Heavy Metal Adaptation

Luis Rafael Herrera-Estrella, CINVESTAV of Irapuato, Irapuato, Guanajuato, Mexico a, CINVESTAV of Irapuato, Irapuato, Guanajuato, Mexico Angel Arturo Guevara-Garc Lo pez-Bucio, CINVESTAV of Irapuato, Irapuato, Guanajuato, Mexico Jose
The adaptive responses of living organisms to heavy metals include physiological, anatomical and ecological mechanisms, some of which are highly conserved in nature. These adaptive mechanisms enable certain species to survive even in the presence of high concentrations of these toxic elements.

Secondary article
Article Contents
. Introduction . Differences in Ecology . Colonizers of Specific Sites . Mechanisms of Metal Uptake, Exclusion, Transport and Storage

Introduction
Heavy metals are dened as the group of elements that have densities higher than 5 g cm 2 3. Approximately 40 chemical elements fall in this category. All heavy metals are toxic at high concentrations, including those that are essential micronutrients, such as copper and molybdenum. Therefore, living species must actively respond to protect themselves from metal poisoning in contaminated sites. Heavy metals are mainly localized as disperse components in rock formations and few ecosystems present natural heavy metal toxicity, such as aluminium toxicity in acid soils. However, in a considerable number of places, heavy metal concentrations in soil increase to toxic levels through agricultural, manufacturing, mining and waste disposal practices. Recent ndings suggest that the adaptive response of living organisms to contaminated environments can be a rapid and ecient process. Insights into the mechanisms involved in heavy metal adaptation are beginning to be obtained.

and grow in the presence of potentially toxic concentrations of heavy metals.

Heavy metal effects in natural populations

Toxic metals are believed to aect natural populations by reducing the abundance and diversity of species and selecting for resistant/tolerant populations. Several studies have shown the high specicity of species distribution in metal-poisoned ecosystems. In fungi, the reduction in number of species has been noted in soils polluted with copper, cadmium, lead, arsenic and zinc. In plants, the formation of endemisms has been documented in dierent ecosystems around the world, for example in the tropical island of New Caledonia (around 16 000 km2), where serpentine soils containing toxic levels of nickel, chromium and cobalt cover about one third of the island surface. These typically infertile soils contain a native ora of approximately 1500 species comprising woody perennials, epiphytic orchids and Cyperaceae growing in xerophytic scrub at low altitude (Morat et al., 1986). In the copper region of Upper Shaba in Zaire, which comprises about 100 coppercobalt ore deposits totalling some 20 km2, disseminated in a metallogenic province, a number of distinct metalliferous habitats exist, such as copper clearings and natural isolated copper hillocks. These bear specic plants, notably small annual herbs and grasses. About 220 taxa are present on these outcrops, of which 42 are endemic. Some of these are located on a single hillock (Malaisse, 1983). In the mining area of Stolberg (South Aachen, Germany), Betula trees grow well on hills of mining smelter ash with a total lead content of up to 10 20 g per kg. In each of these cases, the diversity of plant species is severely restricted, and often edaphic ecotypes, which are tolerant to the specic metal present in excess in the soil, are selected rapidly. Endemisms are typical in areas polluted for fewer than 20 years. At roadsides and beneath galvanized fences are habitats where adaptation takes place rapidly.
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Differences in Ecology
Soil properties and the adaptive response
The distribution of living organisms in ecosystems aected by heavy metal toxicity depends not only on the kind, combination and concentration of metals, but also on environmental factors such as soil chemistry, heterogeneity and, in particular, nutrient status (principally nitrogen, phosphorus and potassium). Gradients of soil conditions often determine areas of increasing stress or disturbance that impose a high selective pressure on the colonizing species. During evolution, adaptations have developed in a very wide range of organisms from all major taxonomic groups. This is reected in the high number of species of some taxa that have become endemic in sites polluted with heavy metals. In each case, these organisms have the ability to survive

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Heavy Metal Adaptation

Symbiosis events in contaminated environments

Ecological associations can be found along polluted habitats. There are some examples that suggest a causal role in the reduction of metal phytotoxicity by mycorrhizal fungi. In ericaceous plants, little or no growth occurs in mycorrhiza-free plants in the presence of copper and zinc; however, two species of ectomycorrhizal fungi, Amanita muscaria and Paxillus involutus, increased zinc tolerance of Betula sp. From these examples, it was suggested that the hyphal complexes of the mycorrhizal fungi bind metals, thus preventing metal translocation to the plant and the resulting toxic symptoms (Bradley et al., 1982).

Colonizers of Specific Sites

The ability of certain bacterial, fungal and plant species to colonize environments polluted by heavy metals has been widely described. Metal-tolerant anaerobic bacteria isolated from contaminated habitats use toxic metals (such as selenium and chromium) as electron acceptors. The bacterial genera Thiobacillus, Streptomyces, Streptococcus and Caulobacter have been isolated from sites polluted by mercury ions. In these cases, the mechanism of detoxication seems to involve the intracellular reduction of toxic forms (Hg2 1 ) to the less toxic, relatively inert metallic form (Hg0) by the activity of specic enzymes (mercury reductases). The accumulation of cadmium in Citrobacter and uranium in strains of Pseudomonas has also been found. In the river Rio Tinto in Spain, which has an exceptionally low pH (around two) and high concentrations of heavy metals (such as iron, arsenic, copper, cadmium and nickel), almost 1300 species have been collected. The bacterium Thiobacillus ferrooxidans and Leptospirillum ferrooxidans are especially abundant and, surprisingly, patches of algae and masses of lamentous fungi living together with several species of yeast and protist have also been found (Ariza, 1998). In soils with highly toxic levels of copper and zinc, the fungi Geomyces and Paecilomyces have been found to be the predominant genera, whereas Penicillium and Oidiodendron spp. decline signicantly. In samples taken from an organomercurial-treated golf green, the species Trichocladium asperum, Trichoderma hamatum, Zygorrhynchus moelleri and Chrysosporium pannorum have been found frequently, whereas the genera Chaetomium, Fusarium, Penicillium and Paecilomyces are greatly reduced. In soil contaminated with cadmium dust, Strobilurus tenacellus, Mycena ammoniaca and Armillaria lutea are the most common basidiomycetes. Some Penicillium species are sensitive towards heavy metals, but one of the best examples of fungal tolerance is
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found in this genus, which underlines the fact that metal responses may be strain-specic. As examples, P. lilacinum comprises 23% of all fungi isolated from soil polluted by mine drainage, and P. ochrochloron is commonly present in industrial euents. In the plant kingdom, it seems clear that tolerance has arisen independently in the full spectrum of families. It is common to nd species of Gramineae, Caryophyllaceae, Lamiaceae and Fabaceae widely distributed along heavymetal contaminated ecosystems. The families Flacourtiaceae, Violaceae and Brassicaceae contain most of the so-called hyperaccumulators, in which the genera Alyssum (50 species) and Thlaspi (about 20 species) have been the most widely characterized. Native vegetation from acid soils of the humid tropics can grow in the presence of concentrations of aluminium that are toxic to species not associated with such habitats. Thus, organisms can evolve mechanisms to cope with excess levels of heavy metals in their environment. In fact, hyperaccumulator plants have not only evolved a mechanism to live in toxic concentrations of heavy metals, but hyperaccumulation could also be functioning as a strategy to prevent predation. As examples, the nickelrich leaves of Strephantus polygaloides prevent the development of the herbivorous larvae of Pieris rapae and avoid the growth of the phytopathogenic bacterium Xanthomonas campestris. A repellent eect of the plant sap from Sebertia acuminata (25% nickel dry wt) was observed on the fruit y Drosophila melanogaster (Sagner et al., 1998).

Mechanisms of Metal Uptake, Exclusion, Transport and Storage

Uptake of some metal solutes from the soil environment may occur through a carrier-mediated system, or as in the case of cations, may be driven largely by the negative potential across the plasma membrane, which is generated in part by proton extrusion mediated by the membrane enzyme H 1 ATPase. Biological exudates, including microbial siderophores and analogous compounds of plants termed phytosiderophores, are known to take part in the mobilization and dierential uptake of certain elements. In this way, molybdenum and copper have been shown to form strong complexes with this class of molecules, facilitating their uptake. Once in the cell, the organism must balance critically the intracellular concentrations of these potentially toxic metals. In some prokaryotes, animals and fungi, a class of small cysteine-rich metal-binding proteins named metallothioneins seems to be of primary importance in metal compartmentalization and tolerance. These metallothionein genes have been used to produce heavy-metal tolerant transgenic plants. In plants, another class of

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Heavy Metal Adaptation

metal-binding ligands named phytochelatins has been described. These proteins are rich in glutamine and cysteine residues and may protect sensitive enzymes by sequestering heavy metals, such as cadmium, lead and zinc (Grill et al., 1987). The majority of organisms that inhabit metalliferous soils are known to exclude toxic metals. The production of extracellular polysaccharides and the excretion of chelating substances, such as organic acids by microbial and plant species, could participate in the immobilization of such toxic elements. Another trait exploited by bacteria is the accumulation of metals as cell-bound metalphosphate compounds. A number of plants, instead of excluding heavy metals, have developed the unusual adaptation of accumulating metals such as nickel, zinc or copper in their above-ground biomass. These plants, termed hyperaccumulators, have gained considerable attention owing to their potential use in biorecovery of contaminated sites. Little is known about the molecular, biochemical and physiological processes that result in the hyperaccumulator phenotype. However, low-molecular weight chelators, such as certain amino acids and organic acids (e.g. citric), have been shown to participate in the transport, compartmentalization and detoxication mechanisms. In the genus Alyssum, in which the nickel concentration can reach 3% of leaf dry biomass, the tolerant response correlates with the increase in the levels of free histidine. The supply of this amino acid to a nonaccumulating species greatly increases both its nickel tolerance and transport to the shoot (Kramer et al., 1996). However, the accumulation of organic acids in roots and leaves of metal-tolerant plants has also been described. Formation of anionic or uncharged zinccitrate complexes resulted in more zinc passing through the excised stem of Pinus radiata and copper in Papyrus stems. These complexes positively aect transport in xylem by reducing adsorption to the vessel walls and by decreasing the rate of lateral escape. A causal relationship between organic acid accumulation and metal tolerance has even been proposed. This is well established for nickel-accumulating plants, which complex nickel with malate, malonate and citrate. Apparently the organic acids bind to heavy metals in the cytoplasm and this complex is nally accumulated in the vacuole. Another way in which organic acids may confer metal tolerance has been found in some aluminium-tolerant cultivars of snapbean, maize and wheat. In these plants, an aluminium-resistant genotype correlates with the exudation of citrate and malate, and it has been shown that organic acids prevent metal toxicity by chelating the aluminium ions in the rhizosphere. This conclusion is supported by the nding that the addition of organic acids to toxic aluminium solutions abates aluminium toxicity in the roots. Even more convincing is the fact that tobacco and papaya transgenic plants, expressing a bacterial citrate synthase gene and overproducing and overexuding citrate, can grow in toxic concentration of aluminium by a

presumptive metal exclusion mechanism (de la Fuente et al., 1997).

Perspectives
Hyperaccumulator plants have been used as indicators for metallic deposits for hundreds of years. Their commercial importance for metal prospecting is indisputable. The shrub Hybanthus oribundus is used as an indicator for nickel in Australia, while a wild variety of the plant Impaticus balsamina, found on leadzinc metal dumps in India, is regarded as a local bioindicator for these metals. In the lichen group, the species Cladonia convoluta, which accumulates high copper concentrations in its tissues, has become a tool for biogeochemical prospecting and can be used to locate mining areas (Aery and Tiagi, 1986). The mechanism of metal hyperaccumulation is the object of intensive investigations, especially with regard to the ecological exploitation of such plants. Gradual depletion of ecosystems encourages researchers not only to investigate the natural response of organisms living on contaminated sites, but also to suggest strategies for environmental clean-up. The lack of aordable eective approaches for heavy-metal remediation has created a major need for the development of novel strategies. The application of genetic engineering has been shown to be highly successful in obtaining heavy-metal resistant species. Transgenic plants tolerant to mercury, cadmium and aluminium have recently been generated. Although there has been some success with heavy-metal resistant plants, the fact that we do not completely understand the limiting factors in increasing uptake, translocation and tolerance to toxic chemical elements makes necessary the continuation of fundamental and applied research. This should eventually make it possible to overcome, at low monetary and environmental cost, the problem of heavy metal contamination.

References
Aery NC and Tiagi YD (1986) Bioindicators and accumulators in geobotanical and biogeochemical prospecting of metals. Acta Biologica Academiae Scientiarum Hungaricae 37: 6778. Ariza LM (1998) River of vitriol. Scientic American 279: 1518. Bradley R, Burt AJ and Read DJ (1982) The biology of mycorrhiza in the Ericaceae.VIII. The role of mycorrhizal infection in heavy metal resistance. New Phytologist 91: 197209. De la Fuente JM, Ram rez V, Cabrera JL and Herrera-Estrella L (1997) Aluminium tolerance in transgenic plants by alteration of citrate synthesis. Science 276: 15661568. Grill E, Winnacker EL and Zenk MH (1987) Phytochelatins, a class of heavy-metal binding peptides from plants are functionally analogous to metallothioneins. Proceedings of the National Academy of Sciences, USA 84: 439443.

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Heavy Metal Adaptation

Kramer U, Cotter-Howells JD, Charnock JM, Baker AJM and Smith JAC (1996) Free histidine as a metal chelator in plants that accumulate nickel. Nature 379: 635638. Malaisse F (1983) Phytogeography of the copper and cobalt ora of Upper Shaba, Za re, with emphasis on its endemism, origin and evolution mechanisms. Bothalia 14: 173180. T, Veillon JM and Mackee HS (1986) Anite s Morat Ph, Jare oristiques et considerations sur lorigine des maquis miniers de la nouvelle caledonie. Adansonia 2: 133182. Sagner S, Kneer R, Wanner G et al. (1998) Hyperaccumulation, complexation and distribution of nickel in Sebertia acuminata. Phytochemistry 47: 339347.

Further Reading
Baker AJM (1987) Metal tolerance. New Phytologist 106: 93111. Cunningham SD, Berti WR and Huang JW (1995) Phytoremediation of contaminated soils. Trends in Biotechnology 13: 393397. Ford T and Mitchell R (1992) Microbial transport of toxic metals. In: Mitchell R (ed.) Environmental Microbiology, pp. 83101. New York: Wiley-Liss. Gadd GM (1993) Interactions of fungi with toxic metals. New Phytologist 124: 2560. Zenk MH (1996) Heavy metal detoxication in higher plants a review. Gene 179: 2130.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net