IntroductionRoots have a central role in plant resource capture and are the interface between the plant and the soil that affect multiple ecosystem processes. Field pennycress (Thlaspi arvenseL.) is a diploid annual cover crop species that has potential utility for reducing soil erosion and nutrient losses; and has rich seeds (30-35% oil) amenable to biofuel production and as a protein animal feed. The objective of this research was to (1) precisely characterize root system architecture and development, (2) understand plastic responses of pennycress roots to nitrate nutrition, (3) and determine genotypic variance available in root development and nitrate plasticity.MethodsUsing a root imaging and analysis pipeline, the 4D architecture of the pennycress root system was characterized under four nitrate regimes, ranging from zero to high nitrate concentrations. These measurements were taken at four time points (days 5, 9, 13, and 17 after sowing).ResultsSignificant nitrate condition response and genotype interactions were identified for many root traits, with the greatest impact observed on lateral root traits. In trace nitrate conditions, a greater lateral root count, length, density, and a steeper lateral root angle was observed compared to high nitrate conditions. Additionally, genotype-by-nitrate condition interaction was observed for root width, width:depth ratio, mean lateral root length, and lateral root density.DiscussionThese findings illustrate root trait variance among pennycress accessions. These traits could serve as targets for breeding programs aimed at developing improved cover crops that are responsive to nitrate, leading to enhanced productivity, resilience, and ecosystem service.