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.

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SummaryMercury is one of the most hazardous heavy metals and is a particular problem in aquatic ecosystems, where organic mercury is biomagnified in the food chain. Previous studies demonstrated that transgenic model plants expressing a modified mercuric ion reductase gene from bacteria could detoxify mercury by converting the more toxic and reductive ionic form [Hg( II )] to less toxic elemental mercury [Hg (0)]. To further investigate if a genetic engineering approach for mercury phytoremediation can be effective in trees with a greater potential in riparian ecosystems, we generated transgenic Eastern cottonwood ( Populus deltoides ) trees expressing modified merA9 and merA18 genes. Leaf sections from transgenic plantlets produced adventitious shoots in the presence of 50 µ M Hg( II ) supplied as HgCl 2 , which inhibited shoot induction from leaf explants of wild-type plantlets.Transgenic shoots cultured in a medium containing 25 µ M Hg( II ) showed normal growth and rooted, while wild-type shoots were killed. When the transgenic cottonwood plantlets were exposed to Hg( II ), they evolved 2 -4-fold the amount of Hg(0) relative to wild-type plantlets.

Plants expressing a modified bacterial mercury reductase, merA, are highly resistant to Hg(II) toxicity as a result of the enzymatically catalyzed electrochemical reduction of Hg(II) to the much less toxic and volatile Hg(0). merA expression may allow plants to manifest a suite of responses to mercury exposure, making them more capable than wild-type plants of interacting with and removing mercury from contaminated soil or water. We have engineered merA-expressing Nicotiana tabacum (tobacco) as a model plant for examining these responses. Mercury resistance was demonstrated by germinating and growing merA tobacco seeds on semi-solid medium spiked with a HgCl 2 concentration acutely toxic to wild-type plants. On similar growth medium, merA plant roots penetrated a highly concentrated, localized Hg(II) zone of HgS (cinnibar) more readily than wild-type roots. In hydroponic medium spiked with HgCl 2 , merA plants maintained higher evapotranspiration activity than wild-type plants. The ability of merA Hg(II)-reductive activity to counter typical plantcatalyzed Hg(0) oxidation to Hg(II) was demonstrated by a lower net foliar absorption of atmospheric Hg(0) than wild-type plants. Mercury translocation through merA plants was examined through reciprocally grafted merA and wild-type tobacco grown on HgCl 2 -spiked hydroponic medium. Elevated mercury concentrations in wild-type shoots grafted to merA roots suggest the vertical movement of mercury within merA tissues or plants may be facilitated by dynamic balance between native Hg(0) oxidation and MerA-catalyzed Hg(II) reduction. These experiments demonstrate that merA-engineered tobacco plants display an array of tissue-level and whole-plant attributes which should allow for more efficient mercury extraction and processing compared to the wild-type.

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).