Role of 2-Oxoglutarate-Dependent Dioxygenases in the Phytoremediation of Polycyclic Aromatic Hydrocarbons (PAHs) Using the Plant Arabidopsis thaliana as a Model System

Nowadays  environmental pollution is an issue that is a major concern.  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 mutagenic and carcinogenic compounds. 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 functions in chemically modifying PAHs.  In our laboratory, we used the 3-ring PAH phenanthrene as a model PAH in physiological stress experiments.  Using bioinformatic tools we identified 52 DOXs in Arabidopsis thaliana.  To further study the function of DOXs in biodegradation, three DOXs cDNAs were cloned, expressed, and purified from Escherichia coli cells.  Current experiments include in vitro enzymatic assays to determine the possible chemical modifications  by these enzymes in phenanthrene.  Preliminary results suggest flavonol synthase, naringenin 3-dioxygenase and, adventitious rooting related oxygenase altered phenanthrene.  Using a genetic approach to study in vivo function, we used “knockout” mutant plant lines of these genes for phenotypic comparison with wild-type plants.  Three mutant lines (Sm_32880, SALK 113321, and RATM 11-1717-1H) showed a hyposensitive response under phenanthrene treatment, suggesting possible roles of these three 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.