Phytoremediation Technologies
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Mercury pollution
poses an immediate threat not only to human health, but also to other plants,
microorganisms and animals in the environment. Methanogenic bacteria
convert ionic and/or elemental mercury to methyl mercury, which is highly
toxic. Other bacteria have been reported to produce enzymes that remove
methyl groups from mercury and reduce ionic mercury to less toxic elemental
mercury Hg (0). Elemental Hg (0) is highly volatile and is readily
converted from liquid to vapor-phase. These bacteria could be used
to volatilize Hg (0), however this process is slow. The genes involved
in bacterial conversion of methyl mercury to ionic mercury Hg+, to elemental
mercury vapor Hg(0) are all a part of a mercury-responsive bacterial operon.
When a bacterium is exposed to mercury, the gene products of
the operon are expressed. These include a mercury responsive regulatory
protein, transport proteins that bind and transport mercury into the cell,
organomercuric lyase, which catalyzes the removal of the methyl group of
methyl mercury converting it into ionic mercury Hg+ (merB), and mercuric
ion reductase which catalyzes the conversion of ionic mercury to volatile
elemental mercury (merA). If the merA and merB are expressed in plants,
then these plants could clean up or phytoremediate a mercury-contaminated
site with relatively low cost compared to current manual ex situ processes. Researchers have isolated the genes encoding these enzymes
and introduced them into plants. The intention behind this research
is to explore the potential of plants to take up methyl mercury and convert
it to volatile Hg(0). The following are summaries of some of the major
contributing research in this area. |
Rugh, C., Dayton Wilde, H., Stack, N., Thompson, D.M., Summers A.O., and Meagher, R.B., (1996) Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene. Proc. Natl. Acad. Sci. 93:3182-3187. The enzyme, mercuric ion reductase, encoded by the gene merA, reduces ionic mercury (Hg+) to the less toxic volatile Hg(0) using NADPH reducing equivalents. Because the merA gene was found to be very G+C rich (~67%) and was suited for expression only in a bacterial system, early attempts to express this gene in plant systems were unsuccessful. Rugh et al. replaced codons 287-336, which constituted 9% of the coding region to contain a sequence of DNA that had codon usage that was more suited to expression in plant systems. Transgenic Arabidopsis thaliana plants containing this modified merApe9 expressed the gene product mercuric ion reductase. Additionally, merApe9 seeds germinated and grew into seedlings on agar plates containing 50 micromolar HgCl2 while control plants did not. Mercury vapor analysis showed that transgenic merApe9 plants volatilized significant amounts (~50 ng Hg(0)/mg tissue of mercury vapor. Finally, Northern blots of total mRNA from transgenic plants confirmed merApe9 gene expression. These data suggest that the potential for plants that volatilize Hg are viable. |
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| Rugh, C., Senecoff,
J., Meagher, R., and Merkle, S. (1998) Development
of transgenic yellow poplar for mercury phytoremediation. Nature
Biotechnology. 16:925-928. Transgenic Arabidopsis plants expressing the merA9 gene construct converted ionic Hg+ to volatile Hg(0). Expression of this type of system in a high biomass plant with potential environmental application, such as yellow poplar (Liriodendron tulipifera) may provide a means for phytovolatilization of mercury pollution. The merA9 sequence was further modified to contain an additional 9% of the coding sequence fragment of DNA with plant-like codon usage. This further modified merA18 sequence was transformed using particle bombardment of yellow poplar proembryonic masses. Transgenic plantlets grew on agar plates containing 25microM and 50microM HgCl2, whereas control plants did not. Additionally significant Hg (0) volatilization was observed by transgenic lines. The demonstrated ability of genetically engineered yellow poplar to grow on increased concentrations of ionic Hg+ may demonstrate the potential for phytovolitazion methods of mercury remediation. However, this research is still in its infancy and future experiments may include growing transgenic poplar plants on mercury-contaminated soils. |
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Bizily, S., Rugh, C., Summers, A., Meagher, R. (1999) Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials. Proc. Natl. Acad. Sci. 96:6808-6813. Mercury deposited into bodies of water is typically converted to methyl-mercury by methanogenic bacteria. Other mercury-resistant bacteria eliminate methyl mercury by producing an enzyme, organomercurial lyase encoded by the gene merB. Because most mercury-contaminated water contains methyl mercury, there would be a benefit to producing a model system in which merB was expressed. Bizily et al., report that transformants of Arabidopsis with merB grow on higher concentrations of methyl mercury-like compounds than control plants. The merB gene that was isolated from mercury-resistant bacteria was modified using PCR techniques to contain flanking regions containing consensus plant sequences and restriction sites. The new merB gene was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation. Transgenic merB plants grew on agar plates containing phenylmercuric acetate or methylmercuric chloride while control plants and transgenic merA plants did not. Additional western blot studies confirmed the expression of significant amounts of the merB gene product, organomercruial lyase. Results suggest that merB was successfully transformed and expressed in Arabidopsis thaliana plants as well as conferring resistance to organomercurials. |
Bizily, S., Rugh, C., Meagher, R. (2000) Phytodetoxification of hazardous organomercurials by genetically engineered plants. Nature Biotechnology. 18:213-217. Methylmercury is found in wetlands and aquatic sediments worldwide. Both ionic mercury and methylmercury are absorbed in the gastrointestinal tract of animals, but methylmercury is retained much longer in the body and is, therefore, is carried up through the food chain more efficiently. Plants engineered with both the merA and merB genes should be able to extract methylmercury from contaminated environments and transpire Hg(0) into the atmosphere. Because Hg(0) resides in the atmosphere for approximately two years, transpired Hg(0) will be diluted to much lower concentrations before being redeposited into terrestrial waters and sediments rather than being concentrated in one area. Additionally the amount of Hg(0) emitted from sites undergoing phytovolitalization can be regulated and will most likely be small in comparison to the concentrations of Hg(0) already in the atmosphere. |
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| Arabidopsis thaliana plants that
had been separately transformed to contain constructs that express merA and
merB, respectively, were crossed. F2 generation plants were analyzed
for expression of both the merA and merB gene products in the same plant.
Plantlets containing merA or merA and merB grew on concentrations of
methylmercury-like compounds (mainly CH3HgCl) up to 5 micromolar. Only
plants expressing the gene products of both merA and merB grew on concentrations
of 10 micromolar methyl mercury. Mercury vapor analysis showed significant
Hg(0) volatilization emitted from merA/merB plants and western blots confirmed
the expression of the gene products of merA and merB. These results
demonstrate that transgenic plants efficiently phytovolatilize methylmercury. |