Progress 07/01/98 to 06/30/04
Outputs
Phytoremediation has the potential to decontaminate soils more cheaply than chemical extraction procedures. However its effectiveness depends upon the ability of plants to survive as long as possible while they are accumulating wastes or toxic metals. This project has been focused on identifying new genes either to sequester metals such as zinc so that it causes less harm, or to protect plants from the damage that the metal can cause. One part of this work involved assessing the role of the rice gene salT in plants either by expressing it from a constitutive promoter or by reducing its expression with antisense and fold-back RNA. For this project, we have recovered 13 rice lines carrying a constitutively expressed version of salt and grew them to maturity so that they could set seed. Seedlings of each were germinated and transferred sterilely to normal growth medium, or to medium supplemented 3 concentrations of ZnCl2. Approximately 30% of the lines appeared to do
better than their untransformed parent. We were recently able to harvest some seeds from the second selfing of most of these lines. These will be screened for homozygous populations so that we can verify the phenotypes and begin to study the cytological and physiological changes occurring in these lines. Over this same period, we began to screen for the effect on growth of expressing different zinc-binding peptides in yeast. By identifying the effects of zinc on this genetically amenable organism, we planned to find ways to trap zinc in specific organelles and thus protect the most sensitive cellular compartments. Once perfected, we planned subsequently to transfer any constructs that increased cell tolerance to zinc into plants. Although we did not find any peptides that improved tolerance, we did find some that made the yeast more sensitive to zinc. We hope to return to study these further once we have clarified how zinc inhibits cell growth (see below). During the course of these
studies, we found a strain of yeast with a galactose-dependent sensitivity to zinc. Zinc is believed to inhibit many unrelated biochemical processes in yeast, but not all. We believe this novel phenotype may reveal at least one of the targets for zinc and thus indicate one of the pathways that needs improving to increase metal tolerance. We are planning to isolate the mutated gene that gives this strain its conditional phenotype, and any additional genes that can suppress it. Simultaneously, we have also begun characterizing some of the physiological changes caused by zinc. Using GFP targeted to either the cytoplasm or the ER, we have obtained evidence that growth in zinc affects either protein import, or the structure of the ER itself. Future studies will focus on identifying the genetic pathways that might be disrupted under these conditions and whether only import processes are similarly affected.
Impacts
Recent interest in phytoremediation of metal-contaminated soils has stimulated studies into the biochemistry of plant tolerance for toxic contaminates. Tolerance depends in part on repairing the cellular damage that inevitably results from the accumulation of metal ions like zinc that can denature or otherwise inactivate a broad range of proteins. Tolerance can also be improved by chelating ions as they enter each compartment before any damage can ensue. Our initial investigations into how plants coped with excess zinc led to the isolation of two rice genes. We found that the expression of antisense transcripts from each reduced the tolerance of plants to both zinc and salt showing that both genes were involved in an abiotic defense process. More recently we have begun to characterize genes that improve the growth of yeast in the presence of inhibitory levels of zinc. If the genes found prove sufficiently novel, this work could reveal biochemical pathways that might
also be affected by zinc in plants, and that might be improved in future genetic engineering programs.
Publications
- Sripo T, Phongdara A, Wanapu C, Caplan AB. 2002. Screening and characterization of aldehyde dehydrogenase gene from Halomonas salina strain AS11. J Biotechnol 95:171-179
- Iyer, S. and Caplan, A. 1998. Products of proline catabolism can function as pleiotropic effectors in rice. Plant Physiol. 116: 203-211.
- Caplan, A., Berger, Ph.H., and Naderi, M. Phenotypic variation between transgenic plants: What is making gene expression unpredictable? (1998) in Somaclonal Variation and Induced Mutations in Crop Improvement. S. M. Jain, D.S.Brar, and B.S. Ahloowahlia (Eds.), Dorderecht, Kluwer Academic Press. pp. 541-564.
- Garcia, A.-B., de Aleida Engler, J., Claes, B., Villaroel, R., Van Montagu, M., Geerts, T., and Caplan, A. 1998. The expression of the salt-responsive gene salT from rice is regulated by hormonal and developmental cues. Planta 207: 172-180.
- Noventa-Jordao, M. A. , Couto, R. M. , Goldman, M. H. S. , Aguirre, J., Iyer, S., Caplan, A., Terenzi, H. F., and Goldman G. H. 1999. Catalase activity is necessary for heat-shock recovery in Aspergillus nidulans germlings. Microbiology 145 : 3229- 3234.
- Cortese, M., Caplan, A., and Crawford, R. 2002. Structural, functional, and evolutionary analysis of moeZ, a gene encoding an enzyme required for the synthesis of the Pseudomonas metabolite, pyridine-2,6-bis(thiocarboxylic acid). BMC Evolutionary Biology 2: 8-
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Progress 01/01/03 to 12/31/03
Outputs
The goal of this research is to enhance the tolerance of plants to toxic metals such as zinc in order to improve their performance as bioremediative agents. In the pursuit of this goal we are attempting to augment the tolerance of rice and tobacco by overproducing two rice genes that are strongly induced by metal treatments. Secondly, we are attempting to develop metal-chelating proteins targeted to different organelles to trap metals as they come into the cell. Finally, we are attempting to isolate genes that suppress an unusual zinc sensitivity shown by one of the yeast strains with which we have been working. Over the last 3 years, we have generated approximately 20 lines of rice transformed with constitutively expressed sense or antisense copies of the rice salT or rezA genes. The copy number of the transgene in each line has been estimated by Southern analysis and plants are currently being selfed to obtained homozygous plants for physiological assessment. Many
of the transgenic lines do not grow as tall as their wild-type parents. Although this has made studies of growth under sodium- or zinc-stressed conditions more challenging, we believe some show increased sensitivity, but none show enhanced tolerance. Northern analysis and more detailed phenotypic scoring will be done once homozygous lines have been obtained. We have also generated chimeric zinc-binding proteins and are testing their ability to improve the growth of yeast on zinc-containing medium. The effects we have seen so far seem small (as expected), but promising as a starting point for further improvements. However, during the course of these experiments, we also observed that the variety of yeast we have been using became hypersensitive to zinc when grown on medium with galactose as the sole-carbon-source. Other strains of yeast showed equal sensitivity to zinc on all media tested. In order to understand the cause of this zinc sensitivity better, we are currently screening a
yeast library for clones that will suppress the effect.
Impacts
Recent interest in phytoremediation of metal-contaminated soils has stimulated an interest in the biochemistry of plant tolerance for toxic contaminates. Tolerance depends in part on repairing the cellular damage that inevitably results from the accumulation of metal ions like zinc that can denature or otherwise inactivate a broad range of proteins. Tolerance can also be improved by chelating ions as they enter each compartment before any damage can ensue. Our initial investigations into how plants coped with excess zinc led to the isolation of two rice genes. We found that the expression of antisense transcripts from each reduced the tolerance of plants to both zinc and salt showing that both genes were involved in an abiotic defense process.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The goal of this research is to enhance the tolerance of plants to toxic metals such as zinc in order to improve their performance as bioremediative agents. In the pursuit of this goal we are attempting to augment the tolerance of rice and tobacco by overproducing two rice genes that are strongly induced by metal treatments. Secondly, we are attempting to develop metal-chelating proteins targeted to different organelles to trap metals as they come into the cell. Over the last year, we have generated approximately 20 lines of tobacco overexpressing either the rice gene salT or the rice gene rezA. Preliminary experiments were done to determine whether one or either of the rice genes affected tolerance levels of the heterologous host. These studies indicated that some of these lines displayed enhanced root and shoot growth when vegetative cuttings were planted on sterile media containing sublethal doses of either NaCl or ZnCl2. We are currently germinating the F1
generation from the inbred lines in order to perform phenotypic tests on the plants and to continue inbreeding for homozygosity. We have also generated and begun inbreeding a number of rice lines transformed with the same constructs. Basic Southern and northern analysis is underway, prior to phenotypic testing of representative transformants. We have continued working on the isolation of artificial zinc-binding proteins generated in vitro, and screened by expression in yeast. Each of these constructions adds one or more motifs to a protein "backbone" encoding an ER-targeting sequence and the beginning of a P450 gene isolated from Arabidopsis. Fusions of this sequence to GFP have shown that the plant targeting sequence is localized correctly in yeast. We have preliminary evidence showing that adding long, randomly-generated peptides to the C-terminal end of this small peptide inhibits yeast growth slightly. Since this effect is not dependent on the concentration of zinc in the medium,
it seems likely that the inhibition either results from clogging of the ER- import machinery, or from the kinds of deleterious effects associated with misfolded proteins. One of the consequences of this phenomenon has been that when we screen for proteins with multiple insertions of zinc-binding motifs, they tend to be the slower growing colonies and not the faster ones on zinc containing media. We are currently characterizing a number of the clones isolated in this way to improve our understanding of the effects of protein structures on yeast growth. These studies will help us establish better parameters for constructing novel proteins.
Impacts
The development of novel metal chelating plant proteins will be important in several ways. By expressing these proteins in the leaves, we will create a sink for zinc that will ensure that much of the metal entering plants accumulates in the organs most easily harvested and removed from the site. Development of new phytoremediation agents could not only help to restore land for normal uses, but also offer a way to "mine" low grade ores for valuable metals. Second, by expressing these proteins in edible parts of the plant, we could enhance the nutritional value of crops grown in normal soils.
Publications
- Cortese, M., Caplan, A., and Crawford, R. 2002. Structural, functional, and evolutionary analysis of moeZ, a gene encoding an enzyme required for the synthesis of the Pseudomonas metabolite, pyridine-2,6-bis(thiocarboxylic acid). BMC Evolutionary Biology 2: 8
- Sripo T, Phongdara A, Wanapu C, Caplan AB. 2002. Screening and characterization of aldehyde dehydrogenase gene from Halomonas salina strain AS11. J Biotechnol 95:171-179
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Progress 01/01/01 to 12/31/01
Outputs
We have isolated 2 zinc-induced rice genes, salT and rezA. In order to obtain more direct evidence that these genes contribute to zinc tolerance, two constructs were built and introduced into rice. One construct was intended to produce a foldback RNA capable of promoting gene silencing, and the other to produce a simpler antisense RNA. Phenotypic analysis was then performed on seedlings of the 2nd or 3rd generation. To do this, 6-9 sterilely grown seedlings were transferred after 1 week to defined plant growth medium supplemented with 0, 50, 75, or 121 mM NaCl, or with 0, 1.0, 3.0, and 6.0 mM ZnCl2. The length of each seedling was measured after day 1 and day 6 of this treatment and compared with the length of untransformed plants (germinated without hygromycin) maintained in the same way. A statistical analysis of 13 lines showed that 1 line behaved like its untransformed progenitor, 9 showed varying degrees of hypersensitivity to both Zn+2 and Na+, and 3 showed
extreme sensitivity to both ions. Plants belonging to this last class were completely inhibited at 75 mM NaCl and 6 mM ZnCl2 whereas the parental line was only 30-35% inhibited at the same concentrations. One plant line was also obtained with an antisense construct using our other gene, rezA. This line showed the same degree of hypersensitivity to both ions as the previous lines. Attempts to obtain transgenic rice, tobacco, or Arabidopsis lines overproducing either rezA or salT have so far failed. The reasons are not clear, but could indicate that the bacteria have lost an essential virulence function, or that overproduction of the proteins is toxic. Studies are currently underway to test each of these possibilities. In another set of studies, we have constructed novel zinc binding proteins in vitro. To do this, oligonucleotide sequences for five zinc-binding motifs have been synthesized and concatamerized in different combinations with each other. Each library was cloned into a yeast
expression vector (pYES2, Invitrogen, Carlsbad, CA) containing one of two 'protein back-bones'. Each backbone encoded an amino-terminus to direct the finished molecule either to the endoplasmic reticulum (ER) or the cell surface, two compartments likely to contain comparatively few zinc-sensitive proteins essential for cell survival. Out of the first libraries made, 2 P450-based clones were selected based on their colony size on 9 mM ZnCl2-supplemented minimal medium. Strains harboring CT3-1and MT2-2 grew like strains with plasmid pYES: P450 when zinc was absent. All of the strains grew slower when zinc was present, however, in the time that it took the latter strain to form colonies with a diameter of 1 mm, CT3-1 and MT2-2 colonies had grown to 1.4 mm. When the inserts were sequenced, CT3-1 had 1 additional base 5' to the cloning site that shifted the reading frame of 2 copies of 1 of the oligonucleotides. MT2-2 (which came from a mixed ligation) had 2 antisense copies of 1
oligonucleotide followed by 1 sense copy of another ligated into a vector that had lost one base 5' to the cloning site.
Impacts
Both sets of studies are intended to improve the capabilities of current phytoremediative agents. The salT and rezA studies have already proved somewhat surprising because of the difficulties of generating overproducing plants. Further studies of these two genes may reveal much about metal accumulation in rice. The second set of studies may have even broader importance. First, comparisons of failed and successful peptides will clarify the biological requirements for engineering proteins to trap metals. It may eventually be possible to create proteins specific to any metal of economic importance. Second, these peptides may be used to reduce zinc accumulation in living cells and identify some of the processes dependent on it.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
The last year has been spent training new personnel to continue the research initiated in 1999. During the summer of 1999, there was a complete turnover of personnel in the lab. The past year has been spent recruiting replacements and training them. Unfortunately, I was only able to recruit temporary help for my technician position until this October. Each of the temporary replacements needed some training, and the individual finally hired for the technician slot had no experience in molecular biology and has had to be trained. Two untrained graduate students from the PRC were brought into the program in August, however, I learned in May that they had never intended to stay here and had made plans to leave to join boyfriends living elsewhere in the country. In spite of this continual interruption of the research program, some analysis was continued of the transgenic rice lines that had been generated using a construct intended to express antisense or fold-back (gene
silencing-promoting) copies of the rice gene, salT and antisense and over-expressing copies of the rice gene, rezA. The undergraduate student hired to generate several additional lines of rice over-producing either rezA or salT was never successful. In May, 1999, a new student was hired and trained. This student has generated several lines of rice from plants that had regenerated in the presence of hygromycin. However, he was unable to continue with this work once school began in September and so additional confirmatory tests for transformation have not yet been conducted. In the meantime, the previously generated lines were allowed to set seed, so that second generation of hygromycin-resistant progeny could then be tested. Plants with the gene-silencing construct produced a range of phenotypes. A number showed no significant increase in sensitivity to three concentrations of either NaCl or ZnCl2. Others showed heightened sensitivity, generally to both salts, while about 7-8% showed
very much heightened sensitivity to both salts. These latter plants are being grown to determine whether the trait segregates with hygromycin resistance. In addition, these plants will be tested by Southern analysis for the presence of the transgenes and by northern analysis for changes in expression in the targeted genes. Some work has continued on a strain of Halomonas salina that was isolated by a visiting student from Thailand. She has returned home and characterized a gene that she had isolated that encoded an aldehyde dehydrogenase. This bacterium displayed great tolerance to both salt and to a variety of toxic organic compounds and it was hoped that some of the genes responsible for these tolerances could be isolated and introduced into plants to improve their ability to survive in stressful environments. A brief paper is being prepared to describe the preliminary work on this strain.
Impacts
The intent of this work is to determine which processes limit the growth of plants in soils contaminated with toxic metals.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
We have introduced antisense copies of a salt-induced gene called salT and a zinc-induced gene called rezA were introduced into rice. Currently, breeding these lines are be devleoped to obtain homozygous lineages that can be used 1) to determine whether the antisense gene is reducing expression of the endogenous copy, and 2) the effect that this has on growth under a variety of ionic conditions. We have also begun to construct modified forms of a well-studied zinc-binding protein, metallothionein. Whether these modified proteins work better than the natural ones when expressed in yeast and later in rice will be investigated.
Impacts
The intent of this work is to determine which processes limit the growth of plants in soils contaminated with toxic metals.
Publications
- Noventa-Jordao, M. A. , Couto, R. M. , Goldman, M. H. S. , Aguirre, J., Iyer, S., Caplan, A., Terenzi, H. F., and Goldman G. H. 1999. Catalase activity is necessary for heat-shock recovery in Aspergillus nidulans germlings. Microbiology 145 : 3229- 3234.
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Progress 01/01/98 to 12/31/98
Outputs
Our major efforts over the last six months have been to construct a new set of vectors for the introduction of genes into rice. We have introduced a modified copy of a stress-induced gene, salT, into rice using Agrobacterium tumefasciens-mediated transformation. The first generation of plants has been allowed to set seeds which are currently being screened in order to identify likely homozygous lines that can be propagated for large-scale phenotypic testing. In the meantime, the parental lines have been analyzed both by PCR and Southern analysis to confirm that transformation did occur and to estimate transgene number. To date, all lines resistant to the selective agent that we used have proven to contain the expected foreign gene sequences, generally in multiple copies. The copy of salT that has been introduced should act as an anti-sense messenger allowing us to determine whether production of the normal gene is critical for plant survival during exposure to
different levels of toxic metals. In addition, we intend to construct an anti-sense version of another gene, rezA, so that the function of that gene can be determined. We are currently constructing a new vector that will express the sense-form of both salT and rezA under the control of the 2' promoter from the octopine T-DNA so that we can assess whether the production of either of these genes is limiting to growth under stress conditions. During this period, we have re-measured many of the biochemical responses to zinc stress that we looked at previously. We have confirmed that growing rice with 1 mM malic acid enhances the accumulation of zinc in the roots. Using higher levels seems not only to enhance zinc accumulation in the roots, but also to inhibit zinc movement into the leaves. This agrees with current, although so far unproven, hypotheses about the role of malate in facilitating zinc compartmentalization. Future studies will attempt to determine whether the metal is indeed
concentrated in vacuoles when plants are given sufficient malate. Finally, in collaboration with a visiting scientist from Thailand, we have cloned a dehydrogenase gene from salt-tolerant bacteria that may be important for the synthesis of osmoprotective compounds. We are planning to modify the gene and to introduce it into rice in order to assess the effect this has on plant survival under stress.
Impacts
(N/A)
Publications
- No publications reported this period
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