Progress 10/01/99 to 09/30/04
Outputs
It was our goal to elucidate and quantify plant processes affecting and involved in trace metal uptake and sequestration ultimately to improve engineered plant-based bioremediation systems (often referred to as phytoremediation). Experiments have been designed and performed to examine Cd uptake both at the whole plant level and at the level of the purified membrane. For the whole plant studies, we designed and built a system to measure real-time changes in uptake parameters under varied environmental conditions in Brassica napus, a plant species that is known to have a fairly high metal tolerance and is also an important agricultural crop (canola oil). We have grown plants hydroponically and have dealt with typical problems associated with these systems (e.g. algal growth, pump failures). We have set up a monitoring station to track temperature and relative humidity in our growth chamber and have optimized a Cd-specific electrode to measure low concentrations of Cd
during the uptake experiments. Preliminary experiments with plants revealed measurable differences in Cd concentration over the duration of the experiment. In addition we have developed protocols to examine transport rates of Cd using purified tonoplast and plasma membrane vesicles. We were unable to demonstrate any transport through the plasma membrane isolated from rice. Thus we switched to examine Cd transport through the tonoplast (into the vacuole). While Cd sequestration in vacuoles is considered an important detoxification strategy in plants, tonoplast transport mechanisms have so far only been studied in oats (Avena sativa L.). For this work, experiments were conducted with tonoplast vesicles isolated from the roots of rice (Oryza sativa L. var. Kaybonnet) and oat seedlings. We looked for evidence of 109Cd transport with or without energy (ATP) and in the presence of specific chelators, glutathione (GSH) and phytochelatin (PC), that are found in plant cells. The oat root
vesicles did exhibit Cd2+/H+ antiport activity and Cd-PC (n=3) complex transport was present in vesicles isolated from oats grown with calcium sulfate, as has been shown previously. However, Cd-PC complex transport was absent in oats grown with nutrient solution and no Cd-GSH transport was observed in oats from either growing condition. None of these transport processes were evident in any of the rice root tonoplast vesicles. We have continued to investigate what particular nutrient limitations are inducing the Cd-PC transport in oats (when grown with only calcium sulfate) and are in the process of examining whether Cd can induce these transport mechanisms. Kinetic parameters from both of these types of experiments, such as reaction velocities and half saturation constants, will ultimately be used to develop sophisticated models of metal uptake and sequestration.
Impacts
Better models of how plants transport and sequester toxic metals will allow us to make better use of plants in engineered systems. Phytoremediation has the promise of being a very cost-effective solution to environmental pollution.
Publications
- No publications reported this period
|
Progress 01/01/01 to 12/31/01
Outputs
Our long-term goal is to elucidate and quantify plant processes affecting and involved in trace metal uptake and sequestration ultimately to improve engineered plant-based bioremediation systems (often referred to as phytoremediation). Experiments have been designed and begun to examine Cd uptake both at the whole plant level and at the level of the purified membrane. For the whole plant studies, we have designed and built a system to measure real-time changes in uptake parameters under varied environmental conditions in Brassica napus, a plant species that is known to have a fairly high metal tolerance and is also an important agricultural crop (canola oil). We have begun growing plants hydroponically and have dealt with typical problems associated with these systems (e.g. algal growth, pump failures). We have set up a monitoring station to track temperature and relative humidity in our growth chamber and have purchased a Cd-specific electrode to measure low
concentrations of Cd during the uptake experiments. Graduate student Aaron Saathoff has been working to improve the sensitivity and response time of the electrode. In addition we are developing protocols to examine transport rates of Cd using purified tonoplast and plasma membrane vesicles. We were unable to demonstrate any transport through the plasma membrane. Thus we switched to examine Cd transport through the tonoplast (into the vacuole). While Cd sequestration in vacuoles is considered an important detoxification strategy in plants, tonoplast transport mechanisms have so far only been studied in oats (Avena sativa L.). For this work, experiments were conducted with tonoplast vesicles isolated from the roots of rice (Oryza sativa L. var. Kaybonnet) and oat seedlings. We looked for evidence of 109Cd transport with or without energy (ATP) and in the presence of specific chelators, glutathione (GSH) and phytochelatin (PC), that are found in plant cells. The oat root vesicles did
exhibit Cd2+/H+ antiport activity and Cd-PC (n=3) complex transport was present in vesicles isolated from oats grown with calcium sulfate, as has been shown previously. However, Cd-PC complex transport was absent in oats grown with nutrient solution and no Cd-GSH transport was observed in oats from either growing condition. None of these transport processes were evident in any of the rice root tonoplast vesicles. We are currently optimizing techniques for the preparation of vesicles from B. napus. In separate experiments, EDTA was used to fix the amount of free cadmium in solution. These experiments demonstrated that the free Cd in solution was controlling the amount of Cd associated with the vesicles when no Cd transport was occurring. The addition of the detergent Triton X-100 did not lower the amount of Cd associated with the vesicles, indicating that the Cd is non-specifically bound to the vesicles. Kinetic parameters from both of these types of experiments, such as reaction
velocities and half saturation constants, will ultimately be used to develop sophisticated models of metal uptake and sequestration.
Impacts
Better models of how plants transport and sequester toxic metals will allow us to make better use of plants in engineered systems. Phytoremediation has the promise of being a very cost-effective solution to environmental pollution.
Publications
- Holtmeier, N. L. 2001 Cadmium transport in plasma membrane and tonoplast vesicles isolated from rice (Oryza sativa L.) and oat (Avena sativa L.) roots. M. S. Thesis. Cornell University.
|
Progress 01/01/00 to 12/31/00
Outputs
The focus of this project is to elucidate and quantify plant processes affecting and involved in trace metal uptake and sequestration ultimately to improve engineered plant-based bioremediation systems (often referred to as phytoremediation). We have begun projects examining Cd uptake both at the whole plant level and at the level of the purified membrane. For the whole plant studies, we have designed a system to study kinetic uptake parameters in Brassica napus, a plant species that is known to have a fairly high metal tolerance and is also an important agricultural crop (canola oil). We have begun growing plants hydroponically and are currently dealing with typical problems associated with these systems (e.g. algal growth, pump failures). We have set up a monitoring station to track temperature and relative humidity in our growth chamber and have purchased a Cd-specific electrode to measure low concentrations of Cd during the uptake experiments. Calibration of the
electrode is underway. Initial short-term Cd exposure experiments have been done to gather base line data on our plants. Plants were exposed to variable Cd concentrations for 1-8 days after which they were harvested for metal and phytochelatin analysis. As expected, higher concentrations and longer exposure times resulted in greater accumulation of Cd and phytochelatin synthesis. Phytochelatin is a polypeptide synthesized intracellularly to detoxify Cd. In addition we are developing protocols to examine transport rates of Cd using purified plasma membrane vesicles. For these experiments we are initially using rice plants since it is easy to get enough root mass, but ultimately we hope to be able to use Brassica napus in these experiments as well. Originally we had planned to use fluorescent reporter chemical, such as BTC-5N (Molecular Probes) but have recently switched to radioactive isotopes (Cd109) to avoid methodological problems that we were experiencing with the BTC-5N. Currently
we are evaluating the non-specific sorption of the Cd to the purified membrane vesicles and testing washing and filtering techniques. Kinetic parameters from both of these types of experiments, such as reaction velocities and half saturation constants, will ultimately be used to develop sophisticated models of metal uptake and sequestration.
Impacts
Better models of how plants transport and sequester toxic metals will allow us to make better use plants in engineered systems. Phytoremediation has the promise of being a very cost-effective solution to environmental pollution.
Publications
- No publications reported this period
|
Progress 01/01/99 to 12/31/99
Outputs
In preparation for experiments on cadmium transport using isolated plasma membrane vesicles, the aqueous polymer two-phase partitioning technique has been used to purify plasma membrane vesicles from rice roots. As judged by the lack of sensitivity of proton transport to the tonoplast ATPase inhibitor, bafilomycin, and sensitivity to the plasma membrane ATPase inhibitor, vanadate, tonoplast vesicles do not significantly contaminate the preparation.
Impacts
(N/A)
Publications
- No publications reported this period
|
|