Progress 10/15/23 to 10/14/24
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the past year, at UNT, 10 undergraduates (including two as part of Texas Academy of Math and Sciences early entrance program) have participated in this project under direction of PI and a graduate student. Most students have been trained in molecular biology and biochemistry techniques to carry out gene characterization within various outlined targets in cyclic fatty acid metabolism and storage. Two undergrad students took part in paid summer internships as part of the grant. One graduate student continues their doctoral work on this project with another graduate student contributing to a summer project. Additionally, one continuing graduate student in Co-PI's lab continues to assist with project. Continuing from last year, Professor Angela Stoeckman from Bethel University and PI have continued to train two additional undergraduates from Bethel that contributed to this project and presented at a national conference How have the results been disseminated to communities of interest?Results have been presented at two conferences by PI including the 2024 International Plant Lipid Symposium and 2024 Beltwide Cotton Conference. Three graduate student presentations were given including UNT Research Day, UNT BioDiscovery Institute Expo, and UNT BioDiscovery seminar series. Two undergraduates in collaboration with Bethel University presented at the American Chemical Society. Results continue to be disseminated via personal communication with leaders at Cotton Inc. (research arm of the co-funding agency, The Cotton Board) and USDA collaborating scientists What do you plan to do during the next reporting period to accomplish the goals?Due to complexities unraveling specific mechanisms for CFA metabolism, we have held off on planned manuscript submissions until late 2024 / early 2025 to coincide with the next reporting period. For Objective 1, we will use the RNA-Seq and proteomics data to revisit our screening list for the CFA desaturase and other CFA metabolism genes. We have also initiated protein-protein interaction assays and CPS characterization to consider novel candidates for these pathways. Students are currently being trained to conduct experiments with radioactivity enabling us to address proposed experiments with methionine labeling. For Objective 2, we will continue to study the individual LD proteins determining the mechanism of LDAP-assisted accumulation. We will also focus on translating these efforts in identifying key LD proteins by expressing them in tobacco and yeast (transiently) to understand whether cotton proteins are specialized relative to other homologous proteins. Finally, we continue our USDA collaboration that focuses on stable transformation of cotton plants to increase value in cottonseed. Based on current progress, a no-cost extension will be requested to finish the outlined objectives at the end of the next reporting period.
Impacts
What was accomplished under these goals?
Objective 1: Efforts continue to characterize the biochemical steps in cyclic fatty acid (CFA) metabolism using vegetative tissues (relatively high CFA levels to seeds) to inform experiments for ultimately altering CFA levels in cottonseeds. Previous experiments using virus-induced gene silencing (VIGS) of cyclopropane synthase (CPS) and fatty acid desaturase 2 (FAD2) showed substantially altered cyclic FA totals (ranging from ~0 to ~40%, respectively) and compositional profiles in cotton petioles demonstrating the plasticity of CFA accumulation in vegetative tissues. Using targeted LC-MS/MS of these VIGS lines, metabolomics profiles of these tissues identified starch, sucrose, fructose, mannose metabolism (and associated phosphorylated forms) as significantly altered metabolic pathways. These results show that carbon remodeling is associated with CFA levels. VIGS tissue samples were also subjected to lipid droplet (LD) isolation of petioles and subsequent proteomics enrichment analysis relative to microsomal and cytosolic fractions. Unfortunately, the yield of protein was too low to yield any conclusive changes on LD protein (and other microsomal) targets. RNA-Seq of these lines has been initiated and we are currently waiting for results to analyze transcriptional changes. Significant effort has been placed on optimizing RNA extractions from petioles and verification of silencing for various gene candidates analyzed through Objs. 1 and 2. We have cloned several additional targets for silencing in cotton and overexpression in tobacco/yeast involved in pathways for CFA modification, glycerolipid assembly and remodeling, and electron shuttling for CFA desaturation. Efforts have also continued in characterizing stable cotton lines generated with collaborator Jay Shockey including CRISPR knockouts and overexpression of CPS. Finally, we have cloned the CPS from cotton, Arabidopsis, and E.coli in multiple expression systems to explore structural variations to verify reports of different methylation reactions (in Arabidopsis versus cotton) and potential protein-protein interaction partners. Objective 2: We have screened several other LD candidate genes (as identified through previous proteomics trials and co-expression analyses) through gene silencing. Most individual genes appear to contribute minimally to CFA accumulation, other than LDAP as identified previously. To verify these results, in a heterologous system, we have generated overexpression constructs for each of these LD genes in tobacco and yeast with testing underway. We have also expanded our proteomics experiments to better characterize petiole and root tissues with results pending after optimizing isolation methods.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Horn PJ. Elucidating Cyclic Fatty Acid Biosynthesis and Compartmentalization in Cotton. International Symposium on Plant Lipids. Lincoln, NE
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Horn PJ. Elucidating Cyclic Fatty Acid Biosynthesis and Compartmentalization in Cotton. Beltwide Cotton Conference. Fort Worth, TX
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
3. Cannon AE, Horn PJ (2024). The Molecular Frequency, Conservation and Role of Reactive Cysteines in Plant Lipid Metabolism. Plant and Cell Physiology- Special Issue in Plant and Algal Lipids. 65 (6), 826-844. doi.org/10.1093/pcp/pcad163
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Progress 10/15/22 to 10/14/23
Outputs
Target Audience:The target audience for this project include cotton breeders, cottonseed researchers, biotechnology and oilseed businesses, plant scientists, and members of the local community interested in agricultural research. Changes/Problems:PI has been approved as of Oct 2023 to conduct radioactive experiments. PI started new lab as part of transfer in Fall 2022. Started first graduate student on project Fall 2023. Having PI and Co-PI at same university has improved productivity on project. What opportunities for training and professional development has the project provided?This project has provided ample training opportunities for undergraduate and graduate students. At UNT, 8 undergraduates (including one as part of Texas Academy of Math and Sciences early entrance program) have participated in this project. Each student has been trained in molecular biology and biochemistry techniques to carry out gene silencing of various targets in cyclic fatty acid metabolism and storage. Of the 8 undergraduates, 3 graduated in spring/summer 2023. All 3 started research technician/assistant positions as a result of their experience on this project. Additionally, 1 new graduate student in PI's lab started in Sept 23 with 1 continuing graduate student in Co-PI's lab working on project. Finally, Professor Angela Stoeckman from Bethel University and PI have established a collaboration to train two additional undergraduates on this project. How have the results been disseminated to communities of interest?Results have been presented to students and scientists as part of three departmental seminars and two conference presentations by PI. As PI started new personnel during this reporting period for project, it is anticipated to have an increased number of presentations and publications within the next reporting period. Additionally, results continue to be disseminated via personal communication with leaders at Cotton Inc. (research arm of the co-funding agency, The Cotton Board) and USDA collaborating scientists. What do you plan to do during the next reporting period to accomplish the goals?We anticipate three article submissions related to our project over the next reporting period (two primary articles and one review on cyclic fatty acid metabolism). For Objective 1, we plan on finishing our characterization of key steps in CFA modification and assembly onto glycerolipids. This will be using a combination of three approaches: gene silencing in cotton, tobacco overexpression, and yeast functional assays. PI has been approved (as of Oct 2023) to conduct experiments with radioactivity enabling us to address proposed experiments with methionine labeling. For Objective 2, we have preliminary evidence of an unusual (unreported) localization of the cyclopropane fatty acid synthase that helps connect our results in Obj. 1 with lipid droplet storage in objective 2. We will further determine the specific contributions of LD-enriched proteins to CFA accumulation, and attempt to understand why cotton LDAP, but not Arabidopsis LDAP, structurally leads to enhanced CFA packaging. We are also currently underway with a large proteomics experiment that will characterize LD proteins in multiple tissues and in VIGS backgrounds with altered CFA levels. For both objectives, we are working towards an engineering application of which combinations of CFA modification/assembly and LD-protein can create targeted compositions in heterologous systems. Finally, we continue our USDA collaboration that focuses on stable transformation of cotton plants to increase value in cottonseed.
Impacts
What was accomplished under these goals?
Objective 1: We have completed detailed fatty acid (FA) analyses in various wild-type tissues of 10-day-old and 4-week-old cotton plants. Most vegetative tissues (petioles, stems, hypocotyl, roots) show higher levels of cyclic fatty acids (CFAs >15% of total FAs) relative to the lower levels accumulated (~1%) in seeds (with trace levels in cotyledons and about 10% CFAs in the embryonic axis). In hypocotyls and roots in both tissue ages there is a gradient change in total CFA content (from 20-to-35%) and CFA composition. The highest CFA levels occur at the hypocotyl-root junction. Petioles and other stem tissue have CFAs with low dihydrosterculic (DHS) content. The higher levels of CFAs may have implications for anti-fungal and/or anti-herbivory activity. Virus-induced gene silencing (VIGS) of different fatty acid desaturase 2 (FAD) isoforms reveals a non-uniform contribution to total CFAs and CFA composition in petiole tissue (where silencing is most effective). DHS content is substantially increased in relative content. Suppression of FAD2 activity also results in a large increase level in lipid droplets (LDs) and has altered the way we approach screening genes important for storage (discussed in Obj. 2). During this reporting period we have screened (through VIGS and yeast overexpression assays) several different types of gene candidates for CFA modification spanning acyltransferases, cytochrome P450s, lipases, and other oxidoreductases. Changes in composition continue to be revealed for specific candidates that have increased the network of genes known to be involved in CFA metabolism. Modulation of these targets in seeds in future engineering experiments may be the key to increasing CFA levels and generating a targeted CFA composition. Objective 2: Shotgun proteomics experiments on lipid droplets (LDs) isolated from cotton seedling roots showed that lipid droplet associated proteins (LDAPs) are highly abundant and enriched proteins on LDs (relative to endoplasmic reticulum, ER, and cytosolic fractions). We have used VIGS to screen several of these proteins, as revealed with proteomics, resulting in a range of changes in CFA content, LD number, and LD morphology. While suppression of FAD2 resulted in an increased number of CFAs and LDs, co-suppression with LDAP1 showed a reduced ability to package CFAs in LDs. Furthermore, in addition to VIGS, production of CFAs in tobacco leaves showed defects in packaging and ER morphology. Overexpression of cotton LDAP1 alleviated these defects and enhanced CFA packaging. It remains unclear of contributions of individual LDAP isoforms and the roles of other LD-storing proteins. These results are currently being prepared for article submission. The objectives above were accomplished as PI started a new research lab in Fall 2022 at UNT.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Horn PJ, Chapman KD. Imaging plants in situ. Journal of Experimental Botany- Special Issue in Plant Metabolism.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Horn P, Stoeckman A, Somasundaram A, Gilmore C, Gutierrez G, Shockey J, Chapman K, Whitehead P, Wise, Salimath S. Elucidating Cyclic Fatty Acid Biosynthesis and Compartmentalization in Cotton. Gordon Research Conference on Plant Metabolic Engineering. Barcelona, Spain
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Borisjuk L, Horn P, Chapman, K, Jakob PM, G�ndel A, Rolletschek H (2023). Seeing plants as never before. New Phytologist Tansley Review 238, 1775-1794. doi.org/10.1111/nph.18871
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