Transgenic Plants in Mercury Phytoremediation
Mercury is a world wide problem as a result of its many diverse uses in industry.  Many of the problem spots have resulted from accidents during the production, use, or storage of mercury.  Although today there is not a large amount of mercury deposition because of better regulatory controls, much of the mercury contaminated areas have not been reclaimed because of high costs and the extent of the problem.  Mercury has been used in bleaching operations (chlorine production, paper, textiles, etc.), as a catalyst, a pigment for paints, for gold mining, as well as a fungicide and antibacterial agent in seeds and bulbs.  Elemental mercury, Hg (0), can be a problem because it is oxidized to Hg2+ by biological systems and subsequently is leached into wetlands, waterways, and estuaries.  Additionally, mercury can accumulate in animals as methylmercury (CH3-Hg+), dimethylmercury ((CH3)2-Hg) or other organomercury salts.  Organic mercury, produced by some anaerobic bacteria, is 1-2 orders of magnitude more toxic in some eukaryotes, is more likely to biomagnify than ionic mercury, and efficiently permeates biological membranes.  Monomethyl-Hg is responsible for severe neurological degeneration in birds, cats, and humans.
Bacteria and the mer Operon
Bacteria are  more exposed than eukaryotic organisms and need an efficient system to manage metals.  Certain bacteria are capable of pumping metals out of their cell, and or oxidizing, reducing, or modifying the metal ions to less toxic species.  One example is the mer operon.  The mer operon contains genes that sense mercury (merB), transport mercury (merT), sequester mercury to the periplasmic space (merP), and reduce mercury (merA).  MerB is a subset of the mer operon and is capable of catalyzing the breakdown of various forms of organic mercury to Hg2+.  MerB encodes an enzyme, organomercurial lyase, that catalyses the protonolysis of the carbon-mercury bond.  One of the products of this reaction is ionic mercury: Hg2+.
R-CH2-Hg+  ----merB---->R-CH3 + Hg(II)
Hg(II) + NADPH  ----merA---->Hg(0) + NADP+ + H+
Hg (0) (elemental mercury) can be volatilized by the cell.


Ionic Mercury (Hg2+) :
Hypothesis:  In 1996, Rugh et. al. hypothesized that stable transformation of the merA gene in Arabidopsis thaliana would increase tolerance to and volatilization of Hg2+.

Rationale:   Mercury ion reductase is known to reduce ionic mercury to elemental mercury, which is easily volatilized.  Therefore, if the plant was able to reduce ionic mercury, and volatilize the elemental mercury product, it would be capable of tolerating higher concentrations of ionic mercury.

Method (a BRIEF synopsis):  Modified merA (identified as merApe9), constructed with overlap extension-PCR reactions, were transformed into Arabidopsis under the 35s promoter via Agrobacterium.

Results:   merApe9 transgenic plants conferred ionic mercury resistance up to 100mM, a concentration lethal to controls without the transgene.  Volatilization was assayed to measure mercury ion reduction in the Arabidopsis transgenics.  MerApe9 plants volatilized more than controls, and mRNA levels and Hg(0) evolution per mg of seedling correlated linearly.  It was concluded that merA did confer increased resistance to ionic mercury because it was reduced and volatilized as elemental mercury (Hg(0)).


Organic Mercury (R-CH2-Hg+):
Hypothesis: Bizily et. al. hypothesized that Arabidopsis transgenic plants expressing the merB organomercurial lyase from bacteria would increase plant tolerance to organic mercury (CH3-HgCl) and phenylmercuric acetate (PMA) compared with control plants not expressing the  transgene.

Rationale:   merB is capable of breaking down organic mercury to ionic mercury (Hg2+), which is less toxic to the plant.

Method (a BRIEF synopsis):   Similar to the previous investigation of merA, Bizily et. al. transformed a modified merB gene under the control of the 35s promoter by vacuum filtration into Arabidopsis.  The merB expressing plants were selected for on kanamycin containing medium and then on either phenylmercuric acetate (PMA) or CH3-HgCl.  Northern blots revealed the presence of merB transcripts and western blot analysis demonstrated that the protein was translated.

Results:   Organic mercury was found to be several fold more toxic than ionic mercury.  It was hypothesized that organic mercury may disrupt membranes in the mitochondria and the chloroplasts.  Furthermore, there may be chelators capable of sequestering some of the ionic mercury conferring a higher tolerance to Hg2+.


merB/A Transgenics
Hypothesis:  Bizily et. al. tested the hypothesis that the bacterial enzymatic pathway for organic mercury detoxification could
be reconstructed in plants conferring resistance to organic mercury.

Rationale:   merB would breakdown organic mercury to ionic mercury which could then be reduced by merA to elemental mercury that is volatilized from the cell.

Method (a BRIEF synopsis):   Previously transformed A. thaliana plants were crossed and the seeds were collected.  After seed sterilization, resistance to PMA up to 10 mM and Hg2+ were determined for merA, merB, merA/B, and control plants.  Additionally, protein expression and evolution of volatile mercury were measured in each line.

Results:   merA/B plants resisted 10-fold higher concentrations of CH3-HgCl than wild type controls.  Additionally, merA/B transgenic plants had a higher resistance than merB plants that can only breakdown organic mercury but are incapable of reducing ionic mercury.

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