Compartmentation of Zinc in Roots and Leaves of the Zinc Hyperaccumulator Thlaspi caerulescens J & C Presl

Thlaspi caerulescens is a metallophyte that is able to hyperaccumulate Zn. In the present study the subcellular compartmentation of Zn was investigated in roots and leaves of this species by means of X-ray microanalysis. Leaves accumulated higher average Zn concentrations than roots. In roots of plants exposed to 10 μM Zn, Zn concentrations in the apoplast were similar to those in vacuoles, while in plants treated with 100 μM Zn considerably higher Zn concentrations were detected in vacuoles than in the apoplast. In epidermal and sub-epidermal cells of leaves of plants from both treatments, Zn mainly accumulated in vacuoles and, to a lesser extent, in the apoplast. In vacuoles from plants exposed to 100 μM Zn, high Zn concentrations were associated with variable amounts of P, Ca and K. In leaves, the highest Zn concentrations (13,600 μg g^?1 d.m.) were found in globular crystals present in many vacuoles of epidermal and subepidermal cells. Smaller deposits with a variable Zn concentration between 1,000 and 18,300 μg g^?1 d.m. were observed in the epidermal and subepidermal cells of roots. Both the high Zn/P element ratios found in the crystals and the absence of Mg indicate that, in contrast to other plant species, myo-inositol hexaphosphate (phytate) is not the main storage form for Zn in Thlaspi caerulescens.     

Sub-cellular localization of Ni in the hyperaccumulator, Hybanthus floribundus (Lindley) F. Muell

The cellular and subcellular distribution of Ni within leaves of Hybanthus floribundus (Lindley) F. Muell, a hyperaccumulator of Ni, was investigated at relatively high spatial resolution using energy‐dispersive X‐ray microanalysis (EDAX). Elemental distribution maps showed that Ni was predominantly localized in the vacuoles of epidermal cells in the leaves. Quantification of Ni revealed concentrations up to 275 mmol kg^?1 (embedded tissue) in some epidermal vacuoles. The accumulation of Ni in these cells was associated with a decrease in the concentration of Na and K. There was no indication that Ni was associated with P, S or Cl in the vacuoles. Ni was also concentrated on the outside of cell walls throughout the leaves, indicating that apoplastic compartmentation is also involved in Ni tolerance and accumulation in this plant.     

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.     

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.     

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J & C. Presl) F.K. Meyer

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state‐of‐the‐art quantitative 2D liquid chromatography/tandem mass spectrometry (2D‐LC‐MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size‐exclusion chromatography/inductively coupled plasma mass spectrometer (SEC‐ICP‐MS) method, in order to identify the Zn‐binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S‐transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn–nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn–nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type‐specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.     

Extracellular complexation of Cd in the Hartig net and cytosolic Zn sequestration in the fungal mantle of Picea abies – Hebeloma crustuliniforme ectomycorrhizas

Compartmentation of heavy metals on or within mycorrhizal fungi may serve as a protective function for the roots of forest trees growing in soils containing elevated concentrations of metals such as Cd and Zn. In this paper we present the first quantitative measurements by X‐ray microanalysis of heavy metals in high‐pressure frozen and cryosectioned ectomycorrhizal fungal hyphae. We used this technique to analyse the main sites of Cd and Zn in fungal cells of mantle and Hartig net hyphae and in cortical root cells of symbiotic Picea abies – Hebeloma crustuliniforme associations to gain new insights into the mechanisms of detoxification of these two metals in Norway spruce seedlings. The mycorrhizal seedlings were exposed in growth pouches to either 1 mM Cd or 2 mM Zn for 5 weeks. The microanalytical data revealed that two distinct Cd‐ and Zn‐binding mechanisms are involved in cellular compartmentation of Cd and Zn in the mycobiont. Whereas extracellular complexation of Cd occurred predominantly in the Hartig net hyphae, both extracellular complexation and cytosolic sequestration of Zn occurred in the fungal tissue. The vacuoles were presumed not to be a significant pool for Cd and Zn storage. Cadmium was almost exclusively localized in the cell walls of the Hartig net (up to 161 mmol kg ^{? 1} DW) compared with significantly lower concentrations in the cell walls of mantle hyphae (22 mmol kg ^{? 1} DW) and in the cell walls of cortical cells (15 mmol kg ^{? 1} DW). This suggests that the apoplast of the Hartig net is a primary accumulation site for Cd. Zinc accumulated mainly in the cell walls of the mantle hyphae (111 mmol kg ^{? 1} DW), the Hartig net hyphae (130 mmol kg ^{? 1} DW) and the cortical cells (152 mmol kg ^{? 1} DW). In addition, Zn occurred in high concentrations in the cytoplasm of the fungal mantle hyphae (up to 164 mmol kg ^{? 1} DW) suggesting that both the cell walls and the cytoplasm of fungal tissue are the main accumulation sites for Zn in P. abies resulting in decreased Zn transfer from the fungus to the root.     

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.     

Physiological responses to Cd and Zn in two Cd/Zn hyperaccumulating Thlaspi species

In a model hyperaccumulation study a Cd/Zn hyperaccumulator Thlaspi caerulescens accession Ganges and a recently reported Cd/Zn hyperaccumulator Thlaspi praecox grown in increasing Cd and Zn concentrations in the substrate and in field collected polluted soil were compared. Plant biomass, concentrations of Cd and Zn, total chlorophylls and anthocyanins, antioxidative stress parameters and activities of selected antioxidative enzymes were compared. Increasing Cd, but not Zn in the substrate resulted in the increase of biomass of roots and shoots of T. praecox and T. caerulescens. The two species hyperaccumulated Cd in the shoots to a similar extent, whereas T. caerulescens accumulated more Zn in the shoots than T. praecox. Cadmium amendment decreased total chlorophyll concentration and glutathione reductase activity, and increased non-protein thiols concentration only in T. praecox, suggesting that it is less tolerant to Cd than T. caerulescens. In the field-contaminated soil, T. caerulescens accumulated higher Cd concentrations; but as T. praecox produced higher biomass, both species have similar ability to extract Cd.     

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.     

Element localization in ultrathin cryosections of high-pressure frozen ectomycorrhizal spruce roots

We present, for the first time, elemental mapping of ultra-thin cryosections from high-pressure frozen ectomycorrhizal roots of Picea abies–Hebeloma crustuliniforme. The maps provide interpretable information on the relationship between elements and the structure of inhomogeneous objects. Cryoultramicrotomy together with energy dispersive X-ray microanalysis (EDX) offers the potential to study the subcellular localization of specific ions and ecologically important tracers (Cs and Sr) in ectomycorrhizal roots under conditions resembling the natural slate as closely as possible. Structural changes of the ectomycorrhizal roots, in particular the absence of a Hartig net at high NH₄⁺ levels in the nutrient solution, were accompanied by elemental modification of Ca in cortical cell walls, where markedly higher concentrations of Ca were found. Cs and Sr applied to the nutrient solution were localized in root and fungal cells of the Hartig net. Cs accumulated mainly in the vacuoles of the Hartig net hyphae and its distribution was very similar to the distribution of K. In contrast to Cs, Sr was found to occur mainly in electron-opaque and P-rich granules. From this study, (here is no indication that Ca is the only ion accompanying P in the P-rich granules. Several elements including Ca, K, Cl, S, Cs and Sr, with highest concentrations for S, can occur together with P in these granules. The occurrence of the P-rich electron-opaque deposits in fungal cells might be the first evidence of polyphosphate granules in the native state, since our specimen preparation technique did not include chemical fixation.     

Effect of Salinity on Zinc uptake by Brassica juncea

Salinity is a major worldwide problem that affects agricultural soils and limits the reclamation of contaminated sites. Despite the large number of research papers published about salt tolerance in Brassica juncea L., there are very few accounts concerning the influence of salinity on the uptake of trace metals. In this study, B. juncea plants divided through soil sets comprising 0, 900 and 1800 mg Zn kg^?1, were treated with solutions containing 0, 60 and 120 mmol L^?1 of NaCl, with the purpose of observing the effect of salt on Zn uptake, and some physiological responses throughout the 90 days experiment. Increasing concentrations of NaCl and Zn produced a decline in the ecophysiological and biochemical properties of the plants, with observable synergistic effects on parameters like shoot dry weight, leaf area, or photochemical efficiency. Nevertheless, plants treated with 60 mmol L^?1 of NaCl accumulated striking harvestable amounts of Zn per plant that largely exceed those reported for Thlaspi caerulescens. It was concluded that salinity could play an important role on the uptake of Zn by B. juncea. The potential mechanisms behind these results are discussed, as well as the implications for phytoremediation of Zn on saline and non-saline soils.     

Nickel and zinc hyperaccumulation by Alyssum murale and Thlaspi caerulescens (Brassicaceae) do not enhance survival and whole-plant growth under drought stress

Nickel and Zn hyperaccumulation by Alyssum murale and Thlaspi caerulescens bear substantial energetic costs and should confer benefits to the plant. This research determined whether metal hyperaccumulation can increase osmotic adjustment and resistance to water stress (drought). Alyssum murale and Thlaspi caerulescens treated with low or high concentrations of Ni or Zn were exposed to moderate (?0·4 MPa) and severe (?1·0 MPa) water stresses using aqueous polyethylene glycol. In the absence of metals both water deficits inhibited shoot growth. Nickel and Zn hyperaccumulation did not ameliorate growth inhibition by either level of water stress. The water stress did not induce major changes in shoot metal concentrations of these constitutive hyperaccumulators. Moreover, metal hyperaccumulation had minimal effects on the osmolality of leaf‐sap extracts, relative water content of the shoots, or rate of evapotranspiration. It is concluded that Ni or Zn hyperaccumulation does not augment whole‐plant capacity for drought resistance in A. murale and T. caerulescens.     

Zinc accumulation by Thlaspi caerulescens from soils with different Zn availability: a pot study

The role of Zn bioavailability in soil on Zn hyperaccumulation by Thlaspi caerulescens was investigated. Thlaspi caerulescens from Prayon, Belgium, and Clough Wood, UK, were grown in pots containing unenriched soil (35 g Zn g^–1), or five treatments enriched with Zn compounds of different solubility (ZnS, Zn₃(PO₄)₂, ZnO, ZnCO₃, and ZnSO₇7H₂O). The Zn-enriched treatments had similar total Zn contents (1000 g Zn g^–1), but differed greatly in their concentrations of extractable-Zn. In the treatments with little extractable-Zn (unenriched and ZnS-enriched) T. caerulescens accessed Zn fractions that were not initially soluble; the mass of Zn accumulated in the shoots on Day 90 was greater than the mass of ammonium nitrate extractable-Zn in the soil on Day 0. Moreover, the decrease in ammonium nitrate extractable-Zn in the unenriched treatment after growth accounted for only 50 and 24% of the Zn accumulated by plants of the Clough Wood and Prayon populations, respectively. Despite accumulation of Zn from the previously non-labile fraction in soil, Zn hyperaccumulation from the unenriched and ZnS-enriched treatments was less than from the four treatments with highly extractable-Zn. The mechanisms involved in the solubilization of Zn were therefore not strong. The dissolution of Zn in the soil might have resulted from the very high root density in the pots either enhancing weak mobilization mechanisms, and/or highly efficient uptake in to the roots coupled with replenishment of the Zn taken up through the soil buffering capacity.     

Zinc hyperaccumulation and cellular distribution in Arabidopsis halleri

Although Arabidopsis halleri ( = Cardaminopsis halleri) is known as a Zn hyperaccumulator, there have been no detailed studies on Zn accumulation, tolerance and cellular distribution in this species. In a hydroponic experiment, A. halleri grew healthily with Zn concentrations varying from 1 to 1000 μ M , without showing phytotoxicity or reduction in root or shoot dry weights. The concentration of Zn in the shoots increased from 300 μ g g ^{? 1} dry weight in the 1 μ M Zn treatment to 32 000 μ g g ^{? 1} in the 1000 μ M Zn treatment. Approximately 60% of the total Zn in the shoots were water‐soluble, and there was no evidence of Zn and P co‐precipitation. Both citric and malic acid concentrations in the shoots were not significantly affected by the Zn treatments, whereas in the roots there was a positive response in both organic acids to increasing Zn in solution. Cellular distribution of Zn, Ca and K in frozen hydrated leaf tissues was examined using energy‐dispersive X‐ray microanalysis. Zinc was sequestered in the base of trichomes, whereas the middle and upper parts of trichomes were highly enriched with Ca. Mesophyll cells appeared to have more Zn than the epidermis, probably because the latter were very small in size. Similarities and differences between A. halleri and the other well‐known Zn hyperaccumulator, Thlaspi caerulescens, are discussed.     

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.     

Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri

The cellular compartmentation of elements was analysed in the Zn hyperaccumulator Arabidopsis halleri (L.) O'Kane & Al-Shehbaz (=Cardaminopsis halleri) using energy-dispersive X-ray microanalysis of frozen-hydrated tissues. Quantitative data were obtained using oxygen as an internal standard in the analyses of vacuoles, whereas a peak/background ratio method was used for quantification of elements in pollen and dehydrated trichomes. Arabidopsis halleri was found to hyperaccumulate not only Zn but also Cd in the shoot biomass. While large concentrations of Zn and Cd were found in the leaves and roots, flowers contained very little. In roots grown hydroponically, Zn and Cd accumulated in the cell wall of the rhizodermis (root epidermis), mainly due to precipitation of Zn/Cd phosphates. In leaves, the trichomes had by far the largest concentrations of Zn and Cd. Inside the trichomes there was a striking sub-cellular compartmentation, with almost all the Zn and Cd being accumulated in a narrow ring in the trichome base. This distribution pattern was very different from that for Ca and P. The epidermal cells other than trichomes were very small and contained lower concentrations of Zn and Cd than mesophyll cells. In particular, the concentrations of Cd and Zn in the mesophyll cells increased markedly in response to increasing Zn and Cd concentrations in the nutrient solution. This indicates that the mesophyll cells in the leaves of A. halleri are the major storage site for Zn and Cd, and play an important role in their hyperaccumulation. Received: 4 April 2000 / Accepted: 16 May 2000     

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.     

Nitrate interference with potassium-selective microelectrodes

Initial attempts to measure K⁺ activity (ak) in vacuoles of barley leaf epidermal cells using triple-barrelled K⁺-selective microelectrodes gave values that were only about one-third of those expected. This was due to high (c. 200 mM) NO3^- concentrations in the vacuoles interfering with the K⁺-sensor. The effect of NO3^- was on 1,2,-dimethyl-3-nitrobenzene (DNB) used as a plasticizer in the K⁺-sensor. Replacing DNB with dibutyl sebacate, but not with 2-nitrophenyl octyl ether, overcame this problem and the modified sensor gave acceptable calibration curves with no interference acceptable calibration curves with no interference from physiological concentrations of other ions. These electrodes were used successfully to measure a mean ak of 235 mM in vacuoles of epidermal cells of K⁺-replete barley leaves.Keywords: Leaf epidermis, potassium activity, potassium-selective microelectrodes, vacuoles, nitrate interference.      

Arabidopsis thaliana and Thlaspi caerulescens respond comparably to low zinc supply

The main objective of this research was to study the response of Arabidopsis thaliana L. and Thlaspi caerulescens J. & C. Presl to different Zn supplies. The A. thaliana plants were exposed to Zn-deficiency (0 and 0.05 μM Zn) and compared to the plants grown on media containing standard Zn (2 μM). T. caerulescens plants were also exposed to Zn-deficiency (0.05 μM Zn), but as this is a Zn hyperaccumulator species, also to high Zn (1,000 μM Zn). Plants were compared to plants grown on standard Zn media (100 μM Zn). Both A. thaliana and T. caerulescens were found to be heavily affected by Zn deficiency, showing similar retarded growth and reduced reproduction phenotypes, and even less reduction in biomass production in T. caerulescens than in A. thaliana. T. caerulescens plants were similarly affected when grown on high Zn concentrations, with comparable effects on reproductive tissues as seen on low Zn supply.     

Physiological responses to Cd and Zn in two Cd/Zn hyperaccumulating Thlaspi species

In a model hyperaccumulation study a Cd/Zn hyperaccumulator Thlaspi caerulescens accession Ganges and a recently reported Cd/Zn hyperaccumulator Thlaspi praecox grown in increasing Cd and Zn concentrations in the substrate and in field collected polluted soil were compared. Plant biomass, concentrations of Cd and Zn, total chlorophylls and anthocyanins, antioxidative stress parameters and activities of selected antioxidative enzymes were compared. Increasing Cd, but not Zn in the substrate resulted in the increase of biomass of roots and shoots of T. praecox and T. caerulescens. The two species hyperaccumulated Cd in the shoots to a similar extent, whereas T. caerulescens accumulated more Zn in the shoots than T. praecox. Cadmium amendment decreased total chlorophyll concentration and glutathione reductase activity, and increased non-protein thiols concentration only in T. praecox, suggesting that it is less tolerant to Cd than T. caerulescens. In the field-contaminated soil, T. caerulescens accumulated higher Cd concentrations; but as T. praecox produced higher biomass, both species have similar ability to extract Cd.