Habitat-specific allocations of elements in Atriplex lentiformis seeds hint at adaptation to metal toxicity.

Self-sustaining vegetation in metal-contaminated areas is essential for rebuilding the ecological resilience and community stability in degraded lands. Metal-tolerant plants originating from contaminated post-mining areas may hold the key to successful plant establishment and growth. Yet, little is known about the impact of metal toxicity on reproductive strategies, metal accumulation and allocation patterns at the seed stage. Our research focused on metal tolerant Atriplex lentiformis, examining the effects of toxic metal(loid) concentration in soils on variability in its reproductive strategies, including germination patterns, elemental uptake, and allocation within the seeds. We employed advanced imaging techniques like synchrotron X-ray Fluorescence Microscopy (XFM; 2D scans and 3D tomograms) combined with ICP-MS to reveal significant differences in metal(loid) concentration and distribution within the seed structures of A. lentiformis from contrasting habitats. Exclusive Zn hotspots of high concentrations were found in the seeds of the metallicolous accession, primarily in the sensitive tissues of shoot apical meristems and root zones of the seed embryos. The findings of this study offer novel insights into phenotypic variability, metal tolerance and accumulation in plants from extreme environments. This knowledge can be applied to enhance plant survival and performance in land restoration efforts.

Quercus rubra invasion of temperate deciduous forest stands alters the structure and functions of the soil microbiome.

Invasive plants can modify the diversity and taxonomical structure of soil microbiomes. However, it is difficult to generalize the underlying factors as their influence often seems to depend on the complex plant-soil-microbial interactions. In this paper, we investigated how Quercus rubra impacts on the soil microbiome across two soil horizons in relation to native woodland. Five paired adjacent invaded vs native vegetation plots in a managed forest in southern Poland were investigated. Soil microbial communities were assessed along with soil enzyme activities and soil physicochemical parameters, separately for both organic and mineral horizons, as well as forest stand characteristics to explore plant-soil-microbe interactions. Although Q. rubra did not significantly affect pH, organic C, total N, available nutrients nor enzymatic activity, differences in soil abiotic properties (except C to N ratio) were primarily driven by soil depth for both vegetation types. Further, we found significant differences in soil microbiome under invasion in relation to native vegetation. Microbial richness and diversity were lower in both horizons of Q. rubra vs control plots. Moreover, Q. rubra increased relative abundance of unique amplicon sequence variants in both horizons and thereby significantly changed the structure of the core soil microbial communities, in comparison to the control plots. In addition, predicted microbial functional groups indicated a predominant soil depth effect in both vegetation plots with higher abundance of aerobic chemoheterotrophic bacteria and endophytic fungi in the organic horizon and greater abundance of methanotrophic and methylotrophic bacteria, and ectomycorrhizal fungi in the mineral horizon. Overall, our results indicate strong associations between Q. rubra invasion and changes in soil microbiome and associated functions, a finding that needs to be further investigated to predict modifications in ecosystem functioning caused by this invasive species.

Diversity and activity of soil biota at a post-mining site highly contaminated with Zn and Cd are enhanced by metallicolous compared to non-metallicolous Arabidopsis halleri ecotypes.

Hyperaccumulators' ability to take up large quantities of harmful heavy metals from contaminated soils and store them in their foliage makes them promising organisms for bioremediation. Here we demonstrate that some ecotypes of the zinc hyperaccumulator Arabidopsis halleri are more suitable for bioremediation than others, because of their distinct influence on soil biota. In a field experiment, populations originating from metal-polluted and unpolluted soils were transplanted to a highly contaminated metalliferous site in Southern Poland. Effects of plant ecotypes on soil biota were assessed by measurements of feeding activity of soil fauna (bait-lamina test) and catabolic activity and functional diversity of soil bacteria underneath A. halleri plants (Biolog® ECO plates). Chemical soil properties, plant morphological parameters, and zinc concentration in shoots and roots were additionally evaluated. Higher soil fauna feeding activity and higher bacterial community functional diversity were found in soils affected by A. halleri plants originating from metallicolous compared to non-metallicolous ecotypes. Differences in community-level physiological profiles further evidenced changes in microbial communities in response to plant ecotype. These soil characteristics were positively correlated with plant size. No differences in zinc content in shoots and roots, zinc translocation ratio, and plant morphology were observed between metallicolous and non-metallicolous plants. Our results indicate strong associations between A. halleri ecotype and soil microbial community properties. In particular, the improvement of soil biological properties by metallicolous accessions should be further explored to optimize hyperaccumulator-based bioremediation technologies.

Zinc accumulation in Atriplex lentiformis is driven by plant genes and the soil microbiome.

Successful phytoremediation of acidic metal-contaminated mine tailings requires amendments to condition tailings properties prior to plant establishment. This conditioning process is complex and includes multiple changes in tailings bio-physico-chemical properties. The objective of this project is to identify relationships between tailings properties, the soil microbiome, and plant stress response genes during growth of Atriplex lentiformis in compost-amended (10 %, 15 %, 20 % w/w) mine tailings. Analyses include RNA-Seq for plant root gene expression, 16S rRNA amplicon sequencing for bacterial/archaeal communities, metal concentrations in both tailings and plant organs, and phenotypic measures of plant stress. Zn accumulation in A. lentiformis leaves varied with compost levels and was the highest in the intermediate treatment (15 %, TC15). Microbial analysis identified Alicyclobacillus, Hydrotalea, and Pseudolabrys taxa with the highest relative abundance in TC15, and these taxa were strongly associated with Zn accumulation. Furthermore, we identified 190 root genes with significant gene expression changes. These root genes were associated with different pathways including, abscisic acid and auxin signaling, defense responses, ion channels, metal ion binding, oxidative stress, transcription regulation, and transmembrane transport. However, root gene expression changes were not driven by the increasing levels of compost. For example, there were 15 genes that were up-regulated in TC15, whereas 106 genes were down-regulated in TC15. The variables analyzed explained 86 % of the variance in Zn accumulation in A. lentiformis leaves. Importantly, Zn accumulation was driven by Zn shoot concentrations, leaf stress symptoms, plant root genes, and microbial taxa. Therefore, our results suggest there are strong plant-microbiome associations that drive Zn accumulation in A. lentiformis and different plant gene pathways are involved in alleviating varying levels of metal stress. Future work is needed to gain a mechanistic understanding of these plant-microbiome interactions to optimize phytoremediation strategies as they will govern the success or failure of the revegetation process.

Copper accumulation in five weed species commonly found in the understory vegetation of Mediterranean vineyards.

Copper (Cu) concentration in agricultural soils often exceeds toxicological limits due to application of Cu-based fungicides. The potential of weeds for their use as functional cover plants in vineyard management and phytoremediation practices is little explored. We identified five weed species widely present in vineyards and assessed their Cu accumulation from eleven Mediterranean vineyards (soil Cu: 60-327 µg g-1) and two adjacent control sites (soil Cu: 15-30 µg g-1). Soils and plants were characterized by their physico-chemical properties and nutrient content. We applied multivariate techniques to analyze relationships between soil properties and leaf nutrient composition. Copper tolerance and accumulation traits were further tested in hydroponics using a series of CuSO4 concentrations (0.1-16 µM). Under field conditions, the highest Cu concentration in both roots and leaves were found in Lolium perenne (221 and 461 µg g-1, respectively), followed by Plantago lanceolata, Rumex obtusifolius and Taraxacum officinale (>100 µg g-1 Cu in leaves). Only one species, Trifolium repens, did not accumulate remarkable Cu concentrations. Overall, and as revealed by the multivariate analyses, leaf Cu concentration was driven by soil Cu content, soil texture, organic matter, nitrogen, and Cu uptake into roots. However, functional regression analysis and controlled experiments suggested that Cu might be additionally absorbed from the deposits on the leaf surface related to the Cu-fungicide treatments and soil dust. Our study highlights the importance of intra-specific variability in Cu accumulation among weed species in Cu-contaminated agricultural soils. Further validation of these findings under controlled conditions could provide essential insights for optimizing management and remediation strategies.

Metalliferous habitats and seed microbes affect the seed morphology and reproductive strategy of Arabidopsis halleri.

Purpose: Plant reproduction in metalliferous habitats is challenged by elevated concentrations of metal trace elements in soil. As part of their survival strategy, metal-tolerant plants have adjusted reproductive traits, including seed morphology, dormancy, and germination rate. These traits are particularly relevant, yet poorly understood, in metal hyperaccumulators that are promising candidates for phytoremediation. Methods: We assessed seed shape characteristics, dormancy, and germination rate in the hyperaccumulating model species Arabidopsis halleri. Seed morphological parameters were evaluated using seeds collected from two metalliferous and two non-metalliferous sites (~ 1000 seeds per location). We also addressed the potential influence of seed surface-associated microbes and endophytic fungi on germination success. Results: Seeds from non-metallicolous populations were on average 18% bigger than those from metal-contaminated post-mining sites, which contrasts the general expectation about reproductive parts in metallicolous plants. Irrespective of their origin, surface-sterilized seeds had up to ~ 20% higher germination rates and germinated earlier than non-sterilized seeds, hinting at a negative effect of seed-associated microbial communities. Surface sterilization also facilitated the emergence of an endophytic fungus (Aspergillus niger) that is a known seed-borne pathogen. Interestingly, A. niger actually promoted germination in surface-sterilized seeds from some locations. Conclusion: Despite species-wide metal tolerance in A. halleri, metalliferous conditions seem to differently affect reproductive traits compared to non-metalliferous environments (e.g., smaller seeds). Yet, higher germination rates in these populations hint at the potential of A. halleri to successfully colonize post-mining habitats. This process is modulated by site-specific interactions with seed microbiota.

Green and Effective Preparation of α-Hydroxyphosphonates by Ecocatalysis.

A green and effective approach for the synthesis of structurally diversed α-hydroxyphosphonates via hydrophosphonylation of aldehydes under solventless conditions and promoted by biosourced catalysts, called ecocatalysts "Eco-MgZnOx" is presented. Ecocatalysts were prepared from Zn-hyperaccumulating plant species Arabidopsis halleri, with simple and benign thermal treatment of leaves rich in Zn, and without any further chemical treatment. The elemental composition and structure of Eco-MgZnOx were characterized by MP-AES, XRPD, HRTEM, and STEM-EDX techniques. These analyses revealed a natural richness in two unusual and valuable mixed zinc-magnesium and iron-magnesium oxides. The ecocatalysts were employed in this study to demonstrate their potential use in hydrophosphonylation of aldehydes, leading to various α-hydroxyphosphonate derivatives, which are critical building blocks in the modern chemical industry. Computational chemistry was performed to help discriminate the role of some of the constituents of the mixed oxide ecocatalysts. High conversions, broad substrate scope, mild reaction conditions, and easy purification of the final products together with simplicity of the preparation of the ecocatalysts are the major advantages of the presented protocol. Additionally, Eco-MgZnOx-P could be recovered and reused for up to five times.

Mechanochemistry and Eco-Bases for Sustainable Michael Addition Reactions.

The Michael addition reaction was revisited with a full focus on sustainability combined with efficiency, using mechanochemistry in mild conditions. First, the synthesis of cyclopentenone derivatives was chosen as a model reaction to find optimal conditions in mechanochemistry while using classical but weak bases. The reaction was efficient (84-95% yields), fast (2-6 h), solvent free, and required 0.1 equivalent of base. Aiming to reach greener conditions, classical bases were then replaced using new bio-sourced bases, called Eco-bases, that were easily prepared from plants and led to heterogeneous catalysts. The composition and structure of Eco-bases were characterized by MP-AES, XRPD, EBSD/EDS, HRTEM/EDX and ion chromatography. Interestingly, a high ratio of potassium was observed with the presence of K2Ca(CO3)2 for the most effective Eco-base. The new Eco-bases were used for the mechanical-assisted construction of functionalized alkenone derivatives. The versatility of the method has been successfully applied with good to excellent yields to different Michael donors and acceptors. Eco-bases were recycled and reused four times with the same performances. Combining Eco-bases and mechanochemistry in Michael addition reactions allowed reaching a maximum degree of sustainability (efficient, rapid, low catalyst loading, solvent-free reactions with bio-sourced catalysts) and participating in the development of mechanochemistry in sustainable chemistry.

Soil microbial community and abiotic soil properties influence Zn and Cd hyperaccumulation differently in Arabidopsis halleri.

Soil contamination with trace metal(loid) elements (TME) is a global concern. This has focused interest on TME-tolerant plants, some of which can hyperaccumulate extraordinary amounts of TME into above-ground tissues, for potential treatment of these soils. However, intra-species variability in TME hyperaccumulation is not yet sufficiently understood to fully harness this potential. Particularly, little is known about the rhizosphere microbial communities associated with hyperaccumulating plants and whether or not they facilitate TME uptake. The aim of this study is to characterize the diversity and structure of Arabidopsis halleri rhizosphere-influenced and background (i.e., non-Arabidopsis) soil microbial communities in four plant populations with contrasting Zn and Cd hyperaccumulation traits, two each from contaminated and uncontaminated sites. Microbial community properties were assessed along with geographic location, climate, abiotic soil properties, and plant parameters to explain variation in Zn and Cd hyperaccumulation. Site type (TME-contaminated vs. uncontaminated) and location explained 44% of bacterial/archaeal and 28% of fungal community variability. A linear discriminant effect size (LEfSe) analysis identified a greater number of taxa defining rhizosphere microbial communities than associated background soils. Further, in TME-contaminated soils, the number of rhizosphere-defining taxa was 6-fold greater than in the background soils. In contrast, the corresponding ratio for uncontaminated sites, was 3 and 1.6 for bacteria/archaea and fungi, respectively. The variables analyzed explained 71% and 76% of the variance in Zn and Cd hyperaccumulation, respectively; however, each hyperaccumulation pattern was associated with different variables. A. halleri rhizosphere fungal richness and diversity associated most strongly with Zn hyperaccumulation, whereas soil Cd and Zn bioavailability had the strongest associations with Cd hyperaccumulation. Our results indicate strong associations between A. halleri TME hyperaccumulation and rhizosphere microbial community properties, a finding that needs to be further explored to optimize phytoremediation technology that is based on hyperaccumulation.

Phytoextraction efficiency of Arabidopsis halleri is driven by the plant and not by soil metal concentration.

The hyperaccumulation trait allows some plant species to allocate remarkable amounts of trace metal elements (TME) to their foliage without suffering from toxicity. Utilizing hyperaccumulating plants to remediate TME contaminated sites could provide a sustainable alternative to industrial approaches. A major hurdle that currently hampers this approach is the complexity of the plant-soil relationship. To better anticipate the outcome of future phytoremediation efforts, we evaluated the potential for soil metal-bioavailability to predict TME accumulation in two non-metallicolous and two metallicolous populations of the Zn/Cd hyperaccumulator Arabidopsis halleri. We also examined the relationship between a population's habitat and its phytoextraction efficiency. Total Zn and Cd concentrations were quantified in soil and plant material, and bioavailable fractions in soil were quantified via Diffusive Gradients in Thin-films (DGT). We found that shoot TME accumulation varied independent from both total and bioavailable soil TME concentrations in metallicolous individuals. In fact, hyperaccumulation patterns appear more plant- and less soil-driven: one non-metallicolous population proved to be as efficient in accumulating Zn on non-polluted soil as the metallicolous populations in their highly contaminated environment. Our findings demonstrate that in-situ information on plant phytoextraction efficiency is indispensable to optimize site-specific phytoremediation measures. If successful, hyperaccumulating plant biomass may provide valuable source material for application in the emerging field of green chemistry.

Zinc allocation to and within Arabidopsis halleri seeds: Different strategies of metal homeostasis in accessions under divergent selection pressure.

Vegetative tissues of metal(loid)-hyperaccumulating plants are widely used to study plant metal homeostasis and adaptation to metalliferous soils, but little is known about these mechanisms in their seeds. We explored essential element allocation to Arabidopsis halleri seeds, a species that faces a particular trade-off between meeting nutrient requirements and minimizing toxicity risks.Combining advanced elemental mapping (micro-particle induced X-ray emission) with chemical analyses of plant and soil material, we investigated natural variation in Zn allocation to A. halleri seeds from non-metalliferous and metalliferous locations. We also assessed the tissue-level distribution and concentration of other nutrients to identify possible disorders in seed homeostasis.Unexpectedly, the highest Zn concentration was found in seeds of a non-metalliferous lowland location, whereas concentrations were relatively low in all other seed samples-including metallicolous ones. The abundance of other nutrients in seeds was unaffected by metalliferous site conditions.Our findings depict contrasting strategies of Zn allocation to A. halleri seeds: increased delivery at lowland non-metalliferous locations (a likely natural selection toward enhanced Zn-hyperaccumulation in vegetative tissues) versus limited translocation at metalliferous sites where external Zn concentrations are toxic for non-tolerant plants. Both strategies are worth exploring further to resolve metal homeostasis mechanisms and their effects on seed development and nutrition.

Endosperm prevents toxic amounts of Zn from accumulating in the seed embryo - an adaptation to metalliferous sites in metal-tolerant Biscutella laevigata.

Seed germination represents the first crucial stage in the life cycle of a plant, and the seed must contain all necessary transition elements for the development and successful establishment of the seedling. Problematically, seed development and germination are often hampered by elevated metal(loid) concentrations in industrially polluted soils, making their revegetation a challenging task. Biscutella laevigata L. (Brassicaceae) is a rare perennial pseudometallophyte that can tolerate high concentrations of trace metal elements. Yet, the strategies of this and other plant species to ensure reproductive success at metalliferous sites are poorly understood. Here we characterized several parameters of germination and used synchrotron X-ray fluorescence microscopy to investigate the spatial distribution and concentration of elements within B. laevigata seeds from two metallicolous and two non-metallicolous populations. We find that average germination time was shorter and the seed weight was lower in the metallicolous compared to the non-metallicolous populations. By allowing for at least two generations within one growth season, relatively fast germination at metalliferous sites accelerates microevolutionary processes and likely enhances the potential of metallicolous accessions to adapt to environmental stress. We also identified different strategies of elemental accumulation within seed tissues between populations. Particularly interesting patterns were observed for zinc, which was found in 6-fold higher concentrations in the endosperm of metallicolous compared to non-metallicolous populations. This indicates that the endosperm protects the seed embryo from accumulating toxic concentrations of metal(loid)s, which likely improves reproductive success. Hence, we conclude that elemental uptake regulation by the seed endosperm is associated with enhanced metal tolerance and adaptation to metalliferous environments in B. laevigata.

Transmembrane transport and stress response genes play an important role in adaptation of Arabidopsis halleri to metalliferous soils.

When plants adapt to local environments, strong signatures of selection are expected in the genome, particularly in high-stress environments such as trace metal element enriched (metalliferous) soils. Using Arabidopsis halleri, a model species for metal homeostasis and adaptation to extreme environments, we identifid genes, gene variants, and pathways that are associated with soil properties and may thus contribute to adaptation to high concentrations of trace metal elements. We analysed whole-genome Pool-seq data from two metallicolous (from metalliferous soils) and two non-metallicolous populations (in total 119 individuals) and associated allele frequencies of the identified single-nucleotide polymorphisms (SNPs) with soil variables measured on site. Additionally, we accounted for polygenic adaptation by searching for gene pathways showing enrichment of signatures of selection. Out of >2.5 million SNPs, we identified 57 SNPs in 19 genes that were significantly associated with soil variables and are members of three enriched pathways. At least three of these candidate genes and pathways are involved in transmembrane transport and/or associated with responses to various stresses such as oxidative stress. We conclude that both allocation and detoxification processes play a crucial role in A. halleri for coping with these unfavourable conditions.

Evolutionary dynamics of quantitative variation in an adaptive trait at the regional scale: The case of zinc hyperaccumulation in Arabidopsis halleri.

Metal hyperaccumulation in plants is an ecological trait whose biological significance remains debated, in particular because the selective pressures that govern its evolutionary dynamics are complex. One of the possible causes of quantitative variation in hyperaccumulation may be local adaptation to metalliferous soils. Here, we explored the population genetic structure of Arabidopsis halleri at fourteen metalliferous and nonmetalliferous sampling sites in southern Poland. The results were integrated with a quantitative assessment of variation in zinc hyperaccumulation to trace local adaptation. We identified a clear hierarchical structure with two distinct genetic groups at the upper level of clustering. Interestingly, these groups corresponded to different geographic subregions, rather than to ecological types (i.e., metallicolous vs. nonmetallicolous). Also, approximate Bayesian computation analyses suggested that the current distribution of A. halleri in southern Poland could be relictual as a result of habitat fragmentation caused by climatic shifts during the Holocene, rather than due to recent colonization of industrially polluted sites. In addition, we find evidence that some nonmetallicolous lowland populations may have actually derived from metallicolous populations. Meanwhile, the distribution of quantitative variation in zinc hyperaccumulation did separate metallicolous and nonmetallicolous accessions, indicating more recent adaptive evolution and diversifying selection between metalliferous and nonmetalliferous habitats. This suggests that zinc hyperaccumulation evolves both ways-towards higher levels at nonmetalliferous sites and lower levels at metalliferous sites. Our results open a new perspective on possible evolutionary relationships between A. halleri edaphic types that may inspire future genetic studies of quantitative variation in metal hyperaccumulation.

Phylogeography of Arabidopsis halleri (Brassicaceae) in mountain regions of Central Europe inferred from cpDNA variation and ecological niche modelling.

The present study aimed to investigate phylogeographical patterns present within A. halleri in Central Europe. 1,281 accessions sampled from 52 populations within the investigated area were used in the study of genetic variation based on chloroplast DNA. Over 500 high-quality species occurrence records were used in ecological niche modelling experiments. We evidenced the presence of a clear phylogeographic structure within A. halleri in Central Europe. Our results showed that two genetically different groups of populations are present in western and eastern part of the Carpathians. The hypothesis of the existence of a glacial refugium in the Western Carpathians adn the Bohemian Forest cannot be rejected from our data. It seems, however, that the evidence collected during the present study is not conclusive. The area of Sudetes was colonised after LGM probably by migrants from the Bohemian Forest.