A 'golden' SNP in CmOr governs the fruit flesh color of melon (Cucumis melo)

The flesh color of Cucumis melo (melon) is genetically determined, and can be white, light green or orange, with -carotene being the predominant pigment. We associated carotenoid accumulation in melon fruit flesh with polymorphism within CmOr, a homolog of the cauliflower BoOr gene, and identified CmOr as the previously described gf locus in melon. CmOr was found to co-segregate with fruit flesh color, and presented two haplotypes (alleles) in a broad germplasm collection, one being associated with orange flesh and the second being associated with either white or green flesh. Allelic variation of CmOr does not affect its transcription or protein level. The variation also does not affect its plastid subcellular localization. Among the identified single nucleotide polymorphisms (SNPs) between CmOr alleles in orange versus green/white-flesh fruit, a single SNP causes a change of an evolutionarily highly conserved arginine to histidine in the CmOr protein. Functional analysis of CmOr haplotypes in an Arabidopsis callus system confirmed the ability of the CmOr orange haplotype to induce -carotene accumulation. Site-directed mutagenesis of the CmOr green/white haplotype to change the CmOR arginine to histidine triggered -carotene accumulation. The identification of the golden' SNP in CmOr, which is responsible for the non-orange and orange melon fruit phenotypes, provides new tools for studying the Or mechanism of action, and suggests genome editing of the Or gene for nutritional biofortification of crops. Significance Statement Our work demonstrates that a change of arginine to histidine in an evolutionarily highly-conserved location in Or proteins, is responsible for b-carotene accumulation in melon fruit and is capable to induce b-carotene accumulation in a heterologous system. This golden SNP could lead not only to better understanding of the mechanism underlying Or-induced carotenoid accumulation, but also to new approaches for biofortification of agricultural products with pro-vitamin A via genome-editing technology.