R. Gabbrielli

Response to cadmium in higher plants

Abstract The paper summarizes present knowledge in the field of higher plant responses to cadmium, an important environmental pollutant. The principal mechanisms reviewed here include phytochelatin-based sequestration and compartmentalization processes, as well as additional defense mechanisms, based on cell wall immobilization, plasma membrane exclusion, stress proteins, stress ethylene, peroxidases, metallothioneins, etc. An analysis of data taken from the international literature has been carried out, in order to highlight possible ‘qualitative’ and ‘quantitative’ differences in the response of wild-type (non-tolerant) plants to chronic and acute cadmium stress. The dose-response relationships indicate that plant response to low and high cadmium level exposures is a very complex phenomenon indeed: cadmium evokes a number of parallel and/or consecutive events at molecular, physiological and morphological levels. We propose that, above all in response to acute cadmium stress, various mechanisms might operate both in an additive and in a potentiating way. Thus, a holistic and integrated approach seems to be necessary in the study of the response of higher plants to cadmium. This multi-component model, which we would call ‘fan-shaped’ response, may accord with the Selyean ‘general adaptation syndrome’ hypothesis. While cadmium detoxification is a complex phenomenon, probably under polygenic control, cadmium ‘real’ tolerance—found in mine plants or in plant systems artificially grown under long-term selection pressure, exposed to high levels of cadmium—seems to be a simpler phenomenon, possibly involving only monogenic/oligogenic control. We conclude that, following a ‘pyramidal’ model, (adaptive) tolerance is supported by (constitutive) detoxification mechanisms, which in turn rely on (constitutive) homeostatic processes. The shift between homeostasis and ‘fan-shaped’ response can be rapid and involve quick changes in (poly)gene expression. Differently, the slow shift from ‘fan-shaped’ response to ‘real’ cadmium tolerance is caused and affected by long-term selection pressure, which may increase the frequency (and promote the expression) of one or a few tolerance gene(s).

Isolation and Characterization of Endophytic Bacteria from the Nickel Hyperaccumulator Plant Alyssum bertolonii

We report the isolation and characterization of endophytic bacteria, endemic to serpentine outcrops of Central Italy, from a nickel hyperaccumulator plant, Alyssum bertolonii Desv. (Brassicaceae). Eighty-three endophytic bacteria were isolated from roots, stems, and leaves of A. bertolonii and classified by restriction analysis of 16S rDNA (ARDRA) and partial 16S rDNA sequencing in 23 different taxonomic groups. All isolates were then screened for siderophore production and for resistance to heavy metals. One isolate representative of each ARDRA group was then tested for plant tissue colonization ability in sterile culture.Obtained results pointed out that, despite the high concentration of heavy metals present in its tissues, A. bertolonii harbors an endophytic bacterial flora showing a high genetic diversity as well as a high level of resistance to heavy metals that could potentially help plant growth and Ni hyperaccumulation.

Response to cadmium in carrot in vitro plants and cell suspension cultures

Abstract In vitro grown plants and cell suspension cultures of carrot ( Daucus carota L.) were treated with various Cd concentrations. Stress ethylene production in carrot plants was highly stimulated by 1 mM Cd. A pre-treatment with buthionine sulfoximine (BSO) did not further increase ethylene production. After being treated with Cd, both plants and cell suspensions produced phytochelatins, and no lipid peroxidation was detected. In cell cultures, the in vitro activity of phytochelatin synthase was assayed in the presence of Cd and glutathione: the first product (PC 2 ) was detected in less than 30 min. Absence of ethylene (after treatment with aminoethoxyvinylglycine (AVG), an inhibitor of ethylene-biosynthesis, or use of ethylene traps) caused both a decrease in the phytochelatin synthase activity of cell suspensions and a strong lowering in the Cd-induced SH groups in plants. However 1-aminocyclopropane-1-carboxylic acid (ACC) supply did not increase either phytochelatin synthase activity or total SH level.

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.

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.

Evolutionary lineages of nickel hyperaccumulation and systematics in European Alysseae (Brassicaceae): evidence from nrDNA sequence data

BACKGROUND AND AIMS Nickel (Ni) hyperaccumulation is a rare form of physiological specialization shared by a small number of angiosperms growing on ultramafic soils. The evolutionary patterns of this feature among European members of tribe Alysseae (Brassicaceae) are investigated using a phylogenetic approach to assess relationships among Ni hyperaccumulators at the genus, species and below-species level. METHODS Internal transcribed spacer (ITS) sequences were generated for multiple accessions of Alysseae. Phylogenetic trees were obtained for the genera of the tribe and Alyssum sect. Odontarrhena. All accessions and additional herbarium material were tested for Ni hyperaccumulation with the dimethylglyoxime colorimetric method. KEY RESULTS Molecular data strongly support the poorly known hyperaccumulator endemic Leptoplax (Peltaria) emarginata as sister to hyperaccumulator species of Bornmuellera within Alysseae. This is contrary to current assumptions of affinity between L. emarginata and the non-hyperaccumulator Peltaria in Thlaspideae. The lineage Bornmuellera-Leptoplax is, in turn, sister to the two non-hyperaccumulator Mediterranean endemics Ptilotrichum rupestre and P. cyclocarpum. Low ITS sequence variation was found within the monophyletic Alyssum sect. Odontarrhena and especially in A. murale sensu lato. Nickel hyperaccumulation was not monophyletic in any of three main clades retrieved, each consisting of hyperaccumulators and non-hyperaccumulators of different geographical origin. CONCLUSIONS Nickel hyperaccumulation in Alysseae has a double origin, but it did not evolve in Thlaspideae. In Bornmuellera-Leptoplax it represents an early synapomorphy inherited from an ancestor shared with the calcicolous, sister clade of Mediterranean Ptilotrichum. In Alyssum sect. Odontarrhena it has multiple origins even within the three European clades recognized. Lack of geographical cohesion suggests that accumulation ability has been lost or gained over the different serpentine areas of south Europe through independent events of microevolutionary adaptation and selection. Genetic continuity and strong phenotypic plasticity in the A. murale complex call for a reduction of the number of Ni hyperaccumulator taxa formally recognized.

Naturally-assisted metal phytoextraction by Brassica carinata: role of root exudates.

Due to relatively high chelant dosages and potential environmental risks it is necessary to explore different approaches in the remediation of metal-contaminated soils. The present study focussed on the removal of metals (As, Cd, Cu, Pb and Zn) from a multiple metal-contaminated soil by growing Brassica carinata plants in succession to spontaneous metallicolous populations of Pinus pinaster, Plantago lanceolata and Silene paradoxa. The results showed that the growth of the metallicolous populations increased the extractable metal levels in the soil, which resulted in a higher accumulation of metals in the above-ground parts of B. carinata. Root exudates of the three metallicolous species were analysed to elucidate their possible role in the enhanced metal availability. The presence of metals stimulated the exudation of organic and phenolic acids as well as flavonoids. It was suggested that root exudates played an important role in solubilising metals in soil and in favouring their uptake by roots.

Evolution of copper-tolerance and increased expression of a 2b-type metallothionein gene in Silene paradoxa L. populations

Two independently evolved cupricolous populations, their ancestral serpentine population, another unrelated serpentine population and a non-metallicolous population of Silene paradoxa L. were compared for copper tolerance and foliar transcript levels of a 2b-type metallothionein gene that was previously shown to be involved in copper tolerance in the congener, S. vulgaris. The levels of copper tolerance and constitutive MT2bexpression, assessed by Northern-blot analysis and semiquantitative RT-PCR, were proportionally increased in the cupricolous populations, as compared to the serpentine and the non-metallicolous populations. Southern-blot analysis revealed amplification of the gene in the cupricolous populations, such as in S. vulgaris, which might account for the higher constitutive expression level. Since the cupricolous populations descended separately from a common serpentine ancestral population, these results imply that MT2b overexpression must have been independently evolved in both of them, again such as previously shown S. vulgaris. Thus, both in S. paradoxa and S. vulgaris, the MT2b locus appears to be a major target for natural selection imposed by soil copper toxicity.

Effects of Cadmium Stress on Hairy Roots of Daucus carota

Summary Axenic hairy root cultures of Daucus carota , treated with cadmium sulfate (100 μmol/L and 1 mmol/L) for 4 days, readily accumulated high concentrations of Cd without any reduction in fresh weight or in total protein content. Hairy roots exposed to Cd: a) showed a stress ethylene production up to 10 fold higher than controls, and at the same time, no differences in lipid peroxidation; b) produced phytochelatins (PC 2 , PC 3 , PC 4 , PC 5 and PC 6 ), which bound Cd ions by means of HMW and LMW complexes; no phytochelatin synthesis was detected in controls or in the presence of buthionine sulfoximine or cycloheximide; and c) showed two protein bands with a molecular mass, respectively, of about 30,000 and 35,000, not present in the uninduced material. The apparent insensitivity of carrot hairy roots to short-term Cd stress is probably due to the prompt induction of such an efficient «protection pool».

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.