Responses to Mg/Ca balance in an Iranian serpentine endemic plant, Cleome heratensis (Capparaceae) and a related non-serpentine species, C. foliolosa

A soil Ca/Mg quotient greater than unity is generally considered necessary for normal plant growth but some serpentine plants are adapted to much lower Ca/Mg quotients, resulting from a major cation imbalance in their substrata. In order to investigate the growth and tolerance responses of serpentine and non-serpentine species to varied Ca/Mg quotients, controlled nutrient solution experiments were performed using an a newly reported Iranian endemic serpentine plant, Cleome heratensis Bunge et Bien. Ex Boiss. and a related non-serpentine species Cleome foliolosa DC. and a Eurasian Ni-hyperaccumulating species Alyssum murale Waldst. and Kit. Seedlings were grown in modified Hoagland’s solutions with varying Ca and Mg concentrations (0.2–2.5 and 0.5–10 mM, respectively) in a fully factorial randomised block design. The yields of the two serpentine plants increased significantly as Mg concentrations in the nutrient solution were increased from 0.5 to 4 mM but decreased in the 10 mM Mg treatment. For C. foliolosa yields decreased significantly from 0.5 to 10 mM Mg, indicating the sensitivity of this non-serpentine plant, and the relative tolerance of the serpentine plants to extremely high levels of Mg. Shoot and root Mg and Ca concentrations in C. heratensis and A. murale were higher than those in C. foliolosa in the low and moderate Mg treatments, supporting the view that many serpentine plants have a relatively high requirement for Mg. Maximum Mg concentrations were found in the roots of C. heratensis. Yields of C. heratensis and A. murale did not change significantly as Ca levels in nutrient solution increased from 0.2 to 2.5 mM Ca, However the yield of C. foliolosa increased significantly from 0.2 to 1.5 mM Ca, indicating sensitivity in this non-serpentine plant and tolerance of the two serpentine plants to low levels of Ca correlated with tissue Ca concentrations, probably because of a greater ability for Ca uptake at low-Ca availability. Calcium deficiency in the low-Ca treatments could be a reason for reduced yield in the non-serpentine plants.     

Nucleolytic activities and appearance of a new DNase in relation to nickel and manganese accumulation inAlyssum murale

Serpentine and non-serpentine plants of Alyssum murale, a nickel (Ni) accumulator plant, from North Greece, were studied in order to examine: (1) The ability of natural plants to accumulate metals; (2) the ability of their seedlings to tolerate increasing concentrations of Ni²⁺ or Mn²⁺ (0, 0.16, 0.32, 0.5 and 1 mmol/L), when grown in nutrient solution; (3) the activities and electrophoretic patterns of root and shoot DNases and RNases under the above conditions. Measurements of metal concentrations in serpentine and non-serpentine natural plants and the respective soils revealed: (1) Very low calcium (Ca)/magnesium (Mg) (0.16) ratio and high concentration of Ni in serpentine soil; (2) very high Ca/Mg (17) ratio and high concentration of manganese (Mn) in non-serpentine soil; (3) the ability of serpentine natural plants to accumulate Ni and the inability of plants of both serpentine and non-serpentine populations to accumulate Mn. A. murale plants grown in nutrient solution with increasing Ni²⁺ or Mn²⁺ concentrations showed a negative correlation between the Ni²⁺ or Mn²⁺ concentrations in the nutrient solution, and the chlorophyll concentration, shoot and especially root length. The accumulation of Ni²⁺ or Mn²⁺ in the plant showed a positive correlation with increasing Ni²⁺ or Mn²⁺ concentrations in the nutrient solution. Application of 0.5 mmol/L Ni²⁺ or Mn²⁺ resulted in the inhibition of DNase activities and the appearance of a new DNase form, in both root and shoot detected by electrophoresis in active ssDNA polyacrylamide gel. The new gel-extracted DNase showed nicking action against plasmid DNA and has been characterised as an endo-DNase. In contrast, electrophoretic patterns and RNase activities were unaffected. According to our studies on growth, both serpentine and non-serpentine plants of A. murale have a constitutive ability to tolerate and accumulate Ni²⁺ or Mn²⁺; they have similar DNase and RNase electrophoretic patterns and show a new DNase form under Ni²⁺ or Mn²⁺ stress. This is the first report on the response of nucleolytic enzymes under metallic elements hyperaccumulation.     

Pollen morphology and localization of Ni in some Ni-hyperaccumulator taxa of Alyssum L. (Brassicaceae)

Pollen morphology of seven Alyssum L. taxa growing on serpentine soils in different places in the European Mediterranean macrobioclimate territory were studied, described and compared. Cluster analysis was performed to show similarity between species and their populations. The shape of the pollen grains varies among the species and among the grains within the same anther. The pollen grains are 3-colpate, prolate, with long and narrow colpi reaching the poles. The ornamentation of the exine varies from micro-reticulate to reticulate between the species. Pollen sterility/fertility was also calculated. The highest percentage of sterile pollen (73.76%) was calculated for Alyssum murale subsp. murale and the lowest (9.54%) for A. bertolonii subsp. bertolonii. All species are representatives of sect. Odontarrhena (C.A. Meyer) Koch well known as Ni-hyperaccumulators. Nickel and other elements present in pollen and stamen were studied by inductively coupled plasma-mass spectrometry. The stamen parts of all species were micromorphologically analyzed by scanning electron microscopy coupled to an energy-dispersive X-ray probe. Accumulation of Ni was detected in the stamens of all studied species and rarely in the pollen grains. The distribution patterns of Ni were similar among the species examined.     

Nickel localization and response to increasing Ni soil levels in leaves of the Ni hyperaccumulator Alyssum murale

We have previously developed phytoremediation and phytomining technologies employing Alyssum Ni hyperaccumulators to quantitatively extract Ni from soils. Implementation of these technologies requires knowledge of Ni localization patterns for the Alyssum species/ecotypes of interest under realistic growth conditions. We investigated Ni uptake and localization in mature Alyssum murale Kotodesh and AJ9ç leaves. Seedlings were grown in potting mix with an increasing series of NiSO₄ addition (0, 5, 10, 20, 40, 80 mmol Ni kg^–1), NiC₄H₆O₄ addition (0, 5, 10, 30, 60, 90 mmol Ni kg^–1), in Ni-contaminated soil from metal refining operations, and serpentine soil. Plants at Ni levels 0, 5, 10, 20 mmolkg^–1 and in native soils grew normally. Plants at 40 mmolkg^–1 exhibited the onset of phytotoxicity, and 60, 80, and 90 mmolkg^–1 were demonstrably phytotoxic, but symptoms of phytotoxicity abated within 6 months. Cryogenic complement fractures were made from frozen hydrated samples. High-resolution scanning electron microscope (SEM) images were taken of one half. The other half was freeze-dried and examined with SEM and semi-quantitative energy dispersive x-ray analysis. Ni was highly concentrated in epidermal cell vacuoles and Ni and S counts showed a positive correlation. Trichome pedicles and the epidermal tissue from which the trichome grows were primary Ni compartments, but Ni was not distributed throughout trichomes. Palisade and spongy mesophyll and guard/substomatal cells contained lesser Ni concentrations but palisade mesophyll was an increasingly important compartment as Ni soil levels increased. Ni was virtually excluded from vascular tissue and trichome rays.     

Evidence of adaptive tolerance to nickel in isolates of Cenococcum geophilum from serpentine soils

Selection for metal-tolerant ecotypes of ectomycorrhizal (ECM) fungi has been reported in instances of metal contamination of soils as a result of human activities. However, no study has yet provided evidence that natural metalliferous soils, such as serpentine soils, can drive the evolution of metal tolerance in ECM fungi. We examined in vitro Ni tolerance in isolates of Cenococcum geophilum from serpentine and non-serpentine soils to assess whether isolates from serpentine soils exhibited patterns consistent with adaptation to elevated levels of Ni, a typical feature of serpentine. A second objective was to investigate the relationship between Ni tolerance and specific growth rates (μ) among isolates to increase our understanding of possible tolerance/growth trade-offs. Isolates from both soil types were screened for Ni tolerance by measuring biomass production in liquid media with increasing Ni concentrations, so that the effective concentration of Ni inhibiting fungal growth by 50% (EC₅₀) could be determined. Isolates of C. geophilum from serpentine soils exhibited significantly higher tolerance to Ni than non-serpentine isolates. The mean Ni EC₅₀ value for serpentine isolates (23.4 μg ml⁻¹) was approximately seven times higher than the estimated value for non-serpentine isolates (3.38 μg ml⁻¹). Although there was still a considerable variation in Ni sensitivity among the isolates, none of the serpentine isolates had EC₅₀ values for Ni within the range found for non-serpentine isolates. We found a negative correlation between EC₅₀ and μ values among isolates (r = −0.555). This trend, albeit only marginally significant (P = 0.06), indicates a potential trade-off between tolerance and growth, in agreement with selection against Ni tolerance in “normal” habitats. Overall, these results suggest that Ni tolerance arose among serpentine isolates of C. geophilum as an adaptive response to Ni exposure in serpentine soils. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Responses of two populations of an Iranian nickel-hyperaccumulating serpentine plant,Alyssum inflatum Nyar., to substrate Ca/Mg quotient and nickel

We selected two geographically close serpentine and non-serpentine populations of a Ni-hyperaccumulating plant (Alyssum inflatum) to investigate the influence of two common factors of serpentine soils: high Ni concentrations and low Ca/Mg quotients. Soils and plants were sampled from serpentine and non-serpentine substrates, and concentrations of Ca, Mg and Ni were measured. A hydroponic culture was used to compare growth and elemental composition responses of serpentine and non-serpentine plants to different Ca/Mg quotients and Ni concentrations in the nutrient solution. The Ca/Mg quotient for non-serpentine soils was 15 times higher than for serpentine soils, but there was no difference in the Ca/Mg quotient of plants from the two populations. In hydroponic culture, plants from both populations were able to survive at high Ca/Mg quotients. This result suggests that serpentine plants of A. inflatum do not necessarily need a substrate with a low Ca/Mg quotient for survival. Decreases in the Ca/Mg quotient in hydroponics decreased growth. The magnitude of this decrease was significantly greater in non-serpentine plants, suggesting a greater resistance of serpentine plants to low Ca/Mg quotients. Total Ni concentration in serpentine soils was 13 times higher than in non-serpentine soils, but ammonium nitrate-extractable concentrations of Ni in both soil types were similar. Ni concentrations in non-serpentine plants from their natural habitat were significantly lower than in serpentine plants, but there was no significant difference in Ni accumulation by plants of the two populations in hydroponic culture. However, increased concentrations of Ni in the hydroponic medium caused similar decreases in growth of both populations, indicating that Ni tolerance of the two populations was similar.     

Response to cytosolic nickel of Slow Vacuolar channels in the hyperaccumulator plant <Emphasis Type="Italic">Alyssum bertolonii</Emphasis>

We applied the patch-clamp technique to investigate the transport properties of the Slow Vacuolar (SV) channel identified in leaf vacuoles of Alyssum bertolonii Desv., a nickel hyperaccumulator plant growing in serpentine soil of the northern Apennines (Italy). SV currents recorded in vacuoles from adult plants collected in their natural habitat showed high sensitivity towards cytosolic nickel. Dose-response analyses indicated half-maximal current inhibition at submicromolar concentrations, i.e. up to three orders of magnitude lower than previously reported values from other plant species. The voltage-dependent increase of residual currents at saturating nickel concentrations could be interpreted as relief of channel block by nickel permeation at high positive membrane potentials. Including young plants of A. bertolonii into the study, we found that SV channels from these plants did not display elevated nickel sensitivity. This difference may be related to age-dependent changes in nickel hyperaccumulation of A. bertolonii leaf cells.     

In-situ phytoextraction of Ni by a native population of Alyssum murale on an ultramafic site (Albania)

Ultramafic outcrops are widespread in Albania and host several Ni hyperaccumulators (e.g., Alyssum murale Waldst. &; Kit.). A field experiment was conducted in Pojske (Eastern Albania), a large ultramafic area in which native A. murale was cultivated. The experiment consisted in testing the phytoextraction potential of already installed natural vegetation (including A. murale) on crop fields with or without suitable fertilisation. The area was divided into six 36-m² plots, three of which were fertilised in April 2005 with (NPK + S). The soil (Magnesic Hypereutric Vertisol) was fully described as well as the mineralogy of horizons and the localisation of Ni bearing phases (TEM-EDX and XRD). Ni availability was also characterised by Isotopic Exchange Kinetics (IEK). The flora was fully described on both fertilised and unfertilised plots and the plant composition (major and trace elements) and biomass (shoots) harvested individually were recorded. The soil had mainly two Ni-bearing phases: high-Mg smectite (1.3% Ni) and serpentine (0.7% Ni), the first one being the source of available Ni. Ni availability was extremely high according to IEK and confirmed by Ni contents in Trifolium nigriscens Viv. reaching 1,442 mg kg^?1 (A new hyperaccumulator?). Total biomass yields were 6.3 t ha^?1 in fertilised plots and 3.2 t ha^?1 in unfertilised plots with a highly significant effect: fertilisation increased dramatically the proportion of A. murale in the plots (2.6 t ha^?1 vs. 0.2 t ha^?1). Ni content in the shoots of A. murale reached 9,129 mg kg^?1 but metal concentration was not significantly affected by fertilisation. Phytoextracted Ni in total harvest reached 25 kg Ni ha^?1 on the fertilised plots. It was significantly lower in unfertilised plots (3 kg Ni ha^?1). Extensive phytomining on such sites could be promising in the Albanian context by domesticating already installed natural populations with fertilisation.     

Hyperaccumulation of nickel by two Alyssum species from the serpentine soils of Iran

Serpentine soils, which contain relatively high concentrations of nickel and some other metals, are the preferred substrate for some plants, especially those that accumulate Ni in their tissues. In temperate regions more Ni-hyperaccumulator plants are found in Alyssum than in any other genus. In this study, serpentine soils of two areas (Marivan and Dizaj) in the west/northwest of Iran and also perennial Alyssum plants growing on these soils were analyzed for Ni and some other metals. The highest concentrations of total metals in the soils of these areas for Ni, Cr, Co and Mn were 1,350, 265, 94 and 1,150 μg g⁻¹, respectively, while concentrations of Fe, Mg and Ca reached 3.55%, 16.8% and 0.585% respectively. The concentration of exchangeable Ni in these soils is up to 4.5 μg g⁻¹. In this study two Alyssum species, A. inflatum and A. longistylum, have been collected from Marivan and Dizaj, respectively. Analysis of leaf dry matter shows that they can contain up to 3,700 and 8,100 μg Ni g⁻¹, respectively. This is the first time that such high Ni concentrations have been found in these species. The concentrations of other metals determined in these species were in the normal range for serpentine plants, except for Ca, which was higher, up to 5.3% and 3.5%, respectively     

Serpentine and non-serpentine Silene dioica plants do not differ in nickel tolerance

Serpentine soils are hostile to plant life. They are dry, contain high concentrations of nickel and have an unfavorable calcium/magnesium ratio. The dioecious plant Silene dioica (L.) Clairv. (Caryophyllaceae) is the most common herb on serpentine soils in the Swedish mountains. It also commonly grows on non-serpentine soils in the subalpine and coastal area. I have compared the germination frequency, plant establishment and growth of serpentine and subalpine non-serpentine populations in serpentine soil under greenhouse conditions. Further more I have studied the specific effect of nickel on root and shoot growth of serpentine and non-serpentine plants from the subalpine and coastal area in solutions with different concentrations of nickel. Plants from serpentine and non-serpentine populations grew well and in a similar fashion in serpentine soil. Moreover, S. dioica plants, irrespective of original habitat, tolerated enhanced concentrations of nickel when grown in solutions. An analysis of metal content in serpentine plants from natural populations shows that S. dioica has a higher nickel concentration in the roots than in the shoots. The growth studies show that S. dioica is constitutively adapted to serpentine, and that all populations have the genetic and ecological tolerance to grow on serpentine.     

Cellular compartmentation of nickel in the hyperaccumulators Alyssum lesbiacum, Alyssum bertolonii and Thlaspi goesingense.

Nickel uptake and cellular compartmentation were investigated in three Ni hyperaccumulators: Alyssum bertolonii (Desv), Alyssum lesbiacum (Candargy) and Thlaspi goesingense (Hálácsy). The three species showed similar hyperaccumulation of Ni, but T. goesingense was less tolerant to Ni than the two Alyssum species. An addition of 500 mg Ni kg(-1) to a nutrient-rich growth medium significantly increased shoot biomass of all three species, suggesting that the Ni hyperaccumulators have a higher requirement for Ni than normal plants. Energy-dispersive X-ray microanalysis (EDXA) was performed on frozen-hydrated tissues of leaves (all species) and stems (Alyssum only). In all species analysed, Ni was distributed preferentially in the epidermal cells, most likely in the vacuoles, of the leaves and stems. In stems, there was a second peak of Ni in the boundary cells between the cortical parenchyma and the vascular cylinder. The non-glandular trichomes on the leaf surfaces of the two Alyssum species were highly enriched with Ca, but contained little Ni except in the base. In the leaves of T. goesingense, the large elongated epidermal cells contained more Ni than the cells of the stomatal complexes. The role of cellular compartmentation in Ni hyperaccumulation is discussed.     

Effects of pH and heavy metal concentrations in solution culture on the proton release,growth and elemental composition ofAlyssum murale andRaphanus sativus L.

The proton release by a species that can hyperaccumulate nickel (Alyssum murale) and by a non-accumulator (Raphanus sativus L.) was studied at different pH and heavy metal concentrations in solution culture. Both factors influenced the growth and composition of the plants.A. murale was more sensitive than radish to a decrease of pH from 7.0 to 6.0 in the growth medium; plant yield and proton production diminished with decreasing pH. However, yields and proton production of radish only decreased at pH 5.5. The differences in the amounts of protons produced between the hyperaccumulator species and radish were not large enough to conclude that decreasing pH in the rhizosphere ofA. murale is a mechanism for heavy metal solubilization.Nickel concentrations inA. murale followed the typical pattern of an accumulator plant — more Ni was accumulated in the shoots than in the roots. Lower concentrations of Zn and Cd occurred in the shoots than in roots ofA. murale, and also of Ni in radish. The concentrations of Co inA. murale shoots were increased when Zn, Ni and Cd were absent from the nutrient solution. However, Co concentrations in radish shoots were independent of the concentrations of other heavy metals in the growth medium.     

Localisation of nickel and mineral nutrients Ca,K, Fe,Mg by Scanning Electron Microscopy microanalysis in tissues of the nickel-hyperaccumulator Alyssum bertolonii Desv. and the non-accumulator Alyssum montanum L.

Plants of the nickel-hyperaccumulator Alyssum bertolonii Desv. and of the non-accumulator A. montanum L. growing on a serpentine site in Tuscany, Italy, and plants of A. montanum from a nearby non-serpentine site were analysed for metal concentration and localisation. The leaves of A. bertolonii contained 160 times more nickel than those of A. montanum from the same site, thus demonstrating its hyperaccumulation capacity towards this metal. On the other hand, both species showed an inversion of the Ca/Mg ratio in their organs relative to the soil. Nickel localisation in plant tissues was examined by Scanning Electron Microanalysis (SEM/EDX). In A. bertolonii, a specific pattern of nickel distribution was detected, with the highest concentrations present in parenchyma and sclerenchyma cells for the roots; in the shoots, the highest amounts of nickel were found in the stem epidermis, the leaf epidermal surface, and the leaf trichome base. This particular nickel tissue distribution pattern was not found in the non-accumulator A. montanum growing on serpentine soil. Other mineral nutrients, namely Mg, Ca, K, Fe, instead, had a similar distribution in the two species. The A. montanum plants from the non-serpentine site had very low nickel levels in their tissues, and these were of the same magnitude as those found in A. bertolonii plants grown in a greenhouse on commercial horticultural soil with low nickel concentration. In A. bertolonii plants, the tissue-specific allocation patterns appeared to depend on the degree of nickel hyperaccumulation, which is, in turn, directly linked to the soil characteristics.     

Effects of calcium on nickel tolerance and accumulation in Alyssum species and cabbage grown in nutrient solution

Nickel (Ni) phytoextraction using hyperaccumulator plant species to accumulate Ni from mineralized and contaminated soils rich in Ni is undergoing commercial development. Serpentinite derived soils have a very low ratio of Ca/Mg among soils due the nature of the parent rock. In crop plants, soil Ca reduces Ni uptake and phytotoxicity, so it is possible that the low Ca of serpentine soils could limit hyperaccumulator plant tolerance of serpentine soils used for commercial phytomining. In this study, we investigated the effects of varied Ca concentration in the presence of high Mg characteristic of serpentine soils on Ni uptake and tolerance by serpentine-endemic species Alyssum murale Waldst. et Kit. and A. pintodasilvae T.R. Dudley in comparison with cabbage (Brassica oleracea L. var. capita) in a nutrient solution study. The levels of Ca and Mg used were based on serpentine and normal soils, and Ni was based on achieving over 1% Ni in Alyssum shoots in preliminary tests. Varied solution concentrations of Ni (31.6–1,000 μM for Alyssum, 1.0–10 μM for cabbage) and Ca (0.128–5 mM) were used in a factorial experimental design; 2 mM Mg was used to mimic serpentine soils. Alyssum spp. showed much greater tolerance to high Ni, high Mg, and low Ca solution concentrations than cabbage. For Alyssum spp., Ni induced phytotoxicity was only apparent at 1,000 μM Ni with relatively low and high Ca/Mg quotient. In the 1,000 μM Ni treatment, shoot Ni concentrations ranged from 8.18 to 22.8 g kg^?1 for A. murale and 7.60 to 16.0 g kg^?1 for A. pintodasilvae. Normal solution Ca concentrations (0.8–2 mM) gave the best yield across all Ni treatments for the Alyssum species tested. It was clear that solution Ca levels affected shoot Ni concentration, shoot yield and Ni translocation from root to shoot, but the relation was non-linear, increasing with increasing Ca up to 2 mM Ca, then declining at the highest Ca. Our results indicate that Ca addition to high Mg serpentine soils with very low Ca/Mg ratio may reduce Ni phytotoxicity and improve annual Ni phytoextraction by Alyssum hyperaccumulator species. Removal of shoot biomass in phytomining will require Ca application to maintain full yield potential.     

Nickel and zinc hyperaccumulation by Alyssum murale and Thlaspi caerulescens (Brassicaceae) do not enhance survival and whole-plant growth under drought stress

Nickel and Zn hyperaccumulation by Alyssum murale and Thlaspi caerulescens bear substantial energetic costs and should confer benefits to the plant. This research determined whether metal hyperaccumulation can increase osmotic adjustment and resistance to water stress (drought). Alyssum murale and Thlaspi caerulescens treated with low or high concentrations of Ni or Zn were exposed to moderate (?0·4 MPa) and severe (?1·0 MPa) water stresses using aqueous polyethylene glycol. In the absence of metals both water deficits inhibited shoot growth. Nickel and Zn hyperaccumulation did not ameliorate growth inhibition by either level of water stress. The water stress did not induce major changes in shoot metal concentrations of these constitutive hyperaccumulators. Moreover, metal hyperaccumulation had minimal effects on the osmolality of leaf‐sap extracts, relative water content of the shoots, or rate of evapotranspiration. It is concluded that Ni or Zn hyperaccumulation does not augment whole‐plant capacity for drought resistance in A. murale and T. caerulescens.     

Improving the growth of Ni-hyperaccumulating plants in serpentine quarry tailings

Phytomining techniques based on metal-hyperaccumulating plants can be implemented in serpentine quarry wastes for Ni recovery. However, strategies must be developed to overcome the unfavourable plant growth conditions that these substrates present and to optimize Ni yields. In this study, the Ni hyperaccumulators Alyssum serpyllifolium, Alyssum inflatum, and Alyssum bracteatum were evaluated for their Ni phytoextraction efficiency from quarry tailings. Effects of two organic amendments, composted municipal sewage sludge and cow manure, on plant growth and physiological status and Ni removal were determined. Organic amendments were incorporated at two addition rates (5% and 20% w/w). The best-performing hyperaccumulators were A. inflatum and A. serpyllifolium. Organic amendments improved plant biomass production, photosynthetic efficiency and nutrition, but reduced shoot Ni concentrations. However, the stimulation in biomass resulted in significantly enhanced Ni yields. The most promising results were found using low addition rates and after manure incorporation.     

Genetic diversity and differential in vitro responses to Ni in <Emphasis Type="Italic">Cenococcum geophilum</Emphasis> isolates from serpentine soils in Portugal

Amplified fragment length polymorphism (AFLP) analysis was used to investigate the genetic diversity in isolates of the ectomycorrhizal fungus Cenococcum geophilum from serpentine and non-serpentine soils in Portugal. A high degree of genetic diversity was found among C. geophilum isolates; AFLP fingerprints showed that all the isolates were genetically distinct. We also assessed the in vitro Ni sensitivity in three serpentine isolates and one non-serpentine isolate. Only the non-serpentine isolate was significantly affected by the addition of Ni to the growth medium. At 30 microg g(-1) Ni, radial growth rate and biomass accumulation decreased to 73.3 and 71.6% of control, respectively, a highly significant inhibitory effect. Nickel at this concentration had no significant inhibitory effect on serpentine isolates, and so the fitness of serpentine isolates, as evaluated by radial growth rate and biomass yield, is likely unaffected by Ni in the field. In all isolates, the Ni concentration in the mycelia increased with increasing Ni concentration in the growth medium, but two profiles of Ni accumulation were identified. One serpentine isolate showed a linear trend of Ni accumulation. At the highest Ni exposure, the concentration of Ni in the mycelium of this isolate was in the hyperaccumulation range for Ni as defined for higher plants. In the remaining isolates, Ni accumulation was less pronounced and seems to approach a plateau at 30 microg g(-1) Ni. Because two profiles of Ni accumulation emerged among our Ni-insensitive serpentine isolates, this result suggests that different Ni detoxification pathways may be operating. The non-serpentine isolate whose growth was significantly affected by Ni was separated from the other isolates in the genetic analysis, suggesting a genetic basis for the Ni-sensitivity trait. This hypothesis is further supported by the fact that all isolates were maintained on medium without added Ni to avoid carry-over effects. However, because AFLP analysis failed to distinguish between serpentine and non-serpentine isolates, we cannot conclude that Ni insensitivity among our serpentine isolates is due to evolutionary adaptation. Screening a larger number of isolates, from different geographical origins and environments, should clarify the relationships between genetic diversity, morphology, and physiology in this important species.     

Transient Influx of Nickel in Root Mitochondria Modulates Organic Acid and Reactive Oxygen Species Production in Nickel Hyperaccumulator Alyssum murale

Mitochondria are important targets of metal toxicity and are also vital for maintaining metal homeostasis. Here, we examined the potential role of mitochondria in homeostasis of nickel in the roots of nickel hyperaccumulator plant Alyssum murale. We evaluated the biochemical basis of nickel tolerance by comparing the role of mitochondria in closely related nickel hyperaccumulator A. murale and non-accumulator Alyssum montanum. Evidence is presented for the rapid and transient influx of nickel in root mitochondria of nickel hyperaccumulator A. murale. In an early response to nickel treatment, substantial nickel influx was observed in mitochondria prior to sequestration in vacuoles in the roots of hyperaccumulator A. murale compared with non-accumulator A. montanum. In addition, the mitochondrial Krebs cycle was modulated to increase synthesis of malic acid and citric acid involvement in nickel hyperaccumulation. Furthermore, malic acid, which is reported to form a complex with nickel in hyperaccumulators, was also found to reduce the reactive oxygen species generation induced by nickel. We propose that the interaction of nickel with mitochondria is imperative in the early steps of nickel uptake in nickel hyperaccumulator plants. Initial uptake of nickel in roots results in biochemical responses in the root mitochondria indicating its vital role in homeostasis of nickel ions in hyperaccumulation.     

Nickel tolerance of serpentine and non-serpentine Knautia arvensis plants as affected by arbuscular mycorrhizal symbiosis

Serpentine soils have naturally elevated concentrations of certain heavy metals, including nickel. This study addressed the role of plant origin (serpentine vs. non-serpentine) and symbiosis with arbuscular mycorrhizal fungi (AMF) in plant Ni tolerance. A semi-hydroponic experiment involving three levels of Ni and serpentine and non-serpentine AMF isolates and populations of a model plant species (Knautia arvensis) revealed considerable negative effects of elevated Ni availability on both plant and fungal performance. Plant growth response to Ni was independent of edaphic origin; however, higher Ni tolerance of serpentine plants was indicated by a smaller decline in the concentrations of photosynthetic pigments and restricted root-to-shoot Ni translocation. Serpentine plants also retained relatively more Mg in their roots, resulting in a higher shoot Ca/Mg ratio. AMF inoculation, especially with the non-serpentine isolate, further aggravated Ni toxicity to host plants. Therefore, AMF do not appear to be involved in Ni tolerance of serpentine K. arvensis plants.     

Quantitative ion localization within Suaeda maritima leaf mesophyll cells

Grown under saline conditions, Suaeda maritima accumulates Na⁺ and Cl^- into its leaves, where individual mesophyll cells behave differently in their compartmentation of these ions. Measurements of ion concentrations within selected subcellular compartments show that freeze-substitution with dry sectioning is a valuable preparative technique for analytical electron microscopy of highly vacuolate plant material. Using this approach, absolute estimates were made of Na⁺, K⁺ and Cl^- concentrations in the cytoplasm, cell walls, chloroplasts and vacuoles of leaf mesophyll cells.Abbreviation TAEM transmission analytical electron microscopy