Grantee Research Project Results
Final 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: Aja, Hayley
Project Period: February 1, 2005 through January 31, 2007 (Extended to January 31, 2008)
Project Amount: $484,700
RFA: Targeted Research Center (2004) Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation
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. Our specific aims are as follows: 1) to engineer glutathione biosynthesis by directed evolution for enhancing cadmium; 2) to test the effect of expressing a zinc-binding protein for improving zinc accumulation; 3) to develop a fertility control system to eliminate transgene flow; and 4) to demonstrate the utility of a chemical gene switch system to control transgene expression in Brassica juncea (Indian mustard).
Summary/Accomplishments (Outputs/Outcomes):
Aim 1: Directed Evolution of Cadmium Tolerance
Glutathione and phytochelatin peptides, which are derived from glutathione, play key roles in heavy metal detoxification by chelating heavy metals. The objective of this aim was to determine if directed evolution of the two enzymes, i.e., glutamate-cysteine ligase (GCL) and glutathione synthetase (GS), both of which are responsible for glutathione formation could be used to engineer protein variants that confer improved tolerance to cadmium toxicity. In addition, 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.
Experiments with GCL and GS did not yield significant improvements in heavy metal tolerance in the screening system; however, engineering of PS did. To engineer improved cadmium tolerance, we targeted the PS from Arabidopsis thaliana for in vitro evolution by random mutagenesis. Screening for suppression of cadmium sensitivity in yeast resulted in the isolation of ~20 PS mutants (Fig. 1) that enhanced cadmium tolerance up to 10-fold versus expression of wild-type protein. Yeast expressing the mutant PS showed up to 6-fold higher phytochelatin content than those expressing the wild-type protein, based on HPLC analysis. In addition, cadmium accumulation in the different yeast strains was determined by atomic absorption spectrometry and showed that the mutant lines accumulated 2- to 5-fold more metal than yeast expressing the wild-type protein.
To test if the evolved PS mutants improve metal tolerance in plants, we generated transgenic Arabidopsis using mutant186 and isolated homozygous lines. Seedlings transformed with either an empty vector control or the mutant186 vector were germinated on plates without cadmium, and then transferred to plates with and without cadmium (Fig. 2). Incorporation of the gene was confirmed by PCR and expression of FLAG-tagged protein verified by Western blot analysis. In parallel, we transformed B. juncea with PS and mut186 vectors and isolated multiple homozygous lines of B. juncea transformed with either AtPCS or an evolved AtPCS variant. As shown, in Figure 3, expression of either enzyme improved cadmium tolerance in B. juncea. More detailed analysis of seedling fresh weight and root length shows improved cadmium tolerance (Table 1). Likewise, accumulation of the heavy metal is increased in the transgenic plants (Table 1).
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 |
Conclusion. Although directed evolution of glutathione biosynthesis did not yield improvements in cadmium tolerance, the selection system successfully produced PS variants that improved cadmium tolerance and accumulation in yeast, Arabidopsis, and B. juncea.
Aim 2: Effect of Overexpressing a Zinc-Binding Protein on Zinc Accumulation
This aim focused on using the metal binding proteins from 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-GFP fusion; to test if expressing either protein increased zinc content; and to use site-directed mutagenesis to change metal specificity in this protein family.
We expressed ZnuA and a ZnuA-GFP fusion protein in tobacco and Arabidopsis. In all cases, the transgenic lines have an overall phenotype the same as wild-type plants. Using both RT-PCR and Western blot analysis, we have shown that the plants express the gene constructs. To test plants for enhanced zinc content, we analyzed the zinc and iron content of tobacco plants expressing ZnuA and the ZnuA-GFP fusion protein. Using atomic absorption spectrometry, the iron and zinc content were measured in dried leaf material. To obtain consistent quantification, we measured both the iron and zinc content. The expressed protein does not bind to iron. The ratios of the iron and zinc content in tobacco overexpressing ZnuA-GFP were calculated shown in Fig. 4. The enhanced zinc content is consistent with the expression level observed in Western blots. These numbers should greatly improve once we have created full homozygous plants.
Because it seemed likely that the expression of the GFP-ZnuA fusion, which is larger than ZnuA, would be at a lower level than ZnuA alone, we made a number of ZnuA expression lines. From the data shown in Fig. 5, expression of ZnuA alone enhances zinc content up to 6-fold. Our results clearly demonstrate that we can increase metal content without affecting plant development using these metal-binding proteins.
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 6 suggests that this is indeed the case. This further suggests that such transgenic plants can absorb metals from the environment.
Our third objective was to explore the versatility of the ABC-type transporter metal binding proteins as a scaffold for altering metal specificity. 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. This deletion, in fact, did not affect metal binding to the high affinity site but did itself bind zinc with ~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. 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+ (Fig. 7). From this analysis, cadmium binds with about 5-10 fold weaker binding affinity than zinc but it nevertheless still binds very tightly (Ka ~ 5x107 M).
Conclusion. We now have transgenic plants expressing ZnuA and will continue these studies on this plant with its superior bioremediation properties. This work demonstrates the effectiveness of combining structural and functional studies to engineer a metal-sink system in plants.
Aim 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::GUS reporter gene revealed that the promoter is active specifically in developing pollen, ovules, and young embryos. The result of the histochemical assay of the PCS1 promoter activity is consistent with that of the Northern blotting analysis that showed that endogenous PCS1 transcript was detected only in developing flowers and young seeds. These results indicate the PCS1 promoter is a good candidate for the fertility control system if its expression pattern in other crop species is similar to that in Arabidopsis.
The first construct for the fertility switch system (FCS) we tried to make contains the Barnase gene under the control of the PCS1 promoter. A synthetic gene encoding Barnase with optimized codon usage for plants was made; however, the toxicity of Barnase has limited our ability to clone the gene into a plasmid or to transform it into plants. We then made an alternative FCS construct in which the CDR1 gene is under the control of the PCS1 promoter. CDR1 is an Arabidopsis gene that encodes an aspartic protease and its activation can lead to induction of defense-related genes and eventually cell death. Transgenic Arabidopsis lines containing the PCS1 p:: CDR1 transgene were generated. Among 12 transgenic lines carrying the fusion construct, 7 showed a partial sterility phenotype. Approximately 70% of pollen grains and 40% of embryos carrying the transgene are not viable, whereas ovules of the transgenic lines appeared normal.
Conclusions:
To determine whether the PCS1 promoter can be used to drive expression of a toxic gene for fertility control in other plant species, we have transformed the PCS1p::GUS construct into B. juncea via the Agrobacterium-mediated gene delivery system. Among 36 putative transgenic lines, 7 were confirmed to carry the transgene because they were resistant to the herbicide Basta which is conferred by the Bar gene in T-DNA. The other lines may not be truly transgenic as they were sensitive to Basta. However, analyses of the transgenic B. juncea lines for GUS activity indicate that unlike in Arabidopsis plants, the PCS1 promoter appears active not only in reproductive tissues such as pollens and ovules but also in other tissues such as sepals and petals.
Conclusion. Partial sterility associated with the transgenic Arabidopsis lines expressing PCS1p::CDR1 indicates that establishing such a fertility control system is feasible. However, expressing the PCS1p::CDR1 transgene is not sufficient enough to cause complete sterility and therefore is not ideal for controlling transgene flow. Expressing the barnase gene is more likely to lead to full sterility. Its high toxicity makes it difficult to clone and transform it into plants. This difficulty may be overcome by using the cre-lox system or other techniques. Alternatively, other toxin-encoding genes may be used to eliminate viable pollen or ovules. The PCS1 promoter is apparently active not only in reproductive tissues but also in other tissues in B. juncea. Other promoters specifically active in pollen and ovules need to be identified for fertility engineering in B. juncea.
Aim 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 aim 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 Aims 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 increasing the transformation efficiency over all.
For this aim, 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 promoter is activated when the VGE receptor and methoxyfenozide ligand complex binds to the five GAL4 DNA binding sites. In this series, 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. Each of these T-DNA constructs in a pCAMBIA 2300 backbone has been introduced into B. juncea. Plant lines carrying pBA100 (CsVMV: VGE) have progressed to the adult (soil) stage. All rooted plant lines thus far evaluated to confirm presence of the transgene show integration of the target gene. These plants were screened for stable germline DNA integration by testing the next generation of plants and confirmed this to be the case. In experiments with the transgenic plants, we applied the ligand, methoxyfenozide, to the leaves of a transgenic line and leaf tissues were collected after 24 hrs and stained for GUS activity. Detection of GUS following addition of ligand, but not in absence of ligand (Fig. 8) confirms that the gene switch system functions well in transgenic B. juncea. Plant lines carrying each of the target genes have progressed to the adult (soil) stage and T2 and T3 lines have been obtained.
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 to soil in which plants are grown. We conducted a limited number of gene induction experiments with T1 and T2 transgenic plant lines, including studies that involved applying the ligand to soil. The plant lines tested [non-homozygous (T1 and T2)] s were induced in a non-uniform manner (Fig. 8). 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 age. Similar, non-uniform, gene switch controlled expression occurred in all plant tissues tested including roots, leaves, stems, flowers and siliques.
Conclusion. We have successfully shown that the chemical gene switch technology is applicable to B. juncea: however, addition research may be required to ensure that the desired level of gene expression is achieved using this technology].
Evaluation of Technical Effectiveness
Heavy metal contamination poses health and environmental challenges with a price tag for clean-up using existing technology estimated at $200 billion in the U.S. Although the costs of growing and harvesting a crop are minimal compared to those of soil replacement, no single plant displays all the necessary traits for efficient remediation of hazardous soils. Therefore, understanding how plants protect themselves from metal toxicity and manipulating these processes are crucial for optimizing plants as agents for environmental clean-up. The objectives of this study were to engineer cadmium and zinc tolerance and accumulation, to test a system to limit pollen and seed development, and to examine the feasibility of using chemical gene switch technology in a phytoremediation plant.
During the course of this work, we successfully engineered cadmium and zinc tolerance and accumulation in yeast and plants. Further work is required to fully develop the potential value of these results, including testing in greenhouse and field conditions. Significant progress was achieved in developing a pollen fertility control switch; however, progress was limited by technical difficulties and did not permit completion of this aim. Lastly, the effectiveness of a chemical gene switch was demonstrated in Indian mustard (B. juncea) seedlings.
These experiments set the stage to incorporate each of these technologies into a phytoremediation plant. The results of this research provide fundamental information on the mechanisms by which plants tolerate and accumulate toxic heavy metals. The validation of a chemical gene switch in different plant species represents a step in demonstrating the versatility of this system. In summary, this work is the initial step towards testing new technologies that are applicable for improving the environment through plant biotechnology.
Quality Assurance and Expenditures
All experimental activities, instrumentation use, and evaluation of data have been performed in accord with the quality assurance guidelines specified in the original proposal. Based on our original evaluation criteria, Aims 1 and 2 have been successful, resulting in engineered proteins that improve tolerance to cadmium (Aim 1) and enhance zinc accumulation (Aim 2) in yeast and plants. For these aims, 100% of the work is completed. Aims 3 & 4 are approximately 75% and 75% complete, respectively. Overall, all of the allocated funds for the four aims have been used.
Journal Articles: 2 Displayed | Download in RIS Format
Other center views: | All 4 publications | 2 publications in selected types | All 2 journal articles |
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Type | Citation | ||
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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) |
Exit Exit |
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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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Supplemental Keywords:
chemistry, structural biology, soil, heavy metals, contaminants in soil, plant-based remediation, zinc transport, transgene contaminant, transcription factor, transgenic plant, chemical gene switch,, RFA, Scientific Discipline, Waste, TREATMENT/CONTROL, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Treatment Technologies, Aquatic Ecosystem, Bioremediation, Ecological Risk Assessment, Ecology and Ecosystems, Agricultural Engineering, gene expression patterns, plant-based remediation, fertility control technology, glutahione biosynthesis, genetically engineered plants, plant uptake studies, ecological consequences, gene transfer, genetic engineering, plant biotechnology, remediation, biochemistry, water quality, cadmium, metals removal, phytoremediationProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.