Abstract Field pennycress ( Thlaspi arvense ) is a new biofuel winter annual crop with extreme cold hardiness and a short life cycle, enabling off-season integration into corn and soybean rotations across the Midwest. Pennycress fields are susceptible to winter snow melt and spring rainfall, leading to waterlogged soils. The objective of this research was to determine if waterlogging during the reproductive stage had a significant effect on gene expression, morphology, physiology, recovery, and yield of two pennycress lines (SP32-10 and MN106). In a controlled environment, total pod number, shoot/root dry weight, and total seed count/weight were significantly reduced in SP32-10 in response to waterlogging, whereas primary branch number, shoot dry weight, and single seed weight were significantly reduced in MN106. This indicated waterlogging had a greater negative impact on seed yield in SP32-10 than MN106. The number of differentially expressed genes (DEGs) between waterlogged and control roots were doubled in MN106 (3,424) compared to SP32-10 (1,767). Functional enrichment analysis of upregulated DEGs revealed Gene Ontology (GO) terms associated with hypoxia and decreased oxygen, with genes in these categories involved in alcoholic fermentation and glycolysis. Interestingly, MN106 waterlogged roots exhibited significant stronger upregulation of these genes than SP32-10. Additionally, downregulated DEGs revealed GO terms associated with cell wall biogenesis and secondary metabolite biogenesis, indicating suppressed growth and energy conservation. Together, these results reveal the reconfiguration of cellular and metabolic processes in response to the severe energy crisis invoked by waterlogging in pennycress.
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