Transgene-free genome editing supports the role of carotenoid cleavage dioxygenase 4 as a negative regulator of β-carotene in banana

Carotenoid cleavage dioxygenases (CCDs) belong to a small gene family and have an important role in the intricate metabolism of carotenoids in plants. In this study we aimed to understand the regulatory mechanism of beta-carotene homeostasis by establishing transgene-free genome editing in banana. We found that the expression patterns of multiple CCDs were correlated with the concentrations of carotenoids in two cultivars with contrasting contents of beta-carotene, Nendran (high) and Rasthali (low). Higher expression of CCD4 in Rasthali (RAS-CCD4) was negatively correlated with beta-carotene accumulation in the fruit-pulp. Docking analysis and enzyme assays of purified RAS-CCD4 suggested that beta-carotene and 10 '-apo-beta-carotenal were the preferred substrates of RAS-CCD4. Bacterial complementation assays demonstrated the role of RAS-CCD4 in beta-carotene degradation, and this was further confirmed by in vivo overexpression of RAS-CCD4 in Arabidopsis, which resulted in significant reduction in beta-carotene concentration. CRISPR/Cas9-mediated editing of CCD4 was conducted in protoplasts and embryogenic cell lines of Rasthali, and carotenoid profiling in the resulting stable mutant lines showed greater increases in beta-carotene accumulation in the roots than in the leaves compared with unedited controls. The differences in expression of carotenoid pathway genes were correlated with differences in metabolites in the edited lines. Overall, our study suggests that carotenoid catabolism is regulated by CCD4 in ways that are tissue- and cultivar-specific, and it also demonstrates the successful use of the genome-editing tool in developing transgene-free biofortified lines of banana.