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Lupinus species have been sporadically reported to be colonized by arbuscular mycorrhizal fungi (AMF). The interactions between AMF and lupine plants could also be non-symbiotic, from positive to negative, as controlled by the stress conditions of the plant. The goal of the study was to reveal the existence of such positive interactions and provide preliminary data for a myco-phytoremediation technology of mining dumps using L. angustifolius as a first crop. The objective was to test the hypothesis that the AMF inoculation of an acidified dump material contaminated with heavy metals would improve the growth of L. angustifolius and decrease oxidative stress. The design consisted of a one-month bivariate pot experiment with plants grown in a mining dump soil inoculated and not inoculated with a commercial AMF inoculum sequestered in expanded clay and watered with acidic and neutral water. There was no AMF root colonization under the experimental conditions, but under neutral and acidic water conditions, the phosphorus concentrations in roots and leaves increased, and the superoxide dismutase and peroxidase activities significantly decreased due to AMF inoculation. The increase in leaf phosphorus concentration was correlated with the decrease in peroxidase activity. The fresh weight of shoots and leaves significantly increased due to the commercial inoculum (under acidic water conditions). At the end of the experiment, the ammonium concentration in the substrate was higher in the inoculated treatments than in the not inoculated ones, and the concentrations of many elements in the dump material decreased compared to the start of the experiment. A comprehensive discussion of the potential mechanisms underlying the effects of the commercial AMF inoculum on the non-host L. angustifolius is completed.

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The idea of linking stressors, services providing units (SPUs), and ecosystem services (ES) is ubiquitous in the literature, although is currently not applied in areas contaminated with heavy metals (HMs), This integrative literature review introduces the general form of a deterministic conceptual model of the cross-scale effect of HMs on biogeochemical services by SPUs with a feedback loop, a cross-scale heuristic concept of resilience, and develops a method for applying the conceptual model. The objectives are 1) to identify the clusters of existing research about HMs effects on ES, biodiversity, and resilience to HMs stress, 2) to map the scientific fields needed for the conceptual model's implementation, identify institutional constraints for inter-disciplinary cooperation, and propose solutions to surpass them, 3) to describe how the complexity of the cause-effect chain is reflected in the research hypotheses and objectives and extract methodological consequences, and 4) to describe how the conceptual model can be implemented. A nested analysis by CiteSpace of a set of 16,176 articles extracted from the Web of Science shows that at the highest level of data aggregation there is a clear separation between the topics of functional traits, stoichiometry, and regulating services from the typical issues of the literature about HMs, biodiversity, and ES. Most of the resilience to HMs stress agenda focuses on microbial communities. General topics such as the biodiversity-ecosystem function relationship in contaminated areas are no longer dominant in the current research, as well as large-scale problems like watershed management. The number of Web of Science domains that include the analyzed articles is large (26 up to 87 domains with at least ten articles, depending on the sub-set), but thirteen domains account for 70-80% of the literature. The complexity of approaches regarding the cause-effect chain, the stressors, the biological and ecological hierarchical level and the management objectives was characterized by a detailed analysis of 60 selected reviews and 121 primary articles. Most primary articles approach short causal chains, and the number of hypotheses or objectives by article tends to be low, pointing out the need for portfolios of complementary research projects in coherent inter-disciplinary programs and innovation ecosystems to couple the ES and resilience problems in areas contaminated with HMs. One provides triggers for developing innovation ecosystems, examples of complementary research hypotheses, and an example of technology transfer. Finally one proposes operationalizing the conceptual methodological model in contaminated socio-ecological systems by a calibration, a sensitivity analysis, and a validation phase.

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The data set consists in a file with two sheets: one includes a matrix of 297 rows and 46 columns, and the second one a matrix of 12 rows and 24 columns. In the first sheet each row is a replicate of an experimental variant with Agrostis capillaris growing on tailing substrate belonging to three experiments witch have the same variants, but are organized at three scales. The data from all experiments are in the same table, with a column indicating by a code the experiment to which they belong. In the second spreadsheet there is a table with the relative plant species cover in the experimental field plots. Experimental design and interpretation of the data are provided in "Implications of spatial heterogeneity of tailing material and time scale of vegetation growth processes for the design of phytostabilisation" [1].

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An anthropic ecosystem from Romania was investigated from acarological, vegetation and chemical point of view. The community structures of two groups of mites were studied (Acari: Mesostigmata, Oribatida) from a tailing pond, using transect method, in correlation with concentrations of heavy metals (As, Cu, Pb, Ni, Mn and Zn), with abiotic factors (altitude, aspect, soil temperature, soil humidity, soil pH) and biotic factor (vegetation coverage). Taking into account the mite communities, in total, 30 mite species were identified, with 1009 individuals and 18 immatures (10 species with 59 individuals, 5 immatures of Mesostigmata and 20 species with 950 individuals, 13 immatures of Oribatida). The investigated habitats from the tailing pond were grouped in five transects, with different degree of pollution, based on total metal loads. Taking into account of the connection between mites communities, abiotic factors and heavy metals, each transect were characterized through specific relationship. Using multivariate statistical analysis, we revealed that the occurrence of some Oribatida species was strongly correlated with vegetation coverage, soil pH and soil humidity, though concentrations of Cu, As, Mn, Ni and Zn also had an influence. Pb and Zn concentrations were shown to influence the occurrence of Mesostigmata mites. The heterogeneity of mites species richness at 2 m2 scale was correlated with a metric related to the heterogeneity of heavy metals at the same scale.

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Phytostabilisation projects for tailing dams depend on processes occurring at spatial scales of 106 m2 and at decadal time scales. Most experiments supporting the design and monitoring of such projects have much smaller spatial and time scales. Usually, they are only designed for one single scale. Here, we report the results of three coupled experiments performed at pot, lysimeter and field plot scales using six sampling periodstimes from 3 to 20 months. The work explicitly accounts for the sampling times when evaluating the effects of amendments on the performance of plants grown in tailing substrates. Two treatments with potentially complementary roles were applied: zeolites to decrease availability of Cd, Cu, Pb and Zn and green fertilizer to increase the availability of nutrients. Zeolites have a positive influence on plant development, especially in the early stages. Analyses of the pooled datasets for all sampling times revealed the possibility of predicting plant physiological variables, such as protein concentrations, pigments and oxidative stress enzyme activities, as a function of the factors extracted by principal component analysis from the metal concentrations in plants, phosphorus concentrations in plants, and sampling times. Two potentially general methodological rules were extracted: account for the spatial geochemical variability of tailings, and cover the broadest possible range of time scales by experiments. The proposed experimental methodology can be of general use for the design of tailing dam remediation technologies with improvements involving the set of measured variables and sampling frequency and by carefully relating the costs to the institutional aspects of tailing dam management.

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The various applications of Ag(I) generated the necessity to obtain Ag(I)-accumulating organisms for the removal of surplus Ag(I) from contaminated sites or for the concentration of Ag(I) from Ag(I)-poor environments. In this study we obtained Ag(I)-accumulating cells by expressing plant metallothioneins (MTs) in the model Saccharomyces cerevisiae. The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) fused to myrGFP displaying an N-terminal myristoylation sequence for plasma membrane targeting were expressed in S. cerevisiae and checked for Ag(I)-related phenotype. The transgenic yeast cells were grown in copper-deficient media to ensure the expression of the plasma membrane high-affinity Cu(I) transporter Ctr1, and also to elude the copper-related inhibition of Ag(I) transport into the cell. All plant MTs expressed in S. cerevisiae conferred Ag(I) tolerance to the yeast cells. Among them, myrGFP-NcMT3 afforded Ag(I) accumulation under high concentration (10⁻50 µM), while myrGFP-AtMT1a conferred increased accumulation capacity under low (1 µM) or even trace Ag(I) (0.02⁻0.05 µM). The ability to tolerate high concentrations of Ag(I) coupled with accumulative characteristics and robust growth showed by some of the transgenic yeasts highlighted the potential of these strains for biotechnology applications.

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In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

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Accumulation of heavy metals without developing toxicity symptoms is a phenotype restricted to a small group of plants called hyperaccumulators, whose metal-related characteristics suggested the high potential in biotechnologies such as bioremediation and bioextraction. In an attempt to extrapolate the heavy metal hyperaccumulating phenotype to yeast, we obtained Saccharomyces cerevisiae cells armed with non-natural metal-binding hexapeptides targeted to the inner face of the plasma membrane, expected to sequester the metal ions once they penetrated the cell. We describe the construction of S. cerevisiae strains overexpressing metal-binding hexapeptides (MeBHxP) fused to the carboxy-terminus of a myristoylated green fluorescent protein (myrGFP). Three non-toxic myrGFP-MeBHxP (myrGFP-H6, myrGFP-C6, and myrGFP-(DE)3) were investigated against an array of heavy metals in terms of their effect on S. cerevisiae growth, heavy metal (hyper) accumulation, and capacity to remove heavy metal from contaminated environments.

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We investigated two Romanian industrial regions- Copsa Mica and Zlatna, to assess the current situation of soil pollution and bioaccumulation of Pb, Cd, Cu and Zn in different vegetable species and possible risks to consumers. Both total and mobile forms of the metals were determined in soil samples, and metal content in the edible parts of root vegetable samples was also assessed. The concentrations of Pb and Zn in soil were higher in Copsa Mica than in Zlatna (566mg/kg vs 271mg/kg for Pb and 1143mg/kg vs 368mg/kg for Zn)·The metal mobility in soil from Copsa Mica decreases in the order Zn>Cu>Cd>Pb (1.88mg/kg, 0.40mg/kg, 0.22mg/kg, 0.16mg/kg, respectively), while in Zlatna, the order was Cu>Zn>Pb>Cd (0.88mg/kg, 0.29mg/kg, 0.04mg/kg, 0.01mg/kg, respectively), apparently depending on metal and soil conditions. In Copsa Mica, the amount of Pb and Cd in vegetable samples exceeded the maximum permissible limits in carrots (median concentration 0.32mg/kg for Pb and Cd) and in yellow onions (median concentration 0.24mg/kg for Cd). In Zlatna region, the content of Cd exceeded the maximum limits in yellow onions (median concentration 0.11mg/kg). The amount of Pb was higher than the maximum acceptable level in carrots from the Zlatna region (median concentration 0.12mg/kg). Cu and Zn levels were within the normal range in all vegetable samples. In the Zlatna region, the transfer factors for Pb and Cd were higher in carrots (median values of 9.9 for Pb and 21.0 for Cd) compared to carrots harvested in Copsa Mica (median values of 4.0 for Pb and 2.0 for Cd). Daily intake rates of metals through local vegetable consumption exceeded the limit values established by the European Food Safety Authority for Pb (1.2 to 2.4 times) and Cd (5.5 to 9.1 times) in both regions, with potential adverse health effects for the local population. The results highlight the need for total soil remediation action before fruit and vegetables produced in these polluted areas can be safely consumed.

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Lanthanides are a group of non-essential elements with important imaging and therapeutic applications. Although trivalent lanthanide ions (Ln³âº) are used as potent blockers of Ca²âº channels, the systematic studies correlating Ln³âº accumulation and toxicity to Ca²âº channel blocking activity are scarce. In this study, we made use of the eukaryotic model Saccharomyces cerevisiae to investigate the correlation between Ln³âº accumulation, their toxicity and their capacity to block the exogenous stress-induced Ca²âº influx into the cytosol. It was found that the Ln³âº blocked the Ca²âº entry into the yeast cells only when present at concentration high enough to allow rapid binding to cell surface. At lower concentrations, Ln³âº were taken up by the cell, but Ca²âº blockage was no longer achieved. At 1 mM concentration, all ions from the Ln³âº series could block Ca²âº entry into cytosol with the exception of La³âº, and to a lesser extent, Pr³âº and Nd³âº. The plasma membrane Ca²âº-channel Cch1/Mid1 contributed to La³âº and Gd³âº entry into the cells, with a significant preference for La³âº. The results open the possibility to obtain cells loaded with controlled amounts and ratios of Ln³âº.

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The involvement of Ca(2+) in the response to high Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Hg(2+) was investigated in Saccharomyces cerevisiae. The yeast cells responded through a sharp increase in cytosolic Ca(2+) when exposed to Cd(2+), and to a lesser extent to Cu(2+), but not to Mn(2+), Co(2+), Ni(2+), Zn(2+), or Hg(2+). The response to high Cd(2+) depended mainly on external Ca(2+) (transported through the Cch1p/Mid1p channel) but also on vacuolar Ca(2+) (released into the cytosol through the Yvc1p channel). The adaptation to high Cd(2+) was influenced by perturbations in Ca(2+) homeostasis. Thus, the tolerance to Cd(2+) often correlated with sharp Cd(2+)-induced cytosolic Ca(2+) pulses, while the Cd(2+) sensitivity was accompanied by the incapacity to rapidly restore the low cytosolic Ca(2+).

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Blueberries (Vaccinium corymbosum L.) are a rich source of antioxidants and their consumption is believed to contribute to food-related protection against oxidative stress. In the present study, the chemoprotective action of blueberry extracts against cadmium toxicity was investigated using a cadmium-hypersensitive strain of Saccharomyces cerevisiae. Four varieties of blueberries were used in the study, and it was found that the extracts with high content of total anthocyanidins exhibited significant protective effect against the toxicity of cadmium and H2O2. Both the blueberry extracts and pure cyanidin exhibited protective effects against cadmium in a dose-dependent manner, but without significantly interfering with the cadmium accumulation by the yeast cells. The results imply that the blueberry extracts might be a potentially valuable food supplement for individuals exposed to high cadmium.

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We performed an experiment at pot scale to assess the effect of plant growth-promoting bacteria (PGPB) on the development of five plant species grown on a tailing dam substrate. None of the species even germinated on inoculated unamended tailing material, prompting use of compost amendment. The effect of inoculation on the amended material was to increase soil respiration, and promote elements immobilisation at plant root surface. This was associated with a decrease in the concentrations of elements in the leaching water and an increase of plant biomass, statistically significant in the case of two species: Agrostis capillaris and Festuca rubra. The experiment was repeated at lysimeter scale with the species showing the best development at pot scale, A. capillaris, and the significant total biomass increase as a result of inoculation was confirmed. The patterns of element distribution in plants also changed (the concentrations of metals in the roots of A. capillaris and F. rubra significantly decreased in inoculated treatments, while phosphorus concentration significantly increased in roots of A. capillaris in inoculated treatment at lysimeter scale). Measured variables for plant oxidative stress did not change after inoculations. There were differences of A. capillaris plant-soil system response between experimental scales as a result of different substrate column structure and plant age at the sampling moment. Soil respiration was significantly larger at lysimeter scale than at pot scale. Leachate concentrations of As, Mn and Ni had significantly larger concentrations at lysimeter scale than at pot scale, while Zn concentrations were significantly smaller. Concentrations of several metals were significantly smaller in A. capillaris at lysimeter scale than at pot scale. From an applied perspective, a system A. capillaris-compost-PGPB selected from the rhizosphere of the tailing dam native plants can be an option for the phytostabilisation of tailing dams. Results should be confirmed by investigation at field plot scale.

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Applied research programs in the remediation of contaminated areas can be used also for gaining insights in the physiological and ecological mechanisms supporting the resistance of plant communities in stress conditions due to toxic elements. The research hypothesis of this study was that in the heavily contaminated but nutrient-poor substrate of mine tailing dams, the beneficial effect of inoculation with arbuscular mychorrizal fungi (AMF) is due to an improvement of phosphorus nutrition rather than to a reduction of toxic element transfer to plants. A concept model assuming a causal chain from root colonization to element uptake, oxidative stress variables, and overall plant development was used. The methodological novelty lies in coupling in a single research program experiments conducted at three scales: pot, lysimeter, and field plot, with different ages of plants at the sampling moment (six subsets of samples in all). The inoculation with AMF in expanded clay carrier had a beneficial effect on the development of plants in the amended tailing substrate heavily contaminated with toxic elements. The effect of inoculation was stronger when the quantity of expanded carrier was smaller (1 % vs. 7 % inoculum), probably because of changes in substrate features. The improvement of plant growth was due mainly to an improvement in phosphorus nutrition leading to an increase of protein concentration and decrease of oxidative stress enzyme activity (superoxide dismutase and peroxidase). In a single data subset, an effect of inoculation on the uptake of several toxic elements could be proved (decrease of As concentration in plant roots correlated with a decrease of oxidative stress independent from the effect of P concentration increase). The multi-scale approach allowed us to find differences between the patterns characterising the data subsets. These subset-specific patterns point out the existence of physiological differences between plants in different development states (as a result of sampling at different plant ages). From an applied perspective, conclusions are drawn with respect to the use of plants in the monitoring programs of contaminated areas and the use of inoculation with AMF in the remediation of tailing dams.

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The planetary importance of microbial function requires urgently that our knowledge and our exploitation ability is extended, therefore every occasion of bioprospecting is welcome. In this work, bioprospecting is presented from the perspective of the UMBRELLA project, whose main goal was to develop an integral approach for remediation of soil influenced by mining activity, by using microorganisms in association with plants. Accordingly, this work relies on the cultivable fraction of microbial biodiversity, native to six mining sites across Europe, different for geographical, climatic and geochemical characteristics but similar for suffering from chronic stress. The comparative analysis of the soil functional diversity, resulting from the metabolic profiling at community level (BIOLOG ECOPlates) and confirmed by the multivariate analysis, separates the six soils in two clusters, identifying soils characterised by low functional diversity and low metabolic activity. The microbial biodiversity falls into four major bacterial phyla: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, including a total of 47 genera and 99 species. In each soil, despite harsh conditions, metabolic capacity of nitrogen fixation and plant growth promotion were quite widespread, and most of the strains showed multiple resistances to heavy metals. At species-level, Shannon's index (alpha diversity) and Sørensen's Similarity (beta diversity) indicates the sites are indeed diverse. Multivariate analysis of soil chemical factors and biodiversity identifies for each soil well-discriminating chemical factors and species, supporting the assumption that cultured biodiversity from the six mining sites presents, at phylum level, a convergence correlated to soil factors rather than to geographical factors while, at species level, reflects a remarkable local characterisation.

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Pho84p, the protein responsible for the high-affinity uptake and transport of inorganic phosphate across the plasma membrane, is also involved in the low-affinity uptake of heavy metals in the Saccharomyces cerevisiae cells. In the present study, the effect of PHO84 overexpression upon the heavy metal accumulation by yeast cells was investigated. As PHO84 overexpression triggered the Ire1p-dependent unfolded protein response, abundant plasma membrane Pho84p could be achieved only in ire1Δ cells. Under environmental surplus, PHO84 overexpression augmented the metal accumulation by the wild type, accumulation that was exacerbated by the IRE1 deletion. The pmr1Δ cells, lacking the gene that encodes the P-type ATPase ion pump that transports Ca(2+) and Mn(2+) into the Golgi, hyperaccumulated Mn(2+) even from normal medium when overexpressing PHO84, a phenotype which is rather restricted to metal-hyperaccumulating plants.

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