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2006 Progress Report: Developing a Molecular System for Phytoremediation
EPA Grant Number: X832201Center: Donald Danforth Plant Science Center
Center Director: Beachy, Roger N.
Title: Developing a Molecular System for Phytoremediation
Investigators: Beachy, Roger N. , Jez, Joseph M. , Smith, Thomas , Xia, Yiji
Institution: Donald Danforth Plant Science Center
EPA Project Officer: Lasat, Mitch
Project Period: February 1, 2005 through January 31, 2007 (Extended to January 31, 2008)
Project Period Covered by this Report: February 1, 2005 through January 31, 2006
Project Amount: $484,700
RFA: Targeted Research Center (2004)
Research Category: Hazardous Waste/Remediation , Targeted Research
Description:
Objective:This proposal aims to develop environmentally safe technologies that enhance cadmium and zinc accumulation in plants and control the risks associated with transgene flow into nature. The specific objectives are to: (1) engineer glutathione biosynthesis by directed evolution for enhancing cadmium; (2) test the effect of expressing a zinc-binding protein for improving zinc accumulation; (3) develop a fertility control system to eliminate transgene flow; and (4) demonstrate the utility of a chemical gene switch system to control transgene expression in Brassica juncea (Indian mustard).
Progress Summary:In the second year of this project, objectives 1 and 2 successfully focused on the generation of homozygous plant lines expressing proteins that improve cadmium tolerance or zinc accumulation and on the molecular characterization of those lines. For objective 3, transgenic B. juncea with the PCS1 promoter were established and are being examined to determine if the promoter is active at all in other organs and tissues of entire plants and whether the fertility control switch system will be in plants. In objective 4, transgenic plant lines (established during year 1) will be used to determine the optimal ligand concentration for gene induction, the time required for induction, the longevity of the signal, and tissue-specific expression.
Objective 1: Directed Evolution of Cadmium Tolerance
Since phytochelatin peptides also protect plants from exposure to heavy metals, we decided to test if the activity of phytochelatin synthase (PS), which uses glutathione as a substrate to synthesize metal-chelating peptides, could be enhanced by directed evolution. As described in last year’s progress report, we employed in vitro evolution of the Arabidopsis PS (AtPCS) to isolate versions of the enzyme conferring improved cadmium tolerance using a yeast selection system. The AtPCS variant conferring the largest enhancement in cadmium tolerance was used to transform Arabidopsis thaliana. These plants also showed improved cadmium tolerance and accumulation compared to either wild-type Arabidopsis or Arabidopsis transformed with AtPCS.
During the second year of this work, we isolated multiple homozygous lines of B. juncea transformed with either AtPCS or an evolved AtPCS variant. As shown in Figure 1, expression of either enzyme improves cadmium tolerance in B. juncea. More detailed analysis of seedling fresh weight and root length (Table 1) shows improved cadmium tolerance. Likewise, accumulation of the heavy metal is increased in the transgenic plants (Table 1).
Figure 1. Comparison of B. juncea and B. juncea Transformed With Either AtPCS or the Evolved Variant. Seedlings were grown on media containing 150 μM CdCl2.
Table 1. Comparison of B. juncea Plants (n = 30 for each)
|
[CdCl2, μM] |
Wild-Type |
AtPCS |
Evolved AtPCS |
Fresh Weight (mg/Seedling) |
0 |
250-300 |
250-300 |
250-300 |
|
150 |
40-45 |
100-120 |
70-80 |
Root Length (mm) |
0 |
100-120 |
100-120 |
100-120 |
|
150 |
3-4 |
10-15 |
6-7 |
Cadmium (μg per mg FW) |
0 |
0 |
0 |
0 |
|
150 |
6-7 |
18-20 |
12-14 |
Objective 2: Effect of Overexpressing a Zinc-Binding Protein on Zinc Accumulation
This objective focuses on using the metal binding proteins from ATP-binding cassette (ABC)-type metal transporters to increase the metal content in plants. As a proof of concept, we used the zinc-binding protein ZnuA. Our plan was to create transgenic Arabidopsis and tobacco plants that expressed ZnuA and a ZnuA-green fluorescent protein (GFP) fusion. If this was successful, we then would test possible protective effects of these proteins and whether they would facilitate metal accumulation. To this end, we have made progress on a number of fronts described below.
Analysis of Metal Binding to ZnuA. From our structural results, ZnuA has a large flexible loop near the metal binding site that is rich in histidine and acidic residues. We initially thought that this loop might be necessary for the high-affinity binding of zinc to the protein. This past year we deleted the loop, determined the structure of the apo and metal bound forms, and measured the effects of the deletion on metal binding. These results are described in a paper in press in Biochemistry. This deletion, in fact, did not affect metal binding to the high-affinity site but did itself bind zinc with about 100-fold lower activity. This may be the reason why we saw significant zinc accumulation in the transgenic plants—there are several zinc binding sites on the protein.
We now have homozygous plants (tobacco and Arabidopsis) expressing ZnuA alone or the ZnuA/GFP fusion protein. From the T2 lines, it is clear that we are getting a large boost in metal accumulation (Figure 2). We are now characterizing these plants and preparing a manuscript describing these results.
Figure 2. Increased Zinc Content in ZnuA Expressing Plants.
The ZnuA expression protects the transgenic plants against the effects of modest zinc toxicity. The results above clearly demonstrated a boost in metal content. We speculated that this “sink” for zinc would afford modest protection against metal toxicity. Figure 3 suggests that this is indeed the case. This further suggests that such transgenic plants can absorb metals from the environment.
Figure 3. Effects of Transgenic Expression of ZnuA on Zinc Toxicity (0.25mM)
The next step is to demonstrate that ZnuA can bind other toxic metals. The natural candidate is Cd since it is chemically similar to Zn. To this end, we performed isothermal titration calorimetry (ITC) on the mutant ZnuA that does not have the long metal binding loop to look only at the high-affinity site. Indeed, ZnuA does bind Cd2+ (Figure 4). From this analysis, cadmium binds with about 5- to 10-fold weaker binding affinity than zinc but nevertheless still binds very tightly (Ka ~ 5x107 M).
![Figure 4. ITC Analysis of Cd[2+] Binding to ZnuA](https://webarchive.library.unt.edu/eot2008/20081105120555im_/http://es.epa.gov/ncer/progress/images/X832201_06_004.gif)
Figure 4. ITC Analysis of Cd2+ Binding to ZnuA
The final step is to put ZnuA into B. juncea. We now have transgenic plants expressing ZnuA and will continue these studies on this plant with superior bioremediation properties.
Objective 3: Development of a Fertility Control System
The goal of this study is to develop a new molecular system for controlling fertility of transgenic plants to prevent transgene flow via pollen and seeds. To achieve our goal, the promoter of the Arabidopsis PCS1 gene (PCS1p) will be used to express a detrimental gene product (such as barnase) specifically in gametophytic cells and embryonic cells so that the transgenic plants do not produce viable pollen or seeds. For the purpose of plant propagation, a chemical inducible system will be used to restore the fertility of the transgenic plants.
Analyses of the transgenic Arabidopsis plants expressing the PCS1 promoter::β-glucuronidase (GUS) reporter gene have revealed that the promoter is active specifically in developing pollen, ovules, and young embryos. To determine whether the PCS1 promoter can be used to drive expression of a toxic gene for the fertility control system in other plant species, we have transformed the PCS1p::GUS construct into B. juncea via the Agrobacterium-mediated gene delivery system. However, histochemical analyses for GUS expression patterns of the first three B. juncea transgenic lines showed that, in addition to the floral tissues and seeds, two of these lines also exhibited significant GUS activity in other tissues, including leaves and stems. The result on the third line is not clear due to a very low level of GUS expression even in the floral tissue. Although the preliminary results suggest that the PCS1 promoter may not be useful for the fertility control system, in order to get more conclusive results on cell specificity of the PCS1 promoter in B. juncea, we will analyze expression patterns of the reporter gene in 20 additional transgenic B. juncea lines carrying this reporter construct that we have recently generated. If the PCS1 promoter is not suitable for the purpose, an alternative approach is to clone the promoter of the orthologous gene of AtPCS1 from B. juncea which may show tissue specificity in B. juncea similar to that of PCS1 in Arabidopsis.
During our efforts to construct fertility switch constructs, the difficulty of cloning the barnase gene in bacteria and transforming it into plants due to its toxicity and the potential advantage of using a natural plant gene to manipulate fertility, have prompted us to find an alternative approach to making a fertility switch construct. We have previously shown that ectopic expression of the Arabidopsis CDR1 gene, which encodes another aspartic protease, could cause spontaneous cell death. We reason that ectopic expression of CDR1 in gametophytes or embryos may also cause abortion of these tissues, leading to infertility. We have made a construct in which expression of the CDR1 gene is under the control of the PCS1 promoter. T1 Arabidopsis seeds carrying this construct have been obtained. The transgenic lines expressing the PCS1p::CDR1 will be analyzed to determined fertility of these plants.
Objective 4: Characterization of the Gene Switch System in B. juncea
The development of gene control systems that are regulated by small molecules has potential biotechnology applications. The Beachy lab has been testing the use of an ecdysone-inducible system to control gene expression. The goal of this objective is to demonstrate that the ecdysone/methoxyfenozide gene switch system can activate gene expression in roots, leaves, and floral parts of B. juncea.
As the initial step to analyze the gene switch technology, B. juncea transformation was successfully established at the Danforth Center’s Plant Tissue Culture and Transformation core facility. With modifications to published protocols, we produced transgenic T0 plants using constructs described in objectives 1 and 3 and have grown plants to the flowering stage. In recent experiments, we increased the number of plants that rooted in vitro and survived when transplanted to soil. Future experiments are planned to obtain more transgenic lines and to further enhance the transformation system by increasing the rooting and recovery efficiencies and by increasing the transformation efficiency overall.
As reported for year 1, we continued to make progress to confirm that the gene switch system can be successfully applied to control gene expression in a tissue or whole organism, in particular in B. juncea. As proof-of-concept regarding global and tissue-specific gene switch gene regulation in B. juncea, we produced three T-DNA-based gene switch vectors. Each vector contains a uidA (GUS) reporter gene under control of the 5xGm35S promoter. The constitutive promoter from CsVMV (pBA100), the green tissue-specific promoter from the Cab gene (pBA101), and the root-specific promoter from the acidic chitinase gene (pBA106), respectively, control VGE expression. Plant lines carrying each of the target genes have progressed to the adult (soil) stage and T2 and T3 lines have been selected; this activity requires calendar time, but little direct experimental effort on analysis was expended.
In year 1, we found that ligand applied to leaves induces expression of the transgene; however, there is no evidence that ligand applied to the leaves moves systemically, unlike the systemic transport of ligand applied via root drench. During year 2, we conducted a limited number of gene induction experiments with T1 and T2 transgenic plant lines; those that were conducted involved applying the ligand to soil in which transgenic plants were growing. The non-homozygous (T1 and T2) plant lines were induced in a nonuniform manner, as shown in Figure 5. The data indicate that systemic induction of gene expression is not uniform, and may suggest that the ligand is not uniformly distributed in these plant lines. Alternatively, expression of the receptor gene is not uniform in leaves of different ages.
Figure 5. Chemical Gene Switch in B. Juncea. (A) Leaves from different plants of Line 69 (pBA100; CsVMV:VGE) T1 generation, stained with Red-Gal 24 hours after soil drench application of methoxyfenozide. (B) Leaves from different plants of Line 213 (pBA100); T1 generation treated as in A.
During the remaining period of no-cost extension of this grant, we expect to complete the analysis of plant lines produced during years 1 and 2. Analysis will include 6–8 lines each in which the receptor is expressed from the CsVMV, CAB, or AC promoters. Analysis is expected to be completed by the end of August 2007. This will complete this aspect of the project as described in the original proposal.
Journal Articles: 5 Displayed | Download in RIS Format
| Other center views: | All 15 publications | 5 publications in selected types | All 5 journal articles |
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Brendza KM, Haakenson W, Cahoon RE, Hicks LM, Palavalli LH, Chiapelli BJ, McLaird M, McCarter JP, Williams DJ, Hresko MC, Jez JM. Phosphoethanolamine N-methyltransferase (PMT-1) catalyses the first reaction of a new pathway for phosphocholine biosynthesis in Caenorhabditis elegans. The Biochemical Journal 2007;404(3):439-448. |
X832282 (2006) |
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Jez JM. Phosphatidylcholine biosynthesis as a potential target for inhibition of metabolism in parasitic nematodes. Current Enzyme Inhibition 2007;3(2):133-142. |
X832282 (2006) |
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Palavalli LH, Brendza KM, Haakenson W, Cahoon RE, McLaird M, Hicks LM, McCarter JP, Williams DJ, Hresko MC, Jez JM. Defining the role of phosphomethylethanolamine N-methyltransferase from Caenorhabditis elegans in phosphocholine biosynthesis by biochemical and kinetic analysis. Biochemistry 2006;45(19):6056-6065. |
X832282 (2005) X832282 (2006) |
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Romanyuk ND, Rigden DJ, Vatamaniuk OK, Lang A, Cahoon RE, Jez JM, Rea PA. Mutagenic definition of a papain-like catalytic triad, sufficiency of the N-terminal domain for single-site core catalytic enzyme acylation, and C-terminal domain for augmentative metal activation of a eukaryotic phytochelatin synthase. Plant Physiology 2006;141(3):858-869. |
X832201 (2006) X832201 (Final) |
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Wei B, Randich AM, Bhattacharyya-Pakrasi M, Pakrasi HB, Smith TJ. Possible regulatory role for the histidine-rich loop in the zinc transport protein, ZnuA. Biochemistry 2007;46(30):8734-8743. |
X832201 (Final) |
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chemistry, structural biology, soil, heavy metals, contaminants in soil, plant-based remediation, zinc transport, transgene contaminant, transcription factor, transgenic plant, chemical gene switch,
,
Ecosystem Protection/Environmental Exposure & Risk, TREATMENT/CONTROL, Scientific Discipline, Waste, RFA, Aquatic Ecosystems & Estuarine Research, Ecological Risk Assessment, Aquatic Ecosystem, Agricultural Engineering, Ecology and Ecosystems, Treatment Technologies, Bioremediation, remediation, water quality, plant uptake studies, phytoremediation, plant-based remediation, plant mediated contaminants, gene expression patterns, fertility control technology, cadmium, genetic engineering, plant biotechnology, gene transfer, glutahione biosynthesis, biochemistry, genetically engineered plants, metals removal
Progress and Final Reports:
Original Abstract
Final Report