Comparison of Physiological and Metabolomic Responses to Drought Across Pennycress CRISPR Mutants and Natural Accessions
Jason Thomas1* (email@example.com), Charles Hawkins1, Liza Gautam2, Xinxin Ding3, Chaevien Clendinen3, Carol Kiam Assato2, John Lagergren4, Daniel Jacobson4, John Sedbrook2 (firstname.lastname@example.org), Pubudu Handakumbura3, and Seung Y. Rhee1
1Carnegie Institution for Science, Stanford, CA; 2Illinois State University; 3Pacific Northwest National Laboratory; and 4Oak Ridge National Laboratory
This project employs evolutionary and computational genomic approaches to identify key genetic variants that have enabled Thlaspi arvense L. (Field Pennycress; pennycress) to locally adapt and colonize all temperate regions of the world. This, in combination with knowledge of metabolic and cellular networks derived from first principles, is guiding precise laboratory efforts to create and select high-resilience lines, both from arrays of random mutagenesis and by employing cutting-edge CRISPR genome editing techniques. This project will deliver speed-breeding methods and high-resilience mutants inspired by natural adaptations and newly formulated biological principles to be introduced into a wide range of commercial pennycress varieties to precisely adapt them to the desired local environments.
Field pennycress is an overwintering bioenergy cover crop that is rapidly being domesticated. It produces oilseeds that can be converted into various products from cooking oil to renewable diesel and sustainable aviation fuel (SAF). By replacing fossil fuels, pennycress directly combats climate change. However, abiotic stresses brought by climate change threatens stable production of many crops including pennycress. Pennycress’s drought response has not been studied in much detail. Here researchers explored the drought responses of wild-type, gene-edited, and natural accessions by physiological assays, metabolic profiling, and data integration via a genome-scale metabolic pathway database for pennycress. Team members first measured phenotypes relevant to drought stress in the reference line Spring32-10 seedlings and plants including yield, biomass, stomatal conductance, and photosynthetic efficiency over various drought conditions. Researchers subjected plants to drought and control conditions and then collected above- and belowground tissues for metabolomic analyses via liquid chromatography coupled mass spectrometry, which allows detection of changes in plant metabolism during drought stress. These data will be analyzed in the context of the PennycressCyc database the team created for the pennycress community. Additionally, team members generated pennycress single, double, and triple mutants using CRISPR-Cas9 mutagenesis targeting 10 genes important for drought responses in other species. These mutants, along with a subset of 800 pennycress worldwide natural accessions predicted to have varied levels of drought resilience based on the team’s climatype predictions, were subjected to drought conditions and phenotyped to identify relative differences in drought responses. Taken together, this work helps decipher how pennycress uniquely responds to drought stress and is identifying natural and induced genetic changes that could improve pennycress drought resilience.
This research is supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program grant no. DE-SC0021286.