Ding, X.; Duckworth, S.; Southworth, M.; Gautam, B.; Lipton, A.; Velickovic, D.; Sedbrook, J. C.; Handakumbura, P.

Pennycress is a winter annual intermediate crop with approximately 30% seed oil content suitable for producing biofuels. Here, we evaluated seed development, anatomy, and agronomically relevant traits of a transparent testa 8 knockout mutant (tt8-2bp) generated by CRISPR genome editing to improve seed quality. We performed histochemical analyses on wild-type and tt8-2bp seeds at different developmental stages. No visible anatomical defects were observed in embryos and endosperm of tt8-2bp seeds. However, tt8-2bp seed coats completely lost proanthocyanidins which were accumulated in an inner integument cell layer and in the thickened cell wall of an outer integument cell layer of wild-type seed coats. Based on spatial metabolomic and solid-state NMR analyses, tt8-2bp seed coats had decreased aromatic compounds and cell wall polysaccharides compared to wild-type seed coats. Additionally, tt8-2bp seeds had reduced seed coat dry weights and increased embryo dry weights compared to wild-type seeds, indicating changes in macronutrient partitioning during seed development. Mature tt8-2bp seeds exhibited increased imbibition rates and seed coat permeability to water-soluble molecules, suggesting a higher seed coat hydrophilicity than wild-type seeds. In conclusion, we did not find defects in tt8-2bp mutant seeds that were unfavorable agronomically, supporting that TT8 is an attractive target for pennycress domestication.