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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).

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.

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.

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).

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 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.

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.

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

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.