Final Report: Using Genetically Engineered Plants to Elucidate Factors Controlling Heavy Metal Tolerance and Sequestration and to Improve Heavy Metal Phytoremediation Efficiency

EPA Grant Number: R827104
Title: Using Genetically Engineered Plants to Elucidate Factors Controlling Heavy Metal Tolerance and Sequestration and to Improve Heavy Metal Phytoremediation Efficiency
Investigators: Pilon-Smits, Elizabeth
Institution: Colorado State University , University of California - Berkeley
EPA Project Officer: Hahn, Intaek
Project Period: July 1, 1999 through June 30, 2001 (Extended to April 14, 2002)
Project Amount: $252,006
RFA: Exploratory Research - Environmental Biology (1998) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Health , Ecosystems


The overall objective of this research project was to elucidate factors that control heavy metal tolerance and sequestration and improve heavy metal phytoremediation efficiency. To meet this objective, we made use of genetically engineered Brassica juncea plants with enhanced levels of metal binding peptides. Three types of transgenic plants were obtained that overexpress different enzymes involved in the production of the metal-binding peptides glutathione and phytochelatins: ATP sulfurylase (APS), glutamylcysteine synthetase (ECS), or glutathione synthetase (GS). The specific objectives of the research project were to: (1) determine which heavy metals are tolerated and/or accumulated best by these transgenic APS, ECS, and GS plants; (2) determine how heavy metals affect plant physiology and biochemistry in transgenics and wildtype plants; and (3) determine the efficiency of transgenic and wildtype plants in removing heavy metal mixtures from polluted soil collected in situ.

Summary/Accomplishments (Outputs/Outcomes):

Metal tolerance was first analyzed at the seedling level, using agar medium spiked with As (III), Cd, Cr, Cu, Hg, Mo, Mn, Pb, Ni, or Zn. All three types of transgenics, APS, ECS, and GS were more tolerant to As, Cd, and Cr. GS plants also were more tolerant to Cu, Hg, Mo, and to a lesser extent, Zn. ECS plants also were more tolerant to Mo, Mn, Ni, and to a lesser extent Cu, Pb, and Zn. APS plants also were more tolerant to Ni, but less tolerant to Mo.

Mature plant tolerance and accumulation were then analyzed using a nutrient film technique hydroponic setup, and a number of selected metals, based on the seedling experiments. The GS and ECS transgenics were treated with As, Cd, Cr, and Mo. The GS plants showed enhanced tolerance to As and Cd, and ECS plants tolerated As, Cd, and Mo better than the wildtype controls. The transgenic plants also showed higher accumulation of these metals. The APS transgenics were tested for tolerance and accumulation of 9 elements. The APS8 plants accumulated Cd, Cr, Cu, and Mo significantly more than wildtype (not As, Hg, Mn, Ni, Zn). Under most treatments, the APS8 plants also contained higher levels of the essential elements Mo, Mg, and S. There were no significant differences in growth under control or metal-treatment conditions.

During our investigation, we found that the transgenic ECS and GS plants contained up to 25-fold higher levels of glutathione (GSH), phytochelatins (PC1, PC2, PC3, and PC4), and total non-protein thiols (NPT) than wildtype, in the presence of arsenic.

Metal-metal interactions were analyzed in seedlings treated with various metals. In general, plant Cu levels decreased after treatment with Cd, Mo, Zn, and Hg, while Mo levels were lowered by Cu, Hg, and Cd. Mg levels were affected by Cu, Hg, Mo, and Zn, while Mn levels were affected by Cu, Hg, Mo, and Zn. Fe levels were affected by Cd and Cu, and Zn levels were reduced by Cd, Mo, Hg, and Cu.

We have done three experiments with the transgenics using contaminated water or soils. In one experiment, a rhizofiltration setup was used in the Leadville Mine Drainage Tunnel. Metal levels in/on the roots were as high as 27 percent of root dry weight (DW) (Fe), 1.2 percent of root DW (Zn), or 0.3 percent of root DW (Mn). Shoot levels of Fe, Zn, and Mn were 100-400 ppm (~1,000-fold higher than in the substrate). The transgenics showed higher shoot levels of Fe, Mn, and Zn, while the root levels of these elements tended to be lower.

In a second experiment, APS8, cytGS7, cpECS2, and wildtype B. juncea were sown on metal-contaminated soil collected from a U.S. Environmental Protection Agency (EPA) Superfund site (Upper Arkansas Fluvial Tailings, site QN). The ECS plants contained significantly higher shoot levels of Cd, Cr, Cu, Pb, and Zn, while the GS plants contained higher levels of Cd and Zn, all compared to wildtype. The APS plants had higher Cd levels, but this was not significantly different from wildtype. Root metal levels and biomass production were not significantly different between all Brassica lines. Importantly, all three types of transgenics removed significantly more of all metals from the soil compared to the wildtype or unplanted control.

In a third experiment, the APS8 and wildtype B. juncea were sown on Serich environmental soil collected from a shale rock formation near Fort Collins, CO. The transgenic APS plants contained significantly (three-fold) higher Se levels in their shoot than wildtype. The APS and wildtype plants grew equally well in the Se soil.

Our research has shown that the overexpression of the enzymes APS, ECS, and GS, all involved in the production of metal-binding peptides, led to greater tolerance and accumulation of several heavy metals, including As, Cd, Cu, Mo, and others. This was found not only using a controlled hydroponic system, but all three types of transgenics removed significantly more metals from environmental soil (Leadville Superfund site) contaminated with a mixture of metals, compared to wildtype plants. Also, the APS transgenics accumulated significantly more Se from an environmental, Se-rich soil.

Factors That Control Heavy Metal Tolerance and Sequestration. The transgenic plants, with enhanced levels of glutathione and phytochelatins, showed enhanced tolerance and accumulation of a wide range of metals and metalloids. This supports the view that these metal-binding peptides play a pivotal role in plant tolerance and accumulation of a variety of different metals.

Relevance for Phytoremediation. These results are significant for phytoremediation because enhanced tolerance and accumulation will enhance a plant's capacity to phytoremediate metals. The multi-metal tolerance and accumulation displayed by these transgenics is especially attractive since most polluted sites are contaminated with multiple metals. These results are especially exciting because this study is the first to show enhanced phytoremediation potential of transgenic plants using aged contaminated soil. These studies also show that hydroponic systems are a valid model to evaluate a plant's phytoremediation potential because corresponding results were obtained using a hydroponic system or environmental soil.

Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other project views: All 8 publications 4 publications in selected types All 4 journal articles
Type Citation Project Document Sources
Journal Article Bennett LE, Burkhead JL, Hale KL, Terry N, Pilon M, Pilon-Smits EAH. Analysis of transgenic Indian mustard plants for phytoremediation of metal-contaminated mine tailings. Journal of Environmental Quality 2003, Volume: 32, Number: 2 (MAR-APR), Page: 432-440. R827104 (Final)
not available
Journal Article Hale KL, McGrath SP, Lombi E, Stack SM, Terry N, Pickering IJ, George GN, Pilon-Smits EAH. Molybdenum sequestration in Brassica species. A role for anthocyanins? Plant Physiology 2001;126(4):1391-1402 R827104 (2001)
R827104 (Final)
not available
Journal Article Hale KL, Tufan HA, Pickering IJ, George GN, Terry N, Pilon M, Pilon-Smits, EAH. Anthocyanins facilitate tungsten accumulation in Brassica. Physiologia Plantarum 2002, Volume: 116, Number: 3 (NOV), Page: 351-358. R827104 (Final)
not available
Journal Article Pilon-Smits EAH, Pilon M. Phytoremediation of metals using transgenic plants. Critical Reviews in Plant Sciences 2002, Volume: 21, Number: 5, Page: 439-456. R827104 (Final)
not available

Supplemental Keywords:

soil, water, metal, remediation, phytoremediation,biology, Colorado, CO, EPA region 8, plant, Indian mustard, Brassica juncea, As, Cd, Cr, Cu, Hg, Mo, Mn, Pb, Ni, Se, W, V, Zn, transgenic, biotechnology, phytochelatin, glutathione, ATP sulfurylase, glutamylcysteine synthetase, glutathione synthetase., Scientific Discipline, Waste, Sustainable Industry/Business, Bioremediation, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, bioengineering, biodegradation, genetically engineered plants, biotechnology, remediation, genetic engineering, plant biotechnology, phytoremediation, bioacummulation, heavy metals

Relevant Websites: Exit

Progress and Final Reports:

Original Abstract
  • 2000
  • 2001 Progress Report