Role of dioxygenases in the phytoremediation of polycyclic aromatic hydrocarbons (PAHs) using the plant Arabidopsis thaliana as a model system

Pollution is an issue with very severe consequences to the environment. Polycyclic aromatic hydrocarbons (PAHs) are a group of contaminants with two or more benzene rings fused together. These structures are very stable and resistant to degradation.   PAHs are considered one of the most toxic compounds with mutagenic and carcinogenic properties. Current  bioremediation strategies use soil bacteria such as Pseudomonas to degrade these contaminants. These bacteria contain enzymes, monooxygenases and dioxygenases (DOXs) that modify PAHs by oxidizing their structure, increasing their reactivity. The  objective of this research is to determine if plant DOXs have  similar ability to detoxify PAHs, specifically phenanthre, a three-ring model PAH. Bioinformatically we identified 52 DOXs genes in Arabidopsis thaliana. Three DOXs cDNAs were cloned and expressed in Escherichia coli BL-21 cells.  After purification in vitro enzymatic assays tested the possibility of these proteins to chemically modify PAHs.  Preliminary results suggest that modifications of phenanthrene took place with Arabidopsis enzymes oxidoreductase and naringenin 3-dioxygenase.  Moreover, we tested in vivo function using “knockout” mutants plant lines of these genes by phenotypic comparison with wild-type plants under PAH exposure.  Two mutant lines (GT_5_94951 and SALK 113321) showed a hyposensitive response under phenanthrene treatment, suggesting possible roles of these two genes in PAH degradation in vivo.  The long-term goal of this project is to over-express such enzymes in plants and use them as a possible strategy in the remediation of contaminated environments.