Progress 10/01/03 to 09/30/07
Outputs
OUTPUTS: The main output of this project was the isolation of two mutant Arabidopsis lines that carry suppressor mutations that modifiy the cell division defects that are caused by the anp2/anp3 double-mutant background. We have described these genetic resources to colleagues at conferences and at on-campus seminars.
PARTICIPANTS: Maria Cristina Suarez-Rodriguez. Graduate Student in the Plant Breeding and Genetics Program. Maria Cristina graduated with her PhD in August of 2007.
TARGET AUDIENCES: The target audience for this research was research scientists who are working on understanding the mechanisms of signal transduction in plants, specifically those interested in the model plant Arabidopsis thaliana.
Impacts
The overall goal of this four-year research project was to investigate the functions of a group of three MAPKKK genes in Arabidopsis named ANP1, ANP2, and ANP3. We had previously shown that anp2/anp3 double-mutant plants are defective in their ability to perform cytokinesis. This observation indicated that the ANP genes participate in a signal transduction pathway that carries information relating to the control of cell division. Because the control of cell division is of fundamental importance to the growth and development of plants, it is of interest to characterize the genetic pathway(s) in which the ANP genes participate. In particular, we wanted to determine the identity of upstream and downstream signaling components through which the information relating to the control of cell division flows. To experimentally address this question we performed an extragenic suppressor/enhancer screen to identify genes that modify the anp2/anp3 phenotype. Such loci would
potentially participate in the same pathway as the ANP genes. For this study we used an activation-tagging approach to identify potential suppressor mutations. We initially identified ten potential suppressor lines, but following more careful analysis of these lines we narrowed our focus to two independent mutant lines that showed the strongest level of phenotypic suppression. After extensive molecular/genetic analysis we determined that the suppressor loci in these lines were not T-DNA tagged. This situation made the process of identifying the mutant loci responsible for the suppressor phenotypes much more difficult than if the loci were T-DNA tagged. In order to determine which loci are responsible for these suppressor phenotypes, it will be necessary to perform map-based cloning. In order to do this, a number of additional experimental steps needed to be taken. To begin with, we needed to obtain T-DNA mutant alleles of the anp2 and anp3 loci in a different genetic background from
the Ws background used for identifying the suppressor loci. We were able to find two such alleles in the SALK T-DNA collection, and have constructed anp2/anp3 double-mutant lines in the Columbia ecotype. These plants display the same cytokinesis and dwarf-plant phenotypes observed for the anp2/anp3 combination in the Ws background. The next step in the map-based cloning procedure is then to cross the suppressor lines to the Columbia anp2/anp3 mutant background and obtain a population of progeny that is segregating the suppressor loci in the hybrid Columbia/Ws background. We performed the crosses between the respective lines for this experiment and are now at the stage where phenotype screening of the segregating population should be possible in order to identify individuals showing the suppressor phenotype. Since the funding for this project has expired, the project is currently "on hold" until personnel and funding become available to enable us to move forward. The next steps in the
project will be to try and identify a specific region of the chromosome that co-segregates with the suppressor phenotype, and from there identify the genetic lesion responsible for the observed phenotype.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
This project is investigating the functions of a group of three MAPKKK genes in Arabidopsis named ANP1, ANP2, and ANP3. We have previously shown that anp2/anp3 double-mutant plants are defective in their ability to perform cytokinesis. This observation indicates that the ANP genes participate in a signal transduction pathway that carries information relating to the control of cell division. Because the control of cell division is of fundamental importance to the growth and development of plants, it is of interest to characterize the genetic pathway(s) in which the ANP genes participate. In particular, we would like to determine the identity of upstream and downstream signaling components through which the information relating to the control of cell division flows. To experimentally address this question we performed an extragenic suppressor/enhancer screen to identify genes that modify the anp2/anp3 phenotype. Suppressors or enhancers of anp2/anp3 will be candidates
for loci that participate in either the same pathway as the ANP genes, or in pathways that interact with the ANP pathway. As previously described, we used an activation-tagging approach to identify potential suppressor mutations. We initially identified ten potential suppressor lines, but following more careful analysis of these lines we have narrowed our focus to two independent mutant lines that show the strongest level of phenotypic suppression. Because our initial analysis has indicated that the suppressor loci in these lines are not T-DNA tagged, we are currently in the process of setting up mapping populations in order to pursue map-based cloning of the loci responsible for the suppressor phenotype. In order to do this we are first generating anp2/anp3 double-mutant plants in the Columbia ecotype. SALK T-DNA lines are available for each of these loci and we have done the genetic crosses to make the Columbia double-mutant. We have now completed the generation of anp2/anp3
double-mutant plants carrying the Salk alleles of these two genes in the Columbia ecotype background. We have performed phenotypic analysis of this Columbia ecotype double-mutant and have confirmed that the cell division phenotype and dwarf plant phenotype observed in the Ws background also occurs in Columbia to a similar degree of severity. This situation will make mapping the suppressor mutations more straightforward since there do not appear to be any ecotype-specific modifiers of the anp2/anp3 phenotype. We have also crossed two of our anp2/anp3 suppressor lines in the Ws ecotype with the Columbia anp2/anp3 lines and are now in the process of generating a mapping population so that we can pursue map-based cloning of the two suppressor alleles that we generated in our original screen. Over the course of the past year we also performed in-gel kinase assays to measure downstream MAP kinase activation in the anp2/anp3 double-mutant background. These experiments revealed the normal
patterns of MAP kinase activation are observed in the anp2/anp3 plants in response to UV-stress and wounding.
Impacts
The work that we are performing should improve our understanding of the signal transduction pathways that make use of the ANP proteins in Arabidopsis. This information will provide plant scientists with a foundation for understanding related signaling pathways in all crop species. Because the ANP pathways seem to be involved in plant division as well as stress responses, it is likely that this fundamental information will have practical applications in the future.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
This project is investigating the functions of a group of three MAPKKK genes in Arabidopsis named ANP1, ANP2, and ANP3. We have previously shown that anp2/anp3 double-mutant plants are defective in their ability to perform cytokinesis. This observation indicates that the ANP genes participate in a signal transduction pathway that carries information relating to the control of cell division. Because the control of cell division is of fundamental importance to the growth and development of plants, it is of interest to characterize the genetic pathway(s) in which the ANP genes participate. In particular, we would like to determine the identity of upstream and downstream signaling components through which the information relating to the control of cell division flows. To experimentally address this question we performed an extragenic suppressor/enhancer screen to identify genes that modify the anp2/anp3 phenotype. Suppressors or enhancers of anp2/anp3 will be candidates
for loci that participate in either the same pathway as the ANP genes, or in pathways that interact with the ANP pathway. As previously described, we used an activation-tagging approach to identify potential suppressor mutations. We initially identified ten potential suppressor lines, but following more careful analysis of these lines we have narrowed our focus to two independent mutant lines that show the strongest level of phenotypic suppression. Because our initial analysis has indicated that the suppressor loci in these lines are not T-DNA tagged, we are currently in the process of setting up mapping populations in order to pursue map-based cloning of the loci responsible for the suppressor phenotype. In order to do this, we are first generating anp2/anp3 double-mutant plants in the Columbia ecotype. SALK T-DNA lines are available for each of these loci and we have done the genetic crosses to make the Columbia double-mutant. Once these lines have been characterized, we will be
able to cross our two lines carrying suppressor mutations (which are in the Ws ecotype) with the Columbia double-mutant lines in order to perform map-based cloning. A large database of polymorphisms between these ecotypes is available on the web. This should make the map-based cloning project fairly straightforward. We are also pursuing one additional line of experiments to investigate another possible function of the ANP proteins independent of cell division. In particular, we are using an in-gel kinase assay to determine if the double-mutants are impaired in their ability to activate down-stream MAP kinase proteins in response to oxidative stress. These experiments are being performed to test a model that has been proposed in the literature, but for which no direct experimental data has been collected. We have both the genetic and biochemical tools available in the lab to directly test this model, and we plan on completing those experiments within the next three months.
Impacts
The work that we are performing should improve our understanding of the signal transduction pathways that make use of the ANP proteins in Arabidopsis. This information will provide plant scientists with a foundation for understanding related signaling pathways in all crop species. Because the ANP pathways seem to be involved in plant division as well as stress responses, it is likely that this fundamental information will have practical applications in the future.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
This project is investigating the functions of a group of three MAPKKK genes in Arabidopsis named ANP1, ANP2, and ANP3. We have previously shown that anp2/anp3 double-mutant plants are defective in their ability to perform cytokinesis. This observation indicates that the ANP genes participate in a signal transduction pathway that carries information relating to the control of cell division. Because the control of cell division is of fundamental importance to the growth and development of plants, it is of interest to characterize the genetic pathway(s) in which the ANP genes participate. In particular, we would like to determine the identity of upstream and downstream signaling components through which the information relating to the control of cell division flows. To experimentally address this question we performed an extragenic suppressor/enhancer screen to identify genes that modify the anp2/anp3 phenotype. Suppressors or enhancers of anp2/anp3 will be candidates
for loci that participate in either the same pathway as the ANP genes, or in pathways that interact with the ANP pathway. In either case, the information that is gathered from such a screen would improve our understanding of the signaling pathways that control cytokinesis in Arabidopsis and what role the ANP genes play in the process. We performed this analysis by generating a large population of T-DNA transformed anp2/anp3 plants by using an activation-tagging T-DNA vector. Transformed plants were visually screened in the greenhouse to identify rare individuals in which the characteristic small size of the anp2/anp3 plants has been restored to a more wild-type level. We identified 36 putative suppressor lines from this primary genetic screen. In order to determine the identity of the genes responsible for the genetic suppression of the anp2/anp3 phenotype we performed extensive TAIL PCR analysis to locate the position of the activation tagging T-DNA construct in the genome. After
many months of careful follow-up investigations we were not able to demonstrate genetic linkage between any of the activation tag insertions and the suppressor phenotype. We are continuing to pursue this mapping project in order to identify the genetic lesion responsible for the suppressor phenotype. We have also imitated a second line of experiments in which we are investigating the potential involvement of the ANP proteins in abiotic stress response. Transient expression studies performed by other laboratories have suggested that the ANP proteins may be involved in abiotic stress response, but no genetic analysis has been reported. Our preliminary studies have indicated that Arabidopsis lines homozygous for ANP mutant alleles are hypersensitive to elevated temperature and osmotic stress. Further studies will be performed in order to determine if these phenotypes co-segregate with the ANP mutations.
Impacts
The work that we are performing should improve our understanding of the signal transduction pathways that make use of the ANP proteins in Arabidopsis. This information will provide plant scientists with a foundation for understanding related signaling pathways in all crop species. Because the ANP pathways seem to be involved in plant division as well as stress responses, it is likely that this fundamental information will have practical applications in the future. Our preliminary data has indicated that in addition to their proven role in regulating cell division, the ANP proteins may also be involved in abiotic stress tolerance. Understanding how plants react to abiotic stress may be useful for improving the ability of crop species to survive sub-optimal growth conditions.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Because this project initiated 3 months ago we are still in the early stages of implementing the experimental plan outlined in the proposal. We have performed the inital phenotypic suppressor screen using an activation-tag T-DNA mutagenesis approach. From the inital T-1 generation of transformed plants we have identified four candidate lines that seem to suppress the mutant phenotype present in anp2/anp3 double-mutant lines. We are in the process of further characterizing the phenotype of these putative suppressor lines as well as analyzing the genetic transmission of the suppressor phenotype. In addition, we have begun to employ plasmid rescue and TAIL PCR as means of locating the T-DNA elements that are responsible for the observed phenotypes.
Impacts
This research should enhance our understanding of the signal transduction pathways that regulate cell division in plant cells. This knowledge should be useful to scientists who are trying to manipulate the growth and development of important crop plants.
Publications
- No publications reported this period
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