Variations in plant metallothioneins: the heavy metal hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> as a study case

Plant metallothioneins (MTs) are extremely diverse and are thought to be involved in metal homeostasis or detoxification. Thlaspi caerulescens is a model Zn/Cd hyperaccumulator and thus constitutes an ideal system to study the variability of these MTs. Two T. caerulescens cDNAs (accession: 665511; accession: 665515), that are highly homologous to type 1 and type 2 Arabidopsis thaliana MTs, have been isolated using a functional screen for plant cDNAs that confer Cd tolerance to yeast. However, TcMT1 has a much shorter N-terminal domain than that of A. thaliana and so lacks Cys motifs conserved through all the plant MTs classified as type 1. A systematic search in plant databases allowed the detection of MT-related sequences. Sixty-four percent fulfil the criteria for MT classification described in Cobbett and Goldsbrough (2002) and further extend our knowledge about other conserved residues that might play an important role in plant MT structure. In addition, 34% of the total MT-related sequences cannot be classified strictly as they display modifications in the conserved residues according to the current plant MTs’ classification. The significance of this variability in plant MT sequences is discussed. Functional complementation in yeast was used to assess whether these variations may alter the MTs’ function in T. caerulescens. Regulation of the expression of MTs in T. caerulescens was also investigated. TcMT1 and TcMT2 display higher expression in T. caerulescens than in A. thaliana. Moreover, their differential expression patterns in organs and in response to metal exposure, suggest that the two types of MTs may have diverse roles and functions in T. caerulescens.     

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens

To study the role of metallothioneins (MTs) in Zn accumulation,the expression of TcMT2a, TcMT2b, and TcMT3 was analysed inthree accessions and 15 F₃ families of two inter-accession crossesof the Cd/Zn hyperaccumulator Thlaspi caerulescens, with differentdegrees of Zn accumulation. The highest expression levels werefound in the shoots of a superior metal-accumulating calamineaccession from St Laurent le Minier, with >10-fold TcMT3expression compared with another calamine accession and a non-metallicolousaccession. Moreover, F₃ sibling lines from the inter-accessioncrosses that harboured the MT2a or MT3 allele from St Laurentle Minier had higher expression levels. However, there was noco-segregation of TcMT2a or TcMT3 expression and Zn accumulation.To examine the functions of TcMTs in plants, TcMT2a and TcMT3were ectopically expressed in Arabidopsis. The transformantlines had reduced root length in control medium but not at highmetal concentrations, suggesting that the ectopically expressedproteins interfered with the physiological availability of essentialmetals under limited supply. The Arabidopsis transformant linesdid not show increased tolerance to Cd, Cu, or Zn, nor increasedCd or Zn accumulation. Immunohistochemical analysis indicatedthat in roots, MT2 protein is localized in the epidermis androot hairs of both T. caerulescens and Arabidopsis thaliana.The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarilyinvolved in Zn accumulation as such. However, the elevated expressionlevels in the metallicolous accessions suggests that they docontribute to the metal-adapted phenotype, possibly throughimproving Cu homeostasis at high Zn and Cd body burdens. Alternatively,they might function as hypostatic enhancers of Zn or Cd tolerance. Key words: Cd, crosses, metallothionein, protein, quantitative real-time PCR, Thlaspi caerulescens, Zn Received 14 August 2008; Revised 14 October 2008 Accepted 15 October 2008     

超富集植物遏蓝菜对重金属吸收、运输和累积的机制

遏蓝菜Thlaspi caerulescens可以在其地上部累积大量重金属如锌、镉等,是公认的超富集植物。由于该植物生物量小,不宜直接用于重金属污染的土壤植物修复,而被广泛作为一种模式植物来进行重金属富集机制研究。遏蓝菜对重金属离子的累积大致经过螯合剂解毒、地上部长距离运输以及在液泡中的储存等生理过程。已经发现的植物体内的金属螯合剂——有机酸、氨基酸、植物络合素(PCs)、金属硫蛋白(MT)和尼克烟酰胺NA等,区室化以及长距离运输相关的转运蛋白——ZIP(ZRT/IRTlike protein)、CDF(Cation diffusion facilitator)、Nramp(Natural resistance and macrophage protein)和HMA(Heavy metal ATPase)等家族,以上各种基因、多肽与蛋白等共同参与了植物对金属累积与耐受过程并发挥各自重要的作用。以下主要介绍了遏蓝菜重金属超富集相关的基因、多肽和蛋白,以及它们在重金属螯合作用和运输过程中的功能。     

Soil microbial diversity as affected by the rhizosphere of the hyperaccumulator Thlaspi caerulescens under natural conditions

It is hypothesized that metal hyperaccumulator plants have specific rhizosphere conditions, potentially modifying the bioavailability of soil metals. This article aims to further the knowledge about the rhizosphere of the hyperaccumulator Thlaspi caerulescens, focusing on its microflora isolated from metalliferous soils collected in situ where the plants grow naturally. We characterized the cultivable microbial communities isolated from the rhizosphere of one population of this Ni hyperaccumulator species grown on a serpentine soil. The rhizosphere soil harbored a wide variety of microorganisms, predominantly bacteria, confirming the stimulatory effect of the T. caerulescens rhizosphere on microbial growth and proliferation. We tested the hypothesis that the rhizosphere of T. caerulescens influences (1) the metabolic diversity of the bacterial community and (2) the bacterial resistance to metals. The principal component analysis of the Biolog plate's data confirmed a structural effect of the rhizosphere of T. caerulescens on bacterial communities. The percentage of Ni-resistant bacteria was higher in the rhizosphere than in the bulk soil, suggesting a direct effect of the rhizosphere on Ni tolerance, reflecting a greater bacterial tolerance to Ni in the rhizosphere.     

Cadmium tolerance and antioxidative defenses in hairy roots of the cadmium hyperaccumulator,Thlaspi caerulescens

Plant species capable of hyperaccumulating heavy metals are of considerable interest for phytoremediation and phytomining. This work aims to identify the role of antioxidative metabolism in heavy metal tolerance in the Cd hyperaccumulator, Thlaspi caerulescens. Hairy roots of T. caerulescens and the non-hyperaccumulator, Nicotiana tabacum (tobacco), were used to test the effects of high Cd environments. In the absence of Cd, endogenous activities of catalase were two to three orders of magnitude higher in T. caerulescens than in N. tabacum. T. caerulescens roots also contained significantly higher endogenous superoxide dismutase activity and glutathione concentrations. Exposure to 20 ppm (178 microM) Cd prevented growth of N. tabacum roots and increased hydrogen peroxide (H(2)O(2)) levels by a factor of five relative to cultures without Cd. In contrast, growth was maintained in T. caerulescens, and H(2)O(2) concentrations were controlled to low, nontoxic levels in association with a strong catalase induction response. Treatment of roots with the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), exacerbated H(2)O(2) accumulation in Cd-treated N. tabacum, but had a relatively minor effect on H(2)O(2) levels and did not reduce Cd tolerance in T. caerulescens. Lipid peroxidation was increased by Cd treatment in both the hyperaccumulator and non-hyperaccumulator roots. This work demonstrates that metal-induced oxidative stress occurs in hyperaccumulator tissues even though growth is unaffected by the presence of heavy metals. It also suggests that superior antioxidative defenses, particularly catalase activity, may play an important role in the hyperaccumulator phenotype of T. caerulescens.     

Dynamics of interdomain and intermolecular interactions in mammalian metallothioneins.

The structures of mammalian metallothioneins (MTs), as solved by X-ray crystallography and NMR spectroscopy, all show seven divalent metals bound in two separate domains. The marked differences in metal-mobilities found for the two domains has led to the proposal for a dual role for the two MT metal domains. The tight metal binding in the C-terminal alpha-domain supposedly constitutes the basis for the detoxification of excess heavy metals, while the more labile metals in the N-terminal beta-domain function in the homeostasis of the essential elements zinc and copper. In this overview, we compare the two types of dimers found for MTs and their influence on metal-mobilities. In the presence of excess metal, the N-terminal domain is responsible for the formation of metal-bridged dimers while under aerobic conditions, a specific intermolecular disulfide is formed between the C-terminal domains. Both forms of dimers not only involve different domains for their intermolecular protein interactions, they also exhibit radical differences in the reactive properties of their respective cluster bound metal ions. Since the metal exchange within each domain is also influenced by interdomain interactions, the relative orientation of the domains is also most likely important for MT functions. Thus far, the relative orientation of the two domains could only be obtained from the crystal structure. Here, we present evidence for increased mobility in the linker region as the reason for the lack of interdomain constraints in the solution NMR studies of mammalian MTs.     

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.     

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.     

Heavy metal tolerance and accumulation in metallicolous and non-metallicolous populations of Thlaspi caerulescens from continental Europe

In continental Europe, the heavy metal hyperaccumulator Thlaspi caerulescens occurs both on heavy-metal polluted soils (subsp. calaminare) and on soils with normal heavy metal content (subsp. caerulescens). In order to assess the extent and partitioning of variation in heavy metal tolerance and foliar mineral composition, twelve families from two populations of each subspecies were grown in pots in four soil treatments differing in heavy metal (Zn, Pb) and macronutrient concentrations. The two subspecies differed systematically in many respects. Subsp. calaminare had a higher survival at high levels of heavy metals and a higher tolerance index in all treatments. It also had three times lower foliar zinc and lead concentrations when grown at moderate levels of heavy metals. This, together with a negative correlation of foliar Pb concentration with growth in subsp. caerulescens, suggests that heavy metal accumulation per se is not a mechanism of tolerance in this species. Variation among families within populations accounted for a larger proportion of total variance in growth and mineral composition than variation between populations. Additionally, within population variation in heavy metal tolerance and accumulation was significantly lower in subsp. calaminare. This suggests that, adding to a background constitutive tolerance at the species level, natural selection has increased heavy metal tolerance in metallicolous populations of Thlaspi caerulescens.     

In Appreciation

Marine animals can induce metallothioneins (MTs) in their responses to exposure to certain trace metals in the environment. MTs generally function as metal storage/detoxification or homeostatic regulation of both essential and non-essential metals. This review discusses the important roles of MTs in metal biokinetics other than metal detoxification and homeostasis in marine animals. Recent studies have revealed the complicated cellular and biochemical processes involving intracellular ligands (cytosolic proteins and insoluble deposits) during metal uptake and elimination. The responses of metal biokinetics to MT induction are metal- and organism-specific. Depending on the different marine animals and metals, all biokinetic parameters such as dissolved metal uptake rate, dietary assimilation efficiency and elimination (efflux) rate can be significantly impacted by MT induction. Among the different metal biokinetic parameters, dietary assimilation efficiency and elimination rate appear to be most impacted by MT induction. MT turnover kinetics can also significantly affect metal uptake kinetics, but again, such influence is also dependent on the organism, particularly its predominant pathway of metal detoxification. Even though the total MT pool in aquatic animals may remain constant, the turnover of MTs, involving MT synthesis and breakdown, can potentially lead to a major change of metal accumulation biokinetics. We propose several issues that need to be further addressed in studying the interaction between MT induction and metal accumulation biokinetics.     

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.     

Metallothionein, zinc, and mercury levels in tissues of young rats exposed to zinc and subsequently to mercury

Several studies have described mercury toxicity and the role of metallothioneins (MT) in the detoxification and regulation of metal homeostasis. However, little data exist on this topic during the specific post-natal developmental phase in young mammals. This developmental phase is particularly important since young animals are more sensitive to toxicants than adults. The objective of this work was to investigate whether MT participates in the mechanism of protection conferred by zinc pre-treatment on the toxic effects induced by mercury in neonate rats. Pups were exposed to ZnCl(2) (5 doses of 27 mg/kg/day, s.c.) and subsequently to HgCl(2) (5 doses of 5 mg/kg/day, s.c.); metal (Zn and Hg) and MT contents were analyzed in the liver, kidney, and blood. MT was induced in the liver and kidney of pups of both Zn-sal and Zn-Hg groups, although the greatest increase was in neonates exposed to Zn only. A direct relationship exists between MT and metals for both hepatic and renal tissues, which indicates that the increase in metal levels occurs in parallel to the increase in MT content. Although the heat-treated cytosolic fraction is rich in MT and metals, higher Zn and Hg contents were detected in the insoluble fraction of all tissues. These results suggest that MT is, at least in part, responsible for preventing Hg accumulation in the liver and blood and decreasing renal toxicity.     

Influence of the zinc hyperaccumulator Thlaspi caerulescens J. & C. Presl. and the nonmetal accumulator Trifolium pratense L. on soil microbial populations

Metal hyperaccumulator plants like Thlaspi caerulescens J. & C. Presl. are used for phytoremediation of contaminated soils. Since little is known about the rhizosphere of hyperaccumulators, the influence of T. caerulescens was compared with the effects of Trifolium pratense L. on soil microbes. High- and low-metal soils were collected near a zinc smelter in Palmerton, Penn. Soil pH was adjusted to 5.8 and 6.8 by the addition of Ca(OH)2. Liming increased bacterial populations and decreased metal toxicity to levels allowing growth of both plants. The effects of the plants on total (culturable) bacteria, total fungi, as well as cadmium- and zinc-resistant populations were assessed in nonrhizosphere and rhizosphere soil. Both plants increased microbial populations in rhizosphere soil compared with nonrhizosphere soil. Microbial populations were higher in soils planted with T. pratense, but higher ratios of metal-resistant bacteria were found in the presence of T. caerulescens. We hypothesize that T. caerutescens acidifies its rhizosphere. Soil acidification in the rhizosphere of T. caerulescens would affect metal uptake by increasing available metals around the roots and consequently, increase the selection for metal-resistant bacteria. Soil acidification may be part of the hyperaccumulation process enhancing metal uptake from soil.     

Organic acid complexation,heavy metal distribution and the effect of ATPase inhibition in hairy roots of hyperaccumulator plant species

Heavy metal uptake and distribution were investigated in hairy roots of the Cd hyperaccumulator, Thlaspi caerulescens, and the Ni hyperaccumulator, Alyssum bertolonii. Hairy roots of both species contained high constitutive levels of citric, malic and malonic acids. After treatment with 20 ppm Cd or 25 ppm Ni, about 13% of the total Cd in T. caerulescens roots and 28% of the total Ni in A. bertolonii were associated with organic acids. T. caerulescens and A. bertolonii hairy roots remained healthy and grew well at high concentrations of Cd and Ni, respectively, whereas hairy roots of the non-hyperaccumulator, Nicotiana tabacum, did not. Most of the Cd in T. caerulescens and N. tabacum roots was localised in the cell walls. In contrast, 85-95% of the Ni in A. bertolonii and N. tabacum was associated with the symplasm. Growth of T. caerulescens and A. bertolonii hairy roots was severely reduced in the presence of diethylstilbestrol (DES), an inhibitor of plasma membrane H(+)-ATPase. Treatment with DES increased the concentration of Cd in the symplasm of T. caerulescens about 6-fold with retention of root viability, whereas viability and Ni transport across the plasma membrane were both reduced in A. bertolonii. These results suggest that the mechanisms of Cd tolerance and hyperaccumulation in T. caerulescens hairy roots are capable of withstanding the effects of plasma membrane depolarisation, whereas Ni tolerance and hyperaccumulation in A. bertolonii hairy roots are not.     

Arabis gemmifera is a hyperaccumulator of Cd and Zn

Hyperaccumulators are essential for phytoremediation of heavy metals. In Europe and North America, many studies have been conducted to find more effective plants for phytoremediation of various pollutants. In Japan, this field of research has just recently come more into focus. A type of fern in Japan, Athyrium yokoscense, is well known as a hyperaccumulator of Cd and Zn. However, it is not suitable for phytoremediation because it is a summer green and grows slowly. Therefore, in order to find hyperaccumulators other than from A. yokoscense, we surveyed plants growing at polluted sites in Japan. We found that the Brassicae Arabis gemmifera is a hyperaccumulator of Cd and Zn, with phytoextraction capacities almost equal to Thlaspi caerulescens.     

Nickel translocation via the phloem in the hyperaccumulator Noccaea caerulescens (Brassicaceae)

Plant and Soil - This study evaluated the effect of phloem translocation on Ni accumulation in the hyperaccumulator Noccaea caerulescens. The first experiment assessed the metal and organic...     

Hyperaccumulation of cadmium by hairy roots of Thlaspi caerulescens

Hairy roots were used to investigate cadmium uptake by Thlaspi caerulescens, a metal hyperaccumulator plant with potential applications in phytoremediation and phytomining. Experiments were carried out in nutrient media under conditions supporting root growth. Accumulation of Cd in short-term (9-h) experiments varied with initial medium pH and increased after treating the roots with H(+)-ATPase inhibitor. The highest equilibrium Cd content measured in T. caerulescens roots was 62,800 microg g(-1) dry weight, or 6.3% dry weight, at a liquid Cd concentration of 3710 ppm. Cd levels in live T. caerulescens roots were 1.5- to 1.7-fold those in hairy roots of nonhyperaccumulator species exposed to the same Cd concentration, but similar to the Cd content of autoclaved T. caerulescens roots. The ability to grow at Cd concentrations of up to 100 ppm clearly distinguished T. caerulescens hairy roots from the nonhyperaccumulators. The specific growth rate of T. caerulescens roots was essentially unaffected by 20 to 50 ppm Cd in the culture medium; in contrast, N. tabacum roots turned dark brown at 20 ppm and growth was negligible. Up to 10,600 microg g(-1) dry weight Cd was accumulated by growing T. caerulescens hairy roots. Measurement of Cd levels in whole roots and in the cell wall fraction revealed significant differences in the responses of T. caerulescens and N. tabacum roots to 20 ppm Cd. Most metal was transported directly into the symplasm of N. tabacum roots within 3 days of exposure; in contrast, T. caerulescens roots stored virtually all of their Cd in the wall fraction for the first 7 to 10 days. This delay in transmembrane uptake may represent an important defensive strategy against Cd poisoning in T. caerulescens, allowing time for activation of intracellular mechanisms for heavy metal detoxification.     

Hyperaccumulation of metals by Thlaspi caerulescens as affected by root development and Cd-Zn/Ca-Mg interactions

The aim of this work was to study, in a rhizobox experiment, the phytoextraction of metals by the hyperaccumulator plant Thlaspi caerulescens in relation to the heterogeneity of metal pollution. Six treatments were designed with soils containing various levels of metals. Homogeneous soils and inclusions of soils in other soil matrices were prepared in order to vary metal concentration and localization. Growth parameters of the plant (rosette diameter and shoot biomass) and localization of roots and shoot uptake of Zn, Cd, Ca, and Mg were determined after 10 weeks of growth. The plants grown on the polluted industrial soils provided a larger biomass and had lower mortality rates than those grown on the agricultural soil. Moreover, these plants accumulated more Zn and Cd (up to 17,516 and 375 mg kg(-1) DM, respectively) than plants grown on the agricultural soil (up to 7300 mg Zn kg(-1) and 83 mg Cd kg(-1) DM). The roots preferentially explored metal-contaminated areas. The exploration of polluted soil inclusions by the roots was associated with a higher extraction of metals. Zinc and Cd in the shoots of Thlaspi caerulescens were negatively correlated with Ca and Mg concentrations; however, the soil supply for these two elements was identical. This suggests that there is competition for the uptake of these elements and that Zn is preferentially accumulated.