Discovery and Deployment of Herbicide Tolerance in Domesticated Pennycress ( Thlaspi arvense L.) to Enable Modern Agronomic Integration

ABSTRACT Modern agronomic systems for large‐scale oilseed production rely heavily on chemical weed control, presenting a barrier to the adoption of newly domesticated crops that lack herbicide tolerance. Pennycress ( Thlaspi arvense L.), a domesticated winter oilseed developed to increase land productivity and support sustainable biofuel production, is highly sensitive to commonly used herbicides targeting acetolactate synthase (ALS) and 4‐hydroxyphenylpyruvate dioxygenase (HPPD). Here, we report complementary strategies for identifying and deploying herbicide‐tolerant alleles in pennycress. ALS inhibitor tolerance was identified through Ethyl methanesulfonate (EMS)‐derived chemical mutagenesis, whereas HPPD inhibitor tolerance was explored using a microbial‐based rational enzyme design platform as proof of concept. Selected alleles were introduced into advanced germplasm via homology‐directed repair (HDR) and evaluated across developmental stages. Plants carrying the P187S mutation in the ALS gene exhibited robust tolerance at both seedling and rosette stages without detectable penalties to growth or seed yield. In contrast, the G419A allele in the HPPD gene conferred tolerance to residual herbicide activity when overexpressed but showed limited efficacy when expressed from its native locus, indicating context‐dependent functionality. Collectively, this study demonstrates the feasibility of integrating herbicide tolerance into a newly domesticated oilseed crop and highlights the utility of combining mutagenesis‐based discovery, rational enzyme engineering, and precision genome editing to support agronomic compatibility and crop deployment.