Progress 05/01/20 to 04/30/25
Outputs
Target Audience:The target audience for this project is four-fold. The first is synthetic biologists working to manipulate plant metabolism and nutritional content. The second is general plant biologists looking to understand how plant metabolism changes between species. The final audience is companies looking to alter plant metabolism either through synthetic biology, crop engineering or traditional breeding. The most specific audience is brassica breeders and crop developers looking to engineer glucosinolate metabolism in their vegetable or oilseed crop. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project impacted training and professional development at the post-doctoral, graduate and undergraduate student levels. At the post-doctoral level, this project has provided training in biochemical analytical techniques to Dr. Singh. Additionally, this provided her an ability to mentor undergraduate students. For this, she mentored an undergraduate working on the chemical analysis and Dr. Kliebenstein met with both of them weekly, as a group and individually, to help assess how the project was going and see if there was any guidance to provide on mentoring. The main impact on training and professional development was at the graduate level where Mrs. Ramos (Soon to be Dr.) largely guided the entire project on her own and is the senior author on the capstone paper resulting from this project. This trained her in a braod array of biochemical, genetic and evolutionary techniques and analysis approaches. This has led to a highly integrative training where she can move between these fields with ease. Reflecting this, she has given seminars on her work at five international meetings and multiple local meetings in the past three years. This has allowed her to develop strong support and academic networks.Additionally, this provided her an ability to mentor undergraduate students. For this, she has mentored five different undergraduate students over the course of the project. Dr. Kliebenstein met with both of them weekly, as a group and individually, to help assess how the project was going and see if there was any guidance to provide on mentoring. At the undergraduate level, this project has contributed the ability to train six undergraduate students on the project. These students were involved in both the genetic and biochemical analysis of the project and are co-authors on the capstone paper with each having contributed a figure to the paper. The students were indpendently in charge of creating and implementing their experimental contribution to help train them in how to develop, design, implement and analyze experiments. Additionally, each of the students presented posters at the UC Davis undergraduate resarch conference.Five of these students have graduated with two in medical school, two in graduate school and one working in the biotech industry. How have the results been disseminated to communities of interest?The results from this project have been disseminated in three predominate routes. The first was through Dr. Kliebenstein and Mrs. Ramos each presenting several seminars per year to academic conferences or universities on this work. This included presentations at the Botany conference, International Conference Arabidopsis, Koln Spring Ecology Meeting, Kansas State University, University of Nevada, Reno, Iowa State University, Pennsylvania State University and others. In addition there have been a number of journal publications generated reporting the work in this project. Finally, Dr. Kliebenstein and Mrs. Ramos both presented the work to multiple industry groups including Pairwise Genetics, Harvest.org, BASF, Bayer and others. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Ultimately, we developed new phylogenetic and high-throughput enzymatic validation approaches that allowed usto identify and validate the presence or absence of the GS-OH gene in 100 different Brassicaceae species including all major vegetable and oilseed crops in this family. This has led to an ability to precisely breed or engineer this gene in any of these species and developed new methodologies and insights that could shape how similar projects are done with other genes, metabolites or in other plant families. The first major innovation was the ability to develop a high-throughput enzymatic validation system for complex metabolic pathways. While some pathways can be transiently expressed in Nicotiana, more complex pathways fpr important nutritional or medical metaboliteslike glucosinolates, flavonoids, etc cannot be transiently studied. To address this complexity, we turned the model plant Arabidopsis thaliana into a rapid stable expression system using its ease of transformation. This allowed us to explicitly test the GS-OH gene from all the Brassicaceae species for function. This was important as it allowed us to show that in about 1/3rd of the species, the gene is present but non-functional due to missense variation. It also allowed us to identify two novel GS-OH gene families that had been previously missed in both the Arabidopsis and Brassica literature. The second major innovation was the development of a phylo-functional survey methodology to identify and validate gene family function. Initial work showed that using the traditional sequence similarity approach was not properly identifying the GS-OH gene. So we developed an automated pipeline approach that surveys all available genomes for all sequences with any similarity to the target gene. This pipeline then automatically develops phylogenetic relationships between the target genes and allows the ability to identify specific gene families as likely candidates rather than specific genes for use in the high-throughput pipeline. This was critical as it showed that the Brassicaceae were not using the same syntenic gene for GS-OH function across the species and that the sequence similarity approach would fail to find the GS-OH gene. This is likely true in other specialized metabolite genes and families and will help to provide more precision in identifying genes to manipulate for nutritional content more broadly. Using these new approaches, we were able to make several new findings that have ramifications for both applied and fundamental efforts in plant metabolism. The first was that plant specialized metabolism is evolving more quickly than expected and this involves a combination of gene loss and convergent enantiomeric specificity shifts. Over half of the Brassicaceae had lost the GS-OH gene and this conservatively represents more than 20 independent gene losses. Even in the species that kept the gene, there were at least five independent convergent shifts between making the R or the S enantiomer of the 2-hydroxy-but-3-enyl glucosinolate product. This means that Brassica oleraceae makes the goiterogenic precursor progoitrin while using essentially the same gene, Sinapus alba and Crambe abyssinica make the enantiomeric epi-progoitrin that does not have goiterogenic properties. Thus, by surveying plant families, it may be possible to rapidly identify enzyme variants that shift enantiomeric properties of the metabolite that is attempting to be engineered. Another key finding from this work was that whole genome duplications do not appear to contribute to the evolution of specialized metabolite novelty. This had long been a key assumption of the research community and shaped a lot of how searches for novel enzymes were conducted. Instead, we found that distal gene duplications and segmental gene duplications were the main source of new GS-OH families. This suggests that it may be more efficient to search for novel enzymatic activities in plants by conducting genome wide scans for enzymes that have moved to new non-syntenic positions. To test this idea, we used new Camelina sativa genomes that we created as a part of this project. Camelina has novel glucosinolates and we could identify the causal genes behind this chemical novelty by looking for non-syntenic gene copies and these appear to have laid the groundwork for the shift in chemistry between species. This creates a new paradigm of how plant specialized metabolism evolves and lays the groundwork for more efficient identification of novel enzymes to enable metabolic engineering or breeding of crops. The final main finding of the project was enabled using the high-throughput stable transgenic line methodology. The identification of rapid evolution in the GS-OH enantiomeric properties led us to question if there were different biotic properties in the progoitrin or epiprogoitrin products. We were able to identify lines that had the precursor butenyl glucosinolate, progoitrin or epiprogoitrin and use these for biotic resistance assays. This allowed us to show that each of the three compounds had different impacts on resistance to lepidopteran herbivores and fungal pathogens. This was such that each compound was the optimal compound against at least one biotic attacker and the least effective compound against at least one other biotic attacker. Additionally, blends of the compounds appeared to be the least effective. This explains the high level of convergent evolution and gene loss as the species are encountering fluctuating biotic pressures, and each must make a different solution. More importantly, this influences how we need to think about how to engineer or breed plant metabolism to improve crop pest resistance. First, stacking more and more compounds into a plant may lead to weaker over-all resistance. Secondly, it is key to identify the suite of biotic pressures on a crop and then choose the specific compound that would be optimal for that blend of biotic attackers. Together, the work on identifying and validating the GS-OH gene across 100 Brassicaceae species, including all major crops, has led to a set of technical innovations and conceptual insights that will influence both fundamental and applied efforts at engineering or manipulating plant specialized metabolism. This includes having developed the potential to specifically target goitrin formation within the Brassicaceae crops.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Caseys, C., Muchich, A.J., Vega, J., Ahmed, M., Hopper, A., Kelly, D., Kim, S., Madrone, M., Plaziak, T., Wang, M. and D.J. Kliebenstein. (2024) Leaf abaxial and adaxial surfaces differentially affect the interaction of Botrytis cinerea across several eudicots. Plant J. 120(4):1377-1391
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Bird, K.A., Ramos, A.A. and D.J. Kliebenstein (2025) Phylogenetic and genomic mechanisms shaping glucosinolate innovation. Curr. Opin. Plant Bio. 85:102705
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Caseys, C. and D.J. Kliebenstein (2025) Polygenic strategies for host-specific and general virulence of Botrytis cinerea across diverse eudicot hosts
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Bird, K.A., Brock, J.R., Grabowski, P.P., Harder, A.M., Healy, A.L., Shu, S., Barry, K., Boston, L., Daum, C., Guo, J., Lipzen, A., Walstead, R., Grimwood, J., Schmutz, J., Lu, C., Comai, L., McKay, J.K., Pires, J.C., Edger, P.P., Love, J.T. and D.J. Kliebenstein. (2025) Allopolyploidy expanded gene content but not pangenomic variation in the hexaploid oilseed Camelina sativa. Genteics 229(1)iyae183
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Ramos, A.A., Bird, K.A., Jain, A., Phillip, G., Okegbe, O., Holland, L. and D.J. Kliebenstein (2025) Convergence and constraint in glucosinolate evolution across the Brassicaceae. BioRxiv
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:Our target audience for the past year have largely been internal to the university given the pandemic. We have also coordinated our efforts with companies working to develop new Brassicales crops such as Pennycress and companies working to genetically manipulate Brassica vegetable flavor. In the past year, this has expanded to assisting engineering companies like Pairwise genetics in using this information to modify green vegetable flavor profiles. In the future, we plan to expand these corporate outreach efforts to more general Brassicales vegetable companies. Further, we have begun communicating our findings in Academic forums like international meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We were able to use the resources of the project to enable Miss Agosto Ramos to attend international plant research conferences over the past year. At both the Botany and Phytochemical Society Meetings she gave seminars on the project. This has given her extensive networking exposure. Additionally, the project has been used to help train four undergraduate researchers. This gave them both research experience as well as mentoring experience for Miss Agosto Ramos. How have the results been disseminated to communities of interest?The results have been presented at Pennsylvania State University as well as the 2023 Botany conference and 2023 Phytochemical Society Meetings. Additionally, the undergraduate researchers presented multiple posters on the project at the annual UC Davis Undergraduate Research Conference. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will publish the findings from the project on both GS-OH evolution and Camelina glucosinolate evolution. Both papers are currently being written and will be submitted in the next couple of months to a journal and as a BioRxiv submission.
Impacts
What was accomplished under these goals?
In collaboration with JGI DOE, we have sequenced 10 teleomere-to-teleomere Camelina sativa genomes and transcriptomes transcriptomes. These have been completely annotated and were released this year to the public. Using these genomes, we have classified the GS-OH evolution and more importantly derived a new model for how novel glucosinolates/specialized metaoblites may arise. Specifically a phylogenetic analysis showed that the glucosinolate gene families have evolved at least four novel enzymes in Camelina that are critical for the novel glucosinolates. This suggests that there is a need to learn how to apply phylogenetics to properly translate research from model to non-model systems. We currently have 10 genomes that have been completed and are being annotated for Thlaspi arvense (Pennycress) with annotation soon to be implemented. These will be available in 2024.The sequencing for Streptanthus tortuosus, Brassica oleracea and Brassica nigra which also contain the GS-OH enzyme are in the pipeline. These are helpful as it will allow us to include an additional species into the pipeline as they species also have the GS-OH enzyme. In the past year, we have used our transgenic pipeline to synthesize and test 30 potential GS-OH genes from the phylogenetic approach. This shows that we have identified at least 20 functional GS-OH genes. This includes two copies from Allysum linfolium that have neo-functionalized to produce the opposite enantiomers with one Alyssum gene making the R-progoitrin and the other gene making the S-epiprogoitrin. Additionally, this has shown that we have been able to identify the confusion within the Brassica lineage. There was a unidentified ancestral duplication such that one copy evolved to a new function while one maintained the GS-OH function. However, reciprocal pairwise BLAST was unable to resolve this. Using a newly developed phylogenetic pipeline, we were able to pull in sequences from 40 different new Brassica genomes to resolve the phylogeny. The extended phylogeny also shows that this glucosinolate gene does not evolve from a primary metabolic enzyme as is often theorized but instead is a member of a broader gene family whose members have no known function. Gene tests showed that these genes do not function as a GS-OH. However, it is likely that the whole gene family is encoding specialized metabolite functions as there is extensive Presence-absence variation both within and between species across the entire gene family. This raises the idea that there may be specific specialized metabolite enzyme gene families that are built to enable the rapid evolution of specialized metabolism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Kliebenstein, D.J. (2024) Specificity and breadth of plant specialized metabolite-microbe interactions. Current Opinion in Plant Biology 102459. doi.org/10.1016/j.pbi.2023.102459
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Singh, R., Caseys, C. and D.J. Kliebenstein (2024) Genetic and molecular landscapes of the generalist phytopathogen Botrytis cinerea. Molecular Plant Pathology 25(1)e13404 doi.org/10.1111/mpp.13404
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Agosto-Ramos, A., Muhich, A.J. and D.J. Kliebenstein (2024) Convergently evolved metabolites are new to me but not to my attackers. New Phytologist v(i)pp-pp doi.org/10.1111/nph.19672.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Kliebenstein, D.J. (2023) Is specialized metabolite regulation specialized. Journal of Experimental Botany 74(17)4942-4948. doi.org/10.1093/jxb/erad209
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Kvitko, B. and D.J. Kliebenstein (2023) Better living through phytochemistry: phytoavengins and reappraising the production-focused dichotomy for defensive phytochemicals. Physiological and Molecular Plant Pathology 125(5)101978. doi: 10.1016/j.pmpp.2023.101978
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:Our target audience for the past year have largely been internal to the university given the pandemic. We have also coordinated our efforts with companies working to develop new Brassicales crops such as Pennycress and companies working to genetically manipulate Brassica vegetable flavor. In the past year, this has expanded to assisting engineering companies like Pairwise genetics in using this information to modify green vegetable flavor profiles.In the future, we plan to expand these corporate outreach efforts to more general Brassicales vegetable companies. Further, we have begun communicating our findings in Academic forums like international meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Given the pandemic, we were able to use the resources of the project to enable Miss Agosto Ramos to attend international plant research conferences over the past summer because of the online format of these conferences. This would not typically have been feasible for early graduate students given the cost of travel to attend meetings. For the upcoming year, we are targeting more specific small conferences for Miss Agosto Ramos to attend in person to transmit her findings. How have the results been disseminated to communities of interest?The results of this work were presented in an on campus seminar at University of Nevada, Reno and in communication with various companies interested in engineering brassica flavor profiles. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will finish the original 10 phylogenetics GS-OH homologue analysis and extend this to include an additional 10 depending on these results. The preliminary analysis of this work suggests that there is a functional GS-OH in some species reported to not make progoitrin. From work with Brassica it suggests that progoitrin may be enriched in roots and these species will be queried if there is progroitrin in the roots. Using the Brassica and Crambe transcriptomes we will conduct transcriptome wide association analysis to identify the top 10 additional candidates from this method and incoprorate them into the in planta expression system.
Impacts
What was accomplished under these goals?
In collaboration with JGI DOE, the Camelina sativa genome sequences, transcritpome and annotation have all been completed. These will not be publically available until 2024 per new JGI DOE guidelines. DNA and RNA are currently undergoing analysis for Thlaspi arvense (Pennycress) with annotation soon to be implemented. These will likely also be available in 2024.The sequencing forStreptanthus tortuosus which also contains the GS-OH enzyme is in the pipeline.. This is helpful as it will allow us to include an additional species into the pipeline as this species also has a GS-OH that appears independently evolved from the Brassica, Crambe, Camlinaand Raphanus sequences. In the past year, we have begun to speed up the transgenic pipeline to identify GS-OH genes from the phylogenetic approach. Specifically we know have 10 enyzmes/genes from multiple Brassicales species including Brassica rapa synthesized and expressed within Arabidopsis. Full results are available on a pair of genes from Allysum linfolium, a near relative of Arabidopsis, that shows that the closest homologue of the Arabidopsis GS-OH is a functional GS-OH enzyme in this species. Interestingly, the two copies have neo-functionalized to produce the opposite enantiomers with one Alyssum gene making the R-progoitrin and the other gene making the S-epiprogoitrin. All other plants are currently being grown and we should have final data on all ten genes by the summer. At which time we will reassess the representation across the phylogeny and identify 10 new genes to include within the next year now that the pipeline is fully established. Within Brassica rapa and Brassica oleracea, we have identified genotypes, tissues and environments that represent a blend of synthesis abilities for GS-OH production. We are currently extracting RNA across this collection to develop transcrpitomics libraries to identify likely candidates for GS-OH using transcript wide association methods and we will synthesize the 10 top candidates and express them within the next year. After further work, we have realized that the report ofprogoitrin (2-hydroxy-3-butenyl) glucosinolate within Raphanus sativus may be an artifact of the majority of labs moving to mass-spectral approaches without developing standards. After nearly a year of work where we could not find any genotype, tissue or condition with progoitrin within Raphnus, we shifted to a LC-MS pipeline from our LC-DAD pipeline and realized that there is a signal but it is of extremely low abundance. Further, a reanalysis of the Raphanus genome and the AOP2 enzyme (produces the precursor for progoitrin) showed that this gene does not function in Raphanus. As such, it is likley that the reports of progoitrin in Raphanus is a technological artifact which we are testing by taking theGSR1, from Raphanus into Arabidopsis to allow testing this hypothesis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Muhich, A.J., Agosto-Ramos, A. and D.J. Kliebenstein (2022) The ease and complexity of identifying and using specialized metabolites for crop engineering. Emer. Top. Life. Sci. 6(2)153-162. doi.org/10.1042/ETLS20210248
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Progress 05/01/21 to 04/30/22
Outputs
Target Audience:Our target audience for the past year have largely been internal to the university given the pandemic. We have also coordinated our efforts with companies working to develop new Brassicales crops such as Pennycress and companies working to genetically manipulate Brassica vegetable flavor. In the future, we plan to expand these corporate outreach efforts to more general Brassicales vegetable companies. Further, we will begin communicating our findings in Academic forums like international meetings. Changes/Problems:The only major change in the past year was due to the finding that Raphanus satvius appears to use a species specific glucosinolate as a precursor for making progoitrin (2-hydroxy-3-butenyl) glucosinolate. Fortunately, the enzyme (GSR1)for making this precursor has been previously published and we have synthesized the gene and are currently introducing it into Arabidopsis to facilitate the goals of this project. What opportunities for training and professional development has the project provided?Given the pandemic, we were able to use the resources of the project to enable Miss Agosto Ramos to attend international plant research conferences over the past summer because of the online format of these conferences. This would not typically have been feasible for early graduate students given the cost of travel to attend meetings. For the upcoming year, we are targeting more specific small conferences forMiss Agosto Ramos to attend in person to transmit her findings. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?First, we will analyze the transgenic Arabidosis plants containing putative GS-OH homologues identified by the phylogenetic approach across the Brassicales including the key crop species focused on in this project. Second, we will analyze the Brassica and Crambe transcriptomes from the sequencing efforts to identify additional GS-OH candidates and place these within our pipeline to test within the year. Third, we will use the de novo sequencing of the Raphanus sativa transcriptome to identify the most likely GS-OH candidates in that species and again place it into the transgenic pipeline to test using the newly developed model biochemical background genotype. In combination the goal in the next period is to identify the most likely candidates in each of the key species of the projects to allow us to begin developing the necessary CRISPR lines to validate their in planta potential.
Impacts
What was accomplished under these goals?
In the past year, we have made progress on several major goals. First, the Brassica oleraceae and Crambe abyssinica transcript and genomic sequence has been completed. These are currently being compiled into completed genomes with full annotation at JGI. The raw data is currently released to the public via the JGI website and it is hoped that the annotated genomes/transcriptomes will be readiy in the next year. The Thlaspi arvense (Pennycress) materials was submitted to JGI as part of the community sequencing project in early 2022. In addition we were able to get a new species, Streptanthus tortuosus, included into the sequencing efforts. This is helpful as it will allow us to include an additional species into the pipeline as this species also has a GS-OH that appears independently evolved from the Brassica, Crambe and Raphanus sequences. We have continued our analysis of the phylogenetic approach to identify possible GS-OH genes. Specifically, we had to take the past year to optimize some unexpected complexities from our gene synthesis/expression pipeline. Speifically, the original plasmids we obtained for this project were not the actual plasmids. After full plasmid sequencing, we have now finally gotten the correct expression system. We have knowsynthesized these genes and placed them into expression vectors and they are currently being transformed into Arabidopsis thaliana as proposed. This system appears to be working more efficiently than expected and we are transforming in a number of genes to test the evolution of this gene family more broadly. We have also used the past year to optimize the Raphanus sativus system as the literature had some unanticipated gaps. We have been able to show that the GS-OH enzyme has a narrower expression pattern then we had been led to anticipate. This however, will allow us to optimize our co-expression approach to finding GS-OH as we can use both within and between genotype libraries that have explicit presence and absence of the gene/enzyme. These libraries will be made and analyzed by the end of the summer. An unexpected aspect of the Raphanus sativus optimization is that combining the genetics and biochemistry has shown us that the enzyme producing the progoitrin (2-hydroxy-3-butenyl) glucosinolate in Raphanus likely uses a completely different precursor than the enzyme within Brassica, Crambe and Arabidopsis. This observation has led us to begin creating a new model genotype that expresses this Raphanus specific precursor to allow for our gene synthesis approach to be successful. We have had to move a separate enzyme, GSR1, from Raphanus into Arabidopsis to allow for this biochemistry to be tested.
Publications
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