Transgene-free genome editing supports CCD4 role as a negative regulator of β-carotene in banana

This study aims to understand the regulatory mechanism of the β-carotene homeostasis by establishing transgene-free genome editing in banana. Carotenoid cleavage dioxygenases (CCDs) belong to a miniature gene family having an imperative role in the intricated carotenoid metabolism in plants. Here, the expression pattern of multiple CCDs was correlated with the levels of carotenoid accumulation in two contrasting cultivars, viz., Nendran (high β-carotene) and Rasthali (low β-carotene). The higher expression of the RAS-CCD4 inversely correlated with β-carotene accumulation in fruit-pulp of the Rasthali. The docking analysis followed enzyme assay of purified RAS-CCD4 suggested β-carotene and 10-apo-β-carotenal as its preferred substrates. Bacterial complementation assay affirmed RAS-CCD4 role in β-carotene degradation and then overexpression of the RAS-CCD4 in the Arabidopsis thaliana further validated results in-vivo by the significant reduction in β-carotene. Subsequently, CRISPR/Cas9 mediated editing of CCD4 was demonstrated in the protoplasts and embryogenic cell lines of Rasthali. The carotenoid profiling in stable mutant lines revealed higher fold β-carotene accumulation in non-green tissue (roots) than in green tissue (leaf) compared with the unedited control plants. The differential expression of carotenoid pathway genes was correlated with the metabolites in the edited lines. The study suggests that carotenoid catabolism is regulated by the CCD4 in tissue and cultivar specific manners, and also demonstrated the use of the genome editing tool in developing transgene-free biofortified banana.