Minisymposium 19: Gene regulation

Abs # 34005: Designing Plants that Accumulate Mercury Aboveground Using an Engineered Root-Specific Expression System

Presenter:	Kim, Tehryung , trkim@phygen.co.kr
Authors	Kim, Tehryung  (A)   Balish, Rebecca S (A)   Heaton, Andrew  (A)   McKinney, Elizabeth  (A)   Dhankher, Om Parkash  (A)   Meagher, Richard B (A)
Affiliations:	(A): University of Georgia

We have been exploring various scenarios using engineered plants to clean environmental pollutants from soil and water. One strategy for the phytoremediation of mercury species involves the electrochemical reduction of Hg(II) to Hg(0) in roots, movement of Hg(0) up the transpiration system to leaves, followed by reoxidation, and trapping of Hg(II) for later harvest. This hyperaccumulation strategy requires that the bacterial mercuric ion reductase gene merA be strongly expressed in all root tissues, but not significantly in above ground organs. We engineered a constitutive Arabidopsis actin 2 gene expression cassette, A2pt, to contain two 25 bp lacO operator sequences from the bacterial lactose operon creating A2pot. We also constructed a gene encoding lacIn, a modified bacterial repressor, with plant nuclear localization signals. The lacIn gene was expressed from a light-induced rubisco small subunit expression cassette, S1pt. Localization of LacI was confirmed by immunolocalization using a LacI-specific antibody. To test the system, β-glucuronidase (GUS) was used as a reporter to determine its expression patterns and levels. GUS expression was limited to root tissue in A2pot:GUS/S1pt:lacIn plants, while it was strongly expressed in all vegetative organs and tissues of A2pot:GUS plants. MUG assay showed that GUS activity was significantly higher in roots (~100 fold) than in leaves of individual plants. The merA gene was then cloned into A2pot to pursue a mercury hyperaccumulation strategy. Immunoblotting with MerA-specific monoclonal antibody also confirmed that MerA activity was significantly higher (~10 fold) in roots than leaves in the presence of S1pt:lacIn, while MerA was high in both leaves and roots of A2pot:merA plants lacking the LacI repressor. These A2pot:merA/S1pt:lacIn plants were highly resistant to mercury. Elemental analysis will provide insight into the hyperaccumulation of mercury in aboveground tissues. This engineered root specific expression system has applications to many areas of plant science and biotechnology including phytoremediation, control of plant root diseases, plant nutrition, and food safety. We plan to construct mercury hyperaccumulator species for field use and phytoremediation.