http://www.isb.vt.edu/news/2003/artspdf/nov0304.pdf
ISB NEWS REPORT NOVEMBER 2003
1
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HEAVY METAL TOLERANT TRANSGENIC PLANTS
Brian R. Shmaefsky
Heavy metals such as cadmium, chromium, copper, lead, arsenic, nickel, and zinc contaminate urban soils at
many sites throughout the world. Nations that continue to use leaded gasoline find toxic levels of lead in
agricultural areas, making it difficult to raise animals and crops. Other heavy metals accumulate in soil and
water from mining operations, industrial manufacturing facilities, recycling plants, and solid waste disposal
sites. Military munitions are also major worldwide sources of groundwater and soil heavy metal contaminants,
which wind or rain can sometimes disperse great distances from their point of use or disposal.
Traditional methods of removing heavy metals from soil and water are expensive and laborious, and often
disrupt the environment. Contaminated soils can be excavated from the site and placed in a sanitary landfill, at
a cost of approximately $2,000,000 per acre. Not only is this method of heavy metal removal expensive, it may
risk the spread of contaminated soil during removal. Alternatively, heavy metals can be stabilized and somewhat
detoxified in situ using chelators. An example would be the addition of phosphate to soils contaminated
with lead, forming an insoluble pyromorphite compound that remains inert in the soil. The cost of this fixation
method is about half that of excavation. Another alternative is the use of plants to remediate heavy metals from
soils. Phytoremediation can cost less than a quarter of the price of removal or fixation; however, the process
can take much longer to be effective. Excavation and fixation of contaminated soils both require six to nine
months on average for completion; by comparison, phytoextraction can take between 18 and 60 months.
Studies by the U.S. Environmental Protection Agency and the U.S. Military investigated the feasibility of
phytoremediation for heavy metals cleanup1. Their research looked at four mechanisms of heavy metal uptake
by plants: phytoextraction, phytovolatilization, phyto-stabilization, and rhizofiltration.
The process of phytoextraction uses plants to absorb, concentrate, and precipitate heavy metals from soil. The
metals accumulate in plant tissues where they are permanently stored. Plants called hyperaccumulators are
preferred because they take up 100 times the concentration of metals over other plants. The plants are then
discarded or processed to reclaim the metals.
Phytovolatilization is used to extract volatile metals such as mercury and selenium from sludge and soils and
release them through transpiration to the atmosphere as a detoxified vapor.
Phytostabilization is used in sludge, soils, and spoils matrices. Plants are used to stabilize the metals by reducing
water and wind erosion. In addition, the mobility of the contaminants is reduced by either being concentrated
in root tissue, adsorbed onto roots, or precipitated in the root zone. Secretions into the rhizosphere
precipitate the metals and bind them to solid particles in the matrix. The plants also dehydrate the matrix
reducing the bunk needed for disposal. This procedure works well for keeping arsenic, cadmium, and lead from
leaving the contaminated matrix.
Water is cleared of heavy metal contaminants using rhizofiltration. Plants growing in an ex situ or in situ
hydroponics system are used to absorb, concentrate, and precipitate the metals, which remain in the roots. This
technique works best with water tolerant plants having fibrous root systems. Cadmium and lead have been
removed from contaminated water using this technique.
A recent summary of heavy metal phytoremediation concluded that the process could significantly decrease
contamination over traditional methods, producing a 95% reduction in contaminated material disposed in
landfills. However, the method has limitations. Natural plants have limited feasibility for remediation because
of the toxicity of the metals to the plants and other inadequate growing conditions. In addition, because
phytoremediation is confined to the area covered by the depths of the roots, the method is restricted to shallow contamination sites and does not fully prevent the leeching of contaminants into groundwater. Also, complete
remediation is a prolonged procedure because of the slow uptake of metals and small biomass of the plants.
Finally, an increased threat of bioaccumulation occurs if the plants enter the food chain in the ecosystem.
Much of the earlier research on improving phytoremediation focused on finding hyperaccumulating plants. The
research of Charles Rhyne and Sumita Ghosh at Jackson State University in Mississippi illustrates the criteria
for plants regarded as cadmium and lead hyperaccumulators (refer to http://www-esd.lbl.gov/CEB/BEST/
ann_rpt99/inter_9story.html). However, hyperaccumulators specific for particular compounds are difficult to
find, and many of the plants take up the compounds under prescribed conditions that restrict their use in the
field.
Youngsook Lee at the National Research Laboratory for Phytoremediation in Pohang, Korea, devised a strategy
for reducing some of the limitations of heavy metal phytoremediation2. Her team developed transgenic
plants capable of tolerating high levels of accumulated cadmium and lead. These plants take up heavy metals
more rapidly than traditional bioremediation plants, making them potential hyperaccumulators with application
for phytoextraction and rhizofiltration in the field.
Lee observed that certain Saccharomyces cerevisiae, which possess the YCF1, or yeast cadmium factor 1
protein, is known to pump cadmium (Cd(II)) into vacuoles, and tested whether YCF1 would also confer
resistance to lead (Pb(II)). Also known as vacuolar glutathione S-conjugate transporter, YCF1 belongs to the
ATP-binding cassette superfamily2,3. Lee’s team confirmed that YCF1 gene expression permitted S. cerevisiae
to withstand the toxic effects of 3 mM lead (Pb II) and 0.1 mM cadmium (Cd II) concentrations in growth
media. This protection against lead and cadmium toxicity was due to the uptake and storage of the heavy
metals in yeast vacuoles. Next, Lee’s group attempted to determine if YCF1 expression in plants produced the
same results.
Arabidopsis thaliana was investigated as a model for YCF1 expression. First, the YCF1 gene was created
using RT-PCR from YCF1 expressing yeast. For expression in A. thaliana, Lee and colleagues subcloned the
YCF1 gene into two vectors—PBI121 and pCambia1302. To enhance expression in plants, the pCambia1302
vector was cloned with four copies of the CaMV 35S promoter. Agrobacterium tumefaciens was used for
transformation of A. thaliana. Green fluorescent protein tagged to YCF1, used as an expression reporter,
indicated the presence of YCF1 protein in the vacuolar as well as in the plasma membrane of the transformed
Arabidopsis cells.
Lee and coworkers investigated the uptake and sequestering of lead and cadmium in the plants. Transformed A.
thaliana was grown on gravel supplemented with half-concentration Murashige-Skoog agar medium containing
0.75 mM lead or 70 uM cadmium. After three weeks, the plant tissues were analyzed for metal uptake using
atomic absorption spectroscopy. Lee’s findings showed that the transgenic plants were as effective as naturally
occurring hyperaccumulators. Although the transgenic plants accumulated less than two fold higher concentrations
of Cd and Pb compared to wild type, this is likely much less than the hyperaccumulator plants (mentioned
above).
Plants used for metal phytoremediation have few purposes after they have done their work, as the levels of
metal taken up by the tissues may make them unsuitable for agricultural use, although they may have value if
methods for inexpensively reclaiming the metals from the plant tissues are refined. Alternatively, heavy metal
accumulating plants can be incinerated and the ashes disposed, which is much easier than excavating and
disposing the contaminated soil. Although it is highly unlikely heavy metal accumulating plants will ever be
used for food, production of non-toxic crops in heavy metal-contaminated areas may be developed from plants
that exclude heavy metals, as recently described in Plant Physiology by Lee and colleagues5.
References
1. Henry JR. (2000) An overview of the phytoremediation of lead and mercury. A report prepared for the U.S. Environmental
Protection Agency Office of Solid Waste and Emergency Response Technology Innovation Office. http://
www.clu-in.org/download/remed/henry.pdf
2. Song W-Y et al. (2003) Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nature
Biotechnology 21(8): 914-919.
3. Li Z-S et al. (1996) The yeast cadmium factor protein (YCF1) is a vacuolar glutathione s-conjugate pump. Journal
of Biological Chemistry 271(11): 6509-6517.
4. Lu Y-P, Li Z-S, & Rea PA. (1997) AtMPR1 gene of Arabidopsis encodes a glutathione S-conjugate pump: isolation
and functional definition of a plant ATP-binding cassette transporter gene. PNAS 94(15): 8243-8248.
5. Lee J et al. (2003) Functional expression of a bacterial heavy metal transporter in Arabidopsis enhances resistance
to and decreases uptake of heavy metals. Plant Physiol. 133: 589-596.
Brian R. Shmaefsky
Department of Biology and Environmental Sciences
Kingwood College, Kingwood, TX
brian.shmaefsky@nhmccd.edu