Nickel and zinc isotope fractionation in hyperaccumulating and nonaccumulating plants.

Until now, there has been little data on the isotope fractionation of nickel (Ni) in higher plants and how this can be affected by plant Ni and zinc (Zn) homeostasis. A hydroponic cultivation was conducted to investigate the isotope fractionation of Ni and Zn during plant uptake and translocation processes. The nonaccumulator Thlaspi arvense, the Ni hyperaccumulator Alyssum murale and the Ni and Zn hyperaccumulator Noccaea caerulescens were grown in low (2 µM) and high (50 µM) Ni and Zn solutions. Results showed that plants were inclined to absorb light Ni isotopes, presumably due to the functioning of low-affinity transport systems across root cell membrane. The Ni isotope fractionation between plant and solution was greater in the hyperaccumulators grown in low Zn treatments (Δ(60)Ni(plant-solution) = -0.90 to -0.63‰) than that in the nonaccumulator T. arvense (Δ(60)Ni(plant-solution) = -0.21‰), thus indicating a greater permeability of the low-affinity transport system in hyperaccumulators. Light isotope enrichment of Zn was observed in most of the plants (Δ(66)Zn(plant-solution) = -0.23 to -0.10‰), but to a lesser extent than for Ni. The rapid uptake of Zn on the root surfaces caused concentration gradients, which induced ion diffusion in the rhizosphere and could result in light Zn isotope enrichment in the hyperaccumulator N. caerulescens. In high Zn treatment, Zn could compete with Ni during the uptake process, which reduced Ni concentration in plants and decreased the extent of Ni isotope fractionation (Δ(60)Ni(plant-solution) = -0.11 to -0.07‰), indicating that plants might take up Ni through a low-affinity transport system of Zn. We propose that isotope composition analysis for transition elements could become an empirical tool to study plant physiological processes.

Histidine-mediated xylem loading of zinc is a species-wide character in Noccaea caerulescens.

Histidine plays a crucial role in nickel (Ni) translocation in Ni-hyperaccumulating plants. Here, we investigated its role in zinc (Zn) translocation in four accessions of the Zn hyperaccumulator, Noccaea caerulescens, using the related non-hyperaccumulator, Thlaspi arvense, as a reference. We compared the effects of exogenous histidine supply on Zn xylem loading, and of Zn-histidine complex formation on Zn uptake in energized tonoplast vesicles. The Zn distribution patterns over root tissues were also compared. Exogenous histidine supply enhanced Zn xylem loading in all the N. caerulescens accessions, but decreased it in T. arvense. Zn distribution patterns over root tissues were similar, apart from the accumulation in cortical and endodermal cells, which was much lower in N. caerulescens than in T. arvense. Zn uptake in energized tonoplast vesicles was inhibited significantly in N. caerulescens, but not affected significantly in T. arvense, when Zn was supplied in combination with histidine in a 1:2 molar ratio. Histidine-mediated Zn xylem loading seems to be a species-wide character in N. caerulescens. It may well have evolved as a component trait of the hyperaccumulation machinery for Zn, rather than for Ni.

Effect of microbial siderophore DFO-B on Cd accumulation by Thlaspi caerulescens hyperaccumulator in the presence of zeolite.

Hyperaccumulators are grown in contaminated soil and water in order that contaminants are taken up and accumulated. Transport of metals from soil to plant is initially dependent on the solubility and mobility of metals in soil solution which is controlled by soil and metal properties and plant physiology. Complexation with organic and inorganic ligands may increase mobility and availability of metals for plants. In this work the influence of desferrioxamine-B (DFO-B), which naturally is produced in the rhizosphere, and zeolite on Cd accumulation in root and shoot of Thlaspi caerulescens (Cd hyperaccumulator) was investigated. Plants were grown in pots with clean quartz sand, amended with 1% zeolite; treatment solutions included 0, 10, and 100 µM Cd and 70 µM DFO-B. Addition of zeolite to the quartz sand significantly reduced Cd concentration in plant tissues and translocation from root to shoot. On contrary, DFO-B considerably enhanced Cd sorption by roots and translocation to aerial part of plants. Treating the plants with zeolite and DFO-B together at 10 µM Cd resulted in reduction of the bioaccumulation factor but enhancement of Cd translocation from root to shoot at the rate of 13%. In contrast, at 100 µM Cd in the solution both bioaccumulation and translocation factors decreased. Total metal accumulation as a key factor for evaluating the efficiency of phytoremediation was highly influenced by treatments. Presence of zeolite in pots significantly decreased total Cd accumulation by plants, whereas, DFO-B clearly enhanced it.

Differential regulation of serine acetyltransferase is involved in nickel hyperaccumulation in Thlaspi goesingense.

When growing in its native habitat, Thlaspi goesingense can hyperaccumulate 1.2% of its shoot dry weight as nickel. We reported previously that both constitutively elevated activity of serine acetyltransferase (SAT) and concentration of glutathione (GSH) are involved in the ability of T. goesingense to tolerate nickel. A feature of SAT is its feedback inhibition by L-cysteine. To understand the role of this regulation of SAT by Cys on GSH-mediated nickel tolerance in T. goesingense, we characterized the enzymatic properties of SATs from T. goesingense. We demonstrate that all three isoforms of SAT in T. goesingense are insensitive to inhibition by Cys. Further, two amino acids (proline and alanine) in the C-terminal region of the cytosolic SAT (SAT-c) from T. goesingense are responsible for converting the enzyme from a Cys-sensitive to a Cys-insensitive form. Furthermore, the Cys-insensitive isoform of SAT-c confers elevated resistance to nickel when expressed in Escherichia coli and Arabidopsis thaliana, supporting a role for altered regulation of SAT by Cys in nickel tolerance in T. goesingense.

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.

Cadmium uptake and sequestration kinetics in individual leaf cell protoplasts of the Cd/Zn hyperaccumulator Thlaspi caerulescens.

Hyperaccumulators store accumulated metals in the vacuoles of large leaf epidermal cells (storage cells). For investigating cadmium uptake, we incubated protoplasts obtained from leaves of Thlaspi caerulescens (Ganges ecotype) with a Cd-specific fluorescent dye. A fluorescence kinetic microscope was used for selectively measuring Cd-uptake and photosynthesis in different cell types, so that physical separation of cell types was not necessary. Few minutes after its addition, cadmium accumulated in the cytoplasm before its transport into the vacuole. This demonstrated that vacuolar sequestration is the rate-limiting step in cadmium uptake into protoplasts of all leaf cell types. During accumulation in the cytoplasm, Cd-rich vesicle-like structures were observed. Cd uptake rates into epidermal storage cells were higher than into standard-sized epidermal cells and mesophyll cells. This shows that the preferential heavy metal accumulation in epidermal storage cells, previously observed for several metals in intact leaves of various hyperaccumulator species, is due to differences in active metal transport and not differences in passive mechanisms like transpiration stream transport or cell wall adhesion. Combining this with previous studies, it seems likely that the transport steps over the plasma and tonoplast membranes of leaf epidermal storage cells are driving forces behind the hyperaccumulation phenotype.

Life history traits of the pseudometallophyte Thlaspi caerulescens in natural populations from Northern Europe.

We examined recruitment, survival, life cycle and fecundity of two metallicolous (M, on metalliferous calamine soils) and two non-metallicolous (NM, on normal soils) populations of Thlaspi caerulescens in Belgium and Luxemburg. In each population, permanent plots were monitored over two reproductive seasons. In M populations, plots were located in two contrasting environments (grass versus grove) in order to test the influence of vegetation cover on life strategy. Our results show that the monocarpic life cycle is dominant in all populations of T. caerulescens. However the length of the pre-reproductive period varies from several months (winter annuals) to 1 year or more (perennials), and is partly related to plant origin (M versus NM). Most plants growing in metalliferous environments were annuals, whereas NM plants were mostly perennials. These differences in life cycle were related to differences in survival during summer, which was better in NM than in M populations. Within each M population, different survival conditions and life cycles were observed according to vegetation cover. Plants growing in grass areas were mostly annuals and had a low survival rate in summer whereas grove plants were mostly perennials and survived better in summer. Our results suggest the selection of stress avoiders (shortening of life cycle) in M populations of T. caerulescens but only for individuals growing in grass areas. Summer survival seems to play a key role in selection of life strategy in T. caerulescens.

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 microM) 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 microM likely due to excessive levels of phytotoxic Cd. SOD activity exhibited almost a similar variation pattern to GR and decreased also at 100 microM Cd. For both plant species, fluorescent PTS uptake (8-hydroxy-1,3,6-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.

Comparison of protein variations in Thlaspi caerulescens populations from metalliferous and non-metalliferous soils.

In this work we analysed the protein variations which occurred in two Thlaspi caerulescens populations when subjected to 0 and 10 microM nickel (Ni) treatments: the Ni hyperaccumulator T. caerulescensfrom a metalliferous soil in Italy and T. caerulescens from Czech Republic, adapted to grow on a non-metalliferous soil. Ni accumulation in roots and shoots and the effect on growth and morphology were examined. Leaves proteins profiles of Ni treated and untreated samples were analysed by two dimensional liquid chromatography technique. From the comparison of more than 500 proteins, few differences were observed between treated and untreated plants of the same population. Differences were found between the two Thlaspi populations, instead. Proteins involved in transport, metal chelation, and signal transduction increased in abundance in the 10 microM Ni treated samples while, in condition of absence of Ni, proteins involved in sulphur metabolism, protection against reactive oxygen species and stress response showed to increase in abundance in the two populations. These proteins can be used as biomarkers both for monitoring biodiversity in indigenous plants and for selection of Ni phytoremediation plants.

Metal hyperaccumulation armors plants against disease.

Metal hyperaccumulation, in which plants store exceptional concentrations of metals in their shoots, is an unusual trait whose evolutionary and ecological significance has prompted extensive debate. Hyperaccumulator plants are usually found on metalliferous soils, and it has been proposed that hyperaccumulation provides a defense against herbivores and pathogens, an idea termed the 'elemental defense' hypothesis. We have investigated this hypothesis using the crucifer Thlaspi caerulescens, a hyperaccumulator of zinc, nickel, and cadmium, and the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm). Using leaf inoculation assays, we have shown that hyperaccumulation of any of the three metals inhibits growth of Psm in planta. Metal concentrations in the bulk leaf and in the apoplast, through which the pathogen invades the leaf, were shown to be sufficient to account for the defensive effect by comparison with in vitro dose-response curves. Further, mutants of Psm with increased and decreased zinc tolerance created by transposon insertion had either enhanced or reduced ability, respectively, to grow in high-zinc plants, indicating that the metal affects the pathogen directly. Finally, we have shown that bacteria naturally colonizing T. caerulescens leaves at the site of a former lead-zinc mine have high zinc tolerance compared with bacteria isolated from non-accumulating plants, suggesting local adaptation to high metal. These results demonstrate that the disease resistance observed in metal-exposed T. caerulescens can be attributed to a direct effect of metal hyperaccumulation, which may thus be functionally analogous to the resistance conferred by antimicrobial metabolites in non-accumulating plants.

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 100 microM). 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 10 microM Cd presented a statistically significant increase in FPCV values in comparison with the control group. Furthermore, roots exposed to 100 microM 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 100 microM of Cd.

Complexation and toxicity of copper in higher plants. II. Different mechanisms for copper versus cadmium detoxification in the copper-sensitive cadmium/zinc hyperaccumulator Thlaspi caerulescens (Ganges Ecotype).

The cadmium/zinc hyperaccumulator Thlaspi caerulescens is sensitive toward copper (Cu) toxicity, which is a problem for phytoremediation of soils with mixed contamination. Cu levels in T. caerulescens grown with 10 microm Cu(2+) remained in the nonaccumulator range (<50 ppm), and most individuals were as sensitive toward Cu as the related nonaccumulator Thlaspi fendleri. Obviously, hyperaccumulation and metal resistance are highly metal specific. Cu-induced inhibition of photosynthesis followed the "sun reaction" type of damage, with inhibition of the photosystem II reaction center charge separation and the water-splitting complex. A few individuals of T. caerulescens were more Cu resistant. Compared with Cu-sensitive individuals, they recovered faster from inhibition, at least partially by enhanced repair of chlorophyll-protein complexes but not by exclusion, since the content of Cu in their shoots was increased by about 25%. Extended x-ray absorption fine structure (EXAFS) measurements on frozen-hydrated leaf samples revealed that a large proportion of Cu in T. caerulescens is bound by sulfur ligands. This is in contrast to the known binding environment of cadmium and zinc in the same species, which is dominated by oxygen ligands. Clearly, hyperaccumulators detoxify hyperaccumulated metals differently compared with nonaccumulated metals. Furthermore, strong features in the Cu-EXAFS spectra ascribed to metal-metal contributions were found, in particular in the Cu-resistant specimens. Some of these features may be due to Cu binding to metallothioneins, but a larger proportion seems to result from biomineralization, most likely Cu(II) oxalate and Cu(II) oxides. Additional contributions in the EXAFS spectra indicate complexation of Cu(II) by the nonproteogenic amino acid nicotianamine, which has a very high affinity for Cu(II) as further characterized here.

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.

Functional characterization of NRAMP3 and NRAMP4 from the metal hyperaccumulator Thlaspi caerulescens.

The ability of metal hyperaccumulating plants to tolerate and accumulate heavy metals results from adaptations of metal homeostasis. NRAMP metal transporters were found to be highly expressed in some hyperaccumulating plant species. Here, we identified TcNRAMP3 and TcNRAMP4, the closest homologues to AtNRAMP3 and AtNRAMP4 in Thlaspi caerulescens and characterized them by expression analysis, confocal imaging and heterologous expression in yeast and Arabidopsis thaliana. TcNRAMP3 and TcNRAMP4 are expressed at higher levels than their A. thaliana homologues. When expressed in yeast TcNRAMP3 and TcNRAMP4 transport the same metals as their respective A. thaliana orthologues: iron (Fe), manganese (Mn) and cadmium (Cd) but not zinc (Zn) for NRAMP3; Fe, Mn, Cd and Zn for NRAMP4. They also localize at the vacuolar membrane in A. thaliana protoplasts. Inactivation of AtNRAMP3 and AtNRAMP4 in A. thaliana results in strong Cd and Zn hypersensitivity, which is fully rescued by TcNRAMP3 or TcNRAMP4 expression. However, metal tolerance conferred by TcNRAMP expression in nramp3nramp4 mutant does not exceed that of wild-type A. thaliana. Our data indicate that the difference between TcNRAMP3 and TcNRAMP4 and their A. thaliana orthologues does not lie in a different protein function, but probably resides in a different expression level or expression pattern.

Plant response to heavy metal toxicity: comparative study between the hyperaccumulator Thlaspi caerulescens (ecotype Ganges) and nonaccumulator plants: lettuce, radish, and alfalfa.

Thlaspi caerulescens (alpine pennycress) is one of the best-known heavy metal (HM) hyperaccumulating plant species. It exhibits the ability to extract and accumulate various HM at extremely high concentrations. In this hydroponic study, the performance of T. caerulescens (ecotype Ganges) to accumulate Cd, Zn, and Cu was compared with that of three nonaccumulator plants: alfalfa (Medicago sativa), radish (Raphanus sativus), and lettuce (Lactuca sativa). Plants were exposed to the separately dissolved HM salts for 7 days at a wide range of increasing concentrations: 0 (control: 1/5 Hoagland nutrient solution), 0.1, 1, 10, 100, and 1000 microM. The comparative study combined chemical, physiological, and ecotoxicological assessments. Excessive concentrations of HM (100 and 1000 microM) affected plant growth, photosynthesis, and phytoaccumulation efficiency. Root exudation for all plant species was highly and significantly correlated to HM concentration in exposure solutions and proved its importance to counter effect toxicity. T. caerulescens resisted better the phytotoxic effects of Cd and Zn (at 1000 microM each), and translocated them significantly within tissues (366 and 1290 microg g(-1), respectively). At the same HM level, T. caerulescens exhibited lower performances in accumulating Cu when compared with the rest of plant species, mainly alfalfa (298 microg g(-1)). Root elongation inhibition test confirmed the selective aptitude of T. caerulescens to better cope with Cd and Zn toxicities. MetPLATE bioassay showed greater sensitivity to HM toxicity with much lower EC(50) values for beta-galactosidase activity in E. coli. Nevertheless, exaggerated HM concentrations coupled with relatively short exposure time did not allow for an efficient metal phytoextraction thus a significant reduction of ecotoxicity.

Expression differences for genes involved in lignin, glutathione and sulphate metabolism in response to cadmium in Arabidopsis thaliana and the related Zn/Cd-hyperaccumulator Thlaspi caerulescens.

Cadmium (Cd) is a widespread, naturally occurring element present in soil, rock, water, plants and animals. Cd is a non-essential element for plants and is toxic at higher concentrations. Transcript profiles of roots of Arabidopsis thaliana (Arabidopsis) and Thlaspi caerulescens plants exposed to Cd and zinc (Zn) are examined, with the main aim to determine the differences in gene expression between the Cd-tolerant Zn-hyperaccumulator T. caerulescens and the Cd-sensitive non-accumulator Arabidopsis. This comparative transcriptional analysis emphasized the role of genes involved in lignin, glutathione and sulphate metabolism. Furthermore the transcription factors MYB72 and bHLH100 were studied for their involvement in metal homeostasis, as they showed an altered expression after exposure to Cd. The Arabidopsis myb72 knockout mutant was more sensitive to excess Zn or iron (Fe) deficiency than wild type, while Arabidopsis transformants overexpressing bHLH100 showed increased tolerance to high Zn and nickel (Ni) compared to wild-type plants, confirming their role in metal homeostasis in Arabidopsis.

Cadmium-induced inhibition of photosynthesis and long-term acclimation to cadmium stress in the hyperaccumulator Thlaspi caerulescens.

Acclimation of hyperaccumulators to heavy metal-induced stress is crucial for phytoremediation and was investigated using the hyperaccumulator Thlaspi caerulescens and the nonaccumulators T. fendleri and T. ochroleucum. Spatially and spectrally resolved kinetics of in vivo absorbance and fluorescence were measured with a novel fluorescence kinetic microscope. At the beginning of growth on cadmium (Cd), all species suffered from toxicity, but T. caerulescens subsequently recovered completely. During stress, a few mesophyll cells in T. caerulescens became more inhibited and accumulated more Cd than the majority; this heterogeneity disappeared during acclimation. Chlorophyll fluorescence parameters related to photochemistry were more strongly affected by Cd stress than nonphotochemical parameters, and only photochemistry showed acclimation. Cd acclimation in T. caerulescens shows that part of its Cd tolerance is inducible and involves transient physiological heterogeneity as an emergency defence mechanism. Differential effects of Cd stress on photochemical vs nonphotochemical parameters indicate that Cd inhibits the photosynthetic light reactions more than the Calvin-Benson cycle. Differential spectral distribution of Cd effects on photochemical vs nonphotochemical quenching shows that Cd inhibits at least two different targets in/around photosystem II (PSII). Spectrally homogeneous maximal PSII efficiency (F(v)/F(m)) suggests that in healthy T. caerulescens all chlorophylls fluorescing at room temperature are PSII-associated.

Expression and functional analysis of metal transporter genes in two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens.

Zinc (Zn) hyperaccumulation is a constitutive property of Thlaspi caerulescens, whereas cadmium (Cd) hyperaccumulation varies greatly among different ecotypes. The molecular basis of this variation is unknown. Ecotypic differences in the sequences and expression of four representative ZIP family transporter genes were investigated. Genome analysis indicated the presence of at least two closely related copies of the TcIRT1 gene in both Ganges (high Cd accumulating) and Prayon (low Cd accumulating) ecotypes, with different copies being expressed in each, and, furthermore, the two genes potentially encode different length transcripts. The predominant transcript in Prayon was truncated, missing sequence coding for the putative metal-binding site and the five C-terminal transmembrane helices. The two ecotypes were grown hydroponically +/-Fe and Cd, and mRNA abundance determined for four ZIP genes. The four ZIP genes studied (TcIRT1, TcIRT2, TcZNT1, and TcZNT5) were expressed in roots only. TcIRT1 expression (full-length in Ganges, TcIRT1-1G; truncated in Prayon, TcIRT1-2P) was enhanced by Fe deficiency or by exposure to Cd. TcIRT2 expression was induced by Fe deficiency, but was unaffected by Cd exposure. TcZNT5-G showed greater expression in Prayon compared with Ganges. The functions of TcIRT1 from Ganges and Prayon and the Arabidopsis homologue were analysed by heterologous expression in yeast. All three IRT1 genes were able to facilitate growth on low Fe concentrations. Cd sensitivity of yeast was conferred in the order AtIRT1>TcIRT1-1G>TcIRT1-2P (truncated). Cd uptake after 4 h was only detectable following complementation by AtIRT1. The results suggest that although TcIRT1-G may be involved in Cd hyperaccumulation in the Ganges ecotype of T. caerulescens, the transporter expressed in yeast does not have an enhanced ability to transport Cd compared with AtIRT1. Therefore, the unique Cd-accumulating ability of the T. caerulescens Ganges ecotype must be due to the levels of expression of the protein or to other factors such as interacting proteins.

Reaction norms of life history traits in response to zinc in Thlaspi caerulescens from metalliferous and nonmetalliferous sites.

* We examined phenotypic plasticity of fitness components in response to zinc (Zn) in the Zn hyperaccumulator, Thlaspi caerulescens. * Two populations from Zn-enriched soils (M) and two populations from normal soils (NM) were grown in pots at three Zn concentrations (0, 1000 and 8000 mg kg(-1) Zn), for an entire life cycle. Growth, Zn accumulation and fitness components were assessed. * Based on vegetative growth, M and NM populations had similar Zn tolerance at 1000 mg kg(-1) Zn. However, reproductive output was markedly decreased in NM at 1000 and 8000 mg kg(-1) Zn. In M populations, Zn did not affect fitness. However, low Zn status enhanced reproductive output in year 1 compared with year 2 and decreased survival after the first flowering season. * M populations are able to achieve equal fitness across a broad range of Zn concentrations in soil by different combinations of fecundity and longevity. No cost of higher tolerance was demonstrated in M populations. Reproductive traits appeared to be a more sensitive indicator of tolerance than vegetative growth.