Metallicolous Populations Research Articles

Species-Specific and Pollution-Induced Changes in Gene Expression and Metabolome of Closely Related Noccaea Species Under Natural Conditions

Hyperaccumulators within the Noccaea genus possess many promising genetic and metabolic adaptations that could be potentially exploited to support phytoremediation efforts and/or crop improvement and biofortification. Although hyperaccumulation is very common in this genus, individual species display specific traits as they can accumulate different elements (e.g., zinc, cadmium, and/or nickel). Moreover, there appears to be some populational variability with natural selection increasing the metal tolerance in metallicolous populations. Therefore, employing robust methods, such as integrated analysis of the transcriptome and metabolome, is crucial for uncovering pivotal candidate genes and pathways orchestrating the response to metal stress in Noccaea hyperaccumulators. Our study highlights several species-specific traits linked to the detoxification of metals and metal-induced oxidative stress in hyperaccumulating N. praecox when compared to a closely related model species, N. caerulescens, when grown in the field. Transcriptome analysis revealed distinct differences between the three studied natural Noccaea populations. Notably, we observed several pathways frequently connected to metal stress, i.e., glutathione metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis, which were enriched. These differences were observed despite the relative evolutionary closeness of studied species, which emphasizes the importance of further expanding our knowledge on hyperaccumulators if we want to exploit their mechanisms for phytoremediation efforts or food quality improvements.

Lead and Zinc tolerance and accumulation in metallicolous and non-metallicolous populations of Peganum harmala L: potential use in phytostabilization

Peganum harmala L., commonly known as Harmal, is an aromatic and medicinal plant that grows in North Africa. P harmala is a pseudometallophyte that occur both in heavy metals contaminated and non contaminated soils . In order to identify the best adapted and suitable populations within P. harmala species, a comprehensive analysis and evaluation of the morphological, physiological, and biochemical responses to lead (Pb) and zinc (Zn) were performed in three metallicolous populations (MP) and a non-metallicolous one (NMP). In Zaida, Mibladen, and Aouli Morrocan sites, P. harmala growing soils show a low organic matter content, high Pb and Zn concentrations, and a pollution index higher than 1, indicating a significant contamination level. The effects of Pb and Zn on growth, physiological, antioxidant enzyme activities, and Pb/Zn accumulation capacity were compared between a NMP and three MP of P. harmala. Plants were grown in hydroponic culture with 100 µM of ZnSO4 or 100 µM of Pb(NO3)2 for 15 days. Our results show that Pb and Zn significantly decreased the aboveground biomass in the NMP, MP-Mibladen, and MP-Aouli compared to MP-Zaida. Pb and Zn decreased significantly the root biomass in the NMP compared to the MP. There is no significant difference in chlorophyll a and b contents between NMP and MP. Under Pb exposure, anthocyanin content is shown to be significantly higher in MP-Zaida than in other populations. In contrast, there is no difference between NMP and MP under Zn stress. Proline content as well as the catalase, peroxidase, and ascorbate peroxidase activities was significantly higher in NMP than in MP under Pb and Zn exposure. In particular, lower proline content, and antioxidant activities were observed in MP-Zaida under the Pb and Zn exposure. Pb and Zn accumulations were significantly higher in the NMP than in the MP, both in roots and aboveground parts, showing that the NMP plants accumulated highly but tolerated less compared to the MP. In the present study, the MP-Zaida accumulated less Pb and Zn in their tissues and tolerated more than the other populations, suggesting this population as a good candidate for a phytostabilization strategy.

Detoxification role of amino acids and phytochelatins on two populations of harmel plant under silver stress.

Two non-metallicolous and metallicolous populations of harmel plants were compared regarding the role of proline, cysteine, reducing sugars, hydrogen peroxide (H2O2), glutathione, thiol compounds, organic acids, total free amino acids, and lipid peroxidation in detoxification and tolerance of silver stress (0, 1, 2.5, 5, 10 ppm Ag). The results of the present research state that the effects of Ag were increased total free amino acids, glutathione, organic acids, proline, reducing sugars, thiol compounds, and cysteine, so the accumulation of these compounds was higher in metallicolous populations than non-metallicolous. On the other hand, non-metallicolous populations showed higher content of lipid peroxidation and H2O2 than metallicolous populations under Ag stresses. Also, the accumulation of phytochelatins (PC) was observed with increasing Ag concentration, which shows that compared to glutathione, non-protein thiols have a higher concentration. The number of organic acids (malic acids, fumaric, oxalic, and citric) except acetic acid increased in the leaves of harmel in both populations. According to the results of this research, the harmel metallophilic population has a crucial role in the tolerance and detoxification of Ag stress, so the antioxidant responses of the plant against Ag stress in the non-metallicolous population were lower than the metallicolous population. Based on the above results, it can be concluded that the harmel plant has a detoxification mechanism to deal with high concentrations of Ag.

Seed Bank Conservation and Incipient Seed Development in Orchids Colonizing Mining Wastes: Results of a Field Pilot Experiment.

As they represent actual or potential risks to human and environmental safety and health, abandoned mines are a major global problem. The heavy metal-polluted tailings dump of Barraxiutta (Domusnovas, southwestern Sardinia, Italy) is home to a metallicolous population of Epipactis tremolsii (Orchidaceae). A reclamation of the abandoned mine area seems to be approaching, and such an intervention may pose a serious risk for the maintenance of the unique orchid population colonizing the mine wastes. In the present work, the seed packet technique was implemented for the first time to observe orchid seed development in mine wastes. This approach allowed us to explore different seed-based conservation options for the metallicolous orchid population and to gain a deeper grasp of population dynamics and ecology. Four different sowing treatments were set up in the tailing dump and in a near unpolluted site (control site). The field phase of the experiment lasted for 10 months, a period in which the experimental seed bank preservation and incipient seed development were observed and statistically approached. Our findings observed no significant seed loss happening during the experiment, demonstrating the suitability of the seed packet technique to also explore seed bank conservation and development in extreme environmental conditions (i.e., polluted mine wastes). This field method will be a useful tool to further explore the more effective translocation and quasi in situ conservation alternatives for the E. tremolsii metallicolous population. Incipient and site-specific seed development (non-mycorrhizal stage) was observed during the experiment. A plant-soil fungus interaction at the seed level was also observed, the nature of which remains to be ascertained in further studies providing a longer duration for the field phases.

Investigation of the Metal Bioremediation Ability of Two Populations of Peganum Harmala

The present study aimed to investigate the effects of zinc exposure (0, 1, 5, 15, 30 mg/L zinc) on the biochemical and physiological parameters of Peganum harmala seedlings. Two populations (metallicolous and non-metallicolous) were compared in Zn tolerance, Zn accumulation, photosynthetic pigments, and enzymatic antioxidant activities. Plants were treated with Zn at concentrations of 0, 1, 5, 15, and 30 mg/L for 14 days. The study results showed that the increase of Zn concentration in the nutrient solution reduced shoot length, root length, root dry weight, shoot dry weight, chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid in both populations; however, the accumulation was more pronounced in metallicolous populations (M) than in non-metallicolous (NM) ones. In response, the activities of antioxidant enzymes such as guaiacol peroxidase, lipoxygenase, superoxide dismutase, catalase, and ascorbate peroxidase were enhanced Zn exposure in both populations. Moreover, it was found that the metallicolous population of P. harmala had a greater capacity to adapt to oxidative stress caused by Zn than the non-metallicolous population, and antioxidative defense in the metallicolous population of P. harmala might have played an essential role in Zn tolerance. Therefore, P. harmala seemed to be a suitable candidate for accumulation; however, it was recommended that further investigations be carried out to explore its metal remediation ability. It is concluded that P. harmala can be a potential candidate for bioremediation of Zn contaminated soils.

Heavy metal tolerance strategies in metallicolous and non-metallicolous populations of mosses: Insights of γ+β-tocopherol regulatory role

As bryophytes present the capacity to colonize a wide range of habitats, including extreme environments (e.g., mine areas), we first checked the global response of bryophytes to heavy metals (HM) by a systematic review. This analysis found i) the lack of studies in a polluted environment and ii) resulted in a global trend of photochemical efficiency decrease when bryophytes faced heavy metals. Secondly, we characterized the photoprotective responses of four metallicolous populations of mosses ( Lewinskya rupestris , Polytrichum commune , Ptychostomum compactum and Rhynchostegium confertum ) naturally growing in an abandoned mine area by an experimental field approach. As a result, we found markedly species-specific tolerance patterns: i ) less tolerant species, presenting high pollutants content accompanied by marked dissipative and antioxidative strategies. This strategy was depicted by the species R. confertum that result to be a good sentinel species due to the high pollutant content and high physiological sensitivity.; ii ) intermediate tolerant species presenting a medium content of pollutants and less marked photoprotective mechanisms, and iii ) most tolerant species minimizing pollutants content and consequently presenting no changes in their physiological performance. This evidence support that species’ bryological attributes have a key role in determining species’ tolerance towards environmental heavy metals, and should be taken into consideration in future studies. The findings of this study also pointed out that the higher levels of γ + β-tocopherol in metallicolous populations could play a regulatory role in metal stress tolerance in mosses and it could be a suitable functional responsive trait for environmental response prediction to heavy metals in polluted environments. • Bryophytes' adaptive mechanisms to metalliferous environments are poorly studied. • Photoprotective strategies are the result of species' bryological attributes and pollutant content. • Changes in photoprotective compounds levels mark bryophytes’ tolerance to environmental heavy metals. • Increasing γ + β-tocopherol is a functional trait in mosses copying with heavy metals.

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.

Structural traits of leaf epidermis correspond to metal tolerance in Rumex acetosella populations growing on metal-contaminated soils

The pseudometallophyte Rumex acetosella L. occupies habitats with normal and high soil concentrations of zinc (Zn), lead (Pb), and copper (Cu). It remains unclear if the plants respond to the toxic metals by altering their morphology and increasing the resilience of their cells. We compared plants growing on soils contaminated with Zn/Pb (populations Terézia, Lintich), or Cu (populations Špania Dolina, Staré Hory), with those from non-contaminated soil (Dúbravka) in Slovakia, and analysed leaf structure, physiology, and metal contents by light and electron microscopy, element localization by energy-dispersive X-ray analysis (EDX) in scanning electron microscope, and by specific fluorescence dyes. In control population, the epidermis of the amphistomatic leaves of R. acetosella contained capitate glandular trichomes, consisting of four head (secretory), two stalk, and two basal cells. The ultrastructure of secretory cells revealed fine wall ingrowths bordered by plasma membrane protruding into the cytoplasm. The metallicolous populations had higher contents of Zn and Cu in the epidermal and glandular cells, and a higher density of both stomata and trichomes. Extensive cell wall labyrinth was present in the trichome secretory cells. Their abnormal number and elevated metal contents might indicate effects of heavy metals, especially of Cu, on mitosis and cell plate formation. Differences in leaf physiology were indicated by significantly higher cytoplasmic tolerance to Zn and Cu in metallicolous populations and by structural properties of glandular heads suggesting secretion of toxic metals. Our findings are suggestive of plant reactions to metal stress, which facilitate the populations to occupy the metal-contaminated sites.

Adaptation of Arabidopsis halleri to extreme metal pollution through limited metal accumulation involves changes in cell wall composition and metal homeostasis.

Metallophytes constitute powerful models for the study of metal homeostasis, adaptation to extreme environments and the evolution of naturally selected traits. Arabidopsis halleri is a pseudometallophyte which shows constitutive zinc/cadmium (Zn/Cd) tolerance and Zn hyperaccumulation but high intraspecific variability in Cd accumulation. To examine the molecular basis of the variation in metal tolerance and accumulation, ionome, transcriptome and cell wall glycan array profiles were compared in two genetically close A.halleri populations from metalliferous and nonmetalliferous sites in Northern Italy. The metallicolous population displayed increased tolerance to and reduced hyperaccumulation of Zn, and limited accumulation of Cd, as well as altered metal homeostasis, compared to the nonmetallicolous population. This correlated well with the differential expression of transporter genes involved in trace metal entry and in Cd/Zn vacuolar sequestration in roots. Many cell wall-related genes were also more highly expressed in roots of the metallicolous population. Glycan array and histological staining analyses demonstrated that there were major differences between the two populations in terms of the accumulation of specific root pectin and hemicellulose epitopes. Our results support the idea that both specific cell wall components and regulation of transporter genes play a role in limiting accumulation of metals in A. halleri at contaminated sites.

Heavy metals modulate DNA compaction and methylation at CpG sites in the metal hyperaccumulator Arabidopsis halleri.

Excess heavy metals affect plant physiology by inducing stress symptoms, however several species have evolved the ability to hyperaccumulate metals in above-ground tissues without phytotoxic effects. In this study we assume that at subcellular level, different strategies were adopted by hyperaccumulator versus the non-accumulator plant species to face the excess of heavy metals. At this purpose the comet assay was used to investigate the nucleoid structure modifications occurring in response to Zn and Cd treatments in the I16 and PL22 populations of the hyperaccumulator Arabidopsis halleri versus the nonaccumulator species Arabidopsis thaliana. Methy-sens comet assay and RT-qPCR were also performed to associate metal induced variations in nucleoids with possible epigenetic modifications. The comet assay showed that Zn induced a mild but non significant reduction in the tail moment in A. thaliana and in both I16 and PL22. Cd treatment induced an increase in DNA migration in nuclei of A. thaliana, whereas no differences in DNA migration was observed for I16, and a significant increase in nucleoid condensation was found in PL22 Cd treated samples. This last population showed higher CpG DNA methylation upon Cd treatment than in control conditions, and an up-regulation of genes involved in symmetric methylation and histone deacetylation. Our data support the hypothesis of a possible role of epigenetic modifications in the hyperaccumulation trait to cope with the high Cd shoot concentrations. In addition, the differences observed between PL22 and I16 could reinforce previous suggestions of divergent strategies for metals detoxification developing in the two metallicolous populations.

Transcription factor behaves differently in metallicolous and non-metallicolous populations of

In Arabidopsis thaliana (L.) Heynh., MYB59 transcription factor participates in regulating Ca homeostasis and signal transduction and is induced by Cd excess. To investigate its role in the facultative metallophyte Arabidopsis halleri ssp. halleri (L.) O'Kane and Al-Shehbaz, MYB59 expression was investigated under Cd treatment or Ca depletion in three populations belonging to distinct phylogeographic units (metallicolous PL22 and I16 and non-metallicolous I29), and compared with the expression in A. thaliana. In control conditions, MYB59 transcription in A. thaliana and the non-metallicolous population I29 follow a comparable trend with higher expression in roots than shoots, whereas in metallicolous populations I16 and PL22 its expression is similar in roots and shoots, suggesting a convergent evolution associated with adaptation to metalliferous environments. After 6 h of Ca depletion, MYB59 transcript levels were very high in I16 and PL22 populations, indicating that the adaptation to metalliferous environments requires tightly regulated Ca homeostasis and signalling. Cd treatment caused variability in MYB59 expression. In I29, MYB59 expression, as in A. thaliana, is likely associated to stress response, whereas its modulation in the two metallicolous populations reflects the different strategies for Cd tolerance and accumulation. In conclusion, MYB59 regulation in A. halleri is part of the network linking mineral nutrition and Cd tolerance/accumulation.

OPDA and ABA accumulation in Pb-stressed Zygophyllum fabago can be primed by salicylic acid and coincides with organ-specific differences in accumulation of phenolics

Salicylic acid (SA) is a well-known priming agent that is widely used to protect plants against stressing agents, including heavy metals as Pb. A better understanding of the mechanisms that enable plants to counteract Pb toxicity would help to select strategies for land reclamation programs. Here we used a metallicolous population of Zygophyllum fabago to assess the extent to which SA pretreatment modulates Pb-induced changes in phenol metabolism and stress-related phytohormone levels in roots and leaves. Our data revealed that accumulation of different phytohormones, lignin, soluble and wall-bound phenolics as well as peroxidase (PRX) activity in Pb-stressed plants differed after SA-pretreatment. Exposure to Pb led to the induction of soluble and cell wall-bound PRX activities, particularly those involved in the oxidation of coniferyl alcohol and ferulic acid, while pretreatment with SA reduced the Pb-induced stimulation of PRX activities in roots but increased them in leaves. SA-treatment by itself induced accumulation of ABA and the JA-precursor 12-oxo-phytodienoic acid (OPDA) in the roots. Pb in turn inhibited these SA-induced effects with the exception of OPDA accumulation that was primed by the pretreatment. The SA treatment also induced accumulation of OPDA in leaves but suppressed the accumulation of JA-Ile although with relatively small absolute changes. Notably, Pb-induced accumulation of ABA was primed in the leaves of SA-pretreated plants. Together our data suggest that priming of OPDA accumulation in the roots and of ABA in the leaves by SA-pretreatment may play important regulatory roles, possibly via regulating PRX activities, for Pb stress in plants.

Differences in the functioning of photosynthetic electron transport between metallicolous and non-metallicolous populations of the pseudometallophyte Viola tricolor

The objective of this study was to assess the effect of metalliferous conditions on the functioning of photosynthetic electron transport in waste heap populations of a pseudometallophyte, Viola tricolor L. Measurements of chlorophyll a fluorescence and the absorbance changes at 830 nm enabled a non-invasive assessment of photosynthetic apparatus performance. This was complemented by the evaluation of the chlorophyll content. Low temperature chlorophyll fluorescence emission spectra were also recorded. Based on the OJIP test performed in situ, we demonstrated a disturbed condition of photosystem II (PSII) in three metalliferous populations in comparison with a non-metallicolous one. The combined effects of elevated concentrations of zinc, cadmium and lead in soil resulted in the decline of some parameters describing the efficiency and electron flow through PSII. The differences between waste heap populations seemed to be partly correlated with the concentration of heavy metals in the soil. The characteristic of electron transport at photosystem I (PSI) in the light-adapted state revealed increased values of PSI donor-side limitation (YND) and a declined PSI quantum efficiency (YI). It was also demonstrated that the waste heap conditions negatively affect the total chlorophyll content in leaves and led to an increased ratio of fluorescence emission at 77 K (F730/F685). The obtained data indicate that, regardless of the high adaptation of metallicolous populations, photosynthetic electron transport is hampered in V. tricolor plants at metal polluted sites.

Does slow and steady win the race? Root growth dynamics of Arabidopsis halleri ecotypes in soils with varying trace metal element contamination

Hyperaccumulating plants possess complex traits, allowing them to thrive in soils with high concentrations of trace metal elements (TME). Accordingly, their TME hypertolerance and hyperaccumulation capacities have been intensely studied from physiological, evolutionary, and ecological perspectives. Little is known, however, about their root system development in TME enriched vs natural soils. We assessed temporal and quantitative changes in root systems of the model species Arabidopsis halleri, using a novel combination of root phenotyping in rhizoboxes and multitemporal digital imaging. We continuously monitored root growth of two non-metallicolous and two metallicolous populations in different substrate treatments, including homogeneous and horizontal layer applications of metalliferous and non-metalliferous soils. Non-metallicolous plants on non-metalliferous soils produced deep-reaching and wide roots, whereas metallicolous plants on metalliferous soil had smaller roots. This pattern was reversed when transplanting seedlings to foreign substrates, indicating that environment rather than ecotype determines root growth in A. halleri. Dampened root development in metalliferous and favored root proliferation in non-metalliferous soils indicate cost of tolerance and TME foraging, respectively. Importantly, root propagation into metalliferous soil was strongly promoted by a non-metalliferous “capping” layer that facilitated initial plant development. Hence, growing on non-polluted substrate in the early stage provides plants with a robust and optimal root system that facilitates seedling establishment and subsequent development under TME enriched conditions. These findings improve our understanding of plant performance in metalliferous environments and can help refine management practices for the sustainable reclamation of degraded lands.

Toxic Effects of Cd and Zn on the Photosynthetic Apparatus of the Arabidopsis halleri and Arabidopsis arenosa Pseudo-Metallophytes.

Hyperaccumulation and hypertolerance of Trace Metal Elements (TME) like Cd and Zn are highly variable in pseudo-metallophytes species. In this study we compared the impact of high Cd or Zn concentration on the photosynthetic apparatus of the Arabidopsis arenosa and Arabidopsis halleri pseudo-metallophytes growing on the same contaminated site in Piekary Slaskie in southern Poland. Plants were grown in hydroponic culture for 6 weeks, and then treated with 1.0 mM Cd or 5.0 mM Zn for 5 days. Chlorophyll a fluorescence and pigment content were measured after 0, 1, 2, 3, 4, and 5 days in plants grown in control and exposed to Cd or Zn treatments. Moreover, the effect of TME excess on the level of oxidative stress and gas-exchange parameters were investigated. In both plant species, exposure to high Cd or Zn induced a decrease in chlorophyll and an increase in anthocyanin contents in leaves compared to the control condition. After 5 days Cd treatment, energy absorbance, trapped energy flux and the percentage of active reaction centers decreased in both species. However, the dissipated energy flux in the leaves of A. arenosa was smaller than in A. halleri. Zn treatment had more toxic effect than Cd on electron transport in A. halleri compared with A. arenosa. A. arenosa plants treated with Zn excess did not react as strongly as in the Cd treatment and a decrease only in electron transport flux and percentage of active reaction centers compared with control was observed. The two species showed contrasting Cd and Zn accumulation. Cd concentration was almost 3-fold higher in A. arenosa leaves than in A. halleri. On the opposite, A. halleri leaves contained 3-fold higher Zn concentration than A. arenosa. In short, our results showed that the two Arabidopsis metallicolous populations are resistant to high Cd or Zn concentration, however, the photosynthetic apparatus responded differently to the toxic effects.

Alternative responses to fungal attack on a metalliferous soil: Phytohormone levels and structural changes in Silene paradoxa L. growing under copper stress

In this work, a non-metallicolous and a metallicolous population of S. paradoxa were exposed to copper excess and fungal elicitation, and investigated for phytohormone production and cytological alterations.Under the stress applied separately and in combination, S. paradoxa plants varied phytohormone concentration in a population-specific way, suggesting a different signalling in response to biotic and abiotic stimuli according to the environment of origin. Generally, the stress responses consisted in increased levels of salicylic acid, auxin, and gibberellin in the non-metallicolous population, and of jasmonic and abscisic acid in the metallicolous one. Interestingly, the metallicolous population increased the level of such phytohormones following exposure to the fungal elicitor only in the presence of copper. This alternative hormonal signalling could derive from the incompatibility between the ordinary ROS-mediated response to pathogens and the acquired mechanisms that prevent oxidative stress in the population from the metal-rich soil. Furthermore, stress-induced autophagic phenomena were more evident in the non-metallicolous plants than in the metallicolous ones, suggesting that the adaptation to the metalliferous environment has also affected autophagy intensity and signalling in response to copper excess and fungal elicitation.

Evaluation of heavy metal-induced responses in Silene vulgaris ecotypes

Silene vulgaris is a pseudometallophyte that spontaneously occurs in various ecological niches. Therefore, three ecotypes of this species representing calamine (CAL), serpentine (SER), and non-metallicolous (NM) populations were investigated in this study. Owing to the presence of Pb or Ni ions in natural habitats from metallicolous populations originated, we used these metals as model stressors to determine the survival strategy of tested ecotypes and analyze metal distribution at various levels of organism organization. We focused on growth tolerance, non-enzymatic antioxidants, and photosynthetic apparatus efficiency as well as anatomical and ultrastructural changes occurred in contrasting ecotypes exposed in vitro to excess amounts of Pb2+ and Ni2+. Although Ni application contributed to shoot culture death, the study revealed that the mechanisms of Pb detoxification differed between ecotypes. The unspecific reaction of both metallicolous specimens relied on the formation of effective mechanical barrier against toxic ion penetration, while the Pb appearance in the protoplasts led to the activation of ecotype-specific intracellular defense mechanisms. Hence, the response of CAL and SER ecotypes was almost unchanged under Pb treatment, whereas the reaction of NM one resulted in growth disturbances and physiological alternations. Moreover, both metallicolous ecotypes exhibited increase generation of reactive oxygen species (ROS) in leaves, even before the harmful ions got into these parts of plants. It may implicate the potential role of ROS in CAL and SER adaptation to heavy metals and, for the first time, indicate on integral function of ROS as signaling molecules in metal-tolerant species.

Effects of manganese stress on phenology and biomass allocation in Xanthium strumarium from metalliferous and non-metalliferous sites

Xanthium strumarium is an annual pseudometallophyte. To reveal the mechanisms of this species to adapt to metallicolous environmental conditions, phenological traits and biomass allocation of metallicolous and non-metallicolous populations of X. strumarium under six Mn2+ concentrations by pot culture experiments were performed. The results showed that both time to bolting and time to fruit setting in the metallicolous population were earlier than those in the non-metallicolous population. The number of flowers, fruits, seeds and 1000-seed weight in the metallicolous population were higher than those in the non-metallicolous population under Mn stress. Reproductive allocation and harvest index in the metallicolous population were higher than those in the non-metallicolous population. Furthermore, all the Mn concentrations in leaves, stems, roots, and fruits of the metallicolous population were higher than the counterparts of non-metallicolous population. These results suggested that metallicolous population had higher tolerance to Mn stress than non-metallicolous population, the earlier flowering and fruiting, and the enhancement in reproductive allocation may contribute to plant tolerance to Mn toxicity for X. strumarium.

Antimony tolerance and accumulation in a metallicolous and a non-metallicolous population of Salvia spinosa L.

Aims: Antimony (Sb) is locally found at potentially toxic concentrations in mineralized soils, usually together with arsenic (As). However, local adaptation of plant populations to Sb toxicity has never been shown thus far. Here we compared Sb tolerance and accumulation between a non-metallicolous (NM) population of Salvia spinosa, and a con-specific metallicolous (M) population from a strongly Sb/As-enriched soil in Dashkasan, Iran. Methods: Plants were exposed in hydroponics to a series of Sb[III] and Sb[V] concentrations. After 3 weeks the dry weights and Sb concentrations of roots and shoots were determined. Results: Estimated from the effects on shoot dry weight, the M population was more tolerant than the NM one, particularly to Sb[V], but to a lower degree also to Sb[III]. In both populations Sb[III] was taken up and translocated at higher rates than Sb[V]. The Sb concentrations in roots and shoots were slightly, but significantly higher in the M than in the NM population. Conclusions: Since Sb[V] and As[V] seem to follow very different detoxification pathways, it can be argued that the superior tolerance to Sb[V] in M represents a local adaptation to Sb[V] toxicity itself, rather than being a mere by-product of hypertolerance to its chemical analogue, As[V]. Since Sb[III] and As[III] or As[V] share common detoxification pathways, the apparent Sb[III] hypertolerance in the M population might represent a by-product of As hypertolerance.

Can zinc pollution promote adaptive evolution in plants? Insights from a one-generation selection experiment.

Human activities generate environmental stresses that can lead plant populations to become extinct. Population survival would require the evolution of adaptive responses that increase tolerance to these stresses. Thus, in pseudometallophyte species that have colonized anthropogenic metalliferous habitats, the evolution of increased metal tolerance is expected in metallicolous populations. However, the mechanisms by which metal tolerance evolves remain unclear. In this study, parent populations were created from non-metallicolous families of Noccaea caerulescens. They were cultivated for one generation in mesocosms and under various levels of zinc (Zn) contamination to assess whether Zn in soil represents a selective pressure. Individual plant fitness estimates were used to create descendant populations, which were cultivated in controlled conditions with moderate Zn contamination to test for adaptive evolution in functional traits. The number of families showing high fitness estimates in mesocosms was progressively reduced with increasing Zn levels in soil, suggesting increasing selection for metal tolerance. In the next generation, adaptive evolution was suggested for some physiological and ecological traits in descendants of the most exposed populations, together with a significant decrease of Zn hyperaccumulation. Our results confirm experimentally that Zn alone can be a significant evolutionary pressure promoting adaptive divergence among populations.