both in non-adapted and As-hypertolerant plants, showing that PC-based sequestration is essential for both normal constitutive tolerance and adaptive hypertolerance to this metalloid. The primary function of PC synthase in plants and algae remains elusive.     

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.     

Growth and mineral element composition in two ecotypes of Thlaspi caerulescens on Cd contaminated soil

The heavy metal hyperaccumulator Thlaspi caerulescens occurs both on heavy metal polluted soils (metallicolous ecotype MET) and on soils with normal heavy metal content (non-metallicolous ecotype: NMET). In order to assess the extent and structure of variation in growth, shoot accumulation of Cd, Zn and mineral element (Ca, Mg, K, Fe), a MET ecotype from Belgium and a NMET ecotype from Luxembourg were studied. Seven maternal families from two populations of each ecotype were grown on both Cd and Zn contaminated soil. Although both ecotypes presented a similar heavy metal tolerance in the experimental conditions tested, they differed in several points. The MET populations had markedly higher biomass and higher root:shoot ratio compared to NMET populations. The Zn, and at lesser extent, the Cd hyperaccumulation capacity tended to be higher in the NMET populations. The same trend was observed for the foliar concentrations of Mg, Ca and Fe with NMET populations having higher concentrations compared to MET ones. Cd and Zn concentrations were negatively correlated with the biomass of both ecotype. However, the negative correlation between the Zn and biomass was much lower in MET ecotype suggesting a tighter control of internal Zn concentration in this ecotype. Finally, although the Cd phytoextraction capacity was similar in both ecotype, a higher Zn phytoextraction capacity was detected in NMET ecotype when these plants grow on moderate Cd and Zn concentrations.     

Natural variation among Arabidopsis accessions reveals malic acid as a key mediator of Nickel (Ni) tolerance

Plants have evolved various mechanisms for detoxification that are specific to the plant species as well as the metal ion chemical properties. Malic acid, which is commonly found in plants, participates in a number of physiological processes including metal chelation. Using natural variation among Arabidopsis accessions, we investigated the function of malic acid in Nickel (Ni) tolerance and detoxification. The Ni-induced production of reactive oxygen species was found to be modulated by intracellular malic acid, indicating its crucial role in Ni detoxification. Ni tolerance in Arabidopsis may actively involve malic acid and/or complexes of Ni and malic acid. Investigation of malic acid content in roots among tolerant ecotypes suggested that a complex of Ni and malic acid may be involved in translocation of Ni from roots to leaves. The exudation of malic acid from roots in response to Ni treatment in either susceptible or tolerant plant species was found to be partially dependent on AtALMT1 expression. A lower concentration of Ni (10?μM) treatment induced AtALMT1 expression in the Ni-tolerant Arabidopsis ecotypes. We found that the ecotype Santa Clara (S.C.) not only tolerated Ni but also accumulated more Ni in leaves compared to other ecotypes. Thus, the ecotype S.C. can be used as a model system to delineate the biochemical and genetic basis of Ni tolerance, accumulation, and detoxification in plants. The evolution of Ni hyperaccumulators, which are found in serpentine soils, is an interesting corollary to the fact that S.C. is also native to serpentine soils.