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summary This paper is the first to report cadmium tolerance in a dicotyledonous species, Silene vulgaris (Moench.) Garcke. The response to cadmium of five populations originating from one uncontaminated and various heavy-metal contaminated sites was examined under standardized conditions for three weeks. The tolerance index (TI), based on the mean relative growth rate (R), was determined. Populations originating from cadmium-contaminated sites showed a distinct tolerance to cadmium. A population from a site enriched only with copper also exhibited a marked co-tolerance to cadmium. A clear difference in biomass production between the sensitive and tolerant populations was attained at 1 μM cadmium. An optimum biomass production in tolerant populations at a metal concentration higher than in the control, as demonstrated for zinc and copper, could not be established for cadmium. The pattern of cadmium uptake and translocation differed between tolerant and sensitive populations. All tolerant populations accumulated cadmium in the roots and showed some degree of restricted transport to the shoots. The effect of cadmium on the elemental distribution in roots and shoots was population-independent for some elements (copper, zinc, potassium) and population-specific for others (phosphorus, magnesium and sodium). The phenomenon of co-tolerance to cadmium is discussed in relation to possible tolerance mechanisms, especially with regard to metal-binding compounds (metallothioneins, phytochelatins).

We expressed the AtMT2b gene under the 35 S cauliflower mosaic virus promoter in Nicotiana tabacum (Sr1), using leaf disc transformation. Arsenite tolerance and uptake, as well as arsenite-induced phytochelatin (PC) accumulation in roots were measured in transgenic lines, and compared to untransformed ('wild type') tobacco. Measured after 5 days of exposure, arsenite tolerance was slightly but significantly decreased in the transgenic lines compared to wild type. The highest AtMT2b expressing line exhibited a significantly decreased arsenic accumulation in roots, but an increased accumulation in shoots, while the total amount of arsenic taken up remained unchanged, suggesting that AtMT2b expression enhanced the arsenic root to shoot transport. The same transformant line also exhibited a decreased rate of phytochelatin accumulation in the roots, but the phytochelatin-SH to As molar ratio was higher than in wild type, suggesting that the lower arsenite tolerance in the transformant lines was not due to a potential shortage of cysteine for PC synthesis, imposed by expression of the transgene. © 2009 Elsevier B.V. All rights reserved.

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. © 2007 Elsevier Ltd. All rights reserved.

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. © 2008 Elsevier Ltd. All rights reserved.

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 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 β-glucosidases, resulting in a rapid degradation of malonylated glucosides into their corresponding aglucones. In this study, Tris was identified as a suitable β-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 β-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. © 2005 American Chemical Society.

Zinc tolerant and non-tolerant ecotypes of Silene vulgaris (Moench) Garcke were examined for their suitability to provide an efficient and reproducible callus formation and regeneration system. Successful and rapid regeneration of adventitious shoots from callus was achieved in leaf tissue but not root or apical meristematic tissue using concentrations of plant growth regulators that spanned a concentration range of (0.05–1 mg l−1) NAA and (0.5–10 mg l−1) BAP respectively. Large differences were observed between ecotypes regarding both callus formation and shoot regeneration on the different hormone concentrations. Leaf explants incubated on basal media with different concentrations of auxin/cytokinin demonstrated initial callus formation after 3 weeks of incubation. Callus initiation was seen to develop from the wounded margins of the leaf explants and, after 2 weeks the initially dark callus became more swollen and green. A mean of 6–8 shoots per leaf explant was observed and the survival rate of these regenerates was seen to be 90%. All regenerated plants that were transferred to soil after the emergence of roots, were seen to have no disturbed morphological characteristics. This study demonstrates the stability of the zinc tolerance traits in the regenerated explants and the potential use of this calli formation and regeneration system in Silene vulgaris. Further, this study is a necessary pre-requite for the development of a genetic transformation system with which to study the genetic basis of zinc and, other heavy metal tolerances in a species with a naturally selected high-level tolerance.

Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in S. vulgaris, we screened a cDNA library derived from a highly copper-tolerant population using Arabidopsis-based MT probes and identified an MT2b-like gene. When expressed in yeast, this gene,SvMT2b, restored cadmium and copper tolerance in different hypersensitive strains. Northern-blot analysis and quantitative reverse transcriptase-PCR showed that plants from the copper-tolerant S. vulgaris populations had significantly higher transcript levels of SvMT2b than plants from the copper-sensitive populations, both in roots and shoots and with and without copper exposure. Southern-blot analysis suggested that the higher expression of the latter allele was caused by gene amplification. Segregating families of crosses between copper-sensitive and copper-tolerant plants exhibited a 1 to 3 segregation forSvMT2b expression. Allele-specific PCR showed that low-expression F3 plants were homozygous for the allele inherited from the copper-sensitive parent, whereas high-expression plants possessed at least one allele from the tolerant parent.SvMT2b expression did not cosegregate with copper tolerance in crosses between sensitive and tolerant plants. However, a significant cosegregation with copper tolerance did occur in families derived from crosses between moderately tolerant F3 plants with different SvMT2b genotypes. Thus, overexpression ofSvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant.

The mixture effects of arsenate and cadmium were studied in Silene vulgaris (Moench) Garcke. The results show that the toxicity of the mixtures, measured as inhibition of root elongation, increased with increasing concentrations, ranging from nonadditive or slightly additive to synergistic. The elements did not influence each other's uptake. This was as expected, considering the differences in uptake mechanisms between both elements. Concentrations of detoxifying phytochelatins (PCs) were additive, with respect to both external and root-internal metal concentrations. However, at high exposure concentrations, the plants metabolism collapsed, resulting in very low PC concentrations.

Phytochelatins (PCs) are a family of thiol-rich peptides, with the general structure (gamma-Glu-Cys)(n)()-Gly, with n = 2-11, induced in plants upon exposure to excessive amounts of heavy metals and some metalloids, such as arsenic. Two types of PC analyses are currently used, i.e., acid extraction and separation on HPLC with either precolumn derivatization (pH 8.2) with monobromobimane (mBBr) or postcolumn derivatization (pH 7.8) with Ellman's reagent [5, 5'-dithiobis(2-nitrobenzoic acid), DTNB]. Although both methods were satisfactory for analysis of Cd-induced PCs, formation of (RS)(3)-As complexes during extraction of As-induced PCs rendered the DTNB method useless. This paper shows that precolumn derivatization with mBBr, during which the (RS)(3)-As complexes are disrupted, provides a qualitative and quantitative analysis of both Cd- and As-induced PCs. In addition, derivatization efficiencies of both methods for the oligomers with n = 2-4 (PC(2)(-)(4)) are compared. Derivatization efficiency decreased from 71.8% and 81.4% for mBBr and DTNB derivatization, respectively, for PC(2) to 27.4% and 50.2% for PC(4). This decrease is most likely due to steric hindrance. Correction of measured thiol concentration is therefore advised for better quantification of PC concentrations in plant material.

Metal concentrations in soils are locally quite high, and are still increasing due to many human activities, leading to elevated risk for health and the environment. Phytoremediation may offer a viable solution to this problem, and the approach is gaining increasing interest. Improvement of plants by genetic engineering, i.e. by modifying characteristics like metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. So far, only a few cases have been reported where one or more of these characteristics have been successfully altered; e.g. mercuric ion reduction causing improved resistance and phytoextraction, and metallothionein causing enhanced cadmium tolerance. These, together with other approaches and potentially promising genes for transformation of target plants are discussed. Copyright (C) 2000 Elsevier Science Ltd.

We describe the isolation of an Arabidopsis gene that is closely related to the animalZnT genes (Zntransporter). The protein encoded by the ZAT (Zn transporter ofArabidopsisthaliana) gene has 398 amino acid residues and is predicted to have six membrane-spanning domains. To obtain evidence for the postulated function of the Arabidopsis gene, transgenic plants with the ZAT coding sequence under control of the cauliflower mosaic virus 35S promoter were analyzed. Plants obtained with ZAT in the sense orientation exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better understanding of the mechanism of Zn homeostasis and resistance in plants.

Production of phytochelatins (PCs) in response to cadmium uptake in the benthic macroalgae Vaucheria compacta and V. debaryana (Xanthophyceae) was investigated. The algae synthesized thiol-containing peptides, identified by means of high-performance liquid chromatography as PC2, PC4 and PC4. In both algal species the level of PCs increased with time of cadmium exposure and was dependent on external cadmium concentration. V. compacta synthesized significantly more PCs than V. debaryana. Exposure of the algae to cadmium at low temperature and in darkness resulted in a significant decrease in PC content over the time interval tested (3 days).

Summary Compartmentalization of zinc in the vacuole has been proposed to explain enhanced zinc tolerance in higher plants. In order to determine whether the vacuolar membrane is involved in differential zinc tolerance, tonoplast vesicles were isolated from roots of zinc tolerant and sensitive Silene vulgaris. In vitro transport studies with K + /nigericin energized tonoplast vesicles showed that zinc (supplied as zinc citrate) was transported across the tonoplast only in the presence of MgATP. Zinc transport was about 2.5 times higher in tonoplast vesicles derived from zinc tolerant Silene vulgaris compared with those derived from sensitive Silene vulgaris at high zinc concentration (0.8 mmol/L). At low zinc concentrations (0.1–0.3 mmol/L) there was no significant difference in zinc transport between both types of plants. The difference in zinc transport at high external zinc concentrations could be due to an additional transport system with lower zinc affinity that may operate at higher zinc concentrations. Overall, these data strongly suggest that the tonoplast plays an important role in naturally selected zinc tolerance.

Germination rate and viability ofStriga hermonthica seeds in the field in the Republic of Benin decreased steadily in the course of the rainy season. The percentage of germinating seeds decreased from 82% on April 13, 1993 to 16% on October 13, 1993. The percentage of viable seeds, according to the tetrazolium colour test, decreased in the same period from 90% to 15%. In the subsequent rainy season these values further decreased and it was concluded that the seeds do not enter a stage of secondary dormancy. This implies that if host plants (including wild hosts) are consistently kept away from infested fields for one or two year periods, this will lead to a dramatic decrease in the number of viableS. hermonthica seeds in the soil.

We expressed the AtMt2b and AtHMA4 genes under the 35S cauliflower mosaic virus promoter simultaneously in Nicotiana tabacum (SR1), using leaf disc transformation. A single AtMT2b tobacco T2 line was used for re-transformation with AtHMA4 to obtain the double transformant. Cadmium (Cd) and zinc (Zn) tolerance, uptake and translocation were measured in the double transformant, and compared to untransformed ('wild type') tobacco and single gene transformants. The double transformant exhibited enhanced Cd-tolerance, enhanced Cd and Zn root to shoot transport, but unaltered Zn tolerance and Cd and Zn uptake, compared with wild type.The single transformant lines did not show significant phenotypes. Our results suggest that the phenotypes of the double transformant are due to synergistic interaction between the transgenes. Except for Cd tolerance, the phenotypes were moderate for Cd and Zn root to shoot transport, which may be due to use of the 35S promotor, resulting in incorrect tissue-specificity. © 2010 Elsevier B.V.

In response to external Cd supply Cd-tolerant Silene vulgaris plants produce three times less phytochelatins than Cd-sensitive ones. Phytochelatin synthase activity in roots of both ecotypes was measured in order to determine whether a lower enzyme activity could be responsible for the lower phytochelatin production. The phytochelatin synthase activity in tolerant plants, grown in the absence or in the presence of Cd, tended to be slightly lower than that in sensitive ones. However, the difference was too small to account for the differential phytochelatin contents observed. Observations on phytochelatin synthesis in excised roots and on the recovery of the level of glutathione, the precursor of phytochelatins, after arresting the Cd exposure, suggested that tolerant plants did not exhibit a decreased capacity to synthesize glutathione. Furthermore, the relative decrease in the phytochelatin concentration after arresting the Cd-exposure was identical for both populations, indicating that the difference in phytochelatin concentrations was not the result of a differential rate of phytochelatin degradation.

SUMMARY The possible role of low molecular weight organic acids in the mechanism of increased zinc tolerance is investigated in Silene vulgaris (Moench) Garcke. In the leaves, the malate concentration is higher in zinc-tolerant plants than in zinc-sensitive ones, but no consistent differences are observed in the roots. Therefore, since the mechanism of increased zinc tolerance operates also in the roots themselves, a primary role for malate in the mechanism of increased zinc tolerance is doubtful. Exposure to zinc does not affect the malate concentration in the plants. The citrate concentration in both the roots and leaves is higher in tolerant plants than in sensitive ones. A clear-cut effect of zinc on the concentration of citrate in the plants is not found. Citrate does not seem to play an important role in the sequestration of zinc in root cells, in view of its low concentration in the roots of tolerant plants. The possible role of citrate as a carrier for zinc in the cytosol is discussed. Oxalate, the most abundant organic acid, might play an important role in the sequestration of zinc, because of its high concentration in the plants. However, this cannot explain increased zinc tolerance, because the oxalate concentration is higher in the sensitive plants than in the tolerant ones. Neither succinate nor maleate play a prominent role in the mechanism of increased zinc tolerance. Malate and citrate are the only organic acids detected in the xylem fluid. The malate concentration in the xylem fluid is the same in sensitive and tolerant plants and it is not affected by the concentration of external zinc. The citrate concentration in the xylem fluid is about three times higher in the tolerant control plants than in the sensitive ones, but it decreases to the same level as in the sensitive plants after exposure to zinc. Differences in the citrate concentrations in the xylem fluid of sensitive and tolerant plants are not related to differences in the allocation of zinc.

Summary The uptake and transport of zinc in zinc-sensitive and zinc-tolerant Silene vulgaris were compared in a short-term experiment. Although the roots of tolerant plants contained more CaCl2-exchangeable zinc, tolerance can not result from exclusion through binding of zinc to the cell wall, because the total uptake of zinc was not significantly different between the populations. Compared at equal levels of zinc injury, measured by root-length growth inhibition, the uptake of zinc was much higher in the tolerant plants. Up to the NOEC for root-length growth of sensitive plants, an equal zinc concentration of the roots was found in sensitive and tolerant plants, but at a higher supply the zinc concentration of the roots was higher in tolerant plants. In sensitive plants the zinc concentration of the shoot was higher than in tolerant ones, except after 3 and 7 days at the highest zinc supply, the percentage of zinc allocated to the shoot was higher at all external zinc concentrations. The results of a split-root experiment clearly show that zinc has a direct inhibitory effect on root growth. Zinc tolerance seems to result from an efficient sequestration of zinc inside the root cells. This probably causes a lower transport of zinc to the shoot.

summary The production of phytochelatins in roots of both cadmium–sensitive and cadmium–tolerant plants of Silene vulgaris (Moench) Garcke in response to Cd supply was recorded over a 7–day growth period. At the same level of Cd, sensitive plants produced more phytochelatins than tolerant ones. In sensitive roots this higher production of PCs was associated with a higher Cd content within one day. From the third day, however, Cd contents were similar, but the difference in PC production persisted. Exposure to buthionine sulphoximine strongly inhibited the production of phytochelatins in roots during cadmium treatment in both sensitive and tolerant plants, but only affected root growth of sensitive plants. Sensitive plants exhibited a higher Cd uptake, which was manifested in a higher Cd accumulation in the shoot. It is concluded that differential cadmium tolerance mechanism in Silene vulgaris is not due to differential production of phytochelatins.

1. In order to evaluate the role of metal-binding proteins in the tolerance mechanism of Porcellio scaber to heavy metals, a comparative study was made using isopods from three locations: a zinc-lead mine (Plombieres), a zinc smelter (Budel) and a reference wood (Spanderswoud). The Cu, Zn and Cd concentrations and the protein composition were determined in the haemolymph and hepatopancreas from the isopods. 2. A constant Cu/Zn molar ratio of about 5 was found in the haemolymph of all populations and no correlation was found between hepatopancreas and haemolymph Cu and Zn content. 3. Using fast-performance liquid chromatography (FPLC), most of the haemolymph Cu and Zn appeared to be associated with a single UV absorbing peak corresponding with an apparent molecular weight of ± 70 kD; this peak is probably the monomer of hemocyanin. 4. The hepatopancreas Zn and Cd concentration were elevated compared to the hepatopancreas of the smelter and mine isopods; after homogenization and centrifugation 70–80% of the metals were found in the supernatant. 5. In all populations the hepatopancreas Cu-, Zn- and Cd-binding compounds eluted in separate peaks of low molecular weight, suggesting the absence of an MT-like compound in Porcellio scaber. 6. The similarity of the protein profiles in haemolymph, and the similar distribution of the metals over the fractions in haemolymph and hepatopancreas suggests that inducible metal binding compounds are not involved in metal tolerance differences between populations.

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.

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. © 2005 Elsevier Ltd. All rights reserved.

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.

Germination and longevity of purple witchweed seeds stored in nylon gauze bags in the soil in situ were tested in northern Bénin over a 2-yr period, covering the rainy seasons in 1994 and 1995. The seeds were collected at Ina Station in November 1993 from corn and sorghum fields. It appeared that germination percentages of the seeds, which were stimulated by GR24 to germinate, as well as their viability according to a tetrazolium test, decreased steadily in wet soil. During the 1994 rainy season, germination percentage of seeds, which reached maximum values of 30 to 74%, decreased to values of 11 to 17%. During the 1995 rainy season, the number of germinating seeds decreased further, and at the end of this season the germination percentage approached zero. Seed viability also decreased in line with the decrease in germination. In addition to the study on longevity under field conditions, seeds also were exposed to various water regimens in pots. In the pot experiment, purple witchweed seed viability and germination declined in moist soil treatments. The dying-off process observed contradicts the common opinion on longevity ofStrigaseeds in their natural environment. “Wet dormancy” was not observed in the course of the rainy season.

In plants, various classes of metal-chelating agents are known, among which are metallothioneins (MTs), phytochelatins (PCs), phytins, organic acids and amino-acids. MTs and PCs are the best characterized sulphur-containing metal-binding ligands and are possibly contribute to metal homeostasis and detoxification by buffering cytosolic metal concentrations.

Phytostabilisation of bare heavily contaminated substrate, such as abandoned mine sites, is considered a very appropriate technology in order to diminish erosion and dispersion of contaminants into the surroundings. In this short-term pot study, application of industrial sugar residue (ISR), a waste product of the sugar industry, proved to ameliorate spoils conditions for plant performance by elevating pH and immobilising several metals. Although arsenate concentrations were positively correlated to spoil pH and spoil treatment with ISR mobilised As, growth of both Phaseolus vulgaris and Holcus lanatus improved significantly after applications of 3.75 g ISR kg −1 dry spoil. Nutrient uptake from the substrate, with the exception of potassium, was elevated by ISR. As a remediation technique ISR application could be effective although in As-contaminated sites application might be restricted to areas where leaching to (ground) water does not form a risk.

A few plant species, like Silene vulgaris, have evolved ecotypes, that are capable of growing on metal-enriched soils. These tolerant ecotypes exhibit metal-specific resistance mechanisms, which are not completely understood to date. Resistance to copper, zinc and cadmium seems to be regulated by different major genes and some hypostatic modifiers. Metallothionein (MT2b) expression is involved in the level of copper tolerance, while an enhanced ATP-dependent copper efflux across the root cell plasma membrane may result in a decreased copper-accumulation and contributes to the higher resistance of mine plants compared to the copper-sensitive plants. Given the lack of evidence for the significance of similar strategies for zinc and cadmium (such as reduction of uptake and increased binding to intracellular molecules) vacuolar compartmentation is generally believed to be the basic mechanism of tolerance of zinc and cadmium in higher plant species. An enhanced tonoplast transport of zinc in roots plays a role in zinc tolerance, but the existence of a similar tolerance mechanism for cadmium remains to be demonstrated. In our present research, the use of molecular biological techniques to study the presence and expression of (transporter) genes (like ZAT, ZNT and other families) has been initiated. Introduction

Terrestrial metal-toxic soils are found all over the world. They originate either from geological anomalies (e.g., ore outcrops, serpentines), or from human activities (e.g., mining, smelting, agricultural use of metal containing fertilizers, pesticides, sludges, etc.). Even extremely metal-toxic soils eventually become vegetated, though often sparsely and without trees. These vegetations are usually poor in species, particularly in parts of the world that have been glaciated during the Pleistocene (Brooks et al., 1985), most probably mainly due to a strong selective effect of metal-toxicity as such (Schat and Verkleij, 1998).

At the end of the cropping season in November 1994, Striga hermonthica seed populations were collected in northern Benin (in the Atacora and Borgou departments). Host crops included Zea mays L. (corn), Pennisetum americanum (pearl millet), and Sorghum bicolor (L.) Moench (sorghum). The seed populations were kept dry in the laboratory, and germination was tested regularly after 2 wk conditioning in the laboratory. The seeds passed through a state of primary dormancy, which was not the same for seed populations collected from Z. mays and S. bicolor fields and those collected from P. americanum fields. The length of the primary dormancy was approximately 6 mo. After passing through primary dormancy (after-ripening), the seeds later went through annual, recurrent states of secondary dormancy. Primary dormancy coincided with the dry season directly after maturity (i.e., between December 1994 and April 1995), and secondary dormancy coincided more or less with the subsequent 1995/1996 and 1996/1997 dry ...

It was demonstrated recently that isolated tonoplast vesicles derived from plants of a Zn-tolerant ecotype of Silene vulgaris accumulate more Zn than vesicles derived from a Zn-sensitive ecotype. We have now characterized the tonoplast-transport system that causes this uptake difference and demonstrated its genetic correlation to Zn tolerance using plant crosses. We conclude that the tonoplast Zn uptake system of the tolerant ecotype differs greatly in its characteristics from that of the sensitive one, with the most prominent differences being its insensitivity to protonophores and ortho-vanadate and its stimulation by Mg-GTP. These differences in characteristics are most likely due to the fact that Zn can be taken up by two or more parallel pathways, which are not present in the same proportions in both ecotypes. In both ecotypes, Zn is actively transported across the tonoplast (temperature coefficient > 1.6), most likely as a free ion, since citrate does not accumulate in vesicles. Most importantly, the uptake difference found using the ecotypes was also found between homozygous Zn-tolerant and Zn-sensitive F3 plants, proving the genetic correlation between increased tonoplast Zn transport and naturally selected Zn tolerance in S. vulgaris.

The results presented in this paper describe the short- and long-term toxicity of arsenate in Silene vulgaris. Short-term toxicity, measured as inhibition of root elongation, depended on phosphate nutrition, arsenate being much less toxic at high phosphate supply. At low phosphate levels more arsenic was taken up by the plants. Under chronic exposure, toxicity (measured as inhibition of biomass production) did not increase with time. In addition, the accumulation of phytochelatins (PCs) as a function of toxicity and duration of exposure was studied. Short-term PC accumulation (over a 3 d period) was positively correlated with exposure. Isolation of peptide complexes from prolongedly exposed plants showed that PC

Summary The allocation patterns of Zn and Cd were studied in a tolerant and a sensitive ecotype of Silene vulgaris . Within mature leaves, the allocation pattern of Zn was similar to that reported earlier for Cd, with the highest concentrations found in the lower epidermis. Part of the Zn present in the leaf seemed to be bound to cell walls of the mesophyll tissue, which is different from the allocation of Cd. At low concentrations, both metals inhibited the growth of the sensitive plants, and the tolerant ecotype was also affected at higher concentrations and longer exposure times. However, Zn and Cd had different effects on morphological parameters such as root/shoot ratio and leaf thickness. The various metal concentrations found in the leaves of different ages could for the most part be explained by these effects on plant growth. It is concluded that the allocation of metals in leaves of S. vulgaris can be largely explained by the different effects of the metals on plant growth and leaf morphology, and not primarily as a specific result of tolerance mechanisms.

In response to a range of Cd concentrations, the root tips of Cd-tolerant plants of Silene vulgaris exhibit a lower rate of PC production accompanied by a lower rate of longer chain PC synthesis than those of Cd-sensitive plants. At the same Cd exposure level, stable PC-Cd complexes are more rapidly formed in the roots of Cd-sensitive plants than in those of tolerant plants. At an equal PC concentration in the roots, the PC composition and the amount of sulfide incorporated per unit of PC-thiol is the same in both populations. Although these compounds might play some role in mechanisms that contribute to Cd detoxification, the ability to produce these compounds in greater amounts is not, itself, the mechanism that produces increased Cd tolerance in tolerant S. vulgaris plants.

There is a duality in plant tolerance to pollutants and its response to the pollutants’ stress. On the one hand some plants, (hyper)tolerant to heavy metals, are able to hyperaccumulate these metals in shoots, which could be beneficial for phytoremediation purposes to clean-up soil and water. On the other hand tolerant food crops, exposed to heavy metals in their growth medium, may be dangerous as carriers of toxic metals in the food chain leading to food toxicity. There is an additional duality in plant tolerance to heavy metals and that is in food crops that are tolerant and/or hyperaccumulators, which could be used on one hand for phytoremediation, under controlled conditions and on the other hand for food fortification with essential metals. Similarly, plants are also exposed to a large number of xenobiotic organic pollutants. Because they generally cannot avoid these compounds, plants take up, translocate, metabolize and detoxify many of them. There is a large variability in tolerance (defence) mechanisms against organic pollutants among plant species. This includes production of reductants but also scavenger molecules like ascorbate and glutathione and expression of the P-450 defence system, and superfamilies of the enzymes glutathione- and glucosyl-transferases. Again, with view to organic pollutants, plant detoxification mechanisms might well protect the plant itself, but produce compounds with some deleterious potential for other organisms. In this review we discuss these dualities on the basis of examples of agricultural and ‘wild’ species exposed to metal contaminants (mainly Cd) and organic pollutants. Differences in uptake and translocation of various pollutants and their consequences will be considered. We will separately outline the effects of the organic and non-organic pollutants on the internal metabolism and the detoxification mechanisms and try to indicate the differences between both types of pollutants. Finally the consequences and solutions of these dualities in plant tolerance to pollutants will be discussed.