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

Self-sustaining vegetation in metal-contaminated areas is essential for rebuilding 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 the metal tolerant Atriplex lentiformis. Specifically, we examined 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 (2D scans and 3D tomograms) combined with inductively coupled plasma mass spectrometry 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. Our findings offer novel insights into phenotypic variability and metal tolerance and accumulation in plants from extreme environments. This knowledge can be applied to enhance plant survival and performance in land restoration efforts.

Dianthus sylvestris subsp. sylvestris as a promising candidate for phytostabilization of copper-contaminated post-mining sites in Alpine ecosystems.

The lack of remediation of inactive mine sites is a serious global concern, as they pose risks to the environment, human health, and safety. The potential of Dianthus sylvestris subsp. sylvestris to remediate post-mining sites contaminated with copper (Cu) at high altitudes, which is a challenging task for most management strategies, was explored in this study. More than 1300 mg Cu kg-1 in shoots were found in plants collected at the Monte Avanza legacy mine site (Alps). However, it is unclear whether this is due to hyperaccumulation or foliar contamination. To address this gap, field samples were washed with two different protocols, and a controlled Cu-tolerance test was conducted. While very high Cu concentrations, exceeding the Cu hyperaccumulation threshold of 300 mg kg-1, were found in samples washed with water, results for the plants cleaned with a more rigorous approach with EDTA suggested Cu exclusion strategy. Under controlled conditions, the plant showed Cu hypertolerance but did not hyperaccumulate Cu. Thus D. sylvestris presents a Cu exclusion strategy rather than hyperaccumulation potential, making it a suitable candidate for Cu phytostabilization at high altitude legacy mine sites. The study emphasizes the need for experiments under controlled conditions when assessing the phytoremediation potential.

Response to Cadmium in Silene vulgaris Ecotypes Is Distinctly Affected by Priming-Induced Changes in Oxidation Status of Macromolecules.

This study investigated the impact of several priming agents on metal-tolerant and sensitive Silene vulgaris ecotypes exposed to environmentally relevant cadmium dose. We analyzed how priming-induced changes in the level of lipid, protein, and DNA oxidation contribute to calamine (Cal) and non-calamine (N-Cal) ecotype response to Cd toxicity, and whether the oxidative modifications interrelate with Cd tolerance. In non-primed ecotypes, the levels of DNA and protein oxidation were similar whereas Cal Cd tolerance was manifested in reduced lipid peroxidation. In both ecotypes protective action of salicylic acid (SA) and nitric oxide (NO) priming was observed. SA stimulated growth and reduced lipid and DNA oxidation at most, while NO protected DNA from fragmentation. Priming with hydrogen peroxide reduced biomass and induced DNA oxidation. In N-Cal, priming diminished Cd accumulation and oxidative activity, whereas in Cal, it merely affected Cd uptake and induced protein carbonylation. The study showed that priming did not stimulate extra stress resistance in the tolerant ecotype but induced metabolic remodeling. In turn, the lack of adaptive tolerance made the sensitive ecotype more responsive to the benefits of the primed state. These findings could facilitate priming exploitation with a view of enhancing metallophyte and non-metallophyte suitability for phytoremediation and land revegetation.

Micro-Evolutionary Processes in Armeria maritima at Metalliferous Sites.

Tolerance to heavy metals in plants is a model process used to study adaptations to extremely unfavorable environments. One species capable of colonizing areas with high contents of heavy metals is Armeria maritima (Mill.) Wild. A. maritima plants growing in metalliferous areas differ in their morphological features and tolerance levels to heavy metals compared to individuals of the same species growing in non-metalliferous areas. The A. maritima adaptations to heavy metals occur at the organismal, tissue, and cellular levels (e.g., the retention of metals in roots, enrichment of the oldest leaves with metals, accumulation of metals in trichomes, and excretion of metals by salt glands of leaf epidermis). This species also undergoes physiological and biochemical adaptations (e.g., the accumulation of metals in vacuoles of the root's tannic cells and secretion of such compounds as glutathione, organic acids, or HSP17). This work reviews the current knowledge on A. maritima adaptations to heavy metals occurring in zinc-lead waste heaps and the species' genetic variation from exposure to such habitats. A. maritima is an excellent example of microevolution processes in plants inhabiting anthropogenically changed areas.

Constitutively enhanced genome integrity maintenance and direct stress mitigation characterize transcriptome of extreme stress-adapted Arabidopsis halleri.

Heavy metal-rich toxic soils and ordinary soils are both natural habitats of Arabidopsis halleri, a diploid perennial and obligate outcrosser in the sister clade of the genetic model plant Arabidopsis thaliana. The molecular divergence underlying survival in sharply contrasting environments is unknown. Here we comparatively address metal physiology and transcriptomes of A. halleri originating from the most highly heavy metal-contaminated soil in Europe, Ponte Nossa, Italy (Noss), and from non-metalliferous (NM) soils. Plants from Noss exhibit enhanced hypertolerance and attenuated accumulation of cadmium (Cd), and their transcriptomic Cd responsiveness is decreased, compared to plants of NM soil origin. Among the condition-independent transcriptome characteristics of Noss, the most highly overrepresented functional class of 'meiotic cell cycle' comprises 21 transcripts with elevated abundance in vegetative tissues, in particular Argonaute 9 (AGO9) and the synaptonemal complex transverse filament protein-encoding ZYP1a/b. Increased AGO9 transcript levels in Noss are accompanied by decreased long terminal repeat retrotransposon expression. Similar to Noss, plants from other highly metalliferous sites in Poland and Germany share elevated somatic AGO9 transcript levels in comparison to plants originating from NM soils in their respective geographic regions. Transcript levels of Iron-Regulated Transporter 1 (IRT1) are very low and transcript levels of Heavy Metal ATPase 2 (HMA2) are strongly elevated in Noss, which can account for its altered Cd handling. We conclude that in plants adapted to the most extreme abiotic stress, broadly enhanced functions comprise genes with likely roles in somatic genome integrity maintenance, accompanied by few alterations in stress-specific functional networks.

Phylogenetic analysis of hyperaccumulator plant species for heavy metals and polycyclic aromatic hydrocarbons.

Increasing concentration of heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) in the soil may impose a serious threat to living organisms due to their toxicity and the ability to accumulate in plant tissues. The present review focuses on the phylogenetic relationships, sources, biotransformation and accumulation potential of hyperaccumulators for the priority HMs and PAHs. This review provides an opportunity to reveal the role of hyperaccumulators in removal of HMs and PAHs from soils, to understand the relationships between pollutants and their influence on the environment and to find potential plant species for soil remediation. The phylogenetic analysis results showed that the hyperaccumulators of some chemicals (Co, Cu, Mn, Ni, Zn, Cd) are clustered on the evolutionary tree and that the ability to hyperaccumulate different pollutants can be correlated either positively (Cd-Zn, Pb-Zn, Co-Cu, Cd-Pb) or negatively (Cu-PAHs, Co-Cd, Co-PAHs, Ni-PAHs, Cu-Ni, Mn-PAHs). Further research needs to be extended on the focus of commercializing the techniques including the native hyperaccumulators to remediate the highly contaminated soils.

Arabidopsis halleri shows hyperbioindicator behaviour for Pb and leaf Pb accumulation spatially separated from Zn.

Lead (Pb) ranks among the most problematic environmental pollutants. Background contamination of soils is nearly ubiquitous, yet plant Pb accumulation is barely understood. In a survey covering 165 European populations of the metallophyte Arabidopsis halleri, several field samples had indicated Pb hyperaccumulation, offering a chance to dissect plant Pb accumulation. Accumulation of Pb was analysed in A. halleri individuals from contrasting habitats under controlled conditions to rule out aerial deposition as a source of apparent Pb accumulation. Several elemental imaging techniques were employed to study the spatial distribution and ligand environment of Pb. Regardless of genetic background, A. halleri individuals showed higher shoot Pb accumulation than A. thaliana. However, dose-response curves revealed indicator rather than hyperaccumulator behaviour. Xylem sap data and elemental imaging unequivocally demonstrated the in planta mobility of Pb. Highest Pb concentrations were found in epidermal and vascular tissues. Distribution of Pb was distinct from that of the hyperaccumulated metal zinc. Most Pb was bound by oxygen ligands in bidentate coordination. A. halleri accumulates Pb whenever soil conditions render Pb phytoavailable. Considerable Pb accumulation under such circumstances, even in leaves of A. thaliana, strongly suggests that Pb can enter food webs and may pose a food safety risk.

Structural, physiological and genetic diversification of Silene vulgaris ecotypes from heavy metal-contaminated areas and their synchronous in vitro cultivation.

MAIN CONCLUSION: Results provide significant comparison of leaf anatomy, pigment content, antioxidant response and phenolic profile between individuals from miscellaneous populations and describe unified cultivation protocols for further research on stress biology. The plant communities growing on heavy metal-polluted areas have attracted considerable attention due to their unique ability to tolerate enormous amounts of toxic ions. Three ecotypes of Silene vulgaris representing calamine (CAL), serpentine (SER) and non-metallicolous (NM) populations were evaluated to reveal specific adaptation traits to harsh environment. CAL leaves presented a distinct anatomical pattern compared to leaves of SER and NM plants, pointing to their xeromorphic adaptation. These differences were accompanied by divergent accumulation and composition of photosynthetic pigments as well as antioxidant enzyme activity. In CAL ecotype, the mechanism of reactive oxygen species scavenging is based on the joint action of superoxide dismutase and catalase, but in SER ecotype on superoxide dismutase and guaiacol-type peroxidase. On the contrary, the concentration of phenylpropanoids and flavonols in the ecotypes was unchanged, implying the existence of similar pathways of their synthesis/degradation functioning in CAL and SER populations. The tested specimens showed genetic variation (atpA/MspI marker). Based on diversification of S. vulgaris populations, we focused on the elaboration of similar in vitro conditions for synchronous cultivation of various ecotypes. The most balanced shoot culture growth was obtained on MS medium containing 0.1 mg l-1 NAA and 0.25 mg l-1 BA, while the most abundant callogenesis was observed on MS medium enriched with 0.5 mg l-1 NAA and 5.0 mg l-1 BA. For the first time, unified in vitro protocols were described for metallophytes providing the opportunity to conduct basic and applied research on stress biology and tolerance mechanisms under freely controlled conditions.

Accumulation of heavy metals in metallophytes from three mining sites (Southern Centre Morocco) and evaluation of their phytoremediation potential.

The current study aims to perform a field survey of three abandoned mining sites in the southern centre of Morocco to assess the recent metal pollution in soils and accumulation potential of plant species. Native plants and soils were sampled at several sites in the studied mines and analysed for Cu, Zn, Pb and Cd concentrations. Soils in the investigated sites proved to be deficient in major macronutrients and to contain toxic levels of Cu, Zn, Pb and Cd. Botanical survey of the prospected sites showed the abundance of diverse plant communities (46 species and 19 families), with no obvious toxicity symptoms. Results showed that the concentrations of heavy metals were different in the same plant species and from plant species to another. Eight plants of 46 species namely Hirschfeldia incana (L.) Lagr.-Foss, Citrullus vulgaris (L.) Schradi, Portulaca oleracea L., Stipa capensis Thunb., Lactuca viminea (L.) J.Presl & C.Presl, Forsskaolea tenacissima L., Lycium intricatum Boiss. and Hammada scoparia (Pomel) Iljin were considered as the best-performing specimens due to their high ability to accumulate multiple metals in their shoots and roots without being affected by excessive metal contents. This was confirmed by the transfer factors generally higher than 1. Consequently, these tolerant and native plant species could be used as tools for an effective phytorestoration of metal-contaminated sites.

Dual Role of Metallic Trace Elements in Stress Biology-From Negative to Beneficial Impact on Plants.

Heavy metals are an interesting group of trace elements (TEs). Some of them are minutely required for normal plant growth and development, while others have unknown biological actions. They may cause injury when they are applied in an elevated concentration, regardless of the importance for the plant functioning. On the other hand, their application may help to alleviate various abiotic stresses. In this review, both the deleterious and beneficial effects of metallic trace elements from their uptake by roots and leaves, through toxicity, up to the regulation of physiological and molecular mechanisms that are associated with plant protection against stress conditions have been briefly discussed. We have highlighted the involvement of metallic ions in mitigating oxidative stress by the activation of various antioxidant enzymes and emphasized the phenomenon of low-dose stimulation that is caused by non-essential, potentially poisonous elements called hormesis, which is recently one of the most studied issues. Finally, we have described the evolutionary consequences of long-term exposure to metallic elements, resulting in the development of unique assemblages of vegetation, classified as metallophytes, which constitute excellent model systems for research on metal accumulation and tolerance. Taken together, the paper can provide a novel insight into the toxicity concept, since both dose- and genotype-dependent response to the presence of metallic trace elements has been comprehensively explained.

Copper and cobalt accumulation in plants: a critical assessment of the current state of knowledge.

This review synthesizes contemporary understanding of copper-cobalt (Cu-Co) tolerance and accumulation in plants. Accumulation of foliar Cu and Co to > 300 µg g-1 is exceptionally rare globally, and known principally from the Copperbelt of Central Africa. Cobalt accumulation is also observed in a limited number of nickel (Ni) hyperaccumulator plants occurring on ultramafic soils around the world. None of the putative Cu or Co hyperaccumulator plants appears to comply with the fundamental principle of hyperaccumulation, as foliar Cu-Co accumulation is strongly dose-dependent. Abnormally high plant tissue Cu concentrations occur only when plants are exposed to high soil Cu with a low root to shoot translocation factor. Most Cu-tolerant plants are Excluders sensu Baker and therefore setting nominal threshold values for Cu hyperaccumulation is not informative. Abnormal accumulation of Co occurs under similar circumstances in the Copperbelt of Central Africa as well as sporadically in Ni hyperaccumulator plants on ultramafic soils; however, Co-tolerant plants behave physiologically as Indicators sensu Baker. Practical application of Cu-Co accumulator plants in phytomining is limited due to their dose-dependent accumulation characteristics, although for Co field trials may be warranted on highly Co-contaminated mineral wastes because of its relatively high metal value.

ECO-physiological response of S. vulgaris to CR(VI): Influence of concentration and genotype.

The objective of this work is to study the response of Silene vulgaris to a range of environmentally relevant concentrations of Cr(VI) in order to evaluate its potential use in the phytomanagement of Cr polluted sites. Cuttings of six homogenous genotypes from Madrid (Spain) have been used as plant material. The eco-physiological response of S. vulgaris to Cr(VI) changed with the genotype. The yield dose-response curve was characterized by stimulation at low doses of Cr(VI). The effects of metal concentration were quantified on root dry weight, water content and chlorophyll content, determined by SPAD index. The response was not homogeneous for all studied genotypes. At high doses of Cr(VI), plants increased micronutrient concentration in dry tissues which suggested that nutrient balance could be implicated in the alleviation of Cr toxicity. This work highlights the importance of studying the eco-physiological response of metallophytes under a range of pollutant concentrations to determine the most favorable traits to be employed in the phytomanagement process.

Symphyotrichum ericoides populations from seleniferous and nonseleniferous soil display striking variation in selenium accumulation.

Symphyotrichum ericoides (Asteraceae) from naturally seleniferous habitat (Pine Ridge) was shown previously to have selenium (Se) hyperaccumulator properties in field and glasshouse studies, and to benefit from Se through protection from herbivory. To investigate whether Se hyperaccumulation is ubiquitous in S. ericoides or restricted to seleniferous soils, the S. ericoides Pine Ridge (PR) population was compared with the nearby Cloudy Pass (CP) population from nonseleniferous soil. The S. ericoidesPR and CP populations were strikingly physiologically different: in a common garden experiment, PR plants accumulated up to 40-fold higher Se concentrations than CP plants and had 10-fold higher Se : sulfur (S) ratios. Moreover, roots of S. ericoidesPR plants showed directional growth toward selenate, while CP roots did not. Growth of both accessions responded positively to Se. Each accession grew best on its own soil. Rhizosphere soil inoculum from the S. ericoidesPR population stimulated plant growth and Se accumulation in both S. ericoidesPR and S. ericoidesCP plants, on both PR and CP soils. While the S. ericoidesPR population hyperaccumulates Se, the nearby CP population does not. The capacity of S. ericoidesPR plants to hyperaccumulate Se appears to be a local phenomenon that is restricted to seleniferous soil. Mutualistic rhizosphere microbes of the S. ericoidesPR population may contribute to the hyperaccumulation phenotype.

Copper mediates auxin signalling to control cell differentiation in the copper moss Scopelophila cataractae.

The copper (Cu) moss Scopelophila cataractae (Mitt.) Broth. is often found in Cu-enriched environments, but it cannot flourish under normal conditions in nature. Excess Cu is toxic to almost all plants, and therefore how this moss species thrives in regions with high Cu concentration remains unknown. In this study, we investigated the effect of Cu on gemma germination and protonemal development in S. cataractae. A high concentration of Cu (up to 800 µM) did not affect gemma germination. In the protonemal stage, a low concentration of Cu promoted protonemal gemma formation, which is the main strategy adopted by S. cataractae to expand its habitat to new locations. Cu-rich conditions promoted auxin accumulation and induced differentiation of chloronema into caulonema cells, whereas it repressed protonemal gemma formation. Under low-Cu conditions, auxin treatment mimicked the effects of high-Cu conditions. Furthermore, Cu-induced caulonema differentiation was severely inhibited in the presence of the auxin antagonist α-(phenylethyl-2-one)-indole-3-acetic acid, or the auxin biosynthesis inhibitor l-kynurenine. These results suggest that S. cataractae flourishes in Cu-rich environments via auxin-regulated cell differentiation. The copper moss might have acquired this mechanism during the evolutionary process to benefit from its advantageous Cu-tolerance ability.

Genetic and morphological evidence support the specific status of the endemic Ericaandevalensis (Ericales, Ericaceae).

Assessing the taxonomic status of closely related taxa is crucial in plant systematics and can have important implications for conservation and human plant use. Ericaandevalensis Cabezudo & Rivera is a metallophyte endemic species from highly metal-polluted soils of SW Iberian Peninsula, an area with a mining history going back more than 5,000 years. Ericaandevalensis is closely related to Ericamackayana Bab., a northern Iberian species also present in western Ireland. The status of E.andevalensis as a species or subspecies subordinated to E.mackayana is subject to debate. Here, we assessed the genetic and phenotypic relationship between both species, including the population structure of E.andevalensis. We used high throughput sequencing to determine genome-wide Single Nucleotide Polymorphisms (SNPs), and morphometric analyses from 35 reproductive and vegetative traits. The morphological analysis showed at least eight characters that can discriminate the two species, from which ovary hairiness and the size of leaf glandular hairs were the most informative. Genetic analyses showed that each species formed a monophyletic cluster with full support, separated by an interspecific genetic distance >4-fold higher than intra-specific distance. Population genetic analyses of E.andevalensis shows that populations are highly structured, with the Portuguese one as the most isolated and less variable. These results support the recognition of E.andevalensis as a distinct species with a highly constrained ecological requirements and a narrow geographic distribution, but with a limited gene flow between populations. We discuss the implications of these outcomes in conservation policies and potential uses of E.andevalensis such as decontamination of polluted soils.

Metal phytostabilization by mastic shrub (Pistacia lentiscus L.) and its root-associated bacteria in different habitats of Sardinian abandoned mining areas (Italy).

Pistacia lentiscus L. is an excluder metallophyte proposed for the revegetation and phytostabilization of metal-contaminated sites in the Mediterranean area. The present study aims at evaluating the linking between bacterial communities and plants spontaneously growing in ecosystems chronically impacted by mining activities. Environmental properties and metal accumulation into hypogeal and epigeal tissues were analyzed in wild plants of two contrasting habitats with extreme metal contamination (> 2300 mg/kg for Zn, > 1100 mg/kg for Pb, > 10 mg/kg for Cd). The community structures of rhizospheric and root endophytic bacteria were fingerprinted by terminal restriction fragment length polymorphism of the 16S rRNA gene. The wild shrubs efficiently restrict the accumulation of the three major contaminants to the epigeal tissues in the two habitats under study (249 ± 68 mg/kg dw for Zn, 43 ± 21 mg/kg dw for Pb, and 1.4 ± 0.5 mg/kg dw for Cd). Evidence was provided that the combined but not individual effect of environmental conditions (moisture, inorganic carbon, pH) and proportion between Zn and Cd in the mine substrate play a role in structuring rhizosphere bacterial communities. The observed changes in community structures of root endophytes were found to be strongly associated with Pb level in roots and substrate properties (inorganic carbon and Zn/Cd ratio). Overall, our study highlights the importance of the analysis of multifactorial interactions among mine substrate, plant, and microbes for understanding how the environmental context affects phytoremediation under real conditions.

Investigation of the Copper Requirements of the Metallophyte Liverworts Cephaloziella nicholsonii Douin and C. massalongoi (Spruce) Müll.Frib.

Former mine sites can provide habitat for many rare specialised bryophyte species that have adapted to metal-rich soil conditions that are toxic to most other plant species. Some of the bryophyte species found in this habitat are facultative metallophytes, and others are regarded as strict metallophytes, the so-called 'copper mosses'. It is a general assumption in the literature that Cephaloziella nicholsonii and C. massalongoi, both categorised as Endangered in the IUCN Red List for Europe, are also strict metallophytes and obligate copper bryophytes. This in vitro experiment investigated the growth and gemma production of these two species from different sites in Ireland and Britain on treatment plates of 0 ppm, 3 ppm, 6 ppm, 12 ppm, 24 ppm, 48 ppm and 96 ppm copper. Results show that elevated copper is not an obligate requirement for optimum growth. Differences in response to the copper treatment levels among populations evident within both species could possibly be due to ecotypic variation. A case is also made for the taxonomic revision of the Cephaloziella genus. Implications for the species' conservation are discussed.

Thallium hyperaccumulation status of the violets of the Allchar arsenic-thallium deposit (North Macedonia) confirmed through synchrotron µXRF imaging.

The abandoned Allchar Mine in the Republic of North Macedonia is a globally unique deposit with the highest known grades of thallium (Tl) and arsenic (As) mineralization. We aimed to determine the distribution of As and Tl in whole dehydrated shoots of the three Viola taxa using synchrotron micro-X-ray fluorescence analysis. Additionally, soil and plant organ samples were collected from all three Viola taxa at the Allchar site and analysed using inductively coupled plasma-atomic emission spectrometry. Concentrations of Tl were extremely high in all three Viola taxa (up to 58 900 mg kg-1), but concentrations of As were highly variable with V. tricolor subsp. macedonica and V. allchariensis having low As (up to 20.2 and 26.3 mg kg-1, respectively) and V. arsenica having the highest concentrations (up to 381 mg kg-1). The extremely high Tl in all three species is endogenous and not a result of contamination. Arsenic in V. tricolor subsp. macedonica and V. allcharensis is strongly affected by contamination, but not in V. arsenica where it appears to be endogenous. The pattern of As enrichment in V. arsenica is very unusual and coincides with Ca-oxalate deposits and Br hotspots. The results of this study could form the basis for more detailed investigations under controlled conditions, including plant dosing experiments.