Transgene-free gene editing of BnaCCD4 homologs significantly improves the carotenoid level of seeds in Brassica napus

Abstract

The absorption of carotenoids, plant pigments with significant health benefits, is promoted when ingested with lipids. However, rapeseed carotenoid contents are relatively low. Our previous study identified that the inactivation of BnaC3.CCD4, a carotenoid cleavage dioxygenase which degrades carotenoids, in rapeseed petals, increased carotenoid accumulation. The present study created loss-of-function single, double, and triple mutants of three other CCD4 copies using a CRISPR/Cas9 system. The results showed that some double and triple mutants exhibited a more than 20-fold increase in seed β-carotene contents without harming quality or yield. In addition, the carotenoid content of BnaC1.ccd4 was close to those of some triple and double mutants. During seed maturation and leaf senescence, BnaCCD4 mutations delayed carotenoid degradation. The reduced power activities and hydroxyl radical scavenging activities of mutant seed oils indicated increased antioxidation. Enzyme activity analysis of BnaCCD4s in Arabidopsis ccd4 mutant seeds confirmed diverging oxidative cleavage activities on carotenoids.