High-level Zn and Cd tolerance in Silene paradoxa L. from a moderately Cd- and Zn-contaminated copper mine tailing.

Cadmium and zinc tolerance were examined in populations of Silene paradoxa, one from uncontaminated calcareous soil (CVD) and one from a mine tailing (FC) (Cd<1-15 ppm, Zn 400-1300 ppm, pH 2-6). The mine population exhibited extremely high Zn and Cd tolerance levels, although the degrees of Cd and Zn enrichment relatively low at the population site. Cd and Zn hypertolerance in FC were associated with reduced rates of accumulation of these metals, both in roots and shoots (Cd), or exclusively in shoots (Zn). However, exclusion potentially explained only a minor part of the superior tolerance in FC. Cd hypertolerance in FC was associated with decreased, rather than enhanced phytochelatin accumulation. The remarkably high levels of Cd and Zn hypertolerance in FC might relate to the low soil pH, due to oxidation of sulphide minerals, and the absence of soil organic matter at the FC site.

Arsenate tolerance in Silene paradoxa does not rely on phytochelatin-dependent sequestration.

Arsenate tolerance, As accumulation and As-induced phytochelatin accumulation were compared in populations of Silene paradoxa, one from a mine site enriched in As, Cu and Zn, the other from an uncontaminated site. The mine population was significantly more arsenate-tolerant. Arsenate uptake and root-to-shoot transport were slightly but significantly higher in the non-mine plants. The difference in uptake was quantitatively insufficient to explain the difference in tolerance between the populations. As accumulation in the roots was similar in both populations, but the mine plants accumulated much less phytochelatins than the non-mine plants. The mean phytochelatin chain length, however, was higher in the mine population, possibly due to a constitutively lower cellular glutathione level. It is argued that the mine plants must possess an arsenic detoxification mechanism other than arsenate reduction and subsequent phytochelatin-based sequestration. This alternative mechanism might explain at least some part of the superior tolerance in the mine plants.

Interaction of heavy metals with the sulphur metabolism in angiosperms from an ecological point of view.

The metabolism of sulphur in angiosperms is reviewed under the aspect of exposure to ecologically relevant concentrations of sulphur, heavy metals and metalloids. Because of the inconsistent use of the term 'metal tolerance', in this review the degree of tolerance to arsenic and heavy metals is divided into three categories: hypotolerance, basal tolerance and hypertolerance. The composition of nutrient solutions applied to physiological experiments let see that the well-known interactions of calcium, sulphate and zinc supply with uptake of heavy metals, especially cadmium are insufficiently considered. Expression of genes involved in reductive sulphate assimilation pathway and enzyme activities are stimulated by cadmium and partially by copper, but nearly not by other heavy metals. The synthesis of the sulphur-rich compounds glucosinolates, metallothioneins and phytochelatins is affected in a metal-specific way. Phytochelatin levels are low in all metal(loid)-hypertolerant plant species growing in the natural environment on metal(loid)-enriched soils. If laboratory experiments mimic the natural environments, especially high Zn/Cd ratios and good sulphur supply, and chemical analyses are extended to more mineral elements than the single metal(loid) under investigation, a better understanding of the impact of metal(loid)s on the sulphur metabolism can be achieved.

Phytoextraction with Brassica napus L.: a tool for sustainable management of heavy metal contaminated soils.

Phytoextraction is a promising tool to extract metals from contaminated soils and Brassica napus L. seems to be a possible candidate species for this purpose. To select accessions with the ability to accumulate cadmium, hydroponically grown 21 day old seedlings of 77 B. napus L. accessions were exposed to 0.2 microM CdSO(4) for an additional 10 days. The effects of Cd on several parameters were quantified i.e.; shoot Cd concentration ([Cd](shoot)), total amount of Cd in shoots (Total Cd) and the shoot to root Cd concentration ratio (S/R ratio). Though generally natural variation was low for [Cd](shoot), Total Cd and S/R ratio, a number of accessions could be selected. Our results indicated that Total Cd and S/R ratio are independent parameters for Cd accumulation and translocation. The selected varieties were then tested in field experiments on two locations nearby metal smelters. The two locations differed in extractable soil Cd, Zn, Ca concentration and pH levels. On both locations B. napus L. accessions showed significant differences in [Cd](shoot) and Total Cd. Furthermore we found significant correlations between Cd and Zn accumulation in shoots. There were site-specific effects with respect to Cd accumulation in the B. napus L. accessions, however, two accessions seem to perform equally well on both sites. The results of the field experiment suggest that certain B. napus L. accessions are suitable for phytoextraction of moderately heavy metal contaminated soils.

Extraction of isoflavone malonylglucosides from Trifolium pratense L.

Extraction of isoflavone malonylglucosides from red clover (Trifolium pratense L.) is a complicated procedure. This is due to the relatively unstable character of the thermolabile glucoside malonates as well as by action of native beta-glucosidases, resulting in a rapid degradation of malonylated glucosides into their corresponding aglucones. In this study, Tris was identified as a suitable beta-glucosidase inhibitor in red clover extracts, optimized at 350 mM Tris in 80% ethanol at pH 7.2. Extraction of fresh red clover leaves using Tris increased the concentration of malonate conjugated isoflavones approximately 13 to 24 times as opposed to extraction without Tris. A comparison of isoflavone profiles obtained after extraction with and without Tris of different plant organs of red clover and several species within the family Fabaceae suggests that the amount and/or activity of the degenerative beta-glucosidase enzymes vary for the different plant parts of red clover and among the species studied. Therefore, the use of standard extraction methods may well result in overestimation of the concentration of aglucones and consequently underestimation of the malonylglucoside isoflavones concentration depending on the plant species and plant part studied.

Plant-specific responses to zinc contamination in a semi-field lysimeter and on hydroponics.

The species Agrostis stolonifera, Brassica napus and Trifolium repens representing different ecological strategies, were selected to study the effect of Zn contamination on Zn tolerance, uptake and accumulation patterns. Parallel tests were carried out with increasing concentrations of Zn in a semi-field lysimeter and hydroponics in the climate chamber. A significant reduction in biomass production or root length and an increase in shoot Zn concentration was observed for all species at increasing external Zn concentrations. However, shoot biomass production, Zn tolerance and Zn accumulation differed significantly among the tested species. The results in both experimental set-ups were quite similar concerning Zn tolerance and accumulation and improved the validity of the findings. The rather specific responses of the different plant species to Zn contamination interfere with the more generic approach used in risk assessment studies. Maximum amounts of Zn in shoot are not likely to cause a risk to herbivores.

Mechanisms of arsenate tolerance in Cytisus striatus.

• Arsenate tolerance, uptake and arsenate-induced phytochelatin (PC) accumulation were compared at different phosphorus supply rates in two populations of the broom, Cytisus striatus , one from an arsenic-enriched gold mine and one from a nonmetalliferous site. • After 7 d of exposure, arsenate tolerance was higher in the mine population. Arsenate uptake was phosphate-suppressible, and much lower in the mine plants. When compared at equal levels of stress, the mine plants and the nonmetallicolous plants exhibited similar arsenic accumulation, suggesting that reduced arsenate uptake is mainly responsible for superior tolerance. • Arsenate-induced PC accumulation occurred in both plant types. The γ-glutamylcysteine synthetase inhibitor, L-buthioninesulfoximine, caused arsenate hypersensitivity in both plant types, suggesting that PC-based arsenic sequestration is essential for both normal and enhanced arsenate tolerance. Mine plants produced longer PCs than the nonmetallicolous plants, possibly due to a differential temporal pattern of arsenate accumulation. • Our results are consistent with a similar mechanism underlying arsenate hypertolerance in C. striatus and grasses, that is reduced arsenate uptake through suppression of phosphate transporter activity.

Revegetation of the acidic, As contaminated Jales mine spoil tips using a combination of spoil amendments and tolerant grasses.

A combination of metal immobilising agents and metal tolerant plants has been utilised in order to reduce the environmental impact of the acidic metal contaminated Jales mine spoil tips. The addition of Beringite (a modified aluminosilicate), steel shots (iron bearing material) and organic matter as spoil amendments resulted in changes in arsenate (As) concentrations and pH of spoil material and improved plant growth. The application of Beringite increased both pH and plant available As concentrations. A 4-year follow up of the spoil analysis demonstrated that the effect of the spoil treatments was stable following treatments, however, the effectiveness did not increase any more after 2 years. The use of metal tolerant grasses in combination with spoil treatments resulted in a rapid and effective revegetation of the As contaminated Jales mine spoils. Colonisation and reproduction of Agrostis castellana and Holcus lanatus was most successful when the substrate contained a combination of all three additives. Plant performances could be enhanced by supplementation of a phosphate fertiliser. The rapid reproduction of the two grass species makes them very suitable for revegetation purposes. Agrostis castellana and Holcus lanatus apparently exhibited a level of metal- and As-tolerance sufficient for survival on untreated spoil, but in the first stages of revegetation the use of spoil amendments was found to be essential. Organic matter in combination with Beringite and/or steel shots resulted in decreased As in the aboveground biomass, protecting possible grazers and predators from undesirable levels of As.

Enhanced ATP-dependent copper efflux across the root cell plasma membrane in copper-tolerant Silene vulgaris.

We studied copper uptake in inside-out plasma membrane vesicles derived from roots of copper-sensitive, moderately copper-tolerant and highly copper-tolerant populations of Silene vulgaris (Amsterdam, Marsberg and Imsbach, respectively). Plasma membrane vesicles were isolated using the two-phase partitioning method and copper efflux was measured using direct filtration experiments. Vesicles derived from Imsbach plants accumulated two and three times more copper than those derived from Marsberg and Amsterdam plants, respectively. This accumulation was ATP-dependent. Also, 9-amino-6-chloro-2-methoxyacridine fluorescence quenching rates upon copper addition decreased in the order Imsbach>Marsberg>Amsterdam. Our results support the hypothesis that efflux of copper across the root plasma membrane plays a role in the copper tolerance mechanism in S. vulgaris.