Reducing the Sinapine Levels of Camelina sativa Seeds Through Targeted Genome Editing of REF1

Sinapine (O-sinapoyl choline) is the major phenolic metabolite typically found in the oil-rich seeds of Brassicaceae such as Camelina sativa and Brassica napus. It imparts a bitter taste to the seeds as a defence mechanism against herbivores, but it also renders them less palatable to livestock. To improve Camelina flour for human consumption or as animal feed, we reduced sinapine content through CRISPR/Cas9-based genome editing of REF1 (REDUCED EPIDERMAL FLUORESCENCE1), which encodes the NADP⁺-dependent coniferaldehyde/sinapaldehyde dehydrogenase (CALDH/SALDH), a key enzyme for sinapine biosynthesis in Arabidopsis thaliana and B. napus. Inactivation of all three homoeologues found in C. sativa lowered the sinapine content in seeds by an overall 56% in two cultivars indicating the presence of a REF1-independent pathway for sinapine biosynthesis. Most importantly however, crucial agronomic seed traits such as total lipid or protein content of the seeds, seed weight or germination were not affected. Hence, the ref1 mutant lines produced here provide a valuable trait, that can be combined with other traits through gene stacking to obtain crops with significantly improved product quality. Furthermore, metabolite fingerprinting by ultra-performance liquid chromatography-electrospray ionisation-quadrupole time-of-flight mass spectrometry of ref1 mutant lines revealed a contrasting phenylpropanoid profile in seeds and leaves, indicating that REF1 oxidises sinapaldehyde to sinapate in seeds and coniferyl aldehyde to ferulate in leaves. In contrast to Arabidopsis however, Camelina accumulates no comparable levels of sinapoyl malate, but substantial amounts of chlorogenic acid, of which an additional chlorogenic acid isomer distinguishes the two different Camelina cultivars as a metabolite marker.