Andrew C. P. Heaton

Phytoremediation of Mercury- and Methylmercury-Polluted Soils Using Genetically Engineered Plants

Inorganic mercury in contaminated soils and sediments is relatively immobile, though biological and chemical processes can transform it to more toxic and bioavailable methylmercury. Methylmercury is neurotoxic to vertebrates and is biomagnified in animal tissues as it is passed from prey to predator. Traditional remediation strategies for mercury contaminated soils are expensive and site-destructive. As an alternative we propose the use of transgenic aquatic, salt marsh, and upland plants to remove available inorganic mercury and methylmercury from contaminated soils and sediments. Plants engineered with a modified bacterial mercuric reductase gene, merA, are capable of converting Hg(II) taken up by roots to the much less toxic Hg(0), which is volatilized from the plant. Plants engineered to express the bacterial organo-mercurial lyase gene, merB, are capable of converting methylmercury taken up by plant roots into sulfhydryl-bound Hg(II). Plants expressing both genes are capable of converting ionic mercu...

Strategies for the engineered phytoremediation of toxic element pollution: mercury and arsenic

Plants have many natural properties that make them ideally suited to clean up polluted soil, water, and air, in a process called phytoremediation. We are in the early stages of testing genetic engineering-based phytoremediation strategies for elemental pollutants like mercury and arsenic using the model plant Arabidopsis. The long-term goal is to develop and test vigorous, field-adapted plant species that can prevent elemental pollutants from entering the food-chain by extracting them to aboveground tissues, where they can be managed. To achieve this goal for arsenic and mercury, and pave the way for the remediation of other challenging elemental pollutants like lead or radionucleides, research and development on native hyperaccumulators and engineered model plants needs to proceed in at least eight focus areas: (1) Plant tolerance to toxic elementals is essential if plant roots are to penetrate and extract pollutants efficiently from heterogeneous contaminated soils. Only the roots of mercury- and arsenic-tolerant plants efficiently contact substrates heavily contaminated with these elements. (2) Plants alter their rhizosphere by secreting various enzymes and small molecules, and by adjusting pH in order to enhance extraction of both essential nutrients and toxic elements. Acidification favors greater mobility and uptake of mercury and arsenic. (3) Short distance transport systems for nutrients in roots and root hairs requires numerous endogenous transporters. It is likely that root plasma membrane transporters for iron, copper, zinc, and phosphate take up ionic mercuric ions and arsenate. (4) The electrochemical state and chemical speciation of elemental pollutants can enhance their mobility from roots up to shoots. Initial data suggest that elemental and ionic mercury and the oxyanion arsenate will be the most mobile species of these two toxic elements. (5) The long-distance transport of nutrients requires efficient xylem loading in roots, movement through the xylem up to leaves, and efficient xylem unloading aboveground. These systems can be enhanced for the movement of arsenic and mercury. (6) Aboveground control over the electrochemical state and chemical speciation of elemental pollutants will maximize their storage in leaves, stems, and vascular tissues. Our research suggests ionic Hg(II) and arsenite will be the best chemical species to trap aboveground. (7) Chemical sinks can increase the storage capacity for essential nutrients like iron, zinc, copper, sulfate, and phosphate. Organic acids and thiol-rich chelators are among the important chemical sinks that could trap maximal levels of mercury and arsenic aboveground. (8) Physical sinks such as subcellular vacuoles, epidermal trichome cells, and dead vascular elements have shown the evolutionary capacity to store large quantities of a few toxic pollutants aboveground in various native hyperaccumulators. Specific plant transporters may already recognize gluthione conjugates of Hg(II) or arsenite and pump them into vacuole.

Toward Detoxifying Mercury-Polluted Aquatic Sediments with Rice Genetically Engineered for Mercury Resistance

Mercury contamination of soil and water is a serious problem at many sites in the United States and throughout the world. Plant species expressing the bacterial mercuric reductase gene, merA, convert ionic mercury, Hg(II), from growth substrates to the less toxic metallic mercury, Hg(0). This activity confers mercury resistance to plants and removes mercury from the plant and substrates through volatilization. Our goal is to develop plants that intercept and remove Hg(II) from polluted aquatic systems before it can undergo bacterially mediated methylation to the neurotoxic methylmercury. Therefore, the merA gene under the control of a monocot promoter was introduced into Oryza sativa L. (rice) by particle gun bombardment. This is the first monocot and first wetland-adapted species to express the gene. The merA-expressing rice germinated and grew on semisolid growth medium spiked with sufficient Hg(II) to kill the nonengineered (wild-type) controls. To confirm that the resistance mechanism was the conversion of Hg(II) to Hg(0), seedlings of merA-expressing O. sativa were grown in Hg(II)-spiked liquid medium or water-saturated soil media and were shown to volatilize significantly more Hg(0) than wild-type counterparts. Further genetic manipulation could yield plants with increased efficiency to extract soil Hg(II) and volatilize it as Hg(0) or with the novel ability to directly convert methylmercury to Hg(0).

EXPRESSION OF ORGANOMERCURIAL LYASE IN EASTERN COTTONWOOD ENHANCES ORGANOMERCURY RESISTANCE

SummaryRelease of inorganic mercury pollutants into shallow aquatic environments has resulted in the bacterial production of a more toxic organic mercury species, methylmercury. The bacterial organomercurial lyase (MerB) catalyses the protonolysis of the carbon-mercury bond and releases Hg(II), a less toxic, non-biomagnified form of mercury. Our objective was to engineer eastern cottonwood (Populus deltoides), a fast-growing tree adapted to growth in riparian environments, with the merB gene to explore its potential for phytoremediation of mercury. We produced multiple eastern cottonwood clones expressing a modified bacterial merB gene, confirmed that the gene was expressed in the transclones and tested the regenerated plants for their ability to tolerate exposure to an organic mercury source, phenylmercuric acetate (PMA), in vitro and in hydroponic culture, compared to wild-type control trees. Transgenic merB plants expressed high levels of MerB protein and showed some evidence of higher resistance to the organic mercury than wild-type plants, producing longer roots under exposure to PMA in vitro, although hydroponic culture results were inconclusive. Our results indicate that in order for merB to be useful in eastern cottonwood trees designed to degrade methylmercury at mercury-contaminated aquatic sites, it will probably need to be combined with other genes such as merA.

Multigene Strategies for Engineering the Phytoremediation of Mercury and Arsenic

Hundreds of millions of people worldwide suffer the consequences their environment, which make them suitable to process related toxic field-adapted plant species that can extract mercury and arsenic from soil or water, process them in high concentrations, and prevent them Z. Xu et al. (eds.), Biotechnology and Sustainable Agriculture 2006 and Beyond, 49–60. of being exposed to toxic levels of mercury and arsenic. Our labophytoremediation of elemental pollutants relies on plants to extract ratory is focused on developing simple genetic engineering stratethem aboveground for later harvest. Plants possess many natural systems to uptake and manage 15 essential elemental nutrients from gies for the phytoremediation of these two toxic elements. The engineering. The long-term goal of our work is to develop vigorous, toxicants from soil and water, manage their toxicity, and concentrate elemental pollutants. These properties may be enhanced via genetic properties, such as using photosynthetic energy and pervasive root 50 R.B. Meagher et al. from entering the food-chain. Our initial research efforts have focused on enhancing several of these properties including: 1) increasing plant tolerance to mercury and arsenic; 2) transforming these toxic elements into the chemical species that promote their shortand longdistance transport from roots to shoots; 3) transforming these toxic elements to the best chemical species for storage in leaves, stems, and vascular tissues above ground; 4) enhancing plant chemical ‘sinks’ that can trap these toxicants aboveground; and 5) enhancing transporters for uptake and storage. It is likely that the next decade can cleanup mercury and arsenic and restore contaminated land and water resources. Introduction Many dangerous, elemental pollutants are present at unacceptably high levels in the environment because of industrial, defense, agricultural, and municipal processes, and they are adversely affecting the health of millions of people worldwide (Nriagu, 1994; Wolz et al., 2003). Elemental pollutants include heavy metals, metalloids, and radionuclides such as mercury, lead, cadmium, arsenic, technetium, tritium, and deuterium. Unlike organic pollutants that can be degraded to harmless small molecules, elemental pollutants are immutable by all biochemical reactions, making them particularly difficult to remediate (Meagher, 2000; Kramer and Chardonnens, 2001). The phytoremediation of some elemental pollution may require genetically engineering several plant properties to enhance plant extraction, tolerance, and hyperaccumulation of these toxicants. Research on engineered phytoremediation is still in its infancy, and a better understanding of natural hyperaccumulators will be a tremendous aid in our understanding of which genes and which cellular and organismal processes can be most effectively manipulated (Baker, 2000; Wang, 2002; Kerkeb and Kramer, 2003). The genes of soil bacteria, also have much to contribute to phytoremediation schemes, because they have evolved many properties that direct the aggressive transformation or management of elemental pollutants (Meagher et al., 2000). Another focal point for research on phytoremediating will see unprecedented progress in developing specialized plants that

The Engineered Phytoremediation of Ionic and Methylmercury Pollution

Our current specific objectives are to use transgenic plants to control the chemical species, electrochemical state, and above ground binding of mercury to (a) prevent methylmercury from entering the food-chain, (b) remove mercury from polluted sites, and (c) hyperaccumulate mercury in above ground tissues for later harvest.