CsBBX24 regulates both carotenoid and anthocyanin biosynthesis in citrus

Abstract Carotenoids and anthocyanins are the two major classes of pigments in plants and are bioactive compounds with nutritional value in fruit crops. The color diversity in citrus (Citrus spp.) is attributed to the varying levels and composition of carotenoids and anthocyanins. In blood orange (Citrus sinensis) fruits, which accumulate both carotenoids and anthocyanins, these pigments exhibit coordinated changes during ripening; however, the mechanisms underlying this accumulation remain unclear. Here, we reveal that B-BOX DOMAIN PROTEIN 24 (CsBBX24) regulates both carotenoid and anthocyanin biosynthesis during the ripening of blood orange fruits. CsBBX24 was co-expressed with the carotenoid biosynthesis gene PHYTOENE SYNTHASE 1 (CsPSY1) and the anthocyanin regulatory gene CsRuby1, which encodes a MYB-domain protein during fruit ripening. Ectopic expression of CsBBX24 in Hongkong kumquat (C. hindsii), tomato (Solanum lycopersicum), and apple (Malus domestica) calli promoted the accumulation of carotenoids and anthocyanins. An electrophoretic mobility shift assay and transient dual-luciferase assays in Nicotiana benthamiana leaves demonstrated that CsBBX24 can bind to and activate the CsPSY1 promoter for carotenoid biosynthesis and the CsRuby1 promoter for regulation of anthocyanin biosynthesis. Our findings have elucidated the role of CsBBX24 in promoting pigment accumulation during fruit ripening in blood orange, providing a valuable target gene for molecular breeding to improve citrus coloration quality.