Cadmium hyperaccumulation and genetic differentiation of Thlaspi caerulescens populations

Although the knowledge on heavy metal hyperaccumulation mechanisms is increasing, the genetic basis of cadmium (Cd) hyperaccumulation remains to be elucidated. Thlaspi caerulescens is an attractive model since Cd accumulation polymorphism observed in this species suggests genetic differences between populations with low versus high Cd hyperaccumulation capacities. In our study, a methodology is proposed to analyse at a regional scale the genetic differentiation of T. caerulescens natural populations in relation to Cd hyperaccumulation capacity while controlling for different environmental, soil, plant parameters and geographic origins of populations. Twenty-two populations were characterised with AFLP markers and cpDNA polymorphism. Over all loci, a partial Mantel test showed no significant genetic structure with regard to the Cd hyperaccumulation capacity. Nevertheless, when comparing the marker variation to a neutral model, seven AFLP fragments (9% of markers) were identified as presenting particularly high genetic differentiation between populations with low and high Cd hyperaccumulation capacity. Using simulations, the number of outlier loci was showed to be significantly higher than expected at random. These loci presented a genetic structure linked to Cd hyperaccumulation capacity independently of the geography, environment, soil parameters and Zn, Pb, Fe and Cu concentrations in plants. Using a canonical correspondence analysis, we identified three of them as particularly related to the Cd hyperaccumulation capacity. This study demonstrates that populations with low and high hyperaccumulation capacities can be significantly distinguished based on molecular data. Further investigations with candidate genes and mapped markers may allow identification and characterization of genomic regions linked to factors involved in Cd hyperaccumulation.     

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.     

Intraspecific variation of metal preference patterns for hyperaccumulation in Thlaspi caerulescens: evidence from binary metal exposures

Metal preferences with regard to accumulation were compared between populations of the heavy metal hyperaccumulator Thlaspi caerulescens, originating from calamine, serpentine and non-metalliferous soils. Plants were exposed for 3 weeks to factorial combinations of concentrations of different metals in binary mixture in hydroponics. The nature and degree of the interactions varied significantly between populations. In the calamine, non-Cd/Ni-hyperaccumulating population, La Calamine, there were no one-sided or mutual antagonistic interactions among the metals with regard to their accumulation in the plant. In three other populations capable of Cd and Ni hyperaccumulation, from calamine, serpentine and non-metalliferous soil respectively, there were one-sided or mutual antagonistic interactions between Cd and Zn, Cd and Ni, and Ni and Zn, possibly resulting from competition for transporters involved in uptake or plant-internal transport. Significant synergistic interactions, probably resulting from regulation of transporter expression, were also found, particularly in the La Calamine population. All the populations seemed to express a more or less Zn-specific high-affinity system. The serpentine and the non-metallicolous populations seemed to posses low-affinity systems with a preference for Cd and Zn over Ni, one of which may be responsible for the Ni hyperaccumulation of the serpentine population in its natural environment. The calamine population from Ganges also seemed to express a strongly Cd-specific high-affinity system which is in part responsible for the Cd-hyperaccumulation phenotype exhibited by this population in its natural environment.     

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.     

QTL analysis of cadmium and zinc accumulation in the heavy metal hyperaccumulator Thlaspi caerulescens

Thlaspi caerulescens (Tc; 2n = 14) is a natural Zn, Cd and Ni hyperaccumulator species belonging to the Brassicaceae family. It shares 88% DNA identity in the coding regions with Arabidopsis thaliana (At) (Rigola et al. 2006). Although the physiology of heavy metal (hyper)accumulation has been intensively studied, the molecular genetics are still largely unexplored. We address this topic by constructing a genetic map based on AFLP markers and expressed sequence tags (ESTs). To establish a genetic map, an F(2) population of 129 individuals was generated from a cross between a plant from a Pb/Cd/Zn-contaminated site near La Calamine, Belgium, and a plant from a comparable site near Ganges (GA), France. These two accessions show different degrees of Zn and, particularly, Cd accumulation. We analyzed 181 AFLP markers (of which 4 co-dominant) and 13 co-dominant EST sequences-based markers and mapped them to seven linkage groups (LGs), presumably corresponding to the seven chromosomes of T. caerulescens. The total length of the genetic map is 496 cM with an average density of one marker every 2.5 cM. This map was used for Quantitative Trait Locus (QTL) mapping in the F(2). For Zn as well as Cd concentration in root we mapped two QTLs. Three QTLs and one QTL were mapped for Zn and Cd concentration in shoot, respectively. These QTLs explain 23.8-60.4% of the total variance of the traits measured. We found only one common locus (LG6) for Zn and Cd (concentration in root) and one common locus for shoot and root concentrations of Zn (LG1) and of Cd (LG3). For all QTLs, the GA allele increased the trait value except for two QTLs for Zn accumulation in shoot (LG1 and LG4) and one for Zn concentration in root (LG1).     

Natural variation in cadmium tolerance and its relationship to metal hyperaccumulation for seven populations of Thlaspi caerulescens from western Europe

Thlaspi caerulescens J. & C. Presl is a distinctive metallophyte of central and western Europe that almost invariably hyperaccumulates Zn to> 1.0% of shoot dry biomass in its natural habitats, and can hyperaccumulate Ni to> 0.1% when growing on serpentine soils. Populations from the Ganges region of southern France also have a remarkable ability to accumulate Cd in their shoots to concentrations well in excess of 0.01% without apparent toxicity symptoms. Because hyperaccumulation of Cd appears to be highly variable in this species, the relationship between Cd tolerance and metal accumulation was investigated for seven contrasting populations of T. caerulescens grown under controlled conditions in solution culture. The populations varied considerably in average plant biomass (3.1‐fold), shoot : root ratio (2.2‐fold), Cd hyperaccumulation (3.5‐fold), shoot : root Cd‐concentration ratio (3.1‐fold), and shoot Cd : Zn ratio (2.6‐fold), but the degree of hyperaccumulation of Cd and Zn were strongly correlated. Two populations from the Ganges region were distinct in exhibiting high degrees of both Cd tolerance and hyperaccumulation (one requiring 3 µM Cd for optimal growth), whereas across the other five populations there was an inverse relationship between Cd tolerance and hyperaccumulation, as has been noted previously for Zn. Metal hyperaccumulation was negatively correlated with shoot : root ratio, which could account quantitatively for the differences between populations in shoot Zn (but not Cd) concentrations. On exposure to 30 µM Cd, the two Ganges populations showed marked reductions in shoot Zn and Fe concentrations, although Cd accumulation was not inhibited by elevated Zn; in the other five populations, 30 µM Cd had little or no effect on Zn or Fe accumulation but markedly reduced shoot Ca concentration. These results support a proposal that Cd is taken up predominantly via a high‐affinity uptake system for Fe in the Ganges populations, but via a lower‐affinity pathway for Ca in other populations. Total shoot Cd accumulated per plant was much more closely related to population Cd tolerance than Cd hyperaccumulation, indicating that metal tolerance may be the more important selection criterion in developing lines with greatest phytoremediation potential.     

Zinc and cadmium hyperaccumulation by Thlaspi caerulescens from metalliferous and nonmetalliferous sites in the Mediterranean area: implications for phytoremediation

Growth, tolerance and zinc and cadmium hyperaccumulation of Thlaspi caerulescens populations from three metal contaminated soils and three normal soils were compared under controlled conditions. Individuals of six populations were cultivated on five soils with increasing concentrations of zinc (50–25000 μg g⁻¹) and cadmium (1–170 μg g⁻¹). There was no mortality of normal soil populations in the four metal-contaminated soils, but plant growth was reduced to half that of populations from metal-contaminated soils. However, in noncontaminated soil, the growth of individuals from normal soils was greater than that of individuals from metal-contaminated soils. Individuals from normal soils concentrated three times more zinc in the aboveground biomass than those from metal-contaminated soils, but the latter accumulated twice as much cadmium. We conclude that populations of T. caerulescens from both normal and metal-contaminated soils are interesting material for phytoextraction of zinc and cadmium, but to optimize the process of phytoextraction it is necessary to combine the extraction potentials of both type of populations.     

Molecular mechanisms of heavy metal hyperaccumulation and phytoremediation

A relatively small group of hyperaccumulator plants is capable of sequestering heavy metals in their shoot tissues at high concentrations. In recent years, major scientific progress has been made in understanding the physiological mechanisms of metal uptake and transport in these plants. However, relatively little is known about the molecular bases of hyperaccumulation. In this paper, current progresses on understanding cellular/molecular mechanisms of metal tolerance/hyperaccumulation by plants are reviewed. The major processes involved in hyperaccumulation of trace metals from the soil to the shoots by hyperaccumulators include: (a) bioactivation of metals in the rhizosphere through root–microbe interaction; (b) enhanced uptake by metal transporters in the plasma membranes; (c) detoxification of metals by distributing to the apoplasts like binding to cell walls and chelation of metals in the cytoplasm with various ligands, such as phytochelatins, metallothioneins, metal-binding proteins; (d) sequestration of metals into the vacuole by tonoplast-located transporters. The growing application of molecular-genetic technologies led to the well understanding of mechanisms of heavy metal tolerance/accumulation in plants, and subsequently many transgenic plants with increased resistance and uptake of heavy metals were developed for the purpose of phytoremediation. Once the rate-limiting steps for uptake, translocation, and detoxification of metals in hyperaccumulating plants are identified, more informed construction of transgenic plants would result in improved applicability of the phytoremediation technology.     

Heavy metal uptake and chemical changes in the rhizosphere of Thlaspi caerulescens and Thlaspi ochroleucum grown in contaminated soils

Thlaspi caerulescens (J. and C. Presl) and Thlaspi ochroleucum (Boiss. ex Heldr) were grown in three different soils containing moderate to high amounts of heavy metals in a pot experiment, using a rhizobag technique. T. caerulescens accumulated significantly more Zn in the shoots than T. ochroleucum. The concentrations of Zn in the shoots of T. caerulescens ranged from 3100 to 8100 mg kg^-1 dry matter (DM), but only from 800 to 1600 mg kg^-1 DM in T. ochroleucum. Total uptake of Zn in the shoots of T. caerulescens was about 5 times that of T. ochroleucum. In contrast, the differences between the two species in the uptake of Cd, Cr, Cu, Ni and Pb were generally small. Concentrations of mobile Zn (extractable with 1M NH₄NO₃) in the rhizosphere and non-rhizosphere soils decreased considerably after growth of both plants, but the decreases were greater with T. caerulescens than with T. ochroleucum. The decreases in the mobile fraction accounted for less than 10% of the total Zn uptake by T. caerulescens indicating that this species was effective in mobilising Zn from less soluble fractions in the soils. The rhizosphere soils tended to have higher concentrations of mobile Zn than the non-rhizosphere soils, probably because of the lower pH in the rhizosphere. The pH in the rhizosphere soils was 0.2-0.4 units lower than that in the non-rhizosphere soils at the end of the experiment. However, there were no significant differences between the two species in the degree of rhizosphere acidification. The results suggest that T. caerulescens has potential for removing Zn from moderately to highly contaminated soils, but that this ability was not related to the pH changes in the rhizosphere.     

Impact of metal pollution and Thlaspi caerulescens growth on soil microbial communities

Soil microorganisms drive critical functions in plant-soil systems. As such, various microbial properties have been proposed as indicators of soil functioning, making them potentially useful in evaluating the recovery of polluted soils via phytoremediation strategies. To evaluate microbial responses to metal phytoextraction using hyperaccumulators, a microcosm experiment was carried out to study the impacts of Zn and/or Cd pollution and Thlaspi caerulescens growth on key soil microbial properties: basal respiration; substrate-induced respiration (SIR); bacterial community structure as assessed by PCR-denaturing gradient gel electrophoresis (DGGE); community sizes of total bacteria, ammonia-oxidizing bacteria, and chitin-degrading bacteria as assessed by quantitative PCR (Q-PCR); and functional gene distributions as determined by functional gene arrays (GeoChip). T. caerulescens proved to be suitable for Zn and Cd phytoextraction: shoots accumulated up to 8,211 and 1,763 mg kg(-1) (dry weight [DW]) of Zn and Cd, respectively. In general, Zn pollution led to decreased levels of basal respiration and ammonia-oxidizing bacteria, while T. caerulescens growth increased the values of substrate-induced respiration (SIR) and total bacteria. In soils polluted with 1,000 mg Zn kg(-1) and 250 mg Cd kg(-1) (DW), soil bacterial community profiles and the distribution of microbial functional genes were most affected by the presence of metals. Metal-polluted and planted soils had the highest percentage of unique genes detected via the GeoChip (35%). It was possible to track microbial responses to planting with T. caerulescens and to gain insight into the effects of metal pollution on soilborne microbial communities.     

Root-to-shoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in the metal hyperaccumulator Thlaspi caerulescens

Plant metal hyperaccumulator species are widely used as models to unravel the heavy metal tolerance and hyperaccumulation mechanisms. Thlaspi caerulescens is capable of tolerating and hyperaccumulating Zn, Cd, and Ni. A search for factors involved in the cellular tolerance to Ni, based on yeast screens, led to isolation of a cDNA encoding a functional nicotianamine (NA) synthase (NAS). The T. caerulescens genome appears to contain a single copy of the NAS gene named TcNAS whose expression is restricted to the leaves. The analysis of dose-response and time-course Ni treatments have revealed that the exposure to Ni triggers the accumulation of NA in the roots. Because neither TcNAS expression nor NAS activity were detected in the roots, the NA accumulation in roots is most probably the result of its translocation from the leaves. Once in the roots, NA, together with Ni, is subsequently found in the xylem, for redirection to the aerial parts. Using liquid chromatography coupled to inductively coupled plasma or electrospray ionization mass spectrometry, it has been shown that part of the Ni is translocated as a stable Ni-NA complex in the xylem sap. This circulation of NA, Ni, and NA-Ni chelates is absent in the non-tolerant non-hyperaccumulator related species T. arvense. Taken together, the results provide direct physiological and chemical evidence for NA and NA-heavy metal complex translocation in a hyperaccumulator species.     

Zinc tolerance and hyperaccumulation in F1 and F2 offspring from intra and interecotype crosses of Thlaspi caerulescens

The relationship between zinc (Zn) tolerance and hyperaccumulation in Thlaspi caerulescens was investigated from F1 and F2 crosses within and among metallicolous and nonmetallicolous Mediterranean populations. F1 offspring were grown on increasingly Zn-enriched soils to test Zn enrichment effects, and many families of F2 offspring were grown on a Zn-rich soil. Tolerance of F1 offspring depended on stress intensity. Tolerance of interecotype crosses was intermediate between that of the intraecotype crosses. No difference in Zn accumulation appeared among the F1 offspring from the three crosses involving metallicolous parents. Otherwise, none of these offspring exceeded the Zn hyperaccumulation threshold (10,000 mg kg(-1)), unlike the nonmetallicolous ones. The latter also showed the highest mortality. In some F2 families from interecotype crosses, hyperaccumulation values exceeded 15,000 mg kg(-1) in nontolerant offspring, whereas tolerant offspring displayed lower values (c. 10,000 mg kg(-1)). There was no difference between tolerant and nontolerant offspring when they showed low hyperaccumulation. Therefore, the relationship between tolerance and hyperaccumulation in F1 and F2 crosses depended on the hyperaccumulation level of plants.