Progress 12/01/08 to 11/30/12
Outputs
OUTPUTS: Beyond the ubiquitous gibberellic acid hormones required for normal growth and development in all higher/flowering plants, the important cereal crop plant rice produces many related diterpenoids. These additional natural products serve an important role as phytoalexins whose biosynthesis is induced by infection either of the devastating blast disease agent Magnaporthe grisea, or the important bacterial leaf blight agent Xanthomonas oryzae pv. oryzae, against which these labdane-related diterpenoids exhibit antibiotic activity. The goal of the current project is elucidation of the complex metabolic network underlying rice diterpenoid biosynthesis, with some additional work in other cereal crop plants. Specifically, in rice we are targeting the oxidative steps following formation of the cyclic diterpene olefins whose biosynthesis we have already defined, as well as investigation of diterpenoid metabolism more generally in other cereal crop plants. We have identified 31 cytochromes P450 mono-oxygenases and 18 short-chain alcohol dehydrogenases that have putative roles in rice diterpenoid metabolism from the rice genome sequence, and are working to elucidate their biochemical function. This involves heterologous recombinant expression of the P450s and dehydrogenases, typically using synthetic, codon-optimized genes, with feeding of potential substrates. In particular, we are able to produce large amounts of the relevant diterpene biosynthetic intermediates using a metabolic engineering system we have recently developed and can co-express individual P450s and the requisite reductase redox partner. From this work we have functionally characterized ten rice P450s that act on the known rice diterpene precursors to produce oxygenated intermediates and, although we are still in the process of producing some of these in sufficient (i.e., larger) quantities for structural analysis, we have already published our analysis of eight of these P450s (Swaminathan et al, 2009; Wang et al, 2011; Wu et al, 2011; Wang et al, 2012a; Wang et al, 2012b), with two more manuscripts in preparation. Moreover, our studies of short-chain alcohol dehydrogenases have yielded results that are nearly ready for publication as well. In addition, we have published our functional characterization of the wheat family of diterpene synthases (Zhou et al, 2012; Wu et al, 2012). PARTICIPANTS: Project director: Reuben J. Peters (Oversight of project, including setting long and intermediate term goals, along with appropriate direction of research). Graduate students: Qiang Wang (graduated 2010) and Yisheng Wu (graduated 2012), carrying out research. Undergraduate student: Taylor Chesnut (Laboratory assistant), learned metabolic engineering system and assisted propagation of transgenic rice plant lines. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our identification of the metabolic roles of the rice diterpene synthases and cytochromes P450 in diterpenoid biosynthesis offers specific targets for alteration of biochemical function and downstream natural product accumulation, with the resulting transgenic rice plants providing direct insight into the impact of the corresponding compounds on the agronomic characteristics of this important crop plant. Indeed, we have just published the first of these studies, providing the first genetic evidence for natural product mediated allelopathy (Xu et al, 2012). Furthermore, the evidence that we have obtained suggesting that our work with rice will apply to other cereal crop plants then offers potential extension of our studies to the improvement of agronomic characteristics of cereal crops more generally, further increasing the expected impact.
Publications
- Wang, Q., Hillwig, M.L., Swaminathan, S., Fulton, D.B., Peters, R.J. (2012) Characterization of CYP76M5-8 indicates metabolic plasticity within a plant biosynthetic gene cluster, J. Biol. Chem., 287(9):6159-6168.
- Wang, Q., Hillwig, M.L., Wu, Y., Peters, R.J. (2012) CYP701A8: A rice ent-kaurene oxidase paralog diverted to more specialized diterpenoid metabolism, Plant Physiol., 158(3):1418-1425.
- Wu, Y., Zhou, K., Toyomasu, T., Sugawara, C., Oku, M., Abe, S., Usui, M., Mitsuhashi, W., Chono, M., Chandler, P.M., Peters, R.J. (2012) Functional characterization of wheat copalyl diphosphate synthases sheds light on the early evolution of labdane-related diterpenoid metabolism in the cereals, Phytochemistry, 84:40-46.
- Zhou, K., Xu, M., Tiernan, M.S., Xie, Q., Toyomasu, T., Sugawara, C., Oku, M., Usui, M., Mitsuhashi, W., Chono, M., Chandler, P.M., Peters, R.J. (2012) Functional characterization of wheat ent-kaurene(-like) synthases indicates continuing evolution of labdane-related diterpenoid metabolism in the cereals, Phytochemistry, 84:47-55.
- Kato-Noguchi, H., Peters, R.J. (2013) The role of momilactones in rice allelopathy, J. Chem. Ecol., 39(2): in press.
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Progress 12/01/10 to 11/30/11
Outputs
OUTPUTS: Beyond the ubiquitous gibberellic acid hormones required for normal growth and development in all higher/flowering plants, the important cereal crop plant rice produces many related diterpenoids. These additional natural products serve an important role as phytoalexins whose biosynthesis is induced by infection either of the devastating blast disease agent Magnaporthe grisea, or the important bacterial leaf blight agent Xanthomonas oryzae pv. oryzae, against which these labdane-related diterpenoids exhibit antibiotic activity. The goal of the current project is elucidation of the complex metabolic network underlying rice diterpenoid biosynthesis, with some additional work in other cereal crop plants. Specifically, in rice we are targeting the oxidative steps following formation of the cyclic diterpene olefins whose biosynthesis we have already defined, as well as investigation of diterpenoid metabolism more generally in other cereal crop plants. We have identified 31 cytochromes P450 mono-oxygenases and 18 short-chain alcohol dehydrogenases that have putative roles in rice diterpenoid metabolism from the rice genome sequence, and are working to elucidate their biochemical function. This involves heterologous recombinant expression of the P450s and dehydrogenases, typically using synthetic, codon-optimized genes, with feeding of potential substrates. In particular, we are able to produce large amounts of the relevant diterpene biosynthetic intermediates using a metabolic engineering system we have recently developed and can co-express individual P450s and the requisite reductase redox partner. From this work we have functionally characterized eight rice P450s that act on the known rice diterpene precursors to produce oxygenated intermediates and, although we are still in the process of producing some of these in sufficient (i.e., larger) quantities for structural analysis, we have already published our analysis of four of these P450s (Swaminathan et al, 2009; Wang et al, 2011; Wu et al, 2011). We are in the process of publishing the description of four more (Wang et al, in press; Wang et al, in press), and have one more manuscript in preparation. Moreover, our studies of short-chain alcohol dehydrogenases have yielded results that are nearly ready for publication as well. In addition, we have finished functional characterization of the wheat family of diterpene synthases, with two manuscripts describing these results from this additional important cereal crop plant under revision for publication in Phytochemistry. PARTICIPANTS: Project director: Reuben J. Peters (Oversight of project, including setting long and intermediate term goals, along with appropriate direction of research). Graduate students: Qiang Wang (graduated Aug. 2010) and Yisheng Wu (current), carrying out research. Undergraduate student: Taylor Chesnut (Laboratory assistant), learning metabolic engineering system and assisting with propagation of transgenic rice plant lines. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our identification of the metabolic roles of the rice diterpene synthases and cytochromes P450 in diterpenoid biosynthesis offers specific targets for alteration of biochemical function and downstream natural product accumulation, with the resulting transgenic rice plants providing direct insight into the impact of the corresponding compounds on the agronomic characteristics of this important crop plant. Indeed, we have just published the first of these studies, providing the first genetic evidence for natural product mediated allelopathy (Xu et al, 2012). Furthermore, the evidence that we have obtained suggesting that our work with rice will apply to other cereal crop plants then offers potential extension of our studies to the improvement of agronomic characteristics of cereal crops more generally, further increasing the expected impact.
Publications
- Wang, Q., Hillwig, M.L., Peters, R.J. (2011) CYP99A3: Functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice, Plant J., 65(1):87-95.
- Wu, Y., Hillwig, M.L., Wang, Q., Peters, R.J. (2011) Parsing a multifunctional biosynthetic gene cluster from rice: Biochemical characterization of CYP71Z6 & 7, FEBS Lett., 585(21):3446-3451.
- Xu, M., Galhano, R., Weimann, P., Bueno, E., Tiernan, M., Wu, W., Gershenzon, J., Tudzynski, B., Sesma, A., Peters, R.J. (2012) Genetic evidence for natural product mediated plant-plant allelopathy in rice, New Phytol., 193(3):570-575.
- Wang, Q., Hillwig, M.L., Swaminathan, S., Fulton, D.B., Peters, R.J. (2012) Characterization of CYP76M5-8 indicates metabolic plasticity within a plant biosynthetic gene cluster, J. Biol. Chem., in press.
- Wang, Q., Hillwig, M.L., Wu, Y., Peters, R.J. (2012) CYP701A8: A rice ent-kaurene oxidase paralog diverted to more specialized diterpenoid metabolism, Plant Physiol., in press.
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Progress 12/01/09 to 11/30/10
Outputs
OUTPUTS: Beyond the ubiquitous gibberellic acid hormones required for normal growth and development in all higher/flowering plants, the important cereal crop plant rice produces many related diterpenoids. These additional natural products serve an important role as phytoalexins whose biosynthesis is induced by infection of the devastating blast disease agent Magnaporthe grisea, as well as other pathogenic fungi, and against which these labdane-related diterpenoids exhibit antibiotic activity. The goal of the current project is elucidation of the complex metabolic network underlying rice diterpenoid biosynthesis, with some additional work in other cereal crop plants. Specifically, in rice we are targeting the oxidative steps following formation of the cyclic diterpene olefins whose biosynthesis we have already defined, as well as investigation of diterpenoid metabolism more generally in other cereal crop plants. We have identified 31 cytochromes P450 mono-oxygenases and 18 short-chain alcohol dehydrogenases that have putative roles in rice diterpenoid metabolism from the rice genome sequence, and are working to elucidate their biochemical function. This involves heterologous recombinant expression of the P450s and dehydrogenases, typically using synthetic, codon-optimized genes, with feeding of potential substrates. In particular, we are able to produce large amounts of the relevant diterpene biosynthetic intermediates using a metabolic engineering system we have recently developed and can co-express individual P450s and the requisite reductase redox partner. From this work we have functionally identified close to 10 rice P450s that act on the known rice diterpene precursors to produce oxygenated intermediates and, although we are still in the process of producing some of these in sufficient (i.e., larger) quantities for structural analysis, we have already published our analysis of two of these P450s (Swaminathan et al, 2009; Wang et al, 2011). We are in the process of publishing the description of several more (Wang et al, under revision), and have two other manuscripts in preparation, with studies of other P450s nearing completion. In addition, we have finished functional characterization of the wheat family of diterpene synthases, with two manuscripts describing these results from this additional important cereal crop plant in preparation. PARTICIPANTS: Project director: Reuben J. Peters (Oversight of project, including setting long and intermediate term goals, along with appropriate direction of research). Graduate students: Qiang Wang (graduated Aug. 2010) and Yisheng Wu (current), carrying out research. Undergraduate student: Taylor Chesnut (Laboratory assistant), learning metabolic engineering system and assisting with propagation of transgenic rice plant lines. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our identification of the metabolic roles of the rice diterpene synthases and cytochromes P450 in diterpenoid biosynthesis offers specific targets for alteration of biochemical function and downstream natural product accumulation, with the resulting transgenic rice plants providing direct insight into the impact of the corresponding compounds on the agronomic characteristics of this important crop plant. The evidence that we have obtained suggesting that our work with rice will apply to other cereal crop plants then offers potential extension of our studies to the improvement of agronomic characteristics of cereal crops more generally, further increasing the expected impact.
Publications
- No publications reported this period
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Progress 12/01/08 to 11/30/09
Outputs
OUTPUTS: Beyond the ubiquitous gibberellic acid hormones required for normal growth and development in all higher/flowering plants, the important cereal crop plant rice produces many related diterpenoids. These additional natural products serve an important role as phytoalexins whose biosynthesis is induced by infection of the devastating blast disease agent Magnaporthe grisea, as well as other pathogenic fungi, and against which these labdane-related diterpenoids exhibit antibiotic activity. The goal of the current project is elucidation of the complex metabolic network underlying rice diterpenoid biosynthesis, with some additional work in other cereal crop plants. Specifically, in rice we are targeting the oxidative steps following formation of the cyclic diterpene olefins whose biosynthesis we defined in the previously funded period. Towards this purpose we have identified 31 cytochromes P450 mono-oxygenases and 18 short-chain alcohol dehydrogenases that have putative roles in rice diterpenoid metabolism from the rice genome sequence, and are working to elucidate their biochemical function. This involves heterologous recombinant expression of the P450s and dehydrogenases, typically using synthetic, codon-optimized genes, with feeding of potential substrates. In particular, we are able to produce large amounts of the relevant diterpene biosynthetic intermediates using a metabolic engineering system we have recently developed and can co-express individual P450s and the requisite reductase redox partner. From this work we have functionally identified more than five rice P450s that act on the known rice diterpene precursors to produce oxygenated intermediates and, although we are still in the process of producing some of these in larger quantities for structural analysis, we have already published our analysis of one such P450 (Swaminathan et al, 2009). We anticipate completion of similar studies for several more of these P450s, with publication of the results in the coming year. In addition, we are finishing functional characterization of the related family of diterpene synthases from wheat, and anticipate publication of these results from this additional important cereal crop plant in the coming year as well. PARTICIPANTS: Project director: Reuben J. Peters Oversight of project, including setting long and intermediate term goals, along with appropriate direction of research. Graduate student: Qiang Wang Senior graduate student carrying out research project, largely independently at this point. Also responsible for writing up results for publication, under the guidance of the project director. Undergraduate student: Mollie Tiernan Laboratory assistant, learning metabolic engineering system and assisting with propagation of transgenic rice plant lines. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our identification of the metabolic roles of the rice diterpene synthases and cytochromes P450 in diterpenoid biosynthesis offers specific targets for alteration of biochemical function and downstream natural product accumulation, with the resulting transgenic rice plants providing direct insight into the impact of the corresponding compounds on the agronomic characteristic of this important crop plant. The evidence that we have obtained suggesting that our work with rice will apply to other cereal crop plants then offers potential extension of our studies to the improvement of agronomic characteristics of cereal crops more generally, further increasing the expected impact.
Publications
- Swaminathan, S., Morrone, D., Wang, Q., Fulton, D.B., Peters, R.J. (2009) "CYP76M7 is an ent-cassadiene C11(alpha)-hydroxylase defining a second multifunctional diterpenoid biosynthetic gene cluster in rice", Plant Cell, 21(10):3315-3325.
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