Progress 04/15/18 to 04/14/20
Outputs
Target Audience:During this reporting period, the PD presentedthe project on campus (Purdue University) and at the American Society of Plant Biology Annual Meeting. Together, these activities should reach the target audience of the plant metabolism, weed science, and biotechnology communities. In addition, the PD has mentored twograduate students in the lab, as well asan undergraduate student from Purdue University and an undergraduate from Icesi University interested in plant science research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has allowed the PD to attend 2national conferences over the course of this award. How have the results been disseminated to communities of interest?The results have been disseminated to the community through a publication in Horticultue Research. Another manuscript is in preparation for submission. The PD also had the opportunity to develop a game theoretic model of allelopathy that is under review.The PD has presented this work orally and in poster format at national conferences and at Purdue. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: Plant natural products are compounds that are predominantly nonessential for growth and development, but which provide key advantages for reproduction, defense, and/or competition with other species. Juglone is a natural product synthesized and secreted by black walnut trees to suppress growth of competing plants via a mode of action unlike any commercial herbicide. Strategies to harness juglone as a novel, natural product-based herbicide are hampered by the lack of knowledge about its metabolism. This project has demonstrated that juglone is made from intermediates in the synthesis of phylloquinone, an essential compound involved in photosynthesis found in all plants and vitamin K1 in humans. In addition, this project has solidified understand of the subcellular architecture of phylloquinone. The gained knowledge will allow the engineering of juglone as anovel natural product-based herbicide in agriculturally important plants. In addition, this project has provided training and professional development opportunities for a graduate student interested in pursuing a career in research and teaching. Research goals: The major goal of this project was to elucidate the biosynthetic pathway of juglone in black walnut. In the first publication from this work, we established that juglone branches off the biosynthetic pathway of phylloquinone, a compound used in all plants as an electron carrier in photosynthesis. We determined that juglone synthesis in black walnut branches off of phylloquinone biosynthesis at the level of the intermediate 1,4-dihydroxy-2-naphthoic acid (DHNA). From DHNA, we propose a DHNA decarboxylase and a 1,4-naphthoquinone (1,4-NQ) hydroxylase are needed to form juglone. Using the same RNA-seq data, candidate decarboxylases and hydroxylases needed to complete the juglone biosynthetic pathway were selected based on expression and metabolite profiling. Moreover, I collaborated to expand my comparative transcriptomic analysis of high and low juglone-producing black walnut tissues by including 19 publicly available datasets. In total, 7 decarboxylases, 16 cytochrome P450 monooxygenases (P450s), and 5 2-oxoglutarate-dependent dioxygenases (2-ODDs) were identified. Of these, 3 decarboxylase, 5 P450, and 3 2- ODD candidates were able to be amplified from black walnut cDNA, cloned into binary expression vectors, and functionally screened via Agrobacterium tumefaciens-mediated transient infiltration. In the functional screen, DHNA or 1,4-NQ were infiltrated with decarboxylase and hydroxylase candidates, respectively. None of the screened candidates were found to exhibit activity consistent with functioning as a DHNA decarboxylase or 1,4-NQ hydroxylase. Given the connection between the juglone and phylloquinone pathways we discovered, I next investigated one of the long-standing questions about phylloquinone pathway architecture in order to gain greater understanding of juglone metabolism.Phylloquinone biosynthesis is thought to be split between the chloroplast and the peroxisome, though the branch point is unknown due to dual-localization of one of the enzymes, o-succinylbenzoate (OSB) CoA ligase. Previous work on the subcellular localization of OSB-CoA ligase used constitutive promoters and masked one of the endogenous signals. To determine whether both signals are functionalin planta, we evaluated the localization under the native promoter with the fluorescent protein in the middle of the protein. We determined that OSB-CoA ligase is targetted to both the chloroplast and the peroxisome inArabidopsis thaliana.To evaluate where the activity is necessary, we complemented the knockout mutant inArabidopsiswith singly-localized OSB-CoA ligase synthetic constructs and determined that OSB-CoA ligase activity is required in both organelles. Finally, we provided genetic evidence for a 1,4-dihydroxy-2-naphthoyl-CoA synthase, the gene encoding the last unknown enzyme in the phylloquinone pathway of plants. Training goals: During my doctoral training, I developed a strong base in molecular biology and biochemistry techniques, as well as experience with both gas and liquid chromatography. I have mentored two graduate students and two undergraduate students in the final year of this project. Additionally, I became an "ambassador" for the Center for Plant Biology at Purdue. My fellow ambassadors and I began a seminar series to feature graduate student and post-docs in the center.I presented this research at a national conference and several on-campus poster sessions this year. Attending these on-campus events as well as two national conferences allowed me to continue to cultivate my professional network.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Auber, R.P., Suttiyut, T., McCoy, R.M. et al. Hybrid de novo genome assembly of red gromwell (Lithospermum erythrorhizon) reveals evolutionary insight into shikonin biosynthesis. Hortic Res 7, 82 (2020). https://doi.org/10.1038/s41438-020-0301-9
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
McCoy, R.M., Widhalm, J.R., McNickle, G.G. Allelopathy as an evolutionarily stable strategy. Journal of Ecology. (submitted).
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Progress 04/15/18 to 04/14/19
Outputs
Target Audience:During this reporting period, the first manuscript for this projectwas published in Horticulture Research. Ialso presented the project at the American Society of Plant Biology Annual Meeting. Together, these activities shouldreach the target audience of the plant metabolism, weed science, and biotechnology communities. I also mentored two first-year graduate students in the lab, as well as two Purdue University undergraduate students interested in plant science research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project allowed me to travel to the annual Plant Biology meeting to present my research, network with other scientists, and attend professional development workshops. How have the results been disseminated to communities of interest?The results have been disseminated through the published manuscript and poster sessions both at Purdue and at a national meeting. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, I will continue cloning and screening juglone biosynthesis candidates. To better understand the subcellular architecture of the juglone/phylloquinone pathway I will reinvestigate unresolved questions about the localization of upstream pathway enzymes. To accomplish my training goals, I will attend two national conferences and at least one Purdue University poster session to disseminate my research and network. I will mentor graduate and undergraduate students in the lab. I will also write up results from this project for publication.
Impacts
What was accomplished under these goals?
? Impact: Plant natural products are compounds that are predominantly nonessential for growth and development, but which provide key advantages for reproduction, defense, and/or competition with other species. Juglone is a natural product synthesized and secreted by black walnut trees to suppress growth of competing plants via a mode of action unlike any commercial herbicide. Strategies to harness juglone as a novel, natural product-based herbicide are hampered by the lack of knowledge about its metabolism. This project has demonstrated that juglone is made from intermediates in the synthesis of phylloquinone, an essential compound involved in photosynthesis found in all plants and vitamin K1 in humans. The gained knowledge will allow the engineering of this novel natural product-based herbicide in agriculturally important plants. In addition, this project has provided training and professional development opportunities for a graduate student interested in pursuing a career in research and teaching. Research goals: The first manuscript for this project was published. In this manuscript, we established that juglone biosynthesis branches off the pathway used to produce phylloquinone, a vital electron carrier involved in photosynthesis. Using RNAseq data from black walnut roots and leaves, I showed that the expression of the early phylloquinone pathway genes upstream of the branchpoint intermediate 1,4-dihydroxy-2-naphthoic acid (DHNA) were expressed to a much higher level than those downstream of DHNA. In addition, the genes encoding enzymes downstream of DHNA were strongly downregulated in roots, where phylloquinone is not present, compared to leaves, while the expression of the genes encoding enzymes upstream of the branchpoint was unchanged in roots compared to leaves. We then labeled isolated roots with 13C-glutamate and recovered labeling in juglone. These results demonstrate that juglone is derived from DHNA produced from the phylloquinone pathway and that juglone can be de novo synthesized in roots. From DHNA, we propose a DHNA decarboxylase and a 1,4-naphthoquinone (1,4-NQ) hydroxylase are needed to form juglone. Using the same RNAseq data, candidate decarboxylases and hydroxylases needed to complete the juglone biosynthetic pathway were selected based on expression and metabolite profiling. Moreover, I collaborated to expand my comparative transcriptomic analysis of high and low juglone-producing black walnut tissues by including 19 publicly available datasets. In total, 7 decarboxylases, 16 P450s, and 5 2-ODDs were identified. Of these, 3 decarboxylase, 5 P450, and 3 2-ODD candidates were able to be amplified from black walnut cDNA, cloned into binary expression vectors, and functionally screened via Agrobacterium tumefaciens-mediated transient infiltration. In the functional screen, DHNA or 1,4-NQ were infiltrated with decarboxylase and hydroxylase candidates, respectively. None of the screened candidates were found to exhibit activity consistent with functioning as a DHNA decarboxylase or 1,4-NQ hydroxylase. As an alternative, the candidates were recloned for recombinant protein production in plants and/or E. coli to test for DHNA decarboxylase or 1,4-NQ hydroxylase activity. A positive control DHNA decarboxylase from crude protein isolated from black walnut roots. Work is currently underway to develop a 1,4-NQ hydroxylase assay.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
McCoy R.M., Utturkar S.M., Crook J.W., Thimmapuram J., and Widhalm J.R. The origin and biosynthesis of the naphthalenoid moiety of juglone in black walnut. Horticulture Research 5, 67. (2018) doi: https://doi.org/10.1038/s41438-018-0067-5
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