Progress 05/01/03 to 09/30/05
Outputs
The objectives for this project are to identify homozygous insertional mutants for Major Facilitator Superfamily (MFS) genes and perform initial phenotypic characterization of the mutants. There are 95 MFS genes in Arabidopsis that are likely to encode metabolite transporter and functions have been identified for very few of these genes. Insertional mutant lines were obtained from Salk Institute and undergraduate researchers funded by this project identified 106 homozygous lines containing T-DNA insertions in specific MFS genes with mutants for around half of the MFS genes. The mutants were analyzed for phenotypes related to growth and elemental composition. Mutations in one gene resulted in an embryo lethal phenotype and therefore revealed a required gene. Twenty seven double mutants were generated for pairs of closely related genes. All of the homozygous mutant lines were confirmed in the next generation, seeds were bulked up, and one mutant per gene was submitted to
the ABRC stock center so that the lines are publicly available. Phenotypic analysis of MFS mutants: MFS mutants defective in metabolite transport could potentially display a biochemical phenotype (altered metabolite level) or differences in growth/development that might be conditional. In general, only a few mutants (5) with phenotypes have been identified so far in this project. This may be due to functional redundancy, which may be more likely for large gene families (such as the MFS). For one line, an obvious embryo lethal phenotype was identified. We performed the following phenotypic tests on the entire set of mutants 1) sensitivity of seedling growth 2% sucrose and 2) hypocotyl elongation in the dark. For each of these tests we have identified one mutant with a phenotype. Elemental analysis of leaf tissue for the 23-mutant set was performed through collaboration with Dr. David Salt (Purdue University) using ICP-MS. One mutant showed elevated sodium content. Sodium sensitivity
tests were performed here in Minnesota and we determined that the mutant was over-sensitive to salt, indicating that the transporter may have some function in sodium tolerance. Generating double mutants: The majority of the mutants we have isolated display no phenotype. This may be due in part to the presence of functionally redundant genes. To address this, we have generated a set of 29 double mutants. The crosses were done by Dr. Anke Reinders who is supervising this project. The undergraduate researchers screened the F1 progeny for the presence of both T-DNA elements, and double-heterozygous plants were obtained for 27 of the 29 crosses. Students funded by the project The research work supported by this grant is conducted exclusively by undergraduate researchers. During the third year, Jennifer Flynn, Mary-Ann Em, Katherine Paulus and Brent Knudson, all undergraduate Biology or Plant Biology majors at the University of Minnesota were supported during the summer (2005). The process
of analyzing gene sequences, designing primers, preparing DNA, performing PCR, analyzing and interpreting the results in order to determine genotypes of the plants proved to be an excellent introduction for them.
Impacts
Although this project is conducted by undergraduates and the objectives are therefore conservative, obtaining mutants for the MFS genes is very important for the long-term objectives of the lab. We are interested in the function of metabolite transporters and in a previously-funded USDA project we cloned cDNAs for MFS genes to study the transport function (substrate specificity, stoichiometry, regulation) by heterologous expression in yeast and Xenopus oocytes. Our long-term goal is to study transport functions for these genes in plants and the mutant lines will be essential for future complementation studies. Therefore this project has a strong impact on funded projects in the Ward lab. The main impact of this project is to provide hands on experience in plant molecular biology and genetics to undergraduate students. Direct laboratory experience is essential for students to fully understand concepts such as genes and gene structure, mutants, phenotypes, linkage, and
other basic concepts of genetics that we often wrongly assume that every biology student understands. Students participating in this research project have a great advantage. Whether they continue on to study plant genetics in graduate school or decide on other directions such as medical school, their understanding of genetics and laboratory skills will be valuable to them.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The objectives for this project are to identify homozygous insertional mutants for Major Facilitator Superfamily (MFS) genes and perform initial phenotypic characterization of the mutants. There are 95 MFS genes in Arabidopsis that are likely to encode metabolite transporter and functions have been identified for very few of these genes. During the first year of the project, undergraduate researchers funded by this project identified 106 homozygous lines containing T-DNA insertions in specific MFS genes. With mutants for around half of the MFS genes, this represents a very solid foundation for further research on this gene family. During the current year of funding, undergraduates funded by the project have focused on 1) preliminary analysis of the mutants for phenotypes and 2) generating targeted double mutants. Phenotypic analysis of MFS mutants: MFS mutants defective in metabolite transport could potentially display a biochemical phenotype (altered metabolite
level) or differences in growth/development that might be conditional. In general, only a few mutants (5) with phenotypes have been identified so far in this project. This may be due to functional redundancy, which may be more likely for large gene families (such as the MFS). For one line, an obvious embryo lethal pheotype was identified. We performed the following phenotypic tests on the entire set of mutants 1) sensitivity of seedling growth 2% sucrose and 2) hypocotyl elongation in the dark. For each of these tests we have identified one mutant with a phenotype. Elemental analysis of leaf tissue for the 23-mutant set was performed through collaboration with Dr. David Salt (Purdue University) using ICP-MS. One mutant showed elevated sodium content. Sodium sensitivity tests were performed here in Minnesota and we determined that the mutant was over-sensitive to salt, indicating that the transporter may have some function in sodium tolerance. Generating double mutants: The majority of
the mutants we have isolated display no phenotype. This may be due in part to the presence of functionally redundant genes. To address this, we have generated a set of 29 double mutants. The crosses were done by Dr. Anke Reinders who is supervising this project. The undergraduate researchers screened the F1 progeny for the presence of both T-DNA elements, and double-heterozygous plants were obtained for 27 of the 29 crosses. We are just starting to screen the F2 populations for double homozygous mutants and only two double mutants have been obtained so far. Students funded by the project The research work supported by this grant is conducted exclusively by undergraduate researchers. During this year, Kristen Shaw and Debby Hsu, both undergraduate Biology majors at the University of Minnesota were supported fulltime during the summer. The process of analyzing gene sequences, designing primers, preparing DNA, performing PCR, analyzing and interpreting the results in order to determine
genotypes of the plants proved to be an excellent introduction for them. Kristen Shaw continued to work on the project during the Fall, 2004 and graduated from the University of Minnesota in December 2004.
Impacts
The main impact of this project is to provide hands on experience in plant molecular biology and genetics to undergraduate students. Direct laboratory experience is essential for students to fully understand concepts such as genes and gene structure, mutants, phenotypes, linkage, and other basic concepts of genetics that we often wrongly assume that every biology student understands. Students participating in this research project have a great advantage. Whether they continue on to study plant genetics in graduate school or decide on other directions such as medical school, their understanding of genetics and laboratory skills will be valuable to them.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The objectives for this project are to identify homozygous insertional mutants for Major Facilitator Superfamily (MFS) genes and perform initial phenotypic characterization of the mutants. There are 95 MFS genes in Arabidopsis that are likely to encode metabolite transporter and functions have been identified for very few of these genes. We obtained 157 lines containing potential T-DNA insertions in specific MFS genes from the Salk Institute. The plants were grown (12-16 plants per line), genomic DNA was extracted, and 2 PCR reactions were performed to identify the genotype at the specific MFS locus. Homozygous plants were identified for 106 (67%) of the lines. T-DNA insertions were not detected for 48 (30%) of the lines and this could be due to errors at the Salk Inst. in identifying the specific site of T-DNA insertion which is more difficult for genes in a large gene family such as the MFS. Three lines produced only heterozygous insertional mutants which may be due
to seedling lethality of the homozygous mutants, which would be a very important and exciting outcome. Further analysis of these mutants is underway in the lab. The 106 homozygous mutants represent insertions in exons in 23 of the 95 MFS genes. This is an excellent starting point for the initial phenotypic characterization experiments that will include measuring root growth on minimal media and leaf starch analysis. Initial results show that one of the homozygous mutants exhibits slower root growth than w.t. on minimal media but normal growth on media supplemented with a carbon source, indicating a role in sugar transport to the root. The research work supported by this grant is conducted exclusively by undergraduate researchers. During the first year of the project Benjamin Brown and Jeremy Allred, both undergraduate Biology majors at the University of Minnesota were supported fulltime during the summer of 2003. Neither student had prior experience in molecular biology. The process
of analyzing gene sequences, designing primers, preparing DNA, performing PCR, analyzing and interpreting the results in order to determine genotypes of the plants proved to be an excellent introduction for them and both performed excellently in the lab. One of the students (Allred) has expressed interest in continuing on the project and the other (Brown) has since moved to Tulane University. Although this project is conducted by undergraduate and the objectives are therefore conservative, obtaining homozygous mutants for the MFS genes is very important for the long-term objectives of the lab. We are interested in the function of metabolite transporters and in another USDA-funded project we are cloning cDNAs for MFS genes and studying the transport function (substrate specificity, stoichiometry, regulation) by heterologous expression in yeast and Xenopus oocytes. Our long-term goal is to study transport functions for these genes in plants and the mutant lines will be essential for
future complementation studies.
Impacts
The main impact of this project is to provide hands on experience in plant molecular biology and genetics to undergraduate students. Direct laboratory experience is essential for students to fully understand concepts such as genes and gene structure, mutants, phenotypes, linkage, and other basic concepts of genetics that we often wrongly assume that every biology student understands. Students participating in this research project have a great advantage. Whether they continue on to study plant genetics in graduate school or decide on other directions such as medical school, their understanding of genetics and laboratory skills will be valuable to them.
Publications
- No publications reported this period
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