Locked up Inside the Vessels: Rare Earth Elements Are Transferred and Stored in the Conductive Tissues of the Accumulating Fern Dryopteris erythrosora.

Rare earth elements (REEs) are strategic metals strongly involved in low-carbon energy conversion. However, these emerging contaminants are increasingly disseminated into ecosystems, raising concern regarding their toxicity. REE-accumulating plants are crucial subjects to better understand REE transfer to the trophic chain but are also promising phytoremediation tools. In this analysis, we deciphered REE accumulation sites in the REE-accumulating fern Dryopteris erythrosora by synchrotron X-ray µfluorescence (µXRF). This technique allows a high-resolution and in situ analysis of fresh samples or frozen-hydrated cross sections of different organs of the plant. In the sporophyte, REEs were translocated from the roots to the fronds by the xylem sap and were stored within the xylem conductive system. The comparison of REE distribution and accumulation levels in the healthy and necrotic parts of the frond shed light on the differential mobility between light and heavy REEs. Furthermore, the comparison emphasized that necrotized areas were not the main REE-accumulating sites. Finally, the absence of cell-to-cell mobility of REEs in the gametophyte suggested the absence of REE-compatible transporters in photosynthetic tissues. These results provide valuable knowledge on the physiology of REE-accumulating ferns to understand the REE cycle in biological systems and the expansion of phytotechnologies for REE-enriched or REE-contaminated soils.

The potential of microorganisms as biomonitoring and bioremediation tools for mercury-contaminated soils.

Mercury (Hg) pollution is a global issue due to the high toxicity and wide dispersion of Hg around the world. Whether due to anthropogenic activities or natural processes, Hg emissions are steadily increasing, with very high levels in some regions, directly threatening human and ecosystem health. However, bacteria and fungi have evolved and adapted in response to Hg-induced stress and have developed tolerance mechanisms, notably based on the mer operon system that is involved in Hg uptake and biovolatilization via Hg reduction reactions. Other processes, such as bioaccumulation or extracellular sequestration, are involved in Hg resistance, and the study of contaminated soils has allowed the isolation of a number of microorganisms capable of these mechanisms, with strong potential for the implementation of bioremediation approaches. In addition to playing an important role in determining the fate of Hg in the biogeochemical cycle, these microorganisms can indeed be applied to reduce Hg concentrations or at least stabilize Hg for the remediation of polluted soils. Moreover, thanks to the development of biotechnological tools, bioremediation based on Hg-tolerant microorganisms can be optimized. Finally, these microorganisms are relevant candidates for biomonitoring, for example, through the engineering of biosensors, because the detection of Hg is a major issue in preserving the health of living beings.

Interactions between Hg and soil microbes: microbial diversity and mechanisms, with an emphasis on fungal processes.

Mercury (Hg) is a highly toxic metal with no known biological function, and it can be highly bioavailable in terrestrial ecosystems. Although fungi are important contributors to a number of soil processes including plant nutrient uptake and decomposition, little is known about the effect of Hg on fungi. Fungi accumulate the largest amount of Hg and are the organisms capable of the highest bioaccumulation of Hg. While referring to detailed mechanisms in bacteria, this mini-review emphasizes the progress made recently on this topic and represents the first step towards a better understanding of the mechanisms underlying Hg tolerance and accumulation in fungal species and hence on the role of fungi within the Hg cycle at Hg-contaminated sites. KEY POINTS: • The fungal communities are more resilient than bacterial communities to Hg exposure. • The exposure to Hg is a threat to microbial soil functions involved in both C and nutrient cycles. • Fungal (hyper)accumulation of Hg may be important for the Hg cycle in terrestrial environments. • Understanding Hg tolerance and accumulation by fungi may lead to new remediation biotechnologies.

Insect Life Traits Are Key Factors in Mercury Accumulation and Transfer within the Terrestrial Food Web.

As plants and associated insects are at the bottom of some terrestrial food webs, they are the primary contributors to mercury (Hg) fluxes in ecosystems. In addition to the trophic position of these organisms, factors related to their life traits have been hypothesized to influence their exposure to Hg. This study investigates the transfer of Hg in a soil-nettle-insect system and the insect-related factors affecting their Hg concentrations in a revegetated chlor-alkali landfill. Twenty-three insect species were identified and classified according to their life traits, their relationship with nettle, and their morphological characteristics. We observed low total mercury (THg) concentrations in nettles, with only 1% methylmercury (MeHg) being detected, while concentrations ranged from 5 to 3700 µg/kg dry wt. in insects with a MeHg percentage of up to 75%. The nettle-related insects were primarily exposed to Hg through the food web with significant biomagnification, particularly at the level of secondary predators. Within the nettle-unrelated group, the insect habitat was the most explanatory factor, with the highest enrichment being for the insects that spent part of their cycle in direct contact with Hg sources. Therefore, these insects require special attention because they are an essential vector of Hg transfer for terrestrial top predators.

Trace Metal(oid) Accumulation in Edible Crops and Poplar Cuttings Grown on Dredged Sediment Enriched Soil.

The development of a biomonitor in the context of multiple-element contamination in urban environments was tested by comparing element transfer in edible crops and poplar ( Torr. × A. Henry cutlivar 'Skado'). A multielemental analysis was performed with various common edible crops (cucumber [ L.], pepper [ L.], cabbage [ L.], and lettuce [ L.]) and the Skado poplar cultivar grown on soils that received sediments dredged from water canals in the 1960s. Sediments were distributed unevenly on the soil, allowing us to sample two types of areas that were either weakly (Area 1) or highly (Area 2) contaminated, mainly by Cd, Pb, and Zn. We registered an accumulation of Cd and Zn in the edible parts of crops, with higher values recorded for leafy vegetables than for fruit vegetables. We did not detect any accumulation of Pb in the plant species studied. We calculated the fresh mass that must be consumed daily to reach tolerable daily intake (TDI) recommendations for each element and found evidence that Cd could be ingested in sufficient amounts to reach the TDI in this context. Poplar and pepper leaves accumulated more Cd and Zn than the edible parts of the study crops grown on both substrates, which suggests that poplar and pepper may be suitable species for biomonitoring element transfer to vegetation in this context.

Co-inoculation of Lolium perenne with Funneliformis mosseae and the dark septate endophyte Cadophora sp. in a trace element-polluted soil.

The presence of dark septate endophytes (DSEs) or arbuscular mycorrhizal fungi (AMF) in plant roots and their effects on plant fitness have been extensively described. However, little is known about their interactions when they are simultaneously colonizing a plant root, especially in trace element (TE)-polluted soils. We therefore investigated the effects of Cadophora sp. and Funneliformis mosseae on ryegrass (Lolium perenne) growth and element uptake in a Cd/Zn/Pb-polluted soil. The experiment included four treatments, i.e., inoculation with Cadophora sp., inoculation with F. mosseae, co-inoculation with Cadophora sp. and F. mosseae, and no inoculation. Ryegrass biomass and shoot Na, P, K, and Mg concentrations significantly increased following AMF inoculation as compared to non-inoculated controls. Similarly, DSE inoculation increased shoot Na concentration, whereas dual inoculation significantly decreased shoot Cd concentration. Moreover, oxidative stress determined by ryegrass leaf malondialdehyde concentration was alleviated both in the AMF and dual inoculation treatments. We used quantitative PCR and microscope observations to quantify colonization rates. They demonstrated that DSEs had no effect on AMF colonization, while AMF colonization slightly decreased DSE frequency. We also monitored fluorescein diacetate (FDA) hydrolysis and alkaline phosphatase (AP) activity in the rhizosphere soils. FDA hydrolysis remained unchanged in the three inoculated treatments, but AMF colonization increased AP activity and P mobility in the soil whereas DSE colonization did not alter AP activity. In this experiment, we unveiled the interactions between two ecologically important fungal groups likely to occur in roots which involved a decrease of oxidative stress and Cd accumulation in shoots. These results open promising perspectives on the fungal-based phytomanagement of TE-contaminated sites by the production of uncontaminated and marketable plant biomass.

Environmental Metabarcoding Reveals Contrasting Belowground and Aboveground Fungal Communities from Poplar at a Hg Phytomanagement Site.

Characterization of microbial communities in stressful conditions at a field level is rather scarce, especially when considering fungal communities from aboveground habitats. We aimed at characterizing fungal communities from different poplar habitats at a Hg-contaminated phytomanagement site by using Illumina-based sequencing, network analysis approach, and direct isolation of Hg-resistant fungal strains. The highest diversity estimated by the Shannon index was found for soil communities, which was negatively affected by soil Hg concentration. Among the significant correlations between soil operational taxonomic units (OTUs) in the co-occurrence network, 80% were negatively correlated revealing dominance of a pattern of mutual exclusion. The fungal communities associated with Populus roots mostly consisted of OTUs from the symbiotic guild, such as members of the Thelephoraceae, thus explaining the lowest diversity found for root communities. Additionally, root communities showed the highest network connectivity index, while rarely detected OTUs from the Glomeromycetes may have a central role in the root network. Unexpectedly high richness and diversity were found for aboveground habitats, compared to the root habitat. The aboveground habitats were dominated by yeasts from the Lalaria, Davidiella, and Bensingtonia genera, not detected in belowground habitats. Leaf and stem habitats were characterized by few dominant OTUs such as those from the Dothideomycete class producing mutual exclusion with other OTUs. Aureobasidium pullulans, one of the dominating OTUs, was further isolated from the leaf habitat, in addition to Nakazawaea populi species, which were found to be Hg resistant. Altogether, these findings will provide an improved point of reference for microbial research on inoculation-based programs of tailings dumps.

Dendrochemical assessment of mercury releases from a pond and dredged-sediment landfill impacted by a chlor-alkali plant.

Although current Hg emissions from industrial activities may be accurately monitored, evidence of past releases to the atmosphere must rely on one or more environmental proxies. We used Hg concentrations in tree cores collected from poplars and willows to investigate the historical changes of Hg emissions from a dredged sediment landfill and compared them to a nearby control location. Our results demonstrated the potential value of using dendrochemistry to record historical Hg emissions from past industrial activities.

New mutualistic fungal endophytes isolated from poplar roots display high metal tolerance.

This study aimed to isolate, identify, and characterise metal-tolerant fungi colonising poplar roots at a metal-contaminated phytoremediation site. Poplar roots were colonised by arbuscular mycorrhizal, ectomycorrhizal, and endophytic fungi, and the species were determined by ITS molecular analyses. Eight different isolates were successfully isolated into pure culture. Three isolates belonging to the Helotiales (P02, P06) and the Serendipita vermifera species (P04) were highly tolerant to metals (Cd, Zn, Pb, and Cu) compared to the mycorrhizal Hebeloma isolates. The three isolates degraded complex carbohydrates, such as xylan and cellulose, indicating that they could partially degrade root cell walls and penetrate into cells. This hypothesis was confirmed by further in vitro re-synthesis experiments, which showed that the three isolates colonised root tissues of poplar plantlets whereas two of them formed microsclerotia-like structures. Taken together, these results suggest an endophytic lifestyle of these isolates. This is the first evidence of S. vermifera as a root endophyte of poplar. A new endophytic putative species belonging to the Helotiales and closely related to Leohumicola is also reported. Interestingly, and when compared to mock-inoculated plants, both P06 and P04 isolates increased the number of root tips of inoculated poplar plantlets in vitro. Moreover, the S. vermifera P04 isolate also increased the shoot biomass. The results are discussed in relation to the potential use of endophytic strains for tree-based phytoremediation of metal-contaminated sites.

Primer pairs, PCR conditions, and peptide nucleic acid clamps affect fungal diversity assessment from plant root tissues.

High-throughput sequencing has become a prominent tool to assess plant-associated microbial diversity. Still, some technical challenges remain in characterising these communities, notably due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are approaches to limit plant DNA contamination. However, a systematic comparison of these factors and their interactions, which could limit plant DNA contamination in the study of plant mycobiota, is still lacking. Here, three primers targeting the ITS2 region were evaluated alone or in combination with PNA clamps both on nettle (Urtica dioica) root DNA and a mock community. PNA clamps did not improve the richness or diversity of the fungal communities but increased the number of fungal reads. Among the tested factors, the most significant was the primer pair. Specifically, the 5.8S-Fun/ITS4-Fun pair exhibited a higher OTU richness but fewer fungal reads. Our study demonstrates that the choice of primers is critical for limiting plant and fungal DNA co-amplification. PNA clamps increase the number of fungal reads when ITS2 is targeted but do not result in higher fungal diversity recovery at high sequencing depth. At lower read depths, PNA clamps might enhance microbial diversity quantification for primer pairs lacking fungal specificity.

Rare earth elements perturb root architecture and ion homeostasis in Arabidopsis thaliana.

Rare earth elements (REEs) are crucial elements for current high-technology and renewable energy advances. In addition to their increasing usage and their low recyclability leading to their release into the environment, REEs are also used as crop fertilizers. However, little is known regarding the cellular and molecular effects of REEs in plants, which is crucial for better risk assessment, crop safety and phytoremediation. Here, we analysed the ionome and transcriptomic response of Arabidopsis thaliana exposed to a light (lanthanum, La) and a heavy (ytterbium, Yb) REE. At the transcriptome level, we observed the contribution of ROS and auxin redistribution to the modified root architecture following REE exposure. We found indications for the perturbation of Fe homeostasis by REEs in both roots and leaves of Arabidopsis suggesting competition between REEs and Fe. Furthermore, we propose putative ways of entry of REEs inside cells through transporters of microelements. Finally, similar to REE accumulating species, organic acid homeostasis (e.g. malate and citrate) appears critical as a tolerance mechanism in response to REEs. By combining ionomics and transcriptomics, we elucidated essential patterns of REE uptake and toxicity response of Arabidopsis and provide new hypotheses for a better evaluation of the impact of REEs on plant homeostasis.

Differential effects of tree species identity on rhizospheric bacterial and fungal community richness and composition across multiple trace element-contaminated sites.

Soil microbial communities play a key role in plant nutrition and stress tolerance. This is particularly true in sites contaminated by trace metals, which often have low fertility and stressful conditions for woody plants in particular. However, we have limited knowledge of the abiotic and biotic factors affecting the richness and composition of microbial communities inhabiting the rhizosphere of plants in contaminated sites. Using high-throughput amplicon sequencing, we studied the rhizospheric bacterial and fungal community structures of 14 woody plant families planted in three contrasting sites contaminated by metals (Pb, Cd, Zn, Mn, Fe, S). The rhizospheric bacterial communities in the given sites showed no significant difference between the various woody species but did differ significantly between sites. The Proteobacteria phylum was dominant, accounting for over 25 % of the overall relative abundance, followed by Actinobacteria, Bacteroidetes and Gemmatimonadetes. Site was also the main driver of fungal community composition, yet unlike bacteria, tree species identity significantly affected fungal communities. The Betulaceae, Salicaceae and Fagaceae families had a high proportion of Basidiomycota, particularly ectomycorrhizal fungi, and the lowest diversity and richness. The other tree families and the unplanted soil harboured a greater abundance of Ascomycota and Mucoromycota. Consequently, for both bacteria and fungi, the site effect significantly impacted their community richness and composition, while the influence of plants on the richness and composition of rhizospheric microbial communities stayed consistent across sites and was dependent on the microbial kingdom. Finally, we highlighted the importance of considering this contrasting response of plant rhizospheric microbial communities in relation to their host identity, particularly to improve assisted revegetation efforts at contaminated sites.

Soil metatranscriptomics for mining eukaryotic heavy metal resistance genes.

Heavy metals are pollutants which affect all organisms. Since a small number of eukaryotes have been investigated with respect to metal resistance, we hypothesize that many genes that control this phenomenon remain to be identified. This was tested by screening soil eukaryotic metatranscriptomes which encompass RNA from organisms belonging to the main eukaryotic phyla. Soil-extracted polyadenylated mRNAs were converted into cDNAs and 35 of them were selected for their ability to rescue the metal (Cd or Zn) sensitive phenotype of yeast mutants. Few of the genes belonged to families known to confer metal resistance when overexpressed in yeast. Several of them were homologous to genes that had not been studied in the context of metal resistance. For instance, the BOLA ones, which conferred cross metal (Zn, Co, Cd, Mn) resistance may act by interfering with Fe homeostasis. Other genes, such as those encoding 110- to 130-amino-acid-long, cysteine-rich polypeptides, had no homologues in databases. This study confirms that functional metatranscriptomics represents a powerful approach to address basic biological processes in eukaryotes. The selected genes can be used to probe new pathways involved in metal homeostasis and to manipulate the resistance level of selected organisms.

OmZnT1 and OmFET, two metal transporters from the metal-tolerant strain Zn of the ericoid mycorrhizal fungus Oidiodendron maius, confer zinc tolerance in yeast.

Two full-length cDNAs (OmZnT1 and OmFET) encoding membrane transporters were identified by yeast functional screening in the heavy metal tolerant ericoid mycorrhizal isolate Oidiodendron maius Zn. OmZnT1 belongs to the Zn-Type subfamily of the cation diffusion facilitators, whereas OmFET belongs to the family of iron permeases. Their properties were investigated in yeast by functional complementation of mutants affected in metal uptake and metal tolerance. Heterologous expression of OmZnT1 and OmFET in a Zn-sensitive yeast mutant restored the wild-type phenotype. Additionally, OmZnT1 expression also restored cobalt tolerance in a Co-sensitive mutant. A GFP fusion protein revealed that OmZnT1 was targeted to the endoplasmic reticulum membrane, a result consistent with a function for OmZnT1 in metal sequestration. Similarly to other iron permeases, OmFET-GFP was localized on the plasma membrane. OmFET restored the growth of uptake-defective strains for iron and zinc. Zinc-sensitive yeast mutants expressing OmFET specifically accumulated magnesium, as compared to cells transformed with the empty vector. We suggest that OmFET may counteract zinc toxicity by increasing entry of magnesium into the cell.

Fungal necromass presents a high potential for Mercury immobilization in soil.

Past industrial activities have generated many contaminated lands from which Mercury (Hg) escapes, primarily by volatilization. Current phytomanagement techniques aim to limit Hg dispersion by increasing its stabilization in soil. Although soil fungi represent a source of Hg emission associated with biovolatilization mechanisms, there is limited knowledge about how dead fungal residues (i.e., fungal necromass) interact with soil Hg. This study determined the Hg biosorption potential of fungal necromass and the chemical drivers of passive Hg binding with dead mycelia. Fungal necromass was incubated under field conditions with contrasting chemical properties at a well-characterized Hg phytomanagement experimental site in France. After four months of incubation in soil, fungal residues passively accumulated substantial quantities of Hg in their recalcitrant fractions ranging from 400 to 4500 µg Hg/kg. In addition, infrared spectroscopy revealed that lipid compounds explained the amount of Hg biosorption to fungal necromass. Based on these findings, we propose that fungal necromass is likely an important factor in Hg immobilization in soil.

Floristic survey, trace element transfers between soil and vegetation and human health risk at an urban industrial wasteland.

This study aimed to determine the trace element accumulation in the soil and plants in an industrial wasteland and to estimate the extent of transfer to humans to measure the effects on and risks to vegetation and human health and find bioindicator plants representative of the levels of the main contaminants. In areas with the highest extractable trace element levels, we observed decreases in plant biodiversity explained by the disappearance of several families, favouring the coverage of tolerant species, such as Urtica dioica and Hedera helix. Trace elements were also found in the leaves of several plants, especially in a dominant species that is poorly studied, Alliaria petiolata. Indeed, this species had the highest contents of Zn (1750 mg.kg-1 DW), Ni (13.1 mg.kg-1 DW), and Cd (18 mg.kg-1 DW) found at the site and is a potential Zn bioindicator since its leaf contents were also representative of the Zn extractable contents in soil (R² = 0.94). The hazard quotient and carcinogen risk revealed that most of the site had an identified or possible risk, mainly due to Pb and As. Native species, especially A. petiolata, could be used in phytoextraction to manage and limit these human and environmental risks.

The arbuscular mycorrhizal status has an impact on the transcriptome profile and amino acid composition of tomato fruit.

BACKGROUND: Arbuscular mycorrhizal (AM) symbiosis is the most widespread association between plant roots and fungi in natural and agricultural ecosystems. This work investigated the influence of mycorrhization on the economically relevant part of the tomato plant, by analyzing its impact on the physiology of the fruit. To this aim, a combination of phenological observations, transcriptomics (Microarrays and qRT-PCR) and biochemical analyses was used to unravel the changes that occur on fruits from Micro-Tom tomato plants colonized by the AM fungus Glomus mosseae. RESULTS: Mycorrhization accelerated the flowering and fruit development and increased the fruit yield. Eleven transcripts were differentially regulated in the fruit upon mycorrhization, and the mycorrhiza-responsive genes resulted to be involved in nitrogen and carbohydrate metabolism as well as in regulation and signal transduction. Mycorrhization has increased the amino acid abundance in the fruit from mycorrhizal plants, with glutamine and asparagine being the most responsive amino acids. CONCLUSIONS: The obtained results offer novel data on the systemic changes that are induced by the establishment of AM symbiosis in the plant, and confirm the work hypothesis that AM fungi may extend their influence from the root to the fruit.

Structure and expression profile of the phosphate Pht1 transporter gene family in mycorrhizal Populus trichocarpa.

Gene networks involved in inorganic phosphate (Pi) acquisition and homeostasis in woody perennial species able to form mycorrhizal symbioses are poorly known. Here, we describe the features of the 12 genes coding for Pi transporters of the Pht1 family in poplar (Populus trichocarpa). Individual Pht1 transporters play distinct roles in acquiring and translocating Pi in different tissues of mycorrhizal and nonmycorrhizal poplar during different growth conditions and developmental stages. Pi starvation triggered the up-regulation of most members of the Pht1 family, especially PtPT9 and PtPT11. PtPT9 and PtPT12 showed a striking up-regulation in ectomycorrhizas and endomycorrhizas, whereas PtPT1 and PtPT11 were strongly down-regulated. PtPT10 transcripts were highly abundant in arbuscular mycorrhiza (AM) roots only. PtPT8 and PtPT10 are phylogenetically associated to the AM-inducible Pht1 subfamily I. The analysis of promoter sequences revealed conserved motifs similar to other AM-inducible orthologs in PtPT10 only. To gain more insight into gene regulatory mechanisms governing the AM symbiosis in woody plant species, the activation of the poplar PtPT10 promoter was investigated and detected in AM of potato (Solanum tuberosum) roots. These results indicated that the regulation of AM-inducible Pi transporter genes is conserved between perennial woody and herbaceous plant species. Moreover, poplar has developed an alternative Pi uptake pathway distinct from AM plants, allowing ectomycorrhizal poplar to recruit PtPT9 and PtPT12 to cope with limiting Pi concentrations in forest soils.

Fate of trace elements during the combustion of phytoremediation wood.

We investigated the fate of trace elements (TE) in poplar wood on the conversion of biomass to heat in a 0.2 MW combustion unit equipped with a fabric filter. The phytoremediation wood was harvested from a TE-contaminated agricultural site planted with a high-density poplar stand. The combustion technology used in the present experiment allows for an efficient separation of the various ash fractions. The combustion process concentrates Cu, Cr, and Ni in the bottom ash, heat exchanger ash, and cyclone ash fractions. Therefore, the impact of the fabric filter is negligible for these elements. Conversely, Cd, Pb, and Zn are significantly recovered in the emission fraction in the absence of the fabric filter above the emission limits. The use of a fabric filter will allow the concentration of these three TEs in the ashes collected below the filter, thus complying with all regulatory thresholds, i.e., those from the large combustion plant EU directive. Because the TE concentrations in the different fractions differed significantly, it is recommended that these fractions be treated separately, especially when recycling of ashes from phytoremediation wood through application in agriculture is envisaged.

Dendrochemistry of multiple releases of chlorinated solvents at a former industrial site.

Trees can take up and assimilate contaminants from the soil, subsurface, and groundwater. Contaminants in the transpiration stream can become bound or incorporated into the annual rings formed in trees of the temperate zones. The chemical analysis of precisely dated tree rings, called dendrochemistry, can be used to interpret past plant interactions with contaminants. This investigation demonstrates that dendrochemistry can be used to generate historical scenarios of past contamination of groundwater by chlorinated solvents at a site in Verl, Germany. Increment cores from trees at the Verl site were collected and analyzed by energy-dispersive X-ray fluorescence (EDXRF) line scanning. The EDXRF profiles showed four to six time periods where tree rings had anomalously high concentrations of chlorine (Cl) as an indicator of potential contamination by chlorinated solvents.