Progress 12/15/00 to 12/31/04
Outputs
We have used the model genetic organism, Arabidopsis to identify how acetyl-CoA is generated in plants. Acetyl-CoA is a crucial intermediate of metabolism from which plants produce large number of agronomically important phytochemicals. These phytochemicals include fats, oils, waxes, natural rubber, fragrances, and essential oils. In addition, Acetyl-CoA is a precursor of a variety of phytochemicals that are crucial to plant growth and development (e.g., membranes, lipid signal molecules, brassinosteroids, flavonoids). At the start of this project it was clear that plants have multiple potential means of producing acetyl-CoA, but the importance of each mechanism was unknown, nor were the genes and enzymes involved in each of these mechanisms well defined. During the course of this project we identified six enzyme systems and the associated genes that could either directly or in combination generate different pools of acetyl-CoA. These enzyme systems are: 1) acetyl-CoA
synthetase (ACS); 2) plastidic pyruvate dehydrogenase complex (ptPDHC); 3) ATP-citrate lyase (ACL); 4) acetaldehyde dehydrogenase (ALDH); 5) pyruvate decarboxylase (PDC); and 6) acetyl-CoA hydrolase (ACH). In Arabidopsis, 21 genes encode these enzyme systems. To elucidate the importance of each enzyme system, we have undertaken two parallel molecular genetic strategies. One approach is to isolate and characterize mutations in genes that encode each of the acetyl-CoA generating enzyme systems. The other approach is to characterize the spatial and temporal pattern of expression of each enzyme system. These complementary strategies would identify the role of each acetyl-CoA generating enzyme system in establishing different acetyl-CoA pools that would be used for the biosynthesis of different sets of phytochemicals. These characterizations have established that ptPDHC generates the major plastidic acetyl-CoA pool that is used for the biogenesis of fatty acids. ACL on the other hand
generates the cytosolic acetyl-CoA pool, which can be used in the biogenesis of waxes, pigments and essentials oils.
Impacts
This research will provided detailed mechanistic understanding of the molecular complexity of acetyl-CoA generation in plants. Furthermore, these studies will lead to a better understanding how plants regulate the biosynthesis and accumulation of a variety of pytochemicals that are important agricultural products e.g., oils, fats, waxes, pigments essential oils.
Publications
- Kirch HH, D Bartels, Y Wei, PS Schnable, AJ Wood (2004) The ALDH gene superfamily of Arabidopsis. Trends in Plant Science. 9:371-377.
- Fatland BL, Nikolau BJ, Wurtele ES. 2005. Reverse genetic characterization of cytosolic acetyl-CoA generation by ATP-citrate lyase in Arabidopsis. Plant Cell. In press; will be in the January, 2005 issue.
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Progress 01/01/03 to 12/31/03
Outputs
The molecular mechanisms by which plants generate the metabolic intermediate, acetyl-CoA, is being investigated. Acetyl-CoA is the activated form of acetic acid, which is the precursor of a large number of phytochemicals that represent either agricultural products or influence the production of agricultural products. Our research is being conducted in the model genetic organism, Arabidopsis, which has proven to be very amenable to these types of investigations. We are investigating how three distinct pools of acetyl-CoA pools are generated. These are generated by three biochemical and associated genetic systems: 1) the plastidic pyruvate dehydrogenase complex, which is encoded by 7 genes; 2) the sequential action of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase, which requires the action of up to 7 genes; and 3) ATP citrate lyase, which requires 5 genes. We have isolated mutations in each of the acetyl-CoA generating genes and are
characterizing the effect of each mutation on acetyl-CoA metabolism. Our studies to date indicate that the plastidic pyruvate dehydrogenase complex is responsible for generating the majority of the acetyl-CoA that is needed in plastids, and this acetyl-CoA pool is essential during embryo development; this acetyl-CoA-pool is the precursor for fatty acid biosynthesis. In addition, our studies indicate that ATP citrate lyase generates the cytosolic pool of acetyl-CoA, which is the precursor for the biosynthesis of waxes, essential oils, and pigments.
Impacts
This research will provided detailed mechanistic understanding of the molecular complexity of acetyl-CoA generation in plants. Furthermore, these studies will lead to a better understanding how plants regulate the biosynthesis and accumulation of a variety of pytochemicals that are important agricultural products e.g., oils, fats, waxes, pigments essential oils.
Publications
- Lin M, Behal RH, Oliver, DJ. 2003. Disruption of plE2, the Gene for the E2 Subunit of the Plastid Pyruvate Dehydrogenase Complex, in Arabidopsis Causes an Early Embryo Lethal Phenotype. Plant Molecular Biology 52: 865-872.
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Progress 01/01/02 to 12/31/02
Outputs
This project is investigating the role of three biochemical systems that can generate acetyl-CoA in plants. These are: 1) the plastidic pyruvate dehydrogenase complex, 2) the sequential action of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase, and 3) ATP citrate lyase. A combined reverse genetic and biochemical studies are being used to investigate these systems. This research is being conducted in the model genetic organism, Arabidopsis, which has proven to be very amenable to these types of investigations. In Arabidopsis we have identified 19 genes that are required for these three potential acetyl-CoA-generating systems. To investigate the role of each gene in acetyl-CoA generation mutants are being isolated for each gene. To date, mutants in 12 of these genes have been identified. Characterization of these mutants is testing three hypotheses: 1) plastidic pyruvate dehydrogenase complex is responsible for generating the majority of the
acetyl-CoA that is needed in plastids; 2) ATP citrate lyase generates the cytosolic pool of acetyl-CoA; and 3) the sequential action of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase generates a third uncharacterized pool of acetyl-CoA.
Impacts
This research will provided detailed mechanistic understanding of the molecular complexity of acetyl-CoA generation in plants. Furthermore, these studies will lead to a better understanding how plants regulate the biosynthesis and accumulation of a variety of pytochemicals that are important agricultural products e.g., oils, fats, waxes, pigments essentail oils.
Publications
- Fatland BL, J Ke, M Anderson, W Mentzen, L-W Cui, C Allred, JL Johnston, BJ Nikolau and ES Wurtele. 2002. Molecular characterization of a novel heteromeric ATP-citrate lyase that generates cytosolic pool of acetyl-CoA in Arabidopsis. Plant Physiology 130:740-756.
- Lutziger I and DJ Oliver. 2000. Molecular evidence of a unique lipoamide dehydrogenase in plastids: analysis of plastidic lipoamide dehydrogenase from Arabidopsis thaliana. FEBS Lett. 484:12-16.
- Lutziger I and DJ Oliver. 2001. Characterization of two cDNAs encoding mitochondrial lipoamide dehydrogenase from Arabidopsis. Plant Physiol. 127:615-623
- Behal RH, M Lin, S Back and DJ Oliver. 2002. Role of acetyl-coenzyme A synthetase in leaves of Arabidopsis thaliana. Arch Biochem Biophys. 402:259-267
- Skibbe DS, F Liu, TJ Wen, MD Yandeau, X Cui, J Cao, CR Simmons and PS Schnable. 2002. Characterization of the aldehyde dehydrogenase gene families of Zea mays and Arabidopsis. Plant Mol Biol. 48:751-764
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Progress 01/01/01 to 12/31/01
Outputs
We are investigating the molecular genetic mechanisms by which plants generate the chemical acetyl-CoA. Acetyl-CoA is the activated form of acetic acid, which as an intermediate of metabolism, is the precursor of a large number of phytochemicals that represent either agricultural products or influence the production of agricultural products. These acetyl-CoA-derived phytochemicals include fats, oils, waxes, essential oils, pigments etc. Our research is being conducted in the model genetic organism, Arabidopsis, which has proven to be very amenable to these types of investigations. We are investigating three biochemical and associated genetic systems that can generate acetyl-CoA in plants. These are: 1) the plastidic pyruvate dehydrogenase complex, which is encoded by 7 seven genes; 2) the sequential action of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase, which requires the action of upto 7 genes; and 3) ATP citrate lyase, which requires
5 genes. The strategy we are pursuing is one of isolating mutant plants that have altered expression of each of the identified genes and characterizing the effect of each mutation on acetyl-CoA metabolism. These data, in combination with detailed studies of the expression of each of the genes that we are studying should address the question of how different acetyl-CoA pools are generated in plants. Our studies to date indicate that the plastidic pyruvate dehydrogenase complex is responsible for generating the majority of the acetyl-CoA that is needed in plastids. This pool of acetyl-CoA is the precursor for fatty acid biosynthesis. In addition, our studies indicate that ATP citrate lyase generates the cytosolic pool of acetyl-CoA, which is the precursor for the biosynthesis of waxes, essential oils, and pigments.
Impacts
This research will provided detailed mechanistic understanding of the molecular complexity of acetyl-CoA generation in plants. Furthermore, these studies will lead to a better understanding how plants regulate the biosynthesis and accumulation of a variety of pytochemicals that are important agricultural products e.g., oils, fats, waxes, pigments essentail oils.
Publications
- Ke J-S, B Behal, S Yunkers, BJ Nikolau, ES Wurtele and DJ Oliver. 2000. The role of pyruvate dehydrogenase and acetyl-CoA synthetase in fatty acid synthesis in developing Arabidopsis seeds. Plant Physiology 123:497-508.
- Nikolau BJ, ES Wurtele, DJ Oliver and PS Schnable. 2000. Molecular biology of acetyl-CoA metabolism. The Proceedings of the 14th International Symposium on Plant Lipids, Cardiff, Wales. Biochem Soc Trans. 28:591-593.
- Fatland B, M Anderson, BJ Nikolau and ES Wurtele. 2000. Molecular biology of cytosolic acetyl-CoA generation. The Proceedings of the 14th International Symposium on Plant Lipids, Cardiff, Wales. Biochem Soc Trans. 28:593-595.
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