Progress 09/15/03 to 09/14/05
Outputs
Over the course of this project, several important discoveries were made that contributed to understanding how coenzyme A (CoA) is produced in plants and how CoA production is related to other aspects of metabolism, especially seed oil metabolism. One of the principle contributions was to more fully characterize plant pantothenate kinase genes. Two pantothenate kinase genes were identified in the oilseed plant Arabidopsis, which we called PANK1 and PANK2, and their involvement in cellular CoA production was monitored by several methods. Real-time PCR measurement of transcript levels of PANK1 and PANK2 showed that both genes have very similar expression patterns with the highest expression in developing seeds and leaf tissue. The similar real-time PCR results suggested that the enzymes have redundant roles in the plant, as opposed to specific roles in different tissues. The possibility of redundancy was supported by phenotypes of PANK1 and PANK2 mutant plants. Analysis
of Arabidopsis mutants showed that loss of PANK1 or PANK2 had very little effect on the growth of the plant. Metabolic processes that are highly dependent on the CoA supply, such as oil production during seed development and oil degradation during seedling growth, were not greatly affected by single mutations, although pank2 mutants showed a slight decrease (10 percent) in seed oil levels. Using an assay method specially designed for measuring CoA levels in plant tissue, we were able to compare the amounts of CoA in the mutant plants relative to the CoA levels in wild-type tissue. CoA levels in flower and silique tissue from wild-type plants were 20.7 and 12.2 nmols/g fresh weight, respectively. Average CoA levels in both mutants dropped only 12-15 percent relative to these wild-type plants, which likely explains why the single mutations did not cause severe perturbations in cellular metabolism. However, a double gene knockout is embryo-lethal, resulting in aborted seeds. The lethal
phenotype suggests that PANK1 and PANK2 are complementary enzymes, having redundant functions. This discovery is significant given the different protein structure of PANK1 and PANK2. Although both enzymes possess a protein domain that is similar to other eukaryotic pantothenate kinases, PANK2 has an additional uncharacterized C-terminal protein domain. Dr. Bill Wedemeyer at Michigan State University performed homology modeling of this uncharacterized domain against the crystal structure of a homologous protein, showing that a subset of conserved residues form a defined cluster that act as a potential binding site for a positively-charged molecule. Whether this domain serves a regulatory function, such as through metal binding, or has an independent catalytic function remains to be determined. Subcellular targeting predictions of PANK1 and PANK2 suggest that both enzymes are cytosolic, which is counter to previous work that indicated all of the pantothenate kinase activity in the plant
cell was in the chloroplast. This presents a scenario in which cellular CoA is produced in the cytosol and shuttled into different organelles.
Impacts
Given the central role that coenzyme A (CoA) plays in cellular metabolism, the discoveries we made in characterizing coenzyme A biosynthetic genes in plants will be helpful in metabolic engineering studies that seek to optimize the yield of specific plant products. This is especially true in engineering oilseed plants, since our results suggest that CoA biosynthesis is an important part of seed development. Particularly useful will be methods we developed for measuring CoA levels out of plant tissue, which has often been complicated by the presence of interfering materials in plant extracts.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The first goal of this project is to characterize the genes encoding the biosynthetic enzymes for coenzyme A (CoA) in the plant Arabidopsis. This includes studying the tissue-specific expression of the genes as well as analyzing plants that have mutations in these same genes. My efforts in this area have focused on four specific genes, including two pantothenate kinase genes (PANK1 and PANK2), a phosphopantetheine adenylyltransferase gene (PPAT), and a dephospho-CoA kinase gene (DPCK). By using real-time PCR, I was able to measure the relative expression levels of both PANK genes in developing Arabidopsis seeds and in germinating seedlings. These measurements demonstrated that there is 5-10 fold more expression of the PANK genes in developing seeds than in germinating seedlings. This is suprising because both stages of Arabidopsis growth require substantial amounts of CoA: for fatty acid biosynthesis in the case of developing seeds and for fatty acid oxidation in the
case of germinating seedlings. While it may be possible that the low expression of the PANK genes during seed germination is sufficient to provide the needed CoA, it is also possible that CoA or the biosynthetic enzymes themselves are produced during seed development and then stored in the seed for later use in germination. In addition to measuring the expression of the PANK genes, I have also isolated mutants for all four of the genes listed above. In the case of the PANK1 and PANK2 single mutants, there is no observable phenotype. However, a double mutation is embryo lethal. Fully homozygous double knockouts could not be found in a segregating population, and heterozygous parent plants have siliques that contain undeveloped seeds. Embryos cannot be detected in these dead seeds, indicating that the double mutation is lethal soon after fertilization. The result is significant because it suggests that PANK1 and PANK2 provide the bulk, if not all, of the pantothenate kinase activity in
the cell. Since PANK1 and PANK2 are likely cytosolic proteins, this places CoA biosynthesis in the cytosol and not in the plastid as was previously believed. Mutations in the PPAT and DPCK genes do not produce any growth phenotypes, which is surprising since there is only one copy of each gene in Arabidopsis. However, expression analysis showed that there was still residual expression of both genes in the mutant plants: 10 percent in the case of PPAT and 35 percent in the case of DPCK. The ability of the plant to grow normally despite such low expression levels shows that either PPAT and DPCK are not the rate-limiting steps of CoA biosynthesis or that the cell requires much less CoA for survival than is produced under normal conditions. In addition to the gene expression and mutant studies, I am also developing methods to obtain reliable measurements of actual CoA levels in plant tissues. This will be important for determining how expression of the CoA biosynthetic genes is correlated
with real levels of CoA in the cell, as well as indicate which tissues and metabolic processes rely most heavily on the CoA supply.
Impacts
Current efforts in biotechnology seek to increase the yield and modify the characteristics of oil in oilseed crops. I have measured the expression of genes in the oilseed plant Arabidopsis that encode the biosynthetic enzymes for coenzyme A (CoA), an important cofactor for oil biosynthesis. Gene expression is highest in developing seeds, where the bulk of oil biosynthesis takes place. Specific mutations in the CoA production pathway are lethal to the plant, which dies in the seed development stage. The importance of CoA in seed development suggests that the CoA supply may be a significant factor to take into account in oilseed engineering.
Publications
- No publications reported this period
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Progress 09/15/03 to 12/31/03
Outputs
I am using several different approaches to study the biosynthesis of coenzyme A in plants. One approach involves analysis of Arabidopsis plants that have mutations in the genes involved in coenzyme A biosynthesis. By screening through populations of available mutants, I have been able to identify and cultivate mutant Arabidopsis plants that have T-DNA insertions in the genes of both known pantothenate kinases (PANK1 and PANK2), the 4-phospopantotheine adenylyl transferase (PPAT), and the dephospho-CoA kinase (DPCK). Only plants carrying mutations in the PANK1 and DPCK genes have been analyzed in a homozygous state, such that there is no normal copy of the gene present. Early results show that the mutant plants do not display any abnormal growth, fertility, or germination characteristics. For the PANK1 mutant, these results suggest that the mutation may be compensated for by increased expression of the other pantothenate kinase gene. As soon as the PANK2 homozygous
mutant becomes available, I will cross the two mutants in order to create a double mutation that will eliminate the expression of both genes. There is only one known DPCK in Arabidopsis, and it is intriguing that the mutant DPCK plants grow normally. Either there is another DPCK in the plant cell that has not been discovered, or there is still some background expression of the known DPCK gene. In order to check the actual expression levels of the CoA biosynthetic genes in tissues of normal and mutant plants, I will use quantitative real time PCR. The primers for analysis of the PANK1, PANK2, and DPCK genes have been tested and proven satisfactory. The next step is to extract RNA from different tissues of wild-type and homozygous mutant plants and compare the amount of mRNA present corresponding to each gene of interest. These quantitative PCR experiments will be important for identifying the tissues where the CoA demand is highest. They will also show whether gene expression has been
totally eliminated in the mutant plants. Along with measuring the expression levels of genes in the CoA biosynthetic pathway, I am currently optimizing an HPLC technique to measure the amount of total CoA in different tissues. This technique has been combined with mass-spectrometry in order to correctly identify the CoA peak on the HPLC plot. Although the mass spectrometer can detect very low amounts of CoA, the linear range for CoA quantification on our system is between 100 pmols and 1.5 nmols. If this is not sensitive enough for measuring tissue levels of CoA, I will combine the extraction method with a process that converts the CoA to a fluorescent derivative. This adds another step to the analysis, but the fluorescent derivative can be detected at very low levels and has a greater linear range. I am also synthesizing radiolabeled CoA to test how effectively CoA is being removed from the plant tissue during the extraction process. With this combination of methods, I expect to be
able to measure the expression of CoA biosynthetic genes in wild-type and mutant plants and correlate those levels with the actual amount of CoA present in the plant.
Impacts
Coenzyme A is a key molecule in the production and breakdown of important plant products like vegetable oil, carbohydrate, and protein. Understanding the genetic aspects that control coenzyme A biosynthesis in the plant cell will place us in a better position to maximize the productivity of crops that generate these important natural products.
Publications
- No publications reported this period
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