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.     

Isolation,characterization, and identification of bacteria associated with the zinc hyperaccumulator Thlaspi caerulescens subsp. calaminaria

We investigated bacterial populations associated with the Zn hyperaccumulator Thlaspi caerulescens subsp. calaminaria grown in a soil collected from an abandoned Zn-Pb mine and smelter in Plombières, Belgium. The bacterial population of the nonrhizospheric soil consisted of typical soil bacteria, some exhibiting multiple heavy-metal resistance characteristics that often are associated with polluted substrates: 7.8% and 4% of the population survived in the presence of elevated levels of Zn (1 mM) and Cd (0.8 mM), respectively. For the bacterial population isolated from the rhizosphere, the comparable survival rates were 88 and 78%. This observation indicates a selective enrichment of the metal-resistant strains due to an increased availability of the metals in soils near the roots compared with nonrhizospheric soil. The endophytic inhabitants of the roots and shoots were isolated, identified, and characterized. Although similar endophytic species were isolated from both compartments, those from the rhizoplane and roots showed lower resistance to Zn and Cd than the endophytic bacteria isolated from the shoots. In addition, root endophytic bacteria had additional requirements. Contrary to the rootresiding inhabitants, the shoot represented a niche rich in metal-resistant bacteria and even seemed to contain species that were exclusively abundant there. These differences in the characteristics of the bacterial microflora associated with T. caerulescens might possibly reflect altered metal speciation in the different soils and plant compartments studied.     

Root development of the Zinc-hyperaccumulator plant Thlaspi caerulescens as affected by metal origin,content and localization in soil

Penetration into and exploitation of contaminated soils by roots of hyperaccumulator plants is a prerequisite for efficient removal of heavy metals, i.e. efficacy of phytoextraction. This work was undertaken to study the development of roots of the Zn-hyperaccumulator Thlaspi caerulescens under various conditions of soil contamination. Rhizoboxes were constructed with a removable plastic front cover, and filled with soils containing different amounts and forms of metals (Zn, Cd and Pb). Treatments were: homogeneous soil profile, superposition of three layers, inclusion of contaminated soil into uncontaminated soil, or inclusion of uncontaminated soil into uniformly contaminated soil. Four seedlings were transplanted into each rhizobox, and development of the root system was periodically recorded for 133 days. At harvest, the biomass and size of the rosette of aerial parts were determined. The aerial biomass/root length fraction as well as the kinetics of root development varied according to the presence and localization of Zn. The distribution and morphology of roots at harvest were strongly dependent upon the metal content and form in soil. Roots exhibited a high affinity for the Zn-contaminated patches and showed two distinct morphologies according to the concentration of Zn in soil. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

The plant CDF family member TgMTP1 from the Ni/Zn hyperaccumulator Thlaspi goesingense acts to enhance efflux of Zn at the plasma membrane when expressed in Saccharomyces cerevisiae

To avoid metal toxicity, organisms have evolved mechanisms including efflux of metal ions from cells and sequestration into internal cellular compartments. Members of the ubiquitous cation diffusion facilitator (CDF) family are known to play an important role in these processes. Overexpression of the plant CDF family member metal tolerance protein 1 (MTP1) from the Ni/Zn hyperaccumulator Thlaspi goesingense (TgMTP1), in the Saccharomyces cerevisiaeDelta zinc resistance conferring (zrc)1Delta cobalt transporter (cot)1 double mutant, suppressed the Zn sensitivity of this strain. T. goesingense was found to contain several allelic variants of TgMTP1, all of which confer similar resistance to Zn in Deltazrc1Deltacot1. Similarly, MTP1 from various hyperaccumulator and non-accumulator species also confer similar resistance to Zn. Deltazrc1Deltacot1 lacks the ability to accumulate Zn in the vacuole and has lower accumulation of Zn after either long- or short-term Zn exposure. Expression of TgMTP1 in Deltazrc1Deltacot1 leads to further lowering of Zn accumulation and an increase in Zn efflux from the cells. Expression of TgMTP1 in a V-type ATPase-deficient S. cerevisiae strain also confers increased Zn resistance. In vivo and in vitro immunological staining of hemagglutinin (HA)-tagged TgMTP1::HA reveals the protein to be localized in both the S. cerevisiae vacuolar and plasma membranes. Taken together, these data are consistent with MTP1 functioning to enhance plasma membrane Zn efflux, acting to confer Zn resistance independent of the vacuole in S. cerevisiae. Transient expression in Arabidopsis thaliana protoplasts also reveals that TgMTP1::green fluorescent protein (GFP) is localized at the plasma membrane, suggesting that TgMTP1 may also enhance Zn efflux in plants.     

Life history variation in the heavy metal tolerant plant Thlaspi caerulescens growing in a network of contaminated and noncontaminated sites in southern France: role of gene flow, selection and phenotypic plasticity

* Here we explore life history differences in a set of neighbouring metallicolous and nonmetallicolous populations of the heavy metal tolerant plant Thlaspi caerulescens. * We contrasted data from field observations and from a common garden experiment, in which soil zinc (Zn) concentration and light availability were manipulated, and data on microsatellite molecular variation. * The two ecotypes showed few differences in life history in the field, but large differences in their response to Zn concentration in the common garden. Soil toxicity affected most characters in nonmetallicolous plants, while it had no effect on metallicolous plants. The two ecotypes responded similarly to light. Genetic differentiation for quantitative characters between ecotypes contrasted with the absence of differentiation for microsatellites. Conversely, populations of the same ecotype showed similar responses to Zn, despite their high differentiation for molecular markers. * We conclude that divergent selection related to soil toxicity has had a predominant role in shaping life history differences between ecotypes, gene flow weakly opposing local adaptation despite geographical proximity.     

Structural element responsible for the Fe(III)-phytosiderophore specific transport by HvYS1 transporter in barley

Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter for Fe(III)-phytosiderophores, involved in primary iron acquisition from soils in barley roots. In contrast, Zea mays yellow stripe 1 (ZmYS1) in maize possesses broad substrate specificity, despite a high homology with HvYS1. Here we revealed, by assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, that the outer membrane loop between the sixth and seventh transmembrane regions is essential for substrate specificity. Circular dichroism spectra indicated that a synthetic peptide corresponding to the loop of HvYS1 forms an alpha-helix in solution, whereas that of ZmYS1 is flexible. We propose that the structural difference at this particular loop determines the substrate specificity of the HvYS1 transporter.     

Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd-hyperaccumulating ecotype of Thlaspi caerulescens

Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.     

The Wingless target gene Dfz3 encodes a new member of the Drosophila Frizzled family

Here we report the identification of Dfz3, a novel member of the Frizzled family of seven-pass transmembrane receptors. Like Dfz2, Dfz3 is a target gene of Wingless (Wg) signalling, but in contrast to Dfz2, it is activated rather than repressed by Wg signalling in imaginal discs. We show that Dfz3 is not required for viability but is necessary for optimal Wg signalling at the wing margin.     

Structure and function of a model member of the SulP transporter family

SHST1 is a sulfate transporter that belongs to a large and diverse family of anion transporters. Little is known about the structure and function of any member of the family. Site-directed mutagenesis of SHST1 is being used to understand the function of particular amino acids. We have mutated highly conserved amino acid residues and the results suggest that the first two helices play an important role in the transport pathway. Furthermore, mutation of equivalent residues to those altered in human genetic diseases produces deleterious effects in SHST1. These results suggest that there are similarities in the molecular mechanism of transport throughout the family and the information obtained with SHST1 may be applicable to the entire family.