Progress 01/15/24 to 01/14/25
Outputs
Target Audience:Target Audience: The main target audience for this project is the plant science research community. This project examined what happens to important plant products under environmental stress. The major research products have been shared through public seminars and the major results are in publications. To pursue this question, this project develops tools and computational models that would also be valuable for formal classroom instruction. PlantCV image analysis tools are used to analyze image data in this project and are used to teach educators, students (K-12), undergraduates, and researchers biocomputing. To that end, we have trained a set of educators from Harris Stowe University (HBCU) in using PlantCV and a course has been designed cross-listed in Math/Bio that uses the tools we developed. We have held virtual workshops to teach researchers to use PlantCV tools that are used in this project. This data collected from this project was used for Summer Research Experiences for Undergraduates (REU Projects). Changes/Problems:No major problems during this no-cost extension, we finished up publications associated with this work. A paper was under review for longer than expected (a common problem), but that paper has moved forward and is currently in revision. ? What opportunities for training and professional development has the project provided?This project has directly trained two postdoctoral researchers and a graduate student as well as five total undergraduate students participating in an REU program and through part time work on the project during the school year. Two technicians have also been trained in a new image analysis method. The postdocs and students have been trained in experimentation, presentations, and writing. The two postdocs have both moved on to promising careers that match their career interests. One postdoc is now the Director of Phenotyping at the Danforth Center, and the other postdoc is now employed in a grant writing position, after discovering that this was the direction she wanted to take with her career. The graduate student Brandon Johnson gave a presentation of his results at a scientific conference and won an award for best poster presentation. This project has trained researchers (undergraduates, researchers) in the use of PlantCV tools developed in part through this project through workshops throughout 2024. How have the results been disseminated to communities of interest?Results have been disseminated through scientific presentations, social media, and through virtual workshops held for the plant science community. The 'Real Time Science' tik tok, which was created for this project now has ~8000 followers and 200 videos on TikTok. These videos disseminate the research to a general public, and have been used in middle school projects via news site Newsela, and continues to be an online source of documented videos for future viewers, and K. Murphy described the project on the Story Collider Podcast. Presentations at conferences and as invited seminars are listed below:? Allen: Modeling Pulse-Chase Radiolabeling Data with a User-Friendly Computational Platform to Assess Lipid Metabolism, 1st International Camelina Conference (ICC), Lincoln, Nebraska Allen: Modeling Pulse-Chase Radiolabeling Data with a User-Friendly Computational Platform to Assess Lipid Metabolism, International Symposium on Plant Lipids (ISPL), Lincoln, Nebraska Bates: University of Alberta, Edmonton, AB, Canada. Seminar: The oily frontier: lipid flux cartography to produce valuable fatty acids in obese plants. Bates: University of Minnesota, Minneapolis, MN. Seminar: The oily frontier: lipid flux cartography to produce valuable fatty acids in obese plants. Bates: Elucidating Diverse Mechanisms of Plant Oil Synthesis to Accumulate Industrially Valuable Fatty Acids. Michigan State University. East Lansing, MI. Gehan: What Phenotypes Matter?: Open Challenges in Plant Phenomics with PlantCV. American Phytopathological Society Annual Meeting. Memphis, Tennessee. Gehan: What Phenotypes Matter?: Open Challenges in Plant Phenomics with PlantCV. Prairie View A&M University Seminar. Gehan: What Phenotypes Matter?: Open Challenges in Plant Phenomics with PlantCV. Multiscale Plant Vascular Biology Gordon Research Conference. Gehan: What Phenotypes Matter?: Open Challenges in Plant Phenomics with PlantCV. Bayer Seminar Series, Saint Louis, MO Gehan: What Phenotypes Matter?: Open Challenges in Plant Phenomics with PlantCV. University of Missouri, Saint Louis, Seminar Series Murphy: University of Missouri, St. Louis. Seminar: Investigating plant responses to climate change with image-based phenotyping Workshops on tools associated with this project: Workshop Name 2/5/2024 Undergraduate Data Science Intern Workshop 2/12/2024 2024 NAPPN Pre-Conference Workshop: PlantCV Machine Learning Lab Workshop 4/2/2024 Embrapa PlantCV Workshop - Campo Grande, Mato Grasso de Sul, Brazil 4/23/2024 PlantCV Bring-Your-Own-Data Workshop 4/29/2024 PlantCV Workflow Fundamentals 4/30/2024 PlantCV Workflow Fundamentals 5/7/2024 Multiple Plant Image Analysis & Workflow Parallelization 5/14/2024 "This Machine's Bean Learning" (Random Forest Classifier Workshop) 5/21/2024 Leaf Disease/Lesion Lab (naive Bayes classifier Workshop) 9/30/2024 PlantCV Workflow Fundamentals 9/30/2024 PlantCV Hyperspectral Workflow Tutorial 11/19/2024 Water Use Phenotyping Workshop What do you plan to do during the next reporting period to accomplish the goals?We are currently in a no-cost extension for this project, so we worked on finishing up publications associated with this project. A paper that was authored by the postdoctoral researcher and graduate student on this project is currently under revision at The Plant Journal (revision submitted December 2024).
Impacts
What was accomplished under these goals?
1.2: Heat stress: Interactive annotation modules were further expanded and improved in the open-source image analysis platform PlantCV, to analyze fluorescent images to analyze photosynthetic efficiency on entire plants with spatial resolution. Next, various temperatures and durations of heat stress were applied to high oil and wild-type plants, and photosynthetic efficiency (including Fv/Fm, Fq'/Fm', and NPQ), were analyzed to determine the temperature, duration, and sampling time points of heat stress.High-oil tobacco and wild-type plants were also analyzed using light and confocal microscopy to determine differences in cellular and subcellular characteristics under control and heat stress conditions. High-oil tobacco was found to have less trichomes and shorter trichomes than WT plants, found through a project of an REU student. High-oil plants had substantial oil accumulated, as expected, but were unexpectedly also found to have large oil droplets in stomatal guard cells. Thermal imaging found that high-oil plants had higher leaf temperature. Stomatal imaging found that high-oil plants had smaller stomatal aperture and fewer stomata, which fits with the thermal data that found higher leaf temperatures. This was measured by a new tool added to the open-source open-development package PlantCV. This data was included in a publication currently under revision at the Plant Journal (submitted 2024). 1.3: Using the new photosynthesis module in PlantCV (Aim 1.1), chlorophyll content was assayed spatially using a chlorophyll red-edge index. High-oil tobacco was found to have reduced chlorophyll content than WT under control and heat-stress conditions. Heat stress increased the chlorophyll content of both WT and high-oil tobacco, but was increased to a greater degree in WT. This data was included in a publication currently under revision at the Plant Journal (submitted 2024). 2.1: Galactolipids (the main components of the photosynthetic thylakoid membranes) were quantified between WT, the 15% oil leaf, and 30% oil leaf tobacco lines (HO and LEC2, respectively) first under normal growth conditions. In the 15% oil leaves galactolipids were reduced ~43% from WT, suggesting fatty acids used for membranes were partitioned into TAG, however in the 30% oil lines there was not a further drop galactolipid content, suggesting expression of LEC2 altered metabolism to increase oil without further reducing the availability of photosynthetic membranes. A lipidomics study was conducted to compare the changes in MGDG and DGDG lipid species under chilling and control conditions between WT and LEC2 to elucidate differences in chloroplast lipid remodeling. Significant increases were the most notable in MGDG 34:6 and 36:6 lipids, as well as DGDG 36:6 lipids, notable for the high degree of unsaturation that is necessary to prevent chloroplastic membranes from freezing together. Additional analysis revealed that under chilling conditions DGDG lipid levels increase by approximately 12% in WT, whereas DGDG levels only increase by 5.5% in LEC2. When chloroplast membranes respond to cold, they often increase their DGDG lipid composition, as the head groups of DGDGs are larger and change membrane conformation shapes to a lamellar bilayer phase that prevents freezing injury to the chloroplast. These findings suggest that lipid metabolism and remodelling is occurring at different rates in LEC2 when compared to WT, most likely due to the pull of fatty acids into TAG from the overexpression of DAG. It is possible that WT tobacco is better equipped to respond to chilling stress at the membrane level than LEC2. A phenomics approach was utilized to analyze the efficiency of photosynthesis in intact WT, HO, LEC2 plants and an additional line that accumulates oil near LEC2, SDP1 (which is the HO background with RNAi knockdown of SDP1 triacylglycerol lipase). The initial results suggest the LEC2 photosynthetic electron transport (measured as photochemical quantum efficiency change, Phi2) significantly is reduced compared to WT. This correlates with a higher reduction level of the intersystem electron transport system measured as lower qL parameter. This suggests that metabolic consumption of photosynthetic generated reducing power is less efficient in LEC2. As potential consequence of this less efficient electron transport in LEC2 we observed partial photoinhibition in this line indicated by a lower maximal photochemical quantum efficiency parameter (Fv/Fm) and higher slow relaxing NPQ component (qI). In contrast, both HO and SDP1 lines behave similarly to WT. 3.1: To better understand how the LEC2 line accumulates more oil without a further drop in galactolipid content from that of the HO line as measured in Aim 2, we performed 14CO2 pulse-chase metabolic labeling over a 145 hour time course on WT and each oil line. A key difference between HO and LEC2 tobacco lines was that in HO the TAG was under a futile cycle of synthesis and degradation, such that by the end of the 145 hours the TAG produced during the pulse was mostly gone, whereas the the oil produced in the LEC2 line was stable. Therefore, oil stability is a key part of accumulation of high levels of oil accumulation in the LEC2 line without further depletion of photosynthetic membranes. Additionally, the TAG turnover in HO led to a futile cycle with starch synthesis/degradation and altered carbon flux through metabolism including induction of the glyoxylate cycle which produces intermediates that can inhibit carbon capture and likely partially explains the reduced CO2 uptake in the HO lines. The increased oil accumulation and CO2 uptake in the next generation LEC2 lines is likely associated with the reduced TAG turnover induced by the LEC2 transcription factor upregulating oil body packaging proteins. Together these results suggest that in vegetative tissues TAG stability is key to limit the adverse effects of TAG turnover on plant growth. 3.2: Refining methods for acyl-ACPs in other systems has continued and will benefit this project as well. We adapted methods to assess acyl-ACP pools in the chloroplast from studies on other systems but measurements with tobacco have been challenging because WT leaves have very little lipid production and thus do not give quantitative results consistently. New methods with additional purifications of ACP proteins and rapid testing of method ideas in simple microbial systems are being worked out to overcome the issues and establish a robust standard operating protocol. We anticipate that the effort will enable a number of studies on fatty acid and lipid analysis wherein the plant system does not make as much oil as a seed and is therefore more challenging to evaluate. Methods are near complete; and no ACP standard is commercially available, standards have been produced through overexpression of ACP in E. coli that is supplied 15N and through overexpression of a transferase enzyme that is used to link expressed ACP with acyl chains of varied lengths that serve as standards. Biomass reflecting cold or heat stress has been collected and stored at -80 until the methods are finalized.? 4.1: A software engineer graduated from WUSTL and has taken up full-time employment but has continued to troubleshoot 'bugs' in the software on a part time basis and worked on the GUI to enable ease of use. In prior years a computational framework was developed to model acyl lipid fluxes in plants with data from radiolabeling experiments for the 15% oil leaf line in comparison to the WT. Efforts to write up a manuscript for publication on this tool will continue.?
Publications
- Type:
Other
Status:
Under Review
Year Published:
2025
Citation:
Murphy KM , Johnson BS, Harmon C, Gutierrez J, Sheng H., Kenney S., Gutierrez-Ortega K., Wickramanayake J, Czymmek KJ, Bates PD, Allen DK, Gehan M (Under Revision at Plant Journal, 2024) Excessive leaf oil modulates the plant abiotic stress response via reduced stomatal aperture in tobacco (Nicotiana tabacum) .
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Johnson BS, Allen DK, Bates PD (2024) Triacylglycerol stability limits futile cycles and inhibition of carbon capture in oil-accumulating leaves. Plant Physiol. doi:10.1093/plphys/kiae121
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Koley S, Jyoti P, Lingwan M, Allen DK (2024) Isotopically Nonstationary Metabolic Flux Analysis of Plants: Recent Progress and Future Opportunities. New Phytologist Tansley Insight 242: 19111918 (2024)
|
Progress 01/15/21 to 01/14/25
Outputs
Target Audience:Target Audience: The main target audience for this project is the plant science research community. This project examined what happens to important plant products under environmental stress. The major research products have been shared through public seminars and the major results are in publications. To pursue this question, this project develops tools and computational models that would also be valuable for formal classroom instruction. PlantCV image analysis tools are used to analyze image data in this project and are used to teach educators, students (K-12), undergraduates, and researchers biocomputing. To that end, we have trained a set of educators from Harris Stowe University (HBCU) in using PlantCV and a course has been designed cross-listed in Math/Bio that uses the tools we developed. We have held virtual workshops to teach researchers to use PlantCV tools that are used in this project. This data collected from this project was used for Summer Research Experiences for Undergraduates (REU Projects). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has directly trained two postdoctoral researchers and a graduate student as well as five total undergraduate students participating in an REU program and through part time work on the project during the school year. Two technicians have also been trained in a new image analysis method. The postdocs and students have been trained in experimentation, presentations, and writing. The two postdocs have both moved on to promising careers that match their career interests. One postdoc is now the Director of Phenotyping at the Danforth Center, and the other postdoc is now employed in a grant writing position, after discovering that this was the direction she wanted to take with her career. The graduate student Brandon Johnson gave a presentation of his results at a scientific conference and won an award for best poster presentation. This project has trained researchers (undergraduates, researchers) in the use of PlantCV tools developed in part through this project through workshops throughout this project's tenure. ? How have the results been disseminated to communities of interest?Results have been disseminated through scientific presentations, social media, and through virtual workshops held for the plant science community. The 'Real Time Science' tik tok, which was created for this project now has ~8000 followers and 200 videos on TikTok. These videos disseminate the research to a general public, and have been used in middle school projects via news site Newsela, and continues to be an online source of documented videos for future viewers, and K. Murphy described the project on the Story Collider Podcast. In previous annual reports we have listed talks and workshops that supported the dissemination of this project. ? What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In general, we accomplished the targeted goals of this project, which will broadly impact the scientific community. Here is a summary of accomplishments of this project. Aim 1.1 We determined that there were no differences in circadian phase or period between high oil and wild type plants, which suggested that differences found in photosynthetic efficiency were not due to differences in the circadian clock. Through this project Interactive annotation modules were further expanded and improved in the open-source image analysis platform PlantCV, to analyze fluorescent images to analyze photosynthetic efficiency on entire plants with spatial resolution. These tools are open-source and have documented use by other researchers in the community. Aim 1.2 In previous reports we described in more detail the work that went into determining the duration and level of heat stress to apply to high oil and wild-type plants. This analysis suggested 42C day/32C night heat stress conditions over 7 days produced a photosynthetic response in both high-oil and wild-type plants. We found that there were significant differences in the heat response of high-oil tobacco compared to wild-type. Specifically, we found that high-oil plants showed a greater and more immediate reduction in photosynthetic efficiency due to heat stress than wild-type. Total oil decreases rapidly over the first two days of the stress and then reaches a steady level over the remaining stress period but at only ? the level of leaf oil as non-stressed plants. High-oil tobacco and wild-type plants were also analyzed using light and confocal microscopy to determine differences in cellular and subcellular characteristics under control and heat stress conditions. High-oil tobacco was found to have less trichomes and shorter trichomes than WT plants, found through a project of an REU student. High-oil plants had substantial oil accumulated, as expected, but were unexpectedly also found to have large oil droplets in stomatal guard cells. Thermal imaging found that high-oil plants had higher leaf temperature. Stomatal imaging found that high-oil plants had smaller stomatal aperture and fewer stomata, which fits with the thermal data that found higher leaf temperatures. These results were submitted for publication to The Plant Journal and are currently under revision. Aim 1.3 Using the new photosynthesis analysis tools developed through this project, chlorophyll content was assayed spatially using a chlorophyll red-edge index. High-oil tobacco was found to have reduced chlorophyll content than WT under control and heat-stress conditions. Heat stress increased the chlorophyll content of both WT and high-oil tobacco, but was increased to a greater degree in WT. This data was included in the publication currently under revision at the Plant Journal (submitted 2024). Aim 2.1: Galactolipids (the main components of the photosynthetic thylakoid membranes) were quantified between WT, the 15% oil leaf, and 30% oil leaf tobacco lines (HO and LEC2, respectively) first under normal growth conditions. In the 15% oil leaves galactolipids were reduced ~43% from WT, suggesting fatty acids used for membranes were partitioned into TAG, however in the 30% oil lines there was not a further drop galactolipid content, suggesting expression of LEC2 altered metabolism to increase oil without further reducing the availability of photosynthetic membranes. A lipidomics study was done to compare the changes in MGDG and DGDG lipid species under chilling and control conditions between WT and LEC2 to elucidate differences in chloroplast lipid remodeling. Significant increases were the most notable in MGDG 34:6 and 36:6 lipids, as well as DGDG 36:6 lipids, notable for the high degree of unsaturation that is necessary to prevent chloroplastic membranes from freezing together. Additional analysis revealed that under chilling conditions DGDG lipid levels increase by approximately 12% in WT, whereas DGDG levels only increase by 5.5% in LEC2. When chloroplast membranes respond to cold, they often increase their DGDG lipid composition, as the head groups of DGDGs are larger and change membrane conformation shapes to a lamellar bilayer phase that prevents freezing injury to the chloroplast. These findings suggest that lipid metabolism and remodelling is occurring at different rates in LEC2 when compared to WT, most likely due to the pull of fatty acids into TAG from the overexpression of DAG. It is possible that WT tobacco is better equipped to respond to chilling stress at the membrane level than LEC2. Aim 2.2 A phenomics approach was utilized to analyze the efficiency of photosynthesis in intact WT, HO, LEC2 plants and an additional line that accumulates oil near LEC2, SDP1 (which is the HO background with RNAi knockdown of SDP1 triacylglycerol lipase). The results suggest the LEC2 photosynthetic electron transport (measured as photochemical quantum efficiency change, Phi2) significantly is reduced compared to WT. This correlates with a higher reduction level of the intersystem electron transport system measured as lower qL parameter. This suggests that metabolic consumption of photosynthetic generated reducing power is less efficient in LEC2. As potential consequence of this less efficient electron transport in LEC2 we observed partial photoinhibition in this line indicated by a lower maximal photochemical quantum efficiency parameter (Fv/Fm) and higher slow relaxing NPQ component (qI). In contrast, both HO and SDP1 lines behave similarly to WT. Aim 3.1: To understand how the LEC2 line accumulates more oil without a further drop in galactolipid content from that of the HO line as measured in Aim 2, we did 14CO2 pulse-chase metabolic labeling over a 145 hour time course on WT and each oil line. A key difference between HO and LEC2 tobacco lines was that in HO the TAG was under a futile cycle of synthesis and degradation, such that by the end of the 145 hours the TAG produced during the pulse was mostly gone, whereas the the oil produced in the LEC2 line was stable. Therefore, oil stability is a key part of accumulation of high levels of oil accumulation in the LEC2 line without further depletion of photosynthetic membranes. Additionally, the TAG turnover in HO led to a futile cycle with starch synthesis/degradation and altered carbon flux through metabolism including induction of the glyoxylate cycle which produces intermediates that can inhibit carbon capture and likely partially explains the reduced CO2 uptake in the HO lines. The increased oil accumulation and CO2 uptake in the next generation LEC2 lines is likely associated with the reduced TAG turnover induced by the LEC2 transcription factor upregulating oil body packaging proteins. Together these results suggest that in vegetative tissues TAG stability is key to limit the adverse effects of TAG turnover on plant growth. Aim 3.2: We adapted methods to assess acyl-ACP pools in the chloroplast from studies on other systems but measurements with tobacco have been challenging because WT leaves have very little lipid production and thus do not give quantitative results consistently. New methods with additional purifications of ACP proteins and rapid testing of method ideas in simple microbial systems are being worked out to overcome the issues and establish a robust standard operating protocol. No ACP standard is commercially available, therefore standards have been produced through overexpression of ACP in E. coli that is supplied 15N and through overexpression of a transferase enzyme that is used to link expressed ACP with acyl chains of varied lengths that serve as standards. Aim 4: A computational framework was developed to model acyl lipid fluxes in plants with data from radiolabeling experiments for the 15% oil leaf line in comparison to the WT. Efforts to write up a manuscript for publication on this tool will continue.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Murphy, K.M., Johnson, B.S., Harmon, C., et al. (2025) Excessive leaf oil modulates the plant abiotic stress response via reduced stomatal aperture in tobacco (Nicotiana tabacum). Plant J., 121, e70067. Available at: https://onlinelibrary.wiley.com/doi/abs/10.1111/tpj.70067 [Accessed April 2, 2025].
|
Progress 01/15/23 to 01/14/24
Outputs
Target Audience:Target Audience: The main target audience for this project is the plant science research community. This project examined what happens to important plant products under environmental stress. To pursue this question, this project develops tools and computational models that would also be valuable for formal classroom instruction. PlantCV image analysis tools are used to analyze image data in this project and are used to teach educators, students (K-12), undergraduates, and researchers biocomputing. To that end, we have trained a set of educators from Harris Stowe University (HBCU) in using PlantCV and a course has been designed cross-listed in Math/Bio that uses the tools we developed. We have held virtual workshops to teach researchers to use PlantCV tools that are used in this project. This data collected from this project was used for Summer Research Experiences for Undergraduates (REU Projects). Changes/Problems:One of the challenges was the loss of postdoctoral personnel that obtained jobs in the plant science field that fit their career interests. As already noted one is now the Director of Phenotyping at the Danforth Center and the other is a grant writer. Our expectation is to train the next generation of scientists and also contributors to the plant science field and to enable people to be productive by finding meaningful work that they are passionate about. As a consequence, as our grant enters the no cost extension, we are using the remaining funds to complete manuscripts, software, and possibly some final measurements dependent on methods that are being established on other projects that we can leverage to tobacco. Though progress has slowed we are near submission on a manuscript and are completing efforts towards others. What opportunities for training and professional development has the project provided?This project has directly trained a total of two postdoctoral researchers and a graduate student as well as five total undergraduate students participating in an REU program and through part time work on the project during the school year. The postdocs and students have been trained in experimentation, presentations, and writing. The two postdocs have both moved on to promising careers that match their career interests. One postdoc is now the Director of Phenotyping at the Danforth Center, and the other postdoc is now employed in a grant writing position as a result of writing a grant during 2023, after discovering that this was the direction she wanted to take with her career. The graduate student Brandon Johnson gave a presentation of his results at a scientific conference and won an award for best poster presentation. This project has trained researchers (undergraduates, researchers) in the use of PlantCV tools developed in part through this project through workshops throughout 2023. Postdoctoral researchers have played a major role in disseminating results of this project through scientific presentations. ? How have the results been disseminated to communities of interest?Results have been disseminated through scientific presentations, social media, and through virtual workshops held for the plant science community. The 'Real Time Science' tik tok, which was created for this project now has ~8000 followers and 200 videos on TikTok. These videos disseminate the research to a general public, and has been used in middle school projects via news site Newsela. Presentations at conferences and as invited seminars are listed below: Gehan: Donald Danforth Plant Science Center, Todd C. Mockler Memorial Lecture, 2023 Gehan: University of Minnesota, Seminar, 2023 Gehan: What Phenotypes Matter? Open Challenges in Plant Phenomics with PlantCV. Single-Cell Approaches Gordon Research Conference. Ventura, California, 2023 Gehan: What Phenotypes Matter? Open Challenges in Plant Phenomics with PlantCV. Washington University in St. Louis, Seminar, 2023 Gehan: What Phenotypes Matter? Open Challenges in Plant Phenomics with PlantCV. Northeastern State University Seminar Series, 2023 Gehan: What Phenotypes Matter? Open Challenges in Plant Phenomics with PlantCV. Iowa State University Plant Breeding Symposium, 2023 Chu: Metabolic characterization of non-transitory starch tradeoff for lipid production in mature tobacco leaves. 2023 Gordon Research Conference on Plant Lipids: Structure, Function and Metabolism. Galveston, TX. Chu: Metabolic characterization of non-transitory starch tradeoff for lipid production in mature tobacco leaves. 2023 Donald Danforth Plant Science Center Scientific Retreat. St. Louis, MO Allen: Lipid Metabolism in Vegetative Tissues, NC1203 Lipids in Crops Meeting. University of Nebraska. Lincoln, NE. Bates: Elucidating Diverse Mechanisms of Plant Oil Synthesis to Accumulate Industrially Valuable Fatty Acids. Michigan State University. East Lansing, MI. Johnson: Phenomics identifies unexpected adaptations to engineering oil accumulation in plant leaves. Life Science Innovation Northwest 2023. Seattle, WA. Murphy: High-throughput microscopy images of plant stomata. NAPPN Conference. February 2023. Workshops on tools associated with this project:? Date Workshop Name 6/1-6/2/2023 2023 Data Science and Phenotyping PlantCV REU Workshop 7/18/2023 Private PlantCV Workshop: Eman, Bhatia, Meyer Group 7/24-7/28/2023 PlantCV Workshop Series: Unique Attendees 7/24/2023 PlantCV Workshop Series: Intro to Image Data and PlantCV 7/25/2023 PlantCV Workshop Series: Phenovation CropReporter 7/26/2023 PlantCV Workshop Series: Handling Bellwether Data using pcvr 7/27/2023 PlantCV Workshop Series: This Machine Bean's Learning 7/28/2023 PlantCV Workshop Series: Bring-Your-Own-Data 8/28/2023 PlantCV Workshop @ Universidade Federal de Minas Gerais - remote assistance 10/26/2023 PlantCV Workshop 11/17/2023 Upskilling your Data Management in R: Using pcvr to Analyze Your Data 12/11-12/14/2023 PlantCV Workshop Series: Unique Attendees 12/11/2023 PlantCV Workshop Series: PlantCV Workflow Fundamentals 12/12/2023 PlantCV Workshop Series: Multiple Plants & Seeds Analysis 12/13/2023 PlantCV Workshop Series: Machine Learning Lab Activity 12/14/2023 PlantCV Workshop Series: PlantCV Installation & Bring-Your-Own-Data What do you plan to do during the next reporting period to accomplish the goals?We are working on developing a graphical user interface for the software in Aim 4 so that it is more user-friendly and less prone to error. Currently the software is functional and the GUI is near completion, we expect to test it on 14C tobacco labeling data from this project very soon and anticipate submitting a manuscript in the late spring or summer of 2024. ?
Impacts
What was accomplished under these goals?
1.2: Heat stress: First, interactive annotation modules were expanded and improved in the open-source image analysis platform PlantCV, to analyze fluorescent images to analyze photosynthetic efficiency on entire plants with spatial resolution. Next, various temperatures and durations of heat stress were applied to high oil and wild-type plants, and photosynthetic efficiency (including Fv/Fm, Fq'/Fm', and NPQ), were analyzed to determine the temperature, duration, and sampling time points of heat stress. This analysis suggested 42C day/32C night heat stress conditions over 7 days produced a photosynthetic response in both high-oil and wild-type plants. Furthermore, high-oil plants showed a greater and more immediate reduction in photosynthetic efficiency due to heat stress than wild-type. During the 7 day heat stress total oil decreased rapidly over the first two days of the stress and then reaches a steady level over the remaining stress period but at only ? the level of leaf oil as non stressed plants. An Electrolyte Leakage Assay (ELA) did not demonstrate measurable differences in cellular damage between high oil and wild-type plants under control or heat stress conditions.High-oil tobacco and wild-type plants were also analyzed using light and confocal microscopy to determine differences in cellular and subcellular characteristics under control and heat stress conditions. High-oil tobacco was found to have less trichomes and shorter trichomes than WT plants, found through a project of an REU student. High-oil plants had substantial oil accumulated, as expected, but were unexpectedly also found to have large oil droplets in stomatal guard cells. Thermal imaging found that high-oil plants had higher leaf temperature. Stomatal imaging found that high-oil plants had smaller stomatal aperture and fewer stomata, which fits with the thermal data that found higher leaf temperatures. This was measured by a new tool added to the open-source open-development package PlantCV. 1.3: Using the new photosynthesis module in PlantCV (Aim 1.1), chlorophyll content was assayed spatially using a chlorophyll red-edge index. High-oil tobacco was found to have reduced chlorophyll content than WT under control and heat-stress conditions. Heat stress increased the chlorophyll content of both WT and high-oil tobacco, but was increased to a greater degree in WT. 2.1: Galactolipids (the main components of the photosynthetic thylakoid membranes) were quantified between WT, the 15% oil leaf, and 30% oil leaf tobacco lines (HO and LEC2, respectively) first under normal growth conditions. In the 15% oil leaves galactolipids were reduced ~43% from WT, suggesting fatty acids used for membranes were partitioned into TAG, however in the 30% oil lines there was not a further drop galactolipid content, suggesting expression of LEC2 altered metabolism to increase oil without further reducing the availability of photosynthetic membranes. The evaluation of membrane lipid content under adverse growth conditions is ongoing.A lipidomics study was conducted to compare the changes in MGDG and DGDG lipid species under chilling and control conditions between WT and LEC2 to elucidate differences in chloroplast lipid remodeling. Significant increases were the most notable in MGDG 34:6 and 36:6 lipids, as well as DGDG 36:6 lipids, notable for the high degree of unsaturation that is necessary to prevent chloroplastic membranes from freezing together. Additional analysis revealed that under chilling conditions DGDG lipid levels increase by approximately 12% in WT, whereas DGDG levels only increase by 5.5% in LEC2. When chloroplast membranes respond to cold, they often increase their DGDG lipid composition, as the head groups of DGDGs are larger and change membrane conformation shapes to a lamellar bilayer phase that prevents freezing injury to the chloroplast. These findings suggest that lipid metabolism and remodelling is occurring at different rates in LEC2 when compared to WT, most likely due to the pull of fatty acids into TAG from the overexpression of DAG. It is possible that WT tobacco is better equipped to respond to chilling stress at the membrane level than LEC2. A phenomics approach was utilized to analyze the efficiency of photosynthesis in intact WT, HO, LEC2 plants and an additional line that accumulates oil near LEC2, SDP1 (which is the HO background with RNAi knockdown of SDP1 triacylglycerol lipase). The initial results suggest the LEC2 photosynthetic electron transport (measured as photochemical quantum efficiency change, Phi2) significantly is reduced compared to WT. This correlates with a higher reduction level of the intersystem electron transport system measured as lower qL parameter. This suggests that metabolic consumption of photosynthetic generated reducing power is less efficient in LEC2. As potential consequence of this less efficient electron transport in LEC2 we observed partial photoinhibition in this line indicated by a lower maximal photochemical quantum efficiency parameter (Fv/Fm) and higher slow relaxing NPQ component (qI). In contrast, both HO and SDP1 lines behave similarly to WT. 3.1: To better understand how the LEC2 line accumulates more oil without a further drop in galactolipid content from that of the HO line as measured in Aim 2, we performed 14CO2 pulse-chase metabolic labeling over a 145 hour time course on WT and each oil line. A key difference between HO and LEC2 tobacco lines was that in HO the TAG was under a futile cycle of synthesis and degradation, such that by the end of the 145 hours the TAG produced during the pulse was mostly gone, whereas the the oil produced in the LEC2 line was stable. Therefore, oil stability is a key part of accumulation of high levels of oil accumulation in the LEC2 line without further depletion of photosynthetic membranes. Additionally, the TAG turnover in HO led to a futile cycle with starch synthesis/degradation and altered carbon flux through metabolism that may be related to the decreased growth phenotypes of the HO background. Additionally, targeted [14C]acetate pulse-chase labeling was performed prior to induction of heat stress during the chase period to determine the metabolic fate of fatty acids during the heat stress. Analysis of these samples are ongoing. 3.2: We adapted methods to assess acyl-ACP pools in the chloroplast from studies on other systems but measurements with tobacco have been challenging because WT leaves have very little lipid production and thus do not give quantitative results consistently. New methods with additional purifications of ACP proteins and rapid testing of method ideas in simple microbial systems are being worked out to overcome the issues and establish a robust standard operating protocol. We anticipate that the effort will enable a number of studies on fatty acid and lipid analysis wherein the plant system does not make as much oil as a seed and is therefore more challenging to evaluate. Methods are near complete; and no ACP standard is commercially available, standards have been produced through overexpression of ACP in E. coli that is supplied 15N and through overexpression of a transferase enzyme that is used to link expressed ACP with acyl chains of varied lengths that serve as standards. Biomass reflecting cold or heat stress has been collected and stored at -80 until the methods are finalized. 4.1: Software development has continued. In prior years a computational framework was developed to model acyl lipid fluxes in plants with data from radiolabeling experiments for the 15% oil leaf line in comparison to the WT. Efforts to make the software more user friendly have included making graphical user interface components. We anticipate submitting a manuscript of the tools and their application to the tobacco lines within 2024..
Publications
|
Progress 01/15/22 to 01/14/23
Outputs
Target Audience:The main target audience for this project is the plant science research community. This project develops image analysis tools and computational models that would be valuable for formal classroom instruction. PlantCV image analysis tools are used to analyze image data in this project and are used to teach educators, students (K-12), undergraduates, and researchers biocomputing. To that end, we have started to train a set of educators from Harris Stowe University in using PlantCV and are planning to use data collected in this project for future lessons. We have held virtual workshops to teach researchers to use PlantCV tools that are used in this project. This data collected from this project was used for four Summer Research Experiences for Undergraduates (REU Projects). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has directly trained two postdoctoral researchers and a graduate student as well as four total undergraduate students participating in an REU program and through part time work on the project during the school year. This project has trained researchers (undergraduates, researchers) in the use of PlantCV tools developed in part through this project through virtual workshops throughout 2022. Postdoctoral researchers have played a major role in disseminating results of this project through scientific presentations. How have the results been disseminated to communities of interest?Results have been disseminated through scientific presentations, social media, and through virtual workshops held for the plant science community. The 'Real Time Science' tik tok, which was created for this project now has ~8000 followers and 200 videos on TikTok. These videos disseminate the research to a general public, and has been used in middle school projects via news site Newsela. Presentations at conferences and as invited seminars are listed below: Gehan: What Phenotypes Matter? Open Challenges in Plant Phenomics with PlantCV. Southern Illinois University, Edwardsville Seminar Series Gehan: What Phenotypes Matter? Open Challenges in Plant Phenomics with PlantCV. University of Illinois Urbana-Champaign Seminar Series Gehan: Utilizing Natural Variation and High-Throughput Phenotyping for Crop Improvement. Gordon Research Conference, Salt and Water Stress, Switzerland. Gehan: NAPPN Career Award Presentation: What Phenotypes Matter? Open Challenges in Plant Phenomics with PlantCV. North American Plant Phenotyping Network Conference. Gehan: US Botanical Garden. Plant Science Conversations Gehan: PlantCV Plant Imaging Workshop. NAPPN Annual Conference, Athens, Georgia. ?Murphy: The Heat Is On: Tobacco and Heat Stress. Interdisciplinary Plant Group Symposium University of Missouri Murphy: The Heat Is On: Tobacco and Heat Stress.Plant Biology Conference Murphy: The Heat is On: High oil tobacco and heat stress. University of Minnesota, Plant Pathology Seminar Series Murphy: The Heat is On: High oil tobacco and heat stress. Merrimack College Murphy: The Heat is On: High oil tobacco and heat stress. University of Hawaii, Plant and Soil Science Seminar Series, Allen: Isotopic Labeling & Metabolic Flux Analysis: Understanding Spatial and Temporal Metabolism for Crop Design. Research Triangle Park Consortium Allen: Isotopic Labeling & Metabolic Flux Analysis: Understanding Spatial and Temporal Metabolism for Crop Design.Washington State University Pullman Chu: Metabolic characterization of non-transitory starch tradeoff for lipid production in mature tobacco leaves. Interdisciplinary Plant Group Symposium. University of Missouri. Bates: Elucidating Diverse Mechanisms of Plant Oil Synthesis to Accumulate Industrially Valuable Fatty Acids. Michigan State University. East Lansing, MI. What do you plan to do during the next reporting period to accomplish the goals?We are working to make the software of Aim 4 more user-friendly and less prone to error. At the moment it is laborious and intensive with many steps to input and evaluate flux models for radioactivity. Our goal is to recruit some help with GUI design that can enable the process and to get a manuscript published comparing the WT and high lipid tobacco. ?
Impacts
What was accomplished under these goals?
1.1:Neither the circadian phase nor period was different between high oil and wild-type plants, suggesting that any differences found between photosynthetic efficiency parameters between these plants is not due to differences in the circadian clock. 1.2: Heat stress: A new module was made inPlantCV to analyze photosynthetic efficiency. Various temperatures and durations of heat stress were applied to plants, and photosynthetic efficiency, were analyzed. This analysis found 42C day/32C night heat stress conditions over 7 days produced a photosynthetic response in both high-oil and wild-type plants. High-oil plants showed a greater and more immediate reduction in photosynthetic efficiency due to heat stress than wild-type. During the 7 day heat stress total oil decreased rapidly over the first two days of the stress and then reaches a steady level over the remaining stress period but at only ? the level of leaf oil as non stressed plants.High-oil tobacco and wild-type plants were also analyzed using light and confocal microscopy to determine differences in subcellular characteristics under control and heat stress conditions. High-oil tobacco was found to have less trichomes and shorter trichomes than WT plants.High-oil plants had substantial oil accumulated but were unexpectedly also found to have large oil droplets in stomatal guard cells. Thermal imaging found that high-oil plants had higher leaf temperature. Stomatal imaging found that high-oil plants had smaller stomatal aperture. Chilling Stress: Four plants of each genotype were grown under normal and chilling temperatures of 10oC. Images were taken for both treatments at 0, 2, 4, 24, 48, 72, 96, 120, and 144 hours using a CropReporter system, and were analyzed using PlantCV software to extract Fv/Fm values for evaluation of PSII efficiency under chilling stress. At both temperatures, there are no significant differences in Fv/Fm values between genotypes, indicating that both have similar PSII efficiency during chilling stress over 144 hours. After one chilling experiment, plants in the 10oC growth chamber were left in chilling conditions for an additional 144 hours, with PSII efficiency measurements taken again at the 288 hour time point. At this time, LEC2 had decreased PSII efficiency (Fv/Fm: 0.72) compared to WT (Fv/Fm: 0.76) at10oC, which indicates that longer time points are necessary to see Fv/Fm values typically associated with plant stress and decreased PSII efficiency.Additionally, there significant differences (p < 0.001) in chlorophyll content between both WT and LEC2. Analysis of NDVIand mean chlorophyll index red edge (Ci), which are measurements indicative of chlorophyll content. WT has consistently higher NDVI values of approximately 0.55, while LEC2 has NDVI values of approximately 0.51.The values for mean chlorophyll index red edge (Ci) between genotypes are also significantly different (p < 0.001): approximately 1.75 and 1.6 for WT and LEC2 at normal conditions, respectively. As chilling stress progresses through 144 and 288 hours, Ci continues to decrease in LEC2 to below 1.5, while Ci remains relatively constant in WT tobacco. Results indicate that LEC2 chlorophyll concentration decreases at a greater rate than WT under chilling stress.? 1.3: Using the photosynthesis module in PlantCV, chlorophyll content was assayed using a chlorophyll red-edge index. High-oil tobacco was found to have reduced chlorophyll content than WT under control and heat-stress conditions. Heat stress increased the chlorophyll content of both WT and high-oil tobacco, but was increased to a greater degree in WT. 2.1: Galactolipids (the main components of the photosynthetic thylakoid membranes) were quantified between WT, the 15% oil leaf, and 30% oil leaf tobacco lines (HO and LEC2, respectively) first under normal growth conditions. In the 15% oil leaves galactolipids were reduced ~43% from WT, suggesting fatty acids used for membranes were partitioned into TAG, however in the 30% oil lines there was not a further drop galactolipid content, suggesting expression of LEC2 altered metabolism to increase oil without further reducing the availability of photosynthetic membranes. A lipidomics study was conducted to compare the changes in MGDG and DGDG lipid species under chilling and control conditions between WT and LEC2 to elucidate differences in chloroplast lipid remodeling. Significant increases were the most notable in MGDG 34:6 and 36:6 lipids, as well as DGDG 36:6 lipids, notable for the high degree of unsaturation that is necessary to prevent chloroplastic membranes from freezing together. Additional analysis revealed that under chilling conditions DGDG lipid levels increase by approximately 12% in WT, whereas DGDG levels only increase by 5.5% in LEC2. When chloroplast membranes respond to cold, they often increase their DGDG lipid composition, as the head groups of DGDGs are larger and change membrane conformation shapes to a lamellar bilayer phase that prevents freezing injury to the chloroplast. These findings suggest that lipid metabolism and remodelling is occurring at different rates in LEC2 when compared to WT, most likely due to the pull of fatty acids into TAG from the overexpression of DAG. It is possible that WT tobacco is better equipped to respond to chilling stress at the membrane level than LEC2. 3.1: To better understand how the LEC2 line accumulates more oil without a further drop in galactolipid content from that of the HO line as measured in Aim 2, we performed 14CO2 pulse-chase metabolic labeling over a 145 hour time course on WT and each oil line. A key difference between HO and LEC2 tobacco lines was that in HO the TAG was under a futile cycle of synthesis and degradation, such that by the end of the 145 hours the TAG produced during the pulse was mostly gone, whereas the the oil produced in the LEC2 line was stable. Therefore, oil stability is a key part of accumulation of high levels of oil accumulation in the LEC2 line without further depletion of photosynthetic membranes. Additionally, the TAG turnover in HO led to a futile cycle with starch synthesis/degradation and altered carbon flux through metabolism that may be related to the decreased growth phenotypes of the HO background. Additionally, targeted [14C]acetate pulse-chase labeling was performed prior to induction of heat stress during the chase period to determine the metabolic fate of fatty acids during the heat stress. 3.2: Methods to assess acyl-ACP pools in the chloroplast were adapted from other systems but have been challenging to use with the tobacco because WT leaves have very little lipid production and thus do not give quantitative results consistently. New methods with additional purifications of ACP proteins are being worked out to overcome the issues. We anticipate that the effort will enable a number of studies on fatty acid and lipid analysis wherein the plant system does not make as much oil as a seed and is therefore more challenging to evaluate. Methods are near complete; however because no ACP standard is commercially available, a standard is also being produced through overexpression of ACP in E. coli that is supplied 15N and through overexpression of a transferase enzyme that is used to link expressed ACP with acyl chains of varied lengths that serve as standards. 4.1: A computational framework developed in the prior year that can compare acyl lipid fluxes in plants by modeling radiolabeling data generated from the 15% oil leaf line in comparison to the WT was advanced by making the software more user friendly. There still remain challenges with the usability of the code and we are eliciting some help to make GUIs to the extent possible prior to publication of the tools. The MATLAB-based code can model 14C transient labeling data to assess different potential metabolic networks.?
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Chu, K. L. et al. Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves. Metab. Eng. 69, 231248 (2022)
|
Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The main target audience for this project is the plant science research community. This project develops image analysis tools and computational models that would be valuable for formal classroom instruction. PlantCV image analysis tools are used to analyze image data in this project and are used to teach educators, students (K-12), undergraduates, and researchers biocomputing. To that end, we have started to train a set of educators from Harris Stowe University in using PlantCV and are planning to use data collected in this project for future lessons. We have held virtual workshops to teach researchers to use PlantCV tools that are used in this project. This data collected from this project was used for three Summer Research Experiences for Undergraduates (REU Projects). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has directly trained two postdoctoral researchers and a graduate student as well as three undergraduate students participating in an REU program. This project has trained researchers (undergraduates, researchers) in the use of PlantCV tools developed in part through this project through virtual workshops throughout 2021. DEI Activity: Katie Murphy and Doug Allen proposed and participated on the Organizing Committee for Picture a Scientist movie Viewing at DDPSC, followed by a discussion on women in STEM. Presentation Skills Development: (COCAbiz) Center for Creative Arts Professional Business Training on Presentation Delivery for Doug Allen, Sarah Powers, and Katie Murphy. How have the results been disseminated to communities of interest?Results have been disseminated through publications, book chapters, social media, and through virtual workshops held for the plant science community. The 'Real Time Science' tik tok, which was created for this project now has ~500 followers and 95 videos on TikTok. The project has expanded to post the videos to Instagram and Facebook under the same title, and was profiled in the DDPSC Blog, Washington Post Kids, and Newsela, where middle and elementary school students across the USA read and wrote about the videos for a homework assignment. ? What do you plan to do during the next reporting period to accomplish the goals?We have determined the circadian phase and period of high oil tobacco (Aim 1.1) and have determined the duration and intensity of heat stress to use in further aims (Aim 1.2). In this reporting period we will continue to refine conditions for chilling stress application (Aim 1.2). We have determined that there are differences in chlorophyll content in high oil tobacco (Aim 1.3) and will continue to characterize these differences in more detail in objectives outlined in Aims 2, 3 and 4
Impacts
What was accomplished under these goals?
1.1: The circadian phase and period of high oil and wild-type tobacco was determined using qRT-PCR for clock-associated gene expression, as proposed, over the course of 48 hours of constant light. Neither the circadian phase nor period was different between high oil and wild-type plants, suggesting that any differences found between photosynthetic efficiency parameters between these plants is not due to differences in the circadian clock. Because there is no difference, the optimum time of day to apply stress treatment was identified as the morning. 1.2: Heat stress: A new module was created in the open-source image analysis platform PlantCV to analyze fluorescent images to analyze photosynthetic efficiency on entire plants with spatial resolution. Next, various temperatures and durations of heat stress were applied to high oil and wild-type plants, and photosynthetic efficiency (including Fv/Fm, Fq'/Fm', and NPQ), were analyzed to determine the temperature, duration, and sampling time points of heat stress. This analysis suggested 42C day/32C night heat stress conditions over 7 days produced a photosynthetic response in both high-oil and wild-type plants. Furthermore, high-oil plants showed a greater and more immediate reduction in photosynthetic efficiency due to heat stress than wild-type. An Electrolyte Leakage Assay (ELA) did not demonstrate measurable differences in cellular damage between high oil and wild-type plants under control or heat stress conditions. Data will be included in a manuscript for publication in 2022.High-oil tobacco and wild-type plants were also analyzed using light and confocal microscopy to determine differences in cellular and subcellular characteristics under control and heat stress conditions. High-oil tobacco was found to have less trichomes and shorter trichomes than WT plants, found through a project of an REU student. High-oil plants had substantial oil accumulated, as expected, but were unexpectedly also found to have large oil droplets in stomatal guard cells. The effect of guard cell oil will be investigated in the subsequent years of this project. Chilling Stress: Chilling stress experiments on approximately 21 day old wild-type (WT) and LEC2 tobacco began in October of 2021. Four plants of each genotype were grown under normal and chilling temperatures of 10oC. Images were taken for both treatments at 0, 2, 4, 24, 48, 72, 96, 120, and 144 hours using the Phenovation CropReporter system, and the images were analyzed using PlantCV software to extract Fv/Fm values for evaluation of PSII efficiency under chilling stress. At both temperatures, there are no significant differences in Fv/Fm values between genotypes, indicating that both have similar PSII efficiency during chilling stress over 144 hours. After one chilling experiment, plants in the 10oC growth chamber were left in chilling conditions for an additional 144 hours, with PSII efficiency measurements taken again at the 288 hour time point. At this time, LEC2 had decreased PSII efficiency (Fv/Fm: 0.72) compared to WT (Fv/Fm: 0.76) at 10oC, which indicates that longer time points are necessary to see Fv/Fm values typically associated with plant stress and decreased PSII efficiency.Additionally, there are inherent, significant differences (p < 0.001) in chlorophyll content between both WT and LEC2. Analysis of the normalized difference vegetation index (NDVI) and mean chlorophyll index red edge (Ci), which are measurements indicative of chlorophyll content, was accomplished with the Phenovation CropReporter system and PlantCV software. WT has consistently higher NDVI values of approximately 0.55, while LEC2 has NDVI values of approximately 0.51. During chilling stress at 10oC, WT NDVI values increase to approximately 0.6, while LEC2 NDVI values decrease slightly to 0.5. The values for mean chlorophyll index red edge (Ci) between genotypes are also significantly different (p < 0.001): approximately 1.75 and 1.6 for WT and LEC2 at normal conditions, respectively. As chilling stress progresses through 144 and 288 hours, Ci continues to decrease in LEC2 to below 1.5, while Ci remains relatively constant in WT tobacco. These results indicate that LEC2 chlorophyll concentration decreases at a greater rate than WT under chilling stress. 1.3: Using the new photosynthesis module in PlantCV (Aim 1.1), chlorophyll content was assayed spatially using a chlorophyll red-edge index. High-oil tobacco was found to have reduced chlorophyll content than WT under control and heat-stress conditions. Heat stress increased the chlorophyll content of both WT and high-oil tobacco, but was increased to a greater degree in WT. Data will be included in a manuscript for publication in 2022. 2.1: Galactolipids (the main components of the photosynthetic thylakoid membranes) were quantified between WT, the 15% oil leaf, and 30% oil leaf tobacco lines (HO and LEC2, respectively) first under normal growth conditions. In the 15% oil leaves galactolipids were reduced ~43% from WT, suggesting fatty acids used for membranes were partitioned into TAG, however in the 30% oil lines there was not a further drop galactolipid content, suggesting expression of LEC2 altered metabolism to increase oil without further reducing the availability of photosynthetic membranes. The evaluation of membrane lipid content under adverse growth conditions is ongoing. 3.1: To better understand how the LEC2 line accumulates more oil without a further drop in galactolipid content from that of the HO line as measured in Aim 2, we performed 14CO2 pulse-chase metabolic labeling over a 145 hour time course on WT and each oil line. A key difference between HO and LEC2 tobacco lines was that in HO the TAG was under a futile cycle of synthesis and degradation, such that by the end of the 145 hours the TAG produced during the pulse was mostly gone, whereas the the oil produced in the LEC2 line was stable. Therefore, oil stability is a key part of accumulation of high levels of oil accumulation in the LEC2 line without further depletion of photosynthetic membranes. Additionally, the TAG turnover in HO led to a futile cycle with starch synthesis/degradation and altered carbon flux through metabolism that may be related to the decreased growth phenotypes of the HO background. 3.2: Methods to assess acyl-ACP pools in the chloroplast have been adapted from development with other species and are under refinement to obtain best results for tobacco followed by comparisons between stressed and unstressed plants. 4.1: A computational framework was developed to compare acyl lipid fluxes in plants by modeling radiolabeling data generated from the 15% oil leaf line in comparison to the WT. The data generated as part of this continually funded NIFA project on high oil in leaves included transient 14CO2 pulse-chase incorporation as previously described for each plant line. The modeling framework was built from scratch in MATLAB and an REU student, who has continued as a part time intern, was recruited to improve the software's usability. During the course of this last year a Microsoft Excel interface was incorporated so that descriptions of the metabolic network can be easily imported into MATLAB.The spreadsheet-based accounting is less prone to error and reduces manual, time-consuming mathematical derivations. Transient labeling curves are fitted to the experimental data through a chi-squared approach and the steady state metabolic model can be restarted from many different initial points to explore the potential solution space formulated and constrained by the metabolic network. PDFs of plots are now automatically generated for the best fit model improving the ease of interpretation. Confidence interval calculation has been partially automated to enhance the user experience and a flow chart and accompanying protocol are under development for inclusion with drafting of a manuscript in 2022.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Casto AL, Murphy KM, Gehan MA. Coping with cold: Sorghum cold stress from germination to maturity. Crop Sci. 2021;61: 38943907. doi:10.1002/csc2.20609
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2021
Citation:
Casto A, Schuhl H, Schneider D, Wheeler J, Gehan M, Fahlgren N. Analyzing chlorophyll fluorescence images in PlantCV. Earth and Space Science Open Archive. 2021. doi:10.1002/essoar.10508322.2
- Type:
Book Chapters
Status:
Published
Year Published:
2021
Citation:
Bates PD (2021) The plant lipid metabolic network for assembly of diverse triacylglycerol molecular species. Advances in Botanical Research. In press
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Chu KL, Koley S, Jenkins LM, Bailey SR, Kambhampati S, Foley K, Arp JJ, Morley SA, Czymmek KJ, Bates PD, Allen DK. Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves. Metab. Engr. 2021; In press
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