|Ph.D Student||Chayut Noam|
|Subject||Characterization of the Orange Gene Regulatory Network in|
Cucumis Melo for Carotenoid Biofortification in
|Department||Department of Biology||Supervisors||Professor Emeritus Shimon Gepstein|
|Dr. Joseph Burger|
|Dr. Yaakov Tadmor|
|Full Thesis text|
Background: The Orange (Or) gene of cauliflower (BoOr) represents the only known gene that acts as a dominant molecular switch for chromoplast biogenesis and thus has strong potential for biofortification of crops with pro-vitamin A carotenoids. However, the BoOr large copia element insertion causes a pleiotropic effect, thus studying the exact mechanisms by which Or induces carotenoid accumulation and the proteins/genes in its regulatory network is difficult in cauliflower. As a result, the efficient utilization of Or for carotenoid biofortification in crops is still hindered. Recently, we found that a single nucleotide polymorphism in melon (Cucumis melo) Or (CmOr) governs fruit β-carotene accumulation causing orange mesocarp pigmentation with no pleiotropic effects. This made CmOr an ideal system to study the Or gene mechanism. The goal of this work was to decipher the molecular roles of CmOr and its mode of action by answering the following questions:
1. What metabolic and cellular processes are associated with CmOr allelic variation?
2. Does CmOr regulate the expression of genes in the metabolic pathway leading to β-carotene accumulation?
3. Does CmOr govern the metabolic flux leading to β-carotene accumulation?
Results: We conducted bulked segregants RNA-seq analysis (BSR-seq) of homozygous orange and green fruited F3 families, derived from a cross between orange and green fruited parental lines. Differentially expressed genes were clustered into functional groups to enable the identification of metabolic and cellular processes associated with CmOr allelic variation. The relatively enriched functional groups were those involved in photosynthesis, RNA and protein regulation, and response to stress. Interestingly, carotenogenesis genes expression was not affected by the CmOr allelic variation. Transcriptome analysis of ‘low-β’, a novel induced nonsense (loss of function) mutation in CmOr, showed that CmOr didn’t regulate the RNA levels of the carotenoid metabolic genes, including the rate limiting phytoene synthase (Psy-1). However, CmOr regulated PSY-1 protein levels. Surprisingly, the natural CmOr allelic variation did not affect PSY-1 protein levels. To measure the effect of CmOr on fruit carotenoid metabolic flux in planta, we chemically inhibited enzymes in detached fruit flesh discs, and followed carotenoid accumulation at several time points. We showed that the ‘low-β’ nonsense CmOr mutation lowered carotenoid metabolism rate in developing fruits . Carotenoid analysis of the ‘low-β’ mutant and of CmOr allelic variation under the genetic background of inactive carotenoid isomerase (crtiso) further validated that a metabolic flux inhibition was caused by the ‘low-β’ mutation but not by CmOr natural allelic variation.
Conclusion: The work on CmOr provided melon breeders with a reliable and efficient molecular marker that defines fruit flesh color phenotype. Moreover, our results led us to conclude that CmOr enhances β-carotene accumulation by two mechanisms: stabilizing PSY-1 protein to boost metabolic flux and preventing the turnover of β-carotene in a yet to be revealed manner. More importantly, the results defined a precise target for DNA alteration in the Or gene as a promising new path toward vitamin A biofortification of other crops.