Phytoremediation of organomercurial compounds via chloroplast genetic engineering

Abstract

Mercury (Hg), especially in organic form, is a highly toxic pollutant affecting plants, animals, and man. In plants, the primary target of Hg damage is the chloroplast; Hg inhibits electron transport and photosynthesis. In the present study, chloroplast genetic engineering is used for the first time to our knowledge to enhance the capacity of plants for phytoremediation. This was achieved by integrating a native operon containing the merA and merB genes (without any codon modification), which code for mercuric ion reductase (merA) and organomercurial lyase (merB), respectively, into the chloroplast genome in a single transformation event. Stable integration of the merAB operon into the chloroplast genome resulted in high levels of tolerance to the organomercurial compound, phenylmercuric acetate (PMA) when grown in soil containing up to 400 micro M PMA; plant dry weights of the chloroplast transformed lines were significantly higher than those of wild type at 100, 200, and 400 micro M PMA. That the merAB operon was stably integrated into the chloroplast genome was confirmed by polymerase chain reaction and Southern-blot analyses. Northern-blot analyses revealed stable transcripts that were independent of the presence or absence of a 3'-untranslated region downstream of the coding sequence. The merAB dicistron was the more abundant transcript, but less abundant monocistrons were also observed, showing that specific processing occurs between transgenes. The use of chloroplast transformation to enhance Hg phytoremediation is particularly beneficial because it prevents the escape of transgenes via pollen to related weeds or crops and there is no need for codon optimization to improve transgene expression. Chloroplast transformation may also have application to other metals that affect chloroplast function.

Figure 1.

Bacterial bioassay. A, Schematic representation of the transformed chloroplast genome: The map shows the transgenic chloroplast genome containing the pLDR-MerAB-3′-UTR construct. The site-specific integration between trnI and trnA chloroplast genes is shown by the dotted line, specifying the homologous recombination sequences in the pLDR-MerAB-3′-UTR and pLDR-MerAB. Landing sites for the 3P/3M and 5P/2M primer pairs used in PCR confirmation of integration, and expected sizes of products are shown. BglII restriction digestion sites and the merAB probe used in the Southern-blot analyses are shown. A fragment of 7.96 kb should be produced after restriction digestion of the transgenic chloroplast genome. B, Transformed E. coli grown in 100 μm HgCl₂. i, Transformed E. coli cells containing the vectors pLDR-MerAB; ii, pLDR-MerAB-3′-UTR grown in Luria-Bertani at 100 μm HgCl₂; iii, untransformed control (E. coli). C, Effect of mercuric chloride on E. coli cell proliferation. The transgenic clone pLDR-MerAB and pLDR-MerAB-3′-UTR and the control E. coli cells were grown on liquid Luria-Bertani medium with 25 and 50 μm of HgCl₂ for 24 h at 37°C. The A₆₀₀ was measured.

Figure 2.

PCR analysis of control and putative transformants. A, PCR products (1.65 kb) using 3P/3M primers show integration into the chloroplast genome. Lane 1, Marker; lane 2, pLDR-MerAB transgenic line; lane 3, pLDR-MerAB-3′-UTR transgenic line; lane 4, untransformed wild type. B, PCR products (3.8 kb) using 5P/2M primers confirm merAB integration. Lane 1, Marker; lane 2, pLDR-MerAB transgenic line; lane 3, pLDR-MerAB-3′-UTR transgenic line; lane 4, positive control (pLDR-MerAB plasmid DNA); lane 5, untransformed wild-type tobacco.

Figure 3.

Southern-blot analysis using the flanking sequence probe and the merAB probe. A, The map shows the wild-type chloroplast genome, restriction digestion sites used for Southern-blot analysis, and the 0.81-kb flanking sequence probe. B, Transgenic lines (T₀ generation) for the pLDR-MerAB (lane 2) and the pLDR-MerAB-3′-UTR (lane 3) show the expected size fragment of 7.96 kb; the untransformed control (lane 1) shows the 4.47-kb fragment. C, Lanes 1 and 2, T₁ generation transgenic lines; lane 3, the untransformed control. B and C, The flanking sequence probe was used. D, T₀ transgenic lines, pLDR-MerAB (lane 1), pLDR-MerAB-3′-UTR (lane 2), and their respective T₁ generation transgenic lines (lanes 4 and 5) show the 7.96-kb fragment. Lanes 3 and 6, Untransformed wild type. The merAB probe was used in D.

Figure 4.

Northern-blot analysis. A, The merA probe: transcripts of merAB dicistron (2,332 nucleotides) and the merA monocistron (1,694 nucleotides) are shown by arrows. B, The merB probe: transcripts for the merAB dicistron (2,332 nucleotides), the aadA/merB dicistron (1,448 nucleotides), and the merB monocistron (638 nucleotides) are shown. C, The merAB probe: transcripts of the merAB dicistron (2,332 nucleotides), the merA monocistron (1,694 nucleotides), the aadA/merB dicistron (1,448 nucleotides), and the merB monocistron (638 bp) are shown. D, The aadA probe: transcripts of the aadA/merB dicistron (1,448 nucleotides) and the aadA monocistron (810 nucleotides) are shown. 0, Marker; 1, wild-type, untransformed; 2, pLDR-MerAB transgenic line; 3, pLDR-MerAB-3′-UTR transgenic line.

Figure 5.

Effect of PMA concentration on the growth of wild-type and transgenic lines of tobacco plants. Seeds were germinated in vitro on Murashige and Skoog medium (without Suc and 0.5 g mL^–1 spectinomycin). Seedling plants (10 d from germination) were transferred to a greenhouse and were grown in soil for 6 d. Plants were then treated by adding 200 mL of 0, 50, 100, and 200 μm PMA supplied in Hoagland nutrient solution. Photographs were taken 14 d after treatment. WT, Negative control cv Petit Havana; 5A, pLDR-MerAB transgenic line; 9, pLDR-MerAB-3′-UTR transgenic line.

Figure 6.

Effect of PMA on the total dry weight per plant of 24-d-old wild-type and transgenic tobacco plant lines grown on soil containing 0, 100, 200, 300, and 400 μm PMA for 14 d. WT, Negative control cv Petit Havana; 5A, pLDR-MerAB transgenic line; 9, pLDR-MerAB-3′-UTR transgenic line. se shown, n = 5.

Figure 7.

Effect of PMA on total chlorophyll content (milligrams per gram of dry weight) of 15-mm diameter leaf discs excised from wild-type and transgenic lines of tobacco and treated with 0 and 10 μm PMA for 6 d. WT, Negative control cv Petit Havana; 5A, pLDR-MerAB transgenic line; 9, pLDR-MerAB-3′-UTR transgenic line. se shown, n = 5.