AbstractEnvironmentally-responsive genes can affect fruit red colour via the activation of MYB transcription factors. The apple B-box (BBX) gene, BBX33/CONSTANS-like 11 (COL11) has been reported to influence apple red-skin colour in a light- and temperature-dependent manner. To further understand the role of apple BBX genes, other members of the BBX family were examined for effects on colour regulation. Expression of 23 BBX genes in apple skin was analysed during fruit development. We investigated the diurnal rhythm of expression of the BBX genes, the anthocyanin biosynthetic genes and a MYB activator, MYB10. Transactivation assays on the MYB10 promoter, showed that BBX proteins 1, 17, 15, 35, 51, and 54 were able to directly function as activators. Using truncated versions of the MYB10 promoter, a key region was identified for activation by BBX1. BBX1 enhanced the activation of MYB10 and MdbHLH3 on the promoter of the anthocyanin biosynthetic gene DFR. In transformed apple lines, over-expression of BBX1 reduced internal ethylene content and altered both cyanidin concentration and associated gene expression. We propose that, along with environmental signals, the control of MYB10 expression by BBXs in ‘Royal Gala’ fruit involves the integration of the expression of multiple BBXs to regulate fruit colour.