Progress 03/01/17 to 02/29/20
Outputs
Target Audience:The main communities of interest are academic scientists, postdocs, graduate students in the field of plant metabolism and plant biotechnology, as well as the plant biotech industry. We have reached these audiences through three presentations by the PD, co-PD and postdoc at the 35th Annual IPG Symposium at the University of Missouri: Advances in Plant Metabolism. Columbia, MO. May 31, 2018. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Since the last report this project has provided for training of 1 postdoc, 1 first year graduate student, and 1 undergraduate. How have the results been disseminated to communities of interest?The main communities of interest are academic scientists, postdocs, graduate students in the field of plant metabolism and plant biotechnology, as well as the plant biotech industry. We have reached these audiences through three presentations by the PI, co-PI and postdoc at the 35th Annual IPG Symposium at the University of Missouri: Advances in Plant Metabolism. Columbia, MO. May 31, 2018. Additionally the post doc presented work at the 19th Annual Donald Danforth Plant Science Center Fall Symposium. Both the Danforth Fall Symposium and the Interdisciplinary Plant Group Symposiums feature international renowned speakers and have attendances on the order of 300 people from around the globe. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we aim to publish manuscripts based on the accomplishments in Objectives 4 and 7. We will use the lessons learned from Objectives 4 & 7, to produce new experimental designs for Objective 5 and preform the initial labeling experiments. In addition the Bates lab will continue with the 14CO2 labeling approach to investigate futile cycles in objective 6 and correlate these findings with complimentary 13CO2 labeling approach within the Allen lab. For Objective 1, we will complete our analysis of 13CO2-labeling data from WT and LEC2 plants over development. We will also complete lipid, acyl-ACP, starch, soluble sugar, free amino acid, and protein measurements from WT and LEC2 leaf tissue collected over the photoperiod over development. In addition, we will perform microscopy work to visualize differences in lipid droplets, starch granules, and chloroplast membranes between the WT and LEC2 leaves. Once we have determined the proper plant stage to perform labeling experiments, we will collect the necessary measurements of biomass components and photosynthetic parameters in preparation for modeling. We will use the conclusions drawn from this objective to perform the transient 13CO2-labeling experiments for Objectives 2 & 3 at the appropriate plant age. Once a transient labeling experiment has been conducted on the WT line and the necessary metabolites analyzed, we can begin constructing a computational flux model for central carbon flux in WT leaves for Objective 7.
Impacts
What was accomplished under these goals?
Objective 1. 95% Completed Lipid and starch levels were measured from the top two fully expanded leaves of plants of all four genotypes (WT, HO, SPD1, and LEC2) sampled at five time points from 28-77 days after sowing (DAS). These experiments have established that the highest increase in total lipids occurs between 49 and 63 DAS, with the lipid content of all three transgenic lines appearing to level off after 63 DAS. The increase in lipid is significant in all ages tested wth leaves at 28 DAS accumulating twice the amount of lipid in the LEC2 line than WT. The lipid phenotype is likely present from emergence because the lines have delayed germination and are stunted early possibly reflecting the engineered production and protection of lipid at a time when normally plants metabolize lipid from the seed to produce the initial leaves. In later stages, approaching flowering, we have measured lipid levels matching or exceeding that reported by (Vanhercke et al., 2017) for the HO and LEC2 lines, though we were unable to observe consistently increased lipid content for the SPD1 line compared to the base HO line. Quantification of foliar starch content confirm decreased levels for all transgenic lines relative to WT by 63 DAS, though this difference became less pronounced with age for the HO and SPD1 lines. Somewhat surprisingly, we also found that WT leaves accumulate high levels of starch over development, with younger leaves displaying a diurnal cycling of starch accumulation and breakdown which is not apparent in older leaves. Objective 2.15% Completed A significant number of preliminary 13CO2-labeling of single leaves of the WT line has been conducted to test and optimize our labeling set-up. After investigating the effects of various parameters such as gas flow rate, the presence of a humidifier in the line, the watering status of the plants, and type of labeling chamber, we were able to achieve up to 40% 13C-label incorporation in Calvin Cycle intermediates after 5 minutes of labeling and over 50% after 10 minutes of labeling, an adequate degree of 13C enrichment. Preliminary results have led us to design multiple labeling apparatus for leaves and freeze clamp methods to quickly quench metabolism, that lead to a more consistent volume and accurate results. During the process of analyzing labeled biomass with mass spectrometry we have also extended our capacity to measure additional sugars, and to devise a rigorous method to quantify protein that subverts challenges of prior assays. Both of these techniques will enable a better depiction of metabolism and add to the rigor in our future modeling efforts. Objective 3. 10% Completed As stated above for Objective 1, we are currently conducting brief 13CO2-labeling of leaves of the LEC2 line to 1) confirm the optimum plant age for label incorporation into lipids and other primary metabolites and 2) look for any developmental differences in metabolic flux to pathways of interest. One of the challenges in working with a crop is confronting the development of a canopy over time and the choice of what leaf at what time to monitor. Thus we continue to put considerable effort into characterizing and understanding the metabolism within the architecture prior to pinpointing leaves for flux analysis which is costly, laborious and must be carefully focused to address all of our initial questions. Objective 4. 95% Completed Pulse and pulse-chase metabolic labeling studies utilizing [14C]acetate and [14C]glycerol have been performed in both wild-type and 15% oil containing leaves. These experiments have indicated that both wild-type and 15% oil accumulating tobacco leaves first incorporate nascent fatty acids into the membrane lipid phosphatidylcholine (PC), rather than direct esterification to glycerol-3-phosphate within the direct Kennedy pathway of oil biosynthesis. In addition the glycerol labeling has indicated that significant leaf oil synthesis utilizes a pathway where both the fatty acids and glycerol back bone move through PC prior to oil synthesis, and likely competes with synthesis of MGDG the major lipid of photosynthetic membranes. These results lead to at least two important conclusions: 1) In the current high oil lines, the fatty acid flux through PC indicates that the final fatty acid composition of the leaf oil will involve the accumulation of essential polyunsaturated fatty acids produced in PC. Therefore, engineering high oleic oil (useful for biofuels and frying) in tobacco may require re-routing the flux of fatty acids away from PC and toward a direct oil accumulation pathway. 2) The similarity of fatty acid flux in tobacco with the unrelated model plant Arabidopsis suggests that using the abundant genetic and molecular biology resources in Arabidopsis may provide useful proof-of-concept results prior to the more difficult task of producing transgenic tobacco. Because of this similarity we preformed a similar fatty acid flux experiments in an Arabidopsis triple mutant (act1/lpcat1/lpcat2) to determine if we could alter leaf flux more toward a direct Kennedy pathway. In these plants there was significantly reduced flux of fatty acids directly into PC through the acyl editing cycle, and thus future research utilizing targeted knockouts of these genes in tobacco may help to divert nascent fatty acid flux more toward a direct Kennedy pathway of oil biosynthesis. Manuscripts for both the tobacco and Arabidopsis leaf lipid labeling are currently in preparation. 5. Objective 5. 5% Completed Since the move to WSU, the plants are being grown in new greenhouses and in correlation to Objective 1 the prime growth stage for labeling will be determined prior to starting the labeling experiments for Objective 5. 6. Objective 6.10% completed. Previously preliminary experiments with whole plant 14CO2 pulse-chase labeling have been conducted to determine how different leaves respond to the pulse-chase labeling approach to design long term lipid turnover experiments. Our initial results indicate distinct variations in carbon partitioning based on leaf age. Therefore we have targeted the first several fully expanded horizontal leaves for analysis after whole plant labeling. Our initial results of a 1.5 hr pulse indicate that the initial product of photosynthesis are rapidly transferred out of leaves during the remainder of the first day, and first night. What carbon that remains in the leaf mostly stabilizes in starch, cell wall, protein, and membrane lipids in both wild-type and the HO line. However, the TAG in the HO line declines during the first and second night. Prolonged darkness leads to almost the complete turnover of the labeled TAG. This result indicates that in the HO line the TAG is not very stable during dark periods. Further analysis of the next generation tobacco lines will indicate if the changes have led to more stable oil. 7. Objective 7.10% completed We have created kinetic models based on radioactivity measurements (i.e. 14C) for the HO line of Objective 4. Radiolabeling of leaf disks with 14C-glycerol by the Bates lab is being modeled by the Allen lab. Such models for pulse and pulse-chase data have not been previously reported. Currently we have developed a series of differential equations to model both pulse and chase aspects that can be fitted by minimizing the least squares difference. The optimal solutions result in estimates of flux from radiolabeling. The development of models is ongoing; however current descriptions include multiple DAG and PC pools, consistent with our understanding of lipid metabolism. Importantly the models are able to fit the combination of pulse and pulse-chase data which has not been previously documented. We will continue to work on this and in addition will model aspects of central carbon metabolism from carbon dioxide production to acetyl-CoA biosynthesis with 13C isotopically nonstationary metabolic flux analysis in the coming year (objectives 2 &3).
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
35th Annual IPG Symposium at the University of Missouri: Advances in Plant Metabolism. Columbia, MO. May 31, 2018. Deciphering Connections Between Diacylglycerol Flux Through the Glycerolipid Metabolic Network and the Regulation of Fatty Acid Synthesis. Oral presentation by Philip Bates (PD).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
35th Annual IPG Symposium at the University of Missouri: Advances in Plant Metabolism. Columbia, MO. May 31, 2018. Characterization of source and sink leaves across development in high-oil tobacco lines to perform isotopic labeling for metabolic flux analysis. Poster presentation by Kevin Chu (postdoc).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
35th Annual IPG Symposium at the University of Missouri: Advances in Plant Metabolism. Columbia, MO. May 31, 2018. Modeling Pulse/Pulse-Chase Radiolabeling to Assess Lipid Metabolism. Poster presentation by Doug Allen (co-PD).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
19th Annual Fall Symposium of Donald Danforth Plant Science Center. St. Louis, MO. Sept 28, 2018. Characterization of central carbon metabolism in high oil tobacco lines over development. Poster presentation by Kevin Chu (postdoc).
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Progress 03/01/17 to 02/28/18
Outputs
Target Audience:The main target audience for our research is scientists, postdocs, and graduate students in the field of plant metabolism and plant biotechnology. We have reached these audiences through three presentations (2 oral, 1 poster) at international (2) and regional (1) meetings. The conference presentations are detailed in the products section. In addition Dr. Bates (PD) gave two invited seminars two reach these audiences: Jackson State University, Department of Chemistry Seminar Series. Jackson, MS. Nov. 10, 2017. Deciphering Bottlenecks within Plant Oil Engineering: What Controls Fatty Acid Flux? Presented by Bates (PD). Donald Danforth Plant Science Center. St. Louis, MO. August 10, 2017. Deciphering Bottlenecks within Plant Oil Engineering: What Controls Fatty Acid Flux? Presented by Bates (PD). In addition to research scientists a secondary audience is high school science students. The Bates lab has partnered with the American Chemical Society Project SEED which provides research opportunities for under privileged high school students. A high school student worked in the Bates lab over summer 2017, and presented his research to his peers at the Mississippi Region 1 Science and Engineering Fair and won first prize in the biochemistry category, and was selected to present at the state wide science fair. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided for training of 1 postdoc, 1 first year graduate student, 1 undergraduate (summer research assistant), and one high school student. The high school student was associated with the American Chemical Society Project SEED which provides research experience opportunities for under privileged high school students. The high school student also presented their work at the Mississippi Region 1 Science and Engineering Fair and won first prize in the biochemistry category, and was selected to present at the state wide science fair. In addition he has presented a poster at the 82nd Annual Mississippi Academy of Sciences research conference listed in the products section. How have the results been disseminated to communities of interest?The main communities of interest are scientists, postdocs, and graduate students in the field of plant metabolism and plant biotechnology. We have reached these audiences through three presentations (2 oral, 1 poster) at international (2) and regional (1) meetings. The conference presentations are detailed in the products section. In addition Dr. Bates (PD) gave two invited seminars two reach these audiences: Jackson State University, Department of Chemistry Seminar Series. Jackson, MS. Nov. 10, 2017. Deciphering Bottlenecks within Plant Oil Engineering: What Controls Fatty Acid Flux? Presented by Bates (PD). Donald Danforth Plant Science Center. St. Louis, MO. August 10, 2017. Deciphering Bottlenecks within Plant Oil Engineering: What Controls Fatty Acid Flux? Presented by Bates (PD). What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we aim to publish manuscripts based on the accomplishments in Objectives 4 and 7. We will use the lessons learned from Objectives 4 & 7, to produce new experimental designs for Objective 5 and preform the initial labeling experiments. In addition the Bates lab will continue with the 14CO2 labeling approach to investigate futile cycles in objective 6 and correlate these findings with complimentary 13CO2 labeling approach within the Allen lab. For Objective 1, we will further measure soluble sugars from the leaf tissue used for lipid and starch analysis over plant development and analyze 13CO2-labeling data from LEC2 plants over development. We will also complete lipid, starch, and soluble sugar analysis of leaf tissue collected over the photoperiod. We will use the conclusions drawn from this objective to perform the transient 13CO2-labeling experiments for Objectives 2 & 3 at the appropriate plant age. Once a transient labeling experiment has been conducted on the WT line and the necessary metabolites analyzed, we can begin constructing a computational flux model for central carbon flux in WT leaves for Objective 7.
Impacts
What was accomplished under these goals?
Objective 1. Measure physiological parameters and biomass components to characterize the proper developmental stages for metabolic flux experiments (Year 0-1). - 75% completed Lipid and starch levels were measured from the top two fully expanded leaves of plants of all four genotypes (WT, HO, SPD1, and LEC2) sampled from 42 days after sowing (DAS) until flowering. These experiments have established that the highest increase in total lipids occurs between 49 and 63 DAS, with the lipid content of all three transgenic lines appearing to level off after 63 DAS. We have measured lipid levels matching or exceeding that reported by (Vanhercke et al., 2017) for the HO and LEC2 lines, though we were unable to observe consistently increased lipid content for the SPD1 line compared to the base HO line. Quantification of foliar starch content confirm decreased levels for all transgenic lines relative to WT by 63 DAS, though this difference became less pronounced with age for the HO and SPD1 lines. We are currently optimizing a method for the quantification of soluble sugars in these leaf samples. We are also analyzing the lipid, starch, and soluble sugar levels of leaves from all four lines sampled throughout the photoperiod to confirm midday as a time of pseudo-metabolic steady state for our subsequent labeling experiments. Our results suggest that the transient 13CO2-labeling experiments proposed for Objectives 2 and 3 should occur around 56 DAS, an age where lipid production in the transgenic lines is presumably at its highest and differences in foliar starch levels are the most pronounced. We are currently conducting 5-minute 13CO2-labeling experiments weekly on growing plants of the LEC2 line to confirm 56 DAS as the plant age for maximum label incorporation. These dynamic labeling measurements may reveal developmental differences in flux to metabolic pathways of interest such as lipid biosynthesis and starch breakdown. We will utilize these analyses to plan the optimal age to perform the labeling experiments for later objectives. Our observations of plant development have led us to believe the LEC2 line to be the more promising of the two newer transgenic lines, exhibiting higher lipid levels and lower foliar starch content than the SPD1 line while also exhibiting the least degree of compromised growth and biomass compared to the WT line. Similar growth staging and lipid content characterization has been done with plants grown by the Bates lab at USM, will help us correlate experiments between each lab. Objective 2. Perform transient 13CO2 in planta leaf labeling experiments and measure isotopic enrichment in wild type and base transgenics accumulating 15% oil in leaves (Years 0.5-1.5) - 10% completed Preliminary 13CO2-labeling of single leaves of the WT line has been conducted to test and optimize our labeling set-up. After investigating the effects of various parameters such as gas flow rate, the presence of a humidifier in the line, and the watering status of the plants, we were able to achieve over 30% 13C-label incorporation in Calvin Cycle intermediates after 5 minutes of labeling, an adequate degree of 13C enrichment. We are currently growing WT plants for the first complete labeling experiment scheduled in mid-late March pending the final determination of optimal plant age from Objective 1. Objective 3. Perform transient 13CO2 in planta leaf labeling experiments and measure isotopic enrichment in transgenics accumulating ~30% oil in leaves (Years 1.5-2.5). - 10% Completed As stated above for Objective 1, we are currently conducting weekly brief 13CO2-labeling of leaves of the LEC2 line to 1) confirm the optimum plant age for label incorporation into lipids and other primary metabolites and 2) look for any developmental differences in metabolic flux to pathways of interest. Objective 4. Characterize changes in acyl-lipid pathways and their impact on photosynthetic membranes in wild-type and 15% oil containing leaves (Years 0.5-1.5). - 95% Completed Pulse and pulse-chase metabolic labeling studies utilizing [14C]acetate and [14C]glycerol have been performed in both wild-type and 15% oil containing leaves. These experiments have indicated that both wild-type and 15% oil accumulating tobacco leaves first incorporate nascent fatty acids into the membrane lipid phosphatidylcholine (PC), rather than direct esterification to glycerol-3-phosphate within the direct Kennedy pathway of oil biosynthesis. In addition the glycerol labeling has indicated that significant leaf oil synthesis utilizes a pathway where both the fatty acids and glycerol back bone move through PC prior to oil synthesis, and likely competes with synthesis of MGDG the major lipid of photosynthetic membranes. These results lead to at least two important conclusions: 1) In the current high oil lines, the fatty acid flux through PC indicates that the final fatty acid composition of the leaf oil will involve the accumulation of essential polyunsaturated fatty acids produced in PC. Therefore, engineering high oleic oil (useful for biofuels and frying) in tobacco may require re-routing the flux of fatty acids away from PC and toward a direct oil accumulation pathway. 2) The similarity of fatty acid flux in tobacco with the unrelated model plant Arabidopsis suggests that using the abundant genetic and molecular biology resources in Arabidopsis may provide useful proof-of-concept results prior to the more difficult task of producing transgenic tobacco. Because of this similarity we preformed a similar fatty acid flux experiments in an Arabidopsis triple mutant (act1/lpcat1/lpcat2) to determine if we could alter leaf flux more toward a direct Kennedy pathway. In these plants there was significantly reduced flux of fatty acids directly into PC through the acyl editing cycle, and thus future research utilizing targeted knockouts of these genes in tobacco may help to divert nascent fatty acid flux more toward a direct Kennedy pathway of oil biosynthesis. Manuscripts for both the tobacco and Arabidopsis leaf lipid labeling are currently in preparation. 5. Objective 5. Characterize changes in acyl-lipid pathways and their impact on photosynthetic membranes in transgenics accumulating ~30% oil in leaves (Years 1.5-2.5). - 0% Competed Objective 5 has yet to be started. 6. Objective 6. Assess carbon flux through futile cycles of lipid synthesis and turnover (Years 1.5-2.5). - 10% completed. Preliminary experiments with whole plant 14CO2 pulse-chase labeling have been conducted to determine how different leaves respond to the pulse-chase labeling approach to design long term lipid turnover experiments. Our initial results indicate distinct variations in carbon partitioning based on leaf age, of which will be useful for designing our future experiments to assess the futile cycles of lipid synthesis/turnover over long periods (several days). 7. Objective 7. Build computational models of carbon fluxes in wild-type and high oil leaves (year 3). - 10% completed We have created kinetic models based on radioactivity measurements (i.e. 14C) for the HO line of Objective 4. Radiolabeling of leaf disks with 14C-glycerol and 14C-acetate by the Bates lab is being modeled by the Allen lab. Such models for pulse and pulse-chase data have not been previously reported. Currently we have developed a series of differential equations to model both pulse and chase aspects that can be fitted by minimizing the least squares difference. The optimal solutions result in estimates of flux from radiolabeling. The development of models is ongoing; however current descriptions include multiple DAG and PC pools, consistent with our understanding of lipid metabolism. We will continue to work on this and in addition will model aspects of central carbon metabolism from carbon dioxide production to acetyl-CoA biosynthesis with 13C isotopically nonstationary metabolic flux analysis in the coming year (objectives 2 &3).
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
56th Annual Meeting of the Phytochemical Society of North America. August 7, 2017. Columbia, MO. Title: Deciphering the Eukaryotic Pathway of Leaf Glycerolipid Assembly through Lipid Flux Analysis in Arabidopsis Mutants and Oil Accumulating Tobacco. Oral presentation by Bates (PD).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
8th European Symposium on Plant Lipids. July 4, 2017. Malmo, Sweden. Title: Insights on the Eukaryotic Pathway of Leaf Lipid Synthesis from Acyl Flux Analysis within the Arabidopsis act1/lpcat1/lpcat2 Triple Mutant. Oral presentation by Bates (PD)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
82nd Mississippi Academy of Sciences Annual Meeting. Feb. 22-23, 2018. Hattiesburg, MS. Poster presentation. Title: Biochemical analysis of plant lipid metabolism for enhanced bio-fuel and bio-chemical production. Kimberton Mai (presenter, high school student), Nischal Karki, Philip Bates.
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