Task Progress:
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The funding period March 2020 — March 2021 was one characterized by interruptions and delays due to the SARS-CoV2 pandemic. Much of the work planned for the year was significantly delayed due to closures and shutdown orders implemented by the State of Ohio to help control the spread of the virus. The three objectives for the year included the completion of sequencing on samples from the Plant Gravity Perception (PGP) flight experiments, bioinformatic analysis of the RNA-seq data arising from that sequencing, as well as completion of ground-based controls and mutant analysis. Progress on each of these objectives is described below.
Samples returned from flight, where they were exposed for 36 h to specific artificial gravity treatments using the European Modular Cultivation System (EMCS) centrifuge, were processed to extract RNA. Seedlings were dissected into 4 organs (cotyledons, hypocotyls, mature roots, and root tips) and RNA was extracted separately for each organ across a range of 12 gravity treatments for both WT and starchless (pgm-1) mutants. Each organ-gravity-genotype sample was replicated 4 times in the design to maximize statistical robustness. After confirming the quality of RNA samples via Bioanalyzer chip, they were shipped to a core facility for consistent library preparation using the Takara low-input library kit. Libraries were sequenced on an Illumina NovaSeq 6000 using paired-end, 150 bp reads. Both the library preparations and the sequencing were delayed due to laboratory shutdowns, with the final flight reads completed in November 2020.
Due to the delays in completing the sequencing process, we have only recently started to analyze the RNA-seq data, which totals 400 sample libraries and approximately 2 TB of data. We are coordinating the upload of the raw read files to NASA’s GeneLab platform for widespread availability of the data and analysis with the community. Fortunately, our collaborators, Sarah Wyatt and Al Meyers, have built and tested the analysis pipeline to be used for processing the data, using our previous pilot data set for development and optimization. We anticipate being able to complete the first analysis of these data by Summer 2021. These data will allow us to directly probe the effect of gravity as a continuum on plant seedling growth and establishment from a minimum of 0.006 g up to 1.0 g, representing the most complete and systematic look at the effect of this key variable on gene expression to date.
In addition to the work on preparing and sequencing flight samples, we also made progress on identifying novel components of the gravity signaling pathway in roots. We had previously identified a list of 124 genes showing contrasting expression patterns between WT and pgm-1 mutants under vertical compared to gravistimulated conditions. As a first step toward testing the roles of some of these genes in gravity response, students tested about 35 T-DNA lines with insertions in some of these genes. While the pandemic and ensuing shutdown precluded students from working in the lab over the summer, students made progress in Fall 2020 toward confirming the genotypes and transcript abundance in these mutant lines using RT-qPCR.
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Abstracts for Journals and Proceedings
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Meyers A, Wolverton C, Wyatt S. "Leveraging Molecular Data from Fractional Gravity Environments on Earth and ISS." Gravity Perception and Response in Plants and Fungi: Ground and Space Studies. Committee on Space Research (COSPAR) 2021-Hybrid, 43rd Scientific Assembly, Sydney, Australia, January 28-February 4, 2021. Committee on Space Research (COSPAR) 2021-Hybrid, 43rd Scientific Assembly, Sydney, Australia, January 28-February 4, 2021. , Jan-2021
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Abstracts for Journals and Proceedings
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Wolverton C, Meyers A, Wyatt S. "Searching for Components of the Non-Statolith Gravity Sensing System in Plants." Gravity Perception and Response in Plants and Fungi: Ground and Space Studies. Committee on Space Research (COSPAR) 2021-Hybrid, 43rd Scientific Assembly, Sydney, Australia, January 28-February 4, 2021. Committee on Space Research (COSPAR) 2021-Hybrid, 43rd Scientific Assembly, Sydney, Australia, January 28-February 4, 2021. , Jan-2021
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Articles in Peer-reviewed Journals
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Overbey EG, Saravia-Butler AM, Zhang Z, Rathi KS, Fogle H, da Silveira WA, Barker RJ, Bass JJ, Beheshti A, Berrios DC, Blaber EA, Cekanaviciute E, Costa HA, Davin LB, Fisch KM, Gebre SG, Geniza M, Gilbert R, Gilroy S, Hardiman G, Herranz R, Kidane YH, Kruse CPS, Lee MD, Liefeld T, Lewis NG, McDonald JT, Meller R, Mishra T, Perera IY, Ray S, Reinsch SS, Rosenthal SB, Strong M, Szewczyk NJ, Tahimic CGT, Taylor DM, Vandenbrink JP, Villacampa A, Weging S, Wolverton C, Wyatt SE, Zea L, Costes SV, Galazka JM. "NASA GeneLab RNA-seq consensus pipeline: Standardized processing of short-read RNA-seq data." iScience. 2021 Apr 23;24(4):102361. https://doi.org/10.1016/j.isci.2021.102361 ; PMID: 33870146; PMCID: PMC8044432 , Apr-2021
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