Progress 09/01/07 to 08/31/11
Outputs
OUTPUTS: Throughout the four year funding period we have finalized characterizing key enzymes in the biosynthesis of the insect- and pathogen induced homoterpene volatiles, TMTT and DMNT, in Arabidopsis leaves and roots, respectively. We were able to extent the project by investigating the enzymatic formation of homoterpene volatiles in poplar. Our work has resulted in five publications, one book chapter, and three manuscripts in preparation. The project provided training for postdoctoral researcher Dr. Sungbeom Lee and two Ph.D. students, Jung-Hyun Huh and Reza Sohrabi, and support for a technician (fall 2007 to spring 2008). Ph.D. student Huh has finished her dissertation in August 2011 and student Reza Sohrabi is expected to finalize his dissertation in the summer of 2012. Furthermore, training was provided for a total of five undergraduate students including one minority student (Titi-Mary Omotade). Dr. Lee and the two graduate students presented their research in the form of 9 posters at local/regional, national, and international meetings (including three Gordon Conferences) with two best poster awards to Jung-Hyun Huh and two best poster awards to Reza Sohrabi. Jung-Hyun Huh's work was featured in the ASPB newsletter (2009) and she won best oral presentation at the Molecular Plant Sciences Mini-Symposium at Virginia Tech (2011). Reza Sohrabi was an invited speaker at the Biological Sciences Research Day at Virginia Tech (2010). Six invited talks or oral presentations were given by the P.D.. Studies on the enzymatic mechanism of TMTT biosynthesis resulted in collaborative research with Dr. Christiane Gatz and her former graduate student Dr. Marco Herde (Georg-August University, Goettingen, Germany) and with Dr. David Bevan (Department of Biochemistry, Virginia Tech) and graduate student Somayesadat Badieyan (Department of Biosystems Engineering, Virginia Tech). The latter collaboration resulted in a publication in PNAS and was featured by the Virginia Tech News (http://www.vtnews.vt.edu/articles/2010/11/112310-research-pnastholl. html). Knowledge of the root-infection assays established by student Jung-Hyun Huh for the Arabidopsis-Pythium (root rot) pathosystem was transferred to studies on the role of sugar transporters and jasmonate-binding proteins in root defense in collaboration with Dr. Wolf Frommer (Carnegie Institution for Science, Stanford) and Dr. Christopher Lawrence (Virginia Bioinformatics Institute, Virginia Tech), respectively. Educational outreach activities of the Tholl lab included class experiments with teacher John Kowalski (2007, 2008) and research training of a student (summer 2010) from the Roanoke Valley Governor's School in conjunction with PREP (Partnership for Research and Education in Plants, Virginia Tech). Related to these activities, the P.D. produced two educational videos on the GC/MS analysis of volatile compounds with Dr. David Lally (PREP), which have been posted on YouTube. In addition, the P.D. presented three lectures to Governor's School teacher Steven Smith and students (fall 2008, 2009, and 2010) and provided Steven Smith with materials for collecting plant volatiles. PARTICIPANTS: Project Director: Dr. Dorothea Tholl. The P.D. supervised postdoctoral researcher Dr. Lee and the two Ph.D. students, Jung-Huyn Huh and Reza Sohrabi. Postdoctoral Researcher: Dr. Sungbeom Lee. Dr. Lee discovered and characterized the homoterpene synthase CYP82G1 from Arabidopsis. He is the primary author of one published manuscript and one manuscript in preparation and co-author of two published review articles and one book chapter. Ph.D. students: Jung-Hyun Huh has identified and characterized two root-specific sesquiterpene synthases with an unexpected subcellular localization in mitochondria. She characterized the role of the homoterpene DMNT in root defense against the root-rot pathogen Pythium irregulare. She is coauthor of one review article and first and coauthor of two manuscripts to be submitted in spring 2012 (directly related to the project). Reza Sohrabi studied the pathogen-induced enzymatic formation of DMNT from a triterpene precursor in Arabidopsis roots. He is coauthor of one review article and first author of one manuscript to be submitted in spring 2012 (directly related to the project). Collaborators: Dr. Christiane Gatz and former Ph.D. student Marco Herde (Georg-August University, Goettingen, Germany) were largely involved in identifying the first enzymatic step in TMTT formation. They provided mutant lines that helped indentify the P450 gene CYP82G1. Dr. David Bevan (Department of Biochemistry, Virginia Tech) and Ph.D. student Somayesadat Badieyan (Department of Biological Systems Engineering, Virginia Tech) constructed a homology model (together with Dr. Lee) for CYP82G1 and conducted docking experiments to probe the substrate specificity of the enzyme and predict possible reaction mechanisms. Training was provided for five undergraduate biology and biochemistry majors including minority student Titi-Mary Omotade. TARGET AUDIENCES: Related to this project, the P.D. taught a new graduate/undergraduate course on the chemical communication of plants in fall 2008 and 2010 at Virginia Tech. The P.D. also taught a module on plant chemical defense in the graduate class Molecular Cell Biology and Biotechnology Topics (BIOL6024) at Virginia Tech. The Tholl lab collaborated on a research project on root volatile formation with teacher John Kowalski (2007, 2008) from the Roanoke Valley Governor's School as part of the pre-college education program PREP (Partnership for Research and Education in Plants, Virginia Tech, Dr. Erin Dolan) and provided research training for one Governor's school student (summer 2010). The P.D. presented three lectures on the biology and agricultural application of plant volatiles and provided research material for plant volatile collection to Governor's School teacher Steven Smith and students (fall 2008, 2009, 2010). PROJECT MODIFICATIONS: While we did not encounter major changes, the discovery of a novel pathway for the biosynthesis of DMNT in Arabidopsis roots was an unexpected outcome of the project.
Impacts
OBJECTIVE 1. This objective has been completed by the end of the funding period. To identify the enzymatic steps responsible for the root-rot (Pythium irregulare)-induced formation of the putative DMNT precursor, (E)-nerolidol, in Arabidopsis roots, Ph.D. student Jung-Hyun Huh characterized two root-specific terpene synthases (TPS25 and TPS22), which produce sesquiterpenes including beta-farnesene as the primary product and (E)-nerolidol. However, studies of the respective single or double knockout plant lines of these genes could not provide evidence for their primary role in DMNT formation. Despite this finding, characterization of TPS25 and TPS22 revealed a novel and unexpected subcellular localization of these terpene synthases in mitochondria. Jung-Hyun Huh finalized the characterization of both enzymes in August 2011 and a manuscript will be submitted in spring 2012. Studies by Ph.D. student Reza Sohrabi showed that DMNT is produced via a novel pathway by breakdown of the triterpene precursor arabidiol. With support by this grant until spring 2010, student Sohrabi identified two genes, the cytochrome P450 monooxygenase CYP705A1 and the arabidiol triterpene synthase AtPEN1, which are responsible for DMNT formation. Student Jung-Hyun Huh demonstrated that DMNT as a volatile compound retards mycelium growth and oospore germination of Pythium, and mutant lines of CYP705A1 and AtPEN1 are more susceptible to infection by Pythium. A manuscript is in preparation to be submitted in March 2012. OBJECTIVE 2. We have completed this objective with the characterization of the two-step pathway for the insect-induced biosynthesis of the volatile homoterpene TMTT in Arabidopsis leaves. Postdoctoral researcher Dr. Sungbeom Lee identified the first homoterpene synthase, which produces both volatiles, DMNT and TMTT, from the alcohols (E,E)-geranyllinalool and (E)-nerolidol, respectively, in vitro, and this work has been published in PNAS. Dr. Lee has also identified and characterized inducible homoterpene synthase homologs in poplar that have different product specificity. A manuscript of this work is in preparation. In addition, transgenic Arabidopsis lines have been generated that constitutively produce TMTT and will be used to test the role of TMTT in the attraction of insect parasitoids. OBJECTIVE 3. Based on the elucidation of the homoterpene biosynthesis pathways in leaves and roots (Objective 1 and 2) and studies using pathway inhibitors, we found that both DMNT and TMTT are produced in the cytosol/endoplasmic reticulum but the precursor for leaf-specific TMTT biosynthesis appears to be derived from plastids. In summary, our results have revealed two evolutionary distinct enzymatic pathways for the formation of homoterpenes above- and belowground (summarized in two review articles) and have demonstrated that enzymes homologous to CYP82G1 function as homoterpene synthases in poplar. We have assembled a gene tool kit (CYP82G1 and its precursor gene GES) that allows for metabolic engineering of homoterpene formation to specify and exploit the role of these compounds as components of insect-induced volatile blends in biological pest control.
Publications
- Journal Articles: Herde, M., Gaertner, K., Koellner, T.G., Fode, B., Boland, W., Gershenzon, J., Gatz, C., and Tholl, D. (2008) Identification and regulation of TPS04/GES, an Arabidopsis geranyllinalool synthase catalyzing the first step in the formation of the insect-induced volatile C16-homoterpene TMTT. Plant Cell 20: 1152-1168.
- Lee, S., Badieyan, S., Bevan, D.R., Herde, M., Gatz, C., and Tholl, D. (2010) Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis, Proc. Natl. Acad. Sci. USA 107: 21205-21210.
- Chen, F., Tholl, D., Bohlmann, J., and Pichersky, E. (2011) The family of terpene synthases in plants: A mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom, Plant J. 66: 212-229.
- Tholl, D., Sohrabi, R., Huh, J.-H., and Lee, S. (2011) The biochemistry of homoterpenes, common constituents of floral and herbivore-induced plant volatile bouquets, Phytochemistry 72: 1635-1646.
- Periodicals (peer-reviewed): Tholl, D. and Lee, S. (2010) Elucidating the metabolism of plant terpene volatiles: Alternative tools for engineering plant defense In D. Gang (ed.) Recent Advances in Phytochemistry, Springer, 41, p.159-178.
- Book Chapter: Tholl, D. and Lee, S. (2011) Terpene specialized metabolism in Arabidopsis; The Arabidopsis Book 9:e0143 DOI: 10.1043/tab.0143.
- Abstracts: 1. Tholl, D. (2007). Synthesis and function of volatile terpenes: News from the Arabidopsis model, June 2007, Program and abstract book, 2007 Annual International Meeting and Symposia of the Phytochemical Society of North America, St. Louis, Missouri, June 2007 (invited talk).
- 2. Tholl, D. (2008). Plant volatiles in above and belowground plant-insect interactions: understanding pathways and molecular regulation. Abstract book, The Rank Prize Funds Mini Symposium on Chemical and Visual Ecology of Arthropods: From Genes to Pest Management, September 2008, Windermere, UK (invited talk).
- 3. Huh, J.-H. and Tholl, D. (2008). Volatile terpene formation in Arabidopsis roots in interaction with the soil-borne pathogen Pythium irregulare. Program and abstract book, 25th Mid-Atlantic Plant Molecular Biology Society Conference, Savage, Maryland, August 2008 (poster).
- 4. Sohrabi, R. and Tholl, D. (2008). Biochemical and molecular analysis of the formation of the stress-induced volatile C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene in Arabidopsis roots. Program and abstract book, 25th Mid-Atlantic Plant Molecular Biology Society Conference, Savage, Maryland, August 2008 (poster).
- 5. Vaughan, M., Huh, J.-H., Sohrabi, R., Wray, A., Askew, W., Tholl, D. (2008). Hidden defense metabolism: Volatile terpene formation in Arabidopsis roots. Electronic scientific program, Banff Conference on Plant Metabolism, Banff, Canada, July/August 2008 (oral presentation, M. Vaughan).
- 6. Lee, S., Herde, M., Gatz, C., and Tholl, D. (2009) Unraveling the biosynthesis of volatiles in plant defense: A single CytP450 enzyme is responsible for the conversion of geranyllinalool to the insect-induced homoterpene TMTT in Arabidopsis thaliana. Program and abstract book, 2009 Annual International Meeting and Symposia of the Phytochemical Society of North America, Towson, Maryland, August 2009 (oral presentation, D.Tholl).
- 7. Sohrabi, R. and Tholl, D. (2009) Biochemical and molecular analysis of the formation of the stress-induced C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene in Arabidopsis roots. Program and abstract book, 49th Annual International Meeting and Symposia of the Phytochemical Society of North America, Towson, Maryland, August 2009 (best poster award).
- 8. Tholl, D. (2009) Plant terpene volatiles in above- and below-ground plant-organism interactions: understanding biosynthetic pathways and molecular regulation. Program and abstract book, International Meeting of the American Chemical Society, Washington D.C., Session Process and Stress-induced Changes in Phytochemicals, August 2009 (invited talk).
- 9. Lee, S., Badieyan, S., Bevan, D., Herde, M., Gatz, C., and Tholl, D. (2010) The simplicity of making insect induced homoterpene volatiles: A single Arabidopsis CytP450 enzyme catalyzes the final step in the formation of DMNT and TMTT. Electronic conference proceedings (abstract), Banff Conference on Plant Metabolism, Banff, Alberta, Canada, June, 2010 (oral presentation, D.Tholl).
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: In the third year of funding, research activities were focused on continuing and finalizing the characterization of key enzymes in the biosynthesis of the insect- and pathogen induced homoterpene volatiles, TMTT and DMNT, in Arabidopsis leaves and roots, respectively. Our progress has resulted in two publications, two manuscripts and a book chapter in review, and two manuscripts in preparation. Continued training has been provided for postdoctoral researcher Dr. Sungbeom Lee and two graduate students, Jung-Hyun Huh and Reza Sohrabi (R.S. until May 2010). Graduate student Huh is expected to finish her dissertation in spring 2011. Miss Huh has supervised one minority student (Titi-Mary Omotade) in fall 2009 and spring 2010. Dr. Lee and Miss Huh supervise one other undergraduate student since fall 2010, who assists with their experiments. Dr. Lee and the two graduate students presented their research in the form of posters at a Molecular Plant Sciences Mini-Symposium at Virginia Tech. Reza Sohrabi won best poster award at this symposium. He also was an invited speaker at the Biological Sciences Research Day at Virginia Tech in February 2010 and gave a poster presentation at the Gordon Conference on Plant Molecular Biology, July 18-23, 2010, Holderness, New Hampshire. Studies on the enzymatic mechanism of TMTT formation have resulted in collaborative research with Dr. David Bevan (Department of Biochemistry, Virginia Tech). Dr. Bevan and graduate student Somayesadat Badieyan (Department of Biosystems Engineering, Virginia Tech) conducted modeling and substrate docking on the identified Arabidopsis TMTT synthase. The collaboration resulted in a recent publication in PNAS (see below). The work has been featured by the Virginia Tech News (http://www.vtnews.vt.edu/articles/2010/11/112310-research-pnastholl. html). Knowledge of the root-infection assays established by graduate student Jung-Hyun Huh for the Arabidopsis-Pythium (root rot) pathosystem has been transferred to studies on the role of sugar transporters and jasmonate-binding proteins in root defense in collaboration with Dr. Wolf Frommer (Carnegie Institution for Science, Stanford) and Dr. Christopher Lawrence (Virginia Bioinformatics Institute, Virginia Tech), respectively. Educational outreach activities of the Tholl lab have included research training of a student from the Roanoke Valley Governor's School in the summer of 2010 by graduate student Jung-Hyun Huh on the role of DMNT in Arabidopsis root defense. In addition, the P.I. presented a lecture on the biology and agricultural application of plant volatiles to Governor's School teacher Steven Smith and students in the fall of 2009. The Tholl lab provided Steve Smith with solid phase microextraction devices for trapping volatile compounds in experiments conducted by his students on the emission and role of volatiles in plant defense. PARTICIPANTS: Project Director: Dr. Dorothea Tholl The P.D. supervised postdoctoral researcher Dr. Lee and two graduate students. Postdoctoral Researcher: Dr. Sungbeom Lee (since March 2008). Dr. Lee discovered and characterized the homoterpene synthase CYP82G1 from Arabidopsis. Dr. Lee is the primary author of one manuscript and co-author on two manuscripts and one book chapter. Graduate students: Jung-Hyun Huh has identified and characterized two root-specific sesquiterpene synthases with an unexpected subcellular localization in mitochondria. She studies the role of the homoterpene DMNT in root defense against the root-rot pathogen Pythium irregulare. She is coauthor on one manuscript and will be first and coauthor on two manuscripts to be submitted in spring 2011 (related to the project). Reza Sohrabi studies the pathogen-induced enzymatic formation of DMNT from a triterpene precursor in Arabidopsis roots. He is coauthor on one manuscript and will be first author on another manuscript to be submitted in spring 2011 (related to the project). Collaborators: Dr. David Bevan (Department of Biochemistry, Virginia Tech), Somayesadat Badieyan (Department of Biological Systems Engineering, Virginia Tech). Dr. Bevan and graduate student Somayesadat Badieyan constructed a homology model (together with Dr. Lee) for the P450 CYP82G1 and conducted docking experiments to probe the substrate specificity of the enzyme and predict possible reaction mechanisms. Training was provided for the above-mentioned postdoctoral researcher (Dr. Lee), two graduate students (J.-H. Huh, R. Sohrabi), and two undergraduate biology majors, Titi-Mary Omotade (African American) and Samuel Shon (by Dr. Lee and J.-H. Huh). TARGET AUDIENCES: Related to this project, the project director taught a module on plant chemical defense in the graduate class Molecular Cell Biology and Biotechnology Topics (BIOL6024) at Virginia Tech. The P.D. and graduate student Jung-Hyun Huh provided research training for one student (Screemoyee Som) from the Roanoke Valley Governor's School during the summer of 2010. The P.D. presented a lecture on the biology and agricultural application of plant volatiles and provided research material for plant volatile collection to Governor's School teacher Steven Smith and students in the fall of 2009. PROJECT MODIFICATIONS: While we did not encounter major changes, the discovery of a novel pathway for DMNT formation in Arabidopsis roots was an unexpected outcome of the project.
Impacts
We have completed one objective and are in the process of finalizing the two other objectives. OBJECTIVE 1. To identify the enzymatic steps responsible for the formation of the putative DMNT precursor, (E)-nerolidol, in Arabidopsis roots, we previously characterized two root-specific terpene synthases (TPS25 and TPS22), which produce (E)-nerolidol together with other sesquiterpene volatiles. Studies of the respective single or double knockout plant lines of these genes could not provide evidence for their primary role in DMNT formation. Despite this finding, characterization of TPS25 and TPS22 has revealed a novel and unexpected subcellular localization of these terpene synthases in mitochondria. Graduate student Jung-Hyun Huh is finalizing the characterization of both enzymes in preparation for a manuscript to be submitted in spring 2011. As part of this work, we plan to elucidate the possible reason for mitochondrial targeting of the root-expressed terpene synthases. Studies by graduate student Reza Sohrabi have substantiated the unexpected role of the triterpene arabidiol as the true precursor in root-specific DMNT formation in Arabidopsis. Two enzymes have been targeted - the cytochrome P450 monooxygenase CYP705A1 and the arabidiol triterpene synthase AtPen1, which are co-expressed upon Pythium infection. Yeast expression studies of both enzymes have provided additional proof for their function in DMNT formation. Further characterization of both enzymes and of the function of DMNT in root defense (by student Jung-Hyun Huh) using respective gene mutant lines are currently performed. A manuscript is in preparation to be submitted in spring 2011. OBJECTIVE 2. We have completed this objective with the discovery and characterization of the first homoterpene synthase, CYP82G1 (At3g25180), which is responsible for the insect-induced formation of TMTT in Arabidopsis leaves. The enzyme produces both volatiles, DMNT and TMTT, from the alcohols (E,E)-geranyllinalool and (E)-nerolidol, respectively, in vitro. The work conducted by postdoc Sungbeom Lee has recently been published in PNAS. Homology based-modeling has led to a more detailed analysis of the complex mechanism of the C-C cleavage reaction catalyzed by CYP82G1. In addition, Dr. Lee is characterizing putative homoterpene synthases related to CYP82G1 from other species including crops (e.g. Brassica). OBJECTIVE 3. Based on our previous and recent studies using pathway inhibitors, we have found that both DMNT and TMTT are produced in the cytosol/endoplasmic reticulum with the difference that the precursor for leaf-specific TMTT biosynthesis appears to be derived from plastids. We plan to corroborate these findings using stable-isotope labeled precursors. In summary, our results have revealed two evolutionary distinct enzymatic pathways for the formation of homoterpenes above- and belowground. With the identification of CYP82G1 and its precursor gene (Herde et al., 2008), we have assembled a gene tool kit that allows metabolic engineering of homoterpene formation to specify and exploit the role of these compounds as components of insect-induced volatile blends in biological pest control.
Publications
- Journal Articles: Lee, S., Badieyan, S., Bevan, D.R., Herde, M., Gatz, C., and Tholl, D. (2010) Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis, PNAS, doi: 10.1073/pnas.1009975107.
- Chen, F., Tholl, D., Bohlmann, J., and Pichersky, E. (2011) The family of terpene synthases in plants: A mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom, Plant J. Special Issue on The Plant Genome: An Evolutionary View on Structure and Function, under review.
- Tholl, D., Sohrabi, R., Huh, J.-H., and Lee, S. (2011) The biochemistry of homoterpenes, common constituents of floral and herbivore-induced plant volatile bouquets, Phytochemistry Special Issue on Plant-Insect Interactions, under review.
- Periodicals (peer-reviewed): Tholl, D. and Lee, S. (2010) Elucidating the metabolism of plant terpene volatiles: Alternative tools for engineering plant defense In D. Gang (ed.) Recent Advances in Phytochemistry, Springer, 41, p.159-178.
- Book Chapter: Tholl, D. and Lee, S. (2011) Terpene specialized metabolism in Arabidopsis; The Arabidopsis Book.
- Abstracts: Lee, S., Badieyan, S., Bevan, D., Herde, M., Gatz, C., and Tholl, D. (2010) The simplicity of making insect-induced homoterpene volatiles: A single Arabidopsis CytP450 enzyme catalyzes the final step in the formation of DMNT and TMTT. Electronic conference proceedings (abstract), Banff Conference on Plant Metabolism, Banff, Alberta, Canada, June 24-28, 2010.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: In the second year of funding, research activities have moved beyond the screening of gene candidates and have focused primarily on the characterization of key enzymes in the biosynthesis of the volatile C11- and C16-homoterpenes DMNT and TMTT in roots and leaves of Arabidopsis thaliana, respectively. Major progress has been achieved for all three objectives resulting in one manuscript in review and two manuscripts in preparation. The project continues to provide multidisciplinary training for one postdoc (Dr. Sungbeom Lee) and two graduate students (Jung-Hyun Huh, Reza Sohrabi) who are mentored by the P.I.. Graduate student Jung-Hyun Huh has supervised one undergraduate researcher during the summer of 2009 (Samuel Rutledge) and has begun mentoring a minority student (Titi-Mary Omotade) in the fall of 2009. The postdoc and graduate students have attended international meetings including the Gordon Conference on Plant Metabolic Engineering, 7-12 July 2009, New Hampshire. Graduate student Jung-Hyun Huh won a best poster prize at this meeting and will be featured in the newsletter of the American Society of Plant Biologists. Reza Sohrabi won a travel award and best poster award at the Annual International Meeting of the Phytochemical Society of North America, 8-12 August 2009,Towson, Maryland. The project has stimulated establishing root-infection assays with the soil borne pathogen Pythium irregulare to investigate the function of root-emitted homoterpene volatiles. In line with these functional assays, novel collaborations have emerged (Ben Field, Anne Osbourn, John Innes Center, UK; Wolf Frommer, Carnegie Institution for Science, Stanford) on root-specific molecular and chemical defense responses to Pythium infection. The Tholl lab has continued a research outreach partnership with the Roanoke Valley Governor's School that participates in the pre-college education program, Partnership for Research and Education in Plants (PREP). Novel contacts have been established with teacher Steven Smith and his students. The P.I. has presented a lecture on the biological function and agricultural importance of plant volatiles to Steven Smith's class during a field trip in the fall of 2008. In support of these activities, the P.I. together with Dr. David Lally (PREP) has produced an educational video on the GC-MS analysis of plant volatiles which has been posted on YouTube. PARTICIPANTS: Project Director: Dr. Dorothea Tholl Postdoc: Dr. Sungbeom Lee (since March 2008). Dr. Lee has identified and characterized the CytP450 enzyme responsible for the formation of the C16-homoterpene volatile TMTT. A publication is in preparation to be submitted in December of 2009. Dr. Lee also investigates the participation of the MEP and mevalonate pathways in TMTT biosynthesis. Graduate students: Jung-Hyun Huh has identified and biochemically characterized two root-expressed enzymes that produce the putative precursor nerolidol in C11-homoterpene volatile biosynthesis. A manuscript describing these results is in preparation to be submitted in spring 2010. Reza Sohrabi has identified a CytP450 gene involved in the root-specific biosynthesis of the C11-homoterpene DMNT and continues to characterize this gene and its encoded enzyme. Reza Sohrabi also investigates the role of the plastidial MEP pathway in root-specific homoterpene formation. Collaborators: Dr. Christiane Gatz (Georg-August University, Goettingen, Germany) Training was provided for the above-mentioned post-doctoral fellow (Dr. Lee), two graduate students (J.-H. Huh, R. Sohrabi) and the two undergraduate biology majors, Samuel Rudlege and Titi-Mary Omotade (African American). TARGET AUDIENCES: Related to this project on plant indirect defense biochemistry, the P.I. taught a new graduate/undergraduate course on plant-organism chemical communication in the fall of 2008. In addition, the P.I. collaborated on a research project on root volatile formation and function with teacher Dr. John Kowalski at the Roanoke Valley Governor's School as part of the pre-college education program, Partnership for Research and Education in Plants (PREP, director Dr. Erin Dolan), now to be continued with teacher Steven Smith. The P.I. has presented a lecture on the biological function and agricultural importance of plant volatiles to Steven Smith's class during a field trip in the fall of 2008. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Significant progress has been achieved on all three objectives. OBJECTIVE 1. To identify the enzyme(s) responsible for the formation of the possible precursor (E)-nerolidol in DMNT formation in Arabidopsis roots, two root-expressed terpene synthases, At3g29410 (TPS25) and At1g33750 (TPS22) were isolated (see previous report). TPS22 and TPS25 recombinant enzymes form (E)-nerolidol and two other sesquiterpenes as main products in vitro. Analysis of single and double mutant lines of both genes showed that TPS22 but not TPS25 is in part involved in the formation of DMNT. The results suggest possible redundancies in (E)-nerolidol formation or the existence of alternative DMNT biosynthetic pathways (see below). The role of (E)-nerolidol as a precursor of DMNT was supported by administering deuterium-labeled (E)-nerolidol to Arabidopsis hairy roots and demonstrating a partial conversion of the alcohol to DMNT. OBJECTIVE 2. We have identified CYP82G1 (At3g25180) as the single cytochrome P450 enzyme responsible for the final biosynthetic step in the insect-induced formation of TMTT in Arabidopsis leaves. Complementation a CYP82G1 gene knock-out line in combination with in vivo and in vitro characterization of recombinant, yeast-expressed CYP82G1 confirmed the function of this CytP450 as a novel TMTT synthase. CYP82G1 also converts (E)-nerolidol to DMNT in vitro, which shows that a single CytP450 enzyme can produce both homoterpene volatiles. CYP82G1 promoter-GUS analysis demonstrated that CYP82G1 is expressed locally at the site of insect feeding damage. Using a reversed genetics approach for CytP450s expressed in Arabidopsis roots treated with the defense hormone jasmonic acid, we have identified another CytP450 gene, CYP705A1 (At4g15330) that appears to be responsible for root-specific formation of DMNT. In vivo and in vitro functional characterization of CYP705A1 is in progress. Since CYP705A1 belongs to a gene cluster of triterpene biosynthesis genes and is co-expressed with the triterpene synthase AtPen1, DMNT might be in part derived from a triterpene precursor, which would represent a novel pathway in homoterpene volatile formation. OBJECTIVE 3. Our previous studies provided evidence that root-specific DMNT biosynthesis is dependent on precursors of the cytosolic mevalonate pathway. In contrast to these findings, we have demonstrated by inhibitor application that the formation of TMTT in leaves depends primarily on the plastidial MEP pathway. Since both enzymes involved in TMTT biosynthesis (GES, CYP82G1) are located in the cytosol and/or the ER, we propose that a directional transport of MEP-pathway derived precursors occurs from plastids to the cytosol in Arabidopsis leaves but that this cross-talk is absent in roots. In summary, substantial progress has been made on identifying genes responsible for key biosynthetic steps in homoterpene volatile formation. Our findings have important impact on analyzing related genes in crops such as Brassica. A primary goal is to use these gene tools in the metabolic engineering of homoterpene emissions for indirect plant defense thereby developing alternative pest control strategies above- and belowground.
Publications
- Abstracts: Lee, S., Herde, M., Gatz, C., and Tholl, D. (2009) Unraveling the Biosynthesis of Volatiles in Plant Defense: A single CytP450 Enzyme is responsible for the Conversion of Geranyllinalool to the Insect-induced Homoterpene TMTT in Arabidopsis thaliana. Program and abstract book, 49th Annual International Meeting and Symposia of the Phytochemical Society of North America, Towson, Maryland, 08/8-12/09, oral presentation, D.Tholl.
- Sohrabi, R. and Tholl, D. (2009) Biochemical and Molecular Analysis of the Formation of the Stress-Induced C11-Homoterpene (E)-4,8-dimethylnona-1,3,7-triene in Arabidopsis Roots. Program and abstract book, 49th Annual International Meeting and Symposia of the Phytochemical Society of North America, Towson, Maryland, 08/8-12/09, best poster award.
- Tholl, D. (2009) Plant Terpene Volatiles in Above- and Below-Ground Plant-Organism Interactions: Understanding Biosynthetic Pathways and Molecular Regulation. Program and abstract book, International Meeting of the American Chemical Society, Washington D.C., 08/16-20/09, invited talk at the Session Process and Stress-induced Changes in Phytochemicals.
- Huh, J.-H., Sohrabi, R., and Tholl, D. (2009) Assembling Gene Tools for Engineering Plant Defense Volatile Formation: Dissecting the Biosynthesis of the C11-Homoterpene DMNT (4,8-dimethylnona-1,3,7-triene) in Arabidopsis. Gordon Conference on Plant Metabolic Engineering, Waterville Valley, New Hampshire, 07/12-17/09; J.-H. Huh, best poster award.
- Lee, S., Herde, M., Gatz, C., and Tholl, D. (2009) Towards Metabolic Engineering of Volatiles in Plant Defense: A Multi-Catalytic Arabidopsis CytP450 Enzyme is Responsible for the Conversion of Geranyllinalool to the Insect-Induced Homoterpene TMTT. Gordon Conference on Plant Metabolic Engineering, Waterville Valley, New Hampshire, 07/12-17/09; poster presentation.
- Journal Articles: Tholl, D. and Lee, S. (2009) Elucidating the metabolism of plant volatiles: Alternative tools for engineering plant defenses? Advances in Phytochemistry, in review.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: The aim of this project is to gain insight into the metabolism and regulation of homoterpene volatiles in above- and belowground plant tissues of Arabidopsis thaliana. Homoterpenes play a significant role in indirect plant defense or biological pest control by attracting natural enemies of insect pests. Current activities of this project have focused on using a functional genomics approach to elucidate and localize the enzymatic steps for the formation of the C11- and C16-homoterpenes DMNT (4,8-dimethylnona-1,3,7-triene) and TMTT (4,8,12-trimethyltrideca-1,3,7,11-tetraene) in roots and leaves, respectively. The project provides multidisciplinary training for one postdoc (Dr. Sungbeom Lee) who joined the group in March 1, 2008, and two graduate students (Jung-Hyun Huh, Reza Sohrabi) who are mentored by the P.I. A technician was supported by this grant from September 1, 2007 until March 1, 2008. The project has stimulated a research outreach partnership of the Tholl lab with the Roanoke Valley Governor's School that participates in the pre-college education program, Partnership for Research and Education in Plants (PREP). In the fall of 2007 and spring 2008, teacher Dr. John Kowalski and his class of high school students have investigated the variation of induced terpene volatile formation including DMNT in the roots of different Arabidopsis ecotypes. In support of these activities, the P.I. has produced an educational video on root volatile analysis. PARTICIPANTS: Project Director: Dr. Dorothea Tholl Postdoc: Dr. Sungbeom Lee (since March 2008). Dr. Lee works on the identification of gene candidates with a role in C16-homoterpene volatile formation. Graduate students: Jung-Hyun Huh has identified and biochemically characterized two root-expressed enzymes that produce the putative precursor nerolidol in C11-homoterpene volatile biosynthesis. Reza Sohrabi has verified the roles of CytP450 enzymes and the cytosolic terpenoid pathway in root-specific C11-homoterpene volatile formation. Whitnee Askew (until March 2008) was involved as technical support to establish protocols for enzyme assays, yeast expression and extraction of CytP450 plant enzymes. Collaborators: Dr. Christiane Gatz (Georg-August University, Goettingen, Germany). Training was provided for the above-mentioned post-doctoral fellow (Dr. Lee), two graduate students (J.-H. Huh, R. Sohrabi) and the two undergraduate students, Austin Wray and Ashley Spencer. Graduate student Martha Vaughan (supported by Virginia Tech startup funds) has contributed valuable preliminary results for the project. TARGET AUDIENCES: Related to this project, the P.I. has developed a research outreach partnership project on root volatile formation and function with Dr. John Kowalski at the Roanoke Valley Governor's School. In the fall of 2007 and spring of 2008, Dr. Kowalski and his students have investigated the variation of induced terpene volatile formation including DMNT in the roots of different Arabidopsis ecotypes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Significant progress has been achieved on all three objectives. Objective 1. To identify the terpene synthase(s) responsible for the formation of nerolidol as precursor of the homoterpene DMNT in Arabidopsis roots, we have screened gene knock-out and knock-down lines of 9 terpene synthases (AtTPS) with constitutive and/or jasmonate-induced expression for the absence of DMNT formation. Using this approach, no single gene candidate involved in DMNT formation was identified suggesting possible redundancy of nerolidol biosynthetic enzymes. We then conducted a comparative root-specific AtTPS gene expression analysis among the three ecotypes Col, Ler, and CVi that show differential jasmonate-induced DMNT formation. This strategy led to the identification of the two root-expressed Col AtTPS genes At3g29410 and At1g33750 whose recombinant enzymes form (E)-nerolidol as the main product in vitro. Double gene knock-out plant lines will be generated to evaluate the role of both genes in DMNT biosynthesis. Objective 2. To pinpoint the oxidative enzymes responsible for the degradation of geranyllinalool to TMTT in Arabidopsis leaves, we have used the recently identified Arabidopsis geranyllinalool synthase GES as bait for in silico-targeting of 16 genes including 4 CytP450 enzymes with strong coexpression to GES. In a follow-up approach we determined the absence of expression of the selected gene candidates in a transgenic GES expression line crossed into the background of the jasmonate-insensitive mutant coi-1. This line produces geranyllinalool but no TMTT. CYP82G1 was identified as the most highly GES-co-expressed, coi-1 dependent gene candidate. A knock-out line of this gene appears to produce only geranyllinalool but no TMTT after treatment with the fungal elicitor alamethicin. Therefore, CYP82G1 is a promising gene candidate in TMTT formation. The role of CytP450 enzymes in the biosynthesis of DMNT in Arabidopsis roots was corroborated by a 90-100% reduction in the emission of DMNT from Arabidopsis hairy roots upon application of the specific CytP450 inhibitors miconazole and clotrimazole. We have also confirmed that DMNT formation in Arabidopsis roots is coi-1-dependent. Objective 3. To examine the subcellular regulation of DMNT formation, treatments of Arabidopsis hairy roots with inhibitors of the cytosolic and plastidic terpene biosynthesis pathways were conducted. Evidence was obtained for a primary contribution of the cytosolic mevalonate pathway in DMNT formation. Transit peptide analysis of the two nerolidol synthases At3g29410 and At1g33750 indicates that the nerolidol precursor might be formed in the cytosol and the mitochondria. This project allows improving our knowledge of the biochemistry and regulation of two of the most common herbivore-induced volatile compounds in plants. The work provides new information with significance for engineering indirect defense pathways in an approach to develop alternative pest controls. Future efforts will be undertaken to generate crop plants such as Brassica spp. with enhanced formation of homoterpene volatiles that may aid in attracting parasites or predators of insect pests above- and belowground.
Publications
- Herde, M., Gaertner, K., Koellner, T.G., Fode, B., Boland, W., Gershenzon, J., Gatz, C., and Tholl, D. (2008). Identification and regulation of TPS04/GES, an Arabidopsis geranyllinalool synthase catalyzing the first step in the formation of the insect-induced volatile C16-homoterpene TMTT. Plant Cell, 20, 1152-1168.
- Tholl, D. (2008). Plant volatiles in above and belowground plant-insect interactions: understanding pathways and molecular regulation. Abstract book, The Rank Prize Funds Mini Symposium on Chemical and Visual Ecology of Arthropods: From Genes to Pest Management, September 2008, Windermere, UK (invited talk).
- Huh, J.-H. and Tholl, D. (2008). Volatile terpene formation in Arabidopsis roots in interaction with the soil-borne pathogen Pythium irregulare. Program and abstract book, 25th Mid-Atlantic Plant Molecular Biology Society Conference, Savage, Maryland, August 2008 (poster).
- Sohrabi, R. and Tholl, D. (2008). Biochemical and molecular analysis of the formation of the stress-induced volatile C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene in Arabidopsis roots. Program and abstract book, 25th Mid-Atlantic Plant Molecular Biology Society Conference, Savage, Maryland, August 2008 (poster).
- Vaughan, M. and Tholl, D. (2008). Constitutive and induced volatile terpene formation in Arabidopsis roots in interaction with the root herbivore Bradysia spp. (fungus gnat). Program and abstract book, 25th Mid-Atlantic Plant Molecular Biology Society Conference, Savage, Maryland, August 2008 (poster).
- Vaughan, M., Huh, J.-H., Sohrabi, R., Wray, A., Askew, W., Tholl, D. (2008). Hidden defense metabolism: Volatile terpene formation in Arabidopsis roots. Electronic scientific program, Banff Conference on Plant Metabolism, Banff, Canada, July/August 2008 (oral presentation, M. Vaughan).
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