Progress 09/01/02 to 08/31/05
Outputs
In the period represented by this project, work was completed to further our understanding of how plants tolerate toxic levels of aluminum in their environment. Aluminum (Al) toxicity is a global problem that severely limits crop productivity in acid soil environments. We have previously used a molecular genetic approach to identify Arabidopsis mutants that have increased sensitivity to Al in order to identify genes that are required for either Al resistance or tolerance in plants. It is expected that loss of genes required for Al resistance or tolerance will result in mutant plants that are incapable of growth in a mildly Al toxic environment. One of the outlined goals of this project was to identify the als3-1 mutation, since loss-of-function mutations in this gene give profound sensitivity to Al in Arabidopsis, which suggests that the als3 mutation represents a defect in a gene required for Al resistance or tolerance. We successfully used a map-based cloning approach
for isolation of the als3 mutation, which lies in a gene that encodes a previously uncharacterized ABC transporter that is highly homologous to a bacterial metal resistance gene. From our results, it appears that the ALS3 gene product functions to export Al from or prevent accumulation of Al in root cells in order to protect these cells from the toxic effects of Al. This is the first documentation of a factor that is required by plants for tolerance of internalized Al. It appears that ALS3 may be part of a larger and more complex Al tolerance mechanism, since we have recently found that the ALS1 gene also encodes an ABC transporter required for Al tolerance. Surprisingly, the expression pattern for ALS1 is nearly identical to that of ALS3, with each being found prominently in the phloem through the plant. From our analysis, we believe that ALS1 also may comprise a part of a mechanism required for redistribution of Al away from sensitive tissues for sequestration in less sensitive
areas such as leaves. Finally, we successfully identified the gene that the als7-1 mutation lies in, with ALS7 representing a gene that is required for production of the critical plant hormone jasmonic acid. We are excited by this result since this is the first indication that jasmonic acid is required for a plant to properly cope with Al in its environment. By identifying genes that mediate Al tolerance, we are continuing to further our understanding of this critical phenomenon, which will lead to advances in our understanding of how plants respond to Al in their environment and give strategies for engineering plants with greater Al resistance and/or tolerance.
Impacts
The impact of this work is that it serves to elucidate further a complicated and poorly understood phenomenon that has profound negative consequences for global agriculture. By doing this molecular and biochemical dissection of Al tolerance in plants, we are gaining new tools for genetic engineering of agriculturally relevant plants that can grow in Al toxic soils. Although any one individual advance may not give the means to substantially impact how we deal with this problem, the collection of results generated from study of this phenomenon, both from the aspects of Al toxicity and tolerance, will provide new strategies for generating plants that are well suited for growth in an Al toxic environment.
Publications
- Larsen, P.B., Geisler, Matt J.B., Jones, C.A., Williams, K.M., and Cancel, J.D. (2005). ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis. The Plant Journal 41:353-63.
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Progress 01/01/04 to 12/31/04
Outputs
Since the last reporting period, we have published our work regarding the ALS3 mutation in The Plant Journal. This work documented the isolation and characterization of this locus, which encodes an ABC transporter like protein, along with speculation about its role in Al tolerance in plants. We have also recently identified the als1-1 mutation, which gives a strong Al sensitivity phenotype in both roots and shoots of Arabidopsis. This mutation represents an amino acid substitution in a previously uncharacterized ABC transporter that possesses both a hydrophobic membrane-localized region and a cytoplasmic ATPase domain. We have begun to use biochemical and molecular approaches to investigate the role of ALS1 in Al tolerance. One of these approaches involves the use of yeast as a heterologous system for expression of this gene to determine its actual biochemical function. For this analysis, we are assessing whether expression of ALS1 in yeast results in a significant
change in growth under a range of environmental conditions including presence of the Al3+ ion, pH stress, and ion deficiency. We have found that ALS1 expression in yeast confers a profound increase in the sensitivity of yeast to acidic pH. In a pH 4.0 environment, yeast expressing ALS1 grow in a manner that is indistinguishable from wt yeast. In contrast, in a pH 3.0 environment, yeast expressing ALS1 are completely incapable of growth compared to wt, which is unaffected. This suggests that ALS1 is capable of mediating proton import into yeast and that in Arabidopsis, it may play a role in regulation of pH at the root surface, which would be consistent with a reported mechanism of Al resistance associated with rhizosphere alkalinization. We are currently investigating what other ions, including Al3+, ALS1 is capable of transporting using this system. As part of our ongoing work with identifying genes required for growth in an Al toxic environment, we are continuing to isolate new als
mutants in Arabidopsis. Currently, we have identified four more als mutants with profound root growth inhibition only in the presence of Al. We are currently analyzing these mutants both phenotypically and genetically in order to begin work to isolate the genes affected by the respective mutations. We are also in the process of finishing up our work to isolate the als7 mutation, which is incapable of root growth in the presence of levels of Al that have little or no effect on wild type roots. We are very excited about our contributions to this poorly understood abiotic stress response.
Impacts
It is expected that this work will lead to a greater understanding of how plants cope with toxic levels of aluminum in their environment. This is a critical global problem that leads to severe reductions in plant growth. This work should provide strategies for engineering plants that can grow and be productive in Al toxic environments, which should contribute to efforts to improve global agriculture.
Publications
- Larsen, P.B., Geisler, Matt J.B., Jones, C.A., Williams, K.M., and Cancel, J.D. (2005). ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis. The Plant Journal 41:353-63.
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Progress 01/01/03 to 12/31/03
Outputs
Aluminum (Al) toxicity is a global problem that severely limits crop productivity in acid soil environments. Using a molecular genetic approach, Arabidopsis mutants with increased Al sensitivity were isolated with the goal of identifying genes that are required for either Al resistance or tolerance in plants. It is expected that loss of genes required for Al resistance or tolerance will result in mutant plants that are incapable of growth in a mildly Al toxic environment. One mutant, als3, was found to exhibit extreme sensitivity to levels of Al that have little effect on wt Arabidopsis roots, suggesting that the als3 mutation represents a defect in a gene required for Al resistance or tolerance. Map-based cloning of the als3 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter that is highly homologous to a bacterial metal resistance gene. Northern analysis for ALS3 expression revealed that it is found in all organs
examined, which is consistent with the severe Al sensitivity that is displayed throughout the loss-of-function mutant, and that its expression is increased in roots following Al treatment. GUS fusion analysis and in situ hybridization showed that ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicated that ALS3 specifically accumulates at the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter that is required for Al resistance/tolerance and functions to export Al from or prevent accumulation of Al in root cells in order to protect these cells from the toxic effects of Al. We are also currently pursuing a second Arabidopsis mutant, als7, which has increased sensitivity to Al compared to the wild type. We are using a map-based cloning approach to isolate the als7mutation and have localized this mutation to a very narrow
region on chromosome 1. Currently, we are sequencing candidate genes in this area to identify the als7 mutation. Following identification, we will employ biochemical and molecular approaches to determine the role of ALS7 in mediating Al tolerance. This will include overexpression studies to test whether this gene can be used to confer greater increases in Al resistance in plants. Finally, we are beginning to use a map-based cloning approach to isolate the als1 and als5 mutations, both of which also result in significant increases in Al sensitivity compared to the wild type. Identification of these genes will lead to advances in our understanding of how plants respond to Al in their environment.
Impacts
It is expected that this work will lead to a greater understanding of how plants cope with toxic levels of aluminum in their environment. This is a critical global problem that leads to severe reductions in plant growth. This work should provide strategies for engineering plants that can grow and be productive in Al toxic environments, which should contribute to efforts to improve global agriculture.
Publications
- Cancel, J.D., Jones, C.A., Geisler, M.J.B., Williams, K.M. and Larsen, P.B. "ALS3 encodes a phloem-localized ABC transporter required for aluminum tolerance in Arabidopsis," Keystone symposium on Plant Responses to Abiotic Stress, February 19-23, 2004, Santa Fe, NM.
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Progress 01/01/02 to 12/31/02
Outputs
This work, which began on 9/01/02, has so far resulted in the isolation of the first gene demonstrated to be required for aluminum resistance in plants. We have been actively characterizing this gene, ALS3, in order to determine its role in Al tolerance with the ultimate goal of using ALS3 to confer increased Al resistance in crop plants. We have successfully analyzed ALS3 with regard to its expression, protein localization, and potential role in mediating Al tolerance. Currently, we are working to overexpress ALS3 in a tissue specific manner in order to determine if ALS3 will be useful for genetically engineering plants to cope with this important global problem. We are also initiating an effort to isolate the als1 mutation, which we have previously described as being a defect in a gene that is also required for Al tolerance. Work is centering on utilizing a map-based cloning approach for a chromosome walk to the als1 locus. To achieve this, we are attempting to
build a large mapping population for the eventual isolation of the ALS1 gene. Following its isolation, we will employ similar techniques as were used for analysis of ALS3. By identification of these genes, we hope to be able to determine the molecular mechanisms that are responsible for Al tolerance in plants.
Impacts
We expect that this work will provide both a basic understanding of Al tolerance and at the same time give molecular tools for generation of transgenic crops that will be able to grow in Al toxic environments.
Publications
- No publications reported this period
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