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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Plant, Soil and Nutrition Research » Research » Publications at this Location » Publication #337503

Research Project: Genetic and Genomic Basis of Vegetable and Fruit Biology, Quality and Nutrient Content

Location: Plant, Soil and Nutrition Research

Title: Distinct mechanisms of the ORANGE protein in controlling carotenoid flux in melon fruit

Author
item Chayut, Noam - Agricultural Research Organization Of Israel
item Yuan, Hui - Cornell University - New York
item Ohali, Shachar - Agricultural Research Organization Of Israel
item Meir, Ayala - Agricultural Research Organization Of Israel
item Sa'ar, Uzi - Agricultural Research Organization Of Israel
item Tzuri, Galil - Agricultural Research Organization Of Israel
item Zhang, Yi - Boyce Thompson Institute
item Mazourek, Michael - Cornell University - New York
item Gepstein, Shimon - Agricultural Research Organization Of Israel
item Zhou, Xiangjun - Cornell University - New York
item Portnoy, Vitaly - Agricultural Research Organization Of Israel
item Lewinsohn, Efraim - Agricultural Research Organization Of Israel
item Schaffer, Arthur - Agricultural Research Organization Of Israel
item Katzir, Nurit - Agricultural Research Organization Of Israel
item Fei, Zhangjun - Agricultural Research Organization Of Israel
item Welsch, Ralf - Agricultural Research Organization Of Israel
item Li, Li
item Burger, Joseph - Agricultural Research Organization Of Israel
item Tadmor, Yaakov - Agricultural Research Organization Of Israel

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/11/2016
Publication Date: 1/12/2017
Citation: Chayut, N., Yuan, H., Ohali, S., Meir, A., Sa'Ar, U., Tzuri, G., Zhang, Y., Mazourek, M., Gepstein, S., Zhou, X., Portnoy, V., Lewinsohn, E., Schaffer, A., Katzir, N., Fei, Z., Welsch, R., Li, L., Burger, J., Tadmor, Y. 2017. Distinct mechanisms of the ORANGE protein in controlling carotenoid flux in melon fruit. Plant Physiology. 173:376-389.

Interpretive Summary: Carotenoids are important to human nutrition and health. Orange melon fruit contains high level of beta-carotene, the provitamin A carotenoid. Our previous study reveals that two alleles of Or gene (CmOr) control orange or green/white flesh color in melon fruits. In this work, we isolated an EMS induced nonsense mutation in CmOr ('low-B'), which dramatically lowered beta-carotene level in melon fruit. By comparing 'low-B' to its isogenic progenitor, we show that an active CmOr is required to maintain PSY protein level and activity in developing melon fruit. However, by examining various melon accessions, we found that regulation of PSY level and activity is not the mechanism by which CmOr natural allelic variation governs fruit beta-carotene levels in melon. Our biochemical inhibition and genetic studies suggest that CmOr stabilizes beta-carotene and inhibits its turnover, resulting in beta-carotene accumulation that leads to chromoplast formation as a metabolite derived process. The current study sheds new light on the mechanism of action of the Or gene in melon fruit, which suggests metabolic flux analysis as a necessary criterion when selecting potential superior germplasm for DNA editing to introduce the Or into other crops.

Technical Abstract: Beta-carotene adds nutritious value and determines the color of many fruits including melon. In melon mesocarp, beta-carotene accumulation is governed by the Orange gene (CmOr) ‘golden’ SNP through a yet to be discovered mechanism. In Arabidopsis, OR increases carotenoid levels by posttranscriptionally regulating phytoene synthase (PSY). Here we identified a CmOr nonsense mutation (Cmor-lowB), which lowered fruit B-carotene levels with impaired chromoplast biogenesis. Cmor-lowB exerted a minimal effect on PSY transcripts but dramatically decreased PSY protein levels and enzymatic activity, leading to reduced carotenoid metabolic flux and accumulation. In contrast, the ‘golden’ SNP did not affect PSY protein levels and carotenoid metabolic flux in melon fruit, as shown by carotenoid and immunoblotting analyses of selected melon genotypes and by using chemical pathway inhibitors. The high B-carotene accumulation in ‘golden’ SNP melons was found to be due to a reduced further metabolism of B-carotene. This was revealed by genetic studies with double mutants including crtiso (‘yofi’), a carotenoid-isomerase nonsense mutant, which arrests the turnover of prolycopene. The ‘yofi’ F2 segregants accumulated prolycopene independently of the ‘golden’ SNP. Moreover, Cmor-lowB was found to inhibit chromoplast formation and chloroplast disintegration in fruits from 30 days after anthesis until ripening, suggesting that CmOr regulates chloroplast-to-chromoplast transition. Taken together, our results demonstrate that CmOr is required to achieve PSY protein levels for maintaining carotenoid biosynthesis metabolic flux, but that the mechanism of the CmOr 'golden' SNP involves an inhibited metabolism downstream to B-carotene to dramatically affect both carotenoid content and plastid fate.