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Bioconcentration of heavy metals by plants
Bioconcentration of heavy metals by plants Ilya Raskin, PBA Nanda Kumar, Slavik Dushenkov and David E Salt Rutgers University, N e w Jersey, USA
Certain plants can concentrate essential and non-essential heavy metals in their roots and shoots to levels far exceeding those present in the soil. Metal-accumulating plant species are invariably restricted to metalliferous soils found in different regions around the world. The mechanisms of metal accumulation, which involve extracellular and intracellular metal chelation, precipitation, compartmentalization and translocation in the vascular system, are poorly understood. Interest in these mechanisms has led to the development of phytoremediation--a new technology to use plants to clean up soil and water contaminated with heavy metals. Current Opinion in 8iotechnology 1994, 5:285-290
Introduction
The notion that the elemental composition of plants is very different from that of the soil in which they grow is taken for granted. Most of these differences can be attributed to a plant's ability to fix carbon from the air and to absorb essential macronutrients and micronutrients from the soil. In many ways, living plants can be compared to solar driven pumps that can extract and concentrate certain elements from their environment. The root morphology of terrestrial plants is a good example of h o w plants are able to produce a large surface area to volume ratio in order to maximize this accumulation. The total length of roots (including root hairs) of a single pot-grown rye plant has been estimated to be about 387 miles [1], and can be even larger for fieldgrown plants, The list of elements essential for plant nutrition is still a subject of some controversy (an essential element is usually defined as one without which plants cannot complete their lifecycle). It is certainly true, however, that plants are not only built of molecules and ions that have functional or structural roles in their development. Therefore, the elemental composition of any field-grown plant exceeds that thought to be essential for its survival. This is particularly true for many heavy metals, which can be readily detected in field-grown plants. The term heavy metal is arbitrary and imprecise. For reasons of simplicity, we will broadly define it here as meaning any element that has metallic properties (ductility, conductivity, density, stability as cations, ligand specificity, etc.) and an atomic number >20. A more biologically relevant but complex classification of metals based o n ligand-forming properties was proposed by Nieboer and Richardson [2]. Heavy metals that are considered essential for at least some forms of life in-
clude V, Cr, Mn, Fe, Co, Ni, Cu, Zn, and Mo. Heavy metals required by plants include Mn, Fe, Cu, Zn, Mo, and, possibly, Ni. The phytotoxicity of such relatively c o m m o n heavy metals as Cd, Cu, Hg, and Ni is substantially greater than that of Pb and Zn. Hexavalent Cr is much more toxic to plants than trivalent Cr. Heavy metals are present in soils as natural components or as a result of human activity. Metal-rich mine tailings, metal smelting, electroplating, gas exhausts, energy and fuel production, downwash from p o w e r lines, intensive agriculture, and sludge dumping are the most important human activities that contaminate soils and aqueous streams with large quantities of toxic metals [3]. It should be noted, however, that in some areas, natural background concentrations of heavy metals in soils and waters exceed those that are considered safe by the regulatory agencies [4°]. Natural mineral deposits containing particularly large quantities of heavy metals are present in many regions of the globe. These areas often support very characteristic plant assemblages and species that thrive in these metal-enriched environments. Examples of such distinct plant communities include serpentine (i.e. growing on Ni, Cr, Mn, Mg and Co rich soils), seleniferous (i.e. growing on Se rich soil), uraniferous (i.e. growing on U rich soils), calamine (i.e. growing on Zn and Cd rich soils) and Cr/Co floras. As a result of their association with specific ore deposits, many metallophyte plants are used as so-called indicator species in prospecting for mineral deposits [5,6]. For example, the copper flower (Haumaniastrum katangense) is a classical indicator of Cr and Co ores in central Africa [7"]. In this review, we summarize current knowledge concerning metal accumulation in plants and the potential commercial application of this p h e n o m e n o n in bioremediation.
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Environmentalbiotechnology Plant responses to heavy metals Plants employ three basic strategies for growing on metalliferous soils [8]. Metal excluders effectively prevent metal from entering their aerial parts over a broad range of metal concentrations in the soil; however, they can still contain large amounts of metal in their roots. Metal non-excluders actively accunmlate metals in their above-ground tissues and can be roughly divided into two groups: indicators and hyperaccumulators. Metal levels in the tissues of indicator species generally reflect metal levels in the soil. Hyperaccumulators can concentrate metals in their above-ground tissues to levels far exceeding those present in the soil or in the non-accumulating species growing nearby. One definition proposes that a plant containing more than 0.1% of Ni, Co, Cu, Cr and Pb or 1% of Zn in its leaves on a dry weight basis is called a hyperaccumulator, irrespective of the metal concentration in the soil [8,9]. Almost all metal-hyperaccumulating species known today were discovered on metalliferous soils, either natural or man-made, often growing together with metal excluders. Actually, almost all metal-hyperaccumulating plants are endemic to these soils, suggesting that hyperaccumulation is an important ecophysiological adaptation to heavy-metal stress and one of the manifestations of heavy-metal resistance [9]. The majority of hyperaccumulating species discovered so far are restricted to a few specific geographical locations. Ni hyperaccumulators are found in New Caledonia [9], the Philippines [10], Brazil [11] and Cuba (AJM Baker, personal communication), Ni and Zn accumulators are found in southern and central Europe and Asia Minor [5,6,9], and Cr and Co accumulators in Central Africa [5,6,9]. We still k n o w very little about the biological and evolutionary significance of metal hyperaccumulation. Several hypotheses have been put forward, including the following: tolerance or disposal of metal from plants, drought resistance, inadvertent uptake, and defense against herbivores or pathogens. Of these hypotheses, present evidence favours most strongly the function of hyperaccumulated metals as a defense mechanism against herbivores [12,13]. A fair amount of phenomenological data on hyperaccumulating plants is available from extensive field collections and herbarium studies [5,9]. The list of hyperaccumulating plants is expanding rapidly. Ni hyperaccumulators are most numerous (numbering at least 277 taxa belonging to 36 different families) followed by Co, Cu a n d . Z n hyperaccumulators (AJM Baker, personal communications). Hyperaccumulators of Mn, Pb, Cd, Cr and Se have also been described. So far, the largest numbers of hyperaccumulating species in the temperate zone belong to the Brassicaceae (nmstard family) [9], but in the tropics, the Euphorbiaceae (spurge family) is the best represented. A New Caledonian tree, Sebertia acuminata, which has more than 11% of Ni in its latex (on a dry weight basis) [13], is one of the most striking examples of metal hyperaccumulation. A large number of other Ni hyperaccumulators (containing Ni at a level over 1% of total dry weigh0 belong to
the genera Alyssum and Thlaspi. Many Thlaspi species also accumulate Pb (up to 1% dry weight) and Zn (up to 3% dry weigh0 [9]. Armeria maritima is another prominent European Pb hyperaccumulator (over 1% Pb in dried tissue) [9,13]. The African species Aeollanthus biformifolius and Haumaniastrum katangense may often contain more than 0.1% Cu or Co in dried leaf tissue [7"]. Astragalus species growing on seleniferous soils may accumulate close to 1% Se in dry matter [61. The specificity of metal uptake by hyperaccunmlating plants is poorly understood and requires much more research. Even so, the available field data suggest that all hyperaccumulators may be divided into three groups on the basis of their tendency to accumulate different metals: Cu/Co accunmlators, Zn/Cd/Pb accumulators and Ni accunmlators (AJM Baker, personal comnmnications). The biochemical and molecular bases of such group selectivity is unknown. So far, the search for metal-accunmlating plants has b e e n carried out by a few people in selected parts of the globe. It is likely that many more as yet unidentified metal-accumulating plants growing o n natural and man-made metalliferous soils remain to be discovered by plant scientists. It is vital that rare and endemic metal-accumulating plant species are identified and preserved before they become extinct. To date, few controlled laboratory experiments that quantify, compare, and explain the n~etal-accumulating ability of various plant species have been carried out. The information is most needed in four areas: first, the metal-accumulating ability of various species as a function of soil metal concentrations, physical and chemical soil properties, physiological state of the plant, et cetera; second, the specificity of metal uptake, transport and accumulation; third, the physiological, biochemical and molecular mechanisms of accumulation and hyperaccumulation; and fourth, the biological and evolutionary significance of metal accumulation.
Mechanisms of metal accumulation A large proportion of metals in soils are bound to organic '(humus) soil constituents, inorganic (clay) soil constituents, or alternatively, are present as insoluble precipitates. For plants to accumulate these 'soil-bound' metals, they must first mobilize them into the soil solution. This mobilization of soil-bound metal can b e achieved in a number of different ways. First, metal-chelating molecules (phytosiderophores) can be secreted into the rhizosphere to chelate and solubilize soil-bound metal. For example, mugineic acid, avenic acid and nicotianamine are phytosiderophores that occur in graminaceous species [14"']. These phytosiderophores are released in response to Fe and Zn deficiency and can mobilize Cu, Zn and Mn from soil [15]. Metal-chelating proteins, perhaps related to metallothioneins [16] or y-glutamylcysteinyl-isopeptides (3' EC-isopepticles) [17], may also function as siderophores
Bioconcentration of heavy metals by plants Raskin et al. in plants. Second, roots can reduce soil-bound metal ions by specific plasma membrane bound metal reductases. Pea plants deficient in Fe or Cu have an increased ability to reduce Fe(III) and Cu(II) that is coupled with an increased uptake of Cu, Mn, Fe and Mg [18]. Third, plant roots can solubilize heavy metals by acidifying their soil environment with protons extruded from the roots. A lower pH solubilizes metal precipitates and releases soil-bound metal ions into the soil. A similar mechanism has been observed for Fe mobilization in some Fe-deficient dicotyledonous plants [19]. It should be noted here that all three of the above processes might also b e performed by mycorrhizal fungi or root-colonizing bacteria. Therefore, it is difficult to assess the individual contribution of root cells and rhizosphere microorganisms in metal mobilization by plants. Solubilized metal ions may enter the root either via the extracellular (apoplastic) or via intracellular (symplastic) pathways. Apoplastic transport is limited by the high cation exchange capacity of cell walls, unless the metal ion is transported as a non-cationic metal chelate. Symplastic transport requires that metal ions move across the plasma membrane, which usually has a large negative resting potential of approximately 170mV (negative inside the membrane). This membrane potential provides a strong electrochemical gradient for the inward movement of metal ions. Most metal ions enter plant cells by an energydependent saturable process via specific or generic metal ion carriers or channels [20]. Non-essential heavy metals may effectively compete for the same transmembrane carriers used by essential heavy metals. This relative lack of selectivity in transmembrane ion transport may partially explain w h y non-essential heavy metals can enter cells, even against a concentration gradient. For example, kinetic data demonstrate that essential Cu 2~ and Zn 2+ and non-essential Ni 2+ and Cd 2+ compete for the same transmembrane carrier [20]. Metal--chelate complexes may also be transported across the plasma membrane via specialized carriers, as is the case for Fe-phytosiderophore transport in graminaceous species [19]. Once metal ions have entered the root, they can either be stored or exported to the shoot. The vacuole plays an important role in metal ion storage [21]. Metal ions can be actively transported across the tonoplast as free ions or as metal-chelate complexes. For example, Cd is actively transported across the tonoplast of oat roots as either a free ion via a Cd/H + antiport [22° ] or as a Cd-('~C)3G/Cd-(TEC)2G (one-letter amino acid code) metal-peptide complex energized by the direct hydrolysis of ATP (DE Salt, WE Rauser, unpublished data). Inside the vacuole, metal ions are chelated either by organic acids, such as citric or malic acid, or by enzymatically synthesized (yEC)-isopeptides, commonly called phytochelatins. The metal-binding role of the proteins encoded by metallothionein-like genes recently identified in plants remains unknown [23]. Under certain conditions, metal ions may form insol-
uble precipitates, for example Zn-phytate [24] and CdS [25]. Compartmentalization, chelation and precipitation of metal ions probably comprise the major resistance mechanisms that protect plant cells from the damaging effect of metals. In hyperaccunmlating species, heavy metals accumulate both in the shoot and the root. In these plants, metal transport to the shoot probably takes place in the xylem. Metals may, however, redistribute in the shoot via the p h l o e m [26]. Xylem cell walls have a high cation exchange capacity that would be expected to retard severely the movement of metal cations. Therefore, metal-chelate complexes, such as Cd-citrate, should facilitate metal movement in the transpiration stream [27]. Analysis of the xylem sap of certain hyperaccumulators has demonstrated the involvement of organic acids in metal transport [9]. Recent work also suggests that (TEC)-isopeptides may be involved in metal binding in xylem sap [28]. Nicotianamine, involved in phloem transport of Fe, can also bind Zn, Co, Ni and Cu, and, therefore, may serve as a general heavy metal transporter in the phloem [26]. In addition, metals may be transported in the phloem chelated to either organic acids, (yEC)-isopeptides or metallothioneins. Clearly, more research is needed to understand the biological mechanisms involved in heavy metal movement, accumulation, and detoxification in plants. Hyperaccumulating plants provide an excellent and almost completely unresearched model system for such studies.
Uses for metal-accumulating plants For a long time, the ability of plants to accumulate metals was considered a detrimental trait. Being at the bottom of many natural food chains, metal-accumulating plants are directly or indirectly responsible for a large proportion of the dietary uptake of toxic heavy metals by humans and other animals [29]. Although some heavy metals are required for life, their excessive accumulation in living organisms is always toxic. The danger of heavy metals is aggravated by their almost indefinite persistence in the environment. The acute and chronic toxic effects of consuming metal-contaminated plants in wild animals, cattle and humans are very well documented and lie outside the scope of this review. Recently, several attempts have been made to produce plants that can trap heavy metals in their roots, thus inhibiting their translocation to the harvestable aboveground parts. For example, transgenic tobacco plants expressing the heavy metal chelating protein mouse metallothionein I contained -24% lower and 5% higher Cd levels in shoots and roots, respectively, than control plants [30°]. In spite of the obvious benefits for human health, currently, there are only a few ongoing efforts to select or genetically engineer metal-excluding crop plants or forage grasses. The need for such crops is particularly great in the large areas of Eastern Europe that were contaminated with radioactive fallout from the Chernobyl nuclear reactor.
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Environmentalbiotechnology Only recently has the value of naetal-accumulating plants for environmental remediation been fully realized [13,31,32°',33]. Despite the ever-growing number of toxic metal contaminated sites, the extremely costly process of removal and burial, or simply isolation of the contaminated sites, are still the most commonly used methods for dealing with heavy metal pollution. Water treatment facilities also do a relatively poor job of removing toxic metals from residential and industrial effluents, contributing to the overall problem [34]. The use of metal-accumulating plants for removal of metals from contaminated soils and waters has a number of advantages: the lower costs, generation of a recyclable metal-rich plant residue, applicability to a range of toxic metals and radionuclides, minimal environmental disturbance, elimination of secondary air or water-borne wastes, and public acceptance. The application of plants in environmental cleanup is an emerging technology that has been called phytoremediation (I Raskin, Grant Proposal #R81869, 1991). Three subsets of this technology are being develo p e d in our laboratory. First, phytoextraction, in which metal-accumulating plants are used to transport and concentrate metals from the soil into the harvestable parts of roots and above-ground shoots. Second, rhizofiltration, which uses plant roots to absorb, concentrate and precipitate toxic metals from polluted effluents. Finally, phytostabilization, which involves the use of plants to eliminate the bioavailability of toxic metals in soils. Since phytostabilization does not necessarily involve bioaccumulation, w e do not discuss this concept in this review. In the phytoextraction process, several sequential crops of laboratory-improved hyperaccumulating plants may be used to reduce soil concentrations of heavy metals to environmentally acceptable levels. Preliminary trials with Ni and Zn hyperaccumulator plants from the Brassicaceae family were successful in partially removing heavy metals from soils contaminated by long-terna application of heavy metal containing sludges [32°°]. Dried, ashed or composted plant residues, highly enriched in heavy metals may be isolated as hazardous waste or recycled as bio-metal ore. Although the most heavily contaminated soils do not support plant growth, sites with light to moderate toxic metal contamination might be suitable for growing hyperaccumulating plants for toxic metal cleanup. Plants that accumulate toxic metals can be grown and harvested economically, leaving the soil or water with a greatly reduced level of toxic metal contamination. Recently, there has been a growing interest in the use of metal-accumulating roots and rhizomes of aquatic or semiaquatic vascular plants for the removal of heavy metals from contaminated aqueous streams. For example, water hyacinth (Eicbbornia crassipes) [35], pennywort (Hydrocotyle umbellata) [36], duckweed (Lemna minor) [37,38] and water velvet (Azolla pinnata) [39],
take up Pb, Cu, Cd, Fe, and Hg from contaminated solutions. In a related development, cell suspension cultures of Datura innoxia were found to remove a wide variety of metal ions from solutions [39,40]. Most of the removed metals were tightly chelated by unidentified components of cell walls in a process that did not require metabolic activity. The observation that hydroponically grown roots of terrestrial plants are extremely effective in removing Pb, Cr, Zn, Cd, Cu, and Ni from water has laid the foundation for the development of rhizofiltration in our laboratory. For example, 1.1 g dry weight of either sunflower (Helianthus annuus) or Indian mustard (Brassica juncea) roots, immersed in 400 ml of water containing 300 gtg ml-1 of Pb, brought the Pb concentration to below 1 l.tg m1-1 in 8 hours (S Dushenkov, I Raskin, unpublished data). Disappearance of Pb from the solution was accompanied by a dramatic concentration of Pb in the root tissue, over 10% on a dry weight basis. Initial analysis indicates that, at least in some instances, rhizofiltration may provide an attractive alternative to current methods of chemical and microbial precipitation of heavy metals.
Conclusions The bioconcentration of heavy metals by plants is a fascinating area of research that should be able to provide answers to some of the fundamental questions of plant biochemistry, nutrition, and stress physiology. Studying heavy metal-plant interactions should improve our understanding of the mechanisms of ion uptake, accumulation and resistance. The unique 'metal accumulation' and 'metal resistance' genes of metal-accumulating plants may directly benefit world agriculture and the environment. Phytoremediation, although still in its infancy, may one day b e c o m e an established environmental cleanup technology. Further development of phytoremediation requires an integrated multidisciplinary research effort that combines plant biology, soil chemistry, soil microbiology, as well as agricultural and environmental engineering. As a major renewable resource exploited by man, plants already give us food, energy, construction materials, natural fibers, and various chemicals. The use of plants in environmental cleanup may guarantee a greener and cleaner planet for all of us.
Acknowledgements The authors thank the US Department of EnvironmentalProtection, New Jersey Commission for Science and Technology,New Jersey Agricultural Experimental Station (NJAES) and PhytoTech Inc for supporting the research related to the topic of this review. We are also grateful to Dr AJM Baker for helpful discussions.
Bioconcentration of heavy metals by plants Raskin et al.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ** of outstanding interest 1.
Galston AW, Davis PJ, Satter RL: The Life of the Green Plant. Englewood Cliffs, New Jersey: Prentice-Hall Inc; 1980:69.
2.
Nieboer E, Richardson DHS: The Replacement of the Nondescript Term 'Heavy Metals' by a Biologically and Chemically Significant Classification of Metal Ions. Env Pollution [B] 1980, 1:3-26.
3.
Seaward MRD, Richardson DHS: Atmospheric Sources of Metal Pollution and Effects on Vegetation. In Heavy Metal Tolerance in Plants: Evohaionary Aspects. Edited by Shaw AJ. Boca Raton: CRC Press; 1990:75-92.
4. •
Runnells DD, Shepherd TA, Angino EE: Metals in Water. Determining Natural Background Concentrations in Mineralized Areas. Env Sci Technol 1992, 26:2316-2323. Summarizes the soils and waters naturally rich in heavy metals. Provides a list of such sites worldwide and some description of their mineral composition. 5.
Brooks RR: Biological Methods o f Prospecting for Minerals. New York: John Wiley & Sons, Inc; 1983.
6.
Peterson PJ: Unusual Accumulations of Elements by Plants and Animals. Sci Prog Oxf 1971, 59:505-536.
7. Brooks RR, Baker AJM, Malaisse F: Copper Flowers. Nat • Res Exploration 1992, 8:338-351. An elegant study of unique flora of the world's richest Zaire copper and cobalt belt, with a particular emphasis on Cu and Co accumulating plants. 8.
9.
10.
11.
Baker AJM, Walker PL: Ecophysiology of Metal Uptake by Tolerant Plants. In Heavy Metal Tolerance in Plants: Evolutionary Aspects. Edited by Shaw AJ. Boca Raton: CRC Press; 1990:155--177. Baker AJM, Brooks 1GR: Terrestrial Higher Plants which Hyperaccumulate Metallic Elements - - A Review of their Distribution, Ecology and Phytochemistry. Biorecovery 1989, 1:81-126. Baker AJM, Proctor J, van Balgooy M, Reeves RD: Hyperaccumulation Of Nickel by Flora of the Ultramafics of Palawan, Republic of the Philippines. In The Vegetation of Ultramafic (Serpentine) Soils. Proceedings o f the First International Conference on Serpentine Ecolopxv. Andover, Hampshire: Intercept Ltd, UK; 1993:291-304. Brooks RR, Reeves RD, Baker AJM: The Serpentine Vegetation of Goias State, Brazil. In The Vegetation o f Ultramafic (Serpentine) Soils. Proceedings o f the First International Conference on Serpentine Ecology. Andover, Hampshire: Intercept Lid, UK; 1993:67-81.
12.
Ernst WHO, Schat H, Verkleij JAC: Evolutionary Biology of Metal Resistance in Silene vuigaris. Evol Trends Plants 1990, 4:45-51.
13.
Baker A, Brooks R, Reeves R: Growing for Gold... and Copper.., and Zinc. New Scientist 1989, 1603:44--48.
Kinnersely AM: The Role of Phytochelates in Plant oo Growth and Productivity. Plant Growth Regulation 1993, 12:207-217. A review of various modes of metal chelation employed by plants. Elucidates the different chemical strategies that plants use for mobilization, transport, and detoxification of heavy metals.
Structure, Immunoreactivity and Metal-Binding Function. Int J Biochem 1991, 23:1-5. 17.
Rauser WE: Phytochelatins. Annu 59:61--86.
18.
Welch RM, Norvell WA, Schaefer SC, Shaft JE, Kochian LV: Induction of Iron(III) and Copper(II) Reduction in Pea (Pisum sativum L.) Roots by Fe and Cu Status: Does the Root-Cell Plasmalemma Fe(III)-Chelate Reductase Perform a General Role in Regulating Cation Uptake? Planta 1993, 190:555-561.
19.
Crowley DE, Wang YC, Reid CPP, Szaniszlo PJ: Mechanisms of Iron Acquisition from Siderophores by Microorganisms and Plants. Plant Soil 1991, 130:179-198.
20.
Clarkson DT, L~ittge U: III. Mineral Nutrition: Divalent Cations, Transport and Compartmentation. Prog Botany 1989, 51:93-112.
21.
VOgeli-Lange R, Wagner GJ: Subcellular Localization of Cadmium and Cadmium-Binding Peptides in Tobacco Leaves. Implications of a Transport Function for Cadmium-Binding Peptides Plant Phystol 1990, 92:1086-1093.
22. •
Salt DE, Wagner GJ: Cadmium Transport Across Tonoplast of Vesicles from Oat Roots. Evidence for a Cd/H Antiport Activity. J Biol Chem 1993, 268:12297-12302. The first direct demonstration of the active transport of Cd across the biological membrane via Cd/H antiport mechanism. 23.
Robinson NJ, Evans IM, Mulcrone J, Bryden J, Tommey AM: Genes with Similarity to Metallothionein Genes and Copper, Zinc Ligands in Pisum saiivum L. Plant Soil 1992, 146:291-298.
24.
Van Steveninck RFM, Van Steveninck ME, Femando DR: Heavy-Metal (Zn, Cd) Tolerance in Selected Clones of Duckweed (Lemna minor). Plant Soil 1992, 146:271-280.
25.
Reese RN, White CA, Winge DR: Cadmium-Sulfide Crystallites in Cd-(qtEC)nG Peptide Complexes from Tomato. Plant Physiol 1992, 98:225-229.
26.
Stephan UW, Scholz G: Nicotianamine: Mediator of Transport of Iron and Heavy Metals in the Phloem? Physiol Plant 1993, 88:522-529.
27.
Senden MHMN, Van Paassen FJM, Van Der Meet AJGM, Wolterbeek HTh: Cadmium-Citric Acid-Xylem Cell Wall Interactions in Tomato Plants. Plant Cell Env 1992, 15:71-79.
28.
Przemeck E, Haase NU: On the Bonding of Manganese, Copper and Cadmium to Peptides of the Xylem Sap of Plant Roots. Water Air Soil Pollution 1991, 57-58:569-577.
29.
Kabata-Pendias AK, Pendias H: Trace Elements in Soils and Plants. Boca Raton: CRC Press; 1989.
30. •
Yeargan R, Maiti IB, Nielsen AG, Hunt AG, Wagner GJ: Tissue Partitioning of Cadmium in T r a n s g e n i c Tobacco Seedlings and Field Grown Plants Expressing the Mouse MetaUothionein I Gene. Transgenic Res 1992, 1:261-267. A thorough investigation of the Cd redistribution in transgenic tobacco plants containing the mouse metallothionein I gene. 31.
14.
15.
R6mheld V: The Role of Phytosiderophores in Acquisition of Iron and other Micronutrients in Graminaceous Species: An Ecological Approach. Plant Soil 1991, 130:127-134.
16.
Kay J, Cryer A, Darke M, Kille P, Leesd WE, Norey C, Stark JM: Naturally Occurring and Recombinant Metallothioneins:
Rev Biochem 1990,
Chaney RL: Plant Uptake of Inorganic Waste. In Land Treatment of Hazardous Wastes. Edited by Parr JE, Marsh PB, Kla JM. Park Ridge: Noyes Data Corp; 1983:50-76.
32. o•
Baker AJM, McGrath SP, Sidoli CMD, Reeves RD: The Possibility of in Situ Heavy Metal Decontamination of MetalPolluted Soils using Crops of Metal-Accumulating Plants. Resource Conservation Recycling 1994, in press. The first detailed and scientific summary of a field phytoremediation trial. 33.
Cunningham SD: Plant-Based Environmental Remediation: Progress and Promise. Abstract P-33. In Vitro Cell Dev Biol 1993, 29A:42A.
289
290
Environmental biotechnology 34.
Bubb JM, Lester JN: The Impact of Heavy Metals on Lowland Rivers and the Implications for Man and the Environment. Sci Total Env 1991, 100:207-233.
38.
Mo SC, Choi DS, Robinson JW: Uptake of Mercury from Aqueous Solutions by Duckweed: The Effects of pH, Copper and Humic Acid. J Env Sci Health 1989, A24:135--146.
35.
Kay SH, Hailer WT, Garrard LA: Effects of Heavy Metals on Water Hyacinths (Eichhornla crassipes (Mart.) Solms). Aquatic Toxicol 1984, 5:117-128.
39.
36.
Dierberg FE, DeBusk TA, Goulet Jr NA: Removal of Copper and Lead Using a Thin-Film Technique. In Aquatic Plants for Water Treatment and Resource Recov~. Edited by Reddy KB, Smith WH. Florida: Magnolia Publishing Inc; 1987:497-504.
Jackson PJ, Torres AP, Delhaize E, Pack E, Bolender SL: The Removal of Barium Ions from Solution using Datura lnnoxia Suspension Culture Cells. J Env Quality 1990, 19:64~(,q8.
40.
Jackson PJ, DeWitt JG, Kuske CR: Accumulation of Toxic Metal Ions by Components of Plant Suspension Cell Cultures. Abstract P-34. In Vitro Cell Dev Btol 1993, 29A:42A.
37.
Jain SK, Vasudevan P, Jha NK: Removal of Some Heavy Metals from Polluted Waters by Aquatic Plants: Studies on Duckweed and Water Velvet. Biological Wastes 1989, 28:115-126.
I Raskin, PBAN Kumar, S Dushenkov and DE Salt, Center for Agricultural Molecular Biology, Environmental and Natural Sciences Building, Rutgers, The State University of New Jersey, Cook College, PO Box 231, New Brunswick, New Jersey 08903-0231, USA.
Certain plants can concentrate essential and non-essential heavy metals in their roots and shoots to levels far exceeding those present in the soil. Metal-accumulating plant species are invariably restricted to metalliferous soils found in different regions around the world. The mechanisms of metal accumulation, which involve extracellular and intracellular metal chelation, precipitation, compartmentalization and translocation in the vascular system, are poorly understood. Interest in these mechanisms has led to the development of phytoremediation--a new technology to use plants to clean up soil and water contaminated with heavy metals. Current Opinion in 8iotechnology 1994, 5:285-290
Introduction
The notion that the elemental composition of plants is very different from that of the soil in which they grow is taken for granted. Most of these differences can be attributed to a plant's ability to fix carbon from the air and to absorb essential macronutrients and micronutrients from the soil. In many ways, living plants can be compared to solar driven pumps that can extract and concentrate certain elements from their environment. The root morphology of terrestrial plants is a good example of h o w plants are able to produce a large surface area to volume ratio in order to maximize this accumulation. The total length of roots (including root hairs) of a single pot-grown rye plant has been estimated to be about 387 miles [1], and can be even larger for fieldgrown plants, The list of elements essential for plant nutrition is still a subject of some controversy (an essential element is usually defined as one without which plants cannot complete their lifecycle). It is certainly true, however, that plants are not only built of molecules and ions that have functional or structural roles in their development. Therefore, the elemental composition of any field-grown plant exceeds that thought to be essential for its survival. This is particularly true for many heavy metals, which can be readily detected in field-grown plants. The term heavy metal is arbitrary and imprecise. For reasons of simplicity, we will broadly define it here as meaning any element that has metallic properties (ductility, conductivity, density, stability as cations, ligand specificity, etc.) and an atomic number >20. A more biologically relevant but complex classification of metals based o n ligand-forming properties was proposed by Nieboer and Richardson [2]. Heavy metals that are considered essential for at least some forms of life in-
clude V, Cr, Mn, Fe, Co, Ni, Cu, Zn, and Mo. Heavy metals required by plants include Mn, Fe, Cu, Zn, Mo, and, possibly, Ni. The phytotoxicity of such relatively c o m m o n heavy metals as Cd, Cu, Hg, and Ni is substantially greater than that of Pb and Zn. Hexavalent Cr is much more toxic to plants than trivalent Cr. Heavy metals are present in soils as natural components or as a result of human activity. Metal-rich mine tailings, metal smelting, electroplating, gas exhausts, energy and fuel production, downwash from p o w e r lines, intensive agriculture, and sludge dumping are the most important human activities that contaminate soils and aqueous streams with large quantities of toxic metals [3]. It should be noted, however, that in some areas, natural background concentrations of heavy metals in soils and waters exceed those that are considered safe by the regulatory agencies [4°]. Natural mineral deposits containing particularly large quantities of heavy metals are present in many regions of the globe. These areas often support very characteristic plant assemblages and species that thrive in these metal-enriched environments. Examples of such distinct plant communities include serpentine (i.e. growing on Ni, Cr, Mn, Mg and Co rich soils), seleniferous (i.e. growing on Se rich soil), uraniferous (i.e. growing on U rich soils), calamine (i.e. growing on Zn and Cd rich soils) and Cr/Co floras. As a result of their association with specific ore deposits, many metallophyte plants are used as so-called indicator species in prospecting for mineral deposits [5,6]. For example, the copper flower (Haumaniastrum katangense) is a classical indicator of Cr and Co ores in central Africa [7"]. In this review, we summarize current knowledge concerning metal accumulation in plants and the potential commercial application of this p h e n o m e n o n in bioremediation.
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Environmentalbiotechnology Plant responses to heavy metals Plants employ three basic strategies for growing on metalliferous soils [8]. Metal excluders effectively prevent metal from entering their aerial parts over a broad range of metal concentrations in the soil; however, they can still contain large amounts of metal in their roots. Metal non-excluders actively accunmlate metals in their above-ground tissues and can be roughly divided into two groups: indicators and hyperaccumulators. Metal levels in the tissues of indicator species generally reflect metal levels in the soil. Hyperaccumulators can concentrate metals in their above-ground tissues to levels far exceeding those present in the soil or in the non-accumulating species growing nearby. One definition proposes that a plant containing more than 0.1% of Ni, Co, Cu, Cr and Pb or 1% of Zn in its leaves on a dry weight basis is called a hyperaccumulator, irrespective of the metal concentration in the soil [8,9]. Almost all metal-hyperaccumulating species known today were discovered on metalliferous soils, either natural or man-made, often growing together with metal excluders. Actually, almost all metal-hyperaccumulating plants are endemic to these soils, suggesting that hyperaccumulation is an important ecophysiological adaptation to heavy-metal stress and one of the manifestations of heavy-metal resistance [9]. The majority of hyperaccumulating species discovered so far are restricted to a few specific geographical locations. Ni hyperaccumulators are found in New Caledonia [9], the Philippines [10], Brazil [11] and Cuba (AJM Baker, personal communication), Ni and Zn accumulators are found in southern and central Europe and Asia Minor [5,6,9], and Cr and Co accumulators in Central Africa [5,6,9]. We still k n o w very little about the biological and evolutionary significance of metal hyperaccumulation. Several hypotheses have been put forward, including the following: tolerance or disposal of metal from plants, drought resistance, inadvertent uptake, and defense against herbivores or pathogens. Of these hypotheses, present evidence favours most strongly the function of hyperaccumulated metals as a defense mechanism against herbivores [12,13]. A fair amount of phenomenological data on hyperaccumulating plants is available from extensive field collections and herbarium studies [5,9]. The list of hyperaccumulating plants is expanding rapidly. Ni hyperaccumulators are most numerous (numbering at least 277 taxa belonging to 36 different families) followed by Co, Cu a n d . Z n hyperaccumulators (AJM Baker, personal communications). Hyperaccumulators of Mn, Pb, Cd, Cr and Se have also been described. So far, the largest numbers of hyperaccumulating species in the temperate zone belong to the Brassicaceae (nmstard family) [9], but in the tropics, the Euphorbiaceae (spurge family) is the best represented. A New Caledonian tree, Sebertia acuminata, which has more than 11% of Ni in its latex (on a dry weight basis) [13], is one of the most striking examples of metal hyperaccumulation. A large number of other Ni hyperaccumulators (containing Ni at a level over 1% of total dry weigh0 belong to
the genera Alyssum and Thlaspi. Many Thlaspi species also accumulate Pb (up to 1% dry weight) and Zn (up to 3% dry weigh0 [9]. Armeria maritima is another prominent European Pb hyperaccumulator (over 1% Pb in dried tissue) [9,13]. The African species Aeollanthus biformifolius and Haumaniastrum katangense may often contain more than 0.1% Cu or Co in dried leaf tissue [7"]. Astragalus species growing on seleniferous soils may accumulate close to 1% Se in dry matter [61. The specificity of metal uptake by hyperaccunmlating plants is poorly understood and requires much more research. Even so, the available field data suggest that all hyperaccumulators may be divided into three groups on the basis of their tendency to accumulate different metals: Cu/Co accunmlators, Zn/Cd/Pb accumulators and Ni accunmlators (AJM Baker, personal comnmnications). The biochemical and molecular bases of such group selectivity is unknown. So far, the search for metal-accunmlating plants has b e e n carried out by a few people in selected parts of the globe. It is likely that many more as yet unidentified metal-accumulating plants growing o n natural and man-made metalliferous soils remain to be discovered by plant scientists. It is vital that rare and endemic metal-accumulating plant species are identified and preserved before they become extinct. To date, few controlled laboratory experiments that quantify, compare, and explain the n~etal-accumulating ability of various plant species have been carried out. The information is most needed in four areas: first, the metal-accumulating ability of various species as a function of soil metal concentrations, physical and chemical soil properties, physiological state of the plant, et cetera; second, the specificity of metal uptake, transport and accumulation; third, the physiological, biochemical and molecular mechanisms of accumulation and hyperaccumulation; and fourth, the biological and evolutionary significance of metal accumulation.
Mechanisms of metal accumulation A large proportion of metals in soils are bound to organic '(humus) soil constituents, inorganic (clay) soil constituents, or alternatively, are present as insoluble precipitates. For plants to accumulate these 'soil-bound' metals, they must first mobilize them into the soil solution. This mobilization of soil-bound metal can b e achieved in a number of different ways. First, metal-chelating molecules (phytosiderophores) can be secreted into the rhizosphere to chelate and solubilize soil-bound metal. For example, mugineic acid, avenic acid and nicotianamine are phytosiderophores that occur in graminaceous species [14"']. These phytosiderophores are released in response to Fe and Zn deficiency and can mobilize Cu, Zn and Mn from soil [15]. Metal-chelating proteins, perhaps related to metallothioneins [16] or y-glutamylcysteinyl-isopeptides (3' EC-isopepticles) [17], may also function as siderophores
Bioconcentration of heavy metals by plants Raskin et al. in plants. Second, roots can reduce soil-bound metal ions by specific plasma membrane bound metal reductases. Pea plants deficient in Fe or Cu have an increased ability to reduce Fe(III) and Cu(II) that is coupled with an increased uptake of Cu, Mn, Fe and Mg [18]. Third, plant roots can solubilize heavy metals by acidifying their soil environment with protons extruded from the roots. A lower pH solubilizes metal precipitates and releases soil-bound metal ions into the soil. A similar mechanism has been observed for Fe mobilization in some Fe-deficient dicotyledonous plants [19]. It should be noted here that all three of the above processes might also b e performed by mycorrhizal fungi or root-colonizing bacteria. Therefore, it is difficult to assess the individual contribution of root cells and rhizosphere microorganisms in metal mobilization by plants. Solubilized metal ions may enter the root either via the extracellular (apoplastic) or via intracellular (symplastic) pathways. Apoplastic transport is limited by the high cation exchange capacity of cell walls, unless the metal ion is transported as a non-cationic metal chelate. Symplastic transport requires that metal ions move across the plasma membrane, which usually has a large negative resting potential of approximately 170mV (negative inside the membrane). This membrane potential provides a strong electrochemical gradient for the inward movement of metal ions. Most metal ions enter plant cells by an energydependent saturable process via specific or generic metal ion carriers or channels [20]. Non-essential heavy metals may effectively compete for the same transmembrane carriers used by essential heavy metals. This relative lack of selectivity in transmembrane ion transport may partially explain w h y non-essential heavy metals can enter cells, even against a concentration gradient. For example, kinetic data demonstrate that essential Cu 2~ and Zn 2+ and non-essential Ni 2+ and Cd 2+ compete for the same transmembrane carrier [20]. Metal--chelate complexes may also be transported across the plasma membrane via specialized carriers, as is the case for Fe-phytosiderophore transport in graminaceous species [19]. Once metal ions have entered the root, they can either be stored or exported to the shoot. The vacuole plays an important role in metal ion storage [21]. Metal ions can be actively transported across the tonoplast as free ions or as metal-chelate complexes. For example, Cd is actively transported across the tonoplast of oat roots as either a free ion via a Cd/H + antiport [22° ] or as a Cd-('~C)3G/Cd-(TEC)2G (one-letter amino acid code) metal-peptide complex energized by the direct hydrolysis of ATP (DE Salt, WE Rauser, unpublished data). Inside the vacuole, metal ions are chelated either by organic acids, such as citric or malic acid, or by enzymatically synthesized (yEC)-isopeptides, commonly called phytochelatins. The metal-binding role of the proteins encoded by metallothionein-like genes recently identified in plants remains unknown [23]. Under certain conditions, metal ions may form insol-
uble precipitates, for example Zn-phytate [24] and CdS [25]. Compartmentalization, chelation and precipitation of metal ions probably comprise the major resistance mechanisms that protect plant cells from the damaging effect of metals. In hyperaccunmlating species, heavy metals accumulate both in the shoot and the root. In these plants, metal transport to the shoot probably takes place in the xylem. Metals may, however, redistribute in the shoot via the p h l o e m [26]. Xylem cell walls have a high cation exchange capacity that would be expected to retard severely the movement of metal cations. Therefore, metal-chelate complexes, such as Cd-citrate, should facilitate metal movement in the transpiration stream [27]. Analysis of the xylem sap of certain hyperaccumulators has demonstrated the involvement of organic acids in metal transport [9]. Recent work also suggests that (TEC)-isopeptides may be involved in metal binding in xylem sap [28]. Nicotianamine, involved in phloem transport of Fe, can also bind Zn, Co, Ni and Cu, and, therefore, may serve as a general heavy metal transporter in the phloem [26]. In addition, metals may be transported in the phloem chelated to either organic acids, (yEC)-isopeptides or metallothioneins. Clearly, more research is needed to understand the biological mechanisms involved in heavy metal movement, accumulation, and detoxification in plants. Hyperaccumulating plants provide an excellent and almost completely unresearched model system for such studies.
Uses for metal-accumulating plants For a long time, the ability of plants to accumulate metals was considered a detrimental trait. Being at the bottom of many natural food chains, metal-accumulating plants are directly or indirectly responsible for a large proportion of the dietary uptake of toxic heavy metals by humans and other animals [29]. Although some heavy metals are required for life, their excessive accumulation in living organisms is always toxic. The danger of heavy metals is aggravated by their almost indefinite persistence in the environment. The acute and chronic toxic effects of consuming metal-contaminated plants in wild animals, cattle and humans are very well documented and lie outside the scope of this review. Recently, several attempts have been made to produce plants that can trap heavy metals in their roots, thus inhibiting their translocation to the harvestable aboveground parts. For example, transgenic tobacco plants expressing the heavy metal chelating protein mouse metallothionein I contained -24% lower and 5% higher Cd levels in shoots and roots, respectively, than control plants [30°]. In spite of the obvious benefits for human health, currently, there are only a few ongoing efforts to select or genetically engineer metal-excluding crop plants or forage grasses. The need for such crops is particularly great in the large areas of Eastern Europe that were contaminated with radioactive fallout from the Chernobyl nuclear reactor.
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Environmentalbiotechnology Only recently has the value of naetal-accumulating plants for environmental remediation been fully realized [13,31,32°',33]. Despite the ever-growing number of toxic metal contaminated sites, the extremely costly process of removal and burial, or simply isolation of the contaminated sites, are still the most commonly used methods for dealing with heavy metal pollution. Water treatment facilities also do a relatively poor job of removing toxic metals from residential and industrial effluents, contributing to the overall problem [34]. The use of metal-accumulating plants for removal of metals from contaminated soils and waters has a number of advantages: the lower costs, generation of a recyclable metal-rich plant residue, applicability to a range of toxic metals and radionuclides, minimal environmental disturbance, elimination of secondary air or water-borne wastes, and public acceptance. The application of plants in environmental cleanup is an emerging technology that has been called phytoremediation (I Raskin, Grant Proposal #R81869, 1991). Three subsets of this technology are being develo p e d in our laboratory. First, phytoextraction, in which metal-accumulating plants are used to transport and concentrate metals from the soil into the harvestable parts of roots and above-ground shoots. Second, rhizofiltration, which uses plant roots to absorb, concentrate and precipitate toxic metals from polluted effluents. Finally, phytostabilization, which involves the use of plants to eliminate the bioavailability of toxic metals in soils. Since phytostabilization does not necessarily involve bioaccumulation, w e do not discuss this concept in this review. In the phytoextraction process, several sequential crops of laboratory-improved hyperaccumulating plants may be used to reduce soil concentrations of heavy metals to environmentally acceptable levels. Preliminary trials with Ni and Zn hyperaccumulator plants from the Brassicaceae family were successful in partially removing heavy metals from soils contaminated by long-terna application of heavy metal containing sludges [32°°]. Dried, ashed or composted plant residues, highly enriched in heavy metals may be isolated as hazardous waste or recycled as bio-metal ore. Although the most heavily contaminated soils do not support plant growth, sites with light to moderate toxic metal contamination might be suitable for growing hyperaccumulating plants for toxic metal cleanup. Plants that accumulate toxic metals can be grown and harvested economically, leaving the soil or water with a greatly reduced level of toxic metal contamination. Recently, there has been a growing interest in the use of metal-accumulating roots and rhizomes of aquatic or semiaquatic vascular plants for the removal of heavy metals from contaminated aqueous streams. For example, water hyacinth (Eicbbornia crassipes) [35], pennywort (Hydrocotyle umbellata) [36], duckweed (Lemna minor) [37,38] and water velvet (Azolla pinnata) [39],
take up Pb, Cu, Cd, Fe, and Hg from contaminated solutions. In a related development, cell suspension cultures of Datura innoxia were found to remove a wide variety of metal ions from solutions [39,40]. Most of the removed metals were tightly chelated by unidentified components of cell walls in a process that did not require metabolic activity. The observation that hydroponically grown roots of terrestrial plants are extremely effective in removing Pb, Cr, Zn, Cd, Cu, and Ni from water has laid the foundation for the development of rhizofiltration in our laboratory. For example, 1.1 g dry weight of either sunflower (Helianthus annuus) or Indian mustard (Brassica juncea) roots, immersed in 400 ml of water containing 300 gtg ml-1 of Pb, brought the Pb concentration to below 1 l.tg m1-1 in 8 hours (S Dushenkov, I Raskin, unpublished data). Disappearance of Pb from the solution was accompanied by a dramatic concentration of Pb in the root tissue, over 10% on a dry weight basis. Initial analysis indicates that, at least in some instances, rhizofiltration may provide an attractive alternative to current methods of chemical and microbial precipitation of heavy metals.
Conclusions The bioconcentration of heavy metals by plants is a fascinating area of research that should be able to provide answers to some of the fundamental questions of plant biochemistry, nutrition, and stress physiology. Studying heavy metal-plant interactions should improve our understanding of the mechanisms of ion uptake, accumulation and resistance. The unique 'metal accumulation' and 'metal resistance' genes of metal-accumulating plants may directly benefit world agriculture and the environment. Phytoremediation, although still in its infancy, may one day b e c o m e an established environmental cleanup technology. Further development of phytoremediation requires an integrated multidisciplinary research effort that combines plant biology, soil chemistry, soil microbiology, as well as agricultural and environmental engineering. As a major renewable resource exploited by man, plants already give us food, energy, construction materials, natural fibers, and various chemicals. The use of plants in environmental cleanup may guarantee a greener and cleaner planet for all of us.
Acknowledgements The authors thank the US Department of EnvironmentalProtection, New Jersey Commission for Science and Technology,New Jersey Agricultural Experimental Station (NJAES) and PhytoTech Inc for supporting the research related to the topic of this review. We are also grateful to Dr AJM Baker for helpful discussions.
Bioconcentration of heavy metals by plants Raskin et al.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ** of outstanding interest 1.
Galston AW, Davis PJ, Satter RL: The Life of the Green Plant. Englewood Cliffs, New Jersey: Prentice-Hall Inc; 1980:69.
2.
Nieboer E, Richardson DHS: The Replacement of the Nondescript Term 'Heavy Metals' by a Biologically and Chemically Significant Classification of Metal Ions. Env Pollution [B] 1980, 1:3-26.
3.
Seaward MRD, Richardson DHS: Atmospheric Sources of Metal Pollution and Effects on Vegetation. In Heavy Metal Tolerance in Plants: Evohaionary Aspects. Edited by Shaw AJ. Boca Raton: CRC Press; 1990:75-92.
4. •
Runnells DD, Shepherd TA, Angino EE: Metals in Water. Determining Natural Background Concentrations in Mineralized Areas. Env Sci Technol 1992, 26:2316-2323. Summarizes the soils and waters naturally rich in heavy metals. Provides a list of such sites worldwide and some description of their mineral composition. 5.
Brooks RR: Biological Methods o f Prospecting for Minerals. New York: John Wiley & Sons, Inc; 1983.
6.
Peterson PJ: Unusual Accumulations of Elements by Plants and Animals. Sci Prog Oxf 1971, 59:505-536.
7. Brooks RR, Baker AJM, Malaisse F: Copper Flowers. Nat • Res Exploration 1992, 8:338-351. An elegant study of unique flora of the world's richest Zaire copper and cobalt belt, with a particular emphasis on Cu and Co accumulating plants. 8.
9.
10.
11.
Baker AJM, Walker PL: Ecophysiology of Metal Uptake by Tolerant Plants. In Heavy Metal Tolerance in Plants: Evolutionary Aspects. Edited by Shaw AJ. Boca Raton: CRC Press; 1990:155--177. Baker AJM, Brooks 1GR: Terrestrial Higher Plants which Hyperaccumulate Metallic Elements - - A Review of their Distribution, Ecology and Phytochemistry. Biorecovery 1989, 1:81-126. Baker AJM, Proctor J, van Balgooy M, Reeves RD: Hyperaccumulation Of Nickel by Flora of the Ultramafics of Palawan, Republic of the Philippines. In The Vegetation of Ultramafic (Serpentine) Soils. Proceedings o f the First International Conference on Serpentine Ecolopxv. Andover, Hampshire: Intercept Ltd, UK; 1993:291-304. Brooks RR, Reeves RD, Baker AJM: The Serpentine Vegetation of Goias State, Brazil. In The Vegetation o f Ultramafic (Serpentine) Soils. Proceedings o f the First International Conference on Serpentine Ecology. Andover, Hampshire: Intercept Lid, UK; 1993:67-81.
12.
Ernst WHO, Schat H, Verkleij JAC: Evolutionary Biology of Metal Resistance in Silene vuigaris. Evol Trends Plants 1990, 4:45-51.
13.
Baker A, Brooks R, Reeves R: Growing for Gold... and Copper.., and Zinc. New Scientist 1989, 1603:44--48.
Kinnersely AM: The Role of Phytochelates in Plant oo Growth and Productivity. Plant Growth Regulation 1993, 12:207-217. A review of various modes of metal chelation employed by plants. Elucidates the different chemical strategies that plants use for mobilization, transport, and detoxification of heavy metals.
Structure, Immunoreactivity and Metal-Binding Function. Int J Biochem 1991, 23:1-5. 17.
Rauser WE: Phytochelatins. Annu 59:61--86.
18.
Welch RM, Norvell WA, Schaefer SC, Shaft JE, Kochian LV: Induction of Iron(III) and Copper(II) Reduction in Pea (Pisum sativum L.) Roots by Fe and Cu Status: Does the Root-Cell Plasmalemma Fe(III)-Chelate Reductase Perform a General Role in Regulating Cation Uptake? Planta 1993, 190:555-561.
19.
Crowley DE, Wang YC, Reid CPP, Szaniszlo PJ: Mechanisms of Iron Acquisition from Siderophores by Microorganisms and Plants. Plant Soil 1991, 130:179-198.
20.
Clarkson DT, L~ittge U: III. Mineral Nutrition: Divalent Cations, Transport and Compartmentation. Prog Botany 1989, 51:93-112.
21.
VOgeli-Lange R, Wagner GJ: Subcellular Localization of Cadmium and Cadmium-Binding Peptides in Tobacco Leaves. Implications of a Transport Function for Cadmium-Binding Peptides Plant Phystol 1990, 92:1086-1093.
22. •
Salt DE, Wagner GJ: Cadmium Transport Across Tonoplast of Vesicles from Oat Roots. Evidence for a Cd/H Antiport Activity. J Biol Chem 1993, 268:12297-12302. The first direct demonstration of the active transport of Cd across the biological membrane via Cd/H antiport mechanism. 23.
Robinson NJ, Evans IM, Mulcrone J, Bryden J, Tommey AM: Genes with Similarity to Metallothionein Genes and Copper, Zinc Ligands in Pisum saiivum L. Plant Soil 1992, 146:291-298.
24.
Van Steveninck RFM, Van Steveninck ME, Femando DR: Heavy-Metal (Zn, Cd) Tolerance in Selected Clones of Duckweed (Lemna minor). Plant Soil 1992, 146:271-280.
25.
Reese RN, White CA, Winge DR: Cadmium-Sulfide Crystallites in Cd-(qtEC)nG Peptide Complexes from Tomato. Plant Physiol 1992, 98:225-229.
26.
Stephan UW, Scholz G: Nicotianamine: Mediator of Transport of Iron and Heavy Metals in the Phloem? Physiol Plant 1993, 88:522-529.
27.
Senden MHMN, Van Paassen FJM, Van Der Meet AJGM, Wolterbeek HTh: Cadmium-Citric Acid-Xylem Cell Wall Interactions in Tomato Plants. Plant Cell Env 1992, 15:71-79.
28.
Przemeck E, Haase NU: On the Bonding of Manganese, Copper and Cadmium to Peptides of the Xylem Sap of Plant Roots. Water Air Soil Pollution 1991, 57-58:569-577.
29.
Kabata-Pendias AK, Pendias H: Trace Elements in Soils and Plants. Boca Raton: CRC Press; 1989.
30. •
Yeargan R, Maiti IB, Nielsen AG, Hunt AG, Wagner GJ: Tissue Partitioning of Cadmium in T r a n s g e n i c Tobacco Seedlings and Field Grown Plants Expressing the Mouse MetaUothionein I Gene. Transgenic Res 1992, 1:261-267. A thorough investigation of the Cd redistribution in transgenic tobacco plants containing the mouse metallothionein I gene. 31.
14.
15.
R6mheld V: The Role of Phytosiderophores in Acquisition of Iron and other Micronutrients in Graminaceous Species: An Ecological Approach. Plant Soil 1991, 130:127-134.
16.
Kay J, Cryer A, Darke M, Kille P, Leesd WE, Norey C, Stark JM: Naturally Occurring and Recombinant Metallothioneins:
Rev Biochem 1990,
Chaney RL: Plant Uptake of Inorganic Waste. In Land Treatment of Hazardous Wastes. Edited by Parr JE, Marsh PB, Kla JM. Park Ridge: Noyes Data Corp; 1983:50-76.
32. o•
Baker AJM, McGrath SP, Sidoli CMD, Reeves RD: The Possibility of in Situ Heavy Metal Decontamination of MetalPolluted Soils using Crops of Metal-Accumulating Plants. Resource Conservation Recycling 1994, in press. The first detailed and scientific summary of a field phytoremediation trial. 33.
Cunningham SD: Plant-Based Environmental Remediation: Progress and Promise. Abstract P-33. In Vitro Cell Dev Biol 1993, 29A:42A.
289
290
Environmental biotechnology 34.
Bubb JM, Lester JN: The Impact of Heavy Metals on Lowland Rivers and the Implications for Man and the Environment. Sci Total Env 1991, 100:207-233.
38.
Mo SC, Choi DS, Robinson JW: Uptake of Mercury from Aqueous Solutions by Duckweed: The Effects of pH, Copper and Humic Acid. J Env Sci Health 1989, A24:135--146.
35.
Kay SH, Hailer WT, Garrard LA: Effects of Heavy Metals on Water Hyacinths (Eichhornla crassipes (Mart.) Solms). Aquatic Toxicol 1984, 5:117-128.
39.
36.
Dierberg FE, DeBusk TA, Goulet Jr NA: Removal of Copper and Lead Using a Thin-Film Technique. In Aquatic Plants for Water Treatment and Resource Recov~. Edited by Reddy KB, Smith WH. Florida: Magnolia Publishing Inc; 1987:497-504.
Jackson PJ, Torres AP, Delhaize E, Pack E, Bolender SL: The Removal of Barium Ions from Solution using Datura lnnoxia Suspension Culture Cells. J Env Quality 1990, 19:64~(,q8.
40.
Jackson PJ, DeWitt JG, Kuske CR: Accumulation of Toxic Metal Ions by Components of Plant Suspension Cell Cultures. Abstract P-34. In Vitro Cell Dev Btol 1993, 29A:42A.
37.
Jain SK, Vasudevan P, Jha NK: Removal of Some Heavy Metals from Polluted Waters by Aquatic Plants: Studies on Duckweed and Water Velvet. Biological Wastes 1989, 28:115-126.
I Raskin, PBAN Kumar, S Dushenkov and DE Salt, Center for Agricultural Molecular Biology, Environmental and Natural Sciences Building, Rutgers, The State University of New Jersey, Cook College, PO Box 231, New Brunswick, New Jersey 08903-0231, USA.