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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Progress 05/01/20 to 04/30/21
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:In the past year, the pandemic has largely shut down the wet lab with only limited access since March of 2020. We were able to develop a computational project for Miss Agosto Ramos that lead to the phylogenetic approach being established. This did however lead to a 6 to 9 month delay in where I had hped that the transcriptional analysis would have been under normal conditions. The beneift of having Miss Agosto Ramos win recognition and funding for five years has allowed an additional graduate student to join the project and I'm hoping that the combined efforts will allow us to catch up on the wet lab 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. We will continue to have both Miss Agosto Ramos and Miss Lensink to attend similar conferences this summer. This would not typically have been feasible for early graduate students given the cost of travel to attend meetings. 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?In the next reporting period we will focus on the following steps to accmoplish 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 conduct 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. 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 worked to accomplish these goals by the following efforts. The key accomplishment of the past year is that we were able to attract two graduate students to work on this project. In April of 2020, Amanda Agosto Ramos joined the project and was simultaneously awarded multiple graduate fellowships. This allowed the project the financial flexibility to alsorecruit Mariele Lensik in the past several weeks. Hopefully the combination of the two students will help us to make up time cost by the pandemic lockdowns in California. Experimentally, our first accomplishment this year is thatwe have smapled tissue forBrassica oleraceae and Crambe abyssinica that are being processed for short and long-read sequencing of the transcriptomes and genomesin collaboration with an indpendently funded JGI community sequencing project. This includes 10 genotypes of each species and 10 different tissues for each genotype. The Crambe abyssinica data should be available in the summer of 2021 and the Brassica oleraceae should be available in the Fall or Winter of 2021. We have also been able to leverage this JGI community sequencing project to also extend these efforts to both Thlaspi arvense (Pennycress) and to Brassica nigra. The second experimental accomplishment is that we were able to collect existing genome data into a phylogenetic approach to identify a family of possible GS-OH genes that appear to be independently evolving in distinct lineages. We have synthesized these genes and placed them into expression vectors and they are currently being transformed into Arabidopsis thaliana as proposed. These results will hopefully be available by the middle of the summer.
Publications
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