Abstract

Carotenoids are the main nutrients and coloring matter in the flesh of watermelon. However, the significant role of Orange gene in carotenoid accumulation and chromoplast differentiation remains uncharacterized in watermelon. In this study, the ClOr1 gene was obtained through homologous alignment, identified to be highly expressed in the red-fleshed watermelon LSW-177, and significantly associated with carotenoid content. A single-nucleotide polymorphism in the ClOr1 gene was detected and confirmed to distinguish well between white-fleshed and non-white-fleshed watermelon after cloning the ClOr1 from LSW-177 and W1-60, and aligning 428 germplasm resources of watermelon, which suggested that the ClOr1 could be taken as a key candidate gene to promote carotenoid accumulation in watermelon fruit flesh. Transient expression assay similarly revealed that ClOr1 is localized to the nucleus, and overexpression of ClOr1 accelerated fruit coloration by contributing a 1.8- to 19.2-fold increase of total carotenoid content in transgenic tomato lines. Furthermore, the ClOr1 gene silencing resulted in a prominent reduction in carotenoid accumulation in the fruits of LSW-177 using virus-induced gene silencing. A direct protein-protein interaction was identified between ClOr1 and ClPSY1 by Y2H and BiFC assays, and ClPSY1 was exhibited as a key gene based on its significant expression related to carotenoid content in the experimental materials. Hence, these results suggested that the ClOr1 plays a positive role in regulating carotenoid accumulation during development and ripening in watermelon fruit by increasing the activities of ClPSY1 protein.