Progress 05/01/23 to 04/30/24
Outputs
Target Audience:During this reporting period (May 1, 2023, to April 30, 2024), we presented our research as posters andoral presentations at several UNT research events, including UNT Scholars Day (aresearch symposium for undergraduate students), UNT Research Day (a campus-wide symposium sponsored by UNT's Research Office), and the BioDiscovery Institute seminar series. These served to familiarize our undergraduate and graduate students, postdoctoral scholars, faculty, and administrators with the importance of our research on the formation of cotton bast fiber as a means to modulate carbon capture and increase bio-product utilization. Changes/Problems:In our original budget, Harmanpreet Kaur was going to receive salary support as an ongoing PhD student, but instead, she was supported by UNT teaching assistantships and a summer fellowship while finishing her MSc. Despite her contributions to the project, her financial support came from elsewhere. Other students budgeted to receive financial support from the project also had support from other sources. On August 1st, 2023, we (PD Ayre and Co-PD McGarry; the timeline is important to understand why expenditures have been low) initiated efforts to hire a highly qualified postdoctoral scholar. The candidate earned her MSc and PhD at the University of Michigan and had an ideal combination of expertise in plant biology, histology, microscopy, and molecular biology. She was also productive in writing papers and her letters of recommendation lauded her excellent laboratory citizenship. We restructured the budget of this and another grant to support our ongoing staffing requirements but also to support a full-time postdoctoral scholar to focus on the objectives of this grant. The USDA was consulted on this restructuring in early September 2023 and the postdoc position was set up through UNT's HR system. After advertising for the requisite two weeks, aletter of offer was sent out and accepted by Oct 2nd, 2023, with the start date of Dec 1st , 2023. The candidate had completed her MSc and PhD at the University of Michigan on a Fulbright Scholarship that had a two-year home residency requirement, which she finished earlier in 2023. UNT sponsored her application for an H1B visa. After submitting her paperwork and attending an in-person interview, the consulate told her it may take 90 days to process her visa. When those 90 days elapsed on Feb 15th, 2024, she was informed that it may take an additional 180 days to process her visa, which would put her realistic start date in July or August of 2024.We required a skilled, full-time postdoctoral scholar to make timely progress on this project. It was a gut-wrenching decision, but we rescinded our offer and as of May 20, 2024, hired anotherhighly-ranked candidate as a 100% FTE postdoctoral scholar. In summary, we offered our postdoctoral position to an outstanding candidate trained at the University of Michigan but delayed processing of her H1B visa prevented her from reporting for work. This, and existing graduate students receiving support from other sources, are the principal reasons why expenditures and progress have not occurred at the rate proposed in our original budget. What opportunities for training and professional development has the project provided?Graduate student Harmanpreet Kaur was pursuing a PhD on aspects of this project but a "life event" curtailed her degree plans and she finished with aMSc in Fall 2023. Harmanpreet Kaur generated preliminary results for the submitted proposal and worked on aspects of Objectives 1, 2, and 3 after theproject was funded. Graduate student Yusuf Mustapha joined the laboratory for the Fall semester of 2023 and is pursuing a MSc on components of Objective 3,specificallyWOX4 and WOX14 virus-mediated gain-of-function assays. He is supported financially through teaching assistantships and thus he has not been supported finacially by this grant. Graduate student Matthew Feragne is pursuing aMSc on components of Objectives 1 and 2.Matthew Feragne started as an undergraduate project student in the Spring of 2023 and continued as an hourly laboratory assistant in the Summer and Fall of 2023; he had a partial TA position for the Fall of 2023 and a full TA for the Spring of 2024. Despite his contributions to the project, his financial support has come from elsewhere. Daniel Fu was an undergraduate research student who worked on aspects of this project since the Fall semester of 2022. As an undergraduate researcher, Daniel Fu participated in the project for college credit and he had a summer fellowship. Therefore, he has not been supported financially from the grant. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will continue with the research plan described in our original proposal but we acknowledge that we are behind schedule in both research objectives and expenditures.
Impacts
What was accomplished under these goals?
Graduate student Harmanpreet Kaurused virus-induced gene silencing (VIGS) to conduct loss-of-function analysis on the cotton orthologs of WOX4 and WOX14. Consistent with our hypothesis, silencing WOX4 and WOX14 reduced activity in the vascular cambia of stems and stems were more narrow than controls. RT-qPCR analysis confirmed that VIGS reduced the abundance of WOX4 and WOX14 transcripts. Harmanpreet Kaur initiated gain-of-function analysis, using our virus-based technologies to increase the expression of WOX4 and WOX14. Increased activity at the vascular cambia, when assessed by measuring stem thickness, was not observed. These results are consistent with observations in poplar when poplar orthologs of WOX4 and WOX14 were overexpressed. Tissues are stored at -20 °C in acetone for future microscopy and at -80 °C for transcript analysis by RT-qPCR. Her results on this project, both before and after funding was initiated, contributed to Objectives 1, 2, and 3 of the proposal, and formed the basis of her MSc thesis defense. Graduate student Yusuf Mustapha joined the laboratory for the Fall semester of 2023 and is pursuing a MSc on components of Objective 3. Harmanpreet Kaur mentored him before she defended her thesis and left the research group. Yusuf Mustapha is currently pursuing the WOX4 and WOX14 virus-mediated gain-of-function assays. He is supported financially through teaching assistantships and thus he has not been supported by this grant. As a relatively new student, his TA responsibilities and coursework have limited his progress. Graduate student Matthew Feragne worked on Objective 1. He grew up the parent lines from public-sector efforts to generate RILS, NAMs, and MAGIC populations, sectioned the stems with a vibratome, and analyzed the layers of bast fibers in each line with fluorescence microscopy. He identified statistical differences among the lines. Matthew Feragne is also pursuing aspects of Objective 2 and developed a protocol for treating cotton bark withcellulases and pectinases followed bygently scraping the softened tissues in a CTAB RNA isolation solution. This method isolates ~100 µm of cells from the vascular cambia out toward the developing fiber bundles and includes the cells that we hypothesize are receiving and responding to a signal that initiates their differentiation into sclerenchymatous fiber cells. At the end of this reporting period, he was using left-over reagents fromlow-input RNA-Seq library preparation kits to "practice" the protocol for making high-quality RNA-Seq libraries. Daniel Fu was an undergraduate research student since the Fall semester of 2022. His project was not described in the original proposal. He made a series of lanolin pastes with different hormones and applied these pastes to growing cotton stems. After ~5 weeks of growth, stems were collected and analyzed with epifluorescence microscopy to assess the impact of the exogenous hormones on vascular cambia activity and fiber development. The hormone treatments included different auxins, different cytokinins, an auxin transport inhibitor, GA, ABA, brassinolide, and hydrogen peroxide as a reactive oxygen species. Although the literature from poplar argues that auxin in particular promotes vascular cambium activity, Daniel Fu did not observed a clear impact of the hormones he applied exogenously in a lanolin paste. Drs. Ayre and McGarry were PDs on a USDA Equipment Program Grant for a Leica LMD7 laser microdissection system. This equipment was installed in early February and is being used to advance Objective 2 for high resolution analysis of gene expression patterns in cotton stems. Our submitted proposal describes going off campus to use this equipment at other institutions and having the equipment on the UNT campus has tremendously benefited protocol development and optimization.
Publications
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