Thlaspi caerulescens on nonmetalliferous soil in Luxembourg: ecological niche and genetic variation in mineral element composition

Forty-seven populations of Thlaspi caerulescens in Luxembourg were characterised for population size, soil mineral element composition and other habitat characteristics. Foliar concentrations of eight elements were assessed in 15 populations in the field and in eight populations cultivated in zinc (Zn)-cadmium (Cd)-nickel (Ni)-enriched soil. T. caerulescens favoured stony soil developed on steep, south-facing Emsian shale outcrops. All soil samples were nonmetalliferous. Soil pH ranged from 4.2 to 6.9. Field-growing plants had very high concentrations of heavy metals in the leaves (Zn, 3000-13 000 mg kg(-1); Cd, 11-44 mg kg(-1); Ni, 38-473 mg kg(-1)). Positive soil-plant correlations existed for Zn and Mn. In cultivation, significant genetic variation was found for biomass and six of eight mineral elements. For Cd and Zn, variation range among 48 half-sib families was two-fold (Cd, 183-334 mg kg(-1); Zn, 8030-16 295 mg kg(-1)). Most of the variation occurred among populations, consistent with the selfing mating system of those populations. There was a tight Zn-Cd genetic correlation (r = +0.83, P < 0.0001). The significance of the results to the conservation of T. caerulescens in Luxembourg is briefly discussed.     

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.     

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 accumulation in populations of Thlaspi caerulescens and Thlaspi goesingense

The capacity to accumulate cadmium (Cd) and zinc (Zn) was compared in Thlaspi goesingense and four populations of Thlaspi caerulescens . Two populations of T. caerulescens were grown in hydroponics at five concentrations of Cd. In addition, plants were grown in pots containing compost in which three different concentrations of Cd and two concentrations of Zn were added. A field trial was conducted to compare Zn and Cd uptake by three populations of T. caerulescens on nine selected plots of the Woburn Market Garden Experiment (UK) which had been contaminated to different degrees with heavy metals owing to past applications of sewage sludge. Results show that the four populations of T. caerulescens had the same ability to hyperaccumulate Zn but were significantly different in terms of Cd accumulation. Two populations of T. caerulescens from Southern France accumulated much more Cd than the populations from Prayon (Belgium) and Whitesike (UK). Generally, uptake of Cd was not decreased by increased concentrations of Zn in the substrate. These results indicate that the mechanisms of Cd and Zn hyperaccumulation are not identical in this species. This is the first report of hyperaccumulation of Cd by T. goesingense , but the growth of this species was markedly reduced by the large concentrations of Zn in the substrate. Future work should focus on the differences between Cd and Zn uptake in hyperaccumulator plants at the species and population level.     

Constitutively elevated salicylic acid signals glutathione-mediated nickel tolerance in Thlaspi nickel hyperaccumulators

Progress is being made in understanding the biochemical and molecular basis of nickel (Ni)/zinc (Zn) hyperaccumulation in Thlaspi; however, the molecular signaling pathways that control these mechanisms are not understood. We observed that elevated concentrations of salicylic acid (SA), a molecule known to be involved in signaling induced pathogen defense responses in plants, is a strong predictor of Ni hyperaccumulation in the six diverse Thlaspi species investigated, including the hyperaccumulators Thlaspi goesingense, Thlaspi rosulare, Thlaspi oxyceras, and Thlaspi caerulescens and the nonaccumulators Thlaspi arvense and Thlaspi perfoliatum. Furthermore, the SA metabolites phenylalanine, cinnamic acid, salicyloyl-glucose, and catechol are also elevated in the hyperaccumulator T. goesingense when compared to the nonaccumulators Arabidopsis (Arabidopsis thaliana) and T. arvense. Elevation of free SA levels in Arabidopsis, both genetically and by exogenous feeding, enhances the specific activity of serine acetyltransferase, leading to elevated glutathione and increased Ni resistance. Such SA-mediated Ni resistance in Arabidopsis phenocopies the glutathione-based Ni tolerance previously observed in Thlaspi, suggesting a biochemical linkage between SA and Ni tolerance in this genus. Intriguingly, the hyperaccumulator T. goesingense also shows enhanced sensitivity to the pathogen powdery mildew (Erysiphe cruciferarum) and fails to induce SA biosynthesis after infection. Nickel hyperaccumulation reverses this pathogen hypersensitivity, suggesting that the interaction between pathogen resistance and Ni tolerance and hyperaccumulation may have played a critical role in the evolution of metal hyperaccumulation in the Thlaspi genus.     

Using hyperaccumulator plants to phytoextract soil Ni and Cd

Two strategies of phytoextraction have been shown to have promise for practical soil remediation: domestication of natural hyperaccumulators and bioengineering plants with the genes that allow natural hyperaccumulators to achieve useful phytoextraction. Because different elements have different value, some can be phytomined for profit and others can be phytoremediated at lower cost than soil removal and replacement. Ni phytoextraction from contaminated or mineralized soils offers economic return greater than producing most crops, especially when considering the low fertility or phytotoxicity of Ni rich soils. Only soils that require remediation based on risk assessment will comprise the market for phytoremediation. Improved risk assessment has indicated that most Zn + Cd contaminated soils will not require Cd phytoextraction because the Zn limits practical risk from soil Cd. But rice and tobacco, and foods grown on soils with Cd contamination without corresponding 100-fold greater Zn contamination, allow Cd to readily enter food plants and diets. Clear evidence of human renal tubular dysfunction from soil Cd has only been obtained for subsistence rice farm families in Asia. Because of historic metal mining and smelting, Zn + Cd contaminated rice soils have been found in Japan, China, Korea, Vietnam and Thailand. Phytoextraction using southern France populations of Thlaspi caerulescens appears to be the only practical method to alleviate Cd risk without soil removal and replacement. The southern France plants accumulate 10-20-fold higher Cd in shoots than most T. caerulescens populations such as those from Belgium and the UK. Addition of fertilizers to maximize yield does not reduce Cd concentration in shoots; and soil management promotes annual Cd removal. The value of Cd in the plants is low, so the remediation service must pay the costs of Cd phytoextraction plus profits to the parties who conduct phytoextraction. Some other plants have been studied for Cd phytoextraction, but annual removals are much lower than the best T. caerulescens. Improved cultivars with higher yields and retaining this remarkable Cd phytoextraction potential are being bred using normal plant breeding techniques.     

Testing of outstanding individuals of Thlaspi caerulescens for cadmium phytoextraction

Thlaspi caerulescens is known to hyperaccumulate high quantities of Cd with Cd concentrations up to 3000 mg kg(-1) in some populations from south of France. However, within these populations, the Cd concentrations can vary widely from plant to plant in a way that appears to be not entirely due to variations in soil Cd. The aim of this work was to investigate the variability in the Cd uptake ability of individual plants within a population and among seedlings grown from seeds from a single plant. Ten populations of T. caerulescens plants were selected from four locations (V: Viviez; SF: Saint Félix-de-Pallières; LB: Le Bleymard; CMA: Col du Mas de l'Air) depending of the extent and soil homogeneity of the site. One population from CMA consisted of the progeny of a single maternal plant. Hundred plants of each population were grown for three months in the same homogeneous and lightly Cd-polluted soil (about 20 mg total Cd kg(-1) dry soil). Cadmium uptake behavior of the plants was monitored by labeling the soil with 109Cd. To allow partial plant destruction, radioanalysis was performed on the largest leaf of each plant as an indicator of the total Cd concentration in plant shoots. Results showed significant differences in biomass production and Cd uptake by T. caerulescens between sites and between populations within sites. We observed a wide intra-population variation in biomass, Cd concentration and total Cd uptake. For these properties, 1 to 5 percents of the plants in each population varied by more than a factor of two from the mean values. The mean Cd uptake by the single-plant population from CMA was more than 40% higher than for the population at large. T. caerulescens would respond to traditional selection methods, which would significantly improve the phytoextraction of Cd.     

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.     

Life history traits of the pseudometallophyte Thlaspi caerulescens in natural populations from Northern Europe

We examined recruitment, survival, life cycle and fecundity of two metallicolous (M, on metalliferous calamine soils) and two non-metallicolous (NM, on normal soils) populations of Thlaspi caerulescens in Belgium and Luxemburg. In each population, permanent plots were monitored over two reproductive seasons. In M populations, plots were located in two contrasting environments (grass versus grove) in order to test the influence of vegetation cover on life strategy. Our results show that the monocarpic life cycle is dominant in all populations of T. caerulescens. However the length of the pre-reproductive period varies from several months (winter annuals) to 1 year or more (perennials), and is partly related to plant origin (M versus NM). Most plants growing in metalliferous environments were annuals, whereas NM plants were mostly perennials. These differences in life cycle were related to differences in survival during summer, which was better in NM than in M populations. Within each M population, different survival conditions and life cycles were observed according to vegetation cover. Plants growing in grass areas were mostly annuals and had a low survival rate in summer whereas grove plants were mostly perennials and survived better in summer. Our results suggest the selection of stress avoiders (shortening of life cycle) in M populations of T. caerulescens but only for individuals growing in grass areas. Summer survival seems to play a key role in selection of life strategy in T. caerulescens.     

Multivariate analysis of protein profiles of metal hyperaccumulator Thlaspi caerulescens accessions

Thlaspi caerulescens is increasingly acknowledged as one of the best models for studying metal hyperaccumulation in plants. In order to study the mechanisms underlying metal hyperaccumulation, we used proteomic profiling to identify differences in protein intensities among three T. caerulescens accessions with pronounced differences in tolerance, uptake and root to shoot translocation of Zn and Cd. Proteins were separated using two-dimensional electrophoresis and stained with SYPRO Orange. Intensity values and quality scores were obtained for each spot by using PDQuest software. Principal component analysis was used to test the separation of the protein profiles of the three plant accessions at various metal exposures, and to detect groups of proteins responsible for the differences. Spot sets representing individual proteins were analysed with the analysis of variance and non-parametric Kruskal-Wallis test. Clearest differences were seen among the Thlaspi accessions, while the effects of metal exposures were less pronounced. The 48 tentatively identified spots represent core metabolic functions (e.g. photosynthesis, nitrogen assimilation, carbohydrate metabolism) as well as putative signalling and regulatory functions. The possible roles of some of the proteins in heavy metal accumulation and tolerance are discussed.     

Cloning of three ZIP/Nramp transporter genes from a Ni hyperaccumulator plant Thlaspi japonicum and their Ni2+-transport abilities.

Ni homeostasis is essential for plant cell activity, but the mechanisms of Ni-transport and delivery are unknown. To elucidate the role of ZIP and NRAMP metal-transporters for Ni2+-transport and homeostasis, we cloned their homologous genes from the Ni hyperaccumulator Thlaspi japonicum, and investigated their Ni-transporting abilities by expression in yeast. The deduced amino acid sequences of the two Zip transporter genes (TjZnt1, TjZnt2) and one Nramp transporter gene cloned had high homologies with TcZNT1 and TcZNT2 of Thlaspi caerulescens and AtNRAMP4 of Arabidopsis thaliana, respectively, and were predicted as integral membrane proteins with 6 or 12 transmembrane domains. TjZNT1 and TjZNT2 had two long histidine-rich domains in the putative cytoplasmic domain between transmembrane domains III and IV. TjNRAMP4 conserved a consensus transporter motif between transmembrane domains VIII and IX. The yeast transformed with TjZNT1 or TjZNT2 showed a marked increase in Ni2+ tolerance with the gene expression. In contrast, the expression of TjNramp4 caused elevation of Ni2+ sensitivity and Ni2+ concentration. These data suggest that ZIP/NRAMP transporters participate in Ni2+ homeostasis of Ni hyperaccumulator plants. TjZNT1 had Zn2+-, Cd2+- and Mn2+-transporting abilities and TjZNT2 also had Zn2+- and Mn2+-transporting abilities, but TjNRAMP4 could transport Ni2+ but not Zn2+, Cd2+ or Mn2+.     

Plant Cd2+ and Zn2+ status effects on root and shoot heavy metal accumulation in Thlaspi caerulescens

* In this study we address the impact of changes in plant heavy metal, (i.e. zinc (Zn) and cadmium (Cd)) status on metal accumulation in the Zn/Cd hyperaccumulator, Thlaspi caerulescens. * Thlaspi caerulescens plants were grown hydroponically on both high and low Zn and Cd regimes and whole-shoot and -root metal accumulation, and root (109)Cd(2+) influx were determined. * High-Zn-grown (500 microm Zn) plants were found to be more Cd-tolerant than plants grown in standard Zn conditions (1 microm Zn). Furthermore, shoot Cd accumulation was significantly greater in the high-Zn-grown plants. A positive correlation was also found between shoot Zn accumulation and increased plant Cd status. Radiotracer (109)Cd root flux experiments demonstrated that high-Zn-grown plants maintained significantly higher root Cd(2+) influx than plants grown on 1 microm Zn. It was also found that both nickel (Ni) and copper (Cu) shoot accumulation were stimulated by high plant Zn status, while manganese (Mn) accumulation was not affected. * A speculative model is presented to explain these findings, suggesting that xylem loading may be one of the key sites responsible for the hyperaccumulation of Zn and Cd accumulation in Thlaspi caerulescens.     

No phytochelatin (PC2 and PC3) detected in Salix viminalis

Phytochelatins (PCs) have been detected in a large range of plant species, but their role in heavy-metal tolerance is unclear. Various clones of Salix viminalis are differently tolerant to heavy metals, and the aim of this work was to investigate whether PCs were differently expressed in tolerant compared with sensitive clones. In a long-term study, five clones with high or low metal tolerance were cultivated 21 days without or with Cd (1 or 10 µ M ), Cu (0.3 or 7 µ M ), Ni (15 µ M ), Pb (7 µ M ) or Zn (10 or 100 µ M ). Controls were further used in a short-term study where Cd (1 µ M ) was added and samples were collected 0, 15 and 30 min, 1, 3 and 24 h after start of treatment. PCs were analysed on high performance liquid chromatography (HPLC) using two different methods: post-column derivatization using Ellmans reagent and pre-derivatization with monobromobimane. Thlaspi caerulescens treated with Cd was used as internal PC standard. No PCs could be detected in Salix with either of the two methods in any of the treatments: different clones, metals, concentrations, plant parts or treatment time. The 16 thiol peaks shown were the same in both control and treated plants. Both HPLC methods showed PC peaks when Thlaspi was used but these peaks could not be associated with any of the 16 peaks. The amino acid composition of the 16 peaks was not the expected composition of that of PCs. Thus, Salix viminalis have no detectable levels of PCs, which in turn are not involved in heavy metal tolerance in Salix .     

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.     

The potential of Thlaspi caerulescens for phytoremediation of contaminated soils

Uptake of Cd, Zn, Pb and Mn by the hyperaccumulator Thlaspi caerulescens was studied by pot trials in plant growth units and in populations of wild plants growing over Pb/Zn base-metal mine wastes at Les Malines in the south of France. The pot trials utilised metal-contaminated soils from Auby in the Lille area. Zinc and Cd concentrations in wild plants averaged 1.16% and 0.16% (dry weight) respectively. The unfertilised biomass of the plants was 2.6 t/ha. A single fertilised crop with the above metal content could remove 60 kg of Zn and 8.4 kg Cd per hectare. Experiments with pot-grown and wild plants showed that metal concentrations (dry weight basis) were up to 1% Zn (4% Zn in the soil) and just over 0.1% Cd (0.02% Cd in the soil). The metal content of the plants was correlated strongly with the plant-available fraction in the soils as measured by extraction with ammonium acetate and was inversely correlated with pH. Bioaccumulation coefficients (plant/soil metal concentration quotients) were in general higher for Cd than for Zn except at low metal concentrations in the soil. There was a tendency for these coefficients to increase with decreasing metal concentrations in the soil. It is proposed that phytoremediation using Thlaspi caerulescens would be entirely feasible for low levels of Cd where only a single crop would be needed to halve a Cd content of 10 g/g in the soil. It will never be possible to remediate elevated Zn concentrations within an economic time frame (<10 yr) because of the lower bioaccumulation coefficient for this element coupled with the much higher Zn content of the soils.     

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.