Progress 10/01/02 to 09/30/07
Outputs
Objective 1, to determine the subcellular localization of P450s predicted to be in chloroplasts and determine the necessary and sufficient structural features of the targeting. Progress on this objective included the use of two methods characterizing the location of CYP97A3 in the chloroplasts. The work with peas was done by importing radioactive protein into isolated chloroplasts. A substantial amount of the protein was integral to thylakoid membranes. A portion of CYP97A3 was removed after entry to the chloroplasts. Arabidopsis protoplasts were used to visualize proteins that were transiently expressed fusions of CYP97A3 with GFP. Special constructs of CYP97A3 that were truncated to remove parts of the N-terminal targeting sequence demonstrated that this was essential for the import. This was not the case for an outer membrane protein used as a control. Furthermore, when the N-terminal sequence from CYP97A3 was fused to an ER-targeted P450, the P450 was imported into
Arabidopsis protoplast chloroplasts. This is the first report of a P450 being synthesized in the cytoplasm and imported to- and processed in the chloroplast. Objective 2, to determine the nature and location of the reducing partners for P450s located in the chloroplasts and other possible organelles. We have worked extensively on a third putative reductase with very high amino acid sequence similarity with ATR1 and ATR2. We have prepared yeast expression vectors with the cDNA for ATR3 and discovered that this recombinant protein has cytochrome c reducing activity equivalent to that of ATR1 and ATR2. Using GFP tagged-ATR3 and immunocytolocalization, the ATR3 appears to be located both in the soluble and in the microsomal fractions of the cell. Interestingly, the cytochrome c reductase activity is only expressed in membrane fractions. In-vitro and in-vivo assays, we have not revealed any evidence that this reductase to pass electrons from NADPH to a cytochrome P450. Objective 3, to
characterize genes encoding P450s involved in metabolic pathways responding to environmental and biological stress, including abscisic acid 8-hydroxylase. The CYP72A sub-family of cytochromes P450 was initially identified as ABA hydroxylases by Cutler et al. in Saskatoon, Canada. Upon analysis of the Arabidopsis genome we identified a cluster of genes in the sub-family on Chromosome 3. We analyzed the expression patterns of six of these using RT-PCR, and Real Time PCR. All of them are inducible by ABA, and three are particularly responsive to water stress. These three have been cloned into several vectors in an effort to over-express them and measure their enzyme activity. We have reconstituted an enzyme system using proteins synthesized in E. coli and reductase purified from rat livers. In all three cases we have demonstrated very small rates of conversion of ABA to 8-hydroxy-ABA. The CYP72A sub-family of cytochromes P450 is clearly influenced by ABA, but it appears that they are not
involved in ABA biosynthesis
Impacts
This project contributed to the general understanding of a large superfamily of oxidative enzymes known as cytochromes P450 in plants. These enzymes are responsible for the synthesis and degradation of many biologically important chemicals in plants, including those that regulate growth and development. Arabidopsis contains more than 250 cytochrome P450s, yet this study identified the first example of one to reside in plant chloroplasts. In humans and many other major groups of organisms there is only one P450 reductase, whereas there are two and a potential third in many plants. Although the third reductase in Arabidopsis has many of the properties of a P450 reductase, our studies show that it does not have a general P450 reductase function like the other two. These studies have provided basic information which will assist other researchers in examining the sub-cellular localization and function of cytochromes P450 in plants.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
Our research this year focused on the discovery of a putative cytochrome P450 reductase in Arabidopsis thaliana. The annotation of the genome sequence for Arabidopsis describes two cytochrome P450 reductases that have been extensively characterized (ATR1 and ATR2), and one putative reductase that has not been characterized (ATR3). We have cloned the gene for ATR3 and prepared expression vectors for use within E. coli and Saccharomyces cerevisiae. The proteins expressed in these preparations were capable of using NADPH to reduce cytochrome c. This is a routine assay for reductases, but it does not necessary test the ability of the reductase to transfer electrons to a cytochrome P450. Therefore, we have constructed vectors containing both ATR3 and CYP73A1 (cinnamic acid hydroxylase) from Arabidopsis. We are in the process of assaying the reduction of CYP73A1. Additional experiments have been done on the localization ATR3 in Arabidopsis protoplasts. Although ATR3 does
not have a recognizable chloroplast targeting sequence at its N-terminus, our transient expression studies with GFP fused to the C-terminus of the reductase indicate that it is probably associated with the endoplasmic reticulum.
Impacts
We are contributing to the general understanding of a large superfamily of oxidative enzymes in plants. These enzymes are responsible for the synthesis and degradation of many biologically important chemicals in plants, including those that regulate growth and development. Through new knowledge about how and where in the plant these enzymes function, we may be able to modify plants to produce higher yields, more antioxidants, or even better withstand environmental or biological stress.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/29/04
Outputs
Objective 1, to determine the subcellular localization of P450s predicted to be in chloroplasts and determine the necessary and sufficient structural features of the targeting. Progress on this objective included the use of two methods characterizing the location of CYP97A3 in the chloroplasts. The work with peas was done by importing radioactive protein into isolated chloroplasts. A substantial amount of the protein was integral to thylakoid membranes. Arabidopsis protoplasts were used to visualize proteins that were transiently expressed fusions of CYP97A3 with GFP. Special constructs of CYP97A3 that were truncated to remove parts of the N-terminal targeting sequence demonstrated that this was essential for the import. This was not the case for an outer membrane protein used as a control. Furthermore, when the N-terminal sequence from CYP97A3 was fused to an ER-targeted P450, the P450 was imported into Arabidopsis protoplast chloroplasts. Objective 2, to determine
the nature and location of the reducing partners for P450s located in the chloroplasts and other possible organelles. Similar approaches have been taken with the two known P450 reductases in Arabidopsis. Contrary to a study with poplar reductases, our preliminary results indicate that ATR2 is imported into chloroplasts. We are also working on a third putative reductase with very high amino acid sequence similarity with ATR1 and ATR2. We have prepared yeast expression vectors with the cDNA for ATR3 and are nearly ready to test the over-expressed protein for enzyme activity. Objective 3, to characterize genes encoding P450s involved in metabolic pathways responding to environmental and biological stress, including abscisic acid 8-hydroxylase. The CYP72A sub-family of cytochromes P450 was initially identified as ABA hydroxylases by Cutler et al. in Saskatoon, Canada. Upon analysis of the Arabidopsis genome we identified a cluster of genes in the sub-family on Chromosome 3. We have
analyzed the expression patterns of six of these using RT-PCR, and Real Time PCR. All of them are inducible by ABA, and three are particularly responsive to water stress. These three have been cloned into several vectors in an effort to over-express them and measure their enzyme activity. Although this is very difficult to accomplish with E. coli, because this bacterium does not contain a functional P450 reductase, we have reconstituted an enzyme system using proteins synthesized in E. coli and reductase purified from rat livers. In all three cases we have demonstrated very small rates of conversion of ABA to 8-hydroxy-ABA. The characterization of the product was done by LC-MS. Although another sub-family of P450s (CYP707A) has recently been shown to have substantial ABA 8-hydroxylation activity, we are currently examining the reasons for our poor yields. We have now prepared one of these constructs in yeast with an Arabidopsis ATR, and we are preparing to express the protein and
measure its enzyme activity in this environment.
Impacts
The impact of this research is limited at the present time. This is basic research which lays the foundation for applications that may have a direct impact on plant metabolism, growth and development, and the ability of plants to respond to stress. The localization research could provide enough information that would allow whole biosynthetic pathways to be engineered into a single organelle. This principal might be used to manufacture large quantities of complex organic molecules in cell cultures, or even in yeast fermentation tanks. The other aspect of this research that has potential for improving agricultural productivity is related to the ABA hydroxylase genes (CYP72A sub-family). If these turn out to have substantial enzyme activity, we may be able to artificially regulate this enzyme to assist plants in responding even more effectively to environmental stress.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
We have continued our search for cytochrome P450-dependent monooxygenases that may be localized in the chloroplast. Using in-vitro chloroplast import studies with radioactive proteins we have determined that CYP79B2, an enzyme involved in auxin biosynthesis, is probably not targeted to chloroplasts even though other enzymes in the auxin biosynthetic pathway appear to be located there. On the other hand, we have firm evidence that CYP97A3, an Arabidopsis P450 of unknown function, is located inside the chloroplast and processed presumably to remove the transit peptide. The results from import studies were confirmed by an independent technique, the visual observation of the P450 protein fused to a green fluorescent protein (GFP), after transient expression of a cDNA fusion construct. This is the first report of a monooxygenase that is imported. This research is continuing to determine exactly where inside the chloroplast the enzyme resides. We have enjoyed collaboration
with Dr. John Froehlich at Michigan State University on this project. And, we are almost to the stage of writing a manuscript for publication. Cytochrome P450-dependent monooxygenases that are associated with the chloroplast must also be located near a reductase enzyme to pass electrons to the P450. Our initial studies with the two known P450 reductases in Arabidopsis have not indicated that either of these is associated with the chloroplast. We are currently examining a third possible reductase that is documented in GenBank as having substantial sequence homology to Arabidopsis reductase 1 and Arabidopsis reductase 2. Experiments are underway to express this protein in E. coli and in yeast cells and determine its enzyme activity. We have identified six genes that we suspect may be involved in abscisic acid metabolism in Arabidopsis. We have analyzed the expression profiles of each of these under different forms of environmental stress. These genes have also been cloned into
expression vectors and we are preparing to express them in E. coli and yeast. In the near future we hope to be able to confirm the function of one or more of these genes. Some of the work on this project is being done in collaboration with Dr. Danielle Werck at CNRS in Strasbourg, France.
Impacts
The impact of our work on localization of cytochrome P450 dependent monooxygenases and reductases is on basic research at the present time. Our discovery of a P450 inside chloroplasts provides impetus for searching for others of the 270 Arabidopsis P450s which may be localized in the same place. The same is true for the reductases. It is also of great interest to discover the function of CYP97A3, as this will tell us a great deal about the location of other enzymes and the metabolic pathway related to this P450. The impact of the work on abscisic acid is potentially much more immediate. Abscisic acid is the plant stress hormone. Cloning a gene that encodes the enzyme that directly controls the cellular concentration of this hormone has profound implications for regulating plant responses to stress.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
One of our objectives is to determine the subcellular localization of cytochrome P450 enzymes that are predicted by their putative chloroplast targeting sequences. We identified two of the most likely candidates for chloroplast targeting and cloned them from Arabidopsis. One of them is in the biosynthetic pathway for auxins. If this one is localized in plastids, it would be the second plant hormone that has part of its biosynthetic pathway associated with plastids. The cDNAs isolated in this manner were then used to prepare 35S-labeled proteins. The potential import of these proteins is now under investigation. This work is in collaboration with John Froehlich at Michigan State University. Construction of P450-GFP fusion proteins is in beginning stages and will provide an independent method for determining the subcellular localization. Another objective is to determine the nature of reducing enzymes that would be required for reduction of P450s in chloroplasts. There
are no known reductases in chloroplast membranes. However, working with a French collaborator, Phillipe Urban, we have identified a putative chloroplast P450 reductase, and begun experiments to determine if it is in the plastid. In addition, preparing DNA constructs for P450-P450 reductase fusion proteins that may be expressed in bacterial cells to directly measure enzyme activity of the fused proteins. This work is also in the beginning stages. Some of our previous studies, in collaboration with Ralph Nicholson, led to the isolation of eight cDNA clones for the enzyme chalcone synthase, an enzyme in the pathway producing antifungal compounds in Sorghum. One of the genes was found to be up-regulated by fungal infection of the plant. Cinnamic acid hydroxylase is a P450 near chalcone synthase in the pathway that leads to production of the antifungal compounds. We are interested in determining if there is more than one gene for cinnamic acid hydroxylase, and if one might be co-regulated
with the gene for chalcone synthase. Preliminary experiments indicate that there are three genes for cinnamic acid hydroxylase. If this is confirmed, we will determine if there is differential regulation of any of these in response to fungal infection. Regulation of genes like these that help a plant resist fungal diseases can be very important to the protection of our crops.
Impacts
The impact of our work on the subcellular localization of enzymes involved in production and degradation of plant hormones is so far limited to basic research. Knowing how enzymes in biosynthetic and metabolic pathways are clustered in space can be important in allowing us to promote specific beneficial aspects of plant growth and development. The same is true for the enzymes we are studying that are involved in production of antifungal compounds.
Publications
- Lo, S.C. Clive, Ronald C. Coolbaugh and Ralph L. Nicholson. 2002. Molecular characterization and in silico expression analysis of a chalcone synthase gene family in Sorghum bicolor. Physiological and Molecular Plant Pathology. (61), 179-188.
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Progress 10/01/00 to 09/30/01
Outputs
Based upon the nucleotide sequence of a cytochrome P450 gene located on chromosome 5 of Arabidopsis thaliana, we hypothesized that the protein it encodes may be targeted for the chloroplast. We isolated a cDNA clone for CYP86B1, sequenced it (GenBank accession AF345898), and expressed it in E. coli. Using 35S-labeled proteins and in vitro chloroplast import techniques we demonstrated that CYP86B1 is localized in the outer envelope membrane of the chloroplast in pea seedlings. Of the 273 members of the superfamily of oxidative enzymes known as cytochromes P450, this is the first monooxygenase to be characterized in the chloroplast. As P450-dependent monooxygenases require reduction prior to their reaction catalysis we have now begun a search for a chloroplast-localized P450 reductase. Constructs of the two NADPH-cytochrome P450 reductases from Arabidopsis have been prepared. 35S-labeled reductase import studies into chloroplasts will be supplemented with experiments
involving transient expression of genes encoding reductase-GFP fusion proteins. Another P450 of great interest to us is the cinnamic acid hydroxylase from sorghum seedlings. This enzyme catalyzes one of the first steps in the synthesis of phytoalexins in this species. This work is being done in conjunction with Ralph Nicholson, a specialist in sorghum phytoalexin biosynthesis. We have isolated a clone from a cDNA library and submitted its sequence to GenBank (accession AY034143). We are interested in determining if there is more than one gene in sorghum for this enzyme and studying the regulation of this (these) genes during infection.
Impacts
One of the priorities of the USDA/NRI program is to better understand the compartmentation of biochemical processes in plants, as it affects the regulation of these processes. Since the class of enzymes known as P450-dependent monooxygenases is involved in pesticide metabolism, natural defense against pathogens, and production of plant growth hormones, the subcellular localization of these enzymes may be important in their regulation. We have reported the first example of a P450-dependent monooxygenase localized in the outer membrane of plant chloroplasts.
Publications
- Christy J. W. Watson, John E. Froehlich, Caroline A. Josefsson, Clint Chapple, Francis Durst, Irene Benveniste, and Ronald C. Coolbaugh. 2001. Localization of CYP86B1 in the Outer Envelope of Chloroplasts. PLANT AND CELL PHYSIOLOGY. 42 (8): 873-878.
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Progress 10/01/99 to 09/30/00
Outputs
The first committed step in the pathway of gibberellin biosynthesis occurs in plastids. We are interested in determining the localization of the next few steps in this pathway. Kaurene oxidation is catalyzed by a cytochrome P450-dependent monooxygenase enzyme. Based upon the nucleotide sequence of the gene encoding kaurene oxidase, we hypothesized that this protein and a few others P450s may be targeted for the chloroplast. Using 35S-labeled proteins and antibodies we have now shown that CYP86B1 is localized in the outer envelope membrane of the chloroplast in pea seedlings. Of the nearly 300 cytochromes P450 in plants, this is the first report of a location outside of the endoplasmic reticulum membrane network. Similar experiments are being done with CYP701 (kaurene oxidase) and preliminary studies indicate that it is not localized in the chloroplast.
Impacts
One of the priorities in the USDA/NRI program is to better understand the compartmentation of biochemical processes in plants, as it affects the regulation of these processes. Our recent results represent the first report of a P450 in plants located outside of the cellular membrane system known as the endoplasmic reticulum. It is quite likely that additional P450s of the nearly 300 in plants will be found in similar locations. These results open a new avenue of research related to the regulation of the many and diverse metabolic pathways that include cytochromes P450.
Publications
- Christy J. W. Watson, Caroline A. Josefsson, Clint Chapple, Francis Durst, Irene Benveniste, John E. Froehlich, and Ronald C. Coolbaugh. 2000. Localization of CYP86B1 in the Outer Envelope of Chloroplasts. Submitted to Plant Physiology.
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Progress 10/01/98 to 09/30/99
Outputs
We have cloned a gene from Arabidopsis thaliana which encodes a cytochrome P450 protein. This protein has been named CYP86B1 based upon its sequence homology with CYP86A1, a known fatty acid hydroxylase. A cDNA for CYP86B1 was obtained from a cDNA library, expressed in E. coli, and purified from the bacterial membranes. The purified protein exhibits typical spectral properties of cytochromes P450. The peptide sequence of CYP86B1 contains a putative chloroplast-targeting domain at the N-terminus. If this enzyme is localized in chloroplasts, it will be the first cytochrome P450 shown to reside in this organelle. Current efforts are aimed at confirming the sub-cellular localization of CYP86B1 using antibodies, and in vitro-synthesized CYP86B1 protein. In addition, we are searching for the function of the protein using reconstitution enzyme assays with a variety of substrates, and by searching for a phenotype for knockout mutants of Arabidopsis which are being produced by
transformation with antisense DNA.
Impacts
These procedures have paved the way for future studies which will focus on the functional analysis of a number of P450 genes that have already been cloned and to clone new genes in this family which are key to the production of important plant products.
Publications
- No publications reported this period
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Progress 10/01/97 to 09/30/98
Outputs
The gene identified by the Kazusa Research Institute in Japan as MKD15.2 was cloned. This gene was originally suspected of being GA3, the gene encoding kaurene oxidase in the gibberellin biosynthetic pathway due to it's proximity to the mapped locus of a mutant gene which causes dwarfism in arabidopsis. The cDNA was then expressed in E. coli, and the product was shown to have a carbon dioxide difference spectrum characteristic of cytochromes P450. The protein has been partially purified and efforts are underway to characterize the function and expression pattern of the protein. This gene does not encode kaurene oxidase, the gene for which was cloned this year by a group in Australia. Based upon their sequence, we have now cloned the GA3 gene by PCR and are in the process of studying its expression by RT-PCR using an internal standard. We have reinitiated our search for the next gene in the gibberellin biosynthetic pathway, kaurenoic acid hydroxylase.
Impacts
(N/A)
Publications
- Abstract: Christy Watson, Caroline Josefsson, Clint Chapple, Francis Durst, and Ron Coolbaugh. 1998. Toward cloning the GA3 gene from Arabidopsis thaliana. In "Program and Abstracts of Fourth International Symposium on P450 Biodiversity and Biotechnology," Strasbourg, France.
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Progress 10/01/96 to 09/30/97
Outputs
The Arabidopsis mutant designated ga3 is reported to be blocked in gibberellin biosynthesis at the site step of kaurene oxidase. Our experiments to test the effects of intermediates in this pathway on seed germination tend to confirm the earlier report. Additional experiments to measure enzyme activity of kaurene oxidase in wild-type and mutant plants are underway. A genomic library has been screened with one probe so far, and yielded promising results for the isolation of new candidates for the gene encoding kaurene oxidase. We are searching for other probes which may be used to help identify and clone the locus of the ga3 mutation.
Impacts
(N/A)
Publications
- Coolbaugh, Ronald C. 1997. Cytochrome P450-dependent steps in gibberellin synthesis. Proceedings of the 1997 annual meeting of the Plant Growth Regulation Society of America. (part of a symposium on "Gibberellin Control in Plants: From Molecular Biology to Commercial Application."
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Progress 10/01/95 to 09/30/96
Outputs
Our reserch during the past year has continued toward the objectives of cloning genes for cytochromes P450 which are invloved in both gibberellin biosynthesis and herbicide metabolism. The studies on gibberellin biosynthesis have resulted in the partial sequencing of three more cDNAs which resulted from subtractive hybridization between fungal cells capable and not capable of making gibberellins. These results were presented at the meeting of the Plant Growth Regulation Society of America meeting in August which was held in Calgary Canada in conjuction with the Japanese Plant Growth Substances Society. In addition, we have turned to Arabidopsis thaliana as a plant for isolation of genes in the gibberellin biosynthetic pathway. This plant has obvious advantages over many others, including the availability of gibberellin mutants and cDNAs for about 60% of the genome. We are using one of these which encodes a related cytochrome P450 to screen a genomic library. A
graduate student, David Galitz, completed his M.S. degree thesis on the partial purification of a cytochrome P450-dependent enzyme, flumetsulam hydroxylase, which metabolizes an important herbicide. Studies have been initiated to use the partial amino acid sequence of this enzyme to clone the gene from wheat.
Impacts
(N/A)
Publications
- Jennings, James C., Banks, J.A., Coolbaugh, R.C. 1996. Subtractive Hybridization between cDNAs from Untreated and AMO-1618-Treated Cultures of Gibberella fujikori. Plant and Cell Physiol. 37,847-854.
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Progress 10/01/94 to 09/30/95
Outputs
Our research has continued along two lines related to plant and fungal cytochromes P450: one is related to cloning the gene for kaurene oxidase, or the closely related kaurenoic acid hydroxylase; the other is related to cloning the gene for a herbicide (flumetsulam) metabolizing enzyme from wheat. The PI was on sabbatical leave at the U. Strasbourg in France from January through June, 1995. During this time he continued the work initiated at Purdue by working on sequencing clones previously isolated from the fungus Gibberella fujikuroi. In addition, he made effort to utilize antibodies obtained from Peter Bramley, England, which should have recognized kaurene oxidase from the fungus. An additional project was to try to isolate kaurene oxidase from a cDNA library from Arabidopsis using a clone for a plant cytochrome P450 isolated by a colleague in France. None of these projects has resulted in publication at this time. The project on the herbicide metabolizing enzyme
from wheat has progressed quite well recently. The purified enzyme has now been partially sequenced and a degenerate nucleotide primer has been produced which will be used to screen a cDNA library from wheat. This project was reported recently at the Third International Symposium on Cytochrome P450 in Woods Hole, MA. Oct. 7, 1995.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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Progress 10/01/93 to 09/30/94
Outputs
Our research involves the characterization of plant and fungal cytochromes P450,a class of oxidative enzymes, ubiquitous to living organisms, and important in many agriculturally significant metabolic pathways, e.g. there are cytochrome P450-dependent enzymes involved in the biosynthesis of plant hormones, fatty acids, sterols, lignins, aflatoxins, and other important plant products. Plus, cytochromes P450 are involved in the metabolism of drugs, herbicides, insecticides, aflatoxins, and other toxic substances. Our work on these enzymes began from interest in the biosynthesis of gibberellins and abscisic acid. In the case of gibberellins in plants and fungi, a series of four reactions in the biosynthetic pathway has been shown to be cytochromes P450. We are trying to isolate, purify, and clone the genes for these enzymes. Recent work resulted in the isolation of four cDNAs which are present in wild-type fungi, but are absent in either mutant fungi or cultures which are
chemically treated to lack the oxidative enzyme activity. To date, one of these clones, absent in both mutant and chemically treated fungi, has been sequenced and is being further characterized. We have also initiated a study to isolate and purify a gene from wheat seedlings which metabolizes the herbicide flumetsulam. An enzyme assay has been developed and the enzyme activity has been solubilized with sodium cholate. We are now attempting to resolve the component part of the reaction complex by chromatographic procedures.
Impacts
(N/A)
Publications
- JENNINGS, J.C., J.A. BANKS, and R.C. COOLBAUGH. 1994. Characterization of cDNAs from subtractive hybridization to isolate kaurenoic acid hydroxylase. Plant Physiol. 105: 15 (Abstract # 13). (manuscript in preparation).
- GALITZ, D.S. and R.C. COOLBAUGH. 1994. Partial purification of flumetsulam hydroxylase activity. Poster presented at the 3rd Annual Midwest Symposium on Cytochromes P450. West Lafayette, IN. September 9, 1994.
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Progress 10/01/92 to 09/30/93
Outputs
We are continuing our research on the biosynthesis of plant hormones in plants and fungi. Progress has been made on the isolation, purification and characterization of cytochrome P450-dependent monooxygenases involved in the biosynthesis of gibberellins. We have continued to purify kaurene oxidase from immature seeds of pumpkin. A recent success has been to reconstitute enzyme activity with purified kaurene oxidase, commercially available phosphatidyl choline and cytochrome P450 reductase purified from rat livers. Although results are still preliminary, they are very promising. We have also prepared an expression library from the fungus, Gibberella fujikuroi. This library is being screened with probes which have been produced by subtraction hybridization using wild-type and mutant fungi. The mutant has been shown to be missing kaurenoic acid hydroxylase activity. I attended the Gordon Conference on Cytochromes P450 in Agriculture and 2nd International Symposium on
Plant and Microbial Cytochromes P450. Valuable contacts have resulted in an e-mail network of people interested in plant cytochromes P450. We have obtained clones of three plant and fungal cytochromes P450 from these colleagues and are in the process of using these as probes for our fungal enzyme. We also hosted the second annual Midwest P450 Symposium.
Impacts
(N/A)
Publications
- JENNINGS, J.C., R.C. COOLBAUGH, D.A. NAKATA, and C.A. WEST. 1993. Characterization and Solubilization of kaurenoic acid hydroxylase from Gibberella fujikuroi. Plant Physiology. 101: 925-930.
- HAZEBROEK, J.P., S.E. SCHECHTER, J.D. METZGER, and R.C. COOLBAUGH. 1993. Presence of endogenous ent-kaurene in a microsomal preparation from Cucurbita maxima L. endosperm and implications for kinetic studies of ent-kaurene oxidase.
- JENNINGS, J.C., D.S. GALITZ, and R.C. COOLBAUGH. 1993. Regulation of kaurenoic acid hydroxylase in Gibberella fujikuroi. Plant Physiology. 102S: 65 (Abst. 352).
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