Jos A.C. Verkleij

Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils

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.

Phytochelatins in Cadmium-Sensitive and Cadmium-Tolerant Silene vulgaris (Chain Length Distribution and Sulfide Incorporation)

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.

Synthesis and degradation of phytochelatins in cadmium-sensitive and cadmium-tolerant Silene vulgaris

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.

Uptake and Transport of Zinc in Zinc-sensitive and Zinc-tolerant Silene vulgaris

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.

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.

Evidence for an important role of the tonoplast in the mechanism of naturally selected zinc tolerance in Silene vulgaris

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.

Allocation patterns of zinc and cadmium in heavy metal tolerant and sensitive Silene vulgaris

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.

Metal binding compounds in hepatopancreas and haemolymph of porcellio scaber (isopoda) from contaminated and reference areas

Abstract 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.

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.

Metallothioneins and Phytochelatins: Ecophysiological Aspects

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.