Removal of metals from contaminated substrates by plants

Details Removal of metals from contaminated substrates by plants Removal of metals from contaminated substrates by plants

1998-04-16

2003-12-02

McElwain, Elizabeth F. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S298000, C800S295000, C800S288000, C800S306000, C435S069100, C435S320100, C435S419000, C435S468000, C536S023100, C536S024100, C536S023700

Reexamination Certificate

active

06657106

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is concerned with the phenomenon of hyperaccumulation of metals in plants and the removal of metals from contaminated substrates by plants. The invention relates in particular to methods for removing metals from soil or other environments using modified plants having an increased capacity for metal uptake, and to methods for obtaining such plants. The invention is also concerned with transgenic plants having an increased capacity for metal uptake, and to recombinant vectors and transformed plant cells used to obtain the transgenic plants.
2. Description of the Related Art
Many soils are contaminated by metals that derive from sources such as mine workings, metal-processing industries, atmospheric depositions, the power and fuel industries, deposition of contaminated sewage sludge, and others. These contaminated substrates pose serious human health problems, are deleterious to the environment, and can render crop cultivation either inadvisable (because of the health hazard from the plant products) or impractical (because of the toxicity of the metals towards plants). These problems are becoming more acute in many regions, for example as a result of increased deposition of contaminated sewage sludge on land rather than at sea.
Conventional approaches for decontaminating metal-polluted soils have relied on physical methods such as landfilling (the excavation and removal of soil to a landfill site designated for hazardous waste), fixation or immobilization (such as infiltration with cement or by vitrification), or leaching (for example with strong acids to desorb the metal ions) (see Salt, D. E. et al. (1995) Bio/Technology 13, 468-474). These methods are not only expensive but are environmentally less than ideal, for example because they tend to destroy soil structure (as well as the microbial organisms in the soil), and can give rise to secondary pollution in run-off water. Thus it has been recognized that removal of metals from contaminated soils by metal-tolerant plants might provide an alternative and cost-effective technology for environmental clean-up, a concept known as “phytoremediation” (Chaney, R. L. (1983) In Land Treatment of Hazardous Wastes, eds Parr, J. F., Marsh, P. B. and Kla, J. M., pp 50-76. Noyes Data Corp., Park Ridge, N.J.; Salt et al. (1995), loc. cit.).
To date, the application of plants in phytoremediation has been limited by the severe toxicity of most metal ions to the majority of plant species. However, a number of plant species are known that are both relatively metal tolerant and capable of accumulating metals to high concentrations in their above-ground biomass. These plants are known as “metal hyperaccumulators”, of which several hundred species have so far been described (Baker, A. J. M. and Brooks, R. R. (1989) Biorecovery 1, 81-126). These plants have favourable characteristics for bioremediation, since the above-ground parts (leaves and stems) can be readily harvested, and the metal-rich residues processed in a controlled manner. Successive cycles of crops of these metal-hyperaccumulator plants would be expected to lead to progressive decontamination of the soils, and this has been demonstrated (Baker, A. J. M. et al. (1994) Resources, Conservation and Recycling 11, 41-49; Brown, S. L. et al. (1994) J. Environ. Qual. 23, 1151-1157; Brown S. L. et al. (1995) Soil Sci. Soc. Am. J. 59, 125-133). However, most hyperaccumulator plants are of relatively small stature and are slow-growing, so long periods of time would be needed to decontaminate most metal-polluted substrates to acceptable levels (Baker, A. J. M. et al. (1994) loc. cit.). The definition of “metal hyperaccumulator” varies according to the metal concerned, e.g. for zinc it is >1% by dry weight, while for nickel and cobalt it is >0.1% by dry weight (see Baker, A. J. M. et al. (1994) loc. cit.).
The underlying mechanism of metal-hyperaccumulation in plants has not up to now been understood. Most theories of metal tolerance in plants have assumed that metal ions within the plant are detoxified by chelation with an appropriate high-affinity ligand (Ernst, W. H. O. et al. (1992) Acta Bot. Neerl. 41, 229-248). The most frequently suggested ligands of this type have been cysteine-rich proteins called metallothioneins (Robinson, N. J. et al. (1993) Biochem. J. 295, 1-10), including the lower-molecular-weight phytochelatins (Rauser, W. E. (1990) Annu. Rev. Biochem. 59, 61-86), and organic-acid anions such as malate, citrate and malonate (Reeves, R. D. (1992) In The Vegetation of Ultramafic (Serpentine) Soils, eds Baker, A. J. M., Proctor, J. and Reeves, R. D., pp 253-277. Intercept Press, Andover). However, the concentrations of these potential metal-chelating ligands do not respond in the proportional and metal-specific manner that would be anticipated if they had a fundamental role in the phenomenon of metal hyperaccumulation (Ernst, W. H. O. et al. (1992) loc. cit.; Reeves, R. D. (1992) loc. cit.; de Knecht, J. A. et al.(1994) Plant Physiol. 104, 255-261).
In principle, if the biochemical mechanisms responsible for metal tolerance and metal accumulation in plants were understood, this would permit the development of novel strategies for the application of plants in the clean-up of contaminated soils. For example, genetically modified plants with altered biochemical characteristics can be generated using recombinant DNA techniques (“genetic engineering”). Several reports have been published of plants genetically transformed to express one or other animal metallothionein gene, but these have given variable results. In some (but not all) experiments, an increase in tolerance towards cadmium was observed in plants expressing an animal metallothionein gene, but this appeared to be associated with a decreased cadmium accumulation in the above-ground parts of the plant (Elmayan, T. and Tepfer, M. (1994) Plant J. 6, 433-440, and references therein). Plants genetically modified in this manner thus do not appear to be suitable for the purpose of phytoremediation of contaminated soils.
Novel strategies for the application of plants in the extraction of metals from soils would be possible if the biochemical processes responsible for the phenomenon of metal hyperaccumulation were understood. The so-called “metal hyperaccumulator plants” have the ability to extract metals effectively from the soil (cf. Bernal, M. P. et al. (1994) Plant Soil 164, 251-259), to accumulate high amounts of metals in their above-ground biomass, and to tolerate metal concentrations in the soil that would be toxic to the great majority of plant species (Baker, A. J. M. and Brooks, R. R. (1989) loc. cit.). These properties are all highly desirable in plants to be used for purposes of phytoremediation. Up to now, however, there has been no clear understanding of the cellular factors responsible for these distinctive features of metal hyperaccumulator plants.
Recently, it has been observed that the amino acid histidine increases in the xylem sap of the hyperaccumulator plant
Alyssum lesbiacum
when these plants are exposed to nickel in the root medium (Kramer, U., Baker, A. J. M. and Smith, J. A. C. (1994) Abstracts of the Fifth International Symposium on the Genetics and Molecular Biology of Plant Nutrition, University of California, U.S.A., July 17-24; Smith, J. A. C., Kramer, U. and Baker, A. J. M. (1995) Meeting on Phytoremediation by Hyperaccumulator Plants—Current Research and Future Requirements, Rothamsted, UK, January 25-28; Smith, J. A. C., Kramer, U., Tibbetts, R. A. and Baker, A. J. M. (1995) Second International Conference on Serpentive Ecology, Nouméa, New Caledonia, July 31−August 5). When supplied to the xylem of excised shoots of the non-metal-accumulating species
A. montanum,
histidine apparently reduced the toxicity of nickel; also, when apparently supplied to the root medium of excised roots of this species, histidine again apparently reduced the toxicity of nickel, as manifested by an increased exudation of sap from the cut xylem

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