Progress 10/01/99 to 09/30/04
Outputs
The main goal of this project was to evaluate the effect of plant root exudate production on toxic metal uptake. We have focused on the effects of phytosiderophore production on Cd uptake in maize plants for most of the duration of this grant but in the last year have expanded this work to look at how the interaction between plants and earthworms might also affect Cd uptake into both the plants and the earthworms. Phytosiderophores are iron chelators excreted by graminaceous plants under conditions of iron limitation, but they also complex other metals including Cd; 2'-deoxmugineic acid (DMA) is the predominant phytosiderophore produced by maize. We implemented a High Pressure Liquid Chromatography (HPLC) technique to measure DMA and developed protocols to grow our plants hydroponically in the absence of bacterial contamination. In our experiments, we found that Cd stress caused Fe deficiency that resulted in greater DMA production by maize roots, but the DMA did not
appear to enhance Cd accumulation. In fact, DMA production appears to protect plants from Cd uptake in our experimental systems. Our findings are contrary to previous studies in which plants that produce more phytosiderophores are found to accumulate more Cd when growing in Cd-contaminated soil. In these experiments, it is likely that the phytosiderophores solubilize the Cd from the soil particles increasing the contact of the Cd and the root tissue. Since phytosiderophores are readily degradable, the microbes at the root surface may liberate the Cd from the complex. Future experiments are planned with the continued use of sterile plants and those with manipulated microbial associations. In order to examine the influence that plant root exudates may exert on metal bioavailability with respect to other organisms present in the soil, microcosm experiments have been performed with barley seedlings and the earthworm Eisenia fetida. We have grown plants and introduced earthworms, both
individually and in combination and have examined the metal content of each following four to five weeks of exposure to variable concentrations of Cd (0 to 150 ppm). Preliminary results suggest that the presence of barley plants decreases Cd accumulation in the worms. While, worms appear to have an opposite effect on the plants: there is increased Cd accumulation in the shoots of barley in the presence of worms. However, no differences were observed in the barley roots. Those experiments were done in a soil with high organic matter content and are being repeated in a soil low in organic matter. The addition of synthetic chelators, EDTA and EDDS (an isomer of EDTA reported to have a faster degradation rate), and the resulting uptake of Cd in worms and barley (separately) were also examined. Both were exposed to chelator concentrations ranging from 0.1mmol/kg soil to 10mmol/kg soil. Preliminary results show that Cd accumulation was enhanced in both worms and plants with increasing EDTA
while Cd concentrations either remained the same or decreased slightly with the addition of EDDS. At the highest concentration both chelators are toxic to the worms.
Impacts
Plants species vary widely in the amount of the toxic metal cadmium accumulated in their tissues for reasons that are not well understood. Since cadmium in plant products (especially those intended for consumption) is undesirable, a better understanding of what factors affect cadmium accumulation will ultimately allow us to make better management decisions to decrease cadmium in our food supply.
Publications
- Hill, K. A., L. W. Lion, and B. A. Ahner. 2002. Reduced Cd accumulation in Zea mays: A protective role for phytosiderophores? Environmental Science and Technology 36:5363-5368.
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Progress 01/01/01 to 12/31/01
Outputs
The main goal of this project is to evaluate the effect of plant root exudate production on toxic metal uptake. We have continued to focus on the effect of phytosiderophore production on Cd uptake in maize plants for the last year. Phytosiderophores are iron chelators excreted by graminaceous plants under conditions of iron limitation, but they also complex other metals including Cd; 2'-deoxmugineic acid (DMA) is the predominant phytosiderophore produced by maize. Previously we had implemented a High Pressure Liquid Chromatography (HPLC) technique to measure DMA and developed protocols to grow our plants hydroponically in the absence of bacterial contamination. In our experiments, we found that Cd stress caused Fe deficiency that resulted in greater DMA production by maize roots, but the DMA did not appear to enhance Cd accumulation. Reviews of our research paper based on Koralie Hill's MS thesis (reported in the 2001 CRIS) pointed out some additional experiments that
would serve to strengthen our paper. Yuko Fokudo, an undergraduate researcher, and Molly Moffee, a first year graduate student, have been working on these experiments as part of an Honors Thesis and MS thesis respectively. In order to make the suggested changes to our experimental protocol, we needed at least a partially purified standard. We collected root washings from a set of Fe-deficient wheat plants and used a published purification protocol to concentrate them from solution. We had mass spectroscopy performed on a fraction of our final product to determine whether we had successfully retrieved the DMA. It appeared we had obtained some but our sample contained large amounts of other salts and impurities. We have been experimenting with other varieties and plants (barley and wheat) and growing techniques to most effectively induce and collect phytosiderophores for yet another large scale attempt to purify these chelators. With the partially purified DMA, we will repeat some of
the early experiments in which we were trying to evaluate the direct effects of DMA on Cd uptake. These experiments will involve exposing the plants to Cd in the presence and absence of chelators (one of which is the DMA). In the original set of experiments we had used root washings from Fe-deficient plants, which include not only phytosiderophores, but potentially many other ligands that could compromise the experimental results. Our partially purified DMA will contain fewer competing ligands and thus provide a more definitive result.
Impacts
Plants species vary widely in the amount of the toxic metal cadmium accumulated in their tissues for reasons that are not well understood. Since cadmium in plant products (especially those intended for consumption) is undesirable, a better understanding of what factors affect cadmium accumulation will ultimately allow us to make better management decisions to decrease cadmium in our food supply.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
The main goal of this project is to evaluate the effect of plant root exudate production on toxic metal uptake. To date we have focused on the effect of phytosiderophore production on Cd uptake in maize plants. Phytosiderophores are iron chelators excreted by graminaceous plants under conditions of iron limitation, but they also complex other metals including Cd. The first step of this project entailed developing a dependable technique to measure these compounds and simultaneously we established protocols and methods to germinate and grow our plants hydroponically in the absence of bacterial contamination. Of the three plant species originally proposed we had the most success with maize so we focused primarily on this species. We are employing an HPLC method from the literature to measure 2'-deoxmugeneic acid (DMA), the predominant phytosiderophore produced by maize. This method involves labeling the compounds of interest with the amine labeling reagent PITC. A
reverse phase column is used to separate the phytosiderophore from other compounds and an absorbance detector is used for quantification. Two types of experiments have been performed to date. We examined the effect of Cd on the rate of DMA release under Fe sufficient and Fe limiting conditions (+/- Fe). For both + and - Fe plants the DMA release rate increased with higher Cd stress. The - Fe plants released more DMA while taking up less Cd than the + Fe plants. In other short-term Cd uptake experiments, addition of DMA to the uptake medium reduced Cd accumulation in root tissue, though plants exposed to Cd in the presence of EDTA (employed as a positive control) contained the least Cd. Collectively, these results indicate that Cd stress causes Fe deficiency that results in greater DMA production by maize roots, but the DMA does not appear to enhance Cd accumulation. It remains to be determined whether phytosiderophores would increase dissolution of Cd from sorbed and mineral solid
phase thereby increasing its bioavailability in soil. In addition, last summer an undergraduate researcher examined sulfhydryl-containing organic ligand exudation. We used existing protocols to measure phytochelatins in root washings as a function of time. Phytochelatin is an intracellular metal-binding peptide but we hypothesized that export of metal-phytochelatin complexe may be important. We were unable to conclusively link observations of dissolved phytochelatin to active export from plant roots.
Impacts
Plants species vary widely in the amount of the toxic metal cadmium accumulated in their tissues for reasons that are not well understood. Since cadmium in plant products (especially those intended for consumption) is undesirable, a better understanding of what factors affect cadmium accumulation will ultimately allow us to make better management decisions to decrease cadmium in our food supply.
Publications
- Hill, K. A. 2000. Examination of the role of the phytosiderophore 2'-deoxymugeneic acid in cadmium accumulation by maize plants. M.S. Thesis, Cornell University.
- Hill, K. A., L. W. Lion, and B. A. Ahner 2000. Examination of the role of the phytosiderophore 2'-deoxymugeneic acid in cadmium accumulation by maize plants. Submitted to Environmental Science and Technology.
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Progress 01/01/99 to 12/31/99
Outputs
Plants synthesize phytochelatins to detoxify metals intracellularly. Previous researchers have demonstrated that they are synthesized by plants in increasing amounts upon exposure to toxic metals such as Cd. This project is aimed at determining whether these compounds could be measured in plant tissues as a bioassay for metal bioavailability in soil. In the laboratory, we have grown lettuce hydroponically in a chemically defined liquid medium designed to maintain trace metal concentrations at environmentally relevant concentrations. In the last year we have tested whether this type of medium truly allows us to manipulate metal availability (thus allowing us to calibrate our bioassay). We have also compared metal uptake and phytochelatin production in plants grown in a typical hydroponic system (continually submerged roots) to those grown in our flowing solution system previously described (intermittent wetting of the roots). Our experimental results suggest that many
metals are not well buffered by EDTA. Plant tissue concentrations of redox active metals, such as Fe, Mn and Cu, are dependent on the total metal in the solution (primarily present as an EDTA complex) and not on the free ion concentration. Even the Cd-EDTA complex appears to be available for plant uptake. Thiol production, on the other hand, appears to be dependent on the free Cd ion concentration in solution which suggests that phytochelatin measurements are a better assay for metal stress than total metal measurements. Metal accumulation and thiol production are also dependent on the hydroponic growth technique used. Intermittent wetting of the roots resulted in higher metal concentrations in nearly all tissue types (new and old leaves, roots) for nearly every metal. Our hypothesis is that the more "soil-like" environment in the flow-through system promoted root hair development and thus more root surface area which resulted in enhanced transport of metals. This suggests we must be
cautious when using hydroponically grown plants to extrapolate to soil-grown plants.
Impacts
(N/A)
Publications
- No publications reported this period
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