Progress 03/15/22 to 08/24/22
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 Patrick Horn accepted a new faculty position in April 2022 at the University of North Texas (the current subawardee institution) shortly after this USDA grant was awarded (March 2022). While this position starts officially September 2022, this job transition delayed summer progress on Objective 1 due to lab closeout procedures. This will also likely limit progress of the project over the course of 2022 while the lab is re-established and the grant is transferred to the new institution (UNT). Research will continue at UNT during this transition in a limited manner analyzing the candidates of interest established during this short research period. What opportunities for training and professional development has the project provided?The training opportunities were from the grant period 3/15/2022 to 8/15/2022, prior to termination of this award due to change in institution of PI. At ECU, two masters-level graduate students (Lauren Novotny, Shannon Donnelly) and two undergraduate students with no previous research experience (Shelly Rodriguez, Kate Freakley) were trained in late spring and summer 2022 on virus-induced gene silencing methods in cotton as well as the extraction and chemical analysis of cyclic FA from various tissues. These were new methods for all students and represented their first experiences expanding beyond model (Arabidopsis) to applied systems (cotton). At UNT, this project provided research experiences for a visiting researcher, Professor Angela Stoeckman from Bethel University, and a doctoral-level graduate student (Payton Whitehead). This project enabled Dr. Stoeckman to apply her knowledge of mammalian lipids to plant systems and identify promising gene candidates via proteomics. Payton was able to provide training to Dr. Stoeckman on methods for organelle isolation, proteomics, and confocal microscopy in plant tissues. Ample additional training opportunities will be available upon continuation of this project. How have the results been disseminated to communities of interest?During this short reporting period, results have been disseminated via personal communication with leaders at Cotton Inc. which are the research arm of the co-funding agency, The Cotton Board. Additional results plan to be presented at future conferences such as the Gordon Research Conferences on Plant Lipids and The Beltwide Cotton Conference. These conferences will enable us to reach our targeted audiences that vary from researchers to breeders. Manuscripts related to this data will also be submitted within the near future to disseminate our specific research findings more broadly. What do you plan to do during the next reporting period to accomplish the goals?While this is the Final Report for the current award, the plan is to continue the work as originally proposed as part of a new award.
Impacts
What was accomplished under these goals?
The accomplishments are from the grant period 3/15/2022 to 8/15/2022, prior to termination of this award due to change in institution of PI. Objective 1: Progress has been made towards identifying genes within both cyclic FA production and accumulation. Virus-induced gene silencing (VIGS) has been used to silence the targets cyclopropane synthase (CPS), putative cyclopropane FA desaturases, and putative genes for alpha-oxidation conversion of sterculic to malvalic acid. Mock VIGS treatments of young cotton plants (4-5 weeks old) showed variation in cyclic FA levels among tissue types and within tissues due to age of development (i.e., Stems/Petioles: 17 to 25% relative total cyclic FA levels; roots: 29 to 37%; and young leaves 1 to 5%). VIGS targeting CPS1 and CPS2 expression resulted in a 7 to 87% reduction in total cyclic FA levels depending on the tissue and its developmental stage. The most efficient silencing (based on decrease in % cyclic FA levels relative to mock treatment) occurred in the petioles and stems above the cotyledonary node (55 to 87% reduction) while the least efficient silencing occurring in younger roots (7 to 24% reduction). Initial VIGS testing of targeted candidates described within this proposal varied in results. VIGS of FA desaturase 2 isoform 3 (FAD2-3), which has high relative expression in vegetative tissues and acts on the same substrate as CPS (18:1 on phosphatidylcholine membrane lipid), resulted in up to 50% relative increases in cyclic FAs in stems (roots and leaves also showed increases). This demonstrates the ability to modulate total cyclic FA levels through modulation of CPS and FAD2-3 expression. Additional gene targets are currently being tested to test for shifts in composition and/or amounts. In addition, tissues with changes in cyclic FA levels will be to better understand lipid accumulation via lipid droplet morphology and composition. Objective 2: Shotgun proteomics were performed on isolated cotton lipid droplets from multiple tissues with varying levels of cyclic FA and lipid droplet morphology: germinating and dry cotyledons (very low levels of cyclic FA; small, uniform lipid droplets), embryonic axis (relatively high levels of cyclic FAs; small uniform lipid droplets), and roots (high levels of cyclic FAs; variation in size and clustering of lipid droplets). The larger variation in lipid droplet size is associated with higher levels of cyclic FAs suggesting different protein machinery may be responsible for this altered morphology and enable higher cyclic FA totals. As predicted by transcriptomic studies, lipid droplet associated proteins (LDAPs) were relatively enriched in root tissue lipid droplets relative to seed tissues. Notably, the embryonic axis and roots did not contain the same set and proportions of lipid droplet proteins despite both having enriched levels of cyclic FAs. Additionally, many of the enzymatic activities predicted for alpha oxidation and FA desaturation within Objective 1 (although not always the exact same genes) were relatively enriched in isolated lipid droplets versus total extracted proteins from each respective tissue. It remains to be tested in the future whether these candidates are bona fide lipid droplet proteins and how they mechanistically contribute to the morphology and cyclic FA levels observed.
Publications
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