Distinct Mechanisms of the ORANGE Protein in Controlling Carotenoid Flux.
β-Carotene adds nutritious value and determines the color of many fruits, including melon (Cucumis melo). In melon mesocarp, β-carotene accumulation is governed by the Orange gene (CmOr) golden single-nucleotide polymorphism (SNP) through a yet to be discovered mechanism. In Arabidopsis (Arabidopsis thaliana), OR increases carotenoid levels by posttranscriptionally regulating phytoene synthase (PSY). Here, we identified a CmOr nonsense mutation (Cmor-lowβ) that lowered fruit β-carotene levels with impaired chromoplast biogenesis. Cmor-lowβ exerted a minimal effect on PSY transcripts but dramatically decreased PSY protein levels and enzymatic activity, leading to reduced carotenoid metabolic flux and accumulation. However, the golden SNP was discovered to not affect PSY protein levels and carotenoid metabolic flux in melon fruit, as shown by carotenoid and immunoblot analyses of selected melon genotypes and by using chemical pathway inhibitors. The high β-carotene accumulation in golden SNP melons was found to be due to a reduced further metabolism of β-carotene. This was revealed by genetic studies with double mutants including carotenoid isomerase (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-lowβ was found to inhibit chromoplast formation and chloroplast disintegration in fruits from 30 d after anthesis until ripening, suggesting that CmOr regulates the chloroplast-to-chromoplast transition. Taken together, our results demonstrate that CmOr is required to achieve PSY protein levels to maintain carotenoid biosynthesis metabolic flux but that the mechanism of the CmOr golden SNP involves an inhibited metabolism downstream of β-carotene to dramatically affect both carotenoid content and plastid fate.
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Noam
0000-0002-2695-5521
N
Chayut
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Hui
0000-0002-8309-6276
H
Yuan
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Shachar
S
Ohali
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Ayala
A
Meir
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Uzi
U
Sa'ar
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Galil
0000-0003-2342-8125
G
Tzuri
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Yi
0000-0002-8042-7770
Y
Zheng
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Michael
M
Mazourek
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Shimon
0000-0003-2589-6299
S
Gepstein
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Xiangjun
0000-0002-9086-6996
X
Zhou
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Vitaly
0000-0003-1130-6788
V
Portnoy
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Efraim
E
Lewinsohn
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Arthur A
AA
Schaffer
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Nurit
0000-0001-8858-8395
N
Katzir
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Zhangjun
0000-0001-9684-1450
Z
Fei
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Ralf
0000-0002-2865-2743
R
Welsch
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Li
0000-0002-4352-4061
L
Li
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.).|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.).|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.).|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.).
Joseph
J
Burger
Department of Vegetable Research, Agricultural Research Organization, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel (N.C., S.O., A.M., U.S., G.T., V.P., E.L., A.A.S., N.K., J.B., Y.T.); tadmory@agri.gov.il.|Plant Breeding and Genetics Section, School of Integrative Plant Science (H.Y., M.M., X.Z., L.L.), and Boyce Thompson Institute (Y.Z., Z.F.), Cornell University, Ithaca, New York 14853; tadmory@agri.gov.il.|Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel (N.C., S.G.); tadmory@agri.gov.il.|Department of Vegetable Research, Volcani Center, Agricultural Research Organization, Bet Dagan 50250, Israel (A.A.S.); tadmory@agri.gov.il.|United States Department of Agriculture-Agricultural Research Service Robert W. Holley Center for Agriculture and Health, Ithaca, New York 14853 (Z.F., L.L.); and tadmory@agri.gov.il.|Faculty of Biology II, University of Freiburg, Freiburg 79098, Germany (R.W.) tadmory@agri.gov.il.
Yaakov
0000-0003-3311-2503
Y
Tadmor
D000595:Amino Acid Sequence; D053898:Biosynthetic Pathways / Q000235:genetics; D002338:Carotenoids / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D028463:Cucumis melo / Q000235:genetics / Q000378:metabolism*; D060146:Ecotype; D004843:Epistasis, Genetic; D005020:Ethyl Methanesulfonate; D005638:Fruit / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D014644:Genetic Variation; D005838:Genotype; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase / Q000378:metabolism; D064688:Metabolic Flux Analysis; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010858:Pigmentation / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D012333:RNA, Messenger; D002338:Carotenoids; C100185:(all-E) phytoene; D005020:Ethyl Methanesulfonate; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase