Characterization of the Or regulatory network in melon for carotenoid biofortification in food crops

The general goals of the BARD research grant US-4423-11 are to understand how Or regulates carotenoid accumulation and to reveal novel strategies for breeding agricultural crops with enhanced β-carotene level. The original objectives are: 1) to identify the genes and proteins in the Or regulatory network in melon; 2) to genetically and molecularly characterize the candidate genes; and 3) to define genetic and functional allelic variation of these genes in a representative germplasm collection of the C. melo species.  Or was found by the US group to causes provitamin A accumulation in chromoplasts in cauliflower. Preliminary genetic study from the Israeli group revealed that the melon Or gene (CmOr) completely co-segregated with fruit flesh color in a segregating mapping population and in a wide melon germplasm collection, which set the stage for the funded research.   Major conclusions and achievements include: 1). CmOris proved to be the gene that controls melon fruit flesh color and represents the previously described gflocus in melon. 2). Genetic and molecular analyses of CmOridentify and confirm a single SNP that is responsible for the orange and non-orange phenotypes in melon fruit. 3). Alteration of the evolutionarily conserved arginine in an OR protein to both histidine or alanine greatly enhances its ability to promote carotenoid accumulation. 4). OR promotes massive carotenoid accumulation due to its dual functions in regulating both chromoplast biogenesis and carotenoid biosynthesis. 5). A bulk segregant transcriptome (BSRseq) analysis identifies a list of genes associated with the CmOrregulatory network. 6). BSRseq is proved to be an effective approach for gene discovery. 7). Screening of an EMS mutation library identifies a low β mutant, which contains low level of carotenoids due to a mutation in CmOrto produce a truncated form of OR protein. 8). low β exhibits lower germination rate and slow growth under salt stress condition. 9). Postharvest storage of fruit enhances carotenoid accumulation, which is associated with chromoplast development.   Our research uncovers the molecular mechanisms underlying the Or-regulated high level of carotenoid accumulation via regulating carotenoidbiosynthetic capacity and storage sink strength. The findings provide mechanistic insights into how carotenoid accumulation is controlled in plants. Our research also provides general and reliable molecular markers for melon-breeding programs to select orange varieties, and offers effective genetic tools for pro-vitamin A enrichment in other important crops via the rapidly developed genome editing technology. The newly discovered low β mutant could lead to a better understanding of the Or gene function and its association with stress response, which may explain the high conservation of the Or gene among various plant species.