Exploring element accumulation patterns of a metal excluder plant naturally colonizing a highly contaminated soil.

This work investigates the element distribution in Silene paradoxa growing on the mine dump of Fenice Capanne (Tuscany, Italy). The accumulation of As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn in root apoplast and symplast and in shoot was assessed and compared to the levels of the same metals in the respective rizosphere soils, analyzing both the total and the phytoavailable fractions. Levels of As, Cu, Fe, Pb and Zn, were above toxicity thresholds in both soil and shoot samples. Inter- and intra-element correlations were analyzed in plant and soil using different statistical methods. Soil total and phytoavailable metal concentration were shown not to be dominant in determining metal accumulation by the plant, since no significant positive correlation was found between metal concentration in soils and plants. Moreover, results indicated that S. paradoxa was able to cope with the studied multi-metal contaminated soil excluding the elements from its tissues and preferentially accumulating them into the root compartment, thus suggesting this species as possible good candidate for phytostabilization purposes.

Cadmium phytoextraction potential of different Alyssum species.

This work was planned for providing useful information about the possibility of using serpentine adapted plants for phytoextraction of cadmium, element scarcely represented in such metalliferous environment. To this aim, we investigated variation in cadmium tolerance, accumulation and translocation in three Alyssum plants with different phenotypes: Alyssum bertolonii, that is a serpentine endemic nickel hyperaccumulator, and two populations of Alyssum montanum, one adapted and one not adapted to serpentine soils. Plants were hydroponically cultivated in presence of increasing concentrations of CdSO(4) for two weeks. For the metal concentration used in the experiments, the three different Alyssum populations showed variation in cadmium tolerance, accumulation and content. The serpentine adapted population of A. montanum showed statistically higher cadmium tolerance and accumulation than A. bertolonii and the population of A. montanum not adapted to serpentine soil thus deserving to be investigated for phytoextraction purposes. Furthermore, as for the kinetic parameters of the cadmium uptake system, A. montanum serpentine population presented a low apparent K(m) value, suggesting a high affinity for this metal of its uptake system, whereas the V(max) values were not significantly different among the plants. Present data revealed metallicolous plants are also suitable for the phytoremediation of metals underrepresented in the environment of their initial origin. Nonetheless, field trials on real contaminated soils are essential.

Evolutionary dynamics of nickel hyperaccumulation in Alyssum revealed by its nrDNA analysis.

• Molecular phylogeny based on ribosomal internal transcribed spacer (ITS) sequences was studied to investigate the phyletic relationships among some nickel (Ni)-hyperaccumulating and nonhyperaccumulating species of the genus Alyssum in relation to their geographic distribution and Ni-hyperaccumulating phenotype. • Thirty-seven samples belonging to 32 taxa were analysed by sequencing the polymerase chain reaction-amplified ITS region and performing neighbor joining, maximum parsimony and maximum likelihood phylogenetic analyses. • The ITS region in the sampled species varied from 221 to 307 bp of ITS1 and from 194 to 251 bp of ITS2. A total of 765 characters was used to infer the phylogeny and the average nucleotide variation detected was 15.15%. • Nickel-hyperaccumulation could have been lost or acquired independently more than once during the speciation of the genus. The geographical location of species could not be related to phylogenetic affinities.

Chloroplast genetic diversity and biogeography in the serpentine endemic Ni-hyperaccumulator Alyssum bertolonii.

• Chloroplast microsatellites (cpSSR) were used to analyze the patterns of genetic variation within and among populations of the serpentine endemic, Alyssum bertolonii . • Thirty-five different chloroplast haplotypes were identified in 90 plants sampled from nine populations originating from the four disjunct northern-Italian serpentine regions where the species is found. • High levels of genetic diversity were found within each of the populations sampled. Analysis of Molecular Variance (AMOVA) showed high degrees of differentiation among both different populations of the same serpentine region and different regions (Φ ST = 0.622, Φ CT = 0.252, respectively). • The results indicated that: each population was established by few founders and then subsequently differentiated the existing chloroplast haplotypes; each population is a distinct genetic entity; and populations within the same serpentine region are more related than populations from different regions.

Genetic diversity of heavy metal-tolerant populations in Silene paradoxa L. (Caryophyllaceae): a chloroplast microsatellite analysis.

Eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in uncontaminated soil in central Italy were studied. Genetic diversity was estimated using five polymorphic chloroplast microsatellite loci (cpSSR), identifying 27 different chloroplast haplotypes. The effective number of alleles, the haplotypic diversity and a stepwise mutational model-based parameter (DSH2) were computed. The effective number of alleles observed within populations from copper mine deposits was 20% that of the serpentine neighbouring populations, suggesting the occurrence of a founder effect. Moreover, 13 of the 27 different haplotypes scored were exclusive to only one population, indicating genetic isolation for all tolerant populations. Even the copper-tolerant populations appeared to have evolved independently. Finally, analysis of molecular variance (AMOVA) of the cpSSR markers gave statistical significance to the grouping of populations according to their geographical location. This study demonstrates that cpSSR markers could be a useful complementary tool to isoenzymes or random amplified polymorphic DNA markers for elucidating the pattern of genetic differentiation in heavy metal-tolerant populations.

Characterization of nickel-resistant bacteria isolated from serpentine soil.

In the present study, heterotrophic nickel-resistant bacteria were isolated and characterized from three different serpentine outcrops in central Italy populated by the nickel-hyperaccumulating plant Alyssum bertolonii. Bacteria were isolated from the rhizosphere of the plant and from soil portions at various distances from the plant. The proportion of nickel-resistant cfu was higher in proximity to the plant than in free soil. A total of 138 isolates was collected and grouped into 47 different operational taxonomic units (OTUs) by means of amplified ribosomal DNA restriction analysis (ARDRA) and into 25 heavy-metal resistant phenotypes. The phylogenetic position of strains belonging to 20 OTUs, representing more than the 70% of the total isolates, was determined by 16S rDNA sequencing. These analyses showed that the most represented genera in all three different outcrops were Pseudomonas and Streptomyces. Pseudomonas strains were found to be predominant in the plant rhizosphere, whereas Streptomyces strains were mainly present in the soil.

Genetic diversity and heavy metal tolerance in populations of Silene paradoxa L. (Caryophyllaceae): a random amplified polymorphic DNA analysis.

Metal-contaminated sites can occur naturally in serpentine outcrops or as consequence of anthropogenic activities, such as mining deposits, aerial fallout from smelters and industrial processes. Serpentine outcrops are characterized by high levels of nickel, cobalt and chromium and present a typical vegetation which includes endemisms and plants which also live in uncontaminated soils. These latter metal-tolerant populations provide the opportunity to investigate the first steps in the differentiation of plant populations under severe selection pressure and to select plants to be used in the phytoremediation of industrially contaminated soils. In this report eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in noncontaminated soil in central Italy, were analysed using random amplified polymorphic DNA (RAPD) markers to investigate the pattern of genetic variation. The genetic diversity observed in populations at copper mine deposits was found to be at least as high as that of the neighbouring serpentine populations. Analysis of molecular variance (AMOVA) of the RAPD markers gave high statistical significance to the groupings of populations according: (i) with their geographical location; and (ii) with the metals present in the soil of origin (copper vs. nickel), indicating that RAPD markers detected a polymorphism related to the soil contamination by copper. Finally, two RAPD bands exclusive to copper-tolerant populations were identified.

The effects of nickel, calcium and magnesium on the acid phosphatase activity of two Alyssum species.

Root Surface acid phosphatase activity was assessed in seedlings of two species of Alyssum, one endemic to Tuscan serpentines (Alyssum bertolonii Desv.) and the other of wide occurrence (Alyssum saxatile L.), cultivated in solutions to which various concentrations of Ni2+ , Ca2+ and Mg2+ were added. Treatments were applied in two factorial experiments of type 2n . A concentration of 0.01 mM Ni2+ markedly stimulated phosphatase activity in A. bertolonii, but slightly decreased it in A. saxatile. Increased Ca2+ concentration stimulated enzyme activity in both species, hut in A. bertolonii the highest activity was obtained with low Ca2+ concentrations, whilst the opposite occured in A. saxatile. A. bertolonii instead required high Mg2+ concentrations to increase phosphatase activity significantly. The same concentrations were toxic to A. saxatile. In the serpentine species, favourable conditions for the increase of phosphatase activity were induced by a high Mg2+ /Ca2+ ratio, which also favoured dry mass production. Therefore it is possible that the increase of phosphatase activity might be an aspect of plant adaptation to serpentines.