Exposure to toxic volatile organic compounds (VOCs) continues to pose a significant risk to human health. Halogenated VOCs such as chloroform, carbon tetrachloride (CT) and trichloroethylene (TCE) are among the most common contaminants in water and air. These compounds share a susceptibility to oxidation by mammalian cytochrome P450 2E1. In past NIEHS-funded work, we have demonstrated that TCE and CT are oxidized by axenic poplar cell cultures using a pathway similar to that in mammals, and that poplar trees are able to take up and degrade VOCs. Our work has led to the realization that TCE and CT uptake by wild-type plants is too weak to significantly reduce their concentration in root zone water, motivating a search for plants with increased degradative activity toward VOCs. We have expressed mammalian cytochrome P450 2E1 in plants, achieving greater oxidation of TCE and greater removal of VOCs in several plants, including poplar. Our latest r2E1 aspen clones have up to 132-fold greater metabolism of TCE compared to control aspens. We are presently testing VOC degradation by transgenic poplar to identify superior clones for field-scale study. In the proposed work we will take transgenic poplar to test-bed scale, testing halogenated VOC uptake compared to wild-type by mass balance studies with full size trees. We will also test the ability of transgenic plants to remove VOCs from air. We will also continue analysis of wild-type plant metabolism of halogenated VOCs in poplar using the recently completed poplar genome sequence. Informed by the genome sequence, we will clone the poplar genes that are most similar to the mammalian genes known to be involved in degradation of pollutants. With overexpression of all genes in the pathway for degradation of TCE and other VOCs, we will produce transgenic plants with enhanced ability for remediation of VOCs in water and air. We will also use the genome sequence for analysis of genes that are upregulated in response to pollutants using poplar microarrays. These experiments are expected to yield important clues about which genes are involved in the degradation of pollutants. We expect these experiments to lead to the discovery of useful poplar promoters that will enable expression of detoxifying transgenes only when the substrates are present. New work will focus on the introduction into plants of genes for the degradation of organophosphorus neurotoxins. Mammalian cytochrome P450 isoforms have been shown to degrade organophosphorus compounds. We will use our existing poplar constructs containing CYP3A4 to determine whether chlorpyrifos is taken up and transformed. To optimize the system for degradation of organophosphorous compounds, we will express CYP3A4 and organophosphorus hydrolase, PON1. With our new knowledge of the genes involved in degradation of pollutants, we will be able to design superior plants for phytoremediation.
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