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.     

Nicotianamine Over-accumulation Confers Resistance to Nickel in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Nicotianamine is a methionine derivative involved in iron homeostasis, able to bind various other metals in vitro. To investigate its role in vivo, we expressed a nicotianamine synthase cDNA (TcNAS1) isolated from the polymetallic hyperaccumulator Thlaspi caerulescens in Arabidopsis thaliana. Transgenic plants expressing TcNAS1 over-accumulated NA, up to 100-fold more than wild type plants. Furthermore, increased NA levels in different transgenic lines were quantitatively correlated with increased nickel tolerance. The tolerance to nickel is expressed at the cellular level in protoplast experiments and is associated with an increased NA content. We have also shown that the most NA-over accumulating line showed a high tolerance to nickel and a significant Ni accumulation in the leaves when grown on nickel-contaminated soil. Our results highlight a new potential role for nicotianamine in heavy metal tolerance at the cellular but also at the whole plant level, easily transposable to a non-tolerant non-hyperaccumulator species. These results open new perspectives for the modulation of nicotianamine content in plants for phytoremediation.     

Cadmium tolerance and antioxidative defenses in hairy roots of the cadmium hyperaccumulator,Thlaspi caerulescens

Plant species capable of hyperaccumulating heavy metals are of considerable interest for phytoremediation and phytomining. This work aims to identify the role of antioxidative metabolism in heavy metal tolerance in the Cd hyperaccumulator, Thlaspi caerulescens. Hairy roots of T. caerulescens and the non-hyperaccumulator, Nicotiana tabacum (tobacco), were used to test the effects of high Cd environments. In the absence of Cd, endogenous activities of catalase were two to three orders of magnitude higher in T. caerulescens than in N. tabacum. T. caerulescens roots also contained significantly higher endogenous superoxide dismutase activity and glutathione concentrations. Exposure to 20 ppm (178 microM) Cd prevented growth of N. tabacum roots and increased hydrogen peroxide (H(2)O(2)) levels by a factor of five relative to cultures without Cd. In contrast, growth was maintained in T. caerulescens, and H(2)O(2) concentrations were controlled to low, nontoxic levels in association with a strong catalase induction response. Treatment of roots with the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), exacerbated H(2)O(2) accumulation in Cd-treated N. tabacum, but had a relatively minor effect on H(2)O(2) levels and did not reduce Cd tolerance in T. caerulescens. Lipid peroxidation was increased by Cd treatment in both the hyperaccumulator and non-hyperaccumulator roots. This work demonstrates that metal-induced oxidative stress occurs in hyperaccumulator tissues even though growth is unaffected by the presence of heavy metals. It also suggests that superior antioxidative defenses, particularly catalase activity, may play an important role in the hyperaccumulator phenotype of T. caerulescens.     

Organic acid complexation,heavy metal distribution and the effect of ATPase inhibition in hairy roots of hyperaccumulator plant species

Heavy metal uptake and distribution were investigated in hairy roots of the Cd hyperaccumulator, Thlaspi caerulescens, and the Ni hyperaccumulator, Alyssum bertolonii. Hairy roots of both species contained high constitutive levels of citric, malic and malonic acids. After treatment with 20 ppm Cd or 25 ppm Ni, about 13% of the total Cd in T. caerulescens roots and 28% of the total Ni in A. bertolonii were associated with organic acids. T. caerulescens and A. bertolonii hairy roots remained healthy and grew well at high concentrations of Cd and Ni, respectively, whereas hairy roots of the non-hyperaccumulator, Nicotiana tabacum, did not. Most of the Cd in T. caerulescens and N. tabacum roots was localised in the cell walls. In contrast, 85-95% of the Ni in A. bertolonii and N. tabacum was associated with the symplasm. Growth of T. caerulescens and A. bertolonii hairy roots was severely reduced in the presence of diethylstilbestrol (DES), an inhibitor of plasma membrane H(+)-ATPase. Treatment with DES increased the concentration of Cd in the symplasm of T. caerulescens about 6-fold with retention of root viability, whereas viability and Ni transport across the plasma membrane were both reduced in A. bertolonii. These results suggest that the mechanisms of Cd tolerance and hyperaccumulation in T. caerulescens hairy roots are capable of withstanding the effects of plasma membrane depolarisation, whereas Ni tolerance and hyperaccumulation in A. bertolonii hairy roots are not.     

Soil microbial diversity as affected by the rhizosphere of the hyperaccumulator Thlaspi caerulescens under natural conditions

It is hypothesized that metal hyperaccumulator plants have specific rhizosphere conditions, potentially modifying the bioavailability of soil metals. This article aims to further the knowledge about the rhizosphere of the hyperaccumulator Thlaspi caerulescens, focusing on its microflora isolated from metalliferous soils collected in situ where the plants grow naturally. We characterized the cultivable microbial communities isolated from the rhizosphere of one population of this Ni hyperaccumulator species grown on a serpentine soil. The rhizosphere soil harbored a wide variety of microorganisms, predominantly bacteria, confirming the stimulatory effect of the T. caerulescens rhizosphere on microbial growth and proliferation. We tested the hypothesis that the rhizosphere of T. caerulescens influences (1) the metabolic diversity of the bacterial community and (2) the bacterial resistance to metals. The principal component analysis of the Biolog plate's data confirmed a structural effect of the rhizosphere of T. caerulescens on bacterial communities. The percentage of Ni-resistant bacteria was higher in the rhizosphere than in the bulk soil, suggesting a direct effect of the rhizosphere on Ni tolerance, reflecting a greater bacterial tolerance to Ni in the rhizosphere.     

Nickel translocation via the phloem in the hyperaccumulator Noccaea caerulescens (Brassicaceae)

Plant and Soil - This study evaluated the effect of phloem translocation on Ni accumulation in the hyperaccumulator Noccaea caerulescens. The first experiment assessed the metal and organic...     

Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescens (J. & C. Presl)

Thlaspi caerulescens (J. & C. Presl, "Prayon") is a heavy-metal hyperaccumulator that accumulates Zn and Cd to high concentrations (40,000 and 4,000 mg kg DW-1 respectively) without phytotoxicity. The mechanism of Cd tolerance has not been characterized but reportedly involves vacuolar sequestration. The role of phytochelatins (PCs) in metal tolerance in T. caerulescens and the related non-accumulator T. arvense was examined. Although PCs were produced by both species in response to Cd, these peptides do not appear to be involved in metal tolerance in the hyperaccumulator. Leaf and root PC levels for both species showed a similar positive correlation with tissue Cd, but total PC levels in the hyperaccumulator were generally lower, despite correspondingly higher metal concentrations. The lack of a role for PCs in the hyperaccumulator's response to metal stress suggests that other mechanisms are responsible Cd tolerance. The lower level of leaf PCs in T. caerulescens also implies that Cd in the shoot is sequestered in a compartment or form that does not elicit a PC response.     

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.     

Heavy metal tolerance and accumulation in metallicolous and non-metallicolous populations of Thlaspi caerulescens from continental Europe

In continental Europe, the heavy metal hyperaccumulator Thlaspi caerulescens occurs both on heavy-metal polluted soils (subsp. calaminare) and on soils with normal heavy metal content (subsp. caerulescens). In order to assess the extent and partitioning of variation in heavy metal tolerance and foliar mineral composition, twelve families from two populations of each subspecies were grown in pots in four soil treatments differing in heavy metal (Zn, Pb) and macronutrient concentrations. The two subspecies differed systematically in many respects. Subsp. calaminare had a higher survival at high levels of heavy metals and a higher tolerance index in all treatments. It also had three times lower foliar zinc and lead concentrations when grown at moderate levels of heavy metals. This, together with a negative correlation of foliar Pb concentration with growth in subsp. caerulescens, suggests that heavy metal accumulation per se is not a mechanism of tolerance in this species. Variation among families within populations accounted for a larger proportion of total variance in growth and mineral composition than variation between populations. Additionally, within population variation in heavy metal tolerance and accumulation was significantly lower in subsp. calaminare. This suggests that, adding to a background constitutive tolerance at the species level, natural selection has increased heavy metal tolerance in metallicolous populations of Thlaspi caerulescens.     

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.     

Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase

Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 microg g(-1) Zn and 10,000 microg g(-1) Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils. In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T. caerulescens. A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P1B subfamily of purported heavy metal-translocating ATPases. A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead [Pb], and copper [Cu]) out of the cell. However, in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance. Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens. Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding. When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation. The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.     

The Role of Metal Transport and Tolerance in Nickel Hyperaccumulation by Thlaspi goesingense Halacsy

Metal hyperaccumulators are plants that are capable of extracting metals from the soil and accumulating them to extraordinary concentrations in aboveground tissues (greater than 0.1% dry biomass Ni or Co or greater than 1% dry biomass Zn or Mn). Approximately 400 hyperaccumulator species have been identified, according to the analysis of field-collected specimens. Metal hyperaccumulators are interesting model organisms to study for the development of a phytoremediation technology, the use of plants to remove pollutant metals from soils. However, little is known about the molecular, biochemical, and physiological processes that result in the hyperaccumulator phenotype. We investigated the role of Ni tolerance and transport in Ni hyperaccumulation by Thlaspi goesingense, using plant biomass production, evapotranspiration, and protoplast viability assays, and by following short- and long-term uptake of Ni into roots and shoots. As long as both species (T. goesingense and Thlaspi arvense) were unaffected by Ni toxicity, the rates of Ni translocation from roots to shoots were the same in both the hyper- and nonaccumulator species. Our data suggest that Ni tolerance is sufficient to explain the Ni hyperaccumulator phenotype observed in hydroponically cultured T. goesingense when compared with the Ni-sensitive nonhyperaccumulator T. arvense.     

Hyperaccumulation of cadmium by hairy roots of Thlaspi caerulescens

Hairy roots were used to investigate cadmium uptake by Thlaspi caerulescens, a metal hyperaccumulator plant with potential applications in phytoremediation and phytomining. Experiments were carried out in nutrient media under conditions supporting root growth. Accumulation of Cd in short-term (9-h) experiments varied with initial medium pH and increased after treating the roots with H(+)-ATPase inhibitor. The highest equilibrium Cd content measured in T. caerulescens roots was 62,800 microg g(-1) dry weight, or 6.3% dry weight, at a liquid Cd concentration of 3710 ppm. Cd levels in live T. caerulescens roots were 1.5- to 1.7-fold those in hairy roots of nonhyperaccumulator species exposed to the same Cd concentration, but similar to the Cd content of autoclaved T. caerulescens roots. The ability to grow at Cd concentrations of up to 100 ppm clearly distinguished T. caerulescens hairy roots from the nonhyperaccumulators. The specific growth rate of T. caerulescens roots was essentially unaffected by 20 to 50 ppm Cd in the culture medium; in contrast, N. tabacum roots turned dark brown at 20 ppm and growth was negligible. Up to 10,600 microg g(-1) dry weight Cd was accumulated by growing T. caerulescens hairy roots. Measurement of Cd levels in whole roots and in the cell wall fraction revealed significant differences in the responses of T. caerulescens and N. tabacum roots to 20 ppm Cd. Most metal was transported directly into the symplasm of N. tabacum roots within 3 days of exposure; in contrast, T. caerulescens roots stored virtually all of their Cd in the wall fraction for the first 7 to 10 days. This delay in transmembrane uptake may represent an important defensive strategy against Cd poisoning in T. caerulescens, allowing time for activation of intracellular mechanisms for heavy metal detoxification.     

Plant Cd2+ and Zn2+ status effects on root and shoot heavy metal accumulation in Thlaspi caerulescens

* In this study we address the impact of changes in plant heavy metal, (i.e. zinc (Zn) and cadmium (Cd)) status on metal accumulation in the Zn/Cd hyperaccumulator, Thlaspi caerulescens. * Thlaspi caerulescens plants were grown hydroponically on both high and low Zn and Cd regimes and whole-shoot and -root metal accumulation, and root (109)Cd(2+) influx were determined. * High-Zn-grown (500 microm Zn) plants were found to be more Cd-tolerant than plants grown in standard Zn conditions (1 microm Zn). Furthermore, shoot Cd accumulation was significantly greater in the high-Zn-grown plants. A positive correlation was also found between shoot Zn accumulation and increased plant Cd status. Radiotracer (109)Cd root flux experiments demonstrated that high-Zn-grown plants maintained significantly higher root Cd(2+) influx than plants grown on 1 microm Zn. It was also found that both nickel (Ni) and copper (Cu) shoot accumulation were stimulated by high plant Zn status, while manganese (Mn) accumulation was not affected. * A speculative model is presented to explain these findings, suggesting that xylem loading may be one of the key sites responsible for the hyperaccumulation of Zn and Cd accumulation in Thlaspi caerulescens.     

Variation in HMA4 gene copy number and expression among Noccaea caerulescens populations presenting different levels of Cd tolerance and accumulation

There is huge variability among populations of the hyperaccumulator Noccaea caerulescens (formerly Thlaspi caerulescens) in their capacity to tolerate and accumulate cadmium. To gain new insights into the mechanisms underlying this variability, we estimated cadmium fluxes and further characterized the N. caerulescens heavy metal ATPase 4 (NcHMA4) gene in three populations (two calamine, Saint-Félix-de-Pallières, France and Prayon, Belgium; one serpentine, Puente Basadre, Spain) presenting contrasting levels of tolerance and accumulation. Cadmium uptake and translocation varied among populations in the same way as accumulation; the population with the highest cadmium concentration in shoots (Saint Félix-de-Pallières) presented the highest capacity for uptake and translocation. We demonstrated that the four NcHMA4 copies identified in a previous study are not fixed at the species level, and that the copy truncated in the C-terminal part encodes a functional protein. NcHMA4 expression and gene copy number was lower in the serpentine population, which was the least efficient in cadmium translocation compared to the calamine populations. NcHMA4 expression was associated with the vascular tissue in all organs, with a maximum at the crown. Overall, our results indicate that differences in cadmium translocation ability of the studied populations appear to be controlled, at least partially, by NcHMA4, while the overexpression of NcHMA4 in the two calamine populations may result from convergent evolution.     

Constitutively elevated salicylic acid signals glutathione-mediated nickel tolerance in Thlaspi nickel hyperaccumulators

Progress is being made in understanding the biochemical and molecular basis of nickel (Ni)/zinc (Zn) hyperaccumulation in Thlaspi; however, the molecular signaling pathways that control these mechanisms are not understood. We observed that elevated concentrations of salicylic acid (SA), a molecule known to be involved in signaling induced pathogen defense responses in plants, is a strong predictor of Ni hyperaccumulation in the six diverse Thlaspi species investigated, including the hyperaccumulators Thlaspi goesingense, Thlaspi rosulare, Thlaspi oxyceras, and Thlaspi caerulescens and the nonaccumulators Thlaspi arvense and Thlaspi perfoliatum. Furthermore, the SA metabolites phenylalanine, cinnamic acid, salicyloyl-glucose, and catechol are also elevated in the hyperaccumulator T. goesingense when compared to the nonaccumulators Arabidopsis (Arabidopsis thaliana) and T. arvense. Elevation of free SA levels in Arabidopsis, both genetically and by exogenous feeding, enhances the specific activity of serine acetyltransferase, leading to elevated glutathione and increased Ni resistance. Such SA-mediated Ni resistance in Arabidopsis phenocopies the glutathione-based Ni tolerance previously observed in Thlaspi, suggesting a biochemical linkage between SA and Ni tolerance in this genus. Intriguingly, the hyperaccumulator T. goesingense also shows enhanced sensitivity to the pathogen powdery mildew (Erysiphe cruciferarum) and fails to induce SA biosynthesis after infection. Nickel hyperaccumulation reverses this pathogen hypersensitivity, suggesting that the interaction between pathogen resistance and Ni tolerance and hyperaccumulation may have played a critical role in the evolution of metal hyperaccumulation in the Thlaspi genus.     

Natural variation among Arabidopsis accessions reveals malic acid as a key mediator of Nickel (Ni) tolerance

Plants have evolved various mechanisms for detoxification that are specific to the plant species as well as the metal ion chemical properties. Malic acid, which is commonly found in plants, participates in a number of physiological processes including metal chelation. Using natural variation among Arabidopsis accessions, we investigated the function of malic acid in Nickel (Ni) tolerance and detoxification. The Ni-induced production of reactive oxygen species was found to be modulated by intracellular malic acid, indicating its crucial role in Ni detoxification. Ni tolerance in Arabidopsis may actively involve malic acid and/or complexes of Ni and malic acid. Investigation of malic acid content in roots among tolerant ecotypes suggested that a complex of Ni and malic acid may be involved in translocation of Ni from roots to leaves. The exudation of malic acid from roots in response to Ni treatment in either susceptible or tolerant plant species was found to be partially dependent on AtALMT1 expression. A lower concentration of Ni (10?μM) treatment induced AtALMT1 expression in the Ni-tolerant Arabidopsis ecotypes. We found that the ecotype Santa Clara (S.C.) not only tolerated Ni but also accumulated more Ni in leaves compared to other ecotypes. Thus, the ecotype S.C. can be used as a model system to delineate the biochemical and genetic basis of Ni tolerance, accumulation, and detoxification in plants. The evolution of Ni hyperaccumulators, which are found in serpentine soils, is an interesting corollary to the fact that S.C. is also native to serpentine soils.     

Relationships of nicotianamine and other amino acids with nickel, zinc and iron in Thlaspi hyperaccumulators

Experimental evidence suggests that nicotianamine (NA) is involved in the complexation of metal ions in some metal-hyperaccumulating plants. Closely-related nickel (Ni)- and zinc (Zn)-hyperaccumulating species were studied to determine whether a correlation exists between the Ni and Zn concentrations and NA in foliar tissues. A liquid chromatography-mass spectrometry (LC-MS) procedure was developed to quantify the NA and amino acid contents using the derivatizing agent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. A strong correlation emerged between Ni and NA, but not between Zn and NA. Concentrations of NA and L-histidine (His) also increased in response to higher Ni concentrations in the hydroponic solution supplied to a serpentine population of Thlaspi caerulescens. An inversely proportional correlation was found between the iron (Fe) and Ni concentrations in the leaves. Correlations were also found between Zn and asparagine. The results obtained in this study suggest that NA is involved in hyperaccumulation of Ni but not Zn. The inverse proportionality between the Ni and Fe concentrations in the leaf may suggest that Ni and Fe compete for complexation to NA.