Phytoremediation of Ionic and Methylmercury Pollution Page: 3 of 8
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did not appear to act on mercury were subcloned for strong constitutive plant expression and
transformation into Arabidopsis. Surprisingly, the first 10 transgenic lines expressing two
different ZIP transporters were significantly more resistant to 65 uM mercury in their media than
wild-type. Thus, these genes confer the opposite phenotype to plants from what conferred to
yeast. We believe the mechanism for this unexpected difference is based in the localization of
zinc transporters to multi-membrane endosomal vesicles under conditions of zinc sufficiency.
These transgenic ZIP plants are currently being compared to wild type for the ability to up take,
accumulate aboveground, and volatilize mercury under conditions of zinc limitation.
Transporters that are successful in moving more mercury to aboveground tissues in these assays
will next be tested in the root-specific expression system described next.
In the long run we need to test the expression of these transporters from a strong root-
specific promoter and restrict their expression from tissues where they are not needed. Previous
research on two different root-specific tobacco promoters obtained from other laboratories, as
described in our original EMSP proposal, met with disappointing results. Both tobacco promoters
were weak, not very root specific, and active in only a few root cell types. This resulted in our
developing our own root-specific expression system.
Strong, tissue-specific expression systems are essential tools in phytoremediation as in many
areas of plant biotechnology. We developed a new expression system tool called a "repressor-
operator gene complex" (ROC) that can be used to develop almost any tissue or organ specific
pattern. ROCs have diverse applications in plant biotechnology fields including
phytoremediation, disease resistance, plant nutrition, food safety, and hybrid seed production. To
test this concept we assembled a root-specific ROC. When a modified E. coli lac repressor with a
nuclear localization signal was expressed from a rubisco small subunit expression vector,
Slpt::lacln, LacIn protein was localized to the nuclei of leaf and stem cells, but not to root cells.
A LacIn repressible Arabidopsis actin expression vector pot was assembled containing
upstream bacterial lacO operator sequences, and it was tested using -glucuronidase (GUS) and
mercuric ion reductase (merA) gene reporters. Strong GUS enzyme expression was restricted to
root tissues of A2pot::GUS/Slpt::lacln plants, while GUS activity was high in all vegetative
tissues of plants lacking the repressor. Repression of shoot activity was greater than 1000 fold for
more than 25% of the plants doubly transformed with both constructs. Similarly, MerA was
strongly expressed in the roots, but not the shoots of A2pot::MerA/S1pt::lacln plants, while MerA
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Meagher, Richard. Phytoremediation of Ionic and Methylmercury Pollution, report, January 20, 2006; United States. (https://digital.library.unt.edu/ark:/67531/metadc880224/m1/3/: accessed July 18, 2019), University of North Texas Libraries, Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.