Solubility of zinc and interactions between zinc and phosphorus in the hyperaccumulator Thlaspi caerulescens

The relationship between Zn and P in the Zn hyperaccumulator Thlaspi caerulescens J. & C. Presl was investigated using hydroponic culture. Total concentrations of Zn in the shoots increased from 0·2 to 27 g kg^–1 dry mass when solution Zn increased from 1 to 1000 mmol m^–3. Water-soluble Zn accounted for > 80% of the total Zn in the shoots containing > 5 g Zn kg^–1 dry mass. Total P was maintained at about 3 g kg^–1 dry mass in the shoots containing < 20 g Zn kg^–1 dry mass, but significantly decreased with higher Zn concentrations. Linear regression between insoluble P and insoluble Zn in the shoots produced a small slope, suggesting that co-precipitation of Zn and P was not an important detoxification mechanism in the shoots. In contrast, there was a strong correlation between insoluble P and insoluble Zn in the roots, with a linear slope of 0·3 — close to the P:Zn ratio in Zn₃(PO₄)₂. Foliar sprays of phosphate did not affect shoot dry mass significantly, but decreased root length and root dry mass significantly at Zn concentrations in solution from 10 to 3000 mmol m^–3. Foliar P was translocated to roots to enhance co-precipitation of Zn and P, although this did not enhance Zn tolerance. The results suggest that T.caerulescens possesses mechanisms which allow it to accumulate and sequester huge amounts of Zn in the shoots without causing P deficiency.     

Uptake and transport of zinc in the hyperaccumulator Thlaspi caerulescens and the non-hyperaccumulator Thlaspi ochroleucum

Growth and zinc uptake of the hyperaccumulator species Thlaspi caerulescens J. & C. Presl and the non-hyperaccumulator species Thlaspi ochroleucum Boiss. & Heldr. were compared in solution culture experiments. T. caerulescens was able to tolerate 500 mmol m^?3 (32.5 g m^?3) Zn in solution without growth reduction, and up to 1000 mmol m^?3 (65 g m^?3) Zn without showing visible toxic symptoms but with a 25% decrease in dry matter (DM) yield. Up to 28 g kg^?1 of Zn in shoot DM was obtained in healthy plants of T. caerulescens. In contrast, T. ochroleucum suffered severe phytotoxicity at 500 mmol m^?3 Zn. Marked differences were shown in Zn uptake, distribution and redistribution between the two species. T. caerulescens had much higher concentrations of Zn in the shoots, whereas T. ochroleucum accumulated higher concentrations of Zn in the roots. When an external supply of 500 mmol m^?3 Zn was withheld, 89% of the Zn accumulated previously in the roots of T. caerulescens was transported to the shoots over a 33 d period, whereas in T. ochroleucum only 32% was transported. T. caerulescens was shown to have a greater internal requirement for Zn than other plants. Increasing the supply of Zn from 1 to 10 mmol m^?3 gave a 19% increase in the total DM of this species. liven the shoots from the 1 mmol m^?3 Zn treatment which showed Zn deficiency contained 10 times greater Zn concentrations than the widely reported critical value for Zn deficiency to occur in many other plant species. The results obtained suggest that strongly expressed constitutive sequestration mechanisms exist in the hyperaccumulator T. caerulescens, which detoxify the large amount of Zn present in shoot tissues and decrease its physiological availability in the cytosol. Both T. caerulescens and T. ochroleucum had constitutively high concentrations of malate in shoots, which were little affected by different Zn treatments. Although malate may play a role in Zn chelation because of the high concentrations present, it cannot explain the species specificity of Zn tolerance and hyperaccumulation.     

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.     

Zinc Phytoextraction in Thlaspi caerulescens

The existence of metal hyperaccumulator species demonstrates that plants have the genetic potential to remove toxic metals from contaminated soil. Possibly, one of the best-known hyperaccumulators is Thlaspi caerulescens. This species has been shown to accumulate very high Zn concentrations without manifesting any sign of toxicity. Thus, T. caerulescens represents an excellent experimental system for studying metal hyperaccumulation in plants as it relates to phytoremediation. In this article, we review the results of an investigation into the physiology, biochemistry, and molecular regulation of Zn transport and accumulation in T. caerulescens compared with a nonaccumulator relative T. arvense. Physiological studies focused on the use of ⁶⁵Zn radiotracer flux techniques to characterize zinc transport and compartmentation in the root, and translocation to the shoot. Transport studies indicated that a number of Zn transport sites were stimulated in T. caerulescens, contributing to the hyperaccumulation trait. Thus, Zn influx into root and leaf cells, and Zn loading into the xylem was greater in T. caerulescens compared with the nonaccumulator T. arvense. The 4.5-fold stimulation of Zn influx into the roots of T. caerulescens was hypothesized to be due to an overexpression of Zn transporters in this species. Additionally, compartmental analysis (radiotracer wash out or efflux techniques) was used to show that Zn was sequestered in the root vacuole of T. arvense inhibiting Zn translocation to the shoot in this nonaccumulator species. Molecular studies focused on the cloning and characterization of Zn transport genes in T. caerulescens. Functional complementation of a yeast Zn transport-defective mutant with a T. caerulescens cDNA library constructed in a yeast expression vector resulted in the cloning of a Zn transport cDNA, ZNT1. Expression of ZNT1 in yeast allowed for a physiological characterization of this transporter. ZNT1 was shown to encode a high-affinity Zn transporter that can also mediate low-affinity Cd transport. Biochemical analyses indicated that enhanced Zn transport in T. caerulescens results from a constitutively high expression of ZNT1 in roots and shoots. These results suggest that overexpression of ZNT1 may be linked to an alteration of the Zn tolerance mechanism in this 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.     

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.     

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.     

Cellular Compartmentation of Zinc in Leaves of the Hyperaccumulator Thlaspi caerulescens

Cellular compartmentation of Zn in the leaves of the hyperaccumulator Thlaspi caerulescens was investigated using energy-dispersive x-ray microanalysis and single-cell sap extraction. Energy-dispersive x-ray microanalysis of frozen, hydrated leaf tissues showed greatly enhanced Zn accumulation in the epidermis compared with the mesophyll cells. The relative Zn concentration in the epidermal cells correlated linearly with cell length in both young and mature leaves, suggesting that vacuolation of epidermal cells may promote the preferential Zn accumulation. The results from single-cell sap sampling showed that the Zn concentrations in the epidermal vacuolar sap were 5 to 6.5 times higher than those in the mesophyll sap and reached an average of 385 mm in plants with 20,000 μg Zn g⁻¹ dry weight of shoots. Even when the growth medium contained no elevated Zn, preferential Zn accumulation in the epidermal vacuoles was still evident. The concentrations of K, Cl, P, and Ca in the epidermal sap generally decreased with increasing Zn. There was no evidence of association of Zn with either P or S. The present study demonstrates that Zn is sequestered in a soluble form predominantly in the epidermal vacuoles in T. caerulescens leaves and that mesophyll cells are able to tolerate up to at least 60 mm Zn in their sap.Different mechanisms have been proposed to explain the tolerance of plants to toxic heavy metals (Baker and Walker, 1990; Verkleij and Schat, 1990). Some tolerant plant species, the so-called “excluders,” use exclusion mechanisms by which uptake and/or root-to-shoot transport of heavy metals are restricted. Other tolerant plant species are able to cope with elevated concentrations of toxic metals inside of their tissues through production of metal-binding compounds, cellular and subcellular compartmentation, or alterations of metabolism.An extreme strategy for metal tolerance that is in sharp contrast to metal exclusion is “hyperaccumulation,” a term that was originally used by Brooks et al. (1977) to describe plants that can accumulate more than 1,000 μg Ni g⁻¹ dry weight in their aerial parts. Approximately 400 taxa of terrestrial plants have been identified as hyperaccumulators of various heavy metals, with about 300 being Ni hyperaccumulators (Baker and Brooks 1989; Brooks, 1998). Only 16 species of Zn hyperaccumulators, which are defined as being able to accumulate more than 10,000 μg Zn g⁻¹ in the aboveground parts on a dry weight basis in their natural habitat (Brooks, 1998), have been reported. Thlaspi caerulescens J. & C. Presl (Brassicaceae) is the best-known example of a Zn/Cd hyperaccumulator. Under hydroponic culture conditions T. caerulescens can accumulate up to 25,000 to 30,000 μg Zn g⁻¹ dry weight in the shoots without showing any toxicity symptoms or reduction in growth (Brown et al., 1996a; Shen et al., 1997). Recently, there has been a surge of interest in the phenomenon of heavy-metal hyperaccumulation because this property may be exploited in the remediation of heavy-metal-polluted soils through phytoextraction and phytomining (McGrath et al., 1993; Brown et al., 1995b; Robinson et al., 1997).The mechanisms for metal hyperaccumulation are not fully understood, and this is particularly true in the case of the Zn/Cd hyperaccumulators. To cope with the consequence of hyperaccumulation, plants must also be hypertolerant to the heavy metals that accumulate. Recent studies comparing the different populations of T. caerulescens have shown that hyperaccumulation of Zn is a constitutive property, although the traits are probably separate from those for tolerance (Baker et al., 1994; Meerts and Van Isacker, 1997). Compared with the nonaccumulating species, T. caerulescens possesses an enhanced capacity to take up Zn and transport it from roots to shoots (Baker et al., 1994; Brown et al., 1995a; Shen et al., 1997). Lasat et al. (1996) found that roots of T. caerulescens and the nonaccumulator Thlaspi arvense had similar apparent K_m values for Zn²⁺, but that the V_max in the former was 4.5-fold higher than that in the latter species, indicating that the hyperaccumulator T. caerulescens possessed more Zn²⁺-transport sites in the plasma membranes of root cells. Shen et al. (1997) showed that T. caerulescens was much more effective in exporting the Zn that was accumulated previously in roots to the shoots than an intermediate accumulator species, Thlaspi ochrolucum. Organic acids such as malic acid have been suggested to play a key role in shuttling Zn from cytoplasm to vacuoles (Mathys, 1977). However, the low affinity of malate to chelate Zn (stability constant pK = 3.5 at infinite dilution) does not favor this hypothesis. Moreover, high concentrations of malate found in the shoot tissues of T. caerulescens appear to be a constitutive property (Tolrà et al., 1996; Shen et al., 1997).The extraordinary tolerance of hyperaccumulator plants must also involve compartmentation of toxic metals at the cellular and subcellular levels. Vázquez et al. (1992, 1994) studied localization of Zn in the root and leaf tissues of T. caerulescens using EDXMA. They compared two methods of sample preparation and found that Na₂S fixation was not suitable for preventing the loss of metal ions from the samples. Using cryofixation and freeze substitution, they showed that Zn accumulated mainly in the vacuoles as electron-dense deposits. Many vacuoles of leaf-epidermal and subepidermal cells contained globular crystals that were very rich in Zn. However, it is not known whether the Zn-rich, globular crystal deposits occur inside of the leaf vacuoles in vivo or if they are artifacts caused by sample preparation. Also, the technique used by Vázquez et al. (1992, 1994) allows only semiquantitative determination of Zn concentrations.In this study we used two techniques to investigate cellular compartmentation of Zn in the leaves of T. caerulescens. The first utilized EDXMA of frozen, hydrated tissue to survey the distribution patterns of Zn and other elements across different leaf cells. The second method involved sampling sap from single cells using microcapillaries, followed by fully quantitative determination of Zn and other elements using EDXMA.     

Cadmium hyperaccumulation and reproductive traits in natural Thlaspi caerulescens populations

During the last decade, the metal hyperaccumulating plants have attracted considerable attention because of their potential use in decontamination of heavy metal contaminated soils. However, in most species, little is known regarding the function, the ecological and the evolutionary significances of hyperaccumulation. In our study, we investigated the parameters influencing the Cd concentration in plants as well as the biological implications of Cd hyperaccumulation in nine natural populations of Thlaspi caerulescens. First, we showed that Cd concentration in the plant was positively correlated with plant Zn, Fe, and Cu concentrations. This suggested that the physiological and/or molecular mechanisms for uptake, transport and/or accumulation of these four heavy metals interact with each other. Second, we specified a measure of Cd hyperaccumulation capacity by populations and showed that T. caerulescens plants originating from populations with high Cd hyperaccumulation capacity had better growth, by developing more and bigger leaves, taller stems, and produced more fruits and heavier seeds. These results suggest a tolerance/disposal role of Cd hyperaccumulation in this species.     

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.