Phytoextraction of cadmium with Thlaspi caerulescens

The in situ phytoextraction of cadmium from soils can only be achieved using plants that are both tolerant to high Cd concentrations and able to extract sufficient amounts of the metal. However, very few plant species are capable of remediating Cd polluted soils in a reasonable time frame. This paper aims to show that the population of the hyperaccumulator Thlaspi caerulescens J. & C. Presl. from Viviez (south of France), which has a high Cd-accumulating capability, is an efficient tool to remove Cd from contaminated soils. Roots of T. caerulescensViviez proliferate in hot spots of metals in soils which is particularly advantageous because of heterogeneity of the distribution of metal in polluted soils. Isotopic techniques showed that plants from this population acquire Cd from the same pools as non-accumulating species, but that it was much more efficient than non-hyperaccumulators at removing the metal from the soil labile pool. This is due: to (i) a specific rooting strategy, and (ii) a high uptake rate resulting from the existence in this population of Cd-specific transport channels or carriers in the root membrane. Growth and overall extraction can be improved with appropriate N fertilisation, supplied either as mineral fertilisers or uncontaminated sewage sludge. Selecting bigger plants is possible from within a suitable Cd-accumulating population to improve the phytoextraction process. Growing the Cd-accumulating populations results in a reduction in the availability of Cd and Zn as shown with field and lysimeter experiments conducted for several years. As a result, on a practical aspect, Cd hyperaccumulating populations of T. caerulescens may be used as a tool to efficiently reduce the availability of Cd in soils, providing appropriate populations are used.     

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.     

Soil pH Effects on Uptake of Cd and Zn by Thlaspi caerulescens

For phytoextraction to be successful and viable in environmental remediation, strategies that can optimize plant uptake must be identified. Thlaspi caerulescens is an important hyperaccumulator of Cd and Zn, whether adjusting soil pH is an efficient way to enhance metal uptake by T. caerulescens must by clarified. This study used two soils differing in levels of Cd and Zn, which were adjusted to six different pH levels. Thlaspi caerulescens tissue metal concentrations and 0.1 M Sr(NO₃)₂ extractable soil metal concentrations were measured. The soluble metal form of both Cd and Zn was greatly increased with decreasing pH. Lowering pH significantly influenced plant metal uptake. For the high metal soil, highest plant biomass was at the lowest soil pH (4.74). The highest shoot metal concentration was at the second lowest pH (5.27). For low metal soil, due to low pH induced Al and Mn toxicity, both plant growth and metal uptake was greatest at intermediate pH levels. The extraordinary Cd phytoextraction ability of T. caerulescens was further demonstrated in this experiment. In the optimum pH treatments, Thlaspi caerulescens extracted 40% and 36% of total Cd in the low and high metal soils, respectively, with just one planting. Overall, decreasing pH is an effective strategy to enhance phytoextraction. But different soils had various responses to acidification treatment and a different optimum pH may exist. This pH should be identified to avoid unnecessarily extreme acidification of soils.     

Assessing the potential for zinc and cadmium phytoremediation with the hyperaccumulator Thlaspi caerulescens

Thlaspi caerulescens is a Zn and Cd hyperaccumulator, and has been tested for its phytoremediation potential. In this paper we examine the relationships between the concentrations of Zn and Cd in soil and in T. caerulescens shoots, and calculate the rates of Zn and Cd extraction from soil. Using published data from field surveys, field and pot experiments, we show that the concentrations of Zn and Cd in the shoots correlate with the concentrations of Zn and Cd in soils in a log-linear fashion over three orders of magnitude. There is little systematic difference between different populations of T. caerulescens in the relationship between soil and plant Zn concentrations. In contrast, populations from southern France are far superior to those from other regions in Cd accumulation. Bioaccumulation factors (plant to soil concentration ratio) for Zn and Cd decrease log-linearly with soil metal concentration. Model calculations show that phytoremediation using T. caerulescens is feasible when soil is only moderately contaminated with Zn and Cd, and the phytoremediation potential is better for Cd than for Zn if the populations from southern France are used. Recent progress in the understanding of the mechanisms of Zn and Cd uptake by T. caerulescens is also reviewed.     

Availability of cadmium and zinc accumulated in the leaves of Thlaspi caerulescens incorporated into soil

When grown on contaminated soil, hyperaccumulator plants contain high concentrations of metals which may return to the soil after senescence. This work was undertaken to assess the availability of Cd and Zn associated to the leaves of the hyperaccumulator Thlaspi caerulescens after incorporation into an uncontaminated soil. A Zn- and Cd- accumulator population of T. caerulescens was grown on a Cd- and Zn- contaminated soil previously labelled with ¹⁰⁹Cd. Leaves (TCL) were harvested, dried, ground and incorporated into the soil at a rate of 2.07 mg Cd kg⁻¹ and 51.9 mg Zn kg⁻¹. Then a pot experiment was conducted for 3 months with rye grass (Lolium perenne) and T. caerulescens. Rye grass was harvested monthly and T. caerulescens at the end of the experiment. Plant biomass was measured, along with the concentration of Cd, Zn and ¹⁰⁹Cd. Results showed that water-extractable metals in TCL were 69% for Zn and 33% for Cd. Addition of TCL to soil, depleted growth of rye grass, and improved that of T. caerulescens. At harvest, concentrations of both metals were increased in plants by TCL. Concentrations of Cd in rye grass increased with the cut number, while that of Zn decreased slightly. Rye grass extracted 1.6% of the total Cd and 0.9% of the total Zn, and T. caerulescens extracted up to 22.4% of the Cd and 7% of the Zn. About 94% of the Cd in rye grass and 86% in T. caerulescens was derived from TCL. In conclusion, metals associated with leaves of the hyperaccumulator T. caerulescens were very mobile after incorporation into the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gene Manipulation of a Heavy Metal Hyperaccumulator Species Thlaspi caerulescens L. via Agrobacterium-mediated Transformation

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species.     

Organic Acids Accumulation and Antioxidant Enzyme Activities in Thlaspi caerulescens under Zn and Cd Stress

Growth, organic acid and phytochelatin accumulation, as well as the activity of several antioxidative enzymes, i.e. superoxide dismutase (SOD), ascorbate peroxidase (APX) guaiacol peroxidase (POX) and catalase (CAT) were investigated under Zn and Cd stress in hydroponically growing plants of Thlaspi caerulescens population from Plombières, Belgium. Tissue Zn and Cd concentration increased (the highest concentration of both was in roots) as the concentration of these metals increased in the nutrient solution. Increasing Zn concentration enhanced plant growth, while with Cd it declined compared to the control. Both metals stimulated malate accumulation in shoots, Zn also caused citrate to increase. Zn did not induce phytochelatin (PC) accumulation. In plants exposed to Cd, PC concentration increased with increasing Cd concentration, but decreased with time of exposure. Under Zn stress SOD activity increased, but APX activity was higher at 500 and 1000 μM Zn and CAT activity only at 500 μM Zn in comparison with the control. CAT activity decreased in Cd- and Zn-stressed plants. The results suggest that relative to other populations, a T. caerulescens population from Plombières, when grown in hydroponics, was characterized by low Zn and Cd uptake and their translocation to shoots and tolerance to both metals. The accumulation of malate and citrate, but not PC accumulation was responsible for Zn tolerance. Cd tolerance seems to be due to neither PC production nor accumulation of organic acids.     

Identification of the form of Cd in the leaves of a superior Cd-accumulating ecotype of Thlaspi caerulescens using 113Cd-NMR

Thlaspi caerulescens (Ganges ecotype) is a known Cd hyperaccumulator, however, the ligands which coordinate to Cd ions in the leaves have not been identified. In the present study, the chemical form of Cd was investigated by using ¹¹³Cd-nuclear magnetic resonance (NMR) spectroscopy. Plants were grown hydroponically with a highly enriched ¹¹³Cd stable isotope. Measurements of ¹¹³Cd-NMR with intact leaves showed a signal at the chemical shift of around –16 ppm. Crude leaf sap also gave a similar chemical shift. Purification by gel filtration (Sephadex G-10), followed by cationic and anionic exchange chromatography, showed that Cd occurred only in the anionic fraction, which gave the same chemical shift as intact leaves. Further purification of the anionic fraction, combined with ¹¹³Cd- and ¹H-NMR studies, revealed that only the fraction containing malate showed a chemical shift similar to the intact leaves. These results indicate that Cd was coordinated mainly with malate in the leaves of T. caerulescens. The malate concentration in the leaves was not affected by increasing Cd concentration in the solution, suggesting that malate synthesis is not induced by Cd. Because the Cd-malate complex is relatively weak, we suggest that the complex forms inside the vacuoles as a result of an efficient tonoplast transport of Cd and a constitutively high concentration of malate in the vacuoles, and that the formation of the Cd-malate complex may lead to a decrease of subsequent Cd efflux to the cytoplasm.     

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.     

Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of<Emphasis Type="Italic"> Thlaspi caerulescens</Emphasis>

Thlaspi caerulescens (Ganges ecotype) is able to accumulate large concentrations of cadmium (Cd) and zinc (Zn) in the leaves without showing any toxicity, suggesting a strong internal detoxification. The distribution of Cd and Zn in the leaves was investigated in the present study. Although the Cd and Zn concentrations in the epidermal tissues were 2-fold higher than those of mesophyll tissues, 65–70% of total leaf Cd and Zn were distributed in the mesophyll tissues, suggesting that mesophyll is a major storage site of the two metals in the leaves. To examine the subcellular localisation of Cd and Zn in mesophyll tissues, protoplasts and vacuoles were isolated from plants exposed to 50 M Cd and Zn hydroponically. Pure protoplasts and vacuoles were obtained based on light-microscopic observation and the activities of marker enzymes of cytosol and vacuoles. Of the total Cd and Zn in the mesophyll tissues, 91% and 77%, respectively, were present in the protoplast, and all Cd and 91% Zn in the protoplast were localised in the vacuoles. Furthermore, about 70% and 86% of total Cd and Zn, respectively, in the leaves were extracted in the cell sap, suggesting that most Cd and Zn in the leaves is present in soluble form. These results indicate that internal detoxification of Cd and Zn in Thlaspi caerulescens leaves is achieved by vacuolar compartmentalisation.