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• Plant hyperaccumulation of the essential nutrient manganese (Mn) is a rare phenomenon most evident in the Western Pacific region, and differs from hyperaccumulation of other elements. • Mn hyperaccumulators employ a variety of species‐dependent spatial distribution patterns in sequestering excess foliar Mn, including primary sequestration in both nonphotosynthetic and photosynthetic tissues. • This investigation employed synchrotron X‐ray absorption spectroscopy (XAS) in a comparative study of Mn (hyper)accumulators, to elucidate in situ the chemical form(s) of foliar Mn in seven woody species from Australia, New Caledonia and Japan. • Foliar Mn was found to predominate as Mn(II) in all samples, with strong evidence of the role of carboxylic acids, such as malate or citrate, as complexing ligands. Overall, the X‐ray absorption near‐edge spectroscopy (XANES) and extended X‐ray absorption fine‐structure spectroscopy (EXAFS) data appeared weighted against previous observations that oxalate binds excess Mn in Mn‐(hyper)accumulating species.

Conversion of natural habitats into agricultural and industrial landscapes, and ultimately into degraded land, is the major impact of humans on the natural environment, posing a great threat to biodiversity. The emerging discipline of restoration ecology provides a powerful suite of tools for speeding the recovery of degraded lands. In doing so, restoration ecology provides a crucial complement to the establishment of nature reserves as a way of increasing land for the preservation of biodiversity. An integrated understanding of how human population growth and changes in agricultural practice interact with natural recovery processes and restoration ecology provides some hope for the future of the environment

We investigated Zn compartmentation in the root, Zn transport into the xylem, and Zn absorption into leaf cells in Thlaspi caerulescens, a Zn-hyperaccumulator species, and compared them with those of a related nonaccumulator species, Thlaspi arvense. 65Zn-compartmental analysis concluded with roots of the two species indicated that a significant fraction of symplasmic Zn was stored in the root vacuole of T. arvense, and presumably became unavailable for loading into the xylem and subsequent translocation to the shoot. In T. caerulescens, however, a smaller fraction of the absorbed Zn was stored in the root vacuole and was readily transported back into the cytoplasm. We conclude that in T. caerulescens, Zn absorbed by roots is readily available for loading into the xylem. This is supported by analysis of xylem exudate collected from detopped Thlaspi species seedlings. When seedlings of the two species were grown on either low (1 micromolar) or high (50 micromolar) Zn, xylem sap of T. caerulescens contained approximately 5-fold more Zn than that of T. arvense. This increase was not correlated with a stimulated production of any particular organic or amino acid. The capacity of Thlaspi species cells to absorb 65Zn was studied in leaf sections and leaf protoplasts. At low external Zn levels (10 and 100 micromolar), there was no difference in leaf Zn uptake between the two Thlaspi species. However, at 1 mM Zn2+, 2.2-fold more Zn accumulated in leaf sections of T. caerulescens. These findings indicate that altered tonoplast Zn transport in root cells and stimulated Zn uptake in leaf cells play a role in the dramatic Zn hyperaccumulation expressed in T. caerulescens

A number of terrestrial plant accumulate-large quantities of metals such as zinc, manganese, nickel, cobalt and copper in their shoots. The largest group of these so-called 'metal hyperaccumulators' is found in the genus Alyssum, in which nickel concentrations can reach 3% of leaf dry biomass. Apart from their intrinsic interest, plants exhibiting this trait could be of value in the decontamination of metal-polluted soil. However, the biochemical basis of the capacity for metal-accumulation has not been elucidated. Here we report that exposing hyperaccumulator species of Alyssum to nickel elicits a large and proportional increase in the levels of free histidine, which is shown to be coordinated with nickel in vivo. Moreover, supplying histidine to a non accumulating species greatly increases both its nickel tolerance and capacity for nickel transport to the shoot, indicating that enhanced production of histidine is responsible for the nickel hyperaccumulation phenotype in Alyssum.

Radiotracer techniques were employed to characterize 65Zn2+ influx into the root symplasm and translocation to the shoot in Thlaspi caerulescens, a Zn hyperaccumulator, and Thlaspi arvense, a nonaccumulator. A protocol was developed that allowed us to quantify unidirectional 65Zn2+ influx across the root-cell plasma membrane (20 min of radioactive uptake followed by 15 min of desorption in a 100 micromolar ZnCl2 + 5 mM CaCl2 solution). Concentration-dependent Zn2+ influx in both Thlaspi species yielded nonsaturating kinetic curves that could be resolved into linear and saturable components. The linear kinetic component was shown to be cell-wall-bound Zn2+ remaining in the root after desorption, and the saturable component was due to Zn2+ influx across the root-cell plasma membrane. This saturable component followed Michaelis-Menten kinetics, with similar apparent Michaelis constant values for T. caerulescens and T. arvense (8 and 6 micromolar, respectively). However, the maximum initial velocity for Zn2+ influx in T. caerulescens root cells was 4.5-fold higher than for T. arvense, indicating that enhanced absorption into the root is one of the mechanisms involved in Zn hyperaccumulation. After 96 h 10-fold more 65Zn was translocated to the shoot of T. caerulescens compared with T. arvense. This indicates that transport sites other than entry into the root symplasm are also stimulated in T. caerulescens. We suggest that although increased root Zn2+ influx is a significant component, transport across the plasma membrane and tonoplast of leaf cells must also be critical sites for Zn hyperaccumulation in T. caerulescens

For the sake of cost and potential environmental risk, it is necessary to minimize the amount of chelants used in chemically enhanced phytoextraction. In the present study, a biodegradable chelating agent, EDDS was added in a hot solution at 90°C to the soil in which garland chrysanthemum (Chrysanthemum coronarium L.) and beans (Phaseolus vulgaris L., white bean) were growing. The application of hot chelant solutions was much more efficient than the application of normal chelant solutions (25°C) in improving the uptake of heavy metals by plants. When 1 mmol kg-¹ of EDDS as a hot solution was applied to soil, the concentrations of Cu, Zn and Cd and the total phytoextraction by the shoots of the two plant species exceeded or approximated those in the shoots of plants treated with 5 mmol kg-¹ of normal EDTA solution. The concentrations of metals in the shoots of beans were significantly correlated with the relative electrolyte leakage rate of root cells, indicating that the root damage resulting from the hot solution might play an important role in the process of chelant-enhanced metal uptake. The soil leaching study demonstrated that decreasing the dosage of chelant resulted in decreased concentrations of soluble metals in soils. On the 28th day following the application of chelant, the concentrations of soluble metals in the EDDS treated soil were not significantly different from the concentrations in the control soil to which chelants had not been applied. The application of biodegradable EDDS in hot solutions to soil may be an efficient alternative in chemically-enhanced phytoextraction to increase metal removal and to reduce possible leaching.

The hypothesis that hyperaccumulation of certain metals in plants may play a role in osmotic adjustment under water stress (drought) was tested in the context of nickel hyperaccumulator Stackhousia tryonii. Field-collected mature plants of S. tryonii, grown in native ultramafic soil, were pruned to soil level and the re-growth exposed to five levels of water stress (20, 40, 60, 80 and 100% field capacity; FC) for 20 weeks. Water stress had significant (P<0.05) influence on growth (biomass), water potential and shoot Ni concentrations, with progressively more impact as water stress was increased from 80 to 40% FC. Shoot Ni concentration increased significantly from 3,400 micrograms g-1 dry weight (at 100% FC) to 9,400 micrograms g-1 dry weight (at 20% FC). Assuming that Ni is uniformly distributed through the shoot tissue, the Ni concentration could account for 100% at the 80 and 60% FC conditions, and 50% at the 40 and 20% FC conditions of plant osmotic regulation. The results are consistent with a role of Ni in osmotic adjustment and protection of S. tryonii plants against drought.

Most commonly used hyperaccumulator plants for phytoextraction of metals evolved on soils where moisture is limited throughout much of the year. As these plant species are commercialized for use, they are frequently moved from the point of evolution to locations where environmental conditions may be significantly different. Greatest among these potential differences is soil moisture. The objective of this study was therefore to determine whether these plants could grow in soils with much higher soil moisture and whether they would continue to hyperaccumulate metals as soils approach saturation. We examined extractable soil metal concentrations, plant growth, and metal accumulation for the Ni hyperaccumulators, Alyssum murale and Berkheya coddii and the Zn hyperaccumulators Thlaspi caerulescens cultivars AB300 and AB336. Non-hyperaccumulating control species for each were also examined. In general, extractable soil concentrations of Ni decreased with increasing soil moisture content. Few significant effects related to Zn extractability were observed for any of the soil moisture treatments. The biomass of all tested species was generally greater at higher soil moisture and inhibited at low soil moisture. Further, plants accumulated large amounts of metals from soil at higher soil moisture. Highest foliar concentrations of Zn or Ni were found at the two highest WHCs of 80 and 100%. These results show that hyperaccumulators grow well under conditions of high soil moisture content and that they continue to hyperaccumulate metals. Thus, growing Thlaspi, Alyssum, and Berkheya for commercial phytoextraction under nonnative conditions is appropriate and suggests that this technology may be applied to a wide and diverse range of soil types, climatic conditions, and irrigation regimes.

Zinc (Zn), lead (Pb) and cadmium (Cd) tolerance in populations of seedlings ofPhragmites australisraised from seeds collected from a mine site (Plombières, Belgium) contaminated with Zn, Pb and Cd and three ‘clean’ sites (Felixstowe, UK; Wisbech, UK; and Mai Po, Hong Kong) were studied under glasshouse conditions. Small differences were found between the metal-contaminated population and the three ‘clean’ populations when seedlings were grown in 1.0 μg  ml−1Zn and 10.0 μg ml−1Pb treatment solutions. In general, however, different populations of seedlings showed similar growth responses, metal uptake and indices of Zn, Pb and Cd tolerance when cultured in the same metal-contaminated media for 89 d or in the same metal treatment solutions (ZnSO4:1.0 and 4.0 μg ml−1Zn; Pb(NO3)2: 10.0 and 25.0 μg ml−1Pb; CdSO4: 0.5 and 1.0 μg ml−1Cd) for 3 weeks. There was insufficient evidence to support the hypothesis that the metal-contaminated population has evolved to a Zn-, Pb- or Cd-tolerant ecotype but the results indicated some differentiation between the populations with that from Hong Kong being the least productive under the experimental conditions used. The implications of the findings on selection of provenances for use in constructed wetlands for wastewater treatment are discussed.

• Metal concentrations within reproductive tissues of metallophytes are rarely reported. Here, the spatial distribution of nickel (Ni) within the fruits (seeds) of the Ni hyperaccumulator Stackhousia tryonii was investigated. • Two microanalytical techniques, energy dispersive x-ray spectrometry (EDXS) and nuclear microprobe (micro-proton-induced x-ray emission spectrometry; micro-PIXE) were employed for qualitative and quantitative assessment, respectively, of localized Ni, within the fruits of S. tryonii. The results were compared with quantitative analysis made using inductively coupled plasma-optical emission spectrometry (ICP-OES). • Nickel analysis made using micro-PIXE was consistent with bulk (ICP-OES) analysis (at 1800 μ g g-1 d. wt), however, a beam resolution of approx. 2× 2 μ m2 allowed tissue localization. Nickel was partitioned to the fruit wall (pericarp) (4433 μ g g-1), while endospermic and cotyledonary tissues possessed little Ni (309 and 182 μ g g-1 d. wt, respectively). • This distribution is consistent with the interpretation that principal pathway of Ni movement within the fruit is symplastic rather than apoplastic (as the filial generation lacks symplastic connection with the parent).