Minisymposium 13: Heavy metals

Abs # 26005: Chloroplast genetic engineering facilitates phytoremediation studies and capabilities

Presenter:	Daniell, Henry , daniell@mail.ucf.edu
Authors	Ruiz, Oscar N (A)   Hussein, Hussein S (B)   Terry, Norman  (B)   Daniell, Henry  (A)
Affiliations:	(A): University of Central Florida (B): University of California at Berkeley

Mercury (Hg) is highly toxic and bacteria biomagnify its toxicity through its conversion to methyl-Hg. Organomercurials are neurotoxins easily absorbed into blood and known to harm humans; over 90% of methyl-Hg is absorbed into blood. In plants, the primary target of Hg damage is the chloroplast; Hg inhibits electron transport and photosynthesis. Novel phytoremediation approach via chloroplast genetic engineering was achieved by integrating the native bacterial merA and merB genes, which code for mercuric ion reductase and organomercurial lyase, respectively, into the chloroplast genome in a single transformation event. Stable integration of the merAB operon into the chloroplast genome was confirmed by PCR and Southern blot analyses. Expression of the mer operon resulted in high levels of tolerance to phenylmercuric acetate (PMA) when grown in soil containing up to 400 uM PMA. Northern-blot analyses revealed stable transcripts independently of the presence or absence of a 3’ untranslated region. Chloroplast transgenic lines showed 80% increase in dry weight over wild type (WT) plants when treated with 300 uM PMA, achieving higher dry weight and increasing root length at every concentration tested with either, PMA or HgCl₂. Strikingly, transgenic lines were able to accumulate very high concentrations of Hg in roots; up to 3-fold higher than in the wild type. Total Hg accumulation in roots of transgenic lines was 1780 ug/g, compared to 500 ug/g in WT, and accumulation in the plant shoots was 90 to 100 fold more than in WT.The ability of WT plants to transport Hg to shoots was extremely limited, less than 0.4 ug/g, while in the transgenic lines Hg translocation increase to up to 14.8 ug/g. Transgenic lines treated with 100 uM PMA volatilized Hg[0] at a rate of 6.5 ug Hg[0]/g dry weight / day, removing all available PMA by day 5. The volatilization of Hg[0] is the final step in the detoxification pathway of organic-Hg. The high levels of Hg volatilization observed in chloroplast transgenic lines confirm optimal activity of the chloroplast encoded MerA and MerB enzymes. For the first time, we observed Hg transport to shoots. The high levels of Hg resistance showed by the chloroplast transgenic lines make this system ideal for Hg phytoremediation and possibly other metals that affect chloroplast functions. The work presented here should facilitate studies on the mechanism of Hg uptake/translocation and increase our understanding of phytoremediation.