Hyperaccumulator Thlaspi Caerulescens Research Articles

Phi thickenings in roots: novel secondary wall structures responsive to biotic and abiotic stresses.

Phi thickenings are specialized secondary walls found in root cortical cells. Despite their widespread occurrence throughout the plant kingdom, these specialized thickenings remain poorly understood. First identified by Van Tieghem in 1871, phi thickenings are a lignified and thickened cell wall band that is deposited inside the primary wall, as a ring around the cells' radial walls. Phi thickenings can, however, display structural variations including a fine, reticulate network of wall thickenings extending laterally from the central lignified band. While phi thickenings have been proposed to mechanically strengthen roots, act as a permeability barrier to modulate solute movement, and regulate fungal interactions, these possibilities remain to be experimentally confirmed. Furthermore, since temporal and spatial development of phi thickenings varies widely between species, thickenings may perform diverse roles in different species. Phi thickenings can be induced by abiotic stresses in different species; they can, for example, be induced by heavy metals in the Zn/Cd hyperaccumulator Thlaspi caerulescens, and in a cultivar-specific manner by water stress in Brassica. This latter observation provides an experimental platform to probe phi thickening function, and to identify genetic pathways responsible for their formation. These pathways might be expected to differ from those involved in secondary wall formation in xylem, since phi thickening deposition in not linked to programmed cell death.

Chelant-assisted phytoextraction and accumulation of Zn by Zea mays

Zea mays plants were exposed to soils with concentrations of Zn ranging from 64 to 1800 mg kg−1 dw, and the efficiency of three selected chelating agents (trisodium citrate (CI), disodium oxalate (OX) and disodium dihydrogen ethylene-diamine-tetraacetate (EDTA)) in enhancing metal phytoextraction was compared. Zn concentration in plant tissues increased in conjunction with the metal concentration of the soil. EDTA was found to be the most efficient chelating amendment, increasing concentrations of Zn in shoots from 88 mg kg−1 dw, at 64 mg kg−1 dw soil, to 8026 mg kg−1 dw at 1800 mg kg−1 dw soil. The overall orders of BCFs and TFs which resulted from this study are: EDTA > H2O > OX > CI, and EDTANa2 > OX > CI > H2O, respectively. The more effective uptake of Zn by plants for the control treatment (distilled water only) than for CI and OX was attributed to the neutral or slightly alkaline pH of the two chelant irrigation solutions. Instead, EDTA had a favorable effect on Zn uptake from soil due to its additive chelating and acidifying properties. Among the three chelants, only EDTA significantly increased the Zn phytoextraction potential of Z. mays, while CI and OX induced a low metal uptake from soil by plants. Although Z. mays has a lower Zn accumulation capacity than the hyperaccumulator Thlaspi caerulescens, it could be considered as a potential phytoremediator of soils with elevated Zn concentrations, especially when metal pollution extends to depths greater than 20 cm.

Hyperaccumulation of cadmium in Matricaria chamomilla: a never-ending story?

Chamomile (Matricaria chamomilla L.) is a widely used medicinal plant but may also accumulate metals including cadmium. Because recent paper (Masarovicova et al. 2010 Acta Physiol Plant 32:823–829) contradicts previous results of our research group and suggested Cd “hyper/accumulation” in chamomile, present study examined Cd uptake in a long-term experiment (7 weeks) using low Cd concentration (1.5 μM). Besides, typical Cd hyperaccumulator Thlaspi caerulescens and Ni hyperaccumulator Thlaspi goesingense were also cultured under identical conditions. Data obtained indicate that shoot Cd content and bioaccumulation factor (BAF) in chamomile was ca. 5–10 times lower than that in Thlaspi. In this view, chamomile is not a hyperaccumulator species. Comparison of shoot Cd content, BAF and translocation factor (TF) in selected crops is also provided.

Hyperaccumulator Thlaspi caerulescens (Ganges ecotype) response to increasing levels of dissolved cadmium and zinc

Hyperaccumulation of metals by plants involves at least three processes: efficient absorption by roots, efficient root-to-shoot translocation and hypertolerance through internal detoxification. In this study, Thlaspi caerulescens was separately exposed to Cd and Zn at 0, 50, 100 and 200 μ M for 7 d to monitor plant responses in hydroponics. Significant dose-dependent accumulation was observed for both metals, mainly in roots (up to 3.2 and 9.2 mg g −1 for Cd and Zn, respectively). However, Cd was more phytotoxic in terms of plant growth and photosynthesis. This higher toxicity was also evidenced by MetPLATE bioassay. Root exudation was significantly correlated to Cd and Zn translocation (r>0.85) proving its involvement in facilitating metal uptake. As for antioxidative responses, plants reacted to Cd and Zn by broadly exhibiting an elevation of glutathione reductase activity before declining at 200 μ M due to higher phytotoxicity. By contrast, superoxide dismutase activity was unlikely to be affected by both metals. Root-to-shoot apoplastic flow was traced using a fluorescent dye (trisodium-8-hydroxy-1,3,6-pyrenetrisulfonic acid; PTS), whose concentration in leaves increased to a certain extent with Cd and Zn accumulation, indicating that heavy metals have a comparable effect to drought or salinity in promoting the passive diffusion of water and solutes. Nevertheless, Cd at 200 μ M hindered the diffusion of PTS and consequently affected the apoplastic transport in plants.

TcOPT3, a member of oligopeptide transporters from the hyperaccumulator Thlaspi caerulescens, is a novel Fe/Zn/Cd/Cu transporter.

Background Thlaspi caerulescens is a natural selected heavy metal hyperaccumulator that can not only tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator.Methodology/Principal FindingsWe cloned and characterized a novel gene TcOPT3 of OPT family from T. caerulescens. TcOPT3 was pronouncedly expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3 expressed in the plant vascular systems, especially in the pericycle cells that may be involved in the long-distance transportation. The expression of TcOPT3 was highly induced by iron (Fe) and zinc (Zn) deficiency, especially in the stem and leaf. Sub-cellular localization showed that TcOPT3 was a plasma membrane-localized protein. Furthermore, heterogonous expression of TcOPT3 by mutant yeast (Saccharomyces cerevisiae) complementation experiments demonstrated that TcOPT3 could transport Fe2+ and Zn2+. Moreover, expression of TcOPT3 in yeast increased metal (Fe, Zn, Cu and Cd) accumulation and resulted in an increased sensitivity to cadmium (Cd) and copper (Cu).ConclusionsOur data demonstrated that TcOPT3 might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate. This is the first report showing that TcOPT3 may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation.

Biochemical and biophysical characterisation yields insights into the mechanism of a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens

TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P1B-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10μM), and all three metal ions activated the ATPase at a concentration of 0.3μM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (EA=38kJ⋅mol−1) over the whole concentration range while it increased from 17 to 42kJ⋅mol−1 with rising copper concentration and decreased from 39 to 23kJ⋅mol−1 with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine.

Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco

Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl(2) over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants.

Characterization of the glyoxalase 1 gene TcGLX1 in the metal hyperaccumulator plant Thlaspi caerulescens

Stress tolerance is currently one of the major research topics in plant biology because of the challenges posed by changing climate and increasing demand to grow crop plants in marginal soils. Increased Zn tolerance and accumulation has been reported in tobacco expressing the glyoxalase 1-encoding gene from Brassica juncea. Previous studies in our laboratory showed some Zn tolerance-correlated differences in the levels of glyoxalase 1-like protein among accessions of Zn hyperaccumulator Thlaspi caerulescens. We have now isolated the corresponding gene (named here TcGLX1), including ca. 570bp of core and proximal promoter region. The predicted protein contains three glyoxalase 1 motifs and several putative sites for post-translational modification. In silico analysis predicted a number of cis-acting elements related to stress. The expression of TcGLX1 was not responsive to Zn. There was no correlation between the levels of TcGLX1 expression and the degrees of Zn tolerance or accumulation among T. caerulescens accessions nor was there co-segregation of TcGLX1 expression with Zn tolerance or Zn accumulation among F3 lines derived from crosses between plants from accessions with contrasting phenotypes for these properties. No phenotype was observed in an A. thaliana T-DNA insertion line for the closest A. thaliana homolog of TcGLX1, ATGLX1. These results suggest that glyoxalase 1 or at least the particular isoform studied here is not a major determinant of Zn tolerance in the Zn hyperaccumulator plant T. caerulescens. In addition, ATGLX1 is not essential for normal Zn tolerance in the non-tolerant, non-accumulator plant A. thaliana. Possible explanations for the apparent discrepancy between this and previous studies are discussed.

Native Plant Communities in an Abandoned Pb-Zn Mining Area of Northern Spain: Implications for Phytoremediation and Germplasm Preservation

Plants growing on metalliferous soils from abandoned mines are unique because of their ability to cope with high metal levels in soil. In this study, we characterized plants and soils from an abandoned Pb-Zn mine in the Basque Country (northern Spain). Soil in this area proved to be deficient in major macronutrients and to contain toxic levels of Cd, Pb, and Zn. Spontaneously growing native plants (belonging to 31 species, 28 genera, and 15 families) were botanically identified. Plant shoots and rhizosphere soil were sampled at several sites in the mine, and analyzed for Pb, Zn and Cd concentration. Zinc showed the highest concentrations in shoots, followed by Pb and Cd. Highest Zn concentrations in shoots were found in the Zn-Cd hyperaccumulator Thlaspi caerulescens (mean = 18,254 mg Zn kg−1 DW). Different metal tolerance and accumulation patterns were observed among the studied plant species, thus offering a wide germplasm assortment for the suitable selection of phytoremediation technologies. This study highlights the importance of preserving metalliferous environments as they shelter a unique and highly valuable metallicolous biodiversity.

Response of Antioxidative Enzymes and Apoplastic Bypass Transport in Thlaspi Caerulescens and Raphanus Sativus to Cadmium Stress

A hydroponics experiment using hyperaccumulator Thlaspi caerulescens (alpine pennycress) and non-specific accumulator Raphanus sativus (common radish) was conducted to investigate the short-term effect of increasing Cd concentrations (0, 25, 50, 75, 100 μM) on metal uptake, chlorophyll content, antioxidative enzymes, and apoplastic bypass flow. As expected, T. caerulescens generally showed better resistance to metal stress, which was reflected by higher Cd accumulation within plant tissues with no signs of chlorosis, or wilt. Glutathione reductase (GR) and superoxide dismutase (SOD) activities in fresh leaves were monitored as the plant metal-detoxifying response. In general, both plant species exhibited an increase trend of GR activity before declining at 100 μM likely due to excessive levels of phytotoxic Cd. SOD activity exhibited almost a similar variation pattern to GR and decreased also at 100 μM Cd. For both plant species, fluorescent PTS uptake (8-hydroxy-l,3,6-pyrenetrisulphonic acid) increased significantly with metal level in exposure solutions indicating that Cd has a comparable effect to drought or salinity in terms of the gain of relative importance in apoplastic bypass transport under such stress conditions.

Flow cytometric assessment of Cd genotoxicity in three plants with different metal accumulation and detoxification capacities

Cadmium (Cd) is a widespread environmental contaminant, strongly mutagenic and known to cause DNA damage in plants. In this work, flow cytometry (FCM) was applied to determine if in vivo exposure to Cd would induce genotoxic effects at the genome level. The hyper-accumulator Thlaspi caerulescens (J. & C. Presl), the related non-accumulator Thlaspi arvense L. and the accumulator crop species Lactuca sativa L. were germinated in distilled water and grown in modified Hoagland’s medium with increasing concentrations of Cd(NO3)2 (0, 1, 10 and 100μM). After 28 days of exposure, shoot and root growth was recorded and the tissues were harvested for Cd and FCM analysis. In general, roots from treated plants contained higher content of Cd than leaves and growth inhibition was observed in the treated plants. Nuclear DNA content was estimated and the G0/G1 full peak coefficient of variation (FPCV), as an indicator of clastogenic damage, was recorded. In T. arvense and T. caerulescens no significant differences were detected between control and exposed plants. Leaves of L. sativa exposed to 10μM Cd presented a statistically significant increase in FPCV values in comparison with the control group. Furthermore, roots exposed to 100μM Cd presented a reduction in nuclear DNA content and an increase in FPCV when compared to the control. FCM data indicates that no major DNA damage was induced on both Cd-exposed Thlaspi species and L. sativa leaves. On the contrary, results obtained with L. sativa roots suggests clastogenic damage in these organs exposed to 100μM of Cd.

Remediation of cadmium contaminated irrigation and drinking water: A large scale approach

Cadmium is one of the most troublesome toxic heavy metals. It accumulates in the water reservoirs and agricultural soil as a result of intensive use of Cd contaminated phosphate fertilizers, e.g. in agriculture in the North Central Province (NCP) of Sri Lanka. The hyper-accumulator Thlaspi caerulescens, accumulates up to 1000 ppm Cd in shoots without exhibiting toxicity symptoms. The storage rhizomes of year old Nelumbo nucifera (lotus) natural vegetation in water reservoirs in NCP accumulated 253 ± 12 mg Cd/kg. Seedlings of lotus grown in 5% Hoagland's solution at 0.75, 1.0 and 1.25 ppm cadmium sulphate showed a significant increase in Cd removal of 0.0334–0.121 ppm/week. However the removal rate of Cd from water failed to increase any further at higher concentrations of Cd in water. The slow growth rate and low rate of phytoextraction demands a more effective but an affordable method of remediation in order to combat the prevailing elevated cadmium levels in NCP that causes chronic renal failure (CRF). We have developed a large scale filtering device using rice husk. We have achieved successful results in sequestering Cd using raw rice husk as well as amorphous silica derived from rice husk.

Growth and Metal Uptake by Plants Grown in Mono- and Dual Culture in Metal-contaminated Soils

Plants are able to affect the mobility of heavy metals in the rhizosphere due to root exudates and other mechanisms resulting in a change in their phytoextraction ability. We tested separate (monoculture) and intercropping (dual culture) systems of hyperaccumulator Thlaspi caerulescens and accumulator tree Salix dasyclados in two soils with different levels of contamination. Intercropping did not significantly affect accumulation and removal of Cd, Pb, and Zn in moderately contaminated soil. Separately cropped S. dasyclados continuously died due to phytotoxicity of the highly contaminated soil. We indicated an important positive effect of intercropping in this soil. Intensive uptake of heavy metals by Thlaspi roots decreased relatively toxic concentrations in soil and improved the growth of S. dasyclados. The maximum limit of the phytoextraction potential of T. caerulescens was not reached at the extremely contaminated soil but the efficiency of the intercropping at the extremely contaminated soil still remains limited and alternative remediation techniques can be considered at this particular contaminated site.

Nicotianamine forms complexes with Zn(ii)in vivo

The non-proteinogenic amino acid nicotianamine (NA) is a major player in plant metal homeostasis. It is known to form complexes with different transition metals in vitro. Available evidence associates NA with translocation of Fe, and possibly other micronutrients, to and between different plant cells and tissues. To date, however, it is still extremely challenging to detect metal-ligand complexes in vivo because tissue disruption immediately changes the chemical environment and thereby the availability of binding partners. In order to overcome this limitation we used various Schizosaccharomyces pombe strains expressing a plant NAS gene to study formation of metal-NA complexes in vivo. Tolerance, accumulation and competition data clearly indicated formation of Zn(ii)-NA but not of Cu(ii)-NA complexes. Zn(ii)-NA was then identified by X-ray absorption spectroscopy (XAS). About half of the cellular Zn was found to be bound by NA in NAS-expressing cells while no NA-like ligands were detected by XAS in control cells not expressing NAS. Given the high conservation of eukaryotic metal homeostasis components, these results strongly suggest the possible existence of Zn(ii)-NA complexes also in planta. Reported observations implicating NA in plant Zn homeostasis would then indeed be attributable to direct interaction of Zn(ii) with NA rather than only indirectly to perturbations in Fe metabolism. Re-evaluation of extended X-ray absorption fine structure (EXAFS) spectra for the Zn hyperaccumulator Thlaspi caerulescens showed that NA is as expected not a major storage ligand for Zn. Instead it is hypothesized to be involved in efficient translocation of Zn to above-ground tissues in hyperaccumulators.

Comparison of two ecotypes of the metal hyperaccumulator Thlaspi caerulescens (J. & C. PRESL) at the transcriptional level

This paper investigates differences in gene expression among the two Thlaspi caerulescens ecotypes La Calamine (LC) and Lellingen (LE) that have been shown to differ in metal tolerance and metal uptake. LC originates from a metalliferous soil and tolerates higher metal concentrations than LE which originates from a non-metalliferous soil. The two ecotypes were treated with different levels of zinc in solution culture, and differences in gene expression were assessed through application of a cDNA microarray consisting of 1,700 root and 2,700 shoot cDNAs. Hybridisation of root and shoot cDNA from the two ecotypes revealed a total of 257 differentially expressed genes. The regulation of selected genes was verified by quantitative reverse transcriptase polymerase chain reaction. Comparison of the expression profiles of the two ecotypes suggests that LC has a higher capacity to cope with reactive oxygen species and to avoid the formation of peroxynitrite. Furthermore, increased transcripts for the genes encoding for water channel proteins could explain the higher Zn tolerance of LC compared to LE. The higher Zn tolerance of LC was reflected by a lower expression of the genes involved in disease and defence mechanisms. The results of this study provide a valuable set of data that may help to improve our understanding of the mechanisms employed by plants to tolerate toxic concentrations of metal in the soil.

Identification and functional analysis of two ZIP metal transporters of the hyperaccumulator Thlaspi caerulescens

The heavy metal hyperaccumulator Thlaspi caerulescens expresses several ZIP-like genes at higher levels than their orthologues in non-hyperaccumulator species, but it is not clear why. To elucidate the function of the T. caerulescens orthologues of the Arabidopsis thaliana ZIP5 and ZIP6 genes, full-length cDNAs of TcZNT5-LC and TcZNT6-LC were cloned, their expression was examined and genes were expressed in A. thaliana. Transcript level analysis revealed the constitutively high expression of these two genes in T. caerulescens compared to AtZIP5 and AtZIP6 genes and differential expression of both genes when comparing two accessions of T. caerulescens with different metal accumulation properties. Expression of TcZNT5-LC in A. thaliana did not modify Cd or Zn tolerance, but mildly affected the root and shoot Zn concentrations towards a hyperaccumulator shoot to root concentration ratio. A. thaliana zip5 knock-out mutants showed increased tolerance to Cd and decreased seed mineral concentrations. Expression of TcZNT6-LC enhanced the Cd sensitivity of A. thaliana, but no phenotype was observed for the zip6 mutant. In conclusion, the changes in expression of TcZNT5-LC and TcZNT6-LC upon changes in Zn or Cd exposure indicate both genes act in metal homeostasis, but their CaMV 35S-mediated expression in A. thaliana does not create T. caerulescens hyperaccumulator phenotypes.

Chelation by histidine inhibits the vacuolar sequestration of nickel in roots of the hyperaccumulator Thlaspi caerulescens

* The mechanisms of enhanced root to shoot metal transport in heavy metal hyperaccumulators are incompletely understood. Here, we compared the distribution of nickel (Ni) over root segments and tissues in the hyperaccumulator Thlaspi caerulescens and the nonhyperaccumulator Thlaspi arvense, and investigated the role of free histidine in Ni xylem loading and Ni transport across the tonoplast. * Nickel accumulation in mature cortical root cells was apparent in T. arvense and in a high-Ni-accumulating T. caerulescens accession, but not in a low-accumulating T. caerulescens accession. * Compared with T. arvense, the concentration of free histidine in T. caerulescens was 10-fold enhanced in roots, but was only slightly higher in leaves, regardless of Ni exposure. Nickel uptake in MgATP-energized root- and shoot-derived tonoplast vesicles was almost completely blocked in T. caerulescens when Ni was supplied as a 1 : 1 Ni-histidine complex, but was uninhibited in T. arvense. Exogenous histidine supply enhanced Ni xylem loading in T. caerulescens but not in T. arvense. * The high rate of root to shoot translocation of Ni in T. caerulescens compared with T. arvense seems to depend on the combination of two distinct characters, that is, a greatly enhanced root histidine concentration and a strongly decreased ability to accumulate histidine-bound Ni in root cell vacuoles.

Cadmium sorption, influx, and efflux at the mesophyll layer of leaves from ecotypes of the Zn/Cd hyperaccumulator Thlaspi caerulescens

Differential sorption and transport characteristics of the leaf mesophyll layer of the Prayon and Ganges ecotypes of the hyperaccumulator Thlaspi caerulescens were examined. (109)Cd influx and efflux experiments were conducted with leaf sections, and X-ray absorption near edge structure (XANES) data were collected from leaves as a general comparison of in vivo cadmium (Cd) coordination. There were modest differences in cell wall sorption of Cd between ecotypes. There were obvious differences in time- and concentration-dependent Cd influx, including a greater V(MAX) for Prayon but a lower K(M) for Ganges for concentration-dependent Cd uptake and a notably greater Cd uptake by Ganges leaf sections at 1000 microm Cd. Leaf sections of Prayon had a greater Cd efflux than Ganges. The XANES spectra from the two ecotypes suggested differences in Cd coordination. The fundamental differences observed between the two ecotypes may reflect differential activity and/or expression of plasma membrane and tonoplast transporters. More detailed study of these transporters and the in vivo coordination of Cd are needed to determine the contribution of these processes to metal homeostasis and tolerance.

Effect of nitrogen form on the rhizosphere dynamics and uptake of cadmium and zinc by the hyperaccumulator Thlaspi caerulescens

The effect of N form (NO3− versus NH4+) on growth and uptake of Cd and Zn by the hyperaccumulator Thlaspi caerulescens (Ganges ecotype) was investigated in hydroponic and rhizobox experiments. In the hydroponic experiments, NO3− or NH4+ was supplied to plants with the pH of the nutrient solution being unbuffered or buffered at around 6.0. A moderately contaminated soil was used in the rhizobox experiment with or without additions of NO3−, NH4+ or NH4+ + DCD (dicyanodiamide, a nitrification inhibitor). A higher biomass was obtained when N was supplied as NO3− in both experiments, indicating that T. caerulescens prefers NO3− over NH4+. In the hydroponic experiments, supplying NO3− resulted in a doubling of Cd concentration in the shoots compared with the NH4+ treatment, regardless whether solution pH was buffered or not. The form of N also had a noticeable effect on root Zn concentrations. In the rhizosphere box experiment, rhizosphere pH was markedly influenced by the N treatment. The acidification in the NH4+ and NH4+ + DCD treatments increased the concentrations of extractable Cd and Zn, both of which showed a considerable depletion in the rhizosphere. However, total uptake of Cd and Zn were highest in the NO3− treatment, despite the fact that concentrations of extractable Cd and Zn in the rhizosphere were the lowest in this treatment. The results showed that supplying N as NO3− promoted growth and phytoextraction of Cd and Zn by T. caerulescens compared with NH4+.

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens.

To study the role of metallothioneins (MTs) in Zn accumulation, the expression of TcMT2a, TcMT2b, and TcMT3 was analysed in three accessions and 15 F3 families of two inter-accession crosses of the Cd/Zn hyperaccumulator Thlaspi caerulescens, with different degrees of Zn accumulation. The highest expression levels were found in the shoots of a superior metal-accumulating calamine accession from St Laurent le Minier, with >10-fold TcMT3 expression compared with another calamine accession and a non-metallicolous accession. Moreover, F3 sibling lines from the inter-accession crosses that harboured the MT2a or MT3 allele from St Laurent le Minier had higher expression levels. However, there was no co-segregation of TcMT2a or TcMT3 expression and Zn accumulation. To examine the functions of TcMTs in plants, TcMT2a and TcMT3 were ectopically expressed in Arabidopsis. The transformant lines had reduced root length in control medium but not at high metal concentrations, suggesting that the ectopically expressed proteins interfered with the physiological availability of essential metals under limited supply. The Arabidopsis transformant lines did not show increased tolerance to Cd, Cu, or Zn, nor increased Cd or Zn accumulation. Immunohistochemical analysis indicated that in roots, MT2 protein is localized in the epidermis and root hairs of both T. caerulescens and Arabidopsis thaliana. The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarily involved in Zn accumulation as such. However, the elevated expression levels in the metallicolous accessions suggests that they do contribute to the metal-adapted phenotype, possibly through improving Cu homeostasis at high Zn and Cd body burdens. Alternatively, they might function as hypostatic enhancers of Zn or Cd tolerance.