Progress 06/01/20 to 05/27/25
Outputs
Target Audience:The target audience for this project is plant breeders, the plant metabolism community, plant biochemists and scientists and engineers engaged in manipulating plant oil production for food, fuel or feed stock needs. In addition, the target audience will include graduate students and undergraduates in plant biochemistry and molecular biology who will be able to use publications from these studies to understand plant oil production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate was trained in experimentation and presentations. One postdoc was trained in experimentation, presentations, and scientific writing. The postdoc trained on this project has just accepted an Assistant Professor position at another university. Thus, this project was key in the career progression for this postdoc. How have the results been disseminated to communities of interest?During this reporting period the results were disseminated through two publications, and four presentations at international scientific conferences. Additionally, the PI presented this research at two University seminar series at the University of Minnesota and the University of Alberta. What do you plan to do during the next reporting period to accomplish the goals?This is the final year of this project. The last remaining results will be submitted for publication soon.
Impacts
What was accomplished under these goals?
Objective 4. Identify in planta roles of TAG remodeling candidate genes (Years 2-3). 100% completed.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Parchuri P, Shockey J, Bates PD (oral presentation). Deciphering diacylglycerol enantiomer specificities of DGAT
isoforms: insights into TAG remodeling in lipid metabolism of different species. 5. Gordon Research Seminar: Plant Lipids
Structure, Function & Metabolism. Jan. 25-26, 2025.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Chen G. MECHANISMS OF HYDROXY FATTY ACID BIOSYNTHESIS IN LESQUERELLA (PHYSARIA FENDLERI).
Fernando, A.L., and M.T. Berti (eds.) 2024. Constructing a Sustainable Future - The Role of Industrial Crops and
Products. 35th Annual Meeting of the Association for the Advancement of Industrial Crops (AAIC). Conference Program
and Abstracts. Lisbon, Portugal, September 1-5, 2024
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Garneau MG, Parchuri P, Zander N, Bates PD (2025) Rapid quantification of whole seed fatty acid amount, composition,
and shape phenotypes from diverse oilseed species with large differences in seed size. Plant Methods 21:67.
https://doi.org/10.1186/s13007-025-01388-3
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Bates PD, Shockey J (2025) Towards rational control of seed oil composition: dissecting cellular organization and flux
control of lipid metabolism. Plant Physiology 197: kiae658. https://doi.org/10.1093/plphys/kiae658
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Bates PD (oral presentation). Triacylglycerol remodeling: Discovery and bioengineering to increase hydroxy- and
polyunsaturated fatty acids in Arabidopsis and Camelina seed oils. 26th International Symposium on Plant Lipids. Lincoln,
Nebraska. July 14-19 2024.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Bates PD (poster presentation) Utilization of triacylglycerol remodeling to engineer increased hydroxy- and
polyunsaturated containing fatty acids in Camelina sativa seed oils. 1. 1st International Camelina Conference. July 19-20,
2024. Lincoln, Nebraska.
|
Progress 06/01/23 to 05/31/24
Outputs
Target Audience:The target audience for this project is plant breeders, the plant metabolism community, plant biochemists and scientists and engineers engaged in manipulating plant oil production for food, fuel or feed stock needs. In addition, the target audience will include graduate students and undergraduates in plant biochemistry and molecular biology who will be able to use publications from these studies to understand plant oil production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, two postdoctoral scholars, two technicians, and fiveundergraduate students have been trained in experimentation, data analysis, or manuscript writing. In addition, this project has provided an opportunity for one postdoctoral scholar to present their work through an oral presentation at an international meeting. How have the results been disseminated to communities of interest?In this project period, results have been disseminated through two published peer reviewed journal articles, and one magazine article. Additionally, the university has put out a press release for one of the publications that was picked up by 10 different media outlets. The results were also disseminated through fouroral presentations at three differentinternational scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?During the final year of the project we aim to wrap up all remaining experimental details and publish the results of objective 5.
Impacts
What was accomplished under these goals?
Objective 1 Biochemical characterization of the fatty acid selectivity of candidate lipases involved in TAG remodeling (Years 1-2). 100% completed. Based on experiments including in vitro substrate selectivity assays, in vivo cellular localization, protein-protein interaction (Obj. 3), gene knockdown experiments in Physaria fendleri (Obj. 4), and overexpression experiments in Arabidopsis thaliana and Camelina sativa (Obj. 5) of candidate lipases PfeSDP1, PfeOBL1, and PfeTAGL1 we have concluded that PfeTAGL1 is the most likely lipase the performs TAG remodeling in Physaria fenderli. These results have been published in Nature Communications. Objective 2. Biochemical characterization of the fatty acid selectivity of candidate diacylglycerol acyltransferases (DGATs) involved in TAG remodeling (Years 1-2). 100% completed. As described in the previous report our in vitro assays indicate that PfeDGAT1 and PfeDGAT2 are both involved in Physaria fendleri TAG biosynthesis with PfeDGAT1 utilizing the initial sn-1,2-DAG derived from PC to produce 1HFA-TAG, and then after the PfeTAGL1 action that removes the sn-1 common fatty acid producing a sn-2,3-DAG containing a HFA at the sn-3 position PfeDGAT2 action then produces the final 2HFA-TAG molecule containing the hydroxy fatty acids at the sn-1 and sn-3 positions. These results have been published in Nature Communications. We have expanded our analysis of DAG substrate stereochemistry selectivity of DGATs to other species and have found a wide range of variability. A manuscript on these results is currently in preparation. Objective 3. Protein-Protein interactions to identify hypothesized lipase-DGAT complex (Years 1-2). 100% completed. The protein-protein interaction work was completed in the last report, and has been published in Nature Communications. Objective 4. Identify in planta roles of TAG remodeling candidate genes (Years 2-3). 100% completed. We have now produced at least T2 generation RNAi knockdown lines in Physaria fendleri for all candidate genes of interest including DGAT1, DGAT2, TAGL1. The in vivo phenotypes are consistent with our in vitro enzyme assays that indicate all three genes are involved in Physaria seed oil biosynthesis, and are important for the accumulation of hydroxylated fatty acids in the oil. These results have been published in Nature Communications. In addition, we have developed a novel virus induced gene silencing functional genomics approach that utilizes the inherent self-incompatibility of Physaria as a selectable maker to identify developing seed pods with genes of interest silenced. Through this method we have characterized in planta two key genes for lesquerolic acid synthesis (FAH12 and FAE1), as well as the essential genes which control Physaria self-incompatibility. These results have been published in Plant Biotechnology Journal. Objective 5. Bioengineer TAG remodeling into an oilseed crop (Years 2-3). 99% completed. Top gene candidates from objectives 1-3 (PfeDGAT1, PfeDGAT2 and PfeTAGL1) have been transformed to into hydroxy fatty acid producing Camelina sativa and Arabidopsis thaliana, as single, double, and triple gene stacks. A key outcome here, is that in two different oilseed species over-expression of a TAG lipase (specifically TAGL1) led to increased oil and HFA content rather than decreased as you might expect from production of an enzyme class (TAG lipases) that may be thought of as primarily for TAG turnover. Therefore, our engineering data (together with the data in Obj. 1-4) suggests that TAGL1 likely has a different in vivo function than other characterized TAG lipases such as SDP1. This differential function of TAGL1 in Physaria is for TAG remodeling, and it appears it can be used as an engineering tool to induce TAG remodeling in other plants. Therefore, we have identified a key enzyme that can be used to control seed oil fatty acid compositions. In addition, we show that in wild-type Camelina and Arabidopsis backgrounds increasing TAG remodeling can lead to higher levels of omega-3 fatty acids, and lower levels of saturated fatty acids. Thus, engineering TAG remodeling can also be used to increase the nutritional properties of plant oils. A manuscript on plant oil engineering is currently in preparation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Parchuri P, Bhandari S, Azeez A, Chen G, Johnson K, Shockey J, Smertenko A, Bates PD (2024) Identification of triacylglycerol remodeling mechanism to synthesize unusual fatty acid containing oils. Nature Communications 15 (1):3547. doi:10.1038/s41467-024-47995-x
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2024
Citation:
Azeez A, Bates PD (2024) Self-incompatibility based functional genomics for rapid phenotypic characterization of seed metabolism genes. Plant Biotechnology Journal. DOI: 10.1111/pbi.14383
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
9th Asian-Oceanian Symposium on Plant Lipids. Seoul, Republic of Korea. Oct. 10-13, 2023. Dynamic seed oil assembly: acyl and stereochemical selective enzymes remodel triacylglycerol fatty acid composition after initial oil synthesis. Philip Bates, oral presentation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
10th European Symposium on Plant Lipids. Amsterdam, The Netherlands. July 9-12, 2023. Dynamic seed oil assembly: Acyl and stereochemical selective enzymes remodel triacylglycerol fatty acid composition after initial oil synthesis. Philip Bates, oral presentation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
American Society for Biochemistry and Molecular Biology (ASBMB) Annual meeting. March 23-26, 2024. San Antonio, TX. DGAT tales: DGAT1 and DGAT2 enzymes across species differ in their specificity towards diacylglycerol enantiomers. Parchuri P*, Shockey J, BATES PD. Oral presentation.
- Type:
Other
Status:
Published
Year Published:
2024
Citation:
Parchuri P, Azeez A, Bates Philip D (2024) Novel oil biosynthetic pathway for hydroxy fatty acids. INFORM: International News on Fats, Oils, and Related Materials, vol 35. AOCS, Champain, IL USA
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
9th Asian-Oceanian Symposium on Plant Lipids. Seoul, Republic of Korea. Oct. 10-13, 2023.Biotechnology for Industrial Materials Production: Current Progreass on Improving Hydroxy Fatty Acid Content in Lesquerella (Physaria fendleri). Grace Chen, oral presentation.
|
Progress 06/01/22 to 05/31/23
Outputs
Target Audience:The target audience for this project is plant breeders, the plant metabolism community, plant biochemists and scientists and engineers engaged in manipulating plant oil production for food, fuel or feed stock needs. In addition, the target audience will include graduate students and undergraduates in plant biochemistry and molecular biology who will be able to use publications from these studies to understand plant oil production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, one graduate student, two postdoctoral scholars, one technician, and two undergraduate students have been trained in experimentation, data analysis, or manuscript writing. In addition, this project has provided opportunities for two postdoctoral scholars to each attend two different scientific meetings and present their work through both oral and poster presentations (see listed conference presentations). How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by three scientific publications and five presentations at scientific meetings presented by two different postdocs and by the PI. See products for list of publications and presentations. What do you plan to do during the next reporting period to accomplish the goals?During the final year of the project we plan to complete Obj. 4 and 5, and publish several scientific journal articles on the results from all objectives.
Impacts
What was accomplished under these goals?
Objective 1 Biochemical characterization of the fatty acid selectivity of candidate lipases involved in TAG remodeling (Years 1-2). 100% completed. Based on experiments including in vitro substrate selectivity assays, in vivo cellular localization, protein-protein interaction (Obj. 3), gene knockdown experiments in Physaria fendleri (Obj. 4), and overexpression experiments in Arabidopsis thaliana and Camelina sativa (Obj. 5) of candidate lipases PfeSDP1, PfeOBL1, and PfeTAGL1 we have concluded that PfeTAGL1 is the most likely lipase the performs TAG remodeling in Physaria fenderli. A publication summarizing these findings is currently in preparation. Objective 2. Biochemical characterization of the fatty acid selectivity of candidate diacylglycerol acyltransferases (DGATs) involved in TAG remodeling (Years 1-2). 100% completed. As described in the previous report our in vitro assays indicate that PfeDGAT1 and PfeDGAT2 are both involved in Physaria fendleri TAG biosynthesis with PfeDGAT1 utilizing the initial sn-1,2-DAG derived from PC to produce 1HFA-TAG, and then after the PfeTAGL1 action that removes the sn-1 common fatty acid producing a sn-2,3-DAG containing a HFA at the sn-3 position PfeDGAT2 action then produces the final 2HFA-TAG molecule containing the hydroxy fatty acids at the sn-1 and sn-3 positions. A manuscript for these results is currently in preparation (along with data from Obj. 1 & 3). In addition we were curious if the differential sn-1,2-DAG vs sn-2,3-DAG selectivity of the Physaria fendleri DGATs may extend to other crop species (especially species that accumulate unusual fatty acids). Therefore, we evaluated the biochemical properties of DGAT1 and DGAT2 from both cotton (Gossypium sp.) and litchi (Litchi chinensis). Both species accumulate cyclopropyl fatty acids (CPFA) in seed TAG, which is a valuable industrial chemical feedstock. We first evaluated the fatty acid selectivity of each DGAT by co-expressing them with a CPFA synthase in Arabidopsis thaliana seeds. Each DGAT demonstrated selectivity for CPFAs, and these results were published (Shockey et al, 2023). Next we evaluated the sn-1,2-DAG vs sn-2,3-DAG selectivity of each of the DGATs. For cotton we found that similar to the Physaria results GhDGAT1 only used sn-1,2-DAG but GhDGAT2 could utilize both sn-1,2-DAG and sn-2,3-DAG. For litchi LiDGAT2 was the same as Physaria and cotton, however we have had trouble getting LiDGAT1 active in vitro and these experiments are ongoing. These initial results suggest that the unexpected differential DAG species selectivity demonstrated by Physara fendleri DGATs is also present in other species, which may imply that the TAG remodeling we first discovered in Physaria fendleri may also be present in other important crops species such as cotton. Objective 3. Protein-Protein interactions to identify hypothesized lipase-DGAT complex (Years 1-2). 100% completed. The protein-protein interaction work was completed in the last report and no additional experimentation was done within this reporting period. Objective 4. Identify in planta roles of TAG remodeling candidate genes (Years 2-3). 80% completed. We have now produced at least T2 generation RNAi knockdown lines in Physaria fendleri for all candidate genes of interest including DGAT1, DGAT2, TAGL1 and SDP1. We are currently testing the phenotypes for the DGAT1, DGAT2 and TAGL1 knockdown lines. The SDP1 results were published (Azeez et al, 2022) indicating it is not involved in TAG remodeling, but it did lead to increases in total seed oil and HFA content. Together, these results suggest that control of SDP1 can be a key target for improvement of Physaria fendleri as a crop. In addition, we have been testing a virus induced gene silencing (VIGS) approach in Physaria fendleri as a way to more quickly evaluate gene function. Our VIGS tests with TAGL1 are consistent with our results from objectives 1 and 3, that it is likely the main lipase involved in TAG remodeling. Objective 5. Bioengineer TAG remodeling into an oilseed crop (Years 2-3). 80% completed.? Top gene candidates from objectives 1-3 (PfeDGAT1, PfeDGAT2 and PfeTAGL1) have been transformed to into hydroxy fatty acid producing Camelina sativa and Arabidopsis thaliana. The single gene lines all indicate an increase in seed oil and HFA content. We are currently propagating and analyzing the seed oil content of the double and triple stacked lines. A key outcome here, is that in two different oilseed species over-expression of a TAG lipase (specifically TAGL1) led to increased oil and HFA content rather than decreased as you might expect from production of an enzyme class (TAG lipases) that may be thought of as primarily for TAG turnover. Therefore, our engineering data (together with the data in Obj. 1-4) suggests that TAGL1 likely has a different in vivo function than other characterized TAG lipases such as SDP1. This differential function of TAGL1 in Physaria is for TAG remodeling, and it appears it can be used as an engineering tool to induce TAG remodeling in other plants. Therefore, we have identified a key enzyme that can be used to control seed oil fatty acid compositions.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Azeez A, Parchuri P, Bates PD (2022) Suppression of Physaria fendleri SDP1 Increased Seed Oil and Hydroxy Fatty Acid Content While Maintaining Oil Biosynthesis Through Triacylglycerol Remodeling. Front Plant Sci 13: 931310
- Type:
Book Chapters
Status:
Published
Year Published:
2023
Citation:
Parchuri P, Garneau MG, Roth MR, Tamura P, Durrett TP, Welti R, Bates PD (2023) Chapter Eight - Comparison of TLC, HPLC, and direct-infusion ESI-MS methods for the identification and quantification of diacylglycerol molecular species. In J Jez, ed, Methods in Enzymology, Vol 683. Academic Press, pp 191-224
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Shockey J, Parchuri P, Thyssen GN, Bates PD (2023) Assessing the biotechnological potential of cotton type-1 and type-2 diacylglycerol acyltransferases in transgenic systems. Plant Physiol Biochem 196: 940-951
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2023
Citation:
American Oil Chemists Society Annual Meeting. Denver, CO. April 30 May 3, 2023. Oral presentation by Philip D. Bates. Novel triacylglycerol assembly pathway elucidation and its bioengineering for hydroxy fatty acid accumulation in Physaria fendleri and other oilseeds.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2023
Citation:
Gordon Research Conference: Plant Lipids Structure, Function & Metabolism. Jan 29 Feb. 3, 2023. Galveston, TX. Oral presentation by Prasad Parchuri (postdoc, Washington State University). Triacylglycerol remodeling in Physaria fendleri: A concert between lipases and acyltransferases.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2023
Citation:
Gordon Research Seminar: Plant Lipids Structure, Metabolism, and Function. Galveston, TX. Jan. 28-29, 2013. Oral presentation by Abdul Azeez (postdoc, Washington State University). Self-incompatibility based functional genomics to characterize the seed lipid metabolic network genes in Physaria fendleri.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2023
Citation:
2023 Plant & Animal Genome Conference (PAG 30) Jan. 13-18, 2023, San Diego, CA. Poster presentation by Abdul Azeez (postdoc, Washington State University). Self-incompatibility based functional genomics to characterize the seed lipid metabolic network in Physaria fendleri.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2022
Citation:
Plant Biology 2022, American Society of Plant Biologists annual meeting (Portland, OR) July 9-13. Poster presentation by Prasad Parchuri (postdoc, Washington State University). Biochemical characterization of Novel Triacylglycerol remodeling pathway genes in Physaria fendleri: An industrial crop producing hydroxy fatty acids.
|
Progress 06/01/21 to 05/31/22
Outputs
Target Audience:The target audience for this project is plant breeders, the plant metabolism community, plant biochemists and scientists and engineers engaged in manipulating plant oil production for food, fuel or feed stock needs. In addition, the target audience will include graduate students and undergraduates in plant biochemistry and molecular biology who will be able to use publications from these studies to understand plant oil production. Changes/Problems:The original plan was that all RNAi plants were to be produced by the co-PI at the USDA-ARS pacific west region location. However, due to COVID-19 based restrictions limiting personnel to 25% capacity at USDA facility for the majority of the project so far, and that a research assistant at the USDA facility was unable to be hired until Feb. 28, 2022 (almost two years into the project) some of the RNAi line production (e.g. DGAT1, DGAT2, TAGL1) and their phenotypic evaluation is behind schedule. To circumventthe issue of reduced lab time for production of RNAi lines, a few of the RNAi lines (e.g. SDP1, PDAT2, PDAT1/2) have been produced at WSU instead at the USDA-ARS to ensure the timely production of products, and we are also using a faster VIGS based approach to evaluate additional gene functions in vivo in Physaria fendleri. We plan on requesting a no cost extension to make sure all RNAi lines and their evaluation can be completed prior to the end of this project. What opportunities for training and professional development has the project provided?During the first two years of this project two postdocs and one graduate student have been trained in experimentation, presentation, and manuscript writing. Additionally, three undergraduates and one research assistants have been trained in experimentation. One PhD graduate student dissertation was produced. How have the results been disseminated to communities of interest?The results have been disseminated by threescientific publications and one dissertation.An additional one manuscript has been submitted. In addition 3 presentations to communities of interest. The presentations include poster presentation at an international scientific conference (Plant & Animal Genome Research, San Diego, CA, USA);oneinvited presentation to the German plant science community CEPLAS: Clusters of Excellence on Plant Sciences (zoom seminar, May 3, 2022); and one departmental seminar in the Institute of Biological Chemistry at Washington State University. What do you plan to do during the next reporting period to accomplish the goals?Progress will be made on objectives 1, 4, & 5. In addition, we plan to submit at least three more manuscripts for publication based on current unpublished results and anticipated results of ongoing projects.
Impacts
What was accomplished under these goals?
Objective 1 Biochemical characterization of the fatty acid selectivity of candidate lipases involved in TAG remodeling (Years 1-2). 70% completed. Top candidate lipases TAGL1 and SPD1 have been cloned from Physaria fendleri into E. coli for expression, and further purified. Both Physaria enzymes demonstrated TAG lipase activity. Substrate selectivity lipase assays were performed on SDP1 and indicated that Physaria SDP1 prefers 2HFA-TAG for lipase based TAG degradation over non-HFA-containing TAG. Because 2HFA-TAG is the major TAG species that accumulates in Physaria seeds, SDP1 is likely not the TAG lipase involved in the recently discovered TAG remodeling pathway of 2HFA-TAG production. However, identification of a HFA-selective TAG lipase may prove to be a useful tool for oilseed plant bioengineering. For example previous research has demonstrated that breakdown of HFA-TAGs in germinating seeds of engineered plants is delayed, reducing seedling establishment. Thus the expression of the HFA-selective PfeSDP1 during germination may enhance TAG turnover and seedling establishment in engineered oilseeds. These SDP1 results (together with Obj. 4 SDP1 results) have been submitted for publication. Enzymatic characterization of Physaria TAGL1 is under progress. Objective 2. Biochemical characterization of the fatty acid selectivity of candidate diacylglycerol acyltransferases (DGATs) involved in TAG remodeling (Years 1-2). 100% completed. PfeDGAT1, PfeDGAT2 and PfeDGAT3 have been cloned from Physaria fendleri into the yeast quadruple mutant H1246 unable to produce TAG. The DGAT1 and DGAT2 complimented TAG biosynthesis is yeast, but the DGAT3 did not. Therefore, we focused on the DGAT1 and DGAT2 for further experiments. We synthesized radioactive ([14C]1HFA-DAG, [14C]20:1-OH CoA) and non-radioactive substrates (18:1-OH CoA; 20:1-CoA and 20:1-OH CoA) for in vitro DGAT assays. The results demonstrated distinct substrate selectivity's for each DGAT enzyme. PfeDGAT1 is most probably involved in 1HFA-TAG formation with non-hydroxy DAG as acyl acceptor and hydroxy acyl-CoA as acyl donor, whereas PfeDGAT2 can form 1HFA-TAG with both hydroxy and non-hydroxy DAG acyl acceptor and hydroxy and non-hydroxy acyl-CoA acyl donors. PfeDGAT2 formed 2HFA-TAG more efficiently than PfeDGAT1. In addition we performed DAG regiochemical selectivity assays. DGAT1 is 1,2-sn-DAG specific and DGAT2 can utilize either 1,2-sn-DAG or 2,3-sn-DAG. According to the TAG remodeling hypothesis DGAT1 is likely involved in formation of the initial 1HFA-TAG whereas DGAT2 is involved in 2HFA-TAG formation after the conversion of 1HFA-TAG to the 2,3-sn-DAG by a lipase. The two key points are that TAG remodeling in P. fendleri likely utilizes both DGAT1 and DGAT2 (which is unlike TAG biosynthesis in many species), and that bioengineering TAG remodeling into a new species may require both DGAT1 and DGAT2. A manuscript with these results (and objective 3) is currently in preparation. Objective 3. Protein-Protein interactions to identify hypothesized lipase-DGAT complex (Years 1-2). 100% completed. Y2H based protein-protein interactions have been screened for PfeDGAT1 and PfeDGAT2 vs the lipases PfeTAGL1, PfeSDP1, and PfeOBL1. Strong interaction was found only between PfeDGAT1 and PfeTAGL1, the Y2H interaction was also confirmed in planta by bimolecular fluorescence complementation (BiFC) through transient expression in N. benthamiana leaves. Additionally we utilized protein-GFP fusions to demonstrate that both DGATs localize to the endoplasmic reticulum (ER) whereas SDP1 and OBL1 localize to oil bodies. However, TAGL1 localizes to both ER and oil bodies. Therefore, these results suggest PfeDGAT1 and PfeTAGL1 as likely candidates for a TAG remodeling complex at the ER oil body junction. A manuscript with these results together with the DGAT results in objective 2 is currently in preparation. Objective 4. Identify in planta roles of TAG remodeling candidate genes (Years 2-3). 50% completed. RNAi gene silencing constructs and the production of stably transformed Physaria fendleri plants has been initiated for genes, DGAT1, DGAT2, TAGL1, SPD1, PDAT2, and PDAT1/PDAT2. 15 PfeDGAT1_RNAi T1 lines have been generated and are growing for seed oil analysis. 5 PfeDGAT2_RNAi lines have been generated and more are at the tissue culture stage. 14 PfePDAT2_RNAi T1 lines were generated but failed to develop seed, indicating a potential role of PDAT2 in initial seed development. PfePDAT1-PDAT2_RNAi lines are at tissue culture stage. The PfeTAGL1_RNAi transformation are at the initial tissue culture stage. Nine PfeSDP1_RNAi lines were generated and analyzed for oil phenotypes, the results complement the enzymology studies (Obj. 1) suggesting the major role of PfeSDP1 is for TAG turnover during seed germination and late seed development. A key phenotype of these RNAi knockdown lines is both increased seed oil and increased seed HFA-content, with minimal affects on seed germination. Therefore, PfeSDP1 should be a key target to increase HFA output of Physaria fendleri through future follow up CRISPR based knockout studies for crop improvement. The results on PfeSDP1 RNAi lines are currently submitted for publication, along with the SDP1 enzymes assays from obj. 1. In addition, we have successfully developed a VIGS based approach for RNA knockdown studies in P. fendleri, and demonstrated the key in vivo roles of PfeFAH12 and PfeKCS18 for lesquerolic acid production. The in Physaria confirmation of the function of these genes has not been done before. We have also used this VIGS approach to identify the genes involving self-incompatibility in Physaria fendleri, and demonstrate we can develop self compatible plants that will not be reliant on an insect pollinator for seed production. Thus the identified genes should be key targets to increase seed production Physaria fendleri through future follow up CRISPR based knockout studies for crop improvement. We are currently applying this VIGS approach concomitantly with the RNAi approach for the analysis of the in vivo function of PfeTAGL1. Objective 5. Bioengineer TAG remodeling into an oilseed crop (Years 2-3). 50% completed. Top gene candidates from objectives 1-3 (PfeDGAT1, PfeDGAT2 and PfeTAGL1) have been transformed to into hydroxy fatty acid producing Camelina sativa. PfeTAGL1+PfeDGAT1 and PfeTAGL1+PfeDGAT2 transgenic lines are in the T3 generation. Seed analysis indicates the HFA content of the Camelina was increased approximately 50%, reaching approximately 15% of total fatty acids. Individual gene overexpression controls are underway.In addition, we are attempting to speed up selection of new gene combinations by complimentary experiments in Arabidopsis thaliana. We have obtained similar results of increased HFA accumulation in transgenic Arabidopsis with various individual and double gene constructs. Interestingly, the combination of lipases from Physaria fendleri with DGAT2 from castor bean appears to give the largest increases in HFA content. After selecting the optimial genes from Arabidopsis new Camelina lines will be produced.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Bhandari, S. & Bates, P. D. Triacylglycerol remodeling in Physaria fendleri indicates oil accumulation is dynamic and not a metabolic endpoint. Plant Physiol 187, 799-815, doi:10.1093/plphys/kiab294 (2021).
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
Bates, P.D. (2022) Chapter Six - The plant lipid metabolic network for assembly of diverse triacylglycerol molecular species. In: Advances in Botanical Research (R�beill�, F. and Mar�chal, E. eds), pp. 225-252. Academic Press. https://doi.org/10.1016/bs.abr.2021.07.003
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Azeez A, Parchuri P, Bates PD. Suppression of Physaria fendleri SDP1 Increased Seed Oil and Hydroxy Fatty Acid Content while Maintaining Oil Biosynthesis through Triacylglycerol Remodeling.
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2022
Citation:
Sajina Bhandari, PhD Dissertation
Washington State University
ELUCIDATION AND RECONSTITUTION OF TRIACYLGLYCEROL REMODELING: A NOVEL PATHWAY OF SEED OIL BIOSYNTHESIS IN PHYSARIA FENDLERI
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Craine, W., Lu, C., Garrison Garneau, M., Bates, P., & Hulbert, S. (2022). Registration of WA-LE1, an edible oil camelina cultivar. Journal of Plant Registrations, 1 5. https://doi.org/10.1002/plr2.20205
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Poster presentation at The International Conference on The Status of Plant & Animal Genome Research, San Diego, CA, USA (PAG XXIX, January 8-12, 2022).
Abdul Azeez and Philip Bates, A rapid functional genomics method using Virus-Induced Gene Silencing (VIGS) of the gene of interest by co-targeting self-incompatibility genes.
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:The target audience for this project is plant breeders, the plant metabolism community, plant biochemists and scientists and engineers engaged in manipulating plant oil production for food, fuel or feed stock needs. In addition the target audience will include graduate students and undergraduates in plant biochemistry and molecular biology who will be able to use publications from these studies to understand plant oil production. Changes/Problems:The original plan was that all RNAi plants were to be produced by the co-PI at the USDA-ARS pacific west region location. However, due to COVID-19 based restrictions limiting personnel to 25% capacity at USDA-ARS only one RNAi line was able to be produced in year one. To circumvent this issue RNAi lines are now also being produced at WSU to ensure timely production of products, and we are testing a new and potentially faster VIGS based approach. It is expected that the personnel capacity at the USDA-ARS will be increased by Oct. 2021 and thus this area of the project will speed up to meet original targets. What opportunities for training and professional development has the project provided?During the first year of this project two postdocs and oneundergraduatehave been trained in experimentation, and one graduate student have been trained in experimentation, presentation, and manuscript writing. How have the results been disseminated to communities of interest?The results been disseminated by onejournal article publicationand onepresentation at a national meeting which was held virtually in 2020. What do you plan to do during the next reporting period to accomplish the goals?Progress will be made on all objectives. Additional results from objectives 1-3 will be used to select additional gene targets in for objectives 4-5. If the VIGS approach mentioned in objective 4 is successful and quicker as expected we will proceed with an expanded set of gene targets to knockdown to better understand Physaria fendleri TAG biosynthesis and identify targets for crop improvement.
Impacts
What was accomplished under these goals?
Objective 1 Biochemical characterization of the fatty acid selectivity of candidate lipases involved in TAG remodeling (Years 1-2). 30% completed. Top candidate lipases TAGL1 and SPD1 have been cloned from Physaria fendleri into E. coli for expression, along with the homologous lipases from Arabidopsis thaliana as controls for lipases with different fatty acid selectivity. Activity assays are undergoing. Objective 2. Biochemical characterization of the fatty acid selectivity of candidate diacylglycerol acyltransferases (DGATs) involved in TAG remodeling (Years 1-2). 50% completed. PfeDGAT1, PfeDGAT2 and PfeDGAT3 have been cloned from Physaria fendleri into the yeast quadruple mutant H1246 unable to produce TAG. In addition as controls AtDGAT1, AtDGAT2, and AtDGAT3 were also used. The DGAT1s and DGAT2s from both species complimented TAG biosynthesis is yeast, but the DGAT3s did not. Therefore, future experiments will focus on the DGAT1s and DGAT2s. In yeast fatty acid feeding studies PfeDGAT1 but not PfeDGAT2 could utilize ricinoleic acid and lesquerolic acid containing substrates suggesting DGAT1 may be more selective for hydroxy fatty acids than DGAT2. This situation is the opposite from what has been characterized in castor which also accumulates hydroxy fatty acids. To confirm these yeast feeding studies, we are currently moving to in vitro assays, which requires us to synthesize the substrates which are not commercially available. Objective 3. Protein-Protein interactions to identify hypothesized lipase-DGAT complex (Years 1-2). 80% completed. Y2H based protein-protein interactions have been screened for PfeDGAT1 and PfeDGAT2 vs the lipases PfeTAGL1, PfeSDP1, and PfeOBL1. Strong interaction was found between PfeDGAT1 and PfeTAGL1, the Y2H interaction was also confirmed in planta by bimolecular fluorescence complementation (BiFC) through transient expression in N. benthamiana leaves. This result suggests PfeDGAT1 and PfeTAGL1 as likely candidates for a TAG remodeling complex. Objective 4. Identify in planta roles of TAG remodeling candidate genes (Years 2-3). 30% completed. RNAi gene silencing constructs have been produced for Physaria fendleri genes, DGAT1, DGAT2, TAGL1, SPD1, PDAT2. Production of stably transformed Physaria fendleri plants through tissue culture based methods have begun for the DGAT1, SPD1, and PDAT2 constructs. In addition, testing of a virus-induced gene silencing method that may circumvent the relatively slow tissue culture steps for gene knockdown assays is underway. Initial results involving the incorporation of the VIGS construct into germinating Physaria fendleri seeds has been effect for silencing of genes expressed in leaves. We are currently testing if the VIGS construct will be transferred to developing seeds for knockdown of TAG biosynthetic genes by targeting PfeFAH12 and PfeKCS18. If effective, the VIGS approach will be used on DGATs and lipases. Objective 5. Bioengineer TAG remodeling into an oilseed crop (Years 2-3). 30% completed. Top gene candidates from objectives 1-3 (PfeDGAT1 and PfeTAGL1) have been transformed to into hydroxy fatty acid producing Camelina sativa together and individually. In addition, PfeDGAT2 has been transformed into Camelina with and without PfeTAGL1 as a control to that of PfeDGAT1. Various plant lines are at the T1 and T2 stage and we are currently screening for transformation events that affect the seed oil fatty acid composition and oil about.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Bhandari S, Bates PD (2021) Triacylglycerol remodeling in Physaria fendleri indicates oil accumulation is dynamic and not a metabolic endpoint. Plant Physiology. https://doi.org/10.1093/plphys/kiab294
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Bhandari S, BATES PD. Seed Oil Biosynthesis: Lesquerella Does It a Unique Way. Plant Biology 2020 Worldwide Summit, by the American Society of Plant Biologists. July, 30, 2020. Oral presentation by Sajina Bhandari.
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