Identification of nickel chelators in three hyperaccumulating plants: an X-ray spectroscopic study

We have investigated the accumulation of nickel in a hyperaccumulating plant from the Brassicacae family Leptoplax emarginata (Boiss.) O.E. Schulz. Two supplementary hyperaccumulating plants, which have been the subject of a high number of publications, Alyssum murale Waldst. & Kit and Thlaspi caerulescens J.&C. Presl, and a nonaccumulating species Aurinia saxatilis were also studied for reference. The plants were grown during 4 months in specific rhizoboxes with Ni-bearing minerals as a source of nickel. Nickel speciation was analyzed through X-ray absorption spectroscopy at Ni K-edge (X-ray absorption near edge spectroscopy and extended X-ray absorption fine structure spectroscopy) in the different parts of the plants (leaves, stems and roots) and compared with aqueous solutions containing different organo-Ni(II) complexes. Carboxylic acids (citrate, malate) appeared as the main ligands responsible of nickel transfer within those plants. Citrate was found as the predominant ligand for Ni in stems of Leptoplax and Alyssum, whereas in leaves of the three plants, malate appeared as the chelating organic acid of accumulated metal. Histidine could not be detected either in leaves, stems nor roots of any studied plant sample.     

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.     

Sensitivity of growth and biomass allocation patterns to increasing nitrogen: a comparison between ephemerals and annuals in the Gurbantunggut Desert,north-western China

Background and Aims

Biomass accumulation and allocation patterns are critical to quantifying ecosystem dynamics. However, these patterns differ among species, and they can change in response to nutrient availability even among genetically related individuals. In order to understand this complexity further, this study examined three ephemeral species (with very short vegetative growth periods) and three annual species (with significantly longer vegetative growth periods) in the Gurbantunggut Desert, north-western China, to determine their responses to different nitrogen (N) supplements under natural conditions.

Methods

Nitrogen was added to the soil at rates of 0, 0·5, 1·0, 3·0, 6·0 and 24·0 g N m⁻² year⁻¹. Plants were sampled at various intervals to measure relative growth rate and shoot and root dry mass.

Key Results

Compared with annuals, ephemerals grew more rapidly, increased shoot and root biomass with increasing N application rates and significantly decreased root/shoot ratios. Nevertheless, changes in the biomass allocation of some species (i.e. Erodium oxyrrhynchum) in response to the N treatment were largely a consequence of changes in overall plant size, which was inconsistent with an optimal partitioning model. An isometric log shoot vs. log root scaling relationship for the final biomass harvest was observed for each species and all annuals, while pooled data of three ephemerals showed an allometric scaling relationship.

Conclusions

These results indicate that ephemerals and annuals differ observably in their biomass allocation patterns in response to soil N supplements, although an isometric log shoot vs. log root scaling relationship was maintained across all species. These findings highlight that different life history strategies behave differently in response to N application even when interspecific scaling relationships remain nearly isometric.     

The chemical form and physiological function of nickel in some Iberian Alyssum species

The form of nickel in leaves of Iberian subspecies of Alyssum serpyllifolium Desv. was investigated. Subsp. lusitanicum T. R. Dudley and P. Silva (from N.E. Portugal) and subsp. malacitanum Rivas Goday (from Southern Spain) were hyperaccumulators of nickel (>1000 μg/g Ni) whereas their non-accumulating precursor (subsp. serpyllifolium ) was not. Nickel existed mainly as a water-soluble polar complex in the vacuoles. Small concentrations of nickel did however exist in cell fractions, particularly in the mitochondria where enzyme systems are located. Gas-liquid chromatography on the purified nickel complexes showed that this element is associated principally with malic and malonic acids which are present in high concentrations in the hyperaccumulators but not in subsp. serpyllifolium . It is suggested that production of malic acid is a mechanism whereby hyperaccumulators can tolerate unfavourable edaphic factors such as nickel-rich soils. Presence of nickel in the mitochondria can presumably block the citric acid cycle by deactivating malic dehydrogenase leading to build-up of malic acid in the vacuoles which could then absorb excess nickel by a complexing reaction and lead to its diffusion back into the vacuoles from the mitochondria, hence unblocking the citric acid cycle. It is postulated that the chemical evidence suggests that the two hyperaccumulators are not conspecific and that subsp. malacitanum should be promoted to full specific rank.     

Intra-specific differences in nickel tolerance and accumulation in the Ni-hyperaccumulator Alyssum bertolonii

Populations of the serpentine-endemic nickel-hyperaccumulator Alyssum bertolonii from nine different outcrops were investigated for differences in their nickel tolerance and accumulation levels and shoot:root ratio of nickel concentration.

Plantlets from the different populations were cultivated in hydroponics culture and subjected to a range of nickel concentrations. For populations of this obligate serpentinophyte, the results showed significant intra-specific differences in the degree of both nickel tolerance and nickel accumulation. Among populations there was no correlation between the patterns of shoot and root tolerance, whereas there was a clearly correlated pattern for root and shoot nickel accumulation. The populations of this hyperaccumulator showed a significant variation also in shoot:root ratio in nickel concentration, but there was no relationship between this trait and shoot nickel accumulation levels.

Regarding the still obscure relationship between metal tolerance and hyperaccumulation, our results demonstrated a strong positive linear relationship between these two phenomena in the nickel hyperaccumulator Alyssum bertolonii.     

Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum

The mechanism of nickel uptake into vacuoles isolated from leaf tissue of Alyssum lesbiacum was investigated to help understand the ability of this species to hyperaccumulate Ni. An imaging system was designed to monitor Ni uptake by single vacuoles using the metal-sensitive fluorescent dye, Newport Green. Nickel uptake into isolated vacuoles from leaf tissue of A. lesbiacum was enhanced by the presence of Mg/ATP, presumably via energisation of the vacuolar H(+)-ATPase (V-ATPase). This ATP-stimulated Ni uptake was abolished by bafilomycin (a diagnostic inhibitor of the V-ATPase) and by dissipation of the transmembrane pH difference with an uncoupler. These observations are consistent with Ni(2+)/nH(+) antiport activity at the tonoplast driven by a proton electrochemical gradient established by the V-ATPase, which would provide a mechanism for secondary active transport of Ni(2+) into the vacuole. This study provides insights into the molecular basis of Ni tolerance in Alyssum, and may aid in the identification of genes involved in Ni hyperaccumulation.     

Effects of nickel hyperaccumulation on physiological characteristics of Alyssum murale grown on metal contaminated waste amended soil

A pot experiment was conducted to investigate the effect of nickel concentration on physiological characteristics of Alyssum murale when grown in a soil mixed with sewage sludge (at the rate of 2.8%). Two types of sludge were used: agricultural sewage sludge (S1) and industrial sewage sludge with an increasing nickel concentration (S2, S3, and S4). Results showed that Ni in shoots was higher than Ni in roots. A. murale is able to concentrate up to 12730 mg/kg Ni in leaves. The highest dry matter yield was observed with plants grown with agricultural sewage sludge. An addition of S2 and S3 increased shoot biomass. However, application of S4 reduced 40% shoot dry weight as compared to the control Ni treatment did not affect all chlorophyll fluorescence parameters. The F(v)/F(m) ratio was stable between Ni treatments. Photosynthesis rate (A) increased with agricultural sewage sludge, but remained stable with variable Ni rates from the industrial sludge. The chlorophyll content increased with S1, S2 and S3 but it remains constant with S4 when compared to the control Therefore, high nickel concentration did not affect the function of the photosynthetic machine of A. murale.     

Rhizosphere Microbial Densities and Trace Metal Tolerance of the Nickel Hyperaccumulator Alyssum Serpyllifolium Subsp. Lusitanicum

In this study we determine culturable microbial densities (total heterotrophs, ammonifiers, amylolytics and cellulolytics) and bacterial resistance to Co, Cr, and Ni in bulk and rhizosphere soils of three populations of the Ni-hyperaccumulator Alyssum serpyllifolium subsp. lusitanicum and the excluder Dactylis glomerata from ultramafic sites (two populations in Northeast (NE) Portugal (Samil (S), Morais (M)) and one population in Northwest (NW) Spain (Melide (L)). The relationship between bioavailable metal concentrations (H₂O-soluble) and microbial densities were analysed. Significant differences in microbial densities and metal-resistance were observed between the two species and their three populations. The hyperaccumulator showed higher microbial densities (except cellulolytics) and a greater rhizosphere effect, but this was only observed in S and M populations. These populations of A. serpyllifolium also showed selective enrichment of Ni-tolerant bacteria at the rhizosphere where Ni solubility was enhanced (densities of Ni-resistant bacteria were positively correlated with H₂O-soluble Ni). These rhizobacteria could solubilise Ni in the soil and potentially improve phytoextraction strategies.     

The Accumulation of Zinc and Nickel in Irankoh Indigenous Plant Species on a Contaminated Land

The accumulation of heavy metals by plants determines both the micronutrient content and the toxic metal content of our food. A field survey of higher terrestrial plants growing on a metalliferous site of the Iranian arid mountain in Isfahan was conducted to identify species accumulating exceptionally large concentrations of Zn and Ni in shoots and roots. Plant samples were collected from Irankoh areas near the Bama Pb and Zn mine. Sampling was carried out in Spring 2004 and analyzed for DTPA (Diethylene triamine pentaacetic acid) extractable Zn and Ni by atomic absorption spectrophotometry. Mean total and available Zn in the studied soils were 259.7 μgg^?1 and 5.067 μgg^?1, respectively. Soil total and available Ni were relatively low (58.9 μgg^?1 and 0.143 μgg^?1 respectively). Zinc concentrations were considerably high in shoots of Stachys inflate, Ebenus stellata, and Astragalus glaucanthus (556.88, 508.8, and 449.53 μgg^?1, respectively). Nickel concentrations were markedly high in shoots of Teucrium polium, Alyssum bracteatum, and Ebenus stellata (13.21, 10.98, and 8.84 μgg^?1, respectively). Zinc translocation factor (TF or shoot/root concentration ratio) was higher than Ni TF in most plant species. Zinc and Ni enrichment factors and shoot/root concentration ratios were also significantly high in Stachys inflate, Ebenus stellata, Astragalus glaucanthus Teucrium polium, Stipa barbata, Bromus tectorum, and Alyssum bracteatum. Results suggest that these plants could be good candidates for use in the revegetation and phytoremediation of Zn and Ni contaminated lands in arid regions.