Organic acids are not specifically involved in the nitrate-enhanced Zn hyperaccumulation mechanism in Noccaea caerulescens

Nitrogen form has been shown to affect Zn uptake, translocation and storage in the Zn-hyperaccumulating plant Noccaea caerulescens but the biochemical processes are not fully understood. Organic acids and amino acids have been implicated in Zn transport and storage. This study aimed to examine the effect of N form on concentrations of organic acids and amino acids and how these metabolites correlated with Zn hyperaccumulation. Plants were grown in nutrient solution with NO₃⁻, NH₄NO₃ or NH₄⁺, supplied with 50 or 300 μM Zn, and buffered at either pH 4.5 or 6.5. The metabolomic profile was determined by gas chromatography mass spectroscopy. The concentration of Zn in shoots, xylem and roots was greatest for the NO₃⁻, pH 6.5 and 300 μM Zn treatments. For all N forms, the lower growth-medium pH raised xylem sap pH but had no influence on Zn concentration or exudation rate of the xylem sap. Nitrate enhanced organic acid production while NH₄⁺ increased amino acid production. Organic acids in the xylem were more responsive to changes in growth-medium pH than N form, and did not correlate with Zn concentration in shoots, roots or xylem. Serine might be directly involved in Zn hyperaccumulation. Phosphoric acid was associated with reduced Zn accumulation in the shoots. Malic acid was not detected in the shoots but responded to cation uptake more than to Zn specifically in the roots. Citric acid responded to cation uptake more than to Zn specifically in the shoots but did not correlate with Zn concentration in the roots or the xylem sap, or any other cations in the roots. In conclusion, organic acids in N. caerulescens are not specifically involved in Zn hyperaccumulation but are involved in regulating pH in the xylem and cation–anion balance in plants.     

Nitrate nutrition enhances zinc hyperaccumulation in Noccaea caerulescens (Prayon)

Nitrate fertilization has been shown to increase Zn hyperaccumulation by Noccaea caerulescens (Prayon) (formerly Thlaspi caerulescens). However, it is unknown whether this increased hyperaccumulation is a direct result of NO₃ ^? nutrition or due to changes in rhizosphere pH as a result of NO₃ ^? uptake. This paper investigated the mechanism of NO₃ ^?-enhanced Zn hyperaccumulation in N. caerulescens by assessing the response of Zn uptake to N form and solution pH. Plants were grown in nutrient solution with 300 μM Zn and supplied with either (NH₄)₂SO₄, NH₄NO₃ or Ca(NO₃)₂. The solutions were buffered at either pH 4.5 or 6.5. The Zn concentration and content were much higher in shoots of NO₃ ^?-fed plants than in NH₄ ⁺-fed plants at pH 4.5 and 6.5. The Zn concentration in the shoots was mainly enhanced by NO₃ ^?, whereas the Zn concentration in the roots was mainly enhanced by pH 6.5. Nitrate increased Zn uptake in the roots at pH 6.5 and increased apoplastic Zn at pH 4.5. Zinc and Ca co-increased and was found co-localized in leaf cells of NO₃ ^?-fed plants. We conclude that NO₃ ^? directly enhanced Zn uptake and translocation from roots to shoots in N. caerulescens.     

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.     

Nickel and zinc accumulation capacities and tolerance to these metals in the excluder Thlaspi arvense and the hyperaccumulator Noccaea caerulescens

Representatives of Brassicaceae species—the hyperaccumulator Noccaea caerulescens F.K. Mey and the metal excluder Thlaspi arvense L.—were compared in terms of their ability to accumulate nickel (Ni) and zinc (Zn) and their tolerance to these metals. Four ecotypes of N. caerulescens were used: the ecotypes La Calamine (LC, Belgium) and Saint Felix de Palliéres (SF, France) grow naturally on calamine soils rich in Zn, Cd, and Pb; the ecotype Monte Prinzera (MP, Italy) originates from serpentine soils rich in Ni, Co, and Cr; and the ecotype Lellingen (LE, Luxembourg) inhabits non-metalliferous soils. The plants of N. caerulescens were grown for 8 weeks in a half-strength Hoagland solution supplemented with 25, 100, 200, 300, and 400 μM Ni(NO₃)₂ (ecotypes LC, SF, MP, LE) or 100, 200, 400, 800, and 1000 μM Zn(NO₃)₂ (ecotypes LC, SF, LE); the plants of T. arvense were grown in the presence of 10, 20, 25, and 30 μM Ni(NO₃)₂ or 40, 50, 60, 70, 80 μM Zn(NO₃)₂. The toxic effect of Ni and Zn was assessed from changes in dry matter of roots and shoots of treated plants compared to untreated. The content of metals in roots and shoots was determined by means of atomic absorption spectrophotometry. The Ni-accumulating capacity of N. caerulescens ecotypes increased in the order: LC < SF < LE < MP, and the Zn-accumulating capacity increased in the row: LC < SF < LE. In the hyperaccumulating plant N. caerulescens, the increments of biomass started to decrease at a lower metal content in roots than in shoots, whereas the opposite pattern was observed in the metal excluder T. arvense. Since T. arvense plants accumulated Ni and Zn in roots, whereas N. caerulescens accumulated these metals in shoots, one may assume that the greater sensitivity of root growth compared with shoots in N. caerulescens was determined by more effective mechanisms of metal detoxification in shoots. Conversely, the higher sensitivity of shoot growth compared to root growth in T. arvense was determined by more effective mechanisms of metal detoxification in roots. Being more tolerant to Ni and Zn than T. arvense plants, the N. caerulescens ecotypes differed substantially in terms of metal-accumulating capacity and their tolerance to heavy metals. The ecotype originating from non-metalliferous soils (LE) accumulated larger amounts of Zn, but was less tolerant compared with ecotypes growing naturally on calamine soils (SF and LC), whereas the ecotype occurring on serpentine soils (MP) exhibited a markedly greater tolerance to Ni, compared with other ecotypes examined, as well as the largest accumulation of this metal. The results indicate the existence of different mechanisms responsible for plant tolerance to Ni and Zn; the study of these mechanisms is a promising direction for future research.     

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 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.     

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.     

Investigating heavy-metal hyperaccumulation using Thlaspi caerulescens as a model system

Background

Metal-hyperaccumulating plant species are plants that are endemic to metalliferous soils and are able to tolerate and accumulate metals in their above-ground tissues to very high concentrations. One such hyperaccumulator, Thlaspi caerulescens, has been widely studied for its remarkable properties to tolerate toxic levels of zinc (Zn), cadmium (Cd) and sometimes nickel (Ni) in the soil, and accumulate these metals to very high levels in the shoot. The increased awareness regarding metal-hyperaccumulating plants by the plant biology community has helped spur interest in the possible use of plants to remove heavy metals from contaminated soils, a process known as phytoremediation. Hence, there has been a focus on understanding the mechanisms that metal-hyperaccumulator plant species such as Thlaspi caerulescens employ to absorb, detoxify and store metals in order to use this information to develop plants better suited for the phytoremediation of metal-contaminated soils.

Scope

In this review, an overview of the findings from recent research aimed at better understanding the physiological mechanisms of Thlaspi caerulescens heavy-metal hyperaccumulation as well as the underlying molecular and genetic determinants for this trait will be discussed. Progress has been made in understanding some of the fundamental Zn and Cd transport physiology in T. caerulescens. Furthermore, some interesting metal-related genes have been identified and characterized in this plant species, and regulation of the expression of some of these genes may be important for hyperaccumulation.

Conclusions

Thlaspi caerulescens is a fascinating and useful model system not only for studying metal hyperaccumulation, but also for better understanding micronutrient homeostasis and nutrition. Considerable future research is still needed to elucidate the molecular, genetic and physiological bases for the extreme metal tolerance and hyperaccumulation exhibited by plant species such as T. caerulescens.Key words: Zn, Cd, Ni, Thlaspi caerulescens, hyperacumulator, phytoremediation, heavy metal     

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.     

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea.A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS™ clones with insert sizes ∼20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs.Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter β-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue.This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.     

Elevated nicotianamine levels in Arabidopsis halleri roots play a key role in zinc hyperaccumulation

Zn deficiency is among the leading health risk factors in developing countries. Breeding of Zn-enriched crops is expected to be facilitated by molecular dissection of plant Zn hyperaccumulation (i.e., the ability of certain plants to accumulate Zn to levels >100-fold higher than normal plants). The model hyperaccumulators Arabidopsis halleri and Noccaea caerulescens share elevated nicotianamine synthase (NAS) expression relative to nonaccumulators among a core of alterations in metal homeostasis. Suppression of Ah-NAS2 by RNA interference (RNAi) resulted in strongly reduced root nicotianamine (NA) accumulation and a concomitant decrease in root-to-shoot translocation of Zn. Speciation analysis by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry showed that the dominating Zn ligands in roots were NA and thiols. In NAS2-RNAi plants, a marked increase in Zn-thiol species was observed. Wild-type A. halleri plants cultivated on their native soil showed elemental profiles very similar to those found in field samples. Leaf Zn concentrations in NAS2-RNAi lines, however, did not reach the Zn hyperaccumulation threshold. Leaf Cd accumulation was also significantly reduced. These results demonstrate a role for NAS2 in Zn hyperaccumulation also under near-natural conditions. We propose that NA forms complexes with Zn(II) in root cells and facilitates symplastic passage of Zn(II) toward the xylem.     

Phytoextraction of Zinc: Physiological and Molecular Mechanism

Zinc is an essential trace element, necessary for plants, animals, and microorganisms. Zn is required for many enzymes as a catalytic cofactor, for photosynthetic CO₂ fixation, and in maintaining the integrity of bio-membranes. However, Zn is potentially toxic when accumulated beyond cellular needs. Phytoextraction technique, which is a part of phytoremediation, has opened new avenues for remediation of Zn-contaminated places. Hyperaccumulators like Thlaspi caerulescens and Arabidopsis halleri have been identified, which can accumulate up to 40,000 mg kg^?1 Zn in the aerial parts of the plant body. Carboxylic acids, primarily malate, citrate, and oxalate, and amino acids are found to play an important role in Zn hyperaccumulation. Transmembrane metal transporters are assumed to play a key role in Zn metal uptake, xylem loading, and vacuolar sequestration. Members of CDF (cation diffusion facilitator) and ZIP (zinc-regulated transporter, iron-regulated transporter like protein) family have been implicated in Zn-metal-tolerance mechanisms. A potential metal-binding motif, containing multiple histidine residues, is found in the variable regions of almost all of the ZIP family, including ZIP1, ZIP2, ZIP4, ZRT1, and ZRT2. Overexpression of some Zn metal transporter genes like TcZNT1 (Thlaspi caerulescens Zn transporter1), TcHMA4 (Thlaspi caerulescens heavy metal ATPase) in Thlaspi caerulescens, AhMTP1;3 (Arabidopsis halleri metal transporter1;3) in Arabidopsis halleri, and PtdMTP1(Poplar metal transporter1) from a hybrid poplar confer Zn hypertolerance in Thlaspi, Arabidopsis, and Poplar plant species.     

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 genetic differentiation of Thlaspi caerulescens populations

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

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.     

OsZIP5 is a plasma membrane zinc transporter in rice

Zinc is essential for normal plant growth and development. To understand its transport in rice, we characterized OsZIP5, which is inducible under Zn deficiency. OsZIP5 complemented the growth defect of a yeast Zn-uptake mutant, indicating that OsZIP5 is a Zn transporter. The OsZIP5-GFP fusion protein was localized to the plasma membrane. Transgenic plants overexpressing the gene grew less well. Overexpression of the gene decreased the Zn concentration in shoots, but caused it to rise in the roots. Knockout plants showed no visible phenotypic changes under either normal or deficient conditions. However, they were tolerant to excess Zn and contained less Zn. In contrast, overexpressing transgenics were sensitive to excess Zn. These results indicate that OsZIP5 plays a role in Zn distribution within rice.     

Cadmium tolerance in Thlaspi caerulescens: I. Growth parameters,metal accumulation and phytochelatin synthesis in response to cadmium

A population of the metallophyte, Thlaspi caerulescens, originating from a Cd–Pb–Zn old mining and smelter site at Plombières (Belgium) was studied. T. caerulescens was cultivated hydroponically to investigate Cd uptake and tolerance. Cd was added to Hoagland’s medium at concentration range from 5 to 500 μM. The plants could tolerate 500 μM Cd in the solution showing only minor visible symptoms of toxicity but with a 32% decrease in fresh weight. After 14 days at 500 μM, Cd content in roots and shoots was 707 and 602 mg kg⁻¹ of dry weight (d.w.), respectively. Application of Cd to hydroponically cultivated T. caerulescens induced the accumulation of PCs in plant roots and shoots. Buthionine sulfoximine (BSO) application almost completely reduced (by 98–100%) the accumulation of PCs without simultaneous increase in plants sensitivity to Cd. These results suggest a minor if any role of PCs in tolerance to Cd of the studied population of T. caerulescens in hydroponics. On the other hand, no PC accumulation was detected either in T. caerulescens plants growing in their natural environment at Plombierès or in plants growing in their native soil in a greenhouse. These results suggest that naturally selected tolerance in T. caerulescens population from Plombières is not associated with enhanced PCs synthesis.