| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online June 12, 2003; 10.1104/pp.103.020958 Plant Physiology 132:1344-1352 (2003) © 2003 American Society of Plant Biologists Phytoremediation of Organomercurial Compounds via Chloroplast Genetic Engineering1Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, Florida 32816–2360 (O.N.R., H.D.); and Department of Plant and Molecular Biology, University of California, Berkeley, California 94720–3102 (H.S.H., N.T.)
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 µM PMA; plant dry weights of the chloroplast transformed lines were significantly higher than those of wild type at 100, 200, and 400 µM 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.
Mercury (Hg) pollution of soil and water is a world-wide problem (Dean et al., 1972  ;
Krämer and Chardonnens,
2001). The extent to which Hg is harmful depends on the form of
mercury present in the ecosystem. Inorganic forms of Hg are less harmful than
organic forms partly because they bind strongly to the organic components of
soil. For this reason, Hg does not tend to contaminate the ground water except
when it leaches from a municipal landfill
(U.S. Environmental Protection Agency,
1984). Organomercurial compounds, on the other hand, may be 200
times more toxic than inorganic Hg (Patra
and Sharma, 2000) and methyl-Hg is especially toxic
(Meagher and Rugh, 1997).
The principal forms of organomercurial compounds are alkyl mercurials
(methyl- and ethyl-Hg), aryl mercurials (phenyl-Hg), and alkoxy alkyl Hg
diuretics. The excessive use of organomercurial compounds (e.g. in fertilizers
and pesticides) is known to have severe effects on plants. The main site of
action of Hg damage appears to be the chloroplast thylakoid membranes and
photosynthesis. Organomercurial compounds have been shown to strongly inhibit
electron transport, oxygen evolution
(Bernier et al., 1993
Current remediation methods to clean up heavy metal-contaminated soils
include soil flushing, chemical reduction/oxidation and excavation, retrieval,
and offsite disposal, all of which are expensive, environmentally invasive,
and labor intensive (Kärenlampi et
al., 2000
All of the attempts to genetically engineer plants with improved
phytoremediation have previously been based on transformation of the nuclear
genome. An alternative and novel approach is to engineer the chloroplast
genomes of higher plants. This approach offers several advantages over nuclear
transformation, i.e. very high levels of transgene expression (up to 46% (w/w)
of total protein; De Cosa et al.,
2001 This is the first report where the chloroplast genome was engineered to enhance the capacity of plants for phytoremediation and where a native bacterial operon was used for expression in plants without codon optimization. Phenylmercuric acetate (PMA) was chosen to test the chloroplast transformation method because of the importance of toxicity of organomercurial compounds as environmental contaminants and because the site of action of organomercurial damage is the chloroplast (see above). The approach we used was to integrate a native operon containing the merA and merB genes, coding for mercuric ion reductase and organomercurial lyase, respectively, into tobacco (Nicotiana tabacum) chloroplast genomes. The results show that the chloroplast transgenic plants were substantially more resistant than wild type to the highly toxic organomercurial compound, PMA.
Chloroplast Vectors and Bacterial Resistance Assays
The bacterial native genes, merA (1.69 kb) and merB (638
bp) that encode the mercuric ion reductase and the organomercurial lyase,
respectively, were amplified by PCR from Escherichia coli strains
harboring plasmids NR1 (containing the full-length merA) and R831b
(containing the full-length merB). The PCR gene products were
successively cloned into the pLD-vector, which is a chloroplast-specific
vector used in previous publications from this laboratory
(De Cosa et al., 2001
The transformed bacterial cells harboring pLDR-MerAB and pLDR-MerAB-3'-UTR, and the control untransformed cells (E. coli) were grown on Luria-Bertani medium in the presence of different concentrations of mercuric chloride. Bacterial cells containing the pLDR-MerAB and pLDR-MerAB-3'-UTR were able to grow in concentrations of HgCl2 of up to 100 µM on solid agar plates (Fig. 1B). Untransformed E. coli cells were unable to grow even at a concentration of 25 µM. Although transformed cells were able to grow in liquid broth at concentrations of 25 and 50 µM HgCl2, differences in the rate of growth between the clone transformed with the plasmid containing the 3' terminator and the clone that lacked the terminator region were examined (Fig. 1C). It is known from previous studies that the 3'-UTRs in E. coli are engaged in the termination of transcription. The pLDR-MerAB-3'-UTR was expected to grow better in the presence of Hg because, by terminating effectively, more copies of a shorter transcript containing the merAB operon would be made, in contrast to fewer long transcripts in the case of the pLDR-MerAB clone. The Hg bioassay showed that indeed E. coli cells transformed with the pLDR-MerAB-3'-UTR vector resulted in higher bacterial growth when compared with the bacterial cells containing the vector lacking a 3' psbA-UTR (Fig. 1C).
Chloroplast-transgenic plants were obtained as described
(Daniell, 1997 The primer pair 3P and 3M was used to test integration of the transgene cassette into the chloroplast genome at very early stages during the selection process. The 3P primer lands in the native chloroplast genome and the 3M primer lands in the aadA gene that is present within the gene cassette (Fig. 1A). If integration has occurred, a 1.65 kb PCR product should be obtained (Fig. 2A). The untransformed control and the mutants (caused by the spontaneous mutation of the 16S rRNA gene that confers resistance to spectinomycin) did not show any product, confirming that these plants are negative for integration of transgenes (Fig. 2A). The integration of transgenes (aadA, merA, and merB) was further tested by using the 5P/2M primers and PCR analysis. The 5P and 2M primers annealed to the internal region of the aadA and trnA genes, respectively (Fig. 1A). The product size of positive transgenic clones was 3.89 kb, whereas the mutants and untransformed control did not show any PCR product (Fig. 2B). The DNA from full-grown T0 and T1 generation plants was extracted and used for the Southern-blot analysis (Fig. 3). The 0.81 kb flanking sequence probe that hybridizes with the trnI and trnA genes (Fig. 3A) allowed detection of the site-specific integration of the gene cassette into the chloroplast genome. The transformed chloroplast genome digested with BglII restriction enzyme produced a fragment of 7.96 kb (Figs. 1A, and 3, B and C). The untransformed chloroplast genome digested with BglII yielded a 4.47-kb fragment (Fig. 3, A–C).
The flanking sequence probe also showed that homoplasmy of the chloroplast
genomes was achieved through the selection process. Southern blots confirmed
stable integration of foreign genes into all of the chloroplast genomes
confirming homoplasmy. T0 and T1 generation transgenic
plants only showed a single fragment of 7.96 kb. The absence of any detectable
native untransformed chloroplast genomes not only confirmed homoplasmy, but
also facilitated detection of transgene copy numbers in each cell. It is known
that mature leaf cells in tobacco contain about 10,000 copies of chloroplast
genomes per cell (Bendich,
1987 Total RNA from T0 and T1 plants transformed with the pLDR-MerAB-3'-UTR and the pLDR-MerAB was extracted and used to perform the northern-blot analysis with four different probes (the merA, merB, merAB, and aadA probes). The merA probe clearly showed the dicistron containing the merB and merA genes with sizes of 2,332 nucleotides and also a minor transcript for the merA monocistron of 1,694 nucleotides (Fig. 4A). The merB probe showed the merAB dicistron (2,332 nucleotides) plus a less abundant transcript (1,448 nucleotides) containing the aadA and merB genes, and the monocistron corresponding to the merB (638 nucleotides) transcript (Fig. 4B). The merAB probe helped to visualize different transcripts in a single blot, the merB and merA dicistronic transcript (2,332 nucleotides), the merA monocistron (1,694 nucleotides), the aadA and merB dicistron (1,448 nucleotides), and the merB monocistron (638 nucleotides; Fig. 4C). The aadA probe showed transcripts for the dicistron containing the aadA and merB genes and also the aadA monocistron of 810 nucleotides (Fig. 4D). The northern-blot analyses showed that the most abundant transcript is the dicistron (2,332 nucleotides) containing the merA and merB genes. Less abundant transcripts corresponding to the aadA/merB dicistron (1,448 nucleotides), the merA monocistron (1,694 nucleotides), the merB monocistron (638 nucleotides), and to the aadA monocistron (810 nucleotides) were also detected. The high abundance of the merAB dicistron in the pLDR-MerAB or the pLDR-MerAB-3'-UTR plants is an interesting observation. Contrary to the current dogma in the literature, these transcripts were stable even in the absence of a 3'-UTR believed to be required for transcript stability. In addition, there is an indication that processing occurs in between transgenes in transgenic chloroplasts even though no such processing sequences were engineered. Even though all three transgenes are transcribed from a single promoter, no tricistrons containing the aadA, merB, and merA genes were detected. Observed processing between transgenes might be due to recognition of bacterial intergenic sequences by the chloroplast protein synthesis machinery.
When 16-d-old tobacco plants were grown for 14 d in soil containing PMA concentrations of 0, 50, 100, and 200 µM, the merAB seedlings (pLDR-MerAB and pLDR-MerAB-3'-UTR clones) grew well at PMA concentrations up to 100 µM PMA, and survived the highest PMA concentration of 200 µM (Fig. 5). On the other hand, PMA concentrations of 100 and 200 µM PMA were lethal to wild-type plants, which barely survived 50 µM PMA (Fig. 5). There were no significant differences between transgenic lines with or without the 3'-UTR terminator.
The effect of PMA on plant growth was determined by treating 24-d-old tobacco plants with PMA concentrations of 0, 100, 200, 300, and 400 µM in soil and measuring total plant dry weight at each concentration (Fig. 6). The total dry weight of wild-type plants decreased progressively with each increase in PMA from 0 to 400 µM. On the other hand, in the transgenic plants, there was no decrease in total dry weight with increase in PMA concentration until PMA reached 400 µM. Statistical analysis (unpaired t test) showed that the transgenic lines were substantially more resistant than wild type to concentrations of PMA of 100, 200, and 400 µM (Table I). These results indicate clearly that, compared with the wild type, the insertion of merA and merB into the chloroplast genome substantially increased the resistance of the transgenic plants to the toxic effects of PMA. There was no significant difference between the dry weights of the two clones, pLDR-MerAB and pLDR-MerAB-3'-UTR, at each concentration of PMA tested (Fig. 6).
As discussed in the Introduction, previous research has shown that the main site of damage of organomercurial compounds is the chloroplast, and that chlorophyll synthesis, electron transport, and photosynthesis are all seriously affected. Therefore, the overexpression of merA and merB in the chloroplast should reduce the toxic effects of PMA directly on chloroplast function. To test this idea, we treated 15-mm diameter leaf discs excised from wild-type and transgenic plants with 10 µM PMA for 10 d and measured chlorophyll contents (Fig. 7). The results show that without PMA present, chlorophyll concentration did not differ between wild-type and the two transgenic lines. Surprisingly, when PMA was supplied to the leaf discs, the chlorophyll content was markedly increased in the transgenic lines, whereas in the wild type, chlorophyll content was reduced. These results are consistent with the view that PMA exerts a damaging effect on the chloroplasts of wild-type plants as expected, reducing chlorophyll content substantially, and that overexpression of merA and merB in the chloroplast genome appears to increase chloroplast resistance to PMA toxicity. However, because the overexpression of these genes results in an increase in chlorophyll content of the transgenic chloroplasts, it would appear that PMA could in fact stimulate chlorophyll synthesis in some way in these transgenic plants. In this regard, it is of interest that the leaf discs taken from the transgenic plants increased in size over the 10-d experimental period, whereas discs from the wild type decreased in size. Thus, it is possible that the increase in chlorophyll concentration with PMA in the transgenic plants was associated with an increase in chloroplast number and/or size.
Levels of transgene expression in chloroplasts could be further enhanced by
introducing appropriate UTRs instead of the ribosome-binding site (RBS) used
in the present study. For example, we have recently shown that use of the
psbA 5'-UTR instead of RBS resulted in a 500-fold increase in
the expression of human serum albumin in transgenic chloroplasts
(Fernandez-San Millan et al.,
2003
This is the first report on the use of chloroplast transformation using
multigene engineering for the phytoremediation of toxic compounds. Because of
the containment of transgenes and high levels of expression via chloroplast
genomes, the chloroplast transformation approach is highly suitable for
phytoremediation, especially for toxic agents that affect chloroplast
function. Although 3'-UTR is believed to stabilize chloroplast
transcripts and to be essential for transgene expression, it may not be
necessary for transcript stability in the context of a polycistron. Because
there are more than 60 such polycistrons within the chloroplast genome
(Sugita and Sugiura, 1996
Bacterial Plasmids That Contain Organomercurial and Hg Resistance Genes
Host Escherichia coli cells containing plasmids NR1 and R831b were
kindly provided by Dr. Ann Summers (University of Georgia, Athens). These
plasmids contain the mer operon with the complete and functional merA and merB
genes, respectively (Jackson and Summers,
1982
To amplify the merB gene from the native plasmid, a primer pair was designed to have a PstI restriction site followed by a chloroplast and bacterial functional RBS of sequence GGAGG in the 5' primer, followed by a four-nucleotide spacer region upstream of the start codon. This primer had 20-nucleotide homology with the 5' end of the gene and a total of 35 nucleotides. The 3' primer was designed to have 20-nucleotide homology with the 3' end of the gene and a ClaI restriction site. To amplify the merA gene from the native plasmid, a 5' primer was designed to have a ClaI restriction site followed by the RBS sequence and a four-nucleotide spacer region before the start codon and the 20-nucleotide homology with the merA gene. All primer pairs were designed using the QUICKPRI program of the DNASTAR software. Two PCR reactions were done to amplify the merA and the merB genes individually from the plasmid NR1 that contained the complete and functional merA gene and the plasmid R831b that contained the full-length merB gene. The PCR products were cloned into suitable plasmid vectors.
The functional merAB operon was amplified via PCR from the vector pCR2.1-MerAB and a new set of primers was made. The 5' primer was designed to have an EcoRV site, an RBS, a spacer region of four nucleotides (attt) and 20 bases of homology to the merAB operon starting at the start codon (atg). The 3' primer is a simple primer with 20 bases of homology to the 3' end of the operon. After cloning, correct orientation was verified by restriction analyses.
The bacterial clones pLDR-MerAB, pLDR-MerAB-3'-UTR, and the control E. coli XL1-blue cells were grown for 24 h at 37°C in 50 mL of Luria-Bertani broth with concentrations of HgCl2 of 0, 25, and 50 µM. The growth medium was autoclaved and cooled to 40°C before adding HgCl2, and was mixed thoroughly to provide an even concentration throughout the plate or growth medium. The bacterial clones pLDR-MerAB, pLDR-MerAB-3'-UTR, and the untransformed control E. coli cells were plated in solid Luria-Bertani medium containing HgCl2 concentrations of 0, 50, 100, and 500 µM. Plates were incubated for 24 h at 37°C.
The steps involved in the gene delivery by particle bombardment and the
selection process of the transgenic tobacco (Nicotiana tabacum var
Petit Havana) clones were performed essentially as describe by Daniell
(1997
Plant DNA was isolated using the DNeasy Plant Mini kit (Qiagen, Valencia,
CA). The PCR primer pairs 3P-3M and 5P-2M were used to confirm the integration
of the gene cassette into the chloroplast and the presence of the genes of
interest, respectively, essentially as described elsewhere
(Guda et al., 2000
The total plant DNA was obtained from transgenic T0 and
T1 plants as well as from untransformed tobacco plants following
the protocol previously explained (Daniell et al.,
2001a
The RNeasy Mini kit and protocol was used to isolate total RNA from plant tissues (Qiagen). The merA, merB, aadA, and merAB probes were used to probe different RNA blots. The merA probe was made by cutting out the merA gene from the pCR2.1-MerA vector with EcoRI. The merB probe was made by cutting out the merB gene from the pCR2.1-MerB vector with EcoRI. The aadA probe was amplified by PCR from the pLD-ctv vector with a specific primer pair (5'-ccatggcagaagcggtaatcg/3'-aagatttatttgccgactacctt). The merAB probe was made digesting the pCR2.1-MerAB vector with EcoRI. Restriction fragments were cut out and eluted from the gels. The probe-labeling reaction, prehybridization/hybridization steps, membrane washing step, and autoradiography were performed as explained in the Southern-blot section in "Materials and Methods."
Seeds of wild-type tobacco and two transgenic lines (pLDR-MerAB and
pLDR-MerAB-3'-UTR) were surface-sterilized in 7% (w/v) sodium
hypochlorite containing 0.1% (v/v) Tween 20. Seeds were kept on a rocking
platform for 20 min and were rinsed in sterile distilled water at least three
times. Sterilized seeds were transferred to plates containing
one-half-strength Murashige and Skoog medium
(Murashige and Skoog, 1962
To determine the inhibitory concentration of PMA on seedling germination, three different concentrations of PMA were applied to pots containing 16-d-old plants from wild-type and two transgenic lines in three replicates. PMA stock solutions were prepared as 10 mM in dimethyl sulfoxide. Different PMA concentrations (50–200 µM) were added to each pot in 100 mL of one-half-strength Hoagland solution. Control pots received the same volume of Hoagland solution without PMA. All plants were grown in the greenhouse under the same conditions as described above.
Pots of five replicates representing the wild-type and the two transgenic lines (of approximately the same size) were transferred to Poly Vinyl Chloride plastic trays 3 inches high. Different concentrations of PMA (in micromoles) were prepared (100, 200, 300, and 400) using a stock solution of one-half-strength Hoagland solution. For each treatment, a single tray maintained approximately 200 mL (to about one-half of the pot's height) of the PMA-Hoagland's solution. All plants in the same treatment were exposed to exactly the same concentration of PMA. The control tray was filled with one-half-strength Hoagland solution without metal. After about 14 d, plants were harvested, washed thoroughly with distilled water, and the length of the longest root and shoot of the plants were measured. Shoots and roots were separated and dry weights were determined.
Leaf discs were cut out with a cork-borer (15-mm diameter) from the youngest and fully expanded leaves on 3-week-old plants grown in the soil with no PMA. Discs of wild-type and different transgenic plants were placed in petri dishes containing solidified Murashige and Skoog medium (pH 5.7 with no Suc) supplemented with different concentrations of PMA ranging from 0.1 to 1 µM, 10 to 100 µM, and 200 to 500 µM. Plates with no PMA were used as controls. The effect of Hg stress was assessed by the loss of chlorophyll in leaf discs. Leaf discs were collected after 6 d of exposure to PMA. They were immediately extracted in 80% (v/v) chilled acetone for determination of total chlorophyll content following the protocol from Current Protocols in Food Analytical Chemistry Online (http://www.mrw2.interscience.wiley.com).
We thank Dr. Ann Summers (University of Georgia) for providing bacterial strains used in this study. Received January 24, 2003; returned for revision March 4, 2003; accepted April 1, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.020958.
1 This work was supported in part by funding from Chlorogen Inc. (St.
Louis). * Corresponding author; e-mail daniell{at}mail.ucf.edu; fax 407–823–0956.
Begley TP, Walts AE, Walsh CT (1986) Bacterial organomercurial lyase: overproduction, isolation, and characterization. Biochemistry 25: 7186–7192[Medline] Bendich AJ (1987) Why do chloroplasts and mitochondria contain so many copies of their genome? Bioassays 6: 279–282[CrossRef][Web of Science][Medline] Bernier M, Popovic R, Carpentier R (1993) Mercury inhibition of photosystem II. FEBS Lett 32: 19–23[CrossRef] Bizily S, Rugh CC, Meagher RB (2000) Phytoremediation of hazardous organomercurials by genetically engineered plants. Nat Biotechnol 18: 213–217[CrossRef][Web of Science][Medline]
Bizily S, Rugh CC, Summers AO, Meagher RB
(1999) Phytoremediation of methylmercury pollution: merB
expression in Arabidopsis thaliana plants confer resistance to
organomercurial Proc Natl Acad Sci USA
96:
6808–6813 Daniell H (1997) Transformation and foreign gene expression in plants mediated by microprojectile bombardment. Methods Mol Biol 62: 453–488[Medline] Daniell H (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol 20: 581–586[Web of Science][Medline] Daniell H (2003) Medical molecular pharming: expression of antibodies biopharmaceuticals and edible vaccines via the chloroplast genome. In K Vasil, ed, Plant Biotechnology 2002 and Beyond. Kluwer Academic Publisher, Dordrecht, The Netherlands, pp 371–376 Daniell H, Datta R, Varma S, Gray S, Lee SB (1998) Containment of herbicide resistance through genetic engineering of the chloroplast genome. Nat Biotechnol 16: 345–348[CrossRef][Web of Science][Medline] Daniell H, Dhingra A (2002) Multigene engineering: dawn of an exciting new era in biotechnology. Curr Opin Biotechnol 13: 136–171[CrossRef][Web of Science][Medline] Daniell H, Khan MS, Allison L (2002) Milestones in chloroplast genetic engineering: an environmental friendly era in biotechnology. Trends Plant Sci 7: 84–91[CrossRef][Web of Science][Medline] Daniell H, Lee SB, Panchal T, Weibe PO (2001a) Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic chloroplasts. J Mol Biol 311: 1001–1009[CrossRef][Web of Science][Medline] Daniell H, Muthukumar B, Lee SB (2001b) Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection. Curr Genet 39: 109–116[CrossRef][Web of Science][Medline] Daniell H, Parkinson L (2003) Jumping genes and containment. Nat Biotechnol 21: 374–375[CrossRef][Web of Science][Medline] Dean JG, Bosqui FL, Lanouette VH (1972) Removing heavy metals from waste water. Environ Sci Technol 6: 518–522 De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Hyper-expression of the Bt Cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19: 71–74[CrossRef][Web of Science][Medline]
De Gray G, Rajasekaran K, Smith F, Sanford J, Daniell H
(2001) Expression of an antimicrobial peptide via chloroplast
genome control phytopathogenic bacteria and fungi. Plant
Physiol 127:
852–862 Doucleff M, Terry N (2002) Pumping out the arsenic. Nat Biotechnol 20: 1094–1095[Medline] Fernandez-San Millan A, Mingo-Castel A, Miller M, Daniell H (2003) A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol J 1: 71–79 Guda C, Lee SB, Daniell H (2000) Stable expression of biodegradable protein base polymer in tobacco chloroplasts. Plant Cell Rep 19: 257–262[CrossRef] Heaton ACP, Rugh CL, Wang NJ, Meagher RB (1998) Phytoremediation of mercury and methylmercury polluted soils using genetically engineered plants. J Soil Contam 7: 497–509
Jackson WJ, Summers AO (1982) Biochemical
characterization of HgCl2-inducible polypeptides encoded by the mer
operon of plasmid R100. J Bacteriol
151:
962–970 Kärenlampi S, Schat H, Vangronsveld J, Verkleij JAC, Lelie D, Mergeay M, Tervahauta AI (2000) Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils. Environ Pollut 107: 225–231[CrossRef][Medline]
Kota M, Daniell H, Varma S, Garczynski F, Gould F, Moar WJ
(1999) Overexpression of the Bacillus thuringiensis
Cry2A protein in chloroplast confers resistance to plants against susceptible
and Bt-resistant insects. Proc Natl Acad Sci USA
96:
1840–1845 Krämer U, Chardonnens AN (2001) The use of transgenic plants in the bioremediation of soil contaminated with trace elements. Appl Microbiol Biotechnol 55: 661–672[CrossRef][Web of Science][Medline] Kupper H, Kupper F, Spiller M (1996) Environmental relevance of heavy metal substituted chlorophylls using the example of water plants. J Exp Bot 47: 259–266 Lee SB, Kwon S, Park S, Jeong M, Han S, Byun M, Daniell H (2003) Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol Breed 11: 1–13 Lin ZQ, Hansen D, Zayed AM, Terry N (1995) Biological selenium volatilization: method of measurement under field conditions. J Environ Qualt 28(1): 309–315 McBride KE, Svab Z, Schaaf DJ, Hogen PS, Stalker DM, Maliga P (1995) Amplification of a chimeric Bacillus genes in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. BioTechnology 13: 362–365[CrossRef][Medline] Meagher RB (2000) Phytoremediation of toxic elemental and organic pollutants. Curr Opin Plant Biol 3: 153–162[CrossRef][Web of Science][Medline] Meagher RB, Rugh CL (1997) Phytoremediation of heavy metal pollution: ionic and methyl mercury. In Organization for Economic Cooperation and Development Document: Biotechnology for Water Use and Conservation. The Mexico'96 Workshop, Organization for Economic Cooperation and Development, Paris, pp 305–321 Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Planta 15: 473–497[CrossRef] Nies DH (1999) Microbial heavy metal resistance. Appl Microbiol Biotechnol 51: 730–750[CrossRef][Web of Science][Medline] Ogawa HI, Tolle CL, Summers AO (1984) Physical and genetic map of the organomercury resistance (Omr) and inorganic mercury resistance (Hgr) loci of the IncM plasmid R831b. Gene 32: 311–320[CrossRef][Medline] Patra M, Sharma A (2000) Mercury toxicity in plants. Bot Rev 66: 379–422 Prasad DDK, Prasad ARK (1987) Altered o-aminolevulinic acid metabolism by lead and mercury in germination seedlings of bajra (Pennisetum typoideum). J Plant Physiol 127: 241–249 Rinderle SJ, Booth JE, Williams JW (1983) Mercuric reductase from R-plasmid NR1: characterization and mechanistic study. Biochemistry 22: 869–876[CrossRef][Medline] Rugh CL, Senecoff JF, Meagher RB, Merkle SA (1998) Development of transgenic yellow poplar for mercury phytoremediation. Nat Biotechnol 16: 925–928[CrossRef][Web of Science][Medline]
Rugh CL, Wilde HD, Stack NM, Thompson DM, Summers AO,
Meagher RB (1996) Mercuric ion reduction and
resistance in transgenic Arabidopsis thaliana plants expressing a
modified bacterial merA gene. Proc Natl Acad Sci USA
93:
3182–3187 Salt DE, Smith RD, Raskin I (1995) Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. BioTechnology 13: 468–474[CrossRef][Medline] Sen AK, Mondal NG (1987) Salvia natans as the scavenger of Hg (II). Water Air Soil Pollut 34: 439–446 Sinha S, Gupta M, Chandra P (1996) Bioaccumulation and biochemical effects of mercury in the plant Bacopa monnieri (L). Environ. Toxicol Water Qualt 11: 105–112[CrossRef] Staub JM, Garcia B, Graves J, Hajdukiewicz PTJ, Hunter P, Nehra N, Paradkar V, Schlittler M, Carol JA, Spatola L et al. (2000) High-yield production of human therapeutic protein in tobacco chloroplast. Nat Biotechnol 18: 333–338[CrossRef][Web of Science][Medline] Sugita M, Sugiura M (1996) Regulation of genes expression in chloroplast of higher plants. Plant Mol Biol 32: 315–326[CrossRef][Web of Science][Medline] Terry N, Zayed AM, De Souza MP, Tarun AS (2000) Selenium in higher plants. Annu Rev Plant Physiol Plant Mol Biol 51: 401–432[CrossRef][Web of Science] U.S. Environmental Protection Agency (1984) Health effects assessment of mercury. Environmental Criteria and Assessment Office, Cincinnati, OH This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS AND DISCUSSION
